key: cord-010222-5oxie0zc authors: oldstone, michael b.a. title: virus-induced autoimmunity: molecular mimicry as a route to autoimmune disease date: 2004-04-11 journal: j autoimmun doi: 10.1016/0896-8411(89)90130-3 sha: doc_id: 10222 cord_uid: 5oxie0zc nan viruses are implicated in autoimmunity on the basis of three findings. first, autoimmune responses are made de nova, or those already present are enhanced concomitant with infection by a wide variety of human dna and rna viruses. this point is strengthend by the second finding that, in experimental animals, both acute and persistent virus infections can induce, accelerate, or enhance autoimmune responses and cause autoimmune disease. for example, we have shown that autoimmune manifestations normally present in nzb mice (anti-dna antibodies, antired blood cell antibodies) or their (nzb x w)f, relatives (anti-dna antibodies) are enormously enhanced by persistent infection with dna (polyoma) or rna (lymphocytic choriomeningitis, lcmv) viruses; that is, antibodies form earlier and reach for higher titers in the infected mice than in their uninfected counterparts [ 1, 21. further, the nzw mice, which normally do not develop these autoimmune responses, do so upon polyma or lcmv infections. indeed, the responses in nzb, (nzb x w)f, or nzw mice are so marked that autoimmune diseases occur at a higher incidence with earlier time of death [nzb, (nzb x w)f,] or appear de novo (nzw) [2, 31. a number of viruses, including retroviruses, are now known to perform similarly. third, by evaluating molecular mimicry in human autoimmune disorders (figure l) , we have uncovered a number of potential etiologic agents and mechanisms of autoimmune disease [4] . viruses can induce autoimmune responses in many ways. for example, certain viruses have a mitogenic affect on unique blood lymphocyte subsets and hence can act as polyclonal activators. viruses also direct the release of lymphokines and monokines. these molecules are important modulators of immune responses by acting directly as growth or differentiation factors or by regulating the expression of class i and class ii major histocompatibility molecules (i.e. interferon effect). viruses can also replicate selectively in particular lymphocyte subsets. by their presence, activation or replication they can cause immunosuppression or immunoenhancement (reviewed in [5, 61) . moreover, some viruses and other microbes contain chemical structural components that mimic normal host 'self proteins. an effector immune response, either b (humoral) or t (cytotoxic t cell), directed against the microbe might than also cross-react with 'self' protein, thereby evoking autoimmunity. the events of this scenario, termed molecular mimicry, comprise the body of this report. we define molecular mimicry as similar structures shared by molecules from dissimilar genes or by their protein products. either the molecules' linear amino acid sequences or their conformational fit may be shared, even though their origins are as separate as, for example, a virus and a normal host self determinant. because guanine-cysteine (cc) sequences and introns designed to be spliced away may provide, respectively, false hybridization signals and nonsense homologies, molecular mimicry must be analyzed at the protein level. such homologies between proteins have been detected either by use of immunologic reactants, humoral or cellular, that cross-react with two presumably unrelated protein structures, or by matching proteins described in computer storage banks. regardless of the methods used for identification, it is now abundantly clear that molecular mimicry is common between proteins encoded by numerous dna and rna viruses and host 'self proteins. such events are relevant not only to autoimmunity but also as a likely mechanism by which viral proteins are processed inside cells [7] . examples of molecular mimicry were first described as such in the early 1980s by investigators who found that monoclonal antibodies against sv40 t antigens crossreacted with host cell proteins [8] . however, the importance of this observation became apparent when others realized that the monoclonal antibodies against a battery of viruses were cross-reacting with host determinants. for example, we showed cross-reactivity between measles virus phosphoprotein (72 k molecular weight) and the cytoskeleton component keratin (54 k molecular weight), and between a herpes simplex virus glycoprotein of 140 k and a separate epitope on keratin from that recognized by the measles virus phosphoprotein [9] . further, we to determine the possible frequency of molecular mimicry, srinivasappa and his colleagues [lo] tested over 600 monoclonal antibodies obtained from several laboratories, all raised against viral polypeptides. these investigators then charted the incidence of the monoclonals' cross-reactivity with host proteins expressed in a large panel of normal tissues. in their analysis and our subsequent studies monoclonal antibodies were reactive with 14 different viruses, including such commonly found representatives dna and rna viruses as the herpesvirus group, vaccinia virus, myxoviruses, paramyxoviruses, arenaviruses, flaviviruses, alphaviruses, rhabdoviruses, coronaviruses, and human retroviruses. most important, over 4% of such monoclonals cross-reacted with host-cell determinants expressed on uninfected tissues. some of these monoclonal antiviral antibodies reacted with constituents of more than one organ. hence, from these data, it is clear that molecular mimicry is common and not restricted to any specific class or group or virus. these and other reported cross-reactivities (table 1) raise interesting questions about the etiology and pathogenesis of a number of autoimmune diseases. because the studies described above indicate that many viruses share antigenic determinants (linear or conformation) with normal host proteins, the next step was to determine experimentally whether molecular mimicry could elicit autoimmune diseases. myelin basic protein was chosen as the host component to study because its entire amino acid sequence is known, and its encephalitogenic site of 8-10 amino acids has been mapped in several animal species. by computer-assisted analysis, several viral proteins listed in the dayhoff files showed significant homology with the encephalitogenic site of myelin basic protein. included were similarities and/or fits between myelin basic protein and the nucleoprotein and hemagglutinin of influenza virus, coat protein of polyoma virus, core protein of the adenovirus, polyprotein of poliomyelitis virus, ec-lf2 protein of epstein-barr virus (ebv), hepatitis b virus polymerase (hbvp), and others. however, the best fit occurred between the myelin basic protein encephalitogenic site in the rabbit and hbvp (figure 2 ). interestingly, products of immune responses, both humoral and cellular, generated in rabbits inoculated with the octamer or decamer viral peptide reacted with whole myelin basic protein. further, inoculation of the hbvp peptide into rabbits caused perivascular infiltration localized to the central nervous system, reminiscent of the disease induced by inoculation of either whole myelin basic protein or the encephalitogenic site of myelin basic protein (figure 2) [l 11. this experiment was seminal in that it conclusively showed molecular mimicry could cause both autoimmune responses and autoimmune disease. the most likely explanation for how molecular mimicry causes disease is that an immune response against the determinant shared by host and virus takes the form of a tissue-specific attack, presumably capable of destroying cells and eventually the tissue. the probable mechanism is generation by the pathogen of cytotoxic cross-reactive effector lymphocytes or antibodies that recognize specific determinants of 'self protein' located on target cells. interestingly, the induction of cross-reactivity would not require a replicating agent, and the immunologically mediated injury could occur after removal of the pathogen-a hit-and-run event. clearly, the virus infection that initiates an autoimmune phenomenon need not be present at the time overt disease develops. a likely scenario would be that the virus responsible for inducing a crossreacting immune response is cleared initially, but the components of that immunity continue to assault host elements. the cycle continues as the autoimmune response itself leads to tissue injury that, in turn, releases more self antigen, thereby inducing more antibodies, and so on. such a sequence might account for the viral encephalopathies occurring in humans after measles, mumps, vaccinia, or herpes-zoster virus infections; in these post-infectious diseases, recovery of the inducing agent has been rare. this theory is reinforced by studies showing that, after any of several acute viral infections, mononuclear cells from the peripheral blood or cerebral spinal fluid proliferate in response to host antigens, one of which is myelin basic protein. interestingly, several clonal populations of lymphocytes harvested from central nervous system fluids of humans with encephalitis proliferate in response to the infecting virus as well as to antigens of the nervous system. viruses or microbes also play by other game plans. for example, a virus with the capacity to persist in its host may continuously or cyclically express its antigens. although expression of a viral genome may be restricted so that no infectious virus replicates, production of a viral determinant in common with that of the host might continue. this would allow initiation of an immune response and/or autoimmunity, either one leading to cyclic, chronic, or progressive disease. in any case, molecular mimicry would occur only when the virus and host determinants are sufficiently similar to induce a cross-reactive response yet different enough to break b or t-cell immunologic tolerance. lastly, evidence for the hypothesis that molecular mimicry causes autoimmune disease in humans emanates from studies on pathogenesis of the non-rheumatoid arthritides, ankylosing spondylitis (as) and reiter's syndrome (rs) [12-151, and on myasthenia gravis [15, 161. six consecutive amino acids (qtdred) are identical between the hypervariable domain of hla-b27 and k. pneumoniae nitrogenase (table 2) . sera from a significant proportion of hla-b27 individuals with rs (18 of 34) or as (7 of 24), but not from appropriate controls, reacted with a synthetic peptide containing the homologous region of hla-b27.1 and k. pneumoniae nitrogenase [ 121. these results with american patients were confirmed by analysis of an additional 27 sera obtained from the netherlands [ ivani and oldstone, unpublished results, 19881 . as expected, sera from the latter as and rs patients also reacted with a peptide derived from the k. pneumoniae nitrogenase (cnsrqtdredeli). chen et al. [ 171 found that antibody to the hypervariable region of hla-b27.1 is reactive with yersinia (strain-pseudo tuberculosis), another microbe implicated in the as/rs arthritides. these observations suggest that rs and as are autoimmune diseases, with hla-b27 serving as the autoantigen. induction would be caused by a microbe(s) encoding a protein with sequence homology to hla-b27 (variable region), and the disease would presumably be related to an unusual concentration of hla antigen in certain tissues-like joints. to address this latter issue, material was obtained from the joints of patients with as and others with non-as arthropathies. accordingly, joint tissue from 11 of 12 as patients studied heavily expressed hla-b27 sequences, i.e. qtdred occurred in synovial cells lining the joint space and in endothelial cells in synovial tissue [ 131. in contrast, these sequences were not observed either in synovial tissue of individuals with non-arthropathies or in endothelial and other cells obtained by skin biopsy from non-as but hla-b27 positive individuals. the large majority of patients with the autoimmune disease, myasthenia gravis, characteristically have detectable antibodies against the acetylcholine (achr). the nicotinic achr is composed of multiple subunits responsible for gating ion flow across membranes in response to binding of the neurotransmitter, acetylcholine. the actions of achr antibodies, either experimentally induced or spontaneous, lead to the numerical reduction of available achr and prevention of the neuromuscular junction's ability to transmit signals from nerve fibers to muscle fibers; medically, the outcome is myastheia gravis. for molecular dissection of the physiologically important binding sites, synthetic peptides representing unique regions of the achain of achr have been used in association with a-bungarotoxin to compete with the binding of antibody specific for achr. these interactions also map accessible sites on achr that bind antibodies. utilizing the strategy outlined in figure 1 , we found immunochemically significant cross-reactivity with the human achr a-chain amino acid sequences 160-167 and herpes simplex virus glycoprotein d residues 286-293 (table 2) . for example, antibodies to the herpes simplex virus peptide reacted with both the corresponding achr peptide and with the achr native protein. interestingly, a different herpes simplex virus glycoprotein sequence amino acid 38 l-389 also showed several similar amino acid sequences, but on immunoche-mica1 analysis failed to raise antibodies that cross-reacted with the achr sequence [ 15, 161. afhnity purification of antibodies from patients with myasthenia gravis, using the human achr a-chain 157-170 peptide immobilized on thiopropyl-sepharose, yielded igg antibodies that bound to the native achr and inhibited the binding of a-bungarotoxin to its specific binding site on the receptor. thus, the human achr a-chain 160-167 peptide specifically cross-reacts with a shared homologous domain on herpes simplex virus glycoprotein d, residues 286-293, by binding and inhibition studies, and elicits antibodies in myasthenic patients that bind to the native achr protein; these antibodies are capable of causing a biologic effect. immunologic cross-reactivity of this 'self epitope with herpes simplex virus suggests that this virus may be associated with some cases of myasthenia. the probability that a random six amino acid sequence will be identical in two dissimilar proteins is 1 in 206, assuming that all amino acids are represented equally. nevertheless, the finding of sequence homology is, by itself, insufficient evidence of biologically meaningful mimicry. two examples illustrate this point. first, like the k. pneumoniue nitrogenase, the ebv bblf protein shares six continuous amino acids with hla-b27, but the homology is in the conserved and not the variable domain of the hla-b27.1 molecule. no immunochemically identifiable cross-reactivity occurs between hla-b27-specific sequences and ebv peptides containing the homologous sequence. further, over 50 patients with as or rs and of the hla-b27 haplotype had no antibodies to the ebv peptides [12, 141. thus, whether or not the homology reflects the biologically meaningful domain is important. second, homology alone may not lead to a cross-reacting immune response, especially if the dissimilar amino acid(s) represents a radical substitution or interferes with binding. for example, despite a high degree of similarity between portions of the achr u-chain (pesdqpdl) and polyoma virus middle t antigen (pksdqdql), no cross-reacting antibodies form. yet a more distant similarity between the achr sequence and herpes simplex virus glycoprotein d (pnatqpel) induces strong immunologic cross-reactivity [15] . just as homologies and immunologic cross-reactivities have been found between the hla-b27 variable domain and k. pneumoniae nitrogenase, between the human achr and herpes simplex virus glycoprotein, and between other host and microbial proteins (table 2) , additional similarities will surely emerge as more genes and proteins are analyzed. some of these examples may account for diseases in terms of an autoimmune response provoked by molecular mimicry. however, unless the homology and subsequent immunologic cross-reactivity involve a host protein that can precipitate disease (e.g. the restricted encephalitogenic site of myelin, and immunodominant domain of the insulin receptor or achr), the autoimmune response is unlikely to lead to autoimmune disease. virus-induced autoimmune disease: viruses in the production and prevention of autoimmune disease the effect of induced chronic viral infections on the immunologic diseases of new zealand mice host igg and c3 deposits in the choroid plexus during spontaneous immune complex disease molecular mimicry and autoimmune disease viruses perturb lymphocyte functions: selected principles characterizing virus-induced immunosuppression virus-induced immunosuppression: infections with measles virus and human immunodeficiency virus serologic relatedness between 2 and actin revealed by monoclonal antibody sv40 large t shares an antigenic determinant with cellular protein of molecular weight 68,000 molecular mimicry in virus infection: cross-reaction of measles phosphoprotein or of herpes simplex virus protein with human intermediate filaments molecular mimicry: frequency of reactivity of monoclonal antiviral anitbodies with normal tissues amino acid homology and immune responses between the encephalitogenic site of myelin basic protein and virus: a mechanism for autoimmunity autoantibodies to hla-b27 in the sera of hla-b27 patients with ankylosing spondylitis and reiter's syndrome: molecular mimicry with klebsiella pneumoniae as potential mechanism of atuoimmune disease hla-b27 related antigens in articular tissues of patients with ankylosing spondylitis autoimmune pathogenesis for ankylosing spondylitis (as) and reiter's syndrome (rs): autoantibodies against an epitope shared by hla-b27 and klebsiella pneumoniae nitrogenase in sera of hla-b27 patients with as and rs peptides as probes to study molecular mimicry and virus induced autoimmunity molecular mimicry and myasthenia gravis: uncovering a novel site of the acetylcholine receptor that has biologic activity and reacts immunochemically with herpes simplex virus this is publication number 5566-imm from the department of immunology, scripps clinic and research foundation, la jolla, ca 92037.this work was supported in part by usphs grants ai-07007, ns-12428, and ag-04342. 7. 9. key: cord-007626-n51v4ior authors: dhib-jalbut, suhayl; kufta, conrad v.; flerlage, marjorie; shimojo, naoki; mcfarland, henry f. title: adult human glial cells can present target antigens to hla-restricted cytotoxic t-cells date: 2002-11-11 journal: j neuroimmunol doi: 10.1016/0165-5728(90)90163-h sha: doc_id: 7626 cord_uid: n51v4ior t-lymphocyte recognition of antigen either on antigen-presenting cells (apc) necessary for the generation of an immune response or on target cells during the effector phase of a cellular immune response requires expression of hla molecules. although immune mechanisms operate in many disease processes of the central nervous system (cns), cells of the cns generally express low levels of hla molecules. in this study, the potential for upregulation of hla molecules on adult human glial cells was examined. moreover, the functional implication of this upregulation was assessed by the capacity of glial cells to process and present target antigens to hla class i-restricted influenza-specific and class ii-restrict myelin basic protein (mbp)-specific ctl lines. glial cells cultured from adult human surgical brain specimens or cells from established glioblastoma multiforme cell lines were studied. lysis by antigen-specific ctls was dependent on treatment of the target cell with interferon-γ. the lysis was hla restricted and antigen specific. the results indicate that adult human glial cells can process and present antigen to hla-restricted ctls but require the upregulation of hla molecules. these findings have implications for infectious and autoimmune diseases of the cns. antigen-specific t-lymphocytes are believed to be involved in the production of immunopathological disease of the central nervous system (cns) such as multiple sclerosis (ms) (mokhtarian et al., 1984; fontana et al., 1987) . direct damage of tissue by cytotoxic t-lymphocytes (ctls) requires recognition of a processed antigen on the target cell in the context of the appropriate hla determinants (zinkernagel and doherty, 1974; sun et al., 1988) . this process may be limited in the cns, since brain cells express very low levels of hla molecules (williams et al., 1980) . expression of these molecules can be upregulated in murine glial cell cultures by interferon--/ (ifn--/) (wong et al., 1984) or virus infection (massa et al., 1986) which allows these cells to function as antigenpresenting cells (apc) . animal experiments have demonstrated that mouse endothelial cells (mc-carron et al., 1985) and rat astrocytes (fontana et al., 1984) can present myelin basic protein (mbp) to encephalitogenic t-cell lines. these lines have been shown to lyse mbp-treated rat astrocytes in an antigen-specific and ia-restricted fashion (sun and wekerle, 1986 ). in addition, inducibility of ia on rat astrocytes has been found to be strain dependent (massa et al., 1987) . while these findings indicate that native cells of the newborn murine cns can present self antigen to t-cells, it is uncertain if similar processes occur in cells of the adult human brain and whether these cells can process and present viral antigen to ctls. moreover, there is evidence that certain cells such as keratinocytes and epithelial cells can be induced to express ia molecules but are incapable of processing and presenting antigen to t-cells (geppert and lipsky, 1985) . in this study, the capacity of adult human glial cells to express hla molecules and to acquire antigen-presenting function was examined by their ability to serve as targets for hla class i-and class ii-restricted ctls. adult human glial cell cultures were established from surgical brain specimens obtained from the temporal lobes of patients treated for intractable seizures. after dissecting the meninges, the tissue was washed in phosphate-buffered saline (pbs), minced into 2-3 nun pieces, and treated with 0.2% trypsin and 20/~g/ml dnase (sigma) in a volume of 10 ml for 40 min at 37 °c. the tissue was vortexed every 10 rain for 1 rain. the trypsin digestion was stopped by adding 10% fetal calf serum (fcs). the cell suspension was then transferred to a 25 cm 2 tissue culture flask or onto coverslips precoated with poly-d-lysine (10/~g/ml) (sigma) and left undisturbed for 1 h in a co 2 incubator. dulbecco's modified eagle's medium (dmem) containing 15% heat-inactivated fcs, glutamine, hepes buffer, mem nonessential amino acids, mem vitamins, and penicillin/streptomycin was then added to the cells. medium was changed every 48 h for the first two changes, then every 4 days. glioblastoma multiforme cell lines u-251 mg and u-373 mg were a gift from dr. darrel bigner (duke university, nc, u.s.a.) and the characteristics of these cell lines have been previously described (bigner et al., 1981; wikstrand et al., 1985) . u-251 mg is an established cell line derived from a human glioblastoma multiforme. this cell line continues to produce glial fibriuary acidic protein (gfap) in culture which is suggestive of its glial origin. u-251 mg expresses high levels of class i hla molecules but not class ii (dr) molecules. u-373 mg is another established cell line derived from a human glioblastoma multiforme but does not produce gfap. this cell fine expresses both class i and class ii (dr) hla molecules. cells grown on coverslips were washed with pbs and incubated with anti-hla class i (w6/32), anti-hla class ii-dr (l243) or nonimmune hybridoma supernatant. the cells were then washed twice and a fluorescein-conjugated sheep anti-mouse igg (kappel) was applied. after two washes, the coverslips were fixed in 2% paraformaldehyde for 15 min followed by triton x-100 (0.1%) treatment for 5 min. rabbit antiserum to gfap (dako, ca, u.s.a.) or normal rabbit serum was then applied (1:250 dilution), followed by tetramethylrhodamine b isothiocyanate (tritc)conjugated goat anti-rabbit antibody (sigma). all antibody incubations were carried for 45 rain at 25 ° c. the specificity of the immunostaining was established by the negative results obtained when one of the primary antibodies was omitted or nonimmune serum was applied. elimination of either sheep anti-mouse or goat anti-rabbit antiserum resulted in the appropriate lack of specific staining indicating absence of interspecies crossreactivity. cells were examined on a leitz fluorescent microscope and photographed with kodak ektachrome p800/1600 film. influenza ctl are predominantly restricted by hla class i molecules (biddison, 1982) . these were used to examine class i-restricted antigen presentation by human glial cells. influenza virusspecific ctl were generated as described (dhib-jalbut et al., 1989) . briefly, 4 × 106 peripheral blood lymphocytes (pbl) matched with the primary glial cell targets for class i hla molecules were cultured with influenza virus (a/jap) for 1 h in 2 ml rpmi 1640 supplemented with glutamine in a 24-well tissue culture plate. five percent human ab serum was then added and the cultures were carried for 7 days in a 5% co 2 humidified incubator. generation of influenza virus-specific ctl lines ql15 and 1d3 has been previously described (cowan et al., 1987; nuchtern et al., 1989) . the line ql15 recognizes a synthetic peptide (m1) which corresponds to amino acid sequences 55-73 of the matrix protein of influenza virus a/jap, and is restricted by hla-a2 molecules. the line 1d3 recognizes influenza virus a/jap and is restricted by hla-a3 molecules. mbp-specific t-cell lines were generated from dr2 homozygous donors by repeated stimulation of pbl with mbp (10/lg/ml) in the presence of autologous irradiated feeders (6000 rad) in rpmi 1640 media containing 10% human ab serum. after two passages, 10% human t-cell growth factor (cellular products, buffalo, ny, u.s.a.) was added to the cultures. these lines were cd4 ÷ by facs analysis, and dr2 restricted as determined by lysis of dr2-transfected targets (jaraquemada, d. et ~ al., in preparation) . these lines were used 6 days after stimulation to examine presentation of class ii-restricted antigen by glial cell targets. glial cell targets obtained from primary cultures were treated with ifn-~/ (100 units/ml, genzyme, ma, u.s.a.) for 3 days and then infected with influenza virus (a/jap) for 16 h. targets obtained from the cell lines u-251 mg, u-373 mg, and an ebv-transformed b-cell line (k4b) were infected either with influenza virus a/jap strain or influenza virus b/aa strain or pulsed with the m1 peptide (5 /~g/ml) for 16 h. mbp targets were pulsed with human mbp (100 #g/ml) for 16 h. gfial ceils were removed from the tissue culture flasks by trypsinization and washed with rpmi 1640 containing 5% fcs. the targets were then suspended in 0.3 ml medium containing 100 /~ci/rnl of na[51cr]o4 (new england nuclear, boston, ma, u.s.a.) and incubated for 90 min in a 37 °c water bath. the chromated targets were then washed twice with medium and counted. varying numbers of effector cells were cultured with 5000 slcr-labelled targets in triplicate wells of a 96-well microtiter plate in a volume of 200 #1 of dmem containing 10% fcs supplemented with glutamine. after 4 h of incubation in a humidified, 5% co 2 atmosphere, the plates were spun at 5000 rpm for 5 min, the supernatants were harvested and sacr release measured using a gamma counter. percent target lysis was calculated as (experimental release-spontaneous release)/ (detergent release -spontaneous release) x 100. cells cultured on coverslips were examined 2-3 weeks after tissue dissociation. 70-80% of the cells used in this study were astrocytes as determined by staining with rabbit antiserum to gfap and visualization by immunofluorescence microscopy. the majority of the remaining cells reacted with the monocyte/macrophage marker (leu-m3) and less than 5% reacted with rabbit antiserum to fibronectin. cells that reacted with leu-m3 had morphological characteristics consistent with microglia including irregular rod-like shapes and short branching processes. the presence of oligodendrocytes was not examined in the cultures used in this study. however, examination of subsequent cultures established from different brains under similar conditions indicated the presence of less than 5% of cells that react with antibodies to the oligodendrocyte markers galactocerebroside (gift from dr. m. dubois-dalcq) and mbp (dakopatts). cells reacting with antibodies to von willebrand factor (an endothelial cell marker) were not observed in these cultures. approximately 50% of the gfap-positive cells constitutively expressed low levels of hla class i molecules ( fig. 1a and b) and less than 5% expressed hla class ii (dr) molecules ( fig. 1e and f) , but expression could be upregulated on these cells up to 90% for both class i and class ii dr by treatment with ifn--/ 100 units/ml for 3 days (fig. 1c , d, and g, h). upregulation of hla class i and dr molecules was also observed on cells with a morphological appearance of macrophage/microglia. the capacity of primary adult human glial cells (70% of which were gfap positive) to present influenza a virus antigen to influenza virusspecific class i hla-restricted ctls was examined. following treatment with ifn-3, and subsequent infection with influenza a/jap virus these glial cells were lysed by influenza virus-stimulated, hla class 1-matched pbl from a healthy donor. lysis was not observed with the unstimulated pbls or with the uninfected targets ( fig. 2a) . lysis of mbp-pulsed targets by mbp-specific t-cell lines was used to examine hla class ii-restricted lysis. the mbp t-cell lines used in this study are cd4 + cells and are restricted by hla class ii molecules (martin, r. et al., in preparation) . primary adult human glial cells (80% of which were gfap positive) were obtained from a patient with an hla-dr2 haplotype. following treatment with ifn-y and mbp these cells could be effectively lysed by two human mbp-specific dr2-restricted t-cell lines ( fig. 2b and c) . the lysis was dependent on treatment of the target cells with ifn-7. no lysis was obtained with targets treated with ifn-y but not exposed to mbp (fig. 2b) . similar results were obtained with another mbp-specific dr2-restricted t-cell line from a different donor at serial effector to target ratios (fig. 2c) . the primary adult human glial cell cultures also included nonastrocytic cell types, and therefore, the magnitude of ~acr release attributable to each cell population cannot be ascertained. thus, the cell line u-251 mg which produces gfap was used as a target in the subsequent experiment to establish that similar results could be obtained with cells presumably of glial origin. moreover, the capacity of this glial target to be lysed by ctl in an hla-restricted fashion was also examined. u-251 mg cell line shares with normal adult astrocytes two relevant characteristics: it is gfap positive and constitutively expresses hla class i (a2) but not dr molecules. in contrast, u-373 mg does not express gfap but expresses both hla class i (a3) and dr molecules. u-251 mg was examined for its ability to present target antigens to the influenza virus-specific hla-a2restricted t-cell line ql15. an hla-a2-positive ebv-transformed b-cell line (k4b) was used as a control target. as shown in fig. 3 , ql15 lysed u-251 mg and b-cell targets that were infected with the a/jap strain of influenza virus (fig. 3a and i) or pulsed with the m1 peptide ( fig. 3b and j) but not targets infected with a different strain of influenza virus (b/aa) ( fig. 3c and k) or uninfected targets ( fig. 3d and l) . lysis of u-251 mg target with the mismatched effectors (1d3) was not observed (fig. 3a-d) indicating that the lysis of this target was restricted by hla-a2 molecules. similarly, no lysis was obtained with ql15 on the mismatched target (u-373 mg, hla-a3 positive) but this target could be lysed by 1d3 (fig. 3e-h) indicating that the lysis by these cell lines is hla restricted. in this study, primary glial cell cultures were established from adult human brain. these cultures consisted primarily of astrocytes (70-80%) and microglia/macrophages (15-20%). the microglia/macrophage cells are likely to be derived from the brain rather than from peripheral blood contamination, since peripheral blood monocytes from the same patient cultured under similar conditions were morphologically different from the brain-derived microglia/macrophages. fibroblasts were rarely detected in cultures less than 3 weeks old, but they increased in number, thereafter. consequently, 2-to 3-week-old cultures were employed in order to study antigen presentation by glial cells. under basal conditions, 50% of gfap + cells expressed class i hla molecules but only 5% expressed class ii molecules. both fluorescence intensity and number of cells expressing hla molecules were increased by ifn-y treatment. in general, these findings are in agreement with the observations reported in previous studies (hirayama et al., 1986; grenier et al., 1989) and personal communication with dr. j.p. antel. the capacity of primary glial cells expressing class i and class ii hla molecules to present target antigens to ctl was examined. since both viral and auto-antigens have been implicated in the development of autoimmune disease (fontana et al., 1987) , presentation of these antigens by primary human glial cells was studied. the present data suggest that primary mixed glial cell cultures can function as targets for antigen-specific ctl. as shown in fig. 2b and c, the ability of these cells to function as targets for mbp ctl is dependent on treatment of the targets with ifn-7 which results in upregulation of hla molecules. ifn-y also has been shown to upregulate the expression of adhesion molecules such as intercellular adhesion molecule 1 (icam-1) on routine astrocytes which could contribute to more effective lysis of these cells (frohman et al., 1989) . since the primary adult human glial cells contained nonastrocytic cell types such as microglia, the extent of 51cr release attributable to each cell population was uncertain. therefore, either astrocytes or rnicroglia or both could have functioned as targets for ctl. attempts to separate the astrocytes and 209 microglial cells have been hampered by the limited number of cells that can be obtained from adult brain tissue. for these reasons an established human glioblastoma cell line u-251 mg which produces gfap was used as targets to establish that similar results could be obtained with cells that are likely to be derived from a glial origin. the study performed with this cell line indicated that u-251 mg can process and present viral antigens as efficiently as b-cells, since both targets produced comparable lysis with the influenza virus and the m1 peptide (which does not require processing). moreover, the results obtained with the u-251 mg targets indicated that the lysis of these targets is antigen specific and hla restricted. in autoimmune disease of the cns the evidence that native cells of the cns can present antigen in situ or become targets for the cellular immune response is largely circumstantial. such evidence is derived from the immunohistochemical demonstration of class ii hla molecules on astrocytes, microglia, and endothelial cells in lesions of brains affected with multiple sclerosis or eae (mccarron et al., 1985; hayes et al., 1987; traugott, 1987; grenier et al., 1989) . studies addressing the functional capacity of these cns cells to present antigen to t-cells have been conducted in marine systems and have largely focused on mbp (fontana et al., 1984; mccarron et al., 1985; sun and wekerle, 1986) . the present study suggests that adult human astrocytes and/or microglia can present viral or auto-antigen to cytotoxic t-cells. in addition, the results obtained with the gfap + cell line u-251 mg support a role for astrocytic cells in presenting antigen to t-cells. these findings have implications for pathogenic mechanisms involved in the development of autoimmune diseases of the cns. hla molecules induced on glial cells by ifn--y released during inflammation could render these cells capable of presenting antigen to t-cells. gliai cells could then play a role in the induction of an immune response within the cns or become targets for ctl. in addition, clearance of a virus infection from the brain may be dependent, in part, on ctl lysis of infected cells (oldstone et al., 1986) . this, in turn, is dependent on the upregulation of hla molecules on the infected brain cells and the capacity of these cells to process viral antigens. while this study suggests that adult human glial cells expressing class i hla molecules may process and present viral antigen to hla class i-restricted ctls, it is not known whether similar processes occur in other cell types of the cns. the persistence of certain viruses in the cns may be related to a failure to upregulate hla molecules, and subsequently, failure of ctl recognition of infected cells. finally, there is increasing interest in intracerebral transplantation of fetal tissue for patients with parkinson's disease. experimental evidence in rats suggests that microglia may participate in the immunological reaction to xenografts by functioning as antigen-presenting cells (poltorak and freed, 1989) . moreover, adult human mixed glial cell cultures have been shown to induce an allogeneic t-cell response (grenier et al., 1989) . the present findings suggest that human glial cells potentially can participate in the immunological reaction to intracerebral transplants under conditions that would lead to upregulation of hla molecules, the role of the major histocompatibility complex in cytotoxic t-cell responses to virus infected cells heterogeneity of genotypic and phenotypic characteristics of fifteen permanent cell lines derived from human gliomas induction and functional characterization of class ii mhc (ia) antigens on routine keratinocytes site directed mutagenesis of an hla-a3 gene identitles amino acid 152 as crucial for major histocompatibility complex-restricted and alloreactive cytotoxic t-lymphocyte recognition impaired human leukocyte antigen-restricted measles virus-specific cytotoxic t-cell response in subacute sclerosing panencephalitis astrocytes present myelin basic protein to encephalitogenic t cell lines immune-mediated encephalitis: on the role of antigen-presenting cells in brain tissue astrocytes and intracerebral immune responses antigen presentation by interferon-y-treated endothelial cells and fibroblasts: differential ability to function as antigen-presenting cells despite comparable la expression immunohistochemical studies of adult human glial cells microglia are the major cell type expressing mhc class ii in human white matter induction of human leukocyte antigen-a, b, c, and dr on cultured human oligodendrocytes and astrocytes by human gamma-interferon viral particles induce la antigen expression on astrocytes inducibility of ia antigen on astrocytes by murine coronavirus jhm is rat strain dependent presentation of myelin basic protein by murine cerebral vascular endothelial cells adoptive transfer of myelin basic protein-sensitized t cells produces chronic-relapsing demyelinating disease in mice brefeldin a implicates egress from endoplasmic reticulum in class i restricted antigen presentation cytoimmunotherapy for persistent virus infection reveals a unique clearance pattern from the central nervous system immunological reactions induced by intracerebral transplantation: evidence that host microglia but not astroglia are antigen-presenting cells ia-restricted encephalitogenic t lymphocytes mediating eae lyse autoantigen-presenting astrocytes cytotoxic t cells in autoimmune disease of the central nervous system multiple sclerosis: relevance of dass i and class ii mhc-expressing cells to lesion development antigenic heterogeneity of human anaplastic gliomas and glioma-derived cell lines defined by monoclonal antibodies distribution and quantitation of hla-a, b, c, and dr (ia) antigens on human kidney and other tissues inducible expression of h-2 and ia antigens on brain cells restriction of in vivo t-cell mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogenic system we would like to thank drs. carol wikstrand and william e. biddison for stimulating discussions, ms. toni simonis for the hla typing, the leukophoresis unit at the nih, and mrs. millie fenton for preparing the manuscript. key: cord-007719-3ypv9k9p authors: wang, mingjun; claesson, mogens h. title: classification of human leukocyte antigen (hla) supertypes date: 2014-05-06 journal: immunoinformatics doi: 10.1007/978-1-4939-1115-8_17 sha: doc_id: 7719 cord_uid: 3ypv9k9p identification of new antigenic peptides, derived from infectious agents or cancer cells, which bind to human leukocyte antigen (hla) class i and ii molecules, is of importance for the development of new effective vaccines capable of activating the cellular arm of the immune response. however, the barrier to the development of peptide-based vaccines with maximum population coverage is that the restricting hla genes are extremely polymorphic resulting in a vast diversity of peptide-binding hla specificities and a low population coverage for any given peptide–hla specificity. one way to reduce this complexity is to group thousands of different hla molecules into several so-called hla supertypes: a classification that refers to a group of hla alleles with largely overlapping peptide binding specificities. in this chapter, we focus on the state-of-the-art classification of hla supertypes including hla-i supertypes and hla-ii supertypes and their application in development of peptide-based vaccines. the immune system, including the innate and adaptive as well as overlapping systems, plays a pivotal role in the defense against viral or bacterial infections, immune homeostasis, and cancer surveillance. within the immune system, t lymphocytes are crucial for adaptive immune responses, and are activated upon recognition of peptides displayed by human leukocyte antigen class i (hla-i) or class-ii (hla-ii) molecules at the surfaces of antigen-presenting cells (apcs). t lymphocytes express the t cell receptor (tcr) that recognizes specifi c peptides, which have been processed and presented in combination with an hla molecule. there are two major subtypes of t lymphocytes: cd8 + cytotoxic t cells (ctls) and cd4 + helper t cells. ctls recognize peptides in the context of hla-i molecules, while cd4 + helper t cells recognize peptides associated with hla-ii molecules. the functional activity of these two subsets of t cells is said to be restricted by hla-i and -ii molecules, respectively. it is known that ctls play a major role in killing tumor cells [ 1 , 2 ] and controlling viral or bacterial infections [ 3 -7 ] , while cd4 + t cells are required for priming and expansion of naive cd8 + t cells as well as secondary expansion of cd8 + memory t cells [ 8 -12 ] . it might therefore be of critical importance to incorporate both hla-i-and -ii-restricted epitopes in peptide-based vaccines to obtain participation of both cd4 + and cd8 + t cells for generation of strong and long-lasting immunity. thus, identifi cation of new antigenic peptides, derived from infectious agents or tumor antigens, which may bind to hla-i or hla-ii molecules in exchange with self-peptides normally occupying the hla-binding site ( see below), is important for developing new effective vaccines capable of activating the cellular arm of the immune responses. however, the barrier to development of peptide-based vaccines with maximum population coverage is that the restricting hla genes are extremely polymorphic resulting in a vast diversity of peptide-binding hla specifi cities and a low population coverage for any given peptide-hla specifi city. as of april 2013, it has been reported that there are 7,089 hla-i alleles and 2,065 hla-ii alleles ( http://hla.alleles.org ). undoubtedly, these numbers will be further increased in the future. to reduce this complexity, one option is to group thousands of different hla molecules into clusters of several so-called hla supertypes: a classifi cation that refers to a group of hla alleles with largely overlapping peptide binding specifi cities. in this chapter, we discuss the state-of-the-art classifi cation of hla-i and hla-ii supertypes and their application in development of peptide-based vaccines. the major histocompatibility complex class i (mhc-i) antigens are referred to as the human leukocyte antigens class i (hla-a, -b, and -c) and as h-2 class i antigens (k, d, and l) in mice. hla-i antigens consist of three non-covalently associated components: a 45 kda glycosylated amino acid (aa) heavy chain (hc), a 12 kda light chain (beta 2 microglobulin, β2m), and a short 8-10 aa self-peptide. the heavy chain of hla-i consists of about 340 aa residues, including a cytoplasmic region (about 30 aa residues), a transmembrane region (about 40 aa residues), and an extracellular region composed of three immunoglobulin-like domains (α1, α2, and α3), each consisting of approximately 90 aa. the α1 and α2 domains form a peptide-binding groove and contain the positions contributing to the binding pockets for the peptide and t cell receptors. the binding groove is divided into six distinct pockets (a-f) based on chemical and physical characteristics; the most important pockets for peptide binding are the b and the f pockets. the membrane-proximal α3 domain of the hc contains a binding site for the co-stimulatory molecule cd8 [ 13 ] expressed by ctls, which play an important enhancing role in killing virus-infected cells and cancer cells. the α1 and α2 domains consist of two segmented alpha helices forming the walls and eight antiparallel β strands forming the fl oor-together forming a unique peptide-binding groove, which is the site where the self (or foreign antigen-derived) peptide (8-10 aa) binds to the polymorphic parts of the hc and is presented to peptide-specifi c ctl for scrutiny. β2m is non-covalently associated with the extracellular region of the hla-i heavy chain by non-covalent interactions with α2 and α3 domains [ 14 ] . β2m is essential for the correct conformation of the peptide-binding groove of the heavy chain and stabilizes the hla-i antigen peptide complex on the cell surface. thus, β2m indirectly participates in the antigen presentation to specifi c t-cell receptors of ctl [ 15 -17 ] . the assembly of hla-i peptide complex occurs in the endoplasmic reticulum (er). initially, the hla-i hc associates with the chaperone calnexin (cnx) initiating an early folding and a disulfi de bond formation within the hc. the newly synthesized hla-i hc then associates with β2m to form heterodimer. this heterodimer is rapidly recruited into the peptide-loading complex (plc) consisting of a transporter associated with antigen processing (tap), and the chaperones tapasin, calreticulin (crt), and erp57. the hla-i hc/β2m heterodimer is now ready for peptide loading. peptides, both self-and pathogen-derived, are predominantly generated in the cytosol by the proteasome to degrade cytosolic proteins into short peptides, although a proteasomeindependent peptide produced directly by insulin-degrading enzyme has been recently documented [ 18 ] . thereafter, the peptides are transported into the er by the tap1 and tap2. these peptides are further trimmed by aminopeptidase eraap1 and eraap2 to 8-10 aa, a length appropriate for hla-i binding. once hla-i/hc-β2m dimers, physically associated with plc, bind a subset of high-affi nity peptides, the fully assembled mhc-i peptide complexes are released from plc and transported via the golgi apparatus to the cell surface, where the peptides are presented by hla-i to ctl for scrutiny ( see details in reviews [ 19 , 20 ] ). the hla-ii molecule consists of two chains: α and β chain (each one with two domains: α1 and α2, β1 and β2) and a self-peptide with 13-25 aa located in a cleft formed by the α1 and β1 domains. classical hla-ii molecules include hla-dr, hla-dq, and hla-dp and are expressed mostly in the membrane of the professional antigen-presenting cells, where they present processed extracellular antigenic peptides to cd4 + t cells. in contrast to the antigen-binding groove of hla-i molecule, which is closed at each end, the antigen-binding groove of hla-ii molecules is open at both ends and allows longer peptides (13-25 aa) to be loaded [ 21 , 22 ] . during synthesis of hla-ii molecules in the er, the α and β chains are produced and associate with an invariant chain, which stabilizes the hla-ii molecule and prevents it from binding of intracellular peptides or peptides from the endogenous pathway. the invariant chain directs transportation of hla-ii from the er to the golgi complex, followed by fusion with late endosomes which contain peptides derived from endocytosed, degraded proteins (self or foreign). the invariant chain is then cleaved by cathepsins to form a small fragment known as clip, which occupies the peptide-binding groove of the hla-ii molecules. hla-dm facilitates clip removal and makes the peptide-binding groove of hla-ii ready for peptide loading before the hla-ii-peptide complex migrates to the cell surfaces to be scrutinized by cd4 + t cells [ 23 ] . the concept of supertypes was fi rstly introduced by alessandro sette's group in 1995 [ 24 , 25 ] . the defi nition of an hla supertype is that hla molecules with similar peptide binding features are grouped into one supertype; this means that if a peptide is able to bind to one allele within a supertype, it can also bind to all other alleles in this supertype. in practice, actually only a few peptides that are able to bind to one allele in a supertype can bind to all the other alleles within the supertype. to date, many methods have been used to defi ne hla-i supertypes, including structural similarities, shared peptide-binding motifs, and identifi cation of crossreacting peptides [ 26 -29 ] . based on motifs derived from binding data or sequencing of endogenously bound peptides, along with simple structural analyses, sette and sidney [ 30 ] defi ned nine supertypes (hla-a1, -a2, -a3, -a24, -b7, -b27, -b44, -b58, -b62), which were reported to cover most of the hla-a and -b polymorphisms. subsequently, ole lund's group [ 26 ] constructed hidden markov models (hmms) [ 31 ] for hla-i molecules using a gibbs sampling procedure [ 32 ] and defi ned a similarity measure between these sequence motifs. by using this similarity to cluster alleles into supertypes, ole lund's group [ 26 ] further defi ned three new hla-i supertypes (hla-a26, -b8, and -b39), in addition to the nine supertypes described previously by alessandro sette's group [ 30 ] , which was based on about 100 hla-i peptide interactions. in the past few years, a lot of binding data have been generated; mhc-binding motif information is readily accessible ( http://www.iedb.org ), and mhc sequence data are also available in the imgt (the international immunogenetics information system: http://www.imgt.org ) database. in 2008 alessandro sette's group analyzed the updated list of alleles available through imgt using a simple approach largely based on compilation of published motifs, binding data, and analyses of shared repertoires of binding peptides, in combination with clustering based on the primary sequence of the b and f peptide-binding pockets [ 29 ] . they provided updated supertype assignments, with new assignments for about 1,000 different hla-i alleles, which is about a tenfold increase in the number of alleles compared to their original classifi cation done in 1999 [ 30 ] . in the updated hla-i classifi cation, alessandro sette's group found that about 80 % of the 945 alleles examined were classifi ed into one of the nine supertypes identifi ed previously [ 30 ], and they did not suggest the existence of any other novel supertypes. however, they found that some alleles have specifi cities spanning two different supertypes, nine alleles share features of both the a01 and a03 supertypes, and another ten alleles have a specifi city overlapping the a01 and a24 supertypes [ 29 ] . in addition, some alleles could not be assigned to any supertypes known today on the basis of the criteria mentioned above; thus these unclassifi ed alleles remain to be addressed. in summary, the updated hla-i classifi cation described by alessandro sette's group [ 29 ] is in agreement with those defi ned by other approaches from the other groups [ 26 , 33 , 34 ] including ole lund's group, and is now widely accepted and has been used for development of peptide-based vaccines [ 29 , 35 , 36 ] . the structural composition between hla-i and hla-ii molecules is fundamentally different, thus leading to very different binding characteristics. the binding groove is closed at both ends in an hla-i molecule, while the peptide-binding groove of hla-ii molecules is open at both ends, which allow the binding of longer peptides (13-25 aa residues) than that for hla-i molecules. a deeper understanding of the polymorphism of hla-ii molecules will contribute signifi cantly to hla-ii-binding peptide prediction and classifi cation of supertypes. in contrast to hla-i supertypes, hla-ii supertypes have been less intensively studied, although a few studies about hla-ii supertypes [ 26 , 37 -41 ] have been reported. one important reason is that peptide binding data for hla-ii molecules is less available than those for hla-i molecules due to the complexity of hla-ii structure. nevertheless, studies have suggested that many dr molecules [ 26 , 37 , 38 ] and many dp molecules supported the existence of three main binding supertypes among hla-dp molecules. in 2005, doytchinova et al. [ 37 ] applied a combined bioinformatics approach using both protein sequence and structural data, to 2,225 hla-ii molecules, to detect similarities in their peptide-binding sites for defi nition of hla-ii supertypes. they defi ned 12 hla-ii supertypes, including fi ve drs (dr1, dr3, dr4, dr5, and dr9), three dqs (dq1, dq2, and dq3), and four dps (dpw1, dpw2, dpw4, and dpw6). in 2011, greenbaum et al. [ 41 ] determined the binding capacity of a large panel of non-redundant peptides for a set of 27 common hla dr, dq, and dp molecules. the measured binding data were then used to defi ne class ii supertypes on the basis of shared binding repertoires. seven different supertypes (main dr, dr4, drb3, main dq, dq7, main dp, and dp2) were defi ned. subsequently, according to motif-based supertype classifi cation [ 27 ] , seven different supertypes were defi ned after the analysis of 27 hla ii proteins described in a previous report [ 41 ] . all the molecules belonging to the dp genetic locus (dpb1*0101, dpb1*0201, dpb1*0401, dpb1*0402, dpb1*0501, and dpb1*1401) were grouped into a single supertype; dq proteins were grouped into two different supertypes, each containing three hlas: (dqb1*0301, dqb1*0302, dqb1*0401) and (dqb1*0201, dqb1*0501, dqb1*0602). the motif-based classifi cation of the dr proteins is less defi ned compared with the other loci. the hla-dr can be grouped into four supertypes: (drb1*0401, drb1*0405, drb1*0802, drb1*1101), (drb3*0101, drb3*0202), (drb1*0301, drb1*1302), and the fourth containing the remaining dr proteins. functional and motif-based clustering of 27 defi ned hla-ii molecules revealed the presence of proteins sharing both functional and structural properties, thus supporting the concept of hla-ii supertypes. to date, one of the major drawbacks of a peptide-based vaccine strategy is that the restricting hla genes are extremely polymorphic resulting in a vast diversity of peptide-binding hla specifi cities and a low population coverage for any given peptide-hla specifi city. to increase population coverage, one might include defi ned epitopes for each hla-i allele; however, this would lead to a vaccine comprising hundreds of peptides. as mentioned above, one way to reduce this complexity is to group hla molecules into hla supertypes; a classifi cation that as mentioned above refers to a group of hla alleles with largely overlapping peptide binding specifi cities [ 24 , 25 , 30 ] . ideally this means that a peptide, which binds to one allele within a supertype, has a high probability of binding to other allelic members of the same supertype. the concept of hla supertypes has been successfully applied to characterize and identify t cell epitopes from a variety of different pathogens, including measles-mumps-rubella, sars, ebv, hiv, hcv, hbv, hpv, infl uenza, lcmv, lassa virus, f. tularensis , vaccinia, and cancer antigens as well [ 29 ] . hla supertypes have been utilized as a component in several approaches and algorithms designed for predicting peptide candidates [ 43 -48 ] . the technology behind "reverse immunology" is developing rapidly in order to identify t cell epitopes from tumor antigens and infectious microorganisms [ 44 -51 ] . during the sars epidemic back in 2003, the sars genome was identifi ed in a matter of weeks, and a complete ctl epitope scanning-just barely possible at that time-was completed a few months later [ 43 ] . therefore, "reverse immunology" as a powerful tool to identify t cell epitopes has now reached the stage where genome-, pathogen-, and hla-wide scanning for hla-binding antigenic epitopes become feasible at a scale and speed that makes it possible to exploit the genome information as fast as it can be generated. importantly, a large-scale dataset of measured hla-ii-binding affi nities covering 26 allelic variants, including a total of 44541 affi nity measurements for hla-dr alleles as well as 11 hla-dp and dq molecules [ 52 ] , are available to be used as training data for generating prediction tools utilizing several machine learning algorithms. to date, the computer-based algorithms for predicting peptides binding to hla-i molecules are being developed for hla-ii-restricted peptide epitopes, a development, which is of pivotal importance for understanding the immune response and its effect on host-pathogen interactions [ 32 , 52 -55 ] . those tools will defi nitely lead to fast identifi cation of novel peptides restricted by hla-i and hla-ii supertypes for use in vaccines against infectious agents as well as tumors. in this respect, individual peptides harboring both hla-i and hla-ii binding potentials [ 46 -48 , 56 ] might be of particular importance. in conclusion, classifi cation of hla supertypes reduces complexity of hla polymorphisms and has a signifi cant impact on the development of peptide-based vaccines with maximum population coverage. since cd4 + t cells are required for priming of naïve cd8 + t cells as well as expansion of cd8 + memory t cells [ 8 -12 ] , it is of critical importance to incorporate both hla-i and -ii supertype-restricted epitopes in peptide-based vaccines with maximum population coverage to obtain participation of both cd4 + and cd8 + t cells for generation of strong and long-lasting immunity. peptide-based vaccines for cancer: realizing their potential adoptive cell therapy for the treatment of patients with metastatic melanoma mycobacterium tuberculosis-specifi c cd8+ t cells require perforin to kill target cells and provide protection in vivo mycobacterium tuberculosis-specifi c cd8+ t cells and their role in immunity t-cell epitope discovery for variola and vaccinia viruses cytotoxic t-cell immunity to infl uenza transgenic mice lacking class i major histocompatibility complex-restricted t cells have delayed viral clearance and increased mortality after infl uenza virus challenge cd4+ t-cell help controls cd8+ t-cell memory via trail-mediated activationinduced cell death cd4+ t cells are required for secondary expansion and memory in cd8+ t lymphocytes requirement for cd4 t cell help in generating functional cd8 t cell memory defective cd8 t cell memory following acute infection without cd4 t cell help cd4+ t cells are required for the maintenance, not programming, of memory cd8+ t cells after acute infection a binding site for the t-cell co-receptor cd8 on the alpha 3 domain of hla-a2 structure of the human class i histocompatibility antigen, hla-a2 antigen processing and presentation by the class i major histocompatibility complex the role of beta 2-microglobulin in peptide binding by class i molecules beta 2-microglobulin restriction of antigen presentation production of an antigenic peptide by insulin-degrading enzyme mechanisms of mhc class i-restricted antigen processing and cross-presentation antigen processing by the proteasome predominant naturally processed peptides bound to hla-dr1 are derived from mhcrelated molecules and are heterogeneous in size sequence analysis of peptides bound to mhc class ii molecules the exogenous pathway for antigen presentation on major histocompatibility complex class ii and cd1 molecules binding of a peptide antigen to multiple hla alleles allows defi nition of an a2-like supertype several hla alleles share overlapping peptide specifi cities defi nition of supertypes for hla molecules using clustering of specifi city matrices consensus classifi cation of human leukocyte antigen class ii proteins defi nition of an hla-a3-like supermotif demonstrates the overlapping peptide-binding repertoires of common hla molecules mhc-i-restricted epitopes conserved among variola and other related orthopoxviruses are recognized by t cells 30 years after vaccination ctl epitopes for infl uenza a including the h5n1 bird fl u; genome-, pathogen-, and hla-wide screening hla class i binding 9mer peptides from infl uenza a virus induce cd4 t cell responses highaffi nity human leucocyte antigen class i binding variola-derived peptides induce cd4+ t cell responses more than 30 years post-vaccinia virus vaccination identifi cation of mhc class ii restricted t-cellmediated reactivity against mhc class i binding mycobacterium tuberculosis peptides reverse vaccinology: developing vaccines in the era of genomics major histocompatibility complex class i binding predictions as a tool in epitope discovery identifi cation of t-cell epitopes for cancer immunotherapy peptide binding predictions for hla dr, dp and dq molecules quantitative predictions of peptide binding to any hla-dr molecule of known sequence: netmhciipan mhc class ii epitope predictive algorithms netmhciipan-2.0-improved pan-specifi c hla-dr predictions using a novel concurrent alignment and weight optimization training procedure hla class ii presentation of hla class i binding antigenic 9mer peptides this work was supported by national institute of allergy and infectious disease contracts hhsn266200400083c, hhsn2662 00400025c, eu 6fp 503231, national institutes of health contract hhsn266200400081c, and a grant from the lundbeck foundation, copenhagen, denmark. key: cord-004395-erqmbi2b authors: bugembe, daniel lule; ekii, andrew obuku; ndembi, nicaise; serwanga, jennifer; kaleebu, pontiano; pala, pietro title: computational mhc-i epitope predictor identifies 95% of experimentally mapped hiv-1 clade a and d epitopes in a ugandan cohort date: 2020-02-22 journal: bmc infect dis doi: 10.1186/s12879-020-4876-4 sha: doc_id: 4395 cord_uid: erqmbi2b background: identifying immunogens that induce hiv-1-specific immune responses is a lengthy process that can benefit from computational methods, which predict t-cell epitopes for various hla types. methods: we tested the performance of the netmhcpan4.0 computational neural network in re-identifying 93 t-cell epitopes that had been previously independently mapped using the whole proteome ifn-γ elispot assays in 6 hla class i typed ugandan individuals infected with hiv-1 subtypes a1 and d. to provide a benchmark we compared the predictions for netmhcpan4.0 to mhcflurry1.2.0 and netctl1.2. results: netmhcpan4.0 performed best correctly predicting 88 of the 93 experimentally mapped epitopes for a set length of 9-mer and matched hla class i alleles. receiver operator characteristic (roc) analysis gave an area under the curve (auc) of 0.928. setting netmhcpan4.0 to predict 11-14mer length did not improve the prediction (37–79 of 93 peptides) with an inverse correlation between the number of predictions and length set. late time point peptides were significantly stronger binders than early peptides (wilcoxon signed rank test: p = 0.0000005). mhcflurry1.2.0 similarly predicted all but 2 of the peptides that netmhcpan4.0 predicted and netctl1.2 predicted only 14 of the 93 experimental peptides. conclusion: netmhcpan4.0 class i epitope predictions covered 95% of the epitope responses identified in six hiv-1 infected individuals, and would have reduced the number of experimental confirmatory tests by > 80%. algorithmic epitope prediction in conjunction with hla allele frequency information can cost-effectively assist immunogen design through minimizing the experimental effort. computational algorithms are increasingly utilised in biological modelling and offer the potential to reduce the time and expense of immunological assays. computational algorithms were initially demonstrated as useful tools for predicting potential epitopes that might elicit quality t-cell responses [1, 2] . computational algorithms that predict potential hla binding t-cell epitopes can facilitate the design of vaccines capable of inducing t-cell immunity against hiv-1. the high variability of hiv-1 and the extensive genetic polymorphism of hla molecules can be managed in silico, allowing immunogen optimisation to increase breadth and magnitude of t cell responses in respect of hla allele frequencies and circulating virus strains in different populations. bioinformatics approaches were previously applied as proof of concept for an hiv-1 peptide-based vaccine for the env and gag genes [3] in cynomolgus macaques for a broad spectrum of hiv-1 clades. computational optimisation of immunogens facilitates the development of the multivalent and mosaic vaccines [4] necessary to control recombinant hiv-1 strains, an increasingly common occurrence in the epidemic in uganda [5] . computational approaches aim to identify optimal epitopes relevant to vaccine development and are not isolated to hiv-1 only, but a wide range of pathogens, including ebola virus [6] , therefore various statistical validation approaches have been applied for evaluation of these methods [7] [8] [9] [10] . for hiv-1 vaccine design purposes an important consideration for the suitability of a computational algorithm is the breadth of discrete number of t-cell epitopes it generates that could reach particular levels of coverage [11] of circulating viruses. the higher the number of epitope variants the more the reduction in their requirements to attain optimum coverage levels for any epidemic. previous data has shown that breadth of tcell response is associated to viral set point in chronic hiv-1 infection [12] [13] [14] [15] [16] [17] . in order to translate the computational epitope prediction into vaccine design, the number of discrete epitopes computationally generated from particular hiv-1 proteins is an important metric for further investigation [11] . a reliable pan-hla-specific algorithm netmhcpan4.0 [18] [19] [20] that has been improved by advances in hla binding data, covers 172 mhc class i molecules from human (hla-a, b, c, e), mouse (h-2), cattle (bola), primates (patr, mamu, gogo) and swine (sla) [20, 21] , and can also predict binding to alleles devoid of experimental data basing on similarity to known binders and non-binders [22, 23] . this is an artificial neural network (ann) algorithm for predictions of 8-14aa and capable of predicting epitopes for other hla alleles using data for similar alleles by positional similarity of residues in their binding motifs. netmhcpan4.0 is considered to be the tool of choice for such predictions considering the benchmarking done against other related tools [24] . nevertheless to have a conclusive outcome of the computational performance we compared netmhcpan4.0 to both an older and recent tool, netctl1.2 [25] [26] [27] and mhcflurry1.2.0 [28] respectively. the binding of ctl epitopes to mhc class i molecules is linear, anchoring at residues 2 and 9; hence the interface between ligand and ctl can be determined computationally [29] . validation of such computational applications can be done by comparing their predictions with suitable experimental data. despite the paucity of data validating the performance of computational methods relative to wet laboratory experiments, a few have documented them to achieve an area under the curve auc of over 90% [18, 19, 30, 31] by isolated experimental data. we have not come across a wet experiment that evaluated computational predictors to achieve a robust auc using a single set of wet laboratory experimental data. the previously reported 90% auc is largely based on positional specific scoring algorithms (pssm) for the collective isolated experiments alongside probability models used to establish affinity or binding scores. one study that explored the reliability of in-silico approaches in epitope prediction and its application for vaccine design reported a meagre 22, 44%, and relatively higher 78% match for three computational tools namely yfpeithi, ctlpred and iedb respectively [32] . using experimental epitope mapping data generated from 757 peptides tested on cells of 6 early hiv-1 infected individuals at paired time points, we show that netmhcpan4.0 can be useful for markedly reducing pooled peptide experiments as demonstrated by the 95% experimental and computational concordance. the data used was from an independent study that did not include this analysis in its objectives. experimental data of peptides previously mapped for hiv-1 epitope recognition of 6 individuals for a separate study (table 1) at 2 time points each was used for comparison with the computationally predicted binders. these were from a ugandan early hiv-1 serodiscordant couple cohort approved by the uganda virus research institute (uvri), research and ethics review board and the uganda national council of science and technology (uncst). all participants provided informed consent. six (6) participants whose experimental epitope recognition profile we evaluated were early hiv-1 infections (table 1) , enrolled under the following criteria: (i) detection of hiv-1 p24 antigen with a simultaneous negative hiv-1 antibody [34] .. the experimentally tested peptides totalled 757 (fig. 1) , were 17aa long, overlapping by 11aa and spanning the hiv-1 proteome consensus for subtypes a1 and d. cultured eli-spot assays using 200,000 cells/well as previously documented by obuku ae. et.al [34] . and ex-vivo ifn-γ elispot assay using 100,000 cells/well were used for testing peptide pools and epitope mapping respectively. experimental positive pools were 3 times the background wells and at least 600 spot forming units per million cells. "deconvolute this" software [35] was used to identify possible responding individual peptides from the pools or where it was not possible all the peptides in a pool were tested as single peptides. high resolution reference strand conformation analysis hla class i tissue typing for the early infected subjects was done using methods described elsewhere [36] . hiv-1 subtyping determination was performed on the gag gene [37, 38] using sanger method generated sequences. the sequences were input into the rega hiv-1 automated subtyping tool to determine the hiv-1 clade [39, 40] . hiv-1 subtypes a1 and d consensus sequences were used as inputs for the computational epitope prediction. these peptide sequences were all for the year 2004 downloaded from the los alamos database (hiv.lanl.gov/content/sequence/newalign/align. html). the web version of netmhcpan4.0 [19] (http://www.cbs.dtu.dk/services/netmhcpan/) was configured to predict 9mer through 14mer epitopes for 22 hla class i alleles ( table 1 ) that were expressed by the 6 hiv-1 infected donors. linux version mhcflurry1.2.0 [28] was used to predict 9mer epitopes and an earlier tool netctl1.2 was also used to predict 9mer epitopes for the 22 hla class i alleles expressed by the 6 study individuals. perl version 5.26.2 was used to extract the binders from all the netmhcpan4.0 predictions and also to compare the computational binders to the 93 mapped experimental 17aa peptides for 9mer through 14mer hits using a sliding window. an experimental peptide was considered a hit if any of the computational 9mer through 14mer sequence was contained in the 17 amino acid experimental peptide sequence as well as any of the hla-a, b or c expressed by the individual matched the netmhcpan4.0 hla class i type(s). if multiple computational epitope predictions were contained in a single 17mer experimental peptide they were counted as a single hit. these were determined by a blast search of the computational binders against the derivative experimental peptides to determine computational predictions from the same test peptide. the accession numbers of the sequences used to determine the hiv-1 subtypes for 5 of the 6 study subjects are; kt825896, kt825897, kt825898, kt825899, kt825900, kt825901, kt825902, kt825903, kt825904, kt825905, kt825906, kt825907, kt825908, kt825909, kt825910, kt825911 and kt82512. statistics computations and plots were generated using spss version 24.0.0.0. the netmhcpan4.0 computational performance was evaluated using a confusion fig. 1 elispot peptide consort; the experimental peptide mapping data was generated by culture elispot of multiple peptide pools tested in duplicate wells per time point, followed by ex-vivo elispot of potential candidate epitopes. to experimentally map a single time point required at least 541 assay wells matrix to classify true positives, true negatives, false positives and false negatives that were used for the receiver operator characteristic (roc) plot. the hit rate (sensitivity) and false hit rate (specificity) of binder predictions as determined by the netmhcpan4.0 threshold of peptides within the top 2% (with a score of 2 or less) were calculated and the strength of the model was determined by calculating the area under the curve, auc of the roc plot [41] [42] [43] . pearson's correlation coefficient was used to evaluate the relationship between the number of epitopes with various hiv-1 genes. to evaluate if there were any differences in the early versus late time point peptides for the binding ranking of the experimentally mapped peptides as predicted by the computational score the wilcoxon signed rank test was used. to evaluate if hiv-1 subtypes a1 and d affected the number of computational predictions generated, fisher exact test was used. to determine whether multiple computationally predicted epitope sequences were derived from the same experimental peptide sequence, a local blast database was set up using geneious version 9.0.5. both hiv-1 clades a1 and d experimental consensus sequences were used separately each as a reference sequence for the blast. the computational peptide sequences were then aligned against the consensuses to evaluate those derived from a single 17 amino acid experimental peptide sequence. where an experimental peptide was predicted by multiple or overlapping computational peptides, the average netmhcpan4.0 score was assigned as the computational score for this peptide. this score was also used during the generation of the roc curve and the confusion matrix. to compare the association between elispot spot forming units and netmhcpan4.0 scores or mhcflurry1.2.0 affinities and also the association between the values for experimentally mapped peptides for all participants and their cognate computational core 9-mer and a single 14-mer epitope sequence with scores. peptides shown in italic text were not algorithmically predicted as binders. multiple computational predictions contained in a single experimental peptide were counted as a single hit. participant's identifiers (id) beginning with e or l represent early or late time sampling points respectively the 2 computational tools, pearson's correlation coefficient was used. number of experimental assays compared to computationally guided prediction assay projections to experimentally determine epitopes for 757 peptides spanning the whole hiv-1 proteome for clades a and d as well as both time points of the 6 individuals required a total of 4230 test assay wells. for each test subject these included 9 antigen proliferation wells, 384 culture elispot wells and an average of 164 epitope mapping elispot wells (range; 148-186 test wells). using the 22 hla alleles represented in the study subjects we were able to computationally predict 95% of the experimentally mapped epitopes. this approach could have reduced the test assays by eliminating all the t-cell antigen proliferation and culture elispot steps totalling to 3258 assay wells (77%) and leaving only 972 (23%) epitope mapping assays required. applying a pooling strategy to the computational predictions similar to that used in the experimental pooling where each pool contained approximately 20 peptides with a coverage of 3 per peptide pool, the 923 potential peptides (95% of experimental peptides for epitope mapping elispot derived from the 972 (23%) eligible epitope mapping peptides) would make at most 46 pools. consequently the computational prediction approach could have reduced the experimental assays by at least 80%. the core 9mer epitope sequence was similar across 9mer through 14mer set length except for one 14-mer peptide (hit 72 in table 2 ) magnitude of epitope predictions are variable across hla alleles, hiv-1 proteins and clades the input hiv-1 subtypes a1 and d consensus whole proteome sequences evaluated for potential 9, 10, 11, 12, 13 and 14-mer binders to the 22 hla alleles represented in the six patients, varied in the distribution of predicted binders across hiv-1 genes and hla alleles. all the peptide hits predicted for 10 through 14-mer were also all predicted in the 9-mer set except for two 14-mer peptides. an expected positive correlation for hiv-1 protein length with number of epitopes predicted was observed as illustrated by spearman's rank order correlation; r s = 0.88 (fig. 2, a and b) . netmhcpan4.0 predicted 95% (88/93) ( table 2 ) of the experimentally mapped peptides as binders and missed 5% (5 out of 93) ( table 3) for the 12time points of the 6 participants. mhcflurry predicted 91% (85/93) of the experimental peptides and had a lot of similarity to netmhcpan4.0 for the predicted hla. netctl was the least performing tool with only 15% (14/ 93) predicted experimental peptides ( table 2) . comparison of the various epitope prediction length set showed that the 9mer setting was ideal for netmhc-pan4.0. the number of predictions were 88, 79, 55, 39, 39 and 37 hits out of 93 for 9, 10, 11, 12, 13 and 14-mer epitopes respectively. increasing the prediction length from 9mer through 14mer resulted in a smaller number of predicted binders as illustrated in fig. 3 . since we held the assumption that our wet experimental data was the gold standard we evaluated the sensitivity and specificity of netmhcpan4.0.the computational predictor had more predicted binders than those determined by the experimental mapping as presented in the confusion matrix in table 4 . the experimental positive's count also shown in table 2 under column "hit no" shows the test peptide count (1through 88) that contained the computational 9mer sequence. multiple computational epitopes may be contained in a single experimental peptide, as shown in the column "netmhcpan4.0 9-mer epitope prediction" in table 2 . overall hiv-1 clade a 9-mer predictions were fewer in number than clade d (fig. 2, c) though the difference did not approach statistical significance. the experimental peptide mapping data was derived from a baseline time point corresponding to hiv-1 fiebig stages iv, v and vi (table 1 ) and a later time point. ninety-three (n = 93) epitopes were experimentally mapped of which 12 were recognized at both baseline and later time points, 34 only at baseline and 54 only at the later time point. comparison of the ranked computational score for netmhcpan4.0 binders of early (n = 34) versus later peptides showed that the later time point predictions were stronger binders reaching statistical significance (wilcoxon signed rank p-value = 0.0000005) (fig. 4) . netmhcpan4.0 ranked binders as those predicted to false positive (37) computational negative false negative (5) true negative (627) the total number of peptides experimentally tested were 757 and these are broken down to show the fractions from both the experimental testing and netmhcpan4.0 computational predictions fig. 4 early versus late peptides. experimentally mapped peptides at baseline (n = 34) and at least 12 months later (n = 34) were compared using the 9-mer computational netmhcpan4.0 scores of the hits. the lower the computational score the stronger the predicted binding. late peptides were significantly stronger binders than early peptides (wilcoxon signed rank test, p = 0.0000005) be in the top 2% and assigned a score of 0.2 or below. any binder within the top 0.5% and assigned a score of 0.05 or below was ranked as a strong binder. considering only the 9-mer computational predictions, peptides that were derived from the same 17mer experimental peptide were determined by a blast mapping to their derivative sequences. the 17-mer peptides were then classified into a confusion matrix (table 4 ) as true positives, false positives, true negatives or false negatives. from the classification the true positive rate (sensitivity) was plotted against the false positive rate (1-specificity) using an roc curve and the auc attained reached 0.928 (fig. 5) . only 9-mer length epitopes were considered in the roc analysis as increasing the length to 10-mer through 14mer netmhcpan4.0 predictions neither raised the number of predicted binders nor improved the hit rate as all their predictions contained the sequence already predicted in the 9-mer set except 1, 14-mer peptide (hit 72 in table 2 ). comparison of the elispot magnitude of response (spot forming units) did not show any association to either netmhcpan4.0 scores or mhcflurry1.2.0 affinity values. similarly a comparison of the latter 2 computational predictors did not show any association between their assigned "affinity" values. netmhcpan4.0 registered the highest concordance to the wet experiments followed by mhcflurry1.2.0. in this analysis we showed that the computational method netmhcpan4.0 predicted 95% of previously experimentally mapped hiv-1 epitopes in 6 hiv-1 infected individuals expressing a total of 22 different hla class i alleles. in our ifn-γ elispot assays we evaluated 757 17mer peptides overlapping by 11 amino acids and covering the whole hiv-1 subtype a1 and d consensus proteomes. out of the 5 experimentally determined epitopes missed by the algorithm (table 3 ), 4 were actually computationally predicted as binders but were not included for lack of concordance with the participant's hla alleles. about one third (37) of 125 total positive predictions were not experimentally supported in our tests. these do not necessarily represent false positives, as elispot detection depends on the frequency of specific t cells in the participant's repertoire, and we observed changes in dominant t cell specificities within a given participant between early and later time points after hiv-1 infection. a formal roc evaluation of the score generated by netmhcpan4.0 as a classifier for peptides recognised/not recognised by pbmc in ifng elispot assays, produced an auc of 0.928. thus experimental confirmatory tests cannot be dropped altogether, however the netmhcpan4.0 algorithm could provide a considerable saving of time and resources in verifying just the predicted epitopes. as the participants had been enrolled in the acute/ early phase of hiv-1 infection and we had observed intra-participant changes in epitope recognition between early and late time points after infection, we compared the binding scores of confirmed epitopes at these time points and found a statistically significant change towards recognition of higher binding peptides as the infection entered the chronic phase. this might represent better support of the t-cell response directed at more stable hla/peptide complexes as the infection progresses into chronicity. the netmhcpan4.0 algorithm, which is based on binding affinity and integrates data on eluted naturally processed ligands, reflected optimal hla class i binding for 9-mers, producing a decreasing number of predictions when the peptide size was increased from 9 to 11 amino acids. with a single exception, predicted binders between 11 and 14 amino acids included at least one 9mer predicted to bind on its own, suggesting a destabilizing effect of the extra amino acids beyond the canonical hla class i binding pockets at positions 2 and 9 could account for fewer predictions. important limitations are the lack of predictions of hla class ii restricted epitopes, which might have contributed to a fraction of ifn-γ elispot responses. approximately 5% of the computational predictions may be false positives that only increase the size of planned wet experiments and approximately 1% of true positives may also be missed. in this analysis, using netmhcpan4.0, mhcflurry and netctl to predict previously experimentally mapped epitopes, we demonstrate that the computational methods reliably predict an acceptable portion of binder epitopes. we recommend the use of such computational methods to reduce the size of experiments required cost associated. vaccine design for h5n1 based on b-and t-cell epitope predictions methods for prediction of peptide binding to mhc molecules: a comparative study induction of broad cross-subtype-specific hiv-1 immune responses by a novel multivalent hiv-1 peptide vaccine in cynomolgus macaques first-in-human randomized controlled trial of mosaic hiv-1 immunogens delivered via a modified vaccinia ankara vector analysis of the history and spread of hiv-1 in uganda using phylodynamics mapping hla-a2, −a3 and -b7 supertype-restricted t-cell epitopes in the ebolavirus proteome enhancing in silico protein-based vaccine discovery for eukaryotic pathogens using predicted peptide-mhc binding and peptide conservation scores discovering a vaccine against neosporosis using computers: is it feasible? vacceed: a high-throughput in silico vaccine candidate discovery pipeline for eukaryotic pathogens based on reverse vaccinology a combined prediction strategy increases identification of peptides bound with high affinity and stability to porcine mhc class i molecules sla-1*04: 01 defining epitope coverage requirements for t cell-based hiv vaccines: theoretical considerations and practical applications control of human immunodeficiency virus replication by cytotoxic t lymphocytes targeting subdominant epitopes consistent cytotoxic-t-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities cd8 t-cell recognition of multiple epitopes within specific gag regions is associated with maintenance of a low steady-state viremia in human immunodeficiency virus type 1-seropositive patients induction of multifunctional human immunodeficiency virus type 1 (hiv-1)-specific t cells capable of proliferation in healthy subjects by using a prime-boost regimen of dnaand modified vaccinia virus ankara-vectored vaccines expressing hiv-1 gag coupled to cd8+ t-cell epitopes dynamics of viral evolution and ctl responses in hiv-1 infection broad and gag-biased hiv-1 epitope repertoires are associated with lower viral loads netmhcpan, a method for mhc class i binding prediction beyond humans netmhcpan-4.0: improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data pan-specific mhc class i predictors: a benchmark of hla class i pan-specific prediction methods a community resource benchmarking predictions of peptide binding to mhc-i molecules efficient peptide-mhc-i binding prediction for alleles with few known binders multipred: a computational system for prediction of promiscuous hla binding peptides automated benchmarking of peptide-mhc class i binding predictions an integrative approach to ctl epitope prediction: a combined algorithm integrating mhc class i binding, tap transport efficiency, and proteasomal cleavage predictions largescale validation of methods for cytotoxic t-lymphocyte epitope prediction reliable prediction of t-cell epitopes using neural networks with novel sequence representations mhcflurry: open-source class i mhc binding affinity prediction t-cell antigenic sites tend to be amphipathic structures emerging vaccine informatics prediction of peptide-mhc binding using profiles comparison of experimental fine-mapping to in silico prediction results of hiv-1 epitopes reveals ongoing need for mapping experiments dynamics of hiv viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary hiv infection macrophage inflammatory protein-1 beta and interferon gamma responses in ugandans with hiv-1 acute/early infections optimized determination of t cell epitope responses high resolution hla class i typing by reference strand mediated conformation analysis (rsca) profile of t cell recognition of hiv type 1 consensus group m gag and nef peptides in a clade a1-and d-infected ugandan population frequencies of gag-restricted t-cell escape "footprints" differ across hiv-1 clades a1 and d chronically infected ugandans irrespective of host hla b alleles a standardized framework for accurate, high-throughput genotyping of recombinant and nonrecombinant viral sequences an automated genotyping system for analysis of hiv-1 and other microbial sequences the meaning and use of the area under a receiver operating characteristic (roc) curve receiver operating characteristic (roc) curve: practical review for radiologists smallsample precision of roc-related estimates publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the study participants who provided specimen for the wet laboratory experiments, the aids information centre clinic kampala, uganda that steered the rubicon discordant couple cohort study, the late anthony kebba who initiated the rubicon cohort and christine watera who coordinated the recruitment clinic activities. we thank ruhena sargeant for hla typing, the late harr f. njai for hiv-1 elisa and western blot assays and deogratius ssemwanga for help with genbank submissions. this research is jointly funded by the uk medical research council (mrc) and the uk department for international development (dfid) under the mrc/dfid concordat agreement, the wellcome trust (grant wt078927ma), and edctp (project code: ta_05_40200_40203). most of the relevant data to support the manuscript has been included in the write-up. if any addition data is required will be availed once requested. the rubicon study, from which we derived the experimental data, was reviewed and approved by the uganda virus research institute, research and ethics committee (uvri-rec) and the uganda national council of science and technology (unct). all participants provided informed signed consent accepting to freely participate in this study. not applicable. the authors declare that they have no competing interests. authors' contributions dbl performed the peptide mapping experiments. nn provided hiv-1 subtyping. dbl analysed the data together with pp. dbl wrote the manuscript with major contributions from pp, aeo, pk and js. all authors reviewed the manuscript and or provided useful contributions as well as approved the final manuscript. key: cord-007664-c5snhymz authors: mauerhoff, thekla; pujol-borrell, ricardo; mirakian, rita; bottazzo, gian franco title: differential expression and regulation of major histocompatibility complex (mhc) products in neural and glial cells of the human fetal brain date: 2002-11-13 journal: j neuroimmunol doi: 10.1016/0165-5728(88)90049-5 sha: doc_id: 7664 cord_uid: c5snhymz the cells of the central nervous system (cns) have the peculiarity of physiologically expressing very low levels of hla molecules. in multiple sclerosis (ms), however, as in endocrine autoimmune diseases, there is a marked increase of hla expression in the tissue (i.e. the plaques) and this is attributable not only to infiltrating cells but also to the astrocytes. to gain an insight into the regulation of hla in the different cell types in the cns and to compare it to that observed in the endocrine organs, we have studied the effect of the lympho/monokines interferon (ifn)-α and -γ, tumour necrosis factor (tnf)-α, and interleukin (il)-2 and other agents on this aspect of the biology of human fetal brain cells in culture. a two-colour immunofluorescence technique which combines antibodies to diverse cns cell markers and monoclonal antibodies (moabs) to the non-polymorphic region of hla molecules was used throughout this study. in control cultures, only astrocytes expressed mhc class i, but after incubation with either ifn-γ or tnf-α oligodendrocytes acquired class i expression. surprisingly, astrocytes became spontaneously class ii positive in culture and this was greatly enhanced by ifn-γ. other agents such as il-2, epidermal growth factor, phorbolmyristate acetate and lectins had no effect. the expression of hla molecules in the cells of the cns both in basal conditions and in response to lymphokines is therefore selective and highly heterogenous, thus reflecting their intrinsic biological diversity. these findings may help to explain the features of the immunopathology of ms and also of latent viral infections of neural cells. the level of cell surface expression of class i and class ii products of the major histocompatibility complex (mhc) (hla in humans) varies widely among different cell types and it seems to be determined not only by their ontogenic lineage but to be also influenced by the direct action of several inducers and modulators. of the latter, the best known are lymphokines and in particular those belonging to the interferon (ifn) family (review in pujol-borrell and . recently, tumour necrosis factor (tnf-a) and lymphotoxin (lt or tnf-fl) , produced by macrophages and t lymphocytes respectively, have also been found to induce and/or enhance hla molecule expression in certain systems (collins et al., 1986; pfizenmaier et al., 1987; . our previous work has clearly indicated that human endocrine cells have different sensitivities to the in vitro effect of lympho/monokines with regard to the induction of hla class ii expression. thus viable thyrocytes are easily inducible following incubation with mitogens (pujol-borrell et al., 1983) or ifn-3, , but pancreatic islet cells are much more resistant to the elicitation of this phenomenon when exposed to the same or other putative modulators . in fact, using islet cell cultures, it was only the combination of tnf or lt with ifn-'t which ultimately produced the first positive results . these latter studies were prompted by the interest generated by the hypothesis that endocrine cells inappropriately expressing hla class ii could present their own surface autoantigens to helper t cells and in this way generate an autoirnmune response . this postulation was based on the observation that thyroid cells have the capacity for de novo expression of class ii molecules (pujol-borrell et al., 1983) , and the finding that in thyroid glands affected by autoimmune disorders, the follicular cells strongly expressed class ii ). since the above hypothesis was proposed, the occurrence of inappropriate class ii expression has been documented in the target organs of many organ-specific autoimmune diseases (review in bottazzo et al., 1986) and the ability of class ii-positive thyroid cells to present antigen to t cells has been experimentally demonstrated (londei et al., 1984 (londei et al., , 1985 . the identification of class ii-positive astrocytes in the active 'plaques' of patients with multiple sclerosis (ms) has attracted a great deal of interest and has led to the suggestion that astrocytes inappropriately expressing class ii molecules in vivo could play a role relevant to the pathogenesis of ms (traugott et al., 1985) , a hypothesis which is somewhat parallel to that originally proposed by us . as an extension of our interests, we report here the results obtained in studies on the inducibility of hla molecules on human fetal brain cells in culture and their differential sensitivity to the action of a variety of stimuli known to be potent modulators of hla expression in other cell types. tissue was obtained from 26 fetal brain specimens (provided by the medical research council tissue bank, royal marsden hospital, london, u.k.), after legal terminations carried out by aspiration between 11 and 19 weeks of gestation. the experimental protocol described here was approved by the university college/middlesex hospital ethical committee. the meninges and visible vessels were carefully removed from the brain, tissue was washed with hanks' balanced salt solution containing 1% bovine serum albumin (bss-bsa), minced with scissors and forceps and digested for 15 min at 37°c in a solution which contained 0.25% trypsin (sigma, london, u.k.), and 0.04% dnase (dnase 25, sigma, london, u.k.) in dulbecco's modified eagle's medium (dmem, gibco, paisley, scotland, u.k.). digestion was stopped by the addition of 10% fetal calf serum (fcs, gibco). the undigested tissue was further disrupted by repeated pipetting. the digest was passed through a 300 #m nylon mesh to remove large fragments, pelleted by centrifugation at 800 rpm for 10 min and resuspended in 'complete medium'. this consisted of 45% dmem, 45% f12 (flow, irvine, scotland, u.k.), 10% fcs, glucose 500 mg/100 ml, glutamine 2 mm, gentamicin 40 gg/ml, and penicillin 50 gg/ml. cells were counted and viability, as assessed by differential staining with acridine orange/ethidium bromide under a uv microscope, was always close to 100%. aliquots of 105 cells were plated on glass coverslips (13 mm diameter), placed in 24-multiwell plates (nunc, kamstrud, denmark) and 'complete medium' added to a final volume of 0.5 ml/well. cultures were kept at 37°c in a 5% co 2 humidified incubator and left undisturbed for 4 days. in some instances small fragments of brain tissue were directly snap frozen and stored at -70°c until used for immunofluorescence studies on cryostat sections. given the complexity of the architecture of the brain tissue and the heterogeneity of cell types present in primary cultures prepared from it, a two-colour ifl technique was employed throughout the study combining monoclonal antibodies (moabs) to hla products with monoclonal or polyclonal antibodies to different cell markers (review in . this ensured the precise identification of positive and negative cells under observation. cryostat sections and monolayer cultures were stained similarly following the same protocols. for the identification of hla class i and ii molecules the following staining protocols were applied (for the specificity and source of the antibody used, see table 1 ). hla class l 1st layer: moab w6/32; 2nd layer: tritc-conjugated goat anti-mouse igg; 3rd layer and 4th layer: either rabbit anti-glial fibrillary acidic protein (astrocytes) or rabbit anti-factor viii (endothelial cells) followed by fitclabelled goat anti-rabbit ig or moab 04 (oligodendrocytes) followed by fitc goat anti-mouse igm. hla class 11. (using igg moabs to class ii) 1st layer: moab mid3 (class ii), tu22 (dq specific), b7/21 (dp specific) or vic-y1 (class ii invariant chain specific); 2nd layer: tritc-labelled goat anti-mouse igg; 3rd layer and 4th layer: either rabbit anti-glial fibrillary acidic protein (gfap) followed by fitc-labelled goat anti-rabbit ig or moab 04 followed by fitc-labelled goat anti-mouse igm. hla class ii. (using rfdr1 igm moab to class ii) 1st layer: moab rfdr1 (class ii); 2nd layer: fitc goat anti-mouse igm; 3rd layer: moabs gc (oligodendrocytes), 308 (astrocytes), or rt.97 (neurons); 4th layer: tritc-labelled goat anti-mouse igg. moabs were used at a final concentration of 10 /~g/ml. unstimulated control cultures were always stained in parallel with treated cultures. in addition, nonspecific binding was ruled out by staining parallel stimulated cultures with an unrelated moab of the same ig class. undesirable cross-reactivities among reagents derived from different species were assessed by omitting one of the layers in turn. to determine hla membrane expression, viable cultures were incubated with the corresponding moabs, stained by the appropriate conjugate and then, prior to counterstaining with antibodies to cell-specific cytoplasmic antigens, cultures were fixed with methane/acetone (50:50, v/v) for 10 min. when the second antibody was recolmizing membrane antigens distinct from hla molecules, the fixation step was done at the end of the entire procedure. for the detection of cytoplasmic class i, class ii and class ii invariant chain the cultures were fixed prior to the staining. all preparations were examined with a x 63 oil immersion objective using a zeiss iii uv photomicroscope equipped with epi-illumination and phase contrast. fluorescence intensity in both membrane and cytoplasm was evaluated in 200-500 cells per culture except in the case of oligodendrocytes where, given their small number, only fewer cells (around 50) could be evaluated. ifl intensity was arbitrarily scored from negative to 3 +. table 2 lists the various biological and chemical compounds used to induce hla product expression on human fetal brain cells in culture. • this compound was tested only for its effect on astrocyte mhc expression. in sections from three different human fetuses stained for neurofilaments with the moab rt.97, the soma of neurons was clearly visible among the complex network of fibres, while the antiserum to glial fibrillary acidic protein (anti-gfap) produced a very intricate reticular pattern which was denser around the vessels. in successive sections stained for class i, the only cells clearly positive were the endothelial cells lining the capillaries, these identified by antibodies to factor viii. in spite of careful and intensive search, no cells positive for class ii products were detected in the parenchyma or in the vessels of the brain when sections were stained with the appropriate moabs. cell attachment occurred as early as 24 h by the time most preparations were processed, between days 6 and 12, cultures showed a complex organization similar to that described by dickson et at. (1982) . cells with long processes identified as neurons (rt.97 +) and oligodendrocytes (04 +) (see fig. la-d) laid on a carpet of astrocytes (gfap +) (fig. 2b ) (raft et at., 1979) . the long processes of the neurons were arranged in dearly distinct bundles forming bridges among clusters of cells which seemed to serve as orga~i7jng centres. immature ofigodendrocytes were present in smaller numbers compared to neurons and were often located in the periphery of these clusters, their intricate branching processes completing the complex three-dimensional network in which all the cells were included. the number of neurons decreased during the period of culture but even after 30 days these cells were quite numerous in the monolayers. fibronectinpositive cells (fibroblasts) presumably derived from contaminating meningeal cells, were very scarce in the initial days and, although their number increased with time, they never overgrew the other cell types even after 4 weeks in culture. attempts were not made to identify microglial cells because we lacked specific reagents to recognize them. endothelial cells were not present in our monolayers, probably due to their inability to grow on glass surfaces (zetter, 1981) . (b) basal expression of hla molecules. class i molecules were consistently detected throughout the culture period on astrocytes but not on oligodendrocytes or neurons ( fig. 2a and b) . class ii molecules were seen in less than 1% of the cells in the culture up to day 3. however, astrocytes (identified by gfap-positive staining) became gradually class ii positive, and by day 20, around 50% of them showed a clear and bright staining ( fig. 2c and d, time-course in fig. 3 ). this 'spontaneous' class ii expression was observed in both the protoplasmic and the fibrous type of astrocytes (identified by their morphology under phase contrast and confirmed by gfap + staining) and also in the astrocyte subtype recently defined by the 308 moab (dickson et at., 1983) . astrocyte class ii expression included also hla-dp and dq subregion products and did not depend on the gestational age of the fetal brain. passive absorption of class ii molecules to the membrane of the astrocytes could be excluded by the concomitant detection of the non-secretory class ii invariant chain in the cytoplasm of these cells when they were stained with moab vic-y1. the other cell types (neurons and oligodendrocytes) remained negative throughout the culture period (up to 30 days). for the reasons mentioned above, microghal or endothelial cells could not be detected. table 2 were tested during the initial 8 days of culture when the percentage of spontaneously positive astrocytes was less than 15%, as to minimize the interference of the spontaneous expression of class ii in astrocytes in the reading of the preparations (see above). among the biological and chemical compounds used (table 2) , only ifn-a, ifn-7 and tnf-a had a clear effect on hla expression in the different cell types and the results are summarized in table 3 . neurons and oligodendrocytes both = astrocytes did not express class ii in the early stages of culture, but an increasing percentage were found spontaneously positive after 6 days in culture (see text and fig. 4) . lacked class i expression in basal conditions but, while the former remained always negative, oligodendrocytes acquired a clear membrane staining for class i after culture with either ifn-t or tnf-a (fig. 4) . ifn-a and ifn-t both produced an increase in class i expression in astrocytes and fibroblasts. none of the mediators or chemicals tested were able to induce class ii expression in neurons or oligodendrocytes, and this also applies to the combination of ifn-t and tnf-a. ifn-~, was able to produce a dramatic increase in the number of astrocytes positive for membrane and cytoplasmic class ii and 4 days after the addition of 1000 u/ml to the cultures, all gfap-positive cells were stained for class ii. this effect was dose related (see fig. 5 and legend) and was also detectable with the moab vic-y1 which reacts with the cytoplasmic invariant chain of class ii products. no apparent synergism was observed between ifn-~ and tnf-a in the induction of class ii in astrocytes (fig. 6 ). it has previously been reported that primary cultures of human fetal brain tissue can be a useful substrate for studying the expression of cell type-specific antigens, e.g. gangliosides, tetanus toxin receptors (dickson et al., 1982 (dickson et al., , 1985 , and the present data confirm that they can also be successfully employed to investigate the expression and modulation of hla molecules. our findings that neurons do not express hla class i or class ii products in basal conditions are in agreement with previous reports using a similar culture system of murine origin (wong et al., 1984; dubois et al., 1985; suzumura et al., 1985; tedeschi et al., 1986) . neither ifn-7 nor tnf-a separately or in combination were able to induce class i or class ii expression in human fetal neurons. with regard to class i expression our results differ from those reported by wong et al. (1985) who showed that ifn-y can induce class i in up to 40% of mouse neurons, and from a similar report by lampson et al. (1984 lampson et al. ( , 1986 ) on a human neuroblastoma cell line. stages of development, species differences and neoplastic transformation could account for these apparent discrepancies. referring to class ii molecules, our results agree with the generally accepted concept that cells of the neuronal lineage are refractory to the effect of available modulators, alone or in combinations, on the levels of hla class ii expression regardless of the species employed (wong et ai., 1985) . the inability of ifn-t to induce class i or class ii in neurons contrasts with the strong positive modulation it produces on most cell types including terminally differentiated human cells such as thyrocytes and melanocytes (houghton et al., 1984) . the biological meaning and molecular basis for this differential regulation are at present unknown. the potential of oligodendrocytes to express hla products has been extensively studied, especially as these cells seem to be the target of the postulated autoimmune response in ms and experimental allergic encephalomyelitis (eae). our finding that human oligodendrocytes are constitutively negative for both class i and class ii molecules but express class i after incubation with ifn-t is in agreement with numerous previous studies on cultures of murine brain cells and suggests the presence of receptors for ifn-t on the surface of these cells (wong et al., 1984; suzumura et al., 1985; tedeschi et al., 1986) . more interesting is the observation that tnf-a can also induce class i expression in oligodendrocytes from human fetal brain. reports of a similar action of tnf-a on other cell types (collins et al., 1986) have suggested that this may be one of the mechanisms through which this mediator exerts its anti-tumour action in vivo (pfizenmaier et ai., 1987) . neither ifn-t or tnf-a alone or the combination of the two were able, however, to induce class ii expression in oligodendrocytes in our system. this is in contrast with our recent findings which showed that human pancreatic islet cells, another cell type unresponsive to ifn-t alone, could be induced to express class ii products by the synergistic action of ifn-t and tnf-a or -fl . there is no consensus in the literature on whether adult human oligodendrocytes can express class ii molecules following incubation with ifn-t. lysak et al. (1983) , like us, did not find class ii expression in stimulated primary cultures while kim (1985) and kim et al. (1985) reported 4-16% of class ii-positive oligodendrocytes (although only in half of the culture preparations); the reason for these differences remains at present unclear. regulation of hla expression in astrocytes has also been extensively studied both in rodent (hirsch et al., 1983; wong et al., 1984; dubois et al., 1985; massa et al., 1986) and in human systems (shen et al., 1985; takiguchi et al., 1985) , again due to its potential importance in the pathogenesis of ms. astrocytes expressing class ii are present in the 'active' plaques in the brain of ms patients (traugott et al., 1985) and, most importantly, it has been shown that they can present antigens to t cell clones in vitro (fierz et al., 1985) . the postulate has accordingly been made that by presenting myelin basic protein (mbp) or related antigens to autoreactive t lymphocytes, class ii-positive astrocytes may initiate and possibly perpetuate an autoimmune recognition to mbp which subsequently leads to the demyelination process in active ms (fontana, 1984; sun and wekerle, 1986; takiguchi et al., 1986) . in short-term cultures we found that astrocytes express class i but not class ii molecules and this is in accordance with previous work (fierz et al., 1985) . however, we were surprised by the arousal of a population of astrocytes positive for class ii after 5-6 days while these cells were growing in culture. these results may substantiate the report by kim (1985) which showed that in cultures of human adult brain 9-27% of the astrocytes were class ii positive at day 10. similar findings were described on a human fetal astrocyte cell line, but the spontaneous class ii expression disappeared after subsequent passages (pulver et al., 1987) . this phenomenon seems to be a unique characteristic of the astrocytes since fibroblasts and other cell types present in our cultures remained negative, thus arguing against the possibility that it is mediated by ifns or other non-specific soluble factors generated during the culture period or by the medium employed, as previously suggested (pulver et al., 1987) . this spontaneous induction of class ii expression indicates that in vivo the expression of class ii by astrocytes should be constantly suppressed by still unidentified humoral or locally produced mediators. alternatively, it is also possible that the abundant debris which our cultures initially contained could have stimulated phagocytosis by the astrocytes and this then triggered the expression of class ii, as part of the general activation process undergoing in these cells. in this context, it is of interest to recall that it has recently been shown that the phagocytosis of coronavirus particles induced class ii expression in rat astrocytes (massa et al., 1986) . as expected, ifn-a and ifn-3, both produced an increase in class i expression in astrocytes. the studies of class ii modulation on astrocytes were carried out at the beginning of the culture period while the level of spontaneous expression was still low and in this way we were able to reproduce previous work which has shown that ifn-7 is indeed able to induce a rapid increase in the percentage of class ii-positive human fetal astrocytes (pulver et al., 1987) as well as enhance the intensity of the staining (fig. 5) . microglial cells were not easy to identify in our monolayers and this was most probably due to the early gestational age of the fetal tissue. however, even if these cells were present in small amounts, our lack of any specific marker made their identification, solely on morphological ground, very difficult. in summary, we have presented for the first time data on a large number of human fetal brains both in sections and in primary cultures indicating that the regulation of the expression of hla products in human fetal brain cells varies widely among the different cell types. there is a gradation in both constitutive expression and inducibility which ranges from neurons, which lack hla products completely and are refractory to all inducers and modulators tested so far, to astrocytes which in culture become spontaneously positive for class ii and are highly responsive to ifn-y. this heterogeneity of hla regulation among the brain cells is reminiscent of that observed for class ii in the human pancreas where ductal/exocrine cells are easily induced to express class ii while islet endocrine cells require a two-med~tor signal . considering the analogies between the different cell populations which form these two complex organs it seems that the cells with a high or intermediate turnover (ductal cells, astrocytes) are more easily induced to express hla molecules than those with a low turnover (e.g. islet cells) or no turnover (e.g. neurons). one may speculate on the biological significance of this differential regulation. one possibility is that the lack of inducibility of class ii expression may constitute one of the mechanisms which maintain tolerance to autoantigens present in highly specialized cells cowing, 1985) . on the other hand, absence of class i and inability to express it, may represent a more extreme way of excluding irreplaceable cells from the interaction with the immune system and in particular from the attack by cytotoxic t lymphocytes. this may help to explain the inability of the cellular immune mechanisms to eradicate certain neuronal viral infections, such as herpes simplex. it may well be that it is more advantageous for the organism to have some neurons latently infected than not to have them at all, with the consequence of an irreversible disability. if these postulates are correct, it follows that a better knowledge of the differential regulation of hla protein expression in the central nervous system may contribute to our understanding of the autoimmune processes and latent viral infections occurring in this organ. role of aberrant hla-dr expression and antigen presentation in the induction of endocrine autoimmunity organ-specific autoimmunity: a 1986 overview recombinant human tumour necrosis factor increases mrna levels and surface expression of hla, a, b, c antigens in vascular endothelial cells and dermal fibroblasts in vitro does t cell restriction to ia limit the need for self-tolerance? cell surface antigens of human foetal brain and dorsal root ganglion cells in tissue culture identification of cell surface antigens present exclusively on a subpopulation of astrocytes in human foetal brain cultures cellular distribution of 04 antigen and galactocerebroside in primary cultures of human foetal spinal cord expression of major histocompatibility complex antigens in neonate rat primary mixed glial cultures monoclonal antibody to a plasma membrane antigen of neurons astrocytes as antigen presenting cells. i. induction of la antigen expression on astrocytes by t cells via immune interferon and its effects on antigen presentation astrocytes present myelin basic protein to encephalitogenic t cell lines aberrant expression of hla-dr antigen on thyrocyte in graves' disease: relevance for autoimmunity expression of la antigens by cultured astrocytes with gamma interferon surface antigens of melanoma and melanocytes. specificity of induction of ia antigens by human gamma-interferon antigen expression by glial cells grown in culture expression of ia antigens on the surface of human oligodendrocytes and astrocytes in culture weak hla and beta2-microglobulin expression of neuronal cell lines can be modulated by interferon monoclonal antibody analysis of mhc expression in human brain biopsies: tissue ranging from 'histologically normal' to that showing different levels of glial tumour involvement epithelial ceils expressing aberrant mhc class li determinants can express antigen to cloned human t lymphocytes human t cell clones from autoimmune thyroid glands: specific recognition of autologous thyroid cells cultured human oligodendrocytes and rat schwann cells do not have immune response gene associated antigen (la) on their surface viral particles induce la antigen expression on astrocytes tumour necrosis factor enhanced hla-a, b, c and hla-dr gene expression in human tumour cell inappropriate class ii expression in endocrine cells. is it a primary event lectin induced expression of dr-antigens on human cultured follicular thyroid ceils differential expression and regulation of mhc products in the endocrine and exocrine cells of the human pancreas hla class li induction in human islet cells by ifn-gamma plus tnf or lt cultured human fetal astrocytes can be induced by interferon-y to express hla-dr cell-type specific markers for distinguishing and studying neurons and the major class of ghal cells in culture cellular and antigenic properties of cultured normal and fetal brain and ghoma cells monoclonal antibodies (o1 to 04) to oligodendrocyte cell surface. an immunocytochemical study in the central nervous system la restricted encephalitogenic t lymphocytes mediating eae lyse autoantigen-presenting astrocytes expression of h2 antigen on oligodendrocytes is induced by soluble factors from concanavalin a activated t cells induction of antigen presentation ability in purified cultures of astroglia by interferon-gamma response of glioma cells to gamma-interferon: increase in class ii mrna, protein and mlr stimulating ability astrocytes produce interferon that enhances the expression of h-2 antigens on a sub-population of brain cells laboratory investigation of autoimmune endocrine diseases interferon-gamma induces hla-dr expression by thyroid epithelium on the presence of ia positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation inducible expression of the h2 and ia antigens on brain cells interferon-gamma induces the expression of h-2 and ia antigens on brain cells the endothelial cells of large and small blood vessels we are greatly indebted to all the scientists and commercial firms cited in the text and tables for their generous donations of monoclonal antibodies and other reagents and to the medical research council tissue bank, royal marsden hospital, london for the supply of fetal brain tissue. we thank our colleagues dr. a. belfiore and dr. i. todd for advice and help during the development of this work, professors deborah doniach and ivan roitt for their constant support, and dr. m.l. cuzner and dr. j.g. dickson for very useful discussion. we would also like to thank marian pine for her help in the preparation of the manuscript. key: cord-001247-pxzbirqd authors: sun, lu; zhang, yu; zhao, bao; deng, mengmeng; liu, jun; li, xin; hou, junwei; gui, mingming; zhang, shuijun; li, xiaodong; gao, george f.; meng, songdong title: a new unconventional hla-a2-restricted epitope from hbv core protein elicits antiviral cytotoxic t lymphocytes date: 2014-03-22 journal: protein cell doi: 10.1007/s13238-014-0041-4 sha: doc_id: 1247 cord_uid: pxzbirqd cytotoxic t cells (ctls) play a key role in the control of hepatitis b virus (hbv) infection and viral clearance. however, most of identified ctl epitopes are derived from hbv of genotypes a and d, and few have been defined in virus of genotypes b and c which are more prevalent in asia. as hbv core protein (hbc) is the most conservative and immunogenic component, in this study we used an overlapping 9-mer peptide pool covering hbc to screen and identify specific ctl epitopes. an unconventional hla-a2-restricted epitope hbc141–149 was discovered and structurally characterized by crystallization analysis. the immunogenicity and anti-hbv activity were further determined in hbv and hla-a2 transgenic mice. finally, we show that mutations in hbc141–149 epitope are associated with viral parameters and disease progression in hbv infected patients. our data therefore provide insights into the structure characteristics of this unconventional epitope binding to mhc-i molecules, as well as epitope specific ctl activity that orchestrate t cell response and immune evasion in hbv infected patients. around 350 million people worldwide are chronically infected with hepatitis b virus (hbv). chronic hbv infection is a major cause of cirrhosis, liver failure, and hepatocellular carcinoma (hcc) (lavanchy, 2005; neuveut et al., 2010) . hbv-specific cd8 + t lymphocytes (ctl)-mediated immune response is multi-specific, polyclonal, and vigorous during acute hepatitis b (ahb), which plays a vital role in viral control and viral clearance, as well as disease pathogenesis (yukihiro, 2012; westover and hughes, 2007; tan et al., 2008) . whereas hbv-specific ctl response is minimal or undetectable in chronic hepatitis b (chb) with viral persistence and immune tolerance, indicating the key role of hbvspecific t-cell response in determination of disease progression and outcome (bertoletti and gehring, 2006; . the hbv genome of ∼3.2 kb in length efficiently encodes several overlapping viral proteins, including the polymerase, core, hbe, envelope (pre-s1, s2, s), and x proteins. analysis of ctls specific for viral epitopes within core (sendi et al., 2009; liu et al., 2012; ) , envelope (liu et al., 2008) , polymerase (rehermann et al., 1995) , and x (hwang et al., 2002) proteins showed that the highly conserved hbv core protein (hbc) elicits the strongest ctl responses than other viral proteins, suggesting that hbc-specific t cell response may play a leading role in viral control and clearance. to identify immune-dominant hbv-specific ctl epitopes, especially epitopes from hbc protein, is therefore necessary for monitoring t cell responses during disease progression, as well as for developing epitope-based therapeutic vaccines against chb (inchauspe and michel, 2007; gordon et al., 2013; liu et al., 2013a, b) . so far 60 hbv-specific human leukocyte antigen (hla) class i restricted and 32 hbv-specific hla class ii restricted epitopes have been identified in all 8 hbv genotypes (desmond et al., 2008; liu et al., 2008; guo et al., 2011; chen et al., 2013; tan et al., 2013) . however, most of these identified epitopes are derived from hbv of genotypes a and d, few hla class i restricted epitopes have been defined in virus of genotypes b and c, which are more prevalent in asia. meanwhile, currently the mostly used methods to predict and identify t cell epitopes are either by computer algorithms based on the mode of peptide binding to mhc molecules, or measurement of t cell responses of pbmcs simulated with panels of overlapping peptides (liu et al., 2011) . these epitope identification strategies may ignore unconventional t cell epitopes as computational analysis is largely based on the characteristic of anchor residues. in this study, an overlapping 9-mer peptide pool was used to screen and identify hbv genotypes b-and c-derived t cell epitopes. a new unconventional cd8 + t cell epitope hbc141-149 derived from viral core protein, which shares partial sequence identity with previously reported hbc141-151 (bertoni et al., 1997) and hbc139-148 (lee et al., 1997) , was discovered. its immunological function and clinical relevance were further assessed. identification of a new hla-a*0201-restricted epitope from hbv core protein hbv core protein is the most conservative and immunogenic component of hbv proteins. we used an overlapping 9-mer peptide pool covering the whole length of core protein (aa 1-150) and its variants (totally 191 peptides) for t2 binding assay to screen genotype b-and c virus-derived hbcag-specific t cell epitopes , as shown in fig. 1a . several peptides were found to have high affinity for binding to hla-a*0201 molecules, as evidenced by the fi (0.72 for hbc183-191, 2.41 for hbc141-149, 2.64 for hbc60-68 (v60), 2.47 for hbc18-27, and 0.04 for hbc82-90), as shown in fig. 1b . hbc141-149 spanning from hbc aa 141 to 149 was chosen as the focus of this study due to its high binding affinity among the top hits from screening. to determine the minimal sequence length of the epitope, panels of n-or c-terminally truncated or extended peptides of hbc141-149 were synthesized. the fis of truncated peptide hbc141-148 (stlpettv) and hbc142-149 (tlpettvv) were only 0.124 and 0.151, respectively (fig. 1c) . the fis of decapeptides hbc140-149 (lstlpettvv) and hbc141-150 (stlpettvvr) were only 0.033 and 0.005, respectively (fig. 1d ). all hbc141-149 truncated and extended peptides displayed little binding to hla-a*0201, which indicates that the 9-mer peptide hbc141-149 is the optimized epitope in length. in addition to t2 cell binding assay, the capability of hbc141-149 to bind to hla-a*0201 was observed in the refolding assay (fig. 1e ). the structure of hla-a*0201/hbc141-149 complex next, the complex of hla-a2 and hbc141-149 was prepared for crystallization to characterize the binding features of hbc141-149. the crystal structure of the hla complex was determined to 2.3 å resolution (table 1 ). the structure of hla-a*0201/hbc141-149 shows that hbc141-149 possesses a typical conformation of an hla-a2-restricted 9-mer epitope ( fig. 2a and 2b ). the unambiguous electron densities of hbc141-149 clearly show that position 2 (t2) and position 9 (v9) are buried in pockets b and f, respectively (fig. 2c ). compared to the typical hla-a2-restricted epitopes which have an anchor residue leu or met at position 2, hbc141-149 has thr at position 2. the side chain oh of thr does not disrupt its inserting into the hydrophobic pocket b properly (fig. 2d) . instead, the side chain oh of thr can form a strong hydrogen bond interaction with h atom of glu on the α1 domain of the heavy chain, which helps hbc141-149 binding to the hla-a2 heavy chain and stabilizes the entire complex. the side chains of amino acids p4, e5, and v8 protrude out from the hla-a2 surface and may be involved in t cell receptor (tcr) attachment and recognition. to the best of our knowledge, this is the first structure showing the binding features of hla-a2 to hbc141-149 with an unconventional p2 anchor thr. hbc141-149 peptide generates specific ctl response in hla-a2.1/kb transgenic mice then, the immunogenicity of hbc141-149 epitope was determined in vivo. female hla-a2.1/kb transgenic mice were immunized with hbc dna-prime/hbc141-149 peptide boost regimen three times, using heat shock protein gp96 as adjuvant (li et al., 2011) . hbc141-149-specific ctl was detected by elispot assay 1 week after the last immunization. as can be seen in fig. 3a , similar to the hbc18-27 peptide-immunized mice (positive control), a strong ctl response was observed in splenocytes from hbc141-149 peptide-immunized mice (sfc, 145.4 ± 58.6) . no peptidespecific cd8 + t cell response was detected from hbc82-90 peptide-immunized mice (negative control). similar results were observed in the killing assay using hbv plasmidtransfected 293t cells (fig. 3b ) or hbc141-149 peptidepulsed t2 cells (fig. 3c ) as target cells. to further determine the epitope-specific ctls, fresh pbmcs from hla-a2 + ahb patients were stimulated with hbc141-149 peptide and detected by ex vivo ifn-γ elispot assays. as shown in fig. 3d , a much higher peptide-specific ctl response was observed in pbmcs stimulated with hbc141-149 or the immunodominant peptide hbc18-27 as the positive control than negative control peptide hbc82-90 (119.68 ± 76.66 for hbc141-149 or 164.58 ± 112.41 for hbc18-27 vs. 20.94 ± 17.57 for hbc82-90, both p < 0.01). the high standard deviations observed in elispot assay may reflect the random between-patient variation. taken together, these results indicate that hbc141-149 is an hla-a2-restricted cd8 + tcell epitope and is naturally processed in patients with hbv infection. hbc141-149 epitope elicits antiviral t cell immunity in hla-a2.1/hbv transgenic mice next, we examined whether hbc141-149 epitope was able to induce anti-hbv t cell response using f1 hybrids of hbv transgenic balb/c mice and hla-a2.1/kb transgenic mice as the experimental model, which are hbv immunotolerant. hla-a2.1/hbv transgenic mice were immunized with an hbc dna prime/ hbc141-149 peptide boost formulation. as shown in fig. 4a , compared to mice immunized with negative control peptide, the number of sfcs increased by around 6-fold in hbc141-149 peptide-immunized mice. similar result was obtained in cytotoxicity assay using hbv plasmid-transfected 293t cells as target cells. we then evaluated hbc141-149 peptide-induced tcell response could lead to inhibition of hbv. immunization with hbc141-149 led to a 35.5% decrease in serum hbsag levels (p < 0.05) (fig. 4c) , and a significant reduction of viral dna levels (p < 0.05) (fig. 4d ) at week 8 compared to the negative control. meanwhile, significant lower levels of serum hbsag and viral dna were observed after immunization (at week 8) than those before immunization (at week 0) in hbc141-149-treated mice but not in control peptidetreated mice. and these results indicate that the epitope-specific ctl response induced by the hbc141-149 could significantly inhibit hbv replication in the transgenic mice. in vitro refolding of hbc141-149 peptide with hla-a*0201 heavy chain and β2m. gel filtration chromatography was used to analyze the refolded complexes on a superdex200 16/60 column. the hla complex with the expected molecular mass of 45 kda eluted at the volume of 15.9 ml. the hla complex (peak 2) was analyzed by sds-page electrophoresis and coomassie blue staining (line 2). finally, to address the clinical relevance of the hbc141-149 epitope in chb, mutations within this epitope were analyzed in 197 chb and 64 aclf patients (table 2 ). in chb, compared to patients infected with wild-type isolates (hbc141-149, stlpettvv), patients infected with hbc141-149 mutants had much higher alt levels (247.7 ± 18.62 vs. 545.2 ± 137.7, p < 0.05) (fig. 5a ) and aspartate aminotransferase (ast) levels (187.6 ± 13.78 vs. 1668 ± 700.5, p < 0.05) (fig. 5b) . notably, compared to chb patients infected with hbc141-149 wild-type viruses, patients with hbc141-149 mutants had much higher (approximately 3.3-fold) hbv dna loads (p < 0.05) (fig. 5c ). there were no statistical differences in sex and age between patients infected with the wild-type and mutant isolates. the prevalence of the hbc141-149 mutations increased with the disease progression in hbv-infected patients (fig. 5d ). to investigate the impact of these sequence variations within hbc141-149 epitope on its immunogenicity, three 9-mer peptides containing main epitope variations of hbcv149i, hbct147a, and hbct147c in chb patients were synthesized, respectively, for hla-a2.1/kb transgenic mice immunization. as seen in fig. 5e , compared to the wild-type hbc141-149 peptide-immunized mice (sfc, 96.7 ± 6.02), ctl responses by elispot assay were significantly decreased in hbcv149i mutant peptide-(sfc, 66.3 ± 5.69) or hbct147a mutant peptide-immunized mice (sfc, 73 ± 7.81). taken together, these results suggest that the hbc141-149 mutations associated with necroinflammation and higher hbv levels, and it may also be associated with poor prognosis, which may be due to viral immune evasion. in this study, we identified a new hla-a2-restricted cd8 + t cell epitope hbc141-149 by screening an overlapping 9-mer peptide pool covering hbv core protein. this unconventional hla-a2 restricted epitope was further determined and structurally characterized by hla-a2 transgenic mouse model and crystallographic analysis. moreover, we demonstrated that the hbc141-149 epitope exhibits antiviral capability in hla-a2.1/hbv transgenic mice. finally, our results show that mutations in hbc141-149 epitope correlate with clinically relevant parameters in chb. our work may therefore provide a comprehensive evaluation of the impact of the newly defined epitope on viral specific t cell response and suggests a possible immune evasion for maintenance of viral persistence in patients with hbv infection. the identification of hbv-specific t cell epitopes is mostly based on the binding mode of the peptides to mhc molecules in silico prediction or assessing t cell responses with panels of overlapping peptides in hbv-infected patients. these methods are effective and rapid to purposefully identify epitopes, however, a considerable number of atypical or unconventional epitopes may be ignored or missed by these methods due to the limitation of anchor residues analysis-based computation (liu et al., 2011) . in this study, an overlapping 9-mer peptide pool was used to screen b and c genotype-derived hbc-specific ctl epitopes, and hbc141-149 was identified as a new 9-mer hla-a2restricted t cell epitope. importantly, similar numbers of hbc141-149-specific and immunodominant epitope hbc18-27-specific cd8 + t cells were observed in ahb patients (fig. 3c) , indicating that hbc141-149 is an immunodominant epitope in hbv-infected patients. however, the newly defined epitope hbc141-149 (stlpettvv) in this study possesses thr at position 2. different t cell epitope prediction programs, including syfpeithi (http://www.syfpeithi. de/scripts/mhcserver.dll/epitope-prediction.htm), net-mhc (http://www.cbs.dtu.dk/services/netmhc/), and bimas (http: //www-bimas.cit.nih.gov) show the binding score of the epitope hbc141-149 to hla-a2 is only 0, 1.465, and 0.414, respectively. as hbc141-149 does not get a high score, it could be omitted in conventional screening. interestingly, as for hbc141-149 (stlpettvv), the side chain oh of thr2 can form a stronger hydrogen bond interaction with glu on α1 domain of the heavy chain, which helps its inserting into lu sun et al. pocket b (fig. 2d ) of hla-a*0201 and stabilize the entire complex. therefore, our data indicated that thr may also act as a dominant role as a p2 anchor for hla-a2-binding peptides, which may be taken into consideration in the future designation of these online prediction programs. cd8 + t cells are the main effector cells responsible for viral clearance as well as disease pathogenesis during hbv infection (thimme et al., 2003; harari et al., 2006; ouyang et al., 2013) . as a consequence of long-term interaction between hbv and infected patients, the virus evolves mutations to reduce epitopes for evading immune detection and clearance, especially escape from t cell recognition (maman et al., 2011; westover and hughes, 2007) . indeed, nonsynonymous mutations in hbv epitope have been found to be associated with disease progression in chb (kim et al., 2011; frelin et al., 2009) . consistent to these studies, we found that mutations in the newly defined epitope are positively related to viral parameters and pathogenesis of liver disease. the effect of mutations within hbc141-149 on viral replication capability, as well as the potential impact of the epitope-specific ctls on the interplay of hbv and chronically infected patients remains to be addressed. in summary, this study indicates that hbc141-149 is an immunodominant hla-a2-restricted ctl epitope with atypical binding characteristics to hla-a2 molecules, has potent anti-hbv immune activity and the clinical significance in patients with hbv infection. we further demonstrated that mutations within this epitope may affect disease progression in chb. given the key role of cd8 + t cells in viral clearance, our work provides valuable insight for the functional implications of hbc141-149 epitope-specific t cell response in hbv infection. understanding the epitope-specific ctl function in the complex regulation networks that orchestrate t cell response, viral persistence and immunoevasion in chb will allow to predict disease progression and develop immunotherapeutic approaches against hbv infection. all the enrolled patients' clinical characteristics are described in table 2 . a total of 29 ahb patients were enrolled for blood collection, which were divided into two groups: hla-a2-positive (n = 19) and hla-a2-negative (n = 10). all patients were negative for hcv, hdv, and hiv-1 infection. 10 ml of blood samples were collected from each patient. all patients were hospitalized in beijing 302 hospital from september 2010 to september 2011. all study participants have written informed consent and the study was approved by the ethics committee of beijing 302 hospital. hbv transgenic balb/c mice were purchased from transgenic engineering lab, infectious disease center (guangzhou, china), which were generated with a viral dna construct, phbv1.3, containing 1.3 copies of the hbv genome. serum hbv s antigen (hbsag) and viral dna, as well as hbc expression in hepatocytes in mice's liver, were tested positive for all transgenic mice. the hla-a2.1/kb transgenic mice (zhang et al., 2007) , and t2 cells labeled with cfse were loaded with 20 μg/ml peptide at 37°c for 1 h as target cells (c). the target cells were then mixed with hbc141-149-stimulated splenocytes at different ratios: 1:1, 1:10, and 1:20, or hbc18-27, hbc82-90-stimulated splenocytes served as positive or negative controls. after 4 h, the mixed samples were stained with pi, and the killing of target cells were analyzed by facs. the data shown are the mean ± sd of five mice. (d) ahb patients (n = 29) were divided into hla-a2-positive (n = 19) and hla-a2-negative (n = 10) groups. pbmcs (2 × 10 5 /well) from these patients were stimulated with the indicated peptides for detection of peptide-specific ctls by elispot assay. hbc82-90 peptide served as negative control for background evaluation in elispot assay. pbmcs from hla-a2-patients were used for specificity evaluation of ctls. paired samples t-tests were used for elispot assay in ahb patients. *p < 0.05 and **p < 0.01 by t-test. data are representative of two independent experiments. by professor huang wl (imcas, beijing, china). the hla-a2.1/kb mice and hbv transgenic mice were crossed to gain the f1 hybrids of hla-a2.1/hbv transgenic mice. all f1 hybrids were screened for serum hbsag by elisa, viral dna by real-time pcr, and hla-a2 by pcr-ssp (protrans, deutschland) before experimental manipulations. the wild-type hbc gene was cloned into pcdna3.1 (invitrogen) and the recombinant plasmid was designated pcdna3.1-hbc. phbv1.3 containing 1.3 copies of the full-length hbv genomic sequence was maintained in the lab. the hbc sequences of genotypes b and c were attained using the protein database from ncbi and a total of 171 hbc sequences of genotype b and 159 hbc sequences of genotype c included. the sequences were compared and served as a basis on peptide synthesis. if the variation rate of a certain amino acid (aa) was more than 10%, a series of peptides associated with this variation would be synthesized. we adopted the overlapping method (8-aa overlap) to synthesize a total of 191 nonapeptides (9-mers) covering hbc1-150 aa . all of these peptides were synthesized at jier biological (shanghai, china), and their purity was determined as >95%. the hbc18-27 (flpsdffpsv) and hbc82-90 (rel-vvsyvn) peptides were used as positive and negative controls, respectively. the 293t fig. 3 . fresh splenocytes (5 × 10 5 ) isolated from immunized mice were stimulated with wild-type or mutant peptide, respectively. peptide-specific ctls were detected by ifn-γ elispot assay. by the manufacturer. cells and supernatants were harvested at 24 h, 48 h, and 72 h after transfection, respectively. t2 cells were used to perform mhc stabilization assays as previously described (zhou et al., 2006) . the binding activity of each peptide was calculated as the fluorescent index (fi), and the fi was determined by: (mean fitc fluorescence with the given peptide − mean fitc fluorescence without peptide) (mean fitc fluorescence without peptide). peptides regarded as epitopes with high affinity should meet the following criteria: fi ≥1. refolding, protein crystallography, and structure determination recombinant proteins of hla-a*0201 heavy chain and β2m were expressed in escherichia coli (garboczi et al., 1992.) and the gradual dilution method was performed during refolding process (liu et al., 2012) . then, superdex 200 10/300 gl gel filtration chromatography followed by resource-q anion-exchanged chromatography (ge healthcare) was used for the concentration and purification of the soluble portion of the refolded complex. the hanging-drop vapor diffusion method was performed at 18°c to crystallize the purified complexes. at a final concentration of 10 mg/ml in 0.1 mol/l bis-tris (ph 6.5) and 25% (w/v) polyethylene glycol 3350, hla-a0201/hbc141-149 crystals were obtained. equipped with an r-axis v||++ image-plate detector, rigaku micromax007 rotating-anode x-ray generator was operated at 40 kv and 20 ma (cu κα; λ = 1.5418 å) to collect crystallographic data at 100 k in house. with protein data bank (pdb) entry 1jf1 as the search model, the structure of hla-a*0201/hbc141-149 was determined by molecular replacement with the program molrep. mice (6-8 weeks old) were immunized i.m. with 50 μg of plasmid pcdna3.1-hbc or pcdna3.1 (control) at week 1 and subcutaneously with 50 μg of hbc141-149 peptide bound to 30 μg of heat shock protein gp96 as adjuvant (li et al., 2011) at weeks 3 and 4, respectively. mice were sacrificed 1 week after the last immunization. splenocytes were isolated as previously described (liu et al., 2009) . each group contained five to seven mice. to detect epitope-specific t cells, enzyme-linked immunosorbent spot (elispot) assay was performed according to the manufacturer's instruction. briefly, ninety-six well pvdf plates (bd-pharmigen, san diego, ca) were precoated overnight at 4°c with the coating ab and blocked for 1 h at 37°c. patient pbmcs (2 × 10 5 ) or murine splenocytes (10 6 ) were added to each well together with 50 μg/ml of peptide and incubated at 37°c for 24-48 h with phytohemagglutinin (pha)-stimulated t cells as positive controls. each test was performed at least in triplicate. the spots were counted and analyzed using an elispot reader (cellular technology ltd, usa). 293t cells were labeled with 2 μmol/l cfse as target cells after transfection with phbv1.3 and seeded into a 96-well plate. then ctls were added at different ratios: 1:1, 10:1, and 20:1. plates were incubated for 4-6 h at 37°c, and cfse positive target cells were stained with propidium iodide (pi) using a vybrant apoptosis assay kit (invitrogen, usa). in addition, t2 cells were loaded with 20 μg/ml hbc141-149 peptide at 37°c for 1 h as target cells, and seeded into a 96-well plate. the cytotoxicity assay was performed as described above. each assay was performed in triplicate. detection of hbsag and hbeag by elisa, and hbv dna by realtime pcr elisas and real-time pcr for detection of serum hbsag, hbeag and viral dna copies were performed as described (fan et al., 2013) . differences between groups were determined using student's t-test. pearson's χ test was used to detect the correlation between variation rate and disease progress in hbv infected patients. clinical statistical analyses were performed with spss version 16.0 software (spss inc., chicago, illinois). p values <0.05 were considered significant. the immune response during hepatitis b virus infection human histocompatibility leukocyte antigen-binding supermotifs predict broadly cross-reactive cytotoxic t lymphocyte responses in patients with acute hepatitis an immunodominant hla-a*1101-restricted cd8 + t cell response targeting hepatitis b surface antigen in chronic hepatitis b patients a systematic review of t-cell epitopes in hepatitis b virus: identification, genotypic variation and relevance to antiviral therapeutics increased expression of gp96 by hbx-induced nf-κb activation feedback enhances hepatitis b virus production a mechanism to explain the selection of the hepatitis e antigen-negative mutant during chronic hepatitis b virus infection hla-a2-peptide complexes: refolding and crystallization of molecules expressed in escherichia coli and complexed with single antigenic peptides antiviral therapy for chronic hbv infection and development of hepatocellular carcinoma in a u.s. population identification and functional studies of hla-a0201 restricted ctl epitopes in the x protein of hepatitis b virus functional signatures of protective antiviral t-cell immunity in human virus infections hla-a2 1 restricted peptides from the hbx antigen induce specific ctl responses in vitro and in vivo vaccines and immunotherapies against hepatitis b and hepatitis c viruses number of mutations within ctl-defined epitopes of the hepatitis b virus (hbv) core region is associated with hbv disease progression worldwide epidemiology of hbv infection, disease burden, and vaccine prevention peptide-specific ctl induction in hbv-seropositive pbmc by stimulation with peptides in vitro: novel epitopes identified from chronic carriers hansenula polymorpha expressed heat shock protein gp96 exerts potent t cell activation activity as an adjuvant a mutant hbs antigen (hbsag)183-191 epitope elicits specific cytotoxic t lymphocytes in acute hepatitis b patients treg suppress ctl responses upon immunization with hsp gp96 revival of the identification of cytotoxic t-lymphocyte epitopes for immunological diagnosis identification of hla-a*0201-restricted cd8 + t-cell epitope c64-72 from hepatitis b virus core protein conserved epitopes dominate cross-cd8+ t-cell responses against influenza a h1n1 virus among asian populations cross-allele cytotoxic t lymphocyte responses against 2009 pandemic h1n1 influenza a virus among hla-a24 and hla-a3 supertype-positive individuals immune-induced evolutionary selection focused on a single reading frame in overlapping hepatitis b virus proteins mechanisms of hbv-related hepatocarcinogenesis cd8(low) t-cell subpopulation is increased in patients with chronic hepatitis b virus infection the cytotoxic t lymphocyte response to multiple hepatitis b virus polymerase epitopes during and after acute viral hepatitis ctl escape mutations of core protein are more frequent in strains of hbeag negative patients with low levels of hbv dna host ethnicity and virus genotype shape the hepatitis b virus-specific t-cell repertoire immunoprevalence and immunodominance of hla-cw0801 restricted t cell response targeting the hbv envelope transmembrane region abbreviations aclf, acute on chronic liver failure; ahb, acute hepatitis b; ctl, cytotoxic t lymphocyte; elispot, enzyme-linked immunosorbent spot; fi, fluorescent index; hbc, hbv core protein; hbeag, hbv e antigen; hbsag, hbv s antigen; hbv, hepatitis b virus; sfc, spotforming cells. lu sun, yu zhang, bao zhao, mengmeng deng, jun liu, xin li, junwei hou, mingming gui, shuijun zhang, xiaodong li, george f. gao, and songdong meng declare that they have no conflict of interest. for studies with human subjects in this article: all patients were hospitalized in beijing 302 hospital from september 2010 to september 2011. written informed consent was provided by all study participants. the study was approved by the ethics committee of beijing 302 hospital.for studies with animals in this article: animals received humane care, and the study of mice was in strict accordance with "the this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. key: cord-002686-zzongyfa authors: oany, arafat rahman; pervin, tahmina; mia, mamun; hossain, motaher; shahnaij, mohammad; mahmud, shahin; kibria, k. m. kaderi title: vaccinomics approach for designing potential peptide vaccine by targeting shigella spp. serine protease autotransporter subfamily protein siga date: 2017-09-07 journal: j immunol res doi: 10.1155/2017/6412353 sha: doc_id: 2686 cord_uid: zzongyfa shigellosis, a bacillary dysentery, is closely associated with diarrhoea in human and causes infection of 165 million people worldwide per year. casein-degrading serine protease autotransporter of enterobacteriaceae (spate) subfamily protein siga, an outer membrane protein, exerts both cytopathic and enterotoxic effects especially cytopathic to human epithelial cell type-2 (hep-2) and is shown to be highly immunogenic. in the present study, we have tried to impose the vaccinomics approach for designing a common peptide vaccine candidate against the immunogenic siga of shigella spp. at first, 44 siga proteins from different variants of s. flexneri, s. dysenteriae, s. boydii, and s. sonnei were assessed to find the most antigenic protein. we retrieved 12 peptides based on the highest score for human leukocyte antigen (hla) supertypes analysed by netctl. initially, these peptides were assessed for the affinity with mhc class i and class ii alleles, and four potential core epitopes vtaraglgy, fhtvtvntl, httwtltgy, and ielagtltl were selected. from these, fhtvtvntl and ielagtltl peptides were shown to have 100% conservancy. finally, ielagtltl was shown to have the highest population coverage (83.86%) among the whole world population. in vivo study of the proposed epitope might contribute to the development of functional and unique widespread vaccine, which might be an operative alleyway to thwart dysentery from the world. shigella is a gram-negative, facultative anaerobic, nonmotile, nonspore forming, and rod-shaped true bacteria closely related to salmonella and escherichia coli. the resulting infection by this organism called shigellosis, also known as bacillary dysentery or marlow syndrome, is most typically associated with diarrhoea and other gastrointestinal symptoms in humans. this pathogen is usually found in water that is contaminated with human feces within the setting of poor hygiene among kids of underneath 5 years old and is transmitted via the fecal-oral route. the infection will occur even if there is just a bodily function of only ten to one hundred microorganisms [1] . in each year, 165 million cases of shigella infection are accounted worldwide, of that, 163 million take place in developing countries and ultimately result in millions of death [2] . bangladesh has got the top rates of shigellosis according to the recent global enteric multicenter study (gems) in asia. the output of this study has revealed that the shigella is the third leading reason behind diarrhoea in children [3, 4] . shigella species are usually classified into four serogroups: s. dysenteriae (12 serotypes), s. flexneri (6 serotypes), s. boydii (18 serotypes), and s. sonnei (one serotype) based on the biochemical properties and group-specific o antigens within the outer portion of the semipermeable membrane. s. dysenteriae, s. flexneri, and s. boydii are physiologically similar in distinction to s. sonnei. among them, s. flexneri is the most frequently isolated species globally and accounts for 60% of cases in the unindustrialized countries; s. sonnei causes 77% of cases in the industrialized countries [1] . the underlying therapeutic challenge to manage shigella is its accrued resistance to most often used antibiotics like ampicillin, tetracycline, streptomycin, nalidixic acid, and sulfamethoxazole-trimethoprim [5] . earlier, ciprofloxacin, a third-generation fluoroquinolone antibiotic, has been used effectively for the treatment of bacillary dysentery [6] . however, this antibiotic is no longer helpful for the treatment of bacillary dysentery in south asian countries together with bangladesh, because of the dissemination of fluoroquinolone-resistant variety and its equivalent clones across the countries [7, 8] . hence, it is essential to find a sustainable approach like vaccinomics, which can elicit long-term and consistent immunological responses to fight against shigella. siga is annotated in the she pathogenicity island of shigella, encoding siga protein which belongs to the serine protease autotransporter of enterobacteriaceae (spate) subgroup proteins. the autotransporter proteins of gramnegative bacteria exhibit an n-terminal signal sequence, required for secretion across the inner membrane, and a cterminal domain that forms an amphipathic β-barrel pore that allows passage of the functional domain across the outer membrane. this type of exporter proteins either remains attached to the cell surface or is released from the cell by proteolytic cleavage [9] . siga is a multifunctional protein, able to degrade casein with cytotoxic and enterotoxic effects. moreover, siga is cytopathic for human epithelial type-2 (hep-2) cells, causing morphological changes and loss of integrity of the cell monolayers, important for the pathologic process of shigella [10] . the position of siga in the chromosome made them less vulnerable to loss compare to the other virulence factors harbouring within the plasmid, and more exposure to the immune cells occurred by this secreted toxin [11] . most importantly, this protein has been shown to be immunogenic following infection with shigella [10] . the generalized modules of membrane antigen-(gmma-) based outer membrane proteins including siga were also shown to be highly immunogenic [12] , which prompted us to target siga as one of the best vaccine candidates and to design potential peptide vaccine covering all the shigella spp. and most of the regions of the world. epitope-based immunizing agents are often an inexpensive choice to thwart enteric shigella infection. the identification of specific epitopes derived from infectious pathogens has considerably advanced the event of epitopebased vaccines (evs). higher understanding of the molecular basis of substance recognition and human leukocyte antigen-(hla-) binding motifs has resulted in the advancement of rationally designed vaccines that solely depends on algorithms predicting the peptide's binding to human hla. the traditional process for the development of a vaccine is very complex compared to that of the epitope-based vaccine, and additionally, it is chemically stable, more specific, and free of any infectious or oncogenic potential hazard [13] . however, the invention of a wet laboratory-based candidate epitope is expensive and laborious that requires varied medicine experiments in the laboratory for the ultimate choice of epitopes. hence, the interest for predicting epitopes by computational strategies, alternate in silico approaches among researchers, is growing bit by bit with reduced efforts. vaccinomics is the application of integrated knowledge from different disciplines including immunogenetics and immunogenomics to develop candidate next-generation vaccine and understand its immune response [14] . currently, various vaccinomics databases are accessible for identification of distinctive b lymphocyte epitopes and hla ligands with high sensitivity and specificity [15] [16] [17] . the vaccinomics approach has already proven its potency in identifying the conserved epitope in the case of human immunodeficiency virus [18] , multiple sclerosis [19] , tuberculosis [20] , and malaria [21] with desired results. in our study, we have applied vaccinomics approaches for the screening of potentially conserved epitopes by targeting protein siga. the flow chart summarizing the protocols for the complete epitope prediction is illustrated in figure 1 . the siga protein sequences of different strains of shigella species were retrieved from the ncbi genbank [22] database and analysed in the vaxijen v2.0 [23] server for the determination of the most potent antigenic protein. additionally, the target protein was crosschecked against human pathogens and other similar pathogens to ensure the orthologous entry by using blast-p [24] and orthomcl [25] databases [26] . the epitope prediction for the respective protein and their affinity score with mhc class i and class ii allele was measured following previously used approach [27, 28] . concisely, the netctl v1.2 server [29] was used for predicting potential cytotoxic t-lymphocyte (ctl) epitopes from the most antigenic protein. a combined algorithms including mhc-i binding, transporter of antigenic peptide (tap) transport efficiency, and proteasomal c-terminal cleavage prediction were employed for the t-cell epitope prediction. the epitope with the highest score for 12 mhc class i supertypes was selected. t cell epitope prediction tools from immune epitope database and analysis resource (iedb-ar) were used for the prediction of affinity with mhc class i [30] and mhc class ii [31, 32] . the stabilized matrix method (smm) was used to calculate the half-maximal inhibitory concentration (ic 50 ) of peptide binding to mhc class i with a preselected 9.0-mer epitope. the peptides were also assessed for hla i binding affinity by the software, episoft. for the analyses of mhc class ii binding, the iedb-recommended method was used for the specific hla-dp, hla-dq, and hla-dr loci. fifteen-mer epitopes were designed for mhc class ii binding analysis considering the preselected 9-mer epitope and its conserved region in the shigella strains. for the mhc class i and mhc class ii alleles, the epitopes consisting ic 50 < 250 nm and ic 50 < 100 nm, respectively, were selected for further analysis. the mhc class ii binding prediction tool predivac was also used to assess their affinity with hla_drb_1. alleles. furthermore, the mhccluster v2.0 server [33] was used for the identification of cluster of mhc restricted allele with appropriate peptides to further strengthen our prediction. this is the additional crosscheck of the predicted mhc restricted allele analysis from the iedb analysis resources. the output from this server is a static heat map and a graphical tree for describing the functional relationship between peptides and hlas. epitope conservancy of the candidate epitopes was examined using a web-based epitope conservancy tool available in iedb analysis resource [34] . the conservancy level of each potential epitope was calculated by considering identities in all siga protein sequences of different strains retrieved from the database. multiple sequence alignment (msa) was employed to understand the positions of the table 2 : epitopes for cd8 + t-cell along with their interacting mhc class i alleles with affinity < 250 nm. interacting mhc-i allele (ic 50 color key epitopes within the sequences. as spate family is very much specific for the enterobacteria, specifically, e. coli and shigella, we also include two e. coli sequences (gi|693049347| and gi|699401135|) along with those of four species of shigella for msa construction. the jalview (http://www.jalview.org/) tool was used for this analysis. the conservancy of the selected peptides was also substantiated by the protein variability software (pvs) [35] . population coverage for the epitope was assessed by the iedb population coverage calculation tool [36] . the combined score for mhc classes i and ii was assessed for the analysis of the population coverage. a homology model of the conserved region was obtained by modeller v9 [37] , and the predicted model was assessed by the procheck [38, 39] server. for the disorder prediction among the amino acid sequences, disopred v3 [40] was used. the protein frustratometer server [41] was employed for the detection of the stability and energy differences of the 3d structure of the protein. docking studies were also performed using the best possible epitope following the strategy used in previous studies [27, 28] . autodock vina [42] was used for the docking analysis. in our study, we have selected the hla-e * 01:01 molecule as a candidate for mhc class i and the hla-dqa1 as a candidate for mhc class ii for docking analysis because they are the available hits in the protein data bank (pdb) database. the pdb structure 2esv, human cytomegalovirus complexes with t-cell receptors, vmaprtlil peptide, and 3pl6-structure of autoimmune tcr hy.1b11 in complex with hla-dq1-were retrieved from the research collaboratory for structural bioinformatics (rcsb) protein database [43] . then, the structures were simplified by using pymol (the pymol molecular graphics system, version 1.5.0.4, schrödinger, llc) for the final docking purpose. the pep-fold server [44] was used for the conversion of the 3d structure of the epitope "ielagtltl" for mhc i and the epitope "kaielagtltltgtp" for the mhc ii molecule in order to analyse the interaction with hla alleles. the allerhunter server [45] was used to predict the allergenicity of our proposed epitope for further securing the prediction, and the support vector machine (svm) algorithm was used for the prediction within the server [46] . the predicted t-cell epitope (15-mer) was screened by iedb-ar using a number of web-based tools for the suitability as the b-cell epitope [47] [48] [49] . a total of 44 siga proteins from different variants of s. flexneri, s. dysenteriae, s. boydii, and s. sonnei were retrieved from the genbank database (table s1 in supplementary material available online at https://doi.org/10.1155/2017/6412353). thereafter, analyses with the vaxijen v2.0 server showed the protein with the accession number of gi|745767180| to have the highest antigenicity of 0.6699 (table s1 ). this highly antigenic protein was further analysed to detect the highly immunogenic epitope. no significant entry was found in the orthologous entry search of our targeted protein. identification. the netctlv1.2 server identified the t-cell epitopes, where the epitope prediction was confined to 12 mhc class i supertypes. based on the combined score, the top twelve epitopes (table 1) were listed for further analysis. analysis. iedb analysis resource predicted both mhc class i and mhc class ii restricted allele on the basis of the ic 50 value. all the predicted epitopes in table 1 were assessed for the mhc interaction analysis. epitopes for the mhc class i alleles are presented in table 2 . the peptide ielagtltlt was predicted to have the highest number of mhc class i binding. this peptide was predicted to have the binding affinity with five mhc class i alleles including hla-e * 01:01, hla-b * 40:01, hla-b * 15:02, hla-c * 03:03, and hla-c * 12:03. furthermore, the interacted alleles were reassessed by cluster analysis and are shown in figure 2(a) , as a heat map, and in figure s1a , as a dynamic tree. the peptides were reassessed by the episopt software for the hla i binding, and ielagtltl was found to have affinity with six hla i alleles (table s3) . from this analysis, we selected top four peptides vtaraglgy, fhtvtvntl, httwtltgy, and ielagtltl depending on the affinity with most mhc class i. epitopes for the mhc class ii alleles are presented in table 3 . depending on the ic 50 values as well as on the number of mhc class ii alleles, three 15-mer peptide candidates were selected. the peptides nsgfhtvtvntldat, kaielagtltltgtp, and aaksymsgnykaflt were predicted to have high affinity with mhc-ii allele, which can interact with 32, 29, and 24 mhc class ii alleles. the data has been validated by another software predivac. the predivac scores of the two core peptides fhtvtvntl and ielagtltl have been shown to be promising for their binding to hla_drb_1 (table 3) . accumulating both mhc class i allele-and mhc class ii allele-based analyses, we showed fhtvtvntl and ielagtltl peptides to have the best score to be a vaccine potential. conservancy of all the proposed epitopes was assessed by the iedb conservancy analysis tool and is summarized in table 4 . fhtvtvntl, ielagtltl, nyawvngni, and smyntlwrv were shown to have 100% conserved regions across all the siga proteins. the position of all the predicted epitopes is shown in a multiple sequence alignment of siga proteins in figure 3 . here, we used only our desired sequences for the proper annotation. so, from the most potential candidates, only two, that is, fhtvtvntl and ielagtltl, were found to be fully conserved. the top four epitopes were shown within the protein in figure 4 . the conservancy of both of these peptides were crosschecked by pvs software, and it was found that they were located in the conserved region of the siga protein ( figure s5 ). the epitopes are precisely positioned on the surface of the protein indicating that they would be accessible to the immune system, especially by b-cells. y k s n nq y k s n nq y k s n nq y k s n nq y k s n nq y k s n nq modeller modelled the three-dimensional structure of the targeted protein through the best multiple templatebased modelling approach. the validation of the model was measured by the procheck server through the ramachandran plot and is depicted in figure s2 , where 88.8% amino acid residues were found within the favoured region. furthermore, the predicted model was also assessed for the frustration analysis and is depicted in figure 5 . the disopred server likewise assessed the disorder of the protein sequences in order to get an understanding about the disorder among the targeted sequences, which is shown in figure s3 . 3.6. population coverage analysis. iedb analysis resource predicted both mhc class i-and mhc class ii-based coverage of the selected epitopes for the world population to assess the feasibility of being a potential vaccine candidate. the combined prediction was also assessed. the epitope "ielagtltlt" has the highest population coverage of 83.86% for the whole world population (shown graphically in figure 6 ); however, another potential epitope "fhtvtvntl" was shown to have 50.61% population coverage (table s2) . 3.7. molecular docking analysis. the core epitope (ielagtltl) with 9.0 mer and its 15-mer extension (kaie-lagtltltgtp) were bound in the groove of the hla-e * 01:01 and hla-dqa1 with an energy of −7.8 and −9.7 kcal/mol, respectively. autodock vina generated different poses of the docked peptide, and the best one was picked for the final calculation at an rmsd (root-mean-square deviation) value of 0.0. the docking interface was visualized with the pymol molecular graphics system. the 9.0-mer epitope interacted with arg-61, asn-62, and glu-152 through steric interaction and formed hydrogen bonding with the glu-156 amino acid residues. on the other hand, the 15-mer epitope interacted with asp-55 through electrostatic interaction and glu-66 through steric interaction and formed hydrogen bonding with the gly-58, arg-61, asn-62, and asn-82 amino acid residues. the docking output and the interacted residues are shown in figures 7 and 8 with different orientations. furthermore, the control docking energy was found to be −6.8 kcal/mol and is illustrated in figure s4. 3.8. allergenicity analysis. the allerhunter web server predicted the sequence-based allergenicity calculation very precisely. the allergenicity of the queried core epitope (ielagtltlt) was 0.05 (sensitivity = 98.40%, specificity = 27.4%), and the allergenicity of the 15-mer epitope (kaielagtltltgtp) was 0.05 (sensitivity = 98.4%, specificity = 27.0%). 3.9. b-cell epitope prediction. b-cell epitope prediction was obtained for the peptide kaielagtltltgtp (15 mer) through the sequence-based approaches, and values are anticipated with different parameters, ranging from −0.6464 to 1.137. these values are the different propensity scores and predicted with a threshold ranging from −0.352 to 1.037 ( figure 9 ). the kolaskar and tongaonkar antigenicity scale was employed for evaluating the antigenic property of the peptide with a maximum of 1.072. the antigenic plot is showed in figure 9 (a). peptide surface accessibility is another important benchmark to meet up the criteria of a potential b-cell epitope. henceforth, emini surface accessibility prediction was employed, with a maximum propensity score of 1.137 (figure 9 (b)). to reinforce our provision for the prediction of the epitope to elicit b-cell response, the parker hydrophilicity prediction was also employed with a maximum score of 1.086 and is depicted in figure 9 (c). enteric infections are the foremost cause of sickness and impermanence throughout the world, and only the shigella infections resulted in over a million deaths annually [2] . the ever rising multidrug-resistant (mdr) strains of the shigella bacteria area unit are another international concern for the researchers to search out a brand new resolution for preventing the deaths [50, 51] . recently, there are several studies that focus on the development of the vaccine against shigella and continue in the clinical trial. most of them use attenuated and inactivated preparation of the bacteria for eliciting immune responses which has some potential escape risk [52] [53] [54] . in this study, we have tried to find out alternatives to treat this global burden through vaccinomics approaches and targeting the immunogenic and toxic protein siga. the sequences of different strains of shigella showed that there is a little island of conserved sequence throughout the species [55] , and we have focused on that target for designing the vaccine candidate. the orthologous entry search of our targeted protein revealed no significant similarity with human pathogens and other closely related pathogens. these results further strengthen our prediction through confirming no cross immunity. in recent time, most of the vaccines are grounded on bcell immunity; vaccines based on a t-cell epitope have been invigorated lately. this is often as a result of body substance response from memory b-cells which may be overawed basically by matter drift as time goes on, whereas cell-mediated immunity repeatedly delivers long-run immunity [56, 57] . as a consequence, a t-lymphocyte epitope elicits a robust and distinctive immune response through the cytotoxic lymphocyte-(ctl-) mediated pathway and impedes the spreading of the infectious agents by the ctl through recognizing and killing the infected cells or by secreting specific cytokines [58] . the epitopes vtaraglgy, fhtvtvntl, httw tltgy, and ielagtltl are primarily selected for the designing of vaccine from the initial analysis depending on the affinity with mhc class i and additionally confirmed their presence along with those of the ancestral homologue in e. coli (figure 2 ). finally, through substantiation with different parameters, the core epitopes ielagtltl and fhtvtvntl (in 15.0-mer form, kaielagtltltgtp and nsgfhtvtvntldat, resp.) were found to be the most potential and highly interacting hla candidates for mhc class ii molecule. furthermore, we have used psortb to predict the subcellular localization of siga and found that there is a score of 5.87 for localization in the outer membrane and another score of 4.13 for extracellular localization. the result was quite similar with that for the localization of other spate proteins in the bacterial cell surface as well as in secreted forms. the three-dimensional model built through model-ler and validated by the ramachandran plot with an acceptable range resulted in the display of the perfect position of the epitope on the surface of the structure. as the epitope was found on the surface (figure 4 ) of the model, it would increase the possibility to interact with the immune system earlier. furthermore, the analysis from the dis-opred and frustration analysis servers strengthen our prediction, though there are no disorder and energy frustration in the epitope region of the sequences and model, respectively ( figure 5 and figure s3 ). to get the acceptability, vaccine candidates must have wider population coverage. this is very much important before designing. in our analysis, we have found that our proposed epitope ielagtltl had combined population coverage of 83.86%, whereas the other most potential candidate fhtvtvntl had combined population coverage of 50.61%. this output revealed that the proposed epitopes would have wider coverage in vitro. molecular docking upkeeps the prediction with a higher docking score and the perfectly oriented interactions between the both mhc and the predicted 9.0-mer and 15-mer epitopes. additionally, comparative analysis with the experimentally known peptide-mhc complex-has also revealed the precision of our prediction through the similar binding energy and interacted residues. another significant finding is the conservancy result. through analysis of the whole retrieved sequences, it was found that our predicted epitopes have a 100% conservancy and hopefully they would be potential candidates for treating all of the shigella spp. our proposed epitopes are nonallergenic in nature according to the fao/who allergenicity evaluation scheme. finally, the core epitope "ielagtltl" was also found to be more potential b-cell epitope candidates that were proposed through the sequence-based approaches including the kolaskar and tongaonkar antigenicity scale, emini surface accessibility prediction, and parker hydrophilicity prediction. from the overhead analysis, we envisage that our suggested epitope would also elicit an immune response in vitro. the improved knowledge about antigen recognition at molecular level led us to the development of rationally designed peptide vaccines. the idea of peptide vaccines is based on detecting and chemical synthesis of immunodominant b-cell and t-cell epitopes capable of evoking specific immune responses. in this study, we used different computational tools to identify potential epitope targets against shigella which will help to decrease the cost and time of wet lab experiments more successfully. our bioinformatic analyses speculate that the selected part of the outer membrane and highly immunogenic protein, siga, is a potential candidate for a peptide vaccine. it might also contribute to the reduction in the siga-mediated pathogenicity to the host. however, further wet lab validation is necessary to confirm the efficiency of our identified peptide sequence as an epitope vaccine against shigella. cytotoxic t-lymphocyte tap: transporter of antigenic peptide smm: stabilized matrix method hla: human leukocyte antigen. availability of data and materials. information about the data and their availability is intricately described in methods. as samples from human or animals had not been used in this study, ethical clearance is not applicable. patient consent is not applicable. no potential competing interest was reported by the authors. arafat rahman oany conceived, designed, and guided the study; drafted the manuscript; and analysed the data. tahmina pervin, mamun mia, and motaher hossain carried out the molecular genetic studies, participated in the sequence alignment, and drafted the manuscript. mohammad shahnaij helped in the design of the study. shahin mahmud helped in drafting the manuscript. k. m. kaderi kibria participated in the design and coordination, performed critical revision, and helped in drafting the manuscript. all authors read and approved the final manuscript. university of texas medical branch at galveston global burden of shigella infections: implications for vaccine development and implementation of control strategies a multicentre study of shigella diarrhoea in six asian countries: disease burden, clinical manifestations, and microbiology burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, gems): a prospective, case-control study shifting serotypes, plasmid profile analysis and antimicrobial resistance pattern of shigellae strains isolated from kolkata, india during randomised comparison of ciprofloxacin suspension and pivmecillinam for childhood shigellosis genetic relatedness of ciprofloxacin-resistant shigella dysenteriae type 1 strains isolated in south asia fluoroquinolone resistance linked to both gyra and parc mutations in the quinolone resistance-determining region of shigella dysenteriae type 1 the great escape: structure and function of the autotransporter proteins the immunogenic siga enterotoxin of shigella flexneri 2a binds to hep-2 cells and induces fodrin redistribution in intoxicated epithelial cells the siga gene which is borne on the shepathogenicity island of shigella flexneri 2a encodes an exported cytopathic protease involved in intestinal fluid accumulation high yield production process for shigella outer membrane particles optimizing vaccine design for cellular processing application of pharmacogenomics to vaccines major histocompatibility complex linked databases and prediction tools for designing vaccines mhcpep, a database of mhc-binding peptides: update 1997 syfpeithi: database for mhc ligands and peptide motifs development of a dna vaccine designed to induce cytotoxic t lymphocyte responses to multiple conserved epitopes in hiv-1 a highly immunogenic trivalent t cell receptor peptide vaccine for multiple sclerosis t cell vaccines for microbial infections a synthetic malaria vaccine elicits a potent cd8 + and cd4 + t lymphocyte immune response in humans. implications for vaccination strategies genbank vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines basic local alignment search tool orthomcl-db: querying a comprehensive multi-species collection of ortholog groups genome-wide prediction of vaccine candidates for leishmania major: an integrated approach design of an epitope-based peptide vaccine against spike protein of human coronavirus: an in silico approach highly conserved regions in ebola virus rna dependent rna polymerase may be act as a universal novel peptide vaccine target: a computational approach large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction sensitive quantitative predictions of peptide-mhc binding by a 'query by committee' artificial neural network approach a systematic assessment of mhc class ii peptide binding predictions and evaluation of a consensus approach peptide binding predictions for hla dr, dp and dq molecules mhccluster, a method for functional clustering of mhc molecules development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines pvs: a web server for protein sequence variability analysis tuned to facilitate conserved epitope discovery predicting population coverage of t-cell epitope-based diagnostics and vaccines evaluation of comparative protein modeling by model-ler aqua and procheck-nmr: programs for checking the quality of protein structures solved by nmr the swiss-model workspace: a web-based environment for protein structure homology modelling the disopred server for the prediction of protein disorder protein frustratometer: a tool to localize energetic frustration in protein molecules autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading the protein data bank pep-fold: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides allerhunter: a svm-pairwise system for assessment of allergenicity and allergic cross-reactivity in proteins combining pairwise sequence similarity and support vector machines for detecting remote protein evolutionary and structural relationships a semi-empirical method for prediction of antigenic determinants on protein antigens induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide new hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-ray-derived accessible sites the role of integrons in antibiotic resistance gene capture arginine-dependent acid-resistance pathway in shigella boydii new possibilities for the development of a combined vaccine against etec and shigella safety and immunogenicity of a candidate bioconjugate vaccine against shigella flexneri 2a administered to healthy adults: a singleblind, randomized phase i study safety and immunogenicity of an intranasal shigella flexneri 2a invaplex 50 vaccine genome sequence of shigella flexneri 2a: insights into pathogenicity through comparison with genomes of escherichia coli k12 and o157 cd4 + regulatory t cells: mechanisms of induction and effector function antibody regulation of t-cell immunity: implications for vaccine strategies against intracellular pathogens role of cd8 + t cells in control of west nile virus infection key: cord-007851-v6h1yro7 authors: han, ki-cheol; park, daechan; ju, shinyeong; lee, young eun; heo, sun-hee; kim, young-ae; lee, ji eun; lee, yuna; park, kyong hwa; park, se-ho; lee, hee jin; lee, cheolju; jang, mihue title: streamlined selection of cancer antigens for vaccine development through integrative multi-omics and high-content cell imaging date: 2020-04-03 journal: sci rep doi: 10.1038/s41598-020-62244-z sha: doc_id: 7851 cord_uid: v6h1yro7 identification of tumor antigens that induce cytotoxic t lymphocytes (ctls) is crucial for cancer-vaccine development. despite their predictive ability, current algorithmic approaches and human leukocyte antigen (hla)-peptidomic analysis allow limited selectivity. here, we optimized a method to rapidly screen and identify highly immunogenic epitopes that trigger ctl responses. we used a combined application of this method involving immune-specific signature analysis and hla-associated peptidomics using samples from six patients with triple-negative breast cancer (tnbc) in order to select immunogenic hla epitopes for in vitro testing. additionally, we applied high-throughput imaging at the single-cell level in order to confirm the immunoreactivity of the selected peptides. the results indicated that this method enabled identification of promising ctl peptides capable of inducing antitumor immunity. this platform combining high-resolution computational analysis, hla-peptidomics, and high-throughput immunogenicity testing allowed rapid and robust identification of highly immunogenic epitopes and represents a powerful technique for cancer-vaccine development. . scheme of the rapid high-throughput approach for discovering natural ctl epitopes. preselected til-resident tnbc tumors underwent hla-peptidomic analysis to identify hla-bound peptides. integrated wts data revealed a higher priority to select promising hla-peptides via high-resolution bioinformatics analysis, showing immune-cell-specific signatures and tcr-repertoire diversity in tumors. combined ngs analysis and the use of predictive algorithms for mhc-binding affinity enabled selection of highly immunogenic hla-peptide candidates. analysis of ifnγ-producing cd8 + t cell response using the highcontent imaging system in a 384-well format at the single-cell level for discovery of immunogenic hla epitopes eliciting a ctl response. filtrating lymphocytes) play a significant role in tumor-sites. additionally, large amounts of tils correlate with improved tumor survival 30 ; therefore, we preselected til-resident tnbc tissues for histologic analysis to identify potentially promising cancer epitopes ( fig. 2a and supplementary fig. s1 ) and scored til density by measuring the proportion of the stromal area infiltrated by lymphocytes, as previously described 31 . to select highly immunogenic hla epitopes, we analyzed the intratumoral heterogeneity of the tcr repertoires in til-resident cancers from six patients with tnbc (fig. 2b,c) . the tcr repertoires comprise somatic recombination of the tcrα and β chains, allowing the specificity of each t cell clone to be determined by rearrangement of the v, d, and j segments of the tcrβ chain during generation of the highly variable complementary determining region 32, 33 . to evaluate tcr diversity of tils, we assembled cdr3 sequences using the sequence reads of rna-seq data. a unique cdr3 sequence of tcrα and tcrβ, respectively, was defined as a clone, and the number of clonotypes represents the number of unique clones per sample after normalization with the corresponding rna-seq depth (fig. 2b) . the t cell clonal fraction was defined as the frequency of the top 10% of tcrα or tcrβ clones among total tcr clones (fig. 2c) . the top 10 most abundant tcrα and tcrβ sequences in each patient are shown in supplementary fig. s2 , and expression of the three hla-class i genes (hla-a/b/c) is shown in fig. 2d . we found a linear positive correlation between the number of tcrβ clonotypes and the expression of mhc-class i genes according to pearson's correlation coefficient (r = 0.68) (fig. 2e) . til immunoprofiles and immune-specific signatures. several recent studies demonstrated the strong relationship between significant overall survival (os) and cancer patients harboring a high number of cd8 + t cells and a low number of foxp3 + t cells 34 . in particular, the abundance of regulatory t (treg) cells and macrophages correlated with worse outcomes, whereas the abundance of intratumoral cd8 + t cells and cd4 + t-helper (th)1 cells correlated with better prognosis 35 . additionally, immune-specific signatures in tils are of potential clinical significance; therefore, we estimated the distribution of til types in each patient according to wts data using cibersort computational analysis 36 (fig. 3 and supplementary fig. s3 ). the relative proportion of each infiltrated immune cell was evaluated in each patient by quantifying immune composition from bulk-tissue gene-expression profiles, as enrichment of cd8/cd45ro and th1 cells are considered positive prognostic factors 37 . the results indicated that cd8 + t cells were highly infiltrated in both patients tnbc#2 and tnbc#6, whereas a large proportion of treg cells was observed in patients tnbc#5 and tnbc#6 (fig. 3a-c) . patient tnbc#6 displayed a highly enriched frequency of cd8 + t cells, as well as treg cells, suggesting increased accumulation of tils. interestingly, we found a significantly increased proportion of cd8 + t cells relative to treg cells in patient tnbc#2 as compared with that observed in other patients (fig. 3d) , suggesting the emergence the relationship between the number of tcrβ clonotypes and the expression of mhc class i genes. pearson's correlation was calculated between two groups. of promising tumor-associated antigens. on the other hand, we found elevated levels of immune-suppressive macrophages in patients tnbc#1, tnbc#3, and tnbc#5, which is predictive of a negative outcome (fig. 3e ). to identify naturally existing mhc class i -restricted ligands, we used an immunoproteomic approach involving tissue from six patients with tnbcs and an mhc-і antibody specific for the hla-a, b, and c molecules ( fig. 4 and supplementary fig. s4 ). after immunoprecipitation, high-resolution lc-ms/ms analysis identified and quantified the hla peptide sequences with a 1% false discovery rate (fdr) (supplementary fig. s5) . notably, the number of eluted peptides from each of the six patients were substantially different ( supplementary fig. s6 ), although >96% of the hla peptides analyzed by lc-ms/ ms showed typical properties associated with epitope length. as expected, most of the peptides were nine amino acids long, with only a few having 13 to 15 amino acids, suggesting a high level of consistency ( fig. 4a and supplementary fig. s6 ). clustering of the 9-mer hla peptides showed predominant enrichment of residues at peptide positions 2 and 9 and consistent with the anchor motifs required by the binding groove of each hla molecule 27 (fig. 4b and supplementary fig. s7 ). additionally, we found high numbers of cd8 + t and cd4 + th1 cells infiltrating into the tumor sites of patient tnbc#2 and relative to treg cells, with patient tnbc#2 showing a 4-fold higher number of cd8 + t cells as compared with that in patient tnbc#1 and accompanied by the lowest expression of mhc class i genes, suggesting a higher accumulation of antigen-specific ctls in patient tnbc#2 (supplementary figs. s3,s8). a total of 594 peptides were identified from the tissue of patient tnbc#2 along with elevated expression of hla genes, whereas only five peptides were received from tissue from patient tnbc#1 and all showing the lowest expression of mhc class i genes ( fig. 4c and supplementary fig. s8 ). moreover, we observed a positive correlation between the number of eluted peptides relative to input lysate and the expression of mhc class i genes (fig. 4c) . we then performed kyoto encyclopedia of genes and genomes (kegg) pathway enrichment analysis to investigate the genes associated with the 594 hla-binding peptides in patient tnbc#2 and the homotypic hla-a*11:01 allele (fig. 4d) . interestingly, the high-count genes (n > 10) were significantly enriched in kegg pathways related to cancer, protein processing in the endoplasmic reticulum (er), viral carcinogenesis, and www.nature.com/scientificreports www.nature.com/scientificreports/ antigen processing and presentation. numerous cancer-related genes overexpressed in cancer tissues contribute to cancer-specific or associated epitopes 38 , and hla epitopes require proteasomal digestion and translocation into the er to bind mhc class-і molecules 39 . it would be expected that the expression of genes encoding machinery responsible for antigen processing would be elevated under these circumstances. these results suggested that the eluted hla peptides identified were naturally presented by hla molecules. we further investigated the levels of the eluted peptides based on rna-seq analysis of corresponding mrna from the same sample (fig. 4e ,f). compared with normal breast tissues, 174 of 594 peptides corresponding to proteins from the same sample showed elevated abundances in cancer tissues accompanied by significant differences in mrna expression (≥2 log 2 fold change). subsequent in silico prediction of the hla-binding affinities to the 174 hla peptides and calculation of their respective binding affinity to specific alleles (predicted ic 50 ) 40 yielded a list of the top 20 highest ranking peptides derived from patient tnbc#2 (table 1) . a rapid imaging-based screening method to determine antigen-specific t cell response at the single-cell level. to determine whether the experimentally identified peptides can functionally elicit an immune response, we evaluated cytokine production by the cd8 + t cells. currently, intracellular cytokine staining (ics)-based detection methods for monitoring ex vivo ifnγ response show low throughput relative to the number of candidate antigens being tested. moreover, an individual antigen test requires large amounts of immune cells 41 . therefore, we developed an efficient and comprehensive screening system to test ctl response based on a high-content, high-throughput imaging approach ( supplementary fig. s9 ). this fluorescence-imaging-based screening system allows the use of lower numbers of viable cells up-scaled performance 42, 43 . development of a 384-well format capable of screening mixed populations of t cells for their response against large number of www.nature.com/scientificreports www.nature.com/scientificreports/ peptides enables a cost-effective approach to phenotype analysis. notably, cancer-associated antigens are highly attractive targets for determining their efficacy in triggering a t cell response; however, numerous clinical trials targeting taas for vaccine development have failed to demonstrate clinical efficacy due to immune self-tolerance. to identify highly immunogenic peptides incapable of eliciting self-tolerance, we tested the antigen-specific t cell response in pbmcs from a healthy donor. monitoring ex vivo ifnγ-producing pbmc reactivity using our fluorescence-labelled cell-based screening system ( fig. 5a -c, supplementary figs. s10,s11) revealed significant ifnγ responses to two individual epitopes (eif4a1-p and tcp1-p) in cd8 + t cells labelled with a fitc conjugated anti-human cd8 antibody (fig. 5b) . additionally, treatment with the hla-a*11:01-specific epitopes allowed detection of apc-conjugated ifnγ released from cd8 + t cells (fig. 5a,b) , with pma and ionomycin co-treatment used to trigger t cell activation as a positive control. similarly, pbmcs from two of the three healthy donors were reactive against same two epitopes (fig. 5c) . to further analyze the peptide-induced cd8 + t cells, we generated hla-a*11:01 tetramers targeting specific peptide-reactive cd8 + t cells. facs analysis revealed that 9.99% and 7.50% of t cells were targeted by eif4a1-p and tcp1-p, respectively, and detectable on day 12 of ex vivo t cell expansion (fig. 5d) . these findings suggested the efficacy of our method to screen highly immunogenic ctl epitopes using an imaging system on the detection of intracellular ifnγ levels following peptide stimulation. the two genes associated with the peptide epitopes, the translation initiation factor eif4a1 and tcp1, a member of the chaperonin-containing complex tcp1-containing ring complex (tric), are involved in tumor proliferation and survival. eif4a1 controls translation initiation and is a critical checkpoint protein involved in cell proliferation and tumorigenesis 44 . additionally, tcp1, as a tric member, is involved in tumor survival and growth and an oncogene driver 45 . moreover, these two genes are significantly overexpressed in tumor tissues according to wts data and data from the genotype-tissue expression project public proteomic database, suggesting their potential as therapeutic targets. these results identified two epitopes derived from eif4a1 and tcp1 as potential promising immunogenic antigens to boost t cell response. patients with tnbc, which is defined by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, have a higher tendency for recurrence at ~3 years after diagnosis 46, 47 . there are currently no known therapeutic targets for tbnc patients due to the molecular heterogeneity of the disease 48 . recent accumulation of massive and comprehensive bioinformatics data allowed identification of potential therapeutic targets associated with clinical survival 49 . interestingly, a tnbc subtype characterized by high levels of immune genes involved in t cell function, immune response, and antigen processing, was found to be associated with favorable prognosis, suggesting a close correlation between immune-gene signatures and better clinical outcomes 50 . therefore, it is possible that tils controlling clinical cancer progression represent key factors for preselection of tumors prior to hla-immunopeptidomics. in this study, til-resident tissues were pre-selected comprehensively to investigate a diversity of tcr repertoires and immune profile as predictors of clinical outcome. we then found a positive correlation between tcr diversity, reflecting clonal composition, and the expression of mhc-class і molecules, suggesting that active tumor-antigen presentation promotes the generation of antigen-specific tils. additionally, immune-specific signature analysis can discriminate specific immune-cell types in each patient and thus enhance the efficiency of selective hla-peptidomic approaches. notably, the expressional comparison of cd8 + t cells relative to immune-suppressive treg cells is extremely crucial to select the high antigenicity of antigens reflecting the therapeutic efficacy. thus, the extreme increase in the number of cd8 + t cells relative to that of treg cells in patient tnbc#2 presented a major analytical reason for further in vitro t cell response testing. furthermore, our results indicated a positive correlation between the number of peptides identified via hla-peptidomics and the amount of hla molecules expressed on the surface of cancer cells. it is also possible that the observed difference in the number of eluted peptides might have been influenced by hla-expression levels resulting from active induction of antigen presentation on mhc molecules, which subsequently elicited a strong immune response. thus, these findings suggested that several factors should be considered for successful hla-peptidomic approaches influenced by tcr diversity and elevated expression of hla genes. www.nature.com/scientificreports www.nature.com/scientificreports/ additionally, we showed that integrating hla-peptidomics with imaging-based immunogenicity screening is applicable for the discovery of highly immunogenic ctl epitopes. characterization of antigen-specific cellular immune response is essential to confirm vaccine-related effects specific to a cancer antigen. currently, there are www.nature.com/scientificreports www.nature.com/scientificreports/ only few assays (e.g., the enzyme-linked immunosorbent spot assay) capable of quantifying t cell responses 51, 52 , and the choice of which assay to use depends on the experimental scale, cost, equipment, reproducibility, and required detection sensitivity. a high-throughput imaging system provides an optimal platform for highly sensitive and quantitative analysis of individual t cells at the single-cell level. moreover, ics-based cytokine detection allows identification of specific cell subpopulations, even when using a small number of cells. the immunopeptidome approach serves massive hla-associated peptides as the collection of cancer epitopes; however, there are obstacles to rapidly determining the optimal set of promising epitopes by testing an enormous pool of peptide candidates in a single measurement. in this study, the hla-peptidomics approach combined with comprehensive analysis of immune-specific signatures and tcr repertories showed high selectivity to determine the immunogenic t-cell epitopes. sequentially, the high-content imaging system allowed high-resolution analysis for t cell reactivity. despite the need for discovery of tumor-derived antigens for effective cancer vaccine development, selection of antigens that elicit robust immune response remains challenging. here, we report a smart strategy for streamlined selection of cancer antigens in vaccine development. through integrative multi-omics and high-content cell imaging, we identified highly immunogenic epitopes from patients with tnbc. identification of potential vaccine epitopes coupled with immune-specific signature analysis, hla-peptidomics, and single-cell-based immunogenicity testing offers a discriminative and powerful tool for cancer-vaccine development. analysis of high-throughput sequencing. dna and rna were simultaneously extracted from cryo-pulverized tnbc tissue powder using an allprep kit (qiagen, hilden, germany), and libraries for whole-exome sequencing and total rna sequencing, respectively, were prepared using truseq library prep kits (illumina, san diego, ca, usa). the libraries were sequenced using the hiseq platform (illumina), and raw data were mapped onto hg38 using the bwa mem algorithm (http://bio-bwa.sourceforge.net/). variant calling was performed using the genome analysis toolkit (https://software.broadinstitute.org/gatk/) as previously described 53 . a custom proteogenomic search database was generated for the variants using the proteomics tool quilts (http://www.fenyolab.org/tools/tools.html). for wts data, contaminating adapters and low-quality bases were removed with trimmomatic 54 , and the trimmed data were mapped onto hg38 using star (version 2.5.3a) 55 . gene expression, including that of hla genes, was calculated by rsem (version 1.3.0) 56 , and hla presentation on multiple cancer cells was evaluated using the expression atlas (https://www.proteinatlas.org/humanproteome/ tissue/cancer) 57 . for identification and quantification of tils from wts data, we used mixcr (v.2.1.3; https:// mixcr.readthedocs.io/en/master/) with the alignment parameter -p rna-seq. 58 . hla typing at 4-digit resolution was performed using the hlascan method and wes data (synthekabio, korea). purification of hla-class i peptides. the lc-ms/ms analysis was performed as previously described 59 . hla-class i peptides were purified from tnbc tissues, and frozen tissues were pulverized with a cp02 cry-oprep automated dry pulverizer (covaris, woburn, ma, usa), followed by incubation at 4 °c for 1 h with lysis buffer containing 0.25% sodium deoxycholate, 0.2 mm iodoacetamide, 1 mm edta, 1 mm pmsf, 1% octyl-β-d-glucopyranoside (sigma-aldrich, st. louis, mo, usa), and a protease-inhibitor cocktail (roche, mannheim, germany) in phosphate-buffered saline (pbs). the lysates were cleared by centrifugation for 20 min at 15,000 g and 4 °c. hla-class i molecules were purified using the w6/32 monoclonal antibody bound to amino-link beads (thermo scientific, waltham, ma, usa), as previously described 60 . the anti-hla-class i antibody was purchased from abcam (cambridge, uk). hla-peptide complexes were eluted from the affinity column using five column volumes of 0.1 n acetic acid. the eluted hla-class i proteins and the released peptides were loaded on sep-pak tc18 columns (waters, milford, ma, usa), and the peptide fraction was eluted with 30% acetonitrile in 0.1% trifluoroacetic acid, followed by drying by vacuum centrifugation. the lc-ms/ms analysis was performed as previously described 59 . dried peptide samples were reconstituted in 10 µl of 0.1% formic acid, and an aliquot containing ~4 μl was injected from a cooled (10 °c) autosampler into a reversed-phase magic c18aq column (15 cm × 75 μm (packed in-house); michrom bioresources, auburn, ca, usa) on an eksigent nanolc 2d system at a flow rate of 300 nl/min. prior to use, the column was equilibrated with 95% buffer a (0.1% formic acid in water) and 5% scientific reports | (2020) 10:5885 | https://doi.org/10.1038/s41598-020-62244-z www.nature.com/scientificreports www.nature.com/scientificreports/ buffer b (0.1% formic acid in acetonitrile). the peptides were eluted with a linear gradient from 5% to 30% buffer b over 70 min and 30% to 70% buffer b over 5 min, followed by an organic wash and aqueous re-equilibration at a flow rate of 300 nl/min, with a total run time of 95 min. the high-performance liquid chromatography system was coupled to an ltq-orbitrap xl mass spectrometer (thermo fisher scientific, bremen, germany) operated in data-dependent acquisition mode. full scans (m/z 300-1800) were acquired at a resolution of 60,000 using an automatic gain-control (agc) target value of 1e6 and a maximum ion-injection time of 10 ms. tandem mass spectra were generated for up to 5 precursors by collision-induced dissociation in the ion-trap using a normalized collision energy of 35%. the dynamic exclusion was set to 60 s, and fragment ions were detected at a normal scan mode using an agc target value of 1e5 and a maximum ion-injection time of 500 ms. source ionization parameters were as follows: spray voltage, 1.9 kv; and capillary temperature, 275 °c. data analysis of the hla-class i peptidome. ms data were analyzed using maxquant software (v.1.5.8.3) 61 against the uniprot database (april 40, 2016; https://www.uniprot.org/), and personalized human database from patient-derived wes data. n-terminal acetylation and methionine oxidation were set as variable modifications, enzyme specificity was set as "unspecific, " and fdrs for peptides and proteins were set at 0.01 and 1, respectively. possible sequence matches were restricted to eight to 15 amino acids, a maximum peptide mass of 2000 da, and a maximum charge state of three. main search peptide tolerance was set at 10, and the box for "use ms2 centroids" was checked. hits to the reverse database and contaminants were removed from the "peptide. txt" output file produced by maxquant. peptides were subjected to hla-class i binding and immunogenicity prediction analyses using netmhc (http://www.cbs.dtu.dk/services/netmhc/) and the mhc i immunogenicity portion of the iedb analysis resource (http://tools.iedb.org/immunogenicity/), respectively. the logo-plots were constructed using a seq.2logo method 62 . pbmcs isolated from three hla-a*11:01-positive healthy donors. t cell responses to treatment with individual peptides were monitored after 12 days of in vitro culture, as previously described 11 . briefly, pbmcs were cultured in rpmi-1640 supplemented with l-glutamine, non-essential amino acids, hepes, β-mercaptoethanol, sodium pyruvate, penicillin/streptomycin (gibco; thermo fisher scientific), and 10% human ab serum (gibco), with a total of 5 × 10 4 pbmcs used in each well. for antigen-specific t cell expansion, individual peptides (2 µg/ml) were incubated with pbmcs in the presence of interleukin (il)-7 (20 ng/ml; peprotech, rocky hill, nj, usa) for 3 days. each peptide with >95% purity was synthesized by synpeptide (shanghai, china). for non-specific t cell expansion, t cells were stimulated using a t cell activation/expansion kit (miltenyi biotec, bergisch gladbach, germany), and cells were cultured every 3 days by replacing the medium with fresh half-medium containing il-7 (5 ng/ml) and il-15 (5 ng/ml; peprotech). on day 12, for ex vivo intracellular ifnγ detection, each peptide (10 µg/ml) or phytohemagglutinin (pma) (50 ng/ml; sigma-aldrich) plus ionomycin (1 µg/ml; sigma-aldrich) for the positive control was administered in the presence of brefeldin a (1:1000; biolegend, san diego, ca, usa) for overnight incubation according to the ics protocol. after intracellular fixation using bd cytofix/cytoperm fixation/permeabilization kit (bd biosciences, san jose, ca, usa), cells were stained with antibodies against surface markers and ifnγ at 4 °c for either 1 h or overnight. for visualization of ifnγ-producing t cells, a fluorescein isothiocyanate (fitc)-conjugated anti-human cd8α antibody (r&d systems, minneapolis, mn, usa), an alexa594-conjuated anti-human cd4 antibody (biolegend), and an allophycocyanin (apc)-conjugated anti-human ifnγ antibody (biolegend) were specifically labeled for cd8 + t, cd4 + t, and intracellular ifnγ capture, respectively. cells were then washed with pbs buffer, and high-throughput imaging was performed using the operetta cls high-content analysis system equipped with harmony software (perkinelmer, waltham, ma, usa). to generate the p/mhc tetramer, a biotinylated hla-a*11:01 monomer complexed with each peptide was obtained from immunomax co., ltd. (seoul, korea). peptides with >95% purity were synthesized by synpeptide (china), and their sequences are provided in table 1 . to generate an apc-labeled p/mhc complex tetramer, p/mhc complex monomers were tetramerized in the presence of apc-conjugated streptavidin (bd biosciences). t cells were then stained with 1 ng/µl of the p/mhc tetramer in fluorescence-activated cell sorting (facs) buffer (biolegend) for 10 min at room temperature, and after washing, immunofluorescence was detected by facs. t cells were gated according to the populations of cells labeled with the fitc-conjugated anti-human cd3 antibody (biolegend). neoantigens in cancer immunotherapy vaccines for established cancer: overcoming the challenges posed by immune evasion translating tumor antigens into cancer vaccines antigen-specific vaccines for cancer treatment targeting neoantigens to augment antitumour immunity neoantigens generated by individual mutations and their role in cancer immunity and immunotherapy a 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humans mhc class i/peptide stability: implications for immunodominance, in vitro proliferation, and diversity of responding ctl high-throughput imaging: focusing in on drug discovery in 3d enabling 1536-well high-throughput cell-based screening through the application of novel centrifugal plate washing decreased proliferation of human melanoma cell lines caused by antisense rna against translation factor eif-4a1 two members of the tric chaperonin complex, cct2 and tcp1 are essential for survival of breast cancer cells and are linked to driving oncogenes a prognostic model for patients with triple-negative breast cancer: importance of the modified nottingham prognostic index and age triple negative breast cancer: the kiss of death immunotherapeutic approaches in triple-negative breast cancer: latest research and clinical prospects comprehensive molecular portraits of human breast tumours the immune contexture in human tumours: impact on clinical outcome squalene emulsion potentiates the adjuvant activity of the tlr4 agonist, gla, via inflammatory caspases, il-18, and ifn-gamma assessment of antigen-specific cellular immunogenicity using intracellular cytokine staining, elispot, and culture supernatants the genome analysis toolkit: a mapreduce framework for analyzing next-generation dna sequencing data trimmomatic: a flexible trimmer for illumina sequence data star: ultrafast universal rna-seq aligner rsem: accurate transcript quantification from rna-seq data with or without a reference genome expression atlas update-an integrated database of gene and protein expression in humans, animals and plants mixcr: software for comprehensive adaptive immunity profiling comprehensive analysis of human protein n-termini enables assessment of various protein forms soluble plasma hla peptidome as a potential source for cancer biomarkers maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteomewide protein quantification seq 2logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion we would like to express my special appreciation and thank dr. supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-62244-z.correspondence and requests for materials should be addressed to k.-c.h., c.l. or m.j.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-006273-xcw0kxjg authors: willemze, r; rodrigues, c a; labopin, m; sanz, g; michel, g; socié, g; rio, b; sirvent, a; renaud, m; madero, l; mohty, m; ferra, c; garnier, f; loiseau, p; garcia, j; lecchi, l; kögler, g; beguin, y; navarrete, c; devos, t; ionescu, i; boudjedir, k; herr, a-l; gluckman, e; rocha, v title: kir-ligand incompatibility in the graft-versus-host direction improves outcomes after umbilical cord blood transplantation for acute leukemia date: 2009-01-08 journal: leukemia doi: 10.1038/leu.2008.365 sha: doc_id: 6273 cord_uid: xcw0kxjg donor killer cell immunoglobulin-like receptor (kir)-ligand incompatibility is associated with decreased relapse incidence (ri) and improved leukemia-free survival (lfs) after haploidentical and hla-mismatched unrelated hematopoietic stem cell transplantation. we assessed outcomes of 218 patients with acute myeloid leukemia (aml n=94) or acute lymphoblastic leukemia (n=124) in complete remission (cr) who had received a single-unit unrelated cord blood transplant (ucbt) from a kir-ligand-compatible or -incompatible donor. grafts were hla-a, -b or -drb1 matched (n=21) or mismatched (n=197). patients and donors were categorized according to their degree of kir-ligand compatibility in the graft-versus-host direction by determining whether or not they expressed hla-c group 1 or 2, hla-bw4 or hla-a3/-a11. both hla-c/-b kir-ligandand hla-a-a3/-a11 kir-ligand-incompatible ucbt showed a trend to improved lfs (p=0.09 and p=0.13, respectively). sixty-nine donor–patient pairs were hla-a, -b or -c kir-ligand incompatible and 149 compatible. kir-ligand-incompatible ucbt showed improved lfs (hazards ratio=2.05, p=0.0016) and overall survival (os) (hazards ratio=2.0, p=0.004) and decreased ri (hazards ratio=0.53, p=0.05). these results were more evident for aml transplant recipients (2-year lfs and ri with or without kir-ligand incompatibility 73 versus 38% (p=0.012), and 5 versus 36% (p=0.005), respectively). ucbt for acute leukemia in cr from kir-ligand-incompatible donors is associated with decreased ri and improved lfs and os. supplementary information: the online version of this article (doi:10.1038/leu.2008.365) contains supplementary material, which is available to authorized users. allogeneic hematopoietic stem cell transplantation (hsct) from family, unrelated bone marrow or cord blood donors is a curative treatment for a considerable number of patients with acute leukemia. major histocompatibility complex-restricted donor t cells play an important role in hla-matched as well as hla-mismatched hsct by recognizing differences in minor and major histocompatibility antigens between donor and recipient. 1, 2 a novel mechanism that relies on interactions between donor natural killer (nk) cells and recipient cells was demonstrated in the setting of haploidentical family donor hsct. [3] [4] [5] nk cells express on their surface inhibitory killer cell immunoglobulin-like receptors (kirs) that recognize allotypic determinants ('kir ligands') shared by certain hla class i allele groups. kir2dl1 recognizes hla-c alleles with a lys 80 residue (hla-cw4 and related, 'group 2' alleles), kir2dl2 and kir2dl3 recognize hla-c with an asn 80 residue (hla-cw3 and related, 'group 1' alleles); kir3dl1 is the receptor for hla-b alleles sharing the bw4 supertypic specificity. [3] [4] [5] finally, kir3dl2 was shown to function as a receptor for hla-a3/-a11 alleles when bound to epstein-barr virus (ebv) peptides. 6 the role of kir and kir ligand in the context of hsct has been investigated over the past 10 years. in transplants that are kir-ligand mismatched in the graft-versus-host (gvh) direction, donor nk cells expressing inhibitory kirs, which do not recognize ligand(s) on recipient targets, are released from hla inhibition and mediate alloreactions leading to clinically significant graft-versus-leukemia effects. clinical studies in haploidentical [7] [8] [9] and in some (un)related donor transplants [10] [11] [12] showed that kir-ligand incompatibilities in the gvh direction are associated with a decreased incidence of relapse and improved disease-free and overall survival (os) after hsct. several unrelated donor transplant studies did not, however, report any effect. [13] [14] [15] [16] [17] umbilical cord blood units from unrelated donors constitute another well-established source of stem cells for hsct in patients with leukemias. the main advantages are rapid availability and the possibility to perform hla-mismatched transplantations due to a relatively low risk of severe gvh disease (gvhd) [18] [19] [20] [21] that increases considerably the probability of finding a suitable donor. moreover, outcomes after umbilical cord blood transplants (ucbts) are comparable with those after unrelated bone marrow transplantation in children and adults with acute leukemias. 19, 21 we hypothesized that in the setting of hla-mismatched ucbt, nk-cell alloreactivity might come into play and influence outcomes. from the eurocord database we retrieved all patients who had received a single-unit ucbt for acute leukemia in complete remission (cr). we grouped transplants according to the presence or absence of kir-ligand mismatches that were shown to trigger donor-versus-recipient nk-cell alloreactivity in haploidentical transplantation, that is mismatches for hla-c group 1, hla-c group 2 and the hla-bw4 allele group. moreover, as hla-a3/-a11 kir-ligand mismatches were not encountered in haploidentical transplants, we decided to include them in our evaluation. as hla-a3/-a11 alleles were reported to function as kir ligands when bound to ebv peptides, we hypothesized that, in view of the frequent ebv reactivation after transplantation, ebv peptides were readily available for hla-a3/-a11 binding, with the possible generation of hla-a3/-a11-specific donor nk cells that were alloreactive against target cells from recipients lacking hla-a3 and/or hla-a11. this retrospective analysis is based on data reported to the eurocord registry from european and non-european centers through a standardized questionnaire concerning patient, cord blood units, disease and graft characteristics, as well as transplant outcomes. the collected information was reviewed by two physicians and checked for computer-generated errors to ensure data quality. all patients or their legal guardians gave informed consent for ucbt according to the declaration of helsinki. this study was approved by the eurocord institutional review board. the following criteria were required: (1) acute leukemia in cr, (2) hla high-resolution typing for hla-a, hla-b, hla-c and -drb1 of patients and cord blood units, or hla-a highresolution typing for those cord blood units without complete high-resolution typing for hla-b and/or hla-c, (3) nonexpanded, single, unrelated cord blood unit and (4) complete clinical data during at least 3 months of follow-up. as of 1 october 2007, 218 patient-donor pairs met the eligibility criteria. complete information on high-resolution typing for hla-a, hla-b, hla-c and hla-drb1 was available for 218 patients and 198 donors, and sufficient information on high-resolution typing for hla-a and either hla-b or hla-c was available for 20 donors to determine kir-ligand (in)compatibility in the gvh direction. high-resolution typing of recipient and donor hla-b and hla-c alleles was used to segregate patients and cord blood units in the following kir-ligand groups as described earlier by ruggeri et al. 22 hla-c group 1 alleles; hla-c group 2 alleles; and hla-bw4 group alleles. non-hla-bw4 alleles were grouped in the hla-bw6 allele group, which is not a kir ligand. in addition, patients and cord blood units were grouped according to whether or not they displayed hla-a3/-a11 alleles, which are putative kir ligands. 6 composition of the study group for kir-ligand (in)compatibility in the gvh direction hla-c group 1 or 2 alleles, which were present in the cord blood units, were missing in 41 transplant recipients; hla-bw4 group alleles were missing in 12 and hla-a3/-a11 in 22 patients. combination of the data on hla-a, hla-b and hla-c of donors and recipients resulted in 69 (32%) donor-patient pairs with kir-ligand incompatibility in the gvh direction. fiftyone patients were hla-c, group 1 or 2, 19 patients hla-bw4 and 18 patients hla-a3/-a11 kir-ligand mismatched in the host-versus-graft direction with the donor. pairs in which the donor did not miss a ligand in the patient but did not possess a kir ligand present in the recipient (n ¼ 50) were pooled with the 99 kir-ligand-matched pairs and, for the purpose of data analyses, are henceforth referred to as the kir-ligand-compatible or -matched group, which comprised a total of 149 (68%). the main end points were leukemia-free survival (lfs), which was defined as the time from transplantation to either first relapse or death in cr; relapse incidence (ri), defined as any morphologically proven recurrence of leukemia occurring after the allograft; and os, defined as the time from transplantation until the date of death. patients were censored at the time of relapse or of the last follow-up. other end points were engraftment, non-relapse mortality (nrm) and acute or chronic gvhd. for engraftment, we considered the incidence of neutrophil recovery as defined by a neutrophil count of at least 0.5 â 10 9 /l for three consecutive days and graft failure as no sign of neutrophil recovery as well as transient engraftment of donor cells within 60 days after transplantation. chimerism data were available during the first 3 months after ucbt. full donor chimerism was defined as the presence of more than 95% of the donor cells, mixed chimerism if more than 5% and less than 95% were donor cells and autologous recovery if less than 5% of cells were donor cells. data on the methodology of chimerism detection were not available. acute gvhd at day 100 was diagnosed and graded according to published criteria, 23 with histopathological confirmation when possible. nrm at 2 years was defined as deaths related to transplantation and not to relapse, and chronic gvhd at 2 years was diagnosed according to standard criteria 23, 24 and evaluated in patients who survived at least 100 days with sustained engraftment. data were analyzed from the eurocord database with the existing data as of october 2007. the duration of follow-up was the time to the last assessment for survivors. variables related to the patient, to the disease, to the cord blood donor and to the transplant were compared (according to kir-ligand compatibility status) using the w 2 test for categorical and the mann-whitney test for continuous variables. all p-values are two-sided with type-1 error rate fixed at 0.05. to analyze the outcome, we considered variables associated with the recipient (age, weight at time of transplantation, sex and cytomegalovirus (cmv) status), with the disease (acute myeloid leukemia (aml) and acute lymphoblastic leukemia (all), first, second or subsequent cr, and separately for all and aml: good-, standard-or bad-risk cytogenetics), with the cord blood donor (hla disparity, infused total nucleated cell and cd34 þ cell doses) and with the transplant (year of transplant, reduced-intensity or myeloablative conditioning regimen, use of total body irradiation, use of atg and gvhd prophylaxis). cumulative incidence curves were used for ri and nrm in a competing risk setting, as death and relapse are competing together. relapse and death in remission were considered as competing events to study neutrophil recovery, and acute and chronic gvhd. 25, 26 gray test was used for univariate comparisons. 27 probabilities of lfs and os were calculated using the kaplan-meier estimate; the two-sided log-rank test was used for univariate comparisons. factors associated with a p-value less than 0.10 by univariate analysis were included in multivariate analyses, using cox proportional hazards for lfs and os, and proportional subdistribution hazard regression model of fine and gray 28 for acute and chronic gvhd, neutrophil recovery, ri and nrm. then a stepwise regression was performed using a threshold of 0.05. the diagnosis (aml and all) was added to the final model. all tests are two sided. the type i error rate was fixed at 0.05 for the determination of factors associated with time-toevent outcomes. statistical analyses were performed with spss (spss inc., chicago, il, usa) and s-plus (mathsoft inc., seattle, wa, usa) software packages. as the effects of kir(-ligand) mismatching have been described to be associated with aml, a limited subgroup analysis was performed with respect to kir-ligand incompatibility for patients with aml or all, separately. the study group comprised 218 patients, undergoing a singleunit ucbt for aml or all in cr between 1997 and 2007 (median 2004). the median follow-up time is 14 months. the transplantations were performed in 57 centers. the cord blood units were supplied by 29 cord blood donor banks, mostly belonging to netcord organization. table 1 summarizes patient, donor and transplant characteristics for the patients transplanted with donors that were kir-ligand incompatible (n ¼ 69) or kirligand compatible (n ¼ 149) in the gvh direction. the diagnoses included all and aml, with good-, standard-or bad-risk cytogenetics in the first, second or third cr. the stages of both diseases were divided equally between the two groups. however, among patients with known cytogenetics, 17 aml patients out of 18 with a kir-ligand-incompatible donor and 33 aml patients out of 57 with a kir-ligand-compatible donor had bad-risk cytogenetics (p ¼ 0.02). cmv status was reported in 204 patients (111 positive) and in 202 cord blood donors (11 cmv positive). combined information is available in 187 patient-donor pairs, of which 86 were both negative and 93 were patient positive/donor negative. ebv status of patients was reported in only 74 cases (34% of all patients) of which 58 were positive (78%) and in 78 donors (36% of all donors). cmv and ebv status were equally divided between the two kir-ligand groups. the number of hla disparities between donors and recipients was not significantly different between the two groups. although a number of myeloablative and reducedintensity conditioning regimens with or without atg were used in the 57 participating transplant centers, the frequencies of myeloablative and reduced-intensity regimens and of use of atg were not statistically different between the two kir-ligand groups as well as other patient-, disease-, donor-and transplantrelated factors (table 1) . overall outcomes of the entire study group with respect to neutrophil engraftment, acute and chronic gvhd, non-relapse mortality, ri, lfs and os are shown in table 2 . association of kir-ligand incompatibility in the gvh direction with lfs, os and ri lfs and os. to assess the impact of kir-ligand mismatches that were shown to trigger donor-versus-recipient nk-cell alloreactivity in haploidentical transplantation, we determined lfs at 2 years for the donor-patient combinations that were mismatched (in the gvh direction) for hla-c and/or hla-bw4 kir ligands. it was 48±9 versus 36±4% (p ¼ 0.09) for those who were kir-ligand matched. as we had a relatively large group of 22 donor-recipient pairs that were mismatched for hla-a3/-a11 (in the gvh direction), we were able to assess for the first time the role of this putative kir ligand in transplantation outcomes; lfs was 62 ± 11% for the donor-patient combinations that were mismatched for hla-a3/-a11 versus 35 ± 4% for those who were not a3/a11 mismatched (p ¼ 0.13). as both analyses demonstrated an increased lfs in an almost not overlapping fashion and as only six patients had two incompatibilities consisting of hla-c group and/or hla-bw4 group incompatibilities in combination with an hla-a3 and/or -a11 mismatch, we combined the two groups and performed a common statistical analysis. leukemia-free survival was significantly improved in patients receiving a ucbt from an hla-a, -b or -c kir-ligand incompatible donor compared with those who were transplanted with an hla-a, -b or -c kir-ligand compatible donor (in the gvh direction). the estimates for lfs at 2 years are 55±7 and 31±4% (p ¼ 0.005), respectively, for patients with and without donor kir-ligand incompatibility in the gvh direction ( figure 1a) . overall survival was also improved in patients receiving a ucbt from a kir-ligand-incompatible donor compared with those who were transplanted with a kir-ligand-compatible donor. the estimates for 2-year os were 57±7 and 40±4% (p ¼ 0.02), respectively, for patients with and without donor kirligand incompatibility in the gvh direction ( figure 1b) . in univariate analysis, which included age, sex, cmv status, diagnosis (aml or all), stage of the disease, infused total number of cells or of cd34 þ cells, hla disparities, conditioning regimen, use of total body irradiation or atg and kir-ligand incompatibility (in the gvh direction), only o2 hla disparity was associated with os (p ¼ 0.008). in multivariate analysis, kir-ligand incompatibility was an independent risk factor associated with improved lfs and os. compatible hla grafts or with one hla disparity were also associated with improved os ( table 3 ). the estimates for lfs at 2 years for aml and all were not significantly different. within aml as well as all, lfs at 2 years was not different for the patients with good-, standard-or poor-risk cytogenetics (data not shown). relapse. the 2-year cumulative incidence of relapse was 32 ± 4% for the entire study group, and it was 37 ± 4% for the kir-ligand-compatible group compared with 20±5% for the kirligand-incompatible group (p ¼ 0.03) (figure 1c) . in univariate analysis, other factors associated with ri were type of conditioning (p ¼ 0.05), use of total body irradiation (p ¼ 0.08) and atg (p ¼ 0.09). in a multivariate analysis, myeloablative conditioning regimen (mac) (p ¼ 0.007), two or more hla disparities (p ¼ 0.05) and kir-ligand incompatibility (p ¼ 0.05) were independently factors associated with a decreased risk of relapse ( table 3) . association of kir-ligand incompatibility in the gvh direction with engraftment, gvhd and nrm neutrophil recovery. cumulative incidence of neutrophil recovery at day 60 for the entire group was 80 ± 3%, and it was 81±4% for patients with and 79±3% for those without kir-ligand incompatibility, respectively (p ¼ 0.21). the median time to neutrophil recovery was 21 days (4-40) for kir-ligand-incompatible transplants and 25 (6-60) days for kir-ligand-compatible transplants. in univariate analysis, the incidence of neutrophil recovery was associated with the median number of nucleated cells infused (p ¼ 0.015) and hla disparity (p ¼ 0.05), but not with kir-ligand incompatibility. in a multivariate analysis (table 2) , only a higher number of nucleated cells (43.09 â 10 7 /kg) infused were associated with higher rates of neutrophil recovery (p ¼ 0.015). at 3 months after ucbt, chimerism data were available in 158 patients: 126 patients were full donors (80%), 15 had mixed chimerism (9%) and 17 (10%) had autologous reconstitution. there was no association between kir-ligand incompatibility and full donor chimerism (p ¼ 0.40). on the basis of antigen-level hla-a and hla-b and allele-level hla-drb1 typing. acute and chronic gvhd. sixty-five patients presented grade ii-iv acute gvhd (grade ii, n ¼ 36, 18%; grade iii, n ¼ 14, 7%; grade iv, n ¼ 15, 7%). at day 100, cumulative incidence of grade ii-iv acute gvhd for the entire population was 29±3%, and it was 28 ± 5% for patients with a kir-ligand-incompatible donor and 30±3% for patients with a kir-ligand-compatible donor (in the gvhd direction), respectively (p ¼ 0.82). the 2-year cumulative incidence of chronic gvhd was 19% (n ¼ 29 for 171 patients at risk). there was no association of chronic gvhd with the kir-ligand groups. nrm and non-relapse causes of death. the 2-year cumulative incidence of nrm was 30 ± 3%, and it was 31±4% for patients with a kir-ligand-compatible donor and 25 ± 5% for those with a kir-ligand-incompatible donor (p ¼ 0.34). in a multivariate analysis, only ric and a lower number of hla disparities (compatible or one hla disparity) were associated with decreased nrm (table 3) . death due to opportunistic infections (viral, fungal and parasitic) was more frequent in the kir-ligand-compatible donor group than in the kir-ligand-incompatible donor group (table 4) . unfortunately, more detailed information on the nature of these lethal infections is not available in the eurocord database, but they occurred equally in the groups of cmv-positive and cmvnegative patients. association of kir-ligand incompatibility in the gvh direction with outcomes in patients with aml or all in cr a limited subgroup analysis was performed for 94 patients with aml (47 patients in first cr; 40 patients in second cr and 7 patients in third cr) and 124 patients with all (58 patients in first cr, 51 patients in second cr and 15 patients in third cr). in aml ucbt recipients, 2-year cumulative incidence of relapse was associated with the presence or absence of kirligand incompatibility in the gvh direction (5±4 versus 36 ± 7%, p ¼ 0.005). the 2-year estimates for lfs were 73±10 and 38±7% (p ¼ 0.012) and for os were 70±11 and 36 ± 8% (p ¼ 0.016), respectively (figure 2a) . in spite of the trend of a decreased ri and improved lfs and os for those with a kir-ligand-incompatible donor, there was not a significant statistical association in all patients (figure 2b ). nrm and acute and chronic gvhd were not influenced by the diagnosis (aml or all) or by the kir-ligand-compatibility status. this study demonstrates that inhibitory kir-ligand incompatibility between donor and recipient in the gvh direction is kir-ligand incompatibility and umbilical cord blood stem cell transplantation r willemze et al associated with a decreased ri and an improved lfs and os for patients with acute leukemia transplanted in cr with a singleunit unrelated umbilical cord blood graft. multivariate analysis shows that these findings are independent of the number of hla disparities between the donor and recipient. we confirm our and other previous studies showing that a higher number of hla disparities (two or more) are associated with diminished neutrophil recovery, increased nrm rates and decreased ri. 19, 20 in contrast, kir-ligand incompatibility was not associated with the cumulative incidence of neutrophil recovery within 60 days and nrm. a limitation of our study is the short median follow-up (13 months for the kir-ligand-matched and 15 months for the kirligand-mismatched donor-patient pairs) due to the fact that hla-c allelic typing has just recently been used for both umbilical cords and patients. however, the aim of our study was not to assess the actual risk of relapse after ucbt but to compare the incidence of relapse after ucbt with or without kir-ligand mismatches in the gvh direction. we cannot rule out that a kir-ligand-mismatched combination delays relapse, which would become evident after a longer follow-up. different effects of kir/kir-ligand incompatibilities on the outcome of allogeneic sct have been reported. during the last 10 years, the understanding of nk-cell alloreactivity is rapidly expanding, and thus the methods, utilized through the years, to determine kir mismatch, vary largely. mismatches between donor kir ligand and recipient kir ligand in the gvh direction (the donor-recipient kir-ligand model) have been used by the perugia group and hsu et al., [7] [8] [9] 12, 22 between donor kir and recipient kir by gagne et al. 28 and between donor kir and recipient kir ligand by leung et al. 29, 30 criticizing the donor-recipient kir-ligand model one could argue that, as some donors might not have possessed the relevant kir gene, a minority of donor-recipient pairs might have been misclassified in this study. in fact, donor-versusrecipient nk-cell alloreactivity was predicted on the basis of kir-ligand mismatching without the direct assessment of the donor alloreactive nk-clone repertoire or donor kir genotyping. however, it is worth noting that kir genotyping is required table 3 multivariate analysis for overall survival, leukemia-free survival, relapse incidence, non-relapse mortality and neutrophil recovery kir-ligand incompatibility and umbilical cord blood stem cell transplantation in only a minority of donors as most possess a full complement of inhibitory kir genes and have the potential to exert nk alloreactions. moreover, the risk of misclassification arises when the donor does not possess/express the relevant kir to exert alloreactivity. under this circumstance, kir-ligand mismatching mistakenly classifies a non-nk-alloreactive transplant pair as nk alloreactive. therefore, in our series, such 'false positives' might have 'contaminated/diluted' the nk-alloreactive cohort. thus, the only conceivable consequence of such misclassification is an underestimation of the effects of nk alloreactivity, which reinforces, rather than detracts from, our core observation that nk-cell alloreactivity favorably impacts on outcomes of cord blood transplantation. our findings in ucbt are in agreement with those of the perugia group, which, using haploidentical family donor sct, has shown that kir-ligand incompatibility in the gvh direction is associated with a decreased incidence of graft failure, gvhd and relapse, and an improved survival of patients with aml. 7-9 they explain their clinical results by findings from mouse experiments and human in vitro data in which donor-versus-recipient alloreactive nk cells eliminate residual leukemic cells, ablate host-type dendritic cells responsible for triggering gvhd and attack residual host lymphohematopoietic cells, including t cells, that are responsible for graft rejection. 8 our present analyses show that kir-ligand incompatibility has a stronger effect on ri, lfs and os in aml patients than in all patients. this finding is in accordance with clinical and in vitro data from ruggeri et al., 8 who showed that aml cells were killed in vitro by alloreactive nk clones, whereas only a sub-population of all cells was sensitive to nk-cell alloreactivity. as we observed a small but consistent kir-ligand effect in our all patients, the data suggest that subgroups of all (t-or b-lineage all) may show differential sensitivities to donor nk-cell alloreactivity. this study provides for the first time information on the role of hla-a3/-a11 kir-ligand mismatches in transplantation, which had been lacking until now, as they were not reported in haploidentical transplants. surprisingly, hla-a3/-a11 mismatching in the gvh direction emerged as just as good a predictor of favorable prognosis as hla-c and hla-bw4 group mismatches. our use of hla-a3/-a11 mismatching as a predictor of nk-cell alloreactivity may be criticized, as the role of hla-a3/-a11 as a ligand for kir3dl2 is controversial. as hla-a3/-a11 alleles were reported to function as kir ligands when bound to ebv peptides, 6 it seems reasonable that, in view of frequent ebv reactivation after transplantation, ebv peptides were readily available for hla-a3/-a11 binding, with the possible generation of hla-a3/-a11-specific donor nk cells that were alloreactive against target cells from recipients lacking hla-a3 and/or hla-a11. in our data set, ebv status was reported in only 74 patients (36%), of which 58 (78%) were ebv positive. no data on ebv reactivation are available in the eurocord database. thus, the role of hla-a3/-a11 as a kir ligand cannot be further supported in this study. this analysis shows that donor-versus-recipient nk-cell alloreactivity exerts a beneficial impact not only in haploidentical but also in cord blood transplantation. these two types of sct have in common that the donors are highly hla mismatched with the recipient, whereas after sct, relatively little severe gvhd is observed. the post-transplant period is further characterized by a rapid recovery of nk cells and longlasting t-cell immunoincompetence. as t cells in the cord blood graft are antigen inexperienced, they contain few or no cytotoxic t cells directed against viral and bacterial peptides, which may cross-react with hla alloantigens. the relative lack of post-thymic t cells and the t-cell naivety may be responsible for the lower-than expected frequency of severe gvhd, even in the setting of one or two hla mismatches, and for the delayed recovery of t-cell function after ucbt [31] [32] [33] to the same degree as after haploidentical sct. lack of memory t cells in the graft (due to t-cell depletion in haploidentical and t-cell naivety in cord blood transplants) apparently permits the recovery of fully functional nk cells. interestingly, one study comparing t celldepleted-versus-repleted unrelated donor transplants demonstrated that t cells in the graft adversely affected reconstitution of kir-bearing nk cells and clinical outcomes. 34 additional evidence that t cells antagonize reconstitution of potentially alloreactive, kir-bearing nk cells derives from several other unrelated donor transplant studies, using t-cellreplete grafts. 10, 11, [13] [14] [15] 33, 35 most studies showed no advantage in transplantation from kir-ligand-mismatched donors, [13] [14] [15] 30, 31, [36] [37] [38] whereas a few observed an increased graft-versus-leukemia effect. 10, 11, [39] [40] [41] in haploidentical 29, 30 , matched sibling 12 and unrelated donor transplants, 36 the 'missing ligand' model was proposed as a powerful algorithm for predicting favorable transplant outcomes. it hypothesizes that alloreactions also occur when kir-ligand-matched donors possess kir(s) for which neither donor nor recipient has an hla ligand(s). these donors were shown to carry kir-bearing nk cells in an anergic/regulated state. 42 the 'missing ligand' model proposes that, upon transfer into the recipient, they may become activated and exert a graftversus-leukemia effect. however, in the ucbt setting, as in a recent haploidentical transplant study, 9 no informative results emerged when we analyzed the outcomes of 'missing ligand' transplants (data not shown). in conclusion, in patients with acute leukemia in cr, singleunit ucbt from donors who are kir-ligand mismatched (for hla-c group, hla-bw4 group and/or hla-a3/-a11) in the gvh direction resulted in a lower incidence of relapse and in an improved lfs and os as compared with ucbt from kir-ligandcompatible donors. if these results are confirmed in a larger series of patients, kir-ligand incompatibility in the gvh direction might be considered as a criterion for cord blood donor choice. minor histocompatibility antigens as target of graft-versus-leukemia reactions graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect killer immunoglobulin-like receptors mhc class i molecules and kirs in human history, health and survival recognition of hla-a3 and hla-a11 by kir3dl2 is peptide-specific role of natural killer cell alloreactivity in hla-mismatched hematopoietic stem cell transplantation effectiveness of donor natural killer alloreactivity in mismatched haematopoietic transplants donor natural killer cell allorecognition of missing self in haploidentical hematopoieitc transplantation for acute myeloid leukemia: challenging its predictive value survival advantage with kir ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors genotypic inhibitory killer immunoglobulin-like receptor ligand incompatibility enhances the long-term antileukemic effect of unmodified allogeneic hematopoietic stem cell transplantation in patients with myeloid leukemias improved outcome in hla-identical sibling hematopoietic stem-cell transplantation for acute myelogenous leukemia predicted by kir and hla genotypes evaluation of kir ligand incompatibility in mismatched unrelated donor hematopoietic transplants the effect of kir ligand incompatibility on the outcome of unrelated donor transplantation: a report from the center for international blood and bone marrow transplant research, the european blood and bone marrow transplant registry, and the dutch registry role of kir ligand incompatibility in hematopoietic stem cell transplantation using unrelated donors the impact of donor kir and patient hla-c genotypes on outcome following hla-identical sibling hematopoietic stem cell transplantation for myeloid leukemia detrimental effect of natural killer cell alloreactivity in t-replete hematopoietic stem cell transplantation for leukaemia patients history of the clinical use of umbilical cord blood hematopoietic cells transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia donor selection for unrelated cord blood transplants outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukaemia: a comparison study alloreactive natural killer cells in mismatched hematopoietic stem cell transplantation consensus conference on acute gvhd grading chronic graft-versus-host syndrome in man. a longterm clinicopathologic study of 20 seattle patients estimation of failure probabilities in the presence of competing risks: new representations of old estimators a class of k-sample tests for comparing the cumulative incidence of a competing risk a proportional hazards model for subdistribution of a competing risk relevance of kir gene polymorphisms in bone marrow transplantation outcome comparison of killer ig-like receptor genotyping and phenotyping for selection of allogeneic blood stem cell donors kir-ligand incompatibility and umbilical cord blood stem cell transplantation r willemze et al determinants of antileukemia effects of allogeneic nk cells t-cell alloreactivity dominates natural killer cell alloreactivity in minimally t-cell-depleted hla-non-identical paediatric bone marrow transplantation similar potential to become activated and proliferate but differential kinetics and profiles of cytokine production of umbilical cord blood t cells and adult blood naive and memory t cells immune reconstitution after unrelated cord blood transplantation kir reconstitution is altered by t cells in the graft and correlates with clinical outcomes after unrelated donor transplantation missing kir ligands are associated with less relapse and increased graft-versus-host disease (gvhd) following unrelated donor allogeneic hct kir ligands and prediction of relapse after unrelated donor hematopoietic cell transplantation for hematologic malignancy low number of donor activating killer immunoglobulinlike receptors (kir) genes but not kir-ligand mismatch prevents relapse and improves disease-free survival in leukemia patients after in vivo t-cell depleted unrelated stem cell transplantation japan marrow donor program. donor killer immunoglobulin-like receptor (kir) genotype-patient cognate kir ligand combination and antithymocyte globulin preadministration are critical factors in outcome of hla-c-kir ligand-mismatched t cell-replete unrelated bone marrow transplantation reduced risk for molecular disease in patients with chronic myeloid leukemia after transplantation from a kirmismatched donor comparison between antithymocyte globulin and alemtuzumab and the possible impact of kir-ligand mismatch after dosereduced conditioning and unrelated stem cell transplantation in patients with multiple myeloma successful use of haploidentical stem-cell transplantation with kir mismatch as initial therapy for poor-risk myelodysplastic syndrome human nk cell education by inhibitory receptors for mhc class i this study was supported by drric (delegation de la recherche clinique), université de paris vii. rw, car, eg and vr designed the study, were the principal investigators and wrote the paper; ml was responsible for confirming the statistical analysis; ii, kb, ah and fg were responsible for data collection and quality control of the eurocord database; gs, gm, gs, br, as, mr, lm, mm and jmr were each responsible for 45 cord blood stem cell transplantations in this study; fg, jg, ll, gk, yb, cn and mb represented the cord blood banks that delivered each 45% of the cord blood units in this study. all authors read the paper, gave suggestions and approved its content. a complete list of the participating transplant centers and cord blood banks participating in this study can be found on the leukemia website. supplementary information accompanies the paper on the leukemia website (http://www.nature.com/leu) kir-ligand incompatibility and umbilical cord blood stem cell transplantation key: cord-009323-sfxpchma authors: link, hartmut; kolb, hans-jochem; ebell, wolfram; hossfeld, dieter kurt; zander, axel; niethammer, dietrich; wandt, hannes; grosse-wilde, hans; schaefer, ulrich w. title: die transplantation hämatopoetischer stammzellen: teil i: definitionen, prinzipielle anwendungsmöglichkeiten, komplikationen date: 1997 journal: med klin (munich) doi: 10.1007/bf03044917 sha: doc_id: 9323 cord_uid: sfxpchma the transplantation of hematopoietic and lymphopoetic stem and progenitor cells has become a standard procedure for the treatment of many malignant diseases. autologous stem cells are derived from the patient himself, allogeneic cells from an hla-identical or hla-compatible family or unrelated donor. hematopoetic stem cells can be obtained from bone marrow, blood and fetal cord blood. after 3 to 5 days treatment, the granulocyte-colony stimulating factor (g-csf) mobilizes stemand progenitor cells from the marrow into the blood. this method is now standard in autologous transplantation and is increasingly preferred in allogeneic transplantation. the time to hematopoietic recovery is shorter with blood stem cells than with bone marrow cells. with myeloablative high dose therapy followed by stem cell transplantation, long term disease free survival is possible in many cases and great proportions of patients can be cured (see part ii). improvements of supportive care have reduced toxicity of treatment substantially, however severe complications still occur at oropharynx, gastrointestinal tract, liver, lung, skin, kidney, urinary tract and nervous system. after allogeneic transplantation immunocompetent donor cells can react with the recipients tissue. in hla-identical donor and recipients differences in the minor histocompatibility antigens account for this graft-versus-host-reaction (gvh), which is mainly mediated by transplanted t-cells. the gvh-reaction can affect skin, liver, gut and other organs and cause clinically relevant gvh-disease (gvhd). the gvhd is more severe in hla-mis-matched or unrelated transplantations. immunodeficiency and organ dysfunction due to gvhd may predispose to life threatening infections and impair the outcome of transplantion. unrelated cord blood stem cells may have a minor risk of inducing acute gvhd, as stem and t-cells are immature. after allogeneic stem cell transplantation, the relapse rate of leukemia or lymphoma is significantly reduced by immunoreactive cells: graft-versus-tumor (gvt) or graftversus-leukemia effect (gvl). ie transplantation von progenitor-und stammzellen der h~imatopoese hat die behandlun~m6glichkeiten schwerer krankheiten der h~imatopoese und des immunsystems sowie ron einigen soliden tumoren wesentlich erweitert. bei patienten mit leukfimie, malignem lymphom, schwerer aplastischer anfimie, immundefekt oder mit bestimmten tumoren emlsglicht sie eine therapie mit heilungschance [5, 6, 34, 43, 66, 81, 96, 97, 99] . fª die transplantation ron h~imatopoetischen stammzellen aus knochenmark oder blut werden die wichtigsten indikationen und komplikationen beschrieben. ausgehend von den totipotenten entstehen ª die pluripotenten die determinierten stammzellen. die totipotenten stammzellen sorgen lebenslang fª den nachschub und die neubildung von blutzellen. auch die gewebsmakrophagen, alveolarmakrophagen [95] , langerhansschen zellen [103] , kupfferschen stemzellen [30] , dend¡ omm.sicht zellen [14] , osteoklasten [51] , synoviozyten [63] und mikrogliazellen des nervensystems [48] entstehen ª die zwischenstufe der monozyten aus den knochenmarkstammzellen. bisher ist es beim menschen nicht gelungen, die pluripotenten stamrnzellen exakt zu definieren. man weil3 jedoch, dab sie unter den zellen mit dem cd34+-phlrmtyp enthalten sind, der auch die weiterdifferenzierten proge-nitorzeuen charaktefisiert [8] . die cd34+-zellen, deren anteil ah den mononukle~en zetlen ira knochenmark i bis 2%, ira blut 0,1 bis 0,2% und ira nabelschnurblut 0,8 bis 1,2% be-rroq [49] , ksnnen als indirekter parameter ftir die stammzellen verwendet werden [13] . mit durchflubzytomet¡ und mehrfachmarkierungen ksnnen sehr frª progenitor-bzw. stammzellen des ph~notyps cd34 +, cd38-, dr-oder cd34 +, thy 1+ abgegrenzt werden [3, 8, 20, 39, 49] . nach chemotherapie, mit einem der h~imatopoetischen wachstumsfaktoren g-csf oder gm-csf oder einer kombination von beiden werden vermehrt cd34+-zellen aus dem knochenmark in das blut ausgeschwemmt [88] , so dab konzentrationen von 1 bis 2% der mononukle~ren blutzellen erreicht werden [49] . allogene transplantation bei der auogenen transplantation werden h~imatopoetische und lymphopoetische stammzellen aus dem knochenmark oder blut von einem spender ª sie ersetzen nach einmaliger transplantation permanent die gesamte blutbildung oder teile von ihr. auch das lmmun-und makrophagensystem wird mit der stammzelltransplantation ª mit ausnahme von bestimmten immundefekterkrankungen mub zur vorbereitung der h2imatopoeuschen stammzelltransplantation das immunsystem des patienten ausgelsscht oder unterdrª werden, damit das transplantat eines gesunden spenders ohne ab-stobungsprobleme best~indig anwachsen kann. die stammzelltransplantation erlaubt eine maximale therapie zur zerstsrung maligner oder defekter empffingerzellen, wodurch auch die normale h2imatopoese und lymphopoese ausgelsscht werden [96, 97] . aus zyten das p, isiko der gvhd gering. nach allogener stammzelltransplantation verm_indern immunreaktive zellen das p, ezidiv¡ (graft-versus-tumor-effekt: gvt). klinisch ist dieser effekt bei h~mato-und lymphopoetischen neoplasien sehr bedeutsam: (graft-versus-leuk~mie-effekt: gvl). h2imatopoetische stammzellen -transplantation 9 definition -anwendung -komplikationen med. klin. 92 (1997) , 480-491. summary the transplantation of hematopoietic stem cells. part i: de¡ application, complications the transplantation ofhematopoietic and lymphopoetic stem and progenitor cells has become a standard procedure for the treatment ofmany malignant diseases. autologous stem cells are de¡ from the patient himself, allogeneic cells from ah hla-identical or hla-compatible family or unrelated donor. hematopoetic stem cells can be obtzined from bone marrow, blood and fetal cord blood. after 3 to 5 days treatment, the granulocyte-colony stimulating factor (g-csf) mobilizes stem-and progenitor ceus from the marrow into the blood. this method is now standard in autologous transplantation and is increasingly preferred in auogeneic transplantation. the time to hematopoietic recovery is shorter with blood stem cells than with bone marrow cells. with myeloablative high dose therapy followed by stem cell transplantation, long term disease free survival is possible in many cases and great proportions ofpatients can be cured (see part ii). improvements ofsupportive care have reduced toxicity oftreatment substantiauy, however severe complications still occur at oropharymx, gastrointestinal tract, liver, lung, skin, kidney, u¡ tract and nervous system. after allogeneic transplantation immunocompetent donor ceus can react with the recipients tissue. in hla-identical donor and recipients differences in the minor histocompatibility antigens account for this graft-versus-host-reaction (gvh), which is mainly mediated by transplanted t-cells. the gvh-reaction can affect skin, liver, gut and other organs and cause clinically relevant gvh-disease (gvhd).the gvhd is more severe in hla-mismatched of unrelated transplantations. inmmnodeficiency and organ dysfunction due to gvhd may predispose to life threatening infections and impair the outcome of transplantion. unrelated cord blood stem cells may have a minor risk ofinducing acute gvhd, as stem and t-cells are immature. after auogeneic stem cell transplantation, the relapse rate ofleukemia or lymphoma is significantly reduced by immunoreactive cells: graft-versus-tumor (gvt) or graftversus-leukemia effect (gvl). [47, 50, 52, 84] . es ist m6glich, dal3 diese technik die markentnahme ersetzen wird, da die ersten klinischen transplantationsergebnisse sehr ermutigend sin& die h~imatopoese regeneriert schneller als mit knochenmarkstammzellen allein [47, 52, 53] . fragen wie die inzidenz der akuten und chronischen graft-versus-host-r.eaktion, einflufl aufgraft-versus-leukiimie-effekt und immunrekons¡ mª sen noch in prospektiven vergleichssmdien evaluiert werden. obwohl den spendem ftinf bis sieben tage g-csf injiziert wird, ist die technik der stammzeugewinnung risikoiirmer und angenehmer geworden [52, 53] gewebe die urspriinglichen eigenschaften des empfangers auf. sehr leicht l~il3t sich der chim~irismus bei geschlechtsdifferenz mit spezifischen dna-sonden fª das y-chromosom nachweisen. h~'natound lymphopoese regenerieren 14 bis 28 tage nach der transplanta¡ wobei die h~imatopoetischen wachstumsfaktoren g-csf oder gm-csf diese zeitspanne s252 die granulopoese [21, 32] und erythropoetin f¨ die erythropoese [55] deutlich verkª el immunkompetenz der transplantierten zeuen: nach der transplantation tritt ª eine graft-versus-host-reaktion durch immunreaktire t-zellen und zytokinfreisetzung auf [26] . diese graft-versus-host-r.eaktion ftihrt zusammen mit anderen immunreaktionen bei leuk~mien und malignen lymphomen zu einem graftversus-leuk~mie-(gvl) bzw. graftversus-tumor-effekt, der sich durch eine signifikant geringere kezidivrate manifestiert [93, 105] . bei st~irkerer auspr~igung ftihrt die graft-versus-host-l:(eaktion zur graft-versus-host-disease (gvhd). eine abstobung der transplantierten lymphonimatopoese durch verbliebene immunreaktive zellen des empf.4ngers ist selten, wenn die stammzellen von einem genotypisch hla-identischen geschwisterspender stammen. sie kommt hliufiger bei aplastischer an~mie, t-zell-depletion des stammzelltransplantates, hla-differenzen zwischen spender und empfinger sowie bei nichtverwandten spendem vor [2, 16, 43, 44] b) antimikrobielle therapie und infektionen: amphotericin b, anzahl der fiebertage nach beginn der vorbereitenden therapie [65] . c) vorbereitende therapie: die zytoreduktive therapie ist die hauptursache der vod, jedoch gibt es keinen sicheren hinweis aufbestimmte zytostatika, obwohl die kombination von busulfan und cyclophosphamid oft angeschuldigt wurde [9] . d) sonstige faktoren: mehrere studien haben gezeigt, dal3 die lebervenen-verschlubkrankheit bei autologer und auogener transplantation mit hlaidentischen geschwisterspendem gleich h~iufig auftritt [65] , jedoch dal3 sie bei transplantation mit hla-differenten oder nichtverwandten spendem wesentlich mehr patienten betrifft [65] . infektionen unabh~ingig von der quelle der h~imatopoetischen stammzeuen bleiben alle patienten rir mehrere monate nach transplantation stark immundefizient, weil das immunsystem nur langsam re-gene¡ insbesondere die gvhd verursacht immer eine ausgepr'~gte immundefizienz, die nach transplantation mit nichtverwandten spendem am deuthchsten ist. die immunsuppressive prophylaxe oder therapie der gvhd zum beispiel mit cyclosporin a, steroiden oder antilymphozytenserum ver-st'• zun~ichst die immundefizienz. infekfionen sind daher sehr eng mit der al-iogenen stammzeutransplantation verbunden und wichtige ursachen von morbidifiit und mortalit~it. t6dliche in_fektionen sind besonders eng mit der gvhd korreliert. jedoch auch ohne gvhd und bei der autologen stammzelltransplantation sind infektionen h~iufige todesursachen. nach der stammzelltransplantation k6rmen drei phasen mit unterschiedlichem infektionsrisiko, erregerspektrum und infektionstyp abgegrenzt werden, die parallel zur h~matologischen und immunologischen p,.ekonstitution verlaufen [98] . q granulozytopeniephase: als folge der hochdosierten chemoradiotherapie ist die erste unrnittelbare phase nach stammzelltransplantation charakte¡ siert durch ausgepr~gte granulozytopenie mit werten zwischen 0 und 1000 granulozyten/gl. diese phase dauertje nach art der transplantation zw61fbis 30 tage. zun~ichst treten bakteri~imien auf, gefolgt von infektionen durch sproi3pilze oder das herpes-simplex-virus (hsv) [67] . o intermedi~ire phase nach granulozytenregeneration: diese phase umfaflt den zeitraum ron der erholung der neutrophilen ron tag 15 bis 30 bis tag 100 nach stammzelltransplantation. die bakte¡ und pilzinfektionen der phase i nehmen ab, auch wenn sie oft nicht vollsfiindig ausheilen. es bestehen humorale und zellul~ire immundefekte, die abh~ingig von grundkrankheit, therapiedosis und art der transplantation stark variieren. hinzu kommt bei der allogenen stammzelltransplantation eine bereits existierende oder neu auftretende akute gvhd, die immunsuppressiv behandelt werden mul3. infektionen durch das zytomegalievirus (cmv) geh6ren zu den bedrohlichsten komplikationen nach allogener, aber auch autologer stammzelltransplantation. bei der akuten gvhd ist das r.isiko einer zytomegalievirusinfektion am gr6bten. andererseits treten die meisten schweren zytomegalievimsinfek¡ bei patienten mit gvhd auf [68] . am gravierendsten sind zytomegalievirusinfektionen bei der stammzelltransplantation mit nichtverwandten spendem. durch eine bessere und frª zytomegalievirusdiagnostik mit dem antigennachweis (pp65) in leukozyten fmh~slct-rr oder dem vims-dna-nachweis mit der polymerasekettenreaktion ist eine friihere und gezielte therapie m6glich geworden [15, 24, 28, 54, 83] . dadurch hat in den letztenjahren die rate der letalen zytomegalievimskomplikationen ebenso abgenommen wie durch die prophylakt~sche ganciclovirtherapie. auch die prophylaxe mit zytomegalievirus-seronegativen bluttransfusionen, leukozytenfiltem und immunglobulinen hat die inzidenz der zytomegalievimsinfektion reduziert. sehr oft ist die lunge ron infektionen betroffen, m6glicherweise begfinstigt durch sch~idigende elemente der konditiomerung, wie zum beispiel die ganzk6rperbestrahlung. pulmonale infektionen kt5nnen lokalisiert auftreten und sind h~iufig durch bakterien oder pitze bedingt. interstitieile pneumonien k6nnen durch zytomegalievirus, pneumozystis carinii, adenoviren, herpessimplex-vims, legioneua pneumophila, mycoplasma pneumoniae, aber auch durch candida-spezies, aspergillus und andere erreger entstehen [59] . zytomegalievirus vemrsacht fast die hstfte der interstitiellen pneumonien mit einer letalit~it von 50 bis 90% [60, 62, 68] . insgesamt zshlen lungenerkrankungen zu den bedrohlichsten komplikationen [591. 21 sp/ite phase nach stammzelltransplantation: die dritte phase beginnt 100 tage nach stammzelltransplantation. das risiko, an schweren infektionen zu erkranken, ist geringer. jedoch sind alle patienten noch m~ibig immundefizient, insbesondere patienten mit chronischer gvhd, bei denen antik6rperproduktion und t-zellfunktionen gest6rt sind [110] . nach einer transplantation mit nichtverwandten spendern bleibt das infektionsrisiko deutlich erh6ht [73] . es treten vorwiegend bakte¡ infektionen mit bekapselten erregem wie streptococcus pneumoniae und haemophilus influenzae auf, aber auch vimsinfektionen kommen h~ufig vor. klinisch imponieren bedrohliche interstitielle pneumonien durch zytomega-iievirus, va¡ zoster oder pneumocystis ca¡ nach rekonstitution des immunsystems k6nnen ab dem zweitenjahr nach transplantation impfungen gegen polio, tetanus, diphthe-¡ influenza und haemophilus influenzae erforderlich sein [61 ]. mit der erfolgreichen allogenen stammzelltransplantation werden die h~imatopoetischen zellen des empf• gers durch zeuen des spenders ersetzt [96, 97] . das immunsystem ein-schlie131ich der zellen des makrophagensystems stammt von stammzellen des spenders ab. bei jeder allogenen stammzelltransplantation resultieren daraus interaktionen zwischen transplantierten immunreaktiven zellen und empfiingerorganismus, die als graft-versus-host-reaktion bezeichnet werden [26, 82] . die ursache der graft-versus-host-reaktion sind differenzen bezª der transplantationsantigene zwischen spender und empfiinger, auf die die t-lymphozyten des spenders reagieren und zytokine produzieren [26] . dadurch werden weitere zellen rekrutiert und histokompatibilitiitsantigene exprimiert. die direkte zytolyse durch aktivierte t-zellen und zytokine wie der tumor-nekrose-faktor-ot und interteukin i schsdigen das gewebe von haut, mund-und darmschleim-hsuten, leber und bronchien [102] . auch bei empf:ingern von hlaidentischen stammzellen kann es durch unterschiede zwischen den minor-hi-stokompatibilit~tsantigenen zur lymphozytenaktivierung und akuten graftversus-host-reaktion kommen [36, 46] . das entsprechende krankheitssyndrom mit den bevorzugten zielorganen haut, leber und darm wird graft-versus-host-disease (gvhd) genannt [96, 97] . 21 akute gvhd: nach dem ausmab der organbeteiligung lfibt sich die frª nach stammzelltransplantation auftretende akute gvh_d in vier schweregrade (i bis iv) einteilen [33, 79, 96] (tabelle 4a). je schwerer die gvhd, desto ausgepr'agter ist die immundefizienz, mit einer entspechenden zunahme schwerer infektionen. daher ist die gvhd eineder hauptursachen der transplantationsassozfierten morbidit~it und mortali(~it innerhalb der ersten monate nach stammzelkransplantafion [37] . in einer intemationalen analyse von 2000 patienten nach transplantafion mit hlaidentischen geschwisterspendern war eine akute gvhd der grade ii bis iv bei 22% der pafienten direkte oder indirekte todesursache [29] . das risiko einer zytomegalievirusinfektion und einer t6dlichen zytomegatieviruspneumonie ist bei der akuten gvhd signifikant er-h6ht [68] . nach einer akuten gvhd folgt signifikant h~iufiger eine chronische gvhd. es besteht daher kein zweifel, dab die akute gvhd proph•lak¡ unterdrª werden mub [92] . cyclospo-¡ a flir sechs bis neun monate kombiniert mit niedrigdosiertem methotrexat w~ihrend der ersten tage nach transplantation oder kombiniert mit prednisolon kann die gvhd unterdrª trotz immunsuppressiver prophylaxen tritt eine akute gvhd bei 20 bis 80% der emps von nicht t-zeu-depletiertem hla-identischem knochenmark auf [19, 29, 71, 90, 91] . durch die depletion reifer t-zeuen aus dem transplantat kann die rate der schweren akuten gvhd deudich vermindert werden [45, 80, 89, 100, 111] . jedoch kommt es dann wesentlich h~iufiger zur abstobung oder zum rezidiv der grundkrankheit, so dab der anteil krankheitsffei ª bender patienten in vielen smdien nicht gr6ber ist als bei der rein medikament6sen gvhd-prophylÿ [64, 89, 111] . altemativen sind m6glicherweise die transplantation definierter mengen an t-zellen [53, 100] oder die selektive depletion von cd8+-zellen aus dem transplantat [72] . auch die isola¡ der patienten mit antimikrobieller darmdekontamination und suppression der anaeroben darmkeime vermindert signifikant das risiko der akuten gvhd [12] . auber dem hla-mismatch zwischen spender und empfinger sind folgende risikofaktoren ftir die akute gvhd bekannt: geschlechtsdifferenz zwischen spender und empf• weiblicher spender ffir m~nnlichen empfinger, anderer familienspender als hla-identischer geschwisterspender, nichtverwandter spender, alloimmunisierung des spenders durch transfusionen oder schwangerschaft, h6heres alter des patienten, positiver zytomegalievirus-serostams, scb_lechter allgemeinzustand des patienten [29, 38, 107] . vi chronische gvhd: die chronische gvhd entsteht durch autoreak¡ und auoreaktive t-zeuen, wenn der funktionsuntª thymus diese zellen nicht eliminieren oder vermindem karm. diese t-zellen reagieren mit an-¡ der hla-klasse-ii[79, 96] . chronische gvhd. thalidomid oder uva-strahlen verwendet [26, 101] . gka ft-vep,.sus-le~ie-bzw. graft-versus-tumor.-effekt rezidive von leuk~imie oder malignem lymphom treten nach allogener stammzeutransplantafion signifikant seltener auf als nach syngener [42, 93] oder autologer stammzelltransplantation [57, 106] . dieser graft-versus-leuk~imie-effekt (gvl) der transplantierten zellen wird auch augemein als graft-versus-tumor-effekt (gvt) bezeichnet, obwohl er bisher bei soliden tumoren nicht bewiesen wurde. er wird durch t-zeuen, natª killerzellen, lymphokinaktivierte killerzellen und zytokine wie interleukin-2, tumor-nekrose-faktor-ct und interferon-y vermittelt [38, 93] . der gvl ist mil der gvhd assoziiert [37, 105, 106] , er tritt in ge¡ ausmab auch ohhe klinisch erkennbare gvhd auf [38] . die depletion der t-zellen aus dem allogenen transplantat zur prophylaxe der gvhd vermindert den gvl und er-h6ht das leuk~imierezidivrisiko signifikant, auch irn vergleich zu patienten ohne gvhd [4, 35] . es konnte gezeigt werden, dab ffir den gvl die kritische mindestmenge an t-zellen 1 x 105 pro kilogramm k6rpergewicht betr~igt [100] . entttiuschend blieben die untersuchungen zur adoptiven immuntherapie mil der zusatzlichen transfusion von spenderlymphozyten nach transplantation. obwohl die rezidivrate der malignen erkrankungen zurª ging, wurde dieser vorteil durch mehr akute gvhd mit t6dlichen komplikationen wieder aufgehoben. bei der chronischen myeloischen leuk~imie gibt es daher aro wenigsten rezidive und t6dliche komplikationen mil einer minimalen akuten gvhd des schweregrades i [37] . chr.onische toxizitat und langzeitnebenwirkungen sp~itkomplikationen treten assoziiert mit der transplantation und der vorbereitenden therapie auf. h~iufig liegt eine multifaktorielle genese vor. nachstehende sp~itfolgen wurden h~iufiger beobachtet: erkrankungen der lunge und atemwege, st6rungen der autoimmunif:it, neuroendok¡ stsrungen, ophthalmologische probleme, aseptische knochennekrosen, dentale und peridontale sch~iden, dysfunktionen im urogenitaltrakt, myelodysplasie und sekund~irmalignome [22, 23, 69, 109] . endok¡ st6rungen treten h~iufig auf und betreffen ª die funktion der gonaden. insbesondere bei patientinnen nach der pubert~it mul3 eine hormonsubstitution erfolgen. in abh~ingigkeit von der konditionierung k6nnen bei kindern endokrine st6rungen mit auswirkungen aufdas wachstum und die schilddrª auftreten [23, 27, 31] . ein wichtiges problem ist die steri-lit~it, die bei pa¡ nach ganzk6rperbestrahlung und hochdosierter chemotherapie fast irnmer eintritt. doch gibt es einzelne fallberichte, insbesondere bei frauen, dal3 die fertilif• noch erhalten bleibt oder wiederkehrt. nach bisher vorliegenden ergebnissen kornmen bei kindern, die nach einer solchen therapie gezeugt und geboren wurden, nicht h~iufiger mibbildungen oder maligne erkrankungen vor als bei vergleichbaren kindergruppen, deren e1tern keine transplantation erhalten harten. wegen der noch relativ kurzen zeitspanne, seit die ersten knochenmarktransplantationen durchgefª wurden, ist noch keine endgª aussage ª die h~ufigkeit ron sekund~irmalignomen zu treffen. derzeit wird von einem fª bis zehnfach h/sherem risiko therapieinduzierter neoplasien ausgegangen. bemerkenswert ist, dab die bei organtransplantation beobachtete relativ hohe rate von lymphomen nach der stammzelltransplantation nicht auftritt, sofem die t-zellen aus dem transplantat nicht komplett entfernt worden sind. an den augen k6nnen in abh~ngigkeit von der vorbereitenden therapie sch~iden auftreten. insbesondere nach einer ganzk6rperbestrahlung treten bei 10 bis 20% der patienten katarakte auf. selbst bei patienten mit chemischer konditionierung k6nnen katarakte vorkommen. bei patienten mil chronischer gvhd k6nnen erhebliche oktfl~ire probleme ira rahmen eines sicca-syndroms auftreten. in humerus-oder femurkopf k6nnen bei manchen patienten aseptische knochennekrosen zu beschwerden fª diese nekrosen treten meist als folge der steroidtherapie auf, die bei vieten patienten n6tig ist. akabane: detection of engraftment and chimerism after bone marrow transplantation by in situ hyb¡ using a y-chromosome specific probe effect of hla compatibility on engraftment of bone marrow transplants in patients with leukemia of lymphoma monoclonal antibody 12-8 recognizes a 115-kd molecule present on both unipotent and muitipotent hematopoie¡ colony-forming cells and their precursors bone marrow transplantauon for chronic myeloid leukaemia in first chronic phase: imporlance ofa graft-versus-leukaemia effect treatment ofnon-hodgkin's lymphoma bone marrow transplantatiort risk factors for chronic graft-versus-host disease after hla-identical sibling bone marrow transplantation peault: lsolation ofa candidate human hematopoietic stem-cell population the syndrome ofhepatic v•no-occlusive disease aficr marrow transplantation re~men-related toxicity and eady posttransplant survival in patients undergoing marrow transplantation for lymphoma regimen-related toxiciw in patients undergoing bone marrow transplantatiom evidence that sustained growth suppression of intestinal anaerobic bacte¡ reduces the risk of acute graft-versus-host disease after sibling marrow transplantation to: identification and companson ofcd34-positive ceus and their subpopula¡ from normal peripheral blood and bone marrow using multicolor flow cytometry generauon ofimmunostamulatory dendritic cells from human cd34+ hematopoie¡ progenitor cells of the bone marrow and pe¡ blood cytomegaloviras anr• detecfion in peripheral blood leukocytes afier allogeneic marrow transplantation graft failure after t-ceu-depleted human leukocyte antigen identical marrow transplants for [eukemia: ii. in vitro analyses of host effector mechanisms marrow har-ves¡ from normal donors unretated donor or autologous marrow transplantation for treatment ofacute leukemia cyclosporine, methotrexate, and prednisone compared with cydosporine and prednisone for prophylaxis ofacute gtaft-versus--host disease lamdorp: f_.xpression of thy-1 on human hematopoietic progenitor cells recombinant human granulocyte-macrophage colony stimulanng factor accelerates neutrophil and monocyte recovery after allogeneic tcell-dcpleted bone marrow transplantation delayed complicatiom and long term effects afier bone marrow transplantation report on the intemational workshop of the kind philipp foundation on late effects after bone marrow transp[antation in childhood malignancies muller: polymerase chain reaction to evaluate ant3viral therapy for cytomegalovirus disease autologous peripheral stem ceu transplantation in children grafi-versus-host disease bone marrow transplantation treatment and prevention of cytomegalovirus pneumonia after bone marrow transplantafion: where do we stand? risk factors for acute graft-versus-host disease bone marrow origin of hepatic macmphages severi: role of busulfan and total body irradiation on growth ofprepubertal children receiving bone marrow transplantation and results oftreatment with recombinant human growth hormone yver: placebo-controlled phase lii trial oflenograstim in bone-marrow transplantauon clinical manifestations of graft-versus-hust disease in hurnan recipients ofmarrow from hla-matched sibling donors bone martow transplantation for patients with chronic myeloid leukemia bone marrow tramplantation for chronic myelogenous leukemia in chronic phase: lncreased ¡ for relapse amociated with t-ceu depletion mismatches ofminor histocompatibility antigens between hla-idenª donors and recipients and the development ofgraft-versus-host disease after bone marrow transplantation acute graft-versus=host disease: gr'a= de and outcome in padents with chronic myelogenous leukemia graft-versusdeukemia reactions after bone marrow ttansplantation ter~tappen: formatinn of haematopoietic microenvironment and haematopoietic stem cells from single human bone marrow stem cells link: monitoring of relapse and remission in acute leukemias by dna-fingerprint analysis marrow harvesting for autologous marrow transplantation evidence of a graft-versus=lymphoma effect associated with allogeneic bone marrow transplantation analysis of 462 transplantations from unrelated donors facilitated by the national marrow donor progmam graft failure after t-cell-depleted human leukocyt• antigen identical marrow transplants for leukemia: 1. analysis of risk factors and results ofsecondary transplants clonable t lymphocytes in t cell-depleted bone marrow transplants correlate with development of graft-v=host disease dupont: minor histocompatibility antigens and marrow transplantation champtin: allogeneic blood stem cell transplantation for refractory leukemia and lymphoma: potential advantage ofblood over marrow allo= grafts cells expressing human glucoce= rebrosidase from a retroviral rector repopulate macro= phages and central nervous system microglia after mu= ¡ bone marrow transplantation civin, w. s. may: cd34: structure, biology, and and clinical utility placental blood as a source ofhematopoietic stem cells for transplantafion into unrelated recipients harvesting and en¡ ofhematopoietic progenitor cells mobilized into the pefipheral blood of normal donors by granulocyte-macrophage colony s¡ factor (gm-csf) or g-csf: potential tole in allogeneic marrow transplantation fevotd: identification and characterizadon ofosteoclast progenitorsby cloralanalysis of hematopoiedc cells combined transplantation of allogeneic bone marrow and cd34+ blood cells polq transplantation of allogeneic cd34+ blood cells cytomegalievirusinfektion: pathophysiologie, modeme nachweismethoden und therapie beim immunsupprimierten patienten heinrichs: a controlled trial ofrecombinant human erythropoietin afier bone marrow transphntation a controlled nial of recombinant human granu[ocyte-macrophage colony stimulating factor after total body irradiation, high dose chemotherapy and autologous bone marrow transplantation q acuse lymphoblastic leukemia of ma]ignant lymphoma prospective comparative t¡ ofpostremi~ion therapy in adult patients with acute myeloblastic ieukemia: hig~ dose wtosine arabinoside versus allogeneic and unpurged autologous bone marrow transplantation voraussetzungen fª die tramplantation ron h~imatopoetischen stammzellen ostendorp: lungdiseases after bone marrow transplantation. a study ofclinic, radiology, histology, lung fun•tion and immunology verdonck: cytomegalovirus inters¡ pneumonia in autologous bone marrow transplant recipients zibaud: lmmunisations afier bone marrow transplantation: results ofa european survey and recommendations finta the infectious diseases working part 3' of the european group for blood and marrow transplantation verdonck: treatment ofinterstital pneumoni¡ due to cytomegalo-• virus with ganciclovir and intravenous immune globulin: experience ofeuropean bone marrow transplant group apparent cure of rheumatoid arthritis by bone marrow transplanta¡ ifrah: impact oft-cell depletion on outcome of allogeneic bone marrow transplantation for standard-risk leukaemias clift: veno-occlusive disease of the liver and multiorgan failure after bone marrow transplantadon: a cohort study of 355 patients autologous tramptants for chronic rnyelogenous leukaemia: resutts from eight transplant ~oups lnfection in bone marrow transplant recipients risk factors for cytomegalovirus infection after human marrow transplantation weisdore myelodysplastic syndrome after autologous bone marrow transplantation: an additional late complication of curative cancer therapy forman: the outcome of matched unrelated donor bone marrow transplantation in patients with hematolo~c malignancies using molecular typing for donor selection and graft-versus-host disease prophylaxis regfimen of cyclosporine, methotrexate, and prednisone acute g'raft-versus-host disease: analysis of¡ factors after allogeneic marrow transplantation and prophylaxis with cyclospo¡ and methotrexate selecuve depletion of cd8+ cells for prevention ofgraft-versus-host disease after bone marrow transplantation: a randomized controlled t¡ al weisdorfi late infections after allogeneic bone marrow transplantation: comparison of incidence in related and unrelated donor transplant recipients weisdo~ predictive factors for chronic gxaftversus-host disease after histocompatible sibling donor bone marrow transplantadon immunogenedc marrow donor search for 1012 patients: a retrospec¡ analysis ofstrategies, outcome and costs progress ofunrehted bone marrow donor search at the university hospital of essen (1991-1994) transplantation potential of hematopoietic cells released into the circulation during routine chemotherapy for non-hodgkin's lymphoma meeting report: consensus conference on acute gvhd grading knochenmarktransplantation bei akuter leuk~mie: prophylaktische antiserumbehandlung des knochenmarkes zur unterdrª einer transplantat-gegen-wirt-reaktion eastera cooperative oncology group: recommended guidelines for the management ofautologous and allogeneic bone rnarrow tramplantafion: a report from the eastem cooperative oncology group (ecog) vogelsang: graft versus host reaetions and disease eviews 88. munksgaard, copenhagen 1985 a randomized, controued trial ofprophytactic ganciclovir for cytomegalovirus pulmonary infection in recipients of allogeneic bone marrow transplants; the city of hope-stanford-syntex cmv study group russelh pfimary transplantation ofallogeneic peripheral blood progenitor cells mobilised by filgrastim (gwanulocyte colony stimulating factor) randomised trial of filgrastim-mobilised peripheral blood progenitor cell transplantation versus autologous bone-marrow transplantation in lymphoma patients effect ofperipheral-blood progenitor cells mobilised by filgwas tiro (g-csf) on platelet recovery after high-dose chemotherapy chronic graft versus host syndrome in man. a iong-term clinicopatholo~c study of 20 seattle patients flow cytometry for clinical estimation of circulating hematopoietic progenitors for autologous transplantation in cancer patients prevention of graft-versus-host disease by selective depletion ofcd6-positive t lymphocytes from donor bohe marrow methotrexate and cyclosporine versus cyclosporine alone for prophylaxis of graft-versus-host disease in patients given hla-identical marrow grafts for leukemia: long-term follow-up ofa controlled t¡ what role for prednisone in prevention ofacute grafi-versus-host disease in patients undergoing marrow transplants hyperacute graft-vhost disease in patients not given immunosuppression after auogeneic marrow transplantation neiman: lnfluence ofacute and chronic graft-versus-host disease on retapse and survival after bone marrow transplantation from hla-identical siblings as treatment of acute and chronic leukemia kuroda: collection and transplantation of peripheral blood stem cells in ver,/" small children weighing 20 kg or leas sale: direct evidence fora bone marrow origin of the alveolar macrophage in man bone marrow transplantation bone marrow transplantation lnfections in bone marrow transphnt recipients rehtionship between pattetas ofengra~ment in peripheral blood and immune reconstitution after allogeneic bone marrow transplantafion for (severe) combined immunodeficiency nieuwenhuis: anogeneic bone marrow transplantation with a fixed low number oft ceus in the marrow graft thalidomide for the treatment of chronic g'raft-versus-host disease graft-versus-host disease: new directions tbr a persistent problem cytogenetic identification of allogeneic epidermal langerhans cells in a bone-marrow-graft recipient (letter). new engl allogeneic sibling umbilicalcord-blood transplantat~on in children with malignant and non-malignant disease antileukemic effect ofgraft-versus-host disease in human recipients ofallogeneic marrow gratis antileukemic effect ofchronic graft-ver-sus~host disease fortsetzung auf seite 505. fª die verfasser: prof. dr. hartmut link, zentrum innere medizin, abteilung hiimatologie und onkologie a survey of ant/microbial suscepdbility of climcal isolates of entemcoecus spp. from lrish hospitals the life and t• of the enterococcus l~-lactamase-producing enterococci. antimicrob weimtock: evidence for clonal spread ofsingle strain of [5-1actamase-producing enterococcus faecalis to six hospitals in five states p,eeovery of vancomycin-resistant enterocoeci on fingerprints and environmental surfaces gudiol: cephalosporins as risk factor for nosocomial enterococcus faeealis bacteremm rapid dissemination of ~-lactamase-producing aminoglycoside-resistant enterococcus faecalis among patients and staff on an infant-toddler surgicals ward gaynes: major trends in the microbial etiology of nosocomial infection vancomycin-resistant entemcocci comparison ofpadents with enterococcal bacteremia due to straim with and without high-level resistance to gentamicin low: detection of vancomyein resistance in enterococcus species gfif¡ faecal carriage and nosocomial spread of vancomycin-resistant enteroeoccus faecium uwe panten die steuung der biguanide in der therapie des diabetes meuitus a prospecdve study of 12 months treatment on serum lipids and apolipoproteins a-1 and b in type 2 (non-imulin-dependent) diabetes prospective randomized two-years clinical study comparing additional metformin treatment with reducing diet in type 2 diabetes metformin normalizes insulin binding to monocytes from obese nondiabetic subjecu and obese type 2 diabetic pa-uents uk prospective study oftherapies o fmaturity onset diabetes. 1. effect ofdiet, sulphonylurea, insulin or bibmanide therapy on fasting plasma glucose and body weight over one year uk prospective diabetes study. ii. reduction in hba 1c with basal insulin supplement, sulphonylurea, or biguanide therapy in maturity-onset diabetes ukpds vili. study design, progress and performance ukpds 13. p, elative efficacy ofrandomly allocated diet, sulfonylurea, insulin or metformin therapy in patients with newly diagnosed type 2 diabetes followed for three years metforrmn decreases the high plasminogen activator inhibition capacity, plasma imulin and triglyceride levels in non diabeuc obese subjects antithrombotie effects of mefformin in laser injured artefies reaven: effect of metfnrmin on carbohydrate and lipoprotein metabolism in niddm pauents filipovich: r.isk factors for acute graft-versm-host disease in histocompatible donor bone marrow tramplantation wissenschaftlicher beirat der bunde~irztekammer: richdinien fdr die anogene knochenmarktramplantation mit nichtverwandten spendem smrb: secondary ma]ignancies after marrow transplantadon for leukemia or aplastic anemia thomas: r.ecovery of antibody production in human allogeneic marrow graft recipients: influence of time posttransplantat" the presence of absence ofchmnic graft-versus-host disease, and andthymoeyte globulin treatment cell-depleted allogeneic bone marrow transplantauon in adults with acute nonlymphocytic leukemia in first remission key: cord-002724-gtv9syzi authors: pfortmueller, carmen andrea; meisel, christian; fux, michaela; schefold, joerg c. title: assessment of immune organ dysfunction in critical illness: utility of innate immune response markers date: 2017-10-23 journal: intensive care med exp doi: 10.1186/s40635-017-0163-0 sha: doc_id: 2724 cord_uid: gtv9syzi in critically ill patients, organ dysfunctions are routinely assessed, monitored, and treated. mounting data show that substantial critical illness-induced changes in the immune system can be observed in most icu patients and that not only “hyper-inflammation” but also persistence of an anti-inflammatory phenotype (as in sepsis-associated immunosuppression) is associated with increased morbidity and mortality. despite common perception, changes in functional immunity cannot be adequately assessed by routine inflammatory biomarkers such as c-reactive protein, procalcitonin, or numerical analysis of leukocyte (sub)-counts. cytokines appear also not suited due to their short half-life and pleiotropy, their unexclusive origin from immune cells, and their potential to undergo antagonization by circulating inactivating molecules. thus, beyond leukocyte quantification and use of routine biomarkers, direct assessment of immune cell function seems required to characterize the immune systems’ status. this may include determination of, e.g., ex vivo cellular cytokine release, phagocytosis activity, and/or antigen-presenting capacity. in this regard, standardized flow-cytometric assessment of the major histocompatibility-ii complex human leukocyte antigen (-d related) (hla-dr) has gained particular interest. monocytic hla-dr (mhla-dr) controls the interplay between innate and adaptive immunity and may serve as a “global” biomarker of injury-associated immunosuppression, and its decreased expression is associated with adverse clinical outcomes (e.g., secondary infection risk, mortality). importantly, recent data demonstrate that injury-associated immunosuppression can be reversed—opening up new therapeutic avenues in affected patients. here we discuss the potential scientific and clinical value of assessment of functional immunity with a focus on monocytes/macrophages and review the current state of knowledge and potential perspectives for affected critically ill patients. the immune system is an essential organ in higher life forms, and its dysfunction or "failure" may be life-threatening. in humans, the immune system is ubiquitously distributed within all organs and consists of humoral and cellular components organized in highly complex dynamic social network architecture-like structures [1] . key functions of the immune system embrace injury control in inflammation/infection and tumor recognition/surveillance [1] . despite its paramount importance, however, the immune system or "immune organ" is mostly overlooked on intensive care units (icu) today [2] [3] [4] [5] [6] [7] . this may at least partly be due to the fact that its functional status cannot be adequately assessed by use of routine biomarkers such as c-reactive protein, procalcitonin, or numerical distribution of leukocyte (sub)-sets. nevertheless, numerical assessment of leukocyte (sub-)populations may provide important additional information, e.g., when considerably deranged [8] [9] [10] . the typical initial immune system response to critical illness consists of systemic and local release of inflammatory mediators and cytokines and activation of specific immune and other cells. this may lead to distinct phenotype changes in immune cells [4, 6, 11, 12] . the traditional understanding was that uncontrolled release of proinflammatory mediators (e.g., interleukin (il)-1, tumor necrosis factor (tnf)-α) would determine adverse clinical outcomes in patients with septic shock [4, 11] . consequently, anti-inflammatory such as anti-tnf-α or anti-lipopolysaccharide (lps) strategies were then tested in large-scale clinical trials. however, respective trial results returned negative or indicated increased intervention-related mortality. this highlighted that an anti-inflammatory approach would not provide general benefits for larger populations of patients with sepsis/septic shock [2-5, 7, 12] . thereafter, immune status characterization in larger patient cohorts using novel biomarkers allowed for a more profound understanding. when looking at an individuals' immune response, a high inter-individual variance and highly dynamic changes can be observed over time (figs. 1 and 2) [4] . today, it is well established that many critically ill patients either show signs of co-existing inflammatory and counter-regulatory antifig. 1 injury-associated immunosuppression in critically ill patients. injury-associated immunosuppression (iai) may develop in critical illness. iai was shown to be of importance in cases of persistence for ≥ 2 days. key future potential therapeutical options are listed. monocytic hla-dr expression (mhla-dr, given in bound antibodies per cell) may serve as a global marker of iai inflammatory response early in critical illness [13, 14] or will undergo transition from early pro-to later anti-inflammatory phenotypes (fig. 2) [2, 4, 7, 11, 12] . the "net effect" (i.e., the resulting phenotype) of such profound anti-inflammation was referred to as "sepsis-(or injury-) associated immunosuppression (sai/iai)" and embraces diminished release of pro-inflammatory mediators, reduced phagocytosis, and reduced expression of cellular surface receptors involved in antigen-presenting activity (e.g., major histocompatibility complex (mhc) class ii) (fig. 3) [4, 7, 11, 12] . this may be associated with enhanced immunological tolerance, increased immune cell apoptosis, and altered gene expression profiles [6, 11] . interestingly, recent data show that respective changes are not exclusive to circulating immune cells and that comparable anti-inflammatory phenotypes can be found, e.g., in splenic or lung tissue and other solid organs [11] . from a clinical perspective, it seems pivotal to distinguish temporary from persisting immunosuppression (figs. 2 and 3). data show that patients failing to recover from injury-(or sepsis-) associated immunosuppression are at increased risk for (secondary) infections or non-survival [4, 6, 11, 15] (fig. 2) . this affects patients with post cardiosurgical conditions [16] , trauma [17] , burns [18] , pancreatitis [19, 20] , solid organ transplantation [21] , hepatic [22] or renal injury [23] , stroke [24] , myocardial infarction/ heart failure, and cardiac arrest [25] [26] [27] [28] , as well as sepsis [15] . recent technological advances now allow for better recognition/monitoring of sai/iai-thus opening up new avenues for the recognition, monitoring, and treatment of such functional immune "organ failure" [7] . inter-individual injury-associated response patterns in critically ill patients. patients with critical illness respond differently to injury (e.g., sepsis). whereas patient "a" undergoes a pronounced inflammatory phase (net effects are shown) with regain of immunological homeostasis and subsequent survival, patient "b" enters a persisting phase of injury-associated immunosuppression (iai). in iai, viral reactivation rates, secondary (re-) infection rates, and mortality is increased. this underlines the importance of inter-individual response patterns and need for individual patient characterization before application of interventional therapeutic approaches (adapted from hotchkiss et al., 2013 [4] ) for identification of patients at risk for sai/iai and associated complications, it seems important to briefly summarize key immunologic responses to injury (fig. 3 ). the first response to injury or infection typically consists in local activation of humoral factors (e.g., complement factors) followed by antigen-presenting cells (apcs) that are at the innate-adaptive interface (i.e., monocytes/macrophages or dendritic cells) [6, 29] . when activated, apcs release cytokines (e.g., tnf-α, il-1, il-6) and other mediators that attract and activate even more apcs and neutrophils, enhance phagocytosis, and stimulate adaptive immune cells after migration to draining lymph nodes (e.g., antigen-loaded dendritic cells) [6, 29] . following phagocytosis, apc-derived antigen presentation occurs via upregulation of class ii transactivator (ciita) and relocalization of mhc class ii molecules from intracellular storages [29, 30] . enhanced surface expression of antigen-loaded human leukocyte antigen (-d related) (hla-dr; a key mhc class ii molecule) on monocytes/macrophages and dendritic cells then induces a t cell response via binding to t cell receptors (tcr) and co-stimulatory molecules (e.g., cd86-cd28 and cd40-cd40l) (fig. 3) . over time, a "counter-regulatory" response may occur in monocytes/macrophages and dendritic cells with increased production of antiinflammatory cytokines (e.g., il-10) [31, 32] . as a consequence, monocyte and dendritic cell deactivation with diminished expression of both hla-dr and co-stimulatory infection-induced activation of key immune cells. in sepsis, bacterial infections trigger numerous pathways resulting in activation of key antigen-presenting cells (apcs) (i.e., monocytes/macrophages, dendritic cells). activated apcs predominantly express pro-inflammatory cytokines and present antigens bound to major histocompatibility (mhc) class ii complexes (such as hla-dr). antigen-bound hla-dr triggers t-cell-receptor (tcr) and co-stimulatory molecule (e.g. cd 40-cd40l) binding. adaptive immune responses are initiated resulting in clearance of infection. in, e.g., cases of overwhelming infection, deactivation of monocytes, as in sepsis-associated immunosuppression (sai), may occur. sai is characterized by a shift towards an anti-inflammatory phenotype with predominant expression of il-10 and diminished hla-dr expression, resulting in impaired clearance of infection and increased mortality. in iai, the deactivated phenotype can be observed immediately after injury molecules can be observed as an indicator of reduced phagocytosis, antigen presentation, and diminished induction of adaptive immune responses. furthermore, expansion of myeloid-derived suppressor cells (mdsc), an immature population of myeloid cells with immunosuppressive functions first described in cancer, was also demonstrated in patients with sepsis [33, 34] . very recently, mdsc were shown associated with prolonged immunosuppression, in particular with diminished t cell functions and development of nosocomial infections in patients with sepsis [35, 36] . in addition, critically ill patients commonly show marked apoptosis-induced lymphopenia and impaired lymphocyte function which contribute to sepsis-and injury-associated immunosuppression as recently reviewed elsewhere [37] . key cytokines: serum levels of il-6, il-10, and tnf-α serum cytokine levels are routinely assessed in some institutions for earlier recognition, estimation of prognosis, and (intra-individual) follow-up of critically ill patients. however, it should be noted that they do not reflect immune cell functionality as cytokines are mostly pleiotropic, derived from different cells including non-immune cells, may be counteracted by natural inhibitors (e.g., gp130 for il-6), and have variable clearance rates [4, 6, 11, 12] . in the following, we discuss three cytokines with pathophysiologic and/or diagnostic relevance in critical illness: il-6 is a potent pleiotropic cytokine with mainly pro-inflammatory effector function. il-6 is expressed by monocytes/macrophages, endothelial lineage cells, and fibroblasts and augments immune responses via induction of t cell activation, b cell proliferation and differentiation, and stimulates acute phase protein release (e.g. c-reactive protein) [38] . systemic il-6 is detected rapidly with peak serum levels observed after about 2 h after an inflammatory insult [38] . il-6 is usually assessed via automated enzyme-linked immunosorbent assay (elisa) in specialized laboratories or via point-of-care tests (blood, liquor) [39, 40] . owing to its fast induction and short half-life, serial il-6 assessment may provide timely monitoring of an inflammatory burden when, e.g., compared to serial c-reactive protein measurements. although increased il-6 levels indicate adverse clinical outcomes in adults with sepsis/septic shock [38] , implementing of il-6 measurement in routine diagnostic work up was not shown to improve patientcentered clinical outcomes. nevertheless, il-6 was shown useful for sepsis diagnostics in neonatal/pediatric critically ill patients [41] . il-10 is regarded the most prominent and exemplary anti-inflammatory cytokine. comparable to il-6, il-10 is mainly expressed by monocytes/macrophages, has a short half-life, and can be assessed by elisa. il-10 was evaluated in several studies and functionally linked to the "classical" biphasic response model to severe injury [42, 43] . in contrast to il-6, increased il-10 expression induces antigen tolerance, enhances sai, and increases susceptibility to infection, and il-10 blockade reverses endotoxin tolerance in several preclinical studies, and some reports show a predictive value of il-10 for mortality and/or (secondary) infection [42, 43] . tnf-α is a key pro-inflammatory cytokine predominantly released by monocytes/ macrophages in early sepsis. it auto-stimulates effector functions and enhances the initiation of adaptive immune responses [44] . several studies showed that elevated tnf-α levels are associated with increased mortality. when compared to other systemic inflammatory markers, it appears that tnf-α has lower discriminatory power with respect to outcome prediction [43, 45] . ex vivo lps-induced tnf-α production (e.g., after 4 h of stimulation) in whole blood allows for quantification of production/release of monocytes and dendritic cell-derived tnf-α. diminished ex vivo tnf-α release is a key feature of immunosuppression in critically ill patients [4, 12, 46] . nevertheless, ex vivo tnf-α release may not be a suitable diagnostic marker for cellular immune function as it requires standardized protocols for sample handling and specific stimulation conditions [46] . today, no generally accepted standardized protocol for assessment of ex vivo tnf-α release exists, hindering multicenter studies [46] . recently, whole-blood monocytic intracellular tnf-α assessment by flow cytometry was tested and showed promising results with regard to improved test feasibility [47] . phagocytosis involves recognition and engulfment with subsequent clearance of pathogens [48] . numerous predominantly innate immune cells perform phagocytosis (e.g., neutrophils, monocytes/macrophages, dendritic cells) [48] . diminished phagocytic capability was linked to increased susceptibility for (secondary) infection in rodent models whereas in humans, the direct influence of critical illness on phagocytosis is incompletely understood [49] . phagocytosis of neutrophils may be conserved in patients with sepsis, while in parallel, other neutrophil functions including chemotaxis and/or generation of oxidative burst may be impaired [49] . in general, phagocytosis assays are heterogeneous with varying specificity. standardized laboratory protocols are missing, resulting in high intra-and inter-lab variation. thus, phagocytosis assays may be of limited use for assessment of immune function in both clinical routine and multicenter clinical trials testing immunological interventions. hla-dr is a mhc class ii molecule and predominantly expressed on monocytes/ macrophages, dendritic cells, and b cells [29] . its surface expression is indispensable for antigen presentation [29] . while increased hla-dr expression reflects activation of immune cells, diminished expression thereof exhibits a phenotype with downregulation of antigen-presenting capacity and a shift from pro-to anti-inflammatory cytokine production [4, 12] . surface expression of hla-dr on monocytes/macrophages is crucial for initiation of adaptive immune responses [11, 29] . this signal is paralleled and/or augmented by activation of co-stimulatory molecules (e.g., cd40-cd40ligand binding) (fig. 3) . given the importance of monocytic hla-dr (mhla-dr) expression in respect to induction of adaptive immune responses, the key interplay of monocytes and dendritic cells with t cells was colloquially referred to as "immunological synapsis." assessment of mhla-dr expression was thus proposed to serve as a "global" functional marker of immune function [4, 5, 7, 12] . in fact, the significance of mhla-dr expression was first described about 30 years ago in patients undergoing organ transplantation when patients with low hla-dr expression could be weaned from iatrogenic immunosuppression without transplant rejection [50] . monocytic hla-dr expression is performed via fluorescence-activated cell sorting (facs) from edta samples [51, 52] . facs allows for simultaneous enumeration and assessment of several surface and intracellular antigens on specific immune cell subsets following staining with fluorochrome-labeled antibodies (fig. 4) . in 2005, the quanti-brite™ hla-dr assay was demonstrated as the first standardized method for flowcytometric mhla-dr assessment with low inter-laboratory variability (coefficient of variation (cv) 15%, inter-laboratory cv < 4%) enabling comparison of data sets collected in multicenter studies [51] . previous methods reporting percentages of hla-dr positive cells (%hla-dr) or mean fluorescence intensities (mfi) lacked an internationally accepted analytical standard and precluded between-center comparison of results [51] . in contrast, the quantibrite™-hla-dr assay harnesses calibration beads and a specifically formulated antibody-fluorochrome conjugate which allows the measurement of bound hla-dr antibodies per cell (mab/cell) independently from the combination of flow cytometer or instrument settings used in different laboratories [51] . despite recent progress in standardization, flow cytometry still requires specialized lab equipment and staff, standardized analytical protocols, and timely handling of samples (maximum of 4-6 h in standard edta-tubes at room temperature for mhla-dr) [51] . delayed assessment of samples may induce activation of monocytes resulting in however, cyto-chex®-bct tubes are expensive and not commonly available. stained and fixed samples can be stored for at least 52 h before analysis [51] . thus, mhla-dr assessment as a biomarker for immune function usually requires establishing of the method in nearby hospital laboratories [7, 52] . in addition, blood samples are usually processed during standard laboratory opening hours and not 24/7 [51, 52] . recently, an automated table cytometer was investigated as potential point-of-care tool for bedside mhla-dr assessment which may be an important step to improve the availability of immune monitoring tools for icu clinicians [53] . further, quantification of hla-dr expression and of other markers of innate and adaptive immune (dys)-regulation by real-time or digital pcr may help to overcome some of the above mentioned limitations of flow-cytometric mhla-dr analysis and thus improve identification of patients with sai/iai [54] [55] [56] [57] . however, the utility of theses assays needs further investigation. using the earlier non-standardized method for mhla-dr assessment as percent positive monocytes, most investigators (including our group) have established a cut-off at 30% hla-dr-positive monocytes for severe injury-associated immunosuppression (earlier referred to as "immunoparalysis") [51] . a recent comparison of the conventional method with the standardized quantitative assay for mhla-dr (given in mab/cell) performed by us revealed that the (earlier) cut-off value of 30% hla-dr positive monocytes corresponds to about 5000 mab/cell and 45% mhla-dr to about 8000 mab/cell [51] . the range between 30 and 45% hla-dr positive monocytes was termed "borderline immunosuppression." thus, a cut-off value of 8000 mab/cell may be used to indicate sai/ iai and was used in subsequent interventional clinical trials [58] . importantly, not single diminished values of mhla-dr should be regarded as clinically relevant but rather the persistence of low mhla-dr levels indicating failure for recovery [4, 7, 12, 15] . sepsis is the clinical condition in which the mhla-dr expression is best evaluated. reduced mhla-dr expression on admission [59, 60] , days 1-3 [15, 45, 60] and days 6-8, [45, 59, 61] was significantly associated with increased mortality. some studies show that the outcome-relevant difference in mhla-dr expression is apparent only on days 3-4 (or later) with mhla-dr returning to normal levels in survivors but not in nonsurvivors [15, 62] . two further studies showed that the dynamic change (or recovery slope) in mhla-dr expression between days 3 and 7 post injury is associated with mortality [15, 61, 62] . in one of these studies, it was shown that despite non-significant predictive value for single mhla-dr values at time points 0, 3, and 7, the delta value between measurements days 0-3, 0-7, and 3-7 were highly predictive for mortality [62] . these results were confirmed in both adult [45] and pediatric patients [61] . one explanation for the better predictive value of relative changes in mhla-dr expression than absolute values may be the high inter-individual variability of hla-dr levels on monocytes. monocytic hla-dr expression on days 3-5 and 6-8 also independently predicts development of secondary infections [63] . recovery of mhla-dr may also reflect normalization of key metabolic pathways in sepsis [64] [65] [66] , but further large-scale clinical data is needed. several studies assessed whether reduced mhla-dr expression predicts adverse outcome following major surgical procedures. culprits for post-surgical immune suppression may be surgical trauma, related intraoperative hypotension [67] , and increased perioperative release of corticosteroids or catecholamines [68] . moreover, anesthetic drugs such as fentanyl [69] may contribute to injury-associated immunosuppression (iai). in patients with cardiovascular surgery, use of extracorporeal circuits is typically associated with a substantial pro-inflammatory response [16] . cardiopulmonary bypass may be followed by iai reflected by impaired monocytic ex vivo lps-induced cytokine release and decreased mhla-dr [16, 70, 71] . the nadir of mhla-dr was typically observed on postoperative days 1-3, but diminished mhla-dr expression was shown to persist up to postoperative day 10 in a considerable number of patients [70, 71] . in two larger studies investigating the predictive power of mhla-dr on outcome in pediatric and adult patients post-cardiac surgery, reduced mhla-dr expression on postoperative day 3 was associated with increased length of icu stay/mechanical ventilation and development of postoperative sepsis [71, 72] after adjustment for bypass time, cross clamp time, complexity of surgical procedure, and a pediatric mortality risk score [72] . in adults, mhla-dr expression on postoperative days 1-5 was significantly different between patients who later developed sepsis vs. with an uncomplicated course and was a factor with a high discriminatory power to identify patients with infection post cardiac surgery [16] . in patients with ruptured abdominal aortic aneurysms, mhla-dr expression after surgery was significantly associated with mortality although this was not related to increased postoperative infection rates [73] . diminished mhla-dr expression was observed in many patients with multiple trauma [17, 74] . in a prospective observational trial in 105 severely injured patients (injury severity score, iss > 25), rise in mhla-dr until days 3-4 following trauma, and not at any earlier day, was associated with non-development of severe infection/sepsis after adjusting for confounders [17] . the dynamic effect of mhla-dr recovery was also shown in patients with multiple trauma and iss > 9 [75] . further studies report an association between mhla-dr expression and occurrence of post-trauma sepsis as early as day 2 [75] . monocytic hla-dr expression was also associated with increased intrapulmonary shunting after severe trauma which is associated with increased incidence of pulmonary sepsis and development of acute respiratory distress syndrome (ards) [75] . infection is a common complication in patients after acute cns injury. in particular, pneumonia is associated with worse neurological outcome and remains a leading cause of death. experimental studies demonstrate that cns injury-induced suppression of cellular and humoral immune functions contribute to the high incidence of infections [24] . several clinical studies demonstrated reduced mhla-dr expression in patients after cerebral ischemia, subarachnoid hemorrhage, spinal cord injury, or neurosurgery [76] [77] [78] . importantly, cns-injured patients with subsequent infectious complications showed lower mhla-dr levels than those with an uncomplicated clinical course as early as day 1 after the insult and well before onset of infection [76] [77] [78] indicating that impaired host responses contribute to an increased infection risk after cns injury. very recently, we confirmed in a large prospective multicenter study stroke-induced immunosuppression (as indicated by low mhla-dr expression) as an independent risk factor for the development of pneumonia besides the known neurological risk factors leading, e.g., to dysphagia and higher risk of aspiration [77] . only few data are available in burn patients. one study in patients with severe burn injury (> 30% of body surface) indicates that days 2-3 mhla-dr expression is significantly associated with increased mortality [18] . patients who later developed sepsis had significantly lower mhla-dr expression in the two ensuing days [18] . reduced mhla-dr expression is associated with increased disease severity in patients with severe pancreatitis [19, 20] . suppression of mhla-dr or decreased mhla-dr is associated with development of sepsis [19, 20] . after day 3, failure to recover in mhla-dr expression was associated with decreased survival [19] . the utility of mhla-dr assessment in patients post (e.g., renal) transplantation was investigated more than 25 years ago. increased mhla-dr expression was observed to be associated with an increased rate of transplant rejection [21, 79] and may serve to monitor iatrogenic immunosuppression [50] . failure to recover to normal mhla-dr levels after transplantation is associated with increased rates of late post-transplant pneumonia in pediatric populations [80] . in adults after liver transplantation, reduced mhla-dr expression levels are associated with pneumonia [81] and cytomegaly virus (cmv) reactivation [82] . monocytic hla-dr expression predicts outcome in patients after cardiac arrest (ca) [25] . in 55 patients after out-of-hospital ca from non-shockable rhythm, mhla-dr levels were significantly decreased when compared to healthy controls [25] . in this study, non-survivors showed different mhla-dr dynamics between days 0 to 1 and 1 to 3 when compared to survivors. whereas the slope between days 0 and 1 was steeper in non-survivors, mhla-dr expression continued to decrease from days 1 to 3 in non-survivors (increased after day 1 in survivors) [25] . the predictive value of mhla-dr on outcome of patients with acute kidney injury (aki) was assessed in one study [23] . despite decreased mhla-dr expression in aki patients when compared to controls, the study did not identify a predictive value for mortality [23] . few studies investigated mhla-dr expression in patients with acute or decompensated chronic liver disease [22, 83] . respective studies found a significant association between mhla-dr expression and mortality at admission with an increase in predictive value when dynamic changes over time were investigated [83] . the discriminatory power of mhla-dr for prediction of mortality was either similar [83] or lower than for the model of end-stage liver disease (meld) score [22] . in the light of the potential of mhla-dr for immune monitoring, several interventional biomarker-guided therapeutic strategies were tested in clinical trials. respective approaches included extracorporeal removal of inhibiting factors via selective immunoadsorption [84] , immunostimulation using interferon gamma (ifn-γ) [32] or stimulation with granulocyte-macrophage-colony-stimulating factor (gm-csf) [58, 85, 86] . potential additional approaches embrace interleukin 7 (il-7) or anti-pd ligand 1 molecules (anti pd-l1). future potential immunomodulatory approaches in sepsis are given in fig. 5 . stimulation of ifn-γ receptors, which are ubiquitously expressed, results in activation of numerous pro-inflammatory pathways. in a landmark trial, doecke et al. showed that ifn-γ immunostimulation restores mhla-dr expression in patients with sepsisassociated immunosuppression (sai) [32] . clearance of infection may be enhanced by ifn-γ use in adult patients with invasive fungal sepsis [87] , and in a randomized double-blind clinical trial in trauma, a decreased incidence for ventilatorassociated pneumonia was observed in patients with mhla-dr < 30% receiving inhaled ifn-γ [74] . ifn-γ treatment was shown to reverse sai resulting in higher tnf-α-, decreased il-10, and increased mhla-dr levels indicating reversal of the sai phenotype [44] . whether administration of ifn-γ in iai results in lower mortality of affected patients remains unclear and larger investigations are needed but, importantly, major side effects of ifn-γ-induced immunostimulation were not observed [32, 74] . in a randomized controlled double-blind placebo-controlled trial in 38 patients with sepsis, we could demonstrate reversal of persisting sai following one treatment of subcutaneous gm-csf [58] . in addition to reversal of sai (as defined by mhla-dr expression > 15,000 mab/cell), we observed improvements in relevant patientcentered outcomes such as shortened time of mechanical ventilation [58] . the finding that gm-csf reverses sai is supported by other groups [86] . whether clinical endpoints such as secondary infection rates are affected by therapeutical application of gm-csf is under research (nct02361528).however, smaller studies showed promising results with lower infection rates [88] or shorter duration of infection in immunosuppressed critically ill patients treated with gm-csf. in another randomized-controlled trial in patients with sepsis and severe respiratory dysfunction, oxygenation significantly improved in patients receiving gm-csf [89] . in newborns, we could recently demonstrate that reduced mhla-dr expression may reflect immunological immaturity in very early newborns [90] and a meta-analysis on gm-csf therapy indicated increased survival rates in very-low pre-term infants (< 2000 g) and infants with neutropenia when treated with gm-csf [91] . importantly, none of the clinical studies reported relevant side effects of gm-csf treatment. critical illness may often induce persisting injury-associated immunosuppression with adverse effects on relevant patient-centered outcomes. however, despite the key task of icu physicians to detect, monitor, and follow up on organ dysfunctions, functional failure of the "immune organ" seems currently mostly overlooked as it cannot be adequately assessed via use of routine biomarkers such as numerical distribution of leukocyte (sub)counts or systemic levels of soluble markers such as cytokines, procalcitonin, or acute phase proteins. importantly, quantitative assessment of a given cell population does not per se allow to conclude on its functional status. today, flow-cytometric assessment of the mhla-dr expression may serve as a standardized "global" biomarker to evaluate immune function. persisting reduced mhla-dr expression reflects a distinct immunological phenotype that is associated with adverse clinical outcomes. nevertheless, mhla-dr assessment currently requires specialized laboratories that may not be available in all institutions. 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expression of hla-dr antigens on peripheral blood t lymphocytes and renal graft tubular epithelial cells in association with rejection human leukocyte antigen-dr expression on peripheral blood monocytes and the risk of pneumonia in pediatric lung transplant recipients the th (1997) low hla-dr expression on monocytes as a prognostic marker for bacterial sepsis after liver transplantation early increase of peripheral b cell levels in kidney transplant recipients with cmv infection or reactivation longitudinal monocyte human leukocyte antigen-dr expression is a prognostic marker in critically ill patients with decompensated liver cirrhosis a novel selective extracorporeal intervention in sepsis: immunoadsorption of endotoxin, interleukin 6, and complementactivating product 5a immunostimulation using granulocyte-and granulocyte-macrophage colony stimulating factor in patients with severe sepsis and septic shock reversal of immunoparalysis by recombinant human granulocyte-macrophage colonystimulating factor in patients with severe sepsis interferon-gamma as adjunctive immunotherapy for invasive fungal infections: a case series immunoparalysis and nosocomial infection in children with multiple organ dysfunction syndrome a randomized phase ii trial of granulocytemacrophage colony-stimulating factor therapy in severe sepsis with respiratory dysfunction hla-dr expression on monocyte and dendritic cell subsets indicating impairment of cellular immunity in preterm neonates: a prospective observational analysis administration of recombinant granulocyte colonystimulating factor to neonates with septicemia: a meta-analysis not applicable. not applicable. all authors (cap, cm, mf, jcs) wrote the article and revised it for important intellectual content. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. the authors declare that they have no competing interests. key: cord-002463-qhtj1pef authors: dash, raju; das, rasel; junaid, md; akash, md forhad chowdhury; islam, ashekul; hosen, sm zahid title: in silico-based vaccine design against ebola virus glycoprotein date: 2017-03-21 journal: adv appl bioinform chem doi: 10.2147/aabc.s115859 sha: doc_id: 2463 cord_uid: qhtj1pef ebola virus (ebov) is one of the lethal viruses, causing more than 24 epidemic outbreaks to date. despite having available molecular knowledge of this virus, no definite vaccine or other remedial agents have been developed yet for the management and avoidance of ebov infections in humans. disclosing this, the present study described an epitope-based peptide vaccine against ebov, using a combination of b-cell and t-cell epitope predictions, followed by molecular docking and molecular dynamics simulation approach. here, protein sequences of all glycoproteins of ebov were collected and examined via in silico methods to determine the most immunogenic protein. from the identified antigenic protein, the peptide region ranging from 186 to 220 and the sequence hkegaffly from the positions of 154–162 were considered the most potential b-cell and t-cell epitopes, correspondingly. moreover, this peptide (hkegaffly) interacted with hla-a*32:15 with the highest binding energy and stability, and also a good conservancy of 83.85% with maximum population coverage. the results imply that the designed epitopes could manifest vigorous enduring defensive immunity against ebov. ebola virus (ebov) is an antisense-strand rna virus from the filoviridae family, and it is structurally filamentous. 1 although the initial discovery of ebov was in 1976, till now more than 24 epidemics have been reported from africa, mostly with the zaire species (http://who.int/mediacentre/factsheets/fs103/en/). [2] [3] [4] the genome of ebov enciphers the seven structural proteins, ie, nucleoprotein (np), viral structural proteins (vp35, vp40, vp30, and vp24), glycoprotein (gp), and rna-dependent rna polymerase (l). 5 among these, three different versions of glycoprotein are transcribed by the gp gene. [6] [7] [8] [9] both attachment protein (gp1) and entry/fusion protein (gp2) are expressed from the full length of the gp chain, which are synthesized from messenger rnas (mrnas), containing an additional nontemplated adenosine. the soluble gp (sgp) is synthesized from the unedited rna transcript. on the contrary, small soluble gp (ssgp) is translated during this process by adding two additional adenosine residues. 10 the gps are expressed virally on the virion surface, which plays a crucial role in the catalysis of membrane fusion and amalgamation to host cells. as a result, it is considered not only a crucial component for vaccines but also an essential target for developing inhibitors and antibodies of attachment and fusion. [11] [12] [13] protein sequence retrieval, evaluation analysis, and antigenic protein identification all available sequences of the gp of ebov were extracted from the uniprot database. 20 after that, multiple sequence alignment was performed by using the clustalw2 tool, and a phylogenetic tree was assembled by mega 6.0 21 software. and then, vaxijen v2.0 22 was used to predict most efficient antigenic protein from the available protein sequences. top scored eiptope subjected to 100 ns md simulation **rmsf **rmsd **hydrogen bond occupency analysis secquence, having highest vaxijen score prediction of b cell epitope, using-**t cell epitope prediction by proteasomal c terminal cleavage, tap transport efficiency and mhc class 1 binding **epitopes with ic50 value less than 50 for their binding to mhc class 1 molecule from iedb analysis along with binding to highest number of alleles in both analyses were chosen **epitope conservancy analysis **population coverage analysis **kolaskar and tongaonkar antigenicity scale 48 **emini surface accessibility prediction 47 **karplus and schulz flexibility prediction 49 **bepipred linear epitope prediction 50 **chou and fasman beta turn prediction 52 vaxijen analysis with a threshold score of >0. 5 secquence, having highest vaxijen score vaxijen analysis with a threshold score of >0.5 in silico-based vaccine design against ebov gp t-cell epitope identification and conservancy analysis t-cell identification was done using the netctl 1.2 server, 22 setting thresholds at 0.5, 0.89, and 0.94 for sensitivity and accuracy. mhc-i binding of the identified epitopes and epitope conservancy were then calculated using tools from the immune epitope database (iedb). [24] [25] [26] these tools calculate the half maximal inhibitory concentration (ic 50 ) value of epitope binding to human leukocyte antigen (hla) molecules using the stabilized matrix base method. 26, 27 the restriction for epitope identification was set to 12 mhc-i supertypes. prior to the run, all the alleles were considered, and the length of the peptides was set at 9.0. the population coverage tool from iedb was applied to determine the population coverage for every single epitope by selecting hla alleles of the corresponding epitope. allergenicity of the predicted epitope was calculated using allerhunter, 27 which can predict both nonallergens and allergens with a high level of accuracy , by comparing the input sequence with the sequence of known allergen. 29 molecular simulation analysis of hla allele interaction design of the three-dimensional structure of epitope and hla protein the three-dimensional structures of all the five epitopes were predicted by a pep-fold web-based server. 30 for each sequence, this server predicted the five most provable structures, the best of which, having the lowest energy model, was chosen for further analysis. to validate the binding of identified epitope and hla molecule, we considered the homology modeling as there is no relevant structure available in the protein data bank. we selected homology modeling using the most popular online protein fold recognition server, phyre2, 31 to generate the three-dimensional structure of hla-a*32:15 32 (accession id: am422702). then, modrefiner 33 was used to minimize and correct the hypothetical structure. the validation of the predicted structure was done using procheck, 34 verify 3d, 35 errat, 36 prove, 37 and qmean. 38 molecular docking analysis was performed using autodock vina, 39 by considering hla molecule as a protein and identified epitopes as ligands. first, we used the protein preparation wizard of ucsf chimera 40 to prepare the hypothetical protein for docking analysis by adding hydrogens and gasteiger-marsili charges. 41 the prepared file was then converted into pdbqt format. the parameters used for the docking simulation were set to default. the size of the grid box in autodock vina was kept at 36.3095, 54.3374, and 48.025, respectively, for x, y, and z. the energy range was kept at 4, according to the default setting. autodock vina was implemented via the shell script offered by autodock vina developers. docking results were observed by negative score in kcal/mol, as binding affinity of ligands. 39 binding energy estimation and molecular dynamics (md) simulation the binding free energy of hla-epitope complexes were calculated by using mm (charmm) 42 -generalized born surface area (gbsa) and poisson-boltzmann surface area (pbsa) protocols, implemented in accelrys discovery studio 2.5. using implicit solvent models of gbsa and pbsa, the binding free energy (δg bind ) for each epitope was calculated by maintaining salt concentration of 0.15 m. default value was set for conformational entropy and ligand minimization. the distance cutoff value was set to 14.0 å. the binding energy was calculated by using following equation: the entire dynamics simulation study for the hlaepitope complex was accomplished in yasara dynamics software. prior to simulation, the complex was cleaned and optimized the hydrogen bond network. 43 after that, a cubic simulation cell was created with a periodic boundary condition, and the atoms of the complex were typed using the amber14 44 force field. the pka (acid dissociation constant) values of protein titratable amino acids were calculated and solvated the simulation box using the transferable intermolecular potential3 points (tip3p) water model (density: 0.997 g/l -1 ). the system consistent with 46406 atoms was energy minimized using the steepest gradient approach (5000 cycles) followed by simulated annealing method. restrained and unrestrained all-atom molecular dynamics simulation were performed in solvent using the pme method to describe long-range electrostatic interactions at a cut off distance of 8 å at physiological conditions (298 k, ph 7.4, 0.9% nacl). 45 a multiple time step algorithm together with a simulation time step interval of 2.50 fs was chosen. 46 molecular dynamics simulations of 100 ns long were performed at constant temperature using a berendsen thermostat and constant submit your manuscript | www.dovepress.com dash et al pressure. the md trajectories were saved every 250 ps for analysis. the trajectories generated from the simulation were analyzed for the stability by various evaluative measures viz. rmsd, rmsf (rms fluctuations), and initial and final protein backbone comparisons using yasara structure built in macros and vmd software. to detect b-cell epitope, various tools from iedb were used to identify the b-cell antigenicity, together with the emini surface accessibility prediction, 47 kolaskar and tongaonkar antigenicity scale, 48 karplus and schulz flexibility prediction, 49 and bepipred linear epitope prediction analysis. 50 since antigenic parts of a protein belong to the beta turn regions, 51 the chou and fasman beta turn prediction tool 52 was also used. a total of 46 gp sequences from the different variants of ebov were collected from the uniprotkb database. multiple sequence alignment analysis was then performed, and a phylogenetic tree (figure 2 ) was constructed thereby. using the unweighted pair-group method with arithmetic mean, a phylogram was constructed using the bootstrap with 1,000 replications in mega6. 53 from the multiple sequence alignment analysis, it is clearly seen that protein sequences that isolated from various strains were having a close relationship. also, from the multiple comparison result, the selected sequences of ebov of the same subtype have 78%-99% similarity. this result also confers the possibilities of mutation in glycoprotein of all strains, which demonstrates a good agreement with the results from veljkovic et al. 54 antigenic protein prediction protein sequences in this study were considered to screen out using vaxijen web server for the identification of potent antigenic protein. as a corollary, uniprotkb id: q9ymg2 was identified as the most potent antigenic protein having a maximum total prediction score of 0.5390. here the threshold of 0.5 is considered as the potent antigenicity. 55 this sequence was used for further analysis. on the basis of the high combinatorial score, the five best epitopes were predicted by the netctl server from the selected protein sequence in a preselected environment. the identified epitopes are represented in table 1 . in combination with several methods such as proteasomal cleavage/transporter associated with antigen processing (tap)/mhc-i combined predictor, mhc-i processing of the netctl server calculates an overall score for each peptide's intrinsic potential from a protein for the designing of t-cell epitope. peptides with a higher score represent higher processing capabilities. the five t-cell epitopes were subjected to mhc-i binding prediction, using the stabilized matrix base method. the epitopes that elicited higher affinity (ic 50 <200 nm) were subjected to afterward analysis (table 2) . notably, proteins are transformed into peptides by proteasome complex, which cleaved the peptide bonds. by combining with class i mhc molecules, these peptides were deported to the cell membrane, where they were introduced to t helper cells. as shown in table 2 furthermore, this epitope retained the highest conservancy of 83.85%, according to the iedb conservancy analysis, as tabulated in table 2 . as population coverage in vaccine design generally plays a crucial role, it was calculated in this study. the cumulative percentage of population coverage was obtained for the predicted epitope hkegaffly. as shown in table 3 , the population coverage for east africa was found to be 66.98%; in west and north africa, it was 69.50% and 63.89%, respectively; and for central africa it was observed to be 75.93%. the population coverage was recorded at 55.88% for the east asian region, which was a major hotspot for viral infection. for north america, the population coverage was found to be 58.69%. in current vaccine design pipeline, allergenicity is considered the most prominent barrier in vaccine designing, since most vaccines convert the immune system into an "allergic" reaction 56 by inducting type 2 t helper cells and immunoglobulin e. that is why we predicted allergenicity of the selected epitope by the allerhunter web server, where the probability is >0.06. the epitope hkegaffly was scored 0.00 (sensitivity =91.6%, specificity =89.3%), and was thus considered a nonallergen, according to the food and agriculture organization/world health organization evaluation system of allergenicity prediction. dash et al protein model having >90% of the residues in the core and allowed regions can be considered a high-quality model. 57 the hypothetical model was further analyzed using errat and verify 3d. 58 for a good model, structure should retain an errat score >80.00, against which the model in this study obtained an errat score of 89.859. 55 verify 3d graph indicates that 100.00% of residues of this model had an averaged 3d-1d score of 0.2, which is good. 59 along with the qmean analysis, the protein model in our interest resulted in a z-score of −1.33, and the total score was 0.636. this value denotes a higher quality of the model, where the acceptable score ranges between 0 and 1 ( figure 3b ). 38 on the basis of the results obtained from the aforementioned structural validation programs, the model ( figure 3c ) showed much reliability and was considered for further study. molecular docking simulation revealed that the proposed epitopes bound in the cleft of the hla-a*32:15 ( figure s2 ), where the highest binding affinity was −7.6 kcal/mol (table s2 , observed for the hkegaffly epitope). the chimera 40 program was used to visualize the interactions of docked hla-a-epitope complexes, as shown in figures 4 and s2. then, binding energy calculation was carried out to understand the binding of hla with epitopes. here the binding free energies of mm-gbsa and mm-pbsa are approximate free energies of binding, so a more negative value denotes stronger binding. from mm-gbsa analysis, the highest binding free energy was observed for hla-a*32:15 with epitope (hkegaffly) of -63.89 kj/mol (table s1 ). on the contrary, the lowest binding free energy was obtained for atedpssgy epitope, i.e. -44.86 kj/mol. in contrast of mm-gbsa, the hkegaffly epitope was also resulted the highest binding energy of -38.48 kj/mol, while the lowest binding free energy was seen for tedpss-gyy epitope, -20.98 kj/mol. since hkegaffly epitope obtained the highest docking affinity and binding free energy, its complex subjected for molecular dynamics simulation. table 2 interaction, binding, and conservancy of identified t-cell epitopes validation of predicted t-cell epitope as described in the "materials and methods" section, the hypothetical structure of hla-a*32:15 protein was generated using the homology technique. the structure was then analyzed through various web-based protein validation software. as shown in figure 3 , the ramachandran plot generated by the procheck 34 server showed that about 98.9% of the residues of protein are located in the most favored region, as against 0% in the outlier region and 1.1% in the generously allowed region. it should be noted that the and remained stable in the range from 2.0 å to 3 å. in case of epitope, similar rmsd pattern was observed, where the order of magnitude was seen to fluctuate in some range. the average energy of the simulation was -578125.270 kj/mol; the average coulombic charge and van der waals interactions was -694749.662 kj/mol, 77122.511 kj/mol, respectively. we also calculated the contribution of each residue for both hla and epitope in the simulation, in terms of rmsf and rmsd. as seen in figure 6a , highest rmsd was observed the 100 ns md simulation of hla-epitope (hla-a*32:15-hkegaffly) complex was carried out using amber14 force field, following the energy minimization protocol. the stability of the hla-epitope complex by means of rmsd was calculated and rendered in figure 5a . from the results, it is revealed that the hla molecule was stabilized after 5 ns simulation and tended to remain in plateau phase thereafter for rest of the period. the rmsd value of hla was observed to grow up quickly from 0. for arg residue at the position of 180 in hla, while lowest rmsd observed for cys100. however, this residue was also resulted highest rmsf value of 7.181 å, while the rests of the residues were in lowest fluctuation. in case of epitope, the histidine residue at the first position and the tyrosine residue in 9th position were seen to be very much flexible, as these residues were resulted with highest rmsd and rmsf ( figure 6b ). in the meanwhile, we calculated the number of hydrogen bond formed between the epitope and hla molecule during the simulation. the results represented in figure 5b , showed that hydrogen bond at initial stage was 236, and the range decreased to 160. during the simulation, the number of hydrogen bond was at a range of 160-210, in silico-based vaccine design against ebov gp potentiality to express the b-cell response. furthermore, the surface accessibility of the protein was also analyzed using the emini surface accessibility prediction methods, since a potent b-cell epitope should be accessible through the surface. 47 as shown in figure s4 and table 5 , higher accessibility was found in regions 9-17 and 186-223 amino acid residues. figure s5 represents the β-turns region identified by chou and fasman β-turn methods. 52 according to the result, the region from 200 to 220 (in the region of 200-220 and 105-150) is regarded as β-turns as well as hydrophilic in nature. these are two properties required to be a potent b-cell epitope. 60 experimentally, antigenicity is related to the protein flexibility. 61 that is why we implemented the karplus and schulz flexibility prediction method, where it was evident that the regions of 255-280 and 200-220 were regarded as the most flexible ( figure s6 ). finally, based on the hidden markov model, the bepipred linear epitope prediction tool was utilized to predict linear b-cell epitopes. the predicted result is rendered and tabulated in figure s7 and table 6 . hence, by comparing the foregoing results, the peptide sequences ranging from 186 to 220 are which indicates the strong binding of epitope-hla complex. hence, all analyses lead to the conclusion that hkegaffly is one of the most prominent t-cell epitopes for gp based designing of vaccine. for the identification of potential b-cell epitopes, amino acid scale base methods have been used in this study. consistent with this protocol, we used diverse investigation processes for the calculation of an incessant b-cell epitope. according to the analysis of kolaskar and tongaonkar's 48 antigenicity prediction method, the average antigenicity was 1.028, while 1.225 and 0.894 were the maximum and minimum, respectively. the kolaskar and tongaonkar 48 antigenicity prediction uses a semiempirical method to predict antigenicity on the basis of physicochemical properties of the residues in a protein and their diversity in experimentally known epitopes, where values >1.00 were considered to denote a potential antigen. as summarized in table 4 and figure s3 [62] [63] [64] [65] [66] however, the information representing the population coverage in the worldwide are still limited. in such case, computational based epitiope screening is very much efficient in context of hla class i molecules, 67 and also much safe, high specificity and cost effective. therefore, this study incorporated various immunoinformatics and molecular modelling tools to identify potential epitopes present in ebov gps. initially, a set of 46 glycoprotein sequences from the different strains of ebov has been subjected to perform multiple sequence alignment. previous gp sequences analysis of different strains of each ebov species revealed a high degree of sequence similarity, 68, 69 and thereby, it is believed that targeting gp from old strain could provide strong and cross reactive immunity against the new strain and previous outbreaks in 2014. 70 interestingly, in our molecular analysis, we have found ~98-99% conservation for the amino acid sequences of different strains within the species, which confers the degree of 1 14 14 f 1 2 57 59 lss 3 3 73 106 ngvatdvpsatkrwgfrsgvppkvvnyeagewae 34 4 114 131 kkpdgseclpaapdgirg 18 5 141 148 vsgtgpca 8 6 175 176 tf 2 7 191 193 kdf 3 8 198 215 plrepvnatedpssgyys 18 9 223 229 tgfgtne 7 10 261 270 ytsgkrsntt 10 11 279 285 peidtti 7 1 4 11 tgilqlpr 8 2 17 56 tsfflwviilfqrtfsiplgvihnstlqvsdvdklvcrdk 40 3 63 69 lrsvgln 7 4 76 82 atdvpsa 7 5 89 99 rsgvppkvvny 11 6 118 126 gseclpaap 9 7 132 154 fprcryvhkvsgtgpcagdfafh 23 8 156 172 egafflydrlastviyr 17 9 177 189 aegvvaflilpqa 13 10 194 202 fsshplrep 9 11 211 221 sgyysttiryq 11 12 233 247 lfevdnltyvqlesr 15 13 249 259 tpqfllqlnet 11 14 274 280 iwkvnpe 7 able to provoke the immune response as b-cell epitope for gp-based designing of vaccine. in recent trends, the primary focus of vaccine development is very much rely on gps, as they are involved in cell attachment, fusion and entry as well as assist in invasion; and thus plays the role of pathogenesis of disease. the central role in silico-based vaccine design against ebov gp similarity and support the previous analysis. from this set of gps, the most antigenic protein sequence was determined by vaxijen server. based on auto cross covariance (acc), the vaxijen server transform the protein sequence into uniform vectors of physicochemical properties of proteins. with 91% sensitive, 82% accuracy and 72 specificity, the l00-cv (leave one -out cross validation) was used to identify antigenicity of protein for viral species. 71 the resultant antigenic protein (vaxijen score ≥0.5) was then subjected for various immunoinformatics analysis, followed by iedb web server. at the beginning five potent 9-mer epitopes have been predicted from netctl 1.2 server and selected for further study. using the threshold of 0.5, the netctl 1.2 server predicts maximum number of epitopes without compromising the specificity or sensitivity levels, covering all 12 mhc class i supertypes. 23 the five most potent epitopes are represented in table 1 , and the scores are the predicted mhc class i affinities in the form of -logic50 and ic50 value. for mhc-i binding prediction, peptides with ic50 values <50 nm are considered high affinity, <500 nm for intermediate affinity, and <5000 nm for low affinity. therefore, we selected maximum alleles having binding affinity <200 nm. 72 it is advocated that t-cell epitope binding to specific multiple hla supertypes are termed as promiscuous in vaccine design, since they effectively increase the coverage of higher proportions of human populations. 73, 74 according to the results, both hkegaffly and lfevdnlty bind to the highest number of alleles. however, hkegaffly represents highest conservancy and was hence considered as epitope of choice. we also validated each epitope by molecular docking simulation and mm-gbsa/mm-pbsa studies with hla-a*32:15 protein, as it was found common in the results from mhc-i binding interaction analysis. prior of docking simulation, the three dimensional structure of hla molecule was prepared by using the phyre2, followed by intensive mood. as a result, 179 residues (99%) of hla-a*32:15 modelled at >90% accuracy. in these study, 2bck_chain a, crystal structure of hla-a*2402 showed the highest similarity of 90% ( figure s1 ). the selected model has been chosen from the twenty models generated by phyre2, on the basis of similarity and confidence level. phyre2 is one of the best protein prediction servers that allows remote fold reorganization and homology detection. using hidden markov model (hmm), this server predicts the structure of given protein sequence by constructing backbone, loop modelling and adding side chains. 75 however in intensive mode, additional ab initio approach is used for reconstruction of missing region, backbone and side chain. 75 in docking simulation, among the other epitopes, hkegaffly obtained highest binding affinity (table s1 , supplementary material). in addition, mm-pbsa and mm-gbsa techniques are frequently to re-rank docking poses from molecular docking study, as they achieve a much better performance than docking scoring functions. 76 nevertheless, the success rate of the absolute binding free energy prediction strongly depends on the systems. 77 thence, we used both of these solvation models to predict binding energy more accurately, where the results from mm-pbsa examine the accuracy and reliability of the results from mm-gbsa. results of binding energy calculation are shown in table s1 . the relative magnitude of binding free energy obtained from gb methods is found to be consistent with those calculated using pb method, despite of the differences in the absolute value of salvation energy. as a corollary, these results also demonstrated the consistence with relative stabilities of hla-epitope complexes. in previous published reports regarding in silico epitope identification of ebov, [78] [79] [80] [81] the studies are limited to sequence-based scoring function techniques and some extend to docking simulation. these techniques have certain limitations, though these are very useful. 82 in docking simulation of peptide and protein, it faces problem like peptide's flexibility. 83, 84 whereas, energy based approach like molecular mechanics and interaction energy scoring can add valuable information to sequence based results. 85, 86 therefore, we have performed md simulation study of 100 ns long to enhance the predictive power of the peptide affinity calculations to mhc molecule. in molecular dynamics simulation, both epitope and hla protein were seen to achieve equilibration, while different fluctuations of rmsd were seen by the time evolution. higher rmsd values of epitope indicate the flexibility in binding with hla molecule, during the simulation. these results were further confirmed by the analysis of per residue contributions in dynamics simulation by means of rmsf and rmsd. low values of rmsf indicate the core region of the hla protein was stable, while high values of rmsd demonstrated the motion of the protein during the simulation. in like manner, the rmsd and rmsf profiles of eptiope confirm the synergic conformation changes to accommodate the binding pocket of hla. the hydrogen bond occupancy analysis between the hlaepitope further confirmed the stability of the complex during the simulation. overall, these results evidently demonstrate that both hla and epitope have remarkable conformation changes to facilitate the binding and formed stable complex in thermodynamic environment (figure 7 ). submit your manuscript | www.dovepress.com dash et al it is one of important factors in vaccine design that the distribution of hla varies according to the diverse ethnic groups and geographic regions around the world. therefore, wide range of population coverage must be considered during the designing of an effective design. according to the results from population coverage analysis, the epitope hkegaffly showed wide range of population coverage in different regions of the world (table 3) , where the highest coverage was observed central africa; one of the most ebov infected areas. this result indicates that it will specifically bind with the prevalent hla molecules in the target population, where the vaccine will be employed. in other aspects, the b-cell epitope stimulates minimal immune unity, which is very much strong enough to elicit a potent humoral immune response, causing no harmful side effects to human body. thereby, we are also calculated and found that the sequences ranging from 186-220 as a b-cell epitope, by taking consideration of amino acid property, hydrophilicity, accessibility, flexibility, turns, exposed surface, polarity and antigenic propensity. this study could provide a solid base for vaccine design. in recent years, most vaccines have been developed based on b-cell immunity; however, the current strategy relies mostly on t-cell epitope owing to long-lasting immunity. both b-cell and t-cell epitopes are offered in this study for stimulating immunity in several ways. the resulting peptides showed b-cell and t-cell selectivity, better conservancy, population coverage, and significant interaction with mhc-1 allele with good affinity. above all, the predicted epitopes are anticipated to offer long-term and high protective immunity against ebov. the authors report no conflicts of interest in this work. computational biophysics; chemoinformatics and drug design; in silico adme/tox prediction. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials. php to read real quotes from published authors. dash et al ebola, marburg and disease ebola viral disease: what should be done to combat the epidemic in 2014? treatment of ebola virus disease ebola viral disease outbreak -west africa genome structure of ebola virus subtype reston: differences among ebola subtypes genomic rna editing and its impact on ebola virus adaptation during serial passages in cell culture and infection of guinea pigs ebola virus rna editing depends on the primary editing site sequence and an upstream secondary structure deep sequencing identifies noncanonical editing of ebola and marburg virus rnas in infected cells ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry the multiple roles of sgp in ebola pathogenesis ebolavirus glycoprotein structure and mechanism of entry structure of the ebola virus glycoprotein bound to an antibody from a human survivor prediction and identification of mouse cytotoxic t lymphocyte epitopes in ebola virus glycoproteins a highly conserved wdypkcdra epitope in the rna directed rna polymerase of human coronaviruses can be used as epitope-based universal vaccine design more than one reason to rethink the use of peptides in vaccine design immunogenicity and safety of a novel therapeutic hepatitis c virus (hcv) peptide vaccine: a randomized, placebo controlled trial for dose optimization in 128 healthy subjects conserved epitopes of influenza a virus inducing protective immunity and their prospects for universal vaccine development approaching rational epitope vaccine design for hepatitis c virus with meta-server and multivalent scaffolding construction and immunological evaluation of multivalent hepatitis b virus (hbv) core virus-like particles carrying hbv and hcv epitopes in silico-based vaccine design against ebov gp uniprot: the universal protein knowledgebase clustal w and clustal x version 2.0 vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines sensitive quantitative predictions of peptide-mhc binding by a 'query by committee' artificial neural network approach generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method modeling the mhc class i pathway by combining predictions of proteasomal cleavage, tap transport and mhc class i binding allerhunter: a svm-pairwise system for assessment of allergenicity and allergic cross-reactivity in proteins combining pairwise sequence similarity and support vector machines for detecting remote protein evolutionary and structural relationships pep-fold: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides protein structure prediction on the web: a case study using the phyre server identification and characterization of three novel hla alleles, hla-a*240214, hla-a*3215 and hla-dqb1*060302 improving the physical realism and structural accuracy of protein models by a two-step atomic-level energy minimization aqua and procheck-nmr: programs for checking the quality of protein structures solved by nmr verify3d: assessment of protein models with three-dimensional profiles verification of protein structures: patterns of nonbonded atomic interactions deviations from standard atomic volumes as a quality measure for protein crystal structures qmean: a comprehensive scoring function for model quality assessment autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading ucsf chimera--a visualization system for exploratory research and analysis rotamer libraries in the 21st century charmm general force field (cgenff): a force field for drug-like molecules compatible with the charmm all-atom additive biological force fields assignment of protonation states in proteins and ligands: combining pka prediction with hydrogen bonding network optimization lipid14: the amber lipid force field fast empirical pka prediction by ewald summation new ways to boost molecular dynamics simulations induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide a semi-empirical method for prediction of antigenic determinants on protein antigens prediction of chain flexibility in proteins improved method for predicting linear b-cell epitopes structural evidence for induced fit as a mechanism for antibody-antigen recognition empirical predictions of protein conformation mega6: molecular evolutionary genetics analysis version 6.0 in silico analysis suggests interaction between ebola virus and the extracellular matrix identification of novel potential vaccine candidates against tuberculosis based on reverse vaccinology vaccination and allergic disease: a birth cohort study computational analysis and binding site identification of type iii secretion system atpase from pseudomonas aeruginosa a method to identify protein sequences that fold into a known three-dimensional structure rational design, synthesis, and biological evaluation of 7-azaindole derivatives as potent focused multi-targeted kinase inhibitors turns in peptides and proteins antigenic determinants in proteins coincide with surface regions accessible to large probes (antibody domains) efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination clusterrandomised trial an adenovirus vaccine expressing ebola virus variant makona glycoprotein is efficacious in guinea pigs and nonhuman primates potent neutralizing monoclonal antibodies against ebola virus infection mechanism of binding to ebola virus glycoprotein by the zmapp, zmab, and mb-003 cocktail antibodies safety and immunogenicity of novel adenovirus type 26-and modified vaccinia ankaravectored ebola vaccines: a randomized clinical trial computational prediction and identification of hla-a2. 1-specific ebola virus ctl epitopes conservancy of mab epitopes in ebolavirus glycoproteins of previous and 2014 outbreaks detection and molecular characterization of ebola viruses causing disease in human and nonhuman primates clinical development of ebola vaccines in silico prediction of b-and t-cell epitope on lassa virus proteins for peptide based subunit vaccine design an in silico approach predicted potential therapeutics that can confer protection from maximum pathogenic hantaviruses development of a dna vaccine designed to induce cytotoxic t lymphocyte responses to multiple conserved epitopes in hiv-1 a combined immuno-informatics and structure-based modeling approach for prediction of t cell epitopes of secretory proteins of mycobacterium tuberculosis the phyre2 web portal for protein modeling, prediction and analysis the mm/pbsa and mm/gbsa methods to estimate ligand-binding affinities computations of standard binding free energies with molecular dynamics simulations highly conserved regions in ebola virus rna dependent rna polymerase may be act as a universal novel peptide vaccine target: a computational approach computational elucidation of potential antigenic ctl epitopes in ebola virus epitope-based peptide vaccine design and target site depiction against ebola viruses: an immunoinformatics study a highly conserved geqyqqlr epitope has been identified in the nucleoprotein of ebola virus by using an in silico approach conformational flexibility in designing peptides for immunology: the molecular dynamics approach managing protein flexibility in docking and its applications protein flexibility: multiple molecular dynamics simulations of insulin chain b toward an atomistic understanding of the immune synapse: large-scale molecular dynamics simulation of a membrane-embedded tcr-pmhc-cd4 complex mhc-peptide binding is assisted by bound water molecules key: cord-016413-lvb79oxo authors: efthimiou, petros; yadlapati, sujani title: adult-onset still’s disease date: 2018-07-14 journal: auto-inflammatory syndromes doi: 10.1007/978-3-319-96929-9_19 sha: doc_id: 16413 cord_uid: lvb79oxo adult-onset still’s disease (aosd) is a rare systemic, autoinflammatory disorder that often presents in adolescence and early adulthood with fever, rash, and polyarthritis. there are significant genetic and clinical similarities with systemic juvenile idiopathic arthritis (sjia) with a different chronological disease onset. the disease can have many protean characteristics leading to delays in diagnosis. treatment includes corticosteroids; traditional immunomodulators, such as methotrexate; and targeted biologic treatments that include il-1 and il-6 inhibitors. aosd remains a diagnosis of exclusion, since more common illnesses can present with very similar symptoms. definitive diagnosis should only be made after ruling out infectious, malignant, and other autoimmune and autoinflammatory diseases. yamaguchi criteria are often used to aid in diagnosis and, especially, classification [3, 4] . patients frequently have a favorable prognosis with timely diagnosis, unless life-threatening complications such as macrophage activation syndrome (mas) ensue. prevalence of aosd is estimated to be less than one case per 100,000 people. aosd is rare, and hence there are currently no consensus on its incidence and prevalence in different populations. based on larger reviews from the 1980s, it appears that aosd occurs worldwide and may affect slightly more women than men. this disease characteristically affects younger people between the age of 16 and 35 years of age [5, 6] . a bimodal peak at ages 15-25 and 36-46 without a sex bias has been described in a retrospective study of 62 patients from western france [7] . however, an epidemiological survey from japan described that 67% of the cases presented after the age of 35, the majority (65-70%) being women [8] . aosd affects all ages, and stress has been suggested as an important risk factor for all ages [9] . no familial trend has been reported in the recent literature; however, an association with certain hla alleles has been observed in certain populations. these hla subtypes include hla dr4, b17, b18, b35, dr2, dr5, and dq1. pathophysiology of aosd was largely obscure until the recent past. a myriad of factors such as genetics, infectious (bacterial and viral) agents, and environmental factors have been thought to play a causative role. concurrent elevations of serologic markers of infectious agents have been noted in some patients with still's disease. these infectious triggers include ebv; parvovirus b19; cmv; hhv; hiv; coxsackievirus; mumps; rubella; echovirus; hepatitis a, b, and c viruses; campylobacter jejuni; chlamydia pneumoniae; adenovirus; influenza virus; parainfluenza virus; and mycoplasma [5, 10] . however, to date definite insight in to precise role of infection in aosd is lacking. several associations with distinct hla alleles and aosd have been described thus far. pouchot et al. described a strong association between hla-b17, b18, b35, and dr2 and aosd [11] . in a small study of 25 aosd patients, hla-bw35 was associated with disease susceptibility conferring a favorable prognosis [12] . wouters et al. noted an increased frequency of the hla-dr4 allele in 29 patients with aosd compared to normal controls, with the presence of hla-drw6 being linked to root joint involvement [13] . an association between a chronic articular form of aosd and hla-drb1*1501 (dr2), drb1*1201 (dr5), and dqb1*0602 (dq1) was previously reported, while hla-dqb1*0602 (dq1) have been also associated with the systemic form of the disease in japanese population [14] . statistics from a korean report supported an association between hla-drb1*12 and drb1*15 and aosd, while hla-drb1*04 seemed to be protective. conversely, hla-drb1*14 alleles were more frequently present in patients with the monocyclic systemic type of aosd [15] . hallmark of aosd involves neutrophil and macrophage activation triggered by the pro-inflammatory cytokine il-18. neutrophil (pmn) cd64, a marker of neutrophil activation, has been found to be upregulated in patients with active aosd. calprotectin (calcium-binding protein) secreted by neutrophils and macrophages and macrophage migration inhibitory factor (mif) have been found to be useful markers of disease activity [13] . intercellular adhesion molecule (icam-1) upregulated by il-18 has been implicated as a useful clinical marker whose expression typically reflects the level of disease activity. macrophage colony-stimulating factor, a cytokine which orchestrates proliferation and differentiation of macrophages, also appears to play a role in aosd. more recently, regulation of cytokine production has been noted in patients with aosd. a predominance of th1 subset of cytokines has been seen in peripheral blood and tissues of active untreated aosd patients. th1 immune cascade is characterized by elevated secretion of interferon γ (ifnγ), interleukin-2 (il-2), and tumor necrosis factor α (tnfα) cytokines that direct b cells toward igg2a production, activate macrophages and natural killer (nk) cells, and promote cell-mediated immunity [10] . when compared with controls, serum levels of il6, tnfα, and ifnγ were significantly increased in 12 patients with active aosd [16] . il18 is a pro-inflammatory cytokine that is overproduced in the acute phase of aosd and is believed to be the cytokine initiating the inflammatory cascade that includes ifnγ, il6, and tnfα [17] . genetic polymorphisms of the human il18 gene have been described to confer disease susceptibility in a japanese study [18] . conversely, in another japanese study, serum levels of soluble il2 receptors, il4, and il18 correlated with chronic articular aosd activity, whereas ifnγ and il8 levels were found to be persistently raised, even in disease remission. understanding of the still's disease was also enhanced by the description of autoinflammatory syndromes. these disorders are associated with recurrent bouts of inflammation without an instigating antigenic stimulus. defective interleukin-1 processing, regulation of nuclear factor-b transcription factor, and possible uncharacteristic apoptosis are all anticipated mechanisms that may possibly play a role in the generation and perseverance of an inflammatory cascade. patients with autoinflammatory syndromes, in particular, the typical hereditary periodic fever syndromes, may share certain genetic traits; mefv gene mutation associated with familial mediterranean fever (fmf) and il-1 hypersecretion was seen with augmented frequency in turkish children with sjia. mutation of perforin and the munc13-4 genes have been seen in patients with macrophage activation syndrome (mas), a known severe, life-threatening complication of aosd [3] . mutations in genes encoding the tumor necrosis factor (tnf) receptor and pyrin superfamilies of molecules may result in the endurance of leukocytes that would customarily go through apoptosis [3] . as a result, relatively minor pro-inflammatory triggers may lead to an exaggerated and potentially harmful, inflammatory response. il-1b, the pivotal cytokine in aosd and other autoinflammatory syndromes, activates the thermoregulatory center, resulting in fever; may activate il-1 receptors on the endothelium, resulting in rash; and can also act on the bone marrow to increase mobilization of granulocyte progenitors and mature neutrophils, resulting in peripheral neutrophilia. il-1 also causes an increase in platelet production, which results in thrombocytosis, and decreases the response to erythropoietin, causing anemia. il-1 induces the production of il-6. circulating il-6 stimulates the hepatocytes to synthesize several acute-phase proteins, such as crp, ferritin, and d-dimer. in general, three types of aosd have been described. the monocyclic pattern, which is the most benign form, is characterized by a single episode of aosd without recurrence. in the polycyclic pattern, the patient experiences recurrent attacks, although the subsequent aosd attacks often seem not as severe as the first one. in both the monocyclic and the polycyclic forms, systemic symptoms (rash, fever) are very prominent. the worst prognosis is carried by the chronic articular form, which is thought to be an unfortunate evolution of the polycyclic form. often, the systemic symptoms are absent or so remotely in the past that the patient may not even remember them and the main morbidity is from a chronic articular polyarthritis that mimics ra. common findings of aosd are fever, arthralgia, rash, and sore throat. other accompanying symptoms include, but are not limited to, myalgia, pharyngitis, lymphadenopathy, splenomegaly, and serositis. fever is usually quotidian and often precedes other manifestations. temperature spikes of >39°c frequently occur and are associated with chills and rigors, joint pain, or rash. aosd is one of the main causes of pyrexia of unknown origin (puo). temperature fluctuations can be dramatic. fever may persist between spikes in approximately 20% of cases, and complete defervescence is not always a characteristic of the quotidian fevers [11] . high-grade temperatures, more than 39.5° c, can be associated more strongly with monophasic pattern of aosd [19] . skin rash associated with aosd is a salmon-pink colored, maculopapular eruption that tends to accompany or, more frequently, be exacerbated by fever. rash usually presents centrally at the trunk and the adjacent extremities (arms/thighs). histopathology of the rash often reveals non-specific findings including dermal edema and mild perivascular inflammation in the superficial dermis with lymphocytes and histiocytes. complement deposits (c3) have been described with immunofluorescence. arthralgias and myalgias are common manifestations of aosd. most commonly involved joints include the wrists, ankles, knees, elbows, proximal interphalangeal joints, and shoulders. these manifestations can evolve into more severe and potentially destructive polyarthritis that can mimic other systemic inflammatory arthritides, such as rheumatoid arthritis [20] . myalgia can be debilitating and often associated with fever spikes. the muscle involvement, when severe, may be accompanied by an elevation of serum creatinine kinase and aldolase concentrations. however, muscle biopsy and electromyographic (emg) studies are typically normal. sterile pharyngitis manifesting as throat pain can occasionally precede the development of fever or rash by weeks, or even months, as a prodromal symptom and can often reoccur with disease relapses. hepatomegaly and modest elevation of serum hepatic aminotransferases and alkaline phosphatase are not uncommon in patients with aosd. several cases of fulminant liver failure in association with aosd have been described and may be associated with overexpression of il-18 [11] . myocarditis, pericarditis, and pleural effusions have also been observed in aosd patients, and they seem to respond to anti-inflammatory treatment. uncommonly, some patients may develop severe interstitial lung disease and some progress to acute respiratory distress syndrome (ards). enlarged, symmetrical, cervical nodes are seen in about one half of patients with aosd. lymphadenopathy is often accompanied by fever, leukocytosis creating diagnostic confusion with lymphoma. lymph node biopsy typically shows intense, paracortical immunoblastic hyperplasia [21] . splenomegaly is also seen in up to one third of patients. macrophage activation syndrome (mas) or reactive hemophagocytic syndrome (rhs) is a life-threatening complication of aosd. mortality rate ranges between 10% and 22% [22] [23] [24] [25] , and an incidence of 12-14% has been noted in two recent series, a rate higher than other rheumatic diseases [26, 27] . it is categorized by an uncontrollable activation of the reticuloendothelial system within the bone marrow, reticuloendothelial system, and central nervous system, with successive phagocytosis of hematopoietic cells by tissue macrophages (histiocytes) [25, 28] . patients developing mas present with acute high fever, lymphadenopathy, and hepatosplenomegaly. laboratory findings include pancytopenia, elevated ferritin levels, triglycerides, and liver enzymes, often accompanied by normal erythrocyte sedimentation rate (esr). the most commonly implicated triggers include infections, medications, and disease flares [29] [30] [31] . patients with mas have a decreased ability to eliminate antigen stimulation, thereby inducing t cell activation and proliferation resulting in cytokine secretion (interferon-gamma and granulocyte macrophage colony-stimulating factor) and macrophage hyperactivation. the end result is an uncontrollable increase in cytokines, specifically tnfα, interleukin-1, and interleukin-6 production resulting in severe systemic inflammatory reaction, i.e., "cytokine storm" [25] . there is also a suggestion that certain therapeutic agents, such as nonsteroidal anti-inflammatory drugs, methotrexate, sulfasalazine, penicillamine, and lately tnf-α, il-1, and il-6 inhibitors may be capable of provoking mas, often complicating their therapeutic use [27] . in theory, these therapies may create a state of immunodeficiency resulting in the reactivation of latent viruses (epstein-barr virus or cytomegalovirus) which in turn can stimulate mas. the counter-argument would be that anti-inflammatory medications may not be able to prevent the development of mas, at least in the dosages used in aosd or sjia. early suspicion of mas is most commonly raised by the detection of subtle laboratory changes, whereas clinical symptoms may be delayed. a recent international effort to identify candidate markers using an expert consensus process identified nine criteria that included a falling platelet count, hyperferritinemia, evidence of macrophage hemophagocytosis in the bone marrow, increased liver enzymes, falling leukocyte count, a persistent continuous fever ≥38 °c, a falling erythrocyte sedimentation rate (esr), hypofibrinogenemia, and hypertriglyceridemia [32] . hemophagocytosis, seen in bone marrow aspiration and biopsy, establishes the diagnosis, even though hemophagocytosis could be seen more frequently in biopsies from the liver, spleen, and/or lymph nodes. bone marrow aspiration is considered the gold standard and is usually required in atypical cases, causing a diagnostic dilemma. there is significant overlap between aosd and mas, and these two conditions are often thought to be anchoring the same disease spectrum, with aosd representing the milder form. aosd, unlike other systemic rheumatic diseases driven by adaptive immunity, is not typically associated with rheumatoid factor (rf) or antinuclear antibody (ana) positivity, although several cases have been published with low-level positivity of these autoantibodies. this has been considered in various sets of classification criteria. elevated esr is a common finding in most patients [11, 33] . c-reactive protein (crp) may also be found to be raised. other laboratory abnormalities include leukocytosis, thrombocytosis, and anemia which often accompany increased disease activity. pancytopenia is often an alarming sign of coexisting or developing mas and necessitates prompt intervention. abnormal coagulation testing can rarely be seen and, in extreme cases, may develop into full-blown disseminated intravascular coagulation (dic) which can be fatal [11] . abnormal liver and biliary function tests (increase in lactic dehydrogenase, aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, and bilirubin) can also be seen in up to 75% of patients and often accompany fever and exacerbation of arthritis. mild periportal inflammation with monocyte infiltration may be seen on liver biopsy. serum ferritin has gained attention as both a diagnostic test for aosd and a marker of disease activity (biomarker). in aosd, ferritin is an acute phase reactant involved in inflammation, is linked to initiation of histiocyte-macrophage system and/or augmented release from damaged hepatocytes, and is not related to iron metabolism and storage. ferritin production is driven by cytokines such as il1β, il18, tnfα, and il6 [2] . serum ferritin levels as high as 1000 ng/ml, five times the upper limits of normal, have been described in these patients. levels ranging from 4000 ng/ml to 30,000 ng/ml are not uncommon. fautrel et al. evaluated the validity of hyperferritinemia as a diagnostic tool in a retrospective analysis looking at 49 patients with still's disease [2] . a fivefold increase in serum ferritin had 80% sensitivity and 41% specificity. similar results were seen in a japanese study 57 with 82% sensitivity and 46% specificity [4] . serum ferritin levels are seen in other diseases such as hemochromatosis, gaucher's disease, infections (sepsis, hiv), and malignancies (leukemia, lymphomas). serum ferritin levels correlate with disease activity and often normalize when the disease flare subsides. however, the absence of elevated serum ferritin does not exclude active aosd. some patients with active aosd do not have elevated serum ferritin levels at all, or the rise of serum ferritin may lag behind the symptom presentation. glycosylated fraction is a more specific diagnostic marker than ferritin, albeit not commercially available in the usa. in healthy subjects, 50-80% of ferritin is glycosylated. saturation of glycosylation mechanisms results in a drop in glycosylated fraction to drop to 20-50%. this phenomenon is particularly prevalent in aosd. glycosylated ferritin often remains elevated for months after the disease goes into remission and hence cannot be used to monitor disease activity or response to treatment. improved diagnostic tests are clearly desirable, and new immunological tests, such as il18, may prove useful in the near future for diagnosis as well as monitoring disease activity and response to treatment [17] . currently available tests: complete blood count and differential, esr, crp, ana, and rf (both negative), liver function tests (lfts) and albumin, ferritin, and glycosylated ferritin (if available) are of practical use to most clinicians. radiographs are often of limited significance in the early phase of the disease. images are either normal or show soft tissue swelling, joint effusion, or mild periarticular demineralization. bone scan and gadolinium-enhanced magnetic resonance imaging were assessed in small series and may prove to be more sensitive imaging modalities for early diagnosis and successful treatment in follow-up. in one study, distinctive pattern of intercarpal and carpometacarpal joint space narrowing was seen in 41% of the subjects (bilateral in 69%) that led to pericapitate ankylosis in 25% of the cases [11] . other investigators have also reported a tendency for distal interphalangeal, intertarsal, and cervical zygapophyseal ankylosis. patients who have the chronic articular disease pattern can present with joint erosions making the differential diagnosis from ra problematic, especially in the absence of systemic signs and symptoms. the differential diagnosis of aosd is extensive, especially at presentation, when the systemic symptoms predominate. conditions such as infections, neoplasms, and autoimmune disorders should be ruled out before the diagnosis of aosd can be confidently made. viral syndromes (e.g., rubella, cytomegalovirus, epstein-barr virus, mumps, coxsackievirus, adenovirus) can be excluded if the symptoms persist for more than 3 months and serologies are negative. neoplastic disorders that can mimic aosd include leukemia and lymphoma, such as angioimmunoblastic lymphoma. however, the clinical presentation can differ substantially, with atypical rashes and/or isolated lymph node enlargement. at times bone marrow and/or lymph node biopsy may be needed to differentiate these entities. common mimickers of aosd include reactive arthritis and the other spondyloarthropathies, hemophagocytic syndrome, dermatomyositis, kikuchi's syndrome, sweet's syndrome, granulomatous disorders, and the vasculitides. other differentials of aosd are the periodic fever syndromes and, in particular, familial mediterranean fever, hyper igd syndrome (hids), and tnf receptor-associated periodic syndrome (traps). patients with familial mediterranean fever often present with acute, self-limited episodes of fever accompanied by signs of peritonitis, pleuritis, synovitis, and erysipelas-like erythema. the disease commonly starts in childhood or early adolescence. a significant family history, clinical presentation, and response to colchicine can aid in making the correct diagnosis. this can be verified, in many cases, with genetic analysis for the mefv gene. traps and hids commonly start in childhood and also have strong familial distributions. however, adult-onset cases of those rare syndromes do exist, and the genetic tests that are commercially available can be helpful in excluding them, especially when atypical presentations are present. several classification criteria have been developed from retrospective data available on aosd. one such study attempted to validate these classification criteria: yamaguchi's criteria were found to be the most sensitive (93.5%), followed by cush's (80.6%) and calabro's (80.6%) criteria (table 19 .1) [4, 34] . of late, a french group has proposed a new set of criteria which takes into consideration the two new disease markers: serum ferritin and its glycosylated fraction (table 19 .2). this set provided a sensitivity of 80.6% and a specificity of 98.5%, which remains to be authenticated in a different population before becoming widely accepted [35] . fever of at least 39 degrees c (102.2 f) last at least 1 week arthralgias or arthritis lasting 2 weeks or longer a non-pruritic macular or maculopapular skin rash that is salmon-colored in appearance and usually found over the trunk or extremities during febrile episodes leukocytosis (10,000/microl or greater), with at least 80% granulocytes lymphadenopathy hepatomegaly or splenomegaly abnormal liver function studies, particularly elevations in aspartate and alanine aminotransferase and lactate dehydrogenase concentrations negative tests for antinuclear antibody (ana) and rheumatoid factor (rf) *several classification criteria have been developed from retrospective data available on aosd. one such study attempted to validate these classification criteria: yamaguchi's criteria were found to be the most sensitive (93.5%) therapeutic goals include controlling physical signs and symptoms of inflammation, which is typically associated with the improvement in laboratory parameters. decisions about optimal therapy are influenced by the severity and chronicity of the disease and involve taking into consideration the adverse effect profile of the treatments, both long and short term, and the clinical response to initial therapies. the primary goal of therapy not only involves achieving control of acute symptoms but also preventing end-organ damage, including joint injury and major organ complications. the initial choice of therapy depends upon disease severity and extent of organ involvement. there has been a lack of head-to-head randomized controlled trials comparing different therapeutic modalities given the rarity of disease. treatment options constantly keep evolving as more insight is gained into the pathogenesis of aosd. support for the use of biologics as first-line therapy in severe disease is based upon the published experience in aosd and, especially, more extensive evidence supporting use of these agents in children with sjia, since recent evidence supports that sjia and aosd are different chronological disease onsets of the same "still's disease." patients with mild-to-moderate disease may present with fevers and rash, as well as with arthralgias or mild arthritis. while nsaids, such as naproxen or ibuprofen, were the first medications to be used in aosd, their role today is a very limited one. their use can be justified either as adjunct treatment or as monotherapy in mild, monocyclic cases for symptomatic relief. in addition to adverse effects that are commonly associated with nsaids, an association between the use of nsaids and macrophage activation syndrome (mas) has been described, as well [11] . patients responsive to nsaids alone, who remain asymptomatic for at least a month, could have nsaid doses gradually reduced. glucocorticoids can be started immediately, as first-line treatment, or when nsaids are insufficient to control signs and symptoms of the disease. glucocorticoid dose can be gradually tapered once disease activity is controlled. oral prednisone is typically initiated at a dose of 0.5-1 mg/kg per day, depending on the severity of disease. intravenous, high-dose steroids are reserved for those with refractory disease [2] . approximately 70% of patients respond to glucocorticoids alone or to glucocorticoids used after a trial of nsaids [36] . in addition to systemic steroids, those with one or two inflamed joints despite systemic therapy may benefit from intraarticular glucocorticoid injections. disease-modifying antirheumatic drugs (dmards) are typically used in the event of inadequate response to corticosteroids or as steroid-sparing medications. methotrexate remains the first-line steroid-sparing agent in aosd and is also a useful agent to treat still's arthritis. methotrexate can result in complete remission in up to 70% of patients and is effective in corticosteroid weaning [37] . sulfasalazine is contraindicated because of lack of efficacy and association with mas development. internal organ damage and debilitating joint symptoms with radiographic are characteristic of moderate-to-severe disease. severe disease is defined as refractory disease, especially when terminal organ and/or life-threatening involvement exists, such as mas, severe hepatic injury, cardiac involvement with/or without tamponade, or disseminated intravascular coagulation (dic). biologic therapies such as il-1 inhibitors (anakinra, canakinumab) and il-6 receptor antagonists (tocilizumab) are used as first line or second line for sjia often without any prior use of corticosteroids or traditional dmards and that practice may be justified in moderate-tosevere aosd as well. small series have reported some efficacy of ivig when used early in the course of aosd [38, 39] . target biologic agents have been historically reserved for refractory aosd (fig. 19.1 ) [40] . these agents include a recombinant antagonist of the il-1 receptor (il-1ra, anakinra), a human monoclonal antibody directed against il-1β (canakinumab), and a soluble il-1 trap fusion protein (rilonacept). anakinra is particularly efficient in the rapid relief of the systemic symptoms. several retrospective case series and one open-labeled prospective randomized trial have evaluated the use of anakinra in aosd patients [41, 42] . anakinra is approved as a daily subcutaneous injection (100 mg) in ra. in aosd, higher dose may be necessary, since anakinra has a very short half-life (4-6 h). while anakinra does not work in all aosd cases, for the ones it does work, its onset of action is very rapid. in many cases, resolutions of systemic symptoms and normalization of inflammatory markers were reported within 2 weeks of anakinra use, allowing for rapid tapering of corticosteroids, if they were ever used in the first place. however, relapses are not uncommon with the cessation of these agents. in certain patients, gradual reduction in dose has enabled the weaning of anakinra. daily anakinra injections are often complicated by frequent injection site reactions. if response to anakinra is incomplete or tolerability issues ensue, rilonacept and/or canakinumab can be considered. they have longer half-lives and can be administered at greater intervals, once or twice weekly for rilonacept and every 4-8 weeks for canakinumab, respectively. additional supplemental data is available in the setting of sjia. swart and associates reviewed data pertaining to 140 children with sjia treated with anakinra [43] . systemic symptoms resolved in 98% of the patients, and fatigue and well-being improved in 93% of cases. arthritis improved in 66% of the cases in time. absolute disease remission was mostly detected in patients with systemic symptoms, less arthritis, and a shorter duration of disease. similar findings were corroborated in a large, multicenter, randomized, placebo controlled trial by quartier et al. [44] . sample population included 24 patients with a systemic-onset jia for duration of more than 6 months and steroid dependency. in 1 month, anakinra was effective in 8 out of 12 patients (versus one in the placebo group) who reached the modified american college of rheumatology (acr) pediatric 30 score. after 2 months, majority of patients (9/10) who had been switched to anakinra were also responders. nigrovic and associates studied 46 patients who received anakinra as first-line treatment. rapid resolution of systemic symptoms was observed in about 95% of cases, along with a supplementary preventive effect on refractory arthritis in almost 90% of the patients. based on these results, it was postulated that there could be a benefit for il-1 blockade therapy in initial phase of the disease (i.e., within 6 months after onset) [45] . these findings were also further reinforced by vaster et al. in 2014 [46] . in a prospective series of 20 patients who received anakinra as first-line therapy, 85% of the patients showed an american college of rheumatology pediatric 90 score response or had inactive disease within 3 months. overall, 75% of the patients treated with anakinra achieved remission. these results clearly indicate that il-1 blockade has an early place in the treatment strategy. tocilizumab, humanized monoclonal antibody directed against il-6 receptor, is used in refractory aosd. this agent has been studied with randomized placebocontrolled trials in sjia patients but not yet in aosd. effects of tocilizumab have been described to persist for ≥6 months after its discontinuation. tocilizumab appears to have a marked corticosteroid-sparing effect and has a good safety and tolerance profile [47] . most of the early data was with tocilizumab administered intravenously (iv) at a dosage of 5-8 mg/kg body weight every 2-4 weeks. nevertheless, larger randomized studies are still needed to further determine the optimal therapeutic scheme for tocilizumab, i.e., optimal dosing, interval, and duration of treatment for both the intravenous but also the subcutaneous route of administration. although those agents were the first biologics to be used in refractory aosd, these agents are no longer recommended as first-line treatment in aosd. anti-tnf-α agents, in particular infliximab, etanercept, and adalimumab, have been used to treat refractory aosd, but data on adalimumab are limited to a few cases [48] . although complete resolution of symptoms has been observed, efficacy of the tnf-inhibitors has been mostly limited to still's arthritis. efficacy was better with the monoclonal antibodies, as compared to the soluble receptor, and switching from one agent to another had no additional effect [19] . moreover, in one published series, two patients who were started on etanercept and adalimumab developed mas that could be allied with the initiation of therapy [49, 50] . overall, tnf-α blockers should be considered for the treatment of chronic polyarticular disease, after the use of il-1 and/or il-6 inhibitors. non-refractory disease comprises of monocyclic and polycyclic aosd. nsaids can be used in monocyclic course of aosd without major systemic or articular involvement. preferred nsaid is high-dose indomethacin (150-200 mg/day). corticosteroids should be started promptly once the diagnosis is confirmed. usually, corticosteroid therapy starts at a dosage of 0.5-1 mg/kg/day. pulse dose methylprednisolone is used if there is severe visceral involvement or there is suspicion of mas complicating the clinical presentation. higher dosages seem to be more efficient in controlling the disease and lessening the number of relapses. tapering of corticosteroids can start after 4-6 weeks, when symptoms have resolved, and biological parameters have returned to baseline. methotrexate (mtx) could be considered early for its steroid-sparing effect. typically, methotrexate (7.5-20 mg/week) enables complete remission of the disease (70%) and limits frequent corticosteroid use. blood count and renal and hepatic function should be monitored before initiation of methotrexate and then at monthly intervals. alternative dmards may be use in the event of methotrexate failure; however, more recent literature suggests better results with targeted biologic treatment. an open-label, multicenter, phase ii study of subcutaneous tadekinig alfa (il-18bp) in patients with aosd was recently published and showed promise. tadekinig alfa is the drug name for recombinant human interleukin-18-binding protein (il-18bp). this study was based on the principle that high levels of il-18 were noted during active flares of aosd. ten patients were assigned to receive 80 mg tadekinig alfa, and 13 patients received the 160 mg dose. at week 3, 5 of 10 patients receiving 80 mg and 6 of 12 patients receiving 160 mg achieved the predefined response criteria. the agent was overall well tolerated with the exception of one case of optic neuropathy [51] . it is becoming exceedingly evident that aosd patients fall into two distinct subsets, i.e., those presenting with systemic manifestations and those presenting with prominent articular manifestations. in addition, these findings are also reinforced by molecular evidence, cytokine profiles, clinical course, and response to therapy. predictors for a prominent articular pattern include female sex, proximal arthritis at disease onset, thrombocytosis, and corticosteroid dependency, whereas high fever, transaminitis, or elevated acute phase reactants are more likely to be connected with a systemic pattern of aosd [14, 52] . alternative evidence to identify the systemic subtype of aosd are the following: thrombocytopenia, rhl, and hyperferritinemia. il-18, interferon-γ, il-10, and il-4 are typically associated with systemic aosd, whereas il-6, il-17, and il-23 are associated with arthritic aosd [53] . this dichotomy aids in management as patients fall into one of the two categories and should benefit from different therapies. patients with systemic symptom predominant aosd often benefit from systemic corticosteroid therapy. from traditional dmards, methotrexate has been studied the most and may continue to have a role as steroid-sparing and in the treatment of chronic articular disease. il-1 antagonists should be considered first line in severe or refractory aosd, either alone or in initial combination with systemic corticosteroids when necessary. regularly reported side effects with anakinra include injection site reactions. longer acting il-1 inhibitors (rilonacept or canakinumab) may play a role in refractory disease or when tolerability issues exist with anakinra. tocilizumab, the il-6 receptor antagonist, has shown efficacy in both systemic and articular disease predominant aosd, even in cases where il-1 inhibition has been unsuccessful. in contrast to il-1 and il-6 inhibitors, anti-tnf-α agents typically have less sustained effect on systemic symptoms, but they may have a limited role for the chronic inflammatory polyarthritis. in an effort to standardize therapeutic management and evaluate comparative efficacy in an observational setting, the childhood arthritis and rheumatology research alliance has developed four consensus treatment plans for sjia [54] . this includes glucocorticoid plan, a methotrexate plan, an anakinra plan, and a tocilizumab plan. since no guidelines are available yet in aosd, this consensus may act as a rough guide for its treatment, as it coincides with the published clinical experience. new therapeutic agents are being developed for aosd, and future randomized, controlled trials will fill the knowledge gap that currently exists. on a form of chronic joint disease in children adult-onset still disease adult onset still's disease and autoinflammation preliminary criteria for classification of adult still's disease adult-onset still's disease adult still's disease: review of 228 cases from the literature epidemiology of adult still's disease: estimate of the incidence by a retrospective study in west france estimated prevalence and incidence of adult still's disease: findings by a nationwide epidemiological survey in japan risk factors for adult still's disease diagnosis and management of adult onset still's disease adult still's disease: manifestations, disease course, and outcome in 62 patients hla-bw35 and prognosis in adult still's disease adult-onset still's disease. disease course and hla associations cytokine and immunogenetic profiles in japanese patients with adult still's disease. association with chronic articular disease association between the hla-drb1 gene and clinical features of systemic sclerosis in korea elevated serum interleukin 6, interferon-gamma, and tumor necrosis factoralpha levels in patients with adult still's disease levels of interleukin-18 and its binding inhibitors in the blood circulation of patients with adult-onset still's disease association between adult-onset still's disease and interleukin-18 gene polymorphisms adult-onset still disease: manifestations, treatment, outcome, and prognostic factors in 57 patients adult-onset still's disease. twenty-year follow up and further studies of patients with active disease characterization of lymph node histology in adult onset still's disease macrophage activation syndrome associated with systemic juvenile idiopathic arthritis macrophage activation syndrome following initiation of etanercept in a child with systemic onset juvenile rheumatoid arthritis macrophage activation syndrome and etanercept in children with systemic juvenile rheumatoid arthritis macrophage activation syndrome: a frequent but under-diagnosed complication associated with rheumatic diseases reactive haemophagocytic syndrome in adult onset still's disease: report of 6 patients and review of the literature adult onset still's disease: a study of 14 cases macrophage activation syndrome treated with anakinra reactive haemophagocytic syndrome in adult-onset still's disease: a report of six patients and a review of the literature preliminary diagnostic guidelines for macrophage activation syndrome complicating systemic juvenile idiopathic arthritis macrophage activation syndrome: a potentially fatal complication of rheumatic disorders an international consensus survey of diagnostic criteria for macrophage activation syndrome in systemic juvenile idiopathic arthritis adult-onset still's disease; clinical and laboratory features, treatment and progress of 45 cases adult-onset still's disease proposal for a new set of classification criteria for adult-onset still disease efficacy of traditional and biologic agents in different clinical phenotypes of adult-onset still's disease corticosteroid sparing effect of low dose methotrexate treatment in adult still's disease intravenous immunoglobulin in adult still's disease refractory to non-steroidal anti-inflammatory drugs treatment of still disease in adults with intravenous immunoglobulins adult-onset still's disease-pathogenesis, clinical manifestations, and new treatment options interleukin-1 receptor antagonist (anakinra) treatment in patients with systemic-onset juvenile idiopathic arthritis or adult onset still disease: preliminary experience in france rapid responses to anakinra in patients with refractory adult-onset still's disease the efficacy and safety of interleukin-1-receptor antagonist anakinra in the treatment of systemic juvenile idiopathic arthritis a multicentre, randomised, double-blind, placebo-controlled trial with the interleukin-1 receptor antagonist anakinra in patients with systemic-onset juvenile idiopathic arthritis (anajis trial) anakinra as first-line disease-modifying therapy in systemic juvenile idiopathic arthritis: report of forty-six patients from an international multicenter series effectiveness of first-line treatment with recombinant interleukin-1 receptor antagonist in steroid-naive patients with new-onset systemic juvenile idiopathic arthritis: results of a prospective cohort study randomized trial of tocilizumab in systemic juvenile idiopathic arthritis adalimumab (anti-tnf-alpha) therapy to improve the clinical course of adult-onset still's disease: the first case report exacerbation of adult-onset still's disease, possibly related to elevation of serum tumor necrosis factor-alpha after etanercept administration a rare trigger for macrophage activation syndrome open-label, multicentre, dose-escalating phase ii clinical trial on the safety and efficacy of tadekinig alfa (il-18bp) in adult-onset still's disease clinical manifestations of adult-onset still's disease presenting with erosive arthritis: association with low levels of ferritin and interleukin-18 distinct subsets of patients with systemic juvenile idiopathic arthritis based on their cytokine profiles consensus treatment plans for new-onset systemic juvenile idiopathic arthritis key: cord-013894-1bgvj62a authors: wang, qihui; song, hao; cheng, hao; qi, jianxun; nam, gol; tan, shuguang; wang, junzhi; fang, min; shi, yi; tian, zhigang; cao, xuetao; an, zhiqiang; yan, jinghua; gao, george f. title: structures of the four ig-like domain lilrb2 and the four-domain lilrb1 and hla-g1 complex date: 2019-07-04 journal: cell mol immunol doi: 10.1038/s41423-019-0258-5 sha: doc_id: 13894 cord_uid: 1bgvj62a leukocyte immunoglobulin (ig)-like receptors (lilrs), also known as cd85 and immunoglobulin-like transcripts (ilts), play pivotal roles in regulating immune responses. these receptors define an immune checkpoint that immune therapy can target. through cis or trans interactions with human leukocyte antigen (hla)-g, the two most abundantly expressed inhibitory lilrs, lilrb1, and lilrb2 (lilrb1/2, also known as cd85j/d and ilt2/4), are involved in immunotolerance in pregnancy and transplantation, autoimmune diseases, and immune evasion by tumors. although the discrete domains of lilrb1/2 are clear, the assembly mode of the four extracellular ig-like domains (d1, d2, d3, and d4) remains unknown. previous data indicate that d1d2 is responsible for binding to hla class i (hla-i), but the roles of d3d4 are still unclear. here, we determined the crystal structure of the four ig-like domain lilrb2 and four-domain lilrb1 in complex with hla-g1. the angles between adjacent domains and the staggered assembly of the four domains suggest limited flexibility and limited plasticity of the receptors during ligand binding. the complex structure of four-domain lilrb1 and hla-g1 supports the model that d1d2 is responsible for hla-i binding, while d3d4 acts as a scaffold. accordingly, cis and trans binding models for hla-i binding to lilrb1/2 are proposed. the geometries of lilrb1/2 in complex with dimeric and monomeric hla-g1 suggest the accessibility of the dimeric receptor, which in turn, transduces more inhibitory signals. the assembly of lilrb1/2 and its binding to hla-g1 could aid in the design of immune regulators and benefit immune interference. leukocyte immunoglobulin-like receptors (lilrs/lirs), also called immunoglobulin-like transcripts (ilts) and cd85, are a family of receptors that regulate immune reactions and play pivotal roles in immunological homeostasis. activating lilrs (lilras) contain a short cytoplasmic tail and are associated with the adaptor molecule fcεrγ, which has an immunoreceptor tyrosine-based activation motif (itam). 1 by contrast, inhibitory lilrs (lilrbs) have an immunoreceptor tyrosine-based inhibitory motif in their cytoplasmic domain that interacts with tyrosine phosphatases and inhibits activating signals. 1 among the five lilrbs identified (lilrb1-5), lilrb1 (also called lir1, ilt2, and cd85j), and lilrb2 (also called lir2, ilt4, and cd85d) have been extensively studied. 2, 3 lilrb1 and lilrb2 (lilrb1/2) bind to multiple ligands and are involved in multiple physiological and pathological situations, which have been summarized in several excellent reviews. [1] [2] [3] among the ligands of lilrb1/2, human leukocyte antigen class i (hla-i) is the most widely expressed. upon binding to hla-is, lilrb1/2 generally inhibit the activities of immune cells, including antigen-presenting cells (apcs), 4 cd8 + t cells 5 and b cells. 4, 6 moreover, lilrb1 inhibits the polarization of nk cell lytic granules and therefore the cytotoxicity of nk cells in response to target cells expressing hla-i (in trans binding). 4, 7, 8 lilrb1/2 also associate with hla-is expressed on the same cells to regulate mast cell activation and osteoclast development (in cis binding). 9, 10 blocking the interaction between lilrb1 and hla-i restores the cytotoxic activity of nk cells 11 and potentiates macrophage phagocytosis of tumor cells. 12 in addition to performing important functional regulation of other members in this family, lilrbs serve as crucial immune checkpoints that, like pd-1, pd-l1, and ctla-4, could be targets for drug development to treat cancers. 13 the lilr family contains 13 members, including two pseudogenes. except for lilra3 and lilrb4 which have two domains, lilrs contain four immunoglobulin (ig)-like domains in their extracellular part, including one soluble member (lilra3). 2 usually, the four domains are named domain d1, d2, d3, and d4 from distal-to-proximal relative to the membrane. although lilrs are important regulatory receptors and 20 years have passed since their characterization, whole structures of lilrs with four domains have yet to be solved. previous work has solved the structures of discrete domains of lilrb1/2 (d1d2 and d3d4) and delineated the interaction between d1d2 and hla-i. [14] [15] [16] [17] [18] however, the roles of d3d4 of lilrb1/2 in the interaction with hla are still under debate. no substantial binding between lilrb1/2 d3d4 and hla-i has been detected by surface plasmon resonance (spr). 19 interestingly, variable binding affinities of lilrb2 to hla-b*3501 and hla-b*3503 (which differ in residue 114 or 116 being located in the α1α2 domain of hla-i) have been reported. [20] [21] [22] mutated presented peptides in hla-b27, hla-a11, b8, and b7 were also likely to confer enhanced binding affinity to lilrb2 and might be related to hiv-1 escape. 23, 24 hence, two models have been proposed: 25, 26 one model is that no interaction exists between d3d4 of lilrb2 and hla-i and the other model is that d3d4 bend to interact with α1α2 and the peptides. 25, 26 further studies are needed to determine which model best describes the interaction between lilrb2 and hla-i. here, we determined the structure of four-ig domain lilrb2 and a complex structure of hla-g1 and lilrb1 containing four ig-like domains. for the first time, the hinge region angles between the d2 and d3 domains in lilrb1 and lilrb2 were elucidated and were found to be~60°and~50°, respectively. the arrangements of the four domains in the long axis were determined, and a staggered assembly mode for lilrb1/2 was uncovered. compared with lilrb2, lilrb1 d2d3 displayed more open angles, probably due to the steric hindrance of d3 by w284. in addition, the structure of lilrb1 in complex with hla-g1 provided the first direct structural data supporting the model that d1d2 are responsible for hla-i binding and d3d4 act as a scaffold. accordingly, models for hla-i binding to lilrb1/2 in cis and trans are proposed. the geometries of lilrb1/2 in binding to dimeric and monomeric hla-g1 indicate more accessibility of lilrb1/2 to the dimeric form of hla-g1, leading to the transduction of more inhibitory signals. the structural data reported here could help to better understand the structures and functions of the lilr family, which would aid in the design of immune regulators and support immune interference. to determine the role of d3d4 in the hla-i interaction, we solved the crystal structure of lilrb1 with four ig domains in complex with hla-g1 incorporating riiprhlql (rl9 from histone h2a) at a resolution of 3.3 å (table 1) . two independent copies of the complex were found in the asymmetric unit, with a root mean square deviation (rmsd) of 0.479 å (for 526 cα atoms, without atoms from lilrb1 d3d4), and we chose the copy with the better electron density map for further analysis (fig. s1 ). the overall structure of the complex demonstrates that the angle between hla-g1 and lilrb1 is~60°. in addition, the lengths of the four ig-like domain lilrb1 and hla-g1 are~100 å and~68 å, respectively (fig. 1a) . compared to previous data, each of the four extracellular domains of hla-g1-complexed lilrb1 maintain similar folds, with an rmsd of 0.28-0.59 (d1, compared with pdbs 1g0x, 1p7q, 1vdg, 1ufu, 3d2u, 4no0, and 5knm), 0.34-0.73 (d2, compared with pdbs 1g0x, 1p7q, 1vdg, 1ufu, 3d2u, 4no0, and 5knm), 0.40 (d3, compared with pdb 4ll9), and 0.41 (d4, compared with pdb 4ll9). 14, [16] [17] [18] 27 although they complex with hla-g1, lilrb1 d1, and d2 have interdomain angles that resemble ligand-free d1d2 (pdb 1g0x),~90°. 27 the angle of the d3d4 hinge region is~60°, similar to a previous report. 25 for the first time, the angle between d2 and d3 is determined to be~60°(the angle in the other copy is~55°) (fig. s1 ). the staggered arrangement of the four domains in the long axis of the molecule is also defined. specifically, the angles between the axes formed by two adjacent domains are~140°(the angle between the axis of d1d2 and d2d3) and 130°(the angle between the axis of d2d3 and d3d4) (fig. 1b) . the assembly of the four extracellular domains of lilrb2 was also illustrated in this study (table 1) . each domain preserves the scaffold in the four ig-like structure, as seen in the d1d2 or d3d4 structures, with rmsds ranging from 0.51 to 1.03 for d1 (compared with pdbs 2gw5 and 2dyp), 0.43 to 0.55 for d2 (compared with pdbs 2gw5 and 2dyp), 0.43 for d3 (compared with pdb 4lla), and 0.49 for d4 (compared with pdb 4lla). 15, 25, 28 the angles between lilrb2 d1d2 and d3d4 are~90°and~60°, where f o and f c are the structure-factor amplitudes from the data and the model, respectively. r free is the r factor for a subset (5%) of reflections that were selected prior to the refinement calculations and were not included in the refinement respectively, similar to previous reports. 15, 25, 28 the angle of the d2d3 hinge region is~50°, which is slightly smaller than its counterpart in lilrb1. the four ig-like domains in lilrb2 are also stacked, similar to lilrb1, at angles of~130°(the angle between the axes of d1d2 and d2d3) and~140°(the angle between the axes of d2d3 and d3d4) (fig. 1c) . structural comparisons of lilrb1/2 binding to different hla alleles similar to previous reports, 14,17 d1 interacts with the hla-g1 α3 domain. both d1 and d2, as well as the interdomain hinge region, form contacts with β2m, which noncovalently associates with the heavy chain of major histocompatibility complex class i molecules. (fig. s2) . notably, lilrb1 includes y38, which is conserved among group 1 lilrs (members that bind to hla-is) and makes contacts with hla-g1 (fig. s3) . the phenyl rings of y38 in d1 and f195 in hla-g1 (hla-g1 y197 at the interface between lilrb2 and hla-g1) probably form π-π stacking interactions and contribute to binding (figs. s2a, s4, and s5a). to date, six lilrb-hla complex structures have been solved. these structures include lilrb1 d1d2 bound to two hla-a2 (presenting two different peptides), 14, 18 hla-f, 17 and ul18 (an hcmv-encoded hla-i homologue); 16 lilrb2 d1d2 in complex with hla-g1; 15 and lilrb1 containing four ig-like domains in complex with hla-g1 in this report. structural alignment along d1d2 shows that hlas form different angles with d1d2, with hla-a2 (pdb 1p7q) 14 and hla-g1 (in this study) differing the most, by 15° (fig. s6 ). hla-a2 (pdb 4no0), 18 ul18, 16 hla-f, 17 and hla-g1 (in complex with lilrb2 d1d2) 15 are interspersed, indicating the plasticity of lilrb1 in associating with hla-i ligands (fig. s6) . among the reported complex structures, lilrb1 associates with ul18 with the highest binding affinity, with a nanomolar k d . 16 consistently, ul18 has the largest quantity of residues that interact with lilrb1. in addition, most of buried surface area, and most of h-bonds and van der waals (vdw) contacts are between lilrb1 d1d2 and ul18 (table s1) . notably, lilrb1 binds to other hla-is with similar binding affinities, which decrease by three orders of magnitude (k d = 2-7 μm) ( fig. 2 and table s1 ). compared with ul18, these complexes have reduced buried surface areas at the lilrb1 interface with hla-is, except lilrb2 in complex with hla-g1. however, the binding of lilrb1 to other hla-is involves fewer contacts than the interactions between lilrb1 and ul18, including both vdw contacts and potential h-bonds, which account for the much lower binding affinities (table s1) . no interactions between lilrb1 d3d4 and hla-g1 further analysis of the complex structure between lilrb1 with four domains and hla-g1 shows that there are no interactions between lilrb1 d3d4 and hla-g1 in one asymmetry unit ( fig. 1 ). however, when symmetry mates were generated to analyze the crystal packing, lilrb1 d4 was observed to interact with another hla-g1 peptide-presenting platform as well as the presenting peptide in the adjacent lattice (fig. 3a) . residue t365 of lilrb1 d4 potentially forms a h-bond with e166 of hla-g1, and a367 interacts with r170 of hla-g1 via hydrogen-bonding interactions. residue s366 binds to the rl9 peptide by forming two hydrogen bonds with the r1 residue of this peptide (fig. 3b) . to test whether this interaction is real or artificial due to crystal structure manipulation, we first evaluated the binding between lilrb1 d3d4 and hla-g1. no interactions were detected between the two molecules in a spr assay (fig. 2) , which is consistent with previous results. 19 we also refolded hla-g1 with five different peptides to assess the effect of the peptide on the association with lilrb1. as indicated in fig. 2 , lilrb1 had similar binding affinities to hla-g1 incorporating various peptides, similar to lilrb1 d1d2, indicating that the peptides probably do not make additional contacts with lilrb1. we further deleted residues 365 tsa 367 in lilrb1, which were observed to interact with hla-g1 in another lattice (fig. 3b ), by either deletion or mutation to aaa or ggg. then, the mutated lilrb1 was expressed on the membrane of hek 293t cells and tested for its interaction with the hla-g1 tetramer with rl9 by flow cytometry. however, no significant differences were observed between wild-type and any mutated lilrb1 (fig. 3e) . then, we changed the strategy from reducing the interaction mediated by lilrb1 to increasing the binding, if any, between d3d4 and hla-g1. we introduced hydrophilic residues into d3d4 to form an electrostatic network with hla-g1 and the peptides (fig. 3c, d) . nevertheless, the mutants failed to bind to the hla-g1 tetramer. thus, we conclude that the observed interaction resulted from artificial results and that no interaction exists between lilrb1 d3d4 and hla-g1. the domain interface between lilrb1 d2d3 is formed by interactions between the c-c′ loop, f strand and f-g loop of d3 with the a strand, a-b loop of d2 and connecting region between d2 and d3 (g-a loop) (fig. 4a, b) , respectively. compared to the accessible buried area in the interdomain interface between d1d2 (1233.5 å 2 ) and d3d4 (1120.6 å 2 ), the value of d2d3 was ). in addition, s282 in the d3 f-g loop interacts with w170 of the f strand of d2. r240, located in the d3 c-c′ loop, interacts with multiple residues in d2 (s105, a106, and q107), forming a small hydrophilic patch. (fig. 4b) . due to the low electron densities of three amino acids in the loop linking d2 and d3, the second structure of this region is invisible, indicating the flexibility of this region. compared with lilrb1, more residues and interactions are involved in the lilrb2 d2 and d3 interface. lilrb2 238 erdl 241 and q243, which are in the c-c′ loop, interact with 104 saqp 107 and 109 pv 110 , which are located on the a strand and a-b loop (figs. 4c, d and s5c), respectively. this region confers 94 vdw contacts. on the other patch, the d3 f-g loop, constituted by 278 nlssecsa 285 , makes 75 and 97 vdw contacts with the d2 e-f loop ( 165 pnrrw 169 ) and the interdomain loop linking d2 and d3 ( 194 lvpg 197 ), respectively (fig. 4c, e) . d3 q243 interacts with e192. l241 contacts d2 p109 and v110. in addition, residues r230, v232, and y234 on the d3 c strand contribute to the d2 and d3 interaction through binding to p109, e192, and l194 (fig. 4d) . accordingly, the stronger interaction between lilrb2 d2 and d3 leads to the more compact assembly of d2d3, resulting in a buried area of 1475.4 å 2 in the d2d3 interface. notably, in the center of the d2d3 contact region, the aromatic w284 in lilrb1 is substituted by the less bulky c283 in lilrb2 (fig. 4f) . compared with c283 in lilrb2, w284 contributes more to the hydrophobic interaction. however, it may also confer more steric hindrance to the strands of f and c in d3. as shown in fig. 4f , strand f, together with the loop extending from lilrb1 strand f (f-g loop), shifts away from d2 by~2.5 å and 6.5 å compared with lilrb2. concomitantly, lilrb1 strand c moves away from d2 by~5 å. because of the interaction between antiparallel strands of c and c′, strand c′ and the loop linking the two strands shift away from d2 by~7 å and~2.5 å in lilrb1 (fig. 4f) , respectively. taken together, the residues in the f-g loop, especially those in the center of the interface (w284/c283 in lilrb1/2), affect the conformation of adjacent secondary structural elements, including the c-c′ loop, causing their shift. these shifts in lilrb1 may be synergized, leading to the reduced interaction between d2 and d3 in lilrb1 and, consequently, the more open angle than in lilrb2. structural predictions for other lilr family members due to sequence similarities, domains in other lilr members will fold into tertiary structures similar to those of the ig domains, such as the domains in lilrb1 and lilrb2. thus, the structures of extracellular regions of other lilr proteins, especially those that have four ig-like domains, will depend on the nature of the interfaces between adjacent domains. similar to the previous results of the discrete domains, most residues responsible for d1d2 interactions are conserved among lilrs 27 (fig. s3) . notably, bulky w185/w184 are located in the center of the d1d2 interface of lilrb1/2 (fig. 5a, d) . in addition, h141 in the d2 c-c′ loop interacts with the main chain of p12 (7 vdw contacts with 4.5 å cutoff) and likely helps to stabilize the interaction, thereby stabilizing the angle between d1 and d2 (fig. 5a) . the same pair of residues (h140 and p12) is conserved in lilrb2, but the d2 c-c′ loop in this receptor was invisible due to the low electron density (fig. 5d) . interestingly, w185 and p12 are conserved among the four ig-domains containing lilrs, while h141 is present in group 1 lilrs (fig. s3) . however, the equivalent position is substituted by l141 in group 2 lilrs, which is unable to associate with hla-is (fig. s3) . l141 might hydrophobically interact with the pyrrolidine ring of p12. these data indicate that the angle of d1d2 in four-ig domain lilrs might be conserved to be~90°. as indicated in fig. s3 , the majority of residues in the interface between d2 and d3 are conserved among lilrs. specifically, all four ig-domains containing lilrs include w at the same position as w284 in lilrb1, except lilrb2 (c283) (figs. 5b, e and s3 ). in lilrb1, r240 in the c-c′ loop potentially forms 21 vdw contacts as well as two potential h-bonds with the side chain of q107 and the main chain of a106, respectively (fig. 5b) . similar pairs of h-bonds between r239 and q106 and a105 are present in lilrb2 (fig. 5e) . r239 interacts with most of the residues in strand a (51 vdw contacts) and contributes more interactions than r240 in lilrb1 (fig. 5d) . these strong interactions between the d3 c-c′ loop and the d2 a strand might partially explain why d2d3 has a more closed conformation than d1d2, although both interfaces possess an aromatic w in the center of the contact region. although r240 in lilrb1 is conserved in most lilrs (except lilrb5 with h235 and lilra4 with d235), lilrb1 q107 is substituted by l in four other ig-domain lilrs (fig. s3) . the equivalent r240 might still interact with the main chain atoms of the residues in the a strand, such as the equivalent a106 in lilrb1, but the binding strength decreases. this interaction more closely resembles the counterpart of the d1d2 interface (h141 with the main chain of p12). thus, the interdomain angle of d2d3 in lilra1, a2, a3, a6, and b3 seem to be similar to that of the lilrb1/2 d1d2 interface (~90°). in lilrb5 and a4, due to the substitution of residues with short side chains, the interaction between d2 and d3 might further decrease and exhibit the largest angles. in the interface between d3 and d4, less bulky l385/384 is present in the center of the contact region in lilrb1/2 (fig. 5c, f) . fig. 3 assessment of the interaction between lilrb1 d3d4 and hla-g1. a one symmetry mate of lilrb1 in complex with hla-g1 was generated. lilrb1 d4 was observed to interact with hla-g1 in the adjacent lattice. the region highlighted by the black square is enlarged in b. b the interaction network between d4 and the hla-g1 peptide-presenting platform as well as the peptide. c, d to increase the interaction between d3d4 and hla-g1, hydrophilic residues were introduced. 365 tsah 368 were mutated to rddg (c) or reeg (e). the probable conformations of the mutated residues are shown in c and d. f evaluation of the interaction between tetrameric hla-g1 incorporating rl9 and the indicated molecules was performed. 365 tsa 367 was deleted from lilrb1 either by deletion or mutation to aaa or ggg. rddg or reeg were used to substitute 365 tsah 368 in lilrb1 d3d4. the indicated lilrb1 proteins were transiently expressed on the membrane of hek 293t cells and tagged with egfp at its c-terminus. then, the cells were sequentially collected and incubated with tetrameric hla-g1 (rl9) and streptavidin-apc. egfp-expressing cells were gated, and the proportion of apc + cells was analyzed. the value of the column is the mean of triplicates (n = 3), and the bar represents the sem value. the assays were independently performed twice. in panels a-e, green, gray, magenta, and cyan indicate the hla-g1 heavy chain, β2m, the peptide, and lilrb1, respectively similarly, all of the lilrs display conservation at the interacting positions (fig. s3) , indicating that they form similar hydrophobic associations in the center of the contact region. in addition, l also confers less steric hindrance than w. thus, other lilrs might adopt similar angles in d3d4 as lilrb1/2 (~60°). notably, based on the above analysis, strong interactions are present in each adjacent domain. thus, as observed in the structure of the complex (figs. 1a and 6a) , hla-g1 exclusively binds to d1d2 of lilrb1 and d3d4 of this receptor is unlikely to bend or rotate to interact with the peptide or peptide-presenting c an overview of the binding interface between lilrb2 d2, shown in surface representation, and d3, shown in cartoon representation. the contacting residues (indicated by numbers 1 and 2) are further delineated in d and e. d, e sites of interaction between d2 and d3, indicated with numbers 1 and 2 in c. f the variation of the angles between d2 and d3 in lilrb1 and lilrb2. the two structures are aligned with respect to d2. stick representations of c283 in lilrb2 and w284 in lilrb1 are shown. the structures of lilrb2 are displayed at 50% transparency platform (fig. 6b, c) . similarly, lilrb1/2 likely adopts the same binding mode to interact with other hla-is (fig. s7) , and d3d4 does not contact hla-is. cis and trans interactions between lilrb1/2 and hla-g1 the interactions between lilrb1/2 and hla-is are reported to be either cis or trans interactions. in addition, hla-g1 could form homodimers on cells (pdb 2d31). 29 comparison between monomeric and dimeric hla-g1 indicates that there are no significant conformational changes at lilrb1/2 binding sites (fig. s8) . thus, dimeric hla-g1 can interact with either one or two molecules of lilrb1/2 expressed on other cells through a trans interaction (fig. 6a, d) . as assessed by the spr assay, dimeric hla-g1 presenting variable peptides displayed similar binding strengths for lilrb1 and lilrb2 (with four ig-like domains or d1d2), indicating that the peptides seem to have no effect on the interactions. furthermore, no detectable interactions were observed between d3d4 and dimeric hla-g1 (fig. s9) . of note, for a cis interaction, the c-terminus of d4 of lilrb1/2, which binds to monomeric hla-g1, needs to be elongated by~100 å (fig. 6e) . by contrast, due to dimer formation, the binding sites on dimeric hla-g1 are tilted and the distance of the c-terminus of interacting lilrb1/2 d4 is~70 å, which is shorter than that of monomeric hla-g1 (figs. 6f and s10) . the lilr family is a group of immune receptors that regulate immune reactions and maintain immune hemostasis. [1] [2] [3] structural studies help to understand the functions of these immune receptors. previous studies indicate that d3d4 may bend or turn to interact with the peptide and α1α2 of hla-is. 25, 26 however, the structure of the hla-g1 complex with four-ig domain lilrb1 reported here indicated that there were no interactions between d3d4 and hla-g1. consistently, we did not observe an association between d3d4 (either in lilrb1 or lilrb2) and the respective hla-g1 incorporating five different peptides. the binding affinities of the four-ig domain lilrb1/2 to hla-g1 with different peptides did not display obvious variation. importantly, the structural studies reported here showed that residues in the interface of d2d3 formed both hydrophobic and hydrophilic interactions and thereby stabilized the interdomain angle of d2d3. this stabilization most likely prevents d3 from rotating anticlockwise with respect to d2 to allow either d3 or d4 to make contacts with hla-is. in addition, the results suggest that d3 is prevented from rotating along the d1d2 axis to force the acute angle of d3d4 to face hla-is and interact with them. according to sequence alignment and interdomain interface analysis, all of the lilrs, especially those in group i, probably adopt similar angles in d1d2 and d3d4 as lilrb1/2. although d2d3 is proposed to be relatively flexible in lilra1, a2 and a3, they still might have a similar interdomain angle as lilrb1/2 d1d2 (~90°). thus, they are unlikely to utilize d3d4 to interact with the peptide or hla-i α1α2 domains. the underlying mechanisms by which the mutated peptides that presented by hla-is or polymorphisms in the hla-is α1 or α2 domains modify the immune reactions through lilrb2 require further study. lilrbs are believed to function by binding to hla-is in both cis and trans. 5, 6, 9, 10, 30 the question is the interaction modes by which lilrbs bind to hla-i in cis and trans, especially for hla-g1, which also forms dimers on cells. structural data in this study indicate that through trans interaction, one hla-g1 monomer binds to one lilrb1/2 molecule. one hla-g1 dimer simultaneously interacts with two lilrb1/2 molecules and amplify lilrb-related inhibition signals. notably, the stalk regions (segments from the c-terminus of d4 to the transmembrane domain) of lilrb1 and lilrb2 are relatively longer (consisting of 44 and 43 residues, respectively) than other type i transmembrane proteins, such as hla-i molecules. these stalk regions in the two receptors are full of g, s, p, and t residues, indicating their flexibility. taking the 48 kvehsdl 54 loop in β2m as an example, the main chain of the seven residues is~21 å. theoretically, these 43-44 residues could constitute a loop limited to 120 å, which might support the return and insertion of the c-terminus of lilrb1 and lilrb2 into the membrane. due to the different configurations of hla-g1 between monomers and dimers, interacting lilrb1/2 in cis exhibit different angles relative to the membrane. consequently, the distance of lilrbs' c-terminus to the membrane is different in the two binding modes (~100 å and 70 å, respectively), indicating that hla-g1 dimers are more accessible for lilrb1/2 binding than hla-g1 monomers. this difference might also explain why dimers are the major functional form of hla-g1 and lead to much stronger inhibition of lilrb than monomers. lilrb1 functions as a checkpoint through its interaction with hla-is, relying on the invariant β2m subunit. lilrb1 cannot fig. 6 models of full-length lilr binding to hla-i in cis and trans. the green, yellow, red, and blue colors indicate the four domains in lilrb1/2. the cyan, gray, and magenta represent the hla heavy chain, β2m, and peptide, respectively. a trans interaction between lilrb1/2 and hla-i. b the proposed interaction mode in which d3 and d4 bend to interact with hla-i and the peptide. c the proposed interaction mode in which the acute interchain angle between d3 and d4 faces the hla-i and peptide, allowing the interaction between d3d4 and the peptide as well as hla-i. in b and c, d3d4 in the complex structure between lilrb1 and hla-g1 are displayed at 50% transparency, while those with no transparency indicate d3d4 in the two proposed models. 25, 26 d trans interaction between lilrb1/2 and an hla-g1 dimer. e cis interaction between lilrb1/2 and hla-i. f cis interaction between lilrb1/2 and an hla-g1 dimer interact with β2m-free heavy chains, in contrast to lilrb2. further structural studies have revealed that d1d2 of lilrb1 employs interdomain loops to interact with β2m allocated to different hla-is. the lilrb1 with four ig-like domains reported in this study was shown to use a similar mode to interact with hla-g1. these structural data suggest that the interdomain region could be an important target to block the interaction with β2m, thereby inhibiting the binding of lilrb1 by hla-is. in conclusion, we reported the structures of lilrb1 and lilrb2 with four ig-like domains and observed how the four domains are arranged. the complex structure reported here provides the first direct evidence that d1d2 is responsible for all of the interactions with hla-is, while d3d4 is not the reason to explain why hla-is that carry a single residue substitution in their α1α2 domains or in the presented peptides display variable binding affinities to lilrb2. the assembly of four other ig-domain lilrs was speculated based on sequence and structural comparisons. this structural information will help to better understand the structures of the lilr family and therefore their functions. the coding regions for each protein, including lilrb1 d1d2 , lilrb1 d3d4 (199-394), lilrb2 d1d2 , and lilrb2 d3d4 (197-393), were cloned into pet21a. four extracellular domains of both lilrb1 (1-394) and lilrb2 (1-393) were cloned into the pfastbac1 plasmid and subjected to insect cell expression. an n-terminal gp67 signal peptide and a c-terminal 6× his were added to facilitate protein secretion and purification. the extracellular domain of hla-g1 was cloned into pet21a. the biotinylation tag (glndifeaqkiewhe) was linked to the 3′ end of hla-g1. in addition, c42s was also prepared based on the pet21a expression vector. to evaluate the interaction between four-ig domain lilrb1 or lilrb1 d3d4 and hla-g1, the coding regions for each protein, including lilrb1 (1-627) and lilrb1 d3d4 (199-627) with its original n-terminal signal peptide (mtpiltvliclglslgprthvqa), were cloned into the pegfp-n1 vector to produce proteins fused with egfp expressed on the cell membrane. protein expression and purification recombinant proteins were refolded and purified as previously reported. 25, 31, 32 briefly, inclusion bodies including lilrb1 d1d2, d3d4, lilrb2 d1d2, d3d4, wild-type hla-g1 heavy chains with a biotinylation tag, c42s mutants with or without the biotinylation tag and β2m were isolated from cell pellets by sonication and washed with washing buffer (0.5% triton x-100, 50 mm tris-hcl, ph 8.0, 300 mm nacl, 10 mm edta, 10 mm β-mercaptoethanol (β-me), and 0.1% nan 3 ) and resuspension buffer (50 mm tris-hcl, ph 8.0, 100 mm nacl, 10 mm edta, 10 mm β-me, and 0.1% nan3) and then dissolved overnight in a denaturing buffer (8 m urea, 50 mm tris-hcl ph 8.0, 100 mm nacl, 10 mm edta, 10% (v/v) glycerol, and 10 mm dtt). then, the proteins were refolded by dilution against a refolding buffer (100 mm tris, ph 8.0; 400 mm l-arginine; 5 mm edta-na; 5 mm glutathione; 0.5 mm glutathione disulfide). for hla-g1 refolding, inclusion bodies containing β2m, peptides, and inclusion bodies containing the heavy chain were sequentially added. the peptides used for hla-g1 refolding include rl9 (riiprhlql from histone h2a), ml9 (mqpthpirl from hs1 protein), rll9 (rlpkdfril, unknown), kll9 (klpaqfyil, unknown), and kgl9 (kgppaaltl from cytokine receptor). after 12 h of slow stirring at 4°c, the refolded protein was concentrated and changed to 20 mm tris-hcl (ph 8.0) and 150 mm nacl buffer and further analyzed by gel filtration (superdex ® 200 column, ge healthcare). both lilrb1 (1-394) and lilrb2 (1-393) were expressed and purified as previously reported. 33 briefly, recombinant bacmids were prepared and then transfected into sf9 cells to obtain a baculovirus stock, which was then used to infect high5 cells for protein expression. target proteins in the supernatant were sequentially collected, affinity purified by a histrap hp column (ge healthcare), and further purified via gel filtration (superdex ® 200 column, ge healthcare). hla-g1 with the biotinylation tag was refolded as described above. after purification, hla-g1 was biotinylated using the bira enzyme (avidity). apc-tagged streptavidin (biosource international) was added at a molar ratio of 4:1 to refolded hla-g1, with the biotin-streptavidin interaction causing four hla-g1 monomers to bind to streptavidin and create a tetramer. the hla-g1 tetramers were then used to stain cells. binding analysis using spr soluble lilrb1 and lilrb2 expressed in insect cells and refolded d1d2 and d3d4 of both lilrb1 and lilrb2 were exchanged into hbs-ep buffer (10 mm hepes, ph 7.4, 150 mm nacl, 0.005% tween 20) . monomeric hla-g1 (c42s) or dimeric hla-g1 (wild type), which contained the biotin tag at the c-terminus and incorporated the indicated peptide (rl9, ml9, rll9, kll9, and kgl9), was immobilized on an sa chip to~400 response units. full-length or discrete lilrb proteins were serially diluted and injected. specifically, both lilrb1 and lilrb2 were loaded at concentrations of 0.156, 0.3125, 0.625, 1.25, 2.5, 5, and 10 μm. both lilrb1 d1d2 and lilrb2 d1d2 were injected at concentrations of 0.156, 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 μm. both lilrb1 d3d4 and lilrb2 d3d4 were loaded at concentrations of 0, 10, and 100 μm. the binding responses were recorded. spr experiments were performed using a biacore ® 3000 system (biacore). k d values were calculated using the model of steadystate affinity. data were analyzed by biaevaluation (biacore) and sigmaplot 10. flow cytometry hek 293t cells transiently transfected with the pegfp-n1-lilrb1 and pegfp-n1-d3d4 plasmids and indicated mutants (tsa deletion, tsa-aaa and tsa-ggg in lilrb1; tsah-reeg and tsah-rddg in lilrb1 d3d4) were used for the binding test. at 48 h post transfection, cells (2 × 10 5 ) were collected and stained with tetrameric hla-g1 incorporating rl9 (riiprhlql) at a final concentration of 1 μg/ml on ice for 30 min. after washing, streptavidin-conjugated apc (1 μg/ml) was added. then, the cells were subjected to analysis using a bd facscalibur. only egfp + cells were gated, and the fluorescence shift for apc was analyzed. flowjo 7.6 was used for data analysis. crystallization, data collection, and structure determination crystallization trials were set up with commercial crystallization kits (hampton research and molecular dimensions) using the sittingdrop vapor-diffusion method. normally, 1 μl protein at the corresponding concentrations was mixed with 1 μl reservoir solution. purified hla-g1 (rl9) was mixed with lilrb1 at a molar ratio of 1:1. crystals with reflection at high resolutions were obtained under the condition of 0.2 m imidazole malate, 15% w/v peg 4000, and ph 6.0 at a concentration of 5 mg/ml at 4°c. diffractable crystals of lilrb2 were finally obtained under the condition consisting of 0.1 m tris, ph 8.5, and 25% v/v tert-butanol at a concentration of 10 mg/ml at 4°c. crystals were cryoprotected in reservoir solution containing 20% (v/v) glycerol and flash-frozen in liquid nitrogen. diffraction data were collected at shanghai synchrotron radiation facility (ssrf) bl17u and processed with hkl2000. 34 the structures were determined by molecular replacement using amore in the ccp4 suite with the coordinates of lilrb2 d1d2 and hla-g1 (pdb 2dyp), lilrb1 d3d4 (pdb 4ll9), and lilrb2 d3d4 (pdb: 4lla) as search probes. the atomic models were completed with coot 35 and refined with phenix.refine in phenix, 36 and the stereochemical qualities of the final models were assessed with molprobity. 37 data collection, processing, and the refinement statistics are summarized in table 1 . the accession numbers for the atomic coordinates and diffraction data reported in this paper are pdb 6aed (crystal structure of lilrb2) and 6aee (crystal structure of lilrb1/hla-g1 complex), respectively. the lilr family: modulators of innate and adaptive immune pathways in health and disease regulation of immune and neural function via leukocyte ig-like receptors leukocyte immunoglobulin-like receptors in human diseases: an overview of their distribution, function, and potential application for immunotherapies a common inhibitory receptor for major histocompatibility complex class i molecules on human lymphoid and myelomonocytic cells human inhibitory receptors ig-like transcript 2 (ilt2) and ilt4 compete with cd8 for mhc class i binding and bind preferentially to hla-g inhibitory receptors cd85j, lair-1, and cd152 down-regulate immunoglobulin and cytokine production by human b lymphocytes ilt2/hla-g interaction impairs nk-cell functions through the inhibition of the late but not the early events of the nk-cell activating synapse inhibitory receptors sensing hla-g1 molecules in pregnancy: decidua-associated natural killer cells express lir-1 and cd94/nkg2a and acquire p49, an hla-g1-specific receptor cis binding between inhibitory receptors and mhc class i can regulate mast cell activation inhibitory immunoglobulin-like receptors lilrb and pir-b negatively regulate osteoclast development overexpression of cd85j in tnbc patients inhibits cetuximabmediated nk-cell adcc but can be restored with cd85j functional blockade engagement of mhc class i by the inhibitory receptor lilrb1 suppresses macrophages and is a target of cancer immunotherapy inhibitory leukocyte immunoglobulin-like receptors: immune checkpoint proteins and tumor sustaining factors crystal structure of hla-a2 bound to lir-1, a host and viral major histocompatibility complex receptor structural basis for recognition of the nonclassical mhc molecule hla-g by the leukocyte ig-like receptor b2 (lilrb2/lir2/ilt4/cd85d) structure of ul18, a peptide-binding viral mhc mimic, bound to a host inhibitory receptor human leukocyte antigen f presents peptides and regulates immunity through interactions with nk cell receptors the antigenic identity of human class i mhc phosphopeptides is critically dependent upon phosphorylation status the inhibitory receptor lir-1 uses a common binding interaction to recognize class i mhc molecules and the viral homolog ul18 hla-b*35-px-mediated acceleration of hiv-1 infection by increased inhibitory immunoregulatory impulses effect of a single amino acid change in mhc class i molecules on the rate of progression to aids motif of hla-b*3503 peptide ligands a viral ctl escape mutation leading to immunoglobulin-like transcript 4-mediated functional inhibition of myelomonocytic cells mutational escape in hiv-1 ctl epitopes leads to increased binding to inhibitory myelomonocytic mhc class i receptors crystal structures of the two membrane-proximal ig-like domains (d3d4) of lilrb1/b2: alternative models for their involvement in peptide-hla binding the emerging role of leukocyte immunoglobulin-like receptors (lilrs) in hiv-1 infection crystal structure and ligand binding properties of the d1d2 region of the inhibitory receptor lir-1 (ilt2) crystal structure of lir-2 (ilt4) at 1.8 å: differences from lir-1 (ilt2) in regions implicated in the binding of the human cytomegalovirus class i mhc homolog ul18 efficient leukocyte ig-like receptor signaling and crystal structure of disulfide-linked hla-g dimer a common inhibitory receptor for major histocompatibility complex class i molecules on human lymphoid and myelomonocytic cells crystal structure of myeloid cell activating receptor leukocyte iglike receptor a2 (lilra2/ilt1/lir-7) domain swapped dimer: molecular basis for its non-binding to mhc complexes crystal structure of leukocyte ig-like receptor lilrb4 (ilt3/lir-5/ cd85k): a myeloid inhibitory receptor involved in immune tolerance crystal structure of the swine-origin a (h1n1)-2009 influenza a virus hemagglutinin (ha) reveals similar antigenicity to that of the 1918 pandemic virus processing of x-ray diffraction data collected in oscillation mode coot: model-building tools for molecular graphics phenix: a comprehensive python-based system for macromolecular structure solution molprobity: more and better reference data for improved allatom structure validation we thank the staff of bl17u beamline at ssrf. we are grateful to zheng fan from the institute of microbiology chinese academy of sciences (cas) for technical assistance with the spr experiments. the online version of this article (https://doi.org/10.1038/s41423-019-0258-5) contains supplementary material.competing interests: the authors declare no competing interests. key: cord-000224-2lz03oqb authors: porter, kristen a.; kelley, lauren n.; george, annette; harton, jonathan a.; duus, karen m. title: class ii transactivator (ciita) enhances cytoplasmic processing of hiv-1 pr55gag date: 2010-06-24 journal: plos one doi: 10.1371/journal.pone.0011304 sha: doc_id: 224 cord_uid: 2lz03oqb background: the pr55(gag) (gag) polyprotein of hiv serves as a scaffold for virion assembly and is thus essential for progeny virion budding and maturation. gag localizes to the plasma membrane (pm) and membranes of late endosomes, allowing for release of infectious virus directly from the cell membrane and/or upon exocytosis. the host factors involved in gag trafficking to these sites are largely unknown. upon activation, cd4+ t cells, the primary target of hiv infection, express the class ii transcriptional activator (ciita) and therefore the mhc class ii isotype, hla-dr. similar to gag, hla-dr localizes to the pm and at the membranes of endosomes and specialized vesicular mhc class ii compartments (miics). in hiv producer cells, transient hla-dr expression induces intracellular gag accumulation and impairs virus release. methodology/principal findings: here we demonstrate that both stable and transient expression of ciita in hiv producer cells does not induce hla-dr-associated intracellular retention of gag, but does increase the infectivity of virions. however, neither of these phenomena is due to recapitulation of the class ii antigen presentation pathway or ciita-mediated transcriptional activation of virus genes. interestingly, we demonstrate that ciita, apart from its transcriptional effects, acts cytoplasmically to enhance pr160(gag-pol) (gag-pol) levels and thereby the viral protease and gag processing, accounting for the increased infectivity of virions from ciita-expressing cells. conclusions/significance: this study demonstrates that ciita enhances hiv gag processing, and provides the first evidence of a novel, post-transcriptional, cytoplasmic function for a well-known transactivator. hiv polyprotein gag serves as a scaffold to promote assembly of progeny virions at cellular membranes [1] and recruits components of the vesicular protein sorting pathway to facilitate virus budding [2, 3, 4] . concomitantly, the virally encoded protease begins to cleave gag, which is required for complete virion maturation and infectivity [5, 6, 7] . gag proteins can be detected at both the pm and the membranes of endosomes among different cell types, suggesting that budding is not limited to one cell-type specific locale [8, 9, 10, 11, 12, 13, 14, 15, 16] . further, host factors which participate in targeting gag trafficking to particular membranes are largely unknown. as gag and infectious virus can originate from two cellular locations, two models for gag trafficking have emerged. the first model proposes that following synthesis, gag traffics to endosomal membranes, and upon exocytosis is deposited on the pm, where it serves as the site for productive virus assembly [14, 17] . the second model proposes that gag is first trafficked to the pm, where virus assembly occurs, and then excess gag is internalized to intracellular compartments [14, 18, 19, 20] , that serve as sites of productive virus assembly [15, 21] . mhc class ii heterodimers follow a similar trafficking route, appearing at both the pm and specialized multivesicular bodies (mvbs) called mhc class ii containing compartments (miics) [22] . mhc class ii is utilized by antigen presenting cells (apcs) to present exogenous processed antigen to cd4+ t cells [22, 23, 24] . mhc class ii genes, including: hla-dr, -dp and -dq and the accessory molecules, invariant chain (ii) and hla-dm, are transcriptionally activated by the class ii transactivator (ciita), the global regulator of coordinate class ii mhc gene expression [25, 26] . as ciita is induced in cd4+t cells upon activation, these cells express mhc class ii [27, 28] . upon synthesis, hla-dr heterodimers are assembled in the er and the immature complex (hla-dr+ ii) travels through the secretory pathway to miics, where the specialized hla-dm chaperone loads the hla-dr heterodimer with peptide [22, 29, 30] . interestingly, both immature and mature forms of hla-dr can be found at the pm and can be subsequently internalized to miics due to a di-leucine motif in the cytoplasmic tail of ii (immature hla-dr) and a dileucine motif and/or ubiquitination of conserved lysine residues within the hla-dr b chain (mature hla-dr), respectively [22, 29, 31, 32, 33, 34, 35, 36] . therefore, a connection between hla-dr and gag trafficking would not be surprising as both have an alternative route to intracellular compartments by way of the pm. indeed, expression of hiv-1 nef, vpu and gag have been shown to alter hla-dr trafficking [37, 38, 39, 40] . in addition, hla-dr is preferentially acquired on the viral envelope of budding virions, which enhances virion infectivity and may play a role in bystander apoptosis of t lymphocytes [41, 42, 43] . therefore, hla-dr localization at virus assembly sites is not unexpected. finzi et al. (2006) addressed the contribution of mhc class iiinduced miic formation to pr55gag trafficking by monitoring virus budding in the presence of transiently expressed hla-dr heterodimers [8] . in hek-293t cells expressing hla-dr, there was a marked redistribution of gag to intracellular compartments, and reduced virus release at the pm; mature virus budded into hla-dr containing multivesicular bodies and was retained [8] . we hypothesized that recapitulating endogenous expression of the entire class ii antigen presentation pathway in producer cells via expression of ciita would restore infectious virus release and provide a more physiologically relevant model for hiv-1 assembly studies. as expected, stable or transient expression of ciita did not induce intracellular retention of gag. however, hla-dr induced gag retention could not be alleviated by transient co-expression with hla-dm and/or ii, or ciita-mediated upregulation of the recycling endosome gtpase, rab4b [44] . further, mutating ubiquitinatible lysine residues or complete truncation of the cytoplasmic tails of the hla-dr a and b chains did not restore virus release. rather, limiting the amount of hla-dr dna transfected into cells restored virus release and alleviated gag retention. curiously, ciita expressing cells produced virus that was significantly more infectious than ciita deficient cells, and this was independent of the class ii antigen presentation pathway. ciita enhances infectivity of virions from producer cells through a novel function, by improving maturation through an increase in viral protease-dependent gag processing. using a panel of ciita mutants, we demonstrate that cytoplasmic ciita increases gag-pol polyprotein levels. overall, our work reveals a novel cytoplasmic, post-transcriptional function of ciita, which is expressed upon t cell activation and is constitutively expressed in macrophages and dendritic cells, all known targets of hiv infection. transient expression of the class ii heterodimer, hla-dr, induces intracellular accumulation of gag and a marked reduction in extracellular virus release from producer cells [8] . this previous study focused on hla-dr in the absence of ii and hla-dm, critical components of the antigen processing and presentation pathway which influence mhc class ii trafficking. to determine if expression of the complete mhc class ii pathway could overcome gag and virus retention we used ciita to coordinately express these genes. cells were either transiently (pcciita) or stably (a293t-ciita) transfected with ciita ( figure s1 ) and hiv-1 nl4-3 dna, and gag localization was monitored by immunofluorescence and compared to cells transiently expressing the hla-dr a and b heterodimers (pcdrab1b5), or vector-only (pcdna3.1). as expected, a weak but uniform gag signal was present in vector-only transfected cells (figure 1aa ) and transient hla-dr expression induced a marked redistribution of gag as indicated by a dense, punctuate gag staining pattern (figure 1ad ). conversely, gag accumulation was not observed when ciita was either transiently or stably expressed (figure 1ab or c, respectively). therefore, transient expression of hla-dr in producer cells does indeed lead to gag retention, which is not observed in the presence of ciita. however, recapitulation of the mhc class ii pathway in trans (pcdrab1b5+ii+hla-dmab), also resulted in dense accumulation of gag signal (figure 1ae ), suggesting that ciita-mediated coordinate activation of hla-dr, -dm and ii expression is insufficient to overcome gag retention. flow cytometric analysis confirmed these findings, as cells transfected with hla-dr heterodimers and/or co-transfected with some or all of the components of the class ii antigen presentation pathway stained as gag hi , indicating gag accumulation ( figure 1b and c). however, this population was absent in cells transiently or stably expressing ciita ( figure 1b and c) . therefore, absence of gag accumulation in ciita expressing cells is likely not due to its transactivation of the mhc class ii antigen presentation pathway. to assess whether ciita-mediated alleviation of gag retention in class ii expressing cells correlated with restored virus production, virus particle and infectious virus release were measured by p24 elisa and ghost cell infectivity assays, respectively. virus release, both infectious and particle titers) were reduced when cells were transfected with either hla-dr or other components of the mhc class ii antigen presentation pathway ( figure s2 ), confirming a correlation between gag retention and reduced virus titers in the presence of hla-dr, as previously demonstrated [8] . these assays also demonstrate a dramatic increase in infectious virus release from ciita-expressing cells as compared to cells expressing vector only (figure 2a & s2), despite significantly fewer virus particles being released from a ciitaexpressing cell (as measured by pg/ml of p24 in the culture supernatant, figure 2b & s2). therefore, while fewer virus particles are released from a ciita-expressing cell, those particles are significantly more infectious ( figure 2c ). previously, hla-dr incorporation into the envelope of budding virions was demonstrated to enhance virion infectivity [45, 46] . to determine if ciita-enhancement of virion infectivity was due to hla-dr or other components of the class ii antigen presentation pathway, we measured virion infectivity from cells expressing these proteins. however, virions from these cells were just as infectious as virions released from vector only controls, yet not as infectious as virions from cells either stably or transientlyexpressing ciita ( figure 2d ). therefore, ciita enhancement of virion infectivity is independent of the mhc class ii antigen presentation pathway. together, these data suggest ciita has two effects on the hiv replicative cycle in producer cells, both of which are independent of the mhc ii antigen processing pathway; i) it does not induce hla-dr, mediated intracellular retention of gag and ii) it increases the infectivity of hiv virions. ciita has been shown to upregulate expression of rab4b, a small gtpase involved in endocytic recycling [44, 47] . therefore, we hypothesized that ciita via the action of rab4b, could increase gag recycling back to the pm, thereby alleviating hla-dr-mediated gag retention. commercially available rab4 antibodies cannot distinguish between rab4a and 4b. however, krawczyk et al. (2007) demonstrated by chromatin immunoprecipitation that ciita specifically associates with the rab4b and not the rab4a promoter [44] . immunoblotting demonstrated that there is an increase in rab4 in ciita-expressing cells ( figure 3a ), therefore it is likely that this is in the form of rab4b. however, when rab4b and hla-dr were co-expressed in producer cells with the hiv-1 nl4-3 plasmid, it did not rescue intracellular retention of gag ( figure 3b ). in fact, infectious virus release from cells transiently co-expressing rab4b and hla-dr was further reduced, suggesting that rab4b does not alleviate hla-drmediated retention of gag ( figure 3c ). up until this point, we had not co-expressed ciita with hla-dr in producer cells to determine if ciita could alleviate retention of gag and/or restore infectious virus release as would be expected. interestingly, when ciita was transiently expressed with the hla-dr heterodimers in producer cells, gag still accumulated intracellularly and infectious virus production remained reduced ( figure 3b and c, respectively), suggesting that expression of ciita is not sufficient to compensate for hla-drmediated gag retention. however, when hla-dr is endogenously expressed under the control of ciita, retention is alleviated. [8] . ubiquitination of a conserved lysine residue on the hla-dr b chain induces intracellular accumulation of class ii heterodimers in mhc class ii compartments (miics) [35, 36] . thus, the ubiquitination state of hla-dr, might influence gag retention. to address this idea, lys225arg (pcdrabk225r) and lys222/ 225arg (pcdrabk222/225r) point mutations were made in the hla-dr beta chain. despite these mutations, hla-dr expression on the surface of a293t cells was not significantly altered (flow cytometric data not shown), nor was intracellular retention of gag alleviated or infectious virus release restored ( figure 4a and b, respectively). therefore, we attempted to alleviate gag retention and restore infectious virus release, as demonstrated by finzi et al. (2006) [8] , by complete truncation of the cytoplasmic tails of both hla-dr a and b (pcdradcyto and pcdrbdcyto, respectively). however, loss of either cytoplasmic tail (pcdradcytob and pcdrabdcyto), or both cytoplasmic tails (pcdradcy-tobdcyto), from the heterodimer did not alleviate gag retention and even further reduced infectious virus release from these cells ( figure 4a and b, respectively). our results may differ from finzi's because of the differences in cell type, or hla-dr gene alleles, nevertheless they strongly suggest that transient hla-dr expression in producer cells induces gag retention. further, flow cytometry to monitor surface hla-dr expression demonstrates that transient transfection of hla-dr induces an approximate half-log to log-fold increase in hla-dr as compared to cells stably or transiently expressing ciita, respectively ( figure s1 ). therefore, it is possible that hla-dr induced gag retention is a consequence of hla-dr overexpression in this transient system to test this possibility, virus producer cells were transfected with increasing amounts of hla-dr in the presence of hiv-1 nl4-3 dna, and infectious virus release was monitored. as hla-dr expression increased, the level of infectious virus release decreased ( figure 4c ). further, while not statistically significant, there was a positive correlation (r = 0.9954) between gag accumulation (gag hi ) and increasing hla-dr expression ( figure 4d ), suggesting gag retention is likely related to hla-dr overexpression. similarly, when hla-dm, which is structurally similar to hla-dr, was overexpressed in producer cells, gag retention was also induced ( figure 4d ) and infectious virus release reduced (data not shown). as endogenous expression of the class ii antigen presentation pathway by ciita does not induce gag retention and limiting expression of hla-dr or -dm likewise has a limited effect on gag retention in producer cells, these data collectively suggest that overexpression of the class ii pathway alters gag trafficking in producer cells leading to reduced infectious virus release. however, these data do not provide an explanation for the increased infectivity of virions released from ciita-expressing cells. viral protease cleavage of the gag and gag-pol polyproteins is required for virion maturation and infectivity [5, 6] . figure 5a ) [13, 48, 49, 50, 51] . as there is an increase in virion infectivity from ciita-expressing cells, we hypothesized that the processing of gag polyproteins may be enhanced in these cells. as suspected, analysis of cell lysates from equal numbers of hiv-1 transfected a293t-ciita and a293t cells demonstrated a higher ratio of mature cap24 to pr55gag in ciita-expressing cells ( figure 5 b&c), demonstrating that gag processing in the presence of ciita is enhanced. additionally, increased levels of processing intermediates are present in a293t cell lysates. specifically, the p41 (ma-ca-sp1) and p25 (ca+p2) products were increased (figure 5 b&c) in these cells, indicating that gag processing in the presence of ciita is more efficient. gag processing is also enhanced in cell-free virions from cells either transiently or stably expressing ciita, as demonstrated by the increased levels of cap24, and the loss of the p41 and p25 intermediate products relative to pr55gag ( figure 5d&e ). collectively, these results suggest that ciita increases gag polyprotein processing, leading to enhanced production of infectious virions. hla-dr is incorporated into the hiv virion envelope [52] and is transcriptionally activated by ciita, therefore its contribution to gag processing was assessed. virions from hla-dr-expressing cells exhibited less gag processing than virions from ciitaexpressing cells ( figure 5f , bottom panel). when virions from both cell lines were affinity-purified on hla-dr binding columns, gag processing was reduced in those produced in the hla-drexpressing cells as compared to virions from cells which express ciita ( figure 5f , upper panel and 5g), suggesting that hla-dr alone does not contribute to increased gag processing. similar to our previous results, there are fewer processing intermediates present in virions from ciita-expressing cells, suggesting that ciita expression enhances virion infectivity by increasing maturation through more complete gag processing. cleavage of the gag and gag-pol polyprotein is mediated specifically by the virally-encoded protease [53, 54, 55] . we considered that ciita might increase viral protease processing of gag; thus, we examined gag processing and infectious virus release from ciita-expressing hiv producer cells in the presence of a protease inhibitor. if ciita is increasing protease processing, such an enhancement should be overcome by lower doses of the hiv protease inhibitor, lopinavir. as expected, between concentrations of 0.6 and 1.25 nm of lopinavir infectious virus release from ciita-expressing cells was reduced to that of a293t cells treated with vehicle only ( figure 6a ). to determine if the reduced gag processing correlated with decreased infectious virus release, we monitored gag cleavage by western blotting and as expected, between 0.6 and 1.25 nm of liponavir, gag processing in virions from ciita-expressing cells was returned to that of vehicle treated, vector-only control cells ( figure s3 ). these results directly demonstrate that ciita-mediated enhancement of gag processing and infectious virus release is through the hiv protease. the only known function of ciita is as a transcriptional coactivator; therefore, we thought it likely that it drives the expression of a gene which enhances viral protease-mediated gag processing. therefore, we reasoned that expression of ciita mutants which fail to localize to the nucleus should not mediate increased gag processing or enhanced infectious virus release. to test this idea, a panel of ciita mutants (gtp2, a magnesium ion coordination site mutant and gtp3, a guanine ring-binding domain mutant and l1035p, a point mutant in the c-terminal leucine rich repeat domain, which are all defective for nuclear localization and fail to activate hla-dra transcription [56, 57, 58] ), were individually co-expressed with hiv-1 nl4-3 dna, and infectious virus release and gag processing were monitored. interestingly, producer cells expressing these mutants also produced more infectious virus as compared to vector-only control ( figure 7a ) and had increased gag processing in both cell-bound virions and cell-free virions ( figure 7b and figure s4 , respectively). loss of ciita nuclear localization (and therefore coactivation potential), did not hinder increased gag processing and infectious virus release, strongly suggesting that ciita acts cytoplasmically to increase hiv virus maturation independent of its transactivation function. we further evaluated ciita transactivation potential as a mechanism of enhanced virus release utilizing a different model system. nih 3t3 balb/c cells (expressing human p32 to allow for virus like particle production [59] ) expressing ciita and transfected with an ltr-deficient hiv genome construct (pchiv pal),under control of a cmv promoter had a dramatic increase in virus-like particle production as compared to nih 3t3 cells in the absence of ciita ( figure s5 ). this result provides further evidence that ciita enhancement of virus production is independent of its transactivation potential on the hiv ltr. to determine the mechanism by which ciita mediates protease activity in these cells, we analyzed the expression of the gag and gag-pol in ciita-expressing cells. expression of the hiv protease is extremely toxic to cells, and is thus very tightly regulated during virus replication [60, 61, 62] . the viral protease arises from the alternative gag translation product ( figure 5a ), gag-pol which, accounts for only 2-10% of gag polyproteins and results from ribosomal frameshifting at a conserved heptamer ''slippery sequence'' and a secondary stem-loop structure on gag-pol transcripts [51, 63] . therefore, increased viral protease-mediated processing of the gag polyproteins from ciita-expressing cells may be due to an increase in overall viral protease levels, due to increased gag-pol synthesis. to establish the ability of ciita to mediate the enhanced production of hiv protease, cells were transiently co-transfected with either ciita or pcdna and hiv-1 nl4-3 dna, and were treated with 80 nm of lopinavir to inhibit the majority of gag polyprotein processing. gag-pol levels relative to gag were then determined by western blotting. while 1-2% of all gag polyprotein in cell lysates of vector-only cells was in the form of gag-pol, expression of ciita increased this level to almost 5% ( figure 7c and d) , indicating that ciita increases gag-pol levels in virus producer cells. we also measured the potential of the cytoplasmic ciita mutants to increase gag-pol levels relative to gag and, interestingly, gag-pol made up approximately 13, 20 and 15% of all gag polyproteins in gtp2, gtp3 and l1035p -expressing cells, respectively. this result further demonstrates a novel transcription-independent role for cytoplasmic ciita during hiv infection, where ciita enhances gag-pol protease expression, which subsequently enhances virus maturation and infectivity. [16] . we speculated that complete expression of the antigen presentation pathway, through expression of ciita, would alleviate this phenotype in virus producer cells. interestingly, we noticed two phenomena: i) ciita did not induce intracellular retention of gag or impair virus release in producer cells despite expression of hla-dr and ii) the virus released from cells expressing ciita was significantly more infectious. upon investigation of ciita-mediated alleviation of hla-dr-induced gag retention, we found that this effect was not due to the lack of ii or hla-dm (key components of antigen presentation), as had been expected, nor was it a consequence of rab4b expression in these cells. further, when lysine residues in the hla-dr chain were mutated or both of the hla-dr chain cytoplasmic tails were truncated, gag was still retained and virus release inhibited. in addition, when ciita was expressed in conjunction with hla-dr this effect could not be alleviated, suggesting that gag retention is a consequence of hla-dr overexpression. indeed, retention of gag is directly correlated to levels of hla-dr expression. further, we did not observe gag retention when class ii antigen presentation pathway genes were expressed at endogenous levels via ciita expression. overexpression of hla-dm, which is structurally similar to hla-dr also induced gag retention, suggesting that retention of gag is a likely consequence of altered trafficking of overexpressed class ii antigen presentation pathway components rather than a physiologically relevant phenomenon. despite observing similar hla-dr and gag retention/reduced virus release effects as finzi et al.(2006) , our results differed in that we did not observe the requirement for intact hla-dr a and b-chain cytoplasmic tails for the induction of gag retention. in our hands, when the ubiquitinatible lysine residues in the hla-dr b-chain were mutated or the cytoplasmic tails of the hla-dr dimer were completely removed, there was no alleviation of gag retention or virus release. beyond the obvious differences between the previous work [8] and our own (i.e. provirus construct and cell type), our mutant data may differ from theirs because arginine residues were substituted for lysine 215 and tyrosine 220 residues in the hla-dr a and b chain, respectively, in order to ensure stabilization of the truncated hla-dr molecule at cellular membranes. these differences in mutant constructs may potentially explain discrepancies in our results. we also observed that expression of hla-dm was sufficient to induce gag retention and impede virus release from cells. however, finzi et al.(2006) did not observe retention in the presence of hla-dm or hla-do [8] . this difference may be explained by their use of a bicistronic construct for expression of the hla-dm heterodimer; however, this strategy was also used to express hla-dr, which still induced retention [8] . irrespective of these differences, gag retention and loss of virus release correlates with increasing hla-dr expression. these results do not exclude the possibility that hla-dr and gag trafficking may be linked. indeed, we have demonstrated that gag co-immunoprecipitates with hla-dr from ciita expressing cells and this interaction is independent of the cytoplasmic tails of hla-dr (data not shown). further, other hiv proteins may be linked to class ii trafficking. stumptner-cuvelette et al. (2003) and chaudhry et al.(2009) , have independently demonstrated that hiv nef induces internalization of surface class ii in epithelial and monocytic cell lines, respectively [37, 64] . however, we did not observe downregulation of hla-dr from the cell surface, following transfection of the hiv genome into ciita-expressing epithelial cells (data not shown) or hiv infection of ciita-expressing cd4+ t cells (unpublished data). further, gluschankof and suzan demonstrated that expression of gag-pol restored hla-dr presence at the cell surface in the h78-c10.0 line, a t cell clone deficient for surface hla-dr expression [40] . collectively, our work and that of others suggests a link between hla-dr and gag trafficking, where localization may be cell-type dependent. one of the more interesting findings of this study is that while overall viral titers from ciita-expressing cells decrease the infectivity of these particles is significantly enhanced. increased infectivity was due to improved virion maturation in a viral protease-dependent manner. not only was processing to capsid p24 more complete in ciita expressing cells, but vector-only control cells and those only expressing hla-dr contained increased levels of processing intermediates. the presence of higher levels of processing intermediates in virus produced in these cells may help explain the reduced infectivity, as studies in both mlv [65] and hiv [7] demonstrate that partially cleaved gag products act transdominantly to reduce virion infectivity through reduced reverse transcription of viral rna, despite the presence of functional reverse transcription polymerase [7] . next we demonstrated that ciita increased gag processing through the viral protease and evaluated whether this enhancement was a consequence of ciita-mediated transcriptional activation. the ciita l1035p mutant, which fails to translocate to the nucleus [58] , demonstrated increased gag processing and virus release compared to vector-only control, suggesting a cytoplasmic role for ciita in the later stages of the hiv infection cycle. this does not preclude the possibility that an undetectable level of ciita l1035p might translocate to the nucleus. however, no l1035p was detected in the nucleus after a 24 hour treatment with the nuclear export inhibitor, leptomycin b [58] . further, the predominantly cytoplasmic gtp-binding ciita mutants, gtp2 and gtp3, had a similar increase in infectious virus release versus vector-only expressing cells. previously, it was demonstrated that increased gag-pol levels severely impair virion infectivity through disruption of rna genome dimerization [62] . further, hiv protease is known to be toxic to cells as it leads to the production of the novel procaspase 8 cleavage product, casp8p41, which induces apoptosis through loss of mitochondrial membrane potential [66] . therefore, we would expect that virions from ciita-expressing cells would be reduced in titer and infectivity, as there is increased gag-pol and protease. however, while overall viral particle numbers (as measured by p24 elisa) from ciita expressing cells were decreased, the infectivity of these virions was significantly increased when calculated by infectious units per pg p24. interestingly, at 0.6 nm lopinavir, the infectivity of virions from ciita-expressing cells increased over vehicle-treated controls, despite reduced gag processing. further, the mutants of ciita, which produced the highest level of gag-pol, did not demonstrate a linear increase in gag processing or virion infectivity, which may be explained by previous work demonstrateing that increased gag-pol levels impairs viral infectivity [61, 62] . therefore it is likely that ciita is capable of increasing gag-pol levels to a point which can impede virus maturation, albeit not enough to reduce it to levels observed from vector-only expressing cells. therefore, overall infectivity of virions is increased from cells expressing ciita despite an altered gag to gag-pol ratio. future studies should focus on how ciita can increase this ratio without severely impairing virus release as well the novel mechanism by which ciita increases gag-pol levels. preliminary studies in this laboratory suggest that ciita enhancement gag-pol may be due to increased ribosomal frameshifting (data not shown). finally, it is tempting to speculate that ciita, which is expressed upon cd4+ t cell activation and increases viral protease levels, may also contribute to casp8p41-mediated apoptosis, which may link ciita to the decimation of t cells in the galt during primary viremia [67] . thymic epithelial cells [68] , cervical epithelial cells [69] , human colonic epithelial cells [70] and oral keratinocytes (normal human oral epithelial cells) [71] have all demonstrated in vitro the capability of being productively infected with hiv. infection of epithelial cells provides potential in vivo significance to this study, especially considering that thymic epithelial cells constitutively express mhc class ii (and thus ciita) [28] . in addition, most other cells can be stimulated to express ciita in the presence of ifn-c (i.e. a pro-inflammatory environment), which is induced during hiv infection of the galt [72] . overall, this study demonstrates that the function of ciita may be more broad than previously thought. given this previously undescribed role in enhancement of virion maturation, the precise consequences of ciita expression during the hiv replicative cycle may provide rationale for the development of novel antiviral therapeutics. a293t cells [73] were maintained as previously described [74] and ciita-stable a293t cell lines were generated by cotransfecting pvu i linearized pdna3.flag.ciita8 [75] and pcmv4his, a mammalian selection vector which encodes the histidinol dehydrogenase gene under control of the cmv promoter into the cells. stable clones were selected in dmem medium containing 5 mm l-histidinol (sigma-aldrich., st. louis, mo) and cloned by limiting dilution assay. ghost cell medium was supplemented with 0.2 mg/ml g418, 0.1 mg/ml hygromycin b and 1 mg/ml puromycin (sigma) as previously described [76] . using cdna reverse transcribed from a293t-ciita rna, we amplified hla-dra, hla-drb1 and hla-drb5 sequences (genbank accession nos. nm019111, nm002121, and nm002125, respectively), using primers containing forward restriction site xbai and the reverse restriction site hindiii (table s1 ). the haplotyping of hla-dr isotypes expressed by a293t cells was determined by the transplantation immunology lab, albany medical college. primers were designed to include an intact kozak sequence upstream of the translation start site. hla-dra and b5 plasmids were then used for site-directed mutagenesis using primers indicated (table s1 ). transient transfections of all plasmids, including: pdna3.flag.ciita8 [75] , ciita-gtp2 and -gtp3 [56] and ciita -l1035p [58] , p33-143 (coding for the p33 and p35 isoforms of invariant chain-a kind gift provided by dr. eric o. long, niaid), hla-dma and b (pmcfr-pac and pdmb/mcfrkindly provided by dr. lisa k. denzin, sloan-kettering cancer center), egfp-rab4b (kindly provided by dr. marci scidmore, cornell university), and hiv gag-igfp [77] (kindly provided by dr. benjamin chen, mount siani school of medicine) were performed using a 3:1 ratio fugene hd transfection reagent to dna in serum-free media as suggested by the manufacturer (roche, indianapolis, in). virus plasmid dna provided half of total dna used in transfection reactions. nih 3t3 balb/c cells are transfected with 1.5 mg ciita and 4.5 ml fugene hd (7:2 ratio of fugene to dna for optimal cells growth) and selected for with l-histidionol for two weeks and analyzed for mhc ii expressing using fluorescence-activated cell sorter (facs) with pe-conjugated mouse igg2a (cedar lane) against i-a d and clones were selected by limiting dilution. these cells were co-transfected with p32cdna and pchiv pal (which contains all hiv genes except the ltr sequences) at a 2:1 ratio. the p32cdna was donated by the peterlin laboratory [59] . both mhc ii expressing vlps and mhc ii-negative vlps were produced and concentrated 10-fold in a 100 k molecular weight cut-off filter tube (millipore) for 15 minutes at 4000 rpm prior to titering via hiv-1 p24 ca antigen capture assay kit were performed following the manufacturer's instructions (nih aids research and reference reagent program). cytoplasmic rna from 48 hr cultures of each cell type was collected using the rneasy mini kit according to manufacturer's instructions (qiagen, inc., valencia, ca). 1ug of dna-free rna was run out on a non-denaturing gel to ensure equal concentrations of each sample followed by reverse transcription of 1 mg of each rna sample using the iscript cdna synthesis kit, following manufacturer's instructions (biorad, hercules, ca). 50 ng of cdna was used to amplify hla-dm, ciita, gapdh, and ii from each cell type to confirm expression. forward and reverse primers sequences used in rt-pcr experiments indicated in table s1 . cells were analyzed for hla-dr expression as previously described [56] . briefly, cells were incubated with murine clone l243 [78] . densitometric analysis was performed as previously described [80] and the percentage of total hiv-1 a ca p24 (183-h12-5c) [79] reactive bands was used as a measure of gag processing [81] . intracellular gag staining 10 6 cells were permeabilized with intraprep permeabilization reagent (beckman-coulter, fullerton, ca), following manufacturer's instructions. gag was stained with fitc-conjugated fh190-1-1 (beckman-coulter) for approximately 15 m prior to analysis and data interpretation as performed above, with the exception of the percentage of gag+ cells was determined after gating on hla-dr+ cells. virions were purified using the mmacs streptavidin kit (miltenyi biotec inc., auburn, ca.), according to the manufacturer's instructions using biotinylated-l243 (biolegend). ghost assay [82] to determine infectious virus production and p24 elisas to determine virus particle release using the hiv-1 p24 ca antigen capture assay kit were performed following the manufacturer's instructions (nih aids research and reference reagent program). cells were transfected with either pcciita or empty pcdna vector 4.5 hours prior to lopinavir (nih aids research and reference reagent program) or dmso (vehicle-only) treatment at indicated concentrations. virus and cell supernatants were collected approximately 17 hours post-treatment. for determina-tion of p160gag-pol to p55gag ratios, producer cells were transfected with ciita constructs (pccitia, gtp2, gtp3, l1035p) or vector only control (pcdna) and then transfected with pnl4-3 16 hours later, prior to being treated with 80 nm lopinavir. cell lysates were used to monitor the levels of pr55gag to pr160pol via western blotting with hiv-1 a ca p24 (183-h12-5c) after 17 hours. table s1 primers used in this study. hiv-1 gag proteins: diverse functions in the virus life cycle tsg101: hiv-1's ticket to ride tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding hiv gag mimics the tsg101-recruiting activity of the human hrs protein the activity of the protease of human immunodeficiency virus type 1 is initiated at the membrane of infected cells before the release of viral proteins and is required for release to occur with maximum efficiency partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles human immunodeficiency virus (hiv-1) gag processing intermediates trans-dominantly interfere with hiv-1 infectivity major histocompatibility complex class ii molecules promote human immunodeficiency virus type 1 assembly and budding to late endosomal/multivesicular body compartments assembly of infectious hiv-1 in human epithelial and t-lymphoblastic cell lines infectious hiv-1 assembles in late endosomes in primary macrophages more than one door -budding of enveloped viruses through cellular membranes retrovirus budding productive human immunodeficiency virus type 1 assembly takes place at the plasma membrane evidence that productive human immunodeficiency virus type 1 assembly can occur in an intracellular compartment the cell biology of hiv-1 virion genesis endosomes, exosomes and trojan viruses intracellular destinies: degradation, targeting, assembly, and endocytosis of hiv gag vpu and tsg101 regulate intracellular targeting of the human immunodeficiency virus type 1 core protein precursor pr55gag plasma membrane is the site of productive hiv-1 particle assembly endosomal trafficking of hiv-1 gag and genomic rnas regulates viral egress mhc class ii transport at a glance regulation of mhc class ii expression, a unique regulatory system identified by the study of a primary immunodeficiency disease genetic control of mhc class ii expression minireview: specificity and expression of ciita, the master regulator of mhc class ii genes genetic control of mhc class ii expression epigenetic control of ciita expression in leukemic t cells function and regulation of mhc class ii molecules in t-lymphocytes: of mice and men trafficking of mhc class ii molecules in the late secretory pathway mhc class ii molecules on the move for successful antigen presentation achieving stability through editing and chaperoning: regulation of mhc class ii peptide binding and expression identification of the hla-dm/hla-dr interface hla-dm induces clip dissociation from mhc class ii [alpha][beta] dimers and facilitates peptide loading the invariant chain is required for intracellular transport and function of major histocompatibility complex class ii molecules surface expression of mhc class ii in dendritic cells is controlled by regulated ubiquitination dendritic cells regulate exposure of mhc class ii at their plasma membrane by oligoubiquitination human immunodeficiency virus-1 nef expression induces intracellular accumulation of multivesicular bodies and major histocompatibility complex class ii complexes: potential role of phosphatidylinositol 3-kinase human immunodeficiency virus type 1 vpu protein interacts with cd74 and modulates major histocompatibility complex class ii presentation down-modulation of mature major histocompatibility complex class ii and up-regulation of invariant chain cell surface expression are well-conserved functions of human and simian immunodeficiency virus nef alleles hiv-1 gag polyprotein rescues hla-dr intracellular transport in a human cd4+ cell line differential incorporation of cd45, cd80 (b7-1), cd86 (b7-2), and major histocompatibility complex class i and ii molecules into human immunodeficiency virus type 1 virions and microvesicles: implications for viral pathogenesis and immune regulation distinct mechanisms of cd4+ and cd8+ t-cell activation and bystander apoptosis induced by human immunodeficiency virus type 1 virions mhc class ii and hiv pathogenesis: lots of data, few conclusions expression of rab4b, a protein governing endocytic recycling, is co-regulated with mhc class ii genes the presence of hostderived hla-dr1 on human immunodeficiency virus type 1 increases viral infectivity the acquisition of host-derived major histocompatibility complex class ii glycoproteins by human immunodeficiency virus type 1 accelerates the process of virus entry and infection in human t-lymphoid cells identification of ciita regulated genetic module dedicated for antigen presentation endosomal trafficking of hiv-1 gag and genomic rnas regulates viral egress vivoprocessing of pr160gag-polfrom human immunodeficiency virus type 1 (hiv) in acutely infected, cultured human t-lymphocytes comparison of human immunodeficiency virus type 1 pr55(gag) and pr160(gag-pol) processing intermediates that accumulate in primary and transformed cells treated with peptidic and nonpeptidic protease inhibitors programmed ribosomal frameshifting in hiv-1 and the sars-cov cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines hiv-1 protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins the regulation of sequential processing of hiv-1 gag by the viral protease complete mutagenesis of the hiv-1 protease gtp binding by class ii transactivator: role in nuclear import gtp-dependent recruitment of ciita to the class ii major histocompatibility complex promoter leucine-rich repeats of the class ii transactivator control its rate of nuclear accumulation human p32 protein relieves a posttranscriptional block to hiv replication in murine cells cell killing by hiv-1 protease overexpression of the gag-pol precursor from human immunodeficiency virus type 1 proviral genomes results in efficient proteolytic processing in the absence of virion production maintenance of the gag/gag-pol ratio is important for human immunodeficiency virus type 1 rna dimerization and viral infectivity the human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy nef promotes endocytosis of cell surface mhc class ii molecules via a constitutive pathway mutant murine leukemia virus gag proteins lacking proline at the n-terminus of the capsid domain block infectivity in virions containing wild-type gag hiv-1 protease processes procaspase 8 to cause mitochondrial release of cytochrome c, caspase cleavage and nuclear fragmentation the gastrointestinal tract is critical to the pathogenesis of acute hiv-1 infection immunopathogenic mechanisms of hiv infection productive infection of a cervical epithelial cell line with human immunodeficiency virus: implications for sexual transmission galactosyl ceramide (or a closely related molecule) is the receptor for human immunodeficiency virus type 1 on human colon epithelial ht29 cells human immunodeficiency virus type 1 infection and replication in normal human oral keratinocytes hiv infection and gut mucosal immune function: updates on pathogenesis with implications for management and intervention characteristics of a human cell line transformed by dna from human adenovirus type 5 separation of human immunodeficiency virus type 1 replication from nef-mediated pathogenesis in the human thymus importance of acidic, proline/serine/ threonine-rich, and gtp-binding regions in the major histocompatibility complex class ii transactivator: generation of transdominant-negative mutants primary human immunodeficiency virus type 2 (hiv-2) isolates, like hiv-1 isolates, frequently use ccr5 but show promiscuity in coreceptor usage predominant mode of human immunodeficiency virus transfer between t cells is mediated by sustained env-dependent neutralization-resistant virological synapses hla-dr alpha chain residues located on the outer loops are involved in nonpolymorphic and polymorphic antibody-binding epitopes macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual v3 envelope sequence homogeneity in comparison with t-cell-tropic isolates: definition of critical amino acids involved in cell tropism functional analysis of glucan binding protein b from streptococcus mutans human immunodeficiency virus (hiv-1) gag processing intermediates trans-dominantly interfere with hiv-1 infectivity characterization of a thymus-tropic hiv-1 isolate from a rapid progressor: role of the envelope key: cord-003472-ml4pbewf authors: manczinger, máté; boross, gábor; kemény, lajos; müller, viktor; lenz, tobias l.; papp, balázs; pál, csaba title: pathogen diversity drives the evolution of generalist mhc-ii alleles in human populations date: 2019-01-31 journal: plos biol doi: 10.1371/journal.pbio.3000131 sha: doc_id: 3472 cord_uid: ml4pbewf central players of the adaptive immune system are the groups of proteins encoded in the major histocompatibility complex (mhc), which shape the immune response against pathogens and tolerance to self-peptides. the corresponding genomic region is of particular interest, as it harbors more disease associations than any other region in the human genome, including associations with infectious diseases, autoimmune disorders, cancers, and neuropsychiatric diseases. certain mhc molecules can bind to a much wider range of epitopes than others, but the functional implication of such an elevated epitope-binding repertoire has remained largely unclear. it has been suggested that by recognizing more peptide segments, such promiscuous mhc molecules promote immune response against a broader range of pathogens. if so, the geographical distribution of mhc promiscuity level should be shaped by pathogen diversity. three lines of evidence support the hypothesis. first, we found that in pathogen-rich geographical regions, humans are more likely to carry highly promiscuous mhc class ii drb1 alleles. second, the switch between specialist and generalist antigen presentation has occurred repeatedly and in a rapid manner during human evolution. third, molecular positions that define promiscuity level of mhc class ii molecules are especially diverse and are under positive selection in human populations. taken together, our work indicates that pathogen load maintains generalist adaptive immune recognition, with implications for medical genetics and epidemiology. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the major histocompatibility complex (mhc) genes in vertebrates encode cell surface proteins and are essential components of adaptive immune recognition [1] . mhc proteins are endowed with highly variable peptide-binding domains that bind short protein fragments. the mhc region is one of the most polymorphic gene clusters in vertebrate genomes [2] . co-evolutionary arms race with pathogens is considered largely responsible for the observed exceptionally high levels of genetic diversity [3] [4] [5] [6] , yet it cannot fully account for the observed geographic differences in human mhc genetic diversity [7, 8] . this indicates that, beyond mhc allelic diversity, other mhc-related factors contribute to the capacity of human populations to withstand pathogens. in this paper, we argue that peptide-binding repertoire size (or, shortly, promiscuity) of mhc alleles is one important factor. recent empirical studies demonstrated that there is a substantial variation in the size of the bound and presented antigen repertoire across mhc class i alleles. certain mhc class i alleles appear to be promiscuous and are capable of binding an exceptionally large set of epitope peptide segments [9, 10] . for example, paul and colleagues carried out bioinformatics analysis to predict the binding capacity of common hla-a and hla-b alleles to a set of 30,000 dengue virus-derived peptides [11] . the analysis revealed over 16-fold variation in the number of peptides bound by the different alleles, indicating significant variation in epitope repertoire size across hla molecules. the authors selected three alleles for further study in an in vivo transgenic mouse model. immunization of the corresponding hla transgenic mouse strains with a set of dengue virus-derived peptides revealed a positive relationship between epitope repertoire size and immunogenicity. similarly, kosmrlj and colleagues computed the fraction of self-peptides that bind to various hla-b molecules, and found that this fraction varies extensively across four hla-b alleles [12] . the authors then demonstrated that the self-peptidebinding repertoire of hla-b shapes the native repertoire of t-cell clones developed in the thymus, with implications for recognizing human immunodeficiency virus (hiv) epitopes. their results could explain why individuals carrying hla-b � 57 alleles can maintain low hiv rna without therapy. remarkably, analogous mhc class i alleles with the hla-b � 27 superfamily is widespread in chinese rhesus macaques, animals which show especially slow progression of simian immunodeficiency virus (siv)/hiv [13] . finally, by focusing on seven chicken mhc class i haplotypes and four human hla-b alleles, chappel and colleagues demonstrated that mhc class i molecules that can bind a wide range of viral epitopes show lowered expression on the cellular surfaces of immune cells, such as monocytes and lymphocytes [9] . the authors suggested that the breadth of epitope-binding repertoire shapes genetic susceptibility to marek's disease virus in chickens and hiv disease progression in humans. more generally, by recognizing more peptide segments, promiscuous mhc molecules may promote immune response against a broader range of pathogens and are hence generalists [9, 10] . prior case studies in chicken indicate that this may be so [14] [15] [16] [17] . however, it remains to be established whether this relationship generally holds across mhc class i and ii alleles and a wide range of infectious diseases. specifically, we propose that in regions of high pathogen diversity, human populations should carry promiscuous mhc alleles. moreover, as migrating human populations have been exposed to changing sets of pathogens [18] , shifts in mhc promiscuity level should have occurred repeatedly and in a rapid manner during the course of human evolution. to test these predictions, we first focused on the human hla class ii drb1 gene, for several reasons. first, drb1 is the most variable hla class ii locus, with over 2,000 registered alleles [19] . together with hla-dra, hla-drb1 encodes the heterodimeric hla-dr protein complex, but hla-dra is basically invariant. second, drb1 shows the strongest general signature of selection among hla class ii loci [20] , while at the same time showing the weakest evidence for divergent allele advantage, an alternative mechanism at the genotype level for presenting a broader set epitopes [21] . third, drb1 has diversified very rapidly in the human lineage [22] . many of the drb1 alleles appear to be human specific and most likely evolved after the migration of ancestral human populations out of africa [22] . these periods have been associated with human populations encountering numerous new pathogens [18, 23] . for other hla class ii loci, the level of genetic diversity is lower [19, 24] , probably driven by selection for functions partly unrelated to pathogens. notably, hla-dq has a fundamental role in the development of immune tolerance [25, 26] , while hla-dp contributes to the presentation of epitopes of intracellular origins [27] [28] [29] . fourth, epitope-binding prediction algorithms show higher accuracy for drb1 than for other hla class ii loci [30, 31] . finally, the abundance of drb1 on the cell surface is especially high compared with other hla class ii molecules [32] [33] [34] . subsequently, we also evaluated promiscuity patterns of hla class i molecules. estimates on epitope-binding promiscuity were derived from two sources: experimental assays that measured individual peptide-mhc interactions in vitro and systematic computational predictions. in a series of analyses, we show that predictions of our hypothesis are upheld, regardless of how hla-drb1 promiscuity level is estimated. given that large-scale experimental assays to measure individual peptide-mhc interactions are extremely tedious, we first employed established bioinformatics tools to predict the binding affinities of experimentally verified epitope peptides for a panel of 162 nonsynonymous hla-drb1 alleles, all of which are present at detectable frequencies in at least one human population [35] [36] [37] . the set of investigated epitopes was derived from the immune epitope database (iedb) and contains 2,691 peptide epitopes of 71 pathogens known to be bound by certain hla class ii alleles [38] (s1 data). epitopes showing high levels of amino acid similarity to each other were excluded from the analysis (see methods). most included epitopes are 15 to 20 amino acids long and are found in only one of the 71 pathogens (s1 fig, s1 data) . the netmhciipan algorithm was used to predict individual epitope-mhc interactions [30] , not least because it outperforms other prediction algorithms [31] . the breadth of epitope-binding repertoire or, shortly, the level of promiscuity of individual hla-drb1 alleles was estimated as the fraction of epitopes with a binding affinity stronger than 50 nm to the given mhc molecule. this threshold corresponds to high-affinity binding, which is frequently observed in mhc molecules displaying immunodominance [39] . we found large variation in promiscuity levels across hla-drb1 alleles (s2 fig, s2 data) . using a smaller dataset with information from both approaches, we show that the computationally predicted and the empirically estimated promiscuity values are strongly correlated with each other (spearman's rho: 0.78, taking advantage of the confirmed reliability of computational predictions, we next investigated the geographic distribution of hla-drb1 alleles. we first collected high-quality hla-drb1 allele prevalence data of 96 human populations residing in 43 countries from two databases and an article [35] [36] [37] . the weighted average of promiscuity level in each population was calculated based on the promiscuity values and allele frequencies of individual alleles in the population (see methods). the analysis revealed a large variation in mean promiscuity across geographical regions and the corresponding human populations (s1 table) . importantly, several distantly related but highly promiscuous alleles contribute to this pattern (s1 table) . notably, an especially high allelic promiscuity level was found in southeast asia, an important hot spot of emerging infectious diseases [40] . to minimize any potential confounding effect of high genetic relatedness between neighboring populations, we merged populations with similar hla allele compositions for all further analyses (see methods). using the global infectious diseases and epidemiology network (gideon), we compiled a dataset on pathogen richness in the corresponding 43 geographic regions [41] . it consists of 95 diseases caused by 168 extracellular pathogens, including diverse bacterial species, fungi, protozoa, and helminthes. using the same protocol, we additionally compiled a dataset on the prevalence of 149 diseases in the same regions caused by 214 viral and other obligate intracellular pathogens. the dataset and methodology employed for the analysis are standardized and have been used previously in similar contexts [7, 8, 42] . we report a strong positive correlation between extracellular pathogen diversity and mean promiscuity: hla-drb1 alleles that can bind epitopes from a broader range of pathogens are more likely to be found in regions of elevated pathogen diversity ( fig 1a) . this pattern is unlikely to be explained by confounding factors, such as country size or hla-drb1 genetic diversity across countries (s2 table) . by contrast, we found no significant association between hla-drb1 promiscuity level and diversity of intracellular pathogens (fig 1b) . we conclude that the geographical distribution of promiscuous hla-drb1 alleles has been mainly shaped by the diversity of extracellular pathogens. the above results hold-and are even stronger-when estimates on promiscuity were derived from empirical in vitro mhc binding data (shortly, in vitro promiscuity), downloaded from the iedb database [38] (fig 1c and 1d , s2 table and s3 data). however, these results do not exclude the possibility that the geographical link between pathogen diversity and promiscuity is indirect. more direct support on the causal relationship between the two variables comes from analysis of prior human genetic studies. to investigate this issue, we focused on two geographically widespread allelic groups with exceptionally high (hla-drb1 � 12) and exceptionally low (hla-drb1 � 03) promiscuity values, respectively, and conducted literature mining on their reported associations with infectious diseases (s3 table) . as expected, hla-drb1 � 12 was associated with protection against at least five infectious diseases, while hla-drb1 � 03 was associated with susceptibilities to eight infectious diseases, which is highly unlikely by chance (fisher test, p = 0.003) (s3 table, s5 data). the data also indicate local adaptation towards elevated promiscuity under diverse pathogen pressure. the hla-drb1 � 12:02 allele is prevalent in specific regions of southeast asia. compared with other alleles detected in this region, hla-drb1 � 12:02 has a relatively high promiscuity value (top . indeed, this allele is associated with protection from recurrent pulmonary tuberculosis, recurrent typhoid fever, and hepatosplenic schistomiasis (s5 data), all of which are endemic diseases in southeast asia [44] [45] [46] . remarkably, the frequency of this allele increases with extracellular pathogen diversity in this region ( fig 2b) . together, these observations support the hypothesis that promiscuous epitope binding of hla-drb1 alleles is favored by selection when extracellular pathogen diversity is high. an important unresolved issue is how promiscuity has changed during the course of human evolution. under the assumption that local pathogen diversity drives the evolution of epitope recognition of hla class ii alleles, promiscuity as a molecular trait should have evolved rapidly as human populations expanded into new territories. to investigate this issue, we combined an established phylogeny of hla-drb1 alleles [47] with predicted epitope-binding promiscuity values. we found that alleles with a high promiscuity level have a patchy distribution across the tree (s6 fig), indicating that high promiscuity has multiple independent origins. to investigate this observation further, we selected a set of 96 hla-drb1 alleles with a detectable frequency in at least one human population and appropriate sequence data (see methods). a comparison of all pairs of these alleles revealed that even very closely related alleles show major differences in promiscuity levels ( fig 3a) . for example, alleles belonging to the hla-drb1 � 13 group show over 98% amino acid sequence identity to each other, but display as much as 57-fold variation in the predicted promiscuity levels. we conclude that the switch between high and low promiscuity levels has occurred repeatedly and in a rapid manner during the allelic diversification of the hla-drb1 locus. we next asked how selection on promiscuity has shaped the genetic diversity along the epitope-binding region of hla molecules. to quantify protein sequence variability at each amino acid position, we calculated the shannon entropy index based on the alignment of the 96 selected hla-drb1 alleles from above. for each position, we also calculated promiscuity fragility, that is, the median impact of single amino acid substitutions on promiscuity (see methods). a strong positive correlation was found between shannon entropy and promiscuity fragility ( the above data suggest a link between allele promiscuity and hla-drb1 diversification, probably as an outcome of selection for locally optimized promiscuity levels. finally, we note that several variable molecular sites in the binding region of hla-drb1 affect epitope-binding characteristics without any major impact on promiscuity per se. for example, our computational analysis indicates that mutations at amino acid site numbers 9 and 47 do not seriously affect promiscuity level ( fig 3b) . however, several mutations at these sites are associated with binding self-peptides and thereby shape vulnerability to specific autoimmune diseases [49, 50] . for all pairs of selected alleles, the predicted promiscuity difference between two hla-drb1 alleles is shown as a function of amino acid distance measured after excluding the epitope-binding region. large differences in promiscuity can be observed even between closely related pairs of alleles (e.g., at zero amino acid distance). as a result, there is no correlation between amino acid distance and promiscuity fold difference (spearman's rho = 0.02, p = 0.19). amino acid distances were binned as shown on the figure (n = 308, 1,168, 564, 654, 1,492). violin plots show the density function of promiscuity fold difference values for allele pairs in the given bin. white circles show median values; bold black lines show the interquartile range. (b) sequence variability of an amino acid site in the epitope-binding region of hla-drb1 (measured as shannon entropy) correlates positively with the site's promiscuity fragility, measured as the median predicted promiscuity fold difference caused by a random amino acid change at the given site (see inset, spearman's rho: 0.76, p = 0.0001). sites that have a larger impact on promiscuity are more diverse in human populations. line in inset represents linear regression between the two variables. the same result was obtained when promiscuity fragility was calculated based on nucleotide substitutions instead of amino acid substitutions (spearman's rho: 0.73, p = 0.0004, see methods) or when sequence variability was measured as nonsynonymous nucleotide diversity (π a ) instead of sequence entropy (s7 fig). sites under positive selection as identified by furlong and colleagues [48] show significantly higher promiscuity fragility (wilcoxon rank sum test, p = 0.0012) and are marked with asterisks (see also s8 fig) . the underlying data for this figure can be found in s4 data. the relationship between pathogen diversity and epitope-binding promiscuity may be more general, as similar results hold for the hla-a locus. hla-a is one of the three types of classical human mhc class i molecules and is mainly involved in the presentation of epitopes from intracellular pathogens [51] . in agreement with expectation, we report a positive correlation between local intracellular pathogen diversity and the hla-a promiscuity level of the corresponding human populations (s9a and s9b fig, s3 data) . no marked positive correlation was found for two other mhc class i genes (hla-b and hla-c, see s9c to s9f fig, and s3 data) . therefore, other unrelated evolutionary forces may shape the geographical distribution of promiscuous hla-b and hla-c alleles (s1 text). central players of the adaptive immune system are the groups of proteins encoded in the mhc. by binding short peptide segments (epitopes), mhc molecules guide both immune response against pathogens and tolerance to self-peptides. the genomic region encoding these mhc molecules is of special interest, for two reasons. it harbors more disease associations than any other regions in the human genome, including associations with infectious diseases, autoimmune disorders, tumors, and neuropsychiatric diseases [52, 53] . a growing body of literature is now revealing that certain mhc class i alleles can bind a wider range of epitopes than others, but the functional implications of this variation remain largely unknown [10] . by recognizing a larger variety of epitopes, such promiscuous mhc alleles promote immune response against a broader range of pathogens at the individual level. therefore, promiscuous epitope binding of mhc molecules should be favored by selection in geographic regions where extracellular pathogen diversity is high. importantly, this mechanism is conceptually distinct from the well-established concept of heterozygote advantage at the mhc [54] , as it concerns individual alleles and not allele combinations or genotypes. to test this hypothesis, we combined data on the geographic distribution of human mhc class ii alleles and prevalence of extracellular pathogens, empirical/computational estimates of epitope-binding promiscuity, and phylogenetic analyses. our main findings, strongly supporting our hypothesis, are as follows. first, in geographical regions of high extracellular pathogen diversity, human hla-drb1 alleles have exceptionally high epitope-binding repertoires. this suggests that the geographical distribution of promiscuous hla-drb1 alleles has been shaped by the diversity of extracellular pathogens. the hla-drb1 � 12:02 allele highlights this point. hla-drb1 � 12:02 is a promiscuous allele that has been associated with protection from certain infectious diseases (s5 data). as expected, this allele is especially prevalent in regions of southeast asia with elevated pathogen load (fig 2b) . it is well established that antigens presented by hla class ii molecules derive mainly from extracellular proteins [1] . however, hla class ii molecules have well-established roles in controlling immune response against viruses [55, 56] . additionally, viral peptides are reported to be processed and presented also by the hla class ii pathway [57] . therefore, it remains to be established why intracellular pathogen diversity has no major impact on the global distribution of hla-drb1 alleles. notably, the relationship between pathogen load and epitope-binding promiscuity may be more general, as similar results hold for the hla-a locus: we found a positive correlation between local intracellular pathogen diversity and the hla-a promiscuity level of the corresponding human populations (s9a and s9b fig, s3 data) . second, a phylogenetic analysis revealed major differences in promiscuity levels of very closely related hla-drb1 alleles. this suggests that high promiscuity level in hla-drb1 has evolved rapidly and repeatedly during human evolution. finally, amino acid positions with a prominent role in shaping hla-drb1 promiscuity level are especially variable in human populations and tend to be under positive selection. in sum, we conclude that hla promiscuity level is a human trait with paramount importance during adaptation to local pathogens. our work has important ramifications for future studies. mhc is the most variable region of the human genome, and the variation is associated with numerous infectious and immunemediated diseases [52, 53, [58] [59] [60] [61] [62] . the impact of mhc promiscuity level on population allelic diversity is an interesting area for future research. in a similar vein, mhc allelic diversity is associated with olfaction-based mating preferences in human and other animals [63] . the roles of mhc promiscuity in mating success and mating preferences are a terra incognita. we note that the most promiscuous hla-drb1 alleles are rare in certain human populations (s1 table; s2 data). this suggest that these alleles are not particularly favored by natural selection in these areas. why should it be so? first, high promiscuity may not be able to cope with the rise of novel and highly virulent pathogens. in such cases, displaying a particular epitope might be the most efficient way to achieve resistance, and high promiscuity might be suboptimal due to a reduced specificity [9, 10] . second, high promiscuity level may elevate the risk of immune reactions against host tissues and non-harmful proteins [9, 64] . clearly, future work should elucidate the evolutionary trade-offs between protection from pathogens and genetic susceptibility to autoimmune diseases. this will require high-throughput experimental methods to determine epitope-binding repertoire [65] , and hla transgenic mice studies on the role of promiscuity in immune response [66] . finally, genetic variation within particular mhc genes influences vaccine efficacy [67] , rejection rates of transplanted organs [68] , susceptibility to autoimmune diseases [49] , and antitumor immunity [28, 69, 70] . our work raises the possibility that, by altering the maturation and functionality of the immune system, the size of the epitope-binding repertoire of mhc alleles itself could have an impact on these processes. the exact role of mhc promiscuity in these crucial public health issues is an exciting future research area. the iedb has collected the results of individual and systematic studies on epitope binding by mhc alleles [38] . the experimental studies include hla-binding assays, t-cell activation assays, and immunopeptidomic studies as well. epitopes of all available viral, bacterial, and eukaryotic pathogens known to be bound by at least one hla-i or hla-ii allele were extracted from iedb. reference proteomes of pathogenic species that carry at least one of the collected epitope sequences were retrieved from the uniprot database (102 for hla-i and 71 for hla-ii epitopes) [71] . only epitopes of these species were analyzed further. all proteomes were scanned for each epitope sequence, and epitope sequences found in only one proteome (i.e., species-specific epitopes) were kept for further analysis. highly similar epitope sequences were identified using clustal omega [72] and excluded as follows. a protein distance matrix was created and epitopes were discarded iteratively. in each iteration, the epitope pairs with the lowest k-tuple distance were identified. then, the epitope with the highest average similarity to all other sequences was excluded. iterations were repeated until distance values less than 0.5 (corresponding to greater than approximately 50% sequence identity) were eliminated from the matrix [73] . note that this filtering procedure was carried out separately for epitope sequences bound by hla-i and hla-ii. binding affinities of the remaining 3,265 hla-i epitope sequences to 346 hla-a, 532 hla-b and 225 hla-c alleles were predicted with the netmhcpan-4.0 algorithm [74] . the binding of 2,691 hla-ii epitope sequences to 162 hla-drb1 alleles was predicted using the netmhciipan-3.1 algorithm [30] . all 162 alleles are present in at least one of the human populations studied here (see below). the "pep" sequence input format was used for both hla-i and hla-ii epitope-binding prediction. a binding affinity threshold of 50 nm was applied, yielding peptides that are likely to be immunodominant [39] . for alternative binding threshold definitions, see s4 fig. for each binding threshold, epitope-binding promiscuity was defined as the fraction of the epitope set bound by a given allele. to determine the epitope-binding promiscuity of hla-drb1 alleles based on previously published experimental data, we used the iedb database [38] . specifically, we downloaded all mhc ligand and t-cell assay data, which was available for 48 hla-drb1 alleles. binding data of 20 alleles screened for at least 100 ligands each were further analyzed. the epitope set of each allele was filtered for highly similar sequences, as described above. as the majority of in vitro assay data were available in a binary format (i.e., presence or absence of binding), promiscuity was calculated as the fraction of positive binding assays for a given allele. to calculate population-level promiscuity values, we obtained hla allele frequency data from the allele frequency net database (afnd) and the international histocompatibility working group (ihwg) populations [35, 36] . haplotype-level data of the 13th international hla and immunogenetics workshop (ihiw) populations were downloaded from dbmhc (national center for biotechnology information [ncbi]; ftp://ftp.ncbi.nlm.nih.gov/pub/mhc/mhc/final %20archive). additionally, allele frequency data of the 14th and 16th ihiw populations, as published by riccio and colleagues [37] , and populations in the afnd were used in the analyses. to avoid potential confounding effects of recent genetic admixture and migration, we focused on native populations, similarly to previous studies (s1 table) [7, 8] . we excluded ihwg populations reported to deviate from hardy-weinberg equilibrium [37]. among the afnd populations and ihwg populations without haplotype-resolution data (14th and 16th ihiw), those comprising less than 100 genotyped individuals or those in which the sum of allele frequencies deviated from 1 by more than 1% were excluded. populations reported in multiple databases were included only once in the analysis. for each hla loci, we calculated mean population promiscuity by averaging promiscuity values of alleles weighted by their relative frequencies in the populations. in all of these calculations, we used standardized (i.e., z-score) promiscuity values to make the in silico and in vitro values more easily comparable. finally, when calculating population-level promiscuity based on in vitro promiscuity data, we excluded populations for which in vitro promiscuity values could be assigned to less than 50% cumulative allele frequency. to tackle the issue of nonindependence of data points, we focused on populations instead of countries and grouped those populations that have highly similar hla allele compositions, based on standard measures of genetic distance (see below). we merged populations with highly similar hla allele compositions, allowing us to avoid pseudoreplication of data points while retaining informative allele frequency differences between populations residing in the same broad geographical areas. to this end, we first generated a genetic distance matrix between populations with the adegenet r library using allele frequency data of the examined locus. we used the rogers' genetic distance measure [75] because it does not assume that allele frequency changes are driven by genetic drift only, an unlikely scenario for hla genes. next, populations were merged using a network-based approach. populations were treated as nodes and two nodes were connected if their genetic distance was under a cutoff value. populations were grouped in an iterative manner. in each iteration, all maximal cliques (i.e., subsets of nodes that are fully connected to each other) in the network were identified. maximal cliques represent groups of populations in which all populations have similar allele compositions to each other. then, mean genetic distance within each clique was calculated. the clique with the lowest average distance was selected and its populations were grouped together. then, this clique was deleted from the network. iterations were repeated until no maximal cliques remained in the network. grouping of populations was carried out using different distance value cutoffs (1st, 5th, 10th, and 15th rank percentile of all distance values). the resulting population groups and the individual populations that remained in the network were treated as independent data points in subsequent statistical analyses. mean promiscuity level in population groups was calculated by averaging population promiscuity values. unless otherwise indicated, all figures are based on population groups using the 15th percentile genetic distance cutoff value. importantly, using different cutoffs has no impact on our results (s3 data). finally, we note that genetic differences among human populations mostly come from gradations in allele frequencies rather than from the presence of distinctive alleles [76] . therefore, traditional clustering of populations based on hla composition would have been ill-suited for our purposes. data on 309 infectious diseases were collected from gideon [41] . for each disease, the number of causative species or genera (when species were not listed for the genus) was determined using disease information in the gideon database, as described previously [42] . causative agents were classified into obligate intracellular and extracellular pathogen groups based on a previous study [7] and literature information. putative facultative intracellular pathogens were excluded from the analysis. diseases caused by agents that could not be clearly classified were also excluded from the analysis. extracellular and intracellular pathogen diversity (richness) of each country was approximated by the number of identified endemic extracellular and intracellular species, respectively. finally, we assigned country-level measures of pathogen and hla diversity to population groups as follows. for each population group, extracellular and intracellular pathogen counts were calculated by averaging the corresponding country-level values across the populations within the group. for example, if a population group contained two populations residing in neighboring countries, then we assigned the average pathogen diversity of the two countries to it. to examine associations between selected hla allele groups and infectious diseases, we carried out a systematic literature search on pubmed database using the following terms: "assoc � drb1 12 02", "assoc � drb1 1202", "assoc � drb1 12 01", "assoc � drb1 1201", "assoc � dr12", "assoc � drb1 � 12", "assoc � drb1 03 01", "assoc � drb1 0301", "assoc � dr3", "assoc � drb1 � 03", "assoc � dr17", "infect � drb1 12 02", "infect � drb1 1202", "infect � drb1 12 01", "infect � drb1 1201", "infect � dr12", "infect � drb1 � 12", "infect � drb1 03 01", "infect � drb1 0301", "infect � dr3", "infect � drb1 � 03", and "infect � dr17". "assoc � " and "infect � " refer to any word beginning with these letters. each resulting paper containing hla association data was examined, and statistically significant associations between allele groups (drb1 � 03 or drb1 � 12) or common alleles in allele groups (drb1 � 12:01, drb1 � 12:02, drb1 � 03:01) and infectious diseases were collected. associations with diseases caused by intracellular pathogens were excluded from the analysis. hla-disease associations were classified as beneficial or detrimental, if all related studies supported the beneficial or detrimental role of hla allele/allele group in the development or course of the given disease. otherwise, the association was classified as controversial. the results were summarized (s3 table) , and statistical association between beneficial/detrimental effects and high/low promiscuity across allele groups was determined by a fisher's exact test. we used amino acid distance as a proxy for phylogenetic distance between pairs of drb1 alleles. to this end, nucleotide sequences of drb1 alleles that contained full exon 2 and 3 regions were downloaded from the ipd-imgt/hla database [19] . to limit our analyses to alleles that have an impact on the inferred promiscuity level of a population, we considered only those sequences that had a nonzero frequency in at least one human population (see above). from allele groups that code for the same protein sequence (synonymous differences, differentiated by the third set of digits in the hla nomenclature), one of the alleles was randomly chosen. this selection procedure resulted in 96 alleles. multiple alignment of nucleotide sequences was performed using the muscle algorithm as implemented in the mega software [77] and converted to protein sequence alignments. amino acid distance was calculated using the jones-taylor-thornton substitution model in mega [77] (fig 3a) . epitope-binding region sites-as defined previously [30]-were excluded when calculating amino acid distance. the rationale behind this exclusion is that these sites are known to be under positive selection [78, 79] and are therefore less informative on evolutionary distance. additionally, by removing these sites, the amino acid distance remains independent of promiscuity predictions. finally, as intragenic recombination may distort the inference of evolutionary distance, we identified such events across all alleles following the protocol of satta and colleagues [80] using gene-conv [81] and rdp algorithms [82] as implemented in the rdp software [83] . recombinant alleles were removed when calculating amino acid distance. we first defined the epitope-binding region of hla-drb1 alleles, as previously [30] . to estimate sequence diversity along the epitope-binding region, we employed two measures: standard shannon entropy [84] and nucleotide diversity (π), a widely employed measure of genetic variation [85] . using the protein sequence alignment of the 96 alleles defined above, we calculated amino acid sequence variability as the shannon entropy of the given amino acid site as follows: where p i is the fraction of residues of amino acid type i at a given site, and m is the number of amino acid types observed at that site. nonsynonymous nucleotide diversity (π a ) measures the average number of nonsynonymous nucleotide differences per nonsynonymous site between two randomly chosen protein coding dna sequences from the same population [85, 86] . π a was calculated for each amino acid site in the epitope-binding region for each population using dnasp software [87] and custom-written r scripts. nucleotide sequences of drb1 alleles were downloaded from the ipd-imgt/hla database [19] . the calculation is based on the work of nei and colleagues [85] using the equation where x i and x j are the frequencies of the ith and jth alleles in the population, respectively, and p a ij is the number of nonsynonymous nucleotide differences per nonsynonymous nucleotide site between the two codon sequences of the given amino acid site in the ith and jth alleles. to calculate π a for each population, allele frequency data of human populations were obtained, as described earlier (see above). an overall nucleotide diversity index was calculated by averaging π a across populations. to calculate each amino acid site's impact on epitope-binding promiscuity (promiscuity fragility), promiscuity was predicted for each 19 possible amino acid change along the epitopebinding region of each of 96 alleles. the fold difference in promiscuity resulting from each amino acid substitution was calculated. the median promiscuity fold difference of each possible allele and amino acid change combination (96 × 19) was used to estimate promiscuity fragility at each amino acid position. as some of the 19 possible amino acid changes are not accessible via a single nucleotide mutation, and the accessible amino acid changes can have different likelihoods based on the codon sequence of the site and the genetic code, we also calculated promiscuity fragility based on each nonsynonymous nucleotide substitution of the codon instead of each amino acid substitution of the site. all statistical analyses were carried out in r version 3.2.0 [88] . smooth curves were fitted using the cubic smoothing spline method [89] . . we selected (i) 216 epitope sequences from this dataset, for which binding affinity data to all the 11 alleles were available, and (ii) 2,665 epitopes used for calculating in silico promiscuity throughout the paper, which were not used for the training of the netmhciipan algorithm. in vitro and predicted promiscuity of the 11 alleles was determined at a 50 nm binding threshold using the selected 216 and 2,665 epitopes, respectively. we used standardized (i.e., z-score) allele promiscuity values for the comparisons. we report a strong correlation between the in silico and in vitro promiscuity . one might speculate that there might be no selection for elevated hla-b promiscuity level due to a dominant balancing selection on this locus (see s1 text) [7, 8] . similarly, (e) the promiscuity level of hla-c molecules showed no significant correlation with intracellular pathogen diversity (spearman's rho: −0.14, p = 0.44). finally, (f) hla-c promiscuity level showed a marginally significant positive correlation with extracellular pathogen diversity (spearman's rho: 0.35, p = 0.04). this is surprising, but this preliminary result needs to be considered with caution, and studied further in future works. for detailed explanation of these results, see s1 text. population groups were created using the 15th percentile genetic distance cutoff (see methods). for a list of populations assigned to each group, see s6 data. for results of multivariate models and obtained upon using alternative distance cutoff values, see s3 data. red curves indicate smooth curves fitted using cubic smoothing spline method in r (see methods). the underlying data for this figure can be found in s4 data. (tif) s1 table. list of populations and their mean hla-drb1 allele promiscuity. table. the relationship between promiscuity and pathogen diversity is independent of hla diversity and country size. table. results of hla association studies suggest a protective role of high allele promiscuity in infectious diseases. (docx) s2(docx) s3 towards a systems understanding of mhc class i and mhc class ii antigen presentation the mhc, disease and selection how pathogens drive genetic diversity: mhc, mechanisms and misunderstandings the importance of immune gene variability (mhc) in evolutionary ecology and conservation from evolutionary genetics to human immunology: how selection shapes host defence genes adaptive value of novel mhc immune gene variants pathogen-driven selection and worldwide hla class i diversity distinct evolutionary strategies of human leucocyte antigen loci in pathogen-rich environments expression levels of mhc class i molecules are inversely correlated with promiscuity of peptide binding generalists and specialists: a new view of how mhc class i molecules fight infectious pathogens hla class i alleles are associated with peptide-binding repertoires of different size, affinity, and immunogenicity effects of thymic selection of the t-cell repertoire on hla class i-associated control of hiv infection peptide-binding motifs associated with mhc molecules common in chinese rhesus macaques are analogous to those of human hla supertypes and include hla-b27-like alleles influences of major histocompatibility complex class i haplotypes on avian influenza virus disease traits in thai indigenous chickens the mhc of the chicken, genomic structure, gene products, and resistance to oncogenic dna and rna viruses. veterinary immunology and immunopathology association of the chicken mhc b haplotypes with resistance to avian coronavirus mhc gene control of growth of avian sarcoma virus-induced tumours in chickens: a study on the role of virus strain natural selection and infectious disease in human populations the ipd and imgt/hla database: allele variant databases current perspectives on the intensity of natural selection of mhc loci divergent allele advantage at human mhc genes: signatures of past and ongoing selection a human-specific allelic group of the mhc drb1 gene in primates understanding rare and common diseases in the context of human evolution intensity of natural selection at the major histocompatibility complex loci cell-surface mhc density profiling reveals instability of autoimmunity-associated hla nonsynonymous substitution rate heterogeneity in the peptide-binding region among different hla-drb1 lineages in humans. g3 (bethesda) diversifying and purifying selection in the peptide binding region of drb in mammals phenome-wide scanning identifies multiple diseases and disease severity phenotypes associated with hla variants hla-drb1*1101: a significant risk factor for sarcoidosis in blacks and whites the mhc class i antigen presentation pathway: strategies for viral immune evasion major histocompatibility complex genomics and human disease excess of deleterious mutations around hla genes reveals evolutionary cost of balancing selection red queen processes drive positive selection on major histocompatibility complex (mhc) genes primary t cell expansion and differentiation in vivo requires antigen presentation by b cells association between an mhc class ii allele and clearance of hepatitis b virus in the gambia endogenous mhc class ii processing of a viral nuclear antigen after autophagy hla class ii sequence variants influence tuberculosis risk in populations of european ancestry class ii hla interactions modulate genetic risk for multiple sclerosis construction of a population-specific hla imputation reference panel and its application to graves' disease risk in japanese widespread non-additive and interaction effects within hla loci modulate the risk of autoimmune diseases polymorphisms of large effect explain the majority of the host genetic contribution to variation of hiv-1 virus load mhc class i peptides as chemosensory signals in the vomeronasal organ does intra-individual major histocompatibility complex diversity keep a golden mean high-throughput engineering and analysis of peptide binding to class ii mhc hla class ii transgenic mice as models of human diseases genetic variants within the mhc region are associated with immune responsiveness to childhood vaccinations indirect recognition of donor hla-dr peptides in organ allograft rejection mhc-i genotype restricts the oncogenic mutational landscape evolutionary pressure against mhc class ii binding cancer mutations uniprot: the universal protein knowledgebase fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega performance comparison between k-tuple distance and four model-based distances in phylogenetic tree reconstruction netmhcpan-4.0: improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data deriving phylogenetic trees from allele frequencies: a comparison of nine genetic distances genetic structure of human populations mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets nucleotide substitution at major histocompatibility complex class ii loci: evidence for overdominant selection natural selection at major histocompatibility complex loci of vertebrates. annual review of genetics mhc class ii dqb diversity in the japanese black bear, ursus thibetanus japonicus statistical tests for detecting gene conversion rdp: detection of recombination amongst aligned sequences rdp4: detection and analysis of recombination patterns in virus genomes bio3d: an r package for the comparative analysis of protein structures mathematical model for studying genetic variation in terms of restriction endonucleases synonymous and nonsynonymous polymorphisms versus divergences in bacterial genomes dnasp v5: a software for comprehensive analysis of dna polymorphism data r: a language for data analysis and graphics pathogen diversity drives the evolution of generalist mhc-ii alleles in human populations we wish to thank jim kaufman for the insightful comments we have received on an earlier version of the manuscript. we are also grateful to károly kovács for his suggestions on data analysis. key: cord-003270-vu9b5a14 authors: panahi, heidar ali; bolhassani, azam; javadi, gholamreza; noormohammadi, zahra title: a comprehensive in silico analysis for identification of therapeutic epitopes in hpv16, 18, 31 and 45 oncoproteins date: 2018-10-24 journal: plos one doi: 10.1371/journal.pone.0205933 sha: doc_id: 3270 cord_uid: vu9b5a14 human papillomaviruses (hpvs) are a group of circular double-stranded dna viruses, showing severe tropism to mucosal tissues. a subset of hpvs, especially hpv16 and 18, are the primary etiological cause for several epithelial cell malignancies, causing about 5.2% of all cancers worldwide. due to the high prevalence and mortality, hpv-associated cancers have remained as a significant health problem in human society, making an urgent need to develop an effective therapeutic vaccine against them. achieving this goal is primarily dependent on the identification of efficient tumor-associated epitopes, inducing a robust cell-mediated immune response. previous information has shown that e5, e6, and e7 early proteins are responsible for the induction and maintenance of hpv-associated cancers. therefore, the prediction of major histocompatibility complex (mhc) class i t cell epitopes of hpv16, 18, 31 and 45 oncoproteins was targeted in this study. for this purpose, a two-step plan was designed to identify the most probable cd8+ t cell epitopes. in the first step, mhc-i and ii binding, mhc-i processing, mhc-i population coverage and mhc-i immunogenicity prediction analyses, and in the second step, mhc-i and ii protein-peptide docking, epitope conservation, and cross-reactivity with host antigens’ analyses were carried out successively by different tools. finally, we introduced five probable cd8+ t cell epitopes for each oncoprotein of the hpv genotypes (60 epitopes in total), which obtained better scores by an integrated approach. these predicted epitopes are valuable candidates for in vitro or in vivo therapeutic vaccine studies against the hpv-associated cancers. additionally, this two-step plan that each step includes several analyses to find appropriate epitopes provides a rational basis for dnaor peptide-based vaccine development. hpvs are a large branch of the papillomaviridae family, grouped in different genera (alpha-, nu-/mu-, beta-and gamma-papillomaviruses), with more than 200 genotypes [1] [2] [3] [4] . the classification of papillomaviruses (pvs) has been based on l1 gene sequence. they are clinically divided into two groups: low-risk hpvs, like hpv 6 and 11, which cause benign lesions (warts and benign papillomas), and high-risk hpvs (hrhpvs), like hpv16 and 18, which are a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 response. however, the addition of cd4+ t cell epitopes can significantly augment its strength and duration [49, 52, 53] . cd8+ ctls commonly recognize intracellular-originated peptides presented by mhc-i molecules. they accommodate peptides with 8-11 residues; the ideal length is 9 residues. while cd4+ helper t lymphocytes (htls) commonly recognize extracellular-originated peptides presented by mhc-ii molecules. they accommodate peptides with 10-30 residues or even more; the ideal length is [12] [13] [14] [15] [16] . the strength of the interaction between a t cell receptor and a peptide-mhc complex (pmhc), depends on the presented peptide and the mhc structure [49, 54] . the binding of a peptide to mhc-i molecule is the most selective stage in the way of peptide presentation [55] . bioinformatics tools can predict the potential immunogenic epitopes from thousands of epitopes in a short time [56] . generally, the algorithms of these tools range from ones programmed to determine peptide-mhc molecule binding data to those based on structural similarity, molecular modeling, and molecular docking [57] . peptides that bind to a specific mhc molecule have sequence similarity. therefore, peptide sequence patterns have been used to predict their binding to mhc molecules [58] . in recent years, the accuracy of these methods has increased strikingly, and more than 90% of natural epitopes have been recognized at a high specificity of 98% [59] . this improvement in performance was achieved by the expanding experimental binding data, available in the immune epitope database (iedb) and analysis resource (http://www.iedb.org/), and by the improvement of machine-learning algorithms [60] . regarding the fundamental importance of epitope prediction in vaccine development, we investigated the best potential cd8+ t cell epitopes from the e5, e6, and e7 oncoproteins of four prevalent hrhpv genotypes (16, 18, 31 and 45) in the world and iran [61] , as shown in a two-step plan was designed to identify the most probable cd8+ t cell epitopes (fig 2) . for the first step, mhc-i and ii binding, mhc-i processing, mhc-i population coverage and mhc-i immunogenicity prediction analyses, and for the second step, mhc-i and ii protein-peptide docking, epitope conservation, and cross-reactivity with host antigens analyses were considered. the second step analyses were performed only for the selected peptides in the first step. in jan 2018, in order of priority, the refseq, reviewed or unreviewed sequences of hrhpv oncoproteins (e5, e6, and e7) were retrieved from the national center for biotechnology information database (ncbi) (http://www.ncbi.nlm.nih.gov/) and uniprotkb/swiss-prot database (http://www.uniprot.org/). the isoform sequences of hpv16, 18, 31, and 45 oncoproteins were retrieved from hpv t cell antigen database (http://cvc.dfci.harvard.edu/hpv/ html/search.php). all the sequences are accessible in supporting information (s1 file). binding of epitopes to mhc-i molecules is an essential step for antigen presentation to ctls. herein, it was predicted by four online servers, as illustrated in table 1 . the hla supertypes and frequently occurring hla-i alleles provided by the servers were included in the analysis. however, when an allele (e.g., hla-b � 14:02) was not provided, but its allele group (i.e., hla-b � 14) was available, we used the allele group instead of the allele. the used human and mouse alleles, or allele groups are provided in supporting information (s1 table) . currently, eight prediction methods are available in the iedb mhc-i binding prediction tool, i.e., iedb recommended [62] , consensus [69] , netmhcpan3 [59, 70] , artificial neural network (ann) [71, 72] , smm with a peptide-mhc binding energy covariance matrix (smmpmbec) [73] , stabilized matrix method (smm) [74] , comblib_sidney2008 [75] , pickpocket [76] , netmhccons [77] and netmhcstabpan [78] . the iedb-recommended and consensus are not independent methods; they use ann, smm and comblib_sidney2008 methods to generate a representative index for each predicted pmhc; the median of percentile ranks (prs) or binding scores obtained from the used methods is reported as a representative pr or consensus score in the iedb-recommended or consensus method respectively. the pr is calculated by comparing the half maximal inhibitory concentration (ic 50 ) of subjected peptide against a group of random peptides from swiss-prot database. the ic 50 value, expressed as nanomolar, shows binding affinity. the lower ic 50 or pr means higher binding affinity. as a rough guideline, peptides with ic 50 values <50 nm are considered as high affinity, 50-500 nm intermediate affinity and more than 500-5000 nm low affinity. no known t cell epitope has got an ic 50 value >5000 nm to date [60] . in this study, iedb recommended method was used. the outputs for each pmhc in this method consisted of a median pr, a method-specific ic 50 , and a method-specific pr. predictions were made against 76 frequently occurring human mhc-i alleles (including 12 hla supertypes) and 6 mhc-i mouse alleles. epitope length was set on 8, 9, 10, and 11mer. peptides with median pr <2.0 are applied for the analysis. netmhcpan4 mhc-i binding prediction. netmhcpan4 server predicts binding of peptides to the known mhc molecules using anns method. it is trained on a combination of naturally eluted ligands (55 human and mouse mhc-i alleles) and binding affinity data (172 mhc molecules from human, mouse, cattle, primates, and swine). besides, the user can perform a prediction against any custom mhc-i molecule by uploading its full-length sequence [66] . in this study, predictions were performed for 8, 9, 10, and 11mer peptides against 76 frequently occurring human mhc-i alleles and 8 mhc-i mouse alleles. pr thresholds for strong and weak binders were set on 0.5 and 2.0, respectively. peptides with pr <2.0 were applied for the analysis. rankpep mhc-i binding prediction. rankpep predicts binder peptides of a given protein sequence or sequence alignments to mhc-i and ii molecules. the algorithm of rankpep based on the comparison of sequence similarities, using position-specific scoring matrices (pssms) method. it employs profiles of a group of aligned peptides recognized to bind to a specific mhc molecule and creates a consensus sequence by determining the most common residue for each position. then, it allocates an optimal score to the consensus sequence, compares the score of the subjected peptide with the optimal score, and gives the peptide a percentile optimal value for comparison. finally, it highlights strong binders in red [67, 68] . herein, the prediction was made against 31 frequently occurring hla-i and 7 h2-i alleles. the server did not provide all common lengths of epitopes for all the mhc alleles. thus, the used alleles and their provided epitope lengths are shown together, as given in supporting information (s1 table) . syfpeithi mhc-i binding prediction. syfpeithi (http://www.syfpeithi.de/0-home. htm/) is a database of over 7000 published and verified peptide sequences of human, mouse, and other organisms, known as natural binders of mhc-i and ii molecules. when syf-peithi analyzes a peptide for binding prediction against a specific mhc-i allele, its scoring system evaluates every residue of the query and gives it an arbitrary value between 1 and 15, according to whether it is an anchor, auxiliary anchor, or preferred residue. it allocates the value 1 to those residues which slightly preferred in that particular position, 15 to the ideal anchor residues, and -1 to -3 to those residues which exhibit an adverse effect on the binding ability. the sum of these values is the score of the peptide. the maximal score could vary between different mhc alleles [54, 79] . herein, the prediction was made against 26 frequently occurring hla-i alleles and 5 h2-i alleles. epitope length was set on 8, 9, 10, and 11mer. every predicted pmhc which got a score less than 70% of the reference sequence score was excluded from the analysis. the allele-specific reference sequence was selected from rankpep's consensus sequence [68] , or our syfpeithi predicted epitopes, whichever got the highest score in syfpeihi server. the reference sequences, their sources, and their scores are given in supporting information (s2 table) . recognition of high immunogenic cd8+ t cell epitopes was the primary aim of this study. therefore, all predictions were primarily made against epitopes with 8-11 residue length. however, it was valuable to determine that which 9mer mhc-i epitope is the core peptide of the mhc-ii epitope(s) too. the core peptide lies on the mhc-ii molecule grooves, and play the central role in constructing pmhc. with this strategy, the short minimal predicted epitopes could be used in designing of synthetic long peptides (slps), resulting in peptide loading to both mhc-i and ii molecules. iedb mhc-ii binding prediction. in this study, the mhc-ii binding prediction was made by iedb mhc-ii binding predictor (http://tools.iedb.org/mhcii/) [60, 63, 64] . iedb possess seven prediction methods for mhc-ii binding prediction: iedb-recommended, consensus [63] , netmhciipan [80] , nn-align [81] , smm-align [82] , combinatorial libraries [75] and sturniolo's method [83] . herein, the iedb-recommended method was used, and all peptides with pr<2.0 were selected for the analysis. the prediction was made against 35 human alleles (iedb reference set) and three mouse alleles, given in supporting information (s3 table) . the server has fundamentally set the epitope length on 15mer. each iedb-recommended method participated in the prediction process offered a core peptide (9mer) for each predicted epitope (15mer). we associated the 9mer mhc-ii core peptides with the 9mer mhc-i predicted epitopes to determine that which mhc-i epitope is the core peptide of the mhc-ii epitope(s) too. mhc-i t cell epitope processing predictions of e5, e6, and e7 oncoproteins are made by the iedb combined predictor (http://tools.iedb.org/processing/). this tool combines predictors of three main steps of mhc-i antigen presentation pathway (proteasomal processing, transporter associated with antigen processing (tap) transport, and mhc-i binding) and calculates a total processing score for each predicted epitope. it allows the user to choose a method from ann, smm, smmpmbec, comblib_sidney2008, netmhcpan, netmhccons and pickpocket methods for the binding prediction. in the current update (2018), the iedb team has changed the choice of the recommended prediction method for the processing tool to be netmhcpan 3.0 rather than a consensus, since the processing tools requiring an ic 50 value, which the consensus method does not provide. furthermore, netmhcpan 3.0 has provided all mhc alleles and has performed the predictions very well in recent comparisons [65] . there are two types of proteasomes, the housekeeping types which are expressed instinctively, and immuno types which are provoked by ifn-γ secretion. the immunoproteasomes are believed to improve the efficiency of antigen presentation [62, 65] . in this study, the immunoproteasome option was selected. the program outputs for every predicted epitope consisted of proteasome score, tap score, mhc score, processing score (proteasome + tap score), total score (proteasome + tap + mhc score), and mhc-i ic 50 . the tap scoring system calculates a-log (ic 50 ) value for the binding of a peptide (or n-terminal of its precursors) to the tap molecules. the higher tap score, the higher transport rate. [62, 65, 84] . herein, the analysis was made against the human and mouse mhc-i alleles used later in the iedb binding prediction, with the iedb-recommended method and other default settings of the program. epitopes with ic 50 <1000 nm for hla-i alleles and <5000 nm for h2-i alleles were included in the analysis. several factors could clarify the difference between epitope and non-epitope peptides; an essential factor is epitope immunogenicity, i.e., it could be recognized by t cells. some amino acids, particularly those with large and aromatic side chains (especially tryptophan, phenylalanine, and isoleucine), are associated with immunogenicity. moreover, the positions p4-6 of a peptide are more critical for immunogenicity [85] . in this study, the mhc-i immunogenicity of all predicted epitopes was determined by the iedb web server (http://tools.iedb.org/immunogenicity/) [85] . this tool uses the properties of amino acids and their locations to predict the immunogenicity of a pmhc. the default option was selected to specify which positions of the query peptide to be masked from the analysis, because it masked positions which are also suggested for the most frequent human mhc-i allele, hla-a � 02:01. iedb population coverage prediction tool (http://tools.iedb.org/population/) [86] is used to predict the hla-i population coverage of all 8-11mer predicted epitopes in the first step. this tool can accept a target population by two query levels: 1) area-country-ethnicity and 2) ethnicity alone. it can integrate allele frequency information retrieved from the allele frequency net database (afnd) (http://www.allelefrequencies.net/default.asp) [87] . iedb also accepts custom populations with allele frequencies defined by users. since, hla-i and hla-ii t cell epitopes elicit immune responses from two different t cell populations (ctl and htl, respectively), the server provided three different population coverage modes: 1) hla-i lonely, 2) hla-ii lonely, and 3) hla-i and hla-ii together. herein, the mhc-i promiscuous predicted epitopes and their binding hla-i alleles (ic 50 <500 nm or pr<2.0) were entered as inputs for the analysis against the world population. the primary aim of molecular docking is the prediction of the binding site of a ligand at a protein receptor surface, and then docking and modeling the ligand into the recognized site. in this study, the binding ability of the first step selected peptides to human and mouse mhc molecules, was analyzed by cabs-dock (http://biocomp.chem.uw.edu.pl/cabsdock/) server. the server uses a multistage procedure that involves multiple programs, with the cα-cβ-side chain (cabs) model at its heart. the detailed information about these stages is given in supporting information (s2 file) [88, 89] . also, fig 3 shows the pipeline of cabs-dock protocol [88] . cabs-dock gets the 3d structure of the receptor and the sequence of the peptide as obligatory inputs. furthermore, there are some non-obligatory inputs as recommendations which could improve outputs. in this study, duplicate dockings for each peptide (6240 dockings in total) were done against the most significant human/mouse mhc-i and ii molecules which had at least one well-structured protein data bank (pdb) file in the rcsb protein data bank (https://www.rcsb.org/), as shown in table 2 . these pdb files are in the complex with their peptidic ligand and some x-ray crystallography solution molecules (heteroatoms). thus, these excess molecules, as well as redundant mhc molecules were removed before executing docking process. since, the binding site of epitopes on the mhc molecules was well-known previously, the unlikely regions to bind masked before the analysis. cabs-dock returns ten representative models (medoids) as the best-simulated models and ranks them by cluster density (cd). cluster density is equal to the number of elements in a cluster divided by their average ligand root mean square deviation (rmsd). the higher cd value implies greater accuracy. ligand rmsd value shows the differentiation measure between cluster elements. as a guideline; rmsd < 3.0 å means high accuracy; rmsd � 3.0 and � 5.5 å means medium accuracy and rmsd > 5.5 å means low accuracy [88] . herein, the rmsd and cd of the best-simulated models were selected for the analysis. the best model, which has the highest cd value, is not necessarily the top-ranked model, because, in some cases, peptides were not attached to their binding site properly. thus, these malformed models were excluded from the analysis. it is important to note that, due to the different frequency of mhc alleles in human populations, the equal cd value of different mhc alleles, don't have equal value regarding population coverage. thus, to involve the effect of population coverage, the cd value of every model was multiplied by its allele population coverage (divided by hundreds for more facility) to obtain a weighted index. then, the sum of all hla-i or ii weighted indexes of each peptide was calculated to get a total docking score (tds), used as a score to compare the candidate peptides. it is the first time that the tds has been formulated and used for this purpose. this formula is also applicable to the similar docking scores obtained from other servers. the use of highly conserved epitopes in a vaccine formulation reduces the risk of tumor immune escape and provides broader protection against different virus strains or genotypes. thus, the conserved areas are preferred to use in therapeutic vaccines, if they are appropriate epitopes. herein, the epitope conservancy analyses for the first step selected peptides were done in three levels: 1. inter-isoform conservancy: the percent of conservancy between all isoforms of each e5, e6, or e7 oncoprotein. 2. inter-type conservancy: the percent of conservancy between hpv16 and 31 (alpha-papillomavirus 9), as well as between hpv18 and 45 (alpha-papillomavirus 7). 3. inter-hrhpv conservancy: complete (100%) conservancy between all hrhpvs (hpv16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59). the selected peptides in the first step were analyzed to find inter-strains and inter-types conservancy percentages by iedb tool, conservation across antigens, (http://tools.iedb.org/ conservancy/). the inter-hrhpvs conservancy analysis was done by the iedb and expasy clustalw servers (https://embnet.vital-it.ch/software/clustalw.html). cross-reactivity with host antigens can cause adverse immune responses. therefore, the selected peptides in the first step were checked for similarities with the mouse and human proteomes by the ncbi blastp tool (https://blast.ncbi.nlm.nih.gov/blast.cgi). regarding the studies, different peptides usually get different scores/ranks in different analyses. this inconsistency indicates that these results needed to be analyzed with an integrated approach. indeed, integrated approach is more practical and efficient in such conditions in comparison with analysis by analysis filtering approach, in which those epitopes are chosen for the next analysis that have gotten an acceptable score in the previous analysis. herein, the integrated approach was applied in both steps of epitope selection. since the ultimate goal of the discovery of therapeutic epitopes is to use them in human vaccines, only the scores/ranks of human alleles were used to rank epitopes in some studies. however, investigators usually test therapeutic vaccines on mouse species in preclinical trials, thus in the current study, the binding status of the predicted epitopes to mouse mhc-i alleles was also studied by several binding predictors and molecular docking, as well. as stated above, ctl-mediated responses play a crucial role in killing the malignant cells. besides, the binding of epitopes to mhc-i molecules is the most selective step for antigen presentation to ctls. therefore, in the first step, the selection was made primarily by the comparison of obtained mhc-i binding, processing and immunogenicity scores/ranks, and population coverage percentages. however, the mhc-ii binding ranks were actually of secondary importance to the selection process as an added advantage. additionally, the population coverage has a dual application. first, it determines the coverage of a given peptide in the target population. second, it is the best index for summarizing and evaluating of the hla-i binding predictions too, since it is calculated from the results of hla-i binding prediction analyses. in the first step, ten peptides (tables 3-5 ) from each hpv genotype oncoprotein (120 peptides in total), which got better results in the first step analyses were selected for the second step analyses, including protein-peptide molecular docking, epitope conservation, and crossreactivity with host antigens. the individual detailed results of the mhc-i and ii binding (s3 file), mhc-i immunogenicity (s4 file) and mhc-i population coverage (s5 file) predictions, as well as, mhc-i and ii molecular docking (s6 file) and epitope conservation (s7 file) analyses are given in supporting information, as 15 excel files. indeed, cabs-dock returns ten representative medoids as the best-simulated models and ranks them by cluster density (cd). cluster density is derived from two factors (the number of elements in a cluster and their average ligand rmsd) that is an advantage for this server. in the second step, five peptides out of ten selected peptides in the first step (tables 6-8) , which got better results in all analyses of both steps, were selected as the final-predicted epitopes. none of the final predicted epitopes showed more than 90% sequence similarity with mouse and human proteomes. high prevalence and mortality of oncogenic infectious pathogens such as hpv and helicobacter pylori have caused serious problems for humans. currently, people who are infected with hrhpvs but show normal cytology or precancerous lesions do not have any treatment option, causing the disease progress toward invasive carcinoma in some cases. unfortunately, no fda-approved immunotherapy exists for pre-existing hpv infections or their related cancers to date. immunotherapy of hpv-associated cancers by dna or peptide-based vaccines, depends on the recognition of highly immunogenic epitopes, inducing robust and specific immune responses, particularly cell-mediated responses against the malignant cells. the primary aim of this study was the prediction of cd8+ t cell epitope from the e5, e6 and e7 oncoproteins, using a comprehensive two-step selection plan. these proteins chose because they play a pivotal role in the cell transformation, immune evasion, and maintenance of malignancy, as well as, their permanent expression (e6 and e7) by the malignant cells [24] [25] [26] . expression of e5 oncoprotein occurs in the early phase of hpv infection. evidence indicates that e5 play a prominent role in the genesis of hpv-associated cancers, but is not essential for cancer progression [90] , since when hpv genome integrates into the host genome, it usually results in the disruption of e1, e2, and e5 genes. therefore, targeting e5 protein provides an opportunity for treatment of hpv infections and preventing the precancerous lesions from the progression to established carcinomas [20, 91] . some genotypes of hrhpvs are more involved in the genesis of epithelial tissue malignancies [61] . thus, in this study, hrhpv16, 18, 31 and 45 were targeted due to their high prevalence in the hpv-associated cancers, especially cervical carcinoma. there are several limitations for epitope prediction: 1) the major drawback of peptidebased vaccines is low immunogenicity [92, 93] . many studies have focused on enhancing immunogenicity using immune stimulating agents or adjuvants to avoid this problem. another solution is the use of agonist epitopes [94] . epitope immunogenicity is a crucial factor in vaccine development. however, many of known natural epitopes when are analyzed in silico by iedb mhc-i immunogenicity predictor, do not obtain a high score. therefore, in this study, epitope selection was based on the integrated approach, in which one analysis does not play an important role alone. 2) there are certain drawbacks associated with the function of each method invented for the mhc-peptide binding prediction [95] . for this reason, several predictors and a molecular docking program were used to augment the prediction accuracy. 3) some web tools have been developed for mhc-ii epitope prediction. since mhc-ii groove can bind to peptides with variable lengths, and different peptides have the different number of residues between their n-terminus and first anchor [54], the exact assignment of mhc-ii core peptide would be a difficult problem which reduces the success rate of these prediction tools. therefore, most mhc-ii prediction tools did not usually make epitope predictions as accurately noted for mhc-i molecules [64, 96] . in cancer immunotherapy, the ctl-mediated responses play the central role in eradication of malignant cells, and the binding of epitopes to mhc-i molecules is an essential step for antigen presentation to ctls. thus, in this study, predicted epitopes were primarily selected by their mhc-i binding and processing scores. however, the mhc-ii binding scores were actually of secondary importance to the epitope selection process as an extra advantage. additionally, there are several other essential determinants which significantly affect the outcomes, such as antigen processing, immunogenicity, population coverage, conservancy and cross-reactivity with host antigens. vaccine development requires a comprehensive approach to cover all these effectual elements, covered in this study. the primary aim of molecular docking is the recognition of binding site of a ligand at a protein receptor surface, and docking and modeling the ligand into this recognized site. in this study, cabs-dock server was used for molecular docking analyses. cabs-dock has several main advantages: 1) the method does not require any data about the peptide structure and its binding site. 2) during docking process, peptide conformation is entirely flexible. 3) it is possible to apply dynamic conformational changes in the receptor structure and 4) to exclude some receptor regions from the docking search, leading to the more efficient search in the vicinity of the binding site at a sensible time. [88, 89] . in comparison with protein-ligand (small molecules) docking, protein-peptide docking analysis is more problematic, since significant conformational changes occur during the process. as a general rule, how much the length of the query peptide to be longer, there are more torsions and conformational flexibilities. additionally, in comparison to protein-protein interactions, protein-peptide dockings are more transient, and their binding affinities are notably weaker [88] . these factors make structural predictions of long peptides very challenging. therefore, in this study, 9mer peptides were preferred for selection compared to other possible lengths. they are also preferred by all mhc-i molecules as epitope and by mhc-ii molecules as the core peptide of epitopes. moreover, expansion of 9 or 10mer ctl epitopes to longer peptides may create a practical alternative, containing both cd4+ htl and cd8+ ctl epitopes; especially, when cd4+ htl epitopes, covering ctl epitopes, are not recognized [97] . [94, 96, [98] [99] [100] [101] [102] [103] . however, the prediction of t cell epitopes inducing strong responses has remained a big challenge. for therapeutic hpv vaccines, many candidates have been designed to trigger the activation of ctls or htls, mostly by targeting two major hpv oncoproteins, e6 or e7 [104] , and in a few studies, e5 oncoprotein [98, 99] . as well as, several clinical trials have been launched for immunotherapy of hpv-associated cancers [46], although, they have not been so immunogenic, to induce a sufficient cellular immunity and eradicate malignant cells completely. some studies have suggested that the use of e6 and e7 slps, containing both cd4+ htl and cd8+ ctl epitopes, led to more potency and durability of cd8+ t cell reactivity in vivo, in comparison with the minimal ctl epitopes [97, 105] . in 1993, as pioneers in hpv epitope studies, feltkamp et al. recognized the hpv16-e7 sequence rahynivtf as an mhc-i epitope that can provoke ctl-mediated responses and eradicates established hpv l6-induced tumor cells in mice [106, 107] . this sequence is the first hpv16-e7 predicted epitope in our study as well. in 2015, kumar et al. studied hpv16-e5 oncoprotein to predict the candidate t-cell and bcell epitopes [98] . they have screened 11 potent epitopes for mhc-i molecules according to pr and the immunogenicity score, using iedb mhc-i binding and immunogenicity predictors. they found a 14mer potent epitope, safrcfivyiifvy, having the lowest pr and the highest immunogenicity score, i.e., 0.5 and 0.70, respectively. notably, our second hpv16-e5 predicted epitope, safrcfivy, is the n-terminal part of safrcfivyiifvy, and our first predicted epitope, flihtharf, is the c-terminal part of the third epitope of their study, vyiplflihtharf. in 2017, tsang et al. scanned the hpv16-e6 and e7 oncoproteins for the match peptides with the consensus motif of hla-a2 binding peptides [94] . the bimas algorithm [108] was employed to rank probable binding peptides according to the predicted one-half-time dissociation of pmhcs. three potential ctl predicted epitopes of the e6 protein (klpqlctel, kiseyr-hyc, and qqynkplcdl) and three of the e7 protein (ymldlqpet, tlheymldl, and rtledllmgt) were selected. they showed the immunogenicity of these peptides was enhanced when their agonist epitopes were used. the klpqlctel and tlheymldl sequences are the seventh and the fifth predicted epitopes of hpv16-e6 and hpv16-e7 in our study, respectively. experimental evidences about hrhpv-derived epitopes in literatures are mostly limited to e6 and e7 oncoproteins of hpv16 and 18. among our first-step predicted epitopes: fllcfcvll and yiifvyipl from the e5-derived epitopes [109] , fafrdlcivy [110] , cyslygttl [111] , vydfafrdl [111, 112] , kfyskisey [113] , klpqlctel [114] [115] [116] , iseyrhycy [117] , eyrhycysl [111] , klpdlctel [116, [118] [119] [120] , fafkdlfvv [119, 120] and klpdlctel [116, [118] [119] [120] from the e6-derived epitopes, rahynivtf [121] , ledllmgtl [122] , tlheymldl [115, [122] [123] [124] , llmgtlgiv [115, 116, 125, 126] , qaepdrahy [117] , gtlgivcpi [115, 126] , fqqlflntl [127] and tlqdivlhl [119] from the e7-drived epitopes were reported as t-cell epitopes experimentally. besides, ivyrdgnpy, cyslygttl, klpqlctel and iseyrhycy from the e6-derived epitopes, and rahynivtf and gtlgivcpi from the e7-derived epitopes were also reported as hla ligands [128] . others are novel epitopes that they also require experimental studies for validation. as far as we know, this is the first time that in a laborious in silico study for epitope prediction, e5, e6 and e7 oncoproteins of hrhpv16, 18, 31 and 45 have been investigated altogether. moreover, in previous studies, usually only one predictor tool was used for making epitope prediction, or if several tools were used, no integrated approach was employed to make the conclusion. we believed that our predicted epitopes are valuable candidates for further in vitro and in vivo therapeutic vaccine studies. additionally, the introduction of the ten epitopes for each hpv genotype oncoprotein in the first step of the study shows which region of each oncoprotein is rich of the epitope, and thus, is more suitable for use in the design of slps. notably, the previous in vivo studies have been conducted using slps of hrhpv-e6 and/or-e7 oncoproteins, in particular hpv16 oncoproteins [92, [129] [130] [131] [132] [133] . furthermore, the two-step plan of this in silico study, which each step includes several analyses to find proper epitopes by an integrated approach, would provide a basis for rational epitope prediction. however, it could be more efficient by adding other useful analyses. further studies are recommended on the peptide binding assays, the design of polyepitope constructions including e5, e6 and e7 epitopes, the expansion of the minimal ctl epitopes to longer peptides (slps), the use of various adjuvants, involvement of delivery routes, mouse immunization with the designed constructs, evaluation of immune responses such as cytokines, antibodies, ctls and tumor growth for finding the best construct for clinical trials. it is important that improper vaccine design and immunosuppressive microenvironment were known as the main reasons of the failure in cancer immunotherapy by therapeutic cancer vaccines [134] . cross-roads in the classification of papillomaviruses hpv vaccine: current status and future directions the natural history of human papillomavirus infection. best practice & research clinical obstetrics & gynaecology classification of papillomaviruses (pvs) based on 189 pv types and proposal of taxonomic amendments global burden of human papillomavirus and related diseases carcinogenic human papillomavirus infection human papillomavirus molecular biology and disease association tumour virus vaccines: hepatitis b virus and human papillomavirus global burden of cancers attributable to infections in 2008: a review and synthetic analysis. the lancet oncology worldwide burden of cancer attributable to hpv by site, country and hpv type comprehensive control of human papillomavirus infections and related diseases human papillomavirus genome variants human papillomavirus types in 115,789 hpv-positive women: a meta-analysis from cervical infection to cancer human papillomaviruses. iarc monographs on the evaluation of carcinogenic risks to humans e5 protein of human papillomavirus 16 downregulates hla class i and interacts with the heavy chain via its first hydrophobic domain the bovine papillomavirus oncoprotein e5 retains mhc class i molecules in the golgi apparatus and prevents their transport to the cell surface the e6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53 degradation of the retinoblastoma tumor suppressor by the human papillomavirus type 16 e7 oncoprotein is important for functional inactivation and is separable from proteasomal degradation of e7 human papillomavirus and related diseases in the world modeling the mhc class i pathway by combining predictions of proteasomal cleavage, tap transport and mhc class i binding peptide binding predictions for hla dr, dp and dq molecules a systematic assessment of mhc class ii peptide binding predictions and evaluation of a consensus approach immune epitope database and analysis resource (iedb) 3.0. mhc-i processing predictions-tutorial netmhcpan-4.0: improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data prediction of peptide-mhc binding using profiles. immunoinformatics: predicting immunogenicity in silico prediction of mhc class i binding peptides using profile motifs a consensus epitope prediction approach identifies the breadth of murine t(cd8+)-cell responses to vaccinia virus netmhcpan, a method for mhc class i binding prediction beyond humans gapped sequence alignment using artificial neural networks: application to the mhc class i system netmhc-3.0: accurate web accessible predictions of human, mouse and monkey mhc class i affinities for peptides of length 8-11 derivation of an amino acid similarity matrix for peptide: mhc binding and its application as a bayesian prior generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method quantitative peptide binding motifs for 19 human and mouse mhc class i molecules derived using positional scanning combinatorial peptide libraries the pickpocket method for predicting binding specificities for receptors based on receptor pocket similarities: application to mhc-peptide binding netmhccons: a consensus method for the major histocompatibility complex class i predictions pan-specific prediction of peptide-mhc class i complex stability, a correlate of t cell immunogenicity information on syfpeithi. institute for cell biology-department of immunology-heidelberg accurate pan-specific prediction of peptide-mhc class ii binding affinity with improved binding core identification nn-align. an artificial neural network-based alignment algorithm for mhc class ii peptide binding prediction prediction of mhc class ii binding affinity using smm-align, a novel stabilization matrix alignment method generation of tissue-specific and promiscuous hla ligand databases using dna microarrays and virtual hla class ii matrices identifying mhc class i epitopes by predicting the tap transport efficiency of epitope precursors properties of mhc class i presented peptides that enhance immunogenicity predicting population coverage of tcell epitope-based diagnostics and vaccines allele frequency net 2015 update: new features for hla epitopes, kir and disease and hla adverse drug reaction associations modeling of proteinpeptide interactions using the cabs-dock web server for binding site search and flexible docking cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site human papillomavirus type 16 e5 protein as a therapeutic target recombinant adeno-associated virus expressing human papillomavirus type 16 e7 peptide dna fused with heat shock protein dna as a potential vaccine for cervical cancer immunotherapy of established (pre) malignant disease by synthetic long peptide vaccines more than one reason to rethink the use of peptides in vaccine design identification and characterization of enhancer agonist human cytotoxic t-cell epitopes of the human papillomavirus type prediction of mhc-peptide binding: a systematic and comprehensive overview prediction of epitope-based peptides for vaccine development from coat proteins gp2 and vp24 of ebola virus using immunoinformatics cd8+ ctl priming by exact peptide epitopes in incomplete freund's adjuvant induces a vanishing ctl response, whereas long peptides induce sustained ctl reactivity identification of immunotherapeutic epitope of e5 protein of human papillomavirus-16: an in silico approach computational prediction of linear b-cell epitopes in the e5 oncoprotein of the human papillomavirus type 16 using several bioinformatics tools multi epitope peptide vaccine prediction against sudan ebola virus using immuno-informatics approaches a systematic bioinformatics approach for selection of epitope-based vaccine targets a novel multi-epitope peptide vaccine against cancer: an in silico approach epitope-based vaccine target screening against highly pathogenic mers-cov: an in silico approach applied to emerging infectious diseases advances in peptide-based human papillomavirus therapeutic vaccines therapeutic vaccination with papillomavirus e6 and e7 long peptides results in the control of both established virus-induced lesions and latently infected sites in a pre-clinical cottontail rabbit papillomavirus model vaccination with cytotoxic t lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells cytotoxic t lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type 16-induced tumors scheme for ranking potential hla-a2 binding peptides based on independent binding of individual peptide side-chains cytotoxic t-lymphocyte responses to human papillomavirus type 16 e5 and e7 proteins and hla-a*0201-restricted t-cell peptides in cervical cancer patients hla class i binding promiscuity of the cd8 t-cell epitopes of human papillomavirus type 16 e6 protein novel oligomannose liposome-dna complex dna vaccination efficiently evokes anti-hpv e6 and e7 ctl responses identification of an hla-a24-restricted cytotoxic t lymphocyte epitope from human papillomavirus type-16 e6: the combined effects of bortezomib and interferon-gamma on the presentation of a cryptic epitope identification of human papillomavirus 16-e6 protein-derived peptides with the potential to generate cytotoxic t-lymphocytes toward human leukocyte antigen-a24+ cervical cancer human papillomavirus 16 e6-specific cd45ra+ ccr7+ high avidity cd8+ t cells fail to control tumor growth despite interferon-gamma production in patients with cervical cancer a conserved e7-derived cytotoxic t lymphocyte epitope expressed on human papillomavirus 16-transformed hla-a2+ epithelial cancers immunization with a poly (lactide co-glycolide) encapsulated plasmid dna expressing antigenic regions of hpv 16 and 18 results in an increase in the precursor frequency of t cells that respond to epitopes from hpv 16, 18, 6 and 11 identification in humans of hpv-16 e6 and e7 protein epitopes recognized by cytolytic t lymphocytes in association with hla-b18 and determination of the hla-b18-specific binding motif up-regulation of hla class-i antigen expression and antigen-specific ctl response in cervical cancer cells by the demethylating agent hydralazine and the histone deacetylase inhibitor valproic acid human t-cell responses to hla-a-restricted high binding affinity peptides of human papillomavirus type 18 proteins e6 and e7 synthetic peptides of human papillomavirus type 18 e6 harboring hla-a2.1 motif can induce peptide-specific cytotoxic t-cells from peripheral blood mononuclear cells of healthy donors establishment of an hla-a*0201 human papillomavirus type 16 tumor model to determine the efficacy of vaccination strategies in hla-a*0201 transgenic mice different methods of identifying new antigenic epitopes of human papillomavirus type 16 e6 and e7 proteins naturally processed and hla-b8-presented hpv16 e7 epitope recognized by t cells from patients with cervical cancer human ctl epitopes encoded by human papillomavirus type 16 e6 and e7 identified through in vivo and in vitro immunogenicity studies of hla-a* 0201-binding peptides dendritic cell-based tumor vaccine for cervical cancer ii: results of a clinical pilot study in 15 individual patients human ctl epitopes encoded by human papillomavirus type 16 e6 and e7 identified through in vivo and in vitro immunogenicity studies of hla-a*0201-binding peptides identification of a naturally processed hla-a*0201 hpv18 e7 t cell epitope by tumor cell mediated in vitro vaccination role of hla-a motifs in identification of potential ctl epitopes in human papillomavirus type 16 e6 and e7 proteins prospects of combinatorial synthetic peptide vaccine-based immunotherapy against cancer experience with synthetic vaccines for cancer and persistent virus infections in nonhuman primates and patients mechanisms of peptide vaccination in mouse models: tolerance, immunity, and hyperreactivity therapeutic vaccination against human papilloma virus induced malignancies immunologic treatments for precancerous lesions and uterine cervical cancer therapeutic cancer vaccines the authors sincerely thank dr. ali namvar and miss elnaz agi for their valuable guidance and comments during preparation of the paper. key: cord-002704-cpa2sl50 authors: el bissati, kamal; zhou, ying; paulillo, sara maria; raman, senthil kumar; karch, christopher p.; roberts, craig w.; lanar, david e.; reed, steve; fox, chris; carter, darrick; alexander, jeff; sette, alessandro; sidney, john; lorenzi, hernan; begeman, ian j.; burkhard, peter; mcleod, rima title: protein nanovaccine confers robust immunity against toxoplasma date: 2017-09-05 journal: npj vaccines doi: 10.1038/s41541-017-0024-6 sha: doc_id: 2704 cord_uid: cpa2sl50 we designed and produced a self-assembling protein nanoparticle. this self-assembling protein nanoparticle contains five cd8(+) hla-a03-11 supertypes-restricted epitopes from antigens expressed during toxoplasma gondii’s lifecycle, the universal cd4(+) t cell epitope padre, and flagellin as a scaffold and tlr5 agonist. these cd8(+) t cell epitopes were separated by n/kaaa spacers and optimized for proteasomal cleavage. self-assembling protein nanoparticle adjuvanted with tlr4 ligand-emulsion gla-se were evaluated for their efficacy in inducing ifn-γ responses and protection of hla-a*1101 transgenic mice against t. gondii. immunization, using self-assembling protein nanoparticle-gla-se, activated cd8(+) t cells to produce ifn-γ. self-assembling protein nanoparticle-gla-se also protected hla-a*1101 transgenic mice against subsequent challenge with type ii parasites. hence, combining cd8(+) t cell-eliciting peptides and padre into a multi-epitope protein that forms a nanoparticle, administered with gla-se, leads to efficient presentation by major histocompatibility complex class i and ii molecules. furthermore, these results suggest that activation of tlr4 and tlr5 could be useful for development of vaccines that elicit t cells to prevent toxoplasmosis in humans. toxoplasma gondii infects all mammals. it can cause severe brain and eye damage in the fetus, in newborn infants, and in immunecompromised individuals. 1 although anti-parasitic medicines such as sulfadiazine and pyrimethamine are available, some patients experience side effects including toxicity and hypersensitivity. latent, encysted parasites are not eliminated by these treatments. 2 therefore, development of a potent, safe, effective vaccine is greatly needed. one approach for toxoplasmosis vaccine development is an epitope-based vaccine designed to enhance host immunity. protection is achieved through stimulation of cd4 + helper t lymphocytes and cd8 + ifn-γ producing t lymphocyte responses. these cd8 + t cells recognize octamer/nonamer peptides presented on hla supermotif molecules on infected cells. previously, our laboratory (rm, ke) identified epitopes eliciting cd8 + t cells derived from proteins expressed during different phases of the toxoplasma life cycle. hla-a02, a03-11 and b07 human, supermotif, major histocompatibility complex (mhc) molecules are present in~90% of humans, [3] [4] [5] [6] and therefore are capable of presenting these epitopes. as the discovery of such protective peptide epitopes accumulates, mechanisms are needed to effectively present these epitopes to the immune system of the host. we have pioneered a platform known as self-assembling protein nanoparticles (sapns). [7] [8] [9] [10] [11] [12] sapns induce a strong immune response due to the repetitive display of antigens. 7, 10, 12 they promote immune responses by cd4 + as well as cd8 + t cells by incorporating the t cell epitopes into the core architecture of the nanoparticle. 8, 9, 11 they trigger a strong innate immune response by activating the tlr5 pathway through the adjuvant flagellin. 13 because of their size and shape they have the potential to reach follicular dendritic cells that are critical for antigen presentation and processing. 14 although macrophages play a role in immunity, interactions between sapn and macrophages were not studied. sapns induce immune response that are orders of magnitudes stronger than keyhole limpet hemocyanin, which is a standard vaccine carrier. we previously designed sapn-based vaccine candidates for various infectious diseases including malaria, 10, 11, 14, 15 hiv, 16 sars, 17 and influenza. 18 earlier findings, and recent parallel work with a recombinant polypeptide, sapns, and gla-se ( fig. 1 and unpublished data [dl] ) provide the foundation for our present studies. these earlier findings provide a basis for use of immunosense selected peptides from different genetic isolates of t. gondii (fig. 1a) , a flagellin scaffold, 7, 8, 13, 19 and adjuvanting with gla-se. [20] [21] [22] [23] earlier studies from the walter reed army institute of research with malaria based sapns demonstrated that flagellin molecules improved immunogenicity (dl, pb, unpublished work). initially, this was the basis for using flagellin as a sapn scaffold in our t. gondii studies (fig. 1b) . this approach was also used in our work with influenza. 24 this work suggested that flagellin would be helpful as a scaffold and immunogen in our newest t. gondii work. in experiments that provided a significant part of the foundation for our approach with sapn to protect against toxoplasmosis, we (dl, pb, unpublished work) found the following: 1) gla-se or gla-se-like adjuvant was needed to produce significant titers of anti-nanoparticle antibody; 2) purified igg from immunized monkeys completely protected naïve mice (100%), when they were challenged with a lethal dose of 5000 sporozoites that express full-length plasmodium. falciparum circumsporozoite protein. purified igg from a control monkey did not protect any mice; 3) purified igg from immunized monkeys, mixed with p. falciparum sporozoites, prevented the sporozoite from infecting primary hepatocytes from human liver in tissue culture. igg from control monkeys did not. thus, we used this preliminary, foundational data when we chose gla-se as the adjuvant for our studies herein. gla-se has two components, gla and se. gla is too hydrophobic to be used alone and any formulation of gla would have other excipients making the formulation nonequivalent to gla. earlier studies demonstrated that the emulsion, called "se", did not adjuvant most proteins when administered alone. at present, gla-se is in pre-clinical studies or clinical trials as an adjuvant to prevent cancer, herpes, leishmania, and mycobacterium tuberculosis infections. our earlier studies also demonstrated that gla-se was superior to alum as an adjuvant for our polypeptide. 25 gla-se was also superior to alum in primates immunized with sapn. in fact, alum diminished the response to gla-se plus sapn (dl, pb, unpublished work). in our previous studies with t. gondii, we constructed sapns displaying the dense granule epitope (gra7 20-28 ) and pan-dr binding epitope padre. 26 we evaluated these vaccine components in hla-b*0702 transgenic mice. 9 immunization of these mice activated gra7-specific cd8 + t cells that produced ifn-γ. thereby, these mice were protected against subsequent challenge with high inocula of type i and type ii parasites. these initial results highlighted the potential to protect against toxoplasmosis with a sapns vaccine approach. in the present study, five epitopes which bind to hla-a11-01 were evaluated for their efficacy in a sapn-vaccine in hla-a11-01 transgenic mice. 5 these included epitopes from the surface antigen (sag1), the dense granule proteins (gra5 and gra6), and the surface antigen-1-related sequences (srs52a). 5 in these constructs, the cd8 + hla-a03-11 supertypes-restricted epitopes were linked by n/kaaa spacers. they were conjugated with padre, a universal cd4 + helper t lymphocyte epitope. 26 this synthetic polypeptide is effective in mice and more effective than the pooled peptides separately. 5, 23 padre binds promiscuously to mhc class ii variants, and augments effector functions of cd8 + t cells through stimulation of il2 production by cd4 + t helper cells. 27, 28 epitopes eliciting both cd4 + and cd8 + t cells are important components in the formulation of successful vaccines that drive protective responses. 29 our data show that incorporating padre into the sapn constructs and delivering it in tlr4 ligand emulsion adjuvant (gla-se), resulted in activation of cd8 + t cells. this vaccine formulation led these cells to produce ifn-γ. they protected against subsequent challenge with type ii parasites given as a high inoculum. thus, our work highlights the potential for the use of sapn as a platform for the delivery of cd8 + and cd4 + -restricted epitopes, formulated with the gla-se adjuvant, to protect against toxoplasmosis. preparation and characterization of cd8 + -sapn and empty-sapn the sapn constructs were expressed, purified and folded to form nanoparticles ( fig. 1c-e) . the protein has a relative molecular weight of about 48 kda on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) (fig. 1c , e). transmission electron microscopy (fig. 1d) showed a relatively uniform distribution of non-aggregated nanoparticles of about 30 nm in diameter. in vivo immunogenicity of cd8 + t cell-eliciting sapns hla-a*1101-transgenic mice were immunized intramuscularly with cd8 + t cell-eliciting sapns combined with gla-se. mice were immunized three times intramuscularly at 2 week intervals. empty-sapns plus gla-se or pbs were used in sham immunizations of control mice. cd8 + -t cell-eliciting sapn-gla-se vs. empty-sapn-gla-se were compared in hla-a*1101 transgenic mice as described. spleen cells were obtained from immunized hla-a*1101 transgenic mice 2 weeks after final immunization. ifn-γ produced by splenocytes cultured with the pool of peptides was measured. figure 2 shows ifn-γ secretion is high in mice immunized with cd8 + -t cell-eliciting sapn plus gla-se when stimulated with padre or the gra6 peptide. the other peptides also elicited ifn-γ production. in our earlier work, 25 and herein, effects of the separate peptides were additive (figs. 2b and 3). the polyepitopes elicited the best response earlier 25 and herein (fig. 3) . figure 3a and b indicate that ifn-γ secretion in cultures with the sag1, gra6, gra3, and srs52a peptides was significantly enhanced by immunization with these peptides but not empty-sapn or pbs. significantly more ifn-γ secretion was observed when cells were stimulated with these pooled peptides plus padre. thus, the association of cd8 + t cell-and cd4 + t cellrestricted peptides contributes to ifn-γ production in hla-a*1101 transgenic mice. in vitro tlr5 stimulation the seaporter tlr5 cell-line was exposed to varying concentrations of the sapns. the sapn included: empty-sapn that do not contain the cd8 + epitopes but still have flagellin; cd8 + -sapn containing the polypeptide with the five restricted cd8 + epitopes; recombinant polypeptide; and recombinant flagellin (as control). the concentrations of sapns used were 0.01, 0.1, 1, 10, 100, and 1000 ng/ml. fold increase in seap expression for each protein sample over non-treated controls reflected level of tlr5 stimulation. as shown in fig. 4a -c, tlr5 activity was fig. 1 assembly of cd8-sapns. a phylogenetic tree showing 62 genetic isolates of toxoplasma analyzed herein. these are in the multisequence alignments of proteins, and peptides derived from them, utilized to create our artificial immunogenic ("smart") protein. b flagellin is used as a scaffold into which epitopes are intercalated from toxoplasma. earlier logic for inclusion of flagellin as adjuvant and scaffold came from work with malaria (http://www.internationalinnovation.com/build/wp-content/uploads/2016/05/david_lanar_intl_innovation_ infectious_diseases_research_media_lr.pdf), 19 as well as with influenza. 24 computer model of the prototype. the core particle composed of the pentameric and trimeric coiled coils is shown in green and blue, respectively. the following are attached to the trimeric coiled coil: the trl5 agonist flagellin (d0 and d1 domains) (purple) with the a11 epitopes (yellow) and a cd4 epitope string (magenta). c sds-page of the purified protein. lanes are as follows: lane 1: mw (molecular weight markers). lane 2-5: elution fractions were from 19 to 22. samples derive from the same experiment and the gels/blots were processed in parallel. d transmission electron microscopy of the nanoparticle preparation. e sds-page 4-20% of the purified protein. lane 1: mw (molecular weight markers). lane 2: cd8-sapn. lane 3: empty-sapn. samples derive from the same experiment and the gels/blots were processed in parallel significantly enhanced by the empty-sapns and the cd8 + -t celleliciting sapns, but not the control polypeptide. surprisingly, flagellin in empty-sapn particles have higher tlr5 activity than recombinant flagellin alone. sapns with gla-se adjuvant confer robust protection against t. gondii in hla-a*1101 transgenic mice in the results shown in fig. 5 , we had immunized mice with either cd8 + t cell-eliciting sapn with gla-se adjuvant, or empty-sapn with gla-se adjuvant, or adjuvant alone, or padre alone, or pbs. we then challenged 2 weeks after the last immunization with type ii strains of t. gondii expressing luciferase. brains from these mice were imaged with a xenogen camera 21 days after challenge with 2000 me49-fluc tachyzoites. figure 5a and b show that luminescence from t. gondii in mice immunized with cd8 + t cell-eliciting sapn plus gla-se was significantly lower than in mice immunized with control empty-sapn plus gla-se, gla-se alone, padre alone, or pbs. this finding correlates with a reduction of the number of cysts per brain in mice that received cd8 + -t cell-eliciting sapn plus gla-se adjuvant (fig. 5c ). improved vaccination and delivery approaches to elicit cellular immune responses against t. gondii are needed. in our previous studies we defined a panel of octamer/nonamer peptides restricted by mhc class i molecules. these peptide epitopes bind to and elicit ifn-γ responses from cd8 + t cells isolated from hla a02, a03, and b07 individuals. these class i supermotifs are present in essentially all the human population worldwide, but with different frequency in different regions. when given with the gla-se adjuvant, these pooled peptides were able to protect graph shows the count of spots for splenocytes of untreated, empty-sapn + gla1 cd8-sapn + gla-se group of mice. gla designates gla-se in this figure. *p < 0.05 for all ifn-γ elispots compared to controls haplotype specific hla supermotif transgenic mice. this protection was measured as survival and reduced parasite burden. our capability to control the ability of peptides and proteins to self-assemble into particles which have a well-defined size and shape allows us to design mechanically and chemically stable particles. these sapns combine strong immunogenic effects of live attenuated vaccines with high specificity in eliciting immune responses of protein-based vaccines because they resemble virus capsids. it is apparent that the sapns have a great potential to serve as a platform for vaccines beyond their ability to present antigens in a repetitive manner. in contrast to live attenuated vaccines, sapn-derived vaccines pose no significant risk of infection. they are very versatile and flexible in their design leading to better biophysical and immunologic properties. furthermore, bacterial protein expression, purification, and selfassembly into nanoparticles reduces the time needed for largescale vaccine production. herein, we used the sapns to present immunogenic peptide epitopes to a host's immune system based on the assembly of five protective cd8 + ctl hla-a03-11 restricted supertypes in addition to the universal helper epitope, padre. all epitopes were flanked at the c-terminus by n/kaaa spacers, which promote optimal immunogenic processing. our data showed potent immunogenicity (high ifn-γ secretion) when splenocytes were stimulated by these peptides through immunization in vivo, and then exposure in vitro. in separate studies, 24 we found that sapn, which contains flagellin, protected better against influenza than sapn without flagellin. this flagellin scaffold then became our sapn platform going forward. in our tlr5 activity assay, the sapn with the flagellin scaffold shows good stimulation of tlr5. however, the activity is reduced compared to the empty-sapn. this could be due to some interference with tlr5-binding and the presentation of the cd8 + t cell-restricted epitopes because the cd8 + epitopes string was engineered into the flagellin molecule to replace the d2 and d3 flagellin domains. thus, our future work will utilize this approach to engineer different sapn constructs with optimized processing and immunogenicity for all our vaccine constituents. the proposed mechanisms for inducing innate immunity by our sapn is the ligation of tlr4 by gla in an emulsion 30 and tlr5 by flagellin on the surface of the sapn. 13 mccoy et al.'s data suggested cross presentation 14 of cd8 + stimulating epitopes in sapns (fig. 1a) . gla-se has been used with sapn to successfully immunize against p. falciparum by eliciting antibody and t cells, whereas sapn without gla-se was not effective (dl, pb, unpublished results). the adjuvant was safe in primates and now is entering clinical trials in humans. despite remarkable protection provided by our sapn vaccination in this study, some brain cysts were still detected. thus, potential improvements in induction of protective immune responses could be made with the addition of separate nanoparticles with other cd4 + and cd8 + t cell-eliciting epitopes of various t. gondii proteins from several parasite life stages and fig. 4 seaporter tlr5 cell line responses to flagellin and sapn. seaporter tlr5 cell-line was exposed to varying concentrations of each indicated protein, and the level of tlr5 stimulation was determined by the level of seap expression. fold increase in seap expression for each protein sample over non-treated controls, error bars are standard error of the means. a two-way anova model was fit with protein concentration and type as factors. there was a significant protein concentration by type interaction (p < 0.001); this indicated that the differences across types depended on the concentration and that the differences across concentrations varied by type. specifically, there weren't statistically significant differences across types at the two lowest concentrations (0.01 and 0.1), but there were significant differences between types at the 1, 10, 100, and 1000 ng/ml concentrations (p < 0.001 for all). subsequent pairwise contrasts at these 4 concentrations found that the 5a11 restricted cd8 + group (the recombinant protein without flagellin) was significantly different from the other 3 groups in all cases except for the 5a11 restricted cd8 + vs. cd8-sapn comparison at the 1 ng/ml concentration. in addition, at each of these 4 concentrations, the empty-sapn was significantly different (greater than) from the cd8-sapn. there was a significant concentration effect for all protein types (p < 0.001) except 5a11 restricted cd8 + (p > 0.9) prevention of toxoplasmosis in humans by nanovaccines k el bissati et al. potentially b-cell epitopes to stimulate a potent antibody response. cell-mediated immunity, with cytolytic t cells and ifnγ production, is considered to be the desired primary, protective, immune response. 28, 31 nonetheless, antibodies may contribute to protection. addition of the micronemal proteins (mics) or other proteins that induce antibodies that are neutralizing, adhesion or invasion blocking, or complement fixing, could further improve protection, if they play a significant part in attachment to or penetration of the host cell by the parasite. mics have recently been used as recombinant vaccines and showed promising protection levels. 32 mic1 also stimulates il 12 production in mice. possibly, these proteins could also be engineered in separate sapns to yield a multi-sapn vaccine to protect against toxoplasmosis. earlier studies provide support for using gla-se as an adjuvant for a wide variety of protein vaccines, including our own. we evaluated a p. falciparum sapn vaccine and demonstrated gla-se was essential, or improved immunogenicity, vs. a related sapn (dl, pb, unpublished). this study involved presenting antigens to protect against the phylogenetically related apicomplexan malaria parasite in non-human primates. it was found that gla-se was needed for immunization of primates even when this was not the case in mice. in other earlier work, separate constituents of gla-se were used. gla alone was called "gla-af" when it was prepared in an aqueous formulation. this formulation did not contain an emulsion or extra excipients so it is not equivalent to the gla in gla-se. gla-af, se alone and gla-se formulations were compared in some of these earlier studies. in almost all of these different systems the value of using both gla and se together was proven (table 1) . [20] [21] [22] [23] [33] [34] [35] [36] this work has been advanced to the clinic, demonstrating both efficacy and safety in studies with gla and se formulated and administered together. we leveraged this extensive, earlier experience to produce our gla-se adjuvanted sapn vaccine for t. gondii. in our work with this new sapn-design, the flagellin molecule itself is an integral part of the sapn scaffold (fig. 1b) , with or without the a11 cd8 + t cell-eliciting epitopes. this sapn scaffold lacking the cd8+ epitopes conferred only a small amount of protection compared with the scaffold with the inclusion of the a11 peptides. it is not possible to create a relevant separate control without flagellin because in this scaffold the hla a11 binding peptides are intercalated into the flagellin molecule itself as shown in fig. 1 . the location of cd8 + epitopes within a protein sequence has been shown to be critical. our arrangement of peptide epitopes into a polypeptide induced robust immunity. 25 deconvolution of peptide components has shown that certain epitopes alone may have different toxicity when separated from other peptides (el bissati, mcleod, et al., in preparation). we already know that the adjuvanted polypeptide can protect in studies that are described in a separate manuscript. 25 we also found that the hla class 1, a*1101 interacting peptides are specific for hla a*1101 and not to other hla supermotifs b7 or a2. 25 further, we demonstrated that the mouse c57bl6 macrophages cannot present these peptides to hla a*1101 t cells. 25 further, these five cd8 + epitopes, as well as full-length proteins from which they originate, were characterized to determine how well conserved the proteins, and especially the specific peptides we included, are across multiple strains of genetically divergent parasites from different geographic regions (tables 2-4 genetically divergent strains (fig. 1a) supports the use of an immunosense approach. this approach creates a single protein which contains the relevant epitopes but does not include extraneous epitopes that are potentially harmful, as certain t. gondii epitopes are known to be. there were far fewer polymorphisms in our smaller octamer nonamer peptides (tables 2-4 ) than in the full-length proteins (table 3 , figs. s1-s5). there were good binding octamer/nonamers for three a11 epitopes among all the different genetic isolates ( table 4 ). the predicted binding scores for the peptides from the many genetically polymorphic strains were high for all but two peptides (table 4 ). in contrast, it would take many full-length variants to obtain good geographic coverage of either of these polymorphic proteins if peptides other than our octamer/nonamer were critical to that protection. this provides further conceptual support for using an immunosense approach to make a vaccine with, potential to work well, and most parsimoniously, in many geographic areas. there are recent data concerning the unique processing of t. gondii proteins in human cells. quite remarkably, there are longer (likely decoy) peptides that bind hla-a2 in the natural infection of thp1 cells. 37 this makes our targeted immunosense approach, beginning with human cells, then using hla transgenic mice, especially valuable in creating a vaccine for people, rather than mice. these are critical considerations important beyond a vaccine protective against just this organism, when one wants to have broad utility across different demographics, worldwide. this is what we are working toward because of the substantial disease burden of toxoplasmosis as a global clinical problem. 38 data from studies of human cells and mice, in our work, considered together, demonstrate the robust and practical use of this model system. [3] [4] [5] [6] 25 antigen processing and presentation in humans and mice have differences that are well known. for example, as shown elegantly, recently, 39 human and murine tapasin diverge in sequence. these tapasins are chaperones of mhc class i molecules. newer murine models with human tapasin and proteasome might improve vaccine potency and be more relevant to human vaccine development. 39 alternatively, our optimized cleavage sites, based on human infection, using human pbmc, may work as effectively in our hla-a*1101 mice. this comparison will be tested in future studies. further, there are other significant differences between mice and humans. murine models have been imperfect predictors of vaccine performance in humans, apart from differences in hla, tapasin or variants of erap. differences in erap between mice and humans does not cause differences in processing and presentation. other differences between mice and humans include very different total blood volume, rate of metabolism, skin composition/absorption rate, different tlr specificities, as well as other basic differences between murine and human cells. for example, human t cells, express class ii mhc, while mouse t cells do not. nonetheless, relating to the question of whether hla transgenic mice are a good approximation of human responses, sette et al. made the following comment: despite the differences, when this issue was considered in detail, practical studies confirm that "although there ought to be a difference, actual data in peer reviewed studies, show that there is little difference". 40 humanized mice with blood and liver transplants and other modifications are also still imperfect. these humanized mouse systems have not yet worked really well to demonstrate practical feasibility. for vaccine development, the fda requires animal in vivo immunogenic data for ind submission. vaccine developers have followed this requirement for the vaccines they are creating, which include viral vector or vlp-based vaccines. to date, there is no system that allows skipping the animal testing step. most vaccines (e.g. viral, virus-like particles, proteins, bacterial) generate an immune response in an animal model. these immune responses provide preliminary data prior to studies in non-human primates and then in humans. this approach permits one to conclude that the vaccine is safe and 'active', even if the animal model is not absolutely predictive of precise human correlates, although that would be ideal. we have found that our approach appears to provide insight and to work effectively. [3] [4] [5] 25 this approach includes using bioinformatics, testing human cells for immunogenicity, and then testing those down-selected peptides re-assembled into a protein with linkers designed for proper cleavage. 41 this is followed by testing for efficacy and safety using hla transgenic mice. [3] [4] [5] 25 this approach is shown in our data herein, in our previous foundational experiments, 25 and also by many others using other systems. this is for both immunogenicity of peptides or polypeptides or dna or rna in human cells first. this is then extended to murine cells, followed by protection measured as reduced parasite burden and enhanced survival. although imperfect, there is considerable prior support, and support in these recent studies. the use of hla transgenic mice can obviate problems of heterogeneity, both for mhc supermotifs, and parasite isolates. this is in a proven practical manner in vaccine development. 19, 37, [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] the issues of genetic polymorphism, as well as potential harmful constituents, are amplified in full-length natural proteins (tables 1-4 ; fig. 1 , s1-s5). thus, the choice of peptides that are sufficient to interact with hla molecules that are present in more than 90% of the human population can be made using bioinformatics in a rational and parsimonious manner. this approach considers parasite genetic variation in an inductive, immunosense manner that is proving to be valuable for development of vaccines for humans. the inclusion of flagellin into immunogens can serve as a potential adjuvant. [53] [54] [55] [56] [57] there is experience where flagellin has been safe and effective as an adjuvant in pre-clinical animal studies. [53] [54] [55] [56] [57] it also has been effective when used for immunizations of both younger and older persons in clinical trials for influenza vaccines. [53] [54] [55] [56] [57] there is a robust literature which describes studies of the mechanisms whereby this tlr5 ligand functions as an adjuvant. [53] [54] [55] [56] [57] in summary, our study showed that a sapn-protein chain with five cd8 + t cell-eliciting mhc class i epitopes from t. gondii, and the mhc class ii epitope padre, can be refolded to form a nanoparticle. using hla-a*1101 transgenic mice, we demonstrate that the sapn emulsified in gla-se adjuvant elicits a protective mhc class i response. thus, our work demonstrates that we have developed an improved assembly of peptides for cross presentation of cd8 + t cell eliciting epitopes (fig. 6 ) in vaccines to prevent toxoplasmosis. ksfkdilpk (sag1 224-232 ), stfwpcllr (sag2c [13] [14] [15] [16] [17] [18] [19] [20] [21] , avvsllrllk (gra5 89-98 ), ssayvfsvk (srs52a 250-258 ), amltafflr (gra6 164-172 ) 25 and padre, a universal cd4 + helper epitope (akfvaawtlkaaa) 26 were used in the vaccine constructs. 25, 52 infectious diseases research institute (seattle, washington) synthesized the tlr4 agonist adjuvant called gla-se. 3-6, 20-23, 25 this was prepared and used as a stable oil-in-water emulsion. the methods using dna coding for the nanoparticle constructs were similar to those described in our earlier work. 18 briefly, they were prepared using standard molecular biology procedures as described in our earlier work from our laboratory by babapoor et al. 18 specifically, plasmids containing the dna coding for the protein sequence were used. 18 they were constructed by cloning into restriction sites in the sapn expression plasmid. 18 we used a sapn construct we had developed and described earlier. 18 briefly, this construct is composed of a pentameric coiled-coil tryptophan zipper. 18 this zipper is linked by a glycine residue to a trimeric de-novo designed leucine zipper coiled coil. 18 in this construct, a flagellin construct composed of the d0 and d1 domains (residues 1-177 and 249-372) of salmonella enterica flagellin from the structure with pdb-code 3v47 from the rcsb protein data bank is used to extend the protein chain at the c-terminus 18 (fig. 1) . the cd8 + -peptide sequence avvsllrllknamltafflrnaaaksfkdilpk-kaaassayvfsvkkaaakfvaawtlkaaakstfwpcllr with the five cd8 + epitopes 25 also containing padre 25, 26 was next inserted into the d1 domain of flagellin. 18 this polypeptide completely replaces the d2 and d3 domains to generate the cd8 + t cell-eliciting sapn called "cd8-sapn". 18 overall, the positive charge of this epitope string is balanced with stretches of negative charges at both ends of the epitope sequence. 18 our empty-sapn was generated using the short linker kykdgkgddk to replace the d2 and d3 domains of flagellin. this was performed exactly as we had performed and described in our earlier work from our laboratory by babapoor et al.: 18 plasmids were transformed into escherichia coli bl21 (de3) cells. 18 e. coli were grown at 37°c in luria broth with ampicillin. 18 we induced expression using isopropyl β-d-thiogalacto-pyranoside. cells were removed from 37°c 4 h after induction. 18 they were harvested by centrifugation at 4000 x g. we stored the cell pellet at −80°c. we then thawed the cell pellet, keeping it on ice. 18 we then suspended the pellet in a lysis buffer consisting of 9 m urea, 100 mm nah 2 po 4 , 10 mm tris ph 8, 20 mm imidazole, and 0.2 mm tris-2-carboxyethyl phosphine (tcep). sds-page was used to assess our protein expression level. 18 the same methodology we used earlier was used. 18 briefly, sonication was used to lyse cells, as described from our laboratory earlier. 18 centrifugation at 30,500 × g for 45 min 18 was used to clarify the lysate. then, for at least 1 h, our cleared lysate was incubated with ni-nta agarose beads (qiagen, valencia, ca, usa). next, the column was washed with lysis buffer. this was followed by a wash with a buffer containing 9 m urea, 500 mm nah 2 po 4 , 10 mm tris ph 8, 20 mm imidazole, and 0.2 mm tcep. a ph gradient was used to purify the protein while bound to the column. the ph gradient for these wash steps was created as follows: 9 m urea, 100 mm nah 2 po 4 , 20 mm citrate, 20 mm imidazole, and 0.2 mm tcep, 18 with subsequent washes performed at ph 6.3, 5.9, and 4.5. 18 to elute the protein, we used the lysis buffer, after the ph gradient, with a gradient of increasing imidazole concentrations. 18 we used methodology we have described in our earlier work. 18 specifically, for refolding, our protein was first rebuffered to the following conditions: 9 m urea, 20 mm tris ph 8.5, 50 mm nacl, 5% glycerol, 2 mm edta. 18 4 µl of a solution with a concentration of 1.8 mg/ml protein was added to the same buffer solution without urea to a final concentration of 0.05 mg/ml for quick refolding of a first screen. 18 we used this because this quick dilution from denaturing (urea) to native (no urea) buffer conditions triggers refolding of protein. 18 we then used negative stain transmission electron microscopy at different resolutions to analyze our solution. 18 next, we used further screens for optimal refolding conditions. 18 these were performed with smaller sampling sizes of the ph and ionic strength. 18 in vitro tlr5 response assay the methods were the same as those used in our recent work. 28 activation through tlr5 was assessed for sapn as we described recently. 24 testing was done using tlr/nf-κb/seaporter™ stably transfected hek 293 cell lines (novus biologicals, littleton, co; tested for mycoplasma but not authenticated by str profiling) as follows: all cell lines were stably cotransfected cell lines which express tlr5 and have a secreted alkaline phosphatase (seap) reporter gene under transcriptional control of an nf-κb response element. fourteen thousand cells per well were seeded in a 96-well plate at passages 5-9. 20-4 h later, we removed growth media. growth media was replaced with dmem high glucose (hyclone, logan, ut). this contained either a sapn, or recombinant flagellin (novus biologicals), at concentrations of 0.1, 1, 10, 100, 1000 ng/ml, each in triplicate. media alone was present in control wells. wells were exposed to the stimulus for 24 h. then, supernatant was collected and used to determine whether seap was present. this was determined with a reporter assay kit for seap (novus biologicals). this was done using the manufacturer's instructions. media-only controls were used to normalize seap activity. this was used to determine each construct's ec50. triplicate determinations were utilized for each experimental condition. the mice were those we created and described earlier. 5, 23 the methods were identical to those used in our earlier work. [3] [4] [5] [6] 23 specifically, "hla-a*1101/k b transgenic female mice were generated and then bred/ produced at pharmexa-epimmune (san diego, ca). 23 they were then embryo-rederived at taconic and jax laboratories. 23 colonies were then expanded and they were then maintained and produced in isolators at the university of chicago. 23 these mice express a chimeric gene called hla-a*1101/k b transgene. 23 this chimeric gene consists of the 1st and 2nd domains of hla-a*1101 and the 3rd domain of h-2k b . 23 mice were between 10 and 14 weeks of age in experiments. mice were maintained in spf conditions throughout. 23 all of our studies were performed with the institutional animal care and use committee at the university of chicago's review, approval, and oversight. to assess the immunogenicity of the sapns, mice with the hla-a*1101 transgene were inoculated intramusculary. in this injection, 20 μg sapn was emulsified in the tlr4 agonist, i.e., 5 μg of gla-se. the immunizations were administered three times at 2 weeks intervals. for the experiments in which these mice were challenged, challenge was at 14 days postimmunization. 3 specifically, they were challenged intraperitoneally using 2000 type ii (me49-fluc) parasites. 23 elispot assay to determine murine splenocyte immune responses this was performed as described in our earlier work which provided the foundation for our own present studies. [3] [4] [5] [6] 25 specifically, spleens were harvested 14 days after immunization 3-6, 25 as follows: initially, they were pressed through a 70 µm screen. [3] [4] [5] [6] 25 this allowed for formation of a suspension of single-cells. erythrocytes were depleted from this suspension. akc lysis buffer (160 mm nh 4 cl, 10 mm khco 3 , 100 mm edta) was used to deplete the rbcs. [3] [4] [5] [6] 25 hank's balanced salt solution (hbss) was used to wash splenocytes twice. [3] [4] [5] [6] 25 then the splenocytes were resuspended in rpmi-1640 supplemented with 2 mm l-glutamax. [3] [4] [5] [6] 25 murine splenocyte elispot assays were performed as described earlier. [3] [4] [5] [6] 25 this was done using anti-mouse ifn-γ mab (an18) and biotinylated antimouse ifn-γ mab (r4-6a2). [3] [4] [5] [6] 25 in each well, 2.5-5 × 10 5 splenocytes were plated. [3] [4] [5] [6] 25 mabtech (cincinnati, oh) was the source of all of the antibodies and all of the reagents used to perform elispot assays. [3] [4] [5] [6] 25 a minimum of three replicate wells were used to plate cells for each condition, 3-6, 25 as described earlier, to measure spot-forming cells per 10 6 murine splenocytes. [3] [4] [5] [6] 25 bioluminescence imaging to determinine outcomes of type ii parasite challenge we imaged mice infected with 2000 fluc tachyzoites of the me49 strain of t. gondii as described in our earlier work. 25 twenty-one days after challenge, an in vivo imaging system (ivis; xenogen, alameda, ca) 25 allowed us to visualize luciferin injected retroorbitally interacting with luciferase in the parasites. 25 these mice were anesthetized. anesthesia was performed in an o 2 -rich induction chamber with 2% isoflurane. 25 imaging took place 12 min after receiving luciferin. 25 living image ® 2.20.1 software (xenogen) was used for assessment of photonic emissions. 25 pseudocolor representations of light intensity and mean photons/region of interest represent parasite burden in the imaging. 25 all these mouse experiments were replicated a minimum of two times, as in our earlier work. 25 in each group we used five mice. enumeration of cysts in mouse brains after type ii parasite challenge mouse brains were collected at day 21, homogenized in 1 ml of saline (0.85% nacl), and 50 µl of the homogenate was used to count the tissue cysts, microscopically, as described earlier. 25 cyst count was then multiplied by 20. this product then was used to determine the number of tissue cysts per brain. statistical analyses and additional detail concerning animal models differences between the groups, as we previously described. 6 means ± sd are used to express data. a p value <0.05 was considered to be statistically significant for our results. 6 sample size in the in vivo studies was selected to be able to detect significant differences in luminescence based on our prior studies. 25 with 5 per group, there is 80% power to detect a 2standard deviation difference between groups. with 3 per group, there is 80% power to detect a 2.7-standard deviation difference between groups. all female mice we bred were utilized. they were randomly selected for the different groups but age-matched in the different groups within the experiment. there was no blinding in this experiment. in all in vivo experiments, there were 5 mice per group. in all in vitro experiments, there were 3 mice per group that provided splenocytes. all experiments were replicated at least twice. representative experiments, of at least 2 separate trials, are shown. there was no data excluded from analyses. the data that support the findings of this study are available from toxodb (http://toxodb.org/toxo/) and the corresponding author on reasonable request. why prevent, diagnose and treat congenital toxoplasmosis? early and longitudinal evaluations of treated infants and children and untreated historical patients with congenital toxoplasmosis: the chicago collaborative treatment trial identification of t. gondii epitopes, adjuvants, and host genetic factors that influence protection of mice and humans toxoplasma gondii hla-b*0702-restricted gra7(20-28) peptide with adjuvants and a universal helper t cell epitope elicits cd8(+) t cells producing interferon-gamma and reduces parasite burden in hla-b*0702 mice human immunome, bioinformatic analyses using hla supermotifs and the parasite genome, binding assays, studies of human t cell responses, and immunization of hla-a*1101 transgenic mice including novel adjuvants provide a foundation for hla-a03 restricted cd8+ t cell epitope based, adjuvanted vaccine protective against toxoplasma gondii towards an immunosense vaccine to prevent toxoplasmosis: protective toxoplasma gondii epitopes restricted by hla-a*0201 peptidic nanoparticles as drug delivery and antigen display systems self-assembling peptide nanoparticles useful as vaccines. us patent us8,546 effectiveness of a novel immunogenic nanoparticle platform for toxoplasma peptide vaccine in hla transgenic mice a nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria protective antibody and cd8+ t-cell responses to the plasmodium falciparum 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vaccines tepitool: a pipeline for computational prediction of t cell epitope candidates hla-drb1 alleles are associated with different magnitudes of dengue virus-specific cd4 + t-cell responses a quantitative analysis of complexity of human pathogen-specific cd4 t cell responses in healthy m. tuberculosis infected south africans immunodominant dengue virus-specific cd8+ t cell responses are associated with a memory pd-1 + phenotype t-cell recognition is shaped by epitope sequence conservation in the host proteome and microbiome the length distribution of class i-restricted t cell epitopes is determined by both peptide supply and mhc allele-specific binding preference an ontology for major histocompatibility restriction characterization of the peptide binding specificity of the hla class i alleles b*38:01 and b*39:06 deciphering the human antigenome automatic generation of validated specific epitope sets development of high potency universal dr-restricted helper epitopes by modification of high affinity dr-blocking peptides induction of a potent immune response in the elderly using the tlr-5 agonist, flagellin, with a recombinant hemagglutinin influenza-flagellin fusion vaccine (vax125, stf2.ha1 si) development of vax128, a recombinant hemagglutinin (ha) influenza-flagellin fusion vaccine with improved safety and immune response safety and immunogenicity of a recombinant m2e-flagellin influenza vaccine (stf2.4xm2e) in healthy adults safety and immunogenicity of a recombinant hemagglutinin influenza-flagellin fusion vaccine (vax125) in healthy young adults immunopotentiators in modern vaccines we thank and are grateful to nihlab shastri (university of california, berkeley) and lo vang (paxvax) for critical advice and suggestions. we gratefully acknowledge the mann-cornwell, morel, engel, rooney-alden, pritzker, langel, drago, and rosenthal families for their support of this work. this project has been funded in whole or part with federal funds from the national institute of allergy and infectious diseases, national institutes of health, department of health and human services under award numbers dmid-niaid u01 ai77887 (to rm), r01 ai027530 (to rm), and u19 ai110819 (to hl). the research was also supported by the knights templar eye foundation (to ke) and the research to prevent blindness foundation (to rm). k.e., p.b., s.r., d.e.l., h.l., and r.m. designed research; k.e., y.z., i.j.b., s.p., s.k.r., j.s., and c.k. performed research; k.e., y.z., s.p., s.k.r., c.k., c.r., d.e.l., s.r., c.f., d.c., j.a., a. s., h.l., p.b., and r.m analyzed data; and k.e., p.b., and r.m. wrote the paper. all authors read and approved the final manuscript version. supplementary information accompanies the paper on the npj vaccines website (doi:10.1038/s41541-017-0024-6).competing interests: p.b. has an interest in alpha-o peptides, a company with a focus on sapn. r.m. agreed to be on the scientific advisory board for this company. this company has patents or patents pending on relevant technology. the following authors (r.m., k.e.b., y.z., p.b., j.a., a.s., j.s., s.r., c.f., d.c., c.w.r.) have submitted a patent application covering much of our vaccine work over the past years, including the work described in this manuscript. this is with the ultimate goal of making a vaccine that can be moved into the clinic to prevent suffering from human toxoplasmosis, so that this vaccine can be widely available for humans. the other authors declare no competing interests. funding sources had no influence in study design; nor in the collection, analysis and interpretation of data; nor in the writing of this report; and no interest in the decision to submit the paper for publication.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-000488-x5ardo5j authors: pedersen, lasse eggers; harndahl, mikkel; rasmussen, michael; lamberth, kasper; golde, william t.; lund, ole; nielsen, morten; buus, soren title: porcine major histocompatibility complex (mhc) class i molecules and analysis of their peptide-binding specificities date: 2011-07-08 journal: immunogenetics doi: 10.1007/s00251-011-0555-3 sha: doc_id: 488 cord_uid: x5ardo5j in all vertebrate animals, cd8(+) cytotoxic t lymphocytes (ctls) are controlled by major histocompatibility complex class i (mhc-i) molecules. these are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating ctls. the polymorphism of the mhc effectively individualizes the immune response of each member of the species. we have recently developed efficient methods to generate recombinant human mhc-i (also known as human leukocyte antigen class i, hla-i) molecules, accompanying peptide-binding assays and predictors, and hla tetramers for specific ctl staining and manipulation. this has enabled a complete mapping of all hla-i specificities (“the human mhc project”). here, we demonstrate that these approaches can be applied to other species. we systematically transferred domains of the frequently expressed swine mhc-i molecule, sla-1*0401, onto a hla-i molecule (hla-a*11:01), thereby generating recombinant human/swine chimeric mhc-i molecules as well as the intact sla-1*0401 molecule. biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules. a pan-specific predictor of peptide–mhc-i binding, netmhcpan, which was originally developed to cover the binding specificities of all known hla-i molecules, was successfully used to predict the specificities of the sla-1*0401 molecule as well as the porcine/human chimeric mhc-i molecules. these data indicate that it is possible to extend the biochemical and bioinformatics tools of the human mhc project to other vertebrate species. electronic supplementary material: the online version of this article (doi:10.1007/s00251-011-0555-3) contains supplementary material, which is available to authorized users. major histocompatibility complex class i (mhc-i) molecules are found in all vertebrate animals where they play a crucial role in generating specific cellular immune responses against viruses and other intracellular pathogens. they are highly polymorphic proteins that bind 8-11 amino acid long peptides derived from the intracellular protein metabolism. the resulting heterotrimeric complexes-consisting of the mhc-i heavy chain, the monomorphic light chain, beta-2 microglobulin (β 2 m), and specifically bound peptides-are translocated to the cell surface where they displayed as target structures for peptide-specific, mhc-irestricted ctls. if a peptide of foreign origin is detected, the t cells may become activated and kill the infected target cell. mhc-i is extremely polymorphic. in humans, more than 3,400 different human leukocyte antigen class i (hla-i) molecules have been registered (as of january 2011), and this number is currently growing rapidly as more efficient hla typing techniques are employed worldwide. the polymorphism of the mhc-i molecule is concentrated in and around the peptide-binding groove, where it determines the peptide-binding specificity. due to this polymorphism, it is highly unlikely that any two individuals will share the same set of hla-i molecules thereby presenting the same peptides and generating t cell responses of the same specificities-something, that otherwise would give microorganisms a strong evolutionary chance of escape. rather, this polymorphism can be seen as diversifying peptide presentation thereby individualizing t cell responses and reducing the risk that escape variants of microorganisms might evolve. in 1999, we proposed that all human mhc specificities should be mapped ("the human mhc project") as a preamble for the application of mhc information and technologies in humans (buus 1999) . since then, we have developed large-scale tools that are generally applicable towards this goal: production, analysis, prediction and validation of peptide-mhc interactions (ferre et al. 2003; harndahl et al. 2009; hoof et al. 2009; larsen et al. 2005; lundegaard et al. 2008; nielsen et al. 2003 nielsen et al. , 2007 ostergaard et al. 2001; pedersen et al. 1995; stranzl et al. 2010; stryhn et al. 1996) , and a "one-pot, read-and-mix" hla-i tetramer technology for specific t cell analysis (leisner et al. 2008) . here, we demonstrate that many of these tools can be transferred to other vertebrate animals as exemplified by an important livestock animal, the pig. we have successfully generated a recombinant swine leukocyte antigen i (sla-i) protein, sla-1*0401, one of the most common sla molecules of swine (smith et al. 2005) . using this protein, we have developed the accompanying biochemical peptide-binding assays and demonstrated that the immunoinformatics tools originally developed to cover all hla-i molecules, despite the evolutionary distance, can be applied to sla-i molecules. we suggest that the "human mhc project" can be extended to cover other species of interest. all peptides were purchased from schafer-n, denmark (www.schafer-n.com). briefly, they were synthesized by standard 9-fluorenylmethyloxycarbonyl (fmoc) chemistry, purified by reversed-phase high-performance liquid chromatography (to at least >80% purity, frequently 95-99% purity), validated by mass spectrometry, and quantitated by weight. positional scanning combinatorial peptide libraries (pscpl) peptides were synthesized using standard solidphase fmoc chemistry on 2-chlorotrityl chloride resins. briefly, an equimolar mixture of 19 of the common fmoc amino acids (excluding cysteine) was prepared for each synthesis and used for coupling in 8 positions, whereas a single type of fmoc amino acid (including cysteine) was used in one position. this position was changed in each synthesis starting with the n-terminus and ending with the c-terminus. in one synthesis, the amino acid pool was used in all nine positions. x denotes the random incorporation of amino acids from the mixture, whereas the single letter amino acid abbreviation is used to denote identity of the fixed amino acid. the peptides in each synthesis were cleaved from the resin in trifluoroacetic acid/1,2-ethanedithiol/triisopropylsilane/water 95:2:1:3 v/v/v/v, precipitated in cold diethylether, and extracted with water before desalting on c18 columns, freeze drying, and weighting. recombinant constructs encoding chimeric and sla-1*0401 molecules a synthetic gene encoding a transmembrane-truncated fragment encompassing residues 1 to 275 of human hla-a*11:01 alpha chain followed by a fxa-bsp-hat tag (fxa = factor xa cleavage site comprised of the amino acid sequence iegr, bsp = biotinylation signal peptide, hat = histidine affinity tag for purification purposes; see online resource 1) had previously been generated and inserted into the pet28 expression plasmid (novagen) (ferre et al. 2003) . synthetic genes encoding the corresponding fragments of the sla-1*0401 alpha chain (α 1 α 2 ) and α 3 , respectively, (sullivan et al. 1997) were purchased from genscript. to exchange domains and generate chimeric human/swine mhc-i gene constructs, a type ii restriction endonuclease-based cloning strategy (seamless® strategene; cat#214400, revision#021003a), with modifications, was used. all primers were purchased hplc-purified from eurofins mwg operon (ebersberg, germany), and all pcr amplifications were performed in a dna engine dyad pcr instrument (mj research, mn, usa). all constructs were validated by dna sequencing. the following mhc-i heavy chain constructs were made hhh, hhp, hpp, php, and ppp, where the first, second, and third letter indicates domains α 1 (positions 1-90), α 2 (positions 91-181), and α 3 (positions 182-275), respectively, and h indicates that the domain is of hla-a*11:01 origin, whereas p indicates that it is of sla-1*0401 origin. constructs were transformed into dh5α cells, cloned, and sequenced (abi prism 3100avant, applied biosystems) . validated constructs of interest were transformed into an escherichia coli production cell line, bl21(de3), containing the pacyc184 expression plasmid (avidity, denver, usa) containing an isopropylβ-d-1-thiogalactopyranoside (iptg)-inducible bira gene to express biotin-ligase. this leads to almost complete in vivo biotinylation of the desired product (leisner et al. 2008 ). to maintain the pet28-derived plasmids, the media was supplemented with kanamycin (50 μg/ml) throughout the expression cultures. when appropriate, the media was further supplemented with chloroamphenicol (20 μg/ml) to maintain the bira containing pacyc184 plasmid. e. coli bl21(de3) cells transformed with appropriate plasmids were grown for 5 h at 30°c, and a 10-ml sample adjusted to od (600) =1 was then transferred to a 2-l fedbatch fermentor (labfors®). to induce protein expression, iptg (1 mm) was added at od (600) ∼25 and the culture was continued for an additional 3 h at 42°c (for in vivo biotinylation of the product, the induction media was further supplemented with biotin (sigma #b4501, 125 μg/ ml)). samples were analyzed by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) before and after iptg induction. at the end of the induction culture, protease inhibitor (pmsf, 80 μg/l) was added, and cells were lysed in a cell disrupter (constant cell disruptor systems set at 2,300 bar) and the released inclusion bodies were isolated by centrifugation (sorval rc6, 20 min, 17,000×g). the inclusion bodies were washed twice in pbs, 0.5% np-40 (sigma), and 0.1% deoxycholic acid (sigma) and extracted into urea-tris buffer (8 m urea, 25 mm tris, ph 8.0), and any contaminating dna was precipitated with streptomycin sulfate (1% w/v). the dissolved mhc-i proteins were purified by ni 2 +/ida metal chelating affinity column chromatography followed by q-sepharose ion exchange column chromatography, hydrophobic interaction chromatography, and eventually by superdex-200 size exclusion chromatography. fractions containing mhc-i heavy chain molecules were identified by a280 absorbance and sds-page and pooled. throughout purification and storage, the mhc-i heavy chain proteins were dissolved in 8 m urea to keep them denatured. note that the mhc-i heavy chain proteins at no time were exposed to reducing conditions. this allowed purification of highly active pre-oxidized moieties as previously described (ostergaard et al. 2001) . protein concentrations were determined by bicinchoninic acid assay. the degree of biotinylation (usually >95%) was determined by a gel-shift assay (leisner et al. 2008 ). the pre-oxidized, denatured proteins were stored at −20°c in tris-buffered 8 m urea. recombinant constructs encoding human and porcine beta-2 microglobulin recombinant human β 2 m was expressed and purified as described elsewhere (ostergaard et al. 2001) , (ferre et al. 2003) . using a previously reported e. coli codonoptimized gene encoding human β 2 m as template , a gene encoding porcine β 2 m was generated by multiple rounds of site-directed mutagenesis (quikchange® stratagene, according to the manufacturer's instructions) (online resource 2). briefly, the genes encoding human or pig β 2 m were n-terminally fused to a histidine affinity encoding tag (hat) followed by a restriction enzyme encoding tag (fxa), inserted into the pet28 vector and expressed in inclusion bodies in e. coli. the fusion proteins were extracted into 8 m urea, purified by immobilized metal affinity chromatography (imac), and refolded by dilution. the fusion tags were then removed by fxa restriction protease digestion. the liberated intact and native human or pig β 2 m were purified by imac and gel filtration chromatography, analyzed by sds-page analysis, concentrated, and stored at −20°c until use (fig. 1 ). purification and refolding of recombinant porcine β 2 m proteins porcine β 2 m was purified in the same way as human β 2 m (ostergaard et al. 2001; ferre et al. 2003) . briefly, the ureadissolved β 2 m protein was purified by ni 2 +/ida metal chelating affinity column chromatography, refolded by drop-wise dilution into an excess refolding buffer under stirring (25 mm tris, 300 mm urea, ph 8.00), and then concentrated (vivaflow, 10 kda). the refolded product was purified by ni 2 +/ida metal chelating affinity column chromatography again (this time in aqueous buffer, i.e., without urea). fractions containing hat-pβ 2 m were iden-tified by sds-page and pooled. removal of the hat tag was performed by cleavage with factor xa restriction protease (fxa) followed by renewed purified by ni 2 +/ ida metal chelating affinity and superdex200 gel filtration column chromatography, concentrated by spin ultrafiltration (10 kda), mixed 1:1 with glycerol, and stored at −20°c. protein samples were mixed 1:1 in sds sample buffer (4% sds, 17.4% glycerol, 0.003% bromophenol blue, 0.125 m tris, 8 mm iaa (iodoacetamide)) with or without reducing agent (2-mercaptoethanol) as indicated, boiled for 3 min, spun at 20,000×g for 1 min, and loaded onto a 12% or 15% running gel with a 5% stacking gel. gels were run at 180 v, 40 ma for 50 min. peptide-mhc class i interaction measured by radioassay and spun column chromatography a hla-a*11:01-binding peptide, kvfpyalink (nonnatural consensus sequence a3con1 ), was radiolabeled with iodine ( 125 i) using a chloramine-t procedure (hunter and greenwood 1962) . dose titrations of mhc-i heavy chains (hhh or hhp) were diluted into refolding buffer (tris-maleate-pbs) and mixed with β 2 m (human or porcine) and radiolabeled peptide, and incubated at 18°c overnight. then binding of radiolabeled peptide to mhc-i was determined in duplicate by sephadex™ g50 spun column gel chromatography as previously described . mhc bound peptide eluted in the excluded volume, whereas free peptide was retained on the microcolumn. both fractions were counted by gamma spectroscopy, and the fraction peptide bound was calculated as excluded radioactivity divided by total radioactivity. to examine the affinity of the interaction, increasing concentrations of unlabeled competitor peptide were added. when conducted under limiting concentrations of mhc-i molecule, the concentration of competitor peptide needed to effect 50% inhibition of the interaction, the ic 50 , is an approximation of the affinity of the interaction between mhc-i and the competitor peptide. peptide-mhc class i interaction measured by an enzyme-linked immunosorbent assay peptide-mhc-i interaction was also measured in a modified version of a previously described enzyme-linked immunosorbent assay (elisa) (sylvester-hvid et al. 2002) . briefly, denatured biotinylated recombinant mhc-i heavy chains were diluted into a renaturation buffer containing β 2 m and graded concentrations of the peptide to be tested and incubated at 18°c for 48 h allowing equilibrium to be reached. we have previously demonstrated that denatured mhc molecules can de novo fold efficiently, however, only in the presence of appropriate peptide. the concentration of peptide-mhc complexes generated was measured in a quantitative sandwich elisa (using streptavidin as capture layer and the monoclonal anti-β 2 m antibody, bbm1, as detection layer) and plotted against the concentration of peptide offered (sylvester-hvid et al. 2002) . a prefolded, biotinylated flpsdyfpsv/ hla-a*02:01 (kast et al. 1994 ) complex was used as standard. because the effective concentration of mhc (3-5 nm) used in these assays is below the equilibrium dissociation constant (k d ) of most high-affinity peptide-mhc interactions, the peptide concentration, ed 50 , leading to half-saturation of the mhc is a reasonable approximation of the affinity of the interaction. the experimental strategy of pscpl has previously been described (stryhn et al. 1996) . the construction of the sublibraries and the elisa-driven quantitative measurements of mhc interaction are as given above. briefly, the relative binding (rb) affinity of each pscpl sublibrary was determined as rb (pscpl)=ed 50 (x 9 )/ ed 50 (pscpl) (where ed 50 is the concentration needed to half-saturate a low concentration of mhc-i molecules) and normalized so that the sum of the rb values of the 20 naturally occurring amino acids equals 20 (since peptides with a given amino acid in a given position are 20 times more frequent in the corresponding pscpl sublibrary than in the completely random x 9 library). a rb value above 2 was considered as the corresponding position and amino acid being favored, whereas a rb value below 0.5 was considered as being unfavorable (these thresholds represent the 95% confidence intervals). an anchor position (ap) value was calculated by the equation ∑(rb−1) 2 . a primary anchor position is characterized by one or few amino acids being strongly preferred and many amino acids being unacceptable. we have arbitrarily defined anchor residues as having an ap value above 15 ). the peptide-sla-i*0401 binding activity of each sublibrary was determined using previously published biochemical binding assay (elisa) (sylvester-hvid et al. 2002 ) (with the modifications described above). sequences logos describing the predicted binding motif for each mhc molecule were calculated as described by rapin et al. (2010) . in short, the binding affinity for a set of 1,000,000 random natural 9mer peptides was predicted using the netmhcpan method, and the 1% strongest binding peptides were selected for construction of a position-specific scoring matrix (pssm). the pssm was constructed as previously described including pseudo-count correction for low counts. next, sequence logos were generated from the amino acid frequencies identified in the pssm construction. for each position, the frequency of all 20 amino acids is displayed as a stack of letters. the total height of the stack represents the sequence conservation (the information content), while the individual height of the symbols relates to the relative frequency of that particular symbol at that position. letter shown upside-down are underrepresented compared to the background (for details see rapin et al. (2010) ). mhc distance trees were derived from correlations between predicted binding affinities. for each allelic mhc-i molecule, the binding affinity was predicted for a set of 200,000 random natural peptides using the netmhcpan method. next, the distance between any two alleles was defined, as d = 1− pcc, where pcc is the pearson correlation between the subset of peptides within the superset of top 10% best binding peptides for each allele. in this measure, two molecules that share a similar binding specificity will have a distance close to 0 whereas two molecules with non-overlapping binding specificities would have a distance close to 2. using bootstrap, 100 such distance trees were generated, and branch bootstrap values and the consensus tree were calculated. we have previously generated highly active, recombinant human mhc-i (hla-i) molecules and accompanying highthroughput assays and bioinformatics prediction resources. here, we transfer the underlying approaches to an important domesticated livestock animal, the pig, and its mhc system, the slas. mhc-i molecules are composed of a unique and highly variable distal peptide-binding platform consisting of the alpha 1 (α 1 ) and alpha 2 (α 2 ) domains of the mhc-i heavy chain (hc) and a much more conserved proximal immunoglobulin-like membrane attaching stalk consisting of the alpha 3 (α 3 ) domain of the hc noncovalently associated with the soluble mhc-i light chain (β 2 m). a priori, the establishment of recombinant sla molecules is complicated by the lack of validated reagents. any failure could therefore be caused either by real technical problems in generating sla molecules, or merely by a lack of information about strong peptide binders to the sla in question. to reduce this uncertainty, we decided to migrate from human to pig mhc-i in a step-wise manner and generate an intermediary chimeric mhc-i molecule composed of a well-known human peptide-binding platform attached to a sla stalk, which might allow us to assess whether we could generate a functional sla stalk consisting of sla-1*0401 α 3 hc and pig β 2 m. to this end, we used the α 1 α 2 domains of the hla-a*11:01 molecule, which we expected should be able to bind a known highaffinity hla-a*11:01-binding peptide (kvfpyalink). this peptide could be 125 i radiolabeled and used in a very robust peptide-binding assay testing whether the human stalk could be replaced with the corresponding sla stalk. once that had been successfully established, the entire sla-1*0401 molecule would be constructed and tested. we have previously expressed and purified the extracellular segment spanning positions 1-275 of the human hla-a*11:01 in a denatured and pre-oxidized version that rapidly refold and bind appropriate target peptides (ostergaard et al. 2001; ferre et al. 2003) . codon-optimized genes encoding the corresponding segments of sla-1*0401 (α 1 α 2 ) and sla-1*0401 (α 3 ) were constructed as described in the "materials and methods" section and used to replace the hla-a*11:01 gene segment in the above construct generating a new construct allowing for the expression of sla-1*0401. for the generation of hla-a*11:01/sla-1*0401 chimeras, the genes encoding α 1 (spanning positions 1-90), α 2 (spanning positions 91-181), and/or α 3 (spanning positions 182-275) domains of hla-a*11:01 and sla-1*0401 were exchanged using seam-less and touch-down cloning strategies. genes encoding the extracellular segments 1-275 of the above natural or chimeric mhc-i molecules were c-terminally fused to a biotinylation tag (as indicated for sla-1*0401 in online resource 1), inserted into pet28, and expressed in inclusion bodies in e. coli (fig. 2 shows sds-page of lysates of recombinant e. coli before and 3 h after iptg induction). the fusion proteins were extracted into 8 m urea (without any reducing agents), purified by ion exchange, hydrophobic and gel filtration chromatography (all conducted in 8 m urea, without any reducing agents) (fig. 3 shows sds-page of the purified sla-1*0401 after gel filtration), concentrated, and stored in urea at −20°c. testing a chimeric molecule consisting of a sla-1*0401 stalk and a hla-a*11:01 peptide-binding platform-comparing human versus porcine β 2 m to test the proximal immunoglobulin-like membrane attaching sla stalk, we generated recombinant porcine β 2 m and a chimeric human/porcine mhc-i heavy chain molecule where the α 1 α 2 were derived from the human hla-a*11:01, and the α 3 was derived from the porcine sla-1*0401. since this construct contains the entire peptide-binding platform of hla-a*11:01, we reasoned that the binding of the hla-a*11:01 restricted peptide, kvfpyalink, could be used as a functional readout of the refolding, activity, and assembly of the entire chimeric molecule including the porcine sla stalk. for comparison, we tested the supportive capacity of human β 2 m and folding ability of the entirely human hla-a*11:01. a total of four combinations could therefore be tested: porcine or human β 2 m in combination with either hhp or hhh (where the first letter indicates the origin of the α 1 domain (human hla-a*11:01 or porcine sla-1*0401), the second letter the origin of the α 2 domain, and the third letter the origin of the α 3 domain). a concentrationtitration of heavy chain was added to a fixed excess concentration (3 μm) of β 2 m and a fixed trace concentration (23 nm) of radiolabeled peptide. as shown in figs. 4 and 5, the four combinations gave almost the same heavy chain dose titration with a half-saturation occurring around 1-2 nm heavy chain. porcine β 2 m supported folding of the chimeric (hhp) α chain slightly better than it supported folding of the human (hhh) α chain. human β 2 m supported folding of hhp and hhh equally well. thus, a recombinant sla stalk can fold and support peptide binding of the peptide-binding platform. these results also suggest that human β 2 m can support folding and peptide binding of porcine mhc-i heavy chain molecules. using a positional scanning combinatorial peptide library approach to perform an unbiased analysis of the specificity of sla-1*0401 and human-pig chimeric mhc class i molecules using human β 2 m to support folding, the recombinant sla-1*0401 and human-pig chimeric mhc-i molecule were tested for peptide binding. we have previously described how pscpl can be used to perform an unbiased analysis of mhc-i molecules (stryhn et al. 1996) . a pscpl consists of 20 sublibraries for each position where one of each of the 20 natural amino acids have been locked and all other positions contain random amino acids. analyzing how much of each pscpl sublibrary is needed to support mhc-i folding (see examples in fig. 6 ) and comparing each sublibrary with a completely random library, the effect of any amino acid in any position can be examined and expressed as a rb value. further, an ap value calculated as the sum of squared deviations of rb values for each position can be used to identify the most prominent anchor position (see "materials and methods" for calculations). thus, the specificity of a nonamer binding mhc-i molecule can be analyzed comprehensively with 9×20+1 completely random library=181 sublibraries (stryhn et al. 1996) . here, this approach was used to perform a complete experimental analysis of sla-1*0401 and a limited analysis of the chimeric hpp and php molecules. a nonamer pscpl analysis of sla-1*0401 can be seen in table 1 . ap values identified positions 9, 3, and 2 (in that order of importance) as the anchor positions of sla-1*0401. in position 9, the amino acid preferences were dominated by the large and bulky aromatic tyrosine (y), tryptophane (w), and phenylalanine (f), all having rb values above 4 (table 1 ). in the almost equally important position 3, preferences for negatively charged amino acids glutamic acid (e) and aspartic acid (d) were observed. in the lesser important position 2, the most preferred amino acids were the hydrophobic amino acids valine (v), isoleucine (i), and leucine (l), followed by the polar amino acids threonine (t) and serine (s). finally, a very limited pscpl analysis was performed for the two chimeric human hla-a*11:01/porcine sla-1*0401 mhc-i molecules, hpp and php (table 2) . for both chimeric molecules, it could be demonstrated that position 9 is a strong anchor position. the positively charged amino acids, arginine (r) and lysine (k), were preferred in position 9 of the chimeric hpp molecule, whereas the aromatic amino acid, tyrosine (y), was exclusively preferred in position 9 of the chimeric php molecule. the positively charged amino acids, arginine (r) and lysine (k), were preferred in position 9 of the chimeric hpp molecule similar to the position 9 specificity of the hla-a*11:01 molecule. in contrast, the aromatic amino acid tyrosine (y) was preferred in position 9 of the chimeric php molecule similar to the position 9 specificity of the sla-1*0401 molecule. using netmhcpan to predict peptides that bind to sla-1*0401 or to human-pig chimeric mhc class i molecules our recently described neural network-driven bioinformatics predictor, netmhcpan (version 2.0), has been trained on about 88,000 peptide-binding data points representing more than 80 different mhc-i molecules (primarily hla-a and hla-b molecules). we have previously shown that netmhcpan is an efficient tool to identify peptides that bind to hla molecules where no prior data exist (nielsen et al. 2007 ) and demonstrated that netmhcpan can be extended to mhc-i molecules of other species 1 (hoof et al. 2009 ). we applied netmhcpan to our peptide repository of about 10,000 peptides, which over the past decade have been selected to scan infectious agents (e.g., sars and influenza, sylvester-hvid et al. 2004; wang et al. 2010) , improve coverage of mhc-i specificities (e.g., buus et al. 2003; christensen et al. 2003) , etc. we extracted 29 peptides as predicted binders to either the sla-1*0401, the hpp, or the php human/porcine chimeric class i molecules (some of the peptides were predicted to bind to two or even all three of these molecules). all these peptide-mhc-i combinations were tested for binding (table 3) ; 13 of 14 peptides bound to the sla-1*0401 molecule with an affinity (ic 50 value) better than 500 nm (6 with an affinity less than 50 nm); all 13 peptides tested on the php molecule were strong binders with ic 50 values below 50 nm; and 3 of 12 peptides tested on the hpp molecule bound with an affinity better than 500 nm. of the 39 peptide-mhc-i combinations tested, 20 (51%) were found to be good binders, 9 (23%) were average binders, and 10 (26%) did not bind well (table 3) . this is in stark contrast to the 0.5% frequency of binders among randomly selected peptides (yewdell and bennink 1999) . next, the netmhcpan method was used to generate pssms and sequence logos from the corresponding amino acid frequencies as described by nielsen et al. (2004) . for each position, the frequencies of all 20 amino acids were displayed as a stack of letters showing the sequence conservation/information content (the height of the entire 1 a preliminary report of sla-1*0401 binding was given in hoof et al. (2009) the normalized relative binding (rb) value indicates whether an amino acid is favored (rb>2, bold numbers) or disfavored (rb<0.5, italic numbers) in a given peptide position. the anchor position (ap) value is given by the equation ∑(rb−1) 2 . the important anchor positions 2, 3, and 9 for sla-1*0401 are underlined stack) and the relative frequency of amino acids (the height of the individual amino acids). figure 7 shows a specificity tree clustering of the sla-1*0401 molecule compared to prevalent representatives of the 12 common hla supertypes that netmhcpan originally intended to cover . by this token, sla-1*0401 most closely resembles that of hla-a*01:01. the limited pscpl analysis of the chimeric mhc-i molecules revealed strong p9 signals with specificities that seemed to be determined by the origin of the α 1 domain: the hpp chimera showed an hla-a*11:01-like p9 specificity, whereas the php chimera showed a sla-1*0401/hla-a*01:01-like specificity. since the netmhcpan predictor successfully captured these chimeric specificities (see above), we reasoned that the predictor might also be used to perform in silico dissection of these specificities and used the p9 specificity as an example of such an in silico analysis. the netmhcpan predictor considers a pseudo-sequence consisting of 34 polymorphic positions, which contain residues that are within 4.0 å of the atoms of bound nonamer peptides (nielsen et al. 2007 ). of the 34 positions of the pseudosequence, 10 delineates the p9 binding pocket; however, only 3 of these, positions 74, 77, and 97, differ between sla-1*0401 and hla-a*11:01. to explore the effect of these three residues, we performed in silico experiments where we examined single substitutions y74d, g77d, and s97i (the letter before the position number indicates the sla-a*0401 single letter residue, whereas the letter after indicates the hla-a*11:01 residue) as well as the corresponding triple substitution (ygs-ddi). as described above, pssms were generated for each of the in silico molecules followed by a specificity tree clustering (including sla-a*0401, hla-a*01:01, and hla-a*11:01). figure 8 shows this tree along with the sequence logo plots showing the predicted binding specificity of each in silico mhc-i molecule. albeit the y74d and g77d single substitutions showing some of the positively charged p9 peptide residue preference of hla-a*11:01, they still clustered with hla-a*01:01. in contrast, the in silico (ygs-ddi) triple substitution clustered with the hla-a*11:01. this suggests that the netmhcpan method is capable of defining the residues of the f pocket that determine the specificity of position 9. we have previously suggested that the specificities of the entire human mhc-i system should be solved ("the human rb and ap values are defined as described in table 1 mhc", buus 1999; lauemoller et al. 2000) . however, due to the extreme polymorphism of the mhcs, any attempt to address the specificity of the entire mhc system is a significant experimental undertaking. during the past decade, we have established a series of technologies to support a general solution of human mhc class i and ii specificities. for mhc-i, this includes (1) a highly efficient e. coli expression system for production of recombinant human and mouse mhc-i molecules (both heavy chain and light chain (β 2 m) molecules) ostergaard et al. 2001) , (2) a purification system for obtaining the highly active pre-oxidized mhc-i heavy chain species (ferre et al. 2003) , (3) a high-throughput homogenous peptide-mhc-i binding assay for obtaining large data sets on peptide-mhc-i interactions (harndahl et al. 2009 ), (4) a positional scanning combinatorial peptide library approach for a robust and unbiased analysis of the specificity of any mhc-i molecule (stryhn et al. 1996) , (5) an immunobioinformatics approach to generate predictors of the peptide-mhc-i interaction, netmhcpan, that allows predictions to be made for any human mhc-i molecules, hla-i, even those that have not yet been covered by existing data set (hoof et al. 2009; nielsen et al. 2007) , and finally (6) we have demonstrated that pre-oxidized mhc-i molecules can be used to generate mhc-i tetramers in a simple "one-pot, mix-and-read" manner (leisner et al. 2008) . here, we propose that the next goal should be to extend the overall approach to mhc-i molecules of other species of interest. mouse and rats have been extensively studied in the past, but much less reagents and information have accrued for the mhc-i molecules of other species. here, we have used an important livestock animal, the pig, as a model system and demonstrated that it indeed is possible to transfer the original human approach to other species. before attempting to generate a recombinant version of the entire porcine sla-1*0401 molecule, we grafted the more conserved membrane-proximal "stalk" (the immunoglobulin-like class i heavy chain α 3 and β 2 m domains) of porcine sla-1*0401 onto the peptide-binding domain of hla-a*11:01 generating a chimeric human/ porcine mhc-i molecule. this chimeric molecule retained the peptide-binding specificity of the hla-a*11:01 molecule, and it clearly demonstrated that the recombinant porcine stalk was functional and, by inference, also properly folded. it also suggests that the peptide-binding specificity of the distal domains do not crucially depend upon the identity of the proximal stalk. further, comparing the ability of human and porcine β 2 m to support mhc-i complex formation using either a human or a porcine mhc-i stalk, we demonstrated that every combination (porcine β 2 m/human-α 3 , porcine β 2 m/porcine-α 3 , human β 2 m/human-α 3 , and human β 2 m/porcine-α 3 ) showed almost the same heavy chain dose titration with identical half-saturations. these results illustrate the ability for porcine and human β 2 m to support complex formation of sla molecules and vice versa and suggest evolutionary that the stalk is quite conserved. next, we generated the entire sla-1*0401 heavy chain and succeeded in generating complexes using human β 2 m as the light chain and pcspl as peptide donors. the latter solved the a priori problem of not knowing which peptides would be needed to support proper folding of sla-1*0401, and it did so in an unbiased manner. furthermore, this approach is highly efficient since it readily establishes a complete matrix representing the amino acid preference for each amino acid and each position of a nonamer peptide. the specificity of sla-1*0401 shows two primary anchors: one in positions 9 with a preference for aromatic amino acids and another in position 3 with a preference for negatively charged amino acids. in addition, the sla-1*0401 features a secondary anchor in position 2 with hydrophobic or polar amino acid preferences. an alternative approach to solve the problem of identifying peptides that support folding of mhc-i molecules of so far unknown specificity is to use our recently developed panspecific predictor, netmhcpan. the successful use of this predictor to initiate peptide-binding studies was recently fig. 7 specificity tree clustering of the sla-1*0401 molecule compared to prevalent representatives of the 12 common hla supertypes ). the distance between any two mhc molecules and the consensus tree is calculated as described in "materials and methods". all branch points in the tree have bootstrap values of 100%. sequence logos of the predicted binding specificity are shown for each molecule. in the logo, acidic amino acids [de] are shown in red, basic amino acids [hkr] in blue, hydrophobic amino acids [acfilmpvw] in black, and neutral amino acids [gnqsty] in green. the axis of the logos indicates in all case positions one through nine of the motif, and the y-axis the information content (see materials and methods) demonstrated for hla-a*3001 . although originally developed to cover all hla-a and hla-b molecules, it has also been shown to extend to mhc-i molecules of other species (hoof et al. 2009 ). here, we demonstrate that the netmhcpan predictor is capable of extracting mhc-i sequence information across species and correctly relate this to peptide binding even in the absence of any available data for the specific query mhc-i molecule, i.e., the sla-1*0401 as well as the chimeric hpp (hα 1 pα 2 pα 3 ) and php (pα 1 hα 2 pα 3 ) molecules. it is not clear why binding of the php chimera was more efficiently predicted than binding of the hpp chimera. one could speculate that netmhcpan has not captured the effect of the different positions of the pseudo-sequence equally well and not all positions and pockets (and by inference-not all chimeric molecules) are therefore predicted equally well. using the netmhcpan predictor to cluster sla-1*0401 and representative molecules of 12 human hla supertypes according to predicted peptide-binding specificities, the sla-1*0401 specificity closely resembled that of hla-a*01:01 (iedb, http://www.immuneepitope.org/mhcalleleid/142, accessed march 9th 2011). this result was also obvious from an inspection of the pscpl analysis of the sla-1*0401. the pscpl analysis of the p9 specificity of the sla-1*0401 and the two chimeric molecules suggested that the p9 specificity primarily was determined by the α 1 domain. this contention was further strengthened by a netmhcpan-driven in silico analysis of the residues delineating the f pocket, which interacts with p9. this suggests that netmhcpan can be used to design and interpret detailed experiments addressing the structurefunction relationship of peptide-mhc-i interaction. in the case of sla-1*0401, netmhcpan suggests that y74, g77, and s97 play a prominent role in defining the p9 f pocket. whereas the netmhcpan readily captured the p9 anchor residue of sla-1*0401, it did not capture the p3 anchor (at least not in the 2.4 version). we surmise that this shortcoming is due to insufficient examples of the use of p3 anchors within the currently available peptide-mhc-i binding data. inspecting the pseudo-sequence of sla-1*0401 and hla-a*01:01 vs. hla-a*11:01 suggests that the presence of an arginine in position 156 might fig. 8 comparison of specific in silico mutations of the sla-1*0401 molecule and comparison with the two hla molecules: hla-a*11:01 and hla-a*01:01. the distance between any two mhc molecules and the consensus tree is calculated as described in "materials and methods". all branch points in the tree have bootstrap values of 100%. the sla-1*0401 mutations are indicated as y74d, g77d, and s97i, where the letter before the position number indicates the sla-1*0401 single letter residue and the letter after indicates the hla-a*11:01 residue. ygs-ddi is the corresponding triple substitution. sequence logos are calculated and visualized as described in fig. 7 . the axis of the logos indicates in all case positions one through nine of the motif, and the y-axis the information content (see materials and methods) explain the preference for negatively charged amino acid residues in p3. future netmhcpan-guided experiments could pointedly address this question, and the resulting data could complement existing data and be used to update and improve the netmhcpan predictor. all in all the two complementary approaches, pscpl and netmhcpan, agreed on the specificity of the sla-1*0401 molecule, as well as of the two chimeric mhc-i molecules. thus, the specificity of sla-1*0401 appear to be well established. this specificity has successfully been used to search for foot-and-mouth disease virus (fmdv)specific ctl epitopes in fmdv-vaccinated, sla-1*0401positive pigs, and the recombinant sla-1*0401 molecules have been used to generate corresponding tetramers and stain pig ctls (patch et al. 2011) . in conclusion, we here present a set of methods that can be used to generate functional recombinant mhc-i molecules, map their specificities and identify mhc-i-restricted epitopes, and eventually generate peptide-mhc-i tetramers for validation of ctl responses. this suite of methods is not only applicable to humans, but potentially to any species of interest. description and prediction of peptide-mhc binding: the 'human mhc project receptorligand interactions measured by an improved spun column chromatography technique. a high efficiency and high throughput size separation method sensitive quantitative predictions of peptide-mhc binding by a 'query by committee' artificial neural network approach selecting informative data for developing peptide-mhc binding predictors using a query by committee approach purification of correctly oxidized mhc class i heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding high-throughput polymerase chain reaction cleanup in microtiter format peptide binding to hla class i molecules: homogenous, high-throughput screening, and affinity assays netmhcpan, a method for mhc class i binding prediction beyond humans preparation of iodine-131 labelled human growth hormone of high specific activity role of hla-a motifs in identification of potential ctl epitopes in human papillomavirus type 16 e6 and e7 proteins the peptide-binding specificity of hla-a*3001 demonstrates membership of the hla-a3 supertype an integrative approach to ctl epitope prediction: a combined algorithm integrating mhc class i binding, tap transport efficiency, and proteasomal cleavage predictions identifying cytotoxic t cell epitopes from genomic and proteomic information: "the human mhc project one-pot, mix-and-read peptide-mhc tetramers definition of supertypes for hla molecules using clustering of specificity matrices netmhc-3.0: accurate web accessible predictions of human, mouse and monkey mhc class i affinities for peptides of length 8-11 reliable prediction of t-cell epitopes using neural networks with novel sequence representations improved prediction of mhc class i and class ii epitopes using a novel gibbs sampling approach netmhcpan, a method for quantitative predictions of peptide binding to any hla-a and -b locus protein of known sequence efficient assembly of recombinant major histocompatibility complex class i molecules with preformed disulfide bonds induction of footand-mouth disease virus-specific cytotoxic t cell killing by vaccination the interaction of beta 2-microglobulin (beta 2m) with mouse class i major histocompatibility antigens and its ability to support peptide binding. a comparison of human and mouse beta 2m the mhc motif viewer: a visualization tool for mhc binding motifs peptide binding to the most frequent hla-a class i alleles measured by quantitative molecular binding assays nomenclature for factors of the sla class-i system netctlpan: pan-specific mhc class i pathway epitope predictions peptide binding specificity of major histocompatibility complex class i resolved into an array of apparently independent subspecificities: quantitation by peptide libraries and improved prediction of binding analysis of polymorphism in porcine mhc class i genes: alterations in signals recognized by human cytotoxic lymphocytes establishment of a quantitative elisa capable of determining peptide-mhc class i interaction sars ctl vaccine candidates; hla supertype-, genome-wide scanning and biochemical validation hla class i binding 9mer peptides from influenza a virus induce cd4 t cell responses immunodominance in major histocompatibility complex class i-restricted t lymphocyte responses acknowledgments we thank lise lotte bruun nielsen, anne caroline schmiegelow, and iben sara pedersen for their expert experimental support. this work was in part supported by the danish council for independent research, technology and production sciences (274-09-0281) and by the national institute of health (nih) (hhsn266200400025c). key: cord-003143-n6b0r92e authors: zhao, min; liu, kefang; luo, jiejian; tan, shuguang; quan, chuansong; zhang, shuijun; chai, yan; qi, jianxun; li, yan; bi, yuhai; xiao, haixia; wong, gary; zhou, jianfang; jiang, taijiao; liu, wenjun; yu, hongjie; yan, jinghua; liu, yingxia; shu, yuelong; wu, guizhen; wu, aiping; gao, george f.; liu, william j. title: heterosubtypic protections against human-infecting avian influenza viruses correlate to biased cross-t-cell responses date: 2018-08-07 journal: mbio doi: 10.1128/mbio.01408-18 sha: doc_id: 3143 cord_uid: n6b0r92e against a backdrop of seasonal influenza virus epidemics, emerging avian influenza viruses (aivs) occasionally jump from birds to humans, posing a public health risk, especially with the recent sharp increase in h7n9 infections. evaluations of cross-reactive t-cell immunity to seasonal influenza viruses and human-infecting aivs have been reported previously. however, the roles of influenza a virus-derived epitopes in the cross-reactive t-cell responses and heterosubtypic protections are not well understood; understanding those roles is important for preventing and controlling new emerging aivs. here, among the members of a healthy population presumed to have previously been infected by pandemic h1n1 (ph1n1), we found that ph1n1-specific t cells showed crossbut biased reactivity to human-infecting aivs, i.e., h5n1, h6n1, h7n9, and h9n2, which correlates with distinct protections. through a t-cell epitope-based phylogenetic analysis, the cellular immunogenic clustering expanded the relevant conclusions to a broader range of virus strains. we defined the potential key conserved epitopes required for cross-protection and revealed the molecular basis for the immunogenic variations. our study elucidated an overall profile of cross-reactivity to aivs and provided useful recommendations for broad-spectrum vaccine development. using the crystal structures of the major humoral antigen ha, we investigated the humoral antigenic variations of different aivs compared to a(h1n1)/california/04/2009 (fig. 1c) . humoral immunogenic sites on the ha protein of influenza a virus are mainly distributed over five conformational sites (fig. 1d) , including four humoral antigenic sites (sa, sb, ca, and cb) on the ha "head" and a subdominant b-cell epitope on the ha "stem." structure-based conservancy analysis of these epitopes revealed that the four antigenic sites on the ha head of the four aivs a(h5n1)/vietnam/1194/2004, a(h6n1)/ taiwan/2/2013, a(h7n9)/anhui/1/2013, and a(h9n2)/hong kong/1073/99 were distinct from those of a(h1n1)/california/04. the stem-derived epitope in the has of each aiv was comparatively conserved but still contained specific substitutions. to expand our discovery to other aiv strains, we performed phylogenetic analyses based on ha sequences from different human-infecting h5n1, h6n1, h7n9, and h9n2 strains (fig. 1e ). different clusters of aiv has were generated. though h1 and h5 are adjacent to each other in phylogeny, there was no cross-reactive serological reactivity to h5n1 among the putative ph1n1-infected population. thus, the results are consistent with respect to comparisons between the divergent b cell epitopes on the humoral ha antigens of different aivs and the phenomenon that no cross-reactive antibody titers were observed between the tested aivs and ph1n1. biased t-cell cross-reactivities. to investigate potential preexisting t-cell responses to different aivs among the ph1n1-infected subjects, we stimulated freshly isolated pbmcs from the ph1n1 ab ϩ cohort via an enzyme-linked immunosorbent spot (elispot) assay with live influenza viruses as stimulators. we found that certain levels of cross-reactive t-cell responses against h5n1, h6n1, h7n9, and h9n2 viruses were detected. cross-reactive t-cell responses against h5n1 (36.8 spot-forming cells [sfcs]/ 10 5 pbmcs) were found at levels similar to those determined for ph1n1-specific t-cell responses (34.9 sfcs/10 5 pbmcs), but the levels for both were higher than those seen with other aivs, i.e., h6n1 (15.1 sfcs/10 5 pbmcs), h7n9 (16 sfcs/10 5 pbmcs), and h9n2 (17.7 sfcs/10 5 pbmcs) ( fig. 2a) . lee et al. synthesized the overlapping peptides covering all proteins and found that m1 and np possess the major immunogenic sites (20) . we evaluated cross-reactive t-cell responses to m1 proteins from different aivs among the ph1n1 m1-cultured pbmcs for 9 days. though cross-t-cell reactivity was observed for all four tested aivs, the cross-reactivity to m1 of h5n1 (497 sfcs/10 5 pbmcs) shown by the ph1n1-specific t cells was higher than that to other aivs (for h6n1, 404 sfcs/10 5 pbmcs; for h7n9, 359 sfcs/10 5 pbmcs) (fig. 2b ). in intracellular cytokine staining (ics) assays, both the cross-cd8 ϩ t cells (4.37% gamma interferon positive [ifn-␥ ϩ ] cd8 ϩ cd8 ϩ ) and cd4 ϩ t cells (0.27% ifn-␥ ϩ cd4 ϩ cd4 ϩ ) displayed a bias with respect to h5n1; in contrast, h7n9 presented the lowest cross-t-cell reactivity level, with 1.08% ifn-␥-secreting cells in cd8 ϩ t cells and 0.14% ifn-␥secreting cells in cd4 ϩ t cells (fig. 2c to f), while the t-cell responses of ph1n1antibody negative individuals did not show such differences in statistics (see fig. s1a in the supplemental material). m1 is the dominant contributor to biased t-cell reactivities. the cross-reactivity to h7n9 was significantly lower than that to other aivs among the ph1n1 antibodypositive (ab ϩ ) subjects. thus, to investigate the contribution of different cellular antigens of influenza viruses to biased cross-t-cell reactivities, we compared t-cell responses to h7n9 and ph1n1 among the members of the healthy population using overlapping peptides covering the m1 and np proteins. we found that t-cell reactivity to h7n9 np was of the same level as the t-cell reactivity to ph1n1 np as detected in the pbmcs ex vivo by elispot assay (fig. 2i ). subsequently, we established h7n9-and ph1n1-specific t-cell lines in vitro by stimulating the pbmcs with an np peptide pool for 9 days. the responses to nps of either h7n9 (354 sfcs/10 5 pbmcs) or ph1n1 (398 sfcs/10 5 pbmcs) of the t-cell lines remained at the same level (fig. 2j) . in ics assays, the results seen with ifn-␥-secreting cells in both cd8 ϩ and cd4 ϩ t cells were similar under conditions of stimulation of h7n9 np (4.49% ifn-␥ ϩ /cd8 ϩ ) and ph1n1 np (4.34% ifn-␥ ϩ /cd4 ϩ ) ( fig. 2k and l) . in contrast, the cross-reactivity to h7n9 m1 (203 sfcs/10 5 pbmcs) was significantly lower than the reactivity to ph1n1 m1 (368 sfcs/10 5 pbmcs) itself ( fig. 2g and h) . subsequently, six immunogenic individual peptides from the ph1n1 m1 pool were identified as responsible for the m1-specific t-cell responses among the subjects via a matrix assay. a biased responsive magnitude against the corresponding peptides from different aivs was observed, consistent with the diverse substitutions in these peptides (see fig. s2 ). further analysis indicated that these peptides contained previously identified hla class i-or class ii-restricted epitopes (see fig. s2d and h) and that the immunogenicity can be influenced by the substitutions in different aivs. phenotypes of the cross-reactive t-cell. to further investigate the functional subset of the cross-reactive t cells, we detected the memory phenotypes of ph1n1specific ifn-␥ ϩ t cells. memory phenotypes of the ifn-␥-secreting cells with respect to all of the different antigens, including live ph1n1 virus and the m1 pool and np pool of ph1n1, were determined by analysis of the cd45ra and cd62l data ( fig. 3a and b) . the results showed that for all the antigens, the effector memory (cd45ra ϫ cd62l ϫ ) t-cell subset dominated the ifn-␥-secreting cells both among cd8 ϩ and cd4 ϩ t cells. for instance, 75.9% (median) of the ifn-␥ ϩ cd8 ϩ t cells and 81.3% (median) of the ifn-␥ ϩ cd4 ϩ t cells specific for m1 presented an effector memory phenotype (fig. 3c) . . t-cell responses were investigated using freshly isolated pbmcs from individuals (n ϭ 11) through ifn-␥ elispot assays with live viruses. (b) ph1n1-specific t cells in pbmcs from individuals (n ϭ 20) were expanded by stimulation with the ph1n1 m1 peptide pool for 9 days, and the t-cell responses were determined through ifn-␥ elispot assays using pools of overlapping peptides derived from influenza virus m1 protein. (c and d) the ratios of influenza virus-specific ifn-␥-secreting cells in cd8 ϩ (c) and cd4 ϩ (d) t-cell levels were determined using ics and flow cytometry. the frequencies of virus-specific t-cell responses to aivs were compared with the frequencies of responses to the ph1n1 m1 pool (n ϭ 8). (e and f) representatives of virus-specific t cells in cd8 ϩ (e) and cd4 ϩ (f) t cells are shown. (g to j) t-cell responses to the m1 antigen and np antigen of ph1n1 and h7n9 before and after culturing with m1 pools or np pools of ph1n1 and h7n9 were compared through ifn-␥ elispot and ics assays. (i) the t-cell responses of freshly isolated pbmcs from the healthy donors to the ph1n1 np pool and h7n9 np pool were compared using ifn-␥ elispot assays. (j) pbmcs were cultured with the ph1n1 np pool and tested on the ninth day using ifn-␥ elispot assays. the t-cell responses to m1 pools of these two viruses were tested at the same time as the control (g, fresh pbmcs; h, pbmcs cultured in vitro with m1). (k and l) np-specific t-cell responses to ph1n1 and h7n9 tested in ics assays using pbmcs cultured in vitro with the ph1n1 np pool for 9 days. data in panels a, b, and g to j are shown as means ϩ sem (standard errors of the means), and data in panels c and d are shown as medians. the differences among multiple groups were compared using anova (a to d), and differences between two groups were compared using student's t test (e to h). *, p ͻ 0.05; **, p ͻ 0.001; ***, p ͻ 0.0001. immunity plays an important role in protection from heterosubtypic influenza infections when antibodies do not work well. to better investigate the correlation of the internal proteins and t-cell cross-reactivities, we performed phylogenetic analyses of the predominant influenza virus t-cell immunogens, i.e., m1, np, and pb1, based on the full-length protein sequences ( fig. 4a and c and e). we found that the ha relationship of the aivs did not reflect the biased cross-t-cell reactivities. the m1 protein from different h5n1 strains and that from ph1n1 were not very clearly closely related to each other, and neither were np and pb1. cross-reactive t cells targeted different cd8 ϩ or cd4 ϩ t-cell epitopes of aivs covering special sequences of the antigens. thus, we hypothesized that the phylogenetic relationship in terms of the t-cell epitopes from different aivs may correspond to the biased cross-t-cell reactivities. we retrieved the previously defined hla class i-restricted peptides located within the internal m1, np, and pb1 proteins of ph1n1 and the corresponding peptides within different aivs and plotted their phylogenetic relationship ( fig. 4b and d and f) . the t-cell epitope evolution of ph1n1 is close to that of h5n1 but distant from those of h7n9, h9n2, and h6n1, which is consistent with the biased cross-t-cell reactivity to h5n1 but not to the other strains. to trace the full view of cd8 ϩ immunogenic features of ph1n1 and aivs, we performed bioinformatic analyses of potential cellular antigenicity for different viruses based on previously determined t-cell epitopes available from the immune epitope data base (iedb). we analyzed 38 representative human-infecting aiv strains as well as the recent seasonal h1n1 stains. a total of 266 ctl epitopes were retrieved from iedb and then mapped to each strain (up to 30 december 2016). we filtered out 129 internal protein epitopes to perform further analysis (see table s2 ). clustering analysis indicated that the h1n1 stains are located in one cluster (see fig. s3a ). for different humaninfecting aiv strains, h5n1 strains remained close to ph1n1 compared to the distance of other aivs (i.e., h7n9, h6n1, h9n2) to ph1n1 (fig. 4g) . the h7n9 and h9n2 strains were intermixed with each other, which may have been related to the shared internal genes of these aiv subtypes in china (5) . seasonal h1n1 influenza viruses before 2009, such as a(h1n1)/brisbane/59/07, were located in a cluster that was adjacent to but distinct from that of ph1n1, indicating a cellular antigenic transition of ph1n1 compared to previous h1n1 stains. table s6 and s7. eleven epitopes that are conserved in h5n1 and ph1n1 strains but that different from those in other aivs are framed with a black rectangle. (h) the sequences of each strain were compared to the sequence of a(h1n1)/california/04/2009 virus, and mutant sites are highlighted in red (potential tcr docking sites) or yellow (anchor residue sites). peptide m1 (99 -109; underlined) indicates peptide h1-p25. the sequence information from the 11 peptides is presented in table s3 . ph1n1, 2009 pandemic influenza virus cluster; sh1n1, seasonal h1n1 strains before 2009 (but the data also include the 1918 h1n1 strain). comparing the different variations of the t-cell epitopes between ph1n1 and various aivs, a cluster that included 11 t-cell epitopes was found to be conserved in h5n1 and ph1n1 strains but presented different variations in other aivs, either in major histocompatibility complex class i (mhc-i)-anchoring or t-cell receptor (tcr)-docking positions (30) (fig. 4g and h; see table s3 ). this cluster of epitopes may have a key role in determining the bias of cross-t-cell reactivities to different aivs in the healthy population. we also performed bioinformatic analyses using influenza virus-derived t-cell epitopes with restrictions of different mouse mhc alleles (h-2d b , h-2k b ). both the clustering and phylogenetic analyses (see fig. s3b and c) showed that the mouse epitope-based h1n1 lineage was still adjacent to h5n1 but far from other aivs, including h7n9. molecular basis of the biased t-cell cross-reactivities. as mentioned above, there was a region covering 11 nonconserved short peptides which was highlighted in the heat map (fig. 4h ). the 11 epitopes had a common feature: the amino acids which showed substitutions at the anchoring positions and/or at the exposed positions were substituted frequently (see table s3 ). this indicates that the variable antigenicity of the nonconserved peptides may be contributed by substitution-dependent intervention of mhc binding and/or tcr recognition. in this study, more than half (54.5%) of the subjects had the hla-a*2402 allele, and m1 protein-derived peptide h1-p25 (m1 99 -109; lykklkreitf in ph1n1) with hla-a*2402 restriction is 1 of the 11 peptides. p25 is conserved between h1n1 and h5n1 (named peptide h1-p25) but has a dominant mutation with substitutions at position 9 from ile to met (i9m) (named peptide h7-p25) in h7n9 and h9n2 and ile to val (i9v) in h6n1. in hla-a*2402 ϩ individuals, we confirmed that peptide h7-p25 could induce lower cross-reactivity than peptide h1-p25 only in ph1n1-specific t cells (fig. 5a) . to characterize the binding of peptide h1-p25 derived from ph1n1 (or h5n1) and h7/h9 variant h7-p25 to hla-a*2402, in vitro renaturing (fig. 5b ) and circular dichroism (31) assays of the hla-a*2402/peptide complexes were performed (fig. 5c ). the refolding efficiencies of both the hla-a*2402/h1-p25 and hla-a*2402/h7-p25 complexes were high (fig. 5b) . further, the hla-a*2402/h1-p25 complex was more thermally stable (with a melting temperature [t m ] of 48.8°c) than the hla-a*2402/h7-p25 complex (with t m ϭ 46.4°c) (fig. 5c ). despite the fact that the i9m mutation is not located in the traditional primary anchoring positions of hla i binding peptides, peptide h7-p25 displayed a minor decreased binding affinity for hla-a*2402 compared to h1/h5-derived peptide h1-p25. to further interpret the immunogenicity transition at the molecular level, we determined the crystal structures of hla-a*2402 in complex with peptide h1-p25 or h7-p25 (data set 1) (see table s4 ). the overall conformations of main chains were similar in the two structures ( fig. 5d and e) . however, in the hla-a*2402/h1-p25 structure, residue ile9 inserts inside the e pocket of the hla-a*2402 groove (fig. 5f ). in contrast, in the hla-a*2402/h7-25 complex, mutated residue met9 is too large to be accommodated in the e pocket of hla-a*2402 and, thus, its side chain protrudes from the peptide binding groove and might be contacted by the tcr (fig. 5g) . another structural variation is contributed by lys4. although this residue is conserved between the p25 peptides of ph1n1 and h7n9, the salt bridge formed between lys4 and glu8 in hla-a*2402/h1-p25 is not present in the structure of hla-a*2402/h7-p25. without the constraint of the salt bridge, lys4 of peptide h7-p25 in hla-a*2402/h7-p25 is exposed to solvent instead of being partially buried in the c pocket as in the hla-a*2402/h1-p25 structure. due to the low resolution (3.3 å) of the first data set from hla-a*2402/h7-p25, we collected another data set which had higher resolution (2.3 å) (see table s4 ). though no clear electron density for residues in the middle portion of h7-25 was observable on the basis of data set 2 of hla-a*2402/h7-p25, the conformations of residues lys4 and met9 from h7-p25 in data set 2 were still different from those from h1-p25 but were similar to those from h7-p25 in data set 1 (see fig. s1b ). taking these data together, though the h7-p25 peptide has only one dominant i9m mutation, this mutation influences the antigenicity of the peptide, most likely by altering both hla binding and tcr recognition. this may illustrate a common mode of antigenic variation of the 11 nonconserved peptides between the h1n1/h5n1 cluster and other aivs (h6n1, h7n9, and h9n2) that induce a biased scale of cross-reactive t-cell responses. biased t-cell cross-reactivities provided distinct cross-protection efficacies against aivs. considering the unequal cross-reactivities to different aivs exhibited by ph1n1-specific t cells, especially with respect to the difference between h5n1 and h7n9, the next issue that we addressed was whether these t-cell immunogenicity variations can lead to different heterosubtypic protections against these aivs. we used ph1n1 virus at sublethal doses to prime mice, and 28 days later, the mice were intranasally infected with lethal doses of ph1n1, h5n1, or h7n9. for homologous challenge of ph1n1, the protection ratio was 100% (fig. 6a) . the rate of heterosubtypic protection against h5n1 challenge was also 100% (fig. 6d ), but the rate of protection against h7n9 challenge was 89% (fig. 6g) . the mice in the homologous ph1n1 challenge group (fig. 6b ) and the ph1n1 primed-h5n1 challenge group (fig. 6e ) n and o) ph1n1-specific cd8 ϩ t cells of ph1n1-primed mice were stained with tetramers h1-np 366ϫ374 (complex between h-2d b and peptide from h1n1), h5/h7-np 366ϫ374 (complex between h-2db and peptide from h5n1/h7n9), h1-pa 224ϫ233 (complex between h-2d b and peptide from h1n1), and h1-pa 224ϫ233 (complex between h-2d b and peptide from h5n1/h7n9). cd8 ϩ t cells were subsequently selected for the analysis of tetramers (n ϭ 4). differences between two groups were compared using student's t test (j to l and n), and differences among multiple groups were compared using anova (m). *, p ͻ 0.05. displayed no or minor body weight losses, while the mice in the ph1n1 primed-h7n9 challenge group (fig. 6h ) had a body weight loss of ͼ10% in the first 7 days after h7n9 challenge. concerning virus shedding in the lungs after infection, no virus was detected by 3 days postinfection (d.p.i.) or afterward for the homologous challenge of ph1n1 (fig. 6c) . as for the ph1n1-primed h5n1 group (fig. 6f) , the h5n1 virus load was much lower than that seen with the unprimed group by 3 dpi and disappeared by 7 dpi. in the h7n9 groups (fig. 6i ), the primed group had a detectable virus load by 3 and 7 dpi. to distinguish the contributions of neutralizing antibodies and t-cell responses in the heterosubtypic protection against aivs, we performed mn and t-cell response detection assays in mice infected with different influenza viruses at sublethal dosages. in the ph1n1-infected group, ph1n1-specific neutralizing antibodies were detectable on day 14, and no cross-neutralization antibody titers to h5n1 or h7n9 could be detected (fig. 6j) and similarly, there was no cross-neutralization antibody titers to ph1n1 detected in serum of the h5n1-or h7n9-infected group (fig. 6k and l) . the ph1n1-specific t-cell response in the mice was found to be 363 sfcs/10 6 splenocytes at 14 dpi with ph1n1 (fig. 6m ). meanwhile, cross-reactive t-cell responses to either h5n1 or h7n9 were found in the ph1n1-infected mice. however, the cross-reactive t-cell responses to h7n9 (273 sfcs/10 6 splenocytes) were lower than those against h5n1 (361 sfcs/10 6 splenocytes). these results indicated that bias of cross-reactive t-cell responses induced by ph1n1 may lead to different protection efficacies against h5n1 and h7n9. we also prepared the tetramers of two immunodominant h-2d brestricted t-cell epitopes, np 366 -374 and pa 224ϫ233 , and their h5n1/h7n9 mutants. the tetramer staining of the splenocytes from the mice infected by h1n1 for 28 days showed that the mice possessed robust h1-np 366ϫ374 and h1-pa 224ϫ233 peptidespecific cd8 ϩ t cells (1.2% and 0.9%), whereas certain t cells cross-recognizing the h5n1/h7n9 mutants could also be detected (0.31% and 0.40%) (fig. 6n and o) . however, the ratios of mutant peptides h5/h7-np 366ϫ374 and h1/h7-pa 224ϫ233 were lower, which may still have contributed to the cross-protection. here, we found that ph1n1-specific t cells had biased cross-reactivities to different human-infecting aivs, while no preexisting neutralizing antibodies were detected. the cross-cellular immune response to h5n1 in previously ph1n1-infected subjects was higher than those to h6n1, h7n9, and h9n2, correlating with heterosubtypic protection in an animal model. epitope-based phylogenetic analysis demonstrated that the h5n1 subtype possesses a cluster of conserved epitopes with ph1n1 that may lead to the observed cross-antigenicity. peptide-mhc (pmhc) structure determination indicated a molecular basis for the immune cross-reactivity between ph1n1 and h5n1, as well as the lack of cross-reactivities toward other aivs, especially h7n9. antibody-mediated neutralization is the direct inhibition of viral infection (32) . the elicitation of a neutralizing-antibody response is a correlate of protection for vaccines and contributes to protection against many viral infections (33). in particular, ha imprinting in childhood could provide lifelong protection against severe infection and death from emergent viruses (34) . however, a previous study showed that no h7n9specific antibody titers were detected in the 1,544 serum samples collected before the emergence of h7n9 from poultry workers, most of whom were born after 1968, when h3n2 emerged (18) . this indicated that cross-reactive serological immunity against h7n9 virus did not preexist among healthy young populations. in our study, the levels of neutralizing antibodies that cross-recognized h5n1 and h7n9 were also undetectable, which may indicate a critical role of t cells in heterosubtypic protection. recent studies indicated that ha-specific cd4 ϩ t cells do not possess a dominant role compared to the internal proteins such as m1 and np (20, 35) . therefore, ha-specific immunity may not be sufficient to explain the cross-protection against aiv by the immunity to previous seasonal influenza viruses. according to our bioinformatics analyses, the heat map of all available t-cell epitopes among human-infecting viruses indicated that the accumulation of varied epitopes may hinder cross-t-cell reactivities. in particular, a cluster of conserved t-cell epitopes shared by h5n1 and h1n1 may be related to cross-protection against h5n1. these epitopes could be considered for use in the development of vaccines preventing h1n1 and h5n1 infections. in contrast, t-cell recognition of the mutant viral epitopes in h7n9 was significantly decreased due to the poor t-cell activation threshold and disrupted peptide-hla interactions. although low cross-reactivity of the variable peptides may also exist due to tcr conformational plasticity, their protective effect remains to be determined (36) . we also detected a certain level of cross-reactive t-cell responses against aivs existing among different individuals, which may be contributed by conserved t-cell epitopes. greenbaum et al. showed that a large fraction of conserved t-cell epitopes in seasonal influenza virus could induce significant t-cell responses; as such preexisting t-cell immunity may decrease the severity of a variant strain infection (37) . our previous work also confirmed a dominant role for conserved t-cell epitopes in anti-influenza virus responses (30) . although there have been hundreds of influenza virus-specific epitopes identified across proteins using a range of epitope identification techniques, the majority of the conserved epitopes are derived from the internal proteins m1, np, and pb1 (37) (38) (39) . lee and others reported that m1 and np possess the major immunogenicities among the internal proteins, followed by pb1 and pa (20) . they also found that the recognition frequency of m1 protein was higher than those of the other internal proteins. however, the identity of the internal proteins which possess a dominant influence on t-cell cross-reactivity was still unknown. on the basis of previous researches, we compared the t-cell responses against h1n1 and h7n9 among healthy donors with overlapping peptide pools of np and m1, respectively, in the present study. np-specific t-cell cross-reactivity against h7n9 showed a high level among the members of the ph1n1 ab ϩ population. interestingly, we found biased t-cell cross-reactivities in responses to m1 proteins derived from different aivs. the similarity of the m1 sequences in the two strains was lower (92%) than those of np (93%) and other internal proteins (pb1 [96%], pb2 [97%], and pa [96%]). besides, the influences of the substitutions on immunogenicity would be different among different proteins, which may also contribute to varied cross-reactivity of the internal proteins between h1n1 and h7n9. mutations in the m1 protein might have a larger influence on its immunogenicity. considering the immunogenicity and dissimilarity conservation of these two proteins, we proposed that m1 might have a more dominant influence in eliciting t cell immunity among influenza viruses and chose m1 as the stimulus in the experiments that followed that assessed t cell cross-reactivity and heterogeneous protection. thus, like the humoral response in ha, minor variations among the immunodominant epitopes may impact cross-t-cell reactivities, which should be carefully considered during the development of universal vaccines based on the m1 proteins of influenza viruses. the activation of t cells could be affected mainly by two factors: the stability of the peptide-mhc (pmhc) complex and the interaction between tcr and pmhc. minor substitutions of the residues on the middle bulged region of the peptides can abrogate t-cell recognition (40) . also, the thermal stability of pmhc could influence t-cell activation. peptide gag 180 -188 derived from hiv could be recognized by hla-b*07:02, hla-b*42:01, hla-b*42:02, and hla-b*81:01. however, gag 180 -188 showed poor thermal stability with hla-b*42:02 and elicited weaker t-cell responses (41) . also, the substitutions of the primary or secondary anchor residues may completely change the antigenicity of the peptides (42) . as previously determined in structural studies, mutational escape in t-cell epitopes contributed to the antigenic disaccord (43, 44) . gras et al. investigated the cross-reactive t-cell responses against hla-b7 supertyperestricted variable epitope np 418ϫ426 in humans (43) . they found that cross-reactive cd8 ϩ t-cell immunity did not exist between the ph1n1 virus and recent seasonal influenza viruses due to structural variation of the solvent-exposed residues in t-cell epitopes that can be recognized by tcrs. mutations of anchor residues (or partially solvent-exposed secondary anchors) can also dramatically decrease cd8 ϩ t-cell responses and result in delayed viral clearance (45) . moreover, the substitutions may also lead to an induced fit on the helices of mhc-i that form the peptide-binding groove (46) and thus impact tcr recognition (47) . in the structures of hla-a*2402 with h1/h5derived peptide h1-p25 and h7/h9-specific epitope h7-p25, the i9m substitution influenced the solvent-exposed surface and decreased the force of anchoring of the peptide to hla-a*2402. this indicated that variable antigenicities of nonconserved peptides could be largely influenced by both exposed positions for tcr docking and anchoring positions for mhc binding, which should be investigated in the hla-a*2402 cohort. influenza virus-specific effector memory t cells were shown to be able to efficiently reduce the pulmonary viral titer early during the secondary infection as they accumulated in the lungs with rapid kinetics (48) . a previous study reported the correlation of preexisting t cells with memory phenotype to the conserved ph1n1 epitopes in core proteins m1 and np with clinical outcomes after incident ph1n1 infection (12) . we also found that most of the functional cross-reactive t cells were dominated by the effector memory phenotype. in recent years, while hundreds of aiv-infected cases had been reported, a large number of latent aiv infections also existed among lpm workers, as revealed by serological surveillance (49) . our study showed that prior ph1n1 infection led to heterosubtypic protection, and others indicated that the cross-reactive memory t cells were critical in heterosubtypic protection against h7n9 with different hierarchies in mice (50, 51) . determining whether the memory t cells induced by seasonal influenza viruses provide cross-protection against aiv infection in humans requires further investigation. overall, our study revealed preexisting but biased t-cell reactivity of ph1n1 influenza virus to human-infecting aivs which provided distinct protection toward each subtype. this cross-reactive t-cell recognition had a regular pattern depending on the t-cell epitope matrix derived from aivs and seasonal influenza viruses. thus, efforts to develop heterosubtypic protection-oriented universal vaccines against influenza viruses should consider the pattern of cross-t-cell immunity. ethics statement. the ethics review committee, national institute for viral disease control and prevention, chinese center for disease control and prevention (china cdc), approved this study [project approval number ivdc2014(005)]. all of the subjects provided written informed consent for the studies performed on their samples and publication of their cases. the donors were identified anonymously as donor 1 to donor 35. animals and eggs used in this study (6-to-8-week-old female c57bl/6 mice and 9-to-11-day-old embryonated chicken eggs) were bought from beijing vital river laboratory animal technology co., ltd. animal experiments were approved by animal experimental ethics board, china cdc (approval number 20140225011). the study was conducted in accordance with the principles of the declaration of helsinki and the standards of good clinical practice (as defined by the international conference on harmonization). subjects and samples. a total of 35 healthy volunteers were recruited from november 2013 to april 2014. serum samples were collected from coagulation-promoting tubes (bd vacutainer) from all donors, and pbmcs were isolated from anticoagulant blood by ficoll-paque density centrifugation from 30 of the samples (see table s1 in the supplemental material). volunteers were given questionnaires to confirm whether they had received influenza vaccination or had caught cold. none of the subjects had previously received influenza vaccines. and no symptoms of influenza virus infection were reported during the sampling period. hla class i genotyping of the donors was performed using labtype sso (one lambda). (52) . the peptides with substitutions within these viruses were divided into corresponding pools (see table s5 ). overlapping peptides covering nps of a(h1n1)/california/4/2009 and a(h7n9)/anhui/1/2013 were synthesized according to a similar strategy. heterosubtypic protections to aivs and t-cell immunity ® pbmcs from donors were incubated with influenza virus peptide pools in rpmi 1640 (gibco) containing 10% fetal bovine serum (hyclone) at 37°c with 5% co 2 at a density of 2.5 ϫ 10 6 cells/ml in a 24-well plate. on day 3, 20 u/ml recombinant human il-2 (rhil-2) was added to the medium (53) . half of the medium was changed on day 7 with supplementation by rhil-2. cells were harvested and tested for the presence of influenza virus-specific t cells on day 9. elispot assay. antigen-specific t lymphocyte responses were detected via an ifn-␥-secreting elispot assay (bd). briefly, 96-well elispot plate membranes were coated with anti-human ifn-␥ antibody at 4°c overnight before use. pbmcs from donors were incubated in wells (2.5 ϫ 10 5 /well ex vivo for freshly isolated pbmcs and 5 ϫ 10 4 /well for in vitro-cultured t-cell lines) along with viruses (multiplicity of infection [moi] of 3) or peptide pools (2 m for individual peptides) for 18 h at 37°c with 5% co 2 , as well as with phytohemagglutinin (pha) as a positive control for nonspecific stimulation. cells incubated without stimulation were employed as a negative control. after incubation, cells were removed and, in turn, plates were processed with biotinylated ifn-␥ detection antibodies, streptavidinhorseradish peroxidase (hrp) conjugate, and substrate. the development of the spots was stopped by thoroughly rinsing with water. the numbers of the spots were captured and quantified with an automatic elispot reader and image analysis software (cellular technology limited). the threshold of the positive responses set for ex vivo elispot was ն20 sfcs/10 6 pbmcs. intracellular cytokine staining and flow cytometry. after ph1n1-specific t cells were expanded in vitro for 9 days, t-cell lines were washed and rested for 2 h. these cells were then stimulated with a specific peptide pool for 2 h and incubated with golgistop/monensin (bd bioscience) for an additional 4 h at 37°c in 5% co 2 . then, cells were harvested and stained with a panel of surface mabs in fluorescence-activated cell sorter (facs) buffer (0.5% bovine serum albumin) for 30 min on ice, including fluorescein isothiocyanate (fitc)-anti-cd8 (bd pharmingen 555366), peridinin chlorophyll protein (percp)-anti-cd4 (biolegend 317432, clone okt4), phycoerythrin (pe)-anti-cd62l (bd pharmingen 555544), and v450-anti-cd45ra (bd biosciences 560362). subsequently, cells were fixed with bd fix/perm buffer on ice for 20 min and then stained with the intracellular marker allophycocyanin (apc)-anti-ifn-␥ (bd pharmingen 554702). after two washes, cells were resuspended in 4% paraformaldehyde (pfa) facs wash buffer for flow cytometry (bd influx). pbmcs stimulated with ph1n1 virus were prepared as described above. samples were analyzed with flowjo software. tetramer preparation and staining. h-2d b -restricted tetramers of peptides np 366ϫ374 and pa 224ϫ233 and hla-a*24-restricted tetramers of peptides h1-p25 and h7-p25 were prepared as previously described (30) . preparation and staining of h-2d b -restricted tetramers were performed as follows. briefly, to produce biotinylated peptide-mhc protein, h-2d b was modified by the addition of a substrate sequence for biotinylating enzyme bira at the c terminus of the ␣3 domain. in vitro-renatured h-2d b / peptide complexes were then purified and biotinylated by incubation with d-biotin, atp, and the biotin protein ligase bira (avidity) at 4°c for 12 h. the biotinylated h-2db was further purified using a superdex 200 10/300 gl gel-filtration column (ge healthcare) to remove excess biotin and then mixed with pe-streptavidin (sigma). cells from the subjects were stained with pe-tetramer, fitc-conjugated anti-cd3 antibody, and percp-cy5.5 anti-cd8 antibody. all samples were analyzed with a facscalibur flow cytometer (becton, dickinson) after staining. six-to-8-week-old female c57bl/6 mice were used for virus infections. for the primary infection, mice were lightly anesthetized by inhalation of drikold and intranasally (i.n.) infected with 10 3.8 50% tissue culture-infective doses (tcid 50 ) of ph1n1 in 40 l of phosphate-buffered saline (pbs). the same operation was performed on the control group of mice with 40 l of pbs. for the secondary challenge, mice were challenged with a high dose of h1n1 (10 5.8 tcid 50 ) and a lethal dose of h5n1 (10 5.2 tcid 50 ) or h7n9 (10 5.6 tcid 50 ) or with pbs 4 weeks later and grouped as h1n1-pbs, h1n1-h1n1, h1n1-h5n1, h1n1-h7n9, pbs-pbs, pbs-h1n1, pbs-h5n1, and pbs-h7n9. inoculated animals were assessed daily. the mice with severe manifestations after the virus challenge (ͼ20% weight loss plus severe clinical impairment) were humanely euthanized according to our approved protocol. tissue sampling and cell preparation. three mice from each group were euthanized at 0, 3, 7, and 14 dpi after secondary challenge. lungs were collected, weighed, and homogenized in 1 ml of cold dmem using an ika t10 homogenizer under sterile conditions. then, solid debris was pelleted by centrifugation at 5,000 ϫ g for 10 min, and the homogenates were used for virus titrations in mdck cells. splenocytes were filtered through cell strainers and were lysed with 0.83% ammonium chloride lysis solution to remove erythrocytes (54) . immunoinformatics. human host and mouse b6 host mhc-i t-cell epitopes of internal proteins of influenza a viruses were downloaded from iedb (55) . epitopes with peptide lengths of ͻ12 residues were selected, and their positions in proteins were renumbered using isolate a/california/04/2009 as a reference. after merging of duplicates was performed, 129 unique human-host mhc-i t-cell epitopes and 122 unique mice b6-host mhc-i t-cell epitopes were obtained (see table s2 and s6). protein sequences of representative strains were downloaded from the gisaid epiflu database, and 38 strains were analyzed (see table s7 ). subsequently, multiple-sequence alignment was performed for each protein with the alignment tool muscle v3.8.31 (56) . peptides were extracted as predicted t-cell epitopes of the representative sequences according to the unique epitopes mentioned above. the numbers of different amino acids of each predicted t-cell epitope compared with those from strain a/california/04/2009 were counted. the maximum-likelihood phylogenetic trees for full-length proteins and epitope joint sequences were constructed using molecular evolutionary genetics analysis mega6 software (57) with the jtt model and 1,000 bootstrap replicates. protein expression, refolding, and purification. the ectodomain of the hla-a*2402 heavy chain and human ␤ 2 microglobulin (␤ 2 m) were expressed in escherichia coli as inclusion bodies and subsequently refolded in vitro in the presence of different peptides. briefly, the dissolved hla heavy chain, ␤ 2 m inclusion body, and peptides were diluted at a molar ratio of 1:1:3, respectively, into a refolding buffer (100 mm tris-hcl, 400 mm l-arginine, 2 mm edta-na, 5 mm glutathione [gsh], 0.5 mm glutathione disulfide [gssg] ) and slowly stirred for 12 h at 4°c. the refolded complexes were then concentrated and purified by superdex 200 10/300 gl (ge healthcare) chromatography, further purified on an ionexchange resource q column (ge healthcare), and manipulated for crystal screening. thermal stability assay. the stability of each hla/peptide complex was tested using circular dichroism (cd) spectroscopy. all complexes were refolded and purified as described above and measured at 150 g/ml in a solution consisting of 20 mm tris-hcl (ph 8.0) and 50 mm nacl. cd spectra at 218 nm were measured on a chirascan spectrometer (applied photophysics) using a thermostatically controlled cuvette at temperature intervals of 0.1°c at a rate of 1°c/min between 25 and 90°c. the denaturation curves were generated by nonlinear fitting with origin 8.0 software. crystallization, data collection, and structure determination. crystals of hla-a*2402/h1-p25 and hla-a*2402/h7-p25 were grown by the sitting-drop, vapor diffusion method at 18°c with a protein/ reservoir drop ratio of 1:1 and at a concentration of 10 mg/ml in a mixture containing 20 mm tris-hcl (ph 8.0) and 150 mm nacl. the hla-a*2402/h1-p25 complex crystal grew in a mixture of 0.1 m imidazole (ph 7.0) and 12% (wt/vol) polyethylene glycol 20000. the hla-a*2402/h7-p25 complex crystal grew in a reaction mixture containing 0.1 m mes (morpholineethanesulfonic acid) monohydrate (ph 6.0) and 14% (wt/vol) polyethylene glycol 4000. reservoir solutions containing 20% glycerol were used for cryoprotection. the x-ray diffraction data were collected at the shanghai synchrotron radiation facility (ssrf) 17u beamline. data were indexed and scaled using denzo and the hkl2000 software package. the structures were determined using molecular replacement with the program cns with the structure of protein data bank (pdb) code 3i6l as the model. extensive model building was performed by hand using coot, and restrained refinement was performed using refmac5. additional rounds of refinement were performed using the phenix.refine program implemented in the phenix software package with anisotropic displacement parameter (adp) refinement and bulk solvent modeling. the stereochemical quality of the final model was assessed with the program procheck. the structure-based antigenic analyses of ha proteins ph1n1, h5n1, h6n1, h7n9, and h9n2 were performed using the structures with pdb codes 4jtv for h1n1 (58), 3znk for h5n1 (59), 4yy9 for h6n1 (55) , 4kol for h7n9 (60) , and 1jsd for h9n2 (61) . structure-related figures were generated using pymol (http://www.pymol.org/). statistical analysis. one-way analysis of variance (anova) was used in multiple comparisons. the two-tailed student's t test was used to compare data that were normally distributed and the mann-whitney test for nonparametric analyses. asterisks in each figure indicate statistical significance (*, p ͻ 0.05; **, p ͻ 0.01; ***, p ͻ 0.001; ****, p ͻ 0.0001). analyses were performed with graphpad prism 6 software (graphpad software, inc., la jolla, ca). accession number(s). the coordinates and structure factors of peptides complexed to hla-a*2402 have been deposited in the protein data bank under accession codes 5wwu (hla-a*2402/h1-p25), 5wxd (hla-a*2402/h7-p25, data set 1), and 5wxc (hla-a*2402/h7-p25, data set 2). supplemental material for this article may be found at https://doi.org/10.1128/mbio .01408-18. two years after pandemic influenza a/2009/h1n1: what have we learned? influenza vaccine effectiveness against 2009 pandemic influenza a(h1n1) virus differed by vaccine type during 2013-2014 in the united states estimating age-specific cumulative incidence for the 2009 influenza pandemic: a meta-analysis of a(h1n1)pdm09 serological studies from 19 countries human infection with influenza h9n2 phylogenetics of varied subtypes of avian influenza viruses in china: potential threat to humans influenza and the live poultry trade genesis, evolution and prevalence of h5n6 avian influenza viruses in china highly pathogenic h5n1 influenza virus infection in migratory birds preexisting influenza-specific cd4 ϩ t cells correlate with disease protection against influenza challenge in humans preexisting cd8 ϩ t-cell immunity to the h7n9 influenza a virus varies across ethnicities universal immunity to influenza must outwit immune evasion cellular immune correlates of protection against symptomatic pandemic influenza human t-cell immunity against the emerging and re-emerging viruses multipronged cd4( ϩ ) t-cell effector and memory responses cooperate to provide potent immunity against respiratory virus 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dendritic cells examination of influenza specific t cell responses after influenza virus challenge in individuals vaccinated with mva-np ϩ m1 vaccine lack of prominent peptide-major histocompatibility complex features limits repertoire diversity in virus-specific cd8 ϩ t cell populations cross-immunity against avian influenza a(h7n9) virus in the healthy population is affected by antigenicity-dependent substitutions protective t cell responses featured by concordant recognition of middle east respiratory syndrome coronavirus-derived cd8 ϩ t cell epitopes and host mhc induction of broadly neutralizing h1n1 influenza antibodies by vaccination conserved epitopes dominate cross-cd8 ϩ t-cell responses against influenza a h1n1 virus among asian populations differential neurovirulence of african and asian genotype zika virus isolates in outbred immunocompetent mice a model for neutralization of viruses based on antibody coating of the virion surface correlates of protection induced by vaccination potent protection against h5n1 and h7n9 influenza via childhood hemagglutinin imprinting high levels of virus-specific cd4 ϩ t cells predict severe pandemic influenza a virus infection molecular basis for universal hla-a*0201-restricted cd8 ϩ t-cell immunity against influenza viruses pre-existing immunity against swine-origin h1n1 influenza viruses in the general human population systematic identification of immunodominant cd8 ϩ t-cell responses to influenza a virus in hla-a2 individuals nucleoprotein of influenza a virus is a major target of immunodominant cd8 ϩ t-cell responses acute emergence and reversion of influenza a virus quasispecies within cd8 ϩ t cell antigenic peptides a molecular switch in immunodominant hiv-1-specific cd8 t-cell epitopes shapes differential hla-restricted escape the l60v variation in hepatitis b virus core protein elicits new epitope-specific cytotoxic t lymphocytes and enhances viral replication cross-reactive cd8 ϩ t-cell immunity between the pandemic h1n1-2009 and h1n1-1918 influenza a viruses protective efficacy of cross-reactive cd8 ϩ t cells recognising mutant viral epitopes depends on peptide-mhc-i structural interactions and t cell activation threshold modification of mhc anchor residues generates heteroclitic peptides that alter tcr binding and t cell recognition loss of t cell antigen recognition arising from changes in peptide and major histocompatibility complex protein flexibility: implications for vaccine design t cell receptor cross-reactivity directed by antigen-dependent tuning of peptide-mhc molecular flexibility t cell responses to influenza virus infection: effector and memory cells antibodies against h5 and h9 avian influenza among poultry workers in china diverse heterologous primary infections radically alter immunodominance hierarchies and clinical outcomes following h7n9 influenza challenge in mice memory t cells generated by prior exposure to influenza cross react with the novel h7n9 influenza virus and confer protective heterosubtypic immunity comparison of overlapping peptide sets for detection of antiviral cd8 and cd4 t cell responses screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes generation of murine ctl by a hepatitis b virus-specific peptide and evaluation of the adjuvant effect of heat shock protein glycoprotein 96 and its terminal fragments structure and receptor binding of the hemagglutinin from a human h6n1 influenza virus muscle: multiple sequence alignment with high accuracy and high throughput mega6: molecular evolutionary genetics analysis version 6.0 molecular basis of the receptor binding specificity switch of the hemagglutinins from both the 1918 and 2009 pandemic influenza a viruses by a d225g substitution recognition of sulphated and fucosylated receptor sialosides by a/vietnam/1194/2004 (h5n1) influenza virus structures and receptor binding of hemagglutinins from human-infecting h7n9 influenza viruses h5 avian and h9 swine influenza virus haemagglutinin structures: possible origin of influenza subtypes gao contributed to the overall concept and the experimental design and hypothesis and wrote the manuscript.this study was supported by grants from national natural science foundation of china (81401312, 81373141 the funding sources had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. we declare no competing financial interests. we thank tong zhao at institute of microbiology, chinese academy of sciences, for support in flow cytometry assays. key: cord-004435-l66ost6q authors: oli, angus nnamdi; obialor, wilson okechukwu; ifeanyichukwu, martins ositadimma; odimegwu, damian chukwu; okoyeh, jude nnaemeka; emechebe, george ogonna; adejumo, samson adedeji; ibeanu, gordon c title: immunoinformatics and vaccine development: an overview date: 2020-02-26 journal: immunotargets ther doi: 10.2147/itt.s241064 sha: doc_id: 4435 cord_uid: l66ost6q the use of vaccines have resulted in a remarkable improvement in global health. it has saved several lives, reduced treatment costs and raised the quality of animal and human lives. current traditional vaccines came empirically with either vague or completely no knowledge of how they modulate our immune system. even at the face of potential vaccine design advance, immune-related concerns (as seen with specific vulnerable populations, cases of emerging/re-emerging infectious disease, pathogens with complex lifecycle and antigenic variability, need for personalized vaccinations, and concerns for vaccines' immunological safety -specifically vaccine likelihood to trigger non-antigen-specific responses that may cause autoimmunity and vaccine allergy) are being raised. and these concerns have driven immunologists toward research for a better approach to vaccine design that will consider these challenges. currently, immunoinformatics has paved the way for a better understanding of some infectious disease pathogenesis, diagnosis, immune system response and computational vaccinology. the importance of this immunoinformatics in the study of infectious diseases is diverse in terms of computational approaches used, but is united by common qualities related to host–pathogen relationship. bioinformatics methods are also used to assign functions to uncharacterized genes which can be targeted as a candidate in vaccine design and can be a better approach toward the inclusion of women that are pregnant into vaccine trials and programs. the essence of this review is to give insight into the need to focus on novel computational, experimental and computation-driven experimental approaches for studying of host–pathogen interactions and thus making a case for its use in vaccine development. vaccination has been undeniably very helpful in promoting a healthy global population. it has severally saved lives, reduced healthcare costs and raised man's quality of life. 1 it greatly reduces disease burden, disability and death. however, newly emerging and reemerging infectious diseases (erid), infectious agents with complex lifecycle and antigenic variability and the need for personalized vaccination present additional challenges in vaccine development. 2, 3 for many pathogens (especially the emerging and those with antigenic variability), their genomes are known but their immune correlates of protection remain unclear. 1, 4 some of these reasons are why vaccine development for erid and multi-lifecycle pathogenic diseases is a tall order. serendipitous discoveries in immunology coupled with knowledge of bioinformatics tools for epitope predictions have resulted in the emergence of new pattern of vaccine design. 5, 6 the art and science of efficient and comprehensive information extraction and analysis of data deposited in relevant databases is now increasingly essential in researches related to immunology. 7 even with this capacity (efficient information extraction), some challenges in the application of bioinformatics in immunology include structure and/or function analysis and immune process analyses as concern the immune interaction specificity. fortunately, although researches in immunology are experimentally costly and very intensive, colossal amounts of data are usually generated. such data can only be analyzed with high precision and speed using bioinformatics tools. for instance, genome sequencing as well as in vitro t-cell confirmation is done in few months as opposed to years using the conventional vaccine design. 8 also, computational immunological methods drastically reduce both time and labor needs in epitopes screening. 5, 9 with computational immunology techniques, it is possible to discover vaccine candidate epitopes simply by scanning the protein sequences in a pathogen of interest. 5 many of these proteins are yet to be isolated or at least cloned. being pathogens specific and unique, they present ready candidates in vaccine construct. this review describes the need to use immunoinformatics-based techniques to unveil vital determinants of immunity made available in the genome sequence database and design vaccines. also, this review gives insight into the need to focus on novel computational, experimental and computation-driven experimental approaches for studying of host-pathogen interactions and thus making a case for its use in vaccine development. this review will further show the need for new approaches for effective drugs or vaccine design so as to combat the antigenic variability of some pathogens. the process of generating vaccine-induced immunity is somewhat challenging in immunology. current conventional vaccines came empirically when there were vague or no knowledge of vaccine immune system activation. a lot of research [10] [11] [12] has been geared toward the understanding of this challenge, but the complexity of it requires a different dimension of approach. 13 an approach that must accommodate many factors affecting vaccine development like pathogen antigenic variability, the emergence of infectious disease, human genetic variation is the goal of immunoinformatics [ figure 1 ]. activation of the immune system involves, among many processes, induction of the immune memory. the strength of this induction determines the efficacy of a vaccine. hence, vaccine efficacy in the long run is influenced by the determinants of immunological memory stimulation, persisting antibodies and kind and type of immune memory cells induced. 14 the primary vaccine-mediated immunological effectors (table 1 ) are mainly the antibodies (from b lymphocytes/ cell) 15, 16 and sometimes cd8+ and cd4+ t cells. these antibodies bind specifically to a particular kind of toxin or pathogen. vaccines and most antigens evoke humoral as well as cell-mediated immune responses. 17 vaccines that mediate immune responses through these systems (b and t cell responses) are said to be more effective. although b cells are regarded as the primary vaccine immune effectors, t cells induce immune memory cells and high-affinity antibodies. studies in reverse vaccinology and immunomics had also proved t cells as prime immune effectors following the discovery of novel vaccine targets with epimatrix. [18] [19] [20] this change of immune target has led to successful advances in vaccine design. even at the face of potential vaccine design advances, immune-related questions are now focused on specific vulnerable populations such as the young, elderly and immunocompromised. 21, 22 these concerns have propelled a better understanding of the efficacy of current vaccines on this vulnerable population and have also paved way for the application of new approaches that can put into consideration the differences of population and better targets that can generate optimum immune induction [23] [24] [25] with the exception of type ii t-cell-independent (ti-2) antigens (i.e., polysaccharide antigens). antigens that could provoke the b lymphocytes as well as the t lymphocyte responses stimulate the germinal centers causing antigen-specific highly efficient b-cell multiplication and eventual differentiation into antibodyforming plasma cells and memory b cells. all existing protein and dna antigens induce immunological memory b cells unlike type ii t-cell-independent (ti-2) antigens (i.e., polysaccharide antigens). these polysaccharide antigens do not generate memory b cells but can induce longlasting humoral immunity even when recall responses are lacking. 26 vaccine efficacy may be short term 27 if only the b cells are activated. the traditional approach for developing vaccines for infectious disease threats has shifted to include other vaccine design techniques like cloning and expressing major surface antigens 28 although this frequently resulted in the formulation of vaccines with poor immunogenicity, requiring strong adjuvantation. 29 this approach is particularly likely to be less specific for pathogens with complex lifecycles (e.g., parasites) or very high mutability (e.g., rna viruses). these pathogens do not depend on one route for their virulence of pathogenesis in human and thus to alter this process, increasing the specificity of the vaccine should be the aim and not just the effectiveness as seen in the current conventional vaccines. 28, 29 vaccines for several neglected tropical diseases are in various stages of development, 30 thanks to mega drug companies that have continued to demonstrate a willingness to invest money in the research and development as regards to diseases plaguing the developing nations. 16, [30] [31] [32] it is very pertinent to invest in researches that have an interest in vaccine specificity on the pathogen antigens than totally 33 computational vaccinology may now be applied to screen these genomes for possible vaccine target. with these tools, many proteins of virulence interest can be sequenced and the most essential gene of interest modeled for a potential vaccine candidate specific for that pathogen. immunoinformatics is the way forward in the identification of vaccine candidates for these tropical erid, for pathogens with varying antigens and for individualized therapy. immunology studies produce data in colossal quantities. also, with proteomics and genomics projects, extensive screening of pathogens and/or pathogen-host interaction, it has become increasingly necessary to store, manage and analyze these data, hence the birth of immunoinformatics. immunoinformatics deals with computational techniques and resources used to study the immune functions. statistical, computational, mathematical and biological knowledge and tools are applied in immunoinformatics in order to accurately and specifically store, and analyze data concerning the immune system and its functions. to handle evidence diversity, immunoinformatics uses tools that cut across several aspects of bioinformatics such as creation and management of databases, 34, 35 use and definition of both structural and functional signatures and the formation and application of predictive tools. [35] [36] [37] these strategies can synergize toward a better understanding of the immune system of both man and animals and fight against some less predictable pathogenesis. the complex nature of vertebrates' immune system, the variable nature of pathogens and environmental antigens coupled with the multi-regulatory pathways show that colossal quantities of data will be needed to unveil how the human immune systems work. conventionally, much cannot be achieved based on the complexity of the immune system and the virulent antigen but with the application of computational vaccinology, researches on vaccine design have been made easier, accurate and specific. applying immunoinformatics in disease study (table 2 ) requires the knowledge of disease pathogenesis, the immune system dynamics, and computational vaccinology, painstaking searches of the database, sequence comparison, structural modeling as well as motif analysis. 35, 38 these methods can go a long way in analyzing the pathogenesis of a disease and identification of vaccine candidates. in order to help understand complex pathogenrelated processes, computational models were developed for viral 46, 47 bacterial, 48 parasitic 49 and fungal pathogens. 50 the bioinformatics tools (table 3 ) are used to identify possible epitopes for vaccine formulation. each tool can screen protein sequences and identify aggregates of mhc binding and supertype motifs for possible use in epitopebased vaccine development and for use among human populations with genetic variability. there are several databases ( table 4 ) that can provide a wide range of information for all forms of immunological studies. generated data from the studies are further organized and stored in the databases (table 4) to provide a means for the development and advance in immunotargets and therapy 2020:9 immunological research. a tour on these databases will actually stimulate some interest in the vaccinology of emerging and re-surging disorders attributable to pathogen including cancer. emerging infections (eis) include infections that are entirely new in a population or that may have existed before in the population but are now gaining rapid and continued spread and/or wide geographical range. re-emerging or re-surging infections represent the infections that were previously of historical relevance but are now quickly becoming relevant because of either increasing incidence or increasing geographical and/or human host range while emerging infections represent the infections that were not originally observed in man. 66 several factors such as human behavioral changes, environmental changes, and host/intermediate factors, animal-human switching and microbial genetic changes all affect infectious disease emergence and spread. 67 these factors interact to promote the evolution of pathogens into new ecosystems, infect, spread and thrive in their new hosts. 68 the overall consequences of these are continued epimatrix this is an in-silico product of epivax developed for predicting and identifying the immunogenicity of therapeutic proteins and epitopes. it is also used to re-design proteins and in designing t-cell vaccine 51 has been applied in comparing strings from different strains of same pathogens and for pathogens identification. configuration of conservatrix allows for amino acid replacement at unusual positions. highly conserved t-cell epitopes in variable genomes such as some viruses are amenable to the algorithm 51,52 clustimer potential t-cell epitopes usually aggregate in specific immunogenic consensus sequence (ics) regions as clusters of 9-25 amino acids with 4-40 binding motifs instead of randomly distribute throughout protein sequences. in combination with epimatrix, the clustimer algorithm may be used to identify those peptides with epimatrix immunogenicity cluster scores ≥ +10. such peptides are usually immunogenic and tend to make a promising vaccine candidate. blastimer using blastimer program, one may also choose to automatically blast "putative epitopes against the human sequence database at genbank". blasting screens off those epitopes with possible autoimmunity and cross reactivity questions and locates the epitopes that can safely be used in developing human or animal vaccine. blastimer can also blasts sequences against pdb, swissprot, pir, prf and non-redundant genbank cds translations. vaccine cad this algorithm evaluates junctional epitopes for possible immunogenicity and inserts "spacers and breakers into the design of any string-of-beads construct". infectious disease emergence and re-emergence, epidemics and public health challenges. emerging infections and multi-antibiotic-resistant strains of pathogenic bacteria usually surge from one geographic location from where it spreads to other places due to immigration of people. 67, 69 most emerging infections originate from a specific population and can spread to a new population or become selectively advantaged that it can lead to the emergence of new strains of the pathogen. 67, 70, 71 also, there could be microbial traffic, in which case, an infectious agent transfers from animals to humans or spreads from isolated groups to new populations. 67, 71, 72 several factors, including ecology, are known to be associated with infectious disease outbreaks. these factors bring man into close contact with a natural disease reservoir/host. 70 with an increasing world population and poor infection control, the emergence of infection and increased microbial populations are sure. the human growth population will only increase the spread of the infection across populations. the information provided in table 5 is the list of remerging infections and current emerging diseases put forward during the who 2018 annual review. 73 the review noted that these infections, if not well controlled, can cause disease outbreaks, bioterrorism and similar occurrences requiring urgent public health attention and that with the dearth of efficacious medicines or vaccines, there is a compelling demand for continuous as well as accelerated research and development in those areas. advances in genomics, proteomics, immunomics, vaccinomics and nanotechnology are being continually exploited in diagnostic, therapeutic and in rational drug and vaccine development. these advances have also served in the control of the afore-mentioned emergences. 74, 75 the knowledge of the emerging pathogen's genome, protein make-up, pathogen-immune system interactions and researching the possible therapeutics will go a long way in directing the optimum path to containing the infection spread and controlling potential re-emergence or emergence in a different population. approaches in direct and computer-based structural determinations, 76 protein-protein interactions predicting, and bioinformatics tools now exist and are used in modern-day development of drug and biologics. 77 vaccine development has been sped up through the advance in the knowledge of the immune system of man. researches in the traditional targets of vaccines (adaptive immune response) and the less specific and fast-acting innate immune responses have been clear evidences for this advance. [78] [79] [80] as our understanding of the intercourse between innate and adaptive immunity increases, reasons and opportunities for more effective vaccine adjuvants will open up. this can be a step forward in solving a critical world's health challenge per population. following the conventional approach of vaccine design, much cannot be achieved but when the knowledge of immunoinformatics is applied, population safety and disease control can be achieved through pathogen's genome sequencing leading to optimum new vaccine design or development of a novel vaccine for the infection. antigenic variability is an important mechanism pathogens use to evade their host immunity. the surface proteins of pathogens are normally variable. this assists them to escape recognition by the immune system. a successful infectious agent presents to the host immune system information that differs from that of its virulence. pathogenic organisms have organized systems of escaping destruction by the immune system of their hosts. for instance, toxoplasma invades and appropriates the host cells thereby circumventing phagocytosis and then spread within their host to establish infection. 81 vertebrates on their own are endowed with immune system robust enough to efficiently and effectively surmount the non-self-attacks. yet the more the host's immune system elaborates, the better the organisms in their evasion of immune effector cells. antigenic variation refers to a pathogen's ability to modify its surface proteins such that it can circumvent the host's immunological attacks. it involves several mechanisms including the varying of surface protein's phase, shifting and drifting of surface protein antigens and/or any other form of alteration of antigenic protein. 82 antigenic variation plays significant roles in the pathogenicity of microorganisms by evasion of the host immune responses and establishment of re-infection. when a pathogen alters its surface antigens, it can evade the host's adaptive immunity and so reestablishes infection. the immune system may battle to generate new immunoglobulins against the new antigen. certain bacteria like neisseria gonorrhea, neisseria meningitides, mycoplasma and species of the genus streptococcus show antigenic diversity. 83 in eukaryotic pathogens, antigenic variation is shown by trypanosoma brucei and plasmodium falciparum. 81, 84 another vital cause of antigenic variation in bacteria is horizontal gene transfer (more important than point mutation) through plasmid acquisition and transduction via bacteriophages. virulence genes are normally acquired by non-virulent organisms via these routes. once this occurs, the new bacteria may quickly get established and cause fresh epidemic outbreak. species of the genus neisseria are champions in the rapid change of surface antigens amongst bacterial pathogens. pathogenic forms exhibit an amount of phenotypic variability not found in the commensal species. the pathogenic forms are implicated in std and meningitis. they employ amazing varieties of antigenic variability mechanisms. • they can recombine their pilin genes in a similar manner that eukaryotes recombine their own genes, such that they can express variable surface protein. 85 • some cell-surface proteins and enzymes synthesizing bacterial cell-surface carbohydrates are expressed in a variety of ways. this is as a result of replication slipping or slippage errors and repairs of simple tandem nucleotide repeats involving either the di-, or tri-or tetra-nucleotides. 86 • neisseria is able to take up and incorporate environmental dna into its genomes. 83 these are why an effective vaccine against neisseria infections is not yet developed. neisseria may be considered as an extreme example. however, many other bacterial pathogens like streptococcus and mycoplasma in promoting their antigenic variation tend to utilize one or more of these techniques. additionally, there are reports that dna-related defects have a much greater association with bacterial pathogen from symptomatic patients than samples of the same bacterial species isolated from environmental sources. [87] [88] [89] pneumococcus streptococcus pneumoniae, gram-positive cocci bacteria that cause otitis media, bacteremia and pneumonia, are a public health concern, causing morbidity and mortality in adults and children. 90 two forms of vaccines (polysaccharide and conjugate vaccines) are currently marketed for the prevention of pneumococcal infections. while the polysaccharide vaccines are for vaccination in the adult population, conjugate vaccines have an added immunogenic non-pneumococcal protein conjugated to the pneumococcal polysaccharides for enhanced immunogenicity in children. it is not yet known that these vaccines can evoke complete immunity against the infection. a polysaccharide capsule is a major virulence factor in the bacteria. several of these capsule types have been identified, and these form the basis of pathogen's antigenic serotyping. 91, 92 current pneumococcal vaccines are combinations of various capsular (polysaccharide) antigens from the serotypes most common in a particular population. currently, over 100 different serotypes are known but are not all covered in the available vaccines. 92 the discovery of a common antigen(s) will produce an effective vaccine. knowledge of the genome of the organism and the different strains has led to a possible advance in driving the pneumococcal potential vaccine search through a different approach. and this consideration will help solve a lot of concerns about the current vaccines. with this knowledge, many methods are been tried to determine whether they can be a source of effective vaccine design that can accommodate all the serotypes of the organism. search for antigen that is common to all the serotypes can be achieved with the knowledge of genomics and immunoinformatics. the introduction of genomic and computational technologies has given new directions in the study of bacterial pathogenesis and vaccine design. 93, 94 plasmodium plasmodium falciparum undergoes two life cycles: one in humans and the other in mosquitoes. the human host's erythrocytes and hepatocytes usually display modified parasite proteins called plasmodium falciparum erythrocyte membrane protein 1 (pfemp1) and plasmodium falciparum hepatocyte membrane protein 1 (pfhmp1), respectively. these proteins function to assist the parasite to evade destruction by the host immune systems. 95, 96 the pfemp1 proteins were identified as the prime ligands responsible for cytoadherence and resetting. 97 they cause the infected rbcs of host tissues to sequester thus helping the parasite to circumvent clearance by the host's spleen. 98 the membrane proteins also shield infected host cells from destruction by the spleen by adhering to the endothelium. luckily, if the pfemp1 protein is expressed for a long while; it comes under attack by the naturally acquired immunity. 98, 99 in defence to this, the parasite has expanded the var genes coding for pfemp1 such that the genes can exist as a polymorphic family of as much as over 50 members in every genomic haploid. antigenic switches work well here in that members of the polymorphic family (also called antigenic-variant-protein family) can be interchanged and cannot express their proteins at the same time. in this way, only one particular protein at the surface of the infected rbc is expressed at any given time. 97, 100 when studying antigenic-variant-protein families, it is pertinent to understand if grouping them into single-family results in any meaningful antigenic activity. studies have tried to understand the "languages" of the antigenic variant of pfemp1 proteins. 97, 101, 102 they sought to know the pfemp1 proteins binding properties or search to understand the correlation between motifs and infection severity. the vardb database is a repository for protein sequences involved in antigenic variation and their associated functionalities. 103 antigenic variant data obtained from several pathogens may be regrouped into a unified database. this will enable researches from several multicopy gene families to be accessed and compared swiftly in a single moment. updated vardb database contains close to 10,000 dna sequences, several protein translations, tens of infectious diseases and pathogens with their gene families. with a novel sequencing-based approach, pacbio, the different pfemp1 proteins can be sequenced and the related sequences used as potential vaccine targets. 104, 105 trypanosoma for many pathogens, antigenic variability occurs during the infection pathogenesis and is to enable them to escape destruction by the host antibodies. for instance, some eukaryotic parasites take to genetic assortment and rearrangement thereby changing their surface antigens. a ready example is seen in trypanosoma brucei, the causative organism for sleeping sickness. trypanosoma brucei replicates in the bloodstream (outside the cells) of their host, but at maturity, it crosses the blood-brain barrier to cause several fatal complications. during replication in the bloodstream, the parasites are subjected to humoral as well as cellular immune responses. it evades the host defenses by encasing itself in homogeneous coat of glycoprotein called the variant surface glycoprotein (vsg). 106, 107 though at initial invasion, the protein coat tends to protect the microbe from the immune system but on constant exposure, the coat will be identified as a foreign matter, and at this point the immune effectors can launch an attack against it. in a particular trypanosoma brucei, there are diversities of the vsg protein being coded by more than a thousand genes in the parasite's genome. unfortunately for the host, the expression of these genes is mutually exclusive. expressed vsg gene is normally due to genetic reassortments causing new alleles to be copied into the sites of expression. some trypanosomes with these abnormal vsg genes evade humoral immunity and multiply thereby causing re-infection and chronic recurring infections. 107 influenza is a viral infectious disease due to infection by any of the three types of rna viruses, namely influenza types a, b and c. current vaccines contain double type a and single type b strains and induce strong antibody responses to neuraminidase and the surface glycoprotein hemagglutinin. these vaccines, however, cannot effectively protect against newly emerging viruses with antigenic shift and drift. 108, 109 antigenic drift results in changes in the antigenic site (a minor change) while antigenic shift results in a new virus subtype. hemagglutinin and neuraminidase are the two enzymes dictating the antigenic properties of the viruses. while inside its host, defined host proteases break the peptide bonds in the hemagglutinin molecule to form hemagglutinin 1 and 2 subunits. virulence tendencies are decreased when the amino acids at the cleavage sites are lipophobic, the virus exhibits high virulence tendencies. 110 the surface glycoprotein can be regarded as antigen and hence can serve as a target for the immune system which if sequenced, using the new immunoinformatics approach and a common site for the varying proteins identified, a potent vaccine can be developed which can accommodate the antigenic drift/shifts of the virus. influenza viruses are able to thrive for a long while in a given human population. 111, 112 the virus has a high mutation rate such that a once effective vaccine can easily lose efficacy. antigenic variability is only one of the evidences of phenotypic variation in the biology of the influenza virus. the use of immunoinformatics in vaccine development has been accelerated toward the design of a multiepitope vaccine construct which has and will fully address the challenges faced with pathogens with mutagenic antigens. previous vaccines developed by conventional approaches consist of several proteins or a whole pathogen. this constitutes unwarranted antigenic load and increases the chances of inducing allergy. the use of peptide-based vaccines surmounts these challenges. the vaccines are made from short peptide fragments capable of eliciting highly specific immune responses, precision targeting and multiepitope constructs, in the case of varying antigenic peptides, which has been made feasible with the advancements in the field of computational biology. [113] [114] [115] [116] vaccines for pathogens with immune escape potentials can basically be constructed by using most, if not all, of their immunogenic peptides 116, 117 because such vaccines prove to be better than single-epitope and classical vaccines. multiepitope vaccines enjoy the following advantages over single-epitope and classical vaccines: a) they are an assemblage of several epitopes obtained from distinct protein targets/antigens of an intended infection; b) the multiple t-cell receptors (tcrs) in the vaccine recipient can easily recognize vaccines with multiple hla epitopes; c); they can be easily adjuvanted to improve their immunogenicity; d) they can activate antibody-mediated and cell-mediated immunological responses because of their overlapping helper t lymphocytes (htl), cd8+ t-cell and b-cell epitopes; and e) unwanted protein antigens are excluded in such construct thereby reducing the chances of untoward effects and/ or immune responses likely to cause disease(s). [118] [119] [120] [121] [122] [123] thus producing a vaccine with these qualities can provide chances of combating most infections such as streptococcus pneumoniae and hiv infections. immunoinformatics can be employed in the docking of single and multiepitope vaccines and subsequently to predict their properties (physicochemical, allergenic and antigenic). this approach has seen the use of diverse tools and database in the analysis of ligands with their targets and has greatly helped to predict the binding score of antigenic peptides with the immune proteins like hla. peptides and hla allele modeling can be done by the 3d structural designing of the epitopes using pepfold3 (an online server), 124 retrieving from protein data bank (pdb) the x-ray crystallographic structure of human population most occurring hla alleles (hla-drb1 01:01, 15:01 and hla-a 02:01) followed by filtration of previously bound ligands. the following is a step-wise detail on how to construct a single or multiepitope-based vaccine and its property prediction; • molecular docking analysis: to determine the interaction pattern of the screened out epitopes with the hla alleles by employing cluspro v.2 (a proteinprotein docking web server). this server performs this task by energy minimization, calculation of both the binding energy scores of the docked complex and electrostatic/shape complementarity. • target-protein comparative modeling and associated structure validation: the sequence of the amino acid in the target protein (e.g., tlr-9) can be retrieved from uniprot and the tertiary structure with raptor-x and i tasser (online comparative modeling tools). the server constructs and creates a 3d model immunotargets and therapy 2020:9 (mathematical representation) of the target protein using hierarchical algorithms. 118 personalized vaccine refers to vaccines "targeted" toward an optimized outcome. immunogenicity is maximized while either the risk of vaccine failure or reactogenicity and side effect is minimized. personalized are developed in the following cases; the individual level vaccines are developed to take care of haplotype and polymorphism knowing that they can retard the formation of a protective immune response or become pointers to the risk of an adverse vaccine reaction. this is needed when it is clear that females produce a higher antibody titre against a particular vaccine antigen than do their male counterparts. where it is clear that a particular human race or ethnic group has a higher or lower immune response to a particular vaccine antigen. personalized vaccines arise due to known complex interactions between host environmental, genetic and some other factors that may be influencing the vaccine immune responses. the associations between the immune response gene polymorphisms and variations in immune responses to a particular gene must be pine-pointed when it is clear that a particular drug either suppresses or augments the transcription of an immune response gene. 127, 128 this could help in designing vaccines or vaccine adjuvants that can circumvent restrictions due to immunological differences arising from varying genetic compositions. 129, 130 personalized vaccines stem from our understanding of how, within the human leukocyte antigen (hla) systemalso referred to as the major histocompatibility complex (mhc), the t cells are able to recognize peptides of pathogenic origin. 131, 132 hla molecules enjoy the double advantages of having stable polymorphisms and being fully characterized. 133 these advantages make good candidates for personalized vaccine design. hla polymorphism, although stable, is complex. for instance, more than 12,000 alleles of hla class i molecules and greater than 4000 class ii molecules have been identified among human populations. 133, 134 hla class i and ii molecules have heterodimeric character comprising of α and β chains, three highly variable extracellular domains (α1, α2, and α3) and then transmembrane and intracytoplasmic domains that are less variable. 133, 135 hla genes contain eight exons. exon 1 is responsible for producing the leader peptide; exons 2,3,4 produce α1, α2, and α3 extracellular domains, respectively, for mhc class 1 or α1, β1and α3, respectively, for mhc class ii; exon 5 encodes transmembrane anchor; exons 6 and 7 encodes the cytoplasmic tail while exon 8 encodes the 3ʹ-untranslated region. 135 most of the several forms associated with the class i and ii genes are seen in α-1 and α-2 (as known as class i) and in α-1 and β-1 (as known as class ii) domains. 133 mhc i and ii bind and present the peptide to t cells. t cell responses to viral pathogens differ from one patient to the other, basically because of the expression of differing hla (mhc) alleles which determine the several viral amino acid sequence brought to the t cells to read. 136, 137 it is most likely that during an infection, diverse epitopes are usually presented to the t cells to read owing to the several forms of hla alleles and also because each human person expresses six hla class i and six hla class ii alleles. 138 now, antibody-binding sites in a given hla (mhc) molecule are mostly prediction-servers predetermined on the basis of particular binding motifs and the anchor residues. 139, 140 these residues refer to known amino acids located at defined locations in the peptide chain and which are peculiar to each mhc molecule. 141, 142 prediction-server database of peptide motifs and/or of mhc ligands may be obtained from web-based and/or from prediction-servers dedicated to netmhc family. 143, 144 in another example, sequence analysis of lassa fever virus (the lasv) and other viruses' immunoproteomic was used to identify the best immunogenic protein predicting t-cell as well as b-cell epitopes and also target sequence and binding sites. 145, 146 the ssnlykgvy peptide sequence at aa41-49 of glycoprotein 1 (produced by the l segment) was the best candidate epitope for the induction of humoral as well as the cell-mediated immunity for lassa fever vaccine construct. 17 hla-i and 16 hla-ii molecules have been proven in sizable african populations and their combination with the ssnlykgvy peptide sequence may prove useful in such lassa fever virus endemic areas. 145 this approach will strongly improve individualized vaccination and help combat emerging infections. the hla region is suspected to contribute, to a large extent, to genetic propensity to infections and differences in vaccine expected immune responses. 132, 147 studies show that females exhibit stronger immune responses to immunization compared to males. 148, 149 there are differential antibody responses to rubella and measles viral protein between males and females and that both hormonal and genetic difference may be influencing the immune responses. 148, 150, 151 practical issues may stand in the way of achieving this new development (personalized vaccinology). having to use different vaccines for different persons based because of personal genetic composition requires more time and labor during the vaccination process. also, screening for individual factors for targeted vaccination can significantly increase vaccination cost. but with all these challenges, personalized vaccination is the new age approach in achieving an optimum immunization that takes into consideration the individual immune differences in a particular population and it is a new dawn for vaccine development. personalized vaccine development is strongly improved by vaccinomics. the field of vaccinomics looks at how immune response gene polymorphisms affect the cellmediated, humoral and innate immune responses to vaccine antigens at population and specific individual levels. "vaccinomics" encompasses both immunogenomics and immunogenetics as it concerns immune responses to vaccine antigen. 152 the fields of personalized vaccinology and vaccinomics were the products of phase i of the international hapmap and that of the human genome project. also, modern molecular assay techniques permitting highthroughput detection of variations at gene level, in particular linkage disequilibrium maps and single nucleotide polymorphism (snp), played significant roles in the development of personalized vaccinology and vaccinomics. it has also been shown that polymorphisms at vital immune response genes can bring about differing immune responses to biopharmaceutical products including vaccines. [152] [153] [154] newer, accurate, cheap and reproducible sequencing technologies; validated databases containing genotypephenotype data; statistical and bioinformatics tools are needed in order to analyze and interpret data that will help and improve vaccine adverse and immune response quantifiability and predictability. 155 the information will enhance clinical practice and accelerate rational and directed vaccine development. safe vaccines are a critical requirement for any immunization program. 156 conventional vaccination has been an approach targeted at all groups and individuals but has failed toward the enrolment of pregnant women into vaccination programs because of presumed fetal and maternal harms. 157, 158 evidence on the safety of vaccination in pregnancy is very small because pregnant women were usually excluded from participating in vaccine trials. 159 pregnancy can alter the maternal as well as fetal immunological responses. 160 it is pertinent to explore research opportunities presented in advanced vaccine designs such as immunoinformatics (multiepitope vaccine docking) by studying human immune system functions and responses specific to pregnant women and their unborn children. 157 according to a report 161 from the dominican republic of congo, during the 2016-2017 zika virus outbreaks, over a thousand pregnant women were suspected of being infected with the virus and a sizable number were at their first trimester. the report further stated that fetal loss was approximately one-tenth of the pregnancies and that there were up to 3 cases of fetus with head circumferences smaller than normal. the widespread morbidity during the epidemic showed that zika virus infection adversely affects pregnancy outcome. 160, 161 currently, there is no proof that pregnancy predisposes to ebola virus infections in comparison with the non-pregnant population, but there is some evidence suggesting pregnancy to worsen the disease prognosis including fetal loss. also, evidence showed that the virus can pass the placental barriers to establish infection in the unborn child. 162 designing, implementing and enrolling pregnant women as well as perspective pregnant women into vaccine trials and programs is imperative in order to protect this group and ensure good vaccine uptake by them during infection outbreaks and epidemics. 157, 163 these recommendations will give an informed decision to be investigated using the immunoinformatic tools to determine the immunogenic responses worthy of safe vaccine development for the pregnant women and perspective pregnant women group. maternal immunization offers palpable benefits in several ways. some vaccines are primarily administered to shield these pregnant women from morbid conditions and/ or death including fetal death. 164, 165 pregnant women stand the risk of being exposed to virulent pathogens and may be at a higher risk of morbidity and/or mortality when compared to the general population. 166 there has been an explosion of new immunological data (table 4 ) due to an increase in research to understand the immune system pathway in infectious disease pathogenesis and the application of the knowledge of bioinformatics has led to a better exposition of the immune system importance through immunoinformatics. the knowledge of immune system and the cost-effective, specific and effective approach like immunoinformatics, the concerns for emerging and re-surging diseases caused by pathogenic organisms, antigenic variability/complex lifecycle of pathogens and the need of personalized vaccination can be combated on a molecular level. the future of immunological research is sharpened by the ability to make discoveries in biologics (e.g., vaccines) more effectively and efficiently. this will mean reduction and better targeting of wet laboratory experiments and will only be possible if wet laboratory experimentation is combined with bioinformatics techniques. • immunoinformatics depends on experimental science (wet laboratory) to produce raw data for analysis. the predictions are not formal proofs of any concepts. they do not replace the traditional experimental research methods of actually testing hypotheses. • the quality of immunoinformatics predictions depends on the quality of data and the sophistication of the algorithms being used. sequence data from high-throughput analysis often contain errors. if the sequences are wrong, or annotations incorrect, the results from the downstream analysis are misleading as well. 167 immunotargets and therapy is an international, peer-reviewed open access journal focusing on the immunological basis of diseases, potential targets for immune based therapy and treatment protocols employed to improve patient management. basic immunology and physiology of the immune system in health, and disease will be also covered. in addition, the journal will focus on the impact of management programs and new therapeutic agents and protocols on patient perspectives such as quality of life, adherence and satisfaction. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. immunotargets and therapy 2020:9 time for t? immunoinformatics addresses vaccine design for neglected tropical and emerging infectious diseases vaccinology in the third millennium: scientific and social challenges antigenic variability: obstacles on the road to vaccines against traditionally difficult targets complex immune correlates of protection in hiv-1 vaccine efficacy trials immunoinformatics approach to design a novel epitope-based oral vaccine against helicobacter pylori immunoinformatics approach for multiepitopes vaccine prediction against glycoprotein b of avian infectious laryngotracheitis virus transcriptome and proteome: the rise of omics data and their integration in biomedical sciences neoantigen vaccine: an emerging tumor immunotherapy designing of cd8 + and cd8 + -overlapped cd4 + epitope vaccine by targeting late and early proteins of human papillomavirus immunization with a recombinant antigen composed of conserved blocks from tsa56 provides broad genotype protection against scrub typhus vaccine mediated protection against zika virus-induced congenital disease vaccination with sporozoites: models and correlates of protection novel approaches for the design, delivery and administration of vaccine technologies vaccination to gain humoral immune memory humoral and cell-mediated immune responses after a booster dose of hbv vaccine in hiv-infected children, adolescents and young adults an evaluation of the cold chain technology in south-east, nigeria using immunogenicity study on the measles vaccines a review of the immunological mechanisms following mucosal vaccination of finfish reverse vaccinology 2.0: human immunology instructs vaccine antigen design large screen approaches to identify novel malaria vaccine candidates reverse vaccinology and subtractive genomics reveal new therapeutic targets against mycoplasma pneumoniae: a causative agent of pneumonia evolution of the immune system in humans from infancy to old age the twilight of immunity: emerging concepts in aging of the immune system pneumococcal vaccination strategies. an update and perspective progress and challenges in tb vaccine development vaccines for the elderly: current use and future challenges remembrance of things past: long-term b cell memory after infection and vaccination global practices of meningococcal vaccine use and impact on invasive disease new vaccine technologies to combat outbreak situations what are the most powerful immunogen design vaccine strategies? reverse vaccinology 2.0 shows great promise the new malaria vaccine program for african children is promising but still quite limited. quartz africa merck's ebola vaccine helps combat deadly outbreak in the congo as the virus spreads. biotech and pharma innovation for the 'bottom 100 million': eliminating neglected tropical diseases in the americas emerging and neglected infectious diseases: insights, advances, and challenges exploitation of reverse vaccinology and immunoinformatics as promising platform for genome-wide screening of new effective vaccine candidates against plasmodium falciparum the use of databases, data mining and immunoinformatics in vaccinology: where are we? systems vaccinology and big data in the vaccine development chain biochemical functional predictions for protein structures of unknown or uncertain function reverse vaccinology: the pathway from genomes and epitope predictions to tailored recombinant vaccines universal genotyping for tuberculosis prevention programs: a 5-year comparison with on-request genotyping integrating whole-genome sequencing data into quantitative risk assessment of foodborne antimicrobial resistance: a review of opportunities and challenges bioinformatics and drug discovery new technologies in predicting, preventing and controlling emerging infectious diseases how genomics can be used to understand host susceptibility to enteric infection, aiding in the development of vaccines and immunotherapeutic interventions in silico analysis of epitope-based vaccine candidates against hepatitis b virus polymerase protein advances in designing and developing vaccines, drugs and therapeutic approaches to counter human papilloma virus computational approaches and challenges to developing universal influenza vaccines. vaccines (basel) experimental and computational analyses reveal that environmental restrictions shape hiv-1 spread in 3d cultures dynamic computational model of symptomatic bacteremia to inform bacterial separation treatment requirements a model of plasmodium vivax concealment based on plasmodium cynomolgi infections in macaca mulatta a computational modeling approach predicts interaction of the antifungal protein afp from aspergillus giganteus with fungal membranes via its γ-core motif. msphere an overview of bioinformatics tools for epitope prediction: implications on vaccine development bioinformatics tools for identifying class i-restricted epitopes in silico-accelerated identification of conserved and immunogenic variola/vaccinia t-cell epitopes nhlbi-abdesigner: an online tool for design of peptide-directed antibodies ivax: an integrated toolkit for the selection and optimization of antigens and the design of epitope-driven vaccines from genome to vaccine: in silico predictions, ex vivo verification designing string-of-beads vaccines with optimal spacers hiv vaccine development by computer assisted design: the gaia vaccine nerve: new enhanced reverse vaccinology environment jennerpredict server: prediction of protein vaccine candidates (pvcs) in bacteria based on host-pathogen interactions genome-wide prediction of vaccine target of human herpes simplex viruses using vaxign rv vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines vacsol: a high throughput in silico pipeline to predict potential therapeutic targets in prokaryotic pathogens using subtractive reverse vaccinology panrv: pangenome-reverse vaccinology approach for identifications of potential vaccine candidates in microbial pangenome understanding emerging and re-emerging infectious diseases. bethesda (md: national institutes of health (us) the consequences of human actions on risks for infectious diseases: a review institute of medicine (us) forum on microbial threats. microbial evolution and co-adaptation: a tribute to the life and scientific legacies of joshua lederberg: workshop summary trends in antimicrobial resistance of bacterial pathogens in harare understanding emerging and re-emerging infectious diseases. bethesda (md): national institutes of health (us) evolution and emergence of infectious diseases in theoretical and real-world networks carbapenem-resistant enterobacteriaceae posing a dilemma in effective healthcare delivery annual review of diseases prioritized under the research and development blueprint the role of nanotechnology in the treatment of viral infections proteomics for development of vaccine computational methods in drug discovery computational approaches for prediction of pathogen-host protein-protein interactions new approaches to understanding the immune response to vaccination and infection adaptive immune responses mediated by natural killer cells friend retrovirus studies reveal complex interactions between intrinsic, innate and adaptive immunity from entry to early dissemination-toxoplasma gondii's initial encounter with its host evolution and divergence of h3n8 equine influenza viruses circulating in the united kingdom from neisseria genomics: current status and future perspectives antigenic variation in plasmodium falciparum malaria involves a highly structured switching pattern neisseria gonorrhoeae muts affects pilin antigenic variation through mismatch correction and not by pile guanine 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rep global genetic diversity of var2csa in plasmodium falciparum with implications for malaria in pregnancy and vaccine development how does the vsg coat of bloodstream form african trypanosomes interact with external proteins? variant surface glycoprotein density defines an immune evasion threshold for african trypanosomes undergoing antigenic variation towards a universal influenza vaccine: different approaches for one goal the emerging influenza virus threat: status and new prospects for its therapy and control influenza a virus hemagglutinin antibody escape promotes neuraminidase antigenic variation and drug resistance a comprehensive review on equine influenza virus: etiology, epidemiology, pathobiology, advances in developing diagnostics, vaccines, and control strategies novel platforms for the development of a universal influenza vaccine computer aided epitope design as a peptide vaccine component against lassa virus genome-wide identification of novel vaccine candidates for plasmodium falciparum malaria using integrative bioinformatics approaches. 3 biotech in-silico design of a multi-epitope vaccine candidate against onchocerciasis and related filarial diseases antigenic variation and immune escape in the mtbc vaccinomics approach for designing potential peptide vaccine by targeting shigella spp. serine protease autotransporter subfamily protein siga designing a multi-epitope based vaccine to combat kaposi sarcoma utilizing immunoinformatics approach efficient control of chronic lcmv infection by a cd4 t cell epitope-based heterologous prime-boost vaccination in a murine model a novel multi-epitope vaccine from mmsa-1 and dkk1 for multiple myeloma immunotherapy evaluation of tandem chlamydia trachomatis momp multi-epitopes vaccine in balb/c mice model development of a multi-epitope peptide vaccine inducing robust t cell responses against brucellosis using immunoinformatics based approaches identification of a cd4 t-cell epitope in the hemagglutinin stalk 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vaccine development: from the identification of vulnerable individuals to the formulation of invulnerable vaccines advances in genetics and genomics: use and limitations in achieving malaria elimination goals. pathog glob health safety evaluation in mice of the childhood immunization vaccines from two south-eastern states of nigeria prevent working group. pregnant women & vaccines against emerging epidemic threats: ethics guidance for preparedness, research, and response vaccinations for pregnant women efficacy and safety of pertussis vaccination for pregnant women -a systematic review of randomised controlled trials and observational studies beyond passive immunity: is there priming of the fetal immune system following vaccination in pregnancy and what are the potential clinical implications? front immunol zika virus epidemic in pregnant women ebola virus disease in pregnancy: clinical, histopathologic, and immunohistochemical findings the ethics working group on zikv research & pregnancy. pregnant women & the zika virus vaccine research agenda: ethics guidance on priorities, inclusion, and evidence generation maternal immunization maternal immunization: where are we now and how to move forward strengthening maternal immunisation to improve the health of mothers and infants immunoinformatics: in silico approaches and computational design of a multi-epitope, immunogenic protein while this review has not been funded directly by them, we gratefully acknowledge the drug design laboratory of faculty of pharmaceutical sciences, nnamdi azikiwe university, nigeria, and drug discovery africa. all the needed data are included in this manuscript. all authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work. the authors report no funding and no conflicts of interest in this work. key: cord-009836-7o6htufh authors: borrow, persephone; shaw, george m. title: cytotoxic t‐lymphocyte escape viral variants: how important are they in viral evasion of immune clearance in vivo? date: 2006-04-28 journal: immunol rev doi: 10.1111/j.1600-065x.1998.tb01206.x sha: doc_id: 9836 cord_uid: 7o6htufh summary: although viral variants which are not recognized by epitope‐specific cytotoxic t lymphocytes (ctl) have been shown lo arise during a number of persistent virus infections, in many cases their significance remains controversial: it has been argued that the immune response is sufficiently plastic to contain their replication. in this review, we describe the mechanisms by which amino acid changes in viral proteins may affect epitope recognition by virus‐specific ctl, and discuss the viral and immunological basis for the emergence of viral variants bearing such amino acid changes during infection. we then consider the impact that viral variation may have on the host ctl response and its ability to contain virus replication. we argue that the emergence of a viral variant demonstrates that it must have an in vivo replicative advantage, and that as such, the variant must tip the balance between virus replication and immune control somewhat in favor of the virus. further, we suggest that although the immune response can evolve to recognize new viral epitopes, the ctl generated following such evolution frequently have a reduced ability to contain virus replication. we conclude that this escape mechanism likely does make a significant contribution to persistence/pathogenesis during a number of different virus infections. following virus infection, a series of complex interactions occur between the virus and the host immune system. the host aims to ehminate the infection and minimize associated pathological consequences, whilst the virus tries to avoid clearance by the host immune response so that it can persist and be disseminated to other hosts over a long time period. viruses have evolved a variety of different strategies for avoiding clearance by the host immune response; a single virus often employs multiple strategies simultaneously to increase its chances of persisting in the face of an adaptive host defense system with an array of different effector mechanisms (1). as cell-mediated immime responses, in particular the virus-specific cd8+ cytotoxic t-lymphocyte {ctl) response, frequently play a key role in the ehmination of established virus infections, many viral evasion strategies are targeted to this arm of the immune response. viral immune evasion strategies may broadly be divided into two categories: those that enable the virus to avoid detec-jrrow & shaw • in vivo importance of ctl escape viruses tion by the host immune response, and those that impair the functioning of the host immune system. viral impairment of the functioning of the host immune system may be non-antigen-specific, as exemplified by the production of homologs of host cytokines or rheir receptors that mimic (in the case of immune downregulatory) or block (in the case of immunestimulatory or antiviral effector) the actions of host cytokines (2), or hy infection of cells of the immune system, causing either impairment of their functions or resulting in their destruction; the latter may occur either directly or hy the cells being rendered targets for immune lysis (3). however, as too severe a generalized impairment of host immune functions can lead to the death of the host, thus reducing the opportunity for virus spread, a preferable strategy is impairment of the virusspecific immune response. examples of how this may be achieved are by viruses infecting and targeting the destruction of antigen-specific b cells (4) or inducing exhaustion of highaffmity clones of virus-specific t cells (5), strategies viruses may employ to avoid detection hy the host immune response include i) the establishment of latent infections during v\'hich viral protein production is minimized, a strategy commonly employed hy herpesviruses and perfected by herpes simplex virus (6); ii) infection of immune-privileged sites (to which access of the immune system is restricted and where levels of major histocompatibility complex (mhc) antigen expression are iow and immune downregulatory mediators may be present), the best example being the hrain where a surprisingly large number of different viruses persist (7); iii) virus-induced downregulation of levels of mhc antigens or adhesion molecules on the surface of infected cells so :hat they do not trigger host t-cell recognition (8); and iv) evolution of antigenic variants whose recognition by the specific immune response is impaired, antigenic variation was initially documented as a means for viral escape from antibody neutrahzation (9), but more recently has also heen shown to confer viral escape from ctl control (10). although the above immune evasion mechanisms all have [he potential to promote viral persistence, the contribution that some of these mechanisms in fact make to persistence in vivo, particularly during human virus infections, is not completely dear. for example, although viral variants which are not recognized by epitope-specific ctl bave been found during a number of persistent virus infections (11), in many cases their significance remains controversial: it has heen argued that the immune response is sufficiently plastic to contain their rephcation (12), in this review, the mechanisms hy which amino acid changes in viral proteins may affect epitope recognition by virus-specific ctl are described, and the viral and immunoiog-ical basis for the emergence of viral variants bearing such amino acid changes during infection is discussed. the impact that viral variation may have on the antiviral ctl response during the course of virus infections is then considered, and the contrihution that this escape mechanism may make to persistence/pathogenesis in different virus infections is discussed. mechanisms by which amino acid changes may affect epitope recognition by virus-specific ctl for a viral epitope to be recognized by cd8-^ t cells, it must be processed and presented on the cell surface in association with mhc class i molecules. amino acid changes may thus affect epitope recognition by specific cd8+ t cells hy altering any of the steps involved in this pathway, from the initial generation of the epitopic peptide to its formation of a stable complex with mhc, they may of course also affect the interaction of the peptide-mhc complex with the t-cell receptor (tcr), either ablating tcr binding altogether, or altering the signahng which occurs via the tcr, as described below, examples have been found of amino acid changes in viral proteins that act via each of these mechanisms. alteration ofantigen processing/peptide transport the efficiency of processing and presentation of ctl epitopes is determined not only by the sequence of the epitope itself, but also by the residues which surround it in the protein. amino acid mutations in flanking sequences may thus affect the recognition of viral ctl epitopes, as was originally demonstrated in experiments using cytomegalovirus and influenza virus-specific ctl (13, 14), the precise mechanism by which antigen processing was affected was not defmed in these studies or a subsequent paper describing mutations in the nef protein of human immunodeficiency virus type 1 (hiv-1) which also affected presentation of a nearby ctl epitope (15), however, in a more recent report, alteration of proteasome-mediated degradation due to a single amino acid difference within a ctl epitope was shown to form the basis of lack of presentation of this epitope by infected cells (16). in mice infected with akv/mcf type murine leukemia virus, an epitope in the pi 5e transmembrane viral envelope protein constitutes the immunodominant sequence recognized by virus-specific ctl, these ctl do not recognize cells infected with friend/moloney/rauscher type murine leukemia virus, where the epitope sequence differs by a single residue (17); this is because the amino acid difference causes epitope destruction by specific proteasomal cleavage. mice infected with the latter virus thus do not mount a ctl response to this epitope, amino acid changes that alter the processing of ctl epitopes can therefore have a dramatic effect on the in vivo antiviral ctl response. it is unclear how commonly such mutations are selected for during persistent virus infections. that relatively few examples of amino acid changes in viral proteins which affect ctl recognition via this mechanism have been described could be a reflection of the fact that such mutations are not readily picked up by the methods most frequently used to analyze antiviral ctl responses. alternatively, the requirements for antigen processing/peptide transport may be so much less stringent than those for peptide interactions with mhc and the t-cel! receptor that amino acid changes which affect the former arise less often. amino acid mutations within epitopic peptides may ablate peptide binding to mhc altogether, or reduce the affmity of peptide-mhc interaction so that the peptide-mhc complex has an extremely short half-hfe and is unlikely to trigger t-cell activation. viral mutations which alter peptide interactions with mhc class i molecules in each of these ways have been described; examples in hiv were recently reviewed (19). as mutations which prevent the stable association of peptides with mhc confer escape from recognition by all epitope-specific t cells regardless of their tcr usage, this is an efficient mechanism for viruses to avoid ctl recognition. it is of interest that the human leukocyte antigen (hla)-a 11 epitope loss from epstein-barr virus (ebv) isolates derived from populations where the frequency of hla-al 1 is extremely high is conferred via mutations in anchor residues of the epitope that are important for binding to hla-al 1(19, 20). ebv is a genetically stable dna virus which generates mutants at a relatively low frequency; mutants will thus only come to prevail in the population if they confer advantages for the spread of virus to new hosts, in which very different tcrs may he used to recognize the same peptide-mhc complex. mutations that ablate binding of the immunodominant hla-a1 ] -restricted ebv ctl epitope to mhc will allow escape from recognition from epitope-specific t cells in any host. alteration of peptide interaction with the tcr amino acid changes in a peptide which do not prevent it from being presented in association with mhc may result in alterations to the surface recognized by the tcr. this surface may be altered by changes in the tcr contact residues, or by changes in other residues in the epitope that cause the peptide to bind to mhc in a distorted conformation (21). epitopes bearing mutations that cause alterations in the tcr contact sur-face have been termed altered peptide ligands (apl) (22). epitope-specific t cells bearing distinct tcrs may be affected by apl in different ways. the altered peptide-mhc complex may fail to interact with a particular tcr altogether; or it may be recognized, but the t cell may receive a reduced or even a different type of signal when it recognizes an apl-bearing cell (23-26), the responding t cell may thus be only partly activated (e.g. to proliferate but not induce cell lysis (27)), or even anergized (28). presentation of certain apl to t cells can therefore inhibit the response to the index peptide not just by competing with it for binding to mhc, but by negatively signaling the responding t-cell population: this phenomenon is known as t-cell antagonism. the abihty of apl to act as ctl antagonists can be tested by measuring the capacity of cells presenting the apl to inhibit lysis of labeled target cells presenting the index peptide (29), using this technique, mutations which confer antagonistic properties on epitopic sequences have been shown to exist in the persisting virus population in individuals chronically infected with both hepatitis b virus (hbv) (30) and hiv-1 (31), although viral mutations which result in the generation of apl may not provide as efficient a means of simple escape from immune recognition as mutations which abrogate epitope presentation altogether (because the apl may still be immunogenic to a proportion of host t cells which could be expanded following emergence of the mutant), they could potentially have powerful effects on the overall control of virus replication via other mechanisms. if they are able to act as t-cell antagonists, they may not only inhibit the lysis of cells on which the mutant epitope is presented, but also that of cells in which nonmutant virus is replicating, hence conferring protection from immune control on the entire viral quasispecies. further, as some apl are able to stimulate and sustain the growth of ctl despite the fact that they do not induce ctl lysis (27), they could drive an ineffectual ctl response and hence modulate the ctl repertoire in a detrimental fashion, reducing the overall efficiency of ctl control of virus replication. genetic variation is a strategy viruses exploit to promote their survival not just in the face of the host immune response, but under any environmental conditions they may encounter. they can achieve variation by a number of different mechanisms, including mutation, homologous and non-homologous recombination and (for viruses with a segmented genome) reassortment: different virns families utihze these to different extents. rna viruses (including retroviruses) and dna viruses such as immtinologicd reviews 164/1998 bepadnaviruses, whose genome replication involves an rna intermediate, have extremely high mutation rates. this is due in large part to the absence or very low efficiency of proof-reading-repair activities associated with rna replicases and transcriptases (32), and the lack of post-rephcative error correction mechanisms such as those diat normally operate during rephcation of cellular dna. misinsertion errors during rna rephcation and reverse transcription have been estimated to be in the range of 10""^ to 10"^ substitutions per nucleotide per round of copying; for a 10 kb genome, this w(mld result in each progeny rna strand including an average of 0.1 to 10 mutations (33). rna viruses thus exist not as homogeneous populations, but as complex, dynamic mixtures of heterogeneous sequences termed quasispecies (34, 35). if a virus is replicating under a constant set of environmental conditions to which it is optimally adapted, although the precise composition of the quasispecies will continually be fluctuating, the average sequence of the viral population will remain unchanged. however if conditions alter, mutations which confer an increase in fitness (the relative ability of the virus to produce infectious progeny) will be selected for and come to predominate in the viral population. the quasispecies nature of rna viruses, conpled with the short replication times and high viral yields they frequently exhibit, favors rapid adaptation to environmental changes. dramatic examples of viral evolution in response to environmental pressure have been provided by the emergence of drug-resistant viral variants in hiv-1-infected patients treated with antiretroviral agents (e.g. (36-38)), hiv is a retrovirus with an in vivo mutation rate of approximately 3x10"^ nucleotides per rephcation cycle (39). human infection with this virns is characterized by high levels of persisting virus, much of which is actively replicating and turning over at remarkable rates (40-42). as many as 10"^ virions may be produced per day (42), allowing great potential for variation (43), although not all of these will be infectious, and the effective population size may be much smaller (44). w^here a single nucleotide change is sufficient to confer a high level of resistance to antiretroviral treatment, resistance may be selected for very rapidly eor example, a single nucleotide change can reduce hiv's susceptibihty to the non-nucleoside reverse transcriptase inhibitor nevirapine by 100-1,000-fold. viral variants bearing this mutation have been shown to pre-exist in the plasma hiv rna in untreated patients (45), and in some patients high level resistance to this drug may be acquired within weeks of starting therapy (3 7), in cases where multiple mutations are required to achieve drug resistance, either because a series of mutations needs to occur before a high level of resistance to one antiretrovirai agent is achieved (38, 46) or because the patient is being treated simultaneously with a combination of several antiretroviral agents (47, 48), it takes longer for resistance to evolve. indeed, in some patients, particularly where virus replication has been reduced to very low levels, it may not do so (49, 50). however, these observations illustrate the tremendous potential for rna viruses to overcome controlling forces during the course of an infection via selection for resistance-conferring mutations. under what conditions are ctl escape viral variants selected in vivol the above outline of how mutations are selected for in a virus population allows a number of predictions to be made about the in vivo conditions under which ctl escape virns variants are likely to emerge. firstly, as a mutation will only be selected for if it confers a fitness advantage on the virus hearing it, ctl escape virus variants will only grow out in infections where ctl pressure exerts significant control over virus replication. it is thus perhaps not surprising that the first report of the in vivo emergence of a ctl escape virus variant came from a study performed using lymphocytic choriomeningitis virus (lcmv) (10), a murine virus: during infection with lcmv the cd8+ ctl response is well known to be the critical determinant of control of virus rephcation (reviewed in (51)), conversely, demonstration that viral variants bearing mutations which confer escape from cd8"^ t cells come to dominate the viral quasispecies during a particular virus infection can be used to provide evidence for the importance of this arm of the immune response in controlling the infection, which in human virus infections may otherwise be difficult to demonstrate convincingly (52) (53) (54) . secondly, as the stronger the control over virus rephcation exerted by the ctl response directed against a particular epitope, the greater the selective pressure it will exert for escape mutations in this epitope to emerge in the viral population, the frequency of ctl directed against a particular epitope and the avidity of their target cell interaction will clearly influence the selection of escape mutations in this epitope. the original demonstration of ctl escape virus selection in the lcmv system was actually made in tcr transgenic mice where cd8+ t cells specific for the epitope in which escape mutations were observed made up 75-90% of the peripheral t-cell population prior to infection (10), this was an artificial system; however, due to advances in methods for quantitating epitope-specific t cells, it has recently become apparent that epitope-specific cd8"^ t cells can reach extremely high frequencies in natural virus infections in both mice and humans, particularly during the primary immune response (55) (56) (57) . primary infection is thus clearly a setting under which escape-conferring mutations may potentially emerge very rapidly although lower frequencies of epitope-specific ctl will also have the capacity to drive the selection of escape viral variants, this selection will occur more gradually over the course of a greater number of rounds of viral rephcation. the greater the selective advantage conferred hy a particular viral mutation, the faster this mutation will be selected for in the viral quasispecies. thus, not only the strength of the ctl response to a particular epitope hut also the immunodominance of this response will have a great impact on the likelihood that escape mutations will be selected for within the epitope. escape mutations in subdominant or even co-dominant ctl epitopes may confer such a shght selective advantage on the viral variants bearing them (whose replication will still he controlled by the more dominant or co-dominant ctl responses in the host (58, 59) ) that they may never emerge in vivo. virus rephcation must clearly be ongoing for viral variants to be generated and selected. particularly where the selective advantage conferred by a certain amino acid mutation is not very great, many cycles of virus replication may need to occur before viruses bearing it become predominant in the viral quasispecies. how dominant the ctl response to a certain viral epitope is in the overall control of virus replication is thus also an important determinant of the emergence of escape mutations in this epitopic sequence from this perspective. if other arms of the immune response or cd8+ t-cell responses to other epitopes in the virus which are not affected by the mutation concerned are able to reduce virus replication to very low levels or mediate viral clearance, the escape mutation will not have time to hecome fixed in the viral population during the course of infection. ctl escape virus variants will thus be most likely to emerge in the face of a host ctl response which is highly dominated by t cells of a single epitope specificity; even a very strong response to a particular epitope is unhkely to have the chance to select for escape viral variants in the context of strong responses to other epitopes. this is illustrated by observations made in the murine lcmv infection model. the ctl response moanted to lcmv by infected c57bl/6 mice has a broad epitope specificity: in the viral glycoprotein (gp) and nucleoprotein (ne) alone, at least five different ctl epitopes have heen identified (60, 61). although the response to some of these epitopes is extremely strong (t cells recognizing the epitope at ne 396-404 constitute up to 40% of the cd8-^ t-cell population, and those recognizing the epitope at ge 33-41 up to 29% at the peak of the antiviral immune response (56)), the infection is rapidly cleared, and ctl escape viral variants do not emerge. if mice are immunized with one of these epitopes in a carrier protein prior to infection, the ctl response mounted upon lcmv infection is then biased in favor of this epitope. viral clearance still occurs; however, if virus is isolated from the mice just prior to clearance and grown up in vitro, it is found to contain escape mutations in the epitope to which the ctl response was biased (62) , escape viral variants thus arise in the context of this more immunodominant ctl response, but they do not grow out in vivo as the infection is cleared too rapidly the lcmv tcr transgenic mice discussed above represent a situation of epitope immunodominance at the extreme of the spectrum: their t-cell repertoire is so dominated by transgene-expressing cells recognizing the lcmv ge 33-41 epitope that ctl responses cannot be effectively mounted to other viral epitopes. thus, when they are infected with lcmv and viral mutants arise which cannot be recognized by the ge 33-4]-specific ctl, their replication is not contained by ctl of other specificities and they emerge in the context of viral persistence (10). the ahove predictions that ctl escape viral variants are most likely to. emerge in the context of a strong host ctl response which is highly focused on a single viral epitope are supported by the observation that one of the clearest examples of the emergence of ctl escape virus variants during a human virus infection occurred under just these conditions. both we and others have shown that strong cd8+ ctl responses are mounted very early following infection with hiv-1, prior to seroconversion, and have hypothesized that they play an important role in containing viral replication (63, 64), in one patient we studied (53), the early ctl response (16-20 days following the onset of symptoms indicative of acute hiv-1 infection, which occurred 20 days after the homosexual encounter during which he initially contracted the virus) appeared to he strongly directed against a single viral protein, gpl60. epitope mapping performed using the gpl 60 sequence of the patient's autologous early hiv-1 population indicated that this response was in fact extremely focused on a single epitope encompassing gpl60 amino acids 30-38(9), recognized in association with hla-b44, the frequency of epitope-specific ctl was extremely high: at the earhest timepoint available for study, which may have been shghtly after the peak of the primary immune response, 1 in 1 7 peripheral blood mononuclear cells (ebmcs) were found to score as virus-specific ctl precursors by limiting dilution analysis, a technique which has recently been shown to greatly underestimate the total numher of epitope-specific t cells (55, 56) , as shown in fig, 1 , viral variants bearing mutations in the epitopic sequence which conferred escape from recognition by epitope-specific ctl rapidly appeared in this patienc, and then increased in frequency until 164/1998 they had cotnpieteiy repiaced the transmitted virai strain. interestingly, the variants which came to predominate in the virai quasispecies aii possessed changes at gpi 60 amino acid 3 i, the position which constituted the major anchor residue for hla-b44. as discussed above, mutations which abiate peptide binding to mhc confer escape from recognition by aii epitope-specific t ceiis, and indeed, not oniy individual ctl ciones but aiso bulk ctl derived from this patient were unabie to recognize peptides corresponding to the mutant virus sequences in in vitto assays (fig. 1) . thus in both human and murine systetns, there is evidence that mutations which confer resistance to controi by epitopespecific ctl are most iiiieiy to be seiected for in highiy immunodominant epitopes. under conditions of epitope immuno-dominance, a singie mutation which provides escape from recognition by ctl of one specificity wiii confer a greater repiicative advantage on the mutant virus, thus the seiection for it wiii be stronger; pius there wiii be a greater chance that virus replication wiii be abie to continue for iong enough for the mutation to compieteiy repiace the index residue in the virai quasispecies. evolution of the ctl profile in response to the emergence of escape viral variants the virai quasispecies can ciearly evoive in response to host immune pressure: as the host immune response is aiso adaptive, can it in turn evoive in response to the emergence of escape viral variants? the specific immune response is nltimately driven by antigen; the emergence of a ctl escape mutation in a virus population during the conrse of an infection could potentially affect the antigen to wbicb tbe immune system is exposed, and hence the ctl profile, in several ways. firstly, unless the mutation selectively affects ctl stimulation so that only effector functions are diminished, the level of ctl specific for the index epitope will decline as the level ofantigen available to stimulate them decreases. secondly, if the mutant sequence is presented to and can stimnlate novel populations of host ctl, these will increase in frequency. thirdly if the mutation reduces the efficiency with which the epitope is presented, this may allow the level of presentation of other viral epitopes to increase, and hence ctl of different specificities to increase in frequency, or novel populations of host ctl to be stimulated. finally, as tbe mutant virus must bave a rephcative advantage over tbe original virus in order to be selected, the antigen load will increase, which will drive the overall expansion of virusspecific ctl. as discussed earlier, ctl escape mutations are most likely to be selected for in immunodominant ctl epitopes. the end result of the above effects will be that the breadth of tbe ctl response will increase, with the previously dominant t-cell clones declining in frequency, and subdominant or novel responses being stimulated. sucb broadening of ctl specificity in response to tbe emergence of viral variants able to escape recognition by ctl directed against a highly immunodominant epitope was apparent in the hiv patient described in the previous section (53) . as illustrated in fig. 2 , whereas pbmc derived from tbe patient very early during tbe infection mediated detectable lysis after in vitro restimulation of only target cells expressing the immunodominant gpl60 30-38(9) epitope, as viral variants bearing mutations in this epitope emerged, responses to epitopes elsewhere in gpl60 and in other viral proteins became apparent. a decline in the frequency ofctl directed against the gpl 60 30-38(9) epitope occurred simultaneously (53) . this patient clearly bad the capacity to make ctl responses to a broad range of epitopes in hiv; why then was his initial response so highly foctised on a single epitope? how epitope immunodominance is dictated dnring an antiviral immune response is not clear: both the level of presentation of different viral epitopes and the available t-cell repertoire impact on this. the former can be affected by epitope processing (65), peptide transport into tbe endoplasmic reticulum (66, 67) , tbe binding affinities of peptides to class i molecules (68) and the stabihty of peptide-mhc complexes on the cell surface (69), if ctl epitopes overlap, different mhc molecules may compete for their presentation (70) . the t-cell repertoire is initially deter-mined by positive and negative selection in tbe tbymus, and subsequently reshaped in tbe periphery during the course of successive immune responses. tbe influence that tbe previous infection history may have on the subsequent immune response to a virus infection bas been illustrated in mnrine arenavirus infections (71) . in the hiv patient discussed above, both properties of the epitopic sequence and the t-cell repertoire at the time of infection may bave contributed to the extreme immunodominance of the early ctl response, tbe gpl 60 30-38(9) peptide may have had advantages over other potential epitopic sequences in its processing and transport: there appear to be differences in the processing of transmembrane and cytoplasmic proteins for presentation with class i whicb may give tbe former a presentation advantage under certain circumstances (72); further, it is of interest that during the natural processing of gpl60, signal peptide cleavage occurs between amino acids 29 and 30, thus generating the same n terminns as the 30-38(9) epitope. in addition, this epitope is predicted to have a very high binding affmity to hla-b44. t-cell repertoire effects may also bave played a role: it is possible tbat prior unrelated infections in this patient left bim with a population of memory t cells that cross-recognized this epitope, and that these cells, being present at higher frequency and more readily activated than naive t cells, dominated the initial hiv-1-speciflc immune response. this cannot be demonstrated conclusively as no samples are available from tbis patient prior to infection; bowever, the fact that epitope-specific ctl were contained in an ohgoclonal population of vp 19+ t cells which underwent a massive expansion very early after infection in this patient (73) would be consistent with this. another study in the hiv system wbich emphasized the effect that escape mutations in ctl epitopes may bave on ctl specificity focused on the ctl responses of two hla-identical hemophiliac brothers who were exposed to the same batch of contaminated factor viii and became seropositive within 10 weeks of one another (74) . their ctl responses were very dissimilar: one made strong responses to two hla-a2 and hla-a3-restricted epitopes in gag, whilst the other instead responded to two hla-b 7-restricted ctl epitopes. mutations in botb immunodominant epitopes of the gag responder were seen in provirai sequences from the non-responder; it was thus very hkely that be had initially made a ctl response to these epitopes, and tbat following the emergence of escape mutant viruses, tbis response had been downregulated and the b7restricted responses stimulated. in this individual, the escape mutant viruses did not revert to the index sequence when the ctl response against them diminished, implying tbat the mutations they bore were not detrimental to viral replication. in other cases, however, ctl escape mutations may reduce viral fitness (75) . if they impair virus replication too severely, tbey will not be selected for in the first place; this may contribute to the fact that in some studies escape variants have not been seen to arise in particular ctl epitopes despite the fact tbat ctl pressure is exerted on them over long periods of time (76. 77). if they have a smaller impact on viral fitness, they will emerge when epitope-specific ctl are exerting a strong pressure on viral replication, as overall they will confer an advantage on the virus, but as the frequency of epitope-specific ctl declines, they will tend to be replaced in the viral quasispecies by the index sequence. this in fact occurred in the hiv patient we described, where a strong selection occurred in tbe viral quasispecies for viral variants bearing mutations at the residue w-bich constituted tbe anchor motif for the hla-b44-restricted ctl epitope to which a dominant response was made in tbe early phase of the infection. later in tbe infection, wben the frequency of epitope-specific ctl bad declined significandy, clones bearing the index sequence again began to emerge in tbe viral quasispecies (x. wei, p borrow, . epitope immunodominance and mutant virus frequency tbus drive one another, which can lead to complex fluctuations in the makeup of tbe viral quasispecies and ctl specificity over the course of a persistent infection, sucb fluctuations have been observed in patients cbronically infected with hiv-1, and mathematical models developed to describe them (78-80), these models predict tbat antigenic variation in immunodominant epitopes can shift responses to weaker epitopes and tbereby reduce immunological control of the virus. that this certainly can occur bas been illustrated in experiments performed in the lcmv model system, ctl clones directed against the three most dominant epitopes recognized during the antiviral ctl response in c57bl/6 mice were used to select in vitro for lcmv variants bearing escape-conferring mutations in one, two or all three of these epitopes (81) (82) (83) . wben cs7bl/6 mice were infected with tbe mutant viruses, they mounted ctl responses to subdominant epitopes including epitope(s) in the viral polymerase wbich are not readily apparent during tbe response to wild-type virus, but the responses driven by the in vitro-generated escape viral variants controlled virus replication less efficiently than the response to wild-type virus (58, 59) . these experiments indicate tbat although tbe immnne response is extremely plastic, its capacity to evolve to efficiently contain the replication of escape mutant viruses may be limited, particularly in inbred mice where the diversity of mhc alleles is more restricted than in the outbred human population. the evolution of escape viral variants may thus make a significant contribution to viral persistence during some infections by driving a suboptimal immune response. for a ctl escape viral variant to emerge, it must tip the balance between virus replication and the host antiviral immune response at least somewhat in favor of the virus. this replicative advantage can in itself affect the course of a virus infection, as was seen in tbe experiments originally demonstrating the in vivo selection of ctl escape viral variants in lcmv epitope-specific tcr transgenic mice: these animals developed a persistent infection following inocnlation with a dose of virns whicb non-transgenic mice were rapidly able to clear (10). however, in tbis system, the tcr repertoire was artificially restricted, leaving open the question of whether ctl escape viral variants may also have biologically significant effects on the course of infection under more natural circumstances, where the immune system has the capacity to respond to a mucb broader range of viral epitopes and, as discussed above, the ctl response may co-evolve with the viral quasispecies. mutations which affect epitope-specific ctl lysis in vitro bave been described in a number of different viruses; here, their likely significance in some of these virus infections is discnssed. mouse hepatitis virus-strain jhm (mhv-jhm) is a coronavirus which produces an acute fatal encephalitis in most inbred strains of mice. if c57bl/6 mice are infected wbilst suckling on dams previously immunized to the virus, tbey are protected from tbe acute encephahtis; a proportion of mice then control the infection, but the majority develop a persistent infection associated with chronic demyelinating encephalomyehtis (84), tbe virusspecific cd8+ t-cell response is important in controlling this infection; in c57bl/6 mice this is directed against two epitopes in the viral surface gp (s), an immunodominant epitope at s 510-518, and a subdominant epitope at s 598-60s (85), a recent study showed that mutations which cause a loss of recognition are present in tbe immnnodominant epitope sequence in almost all virus sampled from symptomatic mice, but not in other t-cell epitopes (86) , mutations in this epitope were not detected in mice witb acute encephalitis (whicb die very soon after the antiviral t-cell response is mounted) and were fonnd at only low frequency in the residual viral rna in the central nervous system of animals which had cleared infectious virus and remained asymptomatic at late times after infection. further, when mhv-jhm variants bearing mutations in epitope s 510-518 isolated from infected mice were used to infect naive mice, they produced an increased morbidity and mortahty (87) . this is thus a convincing example of an infection in wbicb ctl escape variant selection does appear to be a key factor influencing virus pathogenesis. this infection may represent a situation where the host's abihty to control virus rephcation is tenuous (cd8"^ t-cell control of virus replication in the central nervous system may not be tbat efficient), and the rephcative advantage conferred on the virus by escape mutations may tip the balance firmly in favor of the virus. ebv is a human gamma herpesvirus that is carried by the majority of individuals as a hfelong asymptomatic infection, but has oncogenic potential and is implicated in the pathogenesis of a range of malignancies. the increased risk of development of ebv-positive mahgnancies associated with immune suppression illustrates the importance of immune control in maintaining a non-pathogenic equilibrium between this potentially oncogenic virns and its host; the potent antiviral ctl response is thought to play an important role in controlling virus replication and spread and the development of disease (20, 88, 89) . the abihty of tbis virus to evade clearance from the infected host has been attributed to a number of different mechanisms (reviewed in (20)), but ctl escape variant selection is not thought to play a major role. unlike tbe other viruses discussed in this section, fbv is a genetically stable dna virus; mutations whicb may confer escape from the prevailing host ctl response are tbus hkely to arise too infrequently for escape variant generation and selection to constitute an efficient means ofimmune evasion within a given host. mutations have been documented in the virus population prevaihng in certain areas wbich confer escape from recognition by ctl directed against epitopes whicb are presented by hla alleles possessed by a high proportion of the local populace. tbe best known example involves hla-a11 -restricted ctl epitopes in the ebna3b protein which are conserved in tbe majority of type 1 ebv isolates from caucasian and african populations, where the hla-a 11 allele frequency is low, but are mutated in virtually all isolates from highly all-positive immunologicoj reviews 164/1998 populations in china and coastal new guinea (19, 90) . the majority of these mutations are located within anchor residues and abrogate epitope binding to the hla-al 1 molecule; as discussed earher, such mutations would provide escape from epilope-specific ctl regardless of their tcr usage, suggesting that they may have conferred a selective advantage on the virus in the a11 -positive populations from which they were isolated. however, fbv isolates from south-east asia differ from caucasian and african isolates at a number of loci, and other polymorphisms have been shown to affect the antigenicity of epitopes that are not present at high frequency in south-east asian populations (91) , raising the possibility that the changes observed in the al 1-restricted epitopes may be coincidental. support for the hypothesis of specific epitope loss was not obtained when ebv isolates from a highly hla-b35-positive african population were sequenced across a b35-restricted epitope-containing region of the ebna3a protein (92), however, the hla-b35 frequency in this population was only about half that of the hla-a11 frequency in the south-east asian populations where a11 epitope mutations were detected; further, the hla-b3 5-restricted ctl response to ebv is not usually as strong as that restricted by hla-a 11 (92) . whether the ebvspecific ctl response does in fact select for viral variants with a growth advantage within human populations bearing certain hla alleles thns remains an unresolved issue. hbv is a hepadnavirus which, although it has a dna genome, replicates via reverse transcription from a pregenomic rna and thus has a high mutation rate. this virus produces acute and chronic infections in man during which the cd8"^ ctl response plays a key role both in virus clearance and in the pathogenesis of the associated liver disease (93) . during acute hbv infection, most patients develop a strong polyclonal ctl response against multiple epitopes in different viral proteins (93) . as discussed above, the likelihood of selection of escape mutant viruses under these conditions is prohably low, because a mutation which provides escape from ctl of just a single specificity may not confer a significant selective advantage on the virus bearing it in the presence of strong ctl responses to many other epitopes. in contrast, the ctl response is usually much weaker during chronic hbv infection (93) . in some patients it may also be more ohgospecific, providing greater opportunity for the selection of epitope-specific escape mutations. indeed, there is one report (94) of two patients who showed strong hla-a2-restricted ctl responses narrowly focused on an epitope in the hbv core protein at amino acids 18-27 and failed to respond to any of the other hla-a2-restricted ctl epitopes in hbv that are frequently recognized in acutely infected patients. when the persisting virus in these patients was sequenced, the quasispecies was found to be dominated by variant virus carrying mutations within the hbv core 18-2 7 epitope that affected epitope recognition by the patients' ctl and could antagonize the response of certain ctl clones to the index epitope (30). although conversion from a wild-type to the mutant sequence was not demonstrated in these patients, it is very likely that this does represent an example where escape variants were selected by the strong epitopespecific ctl response. ctl responses restricted by other hla alleles were not analyzed in these patients, and no information is available about how the ctl repertoire may have evolved in response to the emergence of the mutant viruses. it is thus difficult to assess how the escape variant may have impacted on the overall efficiency of ctl-mediated control of virus rephcation in these individuals. other reports suggest that the emergence of virus variants bearing ctl escape mutations may not be a common event during chronic hepatitis b infection: no escape mutations were identified in a stndy where the ctl response against eight different hla-a2-restricted epitopes defined in patients with acute hbv infection and the sequence of these regions in the in vivo viral quasispecies were analyzed in parallel in 12 patients chronically infected with hbv (95) . virus-specific ctl responses were undetectable in eight of these patients and weak in the other four; ctl-mediated pressure may thus have been too low to select for escape variants. whether selection of escape mutations plays a critical role in hbv persistence is thus questionable (96) ; it is likely that the development of a weak antiviral immune response is ol much greater importance in determining the course of this infection (93) . hcv is a positive-stranded rna virus, assigned to a new genus in the flaviviridae, which is well adapted for persistence in its human host: at least 60% of infected individuals develop a chronic infection that is frequently associated with clinical hepatitis (97) , as in hbv infection, the virus-specific cds-' ctl response is thought to play an important role in limiting virus replication, and simultaneously to contribute to disease pathogenesis. however, unlike the situation in hbv infection, where cell-mediated immune responses to the virus are generally low/undetectable in chronically infected individuals, hcv persistence in the presence of strong virus-specific ctl responses has been observed in both humans and experimentally infected chimpanzees (reviewed in (98) ). the highly mutable nature of this virus's rna genome raises the possibility that antigenic variation may he one of the mechanisms via which it aciiieves persistence in the face of the antivirai immune response. indeed, there is one report that in a chimpanzee which became persistentiy infected with hcv tiie virai quasispecies underwent compiete repiacement with virai variants hearing a conservative amino acid suhstitution at residue 4 of an epitope in non-structural protein 3 (ns3) recognized hy iiver-derived ctl iines from this animal; this mutation ahrogated recognition of the epitope by the animai's ctl (99) . the ctl response in this animai recognized epitopes in mtiltipie virai proteins (ioo); whetiier mutations were seiected for in any of the other epitopes, how epitope specificity/immunodominance may iiave ciianged over the course of infection in response to virai variation, and what impact escape variant seiection may have had on the overali efficiency of controi of virus repiication were not determined. however, tiiis study iliustrates that even though the ctl response to hcv is frequentiy poiycionai, escape virai variants can be seiected during hcv infection. as discussed beiow for hiv, where more information ahout the co-evoiution of the virai quasispecies and tiie immune response is avaiiahie, escape mutations iikeiy do compromise immune control of hcv infection, and are probabiy one of the factors which contrihute to the persistence of this virus in its human host (98) . hiv ehcits strong ctl responses in most infected individuals. the eariy cd8+ ctl response is temporally coincident with the reduction in acute plasma viremia (63, 64); as discussed ahove, virus-specific ctl iikeiy expand to extremeiy iiigh frequencies during the primary immune response (53, 73) . the frequency of virus-specific ctl then deciines somewhat; however, high ieveis of activated ctl are commoniy detected in chronicaiiy infected asymptomatic individuais, even in the setting of an extremeiy iow virai ioad (77, i 01 -104). as disease progression occurs, virus-specific ctl frequencies may initialiy increase in response to increasing viral ioad, but generaily faii to imdetectabie ieveis in the end stages of the infection (104) . a number of hnes of both direct and indirect evidence (discussed in (53) ) indicate that the cd8+ ctl response does piay an important role in controiiing virus repiication in hiv-1 -infected individuais, and a variety of mechanisms have been proposed to contribute to virus persistence in the face of the host immune response (105) (106) (107) (108) (109) (110) (111) . we focus here just on the role that the selection of ctl escape virus variants may piay during this infection. the high virai turnover during hiv-1 infection, rapid mutation rate of this virus and strong virus-specific ctl responses provide conditions under which ctl escape mutants wouid be iikeiy to emerge. indeed, as indicated earher. there have been numerous reports of variant viruses within the quasispecies in different patients w-hich are able to escape recognition by epitope-specific ctl. escape virus variants may be selected at different stages of infection and correspondingly impact on the virus-host baiance in different ways. during primary hiv-1 infection, when the virus undergoes an intensive hurst of rephcation and ctl directed against dominant epitopes may reach extremeiy high frequencies, escape variant seiection may be particuiariy favored. we have observed repiacement of the virai quasispecies hy variants with mutations in an epitope targeted by tiie primary ctl response in the eariy stages of infection in two patients anaiyzed (53) , and there has aiso been another study documenting ctl escape variant emergence in a seroconverter (54) . interestingly, the two patients in whom we observed eariy seiection of ctl escape variant viruses botii underwent rapid disease progression: studies of larger numbers of patients are required to reveai how frequentiy eariy seiection of mutations in epitopes recognized during the primary ctl response in fact occurs, and whether this phenomenon is aiways associated with rapid progression to aids. the emergence of virai variants with mutations that confer resistance to controi by dominant ctl responses in the eariy stages of infection may potentialiy impact on the suhsequent course of infection in two ways. first, as a virai variant wiii oniy be seiected for if it has a rephcative advantage, the emergence of ctl escape variants must he associated with an increase in eariy virus rephcation and spread. consequences of this may inciude increased ioss of cd4+ t cells and estabhshment of a lareo er pooi of iatentiy infected ceiis or a higiier setpoint ievei of persisting virus: the iatter has been demonstrated to be a good predictor of the subsequent rate of disease progression (112) . second, because (as discussed above) mutations in immunodominant ctl epitopes may promote evoiution of the immune response to recognize subdominant epitopes, the host may he left after tiie acute phase of the infection not oniy with a higher ievei of persisting virus, but aiso with a ctl response that is iess adequate to contain it. in mid-hiv infection, mutations which affect recognition by epitope-specific ctl have been demonstrated in the persisting virai quasispecies in a numher of studies (e.g. (15. 77, 113)); however, clear examples of such variants being selected for and fixed in the virai quasispecies over time are difficuit to find. this is hkeiy hecause ctl responses to muitipie virai epitopes are frequendy present and, as discussed earher, under such conditions a complex cycie of fluctuations in the domi-nance of ctl responses directed against different viral epitopes and corresponding shifts in the epitopic composition of the viral quasispecies may occur (78, 79) , there are also examples of strong ctl responses being exhibited over long periods of time in the absence of appearance of mutations in the epitopes to which they are directed {76, 77). ctl epitope mutation during chronic hiv infection tbus does not seem to be essential for the continued persistence of virus; however, when it does occnr it may allow higher levels of virus replication, and bence promote disease progression. in the later stages of the infection, when the immune system is failing, the impact of viral variation on immune control may become more pronounced. the capacity of the immune response to evolve to focus on tmmutated epitopes as escape variants are selected may be more limited at this time. simultaneously, the rehance of the host on ctl lysis as a means of controlling virus replication and spread may be increasing, particularly if syncytium-inducing viruses resistant to control by chemokines such as rantes, miela and mip1p appear. ctl escape variants may thus grow out and contribute to tbe escalating viremia. a recent paper described two examples of patients where mutations conferring evasion from a previously stable immunodominant ctl response became fixed in the viral quasispecies in the late stages of the infection (80), providing a clear illustration that tbis can occur ctl escape viral variants may thus influence the immune system's abihty to control virus replication at all stages of hiv infection, and likely do bave an important impact on the overall disease course in at least a proportion of infected individuals. the preceding discussion illustrates tbat viral variants whicb are able to escape recognition by host ctl emerge during a number of different virus infections, and clearly have a biologically significant impact on tbe balance between virus replication and its control by the immune response in at least some of these cases. the latter include several infections that are of clinical significance in humans; it is thus important that immunebased propbylactic and therapeutic strategies to combat these infections should be designed to minimize the likelihood of ctl escape variant selection. there are examples in the literature of both prophylactic (114) and therapeutic strategies (115) wbich have led to the selection of viral variants able to escape recognition by the ctl response wbich was intended to mediate a beneficial effect. as described in this review, studies in both human virus infections and murine model systems have illustrated the conditions under which ctl escape viral variants are likely to emerge: this information should be considered in future vaccine/therapy design to prevent a similar outcome from occurring. given that escape-conferring mutations will not be selected for if they have detrimental effects on virus replication, and that escape mutations emerge most rapidly wben ctl pressure is predominantly focused onto a single bighly immunodominant epitope, vaccines should aim to induce broad immune responses that recognize multiple co-dominant viral epitopes, at least some of which should lie in conserved regions of viral proteins on which there are stringent functional constraints. eeptide or minigene-based vaccination strategies are probably not the optimal choice for use in such infections: even if they include a carefully chosen cocktail of epitopes that can be recognized in association with multiple hla alleles, there is a bigh probability that they will induce mono-or oligospecific immune responses in individuals of certain hla types. particularly if the infecting virus shows sequence variation compared to the antigens used for vaccination, there will be a danger that the vaccine might prime for an immune response that could be more detrimental than the immune response that may have been ehcited naturally following infection. vaccines that inclnde multiple viral antigens are preferential immunogens for the induction of beneficial multispecific antiviral immune responses. viral pathogenesis. philadelphia: lippincott-raven publishers one-step ahead of the gameviral immunomodulatory molecules viral pathogenesis. philadelphia: lippincott-raven publishers; i 997 specific cytotoxic t cells eliminate cells 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antiretroviral therapy lymphocytic choriomeningitis virus reduction of hiv concentration duiing acute infection: independence from a specific immune response antiviral pressure exerted by hiv-1 -specific cytotoxic t lymphocytes (ctls) during primary infection demonstrated by rapid selection of ctl escape virus positive selection of hiv-1 cytotoxic t lymphocyte escape variants during primary infection massive expansion of an tig en-specific cds -i-t cells during an acute virus infection counting antigenspecific cds t cells; a reevaluation of bystander activation during viral infection direct visualisation of antigen-specific cd8+ t cells during the primary response to epstein-barr virus in vivo ctl escape virai variatits ii. biologic activity in vivo optimal lymphocytic choriomeningitis virus sequences restricted by h-2db major histocompatibihiy complex class i molecules and presented to cytotoxic t lymphocytes identification of db and kb-restricted subdominant cytotoxic tcell responses in lymphocytic choriomeningiiis virus-infected mice virus-specific cd8+ cytotoxic t-iymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type i syndrome contribution of proteasome-mediated proteolysis to the hierarchy of epitopes presented by major histocompatibility class i molecules peptide iranslocation by variants of the transporter associated with antigen processing tap-1-dependent peptide translocation in vitro is atp dependent and peptide selective determinant selection of major histocompatibility complex class i-restricted antigenic peptides is explained by class ipeptide affinity and is strongly influenced by non-dominant anchor residues the hfe span of major histocompatibility complex-peptide complexes influences the efficiency of presentation and immunogenicity of two class i-restricted cytotoxic t lymphocyte epitopes in the epstein barr virus nuclear antigen 4 different mhc class i alleles compete for presentation of overlapping viral epitopes siliciano re, soloski mj, mhc class irestricted processing of transmembrane proteins -mechanism and biological significance major expansion of cd8-i-t cells with a predominant vb usage during the primary immune response to hiv patterns of immunodominance in hiv-1 -specific cytotoxic t lymphocyte responses in two human histocompaiibilicy leukocyte antigens (hla)-identical siblings with hla-a*0201 are influenced by epitope mutation sequence constraints and recognition by ctl of an hla-b27-restricted hiv-1 gag epitope in vivo persistence of a hiv-1 -encoded hla-b2 7 -restricted cytotoxic t lymphocyte epitope despite specific in vitro reactivity cytotoxic t lymphocytes in asymptomatic long-term non progressing hiv-1 infection antigenic oscillations and shifting immunodominance in hiv-1 infections late escape from an immunodominant cytotoxic t-lymphocyte response associated with progression to aids in vitro selection of lymphocytic choriomeningitis virus escape mutants by cytotoxic t lymphocytes ctl escape viral variants, i. generation and molecular characterization discriminated selection among viral peptides with the appropriate anchor residues: implications for the size of the cytotoxic t-lymphocyte repertoire and control of viral infection late onset, symptomatic, demyelinating encephalomyelitis in mice infected with mhv-jhm in the presence of maternal antibody cd8+ t cell epitopes within the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenicity cytotoxic t cell-resistant variants are selected in a virus-induced demyelinating disease ctl escape mutants cause increased mortality and morbidity ln mice infected with mouse hepatitis virus, strain jhm. keystone symposium on molecular aspects of viral immunity cytotoxic t lymphocyte responses to epstein-bair virus human cytotoxic t lymphocyte responses to epstein-barr virus infection boftow & shaw • in vivo importance of ctl escape viruses t cell responses and virus evolution: lossof hla al 1-restricted ctl epitopes in epstein-barr virus isolates from highly al 1 -positive populations by selective mutation of anchor residues unusuaey high frequency of epstein-barr virus genetic variants iji papua new guinea that can escape cytotoxic t-cell recognition: implications for virus evolution epstein-barr virus isolates with the major hla b35.01-restricted cytotoxic t lymphocyte epitope are prevalent in a liighly b35.01-positive african population hepatitis b virus immunopathogenesis cytotoxic t lymphocyte response to a wild-type hepatitis b virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope chisari fv hepatitis b virus (hbv) sequence variation in cytotoxic t lymphocyte epitopes is not common in patients with chronic hbv infection chisari fv is antigenic variability a strategy adopted by hepatitits b virus to escape cytotoxic t-lymphocyte surveillance? hepatitis c viruses. in: fields bn, ed. fields virology cytotoxic t-lymphocyte responses to the hepatitis c virus in humans and chimpanzees persistent hepatitis c virus infection in a chimpanzee is associated with emergence of a cytotoxic t lymphocyte escape variant hepatitis c virus-specific ctl responses in the liver of chimpanzees with acute and chronic hepatitis c hiv specific cytotoxic t lymphocytes in seropositive individuals aids virus specific cytotoxic t lymphocytes in lung disorders high levels of anti-human immunodeficiency virus type 1 (hiv-1) memory cytotoxic t-lymphocyte activity and low viral load are associated with lack of disease in hiv-1 -infected long-term nonprogressors kinetics of gag-specific cytotoxic t lymphocyte responses during the clinical course of hiv-1 infection: a longitudinal analysis of rapid progressors and long-term asymptomatics why can't cytotoxic t cells handle hiv? hiv-!-specific cytotoxic t lymphocytes and the control of hiv-1 reph cation evidence for rapid disappearance of initially expanded hivspecific cd8-(-t cell clones during primary hiv infection vigorous hiv-1-specific cd4-i-t cell responses associated with control of viremia hiv versus cytotoxic t lymphocytes -the war being lost what can we learn about human immunodeficiency virus infection from a study of lymphocytic choriomeningitis virus? immunoirev hiv-1 nef protein protects infected primary cells against killing by cytotoxic t lymphocytes prognosis in hiv-1 infection predicted by the quantity of virus iji plasma human immunodeficiency virus genetic variation that can escape cytotoxic t cell recognition transfer of hiv-1 -specific cytotoxic t lymphocytes to an aids patient leads to selection for mutant hiv variants and subsequent disease progression bourgault-villada l selection of virus variants and emergence of virus escape mutants after immunization with an epitope vaccine the authors are supported by grant numhers ro i ai37430-03 (pb)and uo1 ai41530 (pb and gms) from the nih and by core funding from the edward jenner institute for vaccine research key: cord-007301-5m269nzi authors: lundegaard, claus; lund, ole; keşmir, can; brunak, søren; nielsen, morten title: modeling the adaptive immune system: predictions and simulations date: 2007-12-15 journal: bioinformatics doi: 10.1093/bioinformatics/btm471 sha: doc_id: 7301 cord_uid: 5m269nzi motivation: immunological bioinformatics methods are applicable to a broad range of scientific areas. the specifics of how and where they might be implemented have recently been reviewed in the literature. however, the background and concerns for selecting between the different available methods have so far not been adequately covered. summary: before using predictions systems, it is necessary to not only understand how the methods are constructed but also their strength and limitations. the prediction systems in humoral epitope discovery are still in their infancy, but have reached a reasonable level of predictive strength. in cellular immunology, mhc class i binding predictions are now very strong and cover most of the known hla specificities. these systems work well for epitope discovery, and predictions of the mhc class i pathway have been further improved by integration with state-of-the-art prediction tools for proteasomal cleavage and tap binding. by comparison, class ii mhc binding predictions have not developed to a comparable accuracy level, but new tools have emerged that deliver significantly improved predictions not only in terms of accuracy, but also in mhc specificity coverage. simulation systems and mathematical modeling are also now beginning to reach a level where these methods will be able to answer more complex immunological questions. contact: lunde@cbs.dtu.dk supplementary information: supplementary data are available at bioinformatics online. the adaptive immune system of vertebrates is thought to be only 400 million years old and exists in most fish, amphibians, reptiles, birds and mammals (thompson, 1995) . adaptive immunity is induced by lymphocytes and can be classified into two types: humoral immunity, mediated by antibodies, which are secreted by b lymphocytes and can neutralize pathogens outside the cells; and cellular immunity, mediated by t lymphocytes that eliminate infected or malfunctioning cells, and provide help to other immune responses. diversity is the hallmark of the adaptive immune systems. both the b and t lymphocyte-specific receptors for antigen recognition are assembled from variable (v), diversity (d), and joining (j) gene segments early in the lymphocyte development. there are multiple copies of v, d and j segments, and a huge repertoire of t and b cells is generated by the recombination of these segments, reviewed by li et al. (2004) . another task faced by the immune system is the tolerance to self, which is handled by continuously removing receptors that react to self-epitopes. special immunoglobulin molecules (antibodies) mediate the humoral response. as mentioned above, the antibodies are produced by b lymphocytes that bind to antigens by their immunoglobulin receptors, which is a membrane bound form of the antibodies. when the b lymphocytes become activated, they start to secrete the soluble form of this receptor in large amounts. the antibody is y-shaped, and each of the two branches functions independently and can be recombinantly produced and is then known as fabs. the highly variable tip of the fab, which can bind to epitopes is called the paratope and is made up of the so-called complementary determining regions (cdrs) . antibodies can coat the surface of an antigen such as a virus, so that it cannot function or infect cells, reviewed by burton (2002) . antibody-covered viruses or bacteria are easily phagocytosed and destroyed by scavenger cells of the immune system, e.g. the macrophages. antigenic proteins can be recognized by the antibodies in their native form without any cleavage or interactions with other molecules. thus the humoral immune response reacts to extracellular pathogens, and the response is crucial in the defense against most pathogens. b-cell epitopes are normally classified into two groups: continuous and discontinuous epitopes. a continuous epitope, (also called a sequential or linear epitope) is a short peptide fragment in a protein that is recognized by antibodies specific for that protein. a discontinuous epitope is composed of residues that are not adjacent in the primary structure (amino acid sequence), but are brought into proximity by the folding of the polypeptide. the classification is not clear-cut as discontinuous epitopes may contain linear stretches of amino acids, and continuous epitopes may show conformational preferences. the cellular arm of the immune system consists of two parts; cytotoxic t lymphocytes (ctl), and helper t lymphocytes (htls). ctls destroy cells that present non-self peptides (epitopes). htls are needed for b cells activation and proliferation to produce antibodies against a given antigen. ctls on the other hand perform surveillance of the host cells, and recognize and kill infected cells, generally explained in janeway et al. (2001) . both ctl and htl are raised against peptides that are presented to the immune cells by major histocompatibility complex (mhc) molecules, which are the most polymorphic of mammalian proteins. the human versions of mhcs are referred to as the human leucocyte antigen (hla). the cells of an individual are constantly screened for such peptides by the cellular arm of the immune system. in the mhc class i pathway, class i mhcs presents endogenous antigens to t cells carrying the cd8 receptor (cd8þ t cells). to be presented, a precursor peptide is normally first generated by the large cytosomal protease complex called the proteasome (loureiroa and ploegha, 2006) . generally, it then binds to the transporter associated with antigen processing (tap) for translocation into the endoplasmic reticulum (er), reviewed by abele and tampe´(2004) , but some peptides can enter the er independently of tap. this should be considered when dealing with virus-infected cells or tumors cells that might have reduced or absent tap function. there are several ways that the peptide can enter the er without tap function depending on the origin and properties of the peptide. the most well-established model, however, is for proteins containing a signal peptide. such proteins are translated directly into the er through the sec61 transporter complex and sometimes the cleaved-off signal peptide will end up in er. this model is especially relevant for peptides binding to hlas belonging to the abundant a2 hla serotype where tap-independent presentation is responsible for up to 10% of the a2 restricted epitopes, reviewed in . during or after the transport into the er the peptide must bind to the mhc class i molecule (stoltze et al., 2000; zhang and williams, 2006) before it can be transported to the cell surface through the golgi system. the most selective step in this pathway is binding of a peptide to the mhc class i molecule. in an older review, yewdell and bennink (1999) states that only 1 in 200 binds with an affinity strong enough to generate an immune response. this has been challenged, and it might be that up to 3% of the possible peptides bind strong enough to generate a subsequent immune response (assarsson et al., 2007) . in another recent work of moutaftsi et al. (2006) , however, it is found that of the 49 epitopes that are responsible for 95% of the total cd8þ t-cell response against a vaccinia challenge in mouse 90% binds mhc with an affinity stronger than 500 nm. in any case a peptide must go through the processes in a greater number than competing peptides to be immunodominant. the mhc is the most polymorphic gene system known. this polymorphism is a huge challenge for t-cell epitope discoveries, enhancing the need for bioinformatical analysis and resources. however, it also highly complicates immunological bioinformatics, as predictive methods for peptide mhc binding have to deal with the diverse genetic background of different populations and individuals. on a population basis, hundreds of alleles have been found for most of the hla encoding loci (1839 in release 2.17.0 of the imgt/hla database, http://www.ebi.ac.uk/imgt/hla/). in a given individual either one or two different alleles are expressed per locus depending on whether the same (in homozygous individuals) or two different (in heterozygous individuals) alleles are coded for on the two different chromosomes. the number of mhc expressing loci, however, differs highly among species. while a fully heterozygous human has six different mhc class i genes, a rhesus macaque may host up to 22 active mhc class i genes (daza-vamenta et al., 2004) . each mhc allele binds a very restricted set of peptides and the polymorphism affects the peptide binding specificity of the mhc; one mhc will recognize one part of the peptide space, whereas another mhc will recognize a different part of this space. the very large number of different mhc alleles makes reliable identification of potential epitope candidates an immense task if all alleles are to be included in the search. however, many mhc alleles share a large fraction of their peptide-binding repertoire, and it is often possible to find promiscuous peptides, which bind to a number of hla alleles. a way of reducing the problem is to group all the different alleles into supertypes in a manner so that all the alleles within a given supertype have roughly the same peptide specificity (hertz and yanover, 2007; reche and reinherz, 2004; sette and sidney, 1998, 1999) . this allows the search to be limited to a manageable representative set. representing a supertype by a well-studied allele might lead to selection of epitopes that is very restricted to this allele, but not to any other alleles within the supertype. thus another, and potentially more rational approach, would be to select a limited set of peptides restricted to as many alleles as possible. this should be within reach with new methods that directly predict epitopes that can bind to different alleles (promiscuous epitopes) (brusic et al., 2002) , or pan-specific approaches that can make predictions for all alleles where the sequence is known (jojic et al., 2006; nielsen et al., 2007a) . when the peptide-mhc complex is presented on the surface of the cell, it might bind to a cd8þ t cell with a fitting t-cell receptor (tcr). if such a tcr clone exists depends on, among other factors, if the tcr-peptide complex is too similar to mhc-peptide complexes generated with peptides from the host proteome (selfpeptides). this effect is called tolerance and might be broken by so-called self-epitopes, reviewed by andersen et al. (2006) . b cells must be activated to produce antibodies against a given antigen, and helper t cells specific for peptides from the antigen must be activated to get a strong b-cell response. the epitope recognized by the helper t cell is usually somehow connected to the epitope that is recognized by the b cell, but the two cells do not necessarily recognize overlapping epitopes. t cells can recognize internal peptides that do not need to be a part of the surface-surface interactions with the b-cell receptor. actually, the t-cell and the b-cell epitopes might not even come from the same protein (janeway et al., 2001) . the peptides recognized by the cd4þ t cells are presented by the mhc class ii molecule, and peptide presentation on mhc class ii molecules follow a different path than the mhc class i presentation pathway (castellino et al., 1997) : mhc class ii molecules associate with the invariant chain (ii) in the er and the mhc-ii complex accumulates in endosomal compartments. here, ii is degraded, while another mhc-like molecule, called hla-dm in humans, loads the mhc class ii molecules with the best available ligands originating from endocytosed antigens. the peptide-mhc class ii complexes are subsequently transported to the cell surface for presentation to t helper cells. immunological predictions and simulations have been demonstrated highly useful in applied immunology in general, and in vaccinology in particular. it can be used as an efficient tool to lower the experimental workload in epitope discovery for use in rational vaccine design, immunotherapeutics and development of diagnogstic tools. a number of recent publications describe in great detail the values and benefits obtained by the use of immunoinformatics and predictions in applied immunology and vaccinology (davies and flower, 2007; de groot, 2006; de groot and moise, 2007; korber et al., 2006; lund et al., 2005; petrovsky and brusic, 2006; tong et al., 2007) . here, we will not engage in this discussion, but rather limit ourselves to describing the available methods for making such predictions, and deliver some of the background information needed to be able to choose the appropriate method for a given task. a large variety of machine-learning techniques are commonly used in the field of immunological bioinformatics ranging from the conventional techniques of position-specific scoring matrices (pssms) (altschul et al., 1997) , gibbs sampling (lawrence et al., 1993; nielsen et al., 2004) , artificial neural networks (anns) described in baldi and brunak (2001) , hidden markov models (hmms) explained in hughey and krogh (1996) , and support vector machines (svms) described in cortes and vapnik (1995) , to more exotic methods like ant colonies (karpenko et al., 2005) and other motif search algorithms (bui et al., 2005; chang et al., 2006; murugan and dai, 2005) . anns and svms and are ideally suited to recognize non-linear patterns, which are believed to contribute to, for instance, peptide-hla-i interactions (adams and koziol, 1995; brusic et al., 1994; buus et al., 2003; gulukota et al., 1997; nielsen et al., 2003) . in an ann, information is trained and distributed into a computer network with an input layer, hidden layers and an output layer all connected in a given structure through weighted connections (baldi and brunak, 2001) . in a pssm on the other hand, all positions in the motif are assumed to contribute in an independent manner, and the likelihood for matching a motif is calculated as a sum of individual matrix scores. the gibbs sampler method is a particular implementation of the pssm search algorithm, where the optimal pssm is determined by a search for a sequence alignment that provides maximal information content for a given motif length. conventionally pssms are log-odds matrices (altschul et al., 1997) , where the weight matrix elements are estimated from the logarithm of the ratio of the observed frequency of a given amino acid to the background frequency of that amino acid. however, many other techniques including the stabilization matrix method (smm) (peters and sette, 2005) , and evolutionary algorithm (brusic et al., 1998) exist to construct a pssm. the pssms might also be coupled with other information available to compensate for lack of data (lundegaard et al., 2004) . finally, hmms have been used in the field of immunological bioinformatics. these are well suited to characterized biological motifs with an inherent structural composition, and have been used in the field of immunology to predict for instance peptide binding to mhc class i (mamitsuka, 1998) and class ii (noguchi et al., 2002) molecules. beside machine-learning techniques, also (empirical) molecular force field modeling techniques (logean et al., 2001) and 3d quantitative structure-activity relationship (3d-qsar) (doytchinova and flower, 2002; zhihua et al., 2004) analysis have been used to predict features of the immune system. as an evaluation of the general quality of a prediction method a measure describing this quality is needed. however, no single measure can capture all qualities of a prediction, and not all types of data and predictions can be reasonably described by the same measure. so to be able to compare different systems, it is often needed to present several measures of quality. most measures need the data to be classified into two groups, i.e. positives and negatives. the number of classified (experimentally measured) positives is often designated as actual positives (ap), and the number of negatives, actual negatives (an), the number of predicted positives (pp), predicted negatives (pn), truly predicted positives (tp), falsely predicted positives (fp), truly predicted negatives (tn), and falsely predicted negatives (fn). some of the most often used measures are briefly described here. the equations for the mentioned measures are given at the end of the section. the fraction correct predicted (fcp) is the fraction of the total predictions that falls into the correct group. this measure is intuitively easily captured, but has the weakness that if a large fraction of the total evaluation data falls into a single group one will get high performance by just blindly predicting most or even everything to belong to this category. the positive predicted value (ppv) is the fraction of the positive predictions that actually falls into the positive class. the sensitivity is the fraction of the ap that is predicted as positives using a given threshold. the specificity is the fraction of the an that is predicted as negatives. the three latter measures are also easily grasped, however they are all dependent on the chosen prediction cutoff classifying the data into positive and negative predictions. a high sensitivity can be obtained by setting your prediction cutoff so that most of your evaluation data will fall into the positive group, but this will then be at the expense of the specificity and the ppv. which cutoff to use is determined by the purpose of the prediction, i.e. how many verified epitopes is needed versus the resources available for experimental validation. a plot of the sensitivity against the false positive rate (1-specificity) is called a receiver operating characteristic (roc) curve (swets, 1988) . such a plot can be a help to set the best prediction cutoff. one of the best ways of measuring the predictive power of a method is to calculate the area under the roc curve (auc) since this is a threshold-independent measure. another robust measure is the pearson correlation coefficient (pcc), which is a measure of how well the prediction scores correlate with the actual value on a linear scale. in situations where the correlation is not necessarily linear, the spearman's rank correlation coefficient (src) is more appropriate. in this measure each prediction is ranked on the basis of the prediction score and the pcc is calculated on the basis of this rank rather than the prediction score. the src, like the auc, is a threshold-independent measure of how well the predictor ranks the data when compared with the actual ranking. when comparing different methods, the thresholdindependent measures are to be preferred. otherwise a threshold has to be set under the same assumptions for all predictors. as an example one can estimate the specificity for each predictor by setting the threshold for the given predictor to a value where the sensitivity will be 0.5 (i.e. half of the total available positives is over the threshold), or estimate the sensitivity at a threshold where the specificity will be 0.8 (i.e. 80% of the an are predicted as negatives). the choice of an evaluation set is also absolutely crucial and several considerations must be taken. a large and diverse dataset is to be preferred to avoid any biases in prediction space. extreme care should also be taken to ensure that none of the predictors have been trained on the data used for evaluation even though that might not always be possible. to make the evaluation as broad as possible cross-validation is often used, i.e. the method is trained on a large part of the available data and a smaller part is left out for evaluation. this is done until all data has been included in the evaluation set and in this way it is possible to estimate the performance on the complete dataset. caution has to be taken, however, that the part used for training is not too similar to the evaluation part, as this will lead to an overestimation of the performance due to overtraining. this is especially true when using the leave-one-out version of cross-validation where everything except one data point is used for training, and the evaluation is then performed on the ensemble of the left out data points. equations are as follows: the state-of-the-art class i t-cell epitope prediction methods are today of a quality that makes it highly useful as an initial filtering technique in epitope discovery. studies have demonstrated how it is possible to rapidly identify and verify mhc binders from upcoming possible threats such as the sars virus (sylvester-hvid et al., 2004) with high reliability, and take such predictions a step further and validate the immunogenecity of peptides with limited efforts, as has been shown with the influenza a virus (wang et al., 2007) . it is also possible to identify the vast majority of the relevant epitopes in a rather complex organism as the vaccinia virus using class i mhc binding predictions and only have to test a very minor fraction of the possible peptides in the virus proteome (moutaftsi et al., 2006) . mhc class ii predictions can be made fairly reliable for certain alleles, and a number of helper epitopes have been identified by the help of bioinformatical approaches (consogno et al., 2003) . b-cell epitopes are still the most complicated task. however, some consistency between predicted and verified epitopes is starting to emerge using the newest prediction methods (dahlback et al., 2006) . in the following, we describe some of the best-performing prediction methods within each area. b-cell epitope prediction is a highly challenging field due to the fact that the vast majority of antibodies raised against a specific protein interact with discontinuous fragments (van regenmortel, 1996) . the prediction of continuous, or linear, epitopes, however, is a somewhat simpler problem, and may be still useful for synthetic vaccines or as diagnostic tools (regenmortel and muller, 1999) . moreover, the determination of continuous epitopes can be integrated into determination of discontinuous epitopes, as these often contain linear stretches (hopp, 1994) . in the early 1980s, hopp and woods (hopp and woods, 1981, 1983 ) developed the first linear epitope prediction method. this method takes the assumption that the regions of proteins that have a high degree of exposure to solvent contain the antigenic determinants. according to the hydrophilicity scale generated by levitt (1976) , hopp and woods (1981) assigned the hydrophilicity propensity to each amino acid in a sequence and looked at groups of six residues. this gave promising results and a number of methods have since been developed with the aim of predicting linear epitopes using a combination of different amino acid propensities (alix, 1999; debelle et al., 1992; jameson and wolf, 1988; maksyutov and zagrebelnaya, 1993; odorico and pellequer, 2003; parker et al., 1986) . , pellequer et al. (1993 proposed an evaluation set containing 85 continuous epitopes in 14 proteins and found that the method based on turn propensity (i.e. the propensity of an amino acid to occur within a turn structure) had the highest sensitivity using this set. seventy percent of the residues predicted to be in epitopes by this method were actually part of epitopes. the sensitivity for methods based on other propensities was in the range of 36-61% (pellequer et al., 1991) . analyzing the epitope regions in the pellequer dataset reveals that almost all the hydrophobic amino acids are underrepresented, supporting the assumption that linear b-cell epitopes will occur in hydrophilic regions of the proteins. an extensive study of linear b-cell epitope prediction methods was published by blythe and flower (2005) . to test how well peaks in single amino acid scale propensity profiles are (significantly) associated with known linear epitope locations, 484 amino acid propensities from the aaindex database (http://www.genome.ad.jp) (kawashima and kanehisa, 2000) were used. as test set they used 50 epitope-mapped proteins defined by polyclonal antibodies, which were the best non-redundant test set available. blythe and flower (2005) found, however, that even the predictions based on the most accurate amino acid scales were only marginally better than random, suggesting that more sophisticated approaches is needed to predict the linear epitopes. bepipred , an algorithm that combines scores from the parker hydrophilicity scale (parker et al., 1986 ) and a pssm trained on linear epitopes, shows a small, but significant, increase in auc over earlier scale-based methods. the sequence parametrizer algorithm (sollner, 2006; sollner and mayer, 2006) , along with its associated machine-learning methods uses the common single amino acid propensity scales, but also incorporates neighborhood parameters reflecting the probability that a given stretch of amino acids exists within a predefined proximity of a specific amino acid residue. training and testing on epitope sequences pulled from a high-quality proprietary database, as well as several publicly accessible databases, yields a degree of accuracy that is greatly increased over single-parameter methods. different experimental techniques can be used to define conformational epitopes. probably the most accurate, and easily defined is using the solved structures of antibody-antigen complexes (fleury et al., 2000; mirza et al., 2000) . the amount of this kind of data is unfortunately still scarce, compared to linear epitopes. furthermore, very few antigens have been studied in a way where all possible epitopes on a given antigen has been identified. unidentified epitopes within the dataset will lower the apparent performance of an accurate prediction method by increasing the apparent false positive rate. the simplest way to predict the possible epitopes in a protein of known 3d structure is to use the knowledge of surface accessibility (novotny et al., 1986; thornton et al., 1986) . two newer methods using protein structure and surface exposure for prediction of b-cell epitopes have been developed. the cep method (kulkarni-kale et al., 2005) calculates the relative accessible surface area for each residue in the structure. then it is determined which parts of the protein that are exposed enough to be antigenic determinants. regions that are distant in the primary sequence, but close in three-dimensional space are considered as one epitope. the tool was tested on a dataset of 63 antigen-antibody complexes and the algorithm correctly identified 76% of the epitope residues. discotope (haste et al., 2006) uses a combination of amino acid statistics, spatial information and surface exposure. it is trained on a compiled dataset of discontinuous epitopes from 76 x-ray structures of antibody-antigen protein complexes. this method outperforms methods that predict linear epitopes. recently a workshop was held on the subject of b-cell epitope predictions attended by a broad range of the current method developers. the workshop resulted in a published review containing conclusions on the present common ground, and suggestions for the future especially concerning coordination and evaluation (greenbaum et al., 2007) . different ways of measuring the accuracy of b-cell epitope predictions have been suggested (hopp, 1994; van regenmortel and pellequer, 1994) . pellequer suggested using the specificity as a measure of accuracy, while hopp suggested using the ppv, but, as described earlier, neither measure will alone give a good description of the performance. in accordance to this the recent workshop concluded that the auc measure is to be preferred (greenbaum et al., 2007) . another issue is whether to make the statistics on a per-residue or on a per-epitope basis. however, as the latter have the additional complications of defining how much of an epitope that must be included in a prediction to be considered correct, and how much extra included residues is allowed, the per residue measure is to be preferred. epitope mapping can be performed experimentally by other methods than structure determination, e.g. by phage display (jesaitis et al., 1999; smith and petrenko, 1997) . the low sequence similarity between the mimotope [i.e. a macromolecule, often a peptide, which mimics the structure of an epitope, (meloen et al., 2000) ] identified through phage display and the antigen complicates the mapping back onto the native structure of the antigen. a number of methods have been developed to facilitate this (batori et al., 2006; enshell-seijffers et al., 2003; halperin et al., 2003; huang et al., 2006; moreau et al., 2006; mumey et al., 2003; schreiber et al., 2005; tarnovitski et al., 2006) . however, these are to be considered as interpreters of experimental data rather than predictors, which are the main focus of this review. a number of methods for predicting the binding of peptides to mhc molecules have been developed (schirle et al., 2001) since the first motif methods were presented (rothbard and taylor, 1988; sette et al., 1989) . the majority of peptides binding to mhc class i molecules have a length of 8-10 amino acids. position 2 and the c-terminal position have turned out generally to be very important for the binding to most class i mhcs and these positions are referred to as anchor positions (rammensee et al., 1999) . for some alleles, the binding motifs further have auxiliary anchor positions. peptides binding to the human hla-a*0101 allele thus have positions 2, 3 and 9 as anchors (kondo et al., 1997; kubo et al., 1994; rammensee et al., 1999) . the importance of anchor positions for peptide binding and the allele-specific amino acid preference at the anchor positions was first described by falk et al., 1990 . the discovery of such allele-specific motifs led to the development of the first reasonable accurate algorithms (pamer et al., 1991; rotzschke et al., 1991) . in these prediction tools, it is assumed that the amino acids at each position along the peptide sequence contribute a given binding energy, which can independently be added up to yield the overall binding energy of the peptide (meister et al., 1995; parker et al., 1994; stryhn et al., 1996) . similar types of approaches are used by the epimatrix method (schafer et al., 1998) , the bimas method (parker et al., 1994) , the syfpeithi method (rammensee et al., 1999) , the rankpep method (reche et al., 2002) and the gibbs sampler method (nielsen et al., 2004) . several of these matrix methods use an approach in the development where the method is build using exclusively positive examples defined after certain criteria, like eluted peptides and interferon gamma response data. this data can be used in training as well as affinity binding data defining binding stronger than a certain threshold (usually 500 nm). other matrix methods, like the smm method, aim at predicting an actual affinity and thus use exclusively affinity data. as described earlier, matrix-based methods cannot take correlated effects into account, i.e. where the contribution to the binding affinity by a given amino acid at one position is influenced by amino acids at other positions in the peptide. higher order methods like anns and svms, on the other hand, are ideally suited to take such correlations into account. these methods can be trained with data either in the format of binder/non-binder classification, or as real affinity data. some of the recent methods combine the two types of data and prediction methods, either by averaging over predictions made by either (bhasin and raghava, 2007) , or by feeding the predictions from the positive data-trained pssms to anns together with sequence/affinity data (nielsen et al., 2003) . a study by yu et al. (2002) clearly shows the influence of having a large dataset on the performance of the resulting method. however, including knowledge of important positions reduce the need for data significantly (lundegaard et al., 2004) . several prediction methods have been made publicly available, and when selecting between these several cautions should be taken. the published performance, and how it is evaluated should be examined, but it is also very important that the method is able to generate predictions for the actual allele of interest. a major study comparing the predictive performance of a large part of the available methods was recently performed by peters et al. (2006) showing that in general the smm and the ann methods (table 1 ) perform the best, even when taken into account the number of training data for each method. the cross-validated performance of these methods for several human and mouse mhc class i alleles was compared with the best performing other method available as web tool. the full results of this work are listed in supplementary table 1 . the tools and urls are listed in table 1 . it should be mentioned, however, that tools known to be trained on a significant part of the test set were excluded from this comparison. to achieve binding predictions for an allele with uncharacterized specificity, the supertype concept (sette and sidney, 1998) can be used for the limited number of alleles with well-defined supertype relationships (lund et al., 2005) . note, however, that predictions with methods predicting the specific allele is most often to be preferred, as the accuracy of these will be better (nielsen et al., 2007a) . in general, hla-i binding predictions depend on sufficient experimental data being available for the exact hla-i molecule in question. unfortunately,510% of the 1500 registered hla-i proteins (lefranc, 2005) have been examined experimentally, and 55% have been characterized with more than 50 examples of peptide binders (rammensee et al., 1999; sette et al., 2005) . several groups have suggested prediction strategies to span these 'uncharacterized' regions of the hla diversity (brusic et al., 2002; jojic et al., 2006; nielsen et al., 2007; zhu et al., 2006) . in different forms, all these methods exploit both peptide and primary hla sequence as input information for training, aiming at simultaneously incorporating all hla specificities. in a recent paper (nielsen et al., 2007a) , it is successfully demonstrated that such an approach can, to a very high degree, accurately characterize the binding motif for previously untested hla-i molecules. unlike the mhc class i molecules, the binding cleft of mhc class ii molecules is open-ended, which allows for the bound peptide to have significant overhangs in both ends. as a result mhc class ii binding peptides have a broader length distribution even though the part of the binding peptide that interacts with the mhc (the binding core) still includes only 9 amino acid residues. this complicate binding predictions as identification of the correct alignment of the binding core is a crucial part of identifying the mhc class ii binding motif (nielsen et al., 2004) . the mhc class ii binding motifs have relatively weak and often degenerate sequence signals. while some alleles like hla-drb1*0405 show a strong preference for certain amino acids at the anchor positions, other alleles like hla-drb1*0401 allow basically all amino acids at all positions (rammensee et al., 1999) . however, there are other issues affecting the predictive performance of most mhc class ii binding prediction methods. the majority of these methods take as a fundamental assumption that the peptide-mhc binding affinity is determined solely from the nine amino acids in binding core motif. this is clearly a large oversimplification since it is known that peptide flanking residues (pfr) on both sides of the binding core may contribute to the binding affinity and stability (godkin et al., 2001) . some methods for mhc class ii binding have attempted to include pfrs indirectly, in terms of the peptide length, in the prediction of binding affinities (chang et al., 2006) . recently, nielsen et al. (2007b) published a method for mhc class ii prediction that directly include pfrs and demonstrated that these pfrs improves the prediction accuracy. most of the methods for mhc class ii binding predictions have been trained and evaluated on very limited datasets covering only a single or a few different mhc class ii alleles, making it very difficult to compare the different performance values and generality of the methods. nielsen et al. (2007b) have made available a large-scale benchmark set-up for evaluating mhc class ii peptide binding affinity prediction algorithms. the benchmark covers 14 hla-dr (human mhc) and three mouse h2-ia alleles, and consists of peptide/ic50 affinity data downloaded from the publicly available iedb database , and could set the start for large-scale unbiased evaluations of novel methods for mhc class ii prediction. successful prediction of the proteasome cleavage site specificity should provide valuable additional information useful in the design of treatments based on ctl responses. however, the complexity of proteasomal enzymatic specificity complicates such predictions. the proteasome have a highly stochastic element, exemplified by the observation that only $80% of the cleavage sites observed in one in vitro experiment can be verified in a second identical experiment (hansjo¨rg schild, personal communication). it is thus expected that the accuracy for prediction of proteasomal activity will be relatively low when compared to that of methods for mhc peptide binding. fragpredict, which is publicly available as a part of mappp service (http://www.mpiibberlin.mpg.de/mappp/), combines proteasomal cleavage predictions with mhc-and tap-binding predictions. fragpredict consists of two algorithms. the first algorithm uses a statistical analysis of cleavage-enhancing and -inhibiting amino acid motifs to predict potential proteasomal cleavage sites (holzhutter et al., 1999) . the second algorithm, which uses the results of the first algorithm as an input, predicts which fragments are most likely to be generated. this model takes the time-dependent degradation into account based on a kinetic model of the 20s proteasome (holzhutter and kloetzel, 2000) . at the moment, fragpredict is the only method that can predict fragments, instead of only possible cleavage sites. paproc (http://www.paproc.de) is a prediction method for cleavages by human as well as wild type and mutant yeast proteasomes. the influences of different amino acids at different positions are determined by using a stochastic hillclimbing algorithm (kuttler et al., 2000) based on the experimentally in vitro verified cleavage and non-cleavage sites (nussbaum et al., 2001) . both the fragpredict and paproc methods make use of the limited in vitro proteasomal digest data available. fragpredict is a linear method, and it may not capture the non-linear features of the specificity of the proteasome. the netchop (kesmir et al., 2002) method tries to address these two issues. the prediction system is a multilayered ann and uses naturally processed mhc class i ligands to predict proteasomal cleavage. since some of these ligands are generated by the immunoproteasome, and some by the constitutive proteasome, such a method should predict the combined specificity of both forms of proteasomes. in 2003, netchop-2.0 were evaluated to be the best-performing predictor on an independent evaluation set (saxova´et al., 2003) . pcleavage is another web accessible proteasomal cleavage predictor, which is svm based and have a published performance comparable to netchop-2.0 (bhasin and raghava, 2005) . an update of the netchop method [netchop-3.0, nielsen et al. (2005) ] consists of a combination of several anns, each trained using a different sequence-encoding scheme of the data. netchop 3.0 has an increase in the prediction sensitivity as compared to netchop 2.0, without lowering the specificity, and is thus probably the current best predictor of proteasomal cleavage. tenzer et al. (2004) have published a weight matrix based method for prediction of both constitutive-and immunoproteasomal cleavage specificity. both matrices are trained on in vitro digest data. relatively few methods have been developed to predict the specificity of tap. daniel et al. (1998) have developed anns using peptide 9mers for which tap affinity was determined experimentally. surprisingly, they found that some mhc alleles have ligands with very low tap affinities, e.g. hla-a2. however, it has been shown that tap ligands can be trimmed in er before binding to mhc molecules (fruci et al., 2001) , i.e. a tap ligand might be an epitope precursor and thus does not need to be 9 amino acids long. hla-a2 might easily have precursors of its optimal ligands, which are also good tap binders. peters et al. (2003) used an smm to predict tap affinity of peptides. this method has the advantage of not being bound to only 9mers but can also be used for longer peptides. the method assumes that only the first three positions in the n-terminal and the last position at the c-terminal influences the tap binding. the method is very well evaluated and the accuracy is high. the significance of tap binding in the epitope presentation pathway is much lower than the mhc binding (see later) and the auc value when this method is used alone as an epitope predictor of 0.79 is thus significantly lower than most mhc-binding prediction methods. two methods were published in 2004. bhasin and raghava (2004) published a method for which they do only compare to the method of daniel et al. (1998) and it is not determined how it performs compared to the peters' method. the method of doytchinova et al. (2004) is evaluated by comparing the resulting method (matrix) with other matrices. from such a comparison it can only be concluded that this method is closer to peters' model than to the model of bhasin and raghava (2004) but not how it actually performs. recently a new tap predictor, predtap, have been published . this method does not have an auc value for the methods performance in epitope prediction making a direct comparison to other models impossible. with increasing numbers of tap ligands available on the internet (e.g. jen-pep database, http://www.jenner.ac.uk) (blythe et al., 2002) , it will likely soon be possible to obtain more accurate tap predictions. with respect to tap-independent transport and cleavage of peptides, the most established model is especially connected to the most abundant hla supertype (a2) and is related to the signal peptides and the processing of such . prediction of potential signal peptides that can be transported by sec61 can be made with tools for prediction of signal peptides, and some of these will also predict the signal peptidase cleavage site (bendtsen et al., 2004; kall et al., 2004; zhang and henzel, 2004) , but the value in the context of cd8þ t-cell epitope predictions remains to be elucidated. the tcrs are generated by highly stochastic processes that secures that the tcrs in general will be able to recognize the entire probable space of mhc-peptide complexes. however, tcrs that recognize self-peptides will be eliminated so peptides that form complex with mhc are indistinguishable from self-peptides will not be recognized. it is still not clear how close peptides must be to the self to be able to escape recognition in this way (louzoun et al., 2006) . reliable predictions of immunogenic peptides can reduce the experimental effort needed to identify new epitopes, and though reliable predictions of the mhc binding alone can indeed be used to rank the possible epitopes very accurately, even better predictions should be possible if the other steps in the pathway were integrated in the predictions. accordingly, many attempts have been made to predict the outcome of the steps involved in antigen presentation, mapp (hakenberg et al., 2003) , netctl (larsen et al., 2005) , mhcpathway (tenzer et al., 2005) , epijen (doytchinova et al., 2006) and wapp (donnes and kohlbacher, 2005) . all these methods attempt to predict antigen presentation by integrating peptide-mhc binding predictions with one or more of the other events involved in the antigen presentation pathway. to benchmark these, a set of verified epitopes can be used as the positive dataset. negative examples (peptides that cannot induce an immunologic response) are hard to identify, as it is very hard to determine that a peptide will never be an epitope in any persons with a given hla haplotype. instead, epitopes from well-studied pathogens (e. g. hiv) are often used as the positive set, and all other peptides from the genome of the same pathogen that have never been shown to be an epitope are assumed negative as they have a very low probability of being an epitope. running a large-scale benchmark calculation comparing the predictive performance of several publicly available mhc-i presentation prediction methods evaluated on a large set of known hiv epitopes (http://www.cbs.dtu.dk/suppl/immunology/ctl-1.2/ hiv_dataset) reveals that the updated netctl and mhcpathway methods have the highest predictive performance with 475% if the epitopes being within the top 5% peptides with the highest prediction scores (mette volby larsen, personal communication). improved understanding of the immune systems, and its population-wide variation, is one of the major challenges in the next decade within biology and medicine. many of the steps by which the immune system deal with infectious agents and disease can now successfully be modeled by computational techniques, and it is clear that the theoretical approaches will be a major player in this area, adding a systems view to the massive experimental effort being carried out at the moment. in this review, we have summarized how a number of bioinformatics tools that use genomic sequences as input to predict epitopes, have been developed over the past decade. at the same time, theoretical models have been developed that describe the dynamics of different immune-cell populations and their interactions with microbes (borghans and de boer, 2007; carneiro et al., 2007; davenport et al., 2007) . these models have been used to interpret experimental findings where timing is of importance, such as the interval between administration of a vaccine and infection with the microbe that the vaccine is intended to protect against. moreover, these dynamic models allowed for generating a quantitative picture of immune system kinetics and diversity during health and disease. the quantitative approach is necessary to understand the functioning of the immune system, which consists of many different cell types and molecules interacting in complicated regulatory pathways involving positive and negative feedback loops. surprisingly little is known about the population dynamics, i.e. the production rates, division rates and distribution of life spans of mouse or human lymphocyte populations. as a consequence, fundamental questions like the maintenance of memory, the maintenance of a diverse naive repertoire and the role of homeostatic mechanisms, remain largely unresolved. having so little insight in the normal lymphocyte population dynamics also hampers our understanding of immune responses during disease and immune reconstitution after therapeutic interventions such as chemotherapy, irradiation and/or bone marrow transplantation. several areas in immunology call for a better interpretation of data by means of theoretical models. a simple pubmed search reveals that at least 10% of the recent papers in the immunological literature involve labeling experiments in which lymphocytes are labeled radioactively, with deuterium, or with dyes. however, the interpretation of such labeling data is controversial and is notoriously difficult (boer et al., 2003a, b; deenick et al., 2003; gett and hodgkin, 2000; hellerstein, 1999; mohri et al., 1998; mohri et al., 2001; revy et al., 2001; ribeiro et al., 2002) , which emphasizes the enormous demand to develop a quantitative mathematical approach to immunology. similar examples of how difficult it is to properly interpret kinetic data come from the attempts to characterize the division history of cells from the length of the telomeres, or from the presence of autosomal dna circles (trecs) that are formed in the thymus (boer and noest, 1998; douek et al., 1998; dutilh and de boer, 2003; hazenberg et al., 2000; hazenberg et al., 2003) . integrating the dynamic (using mathematical models and computer simulations) and bioinformatics approaches clearly could lead to a better understanding of the immune responses and their role during normal, disease and reconstitution states, where both timing and sequence specificity are highly significant. diseases that are characterized by complex interactions between the host cellular immune system and evolving pathogens such as hiv infection, or diseases where molecular similarities between self and non-self are important such as in autoimmune diseases could be investigated in such integrated models. complex generalized cellular automata have been proposed as models of the immune system (kohler et al., 2000; seiden and celada, 1992) . these methods have now developed to a stage where it is possible successfully to simulate the outcome of cancer vaccine protocols using a mouse simulation model (castiglione and piccoli, 2007; lollini et al., 2006; motta et al., 2005; pappalardo et al., 2006) . in a recent paper, rapin et al. 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binding peptides based on independent binding of individual peptide side-chains predicting location of continuous epitopes in proteins from their primary structures correlation between the location of antigenic sites and the prediction of turns in proteins generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method identifying mhc class i epitopes by predicting the tap transport efficiency of epitope precursors the immune epitope database and analysis resource: from vision to blueprint a community resource benchmarking predictions of peptide binding to mhc-i molecules bioinformatics for study of autoimmunity syfpeithi: database for mhc ligands and peptide motifs modelling the human immune system by combining bioinformatics and systems biology approaches definition of mhc supertypes through clustering of mhc peptide binding repertoires prediction of mhc class i binding peptides using profile motifs synthetic peptides as antigens functional antigen-independent synapses formed between t cells and dendritic cells modeling deuterated glucose labeling of t-lymphocytes a sequence pattern common to t cell epitopes exact prediction of a natural t cell epitope predicting proteasomal cleavage sites: a comparison of available methods prediction of well-conserved {hiv}-1 ligands using a matrix-based algorithm combining computer algorithms with experimental approaches permits the rapid and accurate identification of t cell epitopes from defined antigens 3d-epitope-explorer (3dex): localization of conformational epitopes within three-dimensional structures of proteins a model for simulating cognate recognition and response in the immune system hla supertypes and supermotifs: a functional perspective on hla polymorphism nine major hla class i supertypes account for the vast preponderance of hla-a and-b polymorphism prediction of major histocompatibility complex binding regions of protein antigens by sequence pattern analysis phage display selection and combination of machine learning classifiers for prediction of linear b-cell epitopes on proteins machine learning approaches for prediction of linear b-cell epitopes on proteins two new proteases in the mhc class i processing pathway peptide binding specificity of major histocompatibility complex class i resolved into an array of apparently independent subspecificities: quantitation by peptide libraries and improved prediction of binding measuring the accuracy of diagnostic systems sars ctl vaccine candidates; hla supertype-, genome-wide scanning and biochemical validation mapping a neutralizing epitope on the sars coronavirus spike protein: computational prediction based on affinity-selected peptides modeling the mhc class i pathway by combining predictions of proteasomal cleavage, tap transport and mhc class i binding quantitative analysis of prion-protein degradation by constitutive and immuno-20s proteasomes indicates differences correlated with disease susceptibility new insights into {v}({d}){j} recombination and its role in the evolution of the immune system location of 'continuous' antigenic determinants in the protruding regions of proteins methods and protocols for prediction of immunogenic epitopes predicting antigenic determinants in proteins: looking for unidimensional solutions to a three-dimensional problem? mapping epitope structure and activity: from one-dimensional prediction to four-dimensional description of antigenic specificity ctl epitopes for influenza a including the h5n1 bird flu hla-wide screening immunodominance in major histocompatibility complex class i-restricted t lymphocyte responses methods for prediction of peptide binding to mhc molecules: a comparative study pred(tap): a system for prediction of peptide binding to the human transporter associated with antigen processing assembly of mhc class i molecules within the endoplasmic reticulum signal peptide prediction based on analysis of experimentally verified cleavage sites toward the quantitative prediction of t-cell epitopes: qsar studies on peptides having affinity with the class i mhc molecular hla-a*0201 improving mhc binding peptide prediction by incorporating binding data of auxiliary mhc molecules conflict of interest: none declared. key: cord-255069-9xueqdri authors: leary, shay; gaudieri, silvana; chopra, abha; pakala, suman; alves, eric; john, mina; das, suman; mallal, simon; phillips, elizabeth title: three adjacent nucleotide changes spanning two residues in sars-cov-2 nucleoprotein: possible homologous recombination from the transcription-regulating sequence date: 2020-04-11 journal: biorxiv doi: 10.1101/2020.04.10.029454 sha: doc_id: 255069 cord_uid: 9xueqdri the covid-19 pandemic is caused by the single-stranded rna virus severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a virus of zoonotic origin that was first detected in wuhan, china in december 2019. there is evidence that homologous recombination contributed to this cross-species transmission. since that time the virus has demonstrated a high propensity for human-to-human transmission. here we report two newly identified adjacent amino acid polymorphisms in the nucleocapsid at positions 203 and 204 (r203k/g204r) due to three adjacent nucleotide changes across the two codons (i.e. agg gga to aaa cga). this new strain within the lgg clade may have arisen by a form of homologous recombination from the core sequence (cs-b) of the transcription-regulating sequences of sas-cov-2 itself and has rapidly increased to approximately one third of reported sequences from europe during the month of march 2020. we note that these polymorphisms are predicted to reduce the binding of an overlying putative hla-c*07-restricted epitope and that hla-c*07 is prevalent in caucasians being carried by >40% of the population. the findings suggest that homologous recombination may have occurred since its introduction into humans and be a mechanism for increased viral fitness and adaptation of sars-cov-2 to human populations. evidence of viral adaptation to selective pressures as it spreads among diverse human populations has implications for the ongoing potential for changes in viral fitness over time, which in turn may impact transmissibility, disease pathogenesis and immunogenicity. geographic differences in viral sequence diversity and epidemiological profiles of disease are likely to reflect the spread of founder viruses, which first entered different sars-cov-2 naïve populations. however, the extent to which selection pressures operating within those populations also impact sars-cov-2 diversity is currently not known. functional effects of new genetic changes need to be considered in ongoing public health measures to contain infection around the world and in the development of universal vaccines and antiviral therapy. here we describe a new emerging strain of sars-cov-2 within the lgg clade that appears to be the result of a homologous recombination event that introduced three adjacent nucleotide changes spanning two residues of the nucleocapsid protein. that strain expanded rapidly in europe in march 2020. this protein forms an integral part of the virus lifecycle and is known to be highly immunogenic. we utilized publicly available sars-cov-2 sequences from the gisaid database table 1) . of these polymorphisms, three were the polymorphisms l84s in orf8, d614g in surface glycoprotein (s) and g251v in ns3 (orf3a) that mark the major worldwide clades s, g and v, respectively. two newly identified adjacent polymorphisms (r203k and g204r) in the nucleocapsid protein occur in approximately 13.4% of deposited strains and form one of the main strains emerging from europe ( figure 1a ). other common polymorphisms include q57h in ns3, t85i in nsp2, l37f in nsp6, p323l in the rna-dependent rna polymerase, t175m in the membrane glycoprotein and p504l and y541c in the helicase. current low frequency polymorphisms at <5% of deposited sars-cov-2 sequences include s193i in the nucleocapsid, h93y in ns3, and the following polymorphisms v378i, g392d, i739v, p765s, a876t, f3071y, g3278s and k3353r in orf1ab (supplementary table 1 ). the polymorphisms are present in strains sequenced using different next generation sequencing (ngs) platforms (e.g. nanopore, illumina) and the sanger-based sequencing method making it unlikely that the new changes are sequence or alignment errors. in addition, different laboratories around the world have deposited sequences with these polymorphisms in the database and examination of individual sequences in the region does not find obvious insertions/deletions likely representing alignment issues or homopolymer slippage. for the two newly identified adjacent polymorphisms in the nucleocapsid at positions 203 and 204, there were no strains in the database that had only one of the two changes. the sars-cov-2 sequences deposited into the gisaid database are consensus strains predominantly generated from ngs platforms that can typically identify low frequency variants. we did not have access to the original sequence files from the contributing laboratories in order to assess if there was evidence of strains that harbored only one of the polymorphisms at lower frequencies. however, no circulating strain has so far been captured that contains only one of the two nucleocapsid polymorphisms as the consensus sequence. the rapid emergence of these closely linked polymorphisms in viruses may reflect strong selection pressure on this region of the genome in which the original mutation incurred a replicative capacity, or other fitness cost, which could be restored by a linked compensatory mutation. evidence for such adaptations with closely linked compensatory mutations are known to occur under host immune pressure as is well established for other adaptable rna viruses such as hiv 1,2 and hepatitis c virus (hcv) 3 . these viruses have such a high rate of viral replication and error-prone reverse transciptase that a massive swarm of viral variants with ongoing recombination between residues is generated continuously. as a result selection pressure exerted by immune responses or other selective pressures effectively operate on each separate residue independently 4 . in contrast, coronaviruses encode proofreading machinery and have a propensity to adapt by homologous recombination between viruses rather than classic step-wise individual mutations driven by selective pressures operating on single viral residues. this, together with the routine nature of their cross-species transmission 5 , led graham and baric 6 to presciently warn in 2010 that it was a matter of when, rather than if, a pathogenic coronavirus pandemic would occur in humans. also of note, the phenomena of compensatory fixation has been described in the area of hiv antiviral resistance in which the linked mutations cannot revert to wild type when the selective pressure is removed as the virus cannot negotiate the fitness valley to return to its previous optimal state 7 . we therefore predict that the k203/r204 (aaa cga) change is likely to remain fixed and intermediates to the wild type are unlikely to be found. it will be critical to determine if the introduction of the aaacga motif results in a replicative or other fitness cost to the virus, creates an alternative subgenomic mrna transcript or rna secondary structure or increases nucleocapsid activity as this could indicate that there may be viral attenuation as passage occurs globally through populations of diverse immunogenetic background. as further evidence of the likelihood of a homologous recombination event, the r203k polymorphism involves a two-step process from agg to aaa. however, strikingly, the position shows no evidence to date of alternative codon usage, all viral strains that contain an r at this position have the agg codon, and similarly those as of march 31, 2020 there appears to be only a small proportion of strains with krlgg in the us, likely reflecting that deposited sequences have been mainly from the west coast of the us that experienced initial importation of asian strains of sars-cov-2. it will be of great interest to see sequences from the east coast of the us given the early importation of sars-cov-2 from northern europe as well as asia and the widespread community transmission that has followed ( figure 1a) . interestingly, the m175 polymorphism in the membrane glycoprotein appears to only be present on the kr-lgg combination (of the 132 sequences with this polymorphism 131 are from europe and 1 from north america) (supplementary table 2 ). when the other common polymorphisms (>5%) observed in the nsp2, nsp6, rna-dependent rna polymerase (rdrp), membrane glycoprotein and helicase are taken into account, there are at present eight main circulating strains at >5% frequency in the database all within one to three amino acid polymorphism networks (supplementary table 2 ). of note, our current knowledge of the global circulating strains is dependent on the ability of laboratories in different countries to deposit full genome length sars-cov-2 sequences and may be subject to ascertainment bias. as such, the frequencies of specific strains shown in figure 1 may not reflect the size of the outbreak. however, the data does provide the opportunity to predict the presence of specific strains in areas given the known epidemiology within different countries and regions. currently the possible functional effect(s) of the introduction of the aaacga motif into the nucleocapsid are not known. the nucleocapsid protein is a key structural protein critical to viral transcription and assembly, suggesting that changes in this protein could either increase or decrease replicative fitness. however, it is also possible these changes could be functionally compensated by linked polymorphism in the virus and/or counterbalanced by some other host fitness benefit. however, we have not found any other polymorphism linked to the k203/r204 change to date. selection of viral adaptations to polymorphic host responses mediated by t cells, nkcells, antibodies and antiviral drugs are well described for other rna viruses such as hiv and hcv 4, 11 . hiv-1 adaptations to human leucocyte antigen (hla)-restricted t-cell responses have also been shown to be transmitted and accumulate over time 12, 13 . as previously shown for sars-cov, t-cell responses against sars-cov-2 are likely to target the nucleocapsid 14 . notably, sars-cov-2 r203k/g204r polymorphisms modify the predicted binding of the hla-c*07 allele to a putative tcell epitope containing these residues. escape from hla-c-restricted t-cell responses may conceivably confer a fitness advantage for sars-cov-2, particularly in european populations where hla-c*07 is prevalent and carried by >40% of the population (www.allelefrequencies.net). the replication characteristics and plasticity of small, highly mutable viruses such as hiv and hcv are distinct from sars-cov-2, which is significantly less variable. transmission and accumulation of ctl escape variants drive negative associations between hiv polymorphisms and hla hiv evolution: ctl escape mutation and reversion after transmission molecular footprints reveal the impact of the protective hla-a*03 allele in hepatitis c virus infection evidence of hiv-1 adaptation to hla-restricted immune responses at a population level cross-species transmission of the newly identified coronavirus 2019-ncov recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission hiv protease resistance and viral fitness the minimum amount of homology required for homologous recombination in mammalian cells evidence of viral adaptation to hla class i-restricted immune pressure in chronic hepatitis c virus infection extensive host immune adaptation in a concentrated north american hiv epidemic rapid hiv-1 disease progression in individuals infected with a virus adapted to its host population long-lived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sars-recovered patients cytotoxic t-cell immunity to influenza key: cord-013093-aa4cf44u authors: cassotta, antonino; paparoditis, philipp; geiger, roger; mettu, ramgopal r.; landry, samuel j.; donati, alessia; benevento, marco; foglierini, mathilde; lewis, david j.m.; lanzavecchia, antonio; sallusto, federica title: deciphering and predicting cd4(+) t cell immunodominance of influenza virus hemagglutinin date: 2020-07-09 journal: j exp med doi: 10.1084/jem.20200206 sha: doc_id: 13093 cord_uid: aa4cf44u the importance of cd4(+) t helper (th) cells is well appreciated in view of their essential role in the elicitation of antibody and cytotoxic t cell responses. however, the mechanisms that determine the selection of immunodominant epitopes within complex protein antigens remain elusive. here, we used ex vivo stimulation of memory t cells and screening of naive and memory t cell libraries, combined with t cell cloning and tcr sequencing, to dissect the human naive and memory cd4(+) t cell repertoire against the influenza pandemic h1 hemagglutinin (h1-ha). we found that naive cd4(+) t cells have a broad repertoire, being able to recognize naturally processed as well as cryptic peptides spanning the whole h1-ha sequence. in contrast, memory th cells were primarily directed against just a few immunodominant peptides that were readily detected by mass spectrometry–based mhc-ii peptidomics and predicted by structural accessibility analysis. collectively, these findings reveal the presence of a broad repertoire of naive t cells specific for cryptic h1-ha peptides and demonstrate that antigen processing represents a major constraint determining immunodominance. cd4 + t lymphocytes orchestrate adaptive immune responses by secreting cytokines that promote multiple types of inflammatory responses in tissues and by providing help to b cells and cd8 + t cells (sallusto et al., 2010) . for antigen recognition, cd4 + t cells rely on the interaction with antigen-presenting cells (apcs) that take up, process, and present antigen in the form of short linear peptides bound to mhc class ii (mhc-ii) molecules (roche and furuta, 2015; unanue et al., 2016) . typically, only a small fraction of the multitude of potentially immunogenic peptides contained in a complex foreign antigen are able to induce a measurable t cell response, with some peptides recognized with higher magnitude and/or frequency and thus arising as immunodominant, and others that remain subdominant or even cryptic (sercarz et al., 1993; yewdell and bennink, 1999; yewdell and del val, 2004) . given the complexity and tight connection between antigen presentation and recognition, many factors may pertain to peptide and t cell immunodominance. some of those reflect the biochemical rules of antigen processing and mhc presentation, such as the molecular context in which the peptides are embedded (graham et al., 2018; kim and sadegh-nasseri, 2015; landry, 2008; mirano-bascos et al., 2008) , the affinity of the generated peptides for mhc-ii binding, the resistance to hla-dm-mediated editing of newly formed peptide mhc-ii (pmhc-ii) complexes mellins and stern, 2014) , or their kinetic stability on the cell surface of apcs (sant et al., 2005) . furthermore, the heterogeneous set of proteolytic enzymes and endogenous inhibitors that different kinds of apcs are equipped with (unanue et al., 2016) , as well as the interactions with molecular partners that facilitate antigen uptake, such as b cell receptors (bcrs) or soluble antibodies (simitsek et al., 1995; watts and lanzavecchia, 1993) , can affect the antigen processing and the composition of the mhc-ii-presented peptidome. other variables influencing t cell immunodominance depend on the architecture of the t cell repertoires and the mechanisms of antigen recognition (yewdell, 2006) , such as the availability of antigen-specific naive precursors (jenkins and moon, 2012; moon et al., 2007) , the interaction affinity of their tcrs with pmhc-ii complexes (malherbe et al., 2004) , or the occurrence of tcr cross-reactivity to similar antigenic peptides (campion et al., 2014; nelson et al., 2015; su et al., 2013) . in this study, we chose influenza a virus as a model infectious agent that triggers complex adaptive immune reactions comprising both humoral and cellular responses. every year, influenza viruses infect more than a billion people worldwide, and are the cause of prominent economic loss as well as significant morbidity and mortality, especially in children <5 yr old and adults >65 (krammer et al., 2018; lee et al., 2019; zens and farber, 2015) . despite great efforts in research, vaccines are only moderately effective against seasonal strains and are challenged by the rapidly evolving nature of influenza viruses that occasionally emerge as new strains causative of serious epidemics or pandemics (angeletti and yewdell, 2018; krammer et al., 2018; webster and govorkova, 2014; zens and farber, 2015) . we focused our attention on hemagglutinin (ha), which represents the main target of antibody response to influenza virus upon vaccination or infection (angeletti and yewdell, 2018; corti et al., 2011; krammer et al., 2018; lee et al., 2019; pappas et al., 2014) . the detailed and unbiased characterization of ha-reactive memory and naive cd4 + t cell repertoires, paralleled by a deep analysis of the naturally presented repertoire of mhc-ii-binding ha peptides by mass spectrometry (ms)-based immunopeptidomics, allowed us to shed new light on the factors governing cd4 + t cell clonal selection and immunodominance to influenza ha in humans. to capture the entire repertoire of memory t cells specific for influenza ha, we obtained multiple and large blood samples from a donor (hd1) after vaccination with the 2013/14 seasonal inflexal v vaccine containing ha from the pandemic a/california/07/2009 h1n1 strain (h1-ha). central memory (tcm), effector memory (tem), and circulating follicular helper (ctfh) cd4 + t cells were isolated by cell sorting, labeled with cfse, and stimulated with inflexal v. when analyzed on day 6, proliferating cfse lo t cells were detected in all three memory subsets from samples obtained 6 and 12 mo after vaccination ( fig. 1 a) . to select h1-ha-reactive t cells, the cfse lo t cells were sorted, relabeled with cfse, and stimulated with h1-ha ( fig. 1 b) . t cells proliferating in the secondary stimulation were cloned, and 456 h1-ha-specific clones were isolated (fig. 1 c and table s1 ) and characterized for peptide specificity, mhc restriction (hla-dr, hla-dp, or hla-dq), and tcr vβ usage. strikingly, >85% of the clones isolated (393 of 456) recognized two overlapping peptides (h1-ha 401-420 or h1-ha 411-430 ; fig. 1 d) , thus defining, in this individual, a highly immunodominant region. t cells specific for the immunodominant h1-ha 401-430 region were found in all three memory subsets (94 clones in tcm, 112 clones in tem, and 187 clones in ctfh) and were hla-dr restricted, as shown by antibody blocking experiments (fig. s1 a) . several t cell clones specific for subdominant h1-ha regions were also hla-dr restricted, with a minority being hla-dq or hla-dp restricted (fig. s1 , b and c). tcr vβ sanger sequencing performed on 274 t cell clones showed that the response to h1-ha was highly polyclonal, comprising 88 distinct clonotypes, even when directed against the immunodominant region (table s2) . for instance, 62 t cell clones specific for the immunodominant peptide h1-ha 401-420 comprised 16 different clonotypes, and 157 t cell clones specific for the immunodominant peptide h1-ha 411-430 comprised 39 different clonotypes ( fig. 1 e and table s2 ). of note, 26 of the 39 h1-ha 411-430 -specific clonotypes used the trbv19 gene, suggesting a preferential tcr cdr1 and cdr2 usage that might facilitate cognate interaction with the peptide-mhc complex. tracking of h1-ha-reactive t cell clonotypes within the cfse lo t cell population responding to inflexal (fig. 1 a) showed that those against the immunodominant h1-ha region were among the most represented and that some were also found in the tcm, tem, or tfh repertoire ex vivo (fig. 1 f) . strikingly, several of these clonotypes were still detected in memory t cell subsets isolated from donor hd1 48 mo later (fig. 1 g) . collectively, these findings indicate that in an inflexal-immunized donor, a polyclonal repertoire of h1-ha-specific tcm, tem, and ctfh cells is highly focused on a small immunodominant region. memory t cells are focused against immunodominant regions, while naive t cells recognize multiple peptides spanning the entire h1-ha sequence we next investigated whether the immunodominance observed in the memory repertoire is a general phenomenon and whether it is reflected in the naive repertoire of a given individual. to address these questions, we used the highly sensitive t cell library method (geiger et al., 2009) to screen naive and total memory cd4 + t cells from hd1 and three other immune donors with a diverse hla background (table s3) . for each donor, naive and memory t cells were polyclonally expanded in multiple cultures (each containing 1,000-2,000 cells) in the presence of phytohemagglutinin, il-2, and feeder cells. for a broad and unbiased screening of t cell reactivity against h1-ha, the t cell libraries were then screened using overlapping 15mer peptides covering the entire h1-ha sequence. in all four donors tested, h1-ha peptide-specific t cell clones were readily detected in naive and memory libraries although, as expected, their frequencies measured in the naive libraries were lower compared with that measured in the memory libraries (fig. 2, a and b) . epitope mapping of memory t cell clones confirmed in all four donors a skew toward one or two immunodominant regions, which for donor hd1 coincided with those detected by antigen-driven proliferation of memory t cell subsets (fig. 2 c) . strikingly, however, epitope mapping of naive t cell clones showed that these cells covered a broad range of peptide specificities (fig. 2, c and d) . this pattern was particularly evident for donor hd1, which was analyzed at high depth. in this donor, naive t cells with diverse tcr vβs recognized peptides spanning virtually all the h1-ha sequence (fig. 2 c and table s4 ). collectively, these findings demonstrate that the naive t cell repertoire has a very broad coverage of the h1-ha sequence and that only a fraction of this repertoire is selected in the memory repertoire. naive and memory t cell clones show different functional avidities for peptide and naturally processed h1-ha a plausible explanation for the selection of immunodominant peptides is their binding affinity to mhc molecules and/or tcrs. we therefore measured the binding affinity of dominant figure 1 . clonally expanded memory cd4 + t cells target an immunodominant region of influenza h1-ha. (a) memory cd4 + tcm, tem, and ctfh cell subsets were isolated from blood samples of donor hd1 6 and 12 mo after inflexal v vaccination. cells were labeled with cfse and stimulated with the inflexal v vaccine in the presence of autologous monocytes. shown is the cfse profile and the percentage of proliferating cfse lo cells on day 6 in the 12-mo sample. percentage of cfse lo in the 6-mo sample was 38% (tcm), 65% tem, and 57% ctfh. (b) the inflexal v-reactive cfse lo t cells were sorted, relabeled with cfse, and stimulated with recombinant h1-ha in the presence of autologous monocytes. after 5 d, cfse lo proliferating t cells were sorted and cloned by limiting dilution. shown is the experiment performed with the 12-mo sample; comparable results were obtained with the 6-mo sample. (c) a total of 456 h1-ha-specific cd4 + t cell clones were isolated from the tcm, tem, and ctfh cultures based on the proliferative response (stimulation index ≥3) to a pool of overlapping peptides spanning the entire h1-ha sequence. proliferation was assessed on day 3 after a 16-h pulse with [ 3 h]thymidine and expressed as counts per minute. the data are representative of at least two independent experiments. (d) epitope mapping of the 456 h1-ha-specific t cell clones. epitopes were identified by screening the t cell clones with the individual h1-ha peptides in at least three independent experiments, with consistent results. the x axis indicates h1-ha amino acid sequence; each color-coded segment represents the sequence recognized by individual clones isolated from tcm (black), tem (green), or ctfh (red) cultures. the numbers of h1-ha-reactive t cell clones isolated from each subset are reported. the chart at the bottom indicates ha1 and ha2 domains colored in blue and red, respectively (fp, fusion peptide; tm, transmembrane). (e) rearranged tcr vβ sequences of h1-ha-specific t cell clones were determined by rt-pcr followed by sanger sequencing. shown in the pie charts are the repertoires of rearranged tcr vβ sequences of t cell clones recognizing immunodominant h1-ha 401-420 and h1-ha 411-430 epitopes. each slice of the chart indicates a different tcr vβ clonotype (h1-ha 401-420 , n = 16; h1-ha 411-430 , n = 39); the number of sister clones bearing the same tcr vβ sequence are reported for each slice. the total number of clones sequenced is reported at the center. (f and g) to evaluate the clonal expansion of h1-ha-reactive memory t cell clones, tcr vβ deep sequencing was performed on inflexal and subdominant peptides to the recombinant hla-dr molecules of donor hd1 (alleles drb1*01:01 or *08:01). in vitro refolding assays performed in the presence of titrated peptides showed that the immunodominant h1-ha 401-420 and h1-ha 411-430 peptides bound to hla-drb1*08:01 molecules with an affinity that was comparable to that of subdominant peptides binding to the same hla-dr ( fig. s1 c) , indicating that immunodominance of these peptides is not explained by preferential binding to mhc class ii molecules. immunodominance to ha antigen may be also the result of repeated exposure through natural infection or vaccination that can select cross-reactive t cells. however, as shown in fig. s2 , t cell clones responding to immunodominant or subdominant h1-ha peptides were both capable to cross-react, to variable but similar extents, to h1-ha and/or h3-ha from the widespread strains a/brisbane/59/2007 (h1n1) or a/brisbane/10/2007 (h3n2). a few clones cross-reacted to h5-ha from the highly pathogenic subtype a/viet nam/1203/2004 (h5n1). we then selected a large number of h1-ha-specific t cell clones derived from naive or memory t cells of donor hd1 and determined their functional avidity by measuring the proliferative response to autologous monocytes pulsed with different concentrations of the h1-ha peptides or h1-ha protein, which need processing for presentation on mhc-ii molecules. functional avidity, measured in response to peptide stimulation and expressed as half-maximal concentration (ec 50 ) value, was spread over almost 3 logs for both types of t cell clones, with clones from memory t cells being enriched for high-avidity cells ( fig. 3 a and fig. s3 , a and c), consistent with previous observations on the response to tetanus toxoid (geiger et al., 2009) . when the same clones were tested for their response to h1-ha protein, several observations were made. first, clones from memory t cells responded to h1-ha protein, and there was an overall correlation between ec 50 values for protein and peptide, with the immunodominant clones, such as m1 specific for h1-ha 411-430 , showing the highest avidity for both peptide and protein (red dots in fig. 3 b) . however, there were a few notable exceptions. for instance, the subdominant clones m2 and m3, specific for h1-ha 396-410 and h1-ha 526-540 , had high functional avidity for peptide, comparable to clone m1, but showed 1,000-fold lower avidity for the h1-ha protein (fig. 3 b) . second, memory t cell clones restricted by hla-dp (m4 specific for h1-ha 121-140 ) or by hla-dq molecules (m5 and m6 specific for h1-ha 186-200 and h1-ha 446-460 , respectively) recognized peptides and protein with functional avidity lower than the median of the distribution (fig. 3, a and b) . third, several clones from naive t cells, including some with intermediate avidity for peptides, did not proliferate in response to h1-ha protein (fig. 3 c) . finally, and importantly, the differential recognition of dominant and subdominant epitopes in response to h1-ha was also observed for clones derived from naive t cells. for instance, clone n1, specific for the immunodominant h1-ha 411-430 peptide, had high functional avidity for both peptide and naturally processed h1-ha, comparable to that of memory clone m1, while clones n2 and n3 (specific for the subdominant peptides h1-ha 521-540 and h1-ha 536-555 , respectively) had high avidity for peptide but 100-fold lower avidity for protein, similar to what was observed for memory clones m2 and m3 (fig. 3 c) . collectively, these findings indicate that in both the naive and memory repertoires, immunodominant peptides are recognized with high avidity by t cells. they also reveal the presence of a broad repertoire of naive t cells for cryptic ha peptides. finally, they show that the functional avidity of peptide recognition is not predictive of the response to the naturally processed antigen, suggesting a major role for antigen processing in determining the abundance of the processed peptide generated. the mhc class ii peptidome defines immunodominant regions and reveals modulation for antigen processing by antibodies based on the above results, we hypothesize that cd4 + t cell immunodominance could be primarily related to the yield of peptides generated by antigen processing by apcs. we therefore used ms-based immunopeptidomics to identify peptides naturally presented on mhc-ii molecules by different types of apcs. briefly, monocyte-derived dendritic cells (dcs) from donor hd1 and ebv-immortalized b cell clones carrying surface bcrs specific for h1-ha from all four donors were pulsed overnight with recombinant h1-ha. mhc-ii molecules were isolated from lysed cells using a pan-anti-mhc-ii antibody, and peptides were purified by reversed-phase chromatography. using this approach, we identified thousands of total mhc-ii bound individual peptides derived from self-molecules as well as 3-35 h1-ha-derived peptides (median 9) with the expected length peaking at 12-15 aa (fig. 4, a and b) . remarkably, almost 60% of the h1-ha-derived peptides presented by dcs of donor hd1 corresponded to a rich set of nested peptides overlapping the immunodominant regions h1-ha 401-430 targeted by memory t cells, suggesting that peptides in this region are presented in high abundance and are recognized by t cells with high functional avidity (fig. 4 c and table s5) . a similar correspondence between immunodominant peptides recognized by memory t cells and peptides presented on mhc-ii molecules was found in the other three donors analyzed (fig. 4 c and table s6 ), although the breadth of the analysis in these donors was limited by the amount of peptides that could be retrieved from b cells. a notable exception, however, was observed for peptide h1-ha 326-340 that was presented at high abundance by b cells of donor hd4 that lacked memory t cells specific for the same peptide. the antibody response to influenza a virus is directed against several regions of ha, such as the highly variable globular head or the conserved stem region, that can be the target of neutralizing antibodies with high breadth for multiple viral subtypes (corti et al., 2011; lee and wilson, 2015) . the mhc-ii peptidome of h1-ha-pulsed b cell clones with distinct epitope specificity isolated from donor hd1 revealed that a clone specific for the ha globular head presented the h1-ha immunodominant region in a similar manner as dcs, whereas a b cell clone specific for the ha stem region was a poorer presenter of h1-ha-derived peptides (fig. s4, a and b) . indeed, even if we resolved a comparable total number of mhc-ii presented peptides in the two kinds of b cells ( fig. s4 a) , from the anti-stem clone we did not detect any h1-ha-derived peptides corresponding to the dominant h1-ha 401-420 region, nor any subdominant peptides from the ha1 domain ( fig. s4 b) . consistently, presentation of the recombinant h1-ha by anti-stem b cells resulted in a much lower activation and proliferation of t cell clones specific , calculated based on the number of t cell clones recognizing each particular h1-ha peptide. inverse simpson index (1-d) ranges between 0 and 1, and reflects the probability that two h1-ha-specific t cell clones randomly selected from a repertoire recognize different h1-ha epitopes. **, p = 0.0097 as determined by two-tailed paired t test. for h1-ha 401-420 or h1-ha 241-260 (both hla-dr restricted), resulting in a 10-fold reduction of their functional avidity compared with antigen presentation by anti-head b cells (fig. s4 , c and d). conversely, hla-dr-restricted t cell clones specific for h1-ha 416-430 or h1-ha 386-400 , which were detected by mhc-ii immunopeptidomics on both anti-head and anti-stem b cell clones, showed comparable functional avidity when stimulated with either kind of apc (fig. s4 , c and d). taken together, these data show that the mhc-ii peptidome presented by professional apcs defines the immunodominant regions of ha recognized by memory cd4 + t cells. moreover, the spectrum of peptides naturally presented by b cells can be modulated by antibody binding to ha, thus potentially being an additional variable affecting b cell clonal selection during t cell-dependent immune responses. epitope prediction can be improved by combining mhc binding affinity prediction and antigen processing likelihood (apl) the binding of processed peptides to the groove of mhc-ii molecules follows precise rules that have been instrumental to the development of algorithms capable of predicting binding affinity with high accuracy (jensen et al., 2018; unanue et al., 2016; wang et al., 2010) . using the iedb tool for mhc-ii binding prediction, we found that virtually all the h1-ha 15mer peptides predicted as good binders for the mhc-ii alleles carried by the four donors analyzed were recognized by t cell clones isolated from either the naive or the memory compartment (fig. s5) . nevertheless, it was surprising to note that the immunodominant h1-ha peptides did not show stronger binding to mhc-ii compared with subdominant or cryptic peptides, by either in silico prediction or, as demonstrated before, in vitro measurement of mhc-ii binding. these data suggest that the binding to mhc-ii is a necessary, but not sufficient, feature for t cell immunodominance. we therefore set out to explore further parameters that might improve the in silico prediction of haderived t cell epitopes. the molecular context in which a peptide is embedded and its structural accessibility might influence the propensity of unfolding during the progressive ph acidification that occurs in the endocytic pathway, therefore affecting the exposition of denatured stretches of the antigen to the proteolytic environment of the late endosomes (graham et al., 2018; kim and sadegh-nasseri, 2015; landry, 2008) . to evaluate the role of structural constraints of ha in influencing the immunodominance observed in the memory repertoires, we adopted a recently developed algorithm that uses antigen conformational stability to estimate the likelihood of antigen processing (mettu et al., 2016) . in brief, an aggregate z-score of conformational stability was determined for each h1-ha residue by integrating four structural parameters obtained from the 3d structure of postfusion ha resolved by x-ray diffraction (pdb codes: 3lzg for ha1 domain [xu et al., 2010] ; 1htm for ha2 domain in the postfusion conformation [bullough et al., 1994] ). the z-score statistic was then used to calculate an apl for each theoretical h1-ha 15mer peptide (fig. 5 a) , following the rationale that the liberation of antigenic peptides might be facilitated by surrounding unstable regions that are readily unfolded and targeted by endosomal proteases. as shown in fig. 5 a, several peptides recognized by memory t cells in donors hd1-hd4 were found in regions of high apl. we then evaluated the performance of the apl and mhc binding predictive algorithms by computing the receiver operating characteristic (roc) curves using the set of epitopes recognized by memory t cells of each donor as true positives; the performance was assessed using the area under the roc curve (auroc) metric (fig. 5 b) . we also built a combined predictor by iteratively weighting the contributions of apl and mhc binding until we could maximize the auroc value of the predictor. with this approach, we found that in all donors the t cell clones specific for the immunodominant h1-ha 411-430 epitope are reported as red dots. lines represent the median and quartiles. ***, p < 0.001 as determined by two-tailed mann-whitney u test. (b and c) scatter plots of reciprocal ec 50 values of t cell clones from the memory (b) or naive (c) compartment, stimulated in parallel with recombinant h1-ha (x axis) and synthetic peptides (y axis). ec 50 values below the detection limit for stimulations with recombinant h1-ha were set arbitrarily to 20 µg/ml; the corresponding t cell clones are reported as white dots. spearman correlation was calculated based on ec 50 pairs from t cell clones responding to both peptides and recombinant h1-ha (b, n = 36; c, n = 29). thresholds of functional avidity were set arbitrarily at ec 50 values of 10 µg/ml, 200 ng/ml, and 10 ng/ml of antigen. n.d., not detected. cassotta et al. journal of experimental medicine combined predictor achieved a better auroc value, outperforming the predictors based solely on apl or mhc binding (fig. 5 b) . as shown in fig. 5 c, there are a number of ways to combine apl and mhc binding weights to achieve the highest auroc (0.75 in hd1 and hd2, 0.79 in hd3 and hd4), giving an estimate of the maximum and minimum weight of the two scores for each donor. importantly, all the immunodominant peptides identified in the memory repertoire of the four donors analyzed were grouped within a peak of high apl (as defined by a threshold of 0.6; fig. 5 d) . on average, the 20 top-scoring peptides predicted by apl accounted for >50% of the haspecific memory t cell clones, with a sensitivity comparable to the set of peptides measured by mhc-ii immunopeptidomics (fig. 5 e) . thus, a combination of apl and mhc-ii peptide binding affinity may be helpful to improve prediction of immunogenicity and immunodominance in cd4 + t cell responses. in this study, we report the identification of influenza h1-ha peptides recognized by naive and memory cd4 + t cells in individuals with different hla haplotypes. whereas naive t cells recognized a variety of peptides spanning the whole h1-ha sequence, memory t cells were highly focused on just a few peptides. these immunodominant peptides were readily identified by ms-based analysis of peptides eluted from mhc class ii molecules isolated from dcs or h1-ha-specific b cell clones and could be better predicted taking into account the ha structural accessibility to proteolytic cleavage. collectively, these findings indicate that processing of native proteins represents a major constraint determining the immunodominance to influenza ha and delineate new methods to identify immunodominant and cryptic t cell epitopes. the identification and characterization of antigen-specific t cells in the naive and memory repertoire is of both fundamental and practical relevance. the high-throughput t cell library method used in this study can rapidly identify, in different individuals, the range of peptides that can be recognized by t cells, thus determining, with a simple assay, both peptide binding to mhc-ii and the presence of specific tcrs (campion et al., 2014; geiger et al., 2009; latorre et al., 2018) . considering the small size of the naive t cell libraries analyzed (7 × 10 5 t cells) compared with the total naive t cell pool, we can estimate that the naive repertoire contains a large number of diverse ha-specific t cells, with frequencies ranging from 10 −5 to 10 −6 for each epitope and with a range of functional avidities. importantly, we showed that, in humans, naive t cells recognize multiple peptides spanning the whole h1-ha sequence. the diversity of epitope recognition observed in the naive compartment indicates that a multiplicity of ha-derived peptides can potentially trigger t cell activation, underlining the redundancy of the mhc-ii system in accommodating and presenting a large variety of different peptides. nevertheless, many t cell clones isolated from the naive compartment recognized peptides but not naturally processed protein. this finding suggests that the naive repertoire retains t cell precursors recognizing peptides that fail to be generated and/or presented by professional apcs or that are produced in amounts insufficient to trigger priming of cognate naive t cells. it remains to be established whether these cryptic peptides can be generated through unconventional antigen processing, as suggested in some cases of tissue antigens (mohan and unanue, 2012; sadegh-nasseri and kim, 2015) , or by nonprofessional apcs, such as epithelial cells in the respiratory tract that are main targets of influenza viruses. epithelial cells readily up-regulate mhc-ii molecules in response to inflammatory cytokines or viral infection (gao et al., figure 5 . immunodominant epitopes localize in h1-ha regions predicted as promptly liberated by endosomal proteases. (a) apl based on structural accessibility was calculated for each theoretical h1-ha 15mer peptide (upper panel). sets of h1-ha epitopes mapped in the memory cd4 + t cell repertoire of each donor (lower panels); each segment indicates a peptide recognized by at least one t cell clone isolated from memory cd4 + t cells. note that apl could not be calculated in the n-terminal ha region, corresponding to ha2 transmembrane region, since this is not included in the h1-ha crystal structures. (b) performance of in silico predictors was benchmarked by computing roc curves using the sets of h1-ha memory epitopes reported in a as true positives; the performance of each method was assessed using the auroc metric. shown are the maximal auroc values achieved by single predictors based uniquely on apl (orange bars) or mhc-ii binding (green bars) and the optimized combined predictor (black bars). (c) contribution of apl (orange) and mhc-ii binding (green) in the different combinations all resulting in the highest auroc values for each donor (hd1 and hd2, 0.75 ± 0.05; hd3 and hd4, 0.79 ± 0.05). the relative contribution of apl and mhc binding would differ across donors, since each one has a distinct hla background and a distinct set of peptides recognized by memory t cells. (d) apl (black curve) and peptide specificity (blues bars) of memory cd4 + t cell clones of each donor. (e) percentage of memory t cell clones for which the cognate peptide was identified by ms-based mhc-ii peptidomics (ms pept., orange bar) or by apl at different thresholds (green bars). each symbol represents a different donor. 1999; reith and mach, 2001) and, although they have limited endocytic potential, they can generate peptide ligands for mhc-ii presentation through endogenous degradation pathways, such as autophagy and macroautophagy (dengjel et al., 2005; schmid et al., 2007) . the analysis performed on memory t cell libraries and on ex vivo-stimulated memory t cells revealed the presence, in each individual, of one or two immunodominant sites targeted by polyclonal and clonally expanded t cells as well as a few subdominant sites. based on the findings from the analysis of the naive repertoire, this immunodominance cannot be explained by holes in the repertoire. importantly, our study shows that the immunodominant peptides correspond to those that are found most abundantly in the mhc-ii peptidome from h1-ha-pulsed dcs and identified also on h1-ha-specific b cells, although in the latter case, the assay had a limited sensitivity. these findings point to a simple model whereby immunodominance is determined by the abundance of a given peptide-mhc complex generated by processing followed by selection and clonal expansion of high avidity t cells. the functional avidity of memory t cell clones was on average 10-fold higher compared with that of naive t cell clones, consistent with the notion that high-avidity t cells are selected in the memory pool, as originally reported for mouse cd8 + t cells (busch and pamer, 1999; mcheyzer-williams et al., 1999; savage et al., 1999) . the memory repertoire contains also subdominant memory t cell clones that showed very high functional avidity when stimulated by peptides, but not by naturally processed h1-ha protein, suggesting that subdominance may be simply due to a lower abundance of the naturally processed peptide. collectively, our data reveal that only a small fraction of the peptides that bind to mhc-ii molecules and can be recognized by t cells are generated by antigen processing, and even in this case, the yield of processed peptide can vary ≥100-fold between immunodominant and subdominant peptides. previous studies using tetanus toxoid as a model antigen showed that antibodies can modulate antigen processing by enhancing or suppressing the generation of different t cell epitopes (simitsek et al., 1995; watts and lanzavecchia, 1993) . these findings are extended by the analysis of donor hd1, where a b cell clone with a bcr specific for the h1-ha globular head generates the same sets of immunodominant peptides as dcs, while a b cell clone specific for the h1-ha stem generates a different set of peptides, as demonstrated by peptidomics and activation of specific t cell clones. at this stage, we do not have a mechanistic explanation for these findings, since the structural characterization of the anti-stem antibody and anti-head antibody in complex with ha is not available, and the antigen used was an uncleaved ha0 that is not fusion competent. we can only speculate that by stabilizing a protein domain or by locking ha in the prefusion conformation, certain antibodies might change processing of ha by endosomal cathepsins, leading to decreased production of relevant t cell peptides (corti et al., 2011; lee and wilson, 2015) . further experiments using native antigens and well-characterized antibodies or b cells will be necessary to address the impact of anti-head and anti-stem antibodies in ha processing and presentation to t cells and its physiological relevance in the context of the response to influenza virus infection or vaccination. the high-resolution epitope mapping of naive and memory t cells from donors with a diverse mhc background offered us the possibility to benchmark currently available in silico predictors of cd4 + t cell immunogenicity. indeed, we found that, although being a prerequisite for recognition by t cells, peptide-mhc-ii binding affinity, either predicted in silico or measured in vitro, is a weak correlate of t cell recognition and in particular of immunodominance. along with the protein antigen expression level and subcellular localization, the position within the 3d structure of the native antigen may profoundly influence the amount of processed peptides (abelin et al., 2019; graham et al., 2018) . recent reports have shown that t cell epitopes from viral antigens tend to localize adjacent to highly flexible, surface-exposed regions of the protein that could act as sites of initial proteolytic cleavage (koblischke et al., 2017; landry, 2008; mirano-bascos et al., 2008) , suggesting that the physical accessibility within the tertiary structure is a requirement for efficient peptide release by proteases. by analyzing the 3d conformation of ha, we found that immunodominant epitopes are embedded in regions predicted as readily accessible targets of endosomal proteases. furthermore, combined in silico analysis of both peptide-mhc-ii binding affinity and apl yielded higher predictive values for the set of ha memory epitopes here described, thus indicating a possible strategy to develop more accurate predictive algorithms for t cell immunogenicity of protein antigens. in conclusion, the findings here reported suggest a model for epitope selection by antigen processing based on a trade-off between multiple factors, including the structural accessibility to proteases and the binding affinity of liberated peptides for the groove of mhc-ii molecules. structural constrains might define regions prone to be liberated at a higher rate by protease cleavage, thus being a property intrinsic of the antigen tridimensional structure, whereas the mhc-ii allelic background of each individual would select the final sequences of high-affinity binder peptides. the implications of this model are that highly accessible epitopes could be presented at high abundance on the apc surface even if they are not strong mhc-ii binders, and conversely, potentially strong binder peptides could never be presented at relevant amounts if they are not accessible to proteolytic liberation or if they are destroyed by cathepsin cut (resulting in crypticity). the net result of such complex processes would be the differential abundance of some mhc-ii presented peptides, which might drive more prominent clonal expansion of cognate naive t cells, leading to immunodominance. perturbation of the antigen structure, for instance by bound immunoglobulins, might further alter the substrate for endosomal proteolysis and therefore influence the antigen presentation and interaction with cognate t cells. cell purification and sorting serial blood samples from healthy donor 1 (hd1) vaccinated with inflexal v 2013/14 (crucell) and from blood donors from the swiss red cross were used in compliance with the federal office of public health (authorization no. a000197/2 to f. sallusto) and approval from the ethical committee of canton ticino (authorization no. 2018-02166/ce 3428). blood from hd2, hd3, and hd4 vaccinated with fluarix tetra 2015/16 (glaxosmithkline) was obtained from the surrey clinical research centre (university of surrey, uk). these studies were conducted in compliance with relevant local guidelines, approved by the london-surrey borders research ethics committee (reference 15/lo/1649) and registered on clinicaltrials.gov (reference nct02557802). written informed consent was obtained from all subjects participating in the study. peripheral blood mononuclear cells (pbmcs) were isolated with ficoll-paque plus (ge healthcare). monocytes were isolated from pbmcs by positive selection using cd14 magnetic microbeads (miltenyi biotech). cd14-depleted fractions were stained at 37°c for 15 min with a primary anti-human cxcr5 (clone 51505; mab190) from bio-techne, followed by staining with a biotinylated secondary goat anti-mouse igg 2b (1090-08) from southern biotech. after washing, cells were stained on ice for 30 min with pe/cy7-streptavidin (405206) from biolegend, and with the following fluorochrome-labeled mouse monoclonal antibodies: cd8-pe-cy5 (clone b9.11; a07758), cd25-pe-cy5 (clone b1.49.9; im2646) from beckman coulter, cd22-fitc (clone hib22; 555424) from bd biosciences, cd4-pe-texas red (clone s3.5; mhcd0417), cd45ra-qdot 655 (clone mem-56; q10069), cd95-percp-efluor 710 (clone dx2; 46-0959-42) from thermo fisher scientific, ccr7-bv421 (clone g043h7; 353208) from biolegend, and alexa fluor 647-conjugated goat anti-human igg (109-606-170) from jackson immunoresearch. naive and memory cd4 + t cells were sorted to >98% purity on a facsaria iii (bd) after exclusion of cd8 + , cd22 + , and cd25 bright cells. naive t cells were sorted as cd4 + cd45ra + ccr7 + cd95 − ; the remaining cd4 + t cells were sorted as total memory cells. in some experiments with donor hd1, total memory cd4 + t cells were divided in ctfh (sorted as cxcr5 + cells), tcm (sorted as ccr7 + cxcr5 − cells), and tem (sorted as ccr7 − cxcr5 − cells). igg + memory b cells and igg − b cells were sorted to >98% purity after gating on cd22 + cd4 − cd8 − cd25 − cells. cell culture t cells were cultured in rpmi 1640 supplemented with 2 mm glutamine, 1% (vol/vol) nonessential amino acids, 1% (vol/vol) sodium pyruvate, penicillin (50 u/ml), streptomycin (50 µg/ml; all from invitrogen), and 5% human serum (swiss red cross). for some experiments, medium was supplemented with il-2 (500 iu/ml). b cells were cultured in rpmi 1640 supplemented with 2 mm glutamine, 1% (vol/vol) nonessential amino acids, 1% (vol/vol) sodium pyruvate, penicillin (50 u/ml), streptomycin (50 µg/ml; all from invitrogen), and 10% fbs (hyclone, characterized, ge healthcare life science). sorted igg + memory b cells were immortalized with ebv and plated in single-cell cultures in the presence of cpg-dna (2.5 µg/ml) and irradiated pbmc-feeder cells, as previously described (traggiai et al., 2004) . 2 wk after immortalization, the culture supernatants were screened by high-throughput elisa for binding to h1-ha or h5-ha as described (pappas et al., 2014) . ebv-immortalized b cell (ebv-b) cell clones that resulted positive for binding to h1-ha and/or h5-ha were isolated and expanded. igg − b cells to be used as apcs for t cell libraries were expanded with cd40l according to an established protocol (zand et al., 2005) . autologous monocyte-derived dcs were generated by culture in complete medium containing 10% fbs (hyclone) supplemented with recombinant gm-csf (gentaur) and il-4 (immunotools), as previously described (sallusto and lanzavecchia, 1994) . sorted naive or memory cd4 + t cells were polyclonally stimulated with 1 µg/ml phytohemagglutinin (remel) in the presence of irradiated (45 gy) allogeneic feeder cells (5 × 10 4 per well) and il-2 (500 iu/ml) in a 96-well plate. the size of the library (number of wells and initial input of cells seeded per well) depended on the number of cells isolated from each donor. t cell lines were expanded as previously described (geiger et al., 2009) . library screening was performed 14-21 d after initial stimulation, by culturing thoroughly washed t cells (2.5 × 10 5 per well) with autologous irradiated b cells (2.5 × 10 4 per well), untreated or pulsed with a pool of ha overlapping peptides (2 µm per peptide) composed of 15mers (112 peptides, 15mers overlapping of 10) and 20mers (56 peptides, 20mers overlapping of 10) covering the h1-ha sequence. proliferation was assessed on day 4, after incubation for 16 h with 1 µci/ml [methyl-3 h] thymidine (perkinelmer). data were expressed as counts per minute. stringent criteria were used to score positive t cell lines based on two cutoff values: (1) a δcpm value ≥3 × 10 3 (cpm with antigen and apcs − cpm with apcs only) and (2) a stimulation index ≥3 (cpm with antigen and apcs ÷ cpm with apcs only). this threshold was chosen based on previous observations made across multiple negative and positive samples assessed by the t cell library technique and with a variety of donors and antigens (campion et al., 2014; geiger et al., 2009; latorre et al., 2018; lindestam arlehamn et al., 2013) . the specificity of positive cultures was confirmed in subsequent independent experiments of epitope mapping. precursor frequencies were calculated based on numbers of negative wells, assuming a poisson distribution (geiger et al., 2009) , and are expressed per million cells within each subset. inverse simpson index of diversity (1-d) was calculated for each donor by considering the number of individual t cell clones recognizing each particular h1-ha peptide (simpson, 1949) . the inverse simpson index (1-d) quantifies the richness and evenness of populations, ranges between 0 and 1, and can be interpreted as the probability that two h1-ha-specific t cell clones randomly selected from a repertoire will recognize different epitopes. isolation of h1-ha-specific t cell clones sorted memory cd4 + t cell subsets from donor hd1 were labeled with cfse and cultured at a ratio of 2:1 with irradiated autologous monocytes untreated or pulsed with inflexal v 2013/2014 (3 µg/ml). after 6 d, cells were stained with antibodies to cd25-pe (clone m-a251; 555432) from bd biosciences and icos-apc (clone c398.4a; 313510) from biolegend. proliferating activated t cells were facs-sorted as cfse lo cd25 + icos + and expanded in vitro in the presence of il-2 (500 iu/ml). to select haspecific t cells, inflexal v-reactive cfse lo cultures were relabeled with cfse and stimulated with irradiated autologous monocytes untreated or pulsed with recombinant h1-ha (5 µg/ ml). after 5 d, proliferating activated t cells were sorted as cfse lo cd25 + icos + and cloned by limiting dilution. in some experiments, positive cultures from t cell libraries were labeled with cfse and cultured at a ratio of 2:1 with irradiated autologous monocytes untreated or pulsed with h1-ha peptide pool (2 µm per peptide). after 5 d, proliferating activated t cells were sorted as cfse lo cd25 + icos + and cloned by limiting dilution. t cell clone reactivity was determined by stimulation with h1-ha peptide pool (2 µm per peptide) or recombinant h1-ha (5 µg/ml) in the presence of irradiated autologous monocytes or b cells as apcs. in some experiments, h1-ha peptides or recombinant h1-ha were titrated by serial dilution. epitope mapping was performed by stimulation of t cell clones with irradiated autologous ebv-b clones, untreated or prepulsed for 2-3 h with individual peptides (15mers overlapping of 10 or 20mers overlapping of 10) covering the entire sequence of h1-ha (2 µm per peptide). to determine mhc restriction of t cell clones, autologous apcs were pulsed for 4 h with recombinant h1-ha, washed extensively, and then cultured with t cells in the absence or presence of blocking anti-mhc-ii monoclonal antibodies produced in-house from hybridoma cell lines (anti-hla-dr, clone l243 from atcc, hb-55; anti-hla-dq, clone spvl3 [spits et al., 1983] ; anti-hla-dp, clone b7/21 [watson et al., 1983] ). in all experiments, proliferation was assessed on day 3, after incubation for 16 h with 1 µci/ml [methyl-3 h]thymidine (perkinelmer). data were expressed as counts per minute. ex vivo-sorted memory cd4 + t cell subsets and cfse lo fractions of inflexal v-stimulated memory cd4 + t cell subsets from donor hd1 were analyzed by deep sequencing. in brief, 2.5-5 × 10 5 t cells were centrifuged and washed in pbs, and genomic dna was extracted from the pellet using qiaamp dna micro kit (qiagen), according to manufacturer's instructions. genomic dna quantity and purity were assessed through spectrophotometric analysis. sequencing of tcr vβ cdr3 was performed by adaptive biotechnologies using the immunoseq platform. in brief, after a multiplex pcr reaction designed to target any cdr3 vβ fragments, amplicons were sequenced using the illumina hiseq platform. raw data consisting of all retrieved sequences of 87 nucleotides or corresponding amino acid sequences and containing the cdr3 region were exported and further processed. the assay was performed at deep level for ex vivosorted total memory cd4 + cells (detection sensitivity, 1 cell in 200,000) and at survey level for cfse lo inflexal v-reactive cultures (detection sensitivity, 1 cell in 40,000). each clonotype was defined as a unique productively rearranged tcr vβ nucleotide sequence; data processing was done using the productive frequency of reads provided by immunoseq analyzer v3.0. sequence analysis of rearranged tcr vβ genes of ha-specific t cell clones from donor hd1 was performed as previously described (latorre et al., 2018) . briefly, cdna from individual t cell clones was obtained by reverse transcription of total rna from 10 3 -10 4 cells per reaction. rearranged tcr vβ genes were pcr amplified using a forward primer pool targeting vβ genes and reverse primer pairing to c1-c2 β-chain constant region. sequence amplification was assessed through agarose gel electrophoresis; successfully amplified fragments were sequenced by sanger method, and tcr sequence annotation was performed by using imgt/v-quest algorithm (lefranc et al., 2009) . hla typing and peptide-mhc-ii binding affinity measurement hla genotype of the patients was determined by reverse sequence-specific oligonucleotide probes dna typing (labtype; one lambda) performed at the irccs san matteo hospital foundation (pavia, italy). affinity measurements of h1-ha 15mer peptides recognized by hla-dr-restricted t cell clones from donor hd1 to recombinant hla-drb1*01:01 or hla-drb1*08:01 molecules was performed by immunitrack (copenhagen, denmark), as previously described (justesen et al., 2009) . briefly, recombinant hla-drb1 isoforms were refolded in vitro in the presence of recombinant hla-dra and increasing concentrations of h1-ha 15mer peptides. titrated pan-hla-dr-binding epitope was used as positive control. after 24-h incubation at room temperature and ph 7, correctly folded heterotrimeric pmhc-ii complexes were detected by elisa; data were analyzed using graphpad prism 8 software. purification of mhc-ii presented peptides dcs generated from donor hd1 were pulsed for 2 h with 10 µg/ ml recombinant h1-ha at a cellular density of 3 × 10 6 cells/ml and matured overnight with 100 ng/ml lps (enzo life sciences) at a cellular density of 1 × 10 6 cells/ml. ha-specific ebv-b cell clones isolated from igg + memory b cells of each of the four donors were pulsed overnight with 200 ng/ml recombinant h1-ha at a cellular density of 5 × 10 6 cells/ml. mhc-ii complexes were purified from ∼3 × 10 7 ha-pulsed dcs or 10 9 ha-pulsed ebv-b cells with a protocol adapted from bassani-sternberg et al. (2015) . briefly, the b cells were lysed with 0.25% sodium deoxycholate, 1% octyl-β-d-glucopyranoside (sigma-aldrich), 0.2 mm iodoacetamide, 1 mm edta, and complete protease inhibitor cocktail (roche) in pbs at 4°c for 1 h. the lysates were cleared by 20-min centrifugation at 18,000 g at 4°c, and mhc-ii complexes were purified by immunoaffinity chromatography with the anti-hla-dr/dp/dq hb-145 monoclonal antibody produced in-house from hybridoma cell line iva12 (atcc, hb-145) and covalently bound to protein a sepharose beads (thermo fisher scientific). the cleared lysates were loaded three times into the affinity columns at 4°c and subsequently washed at 4°c with 10-column volumes of 150 mm nacl, 20 mm tris•hcl, ph 8.0 (buffer a); 10-column volumes of 400 mm nacl, 20 mm tris•hcl, ph 8; 10-column volumes of buffer a; and finally 10-column volumes of 20 mm tris•hcl, ph 8. the mhc-ii complexes were eluted at room temperature by addition of 500 µl of 0.1 m acetic acid, in total five elutions for each sample. small aliquots of each eluted fraction were analyzed by 12% sds-page to evaluate yield and purity of mhc-ii complexes. sep-pak tc18 (waters) cartridges were used for further separation of peptides from mhc-ii subunits. the cartridges were prewashed with 80% acetonitrile (acn) in 0.5% formic acid, followed by 0.2% tfa, and subsequently loaded three times with each fraction eluted from the immunoaffinity column. after loading, the cartridges were washed with 0.2% tfa, and the peptides were separated from the more hydrophobic mhc-ii chains by elution with 30% acn in 0.2% tfa. the peptides were further purified using a silica c18 column tip (harvard apparatus) and eluted again with 30% acn in 0.2% tfa. finally, the peptides were concentrated by vacuum centrifugation and resuspended in 2% acn, 0.1% tfa, and 0.5% formic acid for ms analysis. liquid chromatography-tandem ms and data analysis mhc-ii peptides were separated on an easy-nlc 1200 hplc system coupled online to a q exactive mass hf spectrometer via a nanoelectrospray source (thermo fisher scientific). peptides were loaded in buffer a (0.1% formic acid) on in-house packed columns (75-µm inner diameter, 50-cm length, and 1.9-µm c18 particles from dr. maisch) and eluted with a nonlinear 120-min gradient of 5-60% buffer b (80% acn and 0.1% formic acid) at a flow rate of 250 nl/min and a column temperature of 50°c. the q exactive was operated in data-dependent mode with a survey scan range of 300-1,650 m/z and a resolution of 60,000 at m/z 200. up to 10 most abundant isotope patterns with a charge ≥1 were isolated with a 1.8-th-wide isolation window and subjected to higher-energy c-trap dissociation fragmentation at a normalized collision energy of 27. fragmentation spectra were acquired with a resolution of 15,000 at m/z 200. dynamic exclusion of sequenced peptides was set to 30 s to reduce the number of repeated sequences. thresholds for the ion injection time and ion target values were set to 80 ms and 3e6, respectively, for the survey scans and 120 ms and 1e5 for the tandem ms scans. data were acquired using xcalibur software (thermo fisher scientific). maxquant software was used to analyze ms raw files. tandem ms spectra were searched against the a/california/07/2009 (h1n1) ha sequence (uniprotkb: a0a075exw1), the bovine uniprot fasta database, the human uniprot fasta database, and a common contaminants database (247 entries) by the andromeda search engine (cox et al., 2011) . n-terminal acetylation and methionine oxidation were set as variable modifications; no fixed modifications were selected; the enzyme specificity was set to unspecific, with a minimum peptide length of 8 aa. a false discovery rate of 1% was required for peptides. peptide identification was performed with an allowed precursor mass deviation of ≤4.5 ppm and an allowed fragment mass deviation of 20 ppm; "match between runs" option was disabled. the ms proteomics data have been deposited to the proteomexchange consortium via the pride (perez-riverol et al., 2019) partner repository with the dataset identifier pxd018151. in silico analysis mhc-ii binding affinity of each theoretical h1-ha-derived 15mer peptide was calculated using the iedb tool for mhc-ii binding prediction (http://tools.iedb.org/mhcii/; paul et al., 2015) . donor-tailored analyses were performed using iedb's recommended method and considering the set of mhc-ii alleles carried by each donor at the following loci: hla-drb1, hla-drb3/4/5 (if associated), hla-dqa1/dqb1 in cis-or transpairing, and hla-dpa1/dpb1 in cis-or trans-pairing. top scoring h1-ha 15mer peptides for each donor were selected based on percentile rank calculated by comparison to a large set of random natural peptides. apl was computed as described in mettu et al. (2016) . briefly, an aggregate z-score of conformational stability was determined for each h1-ha residue by integrating four structural parameters obtained from the 3d structure of postfusion ha resolved by x-ray diffraction (pdb codes: 3lzg for ha1 domain [xu et al., 2010] ; 1htm for ha2 domain in the postfusion conformation [bullough et al., 1994] ). the z-score statistic was then used to calculate an apl for each theoretical h1-ha 15mer peptide, following the rationale that the liberation of antigenic peptides might be facilitated by surrounding unstable regions that are readily unfolded and targeted by endosomal proteases. for the optimization of combined predictors, we systematically performed peptide binding affinity predictions of each theoretical h1-ha 15mer peptide using iedb for the mhc-ii alleles carried by each donor, considering for each peptide the best scoring affinity within each group of mhc-ii alleles. using the set of epitopes recognized by memory t cells of each donor as true positives, we computed roc curves for each predictive model and calculated the corresponding auroc. combined predictors for each donor were then built by iteratively weighting the contributions of epitope likelihood based on structural accessibility and peptide binding affinity to mhc-ii, until we could maximize the auroc value. statistical analyses were performed using graphpad prism 8 software or r software v3.5.1. ec 50 (ng/ml) and k d (ng/ml) values were calculated by nonlinear regression curve fit (4pl with automatic outlier elimination) using graphpad prism 8 software. significance was assigned at p < 0.05, unless stated otherwise. specific tests are indicated in the figure legends for each comparison. online supplemental material fig. s1 shows in representative t cell clones blocking experiments with anti-hla-dr, anti-hla-dp, and anti-hla-dq antibodies to determine mhc-ii restriction. it also shows mhc-ii binding affinity of h1-ha peptides recognized by hla-dr-restricted t cell clones from donor hd1 as measured in vitro. fig. s2 shows the ability of a panel of h1-ha-reactive t cell clones to cross-react to has from different influenza a strains. fig. s3 shows the functional avidities for peptide and naturally processed h1-ha of t cell clones isolated from the memory or the naive compartment determined by stimulation with titrated doses of peptides or recombinant h1-ha. fig. s4 reports an analysis of mhc-ii eluted h1-ha peptides measured by ms and of mhc-ii h1-ha presented peptides to t cell clones of anti-head or anti-stem ebv-b cell clones. fig. s5 reports the theoretical h1-ha 15mer peptide predicted to bind to selected mhc-ii alleles using the iedb tool and a the number of iedb-predicted peptides and mhc-ii eluted peptides measured by ms-based peptidomics found to be recognized by t cells. table s1 shows epitope mapping of h1-ha-reactive t cell clones isolated from memory cd4 + t cell subsets of donor hd1. table s2 shows tcr vβ sequence and epitope specificity of h1-ha-reactive t cell clones isolated from cd4 + memory (tcm, tem, or ctfh) t cell compartment. table s3 shows hla class ii typing of the four hds included in this study. table s4 shows tcr vβ sequence and epitope specificity of h1-ha-reactive t cell clones isolated from the cd4 + naive t cell compartment. provided online are six tables. table s1 shows epitope mapping of h1-ha-reactive t cell clones isolated from memory cd4 + t cell subsets of donor hd1. table s2 shows tcr vβ sequence and epitope specificity of h1-ha-reactive t cell clones isolated from cd4 + memory (tcm, tem, or ctfh) t cell compartment. table s3 shows hla class ii typing of the four hds included in this study. table s4 shows tcr vβ sequence and epitope specificity of h1-ha-reactive t cell clones isolated from the cd4 + naive t cell compartment. table s5 lists h1-ha peptides identified by ms-based mhc-ii peptidomics in donor hd1. table s6 lists h1-ha peptides identified by ms-based mhc-ii peptidomics in donors hd2-hd4. figure s5 . peptide binding to mhc-ii is a necessary but not sufficient condition to define immunodominance. mhc-ii binding affinity of each theoretical h1-ha 15mer peptide was calculated using the iedb tool for mhc-ii binding prediction (http://tools.iedb.org/mhcii/). personalized analyses were performed by considering the mhc-ii alleles carried by each donor (hla-drb1, hla-drb3/4/5, hla-dqa1/dqb1 in cis-or trans-pairing, hla-dpa1/dpb1 in cis-or transpairing). top scoring h1-ha 15mer peptides for each donor were selected based on percentile rank calculated by comparison to a large set of random natural peptides. (a) sets of h1-ha peptides predicted as mhc-ii binders at different thresholds for each donor. the x axis indicates h1-ha amino acid sequence. the sets of top predicted mhc-ii binder peptides are reported as color-coded segments. the immunodominant regions targeted by memory cd4 + t cells of each donor are reported with color-coded shadows. (b and c) iedb-predicted peptides and mhc-ii eluted peptides measured by ms-based peptidomics defined discrete h1-ha regions. the tables summarize the number of ha regions found presented on mhc-ii by ms-based peptidomics, or predicted as mhc-ii binders in different donors. the corresponding number of ha epitopes recognized by at least one t cell clone regardless of the subset of origin (b) or isolated from the memory compartment (c) are reported. identification of immunodominant epitopes is marked by an asterisk. (d) sensitivity was evaluated in terms of percentage of memory t cell clones for which the cognate peptide was identified by ms-based mhc-ii peptidomics (ms pept., orange bar) or by mhc-ii binding predictions (iedb) at different thresholds (iedb, green bars). each symbol represents a different donor. advanced immunization technologies). f. sallusto and the institute for research in biomedicine are supported by the helmut horten stiftung. author contributions: a. cassotta: conceptualization, methodology, validation, investigation, data curation, formal analysis, visualization, software, writing -original draft lewis: resources, writingreview & editing. a. lanzavecchia: writing -original draft, writing -review & editing. f. sallusto: supervision, conceptualization, project administration, data curation, visualization, funding acquisition, writing -original draft defining hla-ii ligand processing and binding rules with mass spectrometry enhances cancer epitope prediction is it possible to develop a "universal" influenza virus vaccine? outflanking antibody immunodominance on the road to universal influenza vaccination mass spectrometry of human leukocyte antigen class i peptidomes reveals strong effects of protein abundance and turnover on antigen presentation structure of influenza haemagglutinin at the ph of membrane fusion t cell affinity maturation by selective expansion during infection proteome-wide analysis of hiv-specific naive and memory cd4(+) t cells in unexposed blood donors a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins andromeda: a peptide search engine integrated into the max-quant environment autophagy promotes mhc class ii presentation of peptides from intracellular source proteins human parainfluenza virus type 3 up-regulates major histocompatibility complex class i and ii expression on respiratory epithelial cells: involvement of a stat1-and ciitaindependent pathway human naive and memory cd4+ t cell repertoires specific for naturally processed antigens analyzed using libraries of amplified t cells antigen discovery and specification of immunodominance hierarchies for mhcii-restricted epitopes the role of naive t cell precursor frequency and recruitment in dictating immune response magnitude improved methods for predicting peptide binding affinity to mhc class ii molecules functional recombinant mhc class ii molecules and high-throughput peptide-binding assays determinants of immunodominance for cd4 t cells protein structure shapes immunodominance in the cd4 t cell response to yellow fever vaccination is it possible to develop a "universal" influenza virus vaccine? potential target antigens and critical aspects for a universal influenza vaccine three-dimensional structure determines the pattern of cd4+ t-cell epitope dominance in influenza virus hemagglutinin t cells in patients with narcolepsy target self-antigens of hypocretin neurons persistent antibody clonotypes dominate the serum response to influenza over multiple years and repeated vaccinations structural characterization of viral epitopes recognized by broadly cross-reactive antibodies imgt, the international immunogenetics information system memory t cells in latent mycobacterium tuberculosis infection are directed against three antigenic islands and largely contained in a cxcr3+ccr6+ th1 subset clonal selection of helper t cells is determined by an affinity threshold with no further skewing of tcr binding properties evolution of antigen-specific t cell receptors in vivo: preimmune and antigen-driven selection of preferred complementaritydetermining region 3 (cdr3) motifs hla-dm and hla-do, key regulators of mhc-ii processing and presentation cd4+ t-cell epitope prediction using antigen processing constraints antigen structure influences helper t-cell epitope dominance in the human immune response to hiv envelope glycoprotein gp120 unconventional recognition of peptides by t cells and the implications for autoimmunity naive cd4(+) t cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude t cell receptor cross-reactivity between similar foreign and self peptides influences naive cell population size and autoimmunity rapid development of broadly influenza neutralizing antibodies through redundant mutations development and validation of a broad scheme for prediction of hla class ii restricted t cell epitopes the pride database and related tools and resources in 2019: improving support for quantification data the bare lymphocyte syndrome and the regulation of mhc expression the ins and outs of mhc class ii-mediated antigen processing and presentation efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/ macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha from vaccines to memory and back the relationship between immunodominance, dm editing, and the kinetic stability of mhc class ii:peptide complexes antigen-loading compartments for major histocompatibility complex class ii molecules continuously receive input from autophagosomes dominance and crypticity of t cell antigenic determinants modulation of antigen processing by bound antibodies can boost or suppress class ii major histocompatibility complex presentation of different t cell determinants measurement of diversity characterization of monoclonal antibodies against cell surface molecules associated with cytotoxic activity of natural and activated killer cells and cloned ctl lines virus-specific cd4(+) memory-phenotype t cells are abundant in unexposed adults an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus variations in mhc class ii antigen processing and presentation in health and disease peptide binding predictions for hla dr, dp and dq molecules detection of a novel human class ii hla antigen suppressive effect of antibody on processing of t cell epitopes continuing challenges in influenza structural basis of preexisting immunity to the 2009 h1n1 pandemic influenza virus confronting complexity: real-world immunodominance in antiviral cd8+ t cell responses immunodominance in major histocompatibility complex class i-restricted t lymphocyte responses immunodominance in tcd8+ responses to viruses: cell biology, cellular immunology, and mathematical models a renewable source of donor cells for repetitive monitoring of t-and b-cell alloreactivity memory cd4 t cells in influenza proliferation was assessed on day 3 after a 16-h pulse with [ 3 h]thymidine and expressed as counts per minute. mhc-ii restriction was defined based on inhibition of t cell proliferation >80%. shown are representative (30 of 85 analyzed) t cell clones specific for immunodominant (a) or subdominant (b) h1-ha epitopes. data are grouped based on the epitope specificity of the t cell clones tested, reported on top of each plot. (c) summary of mhc-ii restriction of h1-ha-specific t cell clones isolated from donor hd1. the x axis indicates h1-ha amino acid sequence; each color-coded segment represents the peptide recognized by t cell clones restricted by hla-dp (pink), hla-dq (blue), or hla-dr (green). the immunodominant h1-ha 401-430 region identified in the memory compartment of donor hd1 is highlighted with a red shadow. (d) mhc-ii binding affinity of h1-ha peptides recognized by hla-dr-restricted t cell clones from donor hd1 was measured in vitro. briefly, recombinant hla-drb1 isoforms were refolded in the presence of recombinant hla-dra and increasing concentration of peptides, at room temperature and ph 7. k d values were calculated by nonlinear regression fitting of pmhc-ii refolding curves we thank the blood donors for their participation in the study. we thank david jarrossay for cell sorting, sandra jovic and chiara silacci for technical assistance, and the servizio tipizzazione of the fondazione irccs policlinico san matteo, pavia, italy, for hla typing.this study has been carried out with financial support key: cord-023675-sidvbzqy authors: oldstone, michael b.a. title: virus-induced autoimmunity: molecular mimicry as a route to autoimmune disease date: 2013-11-17 journal: t-cell activation in health and disease doi: 10.1016/b978-0-12-252682-4.50023-8 sha: doc_id: 23675 cord_uid: sidvbzqy nan viruses are implicated in autoimmunity on the basis of three findings. first, autoimmune responses are made de novo, or those already present are enhanced concomitant with infection by a wide variety of human dna and rna viruses. this point is strengthend by the second finding that, in experimental animals, both acute and persistent virus infections can induce, accelerate, or enhance autoimmune responses and cause autoimmune disease. for example, we have shown that autoimmune manifestations normally present in nzb mice (anti-dna antibodies, antired blood cell antibodies) or their (nzb x w)fj relatives (anti-dna antibodies) are enormously enhanced by persistent infection with dna (polyoma) or rna (lymphocytic choriomeningitis, lcmv) viruses; that is, antibodies form earlier and reach for higher titers in the infected mice than in their uninfected counterparts [1, 2] . further, the nzw mice, which normally do not develop these autoimmune responses, do so upon polyma or lcmv infections. indeed, the responses in nzb, (nzb x w)fj or nzw mice are so marked that autoimmune diseases occur at a higher incidence with earlier time of death [nzb, (nzb x w)fj or appear de novo (nzw) [2, 3] . a number of viruses, including retroviruses, are now known to perform similarly. third, by evaluating molecular mimicry in human autoimmune disorders (figure 1) , we have uncovered a number of potential etiologic agents and mechanisms of autoimmune disease [4] . viruses can induce autoimmune responses in many ways. for example, certain viruses have a mitogenic affect on unique blood lymphocyte subsets and hence can act as polyclonal activators. viruses also direct the release of lymphokines and monokines. these molecules are important modulators of immune responses by acting directly as growth or differentiation factors or by regulating the expression of class i and class ii major histocompatibility molecules (i.e. interferon effect). viruses can also replicate selectively in particular lymphocyte subsets. by their presence, activation or replication they can cause immunosuppression or immunoenhancement (reviewed in [5, 6] ). moreover, some viruses and other microbes contain chemical structural components that mimic normal host 'self proteins. an effector immune response, either b (humoral) or t (cytotoxic t cell), directed against the microbe might than also cross-react with 'self protein, thereby evoking autoimmunity. the events of this scenario, termed molecular mimicry, comprise the body of this report. we define molecular mimicry as similar structures shared by molecules from dissimilar genes or by their protein products. either the molecules' linear amino acid sequences or their conformational fit may be shared, even though their origins are as separate as, for example, a virus and a normal host self determinant. because guanine-cysteine (gc) sequences and introns designed to be spliced away may provide, respectively, false hybridization signals and nonsense homologies, molecular mimicry must be analyzed at the protein level. such homologies between proteins have been detected either by use of immunologic reactants, humoral or cellular, that cross-react with two presumably unrelated protein structures, or by matching proteins described in computer storage banks. regardless of the methods used for identification, it is now abundantly clear that molecular mimicry is common between proteins encoded by numerous dna and rna viruses and host 'self proteins. such events are relevant not only to autoimmunity but also as a likely mechanism by which viral proteins are processed inside cells [7] . examples of molecular mimicry were first described as such in the early 1980s by investigators who found that monoclonal antibodies against sv40 t antigens crossreacted with host cell proteins [8] . however, the importance of this observation became apparent when others realized that the monoclonal antibodies against a battery of viruses were cross-reacting with host determinants. for example, we showed cross-reactivity between measles virus phosphoprotein (72 k molecular weight) and the cytoskeleton component keratin (54 k molecular weight), and between a herpes simplex virus glycoprotein of 140 k and a separate epitope on keratin from that recognized by the measles virus phosphoprotein [9] . further, we virus-induced autoimmunity 189 'immunochemical or immunofluorescent evidence of cross-reactivity, speculation on disease on. [7] found homology between vaccinia virus hemagglutinin and the cytoskeleton protein vimentin. to determine the possible frequency of molecular mimicry, srinivasappa and his colleagues [10] tested over 600 monoclonal antibodies obtained from several laboratories, all raised against viral polypeptides. these investigators then charted the incidence of the monoclonals' cross-reactivity with host proteins expressed in a large panel of normal tissues. in their analysis and our subsequent studies monoclonal antibodies were reactive with 14 different viruses, including such commonly found representatives dna and rna viruses as the herpesvirus group, vaccinia virus, myxoviruses, paramyxoviruses, arenaviruses, flaviviruses, alphaviruses, rhabdoviruses, coronaviruses, and human retroviruses. most important, over 4% of such monoclonals cross-reacted with host-cell determinants expressed on uninfected tissues. some of these monoclonal antiviral antibodies reacted with constituents of more than one organ. hence, from these data, it is clear that molecular mimicry is common and not restricted to any specific class or group or virus. these and other reported cross-reactivities (table 1) raise interesting questions about the etiology and pathogenesis of a number of autoimmune diseases. because the studies described above indicate that many viruses share antigenic determinants (linear or conformation) with normal host proteins, the next step was to determine experimentally whether molecular mimicry could elicit autoimmune diseases. myelin basic protein was chosen as the host component to study because its entire amino acid sequence is known, and its encephalitogenic site of 8-10 amino acids has been mapped in several animal species. by computer-assisted analysis, several viral proteins listed in the dayhoff files showed significant homology with the encephalitogenic site of myelin basic protein. included were similarities and/or fits between myelin basic protein and the nucleoprotein and hemagglutinin of influenza virus, coat protein of polyoma virus, core protein of the adenovirus, polyprotein of poliomyelitis virus, ec-lf2 protein of epstein-barr virus (ebv), hepatitis b virus polymerase (hbvp), and others. however, the best fit occurred between the myelin basic protein encephalitogenic site in the rabbit and hbvp (figure 2 ). replicates, production of a viral determinant in common with that of the host might continue. this would allow initiation of an immune response and/or autoimmunity, either one leading to cyclic, chronic, or progressive disease. in any case, molecular mimicry would occur only when the virus and host determinants are sufficiently similar to induce a cross-reactive response yet different enough to break b or t-cell immunologic tolerance. lastly, evidence for the hypothesis that molecular mimicry causes autoimmune disease in humans emanates from studies on pathogenesis of the non-rheumatoid arthritides, ankylosing spondylitis (as) and reiter's syndrome (rs) [12] [13] [14] [15] , and on myasthenia gravis [15, 16] . six consecutive amino acids (qtdred) are identical between the hypervariable domain of hla-b27 and k. pneumoniae nitrogenase ( table 2) . sera from a significant proportion of hla-b27 individuals with rs (18 of 34) or as (7 of 24), but not from appropriate controls, reacted with a synthetic peptide containing the homologous region of hla-b27.1 and k. pneumoniae nitrogenase [12] . these results with american patients were confirmed by analysis of an additional 27 sera obtained from the netherlands [ivani and oldstone, unpublished results, 1988] . as expected, sera from the latter as and rs patients also reacted with a peptide derived from the k. pneumoniae nitrogenase (cnsrqttxriideli). chen et al. [17] found that antibody to the hypervariable region of hla-b27.1 is reactive with yersinia (strain-pseudo tuberculosis), another microbe implicated in the as/rs arthritides. these observations suggest that rs and as are autoimmune diseases, with hla-b27 serving as the autoantigen. induction would be caused by a microbe(s) encoding a protein with sequence homology to hla-b27 (variable region), and the disease would presumably be related to an unusual concentration of hla antigen in certain tissues-like joints. to address this latter issue, material was obtained from the joints of patients with as and others with non-as arthropathies. accordingly, joint tissue from 11 of 12 as patients studied heavily expressed hla-b27 sequences, i.e. qtdred occurred in synovial cells lining the joint space and in endothelial cells in synovial tissue [13] . in contrast, these sequences were not observed either in synovial tissue of individuals with non-arthropathies or in endothelial and other cells obtained by skin biopsy from non-as but hla-b27 positive individuals. the large majority of patients with the autoimmune disease, myasthenia gravis, characteristically have detectable antibodies against the acetylcholine (achr). the nicotinic achr is composed of multiple subunits responsible for gating ion flow across membranes in response to binding of the neurotransmitter, acetylcholine. the actions of achr antibodies, either experimentally induced or spontaneous, lead to the numerical reduction of available achr and prevention of the neuromuscular junction's ability to transmit signals from nerve fibers to muscle fibers; medically, the outcome is myastheia gravis. for molecular dissection of the physiologically important binding sites, synthetic peptides representing unique regions of the achain of achr have been used in association with a-bungarotoxin to compete with the binding of antibody specific for achr. these interactions also map accessible sites on achr that bind antibodies. utilizing the strategy outlined in figure 1 , we found immunochemically significant cross-reactivity with the human achr a-chain amino acid sequences 160-167 and herpes simplex virus glycoprotein d residues 286-293 (table 2) . for example, antibodies to the herpes simplex virus peptide reacted with both the corresponding achr peptide and with the achr native protein. interestingly, a different herpes simplex virus glycoprotein sequence amino acid 381-389 also showed several similar amino acid sequences, but on immunochemical analysis failed to raise antibodies that cross-reacted with the achr sequence [15, 16] . affinity purification of antibodies from patients with myasthenia gravis, using the human achr a-chain 157-170 peptide immobilized on thiopropyl-sepharose, yielded igg antibodies that bound to the native achr and inhibited the binding of a-bungarotoxin to its specific binding site on the receptor. thus, the human achr a-chain 160-167 peptide specifically cross-reacts with a shared homologous domain on herpes simplex virus glycoprotein d, residues 286-293, by binding and inhibition studies, and elicits antibodies in myasthenic patients that bind to the native achr protein; these antibodies are capable of causing a biologic effect. immunologic cross-reactivity of this 'self epitope with herpes simplex virus suggests that this virus may be associated with some cases of myasthenia. the probability that a random six amino acid sequence will be identical in two dissimilar proteins is 1 in 20 6 , assuming that all amino acids are represented equally. nevertheless, the finding of sequence homology is, by itself, insufficient evidence of biologically meaningful mimicry. two examples illustrate this point. first, like the k. pneumoniae nitrogenase, the ebv bblf protein shares six continuous amino acids with hla-b27, but the homology is in the conserved and not the variable domain of the hla-b27.1 molecule. no immunochemically identifiable cross-reactivity occurs between hla-b27-specific sequences and ebv peptides containing the homologous sequence. further, over 50 patients with as or rs and of the hla-b27 haplotype had no antibodies to the ebv peptides [12, 14] . thus, whether or not the homology reflects the biologically meaningful domain is important. second, homology alone may not lead to a cross-reacting immune response, especially if the dissimilar amino acid(s) represents a radical substitution or interferes with binding. for example, despite a high degree of similarity between portions of the achr a-chain (pesdqpdl) and polyoma virus middle t antigen (pesdqdql), no cross-reacting antibodies form. yet a more distant similarity between the achr sequence and herpes simplex virus glycoprotein d (pnatqpel) induces strong immunologic cross-reactivity [15] . just as homologies and immunologic cross-reactivities have been found between the hla-b27 variable domain and k. pneumoniae nitrogenase, between the human achr and herpes simplex virus glycoprotein, and between other host and microbial proteins ( table 2) , additional similarities will surely emerge as more genes and proteins are analyzed. some of these examples may account for diseases in terms of an autoimmune response provoked by molecular mimicry. however, unless the homology and subsequent immunologic cross-reactivity involve a host protein that can precipitate disease (e.g. the restricted encephalitogenic site of myelin, and immunodominant domain of the insulin receptor or achr), the autoimmune response is unlikely to lead to autoimmune disease. virus-induced autoimmune disease: viruses in the production and prevention of autoimmune disease the effect of induced chronic viral infections on the immunologic diseases of new zealand mice host igg and c3 deposits in the choroid plexus during spontaneous immune complex disease molecular mimicry and autoimmune disease viruses perturb lymphocyte functions: selected principles characterizing virus-induced immunosuppression virus-induced immunosuppression: infections with measles virus and human immunodeficiency virus serologic relatedness between thyl.2 and actin revealed by monoclonal antibody sv40 large t shares an antigenic determinant with cellular protein of molecular weight 68,000 molecular mimicry in virus infection: cross-reaction of measles phosphoprotein or oldstone of herpes simplex virus protein with human intermediate filaments molecular mimicry: frequency of reactivity of monoclonal antiviral anitbodies with normal tissues amino acid homology and immune responses between the encephalitogenic site of myelin basic protein and virus: a mechanism for autoimmunity autoantibodies to hla-b27 in the sera of hla-b27 patients with ankylosing spondylitis and reiter's syndrome: molecular mimicry with klebsiella pneumoniae as potential mechanism of atuoimmune disease hla-b27 related antigens in articular tissues of patients with ankylosing spondylitis autoimmune pathogenesis for ankylosing spondylitis (as) and reiter's syndrome (rs): autoantibodies against an epitope shared by hla-b27 and klebsiella pneumoniae nitrogenase in sera of hla-b27 patients with as and rs peptides as probes to study molecular mimicry and virus induced autoimmunity molecular mimicry and myasthenia gravis: uncovering a novel site of the acetylcholine receptor that has biologic activity and reacts immunochemically with herpes simplex virus this is publication number 5566-imm from the department of immunology, scripps clinic and research foundation, la jolla, ca 92037.this work was supported in part by usphs grants ai-07007, ns-12428, and ag-04342. key: cord-016235-2lhrkmrv authors: roden, anja c.; tazelaar, henry d. title: lung date: 2010-05-17 journal: pathology of solid organ transplantation doi: 10.1007/978-3-540-79343-4_7 sha: doc_id: 16235 cord_uid: 2lhrkmrv experiments with animals in the 1940 and 1950s demonstrated that lung transplantation was technically possible [33]. in 1963, dr. james hardy performed the first human lung transplantation. the recipient survived 18 days, ultimately succumbing to renal failure and malnutrition [58]. from 1963 through 1978, multiple attempts at lung transplantation failed because of rejection and complications at the bronchial anastomosis. in the 1980s, improvements in immunosuppression, especially the introduction of cyclosporin a, and enhanced surgical techniques led to renewed interest in organ transplantation. in 1981, a 45-year-old-woman received the first successful heart–lung transplantation for idiopathic pulmonary arterial hypertension (ipah) [106]. she survived 5 years after the procedure. two years later the first successful single lung transplantation for idiopathic pulmonary fibrosis (ipf) [128] was reported, and in 1986 the first double lung transplantation for emphysema [25] was performed. experiments with animals in the 1940 and 1950s demonstrated that lung transplantation was technically possible [33] . in 1963, dr. james hardy performed the first human lung transplantation. the recipient survived 18 days, ultimately succumbing to renal failure and malnutrition [58] . from 1963 through 1978, multiple attempts at lung transplantation failed because of rejection and complications at the bronchial anastomosis. in the 1980s, improvements in immunosuppression, especially the introduction of cyclosporin a, and enhanced surgical techniques led to renewed interest in organ transplantation. in 1981, a 45-year-old-woman received the first successful heart-lung transplantation for idiopathic pulmonary arterial hypertension (ipah) [106] . she survived 5 years after the procedure. two years later the first successful single lung transplantation for idiopathic pulmonary fibrosis (ipf) [128] was reported, and in 1986 the first double lung transplantation for emphysema [25] was performed. over the following years, the number of lung transplants rapidly increased, and the operation became an accepted treatment for an end-stage lung disease. today, there are four major surgical approaches to lung transplantation: single and bilateral lung transplantation (blt), heart-lung transplantation, and transplantation of lobes of lungs from living donors. in 2007, 2,708 lung transplantation procedures were reported worldwide to the registry of the international society for heart and lung transplantation (ishlt) in adults, the highest number for any year until then [21] . in the same year, 93 lung transplantations were reported in children, the majority in adolescents (12-17 years old) [6] . although the number of single lung transplantations has been relatively stable, blts have continuously increased within the past 15 years. in fact, in 2007, blt was the most common lung transplantation procedure performed with 69% of all lung transplantation procedures, largely due to transplantation for cystic fibrosis and chronic obstructive lung disease/ emphysema which made up for 26.6 and 25.7% of all blts between 1995 and 2008 [21] . the mean age of transplant recipients has consistently increased since 1989 rising to an all time high of 50.8 years in 2008 [21] . the most common indications for lung transplantation in adults are chronic obstructive pulmonary disease (copd)/emphysema, ipf, cystic fibrosis and alpha-1 antitrypsin deficiency emphysema (aat) (see table 7 .1) [21] . indications for pediatric lung transplantation vary by age (see table 7 .1). in children over 5 years old, cystic fibrosis is the most common indication [6] , followed by ipah. in contrast, in infants and preschool children, lung transplantations are usually performed for ipah, congenital heart disease, idiopathic interstitial pneumonitis, and surfactant protein deficiency. well-selected patients with systemic diseases such as sarcoidosis, lymphangioleiomyomatosis, and pulmonary langerhans' cell histiocytosis have also had satisfactory results after lung transplantation [27, 71, 91, 99, 119] as have selected patients with scleroderma [84, 110, 113] . multiple cases of incidental t1n0m0 or even stage iiia non-small cell carcinoma in the excised native lungs of transplant recipients have been reported [14, 30, 124] . although one patient with stage iiia poorly differentiated squamous cell carcinoma died 6 months after transplantation of a neoplastic thromboembolus, patients with t1n0m0 carcinoma are generally free of recurrence. currently, only patients with near end-stage lung disease and a limited life expectancy should be considered for lung transplantation [95] . however, since lung transplantation is a rapidly evolving field, there are no hard and fast rules about who may be transplanted. when choosing a transplantation procedure, several issues are considered including the shortage of organ donors, the original disease, and the center's experience with graft and patient survival. general guidelines for the selection of the procedure have been proposed [36] and are based on the nature of the underlying lung disease. while blts are mandatory for cystic fibrosis [6, 21] ipf idiopathic pulmonary fibrosis; aat alpha1-antitrypsin deficiency; ipah idiopathic pulmonary arterial hypertension; lam lymphangioleiomyomatosis; ob obliterative bronchiolitis [35] , this procedure has also become more popular for indications such as aat, copd, ipf, and ipah. singlelung transplantation is usually performed in patients with restrictive fibrotic lung disease, eisenmenger syndrome with reparable cardiac anomaly, and older patients with copd. heart-lung transplantation is considered in patients with eisenmenger syndrome with irreparable cardiac defect, pulmonary hypertension with cor pulmonale, or end-stage lung disease with concurrent severe cardiac disease [83, 89] . transplantation of lobes from living donors is a recently developed technique involving bilateral implantation of the lower lobes usually from two blood group-compatible living donors. the procedure has been performed in patients with cystic fibrosis, although the indications have been recently broadened. the functional and survival outcomes are similar to those achieved with conventional transplantation of cadaveric lungs. donation of a lobe decreases the donor's lung volume by an average of approximately 15%, which is not associated with long-term functional limitation. other factors of the recipient that must be taken into consideration on an individual basis include ventilator dependence, previous cardiothoracic surgery, and preexisting medical conditions (e.g., hypertension, diabetes mellitus, osteoporosis) since posttransplantation medical regimen can worsen these illnesses. severe coronary artery disease is a contraindication to lung transplantation. however, coronary artery bypass grafting at the same time as lung transplantation has been performed with a reasonably good outcome in some centers, although less invasive preoperative interventions, such as percutaneous transluminal coronary angioplasty and stenting, are preferred. although the donor selection criteria may vary amongst centers, generally acceptable donor criteria include age of donor <65 years for lung transplantation and <45 years for heart-lung transplantation. in 2008, the average donor age was 35.5 years [21] . other donor criteria include the absence of severe chest trauma or infection, no prolonged cardiac arrest (heart-lung transplantation only), minimal pulmonary secretions, negative screens for hiv, hepatitis c, and hepatitis b and blood type (abo) compatibility. a close match of lung size between donor and recipient, pao 2 > 300 mmhg on 100% fraction of inspired oxygen (fio 2 ), clear chest radiograph and no history of malignant neoplasms are also required. most transplant centers will use lungs from a cytomegalovirus (cmv)-positive donor for transplantation into a cmv-negative donor given an adequate postoperative cmv prophylaxis. with the current techniques, satisfactory graft function can be obtained after an ischemic interval of as long as 6-8 h. for pulmonary preservation, systemic heparinization of the donor and hypothermic flush perfusion of the allograft are most commonly used in clinical practice. most flush solutions are administered at a temperature of 4°c, while topical cooling is carried out by filling the pleural cavity with iced crystalloid solution. the harvested lungs are then immersed in crystalloid solution, packed in ice, and transported at a temperature of 1-4°c. the infusion and transport is performed during active ventilation and static inflation with o2, respectively. acute and chronic alloreactive injury to the donor lung affects both the vasculature and the airways [123] . usually, rejection is evaluated on transbronchial biopsies (see below sect. 7.3). on only rare occasions, wedge biopsies are performed. other specimens might include explants for retransplant or autopsy specimens. acute rejection is characterized by perivascular mononuclear cell infiltrates, which may be accompanied by sub-endothelial chronic inflammation (e.g., endotheliitis or intimitis), and also by lymphocytic bronchiolitis. in contrast, chronic rejection is manifest by fibrous scarring, involving the bronchioles and sometimes associated with accelerated fibrointimal changes affecting pulmonary arteries and veins. the presence of presumed irreversible dense eosinophilic hyaline fibrosis in airways and vessels remains the key histologic discriminator between acute and chronic rejection of lung. the histologic changes are divided into grades based on intensity of the cellular infiltrate, and the presence and absence of fibrosis. hyperacute rejection occurs within minutes to a few hours after the newly transplanted organ begins to be perfused. it is a type ii hypersensitivity reaction, mediated by preexisting antibodies to abo blood groups, human leukocyte antigens (hla) class i, or other antigens on graft vascular endothelial cells. preexisting antibodies can result from previous pregnancies, blood transfusions, or a previous transplant. antibody binding provokes complement and cytokine activation leading to endothelial cell damage and platelet activation with subsequent vascular thrombosis and graft destruction. the outcome is usually fatal. in the lungs, hyperacute rejection grossly presents by edema and cyanosis of the graft. histologically, platelet thrombi, neutrophilic infiltration, fibrin thrombi, necrosis of vessel wall, and morphologic features of diffuse alveolar damage (dad) are observed [29] . although hyperacute rejection is a well-known complication in kidney and heart transplantations, in lung transplantation it appears to be rather rare with only five cases reported. one patient reported presented with severe hypoxia, high fever, hemodynamic instability and developing acute renal failure 1 h after completion of the anastomoses [29] . chest radiograph displayed a completely opacified left lung, with homogenous infiltrates. bronchoscopy revealed abundant pink frothy fluid draining from the allograft. mean pulmonary artery pressure increased to 29 mmhg. the patient died 24 h later. at autopsy, the vascular and bronchial anastomoses appeared patent without signs of injury. the transplanted lung showed red hepatization and a firm consistency. microscopically, signs of acute lung injury were evident. although a pretransplant panel-reactive antibody (pra) was negative, flowcytometry revealed 56 and 45% reactivity against hla class i and ii, respectively with anti-a2 detected among the preformed antibodies. three other reported patients with hyperacute rejection died within 4 h to 13 days after transplantation [11, 19, 43, 116] . although in three of the five reported patients pretransplant pras were negative, crossmatch was positive in all cases with anti-a2 the most common identified antibody. collectively, although hyperacute rejection is rare after lung transplantation, one should keep this reaction in mind given that false-negative pras may occur and pretransplantation cross match is not often possible [29] . acute rejection is the host's response to the recognition of the graft as foreign. most patients develop at least one episode of acute rejection within the first 3 weeks following transplantation, typically in the first 5-10 days, with 36% of patients experiencing at least one episode in the first year [21] . obliterative bronchiolitis (ob) is the most common late cause of mortality and morbidity after lung transplantation occurring in 28% by 2.5 years and 74% by 10 years in patients who survive at least 14 days [21] . it also has a significant negative impact on quality of life parameters. risks for acute rejection include hla mismatching, type of immunosuppression, infection, and recipient factors. it is generally thought that the intensity of host alloimmune response is related to recipient recognition of differences with the donor hla antigens and that this process drives acute lung allograft rejection. a higher degree of mismatch increases the risk of acute rejection [101, 115, 141] . however, this effect is not consistent across all hla loci or studies. mismatches at the hla-dr, hla-b [115] , and hla-a [101] loci, as well as a combination of all three loci [141] , appear important. in addition, the ishlt registry has not found a correlation between hla mismatching and survival [130] . thus, while hla mismatching between donor and recipient likely contributes to the immunologic basis for acute rejection, it is difficult to discern if a mismatch at a particular locus or if different degrees of mismatch significantly alter the overall risk for acute rejection. viral infections have been thought to modulate the immune system and heighten alloreactivity. indeed, a high incidence of acute rejection has been found in lung transplant recipients after community-acquired respiratory tract infections with human influenza virus, respiratory syncytial virus (rsv), rhinovirus, coronavirus, and parainfluenzavirus [44, 73, 137] . although cmv is considered a potential risk factor for ob, studies directly linking cmv infections or cmv prophylaxis strategies with acute rejection have been inconsistent [118] . in one study, chlamydia pneumoniae infection was linked to the development of acute rejection and ob [50] . several host genetic characteristics have been suggested to modulate acute lung rejection. for instance a genotype leading to increased il1-production may protect against acute rejection [147] and a multidrug-resistant genotype (mdr1 c3435t) appears to predispose to persistent acute rejection resistant to immunosuppressive treatment [148] . the effect of age on acute rejection appears to be bimodal, with the lowest incidence of acute rejection in infancy (<age 2) [61] and increased risk during childhood as compared with adulthood [117] . the incidence of acute rejection in older lung transplant recipients (age 65 and higher) does not seem to change [32] ; in fact, an increased rate of infections in older lung transplant recipients is thought to contribute to an increased mortality detected at one center arguing for reduced immunosuppression in these patients [55] . the clinical course of acute rejection can be variable. patients with acute rejection might present with dyspnea, fever, leukocytosis, and a widened alveolararterial oxygen gradient. higher-grade rejection appears to cause more severe symptoms and can lead to acute respiratory distress [32] . in patients with rejection, pulmonary function testing may show a decrease in forced expiratory volume in 1 s (fev 1 ) and vital capacity (vc). although spirometry has a sensitivity of greater than 60% for detecting infection or rejection grade a2 and higher, it cannot differentiate between the two [133] . the usefulness of spirometry is diminished in single lung transplant recipients, as the contralateral native lung dysfunction confounds the pulmonary function test results [7] . pulmonary function testing should therefore be used only as an adjunct to clinical evaluation. although in approximately half of the cases of acute rejection, the findings on chest radiograph are normal, chest radiographs may reveal ill-defined perihilar and lower lobe opacities, along with septal lines and pleural effusions. findings on ct scan might include ground-glass opacities, septal thickening, volume loss, nodules and consolidations, and pleural effusions. infiltrates observed on imaging studies during the first week after lung transplantation usually are caused by reimplantation response (i.e., reperfusion edema). persistent infiltrates beyond the first week suggest acute rejection or infection. however, although early small studies attempted to demonstrate the usefulness of chest x-rays and chest ct scans in the diagnosis of rejection, more recent data show very low sensitivity for acute rejection (as low as 35%) and no discriminatory value between rejection and other processes [51] . exhaled nitric oxide (no) is also an attractive marker of lung injury; it has been correlated with lymphocytic bronchiolitis [31] and acute rejection [120] . furthermore, in a study of inert gas single breath washout, the slope of alveolar plateau for helium had a sensitivity of 68% for acute rejection [133] . although lung transplantation has come of age, the development of ob remains the biggest hurdle preventing long-term survival in many patients [136] . because of its low sensitivity (28%) and specificity (75%), ob remains difficult to prove pathologically with transbronchial biopsy [24, 72] . as a consequence, a clinical definition, called bronchiolitis obliterans syndrome (bos) was proposed [24] . this is based on pulmonary function criteria, initially fev 1 evolution, and more recently, mid-expiratory flow rate (fef ) [38] . the onset of bos-symptoms is usually insidious, with progressive exertional dyspnea, often accompanied by cough, which may be dry or productive. the incidence of bos is highest after the first year following lung transplantation. however, the risk of bos increases to 60-80% 5-10 years after the lung transplantation procedure. it remains the leading cause of morbidity and death after lung transplantation, accounting for about 19-29% late mortality and some 35% of patients affected by the condition 5 years after transplantation [21] . as discussed above, an acute immunologic event (acute rejection) may trigger the onset of ob [63] . however, nonalloimmune injury such as a respiratory tract infection (cmv or non-cmv viral infection) is increasingly recognized as also having an important impact on the development of ob [41, 46] . in fact, cmv pneumonitis affects over 20% of lung transplant recipients. despite treatment, it increases the risk for ob and death. early detection of ob in a preclinical stage is ideal so that aggressive attempts can be made to prevent a fully developed syndrome. however, to date, no particular marker to indicate ob, either from the peripheral blood or bronchoalveolar lavage (bal) fluid, has predicted a risk for this disease. an association between chronic aspiration and ob after heart-lung transplantation has also been observed [105] , and there is a high frequency of gastroesophageal reflux with resultant aspiration following lung transplantation [28, 57, 97, 105, 108, 143] . for instance, one study described gastrointestinal complications including gastroesophageal reflux in 51% of patients who underwent lung transplantation [80] . other than leading to an increase in acute rejection by exacerbating the alloimmune response, aspiration of gastric contents might act independently of the alloimmune response to promote allograft injury and the development of ob. experiments in rats have further substantiated the association between chronic aspiration and ob [79] . despite the common clinical impression that lymphocytic pleural effusions are a hallmark of acute rejection, published data are inconclusive [65] . collectively, although the presentation of the patient and several ancillary studies might suggest a transplant rejection process, tissue diagnosis is necessary for definitive diagnosis. in 1990, the ishlt sponsored the lung rejection study group (lrsg), a workshop to develop a "working formulation" for the diagnosis of lung rejection by transbronchial biopsy [9] . the proposed grading scheme found wide acceptance. due to developments in the field and experience with its use, lrsg has remet twice since to assess the merits of the grading scheme. the revisions were published in 1996 [145] and 2007 [123] , respectively. the grading scheme is strictly pathologic and does not consider any clinical parameters. due to overlapping histologic features between acute rejection and infection, the grading scheme relies on the absence of concurrent infection. the most recent classification of lung allograft biopsies is the 2007 ishlt revised consensus classification of allograft rejection [123] (see table 7 .2). this classification was revised from the 1996 ishlt consensus classification. there were no significant changes in the grading of acute rejection in the latest classification, but it was acknowledged that minimal acute rejection is not solely a high power diagnosis but can also be recognized at low power in an adequately alveolated biopsy. in the 1996 grading system, small airways rejection was graded in a four-tiered system, b1 through b4, respectively (see table 7 .2). the 2007 classification consolidated those into low grade and high grade small airways inflammation to make the evaluation easier. the "r" behind b1 and b2 denotes the revised 2007 classification. chronic airways rejection is not divided into active and inactive anymore but only into present and not present. there are no differences in the classification of chronic vascular rejection between the 1996 and 2007 classification. r denotes revised an attempt should be made to accurately distinguish the grade of rejection since treatment is largely dependent on the histologic grade. furthermore, an effort should be made to review the report of the previous biopsy and to comment on whether rejection is ongoing or resolving. although in general, patients with grade a1 biopsies will not receive increased immunosuppression, surveillance might be intensified and the time to the next biopsy shortened. in contrast, a2 biopsy patients will receive an increase in immunosuppression. grade a3 and a4 patients will be treated similar and receive more immunosuppression. inter-and intraobserver variability in grading can impact treatment and outcome [16, 22] . studies have evaluated the inter-and intraobserver variability of the 1996 grading scheme. two studies found relatively good interobserver agreements for the a-grades (kappa of 0.65 and 0.73) [16, 22] , but this could not be replicated in another study in which the kappa was 0.47 in spite of dichotomization of the a-grades to a0/a1 vs. a2-4 [122] . intraobserver agreement for acute rejection has been found to be good: kappa values of 0.65 and 0.795 [16, 122] . infection can complicate the diagnosis of acute rejection. viral infection in particular can cause mononuclear inflammation [126] . in addition, alveolar damage with macrophage and fibrin accumulation was found to be present in 80% of transbronchial biopsies in the first 6 months after transplantation, further increasing interobserver pathologist discordance. therefore, in general, the lrsg recommends grading rejection only after the exclusion of infection. in contrast to the a-grade, the interobserver variability for grading airways inflammation (b-grades) was only fair with kappas of app 0.3 [16, 122] . for this reason, the lrsg has now simplified b-grading to two possible grades. this nomenclature is to be used for grading of noncartilagenous small airways only after rigorous exclusion of infection [123] . eosinophils can accompany acute lung rejection and are recognized in the 2007 ishlt classification in high grade rejection (grades a3/a4) [123] . the presence of eosinophils has been suggested as a risk factor for bos in small single-center studies [114] . furthermore, high proportions of b cells have been shown to accompany steroid-resistant rejection in lung transplant patients [146] . the mechanisms by which b cells contribute to refractory rejection are not entirely clear, although they may reflect ongoing humoral rejection, perhaps explaining the diminished responsiveness to standard immunosuppression. mast cells have been identified in acute rejection biopsies of increasing a-grade but their role has not been elucidated [144] . rejection and chronic airways inflammation should be distinguished from bronchiolar associated lymphoid tissue (balt). balt is found in the vicinity of airways, usually contains black anthracotic pigment and presents as a rather nodular collection of chronic inflammatory cells which does not surround a vessel. acute rejection is defined by the presence of perivascular mononuclear cell infiltrates with or without endotheliitis. with progression, this infiltrate becomes more widespread and extends into the alveolar septa and, subsequently, into the alveoli. the majority of the mononuclear cells in acute rejection are t cells, although a few studies have described increased populations of b cells or eosinophils [104, 123, 146] . in grade a0 normal pulmonary parenchyma is present. scattered infrequent blood vessels, particularly venules, in the alveolated lung parenchyma are surrounded by a relatively thin chronic mononuclear infiltrate (see fig. 7 .1). the lymphocytic rim is rather small and does not spill into the adjacent interstitium. endotheliitis and eosinophils are absent. although previous classification systems commented on a certain number of lymphocyte layers, the current ishlt classification only requires a complete rim of vessels by lymphocytes. in adequately alveolated and artifact-free specimens, the lymphocytic infiltrates might be detected at low magnification. although in mild acute rejection the perivascular infiltrate of lymphocytes is essentially confined to the perivascular adventitia without infiltrating the interstitium, there are more layers of lymphocytes surrounding the vessel and lymphocytes might focally, minimally spill into the adjacent interstitium (see fig. 7 .2). more frequent perivascular mononuclear infiltrates are seen surrounding venules and arterioles. they are readily recognizable at low magnification. these infiltrates usually consist of a mixture of small round lymphocytes, activated lymphocytes, plasmacytoid lymphocytes, macrophages, and eosinophils. there is frequently subendothelial infiltration by mononuclear cells which may be associated with hyperplastic or regenerative changes in the endothelium, i.e., endotheliitis. concurrent lymphocytic bronchiolitis may be seen in association with mild acute rejection. grade a3 acute rejection shows easily recognizable cuffs of venules and arterioles by dense perivascular mononuclear cell infiltrates which are commonly associated with endotheliitis (see fig. 7 .3). eosino phils a b and even occasional neutrophils are common. this grade is defined by the extension of the inflammatory cell infiltrate into perivascular and peribronchiolar alveolar septa which broadens the interalveolar septa and airspaces are associated with collections of intraalveolar macrophages in the zones of septal infiltration. type ii pneumocyte hyperplasia and histologic features of acute lung injury may become apparent. in severe rejection there are diffuse perivascular, interstitial, and air space infiltrates of mononuclear cells with prominent alveolar pneumocyte damage and endotheliitis (see fig. 7 .4). this may be associated with intra-alveolar necrotic epithelial cells, macrophages, eosinophils, hemorrhage, and neutrophils and usually some evidence of acute lung injury in form of organizing pneumonia or hyaline membranes. parenchymal necrosis, infarction, or necrotizing vasculitis might be identified; however, these features are more evident on surgical rather than transbronchial lung biopsies. it should be noted that a paradoxical diminution of perivascular infiltrates can occur as cells extend into alveolar septa and spaces where they are admixed with macrophages. this grade can sometimes be difficult to distinguish from an infectious process, harvest/reperfusion injury, or drug toxicity, however, the presence of perivascular inflammation is helpful in establishing the diagnosis. this grade applies only to small airways such as terminal or respiratory bronchioles. bronchi, if present, should be described separately. it is important to mention in the pathology report whether or not small airways are present. the r behind grades 1 and 2 denotes the revised 2007 version. the small airways appear unremarkable without evidence of bronchiolar inflammation. low grade inflammation is characterized by lymphocytes within the submucosa of the bronchioles (see fig. 7 .5). the lymphocytic infiltrates can be infrequent and scattered or forming a circumferential band, however, intra-epithelial lymphocytic infiltration is not present. although occasional eosinophils may be seen within the submucosa, there is no evidence of epithelial damage, neutrophils, necrosis, ulceration, or significant amount of nuclear debris. this grade combines and replaces the 1996 working formulation b1 and b2 grades. in high grade small airways inflammation there is marked lymphocytic infiltrate of the airway epithelium and airway wall. the mononuclear cells in the submucosa appear larger and activated with greater numbers of eosinophils and plasmacytoid cells. in addition, there is evidence of epithelial damage including necrosis, metaplasia, and marked intra-epithelial lymphocytic infiltration. in its most severe form, high grade airways inflammation is associated with epithelial ulceration, fibrino-purulent exudate, cellular debris, and neutrophils. it is important to exclude an infectious process. in this grade the changes are ungradeable due to sampling problems, infection, tangential cutting, artifact etc. the working formulation for pulmonary rejection has equated ob with one type of chronic rejection, the c-grade. the term as used in the consensus classification is restricted to submucosal and intraluminal scarring of membranous and respiratory bronchioles. when large tissue sections of lung are examined, the process of ob is pan-lobar but patchy. the small airways appear similar in size to the accompanying artery with a ragged inner surface. fibrosis is not present. narrowing of the airways due to fibrosis in the airway wall is seen. the fibrosis is usually eccentric (see fig. 7 .6). ob may be manifest by a histopathologic spectrum of changes depending on the acuteness of the process, the degree of organization and the amount of accompanying inflammation. in the acute phase there is usually loose myxoid granulation tissue with variable numbers of inflammatory cells filling or partially obstructing the airway lumen, resembling "organizing pneumonia." in later phases, ob may consist of eccentric, occasionally confluent plaques of dense hyalinized collagen applied to the wall of bronchioles. metaplastic squamous or cuboidal epithelium may cover these bronchiolar scars. in other airways a slit-like lumen may remain as a result of a confluent submucosal scar or intraluminal polyps of scar tissue. capillaries supplying these intraluminal masses of collagen are occasionally prominent. in the most severe cases the bronchiolar lumen can be entirely occluded by dense scar tissue and be recognizable only with the aid of an elastic stain, its location adjacent to an artery, and by the presence of residual circumferential smooth muscle. in the later phases, inflammation may be minimal. usually, the scarring process is confined exclusively to respiratory bronchioles and terminal bronchioles, although it may occasionally involve adjacent alveoli. the pulmonary arteries appear of a similar size as the accompanying airway. the intima is slender, the media not thickened. chronic vascular rejection rarely is identified on biopsies since they usually lack vessels of sufficient size. wedge biopsies, explants, or autopsy material may reveal it. in the typical case, pulmonary arteries and more often veins are thickened by fibrointimal connective tissue (see fig. 7.7) . also, thickening is usually concentric. chronic vascular rejection may be patchy and typically involves smaller vascular arteries and veins. in the early stages of chronic vascular rejection, the intimal proliferation occurs on the luminal aspect of an intact elastic lamella. subsequently, the internal elastica may become fragmented and discontinuous. occasionally the underlying muscular wall becomes thinned. in approximately half of the reported cases a concurrent endovasculitis has been observed. the process is similar in pulmonary veins, although the intimal deposits may be less cellular and more waxy, eosinophilic, and sclerotic. chronic vascular rejection should be distinguished from recanalizing thrombi. unlike the situation with heart transplant recipients, chronic vascular rejection in lung transplants has not resulted in graft loss; however, some patients develop pulmonary hypertension particularly those with bos [92, 111] . mimickers of severe acute rejection include conditions that might present with acute lung injury or dad. these conditions include infection, drug toxicity, antibody mediated rejection (amr), or harvest/reperfusion injury. therefore, careful slide review for perivascular inflammation should be performed, stains for microorganisms including gomori-grocott methenamine silver stain (gms) and acid fast bacilli (afb) might be added and careful search for viral inclusions should be undertaken. although perivascular mononuclear infiltrates are helpful to identify rejection, these are not entirely specific for acute rejection and many other conditions may simulate or mimic alloreactive lung injury [126] . differential diagnostic considerations include cmv pneumonitis, pneumocystis jiroveci pneumonia and posttransplantation lymphoproliferative disease (ptld). further differential diagnosis of perivascular and interstitial infiltrates include recurrent primary diseases. amr or humoral rejection is well established in other solid organ transplantation. it was originally recognized in kidney transplant patients who presented with acute allograft rejection, antidonor antibodies, and poor prognosis [125] . amr is thought to be due to circulating antibodies that are either preformed because of pregnancy, blood transfusion, or previous organ transplantation or arise de novo after transplantation due to hla-mismatch. although amr is an increasingly recognized entity in lung transplantation, it is still under investigation. early observations were based on the phenomenon of hyperacute rejection, where preexisting donor-specific antibodies lead to complement activation and rapid graft loss. later it has been recognized that lung transplant recipients also can develop antibodies after transplantation to the allograft that might lead to amr. evidence suggests that amr occurs to donor major histocompatibility (mhc) antigens, although other endothelial and epithelial antigens expressed in the lung may become antibody targets as well. the recent development of very sensitive and specific solid phase flowcytometry and luminex-based methodologies has allowed for accurate detection of antibody specificities in sensitized recipients and it has become clear that more patients than previously expected present with preformed anti-hla antibodies. immune stimulation by prior infections or autoimmunity might contribute to the development of antibodies to allomhc in those patients with no identifiable risk factors. these preexisting antibodies can react with donor antigens, leading to immediate graft loss (hyperacute rejection) or accelerated humoral rejection and bos [23] . furthermore, recent studies have consistently demonstrated an increased incidence of acute rejection (a threefold increase in one study) [48] , persistent rejection, increased bos [96] or worse overall survival [56] in patients with anti-hla antibodies. this effect is apparent both with pretransplant hla sensitization and with the development of de novo anti-hla donorspecific antibodies after transplantation [96] . about 10-15% of lung transplant recipients are presensitized to hla antigens [3] . even though "unacceptable antigens" are avoided during the virtual crossmatch, patients with positive pretransplant pra are at higher risk for posttransplant complications. their posttransplant pra can stay stable or increase via generation of either donor-specific or nondonor-specific anti-hla antibodies. similarly, patients that had negative pra screening tests before transplantation can develop de novo nondonor-specific or donor-specific anti-hla antibodies after transplantation. the mechanisms by which antibody promotes lung allograft injury remain poorly understood. antibody binding to allomhc or other endothelial or epithelial targets in the lung could lead to activation of the complement cascade with complement deposits leading to endothelial cell injury, production of proinflammmatory molecules, and recruitment of inflammatory cells. com plement independent antibody-mediated mechanisms can also induce endothelial cell activation without cell injury, leading to increased gene expression and subsequent proliferation [23] . as demonstrated by in vitro studies, anti-hla antibodies can cause proliferation of airway epithelial cells as well, producing fibroblast-stimulating growth factors [64] , potentially contributing to the generation of obliterative airway lesions. recent studies have attempted to evaluate immunoglobulins (ig) and complement deposits in the subendothelial space. septal capillary deposits of igs and complement products such as c1q, c3d, c4d, and c5b-9 have been described in association with anti-hla antibodies [62, 90] as well as allograft dysfunction and bos [82, 139] . the concept of specific histopathologic features associated with humoral rejection remains controversial in lung transplantation. recent studies question the relation between complement or ig staining and allograft rejection [112, 138] . others demonstrate that c3d and c4d staining can occur in lung transplant recipients with nonalloimmune lung injury such as infection and primary graft dysfunction (pgd) with no evidence of anti-hla antibodies [139] . differences in staining techniques between different laboratories may further explain some of the inconsistencies in the published data. the 2007 ishlt revised consensus classification did not agree upon any histopathologic features that might be specific for amr in lung [123] . although capillary injury/small vessel intimitis might raise the suspicion of amr, these are nonspecific findings that can also occur in severe acute rejection, infection, or harvest/reperfusion injury. furthermore, although often the term "capillaritis" is mentioned, ishlt recommends to use the term "capillary injury" which allows for a broader spectrum of morphological changes of the vessels including intimitis, whole wall thickness inflammation, neutrophilic infiltration (which should be distinguished from neutrophilic margination), thrombosis, and necrosis. furthermore, signs of dad and intra-alveolar hemorrhage can occur with amr although again, they are not specific. given the lack of specific histologic findings of amr in lung transplantation, a multidisciplinary approach to diagnosis is recommended that includes the following: (1) the presence of circulating antibodies (hla antibodies, antiendothelial and antiepithelial antibodies), (2) focal or diffuse c4d deposition (see fig. 7 .8), (3) histologic features of acute lung injury or hemorrhage (dad, capillary injury with neutrophils and nuclear debris) and (4) clinical signs of graft dysfunction. if amr is clinically, immunopathologically, or histologically suspected, one might perform immunostains for c3d, c4d, cd68, and cd31. however, these stains are extrapolates from kidney and heart transplantation and although there have been several studies advocating their use [47, 62, 123, 139] , there are no large scale studies validating their use. c4d has been studied extensively in kidney and heart transplant and has been identified as a useful adjunct in the diagnosis of amr in these organs. however, in lung that is not the case. one of the reasons for the difficulties in lung is the relatively high background that is encountered in immunohistochemistry (ihc) as well as immunofluorescence (if). often, c4d binds to elastic laminas or shows other nonspecific binding. staining is commonly only focal and therefore sensitivity and specificity have not been established yet given the limited sample size of transbronchial biopsies. only linear, continuous subendothelial staining of capillaries, arterioles, and/or venules count as positive by ihc. also, c4d is not specific to amr but also can be seen in infection, harvest/ reperfusion injury, or even in severe acute rejection, basically in any process that is associated with complement activation. there is no ihslt recommendation at this time regarding to the coexistence of amr and acute rejection. although clinical evaluation of the patient may suggest the possibility of rejection, tissue evaluation is necessary for a definitive diagnosis. initially, investigators were hesitant to routinely utilize transbronchial biopsy to monitor the graft as it was thought that the amount of tissue obtained would be too small to make a definitive diagnosis. there was also concern about passing a bronchoscope over the anastomosis. however, transbronchial biopsy is now a routine procedure and has become the gold standard to evaluate the graft for acute and chronic rejection, infection, and possible recurrent disease since currently, no surrogate markers have been sufficiently validated as means to reproducibly identify patients with acute rejection. specifically, there is no serological marker to indicate rejection is occurring. after lung transplantation the total bal fluid cell count is constantly increased even in periods with no evidence of infection or rejection [127] . in the early posttransplantation period (first 4 weeks), there is a dominance of neutrophils in the bal fluid (up to 25-50% of total cell count) until about 3 months later when the cell count normalizes [127] . acute rejection has been associated with elevated cd8+ t cells, activated cd4+ t cells, a trend toward increased nk t cells, increased b cells, and decreased nk cells in the bal [53] . nevertheless, no study has proven the bal cellular composition to be adequately sensitive or specific in the discrimination of rejection from infection [107] . small studies have found a correlation between acute rejection and elevation of il17 [134] , il15 [10] , and ifn gamma in the bal [10] . recent advantages in genomics offer the potential for more specific means of diagnosing rejection in lung transplantation. a pilot study of gene expression in the bal of lung transplant recipients found that gene expression signatures related to t-lymphocyte function, cytotoxic cd8 activity, and neutrophil degranulation correlate with acute rejection [98] . however, these studies have no clinical use at the present time. recently published studies in heart transplantation describe the use of peripheral blood gene expression profiling to identify future risk of cardiac allograft rejection [88] . a similar study is now underway in lung transplantation, known as the lung allograft rejection gene expression observational (largo) study. preliminary data from almost 900 patients, similar to the cargo results, show differential gene expression in the lymphocyte priming and neutrophil homeostasis pathways for a0 vs. ³a2 acute lung rejection [66] . such testing may hold promise for a noninvasive technique to monitor the status of the transplanted organs. another study described marked serum elevations of hepatocyte growth factor (hgf) in lung transplant . the high power view shows capillaritis in a case of amr characterized by focally thickened interstitium due to neutrophils and nuclear debris with capillary destruction. the insert reveals c4d immunoperoxidase stain (see arrow) focally lining the endothelium of capillaries and small vessels (magnification × 400, insert × 600) recipients with acute rejection. however, smaller elevations in hgf also occurred with lung infection, and additional studies are needed to validate specificity and sensitivity of hgf for acute rejection [1] . in conclusion, although there are a few promising noninvasive techniques, these are early studies that need to be confirmed in larger studies before being considered for widespread clinical use. therefore, microscopic tissue evaluation remains the gold standard for diagnosing rejection. bronchoscopy and biopsy postlung transplantation are generally performed if there is any clinical suspicion of rejection, infection, recurrent disease, or ptld. in addition, many transplant centers now follow protocol biopsies. however, the pros and cons of surveillance bronchoscopies with biopsy are still debated and therefore, there are no guidelines established in regards to the timing of postlung transplantation surveillance bronchoscopy and biopsy. one institution reports surveillance biopsies in pediatric lung transplant recipients at 1 week and 1, 3, 6, and 12 months posttransplantation [8] . the rational for surveillance biopsies includes the occurrence of clinically silent acute rejection, inadequate surrogate markers for acute rejection, and the relatively low risk of the bronchoscopy procedure. the yield for acute rejection by transbronchial biopsies was reported 6.1-31% and 25% or greater in studies performing surveillance transbronchial biopsies [17, 60] , clinically indicated and follow-up bronchoscopies [18] . grade a2 and higher acute rejection has been found in a relatively high percentage of asymptomatic patients, ranging from 22 to 39% [54, 131] . silent acute rejection appears most common within the first 3 months of transplantation (24.8% at 0-3 months; 16.7% at 3-12 months; 2.7% after 1 year) [87] . the rate of unsuspected but clinically significant infection was highest between 3 and 12 months posttransplantation but a relatively high rate (18.9%) was also detected after 1 year. one small study questioned the benefits of performing surveillance bronchoscopy with biopsy, suggesting that the benefits do not outweigh the procedural risks [132] . in this study, changes in management based on transbronchial biopsy and bal results were significantly higher in the clinically indicated group (65%) compared with the surveillance group (13%) and were mostly related to infection. interestingly, no silent acute rejection episode requiring treatment was detected in the surveillance group of this study. collectively, these findings may reflect differences in programs and operators. although it has been shown that transbronchial biopsy is often the only mean to reveal silent acute rejection of the allograft, definitive evidence that treatment of such episodes have a positive impact on survival or prevention of bos has yet to be demonstrated. however, based on the link between acute rejection and development of bos, surveillance transbronchial biopsies in asymptomatic lung transplant recipients has become common practice in many large lung transplantation centers because evidence suggests that patients who have multiple episodes of low grade (a1) lesions within the first 12 months posttransplantation develop early onset bos. however, there were no differences in overall survival in patients with and without a1 acute rejection [59] . there is also evidence that a1 rejection increases the risk of higher-grade subsequent rejections (³a2) [13, 34] . the finding of a solitary perivascular monocytic infiltrate was followed by worsening acute rejection in four untreated patients in one study, while the treatment of such a solitary infiltrate in nine patients resulted in improvement of the rejection score [67] . the presence of severe lymphocytic bronchiolitis also has been associated with increased risk of bos and death after lung transplantation, independent of the presence of acute rejection [49] . a study [49] in which surveillance transbronchial biopsies were performed at 3, 6, 9, and 12 weeks posttransplantation, at the time of symptoms, and for follow-up of acute rejection or cmv pneumonia showed that patients who develop acute small airways rejection within the first year after transplantation are at risk of development of bos at 1.76, 3.3, and 5.5 years after detection of b3/ b4 lesion (by 1996 ishlt criteria, see table 7 .2), b2 lesion or b0/b1 lesion, respectively. this study strongly suggests that the main utility of surveillance biopsies may be to use lymphocytic bronchiolitis as a surrogate predictor of long-term outcome. however, the impact of therapy for lymphocytic bronchiolitis has yet to be assessed. although complications related to bronchoscopy occur, including a small risk of severe complications, the majority of cases have a favorable outcome. adverse events reported with bronchoscopy in lung transplant recipients include transient hypoxemia, bleeding, pneumothorax, arrhythmia, and anesthesia related complications [74, 87] . bronchoscopy may have contributed to faster deterioration of one patient's already critical condition; however, in the posttransplantation setting, -bronchoscopy has no reported mortality [18, 60] , although there are anecdotal reports of death related to transbronchial biopsy. the 2007 ishlt revised consensus classification of acute allograft rejection [123] requires the evaluation of at least five pieces of well-expanded alveolated parenchyma. however, the bronchoscopist may need to submit more than five pieces to provide this minimum number. further biopsies may improve the detection of ob, although there is no specific number of small airways required by the ishlt consensus classification to exclude this diagnosis. gentle agitation in formalin to open up the alveoli may improve the histologic appearance of the fragments. histologic examination should include three levels from the paraffin block for hematoxylin and eosin (h&e) staining [123] . connective tissue stains such as trichrome or verhoeff-van gieson (vvg) stain to evaluate airways for the presence of submucosal fibrosis are essential for the diagnosis of ob, and atherosclerosis. silver stains such as gms can be performed for fungi, including pneumocystis, but have not been routinely mandated by the 2007 ishlt revised consensus classification, given the numerous microbiologic, serologic, and molecular techniques available for the diagnosis of opportunistic infections. bal may be performed at the time of biopsy and is useful for the exclusion of infection, but currently has no clinical role in the diagnosis of acute rejection (see above). infection is the leading cause of death in lung transplant recipients. factors that increase a patient's susceptibility to infection after transplantation include immunosuppression, reduced mucociliary clearance, decreased cough reflex resulting from denervation, and interruption of lymphatic drainage. bacterial pneumonias are the most common infections following lung transplantation [26] and occur in more than 35% of patients during the first year after transplantation (highest incidence is during the first month posttransplantation) (see fig. 7 .9). furthermore, bacterial pneumonia remains a major infectious complication throughout the patient's life. the donor lung is affected most often. gram-negative organisms are most common, especially enterobacter and pseudomonas. bronchitis secondary to pseudomonas species or staphylococcus aureus infection also is observed. bacterial pneumonia typically manifests radiographically as a lobar or multilobar consolidation. viral pneumonias develop in approximately 11% of patients who have undergone lung transplantation. they occur at any time following transplantation. cmv is the second most common cause of pneumonia in patients who have received lung transplants, and it is the most common opportunistic infection (35-60% of opportunistic infection) [26] . it represents the most significant viral infection, and usually occurs 1-4 months after transplantation. primary infection is the most serious form and is observed in 50-100% of seronegative patients who received a graft from a seropositive donor. in patients who are seropositive, secondary cmv infection develops from reactivation of latent disease following the institution of immunosuppressive therapy or from infection with a different strain of cmv. infected patients may be asymptomatic or may develop a fulminant pneumonia, possibly with extrathoracic findings such as retinitis, hepatitis, and gastritis. presenting symptoms include dyspnea, fever, and cough. the most common finding on chest radiographs in patients with cmv infection is diffuse parenchymal haziness. ct scan findings include areas of ground-glass attenuation; reticulation; multiple, small, ill-defined 1-to 3-mm nodules; and, even less commonly, areas of dense consolidation. the diagnosis of cmv pneumonia can be made by bronchoscopy with lavage and biopsy. cytopathic changes associated with cmv infection include cytomegaly, multiple small basophilic cytoplasmic inclusions, and a large nuclear inclusion surrounded by a halo with thickened nuclear membrane (see fig. 7 .10). prophylactic therapy with acyclovir and immune globulin has not reduced the incidence of cmv infections in patients who have undergone transplant procedures. a less common cause of viral infection includes the herpes simplex virus (hsv) infection. patients with hsv infection present with fever, cough, and dyspnea, but they demonstrate symptomatic improvement after therapy with intravenous acyclovir. radiographic findings may be absent or may demonstrate diffuse groundglass opacities. histopathologically, the classic hsv inclusion consists of a dense, eosinophilic mass within the nucleus that is surrounded by a clear halo and peripherally marginated, beaded nuclear chromatin. hsv may form multinucleated cells. opportunistic fungal infections are less common than viral infections, but they are associated with higher mortality. fungal pneumonias usually occur 10-60 days following transplantation and more commonly involve the transplanted lung. however, they also can involve the native lung in single lung transplantation cases, especially in patients with copd. ct imaging studies most commonly reveal a combination of nodules (multiple, variable sizes, irregular margins), consolidation, and ground-glass opacification. pleural effusions are also common (63% of cases). gms will help to identify fungal organisms. however, even in the absence of identifiable organisms on gms, infection might be considered and cultures and serology should be attempted. locally invasive or disseminated aspergillus infection accounts for 2-33% of posttransplantation infections and 4-7% of deaths in patients who undergo lung transplantation. aspergillus infection most commonly is characterized by local invasion of a necrotic bronchial anastomosis (i.e., ulcerative aspergillus infection, when involving the lung parenchyma, tends to cavitate and has an upper-lobe predominance. histopathologically, the hyphae of aspergillus sp are septated and branching. they appear uniform and grow in parallel fashion with septae at regular intervals. the branching is dichotomous and usually at 45°angle. inhaled amphotericin b is often used in the immediate posttransplantation period to help eliminate this complication [26] . patients are also discharged on voriconazole as daily prophylaxis for the first year. patients who have undergone lung transplant procedures have an increased susceptibility to p jiroveci infection, but prophylaxis with trimethoprim-sulfamethoxazole is effective in preventing the infection (incidence is nearly 0%). without prophylaxis, the incidence of p jiroveci infection approaches 90%. histologically, cysts of p jiroveci are round to oval, measure 5-7 mm in diameter, and often have very prominent grooves or folds. p jiroveci pneumonia classically consists of an intra-alveolar foamy exudate in which the organisms appear as small "bubbles" in a background of proteinaceous exudate, although a variety of other reactions can occur as well [129] . the incidence of ptld is significantly higher after thoracic organ transplantation compared with any other solid-organ transplant [94] . they develop in 4-10% of lung transplant recipients, as opposed to an approximate 2% incidence in other solid organ recipients, with 60% of the ptlds presenting in the allograft. in adults, ptlds are the most common neoplasms at 1 year posttransplantation (peak, 3-4 months posttransplantation) [21] . in children [6] , lymphomas are by far the most common posttransplantation malignancy at any time. in fact, ptld is a major cause of morbidity and mortality in pediatric lung transplant recipients [12] . risk factors for ptld after solid organ transplantation include patient's age, type of organ transplanted, ebv infection status of host before transplantation, and the immunosuppressive regimen [4, 77, 93, 140] . ptlds are a clinically, morphologically, and molecularly heterogeneous spectrum of lymphoproliferative disorders, ranging from early, ebv-driven, polyclonal, polymorphic proliferations (infectious, "mononucleosis-like") to ebv-positive or ebv-negative monomorphic tumors [15, 69, 70, 100] . ptlds most commonly are associated with ebv infection of b-cells either by reactivation of latent virus or primary ebv, most commonly acquired from donor organs [12] . a recent study showed [37] that five of seven lung-transplanted children who developed ptld were ebv-negative recipients who received ebv-positive lungs. in a series [81] of 988 heart and/or lung transplant recipients, 17 ptlds were identified. amongst the 17 ptld cases, there were two b-cell monoclonal polymorphic ptlds and 15 b-cell monomorphic ptlds (see fig. 7 .11) (13 diffuse large b-cell lymphomas (dlbcl) and two burkitt lymphomas). ebv was detected in 9 of the 17 patients. all cases showed monoclonal igv gene rearrangements; igv somatic hypermutation was found in 88% of cases, indicating a prevalent origin from germinal center-experienced b cells. given the association between ebv infection and ptld, elevation in ebv viral load measured in peripheral blood has been proposed as a marker for the development of ptld [109] . in addition, monitoring the degree of immunosuppression after diagnosis of ptld with immune function assays and immunosuppressive drug levels may aid clinicians in assessing the risk for ptld and monitoring treatment after diagnosis [45] . t-cell ptlds tend to occur later and tend not to be associated with ebv infection. t-cell ptlds are associated with a worse prognosis than b-cell ptld. patients with ptlds may be asymptomatic, or they may have nonspecific complaints such as fever, weight loss, dyspnea, and lethargy. solitary or multiple pulmonary nodules ranging in size from 0.1 to 5 cm are the most common pulmonary manifestation of patients with ptlds. mediastinal and hilar adenopathy also can be observed in 22-50% of cases. patients who present with a solitary pulmonary nodule have a better overall prognosis. however, most ptlds have a rapid onset, a predilection for extranodal sites, and an aggressive clinical behavior with poor outcome [15, 69, 70, 78, 100] . as a result of the high mortality associated with ptld, treatment often poses a challenge. the most common treatment of ptld has included decreasing immunosuppression in combination with surgical resection, antiviral agents, radiation, and chemotherapy [45, 103, 109] . the reduction or withdrawal of immunosuppressive therapy may result in a partial or complete regression of the ptld [69] . a higher risk of graft rejection associated with ptld treatment is one reason for the need for early detection of the disease [45, 102, 103, 109] . in adults, malignancy remains a common complication after lung transplantation [21] . among lung transplantation survivors, 3.5, 12.6, and 28.1% have a malignancy at 1, 5, and 10 years posttransplantation, respectively. although lymphoproliferative disorders are most common at 1 year posttransplantation, nonmelanoma skin malignancy is the leading malignancy at 5 and 10 years posttransplantation. in children [6] , the incidence of posttransplantation malignancy is higher than in adults early after transplantation with 5.9% of children developing them within the first year after transplantation. at 5 years posttransplantation, 13.1% were found to have a malignancy. at any time posttransplantation, lymphomas are by far the most common posttransplantation malignancy in children. the risk for cancer is higher following lung transplantation than after kidney transplantation [110, 121] . lung transplant patients have unique characteristics, as their baseline genetic and environmental background, and immunosuppressive regimens are different from patients receiving kidney transplant, all of which may contribute to the increased risk [2] . besides nonmelanoma skin cancer and lymphoproliferative disorders, other malignancies repor ted to be associated with solid organ transplantation include: kaposi's sarcoma, anogenital cancers, oral cavity malignancies, esophageal and urinary bladder cancer, hepatocellular carcinoma, and sarcomas [110, 121] . acute graft vs. host disease (gvhd) is an uncommon and usually fatal complication of lung transplantation for which no effective therapy exists. among the ten reported patients, eight had grade 3 to 4 acute gvhd and died within 208 days. there is one recent case report [42] of a patient with grade 3 to 4 acute gvhd after blt who was successfully treated with highdose corticosteroids after basiliximab and extracorporeal photopheresis were unsuccessful. after 26 days the patient had developed low grade fever, chills, and pruritic maculopapular rash on the chest that spread to the proximal extremities in the next 4 days. liver function tests were elevated. a skin biopsy showed dermal inflammation, patchy loss of basal layer with vacuolar degeneration, apoptosis, and cell necrosis suggesting gvhd. a few days later, profuse, green, watery diarrhea developed with abdominal pain and paralytic ileus. colonoscopy showed ulceration of mucosa and destruction of intestinal crypts, features compatible with gvhd. there was leucopenia, severe anemia, and deterioration of liver function. the diagnosis was confirmed by chimerism study that revealed a recipient/donor lymphocyte ratio of 50:50. a second case of acute gvhd after lung transplantation was successfully treated with corticosteroids [5] . this patient presented with mild symptoms (pruritic rash and blurry vision) suggesting low-grade gvhd. the diagnosis was confirmed when a small dose of prednisone resulted in the resolution of gvhd within 3 weeks. lung harvest/reperfusion (ischemia/reperfusion, i/r) injury after transplantation remains the most common cause of early posttransplantation respiratory failure and manifests typically during the first 72 h after transplant [75] . reported rates are as great as 41% [52] . the 30-day mortality of patients with i/r injury is about 40%, compared with 7% in patients without i/r injury [86] . the clinical equivalent to i/r injury is pgd. the ishlt working group on pgd proposed a four-tiered grading scheme of pgd based on pao 2 /fio 2 (p/f) ratio and radiological chest infiltrates assessed at time points up to 72 h: t (0-within 6 h of reperfusion, 24, 48, and 72 h) [20] . i/r injury usually presents with the immediate impairment in lung function after transplantation accompanied by rapid development of pulmonary edema, increased pulmonary vascular resistance, and decreased airway compliance. patients with i/r injury require prolonged mechanical ventilation with greater hospital stays and are at an increased risk of multiorgan failure. lung i/r injury has long-term consequences and is a risk factor for late graft failure (ob) [39, 40] . i/r injury can occur due to prolonged ischemia during transplantation. ischemic injury to the pulmonary vascular endothelium increases permeability and results in pulmonary edema. histologically, i/r injury presents as acute lung injury pattern including dad (see fig. 7 .12). perivascular infiltrates are usually not present and distinguish it from acute rejection. however, infection can present similarly and needs to be excluded with stains, cultures, and serology. the pathophysiology of lung i/r injury remains incompletely understood. the lungs are particularly susceptible to i/r injury, likely owing to the rich vascularity and relatively large surface area over which blood-borne components interact with the endothelium. the mechanisms of i/r injury are diverse and include generation of reactive oxygen species (ros), leukocyte activation/recruitment, complement and platelet activation, abnormalities in pulmonary vascular tone, and increased procoagulant activity. the production of proinflammatory cytokines is increased considerably in the lung after i/r. expression of cytokines in the lung after i/r may not only cause immediate tissue injury, but may predispose the lung allograft to rejection. several studies suggest that lung i/r injury is biphasic, with a distinct, acute injury characterized by macrophage activation followed by a later, neutrophildependent injury [39] . although recurrent disease in the allograft has been reported in some of the transplantation cases, in general, this has not been clinically significant. diseases for which recurrences in the allograft have been reported include giant cell interstitial pneumonia, sarcoidosis, lymphangioleiomyomatosis, langerhans' cell histiocytosis, allergic bronchopulmonary aspergillosis, desquamative interstitial pneumonia, bronchioloalveolar carcinoma, alveolar proteinosis, and diffuse pan bronchiolitis. morphological features are identical to the disease in nontransplanted lung. bronchial dehiscence is the most common anastomotic airway complication in the early postoperative period. it occurs in 2-3% of cases. ischemia at the anastomotic site is the major factor in the development of this complication. dehiscence probably is best assessed by bronchoscopy; however, ct scans typically demonstrate the presence of extraluminal gas, which is 100% sensitive and 72% specific for dehiscence. patients with telescoping anastomoses also may develop small anastomotic diverticula, which appear as smooth rounded air collections at the inferior-medial aspect of the anastomosis. anastomotic stricture occurs in approximately 10% of cases, and the risk for stenosis may be increased with a telescoping anastomosis. stenoses often manifest with progressive airflow obstruction that can be difficult to differentiate from other causes, such as acute rejection or bos. stricture probably is best evaluated by bronchoscopy; however, ct scans often demonstrate the area of narrowing. treatment is stenting, typically with an expandable metallic stent. more recently, balloon dilatation has obviated the need for stents in some centers. app 43% of lung transplantations between 1995 and 2008 were single lung transplants (slt) [21] . copd, aat, and ipf accounted for app 85% of the indications for slts [21] . advantages of slt over blt include a technically easier procedure, shorter surgery time, and the ability to transplant two patients from one donor. however, there are disadvantages to slt. for instance, survival rates for slt and blt recipients are different, diverging most obviously in later years after transplantation. 5-year survival for slt was 46% compared with 54% for blt [130] . other disadvantages of slt include less pulmonary reserve, and the propensity for complications related to the residual native lung. native lung complications have previously been reported in 13.8-50% of patients. they may be associated with significant morbidity and mortality [85, 135] and may partly explain why outcomes with slt are inferior to those of blt. a recent study [68] reported the occurrence of pneumothoraces, malignancy, aspergilloma, pneumonia, bronchopleural fistulas, and pulmonary embolism. some forms of advanced lung disease, including copd and ipf, are associated with increased risk for lung cancer, a complication that can arise in the native lung after transplantation [76, 142] . patients also often have structural damage to the native lung from their underlying disease process, which can increase the risk of other complications such as aspergilloma and pneumothoraces. the median time from transplantation to major native lung complication was 1.28 years (range, 0.04-5.1 years). in one study [68] , 8 of 18 patients with native lung complications died thereof. median posttransplant survival was lower in slt recipient with significant native lung complications (3.2 vs. 5.3 years, p = 0.004). native lung pneumonectomy was performed in 11 patients. in patients with native lung complications there was a trend toward improved posttransplant survival in patients who underwent native lung pneumonectomy compared with those who did not undergo pneumonectomy. however, there was no difference in survival between patients who underwent native lung pneumonectomy to those who had no native lung complication. the majority of patients with ob also have severe bronchiectasis. by specimen bronchograms, the bronchial tree shows alternating areas of dilatation and constriction. microscopically, the bronchiectasis may be associated with areas of mucous plugging, goblet cell hyperplasia, squamous metaplasia, denudation of the bronchial epithelium, submucosal scarring, and acute and chronic inflammation of the bronchial wall. occasionally foreign body giant cells are present, probably representing a manifestation of aspiration. obliteration of the terminal respiratory bronchioles is often observed distal to these areas. the bronchiectasis of lung allografts is probably the result of several factors including immune-related injury, infection, mucostasis, aspiration, and loss of innervation. despite advances in operative management, lung preservation, critical care, and immunosuppression, longterm survival in lung transplantation remains limited. in fact, outcomes for lung transplantation are the worst of any solid organ transplant [75] . the main obstacles to present day lung transplantation involve: (1) lack of donor organs, (2) i/r injury, (3) acute rejection, and (4) development of ob. the increased susceptibility of the lung to injury, infection, and constant environmental exposure with local innate immune activation likely contributes to the high rates of rejection. the ishlt registry reports a 1-year survival rate of 78% and 5-year survival rate of 52% [21] . mortality is highest in the first year, which consistently decreases across subsequent time periods (see table 7 .3). in the first 30 days, graft failure, non-cmv infections, cardiovascular complications, and technical problems account for most of the mortality. after the first year, bos and non-cmv infections were the predominant causes of death. by 5 years, malignancies and cardiovascular causes account for almost 17% of reported causes of death. however, compared with data beginning in 1988, overall survival has consistently improved by era. the improvement in survival is largely due to improvement in the 1 year survival. long term survival has improved as well, although to a seemingly lesser degree. evidence suggests that age, pretransplant diagnosis, and donor cmv status play important prognostic roles. for instance, the survival half-life for patients older than 65 years of age was 3.2 years compared with 6.3 years for those aged 35-49. overall survival rates at 3 months after transplantation are lowest for ipf (86%) and ipah (78%) and highest for cf (91%) and copd (91%), most likely due to differences in early complications, including pgd [21] . cmv seropositivity of the donor is associated with worse survival; however, the underlying reasons for this association are not entirely clear. inducing a state of immune suppression is the key to successful clinical lung transplantation. the immunosuppressive regimens used for lung transplantation are based on successful protocols that have evolved for renal and heart transplantation. posttransplantation morbidities (see table 7 .4) present at 5 years are those commonly caused or exacerbated by immunosuppressive medicines, including hypertension, renal dysfunction, and dyslipidemia. bos and malignancies are other common posttransplantation morbidities. for instance, bos had developed in 28% of patients by 2.5 years after transplantation and in 74% by 10 years. however, most surviving patients reported no activity limitations at 1, 3, 5, and 10 years (>80% at each time point). furthermore, 13% of survivors reported at least one malignancy at 5 years after transplantation, and 28% were affected by malignancies at 10 years. survival after pediatric lung transplantation is similar to that reported in adults with a median survival of 4.5 years for the period 1990-june 2007. but, results are clearly improving [6] . one and 5-year survival rates for pediatric recipients transplanted in the most ob obliterative bronchiolitis recent era (2002-6/2007) are 83 and 50%, respectively, compared with 67 and 43% for recipients transplanted between1988 and 1994. graft failure, technical issues, cardiovascular failure, and infection are the most common causes of pediatric death in the early posttransplant period whereas infection, graft failure and bos are the most common causes of late death. the prevalence of bos steadily increases with time posttransplantation. as expected, the cumulative incidence of malignancy also increases with time after transplantation, with lymphoproliferative disorders making up the great majority of reported malignancies in children. despite the complications, the functional status of the great majority of long-term pediatric survivors is very good, with 84% of 5-year survivors reporting no limitations in activity. a total of 57 pediatric retransplant procedures were reported between january 1994 and june 2008. the majority of these procedure were performed >12 months after the initial transplantation. survival over this period was slightly poorer than for primary transplantations, being 41% at 5 years. prediction of lung-transplant rejection by hepatocyte growth factor development of malignancy following lung transplantation utility of peritransplant and rescue intravenous immunoglobulin and extracorporeal immunoadsorption in lung transplant recipients sensitized to hla antigens posttransplant lymphoproliferative disease in thoracic organ transplant patients: ten years of cyclosporine-based immunosuppression graft-vs.-host disease in lung and other solid organ transplant recipients registry of the international society for heart and lung transplantation: twelfth official pediatric lung and 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immunohistological analysis of transbronchial biopsy specimens predict responder status in early acute rejection of lung allografts? interleukin-10 production genotype protects against acute persistent rejection after lung transplantation the impact of pharmacogenomic factors on acute persistent rejection in adult lung transplant patients key: cord-034402-64xz1j9i authors: srivastava, shivani; sharma, suraj kumar; srivastava, vivek; kumar, ajay title: proteomic exploration of listeria monocytogenes for the purpose of vaccine designing using a reverse vaccinology approach date: 2020-10-29 journal: int j pept res ther doi: 10.1007/s10989-020-10128-1 sha: doc_id: 34402 cord_uid: 64xz1j9i listeriosis is a major foodborne infection provoked by a bacterium known as listeria monocytogenes. it is one of the predominant causes of death in pregnant women, infants, and immunocompromised persons. despite such fatal effects, until now there is no proper medication or drug available for such a serious foodborne infection. one of the most promising ways to deal with this challenge is vaccination. this present study aims at the prediction of b cell epitopes for subunit vaccine designing against listeria monocytogenes using a reverse vaccinology approach. among screened out 299 epitopes of strain f2365 of listeria monocytogenes, based on the vaxijen score, the top 20 epitopes were selected. 3d modeling of epitopes and alleles was generated by pepstrmod and swiss model respectively. molecular docking reveals 4 epitopes viz., mkflfplkl, ceetfgirl, flkidppil, and vrhhggghk based on binding energy. all 4 epitopes were investigated for non-toxicity, binding affinity, and population coverage. after vigorous investigation, epitope flkidppil was anticipated as the best vaccine contender. the stability of the flkidppil-hla drb1 _0101 complex was proved by performing the simulation. here, predicted peptide through the insilico approach may become a potential remedy against listeriosis, after the wet-lab approach and clinical trials. changing food habits, advancement in technology regarding the preservation of food products for a longer time, and the ability of microorganisms to grow in adverse conditions are leading to the emergence of the foodborne infection, known as listeriosis. the genus listeria consists of seventeen species. only the three hemolytic species viz., listeria monocytogenes, listeria seeligeri, and listeria ivanovii are considered pathogens. of these, listeria monocytogenes is consistently pathogenic and is involved in foodborne outbreaks of listeriosis (abdelhamed et al. 2019 ). based on gram-staining, listeria monocytogenes comes under the category of gram-positive. it shows extreme resistance in conditions like very high temperatures or very low temperatures. these bacteria have a rod-like shape and can form small chains (sallami et al. 2006) . listeria monocytogenes mainly affects women who are pregnant, infants, elders above 65 years of age, and immunocompromised people (cdc 2019) . foodborne infection in humans occurs through the consumption of contaminated foods, particularly unpasteurized milk, soft cheeses, vegetables, and prepared meat products. listeria monocytogenes show completely different behavior in comparison to all other pathogens that cause food contamination. it can multiply at low temperatures in contaminated food. it can be easily transmitted between pregnant women and her newborn either at the time of pregnancy or during delivery (who 2019). pyrexia, cough, cold, headache, and body ache, etc. are the usual symptoms experienced by the patients (department of health 2017). worldwide many countries where food production takes place in absence of proper and better microbiological vigilance and where the percentage of immunocompromised persons are immensely high, listeria monocytogenes loomed as one of the dominant foodborne pathogens (thomas et al. 2020 ). thus, poor surveillance during the production process affects approximately 1600 people every year, and around 260 experience the afterlife (cdc 2019). listeria monocytogenes consists of two genes viz., chia and chib. these two genes play an important role in virulence. a regulatory factor hfq plays a very important role in the formation of biofilm, colony formation, and virulence (yao et al. 2018) . the zipper is the name of the mechanism through which listeria monocytogenes get access to the host cell. in this process, ligands on the surface of bacteria communicate with receptors of the host cells. internalin a and internalin b are the ligands on the bacterial surface and e-cadherin and met are the receptors on host cells with which bacterial ligands interact. this collaboration leads to the rearrangement of actin filaments and invasion of bacteria (hamon et al. 2006) . when internalin b-met interacts together, processes like ubiquitinylation and autophosphorylation takes place (veiga and cossart 2005) . in the year 2018, australia had witnessed around 20 cases of listeriosis between january to april. this minor outbreak had faced around 7 deaths and a single spontaneous abortion (who 2018a, b) . national institute of communicable diseases (nicd) has proclaimed 978 listeriosis cases between 2017 and 2018 from all provinces of south africa, gauteng, western cape, and kwazulu-natal were mainly hit by this fatal disease known as listeriosis. around 78% of cases have been reported from the above-mentioned places of south africa. out of 674 affected people, 27 have faced death. all these data revealed about the threat of this bacteria and its effect on mankind and society. the percentage of infants that get affected during an outbreak is around 42% (who 2018a, b) . pregnant women can easily get infected with listeriosis through the placenta, still, the establishment of neurolisteriosis is completely occasional. listeriosis infection in pregnant women is because of the alliance of the quashed immune system and the specificity of bacteria for the placenta (charlier et al. 2017) . even after the birth of infants infected with bacteria listeriosis monocytogenes endurance is possible only with the help of extracorporeal membrane oxygenation (ecmo) (lee et al. 2019 ). according to a report of who, in india miscarriages and other pregnancy-related disorders is mainly the result of foodborne infection known as listeriosis. listeriosis is still under-reported in many countries. the ability of listeria monocytogenes to survive even in harsh conditions is one of the major threats regarding the outbreak of the disease. high fatality rate and frequent outbreaks demand the designing of a vaccine against listeria monocytogenes, by using the immunoinformatics approach. this study is mainly based on the anticipation of b cell epitopes for the utility of vaccine designing against listeriosis. previously, a study regarding computational identification and characterization of epitopes has been carried out in the case of the zika virus, nipah virus, and bacteria like e. coli (sharma et al. 2020; kaushik 2019; khan et al. 2019) . considering this approach in this research work, all proteins except hypothetical, putative, and non-structural were retrieved from the uniprotkb database. a potential epitope must not possess any allergic property; therefore, first and foremost allergenicity was checked by using the algpred server. netmhcii 2.3 and vaxijen server was used to identify b cell epitopes that could bind to mhc ii molecules with great stability. only the top 20 epitopes were selected for further exploration. this selection was done based on the vaxijen score. 3d modeling of both the epitopes and alleles was performed using pepstrmod and swiss model. epitope-allele pair having low binding energy should be selected for the next sequential refining. to do this, molecular docking was performed using autodock vina software. next to check toxicity, binding affinity, and population coverage toxin pred, mhc pred, and immune epitope database tools were used. the stability of the epitope-allele complex was substantiated by simulation studies. the strategy of the development of subunit vaccines has an upper hand in comparison to traditional vaccines. these next-generation vaccines are extremely specific in eliciting the immune system of the host, can be produced easily in large quantities, and at a comparatively moderate cost. moreover, peptides consisting of epitopes can be manufactured, purified, and processed easily (poland et al. 2011 ). for computational identification and characterization of epitopes for the preparation of subunit vaccine designing, complete proteome analysis of listeria monocytogenes f2365 strain (genbank accession number ae017262.2) was performed using the uniprotkb database. in comparison to other serotype strains, listeria monocytogenes strain f2365 belongs to the 4b serotype group and multiplicates more rapidly in monocytes or macrophages (hasebe et al. 2017 ). presence of a virulence factor viz. listeriolysins (lls) in the f2365 strain accelerates infection in the intestine and other organs (quereda et al. 2016) . listeria monocytogenes f2365 strain is a member of lineage i and comprises a factor known as internalin b which plays a crucial role in nonpregnant infected animals (quereda et al. 2018) . all these remarkable features contribute to the pathogenicity of this strain and hence lead to its selection for the study. excluding hypothetical, putative, and non-structural proteins total of 529 proteins were registered in the uniprotkb database, derived from the different research literature. all these sequences were saved in the fasta format for further examination. the length of the genome of the f2365 strain is 3,021,822 bp, with gc content of 37.9% approximately (briers et al. 2011 ). one of the most eminent features in epitope-based vaccine design is that the particular epitope must elicit an immune response only against the target pathogen. taking this point into consideration, the screened epitope must be non-allergen and thus retrieved proteins were differentiated into allergens and non-allergens by using the algpred server (saha and raghava 2006) this server segregates non-allergens from allergens and − 0.4 was selected as the cut-off value. anticipation was done with high accuracy along with sensitivity and specificity of 88.87% and 81.86% respectively. non-allergens were chosen for another characterization and exploration of antigenic sites for the utility of vaccine designing from the proteome of listeria monocytogenes. b cell epitopes are typical peptide remnants that bind to the immunoglobulin and thus it becomes immensely important to screen out such epitopes from complete proteome sequence. to accomplish this objective, netmhcii 2.3 server was used (jensen et al. 2018) . by making use of artificial neural networks, this server predicts the binding of b cell epitopes with hla alleles. in this study, three alleles viz. drb1_0101, drb1_0701, and drb1_1301 and locus hla-dr was chosen. the peptide length was taken at 9 with a threshold set to − 99.9. the potential b cell epitopes were subjected to the vaxi-jen server to select those candidates that can strongly bind with mhc ii molecules (doytchinova and flower 2007) . only epitopes with a score greater than or equal to 1.1 can bind with mhc ii molecules with extreme affinity and be selected. to further proceed with the reverse vaccinology approach, only the top 20 peptides or antigenic sites were chosen. this selection was based on the vaxijen score. following allergenicity and prediction of b cell epitopes, modeling of both epitopes and hla alleles was performed. for the generation of the 3d structure of the selected epitopes, the pepstrmod server was used. it offers exclusive advantages to the users to predict the structures of peptides having natural residues, some modified residues, posttranslational modifications, etc. (singh et al. 2015) . in this research work, filtered epitopes were modeled and saved in the protein data bank (pdb) format for the next sequential investigation. the first fully automated protein homology modeling server known as the swiss model was used for modeling of hla alleles (waterhouse et al. 2018) . the building of models using this server requires four sequential steps. these 4 steps comprise of template selection, its alignment with the target sequence, model building, and its evaluation. in this study 3d structures of three hla alleles have been performed viz., drb1_0101, drb1_0701, and drb1_1301. to better understand the relationship between anticipated epitopes and their respective alleles, autodock vina software was used to perform molecular docking. it helps us to interpret the synergy between antigenic sites and their corresponding alleles (trott and olson 2010) . one of the prerequisites before performing docking is certain modifications both in ligand as well as the receptor, which was performed by autodock mgl tools. hla alleles were selected as receptors viz., drb1_0101, drb1_0701, and drb1_1301. 4ah2, 3c5j, and 6cql are the crystal structure of these receptors and were retrieved from the research collaboratory for structural bioinformatics (rcsb) protein data bank. molecules of water were removed from these receptors and polar hydrogen as well kollman charges were added to the structure. after modification, the molecule was saved in pdbqt format. changes were also performed in all 20 ligands and were saved in pdbqt files. all these alterations were performed by autodock mgl tools. to perform molecular docking in autodock vina software, 40, 40, 40 were taken as grid box dimensions and energy was calculated at 0 å. the docking result can be analyzed by a visualization tool called pymol. 4 epitopes were selected for succeeding rounds of analysis based on negative binding energies where low binding energy implies good stability. to evaluate the non-toxicity behavior of epitopes toxin pred server was used (gupta et al. 2013) . it is based on machine learning techniques and quantitative matrix scores. along with toxicity prediction, calculation of physicochemical properties is one of the most notable features of this server. mhc pred server was used to vaticinate the binding affinity of epitopes with mhc ii molecules. mhc pred is composed of several models based on structures and its activity, a sturdy multivariate statistical method. results with articulated by giving ic 50 values (guan et al. 2003) . ic 50 values less than 500 are considered to be good binders and were chosen for the next and last analysis. because of the exceptionally heterogeneous behavior of hla alleles, their frequency of expression varies greatly across the globe, and therefore population coverage analysis becomes the utmost important step in computational vaccine designing. it was performed using the immune epitope database (iedb) population coverage analysis tool (bui et al. 2006 ). it is extremely essential to understand the stability of the peptide-allele complex and to analyze that in this research work md web server was used (hospital et al. 2012) . simulation of 10 ns with an output frequency of 500 steps was set to equilibrate the system. coarse-grained brownian dynamics were analyzed for trajectory and output was given in the terms of root mean square deviation (rmsd) and b-factor values. both rmsd and b-factor plots corroborate the stability of epitope-allele complex. with time, the world has acknowledged extreme advancement in medicine and technology thus combating some deadly diseases, but still, diseases like listeriosis were left unnoticed. despite several outbreaks in different parts of the world, there is no legitimate treatment or drug or vaccine available for it. therefore, it becomes extremely important to predict and characterize some potential vaccine contenders that can evoke a strong immune response and this study is one such step in this direction. here we have used computational tools to predict b cell epitopes that can elicit an immune response. the first requirement in the reverse vaccinology approach of vaccine designing is to eliminate all non-allergic proteins from a complete proteome set of bacteria, listeria monocytogenes. the algpred server was used to predict allergenicity of retrieved proteins, to get the most capable subunit vaccine candidate. a total of 529 protein sequences of listeria monocytogenes f2365 strain was retrieved from the uniprotkb database (excluding hypothetical, putative, and non-structural proteins) and were saved in the fasta format for further analysis. after examination by the algpred server, out of 529, a total of 172 proteins were proved to be non-allergens (table 1) . the result has been summarized in table 1 . table 1 consists of protein id, protein names, and scores of all non-allergens. non-allergic proteins were analyzed further by using netmhc ii 2.3 server. by selecting peptide lengths 9 and threshold value − 99.9. b cell epitopes were selected. these chosen epitopes were next investigated by the vaxijen server and the cut-off value was 1.1 å total of 299 epitopes were found to bind with mhc ii molecules ( table 2 ). all 299 epitopes have a vaxijen score of ≥ 1.1 and can bind with mhc ii molecules with great stability. among these epitopes, the majority of epitopes were found to bind with drb1_1301. based on the high vaxijen score, among 299 epitopes, only the top 20 epitopes were selected for modeling. the generation of 3d structures of epitopes was performed by pepstrmod. 3d modeling of the hla allele's viz. drb1_0101, drb1_0701, and, drb1_1301 were performed by the swiss model ( fig. 1) . for the generation of tertiary structures of drb1_0101, drb1_0701 and, drb1_1301 alleles, proteins having pdb id 4ah2, 3c5j, and 6cql were used as templates, respectively. all tertiary structures of hla alleles were visualized by the pymol visualization tool. 3d models have been represented in fig. 1 . autodock vina software was used to perform molecular docking between 20 nonallergic and antigenic epitopes with their respective alleles. the lowest binding energy was obtained for epitope flkidppil-drb1_0101 (− 7.3 kcal/ mol) and the highest binding energy was obtained for epitopes mkgqagskk-drb1_1301 (− 5.1 kcal/mol). as low binding energies imply, high stability of the complex, therefore 4 epitopes based on low binding energy was selected viz., ceetfgirl, mkflfplkl, flkidp-pil, and vrhhggghk (table 3 ). the stable complex of ceetfgirl-3c5j shows the energy of − 6.7 kcal/mol and 6 hydrogen bonds ( fig. 2 ) complexes viz. mkflfplkl-4ah2 and flkidppil-4ah2 shows binding energy of − 6.9 kcal/mol and − 7.3 kcal/mol along with 2 and 6 hydrogen bonds respectively (figs. 3 and 4). the energy of − 6.7 kcal/mol and 6 hydrogen bonds was shown by epitope vrhhggghk along with its receptor 6cql (fig. 5) . most promising vaccine aspirants must not cause any kind of toxicity or vigorous reaction inside the host. so, checking of toxic nature of epitopes is notably important. this prominently important step was performed by toxin pred. it was found that all 4 selected epitopes were non-toxic (table 4 ). all epitopes along with their result of toxicity analysis and physicochemical properties like hydrophobicity, hydrophilicity, and molecular weight were summarized in table 4. mhc pred server was used to study the binding affinity of four non-allergic, non-toxic, and antigenic peptides with allele's viz., hla drb1_0101, hla drb1_0401, and hla drb1_0701. binding affinity was depicted in terms of ic 50 value (table 5) . epitopes showing ic 50 value less than 500 nm were considered to be good binders. epitopes viz., ceetfgirl and vrhhggghk were found to bind with hla drb1_0101 and hla drb1_0401, respectively. both flkidppil and mkflfplkl were found to bind with hla drb1_0101 and hla drb1_0701. most eligible vaccine contenders must show satisfactorily population coverage in different parts of the world. both the (fig. 6) . epitope ceetfgirl and vrhhggghk shows population coverage of 11.53% and 11.21% worldwide respectively (figs. 7 and 8) . the final selection of best and most promiscuous vaccine bidders depends on two main factors, one is low binding energy and another one is high population coverage worldwide. based on these two factors, epitope flkidppil was refined. to check the stability of complex flkidppil-4ah2, molecular dynamics simulation was performed by md web simulation. rmsd value of flkidppil-4ah2 was given in between 0.1 and 1.0 å (fig. 9) and b factor scores between 1 and 25 å 2 (fig. 10 ). both rmsd values and b factor plot of complex viz., flkidppil-4ah2 confirm the stability of the epitope. reverse vaccinology is known by different names like computational biology, immunoinformatics, and many more. it is a combination of immunological research as well as experimental and computational science. it includes computational tools and software to study the immune response of the host against various infectious diseases. immunoinformatics helps us to understand antigen presentation in host cells, the behavior of the host during the infection cycle, and thus enriches the knowledge about the disease that affects the immune system and its control (brusic and petrovsky 2005) . with the help of insilico tools, antigenic regions can be mapped easily (davies and flower 2007) . previously, finding these antigenic regions are extremely costly and time-consuming methods like nuclear magnetic resonance (nmr) were used. but today, computational vaccinology had made it possible to predict these antigenic regions in a short period and also with extreme accuracy (potocnakova et al. 2016) . in this exploration and investigation, the prediction of b cell epitopes has been performed by the authors for the designing of the vaccine against listeriosis by using a reverse vaccinology approach. this research work started with the retrieval of a complete proteome sequence of listeria monocytogenes f2365, from the uniprotkb database. most promiscuous b cell epitopes must not show allergic properties. therefore, to remove all allergic proteins from the investigation algpred server was used. a total of 529 proteins of the f2365 strain of listeria monocytogenes have been proclaimed from the uniprotkb database. out of 529 proteins, 172 have shown non-allergenicity. these 172 non-allergic proteins have been used to find out the best antigenic regions or peptides that can provoke great immune inflammation in the human body, by using netmhcii 2.3 server. 299 epitopes have been identified by the vaxijen server that could bind with mhc ii molecules with great stability. based on the vaxijen score, only the top 20 b cell epitopes were selected for succeeding refining. 3d modeling of all 20 epitopes has been performed by pepstrmod and all these tertiary structures have been saved in pdb format. tertiary structure modeling of alleles was generated with the help of hla alleles were performed based on low binding energy, 4 peptides were selected viz., ceetfgirl, mkflfplkl, flkidppil, and vrh-hggghk. ceetfgirl showed the energy of − 6.7 kcal/ mol and 6 hydrogen bonds. mkflfplkl showed the energy of − 6.9 kcal/mol and 2 hydrogen bonds. flkidp-pil showed the energy of − 7.3 kcal/mol and 6 hydrogen bonds. vrhhggghk showed the energy of − 6.7 kcal/ mol and 6 hydrogen bonds. these 4 epitopes were selected on low binding energy as low energy means high stability. most promiscuous b cell epitope which is a nano peptide, must not be toxic and therefore toxicity analysis must be performed. toxin pred server is used for this analysis. this server also anticipates various physicochemical properties of the epitopes like molecular weight, hydrophobicity, and coverage, worldwide epitope flkidppil was selected. to check the binding energy of epitope flkidppil with its corresponding 4ah2 receptor molecular dynamics simulation study was performed by using md web. rmsd and b factor plot was used to interpret the result of the simulation. after all these vigorous steps of the investigation, epitope flkidppil proved to be the most eligible candidate that should be used for vaccine designing. reverse vaccinology has been proved as one of the most powerful weapons to combat some deadly bacterial diseases and had shown tremendous results also. first and foremost, a peptide-based vaccine using the reverse vaccinology approach was created against e. coli in the year 1985 (jacob et al. 1985) . it has been proved effective against tuberculosis (mustafa 2013) and many more pathogenic diseases. the identification of antigenic peptides by using a reverse vaccinology approach has been found effective against staphylococcus aureus (oyama et al. 2019) . from this research work, we found during the identification and characterization of epitopes for the utility of vaccine designing against listeria monocytogenes, the epitope flkidppil was non-allergic, non-toxic, highly antigenic, and can provoke a better immune response. despite major advancements in the field of technology, society and mankind have been plagued by several kinds of lifethreatening diseases. although vigorous research is going on, on several deadly diseases in various parts of the world. but still, some foodborne diseases are under-reported and listeriosis is one of them. in such conditions, computational vaccine technology is one of the best alternatives to deal with such diseases. computational vaccine technology is a boon in research domains as it accelerates the process of epitope screening and vaccine designing and development. it is a branch of vaccinology that is based on the central idea of solving vaccine development by using a computerdriven algorithm. listeriosis is still under-reported in many countries of the world. computational vaccine technology is going to create some awareness and will bring out the best treatment and remedy for the disease. in this research work, after performing molecular docking, 4 epitopes were screened out. these 4 epitopes viz., ceetfgirl, mkflf-plkl, flkidppil, and vrhhggghk were screened as the most promiscuous b cell epitopes among 299 antigenic sites identified. low binding energy and population coverage analysis predicted flkidppil as the most potent epitope. epitope flkidppil can elicit a strong immune response in the host against listeriosis. further wet lab trials can assure the stability as well as the response of the epitope in vitro and in vivo. reverse vaccinology can be proved as the most powerful approach to find remedies against diseases like listeriosis. validation of predicted virulence factors in listeria monocytogenes identified using comparative genomics genome sequence of listeria monocytogenes scott a, a clinical isolate from a food-borne listeriosis outbreak immunoinformatics and its relevance to understanding human immune disease predicting population coverage of t-cell epitope-based diagnostics and vaccines clinical features and prognostic factors of listeriosis: the monalisa national prospective cohort study harnessing bioinformatics to discover new vaccines vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines mhcpred, a server for quantitative prediction of peptide-mhc binding in silico approach for predicting toxicity of peptides and proteins listeria monocytogenes: a multifaceted model listeria monocytogenes serotype 4b strains replicate in monocytes/macrophages more than other serotypes mdweb and mdmoby: an integrated web-based platform for molecular dynamics simulations priming immunization against cholera toxin and e. coli heat-labile toxin by a cholera toxin short peptide-beta-galactosidase hybrid synthesized in e. coli improved methods for predicting peptide binding energy to mhc class ii molecules silico identification of epitope-based peptide vaccine for nipah virus computational identification and characterization of potential t-cell epitopes for the utility of vaccine design against enterotoxigenic escherichia coli outcomes of neonates with listeriosis supported with extracorporeal membrane oxygenation from 1991 to 2017 listeria (listeriosis) | listeria | cdc in silico analysis and experimental validation of mycobacterium tuberculosis-specific proteins and peptides of mycobacterium tuberculosis for immunological diagnosis and vaccine development in silico identification of novel peptides with antibacterial activity against multidrug-resistant staphylococcus aureus vaccinomics and a new paradigm for the development of preventive vaccines against viral infections an introduction to b-cell epitope mapping and in silico epitope prediction bacteriocin from epidemic listeria strains alters the host intestinal microbiota to favor infection reassessing the role of internalin b in listeria monocytogenes virulence using the epidemic strain f2365 algpred: prediction of allergenic proteins and mapping of ige epitopes heat inactivation of listeria monocytogenes and salmonella enterica serovar typhi in a typical bologna matrix during an industrial cooking-cooling cycle kaushik v (2020) in-silico prediction of a peptide-based vaccine against zika virus pepstrmod: structure prediction of peptides containing natural, non-natural and modified residues outbreak of listeriosis in south africa associated with processed meat autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading listeria hijacks the clathrin-dependent endocytic machinery to invade mammalian cells swiss-model: homology modeling of protein structures and complexes an essential role for hfq involved in biofilm formation and virulence in serotype 4b listeria monocytogenes the authors hereby declare they that have no conflict of interest. the authors did not perform any experiments on human or animals. key: cord-017856-4fccnygg authors: roden, anja c.; tazelaar, henry d. title: pathology of lung rejection: cellular and humoral mediated date: 2018-04-24 journal: lung transplantation doi: 10.1007/978-3-319-91184-7_13 sha: doc_id: 17856 cord_uid: 4fccnygg acute rejection is an important risk factor for bronchiolitis obliterans syndrome, the clinical manifestation of chronic airway rejection in lung allograft recipients. patients with acute rejection might be asymptomatic or present with symptoms that are not specific and can be also seen in other conditions. clinical tests such as pulmonary function tests and imaging studies among others usually are abnormal; however, their results are also not specific for acute rejection. histopathologic features of acute rejection in adequate samples of transbronchial lung biopsy of the lung allograft are currently the gold standard to assess for acute rejection in lung transplant recipients. acute alloreactive injury can affect both the vasculature and the airways. currently, the guidelines of the 2007 international society of heart and lung transplantation consensus conference are recommended for the histopathologic assessment of rejection. there are no specific morphologic features recognized to diagnose antibody-mediated rejection (amr) in lung allografts. therefore, the diagnosis of amr currently requires a “triple test” including clinical features, serologic evidence of donor-specific antibodies, and pathologic findings supportive of amr. complement 4d deposition is used to support a diagnosis of amr in many solid organ transplants; however, its significance for the diagnosis of amr in lung allografts is not entirely clear. this chapter discusses the currently recommended guidelines for the assessment of cellular rejection of lung allografts and summarizes our knowledge about morphologic features and immunophenotypic tests that might help in the diagnosis of amr. acute rejection is the host's response to the recognition of the graft as foreign. it can occur days, months, or even years after transplantation. rejection can be divided into cellular and humoral forms. acute cellular rejection is the predominant type of acute rejection of lung allografts. it is mediated by t lymphocytes that recognize foreign human leukocyte antigens (hla) or other antigens [1, 2] . humoral rejection is mediated by preformed or de novo recipient antibodies (therefore, also referred to as antibody-mediated rejection [amr]) against antigens of the donor organ cells. acute rejection is an important complication in patients with lung allografts. twenty-nine percent of adult patients have at least one episode of treated acute rejection between discharge from the hospital and 1 year after transplantation [3] . moreover, 3.6% and 1.8% of all deaths that occur within the first 30 days or between 30 days and 1 year following lung transplantation are due to acute rejection, respectively [3] . in addition, the frequency and severity of acute rejections are thought to represent the major risk factor for the subsequent development of bronchiolitis obliterans syndrome (bos) [1, [4] [5] [6] . hla mismatch, genetic and recipient factors, type of immunosuppression, vitamin d deficiency, and infection are risk factors of acute rejection. for instance, the recipient alloimmune response is thought to be related to the recognition of differences to donor antigens leading to acute lung allograft rejection. indeed a higher degree of hla mismatch has been shown to increase the risk of acute rejection although this effect is not consistent across all hla loci or studies [4, [7] [8] [9] [10] . mismatches at the hla-dr, hla-b [7] , and hla-a [8] loci, as well as a combination of all three loci [9] , appear specifically important. for instance, acute rejection within 2 months after transplantation has been shown to be associated with hla-dr mismatch, while acute rejection at 4 years has been found to be associated with hla-b mismatch [11] . several host genetic characteristics have been studied that may modulate acute lung rejection. for instance, a genotype leading to increased il1-production may protect against acute rejection [12] , while a multidrug-resistant genotype (mdr1 c3435t) appears to predispose to persistent acute rejection that is resistant to immunosuppressive treatment [13] . the incidence of acute rejection appears to be age-dependent, with the lowest incidence of acute rejection in infants (< age 2) [14] . however, children have a higher risk for acute rejection than adults [15] . furthermore, the registry of the international society of heart and lung transplantation (ishlt) showed that the incidence of acute rejection between discharge and 1-year follow-up was slightly higher in younger adult lung allograft recipients (age 18-34 years) (36%) [16] when compared to the entire adult population in which 29% had at least one acute rejection episode [3] . the incidence of acute rejection does not seem to change in older lung transplant recipients (age 65 and higher) [17] . regimens of immunosuppression might also play a role in acute rejection. for instance, the rate of acute rejection in the first year after transplantation was highest among recipients who were on cyclosporine-based regimens and lowest among those on tacrolimus-based regimens [18] . vitamin d deficiency might also play a role in acute rejection. a study found that 80% of lung recipients were 25(oh)d deficient around the time of transplantation and that vitamin d-deficient recipients had more episodes of acute cellular rejection and infection [19] . a similar association between vitamin d deficiency and acute rejection has been described in other solid organ recipients including the liver, kidney, and heart. although the exact mechanism for this phenomenon is not entirely clear, it is speculated that (1) vitamin d might slow down the maturation of antigen-presenting cells as in vitro studies have shown, (2) vitamin d might induce dendritic cells to acquire tolerance, and/or (3) a synergistic effect between vitamin d analogs and immunosuppressants occurs [19] . viral infections have also been thought to modulate the immune system and to increase alloreactivity. indeed, a high incidence of acute rejection has been found in lung transplant recipients after community-acquired respiratory tract infections with human influenza virus, respiratory syncytial virus, rhinovirus, coronavirus, and parainfluenza virus [20] [21] [22] . chlamydia pneumoniae infection has also been linked to the development of acute rejection in one study [23] . the significance of cmv infections and the impact of cmv prophylaxis strategies on acute rejection frequency are not clear at this time [24] . the clinical course of acute rejection can be variable. acute rejection is often identified on surveillance transbronchial biopsy in an asymptomatic patient. if symptoms occur, they might be non-specific and overlap with those seen in other complications and diseases in this patient population. these symptoms might include dyspnea, fever, leukocytosis, and a widened alveolar-arterial oxygen gradient. higher-grade rejection appears to cause more severe symptoms and can lead to acute respiratory distress [17] . in patients with rejection, pulmonary function testing may show a decrease in forced expiratory volume in 1 s (fev 1 ) and vital capacity (vc). although spirometry has a sensitivity of greater than 60% for detecting infection or rejection of grade a2 and higher, it cannot differentiate between the two [25] . furthermore, the usefulness of spirometry is diminished in single lung transplant recipients, as the dysfunction of the native lung confounds the pulmonary function test results [26] . although in approximately half of the cases of acute rejection, chest x-ray studies are normal, ill-defined perihilar and lower lobe opacities, along with septal lines and pleural effusions, may be seen. findings on ct scan might include ground-glass opacities, septal thickening, volume loss, nodules and consolidation, and pleural effusions. infiltrates observed on imaging studies during the first week after lung transplantation are usually caused by the reimplantation response, i.e., reperfusion edema and other factors. infiltrates that persist beyond the first week following transplantation suggest acute rejection or infection. however, although early, the authors of small studies have attempted to demonstrate the usefulness of chest x-rays and chest ct scans in the diagnosis of rejection, more recent data show a very low sensitivity for acute rejection (as low as 35%) and no discriminatory value between rejection and other processes [27] . exhaled nitric oxide (no) can also serve as a marker of lung injury; it is often increased in patients with lymphocytic bronchiolitis and acute rejection [28] [29] [30] . furthermore, in a study of inert gas single-breath washout, the slope of alveolar plateau for helium had a sensitivity of 68% for acute rejection [25] . although the presentation of the patient and several ancillary studies may suggest the presence of acute allograft rejection, none of these findings are specific. therefore, tissue diagnosis is necessary for a definitive diagnosis. histopathology of adequate lung biopsy samples obtained from transbronchial biopsy is currently the gold standard to assess lung allografts for rejection and to distinguish rejection from its clinical mimickers such as aspiration, infection, drug toxicity, and recurrent disease. recently, the transbronchial cryobiopsy technique was introduced which yields larger biopsies containing more alveoli, small airways, and veins and venules while exhibiting less procedural alveolar hemorrhage and crush artifact than conventional forceps transbronchial allograft biopsies [31] [32] [33] . although cryobiopsies appear to be as safe as forceps biopsies, complications can occur which is one of the reasons that this technique has so far not been universally performed for this purpose [31] . other lung tissue specimens from lung allografts include wedge biopsies, explants for retransplant, or autopsy specimens from lung transplant recipients. wedge biopsies, although seldom obtained in clinical practice, and specimens from explants provide useful histopathologic insights into the etiology of lung allograft dysfunction in advanced stages following all possible medical interventions. cellular alloreactive injury to the donor lung affects both the vasculature and the airways [34] . perivascular mononuclear cell infiltrates are the hallmark of acute cellular rejection. these infiltrates may be accompanied by subendothelial chronic inflammation (e.g., endotheliitis or intimitis) and also by lymphocytic bronchiolitis, which is characteristic of small airway rejection. the histologic changes are divided into grades based on intensity of the cellular infiltrate and the occurrence of an accompanying acute lung injury pattern. in 1990, the ishlt sponsored the lung rejection study group (lrsg), a workshop to develop a "working formulation" for the diagnosis of lung rejection by transbronchial biopsy [35] . since then the grading scheme has been revised twice, in 1996 [36] and 2007 [34] . the grading scheme is strictly pathologic, based on morphologic features recognized in transbronchial biopsies of the allograft. clinical parameters are not considered. due to overlapping histologic features between acute rejection and infection, the grading scheme relies on the absence of concurrent infection. furthermore, infection and rejection may occur together. therefore, the lrsg recommends grading rejection only after the rigorous exclusion of infection [34] . the most recent classification of lung allograft biopsies is the 2007 ishlt consensus classification of allograft rejection [34] (table 13.1 ). an attempt should be made to accurately distinguish the grade of rejection since treatment is largely dependent on the histologic grade assessed by an experienced pulmonary pathologist familiar with the histopathologic features and criteria used for grading. however, inter-and intra-observer variability in grading can impact treatment and outcome [37, 38] . two studies using the 1996 grading system found relatively good interobserver agreements for the a grades (kappa of 0.65 and 0.73) [37, 38] ; however, these results could not be replicated in another study in which the kappa was 0.47 in spite of dichotomization of the a grades to a0/a1 versus a2-4 [39] . intraobserver agreement for acute rejection has been found to be good with kappa values of 0.65 and 0.79 [37, 39] . using the revised 2007 ishlt classification, bhorade and colleagues showed an overall concordance rate of 74% for grade a and 89% for grade b specimens between a site pathologist and a central pathologist [40] . however, the weighted kappa scores in that study showed only fair to moderate agreement for a grades (kappa values varied between 0.22 and 0.48) and less than a chance agreement to moderate agreement for b grades (kappa values varied between −0.04 and 0.46). interestingly, the kappa values for a and b grades were dependent on the time that had elapsed between transplantation and biopsy. the best agreement occurred in biopsies taken within 6 weeks of transplant. slightly higher agreements (81% and 93%, for a and b grades, respectively) were shown in a study that evaluated the interobserver agreement between two transplant pathologists from the same institution using the 2007 revision grading scheme [31] . although cryobiopsies are larger and appear to be easier interpretable, interobserver reproducibility did not improve with the use of cryobiopsies in that study [31] . acute rejection is defined by the presence of perivascular mononuclear cell infiltrates with or without endotheliitis [34] . with progression, this infiltrate becomes more widespread and extends into the alveolar septa and, subsequently, into the alveoli. the majority of the mononuclear cells in acute rejection are t cells, although a few studies have described increased populations of b cells or eosinophils [34, 41, 42] . the histologic features of rejection are summarized in table 13 .1. features of acute cellular rejection are lacking, although the biopsy may not be entirely normal. scattered infrequent blood vessels, particularly venules, in the alveolated lung parenchyma are surrounded by a relatively thin (ring of two to three layers) chronic mononuclear cell infiltrate ( fig. 13.1a , b). the lymphocytic rim can be loose or compact and is in general circumferential but does not spill into the adjacent interstitium. endotheliitis and eosinophils are absent. in adequately alveolated and artifact-free speci-mens, the lymphocytic infiltrates may be detected at low magnification, but often higher power study is needed to identify the infiltrates. although in mild acute rejection the perivascular infiltrate of lymphocytes is still confined to the perivascular adventitia without infiltrating the adjacent interstitium or air spaces, there are more layers of lymphocytes surrounding vessels ( fig. 13.2a, b ). in addition, the perivascular mononuclear infiltrates surrounding venules and arterioles are more frequent than in grade a1. they are typically recognizable at low magnification. these infiltrates usually consist of a mixture of small round lymphocytes, activated lymphocytes, plasmacytoid lymphocytes, macrophages, and eosinophils. the cellular infiltrates can be compact or loose. subendothelial infiltration by mononuclear cells may be noted which can be associated with hyperplastic or regenerative changes in the endothelium. concurrent lymphocytic bronchiolitis may be seen. venules and arterioles are cuffed by easily recognizable dense perivascular mononuclear cell infiltrates that are commonly associated with endotheliitis ( fig. 13 .3a-c). eosinophils and even occasional neutrophils are common. in a b moderate acute rejection, the inflammatory cell infiltrate extends into the adjacent alveolar septa where it can be associated with type ii pneumocyte hyperplasia. the inflammatory infiltrate can also extend into adjacent airspaces and be associated with collections of intra-alveolar macrophages and lymphocytes. histologic features of acute lung injury may become apparent in the form of airspace fibrin. in severe rejection, there are diffuse perivascular, interstitial, and air space infiltrates of mononuclear cells with prominent alveolar pneumocyte damage and endotheliitis ( fig. 13 .4a-f). this may be associated with necrotic intra-alveolar epithelial cells, hemorrhage and neutrophils, and usually morphologic evidence of acute lung injury in the form of organizing pneumonia, fibrin deposition, or hyaline membranes. parenchymal necrosis, infarction, or necrotizing vasculitis may be identified; however, these features are more evident on surgical rather than transbronchial lung biopsies. it should be noted that a paradoxical diminution of perivascular infiltrates can occur as cells extend into interalveolar septa and air spaces where they are admixed with macrophages. protocol surveillance biopsies of lung allografts are performed in many institutions. even though these patients are in general asymptomatic and clinically stable, one study showed that 39% of surveillance biopsies reveal acute cellular rejection with 43% showing features of minimal rejection, 49% mild rejection, and 8% moderate rejection [43] . a more recent prospective study identified morphologic findings of acute cellular rejection only in 6% of surveillance biopsies [44] , while a retrospective study of 592 a b surveillance biopsies taken within 400 days of transplantation revealed histologic findings of either acute cellular rejection or obliterative bronchiolitis in 31% of biopsies with 36% within the first 100 days and 25% between 100 and 400 days following transplantation [45] . evidence suggests that acute cellular rejection is an important risk factor for the development of bos [24] . indeed, studies have demonstrated an increased risk of bos with single episodes, increased frequencies, and increased severity of acute cellular rejection. moreover, patients with multiple episodes of even minimal acute cellular rejection were shown to be at increased risk for bos [46] , and yet a single episode of minimal acute rejection without recurrence or subsequent progression to a higher grade has been identified as an independent significant predictor of bos [47] . because of these findings, patients who are asymptomatic but are found to have acute cellular rejection (even minimal acute cellular rejection) on a surveillance allograft biopsy might be treated accordingly. however, several centers do not utilize surveillance transbronchial lung biopsies and/or treat asymptomatic patients with no clinical evidence of allograft dysfunction. prospective well-designed clinical studies are needed to provide evidence to support surveillance transbronchial lung biopsies and therapeutic interventions. this grade applies only to small airways such as terminal or respiratory bronchioles. bronchi, if present, should be described separately. it is important to mention in the pathology report whether or not small airways are present. if no small airways are identified or the biopsy has obvious infection, the grade "bx" should be used. the r behind grades 1 and 2 denotes the revised 2007 version. the small airways appear unremarkable without evidence of bronchiolar inflammation. low-grade inflammation is characterized by lymphocytes within the submucosa of the bronchioles ( fig. 13 .5a-c). the lymphocytic infiltrates can be infrequent and scattered or form a circumferential band; however, intraepithelial lymphocytic infiltration is not present. occasional eosinophils may be seen within the submucosa. there is no evidence of epithelial damage, neutrophils, necrosis, ulceration, or significant amount of nuclear debris. in high-grade small airway inflammation, there is marked lymphocytic infiltrate of the airway epithelium and airway wall. the mononuclear cells in the submucosa appear larger, and a greater number of eosinophils and plasmacytoid cells can be seen (fig. 13.6a-c) . in addition, there is evidence of epithelial damage including necrosis, metaplasia, and marked intraepithelial lymphocytic infiltration. in its most severe form, high-grade airway inflammation is associated with epithelial ulceration, fibrinopurulent exudate, cellular debris, and neutrophils. it is important to exclude an infectious process, especially if the number of neutrophils is disproportionally high when compared to other mononuclear cells within the airway wall. small airways might not be evaluable for several reasons including lack of small airways due to sampling problems, infection, tangential cutting, artifact, etc. in patients who are known to have an infection that could cause lymphocytic bronchiolitis, the allograft biopsy should also be classified as ungradeable for small airway rejection. chronic airway rejection is restricted to submucosal and intraluminal scarring of small airways including terminal and respiratory bronchioles. when large tissue sections of the lung are examined, obliterative bronchiolitis may be recognized as a panlobar process but is usually patchy. the small airways appear similar in size to the accompanying artery with a ragged inner surface. fibrosis is not present. narrowing of the small airways due to fibrosis in the airway wall is the hallmark of chronic airway rejection. the fibrosis may be eccentric or concentric. the type of fibrosis depends on the acuteness of the process, the degree of organization, and the amount of accompanying inflammation. the fibrosis can range from loose myxoid granulation tissue with variable numbers of inflammatory cells filling or partially obstructing the airway lumen in the more acute phase (fig. 13 .7a) to dense hyalinized collagen in the wall of bronchioles that is a characteristic of the chronic phase ( fig. 13.7b) . metaplastic squamous or cuboidal epithelium may overly the bronchiolar fibrosis. sometimes, only a slit-like lumen of the airway may remain as a result of a confluent submucosal scar or intraluminal polyps of scar tissue. there may be rather prominent capillaries supplying the intraluminal fibrotic areas. ultimately, the bronchiolar lumen might be entirely occluded by dense scar tissue (fig. 13.7c, d) . in these cases, only an elastic stain highlighting residual elastic tissue, the vicinity of the scar to a pulmonary artery, and residual smooth muscle may indicate that a small airway has been replaced by fibrotic scar. in the chronic phase, inflammation may be minimal or absent. usually, the scarring process is confined exclusively to respiratory bronchioles and terminal bronchioles, although it may occasionally involve adjacent alveoli. obliterative bronchiolitis is only infrequently identified in lung allografts by transbronchial biopsy, and the sensitivity of this morphologic finding for the presence of chronic rejection is only between 15 and 28% [48] [49] [50] . in a recent study, all seven conventional transbronchial biopsies that were included from patients clinically known to have bos, the clinical equivalent to morphologic obliterative bronchiolitis, failed to reveal morphologic findings of obliterative bronchiolitis [31] . although cryobiopsies contained more small airways, all nine cryobiopsies that were also included in that study from patients with clinically proven bos did not reveal obliterative bronchiolitis in the tissue [31] . this low sensitivity is largely due to sampling and its patchy nature. therefore, bos is used and more reliable for the clinical assessment of chronic airway rejection. bos is calculated as <80% fev 1 in at least two consecutive lung function tests of the patient's maximum fev 1 posttransplantation [51] . despite the low sensitivity of transbronchial biopsies for obliterative bronchiolitis, the specificity of this morphologic finding in an allograft biopsy is high, ranging from 75 to 94% [49, 50] . therefore, an attempt to diagnose obliterative bronchiolitis should be made in lung allograft biopsies. the pulmonary arteries appear of a similar size as the accompanying airways. the intima is slender and the media not thickened. chronic vascular rejection rarely is identified on biopsies since they usually lack vessels of sufficient size. wedge biopsies, explants, or autopsy material may reveal it. therefore, according to the ishlt, the d grade of rejection is not applicable to allograft transbronchial biopsies. although cryobiopsies contain a higher number of venules and small veins, in a recent small study, no difference was found in the number of cases with possible vascular rejection when compared to transbronchial biopsies [31] . vascular rejection is characterized by thickened pulmonary arteries and more often veins, due to fibrointimal connective tissue ( fig. 13.8a, b) . also, thickening is usually concentric. chronic vascular rejection may be patchy. chronic vascular rejection usually starts with intimal proliferation. subsequently, the internal elastic lamina may become fragmented and discontinuous. occasionally the underlying muscular wall becomes thinned. in approximately half of the reported cases, a concurrent endovasculitis has been observed. the process is similar in pulmonary veins, although the intimal deposits may be less cellular and more waxy, eosinophilic, and sclerotic. recanalized thrombi may mimic chronic vascular rejection. in contrast to heart allografts, chronic vascular rejection in lung transplants has not resulted in graft loss; however, some patients develop pulmonary hypertension particularly those with bos [52, 53] . infection can mimic acute cellular rejection. for instance, viral infection, particularly cmv ( fig. 13 .9a-e) but also pneumocystis jirovecii pneumonia, can be associated with perivascular mononuclear cell inflammation mimicking acute cellular rejection [54] . infection can also cause small airway inflammation imitating lymphocytic bronchiolitis. mimickers of severe acute rejection include conditions that might present with acute lung injury or diffuse alveolar damage. these conditions include infection, drug toxicity, aspiration, amr, or harvest/reperfusion injury. the presence of perivascular inflammation is helpful in establishing the diagnosis of rejection. however, perivascular inflammation is not entirely specific for acute rejection, and many other conditions may simulate or mimic alloreactive lung injury [54] . marked perivascular and/or peribronchiolar mononuclear infiltrates might also raise the possibility of posttransplantation lymphoproliferative disease (ptld), and in such cases, an appropriate workup should be performed, including doing studies for epstein-barr virus, which is ubiquitous in ptld. further differential diagnosis of perivascular and interstitial infiltrates include recurrent primary diseases. small airway rejection and the perivascular infiltrates of grade a rejection should be distinguished from bronchiolar-associated lymphatic tissue (balt) . balt is found in the vicinity of airways, usually contains black anthracotic pigment, and presents as a rather nodular collection of chronic inflammatory cells which does not surround a vessel (fig. 13.10 ). epithelial injury, neutrophils, or eosinophils should not be seen in balt collections [34] . originally recognized in kidney transplant patients who presented with acute allograft rejection, anti-donor antibodies, and poor prognosis [55] , amr is now well established in kidney and heart allografts. in lung transplantation, amr is still an evolving concept but likely explains acute and chronic graft dysfunction/failure in a subset of patients. evidence suggests that amr occurs due to circulating antibodies that are either (1) preformed because of pregnancy, blood transfusion, or previous organ transplantation or (2) arise de novo after transplantation due to hla mismatch. furthermore, the recent development of very sensitive and specific solid-phase flow cytometry and luminex-based methodologies has allowed for more accurate detection of antibody specificities in sensitized recipients, and it has become clear that more patients than previously expected have or develop preformed anti-hla antibodies. immune stimulation by prior infections or autoimmunity may also contribute to the development of antibodies in those patients with no identifiable risk factors. overall, these preexisting or de novo antibodies can react with donor antigens, leading to immediate graft loss (hyperacute rejection), accelerated humoral rejection, and/or bos [56] . in addition, recent studies have consistently demonstrated an increased incidence of acute rejection (a threefold increase in one study) [57] , persistent rejection, increased bos [58] , or worse overall survival [59] in patients with anti-hla antibodies. this effect is seen both with pretransplant hla sensitization and with the development of de novo anti-hla donor-specific antibodies after transplantation [58] . about 10-15% of lung transplant recipients are pre-sensitized to hla antigens [60] . even though "unacceptable antigens" are avoided during the virtual crossmatch, patients with positive pretransplant pra are at higher risk for posttransplant complications. their posttransplant pra can stay stable or increase via generation of either donor-specific or non-donor-specific anti-hla antibodies. similarly, patients that had negative pra screening tests before transplantation can develop de novo non-donor-specific or donor-specific anti-hla antibodies after transplantation. the mechanisms by which antibodies promote lung allograft injury remain poorly understood. antibody binding to allo-hla or other endothelial or epithelial targets in the lung allograft can activate the complement cascade. complement deposits lead to endothelial cell injury, production of proinflammatory molecules, and recruitment of inflammatory cells. complement-independent antibody-mediated mechanisms can also induce endothelial cell activation without cell injury, leading to increased gene expression and subsequent proliferation [56] . furthermore, as demonstrated by in vitro studies, anti-hla antibodies can cause proliferation of airway epithelial cells as well, producing fibroblast-stimulating growth factors [61] , potentially contributing to the generation of obliterative bronchiolitis. although the diagnosis of amr in lung allograft biopsies remains challenging, when the triple test criteria are met (graft dysfunction, positive panel reactive antibodies, and evidence of complement deposition in the graft), the disease can be life-threatening, and prognosis can be poor. although the optimal treatment of amr in the lung is currently not known due to the lack of clinical trials, treatment is typically comprised of plasmapheresis, possibly intravenous immunoglobulin (ivig), and medications such as rituximab and bortezomib, among others. as such, the associated histopathologic and clinical parameters are the subject of intense investigation. deposition of complement 4d (c4d), a complement split product, on the capillary endothelium has been suggested as a surrogate marker for amr in heart, kidney, and pancreas transplants [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] . however, the role of c4d deposition in the diagnosis of amr in lung allografts is still unclear. moreover, reproducibility of c4d deposition in allograft lung tbbx is problematic, even among pathologists who routinely evaluate c4d in lung allograft biopsies [72] . furthermore, there are currently no specific or sensitive morphologic features of amr in lung allografts, although some features that are more commonly identified in these patients have emerged in some recent studies [73] . studies have attempted to evaluate immunoglobulins (ig) and complement deposits in the subendothelial space. septal capillary deposits of igs and complement products such as c1q, c3d, c4d, and c5b-9 have been described in association with anti-hla antibod-ies [74, 75] as well as allograft dysfunction and bos [76, 77] . however, except for c4d and in some institutions c3d, these studies have in general not been implemented for the workup of lung transplant biopsies for possible amr. one of the reasons for the difficulties in lung is the relatively high background that is encountered in immunohistochemical as well as immunofluorescence studies. often, c4d binds to the vascular elastic lamina or shows other non-specific binding such as intracapillary serum. staining is commonly only focal, and, therefore, sensitivity and specificity have not been established. only linear, continuous luminal endothelial staining of capillaries, arterioles, and/or venules by c4d should be interpreted as positive. in addition, c4d is not specific to amr but also can be seen in infection, and harvest/reperfusion injury, or any process that is associated with complement activation. in general, the concept of specific histopathologic features associated with amr remains controversial in lung transplantation. the 2007 ishlt revised consensus classification [34] did propose histopathologic features that might be specific for amr. because of the lack of specific histologic findings of amr, a multidisciplinary approach to the diagnosis was recommended that includes the following: (1) the presence of circulating antibodies (hla antibodies, anti-endothelial and anti-epithelial antibodies), (2) focal or diffuse c4d deposition (fig. 13 .11a-c), (3) histologic features of acute lung injury or hemorrhage (diffuse alveolar damage, capillary injury associated with neutrophils and nuclear debris, i.e., capillaritis), and (4) clinical signs of graft dysfunction [78] . in 2013, the pathology council of the ishlt published findings in a summary statement with recommendations for the pathologic evaluation of amr [78] . this report included suggestions for protocol biopsies with serologic evaluation for donor-specific antibodies (dsas) at or near time of biopsy. in addition, this statement included recommendations for histopathologic patterns in amr (fig. 13. 12a-e) and indications for immunohistochemical or immunofluorescence studies to further elucidate findings in amr (box 13.1). the morphologic features were confirmed by the 2016 consensus report of the ishlt [79] . the 2016 consensus report confirmed the need for a multidisciplinary approach to establish a diagnosis of amr in the lung that "integrates the clinical presentation with available immunologic and pathologic diagnostic tools" [79] . an amr staging was also proposed (table 13. 2) [79] . recently, wallace and colleagues reported findings of the banff study of the pathology of allograft lungs with dsa [73] . nine experienced lung transplant pathologists from multiple institutions performed digital slide interpretation to study transbronchial biopsy specimens from patients with known antibody status (established within 30 days of biopsy) and negative infectious workup. the study demonstrated that biopsies from patients with dsa more commonly showed morphologic features of acute lung injury with or without diffuse alveolar damage than biopsies from patients with non-dsa or no circulating antibodies. endotheliitis was more common in patients with dsa than patients without circulating antibodies. however, there was no difference in occurrence of endotheliitis between biopsies from patients with circulating non-dsa vs dsa or non-dsa vs no circulating antibodies. specimens associated with dsa had a significant higher frequency of capillary inflammation, including neutrophilic margination, increased neutrophils, or capillaritis with karyorrhexis than patients with non-dsa or no circulating antibodies. c4d staining was positive in less than 50% of capillaries in 14% of biopsies and in more than 50% of capillaries in 7% of biopsies. while there was no difference between the groups in biopsies with <50% staining, biopsies with dsa more often had over 50% capillaries staining for c4d than biopsies without any circulating antibodies. there were no significant differences identified between hla classes of the dsa and any of the evaluated pathologic findings. taken together, this study identified capillary inflammation, acute lung injury, and endotheliitis as morphologic features in lung allograft biopsies that correlate with the presence of circulating dsa. however, none of these histopathologic features were specific to patients with dsa. morphologic findings of acute lung injury with diffuse alveolar damage had the highest odds ratio for the presence of circulating dsa. this study also cautioned the usefulness of c4d immunohistochemical stain for the diagnosis of amr in lung allografts because of its infrequent diffuse positivity. although the study shows that some morphologic features correlate with the presence of circulating dsa and, therefore, might be histopathologic markers to at least suggest the possibility of amr, the reproducibility of these morphologic features is quite problematic even among experienced lung transplant pathologists. in fact, the interobserver reproducibility kappa values ranged between 0.14 and 0.4, indicating a less than a chance to moderate agreement. the lowest agreement was noted for suspicion for aspiration (median kappa, 0.14) and the highest for acute cellular rejection, alveolar hemosiderosis, and c4d staining (median kappa, 0.4, all). although a definite diagnosis of amr seems to elude pathologic interpretation at the current time, in a fully contextualized clinical environment, the findings from the biopsy specimen may aid the clinician to make a reasonable diagnosis of amr if other relevant clinical and serologic features are present. the proposed "triple test" [78] of clinical features, serologic evidence of dsa, and pathologic findings supportive of amr including capillary inflammation, acute lung injury with or without diffuse alveolar damage, and endotheliitis may currently be the best guide to the diagnosis of amr. there is no ihslt recommendation at this time regarding the coexistence of amr and acute rejection, but it clearly does occur. hyperacute rejection is a severe form of amr mediated by preexisting antibodies to abo blood groups, hla class i or ii, or other antigens on graft vascular endothelial cells. this rejection occurs within minutes to a few hours after the transplanted organ begins to be perfused. as in any form of amr, the preexisting antibodies can result from previous pregnancies, blood transfusions, or previous transplant, and their binding to donor antigens provokes complement and cytokine activation resulting in endothelial cell damage and platelet activation with subsequent vascular thrombosis and graft destruction. the outcome is commonly fatal. in hyperacute rejection, lungs are edematous, cyanotic, and heavy, have a firm consistency, lack crepitation, and show red hepatization [80] [81] [82] [83] . the cut surface reveals patchy poorly defined areas of hemorrhagic consolidation. anastomoses are intact and typically widely patent. histologically, alveolar hemorrhage, platelet and fibrin thrombi, neutrophilic infiltration, necrosis of vessel walls, and diffuse alveolar damage are observed [76-80, 83, 84] . c4d deposition has been described. although hyperacute rejection is a wellknown complication in kidney and heart transplantations, in lung transplantation, it appears to be rather rare with only eight cases reported. six patients died within 1 h and 13 days after transplantation [80] [81] [82] [83] [84] [85] . only two patients survived [86, 87] . one of these two patients was treated with plasmapheresis, antithymocyte globulin, and cyclophosphamide immediately after hyperacute rejection was diagnosed [86] . the other patient was highly presensitized when he underwent double lung transplantation [87] . this patient was treated with multiple plasma exchanges and intravenous immunoglobulin pre-and posttransplantation together with posttransplant rituximab and bortezomib and later with anti-c5 antibody and eculizumab. although in pretransplant, panel reactive antibodies (pras) were negative in four of the eight reported patients, crossmatch was positive in all reported cases. collectively, although hyperacute rejection is rare after lung transplantation, one should keep this reaction in mind given that false-negative pras may occur and pretransplantation crossmatch is not often possible [80] . at least five pieces of well-expanded alveolated parenchyma are required for adequate evaluation of a transbronchial lung allograft biopsy specimen for acute rejection by the lrsg [34] . this specimen requirement was based on the "uniform opinion of the consensus meeting." to ensure that the minimum number of required pieces of alveolated lung parenchyma is available for pathology review, it is recommended that the bronchoscopist needs to take more than five pieces. even more pieces might be necessary to provide small airways for review. interestingly, a prospective 12-month single-operator study by scott and colleagues [88] including 219 transbronchial allograft biopsies with 6 to 56 samples per procedure (mean 17.3 samples per procedure) taken from 3 lobes (or 2 lobes and the lingula of 1 lung) of 54 heart-lung transplant and 2 single lung transplant recipients revealed a sensitivity of 94% and a specificity of 90% for identification of rejection by histopathology. this study estimated that 18 samples per procedure are needed to have a 95% confidence of finding rejection. therefore, false-negative results due to patchy distribution of acute rejection are likely not uncommon. the absence of histologic and immunophenotypic features of acute rejection or antibody-mediated rejection requires clinicopathologic correlation as a negative biopsy does not necessary rule out rejection. furthermore, the bronchoscopist should be familiar with imaging studies, especially high resolution computed tomography studies if available, and aim to sample radiologically abnormal bronchopulmonary segments. if such imaging was not recently performed or the results are normal, then samples should be obtained from different lobes to try to minimize sampling error. specimens should be gently agitated in formalin to open up the alveoli. there is currently no recommendation for cryobiopsies. in a recent study using cryobiopsies to evaluate rejection in lung allografts, a median of three pieces provided twice as many alveoli and small airways than a median of ten pieces by conventional forceps biopsy [31] . the ishlt recommends a minimum of three levels from the paraffin block for hematoxylin and eosin (h&e) staining for histologic examination [34] . in addition, "connective tissue stains" such as trichrome or verhoeff-van gieson (vvg) stain are recommended to evaluate airways for the presence of submucosal fibrosis and vessels for graft vascular disease. stains for microorganisms including gomori-grocott methenamine silver stain (gms) and acid fast bacilli (afb) may be added. while silver stains are routinely performed on lung allograft biopsies in some institutions, they are currently not mandated by the lrsg because many microbiologic, serologic, and molecular techniques are available and used to identify infections in these patients [34, 89] . bal may be performed at the time of biopsy and is useful for the exclusion of infection but currently has no clinical role in the diagnosis of acute rejection. the transbronchial allograft biopsy is currently the gold standard to evaluate the graft for cellular rejection and to exclude its clinical mimickers in lung transplant patients. when reviewing transbronchial biopsy material of these patients, attention must be paid not only to features of rejection but also to its morphologic mimickers, especially infection, ptld, and abnormal drug effect. before a diagnosis of acute cellular rejection can be rendered, an infectious process should be excluded by using stains for microorganisms and/ or clinical tests including cultures of bal and/or tissue and serology. while studies to identify histopathologic and immunophenotypic features of amr are evolving, there are currently no specific morphologic findings, and clinical and serologic correlations are required for the diagnosis. prospective, well-designed long-term studies with longitudinal data of therapeutic intervention of acr on histopathology in totally asymptomatic patients with no physiological or hrct evidence of allograft dysfunction are needed to determine the clinical significance and relevance of such interventions. acute rejection and humoral sensitization in lung transplant recipients evidence for immune responses to a self-antigen in lung transplantation: role of type v collagen-specific t cells in the pathogenesis of lung allograft rejection the registry of the international society for heart and lung transplantation: thirty-second official adult lung and heart-lung transplantation report-2015; focus theme: early graft failure acute allograft rejection: cellular and humoral processes severity of lymphocytic bronchiolitis predicts long-term outcome after lung transplantation bronchiolitis obliterans syndrome and restrictive allograft syndrome: do risk factors differ? mismatches at the hla-dr and hla-b loci are risk factors for acute rejection after lung transplantation does human leukocyte antigen matching influence the outcome of lung transplantation? an analysis of 3,549 lung transplantations influence of human leukocyte antigen matching on long-term outcome after lung transplantation the registry of the international society for heart and lung transplantation: twenty-seventh official adult lung and heart-lung transplant report-2010 predictors of acute rejection after lung transplantation interleukin-10 production genotype protects against acute persistent rejection after lung transplantation the impact of pharmacogenomic factors on acute persistent rejection in adult lung transplant patients rejection is reduced in thoracic organ recipients when transplanted in the first year of life paediatric incidence of acute rejection and obliterative bronchiolitis: a comparison with adults the registry of the international society for heart and lung transplantation: thirtieth adult lung and heart-lung transplant report-2013; focus theme: age are symptom reports useful for differentiating between acute rejection and pulmonary infection after lung transplantation? heart lung the registry of the international society for heart and lung transplantation: 29th adult lung and heart-lung transplant report-2012 low vitamin d levels are associated with increased rejection and infections after lung transplantation clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant parainfluenza virus infection in adult lung transplant recipients: an emergent clinical syndrome with implications on allograft function influenza pneumonia in lung transplant recipients: clinical features and association with bronchiolitis obliterans syndrome chlamydia pneumoniae infection after lung transplantation risk factors for bronchiolitis obliterans: a systematic review of recent publications role of pulmonary function in the detection of allograft dysfunction after heart-lung transplantation limitations of spirometry in detecting rejection after single-lung transplantation acute rejection following lung transplantation: limitations in accuracy of thin-section ct for diagnosis exhaled nitric oxide in human lung transplantation: a noninvasive marker of acute rejection serial monitoring of exhaled nitric oxide in lung transplant recipients inhaled corticosteroids and the treatment of lymphocytic bronchiolitis following lung transplantation transbronchial cryobiopsies in the evaluation of lung allografts: do the benefits outweigh the risks? cryoprobe transbronchial lung biopsy in patients after lung transplantation: a pilot safety study transbronchial cryobiopsy in lung transplantation patients: first report revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: lung rejection study group. the international society for heart transplantation revision of the 1990 working formulation for the classification of pulmonary allograft rejection: lung rejection study group reliability for grading acute rejection and airway inflammation after lung transplantation analysis of the different histologic lesions observed in transbronchial biopsy for the diagnosis of acute rejection. clinicopathologic correlations during the first 6 months after lung transplantation interpretation of transbronchial lung biopsies from lung transplant recipients: inter-and intraobserver agreement interobserver variability in grading transbronchial lung biopsy specimens after lung transplantation can immunohistological analysis of transbronchial biopsy specimens predict responder status in early acute rejection of lung allografts? alemtuzumab in the treatment of refractory acute rejection and bronchiolitis obliterans syndrome after human lung transplantation the role of transbronchial lung biopsy in the treatment of lung transplant recipients: an analysis of 200 consecutive procedures prospective analysis of 1,235 transbronchial lung biopsies in lung transplant recipients yield of surveillance bronchoscopy for acute rejection and lymphocytic bronchitis/bronchiolitis after lung transplantation association of minimal rejection in lung transplant recipients with obliterative bronchiolitis the significance of a single episode of minimal acute rejection after lung transplantation the diagnosis of obliterative bronchiolitis after heart-lung and lung transplantation: low yield of transbronchial lung biopsy evaluation of transbronchial biopsy in the diagnosis of bronchiolitis obliterans after lung transplantation transbronchial biopsy in heart and lung transplantation: clinicopathologic correlations an international ishlt/ats/ers clinical practice guideline: diagnosis and management of bronchiolitis obliterans syndrome pulmonary hypertension associated with lung transplantation obliterative bronchiolitis and vascular remodeling of the allograft pulmonary hypertension in patients with bronchiolitis obliterans syndrome listed for retransplantation perivascular inflammation in pulmonary infections: implications for the diagnosis of lung rejection national conference to assess antibody-mediated rejection in solid organ transplantation antibody-mediated organallograft rejection hla-specific antibodies are associated with high-grade and persistentrecurrent lung allograft acute rejection development of an antibody specific to major histocompatibility antigens detectable by flow cytometry after lung transplant is associated with bronchiolitis obliterans syndrome pretransplant panel reactive antibody in lung transplant recipients is associated with significantly worse posttransplant survival in a multicenter study utility of peritransplant and rescue intravenous immunoglobulin and extracorporeal immunoadsorption in lung transplant recipients sensitized to hla antigens anti-hla class i antibody binding to airway epithelial cells induces production of fibrogenic growth factors and apoptotic cell death: a possible mechanism for bronchiolitis obliterans syndrome peritubular capillary c4d deposition and renal outcome in post-transplant iga nephropathy capillary deposition of complement c4d and c3d in pediatric renal allograft biopsies immunohistochemistry staining of c4d to diagnose antibody-mediated rejection in cardiac transplantation acute humoral rejection in kidney transplantation: ii. morphology, immunopathology, and pathologic classification chronic humoral rejection: identification of antibody-mediated chronic renal allograft rejection by c4d deposits in peritubular capillaries antibodymediated rejection in human cardiac allografts: evaluation of immunoglobulins and complement activation products c4d and c3d as markers c4d deposition in cardiac allografts correlates with alloantibody update on cardiac transplantation pathology pancreas allograft biopsies with positive c4d staining and anti-donor antibodies related to worse outcome for patients reproducibility of complement 4d deposition by immunofluorescence and immunohistochemistry in lung allograft biopsies banff study of pathologic changes in lung allograft biopsy specimens with donor-specific antibodies c4d deposition in lung allografts is associated with circulating anti-hla alloantibody acute humoral rejection of human lung allografts and elevation of c4d in bronchoalveolar lavage fluid c3d and the septal microvasculature as a predictor of chronic lung allograft dysfunction c3d and c4d deposition early after lung transplantation pathology of pulmonary antibody-mediated rejection: 2012 update from the pathology council of the ishlt antibody-mediated rejection of the lung: a consensus report of the international society for heart and lung transplantation hyperacute rejection after single lung transplantation: a case report fulminant hyperacute rejection after unilateral lung transplantation hyperacute rejection of a pulmonary allograft. immediate clinical and pathologic findings susceptibility of lung transplants to preformed donor-specific hla antibodies as detected by flow cytometry hyperacute rejection following lung transplantation hyperacute rejection after lung transplantation caused by undetected lowtiter anti-hla antibodies hyperacute rejection in single lung transplantation-case report of successful management by means of plasmapheresis and antithymocyte globulin treatment treatment of hyperacute antibody-mediated lung allograft rejection with eculizumab prospective study of transbronchial biopsies in the management of heartlung and single lung transplant patients practical applications in immunohistochemistry: evaluation of rejection and infection in organ transplantation key: cord-016998-6n662amh authors: nan title: nierentransplantation date: 2007 journal: praxis der nephrologie doi: 10.1007/978-3-540-48556-8_13 sha: doc_id: 16998 cord_uid: 6n662amh die nierentransplantation ist die effektivste behandlungsmethode der chronischen terminalen niereninsuffizienz. seit den 1960er jahren entwickelte sie sich zu einer standardtherapie. wichtige voraussetzungen waren die entdeckung des hla-systems, die entwicklung der immunsuppressiva sowie die technische perfektionierung des organerhaltes außerhalb eines lebenden körpers. die 5-jahres-überlebensrate für allotransplantate beträgt etwa 65%, diejenige von lebendspenden 79%. immunsuppression -293 13 von klasse-1-hla-genen kodierte antigene sind auf allen kernhaltigen zellen vorhanden, von klasse-2-hla-genen kodierte antigene hauptsächlich auf b-lymphozyten und monozyten, also zellen des abwehrsystems. klasse-i-und klasse-ii-antigene bestehen aus zwei ketten, die als α und β-kette bezeichnet werden und ihre letztliche konfiguration durch dimerisierung bilden. die klasse-i-antigene enthalten eine polymorphe antigenspezifische kette (»heavy chain«) und kommen auf der zelloberfläche immer mit einem β2-mikroglobulinmolekül (»light chain«) assoziiert vor. durch kristallisation konnte die struktur der klasse-i-moleküle des hla-komplexes sichtbar gemacht werden. auf der moleküloberfläche findet sich eine 2×1×1 nm große grube, in welcher fremdantigene von 8-9 aminosäuren größe gebunden werden können. bei den klasse-ii-antigenen bilden immer eine α-(»heavy chain«) und β-kette (»light chain«) zusammen eine antigenbindende tasche. ▬ das langzeitüberleben eines transplantates hängt u. a. vom ausmaß der hla-übereinstimmung ab. die auslösung einer frühen abstoßung hängt mehr von patientenspezifischen faktoren und der immunsuppression ab. eine analyse aus den usa von 1994 fand eine halbwertszeit von 24 jahren für das transplantatüberleben bei hlaidentischer lebendspende (zwillinge), bei leichennierentransplantation von 20 jahren bei 6facher hla-übereinstimmung, von 9 jahren bei zufallsmatching. primär glomeruläre erkrankungen schwächen die prognostische aussagekraft des hla-matches aufgrund der möglichen rezidive im transplantat. hla-a-, hla-b-und hla-dr-region findet man eine transplantathalbwertszeit von 20 jahren und ein 10-jahres-transplantatüberleben von 65-70%. sechs identische hla-antigene führen zu dem besten 1-jahres-(88%) und auch langzeittransplantatüberleben. ein mismatch in der hla-a-, hla-b-oder hla-dr-region ist mit einer transplantathalbwertszeit von 10 jahren und einem 10-jahres-transplantatüberleben von 40-50% verbunden. stimmen mehrere hla-antigene nicht überein, liegt die transplantathalbwertszeit bei 7-9 jahren und das 10-jahres-überleben bei 30-35%. diese erkenntnisse sind der verdienst großer datenbanken, die überregional, z. t. sogar weltweit daten gesammelt und ausgewertet haben. problematisch bei der datenauswertung ist die weiterentwicklung sowohl der testsysteme als auch die einführung neuer immunsuppressiva. wichtige transplantationsorganisationen: ▬ cts -collaborative transplant study, prof. opelz, heidelberg ▬ eurotransplant, leiden ▬ united kingdom transplant service ▬ unos -united network for organ sharing, nordamerika ▬ seopf -american southeast organ procurement foundation toleranz gegenüber nichteigenen hla-antigenen erwirbt man vermutlich pränatal und in der stillzeit. untersuchungen an transplantatempfängern, die vor der transplantation z. b. über bluttransfusionen mit spenderantigenen konfrontiert wurden, deuten auf die möglichkeit einer induktion von immuntoleranz hin. die bildung von hla-antikörpern wird durch schwangerschaft, geburt und vorherige transplantationen stark, durch bluttransfusionen in geringerem ausmaß induziert. eine erfolgreiche transplantation ist nahezu ausgeschlossen, wenn beim empfänger zytotoxische antikörper gegen folgende antigene vorhanden sind: ▬ blutgruppenantigene (ab0-unverträglichkeit) des spenders ▬ klasse-i-oder -ii-hla-antigene des spenders ▬ endotheliale oder monozytäre antigene des spenders ein positiver t-zell-crossmatch (s. unten) z. b. führt zu einer hyperakuten abstoßung. komplementsystem und gerinnungskaskade werden aktiviert, eine anaphylaktische reaktion tritt ein und polymorphkernige leukozyten und mononukleäre zellen wandern in das transplantat ein. dies führt innerhalb von minuten bis stunden zu einer fibrinoiden nekrose der blutgefäße des transplantates und ischämischer nekrose des nierenparenchyms. in japan wurde aufgrund extremen organmangels blutgruppeninkompatibel transplantiert. durch immunadsorption in kombination mit b-zell-antikörpern konnte im vergleich mit gematchten patienten ein gleiches langzeitüberleben des transplantates erreicht werden. ähnliche ergebnisse wurden bei der transplantation 13.1 · transplantationsimmunologie von organen mit der gering immunogenen blutgruppe a 2 in empfänger der blutgruppen 0 und b erreicht. t-und b-zell-crossmatch. beim t-zell-crossmatch und b-zell-crossmatch wird die stimulation der b-oder t-lymphozyten durch empfängerserum getestet. bei der gemischten lymphozytenkultur (»mixed lymphocyte culture«, mlc) wird die stimulation von empfänger und spenderlymphozyten getestet, die miteinander inkubiert werden. stimulation kann anhand der anzahl von blasten oder des einbaus radioaktiver nukleinsäuren getestet werden. »panel reactive antibodies«, pra. bei diesem test wird das serum des empfängers auf das vorhandensein zytotoxischer antikörper gegen eine große zahl von häufigen (»populären«) antigenen getestet. hohe sensibilisierung zeigt sich in reaktivität gegen einen hohen prozentsatz der angebotenen antigene und wird als prozent-panel-reaktivität ausgedrückt. patienten mit hoher panel-reaktivität haben eine geringere transplantationschance, da sie häufig im crossmatch positiv sind. in einem amerikanischen zentrum z. b. war die wartezeit bis zur transplantation für patienten mit einer panel-reaktivität über 50% 5-mal so lange als bei einer reaktivität unter 10%. die 1-und 2-jahres-überlebensraten des transplantates waren ebenfalls geringer. auch »alte« tests mit hoher reaktivität (>6 monate vor transplantation) verschlechtern das transplantatüberleben, auch wenn das ergebnis unmittelbar vor transplantation besser war. ein großes problem besteht in der interpretation von positiven antikörpertests, denn nicht alle zytotoxischen antikörper des empfängers gegen spenderantigene sind für den transplantationserfolg von bedeutung. die bei der grunderkrankung lupus erythematodes vorhandenen multiplen autoantikörper können die testergebnisse verfälschen. positiver b-zell-crossmatch bei negativem t-zell-crossmatch führt in der regel nur bei anti-körpern gegen klasse-i-hla-antigene zu einer häufung frühen transplantatversagens. positiver b-zell-crossmatch führt zu einer 5% geringeren 2-jahres-überlebensrate bei ersttransplantation und 10% geringeren bei retransplantation. flow-cytometry-test. ein positiver flow-cytometry-test ist sowohl bei erst-als auch bei retransplantation ein negativer prognostischer faktor. dieser sehr empfindliche test wird auch bei vorhandensein, niedrig titriger, nicht komplementaktivierender, inkompatibler hla-antikörper positiv. viele zentren führen unmittelbar vor der transplantation einen crossmatch-test zwischen spender und empfänger durch, um eine hyperakute abstoßung auszuschließen. dieser dauert aber ca. 4 h und verlängert damit die kalte ischämiezeit. bei patienten mit 0% panel-reaktivität kann dieser letzte test vermutlich unterlassen werden. der wachsende anteil von zweit-und drittnierentransplantierten und die damit höhere zahl von hochimmunisierten patienten gewinnt zunehmend an bedeutung. eurotransplant hat den hochimmunisierten transplantatempfänger folgendermaßen definiert: »panel reactive antibodies« (pra) >85%; d. h., dass hla-antikörper im patientenserum mit >85% unselektierter patienten im crossmatch reagieren. das vorhandensein donor-spezifischer hla-antikörper (dsa) resultiert aus einer früheren exposition gegenüber fremden hla-antigenen durch bluttransfusionen, vorausgegangenen berücksichtigt wird die summe der mismatches bzw. der übereinstimmenden hla-antigene. dies wird in einer punktzahl ausgedrückt und hat eine gewichtung von 40%. sie bezeichnet die wahrscheinlichkeit, ein in den hla-merkmalen weitgehend übereinstimmendes daten des ucla-registers (»ucla tissue typing laboratories«) zeigen eine deutlich schlechtere langzeitprognose für nierentransplantationen bei diabetikern. nach 5 jahren werden patientenüberlebensraten von 45-75% beschrieben! diese liegen jedoch deutlich über der 5-jahres-überlebensrate von diabetischen dialysepatienten von 0-35%. im usrds (»united states renal data system«) wurden 7200 transplantierte diabetiker mit 15000 diabetischen dialysepatienten auf der warteliste verglichen: das mortalitätsrisiko nach 18 monaten war bei den dialysepatienten signifikant höher. diese sehr hohe mortalität wird zu einem großen teil durch extrarenale gefäßerkrankungen verursacht. vermutlich ist die elimination von age-proteinen (advanced glycation endproducts, kap. 9) durch das transplantat mit ausschlaggebend für die bessere gefäßsituation der transplantierten diabetiker. in der vorbereitungsphase der nierentransplantation ist eine invasive abklärung der koronarien mittels koronarangiographie unumgänglich. wenn dabei die notwendigkeit einer acvb-operation zu tage tritt, aber nicht zugemutet werden kann, ist eine transplantation vermutlich ebenfalls ein nicht zumutbarer eingriff. die u. a. aus neurologischen gründen gehäuften harnwegsinfekte der diabetiker zwingen zu einer gründlichen urologischen abklärung, oft sind langzeitprophylaxen mit antibiose indiziert. proteinurie und langsamer funktionsverlust können die entwicklung einer diabetischen nephropathie im transplantat anzeigen. auslöser der diabetischen nephropathie im transplantat ist ebenfalls die blutzuckerentgleisung. beim jüngeren patienten ohne ausschlusskriterien sollte eine 13.3 · vorbereitung der transplantation kombinierte pankreas-nieren-transplantation angestrebt werden ( kap. 9). die transplantation von organen nicht verwandter lebendspender sowie die akzeptanz alter spender sind versuche, die bestehende lücke zwischen organangebot und nachfrage zu füllen. gründe für das schlechte ansehen der nicht-verwandten lebendspende sind ungelöste ethische probleme: ▬ wo liegt die wahre motivation der spende? (psychologische evaluierung und betreuung sicher sinnvoll) ▬ wenig akzeptable morbidität und mortalität von spender und empfänger ▬ schlechtes transplantatüberleben während die anzahl der amerikanischen transplantationszentren, die nicht-verwandte lebendspenden akzeptieren ständig zunimmt, ist die nicht-verwandte lebendspende in den europäischen ländern unüblich. auch bei nicht verwandten lebendspendern ist die ab0-kompatibilität ausgangstest für alle weiteren abklärungsuntersuchungen, die denen der lebendspende durch verwandte entsprechen. das 1-jahres-überleben in einigen studien lag bei 92-95%, nach 2 jahren funktionierten in einer studie noch 83% der transplantate. somit liegt die rate funktionierender transplantate nach 1 jahr näher bei derjenigen verwandter lebendspender, als bei derjenigen von leichennierentransplantationen. dies ist leicht mit den kürzeren ischämiezeiten und planbaren operationsumständen zu begründen. nieren von ehepartnern hatten in einer studie eine halbwertsüberlebenszeit von 12 jahren. im jahr 2005 wurden in deutschland 165 pankreastransplantationen an 23 kliniken durchgeführt, davon 144 in kombination mit einer niere (»simultanous pancreas-kidney transplantation«, spk). die zahl der kombinierten pankreas-(nieren)-transplantationen und auch die zahl der neuanmeldungen zur transplantation nahm leicht ab. die patientenüberlebensrate und das überleben der niere entsprechen derjenigen der alleinigen nierentransplantation. patienten über 45 jahre haben ein doppelt so hohes risiko des transplantatverlustes und ein 3faches mortalitätsrisiko. ihnen ist eine alleinige nierentransplantation anzuraten. besteht das angebot einer hla-identischen oder zumindest sehr gut passenden niere, so sollte die einzeltransplantation der organe (»pancreas after kidney«, pak) in erwägung gezogen werden. viele zentren fordern eine koronarographie vor aufnahme auf die warteliste, da koronare komplikationen die mortalität der potentiellen transplantatempfänger vervierfacht. blindheit, hochdruck, periphere bypässe, amputationen sowie zerebrovaskuläre komplikationen haben keinen einfluss auf das transplantatüberleben. unter einer induktionstherapie versteht man die einleitende, meist bereits präoperativ beginnende immunsuppression. prinzipiell unterscheidet man protokolle mit antikörpern gegen t-zellen in kombination mit niedrig dosierten konventionellen immunsuppressiva von protokollen mit hochdosierten konventionellen immunsuppressiva (ohne antikörper). als antikörper finden einsatz: ▬ antilymphozytenserum (atg = antithymozytenglobulin = anti-t-lymphozytenglobulin) ▬ basiliximab, daclizumab: ursprünglich in der maus gezüchtete, humanisierte il-2-rezeptorantikörper eine akute abstoßung ist oft schwer abgrenzbar von einer verzögerten funktionsaufnahme. manche zentren befürworten dann die gabe von atg, um eine okkulte abstoßung nicht untherapiert zu lassen. die transplantatbiopsie ist zur diagnosesicherung erforderlich. es gibt derzeit keine konsensusempfehlungen für die induktionstherapie. die derzeit wichtigsten medikamente zur erhaltungsimmunsuppression nach nierentransplantation sind steroide, azathioprin, mycophenolat, ciclosporin und tacrolimus sowie sirolimus. in den ersten 3 monaten ist das risiko einer akuten abstoßung am höchsten. man setzt deswegen in dieser zeitspanne höhere dosen der immunsuppressiva ein. langfristig werden jedoch möglichst niedrige dosierungen angestrebt, da malignomund infektionsrisiko mit der gesamtdosis der immunsuppression korrelieren. die dosis der immunsuppression wird außerdem höher angesetzt bei: ▬ vorhandener sensibilisierung ▬ retransplantation (höhere dosen als bei ersttransplantation) ▬ hoher anzahl von abstoßungen bei der ersttransplantation ▬ geringer hla-übereinstimmung auch bei völliger hla-übereinstimmung wird mit steroiden und azathioprin oder ciclosporin weiterbehandelt. ein komplettes absetzen kann nicht empfohlen werden, da es auch spät noch zu akuten abstoßungen oder beschleunigter chronischer abstoßung kommen kann. ganz selten kann die entwicklung einer spenderspezifischen toleranz beobachtet werden. glukokortikoide hemmen die aktivierung von t-lymphozyten. sie beeinflussen die zellulären immunreaktionen über eine synthesehemmung von zytokinen (interleukin 1, interleukin 2). die antikörperbildung wird nur bei der gabe hoher dosen beeinflusst. außerdem lagern sich hochdosierte steroide in die zellmembran ein und verändern die struktur und damit funktion der oberflächenproteine durch störung der membranintegrität. die hohe steroiddosis der induktionstherapie wird in abhängigkeit vom verlauf langsam reduziert. ab etwa 0,5 g/kg kg prednisolonäquivalente können transplantierte aus der stationären behandlung entlassen werden. die dosis sollte wenn möglich auf unter 10 mg/24 h bzw. 0,1 mg/kg kg reduziert werden, um langzeitnebenwirkungen der steroide wie cushing-syndrom, osteoporose, aseptische knochennekrosen, muskelatrophie und steroiddiabetes zu vermeiden. erfreuliche nebenresultate der steroidreduktion sind blutdruckabfall, absinken des gesamtcholesterols, erleichterung der diabeteseinstellung und stabilisierung der knochensituation. ein frühes völliges absetzen der steroide ist mit einer deutlichen zunahme akuter abstoßungen, spätes absetzen mit einer reduktion der nierenfunktion und zunahme der proteinurie verbunden. gibt man die steroiddosis jeden 2. tag (sog. »alternate day regimen«), wird die störung der hormonellen feedbackmechanismen der nebenniere bzw. der hypothalamus-hypophysen-nebennierenachse reduziert und die gefahr eines cushing-syndroms sinkt. fieber, schwäche, myalgien, arthralgien und gewichtsverlust können zeichen einer subtilen nebenniereninsuffizienz sein, die mit einem falschnormalen acth-test einhergeht. azathioprin (imurek) führt als antimetabolit der purinbiosynthese zur suppression zellulärer immunreaktionen hauptsächlich der t-lymphozyten. die erhaltungsdosis liegt bei 1,5-2,5 g/kg kg. hohe initialdosen können die inzidenz akuter abstoßungen reduzieren. schwerste nebenwirkung ist die leukopenie. bei leukozytenzahlen unter 3000/mm 3 muss eine behandlungspause unter fortlaufender blutbildkontrolle erfolgen. sinken die leukozytenzahlen unter 1000/mm 3 , muss eine stationäre aufnahme, unter 700/mm 3 eine isolation erfolgen. bei weiter sinkenden leukozytenzahlen sollte granulozytenstimulierender wachstumsfaktor (g-csf=neupogen) verabreicht werden. beim wiedereinsetzen wählt man eine niedrigere dosis. eine häufige nebenwirkung ist die hepatotoxizität, die sich klinisch in oberbauchbeschwerden äußert, welche von einer enzymatischen cholestase und transaminasenanstieg begleitet sein können. allopurinol erhöht den plasmaspiegel von azathioprin über eine hemmung der xanthinoxidase und darf deswegen nicht gleichzeitig verabreicht werden. bei schwerer gicht kann durch umsetzen auf mycophenolat die gabe von allopurinol ermöglicht werden. neoplasmen der haut sind bei nierentransplantierten unter azathioprin vermutlich ebenfalls gehäuft. direkte sonneneinstrahlung muss gemieden bzw. sonnencreme mit hohem lichtschutzfaktor verwendet werden. azathioprin ist heute wegen der besseren wirksamkeit und geringeren myelosuppression weitgehend durch mmf ersetzt worden. mycophenolat-mofetil (mmf, cellcept) blockiert die purinbiosynthese über eine inhibition der inosinmonophosphat-dehydrogenase. es wird als ersatz von azathioprin und zur »rescue«-therapie bei okt 3 resistenten abstoßungskrisen eingesetzt. es ist nicht nephrotoxisch und weniger knochenmarksupprimierend als azathioprin. häufig sind gastrointestinale nebenwirkungen mit diarrhö und gastritis. unter tripletherapie mit steroiden und ciclosporin a (oder tacrolimus) treten akute abstoßungen wesentlich seltener auf als unter zweifachtherapie mit steroiden und ciclosporin a (oder tacrolimus) alleine. bei dem vergleich von 2 und 3 g mycophenolat/tag vs. azathioprin (beide gruppen mit steroiden und ciclosporin a) waren nach 6 monaten transplantatverluste und abstoßungen unter mycophenolat seltener, antilymphozytenglobulin (alg) musste seltener eingesetzt werden und die 1-jahres-transplantatüberlebensrate war tendenziell höher. diese ergebnisse bestätigten sich nach 3 jahren. trotz der höheren therapiekosten war mycophenolat durch die selteneren abstoßungsbehandlungen kostengünstiger als azathioprin. der im tierexperiment gefundene günstige effekt auf chronische abstoßung konnte beim menschen noch nicht nachvollzogen werden. unter azathioprin stabile patienten werden im allgemeinen nicht auf mycophenolat umgesetzt. es ist bisher unklar, ob das absetzen von steroiden unter ciclosporin und mycophenolat möglich ist. ciclosporin ist ein lipophiles peptidantibiotikum, welches von dem pilz tolypodadium inflatum gebildet wird. es hemmt die zelluläre immunantwort über eine bindung an intrazelluläre cyclophylline. dies führt zu einer synthesestörung von interleukin-2 und anderen zytokinen. seit den frühen 1980er jahren hat ciclosporin seinen festen platz in der erhaltungsimmunsuppression. die kombination von ciclosporin a mit steroiden und azathioprin bezeichnet man als »tripletherapie«. die meisten nierentransplantierten patienten erhalten derzeit diese tripletherapie oder ciclosporin a mit entweder steroiden oder azathioprin/mycophenolat. gelegentlich wird ciclosporin auch als einziges immunsuppressivum eingesetzt. auch spätes absetzen von ciclosporin führt gehäuft zu akuten abstoßungen und dadurch schlechterem transplantatüberleben. die inzidenz chronischer abstoßung wird allerdings von ciclosporin nicht vermindert. viele medikamente beeinflussen den abbau von ciclosporin a und können so den plasmaspiegel verändern. der talspiegel sollte in der erhaltungsphase zwischen 50 und 150 ng/ml liegen, der vollblutspiegel zwischen 150 und 300 ng/ml. niedrigere dosen werden bei stabiler transplantatfunktion toleriert. manche patienten sind sehr ciclosporinempfindlich und kommen mit spiegeln um 35 ng/ml gut zurecht. es senkt die rate von zytomegalievirus-infektionen und ermöglicht über eine reduktion der ciclosporin-dosis auch die minimierung der nephrotoxischen effekte von ciclosporin. als teil einer tripeltherapie, zusammen mit ciclosporin-mikroemulsion (sandimmun optoral) und steroiden, hat es sich als ähnlich effektiv erwiesen wie mycophenolatmofetil, das in den letzten jahren am häufigsten in einer ciclosporin-basierten tripeltherapie eingesetzt wurde. die therapie mit in der maus gezüchteten antikörpern führt beim menschen zu durch zytokinausschüttung von t-zellen bedingten nebenwirkungen (fieber, schüttelfrost, übelkeit, erbrechen, diarrhö, hypotonie, thoraxschmerzen, dyspnoe). die gentechnische herstellung von hybridantikörpern aus der antigenbindenden region des murinen antikörpers und dem grundgerüst menschlichen iggs konnte sowohl die immunogenität der antikörper vermindern als auch die halbwertszeit der antikörper verlängern. sie werden zur induktionstherapie und bei der akuten abstoßung eingesetzt. die nebenwirkungen sind geringer als bei okt 3. der erste zugelassene monoklonale antikörper war muromonab-cd3 (okt 3). okt 3 ist ein sehr potentes immunsuppressivum, wird heute aber selten verwendet, da die moderneren antikörper und neuere immunsuppresiva okt 3 mit seinen z. t. heftigen nebenwirkungen weitgehend verdrängt haben. die monoklonalen rekombinanten antikörper, die bisher in der transplantationsmedizin eingesetzt werden, binden an die α-kette des il-2-rezeptors und verhindern die bindung von il-2 an die aktivierte t-zelle. derzeit sind basiliximab (simulect) und daclizumab (zenapax) erhältlich. die prohylaktische gabe als induktionstherapie reduziert die rate akuter abstoßungen, die 1-jahres-funk-tionsrate unterscheidet sich jedoch nicht zur induktionstherapie ohne antikörper. vorwiegender einsatz bei immunologischen risikopatienten. tagesdosen von 8-10 mg/kg kg ciclosporin-mikroemulsion zusammen mit 2-maliger gabe von 1 g mycophenolat und niedrig dosierten steroiden ist die derzeit am häufigsten eingesetzte induktionstherapie. als erhaltungstherapie bieten sich 3-5 mg/kg kg ciclosporin-mikroemulsion, mycophenolat cyclophosphamid und mercaptopurin sind im tierexperiment teratogen und sollten beim menschen vermieden werden. methotrexat ist sicher embryotoxisch, führt zum abort und wird als substanz zur beendigung ektoper schwangerschaften getestet. für mycophenolat gibt es keine anwendungsuntersuchungen in der schwangerschaft. gleiches gilt für den antikörper okt 3. die federal drug administration (fda) stuft beide substanzen bezüglich ihrer teratogenität als sogenannte c-klasse ein (⊡ tab. 13.2). dies bedeutet, dass adäquate studien fehlen und risiken nicht ausgeschlossen werden können, eine vitale indikation die gabe aber rechtfertigen kann. der zeitpunkt des auftretens einer transplantatfunktionsstörung ist oft charakteristisch für die auslösende ursache. polyomaviren sind eine klasse von dns-viren aus der familie der papovaviridae, die zu opportunistischen infektionen führen können. die durchseuchung ist hoch, es kommt jedoch nur selten zum krankheitsausbruch. das zu den polyomaviren zählende jakob-creutzfeld-virus z. b. ist wahrscheinlich die ursache der progressiven multifokalen leukenzephalopathie. bei knochenmarktransplantierten wurde eine assoziation mit dem auftreten einer hämorrhagischen zystitis, bei nierentransplantierten mit dem auftreten einer ureterstenose beobachtet. mittels zytologie, urinkulturen und elektronenmikroskopie konnten polyomaviren im urin von 10-45% der patienten nach nierentransplantation nachgewiesen werden. es kann vermutlich sowohl im rahmen der immunsuppression reaktiviert als auch mit dem transplantat übertragen werden. tritt etwa 11 monate nach transplantation eine akute verschlechterung der transplantatfunktion mit den zeichen einer interstitiellen nephritis auf, so bildet die polyomavirusinfektion eine wichtige differentialdiagnose. in der bisher größten untersuchung an 22 patienten konnte das transplantat bei den 8 patienten, deren immunsuppression reduziert wurde, erhalten werden, während 8 von 12 patienten, die unter der annahme einer abstoßung verstärkt immunsuppressiv therapiert wurden, ihr transplantat verloren. mit steigender viruslast ist das virus zuerst im urin, dann im plasma und schließlich in der niere nachweisbar. die häufigsten ursachen einer langsam progredienten verschlechterung der transplantatfunktion sind: ▬ chronische abstoßung ▬ ciclosporintoxizität ▬ hypertensive nephrosklerose als folge mangelhafter blutdruckkontrolle ▬ obstruktion der ableitenden harnwege ▬ rezidiv der grunderkrankung oder neue nierenerkrankung unter transplantatabstoßung versteht man die immunologischen abwehrreaktionen des empfängers gegen das transplantat. diese wird durch vorgebildete, zytotoxische antikörper gegen klasse-i-antigene oder durch blutgruppeninkompatibilität hervorgerufen. diese form der abstoßung ist durch den ausschluss einer vorbestehenden sensibilisierung des empfängers gegen spenderantigene selten geworden. die aktivierung von komplement-und die gerinnungskaskade führt innerhalb von minuten nach öffnung der gefäßklemmen zur mikroembolisa-13.6 · transplantatabstoßung tion hauptsächlich mittlerer und kleiner gefäße und nekrose des transplantats (⊡ abb. 13.1). eine therapie ist nicht möglich. unter einer akuten abstoßung versteht man eine akute funktionsverschlechterung des transplantates, die mit charakteristischen, histologischen veränderungen einhergeht. sie tritt bei 30% der leichennieren-ersttransplantationen, 27% der lebendspenden und 37% der zweittransplantationen auf. mehr als die hälfte der transplantierten erleidet mindestens eine akute abstoßung. tritt diese in den ersten beiden monaten nach tpl auf, so besteht ein höheres risiko für eine chronische abstoßung. histologisch unterscheidet man zwischen zellulärer und vaskulärer abstoßung. zeichen zellulärer abstoßung sind interstitielle infiltration mit mononukleären, zellen, sowie ruptur der tubulären basalmembran. neutrophile im interstitium deuten dagegen eher auf eine infektion hin. zeichen der antikörpervermittelten, humoralen abstoßung (früher als »vaskulär« bezeichnet) sind endothelschwellung, fibrinoide nekrosen der arteriolen, fibrinthromben in den glomerulären kapillarschlingen und in schweren fällen eine nierenrindennekrose. glomeruläre beteiligung ist ein schlechtes prognostisches zeichen. sind gleichzeitig interstitielle, mononukleäre infiltrate vorhanden, so spricht man von einer gemischten abstoßung. mit hilfe der »banff-97-klassifizierung« können akute abstoßungen standardisiert eingestuft werden. dies ist wichtig, um therapieschemata vergleichen zu können (⊡ tab. 13.3). die erfüllung der »borderline«-kriterien wird nicht als abstoßung gewertet. beweisend für eine abstoßung ist bisher allein die histologische analyse. ein idealer abstoßungsmarker in form eines spezifisch und sensitiv auf abstoßung reagierenden messwerts im blut oder noch besser im urin steht derzeit nicht zur verfügung. geforscht wird nach molekularbiologischen nachweismethoden einer erhöhten expression abstoßungsspezifischer zytokine (z. b. il-7, il-10, il-15, fas ligand, perforin und granzym b). eine akute abstoßung tritt bei 10-35% der nierentransplantierten auf. zur behandlung stehen die steroidstoßtherapie, atg (s. unten) und deren kombination zur verfügung. bei hochgradigem klinischen verdacht sollte bereits vor erhalt des biopsieergebnisses zumindest mit der steroidtherapie begonnen werden. die steroidstoßtherapie besteht aus der i.v.-verabreichung von 3-5 mg/kg kg methylprednisolon über 3-5 tage mit konsekutivem raschem ausschleichen bis zur ursprünglichen erhaltungsdosis. bei bisher eher niedrigem spiegel kann auch die ciclosporindosis angehoben werden. nebenwirkungen der steroide sind erhöhte infektanfälligkeit, blutzuckerentgleisungen, hypertonie, ulkuserkrankung und steroidpsychosen. eine prophylaktische, antimykotische therapie für den gastrointestinaltrakt in form von dünndarmlöslichen amphotericin-b-dragees und -emulsion (ampho-moronal für mund und speiseröhre), sowie eine ulkusprophylaxe mit einem h 2 -blocker wird empfohlen. wenn nach 5-7 tagen kein abfall des serumkreatinins sowie steigerung der urinausscheidung eingetreten ist, spricht man von steroidresistenter abstoßung. antilymphozytenserum wird durch die immunisierung von kaninchen oder pferden mit menschlichen lymphozyten aus dem thymus (deswegen atg = antithymozytenglobulin) oder aus b-zellkulturen gewonnen. eine typische dosierung wäre 10-15 mg/kg kg/24 h. nach einigen tagen bis einer woche kommt es in 75-100% zu einer rückkehr des kreatinins zum ausgangswert. nachteilig ist die serienabhängig unterschiedliche wirkstärke, die aufwendige produktion, die fehlende spezifität sowie die notwendigkeit eines zentralen zuganges zur applikation. um die infektionsgefahr nicht zu groß werden zu lassen, wird die dosis von ciclosporin, tacrolimus, azathioprin und mycophenolat während atg-therapie reduziert. gleichzeitig werden prophylaktisch antibiotika, virostatika und antimykotika gegeben. bei der infusion von atg treten als zeichen der reaktion auf fremdeiweiß fieber und schüttelfrost auf, selten kommt es auch zu anaphylaktischen reaktionen. viele zentren versuchen diesen begleitreaktionen durch gabe eines »cocktails« von steroiden, antihistaminika und antipyretika vorzubeugen. okt 3 wird wegen der starken nebenwirkungen kaum noch oder nur noch als reservemedikament eingesetzt. bei irreversiblem funktionsverlust des transplantates muss die immunsuppression abgesetzt werden. die infektionsgefahr aufgrund von medikamenten und zunehmender urämie potenziert sich. mit zunehmender niereninsuffizienz addiert sich auch die neurotoxizität von urämie und ciclosporin. beim absetzen der immunsuppression kann trotz terminalen transplantatversagens eine abstoßung auftreten, die zu einer nephrektomie zwingen kann. dies ist besonders, wenn ein transplantatversagen innerhalb des 1. jahres eintritt, der fall. ein häufig gewähltes vorgehen ist das sofortige absetzen von ciclosporin, tacrolimus, azathioprin und mycophenolat gefolgt von einem ausschleichen der steroiddosis. kommt es wiederholt zu abstoßungskrisen und nimmt das transplantat an größe zu, besteht die gefahr der transplantatruptur. nach vorübergehender restitution einer höheren steroiddosis muss das transplantat dann entfernt werden. vier fünftel aller nierentransplantierten erleiden mindestens eine infektion im 1. jahr nach transplantation. das spektrum der auslöser von infektionen ist bei immunsupprimierten patienten um die opportunistischen keime erweitert. je stärker die immunsuppression, umso geringer die abstoim 1. monat nach transplantation kommen die typischen, auch bei nicht transplantierten auftretenden, postoperativen infektionen gehäuft vor. dazu gehören wundinfektionen durch bakterien oder pilze, pneumonien, katheterinfektionen etc. erstaunlicherweise sind trotz der in dieser zeit maximalen immunsuppression die opportunistischen infektionen ( pneumocystis jiroveci, nocardiose, listeriose) selten. zwischen 4 wochen und 6 monaten nach transplantation kommen infektionen mit immunmodulierenden viren -insbesondere cmv -besonders häufig vor. diese können den boden für eine weitere infektion bereiten. in dieses intervall fallen auch infektionen durch hsv, hzv, hepatitisviren, mykobakterium tuberculosis und ebv. ebv kann die entwicklung lymphoproliferativer erkrankungen induzieren. infektionserkrankungen, die später als 6 monate nach transplantation auftreten, entsprechen bezüglich der erreger weitgehend den die allgemeine bevölkerung betreffenden infektionen. es gibt jedoch auch chronische virusinfekte, die dann erst klinisch manifest werden (aids, chronische hepatitis, lymphoproliferative erkrankungen nach ebv, chorioretinitis durch cmv). die meisten zentren verabreichen bereits bei der transplantation eine perioperative antibiotikaprophylaxe mit breitem wirkspektrum sowie eine cmv-prophylaxe mit gancyclovir. trimethoprim-sulfamethoxazol wird zur prophylaxe von harnwegsinfekten und speziell zu pneumocystisprophylaxe eingesetzt. bei rezidivierenden harnwegsinfekten, anomalien der ableitenden harnwege oder neurogener blasenentleerungsstörung wird es als dauerantibiose verabreicht. alternativ können gyrasehemmer (z. b. ciprofloxazin, ofloxazin) eingesetzt werden. das zytomegalievirus ist eines der vier herpesviren. die infektionsrate der bevölkerung mit cmv steigt mit dem alter. die transfusion von blut ist eine potentielle infektquelle, der gebrauch von leukozytenfiltern kann die virustransmission deutlich verringern. mehr als zwei drittel aller spender und empfänger von organtransplantaten haben antikörper gegen cmv als zeichen einer durchgemachten, wenn auch klinisch vielleicht nicht manifesten infektion. man unterscheidet eine cmv-infektion von einer cmv-erkrankung. von infektion spricht man bei: ▬ serokonversion von igg zu igm ▬ bei einem 4fachen anstieg des igg titers ▬ beim nachweis von cmv-antigen in infizierten zellen ▬ bei isolation des virus aus kulturen klinik cmv-erkrankung bedeutet das auftreten von klinischen symptomen wie fieber, leukopenie und organmanifestationen (z. b. pneumonitis, hepatitis, pankreatitis, kolitis, meningoenzephalitis). eine symptomatische cmv-infektion tritt meist zwischen 1 und 4 monaten nach transplantation auf, die cmv-chorioretinitis gelegentlich auch später. kardinalsymptom ist eine leukopenie, die bei protokollen mit azathioprin zu einer dosisreduktion oder umsetzen auf mycophenolat zwingt. die häufigste klinische manifestation ist ein der mononukleose ähnliches bild mit fieber, myalgien, arthralgien, schwäche, leukopenie, milder lymphozytose und gelegentlich leichtem transaminasenanstieg. unter cmv-induzierter transplantatglomerulopathie versteht man ein klinisches bild mit verschlechterter transplantatperfusion und akuter tubulusnekrose. dieses syndrom ist weder in seiner klinischen signifikanz, noch in seiner abgrenzung von einer durch eine cmv-infektion getriggerten abstoßung, noch in seiner häufigkeit gesichert. die behandlung richtet sich insgesamt nach der schwere der erkrankung. okt 3 muss abgesetzt, azathioprin reduziert und ggf. durch mycophenolat ersetzt werden. die i.v.-gabe von gancyclovir wird an die nierenfunktion angepasst. man behandelt normalerweise über 3 wochen und verabreicht ggf ergänzend hyperimmunglobulin (s. unten). eine gleichzeitige infektion mit opportunistischen keimen (pneumocystis jiroveci, nocardiose, listeriose) muss ausgeschlossen werden. eine cmv-prophylaxe ist insbesondere in folgenden situationen wichtig: ▬ bei der transplantation von organen cmvpositiver spender auf cmv-negative empfänger zur vermeidung einer neuinfektion ▬ bei cmv-positiven empfängern zur vermeidung einer reaktivierung ▬ bei cmv-positiven empfängern und cmvpositiven spendern zur vermeidung einer infektion mit einem anderen virussubtyp die meisten zentren geben derzeit gancyclovir als prophylaxe. da die bioverfügbarkeit oralen gancyclovirs nicht besonders gut ist, wird primär die i.v.-applikation gewählt, eventuell gefolgt von einer oralen therapie. acyclovir vermindert zwar auch die erkrankungsrate, aber im unterschied zu gancyclovir ist die infektionsrate unverändert. valacyclovir, eine weiterentwicklung von acyclovir, hat interessanterweise im unterschied zu letzterem gute prophylaktische wirkung gegen cmv und eine bessere bioverfügbarkeit als gancyclovir. eine cmv-prophylxe mit cmv hyperimmunglobulin (cytoglobin, cytotect biotest) existiert bereits seit den 1980er jahren. eine infektion wird zwar nicht verhindert, aber die rate an cmv-erkrankungen, an parasitären oder pilzinduzierten superinfektionen sowie an leukopenie wird signifikant reduziert. veränderungen der leberwerte findet man bei 7-24% von organempfängern. leberversagen ist die todesursache bei 8-28% nierentransplantierter patienten. etwa die hälfte der lebererkrankungen bei nierentransplantierten wird durch hcv ver-13.7 · infektionen bei nierentransplantierten ursacht, der rest durch hbv, cmv, ebv, medikamentennebenwirkung (azathioprin, ciclosporin a), alkohol oder hämosiderose. empfänger hcv-positiver organe entwickeln 4-mal so häufig eine hepatitis. die auswirkung auf die überlebensrate sowie die transplantatüberlebensrate ist jedoch umstritten. britische und amerikanische untersuchungen zeigten, dass die nierentransplantation auch beim anti-hcv-positiven patienten der dialysebehandlung überlegen ist. bei der transplantation zwischen hcv-positivem spender und empfänger können gleiche oder unterschiedliche genotypen des hcv-virus vorliegen. nach aktueller datenlage hat dies keine auswirkung auf die schwere einer hepatitis. hcv-positive empfänger entwickeln signifikant häufiger eine hepatitis, meist eine chronisch aktive hepatitis. selten kommt es auch zu einer rasch progredienten, mit cholestase und schwerer fibrose einhergehenden sog. »fibrosierenden cholestatischen hepatitis«. endgültige sicherheit über den hcv-status des spenders gibt nur der rna-test, auch die elisa-systeme der 2. generation haben noch eine geringe rate falsch-positiver ergebnisse. eine leberbiopsie zur bestimmung des ausmaßes der hepatitis ist sehr gut geeignet, die qualität der prognostischen aussage bezüglich des verlaufes nach transplantation bei anti-hcv-positiven patienten zu verbessern. für die prognose bei normaler leberhistologie fehlen studien. bei ausgeprägten leberparenchymschäden, die bei dialysepatienten auch bei fast normalen transaminasen vorkommen können, sollte die entscheidung zur transplantation mit vorsicht gewählt werden. nierengesunde patienten mit hepatitis c können mit interferon-α und bei einer kreatininclearance über 50 ml/min zusätzlich mit ribavirin behandelt werden. da die gabe von zytokinen abstoßungen triggert, ist eine nierentransplantation eine kontraindikation für interferon-α. derzeit wird die auswirkung einer interferon-α-therapie bei dialysepatienten mit hepatitis c vor nierentransplan-tation auf das auftreten eines rezidiv der erkrankung nach transplantation untersucht. ribavirin kann bei ausreichender transplantatnierenfunktion die viruslast mindern. proteinurie beim nierentransplantierten mit hcv-infektion kann auf eine hcv-assoziierte erkrankung des transplantates hinweisen. rezidiv der grunderkrankung 13.8.1 primäre nierenerkrankungen die rezidivrate der iga-glomerulonephritis liegt bei etwa 50%, wobei lebendspenden häufiger rezidive erfahren (bis zu 83%). nicht jede histologische läsion führt zu klinischen symptomen. der verlauf ist ähnlich den iga-glomerulonephritiden in nativen nieren langsam, die transplantatverlustrate durch das rezidiv ist gering (etwa 75% 5-jahres-transplantatüberleben). lebendspenden werden daher als ethisch akzeptabel erachtet. ciclosporin a hat keinen einfluss auf häufigkeit, schwere und verlauf der rezidive. insgesamt ist das transplantatüberleben (leichenniere) von patienten mit iga-glomerulonephritis vermutlich besser als bei anderen grunderkrankungen. hier liegt die rezidivrate bei etwa 20%. es existieren jedoch abgrenzungsprobleme zu den sekundären glomerulosklerosen. patienten unter 20 jahren mit rasch progredienter niereninsuffizienz durch fsgs, haben im transplantat eine rezidivrate von fast 50% und sind oft nach etwa 3 jahren erneut dialysepflichtig. das rezidiv präsentiert sich oft mit nephrotischer proteinurie. die patienten erleiden häufig akute abstoßungen und ein anv in der ersten woche nach transplantation. ein rezidiv im ersttransplantat erhöht das risiko eines rezidivs in folgetransplantaten. geschwindigkeit und kontinuität der rezidive lässt einen im serum zirkulierenden pathogenen faktor vermuten. temporäre verbesserungen der proteinurie nach behandlung mit proteinadsorptionssäulen (plasmaseparation) unterstützen diese these. aufgrund der hohen wahrscheinlichkeit eines rezidivs sollte bei der aggressiven form der fsgs und bei patienten mit fsgs-rezidiv im ersttransplantat keine lebendspende durchgeführt werden. eine membranoproliferative glomerulonephritis rezidiviert häufig im transplantat. bei typ i in 20-30%, bei typ ii in 50-100%. für den seltenen typ iii liegen nur einzelfallstudien mit rezidiven vor. klinisch zeigt sich eine proteinurie und bei typ i auch häufig hämaturie. rezidive bei typ i führen in 30-40%, bei typ ii in 10-20% (mindestens) zum transplantatverlust. eine effektive therapie ist nicht bekannt. eine hepatitis-c-assoziierte mpgn kann ebenfalls im transplantat auftreten. die membranöse glomerulonephritis rezidiviert eher selten (3-5%). betroffen sind hauptsächlich patienten mit rasch progredientem verlauf der primären erkrankung. die rezidive treten nach etwa 10 monaten auf und führen bei 30-50% zum transplantatverlust. häufiger als das rezidiv ist eine mit chronischer abstoßung assoziierte »de-novo«-membranöse glomerulonephritis, die 18-21 monate nach tpl auftritt und klinisch als proteinurie auffällt. die antikörperproduktion bei diesem krankheitsbild ist normalerweise selbstlimitierend. nach dem 13.8 · rezidiv der grunderkrankung verschwinden der antikörpertiter (meist 1 jahr) ist die rezidivrate verschwindend gering. allerdings findet man in bis zu 50% der transplantierten organe lineare igg-ablagerungen an der glomerulären basalmembran. bei nach dem verschwinden der antikörpertransplantierten patienten mit rezidiv findet man hämaturie und proteinurie, der transplantatverlust ist jedoch gering. patienten mit alport-syndrom können nach nierentransplantation eine de-novo-anti-gbm-glomerulonephritis entwickeln. in ihren eigennieren ist nämlich das goodpasture-antigen nicht nachweisbar, so dass eine »gesunde« spenderniere sozusagen ein goodpasture-mismatch mit sich bringt, gegen welches dann vom empfänger antikörper produziert werden. hus-rezidive im transplantat sind für 20-50% der patienten beschrieben. unklar ist jedoch, wie hoch der anteil sekundärer hämolytisch-urämischer syndrome durch vaskuläre abstoßung oder ciclosporintherapie ist. risikofaktoren für ein rezidiv sind infektiöse diarrhö, primäres hus vom autosomal-rezessiven typ, höheres alter, lebendspende, gabe von ciclosporin oder tacrolimus und kurzes intervall zwischen primärem hus und transplantation. therapeutisch kann sowohl atg als auch ciclosporin (mit vorsicht) eingesetzt werden. plasmapherese wurden ebenfalls erfolgreich eingesetzt. beim auftreten eines hus unter ciclosporin sollte dieses reduziert werden. vermutlich gilt gleiches für die extrem selten vorkommende ttp. ein hämolytisch-urämisches syndrom im transplantat bei anderer grunderkrankung ist meist mit ciclosporintoxizität, akuter vaskulärer abstoßung oder hiv assoziiert. prophylaktisch kann niedrig dosierte acetylsalicylsäure oder dipyridamol verabreicht werden. bisher sind nur etwa 80 fälle von nierentransplantation bei sklerodermieerkrankung beschrieben. die transplantatüberlebenszeit ist kürzer als bei anderen grunderkrankungen, nach 5 jahren beträgt sie z. b. nur 47%. die rezidivrate ist aufgrund der ähnlichkeit der renalen sklerodermieläsionen mit den veränderungen bei vaskulärer abstoßung schwer einzuschätzen. sie wird in der literatur mit 20% angegeben und hat meist einen aggressiven verlauf. eine nierentransplantation führt nur selten zu einer reaktivierung der erkrankung. zeichen der lupusnephritis nur in 2-9%. diese rekurrieren in bis zu 20%. anca-titer sagen das risiko der rekurrenz nicht voraus. typische histologische veränderungen der diabetischen nephropathie im transplantat sind die verdickung der basalmembran und eine verbreiterung des mesangiums, die klassischen nodulären läsionen der glomeruli fehlen meist. nur knapp 2% der transplantatverluste sind auf die diabetische nephropathie zurückzuführen, da sie sich sehr langsam entwickelt. ihr wiederauftreten wird durch die kombinierte pankreas-nieren-transplantation verhindert. die meisten chirurgischen und auch urologischen probleme treten relativ früh nach transplantation, oft noch während der stationären nachbehandlung auf. eine erfolgreiche transplantation kann viele der durch die terminale niereninsuffizienz hervorgerufenen knochenstoffwechselstörungen beheben. die plasmaspiegel von phosphat, ap, β 2 -mikroglo-bulin und parathormon fallen, die kalzifikationen nehmen ab. aluminiumosteopathie, hyperparathyreoidismus, β 2 -ablagerungen und diabetische knochenstörungen können jedoch persistieren. die persistenz eines hyperparathyreoidismus ist häufig. sie beruht meist auf einer hyperplasie der nebenschilddrüsen, die jahre bis zur rückbildung benötigen kann. nur selten ist sie zeichen eines adenoms. die meist etwa 10 tage nach tpl auftretende hyperkalzämie beruht auf der resorption von gewebekalzifikationen und der normalisierten vitamin-d-produktion (und gelegentlich auch auf dem albuminanstieg). in extremfällen kann eine kalziphylaxie auftreten. falls die korrigierte kalziumkonzentration nach einem längeren zeitraum (max. 1 jahr) noch über 3,1 mmol/l liegt und die ipth-werte nicht adäquat absinken, muss eine parathyreoidektomie erwogen werden. die rückbildung der aluminiumosteopathie nach transplantation ist auch durch dauertherapie mit desferal nicht zu erreichen. hypophosphatämie nach transplantation wird durch hyperparathyreoidismus oder durch tubulären renalen phosphatverlust verursacht. folgen sind muskelschwäche und osteomalazie. die progression der β 2 -amyloidose wird durch erfolgreiche transplantation unterbrochen, da jetzt wieder ausreichend β 2 -mikroglobulin renal eliminiert wird. alte knochenzysten bleiben jedoch bestehen. osteopenie und osteonekrosen sind die beiden wichtigsten knochenkomplikationen nach transplantation. ursächlich sind fortbestehende störungen des kalziumstoffwechsels und die auswirkungen der immunsuppressiva -insbesondere der steroide -auf den mineralstoffwechsel. zwar konnte durch ciclosporin die steroiddosis gesenkt werden, die längere lebensdauer führt jedoch zu einer höheren gesamtdosis an steroiden. die inzidenz osteopenisch bedingter frakturen bei nierentransplantierten beträgt bis zu 22%. der verlust der knochenmasse ist kurz nach transplantation am höchsten. nach etwa 1,5 jahren liegt die knochendichte von 60% der patienten unterhalb der frakturschwelle. mit hilfe der dexa (dual energy x-ray absorptionsosteometrie) können verlaufskontrollen der knochendichte durchgeführt werden. lymphoproliferative erkrankungen treten nach organtransplantation 30-bis 50-mal häufiger auf, ihre inzidenz beträgt 1%. ihre verläufe und verteilung unterscheiden sich von denen der normalbevölkerung. zum beispiel sind normalerweise ca. zwei drittel der lymphome non-hodgkin-lymphome, bei transplantierten dagegen sind es 93%. extranodales auftreten, beteiligung des zentralen nervensystems und infiltration des transplantats sind häufig. ebv-induzierte b-zellproliferation spielt eine wichtige ursächliche rolle. insgesamt kommen drei formen der ebv-assoziierten lymphoproliferativen erkrankungen bei transplantierten vor: ▬ in 55% eine gutartige polyklonale b-zell-lymphoproliferation mit mononukleoseartigem krankheitsbild und normalen zytogenetischen parametern ohne hinweis auf maligne transformation. ▬ in etwa 30% liegt das gleiche mononukleoseartige bild auch mit polyklonalen b-zellproliferationen, allerdings mit zeichen früher maligner zellentartung vor. ▬ am seltensten ist ein ausschließlich extranodales wachstum solider tumoren von monoklonaler b-zellherkunft mit malignen charakteristika in der zytogenese. es gibt jedoch auch lymphome ohne ebv-assoziation, d. h. eine negative ebv-serologie gibt keine sicherheit. eine nach transplantation erworbene ebv-infektion stellt aufgrund der fehlenden immunität ein größeres risiko dar als eine vorbestehende infektion. weitere risiken für eine lymphoproliferative erkrankung sind therapiezyklen mit okt 3 oder ein sog. cmv-mismatch (spender positiv, empfänger negativ). alle drei risikofaktoren zusammen -fehlende ebv-immunität, cmv-mismatch, okt 3-gabe -erhöhen das risiko einer lymphoproliferativen erkrankung um den faktor 654. ergebnisse der nierentransplantation 13.11.1 transplantatüberleben unter kurzeitüberleben wird üblicherweise die 1-jahres-transplantatüberlebensrate verstanden. diese ist seit einführung von ciclosporin a, also in etwa den letzten 25 jahren deutlich gestiegen. dies gilt allerdings hauptsächlich für die nicht vorsensibilisierten patienten. die ergebnisse für stark hlavorsensibilisierte patienten haben sich leider nicht wesentlich verbessert. im schnitt lag die 1-jahres-transplantatüberlebensrate ende der 1970er jahre bei etwa 55%, in der ersten hälfte der 1980er jahre bei etwa 75% und in der zweiten hälfte bei fast 90%. dabei hatten 16-bis 35-jährige transplantationskandidaten besonders gute resultate. das gleiche gilt für eine kurze kalte ischämiezeit und gute initiale transplantatfunktion. die 1-bis 5-jahres-transplantatüberlebensraten für lebendspenden und postmortale organspenden sind in ⊡ abb. 13.4 dargestellt. die aktuellen transplantationsstatistiken werden im jeweiligen jahresbericht der dso (deutsche stiftung organtransplantation) veröffentlicht. die transplantationshalbwertszeit ist die zeit, innerhalb derer die hälfte der zu einem bestimmten zeitpunkt verpflanzter organe nicht mehr funktioniert. sie wird erst ab dem 1. jahr nach transplantation berechnet. dass sie in den letzten beiden jahrzehnten nicht wesentlich gestiegen ist, bedeutet, dass der effekt von ciclosporin auf das kurzzeitüberleben von nierentransplantaten nicht auf das langzeitergebnis übertragbar ist. letzteres wird negativ beeinflusst durch: ▬ hypertensive nierenschädigung ▬ chronische ciclosporintoxizität ▬ rezidiv der grunderkrankung ▬ hyperfiltration eventuell positiv wirken dagegen vermutlich ace-hemmer. management von patienten auf der transplantationsliste unter besonderer beachtung immunologischer aspekte, mitteilungen de deutschen arbeitsgemeinschaft für klinische nephrologie,xxxv long-term pancreas allograft outcome in simultaneous pancreas-kidney transplantation: a comparison of enteric and bladder drainage mycophenolate mofetil, together with ciclosporin a, prevents anti-okt 3 antibody response in kidney transplant recipients immunosuppression in organ transplantation evaluation of pathologic criteria for acute renal allograft rejection: reproducibility, sensitivity, and clinical correlation the humoral immune response towards hla class ii determinants in renal transplantation development of sang-35: a ciclosporine formulation bioequivalent to neoral infection in organ-transplant recipients glomerular diseaseduring hcv infection in renal transplantation clinical practice guidelines: prevention of cytomegalovirus disease after renal transplantation immunosuppressive effects and safety of a sirolimus/ciclosporine combination regimen for renal transplantation the impact of an acute rejection episode on long-term renal allograft survival (t1/2) analysis of hla-dr matching in dna-typed cadaver kidney transplants prospective evaluation of pretransplant blood transfusions in cadaver kidney recipients plasma exchange and tacrolimus-mycophenolate rescue for acute humoral rejection in kidney transplantation a preliminary report of diltiazem and ketoconazole. their ciclosporine-sparing effect and impact on transplant outcome epstein-barr virusinduced posttransplant lymphoproliferative disorders task force and the mayo clinic organized international consensus development meeting transplantation tolerance -the search continues köhler a für die deutsche stiftung organtransplantation (2000) organspende und transplantation in deutschland international standardization of criteria for the histologic diagnosis of renal allograft rejection: the banff working classification of kidney transplant pathology advantage of antithymocyte globulin induction in sensitized kidney recipients: a randomized prospective study comparing induction with and without antithymocyte globulin maintenance pharmacological immunosuppressive strategies in renal transplantation reduced kidney transplant rejection rate and pharmacoeconomic advantage of mycophenolate mofetil das ausmaß der immunsuppression spielt ebenfalls eine rolle. die entwicklung der lymphoproliferativen erkrankung findet hauptsächlich im ersten jahr statt, denn zu diesem zeitpunkt ist die totale immunsuppression am höchsten.die diagnose einer ebv-assoziierten lymphoproliferativen erkrankungen setzt höchste aufmerksamkeit und misstrauen allen auffälligkeiten gegenüber voraus. sie wird gestellt, wenn aus dem tumor entnommenes gewebe lymphoid ist und folgende bedingungen erfüllt, die in einer konsensuskonferenz (paya et al. 1999 ) festgelegt wurden: ▬ ebv-infektion in vielen zellen ▬ mono-oder oligoklonale zellpopulationen ▬ zerstörung der normalen gewebestruktur durch den lymphoproliferativen prozessdurch eine perioperative gabe von gancyclovir sowie reduktion der tacrolimusdosis konnte bei hochrisikopatienten mit ebv-mismatch die inzidenz der ebv-assoziierten lymphoproliferativen erkrankungen signifikant gesenkt werden. angeregt durch retrospektive analysen wird derzeit die prophylaktische gabe von gancyclovir parallel zu okt 3 getestet. neue entwicklungen umfassen die gabe von b-zell-antikörpern, ein in pilotstudien bereits erfolgreich eingesetztes therapieprinzip. die immuntherapie mit lymphokinaktivierten, autologen killerzellen ist noch umstritten. die infusion von spenderleukozyten unter der rationale des vorhandenseins von gegen ebv sensibilisierten zytotoxischen t-zellen in den leukozytenseparationen hatte dagegen bei 3 von 5 knochenmarktransplantierten einen 10-bis 16-monatigen teilremissionserfolg.bezüglich der photochemotherapie gibt es noch keine empfehlungen, lediglich positive erfahrungen bei lungentransplantationen. es werden photosensibilisierende substanzen verabreicht, die sich bevorzugt in malignen stoffwechselaktiven lymphozyten anreichern. die leukozyten werden dann nach leukapherese extrakorporal bestrahlt, maligne lymphozyten sterben bevorzugt ab.bei lymphoproliferativen erkrankungen nach transplantation kann von einer 25-35% überlebensrate ausgegangen werden. t-zell-lymphome haben eine sehr schlechte prognose. key: cord-265277-ymvrserl authors: crooke, stephen n.; ovsyannikova, inna g.; kennedy, richard b.; poland, gregory a. title: immunoinformatic identification of b cell and t cell epitopes in the sars-cov-2 proteome date: 2020-05-14 journal: biorxiv doi: 10.1101/2020.05.14.093757 sha: doc_id: 265277 cord_uid: ymvrserl a novel coronavirus (sars-cov-2) emerged from china in late 2019 and rapidly spread across the globe, infecting millions of people and generating societal disruption on a level not seen since the 1918 influenza pandemic. a safe and effective vaccine is desperately needed to prevent the continued spread of sars-cov-2; yet, rational vaccine design efforts are currently hampered by the lack of knowledge regarding viral epitopes targeted during an immune response, and the need for more in-depth knowledge on betacoronavirus immunology. to that end, we developed a computational workflow using a series of open-source algorithms and webtools to analyze the proteome of sars-cov-2 and identify putative t cell and b cell epitopes. using increasingly stringent selection criteria to select peptides with significant hla promiscuity and predicted antigenicity, we identified 41 potential t cell epitopes (5 hla class i, 36 hla class ii) and 6 potential b cell epitopes, respectively. docking analysis and binding predictions demonstrated enrichment for peptide binding to hla-b (class i) and hla-drb1 (class ii) molecules. overlays of predicted b cell epitopes with the structure of the viral spike (s) glycoprotein revealed that 4 of 6 epitopes were located in the receptor-binding domain of the s protein. to our knowledge, this is the first study to comprehensively analyze all 10 (structural, non-structural and accessory) proteins from sars-cov-2 using predictive algorithms to identify potential targets for vaccine development. significance statement the novel coronavirus sars-cov-2 recently emerged from china, rapidly spreading and ushering in a global pandemic. despite intensive research efforts, our knowledge of sars-cov-2 immunology and the proteins targeted by the immune response remains relatively limited, making it difficult to rationally design candidate vaccines. we employed a suite of bioinformatic tools, computational algorithms, and structural modeling to comprehensively analyze the entire sars-cov-2 proteome for potential t cell and b cell epitopes. utilizing a set of stringent selection criteria to filter peptide epitopes, we identified 41 t cell epitopes (5 hla class i, 36 hla class ii) and 6 b cell epitopes that could serve as promising targets for peptide-based vaccine development against this emerging global pathogen. in december 2019, public health officials in wuhan, china, reported the first case of severe 60 respiratory disease attributed to infection with the novel coronavirus sars-cov-2 (1). since its 61 emergence, sars-cov-2 has spread rapidly via human-to-human transmission (2), threatening to 62 overwhelm healthcare systems around the world and resulting in the declaration of a pandemic by the 63 world health organization (3). the disease caused by the virus is characterized by fever, 64 pneumonia, and other respiratory and inflammatory symptoms that can result in severe inflammation of 65 lung tissue and ultimately death-particularly among older adults or individuals with underlying 66 comorbidities (4-6). as of this writing, the sars-cov-2 pandemic has resulted in 4 million confirmed 67 cases of covid-19 and over 280,000 deaths worldwide (7). 68 sars-cov-2 is the third pathogenic coronavirus to cross the species barrier into humans in the 69 past two decades, preceded by severe acute respiratory syndrome coronavirus (sars-cov) (8, 9) and 70 middle-east respiratory syndrome coronavirus (mers-cov) (10). all three of these viruses belong to the 71 β -coronavirus genus and have either been confirmed (sars-cov) or suggested (mers-cov, sars-72 cov-2) to originate in bats, with transmission to humans occurring through intermediary animal hosts 73 (11) (12) (13) (14) . while previous zoonotic spillovers of coronaviruses have been marked by high case fatality rates 74 (~10% for sars-cov; ~34% for mers-cov), widespread transmission of disease has been relatively 75 limited (8,098 cases of sars; 2,494 cases of mers) (15). in contrast, sars-cov-2 is estimated to have 76 a lower case fatality rate (~2-4%) but is far more infectious and has achieved world-wide spread in a 77 matter of months (16). 78 as the number of covid-19 cases continues to grow, there is an urgent need for a safe and 79 effective vaccine to combat the spread of sars-cov-2 and reduce the burden on hospitals and healthcare 80 systems. no licensed vaccine or therapeutic is currently available for sars-cov-2, although there are 81 over 100 vaccine candidates reportedly in development worldwide. seven vaccine candidates have 82 peptide was removed from the structure using chimera 1.14 (university of california-san francisco) (50) 158 prior to running simulations. ten models of each peptide-hla complex were generated on the basis of 159 minimized energy scores, and the top model for each complex was selected for comparative analysis. 160 prediction and structural modeling of sars-cov-2 b cell epitopes 161 linear b cell epitope predictions were performed on the three exposed sars-cov-2 structural 162 proteins: s (genbank accession: qhd43416), m (qhd43419), and e (qhd43418) using the bepipred 163 1.0 algorithm (51). epitope probability scores were calculated for each amino acid residue using a 164 threshold of 0.35 (corresponding to > 0.75 specificity and sensitivity below 0.5), and only epitopes > 5 165 amino acid residues in length were further analyzed. the structure of the sars-cov-2 s protein was 166 accessed from the protein data bank (pdb id: 6vsb) (52). discontinuous (i.e., structural) b cell epitope 167 predictions for the s protein structure were carried out using discotope 1.1 (53) with a score threshold 168 greater than -7.7 (corresponding to > 0.75 specificity and sensitivity below 0.5). the main protein 169 structure was modeled in pymol (schrödinger, llc), with predicted b cell epitopes identified by both 170 bepipred 1.0 and discotope 1.1 highlighted as spheres. 171 genetic similarity of sars-cov-2 isolates 173 the primary goal of our study was to identify peptide epitopes that would be broadly applicable 174 in vaccine development efforts against sars-cov-2. we identified 64 point mutations and 4 deletions 175 across the genomes of 44 clinical isolates, with all deletions and the majority of mutations (n=45) 176 occurring in the orf1ab polyprotein (supp. figure s1 ). single-point mutations were also found in the s 177 protein (n=5), n protein (n=5), orf8 protein (n=3), orf3a protein (n=2), orf10 protein (n=2), e 178 protein (n=1), and m protein (n=1). despite the genetic diversity introduced by these events (figure 1d) , 179 matrix analysis determined that > 99% sequence identity was maintained across all viral genomes. based 180 on these findings and for study feasibility, the genome from the original virus isolate 181 genbank: mn908947) was selected as the consensus sequence for all further analyses. 182 we next identified potential cd8 + t cell epitopes from all proteins in the sars-cov-2 184 proteome. using the netctl 1.2 predictive algorithm, we analyzed the complete amino acid sequence of 185 each viral protein to generate sets of 9-mer peptides predicted to be recognized across at least one of the 186 major hla class i supertypes (figure 2a, supp. figure s2) . this approach yielded a significant number 187 of potential epitopes from each viral protein (orf10: 9, orf6: 17, orf8: 23, e: 25, orf7: 39, n: 80, 188 m: 87, orf3a: 87, s: 321, orf1ab: 2814) , with the number directly related to the size of the parent protein. we used the netmhcpan 4.0 server to further refine the list of potential cd8 + t cell epitopes by 190 predicting binding affinity across representative hla class i alleles (see methods) and assigning 191 percentile scores to quantify binding propensity. peptides with percentile rank scores < 0.5% (i.e., strong 192 binders) were filtered using a 500 nm threshold for binding affinity to further delineate 740 candidate 193 hla class i epitopes from the viral proteome (54). for feasibility reasons, we refined our selection to 83 194 candidate epitopes by excluding peptides predicted to bind only one hla molecule (supp . table s1 ). 195 the resultant peptides were enriched for predicted binders to hla-b molecules 196 hla-b*58:01=32; hla-b*08:01=31) ( figure 2b) . a final round of selection on the basis of hla 197 promiscuity (i.e., predicted binding to > 3 hla molecules) and predicted antigenicity scoring using the 198 vaxijen 2.0 server produced a subset of five candidate peptides (four orf1ab, one s protein) as potential 199 targets for vaccine development (table 1) with the hypothesis that increased hla binding promiscuity 200 meant broader population base coverage by those peptides. these peptides were predicted to provide 74% 201 global population coverage and had higher predicted binding affinities for hla-b molecules 202 (b*08:01=42.6 nm; b*15:01=67.7 nm; b*58:01=110.3 nm) compared to hla-a molecules 203 (a*01:01=238.6 nm; a*24:02=142.9 nm), with the exception of one orf1ab-derived peptide 204 (mmisagfsl) that was predicted to bind hla-a*02:01 with high affinity (ic 50 = 6.9 nm) ( figure 2c) . 205 we also sought to identify potential hla class ii peptides from sars-cov-2, as the stimulation 207 of cd4 + t-helper cells is critical for robust vaccine-induced adaptive immune responses. using the 208 netmhciipan 3.2 server, we identified 801 candidate hla class ii peptides from the viral proteome 209 predicted to have high binding affinity (< 500 nm) and percentile rank scores < 2% across a reference 210 panel of hla molecules covering > 97% of the population (33, 45). similar to hla class i epitope 211 predictions, the number of class ii epitopes identified for each viral protein (orf10: 4, e protein: 7, 212 orf7: 8, orf8: 10, orf6: 14, n: 15, m: 29, orf3a: 31, s: 96, orf1ab: 587) was largely proportional 213 to protein size. after excluding peptides predicted to bind to only a single hla molecule in our panel, we 214 refined our selection to 211 peptides (supp . table s2 ), which were enriched for binding to hla-drb1 215 molecules (n=142) ( figure 2d ). filtering on hla promiscuity and predicted antigenicity scores yielded 216 a subset of 36 peptides (24 orf1ab, 5 s protein, 2 m protein, 2 orf7, 1 orf3a, 1 orf6, 1 orf8) as 217 cd4 + t cell epitopes for further study ( table 1) . these peptides were predicted to collectively provide 218 99% population coverage and have significantly higher average binding affinities for hla-dr alleles 219 (drb1=56.4 nm; drb3=50.9 nm; drb4=70.1 nm; drb5=18 nm) compared to hla-dp (155.9 nm) 220 or hla-dq (238.6 nm) molecules ( figure 2e) . 221 characterization of hla class i peptide docking with hla-b*15:01 the five candidate hla class i peptides identified by our computational approach were predicted 223 to provide coverage across six hla alleles (a*01:01, a*02:01, a*24:02, b*08:01, b*15:01, b*58:01). 224 the peptide famqmayrf was the only candidate predicted to bind to a*24:02 molecules, whereas 225 mmisagfsl was predicted to uniquely bind a*02:01 and b*08:01 molecules. four of the five peptides 226 were predicted to bind a*01:01 and b*58:01 molecules, but all were predicted to bind with relatively 227 high affinity (average ic 50 = 67.7 nm) to hla-b*15:01. therefore, we performed molecular docking 228 studies of each peptide with the molecular structure of hla-b*15:01 (pdb: 3c9n). 229 all peptides were predicted to bind within the peptide binding groove, forming hydrogen bond 230 contacts with numerous amino acid side chains ( figure 3a) . the binding motif for hla-b*15:01 is 231 highly selective for residues at the p2 and p9 anchor positions, with a preference for bulky hydrophobic 232 amino acids at the c-terminus ( figure 3b ) (55). all candidate peptides possessed terminal residues (phe, 233 tyr, leu) that fit into the hydrophobic binding pocket of the hla groove, further supporting that these 234 peptides should be strong binders of hla-b*15:01 and promising candidates for vaccine development 235 studies. 236 an effective vaccine should stimulate both cellular and humoral immune responses against the 238 target pathogen; therefore, we also sought to identify potential b cell epitopes from sars-cov-2 239 proteins. we limited our analysis to the primary structural proteins exposed on the virus capsid (s, n, m, 240 and e), as these are the most accessible antigens for engaging b cell receptors. using the bepipred 1.0 241 algorithm, we identified 26 potential linear b cell epitopes in the s protein, 14 potential epitopes in the n 242 protein, and 3 potential epitopes in the m protein ( table 2) . no epitopes were identified in the e protein. 243 studies have previously shown the s protein to be the predominant target of neutralizing antibodies 244 against coronaviruses (56, 57), and, as our findings indicate this to likely be the case for sars-cov-2, 245 we focused all subsequent analyses on the s protein. while the n protein is also a major target of the 246 antibody response (58), it is unlikely these antibodies have any neutralizing activity based on the viral 247 structure. as epitope conformation can significantly influence recognition by antibodies, we also 248 employed discotope 1.1 to identify discontinuous b cell epitopes in the protein structure. our analysis 249 identified 14 potential structural epitopes in the s protein (7 in the s1 domain, 7 in the s2 domain), with 250 six regions having significant overlap with our predicted linear epitopes ( table 2) . antigenic regions 251 identified in both analyses were modeled using the recently published structure of the sars-cov-2 s 252 protein (52) to examine their accessibility for antibody binding. epitopes in the s2 domain (p792-d796; 253 y1138-d1146) were clustered near the base of the spike protein, whereas regions in the s1 domain 254 (d405-d428; n440-n450; g496-p507; d568-t573) were exposed on the protein surface (figure 4 ). in the face of the covid-19 pandemic, it is imperative that safe and effective vaccines be rapidly 257 developed in order to induce widespread herd immunity in the population and prevent the continued 258 spread of sars-cov-2. our study identified probable peptide targets of both cellular and humoral 259 immune responses against sars-cov-2 using computational methodologies to investigate the entire viral 260 proteome a priori. studies such as these are paramount during the early stages of pandemic vaccine 261 development given the relative scarcity of biological data available on the viral immune response, and we 262 employed an approach that allowed us to systematically refine our predictions using increasingly stringent 263 criteria to select a subset of the most promising epitopes for further study. the data we have curated could 264 inform the design of a candidate peptide-based vaccine or diagnostic against sars-cov-2. 265 as selective pressures are known to introduce viral mutations that promote fitness and can lead 266 to evasion of immune responses (59, 60), we first sought to investigate the genetic similarity of all 267 reported sars-cov-2 clinical isolates and identify a consensus sequence for use in our epitope 268 prediction studies. we identified 68 mutations/deletions across the 44 genomes of clinical isolates 269 reported as of 27 february 2020. despite these variations, the viral genomic identity was > 99% 270 conserved across all isolates. as the protein coding sequences were largely conserved, the genome of the 271 original virus isolate (wuhan-hu-1) was deemed a representative consensus sequence for analysis of the 272 sars-cov-2 proteome. 273 cd4 + and cd8 + t cell responses will likely be directed against both structural and non-structural 274 proteins during antiviral immune responses, as all viral proteins are accessible for processing and 275 presentation on the hla molecules of infected cells. therefore, we sought to identify t cell epitopes 276 across the entire viral proteome. our analysis identified 83 potential cd8 + t cell epitopes (supp. table 277 s1) and 211 potential cd4 + t cell epitopes (supp . table s2) , with stringent filtering for more 278 promiscuous peptides with high predicted antigenicity yielding a subset of 5 cd8 + t cell epitopes and 36 279 cd4 + t cell epitopes ( table 1 ) as potential targets for vaccine development. a single study by grifoni 280 and colleagues has recently reported the computational identification of 241 cd4 + t cell epitopes from 281 sars-cov-2 (35), and 22 peptides from our analysis shared sequence homology or were nested within 282 peptides identified in their study. moreover, seven peptides from this initial report were replicated in our 283 final subset of hla class ii epitopes, supporting that these peptides may be promising vaccine targets. 284 an increasing number of studies have employed predictive algorithms to identify potential hla 285 class i epitopes for sars-cov-2, although relatively few have comprehensively analyzed the entire viral 286 proteome. a report from feng et al. recently outlined the identification of 499 potential class i epitopes in 287 the main structural proteins from sars-cov-2 but did not consider any non-structural proteins (38). 288 grifoni and colleagues conducted a more rigorous analysis, identifying 628 unique cd8 + t cell epitopes across all sars-cov-2 proteins but focusing their analyses solely on peptides with sequence homology 290 to known sars-cov epitopes (35). our approach initially identified ~ 3,500 potential cd8 + t cell 291 epitopes across all viral proteins, which we refined to a subset of 5 peptides (table 1) . one peptide 292 derived from orf1ab (mmisagfsl) was predicted to bind hla-a*02:01 with high affinity (ic 50 = 6.9 293 nm) ( figure 2c) . given the prevalence of this allele in the american and european populations (25-60% 294 frequency) (61), mmisagfsl may represent a promising epitope capable of providing broad vaccine 295 population coverage. 296 we also observed a notable enrichment of epitopes predicted to bind hla-b molecules-297 particularly hla-b*15:01-as we imposed more stringent selection criteria ( figure 2b ). all five peptides 298 identified by our approach were predicted to be relatively strong binders for this allele (ic 50 = 67.7 nm), 299 with molecular docking simulations illustrating strong contacts with amino acid residues in the peptide 300 binding groove (figure 3 a, b) . a recent computational study identified another hla-b allele (b*15:03) 301 as having a high capacity for presenting epitopes from sars-cov-2 that were conserved among other 302 pathogenic coronaviruses (62). these data collectively suggest the hla-b locus may be significantly 303 associated with the immune response to sars-cov-2 (and potentially other coronaviruses), with further 304 biological studies warranted to determine the true role of host genetics in sars-cov-2 immunology. 305 lastly, we analyzed the primary structural proteins of sars-cov-2 (s, n, m, e proteins) for 306 potential b cell epitopes, as an ideal vaccine would be designed to stimulate both cellular and humoral 307 immunity. our analysis identified potential linear b cell epitopes in all proteins except for the e protein 308 ( table 2 ). the greatest number of epitopes were predicted in the surface-exposed s protein (n=26), but a 309 significant number of epitopes were also predicted for the n protein (n=14). this is not surprising, as 310 previous reports identified the n protein as a significant target of the humoral response to sars-cov 311 (63, 64). as the s protein is the predominant surface protein and has been the primary target of 312 neutralizing antibody responses against other coronaviruses (56, 57), we elected to focus our subsequent 313 analyses solely on antigenic regions in the s protein. we identified 14 potential structural epitopes in the 314 s protein structure and referenced against our linear epitope predictions to identify six regions that were 315 independently identified by both analyses (table 2, figure 4) to further evaluate the potential of these six antigenic regions as targets for antibody binding, we 319 modeled their surface accessibility on the crystal structure of the sars-cov-2 spike protein (52). four 320 regions in the s1 domain (d405-d428; n440-n450; g496-p507; d568-t573) were solvent exposed 321 (figure 4 a, b) , with minimal steric hindrance for antibody accessibility. the s1 domain contains the 322 residues (n331-v524) important for virus binding to angiotensin converting enzyme 2 (ace2) on the cell surface (65), and studies have shown that antibodies with potent neutralizing activity against sars-cov 324 target this domain (66-68). indeed, three of the four s1 epitopes identified in our analyses are located in 325 the ace2-binding region, supporting their potential utility in vaccine development against sars-cov-2. 326 two regions were identified in the s2 "stalk" domain of the s protein (figure 4 a, c) . while y1138-327 d1146 is located at the base of the s protein and likely inaccessible to antibodies, p792-d796 is on the 328 outer face of the protein and has been previously identified as part of a larger b cell epitope that is 329 conserved with sars-cov (35). as sars-cov s2-specific antibodies have previously been shown to 330 possess antiviral activity (66), it is interesting to speculate whether a strategy similar to targeting the 331 influenza hemagglutinin protein stalk could be employed for developing a broadly reactive coronavirus 332 vaccine. 333 our study possessed several strengths and limitations. rather than restricting our analyses of 334 hla class i and class ii epitopes to specific proteins based on prior studies of sars-cov immunology, 335 we investigated the complete proteome of sars-cov-2 using an unbiased approach. furthermore, we 336 employed a multi-tiered strategy for identifying putative b cell and t cell epitopes from all viral proteins 337 studied. our initial analyses were performed with liberal thresholds for epitope identification, and at each 338 additional step, we imposed more stringent selection criteria to filter these peptides to a subset of b cell 339 and t cell epitopes for further study. nevertheless, the results of this study are derived purely from 340 computational methods, and it should be noted that computational algorithms can fail to capture a 341 significant number of antigenic peptides (69). experimental validation with biological samples will 342 ultimately be needed. 343 during the early stages of a pandemic, access to sufficient biological samples may be extremely 344 limited, so we must continue to utilize methodologies-such as computational predictive algorithms-345 that allow us to explore the epitope landscape for experimental vaccine development. our approach in this 346 study allowed us to identify and refine a manageable subset of t cell and b cell epitopes for further 347 testing as components of a sars-cov-2 vaccine. based on our results, our proposed sars-cov-2 348 vaccine formulation could contain the following: 1) one or more b cell peptide epitopes from the s 349 protein to generate protective neutralizing antibodies; and 2) multiple hla class i and class ii-derived 350 peptides from other viral proteins to stimulate robust cd8 + and cd4 + t cell responses. based on global 351 allele frequencies, these class i and class ii peptides would be expected to collectively provide 74% and 352 99% population coverage, respectively. while such a vaccine could be readily formulated as a synthetic 353 polypeptide or an adjuvanted peptide mixture, these strategies may not retain the epitope structural 354 features necessary to induce a robust antibody response. recombinant nanoparticles and assembly into 355 vlps represent promising alternative vaccine platforms, as they have been extensively used for the 356 controlled display and delivery of peptide-based vaccine components (70-73). by omitting whole viral 357 proteins from the vaccine formulation, a peptide-based sars-cov-2 vaccine should have a well-358 tolerated safety profile and avoid the adverse events previously observed with experimental sars-cov 359 vaccines (19) (20) (21) (22) . 360 in summary, we have identified 41 potential t cell epitopes (5 hla class i, 36 hla class ii) and 361 6 potential b cell epitopes from across the sars-cov-2 proteome that are predicted to have broad 362 population coverage and could serve as the basis for designing investigational peptide-based vaccines. 363 further study on the biological relevance and immunogenicity of these peptides is warranted in an effort 364 to develop a safe and effective vaccine to combat the sars-cov-2 pandemic. 365 the authors would like to thank caroline l. vitse for editorial assistance with this manuscript. 367 the research presented here was not supported by any specific funding source. 368 :2020.2003.2022.20040600. 532 63. huang lr, et al. (2004) evaluation of antibody responses against sars coronaviral nucleocapsid 533 or spike proteins by immunoblotting or elisa. identification workflow illustrating the algorithms used (33, 40-43, 45-47, 51, 53) and filtering criterion 580 applied to refine peptide selection. (d) cladogram illustrating the genetic relationship of sars-cov-2 581 isolates. the original viral isolate and consensus sequence (wuhan-hu-1) is highlighted in red. 582 583 figure 2 . immunogenicity scoring of peptides in the sars-cov-2 proteome with predicted hla class i 584 and ii coverage and binding affinities. discotope prediction algorithms highlighted on the trimeric structure of the s glycoprotein. inset panels 604 show the s1 domain (upper) and s2 domain (lower a new coronavirus associated with human respiratory disease in china a familial cluster of pneumonia associated with the 2019 novel 392 coronavirus indicating person-to-person transmission: a study of a family cluster who declares covid-19 a pandemic epidemiological and clinical characteristics of 99 cases of 2019 novel 397 coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with clinical features of patients infected with coronavirus disease (covid-19) situation report -113 identification of a novel coronavirus in patients with severe acute 407 respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome. 409 isolation of a 411 novel coronavirus from a man with pneumonia in saudi arabia bats are natural reservoirs of sars-like coronaviruses a pneumonia outbreak associated with a new coronavirus of probable bat 416 origin middle east respiratory syndrome coronavirus in bats middle east respiratory syndrome coronavirus in dromedary camels: 420 an outbreak investigation structure, function, and antigenicity of the sars-cov-2 spike 422 glycoprotein covid-19: knowns, unknowns, and questions. msphere 5(2). 424 17. world health organization (2020) draft landscape of covid-19 candidate vaccines tortoises, hares, and vaccines: a cautionary note for sars-cov-2 vaccine 428 development immunization with sars coronavirus vaccines leads to pulmonary 430 immunopathology on challenge with the sars virus vaccine efficacy in senescent mice challenged with recombinant sars-432 cov bearing epidemic and zoonotic spike variants prior immunization with severe acute respiratory syndrome (sars)-434 associated coronavirus (sars-cov) nucleocapsid protein causes severe pneumonia in mice 435 infected with sars-cov characterization of the receptor-binding domain (rbd) of 2019 novel 537 coronavirus: implication for development of rbd protein as a viral attachment inhibitor and 538 vaccine quantitative comparison of the efficiency of antibodies against s1 and s2 540 subunit of sars coronavirus spike protein in virus neutralization and blocking of receptor 541 binding: implications for the functional roles of s2 subunit neutralizing epitopes of the sars-cov s-protein cluster independent of 543 repertoire, antigen structure or mab technology identification and characterization of novel neutralizing epitopes in the 545 receptor-binding domain of sars-cov spike protein: revealing the critical antigenic 546 determinants in inactivated sars-cov vaccine discovery of 548 naturally processed and hla-presented class i peptides from vaccinia virus infection using mass 549 spectrometry for vaccine development development of autologous c5 vaccine nanoparticles to reduce 551 intravascular hemolysis in vivo plug-and-display: decoration of virus-like particles via isopeptide 553 bonds for modular immunization a novel candidate hpv vaccine: ms2 phage vlp displaying a tandem hpv 555 l2 peptide offers similar protection in mice to gardasil-9 targeted 557 immunomodulation using antigen-conjugated nanoparticles scoring function for automated assessment of protein structure 560 template quality galaxypepdock: a protein-peptide docking tool based 562 on interaction similarity and energy optimization key: cord-031937-qhlatg84 authors: verma, anukriti; sharda, shivani; rathi, bhawna; somvanshi, pallavi; pandey, bimlesh dhar title: elucidating potential molecular signatures through host-microbe interactions for reactive arthritis and inflammatory bowel disease using combinatorial approach date: 2020-09-15 journal: sci rep doi: 10.1038/s41598-020-71674-8 sha: doc_id: 31937 cord_uid: qhlatg84 reactive arthritis (rea), a rare seronegative inflammatory arthritis, lacks exquisite classification under rheumatic autoimmunity. rea is solely established using differential clinical diagnosis of the patient cohorts, where pathogenic triggers linked to enteric and urogenital microorganisms e.g. salmonella, shigella, yersinia, campylobacter, chlamydia have been reported. inflammatory bowel disease (ibd), an idiopathic enteric disorder co-evolved and attuned to present gut microbiome dysbiosis, can be correlated to the genesis of enteropathic arthropathies like rea. gut microbes symbolically modulate immune system homeostasis and are elementary for varied disease patterns in autoimmune disorders. the gut-microbiota axis structured on the core host-microbe interactions execute an imperative role in discerning the etiopathogenesis of rea and ibd. this study predicts the molecular signatures for rea with co-evolved ibd through the enveloped host-microbe interactions and microbe-microbe ‘interspecies communication’, using synonymous gene expression data for selective microbes. we have utilized a combinatorial approach that have concomitant in-silico work-pipeline and experimental validation to corroborate the findings. in-silico analysis involving text mining, metabolic network reconstruction, simulation, filtering, host-microbe interaction, docking and molecular mimicry studies results in robust drug target/s and biomarker/s for co-evolved ibd and rea. cross validation of the target/s or biomarker/s was done by targeted gene expression analysis following a non-probabilistic convenience sampling. studies were performed to substantiate the host-microbe disease network consisting of protein-marker-symptom/disease-pathway-drug associations resulting in possible identification of vital drug targets, biomarkers, pathways and inhibitors for ibd and rea. our study identified na((+))/h((+)) anti-porter (nhaa) and kynureninase (kynu) to be robust early and essential host-microbe interacting targets for ibd co-evolved rea. other vital host-microbe interacting genes, proteins, pathways and drugs include adenosine deaminase (ada), superoxide dismutase 2 (sod2), catalase (cat), angiotensin i converting enzyme (ace), carbon metabolism (folate biosynthesis) and methotrexate. these can serve as potential prognostic/theranostic biomarkers and signatures that can be extrapolated to stratify rea and related autoimmunity patient cohorts for further pilot studies. www.nature.com/scientificreports/ approach has advantages over the traditional approach for network analysis that can help to simultaneously characterize several protein interaction modules and has the potential to study complex diseases. the vital information obtained in our study from in-silico analysis is cross-validated through targeted gene expression experimental analysis on patient cohorts. this study will help us to obtain clinico-molecular informatics-based outcomes and expand our knowledge regarding the understanding of biological functions for ibd co-existent rea. text mining: data screening and selection. systematic data search and organization was carried out incorporating data identification, data screening and data selection to find target microorganisms involved in inflammatory bowel disease (ibd) and reactive arthritis (rea). data identification was carried out to obtain records through data sources utilising keywords (e.g. "microorganism and inflammatory bowel disease and reactive arthritis") incorporating boolean operators (and/or/not). data screening and selection were carried as part of the manual curation through primary and secondary screening scrutinizing collected data records to obtain organized records relevant for the autoimmune and enteric disorders triggered by microorganisms, especially ibd and rea and the microbial triggers implicated in ibd and rea that were utilised for further metabolic network reconstruction. bottom-up approach consisting of draft reconstruction and manual reconstruction refinement was followed to create metabolic networks of obtained target microorganisms. genome-scale metabolic models simulation, reconstruction and visualization (gemsirv) software 51 that includes reciprocal basic local alignment search tool (blast) of target microorganisms against a template metabolic network of its phylogenetic neighbour and incorporates information from national center for biotechnology information (ncbi), kyoto encyclopedia of genes and genomes (kegg) and transport db was used for creating draft reconstructs. the manual curation of missing links or gaps in the draft reconstruct was done by mapping the incomplete information to other databases such as expert protein analysis system (expasy) 52 and integrated relational enzyme database (intenz) 53 . this fully connected and annotated network was used for further simulation studies 54 . the metabolic networks thus obtained were visualized using celldesigner, a tool for modelling and editing biochemical and gene-regulatory networks. simulation analysis was carried by converting the metabolic networks obtained into a mathematical model and performing the gene deletion analysis to retrieve essential genes. model conversion was through generation of stoichiometric based matrixes consisting of reactions (columns) and metabolites (rows) corresponding to respective genes. upper boundary and lower boundary fluxes i.e. movement of matter across a system were generated for the gene associated reactions and metabolites that was extracted in systems biology markup language (sbml) format. the next step was gene deletion analysis done using the constraint based reconstruction and analysis toolbox (cobra) that runs in matrix laboratory (matlab) 55 for finding the essential genes based upon the gene-reaction matrix and boolean relationship between genes and reactions 56 . the purpose of data filtering is to remove repeats and homologs from essential genes of target microorganisms associated with ibd co-existent rea. the non-homologous protein sequences corresponding to the essential genes of target microorganisms were extracted from pathosystems resource integration (patric) database 57 . refinement of protein sequences was further done using cluster database at high identity with tolerance (cd-hit) 58 suite so as to have 60% identity non-repeat sequence tolerance stringency. blast-p was further used to remove the homologs from such non-repeats against human database at e-value of 10 -4 to obtain nonhomologous protein sequences used for further in-silico analysis. essential host-microbe and microbe-microbe interactions. the host-microbe interactions of the non-homologous proteins for the selected target microorganisms were obtained using host-pathogen interaction database (hpidb) 59, 60 . the host-microbe interactions were visualised using cytoscape. simulation analysis (gene essentiality) was done to obtain the essential host proteins interacting with common microbe proteins of microorganisms triggering ibd and rea utilising the human metabolic model hmr 2, a cobra compliant metabolic model of human consisting of around 3,765 genes, 8,000 reactions and 3,000 metabolites 61 . this led to profiling of the common host-microbe and microbe-microbe interactions comprehending the complex 'interspecies communication' as complex interaction maps, executed using search tool for the retrieval of interacting genes/proteins (string) 62,63 . host-microbe disease network and molecular mimicry studies. the host-microbe disease network is a multilayered archetype that connects the protein-marker-symptom/disease-drug-pathway associations. the contributions of the microorganisms in the co-evolved ibd and rea as part of the disease network was created through the interactive maps of the essential host interaction proteins (verified using literature survey) and the information processed through gene expression data analysis 64 . the information patronised here is mostly scored through the available non-specific protein diagnostic markers of both ibd and rea e.g. c-reactive protein (crp), interleukin 6 (il6) and toll like receptor 4 (tlr4), major histocompatibility complex, class i, b (hla-b) and major histocompatibility complex, class ii, dr beta 1 (hla-drb1) with the essential host proteins determined using string 65 . database genecards 66 was used to assess the role of these interacting partners aka proteins further with symptoms/diseases associated with ibd and rea. the pathways of the above host interacting proteins were found out using kegg database that provides ontologies for proteins related to biological processes 67 www.nature.com/scientificreports/ subsequently, the role of drugs or inhibitors used to suppress the effect of ibd and rea such as indomethacin, prednisone, ciprofloxacin, sulfasalazine, azathioprine, methotrexate and hydroxychloroquine was scored in the disease network through their docking studies against the potential targets (both host as well microbial targets) as per published methodologies 68, 69 . the host-microbe disease network which is an amalgamation of all the above patterned associations was visualized using cytoscape software 70 . molecular mimicry analysis between the vital targets triggering ibd co-evolved rea, essential human proteins including hla-b27, hla-b51 and hla-drb1 was done using data repository expasy. this led to retrieval of microbe relayed protein sequences that have been implicated in disease development after sequence alignment performed using emboss 71 . experimental evidences to identify the signature molecules in patient samples. the cross-validation of vital in-silico targets was done in rea patient cohort cases via targeted gene expression analysis. scientific and ethical clearance was taken from amity university ethics committee and institutional ethics committee, fortis noida for handling the patient samples. all experiments were performed in accordance with indian council of medical research (icmr) guidelines constituting the ethics committees. the study was carried out for 6 months on the rare disorder rea patients, with the inclusion criteria as patients having rea according to european spondyloarthropathy study group (essg) 72 and exclusion criteria as patients undergoing treatment from last 3-6 months and healthy controls (hc). the participants were inducted in the study design with an informed consent form along with a questionnaire containing information regarding symptomatic and diagnostic history of patient and linked disorders. blood (5 ml) was drawn from participants in ethylenediaminetetraacetic acid (edta) vacutainers. these were transported to the laboratory for further analysis. the processing of the samples was done within 2-4 h of procurement 73 . peripheral blood mononuclear cells (pbmc's) were isolated from blood using density gradient centrifugation 74 . rna was isolated from pbmc's using trizol method 75 . the quantification of rna was done using nano-drop 76 . the high capacity cdna reverse transcription kit (applied biosystems™) was used for conversion of rna to single-stranded cdna as per the standard protocol 77 . quantitative pcr analysis of target gene was executed using biorad cfx96 real time-pcr taking human housekeeping gene, gapdh as a reference. previously reported primers for qpcr analysis of target and reference gene were selected for this study 78, 79 following the standard protocol 80 . relative gene expression analysis from qpcr data was performed using the relative expression software tool (rest® 2009) 81 that utilises the expression of reference genes to normalize expression of target genes in different samples. the schematic representation of methodology involved in our combinatorial analysis is provided in fig. 1 . text mining: data screening and selection. a systematic literature mining and curation for our thematic connecting autoimmune disorders, inflammatory bowel disease (ibd) and reactive arthritis (rea) was carried out. data identification extracted 1,071 records (articles in journals, book chapters, conference papers etc.) corresponding to autoimmune and enteric disorders. data screening extracted 426 records of autoimmune and enteric disorders triggered by microorganisms that belong to class of bacteria, fungi, protozoan, mites, virus, yeast and nematode. data selection yielded 48 ibd, 32 rea and 5 ibd co-evolved rea records. data selection was directed towards the microbial contenders implicated here resulting in 6 target microorganisms namely campylobacter jejuni, escherichia coli o157:h7, klebsiella oxytoca, salmonella typhimurium, shigella dysenteriae and yersinia enterocolitica, whose genome information was available. the etiopathogenesis in the co-evolved disorders have been documented through gut microbiome associated host-pathogen interactions studies, perpetuating where pathogen microorganisms involve in dysbiosis leading to autoimmunity. the results of text mining are provided in fig. 2 . the list of microorganisms is provided in supplementary table s1 online. ing of genes along with their corresponding proteins, reactions and metabolites for the selected microorganisms serve as primary set of partial metabolic network information. the missing data persistent in the draft reconstruct obtained through genome-scale metabolic models simulation, reconstruction and visualization (gem-sirv) was manually refined. entirely associated metabolic networks of target microorganisms were obtained (genes, proteins and reactions). the essential genes of microorganisms (vital for survival. sustenance and growth) were obtained after performing simulation on mathematical models consisting of gene associated reactions and metabolites (metabolites, inner cell reactions, exchange reactions and essential genes). due to lack of availability of exchange reactions for campylobacter jejuni, simulation analysis on the partial metabolic network could not be carried out and essential genes could not be retrieved. an alternative approach for finding essential genes of campylobacter jejuni was carried out. the essential genes of campylobacter jejuni were taken from our previous published report and were found out to be 228 69 . table 1 portrays the results of metabolic network reconstruction and simulation of target microorganisms. the metabolic network and simulation analysis data of target microorganisms is provided in supplementary table s2 online. the proteins corresponding to essential genes, non-repeats and non-homologs were obtained as stated below according to the parenthesis {proteins corresponding to essential genes, non-repeats, non-homologs}. the essential genes, their corresponding proteins, reactions and metabolites from the curated dataset were refined to create a list of most relevant molecular indicators to assess their coveted role in disease establishment. the non-redundant filtered proteins were utilised further in the computational work-pipeline canvassing the drug targets and signatures in the interspecies communication. essential host-microbe and microbe-microbe interactions. the central mechanism of hostmicrobe/microbe interface conferred through gut microbiome was correlated for the selected microbial species and processed to obtain the common signatures so as to follow the core system of metabolic changes affecting the host harbouring them as either commensal or pathogenic loads. the interactors between human and target microorganisms were obtained. the interactors of escherichia coli o157:h7 were 136; klebsiella oxytoca were 141; salmonella typhimurium were 136; shigella dysenteriae were 117 and yersinia enterocolitica were 133. there were no interactors for campylobacter jejuni (supplementary table s3 -s7 online). table 2 shows the results of filtering and host-microbe interactions of protein sequences corresponding to essential genes of target microorganisms. www.nature.com/scientificreports/ the host-microbe interactors were analysed for all the target microbial species and processed to obtain the common signatures. 43 proteins were found between all target microorganisms having interaction among themselves and with 130 human proteins. the essential host correlative targets to the microbial gene targets were followed by obtaining host essential genes and corresponding proteins from human metabolic model hmr 2. there were 1,401 essential proteins (supplementary table s8 online) the essential human protein was found out to be kynu having interaction with essential microbial protein nhaa (fig. 3) . nhaa was also having interactions with non-essential hcls1 associated protein x-1 (hax1), prolyl endopeptidase-like (ppcel), biogenesis of lysosomal organelles complex 3 subunit 1 (hps1) and eukaryotic translation initiation factor 2 alpha kinase 1 (e2ak1) proteins of human host. kynu was further mapped with host proteins (direct and indirect) resulting in 1994 interactions. out of these the single connected essential protein interactions were 988 and protein interactors were 412 ( fig. 4 and see supplementary table s9 online). the research design here followed to assess the interaction map of essential proteins in human host to indicate the clinical insights in pathophysiological trends in the autoimmune development. host-microbe disease network and molecular mimicry. the human essential proteome complement with its interacting proteins were analysed further as part of the disease network. 394 human essential protein interactors were found to be associated with ibd and similarly 3 essential protein interactors namely adenosine supplementary table s10 online) . these 397 proteins can be postulated as probable contenders transcending their role in the simulated network as important regulators in the co-existent disorders. the composite associations of the above 397 proteins with non-specific protein diagnostic markers of ibd and rea were obtained (see supplementary table s11 online) . this gave rise to a single connected protein network consisting of 402 proteins and 13,350 interactions. the association of above 402 with symptoms and diseases linked with ibd and rea were obtained (see supplementary table s12 online) . apart from non-specific diagnostic markers, the major protein linked with majority of symptoms/diseases is angiotensin i converting enzyme (ace). 78 pathways of the 402 proteins were obtained (see supplementary table s13 online) in total out of which the pathway associated with majority of proteins was carbon metabolism. another layer of disease network substantiates the role of therapeutic regime followed in the studied autoimmune diseases, so the docking analysis of drugs used to suppress the effect of ibd and rea against nhaa of target microorganisms and kynu of human host was done. the docking analysis resulted in docking scores that represent binding of drugs with host kynu and microbial nhaa of all 5 microorganisms selected in our study. higher the negative docking score more is the binding 68 . escherichia coli o157:h7 nhaa shows highest and lowest docking score with methotrexate (− 7.362) and azathioprine (− 3.491); klebsiella oxytoca nhaa with methotrexate (− 5.083) and azathioprine (− 3.459); salmonella typhimurium nhaa with ciprofloxacin (− 5.135) and hydroxychloroquine (− 2.597); shigella dysenteriae nhaa with methotrexate (− 8.059) and azathioprine (− 3.847); yersinia enterocolitica nhaa with hydroxychloroquine (− 7.47) and azathioprine (− 3.451) and human kynu with hydroxychloroquine (− 5.357) and indomethacin (1.113). our results portray methotrexate to have highest docking scores with maximum proteins and therefore can be considered as a vital drug for ibd associated rea. the resultant docking scores are provided in fig. 5 . the extensive interaction pattern of nhaa with kynu along with 396 proteins, 5 markers, 66 symptoms/ diseases, 78 pathways and 7 drugs give rise to a host-microbe disease network of ibd co-existent rea (fig. 6 and see supplementary table s14 online) . the final league of information processed in this study design was to accommodate the concept of molecular mimicry between the essential host proteins and selected microorganisms. nhaa protein of target microorganisms shows homology with human hla-b27, hla-b51 and hla-drb1 (fig. 7) . peptides homologous to hla-b27: peptides homologous to hla-drb1: experimental evidences to identify the signature molecules in patients. the in-silico analysis followed for the molecular signature identification till far through gene expression datasets and curated metabolic reconstructs strongly indicate the host protein, kynu being the singular common predictive markers for all pathogenic microbes. kynu has also been indicated in the expression data of inflammatory linked disorder, www.nature.com/scientificreports/ ibd. there is lack of data available regarding kynu differential expression in rea, therefore the experimental evaluation of kynu through targeted expression analysis in rea patients was carried out. a non-probabilistic convenience sampling was followed for our single blind study. this study encompassed 15 individuals: 60% male with mean age of 45.7 and 40% female with mean age of 38 (9 males and 6 females). out of these cases were: 10 with rea and controls were: 3 currently undergoing treatment, 1 with poncet's disease (pd) and 1 healthy control (hc). the clinical characteristics of the patients recruited in the study included inflammatory back pain in 33%, fatigue in 60%, fever in 27%, swollen joint in 47%, ankylosing spondylitis (as) that affects spine in 7%, dactylitis that is inflammation in finger or toe in 7% and poncet's disease (pd) in 7% of participants. the clinical characteristics of the recruits are provided in table 3 . the expression of kynu in peripheral blood mononuclear cells (pbmc's) of rea cases vs controls was evaluated using relative expression software tool (rest) software that estimated a sample's relative expression ratio in relation to the control housekeeping gene (here gapdh) by calculating an intermediate absolute concentration value: where cp = point at which fluorescence escalates considerably above the background fluorescence. here the cp values for reference and target genes are collectively redistributed to control and sample groups and the expression ratios are calculated based on the mean value. a pair wise fixed reallocation randomisation test is followed for normalisation of the target genes with a reference gene and for calculating the statistical difference of variation between 2 groups 81 . it utilises a bootstrapping technique providing a 95% confidence interval for expression ratios. it uses a p(h1) test for testing the significance between the samples and controls. according to our analysis, kynu sample group is different to control group where p(h1) = 0.025. kynu was found to be downregulated in sample group (in comparison to control group) by a mean factor of 0.115 (standard error range is 0.018-0.837) as depicted in the whisker-box plot (fig. 8) . kynu expression showed a ~ ninefold decline in rea cases as compared to controls. gut microbiome is pitched to be the central theme housing enormous diversity of microbial species, characterizing the fine balance between healthy and diseased states. the physiological drifts from healthy to diseased and vice-versa is tuned to sophisticated interactive networks of human host and the microbial flora residing the gut. the autoimmune conditions reactive arthritis (rea) and inflammatory bowel disease (ibd) have been linked to prevalent dysbiosis of the gut, where disease development occurs as a perceptive reaction due invading population of microbes. to find out the basal networks of interactions at the host-microbe interface, common microbes affecting the co-evolved diseases with shared characteristics were studied. these involved comprehensive analysis of the bimolecular functional networks including the gene, protein, metabolite molecular signatures engraved at the host-microbe and microbe-microbe interface. this 'interspecies communication' have been linked now with immuno-pathogenesis of most human autoimmune disorders 82, 83 . www.nature.com/scientificreports/ the etiopathology of these interactions have remained elusive leading to non-specific diagnostic criteria and therapeutic regimes. it is suggested that microbial dysbiosis, pathogenic infection and host-microbe interactions cause incidence of rea. in this study, utilising the combinatorial approach we have compiled a repertoire of microorganisms, biomolecules and pathways that are possibly involved in triggering co-evolved autoimmune disorders ibd and rea. in our study, text mining results convey the presence of microorganisms namely campylobacter jejuni, escherichia coli o157:h7, klebsiella oxytoca, salmonella typhimurium, shigella dysenteriae and yersinia enterocolitica implicated in both the disorders. the thematic concepts for microbe contribution in host immunity have been explored in our previous analysis of metabolic reconstruction and simulation of campylobacter jejuni and salmonella enterica 69, 84 . in our current study, we used a designated work-pipeline for metabolic network reconstruction and simulation of target microorganisms. the analysis conducted extracted the information via constraint-based bottom-up approach that was filtered and utilised for further computational analysis. the essential genes, proteins and metabolites of microorganisms represent the promising drug targets as these are speculated to contribute towards infection triggered host physiological drifts leading to development of the co-evolved pattern of autoimmunity in ibd and rea. a thorough curation pattern followed led to provide robust molecular cues in terms of essential proteins and biological networks that are correlated to the 'interspecies communication' using the host-microbe and microbemicrobe interaction profiling. the most closely associated common protein observed in all the selected common microbial species involved in both ibd and rea is na (+) /h (+) antiporter (nhaa), microbial integral membrane protein, catalyzing the exchange of 2 h (+) per na (+)85 and involved in processes crucial for cell viability. similarly, the common host interacting protein with nhaa is kynureninase (kynu), involved in tryptophan metabolism and whose differential expression (upregulation and downregulation based on the control samples) have been followed in ibd patient cohorts [86] [87] [88] . as per the scientific discourse presented in the studied disorders, the pathological mechanism hypothesizes that after bacterial infection, antigen-presenting cells transport bacterial antigens/peptides into the synovial membrane, where the bacterial components persist causing inflammation. it is suggested that in host-microbe interactions, bacterial proteins entering host cells interact with host proteins and inject their effector components, but has not been proven in rea and ibd. so, this formed a basis of one of the parameters in our study design where we found the physical interactions between nhaa and kynu and predicted that these might be the early host-microbe interactors for establishing pathogenesis in ibd associated rea. this could assist to comprehend the very few reports indicated in the rare autoimmune rea, where gene expression datasets of the co-evolved disorder ibd can serve to incorporate the larger theme of gut-microbiome associations. the theme of gut-microbiome paradigm shifts thus contemplates the vital cues in triggering autoimmunity with indirect linkages to diet and environmental triggers. this is indicative of the identified target molecular signature, kynu, found to be differentially regulated in the patient cohorts with history of infection triggered or ibd co-evolved rea. kynu and nhaa could serve as the robust early and essential host-microbe interacting targets and molecular indicators involved in interspecies communication in ibd associated rea. the investigations further were targeted for parallel analysis of other host-essential protein partners enmeshed to have interaction with host protein kynu indicating the intricate details of host-microbe interaction information. the disease network constructed through our approach consists of 412 single connected essential protein interactors of kynu, where 394 human essential protein interactors are found to be associated with ibd, while 3 of them (adenosine deaminase (ada), catalase (cat) and superoxide dismutase 2 (sod2)) are associated with both ibd and rea. ada protein has been reported in juvenile idiopathic arthritis and rea patient cohorts in serum samples 89 . similarly, cat and manganese superoxide dismutase (sod) genes polymorphisms were observed in rea patient cohorts 90, 91 . these become part of the host-microbe disease network where such molecular elements and co-regulatory pathways represent the intricate biological cross-talk followed during disease development. pathological conditions can also trigger immune cells such as il's and tlr's and various cytokines leading to immune cell infiltration in host and higher levels of inflammation. genetic factors such as hla alleles encode susceptibility, contribute to bacterial persistence and increase risk in rea cases. based on this we also found the interactions of important targets in our study with immunogenic and genetic factors. the host harboured assorted essential proteins were further probed for their association with non-specific protein diagnostic markers as well as with symptoms/diseases linked with ibd and rea, accruing towards a single connected network consisting of 402 interdependent proteins. the reciprocation of these integrated protein indicators to the disease development is conveyed through metabolite monitoring as in the study, angiotensin i converting enzyme (ace) was found to be linked with maximum symptoms/diseases. ace is involved in catalyzing the conversion of angiotensin i into angiotensin ii that is a potent vasopressor and aldosterone-stimulating peptide that controls blood pressure and fluid-electrolyte balance 92 . this could be the indicator of involvement of microbe triggered host physiological drifts. subsequently, the pathways associated with the proteins ramified into 78 pathways of human host speculated to give details of metabolic regulatory checkpoints where carbon metabolism is found to be associated with majority of deduced proteins. carbon metabolism pathway implicated here as the vitally generic pathway for ibd co-related rea confers how diet, balance of gut microbiome, antibiotic exposures can have layered impact on autoimmune disease progression and remissions. kynu is found to be downregulated in rea patients as compared to controls through our targeted gene expression analysis. collectively, the disease network followed here confers interaction of microbial nhaa with host kynu, that is further correlated to 396 proteins, 5 markers, 66 symptoms/diseases, 78 pathways and 7 drugs. docking analysis of drugs used to suppress the effect of ibd and rea predicts methotrexate as an important drug that could be useful for early treatment of ibd co-evolved rea. www.nature.com/scientificreports/ genetic factors found common in both rea and ibd are hla-b27, hla-b51 and hla-drb1. the most important mechanism of susceptibility of hla in rea is molecular mimicry that is microbial peptides mimicking hla autopeptides of human host leading to autoimmunity. this mechanism has been observed in rea where reports have predicted microorganism peptides such as chlamydial proteins (clpc, nqra and dnap) and yersinia pseudotuberculosis peptides (yoph) showing homology with human hla-b27 via bioinformatic analysis 14 . similarly, molecular mimicry has also been observed in ibd cases having extraintestinal manifestations. we performed targeted molecular mimicry analysis in our study using our robust microbial protein (nhaa) with hla-b27, hla-b51 and hla-drb1, enhancing the importance of nhaa acting as a trigger for generating ibd associated rea. we generate a putative hypothesis amalgamating key findings with literature. we state that the initial hostmicrobe triggers for ibd associated rea is when pathogenic microbial protein nhaa interacts with host protein kynu that further interacts with human proteins ada, sod2, cat and ace and carbon metabolism involving the above host proteins is hampered. methotrexate regulates carbon metabolism and the associated host-microbe proteins reducing effect of ibd associated rea. since carbon metabolism is the most basic aspect of life and therefore an extensive network consisting of sub-pathways, we narrowed down our findings towards a consequentially central and a significant pathway that embrace the carbon metabolism pathway involving the molecular signatures kynu, ada, sod2, cat and ace, further is also effectuated by potential drug methotrexate and is associated with ibd/ rea/ ibd and rea cohorts. it is reported that methotrexate is incorporated intracellularly interfering with adenosine concentrations and affecting proinflammatory cytokines in ibd reducing inflammation 93 . in inflammatory arthritis, the mechanisms reported by which methotrexate reduces inflammation include enhanced adenosine release, de novo synthesis of purines and pyrimidines, inhibition of transmethylation reactions, diminished accumulation of polyamines and nitric oxide synthase uncoupling. most of the mechanisms are associated with folate biosynthesis, a type of carbon metabolism 94 . kynu, ada, sod2, cat and ace are also found to be involved in folate biosynthesis and metabolism from genecards. apart from the above targets, parallel interactors, pathways and drugs for ibd co-evolved rea obtained in our host-microbe disease network can be utilised further as disease determinants. the experimental validation of these targets in patient cohorts need to be performed on a pilot scale in future to increase the robustness of this network. the intertwined information processed through the knowledge-base created for the linked disorders have given the most elaborate layout of patterns observed in disease diagnosis and analysis. the major information after processing the gene expression profiles, protein markers, molecular networks and metabolic networks involved here have led to chalk out as well as connect the strings for robust gut microbiome paradigm shifts. the current work on host-microbe interactions provides a starting point for researchers and clinicians to investigate inflammatory bowel disease (ibd) associated reactive arthritis (rea). in this study a combinatorial approach is utilised to reveal the interactions of gut microbes with human host extensively sketched through the work-pipeline providing the vital insights for the drug targets, biomarkers, pathways and inhibitors for etiology, prognosis, diagnosis and treatment attributes of pathogenic rheumatic autoimmunity. the information sorted through the combinatorial study will be useful in deciphering the etiopathogenesis of the co-linked disorders especially for the rare rea, from synonymous analyses of ibd datasets, conferred through common microbial triggers. these predictions substantially furnish the intricate details of the cross-talk between post-infectious inflammatory reactions with shared patho-immunogenesis as the starting point for researchers and clinicians for detailed and newer experimental analysis. future studies are required on larger cohort of patients having rea due to ibd in order to 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genes polymorphisms in reactive arthritis coronavirus disease 2019 (covid-19): do angiotensin-converting enzyme inhibitors/angiotensin receptor blockers have a biphasic effect the current role of methotrexate in patients with inflammatory bowel disease methotrexate and its mechanisms of action in inflammatory arthritis the authors are grateful to amity institute of biotechnology, amity university uttar pradesh, noida and department of biotechnology, teri school of advanced studies, new delhi for providing the facility and technical support during the preparation of the manuscript. we also thank fortis hospital, noida for providing the patient samples. s.s. and b.r. conceived the study concept; s.s., b.r. and p.s. jointly designed and supervised the work; b.d.p. supervised the clinical setting and recruitment of participants; b.d.p. and a.v. recruited the participants and contributed to the sample collection and preparation; a.v. performed the experiments; s.s., b.r., p.s. and a.v. contributed to the analysis and interpretation of data; a.v. generated all figures and tables; a.v. wrote the first draft of the manuscript; s.s., b.r., p.s. and b.d.p. critically reviewed and edited the manuscript; all authors reviewed and approved the final version of the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-71674 -8.correspondence and requests for materials should be addressed to s.s.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-266902-wuty839o authors: wang, yan; sun, meiyi; he, min; cui, honglian; zhang, junxian; shi, limin; wang, wei; xu, wenjiong; gao, bin; ding, jie title: weak binder for mhc molecule is a potent mycobacterium tuberculosis-specific ctl epitope in the context of hla-a24 allele date: 2012-10-31 journal: microbial pathogenesis doi: 10.1016/j.micpath.2012.07.002 sha: doc_id: 266902 cord_uid: wuty839o abstracts tuberculosis causes serious health problem for the world population. antigenic peptides selected by pathogen-specific cytotoxic t lymphocytes (ctls) are presented by major histocompatibility complex (mhc; or human leukocyte antigen [hla] in humans) molecules, and hla-a restricted responses may be of interest for vaccine development and the understanding of cellular immunity. a series of peptides derived from the 10-kda culture filtrate protein (cfp10) and the 6 kda early secretory antigenic target (esat-6) in the mycobacterium tuberculosis (mtb) have been screened and a ctl epitope restricted by the human leukocyte antigen hla-a24, a common hla allele in asian people, has been identified. in this study, we studied a panel of cfp10 and esat-6-derived peptides to identify those with binding motifs for hla-a24 molecules. the antigenicity of candidate peptides was assessed with in vitro refolding tests and an enzyme-linked immunospot (elispot) assay, and by tetramer staining to determine the capacity to stimulate ctls from peripheral blood mononuclear cells (pbmcs) of hla-a24-positive tb patients. we report that one novel candidate peptide at positions 5–14 of esat-6 of mtb could induce peptide-specific ctls from pbmcs of hla-a24-positive patients, but not from hla-a24-negative patients and hla-a24-positive healthy controls. identified epitope is a weak binder for hla-a24 molecule in a mini mhc refolding assay. since the peptide is presented by a common hla class i molecule, it may be useful for immunotherapy against mtb infection and vaccine development in the large population of mtb-infected patients. currently, tuberculosis remains one of the top three fatal infectious diseases, together with acquired immune deficiency syndrome (aids) and malaria. in china, it is estimated that 550 million (about 44.5% of the population) individuals latently have been infected with mycobacterium tuberculosis (mtb), and 2 million are with smear-positive or culture-positive pulmonary tuberculosis (tb) patients [1] . although the mechanisms of protection against tuberculosis are not completely understood, many lines of evidence have shown that antigen-specific cytotoxic t lymphocytes (ctls) might be effective in limiting the spread of mtb and clearing mtb during infection. to control mycobacterial growth, mhc class i-restricted cd8þ ctls are expected to recognize and react to target epitopes presented by antigen presenting cells during the course of infection. moreover, the cytokine interferon gamma (ifn-g) produced by t cells has a critical role in the protective immunological response against primary infection. however, because the antigen specificity of the human t cell response is known to be strongly disciplined by hla polymorphism [2] , the immunogenic potential of candidate vaccines needs to be elucidated in the context of major hla polymorphisms. abbreviations: mtb, mycobacterium tuberculosis; ctls, cytotoxic t lymphocytes; ifn-g, interferon gamma; pbmcs, peripheral blood mononuclear cells; cfp10, 10-kda culture filtrate protein; esat-6, 6 kda early secretory antigenic target; mhc, major histocompatibility complex; hla, human leukocyte antigen; tb, tuberculosis; b2m, b2 microglobulin; sfcs, spot-forming cells; hplc, high performance liquid chromatography; pha, phytohaemagglutinin; rhil-2, recombinant human interleukine-2; rhil-7, recombinant human interleukine-7; afb, acid-fast bacilli; pe, phycoerythrin; bcg, bacillus calmette-guérin ltbi, latent tb infection. therefore, the identification of specific ctl epitopes in the context of major hla alleles remains a critical step in both understanding mycobacterial control mechanisms and evaluating tb vaccines. the antigenicity of tb seems to be largely dependent upon two important secreted proteins: the 6-kda early secretory antigenic target (esat-6) and the 10-kda culture filtrate protein (cfp10). it has been previously demonstrated that esat-6 and cfp10 are able to stimulate t cells to produce ifn-g and display cytolytic ctl activity in humans infected with mtb, suggesting them as potential candidates for application in an anti-tuberculosis subunit vaccine [3, 4] . a series of epitopes for cd8þ t cells, restricted by certain mhc molecules, have been identified [4e7] . according to the report of the frequency of hla alleles in humans, hla-a24 is one of the most common hla-a alleles worldwide, especially in asian populations [8] . nevertheless, relatively few epitopes presented by this molecule have been defined for human cd8 t cells in mtb infection. in the present study, we adapted an approach utilizing a panel of synthetic peptides containing the hla-a24-binding motifs for identification of ctl epitopes in esat-6 and cfp10 proteins. seven tb patients (nos. 1e7) and two healthy controls were examined for the induction of tb-specific ctls ( table 1 ). all patients were recruited from nanjing chest hospital and the people's hospital of luhe, nanjing. the diagnosis of tuberculosis was confirmed in all cases by one of the diagnostic standards developed by the ministry of health of the people's republic of china: (1) two positive acid-fast bacilli (afb) smears, or positive sputum culture for m. tuberculosis, and chest x-rays showing radiographical abnormalities characteristic of pulmonary tuberculosis; or (2) at least two sputum specimen negative for afb and culture and chest x-rays showing signs of active pulmonary tuberculosis, accompanied with symptoms such as cough, cough with blood-tinged sputum, coughing up blood, chest distress, chest pain, and tiredness. four patients (nos. 1e4) were hla-a24positive, and all other patients (nos. 5e7) were hla-a24-negative. both of the two healthy controls were hla-a24-positive and had no history of tuberculosis and no history of contact with patients with tuberculosis. all patients were hiv negative. the tb patient group included three women and four men. the purpose and performance of the study were fully explained to all participants from nanjing chest hospital and the people's hospital of luhe. collection of peripheral blood mononuclear cell (pbmc) samples was authorized by the hospital ethics review committees. fifteen milliliters of peripheral blood was obtained from each subject, and the pbmcs were prepared by ficolleconray density gradient centrifugation. all of the samples were cryopreserved in liquid nitrogen until use in the experiments. the expression of hla-a molecules on the pbmcs was determined as follows: hla typing of the subjects was done using fresh pbmcs. dna was extracted from pbmcs using relaxgene blood (tiangen, beijing, china). low resolution hla typing was performed using pcr with sequence-specific primers (protrans, germany). mhc alleles were identified with the 20090122 version of score virtual sequencing software. the results of the hla typing of the subjects are listed in table 1 . peptides were purchased from sbs genetech (beijing, china). precise information regarding these peptides is provided in table 2 . the purities of all the peptides used here were >90%, as determined by high performance liquid chromatography (hplc). all peptides were dissolved in dimethylsulfoxide (sigma, st. louis, mo, usa) at a concentration of 10 mg/ml and stored at à70 c. human recombinant il-2 and human recombinant il-7 were purchased from r&d systems (minneapolis, mn, usa). refolding was performed as described previously with minor modifications [9] . briefly, the hla-a24 heavy chain and b2m were expressed in a prokaryotic expression system. hla-a24 heavy chain, b2m and peptide were subsequently added into refolding buffer (100 mm tris/hcl, 400 mm l-arginine-hcl, 2 mm sodium edta, 0.5 mm oxidized glutathione, 5 mm reduced glutathione, 1 mm pmsf, 1 mg/ml pepstatin, and 1 mg/ml leupeptin). after stirring at 4 c for 48 h, the soluble refolded portion was concentrated and then purified by superdex g200 gel filtration chromatography (ge healthcare bio-sciences ab) to indicate the correctly refolded peptideehla complex. subjects 1e4 are hla-a24-positive tb patients; subjects 5e7 are hla-a24-negative tb patients; subjects 8 and 9 are hla-a24-positive healthy controls. predicted hla-a*24-restricted peptides for tb epitopes. a 96-well plate membrane was pre-coated with 10 mg/ml anti-ifn-g mab (epigen, beijing, china) overnight at 4 c. pbmcs (2 â 10 5 cells per well) were added in duplicate wells. candidate peptides were used at concentrations of 10 mg/ml to stimulate the effector cells. as a positive control, 5 mg/ml phytohaemagglutinin (pha) was also added to pairs of elispot wells. tb-stimulants was provided by epigen. after incubation (37 c/5% co 2 ), the medium was discarded, and the wells were washed prior to the addition of anti-human biotinylated interferon-g (epigen, beijing, china) at 2 mg/ml per well. the plates were incubated at room temperature for 2 h and then washed, followed by the addition of streptavidinealkaline phosphatase and then the chromogenic substrate nbt/bcip (epigen, beijing, china). after development, sfcs were counted using an elispot reader (autoimmun diagnostika gmbh, strasberg, germany). typically, the negative control will have less than 5 spots. in this case, a positive or reactive sample will have at least 6 spots more than the negative control. if the negative control has 6 or more spots, a response was considered positive if the well contained more than two times the mean sfcs of the negative controls. pbmcs were separated from the whole blood of two hla-a24 þ and one hla-a24 à tb patients, as well as one hla-a24 þ healthy control. pbmcs (2 â 10 6 /ml) were cultured with p5 peptide at a concentration of 10 mm in 1 ml of medium consisting of rpmi 1640 supplemented with 10% fetal calf serum, amino acids, pyruvate, antibiotics, 20 ng/ml rhil-7, and 20 units/ml rhil-2 (eurocetus, amsterdam, netherlands) in 24-well tissue culture plates. half of the medium was then changed every 3 days with supplementation of rhil-2 at a final concentration of 20 u/ml. at day 10, the cells were harvested and tested for the presence of peptide-specific cd8 þ t cells by an ifn-release elispot assay. tetrameric hla-a24epeptide complexepeptide molecules containing candidate peptides were constructed using the previously described method [10] . the cells were incubated at 37 c for 20 min with the pe-labeled tetrameric complex and washed once with pbs prior to incubation at 4 c with fitc-labeled anti-cd8 mab (ebioscience, san diego, ca, usa). the samples were detected by flow cytometry (guava easycyte). tetramer-positive cells gated from cd8þ t lymphocytes were counted as epitope-specific ctls. statistical analysis was performed using the unpaired two-tailed student's t-test to compare cytokine release. differences were considered significant when the p was <0.05. 1. an hla-a24-restricted epitope was identified by ex vivo color ifng elispot assay and tetramer staining with freshly isolated pbmcs to identify peptides recognized by tb-specific ctls in the context of hla-a24 molecules, the ifn-g elispot assay was performed using freshly isolated pbmcs. pbmc samples obtained from 20 donors who had recovered from tb were typed for hla-a24 expression. the samples of 4 donors were hla-a24-positive. each of the 8 candidate peptides from tb-esat-6 and cfp10 were tested for their capacity to stimulate ifn-secretion in the pbmcs from the four hla-a24 þ tb donors ex vivo. the induction of ifn-secretion was revealed by a 24-h direct elispot assay with freshly isolated pbmcs. as shown in fig. 1 , p5 significantly elicited specific ifn-producing ctls from the pbmcs of all hla-a24 þ tb donors compared to the negative controls (40.6 ae 17.8 vs. 10.7 ae 3.7 spot-forming cells (sfcs)/2 â 10 5 pbmcs, p < 0.05). such a t cell response was not observed from the pbmcs stimulated by the other tested peptides except p8 (20.1 ae 17.9 vs. 10.7 ae 3.7). however, there was no statistical difference in ifnproducing cell numbers between the p8 group and negative control wells. both hla-a24 à tb donors and hla-a24 þ healthy donors had similar levels of sfcs in the p5 group as in the negative controls. consequently, an hla-a24/p5 tetramer was prepared and used to confirm the existence of p5-specific ctls. fresh pbmcs from hla-a24 þ healthy donors, hla-a24 þ tb donors, and hla-a24 à tb donors were stained with hla-a24/p5 tetramer, and the proportion of p5-specific cd8 þ t cells was detected by flow cytometry. the results showed that the percentages of p5-specific ctls detected from all hla-a24 þ tb donors were an average of three times higher than those in the pbmcs of all tested hla-a24 þ healthy controls and hla-a24 à tb donors (fig. 2) . the pbmcs were stimulated for 10 days in the presence of p5 peptide and 20 u/ml recombinant human interleukine-2(rhil-2) and 20 ng/ml recombinant human interleukine-7(rhil-7). the induction of ifn-secretion was revealed by the elispot assay in the presence of p5. we adopted the assay because it can test many samples simultaneously in a few plates using relatively small numbers of ctls. as shown in table 3 , the polyclonal tb-specific cd8 þ t cells established from pbmcs of the hla-a24 þ tb donor produced significant numbers of ifn-g spots when incubated with p5 peptide, while the responders generated from the controls produced small numbers of spots. peptide p5 significantly elicited specific ifn-producing ctls from the pbmcs of all hla-a24 þ tb donors, compared to the hla-a24 þ healthy controls or hla-a2 þ tb donors. to test the binding affinity of the candidate peptides to hla-a24 molecules, we adopted a peptide-induced stabilization assay of the hla-a24 heavy chain and light chain in vitro. all the 8 predicted peptides could refold with hla-a24 h chain and b2m molecules with varying avidities (fig. 3 ). among these, p1, p3, p4, and p6 had stronger avidities. compared to the positive control peptide and those peptides with higher avidities, p5 had a lower binding efficiency to hla-a24 molecules. peptide n1(qfkdnvill) acted as the positive control and showed a high binding capacity to hla-a24 molecules. hla-a24 heavy chain and light chain could not form the mhc complexes in the presence of the hla-a2-restricted peptide gilgfvftl. m. tuberculosis, which is the leading cause of death from a single bacterial species, severely impacts global health [11] . vaccination with mycobacterium bovis bcg (bacillus calmette-guérin) reduces the severity of tuberculosis in children; however, there is limited evidence of the efficacy of bcg vaccination when applied to adults [12] . furthermore, vaccination may cause severe disease in immunocompromised patients, such as those infected with hiv or suffering from tumors [13] . t cells play a vital role in fighting against tuberculosis. it has been proven that cd4þ t cells are essential for immunity and that cd8 t cells are indispensable parts of the antibacterial immunity [5] . considering that bcg is a powerful stimulator of cd4 t cells and a poor stimulator of cd8 t cells, further work should focus on eliciting both potent cd8 t cell and cd4 t cell responses to achieve better immunity [14] . in this study, we used an approach starting with a computer motif prediction. the predicted binding affinity in the computer algorithm is reported as the ic50 value. a panel of 9-and 10-mer peptides derived from tb esat-6 and cfp10 proteins were selected for screening based on computer algorithms [15] . all the peptides could bind with the h and l chains of hla-a24 molecules with various avidities in vitro, as determined by dilution refolding. the candidate peptides were previously tested for their capacity to bind to hla-a24 molecules on the cell surface [15] . we observed that the affinities of these peptides were not entirely consistent when measured by the two assays. peptides p4, p6 and p9 could refold with the hla-a24 heavy chain and light chain; however, no binding affinity was observed with the three peptides in the cell surface stabilization assay [15] . it has been suggested that factors such as temperature or the conformation of the heavy chain of mhc class i may affect the formation of mhc-i molecular complexes, which may account for this discrepancy [16] . to verify the results, we assayed pbmcs from tb donors to determine the cd8 þ t cellspecific response to the candidate peptides. only peptide p5specific ctls from hla-a24 þ tb donors were significantly detected by stimulated elispot assays and tetramer staining. our study demonstrates that, although in vitro refolding is an effective approach to identify hla class i-restricted t cell epitopes, the peptides which have refolding capacity with the corresponding hla allele, for example p4, may not react well with t cells in further assays, such as the elispot assay and tetramer staining. while the peptides which present lower binding capacity with the target hla allele, for example p5, may distinctly induced peptide-specific ctls. we assume that some potential t cell epitopes may be missed if we use the refolding test solely to identify peptides because there may be some peptides that bind hla with relatively low affinity but still bind tcr with high affinity. therefore, establishing that an immunodominant hla-a24-restricted, tb-specific cd8þ t cell epitope exists would require screening by an ifn-g elispot assay and tetramer staining. this study demonstrates the presence of hla-a24-restricted cd8 ctl specific for an m. tuberculosis protein antigen in infected individuals. different levels of epitope-specific cd8 t cells may be found between subjects with active mtb infection and those who contain infection (ltbi individuals). during active disease, in which antigen load is high, large numbers of cd8 t cells are indeed sequestered in the lung, and not only in peripheral blood [17] . the results obtained with pbmc in humans may not necessarily reflect the local immune response to the infection. the ctl epitope discovered could be further investigated for the subjects with latent tb where either elevated t cell frequencies could be the major force for the containment of infection, or the consequence of the persistence of pathogens inside macrophages for a long time [18] . in conclusion, we provide evidence that esat-6-derived peptide p5 (qwnfagieaa) may be a novel, naturally processed hla-a24restricted ctl epitope, which adequately sensitized the target for recognition by ctl in the mtb-infected patients carrying hla-a24. peptide p5 was a weak binder for hla-a24 molecule and light chain in the refolding assay, and showed medium affinity in the mhc stabilization assay [15] . this sequence, encompassing residues qwnfagieaa, is located in the n-terminal side of the esat-6 protein, whose corresponding gene (esat-6) is located in rd1, which is a 10-kb dna region deleted in the attenuated tuberculosis vaccine bcg [19] . it is well known that esat-6 is a dominant target for cell-mediated immunity and is frequently recognized during early tb infection [20] . in this regard, the hla-a24-restricted ctl epitope identified in this study may be useful to enhance the ctl activity specific for mtb protein to prevent the spread of mycobacteria in a large population of mtb-infected patients. none of the authors has a conflict of interest related to this study. national technical steering group of the epidemiological sampling survey for tuberculosis identification of hla class ii-restricted determinants of m. tuberculosisderived proteins using hla-transgenic, class ii deficient mice antigenic equivalence of human t-cell responses to mycobacterium tuberculosis-specific rd1-encoded protein antigens esat-6 and culture filtrate protein 10 and to mixtures of synthetic peptides human cytolytic and interferon g-secreting cd8þt lymphocytes specific for mycobacterium tuberculosis characterization of a mycobacterium tuberculosis peptide that is recognized by human cd4þ and cd8þ t cells in the context of multiple hla alleles identification of major epitopes of mycobacterium tuberculosis ag85b that are recognized by hla-a*0201-restricted cd8þt cells in hla-transgenic mice and humans induction of cd8 t cells against a novel epitope in tb10.4:correlation with mycobacterial virulence and the presence of a functional region of difference-1 prominent roles of secondary anchor residues in peptide binding to hla-a24 human class i molecules hla-a2-peptide complexes: refolding and crystallization of molecules expressed in escherichia coli and complexed with single antigenic peptides phenotypic analysis of antigen-specific t lymphocytes review: multidrug-resistant tuberculosis: public health challenges current medical treatment for tuberculosis tuberculosis vaccination versus isoniazid preventive therapy: a decision analysis to determine the preferred strategy of tuberculosis prevention in hiv-infected adults in the developing world analysis of predicted cd8þ t cell epitopes from proteins encoded by the specific rd regions of mycobacterium tuberculosis for vaccine development and specific diagnosis identification of hla-a24-binding peptides of mycobacterium tuberculosis derived proteins with beta 2m linked hla-a24 single chain expressing cells screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes analysis of mycobacterium tuberculosis-specific cd8 t cells in patients with active tuberculosis and in individuals with latent infection highly focused t cell responses in latent human pulmonary mycobacterium tuberculosis infection a mycobacterium tuberculosis operon encoding esat-6 and a novel lowmolecular-mass culture filtrate protein (cfp-10) human t cell responses to the esat-6 antigen from mycobacterium tuberculosis the authors acknowledge the following supports: key: cord-017702-v46ye328 authors: ganguly, nirmal kumar; saha, gautam kumar title: pharmacogenomics and personalized medicine for infectious diseases date: 2013-06-11 journal: omics for personalized medicine doi: 10.1007/978-81-322-1184-6_27 sha: doc_id: 17702 cord_uid: v46ye328 humans have been plagued by the scourge of invasion by pathogens leading to infectious diseases from the time in memoriam and are still the cause of morbidity and mortality among millions of individuals. trying to understand the disease mechanisms and finding the remedial measures have been the quest of humankind. the susceptibility to disease of an individual in a given population is determined by ones genetic buildup. response to treatment and the disease prognosis also depends upon individual’s genetic predisposition. the environmental stress induces mutations and is leading to the emergence of ever-increasing more dreaded infectious pathogens, and now we are in the era of increasing antibiotic resistance that has thrown up a challenge to find new treatment regimes. discoveries in the science of high-throughput sequencing and array technologies have shown new hope and are bringing a revolution in human health. the information gained from sequencing of both human and pathogen genomes is a way forward in deciphering host-pathogen interactions. deciphering the pathogen virulence factors, host susceptibility genes, and the molecular programs involved in the pathogenesis of disease has paved the way for discovery of new molecular targets for drugs, diagnostic markers, and vaccines. the genomic diversity in the human population leads to differences in host responses to drugs and vaccines and is the cause of poor response to treatment as well as adverse reactions. the study of pharmacogenomics of infectious diseases is still at an early stage of development, and many intricacies of the host-pathogen interaction are yet to be understood in full measure. however, progress has been made over the decades of research in some of the important infectious diseases revealing how the host genetic polymorphisms of drug-metabolizing enzymes and transporters affect the bioavailability of the drugs which further determine the efficacy and toxicology of the drugs used for treatment. further, the field of structural biology and chemistry has intertwined to give rise to medical structural genomics leading the way to the discovery of new drug targets against infectious diseases. this chapter explores how the advent of “omics” technologies is making a beginning in bringing about a change in the prevention, diagnosis, and treatments of the infectious diseases and hence paving way for personalized medicine. over the millennia with the progression of human civilization, the condition of human health has changed considerably. the lifespan of the human has increased considerably with the advent of vaccines against several diseases which has eradicated small pox, and now we are embarking on the global campaign to eradicate poliomyelitis and have controlled the disease in most parts of the world. the treatment of infectious diseases got a boost with the discovery of penicillin by alexander fleming in the earlier part of the twentieth century. as a range of antibiotics were later discovered, infectious diseases such as meningitis, bacterial pneumonia, sepsis, and other lifethreatening bacterial infection were treatable. also the survivability of patients undergoing operative procedures and aggressive chemotherapy was feasible and their recovery high. the last 50 years of the twentieth century have been eventful with the discovery of antimicrobials that had given us the hope that we shall eradicate all infectious diseases. despite all the efforts and progress we have made in medical science, infectious disease remained a major health problem. but the golden era of the antibiotics will not be long if we go on unregulated administering and promoting rampant use of antibiotics, as is evident from the rise of antibiotic resistance (caramia and ruffi ni 2012 ) , and the new emerging diseases have posed major challenge (table 27 .1 ). in the last few decades with the advent of fi eld of genomics, there is a new hope in prevention, diagnostics, and treatment of infectious diseases. we will explore in this chapter the table highlights the organisms that are of public health importance and their year of discovery source : world health organization: newly discovered organisms of public health importance: page 6. from who regional offi ce east-asia: combating emerging infectious diseases in south east asia region (2005) how the host genetic polymorphisms of drug-metabolizing enzymes and transporters affect the bioavailability of the drugs which further determine the effi cacy and toxicology of the drugs used for treatment. further, the fi eld of structural biology and chemistry has intertwined to give rise to medical structural genomics leading the way to the discovery of new drug targets against infectious diseases. this chapter explores how the advent of "omics" technologies is making a beginning in bringing about a change in the prevention, diagnosis, and treatments of the infectious diseases and hence paving way for personalized medicine. how the advent of new technologies is bringing about a change in medical treatment of the infectious diseases. for the use of "omics" technology to be successful requires considerable information of pathogen genome as well as genome information of the host. the pathogen genomic and proteomic information helps to identify antigens that can give us information necessary for making a diagnostic tool and vaccine design. the pathogen genome on one hand gives us the information about the important genes conferring disease pathogenesis as well as drug resistance, while the genome of the host on the other hand will reveal the susceptibility genes, and the further knowledge of polymorphisms in genes of the host metabolic and immune system will lead to the new vaccine strategies, drugs targets, and also their treatment outcomes. rapid advances of biotechnological and informatics tools in the past few decades mainly in fi elds of genetics, genomics, and proteomics are leading the way in identifying treating and thus improving the health of human beings. the effective treatment in a patient can only be achieved by fi rst rapid diagnosis of the disease and also identifying its causative agent that is particularly important in the cases of infectious diseases. new insights gained by the analysis of genome and structural feature of pathogen macromolecules have brought about new hope in the treatment of the dreaded diseases. the knowledge of system biology in respect to the microbial infections is still in development, and data available is mostly for few human infections. the rapid development of new generation sequencing technologies have led to generation of new knowledge base and with more advancement of such technologies in the coming years has brought in a hope that all diseases will conquered. in the near future, we will have complete sequences of the total transcriptomes, like genome sequences a decade earlier, and proteomic technologies will attain the throughput and sensitivity of microarrays. other technologies like metabolomics, glycomics, lipidomics, and phosphoproteomics when referred to in the context of infectious diseases are still in various stages of development, but we are taking the right steps in the direction of development of such technologies (antony et al. 2012 ). the technologies of transcriptomics and functional genomics are transforming our understanding of microbial infections and helping us decipher the reason of infections susceptibility in the humans. transcriptomics have been developed and used by scientists to broaden our understanding of infectious diseases. to elucidate the hostpathogen interaction, cdna microarrays have been widely used. the studies have focused on how the pathogens effect the host cell gene expression. the wild-type virulent strains and isogenic mutants have been used to gauge the responses of the host cell. the major fi ndings of these studies have shown how the pathogen virulence factors modify host cell factor expression (roy and mocarski 2007 ) . the role of pathogen recognition receptors (prr) affecting hostpathogen interaction has been studied. these studies have shown that the host cell responses have an alarm signal (jenner and young 2005 ) . studies have also shown that gene cluster signals are responsible for generating the alarm signals that are the target of attack by the invading pathogens (hamon and cossart 2008 ) . array technologies are being used with molecular probes of host human (also animals/plants) and microbial genes to monitor and at the same time point the expression of genes from host cells and those of the pathogens to better understand the full complexity of host-pathogen interaction. functional genomics have also led to the development of tools to decipher infectious diseases by manipulating the cellular mechanisms. the technology of the rna interference (rnai) has undergone tremendous development in the last decade which has led to the large-scale reverse genetic screens in human cells and model organisms (boutros and ahringer 2008 ) . rnai technology uses double-stranded ribonucleic acid (dsrna) with having complementary sequence to the target mrna sequence and is used to silence or downregulate the gene expression of its target. long dsrna induces interferons or other unspecifi c responses in mammalian cells which is avoided by the use of small interfering dsrna (sirna) directly or small hairpin rna (shrna). rnai screening using rna probes, which induces loss of function of host genes, leads to discovery of host resistance factors (hrf). it is made possible when silencing this restrictive factors leads to invading pathogen replication enhancement and may also identify host susceptibility factors (hsf) and also identify permissive factors, that when silenced will decrease the pathogen replication. the rnai screens still have some limitations due to offtarget sirna effect (echeverri et al. 2006 ) . thus, the primary screening validation is made by using additional sirna screens. from the several rnai screenings in human and in fruit fl y cells, only 300 host factors were validated from about 10,000 and 20,000 targets identifi ed in the initial screen (agaisse et al. 2005 ; brass et al. 2008 ; konig et al. 2008 ; krishnan et al. 2008 ; zhou et al. 2008 ) . to make the system biological tools like rnai more effective in fi nding mechanisms in host-pathogen interaction and thus fi nding cure for the microbial infections, there is a need for integrations of all data obtained from the omics technologies. in addition several rounds of biological experimentations are required by using mutant pathogens, cellular rnai knockdowns, or humanized animal models using mice or primate infection model. the resulting inferences from the validated data would help us build predictive models which could lead us to the better understanding of pathogen interactions with the host. it is evident from human history of infectious diseases that not everybody in a given population is affected by an infective disease. for an infective organism to cause an infection, both the virulence of the pathogen and the host susceptibility are important. the identifi cation of genetic factors of host innate and adaptive immunity that determine the protection from pathogen is an important endeavor of scientists. animal models of infectious diseases especially mouse models have been used to fi nd the genetic factors and biochemical mechanism of disease susceptibility (marquet and schurr 2001 ) . the identifi cation of candidate genes responsible for disease susceptibility or resistance and the occurrence of genetic polymorphism in them give us the best possible biological scenario of the disease. researchers have found that in bacterial diseases, tuberculosis and leprosy seem to have similar genetic susceptibility determinants in the host as exemplifi ed from the fi nding that higher incidence of these diseases was found in the monozygotic twins than in dizygotic twins and siblings (abel et al. 1995 ; vidal et al. 1995 ) . mouse model of infections has revealed that gene encoding the natural resistance-associated macrophage protein 1 ( nramp1 ) confers natural resistance to infections caused by mycobacterium , salmonella , and leishmania . nramp1 is found in the membrane of the phagosome of the macrophages where it seems to be probably affecting the replication of the infecting intracellular bacterium (gruenheid et al. 1997 ) . the genomic analysis in humans has found a similar gene to that of mice which is also having similar pattern of expression. hence, it has been inferred that humans too carry similar susceptibility gene. there have been further studies which have shown that polymorphisms found in the nramp1 gene are related to the infectivity of leprosy and tuberculosis (abel et al. 1998 ). in one of the studies, it was found that persons who carry an nramp1 heterozygous variant will be four times more likely to be infected by tuberculosis than persons who are carrying more common variants of the nramp1 (bellamy et al. 1998 ). cell-mediated immunity plays an important role in the context of tuberculosis and is well studied. research has further gone ahead to fi nd links between tuberculosis susceptibility and polymorphism in the gene coding for receptors of interferon-γ and interleukin-12, which are cytokines belonging to t-helper cell type 1 (th 1). the absence of functional copies of either of these genes in families and of isolated patients leads to high susceptibility to m. tuberculosis infection (jouanguy et al. 1996 (jouanguy et al. , 1997 newport et al. 1996 ; altare et al. 1998 ) . the highly polymorphic human leukocyte antigen (hla) system is the name of the major histocompatibility complex (mhc) in humans. the hla class i glycoproteins are highly expressed on the surface of every nucleated human cell, and they present endogenous peptides derived from the cell to the cytotoxic t cells. hla class i glycoprotein plays a major role during the viral infection as it presents intracellular viral peptides on the surface which leads to cellmediated immune response, which further leads to the destruction of virus-infected cells. hla class ii glycoproteins which are present on the surface of antigen-presenting cells (apcs) on the other hand present 9-14 amino acids long peptides that are derived from the engulfed pathogen and displayed on the surface which then are recognized by t cell as foreign antigens, and it will elicit an immune response to the antigen. the length of antigen as well as composition are important in deciding if the antigenic peptide will bind to the antigenic peptide-binding cleft. polymorphisms occur almost solely in the peptide-binding cleft of hla class i and ii in the glycoproteins. the diversity of hla-binding region ensures that some pathogenic peptides will be preferentially presented compared to the others. thus, in a given population, the hla diversity ensures the advantage that some of the hla glycoprotein peptide-binding clefts will be able to bind and present the pathogenic antigen peptide which will lead to an immune response to any invading pathogen. this ensures the survivability of the species against an infection. thus, genomic studies have focused on the identifi cation of susceptibility genes which would lead to the better management of infectious diseases in the population. the tuberculosis susceptibility has been associated with hla class ii genetics. the association is evident from the studies that have been found between pulmonary tuberculosis and class ii hla antigens in several populations (marquet and schurr 2001 ) . as is now clear, the knowledge of the mechanism of action of the pathogen and the identifi cation of the susceptibility genes goes a long way in the management of the disease in context of a public health perspective to prevent, diagnose, identify, and target the vulnerable populations against a given infectious disease. the requirement of the genomic information of both the host and pathogen is important to fully carry out infectious disease management. the fi rst sequence map of human genome being completed in june 2000 (lander et al. 2001 ; venter et al. 2001 ) , followed by discovery of genome-wide single-nucleotide polymorphisms (sachidanandam et al. 2001 ) and further genomic sequencing of several pathogens by institutes like the j. craig venter institute ( www.jcvi.org ) gradually opened the pathway to create new treatment and disease management methods. the ultimate aim of genomic technologies to bring personalized medicine for every infectious disease scenario is still decades away, but here we will focus only on important breakthrough which has shown us the way forward in respect to infectious disease identifi cation and treatment. neisseria meningitidis is a bacterium that can cause meningitis and other forms of meningococcal disease such as meningococcemia, a lifethreaten-ing sepsis ( www.cdc.gov/meningococcal/ ). n. meningitidis is a major cause of morbidity and mortality during childhood in industrialized countries and has been responsible for epidemics in africa and in asia . neisseria meningitidis serogroup b is responsible for causing about one third of infection. the genome sequence of n. meningitidis has opened a new way for disease management (pizza et al. 2000 ; tettelin et al. 2000 ) . the vaccine that was used only contained capsular polysaccharides from the serogroups a, c, y, and w135 only. serogroup b polysaccharide contained elements that resemble human polysialic acid and hence is poorly immunogenic and might generate autoantibodies (hayrinen et al. 1995 ) . in this scenario the n. meningitidis serogroup b was sequenced, and 350 potential antigens from the serogroup b were expressed in escherichia coli to fi nd the potential vaccine candidate. the expressed proteins are injected into mice to fi nd immunogenic antigens that can developed into a vaccine. similar strategies are being used to fi nd vaccine candidates for other serotypes of neisseria species and for other pathogenic organisms. pathogen genomic information is also being used to fi nd the immunologically important peptides for cytotoxic t lymphocytes (ctls) epitopes. the response of ctls is to seek out virally infected cells by recognizing the peptides presented by human leukocyte antigen (hla) glycoproteins on the cell surface and killing the infected cells. ctl epitopes are the viral peptide that is presented by the hla and recognized by ctls. the peptides are of the length of 10 amino acids, and genome sequence is used to fi nd out and synthesize these peptides for immunogenic evaluation. amino acids are divided into segments of peptide, measuring 10 amino acids in length and overlapping the previous peptides 9 amino acids, for example, west nile virus genome translates into 3,433 amino acids which can be segmented into 3,424 peptides that are 10 amino acids in length. immunoinformatics, a fi eld of bioinformatics, is speeding up the fi nding of ctl epitopes for the scientist working in the fi eld. algorithms on computer softwares are being used to match the viral peptides with the hla glycoproteins in silico for binding based on previously known results and are being tested (de groot et al. 2001 ). informatics based algorithms help eliminate 99 % of the peptides that would not be used in the experimental screens. thus, the time and effort to screen for the ctl epitopes have been reduced drastically. ctl epitopes may be used for making subunit-based vaccines and diagnostic tests. virusspecifi c antibody found using ctl epitopes can be used in enzyme-linked immunosorbent assay. it may be even possible to use ctl epitopes to test for the antigen itself. basically as of now only four types of molecular diagnostic tests are carried out to detect infection in laboratory setup. first is by direct detection where the pathogens can be detected directly by imaging technologies of microscopy and cell culture. second method of diagnosis is by the detection of proteins produced by pathogens by the use of specifi c antibodies, like that used in enzymelinked immunosorbent assay (elisa). third method is by detection of the specifi c antibodies iga, igm, and igg against the pathogens and the changes in their titers using antibody capture assay. fourth method uses detection of nucleic acid of the pathogens and amplifying their signal using techniques like polymerase chain reaction. latest diagnostic technologies have been developed on these basic four biotechnological technologies (speers 2006 ) . pathogen genome can also be used to identify the infecting organism itself. microbial dna in the clinical specimen can be used to identify the disease-causing pathogens. human immunodeficiency virus (hiv), hepatitis virus, borrelia burgdorferi (causative agent of lyme disease), and mycobacteria are few examples of pathogens that can be identifi ed by their genomic sequences. mycobacterium tuberculosis antimicrobial resistance strain-caused infections are becoming quite common, and genomic information has deciphered few potential candidates like katg gene mutations in resistant strains (siqueira et al. 2009a , b ; marahatta et al. 2011 ) . the traditional culture test for mycobacterium which is time-consuming and less sensitive is giving way to restriction fragment length polymorphism (rflp), a specifi c technique used in dna fi ngerprinting (van soolingen 2001 ). the technology uses restriction enzymes that cut dna at the places having certain particular nucleotide sequences. the nucleotide pattern that is obtained is then compared to the previously identifi ed specifi c nucleotide pattern of the genome of the pathogen dna. dna patterns can be separated on the basis of length, and the pattern of dna fragments in the dna fi ngerprint is characteristic of particular isolate, and each particular pathogen has a unique pattern. dna fi ngerprint technology is faster and reliable than culturing of the mycobacteria, ideal for discovering new drug-resistant strains from unique genomic sequences of each mycobacterium. the technology is very useful for identification of strains during the time of outbreaks and further epidemiological studies. the knowledge of viral load in patients is also important for dosage determination in drug therapy; hence, detection of the viral pathogenic dna and rna in clinical specimens is of paramount importance. treatment of viral diseases like hiv, chronic hepatitis b, and hepatitis c often depends on the knowledge of viral load (revets et al. 1996 ) . for example, hiv viral loads are detected by enzymatic amplifi cation of the viral nucleic acid and detection of the signal from the labeled probes that hybridizes with them. the signal usually is either a color signal conjugated to the probe or a chemiluminescent probe, and the intensity of the signal corresponds to the number of copies of the nucleic acid rna. capillary electrophoresis detects hybridized probes at a very high sensitivity with detecting as low as 2,000 copies of hiv rna in milliliters of plasma (kolesar et al. 1997 ). the rampant uncontrolled use of antimicrobials has led to increased number of antibiotic bacterial strains. genomic mutations allow the certain bacterial strains to overcome the effects of antimicrobials and are able to propagate in spite of the presence of antibiotics. fluoroquinolones are drugs that act on the bacterial dna replication by binding to bacterial enzymes involved in bacterial dna replication, that is, dna gyrase and topoisomerase. the bacterial resistance to quinolone occurs due to mutation in the quinolone-binding site in the enzymes mentioned above. the mutation leads to change in the amino acid at the site of binding of fl uoroquinolones to the enzymes. if both the bacterial enzymes are mutated, then high-level resistance occurs to the quinolone drug affecting the treatment of infection as compared to when either of the enzymes is mutated (hooper 2001 ) . now genetic test is available to detect antimicrobial resistance in the infecting pathogens. the information is important because it would lead to a better treatment management of the infection. the methicillin-resistant staphylococcus aureus phenotype is detected when cultured in the presence of oxacillin after a period of 24 h. before the era of omics technology, the only means of resistance detection was by culture test which is a very time-consuming test. methicillin resistance in s . aureus is controlled by alternations of penicillin-binding protein pbp2a. gene meca controls the production of pbp2a. polymerase chain reaction test is used to detect the presence of meca in reference laboratories, while commercially developed kit can detect the same using a fl uorescein-labeled meca probe. both dna probe and pcr technology when used for analysis can detect meca -resistant gene in a given sample in less than 3 h. the rapid detection of antimicrobial resistance in pathogens helps patients in providing adequate treatment opportunities (louie et al. 2000 ) . the study of the host genome becomes important to fully understand the drug effects and as such design more effective methods of treatments. although the ultimate goal is to decipher the system biological effect, the trend of single gene effects is also very important. cytokines play a very important role in human immunity (paul and seder 1994 ) . in hepatitis c infection, interferon alpha is used to stimulate cell-mediated immunity against the viral infection and is the primary treatment. however, studies have shown that response to interferon-alpha treatment is only 50 % in some cases even when combined with other antiviral treatment (manns et al. 2001 ) . further studies have shown that if chronic hepatitis c patients have il-10 polymorphism variant, it leads to the reduction in expression of il-10 itself, and they will have fi ve times more chance of effective treatment with interferon alpha than those who do not carry the polymorphism (edwards-smith et al. 1999 ) . interleukin-10 (il-10) is a polymorphic cytokine and is a t-helper cell type ii (th2) cytokine that is associated with the induction of the production of large amount of antibodies in body's immune response. th1 cytokines which promote cell-mediated immunity inhibit th2 response and vice versa. thus, people with high-expressing il-10 genotype if infected and suffering with chronic hepatitis c infection are less likely to respond to interferon-alpha treatment. new treatment regimes have to be developed for patients suffering from chronic hepatitis c infection and carrying il-10 polymorphism associated with high cytokine expression. vaccine responses can be used as a system of gauging the state of immune system ( poland 1999a , b ) . vaccines are administered to large number of population as an integral part of public health system. vaccines are used to mimic the infective disease conditions that induce immunological memory to protect the individual against subsequent exposure to the pathogen and lead to prevention of disease. the phenomenon to gain protective immunity against a pathogen upon being vaccinated for the particular pathogen depends on individual genetic build. as studies have shown, not all healthy individuals are able to generate a protective immune response upon vaccination. it has been observed in the case of measles vaccination that only 10 % of the population was seronegative and clustered in family (poland 1999a , b ; poland et al. 1999c ). both hla i and hla ii class alleles have been responsible for the measles vaccine response, while hla-b7, hla-b51, hla-drb1*13, and hla-dqa1*01 are associated with positive measles vaccine response, and hla-b, hla-dr, and hla-dqa1 have been responsible for the vaccine being noneffective (hayney et al. 1996 (hayney et al. , 1998 poland et al. 1998 ). drugs used for targeting any pathogenic infection can only be successful if we are aware how it is affecting the host and pathogen at genomic level and hence are able to explain the host effi cacy and toxicity. we look at few important infectious diseases where pharmacogenomic research has been bringing a landscape change in the disease treatment. leishmaniasis is a very complex major tropical infection transmitted by the vector sand fl y is all right. the infection is caused by intracellular protozoan parasites of leishmania genus. there are more than 20 species of leishmania . the type of infective species, virulence factors, and host immune responses and depending on the clinical symptoms, the disease is categorized into cutaneous leishmaniasis (cl) and visceral leishmaniasis (vl). vl is also known as kala-azar; the origin of the name is from the eastern and northeastern part of the indian subcontinent where the disease is endemic. depending upon the place where one has acquired the infection, cl is further categorized into "new world" from central america and south america and "old world" if from asia, middle east, africa, or southern europe. more than 1-1.5 million cases of leishmaniasis occur worldwide (about 80 countries are affected) with major countries being the developing nations of asia, africa, and latin america ( www.who.int/topics/leishmaniasis/en/ ) . species of leishmania are several causing different clinical manifestations of the infectious disease. l . donovani produces primary cutaneous disease as well as gives rise to visceral leishmaniasis (vl) and also post-kala-azar dermal leishmaniasis (pkdl) that is manifest after the treatment of the initial visceral disease. visceral leishmaniasis main causative pathogen is the l. donovani complex with old world vl disease being caused by the species l. donovani and l. infantum, and new world disease is mainly caused by different species of l. chagasi in the cases of infection caused by l. braziliensis complex, there is always a chance that the infection dissemination to mucosal region can occur to give rise to mucocutaneous leishmaniasis (mcl) (herwaldt 1999 ) . the complex disease is manifested due to multiple factors ranging from environmental factors such as time and number of exposure with infected vector sand fl ies, species of the infecting leishmania pathogen, to host genetic factors that include immune status of both innate and adoptive immune systems that determine the clinical outcome of the disease. other reasons for leishmania disease susceptibility are malnutrition, immunodefi ciency with hiv coinfection, and young age. the protection against invading pathogenic leishmania protozoa and even the curative resolution of the disease is provided by th1 cytokine response involving cytokine interferon gamma (ifn-γ), interleukin (il)-12, and tumor necrosis factor alpha (tnf-α), whereas th2 response cytokines il-10, transforming (tgf)-β, and il-4 have been implicated in increasing susceptibility to the disease in the experimental animals (reed and scott 1993 ; sacks and noben-trauth 2002 ) . nonhealing lesions and diffused lesions in cl have been implicated to th2 response, while self-healing lesion has been associated with th1 response (melby et al. 1994 ) . however, in some situation il-4 (a th2 cytokine) has been implicated to induce il-12 production and lead to th1 cytokine response, and it has also been found in some cases that th2 response occurs independent of il-4 (alexander and bryson 2005 ; mansueto et al. 2007 ). leishmania infection is a complex infection depending on host factors as well as strain polymorphism. leishmania mexicana cysteine proteases which target il-12 that prevents th1 protective response (buxbaum et al. 2003 ) , while the leishmania analogue of activated c kinase (lack) from the leishmania major induces th2 response that leads to host parasitization (kelly et al. 2003 ) . polymorphism of l. braziliensis also affects disease outcomes (cupolillo et al. 2003 ; schriefer et al. 2004 ). pkdl is a complication arising after treatment of vl, affecting 50 % of vl patients in sudan (study carried out in united sudan) and also 5-10 % patients in india. pkdl has been found to be associated with increased levels of il-10 (zijlstra et al. 2003 ; ganguly et al. 2008 ). the major treatment regime of cl which has propensity of dissemination towards vl and mcl is with parenteral antimonials like sodium stibogluconate or meglumine antimoniate, pentamidine, and oral miltefosine (olliaro et al. 2005 ; ameen 2007 ; amato et al. 2008 ) , whereas cl with low risk of spread is treated with local and physical therapies such as intralesional antimonials, topical paromomycin, cryotherapy, and thermotherapy or by oral azoles. however, when the disease progresses to mcl, treatment is prolonged, and toxicity from such long-duration drug use is a common occurrence (marsden 1986 ; amato et al. 2008 ) . vaccine is still elusive in the case of leishmania. some trials with dna vaccines do have shown a way forward. these vaccines have shown the promise to be effective as they have been able to induce 1l-12 production, which was in response from the persistent antigen exposure from the dna vaccine (requena et al. 2004 ). in venezuela killed leishmania promastigotes along with bacillus calmette-guerin (bcg) used as immunotherapy have shown results with a high cure rate in clinical trials by inducing th1 response (convit et al. 2003 ) . l. major vaccine trial with bcg and parenteral antimony combined have been successfully used for treatment of pkdl (mansueto et al. 2007 ). the search for effective vaccine for leishmania had got a boost with knowledge from genome sequence data of several leishmania strains. more vaccine candidate genes will be evaluated in the future (stober et al. 2006 ). in the absence of an effective vaccine with recurring infection such as pkdl, dissemination infection to mucosa leads to aggravating of the disease. prolonged treatment with parenteral antimonials that give rise to high-level risk of toxicity with high morbidity and mortality from the disease is a problem of concern (convit et al. 2003 ; muse et al. 2008 (samaranayake et al. 2008 ) . l. donovani though normally associated with causing vl is shown in few places, kenya, yemen, cyprus, and the himalayan region of north india, and is the main causative pathogen of cl (mebrahtu et al. 1993 ; pratlong et al. 1995 ; sharma et al. 2005 ; antoniou et al. 2008 ) . to deduce the genetic susceptibility of the leishmania disease, experimental murine animal models along with clonal parasite line (to control environmental variable) have been used to fi nd the genes responsible for disease progression along with their human homologues of disease susceptibility (handman et al. 2005 ) . first genes that were used to deduce from such analysis in murine model were nramp 1 and the h-2 locus had been implicated in l. donovani infection (blackwell et al. 1980 ) . hla class ii antigen hla-pq3 is found to be associated with cl in venezuela (lara et al. 1991 ) and mcl in brazil caused by l. braziliensis (petzl-erler et al. 1991 ) . pcr genotyping studies in mexico on leishmania patients has found an association with hla class ii genes with cutaneous leishmaniasis (cl) (olivo-diaz et al. 2004 ) . high blood tnf has been found to be associated with mcl (castes et al. 1993 ) and acute vl (barral-netto et al. 1991a , b ) . a venezuelan study has implicated that allele 2 of tnf-β polymorphism with high risk of developing mcl caused l. braziliensis and higher frequency of allele 2 of tnf-α polymorphism was also associated with mcl (cabrera et al. 1995 ) . in brazil by using family-based disequilibrium test analysis (tdt), investigation has shown that tnf polymorphism has been linked to l. chagasi infection (karplus et al. 2002 ) . in asymptomatic patients having positive skin test, l. chagasi has been associated with tnf-1 allele of tnf-α gene, while in case of symptomatic l. chagasi vl patients, tnf-2 allele is implicated. due to parasite heterogeneity, this tnf polymorphism association has not been correlated to infection by other leishmania species such as in l. infantum vl (meddeb-garnaoui et al. 2001 ) and l. major cl (kamali-sarvestani et al. 2006 ) . variation in promoter of il-4 and ifn-γ gene polymorphism has been found to be linked to l. major cl disease susceptibility and progression respectively in an iranian study, while in a sudanese vl patient study, il-4 polymorphism has been shown to increase disease susceptibility (mohamed et al. 2003 ) . polymorphism in promoter region of il-10 gene leads to higher il-10 production which has been shown to increase the risk of having skin lesions during an infection of l. braziliensis (salhi et al. 2008 ). il-6 can diminish the high th1 proinfl ammatory response that occurs when l. braziliensis cl progresses to mcl (hatzigeorgiou et al. 1993 ; bacellar et al. 2002 ) . il-6 polymorphism plays an important role in the progression of l. braziliensis cl to mcl, and this fi nding is important since their genetic markers have high prognostic value (castellucci et al. 2006 ) . genome-wide linkage have been performed for l. donovani-infected vl patients in artinga ethnic group in sudan to help identify loci on chromosome 22q12 and is associated with disease susceptibility genes (bucheton et al. 2003a , b ) . il-2 receptor β chain (il2rb) gene is present in the highly susceptible loci on chromosome 22q12 that was identifi ed from this study. il-2 receptor has been detected in high levels during vl infection and plays a critical role in t cell genetic responses (barral-netto 1991b ) . further studies have shown il2rb polymorphism in association with l. donovani vl (bucheton et al. 2007 ) . another candidate gene found is slc11a1 (formerly nrampi) on chromosome 2q35, an innate resistance gene that regulates macrophage activation and contributes to increased vl risk in sudanese population (bucheton et al. 2003a , b ; mohamed et al. 2004 ) as well as increased susceptibility to several intracellular pathogens (blackwell et al. 2001 ) . other studies have shown that genotypes having signifi cantly high level of mannan binding lecithin occur more prominently in patients with clinical vl. an opsonin, mannanbinding protein, is known to enhance pathogen infection. polymorphism in mannan-binding gene has been shown to increase risk of developing l. chagasi vl in brazilian study population (alonso et al. 2007 ) . in pkdl there is elevated level of ifn-γ. polymorphism of ifn-γ receptor 1 from study in sudan has been implicated in pkdl (salih et al. 2007 ). the ifn receptor expression is important for the activation of macrophages via ifn-γ. drug treatments are not very effi cient in the treatment of leishmaniasis disease; more effective treatment regimes can be developed by thoroughly knowing the genetic factors that lead to disease progression. thus, unnecessary drug use and adverse reaction can be avoided. as various genetic susceptibility studies have shown, cytokine response determines the disease progression in leishmaniasis. role of il-10 in pathogenesis of leishmaniasis is known and is well established, and il-10 polymorphisms have shown to increase risk of lesions in l. braziliensis infection. in a study with l. guyanensis infected cl patients from french guiana, high level of mrna il-10 within lesions leads to poor chemotherapy response and treatment failure (bourreau et al. 2001 ) . it is hypothesized that il-10 might be regulating the response to chemotherapy by blocking the th1 response. the increased level of il-10 has been linked to the active vl (nylen and sacks 2007 ) and pkdl (saha et al. 2007 ) and also associated with persistent cl infection occurring from l. major (melby et al. 1996 ) and l. mexicana (louzir et al. 1998 ). success of vl treatment with amphotericin b and the complete elimination of il-10 are associated with one another (saha et al. 2007 ). on the other hand, mcl is associated with low il-10 receptor expression and low il-10 secretion that decrease the ability for modulation of proinfl ammatory response (faria et al. 2005 ) . progress has been made to fi nd the susceptibility genes and will provide further insight into disease pathogenesis and will lead to progress in the fi eld of diagnostic markers, drug targets, and vaccine development to control, treat, and eradicate leishmaniasis. improving the treatment of malaria by pharmacogenomics malaria is vector-borne (mosquito) disease that has been one of the top causes of mortality in the world for generations especially in tropical countries of asia and africa. even after renewed global efforts, still there is high infectivity and mortality. three billion people are at risk with 1-2 million deaths attributed to malaria each year ( www.who.int/topics/malaria/en/ ). four species of protozoan parasite are involved from genus plasmodium, i.e., p. falciparum , p. vivax , p. malariae, and p. ovale . these malaria-causing combination parasitic species occur in human population and occur in infected individuals (gurarie et al. 2006 ) . in respect to prevalence, virulence, and multidrug resistance, p. falciparum has been a major cause of mortality and morbidity. p. falciparum accounts for about 80 % cases of malaria in africa (roca-feltrer et al. 2008 ). next to it is p. vivax which causes 100-300 million cases annually (price et al. 2007 ). the most commonly used drugs are chloroquine (cq) and sulfadoxine-pyrimethamine (s-p fansidar ® ) that are becoming less effective due to the development of resistance in malaria parasite by p. falciparum, and the species has become predominant and become a threat to travelers and people alike (schlagenhauf and petersen 2008 ) . in the absence of vaccine and in addition, development of resistance even in the mosquito vector control against chemical methods using insecticides has thrown new challenges for the researchers (greenwood et al. 2008 ) (fig. 27.4a ). some of the recent developments in malarial treatment using pharmacogenomics are bringing about improvements in the effi cacy of treatment regime of malaria. current treatment regimes have recommended artemisinin combination treatments (acts) in cases of uncomplicated falciparum malaria in nonpregnant adults (lin et al. 2010 ) . the drug regime is highly effi cacious and has reduced development to resistance. in cases of uncomplicated malaria, the act is being used in 88 countries by 2009. in the coming years, a number of patients including women and children will be brought under act therapy regime as per world health organization. like the treatment of hiv and tuberculosis, combination therapy is now being used for malaria treatment too, which reduces resistance among the highly effi cacious drug the artemisinins which rapidly eliminate the parasite from blood and thus limit the number of parasites so that the other more bioavailable drugs given in combination act on the parasite. unrelated mode of action of two or more combination drugs also reduces the chances of resistance (yeung et al. 2004 ). among many other factors which contribute to drug effectiveness, malarial drug bioavailability and tolerability are depended upon the host metabolic mechanisms. the severe drug reaction to primaquine in the 1950s used in antimalarial treatment was instrumental in the discovery of glucose-6-phosphate dehydrogenase (g6pd) defi ciency in 1956; thus, importance of the use of pharmacogenetics in malarial treatment was realized (alving et al. 1956 ). the polymorphism leading to variation g6pd or even its defi ciency is a grave problem in designing the effective drugs. even now during malarial terminal prophylaxis to decrease transmission, primaquines are administered. thus, the g6pd status of patient becomes quite important (luzzatto 2010 ) . knowledge of both the host and parasitic genetics is necessary to designing drugs and dosage for effective treatment regimes. parasitic genetics helps us in deciphering the modes of resistance, and host genetics help us in giving the information about host drug bioavailability and explain adverse reaction to the drugs. g6pd polymorphisms and genetic variation in cyp2c8 can play pivotal role in point of care diagnostics, but these genetic testings will have to be incorporated into the laboratories and national health programs. the knowledge of this important genetic variations in population would ultimately reduce cost and make the treatment regime more effective and with lesser adverse reaction and ultimately reduce the suffering of the patients. the pharmacogenetic drug policy in context of malaria is slowly becoming a reality as per efforts of the who and other agencies. genetics is becoming a guide to new drug policy. amodiaquine was generally known to be tolerated in malarial treatment, but later when it was found in the caucasian population during the decades of 1980 and 1990 to be responsible to cause agranulocytosis with fatalities and also cause hepatotoxicity (hatton et al. 1986 ; raymond et al. 1989 ; phillips-howard and west 1990 ) , the drug was fi rst removed from the list of essential drugs against malaria but then had to be added back to the list as alternate drugs started showing resistance. amodiaquine induced adverse reaction in individuals was attributed to genetic make up of the individual. the genotypes of individuals harboring cyp2c8, cypia1, and cyp1b1 have been reported in studies to show immunogenic adverse reaction to amodiaquine (li et al. 2002 ; kerb et al. 2009 ). some population in africa has shown hepatotoxicity and leucopenia with only two doses given 3 weeks apart (orrell et al. 2008 ) . amodiaquine when administered to an individual with reduced cyp2c8 activity impairs the metabolism of the drug and hence leads to the cause of hepatotoxicity and agranulocytosis. other common variants of the enzymes cyp2c8*2 and cyp2c8*3 have been associated with decrease in the metabolizing activity of cyp2c8 enzyme as is evident from studies with anticancer drugs (dai et al. 2001 ) . individuals with cyp2c8*3 genotype have no cyp2c8 enzymatic activity in vitro (parikh et al. 2007 ). in a study from burkina faso, patients carrying cyp2c8*2 genotype showed common adverse effects to amodiaquine and in addition patients have also reported to experience more abdominal pain when compared to healthy individuals. the study from burkina faso and ghana could not clearly establish the relation between drug efficacy and cyp2c8 genotype (adjei et al. 2008 ) . though the inactivated gene of cyp2c8 is not very high in population, estimates have shown that cyp2c8*2 and cyp2c8*3 occur in about 2.1 % of the population in zanzibar, united republic of tanzania, which was about 30,000 patients of the total malarial patients ~100,000 (cavaco et al. 2005 ) . in ghana it was found that 1.5 % of the population has been estimated to have metabolic variants of cyp2c8. hence, due to high disease burden, the study of pharmacogenomics for drug metabolism was carried out in large patient samples from the population to get a clear correlation between genotype and effi cacy of drug treatment as well as adverse reaction. major active antimalaria metabolite of artemisinin is dihydroartemisinin (dha) (ilett et al. 2002 ) . artesunate is rapidly converted to its active metabolite catalyzed via cyp2a6 which is a major enzyme; conversion to dha also includes minor enzymes cyp2b6, cyp1a1, and cyp1a2 (li et al. 2003 ) . cyp2a6 has about 40 variant forms of which at least 13 have been implicated as slow metabolizing enzymes, and 5 have been reported to show no activity in vitro (di et al. 2009 ) . hence, lower level of cypba6 enzymes in patients will have reduced bioavailability of dha the major antimalarial metabolite and hence have lower antimalarial activity. major endemic areas of malaria like sub-saharan africa, ghana, sabah region of malaysia have been evaluated for the presence of cyp2a6 genotype. among these population of ghana has high wild-type cyp2a6 along with 80 % alleles being cyp2a6*1a (gyamfi et al. 2005 ) , whereas malaysian population has an allele cyp2a6*1a frequency of 32 % with only 8 % wild-type enzyme (yusof and gan 2009 ). other asian populations have been reported to carry several other alleles of cyp2a6 with even alleles that do not show any cyp2a6 enzyme activity at all. no activity variant of cyp2a6 is found about 11.5-20.1 % in japanese, chinese, and thai populations (gyamfi et al. 2005 ) . hence, artesunate is expected to be more effective in population of ghana. in some parts of thailand, about 10 % of patients have shown resistance to artemisinins (white 2008 ) . though it has been found by study that about 14 % frequency of cypz86 alleles have no activity in the thai population and the antibiotic resistance is indicative to be related to cyp2a6 activity and ability to convert artesunate to dha (noedl et al. 2009 ), more studies require to be done to clearly establish the relation between the genotype and resistance to artemisinin-based therapy. several mutations in gene targeted by antimalarial drug have been identifi ed which led to the resistance in vivo of act drug partners such as mefl oquine, lumefantrine, amodiaquine, and chlorproguanil (kerb et al. 2009 ; mehlotra et al. 2009 ). identifi cation of genes and mechanism is important for controlling the infection. research has yielded the information regarding the gene responsible and underlying mechanism of action resistance of some drugs against p. falciparum and p. vivax . chloroquine resistance (cqr) in p. falciparum has been linked to point mutation cq resistance transporter gene (pfcrt chromosome 7). the mutation pfcrt -k76t is a reliable marker for cqr. while cq-sensitive strain carries wild-type allele cvmnk , the variant cqr alleles are s agt vmnt (asia, south america, africa), s tct vmnt (south america), cvmnt (south america, philippines), cviet (southeast asia, africa), and cvmet (colombia). another multidrug resistance gene ( pfmdr1 chromosome 5) is a parasite transporter gene. polymorphism, point mutation, and copy number variation have been implicated in multi drug resistance. in different geographic regions, the pfmdr1 two mutant alleles have been reported, namely, 86y_184y_1034s_104n_1246d found mostly in asia and africa and 86n_184f_1034c_1042d_1246y predominantly from south america (valderramos and fidock 2006 ) . the pfcrt-76 and pfmdr1-86y mutations have been related jointly to contribute in giving rise to cqr phenotype in addition to other likely parasite genes (hayton and su 2004 ) . p . falciparum dhps enzyme ( pf-dhps , chromosome 8) has been linked to resistance to the sulfa class of antimalarial drugs, while mutations in dhfr ( pf-dhfr , chromosome 8) domain have been linked to high level of pyrimethamine resistance. combination of sulfadoxine-pyrimethamine (s-p) treatment failure has been found to be associated with pf-dhps double mutant (437g with either 540e or 581g), combined with the pf-dhfr triple mutant (108n_511_59r) (hayton and su 2004 ; hyde 2007 ) . point mutations in p. vivax ortholog of pfcrt (pcvg10) are associated with clinical cqr. pfmdr1 p. vivax ortholog that is pvmdr1 has been proven and has also been identifi ed. y97cf point mutation of pvmdr1 has been linked to cqr. pv-dhs and pv-dhr gene point mutations have been identifi ed and are suspected to link to clinical resistance in antimalarial s-p treatment (hayton and su 2004 ) . more data is required for new mutations in the parasite genes, and in addition more data is needed for therapy of other act drug partners like sulfadoxine-pyrimethamine and lumefantrine. a new rejuvenation is taking place in pharmacology and pharmacokinetics development of databases of antimalarial pharmacogenetics. worldwide antimalarial resistance network ( http://www.wwarn.org/ ) has set up a module together with high-quality pharmacological research data for optimum drug dosage in light drug resistance information and adverse event reporting. the aim to achieve global cooperation will go a long way to personalized malarial treatment as per population needs. during the last half a century (for about 50 years), the most effective treatment regimes have been the combination therapies of drugs that was because a single drug treatment was found to be in invariantly leading to resistance for the drug, leading to much more severity and complications (crofton 1994 ) . due to rampant and unregulated use of tuberculosis drugs, however, this has led to emergence of multidrug resistant tuberculosis (mdr) (fig. 27.4b ) . now the treatment course is usually for 6 months with the combination of isoniazid, rifampicin, pyrazinamide, and ethambutol for the fi rst 2 months. this has to be followed up by the next 4 months with isoniazid and rifampicin treatment. if the treatment is taken up with diligence and patient completes the whole drug course, then it has been reported that effi ciency of the treatment is very high with more than >95 % patients getting cured and relapse is in less than 5 % of patients (menzies et al. 2009 ). another advantage of multidrug treatment is that the treatment regime helps in treating different population of tubercle bacilli. for the last 20 years, knowledge from the fi eld of genetic molecular basis of drug treatment outcomes has helped us in the better management of and understanding of treatment effi ciency and of drug. the difference in drug response is found among different individuals of the population. the individual person tends to show similar type of response to tuberculosis drugs that do not change over time. thus, in light of above observations, we say that there is a huge variation in drug response among individuals due to variation in genes involved in drug metabolism, drug transporters, and drug targets compared to minimal within-subject variation as found from studies. further studies on drug response revealed that 20-95 % of variation in drug pharmacokinetics is due to genetic factors (kalow et al. 1998 ). the sequence variation in drug-metabolizing enzymes, drug transporters, or drug targets leads to the variation in drug response among individuals (evans and relling 1999 ; evans and johnson 2001 ) . some nongenetic factors such as nutrition organ function, age and other concomitant therapies, nature of disease, and drug interaction can also effect in drug response, but genetic determinant remains constant throughout the lifetime of the individual. pharmacogenomics have played an important role in deciphering therapeutic effi cacy of drug metabolism and occurrence of adverse events. though research is still being pursued to decipher the intricacies of how genetic differences play an important role in regard to clinical application of the drug however, through research we have gained information on the role of genetic polymorphism with respect to drug effi cacy for the treatment of tuberculosis. in this section we will discuss the knowledge we have gained through newer technologies in regard to different drugs being used for tuberculosis. since isoniazid has been in use for antituberculosis treatment since 1952, it is the most well studied of the lot (ellard and gammon 1976 ) . this drug has been found to be tuberculosis specifi c in its action against tubercle bacilli and has relatively minimal toxicity. now pharmacogenomics is playing a very important role in making isoniazid the fi rst-line treatment drug. acetylation of isoniazid takes place mainly in the liver and gut mucosa. for any drug ingested in the body, it is absorbed and metabolized and then its soluble by-products are released or excreted out of the body. the drugs have specifi c retention and metabolizing rates depending upon their chemical composition and the genetic polymorphisms of the metabolizing enzymes. the activation of isoniazid is catalyzed by highly polymorphic enzyme n-acetyltransferase (nat2) and leads to formation of acetyl isoniazid. this is formed by the transfer of acetyl group from the acetyl coenzyme a to acceptor amine leading to formation of an amide. acetyl isoniazid combines with several other cellular compounds to give a variety of metabolites which do not have any antituberculosis activity. the level of acetylating isoniazid that will be subjected to during metabolism in the body determines the disease outcome. the level of bioavailability of the drugs determines whether the drug would be effective for elimination of the invading pathogen or toxic to the human body. acetylation of isoniazid varies from individual to individual depending as per his or her genetic predisposition. genetics determines the amount of active nat2 enzyme that an individual expresses. the metabolism of isoniazid is catalyzed by nat2 enzyme which takes place in liver or gut mucosa. thus, the level of nat2 gene expression is controlled by the type of polymorphism in nat2 that particular individual carries. thus, for the pharmacogenomic and personal medicine in effect to succeed, the dosage for the drugs that are metabolized by nat2 should be tailor made as per the enzymatic activity depending upon the polymorphic variant (roy et al. 2008 ) . the enzymatic activity being highly variable cascorbi and roots (1999) has been studied over the years in human subjects who have been categorized as slow or rapid inactivators ( http://www. brti.co.zw ). the categorization has been based on the measure of capacity of nat2 enzyme to acetylate isoniazid to acetyl isoniazid thus inactivating it. here the rapid inactivators are those who have more concentration of active nat2 enzymes than slow inactivators. based on the new technologies, genotypic studies have led to further classifi cation depending upon enzymatic activity nat2 variant as rapid acetylators, that is, the wild type gene which codes for the completely active enzyme. rapid acetylators are highly active forms of the enzyme denoted by nat2 * 4 allele. patients harboring these alleles can tolerate conventional dosage of drug that is rapidly metabolized by nat2 enzyme. individuals who carry nat2 heterozygous alleles where only one of the allele is active/functional should be administered lower than average drug (those are nat2 metabolized) dosage to get an optimum effective drug response without adverse drug response. mutations in nat2 enzyme in human individuals designated as nat2*5a , nat2 * 5b , nat2*6a, and several others which lead to rendering the nat2 gene activity are termed as slow acetylators which can lead to diminished drug clearance and toxicity. the variation of frequency of slow acetylator gene is depended on the race, population type, and the ethnicity from one country to the next. it is found in a study that 90 % of middle eastern, 60 % in south indian population, caucasian and negroid, and 72 % of the us population harbor slow acetylator gene. in mongoloid populations like the eskimos, japanese, and the chinese, slow acetylators are found in only 10 % of study subjects. in another study carried out in a population of 18 healthy caucasian, there is variability in isoniazid clearance. while isoniazid preparation is responsible for only 2 % variation and body weight accounted for only 3 % variation in isoniazid clearance, the majority variation of 88 % in isoniazid clearance was due to nat2 genotypes (kinzig-schippers et al. 2005 ) . highactivity nat2 allele-carrying individuals have higher isoniazid clearance. other studies have shown that 4-6 times more isoniazid concentration in individual is carrying slow acetylator nat2 genotype (parkin et al. 1997 ) . a study estimating the comparison of urinary isoniazid excretion in japanese patients to normal, healthy individuals showed that persons with higher number of active nat2 alleles had higher level of isoniazid acetylation (kita et al. 2001 ) . the relation between isoniazid concentration in blood with drug effi cacy and toxicity knowledge is important. peak isoniazid concentration to minimum inhibitory concentration ratio has been proposed to serve as a means to outcome tuberculosis treatment (mitchison 1984 ) . mean early bactericidal activity of isoniazid depends on its level in the plasma which in turn depends upon the variant nat2 genotype carried by an individual. comparatively the mean bacterial activity is lower in rapid acetylators than in the slow acetylators (donald et al. 2004 ) . therapeutic failure or relapse of infection is thus attributed to the lower plasma level due to rapid metabolism of isoniazid in rapid acetylator genotypes, while on other hand high level of isoniazid in slow acetylators may lead to the high level of toxicity (weiner et al. 2003 ) . nat2 allele genotyping of tuberculosis patients prior to the treatment with isoniazid is the way forward. dosage adjustment of isoniazid could be carried out depending upon if the patient is harboring none, one, or two alleles nat2 rapid acetylators. thus, isoniazid would be more pharmaceutically viable for treatment of tuberculosis. in pulmonary tuberculosis patients with known acetylator state, the response to isoniazid treatment analysis was carried out when it is administered alone or in combination with p-aminosalicylic acid. the study compared isoniazid response between nat2 slow and fast acetylators, and the study revealed that there is association with treatment response and bacteriological negativity (selkon et al. 1961 ) . tuberculosis treatment trials used dosage regimes of daily, twice weekly (tuberculosis research centre madras, study 1970 , 1973 , or three times weekly drug regimes (ellard and gammon 1976 ) . by means of controlled clinical trials, it was observed that using once a week uptake of isoniazid showed better clinical response to treatment compared to rapid acetylators, with cure rate of 20-35 %. it was postulated that metabolic status of isoniazid may have lesser clinical signifi cance for daily isoniazid treatment regime as compared to thrice weekly or twice weekly treatments. in slow acetylator individuals, the peak concentration of isoniazid was higher than rapid acetylators, and the level of isoniazid decreased more gradually. the effectiveness of a drug in tuberculosis treatment is determined in terms of coverage and the exposure. coverage has been defi ned as the number of hours for which bacteriostatic concentration of isoniazid is (0.2 µg/ml) maintained in the blood, while exposure is defi ned as area under concentration time curve. both parameters have been found to be signifi cantly greater in slow acetylators. hence, in rapid acetylator individuals, there is a suboptimal concentration of isoniazid which leads to failure of once-weekly regime of isoniazid (sarma et al. 1975 ) . other studies using onceweekly isoniazid-rifapentine were compared with twice-weekly isoniazid-rifampicin; treatment also showed that in case of once-weekly treatment regimes, treatment outcome was poor and was associated with isoniazid acetylator status of the patients (weiner et al. 2003 ) . the clinical studies have shown that rapid acetylators having infected from combined tuberculosis and hiv infection are at a further disadvantage since it has been found that antituberculosis drug bioavailability becomes suboptimal in those individuals (gurumurthy et al. 2004 ) . tuberculosis patients having chronic renal failure are also at a risk from adverse drug reactions if they also happen to harbor slow acetylator genotypes of nat2 . studies have shown that slow acetylators have higher peak isoniazid concentration, exposure, and half-life compared to rapid acetylators and healthy subjects ( gurumurthy et al. 1992 ) . hence, in the case of pulmonary tuberculosis patient also suffering from chronic renal failure, the isoniazid dosage should be administered based on their nat2 genotypes status. in adult pulmonary patients, studies were carried out to determine correlation between isoniazid dosage and nat2 genotypic and phenotypic status. determination of isoniazid therapeutic dosage has shown that the fast acetylators need higher drug dosage to have an optimum positive treatment response. fast acetylators tuberculosis patients when administered with 6 mg/kg isoniazid had similar exposure level as 3 mg/kg isoniazid administered to slow acetylators does (donald et al. 2007 ). in a further study in a population of south african tuberculosis patients, it was found that current treatment regimes were causing suboptimal exposure of isoniazid in patients having rapid acetylator status (wilkins et al. 2011 ) . several fi eld studies have further suggested that there is a need for calibration of isoniazid dosage as per the individual tuberculosis patient's age, acetylator status, and disease process for an effective antimicrobial outcome of drug treatment (jeena et al. 2011 ) . in children affected with tuberculosis, it was shown through several studies that the exposure of isoniazid was reduced in the rapid acetylators when compared to the slow acetylators and thus likely to affect the outcome of the treatment of tuberculosis (cranswick and mulholland 2005 ; schaaf et al. 2005 ; mcilleron et al. 2009 ). though isoniazid has been found to be nontoxic during conventional regimes, two types of adverse reactions to isoniazid have been reported. the most common isoniazid toxicity reported is hepatotoxicity which affects 2-28 % of the patients (tostmann et al. 2008 ) . another isoniazid-associated adverse event is peripheral neuropathy. neuropathy usually occurs in slow acetylators due to administration of high doses of isoniazid (devadatta et al. 1960 ) . hepatotoxicity is the major adverse reaction of isoniazid, and the factors that are responsible are nat2 acetylation, oxidation by cytochrome p450s oxidation (cyp) 2ei, and detoxifi cation by glutathione s-transferase (gst) enzyme activity (roy et al. 2008 ) . accumulation of acetyl hydrazine, a toxic metabolite of isoniazid, has been implicated in peripheral neuropathy, and the condition in humans is reversible by concomitant administration of pyridoxine (zilber et al. 1963 ) . further, it has also been deduced that hepatotoxicity occurs due to hydrazine metabolites of isoniazid. rifampicin also causes induction of isoniazid metabolism and inducing isoniazid hydrolase to produce isonicotinic acid and hydrazine. the rifampicin induction is more pronounced in slow acetylators compared to rapid acetylators (sarma et al. 1986 ) . in some populations studies have established association with nat2 acetylator status and isoniazid-induced hepatotoxicity, while in other studies it has not. studies in japanese and taiwanese populations have shown that the acetylator status of nat2 increased the risk factor for hepatotoxicity by 28-fold isoniazidinduced hepatotoxicity (ohno et al. 2000 ; huang et al. 2002 ) . in another study on the korean population, the nat2 slow acetylator status has been implicated to increase isoniazidinduced hepatotoxicity by two-to eightfold, and hence the nat2 acetylator genotype could serve as predictor of hepatotoxicity (cho et al. 2007 ) . nat2*53/5b , nat2*6a/6a , 48it/t, and 590a/a diplotypes have been indicated and could be used as biomarkers for prediction of antituberculosis drug-induced toxicity (ben mahmoud et al. 2012 ) . slow acetylator nat2 alleles have been attributed to increase 3-8-fold to 28-fold higher risk in isoniazid-induced hepatotoxicity. but other studies in tuberculosis patients have not been able to fi nd any association of nat2 acetylator status and the drug-induced hepatotoxicity. case studies of caucasian origin patients with tuberculosis (leiro-fernandez et al. 2011 ) , genotyping in an indian population (roy et al. 2001 ) , and study on heterogeneous population of hispanics, africans, caucasian, south american, and asian have not reported any linkage between nat2 acetylator status and isoniazid-induced hepatotoxicity polymorphisms (vuilleumier et al. 2006 ) . cytochrome p450 2e1 is one of the enzymes of the hepatic microsomal enzyme system. cyp2e1 gene encodes a member of the cytochrome p450 superfamily of enzymes. the cytochrome p450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids, and other lipids. cyp2e1 is an enzyme which brings about conversion of acetyl hydrazine to hepatotoxins, such as acetyl diazone and ketene, and brings about conversion of acetylonium ion (nelson et al. 1976 ) . polymorphism in cyp2e1 has been linked with increasing the risk factor associated with isoniazid-induced liver injury (lee et al. 2010 ) . the enzyme relocates to the endoplasmic reticulum and can be induced by isoniazid or its metabolite hydrazine. in animal model studies using rat, it has been found that cy2pe1 activity is linked to blood isoniazid levels (yue et al. 2004 ). in the presence of variant genotype of cyp2e1, isoniazid could on the other hand inhibit the activity of the cytochrome p450 2e1 enzyme. enhanced cytochrome p450 2e1 activity leads to the increased production of hepatotoxins and hence causing hepatotoxicity. both nat2 and cyp2e1 polymorphisms have been shown to be associated with susceptibility of fi rst-line druginduced hepatitis. cyp2e1 polymorphisms have been related to increase in risk of antituberculosis drug-induced liver toxicity. the common *1a/*1a genotype of cyp2e1, in tuberculosis patients from taiwan, has been linked to increase in the liver damage risk by 2.5 times. presence of both slow nat2 acetylator status and the *1a/*1a genotype further increases risk of hepatotoxicity when compared to presence of either of the single polymorphism (huang et al. 2003 ) . the cyple1 *6 and *1a*6*1d haplotypes in indian pediatric patients have been shown in sep-arate study and have shown to increase the liver toxicity. further, the common *1a allele at cyp2e1 has been implicated to hepatotoxicity in various heterogeneous population comprising of asians, africans, caucasians, hispanics, and south americans (leiro-fernandez et al. 2011 ), but study done on a korean population on the other hand could not fi nd any association between cyp2e1 polymorphism and liver toxicity (huang et al. 2002 ) . the glutathione s-transferases are class of two distinct supergene families of proteins located in cytosolic and membrane-bound forms. glutathione s-transferases are a class of enzymes that are responsible for detoxifi cation of therapeutic medication, carcinogens, therapeutic medication, and toxic chemicals that are mostly electrophilic in nature. gsts are present both in eukaryotes and prokaryotes. at present, eight distinct classes of the soluble and cytoplasmic mammalian glutathione s-transferases have been identifi ed: alpha, kappa, mu, omega, pi, sigma, theta, and zeta. the cytosolic gst enzymes are encoded by at least fi ve different loci coding for gst enzymes, distantly related gene families (designated class alpha, mu, pi, sigma, and theta gst), whereas the membrane-bound enzymes, microsomal gst, and leukotriene c4 synthetase are encoded by single genes and both have arisen separately from the soluble gst (simon et al. 2000 ; strange et al. 2000 ) . glutathione s-transferase catalyzed elimination of toxic chemicals from the human body is carried out by making the toxic chemical soluble by conjugation with glutathione. in context of isoniazid-related hepatotoxicity, studies have indicated that deletions of gst mu 1 ( gstm1 ) and gst theta 1( gstt1 ) are associated with liver damage (cho et al. 2007 ; huang et al. 2007 ) . gst enzymes play an important role in removing the harmful metabolites of isoniazid from the body. the toxic metabolites generated by isoniazid metabolisms are from intracellular free radicals that are scavenged by conjugation with glutathione in reactions catalyzed by gst enzymes. now, studies in indian patients suffering from tuberculosis show that those harboring homozygous gstm1 mutations have higher risk of hepatotoxicity. it was also found in a study on taiwanese tuberculosis that patients have twice the risk of isoniazid-induced hepatotoxicity if they have homozygous gstm1 deletion. thus, it can be inferred from similar studies that identifi cation of gstm1 deletion in patients will lead to the better management of isoniazidinduced hepatotoxicity. reactive oxygen species as we have been aware is a causative agent for damage to hepatic tissue. it has been deduced that level of mitochondrial oxygen species is reduced by the action of manganese superoxide dismutase (msd). as the name suggests, msd catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide and is the fi rst line of defense against reactive oxygen species. polymorphism in the msd enzyme has been found in a study in the population in taiwan, where genotypes having t > c polymorphism in codon 47 lead to variant amino acid valine in place of alanine which increases in risk associated with antituberculosis druginduced hepatotoxicity (huang et al. 2007 ) . the presence of valine at codon 47 causes the increased activity in the enzyme manganese superoxide dismutase which leads to the accumulation of the toxic byproduct hydrogen peroxide which can cause hepatotoxicity. rifampicin has been proven to show concentration-dependent activity against m. tuberculosis and is a very important fi rst-line drug against tuberculosis (ji et al. 1993 ; jayaram et al. 2003 ) . drug transporters p-glycoprotein and oatp1b transporters uptake rifampicin as a substrate and hence play an important role in distributing the drug throughout the body. the drug transporters are transcriptionally regulated by the nuclear receptors, i.e., pregnane x receptor and constitutive androstane receptor. the phenomenon variation in bioavailability of rifampicin among individuals in a population on administration of standard dosage has been subjected to investigation by the scientists. the pharmacokinetics of rifampicin depends upon the uptake of machinery of the cells in the body. it has been found that there is a relation between pharmacokinetics and polymorphisms of genes that is responsible for drug effl ux and infl ux. a study group of individuals suffering from tuberculosis who were categorize as per place of origin africans versus non-africans, it was observed that in drug transporter gene, slco1b1 463 c > a polymorphism leads to reduced rifampicin exposure and bioavailability (weiner et al. 2010 ) . the people of african origin (black subjects) that carry slco1b1 463 c > a gene polymorphisms have been associated with more pronounced reduced rifampicin exposure compared to people from other races. the study thus showed for the very fi rst time that marked interindividual variation in rifampicin exposure can be attributed to slco1b1 polymorphism. another study in south africa has also highlighted that the variant allele of slco1b1 rs 4149032 polymorphism reduced the bioavailability of rifampicin in the body of the patients when present both in the homozygous and heterozygous states (chigutsa et al. 2011 ). this fi nding has been attributed to the observation that there is about 21 % variability in drug clearance among the patients. polymorphisms in the abcb1 , pxr, and car genes have not been found to affect the pharmacokinetics of rifampicin in the patients in any signifi cant manner. researchers have further predicted by means of stimulation that increasing the dosage of rifampicin in patients carrying slco1b1 rs 4149032 would increase the plasma availability of the drug and thus would have a positive impact on the treatment outcome. however, more studies needed to be carried out to know the exact association of slco1b1 gene polymorphisms between rifampicin bioavailability to provide an effective treatment regime. as has been already mentioned, the variation in human leukocyte antigens also is known cause of disease susceptibility and the response to treatment has also in indian patients the lack of human allele hla dqai* 01201, while the presence of dqb1*0207 has been reported to be associated with antituberculosis-induced hepatotoxicity (sharma et al. 2002 ) . aminoglycosides are antibiotics which are molecules that consist of amino-modifi ed sugars, and some of the drugs of this class have been used for treatment of tuberculosis. aminoglycosides such as kanamycin, streptomycin, and amikacin have been used to treat tuberculosis. aminoglycosides have known to cause ototoxicity. ototoxicity is term used when there is damage to the ear (oto-), specifi cally the cochlea or auditory nerve and sometimes the vestibular system due to toxins. the association between aminoglycosideinduced ototoxicity and mitochondrial mutations has been found in a study in chinese family. the deafness phenotype was found to be associated with c > t 1494 12s rrna gene polymorphisms which could be induced with the administration of aminoglycosides or even get more aggravated (zhao et al. 2004 ). there is still no clear relationship of ethnicity and genetic background and response to antituberculosis treatment, and no single variant of nat2 and cyp2e1 genes is associated with signifi cant liver damage (yamada et al. 2009 ). more extensive pharmacogenomic research is still needed for realization of robust personalized medicine for tuberculosis. the antifungal medicine amphotericin b has been found to be effective and well quite toxic. further investigations revealed that the immunomodulatory role of amphotericin b also involves the induction of production of proinfl ammatory cytokine. in human cell the amphotericin-induced higher mrna expression and cytokine production have been detected in studies (rogers et al. 2000 ) . the discovery of induction of proinfl ammatory cytokine production was able to explain the infusion-related toxicity effect like nausea, fever, chills, and hypotension that are characteristics of this cytokine release. it was also able to explain the mechanism of action of amphotericin b since the proinfl ammatory cytokines are responsible for the activation of monocytes, macrophages, and promote chemotaxis that led to enhanced immune response to the infection. since the advent of the era of omics technology, the number of drugs that have been discovered have not delivered as was fi rst predicted especially in the case of infectious disease. some of the shortcoming and the remedial measures have already been discovered in the previous sections. it has been found that even with high-throughput screening of number of drug, candidates have not been successful always (payne et al. 2007 ). the fi eld of scientifi c research which has become all encompassing and interdisciplinary has added strength along the way and opened new avenues. the fi eld of biology is intertwined with structural biology and chemistry has given rise to the fi eld of medical structural genomics. the exact causes of failure of high-throughput screens have not been well defi ned. the fi eld of structure-based drug discovery has tried to overcome these limitations in the availability of chemical libraries and absence of structural information of many of the targets. the fi eld of structure-based drug discovery has its origin from the fi eld of x-ray crystallography and nuclear magnetic resonance (nmr) technology. with the availability of human genome sequences and pathogen genome sequence databases, the fi eld of structural genomics has gained importance, and hence over the past decade, more than 20 such projects have been taken up. the fi eld of structural biology has got a boost with the coming together of robotics and informatics in the biological research sphere (haquin et al. 2008 ) . for the synthesis of an effective drug, by means of medical structural genomics, the protein which drug will affect should be well defi ned experimentally in both structure and functional aspects as the potential target. the protein should not only be well characterized structurally but also should be well defi ned as essential for the survival of the pathogen. once drug and its protein target in a microorganism is identifi ed, the fi eld of medical structural genomics provides rapid mechanisms using high-throughput x-ray crystallography and nmr assay system to fi nd the ligand-bound structures. to identify such drug targets, it is very essential to know the complex host and pathogen interaction. the mode the pathogen uses to cause the infections is very diffi cult to elucidate and is a long process. the technologies of rna interference and other gene knockout techniques should be complimented with experimental chemical biology approaches as microorganisms adopt multiple mechanisms for survival. this has been emphasized for the fact examples of efforts of scientist for targeting the fatty acid biosynthesis pathways of bacteria. at fi rst drugs were found to have high bioavailability and are potent against the bacterial replication in vitro. these compounds were subjected to be tested in animals and have been found to be not effective, the reason being that the bacterium utilizes the fatty acids present in their host vertebrates (brinster et al. 2009 ). hence, this study proves that there is need for more effective screening using the services of scientist from several spectra of biology like microbiologist, biochemists along with structural biologist, and chemical biologists to fi nd effective molecules and compounds which can eliminate the pathogen under proper infective conditions (hoon et al. 2008 ) . in pharmaceutical research scenario, it might also be possible that the drug target for a cell active compound is not known and then medical structural genomics provides a number of purifi ed protein targets which can be assayed for binding interaction with bioactive compound by means of number of biophysical techniques like thermal stability (ericsson et al. 2006 ). such efforts have already been carried out in the fi eld of protozoan pathogens. the program of medical structural genomics of protozoan pathogens ( http://msgpp.org/description.shtml ) has been initiated to screen for drugs for ten protozoan diseases. the initiative has screening of thousands of potential antimalarial drugs against about 67 putative plasmodium falciparum protein targets by expressing them in bacterial expression system in the laboratory and deciphering their 3d structures. further, the com-pounds are assayed for their effectiveness in live organisms and further validated in appropriate disease model. the terms chemical validation and drugability are often used in conjunction in such cases. drugability is meant to be used how tractable a given drug target is for the development of a drug candidate, while chemical validation means that drugs have been found to be active in live organism. drugs which fulfi ll the abovementioned criteria are worth the effort, time, and resources. in the future more collaborative efforts between medical structural genomic centers and the chemical biology institutes would be possible with the availability of collection of phenotypically defi ned compound that would have proven anti-pathogen activity resulting in the synergistic target validation and hit to lead development using structure-based drug design. pharmaceutical industry has now taken fragment-based drug discovery methodology as an alternatively less expensive and at times more effective than high throughput screening. variety of methods like x-ray crystallography, nmr, surface fl uorescence polarization, plasmon resonance yield, and differential thermal denaturation have been used to obtain macromolecular structure to screen libraries of small fragment that are obtained from compounds that are building blocks of drugs and hence can be more drug like. the fragment-based drug discovery is based on the screening libraries of small molecules on the rule of three which has the molecular weight <300 da, the calculated log of octanol/water coeffi cient (clog p) < 3, and 3 ≤ rotatable bonds and hydrogen bonds (rees et al. 2004 ; congreve et al. 2008 ) . protein-protein interactions are important for all biological processes. metabolic activities in the biological system are catalyzed by proteinbased enzyme where in certain cases their activities are regulated by modulation of an equilibrium of an alternate, nonadditive, functionally distinct oligomeric assemblies (morpheeins) that have now been described as mode allostery. the oligomerization from the protein-protein interaction need not lead to gain in free energy, and it has been found that small molecules can block or disrupt any protein-protein interaction that is necessary for biological systems, for example, being in the development of potent peptidomimetic inhibitor of hsv ribonucleotide reductase with antiviral activity (liuzzi et al. 1994 ). the discoveries have opened avenues where structure-based information can be used to develop small novel antimicrobial molecules that can be made which can target protein-protein interfaces (wells and mcclendon 2007 ) . an example of this technology has been used to fi nd small-molecule species-specifi c allosteric drugs for porphobilinogen synthase (pbgs). the oligomeric equilibrium for porphobilinogen synthase (pbgs) consists of high-activity octamers, low-activity hexamers, and two dimer conformations. in silico docking analysis from a small molecule library helped in selecting suitable compounds and molecules that had more affi nity for docking pbgs allosteric site and thus were subjected to testing in vitro . in one compound whose inhibition mechanism is species specifi c, conversion of pbgs octamers to hexamers was thus identifi ed (lawrence et al. 2008 ) . the above fi ndings have led the way of targeting of oligomeric enzymes in pathogenic organism bacteria. prime example is bacterial inorganic pyrophosphatases, which function as hexamer (kankare et al. 1996 ) . on the other hand, the eukaryotic, cytosolic, and mitochondrial pyrophosphatases function as homodimers (oksanen et al. 2007 ) and hence have different interfaces than its bacterial counterparts as evident from the study of evolutionary aspects of inorganic phosphatases. in this context the strategy has been to target the oligomeric state of the bacterial inorganic pyrophosphatase enzymes to inhibit their activity rather than their conserved active site (sivula et al. 1999 ) . the technology has opened a novel pathway where more antibiotics can be developed. the amount of knowledge of protein structures being generated is enormous; the need of the hour is dissemination of the knowledge databases among scientists and academic researchers on a worldwide scale. the protein structural know-how should be in the public domain without any constrains and copyright restrictions; also in addition the databases should be available free of any monetary charge. structural genomic projects the world over have solved the structures of many proteins and have made the knowledge available for world community by submitting the structures to protein data bank {pdb; http://www.wwpdb.org/ }. worldwide protein data bank is the site whose mission is to maintain a single protein structural public database which can be accessed by the global community (berman et al. 2007 ) . there is lot of structural data of protein-ligand complexes that is in private pharmaceutical industries not in the public domain. the economic incentives of drug discovery are driving force for this secrecy, but in this process there are a lot of valuable data that are duplicated and lots of valuable resources and energy efforts. the learning process from failures and successes in pharmaceutical corporate sectors is never known to the scientifi c community, and a major loss is of most valuable time. hence, as we can see, the drug discovery resources are not being adequately utilized across the academia and industry, so there is suggestion to have open-access industry-academia partnerships as possible mechanisms to overcome the problem. a frame work is need where both fi nancial and intellectual properties of the innovators are safeguarded when there structural data are deposited in the databases like pdb. a simple proposition would delay the release date of such structural data so that protection of intellectual property is feasible. policies which can bring into the public domain structural data from the corporate world could only be possible by the concerted efforts of all stakeholders from industry, national, and international research funding agencies from all nations (edwards et al. 2009 ). apart from easier dissemination of structural information related to infectious diseases and collaboration of structural biologist with medical chemist and molecular biologist, there is need for development of automation in several technologies to bring about unprecedented growth in the new drug discovery. fragment-based drug design needs the support of high throughput technologies such that along with structural genomics, there will be more success in the determining protein-ligand structure determination. decades of experience have shown that the infectious diseases would emerge with more vigor and virulence. when the diseases are not controlled, then it would take a considerable toll of human health both in terms of mortality and morbidity. the life would be affected by emerging microbial disease-causing pathogen whatever the region, ethnicity, lifestyle, socioeconomic status, and ethnic background. hence, the threat from infectious diseases is real and the situation is overtly challenging. the great advances in the genetics, genomics, and proteomics have the potential to take up the challenge in the coming decades. it is evident that these technologies have the potential to change the fi eld of diagnostics, treatment, and discovery of drugs and vaccines. the need of the hour is to strengthen the public health programs at both the national and the international levels to strengthen research in the fi eld of omics to fully realize the potential of scientifi c technologies that would usher in the era of pharmacogenomicbased personalized medicine. complex segregation analysis of leprosy in southern vietnam susceptibility to leprosy is linked to the human nramp1 gene effect of concomitant artesunate 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cant association measles antibody seroprevalence rates among immunized inuit, innu and caucasian subjects human cutaneous leishmaniasis caused by leishmania donovani sensu stricto in yemen targeted profi ling of oral bacteria in human saliva and in vitro biofi lms with quantitative real-time pcr fatal acute hepatitis due to amodiaquine t-cell and cytokine responses in leishmaniasis fragment-based lead discovery recent advances in vaccines for leishmaniasis comparative evaluation of nasba hiv-1 rna qt, amplicor-hiv monitor, and quantiplex hiv rna assay, three methods for quantifi cation of human immunodefi ciency virus type 1 rna in plasma estimates of the burden of malaria morbidity in africa in children under the age of 5 years bulletin of world health organization: can pharmacogenomics improve malaria drug policy? amphotericin b induces expression of genes encoding chemokines and cell adhesion molecules in the human monocytic cell line thp-1 pathogen subversion of cell-intrinsic 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patients genetic variations of nat2 and cyp2e1 and isoniazid hepatotoxicity in a diverse population antimalarial drug resistance, artemisinin-based combination therapy, and the contribution of modeling to elucidating policy choices cyp2e1 mediated isoniazid-induced hepatotoxicity in rats high prevalence of cyp2a6*4 and cyp2a6*9 alleles detected among a malaysian population maternally inherited aminoglycoside-induced and nonsyndromic deafness is associated with the novel c1494t mutation in the mitochondrial 12s rrna gene in a large chinese family genome-scale rnai screen for host factors required for hiv replication post-kala-azar dermal leishmaniasis the prevention and treatment of isoniazid toxicity in the therapy of pulmonary tuberculosis. 2. an assessment of the prophylactic effect of pyridoxine in low dosage key: cord-103662-a4ok5wqc authors: tarek, m.; elhefnawi, m.; maricato, j. t.; diaz, r. s.; shytaj, i. l.; savarino, a. title: custommune: a web tool to design personalized and population-targeted vaccine epitopes date: 2020-04-29 journal: nan doi: 10.1101/2020.04.25.20079426 sha: doc_id: 103662 cord_uid: a4ok5wqc computational prediction of immunogenic epitopes is a promising platform for therapeutic and preventive vaccine design. a potential target for this strategy is human immunodeficiency virus (hiv-1), for which, despite decades of efforts, no vaccine is available. in particular, a therapeutic vaccine devised to eliminate infected cells would represent a key component of cure strategies. hiv peptides designed based on individual viro-immunological data from people living with hiv/aids have recently shown able to induce post-therapy viral set point abatement. however, the reproducibility and scalability of this method is curtailed by the errors and arbitrariness associated with manual peptide design as well as by the time-consuming process. we herein introduce custommune, a user-friendly web tool to design personalized and population-targeted vaccines. when applied to hiv-1, custommune predicted personalized epitopes using patient specific human leukocyte antigen (hla) alleles and viral sequences, as well as the expected hla-peptide binding strength and potential immune escape mutations. of note, custommune predictions compared favorably with manually designed peptides administered in a recent phase ii clinical trial (nct02961829). furthermore, we utilized custommune to design preventive vaccines targeted for populations highly affected by covid-19. the results allowed the identification of peptides tailored for each population and predicted to elicit both cd8+ t-cell immunity and neutralizing antibodies against structurally conserved epitopes of severe acute respiratory syndrome coronavirus 2 (sars-cov-2). overall, our data describe a new tool for rapid development of personalized or population-based immunotherapy against chronic and acute viral infections. the rapid development of automated platforms for data generation and analysis are increasingly making precision medicine a concrete option for several diseases. due to its potential for high selectivity and efficacy, immunotherapy is an optimal choice for the design of personalized therapeutic interventions 1 . while most efforts in this direction have focused on cancer 1-3 , viral infections can be a relevant application as well, particularly chronic infections characterized by extensive genetic diversity, in part due to in-host viral evolution. human immunodeficiency virus (hiv-1) is case in point, as the large number of circulating strains and its high replicative mutation rates have hampered the development of effective vaccines, both preventive and therapeutic 4, 5 . several lines of evidence highlight the relevance of immune control in hiv-1 infection. spontaneous long-term control of hiv-1 replication can be accompanied by the presence of broadly neutralizing antibodies 6, 7 or, more frequently, effective cell-mediated immune responses 8 . moreover, protective class i hla alleles have been identified both in people living with hiv/aids (plwha) and macaques infected with the hiv homolog simian immunodeficiency virus (siv) [9] [10] [11] [12] [13] [14] . in line with this, temporary depletion of cd8 + t-cells is associated with a rapid viral load increase, while their replenishment can revert this effect [15] [16] [17] [18] . a therapeutic vaccine based on cell-mediated immunity might offer the advantage of decreasing the number of infected cells. on the one hand, hiv-1 latently infected cells, which constitute the main barrier to a cure [19] [20] [21] , are not targeted by antiretroviral drugs or cd8 + t-cells 22 . on the other hand, effective cell-mediated immune responses could preserve drug-free control of the infection by keeping viral load low/undetectable and by eliminating the infected cells undergoing spontaneous hiv-1 reactivation from latency. such therapeutic vaccines could also be combined with strategies aimed at purging the hiv-1 latent reservoirs by inducing pharmacologic reactivation of latently infected cells 23 . the strong correlation between the host's genetic background and immune-mediated control of the infection suggests that effective immunity is mainly directed against a subset of hiv-1 epitopes. consistently, several studies have shown that cell-mediated immune responses against the hiv-1 gag protein correlate with lower viral loads in plwha and with post-therapy control of the infection in macaques 18, [24] [25] [26] [27] . the peculiar efficacy of anti-gag immunity might be partially explained by the higher fitness cost associated with mutations in this viral protein 28 . in particular, specific regions of gag, which are essential for hiv-1 packaging and assembly, are structurally and evolutionarily conserved, displaying low shannon entropy both in humans and primate lentiviruses 29 . however, it is noteworthy that low diversity is not sufficient per se to induce viral load control, as vaccine approaches designed exclusively by selecting epitopes based on their evolutionary conservation have shown only modest effects 30, 31 . a recent phase ii clinical trial (nct02961829) has attempted to induce anti-gag immunity against conserved epitopes using a personalized approach based on patient hla sequences 32 . although the study enrolled only a small number of plwha and tested multiple interventions, preliminary results suggest that therapeutic vaccination with autologous dendritic cells pulsed with individually designed peptides decreased the viral set point in some patients during analytical treatment interruption (ati) 32 . in the present work we describe and test a new automated, user-friendly web-based tool to design personalized peptides for vaccination. the tool, named custommune, was principally interrogated to develop therapeutic vaccine candidates for hiv-1. to this aim, by intersecting input data from patient-specific viral sequences and hla alleles, custommune provides an output of epitopes of desired length filtered for their predicted specificity, immunogenicity and mutation potential. of note, in our simulations, custommune performance was superior to that of manual vaccine design (applied in clinical trial nct02961829) in terms of prediction of clinical response. one advantage of custommune over traditional vaccine design techniques is the ability to quickly adapt the tool for different targets and strategies. in this regard, we applied custommune to the novel pandemic covid19, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) 33 . due to the acute manifestations of the disease, the design of a population-targeted preventive vaccine was chosen as a more practical approach as compared to a personalized therapeutic vaccine. moreover, to broaden the expected coverage and increase the likelihood of achieving herd immunity 34 , a strategy able to potentially evoke both neutralizing antibodies and cell-mediated immunity was preferred. using input regions of sars-cov-2 identified as viable targets, custommune was able to design vaccine candidates specific for regionally prevalent hla genotypes. in addition, custommune selected those hla class ii restricted epitopes that could induce neutralizing antibodies and thus provide a two-layered protection against the infection. taken as a whole, our results show the potential of the custommune algorithm to quickly design personalized or population-specific peptides for preventive and therapeutic vaccination. due to its intuitive and scalable approach, custommune might provide an effective tool for rapid vaccine development against chronic and acute conditions. the custommune web tool (available at: http://www.custommune.com) was written in python (http://www.python.org) using the django framework (https://www.djangoproject.com) and provides the user with an easy online interface for accessing and downloading prediction datasets without any coding knowledge requirements. the tool utilizes a pipeline ( figure 1 ) to design epitopes for preventive and therapeutic vaccines. for hiv-1 therapeutic vaccine design, custommune crosses input data from patient-specific viral sequences (dna in fasta format or raw dna sequencing inputs) and patient's hla-i and/or hla-ii alleles, providing an output of epitopes of desired k-mer length. to facilitate the allele input step, the tool provides two links directing the user to a list of supported class i and class ii hla alleles, respectively. these lists mirror those of the netmhcpan 4.0 algorithm 35 , for either hla class. although the approach could potentially be extended to encompass entire hiv-1 sequences, we decided to limit the search for viable epitopes to the gag gene only, because of the previously described distinctive efficacy of anti-gag cell-mediated immunity 18, [24] [25] [26] [27] . the tool pipeline ( figure 1 ) starts by translating input gag genomic sequences to protein sequences. custommune then performs multiple sequence alignment using the clustal omega (rest) web service python client 36 and builds a consensus translated sequence. the consensus sequence is then used to predict epitopes restricted to patient-specific hla-alleles for both classes. the hla-specific epitopes provided as final output by custommune are pre-filtered by the algorithm. this pre-filtering follows a set of parameters that compute epitope affinity in terms of sequence variation and conservation degree, allele-restricted affinities, and previous clinical evidence of immune response ( figure 1 ). for calculating evolutionary conservation, each epitope is compared, in terms of similarity, to an internal database of gag amino acid sequences (supplementary file 1) collected mainly from curated alignments retrieved from the los alamos hiv sequence database (http://www.hiv.lanl.gov/). moreover, to verify whether antigenicity has already been reported for the candidate epitopes, the tool compares potential epitopes to those already described in the los alamos hiv immunology site (http://www.hiv.lanl.gov/content/immunology). to further refine the structural assessment of epitope binding to hla-alleles, custommune performs structural epitope modelling followed by epitope-hla docking to determine the structural stability of the hla-predicted epitope binding ( figure 1 ). the custommune pipeline also computes some related physicochemical parameters of the personalized epitope sequence to aid in the assessment of the structural stability of candidate peptides. overall, the tool is optimized to identify immunogenic peptides characterized by the lowest variability (mutation potential). in line with this, the tool specifically highlights potential epitopes that are contained in regions which were previously described as essential for viral fitness 29, 37 . this is a novel and fundamental feature of this approach, as rna viruses are characterized by a high ability to mutate 38 . the custommune pipeline can be applied to other vaccine strategies by following a parallel workflow ( figure 1 ). an example of these applications are acute infections, such as covid-19. in this case, an approach combining neutralizing antibody responses and recognition by hla haplotypes most represented in a given population might provide a reasonable compromise between specificity and scalability. to this aim, using bepipred-2.0 39 , custommune can identify potential neutralizing epitopes which overlap with epitopes consistent with recognition by population-specific class ii hla haplotypes. at the same time, custommune can predict another set of epitopes optimized for recognition by hla class i haplotypes of the same population, thus providing two levels of potential immune recognition. overall, the custommune pipeline provides a flexible and fast tool to generate epitope predictions according to the genetic diversity of the virus and the genetic hla profile/s of the host or susceptible populations. we tested custommune predictions against manual epitope selection using results from an ongoing multi-interventional phase ii clinical trial enrolling plwha (nct02961829) 32 . in this trial, autologous dendritic cells were pulsed with a personalized vaccine designed manually from gag sequences generated from each patient´s circulating virus. in the study groups (g5 and g6) that had received this vaccine (along with other interventions), the patients showed variable responses including two individuals who displayed significant control of viral load during ati 32 . when viral and hla sequences of patients from g5 and g6 were used as input for custommune, the epitopes predicted by the tool generally displayed some overlap with those administered in the study ( figure 2a) . therefore, to investigate the potential therapeutic efficacy of custommune predictions, we stratified patients based on the virologic response during ati, which was defined as > 1 log10 ∆ viral load set point (i.e. the difference between median pre-and post-therapy copies of hiv-1 rna/ml of plasma). of note, non-responders were the only patients for whom there was no overlapping prediction between epitopes calculated by custommune and those administered in vivo ( figure 2b ). conversely, patients who had been administered vaccine epitopes highly overlapping (>50%) with those predicted by custommune, were characterized by higher viral load abatement ( figure 2c ). these data suggest that custommune can predict epitopes with therapeutic potential and could improve both efficacy and speed of personalized vaccine design. as the ongoing covid-19 outbreak is an urgent challenge for vaccine development 40 , we decided to test the potential of custommune for rapid identification of vaccine targets. in order to utilize custommune for sars-cov-2 predictions we first decided to identify the viral regions that could act as optimal input for the tool. due to the recent evolution of sars-cov-2, there is no equivalent of hiv-1 gag, i.e. a validated viral target for effective immunity. however, sars-cov-2 shares approximately 80% sequence identity with sars-cov 41 , the causative agent of an epidemic burst of acute respiratory distress syndrome (ards) in 2003. therefore, we decided to use previously described strategies successfully targeting sars-cov replication as a template to restrict custommune predictions. in particular, our efforts were directed at two validated sub-targets within the s-glycoprotein necessary for viral attachment to host cells 42 : 1) the portion of the s-glycoprotein that mediates the main protein-protein interaction with the cellular entry receptor, i.e. angiotensin converting enzyme 2 (ace2), as this was described as an optimal target for neutralizing antibodies 43 ; 2) the viral s-glycoprotein region binding the glycosylated portion of ace2, an interaction inhibited by pretreatment with chloroquine 44, 45 , a drug recently shown to effectively hamper sars-cov-2 replication in vitro and in patients 46, 47 . in order to translate these approaches into vaccine design: 1) we performed a thorough analysis for molecular complexes of the viral s-glycoprotein with the entry receptor ace2. considering the configuration of ace2, we superimposed complexes of s-glycoprotein/ace2 in both states of the receptor, i.e. free or bound (in this case with the competitive inhibitor mln-4760) 48 . our analyses indicated that the receptor-binding domain (rbd) surface of s-glycoprotein interacting with the bound configuration of ace2 is relatively smaller than (though 100% overlapping with) that interacting with the unbound configuration of ace2 ( figure 3a ,b). in light of this, we decided to restrict the custommune input to the rbd sequence interacting with bound ace2 and the linker amino acids (henceforth rbdp) ( figure 3a ,b). it is expected that this approach will be able to evoke antibodies against the rbdp irrespective of the ace2 bound/unbound configuration. 2) we inspected the possible contribution of oligosaccharide moieties of ace2 to the sglycoprotein/ace2 binding interface. the oligosaccharide moiety of ace2 was described as fundamental for optimal binding of the s-glycoprotein of sars-covs 44 . so far, in published structures, only partial ace2-bound oligosaccharide data is available. therefore, we decided to study this phenomenon by analyzing a published structure of inhibitor-bound ace2 (1r4l), which presents an n-acetylglucosamine (nag) covalently bound to residue asn90 and remaining from the oligosaccharide originally attached to this protein 49 . this evidence suggests that the nag present in the 1r4l structure is a marker of the position of the oligosaccharide originally attached to ace2 before being altered by the crystallization process. by superimposing this structure to the structure of the s-glycoprotein with ace2 and measuring the atomic distances at the binding interface between nag and the s-glycoprotein, we were able to determine the specific segment of the s-glycoprotein rbd that could be responsible for the interaction with the ace2-bound oligosaccharide. two specific residues of the s-glycoprotein (gly416 -lys417) were found to interact with nag, being within a 10 å radius from nag, i.e. a distance associated with significant intermolecular interactions ( figure 3c ,d). using s-glycoprotein gly416 as a starting point, we selected a core peptide spanning 20 amino acids in both directions of the translation frame. this led to the identification of a segment of the s-glycoprotein rbd, which we henceforth name rbdg, as a bona fide target for vaccine epitope design ( figure 4a ). of note, a structure of the sars-cov-2 s-glycoprotein and ace2 interaction (pdb: 6m17) was recently published while the present report was in preparation 50 . the authors concluded that the binding interface to ace2 is similar for sars-cov and sars-cov2, and their conclusions are largely overlapping with the results of the present analyses. overall, this evidence shows that the rbdp and rbdg dna sequences of sars-cov-2 can be used as optimal inputs for custommune. to mimic the approach described for hiv-1, we first analyzed the variability of rbdp and rbdg by multiple alignment of all sars-cov-2 s-glycoprotein sequences available at ncbi and gisaid (including isolates from humans, bats and pangolins) (supplementary file 2, 3 and figure 4a ). in line with the predicted key structural role of rbdp and rbdg, both sequences displayed very limited variability, mostly deriving from non-human isolates (supplementary file 2 and 3) . moreover, every amino acid variant (except one in rbdg) fully preserved the main physicochemical characteristics of the consensus residue (according to the scoring system of 51 ). these results suggest that both rbdp and rbdg represent bona fide equivalents of the conserved gag sequences used as privileged targets for custommune hiv-1 predictions. to adapt custommune predictions to some of the populations most affected by the sars-cov-2 pandemic (at the time at which these analyses were performed), we retrieved the relative hla allele frequencies in individuals from northern italy and south korea (supplementary file 4) (allele frequency net database; http://www.allelefrequencies.net) 52 . moreover, we applied the same approach to hla alleles of individuals from southern china and from the city of wuhan, where the outbreak had initially spread (supplementary file 4). when the rbdp and rbdg sequences were used as inputs along with population-specific hlas, custommune returned a set of epitopes (supplementary file 5) for either class i or class ii hlas. the hla class ii specific epitopes were further filtered to highlight those predicted as targets for . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . neutralizing antibodies using bepipred-2.0 39 . this was done to ensure that a unique peptide may provide the double stimulus necessary for optimal b-cell activation and antibody production (supplementary file 5). in line with the custommune pipeline, and in order to improve the likelihood of immune recognition, the binding stability and affinity of the most promising epitopes was validated by molecular docking. in particular, epitopes were selected for docking if they had been predicted to bind with an ic50 < 600 nm 53 to hla alleles described at four digit resolution for the population of interest in the allele frequency net database (supplementary file 5 and figure 4b ,c). interestingly, the identified epitopes included key residues involved in hydrogen bond formation between the s-glycoprotein of sars-cov-2 and ace2 (e.g. gln 474 in epitope steiyqagstpcngveg, gln498 in epitope lqsygfqp and lys417 in epitope irgdevrqiapgqtgkiadynyklpd of s-glycoprotein, engaged, respectively, in hydrogen bonds with residues gln24, tyr41, asp30 of ace2). since hydrogen bonds were recently described as crucial for the stability of the virus-receptor interaction 50 , epitopes containing the hydrogen-bonding residues might be particularly suitable targets to evoke immunity against structural determinants of sars-cov-2 infection. moreover, in order to ensure the best coverage likelihood of the target population, we also included the predicted epitopes for the most prevalent class i hla antigens. our results show that a peptide set specific for both neutralizing antibody/hla class ii and for hla class i could provide a good population coverage upon simultaneous delivery, potentially achieving herd immunity (fig 4c) . of note, one of the most promising epitope candidates designed by custommune for two of the populations examined (i.e. epitope klpddftgc for southern china/wuhan and northern italy) (supplementary file 5 and figure 4c ) is equivalent to a highly immunogenic peptide previously identified by stimulating cells of patients who had successfully recovered from sars infection 54 . taken as a whole, these results show the application of custommune to predict epitopes for specific populations and highlight a set of vaccine candidates to curb the spread of sars-cov-2 in highly affected areas. the precision medicine era, albeit still in its early stages, is expected to supersede traditional, onesize-fits-all therapeutic approaches. the development of personalized, yet scalable, treatments would allow accounting for the genetic variability of individuals, pathogens, or cancer profiles, and pave the way for more accurate efficacy predictions while reducing side effects. the implementation of our custommune pipeline in the context of hiv/aids shows that the tool algorithm may be used to predict novel immune-based treatments with in-vivo potential. even though the pipeline was applied to the hiv-1 gag protein in the present work, it can potentially be extended to other hiv genomic regions or other chronic viral infections. crucial prerequirements of the personalized custommune approach are the identification of a key structural component of the target pathogen and the obtainment of sequencing data from both the host hla . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . alleles and the infecting virus. while cost considerations might represent a limiting factor in some settings, the quick advances in sequencing technology, coupled to the steep reduction in price 55 , make the approach already feasible in developed countries. moreover, a personalized intervention aimed at a cure could make the cost-benefit analysis attractive also in developing countries, which often bear the main burden of chronic viral infections 56 . in terms of potential efficacy, the custommune approach relies on minimizing epitope diversity while maximizing predicted binding strength and immunogenicity of said epitopes. it is noteworthy that, when compared to a real clinical scenario, epitopes predicted by custommune correlated with treatment response. this was likely aided by the large amount of immunologic data available on hiv-1 (e.g. los alamos hiv immunology site). therefore, due to its low cost and scalability, custommune could be immediately applied to the design of therapeutic hiv peptide vaccines 57 or autologous dendritic cell vaccines pulsed with tailored gag peptides. compared to previous attempts at streamlining vaccine design in the context of cancer 58 , the custommune pipeline includes multiple layers of epitope ranking with scoring parameters accounting for: mutational potential, structural conservation, hla docking, escape mutations, location of the neomutation and previous evidence of antigenicity. these partially redundant filtration stages are envisaged to maximize the chances for durable and potent epitope recognition. moreover, other filters such as predicted epitope processing and cleavage have been included to the pipeline when this manuscript was in preparation, confirming the versatility of the custommune approach. our implementation of custommune was here extended to include vaccine design for sars-cov-2. current predictions suggest that traditional vaccine strategies might be too slow to address the spread of the pandemic and mitigate the death toll 59 . furthermore, immune responses developed during natural infection might be insufficient to provide long-term protection against reinfection 60 . the approach herein proposed is aimed at a flexible response customized for the populations most affected at a given time. as a novel pathogen will necessarily lack the wealth of immunologic data available for heavily studied viruses like hiv-1, our vaccine strategy attempts both induction of cell-mediated immunity and neutralizing antibodies. indeed, early evidence indicates that a broad immune response might correlate with successful clearance of the infection 61 . custommune predicted epitopes would further combine this broad immune stimulation with a design based on the most common hla alleles in the population of interest, potentially providing enough immune coverage for the induction of herd immunity. moreover, the choice of a highly conserved viral target as a source of vaccine epitopes should ensure a broadly effective response in those individuals for whom the vaccine should prove immunogenic. by utilizing custommune, the whole vaccine design process should last less than a working day. therefore, this approach, if successful, could be quickly adopted to blunt the pandemics during its spread or, ideally to preempt it. the binding affinities predicted by custommune for epitopes derived from rbdp and rbdg were generally higher for hla class i alleles in the populations here considered. while this is not sufficient to predict that cell mediated immunity would be preferentially induced by the proposed vaccine, previous evidence in mice suggests that memory cd8 + t-cells might alone be sufficient . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . to provide effective protection against sars-cov 62 . corroborating this hypothesis, one of the peptides designed by custommune was equivalent to an epitope associated with clearance of sars-cov infection during the previous epidemics and with immunogenicity in mice when used as a vaccine 54 . our estimate of the probability to reach herd immunity in the populations considered is based on the assumption that the development of immune responses against each of the vaccine peptides would be per se sufficient to guarantee some level of protection. while this prerequisite might prove optimistic, it is noteworthy that the viral targets selected for vaccine design (i.e. rbdp and rbdg of the s-glycoprotein) display exceptional evolutionary conservation and that no polymorphism in these regions was detected in the viral isolates from either italy, south korea or china. this conservation, coupled with the generally moderate mutation rate of sars-cov 63 and sars-cov-2 64 as compared to other rna viruses, yields credibility to the idea of achieving protection by targeting single immunodominant epitopes 62 . moreover, the expected population coverage of each of the vaccines designed in the present study is theoretically sufficient to achieve herd immunity based on the estimated reproductive number of sars-cov-2 34,65 . in the current work, to simplify administration schedule and increase scalability, we envisage, among other possibilities, a strategy synthesizing one multi-epitope peptide for each target population. this peptide would link class ii hla-restricted and neutralizing antibody epitopes as well as class i hla-restricted cd8 + t-cell epitopes. however, this approach will require empirical validation and could be modified, e.g. by administering hla class i and class ii restricted epitopes in separate formulations. while reduced immunogenicity is a well-known caveat of epitope-based vaccines, recent advances in adjuvant and delivery technology might allow overcoming this limitation 66 . apart from classical adjuvants, the use of an "adjuvant" drug such as chloroquine, is of particular interest for sars-cov-2. this treatment option could enhance vaccine immunogenicity 67,68 while possibly providing per se some protection against the virus 69 . in terms of delivery, carriers such as liposomes and nanoparticles, or strategies employing chemical conjugation or cell-penetrating peptides could increase epitope presentation by antigen presenting cells 66, 70 . finally, in our current model, we envisaged the use of linker sequences with protease cleavage sites between different epitopes 71 . this strategy might increase the chances of presenting peptides of optimal size to both hla alleles of class i and class ii. however, covalent linkage of epitopes has also been described to increase immunogenicity 66 . in-vivo studies will be required to optimize these strategies for inducing immunity against sars-cov-2. due to the ongoing rapid expansion of the epidemics and the relatively good safety profile of peptide vaccines 66 , pilot clinical testing in significantly affected areas might be envisaged. overall, our study describes a novel tool to improve multi-epitope vaccine design specificity while drastically reducing the associated time and cost. the pipeline herein described can be directly applied for testing personalized therapeutic vaccines for hiv-1 and to identify the core epitopes of preventive vaccines aimed at populations heavily affected by sars-cov-2. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . https://doi.org/10.1101/2020.04. 25.20079426 doi: medrxiv preprint the web application of custommune is available at http://www.custommune.com. written in python (v3.7) using django (v2.2.6) custommune is a tool that provides an integrated pipeline (figure 1 ) for prediction and filtration of personalized epitopes. the biopython package 72 is used for translating input sequences. alignment of translated sequences is then performed using the python client of clustal omega (rest) web service 36 . a consensus of the aligned sequences is generated using the biopython module with a 50% similarity cutoff. the biopython "proteinanalysis" function is used to estimate physicochemical parameters and secondary structure of the consensus sequence, including: molecular weight, gravity, specific count of amino acids, isoelectric point and fractions of secondary structures. custommune is connected with restful interface (iedb-api) 73 which serves as a platform for using netmhcpan v4.0 35 for class i and ii hla predictions as well as bepipred v2.0 39 for antibody epitope predictions. the pandas package (mckinney et al. 2010) is then used to structure epitope sorting tables and allow for comparative filtration. the primary filtration is based on ic50 values, a cutoff of 1000 nm is used to prevent loss of potentially false negatives. the los alamos hiv database (http://www.hiv.lanl.gov/content/immunology) was used to create internal hla class-specific datasets of previously reported immunogenic epitopes against hiv gag. using pandas 74 , high-affinity epitopes are compared to these datasets to highlight epitopes with previously described immunogenicity. moreover, another filtration layer is designed to report escape variants by comparing each epitope to an internal database collected from various literature sources including: dataset of hla-associated polymorphisms in hiv-1 gag as reported in ref. 75 , as well as the datasets reported in ref. 76 and the datasets of ctl/cd8 + and t helper/cd4 + epitope variants and escape mutations reported in the los alamos hiv database (http://www.hiv.lanl.gov/content/immunology/). additional filtration is obtained by comparing the epitope location within the gag sequence, to gag regions essential for viral assembly and packaging, which tend to be structurally and evolutionarily conserved, as reported in ref. 29 . to further refine this filtration, custommune computes the degree of conservation for each epitope by comparing the epitope sequence to the hiv sequence compendium database 77 which includes 680 alignments of hiv-1/sivcpz gag protein sequences. the degree of conservation (cscore) of each epitope is calculated as a fraction represented by the subset of sequences{ }in which the epitope scored a local alignment of more than 80% using clustal omega 36 over the total sequences in the internal database. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. the next layer of filtration selects only epitopes that rank high for multiple alleles in case a multiple-allele input was selected by the user for both hla classes. for further assessment of the impact of predictable mutations, custommune computes the effect of these mutations (retrieved from the internal gag sequence database; supplementary file 1) on the binding affinity of epitopes to the patient hlas. this refined analysis is performed only on the top three ranking epitopes initially predicted by the tool. by computing affinities to the same allele the user can estimate the impact of mutations in this specific segment on the affinity to the restricted allele. the degree of deviation of the mutated version is estimated based on , which are calculated as a standard deviation (sd) of the set of ic50 values for the candidate epitope and its mutant versions. the deviation value is therefore considered to negatively reflect the binding stability of this peptide segment to a restricted allele, in respect to a set of predicted mutant versions of the same segment. the python package peptidebuilder 78 is used for generation of 3d models of top epitopes, while the package lightdock 79,80 is implemented to perform epitope-hla docking based on the glowworm swarm optimization (gso) algorithm 81 . solved structures of hla alleles were collected from the phla3d database 82 and the protein data bank (pdb) 83 . homology modelling of structurally unsolved hla alleles was generated using swissmodel 84 . distance-scaled, finite ideal-gas reference (dfire) function 85 is used to calculate mean force potential of all atoms in a residue-specific manner within a resolution of less than 2 å, which has been found to accurately predict stabilities of structural (hla-epitope) complexes. dfire was implemented as a scoring function for lightdock simulations and docking scores were added in the final filtration layer for the highest ranking epitope candidates. for highly ranking epitope candidates, a scoring function is designed to account for each filtration layer. in this function each continuous parameter ( 50, , ) is represented by a quantitative value, according to the following rules: 1) the ic50 value is rescaled by calculating its reciprocal multiplied by a weighting factor of 10 4 ; 2) docking scores are preceded by a negative sign to weight the negative binding energies of the dfire scoring function of lightdock; 3) cscore is considered as a percentile of the cscore fraction weighted by a factor of 10 3 ; 4) sdaffinities are preceded by a negative sign to weight the positive values of deviation values. categorical parameters ( and ) are represented by binary values weighted by a factor of 500 for favorable states while non favorable states are given null values. overall the formula to calculate the final ranking (s) can be calculated as follows: s = 10000 * (ic50) -1 -dfire + escapem *500 + cscore *1000 + locationscore * 500 -sdaffinities + doverlap *500 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . https://doi.org/10.1101/2020.04. 25.20079426 doi: medrxiv preprint the top three epitopes ranked by s score are further analyzed based on their possible overlap with epitope data sets previously associated with: post-art control, efficacy in vaccine studies and the lack of reported escape mutations. finally, predicted antibody epitopes estimated by bepipred 2.0 39 are reported if they overlap with the top candidate epitopes ranked by s score. to allow manual inspection of results, sequence processing data and unfiltered predictions are provided in a separate section of the results page with a downloading link for a text file. s-glycoprotein sequences were retrieved from ncbi (https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/) and gisaid 86 . multiple alignments were performed using clustal omega web service 36 . consensus sequences and sequence conservation scores and histograms were generated with jalview (v. 2.11) 87 according to the amino acid conservation scoring criteria described in 51 . class i and ii hla allele frequencies were retrieved from the allele frequency net database (http://www.allelefrequencies.net/hla.asp) 52 using the "hla classical allele freq search" option. population-specific datasets (shown in supplementary file 4) were employed to identify the most represented hla alleles in areas heavily affected by sars-cov-2 spread, namely northern italy, south korea and china (wuhan and southern china). for all alleles analyzed, each data set provided values of frequency, which were determined as the number of copies of a given allele (x) divided by the total number of alleles in the population (of size n) assayed (i.e. frequency = x/2n). for each population of interest, only hla alleles with frequency ≥ 0.1 in at least one dataset of the same population were considered for further analysis. when a given hla allele was represented in more than one dataset of the same population, a weighted frequency was calculated. specifically, given an allele of interest represented in n datasets with population sizes n1, n2...nn, with a frequency of f1, f2...fn, the weighted frequency (fw) of the allele was calculated as: as the datasets employed included hlas characterized at different resolutions, allele frequencies were considered separately in case a 2 or ≥4 digit resolution 88 was available (supplementary file 4). alleles at 4 digit resolution and ≥ 0.1 (weighted) frequency were used as direct input for custommune. alleles at 2 digit resolution and ≥ 0.1 (weighted) frequency were instead analyzed with custommune by including all potential second field 88 options currently supported by custommune. class i and class ii hla alleles which were predicted by custommune to bind rbdp and rbdg epitopes of sars-cov-2 were used to estimate potential vaccine coverage in the populations of interest. to this aim, only (weighted) frequencies of hla alleles available at four digit resolution in the population of interest were included (supplementary file 4) . moreover, among these alleles, . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . https://doi.org/10.1101/2020.04. 25.20079426 doi: medrxiv preprint only those with high predicted binding affinity (ic50 < 600 nm) for an rbdp or rbdg epitope were included in the vaccine design (supplementary file 5) . to estimate the percentage of individuals (p) of a given population expected to carry an hla allele, the (weighted) frequency of that allele (f) in the same population (supplementary file 4) was used, according to the formula: for heterodimers (e.g. hla-dqa1 and dqb1) an overall frequency of the heterodimer was first calculated as: frequency of heterodimer 1 * frequency of heterodimer 2. this overall heterodimer frequency was then used to calculate p as described above. hla alleles of the population that were predicted to recognize more than one epitope of the vaccine were considered only once in the calculation of m. hiv-1 viral loads of individuals enrolled in trial nct02961829 were measured by q-pcr as described in 89 . clinical data were analyzed by unpaired t-test using graphpad prism (v. 6 graphpad software, la jolla california usa). . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020 following these filtration layers, custommune identifies whether any predicted epitope displays highaffinity to multiple hla alleles and (final epitope filtration) discards any epitopes that have reported escape mutations and/or are not located in an evolutionary conserved region. (affinity robustness) among remaining candidates, custommune restricts further analyses on the three top scoring epitopes for both hla classes. for these, custommune computes the hla binding affinities of potential mutant versions, though not classified as escape mutations, to estimate the impact of these mutations on epitope recognition (sdaffinities). (hla-epitope docking) on the same three top ranking epitopes, custommune computes epitope-hla allele docking scores, calculated using the lightdock 79 python package and scored using the dfire 85 scoring function. (final output and annotation) in a parallel process, the bepipred 2.0 39 algorithm is implemented to predict neutralizing antibody epitopes from the initial consensus sequence, that can be further intersected with class ii restricted epitopes to increase immunogenicity. as a final output, for both class i and ii hlas, custommune ranks the top 3 epitopes according to a score (custoscore) which accounts for all aforementioned filtration parameters. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . by two-tailed student t-test. panel c) ∆ viral load set point in trial participants who received peptides with high or low overlap to custommune predictions (≥ 50% or < 50% overlap, respectively). the ∆ viral load set point was calculated as the difference between pre-and post-therapy viral load set points, with post-therapy viral load set point calculated as the median of all available measurements (up to 9 weeks post-treatment interruption). each data point in panels b and c indicates a trial participant. consensus sequence and evolutionary conservation were calculated based on the multiple sequence alignments in supplementary files 2 and 3 using jalview 87 . the conservation score is based on 51 . (b) example of epitope-hla docking pose generated using lightdock 79 . the custommune-predicted epitope "kiadynykl" (magenta) is shown restricted by the hla class i histocompatibility antigen a-2 α-chain (hla-a*02:01, green), which is highly expressed in northern italy (supplementary file 4) . also shown is the invariant β2-microglobulin (cyan). the docking pose was scored using the dfire function 85 as listed in supplementary file 5. (c) custommune vaccine predictions and expected coverage for each target population. predicted epitopes were selected from those on which docking was performed (supplementary file 5). maximum expected population coverage was calculated based on allele frequencies in each population (listed in supplementary file 4) according to the formula described in the "materials and methods" section. linker regions between vaccine peptides are an example of a vaccine strategy based on a single, multi-epitope, formulation. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april 29, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april 29, 2020. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. 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('o'reilly media human leukocyte antigen-specific polymorphisms in hiv-1 gag and their association with viral load in chronic untreated infection frequent and variable cytotoxic-t-lymphocyte escape-associated fitness costs in the human immunodeficiency virus type 1 subtype b gag proteins peptidebuilder: a simple python library to generate model peptides lightdock: a new multi-scale approach to protein-protein docking lightdock goes informationdriven glowworm swarm optimization for simultaneous capture of multiple local optima of multimodal functions phla3d: an online database of predicted threedimensional structures of hla molecules the protein data bank and the challenge of structural genomics swiss-model: homology modelling of protein structures and complexes distance-scaled, finite ideal-gas reference state improves structurederived potentials of mean force for structure selection and stability prediction data, disease and diplomacy: gisaid's innovative contribution to global health jalview version 2--a multiple sequence alignment editor and analysis workbench key: cord-126015-zc7u3g34 authors: krieger, elizabeth; vissichelli, nicole; leichtle, stefan; kashioris, markos; sabo, roy; brophy, don; wang, xiang-yang; kimbal, pamela; neale, michael; serrano, myrna g.; buck, gregory a.; roberts, catherine; qayyum, rehan; nixon, daniel; grossman, steven; toor, amir a. title: immunological determinants of clinical outcomes in covid-19: a quantitative perspective date: 2020-05-13 journal: nan doi: nan sha: doc_id: 126015 cord_uid: zc7u3g34 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has a variable clinical presentation that ranges from asymptomatic, to severe disease with cytokine storm. the mortality rates also differ across the globe, ranging from 0.5-13%. this variation is likely due to both pathogen and host factors. host factors may include genetic differences in the immune response genes as well as variation in hla and kir allotypes. to better understand what impact these genetic variants in immune response genes may have in the differences observed in the immune response to sars-cov-2, a quantitative analysis of a dynamical systems model that considers both, the magnitude of viral growth, and the subsequent innate and adaptive response required to achieve control of infection is considered. based on this broad quantitative framework it may be posited that the spectrum of symptomatic to severely symptomatic presentations of covid19 represents the balance between innate and adaptive immune responses. in asymptomatic patients, prompt and adequate adaptive immune response quells infection, whereas in those with severe symptoms a slower inadequate adaptive response leads to a runaway cytokine cascade fueled by ongoing viral replication. polymorphisms in the various components of the innate and adaptive immune response may cause altered immune response kinetics that would result in variable severity of illness. understanding how this genetic variation may alter the response to sars-cov-2 infection is critical to develop successful treatment strategies. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has a variable clinical presentation that ranges from asymptomatic, to severe disease with cytokine storm. the mortality rates also differ across the globe, ranging from 0.5-13%. this variation is likely due to both pathogen and host factors. host factors may include genetic differences in the immune response genes as well as variation in hla and kir allotypes. to better understand what impact these genetic variants in immune response genes may have in the differences observed in the immune response to sars-cov-2, a quantitative analysis of a dynamical systems model that considers both, the magnitude of viral growth, and the subsequent innate and adaptive response required to achieve control of infection is considered. based on this broad quantitative framework it may be posited that the spectrum of symptomatic to severely symptomatic presentations of covid19 represents the balance between innate and adaptive immune responses. in asymptomatic patients, prompt and adequate adaptive immune response quells infection, whereas in those with severe symptoms a slower inadequate adaptive response leads to a runaway cytokine cascade fueled by ongoing viral replication. polymorphisms in the various components of the innate and adaptive immune response may cause altered immune response kinetics that would result in variable severity of illness. understanding how this genetic variation may alter the response to sars-cov-2 infection is critical to develop successful treatment strategies. the coronavirus disease 2019 (covid19) pandemic of 2020 has created a challenge to humanity like none other in recent times. with its global reach, infections with the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) have exacted a tremendous toll from populations around the world. this single-stranded rna virus gains access to the host intracellular milieu through interaction with the angiotensin-converting enzyme 2 (ace-2) which is expressed on a variety of epithelial and endothelial cells. 1 2 not only is this novel virus associated with higher mortality than other respiratory viruses, such as influenza, but it also demonstrates a broader variation in its clinical presentation. 3 4 5 6 at disease onset, the symptoms are generally mild and restricted to the respiratory tract. still, later, secondary viremia may involve other organ systems expressing ace-2, including the cardiovascular, nervous, and renal systems. 7 several patients experience an exaggerated immune response in the form of cytokine release syndrome. 8 9 despite this, a vast majority of patients with covid-19 may be asymptomatic or only mildly symptomatic, with the remainder experiencing a range of clinical manifestations, from moderate-to-severe, life-threatening. 7 mortality rates are high in the elderly and those with comorbidities. still, the young and otherwise healthy may also develop severe disease. 10 11 there is wide variability in casefatality rates in different populations around the united states ( figure 1a ) and the world, with rates as high as 10-13% seen in italy, england, and spain, but as low as 0.5-2% in iceland, norway and japan (figure 1 b) . this variation in the global death toll from this infection, despite testing and reporting biases, may suggest a genetic component in disease susceptibility. this variation may represent differences in the relative proportions of tested versus non-tested populations, socio-economic, comorbid conditions, and differential public health practices. it may be postulated that the disparity in disease severity could be at least in part due to genetic differences in the innate and adaptive immune responses that individuals may mount to sars-cov-2. 12 immune response to sars-cov-2, a general overview covid19 symptomatology may be due to a combination of pathogen and host factors. progression to the more severe forms of disease not only dependent on pathogen factors such as viral inoculum and virulence but, most likely, also on host factors that lead to variable immune response. patients with adequate immune function likely contain the virus without over-reaction. 11 however, in those with severe manifestations of covid19, a dysfunctional immune response is frequently observed. 11 13 the immune system has co-evolved with a multitude of historical pathogens, inherent with redundancies to counteract pathogens which have developed multiple mechanism of immune evasion. this has led to the development of genetic differences and polymorphisms which may explain the variability of the immune response in some patients within the host immune pathway (supplementary table 1 ). it is, therefore, critical to explore the intricacies of the pathways of the immune response to understand the differences in disease severity further. the immune response to viral infections is carefully orchestrated between the innate and adaptive responses, and various steps are required to attain control. failure or suboptimal function of any of the components could lead to an inadequate response. the innate response initially recognizes pathogen-associated molecular patterns (pamp) associated with viruses through pattern recognition receptors (prr) in the tissue-resident antigen-presenting cells (apc), such as macrophages and dendritic cells (figure 2) . examples of prrs include toll-like receptor (tlr) -7 & -13, which recognize single-stranded rna, 14 15 and rna-sensing retinoic inducible 1 gene (rig-1). 16 nod-like receptors (nlr) also help by sensing damage-associated molecular patterns. 17 polymorphisms in the tlr and its downstream signaling molecules such as myd88 may alter responses triggered pamp recognition. 18 19 20 the identification of viral pathogens by tissue macrophages initiates a cascade of cytokine secretion. these cytokines inactivate viral replication through type i interferons (ifn-a & ifn-b), and help activate the next line of innate immune defense, the natural killer (nk) cells, with their repertoire of activating and inhibiting receptors. the nk cell receptors include killer immunoglobulin-like receptors (kir) and the lectin-like, nkg2 family of receptors, among others. kir receptors recognize the downregulation of human leukocyte antigen (hla) molecules on infected cells and mediate cytotoxicity. nkg2 receptors recognize non-classical hla molecules, such as hla-e. 21 22 the innate immune effectors trigger a two-pronged hla dependent adaptive immune response. the hla genes exhibit extreme allelic polymorphisms and present viral peptides on host hla molecules to t cells to trigger an adaptive immune response. the t cell response involves both hla class i and ii molecules, which engage cytotoxic t cell (tc) and helper t (th) cell populations, respectively. the latter promotes both the cytotoxic t cell proliferation, as well as, b cell immunity and generation of sars-cov-2 specific antibodies. polymorphisms in the hla molecules result in differential antigen binding and presentation, leading to variability in immune responses. antibody production is the task of the b cell arm of the adaptive immune system, which with their unique b cell receptors, engage antigenic epitopes, either solubilized or directly presented by macrophages and dendritic cells. 23 24 in response, b cells proliferate and differentiate into memory b cells and plasma cells facilitated by interaction with th cells, establishing long term humoral immune response. 25 this humoral immune response is crucial to providing control for any viral infection, including sars-cov-2. a subset of patients may not develop long-lasting antibodies and remains unclear if they are at risk for recurrent infection. 26 polymorphisms in the hla and kir haplotypes may be responsible for the significant worldwide variability in the immune response to covid19. in other viruses, genetic variation among hla alleles and kir confer differing susceptibility. for example, hla-b*46:01 has been linked to the development and increased severity of sars-cov-1. 27 hla-a*02:05 may prevent hiv seroconversion, and hla-b*52:01-hla-c*12:02 had a protective effect on the progression of hiv in japanese patients. 28 a study using in silico modeling to predict viral peptide-mhc class i binding affinity across all known hla -a, -b, and -c genotypes, has shown that hla-b*46:01 has the fewest predicted binding peptides to sars-cov-2. this finding suggests that patients with hla-b*46:01 may be at increased risk for infection with sars-cov-2. 29 thus, global haplotypes variation across populations may partly explain the difference in illness severity and case-fatality rates. a similar variety may be observed in the kir haplotype frequencies. as an example, decreased kir2dl2 expression has been linked to increased susceptibility to sars-cov-1 30 and kir2dl3 homozygosity in association with its ligand hla-c1, is associated with reduced disease progression of hepatitis c virus. 31 the kir2dl2 gene is present in 46% of europeans and 80% of ethiopians. 82% and 72% of these populations respectively are, at least, heterozygous for its ligand, c1. similar hla alleles and hla epitope binding site effects have been observed in dengue fever, transplant outcomes. 32 33 34 3536 t cell and b cell repertoire diversity and symptom severity in covid19 patients in addition to these innate immune pathways, adaptive immunity with its t and b cell repertoire diversity is critical to mount an adequate immune response to viral infections. the mammalian t and b cell repertoire diversity and ability to recognize pathogen-derived antigens, either directly (b cell receptors) or upon presentation by hla (t cell receptors), is derived from a unique process of t and b cell receptor gene rearrangement. each of these genetic loci (for the immunoglobulin heavy and light chain and t cell receptor a & b loci respectively) are comprised of variable joining and diverse gene segments. these segments are recombined and further modified through the addition of non-templated nucleotides to yield an enormous repertoire of t and b cell receptor-bearing clones capable of recognizing the pathogens associated antigens with high precision and fidelity. a decline in repertoire diversity with age may create dominant oligoclonal t and b cell populations and predispose the elderly to develop symptoms with potentially higher severity of illness. 37 38 39 further, inadequate t and b cell clonal responses in immunocompromised patients may put patients at risk for insufficient viral clearance, and a prolonged, severe, clinical course. as noted above, patients with covid-19 develop significant lymphopenia, which may be due to a pan-t cell-suppressive effect. 40 41 42 a reduction in circulating t cells may represent migration to the site of inflammation, hyperfunction, or direct virally mediated lethality, and it is known that t cells also express markers of exhaustion in the face of a severe viral infection. 33 43 inflammatory cytokines during cytokines are key signaling molecules that orchestrate cellular and humoral immune responses to viral infections. the cascade of pro-inflammatory cytokines is triggered by the engagement of tlr on macrophages and by nk cells not finding their cognate autologous inhibitory ligands on infected cells. this first wave of proinflammatory cytokines includes tnf-ail-1, il-2, il-6, il-15, and ifn-g. these stimulate th cells to produce downstream cytokines, such as the pro-inflammatory il-12, and 17, and the anti-inflammatory il-4 and 10. these cytokines provide the critical third signal to trigger both th and tc responses to clear the infected cells. however, a generalized systemic inflammatory response may also be observed in viral infections. while optimal cytokine secretion will trigger an appropriate immune response, excessive cytokine release, i.e., "cytokine storm," may develop in some patients with covid-19, resulting in fulminant disease with high mortality. sars-cov-2 may also infect monocytes and dendritic cells, altering the cytokine expression patterns and contributing to lymphopenia observed. 44 importantly, this results in overproduction of inflammatory cytokines il-6 and downstream release of mcp-1, vegf, and il-8, eventually culminating in a cytokine storm. secondary hemophagocytic lymphohistiocytosis may ensue in these patients. patients with severe disease have higher levels of il-1, il-6, il-8, il-10, and tnf-a compared with patients with milder disease. 45 46 these high cytokine levels are associated with lymphopenia, with a deficit of both th, tc subsets, including naïve t cells and regulatory t cells. 47 48 elevated cytokine levels are also associated with a decrease in hla-dr expression. 42 in parallel, the nk cell population is also depleted, which may be the result of viral replication. 49 while those with severe disease have significantly higher sars-cov-2 rna load and lymphopenia has directly correlated with viral load, 50 these cytokine differences between moderately and severely ill patients may be due to polymorphisms in the cytokines involved. a quantitative approach relating differences in cytokine levels and polymorphisms in the immune response pathways may help identify patients at risk of severe disease. the immune response to infection may be broadly classified into a signaling component and an effector component, with the balance between the two determining the eventual outcome (figure 3) . the signaling function is determined by the cytokine and chemokine secretion by cells of the target tissues and the innate immune system in response to the infection. the effector component, on the other hand, is characterized by a pathogen-specific t and b cell response. the ability to maintain control of the virus and successfully recover from infection, relies on this sequential feed-forward loop nature of the signaling and effector components, with the virus attempting to inhibit these processes simultaneously. mathematical models have been proposed to understand the quantitative nature of these processes. these models generally study the growth of viruses as an exponential function of time. 51 viruses enter their host cells, grow exponentially in these cells, and are released into the surrounding milieu and infect an equal number of cells, where this exponential growth and release are repeated. thus, viral replication exhibits exponential growth occurring in an exponentially rising number of target host cells until a limit is reached by target cell exhaustion. in sars-cov-2, because of the widespread expression of ace-2, a large tissue reservoir is at risk of infection, amplifying the viral burden manifold over time. the corresponding immune response to the viral proliferation in the host has multiple components, and each of these components involves the growth of a population of immune effectors such as dendritic cells, nk cells, t cells, and b cells. the growth of each of these components will have to reach a threshold in an optimal period (e.g., by time, t1, t2, t3 or t, t', t'', t''' and so on for each cell type) to ensure timely and complete clearance of the virus (figure 4) . as the first line of defense, interferons slow down viral replication. virus, driven by their receptor affinities. 52 for nk cells, this represents the balance of inhibitory and activating signals from kir and nkg2 family of receptors. in some individuals with a kir b haplotype and a larger component of activating receptors, this may be a more effective process as opposed to those with a dominant component of inhibitory receptors. for the t cell responses, the ability of the hla haplotype of the individuals to present sars-cov-2 derived peptides will be critical. this encompasses both the antigen-binding affinity of the hla molecules in the host as well as antigen abundance. the requisite t cell response will be proportional to this antigen affinity and the affinity of the t cell receptor to the viral antigen-hla complex. while the nk and t cell subset proliferation may catch up with an exponentially rising number of infected cells, 53 cellular immunity may eventually fail under the pressure of rapidly replicating sars-cov-2, with widespread tissue involvement due to extensive ace-2 expression. thus, in addition to an efficient cellular immune response, a robust humoral immune response is essential to control and eliminate the infection eventually. the humoral response is characterized by a proliferating b cell and plasma cell population. each plasma cell makes large amounts of pathogen-specific antibodies, which finally allows the host to match the growth rate of the virus and neutralize the viral particles being generated. failure or suboptimal rate of any one of these pathways will lead to inadequate immune response and delay in viral clearance. the suboptimal response will potentially lead to earlier components in the pathway over-compensating for the failure of downstream mechanisms, caught in a feedback loop leading to phenomenon such as cytokine storm. the immune pathways are all susceptible to genetic polymorphisms that have functional consequences, such as variability in cytokine expression, antigenbinding affinities, the strength of receptor ligation and downstream signaling. 54 55 56 thus, functionally consequential polymorphisms in these interconnected immune pathways may impede the development of an optimal immune response to covid-19. the immune response to viral infections is a multi-step, precisely coordinated process, which results in viral clearance through initial innate and later adaptive immune mechanisms. this has been modeled mathematically to a high level of precision using ordinary differential equations. 57 58 59 the immune system behaves like a dynamical system when both t cells and nk cells are considered. 60 61 62 63 small changes in parameter values, for instance, in sars-cov-2 antigen -hla binding affinity or the initial il-6 or ifn-levels produced in response to infection, may have a profound impact on the eventual clinical outcome. 64 thus, polymorphisms in critical immune response genes may alter the clinical outcome in a significant manner, mainly when they are considered together in a comprehensive mathematical description of the total immune response. quantifying the number of polymorphisms across variables can, therefore, be used to assess differences in disease risk. as such, it may be assumed that there are a set of variables (innate and adaptive) that confer an advantage in terms of viral clearance without cytokine storm and reduce the risk of severe disease, as compared to the set of variables with no common elements ( figure 5) . in patients who have common elements across the entire set of immune response determinants (overlapping sets), the risk of severe disease may be equivalent when adjusted for age and comorbid conditions such as obesity, lung disease, and diabetes. among patients who either have very few or no overlapping elements, the risk of disease may be significantly different. these considerations become evident in a thought experiment to model the growth of any virus. if rv is the viral growth constant (number of virions replicated, for each cell infected, for each iteration of the growth process), then the total viral burden v at time t can be given by the equation where h is the target tissue reservoir, and ie is the infection efficiency for that virus (proportion of virions that end up infecting a new cell). with an initial inoculum of 1000 viral particles, 100 cells (h) get infected (ie=0.1 or 10%); in the second iteration 100000 virions will be produced with 10,000 cells infected, and it this keeps growing over time t until the limit of h is reached. interferon (ifn) would directly suppress v, by a fraction, sc (cytokine-induced suppression), starting at a later time t' (t' < t), and depending on how ifn much is produced, modifies vt to vt(ir) (diminished viral burden following the immune response) along with ifn production, a cascade of cytokine signaling will also be initiated, by the tissue-resident dendritic cells (dc) as well as the innate immune cells migrating into the tissues, where c is the unit production of cytokine per unit increase in v. 0 are the tissue-resident macrophages, and are the macrophages/inflammatory cells that migrate into the tissues. dc then is the dendritic cell ( ) production of cytokines in response to v. as v gets larger, dc gets higher with t and constitutes the feed-forward loop depicted in figure 3 and figure 6 . unless the overall feedback loop depicted in figure 3 slows down the growth of v over time, this can lead to cytokine storm. it is also important to note that dc is a vector matrix made up of many different cytokines, which all work on their respective receptors in the different target cell populations. the cytokines will trigger an nk cell response, which will slow down v. of note, this variable, and others, represent growth or change over time and is more accurately referred to by dv/dt. still, for simplicity, we will simply consider absolute value vt. nk cell growth and response may be considered as a function of the sum of activating and inhibitory receptors, the kir and the nkg2 family of receptors. these are modeled as vectoroperator equations, where the nk cell with its kir molecules constitutes a 'vector,' and the target with its kirligand molecules, an operator. the 'operator' modifies the 'vector' upon interacting with it; for example, it may either inhibit or activate it. each individual kir-kirl interaction may be described as follows; if an inhibitory kir (ikir) on the nk cell encounters a ligand on its target, this results in an interaction which may be scored, (−1) × (1) = −1, this will give the nk cell an inhibitory signal, assuming constitutively active basal state for nk cells; if there is no ligand for an inhibitory kir, i.e., missing kirl (mkirl), the interaction score will be (−1) × (−1) = +1 because of the abrogation of the inhibitory signal, and finally, activating kir (akir) interacting with its ligands will be scored, (1) × (1) = +1, when the ligand is present, and (1) × (0) = 0, when the ligand is absent since no signal is given. each of these different scores constitute a distinct component of the total kir effect on individual nk cells expressing them, and while akir and ikir function independently the cumulative effect calculated by taking their sum, determining the eventual outcome of nk cell-target interaction. activating kir are not required for a robust nk cell immune response in most instances, evident as individuals with haplotypes containing no functional akir are common and even people with haplotypes that include akir have nk cells in their nk cell repertoire without any akir present. 65 depending on the degree to which the virus down regulates hla class i molecules to evade tc mediated killing, it may lead to ikir-missing kir ligand mediated killing or may not through viral immune escape mechanisms such as, no hla class i down regulation or hla decoy expression. though viral evasion through decoy hla expression can lead to nk cell medicated viral killing as in m157 protein expression by mice with mouse cmv infection. m157 has been shown to strongly associate with an activating mouse ly49 receptor (analogous to kir) and confer host protection against mcmv. 66 so, the nk cell effect may be summarized by here time, t'' is a later period in time at which the nk cell effect becomes manifest, it is later than the time, t when viral proliferation begins. this means that the impact of an immune checkpoint triggered later than t (i.e., t' and t'') will require for those processes to outpace the viral proliferation, which already had a lead on these in time. the another mechanism for cytokine storm in the former situation (with optimal, timely viral control) will be autologous tissue destruction perpetuating the cytokine cascade in a different feedback loop. it is important to recognize that dc is a matrix, where many different cytokines with both suppressive as well as growth promoting effects are represented. 67 thus the overall effect of dc on t cell or b cell growth is not always in the positive direction, rather depending on the cytokines dominating in the milieu at any given time one may observe growth suppression, a phenomenon commonly seen in severely ill covid19 patients. the whole immune cascade may thus be mathematically described as a system of matrix vector operator equations. this schematic depicted in figure 6 . this exercise demonstrates the multiplicity of responses involved in controlling infections in general, as it is in this particular case. further, they indicate the many redundant pathways at work in the immune response to the same. it is also imperative to note that pathogens have evolved mechanisms to evade the immune response at multiple checkpoints. because of genetic variation, not all individuals are equally well endowed with the ability to control infection. a multi-targeted approach may be needed to overcome the redundancies encountered. the notion of performing randomized trials with a single intervention tested at a time is at the heart of medicine, and agents are failing such trials are often discarded for not having met efficacy endpoints. the thought experiment above suggests that for a pathogen that has the capability of outpacing or disabling several different immune mechanisms, a combination of many agents and modalities may be required to control the disease. attention to this aspect of infectious disease management needs to be given in future trial design. a quantitative approach, such 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genetic markers for stratifying the risk of cytomegalovirus infection in kidney transplant recipients diversity of killer cell immunoglobulin-like receptor (kir) genotypes and kir2dl2/3 variants in hcv treatment outcome b cell recognition of membrane-bound antigen: an exquisite way of sensing ligands antigen presentation to b cells transiently antigen-primed b cells return to naive-like state in absence of tcell help the trinity of covid-19: immunology, inflammation and intervention association of hla class i with severe acute respiratory syndrome coronavirus infection accumulation of pol mutations selected by hla-b*52:01-c*12:02 protective haplotype restricted cytotoxic t lymphycotes causes low plasma viral load due to low viral fitness of mutant virueses human leukocyte antigen susceptibility map for sars-cov-2. medrxiv the involvement of natural killer cells in the pathogenesis of severe acute respiratory syndrome consistent beneficial effects of killer cell immunoglobulin-like 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coronavirus disease the pd-1/pd-l1 axis and virus infections: a delicate balance cytokine release syndrome in severe covid-19 dysregulation of immune response in patients with covid-19 in wuhan, china clinical and immunological features of severe and moderate coronavirus disease characteristics of peripheral lymphocyte subset alteration in covid-19 pneumonia the laboratory tests and host immunity of covid-19 patients with different severity of illness complex immune dysregulation in covid-19 patients with severe respiratory failure correlation between relative nasopharyngeal virus rna load and lymphocyte count disease severity in patients with covid-19. viral immunology modeling influenza virus infection: a roadmap for influenza research a qualitatively validated mathematical-computational model of the immune response to the yellow fever vaccine mathematical modeling provides kinetic details of the human immune response to vaccination group as part of the h3africa consortium. classical hla alleles are associated with prevalent and persistent cervical high-risk hpv infection in african women an increased frequency in hla class i alleles and haplotypes suggests genetic susceptibility to influenza a (h1n1) 2009 pandemic: a case-control study cytomegalovirus infection in ireland: seroprevalence, hla class i alleles, and implications mathematical modeling provides kinetic details of the human immune response to vaccination a qualitatively validated mathematical-computational model of the immune response to the yellow fever vaccine modeling influenza virus infection: a roadmap for influenza research dynamical system modeling of immune reconstitution after allogeneic stem cell transplantation identifies patients at risk for adverse outcomes dynamical system modeling to simulate donor t cell response to whole exome sequencing-derived recipient peptides: understanding randomness in alloreactivity incidence following stem cell transplantation dynamical system modeling to simulate donor t cell response to whole exome sequencing-derived recipient peptides demonstrates different alloreactivity potential in hla-matched and -mismatched donor-recipient pairs killer immunoglobulin-like receptor-ligand interactions predict clinical outcomes following unrelated donor transplantations stem cell transplantation as a dynamical system: are clinical outcomes deterministic? understanding how combinations of hla and kir genes influence disease direct recognition of cytomegalovirus by activating and inhibitory nk cell receptors determining the quantitative principles of t cell response to antigenic disparity in stem cell transplantation key: cord-261392-dw56h8vj authors: matijevic, mark; hedley, mary lynne; urban, robert g.; chicz, roman m.; lajoie, christa; luby, thomas m. title: immunization with a poly (lactide co-glycolide) encapsulated plasmid dna expressing antigenic regions of hpv 16 and 18 results in an increase in the precursor frequency of t cells that respond to epitopes from hpv 16, 18, 6 and 11 date: 2011-12-31 journal: cellular immunology doi: 10.1016/j.cellimm.2011.04.005 sha: doc_id: 261392 cord_uid: dw56h8vj abstract a phase ii trial was conducted in subjects with human papillomavirus (hpv) associated high-grade cervical dysplasia testing the safety and efficacy of a microparticle encapsulated pdna vaccine. amolimogene expresses t cell epitopes from e6 and e7 proteins of hpv types 16 and 18. an analysis was performed on a subset of hla-a2+ subjects to test whether cd8+ t cells specific to hpv 16, 18, 6 and 11 were increased in response to amolimogene immunization. of the 21 subjects receiving amolimogene, 11 had elevated cd8+ t cell responses to hpv 16 and/or 18 peptides and seven of these also had increases to corresponding hpv 6 and/or 11 peptides. in addition, t cells primed and expanded in vitro with an hpv 18 peptide demonstrated cross-reactivity to the corresponding hpv 11 peptide. these data demonstrate that treatment with amolimogene elicits t cell responses to hpv 16, 18, 6 and 11. human papilloma virus (hpv) infections are recognized as a primary risk factor in the development of cervical dysplasia and cervical cancer [1] [2] [3] [4] . over 30 different genetic subtypes of the virus have been associated with the disease progression, but most of high-grade dysplasia and cervical cancers are associated with a subset of ''high risk'' viral types; the most frequent being type 16 and type 18 and their closely related subtypes [3, 5, 6] . while the majority of hpv infections are cleared by the host, the probability of developing progressive disease is significantly increased in patients with moderately suppressed immune systems [2] [3] [4] . therapeutic vaccination targeted towards viral antigens has been investigated as a means to enhance the host immune response to hpv and lead to disease resolution. this type of approach could be considered as an alternative to the surgical procedures that are currently used as standard of care [7] . hpv contains a double stranded circular dna genome encoding six early expressing genes (e1, e2, e4, e5, e6 and e7) and two late expressing genes (l1 and l2). the l1 and l2 proteins form the viral capsid and are expressed late in infection [8] . the e1 and e2 viral proteins are required for viral dna replication, while the e4 and e5 proteins are for virus assembly and ampli-fication of the viral genome in the upper layers of the epithelium [9] . the e6 and e7 proteins of high-risk hpv are involved in disregulation of the cell cycle and therefore are oncogenic [10] . the e6 viral protein interacts with an e6-ap to target the p53 protein for degradation [11] , whereas the e7 protein interferes with the normal function of the retinoblastoma protein [12] . collectively, e6 and e7 disrupt the normal cell cycle checkpoint processes. as both proteins are required to maintain the transformed state, e6 and e7 represent potential anti-viral therapeutic drug targets. anogenital warts (condylomata acuminata) are caused by sexually transmitted, non-oncogenic types of hpv. the percentage of global population that develop these warts is somewhere in the range of 0.5-1%, with approximately one million new cases each year in the united states [13] . over 90% of hpv infections resulting in anogenital warts are caused by hpv types 6 and 11. although most individuals who contract the virus will clear the infection or remain asymptomatic, a number of people will develop condylomas. anogenital warts are not life threatening however, there is a negative physical and mental impact on those affected with the disease. since disease resolution is dependent upon cell mediated immune responses, a therapeutic vaccine that can prime and expand hpv 6 and 11 specific t-cells may be a useful treatment option for patients with the disease. amolimogene bepiplasmid (formerly zyc101a) is an investigational immunotherapeutic that contains t cell epitopes from the e6 and e7 proteins of both hpv 16 and 18 that is being tested in 0008-8749/$ -see front matter ó 2011 elsevier inc. all rights reserved. doi:10.1016/j.cellimm.2011.04.005 the clinic as a treatment for high grade cervical dysplasia. amolimogene consists of a plasmid dna (pdna) vector that is encapsulated within poly-lactide co-glycolide (plg) microparticles ($2 micron mean diameter). microparticle encapsulation increases the stability of the pdna vaccine within the host by protecting the pdna from enzymatic cleavage [14] [15] [16] [17] . once injected into the host, the particulate nature of the formulation will cause local inflammation to occur and the resulting influx of antigen presenting cells (apc) will phagocytose the microparticles [18] . the plasmid will then be released inside the activated apc resulting in direct transfection of professional antigen presenting cells that can express the hpv epitopes, in the context of mhc class i and ii molecules, leading to t cell priming and expansion. amolimogene has been tested for safety and efficacy in a randomized, doubleblind, placebo controlled phase ii clinical trial in patients with high-grade cervical dysplasia [19] . the results demonstrated a favorable safety profile and, in a prospectively defined sub group of women under 25 years, clinical resolution of disease was significantly higher in amolimogene treated individuals compared to the placebo control group (70% versus 23%). it is of interest that although amolimogene comprises hpv 16 and 18 sequences, it was observed that resolution of lesions could occur in women with alternate (non 16 or 18) hpv subtypes. amolimogene was designed to induce and expand t cells specific to hpv 16 and 18 antigens. therefore, quantitative analysis of enhanced hpv specific peripheral blood mononuclear cells (pbmc) from patients before, during, and after drug or placebo administration was performed to assess the immunological activity of the patient cohort. we used the ifn-c elispot as a method to detect peripheral t cell activity in hla-a2+ subjects following treatment with amolimogene or placebo control. the elispot assay has the ability to detect low frequency, antigen specific t-cells in the peripheral blood [20] [21] [22] [23] [24] . the work reported here was performed to test whether t cells specific to hpv 16 and 18 were induced by amolimogene immunization, as well as determine if increased levels of t cells specific for non 16/18 subtypes (hpv 6 and 11) were present in the peripheral blood of the patient cohort after immunization. an additional objective of our study was to explore whether t cell clones specific to an hpv 18 peptide can recognize and respond to target cells presenting the corresponding hpv 11 peptide. although not evaluated in this study, the presence of higher levels of t cells with specificity for alternate hpv subtypes may help to explain the clinical observation of resolution of disease in women with non 16 and 18 hpv subtypes. together, these efforts may provide the framework for future studies exploring the diversity and/or cross reactive nature of the t cell response after amolimogene immunization. subjects with histologically confirmed high-grade lesions in the cervix (cervical intra-epithelial neoplasia ii/iii) were enrolled in the institutional review board (irb) approved phase ii clinical study. amolimogene or placebo (saline) was administered to the subjects via intra-muscular injections in the lateral quadriceps. injections occurred at weeks 0, 3 and 6. subjects were closely monitored until the end of the 26 weeks trial, at which point they underwent a loop electrosurgical excision procedure (leep). as described previously, all subjects enrolled in the study were hpv typed using residual sample from subjects' cytologic evalua-tion (thinprep ò system) [25] . gynecologic samples were collected using a cytobrush/spatula cervical sampling device. the sampling device was placed into transport medium and then sent to a central lab for analysis. hpv subtypes 16, 18, 31, 45 and 56 were detected by pcr (method adapted from manos et al.) [26] at the brigham and women's hospital (boston, ma). approximately 50 ml of heparinized venous blood samples were drawn at the clinical site during each subject visit and shipped overnight at ambient temperature to a central lab (covance laboratories, indianapolis, in) for pbmc processing. pbmc were isolated by sedimentation on a ficoll-paque gradient (amersham pharmacia biotech ab, uppsala, sweden) and stored in a freezing medium containing 90% fetal calf serum (fcs) (jrh biosciences, lenexa, ks), 10% dmso (sigma-aldrich, st. louis, mo). all pbmc samples were aliquoted at a concentration of 5 â 10 6 /ml and stored frozen overnight at à80°c before transfer to liquid nitrogen where they were maintained in the vapor phase. aliquots of pbmc were shipped overnight on dry ice to eisai research institute (lexington, ma). all peptides for use in the immunological assays were acquired from quality control biochemicals inc. (hopkinton, ma) or neo-mps inc. (san diego, ca). lyophilized peptides, which were a minimum of 90% pure, were shipped from the manufacturer to eisai research institute. the peptides were suspended in dimethyl sulfoxide (dmso) (sigma, carlsbad, ca) to a final concentration of 20 mg/ml and pipetted into individual aliquots. the peptides were stored at à20°c until the day of assay. peptide selection was based on several criteria. first, only nonamer peptide sequences were considered for the immunological assays. second, candidate peptide sequences were pre-selected based on hla binding restriction such that only the hlaã0201 epitopes encoded within amolimogene with moderate to high affinity binding properties were considered (measured by competitive radioisotope labeled, peptide binding assays; data not shown). third, a predictive algorithm was used to search for sequence homology between the drug encoded sequences and hpv 6 and 11 e6 and e7 amino acid sequences at positions p4, p5, p6 and p7 (the t cell receptor surface accessible side chain positions for a nonamer peptide bound within a hlaã0201 molecule) [27] . there were nine hpv peptides tested in total. each of these peptides was grouped into one of three peptide sets ( table 1) made up of at least one hpv 16 or 18 peptide and its corresponding hpv 6 and/or 11 peptides. a pool of peptides derived from cytomegalovirus, epstein barr virus and influenza (cef pool; anaspec, san jose, ca) was used as a control in the elispot assay [28] . pbmc samples were sent to the american red cross (dedham, ma) for hla typing. the laboratory used a labtype sso typing test (one lambda, inc., canoga park, ca) as a method to determine hla a and b types from each patient. the following method was used to generate antigen specific t cells in vitro. on day 0, dendritic cells from three hla-a2 donors were pulsed with the hpv16 e7 llmgtlgiv or hpv18 e6 nllirclrc peptides and were co-cultured with pbmc from the matching donor in a 24 well plate. the cultures were maintained for 21 days, and were restimulated with the priming peptide loaded onto autologous pbmc at days 7 and 14. il-7 (10 ng/ml), il-10 (10 ng/ml) and il-2 (100 iu/ml) were added at days 1, 9, 11, 15 or 16. culture media was replaced at days 11 and 15. at day 21, the antigen specific t cells were harvested, washed, counted and tested for peptide reactivity in the ifn-c elispot assay. t cell reactivity to both the priming peptide and its corresponding hpv 6 or 11 peptide were tested. an ifn-c elispot assay was employed to enumerate the frequency of hpv specific effector cells within pbmc populations. this assay was designed to directly test for cd8+ t cell reactivity as previously described [24] , or reactivity of antigen specific t cells generated from in vitro immunization cultures. human ifn-c elispot kits were purchased from r&d systems inc. (minneapolis, mn). all steps involving plate development were performed according to the manufacturer's instructions. on the day of assay, either cd8+ t cells enriched from cryopreserved pbmc using cd8+ t-cell enrichment columns (r&d systems inc., minneapolis, mn), or antigen specific t cells from ivi cultures were counted in trypan blue and adjusted to a concentration of 5 â 10 5 /ml. the hla-a2+ t2 cell line served as apc in the elispot assays [29] . t2 were adjusted to a final concentration of 1 â 10 6 /ml and pulsed with 25 lg/ml of peptide for 3-4 h at 37°c/5% co 2 before being plated with the cd8+ t cells (100 ll of each per well). the negative control was elispot wells containing t cells and t2 cells with no peptide, and the positive control was t cells and t2 cells pulsed with cef pool (anaspec, san jose, ca). assays were set using complete pbmc medium [rpmi (jrh lifesciences) with 10% human ab serum (c-six diagnostics, germantown, wi), 1% hepes buffer (life technologies, grand island, ny), 1% l-glutamine (life technologies), 1% penicillin-streptomycin (life technologies) and 0.1% 2-mercaptoethanol (life technologies)]. elispot plates were blocked with pbmc medium for 20 min at room temperature. blocking medium was removed prior to the addition of cells to the plates. duplicate test and control wells were set up for all assays performed in these experiments. cells were incubated in plates for 24 h at 37°c/5% co 2 . the plates were then developed as per the manufacturer's instructions. the developed plates were allowed to dry at rt and then shipped to zellnet consulting inc. (fort lee, nj) for counting via the elispot reader system (carl zeiss vision, germany) with ks elispot 4.0 software. a response to a peptide was considered positive if it was at least 50 spot forming cells (sfc) per million cd8+ t cells (with background subtracted) and 2-fold above the negative control (cd8+ t cells with t2 cells alone). a positive response after treatment initiation was defined as a 2-fold increase above the baseline response to the particular peptide. peptides were selected based on a combination of their experimentally determined binding affinity to hla-aã0201 molecules (data not shown), and their potential cross reactivity as predicted by sequence homology to putative t cell receptor contact residues based on molecular modeling and crystallographic analysis of the t cell receptor/hlaã0201/peptide complex [27] . as shown in table 1 , each of the three peptide sets contains at least one peptide from hpv16 and/or 18 and their corresponding hpv 6 and/or 11 peptide. all peptides were stored frozen until the day of assay. after thawing, the peptides were pulsed onto apc (t2 cells) for use as stimulators in the elispot assay. a multi-center, double-blind, randomized, placebo-controlled phase ii trial was conducted in subjects with high grade cervical neoplasia to test the safety and efficacy of amolimogene and the results of this trial have been described previously [19] . subjects were screened for confirmation of disease and then randomized into three groups. as shown in fig. 1 , the subjects were injected intra-muscularly with either 100 lg of encapsulated pdna, 200 lg of encapsulated pdna, or placebo control (saline) at timepoints week 0, 3 and 6. subjects returned to the clinic for an observation period that included visits at weeks 10, 14, 18, 22 and 26. for this immune response analysis, 26 subjects were selected from the phase ii clinical trial based on hla type (hla-a2+) and sample availability from the pbmc archive. the subjects were assigned random identifying numbers (1-26) for these studies. there had to be at least one pre-treatment sample and one posttreatment sample from each subject. most subjects in this analysis had a sample from baseline (week 0), week 14 (8 weeks post final immunization) and week 26 (20 weeks post final immunization) d l c t e l 13-21 3 hpv 11 e6 s i d q l c k t f 12-20 4 hpv 6b e6 nine hla-a2+ peptides were selected for screening in all elispot assays. all peptides were nine amino acids in length. the hpv 16 and 18 peptides are encoded by amolimogene. a predictive algorithm was used to search for sequence homology between amolimogene encoded 16 and 18 e6 and e7 sequences, and hpv 6 and 11 e6 and e7 amino acid sequences at positions p4, p5, p6 and p7 (highlighted in italic bold), yielding the corresponding hpv 6 or 11 peptides used in these studies. ( fig. 1 ). this provided pbmc from eight subjects that received the 100 lg dose, 13 subjects that received the 200 lg dose and five subjects that received the placebo control. assays were performed in batch format in that all timepoints from one patient were tested on the same day. in order to detect low frequency hpv-specific cytotoxic t-lymphocytes (ctl), an ifn-c elispot protocol that included a cd8+ t cell enrichment step prior to plating was used. cd8+ t cells were incubated with hla-a2+ t2 cells pulsed with hpv peptides to test for recognition and reactivity to the antigenic peptides. in this analysis, enhanced hpv 16/18 specific cd8+ t-cell responses (2-fold greater than the pre-treatment value) were detected in 11 of 21 subjects receiving amolimogene. the results from the subjects that had increased hpv t cell responses post baseline are included in table 2a -c. also listed are those subjects that did not have a measurable increase in hpv immunity (table 2 d). the frequency of hpv peptide responses in the placebo control group was low however, it should be noted that one subject (#22) had a response to the hpv 16 e7, llmgtlgiv and hpv 18 e7, lflntlsfv peptides that was low in overall magnitude, but met the criteria for an enhanced response. of the 11 subjects with increased hpv 16/18 t cell responses, all showed an increase to at least one of the amolimogene encoded hpv 16/18 peptides at the week 14 timepoint, 8 weeks after the final amolimogene immunization. at this timepoint, the most dominant epitopes were llmgtlgiv (hpv 16 e7) and lflntlsfv (hpv 18 e7), with 8 of 11 and 7 of 11 subjects responding to them, respectively (table 2, peptide set 2). in most cases, these t cell responses were lower or not detectable at the week 26 timepoint. in the 11 subjects that demonstrated an enhanced cd8+ t cell response to one of the amolimogene encoded hpv 16/18 peptides, seven showed increases in t cell responses to at least one of the corresponding hpv 6 or 11 peptides (table 2 ). in all but two of these subjects (#13 and #2), the responses were highest in magnitude at week 14 and decreased markedly by their week 26 timepoint, as was the case in the responses to the hpv 16/18 t cell responses. of note, t cell responses detected in subject #13 were higher than any other patient tested in this analysis, and the magnitude of the t cell response in this subject was maintained at the week 26 timepoint. the one subject in the placebo control group who had detectable increases in t cell responses to the hpv 16 and 18 in set #2 (#22) did not have detectable increases in her response to the corresponding hpv 6/11 peptide. three different hla-a2 healthy donors pbmc were tested in cultures designed to prime and expand t cells specific to the hpv 18 e6 nllirclrc peptide. at day 21, after three rounds of peptide stimulation, the t cell clones were collected and tested for cross reactivity by their ability to recognize and react to the corresponding hpv 11 e6 peptide. as shown in fig. 2 , t cells primed with the hpv 18 e6 peptide demonstrate cross reactive properties in 3/3 donors by responding to the hpv 11 e6 peptide. the magnitudes of responses to the hpv 11 e6 kvlircylc peptide in all three cases were similar to or greater than the responses detected to the hpv 18 e6 priming peptide (fig. 2) . these results demonstrate that t cells specific for the hpv 18 peptide nllirclrc are able to crossreact to the corresponding hpv11 peptide kvlircylc. amolimogene is an investigational therapeutic vaccine that consists of pdna encoding antigenic regions of the e6 and e7 proteins from hpv 16 and 18 encapsulated in biodegradable plg microparticles. the ability of amolimogene immunization to elicit t cells that recognize hpv 16 and 18 e6/e7 epitopes, as well as t cells that can recognize and respond to hla-a2 restricted, hpv 6 and 11 peptides containing homologous t cell contact residues was investigated in this study. peripheral blood cd8+ t cell responses were measured from hpv+ subjects with cin 2/3 that were enrolled in a phase ii clinical study [19] . the subjects in this trial were injected with 100 or 200 lg of encapsulated pdna, or a placebo control. cd8+ t cells were enriched from the subject pbmc and ifn-c elispot assays were performed to measure immune responses to the hpv peptide epitopes. the hpv 16 and 18 peptides encoded by the amolimogene pdna formulation were designed to be able to bind hla-a2 molecules [30] . in our current study, increases in immune responses to hpv 16 and 18 peptides were detected in 11 of 21 hla-a2+ subjects that had been immunized with amolimogene regardless of the dose. there was variation in the magnitude of response to the peptides among subjects however, there was consistency in the timepoint at which most responses were detected in that the strongest responses were detected at week 14 [8 weeks post the third (final) immunization]. by week 26, most effector t cell responses had decreased to levels that were near their baseline (week 0) responses. the week 14 t cell responses to the hpv 16 e7 llmgtlgiv and hpv 18 e7 lflntlsfv peptides were highest in magnitude and frequency. it is not entirely clear why we did not observe increased t cell responses in 10/21 subjects that received amolimogene. potential explanations are insufficient dose to prime and expand a measurable response, and/or in-correct blood collection timepoints. we were restricted to testing pbmc that were collected at weeks 0, 16 and 26 in this analysis, and it is plausible that peek immune responses occurred at timepoints far removed from these weeks. t cell responses in hpv infected individuals to the llmgtlgiv peptide are well characterized and this sequence has been used to develop additional hpv immunotherapeutics [31, 32] . it should be noted that of the five placebo control subjects tested, only 1 (#22) demonstrated an increased response to the hpv peptides which was low in magnitude (i.e. 6100 sfc/10e6 cd8+ t-cells). hpv typing was performed on the subjects in this analysis and only 5/24 had changes in their hpv status (by pcr) after their baseline visit. of the five subjects (#9, #13, #15, #20 and #24) only two had elevated immune responses to the hpv peptide sets (#9 and #13). of the remaining 19 subjects without changes in their hpv types, nine had elevated immune responses to hpv, suggesting that elevated immune responses are likely not due to new hpv infections but rather due to immunization with amolimogene. next we were interested to study whether t cells with specificity for other hpv types were induced by amolimogene immunizafig. 2 . results from in vitro immunization (ivi) cultures. t cells generated from pbmc of three hla-a2+ healthy donors were primed with hpv 18 e6 peptide (nllirclrc). the cells were stimulated with the priming peptide at days 0, 7 and 14. at day 21, the cells were harvested and tested using ifn-c elispot for reactivity to the priming peptide and its corresponding hpv 11 e6 peptide (kvlircylc). the negative control peptide was a known hla-a2 binding peptide from the cyp1b1 protein, fldprpltv [42] . the peptide sequences are listed on the x-axis. results are reported as ifn-c sfc/10 6 t cells. tion. subject t cells were tested for their ability to recognize and respond to peptides from hpv 6 and 11, the hpv types associated with genital warts. hpv 6 or 11 peptides were selected based on several criteria, one of which included a predictive algorithm that selected peptides based on sequence homology at the p4, p5, p6 and p7 positions. each hpv 6 or 11 nonomer peptide had as few as one and as many as six amino acid substitutions. increases in t cell responses to hpv 6 and/or 11 were detected in 7 of 21 subjects that had received amolimogene. as was the case in the responses to the hpv 16/18 peptides, most responses to the hpv 6/ 11 peptides peaked at week 14 and were reduced at the week 26 timepoint. these data offer evidence that amolimogene immunization can yield increases in cd8+ t cells with specificity for hpv 6 and 11 subtypes. as previously mentioned, we observed increases in hpv 16/18 t-cell responses at week 14 and 26 in one placebo control subject. interestingly, the t cell response to the corresponding hpv 6/11 peptide in this patient did not increase over time, resulting in 70, 90 and 10 sfc at weeks 0, 14 and 26, respectively. as shown in table 3 , a correlation exists between t cells generated to hpv 16/18 peptides and hpv 6/11 peptides. in most cases (7/11 subjects), when there was a detectable increase in t cell responses to hpv 16 and/or 18 peptides, there was also an increase to the corresponding hpv 6 and/or 11 peptides. this correlation suggests that the same t cell clones that are induced by amolimogene immunization are responding to two different, but homologous, peptides. the one exception was subject #21. this subject had a history of genital warts which may explain why there were detectable responses to the hpv 6b e6 tidqlcktf peptide (60 sfc at week 14) with no corresponding response to the hpv 16 e6 klpqlctel (0 sfc at week 14) or hpv 18 e6 klpdlctel (10 sfc at week 14) peptides. also shown in table 3 is whether the subjects experienced resolution of their disease. we did not observe a correlation between induction of peripheral hpv immune responses to the peptides selected for this study and cin ii/iii resolution. it has been previously reported that patients with cin iii have measurable ctl responses to hpv 16 antigens but do not clear their disease [33] . one potential explanation for this observation is that regulatory t cells could have interfered with or suppressed the effector t cells from effectively targeting the dysplastic tissue. it is known that regulatory t cells are present in and play a key role in hpv associated cervical lesions (persistence and progression to carcinoma) [34, 35] . our analysis on this small number of trial subjects was not designed to further explore this observation but rather to study the induction of hpv t cell responses and their cross reactive properties following immunization with amolimogene. the results of this study indicated that amolimogene can induce t cell responses to multiple t cell epitopes, a phenomenon that has been well documented with other dna vaccines [36] [37] [38] . enhanced cd8+ t cell responses were detected at week 14 (8 weeks after final immunization) to multiple hla-a2 restricted peptides from hpv 16 and 18. not only were the cd8+ t cell responses directed towards hpv 16 and 18, they were also reactive with peptides from hpv 6 and 11 that had sequence homology. based on our data set from the clinical trial subjects it is not clear if the responses we detected were generated from hpv 16/18 specific cd8+ t cells that were cross reacting to hpv 6/11 epitopes, or by distinct cd8+ t cell populations that are specific to unique epitopes from hpv 16/18 and 6/11. we initiated experiments to test whether a t cell line specific for an hpv 18 epitope, encoded by the amolimogene pdna construct, could react to an hpv 11 epitope with similar sequence homology. the results from this demonstrate that t cell lines primed to a single hpv 18 peptide can recognize and respond to an epitope from hpv 11. this offers evidence that suggests the peripheral blood hpv 16/18 t cells that are expanded by amolimogene immunization are capable of cross reacting to hpv 6/11 epitopes. other studies have examined cross reactivity in mouse and man. kreijtz et al. [39] demonstrated a considerable ability of seasonal ctl specific to the h3n2 influenza virus to recognize epitopes from the avian h5n1 influenza virus. this observation is important when assessing the potential impact of a pandemic outbreak caused by h5n1, and suggests that the presence of these cross reactive ctl may benefit the general population. in another study, nilges et al. [40] , showed that ctl reactive to the hpv16 e7 11à19/20 epitope, tmdlqpet, would also recognize the hla-a2 peptide from the human coronavirus ns2. the peptides have 66% sequence homology and the data support the notion that cross reactive cd8+ t cells can recognize immunogenic regions of hpv16 as well as regions of a common pathogen. in work performed by mccarthy and colleagues [41] , the authors show that immunization of hla-a2/k b transgenic mice with hpv 18 and 45 e6 dna yields cross reactive t-cells that recognize other related hpv types. finally, the work reported by williams et al. [42] , demonstrate cd4+ t cell lines, specific to hpv 11 l1, generated from 21 unrelated healthy donors have the ability to recognize and respond to alternate hpv types. our observations of cd8+ t cells capable of cross reactivity offer a potential explanation of how amolimogene induced hpv 16/18 specific t cells could recognize peptide epitopes from other hpv strains. the presence of a cell mediated immune response is helpful in clearing hpv infections [43] . enhancing the number of functional t cells specific to hpv 16, 18, 6 and 11 epitopes within the host may provide a clinical benefit for patients with hpv-mediated diseases. in 21 subjects receiving amolimogene, 11 were found to have a t cell response to the tested hla-a2 restricted hpv 16/18 peptides at either week 14 or week 26 timepoints that was at least 2-fold greater than the baseline value. seven of these 11 subjects had increased t cell responses to the tested hla-a2 restricted hpv 6/11 peptides. the results demonstrate that immunization with amolimogene, an investigational therapeutic vaccine based on hpv 16/18 e6/e7 sequences, elicits t cell responses that also recognize hpv 6/11 epitopes. furthermore, we showed that a t cell line specific to an hpv 18 peptide can recognize the hpv 11 peptide used in our studies. these data, combined with the clinical observation that disease resolution occurred in women that were not hpv 16 or 18 positive, suggest that additional studies designed to further characterize the ability of amolimogene to induce cd8+ and/or cd4+ t cells that react with other hpv types would be of interest. papillomavirus vaccines human papillomavirus infection and cervical carcinoma role of human papillomaviruses in human cancer immunobiology of hpv and hpv vaccines natural history of human papillomavirus infections, cytologic and histologic abnormalities, and cancer human papillomavirus vaccine and cervical cancer prevention cold-knife conization versus the loop electrosurgical excision 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evaluation of the ifn-c elispotassay for quantification of peptide specific t lymphocytes from peripheral blood assays for monitoring cellular immune responses to active immunotherapy of cancer immunization of patients with melanoma peptide vaccines: immunologic assessment using the elispot assay design and validation of an enzyme-linked immunospot assay for use in clinical trials of candidate hiv vaccines use of interferon-c elispot in monitoring immune responses in humans, handbook of elispot dynamics of human papillomavirus infection between biopsy and excision of cervical intraepithelial neoplasia: results from the zyc101a protocol looking for human papillomavirus type 16 by pcr structure of the complex between human t-cell receptor, viral peptide and hla-a2 a panel of mhc class i restricted viral peptides for use as a quality control for vaccine trial elispot assays endogenously synthesized peptide with an endoplasmic reticulum signal sequence sensitizes antigen processing mutant cells to class i-restricted, cell mediated lysis encapsulated plasmid dna treatment for human papillomavirus 16-associated anal dysplasia: a phase i study of zyc101 a phase i trial of a human papillomavirus (hpv) peptide vaccine for women with high-grade cervical and vulvar intraepithelial neoplasia who are hpv 16 positive human papillomavirus-specific cytotoxic t lymphocytes in patients with cervical intraepithelial neoplasia garde iii cd4+cd25hi regulatory t-cell frequency correlates with persistence of human papillomavirus type 16 and t helper cell responses in patients with cervical intraepithelial neoplasia regulatory t cells in a spectrum of hpv-induced cervical lesions: cervicitis, cervical intraepithelial neoplasia and squamous cell carcinoma strategies for effective naked-dna vaccination against infectious diseases dna vaccines against human immunodeficiency virus type 1 dna vaccines: precision tools for activating effective immunity against cancer cross-recognition of avian h5n1 influenza virus by human cytotoxic t lymphocyte populations directed to human influenza a virus human papillomavirus type 16e7 peptide-directed cd8+ t cells from patients with cervical cancer are cross reactive with the coronavirus ns2 protein definition of an hpv 18/45 cross-reactive human t-cell epitope after dna immunization of hla-a2/kb transgenic mice analysis of cd4+ t-cell responses to human papillomavirus (hpv) type 11 l1 in healthy adults reveal a high degree of responsiveness and cross-reactivity with other hpv types the cell-mediated immune response to human papillomavirus-induced cervical cancer: implications for immunotherapy the authors would like to thank susan balthaser for her editorial assistance during preparation of this manuscript. key: cord-254190-bxfne94u authors: tu, wenwei; zheng, jian title: application of humanized mice in immunological research date: 2015-07-07 journal: suppression and regulation of immune responses doi: 10.1007/978-1-4939-3139-2_10 sha: doc_id: 254190 cord_uid: bxfne94u during the past decade, the development of humanized mouse models and their general applications in biomedical research greatly accelerated the translation of outcomes obtained from basic research into potential diagnostic and therapeutic strategies in clinic. in this chapter, we firstly present an overview on the history and current progress of diverse humanized mouse models and then focus on those equipped with reconstituted human immune system. the update advancement in the establishment of humanized immune system mice and their applications in the studies of the development of human immune system and the pathogenesis of multiple human immune-related diseases are intensively reviewed here, while the shortcoming and perspective of these potent tools are discussed as well. as a valuable bridge across the gap between bench work and clinical trial, progressive humanized mouse models will undoubtedly continue to play an indispensable role in the wide area of biomedical research. during the past century, the application of rodent animal models , especially diverse gene-engineered mouse models , provided indispensable platforms and numerous valuable information for the advances in experimental medicine and biological research. however, the gap between species is still the most challenging obstacle for translation of results from rodents to humans . with the great advancement of technology in molecular biology and gene modifi cation, the attempt to establish "humanized" mouse models has made a leap since 1990s [ 1 -3 ] . nowadays, a wide variety of humanized mouse models have been generated and applied in nearly all fi elds of biomedical research [ 4 ] . in this chapter, we briefl y review the history and classifi cation of humanized mouse models and then summarize the current situation and recent advancement of their application in biomedical research, especially in the research of immune-related diseases. in general, the "humanized mice" are composed of three main classes: human gene-expressed transgenic mice ( human genetransgenic mice ), which are modifi ed by gene knock-in or replacement technology to express one or more human specifi c genes; humanized mice carrying human tissue, such as the liver ( humanized liver mice ) in which murine hepatocytes are completely or partly replaced by infused human-original hepatocytes; humanized mice equipped with functional human immune system ( humanized immune system mice ), which are established on immunodefi cient mice by transplanting human immune organs or cells to reconstitute human immune system in mice and thus referred to special "humanized mice." in the following section, we briefl y review the history and current advance of human gene-transgenic mice and humanized liver mice, and then focus on humanized immune system mice. human gene-transgenic mice are closer to gene engineered mice rather than "humanized mice." although the expressions of human gene or protein in transgenic mice provides the platform for studying in vivo role of specifi c human gene or molecule, the value of these data is limited in translational medicine due to the lack of human microenvironment and signal networks in these mice. the most widely used human gene-transgenic mice are human leukocyte antigen (hla)-transgenic mice [ 5 ] . these hlaexpressed transgenic mice represent for a useful tool in studying in vivo tcr-restricted immune responses and thus were adopted in the studies of immune-related diseases during the fi rst 10 years of this century. for example, hla-a0201-transgenic mice were used in inducing cd8 + t cell-restricted type i diabetes (t1d) [ 6 ] and experimental autoimmune encephalitis (eae) [ 7 ] , while hla-drb1-transgenic mice were applied in establishing cd4 + t cellmediated eae [ 8 ], system lupus erythematosus (sle) [ 9 ] and rheumatoid arthritis (ra) models [ 10 ] . more recently, the respective role of hla-dr2 and hla-dq8 in eae [ 11 ] and autoimmune diabetes [ 12 ] was also studied through transgenic mice. meanwhile, transgenic mice with distinct hla subtype expression favor the study of hla-related susceptibility on specifi c diseases, such as eae [ 13 ] , experimental autoimmune uveitis [ 14 ] , arthritis [ 15 -17 ] , allergic bronchopulmonary aspergillosis-like pulmonary responses [ 18 ] , and celiac disease [ 19 ] . although the application of hla-transgenic mice has been reduced due to the simplifi cation of diseases into specialized immune responses, the combination of hla-transgenic technology and reconstitution of human immune system in immunodefi cient mice has re-assigned them vitality in biomedical research, which will be discussed in the next section. other , which had all been blocked by the lack of optimal animal models in "pre-humanized mice time." on the other side, chen et al. tried to stabilize the function of cryopreserved human hepatocytes in immune competent mice through a novel system called "human ectopic artifi cial livers (heals)," which involved juxtacrine and paracrine signal in polymeric scaffolds. they claimed that mice transplanted with heals exhibited persistent normal liver function for weeks and thus provided a window for drugrelated investigation [ 34 ] . however, the effi cacy and value of humanized liver mice in the development of drug are still on debate due to the proposed side effects such as ongoing liver injury caused by transgenic and the infl uences on "normal metabolism" mediated by exogenous treatment [ 35 , 36 ] . apart from these, the potential application of humanized liver mice in immune-related research also deserves further exploration because liver also represents for a critical component of human immune system. the development of humanized immune system mice could be divided into three phases corresponding to the establishment of prkdc scid (protein kinase, dna activated, catalytic polypeptide; severe combined immunodefi ciency) mutation in cb17 mice, the development of nod (non-obese diabetic)-scid mice, and the generation of immunodefi cient mice homozygous for mutation at il (interleukin)-2 receptor γ chain locus [ 2 , 37 ] . each breakthrough mentioned previously signifi cantly improved the engraftment of human immune cells or pluripotent stem cells and stood as milestone on the way to "real humanized mice." the engraftment of multiple human immune components in these mice surpassed conventional human-gene knock-in in breaking the limited viewpoint of studying specifi c molecules under isolated environment. this unique advantage of humanized immune system mice favors their general application in immune-related studies, and opens a window for researchers to observe the interaction among human immune cells in vivo. in the following content, we focus on the characteristics and application of these mouse models and simply refer them as "humanized mice" if not otherwise specifi ed. currently, il2γc −/− mice established on nod/scid and recombination activating gene 2 (rag2) −/− balb/c background were most widely used strains for the reconstitution of human immune system in vivo [ 38 -40 ] . recently, by using bone marrow, liver, thymus (blt) co-transplantation, lavender et al. engrafted high levels of multi-lineage hematopoiesis and organized lymphoid tissues in c57bl/6-rag2 −/− γc −/− cd47 −/− triple-knockout mice. these humanized mice sustained human cell and tissue engraftment as long as 29 weeks post-transplantation without the development of chronic graft-versus-host diseases (gvhd), and thus represented for a new advancement in establishment of humanized mice [ 41 ] . the reconstitution of functional immune system is the key to evaluate the successful establishment of humanized mice. the graft used for reconstituting human immune system includes stem cells [ 42 ] , blt [ 41 ] , and peripheral blood cells [ 43 ] according to specifi c objectives. generally, stem cell and blt transplantation exhibit advantage in establishing stable multi-lineage hematopoietic cells but might need additional treatment for improving development of specifi c cell subpopulations. on the contrary, humanized mice established by peripheral blood cells provide a ready platform for studying the functions of mature immune cells but the length of window appropriate for research is still limited by chronic gvhd and ongoing reduced engraftment. to maximize the potential of humanized mouse model , some progresses have been made recently. firstly, pretreatment or gene-engineering of pluripotent stem cell exhibited satisfactory effects on improving engraftment of immune cells [ 38 , 42 , 44 ] . secondly, human growth factors, cytokines [ 44 , 45 ] or signal regulatory protein alpha (sirpa)expressed [ 46 ] immunodefi cient mice demonstrated superior engraftment for specifi c immune cell subpopulations as well. in the following paragraphs, we briefl y review current status of the reconstitution of specifi c immune cell subpopulations in humanized mice. lymphocytes are most important components of immune system and thus draw a major attention. although human peripheral blood mononuclear cells (pbmc) transplantation led to rapid reconstitution of human lymphocytes in humanized mice, it was found that after initial activation and induction of antibody production, human t cell lymphocytes enter an unresponsiveness status due to loss of human professional antigen-presenting cells (apc), which could be reversed by adoptive transfer of human apc [ 47 ] or activating organ-resident myeloid dendritic cells (dc) through poly(i:c) treating [ 48 ] . meanwhile, stem celltransplanted humanized mice displayed diversifi ed t cell repertoire, but the gap between hla and murine major histocompatibility complex (mhc) molecules prohibited the induction of effi cient t cell-mediated primary immune responses in vivo [ 49 , 50 ]. to overcome these problems, hla-expressed immunodefi cient mice were generated and their effi cacy has been confi rmed [ 51 ] . another concern origins from th1 and th17 immunocompetence in humanized mice [ 52 ] , which supports the utility of their application as surrogate model in transplantation rejection and autoimmunity but might cause some unwanted immune responses against murine tissue antigen as well. distinct from their t cell companion, reconstitution of functional b lymphocytes is generally poor in humanized mice and needed to improve in the future although their primary repertoire were principally unaltered by the differences between mouse and human stromal environments [ 53 ] and their ability to produce antigen-specifi c antibody was partly developed [ 54 ] . as described previously, the reconstitution of myeloid cells not only guarantees immune system intact, but also determines the development and function of both adaptive and innate lymphocytes [ 47 , 48 , 55 ] . unfortunately, monocytes and other myeloid cells usually exhibit immature phenotype and impaired function in humanized mice [ 56 ] , which could be partly rescued by human colony stimulating factor (csf)-1 [ 57 ]. however, the improvements in their survival, differentiation and even migration and residence [ 58 ] are still urgently required. besides leukocytes, other blood components also play important roles during immune response and regulation. recently, hu et al. established the full reconstitution of human platelets in humanized mice after depletion of murine macrophage [ 59 ] , which represents for an interesting attempt in constructing a more "humanized" circulation in mice. in summary, the optimization of humanized mouse model is still on the way and the advances in molecular biology, cellular biology, and system biology will defi nitely bring new era to the development of this useful tool. the applications of humanized mice cover nearly all fi elds of biomedical research and here we concentrate on immune-related studies, especially those aiming at the mechanisms and translational potentials of immune regulation and suppression . we also briefl y summarize the benefi ts brought by these potent models in tumor, infectious diseases, and vaccine studies. cd34 + cd38 lo cd1a − (early t lineage progenitors, etp), and cd34 + cd38 + cd1a + pre-t cells in liver of humanized mice by intrahepatic injection of cd34 + stem cells, establishing a wonderful platform for investigating human t cell development [ 60 ] . however, joo et al. found that human t cells educated by murine mhc in mice without a human thymus differ from normal human t cells marked as higher expression of cd45ro and promyelocytic leukemia zinc fi gure protein (plzf) regardless of similar development stages [ 61 ] . correspondingly, danner et al. generated hla-dr4-expressed nod-rag1 −/− γc −/− mice and demonstrated the critical role of hla class ii molecule for development of functional t cells by infusion with hla-dr-matched human hematopoietic stem cells [ 62 ] . meanwhile, the roles of il-12 [ 63 ] and notch [ 64 ] signals during the development of human cd4 + and cd8 + t cells were evaluated by human hematopoietic stem cell-transplanted mice. moreover, using a human stem cell factor, granulocyte-macrophage colony-stimulating factor (gm-csf) and il-3-expressed nod/scid-γc −/− mice, billerbeck et al. found the increased accumulation of human cd4 + foxp3 + t cells in blood, spleen, bone marrow and liver. most importantly, these cd4 + foxp3 + t cells exhibited potent suppressive capability on t cell proliferation, which made a signifi cant contribution to study of human regulatory t cells (treg) development in vivo [ 65 ] . as described previously, the development of human b cells in humanized mice is relatively weak compared to t cells. in 2011, choi et al. evaluated the effi cacy of busulfan, a chemotherapeutic agent, and claimed that it could effi ciently improve the reconstitution of human specifi c antibody -producing b cells, t cells, macrophage, and even dc from cd34 + cord blood cells with less toxic effects [ 66 ] . on the other hand, kim et al. found that cotransplantation of fetal bone tissue with fetal thymus could facilitate the development and reconstitution of human b cells from fetal liver-derived cd34 + cells together with t cells [ 67 ] . besides adaptive lymphocytes like t and b cells , innate lymphocytes development-related factors were also illustrated in humanized mouse model . as early as in 2008, huntington and di santo made a periodic review on the application of humanized mice in the research of nk cell development [ 68 ] . in 2011, pek et al. further confi rmed the crucial role of il-15 in nk cell development in bone marrow and liver with humanized mouse model [ 69 ] . we believe that more studies in the development of other innate lymphocytes such as nkt, γδ-t cells and innate-like t cells (ilt) will be reported in the near future. myeloid cells are generally regarded as more fragile and diffi cult to survive in "strange environment", which made it attractive and subtle to improve reconstitution of these sensitive cells. addition of human original cytokines such as gm-csf and il-4 was generally accepted as an effi cient way to improve dc maturation [ 70 ] . similarly, the effects of macrophage colony-stimulating factor (m-csf) and fms-related tyrosine kinase (flt)-3 ligand on promoting the development of macrophage [ 71 ] , and cd141 + and cd1c + dc [ 72 ] have also been confi rmed in humanized mouse model respectively. moreover, the development of megakaryocytes was replicated and used as index of dengue virusinfection in humanized mouse model recently [ 73 ] , which also supported the multi-lineage hematopoietic cell development in humanized mice. finally, transplantation of human stem cells from bone marrow of patients with bone marrow failure syndrome into humanized mice provided invaluable tools for evaluating novel gene-targeted therapy before clinical trial [ 74 ] . in summary, the reconstitution of diverse human immune cell populations from their pluripotent progenitors in immunodeficient mice has become a potent platform for investigating the development of human immune system while the next question is how to create a more "humanized" environment in mice for human cells [ 75 ] . the advances in the study of autoimmune diseases in humanized mice, especially those t cell-mediated diseases, are always correlated with development of hla-transgenic technology. in 1999, bachmaier et al. generated a cd4−cd8− double-knockout mice transgenic for human cd4 and hla-dq6 to specifi cally reconstitute the human hla-dq6/cd4 arm in mice and established a dilated cardiomyopathy model [ 76 ] , which was one of the earliest attempt for applying humanized mouse model in the study of autoimmune diseases. using similar strategy, eming et al. established a ra model in a hla-dr4/human cd4/tcr combined transgenic mice with the stimulation of a ra-related human autogenic protein hcgp-39 in 2002 [ 77 ] . however, the lack of human immune system reconstitution in these models constrained their representative for the whole map occurring during autoimmune diseases. on the other side, shultz et al. established t1d model in nod/scid-γc −/− mice by co-transplanting with human stem cell and islet cells [ 78 , 79 ] . importantly, this group pointed out the potential of hla-transgenic immunodefi cient mice in optimization of these models and provided some interesting preliminary data [ 78 ] . soon after, infl ammatory arthritis and type 2 diabetes models were established in hla-transgenic humanized mice by david [ 80 ] and schultz groups [ 81 ] respectively. as we mentioned previously, t cell-mediated immune responses were generally incomplete in humanized mice established on conventional immunodefi cient mice, which usually led to insignifi cant clinical symptoms [ 82 ] and thus limited the application of these models. the involvement of hla not only improves the effi cacy of immune responses, but also provides a platform for study of the relationship between hla subtypes and specifi c diseases susceptibility. nevertheless, the complexity and individuality of hla phenotypes in healthy donors or patients still remain as the biggest challenge in rebuild of physiopathology process in relatively limited hla-expressed humanized mice. due to relatively weak reconstitution of human b cells in humanized mice, the establishment of b cell or antibody -mediated autoimmune diseases seems to be more diffi cult than those t cellmediated autoimmune diseases. kerekov et al. rebuilt the clinical pathogenesis in humanized mice with cells transferred from sle patients and evaluated the potential of b cell-targeted therapy with a chimeric molecule containing a monoclonal antibody against human inhibitory complement receptor type i coupled to a decapeptide that mimic dna antigenicity [ 83 ] . in 2012, another group led by duffi eld recapitulated systemic vasculitis in humanized mice by treating them with anti-proteinase-3 igg isolated from patients [ 84 ] . with the improvement in reconstitution of multiple components of human immune system in humanized mice, it is predictable that the induction of diverse human b cellmediated autoimmune diseases in vivo will be accessible soon. application of humanized mice models in transplantation-related diseases arises as early as the birth of humanized mice but the process is so tortuous till now due to chronic exogenous rejection and ongoing decrease of immune cells . in above three studies, investigators planted solid grafts into immunodefi cient mice before reconstitution of human immune system and induced rejection by infusion of mature human cells. however, the longterm outcome of these models is still not clear. in order to further mimic clinical situation, human cd34 + stem cells were applied in establishing humanized mice. using this strategy, three independent groups reported allogeneic islet transplantation [ 89 ], xenogeneic islet rejection [ 90 ], and xenogeneic skin rejection [ 91 ] models during 2010-2012. unfortunately, insuffi cient development of immune cell populations in these humanized mice still stayed as an obstacle and even led to the failure of rejection [ 89 ] . to solve this problem, some other groups tried to develop a more "mature" human immune system in humanized mice by transplanting human peripheral blood cells. in 2013, our group reported a novel human allogenic gvhd model established on humanized mice reconstituted with human pbmc [ 92 ] . this model reproduced typical clinical process of acute gvhd occurring during allogeneic bone marrow transplantation without apparent interruption of exogenous reactivity. using this model, we evaluated the protective effects of human cd8 + treg induced ex vivo by allogeneic cd40-activated b cells and found that human cd8 + treg could inhibit gvhd and induce long-term tolerance without compromising general immunity and graft-versus-tumor (gvt) activity [ 92 ] . the potent regulatory activity of the cd8 + treg was mainly mediated by the expression of cytotoxic lymphocyte antigen (ctla)-4 on cell surface, while their alloantigen-specifi city and the ability to induce the long-term tolerance favor their clinical application. more importantly, this strategy might reduce clinical dependence on limited hla-match donors and largely improve the survival chance of millions of patients who are waiting for bone marrow transplantation. humanized mouse model undoubtedly brings new hope for transplantation research, but we also need to keep in mind that a lot of questions are still waiting to be answered on this way. as emphasized by brehm and shultz, keys to successful humanized mouse model included available immunodefi cient mouse strains, the choice of tissue to transplant and the specifi c human immune cell population that can be grafted [ 85 ] . besides autoimmune diseases and transplantation-related diseases, humanized mice models are also useful to study some other infl ammatory diseases. in 2002, hammad et al. compared the th2 allergic infl ammation in the lung of humanized mice reconstituted with pbmc. to induce infl ammatory reaction, dcs from home dust mite (hdm)allergic patients or healthy donors were injected intratracheally and mice were then repeated exposed to aerosol of hdm. in contrast to ifn-γ secretion induced in mice receiving normal dcs, those injected with dcs from patients induced il-4 and il-5 production accompanied with the increase of ige production, which represents characteristics of th2 response lymphadenopathy, and other infl ammatory sequelae in humanized mouse model [ 97 ] . in addition to immunopathology study, humanized mouse model was also applied in studying the underlying mechanisms of injury repair. by plating retroviral vector-modifi ed human skin on nude mice and adding human keratinocyte growth factor (kgf) to artifi cial wound in the skin, the re-epithelialization was signifi cantly accelerated [ 98 ] . although this model could not be described as "real" humanized mice because no human immune system was involved in it, this attempt initiated an innovative application of humanized mice. compared to satisfactory reconstitution of circulating blood cells, the successful reconstitution of mucosa immunity in humanized mice is still absent till now. mucosa, especially respiratory and digestive tract surface, plays indispensable role in protection and immune regulation. however, the residence and exchange of immune components in the locus are still diffi cult to rebuild in animal models because the physiological dynamics remains largely unknown the occurrence of humanized mouse model provided a perfect platform for evaluating immunotherapy against tumor. the earliest attempts of inducing antitumor immune responses in humanized mice focused on the generation of specifi c antibody but the outcome varied due to unstable humanization of models [ 104 , 105 ] . in the new century, researchers started to pay more attention on developing complete tumorigenicity, especially metastasis process and its relationship with stromal cells, in immunocompetent humanized mice, and made some signifi cant advances in multiple fi elds like human prostate cancer [ 106 ], mixed-lineage leukemia [ 109 ] . based on these progresses, some novel immunotherapy strategies were evaluated on humanized mouse models, such as inhibitory receptor ig-like transcript (ilt)-3 depletion or blockade in melanoma [ 110 ] and il-15-enhanced nk cell-mediated cytotoxicity against human breast cancer [ 111 ] . recently, our group reported a novel application of pamidronate, a phosphoantigen generally used to treat osteoporosis, in treating epstein-barr virus (ebv)-induced b cell lymphoproliferative disease in humanized mouse model reconstituted with human pbmc [ 112 ] . this "new application of an old drug" was mediated by expanding and activating human vγ9vδ2-t cells, a small cell population of human lymphocytes, which might inspire further exploration of currently available resources. more importantly, the established of donorand tissue-specifi c humanized mouse tumor models will undoubtedly play an indispensable role during the development of individual therapies in the future [ 113 ] . the development of humanized mice represents a milestone in the history of human immunodefi ciency virus (hiv) study. the new generation of humanized mice not only improved our understanding on transmission, latency, and pathogenesis of hiv [ 114 -119 ] , but also provided unprecedented platform for antiviral study. besides further exploration of effi cient virus-specifi c neutralization antibodies [ 120 -125 ] and conventional antiretroviral or antimicrobial therapies [ 126 -128 ] in these models, the effi cacy of vectored immunoprophylaxis [ 129 ] and ccr5-targetd treatment [ 130 -132 ] in preventing hiv transmission were evaluated as well. meanwhile, the crucial roles of hiv-specifi c cd8 + t cells [ 133 , 134 ] and plasmacytoid dc (pdc) [ 135 , 136 ] in the replication of virus and activation of immune responses, and their potentials in targeted therapy were also investigated. other novel immunotherapy assays performed in humanized mouse model included blockade of programmed cell death (pd)-1 receptor [ 137 , 138 ] , engineering hiv-resistant t cells from short-hairpin rna (shrna)-expressing hematopoietic stem/progenitor cells [ 139 ] , and inhibition of hiv replication by a chimera containing an rna aptamer with high binding affi nity to the hiv envelop protein gp120 and virus neutralization properties and a small interfering rna (sirna) triggering sequence-specifi c degradation of hiv rnas [ 140 ] . moreover, a preliminary study on mechanisms underlying viral controlling in hla-b*57 elite controller or suppressor (es) was completed in humanized blt mice and demonstrated that elite suppressors are capable of controlling hiv-1 due to the possession of unique host factors rather than infection with defective virus in vivo [ 141 ] . nowadays, we could even make in-depth study on the cell dynamics in hiv-infected humanized mice model with the help of intravital microscopy [ 142 ] . therefore, it is countable that the future molecular biology will bring more surprise to the efforts of gene therapy against hiv [ 143 ] . except for application in hiv-related studies, humanized mouse model also brought span-new opportunities for other human infectious diseases [ 144 ] , especially those blood-borne pathogencaused diseases such as dengue virus infection [ 145 -149 ] , ebv infection [ 150 -155 ] , hcmv infection [ 156 ] , htlv infection [ 157 ] , and malaria parasite infection [ 158 ] . on the other hand, humanized mouse models for leishmaniasis [ 159 ] , salmonella typhi infection [ 160 , 161 ] , herpesvirus infection [ 162 , 163 ] , mycobacteria infection [ 164 , 165 ] , and group b streptococcus (gbs) infection [ 166 ] have been established. these efforts fi ll in the lacks of suitable animal models for those human-specifi c pathogen-caused diseases and push forward the correlating investigations on development of prevention and treatment , although some technological obstacles like the replication of natural infection and transmission routes are still needed to resolved. in 2011, our group used pbmc-transplanted humanized mouse model to evaluate a novel therapeutic strategy by targeting the host rather than the virus for treating infl uenza virus infection. we demonstrated that aminobisphosphonate can control infl uenza disease through boosting human vγ9vδ2-t cell immunity and this benefi cial effect is active against viruses of varying subtypes and virulence [ 43 ] . nevertheless, differences in the characteristics of molecules, tissues, and organs between human and mice might impair effi ciency of pathogen infection and initiation of specifi c immune responses [ 167 ] . in 2005, lassning et al. increased the susceptibility of mice on human coronavirus by crossing aminopeptidase n (apn), the receptor for human coronavirus (hcov)-229e, and transgenic mice into signal transducer and activator of transcription (stat)-1 null mice [ 168 ] . this work, together with hla-and human cytokines/growth factor-transgenic technology [ 169 ] , provided successful examples for future studying human infectious agents in humanized mice. in the next stage, improvement of versatility and variability of human immune system in humanized mouse model and application of gene-modifi ed pathogens [ 170 ] will defi nitely enhance translational effi ciency of these models. the usage of humanized mice in the development of vaccines targeting human diseases including ebv, hiv-1, dengue virus, infl uenza virus, severe acute respiratory syndrome (sars) corona virus, and carcino-embryonic antigen (cea) has obtained outstanding achievements during the past decade, while the introduction of hla transgenic immunodefi cient mice further accelerated the advancement in this fi eld [ 171 -173 ] . with the improvement of immune cell population reconstitution, more and more novel vaccination protocol will be carried out in humanized mice. compared to conventional mice and non-human primate model, humanized mice exhibit great advantage in translational potential, reproductive capacity and data repeatability, economical and ethical concerns. the increasing applications of diverse humanized mice models in biomedical research during the past two decades significantly improved our understanding on human physiological and pathological, especially immunological process at systemic, cellular, and molecular levels. this further accelerated the development of current translational medicine signifi cantly. nevertheless, there are several major caveats on their development remain to be dealt with, including complete replacement of murine mhc with diversifi ed hla molecules and effi cient methodology to express corresponding growth factors and cytokines at specifi c time and organs [ 174 ] ; how to prolong the maintenance of human engraftment, promote the development of myeloid cells and increase relatively weak quantity and quality of immune cells [ 175 ] ; and the limited development of lymph nodes, inter-organ traffi c of immune cells, and the reconstitution of red blood cells and granulocytes [ 176 ] . in another word, the most important issue is to fi nd the convenient and cost-effective ways to construct appropriate human-like microenvironment including physical structure, intercellular contact and molecular signals transfer in humanized mice. it is foreseeable that knowledge exchange in the age of big data will bring an even more bright future to this advancing tool than ever. analysis of candidate human blood stem cells in "humanized" immune-defi ciency scid mice humanized mice in translational biomedical research mighty mice. scientists are still improving the humanized mouse model but are optimistic about its future role in evaluating aids vaccine candidates humanized mice as a preclinical tool for infectious disease and biomedical research hla transgenic mice as humanized mouse models of disease and immunity prevention of "humanized" diabetogenic cd8 t-cell responses in hla-transgenic nod mice by a multipeptide coupled-cell approach cd8 t cell responses to myelin oligodendrocyte glycoprotein-derived peptides in humanized hla-a*0201 humanized mice for studying human leukocyte integrins in vivo full reconstitution of human platelets in humanized mice after macrophage depletion human t cell development in the liver of humanized nod/scid/ il-2rgamma(null)(nsg) mice generated by intrahepatic injection of cd34(+) human (h) cord blood (cb) cells systemic human t cell developmental processes in humanized mice cotransplanted with human fetal thymus/ liver tissue and hematopoietic stem cells expression of hla class ii molecules in humanized nod. rag1ko.il2rgcko mice is critical for development and function of human t and b cells insuffi cient interleukin-12 signalling favours differentiation of human cd4(+) and cd8(+) t cells into gata-3(+) and gata-3(+) t-bet(+) subsets in humanized mice activation of notch1 promotes development of human cd8(+) single positive t cells in humanized mice development of human cd4+foxp3+ regulatory t cells in human stem cell factor-, granulocytemacrophage colony-stimulating factor-, and interleukin-3-expressing nod-scid il2rgamma(null) humanized mice human b cell development and antibody production in humanized nod/scid/il-2rgamma(null) (nsg) mice conditioned by busulfan co-transplantation of fetal bone tissue facilitates the development and reconstitution in human b cells in humanized nod/scid/il-2rgammanull (nsg) mice humanized immune system (his) mice as a tool to study human nk cell development characterization and il-15 dependence of nk cells in humanized mice gm-csf and il-4 stimulate antibody responses in humanized mice by promoting t, b, and dendritic cell maturation induction of functional human macrophages from bone marrow promonocytes by m-csf in humanized mice flt3-ligand treatment of humanized mice results in the generation of large numbers of cd141+ and cd1c+ dendritic cells in vivo inhibition of megakaryocyte development in the bone marrow underlies dengue virus-induced thrombocytopenia in humanized mice a novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure syndrome and in vivo in humanized mice humanized mice as a model to study human hematopoietic stem cell transplantation generation of humanized mice susceptible to peptideinduced infl ammatory heart disease humanized mice as a model for rheumatoid arthritis humanized nod/ ltsz-scid il2 receptor common gamma chain knockout mice in diabetes research humanized mice for the study of type 1 diabetes and beta cell function role of hla class ii genes in susceptibility/resistance to infl ammatory arthritis: studies with humanized mice humanized mice for the study of type 1 and type 2 diabetes subclinical cns infl ammation as response to a myelin antigen in humanized mice humanized scid mice models of sle anti-proteinase 3 antineutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system targeted activation of human vgamma9vdelta2-t cells controls epstein-barr virus-induced b cell lymphoproliferative disease humanized nod-scid il2rg-/-mice as a preclinical model for cancer research and its potential use for individualized cancer therapies the utility of the new generation of humanized mice to study hiv-1 infection: transmission, prevention, pathogenesis, and treatment generation of hiv latency in humanized blt mice can humanized mice refl ect the complex pathobiology of hiv-associated neurocognitive disorders? blt humanized mice as model to study hiv vaginal transmission hiv-1 infection of hematopoietic progenitor cells in vivo in humanized mice mucosal hiv-1 transmission and prevention strategies in blt humanized mice hiv therapy by a combination of broadly neutralizing antibodies in humanized mice inhibition of in vivo hiv infection in humanized mice by gene therapy of human hematopoietic stem cells with a lentiviral vector encoding a broadly neutralizing anti-hiv antibody inhibitory effect of hiv-specifi c neutralizing iga on mucosal transmission of hiv in humanized mice hiv-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice broadly neutralizing antibodies and viral inducers decrease rebound from hiv-1 latent reservoirs in humanized mice humoral immune responses in humanized blt mice immunized with west nile virus and hiv-1 envelope proteins are largely mediated via human cd5+ b cells long-acting nanoformulated antiretroviral therapy elicits potent antiretroviral and neuroprotective responses in hiv-1-infected humanized mice humanized mice recapitulate key features of hiv-1 infection: a novel concept using long-acting antiretroviral drugs for treating hiv-1 minocycline attenuates hiv-1 infection and suppresses chronic immune activation in humanized nod/ltsz-scidil-2rgamma(null) mice vectored immunoprophylaxis protects humanized mice from mucosal hiv transmission pre-clinical modeling of ccr5 knockout in human hematopoietic stem cells by zinc fi nger nucleases using humanized mice a topical microbicide gel formulation of ccr5 antagonist maraviroc prevents hiv-1 vaginal transmission in humanized rag-hu mice expansion of activated memory cd4+ t cells affects infectivity of ccr5-tropic hiv-1 in humanized nod/ scid/jak3null mice hiv-specifi c cd8(+) t-cell immunity in humanized bone marrow-liver-thymus mice rapid evolution of hiv-1 to functional cd8(+) t cell responses in humanized blt mice effi cient infection, activation, and impairment of pdcs in the bm and peripheral lymphoid organs during early hiv-1 infection in humanized rag2(-)/(-) gamma c(-)/(-) mice in vivo plasmacytoid dendritic cells suppress hiv-1 replication but contribute to hiv-1 induced immunopathogenesis in humanized mice in vivo blockade of the pd-1 receptor suppresses hiv-1 viral loads and improves cd4+ t cell levels in humanized mice pd-1 blockade in chronically hiv-1-infected humanized mice suppresses viral loads engineering hiv-1-resistant t-cells from short-hairpin rnaexpressing hematopoietic stem/progenitor cells in humanized blt mice an aptamer-sirna chimera suppresses hiv-1 viral loads and protects from helper cd4(+) t cell decline in humanized mice hla-b*57 elite suppressor and chronic progressor hiv-1 isolates replicate vigorously and cause cd4+ t cell depletion in humanized blt mice intravital microscopy in blt-humanized mice to study cellular dynamics in hiv infection gene therapy strategies for hiv/aids: preclinical modeling in humanized mice humanized mice for modeling human infectious disease: challenges, progress, and outlook mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice utility of humanized blt mice for analysis of dengue virus infection and antiviral drug testing enhanced humoral and hla-a2-restricted dengue virus-specifi c t-cell responses in humanized blt nsg mice dengue virus infection induces broadly cross-reactive human igm antibodies that recognize intact virions in humanized blt-nsg mice dengue virus tropism in humanized mice recapitulates human dengue fever ebna3b-defi cient ebv promotes b cell lymphomagenesis in humanized mice and is found in human tumors a novel animal model of epstein-barr virus-associated hemophagocytic lymphohistiocytosis in humanized mice epstein-barr virus induces erosive arthritis in humanized mice t cells modulate epstein-barr virus latency phenotypes during infection of humanized mice reproduction of epstein-barr virus infection and pathogenesis in humanized mice adoptive transfer of ebv specifi c cd8+ t cell clones can transiently control ebv infection in humanized mice hcmv infection of humanized mice after transplantation of g-csf-mobilized peripheral blood stem cells from hcmv-seropositive donors adult t-cell leukemia/lymphoma development in htlv-1-infected humanized scid mice of men in mice: the success and promise of humanized mouse models for human malaria parasite infections leishmania major infection in humanized mice induces systemic infection and provokes a nonprotective human immune response humanized nonobese diabetic-scid il2rgammanull mice are susceptible to lethal salmonella typhi infection humanized mice for salmonella typhi infection: new tools for an old problem modeling of human herpesvirus infections in humanized mice human herpesvirus 6a infection and immunopathogenesis in humanized rag2(-)/(-) gammac(-)/(-) mice engrafted human cells generate adaptive immune responses to mycobacterium bovis bcg infection in humanized mice cd4+ cell-dependent granuloma formation in humanized mice infected with mycobacteria humanized mice, a new model to study the infl uence of drug treatment on neonatal sepsis humanized mice for the study of infectious diseases development of a transgenic mouse model susceptible to human coronavirus 229e new generation humanized mice for virus research: comparative aspects and future prospects infectious diseases in humanized mice use of humanized severe combined immunodeficient mice for human vaccine development human immune responses and potential for vaccine assessment in humanized mice broad infl uenza-specifi c cd8+ t-cell responses in humanized mice vaccinated with infl uenza virus vaccines humanized mice: are we there yet? humanized mice: current states and perspectives humanized mice for immune system investigation: progress, promise and challenges this work was supported in part by the area of excellence program supported by the university grants committee of the hong kong sar, china (aoe/m-12/06 and aoe/m-06/08). key: cord-103837-iuvigqdx authors: knierman, michael d.; lannan, megan b.; spindler, laura j.; mcmillian, carl l.; konrad, robert j.; siegel, robert w. title: the human leukocyte antigen class ii immunopeptidome of sars-cov-2 spike glycoprotein date: 2020-11-13 journal: nan doi: 10.1016/j.celrep.2020.108454 sha: doc_id: 103837 cord_uid: iuvigqdx the precise elucidation of the antigen sequences for t-cell immunosurveillance greatly enhances our ability to both understand and modulate humoral responses to viral infection or active immunization. mass spectrometry is used to identify 526 unique sequences from sars-cov-2 spike glycoprotein extracellular domain in a complex with human leukocyte antigen class ii molecules on antigen presenting cells from a panel of healthy donors selected to represent a majority of allele usage from this highly polymorphic molecule. the identified sequences span the entire spike protein and several sequences are isolated from a majority of the donors sampled indicating promiscuous binding. importantly, many peptides derived from the receptor binding domain used for cell entry are identified. this work represents a precise and comprehensive immunopeptidomic investigation with sars-cov-2 spike glycoprotein and allows detailed analysis of features which may aid vaccine development to end the current covid-19 pandemic. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a positive-sense single-strand rna virus that is a novel member of the genus betacoronavirus family coronaviridae responsible for the coronavirus disease 19 that emerged in china late 2019 and became a global pandemic by march 2020. as of date of manuscript preparation, over 34,000,000 cases with more than 1,000,000 fatalities have been reported worldwide (https://coronavirus.jhu.edu/). there are four additional members of the betacoronavius genus; two (hcov-hku1 and hcov-oc43) that cause mild respiratory symptoms associated with the common cold and two (sars-cov and middle east respiratory syndrome-cov) that can cause fatal respiratory tract infections. the sars-cov-2 genomic sequence shares 79.6% identity with sars-cov and 96.2% identity with sarsr-covratg13 isolated from bats supporting a zoonotic origin (zhou et al., 2020a) . at present, no therapeutic interventions to treat or prevent covid-19 have been approved for use (https://milkeninstitute.org/covid-19-tracker). active immunization is an area of intense research with over 100 programs under development and the successful implementation would greatly aid in ending the current pandemic (https://www.who.int/who-documents-detail/draft-landscape-of-covid-19-candidate-vaccines). immunization strategies include the use of live-attenuated virus, inactivated virus, non-replicating viral vector, protein subunit, and nucleic acid-based approaches. at the time of manuscript preparation, at least 43 candidate vaccines have progressed into clinical evaluation. an effective immunization would prevent viral entry into cells. the surface spike glycoprotein is the defining feature of all coronaviruses and is critical for internalization by engaging the host receptor and mediation of virus-host membrane fusion (cavanagh, 1995) . the sars-cov-2 spike protein, as with sars-cov, interacts with human angiotensin-converting enzyme 2 (ace2) (zhou et al., 2020b) , (walls et al., 2020) , (li et al., 2003) . the transmembrane glycoprotein is comprised of two functional subunits and forms homotrimers on the viral cell surface. a defined receptor binding domain (rbd) within the s1 subunit is responsible for binding to ace2 , (li et al., 2005) . the s2 subunit in all coronaviruses contains 2 heptad repeat segments that form a coiled-coil structure and membrane fusion occurs after proteolytic processing and conformational rearrangement (liu et al., 2004) , (millet and whittaker, 2015) . a 4 amino acid polybasic furin cleavage site insertion between the s1 and s2 subunits is a unique feature within the sars-cov-2 spike protein (coutard et al., 2020) , (walls et al., 2020) . efficacious sars-cov-2 vaccine development is dependent on a robust humoral immune response targeting the spike glycoprotein, and j o u r n a l p r e -p r o o f perhaps more specifically the rbd assuming similar results as was observed for sars-cov (buchholz et al., 2004) . a central feature of the adaptive immune response is the presentation of immunogenic peptides for immunosurveillance by cd4+ helper t-cells. computational prediction of t-cell epitope candidates has been applied for vaccine discovery and removal of unwanted immune responses against protein therapeutics (griswold and bailey-kellogg, 2016) . current knowledge of peptide binding motifs is based primarily on data generated using biochemical binding assays (justesen et al., 2009) , (sidney et al., 2013) which are compiled in the immune epitope database (iedb) (vita et al., 2019) . this information is used to train prediction algorithms, such as tepitool resource in iedb (paul et al., 2016) and netmhciipan4 (reynisson et al., 2020) . recently this approach was applied to identify known epitopes from multiple coronaviruses and predict likely b-and t-cell epitopes from several sars-cov-2 proteins and reactive cd4+ t-cells obtained from covid-19 patients were observed when stimulated using a "megapool" comprised of overlapping synthetic 15-mer peptides spanning the entire spike protein sequence (grifoni et al., 2020b) . however, the precise sequences from the sars-cov-2 spike glycoprotein for cd4+ t-cell activation is lacking, and a clear understanding will benefit vaccine development. mass spectrometry-based approaches, often termed immunopeptidomics, have been developed to examine the repertoires of peptides presented by human leukocyte antigen (hla) molecules of the class i or class ii major histocompatibility complex (mhc) used for immunosurveillance by cd8+ or cd4+ tcells, respectively (hunt et al., 1992a) (hunt et al., 1992b) . hla class ii molecules are restricted to antigen presenting cells (apcs) and play an essential role in the development of a humoral adaptive immune response via activation of cd4+ t-helper cells. the hla-ii peptide repertoire is a product of extracellular proteins that are proteolytically processed in the lysosomal compartment after internalization. hla-ii immunopeptidomics has been applied to understand the cd4+ t-cell epitopes for potential vaccine design from pathogens such as mycobacterium tuberculosis (bettencourt et al., 2020) , vaccinia virus (strug et al., 2008) , (lorente et al., 2019) , measles virus (ovsyannikova et al., 2003) and human herpes virus 6b (becerra-artiles et al., 2019). these approaches have been typically limited in scope, restricted to one or two cell lines, and thus sampling only a very limited subset of hla-dr alleles. mhc-associated peptide proteomics (mapps) is a specific extension of hla-ii immunopeptidomics that incorporates the intentional pulsing of dendritic cells (dcs) with an antigen or protein of interest (rohn et al., 2005) . more recently, hla-ii mapps has been implemented to investigate and understand the mechanisms of treatment-emergent immunogenicity for biotherapeutic proteins (cassotta et al., 2019) , j o u r n a l p r e -p r o o f (hamze et al., 2017) , (jankowski et al., 2019) , (sekiguchi et al., 2018) as this approach allows for the facile interrogation of the immunogenic potential from multiple hla class ii alleles. in the present study we sought to identify the naturally processed and presented immunopeptidome of the sars-cov-2 spike glycoprotein from human antigen presenting cells. dcs from a panel of healthy human subjects representing a large percentage of the hla-drb1 allele usage within the united states were treated with recombinant spike glycoprotein extracellular domain (ecd). a subset of donors was also selected to represent common alleles from the asia-pacific geography. hla-ii associated peptides were identified by liquid chromatography and nanoelectrospray ionization tandem mass spectrometry after immunoprecipitation. we observed several clusters, or nested sets of peptides, derived from every domain of the sars-cov-2 spike glycoprotein. we determined the prevalence of these clusters among multiple donors. finally, we sought to compare our observed hla-ii epitopes with those from recent a in silico prediction (grifoni et al., 2020a) and determine regions that are conserved with sars-cov spike protein sequence. the mapps method intentionally pulses human dcs from a panel of donors with a protein of interest. the 4 male and 5 female healthy donors, ranging in age from 21 to 57 years old, used in this study were selected to sample approximately 53% and 46% of the hla-ii drb1 allele frequency from the united states and asian-pacific geographic regions, respectively (table 1) . full hla typing of the donors is also available (supplemental table s1 ). the donors' pbmcs were collected and stored frozen before the outbreak of the sars-cov-2 pandemic and are expected to be unexposed to potential infection. the sequence used for this work was derived from the sars-cov-2 wuhan-hu-1 strain spike glycoprotein ecd spanning residues 1-1213 taken from genbank accession yp_009724390. an r685a mutation was used to prevent cleavage during recombinant protein production with affinity tags added for purification. the recombinant protein was produced from mammalian cells and is expected to have nlinked glycosylation modifications. the method used to profile the hla-ii peptides is outlined in figure 1a . monocyte-derived dcs were generated in culture with a cytokine cocktail. the immature dcs were treated with sars-cov-2 spike glycoprotein ecd and after 24 hours, lipopolysaccharide (lps) was added to mature the dcs. the treated cells were lysed and the hla class ii complex was isolated by immunoprecipitation with a pan-hla class ii antibody. the bound hla-ii peptides were eluted after acidification, filtered to remove high molecular weight co-precipitants, and analyzed by capillary hplc on an orbitrap mass spectrometer. peptide ions were fragmented using multiple fragmentation techniques. peptides were identified using multiple proteomic search engines with a forward and reverse database search. false discovery rate estimates (q-values) were estimated using a null distribution from the reverse database search. the peptides identified from the spike glycoprotein had q-values below 0.07 and all fragmentation spectra matching the spike protein were manually reviewed (see methods for details). a total of 876 hla-ii peptides from the sars-cov-2 spike glycoprotein were identified from the donor panel (supplemental table s2 ). several peptides with identical sequences were identified from multiple donors. removal of these duplicate peptides resulted in 526 unique sequences. in order to minimize both false positive and negative peptide identifications, the database used for peptide identification was intentionally constructed to contain the spike protein along with approximately 2000 background bovine and human proteins previously observed from multiple samples analyzed with this assay system. we also analyzed the data using multiple search engines against a database containing the entire human proteome (downloaded 05apr19) that also included the sars-cov-2 spike glycoprotein and did not find any human protein identifications for the 526 unique sars-cov-2 spike peptides. the primary mass spectrometry data is publicly available for individual analysis (doi:10.25345/c51m7p). the unique peptide sequences had a distribution of lengths consistent with hla-ii peptides with a mean length of 15 residues ( figure 1b ) (kampstra et al., 2019) . there were 169 unique peptides that had modified residues. the modifications observed were consistent with hla-ii peptide processing. the spike protein is heavily glycosylated with 22 putative n-glycosylation sites, presumably to help evade immune detection, and the majority of the regions from the spike ecd not observed were centered around these sites. current limitations of our method prevent detection of glycosylated peptides if, in fact, they were processed and loaded onto hla class ii molecules. interestingly, hla-ii peptides from 3 putative n-linked glycosylation sites were observed which indicates these regions were not modified. we did identify a deamidation modification with the asparagine residue at residue 1098 with our search engines (supplemental table s2 ). this modification is consistent with the conversion of asparagine to j o u r n a l p r e -p r o o f aspartic acid after removal of n-linked glycosylation and is indirect evidence of glycosylation of this residue in the intact protein (khoshnoodi et al., 2007) . every donor examined produced hla-ii peptides derived from the sars-cov-2 spike glycoprotein ( figure 1c ). the number of spike peptides observed per donor ranged from 9 to 203 with a median value of 91 peptides. one donor (40146) did not efficiently produce mature dcs and resulted in a low overall number of peptides relative to all other donors analyzed. repeated analysis with this donor also yielded poor results (data not shown). hla-ii peptides are distributed across entire sars-cov-2 spike protein extracellular domain and have consensus hla-ii clusters hla-ii peptides derived from the sars-cov-2 spike glycoprotein were aligned to the ecd sequence and a peptide density map was generated to help visualize both the breadth and depth of sequence coverage. a schematic listing of the various subunits of the spike glycoprotein ecd and the results obtained with each donor are shown on individual rows ( figure 2 ). the shades of red correspond to the number of overlapping peptides encompassing a given amino acid position. from this presentation of the data, it is evident that hla-ii peptides spanning all segments of the spike glycoprotein ecd were obtained from all donors sampled. the heatmap also reveals that hla-ii peptides from several regions of the spike glycoprotein were observed from multiple donors likely reflecting the more promiscuous hla class ii binding epitopes. an expanded view of the rbd encompassing residues 319-541 is shown in the bottom panel of figure 2 and clearly demonstrates promiscuous epitopes are also contained in this region of the molecule critical for interaction with ace2 cell receptor. groups of nested peptides sharing a common core but with ragged n-and c-termini are generated from the multiple proteases and different temporal patterns of processing that occur in the lysosomal compartment (lippolis et al., 2002) . in order to organize the observed hla-ii peptides from the spike protein into discrete segments for subsequent analysis, we used the iedb epitope cluster analysis tool 1.0 with some manual adjustments as noted in star methods to group these into 73 distinct clusters (supplemental table s3 ). clusters were characterized from both full span and minimal overlap perspectives. the full cluster sequence represents the first start position of the peptides in the cluster to the last position of the peptides in the cluster. the minimum cluster sequence is the smallest common sequence among the peptides in the cluster. clusters with minimum cluster sequences less than 9 residues likely contain 2 overlapping binding cores, which due to their proximity, were unable to be easily separated. as expected, we observed a distribution of the clusters among the donors ( figure 3 ). most of the clusters were observed from 4 or fewer donors and were designated as restricted; whereas, 11 clusters were observed from 5 -7 donors and were designated as consensus ( figure 3a and table 2 ). this arbitrary definition of a consensus cluster was set to reflect those sequences observed in at least 50% of the donors sampled in the study. consensus clusters represent those sars-cov-2 spike glycoprotein sequences with the most promiscuous, but not necessarily highest affinity, binding to the broadest range of hla class ii alleles. no specific cluster was observed in all donors sampled in this study. the median number of clusters per donor was 18 and all donors displayed at least one of the consensus clusters ( figure 3b ). we next examined the distribution in the number of peptides within both restricted and consensus clusters ( figure 3c ). the vast majority of clusters contained less than 10 peptides with most, but not all, of the consensus clusters having 11 or greater members. interestingly, consensus cluster 1, observed in 5 donors, was comprised of a single peptide sequence in the s1 subunit (table 2) . finally, we examined the location of the clusters within discrete segments of the sars-cov-2 spike glycoprotein ( figure 3d ). the clusters were distributed throughout the protein. all regions contained at least one hla-ii peptide cluster with consensus clusters occurring in several different regions. of note, the rbd region responsible for binding ace2 and a target for vaccination strategies contained a total of 16 clusters in which 3 were consensus (table 3) . observed sars-cov-2 spike protein hla-ii peptides have limited overlap with predicted cd4+ t-cell epitopes cd4+ t-cell epitopes from the spike glycoprotein derived from an algorithm designed to predict the dominant hla class ii peptides was recently published (grifoni et al., 2020a) . we sought to compare those predictions with the results from our study. we used the minimum observed cluster sequence for our comparison and considered an overlap of at least 9 residues within the predicted 15 residue peptide sequence as a match with one exception as denoted below. we chose this minimum overlap length based on the number of residues contained within hla class ii peptide binding cleft. we chose to use the minimum cluster sequence in an attempt to reduce matches on the extreme peripheries of the observed peptides that likely do not reflect the likely binding core contained within the cluster. a perfect congruence between prediction and observed results from this donor set would result in 19 matches as one of the predicted peptides resides in the transmembrane portion which was absent from the protein used in this study. in contrast, we observed hla-ii peptide clusters from our donor set that j o u r n a l p r e -p r o o f matched a total of 9 predicted epitopes ( figure 4a and supplemental table s4 ). cluster 27 was deemed to match a predicted epitope located from residues 451 -465 (ylyrlfrksnlkpfe) in the rbd domain even though only a 5-residue overlap was observed using the criteria defined above. this particular cluster was composed of 37 unique sequences, the most out of all the clusters, and likely contains two closely overlapping allele binding sites. since 25 of the observed peptides from this cluster matched the first 9 residues of the predicted peptide, we included this region as overlapping with the prediction (supplemental table s2 for cluster details). we also note two instances in which multiple clusters were deemed to sufficiently overlap with a predicted peptide which skews the venn diagram in figure 4a to show 71 instead of the 73 total observed clusters. comparison of the observed consensus clusters with the predicted peptides reveals that only 2 of the 11 observed promiscuous hla-ii binding regions were predicted (table 2 ). this finding is intriguing given the prevalence of display of these particular sequences and may reflect limitations in the alleles used to generate the predictions and/or the prediction algorithm. the rbd of the sars-cov-2 spike glycoprotein contained 16 observed hla-ii clusters and 4 predicted peptides. we observed 3 of the 4 predicted epitopes (table 3 and supplemental table s3 ). the only predicted peptide that was not observed contained a putative n-linked glycosylation site. as denoted above, our mapps method is unlikely to identify a peptide with this modification. in fact, 6 of the 20 predicted peptides contain a putative nlinked glycosylation site (supplemental table s4 ) possibly reflecting the fact that current predictive algorithms do not consider post-translational modifications. in addition, the glycosylated asparagine residue in many of the predicted peptides is centrally located rather than on the periphery and large complex glycan structures have been shown to interfere with hla or t-cell receptor binding (speir et al., 1999) , (kario et al., 2008) , (malaker et al., 2017) . also striking is the overall number of observed clusters (62), many obtained from multiple donors, which were not predicted. it is worth noting that the algorithm used to predict sars-cov-2 spike protein epitopes is largely trained on hla binding affinity data which do not reflect the authentic processing captured by mapps assay. further expansion of mapps-derived data into these training sets will likely be beneficial. the list of unique peptides for each hla class ii cluster was compared with the spike glycoprotein sequence from sars-cov protein (uniprot accession p59594). the non-identical residues between the two sequences are denoted in red under the spike glycoprotein schematic in figure 4b . the s2 and s2' regions had larger areas of identity between the sequences. the 526 unique hla-ii peptides identified in this study were analyzed for sequence identity with the sars-cov spike glycoprotein. no identical matches were identified in the s1 subunit. however, 14 clusters match the sars-cov sequence in the s2 subunit. interestingly, one of the matches, cluster 49, was a consensus cluster (denoted with blue figure 4b ). these clusters represent sequence regions from both viruses that are potentially presented by hla class ii molecules for t-cell surveillance. the spike proteins from the other coronaviruses known to infect humans (nl63, 229e, hku1, oc43, and mers) were also evaluated and no matches were found. to look for potential cross-reactivity to human proteins, each unique identified peptide from the sars-cov-2 spike glycoprotein was searched against the uniprot human database for an exact match to any human protein. none of the observed peptides had a sequence match (data not shown). finding no matches, we expanded our search to include up to 2 mismatched amino acids without insertions or deletions. a single peptide could be associated with 13 human proteins if 2 residues of non-identity are allowed. these results indicate that the risk from direct sequence cross-reactivity is minimal and any portion of the sars-cov-2 spike glycoprotein associated with hla class ii molecule is unlikely to be subject to previous tolerization in a vaccinated subject or infected patient. however, we do acknowledge that our analysis does not consider cross-reactivity when strictly limited to putative t cell contact residues given the difficulty to reliably predict such registers. cd4+ t-cell participation is vital for a robust humoral response to viral infection or active immunization. a clear delineation of the epitopes presented by apcs for t-cell immunosurveillance greatly enhances our understanding of this process. generally, t-cell lines or pbmcs from recovered patients using peptides derived simply by spanning the entire protein(s) of interest or from hla-ii prediction algorithms have been utilized (meunier et al., 2019) , (grifoni et al., 2020b) , (schulze zur wiesch et al., 2005) . the ability to automate and miniaturize the mapps assay enables facile identification of 1000's of naturally processed and displayed hla-ii peptides from human dcs. using this approach, we were able j o u r n a l p r e -p r o o f to determine the precise regions and sequences of peptides from sars-cov-2 spike glycoprotein ecd derived from a panel of healthy subjects presented for immune surveillance by t-cells. the 9 subjects used in this study enabled sampling of approximately 53% and 46% of the hla-drb1 allele frequency from the united states and asian-pacific geographic regions, respectively (table 1) . this work represents, to our knowledge, the most precise and comprehensive immunopeptidomic investigation with sars-cov-2 spike glycoprotein performed to date and allows detailed analysis of features which may aid vaccine development. we observed a total of 526 unique peptide sequences contained within 73 clusters distributed across each segment of the sars-cov-2 spike glycoprotein ecd presented by human dcs (figure 2 and supplemental table s2 ). two of the clusters were in regions that deviated from the reference sequence. one region was the s1/s2 cleavage site in which the novel furin site was eliminated with mutation to enable production of full-length recombinant protein. we speculate that this region of the spike protein containing the native residue could also be presented from those molecules that are not cleaved during virion particle assembly. the other area of deviation was the c-terminal affinity tags used for purification. of particular interest are those peptides from the spike glycoprotein that are presented by multiple donors as these would be sequences likely to elicit a t-cell response from the greatest number of patients or vaccinated subjects. we observed 11 consensus clusters, defined as being present in 5 or more of the 9 donors analyzed in this study, including 3 within the rbd which is essential for binding to ace2 on host cells (table 3) . a majority of the consensus clusters contained 11 or more nested peptides. in the absence of a dedicated assay to quantify the presented hla-ii peptides, we use this metric as a surrogate for peptide abundance but recognize that even a single specific peptide sequence can be presented in sufficient number to elicit a t-cell response. recent reports leveraging either bioinformatics to predict (grifoni et al., 2020a) or a single-pot peptide pools composed of >150 overlapping peptides spanning the entire open reading frame (grifoni et al., 2020b) , (braun et al., 2020) have been published attempting to elucidate sars-cov-2 t-cell epitopes. unfortunately, the latter approach does not allow any insights into the precise sequences capable of eliciting a response. comparisons of the clusters we observed being presented by apcs reflecting the natural processing hla-ii loading processes with the predicted epitopes is illuminating. one of the predicted epitopes resides in the transmembrane domain which was absent from the protein used for our analysis and omitted from further discussion. of the remaining predictions, roughly 50% (9 of 19) were observed from our panel of donors selected to represent a sizeable percentage of hla-dr allele j o u r n a l p r e -p r o o f usage from multiple geographies (figure 4 and supplemental table s3 ). correspondingly, the vast majority of the observed 73 hla-ii clusters were not predicted. of particular interest are the consensus clusters that were observed in 5 or more of the donors and would be expected to represent those sequences with the most promiscuous hla class ii binding. only 2 of these 11 consensus clusters were predicted with only 1 of 3 consensus clusters contained in the rbd predicted. of note and expanded in more detail below, consensus cluster 49 in the s2 subunit has 100% sequence identity to sars-cov, was predicted, and has been experimentally shown to be a t-cell epitope (yang et al., 2009 ). the "7-allele method" hla class ii reference set used for generating the predicted epitopes is restricted to select drb1/3/4/5 alleles (http://tools.iedb.org/mhcii/) (paul et al., 2015) . the use of the pan-hla class ii antibody used in our study, which would enrich both hla-dq and hla-dp bound peptides, could explain why some, but certainly not all, of the observed clusters were not predicted. however, given the ample evidence which shows the impact that both hla-dq and hla-dp bound peptides have on t-cell activation for a variety of viral antigens (koelle et al., 1997) , (mellins et al., 1987) , (koelle et al., 1994) , (mellins et al., 1987) , (lorente et al., 2019) , (lorente et al., 2020) , we felt it was important to identify as many of those restricted peptides as possible. nevertheless, this disconnect between promiscuously observed hla class ii clusters and predicted t-cell epitopes accentuates what has been highlighted before that the prediction of t-cell epitopes is an imperfect process that may not reflect what hla class ii molecules preferentially bind (paul et al., 2015) , (wantuch et al., 2020) ) and therefore, not the most effective approach for identifying cd4+ t-cell epitopes. (wantuch et al., 2020) , (strug et al., 2008) , (ovsyannikova et al., 2003) ). mapps has been an instrumental method to preclinically assess the immunogenic potential of protein therapeutics (for review, (quarmby et al., 2018) ). multiple reports analyzing different therapeutic antibodies approved for clinical use have shown that many, but not all, hla-ii clusters identified from these molecules can elicit t-cell responses from both drug naã¯ve donors, as well as, patients who developed treatment emergent anti-drug antibody responses (walsh et al., 2020) , (cassotta et al., 2019) , (hamze et al., 2017) ). also, unlike the immunogenicity assessment of a majority of protein biotherapeutics which are engineered to j o u r n a l p r e -p r o o f a great extent to be recognized as a self-protein and therefore generally present a very limited number of non-germline residues for scrutiny, each sars-cov-2 spike protein cluster is fundamentally distinct from any other sequence in the human genome and extremely unlikely to be subject to any tolerization. the list of unique sars-cov-2 spike glycoprotein peptides identified by mapps searched against the human database (uniprot version 2020_2) did not identify any significant matches. nevertheless, the characterization of the activation potential of the sars-cov-2 spike protein clusters with t-cells from healthy donors and, ideally, convalescent patients should be evaluated. the source of dcs used were derived from healthy donors that, due to time of sample collection, had not been exposed to potential sars-cov-2 infection. therefore, the displayed hla-ii clusters reported here could conceivably deviate from the repertoire obtained from infected individuals. this potential discrepancy would require that the internalization of the virus into apcs would fundamentally alter the proteolysis and/or hla-ii molecule loading mechanism. while examples of hla-ii display interference are known for other viruses that can infect and replicate in immune cells (becerra-artiles et al., 2019), the authors are not aware of that attribute with sars-cov-2 at this time and do not consider this to be a fundamental concern. this could be addressed by repeating this study using material obtained from convalescent patients or infection of dcs from healthy donors with live virus. also, the dcs used in this study are derived from monocytes and could potentially have a different processing mechanism from plasmacytoid or follicular dendritic cell lineages. a detailed comparison in the mapps peptides obtained from different dendritic cell types is lacking given the difficulties in obtaining adequate numbers required for investigation. nevertheless, multiple studies have shown monocyte-derived dcs to be as efficient as antigen-specific b-cells in presenting peptides for t-cell surveillance (cella et al., 1997; sallusto and lanzavecchia, 1994) . insights into the immunogenic potential of sars-cov-2 spike glycoprotein can be made from the results obtained in this study. firstly, both the depth and breadth of the hla-ii peptides derived from this critical structural component for viral infectivity indicate that mutational drift as the pandemic continues to spread around the world is not expected to dramatically alter the ability of an infected individual to mount a new b-cell response to replace those antibodies which would be deleteriously impacted by such escape mutations. in the event that a mutation could result in an inability of a particular cluster to bind most hla class ii alleles, the sheer number of clusters distributed across the spike protein, especially the consensus clusters, make the mutation unlikely to enable the virus to escape cd4+ t-cell activation across a wide portion of the population. secondly, sars-cov spike glycoprotein cross-reactive j o u r n a l p r e -p r o o f hla-ii restricted epitopes seem to be limited to the s2 domain as all 14 of the sars-cov-2 clusters identified in this study with complete sequence identity to sars-cov were derived from this region of the molecule (figure 4b , supplemental table s3 ). this result is not surprising given the 90% sequence identity between the two viruses in s2 but only 60% identity in the s1 domain. no significant homology was noted for any of the other human coronavirus spike protein sequences. interestingly, consensus cluster 49 (observed from 6 donors) was one of the 5 clusters from the s2 subunit with complete identity to sars-cov and has previously been identified as a t-cell epitope from healthy donors using a tetramer guided epitope mapping approach (yang et al., 2009) . this epitope has significant, but not complete, overlap with the hla-ii derived cluster and indicates, as denoted above, that the approach of using overlapping peptides may reflect imperfect identifications from those that arise due to natural apc processing. limitations of this study include that the identified peptides are restricted to those with suitable biophysical characteristics for ionization and compatibility with reverse phase liquid chromatography. the total number and wide breadth of coverage spanning the entire extracellular domain of the spike protein indicate that any false negative results obtained with the method outlined in this study are likely a small minority. another limit is focusing the analysis to the spike glycoprotein to the exclusion of the other structural components of sars-cov-2, the membrane and nucleocapsid proteins. undoubtedly many regions from both of those proteins will also be presented for t-cell surveillance; however, the focus of most immunization strategies seems to target the spike glycoprotein. notwithstanding, the "adjuvant-like" potential of some of these presumed clusters to augment the humoral response to the spike glycoprotein cannot be accounted for with the current results and should be targets of future efforts. additional effort to experimentally identify the actual hla-ii peptides presented from the spike glycoprotein, at a minimum, from all other coronaviruses would prove of interest. the ability to direct humoral immune response to discrete segment(s) of the sars-cov-2 spike glycoprotein that confer viral neutralization may potentially enable higher protective titers to be achieved with vaccination and limit antibody-dependent enhancement of infection as reported with other coronaviruses ((tseng et al., 2012) , ). a preliminary report in which the 197 amino acid rbd of sars-cov-2 spike glycoprotein was used for immunization in rodents suggests that robust neutralizing response can be obtained without ade (quinlan et al., 2020) . the approach outlined and the results reported in this study can also be applied to developing novel subunit or nucleic acidbased vaccines and/or monitoring response to such vaccines. it also enables the ability to supply the j o u r n a l p r e -p r o o f immune system with synthetic peptide(s) that mirror natural apcs presentation observed from a broad spectrum of the hla class ii alleles from different geographic regions to maximize t-cell responses. the authors grateful acknowledge richard e. higgs, andrea ferrante, and laurent malherbe for critical review and helpful suggestions during preparation of the manuscript. r.w.s dedicates this work to the memory of karen j. haugh. this work was supported by eli lilly and company. the authors declare no competing interests. the donor id is listed on x-axis and the total number of spike protein peptides from that particular donor denoted on the y-axis. see table 1 and supplemental table s1 for donor information and supplemental table s2 for all identified sars-cov-2 spike protein ecd peptides. the various subunits of the spike protein are denoted at top of the schematic. aligned hla-ii presented peptides are displayed as a heatmap with each of the nine donors on an individual row. the shades of red correspond to the number of overlapping peptides encompassing a given amino acid position. the lightest shade represents a single peptide and the darkest red signifies when at least five peptides overlap an amino acid position. the unlabeled yellow region at the n-term corresponds to the signal peptide and the light green region at the c-term corresponds to the affinity tag used for purification. the s1/s2 cleavage site contains an r685a mutation (denoted with an asterisk). the rbd portion of the heatmap was expanded and displayed in the bottom panel along with numerical markers to indicate location within the sars-cov-2 spike protein ecd. data from each donor in the panel was collected from a single biological and technical replicate. see supplemental table s2 for all identified sars-cov-2 spike protein ecd peptides. s1, subunit 1; s2, subunit 2; ntd, n terminal domain; rbd, receptor binding domain; ctd, c terminal domain. (a) prevalence of peptide clusters across donors. the number of donors from which each of the 73 peptide clusters were observed are shown. the x-axis denotes the number of donors in which a cluster was observed, and the y-axis denotes the number of clusters observed from that particular number of donors. for example, 31 clusters were observed from any given single donor, another 10 clusters were observed from 2 of the 9 donors in the panel, and so forth. peptide clusters that were identified in at least 5 donors were deemed "consensus" and colored red, whereas clusters seen in 4 or less donors are characterized as "restricted" and are colored blue. (b) number and type of cluster by donor. the total number of clusters observed for each donor are shown. the y-axis denotes the total number of clusters from the particular donor listed on the x-axis. clusters designated as restricted or consensus are denoted as blue and red, respectively. the overall median of 18 clusters is indicated with a dashed line. (c) cluster depth. the distribution in the different number of peptides contained within a cluster are shown. the number of peptides within any given cluster are denoted on the x-axis and the number of clusters containing the peptides within the designated bins are denoted on the yaxis. clusters designated as restricted or consensus are denoted as blue and red, respectively. (d) the distribution of the clusters across the spike protein domains. the particular domain is designated on the x-axis and the number of clusters contained with the particular domain denoted on the y-axis. clusters designated as restricted or consensus are denoted as blue and red, respectively. see supplemental table s3 for further cluster details. (a) prediction comparison. the observed sars-cov-2 spike protein clusters were compared to the predicted hla-ii peptides. a venn diagram shows the overlap between the observed minimum cluster sequence and the predicted peptides. to view the details of predicted versus observed clusters, see supplemental table s4 . (b) cluster conservation. the various segments of the sars-cov-2 spike protein are denoted on the top row and sequence mismatches to the sars-cov spike protein are delineated with a red bar in the second row. the third row is a heatmap of the clusters seen from the sars-cov-2 spike protein that contain peptides with an exact sequence match to sars-cov spike protein (darker orange represents overlapping clusters). the blue cluster designates a consensus cluster. s1, subunit 1; s2, subunit 2; ntd, n terminal domain; rbd, receptor binding domain; ctd, c terminal domain further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, robert siegel (siegel_robert@lilly.com). this study did not generate new unique reagents. frozen pbmcs were obtained from 9 informed consent healthy donors (discovery life sciences) according to local ethical practice. protocol was adapted as described (sallusto and lanzavecchia, 1994) with the following modifications. pbmcs were selected from the available inventory to have the broadest hla-drb1 diversity as possible. the cells were lysed on day 6 with ripa lysis and extraction buffer (thermo fisher scientific, cat # 89900, 25 mm trisâ�¢hcl ph 7.6, 150 mm nacl, 1% np-40, 1% sodium deoxycholate, 0.1% sds) containing 1:1000 of 10 unit/ul dnase (roche, cat# 04716728001) and 1 tablet of edta free protease inhibitors (roche, cat# 11836170001) per 10 ml of lysis buffer. the lysates were frozen at -80â°c. an agilent assaymap robot was used to isolate the hla-ii molecules in the lysate. one hundred microliters of 1 mg/ml biotinylated anti-pan hla class ii antibody (in house produced tu39 clone) was immobilized on streptavidin cartridges (agilent, sa-w, 5 ul) by passing over the cartridge at 5 ul/min. the cartridge was washed with 50 ul of pbs three times. lysates were thawed, passed over a 0.45 um filter, and 1 ml of each sample was loaded onto a 96 well polypropylene plate. the lysate was aspirated into the syringes and the antibody loaded cartridge is attached to the syringe tip. the lysate is passed over the affinity cartridge at 5 ul/min at room temperature for 200 minutes. the cartridge is washed 2 x 50 ul with 100mm ammonium acetate at 25 ul/min and once with 50 ul water at 25 ul/min. the cartridge is eluted with 50 ul of 5% acetic acid with 0.1% tfa at 2 ul/min into a 96 well polypropylene pcr plate. the eluted peptides were passed over a 10k mwco spin filter treated with 1 mg/ml bsa (sigma, 05470) and 100ug/ml angiotensin i peptide and washed with 5% acetic acid. the filtered material was loaded in a 96 well polypropylene pcr plate for mass spec analysis. the samples were analyzed with a thermo lumos mass spectrometer using a thermo easy 1200 nlc-hplc system. the separation was carried out with a 75âµm x 7 cm ymc-ods c18 column (new objectives) coupled to a custom nanospray interface with an electrospray potential of 1.2 kv. the solvents were a -0.1% formic acid in water (thermo fisher scientific, optimaâ�¢ lc/ms grade) and b -80% acetonitrile with 0.1% formic acid (thermo fisher scientific, optimaâ�¢ lc/ms grade). the gradient was 65 minutes using a flow rate of 250 nl/min, starting with a 60 min 2-55%b ramp followed by a 1 min 55-100%b ramp and a 4 min hold at 100%b). the lumos was run with a full scan at 240,000 resolution in the orbitrap followed by a 3 second data dependent ms/ms cycle comprised of ion trap rapid scans where +2 ions were fragmented by hcd(ce of 15,22,28) and +3 and +4 ions were fragmented by hcd (ce of 15, 22, 28) and ethcd (calibrated charge-dependent etd parameters and supplemental hcd (ce of 50)). the data were analyzed with the lilly proteomics pipeline (higgs et al. (2008) . the data conditioning steps consisted of extraction from the vendor format, fitting parent ions for data dependent scans to theoretical isotope patterns and correcting the monoisotopic mass and charge of the parent ion, determining the fit of the parent ion isotope to the theoretical isotope pattern and filtering out ms/ms scans if the parent ions did not match the isotope pattern with a score of 0.6 or greater. from the filtered scans, an mgf file was created along with a table of spectral features for each spectrum. the spectral identifications were performed with x! tandem version 2017 and omssa version 2.1.7 search engines. a database was used consisting of the sars-cov-2 spike extracellular domain his-flag tagged protein and 2134 common human and bovine proteins identified from hla-ii bound peptides seen from raji cells, dcs, and bovine proteins in the cell media. the search engine parameters included a no enzyme search with a maximum missed cleavage site setting of 30, 10 ppm tolerance for parent ions, and 0.5 m/z tolerance for the fragment ions. potential amino acid modifications included: cysteine mods of free sh; disulfide; mercaptoethanolation; mono,di, and tri oxidation; and cysteinylation; deamidation of glutamine and asparagine; methionine oxidation; tryptophan oxidation, deoxidation, oxidation to kynurenin. hcd spectra were searched for b-and y-ions and ethcd were searched for c-, z-, b-, and y-ions. false positive identifications were controlled by running the searches against a reversed version of the protein database and estimating false discovery rates. an iterative random forest classifier was trained using search results and spectral features to increase identification sensitivity in a manner similar to the percolator algorithm (kall et al., 2007) . the search results from x! tandem and omssa were pooled and peptides with q-values < 0.20 were assigned to the smallest group of proteins that account for all identified peptides. pepnovo plus (frank and pevzner, 2005) was run on all the spectra with a parent ion tolerance of 10 ppm and a fragment tolerance of 0.5 th. modifications included were methionine oxidation, cysteinylation, and disulfide formation. the output was filtered for peptide tags that matched the sars-cov-2 spike extra cellular domain his flag protein. tag hits were checked against the results from the database search. all tag hits were matched to the database search results indicating that there were no unknown modifications present in the results. the pipeline output was analyzed using knime 3.3 (mazanetz et al., 2012) to merge all the donor search results, manual review of the ms/ms spectra of the identifications to confirm presence of at least 4 contiguous fragment ions to matched peptide sequence, align the peptides to the sars-cov-2 spike extracellular domain his flag protein, and create an excel file with the alignment. the identified peptides from the sars-cov-2 spike glycoprotein were clustered with the iedb epitope cluster analysis tool v1.0 using the default settings. the clustering was manually adjusted to group a continuous amino acid run of at least 9 residues after sorting by donor. this adjustment was reflected in the cluster number as a decimal point after the assigned cluster number from the iedb algorithm. predicted sars-cov-2 spike protein peptides (grifoni et al., 2020a) were matched with the mapps cluster if they shared at least a 9 amino acid overlap to the minimum cluster sequence. the identified hla-ii peptides were checked for exact matches using the unix grep command. the sequences searched were the human database (uniprot version 2020_02), spike proteins from sars-cov (genbank accession # p59594.1), human coronavirus nl63, 229e, hku1, oc43 (genbank accession # qed88040.1, ast12964.1, agw27881.1, aar01015.1), and mers (genbank accession # yp_009047204.1). an imperfect match search was run using the unix command agrep to look for 1 or 2 mismatched amino acids. the searches were done for each peptide with insertion and deletions scores set to 100 to discount any insertion or deletions during the search. the imperfect match search was run against the human database (uniprot version 2020_02). table s2 . hla-ii peptide alignment to sars-cov-2 extracellular domain, related to figure 1 and figure 2 . naturally processed hla-dr3-restricted hhv-6b peptides are recognized broadly with polyfunctional and cytotoxic cd4 t-cell responses identification of antigens presented by mhc for vaccines against tuberculosis sars-cov-2-reactive t cells in healthy donors and patients with covid-19 contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity a single t cell epitope drives the neutralizing anti-drug antibody response to natalizumab in multiple sclerosis patients the coronavirus surface glycoprotein inflammatory stimuli induce accumulation of mhc class ii complexes on dendritic cells the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade pepnovo: de novo peptide sequencing via probabilistic network modeling a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov-2 targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals design and engineering of deimmunized biotherapeutics characterization of cd4 t cell epitopes of infliximab and rituximab identified from healthy donors label-free lc-ms method for the identification of biomarkers characterization of peptides bound to the class i mhc molecule hla-a2.1 by mass spectrometry peptides presented to the immune system by the murine class ii major histocompatibility complex molecule i-ad peptides identified on monocyte-derived dendritic cells: a marker for clinical immunogenicity to fviii products functional recombinant mhc class ii molecules and high-throughput peptide-binding assays semi-supervised learning for peptide identification from shotgun proteomics datasets ligandomes obtained from different hla-class ii-molecules are homologous for n-and cterminal residues outside the peptide-binding cleft n-linked glycosylation does not impair proteasomal degradation but affects class i major histocompatibility complex presentation identification of n-linked glycosylation sites in human nephrin using mass spectrometry antigenic specificities of human cd4+ t-cell clones recovered from recurrent genital herpes simplex virus type 2 lesions preferential presentation of herpes simplex virus t-cell antigen by hla dqa1*0501/dqb1*0201 in comparison to hla dqa1*0201/dqb1*0201 structure of sars coronavirus spike receptor-binding domain complexed with receptor angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus analysis of mhc class ii antigen processing by quantitation of peptides that constitute nested sets interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors the hla-dp peptide repertoire from human respiratory syncytial virus is focused on major structural proteins with the exception of the viral polymerase proteomics analysis reveals that structural proteins of the virion core and involved in gene expression are the main source for hla class ii ligands in vaccinia virus-infected cells identification and characterization of complex glycosylated peptides presented by the mhc class ii processing pathway in melanoma drug discovery applications for knime: an open source data mining platform importance of hla-dq and -dp restriction elements in tcell responses to soluble antigens: mutational analysis impact of human sequences in variable domains of therapeutic antibodies on the location of cd4 t-cell epitopes host cell proteases: critical determinants of coronavirus tropism and pathogenesis naturally processed measles virus peptide eluted from class ii hla-drb1*03 recognized by t lymphocytes from human blood development and validation of a broad scheme for prediction of hla class ii restricted t cell epitopes tepitool: a pipeline for computational prediction of t cell epitope candidates mapps for the identification of immunogenic hotspots of biotherapeutics; an overview of the technology and its application to the biopharmaceutical arena the sars-cov-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement. biorxiv netmhcpan-4.1 and netmhciipan-4.0: improved predictions of mhc antigen presentation by concurrent motif deconvolution and integration of ms mhc eluted ligand data a novel strategy for the discovery of mhc class ii-restricted tumor antigens: identification of a melanotransferrin helper t-cell epitope efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha broad repertoire of the cd4<sup>+</sup> th cell response in spontaneously controlled hepatitis c virus infection includes dominant and highly promiscuous epitopes mhc-associated peptide proteomics enabling highly sensitive detection of immunogenic sequences for the development of therapeutic antibodies with low immunogenicity cell entry mechanisms of sars-cov-2 measurement of mhc/peptide interactions by gel filtration or monoclonal antibody capture crystal structure of an mhc class i presented glycopeptide that generates carbohydrate-specific ctl vaccinia peptides eluted from hla-dr1 isolated from virus-infected cells are recognized by cd4+ t cells from a vaccinated donor immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus the immune epitope database (iedb): 2018 update structure, function, and antigenicity of the sars-cov-2 spike glycoprotein post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms molecular mechanism for antibody-dependent enhancement of coronavirus entry isolation and characterization of new human carrier peptides from two important vaccine immunogens searching immunodominant epitopes prior to epidemic: hla class ii-restricted sars-cov spike protein epitopes in unexposed individuals potential therapeutic targets and promising drugs for combating sars-cov-2 dendritic cells display peptides spanning the entire sars-cov-2 spike protein ii peptides from 11 regions are presented by a majority of the donors analyzed. â�¢ one region with promiscuous hla-ii presentation is conserved with sars-cov â�¢ the correlation of presented to predicted peptides is low pulse dendritic cells derived from healthy human donors with sars-cov-2 spike glycoprotein to determine the precise sequences presented for t-cell surveillance. regions with promiscuous presentation are identified with poor correlation to predicted epitopes. one region with promiscuous presentation is conserved with sars-cov spike glycoprotein sequence key: cord-017629-fuv157f1 authors: de groot, anne s.; moise, leonard; mcmurry, julie a.; martin, william title: epitope-based immunome-derived vaccines: a strategy for improved design and safety date: 2008-07-31 journal: clinical applications of immunomics doi: 10.1007/978-0-387-79208-8_3 sha: doc_id: 17629 cord_uid: fuv157f1 vaccine science has extended beyond genomics to proteomics and has come to also encompass ‘immunomics,’ the study of the universe of pathogen-derived or neoplasm-derived peptides that interface with b and t cells of the host immune system. it has been theorized that effective vaccines can be developed using the minimum essential subset of t cell and b cell epitopes that comprise the ‘immunome.’ researchers are therefore using bioinformatics sequence analysis tools, epitope-mapping tools, microarrays, and high-throughput immunology assays to discover the minimal essential components of the immunome. when these minimal components, or epitopes, are packaged with adjuvants in an appropriate delivery vehicle, the complete package comprises an epitope-based immunome-derived vaccine. such vaccines may have a significant advantage over conventional vaccines, as the careful selection of the components may diminish undesired side effects such as have been observed with whole pathogen and protein subunit vaccines. this chapter will review the pre-clinical and anticipated clinical development of computer-driven vaccine design and the validation of epitope-based immunome-derived vaccines in animal models; it will also include an overview of heterologous immunity and other emerging issues that will need to be addressed by vaccines of all types in the future. the availability of immunome-mining tools has fueled the design and development of vaccines by a process that has come to be termed 'reverse vaccinology,' 'vaccinomics,' 'immunome-derived vaccine' (idv) design, or 'genome-derived vaccine' design (rappuoli and covacci 2003; petrovsky and brusic 2002; pederson 1999; de groot and martin 2003; doytchinova, taylor and flower 2003) . this vaccine concept is based on the identification of a minimal set of antigens that induce a competent immune response to a pathogen or neoplasm. recognition of antigens occurs through the presentation of b cell and t cell epitopes derived from the antigen, in the correct immunological milieu. in its minimal form, an idv would contain only adjuvanated b cell and t cell epitopes in delivery vehicles such as liposomes. when these minimal components are packaged in an appropriate delivery vehicle, the complete package comprises an idv. compared to traditional vaccines, idvs have the potential to be safer and more effective since the vaccine focuses the protective immune response on the most essential antigenic elements of the pathogen/neoplasm. a number of idvs have been tested in clinical trials (elliott 2008; gahery et al. 2006; asj¨o et al. 2002; kran et al. 2004) . because epitope-based idvs are generally considered to be safe, when compared to other vectored or attenuated live vaccines, many have progressed rapidly from pre-clinical concept into clinical trials. in the cancer vaccine field, where epitope-based vaccines are well-established, many such vaccines are currently in phase i/ii clinical trials (pietersz, pouniotis and apostolopoulos 2006) . this chapter will review the development and validation of idvs; it will also provide a step-by-step guide to develop idv using validated immunoinformatics tools. these tools have the potential for dramatically accelerating the development of new and improved vaccines for the emerging and existing infectious diseases. whole-antigen-based idvs will be covered briefly; however, the main focus will be on epitope-based idvs and a description of the immunoinformatics tools that have been developed to accelerate the pre-clinical phase of vaccine discovery. so as to illustrate the process of pre-clinical vaccine development using these tools, two epitope-based idvs case studies will be presented: (i) a genome-derived vaccine for tularemia and (ii) an epitope-based hpv vaccine for adjunctive treatment of cancer. application of molecular biology techniques led to the sequencing of genomes and improved definition of the proteome (expressed proteins). even though the fundamental concept of the 'immunome' (the subset of fragments of expressed proteins that interface with the host immune system) is relatively well accepted, many important questions remain. due to genetic variation and poorly understood determinants of antigen processing, it has become clear that immunomes can vary substantially from one host to the next, even when major histocompatibility complex (mhc) and antibody germline genes are shared. the extent of the overlap between immunomes (in different hosts) and the general size of the immunome-representing epitopes derived from a particular pathogen or cancer remain to be determined. recently published studies are beginning to address these questions, partly due to the availability of algorithms that facilitate the identification of epitopes from whole genomes. the size of the immunome has been puzzling vaccinologists for decades. certainly, there are examples in the literature that 'a single epitope protects.' for example, crowe et al. (2006) recently found that immunization with a single th epitope provided a one-log reduction in influenza viral titers early in infection. a single epitope has also been shown to protect against viral disease in woodchucks (menne et al. 1997) , and multiple single epitopes have protected mice against an array of pathogens (an and whitton 1997 and olsen et al. 2000) . a more diverse set of t cell epitopes appear to be critical to immune response to vaccinia (in mice): 49 class i mhc epitopes were shown to contribute to the large majority of the cd8+ t cell immune response (moutaftsi et al. 2006) . remarkably, it was found that 49 of these predicted epitopes (derived from more than 175,000 candidates) represented over 90% of the vaccinia-specific cd8 t cell repertoire. how this number might be extrapolated to humans is unknown, but the result is relevant because 49 epitopes is few enough to be easily packaged and delivered in a vaccine. an epitope-based idv for genetically diverse populations of humans will almost certainly require more than that number of epitopes, particularly if the vaccine is intended to protect against complex bacteria or viruses, or against solid tumors presenting variable antigenic profiles. harnessing the power of immunoinformatics accelerates the tasks of defining the immunome and of identifying and developing new vaccines for human diseases. the task of developing an epitope-based idv can be deconstructed into a series of achievable steps. after selecting a target organism, the next step in the immunome-to-vaccine process is to identify, from within the target genome, a set of potentially antigenic genes/proteins. these sequences can then be screened using a variety of in silico, in vitro, and in vivo mechanisms or a combination of methods. in the case of small viral genomes, it may be possible to include the entire genome in the search universe. in the case of larger bacterial and viral pathogens, the traditional vaccine targets include surface proteins and secreted proteins (which can easily be found using in silico screening programs), toxins (which can be identified through homology matching), and virulence factors (which can be identified through the use of comparative genomics). however, many other types of proteins may also be worthy of consideration: in particular, proteins expressed in high amounts (such as viral capsid proteins), proteins overexpressed during growth or replication, or proteins overexpressed in response to stress conditions. for cancer vaccines, comparisons between cancerous and normal tissue can uncover cancer-specific genes. one approach involves the use of mrna derived from cancerous and normal tissue to probe dna microarrays, followed by selection of genes that are upregulated in cancerous and not in normal tissue (mathiassen et al. 2001; sepkowitz 2001) . using 'reverse vaccinology', a term recently coined by rino rappuoli, the immune memory of subjects who have successfully encountered and defeated a pathogen can be interrogated to identify the primary targets of a natural immune response. rappuoli and colleagues identified novel vaccine targets using in silico techniques to screen the target genome for 'surface protein-like' sequences. candidate proteins were then expressed in escherichia coli and screened against human sera isolated from pathogen-exposed subjects. reactive proteins were deemed relevant to immune response (pizza et al. 2000) . a related approach for discovering candidate vaccine antigens involves analyzing the target pathogen's proteome in silico, using t cell epitope mapping tools. putative t cell epitopes identified can then be screened against peripheral blood mononuclear cells (pbmc) isolated from human subjects who have been infected with the target pathogen (or who have the target cancer). t cell reaction to a particular peptide epitope, typically measured by elisa or elispot assay, implies that the protein from which the peptide was derived, expressed, processed, and presented to the immune system in the course of a 'natural' immune response. using this method, measuring immune response to an epitope reveals a protein antigen. our group describes this approach as 'fishing for antigens using epitopes as bait.' other common in vitro techniques used to identify expressed or overexpressed genes/proteins include: 2d sds-page, (kaufmann et al. 1992; sonnenberg and belisle 1997; hernychova et al. 2001) , mass spectrometry (tomlinson, jameson and naylor 1996) , and/or tandem mass spectrometry (barnea et al. 2002) . in the context of vaccines against infectious diseases, it may be prudent to exclude proteins that are highly conserved across species; such proteins (including housekeeping genes) may cross-react with unrelated avirulent organisms or with self-proteins to which there may be pre-existing tolerance. however, conservation across species should not be confused with conservation within species variants. sequence conservation within species variants is a highly desirable trait for vaccine components and one that the idv approach is particularly well suited to harness. rna viruses in particular (hcv, hiv, coronaviruses) are highly variable pathogens. in these cases, selecting epitopes that are conserved across variants or subtypes may allow for the development of a more broadly applicable vaccine. alternatively, single proteins that are relatively well conserved, when compared to the balance of the target pathogen, can be selected as vaccine candidates. recently, for example, one team has developed a hexon-epitope vaccine that may be effective against a range of adenoviruses, across serotypes (leen et al. 2008) . once critical antigens have been identified, the next steps in epitope-based idv development are to select epitopes and confirm their immunogenicity. once the adaptive immune system has been engaged, a humoral, or antibodybased response forms the first line of defense against most viral and bacterial pathogens. antibodies recognize b cell epitopes composed of either linear peptide sequences or conformational determinants, which are present only in the three-dimensional form of the antigen. several b cell epitope prediction tools, such as 3dex and cep, have been proposed and are in the process of being refined (enshell-seijffers et al. 2003; schreiber et al. 2005; kulkarni et al. 2005) . unfortunately, the computational resources and modeling complexity required to predict b cell epitopes are enormous. this complexity is due in part to the inherent flexibility in the complementarity determining regions (cdr) of the antibody and in part due to glycosylation, and other post-translational modifications can result in modification of b cell epitopes. although accurate b cell epitope mapping tools remain elusive, the selection of potent b cell antigens can be accelerated using t cell epitope mapping tools. when considering b cell antigens as potential subunit vaccines, it may be important to also consider their t cell epitope content since the quality and kinetics of the antibody response is dependent upon the presence of t help. b cell antigens, which contain a significant t help, may outperform b cell antigens lacking cognate help. and in some cases, an identified t cell epitope may contain a b cell epitope. although different epitopes activate t and b cells, it has been widely reported that b cell epitopes have been shown to co-localize near, or overlap, class ii (th, cd4+) epitopes (graham et al. 1989; rajnavolgyi et al. 1999 ). the adaptive immune system's second line of defense is the t lymphocyte. class i-restricted cytotoxic t cells (cd8+ ctl) directly engage and attack infected host cells. class ii-restricted t helper cells mediate the growth and differentiation of both t effector cells and antibody-producing b lymphocytes. both class i and class ii t cells carry out their roles in response to t cell epitopes, small linear fragments derived from protein antigens, displayed on the surface of antigen-presenting cells (apc) by various alleles of mhc. while b cells and antibodies generally recognize epitopes on surface proteins only, t cells recognize epitopes derived from a variety of proteins. once taken up by apc, antigenic proteins are broken down by digestive enzymes. during this process very large numbers of peptide fragments are released. any one of these fragments could be a t cell epitope, but only about 2% of all the fragments generated can implant themselves in the binding groove of the mhc molecule and be presented on the surface of the apc. one of the critical determinants of t cell epitope immunogenicity is the strength of epitope binding to mhc molecules (lazarski et al. 2005) . peptides binding with higher affinity are more likely to be selected by mhc molecules and to be displayed on the cell surface where they can be recognized by t lymphocytes. using a variety of methods including frequency analysis, support vector machines, hidden markov models, and neural networks, researchers have developed highly accurate tools for modeling the mhc-peptide interface and for accurately predicting t cell epitopes. for a review of t cell epitope mapping tools, see de groot and berzofsky (2004) and the accompanying issue of methods. what all these tools have in common is an ability to quickly screen large volumes of genomic sequences for putative epitopes; this preliminary screen reduces the search space dramatically, typically by at least 20-fold. the ability to accurately predict t cell epitopes from raw genomic data is fundamental to the development of an idv. however, even a highly accurate prediction is still only a prediction. before including predicted epitopes in a candidate vaccine, it is important to validate their immunogenicity in vitro and in vivo. once identified, peptides representing the selected epitopes are then synthesized. hla binding assays can be used to assess whether peptides derived from immunoinformatics analysis can bind to either mhc class i or class ii by measuring the affinities of predicted epitope sequences for the hla alleles in vitro. in vitro evaluation of mhc binding can be performed by quantifying the ability of exogenously added peptides to compete with a fluorescently labeled known mhc ligand (steere et al. 2006 ) and can be adapted for high throughput (mcmurry et al. 2007a) . epivax routinely uses these high-throughput hla binding assays to confirm epitope predictions in vitro. a concordance between hla binding and immunogenicity is often observed (mcmurry et al. 2005 ). peptides are used to measure t cell responses in vitro; they can be of variable lengths (9-25). peptides presented in the context of class i mhc are generally limited to 9 or 10 amino acids in length, although some processing is believed to occur during the t cell assay and so 15-mers are also used for class i assays. in contrast with class i epitopes, which are short and fit tightly in the bounded mhc molecule, class ii (t helper) epitopes lie within an open-ended groove in the mhc ii. as such, a class ii epitope can shift within the groove, thereby accommodating mhc of various haplotypes. the only limit on the size of the peptide is its ability to remain in a linear conformation in the open-ended groove. both mhc class i-and mhc class ii-restricted epitopes (targeting cd4+ and cd8+ t cells, respectively) are believed to be important for the development of effective vaccines. cd4+ t helper cells enhance and amplify cytotoxic t cell (ctl) immune responses and have been shown to be important in the development of cd8+ t cell memory to a range of pathogens (ahlers et al. 2001) . ctls generally play a role in the containment of viral and bacterial infection (plotnicky et al. 2003) , and the prevalence of ctls usually correlates with the rate of pathogen clearance. like peptides, whole antigens too can be used to measure t cell responses in vitro. the recognition of these antigens requires the presence of an apc that is capable of processing and presenting peptides derived from the antigen. if blood from exposed individuals is available, the peptides validated as mhc ligands in binding assays can be tested for their reactivity with t cells, serum, or both. a positive immune response (as measured by elisa, elispot, or intracellular cytokine staining) should be interpreted as a sign that the parent protein interfaces with host immune response in the course of natural infection or disease. following confirmation, the peptides that stimulate a response can be considered vaccine candidates themselves or can be used to select the entire protein for use in a subunit vaccine. these candidates can then be incorporated into a vaccine delivery vehicle with an appropriate adjuvant. elisa and elispot are related methods for detecting t cell responses by the measurement of cytokines secreted by the t cells (gamma interferon, il-2, and il-4 are examples). the expansion (proliferation) of t cells in response to stimulation by peptide:mhc can be measured by (1) the dilution of a fluorescent dye in subsequent generations of cells (cfse) and (2) the incorporation of a radioactive label in the proliferating cell's dna (tritiated thymidine incorporation assay). fluorescence activated cell sorting (facs) and intracellular cytokine staining (ics) are the most precise methodologies available for measuring and defining t cell response. for example, t cells that respond to a particular epitope can be directly labeled using tetramers (comprising mhc class ii: peptide complexes). labeled cells can then be sorted and counted, and the phenotype of t cells that respond to the antigen can be determined using cell surface markers and ics (tobery et al. 2006 ). factors extrinsic to processing, such as the cytokine milieu induced in response to a particular component of a vaccine (krieg et al. 1998) or pathogen (ghosh et al. 1998) , also play a role in the conditioning of the immune response. thus, t cell epitopes may be necessary to drive immune response, but are not sufficient. co-stimulatory molecules that provide a second signal, the right cytokine milieu and other factors directing the nature (th1 vs. th2) of the immune response, are also crucial (shahinian et al. 1993; kuchroo et al. 1995) . adjuvants provide this added 'boost' in the context of vaccines. the same range of delivery vehicles that exist for conventional vaccines can be used for the development of idvs and epitope-based idvs. for example, idvs and epitope-based idvs can be formulated and delivered as pseudo-proteins or peptides in a carrier vehicle such as a liposome or viral-like protein (vlp); alternatively, the sequence of the idv antigens or epitope string can be inserted into a viral or bacterial vector such as adenovirus or salmonella; alternatively, a dna vaccine construct encoding the antigen(s) or epitopes can be developed. the choice of adjuvants for use in humans is relatively extensive and each adjuvant has advantages and disadvantages. the advantages and disadvantages of each type of vaccine delivery vehicle and adjuvant here is beyond the scope of this chapter; readers are referred to a review by fraser et al. (2007) . the next step in the development of epitope-driven idv is to determine whether immunization provides competent immune response. the idv or epitope-based idv is administered and immune responses to the components are evaluated following immunization. even though a range of animal models are used for the evaluation of vaccines, results from immunogenicity studies in these models should be interpreted with caution. although their functions may be similar, the mhc of mice, rodents, and non-human primates differ from human mhc (known as hla in the context of human immune response) at the amino acid level and these differences effect which epitopes can be presented. this helps to explain why different strains of mice (balb/c, c57bl/6) have different immune responses to pathogens as well as vaccines for those pathogens (klitgaard et al. 2006) . in particular, epitope-based vaccines that are developed using predicted human t cell epitope mapping tools can be tested only in murine models that are hla transgenic. fortunately, a number of transgenic mouse strains that express the most common hla a, hla b and hla dr, molecules have been developed. t cell responses in these mice correlate directly with t cell responses observed in infected/vaccinated humans (man et al. 1995; shirai et al. 1995) . hla transgenic mice are now routinely used to assay and optimize (human) epitopedriven vaccines in pre-clinical studies (ishioka et al. 1999; charo et al. 2001; livingston et al. 2001) . despite the limited number of hla class ii alleles for which tg mice have been developed, comparisons of immunogenicity can be done to a high degree of accuracy in the mouse model for selected hla class ii alleles (hla dr 0101, 0301, 0401, 1501). unfortunately, it appears these mice may have difficulty breeding due to poorly understood consequences of their transgenic heritage, limiting the use of this important model system. the final step in the development of any vaccine is experimental validation of the immunogenicity and protective efficacy of computationally selected antigens. currently, a series of experimental vaccines have shown efficacy in animal models and several idvs are being tested in clinical studies. in the context of infectious disease, genome-derived vaccines that have progressed furthest along the vaccine development pipeline are generally based on whole proteins rather than epitopes (rappuoli and covacci 2003; pizza et al. 2000) . however, epitope-based idvs are currently being developed for a range of infectious diseases by the authors' laboratory and by many others (depla et al. 2008 ). while it is common knowledge that subunit-based vaccines can protect against infection, similar success with epitope-based approaches is not as widely known. in addition to the studies previously cited, immunization of balb/c mice with three doses of a peptide construct containing an h-2(d)-restricted cytotoxic t lymphocyte (ctl) epitope from a murine malaria parasite induced both t cell proliferation and a peptide-specific ctl response mediating nitricoxide-dependent elimination of malaria-infected hepatocytes in vitro, as well as partial protection of balb/c mice against sporozoite challenge (franke et al. 2000) . in a separate study, immunization of balb/c and cba mice with measles virus ctl epitopes resulted in the induction of epitope-specific ctl responses and conferred some protection against encephalitis following intracerebral challenge with a lethal dose of virus (schadeck et al. 1999) . these are just a few successful examples of many studies carried out in animal models; however, translation to prevention of disease in humans has been difficult to achieve. in contrast with whole-protein subunit vaccines, idvs and epitope-based idvs have taken longer to make the transition from animal model to the clinic, mainly due to the novelty of the concept and perhaps unfounded concerns that epitopes are not sufficient for the generation of effective immune response. cancer therapy is an exception to this rule. as previously described, eptiopebased idvs have been evaluated in the context of therapy against chronic infection or cancer (ueda et al. 2004; valmori et al. 2003) . in the cancer vaccine field, where the concept of epitope-driven vaccines is well established, many more peptide vaccines have successfully passed pre-clinical tests and are currently in phase i/ii clinical trials (pietersz, pouniotis and apostolopoulos 2006) . new approaches are emerging, which may improve the success rate and, indeed, the results from recent clinical trials prove the principle. one approach is to identify epitopes that are unique to the tumor (prostate, lung, colon) and to pre-screen the patient for response to the peptide. this approach, called personalized vaccination, takes into account the diversity of ctl epitope recognition among patients. whereas the response rates to classical (non-personalized) peptide vaccines have been disappointing, responses to personalized vaccines (in a phase i trial, conducted in japan) have been as high as 11.1% in the advanced cancers and equal to or more than 20% in malignant glioma and cervical cancers, respectively (itoh and yamada 2006) . it is noteworthy that just a few epitope-driven vaccines against viral and microbial pathogens have reached the stage of phase i or ii efficacy trials in humans. for example, bionor immuno's hiv p24 gag peptide vaccine (vacc-4x) was demonstrated to be safe and well tolerated in phase i trials (asj¨o et al. 2002) and dose-dependent and immunogenic in phase ii trials in norway (kran et al. 2004) . similarly, nardin's epitope-based vaccine for malaria is moving along the clinical trial pathway (nardin et al. 2000) . in this section, we describe the immunomics tools developed and used by the epivax vaccine development group in recent collaborations with dr. steve gregory of lifespan, dr. ousmane koita of the university of bamako, mali and dr. david weiner of university of pennsylvania. t cell epitopes are linear peptides that bind to mhc molecules. binding is mediated by the interactions between the r-groups of the amino acids in the peptide ligand and the pockets on the floor of the mhc binding groove. because the mhc:peptide interaction is well characterized, pattern-matching algorithms can be used to screen protein sequences for peptides that will bind mhc. the authors of this report currently use the epimatrix system, a suite of epitope-mapping tools that has been validated by more than a decade of use in selecting putative epitopes for in vitro and in vivo studies (see references (de groot et al. 1997; bond et al. 2001; dong et al. 2004; mcmurry et al. 2005; koita et al. 2006) . the epimatrix algorithm is based on a set of class i and class ii hla matrices wherein individual frequencies of all 20 amino acids (aa) in each hla pocket position are applied to the prediction of overlapping 9-and 10-mer peptides. in a typical analysis, protein antigens are parsed into overlapping 9-mer frames where each 9-mer overlaps the last by eight amino acids. each 9-mer is then scored for predicted binding affinity to one or more class i or class ii hla alleles. in order to compare potential epitopes across multiple hla alleles, epimatrix raw scores are converted to a normalized 'z' scale. peptides scoring above 1.64 on the epimatrix 'z' scale (typically the top 5% of any given sample) are likely to be mhc ligands. since class ii epitopes can be promiscuous, our approach to the prediction of class ii epitopes is to estimate the binding potential of each frame with respect to each of a panel of eight common class ii alleles (drb1*0101, *0301, *0401, *0701, *0801, *1101, *1301, and *1501). taken together, these alleles 'cover' the genetic backgrounds of most humans worldwide (southwood et al. 1998 ) and they also represent the predominant types of 'pockets' for the most common mhc. recently, the epimatrix system has been utilized to measure the potential immunogenicity of whole proteins. in this context, epimatrix assesses the aggregate epitope density of a given protein with respect to the aggregate epitope density of a set of randomly generated pseudo-protein sequences of similar size (de groot 2006) . by correcting for the size and expected epitope density, the potential immunogenicity candidate vaccine antigens can be directly compared. further immunogenicity of low scoring proteins may be enhanced by modifying the immunogenic region amino acid sequence so that it contains more t cell epitopes (see illustration of this approach in the hpv vaccine section, below). peptides predicted to bind to multiple hla alleles are known as promiscuous t cell epitopes. the clustimer algorithm is used to scan the output produced by the epimatrix and identifies the polypeptides predicted to bind to an unusually large number of hla alleles. briefly, the scores of each analyzed 9-mer are aggregated. high-scoring 9-mers are then extended at the n-and c-terminal flanks until the predicted epitope density of the promiscuous epitope falls below a given threshold value. this particular approach to mapping epitopes has also been useful for discovering 'epibars,' which may be a signature feature of highly immunogenic, promiscuous class ii epitopes. an example of a promiscuous t cell cluster containing an epibar (tetanus toxin 830-844) is shown in fig. 1 . a single t cell epitope 'cluster' usually ranges from 9 to about 25 amino acids in length and can contain anywhere from 4 to 40 binding motifs. using epimatrix as described above and clustimer, scores above 10 and, in particular, scores above 15 indicate significant immunogenic potential (de groot 2006) . note the horizontal bar of high z scores at position 308 in fig. 3 . having observed this 'epibar' pattern to be characteristic of promiscuous epitopes, the authors have integrated the pattern into the prospective selection of clusters. promiscuous epitopes also exist, to a certain degree, for class i alleles. some laboratories have demonstrated cross-presentation of peptides within hla 'superfamilies' (such as the a3 superfamily: a11, a3, a31, a33, and a68) described by . the authors have confirmed cross-mhc binding and presentation to t cells in our hiv vaccine studies ). one limitation of conventional vaccination, and to a lesser extent natural infection, is that the immune system focuses strongly on the most mutable immunogen of the virus -typically, the viral envelope. in the case of hiv and other viruses, vaccination with more conserved, subdominant epitopes has been shown to circumvent this hierarchy and potentiate cross-strain protection (ostrowski et al. 2002; nara and lin 2005) . in like manner, a conserved t helper-directed vaccine may provide a more 'democratic' way of stimulating immune response, increasing the number targets for t cell recognition, thereby providing t help to antibody response despite potential viral variability (santra et al. 2002; subbramanian et al. 2003; scherle and gerhard 1988; scherle and gerhard 1986; russell and liew 1979; johansson et al 1987) . the genetic variability of some pathogens constitutes a significant challenge to the efforts to design a vaccine driven by cellular immune response de groot et al. 2002) . the authors have been involved in developing an hiv-1 vaccine that includes highly conserved (cross-clade) t cell epitopes. the conservatrix algorithm, developed for this application, parses input sequences into component strings (the lengths of the strings may be determined ) indicates the potential of a 9-mer frame to bind to a given hla allele. all z scores in the top 5% (>1.64) are considered 'hits'. though not hits, scores in the top 10% are considered elevated; scores below 10% are masked for simplicity. frames containing four or more alleles scoring above 1.64 are colloquially referred to as 'epibars' (see frames 831:yikanskfi and 832: ikanskfig). this band-like pattern is characteristic of promiscuous epitopes. the tetanus toxin peptide scores are extremely high for all eight alleles in epimatrix; the deviation compared to expectation is + 23.82 by the operator) and then searches the input dataset for matching segments. conservatrix may be used to compare strings derived from different strains of the same organism (hepatitis c, for example, or hiv) or to search a given sequence for a user-supplied target sequence. target sequences may be input as specific sequences or as coded patterns. thus, the operator can use 'wild cards', allowing for one or more of the amino acid residues in any given peptide sequence to be any amino acid [x], or a limited set of amino acids such as [l, v] . results of each analysis are stored in a database and may be browsed or exported to another program for analysis. by selecting highly conserved epitopes, regardless of their distance from the ancestral hiv-1 genome, we have identified sequences conserved for structural and functional reasons and are therefore less likely to be modified in the course of further evolution of hiv-1 (peyerl et al. 2004; koibuchi et al. 2005 ). the problem of virus variability also significantly complicates the selection of epitopes that have a population-coverage advantage; such epitopes are termed 'clustered,' 'superfamily,' or 'promiscuous.' to address this problem, the authors developed epiassembler ) to identify sets of overlapping, conserved, and promiscuously immunogenic epitopes and assemble them into extended immunogenic consensus sequences (ics) (see fig. 2 ). in theory, proper processing and presentation of these sequences would allow for the presentation of highly conserved peptides in the context of more than one mhc. the resulting peptide is not a 'pseudosequence' as such, since each constituent epitope occurs in its corresponding position in the native protein. thus, while the full-length 'immunogenic consensus sequence' is not necessarily found in any one variant sequence, the peptide is more representative of the sequence universe. in the case of hiv, for example, we used the ics approach to design a peptide-based vaccine. while the full-composite ics peptides happen to be exactly conserved in a few individual strains of hiv, each peptide represents a significant percentage of circulating strains, because every constituent overlapping epitope is conserved in a large number (range 893 to 2,254) of individual hiv-1 strains. as compared with immunogenic consensus sequences, randomly selected counterparts, on average, contain half as many binding motifs and cover a third fewer isolates. to develop vaccines of equivalent antigenic 'payload,' using conventional methods would be prohibitively expensive as it would require including multiple different variants of each antigen. this approach has been useful for identifying highly immunogenic epitopes for hiv vaccine design . by focusing on conserved, mhc-promiscuous t helper epitopes, the ics approach has the potential to efficiently overcome the genetic variability of both virus and host. one of the advantages of idv is that it is possible to omit deleteriously crossreactive epitopes. perhaps, the most famous example of an adverse effect due to cross-reactivity with self was observed following vaccination for lyme disease with the osp a protein. the vaccine has been recently re-engineered with the cross-reactive epitope removed (willett et al. 2004 ). in the context of our own work, peptides selected for in vitro evaluation are evaluated for homology with human proteins by blasting the sequences against the human sequence database at genbank (http://www.ncbi.nlm.nih.gov/). blastimer automates the process of submitting sequences to the websites featuring search engines such as the blast engine at ncbi (www.ncbi.nlm.nih. gov/blast). by default, blastimer blasts sequences against all non-redundant x x x x x x fig. 2 the ics assembly operation was performed using epiassembler (bill martin, epivax, 2004) . left panel: each variant strain is first analyzed and a highly conserved, putatively promiscuous 9-mer is chosen as the core peptide. mismatches with the selected epitope sequences are represented with the letter x. right panel: additional epitopes are then identified, which overlap with the natural n-and c-terminal flanking regions of the core 9-mer epitope. the overlap length requirement is decreased by one amino acid iteratively until reaching a minimum of three overlapping amino acids. if more than one suitable overlap is identified, the overlapping peptide with the higher overall epimatrix rank is selected. this process is repeated using the extended peptide as the new core sequence. the cycle can be repeated for the length of an entire protein or can be truncated when the peptide reaches a length that can be easily produced synthetically genbank cds translations, pdb, swissprot, pir, and prf. blastimer assesses the homology between the submitted sequence and the sequence of proteins of other organisms. patent blast, on the other hand, targets a database of sequences gleaned from patents. users of either program may control all of the submission options available to interactive users at ncbi. in both cases, results are recorded in a database and can be browsed, exported, or summarized and rendered in a report format. according to the authors' standard practice, any peptide that shares greater than 80% identity with peptides contained in the human proteome is eliminated from consideration in a vaccine. a number of methods for enhancing epitope-based vaccines have been described and implemented (thomson et al. 1998; rodriguez and whitton 2000) . one approach is to align the epitopes in a protein or dna vaccine construct as a 'string of beads' without any intervening sequences or 'spacers' in a dna plasmid encoding the individual epitopes . however, the lack of 'natural flanking sequences' -has raised concern that their proteolytic processing may be compromised, and that junctional epitopes, peptides other that the specific peptides of interest, may be generated as a result of processing (godkin et al. 2001) . to address this concern, the authors developed vaccine-cad (see fig. 3 fig. 3 vaccine-cad, illustrated with three sample epitopes represented by the words 'create,' 'new,' and 'epitopes.' the default arrangement of the words results in unintended sequences, represented by the words 'eaten' and 'ewe,' at the junctions between the intended epitopes. reiterative modifications in the arrangement of the epitopes results in the development of a sequence that has no 'pseudoepitopes' (new epitopes that were not intended) at the junctions of the juxtaposed epitopes junctional epitopes, the insertion of spacers and breakers, the requirements for secretion or processing tags, and the evaluation of epitope strings for potential homologies to human protein fragments. an important component in the epitope-driven vaccine process is the selection of epitopes from the regions of pathogens that are presented by mhc molecules for t cell recognition. 'mhc binding motifs,' first identified by r¨otzschke and falk, are the patterns of amino acids in peptides that are known to promote the binding of peptides containing these patterns to the mhc molecules on the surface of apcs rotzschke et al. 1991) . different mhc molecules have different binding motifs, limiting the set of mhc ligands that can be presented in the context of any given mhc. while consideration of hla alleles may lead to concern about the selection of epitopes for broad coverage of populations, gulukota and delisi (1996) and sette and sidney (1998) have demonstrated that epitope-based vaccines that contain epitopes restricted by selected 'supertype' hla can provide the broadest possible coverage of the human population. furthermore, recent studies by brander and walker indicate that there may be even greater flexibility in the binding of epitopes to mhc than previously recognized; this is also consistent with the data recently presented by frahm et al. (2004) . the inclusion of 'promiscuous epitopes' -epitopes that are recognized in the context of more than one mhc (paina-bordignon et al. 1989; de groot et al. 1991; sette and sidney 1998) in epitope-driven vaccines may therefore overcome the challenge of genetic restriction of immune response. in addition, the repertoire of possible mhc-restricted epitopes recognized by an individual's t cells has been shown to be quite variable, even between hla-matched individuals (jameson, cruz and ennis 1998; gianfrani et al. 2000; betts et al. 2001 ). aggregatrix, a new algorithm that was recently developed at epivax, iteratively searches for the combination of epitopes that achieves maximal cross-clade representation. the authors performed this analysis for our hiv epitopes, as shown in fig. 4 , evaluating each hla-a2-restricted hiv peptide individually and the set in aggregate for coverage of hiv-1 strains by year (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) , by country of origin, and by clade. as can be seen in this figure, the set of highly conserved a2-restricted peptides we have tested and confirmed in elispot assays covered between 54% and 86% of strains in a given year, between 33 and 100% of strains in a given country, and between 0% and 100% of strains in a given clade. the hla a2 peptides cover 85, 78, 78, and 80% of clades a, b, c, and d, respectively. this is a remarkable breadth of coverage for a limited set of coverage of gaia hiv b7 peptides--individually and in aggregate-across time, countries, and clades fig. 4 gaia hiv a2 peptides -individually and in aggregate -percentage coverage of strains by year, country, and clade. each row of the matrix denotes a specific peptide; the peptide's protein of origin is included within the peptide id. each column of the matrix denotes a specific year, country, or clade, grouped as indicated. the percentage coverage of strains is represented on a color gradient, with warm tones indicating values above 50% and cool tones indicating values below 50%. the bottom row of the matrix shows the percentage of each protein set that contains at least one peptide from our pool. black boxes indicate that no isolates of the protein are available for that year, clade, or country. the bottom row represents the aggregate percent coverage for the epitope set. each cell of the matrix represents the percentage coverage per peptide, except for the bottom row cells, which represent the aggregate percentage coverage for the peptide set. column headers are listed here for space considerations: left to right, the year columns are 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, and 2005 ; aggregate coverage of strains by year ranges from 47% (2000) hla a2 epitopes, given the well-known ability of hiv to mutate away from hla (nguyen et al. 2004; iversen et al. 2006) . in studies of immune response to therapeutic proteins, the authors have observed that subject-to-subject variation in t cell response closely relates to (1) subject hla type and (2) the number of protein-derived peptides that match the subjects hla. to describe this relationship, the authors developed a metric that may be useful in the development of epitope-based vaccines or in the clinical assessment of immune response to vaccines, called the 'individualized t cell epitope measure' or item. the item can be calculated for each subject who is responding to a given epitope by summing the epimatrix z-scores for each positive peptide for each hla allele in a given subject's haplotype, thus: item score ¼ ½epimatrix score of peptide for hla type 1 þ ½epimatrix score of peptide for hla type 2 þ . . . the same calculation can be performed for larger peptides and proteins by summing all of the scores for the subject's hla type. this calculated score allows for the individualized potential immunogenicity to be predicted, based on the number of putative epitopes contained in a protein that might be presented to their t cells, based on their hla haplotype. using this score, it is possible analyze the contribution of haplotype to the corresponding t cell response. in our prospective evaluations, significant correlations were found between the ifn-gamma response to a given antigen and the item scores for individual subjects (for data published in koren et al. (2007) r= 0.69, p= 0.0134). in addition, correlations between the item score and patient hla were also observed for their antibody titers. further evaluations of this method for predicting individual responses to vaccines and therapeutic proteins are in progress. defining the immunome by identifying t cell epitopes and confirming their immunogenicity is but the first step of vaccine development. a number of conditions extrinsic to the mhc-ligand interaction may influence the final composition of the epitope ensemble. for example, whether or not a predicted epitope is confirmed is related to: (1) the quantitative expression of the source protein; (2) the number of different epitopes derived from this protein, which are presented on the surface of the apc (wherry et al. 1999) ; (3) the amino acids that flank the epitope (bergmann et al. 1994; shastri, serwold and gonzalez 1995; livingston et al. 2001) ; and (4) proper cleavage and trimming by proteolytic enzymes in the proteasome and processing pathways (van kaer et al. 1994; york et al. 1999; chen et al. 2001; toes et al. 2001) . for example, it is very likely that end-to-end epitope presentation, such as was used in the design of the oxavi hiv gag/epitope vaccine, impaired the presentation of the epitopes in immunogenicity studies. vaccine-cad also takes into account the role of flanking residues: studies conducted in murine models have demonstrated that residues flanking an mhc class i epitope strongly influence the delivery of the intact epitope to tap following proteasome degradation (thomson et al. 1995; hozhutter, frommel and kloetzel 1999; mo et al. 1999 ). in addition, livingston et al. (2001) have tested a standard spacer sequence (-gpgpg-) for vaccine constructs consisting of mhc-ii-restricted, th-cell epitopes; the use of this spacer disrupts junctional epitopes that might compete for degradation or for mhc binding (both g and p are unusual carboxy-terminal anchors for a peptide that binds to class ii mhc). this approach has been used for constructs with up to 20 epitopes, in assays where responses were detected to the majority of epitopes (livingston et al. 2001). 6 methods of confirming idv 6.1 two case studies francisella tularensis is a zoonotic bacterium. it is endemic to certain communities such as martha's vineyard, massachusetts, usa, where it is known as rabbit fever. tularemia represents a potentially dangerous biological weapon owing to its high degree of infectivity, ease of dissemination, and capacity to cause severe illness. despite several decades of research, no vaccine for tularemia is licensed for public use. for a review of tularemia vaccines, see mcmurry et al., 2007b. we have been actively developing an epitope-based tularemia vaccine combining computational immunology with in vitro and in vivo validation (mcmurry et al 2007a) . the starting point of our vaccine was the fully annotated f. tularensis subsp. tularensis (schus4) genome published in by larsson et al. (2005) . a prototype vaccine containing only class ii epitopes has been tested in challenge studies in hla drb1*0101 transgenic mice. for this vaccine, the epimatrix algorithm was utilized to identify highly promiscuous t cell epitopes within the tularemia genome. twenty-five class ii-restricted epitopes were selected, synthesized, and screened in vitro using a recombinant soluble hla class ii competition-binding assay (described above). peptides that bound with high affinity were then tested ex vivo in elispot assays with blood obtained from f. tularensis subsp. tularensis-exposed individuals. search-light analysis was also performed on supernatants derived from human cell culture stimulated with peptide using a panel of nine cytokines. forty-two percent of peptides bound to drb*0101 and are likely to bind to several other alleles (in that they were predicted using the clustimer algorithm). elispot assays showed positive ifn-gamma responses to 21/25 individual peptides and to peptide pools in nearly all of the 23 human study. the number of epitopes recognized per subject ranged from 1 to 17 and averaged 4 per subject; not every peptide was tested for every subject. peptides that elicit a robust memory response, as evaluated by these various assays, were incorporated into a vaccine construct and tested in challenge studies with hla transgenic mice as a possible vaccine against tularemia. immunogenicity studies in hla transgenic drb1*0101 mice were performed using the multi-class ii-epitope dna constructs and/or peptides representing the epitopes, and t cell responses were evaluated in ifn-gamma elispots. hla dr1 transgenic mice were challenged with five times the ld50 of f. tularensis lvs; 57% of vaccinated mice survived, all non-vaccinated mice died (manuscript in preparation). this result demonstrates the potential for a genome-derived epitope-based vaccine to protection from a class a bioterror pathogen. importantly, the protection we observed is accounted for by only 10 of the 14 schus4 epitopes in the vaccine, as they were conserved in the lvs challenge strain. the schus4-specific epitopes that were found to be significantly immunogenic would further contribute to protection in a schus4 challenge. this result is consistent with other findings that a limited set of epitopes may be sufficient to induce a protective immune response (moutaftsi et al. 2006) . studies using schus4 (the wild type tularemia) are planned. the results obtained to date appear to indicate that vaccine design that originates with the whole genome may lead to the development of a protective epitope-based vaccine. cervical cancer is the second leading cause of death afflicting women worldwide; 40% of cervical patients develop persistent, recurrent, or widely metastatic disease. while a preventive vaccine now exists for hpv, there is a need for a therapeutic vaccine to treat existing cases of hpv, especially in resource-poor areas where access to the preventive vaccine is limited. cellular immune responses are believed to be critical for effective immune response to cancer; accordingly, epivax is pursuing the development of an 'immunotherapeutic vaccine' which would focus on the proteins primarily expressed either before or during carcinogenesis (e1 and e2, e6, and e7). in order to maximize immunogenicity across hpv subtypes, our strategy involves analyzing variant strains of hpv protein sequences, using (1) epimatrix to identify class i and class ii hla motif matches, (2) conservatrix to identify those motif matches that are conserved, and (3) epi-assembler to weave together the conserved immunogenic sequences into a full immunogenic consensus sequence (ics) antigen. a full ics vaccine antigen would retain the fundamental structure of its naturally occurring counterparts; however, it will contain more and better epitopes than would occur in any such one counterpart. the authors have identified five conserved epitopes in e1 and e2, which have stimulated significant responses in elispot ifn-g assays. in the proposed vaccine, these epitopes and others will be incorporated in their natural context within the proteins, which could be delivered as dna, proteins, or a primeboost combination. by preserving the natural flanking regions surrounding our epitopes, we hope to retain, in large part, the natural processes surrounding hpv protein degradation, transport, and presentation as they occur during natural infections. the ics approach described here is the same as that illustrated in fig. 5 , but extending over the full natural length of the protein. for a more in-depth review of a similar approach being pursued for influenza, see mcmurry et al. (2008) . the epivax hpv vaccine illustrates yet another aspect of vaccine design: 'megatope' proteins, re-engineered to increase the epitope content. this approach already had some success (okazaki et al. 2006) . in the case of variable viruses such as hcv, influenza, and hiv, one limitation of conventional vaccination, and of natural infection, is that the immune system often focuses strongly on the most mutable immunogens. idvs can be constructed from alternative antigens, which are more conserved or more protective, circumventing this problem (russell and liew 1979; scherle and gerhard 1986; scherle and gerhard 1988; santra et al. 2002; subbramanian et al. 2003) . amino-acid sequences of ns3 proteins from available hpv isolates identification of immunogenic and conserved t-cell epitopes using conservatrix, epimatrix. (epitopes represented by open symbols) ns3 protein is optimized to incorporate the most conserved immunogenic epitopes in their natural context within the protein. illustration of our novel strategy to generate an hpv vaccine candidate. the putative epitopes identified during this analysis will be combined to form several consensus sequences, which retain the fundamental structure of these hpv proteins but which also contain an unnaturally large number of conserved t cell epitopes in addition, broadening the t cell repertoire might make it possible to impair viral escape and decrease viral loads sufficiently to disrupt transmission. epitope-driven vaccines also offer distinct advantages over vaccines encoding whole protein antigens, since epitopes are safe and can be packaged into relatively small delivery vehicles. the epitope-driven approach offers platform independence: a delivery vehicle (peptide, dna, multi-epitope construct) can be modified or selected midway into the development process. multiple conserved epitopes, in addition to augmenting the efficacy of a preventive vaccine, could provide a broad and universal cellular immunity, known to be crucial for containment of infection, although perhaps ineffective for protection against infection. despite these advantages, there are a number of reasons that a given pathogen-directed, epitope-based vaccine might fail to reach clinical trials or protect humans: (1) the limited number of epitopes expressed by the vaccine (i.e., poor payload quantity); (2) limited conservation of epitopes (leading to limited coverage of variant clinical isolates) (3) the limited hla population coverage (i.e., poor payload quality); (4) suboptimal vaccine delivery; and/or (5) the dearth of suitable animal models. in addition, the concept of epitope-driven vaccines is relatively novel. complete genome sequences have been available for only a little more than a decade now and the tools to process the data for vaccine design are only newer. experimental validation needed to push forward these vaccines into clinical trials is now emerging and promises to enable epitope-based vaccines to claim a prominent place in the vaccine world. the technologies needed to identify immunostimulatory antigens and epitopes from pathogen genomes are already well developed. the principle focus of future research in this area will likely be in fine-tuning these technologies and expanding them to tailor immune responses in individuals. for example, development of epitope mapping algorithms for dq and dp class ii hla alleles will make it possible to completely characterize immunomes. this information will make it possible to generate comprehensive individual t cell epitope measures (item) based on an individual's hla genetic make-up and allow researchers to identify a priori clinically important epitopes and screen clinical cohorts for subjects that are more likely to develop targeted immune responses. furthermore, genome-mapping tools that are currently available are not yet useful for discovering b cell epitopes, whether from proteins or from nonprotein components such as carbohydrates or lipid antigens. immunoinformatics tools that are currently available cannot be used to accurately predict conformational (b cell) epitopes that interact with antibody, although such tools are being refined (enshell-seijffers et al. 2003) . thus, the immunogens identified using in silico approaches must be evaluated in vitro and also in appropriate challenge models, prior to progressing to vaccine trials. protective immune response probably also involves some engagement of the innate immune system; it has been impossible to differentiate between effective and non-protective epitopes. cytokine milieu may affect the outcome of immunization; thus, a limited number of toll-receptor agonists (imler and hoffmann 2001) have been identified and these are under study in conjunction with idvs. in the future, toll-receptor signaling 'pathogen-associated molecular patterns' (pamps) might also be modeled and selected using immunoinformatics tools. besides antigen identification, the success of idvs relies heavily on delivery technologies. these areas continue to independently mature and provide important lessons to epitope-based vaccine design. the major areas of research to watch include biological macromolecule (including cytokines), lipopeptide, and polysaccharide adjuvants and particulate (liposomes, exosomes, virosomes, nanoparticles) and cell-based delivery systems. the development of safe and effective vaccines against emerging infectious diseases such as influenza, both seasonal and pandemic, hiv, and tb, in addition to cancers associated with infectious pathogens such as hbv and hcv, is an urgent and achievable public health priority. in addition, vaccines for the prevention and treatment of cancer hold enormous promise for human health. the threat of bioterrorism following the events of september 11, 2001, provided vaccinologists with a persuasive argument for more rapid development of vaccines against viral and bacterial pathogens that are now included on the nih category a-c biopathogen list (http://www3.niaid.nih.gov/topics/ biodefenserelated/biodefense/research/cata.htm). 'emerging infectious diseases' were added to the vaccine wish list following the outbreak of severe acute respiratory syndrome (sars) in guangdong china in 2002. indeed, only a few months following the publication of the sars-coronavirus (sars-cov) genome (marra et al. 2003; rota et al. 2003) , researchers began to map vaccine components using new bioinformatics and immunoinformatics tools, coupled with improved immunology techniques and specialized animal models. new vaccines based on this approach are currently being evaluated in animal models, less than a year from the start of the epidemic. future vaccine approaches may need to move away from 'whole' protein vaccines for a wide range of reasons. multiple antigen or epitope vaccinations such as the approach illustrated here could be one way to elicit the sort of strong th1 response necessary to pathogens following infection, in the context of a therapeutic vaccine. this approach could also be useful for a wide range of pathogens for which genomes have been partially or completely mapped. as described in this chapter, our group is actively pursuing the development of epitope-driven vaccines for hiv koita et al. 2006) , franciscella tularensis, helicobacter pylori, and smallpox. we have progressed from genome-derived epitope mapping to challenge studies in less than one year for some of these vaccine development programs. epitope-based and whole antigen idvs are now just beginning to enter clinical trials, but this relative disadvantage may be cured with the tincture of time. one reason for the relative paucity of idvs in clinical development is that the immunoinformatics tools for developing these vaccines have really only evolved in the last 10 to 15 years. the average length of time to develop a vaccine may be 20 years or more. while immunoinformatics tools are useful for accelerating the discovery and pre-clinical stage of vaccine development, testing vaccines in animal models and developing clinical trials is a lengthy process. it is likely that idv and epitope-based idv will begin to enter clinical trials and emerge on the market in greater numbers in 5 to 10 years. high-affinity t helper epitope induces complementary helper and apc polarization, increased ctl, and protection against viral infection quantitative and qualitative analyses of the immune responses induced by a multivalent minigene dna vaccine a multivalent minigene vaccine, containing b-cell, cytotoxic t-lymphocyte, and th epitopes from several microbes, induces appropriate responses in vivo and confers protection against more than one pathogen phase i trial of a therapeutic hiv type 1 vaccine, vacc-4x, in hiv type 1-infected individuals with or without antiretroviral therapy analysis of endogenous peptides bound by soluble mhc class i molecules: a novel approach for identifying tumor-specific antigens differential effects of flanking residues on presentation of epitopes from chimeric peptides analysis of total human 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allele-specific motifs revealed by sequencing of self-peptides eluted from mhc molecules consistent cytotoxic-t-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities a subdominant cd8(+) cytotoxic t lymphocyte (ctl) epitope from the plasmodium yoelii circumsporozoite protein induces ctls that eliminate infected hepatocytes from culture improving vaccines by incorporating immunological coadjuvants new cd4+ and cd8+ t cell responses induced in chronically hiv type-1-infected patients after immunizations with an hiv type 1 lipopeptide vaccine lipoarabinomannan induced cytotoxic effects in human mononuclear cells human memory ctl response specific for influenza a virus is broad and multispecific naturally processed hla class ii peptides reveal highly conserved immunogenic flanking region sequence preferences that reflect antigen processing rather than peptide-mhc interactions the structural requirements for class ii (i-ad)-restricted t 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vaccine design reduced susceptibility of recombinant polyclonal antibodies to inhibitory anti-variable domain antibody responses limited sequence evolution within persistently targeted cd8 epitopes in chronic human immunodeficiency virus type 1 infection confirmation of immunogenic consensus sequence hiv-1 t cell epitopes in bamako clinical validation of the ''in silico'' prediction of immunogenicity of a human recombinant therapeutic protein hla-and dose-dependent immunogenicity of a peptide-based hiv-1 immunotherapy candidate (vacc-4x) the role of cpg dinucleotides in dna vaccines b7-1 and b7-2 costimulatory molecule activate differentially the th1/th2 developmental pathways: application to autoimmune disease therapy cep: a conformational epitope prediction server the complete genome sequence of francisella tularensis, the causative agent of tularemia the kinetic stability of mhc class ii peptide complexes is a key parameter that dictates immunodominance identification of 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nucleoprotein and evaluation of their protective capacity in mice functional analysis of influenza-specific helper t cell clones in vivo. t cells specific for internal viral proteins provide cognate help for b cell responses to hemagglutinin differential ability of b cells specific for external vs. internal influenza virus proteins to respond to help from influenza virus-specific t-cell clones in vivo 3d-epitope-explorer (3dex): localization of conformational epitopes within three-dimensional structures of proteins tuberculosis control in the 21st century hla supertypes and supermotifs: a functional perspective on hla polymorphism differential t cell costimulatory requirements in cd28-deficient mice presentation of endogenous peptide/mhc class i complexes is profoundly influenced by specific c-terminal flanking residues ctl responses of hla-a2.1-transgenic mice specific for hepatitis c viral peptides predict epitopes for ctl of humans carrying hla-a2.1 definition of mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, n-terminal amino acid sequencing, and electrospray mass spectrometry several common hla-dr types share largely overlapping peptide binding repertoires antibiotic-refractory lyme arthritis is associated with hla-dr molecules that bind a borrelia burgdorferi peptide magnitude and diversity of cytotoxic-t-lymphocyte responses elicited by multiepitope dna vaccination in rhesus monkeys targeting a polyepitope protein incorporating multiple class ii-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by cd4+ cytotoxic t lymphocytes: a novel approach to vaccine design minimal epitoeps expressedin arecombinant polyepitope protein are processed and presented to cd8+ t cells : implications for vaccine design a comparison of standard immunogenicity assays for monitoring hiv type 1 gagspecific t cell responses in ad5 hiv type 1 gag vaccinated human subjects discrete cleavage motifs of constitutive and immunoproteasomes revealed by quantitative analysis of cleavage products strategy for isolating and sequencing biologically derived mhc class i peptides dendritic cell-based immunotherapy of cancer with carcinoembryonic antigen-derived, hla-a24-restricted ctl epitope: clinical outcomes of 18 patients with metastatic gastrointestinal or lung adenocarcinomas simultaneous cd8+ t cell responses to multiple tumor antigen epitopes in a multipeptide melanoma vaccine altered peptidase and viral-specific t cell response in lmp2 mutant mice the induction of virusspecific ctl as a function of increasing epitope expression: responses rise steadily until excessively high levels of epitope are attained an effective secondgeneration outer surface protein a-derived lyme vaccine that eliminates a potentially autoreactive t cell epitope proteolysis and class i major histocompatibility complex antigen presentation key: cord-104099-xhi0oxtr authors: hensen, l.; illing, p. t.; clemens, e. b.; nguyen, t. h. o.; koutsakos, m.; van de sandt, c. e.; mifsud, n. a.; nguyen, a.; szeto, c.; chua, b.; halim, h.; rizzetto, s.; luciani, f.; loh, l.; grant, e. j.; saunders, p.; brooks, a.; rockman, s.; kotsimbos, t. c.; cheng, a. c.; richards, m.; westall, g. p.; wakim, l. m.; loudovaris, t.; mannering, s. i.; elliott, m.; tangye, s. g.; jackson, d.; flanagan, k. l.; rossjohn, j.; gras, s.; davies, j.; miller, a.; tong, s.; purcell, a. w.; kedzierska, k. title: cd8+ t-cell landscape in indigenous and non-indigenous people restricted by influenza mortality-associated hla-a*24:02 allomorph date: 2020-10-05 journal: nan doi: 10.1101/2020.10.02.20206086 sha: doc_id: 104099 cord_uid: xhi0oxtr indigenous people worldwide are at high-risk of developing severe influenza disease. hla-a*24:02 allele, highly prevalent in indigenous populations, is associated with influenza-induced mortality, although the basis for this association is unclear. we defined cd8+ t-cell immune landscapes against influenza a (iav) and b (ibv) viruses in hla-a*24:02-expressing indigenous and non-indigenous individuals, human tissues, influenza-infected patients and hla-a*24:02-transgenic mice. we identified immunodominant protective cd8+ t-cell epitopes, one towards iav and six towards ibv, with a24/pb2550-558-specific cd8+ t-cells cells being cross-reactive between iav and ibv. memory cd8+ t-cells towards these specificities were present in blood (cd27+cd45raphenotype) and tissues (cd103+cd69+ phenotype) of healthy subjects, and effector cd27-cd45ra-pd-1+cd38+cd8+ t-cells in iav/ibv patients. our data present the first evidence of influenza-specific cd8+ t-cell responses in indigenous australians, and advocate for t-cell-mediated vaccines that target and boost the breadth of iav/ibv-specific cd8+ t-cells to protect high-risk hla-a*24:02-expressing indigenous and non-indigenous populations from severe influenza disease. newly emerging respiratory viruses pose a major global pandemic threat, leading to significant morbidity and mortality, as exemplified by 2019 sars-cov2, avian influenza h5n1 and h7n9 viruses, and the 1918-1919 h1n1 pandemic catastrophe. influenza a viruses (iav) can cause sporadic pandemics when a virus reassorts and rapidly spreads across continents, causing millions of infections and deaths 1 . additionally, seasonal epidemics caused by co-circulating iav and influenza b viruses (ibv) result in 3-5 million cases of severe disease and 290,000-650,000 deaths annually 2, 3 . severe illness and death from seasonal and pandemic influenza occur disproportionately in high risk individuals, including indigenous populations. this is most evident when unpredicted seasonal or pandemic viruses emerge in the human circulation. during the 1918-1919 influenza pandemic, 100% of alaskan adults died in some isolated villages, while only school-aged children survived 4 . western samoa was the hardest hit with a total population loss of 19-22% 5 . as many as 10-20% of indigenous australians died from pandemic influenza in 1919 6 in comparison to <1% of other australians, with some reports showing up to 50% mortality in indigenous australian communities 7 . during the 2009 a/h1n1 influenza pandemic, indigenous populations worldwide were more susceptible to influenza-related morbidity and mortality. hospitalization and morbidity rates were markedly increased in indigenous australians 9,10 , with 16% of hospitalised pandemic h1n1 (ph1n1) patients in australia being indigenous. the relative risk for indigenous australians compared to non-indigenous australians for hospitalization, icu admission or death was 6.6, 6.2, or 5.2 times higher, respectively 11 . this was mirrored in indigenous populations globally, including american indians and alaskan native people (4fold higher mortality rate compared to non-indigenous americans) 12 , native brazilians (2fold higher hospitalization rate) 13 , new zealand maori (5-fold higher hospitalization rate) and pacific islanders (7-fold higher hospitalization rate) 14, 15 . although the impact of influenza pandemics is more pronounced in indigenous populations globally, these disproportionate hospitalization rates also occur during seasonal infections. during 2010-2013, indigenous australians had increased influenza-related hospitalizations across all age groups (1.2-4.3-fold higher compared to non-indigenous) 16 . indigenous populations, especially australians and alaskans, are also predicted to be at greater risk from severe disease caused by the avian-derived h7n9 influenza virus, with mortality rates being >30% and hospitalization >99% in china 17 . while higher influenza infection rates could relate to overcrowded living conditions, increased severity and prolonged hospitalization most likely reflect differences in pre-existing immunity that facilitates recovery. however, the underlying immunological and host factors that account for severe influenza disease in indigenous individuals are far from clear. antibody-based vaccines towards variable surface glycoproteins, hemagglutinin (ha) and neuraminidase (na), are an effective way to combat seasonal infections, yet they fail to provide effective protection when a new, antigenically different iav emerges 20 . in the absence of antibodies, recall of pre-existing cross-protective memory cd8 + t-cells minimizes the effects of a novel iav, leading to a milder disease after infection with distinct strains [21] [22] [23] [24] [25] [26] . such pre-existing memory cd8 + t-cells provide broadly heterotypic or crossreactive protection and can recognize numerous the iav, ibv and influenza c viruses capable of infecting humans 27 , promoting rapid host recovery. during the 2013 h7n9 iav outbreak in china, recovery from severe h7n9 disease was associated with early cd8 + tcell responses 24, 28 . patients discharged early after hospitalization had early (day10) robust h7n9-specific cd8 + t-cells responses, while those with prolonged hospital stays showed late (day19) recruitment of cd8 + and cd4 + t-cells. thus, with the continuing threat of unpredicted influenza strains, there is a need for targeting cellular immunity that provides effective, long-lasting and cross-strain protective immunity, especially for high risk groups such as indigenous populations. however, despite the heavy burden of disease in indigenous communities, there is scant data on immunity to influenza viruses in indigenous populations from around the world. as cd8 + t-cell recognition is determined by the spectrum of human leukocyte antigens (hlas) expressed in any individual, and hla profiles differ across ethnic groups, defining t-cell epitopes restricted by hlas predominant in some indigenous populations is necessary to understand pre-existing cd8 + t-cell immunity to influenza. we previously analyzed the hla allele repertoire in indigenous australians 29 and found that hla-a*24:02 (referred to as hla-a24 hereafter), an hla associated with influenza-induced mortality during the 2009-ph1n1 outbreak 30 , is the second most prominent hlas in indigenous australians 29, 31 . hla-a24 is also common to other indigenous populations highly affected by influenza 17 . thus, analysis of prominent influenza-specific cd8 + t-cell responses restricted by hla-a24 is needed to understand the relationship between this allele and disease susceptibility. these specificities will also inform strategies to prime effective t-cell immunity in vulnerable communities. here, we defined cd8 + t-cell immune landscapes against iav and ibv, restricted by the mortality-associated hla-a24 allomorph. we identified iav-and ibv-specific hla-a24 immunopeptidomes and screened . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020. 10 .02.20206086 doi: medrxiv preprint immunogenicity of novel peptides in hla-a24-expressing mice, peripheral blood of indigenous and non-indigenous hla-a24 + healthy and influenza-infected individuals, and human tissues. our studies provide evidence of the breadth of influenza-specific cd8 + t-cell specificities restricted by a mortality-associated risk allomorph hla-a24. these findings have implications for the incorporation of key cd8 + t-cell targets in a t-cell-mediated vaccine to protect indigenous people globally from unpredicted influenza viruses. as hla-a24 has been linked to ph1n1-related mortality 30 , we first determined hla-a24 distribution in indigenous and non-indigenous populations worldwide using the published allele frequency database. compared to the 10% global distribution of hla-a24, the detected frequencies of hla-a24 were the highest in oceania (37%), north-east asia (32.9%), australia (21.4%) and central and south america (20.6%) (fig. 1a) . this was mainly due to a particularly high hla-a24 prevalence in indigenous populations in those regions, especially in the pacific. hla-a24 was highly prevalent in indigenous taiwan paiwan (96.1%), papa new guinea karimui plateau pawaia (74.4%), new caledonia (60.7%), alaskan yupik (58.1%), new zealand maori (38%), american samoans (33%), chile easter island (35.8%) and some australian indigenous people (24%) (fig. 1a) , which highlights its key importance in shaping cd8 + t-cell immunity in indigenous populations. to define influenza-specific cd8 + t-cell responses in indigenous australians, we recruited 127 participants from the northern territory, australia into the lift (looking into influenza t-cell immunity) cohort 29 . the mean age of the participants was 39 years, with a standard deviation of 14 years and 58% of female participants. 36% of the lift donors expressed at least one hla-a24 alleles, with 33% of those being hla-a24 homozygous ( fig. 1b) . notably, hla-a24 was most commonly expressed with hla-a*11:01, -a*34:01, -b*13:01, -b*15:21, -b*40:01, -b*40:02, -b*56:01, -c*04:03, -c*03:03, -c*04:01 and -c*04:03 in indigenous australians, and were less expressed with alleles common in caucasian populations, such as hla-a*01:01, -a*02:01, -b*07:02 and -b*08:01 (fig. 1c) . a handful of iav-specific hla-a24-restricted epitopes have been described [32] [33] [34] [35] (fig. 1d) . we aimed to validate the immunogenicity of these epitopes by probing memory cd8 + t-cells within peripheral blood mononuclear cells (pbmcs) of healthy non-indigenous hla-a24-. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint expressing donors (supplementary table 1 ) using an in vitro peptide stimulation assay ( fig. 1e-g) . only 3 out of 12 peptides (pb1 498-505 , pb1 496-505, pa 130-138 ) induced cd8 + t-cell proliferation and ifn-γ/tnf production in a limited number of donors (3/5, 3/5 and 1/5, respectively) (fig. 1e,f) . we deduced that the minimal epitope for pb1 496-505 responses came from the pb1 498-505 epitope. the specificity of pb1 498-505 -specific cd8 + t-cell responses, observed in multiple donors, was further verified by a24/pb1 498-505 tetramer staining on both in vitro-cultured a24/pb1 498-505 cd8 + t-cell lines and a24/pb1 498-505 + cd8 + t-cells detected directly ex vivo by tetramer enrichment (fig. 1g) . thus, while 3 of the previously published peptides elicited ifn-γ responses in a selected number of hla-a24-expressing individuals, we sought to determine whether as yet unidentified epitopes might provide more robust iavspecific cd8 + t-cell responses in hla-a24-expressing individuals. to identify new a24/influenza-derived epitopes, we utilized an immunopeptidomics approach to sequence hla-bound peptides on influenza-infected cells by liquid chromatography with tandem mass spectrometry (lc-ms/ms) 27 in total, 12 immunopeptidome data sets containing hla-a24-restricted peptides were generated including 3 from uninfected cir.a24 cells, 5 from hkx31-infected and 4 from b/malaysia-infected cir.a24 cells (supplementary fig. 1a, supplementary data 1 ). an additional 3 data sets for endogenous hla-i of cir cells (cir w6/32 isolation of hla-b*35:03 and hla-c*04:01 after 16 hours hkx31 infection; cir.a24 -dt9 isolation of hla-c*04:01 from uninfected cells and after 12 hours hkx31 infection) and 2 data sets for . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint endogenous hla-ii (cir.a24: hla-dr12, -dpb1*04:01,04:02 and -dq7 from uninfected and 12 hours hkx31 infection) were also generated as comparators to help establish true hla-a24 binders (supplementary fig. 1b,c) . comparisons to previously identified b/malaysia-derived hla ligands for cir were also used 27 to distinguish of hla-b*35:03 and hla-c*04:01 binding peptides from those binding to hla-a*24:02. across the 12 data sets, a total of 9051 non-redundant peptide sequences were assigned as hla-i ligands using a 5% false discovery rate (fdr). as expected for hla-i ligands, the majority of peptides were 9-11 amino acids in length but dominated by 9mers (fig. 2a) . consistent with the hla-a24 peptide-binding motif generated by netmhc4.0 40,41 motif viewer, enrichment of tyr/phe at p2 and phe/leu/ile at p9 were observed (fig. 2b) . peptides binding the endogenous hla-i of cir were not removed in this analysis due to the similar preference of hla-c*04:01 for 9mer peptides possessing phe/tyr at p2 and phe/leu at p9 which may result in shared ligands (supplementary fig. 1d ). to maximize identification of potential virus-derived peptides, assignments to the viral proteome or 6-frame translation of the viral genome were considered without a fdr cutoff. instead, lack of appearance in uninfected data sets and predicted binding affinity (netmhcpan4.0 [42] [43] [44] for hla-a24 were used determine likely candidate epitopes. thus, 52 hkx31-derived and 48 b/malaysia-derived peptides were identified as potential hla-a24-restricted epitopes (fig. 2c) , of which 26 iav-derived and 29 ibv-derived peptides were identified at a 5% fdr (supplementary data 1). the identified peptides spanned the viral proteomes including frame-shift proteins, representing 6 iav proteins and 9 ibv proteins (fig. 2d,e) . interestingly, most hkx31-derived ligands mapped to pb2>pb1>ha viral proteins and none were observed from na or m1, while b/malaysia-derived ligands predominantly mapped to np/ha>na. during the time course analyses, broadest peptide identification was achieved for both viruses between 8-12 hours post-infection, while no influenza-derived peptides were identified at 2 hours post-infection, and those identified at 4 hours were of lower confidence (fig. 2f,g, supplementary data 1) . 10 potential hkx31-derived ligands were also identified for each of hla-b*35:03 and hla-c*04:01 based on predicted binding and/or appearance in control data sets ( supplementary fig 1e, supplementary data 1) . furthermore, analysis of the peptides presented by hla-ii molecules showed domination of the virus-derived immunopeptidome by ha ( supplementary fig 1f, supplementary data 1) , as previously observed for ibv 27 . a selection of 48 iav and 41 ibv peptides were synthesised for subsequent screening (supplementary tables 2,3, supplementary data 1) . notably, most synthetic peptides . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint showed highly similar fragmentation patterns and retention times to the discovery data, supporting the original identifications (supplementary data 1). to determine the immunogenicity of novel iav-and ibv-derived peptides during primary and secondary influenza virus infection in vivo, we utilized hla-a24-expressing transgenic (hhd-a24) mice 45 table 2 ). the responses were detected by 5-hr ex vivo peptide stimulation and measurement of ifn-γ production by ics. our data revealed that a24/iav-specific cd8 + t-cell responses were immunodominant (>5% ifn-γ + of cd8 + t-cells) towards 2 pb1and 3 pb2-derived peptides: pb1 216-224 (mean of 10.2% ifnγ + of cd8 + cells in spleen, 16 (fig. 3b) . cd8 + t-cell responses towards the marginal epitopes observed in the primary infection were no longer detected. while the reason for the loss of ha 248-259 could be explained by sequence variation between hkx31 and pr8 (iywtivkpgdvl vs. yywtlvkpgdti) all internal proteins are shared . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint between both viruses. analysis of influenza-specific cd8 + t-cell numbers showed significant reductions in epitope-specific cd8 + t-cells for 9 out of 11 specificities (np [39] [40] [41] [42] [43] [44] [45] [46] [47] (supplementary fig. 2) . thus, our in vivo screening in hhd-a24 mice identified 6 iav derived immunogenic peptides during primary and secondary iav infection, with prominent cd8 + t-cell responses being heavily biased towards pb1-and pb2-derived peptides (supplementary table 2 ). this is of key importance as the current t-cell vaccines in clinical trials focus mainly on internal proteins like np and m1 46-50 which may be poorly immunogenic in hla-a24expressing individuals at risk of severe influenza disease. while the cd8 + t-cell responses towards ibv have been studied in detail for hla-a*02:01expressing individuals 27 , there remains a lack of known cd8 + t-cell epitopes for other hlas. here, we determined the immunogenicity of newly identified ibv-derived peptides is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint of cd8 + t-cells between both variants. all other immunogenic epitopes were conserved between both strains. in contrast to iav infection, the total number of epitope-specific cd8 + t-cells for all immunogenic epitopes after secondary challenge remained comparable in the spleen (supplementary fig. 2) . thus, our data identified prominent a24/cd8 + t-cell responses directed towards ibv during primary and secondary influenza virus infection in hhd-a24 mice (supplementary table 3 ). having identified prominent iav-derived cd8 + t-cell epitopes towards the primary and secondary infection in hhd-a24 mice, it was of key importance to define immunodominant table 4 ) was always included in the same pool as the wildtype peptide identified in the immunopeptidome studies. we observed cd8 + t-cell responses towards pools 1 and 2 via an ifn-γ/tnf ics assay (fig. 4a) , and subsequently cell cultures from those pools were restimulated with individual peptides (+variants) to map the immunogenic epitopes. in 5/5 indigenous donors tested, cd8 + t-cell responses were dominated towards is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint ns2 98-106 in 3/5, pb1 216-224 in 3/5, pb2 549-557 in 1/5, ha 176-184 in 3/5 and pb2 703-710 in 3/5), of which pb1 216-224 , pb2 549-557 and pb2 703-710 were also detected in hhd-a24 mice. interestingly, only 2/4 immunodominant epitopes observed in the indigenous donors elicited comparably robust responses in 5 non-indigenous donors screened (published epitopes: pb1 498-505 median 0.93% vs 1.1%, pb1 496-505 median 0.64% vs 0.8%), while the other two epitopes, pa 649-658 and np [39] [40] [41] [42] [43] [44] [45] [46] [47] were poorly immunogenic in non-indigenous donors who instead responded well to the pb2 549-557 epitope, absent in 4/5 indigenous donors. such differential epitope preference and immunodominance hierarchies between indigenous and non-indigenous donors is perhaps influenced by different hla co-expressions or infection history. following identification of novel ibv-derived peptides in hhd-a24 mice, we defined cd8 + t-cell responses towards ibv peptide pools, comprising 41 peptides identified by mass spectrometry (supplementary table 3 table 4 ), in hla-a24-expressing indigenous and non-indigenous donors. in accordance with the hhd-a24 mouse data, we found broad a24/cd8 + t-cell responses directed towards pool 10, which mapped 6 major immunogenic epitopes spanning 5 different proteins (np 164-173 /np 165-173 , na 32-40, pb2 550-558, pa 457-465 , ha 552-560 and pb1 503-511 ) (fig. 4b) . cd8 + t-cell responses to these peptides were found in 7/9, 8/9, 6/9, 6/9, 5/9 and 5/9 of indigenous and 5/5, 5/5, 4/5, 5/5 and 4/5 of non-indigenous donors, respectively, with comparable ifnγ + cd8 + t-cell frequencies between indigenous and non-indigenous donors. as our experiments examined the immunogenicity of a24/cd8 + t-cells following in vitro peptidedriven expansion, we further verified these novel ibv epitopes in non-indigenous donors by (fig. 4c) . interestingly, the same epitopes were observed in hhd-a24 mice after ibv infection ( fig. 3c.d) . collectively, out of 41 newly identified hla-a24-binding ibv epitopes by immunopeptidomics, we confirmed a total of 9 (22%) immunogenic epitopes after screening hla-a24-expressing mice and humans. of these, 3 were found in both humans and mice (np 164-173 /np 165-173 , na 32-40 , pb2 550-558 ), 3 were only found in humans (ha 552-560 , pa [457] [458] [459] [460] [461] [462] [463] [464] [465] . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint and pb1 503-511 ), and the remaining 3 were only found in mice (pb2 245-253 , na 213-221 & np 392-400 ). the b/malaysia/2505/2004 virus used here is from the victoria (vic) lineage, however, there is another ibv lineage that commonly co-circulates and infects humans called the yamagata (yfspiritf v2 (yam only)) following ics assay (fig. 4d) . likewise cross-reactivity between vic lineage and a variant found in the yam lineage was also observed with the pb2 550-558 variants (tyqwvlknl (variant1, both vic and yam); tyqwvmknl (variant 2, yam only)) in the virus-expansion system, but 3/4 donors did only respond to v1 after peptide expansion (fig. 4d) . we have previously reported cross-reactivity towards iav and ibv (as well as icv) in the hla-a2 model with a single epitope sequence 27 . here, none of the identified hla-a24 iav and ibv epitopes showed 100% sequence identity between strains. instead, we identified a potential hla-a24-restricted iav/ibv cross-reactive candidate, the immunogenic iav pb2 549-557 tyqwiirnw epitope. this epitope shares 55% amino acid is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint respectively (supplementary table 5 ) with clear electron density for each peptide ( supplementary fig. 5 ). the 9mer a/pb2 549-557 peptide adopted a canonical extended conformation within the cleft of hla-a24, with anchor residues at p2-tyr and p9-trp, and a secondary anchor residue at p5-ile. solvent exposed residues were at p4-trp, p6-ile, p7-arg and p8-asn, representing a large surface accessible for tcr interaction. the p9-trp of the peptide formed a network of interactions with hla-a24 tyrosine residues at positions 116, 118 and 123 as well as the leu95 (supplementary fig. 3a-c) , likely assisting with stabilizing the complex reflected in the higher stability observed for the hla-a24-a/pb2 549-557 complex than with other peptides (supplementary table 5 ). the b/pb2 550-558 peptide differed to the a/pb2 549-557 peptide at positions 5 (ile to val), 6 (ile to leu), 7 (arg to lys) and 9 (trp to leu) (fig. 4f) . both peptides shared the same anchor residue at p2-tyr and similar solvent exposed residues (except for p7-lys) but differed at pω (p9). as leu possessed a shorter side chain than tyr at pω, the ibv peptide was not buried as deeply into the f pocket, which may explain the lower stability observed for the b/pb2 550-558 peptide (tm of 57°c) compared to a/pb2 549-557 (tm of 62°c) (supplementary table 5 ). structural overlay of hla-a24 presenting the a/pb2 549-557 and b/pb2 550-558 peptides showed that the antigen-binding cleft and both peptides adopted a similar conformation with an average root mean square deviation (r.m.s.d.) of 0.31 å and 0.37 å, respectively (for cα atoms) (fig. 4f) , consistent with t-cell cross-reactivity observed towards these two peptides. although the 11mer a/pb2 549-559 generated similar responses to the 9mer a/pb2 549-557 in hhd-a24 mice (fig. 3a,b) , it was not immunogenic in peptide-pool screening in humans (supplementary table 2 pool 4) (fig. 4a) as perhaps the minimal 9mer epitope was not exposed for t-cell recognition, due to the two additional c-terminal residues (p10-glu and p11-thr) (supplementary fig. 3a,b) . similar to the 9mer peptide conformation p2-tyr and p9-trp of the 11mer pb2 549-559 act as primary anchor residues buried in the hla-a24 antigen-binding cleft with the structural overlay of the peptides showing an r.m.s.d. of 0.48 å (supplementary fig. 3a,b) .strikingly, the extra p10-glu and p11-thr residues of the 11mer extended outside the antigen-binding cleft, creating an unusual conformation that disturbed the interaction between the peptide and the hla-a24 lys146 at the c-terminal of the cleft. the lys146 residue is a conserved residue in hla molecules that helps stabilise the phla complex 51 . in the 9mer pb2 549-557 peptide, lys146 interacts with the carboxylic group of the . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint pω residue (supplementary fig. 3d) , however this interaction is lost in the 11mer due to the presence of the extra two residues p10-glu and p11-thr. (supplementary fig. 3e) , thereby likely decreasing the phla stability compared to the shorter a/pb2 549-557 peptide (supplementary table 5 ). thus, the bulged conformation of the extra residues in the a/pb2 549-559 may represent a challenge for tcrs interacting with the c-terminal end of the peptide. in compliance with the in vitro data, the structural data support the potential crossreactivity of cd8 + t-cells between the a/pb2 549-557 and the b/pb2 550-558 , verifying our previous findings of broad cd8 + t-cell immunity against influenza virus infections. we observed robust comparable mouse (fig. 3c,d) and human (fig. 4b) fig. 4) . p2-phe and p9-phe anchor residues of the 9mer b/np 165-173 were buried deep inside the hydrophobic b and f pockets of the hla (supplementary fig. 4a) . three residues were exposed to the solvent for possible tcr recognition (p1-tyr, p6-arg, p8-thr). the p5-ile and p7-val of this 9mer peptide were partially-buried (supplementary fig. 4e ). structural overlay of 9mer and 10mer b/np peptides were different due to the 10mer's extra residue at the n-terminus (r.m.s.d. of 1.36 å), which shifted the anchor residues (supplementary fig. 4b,c) . the substitution of p2-tyr (np 164-173 ) for p2-phe (np 165-173 ) occurred without major structural rearrangement, as both residues were large aromatic residues filling the b pocket (supplementary fig. 4f) . however, the additional residue changed the secondary anchor residue at p3 from a small p3-ser (np 165-173 ) to a large p3-phe (np 164-173 ) (supplementary fig. 4g) . the larger p3-phe might stabilize the b pocket of the hla-a24 better than the small p3-ser, and therefore could explain the 7°c higher tm observed for the np 164-173 than the np 165-173 in complex with hla-a24 (supplementary table 5 ), which could also reflect the immunogenicity of this peptide. the largest structural difference between the two peptides was observed at the centre of the peptide (p6/7-arg) with a maximum displacement of 3.9 å for the cα atom (supplementary fig. 4d) . the p7-. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. fig. 4c) and was a prominent feature for potential tcr interaction of the 10mer peptide, contrasting with the hydrophobic nature of the 9mer np 165-173 peptide. thus, the structures of hla-a24 presenting the two np peptides showed that, despite being overlapping peptides that differ only by one residue, the np 164-173 and np 165-173 peptides adopt different structural conformations. as a result, both peptides exposed different residues to the solvent, and hence would most likely be recognized by different tcrαβ repertoires. to determine the protective capacity of the novel cd8 + t cell epitopes in hhd-a24 mice, we performed a proof of principle experiment and vaccinated mice with 3 immunogenic ibv peptides (np 164 , np 392 , na 32 ) using a well-established prime/boost approach 27 , then infected mice i.n. with 1x10 3 pfu b/malaysia (fig. 5a) . vaccination with hla-a24-restricted peptides resulted in significant protection against ibv. this was shown by decreased disease severity on d4, d5 and d6 after ibv infection as measured by the body weight loss (fig. 5b; p<0 .05) as well as a significant ~89% reduction in viral titers in the lung on d7 after ibv infection when compared to the mock-immunized group (p<0.05) (fig. 5c) . additionally, there was a significant decrease (p<0.05) in the levels of inflammatory cytokines (mip-1β, mip-1a, rantes) in d7 bal of peptide-vaccinated mice in comparison to the mock-immunised animals (fig. 5d) . thus, cd8 + t cells directed at our novel hla-a24-restricted ibv-specific epitopes provide a substantial level of protection against influenza disease, as they can markedly decrease body weight loss, accelerate viral clearance and reduce the cytokine storm at the site of infection. having identified the prominent iav and ibv cd8 + t-cell specificities for indigenous and non-indigenous hla-a24 + -individuals, we sought to determine whether cd8 + t-cells specific for our newly identified epitopes were recruited and activated during acute influenza virus infection. we generated peptide/hla-a24-tetramers to the most immunogenic iav (a/pb1 498-505 ) and newly characterised ibv epitopes (np 165-173 and na [32] [33] [34] [35] [36] [37] [38] [39] [40] ). these reagents allow direct ex vivo detection of iav-and ibv-specific cd8 + t-cells using tetramer-. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint associated magnetic enrichment (tame) 52 in both healthy and influenza-infected individuals (fig. 6a, left panels) . in healthy non-indigenous and indigenous donors, ex vivo mean precursor frequencies for a/pb1 498-505 + and b/np 165-173 + cd8 + t-cells, were 4x10 -5 and 1x10 -5 of cd8 + t-cells, respectively (fig. 6b) . non-indigenous b/na 32 + frequencies were 1.8-9x10 -5 of cd8 + t-cells. all tetramer + frequencies fell within the range of previously published frequencies for memory iav-or ebv-specific cd8 + t-cells 52,53 . interestingly, as per our analysis in hla-a*02:01-positive influenza patients 27 6d ). this difference was not observed in the total non-specific cd8 + t-cell population. importantly, hla-a24-restricted influenza-specific cd8 + t-cells against a/pb1 498 and a/np 165 were detected in multiple healthy human tissues directly ex vivo (fig. 6a , right panels). a/pb1 498 -specific cd8 + t-cells were detected in the lung, spleen and tonsil at frequencies ranging 1.5x10 -7 to 4.5x10 -5 of total cd8 + t-cells (n=6 data points), but was not detected in the pancreatic lymph node (panln) of one donor that had detectable a/pb1 498-505 -. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint specific cd8 + t-cells in the spleen (fig. 6e; data not shown) . b/np 165 -specific cd8 + t-cells were found across all the tissues (lung, spleen, tonsil, panln, n=7 data points) at frequencies between 1.1x10 -6 to 1.7x10 -4 . in human lung, iav/ibv-specific cd8 + t-cells had large populations of cd69 -cd103 + and cd69 + cd103 + tissue-resident memory (t rm ) t-cells (a/pb1 498-505 : 75% and 21%, b/np 165-173 : 41% and 47% of tetramer-specific cd8 + t-cells, respectively) (fig. 6f) . secondary lymphoid organs (slos) were predominantly cd69 -cd103circulating effector memory cells (range 23.8-85.7%). our findings demonstrate the presence of highly activated influenza-specific cd8 + tcells against the published a/pb1 498 epitope and the ibv epitopes identified here in hla-a24 + patients with acute influenza infection and memory pools across different human tissues, highly relevant to the indigenous population. binding it was apparent from the tetramer-enrichment assays that some healthy donors contained large populations of hla-a24-tetramer-binding cd8 + t-cells prior to enrichment (up to 6% of cd8 + t-cells) (fig. 7a) . this appeared to be donor-dependent but not entirely cd8 + t-cell specificity-dependent. we found such oversized (0.32%-6.73% in unenriched pbmcs) tetramer + cd8 + t-cell populations for a/pb1 498 in 10 out of 23 donors and in 14 out of 26 donors for b/np 165 tetramers, but not for b/na 32 (0/4 donors), which was further enriched with tame (fig. 7a,b) . it is important to note that our tetramer analyses in figure 5 excluded this oversized low intensity staining tetramer-binding cd8 + t-cell population. such oversized tetramer-binding cd8 + t-cell population could potentially be a unique hla-a24tetramer binding phenomenon occurring in selected donors and hence potentially impair tcr-specific cd8 + t-cell binding. therefore, we sought to better understand hla-a24tetramer binding in donors with conventional and largely oversized hla-a24-tetramer cd8 + t-cell populations. phenotypic analyses comparing tetramer-enriched fractions revealed that tetramer binding cd8 + t-cells of donors with oversized populations were predominantly of the cd45ra + cd27effector (t emra ) phenotype (mean 73.3% and 71.7% for pb1 498 and np 165 , respectively), while those from donors with conventional tetramer + cd8 + t-cells were predominantly t cm (mean 31.6 and 45.5%), t em (10.3 and 8.3%) and t naive (12.5 and 22.6%) in phenotype (fig. 7c) . to determine factors underlying this phenomenon, we performed scrnaseq on single-cell-sorted tame-enriched a/pb1 498-505 + cd8 + t-cell populations from . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint two donors with oversized populations (non-lift 8 and 12) and two donors with conventional-size populations (non-lift 14 and 10) (fig. 7d) . unsupervised hierarchical clustering analysis revealed three gene clusters (fig. 7e) . highly expressed genes from hypothesised that a kir was interacting with the peptide/hla-a24 complex. kir are expressed by a proportion of cd8 + t-cells 56 and kir3dl1 in particular has been previously shown to bind some but not all a24 pmhc tetramers 57 implying a degree of selectivity in the interaction. staining for kir3dl1 revealed its expression on cd27 -cd8 + t-cells, with the highest frequency of kir3dl1 + cells detected in the t emra population in a donor that exhibited strong pre-tame tetramer binding (fig 7f) . co-staining with the a/pb1 [498] [499] [500] [501] [502] [503] [504] [505] tetramer showed that all tetramer-binding cd8 + t-cells were positive for kir3dl1, indicating that kir3dl1 could potentially be binding to the tetramers (fig 7f) . blocking of kir3dl1 prior to tetramer-staining markedly reduced the oversized population after tame enrichment, to the levels of conventional tetramer + cd8 + t cell pools, revealing the true a24/pb1 498 -specific cd8 + t-cell population (fig. 7g) . thus, much of the oversized population comprises tetramer-binding kir3dl1 + cd8 + t-cells with other tcr specificities. future studies are needed to understand whether kir3dl1 binding of peptide-hla-a24 complexes are competing with tcr interactions to mount robust peptide-hla-a24-specific cd8 + t-cell responses, thus impacting on influenza-specific immunity in indigenous and non-indigenous hla-a24-expressing people at risk of severe influenza disease. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. 30 . our data reveal that the variable ha and na viral glycoproteins play a minimal role in hla-a24-restricted cd8 + t-cell immunity to iav. instead, the focus on epitopes from pb1 and pb2 that are well conserved . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint across virus strains circulating in south-east asia and australia suggests that the prominent hla-a24-restricted cd8 + t-cell responses are likely to confer broad cross-reactive immunity to iav. this is of key importance as the current t-cell vaccines in clinical trials focus mainly on structural proteins like np, m1 and m2, and would therefore not elicit crossprotective cd8 + t-cell responses in hla-a24 + individuals at risk of severe influenza disease. in contrast to iav, the protein origins of ibv peptides presented by hla-a24 differed greatly. from 41 ibv-derived peptides, the majority originated from np (9, 22% of total), while 8 were from the ha and na (total of 39% for surface glycoproteins). in terms of immunogenicity, our data from transgenic mice showed that the immunogenic hla-a24binding peptides were predominantly derived from np (40% of response) and na (40% of the response). more importantly, numbers of cd8 + t-cells directed towards our novel epitopes were preserved during secondary ibv challenge, indicating optimal memory establishment and recall, which contrasted with the situation for secondary iav challenge. non-indigenous human donors were also targeted towards np, with np 165-173 and np [164] [165] [166] [167] [168] [169] [170] [171] [172] [173] being prominent cd8 + t-cell specificities alongside cd8 + t-cell epitopes derived from na, ha, pb2 and pa ( table 6 ). the breadth of the hla-a24-restricted ibv response highlights the power of identifying epitopes with our mass-spectrometric approach and might explain, at least partially, why indigenous populations have not been reported to be at risk from severe ibv disease. as for iav, ibv epitope-specific cd8 + t-cells were activated during acute ibv infection in hla-a24 + individuals and were found distributed across tissues including the lung in non-infected individuals. broadly cross-reactive cd8 + t-cell responses that provide universal immunity across multiple strains or subtypes of influenza viruses have a crucial role in protection from severe influenza disease 27 . here we demonstrate cross-reactive responses between ibv lineages for the b/np 165-173 peptide, as well as cross-reactive iav/ibv responses between the a/pb2 549-557 peptide and ibv pb2 550-558 variants in hhd-a24 transgenic mice (data not shown) and humans. the a/pb2 549-557 peptide is conserved between h3n2 and h1n1 iavs 62 , and shares 55% amino acid identity with the cross-reactive ibv pb2 550-558 variants. structures of hla-a24 with a/pb2 549-557 and b/pb2 550-558 showed that the antigen-binding cleft and both peptides adopted a similar conformation, providing a structural basis for t-cell crossreactivity between these epitopes. interestingly, ibv was more effective than iav at expanding cross-reactive cd8 + t-cells, suggesting that infection history may play a role in . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint cross-reactive cd8 + t-cell immunity would provide at least some level of protection against distinct influenza variants, even strains with pandemic potential. such a vaccine would minimise influenza-related deaths in global populations, especially high-risk groups, which includes hla-a24-expressing indigenous and non-indigenous people. our comprehensive analysis of peptide presentation and immunogenicity across mouse and human hla-a24 models defines the candidate ibv and iav peptides needed for a cd8 + t-cell-targeting vaccine that is effective in hla-a24 + individuals. understanding how best to augment these key responses to confer stronger protective immunity is the next step. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. from (d,e) . in all panels, n=number of peptides. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. virus-expanded pbmcs were restimulated by addition of virus-infected cir.a24 cells on day 8 at a 1:10 ratio. cells were then incubated for a total 10-15 days in rf10 media with 10u/ml . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint tablet; roche molecular biochemicals) and incubation at 4°c for 1 hour with slow rotation. lysates were cleared by ultracentrifugation and hla isolated by immunoaffinity purification using protein-a-sepharose-bound antibodies as described 38, 73 . antibodies were either w6/32 (pan class i) alone or sequential dt9 (hla-c specific), w6/32 (pan class i) and mixed class ii (equal amounts lb3.1, spv-l3 and b721, capturing hla-dr, -dq and -dp, respectively). peptide/mhc complexes were dissociated, and fractionated by reversed phase high performance liquid chromatography (rp-hplc) as described 27, 38, 74 . 500µl fractions were collected throughout the gradient, and the peptide containing fractions combined into 9 pools, vacuum-concentrated and reconstituted in 15µl 0.1% formic acid (honeywell) in optima™ lc-ms water. reconstituted fraction pools were analysed by lc-ms/ms using a sciex 5600+ tripletof mass spectrometer equipped with a nanospray iii ion source as previously were based on the best hypothesis for distinct peptides. sequence motifs were generated utilizing peptides assigned at confidences greater than that required for a 5% fdr using seq2logo2.0 69 (default settings). likely hla-a*24:02 binders were determined based on appearance across the experiments/antibodies and predicted binding (netmhcpan4.0). for peptides identified in their native form (and lacking cys residues) that were synthesised for functional analysis, fragmentation patterns and retention times of representative spectra were compared to the synthetic and the quality of the match described (supplementary data 1) . hla-a*24:02 hhd mouse studies. all mouse studies were overseen by the university of melbourne ethics committee (#171408). hhd-a24 mice were generated by françois lemonnier as described previously 42 cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint injected emulsified adjuvant without peptides. two weeks after prime, mice were boosted and challenged with 1x10 3 pfu b/malaysia/2506/2004 intranasally 7 days after boost. on day 6 and 7 after infection lungs were isolated to determine viral load with a plaque assay as described before 27 . cytokines in the bronchoalveolar lavage were assessed with the bd cytometric bead array kit as described elsewhere 27 . crystallization, data collection and structure determination. crystals of the phla-a*24:02 complexes were grown by the hanging-drop, vapour-diffusion method at 20°c with a protein/reservoir drop ratio of 1:1 with seeding at a concentration of 6 mg/ml in the . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 5, 2020. thermal stability assay. thermal shift assays were performed to determine the stability of each phla-a*24:02 complex using fluorescent dye sypro orange to monitor protein unfolding. the thermal stability assay was performed in the real time detection system (corbett rotorgene 3000), originally designed for pcr. each phla complex was in 10 mm tris-hcl ph8, 150 mm nacl, at two concentrations (5 and 10 mm) in duplicate, was heated from 25 to 95°c with a heating rate of 1°c/min. the fluorescence intensity was measured with excitation at 530 nm and emission at 555 nm. the tm, or thermal melt point, represents the temperature for which 50% of the protein is unfolded. tetramer-associated magnetic enrichment in humans tame was performed on pbmcs (7.5x10 6 -2.7x10 8 ) of healthy, iav-or ibv-infected donors, as well as lymphocytes isolated from tonsils, lung and pancreatic lymph nodes (panln) to detect cd8 + t-cells specific for iav and ibv as described previously 25, 27 . pmhc-i monomers were made in-house 79 and conjugated at an 8:1 molar ratio to pe-or apc-labelled streptavidin (sa) to generate tetramers. cells were fcr-blocked and stained with apc or pe conjugated tetramers at a 1:100 dilution for 1h at rt, washed twice then table 6 ). after 30min staining on ice, cells were fixed for 20 min in 1% pfa and acquired by flow cytometry. in some experiments, kir3dl1 blocking was achieved by the addition of anti-human nkb1 antibody (dx9, cat 555964, bd pharmingen) at 1:100 during the fcr-blocking step. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint single-cell mrnaseq. a/pb1 498-505 + cd8 + t-cells were single cell sorted into 96 well plates containing lysis buffer (1µl rnase inhibitor and 19µl triton x-100) after tame on a bd aria iii sorter. libraries were generated as described previously 27 . a nextera xt dna library prep kit was used for the generation of sequencing libraries and sequencing performed on a nextseq500 platform with 150-base par high-output paired-end chemistry for 30 tetramer + cells/donor (120 cells total). gene expression was analysed as previously described 27 . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.02.20206086 doi: medrxiv preprint back to the future: lessons learned from the 1918 influenza pandemic emerging influenza viruses and the prospect of a universal influenza virus vaccine up to 650 000 people die of respiratory diseases linked to seasonal flu each year protective immunity and susceptibility to infectious diseases: lessons from the 1918 influenza pandemic disease and the colonial narrative -the 1918 influenza pandemic in western polynesia disease, health and healing : aspects of indigenous health in western australia and queensland an australian perspective of the 1918-1919 influenza pandemic disproportionate impact of pandemic (h1n1) 2009 influenza on indigenous people in the top end of australia's northern territory h1n1 2009 pandemic influenza in indigenous australians quantifying the risk of pandemic influenza in pregnancy and indigenous people in australia in 2009 deaths related to 2009 pandemic influenza a (h1n1) among american indian/alaska natives -12 states pandemic influenza a (h1n1) 2009: risk factors for hospitalization hospitalizations for pandemic (h1n1) 2009 among maori and pacific islanders influenza surveillance in the pacific island countries and territories during the 2009 pandemic: an observational study australian vaccine preventable disease epidemiological review series: influenza preexisting cd8+ t-cell immunity to the h7n9 influenza a virus varies across ethnicities multimorbidity among aboriginal people in new south wales contributes significantly to their higher mortality risk factors and mitigation of influenza among indigenous children in australia, canada, united states, and new zealand: a scoping review recalling the future: immunological memory toward unpredictable influenza viruses cytotoxic t-cell immunity to influenza cross-recognition of avian h5n1 influenza virus by human cytotoxic t-lymphocyte populations directed to human influenza a virus cellular immune correlates of protection against symptomatic pandemic influenza recovery from severe h7n9 disease is associated with diverse response mechanisms dominated by cd8+ t cells innate and adaptive t cells in influenza disease human cytotoxic t lymphocytes directed to seasonal influenza a viruses cross-react with the newly emerging h7n9 virus human cd8 + t cell cross-reactivity across influenza a, b and c viruses clonally diverse cd38+hla-dr+cd8+ t cells persist during fatal h7n9 disease towards identification of immune and genetic correlates of severe influenza disease in indigenous australians hla targeting efficiency correlates with human t-cell response magnitude and with mortality from influenza a infection hla class i alleles in australian aborigines and their peptide binding profiles immunomic analysis of the repertoire of t-cell specificities for influenza a virus in humans heterosubtypic protections against human-infecting avian influenza viruses correlate to biased cross-t-cell responses cross-allele cytotoxic t lymphocyte responses against 2009 pandemic h1n1 influenza a virus among hla-a24 and hla-a3 supertype-positive individuals identification of broad binding class i hla supertype epitopes to provide universal coverage of influenza a virus negative' mutant cell line c1r expresses a novel hla-b35 allele, which also has a point mutation in the translation initiation codon immunopeptidomic analysis reveals that deamidated hla-bound peptides arise predominantly from deglycosylated precursors mass spectrometry-based identification of mhc-bound peptides for immunopeptidomics downregulation of mhc class i expression by influenza a and b viruses gapped sequence alignment using artificial neural networks: application to the mhc class i system reliable prediction of t-cell epitopes using neural networks with novel sequence representations -nielsen -2009 -protein science -wiley online library netmhcpan-4.0: improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data netmhcpan, a method for mhc class i binding prediction beyond humans netmhcpan-3.0; improved prediction of binding to mhc class i molecules integrating information from multiple receptor and peptide length datasets hla-a*01:03, hla-a*24:02, hla-b*08:01, hla-b*27:05, hla-b*35:01, hla-b*44:02, and hla-c*07:01 monochain transgenic/h-2 class i null mice: novel versatile preclinical models of human t cell responses protective cd8 t cell-mediated immunity against influenza a virus infection following influenza virus-like particle vaccination a novel vaccination strategy mediating the induction of lung-resident memory cd8 t cells confers heterosubtypic immunity against future pandemic influenza virus recombinant vesicular stomatitis virus expressing influenza nucleoprotein induces cd8 t-cell responses that enhance antibody-mediated protection after lethal challenge with influenza virus potent cd8+t-cell immunogenicity in humans of a novel heterosubtypic influenza a vaccine, mva-np+m1 synthetic multi-epitope peptides identified in silico induce protective immunity against multiple influenza serotypes a structural basis for varied peripheral cd8+ t cell characteristics associated with durable responses to immune checkpoint blockade in patients with metastatic melanoma plasticity in the organization and sequences of human kir/ilt gene families biology of t memory type 1 cells cutting edge: allele-specific and peptide-dependent interactions between kir3dl1 and hla-a and hla-b new allele frequency database: www.allelefrequencies.net diversity of hla among taiwan's indigenous tribes and the ivatans in the philippines heterogeneity of taiwan's indigenous population: possible relation to prehistoric mongoloid dispersals hla antigens, alleles and haplotypes among the yup'ik alaska natives: report of the ashi minority workshops, part ii cross-protective peptide vaccine against influenza a viruses developed in hla-a*2402 human immunity model nucleoprotein of influenza a virus is a major target of immunodominant cd8 + t-cell responses contemporary analysis of mhc-related immunodominance hierarchies in the cd8 + t cell response to influenza a viruses immunodominance analysis of ctl responses to influenza pr8 virus reveals two new dominant and subdominant kb-restricted epitopes measuring the diaspora for virus-specific cd8+ t cells coadministration of seasonal influenza vaccine and mva-np+m1 simultaneously achieves potent humoral and cell-mediated responses preliminary assessment of the efficacy of a t-cell-based influenza vaccine, mva-np+m1, in humans seq2logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion circulating t fh cells, serological memory, and tissue compartmentalization shape human influenza-specific b cell immunity genome analysis circlize implements and enhances circular visualization in r nk susceptibility varies inversely with target cell class i hla antigen expression naming of data sets match those in supplementary fig. 1. the predicted binding affinities (nm) and %rank for hla-a*24:02, hla-b*35:03 and hla-c*04:01 for all 8-14mer peptides as calculated by netmhcpan4.0 are shown. for hkx31, data sets derived from the sequential isolation of hla from the same sample are noted. for b/malaysia, previous identification in hla isolations from b/malaysia infected cir and cir.a*02:01 in koutsakos et al. 27 are also noted. colour fill represents isolations with w632 (blue), dt9 (yellow) and mixed class ii antibodies (green). the "best explanation" column denotes the hla hypothesised to present the declaration of helsinki principles and according to the australian national health and medical research council code of practice. all blood and tonsil donors provided written consent prior to study participation. lung tissues, lymph node and spleen samples were obtained from deceased organ donors after receiving written informed consent from next-ofkin. lungs were sourced from the alfred hospital's lung tissue biobank. lymph node and spleen were provided by donatelife victoria to identify epitope-specific cd8 + t-cells that expanded after stimulation, cells from day 10-15 cultures (2x10 5 cells/well) of the presence of brefeldin a (bd golgi plug), monensin (bd golgi stop) and anti-cd107a fitc at 37 . cells were stained with panel 2 (supplementary table 6) and analyzed using flow cytometry for large scale infections, cir or cir.a24 were cultured to high density in rf10 media slowly rotating in 17dm 2 filter-capped roller bottles (corning) at 37°c, 5% co 2 . cells were harvested and infected with influenza a or b virus at a moi of 5 in rpmi at a density of 1 x 10 7 cells/ml in 50ml tubes for 1 hour at 37°c with slow rotation hla expression and infection efficacy were validated by surface staining ~10 6 cells with anti-hla class i remaining cells were harvested by centrifugation in 500ml v-bottom flasks (3283g, 15min, 4°c), washed in pbs, snap frozen as cell pellets in liquid nitrogen, and stored at -80°c until use liquid chromatography-tandem mass spectrometry (lc-ms/ms) analysis of hlabound peptides. cell pellets of 0.7-1.3x10 9 cir or cir.a24 were lysed via cryogenic milling (retsch mixer mill mm 400), resuspension in 0.5% igepal ca-630, 50 mm tris-hcl ph8.0, 150 mm nacl and protease inhibitors to identify immunogenic peptides, spleen and bronchioalveolar lavage (bal) were isolated on day 10 or day 8 for secondary infection, respectively. spleen single cell suspensions were prepared and incubated for 1 h at 37 in affinipure goat anti-mouse for immunization studies, mice were vaccinated with 30nmol of na 32-40 , np 392-400 and np 165-173 emulsified in complete (prime) or incomplete (boost) freund's adjuvant key: cord-022474-xxy83c6u authors: tenorio, grace c.; gupte, snehalata c.; munker, reinhold title: transfusion medicine and immunohematology date: 2007 journal: modern hematology doi: 10.1007/978-1-59745-149-9_22 sha: doc_id: 22474 cord_uid: xxy83c6u blood transfusion is essential and vital in the successful treatment of many malignant and nonmalignant hematological disorders. children with thalassemia, adults with myelodysplastic syndromes, and patients with autoimmune hemolytic anemias, leukemias, or aplastic anemias become chronically dependent on blood transfusions. modern treatment procedures such as high-dose chemotherapy and progenitor cell transplantation require intensive support with blood components and products. the serological basis of blood transfusion, the available blood components and products, and adverse effects of blood transfusion with special emphasis on infectious disease transmission are discussed in this chapter. blood transfusion is essential and vital in the successful treatment of many malignant and nonmalignant hematological disorders. children with thalassemia, adults with myelodysplastic syndromes, and patients with autoimmune hemolytic anemias, leukemias, or aplastic anemias become chronically dependent on blood transfusions. modern treatment procedures such as high-dose chemotherapy and progenitor cell transplantation require intensive support with blood components and products. the serological basis of blood transfusion, the available blood components and products, and adverse effects of blood transfusion with special emphasis on infectious disease transmission are discussed in this chapter. genes for three different blood group systems (abo, hh, and sese) indirectly control the expression of the a, b, and o antigens because antigenic activity is determined by sugars linked to either polypeptides (forming glycoproteins) or lipids (forming glycolipids). each of the a, b, and h genes code for a specific enzyme (glycosyltransferase) that adds a different sugar on a polypeptide or lipid to form the abh antigens. the abo system is the most important system in red cell serology and involves three allelic genes (a, b, and o) in chromosome a. the a and b genes encode glycosyltransferases (enzymes) that produce the a and b antigens, respectively. the o gene is considered to be nonfunctional because it determines no detectable blood group antigen. the expression of the o gene results in loss of production of a functional protein or enzyme; consequently, no product is formed. the red cells of a group o individual lack a and b antigens, but have an abundant amount of h antigen. the precursor h enzyme (fucosyltransferase) specifically adds fucose to a terminal galactose, thus giving h antigenic expression. if the glycosyltransferase adds n-acetyl-d-galactosamine to the terminal d-galactose of an h antigen, then the red cells will have the a antigen on their surface. correspondingly, if the glycosyltransferase adds d-galactose, a b antigen is formed. abo specificity is dependent on both abo and hh genes. table 1 shows the incidence of the four main phenotypes of the abo system. the a blood group has two main subgroups: a 1 and a 2 , which could be distinguished using the dolichos biflorus lectin reagent. a 1 individuals have more a antigen sites than a 2 individuals. the a 2 gene differs from the a 1 gene by one base pair. variability in genes causes variable reactivity as well add after "as well." subgroups of b are very rare and less frequent than a subgroups. in the abo system, naturally occurring antibodies (isoagglutins) are present against a or b antigens. individuals with the blood group a have isoagglutinins against b red cells and vice versa (see table 1 ). these antibodies invariably are immunoglobulin (ig)m that activate complement and cause immediate intravascular hemolysis resulting in severe acute hemolytic transfusion reactions (htr). they are absent at birth and develop within 3-6 mo of age, when the immune system is exposed to abh antigenic determinants present in our environment (i.e., bacteria, plants, dust, and food). antibodies are naturally developed against the abh antigens absent in the individual. generally, the antibody titer increases until the age of 10 yr and progressively falls with increasing age in adults. in acquired immunodeficiency states (e.g., leukemias and lymphomas) the levels may be significantly low. the abh antigens present in red cells have been demonstrated in most tissues of the body, including platelets and leukocytes. the ability to secrete soluble abh antigens is controlled by the secretor (se) gene that is separate from the abh system. about 80% of the population have the dominant secretor (se) gene that controls one's ability to secrete soluble abh antigens. these individuals (secretors) distinctly have soluble abh substances in their plasma and secretions (i.e., saliva, semen, and sweat). the rhesus (rh) system is the second most clinically important and complex blood group system. it consists of some 50 different antigens, but only 5 antigens-d, c, c, e, and e-are inherited in various combinations and account for most of the rh-related problems encountered in practice. the rh antigen with the strongest antigenicity is the rh (d) antigen. as a simple rule, it can be noted that persons whose red cells express the d antigen are rh (d) positive and individuals whose red cells lack the d antigen are rh (d) negative. the different genotypes, their rh status, and the frequency of these genotypes in caucasians are shown in table 2 . about 85% of north american caucasians are rh (d) positive. after the discovery of the rh system in 1940, various theories were postulated to explain the mode of inheritance and different nomenclatures were proposed. the wiener system proposed that the gene product was a single entity with multiple serological specificities. the fisher-race system postulated three sets of closely linked genes and gene products (c and c, d and d, and e and e) . rosenfield proposed a third nomenclature system based on serological reactions, which assigns a number for each rh antigen. the world health organization in genomic studies have revealed the presence of two closely linked genes (rhd and rhce) with considerable homology that refutes both wiener and fisher-race postulates. the rhd gene controls the production of the d antigen and is absent in rh (d)-negative individuals and explains the absence of the "d" antigen. the rhce gene encodes for cc and ee antigens. the d and ce polypeptides differ in only 36 amino acids, the c and c polypeptides differ in four amino acids, and e and e differ in only one amino acid. the approximate molecular weight of a nonglycosylated rh protein is 30 kda. recently, a d protein (a mixture of rh [c] and [e]) has been isolated from rh (d)-negative red cells that differ from the d protein of rh (d)-positive cells. genetic polymorphism may account for the difference. individuals whose red cells give weaker reactions with anti-d reagents are classified as quantitative weak d (red cells that require additional steps to demonstrate d were formerly classified as d variant or d u ). the d antigen has more than 37 epitopes, and if a significant number of epitopes are absent, then the individual is known to have partial d antigen (formerly classified "d mosaic" or "d variant") and can produce an antibody to the portion of the d antigen they lack. partial d phenotypes arise from nucleotide interchange between the rhce and the rhd genes or from single mutations. gene interaction also depends on the position of the genes that ultimately affect the expression of the d antigen. a weak d antigen can also result from the suppressive effect of c in trans position to a d on the opposite chromosome exemplified by cde/cde. a weak d individual because of gene interaction has the entire d antigen and can receive dpositive blood. in contrast, weak d individuals who have partial absence of the d should only be transfused with d-negative blood because they can produce anti-d antibodies. the american association of blood banks (aabb) requires that blood donors be screened for weak expression of the d antigen and to be labeled as rh (d)-positive if the test is positive; however, recipients need not be tested for weak d. on very rare occasions, red cells may lack the expected rh antigens (e.g., d de, cd ). in rh-null individuals, the rh antigens are completely absent. this can arise from the absence of the gene that regulates rh antigen expression or the presence of an amorphic gene at the rh locus. rh-null individuals have a compensated hemolytic anemia and abnormal red cell morphology (stomatocytosis). if transfused, they will produce antibodies against the different rh antigens; therefore, rh null individuals should be transfused only with rh-null cells from the rare donor registry or with autologous red cells. rh antibodies can be acquired during pregnancy or a blood transfusion. the most common rh antibody is anti-d. rhesus immunization during pregnancy or delivery may occur when an rh (d)-negative woman has an rh (d)-positive child. this can be prevented by the prophylactic injection of anti-d igs (rhig). rh antibodies are predominantly igg and react at 37 c. they do not fix complement effectively, but can cause hemolytic disease of newborn (hdn; see chapter 6) and hemolytic transfusion reaction (htr). extravascular hemolysis occurs through the mononuclear phagocyte system. red cells bear antigens of many other blood group systems besides the abo and rh systems (kell, duffy, lewis, i, p, mn, lutheran, kidd, and others). these red cell antigens are not routinely typed and generally are rare causes of hdn. the kell and duffy systems are briefly discussed. for more comprehensive information on the other red cell antigen systems, the reader is referred to reference texts. (see "suggested reading"). the kell blood group system is clinically important, as the k antigen follows the d antigen in immunogenicity and its antibodies can cause hdn and htr. currently, this system includes 24 alloantigens, the most common being the k, k, kp(a), kp(b), js(a), and js(b) antigens. a defective and weak expression of kell antigens (also lack kx) is observed in individuals with the mcleod phenotype. these individuals have a chronic compensated hemolytic anemia and abnormal red cell morphology (acanthocytosis). individuals with mcleod red cells also have neuromuscular and cardiovascular abnormalities (myopathy, areflexia, and cardiomyopathy). rarely, the mcleod phenotype is associated with chronic granulomatous disease and arises from the deletion of the x chromosome that includes both xk and x-cgd loci. the duffy system is unusual in that the antigen frequency varies in different racial groups. the duffy glycoprotein is the receptor for the malarial parasite and serves as an erythrocyte receptor for a number of cytokines, notably interleukin (il)-8. duffy glycoproteins also serve as a sponge for excess chemokines without any adverse effect on the red cell. this system has six antigens; two of these are important and deserve mention. both fy a and fy b antigens have low incidence in africans. in west africa, most probably by natural selection, both antigens are absent in the majority of blacks [fy (a b )]. their red cells exhibit resistance to infection by plasmodium vivax and p. knowlesi. anti-fy a antibody may cause mild hdn and rare but severe htr. infrequently, anti-fy b is associated with either hdn or htr; other antibodies of this system have not been implicated at all. prior to any blood transfusion, the red cell abo and rh(d) blood group (blood type) of the recipient is determined, and the serum is screened for any unexpected red cell antibodies (usually igm and igg antibodies). thereafter, a cross-match is carried out between the donor's red cells and the recipient's serum. blood group antigens or antibodies are determined with agglutination methods. the igm antibodies (i.e., anti-a or anti-b) are usually detected by saline techniques, whereas enzyme, albumin, or antiglobulin methods are employed for the igg antibody detection. low ionic strength solution (liss) is widely used in blood group serology as it shortens the incubation period and is helpful with emergency blood requests. the antiglobulin (coombs') test detects antibodies coated on the red cells. the direct antiglobulin test (dat) detects antibodies that are already bound to red cells in vivo, whereas the indirect antiglobulin test (iat) detects antibodies present in the serum. the direct antiglobulin test may be positive in: (1) autoimmune hemolytic anemias (seen in lymphomas, system lupus erythematosus, cold agglutinin syndrome, and paroxysmal cold hemoglobinuria); (2) alloimmune hemolytic anemias (hdn and htr); and (3) drug-induced hemolytic anemia. figure 22 .1 schematically outlines the determination of the abo blood group with agglutination methods, whereas fig. 22 .2 shows the principles of both dat and iat. human leukocytes bear two types of surface antigens: the human tissue or cell-specific antigens and individual type-specific antigens. the first group is described in the cluster designation (cd) nomenclature. these antigens characterize the lineage, function, or activation state of the individual type of leukocyte (e.g., cd3 for mature t-cells). a list of the most current cd markers is given in appendix 2. the second group, the family of human leukocyte antigens (hlas) (class i, ii, and iii antigens), is encoded by the major histocompatibility complex (mhc) genes on the short arm of chromosome 6. class i and ii antigens are the classic transplantation antigens that define tissue tolerance or rejection and are important for organ transplantation, but their primary role is in immune response regulation. class iii antigens (i.e., complement c2 and c4 or tumor necrosis factor [tnf]-) may be directly or indirectly involved with mhc function. the hla system is expressed on many tissues. the class i antigens (hla-a, -b, and -c molecules) are present on all nucleated cells and platelets. class ii antigens (hla-d molecules) are expressed on b-lymphocytes, antigen-presenting cells (monocytes, macrophages, and dendritic and langerhans cells), and activated t-lymphocytes. hla class i and class ii antigens differ in immunological function. class i antigens interact with cd8 + lymphocytes, which recognize endogenous antigens. class ii molecules on the surface of antigen-presenting cells bring exogenous antigens in contact with cd4 + lymphocytes. class i molecules consist of two glycosylated heavy chains of 44-45 kda and a noncovalently bound 12 kda molecule ( 2 -microglobulin). the class i heavy chain has three extracellular domains, a transmembrane region, and an intracytoplasmic domain. each of the class ii molecules (hla-dr, dq, and dp) consists of two transmembrane noncovalently associated glycosylated polypeptide chains. the -chain has a molecular weight of about 30-34 kda; the -chain has a molecular weight of about 26-29 kda. the inheritance of the hla genes is closely linked, and the entire mhc is inherited as an hla haplotype (half of the genotype) in a mendelian fashion from each parent. for example, a haplotype of a3, b7, cw7, and dr2 may come from the father and the other haplotype of a9, b27, cw1, and dr7 may come from the mother. distances between loci may permit some chance of recombination within the hla system, but this occurs only infrequently (<1%) and usually between dp and dq loci. statistically, siblings have a 25% chance of inheriting the same pairs of hla molecules from the parents (i.e., being hla identical). the hla system is the most polymorphic human antigen system, thereby showing an enormous number of different hla haplotype combinations. the hla antigen pattern varies among different ethnic groups. because of linkage disequilibrium, some haplotypes occur more frequently in certain populations. for example, hla-a1, b8, dr3 is the most common hla haplotype among caucasians with a 5% frequency. the hla complex is divided into three regions indicating the locations of loci. serological and dna techniques differ in the number of alleles that they can identify for each locus. hla-a, hla-c, and hla-b loci have 20, 8, and 30 serologically defined alleles but have 309, 167, and 563 alleles detected by dna analysis. among the class ii antigens, the hla-dr, hla-dp, and hla-dq molecules expressed on the cell membranes are most important with 442, 81, and 127 alleles defined by dna techniques. the allelic variations of dq-a, dp-a, and dp-b can only be defined by dna methods. two hla nomenclatures are currently in use. the older list of specificities is based on detection of epitopes by immunological techniques (serology or mixed leukocyte culture reactions) and the newer molecular nomenclature is based on specific nucleotide sequences of alleles using dna-based methods, now a commonly used technique in hla-typing laboratories. peripheral blood lymphocytes express class i antigens and are used for the serological typing of hla-a, hla-b, and hla-c. class ii antigens are typed using b-lymphocytes. classic serology uses lymphocyte microcytotoxicity tests (by terasaki) utilizing sera from multiparous women who have been immunized against certain hla antigens. clinical molecular techniques have revealed the complexities of both class i and ii antigens. for example, the identities of class ii antigens can be shown by the mixed lymphocyte reaction but hla-d identical pairs remain nonreactive using this method. however, molecular typing is able to distinguish serologically indistinguishable but functionally discrete hla alleles. all current dna-based hla typing assays utilize pcr to amplify the genes of interest. there are three commonly used procedures: sequence-specific primers (pcr-ssp), sequence specific oligonucleotide probes (pcr-ssop), and sequence-based typing (pcr-sbt). dna typing is specific (no batch-to-batch variation in specificity) and flexible (new reagents can be designed as new alleles or new nucleotide sequences are identified). it is highly reproducible (with ssop) and more robust than other techniques because it does not require viable lymphocytes nor is it influenced by the patient's health. dna based methods have the added advantage of hla-typing large numbers of volunteers for donor registries. furthermore, it can detect the full range of hla diversity. hla alleles can specify the hla proteins that are indistinguishable by serology. for example, drb1*0401 and drb1*0412 are allele splits identified by dna typing that belong to the broad specificity dr4 serological type. in addition to the major histocompatibility antigens just described, minor histocompatibility antigens (mhags) have been defined by both class i and ii mhc-restricted t-cells and may affect the outcome of progenitor stem cell and solid organ transplantation. the mhags are immunogenetic peptides bound to class i molecules that stimulate t-cell responses. they are also inherited and the number of minor histocompatibility loci is probably high. to date, the range of polymorphism of these antigens is not well characterized. the disparity of mhags can be associated with graft-vs-host disease (gvhd) in hla-identical transplants (i.e., h-y antigen in a male recipient and a female donor immunized by pregnancy). the frequency of allelic forms, immunogenicity of peptides and tissue-specific expression of proteins will determine the role of mhag disparity in either gvhd or graft rejection. hla testing is used in progenitor stem cell and organ transplantation, disease susceptibility studies, and parentage testing. hla identity is the sine qua non of allogeneic bone marrow transplantation. despite full hla-identical progenitor cell grafts, a substantial number of patients develop graft-vs-host mhag reactivity (details about allogeneic progenitor stem cell and bone marrow transplantation are described in chapter 4). the hla antigens are also important for solid organ transplantation (i.e., kidney or liver). hla-a, hla-b, and hla-dr are considered to be the major transplantation antigens, whereas hla-c, hla-dp, and hla-dq are generally of minor importance. in kidney transplantation, hla matching between donor and recipient is done routinely. hla-matched kidney grafts have better outcomes than unmatched grafts. in contrast to bone marrow transplantation, solid organ transplantation requires abo-compatible grafts. for logistic reasons, other solid organs (heart, liver, lung, and pancreas) are not routinely matched for hla-antigens. hla antibodies play a major role in graft survival and chronic rejection. the presence of cytotoxic hla antibodies in the serum of transplant recipients reactive against a panel of cells (expressed as panel reactive antibodies or [pra]) lowers the graft survival rates and may be a contraindication for kidney transplantation. the pra effect is greater among recipients of a second transplant. multiparous women and patients who receive multiple blood transfusions are frequently alloimmunized to hla antigens. these hla antibodies are broadly reactive. post-transfusion hla alloimmunization is variable and dependent on the patient's diagnosis and therapy. patients with leukemia have lower detectable antibodies (25 to 30%) than patients with aplastic anemia (80%) because they are usually immunosuppressed from intensive chemotherapy when the transfusions are given. leukocyte reduction of blood components to less than 5 10 6 has significantly reduced the development of primary hla alloimmunization. this can be achieved by the use of third-generation leukocyte filters for red blood cells (rbcs) and platelets or by inline leukoreduction systems of blood cell separators used to collect pheresis blood components. as will be discussed later in the chapter, hla antibodies have been implicated in febrile nonhemolytic transfusion reactions and transfusion-related acute lung injury. immunological refractoriness to platelet transfusions results from immune destruction of transfused platelets more often by hla antibodies (class i antigens are expressed on platelets) than by platelet-specific antibodies. nonimmune causes of platelet refractoriness need to be ruled out such as splenomegaly, disseminated intravascular coagulation (dic), bleeding, infection, marrow transplantation, and antibiotics (amphotericin b, vancomycin, ciprofloxacin). detection of hla-or platelet-specific antibodies is usually done by solid phase red cell adherence assay (sprca), flow cytometry, elisa, monoclonal antibody specific immobilization of platelet antigen assay, or mixed passive hemagglutination assay. once immune refractoriness is established, special platelet products are indicated. these patients can receive hla-matched platelets, or platelet crossmatching can be done using sprca or flow cytometry. both methods of crossmatching will detect platelet antibodies against class i and platelet-specific antigens. the efficacy of crossmatched platelets may be as good as hla-matched platelets. platelet-crossmatched units have the additional advantage of being readily available for transfusion. crossmatching is not always practical because alloimmunized patients may have hla antibodies that react to more than 90% of the random population. these patients may also have few broadly reactive antibodies against public epitopes of class i molecules, which will make it harder for a blood center to provide a product because the best hla-matched platelet unit can still have some incompatibility. numerous diseases have a more or less strong association with certain hla antigens. well-known examples are ankylosing spondylitis associated with hla-b27, narcolepsy associated with hla-dr2, hemochromatosis associated with hla-a3, celiac disease with hla-dqb1*02, and type i diabetes mellitus with dr-3 and -4 heterozygotes. the hla-a1, b8, dr3 haplotype is frequently involved in autoimmune disorders. these disease associations indicate the central role of the major histocompatibility complex in determining the susceptibility to disease and immune responsiveness. the hla system is used in parentage testing because of its polymorphism with a low recombination rate and mendelian inheritance. there is a decreasing use of hla typing because it does not provide a high exclusion probability when a case involves a paternal haplotype common in one particular ethnic group. thus, molecular techniques using non-hla genetic systems are widely favored. the use of whole blood is limited and is indicated in massive blood loss to replace the loss of both rbc mass and blood volume. many trauma centers have abandoned the transfusion of whole blood in favor of intravenous solutions in conjunction with rbcs and other blood components. currently, whole blood units serve as source material for blood components and plasma products. rbcs are indicated for replacement of red cell mass in patients who require increased oxygen-carrying capacity to prevent tissue hypoxia. the hemoglobin or hematocrit value, at which a transfusion is given, depends on the clinical circumstances. current information supports a "restrictive strategy." transfusions are indicated when hemoglobin concentration falls below 7 g/dl. the hemoglobin concentration should be maintained between 7 and 9 g/dl. note: in younger patients, it may be necessary to transfuse if the hemoglobin concentration drops below 6 g/dl. in elderly anemic patients with cardiovascular disease (acute myocardial infarction and unstable ischemic syndromes), the threshold for transfusion may be 9 or 10 g/dl. to avoid volume overload, transfusions should be given slowly. in recent years, the recommended hemoglobin value for transfusion during surgery has been lowered from 10 to 7 g/dl. in a typical 70-kg (155-lb) patient, each unit of transfused rbcs is expected to raise the hemoglobin 1 to 1.5 g/dl and the hematocrit by 3 to 5%. two types of platelet components are available for transfusion: "pheresed platelets," derived from single donors using an automated cell separator, and "pooled platelets," derived from whole blood donation and multiple donors. automated cell separators effectively collect platelets (3 to 4 10 11 /u) from donors. the major goal of prescribing platelet transfusions is to effectively and safely prevent and/or treat bleeding in thrombocytopenic patients. platelets may be given prophylactically in severely thrombocytopenic patients who have a hemorrhagic tendency or to patients on intensive myelosuppressive chemotherapy to keep the platelet count above 10 10 9 /l. the success of modern chemotherapy in patients with hematological disorders (i.e., acute leukemias and myelodysplastic syndromes) and progenitor cell transplantation (bone marrow or stem cell transplantation) is largely dependent on effective platelet transfusions. platelet counts for those at risk for spontaneous bleeding (patients with fever, infection, impaired platelet function from drugs, or hepatic or renal failure) should be kept above 15 to 20 10 9 /l. platelets are also indicated in other thrombocytopenic states: consumption coagulopathy, massive transfusion, gvhd, von willebrand disease, and congenital and acquired platelet defects. invasive procedures (i.e., lumbar puncture and liver biopsy) can be performed safely when the platelet count is at least 50 10 9 /l. counts of 100 10 9 /l should be maintained if excessive bleeding cannot be tolerated (i.e., cns or retinal procedures). in autoimmune thrombocytopenia, hypertransfusions of platelets are only indicated in cases of major hemorrhage. platelets are not useful in most other instances, as the autoantibody shortens the survival of both transfused and patient's own platelets. to investigate the mechanism of a poor response to a platelet transfusion, a platelet count is usually obtained within 1 h of the transfusion and the corrected count increment (cci) or percent recovery is calculated. patients who are refractory to platelet transfusions (cci < 7.5 10 9 /l or percent recovery of <15 or 20%) may have a better response if transfused with a sufficient dose of apheresis platelets. for those who become immunologically refractory, hla-matched or crossmatched compatible platelets may offer satisfactory results. however, patients refractory to all available platelet concentrates may benefit from intravenous immunoglobulin (ivig), plasma exchange, massive abo-identical platelet transfusions, and acid-treated platelets (stripped of hla antigens). platelets are stored at 20-22 c (room temperature) with agitation for no more than 5 d. more recently (april 2005), the food and drug administration (fda) extended the shelf-life of apheresis platelets to 7 d only if collected by the trima â® cobe spectraâ�¢ blood separator in conjunction with 100% testing by bacterial culture system, biomerieux bactt/alert microbial detection system release test â® , to monitor for any bacterial contamination. current blood separators can collect granulocytes at high yields (20 to 30 10 9 granulocytes) from donors stimulated with recombinant granulocyte colonystimulating factor (g-csf) and steroids; however, granulocyte transfusions lost popularity between 1985 and 1995 because of reported adverse pulmonary reactions and marginal clinical results. the unimpressive clinical efficacy may be attributed to rapid postcollection neutrophil apoptosis and inadequate doses (because previous donors did not undergo any stimulation/mobilization). renewed interest in granulocyte transfusion stems from the availability of g-csf that not only increases the yield of granulocyte collections but also inhibits neutrophil apoptosis. currently, granulocyte transfusion has limited indications that include refractory fungal or bacterial infections in neutropenic patients and those with qualitative neutrophil defects. granulocyte transfusions may also be beneficial to newborns with sepsis, neutrophil counts of less than 3000/ l or a defective marrow response. granulocytes have no defined regulatory specifications because the fda does not license them. however, the american association of blood banks (aabb) requires that each unit contain at least 1.0 10 10 granulocytes (note: value intended as a goal for adequate collection but not as an adequate clinical dose). at least four consecutive transfusions of 1.0 10 10 granulocytes are recommended. the use of additional transfusions is based on the patient's response or clinical course and the clinician's judgment. granulocyte units are suspended in 200 to 400 ml plasma and contain significant numbers of platelets (1 to 6 10 11 platelets, equivalent to an apheresis platelet unit), rbcs, and viable lymphocytes. thus, they must be abo and rh compatible with the recipient and irradiated to prevent transfusion-associated (ta)-gvhd. granulocytes have to be transfused as soon as possible and, if necessary, stored without agitation at 20-24 c for no more than 24 h. fresh-frozen plasma (ffp) is a single-donor unit of plasma that has been separated from one unit of whole blood or collected by apheresis and frozen at -18 c or lower within 6 to 8 h of collection. it contains coagulation factor levels at 1 u/ml (or 100% activity) and is indicated to treat global or multiple coagulation factor deficiencies (i.e., liver failure and dilutional coagulopathy in massively transfused patients). other uses for ffp include emergent reversal of warfarin therapy (when time does not allow the use of vitamin k) and plasma exchanges in thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. the timing and dose are important. correction of a markedly abnormal prothrombin time and activated partial thromboplastin time requires ffp transfusions immediately before surgery because several of the coagulation factors have very short half-lives (particularly factor vii with a biological half-life of 3 to 6 h). the dose is based on the patient's weight at 10 to 20 ml/kg. ffp contains abo antibodies and therefore must be compatible with the recipient's red cells. cryoprecipitated antihemophilic factor (cryo) is the cold insoluble portion of plasma processed from ffp by cold precipitation. the precipitate is formed as ffp is thawed at 1 to 6 c. the stability of the coagulation factors is maintained for up to 1 yr at -18 c storage. it is a plasma-derived product that contains the highest concentration of fibrinogen (250 mg, primarily used in acquired hypofibrinogenemia of dic), factor viii (80-120 u/concentrate), and a variable percentage of the original plasma concentration of von willebrand factor (40-70%) and factor xiii (30%). it is indicated for the treatment of hemorrhagic disorders resulting from either quantitative or qualitative defects of these factors. however, the availability of factor concentrates and recombinant factors have contributed to its diminished usage. cryo is also used as a source of fibrinogen to form a fibrin glue or sealant (when added to thrombin) and is also indicated to ameliorate the platelet dysfunction in uremia. like ffp, it contains abo antibodies, requiring consideration of recipient red cell compatibility. parenteral igs are manufactured from pooled human plasma and are used in primary or acquired hypogammaglobulinemia to protect against viral and bacterial infections. they consist of all subclasses and allotypes of igg with only trace amounts of igm and iga. polyvalent/multispecific immunoglobulins offer protection against many infections, whereas hyperimmune/specific immunoglobulins are obtained from actively immunized donors and contain high titers of disease-specific antibodies (i.e., hepatitis b virus [hbv]), varicella zoster, rabies, or cell-specific [rhig] ). rigorous donor screening and modified cohn fractionation have made immunoglobulin preparations relatively safe. immunoglobulins can either be administered intravenously (ivig) or intramuscularly (immune serum globulins) and are generally well tolerated. adverse effects are rare (<1%) and include headache, nausea, vomiting, chills, volume overload, and allergic and pulmonary reactions that can be prevented by slow infusion and pretreating with diphenhydramine and/or hydrocortisone. true anaphylactic reactions are very rare and are seen in common variable immune deficiency and iga deficiency. iga deficient products are recommended in these cases. polyvalent immunoglobulins are indicated in hypogammaglobulinemias (congenital or acquired) and for immunomodulation in certain disorders. note: the plasma half-life of igg (the major human immune globulin) is 21-23 d, but longer in hypogammaglobulinemia. in hypogammaglobulinemia, a dose of 0.4-0.6 g/kg body weight is given every month to maintain serum igg levels. in acquired hypogammaglobulinemia (e.g., multiple myeloma), immunoglobulins are recommended in patients with frequent infections. established indications for immunoglobulin replacement or prophylaxis are parvovirus b19 infections (among immunosuppressed patients) and recurrent life-threatening infections (among hiv-infected children). the use of prophylactic ivig in adults with advanced hiv infection has shown equivocal results. high-dose polyvalent immunoglobulins achieve immunomodulatory effects through multiple mechanisms: (1) by suppressing antibody production directly through its effect on b-lymphocytes, (2) by interfering in the interaction between autoantibodies and cellular targets through its anti-idiotypic antibodies, and (3) by blocking the fc-receptors on macrophages. higher doses of ivig (e.g., 0.4 g/ kg 5 d) are recommended for immunomodulation in kawasaki syndrome (a mucocutaneous lymph node syndrome in children) and idiopathic thrombocytopenic purpura. acute hemolytic transfusion reactions (ahtr) involves rapid destruction of blood cells immediately or within 24 h of a transfusion, commonly of rbcs or whole blood. it has a low rate of occurrence but is the most dangerous complication with a high mortality. mortality depends on the amount of incompatible blood infused (25 to 44 % with infusion of less than or more than 1000 ml, respectively). abo incompatibility is the most common cause of immune ahtr and accounts for 74% of all fatal reactions. ahtr is due to preformed antibodies, often igm (commonly anti-a), that bind complement and cause red cell lysis. nonimmune causes of hemolysis mimic immune mediated hemolysis and have to be excluded. chemical, mechanical, thermal, and physical damage of red cells may result from bacterial contamination, mechanical trauma during infusion, thermal hemolysis (from overheating or freezing a unit), and osmotic hemolysis (use of hypotonic reconstituting solutions and co-administration of drugs during transfusions) the patients commonly experience fever and chills, hypotension, and chest pain. other signs and symptoms include low back pain, flushing, dyspnea, hemoglobinuria, gastrointestinal symptoms (abdominal pain, diarrhea, and vomiting), and unexpected bleeding from dic. in patients under anesthesia, no immediate reactions may be recognized. changes in blood pressure, diffuse bleeding, and hemoglobinuria may be the only signs. ahtr may cause oliguria and acute renal failure. the classic laboratory changes include a drop in hemoglobin and hematocrit, hemoglobinemia (free hemoglobin can be demonstrated in serum or plasma), hemoglobinuria, reduced serum haptoglobin, and positive dat. elevations of unconjugated bilirubin, methemalbumin, and lactate dehydrogenase, and an unexpected red cell antibody may be present. the signs and symptoms of an igm-type acute hemolysis result from complement-mediated lysis of red cells and the release of cytokines. binding of complement to the red cell surface is a major factor in cytokine production. the symptoms of igg-type hemolysis also involve several cytokines (il-1, il-6, il-8, and tnf). coagulopathy, frequently seen in hemolytic transfusion reactions, especially due to igm antibodies, results from several mechanisms. the coagulation cascade is activated by (1) antigen-antibody complexes (activates hageman factor); (2) thromboplastic substances of the red cell stroma; (3) platelet factor 3 released by activated platelets; and (4) tissue factor release secondary to hypotension. in order to minimize ahtrs, all transfusions should have clearly defined indications. if an ahtr is suspected, the transfusion must be stopped and immediate steps taken to confirm or exclude this possibility. management of ahtr is dependent on the clinical status of the patient and may include cardiopulmonary support, prevention of renal failure (i.e., fluid resuscitation, vasopressors, and diuretics), and treatment of dic. severe bleeding in dic requires platelets, replacement of procoagulant factors and fibrinogen (ffp and cryo transfu-sions) , and administration of low-dose heparin. heparin limits both hemolysis (by its direct anticomplement activity) and inappropriate activation of procoagulant activity (by enhancing antithrombin iii-neutralizing serine proteases). delayed hemolytic transfusion reaction (dhtr) represents an anamnestic response to a red cell antigen to which the patient has been previously sensitized by pregnancy or previous transfusions. it occurs in patients without identifiable antibody detected in the pretransfusion compatibility testing who experience accelerated red cell destruction of the transfused red cells after an interval of 3 to 10 d from transfusion. such reactions are due to weaker antigen-antibody reactions and may develop over days. because of the low titer of reactivity, the implicated antibody is not detectable at the time of screening or compatibility testing. the sensitivity of the antibody-screening test is important in preventing dhtrs because insensitive tests will miss weak-reacting antibodies. note: these weak-reacting antibodies commonly include rh antibodies (against cece) and other antibodies in the kell (anti-k), kidd (anti-jk a ), and duffy (anti-fy a ) blood group systems. dhtrs often remain asymptomatic and have milder symptoms than ahtrs, including unexplained anemia, jaundice, and fever. in patients with sickle cell disease, dhtrs may precipitate a sickle cell crisis. laboratory findings are similar to those of ahtr, except for the identification of a new alloantibody in the patient's rbc eluate or serum or both. the degree of hyperbilirubinemia will depend on the rate and amount of hemolysis and the patient's liver function. hemolysis is usually extravascular but intravascular hemolysis can occur. both ahtrs and dhtrs can demonstrate a positive, mixed-field, or negative dat. a mixed-field reaction will show a mixture of agglutinated transfused donor cells along with unagglutinated patient's cells. the dat can be negative if all the incompatible transfused donor cells are immediately destroyed. there is a need to differentiate dhtr from delayed serological transfusion reactions (dstrs), in which only serological incompatibility is evident without clinical evidence of hemolysis. dstrs are more common than dhtrs in the multiply transfused patient as more sensitive screening methods are employed and the length of stay for in-patients increases. dhtrs are tolerated well by many patients and may only require close monitoring. typically, fluid loading and diuresis are not indicated unless active intravascular hemolysis is present. complications, such as renal failure and sickle cell crisis, should be treated accordingly. a red cell exchange is indicated if there is a large burden of antigen-positive cells. ivig may be useful because extravascular hemolysis is similar to acute immune hemolytic anemia. transfusion should be avoided until the causative antibody is identified and antigen-negative units are available. withholding transfusions because of the lack of serologically compatible blood in patients with severe anemia is associated with significant morbidity. this can be avoided by good communication between the clinician and transfusion service. febrile nonhemolytic transfusion reactions (fnhtr) are defined as a temperature rise of at least 1 c in association with a transfusion or up to 4 h after that may be accompanied by chills or rigors. such reactions are due to acquired antibodies to donor leukocyte antigens or pyrogenic cytokines (il-1, il-6, il-8, and tnf-) elaborated by leukocytes present in the blood components or products. fnhtrs are less frequent with prestorage leukocyte-depleted blood components. they occur in 0.5-1.4% of all transfusions. the fever is self-limited and resolves after 4-6 h. antipyretics are effective as symptomatic treatment. meperidine can be useful for treating rigors. among patients who have experienced a fnhtr, there is a 15% recurrence rate. allergic reactions are probably the most frequent, occurring in 1-2% of all transfusion reactions. the symptoms range from local or diffuse pruritus, urticaria, erythema, and cutaneous flushing to anaphylactic allergic reactions occurring within minutes of the transfusion. anaphylactoid reactions fall in between the two ends of the spectrum. allergic disorders afflict 30% of donors, and passive transfer of ige antibodies may be involved. uncomplicated allergic reactions are associated with increased histamine (increased during storage), cytokines, mast cell activators (i.e., leukotrienes), and other vasoactive substances (c3 a and c5 a ) produced by donor leukocytes during storage. some allergic reactions only have pulmonary signs and symptoms without cutaneous involvement (10%). severe anaphylactic reactions may occur after infusion of a very small volume (<10 ml) in patients with iga deficiency and are due to preformed, class specific, recipient anti-iga antibodies against infused donor iga proteins. additionally, they may be due to antibodies against complement c 4 and haptoglobin. patients with chido (ch) and rogers (rg) antibodies (against the ch/rg blood group antigens carried by complement c4d of the classic complement pathway) also exhibit anaphylactoid reactions following plasma product transfusions. haptoglobin deficiency is rare in north american populations but more common than iga deficiency among japanese patients experiencing anaphylactic reactions. anaphylactoid reactions are typically associated with subclass, allotypic, or specific anti-iga in patients with normal or demonstrable levels of iga. these reactions may be seen in other transfused products, such as peanut allergen transfused to patients with peanut allergy. mild uncomplicated allergic reactions involving hives (without other symptoms) respond to antihistamines (such as diphenhydramine). the transfusion may be restarted after treatment if there is no recurrence or progression of symptoms. restarting a transfusion is not advisable with more serious reactions; in particular, pulmonary symptoms with airway involvement. treatment of severe anaphylactic reactions is the same as for any anaphylactic reaction and requires immediate cessation of the transfusion, epinephrine, and other supportive care. prevention of anaphylaxis in iga-deficient individuals requires avoidance of all plasma containing products unless collected from a known iga-deficient donor and washing of all red cell and platelet products. the major concern is to make all blood transfusions as safe and effective as possible. several infectious agents, viruses, bacteria, and parasites are transmissible by transfusion of whole blood, blood components, or products. attention will focus on those transfusion-transmissible diseases (ttd) that present risks of chronic infection in the recipient. major strategies available to reduce transmission include: stringent donor selection and laboratory testing; use of autologous blood, pharmacological substitutes, or new transfusion strategies; inactivation of residual infectious agents in the units to be transfused, and limiting the number of donor exposures and allogeneic transfusions. blood donor screening is one of the more important steps in protecting the safety of the blood supply. nonremunerated, voluntary donors with low infectious risks are encouraged and recruited to become blood donors. the scarcity of the blood supply in many countries has spurred the use of paid donors. direct interviews, miniphysical examinations and laboratory testing of all blood collections help to exclude donors who have exposure to transmissible infections or who have the risk factors for ttd. the blood supply is screened or tested for bacterial and viral infections (including tick borne infections), parasites (i.e., malaria, babesia, chagas' disease, and microfilariasis). other infectious agents can be tested or excluded based on the epidemiology of the infectious agent, geographic area, and intended use of the blood product. in countries with endemic malaria, testing for malaria parasites is mandatory. the general safety of the blood supply will be enhanced if these concerns are addressed; however, minimal risks still remain because very recent infections are not detected by recommended/standard laboratory tests (see table 3 ). bacterial contamination resulting in transfusion-associated bacterial sepsis is now believed to be the most common infectious source of morbidity and the most frequently reported cause of transfusion-related fatalities in the united states (accounting for 16% of transfusion fatalities). bacteria are believed to originate from the donor either from the venipuncture site or unsuspected bacteremia. excluding donors with chronic diseases or recent febrile infections and scrupulous disinfection of the skin reduces the risk of bacterial transmission. both gram-positive and -negative bacteria have been implicated. bacterial multiplication is more likely in components stored at room temperature (i.e., platelets) than refrigerated components (i.e., rbcs) or frozen components (ffp and cryo). bacterial contamination of platelet transfusion is one of the most common infectious risks of transfusion, causing life-threatening sepsis in 1 in 100,000 recipients and immediate fatal outcome in 1 in 500,000 recipients. despite limiting platelet storage to 5 d, various pathogens have been implicated (staphylococcus, enterobacteriaceae, streptococcus, bacillus, and pseudomonas) . the low incidence of platelet-associated sepsis may be due to underreporting because (1) they typically occur several hours after transfusion and are not as catastrophic as rbc-associated sepsis; and (2) sepsis is attributed to other causes because platelets are commonly transfused to immunocompromised patients with other complex problems. psychrophilic gram-negative bacteria can multiply in refrigerated blood and components. rbc contamination is primarily from yersinia enterocolitica and serratia liquifaciens. y. enterocolitica proliferates and produces an endotoxin in refrigerated anticoagulated blood because it can grow at temperatures below 37 c in a calcium-free medium even after a long lag period. any bacterial contamination of blood products is potentially serious. p. cepacia and p. aeruginosa are environmental organisms that grow optimally at 30 c and have been isolated from cryoprecipitate and plasma thawed in contaminated water baths. syphilis transmission by blood transfusion is possible but its occurrence is extremely rare because the phase of spirochetemia is short and the infective organism, treponema pallidum, does not survive refrigerated storage for more than 96-120 h. seroconversion occurs after the phase of spirochetemia so testing donor blood by standard sts does not effectively prevent transmission. however, most positive sts results are demonstrated by donors with inadequately treated noninfectious syphilis, or are biological false positives (may be positive for hepatitis, mononucleosis, measles, chickenpox, immunizations, rheumatoid arthritis, and pregnancy). there have only been two cases of transfusion-transmitted syphilis reported in the english literature. another spirochete, borrelia burgdorferi, causing lyme disease (transmitted by the ixodes deer tick) can survive routine storage of rbcs and ffp. however, transfusion-related transmission has not been reported at this time. the phase of spirochetemia is associated with clinical symptoms that would render potential donors ineligible for donation. donors diagnosed with lyme disease are accepted provided they have completed antibiotic therapy and are completely asymptomatic. before the 1980s, the transmission of hepatitis was the major transfusionrelated viral infection. the absolute number of hepatitis infections post-transfusion has decreased significantly, because reliable tests for hbv and hcv were introduced. in the past, donors having elevated alanine aminotransferases were rejected. this surrogate marker is no longer used with the availability of more specific tests for anti-hcv and hcv rna. anti-hbc testing has been retained because it may still detect a few donors with infectious hbv who are negative for hbsag. hepatitis a (hav), an rna virus, is generally transmitted by the oral-fecal route and very rarely transmitted by transfusion. the main concern of hav (and parvovirus b19 as well) is transmission by plasma derivatives (particularly human source factor viii concentrates) because it does not have a lipid envelope and is not inactivated during the manufacturing process. nucleic acid testing (nat) is available and is done only for source plasma (plasma intended for manufacture of blood derivatives/products). nat testing for this virus is currently considered an "in-process control" by the fda so donor notification (for positive tests) is not required. in contrast to persons infected with hbv, persons who are exposed to hav do not develop a chronic carrier state. blood donors are not routinely screened for hav because of the rarity of transfusion transmissible hav and the absence of hav antibody at the time of viremia. the risk for hav is estimated at 1 per 10 million units. hbv, a dna virus, is primarily transmitted by the parenteral route. it can be transmitted within the first 100 d after infection when viremia is present and no protective immunity has yet developed, or in the chronic carrier state. donor screening to detect hbv infection includes assays for the hbsag and for anti-hbc. most infections are asymptomatic and hbsag positivity occurs 2 to 6 wk before the onset of symptoms; thus, an apparently healthy but infectious donor will be eligible for donation. a "window period" for any transfusion-transmissible agent is defined as the period of time that an individual is potentially infectious but demonstrates negative serological tests (without detectable antibodies). a licensed nat test is not available for hbv because of low levels of viremia during the "window phase" resulting from a slow viral doubling time. however, a recent publication reported that the "window period" can be reduced by 25-36 d using single donor nat (sdnat), further reduced by 9-11 d using minipool nat (mpnat) and reduced by 2-9 d using a new and more sensitive hbsag assay. disagreement exists as to whether hbv-nat will be cost effective to further reduce the risk of transfusion-transmissible hbv. hcv, an rna virus, was discovered in 1989 and was linked with most cases of non-a, non-b hepatitis in the past. hcv infections are mild and generally (80%) asymptomatic. the long-term effects are far more serious in that the majority of hcv infected patients develop chronic liver disease with 20% developing cirrhosis. in addition, those with hcv cirrhosis develop hepatocellular carcinoma. it was estimated that the elimination of hepatitis c by anti-hcv antibody testing prevented 40,000-50,000 new cases of post-transfusion hepatitis per year (an additional 10,000-13,000 cases may have been prevented by newer versions of the test). the risks of transfusion-transmissible hcv has further been reduced by hcv-nat. prior to hcv-nat, the window period because of a slower doubling time was 70 d. the newly developed hcv-nat (mpnat/sdnat) has further closed the window phase to 10 d, so that the transfusion-transmissible risk for hcv is reduced to 1:1,935,000 per unit transfused from 1:276,000 in the past (see table 3 ). hepatitis d (or ), another rna virus, exists as a co-infection in patients chronically infected with hbv because the "delta agent" cannot multiply in the absence of hbv. testing for hbv markers eliminates hepatitis d-positive donors. the hepatitis g virus (hgv, also known as gbv-c), a newly discovered rna virus, is potentially transmitted by blood products. among normal donors, the reactivity is 1-4% by pcr techniques. currently, hgv has not been associated with a specific disease entity and its designation as a "hepatitis" virus may have been premature. thus, no routine donor testing is performed. two other hepatitis viruses, hepatitis e virus (hev) and sen virus, are apparently transmitted by transfusion. hev does not occur in the united states but is endemic in other parts of the world, although its true incidence remains unknown. sen virus is the latest virus postulated to cause the remaining non a-e transfusion-transmitted hepatitis. at present, the biological role of sen virus has not been clearly defined. lastly, tt virus, a dna virus, first described by a japanese group was originally postulated to cause non-a-e hepatitis. this virus is prevalent in many countries and is currently not associated with hepatitis. retroviral infections transmitted by blood transfusion include hiv-1 and hiv-2 and human lymphotropic virus (htlv-i and htlv-ii). hiv is transmitted by both cellular blood components and plasma; however, both types of htlv are highly cell-associated and require viable lymphocytes for transfusion transmission. hiv is a cytopathic retrovirus that preferentially infects cd4-positive tlymphocytes. the infection begins as a viremia of cell-free virions that may be clinically manifested by an acute nonspecific flu-like illness. viremia is detectable in the plasma 10 d to 3 wk after infection. as hiv antibodies appear, the disease enters a clinically latent phase. viral replication and dissemination continues and the virus can be transmitted by blood or genital secretions. hiv-2 causes endemic infection in west africa and has apparently spread with population movements. it is indistinguishable from hiv-1 in disease spectrum although hiv-2 has a longer incubation period and is less efficiently transmitted than hiv-1. the aids epidemic in the early 1980s had a catastrophic impact on the safety of blood transfusions but triggered a major impetus for improvement in blood safety. the risk of hiv transmission through blood components/products dramatically decreased with more stringent donor history screening and improvement of donor testing (see table 3 ). combination hiv-1/hiv-2 tests implemented in the united states in 1992 have identified to date three hiv-2 infected donors, none appear to have been infected in the united states. prior to 1992, the window period for hiv averaged 45 d. more sensitive hivantibody screening tests closed this window to 22 d. in 1996, hiv antigen (p24 antigen) testing reduced this infectious window by an estimated 6 d; such that circulating cell-free virions could be detected as early as 16 d following infection. with the newly developed pcr-based nat, this window period was further reduced to about 10 d. although sdnat has a detection sensitivity of less than 50 viral copies/ml, its higher cost prompted many blood centers into using mpnat with 14-16 donors per pool. an estimate of residual risk of hiv infection from repeat donors after nat is 1 per 2,135,000 (per unit transfused). currently, many blood centers have implemented nat and discontinued p24 antigen testing following the fda's licensure of the hiv-nat assay. htlv-i and ii are human retroviruses that can be transmitted by blood, sexual contact (predominantly male-to-female), and through breast milk. it circulates as a provirus that is incorporated into the dna of lymphocytes. no cases of transfusion-transmitted htlv have been reported with noncellular blood components. prolonged storage for more than 10 to 14 d (refrigeration inactivates the lymphocytes) also reduces transmission risk. compared to other viruses, htlv-i and -ii transfusion transmission is less efficient because exposure invariably does not result in infection. look-back studies have shown that one in three htlv-contaminated units transmitted the virus. risk factors for htlv-1 infection are birth or sexual contact in areas where the disease is endemic (japan and certain pacific islands, caribbean basin, sub-saharan africa, central and south america). the natural epidemiology of htlv-ii is not fully known, although a high prevalence is seen in some native american populations. htlv-ii is presumably associated with parenteral transmission with risk factors of intravenous drug use (1 to 20% seroprevalence) and sexual contact with an iv drug user. an excess of infectious syndromes (i.e., bronchitis, pneumonia, and urinary infections) is seen among blood donors infected with htlv-i or -ii. however, most htlv-1-positive individuals remain asymptomatic donors with an extremely long latency period (decades) and a 2-5% life-long risk of developing adult t-cell lymphoma/leukemia or htlv-associated myelopathy/tropical spastic paresis in areas of high endemicity. enzyme immunoassay (eia) for both htlv-i and -ii is used to screen donors, and eia-reactive donors are indefinitely deferred regardless of investigational supplemental tests since there is no licensed confirmatory test. the risk of transfusion-transmissible htlv with the same "51-d window period" has decreased from 1: 514,000 (in 1998-1999) to 1:2,993,000 (in 2000-2001) . such a dramatic risk reduction may be partly attributed to the implementation of universal leukocyte reduction. viral load reduction by removal of infected leukocytes remains controversial. leukocytotropic human herpesvirus (hhv) may contaminate blood components. cytomegalovirus (cmv or hhv-5) and epstein-barr virus (ebv or hhv-4) have the greatest clinical relevance to transfusion medicine. cmv is transmitted by transfusion in the latent, noninfectious state in the genome of leukocytes present in cellular blood components. seropositivity in the general population, united states ranges from 20 to 80%, but only a small fraction of these individuals have circulating virion. exposure and host factors determine symptomatology. among immunocompetent individuals, cmv causes a mononucleosis-like syndrome or an asymptomatic infection and remains latent in tissues and leukocytes. symptomatic cmv infections develop in immunosuppressed, seronegative hosts. human progenitor cell transplant (hpct) recipients (develop cmv pneumonitis), low birth weight neonates (<1500 g) of seronegative/seropositive mothers and hiv-infected patients (develop cmv chorioretinitis, encephalitis, and enteritis) are particularly at risk. among seropositive hpct recipients, viral reactivation is the most common cause of cmv infection (up to 69% in one study). generally, the donor organ is the source among transplant recipients. additionally, the risk of transfusion-transmitted cmv is high in heavily transfused recipients (liver, heart-lung, and pancreas transplants). "cmv-reduced-risk blood components" are recommended to reduce cmv transmission. leukoreduced cellular blood components are comparable to seronegative cellular blood components. nonetheless, there remains a small risk of transmission with either type of component. most centers in the united states, canada, australia, and europe recommend transfusion of cmv-negative blood components to cmv-negative pregnant women and for intrauterine transfusions (to prevent transplacental infection), and to cmv-negative immunosuppressed individuals (i.e., hpct recipients, hiv-positive with aids, and other immunosuppressed individuals). it is also prudent to extend this consideration to cmvnegative candidates for hpct. despite transfusions of "cmv-reduced-risk blood components," a few marrow transplant patients (1 to 4%) still develop primary cmv infection. ebv targets b-lymphocytes causing polyclonal proliferation with t-lymphocyte response and demonstration of "atypical lymphocytes." it causes infectious mononucleosis, the endemic form of burkitt's lymphoma in africa and nasopharyngeal carcinoma. ebv can be transmitted by blood transfusion, but is a rare cause of significant disease in immunocompetent individuals. transfusion-transmitted ebv is usually asymptomatic. rarely, ebv causes posttransfusion hepatitis and "postperfusion syndrome." the latter is characterized as a viral-like illness following massive transfusion of fresh blood during cardiac surgery. ebv contributes to the development of lymphoproliferative disorders among immunosuppressed hpct and organ transplant recipients from a reactivation of a latent infection. the high seropositivity rate (90%) among blood donors and the low risk of acquiring clinical disease among immunocompetent recipients make blood donor screening and laboratory testing less beneficial. parvovirus b19 is the etiological agent of erythema infectiosum ("fifth disease") in children, arthritis in adults, and hypoplastic anemias in hiv-infected individuals. more ominously, it causes an aplastic crisis among patients with chronic hemolytic anemias who rely on active erythropoiesis to offset the shortened red cell survival. the red cell p antigen is the cellular receptor for parvovirus b19 so those who do not possess the antigen are naturally resistant to infection. the presence of parvovirus b19 antibodies (30 to 60% prevalence) and the brief viremia in blood donors make viral transmission uncommon (ranging from 1 in 3300 to 1 in 50,000). the rarity of clinically significant disease or viral transmission has not made donor screening and testing imperative. there are reports of parvovirus b19 transmission by solvent-detergent plasma (solvent detergent viral inactivation is ineffective because the virus lacks a lipid envelop), cellular blood components, and clotting factor concentrates. there are no reports of transmission from ivig and albumin. nat screening has been implemented only for in-process manufacturing control of plasma derivatives. west nile virus (wnv), a flavivirus, is transmitted through mosquito bites (an arthropod-borne-virus), blood transfusions (first reported during the 2002 epidemic), and organ transplants to humans causing encephalitis, meningitis, and very rarely asymmetrical flaccid paralysis. immunocompromised and elderly individuals are at risk of developing severe disease. viremia occurs 1 to 3 d following an infecting mosquito bite and lasts from 1 to 11 d. wnv transfusion-transmission risk (per 10,000 donations) ranges from 1.46 to 12.33 for selected metropolitan areas and from 2.12 to 4.76 for six highincidence states. serological tests are ineffective in donor screening as viremia disappears by the time igm antibodies are detected by elisa tests. the seasonal increase in 2003 prompted mpnat under a clinical protocol in the united states. likewise, donor history questionnaires have been implemented to reduce the risk of wnv transmission. during periods of high endemicity, frozen products have been withdrawn voluntarily from the supply, as cessation of donor collection is not feasible. other blood derivatives do not appear to be at risk for transmission as the wnv is inactivated by heat or solvent-detergent treatment. the severe acute respiratory syndrome (sars) virus that first appeared in guangdong, china is spread by close person-to-person contact and is believed caused by a corona virus that causes the common cold and/or probably a paramyxovirus. its spread to other countries was linked to airline travel, often by health care workers in contact with sars patients. the virus has been isolated from the blood of an infected individual but its risk of transmission through blood transfusion remains unknown. however, because of its highly contagious nature, transfusion transmission is possible if blood collection coincides with the viremic phase of the disease. the fda recommends deferral of at-risk donors for 14 d after a possible exposure and at least a 28-d deferral after resolution of symptoms. likewise, deferral is extended to donors with a history of travel or residence in sars-affected areas. transmissible spongiform encephalopathies (tses) are rare, fatal degenerative neurological disorders caused by infectious agents classified as prions or proteinaceous infectious particles that lack nucleic acid. a prion is an abnormal isoform (prp sc ) of a normal cellular protein (prp c ) that is resistant to inactivation by alcohol, formalin, ionizing radiation, proteases, and nucleases; but is disrupted by autoclaving, phenols, detergents, and extremes in ph. tses have long incubation periods (years to decades). two such tses, classic creutzfeld-jakob disease (cjd) and variant creutzfeld-jakob disease (vcjd) are important from the transfusion medicine perspective. unlike classic cjd, which presents in older patients, vcjd is observed in young adults and has an acute course with rapid progression to death in 2 yr. the majority of classic cjd is sporadic (80%). familial cases (10-15%) are caused by mutations and the rest (10%) arise from iatrogenic transmission (administration of growth hormone and gonadotropic hormone derived from pooled human pituitary tissue, allografts of dura mater and cornea, and reuse of intracerebral electroencephalographic electrodes from such patients). to date, transfusion transmission of cjd has been reported in experimental rodent models, but not in humans. although theoretically possible, there is growing consensus that cjd transmission by blood or its components is unlikely. variant cjd (vcjd), first reported in the united kingdom in 1996, is caused by the same prion responsible for bovine spongiform encephalopathy (bse) but might be entirely different. its potential for transmission by blood and blood components is heightened by the fact that vcjd can spread from cattle to humans (presumably by ingestion) and from human to humans (a recipient developed symptoms 6.5 yr after a red cell transfusion). in december of 2003, a potential case of vcjd associated with blood transfusion was reported in the united kingdom. its presence in lymphoreticular tissue of vcjd patients, determined by animal studies and its association with b-lymphocytes, suggests possible transmission through blood transfusions. as a safety measure, several european countries and canada implemented universal leukocyte reduction for all blood products to prevent lymphocytes from transmitting vcjd. however, the efficacy of such intervention remains uncertain. expanded donor deferral criteria have been implemented to reduce the risk of transmission of both tses. this includes deferral of donors at risk of exposure due to travel or residency in areas with bse epidemics, deferral of donors who received bovine insulin and pituitary-derived human growth hormone or dura transplants since 1980, and deferral of donors with blood relatives diagnosed with cjd. malaria is caused by several species of the intraerythrocytic protozoan plasmodium and can be transmitted by transfusion of parasitemic blood. malarial parasites survive for at least 1 wk in blood components stored at room temperature (i.e., platelets) or at 4 c. they can survive cryopreservation with glycerol. any blood component that contains red cells can transmit infection via the asexual form of the parasite. it is frequently transmitted by red cell transfusions, rarely by platelet transfusions, and is absent in plasma products. it is recognized as a global health problem, but is very rare in the united states. however, it is the most commonly recognized parasitic complication of transfusion and occurs as at an estimated rate of 0.25 cases per 1 million blood units collected. asymptomatic carriers are the general source of transfusion acquired infections. at present, there are no practical serological tests to screen asymptomatic carriers. prevention of malaria transmission is possible by deferral of prospective donors with increased risk of infectivity based on medical and travel history. in western europe and the united states, blood donors are deferred for 12 mo after travel to malaria-endemic areas. individuals born in areas endemic for malaria generally are excluded from blood donation for 3 yr after leaving the area. chagas' disease (american trypanosomiasis) endemic in south and central america is caused by another protozoan parasite, trypanosoma cruzi, transmitted by reduviid bugs (cone-nosed or "kissing" bugs). infection occurs from fecal contamination of the reduviid bug bite wound by the infectious trypomastigote. acute infections are mild to asymptomatic and 20-40% of infected individuals enter a chronic phase of intermittent parasitemia manifested by "megasyndromes" (cardiomegaly, megaesophagus, and megacolon). blood transfusion is a major source of infection in south america, where parasite reduction using chemicals (like gentian violet) on donated blood pose additional risks. six cases of transfusion-transmitted chagas' disease have been reported in the united states (new york, los angeles, texas, and florida) and canada, involving platelet concentrates in at least four cases. currently, there is a 0.1 to 0.2% seroprevalence in areas with high immigrant populations from central and south america. to date, no tests are licensed by the fda for screening, but highly specific confirmatory tests (i.e., western blot assays) are available. close monitoring is needed to define the risk of transfusion transmitted chagas' disease. patients who are transfusion-dependent for aplastic anemias or chronic hemolytic anemias (sickle cell anemia and thalassemias) such as genetic hemochromatosis may develop iron overload (hemosiderosis), which may result in organ failure, primarily of the heart, liver, and pancreas. every milliliter of transfused rbcs contains approx 1 mg of elemental iron. signs of clinical toxicity become evident when total body iron reaches 400 to 1000 mg/kg of body weight. patients who develop iron overload on chronic transfusion are candidates for chelation therapy with parenteral deferoxamine or oral iron chelators (see chapter 23). transfusion of rbc units enriched with a younger cell population or "neocytes" (reticulocytes), advocated by some investigators, potentially increases transfusion intervals and intravascular survival of rbcs (41%). however, its value in reducing hemosiderosis has not yet been established. a transfusion recipient's ability to mount an immune response and inability to reject transfused (donor) t-lymphocytes in cellular blood components is fundamental to the pathogenesis of ta-gvhd. only whole blood and its cellular components (rbcs, granulocytes, and platelets) containing sufficient, viable, cytotoxic t-lymphocytes can meditate ta-gvhd. no cases have been reported following ffp transfusions. this disease has been reported in immunosuppressed patients with hematological and solid malignancies. rarely, it occurs after organ and hpct and in patients receiving myeloablative therapy. immunocompetent transfusion recipients at risk of ta-gvhd share a haplotype with related or unrelated hla homozygous donors (hla haploidentical). ta-gvhd typically appears 2 to 50 d after transfusion. the development of marrow aplasia and progressive pancytopenia distinguishes ta-gvhd from gvhd following hpct. these patients may develop a skin rash, diarrhea, fever, and abnormal liver function accompanied by extensive hepatocellular damage. most cases (>90%) of ta-gvhd are fatal. treatment with immunosuppressive regimens (steroids, cytoxan, and antithymocyte globulin, including okt3) has been ineffective. as a consequence, immunosuppressed patients (with congenital immunodeficiency syndromes, hematological malignancies undergoing myeloablative therapy, allogeneic or autologous hpct) premature neonates (<1200 g), intrauterine transfusions, recipients of blood from biological relatives and hla-matched platelets should receive only irradiated blood. solid organ transplant recipients receiving immunosuppressive therapy or those undergoing chemotherapy/radiation therapy for solid tumors do not require irradiated blood componenets. interestingly, ta-gvhd has not been reported in patients with aids. gamma irradiation with 25-30 gy inactivates all immunoreactive lymphoid cells and prevents ta-gvhd. leukocyte reduction is insufficient to prevent ta-gvhd. the transfusion-related acute lung injury (trali) reaction is rare (1:2000 to 1:5000 units) and occurs during or within the 4-6 h after a transfusion. patients suddenly develop shortness of breath with severe hypoxemia, chills, fever, cough, and tachycardia. noncardiogenic pulmonary edema recognized on chest x-ray as bilateral diffuse interstitial infiltrates results from leukocytic activation and aggregation in the pulmonary bed causing "capillary leak." the pathophysiology of this reaction is thought to be immune-mediated, typically resulting from (1) donor antibodies (granulocytic or lymphocytotoxic antibodies) directed against white blood cell antigens, (2) hla class i and ii antibodies, and (3) lipid activators of neutrophils in donor plasma. in very rare cases, these antibodies may be present in the recipient. most donors associated with trali are multiparous females or donors with multiple exposures to varying hla types. treatment is supportive; however, ventilatory and pressor support may be necessary. resolution occurs within 3 to 7 d in 81% of cases but is fatal in a small proportion of cases. trali is the third leading cause of transfusion-related deaths, accounting for 13% of all transfusion fatalities. an autologous donor donates blood for his or her own future use. for practical reasons, the transfusion of autologous rbcs is an attractive option for patients undergoing elective surgery. autologous blood can be collected from a patient in advance of anticipated need (preoperative collection) or at the start of surgery (acute normovolemic hemodilution); additionally, shed blood can be recovered for reinfusion during surgery (intraoperative collection) or during the postoperative period from drainage devices (postoperative collection). clinicians should be aware that recovered blood does not provide platelets or coagulation factors. autologous blood transfusion avoids the small risk of transfusion-transmitted infectious agents, red cell alloimmunization, adverse reactions resulting from antibody-mediated hemolysis, and leukocyte-associated febrile reactions. in a larger perspective, autologous transfusion supplements and preserves the blood supply for other patients who acutely or chronically need allogeneic transfusions. autologous donors undergoing preoperative collection should be in satisfactory or good health without major cardiac problems or anemia. the predonation hemoglobin requirement is lower (11 g/dl) for autologous donation than for allogeneic donation (12.5 g/dl). autologous donors can donate as often as every 72 h before the scheduled surgery. ideally, supplemental iron is prescribed before the first collection because iron-restricted erythropoiesis is one of the limiting factors in collecting multiple units of blood over a short period of time. oral or parenteral iron supplement enhances recovery of hematopoiesis. rarely, recombinant human erythropoietin must be administered to donors who have insufficient erythropoietic response. in order to justify the cost effectiveness of autologous collections, there should be a high likelihood that at least two units of blood will be used during surgery. hospitals must establish guidelines regarding indications for autologous blood. during a 4-5 wk collection period, 2-4 units of rbcs can be collected. the storage time of autologous blood is comparable to blood from other donors (up to 42 d with additive solutions). unused autologous blood may be discarded or may be used for allogeneic transfusions in facilities that allow "crossover" only after infectious disease testing. only 30% of collections are typically eligible for allogeneic use. many institutions choose not to "crossover" autologous units because of the cost, complexity, and error risks associated with the process. freezing and long-term storage of rbcs is indicated for patients with rare blood group antibodies, making it difficult to find compatible blood (see p.439). scientific basis of transfusion medicine estimated risk of transmission of the west nile virus through blood transfusion in the us american association of blood banks current and emerging infectious risks of blood transfusions current prevalence and incidence of infectious disease markers and estimated window-period risk in the american red cross blood donor population modern blood banking and transfusion medicine blood banking and transfusion medicine standards for blood banks and transfusion services transfusion medicine transfusion therapy: clinical principles and practice the hla system: basic biology and clinical applications clinical practice of transfusion medicine risks of transfusion-transmitted infection rossi's principles of transfusion medicine principles of transfusion medicine request for an exception under 21 cfr (code of federal regulation) 640.120, alternative procedures, to 610.53 (d), dating periods, to use the gambro 7-day elp platelet storage system using apheresis platelets collected with the cobe spectra apheresis system and gambro trima automated blood component collection system us food and drug administration approves gambro bct's 7-day platelet post-market surveillance plan. gambro.bct march 2005 press release key: cord-266204-ipa017wz authors: poland, g. a.; ovsyannikova, i. g.; kennedy, r. b. title: personalized vaccinology: a review date: 2018-08-28 journal: vaccine doi: 10.1016/j.vaccine.2017.07.062 sha: doc_id: 266204 cord_uid: ipa017wz abstract at the current time, the field of vaccinology remains empirical in many respects. vaccine development, vaccine immunogenicity, and vaccine efficacy have, for the most part, historically been driven by an empiric “isolate-inactivate-inject” paradigm. in turn, a population-level public health paradigm of “the same dose for everyone for every disease” model has been the normative thinking in regard to prevention of vaccine-preventable infectious diseases. in addition, up until recently, no vaccines had been designed specifically to overcome the immunosenescence of aging, consistent with a post-wwii mentality of developing vaccines and vaccine programs for children. it is now recognized that the current lack of knowledge concerning how immune responses to vaccines are generated is a critical barrier to understanding poor vaccine responses in the elderly and in immunoimmaturity, discovery of new correlates of vaccine immunogenicity (vaccine response biomarkers), and a directed approach to new vaccine development. the new fields of vaccinomics and adversomics provide models that permit global profiling of the innate, humoral, and cellular immune responses integrated at a systems biology level. this has advanced the science beyond that of reductionist scientific approaches by revealing novel interactions between and within the immune system and other biological systems (beyond transcriptional level), which are critical to developing “downstream” adaptive humoral and cellular responses to infectious pathogens and vaccines. others have applied systems level approaches to the study of antibody responses (a.k.a. “systems serology”), [1] high-dimensional cell subset immunophenotyping through cytof, [2,3] and vaccine induced metabolic changes [4]. in turn, this knowledge is being utilized to better understand the following: identifying who is at risk for which infections; the level of risk that exists regarding poor immunogenicity and/or serious adverse events; and the type or dose of vaccine needed to fully protect an individual. in toto, such approaches allow for a personalized approach to the practice of vaccinology, analogous to the substantial inroads that individualized medicine is playing in other fields of human health and medicine. herein we briefly review the field of vaccinomics, adversomics, and personalized vaccinology. vaccines have been one of the most effective public health strategies in preventing infectious diseases. a decade ago, we described the idea of vaccinomics and adversomics, based on the immune response network theory [5, 6] , which utilizes immunogenetics/imunogenomics and systems biology approaches to understand the basis for inter-individual variations in vaccineinduced immune responses in humans, as well as the basis for adverse side effects from vaccines [7] . vaccinomics and adversomics explore the influence of genetic and non-genetic regulation on the heterogeneity of vaccine-induced immune responses at both the personal and population levels [5] . in particular, vaccinomics and adversomics utilize high-throughput, high-dimensional systems biology approaches, which aim to predict variations in protective and maladaptive innate and adaptive immune responses to vaccines [1] [2] [3] [4] 6, 8] . in this regard, the basis of personalized (and predictive) vaccinology is the assessment of an individual's genetic background, sex, as well as other factors that may impact vaccine immunogenicity, efficacy, and safety [8] [9] [10] [11] . we and others have widely published on the applicability of the tools and concepts of vaccinomics, including immunogenetics and immunogenomics, to the knowledge-based directed development of new and improved vaccine candidates [12] [13] [14] [15] . the application of these concepts is likely to allow for explanation, quantification, and prediction of vaccine-induced protective immune responses-including the http://dx.doi.org/10.1016/j.vaccine.2017.07.062 0264-410x/ó 2017 elsevier ltd. all rights reserved. development of predictive immune signatures in response to vaccines. indeed, we have previously published what we believe is the first draft of a mathematical model and predictive equation describing the non-random events that lead to a pre-determined immune response [6] : b i x iþe y = measure of immune response b o = intercept b i = coefficient for the ith variable x i and indicates the amount of change in y for a 1 unit change in x i ε = random deviations from the model we recognize that such an equation, given the current state of the science, is incomplete and cannot yet predict immune responses. but we present it as an early directional attempt to quantify such an equation. such an approach begins to move us into a 21st-century model of directed vaccine development and an advanced understanding of how, and by what mechanisms, vaccines and vaccine adjuvants trigger both useful and maladaptive innate and adaptive immune responses. we believe that vaccinomics and adversomics represent approaches counter to the standard methods of vaccine development until recently. historically, vaccine development has been empirical, despite many emerging and re-emerging complex, hyper-variable pathogens-many with elaborate immune escape mechanisms. in addition, vaccine coverage rates continue to suffer as society is risk-averse toward vaccines and demands levels of safety that may not be achievable. finally, the ''one-size-fits-all" approach to the practice of vaccinology ignores the complexity and diversity of the human immune system and host genome. thus, the promise of vaccinomics and related paradigms is to identify specific immune response profiles, immunosignatures, and biomarkers that predict vaccine safety and/or efficacy, and which may lead to new vaccine candidates. vaccinomics provides the opportunity to examine not only immune response genes likely to be involved in vaccine response, but also the possibility of identifying the influence of new (uncharacterized) genes on vaccine-induced immunity. in turn, the identification and directed study of such genetic variants allows recognition, often at the molecular level, of the effects of differential binding, processing, and expression/presentation of antigenic viral peptides used in vaccine development, identification of the differential range of presented peptides (genetic restriction), altered secretion patterns (cytokines) in response to vaccines or vaccine adjuvants, altered transcription of important genes (signaling molecules) and gene products, altered binding of virus/antigens by membrane-based receptors (tlrs, other), differential receptor function, expression, and affinities, and the impact of epigenetics on vaccine-induced immune responses. we have utilized this knowledge in our own laboratory to create a research-oriented paradigm of ''discover-validate-characterize-apply," which may be used in new candidate vaccine development ( fig. 1 ) [6] . in this paradigm, we have been able to utilize vaccinomics approaches to discover genetic variants that are significantly associated with subsequent downstream immune responses, validate that such variants are indeed associated, then seek to characterize the mechanism whereby such effects occur and, finally, apply this knowledge-often in functional studies that confirm the effect on immunity. such knowledge can be exploited in developing immune strategies to enhance or circumvent genetic restrictions, for example, in triggering vaccine-associated immune responses, by ''reverse engineering" around a given genetic or other obstacle to generating protective immune responses. there are a growing number of studies reporting unbiased genome-wide assessments of genetic variation and its influence on adaptive (humoral and cellular) vaccine-induced immune responses across multiple viral and bacterial vaccines. for example, candidate and gwas immunogenetic and phamacogenetic studies have identified polymorphisms in hla, kir, mica, and btn genes associated with immune responses to pathogens causing disease in humans, such as hepatitis c [16] , mycobacterium leprae [17, 18] , human immunodeficiency virus [19] , and measles [20] [21] [22] . similar studies have identified novel genes impacting immune responses to vaccines, including hepatitis b, rubella, influenza a, smallpox, anthrax, and mumps [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] . our gene association studies of measles-mumps-rubella (mmr) vaccines have demonstrated that inter-individual variations in measles vaccine virus-induced humoral and cellular responses are significantly associated with polymorphisms in immune response genes and, together with hla alleles, explain 30% of the inter-individual variability in humoral response [5, [34] [35] [36] . these findings, which illustrated the importance of key hla alleles in the adaptive humoral immune response to measles vaccine, led to the identification of naturally processed and presented measles-derived peptides isolated from specific hla polymorphisms associated with vaccine non-and hyper-response [37, 38] . these peptides containing specific components (adjuvants and biodegradable nanoparticles) are now being utilized in a reverse-engineering strategy to develop peptide-based candidate measles vaccines. likewise, homan et al. have attributed diminished protection to differential hla presentation of t and b cell epitopes between vaccine and wild type strains of mumps virus [39] . this diminished efficacy could theoretically be overcome by incorporating defined critical immunogenic peptides into an improved vaccine. tlr genes represent an important link between the innate and the adaptive immune system [40, 41] . as an example, we have demonstrated that measles vaccine-induced humoral responses are significantly associated with coding polymorphisms in the tlr2 (rs3804100) and tlr4 (rs5030710) genes [42] . for the rubella vaccine and tlr3 gene, a tlr3 gene snp rs5743305 was associated with rubella-specific gm-csf production [43] . our recent mumps vaccine study has identified and replicated tlr4 snps associated with a 45% decrease in antibody titer, and a tlr5 snp associated with a 64% increase in t cell response (unpublished data). these data strongly suggest that robust tlr activation by measles, mumps, and rubella viruses is crucial for optimal vaccine response. supporting these findings is a study demonstrating that an inactivated mumps vaccine containing a protollin-based tlr2/4 adjuvant is highly immunogenic in a mouse model; it led to superior total igg levels, higher neutralizing antibody titers, greater mucosal iga production, and enhanced th1/th2 cytokine secretion [44] . one potential application of this finding is to identify the specific and critical interactions between tlrs (and other genes) and virus, leading to advances in our knowledge of the precise mechanisms driving immunity to mmr vaccine. significant sex differences in humoral and cellular immune responses to vaccines are apparent [45, 46] . additionally, local and systemic adverse rates are generally higher in females versus males. protective antibody responses are significantly higher in females than males after vaccination against influenza, yellow fever, measles, mumps, rubella, hepatitis a and b, herpes simplex (hsv) 2, rabies, smallpox, and dengue viruses [47] [48] [49] [50] [51] [52] [53] [54] [55] . sex-based differences in humoral immune responses are observed through various age groups [47] [48] [49] [50] [52] [53] [54] [55] [56] [57] , suggesting that sex steroid hormones are not the singular mediators of sex differences in humoral immune responses to vaccines [45, 58] . this suggests that genetic, or other, factors may be an important driver of sex-related differences in humoral immune response [59] . despite significant evidence of immune response differences between the sexes, for the most part, vaccine studies have not examined and analyzed immune response outcomes by sex [60, 61] . in fact, little information is known about potential mechanisms for sex-based effects, which should be a priority for vaccine research studies. discovery of specific factors involved in sex-based differences in immune response may allow the identification of new correlates of vaccine immunogenicity. in a cohort of 556 older (ages 50-64) and 558 younger (ages 18-49) previously vaccinated individuals, the seasonal trivalent influenza vaccine induced >1.5-fold higher a/h3n2-specific hai antibody titers in women than men across both age groups [47] . similarly, a study of standard seasonal influenza vaccine and high-dose influenza vaccine responses in a sex-balanced cohort of 414 elderly subjects (ages 65-95) demonstrated significantly higher rates of seroconversion in females than in males [48] ; however, no significant differences in antibody measures were found between males and females after seasonal influenza vaccination in another cohort of 158 older adults (ages 50-74) [62] . a study by furman et al. examining gene expression, serum cytokines/ chemokines, cell subsets, and phosphorylation events found several serum markers (lept, il-1ra, crp, gm-csf, and il-5) to be more highly expressed in females than males after influenza vaccine [51] . this same report used a systems biology approach to identify a gene cluster involved in lipid biosynthesis that is regulated by testosterone and significantly correlated with poor humoral responses following influenza vaccination in men [51] . these data suggest that this gene cluster (e.g., genes involved in lipid metabolism) could be an important driver of sex-related differences in humoral immune response. this collective knowledge could substantially assist future personalized vaccine development efforts through the generation of new knowledge and the identification of targets and biomarkers that predict vaccine responses in specific populations (e.g., females vs. males; young vs. old; obese vs. lean). further research is needed to clarify the effects of sex on immune response. identification of molecular immune signatures of sex differences in innate and adaptive immune responses to vaccines may provide evidence necessary for additional efforts in designing personalized vaccination and vaccinomics approaches (i.e., in which males and females might be vaccinated differently using different doses or different vaccines) to provide equal protection while reducing side effects [46, 63, 64] . a significant global public health issue is the aging of the population. as individuals age, immunosenescence develops, leading to poorer immune responses to vaccines. immunosenescence is an age-related dysregulation of the immune system due to ageassociated changes in innate and adaptive immune system components, which leads to impaired immunity and protection following immunization or infection [65] [66] [67] . published data reveal that innate and adaptive immunity is decreased with age, but the systems-level mechanisms for these findings are unclear [66, 68] , particularly in regard to influenza and other viral vaccine responses where the morbidity, mortality, and associated healthcare costs are greater in older individuals [11] . major signs of innate immune dysfunction commonly observed in the elderly include, but are not limited to, altered cytokine secretion; decreased nk cell activity; reduced tlr expression; and a chronic inflammatory state (elevated levels of il-1b, mcp-1, tnf-a, and serum il-6) known as ''inflamm-aging" [8, [69] [70] [71] . age-related humoral immune dysfunction, for example, might be overcome through optimal stimulation of innate and/or th cell-specific genes, which may be different in males and females. for example, adjuvanted zoster subunit vaccine (hz/su) reduced the risks of herpes zoster, and postherpetic neuralgia in immunocompetent persons 70 years of age and older [72] . this hz/su vaccine contains varicella zoster virus glycoprotein e and a novel as01 b adjuvant system aimed to improve and preserve with age zoster-specific cd4+ t cell responses [73] . a tlr4 agonist gla-se (glucopyranosyl lipid adjuvant formulated in a stable emulsion) has been shown to enhance th1 responses to influenza vaccine in older adults [74] , suggesting a potential mechanism for targeting innate receptor agonists (e.g., tlrs) that enhance innate immune responses against influenza. given the substantially diminished efficacy of influenza and other vaccines with age and the importance of developing improved vaccines [75] , data from vaccinomics studies could be used to inform directed and rational development of next-generation influenza vaccines-potentially circumventing immunosenescence-related factors. systems biology approaches provide a unique opportunity to identify biomarkers likely to be involved in immune responses to vaccination [1] [2] [3] [4] 8, 76, 77] . fourati et al. applied a systems vaccinology approach to examine gene signatures and molecular pathways of age-related hyporesponse to hepatitis b vaccine (hbv) in naïve older adults [78] . they observed the b cell signaling pathway (and higher memory b cell frequencies) and inflammatory pathway (and increased frequencies of activated pro-inflammatory innate cells) were strongly correlated with higher and low antibody responses to hbv, respectively. this signature, including serum cytokine profiling and flow cytometric correlates of response, predicted the antibody response to hbv with up to 65% accuracy [78] . this study demonstrates that a systems biology approach can be used to predict age-related immune response to vaccination. obesity is another major public global health concern. in the us, 68% of adults and nearly 32% of children and adolescents are now overweight or obese [79] . weight gains across all countries have been demonstrated to be associated with increasing socioeconomic status. obesity has been shown to be a predictor of impaired immunogenicity (e.g., decreased antibody response) to hepatitis b, tetanus toxoid, rabies, and influenza vaccines [80] [81] [82] [83] , and as such can be considered a marker, or state, of immunosuppression at its extremes. these data suggest that obesity is correlated with poorer vaccine-induced immune responses in humans, and further research is required to understand the immune mechanisms that are altered in obesity. as individuals age, their circulating leptin levels rise with a concomitant reduction in leptin signaling; this results in leptin resistance, which is a finding associated with obesity [84] . leptin resistance has been shown to adversely affect the immune response in obese subjects, including responses to influenza virus [85, 86] . for example, obese individuals demonstrate decreased activation of influenza-specific cd8+ t cells compared to healthyweight persons, including decreased production of ifn-c and granzyme b, suggesting that influenza vaccination may not be as effective in the obese population as in healthy-weight individuals [87] . given only moderate seroprotection of influenza and other vaccines in obese older adults [83] , and the importance of developing improved influenza vaccines [75] , systems biology studies designed to identify the mechanisms for improved immune response are needed. in fact, data from vaccine studies could be used to inform directed and rational development of personalized vaccines that optimally stimulate innate and adaptive immune responses in males and females and overcome immune deficiencies induced by obesity [88] . careful vaccine studies comparing lean and obese persons could provide foundational data used to improve vaccine-induced protection in the obese, a subpopulation with an elevated risk for serious vaccine-preventable illnesses and suboptimal vaccine-induced protective responses [10] . adversomics utilizes tools-much like those used in vaccinomics-to identify, characterize, and predict adverse, or maladaptive, immune responses to vaccines [6, 89, 90] . the promise of adversomics would be to develop or identify either predictors or immune signatures of maladaptive immune responses that lead to harm rather than benefit, and to better understand the generation and mechanisms of such maladaptive immune responses. we have asked the question, as have other scientists, ''does it make sense in the 21st century to give the same vaccine, dose, and at the same frequency to everyone, regardless of age, weight, gender, race, genotype, and medical condition?" for example, we give adult males and females the same dose, and the same number of doses of vaccines, ignoring the findings that females nearly always have superior humoral immune responses to males for all vaccines studied, and yet experience significantly more side effects-more adverse events, of greater duration, and of higher intensity [47, 55, 60] . while the field is young in implementation, research has already revealed associations between specific genes or snps and adverse immune outcomes. for example, associations between cytokine gene expression and fever after smallpox vaccine have been identified [91] . other studies have demonstrated correlations between smallpox vaccine-induced fevers and il1a and il18 snps [92] . other smallpox vaccine-induced adverse events such as fever, rash, and enlarged lymph nodes have been significantly associated with mthfr, irf1, and il4 snps haplotypes [93] . while smallpox vaccine is not used in the general population, such studies stand as examples of the usefulness of vaccinomic approaches. finally, other recent studies have identified generic fever gene networks (tnfa) after vaccine administration [94] , and relationships between mmr vaccine administration and snps in ifi44l, cd46, scn1a, 2a, and tmem16 (ano3) genes [95] . despite the tremendous success of vaccines, vaccinologists face several current challenges, including difficulty in developing vaccines for hypervariable viruses (hiv, rhinovirus, hepatitis c virus, coronavirus) and complex pathogens (malaria, mycobacterium tuberculosis); newly emerging pathogens, such as zika virus (zikv); complications imposed by aging and immunosenescent populations; inadequate understanding of the neonatal and newborn immune systems; increasingly immune deficient or immunocompromised populations due to hiv, cancer, or medications; sexbased differences in vaccine response and adverse-event rates; enhanced scrutiny of vaccine safety; and as noted global increases in age and weight. in addition, vocal and active anti-vaccine groups whose messages are not easily countered by facts or scientific studies have materially and detrimentally affected vaccine coverage rates [96] [97] [98] . vaccinomic approaches can be utilized to better understand these issues; this information can then be used to inform new approaches, new understandings, and new vaccine candidates. just as new technologies have created exciting new opportunities in personalized medicine, they have brought with them novel challenges in addition to those mentioned above. in order for the full potential of personalized vaccines to be achieved, we must overcome additional challenges, such as the need for the following: larger genotype:phenotype datasets (often in the many thousands to ten thousands) integrating increasingly diverse high-throughput, highdimensional data types biomarkers that can reliably distinguish which product patients receive based on the likelihood of their response or an adverse side effect vaccines with different mechanisms of action may require a move away from humoral correlates of protection for licensure; in this regard, correlates of protection based on cellular immune outcomes are likely to play an important role in future vaccines more sophisticated biostatistical and bioinformatics approaches that can identify patterns and causative networks within terabyte levels of extremely high dimensional data types from the economic side: methods of technology transfer and funding mechanisms to move novel vaccines developed through vaccinomic approaches into low and middle-income countries who often most need specific vaccines (malaria, others) we have seen the shift from ''vaccinology 1.0," which is the empirical ''isolate-inactivate-inject" paradigm, to ''vaccinology 2.0"-the use of recombinant technology and novel adjuvants. however, even this paradigm is limited by our incomplete mechanistic understanding of adjuvants and innate immunity. as we adopt approaches such as those listed above, we envision a movement of the field into an era of ''vaccinology 3.0," during which we expect to see the use of vaccinomics and systems-level approaches to develop new vaccines; innovative vaccine-antigen packaging methods; and adjuvant development targeted at the innate response pathways best suited for a given pathogen. a common reaction to this paradigm of personalized vaccinology is questioning cost and economics. at one level, such considerations are simply ''too soon" in the development of the science to effectively answer. however, like progress being made in individualized medicine, it is likely that being able to provide the right vaccine to the right patient-for the right reasons and at the right dose-will lead to improved medical outcomes and reduced costs at the population level. personalized vaccinology is the goal of applying the concept of personalized medicine to vaccines. rapid strides in omics technologies and foundational work applying systems biology, computational immunology and reverse vaccinology have facilitated modern approaches to vaccine design and development enabling us to create vaccine formulations for new and re-emerging pathogens. egg-based influenza vaccines take >6 months to create. the recent licensure of cell culture-based influenza vaccines demonstrate that rapid, scalable processes can now be implemented in order to create vaccine against emerging influenza strains (e.g., h1n1, h5n1, h7n9, h9n2, h7n8) within weeks [99] and can be safely administered to individuals with egg allergies [100] . the ebola outbreak in liberia, sierra leone, and guinea in 2015 provides an example of the need to rapidly develop vaccine candidates [101] . dna vaccines, virus-like particle vaccines, and replicating/nonreplicating viral vector vaccines have all been created and tested. among the most promising are a replication-competent, recombinant vesicular stomatitis virus vector expressing the glycoprotein of ebola zaire (rvsv-zebov), [102] a variety of adenovirusvectored vaccines expressing ebola glycoprotein, [103, 104] a modified vaccinia virus ankara-based vaccine encoding the ebola zaire glycoprotein (mva-bn-filo), [105, 106] and dna-based vaccinesone expressing glycoproteins from both zaire and sudan, and the other expressing the marburg glycoprotein [107] . although the rvsv-based vaccine elicits high titers of neutralizing ab, it is contraindicated in children and those with compromised immune systems. viral vector vaccines present the problem of developing robust immunity to the vector as well as the target immunogen, limiting their usefulness to a single vaccination. the availability of vaccines in multiple vector backbones opens up the possibilities for prime-boost vaccination strategies for ebola, similar to those that have been applied to hiv, malaria, and tuberculosis [108] [109] [110] [111] . in this regard, a prime-boost regimen using the mva-based vaccine as the booster vaccination has shown considerable promise [101] . another example of modern vaccine development being applied to a new pathogen can be seen with the response to zika virus. a purified, formalin-inactivated vaccine (zikv piv) has been developed by the walter reed army institute of research (wrair) [112] and is being evaluated in several clinical trials (nct02963909, nct02952833, nct02937233), while other inactivated vaccines are in preclinical development [113] . two variants of a plasmid dna vaccine containing the prm-env proteins have been developed by niaid and one of the formulations is currently in a phase i clinical trial (nct02840487) [114] . inovio pharmaceuticals developed their own plasmid dna vaccine (also expressing prm-env), which is currently in two clinical trials (nct02809443, nct02887482). rna-based vaccines [115] and a variety of subunit and viral vector-based vaccines are also in development [113, 116, 117] . dna and rna-based vaccines can be rapidly made at minimal costs compared to other formulations and are fairly stable, without the cold-chain requirements of live virus-based vaccines. subunit vaccines are typically safer than whole virus-based products, which represents an active area of investigation not only for pathogens with no existing vaccines, but also for improving on established vaccines. our group and others have identified pathogen-derived epitopes as preliminary steps in the development of safe, stable, and effective peptide-and protein-based vaccines for smallpox, influenza, measles, tuberculosis, staphylococcus, and myriad other viral and bacterial pathogens [38, [118] [119] [120] [121] [122] . parallel efforts by different groups to create new vaccines result in a spectrum of potential products that can be uniquely tailored to specific population groups. live viral vaccines rapidly inducing robust immunity can be used in healthy individuals where time is of the essence (e.g., in outbreak scenarios), while inactivated or subunit vaccines can be used in vulnerable populations such as pregnant women or those with immunocompromising conditions, or in young children where the presence of maternal antibody interferes with whole virus vaccines. vaccines based on different viral vector backbones can be combined into effective primeboost regimens. vaccines with specific adjuvants may be most appropriate for the elderly in order to overcome immunosenescence, or in the very young in order to compensate for immune system immaturity. we, along with increasing numbers of other scientists, believe that personalized vaccinology will revolutionize the practice of vaccinology to the benefit of human health. as part of the development of this field of science, vaccinomics and adversomics will allow us to develop molecular immune signatures of adaptive and maladaptive immune responses to vaccines, develop early biomarkers of vaccine response in vaccine trials, identify who should get what vaccine and at what dose, and increase safety and public confidence in vaccines by reducing the likelihood of serious adverse events related to vaccines. in many ways, however, personalized vaccinology is most challenged by the difficulty in moving the field away from the post-wwii population-level paradigm of ''one dose of every vaccine for everyone," toward an individualized or personalized approach based on the unique factors relevant to a given individual. in his book, the structure of scientific revolutions [123] , thomas kuhn recognized that ''we wrongly believe scientific progress is a process of linear accretion of knowledge, that science is predicated on the belief that the scientific community understands what the world is like, and that we suppress or resist 'fundamental novelties' because they are seen as subversive to our firmly held beliefs of what the world is like." later in his book, he suggests that ''new advances always have and always will reveal that science and medicine includes bodies of belief incompatible with beliefs we hold today, and that advancements come when we reject a time-honored scientific theory in favor of another incompatible with it." these cognitive biases have, in our opinion, been manifest in our discussions with scientific colleagues as we developed this field of science. schopenhaur, the german philosopher, suggested that new discoveries are at first ridiculed, then opposed, and finally accepted as self-evident. vaccinomics and adversomics appear to be moving from the ridiculed and opposed steps, and into the not-yet quite self-evident phase of the continuum. part of the challenge is that often the concept of personalized vaccinology suggests to the reader that a unique vaccine will be developed for each individual. while that is one tactic being used in the cancer-vaccine field, it is neither necessary nor practical for the prevention of infectious diseases. rather, the personalized vaccinology approach would suggest the development of specific vaccines based on factors that relate to overcoming the potential for poor immunogenicity and the potential for adverse events. an excellent example is influenza vaccines. a mere decade or so ago, only a trivalent injectable influenza vaccine was available. quadrivalent vaccines were unavailable. for with one exception, everyone received the same vaccine and dose, regardless of age, weight, immunosuppression state, etc. at the current time in the us, multiple influenza vaccines are available so that the right vaccine, for the right patient, can be given at the right time. for example, laiv (live attenuated influenza vaccine) can be used in younger subjects or the needle-phobic. high-dose or mf59-adjuvanted vaccines can be chosen for the elderly. recombinant vaccines can be chosen for those with egg allergy, and so on. this is the approach that should be taken with all vaccines. in some cases it may mean merely adjusting the dose based on weight, gender, or age. in other cases it may mean utilizing an adjuvanted or non-adjuvanted vaccine based on immune status. other examples include the recently licensed mf59 adjuvanted influenza vaccine (fluad ò ), which has demonstrably higher immunogenicity and efficacy than its nonadjuvanted counterparts, [124] [125] [126] or the highly effective as01adjuvanted zoster glycoprotein e vaccine, which does not contain live virus and may be more broadly suitable for administration to older individuals [72, 73] . thus, the movement toward a new paradigm of vaccine practice, based on a personalized approach, is occurring in the 21st century based on new scientific knowledge, market demand, safety considerations, immunogenicity concerns, public health trends (age, obesity, other), and the simultaneous pull of individualized medicine in other medical arenas. the net result is likely to be higher vaccine coverage rates, increased public confidence in vaccines, improved immunogenicity and adverse event rates, and a reduction or elimination in the morbidity and mortality related to vaccine-preventable diseases. as a result, we anticipate a new era of personalized ''predictive vaccinology," whereby we abandon a ''one size and dose fits all vaccine approach" in order to design and develop new vaccines, and acquire the ability to make the following predictions for each individual: whether to give a vaccine based on likelihood of response (and perhaps need); the likelihood of a significant adverse event to a vaccine; and the number of doses likely to be needed to induce a protective response to a vaccine [63] . current vaccine development is largely empirical. vaccines are tested by trial and error, are mass produced, and given to the entire population using the same antigen dose, route of administration, number of vaccinations, and at the same age. in contrast, the new vaccine-development paradigm begins with the ''discovery" of new knowledge by integrating unbiased, comprehensive analysis of the genome, transcriptome, proteome, metabolome, microbiome, and immunome-along with the assessment of multiple measures of immune function-in order to under-stand and evaluate perturbations of the immune system. findings are then ''validated" in replication cohorts or additional model systems. the new knowledge is then ''applied" to the creation of new vaccine formulations that can undergo additional testing to start a new round of ''discovery," or can move into clinical trials in order to develop vaccine products engineered to elicit (or avoid) specific effects on the immune system. each product is tailored to specific subgroups such that robust, protective immunity can be elicited in the old and young, lean and obese, or male and female, while avoiding inappropriate immune responses due to genetics, metabolism, race, gender, malnutrition, immunosuppression, and other host factors or underlying conditions. r01ai033144, and contract no. hhsn266200400025c (n01ai40065). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. the authors would like to acknowledge the contributions of the centers for disease control and prevention (cdc), which provides financial support to the world health organization initiative for vaccine research (u50 ck000431). dr. poland is the chair of a safety evaluation committee for novel investigational vaccine trials being conducted by merck research laboratories. dr. poland offers consultative advice on vaccine development to merck & co. inc., avianax, dynavax, novartis vaccines and therapeutics, emergent biosolutions, adjuvance, seqirus, and protein sciences. drs. poland and ovsyannikova hold three patents related to vaccinia and measles peptide research. dr. kennedy has received funding from merck research laboratories to study waning immunity to mumps vaccine. these activities have been reviewed by the mayo clinic conflict of interest review board and are conducted in compliance with mayo clinic conflict of interest policies. dissecting polyclonal vaccine-induced humoral immunity against hiv using systems serology cytometry by time-offlight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of cd8 + t cell phenotypes highresolution myogenic lineage mapping by single-cell mass cytometry metabolic phenotypes of response to vaccination in humans heterogeneity in 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response against homologous and heterologous strains elicited by a mf59-adjuvanted influenza vaccine in infants and young children the comparative effectiveness of adjuvanted and unadjuvanted trivalent inactivated influenza vaccine (tiv) in the elderly research reported in this publication was supported by the national institute of allergy and infectious diseases of the national institutes of health under award number u01ai089859, r37ai048793, key: cord-273906-s7l0yxc0 authors: ranga, vipin; niemelä, erik; tamirat, mahlet z.; eriksson, john e.; airenne, tomi t.; johnson, mark s. title: immunogenic sars-cov-2 epitopes: in silico study towards better understanding of covid-19 disease—paving the way for vaccine development date: 2020-07-23 journal: vaccines (basel) doi: 10.3390/vaccines8030408 sha: doc_id: 273906 cord_uid: s7l0yxc0 the emergence of the covid-19 outbreak at the end of 2019, caused by the novel coronavirus sars-cov-2, has, to date, led to over 13.6 million infections and nearly 600,000 deaths. consequently, there is an urgent need to better understand the molecular factors triggering immune defense against the virus and to develop countermeasures to hinder its spread. using in silico analyses, we showed that human major histocompatibility complex (mhc) class i cell-surface molecules vary in their capacity for binding different sars-cov-2-derived epitopes, i.e., short sequences of 8-11 amino acids, and pinpointed five specific sars-cov-2 epitopes that are likely to be presented to cytotoxic t-cells and hence activate immune responses. the identified epitopes, each one of nine amino acids, have high sequence similarity to the equivalent epitopes of sars-cov virus, which are known to elicit an effective t cell response in vitro. moreover, we give a structural explanation for the binding of sars-cov-2-epitopes to mhc molecules. our data can help us to better understand the differences in outcomes of covid-19 patients and may aid the development of vaccines against sars-cov-2 and possible future outbreaks of novel coronaviruses. the ongoing pandemic outbreak of covid-19 has resulted in the declaration of a global health emergency around the world on 30 january 2020 by the world health organization (who) [1] . the first reported case was on 31 december 2019 from the chinese city of wuhan, from which the virus quickly spread to 213 other countries and territories and, as of 17 july 2020, resulted in at least 13.6 million infections and over 585,000 deaths [2] . based on the early epidemiology of the covid-19 disease statistics, the who estimated that the fatality rate of the novel coronavirus is around 4%, significantly higher than the mortality rate caused by common human coronaviruses [3] . there are currently numerous scientific, clinical, and socio-economical efforts aimed at combating the covid-19 disease and its ramifications around the world [4, 5] . peptides are loaded on mhc receptors using a peptide loading complex consisting of tap and several other proteins [23] . in order to narrow down the specific epitopes that could elicit an effective mhc class-i-mediated t cell response, we predicted linear 9-mer immunogenic sars-cov-2 peptides and their prominent interacting hla allotypes using the immune epitope database and analysis resource (iedb) and netctl1.2 web servers. the identified peptides were then analyzed in conjunction with the available experimental data for sars-cov-derived linear t-cell epitopes. the three-dimensional structural models of selected ternary complexes of sars-cov-2 epitope-hla allotype-t cell receptor were created to assess interactions at the structural level. our results can at least partially explain individual differences in the covid-19 severity and could potentially be used for vaccine development against sars-cov-2. all 26 protein sequences encoded by the most up-to-date sars-cov-2 genomic sequence (refseq: nc_045512.2) were retrieved from ncbi refseq database [24] on 27 march 2020 (full accession identifiers in table 1 ). the iedb (http://tools.iedb.org/mhci/) [25] and netctl1.2 (http://www.cbs.dtu.dk/services/ netctl/) [26] web servers were used with default parameters to predict sars-cov-2 epitopes and their binding affinities (expressed as ic 50 ) to different hla allotypes. the iedb server sorts the predicted mhc-i-binding viral epitopes based on the percentile score, which is calculated by comparing the predicted binding affinities of sars-cov-2 peptides and affinities calculated for a large set of peptides, randomly selected from the swissprot database [25] ; the iedb server integrates an artificial neural network (ann), stabilized matrix method (smm) and combinatorial library (comblib). the iedb method is highly accurate in classifying mhc class i epitopes, having an auc for the roc curve greater than 0.9 [27] . the netctl1.2 server integrates prediction of proteasomal cleavage, tap transport and peptide-binding to 12 mhc-i supertypes (see table s1a for a list of hla-a and hla-b allotypes that belong to these 12 supertypes, with data extracted from the published scientific literature [28, 29] ). the netctl1.2 method allows for the identification of class i mhc epitopes with a sensitivity of 0.80 and specificity of 0.97 based on the default filtering threshold score of 0.75 [26] . since the netctl1.2 and iedb servers use different multistep approaches to predict the binding of sars-cov-2 peptides to hlas, we used both servers. mhc-i allotypes are known to bind epitopes with lengths of 8 to 11 amino acids. the optimal epitope length was determined by potting the ic 50 values of the predicted (iedb) top 1 percentile epitopes against 8-to 11-mers. moreover, the immunogenicity of the top 1 percentile epitopes were predicted using the mhc-i immunogenicity server of iedb (http://tools.iedb.org/immunogenicity/) [25] . the epitopes with an immunogenicity score greater than 0.25 were considered for comparison with epitopes predicted using the netctl1.2 server. in order to identify experimental epitopes matching the predicted sars-cov-2 epitopes, the data of experimentally known sars-cov epitopes and their interacting mhc allotypes (validated using t-cell assays and mhc ligand assays) were downloaded from the iedb database on 15 april 2020 (https://www.iedb.org/database_export_v3.php) [25] . the characteristics of each "match" (protein name, sequence, mapped start-end, mhc-i allotype, etc.) were tabulated and are presented in table s8 . the grand average of hydropathicity index (gravy) was calculated using the kyte-doolittle hydropathy index scale [30] . tmhmm2.0 was used to search potential transmembrane helices [31] . the jpred4 web server was used to predict secondary structures in non-transmembrane proteins [32] . the half-lives of the predicted emhc-i complexes was predicted using the netmhcstabpan1.0 web server [33] . data were plotted using graphpad prism (graphpad software, version 8.0.0; https://www.graphpad.com). the crystal structures of an influenza a virus epitope in complex with the hla-a*02:01 allotype (pdb id: 5tez, chain a and c; 1.7 å) [34] and hepatitis b core antigen with hla-a*02:06 (pdb id: 3oxr, chain a and c; 1.7 å) [35] were downloaded from the protein data bank (pdb) [36] (see table s1b for the pdb codes of mhc-i allotype structures indicated in table s1a ). for docking to hla allotypes, the rosetta flexpepdock web server [37] was used, after the 9-mer influenza a epitope was mutated to match selected sars-cov-2 epitopes using pymol (the pymol molecular graphics system, schrödinger, llc). for ternary complex analysis, the t-cell receptor (tcr)-hla-influenza a epitope complex (pdb id:5tez) was used as a template. coordinates of the tcr α and β chains (pdb id: 5tez; chain i and j, respectively) and the docked epitope-hla-a*02:01 complex were saved using pymol, and interacting residues visually inspected at the interface. molecular dynamics (md) simulations followed the general protocol detailed in [38] . briefly, the ternary composite structure of hla-a*02:01 in complex with the sars-cov-2 s protein epitope 1220 fiagliaiv 1228 and tcr was used as a starting structure to perform molecular dynamics simulation. the structure was initially prepared using maestro (schrödinger release 2019-1: maestro, version, schrödinger, llc) by adding hydrogen atoms, determining the protonation states of ionizable side-chains and energy minimizing the structure to remove bad contacts. three independent simulations were carried out using the amber program (version 2018) [39] and the ff14sb protein forcefield [40] . the ternary structure was solvated with tip3p water molecules [41] in an octahedral box, keeping a 12 å distance between solute atoms and the surface of the box. the simulation system was neutralized by adding na + ions, with additional na + /cl − ions incorporated to bring the system salt concentration to 0.15 m. the system was then energy-minimized for 5000 cycles using the steepest descent and conjugate gradient methods. the minimization was carried out in six-stages, where a restraint on solute atoms was gradually lowered from 25 to 0 kcal mol −1 å −2 . subsequently, the system was heated to 300 k during 100 ps with a 10 kcal mol −1 å −2 restraint on solute atoms. next, equilibration was performed for 6 ns in four steps by systematically reducing the restraint force to 0 kcal mol −1 å −2 . finally, a restraint-free 100 ns production simulation was carried out at constant temperature (300 k) and pressure (1 bar). coordinates were saved every 20 ps and the sampled conformations were analyzed using vmd [42] , cpptraj [43] and chimera [44] programs. root-mean-square fluctuation (rmsf) calculation was computed using the cα atoms of the initial structure as a reference. hydrogen bonds were defined with a bond length ≤3.5 å and a bond angle ≥135 • . in order to estimate the potential antiviral cytotoxic t-cell response linked to specific hla allotypes, we predicted the binding affinity of all possible linear 8-to 11-mer peptides derived from the 26 proteins (table 1 ) of the sars-cov-2 proteome (n 8 = 375, n 9 = 2105, n 10 = 1556 and n 11 = 2377) to hla-a and hla-b supertypes using the iedb web server [25] . the hla-c supertype-an extremely good ligand for killer-cell immunoglobulin-like receptor (kir) on natural killer (nk) cells-was not selected for this analysis because it is known to be less effective in presenting antigens to cytotoxic t-cells than either hla-a or hla-b [45] . the class i mhc-epitope (emhc-i) complexes were classified into three different groups based on the predicted epitope-to-mhc binding affinity scores [46] : strong binders (ic 50 ≤ 50 nm), weak binders (50 nm < ic 50 ≤ 500 nm) and non-binders (ic 50 > 500 nm). out of the highest scoring sars-cov-2 epitopes (top 1 percentile), the 9-and 10-mers had, on average, a higher binding affinity to mhc class i supertypes than either the 8-or 11-mer epitopes ( figure 1a ). moreover, there were 52% more 9-mer peptides (3187) predicted to bind to class i mhc receptors (ic 50 ≤ 500 nm) in comparison with 10-mer peptides (2096) ( figure 1b) . consequently, the top one percentile 9-mer peptides were selected for further analysis. the predicted (iedb) sars-cov-2 9-mer epitopes (top one percentile) for 50 different hla allotypes (table s1a) with an immunogenicity score ≥ 0.25 were compared to those predicted using the netctl1.2 server [26] . the mhc class i epitopes identified from this consensus/combined approach were classified using the binding affinity values from iedb prediction method as strong mhc binders (table s2) , weak binders (table s3 ) or non-binders (table s4 ). based on this analysis, many peptides derived from nonstructural proteins (nsp), surface glycoproteins (s) and membrane proteins (m) of sars-cov-2 are likely to be presented by mhc class i receptors ( figure 1c , table s5 ), and hence have a high potential to activate an immune response or the destruction of infected host cells, with many epitopes being derived from the s, e, 3c-like proteinase (3clpro) and 3 -to-5 exonuclease (35exo) proteins. in order to predict the most potent epitopes, including those without experimental data, we screened in silico the proteins of sars-cov-2. we ranked the epitopes based on predicted binding to mhc-i molecules (affinity values; iedb) and "combined score" (netctl1.2), predicting the potency of an epitope to be presented by mhc-i (tables s6 and s7 ). the top-ranked, common epitopes from both prediction methods (table 2) were selected for further analysis; all five of these predicted epitopes are unique to sars-cov-2 and not identical in sars-cov. this comparison, we identified 29 common epitopes of sars-cov/sars-cov-2 (table s8 ) and hla allotypes hla-a*02:01 and hla-a*02:06 molecules as the top antigen presenters; both allotypes having strong binding affinities (ic50 ≤ 50 nm) to six peptide epitopes (table 3) : residues 1220 fiagliaiv 1228 from the s protein, 17 vllflafvv 25 and 20 flafvvfll 28 to the e protein, 204 vlawlyaav 212 to 3clpro, 330 llsagifga 338 to nsp3 and 184 vlwahgfel 192 to 35exo of sars-cov-2. in order to obtain experimental proof that the mhc-i-binding sars-cov-2 epitopes predicted in this study are presented by the mhc class i antigen processing pathway in vivo, we compared the in-silico-identified 9-mer epitopes (n = 166, table s5 ) to the equivalent, experimentally identified epitopes of sars-cov strains (n = 3760; mhc ligand assays data from the iedb database) [25] . in this comparison, we identified 29 common epitopes of sars-cov/sars-cov-2 (table s8) and hla allotypes hla-a*02:01 and hla-a*02:06 molecules as the top antigen presenters; both allotypes having strong binding affinities (ic 50 ≤ 50 nm) to six peptide epitopes (table 3) : residues 1220 fiagliaiv 1228 from the s protein, 17 vllflafvv 25 and 20 flafvvfll 28 to the e protein, 204 vlawlyaav 212 to 3clpro, 330 llsagifga 338 to nsp3 and 184 vlwahgfel 192 to 35exo of sars-cov-2. table 3 . sars-cov-2-derived hla-a*02 supertype-binding epitopes that are identical to the epitopes of sars-cov strains experimentally known to activate cytotoxic t-cells. to examine the evolutionary conservation or "sequence stability" of the six common epitopes of sars-cov/sars-cov-2 binding strongly to hla-a*02:01 and hla-a*02:06, we performed a blastp (ncbi) search against the non-redundant database of sars-cov-2 [47] . only one epitope-330 llsagifga 338 from nsp3-was found to have heterogeneity in its sequence (table 3) , whereas the other five epitopes were fully conserved, i.e., not yet having changed during the evolution of sars-cov-2, suggesting an important role for these peptide sequences for the virus and, at the same time, making these peptides top-candidate antigens for activating the cytotoxic t-cell response against sars-cov-2 itself. the five conserved and experimentally proven epitopes we selected for further analyses. since mhc class i molecules must retain bound epitopes long enough at the cell surface to successfully induce t-cell-specific immune responses [48] , we estimated the half-life in hours for each of the experimentally identified epitope-hla complexes (table 3 ) using the netmhcstabpan1.0 web server [33] . this analysis revealed that the hla-a*02:01 and hla-a*02:06 allotypes have longer predicted half-lives than hla-a*68:02 when in complex with the immunogenic epitopes (table 4 ): all five hla-a*02:01-epitope complexes and three out of five hla-a*02:06-epitope complexes had a predicted half-life longer than 4 h, whereas the single hla-a*68:02-epitope complex had the lowest half-life of less than one hour. table 4 . predicted half-lives of complexes of the conserved sars-cov-2-derived most immunogenic experimentally identified epitopes and hla-a*02 allotypes shown in table 3 . secondary structures, localization within sars-cov-2 and gravy (grand average of hydropathicity index) scores of the epitopes. allotypes in order to examine the distribution of the hla-a*02-antigen complex half-lives in general, we analyzed more than 6500 experimentally known complexes of hla-a*02 supertypes (see table s1a for a list of allotypes) bound to bacterial-and viral-pathogen-derived epitopes, which were extracted from the iedb database [25] . this revealed that immunogenic (ic 50 ≤ 50 nm) hla-a*02:01-epitope complexes with predicted half-lives of more than three hours were almost double in number in comparison to the hla-a*02:06-epitope complexes, whereas no immunogenic hla-a*68:02-epitope complexes were found that would have a similar half-life ( figure s1a ). interestingly, the amino acid sequences of the α chains of hla-a*02:01 and hla-a*02:06 are 99.6% identical; the only difference in the sequences-a phenylalanine to tyrosine substitution-is located at the epitope-binding site and is the likely reason for the difference in the epitope binding affinities and half-lives. out of the five most stable epitopes (table 4) , three-1220 fiagliaiv 1228 (s protein), 17 vllflafvv 25 (e protein) and 20 flafvvfll 28 (e protein)-were derived from transmembrane proteins and were, as expected, more hydrophobic (gravy score > 3) than the 204 vlawlyaav 212 (3clpro) and 184 vlwahgfel 192 (35exo) epitopes originating from intravirion proteins ( table 4 ). the epitopes derived from the s and e proteins respectively map to transmembrane helical segments 1214 wyiwlgfiagliaivmvtimlcc 1236 and 12 livnsvllflafvvfllvtlail 34 that, based on analysis using the tmhmm2.0 web server [31] , are bitopic in nature, i.e., the predicted transmembrane helices span the lipid bilayer only once. in fact, our predicted epitopes share features, such as being membrane associated, with the well-studied and clinically important epitopes in hiv [49] and tuberculosis [50] . this supports the idea that the transmembrane helical epitopes of sars-cov-2 could potentially stimulate cytotoxic t-cell-mediated immune responses. in order to assess whether hydrophobic residues are enriched in the immunogenic epitopes, we compared the gravy score distribution of the immunogenic (n = 1678, ic 50 ≤ 50 nm) and non-immunogenic (n = 2228; ic 50 > 500 nm) hla-a*02-bound epitopes of bacterial and viral pathogens (retrieved from the iedb database) ( figure s1b ). we found that, for epitopes with a gravy score greater than one (having at least seven non-charged residues in a 9-mer epitope), the immunogenic epitopes were more enriched in hydrophobic residues (55%) in comparison to the non-immunogenic epitopes (19%). thus, our analysis suggests that hla-a*02 supertype molecules prefer binding to hydrophobic epitopes (ic 50 ≤ 50 nm), and this agrees with published results [51] . moreover, we obtained similar results from our in-silico-identified sars-cov-2-derived novel epitope-mhc-i complexes ( table 2) : the most potent identified epitopes had long half-lives and were derived from either hydrophobic, transmembrane regions of sars-cov-2 or from intravirion proteins that were also found to be mutated among sars-cov-2 sequences (table 5) . table 5 . predicted half-lives of the novel sars-cov-2-derived, most immunogenic in-silico-identified epitopes in complex with the allotypes shown in table 2 . secondary structures, localization within sars-cov-2, gravy scores and known mutations in the epitopes. in order to compare the interaction patterns adopted by the predicted top five immunogenic epitopes (table 4 ) of mhc-i molecules, we docked the epitopes to the cleft between the α1 and α2 helices of hla-a*02:01 (pdb id: 5tez, chain a) and hla-a*02:06 (pdb id: 3oxr, chain a). this docking analysis agrees with our other prediction data and suggests that the immunogenic epitopes bind to both the hla-a*02:01 and hla-a*02:06 allotypes by adopting a similar backbone conformation, as has been observed for the canonical epitope 1 gilgfvftl 9 of the influenza a virus (pdb id: 5tez, chain c) ( figure s1c,d) . in more detail, we observed that the residues at position (pos) 1, 2, 3 and 9 are fully buried within the antigen-binding cleft of hlas and act as anchoring residues, providing steric constraints to the nand c-terminus of the epitopes (figure 2a,b) . comparison of the root-mean-square deviation (rmsd) of the superposed cα atoms of the epitopes revealed a maximum deviation for the 184 vlwahgfel 192 epitope (35exo), possibly due to the bulky, aromatic side chain of w186 buried at pos3 and two charged residues: h188 at pos5 and e191 at pos8 (table 6) . interactions between the partially solvent-exposed hydrophobic residues at pos4-pos8 of the docked epitope 1220 fiagliaiv 1228 (s protein) and the solvent-exposed residues a69, k66, v76, t80, k146, v152 and q155 of the α1 and α2 helices of hla-a*02:01, complement what is seen in the x-ray crystal structure of the influenza a virus epitope-hla-a*02:01 complex (pdb id: 5tez, chain a and c), and suggest that the sars-cov-2 epitope-hla complex interacts with tcr ( figure 2c ). and cdr3β of the tcr-β chain, recognize the hla-a*02:01-sars-cov-2 fiagliaiv epitope ( figure 2d ); the cooperative interacting nature of residues in these loops could provide specificity towards class i mhc molecules. moreover, the residues l96 and w99 within the cdr3β loop and i96 within the cdr3α loop could be important for antigen-hla complex recognition due to their likely direct contacts with residues l1224, i1225 and i1227 of the 1220 fiagliaiv 1228 epitope and residues a69, v152 and q155 of hla-a*02:01 ( figure 2e ). to understand the molecular basis of tcr binding to the sars-cov-2 antigen-loaded mhc-i cell surface molecules, hla-a*02:01, in complex with the s protein epitope 1220 fiagliaiv 1228 , was superimposed with the hla-a*02:01 allotype of the influenza a virus ternary complex structure (pdb id: 5tez, chain a), and the atomic coordinates of tcr (tcr-α and tcr-β chain; pdb id: 5tez, chain i and j, respectively) were then utilized for visual analysis of binding. this visualization suggests that the loops cdr2α, cdr1α and cdr3α of the tcr-α chain, and loops cdr2β, cdr1β and cdr3β of the tcr-β chain, recognize the hla-a*02:01-sars-cov-2 1220 fiagliaiv 1228 epitope ( figure 2d ); the cooperative interacting nature of residues in these loops could provide specificity towards class i mhc molecules. moreover, the residues l96 and w99 within the cdr3β loop and i96 within the cdr3α loop could be important for antigen-hla complex recognition due to their likely direct contacts with residues l1224, i1225 and i1227 of the 1220 fiagliaiv 1228 epitope and residues a69, v152 and q155 of hla-a*02:01 ( figure 2e) . to assess the conformational and intermolecular interaction dynamics of the hla-a*02:01-1220 fiagliaiv 1228 s protein epitope-tcr complex, a 100 ns simulation was carried out on the ternary structure in triplicate. the global conformational dynamics was assessed by computing the rmsf over the cα atoms ( figure 3a ), which shows a stable tcr and epitope, with an average rmsf of 1.86 ± 0.47 å and 2.36 ± 0.18 å, respectively. the α1 and α2 domains of hla-a*02:01 were also stable (3.16 ± 0.48 å, 3.19 ± 0.46 å), unlike the α3 domain, which exhibits a higher fluctuation (6.4 ± 2.57 å) likely arising from the flexibility of the loop between domains α2 and α3 and the lack of stabilizing β2-microglobulin. figure 3b compares the complex at 0, 50 and 100 ns during the simulation. these observations were consistent among the three independent simulations ( figure s2 ). the intermolecular interactions taking place in the complex structure were also examined by visually inspecting the trajectory and calculating the number of hydrogen bonds formed during the simulation (table s9) . for example, in simulation 1, the highest number of hydrogen bond interactions recorded were between hla-a*02:01 and backbone atoms of the epitope peptide, with t143-v1228 and w147-i1227 interactions topping the list, as they were respectively formed during 98% and 97% of the simulation time. on the other hand, hydrogen bond interactions between the tcr and the epitope were almost exclusively from interactions between w99 (β chain)-i1225 (63%) and q101 (α chain)-i1225 (10%). the hla-a*02:01-tcr hydrogen-bonding interactions were mostly between residues from the cdr1α, cdr2α, cdr3α, cdr1β and cdr2β loops of the tcr and the α1 and α2 helices of hla-a*02:01. visual analysis also revealed that hydrophobic interactions are integral to the interaction the epitope is making, both with hla-a*02:01 and the tcr, as the hydrophobic epitope peptide was tightly enclosed by hydrophobic clusters from the proteins throughout the simulation, reflecting observations made on the basis of the original docked complex. thus, our structural analysis suggests that the s protein epitope 1220 fiagliaiv 1228 of sars-cov-2 (and sars-cov), and the other epitopes listed in table 4 , could form strong complexes with hla-a*02:01 and hla-a*02:06 allotypes, and that the epitope-hla complexes can also be recognized by tcrs to initiate cytotoxic t-cell-mediated immune responses. the intermolecular interactions taking place in the complex structure were also examined by visually inspecting the trajectory and calculating the number of hydrogen bonds formed during the simulation (table s9) . for example, in simulation 1, the highest number of hydrogen bond interactions recorded were between hla-a*02:01 and backbone atoms of the epitope peptide, with t143-v1228 and w147-i1227 interactions topping the list, as they were respectively formed during 98% and 97% of the simulation time. on the other hand, hydrogen bond interactions between the tcr and the epitope were almost exclusively from interactions between w99 (β chain)-i1225 (63%) and q101 (α chain)-i1225 (10%). the hla-a*02:01-tcr hydrogen-bonding interactions were mostly between residues from the cdr1α, cdr2α, cdr3α, cdr1β and cdr2β loops of the tcr and the α1 similar to the docking results obtained from the experimentally known epitopes with the hla-a*02 supertype, we observed that residues at pos1-3 and pos9 (a1507, e1508, w1509 and l1515) of the novel epitope 1507 aewflayil 1515 -derived from a transmembrane segment of the nsp3 protein of sars-cov-2-are fully buried within the antigen-binding cleft of hla-b*40:01 (pdb id: 6iex, chain a). moreover, the location of the partially solvent-exposed residues at pos4-pos8 (l1511, a1512, y1513 and i1514) of the docked epitope along with solvent-exposed residues r62, t69, t73, e76, q155, y159, e163 and w167 of the α1 and α2 helices suggest an interaction of the 1507 aewflayil 1515 -hla-b*40:01 complex with tcr ( figure 2f ). in order to tackle the current covid-19 pandemic, it is critically important to better understand the underlying mechanism that gives rise to the individual differences in disease severity as well as to aid the vaccine development against the causative virus, sars-cov-2. effective vaccinations are needed to eradicate the virus from populations all over the world and knowledge regarding the immunological response should have a significant impact on understanding disease progression. however, due to the limited experimental and clinical data currently available on the specific immune responses against sars-cov-2, the development of an effective vaccine against covid-19 will be a challenge. this study sought to better understand the individual differences in the viral antigen presentation pathway and to aid the development of vaccines against covid-19 by predicting in silico sars-cov-2 immunogenic epitopes. based solely on in silico predictions, the most potent sars-cov-2-derived mhc class i binding epitopes are 1507 aewflayil 1515 and 1505 lvaewflay 1513 in terms of binding affinity, hydrophobicity and stability. however, for these "in silico epitopes", only limited experimental data are available to correlate with. therefore, in this study we mainly focused on potential sars-cov-2 epitopes that were conserved with sars-cov epitopes experimentally known to activate cytotoxic t-cells, and hence could be used in vaccine development. the s glycoprotein-derived epitope 1220 fiagliaiv 1228 binds to the hla-a*02:01 and hla-a*02:06 allotypes with experimental ic 50 values lower than 50 nm (table 3 ). our docking analysis supports these predictions, i.e., that epitope 1220 fiagliaiv 1228 could bind tightly to these allotypes and that ternary complexes with tcrs could form. moreover, recent data demonstrate that patients with a severe form of covid-19 have a stronger t-cell response after stimulation with the sars-cov-2 s-protein peptide pool compared to those with a mild manifestation of the disease [22, 52] . the disease progression of covid-19 is also associated with a higher magnitude of inflammatory cytokine-producing cd8+ t cells [52] . whether these immune responses are due to strong binding of sars-cov-2 epitopes, including 1220 fiagliaiv 1228 , to certain hla allotypes, such as hla-a*02:01 and hla-a*02:06, or whether tight virus epitope-hla interaction in general can actually be harmful for covid-19 patients by causing, e.g., an immunological over-reaction, is not yet fully understood [53, 54] . furthermore, both cd4+ and cd8+ t-cells have been shown to be stimulated by overlapping peptides (15-mers overlapping by 10 amino acids) of the entire s glycoprotein sequence [55] . does this mean that the s protein might function as a double-edged sword-that is, being crucial for viral entry into the host cell, but also important for overstimulating the immune responses, causing severe inflammation that aids the spread of the virus to surrounding cells? this still needs to be answered. the latter "sword" is known to be avoided at least by hiv, which has a sophisticated mechanism to limit the infection rate in order to better avoid immune surveillance [56, 57] . a recent in vivo study shows that epitopes derived from the c-terminus of the s protein had a significantly stronger cd4+ t helper cell response in healthy donors in comparison to those infected with sars-cov-2 [22] . the cd4+ t cells' cross-reactivity to the s protein might represent the key for understanding the different disease manifestations of covid-19, particularly in the asymptomatic infections in children and adolescents. our predicted epitope 1220 fiagliaiv 1228 overlaps with the c-terminal sequence of the s protein containing the s2 subunit, which is internalized after tmprss2 cleavage. therefore, this particular amino acid sequence may also be important for inducing a protective immunological response towards immunity in covid-19. however, canonically t helper cells recognize mhc class ii molecules, whereas our prediction is based on the mhc class i molecules' ability to present viral antigens for a possible cytotoxic t-cell response [58] . indeed, we found four 15-mer epitopes that include the intact 9-mer 1220 fiagliaiv 1228 epitope sequence and showed binding affinities < 500 nm with drb1*01:01 allotype of mhc class ii (http://tools.iedb.org/mhcii/) [25] , an allotype that is common in caucasoid and oriental ethnic backgrounds (https://www.ebi.ac.uk/ipd/imgt/hla/ethnicity.html) [59] . nevertheless, there are a few reports regarding mhc class i-reactive cd4+ t helper cells, including the study where co-cultures of highly purified cd4+ t cells, together with a stimulatory mhc class ii-negative cell line transfected with mhc class i molecules, were used to show the direct interaction of t helper cells with mhc class i molecules [60] . however, whether or not the 1220 fiagliaiv 1228 epitope is presented to both cytotoxic and helper t cells needs to be experimentally verified; a recent study that appeared while the current work was under review suggested that cross-reactivity could affect disease progression in covid-19 [54] . furthermore, the latest experimental reports suggest that the s glycoprotein of sars-cov-2 is both o-and n-glycosylated, especially on the rdb domain, which could mask immunogenic epitopes and may play an important role in sars-cov-2 immune evasion [61] [62] [63] . fortunately, the predicted epitope 1220 fiagliaiv 1228 is part of a transmembrane helix, and consequently is neither o-nor n-glycosylated; the closest glycosylation site is at pos1194, rendering this particular amino acid sequence potentially suitable for vaccine development. intriguingly, the sars-cov-2-derived, membrane glycoprotein epitope 148 hlriaghhl 156 , which has low binding affinity (ic 50 = 1693.63 nm; table s4 ) towards hla-b*15:02, is 78% identical in sequence with the intravirion sars-cov epitope hlrmaghsl also from a membrane glycoprotein and shown to elicit a strong t-cell response in patients with the hla-b*15:02 allotype [19] . furthermore, hla-b*15:02 has been shown to have a protective role against the severe forms of sars-cov [64] . this inspired us to do a separate in silico binding affinity analysis of the sars-cov hlrmaghsl epitope to the hla-b*15:02 allotype: a high binding affinity (ic 50 = 232.85 nm) of the hlrmaghsl epitope with the hla-b*15:02 was predicted and agrees with the reported [19] protective immune response against sars-cov. thus, there seems to be a difference between the highly conserved sars-cov-derived hlrmaghsl and sars-cov-2-derived 148 hlriaghhl 156 epitopes in their potency towards the hla-b*15:02 allotype. moreover, the predicted low binding affinity of the sars-cov-2 epitopes with the hla-b*15:02 allotype (table s4 ) might not be sufficient to induce immune responses, thus rendering the potential of this specific epitope unfavorable for vaccination. based on our predictions, hla-b*46:01 is one of the worst allotypes for presenting sars-cov-2-derived epitopes with an average binding score (ic 50 ) of 2264 nm for the four epitopes predicted to bind (table s4 ). this is in line with similar results from the predictions for sars-cov-2 and previous clinical data from sars-cov patients, demonstrating that this particular hla allotype gives susceptibility to a more severe form of the viral disease [65] . furthermore, our prediction shows that hla-b*46:01 is not binding to either the s or m protein-derived peptides, strengthening the reported general view that hla-b*46:01 is not optimal for eliciting an immune response in covid-19 patients [58] . taken together, identification of the predicted most immunogenic epitopes of sars-cov-2 could aid vaccine development. since the sequences of the "top" epitopes of sars-cov-2 and sars-cov are highly conserved and the sars epitopes are known to elicit an immunological response based on a previous study [66] , a common vaccine protecting against both viruses and potential future strains is possible. moreover, the selected experimentally known epitopes have been shown not to evoke an unwanted t cell cross-reactivity in vitro, further validating the potential use of the conserved sars-cov/cov-2 peptides in vaccine development without disrupting self-tolerance [67] . however, there are many hurdles that need to be addressed; for example, due to the hydrophobic nature of these epitopes, they probably would need to be loaded in a liposome or nanocarrier for efficient vaccine delivery [68] . moreover, as these conserved epitopes are presented by only a few hla allotypes, i.e., a*02:01, a*02:06, a*68:02, which are common in american indian, caucasoid, hispanic and oriental ethnic backgrounds [59] , the estimated world population coverage using these epitopes would only be around 42.1% (http://tools.iedb.org/population/) [25] . therefore, developing a globally effective sars-cov-2 vaccine will probably require a pool of both novel and conserved epitopes, making a globally effective sars-cov-2 vaccine development a challenging task. in the present study, we identified sars-cov-2 epitopes that were predicted to be presented by the mhc class i antigen processing pathway to the cytotoxic t cells. we report five purely in-silico-predicted most potent epitopes unique to sars-cov-2 and five potent sars-cov-2 epitope peptides identical to and experimentally determined for sars-cov. the novel sars-cov-2 epitopes were analyzed for their interaction with hla allotypes using the iedb and netctl1.2 web servers and three-dimensional structural models of selected, molecular dynamics simulation proven ternary complexes of sars-cov-2 phla-tcrs were created to assess interactions at structural level. hla-a*02:01 and hla-a*02:06 were found to have the greatest potential to present the selected epitopes, which are hydrophobic in nature and originated mainly from the transmembrane region of sars-cov-2 proteins. our results could assist in the understanding of the individual and varying disease progression of covid-19, as well as paving the way towards vaccine development against sars-cov-2. supplementary materials: the following material are available online at http://www.mdpi.com/2076-393x/8/3/408/ s1, figure s1 : (a): distribution of the predicted half-lives (log scale) of predicted (iedb) 9-mer epitope-hla-a*02 supertype complexes. the complexes are classified as immunogenic (ic50 ≤ 50 nm) and non-immunogenic (ic50 > 500 nm) based on the epitope binding affinity with the mhc molecule, (b): distribution of the gravy scores of immunogenic (ic50 ≤ 50 nm) and non-immunogenic (ic50 > 500 nm) epitopes, figure s1c and s1d: comparison of the backbone conformation of the five epitopes docked into the cleft of hla-a*02:01 (c) and hla-a*02:06 (d) molecules against the canonical epitope 1gilgfvftl9 of the influenza a virus (pdb id: 5tez, chain c), figure s2 : structural dynamics of the hla-a*02:01-1220fiagliaiv1228 s protein epitope-tcr complex during three replicate 100 ns simulations. (a) cα atom rmsf of the ternary complex. (b) average cα atom rmsf. table s1a : mhc class i allotypes association with supertypes described in published scientific literature [28, 29] . superscript "x" indicates availability of x-ray crystal structure in protein data bank (pdb) [36] , table s1b : pdb codes for the x-ray crystal structures of mhc class i allotypes, table s2 : sars-cov-2-derived mhc class i binding epitopes identified with iedb and netctl1.2 prediction methods as having strong binding affinity (ic50 ≤ 50 nm) with mhc molecules, table s3 : sars-cov-2-derived mhc class i binding epitopes identified with iedb and netctl1.2 prediction methods as having weak binding affinity (50 nm < ic50 ≤ 500 nm) with mhc molecules, table s4 : sars-cov-2-derived mhc class i binding epitopes identified with iedb and netctl1.2 prediction methods as having non-binding affinity (ic50 > 500 nm) with mhc molecules, table s5 : sars-cov-2-derived epitopes (immunogenicity score ≥ 0.25) and their prominent interacting hla allotypes identified with iedb and netctl1.2 prediction methods, table s6 : most potent sars-cov-2-derived epitopes having immunogenicity score ≥ 0.25 and predicted binding affinity (ic50) ≤ 500 nm with their prominent interacting hla allotypes identified with iedb prediction method, table s7 : most potent sars-cov-2-derived mhc class i binding epitopes identified with netctl1.2 (combined score ≥ 2) prediction method, table s8 : most potent sars-cov-2-derived mhc class i binding epitopes that are identical to the experimentally known epitopes (mhc ligand assays data from the iedb database) of sars-cov strains to activate cytotoxic t-cells. predicted half-lives of the epitope-mhc-i complexes, gravy scores and mutations in the epitopes are shown. table s9 world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19 an interactive web-based dashboard to track covid-19 in real time preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies data sharing and outbreaks: best practice exemplified potential inhibitors against 2019-ncov coronavirus m protease from clinically approved medicines a review of coronavirus disease-2019 (covid-19) acute respiratory distress syndrome advances in diagnosis and treatment an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov-2 epidemic: an observational cohort study middle east respiratory syndrome coronavirus (mers-cov) in oman: current situation and going forward emerging coronaviruses: genome structure, replication, and pathogenesis structural genomics of sars-cov-2 indicates evolutionary conserved functional regions of viral proteins universal three-dimensional construction of eleven amino acids near the catalytic nucleophile and base in the superfamily of (chymo)trypsin-like serine fold proteases sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a pneumonia outbreak associated with a new coronavirus of probable bat origin structural basis of receptor recognition by sars-cov-2 functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses human leukocyte antigen (hla) and immune regulation: how do classical and non-classical hla alleles modulate immune response to human immunodeficiency virus and hepatitis c virus infections? front association of hla class i with severe acute respiratory syndrome coronavirus infection memory t cell responses targeting the sars coronavirus persist up to 11 years post-infection hla-a*0201 t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus nucleocapsid and spike proteins efficient induction of cytotoxic t lymphocytes specific for severe acute respiratory syndrome (sars)-associated coronavirus by immunization with surface-linked liposomal peptides derived from a non-structural polyprotein 1a presence of sars-cov-2-reactive t cells in covid-19 patients and healthy donors pathways of antigen processing ncbi viral genomes resource the immune epitope database (iedb): 2018 update large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction the immune epitope database and analysis resource in epitope discovery and synthetic vaccine design definition of supertypes for hla molecules using clustering of specificity matrices hla class i supertypes: a revised and updated classification a simple method for displaying the hydropathic character of a protein predicting transmembrane protein topology with a hidden markov model: application to complete genomes jpred4: a protein secondary structure prediction server pan-specific prediction of peptide-mhc class i complex stability, a correlate of t cell immunogenicity structural basis for clonal diversity of the human t-cell response to a dominant influenza virus epitope structural insights into the binding of hepatitis b virus core peptide to hla-a2 alleles: towards designing better vaccines the rcsb protein data bank: new resources for research and education rosetta flexpepdock web server-high resolution modeling of peptide-protein interactions deciphering the structural effects of activating egfr somatic mutations with molecular dynamics simulation improving the accuracy of protein side chain and backbone parameters from ff99sb comparison of simple potential functions for simulating liquid water visual molecular dynamics software for processing and analysis of molecular dynamics trajectory data ucsf chimera-a visualization system for exploratory research and analysis friends or foes? retrovirology systematically benchmarking peptide-mhc binding predictors: from synthetic to naturally processed epitopes ncbi blast: a better web interface peptide-mhc class i stability is a better predictor than peptide affinity of ctl immunogenicity primary human immunodeficiency virus type 1-specific cd8+ t-cell responses induced by myeloid dendritic cells identification of antigens presented by mhc for vaccines against tuberculosis tcr contact residue hydrophobicity is a hallmark of immunogenic cd8+ t cell epitopes a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov-2 targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals phenotype of sars-cov-2-specific t-cells in covid-19 patients with acute respiratory distress syndrome key words natural killer cells in hiv-1 infection: a double-edged sword structural basis of transmembrane coupling of the hiv-1 envelope glycoprotein human leukocyte antigen susceptibility map for sars-cov-2 major histocompatibility complex class i-restricted alloreactive cd4 + t cells deducing the n-and o-glycosylation profile of the spike protein of novel coronavirus sars-cov-2 site-specific analysis of the sars-cov-2 glycan shield site-specific n-glycosylation characterization of recombinant immunogenetics in sars: a casecontrol study hla studies in the context of coronavirus outbreaks a epitopes described in-immune epitope database (iedb) quantitating t cell cross-reactivity for unrelated peptide antigens harnessing self-assembled peptide nanoparticles in epitope vaccine design conflicts of interest: e.n. has filed a patent application regarding a method for preventing the spreading and lowering the infection rate of pathogens, finnish patent application number 20205382, filing date 09.04.2020. all other authors declare that they have no competing interests. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.vaccines 2020, 8, 408 key: cord-028275-szb45jm2 authors: reza khorramizadeh, m.; saadat, farshid title: animal models for human disease date: 2020-06-26 journal: animal biotechnology doi: 10.1016/b978-0-12-811710-1.00008-2 sha: doc_id: 28275 cord_uid: szb45jm2 this chapter introduces some types of animal models which are used for better understanding the disease mechanisms and its treatment. these experimental models fall into two categories: spontaneous models and induced models. among the diseases, rheumatoid arthritis (ra) as an autoimmune disease was considered. to study the pathogenesis of ra, we explained collagen-induced arthritis as an animal model that reflects a characteristic feature of ra patients. in addition, experimental allergic encephalomyelitis (eae) as an experimental model for multiple sclerosis (ms) was explained in detail to represent a standard method to investigate in its mechanism, finding the way for the amelioration of this incurable neurological disorder. the architecture of human body is comprised in such a manner that cells cannot be considered as a separate entity. physiologically, homeostasis is the reason that these components live and perform their functions within that environment. disruption of this process leads to fatal conditions and is considered a disease. to investigate the mechanism of disease and to find the means to reverse adverse conditions, various strategies are used including cell-based assays and tissue culture studies. although these models can provide useful information, they fail to address various physiological conditions and the complex interactions among different cell types of tissues and organs. ideally a useful animal model for any disease has to have pathology similar to the disease conditions in humans. use of animals in research has a long history that dates back to the fourth century b.c. in the 1600s, william harvey used animals to describe the blood circulatory system. many scientists, such as louis pasteur and emil von behring, have used animal models for experimental purposes to prove their hypotheses. animal models are good for understanding disease mechanisms and treatment and for overcoming the limitations of clinical trials that use human subjects. for example, experimental animal models for diseases like rheumatoid arthritis or multiple sclerosis have been successfully employed to screen new bioengineered, chemical, or herbal therapeutics that might have the potential for the treatment of human patients. so far, more than 550,000 studies have been reported in the ncbi database; they use animal models for different diseases. animal model studies have been the main reason for a better understanding of disease mechanisms. animal models of disease can be divided into two categories (kurkó et al., 2013) : spontaneous disease models and ( van heemst et al., 2014) induced disease models. in the case of induced disease models, induction can occur by various agents, both chemical and biological. this chapter discusses some of the most important animal models. rheumatoid arthritis (ra) is an autoimmune disorder with progressive occurrence that preferentially affects peripheral joints. in spite of the fact that ra is severe and crippling and affects large numbers of people, very little knowledge about its etiology and pathogenesis is available in the literature. rheumatoid arthritis affects about 1% of the population. the ratio of the prevalence of ra in males and females is 1à2.5. ra can occur at any age, but it is mainly reported to affect the 40-to 70-year-old age group. no doubt the incidence has been reported to increase with age. the etiology of ra is unknown, but it has been predicted that genetic and environmental factors play an important role in the onset of ra. recent advances have identified genetic susceptibility markers both within and outside of the major histocompatibility complex (mhc). human leukocyte antigen (hla) genes located on chromosome 6p have been found to have a strong association with rheumatoid arthritis. the contribution of hla to heritability of ra has been estimated to be 11%à37%. individuals carrying hla-dr4 and hla-dr1 alleles have been shown to have a higher risk of ra. apart from the known shared epitope alleles (hla-drb1 ã 01, drb1 ã 04), other hla alleles, such as hla-drb1 ã 13 and drb1 ã 15, have been linked to ra susceptibility (kurkó et al., 2013) . the hla class ii locus is the most important risk factor for anticitrullinated protein antibodies (acpa)-positive ra (acpa 1 ra) (van heemst et al., 2014) . a positive correlation has been suggested for the role of hla in terms of the severity of ra rather than the onset of the disease. the most relevant non-hla gene single nucleotide polymorphisms (snps) associated with ra include ptpn22, il23r, traf1, ctla4, irf5, stat4, ccr6, and padi4 (kurkó et al., 2013; suzuki and yamamoto, 2015; stanford and bottini, 2014) . although the data regarding this conclusion are inconsistent, some of the studies have shown associations between tumor necrosis factor (tnf) alleles and rheumatoid arthritis. other genes like those for corticotrophin-releasing hormone, interferon (ifn)-γ, and interleukin-10 (il-10) have also been implied for ra. it can be concluded that the role of genetic components in ra is modest at the best (viatte et al., 2013) . epigenetics is another important factor that contributes to ra. in the case of identical twins, ra has not been shown to have 100% concordance; therefore, the role of nongenetic factors has also been implicated in the etiology of ra (meda et al., 2011) . throughout the world, rheumatoid arthritis is more common in women than in men. this indicates that hormones may play an important role in the development of the disease. pregnancy has also been considered as a risk factor for rheumatoid arthritis. studies show that the onset of ra is rare during pregnancy, but the risk increases after delivery. smoking is associated with increased incidences of ra, especially in men. on the contrary, populations that consume a diet high in omega-3 fatty acids have been reported to be protected from rheumatoid arthritis. from experimental models in animals, a large number of infectious agents such as viruses and bacteria have also been suggested to trigger or contribute to the development of rheumatoid arthritis. however, no relationship between infectious agents and the development of ra has been found. tissue edema and fibrin deposition are prominent and can manifest clinically as joint swelling and pain. within a short period, the synovial lining becomes hyperplastic, commonly becoming ten or more cells deep and consisting of type a (macrophage-like) and type b (fibroblast-like) synoviocytes that produce glycosaminoglycans (e.g., hyaluronan, as reported to be present in synovial tissue and synovial fluid). the sublining also undergoes alterations for its cellularity, both in cell type and in cell numbers, with prominent infiltration of mononuclear cells, including t cells, b cells, macrophages, and plasma cells. the abundance and activation of macrophages at the inflamed synovial membrane correlates significantly with the severity of the disease. activated macrophages over-express major histocompatibility complex (mhc) class ii molecules and produce pro-inflammatory or regulatory cytokines and growth factors [il-1, il-6, il-10, il-13, il-15, il-18, tnf-α, and granulocyte macrophage colony stimulating factor (gm-csf)], chemokines [il-8, macrophage inflammatory protein 1 (mip-1), monocyte chemoattractant protein 1 (mcp-1)], metalloproteinases, and neopterin. these biomolecules are routinely detected in inflamed joints. most of the t cells infiltrating the rheumatoid synovium express cd45ro and cd4, which is an indication that the t-cell subset present in the synovium is memory helper t cells. surprisingly, 10%à15% of the t cells present in the case of the synovium have granzymes a and perforins. this 10%à15% of cells present in the synovium represents cytotoxic t-cell subsets. therefore, it can be concluded that cd8-expressing cells are infrequent in the synovium. in the synovial fluid of rheumatoid arthritis patients, cd4 and cd8 t cells are equally represented. tcrα/tcrß is expressed on most of the t cells while only a minority of cells show tcrγ/tcrδ expression. it has, however, been found that the expression of tcrγ/tcrδ is increased in the synovium of patients with active ra. synovial-vessel endothelial cells transform into high endothelial venules early during the course of disease. high endothelial venules are specialized post-capillary venules usually present in secondary lymphoid tissue or inflamed nonlymphoid tissues; these venules facilitate the transit of leukocytes from the bloodstream into tissues. the cytokine-mediated events have conventionally been viewed in the milieu of the cd4 1 th1/th2 paradigm. nowadays, newer cytokines of the il-17/il-23 axis and others have changed investigations into the immunopathogenesis of arthritis. both the cd4 1 th17 and γδ-t cells secreted il-17 which is a chemotactic for neutrophils and its response inhibited by il-27 due to ifn-γ induction. the roles of other cytokines such as il-18 and il-33 in arthritis have been clarified further with inhibitors of them (veenbergen et al., 2010; palmer et al., 2009) . recent studies in arthritis models have revealed new aspects toward regulatory t cell (treg) activity. in the cia model, treatment with il-35 induced the regression of arthritis via expansion of regulatory t cells (kochetkova et al., 2010) . the formation of locally invasive synovial tissue (i.e. pannus) is a characteristic feature of rheumatoid arthritis. pannus is involved in the erosion of joints in rheumatoid arthritis. pannus is histologically distinct from other regions of the synovium and shows phases of progression. initially, there is penetration of cartilage by synovial pannus, which is composed of mononuclear cells and fibroblasts, with a high-level expression of matrix metalloproteinases (mmps) by synovial lining cells. in later phases of the disease, cellular pannus can be replaced by fibrous pannus comprised of a minimally vascularized layer of pannus cells and collagen overlying cartilage. the tissue derivation of pannus cells has not been fully elucidated, although they are thought to arise from fibroblast-like cells (type-b synoviocytes). in vitro work shows that these fibroblast-like synoviocytes have anchorageindependent proliferation and loss of contact inhibition, which a phenotype is usually found in transformed cells. however, the molecular pathogenic mechanisms driving pannus formation still remains poorly understood. the range of presentations of rheumatoid arthritis is broad, but the disease onset is insidious in most cases, and several months can elapse before a firm diagnosis can be ascertained. the predominant symptoms are pain, stiffness, and swelling of peripheral joints. although articular symptoms are often dominant, rheumatoid arthritis is a systemic disease. active rheumatoid arthritis is associated with a number of extraarticular manifestations, including fever, weight loss, malaise, anemia, osteoporosis, and lymphadenopathy. the clinical course of the disorder is extremely variable, ranging from mild, self-limiting arthritis to rapidly progressive multisystem inflammation with a profound morbidity and mortality. analyses of clinical course and laboratory and radiological abnormalities have been defined as negative prognostic factors for progressive joint destruction; unfortunately, none of these are reliable enough to allow therapeutic decisionmaking. frequent assessment of disease symptoms and responses to therapy is crucial for a successful and long-term management of rheumatoid arthritis. joint destruction from synovitis can occur rapidly and early in the course of the disorder; radiographic evidence is present in more than 70% of patients within the initial 2 years. more sensitive techniques such as magnetic resonance imaging (mri) can identify substantial synovial hypertrophy, bone edema, and early erosive changes as early as 4 months after the onset of disease. these radiographic changes predate misalignment and functional disability by years; by the time physical deformity is evident, substantial irreversible articular damage has commonly occurred. furthermore, the biopsy analysis of clinically symptomless knee joints in patients with early rheumatoid arthritis shows active synovitis, highlighting the poor correlation between clinical assessment and disease progression, and the rapid development of polyarticular synovitis. the main goal of ra treatment is to stop inflammation, relieve symptoms, prevent joint damage, and reduce long-term complications. the past decade has seen a major transformation in the treatment of rheumatoid arthritis in terms of approach and choice of drugs. the previous therapeutic approach generally involved initial conservative management with nonsteroidal antiinflammatory drugs (nsaids) for several years; disease-modifying antirheumatic drugs (dmards) were withheld until a clear evidence of erosion was seen. dmards were then added individually in slow succession as the disease progressed. this form of treatment has been supplanted by early initiation of dmards and combination dmard therapy in patients with the potential for progressive disease. the idea of early intervention with the conventional disease-modifying antirheumatic drugs (cdmard) has been validated in several randomized trials. cdmards contain medications from different classes of drugs including methotrexate, gold salts, hydroxychloroquine, sulfasalazine, ciclosporin, and azathioprine. dmards are often partly effective and poorly tolerated for long-term therapy. in metaanalyses of dropout rates from clinical trials, 20%à40% of patients discontinued the use of dmards assessed as monotherapy during the duration of the trial; even in clinical practice, the median duration of dmard monotherapy was less than 2 years for nonmethotrexate agents. although there are many reasons for the lack of long-term adherence to treatment, poor efficacy, delayed onset of action, and toxic effects are major limitations. additionally, dmards therapy requires patients to undergo frequent monitoring of blood and physical examinations for toxic effects of treatment protocol. results from clinical trials showed that dmard therapy decreased markers of inflammation such as erythrocyte sedimentation rate and swollen joint counts, and that improved symptoms in a selected subset of patients; however, most patients continued to show progression of irreversible joint destruction on radiography. cdmards is increasingly burdened by side effects or clinical inefficacy, so other immunosuppressive drugs such as tacrolimus that blocks t-cell activation by specifically inhibiting calcineurin pathway and leflunomide have been developed. a new synthetic dmard, iguratimod, which exerts its action by the inhibition of the inflammatory cytokines (tnf-α, interleukin (il)-1 β, il-6, il-8, and il-17), is recently developed. the findings illustrate the consequences of progressive disease and have shown the need for the development of new and more effective therapies based on the therapeutic principles used for oncology; it means that treatment protocols for ra patients require the use of several therapeutic agents from different classes to be used in combination. recent studies have shown that combination therapy of biological dmards like tnfα inhibitors with methotrexate has clear-cut benefits with tolerable toxic effects. treatment with agents that can block tnf-α function has proved to be highly effective against ra. further studies reported downregulation of synovial gm-csf, il-6, and il-8, suggesting that tnf-α supports the production of other pro-inflammatory cytokines. however, the mechanisms behind the clinical effect of the tnf-α-blocking treatment are not fully understood. in an animal model, tnf-α-blocking agents such as etanercept (a soluble tnf-α receptor) and infliximab (a monoclonal antibody) reduce the expression of vascular adhesion molecules and inhibit the spontaneous production of il-1 and il-6. patients with a new onset of symptoms and those with diseases of several years' duration and who had failed previous dmard therapy all benefited. these results suggest that patients in many stages of disease progression can benefit from combination therapy (chiu et al., 2012) . with the approval of tnf-α inhibitors (infliximab, etanercept, adalimumab, certolizumab, and golimumab), non-tnf biologic agents (rituximab, abatacept, tocilizumab, and anakinra), and other biologic agents, determining advances in treatment options of ra were made. rituximab (chimeric monoclonal antibody targeted against cd 20) is a selective b-cell depleting agent for treating refractory rheumatoid arthritis. abatacept selectively modulates t-cell co-stimulation and has shown efficacy in several clinical trials. tocilizumab, a humanized monoclonal antiinterleukin-6 receptor antibody, has proven to be efficacious in patients who did not respond to methotrexate or other synthetic dmards. recently, several clinical trials have focused on a new class of drug: the janus kinase (jak) inhibitors. jaks are a family of nonreceptor tyrosine kinases 156 8. animal models for human disease (jak1, jak2, jak3, and tyk2) involved in the intracellular signal transduction of many cytokines. tofacitinib is a pan-jak inhibitor that primarily inhibits jak1 and jak3. in addition to tofacitinib, other jak inhibitor molecules including baricitinib, peficitinib, and decernotinib have also been studied in rcts. finally, filgotinib is a selective jak1 inhibitor which is currently in clinical development for the treatment of ra (calabrò et al., 2016) . as mentioned before, pro-inflammatory/regulatory cytokines and growth factors play important roles in the pathogenesis of ra. therefore, each of them or their pathway represents an attractive therapeutic target for ra. tocilizumab, a humanized monoclonal antibody targeting il-6 receptor, has already been approved for the treatment of ra in patients who failed to achieve remission with cdmards. another cytokine that plays an important role in the pathogenesis of ra is il-17 in which ixekizumab and brodalumab as humanized monoclonal antibody were developed against il17a and its receptor. a new possible therapeutic target for the treatment of ra is the gm-csf pathway. the efficacy and safety of mavrilimumab (an anti-gm-csf receptor monoclonal antibody) in patients with moderate-to-severe ra has been investigated (takeuchi et al., 2015) . as an increased activation of osteoclasts contributes to bone erosions in ra, the inhibition of rankl that is essential for the osteoclast activation by denosumab (human monoclonal antibody against rankl) can reduce joint destruction in ra patients. takeuchi et al. (2016) , finally, do not forget that certain nutritional components interfere in the pathological inflammatory process, so that they should be considered as coadjuvant in the treatment of ra. it has been mentioned that flavonoids reduce cytokine expression and secretion. in this regard, flavonoids may have a therapeutical potential in the treatment of inflammationrelated diseases as cytokine modulators (rosillo et al., 2016; leyva-lópez et al., 2016) . in order to study the pathogenesis of ra, one can use different animal models. there are many experimental models that resemble ra in different respects. since ra is a heterogeneous disease, there is probably a need for different animal models that each reflect a characteristic feature of a particular subgroup of ra patients or illustrate a particular aspect of the disease. despite the fact that ra is not a spontaneously developing disease, spontaneously developing models for arthritis may be useful to study the role of genetics in the development of the disease. an activated immune response was reported in models such as the human tumor necrosis factor-α transgenic (htnftg), interleukin 1receptor-α (il-1ra) knockout, il-6ractivating mutation knockin, or skg mouse which bears the primary inflammatory response in joints (keffer et al., 1991) . in addition, arthritis can be rapidly induced with an adoptive transfer of t-helper 17 cells in the il-6r knockin mouse. transgenic mice expressing a tcr specific for bovine pancreas ribonuclease develop spontaneous arthritis that is mediated by antibodies (korganow et al., 1999) . this model is particularly interesting because it demonstrates that t cells specific for a ubiquitous antigen may induce an organspecific autoimmune disease. the expression of the gene product causes an upregulation of several cytokines (il-1, il-6, tgf-β1, ifn-γ, and il-2) and subsequent development of arthritis (iwakura et al., 1995) . there are some other spontaneous models for arthritis in nontransgenic mice (bouvet et al., 1990) . arthritis can be induced by complete freunds' adjuvant (cfa). pearson (pearson, 1956) described this model for the first time. subsequently, it was demonstrated that other adjuvants, such as ifa, pristane, or squalene, could also induce arthritis (carlson et al., 2000) . microbially derived products such as lipopolysaccharide (lps), muramyle dipeptide (mdp), and trehalosedimycolate (tdm) can also induce arthritis when given with mineral oil (lorentzen, 1999; kohashi et al., 1980) . collagen-induced arthritis (cia) is normally induced by the immunization of susceptible mouse (e.g., dba/1) or rat (da, lewis) strains at the base of the tail. the inoculum used for immunization contains both adjuvant and collagen type ii. the adjuvant has to be sufficiently strong to cause tissue destruction as well as induction of a strong pro-inflammatory immune response (holmdahl and kvick, 1992; kleinau et al., 1995) . susceptibility to cia is dependent on both mhc (class ii region) and non-mhc genes (lorentzen and klareskog, 1996) . antibodies against collagen ii are essential for the development of cia. this fact has been demonstrated by the passive transfer of anti-cii antibodies, which results in synovitis (svensson et al., 1998) . t cells are also important for cia development during early stages of disease progression. the dependence of both t-and b-cell responses has also been demonstrated in the same model (seki et al., 1988) . in cia, an immune response is being directed against a joint collagen type ii (cii) antigen. inflamed joints in cia are infiltrated by inflammatory cells that accumulate in the synovial membrane and fluid, similar to ra. the most frequent cell type in the synovial fluid is granulocyte. there is also a great infiltration of leukocytes into the synovial membrane. these cells have signs of an activated phenotype of ra since mhc class ii molecules are expressed (klareskog and johnell, 1988 ). in addition, there is an intense production of macrophage-derived cytokines in inflamed joints (e.g., tnf-α and il-1β) (mussener et al., 1997; ulfgren et al., 2000) . a small number of t cells are encountered, and some of these t cells have il-2 receptor α chain upregulated. the disease shows a thickened synovial membrane that subsequently forms a pannus on the cartilage surface (holmdahl et al., 1988; holmdahl et al., 1991) . in both cia and ra, cartilage and bone destruction occurs mainly at the carti-lageàpannus junction. there are some features of the pathology of cia that differ from what is usually observed in ra (e.g., extra-articular manifestation). although the compatibility of the cia model to human ra has been argued, many pathological features of cia are similar to those of rheumatoid arthritis. currently, collagen type ii-induced arthritis in mice and rats is one of the most widely used arthritis models in academia and industry. experimental collagen-induced arthritis was initiated by injecting bovine collagen type ii at the base portion of the tail of the animal (saadat et al., 2005) . male lewis rats weighing about 160à180 g were used. after the induction of cia, animals were divided randomly into four or more groups based on the experimental design. at least four different groups were needed, including a control group without arthritis, animals with collagen-induced arthritis, cia animals with treatment, and cia animals treated with methotrexate as a positive control. sample preparation: bovine collagen type ii (cii) was dissolved in 0.1 m acetic acid at a concentration of 2 mg/ml by stirring overnight at 4 c (the dissolved cii can be stored at 270 c if it has to be used at a later time). before injecting the animals, cii was emulsified with an equal volume of complete freund's adjuvant (cfa). for the induction of cia, on day 1 rats were injected intradermally at the base of the tail with 100 μl of emulsion (containing 100 μg of cii). after 12à16 days, animals showed the development of inflammation at peripheral joints ( fig. 8.1 ). on day 21, a booster injection of cii in cfa was administered. this model was used to evaluate the anti-ra effect by giving intraperitoneally injections of test materials (e.g., chemical or herbal extracts). methotrexate was a control used to evaluate the effect of the test compound and to compare the efficacy of the new compound with methotrexate. in this model, the test compound was given from day 25, where the frequency, route of administration, and dose could be selected as needed. the end point and days for the evaluation of different parameters were selected; one of the most common points was day 35 (flow chart 8.1). the paws and knees were then removed for the histopathological assay. the visual observation can be done by using the macroscopic system as given in table 8 .1. moreover, rats immunized with cfa should be checked for weight gain from the first to the end of experiment at least every other day. the decline in body weight that followed on the onset of arthritis was proportional to the disease severity and, hence, can be used as a measure of disease activity. the scaling to record the observation should be from 0 to 4 for each paw (szabó et al., 1998) . on day 35, animals were anesthetized with sodium pentobarbital (45 mg/kg intraperitoneally) and euthanized. blood was collected by intracardiac puncture, and paws and knees were removed, trimmed, and fixed in 10% buffered formalin, decalcified, and then embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin for the histological examination. joint damage was assessed based on synovial hypertrophy, pannus formation, inflammatory cell infiltration, and cartilage and subchondral bone destruction. joint erosion was graded on a scale of 0à3 for each limb (table 8. 2), according to the severity of damage ( fig. 8.2 ). radiological scoring was performed by an investigator who was blind to the treatment protocol (on day 35). radiographical analysis of affected joints in control rats typically showed soft tissue swelling, joint space narrowing, reduced lucency due to demineralization, and areas of recalcification indicative of new bone formation. a score was assigned to each joint on the basis of the information as listed in table 8 .3. scores were 0à3 per joint (0, normal; 3, maximum joint destruction). multiple sclerosis (ms) is a chronic inflammatory demyelinating disorder of the central nervous system (cns) that affects over 2.3 million individuals worldwide. similar to the affected population in other autoimmune diseases, twice as many women as men have ms. multiple sclerosis, like many other diseases, has existed as long as human life. in the 1860s, the first report by dr. jean-martin charcot certified ms as a disease. a patient of him who suffered an unusual symptom died. after dissection, brain lesions were discovered. he called the disease scleroseen plaques. myelin was subsequently discovered, although its exact role was not recognized. about one century of research resulted in the discovery multiple sclerosis is the most common inflammatory demyelinating disease of the cns in europe and north america. the prevalence of ms in north america and europe is b 80à100 per 100,000 people. however, the prevalence is not globally uniform, geographically decreases in latitudes, and has been observed in only b1à2 per 100,000 individuals in africa and asia. the etiology of ms is unknown. both genetic involvement and environmental factors have been indicated in ms. the only consistent correlation of involvement of the mhc locus is the mhc class ii allele hla-dr2, which reflects a linkage with ms. in addition to associations within the major histocompatibility complex (mhc) region, other non-mhc loci reached a genome-wide significance. they map to the genes l3mbtl3, maz, erg, and shmt1. products of the genes l3mbtl3, maz, and erg play important roles in immune cell regulation. shmt1 encodes a serine hydroxymethyl transferase catalyzing the transfer of a carbon unit to the folate cycle, which is important for establishment and maintenance of epigenetic signatures (andlauer et al., 2016) . some other factors like dietary components (e.g., milk), pathogens like human herpes virus 6 (hhv-6), measles virus, epsteinàbarr virus, and chlamydia have been implied as etiological factors. however, the association between any of these agents with ms is debatable. the presence of cns inflammation is a hallmark of ms. this inflammatory process greatly increases in the cns by the activation and deregulation of different cell types of the immune system. activation and entry of myelin-specific lymphocytes into the cns cause damage to oligodendrocytes, leading to demyelination. most of the cells from the immune system can contribute toward demyelination, but the main process of demylation is mediated by antibody and complement. so far, it has been noticed that antibody and complement are responsible for lesions in 40%à50% of ms patients. myeloid cells may cause axonal damage by releasing molecules, such as glutamate, reactive oxy-gen species, and reactive nitrogen species. besides, these cells decreased the expression of glutamate clearance. as a result of increased glutamate in the cerebrospinal fluid, ms patients would be vulnerable to degeneration (yandamuri and lane, 2016) . in addition, cd8 1 t cells play an important role in ms pathogenesis. cd8 1 t cells make up the largest percentage of lymphocytes found in the brain of ms patients. all neuroectodermal cells in ms lesions express mhc class i molecules, making them an excellent target for cd8 1 t cells. in addition to their proinflammatory properties, cd8 1 t cells can also suppress the immune system and down-regulate inflammation. however, experiments with perforin, an important regulator of cytotoxic damage to immune cells, have made it clear that cd8 1 t cells present at ms lesions cause cytotoxicity, which could be the main source for demyelination and axonal damage (sinha et al., 2015) . bystander cd4 1 t cells do not contribute to the demyelinating process, but once cd4 1 t cells move into the cns and become activated against myelin antigen, these cd4 1 t cells could be contributing directly toward the demyelination of cns (basdeo et al., 2016) . moreover, cd4 th17 effector t cells are postulated to play a crucial role in the pathogenesis of ms (bettelli et al., 2006) . in addition to autoreactive immune cells against myelin and nerves, a progressive loss of the structure and function of neurons occurs. it has been reported that the alterations in the expression of mirnas may play a crucial role in ms pathogenesis (huang et al., 2016) . after demyelination, remyelination is possible, which could further damage the cns. the ratio of demyelination to remyelination determines whether a patient will develop secondary progressive ms (spms) or relapse remitting ms (rrms). if remyelination occurs before axonal damage, irreversible physiological damage can be prevented. none of the fda-approved therapies target oligodendrocytes to stimulate remyelination, but it is a very interesting possibility for future therapeutic intervention (huang et al., 2016) . the majority of symptoms associated with ms can be directly attributed to inflammation, edema, demyelination, and/or axonal damage within the brain, spinal cord, and optic nerves. clinical motor manifestations include weakness, stiffness, and/or pain in arms or legs, abnormal reflex activity, and spasticity. often, the earliest symptoms of ms are somatosensory, including numbness and tingling. in ms, cerebral involvement is often accompanied by symptoms such as ataxia and intention tremor. many individuals with ms complain of increased urinary frequency, urgency, and incontinence. bladder and bowel disturbances remain among the most disabling and embarrassing symptoms experienced by ms patients. sexual symptoms are also very common among both men and women. fatigue, sleep disturbances, depression, and deficits in cognitive functioning are also common. the clinical course of ms is often highly variable and is generally characterized by relapses or exacerbations and deterioration of neurologic function, which entitle relapsing remitting ms. the features of relapsing remitting ms are defined as "episodes of acute worsening of neurologic function followed by a variable degree of recovery, with a stable course between attacks." approximately 80%à85% of patients are initially diagnosed with rrms that evolves from an isolated demyelinating attack, which is characterized by multifocal inflammation along with varying degrees of axonal injury. a patient may experience disease progression with or without relapses and minor remissions; that clinical condition is defined as secondary progressive ms. it has been seen that 75% of rrma patients will eventually develop the spms state. primary progressive ms (ppms) affects b10%à15% of ms patients. ppms is defined as "disease progression from onset, with occasional plateaus and temporary minor improvements in clinical condition." the duration of ms varies significantly among ms patients. some patients will live with ms for several decades, while about 10% will develop an acute, fulminant form of ms. patients with an acute and fulminant form of ms show a rapid deterioration in their clinical signs and symptoms that have fatal consequences; these patients usually die within 1à3 years after the onset of disease. in general, the clinical spectrums among ms patients represent a benign disease and a low relapse rate, and these may never develop into secondary progressive disease. the heterogeneity of the clinical course of ms is shown to have a similar variation in its pathology. multiple sclerosis lesions were recently segregated into four distinct subtypes. the general pathology of ms, the formation of demyelinating lesions in the cns associated with infiltrating cd3 1 t cells, activated macrophages, and microglia-containing myelin debris, and infiltrating b cells, is common to all forms of the disease. it is thought that ms lesions are mediated by soluble factors such as tnf-α and immunoglobulin deposition on the myelin sheath, and the local activation of the complement cascade. the diagnostic criteria for clinically definite ms (cdms) include factors such as clinical history, mri imaging, and csf abnormalities. at present, there are no identifiable biomarkers that can predict the clinical subtype of ms. similarly, there are no factors that can assist in predicting whether a patient diagnosed with ms will develop either a progressive or a benign version of the disease. the clinical and pathological heterogeneity in ms has made it important to either develop or identify reliable biomarkers. several cytokines, immunoglobulins, mmps, markers of axonal/neuronal injury, and apoptotic markers have been suggested to have potential as biomarkers, but these biomarkers need validation by rigorous durability trials. in ms, treatment strategies can be either acute or long term. during a relapse, the goal of acute treatment is to reverse neurological disability as well as to delay further neurological dysfunction, so that the normal function can be restored. this type of treatment for ms patients is in contrast to the goals of long-term treatments. the main objective of long-term treatment for ms patients is to decrease relapses (both severity and frequency), which could lend support to stopping the progression of disability. patients experiencing a relapse, such as optic neuritis or transverse myelitis, are often administered high-dose corticosteroid firstline therapy. during progressive phases of the disease, patients may be prescribed immunosuppressive agents such as cyclophosphamide or mitoxantrone because the progressive phase is often accompanied by worsening inflammatory demyelination and axonal degeneration (rommer et al., 2019) . in 1993, ifn-β was the first agent to demonstrate the significant clinical efficacy among patients suffering with rrms. although the exact disease-modifying effects of ifn-β in ms are unknown, several immunomodulatory mechanisms have been suggested. presently, two forms of ifn-β, including ifn-βla (avonex and rebif) and ifn-βlb (betaseron and extavia), have been prescribed. glatiramer acetate (copaxone) is a synthetic mixture of polypeptides that has been approved to treat rrms. similar to ifn-β, glatiramer acetate is found to be not effective for progressive forms of ms. natalizumab (tysabri) is an alpha-4 integrin antagonist and is the first drug of an entirely new class of immune-directed therapies that has been approved by the fda to treat relapsing ms. natalizumab is a humanized recombinant monoclonal antibody that blocks leukocyte migration into the cns by binding to α-4 integrins; these are components of the very late antigen-4 (vla-4) complex constitutively expressed on the leukocyte surface. in monotherapy trials, natalizumab has been reported to reduce the risk for sustained progression of disability as well as decrease the frequency of relapses. based on the current literature, natalizumab appears to be one of the most effective agents to prevent relapses as well as to stop disease progression. other monoclonal antibodies administered by intravenous (iv) infusion include lemtrada (alemtuzumab) and novantrone (mitoxantrone). currently, numerous other monoclonal antibodies are under investigation as potential therapies for ms; for example, anti-cd25 (daclizumab), anti-cd 20 (rituximab), and so on. a number of other agents are under investigation for possible future use in ms including secukinumab (a humanized monoclonal antibody to il-17), rtl1000 (inhibitor of the activation of myelin-reactive t cells), firategrast (affect on the vla-4 system) and aimspro (neuropeptide stabilizer). moreover, stem cell-based therapy might be considered as another approach for attenuating ms through regulating the immune system, although several challenges should be resolved. more investigations and clinical trials should be designed to assess the effectiveness of several drugs and approaches that can target both inflammatory and degenerative components of ms. these kinds of approaches may offer hope for individuals who are suffering from this debilitating disease (mansoor et al., 2019; agrawal and yong, 2007; hart and bainbridge, 2016) . to gain ideas about ms mechanisms, a number of models have been developed. these experimental models fall into two categories: spontaneous models and induced models. each model reflects characteristic features of ms patients and has its own merits and demerits. myelin basic protein mutant (taiep rat), proteolipid protein mutants (rumpshaker and jimpy mice), as well as gene-knockout animals (the myelin-associated glycoprotein (mag) knockout, thy1-eb3-yfp mice, and thy1-xfp mice) show dysmyelination, altered neurotransmission and, in some instances, clinical disease. these models have frequently been used to study myelination. with chemically induced lesions, viral and autoimmune models are developed to show some evidence of demyelination, which is considered a pathological hallmark of ms. direct injection of ethidium bromide or lysolecithin into the cns produces demyelination. these induced models are usually effectively repaired once macrophages clear the myelin debris. for this reason, these models are rarely used at the present time. besides, local administration of glutamate or nitric oxide donors induces axonopathy in mice and have also been used to understand mechanisms of axonal degeneration and regeneration (luchtman et al., 2016) . a number of viruses, including semliki forest virus, theiler's murine encephalomyelitis virus, and a murine coronavirus have been found to induce disease by neurotrophic infection of the cns, specifically oligodendrocytes (lane and hosking, 2010) . moreover, studies using immunodeficient rag1 2 / 2 mice have indicated that cd4 1 and cd8 1 t lymphocytes as well as macrophages are key contributors to demyelination in coronavirus-infected mice (dandekar et al., 2001) . finally, experimental allergic encephalomyelitis (eae) has received the most attention as a model for ms; this animal model is routinely used for testing different therapeutic strategies. today, eae as the most commonly used preclinical murine model of ms induced actively by the injection of defined encephalitogenic myelin protein epitopes plus cfa, or passively by the transfer of encephalitogenic myelin-sensitized t lymphocytes. some of these eae models also require the administration of the microbial-based immunologic adjuvant pertussis toxin (pt) (yandamuri and lane, 2016) . eae exhibits many clinical and histological features of ms and is caused by autoimmunity induced against antigens that are expressed either naturally or artificially in cns (denic et al., 2011) . the method for eae induction and preparation of antigens to induce eae in c57bl/6 mice was adapted from the method described by kafami et al. (2010) . it is important for the successful induction of eae to follow standard precautions for the use of animals. female c57bl/6 mice that are 4-to 6-week old are used for the induction of eae. animals must adhere to the normal laboratory animal maintenance guide. animals were immunized with the hooke kits (hooke labs, ek-0115, lawrence, ma, usa). it is recommended to follow the manufacturer's instructions. a mesh was dampened in ether and put in a desiccator. the mouse was kept in the desiccator and observed until breathing slowed down to ascertain whether the mouse had been anesthetized. the mouse was removed from the anesthetic chamber and laid on its side. two syringes were filled with 1 ml of myelin oligodendrocyte glycoprotein (mog) emulsion with complete freund's adjuvant. each animal was given an injection of 200 μl. the needle was gently inserted into the subcutaneous space at the base of the tail, and 200 μl of emulsion was injected into the site. since it was difficult to give the mouse a 200-μl injection, every mouse was given a 100-μl injection at two different sites on the same day. immediately, and after 24 hours from the first injection, each mouse was given an intraperitoneal injection of pertussis toxin (100 μl/ animal). the animal was observed until complete recovery, and it could move without a floppy gate. this procedure was repeated for all animals. after 2à3 days, the flanks were bulging in response to the subcutaneous injection (flow chart 8.2). one day before immunization, and from the 7th to the 35th day post-immunization, the animals were evaluated on a daily basis for signs of eae following the 10-point score system (table 8. three different clinical parameters were analyzed to compare the course of eae (fig. 8.3) : (1) severity of disease as the cumulative disease index (cdi) was the mean of the clinical scores of the animals; (2) disease onset, calculated as the mean of the first-day animals showed the signs of the disease in experimental animals; and (3) peak of disease score, which represented the mean of the highest clinical score of disease for all animals in each group. tonicity of the tail and the distal part of the tail was ascertained by touching the tip of the tail. if the distal part of the tail was flaccid, the animal was removed from the base and observed to see if its tail remained erect or fell down (examined with the touch of the finger). after ascertaining tonicity of the tail, the gate of the animal was observed by keeping it in an open area (like a tabletop) and allowing it to walk. after checking the gate, the hind limb was observed by grabbing its tail. after that, the paralysis score was recorded for unilateral paralysis. by holding the animal in the palm of the hand, it was easy to evaluate the type of paralysis (unilateral or bilateral). it was noted whether the mouse rolled spontaneously in its cage or was dead with complete paralysis. after 35 days, animals that had an eae score of 5 and did not change for 3 more days were euthanized by chloral hydrate injection (0.3 ml, ip). for histopathlogical evaluations, different tissues were harvested after dissecting the animals. animals were placed appropriately in the dissection tray. a midline incision was made on the abdomen; the diaphragm was opened while ribbons were cut to expose the beating heart. the needle was inserted into the left ventricle of the heart while a phosphate-buffered saline (pbs) tap was allowed to fill the heart for 2 seconds. the right aorta was cut with small scissors to allow the pbs and pfa to circulate to exit. pbs allowed perfusion until the liver turned from red to yellow (b2à3 minutes). the best sign was when the liquid flowed out of the incised left aorta and turned from red to clear. another indicator was when pbs entered the pulmonary system and emerged through the nose of the animal. then, the pbs tap was closed and the tap was turned on for 4% paraformaldehyde (pfa, ph 7.4 at 37 c) to allow pfa to flow and perfuse the circulatory system for 3 minutes. perfusion was evaluated by involuntary hind limb movement and tail shivering. when the mouse became stiff, it was time to stop pfa perfusion. after the perfusion was complete with pfa, the system was washed with pbs to remove residual pfa. after perfusion, the various tissues of interest were harvested and stored in fresh 4% pfa for 3 days at 4 c. then, these tissues were washed with pbs and the pfa-fixed tissue could be stored in pbs for a few months. these tissues were then available for sectioning and staining (fig. 8.4) . for immunohistochemistry, the three sections showing the highest infiltrations were studied. an area $ 1.5 3 107 μm 2 from the brain/spinal cord was selected and analyzed under 200 3 magnification to assess the average number of positive cells per millimeter square and to quantify it on a computerized imaging system [bx51 microscope (olympus, hamburg, germany) with analysis software (special sis docu; soft imagingsystem)] by planimetry. the inflammatory index had to be calculated as a percentage determined by dividing the number of visual fields with .10 cd3 t cells by the total number of visual fields examined. detection of amyloid precursor protein (app) was performed for acute axonal damage. to assess the content of circulating proinflammatory cytokines like il-6, il-4, il-12, il-10, tnf-α, and ifn-γ, enzyme-linked immunosorbent assay (elisa) was employed. to evaluate the levels of different cytokines, blood was collected into tubes by a retro-orbital plexus method. the collected blood was kept in the tube to clot. after the clotting of the blood serum, it was separated and stored at 220 c. these serum samples were then used for the evaluation of different cytokines using the elisa. in order to quantify the mrna of different proinflammatory cytokines such as tnf-α and ifn-γ, antiinflammatory cytokines like il-10, myelin-deteriorating matrix metalloproteinase mmp-9, and the content of 164 8. animal models for human disease myelin basic protein (mbp 3à4), samples from animals had to be analyzed by real-time pcr. animals were sacrificed with lethal injection and perfused with cold pbs. then, the limbs and muscles were removed with scissors and the skin removed from these organs. a transverse cut was made at the base of the skull and vertebral column to separate them. the nasal bridge was broken with a small scalpel and the eyeballs removed. very thin forceps were used under the skull bones to break it into pieces from the frontal to occipital lobes. the bony connection under the cerebellum was broken to expose the cerebellum. the broken bones of the skull needed to be removed. the nerve root connection with the brain was cut. the brain was removed and stored in liquid nitrogen. for the removal of the spinal cord, an oblique cut was made from the lateral side of the spinal cord (started from the cervical part) to the furthest part of the vertebral column (both sides). the spinal cord was then exposed by cutting the boney flap. the steps above were repeated to get to the coda aquina. the spinal cord was taken out by cutting its adhesion to the base. it was then stored in liquid nitrogen. the frozen tissue sample was used for rna extraction. first, the sample was homogenized by pushing and rotating it with a sterile glass homogenizer. next, the homogenate sample was left on the bench top at room temperature (15 cà25 c) for 5 minutes to promote the dissociation of nucleoprotein complexes. then, 200 μl of chloroform was added to the tube and the tube was shaken vigorously for 15 seconds. the tube containing the homogenate was placed on the bench top at room temperature for 2à3 minutes and then centrifuged again at 15,000 rpm for 15 minutes at 4 c. after centrifugation, the sample separated into three phases: an upper, colorless, aqueous phase containing rna; a white interphase; and a lower, red, organic phase. the upper, aqueous phase was transferred to a new sterile eppendorf tube. one volume (usually 600 μl) of 70% ethanol was added to the tube containing the aqueous phase and mixed thoroughly by vortexing. visible precipitates after the addition of ethanol could then be noticed. up to 700 μl of the sample was processed for total rna extraction by using an rneasy mini spin column (roche germany) according to the kit instructions. after rna extraction, rna was quantified spectrophotometrically and the purity of rna was ascertained by taking out a ration between the od at 260 and 280 nm. a quantitative real-time reverse transcriptase pcr was performed to analyze the levels of mrna of different cytokines using cytokine-specific primers. the first step was to perform cdna synthesis by using a cdna synthesis kit (takara, japan), which was followed by a syber green i real-time pcr master mix kit (takara, japan). a house-keeping gene (like the β-actin gene) was included in the study to compare the results. the use of laboratory animals in research is of major ethical concern. much of the argument revolves around moral values. today, there is a wide spectrum of views on animal rights. this has prompted the establishment of guidelines on the care and use of experimental animal models. the guidelines endorse some essential principles for the care and use of animals for scientific projects. the basis of these principles is to replace animals with other methods such as mathematical models, computer simulations, and in vitro biological systems, thus reducing the number of animals used in order to obtain valid results without unnecessary duplication, and finally, refining projects by selecting appropriate species and techniques to minimize pain or distress to animals using appropriate sedation or anesthesia. as a researcher, one must always assume that procedures that cause pain to humans will cause pain in such situations in animals. surgical procedures should be performed on anaesthetized animals. it should be kept in mind that if the animal would suffer severe pain during a procedure, or if at the end point cannot be alleviated swiftly, the animals must be killed humanely. the transportation, housing, feeding, and handling of animals are also important. housing facilities should be compatible with the needs of the species and equipped to achieve a high standard of animal care. the place should be designed to facilitate control of environmental factors. cages should be comfortable and should fulfill behavioral requirements such as free movement and activity, bedding, contact with others of the same species, lighting, temperature, air quality, appropriate day/night cycles, and protection from excessive noise. the population density of animals within cages should also be considered from an ethical standpoint. this statement refers to the need for the reader to operate in accordance with the guidelines at her/his academy. the concept of translational research is to try to convert the results derived in animal models into a new understanding of disease mechanisms and therapeutics in human beings. it is a bridge from experimental models to clinical medicine. over recent years, the importance of this kind or research has progressively increased. consequently, translational research is considered a key component to finding practical applications, especially within medicine. with the improvement of technologies, significant progress has been made in producing various types of engineered experimental animal models based on a better understanding of the molecular and genetic principles of disease. as a result, any interventions in experimental models are more practical and repeatable when compared to patient-oriented research. various risk factors that are linked to, or even responsible for, differences in clinical results should also be considered as significant for the development of experimental models; this will enhance the translational value of experimental models. these risk factors can be categorized into genetic factors, acquired factors, and health conditions, which can be studied in models in a controlled manner. in medicine, the performance of successful translational research requires data from hospitals. as we mentioned before, rheumatoid arthritis as a progressive debilitating disease is characterized by hyperplasia of synoviocytes leading to joint destruction and permanent deformity. although the definite pathophysiology of ra is ambiguous, some evidence suggests that telomerase is also involved in the pathogenesis of this disease. nobel laureates in physiology/ medicine in 2009, elizabeth blackburn, jack szostak and carol greider, have solved a major problem of the chromosomal protection against degradation during cell divisions. they identified telomerase and a unique dna sequence in the telomeres. telomerase is a ribonucleoprotein enzyme that adds repeated units of ttaggg to the ends of chromosomes. this enzyme is composed of an rna component, called htert which serves as a template for addition of telomeric repeats. although it is now known that the dna sequence in the telomere attracts proteins that form a protective cap around the fragile ends of the dna strands, a number of reports have mentioned a link between the increased telomerase activity of human tumor samples and degree of invasiveness. in patients with ra, an impaired telomerase enzyme and premature cellular ageing (senescence) of thymic naïve and memory t cells was reported. moreover, transfection of rheumatoid arthritis synovial fibroblasts with vectors expressing antisense oligonucleotide against the htert component of telomerase enzyme has led to cytolysis of these cells that exhibit high telomerase activity. taken together, their discoveries have shed light on disease mechanisms and stimulated the development of potential new therapies in experimental models. there are many methods to evaluate telomerase activity, but we measured it by telomere repeat amplification protocol using trapeze telomerase detection kit (intergen, inc., usa) in animals treated with camellia sinensis stew. in detailed, biopsies of synovial tissue were obtained aseptically from the knee joints of rat after the induction of cia. synovial tissue specimens were rinsed, minced, and digested with 0.2% collagenase in high-glucose dmem containing 10% fbs and antibiotics. following overnight incubation at 37 c, cells were collected, plated in culture flask, and allowed to reach confluency at 37 c in a humidified atmosphere of 5% co2. after the lysis of equal number of cells which harvested from synovial tissue with the chaps lysis buffer, the telomerase was first extended for 30 minutes at 30 % oc and then amplified by30 cycles of pcr. the products of pcr were detected by polyacrylamide gels and revealed by silver nitrate staining. telomerase activity was calculated as the ratio of the intensity of telomerase ladders to the intensity of the 36-bp internal standard. in conclusion, we show that c. sinensis stew effectively suppresses collagen arthritis and a potent inhibitory effect on telomerase activity. so, natural products should continue to provide innovative lead compounds currently entering clinical trials. recently, the circular plant peptide kalata b1 (cyclotide) was investigated by thell et al. using the ms mouse model experimental autoimmune encephalomyelitis. according to their findings, treatment of mice with the cyclotide resulted in a significant delay and diminished symptoms of eae by oral administration. taken together, natural product should be considered as a candidate for the future investigations to possible implication for human health. using these above-mentioned models associated with other experimental models gives us such an opportunity to accomplish many findings in human medicine. until now, many progresses in medical sciences have been achieved. the discovery of numerous types of antibiotics for controlling infectious disease and elimination some viral disease like smallpox might be considered as one of researcher and indeed experimental animals honor. also, blood transfusions, open heart surgery, and other life-saving techniques have all been developed. nevertheless, there are many unsolved subjects included cancer, aging, alzheimer's disease, and acquired immunodeficiency syndrome in front of the society. without no doubt, until to find another means for answering human beings dilemma, the use of living animals in scientific research would be the best and applicable procedure. with all those valuable function, it is pivotal to consider ethical concerns over the quality of life of animals when you as a young researcher start to write a proposal. medlineplus is the national institutes of health (nih) site for patients and their families and friends. produced by the national library of medicine, it brings you information about diseases, conditions, and wellness issues in easy-to-understand language. medlineplus offers reliable, up-to-date health information, anytime, anywhere, for free. http://www.ebi.ac.uk/ipd/imgt/hla/ the imgt/hla database provides a specialist database for sequences of the human major histocompatibility complex (hla) and includes the official sequences for the who nomenclature committee for factors of the hla system. the imgt/hla database is part of the international immunogenetics project. adjuvant a substance such as complete freund's adjuvant (cfa) that enhances t-and b-cell activation, mainly by promoting the accumulation and activation of antigen-presenting cells at the site of antigen exposure. adjuvants stimulate the expression of t-cellactivating co-stimulators and cytokines by antigen-presenting cells and may also prolong the expression of peptideàmhc complexes on the surface of these cells. autoimmune disease a disease caused by a breakdown of selftolerance such that the adaptive immune system responds to selfantigens and mediates cell and tissue damage. autoimmune diseases can be organ specific (e.g., thyroiditis or diabetes) or systemic (e.g., systemic lupus erythematosus). cd molecules cell surface molecules expressed on various cell types in the immune system that are designated by the "cluster of differentiation (cd) number." disease-modifying antirheumatic drugs (dmards) they contain medications from different classes including methotrexate, gold salts, hydroxychloroquine, sulfasalazine, ciclosporin, and azathioprine. dmards were often only partly effective and poorly tolerated in long-term therapy of autoimmune diseases. enzyme-linked immunosorbent assay (elisa) a method of quantifying an antigen immobilized on a solid surface by use of a specific antibody with a covalently coupled enzyme. the amount of antibody that binds the antigen is proportional to the amount of antigen present and is determined by spectrophotometrically measuring the conversion of a clear substrate to a colored product by the coupled enzyme. experimental autoimmune encephalomyelitis (eae) this is an animal model of multiple sclerosis, an autoimmune demyelinating disease of the central nervous system. eae is induced in rodents by immunization with components of the myelin sheath (e.g., myelin basic protein) of nerves, mixed with an adjuvant. the disease is mediated in large part by cytokine-secreting cd4 1 t cells specific for the myelin sheath proteins. granulocyte-monocyte colony-stimulating factor (gm-csf) a cytokine made by activated t cells, macrophages, endothelial cells, and stromal fibroblasts that acts on bone marrow to increase the production of neutrophils and monocytes. gm-csf is also a macrophage-activating factor and promotes the differentiation of langerhans cells into mature dendritic cells. granuloma a nodule of inflammatory tissue composed of clusters of activated macrophages and t lymphocytes, often associated with necrosis and fibrosis. granulomatous inflammation is a form of chronic delayed-type hypersensitivity, often in response to persistent microbes or to particulate antigens that are not readily phagocytosed. granzyme a serine protease enzyme found in the granules of ctls and nk cells is released by exocytosis, enters target cells, and proteolytically cleaves and activates caspases and induces target cell apoptosis. homeostasis in the adaptive immune system, the maintenance of a constant number and diverse repertoire of lymphocytes, despite the emergence of new lymphocytes and the tremendous expansion of individual clones that may occur during responses to immunogenic antigens. homeostasis is achieved by several regulated pathways of lymphocyte death and inactivation. human leukocyte antigens (hla) mhc molecules expressed on the surface of human cells. human mhc molecules were first identified as alloantigens on the surface of white blood cells (leukocytes) that bound serum antibodies from individuals previously exposed to other individuals' cells. interferons a subgroup of cytokines originally named for their ability to interfere with viral infections, but that have other important immunomodulatory functions. type i interferons include interferon-α and interferon-β, whose main functions are antiviral; type ii interferon, also called interferon-γ, activates macrophages and various other cell types. interleukins any of a large number of cytokines named with a numerical suffix roughly sequentially in order of discovery or molecular characterization (e.g., interleukin-1 and interleukin-2). some cytokines were originally named for their biological activities and do not have an interleukin designation. lipopolysaccharide (lps) a component of the cell wall of gramnegative bacteria that is released from dying bacteria and stimulates many innate immune responses, including the secretion of cytokines, induction of microbicidal activities of macrophages, and expression of leukocyte adhesion molecules on endothelium. lps contains both lipid components and carbohydrate moieties. major histocompatibility complex (mhc) molecule a heterodimeric membrane protein encoded in the mhc locus that serves as a peptide display molecule for recognition by t lymphocytes. two structurally distinct types of mhc molecules exist. class i mhc molecules are present on most nucleated cells, bind peptides derived from cytosolic proteins, and are recognized by cd8 1 t cells. class ii mhc molecules are restricted largely to dendritic cells, macrophages, and b lymphocytes, bind peptides derived from endocytosed proteins, and are recognized by cd4 1 t cells. matrix metalloproteinase (mmp) mmps are a family of highly conserved endopeptidases dependent on zn2 1 ions for activity. mmps can collectively cleave most extracellular matrix. at present, 25 vertebrate mmps and 22 human homologs have been identified and characterized. mmps participate in many physiological processes, such as embryonic development, organ morphogenesis, blastocyst implantation, ovulation, nerve growth, cervical dilatation, postpartum uterine involution, mammary development, endometrial cycling, hair follicle cycling, angiogenesis, inflammatory cell function, apoptosis, tooth eruption, bone remodeling, and wound healing. myelin oligodendrocyte glycoprotein (mog) mog is a cnsspecific type i membrane glycoprotein of the immunoglobulin superfamily expressed mainly on the outermost layer of the myelin sheath, making it an ideal target for antibody-mediated demyelination. it is highly immunogenic, and unlike other myelin proteins used to induce eae, is unique in inducing both an encephalitogenic t-cell response and a demyelinating response in eae. multiple sclerosis (ms) a chronic inflammatory demyelinating disorder of the central nervous system. the majority of symptoms associated with ms can be directly attributed to inflammation, edema, demyelination, and/or axonal damage within the brain, spinal cord, and optic nerves. nitric oxide (no) a biologic effector molecule with a broad range of activities that in macrophages functions as a potent microbicidal agent to kill ingested organisms. pannus formation of locally invasive synovial tissue is a characteristic feature of rheumatoid arthritis. perforin a protein that is homologous to the c9 complement protein and is present in the granules of ctls and nk cells. when perforin is released from the granules of activated ctls or nk cells, it promotes the entry of granzymes into the target cell, leading to apoptotic death of the cell. rheumatoid arthritis (ra) an autoimmune disease characterized primarily by inflammatory damage to joints and sometimes inflammation of blood vessels, lungs, and other tissues. cd4 1 t cells, activated b lymphocytes, and plasma cells are found in the inflamed joint lining (synovium), and numerous proinflammatory cytokines, including il-1 and tnf, are present in the synovial (joint) fluid. reverse transcriptase (rt) an enzyme encoded by retroviruses, such as hiv, that synthesizes a dna copy of the viral genome from the rna genomic template. purified reverse transcriptase is used widely in molecular biology research for purposes of cloning complementary dnas encoding a gene of interest from messenger rna. th1 cells subset of cd4 1 helper t cells whose principal function is to stimulate phagocyte-mediated defense against infections via secretion of a group of cytokines, including ifn-γ. th2 cells subset of cd4 1 helper t cells whose principal functions are to stimulate ige and eosinophil/mast cell-mediated immune reactions via a particular set of cytokines, including il-4 and il-5. th17 cells subset of cd4 1 helper t cells that are protective against certain bacterial infections and also mediate pathogenic responses in autoimmune diseases. tumor necrosis factor (tnf) a cytokine produced mainly by activated mononuclear phagocytes that stimulates the recruitment of neutrophils to sites of inflammation. tnf-α blocking agents a group of biological disease-modifying antirheumatic drugs such as etanercept (a soluble tnf-α receptor) and infliximab (a monoclonal antibody). very late antigen (vla) the set of integrins that shares a common beta-1 chain. long-answer questions immunopathogenesis of multiple sclerosis novel multiple sclerosis susceptibility loci implicated in epigenetic regulation immunology increased expression of tbet in cd4 1 t cells from clinically isolated syndrome patients at high risk of conversion to clinically definite ms reciprocal developmental pathways for the generation of pathogenic effector th17 and regulatory t cells spontaneous rheumatoid-like arthritis in a line of mice sensitive to collagen-induced arthritis one year in review 2016: novelties in the treatment of rheumatoid arthritis the endogenous adjuvant squalene can induce a chronic t-cell-mediated arthritis in rats access to the next wave of biologic therapies (abatacept and tocilizumab) for the treatment of rheumatoid arthritis in england and wales axonal damage is t cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus the relevance of animal models in multiple sclerosis research current and emerging treatment of multiple sclerosis hla and rheumatoid arthritis: how do they connect? vaccination and genetic experiments demonstrate that adjuvant-oil-induced arthritis and homologous type ii collagen-induced arthritis in the same rat strain are different diseases early appearance of activated cd4 1 t lymphocytes and class ii antigen-expressing cells in joints of dba/1 mice immunized with type ii collagen involvement of macrophages and dendritic cells in synovial inflammation of collagen induced arthritis in dba/1 mice and spontaneous arthritis in mrl/lpr mice micrornas associated with the pathogenesis of multiple sclerosis autoimmunity induction by human t cell leukemia virus type 1 in transgenic mice that develop chronic inflammatory arthropathy resembling rheumatoid arthritis in humans intermittent feeding attenuates clinical course of experimental autoimmune encephalomyelitis in c57bl/6 mice transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis induced expression of class ii transplantation antigens in the cartilage-pannus junction in ra: chronic synovitis as a model system for aberrant t-lymphocyte activation role of adjuvants in turning autoimmunity into autoimmune disease il-35 stimulation of cd39 1 regulatory t cells confers protection against collagen ii-induced arthritis via the production ofil-10 arthritis-inducing ability of a synthetic adjuvant, n-acetylmuramyl peptides, and bacterial disaccharide peptides related to different oil vehicles and their composition from systemic t cell self-reactivity to organ-specific autoimmune disease via immunoglobulins genetics of rheumatoid arthritis -a comprehensive review the pathogenesis of murine coronavirus infection of the central nervous system flavonoids as cytokine modulators: a possible therapy for inflammation-related diseases identification of arthritogenic adjuvants of self and foreign origin susceptibility of da rats to arthritis induced with adjuvant oil or rat collagen is determined by genes both within and outside the major histocompatibility complex in vivo and in vitro effects of multiple sclerosis immunomodulatory ther-apeutics on glutamatergic excitotoxicity the potential use of mesenchymal stem cells for the treatment of multiple sclerosis the epigenetics of autoimmunity cytokine production in synovial tissue of mice with collagen-induced arthritis (cia) inhibition of interleukin-33 signaling attenuates the severity of experimental arthritis development of arthritis, periarthritis and periostitis in rats given adjuvants an update on dietary phenolic compounds in the prevention and management of rheumatoid arthritis effect of pyrimethamine in experimental rheumatoid arthritis type ii collagen-induced murine arthritis. i. induction and perpetuation of arthritis require synergy between humoral and cell-mediated immunity cd8(1) t-cells as immune regulators of multiple sclerosis ptpn22: the archetypal non-hla autoimmunity gene from genetics to functional insights into rheumatoid arthritis b celldeficient mice do not develop type ii collagen-induced arthritis (cia) protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly (adpribose) synthetase efficacy and safety of mavrilimumab in japanese subjects with rheumatoid arthritis: findings from a phase iia study effect of denosumab on japanese patients with rheumatoid arthritis: a doseresponse study of amg 162 (denosumab) in patients with rheumatoid arthritis on methotrexate to validate inhibitory effect on bone erosion (drive)-a 12-month, multicentre, randomized, double-blind, placebo-controlled, phase ii clinical trial interindividual and intra-articular variation of proinflammatory cytokines in patients with rheumatoid arthritis: potential implications for treatment the natural soluble form of il-18 receptor beta exacerbates collagen-induced arthritis via modulation of t-cell immune responses genetics and epigenetics of rheumatoid arthritis imaging axonal degeneration and repair in preclinical animal models of multiple sclerosis use of animals in scientific research. indian council of medical research ministry of health & family welfare new delhi animal models of rheumatoid arthritis and their relevance to human disease animal models of multiple sclerosis-potentials and limitations the rights of animals animal models of multiple sclerosis. neuroinflammation 55à79. glossary adhesion molecule a cell surface molecule (e.g., selectin, integrin, and member of the ig superfamily) whose function is to promote adhesive interactions with other cells or the extracellular matrix describe the significance of animal modeling in biotechnology? how cia is induced and how the ability of medications is evaluated in mice? discuss about different types of animal model to study the pathogenesis of rheumatoid arthritis? why the presence of inflammation in the cns is considered as a hallmark of multiple sclerosis? explain the various methods for evaluation of experimental models of multiple sclerosis? short answer questions what is the reason of reportedly experiencing different animal models for studying the pathogenesis of rheumatoid arthritis? give an example which shows the impact of the epigenetics in the initiation of ra? which types of evaluation should be performed after "collagen-induced arthritis" aroused? 4. what are the "intervening factors after the activation of the immune system, which type of lymphocytes enters into central nervous system answers to short answer questions 1. there are many experimental models that resemble ra in different respects. since ra is a heterogeneous disease there is probably a need for different animal models that each reflect a characteristic feature of a particular subgroup of ra patients or illustrate particular aspect of the disease.2. the epigenetics of ra have also been responsible in the initiation of ra. since the concordance of rheumatoid arthritis in identical twins is not 100% other nongenetic factors also play a role in the disease etiology. 3. daily clinical assessment according to a macroscopic scoring system, histological processing and assessment of arthritis damage, radiographic evaluation by an investigator blinded to the treatment protocol on day 35. 4. age, weight, and possible infectious disease in animals should be considered as the intervening factors. 5. following activation, myelin-specific lymphocytes enter into the cns and oligodendrocytes are damaged.yes/no type questions 8. natalizumab blocks leukocyte migration into the cns by binding to icam. 9. eae induced actively by injection of the microbialbased immunologic adjuvant pertussis toxin. 10. housing facilities should be compatible with the needs of the species and equipped to achieve a high standard of animal care.answers to yes/no type questions 1. yes-the most important risk factor for acpa 1 ra is the hla class ii locus. key: cord-280979-0vaarrji authors: gauttier, v.; morello, a.; girault, i.; mary, c.; belarif, l.; desselle, a.; wilhelm, e.; bourquard, t.; pengam, s.; teppaz, g.; thepenier, v.; biteau, k.; de barbeyrac, e.; kiepferlé, d.; vasseur, b.; le flem, fx.; debieuvre, d.; costantini, d.; poirier, n. title: tissue-resident memory cd8 t-cell responses elicited by a single injection of a multi-target covid-19 vaccine date: 2020-08-14 journal: biorxiv doi: 10.1101/2020.08.14.240093 sha: doc_id: 280979 cord_uid: 0vaarrji the covid-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) which enters the body principally through the nasal and larynx mucosa and progress to the lungs through the respiratory tract. sars-cov-2 replicates efficiently in respiratory epithelial cells motivating the development of alternative and rapidly scalable vaccine inducing mucosal protective and long-lasting immunity. we have previously developed an immunologically optimized multi-neoepitopes-based peptide vaccine platform which has already demonstrated tolerance and efficacy in hundreds of lung cancer patients. here, we present a multi-target cd8 t cell peptide covid-19 vaccine design targeting several structural (s, m, n) and non-structural (nsps) sars-cov-2 proteins with selected epitopes in conserved regions of the sars-cov-2 genome. we observed that a single subcutaneous injection of a serie of epitopes induces a robust immunogenicity in-vivo as measured by ifnγ elispot. upon tetramer characterization we found that this serie of epitopes induces a strong proportion of virus-specific cd8 t cells expressing cd103, cd44, cxcr3 and cd49a, the specific phenotype of tissue-resident memory t lymphocytes (trm). finally, we observed broad cellular responses, as characterized by ifnγ production, upon restimulation with structural and non-structural protein-derived epitopes using blood t cells isolated from convalescent asymptomatic, moderate and severe covid-19 patients. these data provide insights for further development of a second generation of covid-19 vaccine focused on inducing lasting th1-biased memory cd8 t cell sentinels protection using immunodominant epitopes naturally observed after sars-cov-2 infection resolution. statement of significance humoral and cellular adaptive immunity are different and complementary immune defenses engaged by the body to clear viral infection. while neutralizing antibodies have the capacity to block virus binding to its entry receptor expressed on human cells, memory t lymphocytes have the capacity to eliminate infected cells and are required for viral clearance. however, viruses evolve quickly, and their antigens are prone to mutations to avoid recognition by the antibodies (phenomenon named ‘antigenic drift’). this limitation of the antibody-mediated immunity could be addressed by the t-cell mediated immunity, which is able to recognize conserved viral peptides from any viral proteins presented by virus-infected cells. thus, by targeting several proteins and conserved regions on the genome of a virus, t-cell epitope-based vaccines are less subjected to mutations and may work effectively on different strains of the virus. we designed a multi-target t cell-based vaccine containing epitope regions optimized for cd8+ t cell stimulation that would drive long-lasting cellular immunity with high specificity, avoiding undesired effects such as antibody-dependent enhancement (ade) and antibody-induced macrophages hyperinflammation that could be observed in subjects with severe covid-19. our in-vivo results showed that a single injection of selected cd8 t cell epitopes induces memory viral-specific t-cell responses with a phenotype of tissue-resident memory t cells (trm). trm has attracted a growing interest for developing vaccination strategies since they act as immune sentinels in barrier tissue such as the respiratory tract and the lung. because of their localization in tissues, they are able to immediately recognize infected cells and, because of their memory phenotypes, they rapidly respond to viral infection by orchestrating local protective immune responses to eliminate pathogens. lastly, such multiepitope-based vaccination platform uses robust and well-validated synthetic peptide production technologies that can be rapidly manufactured in a distributed manner. the covid-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) which enters the body principally through the nasal and larynx mucosa and progress to the lungs through the respiratory tract. sars-cov-2 replicates efficiently in respiratory epithelial cells motivating the development of alternative and rapidly scalable vaccine inducing mucosal protective and long-lasting immunity. we have previously developed an immunologically optimized multi-neoepitopes-based peptide vaccine platform which has already demonstrated tolerance and efficacy in hundreds of lung cancer patients. here, we present a multi-target cd8 t cell peptide covid-19 vaccine design targeting several structural (s, m, n) and non-structural (nsps) sars-cov-2 proteins with selected epitopes in conserved regions of the sars-cov-2 genome. we observed that a single subcutaneous injection of a serie of epitopes induces a robust immunogenicity in-vivo as measured by ifnγ elispot. upon tetramer characterization we found that this serie of epitopes induces a strong proportion of virus-specific cd8 t cells expressing cd103, cd44, cxcr3 and cd49a, the specific phenotype of tissue-resident memory t lymphocytes (trm). finally, we observed broad cellular responses, as characterized by ifnγ production, upon restimulation with structural and nonstructural protein-derived epitopes using blood t cells isolated from convalescent asymptomatic, moderate and severe covid-19 patients. these data provide insights for further development of a second generation of covid-19 vaccine focused on inducing lasting th1biased memory cd8 t cell sentinels protection using immunodominant epitopes naturally observed after sars-cov-2 infection resolution. covid-19, the infectious disease caused by the zoonotic coronavirus sars-cov-2, is a global pandemic which has infected more than 16 immune responses against this type of respiratory viruses but that antibody responses are shortterm [1] [2] [3] [4] [5] in contrast to the cellular immunity which is still observed 11 and 17 years after the infection 6, 7 . current and previous covs vaccine strategies have been almost exclusively focused on eliciting a humoral immune response, particularly anti-spike neutralizing igg antibody. however, the generation of non-neutralizing antibody responses 8 , insufficient antibody titers 9 , th2-biased immune response or glycosylation changes in the igg fc tail 10 may be associated with vaccine failure, and in the worst case scenario may enhance disease upon viral exposure, either through the induction of enhanced pulmonary macrophage-mediated hyper-inflammation 11 , or fc receptor-mediated antibody-dependent enhancement (ade) 12 . th1-biasing immunization using cd8 t cells optimized peptide vaccination may offer an important alternative and complementary approach with a history of safe administration, may be developed and updated rapidly, and should avoid safety pitfalls in the pursuit of a covid-19 vaccine 8, 13, 14 . the discovery of memory t lymphocytes resident in diverse tissues, in particular mucosal and barrier tissues, has highlighted the importance of site-specific responses and continuous surveillance mediated by a specific tissue-resident memory t cell population (trm) [15] [16] [17] . a couple of studies demonstrated that after induction trm migrate and reside in the lung, skin, and gut long after infection resolution and provide localized protective immunity and immunosurveillance in tissues [18] [19] [20] [21] . trm represents an attractive target population and a growing interest for developing vaccine strategies since they act as immune sentinels in mucosal and barrier tissue and rapidly respond to infection by orchestrating local protective immune responses to eliminate pathogens 22 . previous cd8 t cell-based vaccines against sars-cov-1 23 and influenza a 24 viruses showed lasting virus-specific memory cd8 t cells induction in the spleen, lung and bronchoalveolar fluids (bals) and protection of mice from lethal sars-cov-1 or influenza challenges. expression of chemokine receptors, such as cxcr3, is critical for cd8+ trm to populate the airways after vaccination and protection against influenza a viruses 25 . more recently, a trm-inducing hiv vaccine durably prevented mucosal infection in non-human primates even with lower neutralizing antibody titers 26 . altogether, these data emphasize the interest of tissue-resident memory viral-specific cd8 t cell generation upon vaccination for optimal protection against airways viruses. stimulation of a proper immune response that leads to protection is highly dependent on presentation of epitopes to circulating t-cells via the hla complex. memopi ® is a robust vaccine platform based on selection and immunogenicity optimization of hla-restricted peptides (neo-epitope) technologies 27, 28 and formulation 29 for multi-epitopes and targets combination with a pan-dr helper epitope (padre) providing help for memory cd8 t cell generation 30 . a combination of multi-target antitumor neo-epitopes, tedopi ® , based on this platform already demonstrated good safety profile and efficacy in clinical phase 2 trial 31 and 6 more recently successfully validated in the first step of a phase 3 clinical testing in lung cancer paients 32 . while induction of mucosal immunity using parenteral administration of conventional virus vaccine technologies was challenging, we observed that subcutaneous injection of our neo-epitopes multi-target cancer vaccine promotes th1-biased antigen-specific memory cd8 t cell responses in the lung and bals of vaccinated mice in the absence of tumor. based on this original preclinical observation and the significant survival increase measured during clinical trials in lung cancer patients correlating with epitope responses, we generated and screened individually a large number of immuno-dominant sars-cov-2 epitopes, and their neo-epitopes generated using artificial intelligence (ai) algorithms, covering all sequenced circulating sars-cov-2 strains and derived from 11 structural (including spike) and non-structural proteins with significant homology with previous sars-cov-1 virus. previous research in sars-cov-1 suggests that the structural spike (s) protein is one of the main antigenic component responsible for inducing the host immune responses 33 orf1a/b non-structural proteins (nsp3, nsp4, nsp6, nsp12, nsp13, nsp14, nsp16) (figure 1 and table 1 ). based on our knowledge of key fixed-anchor positions to enhance hla binding and increase their immunogenicity potential, we designed 400 mutated sequences for each individual peptide resulting in 22 000 total analyzed sequences of the 55 selected epitopes. we first screened these potential peptides using in-silico bioinformatic analyses (e.g. iedb immune epitope database, netmhcpan el 4.0 algorithm) and a first series of the most optimized mutant for each epitope was selected (neo-epitopes a). in parallel, 22000 in silico hla-a*0201peptide docking models were generated using computational tools and analyzed using newly europe, then spread worldwide and became the most prevalent form 62, 63 . we eliminated t cell epitopes with recurrent mutation and homoplasic site in order to cover all circulating sars-cov-2 strains and anticipate future evolution of the virus in hotspot mutation regions. 134 wt and mutated peptides (neoepitopes a and b) were produced using synthetic peptide synthesis (proteogenix, france). hla-a2 binding property characterization at 37°c, using uv peptide exchange assay on hla-a*0201 monomer, showed that the majority of selected wt epitopes binds to hla-a2 with good efficacy (figure 2a ) as compared to our memopi ® internal positive neoepitope control (mutated peptide with increased hla-a*0201 binding and in-vivo immunogenicity). hla-a2 binding was increased with several neoepitopes a and/or b, particularly when the corresponding wt peptide showed weak (< 15%: figure 3a) . similarly, broad immunogenicity response was observed by hla-a*0201-tetramer flow cytometry analyses to a higher number of epitopes since 44 out of 60 (73%) evaluated peptides, derived from 10 out of the 11 selected proteins, exhibited significant frequency (0.1-1%) of viral-specific cd8 t cells ( figure 3b ). phenotypic characterization of tetramer+ cells showed that 17 out of 44 (39%) positive peptides elicited viral-specific cd8 t cells with mainly a trm phenotype: co-expressing the memory marker cd44, the cd103 αe integrin, the th1-biased cxcr3 chemokine receptor and to a lesser extent the cd49a α1 integrin (figure 4) . altogether, these data showed that optimized peptide vaccination against selected sars-cov-2 epitopes elicits robust and broad th1-biased immunogenicity against several structural (s, m, n) and non-structural proteins in hla-a2 expressing mice and that several peptides induce viral-specific memory cd8 t cells displaying all characteristics of t lymphocyte sentinels in barrier tissues. in order to identify and select naturally sars-cov-2 cd8 t cell immunodominant epitopes, peripheral blood mononuclear cells (pbmc) from asymptomatic and moderate or severe covid-19 patients with a previously confirmed (at least one month before sampling) and altogether, when we compared convalescent sars-cov-2 individuals to unexposed healthy donors, we identified 25 significantly different cd8 t cell immunodominant epitopes against 3 structural proteins (s, m, n), 1 accessory factor (orf3a) and 7 non-structural proteins (nsp3, nsp4, nsp6, nsp12, nsp13, nsp14, nsp16). 16 of these epitopes are of particular interest for vaccination since they were able to elicit also in-vivo immunogenicity (elispot response) against all 11 structural and non-structural sars-cov-2 proteins after a single peptide injection. finally, we selected a combination of 12 cd8 t cells epitopes based on manufacturing facilities, hla-i coverage, previous covs homology and sars-cov-2 proteins diversity considerations ( table 2) . these 12 epitopes covered the 11 selected proteins, 1 epitope/protein excepting spike for which 2 epitopes (including 1 rbd epitope) have been selected. bioinformatic analyses illustrate these 12 epitopes are not restricted only to the hla-a*0201 allele, hence are predict (netmhcpan score < 1) to bind efficiently to different hla-i (a, b, c) alleles with high genetic coverage in all geographical region of the world. despite hla polymorphism and different worldwide hla-i distribution, the combination of these 12 t cell epitopes should induce at least 1 to 4 positive peptide responses in all individuals globally and achieve the 60-70% 'herd immunity' threshold 66 with at least 4 to 5 positive peptide responses in each geographical region ( table 3 ). here we report a differentiated sars-cov-2 vaccine design based on memory t-cell induction technology. using sequence design through reverse vaccinology selection approach based on previous covs knowledge on immunodominant epitopes and computational immunology optimization, we developed a combination of 12 cd8 t cell synthetic peptides originating from 11 sars-cov-2 structural and non-structural proteins capable to cover hla polymorphism with high coverage globally and to induce immunogenicity to different proteins independently of hla alleles expression. these epitopes are naturally immunogenic after sars-cov-2 infection in recovered individuals and, for most of them, elicit a specialized sub-population of viral-specific memory cd8 t cells with a tissue-resident phenotype hence capable to migrate, stay attached and patrolling in airways barrier tissues. vaccine-induced t cell entry to the lung mucosal compartments [75] [76] [77] [78] [79] . here we showed that, as previously observed with our memopi ® -based neoepitope cancer vaccine approach, several peptides induced viral-specific cd8 t cells expressing the e (cd103) and α1 (cd49a) integrins and the cd44 memory marker, altogether characteristic of trm. these cells express also the cxcr3 marker of chemoattraction, which is also a surrogate marker of th1 cd8 t cells since cxcr3 is transactivate directly by the th1 master gene t-bet 79 animal housing and procedures have been conducted according to the guidelines of the french agriculture ministry and were approved by the regional ethical committee (apafis 25256. t-cell wt and mutated peptides binding property on hla-a2 has been evaluated using the flex-t hla-a*02:01 monomer ultraviolet (uv) exchange assay according to the manufacturer recommendation (biolegend, san diego, usa). hla-a*02:01 monomer (200 µg/ml) were exposed to a 366-nm uv lamp in the presence or absence of 400 µm of peptide. after uvexposure, hla-peptide complexes were incubated at 37°c for 30 min to promote unfolding of peptide-free hla molecule. hla-peptide complexes stability was detected by elisa with β2microglobulin coated antibodies and incubation of 3 ng/ml of complexes for 1h at room temperature under shaking condition. avidin-hrp were used to reveal stable biotinylated hla-peptide complexes and absorbance was monitored at 450 nm. data are expressed as percentage of binding relative to an memopi ® internal positive control neoepitope. a visualization tool was used to determine t-cell and b-cell epitope location in sars-cov-2 genomes according to single nucleotide polymorphism (snps) and homoplasic site (https://macman123.shinyapps.io/ugi-scov2-alignment-screen/) 85 all subjects were enrolled in the covepitwere not considered as an exclusion criterion. pbmc were isolated after a ficoll density-gradient centrifugation and a red blood cell lysis. hla-a2 phenotyping was performed by flow cytometry (clone bb7.2, bd bioscience). exvivo stimulation protocol was adapted from a previously described protocol 86 continuous variables were expressed as the mean ± sem, unless otherwise indicated, and raw data were compared with nonparametric tests: mann-whitney for 2 groups or kruskall-wallis with dunn's comparison when the number of groups was > 2. p values of <0.05 were considered statistically significant. all statistical analyses were performed on graphpad software (graphpad software, san diego, ca). this work was supported by funding from nantes metropole as part of the metropolitan fund to support health innovations linked to the covid-19 health crisis. we thank clinicians and patients involved in the covepit-1 trial as well as the exystat company for biometric expertise and data management for the covepit-1 trial. figure 1 : t-cell epitopes location in sars-cov-2 genome sars-cov-2 genome annotation by the krogran lab 52 and schematic representation of t-cell epitopes location in each encoded proteins. n=4 structural proteins; n=16 non-structural proteins (nsps); n=9 accessory factors. hla-a2 binding characterization of wt and mutated t-cell epitopes (a) wt and mutated peptides were incubated with hla-a*02:01 monomer, exposed to uv for peptide exchange and then hla-peptide complexes stability at 37°c was measured by elisa. data are mean +/-sem (n=4) expressed as percentage of binding relative to an internal memopi ® positive control neoepitope. (b) wt and mutated peptides (25µm) binding to tapdeficient human cell line (t2) expressing hla-a2. data are expressed as percentage of binding relative to an internal memopi ® positive control neoepitope. x mark indicates that the peptide was not tested in the assay. figure 4a ). medium and high response threshold were defined based on 2-fold or 3-fold increase respectively compared to the background frequency measured in the non-vaccinated mice control group. controls+ are memopi ® peptides with previously validated immunogenicity. data are mean +/-sd of pooled female (n=3) and pooled male (n=3) vaccinated mice. medium level: two-fold background. high level: three-fold background ifnγ secretion responses for 48 hours after one week of restimulation of human pbmc from unexposed hla-a2+ healthy donors (n=5), asymptomatic confirmed covid-19 hla-a2+ individuals (n=4) and moderate or severe covid-19 hla-a2+ convalescent patients (n=7) with each of the isolated peptides and hla-a2+ antigen-presenting cells. data were normalized to negative control peptides. data are expressed as mean +/-min to max. *p<0.05 table 1 : t-cell wt epitopes hla-i alleles numbers and regional hla-i coverage were determined using iedb public database and netmhcpan score < 1. duration of antibody responses after severe acute respiratory syndrome lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease longitudinal evaluation and decline of antibody responses in sars-cov-2 infection rapid decay of anti-sars-cov-2 antibodies in persons with mild covid-19 memory t cell responses targeting the sars coronavirus persist up to 11 years post-infection sars-cov-2-specific t cell immunity in cases of covid-19 and sars, and uninfected controls rapid 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in human skin potently neutralizing and protective human antibodies against sars-cov-2 respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine altered reactivity to measles virus atypical measles in children previously immunized with inactivated measles virus vaccines no evidence for increased transmissibility from recurrent mutations in sars-cov-2 spatially resolved analyses link genomic and immune diversity and reveal unfavorable neutrophil activation in melanoma key: cord-280172-6o1gqe8v authors: sanami, samira; zandi, milad; pourhossein, behzad; mobini, gholam-reza; safaei, mohsen; abed, atena; arvejeh, pooria mohammadi; chermahini, fatemeh amini; alizadeh, morteza title: design of a multi-epitope vaccine against sars-cov-2 using immunoinformatics approach date: 2020-07-15 journal: int j biol macromol doi: 10.1016/j.ijbiomac.2020.07.117 sha: doc_id: 280172 cord_uid: 6o1gqe8v severe acute respiratory syndrome coronavirus 2 (sars-cov-2) caused covid-19 disease in china. so far, no vaccine has licensed to protect against infection with covid-19, therefore an effective covid-19 vaccine needed. the aim of this study was to predict antigenic peptides of sars-cov-2 for designing the covid-19 vaccine using immunoinformatic analysis. in this study, t and b-cell epitopes of s protein were predicted and screened based on the antigenicity, toxicity, allergenicity, and cross-reactivity with human proteomes. the epitopes were joined by the appropriate linker. lt-iic as an adjuvant was attached to the end of the structure. the secondary and 3d structure of the vaccine was predicted. the refinement process was performed to improve the quality of the 3d model structure; the validation process is performed using the ramachandran plot and prosa z-score. the proposed vaccine's binding affinity to the hla-a11: 01 and hla-drb1_01: 01 molecule was evaluated by molecular docking. using molecular dynamics, the stability of vaccine-hla complexes was also evaluated. finally, in silico gene cloning was performed in the pet30a (+) vector. the findings suggest that the current vaccine may be a promising vaccine to prevent sars-cov-2 infection. in december 2019, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) caused covid-19 disease in china, which was associated with a cluster of respiratory infections [1] . infection with sars-cov-2 has spread to various countries and created a significant threat to public health. following the outbreak of sars-cov in 2002 and 2003 and mers-cov outbreak in 2012, the sars-cov-2 outbreak is a recurrence of the previous occurrence with new pressure, and the bat is highly probable to be the origin of all types of cov [2] . researchers have confirmed the transmission of this virus from human to human, which has led to a further outbreak of the disease [3] . coronaviruses are classified into four genera: alpha, beta, gamma, and delta coronavirus. alpha and bata coronaviruses infect bats and human [4, 5] , sars-cov-2 is a member of β coronavirus genera [6] . sars-cov-2 is a single strand, positive-sense rna virus with a genome of around 30 kb in length [7] . in all coronaviruses, the structural proteins comprise spike (s), envelope (e), membrane protein (m), and nucleoprotein (n) [8, 9] . sars-cov-2 uses the s protein as the key to binding and entering host cells therefore understanding its structure and function is important. the s protein is a type i transmembrane protein and highly glycosylated. spike glycoproteins assemble into trimers on the viral particle surface. s glycoprotein has three domains including ectodomain, transmembrane, and endodomain. ectodomain subdivided into s1 and s2 subunits, s1 is important for binding to host cell receptors, the amino acid sequences of s1 are more variable than s2 [10] . s protein is an attractive target for vaccine development for the following reasons: s protein is located at the virus surface and uses angiotensin-converting enzyme 2 receptor in respiratory epithelial cells to enter the host cells [11, 12] , it also can induce neutralizing antibodies [13] . the development and production of a vaccine are costly and will take many years to achieve this goal, the various strategies were used to reduce the time and costs of this process [14] . reverse vaccinology or vaccinomics is a new method that combines immunogenomics and bioinformatics to develop novel vaccines [15] . this method has many benefits over conventional vaccinology, it decreases the vaccine development time and costs and it also makes possible to study non-cultivable or risky microorganisms [16] . scientists are working very hard to combat this virus, but still, there is no drug or vaccine available to protect against infection with covid-19 [17] . thus, in this regard, we decided to design a new multiepitope vaccine against sars-cov-2 infection using reverse vaccinology approaches. in this research, first, the ctl, htl, and b-cell epitopes of the s protein were predicted using propred-1, propred, and abcpred servers, respectively, and then were selected base on antigenicity, toxicity, allergenicity, and cross-reactivity with human proteomes. the selected epitopes were joined together using the appropriate linker to construct the main core of the multi-epitope vaccine. since the multi-epitope vaccine has poor antigenicity, lt-iic as an adjuvant to overcome this weakness was attached to the end of the structure. lt-iic is a type ii subfamily of ltbs consisting of 121 amino acids. moreover, when coadministered with a multi-epitope vaccine it shows an increase in cd8+ t cell immune responses [18] . next, the physicochemical properties of the construct were investigated, the 3d structure of the protein was predicted, and finally, its affinity to the mhc i and ii molecules was investigated through docking, following that, was performed the molecular dynamics (md) simulation of docking complexes. finally, the vaccine coding gene expression in escherichia coli (e. coli) was evaluated, and in silico gene cloning was performed in the pet30a (+) vector. the amino acid sequences of sara-cov-2 s protein (qii57328.1) and lt-iic (h6w8f2) were obtained in fasta format from ncbi (https://www.ncbi.nlm.nih.gov/) and uniprot (https://www.uniprot.org/) databases, respectively. the binding epitopes to mhc i alleles were predicted using the propred-1 server (http://crdd.osdd.net/raghava/propred1/). propred-1 server is based on a method of predicting epitopes that bind to 47 different alleles of mhc i [19] . to predict htl epitopes was used the propred server (http://crdd.osdd.net/raghava/propred/) which uses quantitative matrices to predict t-cell epitopes binding to 51 different alleles of mhc ii [20] . in this study, peptide fragments with default parameters for mhc i and ii alleles with the highest coverage of iran's population were predicted. gamma interferon (ifn-γ) is a cytokine that plays a key role in both innate and adaptive immune response; ifnepitope server (https://webs.iiitd.edu.in/raghava/ifnepitope/predict.php) determined the ifn-γ-inducing htl epitopes. abcpred server (http://crdd.osdd.net/raghava/abcpred/) using an artificial neural network predicted the b-cell epitopes. this is the first server developed using fixed-length patterns based on a recurrent neural network. the server predicts peptides 10-20 mer in length and the threshold is among 0.1 to 1. in this study, the server was predicted peptide fragments of 16 mer length, and the threshold was set at 0.89 [21] . antigenicity, toxicity, and allergenicity were evaluated for predicted epitopes. the antigenicity of the epitopes were calculated by vaxijen v2.0 server (http://www.ddgpharmfac.net/vaxijen/vaxijen/vaxijen.html), which is based on the transformation of the protein sequences auto cross-covariance (acc) into uniform vectors of main amino acid properties. the performance accuracy of the vaxijen server varied from 70 to 89%, based on the selected model organism. in this study, the vaxijen v2.0 server determined the epitopes antigenicity based on a threshold of 0.4 and a virus model [22] [23] [24] . the epitopes toxicity were determined using the toxinpred server (http://crdd.osdd.net/raghava/toxinpred/). this server predicts of toxic or non-toxic peptides from a wide variety of peptides, along with their important physical and chemical properties such as hydrophobicity, hydropathicity, amphipathicity, molecular weight, and pi charge [25] . the allertop v. 2.0 server (https://www.ddg-pharmfac.net/allertop/index.html) predicted epitopic allergenicity. the approach used in this server is based on the automatic cross-covariance (acc) transformation of protein sequences into uniform vectors of equal length [26] . the pir peptide matching program (https://research.bioinformatics.udel.edu/peptidematch/index.jsp) was used to investigate the presence or absence of similarity with the human proteome in predicted epitope sequences [27] . the selected t and b-cell epitopes from the previous step were placed in the vaccine structure. kk and gpgpg linkers joined the screened t and b-cell epitopes in a structure of multi-epitope vaccine, respectively. the lt-iic amino acid sequence was attached to the merged epitopes at the n-terminus using the eaaak linker to boost the immunogenicity of the vaccine. an effective vaccine should not only cause strong immune responses but should also have appropriate physicochemical properties during the j o u r n a l p r e -p r o o f development cycle. protparam server (https://web.expasy.org/protparam/) calculated the amino acid content and some of the designed vaccine's physicochemical properties such as molecular weight, theoretical pi, half-life in the mammalian reticulocytes, instability index, aliphatic index, and grand average of hydropathy (gravy) [28] . a standard method for determining the molecular weight of a protein, given the number of amino acids that make it up, is to multiply that number by the average weight of an amino acid, which is 110da [29] . the isoelectric point is the ph at which amino acid or protein loses its charge and is, therefore, unable to move in a direct current electric field. knowing the theoretical pi of a protein can be very useful for selecting and optimizing the methods used for protein purification, including ion-exchange chromatography and isoelectric focusing electrophoresis [30] . the half-life is the time it takes for half of the protein to disappear inside the cell after synthesis [31] . the instability index of proteins indicates their stability in the test tube so that proteins with an instability index of less than 40 are predicted as stable proteins [32] . the aliphatic index of a protein is the relative volume of protein occupied by aliphatic side chains, this factor indicates the stability of the protein against heat [33] . the gravy is computed by dividing the sum of the hydropathic values of all amino acids by the number of residues in the sequence [34] . the gravy demonstrates the amphipathic nature of the proteins, the negative and positive values suggest the hydrophilic and hydrophobic nature of the designed vaccine, respectively [35] . solpro server (http://scratch.proteomics.ics.uci.edu/) predicted the protein tends to be soluble when overexpression in e. coli [36] . antigenpro server (http://scratch.proteomics.ics.uci.edu/) was used to estimate the antigenicity of the vaccine, the accuracy of the server was determined to be 76 % based on cross-validation experiments using the combined dataset [37] . allergenicity and toxicity of the vaccine were determined by allertop v. 2.0 and toxinpred servers, respectively. prabi server (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/npsa/npsa_gor4.html) used gor4 method to predict the secondary structure of the engineered vaccine. gor iv with a mean accuracy of 64.4% uses all possible pair frequencies within a 17 amino acid residues window [38] . 3dpro server (http://scratch.proteomics.ics.uci.edu/) predicted the tertiary structure of the multiepitope vaccine [36] . 3drefine server (http://sysbio.rnet.missouri.edu/3drefine/) improved the quality of the 3d model from the previous stage. 3drefine refining protocol includes a two-step process as follows: (i) optimization of the hydrogen bonding network and (ii) minimization of atomic energy by integrating physics with knowledge-based force fields [39] . model validation is an important step to detect possible errors in crude structures and to compare the quality of models before and after the refinement process [40] , for this purpose, rampage (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) and prosa (https://prosa.services.came.sbg.ac.at/prosa.php) servers were used in this study. ramachandran plot was generated using the rampage server. the most important application of the ramachandran plot is to predict the quality of different protein structures regarding experimental methods (x-ray crystallography, nmr, and cryo-em). acceptable and nonacceptable quality protein structures consisted of a collection of torsional angles in the allowed and disallowed regions, respectively [41] . the prosa j o u r n a l p r e -p r o o f server evaluated the overall quality of the protein models in the form of a z-score. when the predicted model's z-scores are outside the normal range of native proteins, it indicates an incorrect structure [42] . cluspro 2.0 server (https://cluspro.org) performed molecular docking between vaccine candidate and two of the most common binding alleles in the population of iran including hla-a11:01 with pdb entry of 1x7q, and hla-drb1_01:01 with pdb entry of 1aqd. cluspro can discriminate the hypothetical usercreated structures and run any server-compatible docking algorithms. additional functions allow the users to modify the structure, determine the attraction and repulsion, or identify pairwise distance restrictions [43] [44] [45] . md simulation was performed using gromacs 5.1.5 (groningen machine for chemical simulations) software to investigate the behavior of vaccine, hla-a11:01, and hla-drb1_01:01 molecules in docking structure [46] . the input structures were prepared using the amber ff99sb force field. the correct hydrogen status of histidines was established for all the histidines presented in the structure, and the disulfide bonds were defined for the protein. then, the surface charge of the structure was neutralized by adding several sodium and chlorine ions. the protein was inserted into a layer of tip3p water molecules at 10 angstroms using gmx solvate software. energy minimization was performed on structures with the1000-step by steepest descent method to eliminate van der waals interactions and to form the hydrogen bonds between water and complex molecules. in the next step, the system temperature gradually increased from 0 up to 300 k for 200 ps in constant volume and was then equilibrated in constant pressure at 1000 ps. md simulation was performed at 27°c (300 k) for 40 ns. non-bonding interactions with a distance of 10 angstroms were calculated using the pme method. to increase the computational speed, the shake algorithm was used to limit the bonds involved in the hydrogen atom. backtranslation of the amino acid sequence of the multi-epitope vaccine was conducted using the gene infinity server (http://www.geneinfinity.org/sms/sms_backtranslation.html). the codon adaptation index (cai) and gc content of optimized dna were assessed by the genscript server (https://www.genscript.com/tools/rare-codon-analysis). finally, the multi-epitope vaccine sequence was inserted into the pet-30a (+) vector by the snapgene tool. using the propred-1 server, which was set to default parameters, were predicted 15 ctl epitopes (9 mer) for human mhc-i alleles including hla-a*0201, hla-a*1101, hla-b*3501, hla-b*5101 and hla-b*5301 ( table 1 ). the propred server predicted 657 htl epitopes (9 mer) for the alleles of mhc-ii, namely, drb1*01:01, drb1*03:01, drb1*07:01, drb1*11:01, drb1*13:01 and drb1*15:01. next, 52 epitopes capable of binding to at least four types of mhc alleles were assessed using the ifnepitope server for their ability to induce ifn-γ, the result of this step suggested that 23 htl epitopes were capable of inducing ifn-γ (table 2 ). using the default parameters of the abcpred server were predicted j o u r n a l p r e -p r o o f 15 linear b-cell epitopes ( table 3) . the predicted ctl, htl, and b-cell epitopes were evaluated for overlapping, antigenicity, toxicity, allergenicity, and cross-reactivity with human proteomes. it is noteworthy that among the overlapped epitopes were selected epitopes that had a higher antigenicity and improved quality of the second and three-dimensional structure of the vaccine by being in the chimeric structure. finally, 4, 8, and 6 epitopes were selected from ctl, htl, and b-cell epitopes, respectively. the final vaccine structure consists of four domains: lt-iic, linear b-cell, ctl, and htl epitopes, which were placed in the vaccine construct by different linkers (fig. 1) . the protparam results revealed that the multi-epitope vaccine comprised of 380 amino acids. the vaccine's molecular weight was 41.65 kda and its theoretical pi value was 9.96. the half-life of the vaccine was 30 h in mammalian reticulocytes, more than 20 h in yeast, and more than 10 h in e. coli. the instability index, aliphatic index, and gravy were predicted 22.73, 91.08, and -0.035, respectively. the solpro server result showed the vaccine to be soluble. the antigenicity of the final sequence (including the adjuvant ) and the main sequence (without adjuvant) of vaccine were predicted using the antigenpro server to be 0.584792 and 0.512677, respectively. the vaccine sequence was predicted to be non-allergenic and non-toxic using allertop v. 2.0 and toxinpred servers, respectively. the prabi server predicted that 24.47, 25.26 and 50.26% of the total 380 amino acids of vaccine construct were alpha helix, extended strand, and random coil, respectively. vaccine primary 3d model was predicted using the 3dpro server. the 3drefine server performed the process of refining the protein model. five parameters, including 3drefine, gdt-ts, gdt-ha, rmsd, rwplus, and molprobity, ranked the optimized models. the higher gdt-ts, rmsd and gdt-ha scores, and the low scores of 3drefine, molprobity, and rwplus showed a higher quality model. based on these factors, refined model no. 5 was selected, which is indicated in the first row of table 4 , the raw 3d structure and refined model of the vaccine are depicted in fig. 2a and b . the validation process was performed using the ramachandran plot and the prosa z-score. ramachandran plot result revealed that in the primary model, 93.4%, 3.2%, and 3.4% of residues were located in favored, allowed, and outlier regions, respectively (fig. 3a) , while in the refined model, 93.1%, 5%, and 1.9% of residues were found j o u r n a l p r e -p r o o f in favored, allowed, and outlier regions, respectively (fig. 3b) . z-score of the initial model was calculated -0.82 (fig. 4a) , while the z-score of the refined model was estimated -1.08 (fig. 4b ). the interaction of the refined model of vaccine with hla-i and hla-ii molecules was investigated using the cluspro 2.0 server. proteins with pdb entry of 1x7q and 1aqd were identified separately as receptor and vaccine as a ligand to do molecular docking. the server provided 30 clusters from each complex and ranked the clusters based on the amount of energy. cluster no. 0.00 in the results of the interaction between the hla-a11:01 and hla-drb1_01:01 molecules with the multi-epitope vaccine showed the lowest energy weighted score of -1165.4 and-1279.1 kcal/mol, respectively ( fig. 5a and 6a ). the pdbsum server was used to obtain accurate information on the interaction between residues in docking complexes. a total of 41 vaccine residues coupled with 39 residues of chain a from hla-a11:01 molecule. a number of 18 hydrogen bonds were formed between the residues of the chain a from the hla-a11: 01 molecule and the residues of the vaccine, which included pro105-his113, glu53-lys250, glu53-his211, asn174-gln183, glu166-tyr109, arg169-tyr109, lys146-tyr361, asp39-lys259, glu58-lys27, lys176-gly376, lys68-asn112, lys68-ala379, lys68-leu377, arg163-ser18, asp106-asn98, arg75-ile367, arg75-ala368 and thr80-trp360 (fig. 5b ). in the vaccine-hla-drb1_01:01 complex 15 and 23 residues of the vaccine associated with 14 residues of the chain a and 24 residues of the chain b from the hla-drb1_01: 01 molecule, respectively. hydrogen bond of thr162-ser53 was established between the vaccine and chain a from hla-drb1 01:01 molecule and 14 hydrogen bonds, including glu176-lys4, glu128-lys4, gln92-ala34, gly84-arg149, his81-arg149, glu87-arg149, glu87-lys39, thr21-lys39, glu22-gln42, asn19-val41, asn82-gly145, asn82-thr141, thr77-thr141and gln70-arg137 were formed between the chain b from hla-drb1 01:01 molecule and vaccine (fig. 6b ). the best molecular docking complexes were used as inputs to md simulation. at this stage, md simulation of both complexes was performed for 40 ns and the results were evaluated as root mean square deviation (rmsd), root mean square fluctuation (rmsf), and radius of gyration (rg). rmsd between the created structures during md simulation is an appropriate parameter to ensure the stability of the vaccine-hla complexes. rmsd of the hla-a11:01 molecule in the vaccine -hla-a11:01 complex reached 0.2 nm after approximately 1000 ps and showed a slight variation around this amount until the end of the md simulation. the rmsd of the vaccine showed a significant increase from the beginning of the md simulation until 3500 ps, after that, there was a downward trend up to 5,000, from this point to until 36000 ps the trend was approximately increasing (fig. 7a) . the profile of rmsd changes of hla-drb1_01: 01 molecule in the vaccine -hla-drb1_01:01 complex was similar to hla-a11: 01, while the rate of rmsd changes in the vaccine was relatively different. at the beginning of the md simulation, the value of rmsd showed a sharp increase until 20000 ps which reached 2 nm, of this point until the end of the md simulation was observed no significant changes. (fig. 7b) . rmsf determined the flexibility of any residue in the vaccine-hla complexes. rmsf result showed that the rmsf of most amino acids in the chain a of the hla-a11: 01 molecule was high and the maximum and minimum rmsf values were 0.17 and 0.05, respectively (fig. 8a) . two regions on the chain b of the molecule showed relatively high fluctuations, which included regions 12-21 and 70-78. in the range of j o u r n a l p r e -p r o o f 12-21, the rmsf reached 0.23 nm (fig. 8b) . the study of vaccine structure in this complex showed high rmsf in most of its amino acids. the two regions of the vaccine showed very high levels of rmsf of 0.57 and 0.5 nm, which included amino acids in regions 65-80 and 300-320, respectively (fig. 8c ). most parts in chain a of hla-drb1_01: 01 molecule in the vaccine -hla-drb1_01: 01 complex including amino acids in areas 32-38, 48-51, 92-100, 125-130, 154-157 and 167-171 showed high fluctuations (fig. 9a) . in chain b, the lowest and highest value of rmsf was 0.05 and 0.15 nm, respectively (fig. 9b) . the vaccine showed very high fluctuations in most areas and had a maximum rmsf of 0.55 nm (fig. 9c) . rg for the hla-a11: 01 molecule in the vaccine -hla-a11:01 complex was 2.35 nm at the beginning of the simulation and showed no change from the beginning of the simulation to the end (fig. 10a) . while rg of the hla-drb1_01: 01 molecule in the vaccine -hla-drb1_01: 01 complex was 2.4 nm at the beginning of the md simulation and had a slight increase up to 1000 ps, then remained constant until the end of the md simulation (fig. 10b) . in the presence of the hla-a11: 01 molecule, the rg value of the vaccine was 3.7 at the beginning of the simulation, and during the simulation period, a slight increase and decrease were observed in this parameter, and at the end of the simulation time, the rg value reached 3.5 (fig. 10a) . the rg level of the vaccine in the presence of the hla-drb1_01: 01 molecule showed a relatively significant decrease from the beginning of the simulation to 17500 ps then remained constant from that point until the end of the simulation period (fig. 10b ). the gene infinity server conducted backtranslation of the vaccine construct. genscript tool was used to evaluating the key properties of the gene sequence including the codon adaptation index (cai) and gc content. the optimized nucleotide sequence cai value was 1 and the gc content of the optimized sequence was 55.47% in e. coli. recognition sites for bamhi and xhoi restriction enzymes were added to 5′ and 3′ end of the optimized gene, respectively. the adapted codon sequence was inserted into the pet30a (+) vector using snapgene software (fig. 11 ). the covid-19 outbreak demonstrates a pandemic threat to global public health [47] . the sars-cov-2 can be transmitted from person to person, even when the infected individual has no clinical symptoms, and sometimes cannot be detected in the infected individual for several days or even weeks. there are cases where a person has clinical symptoms of covid-19, but the diagnosis test is negative, so the best way to prevent coronavirus from occurring is to get a vaccine [48] , but unfortunately, because of the novelty of the virus, there is still no licensed vaccine for the disease. however, researchers and pharmacists around the world are working at an unprecedented pace to develop an effective virus vaccine, and clinical trials of several candidate vaccines have begun, including mrna-1273, ino-4800, and chadox1 ncov-19 [49] . reverse vaccinology technology has been widely used to determine the epitopes for the development of multi-epitope vaccines in various organisms. in recent months, several reverse vaccinology studies have also been conducted to design the covid-19 vaccine. in a study conducted by enayatkhani et al. the b-cell and t epitopes of n, orf3a, and m proteins were predicted, a multi-epitope vaccine candidate was constructed, then were performed bioinformatics evaluations [50] . peele et al. designed a vaccine candidate using b-cell and t epitopes of s protein by in silico methods [51] . kalita et al. selected n, m, and s proteins as the target antigen for the prediction of t and b-cell epitopes and designed a multi-epitope vaccine against sars-cov-2 [48] . guglielmo lucchese predicted s j o u r n a l p r e -p r o o f protein antigenic peptides from sars-cov-2 using immunoinformatics methods [52] . ahmed et al. predicted a collection of b cell and t cell epitopes derived from the s and n proteins of sars-cov-2 [53] . in each of the studies mentioned above, different proteins have been used to predict epitopes, also, the type of bioinformatics softwares, linkers, and many other factors are different in each study, therefore the laboratory evaluations of each of these designed vaccines show different results. this work focussed on the in silico design of a potential multi-epitope vaccine for covid-19 using s protein. it is important to identify b-cell and t epitopes of the target antigens in order to develop a multi-epitope vaccine. in the current study, we predicted a large number of ctl, ifn-γ-inducing htl, and b-cell epitopes for s protein then selected 18 high-ranked epitopes based on antigenicity, toxicity, allergenicity, and cross-reactivity with human proteomes. linkers play a dual role in the multi-epitope vaccine structure : (i) the prevention of the generation of junctional epitopes (neoepitopes), which is the major concern in designing multi-epitope vaccines, and (ii) promotion of the immune processing and presentation of hla-ii binding epitopes [40, 54] . to design the vaccine construct, the selected t and b-cell epitopes were fused using the kk and gpgpg linkers, respectively. lt-iic was attached to the n-terminal of the multiepitope sequence by an eaaak linker. the designed vaccine was evaluated for its physicochemical features, antigenicity, allergenicity, and toxicity .the vaccine's molecular weight was 41.65 kda, which makes it an acceptable vaccine since proteins with a molecular weight of less than 110 kda are considered to be more appropriate targets for the development of vaccines, as proteins with a lower molecular weight can be easily purified [29] , the vaccine construct was basic in nature and the total numbers of negatively and positively charged residues were 16 and 48, respectively. the vaccine structure has a half-life of 30, 20, and 10 h in mammalian, yeast, and e. coli, respectively, therefore the vaccine is exposed to the immune system for a relatively long time and causes more immune responses [28] . the recombinant vaccine's instability index was calculated at 22.73, and as it was less than 40, it was considered to be a stable protein [28] . the calculated aliphatic index and the gravy were 91.08 and -0.035, respectively. the higher aliphatic index value suggests greater thermostability and the negative gravy value shows the vaccine's hydrophilic nature therefore it can show strong interaction with water molecules [55] . the sequences of vaccine (with and without adjuvant) were both antigenic, and adding the adjuvant to the n-terminal of the vaccine sequence increased the vaccine's antigenicity. results also suggested that the vaccine was soluble, non-allergenic, and non-toxic. to obtain a high-quality 3d structure, the initial 3d structure was subjected to the refinement process. the quality of the initial and refined models were assigned using ramachandran plot and prosa z-score. ramachandran plot data showed that 3.4% of residues of the initial model was located in the outlier region while this value was reduced to 1.9% after the refinement process. in addition, the z-score of primary model was calculated -0.82, while this parameter was determined -1.08 after refining. these results suggest that the three-dimensional structure of the vaccine has improved after refinement. cluspro server conducted the docking process between the multi-epitope vaccine with hla-a11:01 and hla-drb1_01:01 molecules, the value of lowest energy in the selected complexes of vaccine-hla-a11: 01 and vaccine-hla-drb1_01: 01 were -1165.4 and -1279.1, respectively, which indicated that the interaction of the vaccine with the hla-drb1_01:01 to be stronger than its interaction with the hla-a11:01. the results of the rmsd evaluation showed that the vaccine -hla-drb1_01:01 complex reached stability at 20 ns times, while the vaccine -hla-a11:01 complex required more time to stabilize. the rmsf result suggested that the flexibility in the regions between vaccine-hla in the both complex and the results of rg showed that the vaccine structure in both complexes was densified during simulation, but gained more density in the vaccine-hla-drb1_01: 01 complex. in practice, some essential factors including cai and gc content of the gene need to be optimized to achieve an optimal j o u r n a l p r e -p r o o f level protein expression in e. coli. according to the results, the value of the cai was 1, the cai value was greater than 0.8, which suggests a higher probability of protein expression at a high level [56] . gc content was 55.47%, and the ideal percentage of gc content ranges from 30 to 70%. the results of bioinformatics evaluation of the vaccine showed that this vaccine candidate may have a high potency against sars-cov-2 but the in vitro and in vivo studies are needed to confirm these results. the purpose of this research was to develop a covid-19 vaccine using bioinformatics approaches. to achieve this aim were predicted t and b-cell epitopes of s protein and then screened for antigenicity, toxicity, allergenicity, and cross-reactivity with human proteomes. the eighteen high-ranked epitopes and lt-iic were incorporated into the final construct. after evaluating the physicochemical properties of the vaccine construct, its secondary and three-dimensional structure was predicted. structural quality, binding affinity to mhc-i and ii, and stability of the docked complexes were assessed. finally, the expression level of the vaccine in e. coli strain k12 was evaluated. significant results were reported in this research, experimental confirmation of the engineered vaccine required to further assess the efficacy of this vaccine candidate. j o u r n a l p r e -p r o o f clinical features of patients infected with 2019 novel coronavirus in the 2019 novel coronavirus disease (covid-19) pandemic: a zoonotic prospective sars-cov-2 causing pneumoniaassociated respiratory disorder (covid-19): diagnostic and proposed therapeutic options host factors in coronavirus replication, roles of host gene and non-coding rna expression in virus infection discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus a novel coronavirus from patients with pneumonia in china covid-19: a new challenge for human beings the genome sequence of the sars-associated coronavirus unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage mechanisms of coronavirus cell entry mediated by the viral spike protein cryo-em structure of the 2019-ncov spike in the prefusion conformation consider tlr5 for new therapeutic development against covid-19 identification of an antigenic determinant on the s2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies history of vaccination application of pharmacogenomics to vaccines reverse vaccinology probable molecular mechanism of remdesivir for the treatment of covid-19: need to know more lt-iic, a new member of the type ii heat-labile enterotoxin family encoded by an escherichia coli strain obtained from a nonmammalian host propred1: prediction of promiscuous mhc class-i binding sites propred: prediction of hla-dr binding sites prediction of continuous b-cell epitopes in an antigen using recurrent neural network vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines identifying candidate subunit vaccines using an alignmentindependent method based on principal amino acid properties bioinformatic approach for identifying parasite and fungal candidate subunit vaccines in silico approach for predicting toxicity of peptides and proteins dna and peptide sequences and chemical processes multivariately modelled by principal component analysis and partial least-squares projections to latent structures a fast peptide match service for uniprot knowledgebase protein identification and analysis tools on the expasy server exoproteome and secretome derived broad spectrum novel drug and vaccine candidates in vibrio cholerae targeted by piper betel derived compounds isoelectric point separations of peptides and proteins how are substrates recognized by the ubiquitin-mediated proteolytic system correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence thermostability and aliphatic index of globular proteins a simple method for displaying the hydropathic character of a protein development of multi-epitope driven subunit vaccine against fasciola gigantica using immunoinformatics approach scratch: a protein structure and structural feature prediction server high-throughput prediction of protein antigenicity using protein microarray data gor secondary structure prediction method version iv meshi: a new library of java classes for molecular modeling designing an efficient multiepitope peptide vaccine against vibrio cholerae via combined immunoinformatics and protein interaction based approaches prosa-web: interactive web service for the recognition of errors in three-dimensional structures of proteins the cluspro web server for protein-protein docking new additions to the c lus p ro server motivated by capri how good is automated protein docking? gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers sars-cov-2: an emerging coronavirus that causes a global threat design of a peptide-based subunit vaccine against novel coronavirus sars-cov-2 a review of sars-cov-2 and the ongoing clinical trials reverse vaccinology approach to design a novel multi-epitope vaccine candidate against covid-19: an in silico study design of multi-epitope vaccine candidate against sars-cov-2: a in-silico study epitopes for a 2019-ncov vaccine preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies a novel design of a multi-antigenic, multistage and multi-epitope vaccine against helicobacter pylori: an in silico approach exploring dengue genome to construct a multi-epitope based subunit vaccine by utilizing immunoinformatics approach to battle against dengue infection codon adaptation index as a measure of dominating codon bias we would like to thank dr. shahram tahmasebian and dr. sorayya ghasemi for their valuable feedback and suggestions. the authors have declared no competing interest. none table 1 . results of ctl epitope prediction. four ctl epitopes were selected (labeled with *) for incorporation in the proposed vaccine amino acid sequence. key: cord-286968-ud1uerc8 authors: nienhold, r.; ciani, y.; koelzer, v. h.; tzankov, a.; haslbauer, j. d.; menter, t.; schwab, n.; henkel, m.; frank, a.; zsikla, v.; willi, n.; kempf, w.; hoyler, t.; barbareschi, m.; moch, h.; tolnay, m.; cathomas, g.; demichelis, f.; junt, t.; mertz, k. d. title: two distinct immunopathological profiles in lungs of lethal covid-19 date: 2020-06-19 journal: nan doi: 10.1101/2020.06.17.20133637 sha: doc_id: 286968 cord_uid: ud1uerc8 immune responses in lungs of coronavirus disease 2019 (covid-19) are poorly characterized. we conducted transcriptomic, histologic and cellular profiling of post mortem covid-19 and normal lung tissues. two distinct immunopathological reaction patterns were identified. one pattern showed high expression of interferon stimulated genes (isgs) and cytokines, high viral loads and limited pulmonary damage, the other pattern showed severely damaged lungs, low isgs, low viral loads and abundant immune infiltrates. distinct patterns of pulmonary covid-19 immune responses correlated to hospitalization time and may guide treatment and vaccination approaches. isg low samples, and observed a higher frequency of cd8+pd1+ t-cells in the isg low subgroup, potentially indicative of advanced disease (extended data figure 3a,b) . histological analysis of covid-19 lung tissues revealed striking pulmonary damage exclusively in isg low samples, with distinct peri-alveolar foci of infiltrating cd68+ macrophages and cd8+ t cells (figure 1e) . expression of isgs was tightly correlated with pulmonary viral load (figure 2a) , and immunohistochemical staining showed sars-cov-2 nucleocapsid protein mostly in pneumocytes of isg high lungs (figure 2b) . since a cytokine storm has been proposed to cause adverse outcome of covid-19 7 , and cytokines were highly expressed in bronchoalveolar lavages (bals) of covid-19 patients 8 , we investigated expression of a pro-inflammatory cytokine signature (tnf, il6, il1b, ccl2, ifna17, ifnb1, cxcl9, cxcl10, cxcl11) in lung samples from lethal covid-19. the proinflammatory gene signature was significantly enriched in the isg high subset (figure 2c) , but was not associated with alveolar hemorrhage (figure 2d ). within this cytokine signature, co-regulated subgroups (il1b/il6/tnf, ifnb1/ifna17, ccl2/cxcl9/cxcl19/cxcl11) were identified ( figure 2e ). importantly, only the cxcl9/10/11 sub-signature was positively associated with alveolar hemorrhage (figure 2f , extended data figure 4 ). this is in line with observations that these chemokines compromise endothelial integrity via cxcr3 9 , and that cxcl10 is a key determinant of severe covid-19 10 . interestingly, basal levels of cxcl9 or cxcl10 are elevated in elderly, hypertensive and obese individuals, which were strongly represented in our autopsy cohort and are predisposed to severe covid-19 11, 12 . of note, our study could not take extrapulmonary cytokine sources or effects into account. since none of the above pulmonary cytokine sub-signatures was positively associated with diffuse alveolar damage (dad, extended data figure 5) , we investigated which other local immune signature showed this association. we found a strong association of dad with low expression of isgs (figure 2g) , and an activated cd8+ t cell signature (cd38, gzma, gzmb, ccr5, figure 2h ), yet not with pulmonary cd8+ t-cell infiltration (figure 2i ). in addition, the activated cd8+ signature was inversely correlated to viral counts, particularly in isg low cases (figure 2j ). therefore we speculate that activated cd8+ t cells are essential for virus elimination, similar to murine models of coronavirus infection 13 , yet it is possible that they contribute to pulmonary damage as well. of note, isg low samples also expressed elevated p53 and ki67 (figure 2k) , i.e. reactive markers of dad which indicate lung remodeling 14 . since the isg low pattern showed lower viral counts, higher accumulation and activation of cd8+ t cells in tissues and accrual of pulmonary damage and remodeling, the isg low phase may follow an earlier isg high phase during the course of infection. this was supported by significantly 4 instead show low viral loads yet strong complement activation in lungs (figure 2m ) and thus may potentially benefit from complement inhibition 16 . in addition, the isg low pattern suggests that cd8+ t cells are involved in antiviral protection and should be considered for vaccination efforts. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. table 1a . detailed autopsy findings for each patient were recently published, and the identifiers (with the prefix "c") for each covid-19 patient are consistent with the description of this swiss covid-19 autopsy cohort 3 . in this study, we analysed formalin fixed and paraffin embedded (ffpe) lung tissue of distinct areas of the lungs of 16 of these covid-19 patients. all 16 covid-19 patients had positive nasopharyngeal swabs collected while alive. in all covid-19 patients, diagnosis was confirmed by detection of sars-cov-2 in postmortal lung tissues. 5/16 patients were additionally tested by postmortal nasopharyngeal swabs which were positive for sars-cov-2 in all 5 cases. as a control cohort, we selected 6 autopsies performed between january 2019 and march 2020 at the institute of pathology liestal ("normal" patients n1 -n6). these control patients died of other, non-infectious causes and had a similar age, gender and cardiovascular risk profile. patients with infections were excluded from this control cohort. another control cohort consisted of 4 autopsies of patients suffering from various infections mainly with bacteria affecting the lung (patients with lung pathology, p1 -p4). details for both control cohorts are listed in extended data (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. quantification of sars-cov-2 in ffpe tissue samples the oirra is a targeted gene expression assay designed for the ion™ next-generation sequencing (ngs) platform. this gene expression assay was originally designed to interrogate the tumor microenvironment to enable mechanistic studies and identification of predictive biomarkers for immunotherapy in cancer. the assay is optimized to measure the expression of genes involved in immune cell interactions and signaling, including genes expressed at low levels and involved in inflammatory signaling. the 398 genes covered by this assay are listed in extended data table 2 . the ngs libraries were prepared as recommended by the supplier. in brief, 30ng of total rna were (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 19, 2020. immunohistochemical analyses for cd3, cd4, cd8, cd15, cd20, cd68, cd123, cd163, pd-1, mpo, p53, ki67, c3d and c5b-9 were performed on all lung tissue blocks used in this study. antibodies, staining protocols and conditions are detailed in extended data table 4 . qualitative and semiquantitative assessment of histopathological lung damage and neutrophilic infiltration hematoxylin and eosin (h&e) and elastica van gieson (evg) stained sections of all lung tissues used in this study were independently evaluated by two experienced and board certified pathologists (vz and kdm) (extended data table 5 ). both pathologists evaluated the presence of diffuse alveolar damage (dad), and if present, its stage, intra-alveolar edema and hemorrhage. in addition, both all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 19, 2020. . https://doi.org/10.1101/2020.06.17.20133637 doi: medrxiv preprint pathologists evaluated the severity of histopathological changes in covid-19 lungs (1 = mild / discrete alterations, 2 = moderate, 3 = severe changes) based on resemblance between normal and pathologically altered lung tissues. parameters that were taken into account included reduction of alveolar air-filled spaces, typical histologic features of dad with hyaline membrane formation, infiltration of lymphocytes, monocytes and neutrophils into interstitial and alveolar spaces, type 2 pneumocyte hyperplasia, desquamation of pneumocytes, histologic features of organizing pneumonia including intra-alveolar fibrin deposition and fibrosis (acute fibrinous and organizing pneumonia, afop) 17, 18 for cell-level analysis, color deconvolution for dab, ap and hematoxylin channels was performed and nuclear segmentation was optimized using cell-morphometric parameters. marker-positive cells in stromal and epithelial regions were quantified. for cd3, cd4, cd8, cd20, cd68, cd123, cd163 and pd1, staining detection was optimized for the cytoplasmic / membranous compartment and marker expression was measured on a continuous scale at single cell resolution. for assessment of cd8/pd1 double stains, color deconvolution was optimized for separation of dab (pd1) and ap (cd8) staining products. internal controls (non-immune cells) and external controls (tonsil) were used to calibrate the detection limits and cross-validated by visual review. for each tissue sample, the total area of lung tissue in mm 2 , the absolute number of marker-positive cells, cell morphometric parameters and staining intensity were recorded. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 19, 2020. expression of gene signatures was calculated as median of log2(cpm + 1) of selected genes. biological processes enrichment was performed using the enrichgo function of the package clusterprofiler 20 setting all the genes included in the assay as universe. all the analyses and graphical representations were performed using the r statistical environment software 21 and the following packages: ggplot2 22 , circlize 23 , complexheatmap 24 , ggfortify 25 , reshape2 26 and factoextra 27 . correlation between transcripts and viral counts was performed using pearson's correlation. association between continuous and categorical data were tested using wilcoxon rank sum test. box-plots elements indicate the median (center line), upper and lower quartiles (box limits) and show all the data points. whiskers extend to the most extreme value included in 1.5x interquartile range. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 19, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 19, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 19, 2020. . the datasets generated and analysed during this study can be accessed in geo (gse151764) and are available from the corresponding author upon request. competing interests vhk has served as an invited speaker on behalf of indica labs. th and tj are employees of novartis. the other authors declare no competing interests. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. patients were suffering from other infections of the lung (bacterial or viral pneumonia). detailed analysis of individual pathogens is shown in extended data figure 1 . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 19, 2020. . https://doi.org/10.1101/2020.06.17.20133637 doi: medrxiv preprint extended data table 2. oirra gene list abcf1 ccl5 cd40lg ctag2 faslg hif1a ifit1 il3ra adgre5 ccnb2 cd44 ctla4 fcer1g hla-a ifit2 il4 adora2a ccr1 cd47 ctss fcgr1a hla-b ifit3 il6 aif1 ccr2 cd48 cx3cl1 fcgr2b hla-c ifitm1 il7 akt1 ccr4 cd52 cx3cr1 fcgr3a hla-dma ifitm2 il7r alox15b ccr5 cd53 cx3cr1 fcgr3b hla-dmb ifna17 irf1 arg1 ccr6 cd6 cx3cr1 fcrla hla-doa ifnb1 irf4 axl ccr7 cd63 cx3cr1 foxm1 hla-dob ifng irf9 b3gat1 cd14 cd68 cxcl1 foxo1 hla-dpa1 igf1r irs1 bage cd160 cd69 cxcl10 foxp3 hla-dpb1 igsf6 isg15 batf cd163 cd70 cxcl11 fut4 hla-dqa1 ikzf1 isg20 bcl2 cd19 cd74 cxcl13 fyb hla-dqa2 ikzf2 itga1 bcl2l11 cd1c cd79a cxcl8 g6pd hla-dqb2 ikzf3 itgae bcl6 cd1d cd79b cxcl9 gadd45gip1 hla-dra ikzf4 itgal brca1 cd2 cd80 cxcr2 gage1 hla-drb1 il10 itgam brca2 cd209 cd83 cxcr3 gage10 hla-e il10ra itgax bst2 cd22 cd86 cxcr4 gage12j hla-f il12a itgb1 btla cd226 cd8a cxcr5 gage13 hla-f-as1 il12b itgb2 bub1 cd244 cd8b cxcr6 gage2c hla-g il13 itgb7 c10orf54 cd247 cdk1 cybb gata3 hmbs il15 itk c1qa cd27 cdkn2a ddx58 gbp1 icam1 il17a jaml c1qb cd274 cdkn3 dgat2 gnly icos il17f jchain ca4 cd276 ceacam1 dmbt1 gpr18 icoslg il18 kiaa0101 cblb cd28 ceacam8 ebi3 grap2 id2 il1a kir2dl1 ccl17 cd33 ciita efna4 gusb id3 il1b kir2dl2 ccl18 cd37 clec4c egfr gzma ido1 il2 kir2dl3 ccl2 cd38 cmklr1 egr2 gzmb ido2 il21 klf2 ccl20 cd3d coro1a egr3 gzmh ifi27 il22 klrb1 ccl21 cd3e crtam eif2ak2 gzmk ifi35 il23a klrd1 ccl22 cd3g csf1r entpd1 havcr2 ifi44l il2ra klrf1 ccl3 cd4 csf2rb eomes herc6 ifi6 il2rb klrg1 ccl4 cd40 ctag1b fas hgf ifih1 il2rg klrk1 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 19, 2020. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 19, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 19, 2020. at least two different tissue blocks from different areas of the lungs were evaluated for each case. 1 1 = slight to moderate changes; 2 = moderate changes; 3 = severe changes 2 1 = exudative; 2 = proliferating/organizing; 3 = fibrotic 3 1 = yes; 0 = no 4 1 = very few or few; 2 = moderate; 3 = numerous all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 19, 2020. . https://doi.org/10.1101/2020.06.17.20133637 doi: medrxiv preprint corona virus resource center clinical and immunological features of severe and moderate coronavirus references 17 time to consider histologic pattern of lung injury to treat critically ill patients with covid-19 infection pathological findings of covid-19 associated with acute respiratory distress syndrome fitting linear mixed-effects models using lme4 hundreds of genes experienced convergent shifts in selective pressure in marine mammals r: a language and environment for statistical computing ggplot2: elegant graphics for data analysis circlize implements and enhances circular visualization in r complex heatmaps reveal patterns and correlations in multidimensional genomic data ggfortify: data visualization tools for statistical analysis results reshaping data with the reshape package package 'factoextra'. extract and visualize the results of multivariate data analyses key: cord-018595-x3tleomb authors: dodiuk-gad, roni p.; chung, wen-hung; shear, neil h. title: adverse medication reactions date: 2017-04-25 journal: clinical and basic immunodermatology doi: 10.1007/978-3-319-29785-9_25 sha: doc_id: 18595 cord_uid: x3tleomb cutaneous adverse drug reactions (adrs) are among the most frequent adverse reactions in patients receiving drug therapy. they have a broad spectrum of clinical manifestations, are caused by various drugs, and result from different pathophysiological mechanisms. hence, their diagnosis and management is challenging. severe cutaneous adrs comprise a group of diseases with major morbidity and mortality, reaching 30 % mortality rate in cases of toxic epidermal necrolysis. this chapter covers the terminology, epidemiology, pathogenesis and classification of cutaneous adr, describes the severe cutaneous adrs and the clinical and laboratory approach to the patient with cutaneous adr and presents the translation of laboratory-based discoveries on the genetic predisposition and pathogenesis of cutaneous adrs to clinical management guidelines. the world health organization defined an adverse drug reaction (adr) in 1972 as "a response to a drug that is noxious and unintended and occurs at doses normally used in man" [1] . edwards and aronson [2] proposed a different definition in 2000: "an appreciably harmful or unpleasant reaction, resulting from an intervention related to the use of a medicinal product, which predicts hazard from future administration and warrants prevention or specific treatment, or alteration of the dosage regimen, or withdrawal of the product." the terms 'adverse reaction' and 'adverse effect' are interchangeable, except that an adverse reaction is seen from the point of view of the patient and adverse effect is seen from the point of view of the drug. however, both terms must be distinguished from 'adverse event'. an adverse event is an adverse outcome that occurs while a patient is taking a drug, but is not or not necessarily attributable to it [2] . differentiating between serious adr and severe adr is imperative. serious adr is a legal term applied to any untoward medical occurrence that at any dose results in death, is life-threatening, requires inpatient hospitalization or prolongation of existing hospitalization, results in persistent or significant disability/incapacity, or is a congenital anomaly/birth defect [3] . conversely, the term 'severe' is a clinical term used to describe the intensity (severity) of a medical event, as in the grading 'mild', 'moderate', and 'severe'; thus, a severe skin reaction need not be serious [2] . adrs are associated with significant morbidity and mortality and have considerable economic implications. clinical manifestations of an adr are variable and may include cutaneous and or systemic features [4] . when analyzing the type of adrs most encountered, two major groups emerge; common-mild reactions and raresevere reactions. common-severe reactions are not approved for clinical usage and rare-mild reactions are usually not noticed or reported. cutaneous adrs are among the most frequent adverse reactions in patients receiving drug therapy [5] . they accounted for 65 % of all reported adrs in a 4-year retrospective study in taiwan [6] . the prevalence and incidence of cutaneous adrs vary greatly among different populations [7] [8] [9] [10] [11] . in the usa, a 7-year prospective study found that the prevalence of cutaneous adrs was 2.2 % in hospitalized patients [7] ; and an 11-year retrospective study found the annual incidence of cutaneous adrs to be 2.26 per 1,000 persons [11] . in denmark, in a 1-year cross-sectional study the prevalence of cutaneous adrs was 0.33 % in in-patients and 0.14 % in outpatients [8] . in southern china, in an 8-year retrospective study, the prevalence of cutaneous adrs was 0.14 % in hospitalized patients [9] . in india, in a 12-month prospective study, the primary incidence of cutaneous adrs was 2.05 per 1,000 persons [10] . the need to survey adrs in clinical practice is universally recognized. various methods may be employed: spontaneous surveillance, prescription-event monitoring (pem), linkage analysis, case-control surveillance and cohort studies [5] . in 1963, the 16th world health assembly reaffirmed the need for early detection and rapid dissemination of information on adverse reactions due to medications. this affirmation led to the creation of the world health organization (who) programme for international drug monitoring, under whose auspices systems have been created in member states for the collection and evaluation of individual case safety reports (icsrs) [12] . in 1978 the who set up its international drug monitoring programme in sweden at the uppsala monitoring centre (umc) http://www.who-umc. org. the us food and drug administration (fda) provides several options for reporting adverse events. one such option is medwatch, the fda safety information and adverse event reporting program http://www.fda.gov/safety/ medwatch/default.htm, founded in 1993 as a system for both consumers and healthcare professionals to report adverse events. medwatch is intended to detect safety hazard signals for medical products; in the event a signal is detected, the fda can issue medical product safety alerts or order product recalls, withdrawals, or labelling changes to protect the public health [13] . a number of international research groups are investigating severe cutaneous adrs (scars): the regiscar network, an international registry of scar established in 2003, the japanese research committee, j-scar, the asian scar consisting of japan and taiwan scar groups (j-scar and t-scar) established in 2010, and the southeast asia network, sea-scar, with ten member countries: brunei, cambodia, indonesia, laos, malaysia, myanmar, philippines, thailand, singapore, and vietnam. the international serious adverse event consortium (isaec), a non-profit organization formed in 2007, is a pharmaceutical industry-and fda-led international consortium that focuses on identifying and validating dna variants useful in predicting the risk of rare drug-induced serious adverse events [14] . mechanisms of adverse drug reactions (adrs) can be classified into immunologic and non-immunologic etiologies. there are two common types of immune-mediated drug reactions: immediate-type hypersensitivity (type i hypersensitivity) and delayed-type hypersensitivity (type iv hypersensitivity). 1. immediate-type drug hypersensitivity: immediate-type drug hypersensitivity reactions usually occur minutes to hours after drug exposure, with clinical manifestations including pruritus, urticaria, angioedema, and bronchospasm to anaphylaxis. the reaction is mediated mainly by drug-specific ige, the most common causative agents being penicillins, cephalosporins and neuromuscular blocking agents. ige-mediated reactions to drugs are usually thought to be an immune response to a hapten/carrier complex. in the primary drug sensitization, drug-specific ige is formed when plasma cells transformed from activated b cells interact with t cells. in an allergic reaction, drug allergens bind to mast cells with high-affinity fc receptor, to which drug-specific ige is bound, causing mast cells to release mediators, such as histamine, leukotrienes, prostaglandins and cytokines [15] . 2. delayed-type drug hypersensitivity: delayed-type drug hypersensitivity reactions usually take several days to weeks following drug exposure, with variable clinical presentations that may include maculopapular eruption (mpe), fixed drug eruption (fde), acute generalized exanthematous pustulosis (agep), stevens-johnson syndrome (sjs), toxic epidermal necrolysis (ten) and drug reaction with eosinophilia and systemic symptoms (dress). t cell receptor (tcr), cd4+ and cd8+ t cells are involved in different delayed-type drug hypersensitivity reactions [16] . drugs are low molecular weight and usually considered not able to bind to tcrs to activate adaptive immunity. in the case of drug allergy, drug interactions with tcrs may involve a drug-peptide complex presented by human leucocyte antigen (hla) molecules of antigen-presenting cells (apcs). this process is known as the hapten concept; an example is β-lactams that covalently bind to lysine residues [17] . drugs can also interact directly with tcrs without binding to the peptide/hla of the apc in what is known as the p-i concept (pharmacological interaction of drugs with immune receptors) [18] . for example, carbamazepine is not able to bind covalently to peptides or proteins, but can associate with low affinity to tcrs and provoke t cell activation [19] . the immunohistologic characteristics of delayed type drug hypersensitivity are summarized in table 25 .1. the skin of mpe is infiltrated by numerous mononuclear cells (cd4 and cd8 t cells, monocyte/macrophages) and some eosinophils. typically, interface dermatitis is seen with a predominance of cd4+ t cells. these cells are located mainly in the perivascular dermis, and both cd4+ and cd8+ t cells are located at the dermoepidermal junction [20] . skin manifestations of dress may vary from mpe-like to exfoliative dermatitis and are characterized by a heavy infiltration of cd4+ and cd8+ t cells, monocyte/macro-phages and eosinophils [21] . mpe and dress share many pathological features, but dress exhibits more severe dyskeratosis (keratinocyte death in epidermis) and a greater extent of systemic involvement and eosinophilia [26] . immunohistology of skin lesions in agep reveals intraepidermal pustules with infiltration of neutrophils surrounded by il-8 producing t cells [22] . despite very diverse clinical presentations, constant features of delayed-type drug hypersensitivity are the presence of high numbers of drug-specific cd8+ cytotoxic t cells and low numbers of innate nk lymphocytes [20, 27, 28] . cd8+ t cells of cutaneous adrs have classic cytotoxic functions: lysis of autologous lymphocytes or keratinocytes in an mhc class i-restricted and drug-dependent manner [28] . cytotoxic immune cells in sjs/ten drug-induced sjs and ten are severe cutaneous adrs in which cytotoxic t lymphocytes (ctls) and natural killer (nk) cells are activated, and subsequently carry out the cellular immune reactions directed at keratinocytes in a major histocompatibility class (mhc) i-restricted manner. upon activation of these immunocytes, various cytotoxic signals, including granulysin, perforin/granzyme b, fas/fas ligand, and cytokines/chemokines, are relayed to the skin lesions to mediate the disseminated keratinocyte death [23] [24] [25] . it is noteworthy that the number of granulysin-positive cells in fixed drug eruptions was found to be similar to that observed in sjs/ten [27] . non-immune-mediated hypersensitivity is commonly referred to as pseudoallergic reactions because they do not involve a specific immune mechanism -neither ige-mediated (type i) nor delayed (type iv) hypersensitivity. clinical manifestations, which range from milder erythematous to urticarial reactions to severe lethal anaphylaxis, may be indistinguishable from immune system-mediated hypersensitivity reactions. common non-immune-mediated hypersensitivity can be caused by contrast media, vancomycin, non-steroidal anti-inflammatory drugs (nsaids), opiates, plasma expanders, and drugs used in general anesthesia [29] . nsaids-induced pseudoallergic reactions have been attributed to cyclooxygenase-1 inhibition and overproduction of leukotrienes, and may require higher drug doses than are needed for true ige-mediated reactions [30] . mast cell drug-specific t cells mediate skin inflammation in variable clinical presentations of delayed-type drug hypersensitivity through the release and induction of different cytokines and chemokines (table 25 .1) [31] . the heterogeneous cytokines include th1 cytokines (interferon-γ) and th2 cytokines (il-4, il-5) [22] . increased expression of il-5, which is a key cytokine for activation of eosinophils, is commonly seen in delayed-type drug hypersensitivity [32] . the activation of eosinophils can be further enhanced by the chemokines eotaxin and rantes [33] . thymus and activation-regulated chemokine (tarc/ccl17) has been reported to be a dress specific cytokine [34] . in addition to th1 and th2 cytokines, a recent study demonstrated the involvement of il-17aproducing th17 in dress and sjs/ten [35] . elevated expression of the neutrophil-attracting il-8 has been known to be the key cytokine involved in agep. there are several cytokines involved in sjs/ ten. numerous studies have shown tumor necrosis factor alpha (tnf-α) strongly expressed in sjs/ten lesions and correlated with disease severity [24, 36, 37] . tnf-α is a potent cytokine that induces cell apoptosis, cell activation, differentiation, and inflammatory processes [38, 39] . interferon gamma (ifn-γ) is a common cytokine involved in delayed-type drug hypersensitivity, including sjs/ ten. ifn-γ was intensely expressed in the superficial dermis and epidermis of sjs/ten lesions [36, 37] . ifn-γ is also known to promote antigen presentation and thus stimulate the cell-mediated immunity by upregulation of mhc molecules [40] [41] [42] . in addition to tnf-α and ifn-γ, several cytokines and chemokine receptors that are responsible for trafficking, proliferation, and activation of t-cells and other immune cells have been found elevated in the skin lesions, blister fluids, blister cells, pbmcs, or plasma of sjs/ten patients . these cytokines/chemokines include il-2, il-5, il-6, il-10, il-12, il-13, il-15, il-18, ccr3, cxcr3, cxcr4, and ccr10 [24, 36, 37, [43] [44] [45] . the central hypothesis proposed to explain the severe mucocutaneous lesions of sjs/ten is the cd8+ cytotoxic t cell and natural killer (nk) cell-mediated cytotoxic immune reactions. three major cytotoxic signals from cytotoxic cells are reported to be involved in the extensive skin necrosis of sjs/ten, including the fas-fasl interaction, perforin/granzyme b, and granulysin, which can induce keratinocyte apoptosis [23, 28, 46] . granulysin is not only a cytotoxic protein; it is also a chemoattractant and proinflammatory activator that can promote monocyte expression of ccl20 [47] , and is capable of promoting antigen-presenting (dendritic) cells and leukocyte recruitment, and activating specific immune responses, such as il-1b,il-6, il-10, tnf-a [48] . reports on the familial occurrence of severe drug hypersensitivity and cases occurring in identical twins suggest genetic links [49] [50] [51] [52] . the hla genes show strong association with drug hypersensitivity. examples of strong associations of hla alleles with specific drug-induced hypersensitivity reactions include abacavir, nevirapine, carbamazepine, and allopurinol (table 25. 2). the view that hla alleles are the main genetic determinants of sjs/ten was first proposed by roujeau et al. [61] , who reported the weak associations of hla-a29, b12, and mpe maculopapular drug eruption, dress drug reaction with eosinophilia and systemic symptoms, sjs/ten stevens-johnson syndrome/toxic epidermal necrolysis dr7 in sulfonamide-related ten, and hla-a2, b12 in oxicam-related ten in europeans [61] . following the immunological hypothesis, the most striking evidence of genetic susceptibility to sjs/ten was provided by the findings that hla-b*15:02 is strongly associated with carbamazepine-induced sjs/ten [53] , hla-b*58:01 with allopurinol-induced sjs/ten or dress [54] , and hla-b*5701 with abacavir hypersensitivity [62] . the hla association to specific drug-induced hypersensitivity can be ethnic and phenotype-specific. the strength of hla associations with specific drug-induced hypersensitivity in different populations has been found related to the prevalence of the susceptibility allele in the ethnic population. the association of hla-b*15:02 with carbamazepineinduced sjs/ten was replicated in other asian countries, including thailand, hong kong, malaysia, china, vietnam, cambodia, reunion, philippines and indian ethnicities, which carry high hla-b*15:02 allele frequency, but not in europeans, which carry low hla-b*15:02 allele frequency (<1 %) [63] . in contrast, the strong association of hla-b*58:01 with allopurinol-induced sjs/ten is more universal, being found in han chinese in china, thai populations, korean, japanese, and european populations; hla-b*58:01 is the allele common to all these populations [64] . the phenotype-specific characteristics are exemplified by carbamazepine hypersensitivity. while hla-b*15:02 is strongly associated with carbamazepine-induced sjs/ten, it is not associated with carbamazepine-induced dress; in an international study, hla-a*31:01was strongly associated with carbamazepine-induced dress, but not with carbamazepineinduced sjs/ten [65] . phenytoin -an aromatic antiepileptic drug structurally related to carbamazepine -also frequently causes sjs/ten and dress [66, 67] . hla-b*15:02 has been associated with phenytoin-related sjs/ten in asians, although the association is much weaker than that found for carbamazepine-related sjs/ten [68] . a recent genome-wide association study by chung wh et al. turned up cytochrome (cyp) 2cvariants, including cyp2c9*3, that showed a strong association with phenytoin-related scar. the significant association between cyp2c9*3 and phenytoin-related severe cutaneous ards was replicated in different asian populations [69] . similar to delayed-type drug hypersensitivity, genetic predisposing factors have been reported in immediate-type drug hypersensitivity. β-lactam allergy was reported associated with gene variants of il13, il4, and il4ra [70] [71] [72] [73] . several genetic predisposing factors, including gene polymorphisms in cysteinyl leukotriene receptor type 1 (cysltr1) and leukotriene c4 synthase (ltc4s) [74] and high-affinity ige receptor (fcepsilonr1) [75] , were associated with aspirin. cutaneous adrs may be classified in terms of their presumed mechanism, severity of the reaction, histological findings, and cutaneous morphological manifestations. the modern pharmacological classification of adrs differentiates two basic types of reactions; type a, predictable reactions, and type b, unpredictable or idiosyncratic reactions. type a reactions ('augmented') are dose-dependent, common and predictable based on the pharmacology of the drug; about 80 % of all adrs are type a. type b reactions ('bizarre') do not occur at any dose in most patients, but may be dose dependent in susceptible individuals. they are uncommon, affecting a small number of patients based on an individual predisposition that depends on both genetic and environmental factors [76, 77] . the pathogenesis of type a reaction was described in the sixteenth century by paracelsus, the swiss german renaissance physician who founded the discipline of toxicology: "all things are poison, and nothing is without poison; only the dose permits something not to be poisonous" [78] . the pathogenesis of type b reaction was designated in the first century bc didactic poem, de rerum natura (on the nature of things), by the roman poet and philosopher lucretius: "one man's meat is another man's poison" [79] . type b reactions can be categorized into different subtypes according to gell and coombs' classification system [80] . the effector phase of the allergic reaction is classified into four types: type i mediated by drug-specific ige antibodies, types ii and iii mediated by drug specific igg or igm or iga antibodies, and type iv induced by drug-specifc t lymphocytes [81] . this classification system may be helpful in daily clinical practice as a guide to diagnostic and therapeutic decisions. in addition to the basic classification of type a and b reactions, further types of reactions were subsequently added; type c-dose and time-related, 'chronic'; type dtime-related. 'delayed'; type e-withdrawal effects, 'end of use'; and type f-unexpected failure of therapy, 'failure' [2] . the diagnosis of a cutaneous adr must be followed by differentiation between a simple reaction involving only the skin and a complex reaction that includes systemic involvement of organs in addition to the skin [82] . systemic involvement should be explored even in a mild cutaneous eruption due to a drug since the severity of skin manifestation does not necessarily mirror the severity of the systemic involvement. systemic involvement is evaluated by assessing the patient's symptoms, including fever, facial edema, malaise, chills, dyspnea, cough, palpitations, nausea, vomiting, diarrhea, sore throat and arthralgia. further investigation is based on the patient's symptoms. basic laboratory screen, conducted in cases of suspected systemic involvement, includes a full blood count, liver and renal function tests, and urine analysis [83] . skin biopsy is an invaluable diagnostic modality in the assessment of drug eruptions. histologically, drug eruptions can elicit a variety of inflammatory disease patterns in the skin and panniculus, and overlapping reaction patterns. ackerman et al.'s basic patterns of inflammatory skin diseases [84] (table 25. 3) are a helpful guide. the most common pattern of drug eruptions is the perivascular type, while psoriasiform and granulomatous patterns are rarely reported [85] . drug eruptions may also mimic specific skin diseases such as lupus, lichen planus or lymphoma [85] . a single drug may cause a wide range of reaction patterns and no reaction pattern is specific for a particular drug [88] . while the histological changes are not distinctive in many cases of drug eruption, a few important histopathological clues may aid in the diagnosis: (1) overlapping histological patterns in one specimen (e.g., lichenoid and spongiotic). (2) presence of eosinophils (although not mandatory); although eosinophils are an important tell-tale sign of a drug-induced reaction, they may also be conspicuous in skin rashes devoid of a drug association and sparse or absent in some drug exanthems. (3) apoptotic keratinocytes. (4) mismatch between clinical and histomorphological features [85, 86, 88] . in a study assessing the histological pattern of 104 cases of diagnosed drug eruption during a 5-year period in one institution [89] , the majority of the cases (94 %) were morbilliform-type rashes. the most common histological pattern was superficial perivascular and interstitial with interface changes. eosinophils were present in only 50 % of cases, and approximately half (53 %) of the cases exhibited epidermal-dermal interface changes [89] . in view of the large diversity of cutaneous drug reactions, it is helpful to approach them as clinicopathologic entities and to base the diagnosis on a combination of clinical, histological and disease course data [89] . heightened awareness of the possible mimicry of other skin diseases and of the suspicious histopathological clues pointing to drug etiology are key elements to the appropriate histological diagnosis of drug reactions in the skin [85, 88, 89] . a widely accepted approach to diagnosing the type of drug eruption is a simplified method based on the morphology of the primary lesions. the four main categories are maculopapular, urticarial, pustular and blistering [82] . the diagnosis of the drug eruption can be challenging since the same cutaneous morphology can be manifested in a simple reaction involving only the skin and in a complex reaction including systemic involvement in addition to the skin. therefore, there are two major steps in diagnosing drug eruptions: determine the morphology and assess systemic involvement [90] . terminology the term 'maculopapular' is descriptive. morbilliform means measles-like, the rash of measles consisting of macules and papules that tends to confluence. the etymon of 'exanthema' is the greek 'exanthema', which means 'a breaking out'. thus exanthema merely means 'rash', and 'exanthematous rash' literally means 'rash-like rash'. therefore, the terminology is redundant [89] . polymorphous pink-to-red macules and or papules usually in a symmetric distribution that may coalesce to form plaques ( fig. 25 .1) [91] . the eruption begins on the trunk and upper extremities and progressively becomes confluent. in addition, purpuric lesions may appear on the ankles and feet [90] . the drug eruption can also manifest in a scarlatiniform pattern of pinpoint-sized pink-red papules coalescing and giving the skin the texture of sandpaper [92] . frequency the most common drug-induced eruptions, occurring in 1-5 % of first-time users of most drugs [91] . lag period 7-14 days [90] . symptoms pruritus and low-grade fever are common [91] . the eruption usually begins on the trunk and becomes generalized. palms and soles are often involved; mucous membranes are usually spared [90] . histology nonspecific changes consisting of mostly superficial but also deep perivascular and interstitial infiltrate of lymphocytes. eosinophils and epidermal-dermal interface changes appear in approximately half the cases [89] . differential diagnosis viral exanthems, scarlet fever, toxic shock syndrome, acute graft versus host disease (gvhd), kawasaki disease, juvenile idiopathic arthritis [90] . treatment identifying and discontinuing the causative drug are the most important steps in management. symptomatic treatment with antipruritic agents and potent topical glucocorticoids may be helpful [91] . a decision can be made to continue the drug and offer symptomatic treatment if the drug is of paramount importance, but the risk: benefit ratio of this option has to be carefully weighed, and the evolution of the eruption must be meticulously monitored [90] . prognosis the eruption often fades within 7-14 days of discontinuation of the offending drug and scaling and desquamation may follow. re-challenge may lead to reappearance of the reaction within a few days [90] . offending drugs the most common classes of drugs implicated are penicillins, sulfonamides, cephalosporins, and antiepileptics [90] . terminology the term 'urticaria', first introduced by william cullen in the eighteenth century, is derived from urtica urens (common european stinging nettle). one of the earliest descriptions of urticaria comes from china, and is more than erythematous macules and papules coalescent into illdefined plaques on the trunk -maculopapular morphology of cutaneous adr 2,000 years old. in the huangdi neijing, written around 200 bc, urticaria is referred to as feng yin zheng ('wind type concealed rash'). in ancient latin medical literature, urticaria was called 'uredo' (urere means 'to burn'), and in the old persian medical texts, 'essera' (meaning 'elevation') [93] . skin signs urticaria is induced by superficial dermal swelling due to plasma leakage and vasodilation triggered by activation of mast cells. the skin manifestations of this process include erythematous and edematous papules and plaques (wheals) of various sizes that may coalesce to form large plaques [94] . wheals may be characterized by pink or pale center and assume a figurate or polycyclic configuration. linear lesions can be seen with dermatographism [92, 94] . frequency drug-induced urticarial eruptions are the second most common type of cutaneous drug eruption and account for approximately 5 % of all cutaneous drug eruptions [85] . lag period urticaria occurs within minutes to days of drug administration [94] . symptoms a major clinical feature is pruritus, the lack of which should put the diagnosis in doubt. the lesions can also be painful if they occur on the soles, over joints, or in areas where the skin is tightly adhered to subcutaneous tissue [94] . a single lesion lasts less than 24 h and upon resolution leaves normal skin. however, new lesions may continue to arise for various periods of time. acute urticaria is defined when a bout of hives lasts less than 6 weeks; when it lasts longer, it is defined as chronic urticaria [95] . urticaria may be associated with angioedema [93] . angioedema is defined as a deep, dermal, subcutaneous and/or mucous swelling that may involve the intestinal lining and the upper respiratory tract. symptoms include slight heat, burning, pain and sensation of pressure or tightness. however, pruritus is minimal or absent. swelling of gastrointestinal tract mucosa can induce abdominal pain, vomiting and diarrhea. edema of the respiratory tract may induce various symptoms including life-threatening asphyxia. drug-induced angioedema is associated with urticaria in approximately 50 % of cases. some drugs may induce angioedema without urticaria [96] . common sites of involvement lesions of urticaria can appear anywhere on the skin, including the palms, soles and scalp, but not on mucosal surfaces [94] . angioedema most commonly occurs in the head, neck and hands, but can occur anywhere and frequently involves mucosal tissue. swelling may be more prominent in areas of looser skin, such as the scrotum, labia, lips, and eyelids [94] . histology urticarial drug reactions are characterised by dermal edema and a superficial and deep perivascular and interstitial dermatitis. the mixed inflammatory infiltrate comprises lymphocytes, histiocytes, mast cells, eosinophils and neutrophils. the presence of neutrophils and deep vascular plexus involvement may be a clue to the drug-induced nature of the urticaria [86] . the wheals with central red halo of urticaria may resemble the target lesions of erythema multiforme. four clinical signs of urticaria can help distinguish it from erythema multiforme: (1) the central zone consists of normal skin, whereas in erythema multiforme, skin is dusty, bullous or crusted. (2) each lesion is transient, lasting less than 24 h, whereas erythema multiforme lesions are 'fixed' for a few days. (3) new lesions appear daily and in erythema multiforme all lesions appear within the first 72 h. (4) there may be associated swelling of face, hands and feet and in erythema multiforme there is no edema [97] . differential diagnosis of urticaria includes also bullous pemphigoid, urticarial vasculitis and serum sickness-like reaction (sslr). drug-induced urticaria needs to be differentiated from cases of urticaria induced by other etiologies, such as food, environmental allergens, insects, systemic illness, physical stimuli, genetic and idiopathic [94] . urticaria and angioedema are the most common symptoms of anaphylaxis (88 % of cases), and are one of the clinical criteria of the national institute of allergy and infectious disease (niaid) and the food allergy and anaphylaxis network (faan) for the diagnosis of anaphylaxis [98] . therefore, all cases of sudden acute urticaria and angioedema should be evaluated for indications of the anaphylactic type of reaction: presence of respiratory compromise, decreased blood pressure, and end-organ dysfunction (collapse, syncope, incontinence) [98] . treatment the most important step in the management of drug induced urticaria with or without angioedema is withdrawal of the causative agent. in most cases of acute urticaria, when the trigger is removed the rash quickly resolves. h1-receptor blockers are the mainstay of treatment for patients with only cutaneous symptoms. systemic glucocorticoids are indicated in all cases with upper airway edema and should be considered in cases with extensive cutaneous involvement. epinephrine is reserved for angioedema with upper airway involvement [94] . the presence or absence of any airway involvement should be specifically investigated. prognosis both urticaria and angioedema fade without visible sequelae. following resolution, there should be no residual pigmentary changes unless excoriated [94] . offending drugs many drugs can induce acute urticaria, and do so by both immunologic and non-immunolgic mechanisms. the major drugs responsible for immunologically based urticaria are antibiotics, especially penicillins and cephalosporins [90] . the major drugs triggering mast cell release (non-immunolgic mechanisms) are aspirin, nonsteroidal anti-inflammatory drugs (nsaids), opioids and radiocontrast media [90] . viral infections or connective tissue diseases may induce or augment urticarial drug reactions [86] . the national institute of allergy and infectious diseases (niaid) and the food allergy and anaphylaxis network (faan) defined anaphylaxis as a systemic reaction resulting from the sudden release of multiple mediators from mast cells and basophils, often life threatening, and usually unexpected. the world allergy organization (wao) has divided anaphylaxis into immunologic (further divided into immunoglobulin e [ige]-mediated and non-ige-mediated), non-immunologic, and idiopathic causes. drugs are the second most common cause of anaphylaxis after food, which constitutes 20 % of triggers [98] . common medications associated with anaphylaxis include penicillins, nsaids, and biologic response modifiers [99] . the niaid/ faan definition of anaphylaxis has been translated into clinical diagnostic criteria that include an acute onset of illness (minutes to hours) and involvement of the dermatologic, respiratory, cardiovascular, or gastrointestinal systems [98] . epinephrine is the only first-line treatment for anaphylaxis and is the sole effective treatment for an acute reaction. delays in administration have been associated with fatalities. supportive treatment with oxygen, fluids and additional drugs are also necessary according to the cardiopulmonary resuscitation (cpr) anaphylaxis algorithm [98] . • serum sickness-like reaction (sslr) -see severe cutaneous adverse drug reactions. terminology the term pustule originates in classical latin in which pustule means a blister [100] . skin signs pustular drug eruptions are characterized by monomorphic eruption consisting of erythematous papules (mostly follicular) and pustules at the same location lacking comedones. acneiform drug eruptions (acne medicamentosa) the term acneiform is applied to eruptions that resemble acne vulgaris. frequency varies, depending on the drug. the highest incidence involves epidermal growth factor receptor inhibitors (egfris), affecting 60-100 % of patients [101] . lag period the eruption begins after a variable delay; corticosteroids may induce an acneiform eruption from shortly after their introduction (2-4 weeks) to several months [101] . acneiform eruptions induced by egfris usually appear after 1-2 weeks of treatment but can also occur after only a few days [102] . symptoms pruritus, tenderness and pain may occur. in cases of chemotherapy-related side effects, their appearance and severity are part of the criteria used for the classification of the adr [103] . common sites of involvement lesions may be located in and beyond the seborrheic areas, such as the arms, trunk, lower back and genitalia [104] . histology drug-induced acneiform eruptions show histopathologic features similar to acne vulgaris. early lesions most commonly have a corneocytic plug within a widened infundibulum, accompanied by infundibular spongiosis, perifollicular edema, with sparse perivascular and periinfundibular infiltrates of neutrophils and lymphocytes. larger older lesions show similar findings but the infiltrate is denser, with more neutrophils around the involved follicles, and infundibular rupture [85, 88] . in a review of the histological findings of acneiform eruptions induced by egfris [105] , all ten cases showed a superficial, predominantly neutrophilic suppurative folliculitis with ectatic infundibula and a rupture of the epithelial lining. differential diagnosis the main differential diagnosis is acne. the following clinical characteristics of acneiform drug eruptions may aid in differentiating between the two entities: (1) clinical presentation: monomorphic pattern, lack of comedones and cysts and localization on areas beyond the seborrheic area. (2) patient characteristics: age of onset before or after the teens, and absence of past history of acne. (3) resistance to conventional acne therapy. (4) time relationship: onset after recent drug introduction, improvement after drug withdrawal, and recurrence after drug reintroduction [101] . the differential diagnosis also includes folliculitis, rosacea, perioral dermatitis, demodicosis, acne cosmetic, acne mechanica, chloracne, acne necrotica and acneiform presentation of cutaneous lymphomas [104] . treatment the main treatment is withdrawal of the offending drug and the application of topical treatments as needed (benzoyl peroxide topical antibiotics and topical retinoids) [90] . the management of acneiform eruptions associated with chemotherapy differs from all other types of acneiform drug euptions, as acneiform eruption is an expected outcome and discontinuation of the medication is not an option in a patient who is responding to therapy [102, 103, 106, 107] . in fact, continuation of egfri therapy in these patients may be especially favourable in view of studies that have shown an increased survival with increasing severity of rash [102] . the cutaneous reaction serves as an important clinical tool for determining tumor response and survival [102] . the national cancer institute developed a scale for defining the degree of rash and laid down management guidelines for each stage [103] . other management protocols were suggested by bachet et al. [107] , who recommended that unless contraindicated, a tetracycline should be routinely prescribed for the prevention of acneiform eruption in patients treated with an egfri for more than 6 weeks. chiang et al. [106] reported successful treatment with isotretinoin for high grade and refractory cases. prognosis in most patients with acneiform drug eruption, the rash resolves upon discontinuation of the offending drug and the use of topical treatment. in egfri-induced acneiform eruption, prophylactic administration of a tetracycline was associated with significantly lower incidence of grade 2-3 folliculitis and improved quality of life of patients [107] . few cases of drug-induced eosinophilic pustular folliculitis have been reported [88, [108] [109] [110] [111] . drugs reported include chemotherapy (cyclophosphamide, methotrexate, and 5-fluorouracil) [108] , minocycline [109] , carbamazepine [110] , and allopurinol with timedium bromide [111] . clinical presentation includes pruritic follicular papules and pustules on the face, scalp, trunk and arms [88] . histological findings include spongiosis of the follicular epithelium, and an intraand perifollicular lymphohistiocytic infiltrate with numerous eosinophils that form microabscesses within the follicular epithelium [88] . topical steroids are the first line of treatment [108] . acute generalized exanthematous pustulosis (agep) -see severe cutaneous adverse drug reactions. terminology the term pseudoporphyria was coined in 1975 by korting to describe patients with chronic renal failure and a bullous disease resembling porphyria cutanea tarda (pct) [112] . frequency the incidence of pseudoporphyria is unknown. however, in a 6-month prospective study, 12 % (9/74) of children taking naproxen for juvenile idiopathic arthritis developed pseudoporphyria [113] . lag period the skin lesions appear following drug intake combined with exposure to light. various time durations were reported, weeks to months [114] [115] [116] . the clinical features of pseudoporphyria may be identical to those of pct; both exhibit vesicles, bullae, milia, and scarring on sun-exposed skin. in contrast to pct, however, hypertrichosis, hyperpigmentation, sclerodermoid changes, and dystrophic calcification are rarely reported in pseudoporphyria [117] . often, fragility and bruising may be the only clinical signs [116] . in children, facial scarring resembling erythropoietic protoporphyria (epp) may be found [117] . symptoms skin fragility and photosensitivity [116] . the lesions appear on sunexposed skin, particularly the hands and feet, but also on the face and extensor surfaces of legs [116] . histology the histological features are identical to those seen in pct. the blisters are subepidermal and the floor of the blister is typically lined by well-preserved dermal papillae (festooning). there is usually no significant inflammatory component although a light perivascular lymphocytic infiltrate may occasionally be seen in the superficial dermis. thickening of the superficial vessels (highlighted by a pas stain) and dermal sclerosis with elastosis may be apparent. in both pseudoporphyria and pct, direct immunofluorescence reveals granular deposits of igg and c3 at the basement membrane zone and in the perivascular region [115] . differential diagnosis while pseudoporphyria and pct share clinical and histologic features, they can be differentiated by several features. most important, by definition, biochemical porphyrin abnormalities are absent in pseudoporphyria. epidemiologically, pseudoporphyria affects mainly women while there is a male predilection in pct. clinically, hypertrichosis, hyperpigmentation, sclerodermoid changes, and dystrophic calcification are frequently evident in pct and conspicuously absent in pseudoporphyria [117] . the differential diagnosis also includes other types of cutaneous porphyria that manifest with blistering, epidermolysis bullosa acquisita, polymorphous light eruption, and other photosensitive dermatosis [117] . treatment treatment entails discontinuation of suspected agents and sun protection, especially against uva wavelengths, for several months following withdrawal of the drug [114] . prognosis blisters may continue to appear for weeksmonths after discontinuation of the offending drug [117] . offending drugs the most common group of drugs causing pseudoporphyria are nsaids [117] . other groups are antibiotics, diuretics and retinoids. additional culprits are hemodialysis, renal failure, tanning beds and excessive sun exposure [117] . terminology fixed drug eruption (fde) was first reported by boums in 1889 [118] , and the term was coined by brocq in 1894 [119] . frequency the incidence is not known, but is suspected to vary greatly by geographic region [120] . lag period after initial use of the offending agent, a variable refractory period of weeks, months or years may pass before the lesions first appear on the skin of a sensitized individual [121] . repeated exposure to the agent typically results in acute lesions within 30 min to 8 h. a refractory phase may occur following an acute flare in which exposure to the offending drug will not exacerbate the lesion for weeks to months [121] . in its classical form, fde typically presents round or oval, sharply demarcated, red to livid, slightly elevated plaques ranging from several millimeters to over 10 cm in diameter. vesicles or even blisters can develop [122] . usually only a single lesion appears. sometimes, multiple lesions are present and even lead to generalized fde characterized by multiple, sharply defined, deep red macules distributed bilaterally and often symmetrically. generalized bullous fde is characterized by flaccid blisters arising on these macules. mucosal lesions are usually bullous and may appear with or without involvement of other areas of the skin [122] . symptoms patients often complain of burning and itching in the lesions. general symptoms such as fever, nausea, dysuria, abdominal cramps and diarrhea are rare [122] . pruritus and burning may be the only manifestations of reactivation in a postinflammatory hyperpigmentation lesion [121] . common sites of involvement the eruption can occur anywhere on the body, but the lips, palms, soles, genitalia (especially male genitalia), groin and occasionally oral mucosa are favored sites [121] . the diagnostic hallmark of fde is the reappearance of the lesions precisely over the previously affected sites. studies investigating the predilection areas indicate that some specific kind of drugs cause fde predominantly at specific sites: examples are tetracycline and location on the male genital area, and naproxen and fde on the lips [122] . in rare cases, fde manifests in old trauma sites such as bcg vaccination, burn scar, venipuncture site or insect bite. with each recurrence, additional sites may be affected. the presence of numerous lesions is referred to as generalized fde [122] . histology histologically, the acute phase is characterized by marked basal cell hydropic degeneration, with lymphocyte tagging along the dermoepidermal junction and individual keratinocyte necrosis. marked pigmentary incontinence is typical, and may be the sole histological finding in late lesions [121] . differential diagnosis skin lesions can imitate various dermatoses, including lichen planus, erythema multiforme, erythema annulare centrifugum, and pityriasis rosea. in generalized fde, residual pigmentation in healed lesions may be reminiscent of erythema dyschromicum perstans. involvement of oral and genital mucosa raises the possibility of herpes simplex, pemphigus vulgaris, aphthous stomatitis, behçet syndrome, and erosive lichen planus [122] . generalized bullous fde may resemble sjs/ten. the following typical clinical features of generalized bullous fde may aid in differentiating between conditions: (1) blistering usually affects only a small percentage of body surface area, and between the large blisters there are sizable areas of intact skin. (2) erosive mucosal involvement is rare, and when it does occur is rather mild. (3) patients usually do not feel sick or have fever, and generally are in much better overall health than those with sjs/ten. (4) most patients report a history of a similar, often local reaction [123] . treatment for mild lesions, topical corticosteroids usually suffice. in severe involvement, especially generalized bullous fde, systemic corticosteroids may be indicated. strict avoidance of the causative drug and cross-reacting substances is essential for prophylaxis. successful desensitization was reported [122] . prognosis the prognosis of localized fde is good and the lesions fade within a few days to leave a post-inflammatory brown pigmentation [122] . generalized bullous fde does not have this benign nature and the mortality rate was 22 % in a recent case control study of 58 patients [120] . offending drugs the most common groups of drugs implicated are antibiotics, analgesics, antiphlogistics and hypnotics [122] . there is usually only one causative drug (monosensitivity), but sometimes several drugs can induce fde in the same patient (multisensitivity). it has also been claimed that recurrences of fde can be induced in nonspecific fashion by mast cell degranulators such as food, acetylsalicylic acid, bacterial toxins, or physical stimuli [122] . • drug-induced/triggered autoimmune blistering dermatosis (pemphigus, bullous pemphigoid (bp)) and linear iga bullous dermatosis (labd) [127] . cases of autoimmune blistering dermatosis resulting from exposure to drugs present clinical, histologic and immunopathologic features identical or very similar to those seen in idiopathic disease, but are induced by systemic ingestion or local use of certain drugs. there appear to be two main types: drug-induced autoimmune blistering dermatosis proper, the acute and self-limiting type with rapid resolution after withdrawal of the offending agent; and drug-triggered autoimmune blistering dermatosis in which the role played by the drug is only secondary to hereditary and immunologic factors. the drug stimulates a predisposition (hidden susceptibility) to develop the disease and is considered the chronic type in which the disease persists despite withdrawal of the offending agent [128, 129] . frequency unknown. [130, 133, 136] . of note, drug-induced labd patients tend to be older than idopathic type patients [134, 135] . the polymorphic nature of the eruption may mimic other bullous diseases and or drug-induced bullous diseases such as sjs, ten, and fde [136] . treatment treatment consists of discontinuing the offending agent, and, depending on the severity of the disease, systemic immunosuppressive treatment [129] . prognosis drug-induced autoimmune blistering dermatosis remits after the offending drug is withdrawn, while drugtriggered autoimmune blistering dermatosis may persist despite withdrawal of the offending agent and chronic immunosuppressive treatment may be required [129, 130] . two major groups of chemical structures were found in the drugs or their metabolites implicated in pemphigus: sulfhydryl radical drugs (thiol drugs or sh drugs) such as penicillamine, and phenol drugs such as aspirin [128, 137, 138] . bp many drugs were reported [129, 132, 136] , the most frequent being nsaids, cardiovascular agents and penicillin-derived antibiotics [136] . in addition, external use of skin and mucous membrane preparations has been documented to provoke cases of either bp or cicatricial pemphigoid [136] . labd of the various drugs reported, vancomycin is the most common [134, 135, 139] . reactions. the incidence of dress remains to be determined because of variable presentations and lack of universally accepted diagnostic criteria [140] . the estimated risk at first or second prescription of an aromatic antiepileptic drug was 1-4.5 in 10,000 [141] . a slight female predominance was found in the regiscar study (male/female 0.8) [142] . the drugs most commonly inducing dress are anti-convulsants (mainly aromatic anti-convulsants such as carbamazepine), allopurinol, sulfonamides (the anti-infective sulfamethaxazole-trimethoprim, and the anti-inflammatory sulfasalazine), and antibiotics (such as vancomycin and minocycline) [142] . numerous other drugs have been reported [140, 143, 144] . the role of human herpesvirus (hhv) reactivation in the development of this adverse drug reaction is well recognized, especially hhv-6 [145] . hhv-6 reactivation is among the diagnostic criteria of the japanese consensus group for dress/drug-induced hypersensitivity syndrome [146] . the reactivation of other herpesviruses, including hhv-7, cytomegalovirus (cmv), epstein-barr virus (ebv), and human herpes simplex virus was also reported [147] . dress is considered to result from complex interactions between genetic predisposition, exposure to drug and viral reactivation [148] . delayed onset of 2-8 weeks after drug administration followed by a stepwise development of manifestations. rechallenge can result in a reaction within hours to days [26] . the lag period differs between drugs; carbamazepine tended to show a longer latency (median 29 days) than allopurinol (median 20 days) in the regiscar study [142] . dress has multi-organ involvement with cutaneous, mucosal, hematological and solid organ manifestations. the cutaneous involvement in dress is typically extensive and symptomatic (pruritus, burning and pain) [142, 143] . various dermatological features were reported. walsh et al. [143] proposed a classification system based on four distinct patterns: (1) urticated papular exanthema, the most common, (2) morbilliform erythema, (3) exfoliative erythroderma, and (4) erythema multiforme-like (em-like), which was prognostic of more severe hepatic involvement. the extent of skin involvement varies between studies: it exceeded 50 % of the body surface area in most of the patients (79 %) according to the regiscar study [142] ; head and neck edema observed in most patients [26, 142] ; and pustules reported in various studies, predominantly in a facial distribution of the edema [142, 143] . [142] . most frequent were oral lesions including lips, oral cavity and throat [142] . the manifestations of oral lesions in dress include cheilitis, erosions and dysphagia that may appear before skin lesions, and oropharaynx is considered the first site of herpesvirus reactivation in dress [149] . involvement of eyes and genitalia were also reported in the regiscar study [142] . multi-organ involvement is common in dress and may include a wide variety of systems. highgrade fever (38-40 °c) is a typical early manifestation that may last for several weeks; it often precedes the cutaneous eruption by several days [142] . lymphadenopathy is common and has two distinct types: a benign pattern of lymphoid hyperplasia and a pseudolymphoma pattern [150] . hematologic abnormalities are frequent and diverse, the most common being marked leukocytosis, eosinophilia and atypical lymphocytes [142] . however, neutrophilia, monocytosis, thrombocytopenia, anemia, pancytopenia and hemophagocytic syndrome were also reported [140, 142, 143, 151] . hypereosinophilia and activated neutrophils, if persistent, can contribute to organ damage [142] . the liver is the most frequently affected visceral organ in dress; hepatitis with isolated elevation of liver enzymes is common and usually anicteric and without cholangitis. however, severe acute hepatitis with liver failure may result and is the primary cause of mortality in dress [150] . renal involvement is common [150] . involvement of the following organs was also reported: lungs, muscle, heart, pancreas, colon, thyroid, joints, parotid gland and brain [150] . the type of organs involved was found to be related to the eliciting drug [152] . the most common pathological changes found in a study of 32 patients with dress were basket-weave hyperkeratosis (94 %), dyskeratosis (97 %), lymphocytic exocytosis (91 %), spongiosis (78 %), papillary edema (66 %), perivascular lymphocytic infiltration (97 %), eosinophilic infiltration (72 %), and interface vacuolization in the dermoepidermal junction (91 %) [26] . the presence of severe dyskeratosis was correlated with a greater extent of systemic involvement [26] . in a different study assessing the histological findings of 27 cases with dress [143] , the predominant pathological pattern was spongiotic dermatitis with superficial lymphocytic infiltrate (59 %); necrotic keratinocytes were noted in 33 % of cases, and were associated with a worse hepatic involvement [143] . the diverse presentations in dress have hampered efforts to define diagnostic criteria. three diagnostic criteria have been proposed: bacquet et al. [153] , the japanese study group of severe cutaneous adverse reactions to drugs (j-scar) [146] , and the regiscar network [154] . the first step in the management is immediate withdrawal of the culprit drug. the treatment is tailored according to the severity and extent of systemic involvement, and the diagnosis of viral reactivation of herpesviruses (mostly hhv-6) [150, 155, 156] . management protocol for dress based on the consensus of experts was designed by the french society of dermatology [156] , and includes four visceral involvement severity categories and corresponding treatment: counselling both the patient and his family members about drug avoidance is necessary. first-degree relatives have a higher risk of developing the same drug reactions [90] . increased knowledge of hla susceptibility genes enables screening patients with dress for several high risk drugs [148, 157] . symptoms are usually present for several weeks even after discontinuation of the offending agent and appropriate treatment [155] . late complications include the appearance of autoimmune diseases such as lupus erythematosus and autoimmune thyroiditis, with laboratory evidence of autoantibodies [144] . systemic corticosteroids were found beneficial in the prevention of autoimmune disease. however, this effect needs to be counterbalanced against the higher risk of viral reactivation and infection. [144] . in a 1-year follow-up study of 52 affected patients with dress in taiwan, the overall cumulative incidence of long-term sequelae was 11.5 %; four developed autoimmune diseases (graves disease, type 1 diabetes mellitus and autoimmune hemolytic anemia); and the other two developed renal failure and required lifelong hemodialysis. the author concluded that the sequelae of dress can be divided into two major types that appear in different age groups: young patients tend to develop autoimmune diseases; elderly patients are more vulnerable to end-organ failure [158] . mortality in dress has been estimated at 10 %, with most patients dying from liver failure [159] . pancytopenia, leukocytosis, tachycardia, tachypnea, coagulopathy, gastrointestinal bleeding and systemic inflammatory response syndrome were associated with a poor outcome in dress patients [159, 160] . the incidence of sslr is unknown. epidemiology studies in children suggest that the overall frequency induced by cefaclor is 0.024-0.2 % per course of the drug [76] . most reactions were reported in children under 5 years old, mainly during the second and third courses of therapy [161] . cefaclor is the most common cause of sslr in children, inducing 84.1 % of cases [162] . other drugs implicated include other cephalosporins, [163] penicillins, [164] minocycline, [165] insulin, [166] and infliximab [167] . usually 7-14 days (range 0-20 days) [162, 168] . skin the skin is the most frequent finding in sslr, including erythema that progresses to urticarial lesions (pruritic and migratory), urticarial wheals with dusty to purple centers ('purple urticaria') that morphologically resemble erythema multiforme (em) [161] and other cutaneous manifestations including morbilliform or scarlatiniform eruptions [82] . mucous membranes are not involved [161] . systemic involvement joint involvement may be prominent, presenting with edema, decreased range of motion, warmth, pain, and difficulty walking. polyarticular involvement is often observed, with involvement mainly of the wrists, ankles, hips and knees [169] . some authors suggested that joint involvement may be related in part to increased fluid in the skin around affected joints due to urticarial eruption rather than arthritis [161] . fever, malaise, myalgia and lymphadenopathy were also reported. neurologic involvement, gastrointestinal symptoms and renal complications were rarely documented [163] . notable laboratory abnormalities include elevated erythrocyte sedimentation rate (esr), c-reactive protein (crp) and leukocytosis [163, 170] . the histological findings of sslr appear to be in the spectrum of urticaria with no vasculitis [171] . histology can be helpful in differentiating sslr from acute hemorrhagic edema of infancy, which is characterized by vasculitis [171] . there are no diagnostic criteria. the diagnosis is based on clinical findings [161] . withdrawal of the offending agent and symptomatic treatment with oral antihistamines and topical corticosteroids are usually sufficient. a short course of oral corticosteroids may be required in patients with severe symptoms [82] . the disease course is benign and resolves in a few days. however, a few cases lasting several weeks have been described [170] . no long-term morbidity has been reported [172] . the estimated incidence of agep is 1-5 cases per million per year [173] . female predominance was reported in several studies [174] [175] [176] . the majority of cases appear to be related to drugs (>90 %), mainly antibacterials [4] . in a large multinational casecontrol study (the euroscar study), the following agents were highly suspected drugs for agep: prestinomycin, ampicillin/amoxicillin, quinolones, (hydroxy)chloroquine, anti-infective sulfonamides, terbinafine and diltiazem [176] . latent periods fall into two categories, according to the offending drug: median duration of 1 day, associated with antibiotics (including sulphonamides), and median duration of 11 days for all other associated drugs [176] . longer periods of months were reported in a few agep cases with an underlying malignancy [177] . agep is a severe acute pustular cutaneous reaction characterized by a rapid clinical course [174] . skin the typical morphology of agep is an acute edematous erythema with burning and or itching sensation, followed by dozens to hundreds of small (pinhead sized) non-follicular sterile pustules with a predilection for the big folds, or with widespread distribution (fig. 25.2) . sometimes confluence of pustules may mimic a positive nikolsky's sign [176, 178] . additional cutaneous manifestations include marked edema of the face, purpura, blisters and target-like lesions [173, 174, 179] , all of which overlap with manifestation of agep and ten [180, 181] , and acute localized exanthematous pustulosis (alep) [179, 182] . mild, nonerosive mucous membrane involvement of one location (mostly oral) occurs in about 20 % of cases [183] . systemic involvement fever (above 38 °c) and leukocytosis with neutrophilia are almost always apparent. lymphadenopathy, myalgia, headache, mild eosinophilia, elevated crp, slight reduction of creatinine clearance, and mild elevation of aminotransferases were also reported [173, 175] . a 10-year retrospective review of 58 patients with agep [184] turned up 10 patients (17 %) with at least one systemic involvement in the acute phase, 7 with abnormal hepatic function test, 6 with renal insufficiency, two with acute respiratory distress and one patient with agranulocytosis. mean peripheral neutrophil counts and mean c-reactive protein levels were elevated significantly in patients with systemic involvement [184] . biopsy specimen should be obtained from an early pustular lesion [183] . a histopathological study of 102 agep cases [185] found the following histopathological features: (1) all cases demonstrated pustules (sub/intracorneal and or intraepidermal). the agep validation score developed by the euro-scar study group is a standardized scoring system made up of data related to clinical features (morphology and clinical course) and histopathology. based on this score, agep cases can be categorized as no agep, possible agep, probable agep, and definite agep [173] . treatment consists of discontinuation of the causative drug and supportive treatment. although, specific treatment is generally unnecessary, topical and systemic steroids were reported [174, 175] . the treatment of overlapping agep and ten cases is not yet established [180] , although successful treatment with infliximab was documented [181] . after elimination of the causative drug, pustules usually spontaneously disappear in a few days with desquamation, and the reaction fully resolves within 15 days [183] . the overall prognosis is good, although high fever or superinfection of skin lesions can sometimes lead to life-threatening situations in patients of old age or poor general condition [173] . the mortality rate is about 5 % [4] . the annual incidence of sjs and ten is 1.2-6 and 0.4-1.2 per million individuals, respectively [186, 187] . the annual incidence of sjs and/or ten in hiv patients is estimated at 1-2 per 1000 individuals, approximately 1000-fold higher than that of the general population [188] . the incidence of sjs/ten increases with age; children less than 15 years of age account for only 10 % of the samples in most studies [189] . women are two times more likely to be affected by sjs/ten than men in the adult population, while the male to female ratio is about equal in children [189] . drug exposure is the most common cause of sjs/ten [190] , with more than 200 drugs identified [191] . the groups of medications associated with high risk of inducing sjs/ fig. 25.2 multiple, pin-head sized, non-follicular pustules on erythematous skin on the trunk in a patient with agep ten vary according to the population. in the general population in europe, high risk drugs for sjs/ten include allopurinol, carbamazepine, cotrimoxazole and other anti-infective sulfonamides, lamotrigine, nevirapine, oxicam-nsaids, phenytoin, phenobarbital and sulfasalazine [192] . in the pediatric population in europe, they include anti-infective sulfonamides, phenobarbital, carbamazepine and lamotrogone [189] . in africa, they include antibacterial sulfonamides, nevirapine, tuberculosis drugs, nsaids, antiepileptics, aminopenicillin, analgesics and allopurinol [193] . non-medication triggers, implicated mainly in sjs, include infections, contrast media and vaccinations [194] [195] [196] . alden is an algorithm for the assessment of drug causality in sjs/ten developed by the regiscar study group and consists of 6 parameters according to which the drug causality is classified as very unlikely, unlikely, possible, probable and very probable [197] . usually 4-28 days. the median latency was longer (above 30 weeks) for drugs with no associated risk [192] . sjs and ten represent different degrees of a severe, acute and life-threatening mucocutaneous reaction. we will refer to this disease spectrum as a single entity, namely sjs/ ten. the classification of sjs/ten, defined by bastuji-garin et al. [198] , is based on the extent of epidermal detachment and the findings of characteristic skin lesions (table 25 .4). it should be emphasized that only necrotic skin, which is already detached (e.g., blisters, erosions), or detach-able skin (positive nikolsky sign whereby slight rubbing of the skin results in exfoliation of the outermost layer) should be included in the evaluation of the extent of epidermal detachment [190] . the characteristic skin morphology of sjs/ten consists of 'flat, atypical target lesions' and 'spots/macules', which are defined as follows. flat, atypical target lesions are round lesions, with only two zones and/or a poorly defined border, nonpalpable with the exception of potential central blister. 'spots/macules' are nonpalpable, erythematous or purpuric macules with irregular shape and size, often confluent [198] . epidermal necrosis, the hallmark process of sjs/ten, induces flaccid blisters with positive asboe-hansen sign (lateral extension of bullae with pressure), erosions, positive nikolsky sign, and in severe cases extensive skin sloughing [199] . at least 1 % of epidermal detachment is required for the diagnosis of sjs/ten [83] . in rare instances, extensive epidermal necrosis occurs with only widespread erythema and no evidence of 'flat, atypical target lesions' or 'spots/macules'; these cases were classified as 'ten without spots' (table 25 .4). a characteristic sign of sjs/ten is severe pain and tenderness of the skin [83] . mucosal involvement is evident in most of the cases with erythema, erosions and ulceration, due to necrosis of the epithelial lining [199] . sjs/ten involve more than 2 mucosal sites in 17-71 % of cases [200] . most common sites are oral (fig. 25.3) , ocular and genital mucous membranes, although any mucous membrane may be involved, such as respiratory, gastrointestinal and urethral [199] . fuchs syndrome is a unique type of sjs that involves the mucosa without skin lesions and was reported to be associated with mycoplasma pneumoniae, mostly in children and adolescents [201] . table 25 .4 classification of erythema multiforme major (emm), stevens-johnson syndrome (sjs) and toxic epidermal necrolysis (ten) according to bastuji-garin et al. [198] emm sjs a sjs-ten overlap ten ten with spots ten without spots/ten with widespread erythema systemic involvement systemic findings in sjs/ten include: (1) flu-like symptoms (malaise, fever, anorexia) that are usually the initial signs of the disease in the prodromal phase prior to the cutaneous involvement. (2) epidermal barrier breakdown-related symptoms including hypothermia, dehydration and sepsis. (3) organ involvement induced by necrosis of epithelial lining, including respiratory distress syndrome, colitis, hepatitis and nephritis [199] . characteristic histologic features include extensive keratinocyte destruction via apoptosis with separation of the epidermis from the dermis at the dermoepidermal junction. a paucicellular, dermal mononuclear infiltrate has been commonly described. lymphocytes cross the dermoepidermal junction with moderate infiltration of the epidermis. em and sjs often demonstrate less keratinocyte destruction on a background of extensive dermal mononuclear inflammation [202] . in a retrospective analysis of the clinical records and histologic material of 37 patients with ten, the histologic spectrum ranged from sparse to extensive dermal mononuclear inflammation, the extent of which predicts clinical outcome approximately as well as scorten. increased inflammation correlated with a worse prognosis; a mean cell count of dermal mononuclear >215 cells per high-power field predicted a worse prognosis (65 %) vs 24 % mortality in those with <215 cells in patients with 30 % or more total body surface area sloughing [202] . however, in a retrospective study analyzing clinical records and skin biopsy of 108 patients with sjs, sjs/ten overlap and ten, dermal infiltrate severity was not associated with day-1 scorten or hospital death, but full-thickness epidermal necrosis was associated with mortality [203] . diagnostic criteria based on integration of the major clinical characteristics of skin and mucous membrane findings, pathology assessment, lag period and systemic signs remain to be defined. the management of sjs/ten consists of a multidisciplinary approach that includes the following important aspects: 1. identification and withdrawal of the culprit drug: documenting the medication history during the previous 2 months and withdrawal of all suspected and unessential medications [123] . 2. transfer of the patient to intensive care, burn unit or other specialty unit: supportive care including thermoregulation, fluid replacement, nutritional support, monitoring for infection, sedation and pain management, and psychological support [204] . 3. assessment of skin, mucous membranes and systemic involvement and the scorten score: type of lesions in the skin, extent of epidermal detachment, and mucous membranes and systemic involvement. all patients should be evaluated by an ophthalmologist promptly following the diagnosis and at regular follow-up intervals to minimize potential long-term ocular sequelae [205] . possible acute manifestations include the eyelids, conjunctiva and cornea, and result in the classification of ocular involvement as mild, moderate or severe [206] . bringing other specialists in on the patient's care is decided in accordance with the relevant findings. the scorten system, a severity-of-illness score for toxic epidermal necrolysis, developed to stratify severity of illness and predict mortality in patients with ten, includes seven independent risk factors: age, malignancy, tachycardia, initial body surface area of epidermal detachment, serum urea, serum glucose, and bicarbonate [207] . 4. skin treatment: there are no clinical guidelines for the skin care of patients with sjs/ten. debridement of the necrotic epidermis was recommended in past publications [187, 204] . recent publications advise avoiding debridement, which may cause hypertrophic scars, and recommend considering the detached epidermis as a natural biological dressing that favors reepithelialization [14, 205, 208] . various topical treatments reported include bioactive skin substitutes, semi-synthetic and synthetic dressings, and topical antimicrobials [187, 204] . a recent report on the management of sjs/ten in an experienced french referral center described the following treatment; wound care once a day with minimal manipulation to prevent skin detachment, including a bath containing a solution of chlorhexidine 1/5000 (morphine is given prior to the bath and/or equimolar mix of oxygen and nitrogen monoxide during the bath); if bathing is not possible, the chlorhexidine solution is sprayed 2-3 times daily on the skin, blister fluid is aspirated while maintaining the blister roof, vaseline is systemically applied over all detached skin areas, topical sulfa-containing medications are avoided, and hydrocellular or absorbent nonadhesive dressings are applied at least once daily to cover pressure points [205] . 5. mucous membranes treatment: specialized care is essential to prevent lifelong complications [208] . although there is no standardized care for ocular management, the following supportive local treatment is advised: tear replacement solutions, removal of pseudomembranes, lysis of symblepharon, debridement of loosened epithelium, topical antibiotics to prevent secondary infection, topical corticosteroid to prevent scar formation, and cycloplegic drops to relieve pain, photophobia and ciliary spasm [206] . amniotic membrane transplantation was found effective in the acute and chronic stages of sjs/ten [209, 210] . a 'triple-ten' protocol for severe ocular cases was recently reported [211] , comprised of the following: (1) subconjunctival triamcinolone (kenalog 20 mg) administered into each of the fornices to curb the local inflammatory response without compromising systemic immunity. (2) placement of amniotic membrane tissue mounted on a polycarbonate skirt (prokera) over the corneal and limbal regions to facilitate reepithelialization of the ocular surface. (3) insertion of a steeply curved acrylic scleral shell spacer (technovent, sc21) to vault the lids away from the globe and provide a barrier to symblephara formation. this treatment offers an effective therapeutic option, without the need for microsurgical equipment, microscope, or sutures in the critical care setting. oral-the mouth should be rinsed several times a day with an antiseptic or anifungal solution and the lips lubricated with an ointment such as dexpanthenol [123] . genital-wet dressings or sitz baths and lubrication with emollient are recommended to avoid adhesions and strictures of genital erosions in females [123, 205] . a specialist is required in case of involvement of other mucous membranes: respiratory, gastrointestinal and/or urethral. 6 . systemic immunomodulatory treatment: the optimal therapeutic regimen has yet to be established, but according to recent publications, the following conclusions can be drawn: the use of ivig does not yield survival benefits in sjs/ten [212] ; cyclosporine decreased the death rate and the progression of detachment (dosage of 3 mg/kg/ day for 10 days) [213] ; systemic corticosteroids were associated with clinical benefit according to the euroscarstudy [214] and were reported to be the most common treatment for sjs/ten in a recent survey of 50 drug hypersensitivity experts from 20 countries [14] . one of the suggested protocols is iv dexamethasone 1.5 mg/kg pulse therapy (given for 30-60 min) for 3 consecutive days [215] . treatment with anti-tnf biologic treatment was reported to be beneficial [216] [217] [218] . a prospective, randomized, open-label trial currently underway in taiwan [14] comparing etanercept versus systemic corticosteroids in patients with sjs/ten, reported that the average duration to reach maximal skin detachment and complete skin healing was shorter in the etanercept group. in vitro investigations demonstrated that etanercept, steroids or thalidomide significantly decreased granulysin expression of blister cells. etanercept did not, however, increase the cytotoxic effect to keratinocytes found with thalidomide [14] . 7. causality assessment and communication with the patient and his/her family, health-care providers and regulatory agencies: recent discoveries of specific hlas that predict genetic susceptibility to sjs/ten offer a simple, fast, safe and reliable method for establishing clear causality between a drug and a disease [148] . the hlas are specific to a drug and an ethnic background [148] . since these tests are available only for certain drugs and a negative test does not exclude the drug as the offending agent, additional clinical and laboratory methods are available for assessing causality. the mortality rates of sjs/ten are variable. that of ten may approach 30 % [191] , and that of children with sjs/ten is approximately 2-7.5 % [189] . in a large-scale, populationbased, 1-year follow-up study of 460 sjs/ten patients, the 6-week in-hospital mortality rate was 23 %, and the death rate from 6 weeks to 1 year was 14 % [219] . the mortality rate at 1 year in this study was 24 % for sjs, 43 % for sjs and ten overlap, and 49 % for ten. several factors were found to affect mortality: age, severity of reaction, recent malignancy, preexisting severe kidney or liver disorder, and recent infection. the last two factors were recognized for the first time in this study as being independent risk factors for death. all other factors are part of the scorten [207] . the severity of the reaction was a major risk factor for death in the first few weeks, and severe co-morbidities and older age had major impact on mortality after 6 weeks [219] . early and late physical complications are common among patients who survive sjs/ten [219] , with some 80 % experiencing long-term sequelae [220] . complications may affect multiple organ systems including skin, nails, hair, oral and genital mucosal sufaces, eyes, kidneys, gastrointestinal tract, and respiratory system [221] . ocular complications, which can lead to blindness, are the major long-term morbidity [206] . a few studies have dealt with the quality of life of patients surviving sjs/ten [221] [222] [223] , which was found to be lower in every domain from before hospitalization to follow-up and a low rate of return to previous employment was documented [221] . patients reported concerns about social interactions, fear of taking medications, and fear of contracting an illness necessitating medication [223] . insufficient information and support for patients surviving sjs/ ten was also documented [221] [222] [223] . unfortunately, because of the rarity of sjs/ten, most physicians are not aware of the long-term complications of the diseases [220] . there are several methods to approach a patient with a cutaneous adr. the following is the authors' protocol: clinical assessment of drug-induced skin injury: 4ds by dr. shear a cutaneous eruption in a patient taking a medication should immediately raise the suspicion of a cutaneous adr. the physician must then determine whether the patient's clinical symptoms are signs of a cutaneous adr or of another skin disease not related to a drug. the diagnosis of a cutaneous adr is based on three key clinical elements (fig. 25.4) : (1) appearancethe morphology of the cutaneous eruption according to four main categories of the primary lesion: maculopapular, urticarial, bullous and pustular (see section "morphological classification of cutaneous adrs"). (2) systemicextra-cutaneous signs (fever, dyspnea, lymphadenopathy, etc.) that distinguish between a simple reaction involving only the skin and a complex reaction that includes systemic involvement in addition to the skin (see section "severity of cutaneous adrs: skin only (simple) versus skin and systemic involvement (complex)") and (3) histologyhistopathology and direct immunofluorescence studies of skin biopsies to confirm the clinical impression and to distinguish between a drug-induced eruption and other skin diseases (see section "histological classification of cutaneous adrs"). establishing a differential diagnosis that takes into account all possible diagnoses is essential. ranking the approximate likelihood of each condition is encouraged. all medications, regardless of route of administration, must be considered, especially new drugs taken in the 8 weeks prior to the skin reaction. drugs taken intermittently, such as vitamins, sedatives, pain relievers, laxatives and natural products, must also be considered. assessment of the lag period -the time between initiation of the drug and onset of the cutaneous reaction -is crucial in view of the different lag times for different cutaneous drug reactions. a recommended method for drug exposure analysis is to chart a timeline in order to visualize the chronology and facilitate comprehension of the event. the timeline includes the relevant information (starting day, dosage, and discontinuing day) for each drug and the signs and symptoms throughout the period in question [82] . the most important challenge in assessing drug-induced skin injury is establishing whether there is a causal relationship between the suspected drug and the untoward clinical event. the following methods are helpful: (1) patient history: the patient should be questioned about previous cutaneous reactions to drugs, and whether rechallenge with the drug improved the eruption [82] . these data should also be part of the above timeline. (2) good communication strategies will aid in the interactions with the patient and family following a cutaneous adr and decrease the likelihood of lawsuits, especially in cases of severe reactions such as sjs/ten. physicians are advised to follow these steps: (1) express empathy and say "sorry" according to the "apology laws" in an honest and respectful fashion and in a way that protects the physician from having an apology used against him in case of legal action. http:// www.sorryworks.net/. (2) provide disclosure in a "disclosure meeting" planned according to the acronym cones: context -arrange the setting for a quiet, uninterrupted meeting and decide on the participants; opening shot -the first sentence in the meeting explains the aim of the conversation; narrative -lay out the facts; it is advised to avoid using the words "error" and "mistake" since the adr is a result of multiple factors, particularly when the facts are not completely known; emotions -provide an empathic environment; summary (3) provide the patient with clear information on his cutaneous adr, the name of the offending drug, potential cross-reacting drugs, and drugs which can be safely taken as an alternative to the offending drug. in addition, advise the patient to wear a medic-alert bracelet. (4) family counselling is part of the management plan since the predisposition to some cutaneous adrs may be genetic [191] . information on the adverse event must be provided to the family physician and entered in the patient's records. report the cutaneous adr to the manufacturer and regulatory agencies [225] . the strong associations found between hla alleles and specific drug-induced hypersensitivity reactions have fostered pharmacogenetic testing to prevent the development of lifethreatening drug-induced hypersensitivity reactions, such as sjs/ten and dress. the usefulness of such testing is dependent on a number of factors, including the incidence and severity of the adverse event, the sensitivity and specificity of the predictive markers, and the availability of equally effective, alternative medications for individuals who test positive. although the incidence of sjs/ten is relatively low, it is life-threatening and many patients who survive have longterm sequelae, such as ocular complications. hla-b*1502 is a useful and strong predictive marker with high sensitivity and specificity for carbamazepine-induced sjs/ten in asian populations. this genetic association is strong enough that it prompted the usfda and many countries to relabel the genetic information for carbamazepine, and to recommend screening for hla-b*1502 before prescribing the drug for subjects of asian descent. the hla-b*1502 test for carbamazepine-induced sjs/ten has very high sensitivity (near 100 %) and specificity (97 %). with the 0.25 % prevalence rate of carbamazepine-induced sjs/ten among chinese, the hla-b*1502 test has a 7.7 % positive predictive value and 100 % negative predictive value for detecting [226] . in view of the serious consequences of sjs/ten and the availability of alternative drugs, withholding carbamazepine from screened patients who test positive for hla-b*1502 and switching to alternative antiepileptic drugs is reasonable and feasible in the high risk populations, including chinese and south-east asians. abacavir is used in the treatment of hiv infection, and has been associated with drug hypersensitivity syndrome in 8 % of patients [227] . hla-b*5701 is a strong and useful predictive marker with high sensitivity and specificity for abacavir hypersensitivity in caucasians, prompting the usfda and many other countries to recommend screening for it before prescribing the drug. the hla-b*5701 test for immunologically-mediated abacavir hypersensitivity has very high sensitivity (100 %) and specificity (97.4 %) as well as positive predictive value (55 %) and negative predictive value (100 %) [55] . hla-b*5801 is a potentially useful predictive marker for allopurinol-induced sjs/ten or dress, with 3 % positive predictive value and almost 100 % negative predictive value for detecting allopurinol-induced sjs/ten or dress in chinese (table 25. 2). this association was significant in caucasian and other asian populations as well. the recent american college of rheumatology guidelines for the management of gout recommend hla-b*5801 screening for populations with high frequency of the allele [228] . other recently discovered hla alleles related to drug hypersensitivity of potential usefullness in clinic practice are hla-b*1301 for dapsone hypersensitivity [58] , hla-a*3101 for carbamazepine-related dress [65] , and cyp2c9*3 for phenytoin hypersensitivity [69] . the lymphocyte transformation test (ltt) is a widely used in vitro assay for the diagnosis and identification of offending drugs with t cell-mediated drug hypersensitivity [229] . ltt is based on the activation and proliferation of t cells from pbmc obtained from drug-sensitized patients after stimulation, and incubation with the culprit drug in vitro [230] . following in vitro stimulation by specific drugs, drug-specific t cells are activated and release several cytokines that promote proliferation of t cells. this in vitro proliferation of specific drug-activated t cells can be detected by the incorporation of 3h-thymidine during dna synthesis after 6 days of culture. the results of ltt are expressed as the stimulation index (si): the relationship between the 3h-thymidine uptake in cells (counts per minute (c.p.m.)) with and without the drug antigen [229] . the general sensitivity of the ltt is 50-80 %, varying with different drugs and different phenotypes of delayed-type hypersensitivity reactions; thus, a negative result does not exclude the possibility of drug hypersensitivity. extensive studies on ltt for beta-lactam drugs report even higher sensitivity [230] [231] [232] [233] [234] . the specificity of the ltt is 85-100 % in different studies [231] [232] [233] 235] . ltt for the diagnosis of drug hypersensitivity has limitations. because it is measured by radioisotopes, the sensitivity can be very low and negative results are commonly observed for specific drugs (e.g., allopurinol, lamotrigine) and specific phenotypes (e.g., sjs/ten) [236, 237] . several nonradioactive methods have been developed for measuring lymphocyte proliferation or activation in in vitro tests for diagnosis of delayed-type drug hypersensitivity, including the use of carboxyfluorescein succinimidyl ester (cfse) cell staining dye [238, 239] , and measuring cytokines or cytotoxic proteins expression, such as inf-γ, il-2, il-4, il-5, il-13, granzyme-b, and macrophage migration inhibitory factor [240] [241] [242] [243] [244] . flow cytometry-assisted basophil activation test (bat), which measures specific cell makers such as cd69 or cd203c to quantify basophil activation after antigenspecific stimulation, has been widely used in the diagnosis of immediate-type drug hypersensitivity [245] . bat directly measures basophil responses instead of ige sensitization. it has been applied to the diagnosis of different drugs implicated in immediate-type hypersensitivity, including beta-lactam antibiotics, neuromuscular blocking agents, aspirin, nsaids and radiocontrast media [246] [247] [248] . the sensitivity of bat varies in different types of drugs: that for beta-lactam antibiotics ranged from 28.6 to 55 % [249, 250] ; that for nsaids ranged from 30 to 70 % [251, 252] . recent data have shown that the unique interaction between drug, t-cell receptor and hla molecule is a key factor in the development of immune-mediated adverse reactions to drugs. the discovery of strong association of specific hla alleles with specific drug-induced hypersensitivity (e.g., hla-b*1502 to carbamazepine-sjs/ten, hla-b*5801 to allopurinol-sjs/ten/dress, and hla-b*5701 to abacavir hypersensitivity), and studies of the functional role of hla-b* allele (e.g., hla-b*1502) directly interacting with a specific drug (e.g., carbamazepine) and unique t-cell receptor support the hypotheses of the 'pharmacological interaction with immune receptors' (p-i) [18, 19, 253] . in recent years, bioinformatics and computer modeling have been applied to elucidate how drug molecules interact with specific hla in drug hypersensitivity. hla alleles have been associated with liver injury induced by different drugs (such as flucloxacillin). using silico strategies to examine hla haplotype relationships, and bioinformatics tools, alfirevic et al. [254] demonstrated a connection between the different hla alleles associated with drug-induced liver injury caused by therapeutically and structurally different drugs, suggesting a mechanism of peptide binding of one of the associated hla alleles [254] . computer modeling of the molecular interaction between hla-b*1502 and carbamazepine predicted a favorable drug-binding position in the b pocket of the hla-b*1502 protein, where the side chain of arg62 could form a hydrogen bond with the ketone group of 5-carboxamide of carbamazepine ( fig. 25.5 ) [253] . cutaneous adrs have a wide spectrum of clinical manifestations that may be caused by multiple drugs and different mechanisms. in this decade, our understanding of the pathogenesis of cutaneous adrs had progressed greatly. understanding how a drug can possibly cause reactions in the skin has led to an understanding of the cellular immunology, cytokines and immunogenetics. these key insights can help mitigate the risk of reactions by 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drug-induced liver injury: use of a hla-genotyped dna archive from healthy volunteers key: cord-271032-imc6woht authors: schulte-schrepping, jonas; reusch, nico; paclik, daniela; baßler, kevin; schlickeiser, stephan; zhang, bowen; krämer, benjamin; krammer, tobias; brumhard, sophia; bonaguro, lorenzo; de domenico, elena; wendisch, daniel; grasshoff, martin; kapellos, theodore s.; beckstette, michael; pecht, tal; saglam, adem; dietrich, oliver; mei, henrik e.; schulz, axel r.; conrad, claudia; kunkel, désirée; vafadarnejad, ehsan; xu, cheng-jian; horne, arik; herbert, miriam; drews, anna; thibeault, charlotte; pfeiffer, moritz; hippenstiel, stefan; hocke, andreas; müller-redetzky, holger; heim, katrin-moira; machleidt, felix; uhrig, alexander; bosquillon de jarcy, laure; jürgens, linda; stegemann, miriam; glösenkamp, christoph r.; volk, hans-dieter; goffinet, christine; landthaler, markus; wyler, emanuel; georg, philipp; schneider, maria; dang-heine, chantip; neuwinger, nick; kappert, kai; tauber, rudolf; corman, victor; raabe, jan; kaiser, kim melanie; vinh, michael to; rieke, gereon; meisel, christian; ulas, thomas; becker, matthias; geffers, robert; witzenrath, martin; drosten, christian; suttorp, norbert; von kalle, christof; kurth, florian; händler, kristian; schultze, joachim l.; aschenbrenner, anna c.; li, yang; nattermann, jacob; sawitzki, birgit; saliba, antoine-emmanuel; sander, leif erik title: severe covid-19 is marked by a dysregulated myeloid cell compartment date: 2020-08-05 journal: cell doi: 10.1016/j.cell.2020.08.001 sha: doc_id: 271032 cord_uid: imc6woht summary coronavirus disease 2019 (covid-19) is a mild to moderate respiratory tract infection, however, a subset of patients progresses to severe disease and respiratory failure. the mechanism of protective immunity in mild forms and the pathogenesis of severe covid-19, associated with increased neutrophil counts and dysregulated immune responses, remains unclear. in a dual-center, two-cohort study, we combined single-cell rna-sequencing and single-cell proteomics of whole blood and peripheral blood mononuclear cells to determine changes in immune cell composition and activation in mild vs. severe covid-19 (242 samples from 109 individuals) over time. hla-drhicd11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild covid-19. severe covid-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and hla-drlo monocytes. our study provides detailed insights into the systemic immune response to sars-cov-2 infection and it reveals profound alterations in the myeloid cell compartment associated with severe covid-19. immune responses in blood samples in two independent cohorts of covid-19 patients. 122 activated hla-dr hi cd11c hi cd14 + monocytes were increased in patients with mild covid-123 19, similar to patients with sars-cov-2 negative flu-like illness ('fli'). in contrast, 124 monocytes characterized by low expression of hla-dr, and marker genes indicative of anti-125 inflammatory functions (e.g. cd163, plac8) appeared in patients with severe covid-19. 126 the granulocyte compartment was profoundly altered in severe covid-19, marked by the 127 appearance of neutrophil precursors due to emergency myelopoiesis, dysfunctional 128 neutrophils expressing pd-l1, and exhibiting an impaired oxidative burst response. 129 collectively, our study links highly dysregulated myeloid cell responses to severe j o u r n a l p r e -p r o o f results 131 dual center cohort study to assess immunological alterations in covid-19 patients 132 in order to probe the divergent immune responses in mild vs. severe covid-19, we 133 analyzed blood samples collected from independent patient cohorts at two university medical 134 centers in germany. samples from the berlin cohort (cohort 1) (kurth et al., 2020) , were 135 analyzed by mass cytometry (cytof) and single-cell rna-sequencing (scrna-seq) using a 136 droplet-based single-cell platform (10x chromium), while samples from the bonn cohort 137 (cohort 2) were analyzed by multi-color flow cytometry (mcfc) and on a microwell-based 138 scrna-seq system (bd rhapsody). we analyzed a total of 24 million cells by their protein 139 markers and >328,000 cells by scrna-seq in 242 samples from 53 covid-19 patients and 140 56 controls, including 8 patients with fli ( fig. 1a+b , s1a, table s1 ). 141 we first characterized alterations of the major leukocyte lineages by mass cytometry on 142 whole blood samples from 20 covid-19 patients collected between day 4 and day 29 after 143 symptom onset, and compared them to 10 age-and gender-matched controls and 8 fli 144 patients. we designed two antibody panels to specifically capture alterations in mononuclear 145 leukocytes (lymphocytes, monocytes and dcs, panel 1), and in granulocytes (panel 2, table 146 s2). high-resolution spade analysis was performed with 400 target nodes and individual 147 nodes were aggregated into cell subsets based on lineage-specific markers, such as cd14 148 for monocytes and cd15 for neutrophils (fig. s1b) . uniform manifold approximation and 149 projection (umap) analysis revealed distinct clustering of samples from covid-19 patients, 150 fli, and healthy controls, with marked changes of the monocyte and granulocyte 151 compartment (fig. 1c) . leukocyte lineages were compared in the earliest available samples 152 in covid-19 patients (day 4 to 13), fli, and controls (table s1, fig. 1d ). since leukocyte 153 counts were not available for all control samples, we compared the control samples for 154 cytof ('ctrl cytof') to data from our recently published healthy control cohorts ('ctrl flow') 155 (kverneland et al., 2016; sawitzki et al., 2020) . the proportions of all major lineages were 156 highly similar, irrespective of the methodology (fig. 1d) . cell counts of the published cohort 157 could therefore be used as a reference to report absolute cell counts for leukocyte lineages 158 in covid-19 samples. in line with recent reports xintian et al., 2020) , 159 we observed elevated leukocytes and increased proportions of neutrophils in patients with 160 severe covid-19 ( fig. 1d) , whereas only proportional increases in neutrophils were evident 161 in fli and mild covid-19 patients (fig. 1d) . total lymphocytes and t cells were strongly 162 reduced in all covid-19 and fli patients, whereas non-classical monocytes were 163 specifically depleted in covid-19 (fig. 1d) . increased neutrophils in severe covid-19 and 164 loss of non-classical monocytes in both mild and severe disease, were validated in cohort 2 165 by mcfc (fig. s1c, table s1+3 ). given the dramatic changes in various immune cell populations (fig. 1c+d) , we next 171 assessed their composition and activation state by droplet-based scrna-seq in 27 samples 172 from 18 covid-19 patients (8 mild & 10 severe, cohort 1, table s1 ) collected between day 173 3 and day 20 after symptom onset. a total of 48,266 single-cell transcriptomes of pbmc 174 were analyzed together with 50,783 pbmc from publicly available control datasets (21 175 control donors, table s1 ). umap and high-resolution cell type classification identified all cell 176 types expected in the mononuclear compartment of blood with a high granularity in the 177 monocytes, identifying five distinct clusters (cluster 0-4) (fig. 2a+s2a, table s4 ). 178 monocytes in clusters 0-3 expressed cd14, cluster 4 comprised the non-classical 179 monocytes marked by fcgr3a (encoding cd16a) and low expression of cd14. separate 180 visualization of cells in mild and severe cases revealed highly disease severity-specific 181 clusters (fig. 2b) . a distinct subset of cd14 + monocytes (cluster 1)( fig. 2a ) marked by high 182 expression of hla-dra, hla-drb1 and co-stimulatory molecule cd83 (fig. s2d) , 183 engagement of which has been linked to prolonged expansion of antigen-specific t cells 184 (hirano et al., 2006) , was selectively detected in mild covid-19 (fig. 2c ). in addition, we 185 identified another closely related cd14 + hla-dr hi monocyte population (cluster 2), which 186 was characterized by high expression of ifn-stimulated genes (isgs). however, upon closer 187 analysis, this cluster was found to originate from a single donor with mild covid-19 (fig . 188 2a-c, fig. s2d ). both cluster 1 and cluster 2 expressed high levels of isgs ifi6 and isg15 189 (fig. s2d) . in patients with severe covid-19, monocytes showed low expression of dr and high expression of alarmins s100a8/9/12 (cluster 3, fig. 2a-c, fig. s2d ). the most 191 prominent change in severe covid-19 was the appearance of two distinct cell populations 192 (cluster 5+6), absent in pbmc of patients with mild covid-19 and control donors ( fig. 2a) . 193 published markers (kwok et al., 2020; ng et al., 2019) identified cluster 5 and 6 as 194 neutrophils and immature neutrophils, respectively (fig. 2a+b) . immature neutrophils 195 (cluster 6) expressed cd24, pglyrp1, defa3 and defa4, whereas neutrophil cluster 5 196 expressed fcgr3b (cd16b), cxcl8, and lcn2 (lipocalin 2) (fig. 2c, fig. s2a ). their 197 migration within the pbmc fraction on a density gradient marked these cells as low-density 198 neutrophils (ldn). 199 in the second cohort, pbmc from 17 covid-19 patients (8 mild, 9 severe, table s1), 200 sampled between 2 and 25 days after symptom onset, and 13 controls, were collected for 201 scrna-seq on a microwell-based platform (bd rhapsody). high-quality single-cell 202 transcriptomes for 139,848 pbmc were assessed and their population structure was 203 visualized using umap (fig. 2d, table s4 ). data-driven cell type classification (aran et al., 204 2019) and cluster-specific marker gene expression identified all cell types expected in the 205 pbmc compartment and revealed additional clusters and substructures (fig. 2d+s2b) . 206 similar to cohort 1, monocytes exhibited significant plasticity and were subclassified into 5 207 clusters (fig. 2d , clusters 0-4). disease severity-associated changes seen in cohort 1 were 208 validated in cohort 2 (fig. 2e) . immature and mature neutrophil clusters were detected in 209 both cohorts (clusters 5-6) and showed near identical marker gene expression (fig. 2c) . similar to cohort 1, a prominent shift in subpopulation occupancy was observed in the 211 monocyte clusters (fig. 2d+e) . 212 based on the union of the top 50 genes for monocyte and neutrophil clusters, we found a 213 high correlation between the independently defined functional states within the monocyte 214 j o u r n a l p r e -p r o o f compartment, and mature and immature neutrophils in cohort 1 and cohort 2 (fig. s2c) . 215 violin plot representation of important marker genes illustrated distinct phenotypic states and 216 underscored the high similarity of the two cohorts (fig. s2d) . 217 disease-severity dependent alterations of the monocyte compartment and the appearance 218 of two ldn populations were detected in two cohorts of covid-19 patients. 219 predominance of hla-dr hi cd11c hi inflammatory monocytes in mild and hla-220 dr lo cd11c lo cd226 + cd69 + monocytes in severe covid-19 221 the monocyte compartment is particularly affected by covid-19, indicated by a loss of 222 cd14 lo cd16 hi non-classical monocytes (fig. 1c+d) . disease severity-dependent shifts in 223 monocyte activation were identified by scrna-seq (fig. 2) . we further explored the 224 phenotypic alterations of the monocyte compartment using mass cytometry ( fig. 3a,c,d) . increased levels of activated hla-dr hi cd11c hi monocytes in mild covid-19 249 patients was confirmed by mcfc in cohort 2 (fig. 3e) . in severe covid-19, we detected 250 increased expression of cd226 and cd69 (cluster 10) and/or decreased expression of hla-251 dr, and total cd226 + cd69 + monocytes were elevated compared to controls. cluster 10 252 expressed high levels of cd10, which is induced during macrophage differentiation (huang 253 et al., 2020b). thus, an alternative activation pattern of classical monocytes appeared to be 254 covid-19 specific and was associated with severe disease. besides activated lymphocytes, 255 also monocytes upregulate cd69 expression (davison et al., 2017) , which promotes tissue 256 infiltration and retention (cibrián and sánchez-madrid, 2017) . similarly, cd226 expression 257 on alternatively activated monocytes might also promote diapedesis and tissue infiltration 258 j o u r n a l p r e -p r o o f (reymond et al., 2004) . together, this activation pattern may contribute to the reduction of 259 circulating monocytes in covid-19. 260 261 hla-dr lo monocytes persist in severe covid-19 262 next, we dissected covid-19 associated phenotypic alterations of monocytes by scrna-263 seq. marker genes of the monocyte clusters derived from fig. 2a showed that mild covid-264 19 associated clusters 1 and 2 were characterized by an isg-driven transcriptional program 265 (fig. s3a) , and gene ontology enrichment analysis (goea) assigned these clusters to 'type 266 i interferon signaling pathway' (fig. s3b) . a monocyte cluster marked by low expression of 267 hla-dr and high expression of s100a12 and cxcl8 (cluster 3, hla-dr lo s100a hi ) was 268 strongly associated with severe covid-19 (fig. s3a, 2b, s2d) . for further in-depth 269 analysis, we subclustered the monocyte compartment of the pbmc dataset of cohort 2 ( fig. 270 2d, s3c, table s1 ) resulting in 7 subclusters (fig. 4a) . cluster 1 was marked by high 271 expression of hla-dra and hla-drb1 and co-stimulatory molecule cd83 and was 272 therefore designated hla-dr hi cd83 hi activated inflammatory monocytes (fig. 4a+b , 273 s3d+e). we identified two major clusters (0, 2) and a smaller cluster 6 with low hla-dr 274 expression, which were associated with severe covid-19 (fig. 4b, s3d+e) . low hla-dr 275 expression is an established surrogate marker of monocyte dysfunction (venet et al., 2020) 276 which results in reduced responsiveness to microbial stimuli (veglia et al., 2018) , suggesting 277 that cluster 0 and 6 are composed of dysfunctional monocytes. genes of the s100a family 278 were expressed in both hla-dr lo clusters (fig. 4b ), albeit to a higher degree in cluster 0 279 (hla-dr lo s100a hi , e.g. s100a12, fig. s2d , s3e, well as pre-maturation markers like mpo and plac8 (fig. 4b) , recently linked to immature 283 monocyte states in sepsis patients (reyes et al., 2020) . in line with these findings, clusters 284 0, 2 and 6 were significantly enriched in a gene signature derived from sepsis-associated 285 monocytes ( fig. 4c ) (reyes et al., 2020) . moreover, blood monocytes isolated from covid-286 19 patients showed a blunted cytokine response to lps stimulation, particularly monocytes 287 from patients with severe covid-19 (fig. 4d) . accordingly, hla-dr lo monocyte clusters (0, 288 2, 6) were detected almost exclusively in severe covid-19 (fig. 4e) . we next analyzed 289 time-dependent cluster occupancies per patient in cohort 2 (fig. 4e+f) . activated hla-290 dr hi cd83 hi monocytes (cluster 1) were found in all cases of mild covid-19, even at late 291 time points (fig. 4e+f) . in contrast, hla-dr lo cd163 hi monocytes (cluster 2) were present 292 mainly early in severe disease, while hla-dr lo s100a hi monocytes (cluster 0) dominated the 293 late phase of disease (fig. 4e+f) . violin plots of isg (fig. s3d ) and visualization of marker 294 genes ( fig. s3e) indicated differential expression patterns of ifn signature genes in 295 individual monocyte clusters. to reveal the kinetics of isg expression, we plotted the 296 expression of isg15 and ifi6 in the complete monocyte population for all patients that had 297 been sampled at least twice (fig. 4g) . expression levels were highest at early time points 298 and consistently decreased over time, clearly indicating that the ifn response in covid-19 299 is inversely linked to disease severity and time (fig. s3f+g) . in contrast, decreased 300 expression of hla-dra and hla-drb1 in severe covid-19 is evident early on and 301 sustained over time. 302 j o u r n a l p r e -p r o o f transcription factor prediction indicated a stat signaling-driven gene expression program 303 in monocytes in covid-19 (fig. 4h) neutrophils and the remaining clusters as mature neutrophils (fig. s4a) . accordingly, pro-322 and pre-neutrophils were enriched for transcriptional signatures of neutrophil progenitors 323 derived from published single-cell data ( and pro-neutrophils in cluster 4 and 6 showed the highest proportion of cells with a 325 proliferative signature (fig. s4b) . clusters 0, 1, 2 (originally in cluster 4 in fig. 2a ) 326 expressed mature neutrophil markers fcgr3b (cd16) and mme (cd10) (fig. s4a) . including cd24, olfm4, lcn2, and bpi, previously associated with poor outcome in sepsis 341 (fig. 5b, s4a ) (kangelaris et al., 2015) . 342 all ldns also expressed high levels of alarmins s100a8 and s100a9 (fig. 5d) , whereas 343 other s100 genes (e.g. s100a4, s100a12) were strongly induced in selected neutrophil 344 alterations of the neutrophil compartment were further interrogated by mass cytometry of 362 whole blood samples of covid-19 patients (n=8 mild + 9 severe, cohort 1), fli patients 363 (n=8), and age-and gender-matched controls (n=9) (table s1), using a panel designed to 364 detect myeloid cell maturation and activation states as well as markers of 365 immunosuppression or dysfunction (table s2) . unsupervised clustering analysis of all 366 neutrophils in all samples revealed 10 major clusters (fig. 6a ) of immature (cluster 2, 5, 6, 367 7), mature (cluster 1, 3, 4) and remaining clusters of low abundancy (cluster 8, 9, 10). based 368 on their differential expression of cd11b, cd16, cd24, cd34 and cd38, clusters 5 and 6 369 were identified as pro-neutrophils and cluster 2 as pre-neutrophils (kwok et al., 2020; ng et 370 al., 2019). the fourth immature cell cluster (7) showed very low expression of cd11b and 371 cd16, reminiscent of pro-neutrophils, but lacking cd34, cd38 and cd24 (fig. 6a) , 372 suggesting a hitherto unappreciated pro-neutrophil-like population. the mature neutrophils 373 segregated into non-activated (cluster 1), partially activated (cluster 3) and highly activated 374 cells (cluster 4), based on the loss of cd62l and upregulation of cd64, as well as signs of 375 proliferative activity (ki67 + ) (fig. 6a) . 376 neutrophils from covid-19 patients clearly separated from those of controls and also fli 377 patients in umap analysis (fig. 6b) , and neutrophils in patients with severe covid-19 were 378 distinct from those of patients with mild disease (fig. 6b) . cells from control donors 379 accumulated in areas enriched for mature non-activated cells (cluster 1) and immature pre-380 neutrophil-like cells (cluster 2). in contrast, neutrophils from fli patients were mainly mature 381 non-activated (cluster 1) and mature highly activated (cluster 4) cells. neutrophils from 382 covid-19, particularly from patients with severe disease primarily occupied immature pre-383 and pro-neutrophil-like clusters. plotting cell cluster-specific surface marker expression onto 384 the umaps (fig. 6c ) as well as statistical analyses of cell cluster distribution and surface 385 marker expression among different patient groups supported these observations (fig. 386 6d+e) . samples from fli patients contain a high proportion of highly activated mature 387 neutrophils, but barely any immature neutrophils. in contrast, severe covid-19 is 388 associated with the appearance of immature pre-and pro-neutrophils (fig. 6d+6e) . 389 interestingly, immature cell clusters in severe covid-19 showed signs of recent activation 390 like upregulation of cd64 (mortaz et al., 2018) , rank and rankl (riegel et al., 2012) , as 391 well as reduced cd62l expression (mortaz et al., 2018) . in addition to loss of cd62l, 392 immature and mature neutrophils from severe covid-19 showed elevated pd-l1 393 expression compared to control samples (fig. 6e) we next assessed the dynamics of the changes within the myeloid cell compartment over 405 time. we grouped samples according to collection time as 'early' (within the first 10 days) or 406 late (during the following 20 days) after onset of symptoms. in both cohorts, we observed a 407 tendency towards (cohort 1) or significantly higher (cohort 2) proportions of granulocytes in 408 severe vs. mild covid-19 patients, both at early and late time points (fig. s5a) . we 409 observed a persistent release of immature neutrophils (e.g. cluster 6) in severe covid-19 410 (fig. s5b) showing high expression of cd64 and pd-l1, but downregulation of cd62l as a 411 sign of activation, dysfunction and immunosuppression (fig. s5c ). in addition, severe 412 covid-19 patients show further increased frequencies of mature, partially activated 413 neutrophils (cluster 3) at later time periods (fig. s5b) . thus, the neutrophil compartment of 414 severe covid-19 patients is characterized by a combination of persistent signs of 415 inflammation and immunosuppression, which is reminiscent of long-term post-traumatic 416 complications (hesselink et al., 2019) . 417 we also analyzed time-dependent phenotypic changes in the monocyte compartment by 418 mass cytometry. non-classical monocytes started to recover in covid-19 patients during 419 the later stages of the disease (fig. s5a) . hla-dr hi cd11c hi monocyte cell clusters also 420 declined at later time points in mild covid-19 ( fig. s5d,e,f) , which correlates well with the 421 longitudinal changes of ifi6 and isg15 as well as hla-dra, and hla-drb1 expression 422 profiles (fig. 4g+s3f) . in contrast, overall proportions of hla-dr hi cd11c hi monocytes in 423 severe covid-19 remained low throughout the course of the disease. proportions of cd10 hi 424 macrophage-like cluster 10 and cd226 + cd69 + monocytes were generally higher at later 425 stages in severe covid-19, which resembled the kinetics of hla-dr lo s100a hi monocytes 426 identified by scrna-seq (fig. 4f ). this indicates a prolonged alternative activation of 427 monocytes in severe covid-19 (fig. s5e) . table s1 ). integrated visualization of 435 all samples of cohort 2 (fresh/frozen pbmc, fresh whole blood, 229,731 cells, fig. s6a ) 436 revealed the expected blood leukocyte distribution, including granulocytes ( fig. 7a, s6a , 437 table s4 ). cell type distribution identified by scrna-seq profiles (fig. s6b ) strongly 438 correlated with mcfc characterization of the same samples (fig. s6c) . for further analysis 439 of the granulocyte compartment, we first combined the whole blood samples with the fresh 440 pbmc to guide the clustering of all major immune cells resulting in a total of 122,954 cells 441 (fig 7a) . from these samples, we identified all neutrophil clusters and extracted the cells 442 derived from whole blood for subsampling, which revealed a structure of 9 clusters 443 (n=58,383 cells, fig. 7b+c ). 444 using marker-and data-driven approaches as applied to ldn (fig. 5d, s4a) , we identified 445 fut4(cd15) + cd63 + cd66b + pro-neutrophils, itgam(cd11b) + cd101 + pre-neutrophils, along 446 with 7 mature neutrophil clusters ( fig. 7b -d, s6d, table s4 ). heterogeneous expression of 447 various markers involved in mature neutrophil function including cxcr2, fcgr2a (cd32), 448 fcgr1a (cd64) or mme (cd10), pointed towards distinct functionalities within the 449 neutrophil compartment (fig. 7e, s6d+e) . seven of the nine 9 neutrophil clusters identified 450 in whole blood in cohort 2, could also be mapped to the fresh pbmc transcriptomes in 451 cohort 1 (fig. s6f) , indicating that scrna-seq of fresh pbmc in covid-19 patients reveals 452 relevant parts of the neutrophil space. the transcriptional phenotype of pro-and pre-453 neutrophils (cluster 8+9) was corroborated in cohort 2 ( fig. 7b-d, s6d) . 454 heatmap and umap visualization of the cell type distribution identified pro-and pre-455 neutrophils mainly at late time points in severe covid-19 ( fig. 7f-g) . furthermore, mature 456 neutrophils with a high ifn-signature (cluster 1) were associated with severe covid-19 457 (fig. 7e, s6g ). this cluster was also enriched for markers identified by cytof as 458 differentially expressed in patients with severe covid-19 ( fig. 6) , such as elevated 459 expression of cd274 (pd-l1) and fcgr1a (cd64) (fig. 7h ). in addition to cd274, cells in 460 cluster 1 expressed genes indicative of a potentially suppressive or anti-inflammatory state, 461 including zc3h12a (fig. 7e) , which is known to suppress hepatitis c virus replication and 462 virus-induced pro-inflammatory cytokine production (lin et al., 2014) . cluster 2 was also 463 enriched for cells from covid-19 patients, mainly from severe but also mild cases (fig. 7f is mainly driven by e2f family members and pre-neutrophils mainly depend on ets tfs 475 (fig. s6h) . 476 pseudotime analysis strongly supported the differentiation trajectory from pro-neutrophils 477 (cluster 8) via pre-neutrophils (cluster 6) to mature neutrophils in cluster 2 and 1 ( fig. s6i-j) . 478 particularly cd274 (pd-1l) was enriched in cluster 1 compared to cluster 2, supporting the 479 potential of neutrophils to progress towards a suppressive phenotype in severe covid-19 480 (fig. s6j) . interestingly, cd177 is expressed in pre-neutrophils and persisting in cluster 1 481 further highlighting the newly emerging character of this cluster (volkmann et al., 2020) . 482 finally, we studied whether the persistent emergence of immature, potentially dysfunctional 483 neutrophils in severe covid-19 patients can be captured under routine diagnostic 484 conditions. therefore, samples of 32 covid-19 patients ( table s1 , cohort 1) were 485 characterized by routine hematology analyses using a clinical flow cytometry system 486 (sysmex analyzer). indeed, the assumption of rescue myelopoiesis in severe covid-19 was 487 supported by significantly higher counts in the population of immature granulocytes (ig, 488 representing promyelocytes, myelocytes, and metamyelocytes) in this patient group ( fig. 489 7k). we also found significant differences in the neutrophil compartment, when analyzing 490 the width of dispersion with respect to granularity, activity, and cell volume defined as ne-491 wx, ne-wy and ne-wz, respectively. as compared to patients with mild course, severely ill 492 patients displayed increases in width of dispersion of activity and cell volume as surrogates 493 for increased cellular heterogeneity, immaturity and dysregulation in severe covid-19 ( fig. 494 7k), resembling previously described alterations in sepsis patients (stiel et al., 2016) . 495 furthermore, neutrophils of severe covid-19 patients were partially dysfunctional as their 496 oxidative burst upon stimulation with standardized stimuli (e.coli or pma) was strongly 497 impaired in comparison to control and mild covid-19 neutrophils, whereas phagocytic 498 activity was preserved (fig. 7l , table s1 ). 499 collectively, the neutrophil compartment in peripheral blood of severe covid-19 patients is 500 characterized by the appearance of ldn, fut4(cd15) + cd63 + cd66b + pro-neutrophils, and 501 itgam(cd11b) + cd101 + pre-neutrophils, reminiscent of emergency myelopoiesis, as well as 502 cd274(pd-l1) + zc3h12a + mature neutrophils reminiscent of gmdsc-like cells, which might 503 exert suppressive or anti-inflammatory functions. 504 dysfunctional phenotype, plac8 was recently shown to suppress production of il-1β and il-559 18 (segawa et al., 2018) . in fact, we observed that inflammatory cytokine production, 560 including il-1β release, was impaired in monocytes from patients with severe covid-19 561 (fig. 4) . pbmc fractions in severe covid-19 contained immature neutrophils, including pro-and pre-574 neutrophils, which was not observed in mild cases (fig. 5) . these immature ldn showed a 575 surface marker and gene expression profile reminiscent of granulocytic mdscs including 576 genes such as s100a12, s100a9, mmp8, arg1 (uhel et al., 2017) , and olfm4, which has 577 been recently associated with immunopathogenesis in sepsis (alder et al., 2017) . 578 emergence of pro-neutrophils in severe covid-19 was also detected by single-cell 579 proteomics on whole blood samples. strikingly, both immature and the mature neutrophils 580 showed increased expression of cd64 and pd-l1 (fig. 6+s5 ), similar to recently described 581 alterations in sepsis (meghraoui-kheddar et al., 2020). in addition to the altered phenotype, 582 we also observed an altered functionality. neutrophils from patients with severe covid-19 583 showed an impaired oxidative burst response, while their phagocytic capacity was preserved 584 (fig. 7) . 585 single-cell transcriptomics of whole blood samples revealed mature activated neutrophils in 586 both mild and severe covid-19 (fig. 7b, cluster 2) , however, expression of cd274 (pd-l1) 587 was only found in severe covid-19 (cluster 1), and it increased in later stages of the 588 disease. expression of pd-l1 on neutrophils has been associated with t cell suppression 589 (bowers et methodology: js-s, dp, tk, sb, lb, edd, mg, dw, mb, tsk, as, od, hm, ars, cc, dk, ev, 664 cjx, ad, ct, sh, clg, ml, ew, tu, mb, rg, (table s4) . (table s4) . within the monocyte space of cohort 1 (related to fig. 2, table s4 ). cluster ranked by adjusted p-values. 894 c, back-mapping of monocyte clusters of cohort 2 (fig. 4c) onto the pbmc umap of cohort 895 2 (fig. 2d) . the legend shows the association of the colors to the clusters together with the 896 labeling of the clusters based on expressed marker genes (according to fig. 2 and fig. 897 s3d-f). 898 d, violin plots of marker gene expression in the monocyte clusters identified in the complete 899 pbmc space of cohort 2 (fig. 2c,d ) 900 e, dot plot of the top 10 marker genes sorted by average log fold change calculated for the 901 monocyte clusters (fig. 4c) . severe covid-19 patients ( figure 1a+b, table s1 ). information on age, sex, medication, 1020 and co-morbidities is listed in covid-19 patients ( figure 1a+b , table s1 ) were included in the study. in patients who 1030 were not able to consent at the time of study enrollment, consent was obtained after 1031 recovery. information on age, sex, medication, and co-morbidities are listed in table s1 . 1032 covid after one wash in dpbs cells were directly processed for scrna-seq (bd rhapsody) or 1058 multi-color flow cytometry (mcfc). frozen pbmc were recovered by rapidly thawing frozen 1059 cell suspensions in a 37°c water bath followed by immediate dilution in pre-warmed rpmi-1060 1640+10% fbs (gibco) and centrifugation at 300g for 5min. after centrifugation, the cells 1061 were resuspended in rpmi-1640+10% fbs and processed for scrna-seq. antibody 1062 cocktails were cryopreserved as described before (schulz et al., 2019) . 1063 all anti-human antibodies pre-conjugated to metal isotopes were obtained from fluidigm 1066 corporation (san francisco, us). all remaining antibodies were obtained from the indicated 1067 companies as purified antibodies and in-house conjugation was done using the maxpar x8 1068 labeling kit (fluidigm). tlrpure; innaxon, uk). after stimulation, cell-free supernatants were collected and tested 1160 for il-1β, ifnγ, and tnfα, respectively, using the cytokine bead assay legend-plex 1161 mix&match test was used to report differences in ig count, whereas mixed-effect-analysis and sidak's 1182 multiple comparison test was applied to report statistical differences of ne-wx, ne-wy and 1183 ne-wz between mild and severe covid-19 patients. 1184 1185 10x genomics chromium single-cell rna-seq 1186 pbmc were isolated and prepared as described above. afterwards, patient samples were 1187 hashtagged with totalseq-a antibodies (biolegend) according to the manufacturer's protocol 1188 for totalseq tm -a antibodies and cell hashing with 10x single cell 3' reagent kit v3.1. 50µl 1189 cell suspension with 1x10 6 cells were resuspended in staining buffer (2% bsa, jackson 1190 immuno research; 0.01% tween-20, sigma-aldrich; 1x dpbs, gibco) and 5 µl human 1191 trustain fcx tm fcblocking (biolegend) reagent were added. the blocking was performed 1192 for 10min at 4°c. in the next step 1µg unique totalseq-a antibody was added to each 1193 sample and incubated for 30min at 4°c. after the incubation time 1.5ml staining buffer were 1194 added and centrifuged for 5min at 350g and 4°c. washing was repeated for a total of 3 1195 washes. subsequently, the cells were resuspended in an appropriate volume of 1x dpbs 1196 (gibco), passed through a 40µm mesh (flowmi tm cell strainer, merck) and counted, using a 1197 neubauer hemocytometer (marienfeld). cell counts were adjusted and hashtagged cells 1198 were pooled equally. the cell suspension was super-loaded, with 50,000 cells, in the 1199 chromium tm controller for partitioning single cells into nanoliter-scale gel bead-in-1200 emulsions (gems). single cell 3' reagent kit v3.1 was used for reverse transcription, cdna 1201 amplification and library construction of the gene expression libraries (10x genomics) 1202 following the detailed protocol provided by 10x genomics. hashtag libraries were prepared 1203 according to the cell hashing protocol for 10x single cell 3' reagent kit v3.1 provided by 1204 biolegend, including primer sequences and reagent specifications. biometra trio thermal 1205 cycler was used for amplification and incubation steps (analytik jena). libraries were 1206 quantified by qubit tm 2.0 fluorometer (thermofisher) and quality was checked using 2100 1207 bioanalyzer with high sensitivity dna kit (agilent). sequencing was performed in paired-end 1208 mode with a s1 and s2 flow cell (2× 50 cycles) using novaseq 6000 sequencer (illumina). the qubit dsdna hs kit (thermofisher) and the size-distribution was measured using the 1229 agilent high sensitivity d5000 assay on a tapestation 4200 system (agilent technologies). 1230 sequencing was performed in paired-end mode (2*75 cycles) on a novaseq 6000 and 1231 nextseq 500 system (illumina) with novaseq 6000 s2 reagent kit (200 cycles) and 1232 nextseq 500/550 high output kit v2.5 (150 cycles) chemistry, respectively. 1233 data pre-processing of 10x genomics chromium scrna-seq data 1236 cellranger v3.1.0 (10x genomics) was used to process scrna-seq. to generate a digital 1237 gene expression (dge) matrix for each sample, we mapped their reads to a combined 1238 reference of grch38 genome and sars-cov-2 genome and recorded the number of umis 1239 for each gene in each cell. 1240 1241 data pre-processing of bd rhapsody scrna-seq data 1242 after demultiplexing of bcl files using bcl2fastq2 v2.20 from illumina and quality control, 1243 paired-end scrna-seq reads were filtered for valid cell barcodes using the barcode whitelist 1244 provided by bd. cutadapt 1.16 was then used to trim nexterape-pe adapter sequences 1245 where needed and to filter reads for a phred score of 20 or above (martin, 2011) . then, 1246 star 2.6.1b was used for alignment against the gencode v27 reference genome (dobin et 1247 al implemented in seurat. 1258 we excluded cells based on the following quality criteria: more than 25% mitochondrial 1260 reads, more than 25% hba/hbb gene reads, less than 250 expressed genes or more than 1261 5,000 expressed genes and less than 500 detected transcripts. we further excluded genes 1262 that were expressed in less than five cells. in addition, mitochondrial genes have been 1263 excluded from further analysis. 1264 lognormalization (seurat function) was applied before downstream analysis. the original 1266 gene counts for each cell were normalized by total umi counts, multiplied by 10,000 (tp10k) 1267 and then log transformed by log10(tp10k+1). 1268 after normalization, the count data was scaled regressing for total umi counts and principal 1270 component analysis (pca) was performed based on the 2,000 most variable features 1271 identified using the vst method implemented in seurat. subsequently, the scrna-seq data 1272 from cohort 1 was integrated with publicly available 10x scrnaseq data of healthy controls 1273 using the 'harmony' algorithm (korsunsky et benchmarking data from healthy controls and 10x v3.1 scrna-seq data from cohort 1). we 1277 downloaded the count matrices for the publicly available scrna-seq data and filtered the 1278 cells using the above-mentioned quality criteria prior to data integration. for two-dimensional 1279 data visualization we performed umap based on the first 20 dimensions of the 'harmony' 1280 data reduction. the cells were clustered using the louvain algorithm based on the first 20 1281 'harmony" dimensions with a resolution of 0.4. 1282 differential expression (de) tests were performed using findmarkers/findallmarkers 1284 functions in seurat with wilcoxon rank sum test. genes with >0.25 log-fold changes, at 1285 least 25% expressed in tested groups, and bonferroni-corrected p-values<0.05 were 1286 regarded as significantly differentially expressed genes (degs). cluster marker genes were 1287 identified by applying the de tests for upregulated genes between cells in one cluster to all 1288 other clusters in the dataset. top ranked genes (by log-fold changes) from each cluster of 1289 interest were extracted for further illustration. the exact number and definition of samples 1290 used in the analysis are specified in the legend of fig. 2a and summarized in table s1 . 1291 clusters were annotated based on a double-checking strategy: 1) by comparing cluster 1293 marker genes with public sources, and 2) by directly visualizing the expression pattern of 1294 cytof marker genes. 1295 significant degs between each monocyte cluster and the rest of monocyte subpopulations 1297 were identified by findmarkers function from the seurat package using wilcoxon rank sum 1298 test statistics for genes expressed in at least 25% of all monocyte clusters. p-values were 1299 corrected for multiple testing using bonferroni correction and genes with corrected p-values 1300 lower or equal 0.05 have been taken as significant degs for go enrichment test by r 1301 package/clusterprofiler v.3.10.1 (yu et al., 2012) . 1302 to systematically compare the similarity of marker gene expression in the identified 1305 monocyte/neutrophils subpopulations between the two cohorts, the spearman correlation 1306 coefficients were calculated based on the union of the top 50 marker genes of each cluster 1307 sorted by fold change in the two cohorts, based on their average expression of all cells in the 1308 specific subpopulation. the pairwise comparisons were performed, and the correlation 1309 coefficients were displayed using a heatmap. 1310 the neutrophil space was investigated by subsetting the pbmc dataset to those clusters 1312 identified as neutrophils and immature neutrophils (cluster 5 and 6). within those subsets, 1313 we selected top 2,000 variable genes and repeated the clustering using the snn-graph 1314 based louvain algorithm mentioned above with a resolution of 0.6. the dimensionality of the 1315 data was then reduced to 10 pcs, which served as input for the umap calculation. to 1316 categorize the observed neutrophil clusters into the respective cell cycle states, we applied 1317 the cellcyclescoring function of seurat and visualized the results as pie charts. 1318 a gene signature enrichment analysis using the 'aucell' method (aibar et al., 2017) was 1319 applied to link observed neutrophil clusters to existing studies and neutrophils of cohort 2. 1320 we set the threshold for the calculation of the area under the curve (auc) to marker genes 1321 from collected publications and top 30 of the ranked maker genes from each of neutrophil 1322 clusters from cohort 2. the resulting auc values were normalized the maximum possible 1323 auc to 1 and subsequently visualized in violin plots or umap plots. 1324 1325 scrna-seq umi count matrices were imported to r 3.6.2 and gene expression data 1328 analysis was performed using the r/seurat package 3.1.2 (butler et al., 2018) . 1329 demultiplexing of cells was performed using the htodemux function implemented in 1330 seurat. after identification of singlets, cells with more than 25% mitochondrial reads, less 1331 than 250 expressed genes or more than 5,000 expressed genes and less than 500 detected 1332 transcripts were excluded from the analysis and only those genes present in more than 5 1333 cells were considered for downstream analysis. the following normalization, scaling and 1334 dimensionality reduction steps were performed independently for each of the data subsets 1335 used for the different analyses as indicated respectively. in general, gene expression values 1336 were normalized by total umi counts per cell, multiplied by 10,000 (tp10k) and then log 1337 transformed by log10(tp10k+1). subsequently, the data was scaled, centered and 1338 regressed against the number of detected transcripts per cell to correct for heterogeneity 1339 associated with differences in sequencing depth. for dimensionality reduction, pca was 1340 performed on the top 2,000 variable genes identified using the vst method implemented in 1341 seurat. subsequently, umap was used for two-dimensional representation of the data 1342 structure. cell type annotation was based on the respective clustering results combined with 1343 data-driven cell type classification algorithms based on reference transcriptome data (aran 1344 et al., 2019) and expression of known marker genes. 1345 scrna-seq count data of 229,731 cells derived from fresh and frozen pbmc samples 1348 purified by density gradient centrifugation and whole blood after erythrocyte lysis of cohort 2 1349 (bonn, bd rhapsody) were combined, normalized and scaled as described above (see fig. 1350 s6a). after variable gene selection and pca, umap was performed based on the first 20 1351 principal components (pcs). no batch correction or data integration strategies were applied 1352 to the data. visualization of the cells (fig. s6a) showed overlay of cells of the same type 1353 (e.g. t cells clustered within the same cluster, irrespective of cell isolation procedure). in 1354 other words, cell type distribution was unaffected by the technical differences in sample 1355 handling. data quality and information content was visualized as violin plots showing the 1356 number of detected genes, transcripts (umis) and genic reads per sample handling strategy 1357 split by pbmc and granulocyte fraction. 1358 scrna-seq count data of 139,848 cells derived from fresh and frozen pbmc samples of 1360 cohort 2 (bonn, bd rhapsody) purified by density gradient centrifugation were normalized 1361 and scaled as described above. after variable gene selection and pca, umap was 1362 performed and the cells were clustered using the louvain algorithm based on the first 20 1363 pcs and a resolution of 0.4. cluster identities were determined by reference-based cell 1364 classification and inference of cluster-specific marker genes using the wilcoxon rank sum 1365 test using the following cutoffs: genes have to be expressed in more than 20% of the cells of 1366 the respective cluster, exceed a logarithmic fold change cutoff to at least 0.2, and exhibited a 1367 difference of >10% in the detection between two clusters. the exact number and definition of 1368 samples used in the analysis are specified in the legend of fig. 2d and summarized in 1369 table s1 . 1370 to compare shifts in the monocyte and neutrophil populations in the pbmc compartment of 1373 covid-19 patients, the percentages of the cellular subsets -as identified by clustering and 1374 cluster annotation explained above for the two independent scrna-seq data sets (cohort 1 1375 and cohort 2) -of the total number of pbmc in each data set were quantified per sample and 1376 visualized together in box plots. to determine the statistical significance of differences in cell 1377 proportions between the different conditions, a dirichlet regression model was used, due to 1378 the fact that the proportions are not independent of one another. the r/rdirichletreg 1379 (maier, 2014) package was used. the p-values were corrected for multiple testing using the 1380 benjamini-hochberg procedure. 1381 the monocyte space was investigated by subsetting the pbmc dataset to those clusters 1383 identified as monocytes (cluster 0-4), removing cells with strong multi-lineage marker 1384 expressions, and repeating the variable gene selection (top 2,000 variable genes), 1385 regression for the number of umis and scaling as described above. the dimensionality of 1386 the data was then reduced to 8 pcs, which served as input for the umap calculation. the 1387 snn-graph based louvain clustering of the monocytes was performed using a resolution of 1388 0.2. marker genes per cluster were calculated using the wilcoxon rank sum test using the 1389 following cutoffs: genes have to be expressed in >20% of the cells, exceed a logarithmic fold 1390 change cutoff to at least 0.25, and exhibited a difference of >10% in the detection between 1391 two clusters. the exact number and definition of samples used in the analysis are specified 1392 in the legend of fig. 4a and summarized in table s1 . 1393 j o u r n a l p r e -p r o o f for each patient and time point of sample collection, the proportional occupancy of the 1395 monocyte clusters was calculated, and the relative proportions were subsequently visualized 1396 as a function of time. 1397 scrna-seq count data derived from fresh pbmc samples purified by density gradient 1399 centrifugation and whole blood after erythrocyte lysis of cohort 2 (bd rhapsody) were 1400 normalized, scaled, and regressed for the number of umi per cell as described above. after 1401 pca based on the top 2,000 variable genes, umap was performed using the first 30 pcs. 1402 cell clusters were determined using louvain clustering implemented in seurat based on the 1403 first 30 principle components and a resolution of 0.8. cluster identities were assigned as 1404 detailed above using reference-based classification and marker gene expression. 1405 subsequently, the dataset was subsetted for whole blood samples after erythrocyte lysis and 1406 clusters identified as neutrophils and immature neutrophils, and re-scaled and regressed. 1407 after pca on the top 2,000 variable genes, the neutrophil subset data was further processed 1408 using the data integration approach implemented in seurat (stuart et al., 2019) based on the 1409 first 30 pcs removing potential technical biases of separate experimental runs. umap and 1410 clustering were performed as described above on the top 12 pcs using a resolution of 0.3. 1411 differentially expressed genes between clusters were defined using a wilcoxon rank sum 1412 test for differential gene expression implemented in seurat. genes had to be expressed in 1413 >10% of the cells of a cluster, exceed a logarithmic threshold >0.1. the exact number and 1414 definition of samples used in the analysis are specified in the legend of fig. 7a and 1415 summarized in table s1 . 1416 after cell type classification of the combined scrna-seq data set of fresh pbmc and whole 1419 blood samples of cohort 2 described above, 89,883 cells derived from whole blood samples 1420 after erythrocyte lysis were subsetted. percentages of cell subsets in those whole blood 1421 samples of the total number of cells were quantified per sample and visualized in box plots 1422 separated by disease stage and group. 1423 to categorize the cells within the neutrophil clusters into the respective cell cycle states, we 1434 applied the cellcyclescoring function of seurat and visualized the results as pie charts. 1435 trajectory analysis was performed using the destiny algorithm v3.0.1 (angerer et al., 2016) . generate umap representations all events of a given population of interest were down-1473 sampled to 70,000 cells and then embedded using the tumap function (r uwot package, 1474 https://cran.r-project.org/package=uwot) parameterized by local neighborhood 50, 1475 learning rate 0.5, and using the indicated markers ( 3 8 11 14 22 24 3 8 14 6 10 12 13 17 13 22 7 11 16 8 13 13 18 15 20 6 7 6 8 11 7 8 12 5 8 11 5 7 11 19 9 12 16 17 19 23 9 16 9 16 8 15 13 stat3 fkbp5 lgals9 ifitm3 ifit2 isg15 ifi27 mx2 ifi6 ifit1 herc5 oasl mx1 ifih1 ifi44 ifi44l oas2 serpinb1 il1r2 serping1 cd163 rnase1 ifi16 oas3 adar lgals3bp spi1 defa3 defa4 hsp90aa1 mpo elane prtn3 cd24 bpi cd63 clec5a fut4 hexa pde4d c1qbp ceacam8 anxa1 gsn clec12a nlrc4 olfm4 cybb lcn2 lgals3 ltf mmp8 hp cd101 camp s100a8 s100a12 cd177 tspo rab27a s100p itgam s100a9 s100a6 ifi6 isg15 ly6e ifi16 gbp1 ccr1 c3ar1 ifih1 ddx58 fcgr1a aim2 zc3h12a tlr2 abca1 icam1 inpp4b fbxl17 slc38a1 clec2d itga4 sell s100a11 cxcr2 tlr5 clec4e fcgr2a adam8 slc11a1 nlrp12 tlr4 c5ar1 coro1a cmtm6 tnfrsf1a tnfrsf1b rac1 nlrp3 ptprc ptgs2 sirpa ncoa4 mme s100a4 % exp. transcriptome meta-analysis deciphers a 1524 dysregulation in immune response-associated gene signatures during sepsis scenic: single-cell 1528 regulatory network inference and clustering is a candidate marker for a pathogenic neutrophil subset in septic shock destiny: diffusion maps for large-scale single-cell data in r reference-based analysis of lung single-cell 1536 sequencing reveals a transitional profibrotic macrophage gene ontology: tool for the unification of 1539 biology the pathogenicity of sars-cov-2 in hace2 transgenic mice targeting potential 1545 drivers of covid-19: neutrophil extracellular traps myeloid-derived suppressor cell subsets drive glioblastoma growth 1548 in a sex-specific manner human intestinal pro-inflammatory cd11chighccr2+cx3cr1+ 1552 macrophages, but not their tolerogenic cd11c-ccr2-cx3cr1-counterparts, are expanded 1553 in inflammatory bowel disease article immune suppression by neutrophils in hiv-1 infection: role of pd-l1/pd-1 1556 pathway presence of sars-cov-2 reactive t 1559 cells in covid-19 patients and healthy donors l-arginine 1561 metabolism in myeloid cells controls t-lymphocyte functions recommendations for 1564 myeloid-derived suppressor cell nomenclature and characterization standards integrating single-1567 cell transcriptomic data across different conditions, technologies, and species the gene ontology resource: 20 years and 1571 still going strong deciphering myeloid-derived suppressor cells: 1574 isolation and markers in humans, mice and non-human primates neutrophils which migrate to lymph nodes modulate cd4+ t cell response by a pd-l1 1578 dependent mechanism clinical and immunologic features in severe and moderate coronavirus 1581 disease covid-19 severity correlates with airway 1584 epithelium-immune cell interactions identified by single-cell analysis ccl2 promotes colorectal carcinogenesis by enhancing 1588 polymorphonuclear myeloid-derived suppressor cell population and function cd69: from activation marker to metabolic 1591 gatekeeper from mice to monkeys, animals studied for coronavirus answers mafb determines human macrophage anti-inflammatory polarization: relevance for the 1597 pathogenic mechanisms operating in multicentric carpotarsal osteolysis neutrophils with myeloid derived 1601 suppressor function deplete arginine and constrain t cell function in septic shock patients platelet, monocyte and neutrophil activation and glucose tolerance in 1605 favorable anakinra responses in severe covid-19 patients with secondary 1609 star: ultrafast universal rna-seq aligner genomewide association study of severe 1615 covid-19 with respiratory failure cd163 1618 expression defines specific, irf8-dependent, immune-modulatory macrophages in the bone 1619 marrow complex immune dysregulation in covid-19 patients with severe respiratory failure targets of t cell responses to 1626 sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals complex heatmaps reveal patterns and 1629 correlations in multidimensional genomic data impaired type i interferon activity and 1632 exacerbated inflammatory responses in severe covid-19 patients normalization and variance stabilization of single-cell 1635 rna-seq data using regularized negative binomial regression neutrophil heterogeneity and its role in infectious 1639 complications after severe trauma engagement of cd83 ligand induces prolonged 1643 expansion of cd8+ t cells and preferential enrichment for antigen specificity stroke-induced 1647 immunodepression and dysphagia independently predict stroke-associated pneumonia -1648 the predict study dexamethasone in hospitalized patients with covid-1651 19 -preliminary report simultaneous inference in general parametric 1653 models clinical features of patients infected with 2019 novel coronavirus in wuhan induced cd10 expression during monocyte-to-macrophage differentiation 1659 identifies a unique subset of macrophages in pancreatic ductal adenocarcinoma should we stimulate or suppress immune responses in covid-19? 1663 cytokine and anti-cytokine interventions gene list to a gene regulatory network using large motif and track collections heterogeneity among septic shock patients in a 1670 set of immunoregulatory markers human suppressive neutrophils cd16 bright /cd62l dim exhibit decreased 1673 adhesion toward understanding the origin and evolution of cellular organisms increased expression of neutrophil-related genes 1678 in patients with early sepsis-induced ards suppress lymphocyte proliferation through expression of pd-l1 incidence of thrombotic complications in critically ill icu patients with covid-19 fast, sensitive and accurate 1689 integration of single-cell data with harmony web-based analysis and publication of 1691 flow cytometry experiments immunologic 1695 perturbations in severe covid-19/sars-cov-2 infection studying the 1698 pathophysiology of coronavirus disease 2019: a protocol for the berlin prospective covid-1699 19 patient cohort (pa-covid-19) age and gender leucocytes 1702 variances and references values generated using the standardized one-study protocol combinatorial single-cell analyses of granulocyte-monocyte 1706 progenitor heterogeneity reveals an early uni-potent neutrophil progenitor spleen-derived ifn-γ induces generation of pd-l1 + -suppressive neutrophils during 1710 endotoxemia immunophenotyping of covid-19 and influenza highlights the role 1713 of type i interferons in development of severe covid-19 least-squares means: the r package lsmeans pad4 mediated histone hypercitrullination induces heterochromatin decondensation and 1718 chromatin unfolding to form neutrophil extracellular trap-like structures arginine deficiency is involved in thrombocytopenia and 1722 immunosuppression in severe fever with thrombocytopenia syndrome single-cell landscape of bronchoalveolar immune cells in patients with covid-1726 19 the molecular signatures database hallmark gene set collection mcpip1 suppresses hepatitis c virus 1732 replication and negatively regulates virus-induced proinflammatory cytokine responses dysregulated myelopoiesis and 1735 hematopoietic function following acute physiologic insult antibody responses to sars-cov-2 in patients with covid-1738 19 longitudinal analyses reveal immunological misfiring 1741 in severe covid-19 ebola virus disease is 1744 characterized by poor activation and reduced levels of circulating cd16+ monocytes single cell rna sequencing of human liver 1748 reveals distinct intrahepatic macrophage populations dirichletreg: dirichlet regression for compositional data in r cutadapt removes adapter sequences from high-throughput sequencing 1752 reads validation of diagnostic gene sets to identify critically ill patients with sepsis deep immune profiling of 1758 covid-19 patients reveals patient heterogeneity and distinct immunotypes with implications 1759 for therapeutic interventions the innate immune system: fighting on the front 1761 lines or fanning the flames of covid-19? two new 1764 immature and dysfunctional neutrophil cell subsets define a predictive signature of sepsis 1765 useable in clinical practice barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry platinum-conjugated antibodies for 1770 application in mass cytometry the cd14+hla-drlo/neg 1772 monocyte: an immunosuppressive phenotype that restrains responses to cancer 1773 immunotherapy pathological inflammation in patients with covid-19: a 1775 key role for monocytes and macrophages ultra-high-throughput 1778 clinical proteomics reveals classifiers of covid-19 infection frequencies of circulating mdsc 1782 correlate with clinical outcome of melanoma patients treated with ipilimumab neutrophil extracellular traps 1786 (nets) contribute to immunothrombosis in covid-19 acute respiratory distress 1787 syndrome persisting low monocyte human leukocyte 1790 antigen-dr expression predicts mortality in septic shock update on 1793 neutrophil function in severe inflammation different phenotypes of non-classical monocytes associated with 1797 systemic inflammation, endothelial alteration and hepatic compromise in patients with 1798 dengue heterogeneity of neutrophils detection of sars-cov-2-specific humoral and cellular immunity in covid-1803 19 convalescent individuals cytof workflow: differential discovery in high-1806 throughput high-dimensional cytometry datasets a comprehensive single cell transcriptional landscape 1809 of human hematopoietic progenitors immunopathogenesis of coronavirus infections: 1811 implications for sars biological basis and pathological 1813 relevance of microvascular thrombosis a subset of neutrophils in human 1816 systemic inflammation inhibits t cell responses through mac-1 decoding human fetal liver 1819 haematopoiesis extracting a cellular hierarchy from high-1822 dimensional cytometry data with spade mortality rates of patients with covid-19 in 1824 the intensive care unit: a systematic review of the emerging literature immunotherapies for 1827 covid-19: lessons learned from sepsis an immune-cell signature of 1830 bacterial sepsis dnam-1 and pvr regulate monocyte 1833 migration through endothelial junctions human polymorphonuclear neutrophils express rank and are activated by its 1836 ligand, rankl immunosuppression for hyperinflammation in 1838 covid-19: a double-edged sword? convergent antibody responses to 1841 sars-cov-2 in convalescent individuals differential redistribution of activated monocyte and dendritic cell subsets to the 1845 lung associates with severity of covid-19 hepatic acute-phase proteins control innate immune 1849 responses during infection by promoting myeloid-derived suppressor cell function invariant nkt cells reduce the 1853 immunosuppressive activity of influenza a virus-induced myeloid-derived suppressor cells in 1854 mice and humans regulatory cell therapy in kidney 1857 transplantation (the one study): a harmonised design and analysis of seven non-1858 randomised, single-arm, phase 1/2a trials human neutrophils in the 1860 saga of cellular heterogeneity: insights and open questions myeloperoxidase can differentiate between sepsis and non-infectious sirs and predicts 1863 mortality in intensive care patients with sirs emerging principles in myelopoiesis at 1866 homeostasis and during infection and inflammation surface barcoding of live pbmc for multiplexed mass 1868 cytometry minimizing batch effects in mass 1871 production through regulation of autophagy and is associated with adult still disease neutrophil 1877 diversity in health and disease neutrophil fluorescence: a new indicator of cell activation 1880 during septic shock-induced disseminated intravascular coagulation neutrophil activation during septic shock comprehensive integration of single-cell 1886 data human cd62ldim neutrophils 1889 identified as a separate subset by proteome profiling and in vivo pulse-chase labeling interleukin-3 receptor in acute leukemia leukocyte protease binding to nucleic acids promotes nuclear 1895 localization and cleavage of nucleic acid binding proteins type i ifn immunoprofiling in covid-1898 19 patients early expansion of circulating granulocytic 1901 myeloid-derived suppressor cells predicts development of nosocomial infections in patients 1902 with sepsis myeloid-derived suppressor cells coming 1904 of age review-article myeloid cells in sepsis-1906 acquired immunodeficiency expression of dnam-1 1908 (cd226) on inflammatory monocytes kidney injury enhances renal g-1911 csf expression and modulates granulopoiesis and human neutrophil cd177 in vivo clinical characteristics of 138 hospitalized patients with coronavirus-infected pneumonia in wuhan, china dysregulation of the immune response 1917 affects the outcome of critical covid-19 patients a single-cell atlas of the peripheral 1920 immune response in patients with severe covid-19 pathological findings of covid-19 associated with acute respiratory distress 1926 syndrome increased formation of neutrophil extracellular traps is associated with gut 1929 leakage in patients with type 1 but not type 2 diabetes myeloid-derived suppressor cells: their 1931 role in the pathophysiology of hematologic malignancies and potential as therapeutic targets clusterprofiler: an r package for 1934 comparing biological themes among gene clusters clinical course and risk factors for mortality of adult inpatients with covid china: a retrospective cohort study overly exuberant innate immune response to sars-cov-2 infection. 1940 ssrn electron a dynamic immune response shapes covid-19 1943 progression neutrophil extracellular traps in covid-19 cov-2 infection induces profound alterations of the myeloid compartment • mild covid-19 is marked by inflammatory hla-dr hi cd11c hi cd14 + monocytes • dysfunctional hla-dr lo cd163 hi and hla-dr lo s100a hi cd14 + monocytes in severe • emergency myelopoiesis with immature and dysfunctional neutrophils in severe covid-19 analysis of patients with with mild and severe covid-19 reveals the presence of dysfunctional neutrophils in the latter that is linked to emergency myelopoiesis in brief, the neutrophil space was subsetted to only severe patients (early and late) and only 1438 the most prominent clusters of the latter (clusters 1,2,6,8). the normalized data were scaled 1439 and regressed for umis and a diffusion map was calculated based on the top 2,000 variable 1440 genes with a sum of at least 10 counts over all cells. based on the diffusion map, a diffusion 1441 pseudo time was calculated to infer a transition probability between the different cell states 1442 of the neutrophils. subsequently, the density of the clusters along the pseudotime and 1443 marker gene expression for each cluster were visualized. 1444 enrichment of gene sets was performed using the 'aucell' method (aibar et al., 2017 (aibar et al., ) 1445 implemented in the package (version 1.4.1) in r. we set the threshold for the calculation of 1446 the auc to the top 3% of the ranked genes and normalized the maximum possible auc to 1. 1447the resulting auc values were subsequently visualized in violin plots or umap plots. key: cord-015389-vwgai4k9 authors: nan title: publication only date: 2009-03-25 journal: bone marrow transplant doi: 10.1038/bmt.2009.50 sha: doc_id: 15389 cord_uid: vwgai4k9 nan introduction & objectives: literature states that human postnatal dental pulp stem cells (hdpscs) have the ability to differentiate to osteoblastic cells. the purpose of this paper is to present the results obtained in the differentiation of hdpscs with three different media and to compare their osteogenic ability. materials & methods: human dental pulp was extracted from teeth of healthy adult subjects aged 21 to 45 years. the pulp was gently removed and immersed in a digestive solution for 1 h at 37cº. after digestion, cells were cultured and adherent cells were isolated. after the second pass the cells were placed in three different 75 fl asks with three classes of differentiation media. medium 1: osteodiff (miltenyi®); medium 2: alpha-mem supplemented with 15% fetal bovine serum (fbs), 100 u/ml penicillin, 0.1 mg/ml streptomycn, and 0.25 mg/ml amphotericin b; medium 3: alpha-mem medium, supplemented with 20% fbs, 100 mm 2 p-ascorbic acid, 2 mm l-glutamine, 100 u/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 mg/ml amphotericin b. flasks were incubated at 37ºc in a 5% co2 and the medium changed twice a week for 35 days. to quantify the different amount of mineralized nodules the absorbance rate was used. results & discussion: hdpscs were obtained at a good rate and differentiated with any of the three media into osteoblastic cells that developed mineralization nodules (clusters), as revealed by alizarin red staining. this staining was signifi cantly more intense with medium 1 than medium 2 and medium 3 (absorbance values 1.107, 0.576 and 0.325 respectively). conclusions: this study demonstrates the ability of hdpscs to differentiate into osteoblasts. the medium 1 (osteodiff medium, miltenyi®) , was the best to differentiate these cells to the osteogenic lineage. long-term haematopoietic reconstitution and clinical evaluation of autologous peripheral blood stem cell transplantation after cryopreservation of cells at -80°c in a mechanical freezer for longer than 6 months l. calvet, a. cabrespine-faugeras, n. boiret-dupre , e. merlin, c. paillard, m. berger, j.-o. bay, o. tournilhac, p. halle chu (clermont-ferrand, fr) controlled-rate freezing in 5 or 10% of dmso and storage in the nitrogen is the standard technique for cryopreservation of hematopoietic progenitor cells (phs). the main inconveniences are its high cost and dmso toxicity. many teams try to reduce dmso infused by phs concentration before cryopreservation or wash before infusion. however, labor intensive increases the cost and not free of cell loss. we developed an easier and cheaper technique, the cryopreservation of the phs at -80°c, an uncontrolled rate freezing with 2.5% hes, 1% albumin and only 3.5% of dmso allowing infusion without wash. this technique preserves the functional capacities of phs, can produce successful engraftment and reduces toxicity during infusion. does the cryopreservation of the phs at -80 °c allow a long-term hematopoietic reconstitution and clinical course even if storage is greater than 6 months? 239 patients who had undergone 325 autografts (204 adults, 121 children) were studied. the median storage time of the 445 phs cryopreserved was 1.7 months [0. with 9.7% (43/402) preserved more than 6 months (median 13,7 [6-136] ). the median recovery of nucleated cells and cd34+ cells were similar, for the preserved phs <or > 6 months (71% versus 70% , p=0.44) and (104% versus 91% , p=0.11), respectively. only mild infusion-related toxicity was observed in 29.8% (nauseas/vomiting 8.6%, shivers 4.7%). median time to reach 0.5x109/l granulocytes (pn), 20 and 50x109/l platelets (pl) were 13 , 12 and 15 days respectively. delay to reach hematopoietic reconstitution was similar between phs preserved < or > 6 months except for pl > 20x 109/l. this delay was signifi cantly longer for phs kept > 6 months 12 versus 14 [6-46] (n=0.015) with a correlation between cd34+ cells dose and the number of days need to reach 20x109/l pl. in order to assess long term hematopoietic reconstitution, only patients without other treatment (n=128) were studied at 3, 6 and 12 months. median values were 150, 168 and 185x109/l for the platelets and 2,37, 2,43 and 2,8 x109/l for the pn at 3, 6 and 12 months respectively. mortality at 100 post-autograft days was of 5.5%. median overall survival was 54 months and 3 years survival rate was of 55%. the long term hematopoietic reconstitution was satisfactory. this easier and cheaper cryopreservation method leads to successful engraftment even if phs had been cryopreserved more than 6 months. improve mobilization in these patients have been described. another exciting option for these patients is the new cytokine, amd3100. this agent is an inhibitor of sdf1 binding to cxcr4 and appears to promote mobilization of cd34+ cells into the circulation. the use of this amd3100 in combination with g-csf in patients unable to collect adequate cd34+ cells with g-csf alone was recently reported in 280 patients with lymphoma and multiple myeloma (mm) . in this study g-csf was given at a dose of 10 mcg/kg per day and amd3100 was started at 240 mcg/kg on day 4 of mobilization. in contrast, clinical studies showed that aml, cll and pcl cells may also be mobilized by amd3100 via cxcr4 inhibition. due to these concerns, aml, cll and pcl patients are excluded from amd3100 trials. we here report 8 patients (3 female/5 male) with non hodgkins lymphoma (n=4), mm (n=3) and germ cell cancer (n=1) who failed stem cell mobilization after chemotherapy and g-csf administration (patient characteristics table 1 ). patients received 2 x 5 µg/ kg daily of g-csf for 4 days followed by 240 µg/kg of amd3100 given subcutaneously 10-11 hrs before collection on day 5. our aim was to assess the effect of amd3100 on the mobilization of cd34+ cells. administration of g-csf and amd3100 were continued daily until end of collection cycle. adequate collection of cd34+ cells (2.6 and 5.54 x 106 cd34+ cells/kg) were achieved in 5 patients. in 2 patients additional bone marrow collection were performed, 1 patient failed mobilization with amd3100. until now 4 patients underwent autologous transplantation with 1.48, 2.6, 3.35 and 3.58 x 106 cd34+ cells/kg respectively and achieved sustained leukocyte and platelet engraftment. in conclusion, amd3100 in combination with g-csf was generally safe and offers a new treatment to collect cd34+ cells for autologous transplant from poor mobilizers. due to the reported mobilization of leukemic cells, amd3100 should be restricted to patients with lymphomas, mm and solid tumors. evaluating the effect of substance p on expansion of human umbilical cord blood cd34+ haematopoietic stem cells in a serum-free media s. shahrokhi (1) , m. ebtekar (1) , k. alimoghaddam (2) , m. kheirandish (3) , a. pourfathollah (1) , a.r. ardjmand (4), a. ghavamzadeh (2) (1)tarbiat modares university (tehran, ir); (2) hematology, oncology and bone marrow transplantation research center (tehran, ir) ex vivo expansion of cord blood hematopoietic stem cells has been progressively interested as alternative sources for stem cell transplantation. using different combination of growth factors especially cytokines has been investigated in most reports, but there are little evidence about regulatory roles of other factors including neuropeptides in this way, then we choose substance p (sp) to evaluate its effect on expansion. material and methods: cd34+ purifi ed from umbilical cord blood by macs, were cultured in a serum-free liquid culture system. different concentration of sp used in combination with cytokine cocktail of scf, fl, tpo, il3 and il6. phenotypic and functional analysis of the cells produced in culture, was performed by fl owcytometry. count and percentage of cd34+ cells were compared in different groups of treated cells. results: ex vivo expansion cultures of cd34+ cells of ucb were signifi cantly increased, in cells cultivated in "sp + cytokine cocktails" group compared cytokine groups alone. conclusion: consideration of the role of other growth factor such as sp along with cytokines, may enable us to overcome the diffi culties before us in ex vivo expansion of cord blood cells. our studies indicate that sp could act as a superior supplement for expansion of ucb-hsc cytokine cocktails. additional studies are needed to establish the functional activity of expanded ucb-hsc as well as the effects of substance p. standard protocols for cryopreservation of peripheral blood progenitor cells (pbpc) use rate-controlled freezing and storage in liquid nitrogen, which are both time-consuming and expensive. in the last 11 years we used a simplifi ed method (galmes et al 1995) consisting of storage in a mechanical freezer at -80ºc, with dmso as the sole cryoprotectant. this study evaluates the safety of this approach, in terms of infusion-related toxicity and hematopoietic reconstitution, in 385 consecutive autologous transplantations performed from 4/97 to 9/08 in 348 patients (median age 46; underlying disease: lymphoma in 178, myeloma in 131, acute leukaemia in 17, breast cancer in 22). after mobilization with g-csf ± chemotherapy (usually cyclophosphamide 1.5 g/m²) pbpc were collected in a cs3000+ separator (fenwall), mixed in autologous plasma and dmso (to a fi nal concentration of 10%) and frozen in plastic bags (cryocyte, fenwall) at -80ºc. median cd34+ count was 3.6x106/kg and median storage duration was 32 days (6-564). infusion-related toxicity was frequent (25%) and generally mild (transient hypoxemia, broncospasm, hypertension or arrhythmia, and abdominal pain, nausea or diarrhea) but there were 2 cases of acute congestive heart failure and 1 anaphylactic shock (probably related to dmso). engraftment to 500 neutrophils and 20,0 platelets/ul occurred on days +11 and +14 (median). bacteremia occured in 25% transplantations, and grade 3 or 4 toxicity in 20%. median hospitalisation duration was 19 days. mortality at day +30 and +100 was 0.5 and 2.8% respectively. an engraftment delay beyond d+60 was seen in 2 cases. there were no secondary graft failures. with a median follow up of 37 months, 66% patients are alive. these results confi rm the feasibility and safety of this simpler and cheaper cryopreservation methodology. belarus y. isaikina, n. minakovskaya, o. aleinikova belarusian center for ped oncohematology (minsk, by) introduction: recent studies suggest that cotransplantation of mesenchymal stem cells (mscs) can improve the engraftment of allogeneic hematopoietic stem cells and prevent graft-versus-host disease (gvhd) due to their immunomodulatory properties. we analyzed the clinical effect of msc infusion on day +30 after hsct for prophylaxis of gvhd and applying of mscs for treatment of severe steroid-resistant gvhd. patients and methods: eight pts after allogeneic hematopoetic stem cell transplantation (hsct) underwent mscs infusions (median age of pts was 11 years, male/female: 6/2) between 2006 and 2009. diagnoses included:all-4, aml-1, aa-2, mds-1.gvhd prophylaxis for pts with all, mds consist of csa and mtx 10 mg/m² (n=3); for pts with aa -csa+mmf; for pts with aml -csa and mtx 10 mg/m² (n=4). for the treatment of gvhd all pts received metylprednisolon 1-2 mg/kg. mscs were prepared applying technique of expansion in vitro from bone marrow of hla-identical siblings, haplo-identical and haplo-nonidentical family donors and unrelated donors. four pts received mscs once and four -twice. for three pts mscs was used for prophylaxis of gvhd on day +30 after hsct and the median dose was 1,0(0,7-1,5)x106/kg and fi ve pts received mscs for treatment of steroid-resistant gvhd with medium time of mscs infusion after hsct 126(110-151) days and the dose was 2,2(1,3-3,7)x10 6 /kg. results: there was no evidence of early and late side effect of msc infusion. one patient died from pulmonary gvhd 1 month after cotransplantation mscs and seven pts-alive. all pts (n=3), who received mscs on day +30 for prophylaxis gvhd developed grades ii-iv gvhd and needed the secondary mscs infusion and the median time between mscs infusions were 120(90-150) days. four pts out of fi ve with steroid-resistant gvhd showed signifi cant improvement of clinical sign of gvhd that allowed reducing immunosuppressive therapy and stopping the steroids. conclusion: our experience demonstrates the absence of positive gvhd prophylactic effi cacy when infusion of mscs was done on day +30. however, we observed decreasing of gvhd grades from iii-iv to 0-ii, when mscs were used as treatment of steroid-resistant gvhd. clinical characteristics of early-onset acute graft-versushost disease after allogeneic haematopoietic stem cell transplantation t. yamashita, y. najima, t. kikuchi, h. muto, c. sakurai, w. munakata, m. yamamoto, k. ohashi, h. sakamaki, h. akiyama tokyo metropolitan komagome hospital (tokyo, jp) acute graft-versus-host disease (gvhd) is one of the major factors that have infl uence on the outcomes of allogeneic hematopoietic stem cell transplantation (hsct). traditionally, acute gvhd has been defi ned as a syndrome after neutrophil engraftment within the fi rst 100 days following hsct. but in our practice, we sometimes encounter acute gvhd that may occur s367 both early, even before engraftment, and late, beyond day 100. the latter has been defi ned as "late-onset acute gvhd", but the former may not be clearly identifi ed yet. in this retrospective study, we evaluated the incidence, clinical manifestations and outcomes of "early-onset acute gvhd", defi ned as that occurring before engraftment after transplantation, among 117 consecutive myeloablative allogeneic hscts at our hospital. of 117 patients, the median age was 40 years. ninety-three percent of patients received allogeneic hsct for hematologic malignancies. thirty-eight percent of patients received an hlamatched related donor transplant, 40% received hla-matched unrelated donor grafts and 19% received hla-mismatched unrelated donor grafts. the stem cell source was bone marrow in 82% of patients and peripheral blood in 18%. the conditioning regimen was tbi-based for 34% of patients and 60% received busulfan-based conditioning. forty-three percent (n=50) of the 117 cases developed grade ii-iv acute gvhd. of these, 30 (60%) cases were described as early-onset acute gvhd (group e). other 20 cases of acute gvhd occurred after engraftment (group c). the median onset date of acute gvhd is day 10 in group e and day 28 in group c. grade iii-iv acute gvhd was seen in 27% of group e and in 35% of group c (p=0.34). the frequency and severity of each involvement site were comparable in both groups. major primary therapy for acute gvhd was mpsl 2-2.5mg/kg/day, but 41% cases in group e were refractory for this primary therapy and 18% in group c (p=0.05). three-years overall survival (oas) was 58% in group e and 49% in group c (p=0.83). in group c, oas of 19 cases without gi symptoms was 71%, whereas oas of 11 cases with gi involvement was 36% (p=0.02). in group c, oas was not affected by with or without gi-gvhd (p=0.89). in conclusion, early-onset acute gvhd accounts for a substantial proportion of acute gvhd after allogeneic hsct. patients with early-onset acute gvhd tend to be refractory to steroid therapy and will have poor prognosis if gi involvement exists. contrast enhanced ultrasound sonography in intestinal acute graft-versus-host disease e. benedetti (1) a 20 year old female with high risk acute b cell leukemia received a fully ablative peripheral blood stem cell transplant from a 1 allele (at the b locus) mismatched unrelated donor. conditioning consisted of cy/tbi and gvhd prophylaxis of cyclosporine (csa) and short course mtx. on day +19 she developed steroid refractory (biopsy proven) acute skin gvhd. photopheresis was started with major skin improvement. on day +102 she developed nausea, vomiting and profuse diarrhea. standard endoscopy with gastric biopsies showed gvhd. infections were ruled out. a trans-abdominal sonography (ta-us) revealed mucosal oedema and thickening of the terminal ileum (5.1 mm) and the ascending colon. moreover, pillcam capsule endoscopy showed mucosal oedema, erosions and lymphagectasies. infl iximab at 10mg/kg was added and, after 2 doses, despite a major clinical improvement, her terminal ileum was still thickened. to investigate if this thickening was associated with residual active gvhd she underwent a contrast enhanced ultrasound sonography (ceus) using a linear phased-array 7.5-mhz transducer. a sulphur hexafl uoridebased with a phospholipid shell microbubble contrast agent (sonovue®, bracco) was injected i.v. as a bolus (2.4 ml) followed by 5 ml saline fl ush. sonovue® is a blood pool second generation contrast agent. ceus showed an intense and sustained enhancement in the arterial phase involving the whole ileum wall with a late phase wash out. such enhancement pattern has been previously described in active crohn disease. given the clinical improvement, infl iximab was discontinued to reduce the risk of infections. however, as ceus revealed active gvhd she continued on budesonide, beclometasone, csa and prednisone. forty days later her abdominal symptoms had completely resolved and a ta-us showed a normal terminal ileum. four months later her intestinal gvhd (confi rmed by colon biopsies) fl ared. ceus was performed on descending colon (most involved intestinal tract by standard ultrasonography) and showed intense arterial phase enhancement with late phase wash out. rituxan and mmf were added with slow resolution of symptoms and normalisation of us features. in conclusion ceus showed residual gvhd activity despite the improved clinical symptoms. moreover, good concordance with clinical symptoms and standard colonoscopy when gvhd fl ared was also shown. further prospective studies are needed to evaluate its usefulness in monitoring intestinal gvhd. extensive chronic graft-versus-host disease is a frequent complication after peripheral blood stem cell transplantation -results of long-term follow-up d. stamatovic, l. tukic, b. balint, o. tarabar, m. elez, g. ostojic, b. todoric zivanovic, z. tatomirovic, o. tasic, b. cikota, m. malesevic, s. marjanovic military medical academy (belgrade, rs) introduction: many studies have compared effi cacy of allogeneic stem cell transplantation (sct) from peripheral blood (pb) with bone marrow (bm), but fi nal conclusion concerning this treatment modality is still not well defi ned. aim: to compare effi cacy of pbsct with bmt in the treatment of hematological malignancies with respect to engraftment, transfusion need, frequency and severity of acute and late complications and overall survival (os). methods: we have analyzed 132 patients (pts), median age 27 years (9-52), m/f 84/48, with various hematological diseases (saa-18, cml-31, aml-29, all-38, mds-8, mm-2, mh-2, granulocytic sarcoma-2) in whom we perfomed allogeneic sct from 1989 till 2008. in 15 pts we perfomed secondary allogeneic sct in due to graft rejection (2) or relapses (13 pts). pts were divided into two groups concerning sc origin-69 pts in bm group and 63 pts in pb group. all pts had hla-dr sibling transplant (5 singeneic, 121 fully matched, 4 mismatched and 2 haploidentical). sc were collected from bm up to standard method and from pb with one apheresis after fi ve days aplication of granulocytic growth factor. all pts have received unmanipulated suspension of sc. conditioning were adjusted to primary diseases and gvhd prophylaxis was mostly combination of cyclospirine a and metothrexate. prevention of infections were standard. results: pts with sc originate from pb have received signifi cantly more mononuclear cells (10,07±7,31 vs 2,33± 0,79, p<0,001) in comparisson with bm. engraftment was more rapid (p<0,001) in the pb group approximately for 6 days. transfusion requirements were much higher in bm group (p<0,01). those pts had more frequent oropharingeal mukositis grade 3-4 (33,33% vs 9,5%, p<0,05). there were no difference in the incidence of acute (44,4% vs 49,2%, ns) or chronic gvhd (38,6% vs 54,5%, ns). pts with pbsct had signifi cantly more frequent extensive cgvhd (29,5% vs 12,4%, p<0,05). there were no difference considering trm (10,1% vs 15,1%, ns) or relapses (21,7% vs 22,2%, ns). pts with bmt had better overall survival but with no statistical signifi cancy. conclusion: results of this analysis mostly corresponds with other studies showing that pbsct have rapid engraftment and less acute complications. pbsct is connected with more frequent extensive chronic gvhd that is potentialy fatal, making results of this particular treatment option less better. future will bring defi nite estimation of pbsct effi cacy. a preliminary study of human natural killer t-cell recovery post allogeneic stem cell transplantation b. rees (1) , r. morse (1) , s. robinson (2) , j. hows (1) , c. donaldson (1) (1)centre for research in biomedicine, university of the west of england (bristol, uk); (2)university hospitals bristol nhs foundation trust (bristol, uk) natural killer t cells (nkt), defi ned by their cell surface immunophenotype cd3+, v alpha 24+, v beta 11+ and their specifi c activation pathway by the glycolipid alpha-galactosyl ceramide are a unique and small (0.01-0.1%) subset of lymphocytes. these cells may play a key role in the cure of leukaemia after stem cell transplantation (sct) through activation of the graft versus leukaemia (gvl) effect. they have the ability to stimulate both innate and adaptive immune responses through cytokine production and the activation of 'classical' t, b and natural killer (nk) cells. campath, a complement fi xing monoclonal antibody targets the cd52 antigen expressed by t, b and nk cells and may be used in vitro and/or in vivo for donor lymphocyte depletion during stem cell transplantation. our previous work has shown that cd3+, v alpha 24+, v beta 11+ nkt cells also express the cd52 antigen and so are also susceptible to damage by campath. twelve patients (median age 45.5 years, range 21 -57) on the bmt unit, university hospitals bristol were recruited. diagnoses were aml (3), all (1), anll (1), cml (2), mds (2), nhl (2) , and hd (1) . seven received reduced intensity conditioning, 4 tbi and 10/12 received campath. all patients received adult stem cells, 4 from matched siblings, 8 from unrelated donors. nine survived more than 1 year, including the patient with hd who relapsed 6 months post autologous sct and is alive 20 months post matched unrelated sct. the normal range for nkt cell numbers in adult blood was established, mean 0.71 x 106/l (sd 0.92) (n=18). cells stained with cd3-pecy5, v alpha 24-fitc and v beta 11-pe were analysed using the becton dickinson facs vantage se cell sorter with cell quest software. recovery of nkt cells was studied up to 18-24 months post transplant, with mean levels of 0.10 ± 0.04 x 106/l. all individual values were below those in the normal adult population. recovery of other lymphocyte subsets was comparable with those reported in previous studies. nk cells recovered to within their normal range 3 to 6 months post sct, cd8 t cells numbers were within the normal range by approximately 6 months and cd4 t cells only attained values in their normal reference range by 18 months. the slow recovery of nkt-cells has not been previously reported and this may contribute to a reduced gvl effect. n. nakano, a. kubota, m. tokunaga, y. takatsuka, s. takeuchi, t. itoyama, a. utsunomiya imamura bun-in hospital (kagoshima, jp) background: adult t-cell leukemia/lymphoma (atll) has a poor prognosis because of its chemo-resistance. many chemotherapeutic regimens have been created but none of them have shown suffi cient results. we proposed allogeneic stem cell transplantation (allo-sct) for atll patients and showed an improved survival rate. however, relapse or progression of atll is one of the major limiting factors of survival in post sct patients. objectives: in order to establish a better treatment strategy for poor responders after sct for atll, we analyzed the outcome of relapse or progression cases after allo-sct. we paid special attention to the graft versus atll (gvatll) effect. methods: there were 33 atll patients in which allo-sct was performed in imamura bun-in hospital (ibh) from june 1998 to november 2007. twenty seven cases survived over 90 days after sct. sixteen of the 27 patients relapsed. using data in medical records of ibh, we analyzed transplant characteristics and the outcome of these 16 patients retrospectively. results: disease status at sct was cr in 2 pts, 2 pr, 5 sd, and 7 pd. eight patients received conventional stem cell transplantation (cst) and the other eight patients received reducedintensity stem cell transplantation (rist). fourteen patients in 16 obtained remission (9 cr and 5 pr), but the remaining 2 did not (1 sd and 1 pd) after sct. the sites of relapse or progression in 16 were skin in 10 patients, 6 lymph node, 7 peripheral blood, 3 central nervous system, and 1 bone. all patients discontinued immunosuppressants after relapse or progression. eleven patients obtained remission. especially, in 6 out of 11 patients, remission was obtained only by discontinuation of immunosuppressants, and the time to remission after discontinuation of immunosuppressants was between 1 to 14 days. twelve patients were complicated with acute gvhd (grade i-iv). twelve patients died after sct. the causes of death were disease progression of atll in 5 patients, 3 acute gvhd, 3 infectious complications, and 1 interstitial pneumonia. four patients who were complicated with acute gvhd survived over 24 months. conclusions: a certain number of patients obtained remission only by the discontinuation of immunosuppressants. four patients survived more than 2 years with their complication of acute gvhd. these results suggest that the gvatll effect after sct exists and plays an important role in longer survival for poor responders of post allo-sct in atll patients. adoptive immune transfer in paediatric and young adult patients with refractory malignancies p. sovinz, w. schwinger, h. lackner, m. benesch, a. moser, c. urban medical university graz (graz, at) background: patients with metastatic malignancies refractory to or relapsing after conventional ± high-dose chemotherapy have a poor prognosis. graft-versus-tumor (gvt) effects have been reported in small numbers of patients for various solid tumors. patients and methods: eight pediatric and young adult patients (male: female = 3:5; age 1.9 to 22 years) underwent 9 allogeneic hematopoietic stem cell transplantations (allohsct). diagnoses were relapsed/ refractory neuroblastoma (n=3), second relapse of hodgkin's disease, refractory mediastinal large-b-cell-lymphoma, metastatic ewing sarcoma/ osteosarcoma /wilms tumor, respectively. five patients had received high-dose chemotherapy with autologous stem cell rescue. conditioning regimens consisted of fl udarabine (n=8) combined with melphalan ±atg (n=2) or melphalan/thiotepa/okt3 (n=5) or treosulfan/thiotepa/okt3 (n=1); and treosulfan/melphalan (n=1). haploidentical donors (parents, n=6) underwent 2 aphereses: one product was cd3/19 depleted, the other cd34selected; grafts from matched donors (siblings:n=2, unrelated: n=1) were not manipulated. median cd34-number was 12.8 x 106/kg; median cd3-number in haploidentical grafts was 6.35x 104/kg. in the absence of graft-versus-host disease (gvh) immunosuppression was stopped median on day +37. to date, a median of 7 donor lymphocyte infusions (dli; 1-66; dose range:2.5x104 to 3x106) were given to 7/8 patients, starting on median day + 50. results: neutrophil engraftment (>1.0x 109/l) was achieved median on day +9. acute gvh of the skin (i-ii) developed in 3 patients, of skin+liver (iii) in one; chronic gvh occurred in 3 patients (skin:n=3, gut:n=1) there was no transplant-related mortality; 6/8 patients survive for a median of 310 days (range: 64-777) in complete (cr; n=2) or partial remission (pr; n=3) with ongoing regression (disease status not yet evaluated: n=1). two patients who were transplanted in disease progression showed partial response after allohsct but eventually died of progressive disease on day +84 (mediastinal large-b-cell-lymphoma) and +126 (neuroblastoma, after the second allohsct). conclusions: eight heavily pretreated pediatric and young adult patients with poor-prognosis metastatic malignancies tolerated the conditioning regimens well. all patients showed at least transient partial response to allohsct ±dli; six patients in partial remission or better before allohsct survive in cr or pr with evidence of further tumor regression. cmv infection in seropositive patients with haematologic malignancies after allogeneic peripheral blood stem cell transplantation t.-d. tan koo foundation sun yat-sen cancer center (taipei, tw) objective: to investigate the incidence and outcomes of cmv infection in our seropositive population patients after allotransplant as compared with other western patients. we also investigate the impact of post-transplant occurrence of acute graft-vs-host disease and the use of anti-thymocyte globulin upon the outcome of our patients. methods: 68 cmv seropositive patients of various hematologic malignancies underwent allogeneic peripheral blood stem cell transplantation at our institute between march 2001 and november 2008. we used weekly cmv pcr to monitor cmv infection following neutrophil engraftment until day +90 or when any infectious complication occurred. when two consecutive pcrs were positive with >1000 copies present or cmv was found histopathologically, we treated patients with intravenous ganciclovir 5mg/kg q12h for 14 to 21 days. results: 68 patients (median age 38.5, 19~59) of various hematologic malignancies including aml (n=28), cml (n=10), all (n=9), nhl (n=14), hl (n=4), myeloma (n=2), myelodysplastic syndrome (n=1), underwent myeloablative or non-myeloablative allotransplant (51 vs 17). the source of stem cells includes related (48 patients), unrelated (16 patients), and umbilical cord blood stem cell (4 patients). cmv infection or reactivation rate was 21.3% (13 in 61) with median date of occurrence ranges +15 to +267 days with the median of +45 days and the immediate cmv-related mortality rate was 23.1% (4 in 13). the incidence of cmv infection in patients with grade 0~i vs ii~iv acute gvhd are 6.25% vs 42.31%, respectively, with risk ratio 11 (p=0.0039). the occurrence of cmv infection in patients with or without the use of anti-thymocyte globulin use was 26.67% vs 20.0%, respectively, with risk ratio 1.46 (p=0.59). the 5-year event-free survival and overall survival of our patients with or without cmv infection are 38.5% vs 72.2%(p=0.015), and 38.5% vs 73.9%(p=0.004), respectively. conclusions: our cmv seropositive patients do not have higher incidence of cmv infection or reactivation than other lower seropositive patients reported in the western world. there is an increased incidence of cmv infection in the patients who suffer from grade ii~iv acute gvhd, and there are signifi cant differences in efs and os between patients with or without cmv infection. on the contrary, the impact of atg use in our patients is not clear. objectives: patients after hematopoietic stem cells transplantation (sct) have markedly increased susceptibility to moulds infections. according to recent data, the moulds of fusarium spp are emerging as human pathogens associated with significant morbidity and mortality in immunocompromised patients. in current report we are describing disseminated invasive fungal infections caused by fusarium incarnatum in three recipients of allogeneic hematopoietic stem cells, a pathogen not earlier reported for such patients. methods: blood samples were analyzed using automatic bact/ alert system. the culture and identifi cation were performed according to conventional microbiological procedures. the sabouraud agar was used for strain's isolation and the samples were incubated in 30°c for 10 days. the cream to nut-brown mould's colonies were suggestive for fusarium incarnatum. also the microscopic analysis of direct samples revealed microand macroconidias typical for fusarium genus. results: the 46-years-old male and a 28-years-old female patients, with relapsed and refractory acute myelogenous leukemia (aml) have been treated by allogeneic sct from matched unrelated donors after myeloablative conditioning. the third patient, a 51-years-old woman with hodgkin's lymphoma relapsed after autologous sct was transplanted from hla-matched sibling donor after reduced intensity conditioning. all patients suffered from neutropenic fever which did not respond to broad-spectrum antibiotics and fl uconasole. the appearance of nodular, painful skin lesions with characteristic dark red colour and central necrotic area in later stadium suggested skin microembolism caused by infectious microorganism. the mycological analysis confi rmed fusarium incarnatum as a pathogen. i.v. voriconazole in standard doses was started as soon as invasive fungal infection was suspected. the two female patients responded well to voriconazole with gradual resolution of fever and skin lesions. this corresponded with neutrophil engraftment. the male patient with aml died of disseminated fusariosis (autopsy confi rmed) before achieving engraftment. conclusions: we identifi ed fusarium incarnatum as a new mould pathogen which can cause disseminated fatal infections in immunocompromised patients and sct recipients. although the voriconazole was proven to be an effective agent to treat these patients, the hematological recovery seems to be a prerequisite factor needed to survive the disseminated fusariosis. background: infections are the most common complications of stem cells transplantation and chemotherapy induced neutropenia. bacterial infections predominate during the early stage after transplantation. during this phase deep neutropenia and central venous catheter are the most important risk factors. because of high rate of mortality due to gram-negative bacteria, prophylaxis against this microorganisms is mandatory, but this strategy offer gram-positive predomination in all sites of isolation. despite low rate of mortality due to gram-positive bacteria, infections caused by streptococcus today became a real problem. material and methods: during a 8 years period we have performed 144 stem cells transplantation in 134 patients with different hematological malignancies(aml: 74; all: 6; cml: 7; cll: 1, nhl: 13; hodgkin diseases: 16; multiple myelomas: 24; aplastic anaemia: 1;myelofi brosis:1 ewing sarcoma: 1; male:78 female 66. median age: 34 years (12-63). in order to monitoring local micro-fl ora we perform in all patient two times a week: blood-culture, sputum, urine-culture, and simples from central venous catheters. cultures were performed using standard microbiological tools. patients were treated in sterile room conditioned with hepa fi lters, gram-negative prophylaxis with ciprofl oxacine 1,0gr. per day, low bacterial diet. results: gram-positive cocci were predominantly isolated microorganisms (70%), then gram-negative bacteria (20%) and fungi (10%). the most frequent isolated bacteria was staphylococcus coagulaza negative, from central venous catheter, while streptococcus pneumonia was the most common bacteria isolated after day +12, predominantly from sputum. meticillin resistant staphylococcus aureus (mrsa) was isolated in 10% from all gram positive bacteria. we have no vancomicyn-resistant enterococcus isolation. conclusion: the epidemiological pattern of bacterial infection continues to evolve globally and locally at the institutional level, as do patterns of susceptibility and resistance. these trends are often associated with local treatment practices and have a signifi cant effect on the nature of empirical antibiotic prophylaxis and therapy. in our center gram positive bacteria were isolated predominantly. gram-positive prophylaxis is doctrinary used in some centers, but there is a problem with gram-positive resistance. heptavalent pneumococcal vaccination may be reasonable choice. background: invasive fungal infections (ifi) are an important life-threatening complication after allogeneic hematopoietic stem-cell transplant (ahsct). risk factors that further increase the risk of ifi in these patients include prolonged neutropenia, graft failure, immunosupression and graft-versus-host-disease (gvhd). aim: to evaluate the effi cacy and safety profi le of posaconazole as prophylaxis of invasive fungal infection after ahsct. material and methods: in patients at high risk who received posaconazole for prophylaxis we analyzed the incidence of ifi during the treatment period. demographic, clinical, laboratorial and radiologic variables of all patients were studied including age, gender, underlying disease and it´s status at allogeneic transplantation, presence of gvhd, treatment with steroids, adverse events, galactomannan antigen in plasma and high resolution computed tomography (ct-scan). adverse events were also analyzed. results: from a total of 44 patients received posaconazol 37 patients were included in the study, among them 34 received ahsct. during the treatment period there were no proven ifi reported. probable ifi were reported in 1 patient. no serious adverse events related to treatment were reported. during the observational period the overall mortality was 21% (8 patients) and none of them died due to ifi. 19 patients (51,4%) were receiving steroids during the treatment period and none of them developed ifi. the incidence of global gvhd was 65%. acute gvhd incidence was 46%. 3 patients had galactomannan positive and ct-scan were performed in all of them without found ifi in any case. conclusions: posaconazole prophylaxis is a useful and safe approach in order to prevent ifi avoiding systemic antifungal treatment in patients who had undergone ahsct. mucormycosis are an emerging form of invasive fungal infections (ifi) with high mortality rate (60%). early treatment contributes to improve prognosis. posaconazole is a broad spectrum azole that prevents ifi in patients with aml and in patients receiving an immunosuppressive treatment for gvhd. we describe two cases of mucormycosis (cunninghamella bertholletiae) in patients receiving posaconazole prophylaxis. the fi rst received allogeneic haematopoietic stem cell transplantation with reduced-intensity conditioning for myeloma in relapse. because of grade ii cutaneous gvhd, corticosteroids s371 were added to ciclosporine 2 months later associated with posaconazole prophylaxis. however, the patient developed a digestive gvhd. at this date, cunninghammella bertholletiae was found in bronchioalveolar lavage cultures. amphotericin b was added. the patient died with disseminated infection. autopsy confi rmed multiple pulmonary lesions of mucormycosis. the second patient was hospitalised with aml for induction therapy. posaconazole was introduced on the fi rst day. ten days after, a febrile episode occurred without documentation. liposomial amphotericin b was substituted. five days later, mucormycosis was identifi ed in skin biopsy. despite anti-fungal treatment associating amphotericin b and posaconazole, he died 2 months later with disseminated infection. residual concentrations of posaconazole were assessed retrospectively by hplc, using sera conserved at a temperature of 4°c (therapeutic residual plasma concentration: 0.5 and 1 mg/l). for the fi rst patient, the serum concentration was below detection threshold (<0.1mg/l). for the second patient, two sera were collected at prophylaxis and curative treatments (0.5 and 0.6 mg/l, respectively). in both cases, the pathogens were susceptible to posaconazole (in vitro minimal inhibitory concentrations values). our second patient had probably been imunocompromised for several months (long-lasting neutropenia preceding the onset of aml, and history of diabetes). our fi rst patient had an intestinal gvhd with major diarrhoea, which was likely responsible for the very low (undetectable) levels measured when mucormycosis was diagnosed. in conclusion, our report stresses out the necessity to closely evaluate the use of broad spectrum prophylactic antifungal therapy. the prophylaxis in patients with gvhd and/or diarrhea must be used with caution. we recommend to systematically monitor posaconazole levels at least in these cases. inhalation of mold spores can lead in immunocompromised patients to an invasive disease and pneumonia. invasive fungal infection (ifi) has still a high mortality rate. mold-dna can be detected by a polymerase chain reaction (pcr) based method. using it for the bronchio-alveolar lavage (bal) can help to detect an ifi in an early stage. the pcr can discriminate between different mold species and directs the treatment. in our study on 23 patients, a mold pcr from bal was conducted in addition to routine diagnostics. the pcr with primers specifi c for mitochondrial aspergillus-dna and ribosomal 18s dna for zygomycetes. our results show that mold pcr is more sensitive than standard fungal diagnostics. based on these pcr results, an intensifi ed therapy was undertaken successfully. hence, mold pcr from bal is a useful additon of the microbiological investigations. the mould pcr allows the proof of a zygomycosis at an early stage and thereby ensures successful treatment. further investigations are to show if computer-tomography of the lung combined with mold pcr are suffi cient to diagnose for sure a pulmonal mold infection. introduction: cartilage hair hypoplasia (chh)is a rare autosomal recessive disorder caused by mutations in the ribonuclease rna-processing rmrp complex. hsct has resulted in immune restoration, yet fails to correct the chondrodysplasia. we describe a patient with chh and combined immune deficiency who developed granulomatous infl ammation. treatment with anti-tnf-alpha monoclonal antibodies (moab) caused reactivation of jc virus with ensuing progressive multifocal leukoencephalopathy (pml). case report: at age 4y a female chh patient (63c>t and 70 a-g mutation in rmrp) with combined immune defi ciency developed painful non-caseating granulomas. no infectious agent was identifi ed and antibiotic therapies failed. finally at age 17y anti-tnf-á moab(infl iximab) was started with partial response. after the 3rd administration she developed a debilitating intentional tremor of the right hand. mri t2 and flair showed demyelination in the right cerebellum. jc virus pcr was (+)in blood and in cerebrospinal fl uid (csf) and (pml) was diagnosed. 4 weekly administrations of cidofovir, followed by two-weekly administrations for 1 month resulted in a partial response. cidofovir was continued two-weekly. 7 months after diagnosis of pml, hsct with a 9/10 unrelated donor was performed with reduced intensity conditioning according to ebmt-esid guidelines. there was neutrophil engraftment at d+10 and stable donor chimerism of >95% at d+30. at d+60, the patient complained of dizziness, with evidence of a cerebellar syndrome. mri and csf polyoma virus copies were stable. at d+87, she presented with hypertensive encephalopathy including convulsions reminiscent of posterior reversible encephalopathy. discontinuation of ciclosporine led to resolution of the encephalopathy. however, pml progressed despite restoration of t cell function, with increasing cerebellar and brain stem symptoms including ataxia, dysarthria, aphasia, n. facialis and n. glossopharyngeus paralysis with corresponding mri imaging and increase in jc virus pcr copies in the csf. despite intensifi cation of cidofovir treatment, trials of steroids, fl uoroquinolones, mirtazapine, lefl unomide as well as high dose ivig and cytarabine iv, the neurodegeneration was progressive and the patient died of respiratory failure at d+205. conclusion: we describe the fatal course of pml due to jc virus reactivation in a patient with chh, despite successful hsct in terms of myeloid engraftment and restoration of t cell function. a. tomaszewska (1), b. nasilowska-adamska (1), t. dzieciatkowski (2), b. marianska (1) (1 introduction: viral infections still are a serious diagnostic and therapeutic problem in patients undergoing alternative donor transplants. betaherpesviruses (hhv5, hhv6, hhv7) are recognized pathogens in this group of patients. we report a case of hhv6 encephalitis complicated by guillain-barré syndrome (gbs) in a hematopoietic stem cell transplant (hsct) recipient with preceding reactivation of cmv infection. methods: a 43 year-old-man with a history of chronic myeloid leukemia underwent hsct from a matched unrelated female donor in october 2006. sero-status for cmv was igg positive in the recipient and igg negative in his donor. on the day +70 patient developed acute graft-versus-host disease successfully treated with iv methylprednisolone. in march 2007 he was admitted to our unit due to cmv infection reactivation. he started pre-emptive therapy with iv gancyclovir. after 2 weeks of treatment he revealed high fever, uroschesis, paraparesis, impaired consciousness and generalized epileptic seizure. computed tomography of his brain was normal. a lumbar puncture revealed pleocytosis (24/µl) and elevated level of protein (213.2 mg/dl). investigation of cerebrospinal fl uid (csf) by pcr for infective causes of patient's neurological decline including hsv t.1/2, vzv, adenovirus, cmv and dna candida and aspergillus were negative as well as csf culture, real-time pcr revealed in his csf presence of hhv6 dna. according to these fi ndings and neurological status of our patient we made a diagnosis of an hhv6 encephalitis complicated by gbs. the therapy with foscarnet (all symptoms revealed during pre-emptive therapy with gancyclovir) and ivig was started. due to gbs diagnosis we performed 5 procedures of plasmapheresis. we observed gradual improvement in neurological status. after discharging home the therapy was continued with cidofovir given once a week during four weeks. at present, 1.5 year after this episode, the patient remains in a good condition without cmv and hhv6 reactivation, with slight neurological defi ciency. conclusions: betaherpesviruses are emerging pathogens in the hsct setting and may cause central nervous system disease. gbs is a very rare complication among stem cell transplant recipients and usually has been attributed to infection. our successfully diagnosed and treated case of hhv6 neuroinfection complicated by gbs suggests that hsct recipients with cns signs and symptoms should have their csf investigated for hhv6 as well as other pathogens. zygomycosis is a rapidly growing systemic fungal infection, commonly fatal, despite intensive antifungal treatment. it almost always occurs among patients with an immunosuppressive background, diabetes mellitus, prolonged neutropenia, recent chemotherapy and an excessive iron overload. iron is essential for the growth, development and virulence of many fungi, and particularly of the zygomycetes, which are incapable to grow under iron-deprived conditions. we report on a 38-year old male patient, who at the age of 33 was diagnosed with cd10+ b-cell acute lymphoblastic leukemia and achieved a cr following chemotherapy of hyper-cvad type. the patient remained relapse-free for almost 3 years, but when he relapsed, he was treated with the g-mall protocol and a second cr was obtained after 2 cycles of treatment. at that point a fully matched related pbsc allograft, obtained from his 34-year old sister was offered. he engrafted on day+15, and the post-transplant period was complicated by cmv reactivation and mild chronic gvhd. the patient relapsed on day+367 and he was treated with high dose cytosine arabinoside days 1-4 and 24-h infusional mitoxandrone on days +5 and +6. during the aplastic phase he was complicated by histologically proven, extensive left rhinocerebral and pulmonary zygomycosis, with left facial nerve paresis. at that time point he had a transferring saturation of 95% and ferritin 10583 ng/ml. the patient was refractory to initial treatment was surgical debridement and a combination of liposomal amphotericin-b and posaconazole. since no signifi cant improvement was obtained despite a second surgical intervention, deferasirox 30 mg/kg of body weight was added to his antifungal regimen. following 10 weeks of treatment with the triple combination fever was rapidly subsided, as did both, nasal and facial symptoms and lesions. the pulmonary lesions were clearly improved. transferrin saturation decreased to 32% and ferritin to 572 ng/ml. unfortunately, chemotherapy produced a minor response and 2 months later leukemia reappeared. the patient fi nally succumbed from pulmonary hemorrhage, following salvage treatment with clofarabine and cyclophosphamide, without any sign or symptom of recurrence of his previous zygomycosis. introduction: despite the relatively high transplant-related mortality (trm), the management of the end-life care is poorly understood issue and the problems of providing palliative care to patients submitted to stem cell transplantation (sct) may be underestimated. in this regard, the use of palliative sedation therapy (pst) in the sct setting remains a major concern. patients (pts) and methods: in order to address this issue, a retrospective study on the use of pst in our tertiary sct unit was performed. search criteria were: death and previous sct. data regarding symptoms, symptoms control and use of pst were collected. we identifi ed 18 dead pts. last line of therapy before death was sct and a salvage treatment given for a post sct relapse in 11 and 7 patients respectively. near the death, 12/18 patients experienced a total of 18 refractory symptoms and in 6 cases more than one of them was present. intractable symptoms were: excruciating dyspnoea in 8 (67%), agitated delirium in 6 (50%), severe pain in 2 (17%) and massive bleeding in 2 (17%). results: pst was started in all 12 patients, at a median of 2 (1 -4) days before death. the most used sedative drug was midazolam, that was administered to 9/12 pts as single agent and in 2 cases in association with promazine; 1 pt received the latter agent alone. at the start of pst, 8 pts with pain were receiving parenteral morphine. symptoms control was adequate in 12 cases (complete and partial symptoms control in 9 and 2 respectively) and not adequate in 1. conclusion: pst is a controversial issue in palliative medicine, although it has been clearly claimed that when it has the intent to provide symptom relief, pst should be considered a proportionate intervention. sct failure represents a so strongly discouraging event to determine diffi culties to recognize end life status. as a consequence, the risk of an inadequate symptoms assessment and of an inappropriate palliation should be considered. in our experience, in a patient closed to the death, when other treatments failed to relieve the intolerable suffering from refractory and otherwise intractable symptoms, pst represented a valid palliative care option by a reduction in patient consciousness, using appropriate drugs carefully titrated to the patient's comfort. adequate symtom control was obtained in more than 80% (11/12) of pts. an internal operative protocol is under construction to improve those results. donor lymphocyte infusion as therapy for persistent pure red cell aplasia following major abo-incompatible stem cell transplantation a. lübking, i. winqvist, s. lenhoff lund university (lund, se) pure red cell aplasia (prca) after abo-mismatched allogeneic stem cell transplantation (sct) is not uncommon. however, spontaneous remissions within 6 months are frequent. we here report a case of long-lasting prca refractory to multiple therapies that eventually responded to donor lymphocyte infusion (dli). a 36 year old woman received peripheral blood cells from an unrelated hla-identical donor following myeloablative conditioning six months after diagnosis of aml. there was a major abo incompatibility between recipient (0+) and donor (b+). engraftment of granulocytes (>0,5x109/l) and platelets (>20x109/l) was noted on day 25 and 30 respectively. due to the absence of reticulocytes, bone marrow analysis was performed on day 50 showing the total absence of erythroid precursors. initial treatment with steroids, erythropoetin and withdrawal of immunosuppressive therapy was not successful. four doses of rituximab were given from day 265 without any effect. starting on day 545 immunoabsorbtion on three consecutive days was performed followed by methylprednisolone, cyclophosphamide and immunoglobulin infusions. although the igg and igm antidonor isoagglutinins were reduced from 1:128 to1:4 and 1:1 respectively, the prca persisted. from day 638 she received 4 doses of dli within 6 months in escalating doses (1, 5, 15 and 24 million cd3/kg). three months after last dli she developed signs of a mucosal gvhd accompanied by moderate eosinophilia. concomitantly, stable reticulocytosis occurred from day 924 and she became transfusion independent. since residual recipient b-and plasma cells are presumed to be responsible for production of anti-donor-isoagglutinins causing prca, inducing gvhd by withdrawal of immunosuppressive therapy or dli might be a reasonable option. there are previously published cases of successful dli treatment for prca, but in many cases dli was given relatively shortly after transplantation, i.e. when spontaneous remission still was possible and the time between dli and reappearance of reticulocytes varied. in our case stable reappearance of reticulocytes occurred concomitant with signs of gvhd. we therefore fi nd our case highly suggestive of that inducing gvhd with dli can overcome post-sct prca refractory to almost all other therapy options. cystatin c level as a marker of renal function in haematopoietic stem cell transplantation h. muto, k. ohashi, m. ando, r. hanajiri, t. kikuchi, w. munakata, c. sakurai, m. yamamoto, t. kobayashi, t. yamashita, h. akiyama, h. sakamaki tokyo metropolitan komagome hospital (tokyo, jp) hematopoietic stem cell transplantation (hsct) recipients have an increased risk of acute kidney injury (aki) or chronic kidney disease (ckd). however, serum creatinine level may underestimate the prevalence of these renal complications because of decreased lean body mass or concurrent liver disease, which was frequently observed in a hsct setting. cystatin c measurement may be more sensitive for detecting impaired kidney function. we retrospectively reviewed the medical records of 95 hsct (75 allogeneic and 20 autologous) recipients who had at least one chance to monitor serum cystatin c level during last 2 years in our institution, and evaluated cystatin c as a possible new marker which can predict subsequent renal dysfunction. the occurrence of aki was defi ned by the rifle classifi cation and ckd staging was based on kdoqi criteria. of 95 transplant recipients, 35 patients developed aki after median 48 days (range 0-664 days) after hsct, while worsening ckd stage was observed in 24 patients during observational periods. cystatin c level was not infl uenced by autologous transplant (p=0.311), but signifi cantly elevated after allogeneic transplantation (p<0.001). pretransplant advanced disease status also had an infl uence on cystatin c level before transplantation (p=0.004) multivariate analysis disclosed that the use of calcineurin inhibitor was a major cause of cystatin c elevation (odds ratio 7.09, p=0.017). there was also a strong inverse correlation between cystatin c and estimated gfr (r=-0.749, p<0.001). proportional hazard modeling analysis revealed that the episode of aki after transplantation were a great risk for substantially worsening ckd stage (hazard ratio 19.5, p<0.001). cystatin c measurement could be a useful clinical tool to identify hsct recipient at increased risk for ckd. control of severe bleeding from acute gvhd by treatment with tranexamic acid j. hasenkamp (1) acute graft-versus-host disease (agvhd) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. 50% of the cases with intestinal agvhd are refractory to standard treatment regimen. these patients suffer frequently from severe agvhd grades 3 to 4 including massive gastro-intestinal bleedings. we report from clinical courses of two cases treated with tranexamic acid for diffuse, life-threatening gastro-intestinal bleedings caused by steroid-refractory agvhd. the agvhd was confi rmed by biopsy and histopathology. immunosuppression consisted of tacrolimus, mycophenolate mofetil, prednisolone and second line treatment with alemtuzumab. one patient received additionally extra-corporal photopheresis and mesenchymal stem cells. global coagulation and factor xiii plasma levels were kept in normal ranges by substitution. thrombocytopenias were compensated by adequate transfusion of cell separated thrombocytes concentrates. bloody stool volumes of 3 and 5 kg in 24h lead to dropped hemoglobin levels despite massive transfusion of erythrocyte concentrates. because of this persistent, diffuse gastro-intestinal bleedings, both patients were treated additionally with 500 mg tranexamic acid i.v. every 8h. after three infusions of tranexamic acid the bleedings in both patients stopped. treatment with tranexamic acid was discontinued without reoccurrence of the bleedings. there were no adverse events of tranexamic acid observed. local hyperfi brinolysis in the gastro-intestinum may contribute to bleedings from tissue damage caused by agvhd. tranexamic acid is indicated for prophylaxis and treatment of bleedings by systemic and local hyperfi brinolysis after e.g. surgery or plasminogen activator treatment. abortion of hyperfi brinolysis can contribute to stabilization of coagulation. prophylaxis or control of severe agvhd is preferred for prevention of hemorrhage. however, tranexamic acid is a treatment option in otherwise unmanageable gastro-intestinal bleeding caused by agvhd. further studies are desired to charge the signifi cance of tranexamic acid in this indication. a low or high body mass index is not predictive for outcome following allogeneic haematopoietic stem cell transplantation j. auberger, j. clausen, b. kircher, g. gastl, d. nachbaur innsbruck medical university (innsbruck, at) objectives: recently it was hypothesized that a low (<20) bodymass index (bmi) is signifi cantly correlated with an increased transplant-related mortality, decreased survival and relapsefree survival after allogeneic sct (k le blanc, haematologica 2003;88:1044). patients: 208 patients receiving a fi rst allogeneic transplant were studied. underlying diagnoses were acute myeloid leukemia (aml) (n=71), acute lymphoblastic leukemia (all) (n=41), lymphoma (n=11), and other diseases (n= 75). median patient age at time of transplant was 45 (range, 18-76) years. 108 patients were grafted from an hla-identical sibling donor and 110 patients received grafts from volunteer unrelated donors. conditioning was myeloablative in 60 and of reduced intensity in the remaining 148 patients. results: overall survival for the entire cohort was 34% (24%-45%,95% confi dence interval, ci). there was a trend for a poorer outcome in patients with <25% and >75% percentile bmi (i.e. bmi ≤ 21 and ≥ 27) (os 48% vs 34%, p=0.1 log rank test) due to a higher non-relapse mortality in this patient cohort (37% vs. 30%). these differences were observed in both, the myeloablative as well as reduced intensity transplant cohorts. the bmi had no infl uence on relapse incidence in either patient cohort. conclusion: by deviding patients into percentiles bmi had no signifi cant impact on outcome and non relapse mortality neither following myeloablative nor following reduced intensity allogeneic stem cell transplantation. autoimmune thyroiditis after haplo-identical stem cell transplantation for severe combined immunodefi ciency f. dogu (1) introduction: thyroid dysfunction is a well known complication in survivors of hematopoietic stem cell transplantation, and is reported after tbi as well as radiation-free conditioning. the most common disorders after radiation free conditioning are euthyroid sick syndrome(ets) and compansated hypothyroidism. autoimmune thyroiditis is rarely reported after hsct in children and it has never been described after hsct for scid. here we report an autoimmune thyroiditis developed 9 months after the third haploidentical stem cell transplantation for scid. case: a 5-months-old girl was referred to clinic with the diagnosis of t-b-nk+ scid. as she didn't have a fully matched sibling donor and her clinical condition was unstable she received peripheral blood stem cell transplantation(pbsct) from his haploidentical father after cd34+ cell selection without conditioning. engraftment wasn't achieved on day +28 and she received second haploidentical cd34+ selected pbsct from her mother. third transplantation was performed 2 months after the second one, due to graft failure and this time she received bu/cyclo for conditioning and csa for gvhd prophylaxis. myeloid and platelet engraftments were achieved on day+14 and +18 respectively. grade i acute gvhd developed on +26 and treated with corticosteroid for ten days. she was discharged on day+55 with full donor chimerism. thyroid hormone levels which were normal before hsct revealed compansated hypothyroidism at posttransplant 9 months in a routine follow-up visit. elevated antithyroid proxidase (53 iu/ml) and anti-thyroglobulin (478 iu/ml) titers were all consistent with the diagnosis of autoimmune thyroiditis(hashimoto). levothyroxin treatment was started. since the thyroid hormone levels were normal and antithyroid antibodies were negative in her mother, the transfer of autoimmune disorder was excluded. conclusion: regular screening of thyroid functions is important and necessary to detect and treat thyroid illness, especially in young children following hsct. once-daily intravenous busulfan as myeloablative reduced-toxicity conditioning regimen in haematopoietic stem cell transplantation s. santarone, e. di bartolomeo, p. bavaro, p. di carlo, p. olioso, g. papalinetti, p. di bartolomeo bmt center (pescara, it) postulating favorable antileukemic effect with reduced toxicity and improved safety, we used i.v. busulfan (bu) associated with either cyclophosphamide (cy) or fl udarabine (flu) as conditioning therapy for hematopoietic stem cell transplantation (hsct) in 14 patients affected by aml (n=8), mds (n=4), all (n=1) and thalassemia major (n=1) between may 2006 and june 2008. patient age was 1-61 (median 28) years. five of them were older than 50 years. nine patients received flu at a dose of 30 mg/m2/day for 4 days (from -5 to -2) immediately followed by bu given in single i.v. administration over 3 hours at a dose of 3,2 mg/kg day for 4 days (total dose 12,8 mg/kg). five patients received the same dosage of bu from day -7 to day -4 followed by cy 60 mg/kg/day from -3 to -2. donors were hlaidentical (n=6) or 1 antigen mismatched siblings (n=2) and 6 were matched unrelated (mud). the graft-versus-host disease (gvhd) prophylaxis included cyclosporine and short course methotrexate for all patients with the addition of antithymocyte globulin for the mud transplants. eleven patients received bone marrow cells (median dose of nucleated cells 3,76 x108/ kg, range 3,1-10,9) and 3 were given peripheral blood stem cells (median dose 7,4 x106/kg cd34+ cells, range 5,7 -7,7). all patients achieved primary engraftment. the median time to 0.5 x109/l neutrophils and 25 x109/l platelets was 18 (range, 15-32) and 15 days (range, 12-25) respectively. chimerism studies revealed that 13 of 14 were complete chimeras (100% donor) at 1 year post-hsct. acute gvhd was observed in 5 patients (grade i in 2, grade ii in 2, grade iii in 1). two patients had mild to moderate chronic gvhd. there was no death due to the transplant procedure. the transplant-related complications were limited. grade iii who hepatic toxicity occurred in 3 patients, hemorrhagic cystitis in 2, moderate oral mucositis in 1 and a single episode of seizures in 1. six patients developed cmv reactivation between day 6 and 47 post-transplant (median, day 32). two patients relapsed and died. as of december 15 2008, 12 patients (86%) are alive and disease-free after a median follow-up of 250 days (range, 130-641) . although the small number of patients does not permit any fi nal conclusion, our hsct protocol treatment confi rms that i.v. bu, associated either with flu or cy, is a well tolerated reduced-toxicity myeloablative conditioning regimen and deserves further study with more patients and longer follow-up. background: down's syndrome (ds) is associated with higher incidence of both haematological and non haematological neoplastic diseases, if compared with general population. reduced susceptibility to chemo-and radiotherapy and the frequent comorbidities limit the use of high dose treatments, especially required in adult patients. case report: a 19 year old male with ds developed an acute myelogenous leukaemia, fab m2, aml1/eto rearranged, in september 2003. he received standard induction treatment obtaining complete remission (cr), consolidation therapy and, in february 2004, autologuos transplantation with bu-mel conditioning regimen using mobilized peripheral blood stem cells. patient relapsed in february 2007, at the age of 23, was treated with mec schedule, obtaining a second cr. hla typing showed the presence of an identical sibling. a full clinical evaluation revealed mild reduction of the ejection fraction due to corrected congenital fallot tetralogy (ef=55%) and a pulmonary hypertension. a reduced intensity conditioning regimen was proposed, consisting of thiotepa (5 mg/kg iv /d x 2 dd) and fludarabine (25 mg/kg iv /d x 5 dd) followed by allogeneic stem cell reinfusion in june 2007. standard gvdh prophylaxis was given; engraftment was achieved at day +16 for anc >0,5 x 109 /l and +13 for plt >50 x 109/l; grade 4 who was recorded for liver toxicity and grade 1 for mucosal toxicity. full donor chimerism was documented at day +30. the patient developed stage 1 agvhd; however, 4 months after transplantation relapse was diagnosed with immunological features of all. immunosoppression was suspended, although blast percentage increased rapidly to 100% and a salvage therapy with all active drugs was started. discussion and conclusion: few data are reported on allogeneic stem cell transplantation in adult patients with ds. this is the fi rst case of ric allosct in a ds adult. those sporadic data do not allow conclusions about outcome on ds adult. a retrospective analysis on large database and a prospective study would be useful to address this issue, helping physicians on treating adult ds pts, when immunogenic effect of allosct play a crucial role to prevent relapse. severe immune hemolysis and pure red cell aplasia after haploidentical non-myeloablative allogeneic stem cell transplantation g. nair, a. mischo, g. stüssi, u. schanz university hospital (zurich, ch) haploidentical stem cell transplantation (sct) offers potential cure to patients without hla-identical donor. recently nonmyeloablative conditioning regimens with in-vivo t-cell depletion have been introduced. severe immune hemolysis rarely occurs after hla-identical sct, but little is known about the occurrence after haploidentical sct. here, we describe 5 patients receiving haploidentical sct for high-risk or relapsed aml (3), cml after 2 blast crises (1), and as a rescue therapy in a patient with all after primary graft failure following hla-identical mud sct. all patients were in morphological remission at the time of sct. conditioning regimen included fl udarabine (30mg/qm x 4 days), cyclophosphamide (500mg/qm x 4 days), and alemtuzumab (20mg x 5 days) for in-vivo t-cell depletion. gvhd prophylaxis comprised mycophenolate mofetil (day 1-28) and cyclosporine (day -1-60) and all patients received prophylactic antibiotic treatment. g-csf was administered until hematologic recovery. peripheral blood sct was performed over 1-3 days with a median number of 8.12 x 106 (7.97-9.82) cd34 positive cells. the early posttransplant course was uneventful, the median time of aplasia was 3 (0-6) days. acute gvhd occurred in 4/5 patients (i: 2; ii:1; iv:1). four patients experienced 9 posttransplant infectious complications (3 cmv, 3 bk, 2 fungal infections, one pulmonary infection). two patients experienced severe immune hemolytic anemia and concomitant pure red cell aplasia in the bone marrow 4 and 10 months after sct. in both patients relapse was diagnosed shortly before or after the onset of hemolysis. the direct antiglobulin test was positive for igg and c3d. the serum of both patients reacted with all cells in a 11 cell antibody search panel without evidence for cold-reacting antibodies and no antibody specifi city could be evaluated. one patient was treated with steroids, ivig, rituximab, and high-dose cyclophosphamide, but eventually died due to fatal hemolysis. the second patient is currently being treated with steroids, ivig and high dose cyclophospamide with a marked reduction of hemolytic activity. the remaining three patients are currently in complete remission without evidence of hemolysis. in conclusion, nonmyeloablative conditioning regimens in haploidentical sct offer new possibilities for patients without a hla-identical donor. however, physicians should be aware of the potentially fatal complication of severe immune hemolysis. one of the major side effects poorly tolerated, especially in children, is represented by emesis post-chemotherapy. the use of antiemetic during chemotherapy (three to four doses for day) is necessary to reduce this complication. in this work was evaluated using a single dose of palonosetron intravenous for the prevention of nausea and vomiting secondary to chemotherapies. methods: since 2006 we have used the palonosetron in 28 pediatric patients of which 19 males and 9 females, undergoing bone marrow transplantation, 15 allogeneic (both sibling that mud) and 13 autologous. the median age is 10 years (range 1-18) and the median weight is 42 kg (range 8-79 kg). the diseases in young patients are reported in table 1 , the conditioning transplantation are listed in table 2 . the dosage used, including scientifi c literature data, was 5 mcg /kg body weight. the palonosetron was considered effective when the emesis was not more than 2 episodes in 24 hours and nausea no more than 2nd grade. results: it was encouraging, having achieved a good control of nausea and/or vomiting induced by chemotherapy, in fact, only seven patients (25%) was necessary to resort to a second dose of antiemetic, in four of seven (14% of total) was repeated the success with palonosetron a distance of four days after the fi rst dose using the same dosage. in 15 patients (53%) has not been no emetic episode while in the remaining group (22%) episodes were occasional and not have needed any treatment. in all patients was not noted any adverse event or side effect. conclusions: our experience, although on a small sample, it suggests that palonosetron can be considered an effective drug in preventing the nausea induced by chemotherapy, is also a drug that not have adverse events, so well-tolerated and easily manageable, it is necessary a single dose within 24 hours before the start of chemotherapy, not least the assessment of the reduction in costs compared to conventional antiemetic. allogeneic or autologous haematopoietic stem cell transplantation (hsct) is an established mode of treatment of different diseases. loss of protective immunity to pathogens has been consistently demonstrated in patients referred to hsct. impairment of humoral and cell-mediated immunity is commonly seen after transplantation. the degree of immunodefi ciency is determined by many factors, particularly by the type of disease and transplant, the presence of graft-versus-host disease (gvhd) or ongoing immunosuppressive treatment. the aim of the study was to evaluate 1) immunogenicity of a revaccination schedule in pediatric hsct recipients 2) quality of recipient immune reconstitution and protection against ordinary pathogens. patients and methods: twenty one patients (pts) 1.4-22 (average 7.8) years old, 13 boys and 8 girls after autologous (11, 52%) and allogeneic (10, 48%) hsct were included in revaccination program. indications to hsct were: solid tumors -11, hematological malignancies -5, immunodefi cency states -3 and aplastic anemia 2 pts. time interval between hsct and begining of vaccination protocol was 0.8-4 (av. 1.5) years. vaccines used in protocol were as follows: diphtheria and tetanus toxoids, pertussis (for patients <7 years old), hbv, vzv, haemophilus infl uenzae type b conjugate, 23-valent pneumococcal polysaccharide, inactivated infl uenza, inactivated polio and attenuated measles-mumps-rubella vaccines. plasma samples to determine specifi c antibodies by elisa tests were collected before and after vaccinations. results: with the exception of one patients presented with repeated fevers, lymph nodes enlargement, muscles and joints pain, no important side effects of vaccinations were observed. a meningococcial meningitis developed in one patient who refused vaccinations. plasma antibody concentrations before and after vaccinations were as follows: antidiphteria (0-300, mean 62.5; 100-5800, mean 1838), antitetanous (0-500, mean 133; 826-5500, mean 3483) and antihbv (0-135, mean 33; 317-1000, mean 532) iu/ml. conclusions: 1) systemic immunization is necessary at appropriate time intervals following transplantation to re-establish immunity. 2) a signifi cant increase of antibodies titer after hbv, diphtheria and tetanus toxoids was detected. 3) vaccinations in patients after hsct are effi cient and well tolerated. 4) a delay in begining of vaccination can result in life threatening complications. ministry of science rp, grant number 501/g/640. according to the world bank data, released in the 2008 report, romania has an upper-middle-income economy. the hematopoietic stem cell transplantation (hsct) program started in romania in 2001 and more than 200 transplants (auto and allo) were performed. we analyzed the outcome for 26 patients who underwent an allogeneic hematopoietic stem cell transplantation from matched related donor for acute leukemia (24 patients) and aplastic anemia (2 patients). for 20 of the patients the procedure was performed in romania and for 6 patients abroad. for both categories the follow-up after transplant was done in hematology units in romania. the overall survival was 14.69 months, with the longest survival of 60 months and respectively shortest outcome for less than one month. on the 1st november 2008, there were 10 patients alive, between 1 and 60 months from the procedure, with a median survival of 27 months. sixteen patients died, the median survival being 6 months after transplant. four out of 16 patients died during the fi rst month after transplant, and a total of 9 patients died during the fi rst 6 months after transplant. the transplant related mortality was 53.84%, 38.46% died due to relapsed disease and 15.38% died of graft failure. for these results, there could be incriminated the irregular and inadequate drugs and reagents supplies in the romanian health system, an ineffi cient follow-up system and registry and home-care facilities defi ciencies in romania. in conclusion, the gross national income (gni) per capita and the human development index (hdi) are very important factors for the outcome of recipients of hematopoietic stem cell. background: umbilical cord blood stem cell transplantation has many advantages over bone marrow transplantation or peripheral blood stem cell transplantation. but, there are some problems to be solved in order to be applied to adults. the main problem is limitation of volume, which can be collected from one placenta was only between 80ml and 120ml. to overcome this problem, the ex vivo expansion of cryopreserved umbilical cord blood stem cells is needed. the object of this study was to evaluate the effect of cryopreservation on ex vivo expansion potential and viability of umbilical cord blood stem cells. methods: after normal delivery, cord blood was drawn from umbilical cord vein and was used to evaluate the mononuclear cell count, the cell viability and clonogenic capacity of cord blood stem cells before and after cryopreservation. results: before cryopreservation, the mononuclear cell count of umbilical cord blood was 2.92 ± 1.08 x 106/ml, cell viability was 92 ± 2.88%, total colony count was 101.5 ± 23.74 and percentages of cfu-gm, cfu-gemm, bfu-e were 29.5 ± 5.80%, 21.0 ± 1.45% 24.8 ± 5.0%, respectively. the mononuclear cell count of umbilical cord blood cryopreserved for 28 days was 1.42 ± 0.42 x 106/ml and cell viability was 66 ± 3.87%. total colony count of umbilical cord blood cryopreserved for 28 days was 52.5 ± 12.13 and percentages of cfu-gm, cfu-gemm, bfu-e were 28.0 ± 3.45%, 27.2 ± 6.52%, 45.3 ± 4.99%. but, there were few colony count which could be observed after cryopreserving for 7 days. conclusion: there was no difference of clonogenic capacity of umbilical cord blood stem cells before and after cryopreservation. the cell viability of umbilical cord blood stem cells was decreased after cryopreservation but there was no difference between umbilical cord blood cryopreserved for 7 days and 28 days. therefore, it is possible that suffi cient umbilical cord blood stem cells could be obtained by ex vivo expansion of cryopreserved umbilical cord blood in order to be used for adult patient. objective and methods: combined hematopoietic stem cell transplants (hsct) plus solid organ transplants (sot) have been rarely reported. the majority of patients with a previous history of liver transplants were children that underwent hsct for aplastic anemia after viral hepatitis. here we report an adult patient who received a cord blood hsct after a preceding liver transplantation. results: in 1993 a 42 year old man required orthotopic liver transplantation for cirrhosis after b viral hepatitis. in april 2006 acute myeloid leukaemia m1 citotype , normal karyotype, flt-itd positive was diagnosed and a fi rst complete remission was reached after 2 induction and consolidation cycles. at that time the patient was not considered eligible for a transplant program due the previous history of sot. in february 2008 the patient relapsed and came to our centre: he was treated with high-dose cytosine-arabinoside chemotherapy, that was complicated by a pulmonary aspergillosis , but reached a second complete remission. we decided to start a cord blood donor search, since siblings were not available and he could not wait for an unrelated donor search. a cord blood with hla locus a allelic mismatch and locus c antigenic mismatch was identifi ed. patient's comorbidity index according sorror at transplant was 5. in may 2008 a preparative regimen containing treosulfan, fludarabine and atg fresenius was administered and 1,2 x 105/kg cd 34+ cells were reinfused. grade i mucositis and grade ii hepatoxicity were observed. a bacterial pneumonia and cmv reactivation occurred at day 6 and at day 34 respectively and both rapidly resolved. a neutrophil count > 1 x 109/l was reached at day 19 and platelet counts > 20 and > 50 x 109/l platelet count were reached at day 36 and day 43 respectively. no acute and chronic gvhd were observed. a 100% donor chimerism has been reached in whole peripheral blood and in cd3+ cells since 28 days onwards. no minimal residual disease has been detected by marrow immunophenotyping and by wt-1 gene expression until last follow-up, at day 171. conclusion: to our knowledge this is the fi rst report of a successful cord blood allogeneic hsct in an adult patient with a history of liver transplantation. this case might encourage physicians to propose allogeneic hsct by any stem cell source to patients with high-risk haematological diseases, who had previous liver or other sot's. double unit cord blood transplantation(cbt) has been established as an alternative source of donor cells for allogeneic haematopoietic stem cell transplantation (hsct). we reported here an interesting case of long-term mixed full donor chimerism during the regular follow-ups of one year after hsct. a 20year-old woman with acute lymphoblastic leukaemia in second complete remission received two units of cord blood after a myelo-ablative conditioning regimen. the cord blood units were hla 4/6 identical with the recipient (2b mismatches and 1a+1b mismatches). total nuclear cell doses infused were respectively 1.8x107 and 1x107, whereas the cd34+cells number was identical in the 2 cbus and the cd3+cells number was higher (x2) in the fi rst one (table1). neutrophil recovery was observed at day 34 and platelets engraftment at day 55 after cbt. only one event of acute graft versus host disease (gvhd)grade i was reported at day 49. currently the patient does not have chronic gvhd and is disease free. analysis of chimerism was performed by str-pcr or rq-pcr on whole blood and specifi c lineage cells (cd3+, cd15+ and cd19+). follow-up was done at 3, 6, 9 and 12 months post transplant. full donor chimerism (fd) was achieved on day 60. each of the two units contributed at different levels to the donor chimerism in specifi c lineage cells: whole blood and cd15 were about 50% cbu1/cbu2, cd19 cells were preferentially from cbu2 origin (65%), and cd3 cells were preferentially from cbu1 origin (75%). this mixed origin of donor cells was detected early and was constant during regular follow-ups. usually, recipients of double unit cbt were engrafted predominantly with one of the 2 units after 4 months. the mechanism of a long-term mixed full donor chimerism is still unknown for our patient. kir ligand analysis showed an absence of mismatch in gvh direction between recipient (c1-c2) and each cb unit (c1-c1 and c2-c2) while there was a mismatch between the 2 units. in this case, a state of full tolerance settled down between the various lineages, either immune mediated interaction between host/graft or between graft/graft could explain s378 this chimerism pattern, but it will have to be clarifi ed: a specifi c study of treg cells is in progress. optimising cd34 yields in pbsch: a comparative analysis of 4 mobilisation regimens c. black, t. elston, m. streetly, m. kazmi guy's hospital (london, uk) cyclo/g-csf (cyclophosphamide/ granulocyte-colony stimulating factor) has been the mobilisation regime of choice when collecting peripheral blood stem cells (pbscs) for transplantation yet pbsc harvests post chemotherapy produce effi cacious yields. this data seeks to compare and inform current mobilisation strategies in this centre. dhap (p=0.018, and ara-c (p=0.001, t-test) therapy yielded signifi cantly better cd34 results compared to cyclophosphamide. mm patients mean cd34 for cyclo mobilisation (n=11) were 2.18 x 10 6 /kg (range: 0.38-5.42), g-csf only (n=3) 2.62 x 10 6 /kg (range: 1.21-4.95), and ara-c ( n=31) 14.15 x 10 6 /kg (0.86-74.62). myeloma patients post ara-c yielded signifi cantly more cd34+ cells (p=0.001) compared to cyclo than those mobilised with g-csf only. nhl patients mean cd34 harvest results for cyclo mobilisation (n=6) were 2.94 x 10 6 /kg (range: 0.16-7.86), g-csf only (n=7) 1.62 x 10 6 /kg (range: 0.71-3.09), and dhap (n=17) 16.08 x 10 6 /kg (range: 0.55-64.31). cd34 yield of nhl patients mobilised with cyclo compared with those harvested post dhap, a signifi cantly higher harvest result was noted (p= 0.020) than those mobilised with g-csf only (p=0.328). paired mm data (n=6) compared patients fi rst mobilised with cyclo-gsf and post ara-c, (p=0.002, paired t-test). this study suggests that harvesting of patients post ara-c, or post dhap is valuable, giving greater cd34 yields than traditional agents and should be considered. paired data also indicates that ara-c could be used for effective second mobilisation. can type of delivery infl uence cord blood units' quality? g. pucci, a. pontari , d. marcuccio, i. bova, r. monteleone, d. princi, g. gallo, a. dattola, e. spiniello, c. garreffa, t. moscato, p. iacopino ao bianchi melacrino morelli (reggio calabria, it) the cord blood banks use the total nucleated cell (tnc) number as principle to proceed or not to cryopreservation of the cord blood (cb) units. we know that tnc and cd34-positive cells infused on unrelated umbilical cord blood transplantation in haematological disease are fundamental for the engraftment of haematopoietic stem cells (hsc) background: immunomagnetic cd34+ selection is a procedure used both for autologous grafts to perform cellular purging and for allogenic transplant. in aploidentic transplant the purpose of cd34+ is to reduce the quantity of cd3+ and cd19+ cells so as to reduce the incidence rate of the graft versus the host disease (gvhd). aims: in this study we have valued the purity and the cellular recovery after immunomagnetic selection performed with clini-macs automatic system (miltenyi biotec, germany); a group of concentrates has been selected after incubation manually performed, while another group has been submitted to incubation and to the subsequent washings using an automated system (cytomate (baxter oncology, chicago il). methods: in our study we subjected 63 peripheral blood stem cells (pbsc) concentrates taken from 16 donors with microcythemia to immunomagnetic cd34+ selection, in order to perform aploidentic grafts on children affected by beta-thalassemia major. 46 concentrates have been submitted to washings pre and postincubation and to incubation using the automatic system cytomate, while 17 concentrates have manually been worked. cell count of nucleated cells (nc) was performed using an electronic cell counter while cd34+, cd3+, and cd19+ were quantifi ed using fl ow cytometry. results: the following table shows the results. conclusion: immunomagnetic selection in microcythemic donors determines according to our experience, a less recovery in comparison to the data reported in literature, nevertheless in our study results evident, even though casuistry is not very ample, that the use of an automatic system for the washing and the incubation of the cellular concentrates has determined a greater recovery and a greater purity in comparison to the procedures manually performed. allogeneic transplantation from hla identical family donor is a common therapeutic approach in patients with intermediate risk aml in fi rst cr. we present an unusual onset of acute leukemia in a female patient that was a healthy donor of pbsc (previously mobilized with g-csf 10mcg/kg) for her hla identical sister with diagnosis aml (fab-m4). the transplantation was preformed in january 2004 with myeloablative bu-cy conditioning and conventional cy+mtx gvhd prophylaxis. at day +35 acute gvhd gr i/ii was observed and resolved with addition of median dose of corticosteroids. in a period of 4 years after this occasion, acute leukemia (aml-m2 no cytogenetic abnormalities) was diagnosed in the donor and treatment with chemotherapy was started. the induction chemotherapy was provided with dae regimen. with 2 consecutive cycles cr was achieved. the patient followed consolidation treatment with hd-arac and anthracikline. further treatment with allogeneic transplantation was on schedule and the source of stem cells would be taken under consideration. can a person with aml and 4 years surviving in a complete remission become a donor of its own donor (hla dna identical) with the same diagnosis is a question that has to be resolved in a higher number of patients. clinical outcome and characteristics of donor graft failure in 25 patients with haematopoietic disease given donor cell boost, second allogeneic transplantation or no treatment r. ahmed nacer, m. benakli, f. mehdid, r. belhadj, n. rahmoune, m. baazizi, a. talbi, f. kaci, r.m. hamladji pierre and marie curie center (algiers, dz) introduction: donor graft failure (gf) is a life-threatening complication of allogeneic hematopoietc stem cell transplantation(hsct), determined when anc had not reached 0,5 109/l by day 21 (primary gf) or when anc decreased irreversibly after engrafment (secondary gf). frequency of gf is variable in function to hematopoietic disease. material and methods: from may 1998 to december 2007, 811 patients (pts) underwent allogeneic hsct from hla-identical sibling donor. 758 pts are appraisable for this study and gf was diagnosed in 25 pts (3,29%) : primary gf: 18 pts, secondary gf: 7 pts with median time engrafment to ; median age at transplant 18 years (5-49); sex ratio (m/f) 1,5; hematologic non malignant disease (hnmd; n: 17): aplastic anemia: 11/198 (5,6%), major athalassemia: 6/15(40%) and hematologic malignant disease (hmd): 8/492 (1,6%); 15 pts had received more than 20 transfusions before allograft; abo incompatibility between donor/recipient was seen in 11 d/r pair; median interval from diagnosis to transplant 39 months ; 2 pts was multiparous; 24 pts received myeloablative conditioning regimen (mcr ) and one pt reduced intensity conditioning (ric); 18 pts received peripheral blood stem cell (pbsc) and 7 pts bone marrow transplant (bmt). the chimerism testing was performed in 8 cases: predominant host population (host population: 90-100%) in 6 pts and mixed (host population < 85%) in 2 pts. 14 pts were given donor cell boost with no additional conditioning with median time gf to treatment 108 days (30-559) and fi ve pts a second hsct within one a third hsct (mcr:5; ric:1) with median time gf to treatment 296 days (69-683). 6 pts had not been treated. at november 2008 maximal follow up is 127 months and minimal 11 months. results: 10 pts are alive (40%) within 6 pts (donor cell boost:5 pts; second hsct:1 pt) with success engraftment(donor population:100%) after median follow up 101 months (70-124) and 4 pts (second hsct:1 pt; no treatment:3 pts) with autologous reconstitution (donor population:0%). 15 pts died (60%) within 9 pts after given donor cell boost, 3 pts after second hsct and 3 pts before given donor cell boost. os at 9 years is 45%. conclusion: gf is rare but serious and concern hnmd more than hmd. better outcome can be obtained after chimerism testing study to choice treatment : predominant donor population will have donor cell boost and predominant host population second hsct with another donor. mixed haematopoietic chimerism: how the initial dynamics of mixed chimerism correlate with later chimerism status k assing (1), c. heilmann (2) (1)herlev university hospital (herlev, dk); (2)university hospital, rigshospitalet (copenhagen, dk) background: the natural history of mixed (hematopoietic) chimerism (mhc) has been extensively studied in hematopoietic stem cell recipients, in order to fi nd determinants for relapse or graft rejection. methods enabling quantitative prediction of later mhc status have not been devised. methods: 21 recipients, receiving hematopoietic stem cells due to non-clonal disorders and displaying at least 5% donor chimerism at minimum one time point, were serially tested for whole blood chimerism over a median period of 2.6 years (range: 0.7-5.6 years). relative changes in the host fraction (termed alfa4) between the median time points: 3.3 and 17.1 weeks post-transplantation were correlated with later mhc status. the predictivity of alfa4 values for later mhc outcome was assessed in a linear regression model. findings: all recipients engrafted. subsequently, 66.6% became mixed chimera and 28.6% achieved complete donor chimerism. weekly chimerism fl uctuations prior to six months posttransplantation (12.0% points; 0.0-192.3% points) exceeded those after six months time (0.9% points; 0.0-192.3% points, p<0.001). at seventeen weeks, alfa4 values correlated with endpoint mhc levels at 2.6 years (r = 0.87, p<0.001). negative alfa4 values predicted (95% confi dence intervals) the presence of less than 30% host cells, while alfa4 values between: 0.0-107.7, were predictive of mhc with ≤ 70% host cells. the only recipient experiencing rejection (4.8%) displayed the largest alfa4 value and had a predicted mhc outcome of 99.8% host cells (95% ci: 79.3-120.2%). interpretation: we have devised a simple mathematical method enabling us, early post-transplantation, to predict later mhc status and thus determine at an early time point, where intervention is needed in order to prevent rejection or poor graft function. feasibility of out-patient autologous stem cell transplantation for malignant haematologic disorders a. ghavamzadeh, a. allahyari, k. alimoghaddam, a. karimi, r. aboulhasani, a. manookian, m. asadi, a.r. shamshiri hematology-oncology and sct research center (tehran, ir) introduction: high-dose chemotherapy with autologous stem cell support is utilized for the treatment of a variety of malignancies including non-hodgkin's lymphoma, hodgkin's lymphoma, and acute leukemias. the aim of this study was to explore the feasibility and safety of performing autologous stem cell transplantation (asct) on an out-patient basis. material and methods: total of 8 patients affected by malignant hematologic disorders(4 cases of hl, 2 cases of nhl, 2 cases of aml) with median age of 25 y (range :16-41 y) and in complete remission and without medical problem were selected. they received conditioning regimen (ceam for nhl and hl, busulfan and etoposide for aml) and stem cell infusion in hospital. the day after sct, patients were discharged and followed by outpatient sct team; include a general physician, staff nurse and care giver during their neutropenic period, and to be rehospitalized in the case of febrile neutropenia, after sepsis workup and performing chest x-ray, they were received the fi rst dose of antibiotic in hospital and treatment continued in their home. results: median time for wbc recovery was11 days (range: 8-13 days), median time for plt recovery was 15 days (range: 11-66 days), median number of transfused single donor plt was 2.5 units (range: 1-9 unit). mucusitis grade 3 was seen in 2 patients, median duration of neutropenic fever was 6 days (range: 0-10 days), 3 patients was rehospitalized because of the neutropenic fever, median duration of rehospitalization in these patients was 5 days, median follow up of patients was 130 days (range: 20-200 days), all patients were alive and in complete remission. conclusion: results show that out-patient autolgous sct in malignant hematologic disorders (hl, nhl, and aml) is feasible and its complication is manageable. haplo-identical sct as a salvage therapy in haematological malignancies: a single-centre experience o. paina, y. stankevich, i. kazantsev, n. stancheva, a. golovacheva, e. babenko, a. alyanskiy, n. ivanova, e. semenova, p. krugliakov, d. polyntzev, l. zoubarovskaya, b. afanasyev spb state i. pavlov medical university (st. petersburg, ru) background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is the one of curative option for patients (pts) with acute leukemias, though its usage is often limited by lack of matched related donor or the time required for search of unrelated one. usage of haploidentical donors allows to avoid these problems and to perform allo-hsct in time. patients and methods: 24 very high risk pts underwent haploidentical sct: all -10 (42%) pts, aml 11 (44%) pts, jmml -1pt, cml-1 pt, and resistant neuroblastoma-1 pt. the total number of resistant/in progression pts was 16 (66%), 8 (33%) pts were in remission. 19 (79%) pts were children (age 1-18), 5 (21%) were adults (age 21-47). in all cases reduced intensity conditioning regi-mens (ric) were used: fl udarabine and atg with addition of different alkylating agents (busulphan, melphalan or thiotepa). sources of hsc -peripheral blood stem cells (pbsc) and bone marrow. for pbsc cd34+ positive selection clinimacs was used. the mean cd34+ count was 12,8x106 /kg (1, 7) . in 20 pts agvhd prophylaxis consisted of csa and short course of mtx with or without mmf. in 4 pts tacrolimus and mmf were used. in 2 pts at d-1 used mesenchymal stem cells (msc) from third -party donors were used prevention of agvhd, in 3 pts, msc were used for treatment of acute gvhd. results: the incidence and severity of agvhd weren't higher, than in other types of allohsct: 6 (25%) pts had grade iii-iv agvhd with skin and gut involvement, one pt died. when mcs using in conditioning regimen agvhd, i stage was observed. treatment of agvhd with msc was successful: in 3 pts in 2 cr. the toxicity of the conditioning regimen was acceptable, 6 (25%) developed grade ii-iii organ toxicity. 5 (21%) pts had invasive aspergillosis and 8 (33%) pts of them had cmv reactivation. the 1-year os is 62%, with mediam observation terms of 4,6 months (1 to 12 months). 5 pts died in relapse and 3 in cr (infection -1pt, another failed to engraft and acute gvhd of the gut). conclusions: haploidentical hsct with ric is characterized by acceptable toxicity and agvhd control, stable engraftment. it proved to be a good option for the group of pts with poor prognosis. randomized clinical trials are necessary for estimation of therapeutic effect of mscs in haploidentical hsct pts. results: there were 38 donation requests involving 37 donors (18 females,19 males). one donor was contacted but declined to donate dli. one donor donated twice for the same recipient. for 1/37 donors no details of donation are available. the median age at time of donation was 43.8 years (range 12-68 years). there were no failed collection procedures. 6/37 donors experienced mild citrate toxicity. 2/37 donors had a vasovagal episode, but both recovered rapidly and collection was able to be completed.1 donor required central access for dl collection: she had also previously required central access for pbsc donation. a median 1.88 donor blood volumes was processed (range 1.30-2.34). no late donor complications were reported. in total, 36 donors had dl collected. among the 36 prospective recipients (15 female; 21 male), indications for dl were: mixed chimerism(n=17); residual disease(n=3); molecular relapse(n=10); clinical relapse(n=2); ebv reactivation(n=1); pancytopenia of uncertain cause(n=1); no data(n=2). 29 of 34 patients for whom data were available (85%) actually received dl infusions. for the remaining 5, reasons for not proceeding were: spontaneous improvement in blood counts; death from ebv; death from relapsed disease; development of gvhd prior to dli; and spontaneous resolution of mixed chimerism. an escalating-dose regimen was used at 3-monthly intervals depending on response: the median number of doses reinfused was 2 (range 1-5). 9 patients (31%) developed gvhd s381 following dli. the dli was successful in treating the stated indication in 18 patients (46%). there were 10 recipient deaths: relapsed disease(n=4), infection(n=3), gvhd(n=2) and progressive ebv(n=1). only the two gvhd deaths were considered dl-related. conclusions: our single-centre experience confi rms that dl are frequently an effective treatment for mixed chimerism or early relapse post-hsc transplant, and that donor experiences are generally good. although requirement for dl is itself an adverse prognostic factor following hsc transplant, 46% of recipients had a successful outcome. nowadays, haematopoietic stem cell transplantation (hsct) remains the single curative approach to the treatment patients (pts) with the resistant primary and secondary aml. these pts have extremely poor prognosis with the level of relapse at least 40% and the risk of trm 45-60%. as known, high level of blasts to the moment of transplantation infl uences on dfs and os. patients and methods: at the russian children research hospital between october 2005 and june 2008 32 hsct were made in 27 refractory aml pts (20m/7f). the median age was 11 (1-18) years. fab-type: m0 -2 pts, m2 -4 pts, m3 -1 pt, m5 -5 pts, m6 -4 pts, m7 -7 pts, mx -4 pts. primary refractory aml was diagnosed in 8 pts and secondary refractory -in 19 pts. 15 kids were transplanted from msd, 8 pts -from mud (2 mmud) and 9 pts -from mmfd (8 haplo-pbsc) with the usage of cd3/cd19-depletion (7 pts) or cd34-selection (1 pt) of the graft. the median level of blasts in bone marrow prior to hsct was 18% (6% -98%). the myeloablative conditioning regimens were used in 29 hsct and non-myeloablative regimen -in 3 second hsct. 11 pts received double-phase conditioning regimens. a median dose of cd34±cells was 8 (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) x 106/kg. 14 pts received dli on median day 63 (8 -119); 4 pts received prophylactic dlis and 10 pts received treated dlis due to increasing of mrd level or the mixed chimerism. results: the engraftment level was 89% with a median time to neutrophil recovery 15 days (8-33) and to platelets recovery -18 days (11-132). 84% pts achieved cr to day 30, 3 pts had the progression of disease, one pt died before engraftment, and the rejection was documented in 1 pt. acute gvhd developed in 17 pts (55%), chronic gvhd -in 8 (44%) from 18 pts who were alive to day 100 after hsct. 14 pts had the liver toxicity grade 2-3 and two pts had the pulmonary toxicity grade 3-4 (who-classifi cation). trm level was 22%. relapse was diagnosed in 10 pts (dfs 48%). at this time 12 pts are alive in cr, 16 pts died (6 -from relapse, 9 -from gvhd and sepsis and one pt -from dag). os for all pts was 17% with a median time 8 months (2 -37) . but after double-phase conditioning regimen os and dfs were 41% both and trm was 0%. overall rfs depended on the presence of gvhd, the type of donor, the using of dli. conclusion: our results show that even for very high risk aml pts hsct may be performed successfully without the significant increasing of trm particularly with prophylactic dlis. a.a. hamidieh, a. ghavamzadeh, m. jahani, k. alimoghaddam, a. mosavi, m. iravani, b. bahar, a. khodabandeh, m. jalili hematology-oncology and sct research center (tehran, ir) objective: hematopoietic stem cell transplantation (hsct) has been extensively used in the treatment of pediatric leukemia. this is follow-up report of pediatric patients (<15 years) with acute myeloid leukemia (aml) and acute lymphoblastic leukemia (all) whom transplanted in hematology-oncology and stem cell transplantation research center, tehran, iran. methods: 142 pediatric patients 85 boys and 57 girls (median age=11 years) with acute leukemia (96 patients with aml and 46 patients with all) received hsct between 1991 and 2008. the most common conditioning regimen was cyclophosphamide + busulfan. they have received allogeneic (51 aml/40 all) or autologous (45 aml/6 all) hsct from bone marrow (26 aml/9 all), peripheral blood (68 aml/37 all) or cord blood (2 aml). donor type for allogeneic transplantation in 84 patients(47 aml/37 all) were stem cell from hla matched siblings, 5 patients (3 aml/2 all) received from other related (hla-matched confi rms with high resolution method) and 2 patients(aml) from other related with one or more than one antigen mismatch. prophylaxis regimen for graft-versushost disease (gvhd) was cyclosporine a and methotrexate or cyclosporine a alone. results: 104(73.2%) patients are alive, 38(26.8%) patients died (27 aml/11 all). the most common cause of death was relapse of disease in 81.6%. among patients who received allogeneic transplantation acute gvhd occurred in 65% and chronic gvhd in 15.5%. two years overall survival and disease free survival of aml patients were 70% and 67% respectively. two years overall survival and disease free survival of all patients were 74% and 60% respectively. no statistical difference between aml group and all. conclusions: our results of overall survival and disease free survival are compatible with literatures. autologous haematopoietic stem cell transplantation with busulfan and etoposide as conditioning regimen for acute myelogenous leukaemia patients s. mousavi, k. alimoghaddam, f. khatami, m. jahani, m. iravani, b. bahar, a. khodabandeh, a. jalali, a. ghavamzadeh hematology-oncology and stem cell transplantation research center (tehran, ir) introduction: acute myelogenous leukemia(aml) is a potentially lethal disease. hematopoietic stem cell transplantation(hsct) has increased disease free survival (dfs) and overall survival (os) of patients more than conventional treatment. the result of autologous hsct in patients without suitable donor is near to allogeneic transplantation. we performed autologous transplantation with busulfan and etoposide as conditioning regimen for patients who didn't have suitable donor. methods: since january 2003 until oct 2008, 108 patients received autologous transplantation. we included all children and adult with aml in fi rst or second complete remission without suitable donor and end organ failure who can tolerate high dose chemotherapy. mobilization regimen was cyclophosphamide 2g/m² for one day and g-csf 10µg/kg for 7 days. we have done stem cell harvesting when patient's white blood cell (wbc) count raised to 1000/µl then the patients received oral busulfan 4mg/kg(from -4 to -1) and etoposide 15mg/kgiv (from -4 to -3) as conditioning regimen. after that patients have transplanted with their peripheral blood stem cells. results: median age at time of transplantation was 25.5 years (age range:4-68), male/female:57/51. 94 patients were in fi rst complete remission and 14 patients were in second complete remission. median infused mononuclear cells was 5.11 * 10 8 /kg despite of the progress in the treatment of acute myeloid leukemia allogeneic hematopoietic stem cell transplantation (hsct) remains the single curative approach to the treatment of resistant aml. these patients (pts) have extremely poor prognosis with the level of relapse 70-80% and the risk of trm 45-70%. high level blasts to the moment of transplantation infl uences on dfs and os. the usage double-phase conditioning regimens with the aim to reduce the level of blasts maximally to the transplantation date can improve dfs and os without the elevation of trm. patients and methods: we reviewed the records of 12 refractory aml pts (8 m/4 f) who underwent hsct at the russian children research hospital between october 2005 and june 2008. the median age was 5 (1-18) years. fab-type: m0 -2 pts, m5 -3 pts, m6 -2 pts, m7 -3 pts, mx -1 pt and secondary aml -1 pt. primary refractory aml was diagnosed in 7 pts and secondary refractory -in 5 pts. 4 kids were transplanted from msd, 2 pts -from mud, 1 pt -from mmud and 5 pts -from mmfd with cd3/cd19-depletion of graft. the median level of blasts in bone marrow prior to hsct was 58% (6-98). first phase included flam (3 pts), flag/go (2 pts), ham (3 pts), flae (1 pt), go (1 pt) or dacogen (1 pt). mylotarg doses were 3-5 mg/m². the interval between 1-st and 2-nd phase of conditioning protracted 1-12 days. second phase of conditioning was treosulfan-(9 pts) or bu-based (3 pts) . a median dose of cd34±cells was 8,5x10 6 /kg. seven patients received dli on median day 63 (44 -119). results: engraftment was documented in all patients with a median time to neutrophil recovery 17 days (8-33) and to platelets recovery -18 days . 11 patients achieved cr to day 30 and one patients had pr. acute gvhd developed in 6 patients, chronic gvhd -in 3 from 9 patients who were alive to day 100. 5 patients had the liver toxicity grade 2-4, and one patient had the pulmonary toxicity grade 3-4. trm level was 8,3%. relapses were diagnosed in 6 patients (dfs 33%). nowadays 5 pts are alive in cr, 1 pt has bm-relapse, 6 pts died (4 -of the relapse, 1 pt -of viral infection, 1 pt -of vod). os amounted 47% with a median time 6 months. rfs depended on the presence of gvhd and type of donor. conclusion: our results show that the usage of double-phase conditioning regimen generally doesn't increase the level of toxicity and trm and allows to achieve the long-term survival in pts with very high risk aml. allogeneic haematopoietic stem cell transplantation for acute myelogenous leukaemia in other than fi rst complete remission status k. alimoghaddam, f. khatami, a. jalali, m. jahani, s. mousavi, m. iravani, a. khodabandeh, b. bahar, a. alimohammadi, n. bahar, a. ghavamzadeh hematology-oncology and stem cell transplantation research center (tehran, ir) introduction: for patients with aml in whom an initial remission cannot be achieved or for those who have a relapse after chemotherapy, stem cell transplantation from an hla-identical sibling offers the best chance for cure. this study reports the result of 18 years allogeneic hsct for aml in other than fi rst complete remission status. patients and methods: from march 1991 until november 2008, 108 aml patients, 55 male and 53 female with median age of 27 years old (range: 3-56 yrs) received allogeneic hsct. the status of them before transplantation was other than cr1 (including second cr, third or more cr, relapse and primary induction failure). source of hematopoietic stem cells was 97 peripheral blood, 10 bone marrow, and 1 cord blood. result: median time to absolute neutrophil count ≥0.5 * 10 9 l was 12 days post hsct and median time to platelet count ≥20 * 10 9 l was 17 days post hsct. forty-two recipients developed acute graft versus host disease (gvhd) and twenty-four developed chronic gvhd. at present 73 (68%) patients are alive. the most common cause of death was relapse. median follow up period was 10 month (range: 1-99 month). six month disease free survival (dfs) and overall survival (os) were 64% (se=5%) and 75% (se=4%), respectively. 2 year dfs and os were 50% & 64% (se=5%). conclusion: allogeneic hsct for aml in other than fi rst complete remission could be advice and can improve the result of treatment in these high-risk patients. outcome of haematopoietic stem cell transplantation for patients with acquired aplastic anaemia at a cancer center, amman, jordan: experience of a young hsct program in a developing country f. abdel-rahman, i. al-sadi, a. badeeb, h. el taani, a. ahmed, r. rihani, a. al zaben, m. sarhan king hussein cancer center (amman, jo) purpose: to evaluate the outcome of hsct in patients with acquired aplastic anemia at khcc. patients and methods: between (3/2003-10/2008) ,15 patients had allogenic hsct for aplastic anemia. there were 9 adults (60%), and 6 children (40%), with a median age of 20 years (range:5-52 years). there were 10 patients (67%) with severe aplastic anemia and 5 patients (33%) with very severe disease. the source of stem cells was bone marrow in 13 patients (87%), and peripheral blood in 2 patients (13%). the median time from diagnosis to transplantation was 86 days. among the group, 12 patients had a full hla matched-related donor, one had 5/6 matched related donor and 2 had 3/6 donor. the conditioning regimens were cyclophosphamide +antithymocyte globulin (atg) in 10 patients, and different conditioning in the other 5 patients. results: the main end points of the study are overall survival for the whole group, and overall survival according to the age, severity of the disease, occurrence of graft versus host disease, and degree of hla match. the median duration of follow up was 5.5 months (1.1-47.2 months) . the median time for the wbc engraftment was engrafted the wbc or the platelets, and 2 patients never engrafted the platelets. the median survival for the whole group was 10.6 months. from the 15 patient, 8 patients are still alive. from the 7 deaths, 6 patients died from sepsis and one from massive gi-bleeding secondary to gut gvhd. from the 9 adult patients 6 are alive (57%), while from the 6 pediatric patients 2 are alive (33%) from the 10 patients with severe aplastic anemia 5 are alive while from the 5 patients with very severe disease 3 are alive. from the 12 patients who had transplant from 6/6 hla matched related donor, 8 are alive (67%). the three other patients who received mismatched graft died. acute gvhd was associated with increased mortality. six of nine patients who develop gvhd died while only 1 out of 6 patients who did not develop gvhd died. four patients had second transplant, two of them are still alive. conclusion: the important predictors of the outcome are: 1-degree of hla match: survival 67% in 6/6 hla match, versus 0% for the mismatch transplant. 2-occurrence of gvhd: survival is 83% in patients without gvhd, and 33% in patients with gvhd. therefore, the most important factor for predicting survival is the degree of hla match. our plan is not to transplant aa from mismatched donors except according to an international study protocol. (1) diabetes type 1 is caused by immune destruction of insulinproducing b cells of pancreas. it has recently been shown that immunoablation combined with transplantation of autologous hematopoietic stem cells may alter the course of the disease and alleviate exogenous insulin requirement [voltarelli et al. jama, 2007; 297:1568 -1576 . we report a 28 year old patient with an early diabetes type 1 (typical clinical course, presence anti-gad antibodies, diagnosis 6 weeks prior to study inclusion) with sustained presence of c-peptide in the blood, in good clinical condition without other serious comorbidities who has been chosen for treatment after signing informed consent for study protocol earlier accepted by local bioethics committee. treatment consisted of plasmapheresis followed by mobilization with cyclophospamide (2 g/m²) and granulocyte colony stimulating factor (g-csf) analogues at 10 µg/kg from day +1. three x 106 cd34+ cells/ kg were obtained by leukapheresis and were later used for transplantation without further selection. conditioning regiment consisted of cyclophosphamide (50 mg/kg for days -5 to -2 each) with atg (thymoglobuline 4,5 g /kg over days -5 to -1). and was followed by transfusion of collected peripheral blood cells on day 0. results: the transplantation was performed on the 1.05.2008. patient engrafted on day +13. during the cytopenic period no major complications were observed. the patient insulin requirement was: 0,47 iu/kg -before moblization, 0,36 iu/kg on the transplantation day, 0,17 iu/kg on engraftment. insulin was discontinued shortly after the regeneration (+24). glucose monitoring showed normal glucose levels without the need for insulin injections from that day on. hba1c levels at diagnosis were 13,8%, 5,2% after 3 months from transplantation and 5,7% 6 months after the transplantation. continuous glucose monitoring was performed around 5 months after the transplant and showed normal values (glucose 75 -135 mg/dl) -fi gure 1. intravenous glucose tolerance test showed normal values of glucose levels after 120 minutes, however the 1st phase of insulin secretion was not present . conclusion: this case support the notion that immunoablation followed by autologous stem cell transplantation in patients with early diabetes type 1 may at least temporary alleviate insulin requirement with excellent control of glycemia. introduction: the allogeneic stem cell transplantation (hsct) represent an effective curative treatment in cml but treatmentrelated morbidity and mortality can be substantial. with the era of bcr-abl kinase inhibitor the place of sct is in discuss for children. we report the results of myeloablative allogeneic hsct underwent in 25 patients (pts) under 18 years of age. material and methods: from december 1998 to april 2007 (101 months period) 25 pts under 18 years of age with cml (chronic phase: 21, accelerated phase: 4) underwent myeloablative allogeneic hsct from hla-identical sibling donors; median age at transplant 13 years (4-17); sex ratio m/f 0,66; median interval from diagnosis to transplant 13 months (4 to 39). all patients received chemotherapy based conditioning regimen: tutshka (n:19), tutshka with additional vp16 (n : 3) and santos (n: 3). 21 pts received peripheral blood stem cell with median cd34+ cell 7,77 10 6 /kg body weight (bw), 3 pts bone marrow transplant with median nuclear cell ( nc ) 3,97 10 8 /kg bw, one pt blood cord transplant with 3,5 10 7 nc/ kg bw. graft-versus-host disease (gvhd) prophylaxis consisted of association ciclosporin and methotrexate. the molecular bcr-abl transcripts diagnosis concerned 14 pts (m(b2a2): 8, m(b3a2): 5 and double transcripts m (b2a2; b3a2):1). molecular monitoring of disease using real-time quantitative polymerase chain reaction (rq-pcr) concerned 11 pts. at 31 july 2008 maximal follow-up is 128 months and minimal 16 months. results: the median time of aplasia was 14 days ( 6-35). eighteen pts (72%) are alive in complete hematological remission (complete molecular remission: 8; major molecular remission: 3; no evaluated: 7) after median follow-up time 61 months . acute gvhd occurred in 7 pts (28%) with 6 grade ii-iv, and chronic gvhd in 10 pts (43,5%) with 5 extensive. disease relapse occurred in 7 pts (28%) within 2 are in complete remission with imatinib. seven pts died (32%): acute gvhd grade iv (n : 2, trm: 8%) ; relapse (n: 5; 24%). the os and event free survival (efs) at 9 years are respectively 66% and 60%. conclusion: our results confi rm that trm is low in young pts and the mayor problem is still relapse disease. the relapse after graft can be treated with bcr-abl kinase inhibitor (2 pts in our study). the question about using allogeneic hsct or bcr-abl kinase inhibitor in children with cml is still open. reduced-intensity conditioning allogeneic stem cell transplantation in advanced chronic lymphocytic leukaemia. the impact of conditioning regimen on the non-relapse mortality. a single-centre experience j. el-cheikh, c. oudin, l. wang, c. faucher, s. furst, d. blaise institut paoli calmettes (marseille, fr) purpose: a unicentric retrospective study to determine the transplant related toxicity in patients with advanced chronic lymphocytic leukemia (cll) after reduced intensity conditioning hematopoietic stem-cell transplantation (hsct) including or not antithymoglobuline (atg). patients and methods: we studied 19 patients with progressive or relapsing chronic lymphocytic leukemia (cll) treated with hematopoietic stem cell transplantation (hsct) in our cancer centre of marseille. 13 males and 6 females, (median age: 60 years). all patients received a reduced intensity conditioning regimen. we compared 11 patients (58%) receiving a non myeloabaltive conditioning including atg with fl udarabine, busulfan (atg group) to 8 patients (42%) receiving fl udarabine, total body irradiation (tbi 2gy) and anti cd20 without atg (non atg group). 14 patients (74%) had a matched related and 5 patients (26%) a matched unrelated donors. graft-versus-host disease (gvhd) prophylaxis consisted of cyclosporine alone in the atg group or a combination with mycophenolate mofetil (mmf) in the non atg group. results: after a median follow-up of 29 months, 13 patients (68%) still alive and in complete remission (to date). mrd was monitored in those patients with cr; all patients achieved a molecular cr. 13 patients had acute and/or chronic gvhd, (70% in atg group vs 30% in non-atg group). at the last follow up 6 patients died (32%), and the cause of death in all of them was the treatment related complications (infections and/ or gvhd); the trm at 100 days was 0%; 26% at one year and 32% at three years of transplantation. (21% in atg group vs 11% in the non atg group); overall survival (os) at three years was 52% in the atg group vs 66% in the non atg group. the os at one and three years was 69% and 59% respectively. fig1. in conclusion: despite the small effective, we can conclude that hsct after reduced conditioning is effective and has the capacity to induce a long term complete remissions, the real impact of atg should be revaluated on further large multicentric studies. dasatinib: optimal bridge to stem cell transplant in chronic myeloid leukaemia blast crisis a. gozzini, s. guidi, c. nozzoli, b. bartolozzi, r. saccardi, b. scappini, a. bosi bmt unit (florence, it) pts presenting cml-bc have a survival of 3-6 months and scarce response to imatinib. dasatinib (bms-354825) is an oral, multi-targeted kinase inhibitor, currently being used in pts with imatinib-resistant advanced cml or relapsed/refractory ph+ all. most of these pts will be evaluated for sct, even though for them this curative therapy showed higher incidence of gvhd, vod and trm. we report here fi ve pts affected from cml-lb who received dasatinib prior to allosct. donors were matched siblings (2), matched unrelated (2) or blood cord unit (2) . 4 were male and 2 female with a median age of 36,4 (18-55) years. first line therapies included chemotherapy (vcr) plus high dose imatinib. all pts after 2-5 months from diagnosis received dasatinib 70mg bid. t315i mutation occurred in 3 patients, y253 and e255k in 2 patients, and a non codifi ed mutation in 1 patient. dasatinib induced complete hematological response (chr) in 4 pts, and complete (n=2) and partial cytogenetic response (pcyr) (n=1) prior to sct. 3 patients did not achieved a complete haematological response presenting 25% marrow blasts and 65% respectively prior to sct. all pts were conditioned with myeloablative protocol. gvhd prophylaxis consisted of csa and mtx (n=4) or micofenolate association until +30 (n=2). pts received a mobilized peripheral blood stem cell graft with 3,52-11,04x 10 6 cd34+ cells/kg (n=4) and cord blood unit with 0,1x10 6 cd34+ cells/kg (n=2). dasatinib was stopped 6 days before transplant procedure. 6/6 pts successfully engrafted reaching anc >0.5x10 9 /l on day +19 (11-37) and plt >20x10 9 /l on day +21 (11-50). dasatinib was introduced again in 2 patients 30 days after sct. one of them stopped therapy because of haematological toxicity after 2 weeks. 5/6 patients presented chimerism was 97-100%. transplant related toxicities were grade i/ii. no pts developed hyperbilirubinemia or vod. hyperacute extensive gvhd (gr iii) was observed in only 1 pts at +9. five patients are alive, all of them in complete molecular response with a median follow-up of 9.3 (4-19) months, 1 died of agvhd. we may conclude that in pts undergoing sct following dasatinib there is no evidence of adverse effect on sct outcome, organ toxicities. larger studies and longer follow-up are obviously indicated to confi rm our preliminary results. both t315i positive patients are alive in chr. dasatinib represents an effi cient bridge to transplant to improve the outcome of this subset of patients. inmatinib combined myeloablative allogenetic haematopoietic stem cell transplantation for advanced chronic myeloid leukaemia y. luo, y. tan, j. shi, x. han, g. zheng, x. zhu, x. lai, h. huang zhejiang uninversity school of medicine (hangzhou, cn) improved strategies are needed to treat patients with advanced chronic myeloid leukemia (cml) in order to reduce the need for lifelong therapy. we treated 14 patients with advanced cml (5 in ap, 9 in bc) with myeloablative allogeneic stem cell transplantation (allo-sct) combined with pre-transplantation imatinib. the donors included hla-matched and 1-locus mismatched unrelated volunteers (n=7), and hla-matched siblings (n=7). graft-versus-host disease (gvhd) prophylaxis consisted of cyclosporine, mycophenolate mofetil and short-term methotrexate. 6 out of 14 (42.9%) evaluable patientsdeveloped ii-iv agvhd, 3(21.4%) patients suffered from agvhd grade iii-iv. two patients suffered from intensive chronic gvhd. after a median follow-up of 19 months (range 3¨c47 months), the overall survival was (71.4%) 10/14. the ten patients were all in molecular remission. imatinib combined with allo sct could provide a safe, well-tolerated therapeutic option for patients with advanced cml. this conclusion needs to be tested in prospective randomized clinical trials. p-beam group. both groups did not differ in terms of time of hospital stay, days of iv antibiotics, mucositis and infections. with the median follow-up of 6 (5-10) years, the probability of overall survival at 6 years equaled 83% for p-beam and 63% for chopp-cbv group (p= 0.2). the probability of progressionfree survival was 65% and 50%, respectively (p=0.2). conclusions: p-beam and chopp-cbv protocols followed by autopbsct are effective and well-tolerated salvage therapies for patients with advanced hl. prolonged administration of the therapy seems to be appropriate for this group of patients. towards safer autotransplants in patients with non-hodgkin's lymphoma: cardiac pre-evaluation, angiotensin-converting enzyme inhibition in patients with decreased left ventricular function, antimicrobial prophylaxis and vigilant supportive care e. jantunen, s. hämäläinen, t. kuittinen, k. penttilä, m. pyörälä, a. juutilainen, i. koivula, t. nousiainen kuopio university hospital (kuopio, fi) autologous stem cell transplantation (asct) for nhl is associated with an early non-relapse mortality rate of 3-5% most commonly due to sepsis. during 1996-2006 160 nhl patients received asct at our department. seventeen patients (9%) experienced severe sepsis and nine (4.5%) died due to septic shock. severe sepsis was caused by gram-negative bacteria including pseudomonas in a signifi cant proportion of the patients (hämäläinen et al. scand j infect dis 2008). subclinical anthracycline cardiomyopathy may be important in regard to the development of severe sepsis in some nhl patients. since january 2008 we have applied prospectively cardiac pre-evaluation (radiocardiography), angiotensin converting enzyme inhibition in patients with decreased left ventricular ejection fraction (lvef) (< 50%), ciprofl oxacin prophylaxis and start with ceftazidime plus tobramycin in patients with neutropenic fever in nhl patients undergoing asct. febrile patients are observed closely with measurements of pro-brain type natriuretic peptide (bnp) and c-reactive protein (crp) for three days. also blood pressure, blood oxygen saturation, hydration and diuresis are monitored. until nov 2008, altogether 14 patients with nhl (10 m, 4 f) with a median age of 55 years (range 28-65) have received beam followed by pb infusion according to this protocol. lvef was < 50% in six patients (43%) pre-transplant and they received enalapril during the peritransplant period. neutropenic fever was observed in 12 patients (86%). no cases with gram-negative bactereamia or severe sepsis have been observed. the median peak crp value was 139 mg/l (23-333) and was reached in a median of two days after rise of fever. serum bnp values were above normal limit in 3/12 patients with fever on day 0. elevated bnp values were observed in 4/10 patients on day 1, in 9/12 patients on day 2, and in 7/9 patients on day 3, respectively. whether severe sepsis or early deaths could be prevented with this approach remains to be seen in upcoming years with larger number of patients. outcome of refractory/relapsed patients affected by hodgkin's lymphoma treated with or without peripheral blood stem cells autografting: a single-centre experience f. angrilli, s. falorio, f. fioritoni, s. santarone civic hospital (pescara, it) introduction: despite a high curability rate, 10 to 40% of patients (pts) affected by hodgkin lymphoma (hl) fail to respond or relapse after front-line treatment with polychemotherapy alone or combined with radiotherapy. the treatment of choice for refractory or early relapsed pts is high-dose chemotherapy (hdc) followed by peripheral blood stem cells autografting (pbsca), while late relapsed pts may be treated with either conventional therapy or hdc plus pbsca. methods: from 1999 to december 2007, 179 untreated pts with hl have been admitted in our institution. after front-line therapy, 168 (93%) pts obtained a complete remission (cr) and 11 pts (7%) were refractory to standard treatment. overall, 11 pts relapsed within 12 months after diagnosis of hl, while 6 pts experienced late relapse. the aim of this retrospective study is to evaluate the outcome of the our 28 refractory/relapsed pts according to the type of salvage treatment. twenty-six pts received as salvage treatment 3-6 courses of igev (iphosphamide, gemcytabine, vinorelbine), 1 patient 6 courses of coppebvcad (cyclophosphamide, carmustine, melphalan, epirubicin, vinvristine, vinblastine, prednisone) and 1 patient 6 courses of abvd (doxorubicin, bleomycin, vincristine, dacarbazine). today, 27 pts completed salvage chemotherapy. of them, 19 (8 with refractory hl and 11 with relapsed disease) have been submitted to pbsca. conditioning regimen consisted of beam in all cases. results: 15 pts were male and 13 female (m/f ratio 1,15). median age was 34 years (range 16-59). overall, 17 pts obtained a cr (63%) and 10 pts had progressive disease (37%). in particular the cr were 14 (73%) in the group of the pts receiving pbsca and 3 (33%) in the other pts (p <0.05). one patient died in cr of beam toxicity prior pbsca and 10 pts died of progressive hl. after a medium follow-up of 24 months, overall survival was 63% for the pts who received pbsca and 38% for those who received conventional treatment (p=0.05). conclusions: our data confi rm the benefi t of hdc plus pbsca both in relapsed and in refractory patients with hl. nevertheless, a portion of refractory or early relapsed pts fail to respond to pbsca and died of hl. for these pts tandem pbsca or allogeneic stem cell transplantation should be proposed, especially if they are not in cr prior to pbsca. t cell lymphoma is a heterogeneous group of aggressive lymphomas associated with poor prognosis with standard chemotherapy and autologous hematopoietic progenitor cells transplantation (hpct) is offered as consolidation in fi rst remission or at relapse. in this study we conducted a retrospective analisis of 16 patients underwent hpct from december 1993 to august 2008. seven patients had diagnosis of peripheral t-cell lymphoma, four patients of systemic anaplastic large cell, and fi ve patients of linfoblastic lymphoma. five patients were transplanted in fi rst complete or partial response, ten patients in second or beyond complete or partial response and one patients in second refractory disease. median age was 36,5 years; seventy-fi ve percent preesented advanced (iii-iv) ann arbor stage, 50% had b symptoms, 50% had high lactate dehydrogenase. with a median follow-up of 73 months from diagnosis and 31,5 months from transplantation, the 5-year progression-free survival (pfs) and overall survival (os) were 37,5% and 31,2% respectively. based on these preliminary results the hpct as consolidation therapy may offer a durable survival benefi t. the chimeric anti-cd20 monoclonal antibody rituximab offers new therapeutic options in the treatment of b-cell nhl (non-hodgkin's lymphoma). the addition of rituximab to chop (cyclophosphamide, doxorubicin, vincristine, and prednisolone) or cvp (cyclophosphamide, vincristine, and prednisolone) regimen was found to signifi cantly improve the response rates and survival in patients with untreated diffuse large b-cell lymphoma (dlbcl) and is now considered as the standard therapy option. rituximab also has been shown to improve response rates when combined with salvage chemotherapy. there are few studies regarding the effects of rituximab on mobilization. we compared the effi cacy of rituximab plus eshap (etoposide, metil prednisolone, cytosine arabinoside, cisplatin) with eshap alone as mobilization regimen in 34 (40%) hodgkin's and 50 (60%) non-hodgkin's lymphoma patients. 26 (30%) patients were dlbcl. 43 (51%) relapsed and 21 (25%) refractory patients were involved. 19 (23%) patients were treated with r-eshap and 65 (77%) patients with eshap regimen. 228 aphaeresis were evaluated. median number of aphaeresis was 2.64 days for r-eshap patients and 2.71 days for eshap patients. median number of mononuclear cell aphaeresis was 4.75*10 8 per kg (kilogram) and 6.83*10 8 per kg respectively. total number of cd34+ cells was 15.58*10 6 per kg in the r-eshap group and 17.75*10 6 per kg in the eshap group. toxicities were similar in both groups. there were no engraftment delays in the r-eshap group. so we conclude that r-eshap is effective and feasible as eshap regimen for mobilization. total number of cd34+ cell aphaeresis was slightly lower in the r-eshap group but did not have an effect on engraftment. prospective randomized studies are needed to evaluate whether rituximab really decreases mobilization adequacy or not. no benefi t of autologous stem cell transplantation as consolidation for high and high-intermediate risk diffuse large b-cell lymphoma in 1.cr after r-chop therapy -a single-centre experience m. karas, k. steinerová, p. jindra, d. lysák, s. vokurka, v. vozobulová, m. schützová, l. mohammadová, v. koza charles university hospital pilsen (pilsen, cz) objectives: the role of high-dose therapy (hdt) and autologous stem cell transplantation (asct) for patients (pts) with high and high-intermediate (h/hi) risk diffuse large b-cell lymphoma (dlbcl) in 1.cr was not clearly defi ned especially after addition of rituximab (r) to fi rst line chemotherapy (cht) and the use of rituximab also as maintenance therapy. therefore, we retrospectively analysed outcome of pts treated in our transplant centre with hdt and asct for h/hi risk dlbcl in 1.cr after 6-8 cycles of r-chop-21 chemotherapy and we compared their outcome with a control group of pts with h/hi risk dlbcl in 1.cr treated only with chemoimmunotherapy. patients and methods: between 2003 and 2008 (median follow-up 38 months, range 13-64 months) 17 consecutive pts with median of age 48 years (range 24-63 years) with h/hi risk dlbcl in 1.cr after 6-8 cycles of r-chop-21 underwent hdt (beam) and asct. the median of time from diagnosis to asct was 8 months (range 5-13 months). source of stem cells was peripheral blood and median of infused cd34+ cells was 4,36x10 6 /kg (range 3,28-9,94x10 6 /kg). the control group consisted of 11 consecutive pts with h/hi risk dlbcl in 1.cr treated only with chemoimmunotherapy (6-8 cycles of r-chop-21, 45% maintenance therapy with rituximab). the control group except for the older age did not differ in any prognostic parameters. results: in the transplanted group 15 pts (88%) are alive in cr. 2 pts (12%) relapsed and died. no patient died due to transplant-related mortality (trm). the estimated probabilities of 4-years disease-free survival (dfs) and overall survival (os) were 87% and 86%. in the chemoimmunotherapy treated group 10 pts (91%) are alive in cr. 1 patient (9%) relapsed and died. the estimated probabilies of 4-years dfs and os were 75% and 67%. we did not observe between both groups any significant difference in cumulative relapse incidence (p=1,00), dfs (p=0,91) and os (p=0,89). conclusion: our data suggest that hdt with asct in pts with h/hi risk dlbcl in 1.cr after r-chop chemotherapy was well-tolerated with no trm death but in comparison with pts treated only with chemoimmunotherapy we did not observe any improvement of outcome among transplanted pts. of course relatively lower number of evaluated pts and retrospective type of analysis could infl uence our results and only prospective randomized studies can fi nally defi ne the role of frontline hdt with asct for h/hi risk dlbcl in 1.cr after chemoimmunotherapy. kyrcz-krzemien medical university of silesia (katowice, pl) background and aims: autologous peripheral blood stem cell transplantation (autopbsct) is widely used for the treatment of poor-risk patients with hodgkin's lymphoma (hl), however, the optimal preparative regimen has not been established. we assumed that patients with advanced hl may benefi t from receiving intensive pre-transplant therapy with prolonged administration of cytostatics and the addition of oral drugs, such as procarbazine or chlorambucil. therefore, we modifi ed the commonly used beam and cbv protocols by incorporating oral agents and prolonging the distribution of the total doses to 7 and 9 days, respectively. the goal of this pilot study was to evaluate safety and effi cacy of those regimens. patients and methods: 33 patients (20 males and 13 females, median age 27 years, range 17-63) with relapsed hl were included in this study. previous therapy consisted of median 3 (2-5) lines of treatment and 11 (3-32) chemotherapy cycles results: 1/19 patients died due to septic complications in chopp-cbv group, whereas no procedure related mortality was observed among patients treated with p-beam. all remaining patients engrafted. time to achievement anc >0.5 g/l was signifi cantly shorter in p-beam vs. chopp-cbv group: 14 (10-34) days vs zevalin-beam conditioning in transformed follicular lymphoma; acceptable toxicity and possible therapeutic benefi t a hdt-sct) using beam conditioning has become standard therapy for relapsed fl, however recurrent disease especially in transformed follicular lymphoma (t-fl) remains the commonest cause of death. the addition of zevalin (ibritumomab tiuxetan), a cd20 targeted radiolabelled antibody to beam is safe and may improve the effi cacy of hdt we analysed 5 patients aged 42 to 58 with advanced stage t-fl who had received a median of 4 (range 3-4) lines of therapy prior to zevalin-beam sct. the median time from diagnosis to hdt-sct was 43 months (range 17-55.7) and all patients had chemosensitive disease in partial remission bcnu 300mg/m 2 , etoposide 200mg/m 2 , cytarabine 200mg/m 2 , melphalan 140mg/m 2 ) from day -7 to -1. the median stem cell dose was 2 the 3 remaining patients remain stable at a median of 16 months (range 4 -23) post-sct conclusion: the zevalin-beam protocol is as well tolerated as standard beam conditioning. the disease free survival in this small cohort of high risk patients with t-fl is encouraging but needs longer follow-up rituximab or not? a historical comparison of eshap and r-eshap as mobilisation regimen in 84 patients non-myeloablative allogeneic stem cell transplantation in patients with high-risk lymphoma: a multicentre experience g. console (1), g. irrera (1), m. martino (1) a. meliadò (1), c. rigolino (1), t. del vecchio (1), o. iacopino (1), m.c. cannatà (1), p. scaramozzino (1), i. bova (1), d. marcuccio (1), c. stelitano (2), s. molica (3), r. cantaffa (3), l. nocilli (4), a. mele (5) pugliese-ciaccio" (catanzaro, it); (4)osp 44 patients (pts) (23 females and 21 males), median age 43,1 years (range 18-67) underwent nst for high risk hodgkin disease (hd, 14 cases) and non hodgkin lymphoma (nhl, 30 cases) conditioning regimens consisted of fludarabine, thiotepa and cyclophosfamide in 30 cases, tli and atg in 5 cases, fludarabine and cyclophosfamide in 4 cases, fludarabine and thiotepa in 1 case, fludarabine, melphalan, thiotepa and atg in 1 case, campath-1, fludarabine, melphalan and tbi were employed in 2 cases. in 1 case tbi and fludarabine. cyclosporine-a (cya) and methotrexate (mtx) were used as gvhd prophilaxys in 35 cases, in 2 campath-1 and moftil micofenolate (mmf) were combined and in 7 cases cya and mmf were used. a mean number of 5.41x10 6 /kg cd34+ cells (range 2,1-8,7) were infused. pts received a mean of 3.04 (range 0-22) packed red blood cells after a median follow up of 33,2 months, (range 2-84), 31 pts are still alive (22 in cr,4 pr,5 in relapse), experienced cgvhd (4 cutaneos w.h.o. grade1-2,1 pneumonial w.h.o. grade 1,1 liver w.h.o 4). 3 pts died for liver agvhd,1 patient died for cerebral vasculitis at 18 months to the transplant,1 patient died for ards at 2 months from transplant. 1 pts for acute bacterial pneumonia. 2 pts for mof respectively at 1 and 2 months from transplant, 1 for aptt, at 18 months from transplant. 1 patient for interstitial pnemoniae at 20 months to the transplant, 3 pts died for disease recurrence at 10, 17and 36 months post-transplant respectively here we report on a single centre, retrospective analysis evaluating the outcome of patients (pts.) with dlcl treated with high-dose chemotherapy and autologous (auto) or autologous-allogeneic (auto-allo) hsct. patients and methods: in total, 22 (66,7%) male pts. and 11 (33,3%) female pts. with dlcl underwent auto (26 pts.) or autoallo hsct (7 pts) between 01.01.1994 and 30.06.2007. 19 pts. received auto hsct as part of fi rst-line therapy (group 1). in 14 pts auto (7pts, group 2) or auto-allo (7pts., group 3) hsct was performed as second-line therapy. the median patient age was 49 years 1: 3/6/10, group 2: 0/6/1, group 3: 0/2/5. patients who received auto hsct as fi rst-line therapy (group 1) tended to have a better median os (1374 vs. 272d, p=0.232), rfs (1245 vs. 95 d, p=0.025) and 5-ysr-os (65% vs. 45%, p=0.382) compared to pts of group 2. furthermore patients of group 1 had a signifi cant better os and 5-yrs-os in the auto-allo group 6 of 7 pts died (5 pts died from severe infection with multiorgan failure and 1 patient from relapse of disease). in contrast, none patient died from trm after second auto hsct, but 2 died from progressive disease and 1 pt from relapse. conclusion: the survival of patients with relapse of dlcl could not be improved by using the therapeutic approach of auto-allo hsct compared to an auto hsct based regime, due to the high trm in the auto-allo group. however, for interpretation of these results some facts have to taken into account since year 2000, 10 patients with primary myelofi brosis (8 females and 2 males age 29-55y median 46.5) received allo hsct (7 sib and 3 unrelated donors matched at allele level). according to the dupriez prognostic system: 2,6,2 patients were in high, intermediate an low risk of the disease. the diagnosis was proved by trephine biopsy, which revealed that all patients were at advanced stage of fi brosis, all patients had splenomegaly and abnormal blood smear with the presence of erythroblasts. the length of the disease duration was from 7 to 36 months (median 19). six patients were transfusion dependent because of anaemia and thrombocytopenia, three patients were on steroids and six on hydroxycarbamide. splenectomy prior to transplantation was performed in two patients. two patients received myeloablative conditioning (busulfan 16mg/ kg cyclophosphamide 120 mg/kg) and eight reduced intensity conditioning (busulfan 8 mg/kg, fludarabine 120 -150 mg/m² or melphalan 120-140 mg/m² and low dose atg). all patients were transplanted with pbpc with cd34 dose from 1.8 to 11.7 x 10 6 /kg (median 6.2 x 10 6 /kg). two patients died due to transplant toxicity (one with additional ebv reactivation and sepsis and one with vod symptomatology). in other patients toxicity was mild and there was no agvh exceeding grade i. two patients transplanted with major blood group incompatibility developed prca. plasmapheresis and erythropetin were successfully employed in those patients. finally all surviving patients reconstituted haematologically. a trephine biopsy performed 1 months post transplant documented the process of bone marrow remodeling with a normal picture six months post transplant. all patients except one had full chimerism. eight out of ten patients are alive and with normal hematopoesis during observation period from 1 to 104 months (median 24). the post transplantation course was similar in patients having and lacking jak 2 mutation. in conclusion haematopoetic stem cell transplantation in primary osteomyelofi brosis is associated with rather low risk and results in sustained haematological recovery. nutritional assessment of children undergoing haematopoietic stem cell transplantation for primary immunodefi ciency or severe autoimmune disease m. slatter, c. ferguson, e. rogerson, a. yurasova, p. askew, t. flood, m. abinun, a. cant, s. bunn, j a major challenge post hsct is adequate nutrition, as poor nutritional status adversely affects outcome. patients undergoing hsct for pid often fail to thrive pre-hsct due to underlying disease. we aimed to document nutritional intake of pid children undergoing hsct at our centre. 15 children who underwent hsct for pid or severe autoimmune (ai) disease from april 2007 -january 2008 were evaluated. the following prospective data was collected: diagnosis, age, donor, conditioning, presence of infection and growth. nutritional intake, biochemical indices, use of antiemetics and complications were documented on admission, after 2 weeks, then monthly until day 0, +7, +14, +21, +28, +42 then monthly until discharge home. patient characteristics: 8 patients had scid, 5 had other pid. 2 had severe ai disease. age at transplant ranged from 2 months to 16.6 years (median 8 months). 9 were ≤ 1yr. 8 had unrelated (2 cords), 5 matched family, 1 matched sibling and 1 haploidentical donor. all had chemotherapy conditioning -4 bu/cy, 3 flu/melph, 7 treo/flu, 1 treo/cy. results: all received supplementary feeding via nasogastric (14) or percutaneous jejunal tube (1) . only 2 required total parenteral nutrition, 1 with severe ai disease and 1 with persistent norovirus enteritis. all received at least 1 anti-emetic. 4 had viral enteritis -2 norovirus, 2 adenovirus. in 14 patients for whom adequate data was available, all had a reduction in calorie and protein intake in the 2-3 weeks following hsct, because of fl uid restriction. 2 had grade ii skin gvhd, none developed gut gvhd. 7 had mucositis requiring morphine. only 1 patient lost weight overall from time of admission to discharge, one had static weight, but 13 gained weight, by time of discharge. further evaluation of nutritional indices is required. the time around hsct is the most challenging to support adequate nutrition. careful nutritional assessment of patients undergoing hsct is critical and should direct nutritional support. patients should be optimised nutritionally prior to hsct, as the high metabolic demands around the time of hsct are unlikely to be met over the immediate transplant period. thalidomide+dexamethasone and partly b, b+dexamethasone, b+adriamycin+ dexamethasone (pad). the b group had a post-tx maintenance therapy with b 4 weeks: 1,3 mg 2 iv doses weekly + dexamethasone 20 mg 4 days. results: length of survival times (os) without and with b were signifi cantly different. further analysis of the curves in complete remission indicated 100% survival probability and 90% disease free survival (dfs) in 19 patients in a 50-month period. in the very good partial remission (vgpr) group (12 pts) the os was 100%, however, the dfs was only 60%. the survival curves were signifi cantly worse when tx was made in partial remission (os: 55%, dfs: 50% by 23 pts). conclusions: 1. the author›s data support the fi nding that lasting survival can be expected when tx is performed in cr or vgpr. 2. in the interest of this, in cases of a more aggressive disease, the fi rst line pad protocol before tx is the best therapy. after the tx a consolidation therapy with b+dexamethasone is very useful. 3. in a slightly less aggressive disease or with accompanying diseases a thalidomide+dexamethasone fi rst therapy may also be possible. 4. tx performed in partial remission maybe dangerous. at this time needed put in "the therapy arsenal". acute renal failure in myeloma patients during mobilisation procedures for autologous transplantation a. pivkova (1) during the last 30 years blood cell separation, generally referred to aphaeresis, has established a central role in both blood donor programmes and therapeutics. the technological advances in aphaeresis equipment have made procedures safer, faster and more effective. we present 3 cases (2 males and 1 female) with multiple myeloma treated at our department during 2007 until 2008. initial chemotherapy treatment was provided with thalidomide based regimens (c-thal dex 4 cycles or thaldex in 4 cycles) or 4 cycles of vad in one patient. all 3 patients before diagnosis and during initial treatment had normal and stable renal function. after completing remission in all, mobilisation of pbsc was preformed with g-csf 10mcg/kg in duration of 5 days. the number of wbc count prior collection was median 42 x10 9 /l (30-51) with median lymphomonocyte percent 13, 43 (4-22). aphaeresis was preformed at day 5 with cobe spectra cell separator and large volume aphaeresis. in all 3 patients after fi nishing the fi rst procedure we registered increase of renal degradation products in the serum during the fi rst 6 hours post aphaeresis and complete anuria which revealed in acute renal failure (renal type) treated with haemodialysis in several consecutive occasions. one month after resolving the renal impairment the patients continued with second mobilisation procedure with the same regimen and obtained a minimal mnc count of 2,0x10 8 /kg. autologous transplantation followed by melphalan reduced dose conditioning 100mg/m². engraftment was registered for ne>0,5x10 9 /l and plt >20x10 9 /l on median day + 10 (8 to 12). the patients had no need for blood transfusions. all 3 are in cr med 7 mths (3) (4) (5) (6) (7) (8) (9) (10) (11) after transplant. in one patient 4 months after, a double transplant was preformed. concerning the small group of patients, we can evaluate the possible impact of large volume aphaeresis in the renal impairment in these patients or the infl uence of cytokine mobilised cells on renal tubules. key: cord-281141-ouno4jpl authors: mahajan, swapnil; kode, vasumathi; bhojak, keshav; magdalene, coral m.; lee, kayla; manoharan, malini; ramesh, athulya; sudheendra, hv; srivastava, ankita; sathian, rekha; khan, tahira; kumar, prasanna; chakraborty, papia; chaudhuri, amitabha title: immunodominant t-cell epitopes from the sars-cov-2 spike antigen reveal robust pre-existing t-cell immunity in unexposed individuals date: 2020-11-05 journal: biorxiv doi: 10.1101/2020.11.03.367375 sha: doc_id: 281141 cord_uid: ouno4jpl the covid-19 pandemic has revealed a range of disease phenotypes in infected patients with asymptomatic, mild or severe clinical outcomes, but the mechanisms that determine such variable outcomes remain unresolved. in this study, we identified immunodominant cd8 t-cell epitopes in the rbd and the non-rbd domain of the spike antigen using a novel tcr-binding algorithm. a selected pool of 11 predicted epitopes induced robust t-cell activation in unexposed donors demonstrating pre-existing cd4 and cd8 t-cell immunity to sars-cov-2 antigen. the t-cell reactivity to the predicted epitopes was higher than the spike-s1 and s2 peptide pools containing 157 and 158 peptides both in unexposed donors and in convalescent patients suggesting that strong t-cell epitopes are likely to be missed when larger peptide pools are used in assays. a key finding of our study is that pre-existing t-cell immunity to sars-cov-2 is contributed by tcrs that recognize common viral antigens such as influenza and cmv, even though the viral epitopes lack sequence identity to the sars-cov-2 epitopes. this finding is in contrast to multiple published studies in which pre-existing t-cell immunity is suggested to arise from shared epitopes between sars-cov-2 and other common cold-causing coronaviruses. whether the presence of pre-existing t-cell immunity provides protection against covid-19 or contributes to severe disease phenotype remains to be determined in a larger cohort. however, our findings raise the expectation that a significant majority of the global population is likely to have sars-cov-2 reactive t-cells because of prior exposure to flu and cmv viruses, in addition to common cold-causing coronaviruses. uncovering the immunological responses to covid-19 infection will help in designing and developing next-generation therapies and manage the treatment of critical covid-19 patients. many host factors associated with mild or severe disease symptoms have been reported. for example, leukopenia, exhausted cd8 t-cells, higher levels of th2 cytokines in serum, a high titer of neutralizing antibodies, blunted interferon response, dysregulation of the myeloid cell compartment, activated nk cells, and the size of the naïve t-cell compartment is associated with critically ill patients (1) (2) (3) (4) . this wide range of variable factors shares a common immunological underpinningthat of a systemic dysregulation in immune homeostasis due to the failure of the host immune system to clear the virus during the early stages of the infection (5) . many studies have shown that clearance of respiratory viruses requires cd8 t-cell immunity (6) . a delay in the activation of cd8 t-cells and a lack of early ifn- production lead to an increase in viral load triggering overactivation of the innate and the adaptive arm of the immune system leading to a loss of immune homeostasis resulting in severe disease phenotype, including death. therefore, an early wave of strong cd8 t-cell response may delay viral titer build-up, allowing rapid clearance of the virus by the immune system without perturbing immune homeostasis. healthy humans not exposed to covid-19 show pre-existing cd4 and cd8 t-cell immunity to sars-cov-2 antigens (7) (8) (9) . the pre-existing immunity to cd4 and cd8 t-cells was detected against structural and non-structural sars-cov-2 proteins by overlapping 15-mer peptide pools. the existence of a pool of sars-cov-2-reactive t-cells in unexposed individuals is thought to arise from coronaviruses that cause common cold (8, 10) . whether pre-existing immunity provides any protection to sars-cov-2 infection, or contribute to a faster recovery from infection remains speculative. besides, it is unclear whether a pre-existing immunity, involving either cd4 or cd8 t-cells, or both, is required for maximal protection. identifying robust pre-existing immunity against sars-cov-2 in the healthy population can be used as a measure to assess the mode of recovery and also viral spread in the global population. in this study, we identified strong cd8 t-cell-activating epitopes from sars-cov-2 spike protein by a combination of epitope prediction and t-cell activation assays in healthy donors unexposed to sars-cov-2. the rationale for identifying epitopes that favor cd8 t-cell activation was two-fold. first, robust cd8 t-cell activating epitopes can be formulated as second-generation vaccines for short and long-term protection against viral infection. second, detection of preexisting immunity in healthy donors using epitopes that favor cd8 t-cell activation may provide a framework to understand the complex immune responses observed in clinical settings. it may also shed light on the differences in morbidity and mortality in different population groups across the globe. we developed a proprietary algorithm oncopeptvac to predict cd8 t-cell activating epitopes across the sars-cov-2 proteome. oncopeptvac predicts binding of the hla-peptide complex to the t-cell receptor (tcr). we selected a cocktail of eleven 15-mer peptides with a broad class-i and class-ii coverage and favorable tcr engagement predicted by the algorithm. the cocktail of peptides was tested for t-cell activation in healthy donors from the usa and india unexposed to covid-19. we observed higher cd8 t-cell activation by the 11-peptide pool compared to the overlapping 15-mer peptide pools from the spike-s1 and s2 proteins. homology analysis of the selected peptides with other coronavirus spike proteins indicated a lack of significant amino acid identity with any of the 11 peptides, suggesting engagement of one or more peptides in the pool to cross-reactive tcrs from other viruses, not particularly from a coronavirus. bulk and single-cell tcr analysis revealed expanded clonotypes recognizing epitopes from cmv, influenza-a, and other viruses to which most of us are exposed. taken together, our findings support that strong pre-existing cd8 t-cell immunity in unexposed donors is contributed by cross-reactive tcrs from other viruses. significantly, we discovered multiple immunodominant epitopes in our predicted pool of peptides that favored cd8 t-cell activation. finally, we show that our cocktail of 11-peptides induced a robust immune response in convalescent patients demonstrating that these peptides are recognized by infected patients. taken together, our study uncovered strong pre-existing cd8 t-cell immunity against sars-cov-2 using a small set of 11 epitopes that engaged cross-reactive tcrs recognizing epitopes from other viruses, not necessarily common cold viruses belonging to the coronavirus family as hypothesized by other studies. additionally, our findings provide a basis for the generation of herd immunity against covid-19 without prior sars-cov-2 infection. a deep cnn model oncopeptvac was implemented to predict the immunogenicity of the peptide-based only on the peptide and hla sequences. a total of 8,870 immunogenic and nonimmunogenic peptide-hla pairs were obtained from the iedb (11) . the blosum encoding was used to represent the peptide and hla molecules. the blosum substitution scores encode evolutionary and physicochemical properties of the amino acids (12) . in addition, hydrophobicity indices and predicted hla binding scores were also used to represent the peptide and hla sequences. oncopeptvac used the cnn model with multiple 2d convolutional layers combined with maxpooling to confirm the additive effect of different input features on the performance of the model. all the model versions were trained using 5-fold cross-validation. the aucs of the final model was 0.87 based on a blind test dataset ( figure 1a ). the prediction algorithm showed a sensitivity of 0.64 and a specificity of 0.84 based on the score cut-off of 0.2 ( figure 1b) . by increasing the cut-off score to 0.5, the specificity could be further increased to 96.8 with a concomitant loss in sensitivity. oncopeptvac reduced the number of false positives significantly compared to the hla-binding rank (compare figures 1b and c) reducing the number of epitopes that needed to be screened in a t-cell activation assay by 30% to identify true immunogenic epitopes. for example, to identify 50% (119 out of 238) of the immunogenic peptide-hla pairs present in the blind test dataset, 256 top peptide-hla pairs from oncopeptvac prediction needed to be screened, compared to 753 top peptide-hla pairs predicted by netmhcpan-4.0. the prediction algorithm was applied to the sars-cov-2 proteome and screened against 23 class-i hlas covering over 98% of the world population. a schematic of the in silico screening approach is shown in figure 1d . briefly, 9-11-mer peptides from the sars-cov-2 proteome were screened for tcr-binding against 23 hla, and peptides with oncopeptvac score >0.2 were analyzed for class-i hla binding. peptide-hla pairs with a high predicted binding affinity (<1 percentile rank) were selected, their length extended to 15-mer and screened for class-ii hla binding. peptides with favorable tcr binding and class-i/ii hla binding features were selected for further validation. the number of predicted immunogenic epitopes from sars-cov-2 protein-coding genes is shown in figure 1e . the distribution of oncopeptvac scores against different class-i hla genes indicates a higher number of favorable tcr-binding peptides for hla-b and c compared to hla-a ( figure 1f ). natural biases in hla-restrictions have been reported for immunogenic hiv epitopes (13) . we performed t-cell activation assay using the selected 11 epitopes from the sars-cov-2 spike antigen in unexposed donors. the 15-mer peptides are distributed across different segments of the rbd and the non-rbd regions of the spike antigens and few peptides carry ace2 receptor binding sites (figure 2a and tabletable-s1 ). activation of t-cells using the cocktail of 11 peptides (all-peptide) was compared to the responses from spike-s1 (157 peptides) and s2 (158 peptides) pools (see methods for assay details). in a 48h assay, 70% of the unexposed donors responded strongly to the predicted peptide mix by inducing intracellular ifn + in both cd4 and cd8 t-cells. the responses to spike-s1 and s2 peptide pools were weaker ( figure 2b) . a strong 48h ifn- response suggested recall to pre-existing antigen-experienced cd8 t-cells. the peptide-mix also induced a strong 4-1bb response in cd8 t-cells but not in cd4 t-cells ( figure 2b ). both ifn- and 4-1bb levels increased in cd8 t-cells at day-7 by the all-peptide mix compared to the spike peptide pools ( figure 2c ). we observed higher expression of ifn- and 4-1bb in the cd4 t-cells by the spike peptide pools at day-7 suggesting de novo activation ( figure 2c ). although the use of 15-mer peptides is expected to skew the response towards cd4 t-cells, we observed a stronger cd8 t-cell response to the peptide-mix suggesting that the 15-mer peptide though added exogenously, was processed and presented by class-i hlas efficiently. taken together, the results demonstrate that the use of oncopeptvac identified potent cd8 t-cell epitopes in the spike antigen that could not have been detected by using large overlapping peptide pools used in t-cell activation assays. next, we tested individual peptides from the mix to assess their contribution to t-cell activation. the magnitude and kinetics of ifn- induction in cd8 t-cells were variable in different donors ( figure 2d ). in most donors, the maximal response was detected by 7-days, but in donors d142 and d176 the response peaked at 48h and declined ( figure 2d ). we tested the effect of individual peptides in multiple donors as indicated by the arrows to determine their immunogenicity. as shown in figure multiple studies have reported pre-existing t-cell immunity in unexposed donors using spike peptide pools and attributed the response to t-cells recognizing epitopes from common coldcausing coronaviruses to which a large section of the global population is exposed (7, 8, 10) . homology analysis of the selected epitopes (see methods) indicated that 6 out of the 11 peptides share >67% sequence identity with sars-cov and only 1 (peptide-11) out of the 11 peptides has over 70% identity with multiple coronaviruses ( table 2) . peptide-11 is in the s2 domain of the spike protein and showed ≥1% cd8 t-cell response at 48h in 1 out of 7 donors tested (d167, figure 1e ). however, peptides 3, 6, 7, and 9 lacking significant identity to other coronaviruses ( table 2) showed ≥1% cd8 t-cell activation at 48h in at least one donor out of 7 ( figure 2e -f and figure s1 ). peptide-7 induced high cd8 t-cell activation at 48 h in two donors (d089 and d225 ( figure 2f and figure s1 ). taken together, the data suggest that pre-existing t-cell immunity to these peptides may be derived from cross-reactive tcrs recognizing other viruses. to identify cdr3s amplified by individual peptides, or the all-peptide-mix, bulk tcr analysis was performed on antigen-stimulated pbmcs from donors d089 and d225 (see methods). both donors showed a robust ifn- response to pep-7, and the pep-mix, but not to pep-1 ( figure s4 ). diversity and clonal amplification of unique public and private cdr3s were analyzed at three different time points ( figure 3a -b). both the donors showed clonal expansion of multiple public cdr3s recognizing hcmv, human herpes virus-5 (hhv-5), and influenza-a peptides when stimulated with pep-7 and all-peptide mix, but not with pep-1 (table s2 ). hcmv and hhv-5 cdr3s were expanded in donor d089 ( figure 3a , top panel), whereas d225 showed expansion of hcmv and influenza-a cdr3s ( figure 3a , bottom panel). significantly, these cdr3s were not amplified by spike-s1 and s2 peptide pools or by pep-1, the latter failed to activate t-cells in these donors. further, cdr3s recognizing hcmv peptide nlvpmvatv in donors 089 and 225 were different, suggesting that the same antigen engages multiple crossreactive tcrs in different donors. next, we analyzed private cdr3s in these two donors to identify novel sars-cov-2 antigen-specific cdr3 ( figure 3b ). donor 089 showed a lack of specific amplification of private cdr3s suggesting that the robust cd8 t-cell response detected in this donor may be contributed by the amplified public cdr3s ( figure 3b , top panel). in contrast, two private cdr3s were clonally amplified by pep-7 and all-peptide mix in d225 suggesting that the t-cell response is derived from both public and non-public tcrs in this donor ( figure 3b , bottom panel). a list of clonally amplified public and private cdr3s detected in the two donors is given in table s2 . to further investigate the tcr repertoire profile of donors 089 and 225 we analyzed the vdj gene usage in the bulk cdr3 data. in d225, two v segments trvb2 and trvb30 and a j gene trbj2-1 were significantly over-represented in pep7 and peptide-mix treated samples ( figure 3c ), whereas in d089 trbv12-4 and trbj1-2 genes were amplified (fig. 3d ). to characterize the phenotype and functional state of activated t-cells and reveal differences between the different treatments, we performed single-cell sequencing on a 10x platform. single-cell transcriptomics and tcr data obtained from 3500 -4500 cells identified 3000 -3500 unique transcripts (see methods). using graph-based clustering of uniform manifold approximation and projection (umap) we captured transcriptomes of 4 distinct cell types ( figure 4a and table s3 ). our assay method is enriched for the growth and proliferation of t-cells causing depletion of other immune cell types present in pbmc in a 14-day culture. three cell types, cd8, /, and nk-t were detected in all the samples. compared to dmso and pep-7 in which the cd8 t-cell fraction was ~60%, in spike-s1 and spike-s2 the cd8 t-cell fraction was 50% and 38% respectively. conversely, the cd4 cluster was expanded in spike-s1 (12%) and s2 (27%) compared to dmso (7%) suggesting that the spike peptide pools engaged cd4 tcells (table s3 ). the single cell transcriptomic analysis further revealed that pep-7 induced effector phenotype in the cd8 t-cell cluster by the expression of activation markers ifn-, 4-1bb ( figure 4b ) tnfrsf9, fas, and tigit (compare figure s5d with s5a-c). the top 10 pep-7-expanded clonotypes were cd27 + /sellsuggesting transition towards effector memory phenotype ( figure 4b ). spike-s1 and s2 peptides induced cd27 + /sell -t-cells in the cd4 clones 6 and 4 respectively ( figure 4b ). single-cell data revealed amplification of trbv2 (40%) and trbj2-1 (32%) in pep-7 stimulated t-cells ( figure 4c ) confirming the results from the bulk tcr analysis. next, we mapped cdr3- to specific clones from each treatment ( figure 4d ). the dmso, spike-s1, and s2-treated samples shared many clonotypes among themselves in the same frequency range suggesting weak antigen-induced activation and proliferation of t-cells. next, we analyzed the clonal composition and phenotype of t-cells to investigate the dynamics of antigen-specific t-cell response in the treated samples. we analyzed the top-30 clones for their phenotype by the expression of 25 marker genes ( figure s6 ). in all samples, including dmso, cd8 t-cell clonotypes were more frequent ( figure s6a-d) . as expected, the cd4 t-cell compartment was expanded in spike-s1 and s2-treated samples (20 and 25% of all clonotypes respectively) compared to dmso (6.5%) ( figure s6b-c) . the cd4 t-cells expressed tnfsf4 (ox-40) suggesting activation, although they failed to express ifn- ( figure s6b-c) . a few expanded cd4 clones in the spike-s1 and s2 treated samples showed a high expression of il17rb suggesting polarization towards a th17 phenotype ( figure s6b-c) . in the pep-7 treated sample, almost all clonotypes in the top-30 were cd8 t-cells. the highly expanded clones expressed multiple t-cell activation markers ( figure s6d ). interestingly, in addition to the activation markers, these cells expressed higher levels of il2ra (cd25) suggesting differentiation towards an effector memory phenotype ( figure s6d ). cd25 expression was low in the cd8 t-cell compartment in other samples. taken together, the results of the transcriptomic analysis highlighted that the strong immunogenic cd8 t-cell epitope identified in this study preferentially engaged cd8 t-cells pushing them towards an effector and effector memory phenotype. the spike-s1 and s2 peptide pools on the other hand engaged both cd8 and cd4 t-cells and modulated the cd4 t-cells towards a th17 phenotype. to assess whether the predicted epitopes are recognized by covid-19 infected patient t-cells, we tested the all-peptide mix on seven asymptomatic, five with mild-moderate symptoms, and five severe convalescent patients requiring icu admission (table s4 ) and analyzed their cd4 and cd8 t-cell response after 48h. the patients experiencing mild to moderate symptoms exhibited higher induction of ifn- in cd8 t-cells ( figure 5a ). the ifn- induction in cd4 tcells was higher in the presence of the spike-s1 peptide pool compared to the all-peptide mix ( figure 5c ). spike-s2 peptide pool induced stronger 4-1bb induction in cd8 and cd4 t-cells compared to the all-peptide mix ( figure 5b-d) . taken together, our results confirm that the epitopes prioritized by the algorithm were recognized by covid-19 infected patient t-cells, and the ifn- response induced by the all-peptide mix was skewed towards cd8 t-cells. spike peptide pools favored activation of the cd4 compartment in these convalescent subjects in line with our assay results and single cell transcriptome analysis showing a preferential expansion of the cd4 compartment by these peptide pools. a wide array of respiratory viruses induces severe pneumonia, bronchitis, and even death following infection. despite the immense clinical burden, there is a lack of efficacious vaccines with long-term therapeutic benefit. most current vaccination strategies employ the generation of broadly neutralizing antibodies, however, the mucosal antibody response to many respiratory viruses is short-lived and declines with age. in contrast, several studies on respiratory viruses have shown the presence of robust virus-specific cd8-t cell responses which has been shown to last for decades. therefore, vaccine designs for emerging respiratory viruses need consideration and rational inclusion of cd8 epitopes to confer long term resistance (14) . this study demonstrates the existence of strong cd8 t-cell activating epitopes in the spike antigen and uncovers robust pre-existing cd8 t-cell immunity in unexposed donors. several studies have reported pre-existing t-cell immunity in unexposed donors and attributed these to infections by common cold-causing human coronaviruses (7, 8, 10) . other studies, on the contrary, have reported a lack of pre-existing t-cell immunity in unexposed donors (15, 16) . to identify strong cd8 t-cell epitopes, we developed a novel tcr-binding algorithm oncopeptvac that selects epitopes favorable for tcr-binding. in all epitope screening methods, epitope selection is primarily based on class-i and ii hla-binding affinity, which predicts surface presentation of antigen in complex with hla (19) , but not the interaction of the peptide-hla complex with a tcr (20) . by incorporating features that predict tcr-binding of a peptide, our algorithm oncopeptvac successfully identified many cd8 t-cell epitopes in a small pool of 11 peptides used in t-cell activation assays. the tcr-binding algorithm is especially suitable for reducing the number of epitopes that need to be screened to identify robust cd8 t-cell activating epitopes. for example, our algorithm predicted 83 peptides from all sars-cov-2 proteins excluding orf1, which is a much smaller number compared to the number of peptides screened in some of the published studies to identify pre-existing t-cell immunity (7, 8, 15, 21) . a second factor that may have resulted in the identification of strong cd8 t-cell activating epitopes is the avoidance of epitope competition. using a large pool of peptides to screen for t-cell responses ensures broad coverage of all hlas, but has the disadvantage that strong immunogenic epitopes are not detected efficiently. some of the peptides predicted by our algorithm produced >5% cd8 t-cell response in healthy donors by 14-days. in the same donors, the response from spike-s1 and s2 peptide pools containing 157 and 158 peptides respectively was much weaker. a similar finding was reported by mateus et al. where deconvolution of peptide pools identified a single peptide that evoked 5-fold higher t-cell response compared to the pool (8) . also, important to note, that the strategy of using 15-mer peptides with overlapping 10 or 11-mer sequences may not identify immunodominant epitopes. for example, out of the 11-peptides tested in our assay, only three peptides were present in the spike peptide pools. by using a smaller pool of immunodominant cd8 t-cell epitope, our study uncovered a fundamental feature of the host immune response to sars-cov-2the existence of crossreactive tcrs to viruses, such as cmv and influenza that recognizes sars-cov-2 antigen. an early and robust t cell response is driven by the size and the diversity of the tcr repertoire to a given antigen (22) . the pep-7 epitope derived from the rbd domain of sars-cov-2 spike antigen lacking homology to other coronaviruses expanded multiple public cdr3s recognizing immunodominant cmv epitope nlvpmvatv and influenza epitope gilgfvftl. further, tcr analysis demonstrated that although a donor's tcr repertoire contains many cmv-epitopespecific cdr3s, only a few are expanded in the presence of the sars-cov-2 peptide. for example, donor d225 tcr repertoire has 159 cmv and 249 influenza cdr3s of which three and one were expanded respectively. similarly, donor 089 carries 103 nlvpmvatv specific cdr3 of which only two expanded. these findings suggested the specificity of interaction between cross-reactive cdr3s and specific peptides from sars-cov-2. significantly, the expanded cdr3s in the two donors d089 and d225 were different, even though they recognized the same cmv peptides. it has been documented that conserved features within cdr3- allow recognition of the same phla complex within a group of diverse cdr3s (23) . a robust antigen-specific t cell response utilizes a broad range of tcrs and for many viral infections, tcr usage diversity has been positively linked to disease outcomes (24) (25) (26) . a diverse repertoire not only allows increased structural capacity to recognize variant epitopes (27) but increases the chances that high-affinity tcrs may be present in an individual (28) . a recent large-scale study mapped a few immunogenic regions in the sars-cov-2 proteome responsible for expanding many unique tcrs in a large number of convalescent covid-19 patients and unexposed healthy donors (21) . immunodominant epitopes reported in our study cover some of the "hotspot" regions identified by this large-scale study (21) . efforts to identify cross-reactive tcrs recognizing different antigens from diverse infectious organisms can lead to the development of broad-spectrum tcr-based therapeutics against infectious diseases. we compared the all-peptide mix with spike-1 and 2 peptide pools on a small number of convalescent patients and identified a slightly higher cd8/ifn- response by the peptide mix in mild to moderate disease, compared to patients with asymptomatic or severe disease. many studies have indicated that short and long-term protection against respiratory viruses requires cd8 t-cell immunity and antibody response alone is not sufficient (6, 29) . in line with this observation, low plasma titers of neutralizing antibodies are detected in a large fraction of convalescent patients suggesting additional immune protective mechanisms, besides viral neutralization (30) . on the contrary, high levels of neutralizing antibodies were associated with severe disease and icu visits in many covid-19 patients suggesting an imbalanced cd4 t-cell response is not optimal for protection (31) (32) (33) . it has been challenging to demonstrate a strong cd8 t-cell response in covid-19 patients in many studies. however, our findings along with a recent report from peng et al. showed that a higher cd8 t-cell response correlated with a mild disease compared to patients with severe disease (15) . in conclusion, our study demonstrates strong pre-existing cd8 t-cell immunity in many data on 371,865 t cell assays was collected from the iedb (1). there were 105,673 cd8 t cell assays in total with 61,968 cd8 t cell assays with humans as a host. the cd8 t cell assays with hla allele names and peptide lengths ranging 8-14 residues were further selected. hla supertypes were replaced with their representative allele names, for example, hla-a2 was replaced with hla-a*02:01. the immunogenic peptide-hla pairs tested on at least three donors with 100% response frequencies or at least tested on 5 donors with greater than 50% response frequencies were labelled as positive. the non-immunogenic peptide-hla pairs tested on at least 3 donors with 0% response frequency were labelled as negative. the final dataset contained 8,870 unique peptide-hla pairs which were split randomly into 80% training and 20% test datasets. the training dataset had 884 immunogenic and 6,212 nonimmunogenic peptide hla pairs. the test dataset had 238 and 1,536 immunogenic and nonimmunogenic peptide-hla pairs, respectively. a deep convolutional neural network (cnn) was implemented to predict immunogenicity of the peptide-hla pair (provisional patent pending). the hla alleles were represented as pseudosequences described as 34 amino acid residues (2) . the peptide and hla pseudo-sequences were converted to the two-dimensional (2d) feature matrices of 14x20 and 34x20 dimensions using blosum encoding (3) respectively. peptide sequences shorter than 14 residues were padded by zeroes to maintain 14x20 feature matrix dimensions. peptide sequences were also encoded into 1x14 feature vector using the kyte-doolittle hydrophobicity scale (4). the hla binding percentile ranks, and scores for each peptide-hla pair were obtained using netmhcpan-4.1 (5) and were appended to the kyte-doolittle hydrophobicity scale feature vector. the peptide and hla feature vectors were each processed by multiple 2d convolutional filters of two different sizes followed by max-pooling layers of the same sizes serially. the peptide and hla max-pooled layers were concatenated and processed again with multiple 2d convolutional filters followed max-pooling layers. the 2 max-pooled layers were flattened-concatenated and then connected to a dense layer. the output of the peptide and hla dense layer was concatenated with the hydrophobicity and hla binding feature vector and again connected to two dense layers. the final output of dense layer was connected to the output neuron. the cnn was trained using 5-fold cross validation with the training dataset exclusively. the test dataset was solely used for model performance evaluation. model performance was evaluated using auc (area under roc curve) where an auc of 0.5 represents random predictions and auc of 1.0 represents the perfect predictions. the tensorflow library from python programming language was used to implement the models. full length shortlisted peptide sequences of sars-cov-2 were blasted against the spike proteins of other coronaviruses, oc43, nl63, 229e and hku1. an e-value cutoff of 0.01 was used with a minimum cutoff of 11 amino acid residues was used to identify homologous peptides. unexposed donor pbmcs were obtained from the us and india for this study. pbmcs from the us were collected between 2016 -2018 and purchased from stemcell technologies, canada. pbmcs from india were collected between 2015 -2018. covid-19 convalescent patient blood from the us was purchased from ppa research (usa) and the indian samples were collected through hospitals. all participants in this study provided informed consent in accordance with protocols approved by the institutional review board. pbmcs were thawed, counted and analyzed using the diagnostic panel of antibodies (table s5) . pbmcs were rested overnight in rpmi containing 10% human serum (table s5) . for t-cell activation assays, 750,000 pbmcs were incubated either with dmso (negative control) or with different peptide pools in 0.5 ml rpmi (gibco) +10% human ab serum (sigma) + 10 ng/ml il-15 and 10 iu of il-2 (stemcell technologies, canada). the culture media was replenished every three days with fresh media containing 10 iu of il-2 and 10 ng/ml il-15. on days 7, 14 and 21 of incubation, fresh peptides were added to the culture. for intracellular cytokine staining, cells were treated with brefeldin a (bd biosciences) for 5 hours, fixed and permeabilized using bd lysis solution and perm2 solutions respectively followed by staining with t-cell activation panel of antibodies (table s5) . stained cells were analyzed in bd accuri c6 plus to detect the expression of activation markers ifn- and cd137 (4-1bb) on cd4 and cd8 t cells. data was analyzed using bd accuri c6 plus software. 200,000 pbmcs was removed after 48h, 7, 14 and 21 days from the t-cell activation assay and processed for bulk tcr sequencing. tcr repertoire profiling was performed using the smarter tcr α/β profiling kit (takara bio, usa) according to the manufacturer's protocol. rna was isolated using the qiagen rna isolation kit. 10ng rna from antigen-induced pbmcs were used as the starting material. the kit uses smart technology (switching mechanism at 5'end of rna template) with 5'race to capture the entire v(d)j variable regions of tcr transcripts followed by two rounds of seminested pcr to obtain tcr- and the -chain. libraries are prepared analyzed for quality and quantity. sequencing is performed using the 2*300 miseq reagent kit v3 (illumina, inc.). for each sample, raw gene expression matrices were generated by cell ranger(v.3.0.2) coupled to the human reference version grch38. the gene expression data was analyzed by r software (v.3.4.4) with the seurat package (2.3.4) . in brief, low-quality cells were removed if they met one of the following criteria: >75,000 unique molecular identifiers (umis); <500 or >7,500 genes; >10% umis derived from the mitochondrial genome; and >50% of transcripts contributed by top 50 genes ( figure s7 ). after removing low-quality cells, gene expression matrices were normalized by the normalizedata function and features with high cell-to-cell variation were calculated using the findvariablegenes function. next, the expression of the sphase and the g2-m phase genes were used to calculate the cell cycle score for all the cells using the cellcyclescoring feature. to generate unbiased clustering of cells scaledata function was used, regressing out the expression of cell cycle genes, mitochondrial % and number umi from the features ( figure s8 ). the dimensionality of the datasets was reduced by runpca function using variable features identified by findvariablegenes function on lineartransformation scaled data generated by the scaledata function. next, the elbowplot and dimheatmap functions were used to identify the true dimensionality of each dataset. finally, cells were clustered using the findclusters function and nonlinear dimensional reduction with the runumap function using the euclidean distance feature. all details regarding the seurat analyses performed in this work can be found in the website tutorial (https://satijalab.org/seurat/v2.4/pbmc3k_tutorial.html). after nonlinear dimensional reduction and projection of all cells into two-dimensional space by umap, cells were clustered together according to common features. the findallmarkers function in seurat was used to find markers for each of the identified clusters. clusters were then classified and annotated based on expressions of canonical markers of particular cell types. differential gene expression was performed using the findallmarkers function in seurat with default parameters. we selected top-25 upregulated degs with maximum fdr value of 0.01 and annotated the clusters based on the expression of these upregulated genes. next, we used mergeseuratfunction to integrate the four datasets from the four treatment conditions and performed the steps of data normalization, feature extraction, regressing out features as described above. the heatmap and dot plots were generated using the doheatmap and dotplot function in seurat. full-length tcr v(d)j segments were enriched using a chromium single-cell v(d)j enrichment kit according to the manufacturer's protocol (10x genomics). demultiplexing, gene quantification and tcr clonotype assignment were performed using cell ranger (v.3.0.2) vdj pipeline with grch38 as reference. tcr diversity metric, containing clonotype frequency and barcode information, was obtained. cells with at least one productive tcr α-chain (tra) and one productive tcr β-chain (trb) were retained for further analysis. each unique tra(s)-trb(s) pair of tra-trb was defined as a clonotype. the presence of identical clonotypes at least in two cells were considered to be clonal, and the number of cells containing the same tra-trb pairs defined clonal amplification of a clonotype. using barcode information, tcr clonotypes were projected on umap and dot plots. public tcrs were mapped to the iedb and vdjdb annotated databases using the trb sequence. the trinity of covid-19: immunity, inflammation and intervention clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan pathological inflammation in patients with covid-19: a key role for monocytes and macrophages systems biological assessment of immunity to mild versus severe covid-19 infection in humans imbalanced host response to sars-cov-2 drives development of covid-19 news feature: avoiding pitfalls in the pursuit of a covid-19 vaccine targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals selective and cross-reactive sars-cov-2 t cell epitopes in unexposed humans phenotype and kinetics of sars-cov-2-specific t cells in covid-19 patients with acute respiratory distress 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infection at both individual and population levels. medrxiv revealing factors determining immunodominant responses against dominant epitopes identifying specificity groups in the t cell receptor repertoire clonally diverse ctl response to a dominant viral epitope recognizes potential epitope variants narrowed tcr repertoire and viral escape as a consequence of heterologous immunity limited t cell receptor repertoire diversity in tuberculosis patients correlates with clinical severity a molecular basis for the control of preimmune escape variants by hiv-specific cd8+ t cells direct link between mhc polymorphism, t cell avidity, and diversity in immune defense sars-cov-2 t cell immunity: specificity, function, durability, and role in protection convergent antibody responses to sars-cov-2 in convalescent individuals altered cytokine levels and immune responses in patients with sars-cov-2 infection and related conditions disease severity dictates sars-cov-2-specific neutralizing antibody responses in covid-19 high neutralizing antibody titer in intensive care unit patients with covid-19 the immune epitope database (iedb): 2018 update netmhcpan, a method for mhc class i binding prediction beyond humans reliable prediction of t-cell epitopes using neural networks with novel sequence representations a simple method for displaying the hydropathic character of a protein netmhcpan-4.1 and netmhciipan-4.0: improved predictions of mhc antigen presentation by concurrent motif deconvolution and integration of ms mhc eluted ligand data key: cord-286858-zbhtl2yn authors: mishra, b. title: tamasomā jyotirgamaya: seeking the self amidst covids’ cytokine cyclones date: 2020-10-22 journal: j indian inst sci doi: 10.1007/s41745-020-00186-1 sha: doc_id: 286858 cord_uid: zbhtl2yn pondering on pandemics and the promise of purification from the plethora of problems that it has spawned, the paper builds on a game-theoretic model of host–pathogen interaction, and... moves beyond. it highlights how quickly this ‘wicked’ problem has led to deceptive nash equilibria of certain information-asymmetric games as well as their sequels of more complex intertwined games at human scale but without an exit strategy in sight. in the absence of clarity (e.g., access to complete information) and yet facing a capricious and complex conspirator, we overview an exemplary solution, created by rxcovea, and examine how it might help. abstract | pondering on pandemics and the promise of purification from the plethora of problems that it has spawned, the paper builds on a game-theoretic model of host-pathogen interaction, and... moves beyond. it highlights how quickly this 'wicked' problem has led to deceptive nash equilibria of certain information-asymmetric games as well as their sequels of more complex intertwined games at human scale but without an exit strategy in sight. in the absence of clarity (e.g., access to complete information) and yet facing a capricious and complex conspirator, we overview an exemplary solution, created by rxcovea, and examine how it might help. biomolecules constitute an important subclass of molecules and are dominated by phosphorous, carbon, nitrogen, oxygen, hydrogen, etc. the biomolecules interact with each other and with water molecules (e.g., via atp hydrolysis reaction process) it is important that the "cellularizing" ensembles of such biomolecules-very likely membrane separated from the environmentremain lively (via a sequence of endergonic and exergonic reactions) and safe (maintaining right ph level and avoiding h 2 o 2 , etc.). because liveliness and safety conferred to the cells can be abstracted by a "utility" function, cells may be abstracted as individual "biomolecular agents" competing with each other in an evolutionary game in which the fittest survives and replicates. these biomolecules are also able to store useful information via concatenation of atplike subunit monomers to form polymers (e.g., rna, dna, microtubules, etc.) and, thus, symbolically encode molecular patterns, each with its own utility. such polymers form the symbolic world, in which they duplicate (or allow transcription and translation into other polymers mapping homomorphic patterns) and propagate the information stored in molecular patterns. if the duplication process permits variations and a utility-dependent replicator dynamics and pattern-specific information propagation is unidirectional, it sets the stage up for a darwinian evolution and amplifies diversity, giving rise to 1 3 j. indian inst. sci. | vol xxx:x | xxx-xxx 2020 | journal.iisc.ernet.in inter-agent information asymmetry. thus, not all possible molecular patterns would be uniformly (or information-symmetrically) represented in the symbolic world; only an infinitesimally small subset of all possible patterns would encode viable genomes (dna), transcriptomes (rna), or translatomes/proteomes (proteins). interactions among biological macro-molecules may be described using a sender-receiver signaling structure, where an expressed macromolecule, such as a protein or rna, constitutes a signal sent on behalf of a sender agent (e.g., cell). the signal may be modeled as the threedimensional conformation and physicochemical properties of the macro-molecule. a receiver agent (e.g., another macro-molecule) may then bind to the signal macro-molecule, which produces an action (such as an enzymatic reaction). the action produces utility for the participating agents, sender and receiver, and thereby-albeit indirectly-a change in overall fitness of the organism, and hence its genome (in evolutionary game theory, utility and fitness are treated as analogous). when there is common interest, the utility is expected to benefit both sender and receiver and their selectivity, thus driving darwinian co-evolution. if there is neither common identity (i.e., they are "strangers") nor common interest (i.e., they pose "dangers"), the sender and receiver may avoid each other in order not to undermine their common fitness. thus, antibody: an important part of the adaptive immune system. it is a protein usually in the blood and destroys or neutralizes bacteria, viruses, or other harmful toxins. antibody production takes place in response to antigen. they are members of a class of proteins known as immunoglobulins, produced by b cells in response to stimulation by an antigen. interferons: a family of proteins (lymphokines), secreted by infected host cells to protect uninfected cells from viral infections. evolution of processes encoding, recognizing, and acting against pamp (pathogen associated molecular patterns) and damp (damage-associated molecular patterns) plays an important role in the evolution of the multicellular immune systems. note that not all strangers are dangerous (e.g., symbionts such as corona virus in bats) and that some non-strangers could be dangerous (e.g., cytokine storms inducing activated white blood cells). however, since the immune systems also play an important role in defining many more higher order evolutionary processes: e.g., hla diversity, mate-selection, cognitive processes, culture, etc., our very human intellectual and cultural journey has also shaped our secular scientific understanding of the physical universe, albeit, it still remains inconsistent and lacks unification (e.g., quantum and classical mechanics). the resulting body of scientific literature, however, forms only a small part of our body of human knowledge that relates us to a tentative, though conventional, picture of a 'natural world,' containing both symbolic and signaling systems. as humans struggle with their understanding of the genesis and mitigation of a surging pandemic, initiated by a single zoonosis event in a remote asian town, it is worthwhile to meditate on these connections among the three worlds-mundus triplex, further subdivided and re-connected by such subdisciplines as virology, immunology, epidemiology, logical/mathematical inference, and economics-going from nanoscale viruses to global-scale human societies. or, "must we wring the neck of a certain system in order to stuff them into [] pigeon holes for the satisfaction of the analogy mongers 1 :" do the worlds divide neatly into bruno's three magics-sciences into three disciplines: physics, mathematics, and divine? or does it just model a biological gödelian self-referential self-seeking, selfishly greedy computation, hopelessly aspiring to halt with stable (sound and complete) turing decidability notion(s) of self(-ves)? can it reach an ne (nash equilibria), in which self separates from non-self, decoy epitopes from neutralizing, susceptible from infected, symptomatic from asymptomatic, essential economic activities from dispensable? replicator dynamics allow the signaling game to be couched in evolutionary terms 6 . replicator dynamics arise from the increased replication of players with higher utility (fitness). thus, if a cytokine: a signaling molecule, acting as a chemical messenger released by white blood cells, including macrophages, monocytes, or lymphocytes. the cytokines include the interferons, the interleukins, tumor necrosis factor, etc. memory t cell: persistent t cells that bear a receptor for a specific antigen that was previously encountered in the course of illness or vaccination. it plays a role of a verifier to ensure safe execution of the signaling game. the human leukocyte antigen (hla) complex is a group of related proteins, encoded by the major histocompatibility complex (mhc) in humans. hla genes are highly polymorphic which means that they have many different alleles, allowing them to fine-tune the adaptive immune system-differently for different individuals. hlas corresponding to mhc class i (a, b, and c) present peptides from inside the cell. foreign antigens presented by mhc class i attract t lymphocytes called killer t cells. figure 1 : molecules, molecular patterns, and (pathogen/damage) associated molecular patterns: who signals who? we depict the three worlds, important to defining a host's self and protected by a complex immune system, but amenable to invasion by a simple deceptive virus, which, in turn, can be deceived by a vaccine. we develop a signaling game framework to study virology, and immunology-while we hope to extend the framework to asymptomatic patients, with misaligned utilities, and impaired by moral hazards in economy. we suggest that they all conspire to make the covid problem a "wicked" (difficult or impossible to solve) problem. courtesy r.x. covea et al. cell has a strategy that results in increased utility, then it will increase in frequency in a population. for a sender cell, this would entail sending a signal that may result in an increased utility, while, for a receiver cell, this would entail undertaking an action that likewise may result in an improved utility (e.g., growth rate) (fig. 2) . as already suggested, these dynamics represent a process analogous to darwinian (adaptive) evolution or positive selection. in other words, as part of the innate immune system, sender agent, a dendritic cell (dc), in a multicellular organism that recognizes an appropriate damp/pamp combination could signal receiver agent, a macrophage (m ), to engulf and digest foreign substances (associated with the molecular patterns, amp) in an action process called phagocytosis. the innate system is said to be engaged in a signaling game, in which dc is the sender; m , the receiver; they signal using cytokines and phagocytosis which is the action taken by the receiver upon receiving the signal. their utility is determined by protection which they confer against non-self (mostly danger, potentially posed by strangers) and tolerance for self. since, many pathogens (e.g., sars-cov-2) can engage in deception (e.g., entering a host cell by targeting host ace2 receptorby binding to a site somewhat distant from the ace2 canonical binding site), innate immune system may need other auxiliary adaptive agents to be "educated" ex tempore, replicate dynamically and retain memory. these cells belonging to the adaptive immune system are not strategic (e.g., do not confer fitness along the germ line), but play an important role as recommenders (b cells)and verifiers (t cells) in taming the deceptive pathogens that can grow and mutate rapidly to frustrate the innate immune system. helper" t-cell, coordinates much of the adaptive immune response. a thymus derived white blood cell that precipitates a variety of cell-mediated immune reactions. three fundamentally different types of t cells are recognized: helper, killer, and suppresser (each has many subdivisions). they can be modeled as verifiers in the signaling game. b-lymphocytes are blood cells derived from the bone marrow and spleen involved in the production of antibodies. b cells produce antibodies, when primed by t cells. b-cell lymphocytes can later differentiate into plasma and memory cells. in our signaling game, they are modeled as recommenders and accelerate convergence to a separating ne based on the past memory of encounters. dendritic cell: an antigenpresenting immune cell. they have elongated, tentacle like branches to trap foreign objects. a large scavenger cell that ingests degenerated cells and secretes messenger proteins (monokines) involved in inflammatory reactions, lymphocyte activation, and acute systemic immune responses. the consumption and destruction of foreign materials by white blood cells like macrophages. however, in an adversarial chase, pathogens can evolve strategies to frustrate the education of t cells (e.g., antigen presentation), but can be remedied by costly signaling via dc's maturation and chemo-taxis to germinal centers. it is no wonder, most immunology jokes end with the punchline 7 , "the immunologist says, 'the thing is, the immune system is very complicated...' and the cardiologist says, 'just shoot me now. " we hope to simplify the model by formulating the innate immune system as a signaling game 4 , 6 , which, though prone to deception, can be critically tamed by the education, surveillance, and memory acquired by the adaptive immune system; the education of the adaptive immune system, though highly costly, can also be hijacked by a deceptively simple virus and points to a need for a better understanding of how the system behaves among various hosts (bats and humans with diverse hla types). signaling games signaling games are multiplayer (usually, two players) games with incomplete information: specifically, one player is informed and the other player is not. the informed player's strategy set consists of signals contingent on information; uninformed player's strategy set consists of actions contingent on signals. game is played as follows 1. player s is assigned a type t ∈ t 2. s send to player r a signal s ∈ m. 3. r takes an action a ∈ a. payoff function the utility functions for sender and receiver are dependent on the type of the sender, signal sent and action carried out by the receiver: equilibrium concept behavior strategies for senders and receivers: describing biomimicry by a virus to fool the immune system: cell can be of two types t c (self) or t d (non-self viral): they can send c signals honestly or by mimicry, respectively, and may be trusted or rejected by the immune system; similarly, they can send d signals erroneously or honestly, respectively, and may be rejected by the immune system. courtesy s. massey et al. j. indian inst. sci. | vol xxx:x | xxx-xxx 2020 | journal.iisc.ernet.in α(s, a) = probability that r takes action a following signal s. we may then say that the receiver may acquire a subjective probability distribution over t: proposition behavior strategies (α * , µ * ) form a nash equilibrium iff for all t ∈ t: signaling games in nature to assume reasonable utility functions, we use two natural maps that relate the sender's private information to receiver's action that would be desirable via rate distortion functions combining i(·, ·) (mutual information or rate) with a distortion d · (·, ·) combined using suitable lagrange parameters · : • mapping types and actions into signals: the model of signaling games the game thus involves two players, namely: a∈a α(s, a) = 1, for all s. . a) . • their roles can be shared; s may only have partial information; and r may be allowed to perform distributed actions. • a notion of type, t private to s, is captured by a random variable t whose support is given by t (known to sender s). π(·) = probability distribution over t is a prior belief of r that the sender's type is t. • note that the distribution of signals received by r is given by the probability distribution π m , where: • • and the distribution of actions produced by r is given by the probability distribution π a , where: • • clearly, π t and π a are probability distributions on t and a, respectively. if π t is the probability distribution on t induced by π a under the function f r , then: • a natural choice of measure of deception may be given by the relative entropy between the probability distributions π t and π t : this definition describes deception from the point of view of the receiver. to get the notion of deception from the point of view of the sender, one needs to play the game several rounds. thus, signaling games may reach a nash equilibrium that is stable but not deception-free (e.g., immune system may reach a homeostasis that may not be ideal in separating self from non-self). (1) π t (·) := π a (f −1 r (·)). (2) • if, on the other hand, the game reaches a pooling equilibrium: all types t send a single signal s * with probability 1. • convention and deception the divergence between the objective probabilities and the subjective probabilities induced by conventional equilibria. • solution costly signaling; credible and noncredible threat; aligned utilities; augmenting with additional non-strategic playersdenoted as: m recommenders + nverifiers the possibility of deception (e.g., biomimicry by sars-cov2) in the nash equilibrium (ne) of the signaling game may be the reason why the innate immune system (with pamp and damp) is not the ultimate solution; the ne must be stable, but also keep up with rapidly evolving pathogens. it leads to a need for an adaptive immune system, training of t cell by dc, chemo-taxis, and maturation processes that dc must go through, cytokine storms, etc. therefore, "it is very complicated.." and more so if we need to create a vaccine that must deceptively mimic the virus, which may have been already deceptively mimicking the host system, and the vaccine must be carefully analyzed and regulated so as not to catalyze autoimmune disorders or antigen-dependent enhancement ade. covid-19 poses a wicked problem. "why does the repeated game it plays-covid game-have long and branching chains, intertwined hierarchies, plenty of room for deception, and explosive growth rates? why not use similar features, perhaps, needed in the counter measures to tame it 1 ?-" a proportionally rapid and rigorous analysis of the game as well as synthesis of hyper-local policies (e.g., strategies) addressing molecular, individual, and community-wide actions. for argument's sake, assuming that it only manifests as a socio-economic question, it remains unfathomable what economic utilities might be ethically and equitably traded off for loss and depreciation of human capital (fig. 3) . many of the unknown unknowns are exacerbated by the impacts of unquantified-or unquantifiable-(1) proportion of asymptomatic patients, (2) comorbidities and vulnerability of various subpopulations, (3) co-evolution of the disease in the presence of others (flu or multiple strains-with unknown cross immunity) , (4) number and combinatorial structure of the network of super-spreaders, (5) individual boundedly rational compliance, (6) spatio-temporal reaction-diffusion nature of the disease (e.g., turing labyrinth for herd immunity), (7) accuracy, frequency, and scaling of biomedical tests (separating asymptomatic from symptomatic, susceptible from infected and infected from immune, etc.), (8) unpredictability of treatment and preventive measures such as socialdistancing, contact-tracing, repeated lock-downs, and quarantines, (9) allocation of resources for patients across vulnerabilities, demographics, genders and races, etc. rxcovea has sought to develop and openly disseminate mathematical and computational tools and theories to address these problems with the help of modelers (sdes, graph-based, hybrid, cellular automata), game theorists (signaling games, minority games), logicians (formal methods, model checkers, belief revision), algorithmicists (multiobjective decision and optimization problems), statisticians (bandit problems, reinforcement learning), and computational systems biologists (in silico vaccine figure 3 : covid poses a wicked problem affecting different human organ, physical, and social systems at different spatio-temporal scales. note: wiki defines "a wicked problem as a problem that is difficult or impossible to solve because of incomplete, contradictory, and changing requirements that are often difficult to recognize. ... another definition is 'a problem whose social complexity means that it has no determinable stopping point.' moreover, because of complex inter-dependencies, the effort to solve one aspect of a wicked problem may reveal or create other problems.' covid problem started as a healthcare problem, but quickly evolved into an economic problem, triggering various issues related to humans' fundamental rights. courtesy: r.x. covea et al. j. indian inst. sci. | vol xxx:x | xxx-xxx 2020 | journal.iisc.ernet.in and drug discovery). detailed game-theoretic analyses in different contexts, namely, germline, somatic, or population evolution, may be found elsewhere 2 , 3 , 5 . (1) 100 nm scale: virology-a task group ("la famiglia"), consisting of salvatore alaimo, eva bischof, jantine broek, ashley duits, alfredo ferro, naomi maria, alfredo pulvienti, and-valentina rapicavoli, has been studying the systems biology of the virus and host-pathogen interactions. the group has been able to replicate in silico-safely, scalably and inexpensively-a large class of in vivo and in vitro experiments to test a substantial number of hypotheses regarding host-pathogen-drug interactions. rigorous statistical analysis, to correct for multiple hypotheses testing, then points to the strategies (possibly involving deceptive biomimicry) that the virus may evolve and how drugs may be re-purposed to enable the host organs, systems, and pathways to counter safely. the group also aims to investigate how sars-cov2 virus achieves a symbiotic existence with other reservoir hosts (e.g., bats and pangolines) and how its genome mutates in order that hosts' immune system may accommodate such a ("pooling") nash equilibria (perhaps, with constrained antigenic drift). another task group ("the gamers") consisting of william casey and steven massey has developed models of biomimicry (especially the ones achieved by sars-cov-2, mimicking polybasic cleavage site, pcs mutations to interact with host furins) that speaks to intelligent and safe vaccine and drug design. charles cantor studies sophisticated multicolor pcr testing methods, tracking the viral genomic mutations. marco antoniotti studies the phylogeny of the genomic strain mutations. tamar schlick with her molecular modeling group studies the structure and dynamics of the fse (frame-shifting element) of the viral rna genome and its screening in silico to discover novel anti-viral therapy to inhibit protein synthesis (fig. 4) . 1 µ m scale: immunology-another task group ("the adaptors") has been studying the host immune system's response to viral figure 4 : target rna residues for anti-viral therapy in the frame-shifting element of sars-cov-2 determined by molecular modeling. a shows the 3d structure of the frame-shifting element of sars-cov-2 as modeled by md (molecular dynamics) with the key residues which are identified by destroying the pseudoknot and/or a stem using graph theory and genetic algorithms. the identified residues are then used in the drug screening algorithm. other identified residues are shown in b and c. courtesy: t. schlick et al. 2 in yacht's simplest incarnation: a user is recommended a pool in which their saliva can be tested within a time interval: if the user selects to be pool-tested and the pool-test result is negative, the user is given a badge (non-counterfeitable plastic wrist tag, encrypted electronic badge or a crypto-coin tag) that lasts a pre-determined period. during that period with the safe badge, the user may be allowed to enter (resp. exit) certain safe (resp. unsafe) locations. if, on the other hand, the user selects to be pool-tested and the pool-test result for the user is positive, then the user is given an unsafe badge-and recommended a uniformly randomly selected pool (hot-potato protocol) and/or exponentially randomly selected time interval (exponential-back-off protocol) for being retested. the user without a safe badge is virtually quarantined from entering a safe region (or leaving unsafe region-which must have sheltering-in-place or hospitals). also, since the subsequent choices of the pools are (by recommendation) independent, if the user is truly negative, they will be determined so (almost surely) no later than a fixed run-length of "(apparent) false-positive" results. if a user fails to be negative after fixed run-length of "(apparent)-true-positive" results, the user must be quarantined. infection: the group consists of james bannon, charles cantor, ashley duits, john conolloy, kelly ganjei, jia han, and mike lotze, and it analyzes longitudinal adaptomic data from patients, using temporal network analysis using advanced combinatorics and formal methods. it is desired, with the availability of adequate resources, to expand the team to study the genomic markers stratifying the probands, who exhibit a wide range of symptoms-asymptomatic vs symptomatic or super-spreaders or kawasaki/misc disease phenotypes. it is conjectured that the immunogenic biomarkers can be characterized by a gwas (genome-wide association study), if certain genomic regions (e.g., p-arm of chr 6) are mapped haplotypically with long-range single-molecule technology such as nanomapping. possible biomarkers clustering around hla, kir, and tlr regions are being studied by a group consisting of marcin imilienski, sanjan kumar, jason reed, and amir toor. vijay chandru and ramesh hariharan are aiming to initiate a similar project in india, which enjoys a more interesting admixture population, driven by endogamy ("the grandest genetic experiment ever performed on man," according to theodosius dobzhansky). (3) 100 m scale: epidemiology/social immunology-rxcovea's third task group ("the labyrinthers") has focused on various epidemiological models and their parametric control using clever (pool-and individual-) testing methods with low latency: the group comprises ved basu, jantine broek, shireshendu chatterjee, inavamsi enaganti, hilary gao, eva guo, mathew jacobs, and kim mishra (for modeling); charles cantor, vijay chandru, manoj gopalakrishnan, inavamsi enaganti, satyajit mahrana, and rohit nandwani (for testing); the two subgroups cross-pollinate intimately. the subgroups started with standard cellular automata (ca) agent-based model for incorporating geographical distributions of infection and easily captures the notion of discrete pairwise interactions, since the new state of a cellular automaton (ca) is based on its current state and the states of its surrounding cas as well as some common evolving rules. the infection spreads from an infected ca to adjacent neighboring cas, while infected patients also recover and become immune to the reinfection. we have assumed the following states for the neutralizing antibody: an antibody that neutralizes (renders harmless) the infectivity of microorganisms, particularly viruses. population exposed, symptomatic, asymptomatic, susceptible, recovered/immunefurther augmented with such other states as: unknown, tested, vaccinated, quarantined, retested, recurrent, etc., with respect to other sars-covs and flu. rxcovea has also implemented ctmc (continuous time markov chain) and realistic sde (stochastic differential equation) models-the later model obtained using turing's reaction-diffusion equation framework and the analysis of its solutions-e.g., turing labyrinths. we conjecture that the formation of turing labyrinths (possibly further stratified by hlas and cov2-strains) will play a role in the formation of herd immunity, vaccination, and social-distancing policies. these pattern formations can be further moulded by rna, antigen, and immunoglobulin tests (yacht, yet another covid-health testing) 2 , as well as contact tracing (with 1 or 2 degrees of separation) both operated at a large scale with a selected periodicity. the original yacht design motivated by the following game-theoretic model: (i) repeated game: repeated saliva pool testing (involving hundreds of individuals in a pool) using rapid and accurate (duplicated) pcr test is feasible with negligible false negatives and manageably low false positive. (ii) private game/ no intermediary: sample collection must remain safe, unsophisticated, ubiquitous, and unbiased (from self-selection) and, thus, may not involve robotics and technicians. (iii)decentrality: data collection and storage must be reliable, private, and yet not centralized. (iv) factual and counter-factual: the analysis is flexible, hypotheses-testable, easyto-communicate, and adaptive in controlling compliance, prevalence, immunization, and resource usage. yacht's resemblance to ethernet's aloha protocol and pagerank's random surfer approach, especially with small/moderate prevalence, adds to our faith in its robustness. we have simulated a model, with promising results, that partition the population into two regions ("home" encountering both safe and unsafe household members and "work" encountering safe non-household members) and assumes that the household statistics differ from country to country (india, afghanistan, usa, and the netherlands). in the near future, we wish to resimulate the system with one additional region: "challenge region," which may be factual or counter factual (e.g., with vir-immunoglobulin: a protein that acts as an antibody to fight off pathogens. there are five classes: igg, iga, igd, igm, and ige. recombinant and pooled immunoglobulins from blood donations are being used to fight off sars-cov-2 infection. tual viral strains and spreading and testing mechanisms) and allow collection of statistical data for hypotheses testing (fig. 5) . (4) 100 km scale: logic, inference, and learning-the fourth task group ("the model checkers") is a data-science group to delineate the causal structures among various pandemic-related events; the group includes: marco antoniotti, jantine broek, daniel cage, will casey, shirshendu chatterjee, vijay chandru, james edmondson, yaron gvili, stella luna, ramon luna, and larry rudolph. the group is preparing for a near future when massive amount of meta-data, (e.g., involving pools, mobility, and geographic occupancy datafrom foursquare) can be combined with machine learning tools augmented with formal methods to hypothesize and test causal relationship and intervention strategies. since our games include complex subgames involving credible and non-credible threats, monitoring human behavior-without assuming perfect rationality-is likely to play a critical role. we plan to use probprog tools (julia/turing 3 ) to understand the disease etiology, and individuals' strategic choices tempered by the knowledge of causality, time, logic and games, privacy, reputation, trust, deception, and cellularization (fig. 6 ). equilibria-the "open sim," group (including ved basu, jantine broek, will casey, shirshendu chatterjee, and yaron gvili) is studying how to connect components of global data to infer effect of stratification of populations based on demographics, age, gender, genomics (e.g., hla), (co-)morbidity, etc. to understand genuine causal structures only using natural experimental data and without getting bogged down by the bias due to multiple hypotheses testing and simpson's effect. the economic roles played by the disrupted interrelationships among different subgroups (and their possible simulation by digital means, when individuals need to be socially distanced) have a strong impact on the v or w shaped economic recovery. the "burpa group" (comprising kose john, samir saadi, and cheryl qi) has designed a novel market microstructure to enable health t helper cells: lymphocytes responsible for assisting other white blood cells in responding to infection, processing antigen, and triggering antibody production (also known as t4 cells and cd4 cells). a major component of cytotoxic lymphocyte response (ctl), responsible for lysing infected or cancerous cells, and t killer cells (not to be confused with natural killer cells) are a subset of cd8+ lymphocytes. insurance for a patient population (social network) interacting with scientists and clinicians (expert network) to create digital healthcare markets that tame its risks by combining multiple spv's (special purpose vehicles) and securitization (fig. 7) . in a more optimistic future, to which we hope to return soon, rxcovea will focus on better algorithms for randomly surfing explainable ais to create a world-wide adaptive immune system to protect humans and things physiologically, physically, and digitally. the adaptive learning recommenders and verifiers will be able to anticipate and create mitigating plans for future zoonotic events. for now, our collective afforts aim to t suppressor cells: t lymphocytes responsible for turning the immune response off after infection is cleared, a subset of cd8+ lymphocytes. yacht's performance on a university campus with five dorms. the student population is simplified to be heterogeneous with two assumed immunogenic biomarkers: e.g., hla types hla' and hla", where agents of type hla' are less infectious than hla" agents. simulation assumes that there is only one viral strain. a observe that increasing pool sizes decreases the total number of infections while increasing quarantining. after a delay, the quarantined time also reduces. b shows the sir plot of the same simulated system with exactly 15 pools per dorm. observe that the prevalence is easily controlled to below 5%. courtesy i. enaganti et al. provide computational models of host-pathogen interactions that will help to define parameters critical to healthcare policies and methods to control the viral spread-"the hammer and the dance." we hope that optimistic future will also include a successful completion of human haplotypic hla genome project, making it unfashionable among immunologists to say, "the thing is, the immune system is very complicated... !". springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. figure 6 : epidemiological model-e.g., compartmental models such as sir with compartments: s (susceptible), i (infected), and r (removed). the diagram shows why the uncertainties in parameters may make the models unpredictable-especially, when population is of finite size. still, the models can be used fruitfully to stress test various plausible policies and affect boundedly rational agent behaviors. courtesy: j. broek et al. joyce' , in finnegans wake: a symposium -exagmination round his incamination of work in progress deception, identity, and security: the game theory of sybil attacks compliance signaling games: toward modeling the deterrence of insider threats evolutionary dynamics of lewis signaling games: signaling systems vs. partial pooling cancer hybrid automata: model, beliefs and therapy signals immunology is where intuition goes to die. the atlantic in mid-march of 2020, spurred by a hypothesis centered around hla-hetero-zygotic advantages of asymptomatic covid patients, we organically converged to form the "cure covid-19 for ever and for all"(rxcovea) group, a rigorous community of scientists, clinicians, ai specialists, mathematical and computational/data modelists, pharmaceutical, public and digital health intelligence representatives from multiple institutions, countries, and scientific training. within the network, self-organized task forces continue to conduct core projects, structured around hypotheses, and minimal viable products (mvp's), mentored, and examined by experienced senior members.special "thank you"s to ashley, charles, larry, lex, and vijay-privately "typed" as friends, mentors, and mentees-who meticulously avoid social-distancing in all the three worlds: the paper has benefited a lot from their signals and actions! the contributions by bm were supported by nsf grants ccf-0836649 and ccf-0926166, nci physical sciences-oncology center grant u54 ca193313-01, and a us army grant w911nf1810427. the contributions by rxc have been voluntary and unsupported. the work of ts (t. schlick) was supported by nsf rapid award 2030377 and nih award r35gm122562 (nigms). the immune system is a host defense system comprising many biomolecular structures and signaling processes within an organism that protects against pathogens, dangerous (and possibly, stranger) to the organism-from viruses to parasitic worms, and distinguish them from the organism's own healthy tissue. it is possible to study the immune systems as a signaling game whose nash equilibria separate two types: self and non-self. the immune system consists of an innate system coupled with an adaptive system: both following replicator dynamics, but innate system selecting over organisms' germ line and adaptive system adapting over organisms' somatic immunological memory. [adapted from https :// morgr idge.org/outre ach/teach ing-resou rces/virol ogy-immun ology /immun e-syste m-gloss ary/]. b. mishra an educator, an inventor, as well as a mentor to technologists, entrepreneurs, and scientists. prof. mishra founded the nyu/courant bioinformatics group, a multidisciplinary group working on research at the interface of computer science, applied mathematics, biology, biomedicine, and bio/ nano-technologies. he is the author of a textbook on algorithmic algebra and more than 200 archived publications. he has advised and mentored more than 35 graduate students and post-docs in the areas of computer science, robotics and control engineering, applied mathematics, finance, biology, and medicine. he holds 21 issued and 23 pending patents in areas ranging over robotics, model checking, intrusion detection, cyber security, emergency response, disaster management, data analysis, biotechnology, nanotechnology, genome mapping and sequencing, mutation calling, cancer biology, fintech, adtech, internet architecture, and linguistics. prof. mishra's pioneering work includes: first application of model checking to hardware verification; first robotics technologies for grasping, reactive grippers, and work holding; first single molecule genotype/haplotype mapping technology (optical mapping); first analysis of copy number variants with a segmentation algorithm, first whole-genome haplotype assembly technology (sutta), first clinical-genomic variant/base calling technology (totalrecaller), first single-molecule single-cell nanomapping technology, etc. prof. mishra has a degree in science from utkal university, in electronics and communication engineering from iit, kharagpur, and ms and phd degrees in computer science from carnegie-mellon university. he is a fellow of ieee, acm, and aaas, eai, a fellow of national academy of inventors (nai), a distinguished alumnus of iit (kharagpur), and an nystar distinguished professor. key: cord-298169-2133gahl authors: tamouza, ryad; krishnamoorthy, rajagopal; leboyer, marion title: understanding the genetic contribution of the human leukocyte antigen system to common major psychiatric disorders in a world pandemic context date: 2020-10-05 journal: brain behav immun doi: 10.1016/j.bbi.2020.09.033 sha: doc_id: 298169 cord_uid: 2133gahl the human leukocyte antigen (hla) is a complex genetic system that encodes proteins which predominantly regulate immune/inflammatory processes. it can be involved in a variety of immuno-inflammatory disorders ranging from infections to autoimmunity and cancers. the hla system is also suggested to be involved in neurodevelopment and neuroplasticity, especially through microglia regulation and synaptic pruning. consequently, this highly polymorphic gene region has recently emerged as a major player in the etiology of several major psychiatric disorders, such as schizophrenia, autism spectrum disorder and bipolar disorder and with less evidence for major depressive disorders and attention deficit hyperactivity disorder. we thus review here the role of hla genes in particular subgroups of psychiatric disorders and foresee their potential implication in future research. in particular, given the prominent role that the hla system plays in the regulation of viral infection, this review is particularly timely in the context of the covid-19 pandemic. since the first description of an association between human leukocyte antigen (hla)-b and hodgkin lymphoma (amiel, 1967) the highly polymorphic hla gene cluster has been linked to a wide array of immune/inflammatory disorders, including now psychiatric disorders . recently, a strong association between schizophrenia and the major histocompatibility complex (mhc), which hosts the hla gene cluster, was reported by the psychiatry genomic consortium (ripke et al, 2014) . this finding extends the long-standing observation of links between hla and psychiatric disorders (eberhard et al, 1975) . given the prominent role of hla gene cluster in the regulation of immuno-inflammatory processes, as well as in neurodevelopment and neuroplasticity, through microglia regulation and synaptic pruning (moktari and lachman, 2016), it was expected that hla alleles would play a major role in the pathophysiology of psychiatric disorders. convergent results now show that this is particularly relevant for autism spectrum disorders (asd), schizophrenia or bipolar disorders, all known to be associated with chronic low-grade inflammation and comorbid autoimmune diseases (pape et al, 2019 ). the hla system is expected to influence both the early development and the course of psychiatric disorders. the mhc is a four megabases region located on the short arm of the chromosome 6 (6p21.3-22.1) and is one of the most polymorphic and gene dense regions of the human genome (trowsdale and knight, 2013) ,). this region hosts the hla gene cluster, which is physically divided into three functionally distinct sub-regions: i) the hla-class i region includes the classical hla-a, hla-b, and hla-c genes as well as the non-classical hla-e, hla-f and hla-g loci. the three classical hla genes act to regulate antigen presentation to cd8+ t-lymphocytes, whilst the non-classical genes are mainly implicated in different immunomodulatory functions; ii) the hla class ii region encompasses the hla-dpa1, hla-dpb1, hla-dqa1, hla-dqb1, hla-dra, hla-drb1, hla-drb3, hla-drb4 and hla-drb5 genes which are involved in antigen presentation to and iii) the class iii region which encompasses gene loci involved in inflammatory responses, leukocyte maturation and in the complement cascade. while the encoded molecules of the hla-a, -b and-c genes play an essential role in the detection and elimination of virus-infected cells and tumoral cells through cell-mediated cytotoxic processes, their hla class ii counterparts modulate humoral immune responses (klein & sato, 2000) . both gene sets are highly polymorphic with more than 26.000 alleles reported to date, although each locus has only around 10 to 20 dominant alleles (imgt/hla database, 2020), the main function of which is to present self or foreign antigens to t cell receptors (tcr) on effector cells. the hla molecules have long been appreciated to be involved in fine tuning of inflammatory processes as well as in the development of immuno-mediated pathophysiological processes including autoimmunity, a frequent comorbidity of psychiatric disorders (khandaker, dantzer, jones et al, 2017) . recent data also show that hla molecules modulate the development and function of the central nervous system (cns), including core functions such as neuronal/synaptic plasticity, learning, memory and behavior (boulanger, 2009) , as well as more direct modulation of neuron-neuron interactions and neuro-signaling (sterner, weckle, and chugani, 2012) . the highest hla expression levels are detected in post-synaptic hippocampal neurons (goddard, butts and shatz , 2007) . moreover, the hla molecules are pivotal for the anatomical integrity of the cns, as exemplified by the enlarged ventricles observed in hlaclass i deficient murine models (huh et al, 2000) . despite evidence of prominent immune implication in a significant subset of major psychiatric disorder patients such as schizophrenia, bipolar disorder or depression (khandaker, dantzer, jones, 2017) or autism spectrum disorder (meltzer & van de water, 2017) , deciphering the mechanistic link between the hla system and these disorders was difficult, primarily because of the complex genetic architecture of the hla system. recent technological advances now allow a more precise characterization of the hla gene cluster and the identification of its etiological or protective role in psychiatric disorders. it thus timely to review the role of hla genetics in major psychiatric disorders and to describe future research directions including the regulation of the impact of viral infections on aetiology and course of psychiatric conditions in the context of a world pandemic. since the discovery of the hla system by jean dausset (dausset and brecy, 1957) , the analysis of hla polymorphisms has undergone successive technological improvements over 20 years, leading to the gradual incorporation of dna-based molecular approaches, including the high throughput and cost-effective next generation sequencing (ngs) which provides reliable and extensive hla genotyping. however, fine-tuned analysis and understanding of the hla diversity in genetic studies still requires specialized training either in terms of histocompatibility testing or imputation analysis. despite such advances and expertise, hla investigation in disease association studies still faces a number of challenges, including: (i) the extreme rate of polymorphism; (ii) the ethnogeographical-dependent distribution of hla alleles; (iii) the disease-dependent variable pertinence of a given hla allele e.g. the hla-b27 association is prominently evident for ankylosing spondylitis while for other multi-genic and multifactorial disorders, evidence of disease association may not be that apparent as distinct alleles may have shared functions; and (iv) the variability of allele expression (ramsuran et al, 2018; d'antonio et al, 2019; aguiar et al, 2019) . it thus should be stressed that while the candidate gene approach, which may ignore the degree of contribution of other interacting loci to the phenotype, is not a satisfactory way to fully underscore the hla genetic diversity, genome-wide association studies (gwas) followed by hla imputation-based methods, at least in caucasian and asians, may provide clues towards understanding the hla genetic contribution to psychiatric disorders. unfortunately, at the present time, only few post-gwas hla imputation-based studies have been undertaken. an approach to overcome these difficulties is to understand the evolution-based shaping of present-day hla diversity as stemming from a limited, but manageable, number of ancestral haplotypes (ah). various genetic events, viz crossing-overs, recombination and point mutations, have participated in that evolution (price et al, 1999; dawkins et al, 1999) . these ahs were selected under diverse geographic-specific environmental pressures, then conserved and fully or partially transmitted to successive generations (dawkins et al, 1999) . consequently, study of ah distribution in disease association studies have clarified why apparently distinct hla alleles exhibit association with a given disorder. indeed, these alleles, linked to ahs, allow to stratify the patients into an immuno-inflammatory subset for ah associated with steady state inflammation even among healthy subjects. for example, hla-8.1ah is the most associated ah with immune-related disorders, including infections and autoimmunity, whilst also being characterized by a steady state pro-inflammatory background in healthy individuals (price et al, 1999; giambino et al, 2018) . such properties may explain why the 8.1ah can be protective against pathogens and therefore positively selected, while also having a negative health impact due to the association of chronic pro-inflammatory status with high risk for autoimmunity (crespy and go, 2015) . hla functional diversity can also be investigated via allele-dependent expression status which reflects the influence of hla alleles per se. for example, single nucleotide polymorphisms (snp) categorize the hla-dpb1 and hla-c alleles into highly and lowly expressed variants (petersdorf et al, 2015; thomas et al, 2009; apps et al, 2013) . hla-peptide combination is mediated by the polymorphic tcrs on cd8+ t cells, which bind class i molecules, and on cd4 +t cells which bind class ii molecules. the specificity of hla-peptide-tcr tripartite interactions is fundamental in enabling the adaptive immune system to mount an efficient and appropriate response against infection, whilst simultaneously preventing auto-immune processes (creusot, mitchison, terazzini, 2002; woodsworth, castellarin and holt, 2013; morris and allen, 2012). different mechanisms may underpin hla associated diseases, including: 1) atypical hla-peptide-tcr binding orientation; 2) low affinity peptide binding that facilitates thymic escape; 3) tcr-mediated stabilization of weak-peptide-hla-interaction; and 4) presentation of peptides in a different binding register. other mechanisms that may generate autoreactive t cells are driven by epitope variation, including molecular mimicry (yin, li and mariuzza, 2012) . thus, while gwas identified the mhc/hla genetic cluster as being a pivotal region, specific hla-dedicated expertise started to uncover meaningful functional haplotypes associated with specific psychiatric entities. hla analysis in psychiatric disorders will help not only to identify homogeneous subgroups, hla based, but also to decipher their underlying mechanisms. recent gwas and haplotype-based studies have revived and strengthened interest in the roles of the immune system in psychiatric disorders. this section reviews investigations of hla gene candidate association in schizophrenia, bipolar disorders and autism spectrum disorders, focusing on the risk/protection that hla alleles/haplotypes may confer on specific sub-groups. accumulating evidence from epidemiological, immunological, genetic, and imaging studies strongly indicate a role for mhc in schizophrenia risk , dating from the 1970's (eberhard, franzén and löw, 1975; cazzullo and smeraldi, 1979) . previous work has shown a number of associations across different ethnic populations, including the hla-a9, hla-a10, hla-drb1, and hla-dqb1 alleles . in 2009, three gwas and a meta-analysis of these gwas were published in nature, revealing a strong association between the mhc region and schizophrenia, although without any precision as to the specific location (purcell et al, 2009 , shi et al, 2009 stefansson et al, 2009 ). subsequent studies using gwas subjected to hla imputation of classical hla alleles, revealed a marked dominant protective effect conferred by hla-a*01, b*08 and drb1*03 (donnelly et al, 2012) . these alleles are all derived from the so-called 8.1 "autoimmune" ancestral haplotype (8.1ah) (a*01~b*08~cw*07~drb1*03~dqb1*02). the 8.1 ah is the most associated hla haplotype with inflammatory processes and autoimmune diseases, including type1 diabetes, celiac disease, grave's disease, and myasthenia gravis (price et al, 1999) , a situation that may appear at first sight contra-intuitive according to the protecting effect conferred against schizophrenia risk. a deeper exploration of the hla region further revealed a major contribution to schizophrenia risk mediated by increased complement c4a gene copy number with consequent c4-dependant neuro-synaptic pruning (sekar et al, 2016 ). the complement system is not only in first-line defense against pathogens but also a major contributor to synaptic pruning during neurodevelopment (druart and le magueresse, 2019) . however, it is important to signal that the sekar's study not only suffer from absence of replication especially in ethically-distant population groups such as asians (lam et al, 2019) but also from the complexity of long-range imputation statistics and absence of direct inference of c4 haplotypes along their respective expression status. it is evident that the full sequencing of the c4 cluster may help genetic dissection of this complex region. besides these considerations, recent investigations have established a link between the c4 locus and classical hla haplotype diversity in the modulation of schizophrenia risk. indeed, the protective status conferred by the 8.1ah haplotype plausibly arises from this haplotype naturally lacks the c4 locus. we recently showed that a 8.1 ah-derived hla haplotype was significantly less frequent in schizophrenia patients with early onset, whilst gradually increasing in frequency with the age of schizophrenia onset . this would suggest that 8.1 ah, via decreased c4 expression, leads to less synaptic pruning and cortical thinning, thereby delaying the age of schizophrenia onset, whilst concurrently potentially favoring heightened autoimmune processes due to its proinflammatory properties. in contrast, we hypothesized that schizophrenia patients not bearing 8.1 ah-derived hla haplotypes have an active c4 complement and may suffer from a more severe form of the disorder, characterized by early age at onset, increased synaptic pruning and cortical thinning, whilst being less prone to develop autoimmune disorders. this is in line with previous observations that carriers of mhc-linked risk variants (rs2596532) have larger ventricles (agartz et al, 2011) . even if it is assumed that the above-mentioned mechanisms, if proven, would be not the unique disease process, overall hla data may highlight the possibility that hla genetics will help to identify homogeneous sub-groups of schizophrenia patients. although gwas clearly indicate that the mhc region confer an increased autism spectrum overall, these data give some indication of the role that hla haplotypes may play in the abnormal neurodevelopment as well as in the systemic and gastro-intestinal inflammatory processes at work in autism. we recently reported an association between an 8.1 ah derived hla haplotype and bipolar disorder, specifically in patients with severe forms of the disorder defined by rapid cycling and/or personal history of suicidal behaviors . this association might also underline the well-known high frequency of auto-immune disorders in bipolar patients, including elevated levels of anti-thyroid or other organ-specific autoantibodies (padmos et al, 2004; jeppesen et al, 2019) . we also found that two haplotypes namely hla 57.1 ah and 7.1 ah were specifically associated with bipolar disorder having first episodes defined by hypomanic episode or psychotic symptoms. this is of importance as these two haplotypes are also linked to common inflammatory conditions, and might thus contribute to the proinflammatory processes observed in bipolar disorder. although these data, observed in phenotypically well-defined subgroups of patients, may constitute interesting tags of the mhc implication in bd, here again, a recent gwas did not allowed to uncover any mhc-derived signal (stahl et al, 2019) . the contrasting effect of 8.1 ah, being protective in schizophrenia and autism but conferring severity in bipolar disorder, is interesting. andreasen et al. (2015) reported that the same hla allele was shared between schizophrenia and multiple sclerosis (ms), but not between ms and bd. despite the large overlaps of genetic as well as clinical features in bipolar disorder and schizophrenia, these data are suggestive of temporal differences in hla-dependent immunogenetic influences on neurodevelopmental processes (andreassen et al, 2015; bergen et al, 2012) . these two psychiatric disorders may be differentiated by opposite effects mediated by the 8.1 ah possibly through specific complement c4-mediated synaptic pruning processes. the role of hla-driven changes to the temporal neurodevelopmental specificities of bipolar disorder and schizophrenia will be important to clarify in future research. beside schizophrenia, autism spectrum disorders and bipolar disorders, major depressive disorders and attention deficit hyperactivity disorders are also major, common and frequent psychiatric conditions but suffer from scarcity of published/robust data implicating hla genetics. concerning major depressive disorders results from number of small studies, using broad serological typing (now considered as obsolete), generated inconsistent data. besides, a very recent gwas failed to identify any hla-related signals in mdd (glanville et al, 2020) . this is also true for adhd where few previous studies indicated potential association with the mhc non-classical complement c4b, but again, a recent large gwas did not allow to detect any association between adhd and hla (nudel et al, 2019) . nevertheless, the potential genetic association between hla polymorphism and anti-nmda-r encephalitis awaits confirmation. more consistently, other autoantibodies for brain receptor targets have recently been described. in particular, a german study described a strong association between anti-leucine-rich glioma-inactivated1 (lgi1) encephalitis and the hla-drb1*07:01~dqa1*02:01 haplotype (mueller et al, 2018) . in addition, another study, besides replicating the above-mentioned hla association with lgi1-mediated encephalitis, described an additional association between the hla-drb1*11:01~dqa1*05:01~dqb1*03:01 haplotype and anti-contactin-associated protein-2 (caspr2) encephalitis (binks et al, 2018) . hla genetic diversity is also implicated in the modulation of treatment responses in psychiatric conditions, including in the regulation of adverse drug reaction (adrs) and treatment efficacy. both candidate gene studies and gwas have shown that clozapine-induced agranulocytosis is partly mediated by class i and ii hla alleles (numataa et al, 2018) . in a study of treatment response to antipsychotic, we showed that a double amino-acid change in the hla-a peptidebinding groove was associated with a better response to treatment with risperidone in patients with schizophrenia (leclerc et al, 2015) . a recent large survey showed that treatment response to lithium in bd is strongly influenced by both schizophrenia-linked polygenic score and the mhc/hla genetic diversity (amare et al, 2018) . in the latter context the authors identified signals related to antigen presentation pathway (hla-dm region), the main function of hla molecules. such observation could be in line with the notion that differences in the heritability between schizophrenia and bd may lie in the mhc cluster (andreassen et al, 2015) . the where two types of herv family, namely herv-w and herv-k, are respectively implicated (greenig, 2019) . given the role of gene and environment interactions in herv reactivation, and its capacity to induce pro-inflammatory and neurotoxic proteins, herv has been the focus of studies in psychiatric disorders. we and others have found associations between the herv-w type and both schizophrenia and bd at protein and/or at dna/rna levels, further influenced by copy number variations (perron et al, 20121; leboyer et al, 2013) . recent data show herv-k to be a potential risk component for schizophrenia. it is worth mentioning that sekar et al demonstrated that complement c4 long allele, harboring insertion of herv-k, expressed higher levels of c4a molecules, thereby increasing the risk of exaggerated synaptic pruning, cortical thinning and early onset (sekar et al, 2016) . this is an interesting area of investigation as clearly different herv family members can be associated with the same disease, although with different disease pathways. all living organisms have to constantly learnt to deal with environmental insults in order to survive, including various types of pathogens. given the extreme diversity of these environmental insults, evolutionary forces have gradually shaped powerful biological systems characterized by a large number of genes with high allelic diversity adapted to handle these challenges, viz the different wings of the immune system. upon interaction with a given trigger, the immune system first mounts non-specific pro-inflammatory processes and then, if necessary, more adaptive cellular processes directed against the triggering event. in parallel with the shaping of the immune response, counteracting immune-modulatory genetic strategies, limiting uncontrolled inflammation, have emerged and have been positively selected by evolutionary constraints. the hla-class i classical and non-classical molecules represent one of the best examples of such janus-faced system. indeed, within the same hla-class i region lie (i) the classical hlaclass i -a, -b and -c loci, characterized by an extreme polymorphism essential for their antigenpresentation functions, and maintained by balancing selection (heterozygote advantage) to cope up with a large variety of environmental pathogens and (ii) the non-classical hla-e, g and f genes, remarkable due to a very low rate of diversity that reflect broader properties, such as immunomodulation. among the latter, the non-classical hla-g encode cell surface molecules exerting powerful immunomodulatory functions, demonstrated to be essential for the establishment and tolerance between the maternal immune system and the semi-allogeneic fetus at the fetal-placental interface (ferreira et al, 2017) . genetically determined low expression of the tolerogenic hla-g molecules at the fetal-mother interface, possibly led to prenatal immune activation, is associated with asd risk (guerini et al, 2015 (guerini et al, , 2018a (guerini et al, , 2018b (guerini et al, , 2019 . schizophrenia is also widely believed to be a neurodevelopmental psychiatric disorder, although the role of hla-g polymorphism and expression has been less investigated. however, available data does indicate that low levels of hla-g, either circulating or genetically determined, may influence disease onset and phenotype (rajasekaran et al, 2015 , rajasekaran et al, 2016 rajasekaran et al, 2016; shivakumar et al, 2018) . in two studies performed on bipolar disorder patient populations of distant ethnicity, namely french and south indian tamils, data shows that in contrast to autism and schizophrenia, genetically determined hla-g low expression confers protection against bipolar disorder, suggesting the role of distinct hla-g effects in the aetiology of these disorders (debnath et al, 2013; sundaresh et al, 2018) . it is hence possible that in bipolar disorder, the protection conferred by low grade immunomodulation may favor a more efficient and intense pro-inflammatory, anti-infectious response, but outside the neurodevelopmental window. at the time of writing this review, the covid-19 pandemic was devastating the health and economies of countries worldwide, highlighting the powerful influence that viruses have had and have on animal and plant life over the course of evolution. there is an increasing appreciation of the role of viruses in wide array of diverse medical conditions, including cancers and neurodegenerative conditions, but also in psychiatric conditions (avramopoulos et al, 2015) . direct impact of viruses on the central nervous system was illustrated in contemporary history by clinical situations in neuro-psychiatric settings after pandemics such as the spanish flu or the more recent h1n1. while an increased frequency of psychosis (yudofsky sc, 2009) and encephalitis lethargica/parkinsonism (lymphaibool et al, 2019; hoffman & vilensky, 2017) were observed following the spanish flu pandemic, a raised rate of narcolepsy was described after h1n1 pandemic likely triggered by the pandemrix® vaccination (sarkanen et al, 2018) . more recently, comforting early reported deleterious effects of viral infections on cns, large nationwide studies demonstrated association between maternal viral infectious events during the first trimester and subsequent psychiatric diseases in the offspring (brown & derkits, 2009) . it is worth reminding here again, as for the vast majority of viral infections, that the hla system is pivotal in the anti-infectious immune response processes, very likely in the present pandemics too. in the current context, it is important to note that variations in hla/mhc act to regulate viral infections, including influenza infection in humans (dutta et al, 2018) . influenza and other infections prenatally can also increase risk of schizophrenia and autism spectrum disorders in the offspring, suggesting that hla/mhc genetic variations may interact with prenatal infection in the etiology of major psychiatric disorders, although complicated by the immune-suppression that occurs in pregnancy (shah et al, 2010) . the devastating influence of the severe acute respiratory syndrome coronavirus (sars-cov)-2 virus that has led to the covid-19 pandemic raises the question as to the role of hla/mhc in the regulation of the inflammatory processes which is an integral aspect of sars-cov-2 infection, usually referred to as the 'cytokine storm'. this will also be important to investigate in the distinct sub-groups of major psychiatric disorder patients carrying particular variations of their hla alleles. although the literature concerning the immunogenetic aspects of previous pandemic waves of sars or mers-cov (middle east respiratory syndrome) is relatively low, data do show that hla plays a major role in viral infection regulation, and this is also relevant to analyze risk/protection of major psychiatric disorders in the pandemic context. the available data on hla diversity show associations with hla-class ii alleles, notably the hla-drb1*03 variant in two studies from taiwan (wang et al, 2011) , and hong kong (ng et al, 2004 (nguyen et al, 2020) . as acknowledged by the authors, among many other limitations, peptide-mhc binding affinity cannot be alone a predictor of subsequent t-cell responses. the psychiatric inpatient population-group is at high risk for any epidemic threat. increased vulnerability not only arises due their psychiatric condition, associated stressors, and confinement with other patients but also from their comorbid somatic disorders including, cardiovascular disorders, metabolic syndrome, diabetes, autoimmune disorders and respiratory tract dysfunctions. all of these common comorbidities are risk factors for severe infection and almost all are linked to hla genetic diversity (trowsdale and knight, 2013) . as with influenza viruses, the most deleterious event in covid-19 does not seem to be the infectious agent per se, but the overwhelmed reactive inflammation arising from the 'cytokine storm'. future research will have to determine whether variations in hla genetic diversity modulate the susceptibility to severe infection and fatality in major psychiatric disorders subjected to the sars-cov-2 pandemic. the data reviewed above and the long evolutionary history of hla-equivalent loci, including further investigation is required in people from africa, given that the rate of the genetic diversity is the highest in africans amongst all human ethnic groups. in this context, a recent study of a sample of sz patients from south africa, namely the xhosa population-group, revealed not only that the observed overall genetic diversity was more important than that of non-african populations, but importantly uncover mutational events relevant to the origins of schizophrenia (gulsuner et al, 2020) . future studies of long-established populations from where the human genome was shaped by various environmental pressures over time, including a variety of social and microbial pressures, will be pivotal for the understanding of psychiatric disorders. as highlighted in this review hla diversity is integral to variations in the immune responses that form biological underpinnings, among others and likely in subsets of patients, of a wide array of psychiatric presentations as well as to how we, and other animals, have interacted with viruses over the course of evolution. relevant papers were identified through pubmed searches of articles published in english from jan 1, 1955, up to april 30, 2020, using the following search terms (alone or in combination): "hla, mhc, psychiatry, polymorphism, immunogenetics, immuno-psychiatry, covid". additional studies were identified from our own files. the final reference list was generated on the basis of their relevance to the topics covered in this review. the authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. this review was written under the framework of agence nationale de la recherche (i-give anr-13-sama-0004-01), inserm (institut national de la santé et de la recherche médicale) and fondation fondamental however without any role of the above-mentioned institutions in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the review for publication. rt: designed the search strategy and performed it, wrote the manuscript and take the primary responsibility to submit the review. rk and ml: participated to the search strategy, critically reviewed and edited the manuscript. all authors approved the final version.  major psychiatric disorders are associated with genetically-determined immune dysfunctions  human leukocyte antigen (hla) molecules are prominent players of immune and neurodevelopmental processes.  hla molecules are encoded by polymorphic genes located in the major histocompatibility complex (mhc).  recent methodological approaches allowed understanding of the hla genetic complexity and reconcile findings from gwas in psychiatry.  hla haplotypes studies uncover protective and at risk markers for disease development or proxies of severity in schizophrenia, autism and bipolar disorders.  beyond disease risk, the hla genetic diversity is associated with autoimmune encephalitis, treatment response, retrovirology and neurodevelopmental immunomodulation.  hla is also involved in anti-infections processes including against the 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different from that found in europe sequence analysis of t-cell repertoires in health and disease structural basis for self-recognition by autoimmune t-cell receptors contracting schizophrenia: lessons from the influenza epidemic of 1918-1919 key: cord-280641-zqdrzyzl authors: fernández fabrellas, estrella title: epidemiología de la sarcoidosis date: 2007-02-28 journal: archivos de bronconeumología doi: 10.1157/13098420 sha: doc_id: 280641 cord_uid: zqdrzyzl la sarcoidosis es una enfermedad multisistémica que afecta frecuentemente al pulmón. su incidencia y prevalencia han sido ampliamente estudiadas, pero la falta de estandarización del diagnóstico, los diferentes métodos de detección de casos y la escasa sensibilidad y especificidad de las pruebas diagnósticas explican los datos discordantes. el pronóstico es generalmente favorable. gran parte de las personas afectadas no manifestarán nunca síntomas y muchas tienen remisión espontánea. el curso es crónico en el 10-30% de los casos, con un deterioro permanente de la función pulmonar. la enfermedad es el resultado de la acción de un agente externo que desencadena la respuesta inmunitaria característica en individuos genéticamente susceptibles. se han implicado factores ambientales, ocupacionales y genéticos, pero las investigaciones están todavía en los inicios. estudios de casos y controles, así como los avances en biología molecular, ayudarán a definir los factores de susceptibilidad genética y a entender los distintos fenotipos de la sarcoidosis. sarcoidosis is a multisystemic disease in which lung involvement is common. its incidence and prevalence have been extensively studied, but with contradictory results because of the lack of standard diagnostic criteria, variations in the methods for detecting cases, and the low sensitivity and specificity of diagnostic tests. prognosis is generally favorable. many of those affected remain asymptomatic and remission often occurs spontaneously, although between 10% and 30% of the patients have chronic disease and permanent deterioration in lung function. sarcoidosis is caused by an external agent that triggers a characteristic immune response in genetically susceptible individuals. environmental, occupational, and genetic factors have all been implicated, but research is still in the early stages. case-control studies, as well as advances in molecular biology, will help to identify genetic susceptibility factors and to understand the different phenotypes of sarcoidosis. desde que en 1877 jonathan hutchinson, cirujano y dermatólogo, describió el primer caso en londres como una enfermedad dermatológica, la sarcoidosis ha continuado fascinando a clínicos e investigadores. durante décadas se ha conseguido progresar en el conocimiento clínico y en los hallazgos patológicos de la enfermedad, pero se sabe muy poco sobre la epidemiología y los factores genéticos que contribuyen a su desarrollo y forma de expresión, el tratamiento adecuado todavía no está bien definido para todos los pacientes y, lo que es más importante, la causa del proceso continúa siendo desconocida 1 . la sarcoidosis se caracteriza por una respuesta inmunológica inicial de células t helper tipo 1, que lleva al desarrollo de granulomas no caseificantes con afectación multisistémica, siendo los órganos diana más frecuentes el pulmón (90%), la piel y los ojos. durante los siguientes 2-5 años desde el inicio de los síntomas, más del 60% de los pacientes experimentan una resolución completa de la enfermedad, pero en casi el 30% restante puede seguir un curso crónico, que en algunos casos lleva al desarrollo final de fibrosis pulmonar con síntomas respiratorios permanentes. la complejidad de esta enfermedad y la amplia gama de síntomas con que puede presentarse hacen imprescindible muchas veces el abordaje multidisciplinario del paciente. en general, la situación de una enfermedad puede clasificarse por su actividad o gravedad, pero en la sarcoidosis la actividad no indica necesariamente un curso progresivo, un pronóstico fatal o la necesidad de tratamiento. esto supone un reto para el clínico, que afronta muchas dificultades al tratar de categorizar la sarcoidosis en un paciente concreto. el reconocimiento de la raza como un importante factor de riesgo para presentar esta enfermedad apunta claramente a una predisposición genética 2 . de esta manera, las últimas investigaciones se han dirigido a identificar estos factores de riesgo genéticos y a esclarecer cómo el genotipo del paciente determina la presentación y la evolución, es decir, el fenotipo de la enfermedad. desde este punto de vista, la sarcoidosis es una enfermedad compleja, cuya predisposición genética no está determinada por un solo gen 3 . la sarcoidosis es una enfermedad granulomatosa sistémica de distribución mundial, que se presenta en ambos sexos, a cualquier edad y en cualquier raza. ¿por qué, entonces, no se ha encontrado un agente causal? aunque los esfuerzos se han dirigido durante décadas al aislamiento de un posible agente microbiológico, los resultados han sido desalentadores. hay varias razones. la primera es que posiblemente desconozcamos todavía las condiciones óptimas para el aislamiento de ese posible germen causal; otra razón es que la causa no sea infecciosa, y, por último, cabe la posibilidad de que esta entidad nosológica llamada sarcoidosis sea en realidad la suma de más de una enfermedad, cada una de las cuales tiene distinta etiología 4 . podría haber otras muchas razones por las que el agente etiológico de la sarcoidosis permanece oculto, y estarían en relación con los diseños de los estudios realizados a lo largo del tiempo. una es la definición de caso: una cuidadosa revisión de la literatura médica 5 demuestra que los investigadores han usado una definición de caso amplia, imprecisa y variada. esta limitación ha mejorado desde la publicación de las guías de esta enfermedad 1 . otra causa puede estribar en las diferentes formas de reclutamiento de pacientes en los estudios publicados, que dificulta poder comparar los resultados. la heterogeneidad de la propia enfermedad, con una amplia gama de patrones clínicos, aumenta la posibilidad de que pueda no ser una sola enfermedad, de que pueda haber distintos agentes etiológicos o de que uno solo pueda ocasionar diferentes efectos según la susceptibilidad individual basada en factores genéticos 6 . el término "epidemiología" se usa para identificar la distribución de la enfermedad, los factores que la causan y sus características en una población dada. esto también incluye la incidencia, frecuencia, prevalencia y brotes endémicos y epidémicos, además de incorporar estudios y estimaciones de morbimortalidad en áreas geográficas y poblaciones concretas. muchos investigadores han tratado de calcular la incidencia y prevalencia de la sarcoidosis en diferentes poblaciones y con distintas estrategias de estudio -detección de adenopatías mediastínicas en la radiografía simple de tórax, registros nacionales, bases de datos o cuestionarios y revisiones de autopsias-, de manera que los datos disponibles son discordantes y difíciles de extrapolar al resto de la población. hasta hace poco se admitía que la enfermedad es más frecuente en adultos de menos de 40 años, con un pico de mayor incidencia entre los de 20 y 29 años. en los países escandinavos y japón hay un segundo pico de incidencia en mujeres mayores de 50 años. la mayoría de los estudios señalan un ligero predominio en el sexo femenino, y a partir de estudios poblacionales realizados en ee.uu. el riesgo de presentar sarcoidosis se calcula en el 0,85% para la raza blanca y el 2,4% para la raza negra 2 , con una tasa de incidencia anual en dicho país, ajustada por edad, de 35,5 por 100.000 habitantes entre la población negra y 10,9 entre los caucásicos 7 . en nuestro país las tasas de incidencia anual publicadas son inferiores. en el estudio realizado en un área sanitaria de la provincia de león se fijó en 1,37 por 100.000 habitantes 8 , y para todo el país la estimación de la tasa de incidencia anual acumulada es de 1,36 por 100.000 habitantes 9 . el registro de incidencia de enfermedad pulmonar intersticial difusa en españa realizado de octubre de 2000 a septiembre de 2001, con participación de 37 centros, puso de manifiesto que la sarcoidosis es la segunda causa de enfermedad intersticial en nuestro país, por detrás de la fibrosis pulmonar idiopática, con 76 casos registrados, lo que supuso el 14,9% del total de casos de enfermedad pulmonar intersticial difusa 10 . los estudios de prevalencia también muestran resultados dispares, con cifras que varían de 1 a 40 casos por 100.000 habitantes/año. suecos, daneses y afroamericanos parecen tener las tasas de prevalencia más elevadas de la población mundial 11 . el estudio más amplio realizado hasta ahora, y con mejor diseño, es el access (a case control etiologic study of sarcoidosis 12 ), que ha arrojado luz sobre diversos aspectos epidemiológicos y etiológicos de la sarcoidosis. el access es un estudio multicéntrico que se concibió para determinar la etiología de la enfermedad y en el que participaron 10 centros investigadores de ee.uu. desde 1997 a 1999. su relevancia radica fundamentalmente en los criterios de selección y definición de caso, en un intento de obviar la imprecisión de estudios previos. de este modo, la definición de caso requiere la confirmación histológica de granulomas no caseificantes, aunque éstos no son patognomónicos, y que las biopsias sean interpretadas como indicativas del diagnóstico de sarcoidosis, descartándose siempre otras posibles causas (tabla i). todas las piezas histológicas se centralizaron en un solo laboratorio y fueron revisadas por los mismos patólogos designados para el estudio 13 . tras establecer claramente la definición de caso, el estudio access protocoliza el abordaje diagnóstico de manera homogénea para todos los centros, estableciendo criterios claros y definidos para llegar al diagnóstico de afectación de órgano y para el reclutamiento del grupo control 14 . además de investigar la posible etiología de la enfermedad, este estudio examina el situación psicosocial 15 y el curso clínico de 736 pacientes incluidos en los 6 primeros meses desde el diagnóstico histológico de sarcoidosis, y los compara con otros tantos controles pareados por edad, sexo y raza, con un seguimiento de los primeros 215 casos durante los 2 años siguientes a la inclusión 16 . a pesar de su importancia, este estudio tiene limitaciones: posiblemente sobrevalora la afectación pulmonar (encontrada en el 95% de los casos), debido a que los investigadores fueron neumólogos; como el seguimiento es de 2 años, no incluye a pacientes con sarcoidosis crónica, que son los que probablemente presentan la forma más grave. tendremos que esperar un tiempo para conocer resultados evolutivos y de pronóstico de este estudio. aun así, los resultados del access son sorprendentes en muchos aspectos: por una parte, la edad de presentación de los pacientes supera los 40 años, especialmente entre las mujeres, sin que los investigadores puedan explicar este "retraso" respecto a publicaciones previas; por otro lado, llegan a la conclusión de que sólo la afectación pulmonar es independiente de la edad, sexo o raza, mientras que el resto de presentaciones clínicas están vinculadas a estos factores 17 . este estudio se comentará ampliamente en cada uno de sus aspectos a lo largo de esta revisión. el conocimiento epidemiológico de la sarcoidosis está basado principalmente en estudios realizados hace más de 30 años, que ya incidían en un mayor predominio de la raza negra sobre la blanca y de las mujeres sobre los varones 18 . estos hallazgos dieron pie a posteriores investigaciones que confirmaron algunas de estas interesantes asociaciones, pero sin diferencias tan evidentes en función de la raza y el sexo como se apuntaba al principio. en efecto, el riesgo de sarcoidosis entre la población afroamericana es de 3 a 4 veces mayor que en la raza caucásica de ee.uu. las mujeres tienen un riesgo relativo mayor que los varones, aunque no excede el doble, y existe una agregación familiar que indica cierta susceptibilidad genética. esta sarcoidosis familiar es más frecuente también en afroamericanos (17%) que en caucásicos (6%) 19 . la evidencia epidemiológica de estos trabajos indica que hay que considerar factores de riesgo tanto ambientales como genéticos en la etiología de la sarcoidosis, porque probablemente la interacción entre ellos producirá la enfermedad 20 . un reciente estudio poblacional 21 realizado en dinamarca utilizando el registro nacional de pacientes describe diferencias en el momento del diagnóstico en relación con la edad y el sexo, de manera que establece un pico de incidencia en varones de 30-34 años de 14,8/100.000 habitantes, mientras que las mujeres tienen 2 picos: entre los 25 y 29 años (10,5/100.000 habi-tantes), y entre los 65 y 69 años (11,0/100.000 habitantes). la edad media entre los varones fue de 38 años y en mujeres de 45, con un ligero predominio para el sexo femenino de 1,06. en el access, la población de estudio es heterogénea en términos de raza (un 53% de raza blanca y un 44% de raza negra), sexo (un 64% mujeres y un 36% varones) y edad (un 46% son menores de 40 años), pero las características diferenciales son interesantes: las mujeres se presentaban más frecuentemente con afectación neurológica y ocular, eritema nodoso y edad superior a los 40 años, mientras que en los varones las anomalías del metabolismo del calcio fueron más habituales; los pacientes de raza negra tenían afectación cutánea distinta del eritema nodoso, afectación ocular, hepática, de médula ósea y adenopatías extratorácicas con más frecuencia 17 . las mayores diferencias encontradas en este estudio se relacionan con la raza de los pacientes. hay pocos datos epidemiológicos referidos a niños menores de 15 años 22 , población en la que se establece una incidencia anual de 0,29 por 100.000 habitantes, con variaciones desde 0,06 en niños menores de 4 años e incremento progresivo hasta 1,02 en niños de 14 a 15 años, que parecen tener un pronóstico más favorable, similar al de los pacientes adultos jóvenes. a pesar de los datos discordantes de los diferentes estudios, parece claro que la sarcoidosis tiene tendencia a desarrollarse al inicio de la edad adulta (es rara la presentación en la infancia y adolescencia y en mayores de 70 años 23 ), lo que incide en la hipótesis de que la exposición a agentes ambientales, infecciosos o antigénicos ocurriría durante la etapa laboral de los pacientes; por tanto, se especula sobre la contribución de la exposición ocupacional. en este mismo sentido, recordemos que la proporción de afectados por sarcoidosis parece decantarse ligeramente hacia las mujeres. sin embargo, en el único estudio poblacional de incidencia realizado en ee.uu. 7 , la incidencia ajustada por edad era similar entre varones (5,9/100.000 habitantes/año) y mujeres (6,3/100.000 habitantes/año), pero los autores llaman la atención sobre el incremento de la incidencia femenina desde el año 1946 a 1975, que les lleva a plantearse la hipótesis de que la progresiva incorporación al mundo laboral de las mujeres durante dicho período supondría la exposición a antígenos ambientales que inducirían la sensibilización y el desarrollo de la enfermedad. todos los estudios evidencian que la sarcoidosis es más frecuente entre personas de raza negra que entre caucásicos, pero paradójicamente también se ha documentado cierta agregación entre individuos descendientes de países del norte de europa, especialmente en las formas agudas de la enfermedad, como el síndrome de löfgren 24 . los estudios realizados en estos pacientes indican un importante determinante genético que explicaría estas formas de presentación 25 . sin embargo, todavía hay muchas consideraciones que hacer sobre los factores del huésped y ambientales que pudieran influir en la expresión de estos genes. la sarcoidosis tiene cierta tendencia a manifestarse al final del invierno y, sobre todo, al principio de la primavera [26] [27] [28] . si se da por sentado que la latencia entre la exposición al agente causal y el desarrollo de síntomas de sarcoidosis es del orden de unas pocas semanas a pocos meses, como se ha visto en modelos animales experimentales, la exposición tendría que ocurrir en muchos casos durante los meses previos a las manifestaciones clínicas. es atractivo conjeturar que habría un mayor contacto con el agente etiológico, tanto si es antigénico como infeccioso, cuando las personas pasan más tiempo en sitios cerrados como el lugar de trabajo o el domicilio durante los meses más fríos, y se podría llegar a pensar que la sarcoidosis es un tipo de enfermedad "relacionada con los edificios", resultante de la sensibilización a antígenos o a gérmenes vehiculizados por el aire (bioaerosoles), como sucede en otros procesos como la neumonitis por hipersensibilidad o la legionelosis. no todos los estudios llegan a las mismas conclusiones sobre la distribución espacial de la sarcoidosis. sin embargo, a pesar de las discrepancias y de los distintos métodos empleados, la mayoría de los datos publicados apuntan a que esta enfermedad se produce más frecuentemente en regiones geográficas determinadas, lo que ha dado pie a investigar factores meteorológicos y del suelo, plantas, pólenes y proximidad a bosques, utilización de recursos hídricos, uso de leña y exposición a mascotas o a animales de granja como posibles agentes etiológicos o de riesgo 6 . el access encuentra una asociación positiva con ocupaciones concretas (agricultura), exposición a determinados agentes potencialmente tóxicos (insecticidas y polvos orgánicos ambientales) y desempeño del trabajo en ambiente enrarecido y con olores mohosos. este mismo estudio documenta mayor riesgo de sarcoidosis en personas que han vivido en ciudades pequeñas durante su infancia, lo que incide en el ambiente rural como factor de riesgo 29 , hecho que también se ha descrito en nuestro país 30 . además de la asociación geográfica, al parecer tienen cierta tendencia a presentar sarcoidosis las personas con estrecho contacto físico con pacientes o con relaciones muy cercanas dentro de la misma comunidad. el estudio de casos y controles realizado en la isla de man 31 , en reino unido, pone de manifiesto que un 40% de los 96 casos confirmó haber tenido contacto previo con una persona diagnosticada de sarcoidosis, frente al 1-2% de los controles. de estos contactos, 14 ocurrieron en la misma casa, aunque sólo 9 eran familiares consanguíneos. otros 19 habían tenido contacto en el lugar de trabajo, 2 con vecinos y 14 con amigos no convivientes. desde una perspectiva de enfermedad infecciosa, esta asociación espacial y temporal puede indicar que la sarcoidosis es una enfermedad transmisible, pero también podría explicarse por el hecho de que estos casos tienen en común una exposición ambiental o laboral que induce una misma respuesta de hipersensibilidad. la sarcoidosis es una enfermedad benigna. un porcentaje importante de pacientes afectados pueden no tener nunca manifestaciones clínicas y más de un 30% tienen remisión espontánea. el curso crónico ocurre en un 10-30% de los casos, dando lugar a veces a un significativo deterioro de la función pulmonar. se han publicado tasas de mortalidad del 1 al 6% 32 ; la presencia de fibrosis en la radiografía de tórax y una capacidad vital forzada inferior a 1,5 l son predictores de muerte por insuficiencia respiratoria debida a sarcoidosis 33 . un estudio reciente encuentra hipertensión pulmonar en el 40% de los afectados de sarcoidosis sin estadio iv radiográfico, es decir, sin fibrosis. cuando la fibrosis y la hipertensión pulmonar coexisten, se produce un acusado descenso de los parámetros funcionales (capacidad de difusión del monóxido de carbono entre el 30 y el 35%; flujo mesoespiratorio forzado ≤ 30%; volumen espiratorio forzado en el primer segundo < 1,2 l), lo que debería alertar al clínico de esta grave complicación 34 . respecto a la supervivencia, la sarcoidosis tiene mejor pronóstico a los 5 años (91,6%) que otras enfermedades pulmonares intersticiales difusas como la neumonía intersticial no específica o la neumonía intersticial descamativa (85,5%), la neumonitis por hipersensibilidad (84,1%), la enfermedad pulmonar intersticial difusa por colagenopatías (69,7%), formas no definidas de fibrosis pulmonar (69,5%) y la fibrosis pulmonar idiopática (35,4%) 33 . sólo un estudio de casos y controles señala un posible riesgo incrementado para desarrollar neoplasias del tipo linfomas, cáncer de pulmón o cáncer en otros órganos afectados por la enfermedad 35 , pero estos hallazgos no se han confirmado en estudios con seguimiento a largo plazo 36 , que además llegan a la conclusión de que ni la edad del paciente en el momento del diagnóstico ni las manifestaciones clínicas son indicadores de posterior desarrollo de neoplasias. de lo que no hay duda es que el pronóstico de la sarcoidosis está claramente ligado a la gravedad de la enfermedad. está claramente establecido que existe diferente susceptibilidad individual para desarrollar la sarcoidosis. fernández fabrellas e. epidemiología de la sarcoidosis los factores genéticos se asocian con patrones concretos de la enfermedad (fenotipo clínico), con el riesgo de enfermar y con la gravedad y progresión de la sarcoidosis [37] [38] [39] [40] . como consecuencia, incluso si se pudiera identificar un factor ambiental específico, es probable que el riesgo de enfermar se derivara de su interacción con los factores genéticos del huésped y con sus hábitos sociosanitarios 41, 42 . hay buenas razones que sustentan la hipótesis de que la sarcoidosis está causada por antígenos ambientales en individuos genéticamente predispuestos. tanto la piel como los pulmones -órganos más comúnmente afectados-están siempre en contacto con estos antígenos; los estudios sobre la inmunopatogenia de la sarcoidosis apoyan que la enfermedad es el resultado de una superrespuesta inmunitaria y que hay un gran número de potenciales antígenos ambientales que pueden inducir la sensibilización y la consiguiente respuesta mediada por células responsable del desarrollo de granulomas 43 . estos factores ambientales causan multitud de enfermedades que simulan la sarcoidosis (tabla i), como la inhalación de berilio u otros metales (aluminio, titanio, circonio), neumonitis por hipersensibilidad e infecciones como la tuberculosis, micobacterias atípicas y hongos, entre otros. las fibras y polvos inorgánicos (talco, sílice, fibra de vidrio) también son capaces de respuestas inmunológicas similares a las de la sarcoidosis. la lista de agentes inductores de una respuesta granulomatosa en animales es incluso más larga e incluye micobacterias, proteínas aviares, esporas fúngicas, amebiasis, huevos de schistosoma, brucella y leishmania, entre otros 6 . de este modo, hoy día se cree que la sarcoidosis aparece como consecuencia de la exposición a uno o más agentes ambientales que interaccionan con factores genéticos individuales. el reto está en identificar esos agentes ambientales y relacionarlos con la susceptibilidad genética. algunas de las primeras investigaciones epidemiológicas sobre la sarcoidosis planteaban la posibilidad de una exposición común a antígenos inductores de respuesta inmunitaria granulomatosa en el lugar de trabajo, pero hasta hace poco tiempo han sido muy pocos los estudios que han investigado prospectiva y sistemáticamente la exposición laboral o ambiental de los pacientes 44 . publicaciones recientes derivadas del estudio access 45 , más un estudio realizado en carolina del sur 46 y otro que examina los factores de riesgo laborales en familias afroamericanas 47 , han ayudado a afianzar la importancia de este tipo de factores de riesgo. en el estudio de barnard et al 45 , basado en los datos del access, se utilizan los códigos de sic (clasificación industrial estándar) y soc (clasificación ocupacional estándar) para definir la ocupación de los pacientes e investigar la contribución del factor laboral al riesgo de sarcoidosis. el análisis univariado de sus resultados identifica un mayor riesgo entre trabajadores con exposición industrial a polvos orgánicos, especialmente en caucásicos, y entre los trabajadores de industrias de materiales de construcción, ferretería y jardinería. los empleos relacionados con el cuidado de niños se asociaron negativamente con sarcoidosis, así como también la exposición laboral a humos o polvo de metales, de nuevo más evidente entre trabajadores caucásicos. el estudio realizado por kajdasz et al 46 establece otras asociaciones entre los pacientes de raza negra hospitalizados por esta enfermedad: uso de estufas de leña, de chimeneas, consumo de agua no pública (pozos) y vivir o trabajar en una granja. este estudio destaca menos la ocupación agrícola y da mayor importancia al diferente uso de la madera en los municipios rurales de carolina del sur. utilizando un cuestionario derivado del access, kucera et al 47 encontraron asociaciones positivas con ciertas ocupaciones de afroamericanos afectados de sarcoidosis comparados con hermanos sanos. observaron que los que trabajaban con potencial exposición a metales o en lugares con alta humedad ambiental o con olor mohoso (lo que indicaba un ambiente rico en agentes microbianos) podían tener un riesgo incrementado de presentar sarcoidosis. estos autores llaman la atención sobre la complejidad de las posibles exposiciones laborales, que hacen difícil identificar agentes concretos basándose sólo en la filiación del puesto de trabajo, una crítica que es aplicable a la mayoría de las investigaciones sobre exposición de riesgo laboral para sarcoidosis realizadas hasta la fecha. otra vez es el access el estudio más consistente que ha investigado la asociación de la exposición ambiental y ocupacional con la sarcoidosis. los autores elaboraron unos cuestionarios con preguntas específicas sobre posibles exposiciones laborales y no laborales y su duración. los resultados, en parte ya comentados en otros apartados de esta revisión, muestran una asociación positiva entre sarcoidosis y determinadas ocupaciones, como las relacionadas con la agricultura, con el contacto con aves, manufacturas del automóvil, profesorado de secundaria y personal sanitario. la cuidadosa revisión de los individuos con exposición a aves demostró que no eran casos típicos de neumonitis por hipersensibilidad. más asociaciones positivas con sarcoidosis se encontraron también con el uso de insecticidas y con trabajos desempeñados en ambientes con exposición a hongos o mohos, por lo que los autores conjeturan con la posibilidad de inhalación de bioaerosoles microbianos. el modelo estadístico multivariado del access establece una odds ratio (or) elevada para áreas con olores mohosos y exposición a insecticidas, y un efecto protector para los fumadores o ex fumadores, aunque esto puede ser un sesgo metodológico, puesto que muchos de los pacientes dejan de fumar al inicio de los síntomas. en cualquier caso, el access no encuentra un único factor de riesgo predominante para sarcoidosis; por otro lado, aunque las or son altas para un determinado número de factores, en general esas asociaciones fueron débiles 29 . durante el último siglo estuvo candente la hipótesis de que patógenos microbianos eran la causa de la sarcoidosis. concretamente, las principales sospechosas fueron las micobacterias, incluso se llegó a documentar su crecimiento en muestras sanguíneas de casos frente a controles, afirmación que no se ha confirmado recientemente 48 . varios estudios han identificado adn de micobacterias por reacción en cadena de la polimerasa (pcr) 49,50 hasta en la mitad de pacientes frente a controles, y adn de micobacterias no tuberculosas en más del 20% de ellos, lo que indicaría que mycobacterium tuberculosis complex podría tener un papel en la etiología de esta enfermedad. sin embargo, no ha sido posible aislar el germen ni cultivarlo a partir de tejidos de pacientes, lo que es fundamental para poder adscribir la etiología del proceso siguiendo los postulados de henle-koch (aislamiento del patógeno en el paciente, crecimiento en cultivo puro y reproducibilidad de la enfermedad cuando se inocula en un huésped susceptible 6 ). además, el seguimiento durante más de 10 años de pacientes con sarcoidosis y pcr positiva para m. tuberculosis no ha detectado el desarrollo de enfermedad tuberculosa en ninguno de ellos 51 . el antígeno de kveim, que es un extracto proteico obtenido de ganglios linfáticos o bazo de pacientes, provoca una respuesta oligoclonal de células t en pacientes con sarcoidosis, además de producir una infiltración granulomatosa en la piel. aunque el agente activo del antígeno de kveim no se ha identificado, se sabe que este antígeno no contiene adn bacteriano. un estudio reciente ha documentado la presencia de antígenos de micobacterias en tejidos sarcoideos, así como sus anticuerpos en algunos pacientes, lo que una vez más incide en el papel de las micobacterias en la etiología de esta enfermedad 52 . las investigaciones en este sentido continúan porque, aunque no se ha identificado ningún agente infeccioso en cultivos de biopsias de pacientes con sarcoidosis y ni siquiera se han detectado consistentemente con marcadores de arn ribosómico, determinados hechos clínicos y epidemiológicos apuntan a una etiología infecciosa para esta enfermedad. por ejemplo, hay evidencia de la transmisibilidad de la sarcoidosis; en efecto, se ha documentado la denominada "sarcoidosis adquirida del donante", en la cual la enfermedad se desarrolla en el receptor del trasplante de tejidos u órganos provenientes de donantes con sarcoidosis diagnosticada o probable 53 . y a la inversa, la sarcoidosis se ha desarrollado en el pulmón trasplantado en pacientes sarcoidóticos 54 . los animales a los que se ha implantado tejido afectado de pacientes han desarrollado granulomas de tipo sarcoideo 55 . cuando se inoculó este tejido humano en ratones, los granulomas tardaron 15 meses en desarrollarse, pero este efecto no se conseguía si se sometía previamente la muestra de tejido a autoclave, congelación a -20 °c o a radiación. por otra parte, el examen por microscopia electrónica y con técnicas inmunohistoquímicas del granuloma sarcoideo ha identificado estructuras que se asemejan a organismos como leptospira, mycoplasma y propionibacterium 6 , de manera que aún es necesario investigar más sobre la naturaleza de los elementos ultraestructurales que forman el granuloma sarcoideo hasta llegar a algún resultado concluyente. los hallazgos epidemiológicos del access apuntan claramente a que el riesgo de sarcoidosis está ligado a condiciones medioambientales propicias para la formación de bioaerosoles, tanto antigénicos como infecciosos 29 . como ya se ha comentado, las ocupaciones laborales directamente relacionadas con ambientes húmedos y enrarecidos con olor mohoso se asociaron con el riesgo de sarcoidosis en el modelo multivariado del estudio. la mayoría de los hongos exudan, durante su crecimiento, compuestos orgánicos volátiles que causan ese típico olor que se asocia con la contaminación fúngica, y que podría reflejar la presencia del microorganismo incluso cuando no es visible su crecimiento. además, en el access se observó que los casos de sarcoidosis se dieron entre pacientes que utilizaban acondicionadores de aire en su domicilio, con o sin humidificadores. muchos de los microorganismos que se han señalado como agentes etiológicos de la enfermedad, o que producen cuadros clínicos similares a ella, crecen rápidamente en el agua. las condiciones oportunas para aerosolizar partículas antigénicas o agentes infecciosos pueden llevar a la inhalación de estas partículas, su consiguiente depósito pulmonar y el desarrollo de la respuesta inmunitaria característica. otros agentes microbiológicos implicados en la etiopatogenia de la sarcoidosis han sido herpesvirus, retrovirus, chlamydia pneumoniae, borrelia burgdorferi, rickettsia helvetica y últimamente pneumocystis jiroveci 56 , entre otros. sin embargo, ninguno de estos patógenos puede considerarse agente etiológico de la enfermedad, puesto que, igual que las micobacterias, no cumplen los postulados de henle-koch. a pesar de los muchos esfuerzos realizados para encontrar un posible agente microbiológico implicado en la etiología de la sarcoidosis, hasta la fecha no disponemos de suficiente evidencia científica que sustente esta hipótesis, pero tampoco que la descarte. se ha planteado que los microorganismos probablemente actúan como desencadenantes antigénicos, pero no causantes de infección, en una persona predispuesta genéticamente, y que éste sería el inicio de la respuesta granulomatosa de la sarcoidosis 57 . los análisis con técnicas de pcr pueden ayudar a detectar agentes infecciosos en los tejidos de pacientes, aun cuando fallen los cultivos. estas técnicas de pcr han conseguido identificar los agentes etiológicos de otras enfermedades como la angiomatosis bacilar (bartonella henselae), la enfermedad de whipple (tropheryma whippelii) o el síndrome agudo respiratorio grave (nuevo coronavirus). son muchos los estudios que se han ocupado de investigar la agrupación familiar de la sarcoidosis entre parejas de padre e hijo del mismo sexo, parejas de madre e hijo, hermanos del mismo sexo y gemelos monocigóticos 6, 19, 58 . los primeros resultados señalaban que esta agregación familiar era más frecuente entre personas de raza negra que entre caucásicos 59 . las cifras de prevalencia de agregación familiar de la sarcoidosis van desde el 1,7% del reino unido, el 4,3% de japón, el 4,7% de finlandia y el 9,6% de irlanda hasta el 17% de familias afroamericanas de ee.uu. 60 . el access investigó la agregación familiar de la sarcoidosis utilizando datos de 10.862 familiares de primer grado y 17.047 de segundo grado de 706 pares de casos y controles pareados por edad, sexo, raza y ubicación geográfica. las conclusiones fueron que hay un riesgo elevado de presentar sarcoidosis entre familiares de primer y segundo grados de los pacientes comparados con los familiares de primer y segundo grados de los controles. los hermanos tenían el riesgo relativo más elevado (or = 5,8; intervalo de confianza [ic] del 95%, 2,115, 9) , seguidos de los tíos (or = 5,7; ic del 95%, 1,6-20,7), abuelos (or = 5,2; ic del 95%, 1,5-18) y padres (or = 3,8; ic del 95%, 1,2-11,3). utilizando un modelo multivariado con los datos de padres e hijos, el riesgo relativo familiar ajustado por edad, sexo, clase social y factores ambientales comunes fue de 4,7 (ic del 95%, 2,3-9,7), pero los pacientes caucásicos tuvieron un riesgo relativo familiar más elevado que los casos de afroamericanos (18,0 frente a 2,8; p = 0,098) 19 . en otro estudio realizado entre 179 familias afroamericanas, estos mismos investigadores llegan a la conclusión de que los hermanos y padres de estos pacientes tienen un riesgo 2,5 veces mayor de desarrollar la enfermedad 61 . el estudio de reino unido 58 , basado en un cuestionario contestado por 268 pacientes con sarcoidosis, reveló que el 5,91% tenía al menos un familiar de primer, segundo o tercer grados con sarcoidosis demostrada histológicamente. los autores calculan que la ratio de prevalencia de sarcoidosis de hermanos de pacientes respecto a la prevalencia para el resto de la población es de 38-73 (ic del 95%, 21-145), sin encontrar diferencias significativas por etnias, al contrario que el estudio norteamericano 19 . uno de los objetivos investigados con más interés en los últimos años es el factor genético que confiere susceptibilidad para la sarcoidosis. los primeros resultados se consiguieron a través del análisis de los genes del complejo principal de histocompatibilidad (mhc), especialmente de los antígenos de histocompatibilidad (hla). sobre la base de los estudios inmunofenotípicos de los linfocitos t recogidos de muestras de lavado broncoalveolar, se puede afirmar que en la fisiopatología de la sarcoidosis muy probablemente esté afectado el reconocimiento, procesamiento y presentación del antígeno por los macrófagos a las células t 62 . las primeras investigaciones genéticas, que utilizaron técnicas serológicas, evaluaron las posibles asociaciones con genes del mhc localizado en el cromosoma 6p, concretamente con hla de clase i. aunque no se ha encontrado una asociación concluyente, los alelos más frecuentemente vinculados con el riesgo de sarcoidosis han sido hla-b8 y hla-b7. además, se han encontrado ciertas asociaciones hla en pacientes con sarcoidosis pertenecientes a diversos grupos étnicos 59, 63 . las investigaciones más recientes han utilizado técnicas de biología molecular para determinar asociaciones con el mhc de clase ii, concretamente con hla-dr, que parece tener más influencia en la susceptibilidad y pronóstico de la enfermedad que el de clase i. en los últimos años, se han implicado muchos de estos alelos de clase ii en determinados aspectos de la enfermedad: hla-dr5, hla-dr6, hla-dr8 y hla-dr9 parecen conferir riesgo de enfermar entre los japoneses, aunque hla-dr9 confiere protección entre la población escandinava; hla-dr5 se asocia con enfermedad crónica en los pacientes alemanes y hla-dr3 con las formas agudas; de manera similar a éstos, los escandinavos asocian hla-dr14 y hla-dr 15 con las formas crónicas, y hla-dr17 con las autolimitadas 64 . en el estudio ac-cess se identifica una asociación significativa entre alelos hla-drb1 (concretamente hla-drb1*1101) y el desarrollo de la enfermedad, tanto en población negra como caucásica 65 . el único alelo de clase ii con diferente distribución entre estas razas respecto a la enfermedad fue el hla-drb1*1501, que se asoció con controles en los negros y con casos en los blancos. esto indicaría que, en general, alelos similares de hla clase ii pueden asociarse con la sarcoidosis en ambas poblaciones. en este mismo sentido, otras investigaciones señalan alelos específicos del hla-dqb1 como determinantes de susceptibilidad para sarcoidosis entre la población afroamericana 38, 40 ; queda por demostrar cómo estos alelos interaccionan con factores ambientales y con otros genes para determinar el fenotipo de la enfermedad 66 . hay estudios en marcha 67,68 que ayudarán a confirmar si los genes que confieren la susceptibilidad para la sarcoidosis están en esa compleja región del cromosoma 6p. actualmente disponemos de evidencia convincente de que la sarcoidosis es el resultado de desencadenantes ambientales que, al actuar sobre individuos genéticamente susceptibles, ocasionan una superrespuesta inmunitaria con formación de granulomas en los órganos afectos. aunque la enfermedad se ha descrito en casi toda la población mundial, son muchas las variaciones en cuanto a incidencia y prevalencia entre los diferentes fenotipos clínicos. las investigaciones sobre agregración familiar y los estudios de casos y controles sustentan la hipótesis de que la predisposición inmunogenética determina el distinto patrón de afectación orgánica de la sarcoidosis. en este momento, el consenso científico es contundente al señalar la región del cromosoma 6p donde se localiza el mhc de clase ii como el emplazamiento de las asociaciones genéticas más importantes. definiciones más rigurosas de los distintos fenotipos clínicos y los estudios en marcha con grandes cohortes de pacientes, que incluyen la sarcoidosis familiar, combinados con los nuevos avances tecnológicos, ayudarán sin duda a una mejor comprensión de la susceptibilidad genética de la sarcoidosis y sus fenotipos. association of hla class ii alleles with sarcoidosis: results by race and sex of the access study is there a role for microorganisms in the pathogenesis 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interstitial lung diseases: characteristics at diagnosis and mortality risk assessment distinctive clinical, radiographic and functional characteristics of patients with sarcoidosis-related pulmonary hypertension increased risk for cancer following sarcoidosis sarcoidosis and cancer revisited: a long-term follow-up study of 555 danish sarcoidosis patients analysis of mhc encoded antigen-processing genes tap1 and tap2 polymorphisms in sarcoidosis sarcoidosis susceptibility and resistance hla-dqb1 alleles in african americans results from a genome-wide search for predisposing genes in sarcoidosis the major histocompatibility complex gene region and sarcoidosis susceptibility in african americans sarcoidosis: social predictors of severity at presentation health-related disparities: influence of environmental factors immunology and pathophysiology of sarcoidosis trends and occupational associations in incidence of hospitalized pulmonary sarcoidosis and other lung diseases in navy personnel. a 27-year historical prospective study job and industry classifications associated with sarcoidosis in a case-control etiologic study of sarcoidosis (ac-cess) geographic variation in sarcoidosis in south carolina: its relation to socioeconomic status and health care indicators occupational risk factors for sarcoidosis in african-american siblings recovery of cell wall-deficient organisms from blood does not distinguish between patients with sarcoidosis and control subjects detection of mycobacterial dna in sarcoidosis and tuberculosis with polymerase chain reaction a search for mycobacterial dna in granulomatous tissues from patients with sarcoidosis using the polymerase chain reaction high prevalence of mycobacterium tuberculosis dna in biopsies from sarcoidosis patients from catalonia mycobacterial catalase-peroxidase is a tissue antigen and target of the adaptive immune response in systemic sarcoidosis donor-acquired sarcoidosis sarcoidosis recurrence following lung transplantation experimental inoculation of laboratory animals with samples collected from sarcoidal patients and molecular diagnosis evaluation of the results pneumocystis jiroveci colonisation in patients with interstitial lung disease characterization of mycobacterium tuberculosis complex isolates from greek patients with sarcoidosis by spoligotyping epidemiology of familial sarcoidosis in the uk familial sarcoidosis: analysis of 61 families epidemiology of sarcoidosis familial risk ratio of sarcoidosis in african-american sibs and parents lavado broncoalveolar en la enfermedad pulmonar intersticial. últimas noticias antígenos leucocitarios humanos a y b en pacientes turcos con sarcoidosis genetics of sarcoidosis hla-drb1*1101: a significant risk factor for sarcoidosis in blacks and whites the btnl2 gene and sarcoidosis susceptibility in african americans and whites a sarcoidosis genetic linkage consortium: the sarcoidosis genetic analysis (saga) study. sarcoidosis vasc diffuse lung dis genome-wide search for sarcoidosis susceptibility genes in african americans key: cord-275608-joyan7ij authors: sewell, andrew k. title: why must t cells be cross-reactive? date: 2012-08-24 journal: nat rev immunol doi: 10.1038/nri3279 sha: doc_id: 275608 cord_uid: joyan7ij clonal selection theory proposed that individual t cells are specific for a single peptide–mhc antigen. however, the repertoire of αβ t cell receptors (tcrs) is dwarfed by the vast array of potential foreign peptide–mhc complexes, and a comprehensive system requires each t cell to recognize numerous peptides and thus be cross-reactive. this compromise on specificity has profound implications because the chance of any natural peptide–mhc ligand being an optimal fit for its cognate tcr is small, as there will almost always be more-potent agonists. furthermore, any tcr raised against a specific peptide–mhc complex in vivo can only be the best available solution from the naive t cell pool and is unlikely to be the best possible solution from the substantially greater number of tcrs that could theoretically be produced. this 'systems view' of tcr recognition provides a plausible cause for autoimmune disease and substantial scope for multiple therapeutic interventions. supplementary information: the online version of this article (doi:10.1038/nri3279) contains supplementary material, which is available to authorized users. jonathan kipnis's homepage: http://www.medicine.virginia. edu/basic-science/departments/neurosci/faculty/kipnis t cells recognize peptides bound to mhc class i and class ii molecules at the cell surface 1 . the specificity of this recognition is conferred by the clonotypic αβ t cell receptor (tcr), which is made from two separate chains manufactured from variable (v), diversity (d), joining (j) and constant (c) gene fragments through a process of somatic gene rearrangement. this process involves nucleotide insertions and deletions at v(d)j junctions in each chain. the 'randomization' of v(d)j junctions and the fact that the tcr is a heterodimer of two separately rearranged chains results in a theoretical repertoire of >10 15 unique αβ tcrs in the mouse 2,3 . the theoretical number of possible tcrs in humans is likely to be orders of magnitude larger, as humans possess 54 tcrβ variable genes as compared with the 35 genes in mice, with all other variables being comparable 4 . why must t cells be cross-reactive? abstract | clonal selection theory proposed that individual t cells are specific for a single peptide-mhc antigen. however, the repertoire of αβ t cell receptors (tcrs) is dwarfed by the vast array of potential foreign peptide-mhc complexes, and a comprehensive system requires each t cell to recognize numerous peptides and thus be cross-reactive. this compromise on specificity has profound implications because the chance of any natural peptide-mhc ligand being an optimal fit for its cognate tcr is small, as there will almost always be more-potent agonists. furthermore, any tcr raised against a specific peptide-mhc complex in vivo can only be the best available solution from the naive t cell pool and is unlikely to be the best possible solution from the substantially greater number of tcrs that could theoretically be produced. this 'systems view' of tcr recognition provides a plausible cause for autoimmune disease and substantial scope for multiple therapeutic interventions. the diversity of tcrs is based on the six complementarity-determining regions (cdrs), which engage both the peptide and the mhc molecule 5 (fig. 1) . typically, mhc class i and class ii molecules present peptides from endogenous and exogenous antigens, respectively. the mhc class i molecule has a closed-ended peptide-binding groove and binds peptides of 8-14 amino acids in length. longer peptides become increasingly distorted in the central region of the mhc class i molecule as the peptide length increases, resulting in peptide 'bulging' 6, 7 . by contrast, the ends of the mhc class ii peptide-binding cleft are open, allowing even longer peptides to extend beyond this groove without bulging (fig. 1b,c) . the clonal selection theory 8, 9 proposed that individual lymphocytes are specific for a single antigen and that the recognition of alternative ligands is unlikely. for many years the concept of huge numbers of tcrs successfully providing immunity to all foreign peptides in a 'one-clonotype-one-specificity' paradigm was accepted. however, several workers questioned this concept [10] [11] [12] [13] . most notably, don mason called for the abandonment of such a notion in his seminal thesis on the topic (see ref. 10 ). many of the reasons for this paradigm shift were based on the simple arithmetic of effective immunity requiring the recognition of >10 15 potential foreign peptides. indeed, put in the context of 10 15 t cells weighing >500 kilograms, the notion of immune coverage by a naive pool of 10 15 monospecific tcrs as suggested by the clonal selection theory is clearly absurd 10 . there are only 10 12 t cells in a human, and more recent studies have estimated that there are <10 8 distinct tcrs in the human naive t cell pool 14 . in humans, mhc molecules are encoded within the hla locus. the hla locus is the most polymorphic region of the human genome and is known to encode more than 7,000 allelic variants across the population, with a large number of these variants present at appreciable frequencies 15 . some hla loci are among the fastest evolving coding regions in the human genome 16 . each individual expresses six different classical peptide-presenting hla class i molecules (two hla-a, two hla-b and two hla-c) and six hla class ii molecules (two hla-dr, two hla-dq and two hla-dp). the expression of a wide variety of hla molecules ensures that individuals across the population present different antigenic peptides and provides the greatest chance that some individuals may survive any emerging infection. it is extremely difficult to link hla diversity to past pandemics, but evidence of the importance of infectious diseases in driving hla selection can be seen with current emerging infectious diseases. for example, homozygosity at hla class i alleles results in faster disease progression during hiv infection 17 , and some hla class i alleles are associated with lower viral loads and protection from disease 18 . various factors in addition to t cell immunity are thought to contribute to the maintenance of hla diversity, including natural killer cell recognition 19 , mate selection 20,21 and transmissible tumours 22 . overall, the fact that mutations that alter the amino the closed ends of the mhc class i binding groove cause long peptides to 'bulge' out of the binding groove, and this bulging increases with each additional amino acid in the peptide. by contrast, the ends of the mhc class ii binding cleft are open, which allows the accommodation of much longer peptides without the need for peptide kinking. d,e | the images show hla-a*0201 (in grey) presenting the immunodominant glctlvaml peptide (stick model) from epstein-barr virus and hla-dr4 (in grey) presenting a peptide from myelin basic protein (mbp). tcrs dock on a peptide-mhc complex in a diagonal mode that is conserved for binding to mhc class i and class ii molecules. the colours indicate the docking footprints of the as01 tcr 96 and msc-2c8 tcr 97 on their cognate peptide-mhc complexes and show the 'footprints' on the mhc complex of the six cdr loops. in general, the germline-encoded cdr1 and cdr2 loops interact mainly with the mhc molecule itself, whereas the hypervariable cdr3 loops sit over the peptide. however, the small structural database that has been compiled to date already contains examples in which cdr1 and cdr2 make substantial interactions with the peptide and in which cdr3 has an important role in contacting the mhc molecule 98,99 . tcr binding degeneracy and structure the recognition by tcrs of all hla molecules and a roughly conserved diagonal mode of binding on peptide-mhc complexes suggest that tcr interactions conform to some 'rules of engagement' (fig. 1) . such rules have been proffered in the form of a tcr 'interaction codon' 32 that interacts with mhc class ii molecules, and in the form of a 'restriction triad' 7 that consists of three largely conserved residues in mhc class i molecules that interact with tcrs. these rules fit the generally observed arrangement of tcr-peptide-mhc interactions, in which the germline-encoded (that is, non-rearranged) cdr1α, cdr1β, cdr2α and cdr2β elements of the tcr contact the germline element of the mhc molecule, whereas the non-germline (that is, somatically rearranged) cdr3α and cdr3β loops contact the 'random' peptide element (fig. 1) . however, these convenient rules fail to match all the structures of tcr-peptide-mhc complexes that have been generated to date 5 , and mhc mutational studies show that the dependency on fixed pairwise interactions between a tcr and a peptide-mhc complex varies widely between individual tcrs 33 . the peptide-mhc complex itself can also change its confirmation following tcr binding [34] [35] [36] . thus, it is clear that tcr-peptide-mhc interactions are not rigidly conserved but rather allow for considerable flexibility within the confines of some general orientation and binding rules. the tumour-specific dmf4 tcr provides an excellent example of how large changes in tcr orientation can increase t cell cross-reactivity. the dmf4 tcr engages the nine-amino-acid (9-mer) peptide aagigiltv and the 10-mer peptide elagigiltv (which have overlapping sequences) in the context of hla-a*0201 by adopting a different orientation for the two peptide-mhc complexes 37 . tcr-binding plasticity can extend beyond different peptide binding registers or different peptide binding angles on peptide-mhc complexes because the cdr loops can be extremely flexible 38, 39 . the mouse 2c tcr structure has been solved in complex with eqykfysv-h2-k b (ref. 40 ), eqykfysv-h2-k bm3 (ref. 41 ), siyryygl-h2-k b (ref. 42) and box 1 | extensive t cell cross-reactivity and apparent specificity are not incongruous from the 20 proteinogenic amino acids, it is possible to generate vast numbers of peptides of a length that can be presented by mhc molecules (see the table). t cells are specific because any given t cell can recognize only a tiny fraction of the 'universe' of peptides that can be presented by any given mhc molecule, but they are multispecific because the peptide universe is so large. by way of example, a t cell that recognizes 1 million 10-mer (10-amino-acid) peptides will have less than a 1 in 10 million chance of recognizing any 10-mer peptide chosen at random from the entire peptide universe. these numbers indicate that if a t cell that recognizes 1 million different 10-mer peptides was tested for recognition of random 10-mer peptides at a rate of 1 every minute then on average it would take over 20 years before a cross-reaction was seen! even the total number of overlapping peptides that can be made from the entire human proteome is an extremely small fraction of all possible peptides (for example, fewer than 10 7 of the total possible number of 10-mer peptides (>10 13 ) can be made from the human proteome). in the environment in which t cells function, the important number is the frequency of functional recognition of unrelated peptides that can be processed and presented by mhc molecules. assuming that just 1% of possible peptides are presented by an mhc molecule, then the functional recognition of 10 6 10-mer peptides by a single tcr translates into a frequency of cross-reactivity of 1 in 100,000, which is in good accord with an experimental attempt to directly measure this parameter 95 . thus, the sheer size of the possible peptide universe allows t cells to be enormously cross-reactive while appearing to be very specific within the environment in which they are required to operate. acid sequence of hla class i and class ii molecules are clustered around the peptidebinding cleft and often alter the peptide sequence that is preferentially bound by the hla molecule [23] [24] [25] strongly suggests that hla diversity is upheld to increase the variety of peptides displayed. the tcr recognizes peptide antigens presented by all hla variants. unlike the b cell receptor, the protein sequence of the tcr is fixed, and the tcr never undergoes affinity maturation. thus, tcrs expressed by naive t cells are required to respond to all foreign antigens despite never having encountered them before and being unable to adapt to them at the protein sequence level. if the tcr repertoire was unable to recognize virtually all foreign peptides bound to self mhc molecules, then pathogens -which usually evolve many millions of times faster than their vertebrate hosts -would be expected to rapidly evolve to exploit these t cell 'blind spots' and overwhelm the host. it is difficult to conceive of any obvious universal mechanism that might transmit knowledge of 'presentable' epitopes from previous infections between generations within the tcr cdr loops 10 . in the absence of 'prior knowledge' of the epitopes that might be encountered, t cell immunity must provide immune cover for all possible foreign peptides that contain appropriate anchors for binding to self mhc molecules 10 . this universal cover represents a major challenge to the immune system, as the possible array of peptides that can be manufactured from the 20 proteinogenic amino acids of a length that can bind to self mhc molecules is vast (>10 15 ) (box 1). in fact, the theoretical number of possible peptides that t cells might provide immunity to is even greater, as it is possible to raise specific t cell responses to peptides that contain amino acids with post-translational modifications, such as glycosylation 26 , citrullination 27 , phosphorylation 28, 29 , cysteinylation and dimerization 30, 31 . thus, the number of potential foreign peptide-mhc complexes that t cells might encounter dwarfs the number of tcrs available. here, i consider how the challenge of this disparity has been met by compromising on antigen specificity so that individual t cells are capable of responding to enormous numbers of different peptide-mhc complexes. this inevitable, extensive t cell cross-reactivity has some profound consequences, including providing a plausible cause for autoimmune disease. i also discuss how the consequences of tcr binding degeneracy offer substantial scope for multiple therapeutic interventions. the recently described 1e6 tcr -which was isolated from a patient with type 1 diabetes and which recognizes residues 15-24 of the preproinsulin molecule (ppi [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] presented in the context of hla-a*0201 (ref. 45) -does not undergo structural rearrangements following ligand binding 46 but is still hugely cross-reactive. despite a rigid 'lock and key' binding mode, t cells expressing the 1e6 tcr respond to over 1.3 million 10-mer peptides at least as strongly as they respond to the ppi 15-24 peptide 46, 47 . peptides were identified that were >100-fold more potent than ppi 15-24 at activating 1e6 tcr-expressing t cells but that differed from ppi 15-24 at seven of the ten amino acid positions 47 . this promiscuity is explained by the structure of the 1e6 tcr-ppi 15-24 -hla-a2 complex, in which the tcr exhibits peptide-centric binding that is focused on just two amino acids in the peptide 46 . this residue-focused mode of binding presumably allows for substitutions at other positions that, in some cases, must considerably stabilize the interaction. in another example of such peptide-centric binding, a single amino acid interchange within two hiv envelope epitopes was shown to reciprocally swap the specificities of two cd8 + t cell clones 48 , suggesting that a dominant focus on a single amino acid residue in the peptide within a peptide-mhc complex might be reasonably common. indeed, the tcr-peptide-mhc structures that have been described to date show that usually only a few upward-facing residues from the peptide contribute to the inter action of the tcr with the peptide-mhc complex. thus, data from the limited number of tcr structures available indicate that tcrs can exhibit substantial binding degeneracy by being extremely flexible and/or through a focused interaction that is dominated by a few peptide residues (fig. 2) . together, this binding promiscuity at the tcr interface and the flexible mhcbinding 'motifs' 49 that often allow the accommodation of several amino acids at primary mhc anchor positions enable a substantial number of peptides to act as agonists for any given tcr. it is possible to generate vast numbers of peptides of the length recognized by t cells from the 20 proteinogenic amino acids . even conservative estimates predict that substantially more than 1% of these peptides will possess anchors that allow them to bind to any single mhc molecule. taking 10-mer peptides as an example, it is possible to generate >10 13 different peptides of a | macro-level changes enable the t cell receptor (tcr) to bind to peptide-mhc complexes with an altered peptide binding angle (red dotted line) and/or peptide binding register (black dotted line) within a roughly diagonal binding mode 38 . the cartoon shows 'footprints' of the tcr complementaritydetermining region (cdr) loops projected down onto the peptide-mhc platform. b | micro-level cdr loop flexibility enables the accommodation of different peptide-mhc 'landscapes'. the cartoon shows a side view of a tcr engaging a peptide-mhc complex. c | structural studies show that most tcrs focus on two to four upward-facing peptide residues. in this example, the tcr is focused on the two peptide residues shown in red. such residue-focused interaction allows the tcr to tolerate multiple amino acid substitutions at other positions in the peptide (indicated by different colours). the above examples are not mutually exclusive and represent only some of the possibilities. mhc-binding motifs often allow for different residues at primary mhc anchors 49 . it should also be noted that tcrs can change the conformation of the peptide-mhc complex following engagement 34-36 . 10 amino acids in length from the 20 amino acids. assuming that at least 1% (>10 11 ) of these peptides can bind to a given self mhc molecule, a heterozygous human antigenpresenting cell could theoretically present more than 12 × 10 11 different 10-mer peptides on its six mhc class i molecules and six mhc class ii molecules. furthermore, as mhc class ii molecules can present longer peptides that can 'frame-shift' within the open-ended binding groove (fig. 1) , mason calculated that each mhc class ii molecule could theoretically present almost 10 17 different 14-mer peptides, assuming that 3% of all peptides associate with mhc class ii molecules 10 , and this is without even considering the possibility of post-translational modifications. in summary, the number of potential peptide antigens exceeds the number of tcrs available to respond to them by many orders of magnitude, so t cells can only provide comprehensive immune cover if each one is capable of recognizing many peptides. the theoretical arguments of mason suggesting that t cells must each recognize on average at least 1 million individual peptides 10 have recently gained traction as a result of data that demonstrate this level of cross-reactivity and provide plausible structural mechanisms for its occurrence. all t cells are 'auditioned' in the thymus and only those that react weakly with a self peptide-mhc ligand are positively selected 50 . t cells bearing tcrs that react strongly to self antigens are 'culled' at this stage. extensive tcr binding degeneracy and cross-recognition of peptide-mhc molecules by thymocytes has been elegantly demonstrated by studies showing that a remarkably comprehensive t cell repertoire can be selected by a single peptide 51 and that the resulting t cells can be activated by peptides that are unrelated in sequence to the peptide that they were selected on 52 . further compelling evidence that t cells can exhibit extensive cross-reactivity comes from studies with combinatorial peptide libraries that comprise almost all possible peptides of a particular length 11, 47, [53] [54] [55] [56] . these libraries are usually used as a series of sub-libraries laid out in positional-scanning format such that there is a sub-library with each amino acid fixed in each position and with all other positions made up of an equimolar mix of the remaining amino acids (of note, cysteine is generally excluded from the 'random' positions to avoid problems of oxidation) (see supplementary information s1 (figure)). studies with these libraries in t cell activation assays indicate that agonist ligands can contain several different amino acids at many positions. several studies have gone on to use this approach to prove the 'mason hypothesis' and show that individual t cell clones really can recognize over a million different individual peptides in the context of a single mhc molecule 47, 56, 57 . the antigen sensitivity of a t cell and its ability to respond to weaker tcr ligands are inexorably linked. t cell sensitivity to an antigen is not a fixed parameter. memory t cells can recognize concentrations of a peptide antigen that are >50-fold lower than those recognized by naive t cells 58, 59 , and individual t cell clones can generate progeny with both high and low antigen sensitivities 60 . antigen sensitivity can be regulated by changes in tcr expression levels or clustering on the cell surface, by changes in the expression or function of co-stimulatory molecules, by differential control of phosphatase pathways that dampen t cell signalling or by alterations in the glycosylation status of the tcr or other cell-surface molecules (reviewed in ref. 61 ). although these mechanisms may regulate the antigen sensitivity of t cells, and thus the ability of t cells to cross-recognize weak tcr ligands, it is difficult to conceive how they might be used to tune the biophysics of tcr engagement with a specific ligand. by contrast, the cd4 and cd8 glycoproteins have a unique role in 'co-receiving' peptide-mhc molecules by binding to largely invariant sites on mhc class ii and mhc class i molecules, respectively 62 . thus, these coreceptors might possess an ability to differentially regulate the responsiveness of the tcr to the ligand and thereby modulate tcr specificity 63 . indeed, cd8 is known to affect both the on-rate 64,65 and off-rate 66,67 of tcr-peptide-mhc class i engagement and therefore can modulate the kinetics of tcr binding by different peptide-mhc ligands. we have demonstrated how the strength of the peptide-mhc class i-cd8 interaction can have substantial effects on t cell cross-reactivity 53 . it is important to realize that, although the tcr sequence is invariant, tcr sensitivity to agonist ligands (and therefore t cell cross-reactivity) is not fixed and can be varied throughout development by a number of parameters 53 . the idea that immune cover is provided by limited numbers of highly cross-reactive t cells has both positive and negative implications. the presence of pools of cross-reactive t cells that each recognize large numbers glossary altered peptide ligands (apls) . peptide analogues that are derived from an original antigenic peptide. they commonly have amino acid substitutions at residues that contact the t cell receptor (tcr) and alter tcr engagement, resulting in different activation consequences than those induced by the wild-type ('index') antigenic peptide. a measure of how sensitive t cells are to the density of cognate antigen on the antigen-presenting cell surface. t cell receptor (tcr) affinity for a peptide-mhc complex has a large role in antigen sensitivity, but the parameter is also affected by the expression of other molecules that influence cell-cell contact or the downstream signal transduction that results from tcr-peptide-mhc engagement. a theory proffered by niels jerne which states that there is already a vast array of lymphocytes in the body before any infection. any challenge with antigen selects, and clonally expands, a single corresponding lymphocyte (b cell or t cell) from the pre-existing lymphocyte pool of differing specificities, and this clonal lymphocyte population then eliminates the antigen. (cdrs). the regions within antigen receptors that complement the shape of an antigen. the cdrs are the most variable part of the antigen receptor and are largely responsible for the diversity in these molecules. the cdrs allow antibodies and t cell receptors to recognize a vast repertoire of antigens. the term used to describe how an immune response to a pathogen can provide immunity to a non-identical pathogen. heterologous immunity can be mediated by cross-reactive t cells or antibodies. resemblance between epitopes contained in microbial and host proteins, leading to cross-reactivity of t cells in the host. a 'footprint' of immune responses is established during the first exposure to a pathogen. these specific memory t cell populations are preferentially re-expanded when re-exposed to the same antigen or one that is similar, thereby limiting the clonal expansion of new antigenspecific t cells. a similar mechanism has been proposed for b cell responses. the reaction of t cells to more than one distinct peptide-mhc ligand. refers to the promiscuity of t cell receptor (tcr) engagement that allows a single tcr to bind to different peptide-mhc complexes. pathogen-derived peptide self peptide t cell priming cross-recognition autoimmune attack tcr tissue cell of peptides but that do not respond to self peptides in the periphery has a number of positive consequences. first, a cross-reactive t cell repertoire generates a near perfect solution to the huge challenge of providing effective immune cover by allowing a limited number of t cells to provide immunity against virtually all foreign peptides that can bind to self mhc molecules. second, a system with a limited number of hugely cross-reactive t cells is both temporally and spatially favourable, as far fewer t cells are needed to scan any infected cell than if the clonal selection theory was rigidly upheld. third, the corollary of extensive t cell crossreactivity is that several tcrs are likely to recognize any one peptide (and thus that t cell responses are polyclonal). polyclonal recognition of peptide-mhc molecules makes it substantially more difficult for pathogens to escape immune recognition, as a mutation that escapes recognition by one tcr might be recognized by another. fourth, extensive t cell cross-reactivity also provides excellent conservation of resources by generating 'one weapon with several triggers' . several documented examples show that an individual t cell clone can target more than one infection through different peptides, a phenomenon known as heterologous immunity 68 . heterologous immunity between related pathogens is common. it is well known that immunity to cowpox provides cover for smallpox 69 , and the tuberculosis vaccine bacterium mycobacterium bovis bacillus calmette-guérin (bcg) can provide some protection against leprosy 70 . but, the existence of extensive t cell crossreactivity means that heterologous immunity can extend beyond the cross-recognition of pathogens with high sequence similarity to allow, for example, bcg-induced t cells to also provide immunity against poxviruses 71 . similarly, cd8 + t cells specific for the human papillomavirus hla-a2-restricted ymldlqpet peptide also recognize the hla-a2-restricted tmldiqped peptide from coronavirus 72 . indeed, cd8 + t cellmediated heterologous immunity can extend to very dissimilar antigens. for example, cells that are specific for the immunodominant gilgfvftl peptide from influenza virus can often recognize the epstein-barr virus epitope glctlvaml 73 or the immuno dominant hiv-derived slyntvatl antigen 74 (all of which are hla-a2 restricted). the extent of heterologous immunity and its importance to human immunity is not yet fully known. the potential positive outcomes of this phenomenon are clear, but heterologous immunity could also have deleterious effects. documented negative consequences of heterologous immunity include influenza-specific cd8 + t cells contributing to lymphoproliferation in epstein-barr virus-associated mononucleosis 75 or cross-recognizing a peptide derived from hepatitis c virus (hcv) 76 , which increases the severity of hcv-associated liver pathology 77 . it is also possible that heterologous immunity via t cell cross-reactivity could encourage a suboptimal response to the second pathogen owing to 'original antigenic sin' . this antigenic sin could extend beyond the simple case of suboptimal sensitivity to the second antigen to a situation in which the original antigen has established a t helper 1 (t h 1)-t h 2-or t h 17-type response bias that is inappropriate for the second pathogen. however, the most obvious and detrimental consequence of t cell cross-reactivity to vast numbers of individual peptides is the potential such a system has for causing autoimmunity (fig. 3) . although strongly selfreactive t cells are deleted in the thymus 50 , weakly cross-reactive t cells may survive and become activated in the periphery through the cross-recognition of peptides from infectious agents, a phenomenon known as molecular mimicry [78] [79] [80] [81] . memory t cells can be stimulated by peptide concentrations more than 50-fold lower than those required to stimulate naive t cells 58, 59 . it is therefore likely that a memory t cell could be stimulated by a cross-reactive peptide with an affinity for the tcr that is far lower than that of the original pathogen-derived peptide. in such a situation, pathogen-mediated priming would be obligatory before functional crossrecognition of a self peptide, a notion that is consistent with the observation that infection can precipitate autoimmune diseases 79, 82 . the compromise imposed by t cells being hugely cross-reactive in order to provide complete immune cover dictates that an individual tcr-peptide-mhc pairing is highly likely to be suboptimal. thus, it should be possible to improve the binding of any given tcr to its cognate antigen by enhancing the specific molecular matching. indeed, yeast display 83 , phage display 84 and computational design 85, 86 have been used to produce tcrs that bind to peptide-mhc complexes with extremely high affinities (k d <10 pm) and half-lives of many hours. the mhc class i pathway is predicted to present at least one peptide at the cell surface from every internally produced protein 10 . this allows tcrs to potentially target any cell based on its expression of any protein (fig. 4a) . consequently, tcrs might have considerable advantages over regular antibody-based therapies, as they can target a substantially greater number of cellular proteins. furthermore, there is now substantial evidence that it is possible to improve the affinity of almost any peptide antigen for a given natural tcr. thus, there is ample scope for the rational design of therapeutic interventions that exploit the fact that most natural tcr-peptide-mhc interactions can be improved upon. enhanced tcrs in tcr gene transfer therapy. the rigours of thymic selection ensure that natural tcrs bind to ubiquitous self or tumour-associated antigens with substantially lower affinities than they bind to pathogen-derived antigens 87 . natural tcr-peptide-mhc interactions have affinities (measured in terms of k d ) in the 87, 88 . within this range of tcr binding affinities, the affinity and/ or half-life correlates with antigen sensitivity 65, 89 , placing natural antitumour t cells at a distinct disadvantage compared with their pathogen-reactive counterparts. the transfer of tcr genes into recipient host t cells followed by the adoptive transfer of the t cells to patients allows the passive transfer of immunity and can provide a useful mechanism for breaking tolerance to tumour antigens 90 . this strategy has already shown some promise in patients with malignant melanoma 91 , but there is room for improvement. the transfer of genes encoding tcrs that have been affinity matured to bind to tumourassociated peptide-mhc complexes with affinities as high as those of the best antiviral t cells (k d = 100 nm) 87 enhanced tcrs as soluble therapies. highaffinity soluble tcrs provide an efficient means for the cellular targeting of intracellular antigens that are presented by mhc molecules in vivo (fig. 4a) . soluble tcrs can be linked to other molecules, such as antibody fab fragments, and can deliver these molecules to sites of antigen expression in vivo 92 . despite the low copy number of most peptide-mhc molecules (<50 copies per cell), we have recently used a soluble tcr fused to a cd3-specific fab fragment to induce tumour regression in vivo 92 . these bispecific t cell-engaging tcrs function by recruiting polyclonal t cells via the cd3specific fab component but do not by themselves crosslink tcrs or induce t cell activation. once these molecules are bound to a target cell surface, they become potent activators of antigen-experienced cd8 + t cells and promote the lysis of targets expressing as few as ten cognate peptide-mhc complexes 92 (fig. 4b) . a similar approach could be used to dampen autoimmunity by crosslinking inhibitory receptors such as cytotoxic t lymphocyte antigen 4 (ctla4). the fact that any tcr will be capable of recognizing enormous numbers of ligands paves the way for therapies based on altered peptide ligands (apls). apls can have advantages over natural ligands, as they can bind strongly to tcrs and can break tolerance to self ligands (including tumour-derived ligands). previous assumptions about apls, such as the suggestion that altering a buried anchor residue will not substantially alter tcr binding, have proved to be incorrect 93 . nevertheless, combinatorial screening of peptide (or non-peptide) ligands can be used to determine the preferred binding 'landscape' of any tcr and circumvent the requirement for any assumptions. the nature of the system makes it highly likely that each tcr has a different preferred binding landscape. this then enables relatively precise targeting of specific tcrs within populations of antigen-specific t cells through a process termed tcr-optimized peptide skewing of the repertoire of t cells (topsort), which can be used to sort the most effective clonotypes (fig. 5) . the widespread applicability of figure 4 | enhanced tcrs as soluble therapies. a | the mhc class i presentation pathway presents peptides at the cell surface from intracellular proteins. this potentially allows soluble high-affinity 'monoclonal' t cell receptors (tcrs) to target any cell based on its expression of any protein. 'monoclonal' tcrs are able to use the mhc class i presentation pathway to 'see inside' cells and scan them for internal anomalies. this 'x-ray vision' opens up access to a far greater range of disease-relevant antigens than are available for monoclonal antibodies. tcrs can be engineered to deliver a variety of molecules that stimulate or suppress the immune system. potential 'payloads' include antibody fab fragments that then deliver a signal to immune cells. as mhc-bound peptides are often present at low copy numbers (<50 copies per cell), the payloads delivered by tcrs must act at very low concentrations. b | high-affinity tumour-specific tcrs that are manufactured as bispecific t cell-engaging molecules by linking them to cd3-specific fab fragments can direct the lysis of tumour cells by cd8 + t cells and thereby induce the regression of established tumours 92 . these molecules do not activate t cells as monomers at the concentrations used. t cell-engaging tcrs bind to the cognate antigen on the tumour cell surface with long half-lives and 'present' the linked cd3-specific fab fragments. these fab fragments then crosslink tcrs on the surface of antigen-experienced cd8 + t cells, resulting in cellular activation and elimination of the target cell 92 . the delivery of toxins with soluble tcrs is not recommended, as the soluble tcr constructs are taken up by scavenging cells such as macrophages. thus, molecules that deliver a particular signal to a specific effector cell are preferable. for example high-affinity tcrs could be used to downregulate immune responses by signalling through inhibitory receptors such as cytotoxic t lymphocyte antigen 4 (ctla4) (not shown). this approach is dependent on the effective clonotype being 'public' 94 (that is, occurring in all individuals with the restricting hla molecule) or having a public motif that is shared by all individuals with the relevant hla molecule. our own preliminary studies using ex vivo peripheral blood mononuclear cells show that this approach can be used to skew the clonotypes that respond to a tumour antigen (j. ekeruche-makinde et al., unpublished observations). a similar approach could be used to skew the clonotypes induced by a vaccination against hiv towards those that are known to be more difficult for hiv to escape from. accumulating evidence, including direct estimates of the total number of tcrs in a human, supports mason's notion that we should abandon the 'one-clonotypeone-specificity' paradigm suggested by clonal selection theory in favour of a 'one-clonotype-millions-of-specificities' reality. the simple arithmetic of t cell immunity allows t cells to be highly cross-reactive while appearing to be exquisitely specific in the environment in which they are expected to function . however, the realities of t cell immunity dictate that tcrs are very rarely an optimal fit for a real antigen and that real mhc-presented peptide antigens are rarely the optimal agonists for a 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responsiveness human tcr-binding affinity is governed by mhc class restriction control of hiv-1 immune escape by cd8 t cells expressing enhanced t-cell receptor kinetic proofreading in t-cell receptor signal transduction adoptive immunotherapy for cancer: harnessing the t cell response cancer regression in patients after transfer of genetically engineered lymphocytes monoclonal tcr-redirected tumor cell killing modification of mhc anchor residues generates heteroclitic peptides that alter tcr binding and t cell recognition bias in the αβ t-cell repertoire: implications for disease pathogenesis and vaccination quantitating t cell cross-reactivity for unrelated peptide antigens genetic and structural basis for selection of a ubiquitous t cell receptor deployed in epstein-barr virus infection structure of a tcr with high affinity for selfantigen reveals basis for escape from negative selection germ line-governed recognition of a cancer epitope by an immunodominant human t-cell receptor the shaping of t cell receptor recognition by self-tolerance we thank s. smith and n. watson for editing the manuscript. we thank the members of the kipnis laboratory for their valuable comments during multiple discussions of this work. n.c.d. is the recipient of a hartwell foundation postdoctoral fellowship. this work was primarily supported by a grant from the us national institute on aging, national institutes of health (award ag034113 to j.k.). heath park, cardiff, uk. e-mail: sewellak@cardiff.ac.uk my studies in this area were made possible by the generous support of the uk biotechnology and biological sciences research council to my colleagues and myself (grant bb/ h001085/1). i thank d. cole and b. baker for helpful discussions. the authors declare no competing financial interests. the author declares no competing financial interests. andrew k. sewell's homepage: http://www.tcells.org see online article: s1 (figure) key: cord-289711-4ab3d00h authors: yarmarkovich, mark; warrington, john m.; farrel, alvin; maris, john m. title: identification of sars-cov-2 vaccine epitopes predicted to induce long-term population-scale immunity date: 2020-06-08 journal: cell rep med doi: 10.1016/j.xcrm.2020.100036 sha: doc_id: 289711 cord_uid: 4ab3d00h summary here we propose a sars-cov-2 vaccine design concept based on identification of highly conserved regions of the viral genome and newly acquired adaptations, both predicted to generate epitopes presented on mhc class i and ii across the vast majority of the population. we further prioritize genomic regions that generate highly dissimilar peptides from the human proteome, and are also predicted to produce b cell epitopes. we propose sixty-five 33mer peptide sequences, a subset of which can be tested using dna or mrna delivery strategies. these include peptides that are contained within evolutionarily divergent regions of the spike protein reported to increase infectivity through increased binding to the ace2 receptor and within a newly evolved furin cleavage site thought to increase membrane fusion. validation and implementation of this vaccine concept could specifically target specific vulnerabilities of sars-cov-2 and should engage a robust adaptive immune response in the vast majority of the population. the current sars-cov-2 pandemic has precipitated an urgent need for a safe 43 and effective vaccine to be developed and deployed in a highly accelerated timeframe 44 as compared to standard vaccine development processes 1 immunogenicity. we present a list of sars-cov-2 minigenes and propose their use in 57 multivalent vaccine constructs that should generate t and/or b cell epitopes that can be 58 delivered by scalable manufacturing techniques such as dna or nucleoside mrna. 59 sars-cov-2 is the third coronavirus in the past two decades to acquire 60 infectivity in humans and result in regional epidemics, and the first to cause a global 61 pandemic. the spike glycoprotein of coronaviruses mediates host cell entry and dictates 62 species tropism, with the sars-cov-2 spike protein reported to bind its target ace2 63 with 10-20-fold higher affinity than sars-cov in humans 2, 3 . in addition, insertion of a 64 novel protease cleavage site 4 is predicted to confer increased virulence by facilitating 65 the cleavage necessary to expose the fusion peptide that initiates membrane fusion, 66 enabling a crucial step of viral entry into host cells 5, 6 . it is now clear that covid -19 67 disease results when sars-cov-2 infects type ii pneumocytes lining the pulmonary 68 alveoli that co-express ace2 and the tmprss2 protease (ziegler et al., 2020), likely 69 impairing release of surfactants that maintain surface tension. this impairment hinders 70 the ability to prevent accumulation of fluid and ultimately resulting in acute respiratory 71 distress syndrome 7, 8 . the immune response of convalescent covid-19 patients 72 consists of antibody-secreting cells releasing igg and igm antibodies, increased 73 follicular helper t cells, and activated cd4 and cd8 t cells 9 , suggesting that a broad 74 humoral and t cell driven immune response mediates the clearance of infection and 75 that vaccination strategies directed at multiple arms of the immune response can be 76 effective. the large size of the sars-cov-2 (~30 kilobases) suggests that selection of 77 optimal epitopes and reduction of unnecessary antigenic load for vaccination may be 78 essential for safety and efficacy. 79 rapid deployment of antibody-based vaccination against sars-cov-2 raises the 80 concern of accelerating infectivity through antibody-dependent enhancement (ade), 81 the facilitated viral entry into host cells mediated by subneutralizing antibodies (those 82 capable of binding viral particles, but not neutralizing them) 10 . ade mechanisms have 83 been described with other members of the coronaviridae family 11, 12 . it has already been 84 suggested that some of the heterogeneity in covid-19 cases may be due to ade from 85 prior infection from other viruses in the coronavirus family 13 . 86 while the immunogenicity map presented in this study can be used to inform 87 multiple modalities of vaccine development, we present peptide sequences that are 88 expected to be safe and immunogenic for use in t cell based vaccination, and highlight 89 b cell epitopes derived from peptides within the regions of the spike protein involved in 90 infectivity that we expect will minimize the risk of ade. as it has been shown that t 91 helper (t h ) cell responses are essential in humoral immune memory response 14, 15 , we 92 anticipate that the t cell epitopes generated from the peptide sequences presented 93 here will aid the activation of cd4 t cells to drive memory b cell formation and somatic 94 hypermutation when paired with matched b cell epitopes. 95 the potential of epitope-based vaccines to induce a cytolytic t cell response and 96 drive memory b cell formation is complicated by the diversity of hla alleles across the 97 human population. the hla locus is the most polymorphic region of the human 98 genome, resulting in differential presentation of antigens to the immune system in each 99 individual. therefore, individual epitopes may be presented in a mutually exclusive 100 manner across individuals, confounding the ability to immunize a population with 101 broadly presented antigens. while t cell receptors (tcrs) recognize linearized peptides 102 anchored in the mhc groove, b cell receptors (bcrs) can recognize both linear and 103 conformational epitopes, and are therefore difficult to predict without prior knowledge of 104 a protein structure. here we describe an approach for prioritizing viral epitopes derived 105 from a prioritized list of 33mer peptides predicted to safely target the vulnerabilities of 106 sars-cov-2, generate highly immunogenic epitopes on both mhc class i and ii in the 107 vast majority of the population, and maximize the likelihood that these peptides will drive 108 an adaptive memory response. 109 we applied our recently published methods for scoring population-scale hla 112 presentation of all 9mer peptides along the length of individual oncoproteins in human 113 cancer to analyze the population-scale hla presentation of peptides derived from all 10 114 sars-cov-2 genes across 84 class i hla alleles 16 table s1 ). 121 we next tested various peptide sequence lengths to maximize hla presentation 122 on multiple alleles within a single k-mer, finding that 33 amino acids generated maximal 123 population-scale hla presentation. we show that 99.7% of all 9,303 possible 33mers 124 are predicted to generate at least one hla class i epitope, and propose that expression 125 and presentation of these 33mers in dendritic cells is expected to induce an immune 126 response across a significant proportion of the population 18, 19 . we identified viral 127 regions predicted to generate epitopes that would presented across the majority of the 128 population, highlighting a single 33mer isnswlmwliinlvqmapisamvrmyiffasfy 129 containing multiple epitopes predicted to bind 82 of the 84 hlas alleles, suggesting that 130 this single 33mer can potentially induce an immune response in up to 99.4% of the 131 population given proper antigen processing (table s1) predicted to be presented on hla class i and ii across the majority of the population. as 140 hla frequencies vary significantly by population, the frequency of individual hla alleles 141 can be adjusted based on specific populations using the sars-cov-2 immunogenicity 142 map presented here, such as to customize vaccine design for groups with distinct hla 143 allele distributions (table s1) . 144 next, we sought to identify the most highly conserved regions of the sars-cov-145 2 virus, positing that conserved regions are essential to viral replication and maintaining 146 structural integrity, while non-conserved regions can tolerate mutations and result in 147 antigens prone to immune evasion. to do this, we compared the amino acid sequence 148 of sars-cov-2 to 14 closely related mammalian alpha and beta coronaviruses (human, 149 bat, pig, and camel) from the coronaviridae family (table s2) , scoring each amino acid 150 for conservation across the viral strains. additionally, we scored the conservation across 151 the 727 sars-cov-2 genes sequences available at the time of this analysis (table s2) , 152 equally weighing contributions from cross-species and interhuman variation (scores 153 normalized to 0-1, with entirely conserved regions scoring 1). as expected, evolutionary 154 divergence was greatest in the tropism-determining spike protein and lowest in orf1ab 155 which contains 16 proteins involved in viral replication (figure 1b, bottom) . 156 we then compared predicted viral mhc-presented epitopes to self-peptides 157 presented in normal tissue on 84 hla alleles across the entire human proteome as 158 listed in the uniprot database, prioritizing antigens that are most dissimilar from self-159 peptides based on: 1) higher predicted safety based on decreased likelihood of inducing 160 autoimmunity due to cross-reactivity with similar self-peptides presented on mhc; and table s1 ). we find regions of the viral 172 proteome that are identical or highly similar to portions of the normal human proteome 173 predicted to be presented on mhc, suggesting that an immune response mounted 174 against these viral epitopes could result in an autoimmune response, while other high-175 scoring regions are highly dissimilar from self and expected to generate antigens with 176 minimal likelihood of cross-reactivity (table s1) . 177 to assign an overall score for putative t cell antigens, we normalized each of our 178 four scoring parameters (represented in figure 1a and 1b) between 0-1 and summed 179 each metric to obtain a final 33mer peptide score, highlighting the local maxima of 180 potentially generated epitopes scoring in the 90 th percentile (55 top scoring t cell 181 peptides) across 10 sars-cov-2 genes as peptide sequences for vaccination ( figure 182 1c; table s3) . 183 finally, we sought to characterize b cell epitopes, assessing linear epitopes in 184 spike (s), matrix (m), and envelope (e) proteins that are exposed and expected to be 185 accessible to antibodies; we also characterized conformational epitopes in the spike 186 protein for which structural data are available using we combined t cell epitope scores calculated above with available b cell epitope 194 scores derived from the s, m, and e genes, providing a list of 65 peptides predicted to 195 stimulate both humoral and cellular adaptive immunity ( figure 1f ; table s5 ). 196 to estimate the accuracy of our predictions, we compared the 65 unique 33mer 197 peptides presented in table s5 to 92 epitopes derived from the first sars virus cov) in the immune epitope database (iedb: https://www.iedb.org/home_v3.php) 199 shown to elicit t cell responses. we found a significant enrichment in immunogenic table 1) , demonstrating that epitopes selected using this analysis epitopes are more 207 likely to be processed and immunogenic based on previous studies with sars-cov, 208 and supporting the hypothesis that a single 33mer is capable of generating multiple 209 unique epitopes presented by multiple hla alleles. we also found that a significant 210 proportion of the peptides present within prioritized 33mer have been predicted to bind 211 mhc based on structural predictions 22 . bound to the spike protein from sars-cov-2 as compared to sars-cov 23 , we 230 searched for 33mers containing the five acquired residues that increase spike binding to 231 ace2, identifying kpferdisteiyqagstpcngvegfncyfplqs as the highest 232 ranked peptide sequence containing each of these residues (hotspots underlined; table 233 1). additionally, a d614g mutation in the spike protein has been reported as a 234 potentially dominant strain with increased pathogenicity 24, 25 . we thus suggest including 235 the highest scoring 33mer (ntsnqvavlyqgvnctevpvaihadqltptwrv) 236 predicted to present this mutant epitope in a vaccine construct. finally, it is known that 237 mrna transcripts proximal to the 3' end of the coronaviridae family genome show 238 higher abundance consistent with the viral replication process, with s, e, m, and n 239 genes shown to have significantly higher translational efficiency compared to the 5' 240 transcripts, with the highest expression in the n gene, and consistent with the high 241 degree of mhc presentation as described above five immunogenic peptides derived 242 from a single n protein 33mer [26] [27] [28] . we therefore posit that viral epitopes derived from 3' 243 terminus including the s, e, m, and n genes will have a higher representation on mhc 244 and suggest their prioritization in a vaccine construct. table s5 lists the highest priority 245 viral peptides we suggest should be considered for inclusion in vaccine constructs. 246 here we present a comprehensive immunogenicity map of the sars-cov-2 248 virus (table s1) , and propose sixty-five 33mer peptide sequences predicted to generate 249 b and t cell epitopes from a diverse sampling of viral domains across all 10 sars-250 cov-2 genes (tables 1 and s5 ). based on our computational algorithms, we expect 251 that the highest scoring peptides will result in safe and immunogenic t cell epitopes, 252 and that b cell epitopes should be evaluated for safety and efficacy using previously 253 reported methods with validated subsets of these 65 epitopes 11 . dna and mrna 254 vaccines have been shown to be safe and effective in preclinical studies, and can be 255 rapidly and efficiently manufactured at scale 29, 30 . nucleoside modification of rna has 256 been shown to improve efficacy, which has been attributed to a reduction of rna-257 induced immunogenicity 31 . we suggest that multivalent constructs composed of the 258 sars-cov-2 minigenes encoding subsets of the b and/or t cell epitopes proposed 259 here (tables 1, s3, s4, and s5) can be used in a dna on mrna vaccine for 260 expression in antigen-presenting cells. 261 these epitopes can be used in tandem with a tlr agonist such as tetanus 262 toxoid or padre [32] [33] [34] [35] to drive activation of signals 1 and 2 in antigen presenting cells. 263 constructs can be designed to contain a combination of optimal b and/or t cell 264 epitopes, or deployed as a construct consisting of the top scoring t cell epitopes to be 265 used in combination with the vaccines currently being developed targeting spike protein 266 in order to drive the adaptive memory response. dna vaccine sequences can also be 267 codon optimized to increase cpg islands such as to increase tlr9 activation 36 . 268 with the third epidemic in the past two decades underway, all originating from the 269 coronavirus family, these viruses will continue to threaten the human population, and table s1 can be used to design 281 customize multi-valent vaccines based on the hla frequencies of specific populations. 282 although we suggest the use of 33mers based on optimal mhc presentation across the 283 population, these methods can be generalized and applied to the evaluation k-mers of 284 various sizes depending on desired application. since antigens may arise from the 285 junctions between epitopes, the analyses presented here can also be used to evaluate 286 epitope generation at the junction of specific vaccine constructs, such as to engineer 287 linker regions that reduce the potential immunodominant epitopes elicited from 288 irrelevant sequences. we also find up to five peptides reported by grifoni within a single 33mer, and up to 12 302 peptides reported by ahmed contained in the 33mer 303 aqfapsasaffgmsrigmevtpsgtwltytgai described above. taken together, 304 these comparisons show a significant convergence on a subset of epitopes using 305 agnostic analyses, while also reporting unique epitopes in each study. the finding that 306 up to 12 epitopes from previous analyses are represented in a single 33mer from our 307 agnostic analysis further supports the our prediction that cocktails of 33mer epitopes 308 can be used for population-scale vaccination. 309 by narrowing the pool of peptides selected for downstream screening, we expect 310 that the analyses presented here will contribute to maximizing the efficiency of vaccine 311 development. antigenic burden from epitopes that do not contribute to viral protection 312 can cause autoimmune reactions, reactogenicity, detracting from the efficacy of the 313 vaccine, or result in ade. we found that the vast majority of the sars-cov-2 virus is 314 immunogenically silent on mhc class i and ii and suggest these regions should be 315 excluded from vaccine development. though empirical testing is necessary to evaluate 316 ade, we suggest that antibodies directed at the receptor binding domain and furin 317 cleavage sequences may mitigate ade by blocking the processes needed to achieve 318 membrane fusion. to avoid potential t cell cross-reactivities a priori, we selected 319 maximally immunogenic epitopes with the highest degree of dissimilarity to the self-320 proteome with minimal potential of cross-reactivity that can lead to adverse reaction or 321 weaken the efficacy of vaccination. in addition to the predicted safety of these epitopes 322 (stemming from lack of potentially cross-reactive normal proteins), we expect that a 323 greater repertoire of viral antigen-specific t cells will be present due to the absence of and symptoms of anosmia 42, 43 . while cd8 vaccines targeting conserved antigens in 339 influenza did not completely block infection upon challenge with virus, they effectively 340 reduced viral replication, morbidity, and mortality 44, 45 . taken together, these findings 341 suggest that cd8-based immunity can be a viable strategy in quelling sars-cov-2. studies demonstrating protection against multiple influenza strains imply that cd8-343 mediated vaccination may act more broadly than antibody responses in protecting 344 against multiple virus family members through targeting of conserved non-structural 345 proteins critical in the viral life-cycle. 346 currently, targeting cd8 epitopes has been complicated by hla restriction of 347 peptides and antigenic drift resulting from viral regions in which mutation is tolerable. concordance with predicted population-scale presentation 16 . although we expect that a 366 significant fraction of predicted antigens to be presented on mhc, binding predictions 367 alone do not determine which antigens will elicit an immunodominant response. while 368 the dissimilarity scoring predicts that tcrs specific for these antigens are more likely to 369 exists (because these tcrs far less likely to have undergone negative thymic 370 selection), these predictions are confounded by the tcr repertoire of a given individual 371 and the intrinsic immunogenicity of a particular peptide which cannot be predicted 372 without empirical testing. since mhc binding is a prerequisite for antigen 373 immunogenicity, we expect that immunodominant antigens will be contained within our 374 highest scoring epitopes. however, experimental validation will be necessary to 375 determine the contribution of individual antigens to immunity. as a best approximation 376 for our predictions, we show a significant enrichment of peptides previously reported in 377 iedb to be immunogenic in the sars-cov virus, contained within the 65 prioritized 378 epitopes that we present, supporting the concept that multiple antigens derived from 379 33mers can be presented across multiple hla alleles. 380 we expect that the comprehensive immunogenicity map presented here can be 381 used by the scientific community to inform the design of various vaccination modalities. 382 we are presently designing a set of vaccine vectors and validation reagents based on 383 these analyses that we plan to make available to the research community for testing. 384 the 65 epitopes presented here out of the 9,303 possible 33mers derived from sars-385 cov-2 can significantly narrow the focus of vaccine development (table s5) ; these 386 epitopes can be expressed as a single <7kb construct, or more likely tested in various 387 combinations delivered as a cocktail of rna constructs encoding individual 33mers. 388 these vaccine constructs can be rapidly and efficiently tested for the neutralizing 389 potential of antibodies using sars-cov-2 pseudo-virus 47 , the formation of memory b 390 cells, and for induction of t cell activation using methods that we have recently 391 developed for interrogating antigen specificities in a highly multiplexed manner 48 . as 392 sars-cov-2 has precipitated the need to rapidly develop and deploy vaccines in 393 pandemic situations 49 , we suggest that this comprehensive analysis can be 394 incorporated into a process that can be rapidly implemented when future novel viral 395 pathogens emerge. 396 the in silico analysis of the sars-cov-2 genome reported here has yet to be 398 experimentally validated. while it is reassuring that we demonstrate enrichment of 399 predicted epitopes from the original sars virus previously reported in iedb that have 400 been shown to be immunogenic, rigorous experimental validation of our findings is 401 required. computational pmhc binding predictions do not consider critical variables in 402 antigen presentation such as proteasomal degradation and peptide processing. in 403 addition, it is unclear whether the 33mers designed to elicit a b cell will properly fold into 404 conformations resembling the native spike protein such as to elicit a protective antibody 405 response. we have designed multiple dna and mrna constructs containing 406 combinations of 33mers proposed here to test hypotheses that these vaccines can elicit 407 memory and/or cytolytic t cell response and/or protective antibodies against a sars-408 spike-gfp pseudovirus 47 in hla-a2 transgenic mice 50 the population predicted to have at least one of these hlas, normalized dissimilarity scores, normalized conservation 466 scores, across the 33mer, total t cell score, b cell score, and combined b and t cell percentile for 33mers. further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, john m. maris (maris@email.chop.edu). vaccine constructs and testing reagents are available from the lead contact, john m. maris with a completed materials transfer agreement. please email maris@chop.edu. all raw data has been reported in paper and models are described in methods. we identified potential sars-cov-2 epitopes by applying our recently published algorithm for scoring population-scale hla presentation of tumor driver gene, to the sars-cov-2 genome (genbank acc#: mn908947.3) 51 . all possible 33mer amino acid sequences covering every 9mer peptide from the 10 sars-cov-2 genes were generated and we employed netmhc-4.0 to predict the binding affinities of each viral 9mer peptide across 84 hla class i alleles 52 . we considered 9mer peptides with binding affinities <500nm putative epitopes. mhc class ii binding affinities were predicted as described above across 36 hla class ii alleles population using netmhcii 2.3 53 . all 9mers present in a 33mer contribute to the score. 33mer scores calculated by infering population scale hla presentation of all predicted peptides within 9mer on class i and ii. the frequencies of hla class i alleles -a/b/c and hla class ii alleles -drb1/3/4/5 were obtained from be the match bone marrow registry 17 .hla class ii alleles -dqa1/dqb1 and -dpa1/dpb1 were obtained from 54 and 55 , respectively. we obtained all 727 unique protein sequences categorized by each of the 10 sars-cov-2 genes available from the ncbi as of 25 march 2020. all sequences were aligned using clustal omega 56 and each position summed for homology. in addition to human sequences, we scored each amino acid position for homology across 15 species of related coronavirus found in bats, pigs, camels, mice, and humans (sars-cov, sars-cov-2, and mers). each amino acid was scored up to 100% conservation. 33mer peptides were then scored in equation 1: where c is the 33mer conservation score, a is the conservation percentage of an amino acid position, y is the minimum 33mer conservation percentage sum, and z is the maximum 33mer conservation percentage sum. in the same way, we ranked the conservation across 274 sars-cov-2 amino acid sequences available at the time of this study. a final conservation score was generated by averaging the conservation scores from cross-species and interhuman variation and 33mer peptides with the highest score were considered the most conserved. 3,524 viral epitopes were compared against the normal human proteome on each of their mhc binding partners, testing a total of 12, 383 peptide/mhc pairs against the entire human proteome (85,915,364 normal peptides across hlas), assigning a similarity score for each peptide. residues in the same position of the viral and human peptides with a perfect match, similar amino acid classification, or different polarity, were assigned scores of five, two, or negative two respectively. similarity scores were calculated based on amino acid classification and hydrophobicity were determined using non-anchor residues on mhc ( figure s1a) . the canonical tcr-interaction hotspots (residues four through six) were double weighted [57] [58] [59] . the similarity scores generated for each viral peptide were converted to z-scores and peptides with a p <0.0001 were selected for comparison to viral epitopes ( figure s1b ). the overall dissimilarity score for the viral peptide was then calculated using equation 2: where is the overall dissimilarity score for the viral peptide, is the highest possible z-score given a perfect sequence match to the viral peptide, is the highest z-score from the human proteome, % & is the number of statistically significant peptides from the human proteome, and & is the mean z-score from the statistically significant peptides given a p < 0.001. we used bepipred 2.0 and discotope 2.0 20,21 to score individual amino acid residues, assessing linear epitopes in matrix, envelope, and spike proteins, and conformational epitopes for spike protein, based on published structure (pdb 6vyb). to we summed and normalized linear and conformational, using separate normalizations for proteins in which only linear predictions were available. table legends table s1 . immunogenicity map of sars-cov-2, related to figure 1 and table 1 . combined analysis of sars-cov-2 for hla class i & ii binding, dissimilarity scoring, and conservation scoring for peptides beginning at each position in the viral proteome. hla binding reported as percent rank of epitopes and 33mers beginning at each position, with a <=0.5 threshold for listed binders (strong binders). dissimilarity, conservation and b cell (linear and conformational) scores listed for 33mers starting at each peptide. table s2 . sequences used for conservation scoring, related to figure 1 and table 1 . sequences for 14 alpha and beta coronavirus genes and 727 sars-cov-2 genes. table s3 . top scoring sars-cov-2 t cell 33mers, related to figure 1 and table 1 . fifty-five highest scoring 33mer peptides based on a combination of hla class i, class ii, dissimilarity, and homology scoring (contains peptides found to be b cell epitopes as well). table s5 . prioritized list of 65 33mer peptide sequences enriched for population scale immunity, related to figure 1 and table 1 . yarmarkovich et al. report sars-cov-2 peptides for use in multi-epitope vaccines. these peptides are predicted to activate cd4 and cd8 t cells, are highly dissimilar from the self-proteome, and are conserved across 15 related coronaviruses. presented epitopes are expected to drive long-term immunity in the majority of the population. â�¢ selecting optimal epitopes is essential for vaccine safety and efficacy â�¢ we report 65 vaccine peptides predicted to drive long-term immunity in most people â�¢ epitopes contain domains conserved in 15 coronaviruses and newly evolved sars2 regions â�¢ epitopes can be used to generate b and/or t cell vaccines (rna and dna) middle east respiratory syndrome: obstacles and prospects for vaccine development cryo-em structure of the 2019-ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation activation of the sars coronavirus spike protein via 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the prevention of old world arenavirus infection in humans immunogenicity and immune silence in human cancer gapped sequence alignment using artificial neural networks: application to the mhc class i system improved methods for predicting peptide binding affinity to mhc class ii molecules divergent motifs but overlapping binding repertoires of six hla-dq molecules frequently expressed in the worldwide human population balancing selection and heterogeneity across the classical human leukocyte antigen loci: a meta-analytic review of 497 population studies fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega structural bases for the affinity-driven selection of a public tcr against a dominant human cytomegalovirus epitope the structural dynamics and energetics of an immunodominant t cell receptor are programmed by its v&#x3b2; domain unraveling a hotspot for tcr recognition on hla-a2: evidence against the existence of peptide-independent tcr binding determinants key: cord-296007-1gsgd22t authors: mohseni, amir hossein; taghinezhad-s, sedigheh; su, bing; wang, feng title: inferring mhc interacting sars-cov-2 epitopes recognized by tcrs towards designing t cell-based vaccines date: 2020-09-12 journal: biorxiv doi: 10.1101/2020.09.12.294413 sha: doc_id: 296007 cord_uid: 1gsgd22t the coronavirus disease 2019 (covid-19) is triggered by severe acute respiratory syndrome mediated by coronavirus 2 (sars-cov-2) infection and was declared by who as a major international public health concern. while worldwide efforts are being advanced towards vaccine development, the structural modeling of tcr-pmhc (t cell receptor-peptide-bound major histocompatibility complex) regarding sars-cov-2 epitopes and the design of effective t cell vaccine based on these antigens are still unresolved. here, we present both pmhc and tcr-pmhc interfaces to infer peptide epitopes of the sars-cov-2 proteins. accordingly, significant tcr-pmhc templates (z-value cutoff > 4) along with interatomic interactions within the sars-cov-2-derived hit peptides were clarified. also, we applied the structural analysis of the hit peptides from different coronaviruses to highlight a feature of evolution in sars-cov-2, sars-cov, bat-cov, and mers-cov. peptide-protein flexible docking between each of the hit peptides and their corresponding mhc molecules were performed, and a multi-hit peptides vaccine against the s and n glycoprotein of sars-cov-2 was designed. filtering pipelines including antigenicity, and also physiochemical properties of designed vaccine were then evaluated by different immunoinformatics tools. finally, vaccine-structure modeling and immune simulation of the desired vaccine were performed aiming to create robust t cell immune responses. we anticipate that our design based on the t cell antigen epitopes and the frame of the immunoinformatics analysis could serve as valuable supports for the development of covid-19 vaccine. designing of multi-hit peptides vaccine sequence 1 3 7 a set of high immunogenic hit peptides derived from n and s proteins of sars-cov-2 with high binding 1 3 8 events to hla-a0201, hla-b0801, hla-b3501, hla-b3508, hla-b4405, and hla-e were selected 1 3 9 on the basis of their solvent exposed residues and hydrophobicity scales. the aay and gpgpg linkers 1 4 0 were applied for linking the candidate n and s hit peptides together, respectively. additionally, the human 1 4 1 beta defensin 3 was also joined at n-terminus of the vaccine construct using eaaak linker which acts as 1 4 2 adjuvant to improve the immunogenicity of the multi-hit peptides vaccine. the sopma web server (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) was 1 9 8 identified for them, respectively. giving our data, five and ten hit peptide antigen candidates (z-value 1 9 9 cutoff > 4), and 23 and 40 homologous peptide antigens in 16 and 25 organisms by using hla-a0201-2 0 0 peptide-tcr template [pdb entry 2p5e and 1oga] and the experimental peptide database were inferred in 2 0 1 orf3a and s proteins, respectively. no hit peptide antigen candidates with z-value cutoff > 4 were 2 0 2 detected for orf6, orf7a, and orf8 queries by using hla-a0201-peptide-tcr template ( figure s1a ). our results showed that by using hla-b0801 ( figure s1d ), hla-b3501 (figure s1b), and hla-b3508 2 0 4 ( figure s1e ) template, tcr-pmhc models with z-value cutoff > 4 were only predicted for s (5 hit peptide 2 0 5 antigen candidate and 91 homologous peptide antigens in 57 organisms with pdb entry 1mi5), n (3 hit 2 0 6 peptide antigen candidate and 3 homologous peptide antigens in 2 organisms with pdb entry 2nx5), and s 2 0 7 (1 hit peptide antigen candidate and 1 homologous peptide antigens in 1 organisms with pdb entry 2ak4) 2 0 8 proteins, respectively. while, only m and s proteins were used to predict tcr-pmhc complex by using 2 0 9 hla-b4405 ( figure s1c ) with pdb entry 3dxa (15 homologous peptide antigens in 12 organisms). also 2 1 0 only n, orf10, and s proteins were used for prediction of tcr-pmhc complex by hla-e (figure s1f) 2 1 1 with pdb entry 2esv (84 homologous peptide antigens in 41 organisms) with z-value cutoff > 4. no hit 2 1 2 peptide antigen candidates with z-value cutoff > 4 were detected for m, e, and orf3a queries by using 2 1 3 hla-e-peptide-tcr template ( figure s1f ). moreover, model for orf1ab query did not predict due to its 2 1 4 sequence length was ≥ 300. the 3d structure of the tcr-pmhc complex for each hit peptide derived 2 1 5 from each protein was illustrated by swiss-model. detailed information about derived hit peptides for 2 1 6 sars-cov-2 along with bat-cov, mers-cov, and sars-cov were tabulated in table 1 . the binding events among hit peptides derived from n proteins related to hla-a0201 2 1 9 our tcr-pmhc models predicted that position 2 of the homologous peptide antigens ( figure 1a ) related 2 2 0 to tcr-pmhc complex of sars-cov-2 as well as sars-cov and bat-cov n proteins prefers the 2 2 1 hydrophobic amino acid residues (e.g. ile, leu, met, and phe), and the second position of these hit peptides 2 2 2 is an hydrophobic amino acid residue pro forming five strong vdw forces with residues y99, v67, m45, 2 2 3 y7, and f9 and two h-bonds with residues k66 and e63 on mhc molecule ( figure s2a , left). by contrast, 2 2 4 the second position of mers-cov n protein-derived hit peptide is charged residue arg forming three 2 2 5 strong vdw forces with residues m45, f9, and v67 and two h-bonds with residues k66 and e63 (as 2 2 6 same as sars-cov-2) on mhc molecule. surprisingly, position 9 of all homologous peptides antigens 2 2 7 ( figure 1a ) prefers the hydrophobic amino acid residues (e.g. leu, ile, val, and met), and the position 9 of 2 2 8 these hit peptides is hydrophobic amino acid residue leu (in the sars-cov-2, sars-cov, and bat-cov) 2 2 9 and gly (in the mers-cov). our results showed that leu attaches to the mhc with three strong vdw 2 3 0 forces with residues l81, i124, and w147 and three h-bonds with residues d77, y84, and t143, while gly 2 3 1 forms three strong vdw forces with residues l81, v95, and w147 and two h-bonds with residues d77 2 3 2 and v95 on mhc molecule ( figure s2a , left). moreover, positions 4, 6, and 8 of hit peptides in sarscov-2, sars-cov, and bat-cov form one h-bond with residues q52, q52 and d32 in chain e of tcr, with reside s100 in chain d of tcr (table s1 ). visualization of interactions in the atomic level structure 2 3 6 of a tcr-pmhc complex in the hit peptide of sars-cov-2 n protein for hla-a0201 ( figure s3a ) 2 3 7 within 20 and 8 å generated on-the-fly using pymol. accordingly, position 1 of these hit peptides forms two h-bonds with residues y170 and y158 and position 2 4 3 2 of these hit peptides forms a h-bonds with residue e62 and four strong vdw forces with residues a66, 2 4 4 y6, w96, and m44. moreover, position 7 of these hit peptides forms a h-bond with residue n76 and three 1 1 hit peptides lacks any contacts, although the position 5 and 6 of hit peptides form both h-bonds and/or 2 4 7 strong vdw forces on both mhc molecule and tcr ( figure s2a and s2c, center) (table s2 ). visualization of interactions in the atomic level structure of a tcr-pmhc complex in the hit peptide of 2 4 9 sars-cov-2 n protein for hla-e ( figure s3c ) within 20 and 8 å was generated on-the-fly using of the homologous peptide antigens of all queries has no detectable binding to both mhc and tcr. additionally, position 6 of these hit peptides with a h-bond and strong vdw forces connects to the 3 0 5 residues q96 and y96 on tcr, respectively ( figure s2d , right). the hit peptide correlates well with the 3 0 6 amino acid profile on the conserved positions 7 (tyr) that forms one strong vdw forces with residue 3 0 7 w147 on mhc ( figure s2b , right) and four strong vdw forces with residue a97, h47, h32, and l90 on 3 0 8 tcr ( figure s2d , right and table s6 ). visualization of interactions in the atomic level structure of a tcr-3 0 9 pmhc complex in the hit peptide of sars-cov-2 s protein for hla-b0801 ( figure s3d ) within 20 and 8 3 1 0 å was generated on-the-fly using pymol. cov and sars-cov orf3 proteins. our data emphasized the hit peptide derived from sar-cov-2 s 3 2 6 protein by using hla-a0201 had lower immunogenicity than bat-cov, mers-cov, and sars-cov. 1 4 revealed a high degree of immunogenicity between sars-cov-2, sars-cov, and bat-cov but a more 3 3 0 limited immunogenicity with mers-cov. based on the hypothesis that solvent exposed residues via increasing tcr binding can provide appropriate 3 3 2 evidence about peptide immunogenicity, we measured solvent exposed area (sea) for each hit peptide. our results displayed that the sea > 30 å 2 for hit peptide related to hla-b0801, hla-b3501, and hla-3 3 4 b3508 were 5.08, 7.76, and 8.28, respectively. indeed, we found the most solvent accessibility of amino 3 3 5 acids were in the hit peptide m (sea > 30 å 2 : 5.99), s (sea > 30 å 2 : 6.75), and orf10 (sea > 30 å 2 : 6) 3 3 6 for hla-a0201, hla-b4405, and hla-e, respectively. over the past few months, studies in humans are beginning to unravel the underpinnings relationship 3 3 8 between hydrophobicity scales and eradication of immune responses. currently, identification of peptide 3 3 9 regions exposed at the surface has gained much attention in the field of immunogenicity of peptides to hla-e-peptide-tcr ( figure 2e ) and s protein related to hla-b4405 peptide-tcr ( figure 2d ) had 3 5 0 grater hydrophobicity than orf10 and m proteins due to differences at positions 6 and 9, and 5, 3 5 1 3 9 8 predicted as a single domain without disorder. among them, the first model had a better quality in most 4 4 0 cases with c-score = -3.25, estimated tm-score = 0.35±0.12, and estimated rmsd = 11.7±4.5å. however, because of the modelling errors and unavailability of an appropriate template such as angles and 4 4 2 irregular bonds, generation of the 3d models cannot be sufficient to follow the necessary accuracy level for 4 4 3 some biological purpose, especially where experimental data is rare. as such, for modification of local 4 4 4 errors, helping to bring 3d model of vaccine closer to native structures, and growing the accuracy of 4 4 5 primary 3d model, the refinement of 3d structure of the vaccine is vital, particularly for furthering in-silico 4 4 6 studies [20] . therefore, the refinement of the model was performed by using 3d refine tool. on the basis of 4 4 7 the overall quality of the refined model, the model 5 exhibited the best results with rmsd 0.549å ( figure 4 4 8 5b). the quality of the best model of the multi-hit peptides vaccine construct was validated by prosa-web. it is well-known that the z score is in relation with the length of the protein, indicating that negative z-4 5 0 scores are more appropriate for a trustworthy model. in fact, the z score shows the overall quality and (range −20 to 10) and also was located within the space of protein related to x-ray, suggesting that the 4 5 6 obtained model is reliable and closes to experimentally determined structure. evaluation the overall quality 4 5 7 of the finalized model of vaccine construct by ramachandran plot analysis emphasized that 77.5% (93/120) 4 5 8 of all residues in finalized model ( figure 5f ) compared to 54.2% (65/120) of all residues in initial model 4 5 9 ( figure 5e ) were in favored (98%) regions, also, 90.0% (108/120) of all residues in finalized model ( figure 4 6 0 5f) compared to 78.3% (94/120) of all residues in initial model ( figure 5e ) were in allowed (>99.8%) 4 6 1 regions. after that, protein structure and visualization of the measured interactions between atoms 1 0 to our best knowledge, from the standpoint of immunoinformatics approaches, the concept discussed in 4 8 1 this study is the first structural modeling to investigate both the tcr and pmhc interfaces for the sars-4 8 2 cov-2 proteins. our results provide a blueprint for inferring the sars-cov-2-derived hit peptides with 4 8 3 high accuracy towards vaccine development. therefore, it is tempting to speculate that the aforementioned 4 8 4 models will offer valuable framework for identifying specific peptides with a potential to acrivate t cell hit peptide; vdw: van der waals (vdw) forces c-immsim immune simulator web server was used for determining ability of vaccine to induce t cell 4 6 8 immunity. this server yielded results consistent with actual immune responses as evidenced by a general 4 6 9 marked increase in the generation of secondary responses. for better following the effects of the final 4 7 0 vaccine construct for stimulation of t cell immunity, a construct with point mutations on key residues, 4 7 1 replacing of the hydrophobic amino acids with charged amino acids was constructed ( figure 6a ). according to data, after vaccination with native vaccine, there was a consistent rise in th (helper) cell key: cord-288146-xqxznv1r authors: kohyama, shunsuke; ohno, satoshi; suda, tatsuya; taneichi, maiko; yokoyama, shoichi; mori, masahito; kobayashi, akiharu; hayashi, hidenori; uchida, tetsuya; matsui, masanori title: efficient induction of cytotoxic t lymphocytes specific for severe acute respiratory syndrome (sars)-associated coronavirus by immunization with surface-linked liposomal peptides derived from a non-structural polyprotein 1a date: 2009-09-11 journal: antiviral res doi: 10.1016/j.antiviral.2009.09.004 sha: doc_id: 288146 cord_uid: xqxznv1r spike and nucleocapsid are structural proteins of severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) and major targets for cytotoxic t lymphocytes (ctls). in contrast, non-structural proteins encoded by two-thirds of viral genome are poorly characterized for cell-mediated immunity. we previously demonstrated that nucleocapsid-derived peptides chemically coupled to the surface of liposomes effectively elicited sars-cov-specific ctls in mice. here, we attempted to identify hla-a*0201-restricted ctl epitopes derived from a non-structural polyprotein 1a (pp1a) of sars-cov, and investigated whether liposomal peptides derived from pp1a were effective for ctl induction. out of 30 peptides predicted on computational algorithms, nine peptides could significantly induce interferon gamma (ifn-γ)-producing cd8(+) t cells in mice. these peptides were coupled to the surface of liposomes, and inoculated into mice. six liposomal peptides effectively induced ifn-γ-producing cd8(+) t cells and seven liposomal peptides including the six peptides primed ctls showing in vivo killing activities. further, ctls induced by the seven liposomal peptides lysed an hla-a*0201 positive cell line expressing naturally processed, pp1a-derived peptides. of note, one of the liposomal peptides induced high numbers of long-lasting memory ctls. these data suggest that surface-linked liposomal peptides derived from pp1a might offer an efficient ctl-based vaccine against sars. the outbreak of severe acute respiratory syndrome (sars) in early 2003 led to thousands of infected patients and hundreds of deaths (groneberg et al., 2003) . sars is caused by a novel coronavirus termed sars-associated coronavirus (sars-cov) (drosten et al., 2003; ksiazek et al., 2003; peiris et al., 2003) . this is a plusstranded rna virus with an approximately 30 kb long genome encoding replicase gene products and the structural proteins containing spike, envelop, membrane and nucleocapsid (groneberg et al., 2005) . although the viral receptor has been identified (li et al., 2003b) , the pathogenesis of sars remains poorly understood and the apparent latency of sars-cov in animal reservoirs continuously provides us a serious threat of reemergence. spike protein of sars-cov is a major target for neutralizing antibodies because this protein interacts with the cellular receptor (li et al., 2003b) . high titers of neutralizing antibodies to sars-cov were detected in sera of the recovered patients (li et al., 2003a) , and humoral immunity induced by a dna vaccine contributed to the protection against sars-cov challenge in mice (yang et al., 2004) . these data strongly suggest that neutralizing antibodies play a central role in the clearance of sars-cov. on the other hand, a rapid loss of both cd4 + and cd8 + t cells was observed in patients suffering from severe sars, and the cell counts gradually returned to normal ranges as the patients recovered (tang et al., 2003) . furthermore, certain hla class i alleles have been reported to correlate with sars susceptibility (lin et al., 2003; ng et al., 2004) , suggesting that cytotoxic t lymphocytes (ctls) play an important role in the elimination of sars-cov as well. several ctl epitopes have been identified from spike and nucleocapsid proteins of sars-cov (wang et al., 2004a; wang et al., 2004b; chen et al., 2005; tsao et al., 2006; zhou et al., 2006; ohno et al., 2009) . however, ctl epitopes have not been found in non-structural proteins of sars-cov. in general, non-structural proteins are more conserved and synthesized earlier than structural proteins, and therefore, they could be preferable as an antigenic target for ctls. a synthetic peptide vaccine is a potential candidate for a ctl-based vaccine against pathogenic viruses because short peptides rarely cause undesirable responses including general toxicity, immunosuppression and autoimmunity. however, the immunogenicity of this type of vaccine is very weak. liposomes have extensively been investigated as a delivery system for antigen (alving et al., 1995) . in most cases, it has been prepared by antigen entrapment within the aqueous lumen of liposomes. in contrast, we have previously demonstrated that an ovalbumin (ova)-derived peptide, ova 257-264 conjugated on the surface of liposomes induced ova 257-264 -specific ctls in mice more effectively than did liposomes containing ova 257-264 inside (taneichi et al., 2006; nagata et al., 2007) . furthermore, we have recently shown that surfacelinked liposomal peptides derived from nucleocapsid of sars-cov effectively induced sars-cov-specific ctls in mice (ohno et al., 2009) . these data suggest that liposomes would become an excellent adjuvant vehicle for a synthetic peptide vaccine when a peptide(s) is chemically coupled to the surface of liposomes. in the current study, we attempted to identify hla-a*0201restricted ctl epitopes derived from a largest non-structural polyprotein 1a (pp1a) of sars-cov using computational algorithms and hla-a*0201 transgenic mice. there are several reasons why we focused on this protein. first of all, pp1a protein is a regulatory protein, and hence, more conserved and synthesized earlier than spike and nucleocapsid proteins (groneberg et al., 2005) . second, since pp1a is a largest protein composed of 4382 amino acids among sars-cov-associated proteins (groneberg et al., 2005) , it is more likely to find highly immunogenic, dominant epitopes in pp1a protein than in any other proteins of sars-cov. peptides identified were then chemically conjugated on the surface of liposomes and evaluated for their abilities to induce sars-cov-specific ctls. to define potential hla-a*0201-binding peptides derived from pp1a of sars-cov (urbani strain) (genbank accession number: aap13439), we used two computer-based programs, syfpeithi (http://www.syfpeithi.de/) (rammensee et al., 1999) and bimas (http://www-bimas.cit.nih.gov/molbio/hla bind/) (parker et al., 1994) . as shown in table 1 , 30 peptides with superior scores were selected for predicted ctl epitopes. these peptides were synthesized by operon biotechnologies (tokyo, japan). in addition, three known epitopes derived from the sars-cov spike protein (spike-978: litgrlqsl; spike-1167: rlnevaknl; spike-1203: fiagliaiv) (wang et al., 2004a; wang et al., 2004b) were synthesized as well. t2 (salter et al., 1985) is a transporter associated with antigen processing (tap)-deficient, human lymphoblastoid cell line expressing natural hla-a*0201. c1r-a2 is a human b cell line, hmy2.c1r that was transfected with an hla-a*0201 gene (winter et al., 1991) . t2 and c1r-a2 cells were maintained in rpmi-1640 medium (sigma-aldrich, st. louis, mo) supplemented with 10% fetal calf serum (fcs) (jrh biosciences, lenexa, ks) (r-10) and r-10 containing 500 g/ml g418 (sigma-aldrich), respectively. peptide binding assay was performed as described by ohno et al., 2006 . briefly, t2 cells were suspended in aim v serum-free medium (life technologies, rockville, md) containing 100 nm human beta-2 microglobulin (␤2m) (sigma-aldrich) and were incubated with each synthetic peptide at various concentrations overnight at 37 • c. cells were then stained with the anti-hla-a*0201 monoclonal antibody (mab), bb7.2 (parham and brodsky, 1981) , followed by fluorescein isothiocyanate (fitc)-labeled goat anti-mouse igg (sigma-aldrich). the mean fluorescence intensity (mfi) was measured by flow cytometry (facscan, bd biosciences, san jose, ca). the concentration of each peptide that yields the half-maximal mfi of t2 cells pulsed with a control peptide derived from hepatitis c virus, ns3-1585 (ohno et al., 2006) was calculated as the half-maximal binding level (bl 50 ). experiments were performed three times, and data are given as mean values ± standard deviation (sd). mice express a transgenic hla-a*0201 monochain, designated as hhd, in which human ␤2m is covalently linked to a chimeric heavy chain composed of hla-a*0201 (␣1 and ␣2 domains) and h-2d b (␣3, transmembrane, and cytoplasmic domains) (pascolo et al., 1997; matsui et al., 2004) . eight-to twelve-week-old mice were used for all experiments. mice were housed in appropriate animal care facilities at saitama medical university, saitama, japan, and handled according to international guidelines for experiments with animals. surface-coupled liposomal peptides were prepared as described previously by taneichi et al., 2006 via disuccinimidyl suberate (dss) . briefly, 10 ml of anhydrous chloroform solution containing 0.136 mm dioleoyl phosphatidyl ethanolamine (dope) and 24 l of triethylamine was mixed with 26.6 ml of anhydrous chloroform solution containing 0.681 mm dss and stirred for 5 h at 40 • c. the solvent was evaporated, and 18 ml of a 2:1 mixture of ethyl acetate and tetrahydrofuran was added to dissolve the residue. thirty six milliliters of 100 mm sodium phosphate (ph 5.5) and 90 ml of saturated nacl aqueous solution were added to the solution, shaken for 1 min, and allowed to separate. the upper layer was washed with the same buffer again. after evaporation of the solvent, 3 ml of acetone was added to dissolve the residue. ice-cold acetone (100 ml) was added in drops and kept on ice for 30 min to precipitate. crystals were collected and dissolved in 5 ml of chloroform. after evaporation, 34.4 mg of dope-dss was obtained. for preparation of unsaturated liposomes, dope-dss (0.03 mm), dioleoyl phosphatidylcholine (0.18 mm), cholesterol (0.21 mm), and dioleoyl phosphatidyl glycerol (0.06 mm) were dissolved in 10 ml of chloroform/methanol. the solvent was then removed under reduced pressure and 5.8 ml of phosphate buffer (ph 7.2) was added to make a 4.8% lipid suspension. the vesicle dispersion was extruded through a 0.2 m polycarbonate filter to adjust the liposome size. a 2 ml suspension of dss-introduced liposomes and 0.5 ml of 5 mg/ml peptide solution were mixed and stirred for 3 days at 4 • c. the liposome-coupled and -uncoupled peptides were separated using cl-4b column chromatography. liposomes used in the experiments were prepared under the regulation of good manufacturing practice and involved no detectable endotoxin. for identification of ctl epitopes, mice were immunized intravenously (i.v.) with 2 × 10 7 syngeneic spleen cells pre-pulsed with 10 m of each synthetic peptide. in the case of immunization with liposomal peptides, mice were subcutaneously (s.c.) immunized once or several times at one-week intervals with each of (rammensee et al., 1999) at http://www.syfpeithi.de/. b peptide binding scores to hla-a2.1 were determined by the bimas database (parker et al., 1994) at http://www-bimas.cit.nih.gov/molbio/hla bind/. c data of peptide binding assays are shown as bl50, indicating a concentration of each peptide that yields the half-maximal mfi of t2 cells pulsed with a control peptide, ns3-1585. data are given as mean values ± sd of three independent experiments. surface-coupled liposomal peptides (100 g/mouse) together with cpg-odn (5 -tccatgacgttctgatgtt-3 , hokkaido system science, sapporo, japan) (5 g/mouse) in 100 l pbs in the footpad. ics was performed as described by matsui et al., 2005 . briefly, after one week following immunization, 2 × 10 6 spleen cells of immunized mice were incubated with 10 m of a relevant peptide for 5 h at 37 • c in the presence of brefeldin a (golgiplug tm , bd biosciences). after blocking fc receptors with the rat antimouse cd16/cd32 mab (fc block tm , bd biosciences), cells were stained with fitc-conjugated rat anti-mouse cd8␣ mab (bd biosciences) for 30 min at 4 • c. cells were then fixed, permeabilized, and stained with phycoerythrin (pe)-conjugated rat anti-mouse interferon-gamma (ifn-␥) mab (bd biosciences). after washing the cells, flow cytometric analyses were performed. in vivo ctl assay was carried out as described before by suvas et al., 2003 . in brief, spleen cells from naive hhd mice were equally split into two populations. one population was pulsed with 10 m of a relevant peptide and labeled with a high concentration (2.5 m) of carboxyfluorescein diacetate succinimidyl ester (cfse) (molecular probes, eugene, or). the other population was unpulsed and labeled with a lower concentration (0.25 m) of cfse. an equal number (1 × 10 7 ) of cells from each population was mixed together and adoptively transferred i.v. into mice that had been immunized once with a liposomal peptide two weeks earlier. twelve hours later, spleen cells were prepared and analyzed by flow cytometry. to calculate specific lysis, the following formula was used: % specific a multiepitope minigene that encodes multiple predicted epitopes and natural flanking amino acid sequences proximal to the n and c termini of each epitope (fig. 1a) was synthesized by invitrogen japan k.k. (tokyo, japan). this minigene contains the kozak sequence and the 3xflag-epitope-tag sequence upstream of the multiepitope sequence (fig. 1a) . the minigene was then cloned into the nhe i and hind iii sites of a mammalian expression vector, pacgfp1-hyg-n1 (clontech laboratories inc., mountain view, ca). since this vector encodes a green fluorescent protein (gfp) downstream of the multiple cloning site, the resultant pacgfp1-pp1a plasmid expresses a gfp fusion protein containing pp1a-derived ctl epitopes under the control of the cmv promoter in mammalian cells. this plasmid was transfected into c1r-a2 cells by electroporation (gene pulser, bio-rad laboratories inc., hercules, ca). after selection with hygromycin b (invitrogen, carlsbad, ca) at a final concentration of 1.5 mg/ml, the transfectant, c1r-a2-pp1a was confirmed for their gfp expression by flow cytometry (fig. 1b) . 2.10. 51 cr-release assay 51 cr-release assays were carried out as described before by matsui et al., 2004 . in brief, after two weeks following immunization, spleen cells of immunized mice were cultured with 10 m of a relevant peptide for one week, and used as effector cells in standard 51 cr-release assays. c1r-a2-pp1a cells were labeled with 100 ci of na 2 51 cro 4 , and used for target cells. c1r-a2 cells transfected with pacgfp1-hyg-n1 vector were used as a negative target control. after a 4 h incubation, supernatant of each well was harvested and the radioactivity was counted. results were calculated as the mean of a triplicate assay. percent specific lysis was calculated according to the formula: % specific lysis = [(cpm sample − cpm spontaneous )/(cpm maximum − cpm spontaneous )] × 100. spontaneous release represents the radioactivity released by target cells in the absence of effectors, and maximum release represents the radioactivity released by target cells lysed with 5% triton x-100. statistical comparisons between two groups were performed by the student's t test. one-way anova followed by post-hoc tests were used for statistical analyses between multiple groups in fig. 2 . all statistic analyses were performed using graphpad prism software. a value of p < 0.05 was considered statistically significant. the amino acid sequence of pp1a protein of sars-cov was searched for potential hla-a*0201-restricted ctl epitopes by two computer-based programs, syfpeithi (rammensee et al., 1999) and bimas (parker et al., 1994) . based on the scores calculated, 30 nonameric peptides were selected and synthesized (table 1) . these peptides were then evaluated for their binding affinities to hla-a*0201 molecules as described before by ohno et al., 2006 (table 1) . twenty-four out of the 30 peptides were high binders displaying bl 50 values less than 100 m, and four of them were medium binders displaying bl 50 values ranging from 100 to 200 m, suggesting that most epitopes should be properly predicted. in contrast, two peptides showed low affinity binding. to examine whether the predicted peptides could elicit peptidespecific ctls in vivo, hhd mice were immunized i.v. with syngeneic spleen cells pre-pulsed with each of pp1a-derived peptides. one week after immunization, spleen cells of the immunized mice were prepared and stimulated in vitro with a relevant peptide for 5 h. cd8 + t cells were then analyzed for their peptide-induced intracellular expression of ifn-␥. as shown in fig. 2 , significant numbers of ifn-␥-producing cd8 + t cells (p < 0.01) were detected in mice immunized with syngeneic cells pulsed with each of nine pp1aderived peptides including pp1a-2187, -2207, -2340, -2546, -2755, -2990, -3444, -3687, and -3709 , suggesting that these nine peptides may be hla-a*0201-restricted ctl epitopes derived from sars-cov pp1a protein. since the nine pp1a peptides were expected to be ctl epitopes (fig. 2) , these peptides were conjugated on the surface of liposomes. the resultant surface-linked liposomal peptides were then evaluated for their capabilities of ctl induction in mice. after hhd mice were immunized once with one of the nine liposomal peptides, spleen cells of them were prepared, stimulated with a relevant synthetic peptide, and stained for their expression of surface cd8 and intracellular ifn-␥. as shown in fig. 3 , six liposomal peptides including lip-pp1a-2187 (p < 0.05), -2340 (p < 0.05), -2546 (p < 0.05), -2755 (p < 0.05), -2990 (p < 0.01), and -3709 (p < 0.01), were able to significantly induce ifn-␥-producing cd8 + t cells compared with each negative control stimulated without a relfig. 2 . intracellular ifn-␥ staining of cd8 + t cells specific for pp1a-derived peptides in spleen cells of mice immunized i.v. with peptide-pulsed spleen cells. hhd mice were immunized i.v. once with syngeneic spleen cells pre-pulsed with each of pp1a-derived peptides. one week after immunization, spleen cells were prepared and stimulated with or without (no pep) a relevant peptide for 5 h. cells were then stained for their surface expression of cd8 and their intracellular expression of ifn-␥ (y axis). the data are shown as percentages of intracellular ifn-␥ + cells within cd8 + t cells. three mice were used in each group and the data are shown as the mean ± sd of three mice. one-way anova was used for comparison of data between groups in each of the panels (a-f). *p < 0.01 compared to data of negative controls (no pep). evant peptide. however, numbers of ifn-␥ + cd8 + t cells varied among these liposomal peptides (fig. 3) , suggesting a variety of immunogenicity. in particular, lip-pp1a-3709 was most effective for the induction of peptide-specific ifn-␥ + cd8 + t cells (fig. 3) . in contrast, lip-pp1a-2207 and lip-pp1a-3444 marginally elicited ifn-␥-producing cd8 + t cells, and lip-pp1a-3687 failed to induce ifn-␥ + cd8 + t cells in mice. to further assess immunogenicity of the liposomal pp1a peptides, three known ctl epitopes derived from sars-cov spike protein, spike-978, -1167 and -1203 (wang et al., 2004a; wang et al., 2004b) were conjugated on the surface of liposomes (lip-spike-978, -1167, and -1203), and were compared to the pp1a-derived peptides for their induction of ifn-␥-secreting cd8 + t cells. as shown in fig. 3 , high percentages of ifn-␥ + cells within cd8 + t cells were detected in mice immunized with lip-spike-1203. however, this liposomal peptide was likely to be slightly less effective than lip-pp1a-3709 in the induction of ifn-␥ + cd8 + t cells (fig. 3) . on the contrary, either lip-spike-978 or lip-spike-1167 did not elicit ifn-␥-producing cd8 + t cells in mice (fig. 3) . we next examined in vivo killing activities of peptide-specific ctls in mice immunized with liposomal peptides. two weeks after immunization, both peptide-pulsed cfse high and unpulsed cfse low target cells were delivered into the mice via i.v. injection, and then peptide-specific lysis was analyzed by flow cytometry (fig. 4) . in agreement with the ics data in fig. 3 , high killing activities were observed in mice immunized with lip-pp1a-3709 as well as lip-spike-1203 (fig. 4) . lip-pp1a-2990 also induced a high level of killing activity in mice (fig. 4) . on the other hand, modest killing responses were elicited in mice immunized with each of five liposomal peptides involving lip-pp1a-2187 lip-pp1a, -2340 lip-pp1a, -2546 lip-pp1a, -2755 , whereas any significant cell lysis was not observed in mice injected with either lip-pp1a-2207, -pp1a-3444, -spike-978, or -spike-1167 (fig. 4) . these data also suggest that each of the liposomal peptides exhibits different immunogenicity. to address whether the pp1a-derived peptides identified are naturally processed and presented, liposomal peptide-induced ctls were tested for their capacity to lyse c1r-a2-pp1a cells. c1r-a2-pp1a cells carry the multiepitope minigene that encodes multiple predicted epitopes with several natural flanking amino acid residues at the n and c termini of each epitope (fig. 1a) , and thereby express naturally processed epitopes. expression of the multiepitope minigene was confirmed by detection of gfp in c1r-a2-pp1a cells (fig. 1b) . hhd mice were immunized twice with each of seven liposomal pp1a-derived peptides including lip-pp1a-2187 lip-pp1a, -2340 lip-pp1a, -2546 lip-pp1a, -2755 lip-pp1a, -2990 , and -3709 that significantly induced ifn-␥ + ctls (fig. 3) and/or in vivo killing activities (fig. 4) . ctl activities in spleen cells of the mice were then determined by 51 cr-release assays using c1r-a2-pp1a cells as a target. as shown in fig. 5 , c1r-a2-pp1a cells were significantly (p < 0.01 or p < 0.05) lysed by liposomal peptide-induced ctls, whereas c1r-a2 cells were not recognized by them. these data indicate that the seven pp1a-derived peptides are naturally processed and presented to ctls. we next examined whether long-lasting peptide-specific ctls could be elicited by immunization with liposomal peptides. hhd mice were immunized three times at one-week intervals with either lip-pp1a-2990 or lip-pp1a-3709. spleen cells were then prepared at various days after the final immunization, and stimulated in vitro once with a relevant peptide for 5 h at 37 • c. cd8 + t cells were then analyzed for their peptide-induced expression of intracellular ifn-␥. in both of the cases, the total numbers of ifn-␥-producing cd8 + t cells per mouse spleen were slightly increased at day 3 after immunization, rose to a peak at day 7, and gradually decreased as days went by (fig. 6a and b) . as shown in fig. 7 , ifn-␥-producing cd8 + t cells were still detected in mice 75 days after immunization with any of the two liposomal peptides. these results demonstrate that immunization with these liposomal peptides generated long-lasting memory ctls. especially, the frequency of ifn-␥ + cd8 + t cells in mice injected with lip-pp1a-3709 was quite high (fig. 7) , indicating that lip-pp1a-3709 may be an excellent vaccine candidate. in the current study, high-performing computational algorithms have extensively been utilized for the prediction of ctl epitopes derived from pp1a protein of sars-cov. as shown in table 1 , 30 peptides were selected as potential ctl epitopes using syfpei-thi (rammensee et al., 1999) and bimas (parker et al., 1994) . out of them, only nine peptides could significantly induce ifn-␥-producing cd8 + t cells in mice (fig. 2) . furthermore, there was not always a good correlation between peptides identified by the algorithms and their activity in the biological assays. for instance, pp1a-2340 and pp1a-3709 showed relatively inferior scores in bimas (table 1) , whereas both peptides stimulated good ctl responses (figs. 2-4) . thus, currently available algorithms have limited accuracy to find actual epitopes (chentoufi et al., 2008) , fig. 4 . in vivo killing activities specific for peptides derived from pp1a and spike of sars-cov in mice immunized with surface-linked liposomal peptides. hhd mice were immunized once with either each liposomal peptide (lip-peptide) or liposomes alone (lip) together with cpg. one week later, an equal number of a relevant peptide (pp1a-2187 , pp1a-2207 , pp1a-2340 , pp1a-2546 , pp1a-2755 , pp1a-2990 , pp1a-3444, pp1a-3687, pp1a-3709, spike-978, spike-1167 spike-1203)-pulsed cfse high targets (m2) and unpulsed cfse low targets (m1) were transferred into the immunized mice by i.v. injection. after 12 h, cfse-labeled cells were recovered from spleens of recipient mice and analyzed by flow cytometry. the numbers are the percentages of specific lysis shown as mean values ± sd of three independent experiments. however, they are still quite useful because we can easily choose a cluster of promising peptides within a huge protein such as pp1a on the programs. multiple immunological screenings have been advanced to validate predicted ctl epitopes. when used individually, each screen is not sufficient for identifying actual epitopes. however, various combinations of these screens are usually successful (chentoufi et al., 2008; ohno et al., 2009 ). therefore, we performed multiple screenings, including cell surface stabilization of hla-a*0201 molecules on t2 cells, detection of antigen-driven ifn-␥-producing cd8 + t cells, and functional in vivo and in vitro ctl assays. in these experiments, we took advantage of highly reactive hla-a*0201 transgenic mice, termed hhd mice (pascolo et al., 1997) . in hhd mice, the innate h-2d b and mouse ␤2m genes have been disrupted by homologous recombination, and therefore, the only mhc class i molecule on the cell surface, hla-a*0201, is efficiently utilized by hla-a*0201-restricted ctls. as a consequence, six peptides conjugated on the surface of liposomes significantly induced ifn-␥-producing cd8 + t cells (fig. 3) , and seven liposomal peptides including the six peptides primed ctls showing peptidespecific killing activities in mice (fig. 4) . however, it has to be taken account that there may be differences between the immunogenic variation observed in hla class i transgenic mice and that in humans primarily because the antigen processing, presentation and ultimately, immunodominance may differ between them. in fact, it was shown that several hla-a*0201-restricted ctl epitopes derived from human papillomavirus were not processed in hdd mice although these epitopes were naturally processed in hla-a*0201 + humans (street et al., 2002) , indicating that cross-species incompatibility in antigen-processing and presentation machinery skews the presentation of some ctl epitopes. because pp1a-specific ctls induced were generated by stimulation with synthetic peptides, it was necessary to test whether they would recognize naturally processed peptides. to this end, we generated an hla-a*0201 positive c1r-a2-pp1a cell line in substitution for sars-cov-infected target cells because it is quite fig. 5 . recognition of naturally processed epitopes derived from pp1a. hhd mice were immunized twice with either lip-pp1a-2187 , lip-pp1a-2340 , lip-pp1a-2546 , lip-pp1a-2755 , lip-pp1a-2990 , or liposomes alone. two weeks after immunization, spleen cells were prepared and stimulated in vitro with a relevant peptide. after one week, 51 cr release assays were performed at an e:t ratio of 150 with c1r-a2-pp1a cells (gray bars) or c1r-a2 (black bars) as targets. data are shown as the means ± sd of triplicate wells. the experiment was repeated twice with similar results. at least three mice per group were used in each experiment. *p < 0.01; **p < 0.05; ns, not significant. difficult to obtain live sars-cov in japan. c1r-a2-pp1a cells carry the multiepitope minigene that encodes nine predicted epitopes with several natural flanking amino acid residues at the n and c termini of each epitope (fig. 1) . the basic idea to utilize flanking amino acids (fig. 1) comes from the observation that flanking amino acid sequences modulate antigen processing of ctl epitopes (le gall et al., 2007) . in fact, it was shown that several mutations at nterminal (draenert et al., 2004; milicic et al., 2005) and c-terminal (allen et al., 2004; milicic et al., 2005) flanking residues of ctl epitopes disrupt proteasomal processing of hiv gag and nef proteins. therefore, addition of flanking amino acid sequences allows natural antigen processing of ctl epitopes in c1r-a2-pp1a cells. as shown in fig. 5 , ctls induced by seven liposomal peptides recognized hla-a*0201 positive c1r-a2-pp1a cells. these data indicate that the seven peptides are naturally processed epitopes. as a matter of course, it will be necessary to examine whether pp1a-derived peptides can induce protective ctls using sars-cov at the final screening. so far, several ctl epitopes have been identified from spike and nucleocapsid proteins of sars-cov (wang et al., 2004a; wang et al., 2004b; chen et al., 2005; tsao et al., 2006; zhou et al., 2006; fig. 6 . kinetics of pp1a-specific cd8 + t cell responses after immunization with surface-linked liposomal peptides. spleen cells were prepared from mice at various days after immunization with either lip-pp1a-2990 (open circles in a), lip-pp1a-3709 (open circles in b) or liposomes alone (closed circles in a and b). after stimulation with a relevant peptide for 5 h, cells were stained for their surface expression of cd8 and their intracellular expression of ifn-␥. the data indicate the total numbers of intracellular ifn-␥ + cd8 + t cells per mouse spleen, and are shown as the means ± sd of three mice per group. significant (*p < 0.05) difference compared to mice immunized with liposomes alone on the same day post immunization. ohno et al., 2009) . to our knowledge, however, the current study is the first report to demonstrate ctl epitopes derived from a nonstructural protein of sars-cov such as pp1a protein. in fact, several liposomal peptides derived from pp1a induced high frequencies of ifn-␥-producing cd8 + t cells (fig. 3) in comparison with liposomal peptides derived from nucleocapsid which we have recently published (ohno et al., 2009) . in particular, lip-pp1a-3709 turned out to be most effective in the induction of antigen-driven ifn-␥producing cd8 + t cells (fig. 3) , indicating that pp1a-3709 is a highly immunogenic, dominant ctl epitope. it was demonstrated that the surface-linked liposomal peptide was effective for peptide-specific ctl induction in the current study as well as in the previous study (ohno et al., 2009) . it is noteworthy that long-lasting memory ctls were detected in mice 75 days after immunization with liposomal peptides (fig. 7) . these data suggest that the surface-linked liposomal peptide may be an effective tool for ctl-based immunotherapy against infectious diseases such as sars. the surface-linked liposomal peptide might be similar to the lipopeptide, a form of palmitoyl-lipidated peptide that is currently under intense investigation as human vaccines (zhu et al., 2004; zhang et al., 2009) . although both effectively induce peptide-specific ctls, there are several differences between them. the lipopeptide is self-adjuvanting to stimulate peptide-specific ctls via toll-like receptor (trl)-2 (zhu et al., 2004; zhang et al., 2009 ), but the surface-linked liposomal peptide requires external tlr ligands such as cpg (nagata et al., 2007) . however, cpg causes toxicity in humans (davila et al., 2003) , and hence, it is essential to find out a safe adjuvant for clinical use of liposomal peptides. on the other hand, the lipopeptide alone without a cd4 + t-cell epitope fig. 7 . induction of long-lasting ifn-␥-producing cd8 + t cells in mice immunized with surface-linked liposomal peptides. hhd mice were immunized three times at one-week intervals with either lip-pp1a-2990, lip-pp1a-3709 or liposomes alone (lip-no peptide). spleen cells were then prepared 75 days after the final immunization, and stimulated in vitro once with (+) or without (−) a relevant peptide for 5 hours at 37 • c. cells were then stained for their surface expression of cd8 (x axis) and their intracellular expression of ifn-␥ (y axis). the numbers shown indicate the percentages of intracellular ifn-␥ + cells within cd8 + t cells. the experiment was repeated twice with similar results, and the data shown are representative of the two independent experiments. at least three mice per group were used in each experiment, and spleen cells of mice per group were pooled. failed to induce ctl-based protective immunity, whereas a helper peptide is not necessary for the surface-linked liposomal peptide to induce peptide-specific ctls. although we focused this study on ctl epitopes restricted by hla-a*0201 which is the most common hla class i allele in the world, this is just a model system that could be applied to any haplotypes. the hla polymorphism should hinder the development of our system, but the supertypes of hla class i may solve this issue. sette and sidney, 1999 defined only nine hla class i supertypes that almost cover the entire repertoire of hla class i molecules. epitopes related to all of the nine supertypes should be identified and incorporated into the liposomal vaccine. in summary, we have identified seven hla-a*0201-restricted ctl epitopes derived from pp1a protein of sars-cov using computational algorithms, hla-a*0201 transgenic mice and the surface-linked liposomal peptide. it was shown that one of the liposomal pp1a peptides was effective for peptide-specific ctl induction in mice, and efficiently elicited long-lasting memory ctls. these data suggest that surface-linked liposomal peptides derived from pp1a protein may offer an effective and safe ctl-based vaccine against sars. selection, transmission, and reversion of an antigen-processing cytotoxic t-lymphocyte escape mutation in human immunodeficiency virus type 1 infection liposomes as carriers of peptide antigens: induction of antibodies and cytotoxic t lymphocytes to conjugated and unconjugated peptides response of memory cd8 + t cells to severe acute respiratory syndrome (sars) coronavirus in recovered sars patients and healthy individuals hla-a*0201-restricted cd8 + cytotoxic t lymphocyte epitopes identified from herpes simplex virus glycoprotein d generation of antitumor immunity by cytotoxic t lymphocyte epitope peptide vaccination, cpg-oligodeoxynucleotide adjuvant, and ctla-4 blockade immune selection for altered antigen processing leads to cytotoxic t lymphocyte escape in chronic hiv-1 infection identification of a novel coronavirus in patients with severe acute respiratory syndrome molecular mechanisms of severe acute respiratory syndrome (sars) severe acute respiratory syndrome: global initiatives for disease diagnosis a novel coronavirus associated with severe acute respiratory syndrome portable flanking sequences modulate ctl epitope processing profile of specific antibodies to the sars-associated coronavirus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus association of hla class i with severe acute respiratory syndrome coronavirus infection adjuvant activities of novel cytokines, interleukine (il)-23 and il-27 for induction of hepatitis c virus-specific cytotoxic t lymphocytes in hla-a*0201 transgenic mice t-bet is required for protection against vaccinia virus infection cd8 + t cell epitope-flanking mutations disrupt proteasomal processing of hiv-1 nef peptides coupled to the surface of a kind of liposome protect infection of influenza viruses association of human-leukocyte-antigen class i (b*0703) and class ii (drb1*0301) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome synthetic peptides coupled to the surface of liposomes effectively induce sars coronavirus-specific cytotoxic t lymphocytes and viral clearance in hla-a*0201 transgenic mice immunogenic variation between multiple hla-a*0201-restricted, hepatitis c virus-derived epitopes for cytotoxic t lymphocytes partial purification and some properties of bb7.2: a cytotoxic monoclonal antibody with specificity for hla-a2 and a variant of hla-a28 scheme for ranking potential hla-a2 binding peptides based on independent binding of individual peptide sidechains hla-a2.1-restricted education and cytolytic activity of cd8 + t lymphocytes from ␤2 microglobulin (␤2m) hla-a2. 1 monochain transgenic h-2d b ␤2m double knockout mice coronavirus as a possible cause of severe acute respiratory syndrome syfpeithi: database for mhc ligands and peptide motifs genes regulating hla class i antigen expression in t-b lymphoblast hybrids nine major hla class i supertypes account for the vast preponderance of hla-a and -b polymorphism limitations of hla-transgenic mice in presentation of hla-restricted cytotoxic t-cell epitopes from endogenously processed human papillomavirus type 16 e7 protein cd4 + cd25 + t cells regulate virus-specific primary and memory cd8 + t cell responses antigen chemically coupled to the surface of liposomes are cross-presented to cd8 + t cells and induce potent antitumor immunity measurement of subgroups of peripheral blood t lymphocytes in patients with severe acute respiratory syndrome and its clinical significance hla-a*0201 t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus nucleocapsid and spike proteins identification of an hla-a*0201-restricted cd8 + t-cell epitope ssp-1 of sars-cov spike protein t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus spike protein elicit a specific t-cell immune response in patients who recover from sars the 45 pocket of hla-a2.1 plays a role in presentation of influenza virus matrix peptide and alloantigens a dna vaccine induces sars coronavirus neutralization and protective immunity in mice a genital tract peptide epitope vaccine targeting tlr-2 efficiently induces local and systemic cd8 + t cells and protects against herpes simplex virus type 2 challenge screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes lipopeptide epitopes extended by an n ε -palmitoyllysine moiety increase uptake and maturation of dendritic cells through a toll-like receptor-2 pathway and trigger a th1-dependent protective immunity this work was supported by a grant from the ministry of health, labor and welfare of japan. the authors are grateful to dr. f. a. lemonnier (pasteur institute, paris, france) for providing hhd mice. key: cord-326554-iphe3rni authors: joshi, amit; kaushik, vikas title: in-silico proteomic exploratory quest: crafting t-cell epitope vaccine against whipple’s disease date: 2020-05-18 journal: int j pept res ther doi: 10.1007/s10989-020-10077-9 sha: doc_id: 326554 cord_uid: iphe3rni whipple’s disease is one of the rare maladies in terms of spread but very fatal one as it is linked with many disorders (like gastroenteritis, endocarditis etc.). also, current regimens include less effective drugs which require long duration follows up. this exploratory study was conducted to commence the investigation for crafting multi target epitope vaccine against its bacterial pathogen tropheryma whipplei. the modern bioinformatics tools like vaxijen, netmhcii pan 3.2, allergen-fp, patch-dock, toxic-pred, mhcpred and iedb were deployed, which makes the study more intensive in analyzing proteome of t. whipplei as these methods are based on robust result generating statistical algorithms ann, hmm, and ml. this immuno-informatics approach leads us in the prediction of two epitopes: vlmvsafpl and irylaalhl interacting with 4 and 6 hla drb1 alleles of mhc class ii respectively. vlmvsafpl epitope is a part of dna-directed rna polymerase subunit beta, and irylaalhl epitope is a part of membranous protein insertase yidc of this bacterium. molecular-docking and molecular-simulation analysis yields the perfect interaction based on atomic contact energy, binding scores along with rmsd values (0 to 1.5 ǻ) in selection zone. the iedb (immune epitope database) population coverage analysis exhibits satisfactory relevance with respect to world population. george hoyt whipple in 1907 explains whipple's disease, as a multisystemic chronic infectious disease. he identified silver stained rod shaped bacterium in vacuoles associated with macrophages of patients, he initially did not think of them as the cause for the disease rather he think that intestinal lipodystrophy (whipple's disease) was caused due to some novel disturbances in fat metabolic schemes (whipple 1907) . when the first successful treatment started by using antibiotics in 1952, determined that this bacterium might be the major causative agent of this disease (paulley 1952 ). an electron microscopic study in 1960′s provided additional support for this hypothesis (cohen et al. 1960; yardley and hendrix 1961 ). whipple's disease occurs uncommonly, as a multisystemic disorder (inexact annual frequency less than 1 per 1,000,000 populace) that specially affects middle-aged caucasian men (fenollar et al. 2007; ramharter et al. 2014 , dobbins et al. 1981 ). this bacterium was found to mostly affect small children (keita et al. 2015) and sewage workers (schöniger-hekele et al. 2007 ). since, its first portrayal by whipple in very beginning of first decade in twentieth century (whipple 1907) , a limited progresses with in pathogenesis, prognosis, and treatment of the malady have been made. the bacterium gets internalized in to lamina propria of intestine and then make its way to mucosal macrophages, as this bacterium induces the decreased expression of cd11b in such macrophages (cd11b on macrophages frequently mediates the intracellular degradation of bacteria) causes flip in the scenario (inappropriate antigen presentation by such macrophages and dendritic cells). this specially reasons the boom in il-10, tgf-β and ccl-18 expression and decrease in ifn-γ, which in turn causes destroy in maturation of phagosomes and decrease in thioredoxin expression, lead them unable to kill bacterium and antigen presentation (moss et al. 2006 (moss et al. , 2010 . an unseemly development of proficient antigen-presenting cells caused by the presence of interleukin 10 and interleukin 16, and the non appearance of interferon γ and interleukin 12 might lead to inadequate antigen-presentation and hinder the incitement of antigen-specific t-helper 1 cells enhancing growth and systemic spread of tropheryma whipple. the nearby generation of provocative cytokines through macrophages and endothelial cells within the fringe might actuate lymphocyte invasion through a defective endothelial obstruction taken after by central aggravation, indeed in immunologically ensured tissues such as joints or the neuronal domain (schneider et al. 2008) .currently hydroxychloroquine (600 mg/day) and doxycycline (200 mg/day) used for treatment of whipple's disease for 12-18 months, but life time follow up is required (lagier et al. 2014) , so it is time consuming treatment process and only few handful trials were conducted in earlier studies (feurle et al. 2013) . nowadays epitope based vaccines provide better options in search of good treatment strategy for such type of harmful and rare malady, even if the individuals are genetically predisposed as in case of classical whipple's disease (trotta et al. 2017) . this modern approach of putative vaccine determination which involves the use of proteomic databases is very handy and easy to use method not only for rare bacterial pathogens, but also very effective in case of harmful viruses like nipah (kaushik 2019) . tropheryma whipplei was found to be associated with major ailments like gastroenteritis and endocarditis (fenollar et al. 2013) . in this research work, five proteins from proteomic data of t. whipplei were analyzed for allergenicity. non-allergenic proteins were deployed for predicting epitopes. predicted epitopes were subjected for immunogenic properties, structural modeling and the docking with corresponding mhc ii alleles to investigate the strong binding affinity. method is more economic, time efficient, and harmless when compared to the vaccine designing and testing in wet lab and animal testing strategies (kumar et al. 2015) . reverse vaccinology is the suitable approach as well as novel science method that use the genomic data with the utilization of computer for the arrangement of antibodies without culturing bacterium species (kanampalliwar et al. 2013; tang et al. 2012) . it allow the choice in hands of human interface for selecting antigens from pathogenic set of dna and most antigenic areas could be used to synthesize potential immunization to initiate defensive responses against such pathogenic species (ada et al. 2018) . epitopes based antibodies selection and production is explicitly less time consuming, economical and considered safest approach in vaccine designing. earlier computational methods were found to be successful in analyzing genome and prediction of putative drugs for t. whipplei (palanisamy 2018) , such studies provide motivation to craft vaccine targets by deploying in-silico approach. t-cell epitopes were screened out in this study may effectively elicit immune responses against this bacterium, and also similar type of recent study was found to be successful in determining epitope based vaccine agents for sars-cov2 (joshi et al. 2020) . brief flow chart of the study used to determine putative epitope based vaccine candidates against t. whipplei is presented in fig. 1 . proteomes were retrieved in fasta format from ncbi-genbank and uniprotkb databases. five proteins of different functionality were selected with following accession no's: wp_042507409.1 dna-directed rna polymerase subunit beta (rpo-b), wp_033800049.1 co-chaperone groes, wp_038104819.1 terc/alx family metal homeostasis membrane protein, wp_042505650.1 membrane protein the protein sequences were then deployed for further analysis based on allergen fp v 1.0 for predicting allergenicity (dimitrov et al. 2014) . net mhcii pan 3.2 server is used to find and screen out hla alleles which have good interaction with selected non-allergens of pathogenic origin (jensen et al. 2018 ). to bring higher confidence in selecting epitope, vaxijen server is deployed to determine antigenicity with threshold ≥ 0.7 for selected rare bacterium (doytchinova et al. 2007 ). by subjecting proteomic sequences to net mhcii pan 3.2 server we obtained 1147 epitopes for wp_042507409.1, 90 epitopes for wp_033800049.1, 309 epitopes for wp_038104819.1, 302 epitopes for wp_042505650.1, and 510 epitopes for wp_011096746.1,this server was used because of its neural networking algorithm based approaches for fine predictions. 1-log50k (affinity score) ≤ 0.6 is used to screen out possible epitopes presented in table 2 . these epitopes were further subjected for antigenicity analysis based on vaxijen scores. toxicity for putative peptides was designated by using svm scores from toxin pred web server (gupta et al. 2013) . non toxic peptides were finalized for further analysis. the tertiary structure or 3d structure for epitope is determined by using pep-fold 3 web server (lamiable et al. 2016; shen et al. 2014; thévenet et al. 2012) . and predicted human leukocyte antigen alleles 3d structure was obtained from rcsb pdb database (berman et al. 2000) . also ramachandran plot analysis was conducted for verification of results by using molprobity server (williams et al. 2018 ). the docking experiments was conducted by using patch-dock tool (schneidman-duhovny et al. 2005) , the predicted docked models of putative epitope and hla alleles was selected on the basis of score, which relies on highest geometric shape complementarities and atomic contact energy (zhang et al. 1997 ). this allows the best selection of epitope and hla allele interaction. this tool is easy to deploy for all life science domains. immune epitope database (iedb) analysis resource tool of population coverage was used to predict population coverage of the putative epitopes that are exhibiting interaction to hla alleles and based on mhc-ii restriction data (bui et al. 2006) . mhcpred tool was deployed for quantitative prediction of selected epitopes interacting to major histocompatibility complexes (guan et al. 2003 ). epitope-hla allele docked sets were then used for simulation and dynamics analysis by deploying namd (phillips et al. 2005 ) associated with vmd (visual molecular dynamics) tool (humphrey et al. 1996) . total 5 protein sequences were analyzed for allergenicity and depicted as non-allergen in table 1 by using allergenfp tool. net mhcii pan 3.2 server is deployed to identify promiscuous epitopes and probable hla alleles of mhc class ii 3d structural models of selected epitopes were designed by using pep-fold 3 web server and than most common hla drb1 proteins structural models were derived by using rcsb-pdb database. in table 3 pdb id along with hla alleles is exhibited. molprobity results of ramachandran plot analysis results shows satisfactory structural prediction (> 85% residues in favorable region) of epitopes that were finalized at last in fig. 8 . patchdock tool was deployed for interaction between selected structures of epitopes and hla drb1 proteins. then interaction data produced by docked molecules include ace (atomic contact energy) and best model score that leads to the final selection in the way of prediction for each pair. in table 4 the selected models and rejected models both were included to enhance the comparative analysis. the two selected epitopes were vlmvsafpl and irylaalhl interacting with 4 and 6 hla drb1 alleles respectively. vlmvsafpl epitope is a part of dna-directed rna polymerase subunit beta and irylaalhl epitope is a part of murein biosynthesis integral membrane protein of t. whipplei and are major identifiers of this bacterium. figure 2 clearly depicts the good interaction between epitopes and hla alleles in docked results. in fig. 2a docked result of irylaalhl with hla-drb1* 01:01 exhibits perfect hydrogen bond due to presence of tyrosine residue in epitope at 3rd position, while most of the other non polar amino acids of this epitope are depicts vander waals interactions with in the hla model and in fig. 2c docked result of vlmvsafpl with hla-drb1* 04:04 exhibits perfect (schlundt et al. 2012; chen et al. 2013) . the reference docked peptides have great difference in amino acid sequence in comparison to our screened epitopes but exhibits some resemblance alike of our epitopes in interaction towards antigen binding pocket. figure 3a , b represents the free undocked hla-drb1 receptors (4ah2, 4is6 respectively), while fig. 3c , d represents free unbound putative epitopes (irylaalhl, vlmvsafpl respectively) and their side chains. figure 4 graphically represents the selected epitopes and hla alleles of mhc ii on the basis of ace values. predicted epitopes vlmvsafpl and irylaalhl have vaxijen scores 0.9461 and 1.2114 respectively, they are also of non toxic nature as per the study of toxin pred tool and its toxicity scores (svm score) represented in table 5 . in table 6 quantitative estimation of best interaction of epitope with hladrb1 alleles were achieved with upright ic 50 values by using mhcpred tool, this allows confidence of prediction. table 7 shows half-life and instability index for putative epitopes by deploying protparam expasy tool. vlmvsafpl and irylaalhl manifest 28.82% and 37.06% elicitation of immune responsiveness by world population by availing iedb tool. the epitopes vlmvs-afpl and irylaalhl shows greater effect in european population by 29.63% and 42.68% respectively, and correspondingly similar results with north american population coverage analysis. this indicates its greater relevance in treatment of whipple's disease as it is mostly seen in caucasoid population. in figs. 5 and 6 it is clearly represented in a graphical representation. namd was deployed for simulation studies on docked epitope-hla allele sets to obtain rmsd values. maximum value of rmsd for vlmvsafpl and irylaalhl epitopes were analyzed, this gives more confidentiality in selection of vaccine candidate against tropheryma whipplei. figures 7 and 8 shows rmsd plots that indicates clear picture of selection of these two epitopes. immuno-informatics is the suitable approach as well as novel science method that use the proteomic data with the utilization of computer systems for predicting epitopes without culturing bacterium species (kanampalliwar et al. 2013; tang et al. 2012) . it allow the choice in hands of human interface for selecting antigens from pathogenic set of dna and most antigenic areas could be used to synthesize potential immunization to initiate defensive responses against harmful pathogenic species (ada et al. 2018 ). insilico approach was earlier successful in case of staphylococcus aureus (delfani et al. 2015) , mycobacterium -367.28-321.11-353.51-353.51-353.51-349.18-443.31-443.31-226.35-297.99 tuberculei (mustafa 2013 ) and numerous bacterial species, but t. whipplei is still not fully explored in this domain. current regimens include hydroxychloroquine and doxycycline for treatment of whipple's disease for 12-18 months, but life time follow up is required (lagier et al. 2014) , so it is time consuming treatment process and (feurle et al. 2013) . in present study we identified two possible epitopes that can interact with mhc-ii alleles to elicit immune response on individuals namely vlmvsafpl epitope (part of dnadirected rna polymerase subunit beta), and irylaalhl epitope (part of murein biosynthesis integral membrane protein). these epitopes exhibit better interaction with hla drb1 alleles, as confirmed by deploying molecular-docking and molecular-simulation studies (adhikari et al. 2018) . population coverage analysis was found to be satisfactory and in earlier studies it was used in strengthening vaccine prediction aspects (misra et al. 2011) . very similar studies were also conducted successfully for related bacterium mycobacterium avium and found to be successful in predicting epitopes (gurung et al. 2012 − 321.1, − 353.5, − 353.5, − 353.5, − 349.1 respectively) in docking results similar type of methodology was seen in recent studies in screening epitopes for sars-cov-2 (joshi et al. 2020) . both selected epitopes exhibit structural integrity as possess less than 35% instability index score, and half life greater than 20 h for mammalian reticulocytes, this makes the screening criteria more reliable. also, more than 85% residues of selected epitopes come under favorable region in ramachandran plot analysis (fig. 9 ). still no one has used vaccine based treatments for whipple's disease, as it is thought to be rare and possess reduced genome but considered one of the harmful pathogen of human ( raoult et al. 2003; la scola et al. 2001; marth et al. 2016) .the effectiveness of epitope based vaccines for treatment of endocarditis has already been claimed (priyadarshini et al. 2014) . but in our study we found the short peptides that can easily be synthesized and deployed in developing immunity in caucasian populations against whipple's disease. in this study we obtained vlmvsafpl and irylaalhl as predicted epitopes for vaccine crafting. this novel approach in crafting vaccine based treatment of t. whipplei will open new doors in research for creating regimens to treat such harmful bacterium by developing adaptive immune response and eradicating it globally before any future escalations takes place. the predicted epitopes can be deployed in crafting vaccines against t. whipplei bacterium after molecular-wet lab corroboration. current progress of immunoinformatics approach harnessed for cellular and antibody-dependent vaccine design immunoinformatics approach for epitope-based peptide vaccine design and active site prediction against polyprotein of emerging oropouche virus the protein data bank predicting population coverage of t-cell epitope-based diagnostics and vaccines structure-based design of altered mhc class ii-restricted peptide ligands with heterogeneous immunogenicity ultrastructural abnormalities in whipple's disease in silico analysis for identifying potential vaccine candidates against staphylococcus aureus allergenfp: allergenicity prediction by descriptor fingerprints is there an immune deficit in whipple's disease? vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines whipple's disease intravenous seftriaxone, followed by 12 or three months of oral treatment with trimethoprim-sulfamethoxazole in whipple's disease mhcpred: a server for quantitative prediction of peptide-mhc binding in silico approach for predicting toxicity of peptides and proteins in silico identification of epitopes in mycobacterium avium subsp. paratuberculosis proteins that were upregulated under stress conditions vmd-visual molecular dynamics improved methods for predicting peptide binding affinity to mhc class ii molecules epitope based vaccine prediction for sars-cov-2 by deploying immuno-informatics approach reverse vaccinology: basics and applications silico identification of epitope based peptide vaccine for nipah virus high prevalence of tropheryma whipplei in lao kindergarten children protective enterotoxigenic escherichia coli antigens in a murine intranasal challenge model description of tropheryma whipplei gen. nov., sp. nov., the whipple's disease bacillus treatment of classical whipple's disease: from in vitro results to clinical outcome pep-fold3: faster de novo structure prediction for linear peptides in solution and in complex tropheryma whipplei infection and whipple's disease population coverage analysis of t-cell epitopes of neisseria meningitidis serogroup b from iron acquisition proteins for vaccine design reduced peripheral and mucosal tropheryma whipplei specific th1 response in patients with whipple's disease impaired immune functions of monocytes and macrophages in whipple's disease in silico analysis and experimental validation of mycobacterium tuberculosis-specific proteins and peptides of mycobacterium tuberculosis for immunological diagnosis and vaccine development identification of putative drug targets and annotation of unknown proteins in tropheryma whipplei a case of whipple's disease (intestinal lipodystrophy) scalable molecular dynamics with namd genome-based approaches to develop epitope-driven subunit vaccines against pathogens of infective endocarditis makristathis a (2014) prevalence and risk factor assessment of tropheryma whipplei in a rural community in gabon: a community based cross-sectional study tropheryma whipplei twist: a human pathogenic actinobacteria with a reduced genome peptide linkage to the α-subunit of mhcii creates a stably inverted antigen presentation complex whipple's disease: new aspects of pathogenesis and treatment patchdock and symmdock: servers for rigid and symmetric docking tropheryma whipplei in the environment: survey of sewage plant influxes and sewage plant workers improved pep-fold approach for peptide and miniprotein structure prediction the epitopes of foot and mouth disease pep-fold: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides peripheral t-cell reactivity to heat shock protein 70 and its cofactor grpe from tropheryma whipplei is reduced in patients with classical whipple's disease a hitherto undescribed disease characterized anatomically by deposits of fat and fatty acids in the intestinal and mesenteric lymphatic tissues molprobity: more and better reference data for improved all-atom structure validation combined electron and light microscopy in whipple's disease. demonstration of "bacillary bodies" in the intestine determination of atomic desolvation energies from the structures of crystallized proteins key: cord-275677-hbv49e01 authors: ramana, lakshmi narashimhan; anand, appakkudal r.; sethuraman, swaminathan; krishnan, uma maheswari title: targeting strategies for delivery of anti-hiv drugs date: 2014-10-28 journal: j control release doi: 10.1016/j.jconrel.2014.08.003 sha: doc_id: 275677 cord_uid: hbv49e01 human immunodeficiency virus (hiv) infection remains a significant cause of mortality globally. though antiretroviral therapy has significantly reduced aids-related morbidity and mortality, there are several drawbacks in the current therapy, including toxicity, drug–drug interactions, development of drug resistance, necessity for long-term drug therapy, poor bio-availability and lack of access to tissues and reservoirs. to circumvent these problems, recent anti-hiv therapeutic research has focused on improving drug delivery systems through drug delivery targeted specifically to host cells infected with hiv or could potentially get infected with hiv. in this regard, several surface molecules of both viral and host cell origin have been described in recent years, that would enable targeted drug delivery in hiv infection. in the present review, we provide a comprehensive overview of the need for novel drug delivery systems, and the successes and challenges in the identification of novel viral and host-cell molecules for the targeted drug delivery of anti-hiv drugs. such targeted anti-retroviral drug delivery approaches could pave the way for effective treatment and eradication of hiv from the body. cells i.e. cd4 + t cells, monocytes, macrophages, and dendritic cells. the decline in cd4 + t-cells in the more advanced stages of infection is responsible for the profound immune suppression that characterizes the advanced stage of aids. however, the advent of highly active anti-retroviral therapy (haart), a combination of drugs that inhibit hiv-1 replication, has led to reduced viremia and the onset of opportunistic infections in most patients and prolonged survival. chemotherapy has been the main mode of treatment of aids. the common classes of drugs that have been employed in the treatment of hiv infections are designed to inhibit a particular stage in the infectious cycle of hiv. fig. 1 depicts the different targets for the various antiretroviral drugs. entry inhibitors comprising fusion inhibitors and coreceptor inhibitors prevent the entry of hiv into the host cell. while fusion inhibitors interact with the glycoprotein-41 (gp-41) on the viral envelope thereby blocking its fusion with the cell membrane, the coreceptor inhibitors interact with the ccr5 receptor in the host cell and prevent their interactions with the glycoprotein-120 (gp-120) of the virus [2] . protease inhibitors (pis) interfere with the proteolytic processing of viral proteins through binding interactions with the active site of the hiv protease [3] . the reverse transcriptase inhibitors form a major class of anti-retroviral drugs. these molecules disrupt the transformation of the single stranded rna of the viral genome into a double stranded dna by the referred to as nucleoside reverse transcriptase inhibitors (nrtis) and nucleotide reverse transcriptase inhibitors (ntrtis) respectively. these inhibitors binds directly to the reverse transcriptase enzyme and thus block the hiv life cycle. a related class of drugs known as non-nucleoside reverse transcriptase inhibitors (nnrtis) binds to an allosteric site of reverse transcriptase resulting in inhibition of the enzyme activity. a recent addition to the repertoire of anti-retroviral drugs is the integrase inhibitors that interfere with the integration of the viral genome with the host cell. list of anti-hiv drugs are listed in table 1 . anti-retroviral therapy (arv) has significantly reduced aids-related morbidity and mortality. however there are several drawbacks in the current arv therapy. a) drug resistance: one other major problem with anti-hiv drug therapy is that of resistance. the process of hiv replication is rapid and error-prone (~10 billion viral particles are produced on a daily basis), while generating at least one mutation per genome. these genetic mutations enable the virus to develop resistance to anti-hiv drug therapy, especially when monotherapy is employed. an example is the emergence and selection of hiv subtype c virus in india, which contains multiple nf-kb (sites?) and strengthens the promoters of the virus. b) long-term drug therapy: the nature of hiv infection, including viral persistence in reservoirs necessitates long-term uninterrupted multi-drug arv therapy. treatment compliance is critical regardless of whether a patient is treatment-naïve or treatment-experienced since poor patient compliance is often a factor in treatment failure and viral rebound. the lack of patient adherence to complicated drug administration regimens is further exacerbated by the cumulative costs of combined arv therapy. c) toxicity and drug-drug interactions: prolonged treatment with arv drugs has resulted in several side-effects, including constipation, fever, liver disorders, muscular dystrophy, metabolic disorders, and peripheral neuropathy. the use of a combination of drugs in the conventional therapy can also lead to undesirable drug-drug interactions thereby reducing the efficacy of the drugs. for instance, when a combination of the reverse transcriptase inhibitor nevirapine and protease inhibitor saquinavir was used to treat hiv infections, saquinavir levels in the plasma were found to drop rapidly. this is due to the induction of the liver cytochrome p450 by nevirapine leading to the metabolization of saquinavir and its subsequent elimination therefore reducing its availability and efficacy [4] . d) poor bio-availability: arv drugs are burdened by poor pharmacokinetics. most of the arv drugs are formulated as solid dosage forms for oral route of administration. the oral dosage forms offer convenience, but the combined dose of compounds that make up a therapeutic regimen is usually high. high doses are preferred because the treatment objective is to completely inhibit viral proliferation, an effect which is proportional to drug concentrations. the delivery of drugs via oral route suffers from significant first-pass effect, variation of absorption and degradation in the gastrointestinal tract due to enzymes and extreme ph conditions leading to low and erratic bioavailability. for example, the expression of multidrug resistant efflux proteins (mrps) such as p-glycoprotein (p-gp) on the gastrointestinal tract further decreases their oral bioavailability. the metabolism/elimination and transport barriers will substantially reduce the effective amount of anti-hiv drug reaching the target action site. the half-life for several arv drugs is short, which then requires frequent administration of doses leading to poor patient compliance. the short residence time and reduced half-life of the drug in the plasma necessitate frequent administration of booster dosages as well as increased drug dosages contributing to the development of drug resistance. some anti-retroviral drugs such as saquinavir possess poor bioavailability due to their metabolization by liver enzymes [5] . e) lack of access to tissues and reservoirs: most of the anti-retroviral drugs cannot cross the blood-brain barrier (bbb) as a result of which they are ineffective in entering microglial cells such as astrocytes that hoards hiv particles [6] . a major factor is the high protein binding of most anti-hiv drugs that prevent their diffusion across the bbb. also, it has been observed that the anti-retroviral drugs are unable to reach the lymphatic system that harbors hiv. thus the conventional therapy is ineffective in annihilating viral reservoirs as less than 2% of the lymphatic cells are present in systemic circulation [7] . the cells of the lymphatic system like macrophages and dendritic cells are involved in the transmission of the virus to cd4 + t h cells (helper t lymphocytes) [8] and failure to target such reservoirs of hiv increases the risk of a viral relapse post-treatment. 3. emergence of newer approaches to counter problems in conventional arv therapy to circumvent the problems mentioned above and effectively treat the hiv infection, recent anti-aids therapeutic development efforts have been focusing on improving drug delivery and not just on discovering new chemical entities. efforts have been made to design novel drug delivery systems for anti-hiv agents to reduce the dosing frequency, to enhance the bioavailability, to improve the central nervous system (cns) penetration, to inhibit the cns efflux and to deliver them to the target cells selectively with minimal side effects. among the recent approaches of novel drug delivery system for anti-hiv drugs, targeted/intracellular drug delivery only in host cells capable of getting infected with hiv or more specifically hiv-infected cells and reservoirs holds great promise. the main aim of drug targeting is to optimize a drug's therapeutic index by strictly localizing its pharmacological activity to the site or organ of action. the result of the targeting would be a significant reduction in drug toxicity, reduction of the drug dose, and increased treatment efficacy. during hiv infection, the virus infects only specific cells of the immune systems including cd4 + t-cells, macrophages and dendritic cells, which forms a strong rationale for targeting delivery of anti-hiv drugs specifically to these cells for increasing the efficacy of therapy. drug delivery to hiv-infected tissues/ cells with selectivity can be achieved by targeting surface markers on cd4 + t-cells, macrophages, dendritic cells and towards the cns. targeting these receptors can have two effects: i) prophylactic protection of the cell by occupying the receptor necessary for virus-cell interaction, and/or ii) improving the specific uptake of a nanocarrier loaded with anti-viral drugs. in this regard, several molecules of both viral and host cell origin have been described in recent years, that would enable targeted drug delivery in hiv infection. in the following review, we provide a brief overview of the recent developments in the identification of novel viral and host-cell molecules for the targeted drug delivery of anti-hiv drugs. a schematic representation of the different types of viral and host targets investigated for specifically targeting hivinfected cells, thus far is shown in fig. 2 . the conserved regions in hiv envelope glycoproteins gp41 and gp120 are involved in binding of the virus to the host cells [9] . many targeting strategies have focused on binding to these protein sequences to achieve specificity, though the high rates of mutation exhibited by the retrovirus make it challenging to identify a suitable targeting sequence on the envelope [10] . the glycoprotein-120 (gp120) present in the outer envelope of hiv enables the entry of the virus particles into cd4 + t-cells. it mainly binds to the cd4 receptor of the t-lymphocytes (t h cells) and macrophages and aids in the viral entry with the help of the fusion protein gp41 [11] . upon infection, the gp120 is mainly exposed on the surface of the hiv infected cells and hence can be an attractive target for cellspecific delivery of anti-retroviral drugs. an immunoliposomal carrier encapsulating the protease inhibitor p11 and surface modified with anti-gp120 was reported to exhibit excellent specificity towards hiv infected cells due to binding of the anti-gp120 to the exposed domains of gp120 present on the surface of hiv infected cells [12] . actinohivin, a prokaryotic lectin found in actinomycetes was identified to possess anti-hiv activity and was found to specifically target hiv infected cells. this lectin belongs to the family of carbohydrate binding agents (cbas) and displays high affinity binding to the mannose residues of the d1 chains of high mannose-type glycans associated with gp120 of hiv [13] . in an earlier work, a glycosylphosphatidylinositol (gpi) moiety conjugated to gp120 was incorporated in liposomes and was found to selectively bind to cd4 + cells [14] . comparison of the gpi-gp120 liposomes with liposomal vesicles formed from components of the viral envelope revealed differences in the intracellular localization in the two systems. as both carriers were internalized through cd4 mediated endocytosis, it was inferred that differences in the lipid composition in the two systems influenced their intracellular compartmentalization. this facet, however, remains largely unexplored in the context of targeted nanocarrier delivery of anti-retrovirals. mesoporous silica nanoparticles with highly oriented and accessible pores were functionalized with an 18-mer fragment of cd4 designated as scd4 either through amide conjugation or glycosyl linkage [15] . the functionalized nanoparticles exhibited excellent binding to gp120 protein. recently, v3 loop of gp120 has been identified as the region involved in the initial entry of virus into the cell [16] . antibodies against the v3 loop have been found to inhibit the entry of the virions [17] . antibodies against the v3 loop can also be used to target hiv infected cells thereby conferring both target specificity and anti-viral activity. another strategy involving the use of a tri-functional igg-like bi-specific antibody with anti-gp120 and anti-c3d domains has been reported [18] . a tri-functional antibody contains two binding sites for different molecules and has been reported to display nearly 1000-fold enhancement in the targeting specificity when compared with the conventional antibodies that seek only a single target [18] . the bi-specific antibody reported against hiv infected cells was found to bind specifically with the gp120 fragment exposed in the infected cells as well as to the c3d, a component of the complement resulting in the activation of the complement-mediated lysis of the infected cells. a broad activity antibody against gp120, f105 has also been used to target the hiv infected cells [19] . f105 displays higher affinity to gp120 even at nanomolar concentrations. crystal structure analysis of the binding fragment of f105 reveals a h-loop containing a unique sequence of serine and tyrosine residues and a phenyl alanine residue at the apex [19] . it is proposed that this phenyl alanine residue in f105 associates with the binding pocket of gp120 similar to the phenyl alanine residue (phe43) from cd4 of the host cells. the serine and tyrosine residues in the h-loop stabilize the f105-gp120 binding through formation of hydrogen bonds [20] . in a novel approach, a cd4 mimetic peptide covalently linked with heparan sulfate was synthesized to inhibit hiv-1 entry into the host cell [21] . the cd4 mimetic peptide specifically binds to gp120 resulting in a conformation change that exposes the glycoprotein-binding domain of gp120, which interacts with the heparan sulfate moiety thereby blocking further interactions of the viral gp120 with the host cell receptors. a recombinant peptide expressing the cd4 binding region for gp120 designated as cd4(178)-pe40 and pseudomonas toxin was developed to specifically target and destroy hiv infected cells. the peptide was found to specifically bind to the exposed regions of gp120 found on the surface of hiv infected cells [22] . a surfactant protein extracted from the lung surfactant, sp-d, which belongs to the lectin family has been found to display high affinity calcium-dependent binding to the glycosylated residues of gp120 [23] . the use of soluble cd4 and the immunoadhesin molecule cd4-igg for targeting hiv infected cells has also been reported [24] . these molecules were found to bind to the hiv infected cells through the gp120 domain exposed on the surface of the infected cells. as conjugation of large antibodies to the surface of the nanoparticles is tedious and the risk of denaturation or loss of proper orientation conducive to ligand binding is high, smaller molecules that display similar specificity and affinity to the target antigen have been explored. aptamers, a class of small molecules that can be either an oligonucleotide or a peptide, that exhibit high binding affinities to the target molecules can be a suitable alternative to antibodies [25] . a 2-fluoropyrimidine substituted rna binding aptamer that specifically binds to the surface glycoprotein gp120, has been reported to display excellent target specificity [26] . further studies on the use of such aptamer conjugated nanoparticle systems are warranted for improved therapy. fig. 3 depicts a schematic representation of the concept of targeting hiv infected cells through gp120. glycoprotein-41 (gp41) is a trans-membrane protein present in the hiv envelope that facilitates the fusion of the virus with cd4 + cells [27] . the gp41 moiety is attached non-covalently to the larger glycopro-tein120 (gp120), which is involved in the binding of the viral particles to cd4 + cells. the binding of gp120 to the cd4 receptors of the host cells causes conformational changes leading to the release of the stable gp41 [28] . the ectodomain of gp41 aids the fusion between the virus and host cell. the gp41 protein contains various subunits such as fusion peptides, n-terminal heptad repeats, c-terminal heptad repeats and membrane proximal extracellular region [29] . similar to gp120, gp41 is also expressed on the surface of hiv infected cells and hence is an attractive target for therapeutic strategies involving selective delivery of the therapeutic agent to hiv infected cells. antibodies against gp41 have been employed for targeting hiv infected cells [30] . in few cases, radio immunotherapy (rit) using gp41 antibody labeled with 188 re (rhenium) has been employed for targeting hiv infected cells containing viral particles budding on the cell [31] . the radioactive element causes the death of the hiv infected cells. this strategy was successfully demonstrated in vivo with about 99% reduction in the viral load reported. recently, a new strategy has been reported for targeting both gp41 and gp120 using two antigenic determinants in a single antibody known as double variable domain immunoglobulins (dvds). the use of dvds promotes greater interactions with the two glycoprotein domains in the hiv infected cells resulting in higher uptake of the immunoconjugates and thereby leading to superior viral load reduction [32] . in a similar strategy, lu et al. have reported a recombinant bivalent protein 2dlt, which contains the did2 fragment of the cd4 and a peptide t1144 [33] . the did2 domain of the 2dlt binds to the gp120 and then interacts with the nhr trimer repeat of the gp41 through t1144 resulting in the decay of the gp41 thus preventing further fusion of the hiv virions with other uninfected cells. such recombinant proteins may also be used in conjugation with nanoparticles for targeting hiv infected cells, though such approaches have not been explored thus far. many engineered peptides have been developed for targeting gp41. for instance, it has been identified that the peptide t20 binds specifically to the n-terminal heptad region of gp41 and inhibits infection by the virions due to prevention of conformational changes in gp41 responsible for viral entry [34] . the sequence of t20 is derived from the gp41. similar peptides such as c34 and c37 that are also derived from gp41 have been designed to bind and inactivate the interactions between gp120 and the c-heptad repeat (c-hr) domain of gp41 leading to the inactivation of the env gene. these engineered peptides interact with specific exposed regions of the gp41 of hiv and prevent the entry of the virus into the cells [34] . the membrane proximal external region (mper) in gp41 is also a highly conserved region containing 23 amino acids and antibodies against this domain have proved to be successful in targeting the hiv infected cells [34] . surprisingly, though several molecules targeting conserved regions on the virus have been identified, integrating these targeting molecules with anti-retroviral drug containing nanoparticles has not been extensively investigated and remains in its infancy. yet another interesting approach to target hiv-infected cells is to identify unique markers on the host cells that are also sought by the virus. compared with virus-based targets, a relatively greater number of host cell-based targets have been identified. these include cd4 (receptor for hiv), chemokine receptors, cxcr4 and ccr5 (co-receptors for hiv), carbohydrate-binding antigens (cbas), tuftsin, hla-dr, dc-sign and lfa-1. however, recent studies indicate that the most promising targets include hla-dr, dc-sign and lfa-1. in addition to infection with cell-free virions, the importance of cellcell spread across connecting membrane bridges and close cell-cell contacts referred to as virological synapse (vs) for hiv-1 propagation is increasingly being recognized as the predominant mechanism of hiv-1 propagation between t-cells. assembly of the hiv-1 t-cell vs requires engagement of the hiv-1 env surface subunit gp120, expressed on the effector cell, with its cellular receptors cd4 and cxcr4 on the target cell. further recruitment of receptors and hiv-1 proteins to the conjugate interface is a cytoskeleton-dependent process in both target and effector t cells. specifically, the vs is characterized by clustering of leukocyte function-associated antigen 1 (lfa-1, also known as α l β 2 or cd11a/cd18) at the effector-target cell interface. the lymphocyte function associated antigen 1 (lfa-1) belongs to the integrin family of adhesion molecules and is hypothesized to contribute to the formation of a stable adhesive junction between the effector t-cell and the target t-cell. the major cognate ligand of lfa-1 on t cells is icam-1 (cd54). furthermore, integrins have been implicated in cell-cell transmission of hiv-1 from dendritic cells (dcs) to t cells via lfa-1 and dc-sign (dc-specific intercellular adhesion molecule 3 [icam-3]-grabbing nonintegrin), and their probable role in this setting is to maintain robust dc-t-cell contacts. since lfa-1 is a key molecule involved in transmission of hiv during the formation of the vs between t-cells and in dc-tcell interaction, recent studies have targeted lfa-1 as a potential anti-hiv target. lfa-1 is present in t lymphocytes, b lymphocytes, neutrophils and macrophages and its expression is high during hiv infections. in addition, it has also been recognized that the hiv envelope glycoprotein gp120 induces high levels of lfa-1 expression [35] . immunoliposomes modified with antibody targeting the lfa-1 integrin receptor were developed to deliver si-rna against ccr5 gene and were evaluated for its efficacy in vitro and in vivo [36] . the antibody linked liposomes were able to deliver the si-rna successfully into the infected cells. cyclic peptides cibr, cibl and cibc derived from icam-1 were able to bind and internalize into cells that express lfa-1 through the i-domain of lfa-1 and thus can be used to target hiv infected cells [37] . a monoclonal antibody al-57 has also been demonstrated to exhibit high affinity to cells that express lfa-1 [38] . it was observed that the binding of al-57 occurred with lfa-1 present only in lymphocytes activated by the hiv infection while the antibody binding did not occur in inactive cells thus displaying a high degree of selectivity towards hiv infected cells [39, 40] . an emerging paradigm in the use of monoclonal antibodies directed against lfa-1 is their ability to inhibit viral replication. in a recent study focused to understand the mechanism of viral replication inhibition by anti-lfa-1, it was found that the binding of the antibody to free virus did not influence its replication efficiency. however, binding of the antibody with the lfa-1 present in hiv infected cells resulted in the production of a soluble factor that interfered with the viral replication [40] . hence, use of anti-lfa-1 could be doubly beneficial by ensuring specific targeting to hiv infected cells as well as inhibiting viral inhibition. this facet, however, needs to be explored in-depth with additional experiments in vivo. fig. 3 represents the concept of targeting lfa-1 using anti-lfa-1 modified drug loaded nanoparticles. though the above literature reports that studies in targeting lfa-1 with nanoparticles are few, the studies are promising and will surely open up avenues for more detailed studies on targeting lfa-1 as an anti-hiv target. the human leukocyte antigen (hla) receptor belongs to the major histocompatibility class (mhc) of proteins and is involved in presenting of the antigens to the lymphocytes leading to the activation of the immune response [41] . among the various mhc class ii types, hla-dr, hla-dq and hla-dp are heterodimeric cell surface receptors found in macrophages, dendritic cells and b cells and present the antigen to the cd4 + t helper cells. due to their localization on the surface of cells that serve as hiv reservoirs and their ability to attract cd4 + t helper cells, the hla cell surface receptors are an attractive targeting moiety for hiv infected cells. interestingly, hla-dr molecule expression is increased during hiv infection along with cd25, a trans-membrane receptor of interleukin 2 [42] . recently, anti-hla-dr molecule has been used to target the hla-dr expressed on the hiv infected cells [43] . immunoliposomes loaded with the protease inhibitor indinavir and surface modified with anti-hla-dr were found to effectively bind to hiv infected cells and deliver the antiretroviral drug leading to a significant reduction in the viral load [44] . in another study, the biodistribution of anti-hla-dr conjugated immunoliposomes was evaluated using mice models and it was found that the immunoliposomes were concentrated in the secondary lymphoid organs like spleen and lymph nodes which are the main reservoirs of the hiv virions [101] . a unique advantage acquired through use of anti-hla-dr modified liposomes was that it was able to bind both hiv infected cells and also the free hiv virions found entrapped in the follicular dendritic cell network [45] . it has also been reported that the other viral reservoirs such as monocytes, macrophages, and dendritic cells that are difficult to target due to the dormant nature of the viruses, can be easily targeted using the anti-hla-dr due to the high levels of hla-dr expression on these cells and can therefore reduce undesirable toxicity of the anti-retroviral drugs to normal cells. it was also observed that the liposomes with anti-hla-dr preferentially accumulated in the lymph nodes rich in the hiv infected cells [46] . immunoliposomes conjugated with anti-hla-dr loaded with amphotericin b, an inhibitor of hiv replication, were also reported to target hiv infected cells and reduce the viral load [45] . immunoliposomal systems encapsulating si-rna against rev and tat genes of hiv that are involved in the replication process were successfully delivered to the target cells through conjugation of antibodies against hla-b and hla-c that belong to the mhc class i molecules. it is evident from the scan of literature that the anti-hla antibodies conjugated to liposomes alone have been reported while other nanocarriers modified with hla-dr are yet to be explored. one of the major challenges in hla targeting lies in the design of an antibody with high specificity due to the presence of large number of serotypes for hla. furthermore, hla-dr cd38 + cd4 + t cells show not only increased susceptibility to hiv, but once infected also produce support higher viral replication levels compared to other cells [47] . the hiv peptides show high affinity to the hla-dr moieties. a study has also demonstrated the high affinity of certain hiv-derived peptides to the hla-dr on the cell surface [48] . studies indicate that the hla-dr is incorporated into the viral envelope at the budding stage of the hiv cycle and facilitates further infection of the other uninfected cells using the cd4 receptor as the receptor. taken together, these studies indicate that targeting of the hla-dr will not only deliver the anti-retroviral drugs to the possible sites of hiv infection, but may also attenuate the hla-dr-mediated spread of viral infection in the body [49] . the c-type lectin dc-sign (dc specific icam-3-grabbing nonintegrin) has been identified as a cell surface receptor on immature dcs that binds hiv and mediates transfer of virus to cd4 + permissive t cells. contact of hiv-1 with dc-sign is thus the first event in the pathogenic cascade and, therefore, it is the primary target point for therapies aimed at hiv infection prevention. dc-sign binding to hiv results in internalization of virus to a non-endolysosomal compartment. from this compartment, internalized virus moves rapidly to synapses formed by infected dcs and cd4 + t cells. the transmission of virions is mainly due to the interaction of the cd4-dc-sign and followed by tetramer formation resulting in the higher affinity of the dc-sign to the gp120 residues thus resulting in the fusion of the membrane and capture and transfer of the hiv virions by the dendritic cells to the cd4 positive cell [50] . the silencing of the dc-sign expression in hiv infected cells has been shown to reduce the viral load, thus confirming the potential for dc-sign to function as a potential target [50] . some of the molecules which bind dc-sign are important in targeting the hiv infected cells. these include polyman 19 which binds specifically to the dc-sign expressed on the hiv infected t cells. thus dc-sign could be a critical target to combat early hiv infection. 4.2.4.1. cell surface glycoprotein cd4. the glycoprotein cd4 (cluster of differentiation 4) belongs to the immunoglobulin family and is expressed on the surface of several immune cells including t lymphocytes, monocytes, macrophages and dendritic cells [51] . cd4 elicits an immune response through interactions with the antigen presented by the major histocompatibility complex (mhc) molecule in the antigen presenting cells [52] . hiv hijacks cd4 as its primary receptor to invade its target cells. therefore, targeting the cd4 moiety is an attractive stratagem to selectively deliver anti-hiv drugs to its target cells. the targeting of the cd4 + cells can be achieved through antibodies designed to bind specifically to any of the four domains d1 to d2 present in cd4. a liposomal carrier conjugated with antibodies against the d2 or d3 domains of cd4 was developed that specifically bound to the hiv infected cells [53] . similarly, anti-cd4 conjugated liposomes were employed for the delivery of si-rna to silence the rev gene that is implicated in the regulation and expression of virion proteins. a significant amount of viral inhibition was observed due to the effective targeting to cd4 + cells. in a recent work, lipid nanoparticles conjugated with two peptide sequences that were derived from the binding domains of cd4 designated as cd4-bp2 and cd4-bp4 were used for targeted delivery of the protease inhibitor indinavir to hiv infected cd4 + cells with high specificity [54] . one of the major challenges in targeting cd4 + cells is its possible interference with the normal functions of cd4 expressing cells. in normal conditions, cd4 is used by the t lymphocytes to bind to mhc (major histocompatibility complex) class ii proteins for antigen recognition [55] . the binding domain of cd4 involved in its interactions with mhc class ii overlaps with the binding domain involved in its interactions with gp120 of hiv, the differences being the higher contact area and stronger affinity of the gp120 with cd4 [56] . therefore, design of peptidomimetics with greater affinity to cd4 has been explored that may also serve as competitive inhibitors of the gp120 binding to cd4 [57] . the use of such anti-cd4 peptides for targeting drug-loaded nanoparticles however has not been explored thus far. hiv-infected cell targeting has also been achieved using an engineered cytotoxic t lymphocyte (ctl) expressing a chimera cd4 receptor that employs the extracellular portion of cd4 as a targeting moiety. the specific binding of the engineered cell through the cd4 receptor with the cd4 + cells results in the activation of signaling pathways in ctl that causes the lysis of the hiv infected cells [58] . a novel concept that had been experimented during the late 90s was the development of 'viraceuticals' that involved engineered viruses of the rhabdoviridae family devoid of their envelope proteins but expressing the co-receptors cd4 and cxcr4 that enables it to selectively seek gp120 bound cells and destroy them [59] . however, their successful demonstration in a clinical set-up is yet to be realized. in a departure from the conventional concept of targeting cd4 + cells for delivery of anti-retrovirals or inhibit viral entry, a recent report has employed anti-cd4 conjugated gold nanoparticles for visualizing the lymph nodes through x-ray computed tomography [60] . such applications can be employed in the future for development of 'theranostic' systems that enable simultaneous visualization of the infected cells and their treatment using a co-administered therapeutic agent. the viral entry into the cell is facilitated by the presence of co-receptors cxcr4 and ccr5 that have also been employed for targeting hiv infected cells. the ccr5 or c-c chemokine receptor type 5 belongs to the beta chemokine receptor family and acts as the receptor for many chemokines such as ccl5 and macrophage inflammatory proteins (mips) [61] . it is expressed by t cells, macrophages, microglial cells and dendritic cells [62] . it is also an important co-receptor recruited by the hiv virus to gain entry into the host cell. while cxcr4 is believed to be involved in the later stages of the viral infection, ccr5 is involved in the entry of virus during the early phases of the hiv infection [63] . as a result, ccr5 remains among the most extensively investigated chemokine receptor target for controlling the spread of hiv infections. a liposomal system covalently linked with ccr5 and encapsulating the model drug ethylene diaminetetraacetic acid (edta) was targeted to the hiv infected cells expressing gp120 along with soluble cd4 [63] . as ccr5 possesses affinity towards the hiv gp120, the ccr5-modified liposomes bind to the infected cells causing their selective death in the presence of soluble cd4 [64] . monoclonal antibodies developed against ccr5 have also been employed for homing into hiv infected cells. the anti-ccr5 monoclonal antibody pro140 can bind to multiple extracellular domains of ccr5 and therefore can selectively deliver anti-retroviral drugs efficiently to hiv infected cells on conjugation with nanoparticles [65] . the natural ligand for ccr5 is chemokine c-c ligand 5 (ccl5) also referred to as rantes (regulated on activation, normal t cell expressed and secreted) can also be used to deliver the therapeutic moieties to hiv infected cells [65] . synthetic peptides derived from the sequences in the extracellular loop of ccr5 that is involved in interactions with the viral proteins have also been employed for targeting hiv infected cells. these synthetic peptides have been demonstrated to bind to gp120 present in the surface of the virus infected cells [66] . a classic example of this category of peptides is the e51-derived peptide referred to as ccr5mim, which resembles the extracellular loop of ccr5 that has high affinity to gp120 and can therefore be used to target hiv infected cells [67] . in a novel approach, peptide nucleic acids (pnas) with specificity towards the ccr5 gene were encapsulated in biodegradable nanoparticles of plga (poly(l-lactide-co-glycolide)) and delivered to hiv infected cells [68] . the pnas were associated with the ccr5 gene through hoogsteen bonding and resulted in the suppression of the gene expression leading to a condition akin to those found in cells carrying a ccr5-δ32 mutation. these cells turn hiv resistant due to the absence of ccr5 expression on the cell surface thereby preventing the entry of hiv. the cxcr4 (chemokine receptor type 4) also known as fusin, is a trans-membrane protein that belongs to the g protein coupled receptor family and is ubiquitously expressed by several cell types including t lymphocytes, endothelial cells and tumor cells [69] . the natural ligands for this receptor are the stromal derived factor-1alpha (sdf-1α) (cxcl12) that serves as a chemoattractant for lymphocytes [70] and ubiquitin that is implicated in mitigating pro-inflammatory molecules [71] . hiv-1 strains designated as t-tropic gain entry into the host cell through binding interactions with cd4 and cxcr4. it is believed that the primary binding with cd4 facilitates a conformation change in the viral protein that then binds to cxcr4. it is also found that several strains of hiv-2 are able to gain entry even in cd4 − cells through the cxcr4 receptors suggesting that these viruses possess an envelope protein that binds specifically to the chemokine receptor [72] . a small basic bicyclam molecule amd3100 has been synthesized that exhibits high affinity binding to cxcr4 [73] . this molecule has been found to be a useful target in cancer therapy and wound healing apart from potential anti-hiv properties [74] . however, its therapeutic use against hiv infections is limited as the viral load reduction post-administration of this drug was not significant. this is because in most cases the virus was able to gain entry through other chemokine receptors. antibodies and peptide sequences directed against cxcr4 have been used to target the hiv infected cells. peptide sequences derived from the viral macrophage protein vmip-ii designated as dv1, dv3 and dv1-k-(dv3) were investigated for their targeting affinity towards cxcr4 [75] . it was found that dv1 and dv3 exhibited moderate affinity while dv1-k-(dv3) displayed high affinity towards cxcr4 and hence can be explored further for targeting cxcr4 positive cells. in a recent study, a peptide n15p derived from the amino acid sequence 1-15 of the n-terminus of the viral macrophage inflammatory protein (vmip-ii) was encapsulated in a nanoliposomal carrier [76] . this liposomal system was found to inhibit the sdf-1 induced chemotactic activity of peripheral blood mononuclear cells through competitive binding to cxcr4. the antibody 12g5 against cxcr4 has also been used for targeting the hiv infected cells possibly through recognition of a specific sequence in the extracellular loop 2 (ecl2) of cxcr4 [77] . currently, a humanized anti-cxcr4 antibody designated as 515h7 is under preclinical trials to evaluate its targeting efficacy [78] . stromal cell derived factor-1 (sdf-1) which is secreted by the bone marrow stromal cells has also been used for targeting hiv infected cells. two types of sdf namely sdf-1α and sdf-1β have been investigated due to their high affinity and easy binding to the cxcr4 receptors thereby serving as promising targeting moieties towards cd4 + t h cells infected with hiv [79] . conversely, cxcr4 can also be linked to the nanocarrier to target hiv infected cells due to its affinity for the viral gp120 that is present on the surface of hiv infected cells [80] . a trans-membrane protein, tm4 derived from cxcr4 has also been demonstrated to bind to cd4 + cells and can be used for targeting the hiv infected cells [81] . the synthetic pentapeptides 15k and 15d have been employed as inhibitory peptides for hiv infection and have been found to exhibit superior binding affinity towards cxcr4 co-receptor than the anti-cxcr4 monoclonal antibody 12g5 [82] . the selective binding of two synthetic antagonistic polypeptides tn140 and tn14003 to cxcr4 has been demonstrated in different cancer cell types and hence can be a promising moiety to target hiv infected cells [83, 84] . however, these peptides have not been evaluated in hiv infected cells yet. the recent emergence of 'nanobodies', single domain antibodies that contains a single chain of the variable domain binding to the antigen, has resulted in the development of alx-0651 that displayed high affinity binding to cxcr4 comparable to amd3100 [85] . this nanobody that binds to the ecl2 loop of cxcr4 is currently in phase ii clinical trials where its potential as a targeting ligand towards hiv infected cells is being evaluated. . the viral envelope contains many glycan residues that aid in specific binding to the host cells and also serve to evade immune recognition by masking the immune epitopes. mannose residues are important binding moieties on the surface of the hiv glycoprotein gp120 [86] . targeting the sugar moieties can be an effective strategy for specific homing onto hiv infected cells because of the numerous glycan residues that are exposed on the surface of the hiv infected cells. this concept has led to the emergence of carbohydrate binding agents (cbas) that can be broadly classified either as lectins that belong to the protein family or as non-peptidic molecules with specificity towards a particular type of sugar moiety [87] . apart from imparting the ability to selectively bind to the viral envelope thereby blocking its ability to interact with the host cell surface receptors, the repeated use of cbas also induces the virus to delete the glycan residues thereby leading to unmasking of the hidden immune epitopes resulting in immune activation [88] . most of the glycan residues are also involved in the folding of a protein to its native conformation. deletion of the glycan residues by the virus in response to the treatment with cbas can therefore indirectly hamper the function of the glycans in protein folding thereby reducing the virulence of the virus [89] . a host of peptidic cbas, namely, lectins has been identified from different plant, microbial and mammalian sources. apart from their origin, these lectins differ in their size and specificity towards the sugar residues that have an impact on their binding affinity as well as viral inhibition activity [90] . in general, it has been identified that lectins that display specificity towards α-1,2, α-1,3 and α-1,6-mannose oligomers exhibit better viral binding and inhibition when compared with lectins that exhibit specificity to other carbohydrate moieties [91] . an exception to this generalization is the lectin isolated from the stinging nettle urtica dioica referred as uda (u. dioica agglutinin) that displays specificity towards n-acetyl glucosamine moieties and has also displayed significant affinity towards the hiv glycan envelope [92] . the lectin cyanovirin-n (cv-n) extracted from the cyanobacterium nostoc ellipsosporum [93] was found to bind to the α-1,2-mannose residues of gp120 with high affinity. similarly, scyctovirin (svn), a lectin extracted from the cyanobacterium scytonema varium exhibited selectivity to α-1,2-α-1,6-mannose residues and was found to bind to hiv glycoproteins gp41 and gp120 present on the surface of the hiv infected cells [94] . the lectins from sources of eukaryotic origin gsa (gerardia savaglia agglutinin) from the sea coral g. savaglia displays calcium dependent binding to the dmannose residues in the hiv-1 glycoprotein envelope resulting in complete viral inhibition in in vitro conditions [95] . the lectin actinohivin isolated from the actinomycete longisporum albida has also been identified as an important targeting moiety that binds to the mannose residues present in the gp120 at lower concentrations with an affinity greater than anti-gp120 antibodies thereby preventing the interaction of the viral glycoprotein with cxcr4 and ccr5 chemokine receptors [13] . griffithsin, a lectin isolated from the red algae griffithsia species has been identified to possess specific binding to mannose, glucose, fucose and n-acetyl glucosamine moieties thus conferring a broad spectrum of activity to this lectin towards different viral strains [96] . griffithsin has been found to be effective in interfering with the interactions of the viral glycoproteins with cxcr4 as well as ccr5 [97] . it has also shown promise in inhibiting the sars coronavirus by binding to the outer glycoprotein coat of sars (severely compromised acute respiratory distress syndrome) virus. the lectin bca (boodlea coacta agglutinin) extracted from the green algae b. coacta shows structural similarities with the galanthus nivalis agglutinin (gna) isolated from the snowdrop plant and acts as a potent targeting moiety for the hiv infected cells [97, 98] . it has been shown to bind mainly to the α-1,2-mannose type n-glycans present in the hiv envelope glycoprotein gp120 with high affinity of about 3.7 × 10 8 m −1 making it a promising targeting agent against hiv infected cells [97] . the binding affinity of bca was found to increase with increasing number of mannose residues in the cluster. other lectin molecules that have been identified to possess specific affinity towards the mannose residues of the hiv glycoproteins are cvl (chaetopterus variapedatus lectin) isolated from the annelid worm c. variapedatus, npa (narcissus pseudonarcissus agglutinin) from the daffodil plant n. pseudonarcissus, scl (scilla campanulata lectin) from the bluebell plant s. campanulata, cona (concanavalin a) from the jack bean canavalia ensiformis, jacalin from the jack fruit artocarpus integrifolia, mbl (mannose binding lectin) that is found in the serum of mammalian systems and displays calcium-dependent binding to mannose residues, dc-sign (dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin) expressed in dendritic cells of mammalian systems with specificity towards α-1,3 and α-1,6 mannotriose residues, etc. [98, 99] . a unique lectin referred to as mermaid, which is a c-type lectin expressed on the surface of the marine nematode laxus oneistus was identified to possess a structure similar to dc-sign [100] . this lectin binds to gp120 with high affinity and has been found to inhibit the binding of the virus to the dendritic cells as well as to inhibit the transmission of the virus from dendritic cells to cd4 + t lymphocytes [100] . yet another class of cationic cysteine-rich peptides expressed in the leukocytes is the defensins that possess anti-viral properties. different isoforms of defensins namely α, β, and θ have been identified [101] . the defensins promote chemotaxis of the t lymphocytes and have also displayed the ability to inhibit viral entry and replication [102] . the anti-viral activities of α and β defensins have been mainly attributed to their ability to interfere with the pkc (protein kinase c) signaling in hiv infected cd4 + cells as well as enhancement in the c-c chemokine levels in the cells leading to competitive inhibition of the viral binding to the host cells [102] . the θ defensins also known as retrocyclins display similarity with lectins in binding specifically to the glycan residues of the hiv envelope and hence could serve as a glycan-targeting moiety. a recent attempt to encapsulate defensin peptides along with gp41 residue in biodegradable poly(l-lactide-co-glycolide) microparticles was reported [103] . the presence of defensin resulted in an elevated immunogenic response against hiv and hence it was proposed that such strategies could be effective as vaccines against hiv infections. early work on glycan targets have employed mannose as the targeting moiety towards the mannose receptors present in hiv infected cells like macrophages, dendritic cells and astrocytes resulting in increased uptake of the mannose-linked drug-loaded nanoparticles through receptor-mediated endocytosis leading to a decrease in the viral load [104] [105] [106] . antibodies against the glycan residues of hiv have also been used as a targeting moiety. monoclonal antibodies against the oligosaccharides in the glycoproteins gp120/gp41 were reported to bind to the exposed glycan residues on the surface of the hiv infected cells with high affinity [107] . one of the common monoclonal antibody employed to target the glycan residues is 2g12 that binds specifically to hiv gp120 through terminal mannose oligomers [107] . however, any mutation in this region resulting in the deletion of this residue might render the antibody ineffective. such issues are not commonly encountered with the use of cbas because they are capable of binding to multiple residues in the same glycoprotein sequence. as a result, numerous deletions and mutations have to be effected by the virus to achieve resistance against the cbas [108] . this high genetic barrier achieved by the cbas makes them attractive therapeutic moieties that can also be used to achieve target specificity. among the non-peptidic cbas, the antibiotics pradimicin (prm-a) and nanomicin a (bnm-a) from actinomycete species have been found to exhibit calcium dependent binding to the terminal d-mannose of the viral glycoprotein forming a ternary complex [109, 110] . concerted efforts to develop low molecular weight non-peptidic molecules with carbohydrate-specific binding are underway in many research laboratories. a liposomal carrier containing a c-type lectin with high binding specificity towards dc-sign was developed and was found to exhibit excellent target-specific delivery of its cargo, the fluorescent calcein suggesting that such carriers may be useful in the context of site-directed delivery of therapeutic agents [111] . the use of glycan residues for immuno-stimulatory purposes has also been attempted as a strategy to reduce viral infections. in order to achieve highly effective multivalent display of carbohydrate moieties to stimulate the immune system, novel structures such as glycoclusters and glycodendrimers have also been reported in literature [112] . the concept of using cbas as part of the anti-hiv arsenal is however hampered by many factors. the high expense involved in the isolation and purification of the cbas, storage and stability issues, mitogenic nature of the lectins, poor bioavailability and their inability to distinguish between pathogenic glycan targets from native host glycan residues remains to be addressed before these could be used in a clinical set-up. tuftsin is a tetrapeptide derived from the immunoglobin igg in the spleen and consists of the amino acid sequence thr-lys-pro-arg [113] . since its discovery in 1970, tuftsin has found applications in immunotherapy due to its ability to bind to and activate macrophages and dendritic cells [113] . binding of tuftsin to the tuftsin receptors expressed on the surface of the cells of the immune system activates their chemotaxis and phagocytosis. as these cells also serve as viral reservoirs during hiv infections, employing tuftsin to target the infected cells has been explored as a potential treatment strategy against aids. a drug-tuftsin conjugate was developed by covalently linking tuftsin to the reverse transcriptase inhibitor 3′-azido-3′-deoxythymidine (azt) [114] . the conjugate was effectively used for targeting of macrophages, which hoards the hiv virions. a significant decrease in the viral load compared with the free drug was achieved thus illustrating the specific targeting potential of tuftsin. the tuftsin moiety also stimulated the release of the cytokine interleukin 1 (il-1) from macrophages thereby increasing the immune response [114] . ag5poly(propylene imine) (ppi) dendrimer loaded with the reverse transcriptase inhibitor efavirenz was conjugated with tuftsin and evaluated for its anti-viral efficacy in vitro using macrophages [115] . the tuftsin-conjugated dendrimer significantly decreased the viral load when compared with the unconjugated ppi dendrimer suggesting that tuftsin conjugation enhances uptake in infected cells [115] . tuftsin conjugated liposomes have also been extensively investigated for treatment of tuberculosis [116] , malaria [116] , leishmaniasis [117] and fungal infections [118] . however, apart from a few reports of tuftsin conjugated dendrimer systems, studies involving tuftsin-conjugated nanocarriers remain largely unexplored in the context of hiv therapy. transferrin is an important iron-binding glycoprotein present in blood that is primarily involved in the maintenance of iron levels in the biological system [119] . the affinity of iron to transferrin is extremely high at physiological ph but decreases with decrease in ph. transferrin receptors are abundantly present in liver and brain in physiological conditions [120] . as the brain is an important sanctuary for hiv, it is essential to deliver anti-retroviral drugs across the bloodbrain barrier. as the transferrin receptor expression is high in the brain, it is possible to deliver therapeutic agents into the brain through transferrin-conjugated nanocarriers that will be internalized using receptor-mediated endocytosis [121] . transferrin-conjugated plga nanoparticles encapsulating the reverse transcriptase inhibitor nevirapine were successfully transported across the blood-brain barrier into human brain microvascular endothelial cells [122] . a similar strategy was reported to enable delivery of a hiv model antigen cn54gp140 through the epithelial barrier in the mucosal layer [123] . the transferrin-conjugated system was able to elicit higher immune response as evident from the high titers obtained for the antibodies igg and iga in the genital tract of female mice when compared with the unconjugated system. a specific antibody against the transferrin receptor namely ox26 that binds to the cells expressing the transferrin receptor was conjugated to a streptavidin moiety to deliver antisense oligonucleotides against the rev gene of hiv that was modified covalently with a biotin molecule. the biotinylation protected the oligonucleotides from exonuclease degradation and enabled its binding to the transferrin conjugated streptavidin. this system was found to increase the inactivation of the rev mrna in the cerebral region when compared with the free oligonucleotides highlighting the ability of transferrin conjugated systems to cross the blood-brain barrier [124] . albumin nanoparticles encapsulating the nucleoside reverse transcriptase inhibitor azidothymidine (azt) were surface modified with transferrin and were found to cross the blood-brain barrier efficiently and decrease the viral load [125] . the biodistribution studies of the nanoparticulate system in albino rats revealed selective enhancement in the uptake of the nanoparticles in the brain illustrating the targeting efficacy of transferrin. investigations on the relation between iron levels and hiv infections have revealed that hiv infections lead to an increase in the oxidative stress in the cells and also cause the release of intracellular iron stores. as a result of the iron overload, it has been observed that the transferrin receptor expression in the infected cells is reduced. hence, in the advanced stages of infection, it remains to be seen if transferrin receptor could still be an effective target. in a departure from conventional carriers, use of a modified transferrin as a drug carrier was reported as a proof of principle work. the authors had inserted a peptide sequence in transferrin that was recognized by the hiv protease enzyme. this system was successfully demonstrated to enter hiv infected cells and was lysed by the hiv protease enzyme and can be explored for drug delivery to hiv infected cells. quantum rods comprising of a cadmium selenide core and a graded cadmium sulfide-zinc sulfide shell were conjugated with transferrin and complexed with saquinavir for treatment of neuro-aids [126] . these complexes were found to be extremely effective in transporting saquinavir across the blood-brain barrier. 4.2.4.6. aptamers. the challenges in conjugation of antibodies to carriers primarily due to changes in conformation induced by the chemical treatment steps result in a loss of target specificity. this has necessitated the development of novel small molecule ligands that retain the target specificity of antibodies but with greater structural stability. aptamers are small molecules of single stranded oligonucleotides (dna or rna) or peptides that have excellent target specificity. aptamers are developed using selex (systematic evolution of ligands by exponential enrichment) after a series of sequential steps that involve binding, separation, purification and amplification [127] . aptamers also possess the ability to hybridize through watson-crick pairing and hence can be used to form chimeric structures with other molecules such as si-rna and ribozymes that can be used for delivery into desired locations [128] . a chimeric aptamer-si-rna system exhibiting specificity towards gp120 has been reported to enter hiv infected cells through gp120mediated endocytosis [129] . the aptamer-si-rna chimera is cleaved by dicer inside the cell, leading to the release of si-rna, which binds to the mrna of the hiv tat and rev genes and inhibits the hiv life cycle. aptamers that bind specifically to hiv infected cells have been developed. a 2′f-rna aptamer that binds to the gp120 of infected cd4 + cells has been reported and could be employed for targeting hiv infected cells [130] . several aptamers with specificities towards the hiv reverse transcriptase, integrase, nucleoprotein, gp120, gag protein, tat protein, rev protein and transactivation response element (tar) have been reported [131] . novel aptamers with g-rich repeats that spontaneously form g-quadruplexes in the presence of divalent ions through hoogsteen pairing have been developed for hiv integrase and gp120 through terminal modifications. these quadruplex aptamers display extraordinary stability and binding specificity to their target [132] . these aptamers have immense potential in the field of targeted delivery of anti-retrovirals that remain to be explored in-depth. low-density lipoprotein (ldl). low-density lipoprotein (ldl) is a type of lipoprotein that is involved in the transport of lipids into the cells [133] . ldl has the ability to attract macrophages and hence has been explored as a possible targeting moiety against hiv infected macrophages. ldl conjugated azt (azidothymidine) was demonstrated to internalize into macrophages through ldl receptor-mediated endocytosis and deliver the drug [134] . similar studies were carried out using ldl conjugated fluorothymidine, which binds to the macrophages infected with hiv and delivered the drug effectively causing a decrease in the viral load when compared with the free drug [135] . another study had employed acetylated ldl loaded azt for site-specific delivery to macrophages. the uptake of acetylated ldl modified carrier was achieved through scavenger receptors and resulted in a decrease in the viral load [136] . however, it was observed that hiv infections result in a decreased expression of the ldl receptor and hence targeting this receptor during advanced stages of the disease might not be an effective strategy. passive targeting does not involve any chemical modification of the carrier and its internalization into the desired location is mainly driven by specific property of the targeted cells. the concept of passive targeting has been well exploited in cancer therapeutics by utilizing the enhanced permeation and retention (epr) property of cancer cells [137] . in case of hiv infections, the dendritic cells and macrophages having high amount of virions were found to transfer them to the cd4 + t h cells through virological synapses. these macrophages and dendritic cells have been observed to be highly active during hiv infections and exhibit higher phagocytic properties than uninfected cells [138] . this enhanced phagocytic activity of the macrophages can be harnessed to entrap anti-retroviral loaded carriers in the cells that are infected with hiv. this strategy of passive targeting was successfully demonstrated using indinavir encapsulated liposomal nanoparticles prepared using the synthetic phospholipids phosphatidyl choline, phosphatidyl ethanolamine and a stabilizing agent [139] . these nanoparticles exhibited good uptake in hiv infected macrophages and reduced the viral load significantly when compared with soluble indinavir. this difference between the encapsulated and free form of the drug was mainly attributed to the phagocytosis of the nanoparticles by macrophages. in another approach, a self-assembled dual drug conjugate of azt (zidovudine) and didanosine (ddi) separated by adeoxycholyl spacer was synthesized [140] . this zidovudine-phosphoryl-deoxycholyl didanosine (zpdd) conjugate was easily internalized by the phagocytic macrophages and resulted in the destruction of the virions. a schematic representation of nanoparticles internalized in infected cells through passive targeting is shown in fig. 4 . other targeting moieties for hiv positive cells that have been explored are thiamine [141] , glutathione and albumin [142] . these moieties can easily pass through the blood-brain barrier and are efficient in delivering the drugs on conjugating with the nanocarriers. yet another strategy that is still in the 'proof of principle' stage is the use of pluronics® (tri-block copolymer of poly(ethylene oxide) and poly(propylene oxide)) micelle carriers to disable the efflux pumps in the hiv infected cells that are triggered by the infection to achieve multidrug resistance [143] . the pluronics polymer and unimer alter the mitochondrial membrane fluidity thereby disrupting the electron transport chain. this impairs atp production leading to the shutdown of the efflux pumps [144] . this strategy may enable sensitization of hiv infected cells to anti-retrovirals thereby enhancing their therapeutic efficiency. investigations using knockdown experiments and next generation sequencing to unravel complex interactions between hiv and human proteins have helped in identification of potential targets that could be explored in future for therapeutic applications. in a recent report, a novel viral accessory factor, vif has been identified to recruit a human transcription factor cbf-β (core binding factor subunit beta) and uses the ubiquitin-ligase complex to degrade the restriction factors such as retroviral complementary dna deaminase in the human system that can inactivate the viral replication [145] . targeting cbf-β or vif might offer better chances of curbing the viral replication. another novel target could be slit2n, a glycoprotein that has been found to inhibit the replication of both t tropic and m tropic hivs probably through modification of the cytoskeleton dynamics [146] . this could also be explored indepth as a therapeutic target for viral infections. in a novel approach, melittin, an active component of the bee toxin was encapsulated in liposome nanoparticles and evaluated for their anti-hiv activity [147] . melittin is an activator of phospholipase enzyme and can destroy cells through disruption of the lipid coat. this cytolytic agent was found to selectively internalize to a greater extent in hiv infected cells where it destroyed the viral lipid coat. though free melittin also was found to destroy the virus, its cytotoxicity towards normal cells was significantly high when compared to its nanoparticle counterpart, which displayed selective toxicity only to the virus. this system displayed toxicity towards both cxcr4 and ccr5 tropic viruses. as it attacked the lipid coat, the probability of the virus developing a resistance towards this molecule is remote. this system needs to be further evaluated and validated for clinical significance. rna silencing has emerged as an important strategy to combat aids infections. many new viral targets are being identified to be silenced using rnai technology. some of the targets that have been targeted through rnai technology include the structural, regulatory, and accessory genes such as gag, pol, env, rev, tat, tar, vif, nef, vpx, vpr, etc. attempts to silence factors triggered in the host cell as a result of hiv infection such as the golgi transport proteins rab6 and vps53, karyopherin tnpo3, mediator complex med28, akt1, prkaa1, cd97, neil3, bmp2k and serpinb6 have also been made to control hiv infection [148] . however, the success of rnai depends on its complexation with a suitable nanocarrier as well as identification of a targeting moiety to selectively deliver these into the infected cells. also, a new emerging paradigm is that it may be necessary to silence more than one gene target simultaneously to ensure total disruption of the hiv virions. but such studies are yet to be explored in-depth thereby leaving the field wide open for further research. table 2 lists the numerous targeted nanocarriers that have been explored for hiv therapy, indicating the tremendous scope for further investigations employing a combination of therapeutic agents, nanocarriers and targeting agents. there continues to be a vital need for newer agents to confront the emergence of drug resistance and various adverse effects with longterm use of arv therapy. also the half-life for several arv drugs is short, requiring frequent administration of doses, which in turn leads to poor patient compliance. therefore, the usage of novel drug delivery systems is a logical approach to overcome these problems and effectively treat hiv infection. among the recent therapeutic approaches, a fig. 4 . schematic representation of a passive targeting strategy employing nanoparticles. major thrust area is in developing effective drug delivery systems for the existing drugs, which have been tested and proven effective in reducing the viral load. in this review, the need for novel drug delivery, advantages, and recent developments in identification of viral and host surface molecules as markers for targeted drug delivery of antiretroviral drugs were discussed. these studies open new avenues for more indepth studies on the effective use of these targeting strategies for hiv therapy. such a comprehensive approach could ultimately prove effective not only in reducing viral load, but also in eradication of virus from the reservoirs. we envisage that future directions in the field will involve a multipronged strategy to target hiv at various stages of infection. these include the transmission of the virions from dendritic cells and macrophages to the cd4 + t cells with potential targets being hla-dr and dc-sign. furthermore, cell-to-cell transmission of hiv between cd4 + t cells through the virological synapses is also a critical stage of viral transmission in the body. in case of cell-to-cell transmission, lfa-1 is likely to emerge as a potential target for targeted anti-hiv therapy. another key area should involve countering the challenges in targeting latent hiv in multiple reservoirs. in this regard, strategies that combine the release the hiv virions from latently infected cells such as activation of the protein kinase pathway along with anti-hiv drug delivery hold tremendous potential. in summary, recent scientific advances in the development of targeted drug delivery of anti-hiv drugs hold great promise for the development of improved treatment strategies and should certainly pave the way towards global eradication of hiv/aids. chimeric anti-gp120 aptamer rnai in vitro [165] the global impact of hiv/ aids a binding pocket for a small molecule inhibitor of hiv-1 entry within the transmembrane helices of ccr5 pathogenesis of hiv-1-protease inhibitor-associated 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against cxcr4 novel antibodies for the treatment of hiv, in, google patents identification and characterization of the cxcr4 chemokine receptor in human t cell lines: ligand binding, biological activity, and hiv-1 infectivity anti-cxcr4 monoclonal antibodies recognizing overlapping epitopes differ significantly in their ability to inhibit entry of human immunodeficiency virus type 1 dimerization of cxcr4 in living malignant cells: control of cell migration by a synthetic peptide that reduces homologous cxcr4 interactions novel peptides based on hiv-1 gp120 sequence with homology to chemokines inhibit hiv infection in cell culture inhibition of breast cancer metastasis by selective synthetic polypeptide against cxcr4 development of specific cxcr4 inhibitors possessing high selectivity indexes as well as complete stability in serum based on an anti-hiv peptide t140 halting metastasis through cxcr4 inhibition mannose binding lectin (mbl) and hiv carbohydrate-binding agents (cbas) inhibit hiv-1 infection in human primary monocyte-derived macrophages (mdms) and efficiently prevent mdmdirected viral capture and subsequent transmission to cd4 + t lymphocytes exposure of hiv-1 to a combination of two carbohydratebinding agents markedly delays drug resistance development and selects for virus strains with compromised fitness carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp120: a new therapeutic concept to hit the achilles heel of hiv inhibition of hiv entry by carbohydrate-binding proteins differences in the mannose oligomer specificities of the closely related lectins from galanthus nivalis and zea mays strongly determine their eventual anti-hiv activity the mannose-specific plant lectins from cymbidium hybrid and epipactis helleborine and the (n-acetylglucosamine)n-specific plant lectin from urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro 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mucosal molecular targeting to a model hiv-1 trimeric gp140 vaccine antigen drug delivery of antisense molecules to the brain for treatment of alzheimer's disease and cerebral aids targeted brain delivery of azt via transferrin anchored pegylated albumin nanoparticles enhancing the delivery of anti retroviral drug "saquinavir" across the blood brain barrier using nanoparticles development of dna aptamers using cell-selex rna-based therapeutics: current progress and future prospects selection, characterization and application of new rna hiv gp 120 aptamers for facile delivery of dicer substrate sirnas into hiv infected cells novel dual inhibitory function aptamer-sirna delivery system for hiv-1 therapy molecular strategies to inhibit hiv-1 replication investigation of formation, recognition, stabilization, and conversion of dimeric g-quadruplexes of hiv-1 integrase inhibitors by electrospray ionization mass spectrometry transport of lipids from high and low density lipoproteins via scavenger receptor-bi cell specific uptake of antiretroviral drugs: azt coupled to ldl inhibits hiv replication in human macrophages selective endocytosis of fluorothymidine and azidothymidine coupled to ldl into hiv infected mononuclear cells enhanced delivery of azt to macrophages via acetylated ldl enhanced permeability and retention effect for selective targeting of anticancer nanomedicine: are we there yet? macrophage targeting of azidothymidine: a promising strategy for aids therapy* laboratory investigations for the morphologic, pharmacokinetic, and anti-retroviral properties of indinavir nanoparticles in human monocyte-derived macrophages combination anti-hiv therapy with the selfassemblies of an asymmetric bolaamphiphilic zidovudine/didanosine prodrug drug delivery to the central nervous system: a review inhibition of hiv in vitro by antiviral drug-targeting using nanoparticles influence of pluronic® f68 on ceftazidime biological activity in parenteral solutions pluronic® 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loaded galactosylated liposomes lymphatic targeting of zidovudine using surfaceengineered liposomes stavudine-loaded mannosylated liposomes: in-vitro anti-hiv-i activity, tissue distribution and pharmacokinetics reduced hepatic toxicity, enhanced cellular uptake and altered pharmacokinetics of stavudine loaded galactosylated liposomes efficient sirna delivery into primary cells by a peptide transduction domain-dsrna binding domain fusion protein antibody mediated in vivo delivery of small interfering rnas via cell-surface receptors dendritic cells loaded with hiv-1 p24 proteins adsorbed on surfactant-free anionic pla nanoparticles induce enhanced cellular immune responses against hiv-1 after vaccination nanochemistry-based immunotherapy for hiv-1 targeting of efavirenz loaded tuftsin conjugated poly(propyleneimine) dendrimers to hiv infected macrophages in vitro tuftsin-azt conjugate: potential macrophage targeting for aids therapy sustained and specific in vitro inhibition of hiv-1 replication by a protease inhibitor encapsulated in gp120-targeted liposomes dual functional rna nanoparticles containing phi29 motor prna and anti-gp120 aptamer for cell-type specific delivery and hiv-1 inhibition the authors gratefully acknowledge the financial support from the indian council for medical research (icmr, grant no. 35/9/2009-bms) and infrastructural support from sastra university. key: cord-312865-nno2yjae authors: sylvester‐hvid, c.; nielsen, m.; lamberth, k.; røder, g.; justesen, s.; lundegaard, c.; worning, p.; thomadsen, h.; lund, o.; brunak, s.; buus, s. title: sars ctl vaccine candidates; hla supertype‐, genome‐wide scanning and biochemical validation date: 2004-04-23 journal: tissue antigens doi: 10.1111/j.0001-2815.2004.00221.x sha: doc_id: 312865 cord_uid: nno2yjae abstract: an effective severe acute respiratory syndrome (sars) vaccine is likely to include components that can induce specific cytotoxic t‐lymphocyte (ctl) responses. the specificities of such responses are governed by human leukocyte antigen (hla)‐restricted presentation of sars‐derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information (lauemoller et al., rev immunogenet 2001: 2: 477–91). the latter was recently established when a causative coronavirus (sars‐cov) was isolated and full‐length sequenced (marra et al., science 2003: 300: 1399–404). here, we have combined advanced bioinformatics and high‐throughput immunology to perform an hla supertype‐, genome‐wide scan for sars‐specific ctl epitopes. the scan includes all nine human hla supertypes in total covering >99% of all individuals of all major human populations (sette & sidney, immunogenetics 1999: 50: 201–12). for each hla supertype, we have selected the 15 top candidates for test in biochemical binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. eradicated. this would require detection assays that can track the disease and intervention measures that can break the chain of transmission. all of these procedures should be simple, yet effective. unfortunately, no such diagnostic test is currently available, and controlling transmission by containment solely is complicated and extremely costly. further complicating any eradication effort, a nonhuman reservoir appears to exist. thus, a strong case for a sars vaccine can be made. it would be of significant help in any eradication effort and, should that fail, it could protect infected individuals against the disease. the sars-cov infects epithelial cells in the respiratory tract causing interstitial pneumonia (4) . one would therefore expect that an effective vaccine should induce mucosal immunity such as that effected by secretory immunoglobulin a (iga), which specifically prevents an infectious agent from penetrating the mucosal epithelium, and by cytotoxic t lymphocytes (ctls), which specifically eradicate infected cells (5) . iga responses are generally considered the major protective mechanism; however, there are examples of ctls, not antibodies, being responsible for early control of mucosal infection (5) . particularly noteworthy, this is the case for the infectious bronchitis virus of chicks, a prototype of the coronaviridae family, where primary effector cd8 þ ctls play a critical role in the elimination of virus during acute infection and subsequent control of the infection (6-10). human ctls are specific for peptides presented in the context of human leukocyte antigen (hla) molecules [generically known as ''major histocompatibility complex (mhc) molecules'']. prior to presentation, peptides are generated in the cytosol by limited proteolytic fragmentation of all available protein antigens, translocated to the endoplasmic reticulum, specifically sampled by the mhc molecules and exported to the cell surface, where they await ctl scrutiny. importantly, the hla is extremely polymorphic and the peptide binding specificity varies for the different polymorphic hla molecules (1). it has, however, been suggested that the majority of all major human populations can be covered with three to nine ''hla supertypes'', where the different members of each supertype bind similar peptides (3). if one knew exactly how peptides were generated and selected, then genomic/proteomic information could be used to predict the outcome of antigen presentation and forecast immunogenicity. here, we have used advanced immunobioinformatical tools to mimic antigen presentation and, in a highly cost-and timeeffective manner, predicted possible immunogenic epitopes. the complete sars genome/proteome was obtained from gen-bank (nc004718) and virtually digested into all 9862 unique nonamer peptides (2) . thus, close to 10,000 binding predictions were made for each of the nine hla supertypes. artificial neural networks (anns) were used to predict the binding affinity quantitatively when the corresponding data were available [e.g. for a*0201 (11, 12) ]. the performance of the anns is high, as the correlation coefficient between predicted and measured binding is 0.85. the remaining hla bindings were predicted using weight matrices derived from gibbs sampling sequence-weighting methods with pseudocount correction for low counts as well as differential position-specific anchor weighting (nielsen et al. manuscript in preparation). these weight matrices were calculated from available nonamer data from the syfpeithi and mhcpep databases with the peptides clustered into the nine supertypes (a1, a2, a3, a24, b7, b27, b44, b58, and b62). the positive predictive value of the matrix-driven prediction has been found to be around 66%, whereas the negative predictive value has been found to be around 97% (lamberth et al. unpublished observation). proteasomal processing was predicted using netchop 2.0 (13). netchop 2.0 has been found to be superior to other proteasomal prediction algorithms (14) . peptides with a netchop 2.0 score below 0.5 (i.e. poorly predicted proteasomal processing) were excluded from further analysis. finally, we excluded all peptides that did not represent epitopes conserved in all sars isolates. figure 1 shows a representative example for a member of the hla-a3 supertype, the hla-a*1101 (this haplotype is particularly common in southeast asia). for each hla supertype, the 15 top-ranking nonamer peptides were synthesized by standard 9-fluorenylmethyloxycarbonyl (fmoc) chemistry, purified by reversed-phase high-performance liquid chromatography (at least 80%, usually >95% purity) and validated by mass spectrometry. the interaction of these epitope candidates with the appropriate hla was subsequently validated in a biochemical binding assay (15) . briefly, denatured and purified recombinant hla heavy chains were diluted into a renaturation buffer containing hla light chain, b 2 -microglobulin, and graded concentrations of the peptide to be tested, and incubated at 18 c for 48 h allowing equilibrium to be reached. we have previously demonstrated that denatured hla molecules can de novo fold efficiently, however, only in the presence of appropriate peptide (16) . the concentration of peptide-hla complexes generated was measured in a quantitative enzyme-linked immunosorbent assay and plotted against the concentration of peptide offered (15) (fig. 2) . because the effective concentration of hla (3-5 nm) used in these assays is below the equilibrium dissociation constant (k d ) of most high-affinity peptide-hla interactions, the peptide concentration leading to half-saturation of the hla is a reasonable approximation of the affinity of the interaction. an initial screening procedure was employed whereby a single high concentration (20,000 nm) of peptide was incubated with one or more hla molecules. if no complex formation was found, the peptide was assigned as a non-binder to the hla molecule(s) in question, conversely, if complex formation was found in the initial screening, a full titration of the peptide was performed to determine the affinity of binding. the resulting binding isotherms were analyzed by one-site hyperbola regression (prism 1 graphpad) determining the concentration of hla employed (3-5 nm, data not shown), the k d of the interaction (table 1 ) and the goodness of the curve fit (r 2 was always >0.95 and in the majority of cases it was >0.98, data not shown) (15) . in general, intermediate and high-affinity binders have k d s better (i.e. lower) than 500 nm and 50 nm, respectively, and the higher the affinity, the more likely the peptide is going to be a t-cell epitope (17) . table 1 summarizes the data for hla-a*0301 and hla-a*1101. the pepfor the top-ranking peptides, only 22 (or 2-3%) of the 952 combinations involves cross-responses, where a peptide predicted to be a top-ranking binder to one hla molecule turns out to be a binder to a member of another hla supertype, i.e. it supports the contention that hla supertypes effect significant diversification of anti-sars ctl responses. conversely, the overlap between different members of the same hla supertype appears to be extensive. thus, 13 of the 15 peptides predicted to be good binders to a*0301 were found to bind to another member of the a3 supertype, hla-a*1101. similarly, nine of the 15 peptides predicted to be good binders to a*1101 were found to bind to hla-a*0301 (table 1) once all nine supertypes have been tested, we would project to have found well over 100 different vaccine candidates. these would all have been predicted to be successfully processed by the proteasome and biochemically validated for hla binding. therefore, there should be a high probability that these peptides are indeed presented to ctls. once that occurs, there is an approximately 50% chance of being able to raise a ctl response (18) . thus, our data do in all likelihood include some 50 ctl epitopes. to identify these from the >100 binding peptides, one could search for the corresponding ctl reactivities in peripheral blood of sars survivors using robust and reasonably simple technologies such as interferon-g secretion from stimulated whole blood t cells (19) peptide binders to hla-a*0301 (top frame) and hla-a*1101 (bottom frame), sorted according to predicted binding strength, were synthesized and the affinities of binding to a*0301 and a*1101 were determined. the peptide sequence is given in single-letter code and the measured binding affinity is given as the kd. in the near future, the genome of any pathogen can be fully sequenced in a matter of days. the bioinformatics tools currently being developed and perfected will be able to use such genomic information to predict immune epitopes computationally, and the corresponding immunological tools will currently be able to validate these predictions in a matter of weeks to months. we predict that epitope identification in the near future will be as fast as a dna sequencing in handling whole organisms. with the dissemination of these tools, one could envision that clinicians and scientists anywhere would be able to analyze pathogens of their interest (or agent of bioterrorism, or tumor cell) for the purpose of fast identification of immunogenic epitopes (1) . the timeline of the present sars epidemic has demonstrated how fast modern science can identify a pathogen and decipher its genome. using this information in a fast and rational design of vaccines, immunobioinformatics promises to take this development one step further. identifying cytotoxic t cell epitopes from genomic and proteomic information: ''the 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cleavage motifs by neural networks predicting proteasomal cleavage sites: a comparison of available methods establishment of a quantitative elisa-based assay capable of determining peptide-mhc class i interaction efficient assembly of recombinant major histocompatibility complex class i molecules with preformed disulfide bonds the relationship between class i binding affinity and immunogenicity of potential cytotoxic t cell epitopes immunodominance in mhc class i restricted t lymphocyte responses cytokine-based human whole blood assay for the detection of antigen-reactive t cells recombinant polyepitope vaccines for the delivery of multiple cd8 cytotoxic t cell epitopes delivery of multiple cd8 cytotoxic t cell epitopes by dna vaccination immunogenicity of a human immunodeficiency virus (hiv) polytope vaccine containing multiple hla a2 hiv cd8(þ) cytotoxic t-cell epitopes an hla-a2 polyepitope vaccine for melanoma immunotherapy papillomavirus virus-like particles for the delivery of multiple cytotoxic t cell epitopes cytotoxic t cell polyepitope vaccines delivered by iscoms the development of multi-epitope vaccines: epitope identification, vaccine design and clinical evaluation optimization of epitope processing enhances immunogenicity of multiepitope dna vaccines the danish mrc (grant 22-01-0272), the 5th framework programme of the european commission (grant qlgt-1999-00173), the nih (grant ai49213-02), and the danish national research foundation supported this work. support for this work has been obtained from siga technologies. key: cord-287709-y247lan1 authors: navarrete, cristina title: cord blood banking: operational and regulatory aspects date: 2014-12-12 journal: cord blood stem cells and regenerative medicine doi: 10.1016/b978-0-12-407785-0.00015-3 sha: doc_id: 287709 cord_uid: y247lan1 umbilical cord blood (ucb) is an alternative source of hematopoietic stem cells for transplantation in patients with hematological malignancies, bone marrow failures, immunodeficiencies, and inherited metabolic disorders. in order to facilitate these transplants, large repositories of frozen ucb units collected from altruistic unrelated donations have been established and to date there are more than 600,000 units stored in cord blood banks all over the world. these products have been collected, stored, and released for transplantation under stringent quality conditions in order to ensure their safety and efficacy. the development and evolution of the policies and procedures currently in use in cord blood banking have been largely influenced by the clinical outcome of the transplants performed using these units. this review will describe some of the main steps and procedures involved in the clinical banking of unrelated ucb donations starting from the recruitment and selection of the potential donor (the mother) to the final distribution of the unit to the transplant program and its clinical outcome follow-up. the demonstration that umbilical cord blood (ucb) contained cells capable of reproducing hematopoiesis in vitro and that they could be cryopreserved for long periods without significant loss in their function paved the way for the use of this new source of stem cells in the field of clinical hematopoietic stem cell (hsc) transplantation. [1] [2] [3] [4] [5] although the first attempt to transplant ucb was reported in 1972, 6 the first successful related ucb transplant was performed in 1988 in a patient with fanconi anemia. 7 the first unrelated ucb transplants were then performed in 1993 and the report of a large series of patients was published in 1996. 8 these results prompted the establishment of large repositories of frozen unrelated ucb units, i.e., cord blood banks (cbbs) to support this clinical development. the first ucb bank was set up in 1991 by p. rubinstein at the new york center in the united states, 9 and this was followed by the establishment of cbbs in dusseldorf, milan, paris, and london. [10] [11] [12] [13] at present, there are over 130 cbbs all over the world storing over 650,000 unrelated ucb units readily available for transplantation. 14, 15 these cbbs have enabled the performance of over 30,000 unrelated ucb transplants in children and adults with both malignant and nonmalignant diseases. [16] [17] [18] [19] [20] [21] with the increased clinical use and exchange of these ucb units it became clear that it was necessary to standardize practices across the various cbbs. in order to contribute to this, a group of experts involved in ucb banking established the netcord organization in 1998. 22 the initial remit of netcord was to set up an international registry of ucb and to develop procedures and quality standards for the safe collection, exchange, and clinical use of these banked units. these efforts culminated with the development of the net-cord-fact (foundation for the accreditation of cellular therapy) international standards for cord blood collection, processing, testing, banking, selection, and release in 2000 with the last version (5th) published in 2013. 23 these standards form the basis for the netcord-fact accreditation program. the american association of blood banks (aabb) has also developed standards and an accreditation scheme, and since 2004 have been incorporated into the standards for cellular therapy services. 24 different types of ucb banking programs have been established depending on the genetic relationship of the donated ucb unit with the potential recipient, i.e., allogeneic or autologous, and on the funding sources, i.e., public or private. [25] [26] [27] in the allogeneic setting, an additional distinction needs to be drawn between unrelated (also called altruistic) and related donations. unrelated allogeneic ucb banking includes the collection, processing, and storage of altruistically donated ucb, in order to create an inventory of hscs that can be searched for any patient in any part of the world and in need of an unrelated allogeneic hsc donor. these programs are also referred to as public ucb banking. in the allogeneic-related setting, the ucb is collected from a healthy sibling of a patient with a disease that can potentially be treated with a ucb transplant. these collections are performed at the request of the physician treating the patient, with the agreement of the obstetrician looking after the mother. [28] [29] [30] [31] although the standards for the collection of unrelated and related ucb are similar, in the banking of related ucb units, no threshold values for minimum volume collected are required and there is no operational need for volume reduction (vr) of these units, although clinically vr may be beneficial (see below). also, the exclusion from banking due to microbial contamination does not always apply since antibiotic sensitivity tests can be performed if and when the collection is required for transplantation. an important consideration in related ucb banking is that in approximately 70% of cases, the collected ucb unit is not fully human leukocyte antigen (hla)-matched with the patient and it is unlikely that it will ever be used for the intended patient. these units will have to be kept frozen indefinitely unless clear policies regarding their disposal are put in place. with the availability of prenatal genetic diagnosis, including hla typing, it will be possible to collect units selected only from hla-identical siblings, as has already proved possible. 32 so far, the majority of the transplanted related ucb units have been fully hla-matched and in patients with hemoglobinopathies. in fact, in some centers, related ucb transplantation is the first line of treatment for patients with thalassemia major. 33 in most european countries, these two types of allogeneic ucb banking (unrelated and related) are carried out by government-administered institutions and funded by the national health systems available in each country. in the autologous or family ucb banking setting, the collections are normally performed by privately funded institutions at the request of the family of the potential donor, and as its name indicates these collections are mainly for autologous use or for a named recipient normally within the family. worldwide, many private cbbs are collecting and storing ucb for eventual autologous or family use. although there are more than 1 million of these units stored in private cbbs, a very small number have been transplanted, mostly with unknown outcomes. there are anecdotal cases of autologous ucb transplantation, but in general the scientific and clinical arguments for the banking of these units are not universally accepted. 34 there are also a number of ethical issues associated with this practice, which have been extensively reviewed. [35] [36] [37] [38] [39] furthermore, it appears that quality parameters for privately stored units seem to be inferior to those measured in those stored in public cbb, highlighting the fact that if the clinical value of autologous ucb transplantation is established, the issue of the quality of these units will become even more relevant. 40 alternative models of cbb, called hybrid or mixed, have been developed and in these programs the ucb collection is carried out on behalf of (and paid for) the family requesting the collection for either autologous use or for a named person within the family. in one of these models, the collection is split into three and 1/3 is donated to the unrelated public bank and the other 2/3 is stored as a private collection. however, with the increasing evidence on the impact of high total nucleated cell (tnc) and cd34+ cell content on the clinical outcome of ucb transplantation, which is directly correlated with the volume collected, the value of these small volumes of ucb is limited. the other "mixed" model involves the collection and storage of the ucb, for a fee, for autologous or for a named family member, but in this case, the relevant information on the ucb unit is made accessible to unrelated donor registries. once or if the unit is selected for an unrelated patient, the family is then asked to release the stored unit with the consequent refund. to date, no evidence on the application of this model has been presented. the majority, if not all of, the ucb banking procedures and standards were initially developed primarily for the collection and banking of allogeneic unrelated cord blood units (cbus) from altruistic donors. however, in order to ensure the quality and efficacy of all collected units and to safeguard the eventual recipients of these products, netcord-fact and aabb have also developed standards that are applicable to the collection and storage and release of allogeneic related and autologous ucb. the main aspects of an unrelated ucb banking program include the following: 1. promotion, recruitment, donor selection, informed consent, collection, and transportation to processing facilities and donor follow-up 2. processing, testing, cryopreservation, and storage 3. listing, searches, selection, testing, and distribution to a transplant program and posttransplant clinical follow-up all these procedures and activities of a cbb program are supported by comprehensive inventory and quality management systems covering all the various components of ucb banking as described below (see figure 1 ). selection, consent, collection, transportation to processing facilities, and donor follow-up the promotion and model of collection of ucb vary from country to country and depend largely on the nature of the funding and health-care system available in each country. in cbbs funded with public or government monies, the promotion is generally carried out and restricted to antenatal clinics selected as collection sites. the material used in the promotion includes leaflets, videos, seminars, etc. these should provide as much information as possible about the program including the need for consent, the right to withdraw at any step of the process, the clinical use of the collection, and about its potential use in research if the collected unit is not suitable for clinical banking. the promotional material should be clearly understood by the potential mother donors, and it should be translated into various languages if required. the latter is particularly important when recruiting donors are from an ethnic minority background and are not fluent in the language of the country where the recruitment is taking place. 41 the majority of the banked units so far have been collected in countries with a population of predominantly european caucasoid ethnic background and expressing the hla profile of this ethnic group. there is therefore a need to increase the hla diversity of the banked ucb and one way of achieving this is to recruit donors in maternity units with high numbers of deliveries from an ethnically diverse population. a number of cbbs have now been established in countries with an ethnic and hla profile not historically represented in the international registries of unrelated hsc and these units may contribute to expand the hla profile of the internationally available pool of unrelated ucb units. a number of publications have indicated that the volume and tnc content of the units collected are smaller in mothers from afro-caribbean and asian ethnic background compared to those collected from european caucasoid donors. 42, 43 it is therefore crucial to carefully select these donors in order to minimize the waste of these units. the national health service cord blood bank (nhs-cbb), formerly known as the london cord blood bank, was set up in 1996 with the aim of enriching the national and international hsc donor pool with units from ethnic minorities (em). 44, 45 at present, nearly 38% of the banked cbus are from em and ethnically mixed genetic backgrounds, expressing unique hla haplotypes. this is of great benefit to em patients as reflected by the fact that nearly 36% of the units issued for transplantation by the nhs-cbb are from em donors. looking into the future it will be important to try to enrich the cbbs with units from a mixed genetic background. in fact, the majority of patients who have difficulties in finding an hla-matched stem cell donor express a combination of a common and a rare hla haplotype. this is one of the most important aspects of cbb and detailed and comprehensive procedures and policies for the selection and acceptance of mothers donating their ucb are crucial to ensure the quality and safety of the collected units. the responsibility for the selection of an ucb donor lies with the medical director of the cbb who should ensure that an appropriate medical and social history is obtained from the mother in order to prevent the transmission of microbiological infection and/or genetic, malignant, or degenerative disease. donors with a family or personal history of genetic disease, particularly relating to the hematopoietic or immune system, should be asked for details of those suffering from such diseases and their family relationship to the infant donor. further details may be required from the general physician or other professionals. the communicable disease risk history of a surrogate mother who carries an infant not genetically related to her, and of a sperm, egg, or embryo donor shall also be obtained and documented if applicable. travel history of potential donors is also important to assess risks. more recently, a number of publications have indicated other factors such as the ethnicity, gender, and age of gestation which may affect the size/volume of the collections. initially and in order to make the best use of resources, consent was only obtained from mothers from whom a successful collection had been obtained. however, in europe following the implementation of the european union tissues and cells directive (eutcd) 2004/23/ec in april 2006, stating that "procurement of human tissues or cells shall be authorised only after all mandatory consent or authorisation requirements have been met," all collected ucb units need to have a signed consent obtained prior to delivery. 46 at present, in most cbb programs, consent is obtained when the mothers attend their hospital at around 30 weeks of pregnancy or via a "mini consent" form completed before the mother is in established labor. the introduction of the "mini consent" form, which allows for the collection of the cbu, has facilitated the implementation of the eutcd, and it has also increased the efficiency of collections by decreasing the wastage of cbus discarded due to the lack of consent. 47 following the initial mini consent, a full more detailed consent is required and this provides the basis for proceeding to the processing and testing of the collected unit. an important aspect of the consent process is to provide detailed and clear information about the tests required, the intended use of the unit, particularly in relation to the altruistic nature of the donation, and about the potential use of the clinically unsuitable units for research and development. also, it needs to include the consent to contact relevant health professionals in the event of a positive result relevant to their health, to obtain and store samples for future testing, and to store personal information. this also means that for mothers who do not speak the language of the country where they are giving birth, all relevant information and the process of obtaining consent should be performed not only in their own language, but also by somebody able to communicate and answer the relevant questions of the mother. consent should be taken when the mother is able to concentrate on the process and certainly not when the mother is in labor. importantly, the netcord-fact standards also mention that regardless of whether the unit is collected for unrelated or related use, if this unit may potentially be used for reasons other than the initial clinical intent, not only this should be mentioned in the informed consent but also the donor should have given consent with documents and information related to the potential related or unrelated use of the unit. the collection of the ucb is carried out by suspending the placenta, cannulating the vein and allowing the blood to drain by gravity into a specially designed ucb collection bag placed on a shaker in order to avoid the formation of clots. collections can be carried out at fixed and nonfixed sites and in either case an agreement between the cbb and the collection site is required. 23 fixed sites. in this model, the ucb units are collected by trained staff employed by the cbb or by the maternity units in each hospital. in europe, the collection of ucb can only be performed in sites that comply with the regulatory requirement of the eutcd, i.e., in licensed or fixed sites and by trained staff. nonfixed sites. in this case, the collection is performed at any maternity unit by either their own staff or by agency staff. the cbb provides the appropriate kit and instructions for the ucb collection. although this practice facilitates and allows for the collection of altruistically donated units anywhere in the country, it is also associated with a reduced number of units suitable for banking due to an increase in the number of bacterial infections, and low volumes and tnc of the collected units. this may be due to the lack of training or experience of the personnel performing these collections. this practice is not very common in europe due to the stringent training requirement of the eutcd. the current revised version of the netcord-fact standards covers the collection at both fixed and nonfixed sites. when selecting the collection sites, it is important to consider the number of deliveries per year in order to maximize the resources and to maintain the training of the collection staff. the ucb collection can be performed in utero or ex utero, following full-term normal delivery or cesarean section. the minimum gestation period for collection is 34 weeks. in utero collections are performed by a trained member of the delivery team during the third stage of labor before the placenta is delivered. ex utero collections are carried out by cbb trained staff, normally outside the delivery room to avoid interference with the delivery process. in these collections, the risk to the mother or infant is minimal, but the risk of microbial contamination may be higher. initial studies had indicated that in utero collections yielded larger volumes (and tnc doses) than ex utero collections, but more recent studies have shown that, if appropriately trained staff are involved in the collection, there is no significant difference in the volume, or indeed in the contamination rate, with either of these two methods. 47, 48 since the safety of mother and child are paramount and because of the possible diversion of the attention from the mother and newborn to the ucb collection, both the uk royal college of obstetricians and gynaecologists (rcog) (scientific advisory committee for the royal colleges of obstetricians and gynaecologists, 2001) and the royal college of midwives in the united kingdom have recommended that all ucb collections be carried out ex utero. 49 also, following reports on the effect of delayed clamping on infant development and iron status, the uk rcog has also issued guidelines regarding the timing of clamping. [50] [51] [52] once a successful ucb collection has been obtained, blood samples from the mother are taken for communicable disease testing within 7 days before or after collection. a history of the current pregnancy and delivery, and the infant's donor birth data should be obtained and documented including gender, gestational age, and results of any other relevant test. information about the clinical examination or any finding suggestive of disease potentially transmitted through transplantation should also be recorded. the collected units and the associated maternal samples are then transported to the cbb processing centers in a temperature-controlled environment as soon as possible following the collection. an agreement between the collection site and the staff responsible for the transport of these units is required, as well as a documented evidence of training the staff involved in this task. in some cbbs, a follow-up telephone interview is carried out 8-12 weeks postcollection in order to check the health status of the mother and the newborn from whom the ucb was collected. other cbb programs have a policy of contacting the mother when a unit has been reserved or prior to its release to the transplant program. all cbus are quarantine frozen in temporary containers until all relevant test results are reviewed and the units are medically released for long-term storage. counseling resources should be in place to support the donor and family in case of a positive infectious disease marker other than cytomegalovirus. an appropriately signed consent authorizing the processing, testing, and storage of the units and associated samples is required before commencing these procedures. although the viability and functionality of the collected stem cells seem to be preserved for up to 96 h, the majority of publications have shown the benefits of processing the ucb units as soon as possible and ideally within 48 h of collection. [53] [54] [55] [56] the current netcord-fact standards indicate that the cryopreservation of unrelated ucb units should be performed within 48 h of collection in either a closed system or in an environmentally controlled clean room. for related ucb, the cryopreservation should commence within 72 h of collection. 23 the tnc and cd34+ cell content per kilogram of patient's weight (as well as hla matching) are important factors influencing the outcome of ucb transplantation, particularly in improving engraftment rates. 19, 25, 57, 58 as a result, a minimum dose of tnc and cd34+ cell content of the units has been proposed for transplantation into patients with malignant and nonmalignant diseases. [59] [60] [61] furthermore, world marrow donor association (wmda) data indicate that the minimum cut-off tnc and cd34+ count of the transplanted units have increased throughout the years. 14 thus, in order to ensure that the stored units can meet the requirements of transplant center acceptable values for volume, tnc and cd34+ cell content of the ucb units collected (i.e., preprocessing) are established by each cbb program in order to compensate for the cell loss occurring during the processing of the units at various stages of the procedure (approximately 10%). therefore, in order to improve the quality of the units banked and to minimize the costs of cbb, most ucb banks evaluate the collected units for a number of parameters before processing a unit, and these include volume, tnc, cd34, and colony forming unit (cfu) content. [62] [63] [64] [65] [66] [67] [68] the volume of the collected unit was the original parameter used to select those collections that should proceed to processing and subsequent banking and it remains a simple, fast, and cost-effective surrogate marker to be used in the assessment of the collected units. although the collected volume has a strong correlation with the tnc content, most cbbs use the tnc parameter to inform decisions throughout the cbb process and for the selection of the ucb unit for transplantation. the benefits of measuring the cd34+ cell content of the units has been highlighted by studies showing that this marker was a better correlate for engraftment than the tnc dose. 19, 69 however, in the past, not all banked units had cd34 counts performed at banking, and there was no standardized test for the identification and measurement of cd34+ cells. today, this test has become more standardized and it is now also possible to assess simultaneously both the percentage of cd34+ cells and viability. 70 it has been suggested that the potency of the ucb measured by the number of cfus is strongly positively associated with engraftment rates in children. 71 page et al. have shown that the potency of the units (measured by the cfu content) was a strong predictor of engraftment. 72 current methods for assessing the number of cfus are complex and time-consuming, and unless these assays are performed on every single unit banked at a considerable cost bearing in mind that only about 1-2% of banked units are issued for transplantation. also, the results may not be available when the unit is requested (they can take up to 14 days). alternative methods to assess the function or potency of the ucb units have been recently described and are currently undergoing a more extensive clinical evaluation. our own studies have shown a very good correlation between the amount of volume collected with the tnc, cd34, and cfu content of the units (see figure 2 ). because the processing procedures can impact on the recovery of the tnc and cd34 (and cfus), all these parameters have to be measured pre-and postprocessing (or before freezing), and also on the finally selected units released for transplantation. other obstetric factors including birth weight also seem to affect the characteristics of the collected units. [73] [74] [75] more recently, kurtzberg and colleagues have developed a scoring system called the cord blood apgar to optimize ucb unit selection for transplantation. 76 this system considers a number of characteristics of the ucb unit such as volume, tnc, cd34, and cfus before and after processing, and it also includes thawing techniques and other donor or patient variables. initially, all ucb units were frozen without any manipulation but it soon became clear that the long-term storage of large numbers of frozen units would create a space issue. these considerations led to the development of protocols to reduce the volume of the collected ucb units prior to storage. a number of vr methods are currently being used, the majority of which deplete the unit of red cells and plasma, leaving the buffy coats in a standard volume. 77, 78 an important consideration with any vr method is the preservation of a maximum number of viable tncs and cd34+ cells in the stored buffy coat layer. the first semiautomated system for vr, the optipress, was introduced in 1999. at present, most cbus are reduced to a standard volume of 21 ml prior to freezing, using automated systems such as sepax 540 or the autoxpress. through the introduction of two new filters, one for the hydroxyethyl starch/anticoagulant (complete with a small sample bulb to allow for resampling) and a second to add the dimethyl sulfoxide (dmso), the sepax 540 system remains sterile and is referred to as "closed." [79] [80] [81] this means that processing of these units can be undertaken in a grade c room, under a laminar flow cabinet. an additional clinical benefit of vr is that it reduces the amount of dmso contained in the unit, which is particularly beneficial for units that will be infused to small children. initially, due to the large volume of dmso, cb cells had to be washed prior to infusion, especially in the case of small children. nowadays, washing is not essential for volume-reduced units. with the introduction of vr, it is now necessary to perform full blood count, tnc, nucleated red blood cell, and cd34 (and cfus) counts before and after processing, in order to assess the effect of the manipulation on the viability and quality of the unit prior to its long-term storage. the cryopreservation of buffy coats containing the hsc can be performed using manually or automated (bioarchivesystem) controlled rate freezer equipment. the automated system provides a platform to freeze and store cells in the same place minimizing exposure to temperature changes and also allowing the electronic identification of the archived units. thus, when a unit is required for issue, it can be automatically retrieved, through a periscope, without exposing the other units to temperature changes. the vr ucb units are resuspended in 10% dmso cryoprotectant (50% dmso diluted in dextran 40) in a freezing bag with two compartments, which is placed in a metal container for cryopreservation and long-term storage however, both the automated and the manual systems are perfectly adequate, provided the temperatures are regularly monitored and the process is fully evaluated and quality controlled. long-term viability of the frozen cells was also of concern but it is now known that the standard cryopreservation protocols of freezing the cells in 10% dmso in controlled rate freezers and storage below â��135 â°c give an average of 80% recovery of nucleated cells and >90% recovery of progenitor cells, as measured by stem cell surrogate markers, cd34+ cells, and cfu assays. 82-85 in order to maximize the amount of cells stored, all the "waste" components produced during the processing of the units are utilized for testing and archiving. archiving of samples is crucial, in order to be able to perform additional tests in future when a unit is selected for transplantation and, if required, to test for any new marker that may affect the use of the units. at the nhs-cbb, a blood film is prepared from the fresh cb to perform an initial hematological screening of the unit. in addition, a small piece of cord tissue is collected and frozen as a source of dna for future testing, if required. the algorithm for testing the ucb collection is complex. some tests need to be performed upfront before banking (pre-and postprocessing) and others are carried out when the units are listed for searches. some tests are performed on the mothers, others in the ucb unit, and others on both. other tests can (and some must) be performed once the unit has either been reserved or selected for transplantation (see figure 3 ). among the tests required at banking are those performed on the mother's blood, which in the united kingdom at least, is the same as those required for blood donors. with the shortening of the window period of infectivity by the introduction of nucleic acid testing for human immunodeficiency virus/hepatitis b virus/hepatitis c virus, it should be possible to eliminate the need for a second 6-month followup sample from the mother to retest for infectious disease markers. this requirement was one of the important reasons why significant numbers of units had to be discarded, in spite of their compliance with the banking requirements. also, mostly depending on the country of origin of the motherdonors, additional screening, such as tests for malaria, chagas disease, and, more recently, west nile virus and severe acute respiratory syndrome, are required to comply with regulations in each country. the ucb unit is also tested for these markers, once selected for transplantation. the finally processed unit is also tested for both aerobic and anaerobic cultures prior to freezing to assess the presence of bacterial and/or fungal cross-contamination from the birth canal or systemic sepsis in the donormother or infant. initially, lightly contaminated units were kept in the bank provided an antibiotic sensitivity test was performed and the results communicated to the transplant center if required. however, the current netcord-fact standards mandate that bacterially contaminated unrelated units should be discarded. ucb units collected for directed use, either related or autologous, can still be banked provided the above-mentioned tests are performed. all ucb units are tested for abo/rh, tnc, and cd34, and some cbbs also perform cfu on all units post processing. the hla typing of the units is also carried out at the time of banking. current standards indicate that all hla typing should be carried out using dna-based molecular techniques. low-resolution hla-abc and high-resolution hla-drb1 typing should be performed prior to the listing of the units with the relevant ucb or adult unrelated hsc donor registries. a number of recent publications have indicated that if the transplanted ucb unit is a mismatch at one of the noninherited maternal hla alleles, there is an improvement in the outcome of this transplant. 86, 87 as a result, a number of ucb banks are now typing and reporting the maternal hla type of the listed units. the role of the kir receptor/ligand mismatching is still controversial; 88, 89 therefore, the majority of cbbs do not routinely test their units for these markers before listing them in the national or international hsc registries. on completion of the processing and testing, all the information regarding the mother and the cbu must be reviewed by the medical director of the cbb (or a designee) to assess the suitability of the unit for inclusion into the bank. once the units are medically released, they can be listed for searches with both the national and international registries. all units are listed under a unique identifier with the following information: hla type, volume of collected ucb, and tnc (and cd34+, cfus, and maternal hla typing if available) of the final product. the issue as to whether the cd34 count should be included at registration is currently under discussion. some cbbs are also registering the hla typing of the mother of the cbu in order to provide the option of selecting mismatched cbu based on the noninherited maternal antigens (nimas.) at present, there are several international registries, netcord listing only ucb units and asiacord and bone marrow donors worldwide (bmdw), which contain both adult hsc donors and cbus (http://www.bmdw.org). the national marrow donor program (nmdp) also lists units of its associated partners for searches. 90 there are approximately 200,000 cbus in netcord, and over 600,000 registered with bmdw. 15 most units registered with netcord are also in bmdw (see figure 4 ). in future, with the implementation of the european marrow donor information system (emdis) cord, an electronic system designed for the rapid exchange of information and requests between transplant centers and donor registries, this system should speed up the whole process of donor searches and selection, since all relevant information about a ucb unit will be readily available. some of the first cbbs that were established operate as independent registries. however, today, the vast majority of cbbs work through their national registries due to the fact that most transplant centers prefer a combined search report, listing all available, suitably matched adult donors and cbus at the same time. the current netcord-fact standards indicate that all registry aspects of the cbb programs need to operate under the guidelines of the wmda, 91,92 and that these registries should be wmda accredited or in the process of obtaining accreditation. transplant centers initiate a search for cbus in the same way as for adult stem cell donors and once a transplant center receives a match report, it contacts the relevant ucb bank directly to request further information and/ or additional tests such as high-resolution hla data, cfu content, or microbiological markers. the cbb program should have a fully validated electronic record system to enable the listing, searches, and distribution of ucb to the transplant program. when a ucb unit is reserved or released for transplantation, a number of additional tests are performed at the request of the transplant centers. the type and resolution of tests required at this point have changed with the years as a result of the clinical outcome analyses. for instance, the range of required tests for infectious disease markers is expanding and now includes epstein barr virus, human herpesvirus 6, 7, and 8, and toxoplasmosis. the request for cfu assays to assess the functionality or potency of the ucb cells is not consistent and many transplant centers are prepared to go ahead with the procedure in the absence of these results. due to the high cost of cfu assays and since these results can take up to 14 days, most cbbs perform this test at the stage of reservation of the unit. high-resolution hla-a, b, c, and drb1 typing is also performed prior to the release of the unit and on a contiguous segment, if this is not available then another method needs to be used to confirm the identity of the ucb unit. screening of the selected ucb unit for abnormal hemoglobins has also become an additional requirement prior to their release. clear documentation and procedure for the transport of the frozen selected ucb units to the transplant center should be in place. the units are transported in shipment containers by accredited and trained carriers. the clinical follow-up of the released units is an important quality aspect of the operation of cbb. this is normally carried out by a national registry and/or by eurocord 93 and the center for international blood and marrow transplant research (cibmtr). 94 eurocord was established in 1999 and is responsible for collecting and analyzing all clinical outcome data on cb transplantation on behalf of the european blood and marrow transplant group. 95 cib-mtr fulfills a similar role for the transplant activity in the united states and other north and south american countries. eurocord and cibmtr have recently agreed to share information and analyses in order to avoid duplication of the reported data. as mentioned above, all the activities of a cbb program need to be supported by robust electronic inventory and quality management systems and programs. all policies and procedures should be documented and updated regularly to incorporate changes in the relevant standards and/or to the outcome of internal or external audits. the label of the ucb and all associated samples including maternal samples produced throughout the various stages of the process have to be international society of blood transfusion 128 compatible for traceability. since cbb and ucb transplantation activities involve the import and export of a cellular product across different countries, they need to operate within a highly regulated environment in order to ensure that the donations released are safe and of high quality. netcord-fact and the aabb have developed standards and accreditation schemes to support this activity. these standards also state that all laboratories supporting cbb activities need to have the relevant additional accreditations in place, e.g., european federation for immunogenetics or american society of histocompatibility and immunogenetics for the hla aspects and wmda for the registry aspects. 23, 92, 96 internationally, all aspects related to the clinical transplantation of ucb cells are covered by the fact-jacie (joint accreditation committee isct & ebmt) standards and not by the netcord-fact or aabb standards. the regulatory aspects covering the activity of cbb have also increased significantly in the past years. in the european union, the eu directive 2006/17/ec and 2006/86/ec regulates on the quality and safety issues covering the donation, procurement, testing, processing, preservation, storage, and distribution of human tissues and cells. 97 these directives require all member states to have inspection and accreditation systems in place ensuring that all banks providing these services comply with an agreed set of standards. in the united kingdom this is implemented by the human tissue act, set up in 2004 and implemented in april 2006. 98 discussions around cost efficiency and optimal size of ucb units required to provide donors for the majority (80%) of patients in need of an unrelated donor have been ongoing since adult hsc donor registries were first established. the probability of finding an hla-matched unrelated donor depends not only on the number of loci (i.e., 6/6 or 10/10 loci) and resolution (medium vs. high) of the hla matching required, but also on the ethnic background of the patient and the pool of donors to be searched. since the vast majority of donors currently available in the international registries are of european caucasoid ethnic background, the probability of finding a 6/6 (or a 10/10) hla-matched donor for patients from em backgrounds is significantly reduced. 102 in ucb transplantation, a higher degree of hla netcord mismatches could be tolerated and these transplants could be performed with as little as 3/6 hla loci matching between the recipient and the cbu. also, several studies have confirmed that the outcomes of cb transplantation between 4/6 and 5/6 matched donors and recipients seem to be comparable to those seen between fully netcord matched adult donors. these results led to the proposition that the required size of ucb inventory could be smaller than that of adult unrelated hsc donors. most patients could find at least one 4/6 matched donor from the current global cbu inventory. a study published in 2009 demonstrated that, at least for the united kingdom, a minimum number of 50,000 banked ucb should be sufficient to provide a ucb net-cord matched unit to approximately 80% of patients. [103] [104] [105] [106] [107] the role of high resolution (hr) hla matching was found not to be significant. 108 however, it has now been shown that high-resolution hla-a, b, c, and drb1 matching may also influence the clinical outcome of ucb transplantation and if these results are confirmed, a larger pool of donors to select the best compatible ucb will be required. 109, 110 therefore, the question as to whether the exact number of banked ucb units would suffice requires further evaluation. recent data have shown improved transplant outcomes after hla-mismatched ucb transplantation where the mismatched antigen in the patient is matched to the donor nima, suggesting that when a fully hla-matched cbu donor is unavailable, a nima-matched donor could be chosen. 86, 87 if nimas are taken into account, additional "virtual" hla phenotypes of the cbu are available for matching consideration. one locus nima substitution for the current matching guidelines for hla-a, b, or drb1 loci would add six "virtual" phenotypes, two substitutions would provide 12 "virtual" phenotypes, and three substitutions would provide eight "virtual" phenotypes. one cbu could then provide a maximum of 26 "virtual" phenotypes if all hla-a, b, and drb1 loci are included. the nhs-cbb currently has 3000 ucb units listed with maternal hla phenotypes in the british bone marrow registry (bbmr) and bmdw. these ucb units have the potential of adding 18,000 new phenotypes with one nima substitution, 36,000 new phenotypes with two, and 24,000 with three substitutions, giving a total of 78,000 new phenotypes for the cbu registered. if maternal hla typing was performed on the 15,000 nhs-cbb banked and registered cbus, a potential 390,000 additional "virtual" cbu phenotypes would be added to the bbmr. by using information on the hla types of nimas it is possible to increase the number of cbu phenotypes available for searching and consequently increase the probability of finding an appropriately matched donor for a patient. one of the important remaining challenges in ucb transplantation is how to improve engraftment, associated with a slow and often incomplete immune recovery, particularly in adult patients. this limitation, which seems to be primarily due to the insufficient number of hsc and to immunological naã¯vety of the immunological effectors, such as t cells in the ucb collections, has led to the development of a number of approaches to improve these outcomes. of these the most successful so far has been the use of two ucb units for an individual patient in order to increase the number of transplanted hscs. [111] [112] [113] this approach has yielded comparable results to those using a single cbu, and has opened the way for using ucb transplantation in older and heavier patients. the initial protocol developed by the minneapolis group has now been adopted by many centers with or without modifications and has allowed the performance of ucb transplantation in patients not previously considered for the procedure due to their age or weight. other developments include the in vitro expansion of ucb hsc but early attempts were not very successful, since it appears that the majority of the protocols used led to the expansion of mainly mature progenitors. more recently, in vitro and in vivo expansion using sdf-1/cxcl12 associated to diprotin a and/or other cytokines, or using notch-ligand delta 1, or mesenchymal stem cells (mscs) has been described. 114, 115 enhancing the homing capacity of ucb cells by inhibiting the enzymatic activity of cd26/dipeptidylpeptidase iv or by in vivo direct injection of cb cells into the iliac crest has been published and has also gone into phase ii clinical trials. 116 some investigators have now attempted the infusion of ucb intrabone or in conjunction with cd34+ or third party bone marrow-derived mscs, with or without cd34+ cells, with limited improvement in engraftment rates. 117, 118 regardless of these potential new developments, the majority of cbbs are now banking ucb units with greater tnc and a high number of cd34+ cells. the identification of modifiable prognostic factors for engraftment such as choosing the "best" cbu based on cell dose such as hla, diagnosis, and presence of hla antibodies may also contribute to the improvement of this procedure. another challenge is to try to improve the immune reconstitution of cord blood transplant patients in order to reduce infections and/or viral reactivation. 119 unrelated ucb banking is a complex and highly regulated procedure and involves the collection, processing, testing, banking, listing, selection, and release of frozen ucb units to patients in need of an hsc transplant. since its inception, it has undergone a significant evolution driven primarily by the clinical results obtained with the use of the banked units. on the other hand, despite the initial skepticism of many transplant physicians, the success of ucb transplantation that we see today has been aided by development and implementation of stringent standards and international accreditation programs to ensure the safety, quality, and efficacy of these ucb units. in future and if the new experimental protocols for the expansion of hscs and/or immune effectors prove to be successful, further development of the procedures and standards currently used in the banking of ucb will be required. as ucb transplantation continues to increase and with the introduction of new clinical protocols, other genetic and epigenetic factors related to the quality and the efficacy of the ucb units and/or related to the recipient of these units may begin to emerge. also, some of the immunotherapy protocols derived from adult unrelated hsc donors such as expansion of viral-specific t cell, regulatory t cells, nk cells, or mscs could be applied to ucb transplantation. if this is the case, cbbs will have to consider the operational changes that will be required to collect, process, store, and release these new associated products. in vitro growth of granulocytic colonies from circulating cells in human cord blood granuloerythropoietic colonies in human bone marrow, peripheral blood and cord blood haematopoietic stem cells (cfuc) in human cord blood human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults hematopoietic transplantation by means of fetal (cord) blood. a new method hematopoietic reconstitution in a patient with fanconi's anemia by means of umbilical-cord blood from an hlaidentical sibling placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients unrelated placental blood for bone marrow reconstitution: organization of the placental blood program outcome of cord-blood transplantation from related and unrelated donors the london cord blood bank hematopoietic transplant potential of unrelated cord blood: critical issues the milan cord blood bank and the italian blood network wmda annual report cord blood banks/registries. 2012. available on request at mail@wmda.info number of donors/cb per registry in bmdw combined effect of total nucleated cell dose and hla match on transplantation outcome in 1061 cord blood recipients with hematologic malignancies outcomes among 562 recipients of placental-blood transplants from unrelated donors a review of factors influencing the banking of collected umbilical cord blood units current thawing and infusion practice of cryopreserved cord blood: the impact on graft quality, recipient safety, and transplantation outcomes unrelated donor umbilical cord blood transplantation for inherited metabolic disorders in 159 pediatric patients from a single center: influence of cellular composition of the graft on transplantation outcomes comparative single-institute analysis of cord blood transplantation from unrelated donors with bone marrow or peripheral blood stem-cell transplants from related donors in adult patients with hematologic malignancies after myeloablative conditioning regimen the international netcord foundation international standards for cord blood collection, banking and release for administration accreditation manual aabb standards for cellular therapy product services umbilical cord blood transplantation and banking cord blood banking and transplantation: a promising reality collection and preservation of cord blood for personal use comprehensive banking of sibling donor cord blood for children with malignant and non-malignant disease directed sibling cord blood banking for transplantation: the ten year experience in the national blood service in england family-directed umbilical cord blood banking long-term follow-up and factors influencing outcomes after related hla-identical cord blood transplantation for patients with malignancies: an analysis on behalf of eurocord-ebmt successful hematopoietic stem cell transplantation for fanconi's anemia from an unaffected hla-genotype-identical sibling selected using preimplantation genetic diagnosis related umbilical cord blood transplantation in patients with thalassemia and sickle cell disease the successful treatment of severe aplastic anemia with autologous cord blood transplantation ethical considerations related to the collection and distribution of cord blood stem cells for transplantation to reconstitute hematopoietic function umbilical cord blood banking: economic and therapeutic challenges private cord blood banking: experiences and views of pediatric hematopoietic cell transplantation physicians high acceptance rate of hybrid allogeneic-autologous umbilical cord blood banking among actual and potential swiss donors ethical issues in umbilical cord blood banking: a comparative analysis of documents from national and international institutions differences in quality between privately and publicly banked umbilical cord blood units: a pilot study of autologous cord blood infusion in children with acquired neurologic disorders cord blood banking in london: the first 1000 collections does ethnicity affect cell dose in cord blood donation? racial diversity with high nucleated cell counts and cd34 achieved in a national network of cord blood banks the london cord blood bank: analysis of banking and transplantation outcome paucity of hla-identical unrelated donors for african-americans with hematologic malignancies. the need for new donor options introduction of mini consent process for the collection of cord blood following implementation of eu directive 2004/23/ec comparison between two strategies for umbilical cord blood collection in utero or ex utero cord blood collection: which is better? scientific advisory committee opinion paper 2 effect of delayed versus early umbilical cord clamping on neonatal outcomes and iron status at 4 months: a randomised controlled trial effect of timing of umbilical cord clamping of term infants on maternal and neonatal outcomes optimal timing for processing and cryopreservation of umbilical cord haematopoietic stem cells for clinical transplantation viability of umbilical cord blood mononuclear cell subsets until 96 hours after collection storage time affects umbilical cord blood viability viability of umbilical cord blood mononuclear cell subsets until 96 hours after collection. transplantation and cellular engineering factors associated with outcomes of unrelated cord blood transplant: guidelines for donor choice outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukaemia: a comparison study history of the clinical use of umbilical cord blood hematopoietic cells recommendations for a standard uk approach to incorporating umbilical cord blood into clinical transplantation practice: conditioning protocols and donor selection algorithms how i treat: the selection and acquisition of unrelated cord blood grafts human uc-blood banking: impact of blood volume, cell separation and cryopreservation on leukocyte and cd34+ cell recovery factors associated with parameters of engraftment potential of umbilical cord blood initial cord blood unit volume affects mononuclear cell and cd34+ cell-processing efficiency in a non-linear fashion evaluation of volume and total nucleated cell count as cord blood selection parameters factors affecting umbilical cord blood stem cell suitability for transplantation in an in utero collection program optimizing donor selection for public cord blood banking: influence of maternal, infant and collection characteristics on cord blood unit quality review of factors influencing the banking of collected umbilical cord blood units viable cd34+ stem cell content of a cord blood graft: which measurement performed before transplantation is most representative? single platform flow cytometric absolute cd34+ cell counts based on the ishage guidelines. international society of hematotherapy and graft engineering the impact of post-thaw colony-forming units-granulocyte/macrophage on engraftment following unrelated cord blood transplantation in pediatric recipients total colony-forming units are a strong, independent predictor of neutrophil and platelet engraftment after unrelated umbilical cord blood transplantation: a single-centre analysis of 435 cord blood transplants obstetric predictors of placental/umbilical cord blood volume for transplantation bigger is better: maternal and neonatal predictors of hematopoietic potential of umbilical cord blood units association among birth weight, placental weight, gestational period and product quality indicators of umbilical cord blood units the cord blood apgar: a novel scoring system to optimize selection of banked cord blood grafts for transplantation cord blood banking: volume reduction of cord blood units using a semi-automated closed system cord blood processing: volume reduction cord blood banking for clinical transplantation pre-transplant manipulation processing of umbilical cord blood units: efficacy of rubinstien's thawing technique used in 40 transplantation procedures qualitative and quantitative cell recovery in umbilical cord blood processed by two automated devices in routine cord blood banking: a comparative study comparison of cord blood thawing methods on cell recovery, potency and infusion methods of freezing cord blood hematopoietic stem cells successful short-term cryopreservation of volume-reduced cord blood units in a cryogenic mechanical freezer: effects on cell recovery, viability, and clonogenic potential current thawing and infusion practice of cryopreserved cord blood: the impact on graft quality, recipient safety and transplantation outcomes reexposure of cord blood to noinherited maternal hla antigens improves transplant outcome in hematological malignancies effect of hla-matching recipients to donor non-inherited maternal antigens on outcomes after mismatched umbilical cord blood transplantation for hematologic malignancy kir-ligand incompatibility in the graft-versus-host direction improves outcomes after umbilical cord blood transplantation for acute leukemia negative effect of kir alloreactivity in recipients of umbilical cord blood transplant depends on transplantation conditioning intensity international exchange of cord blood units: the registry aspects a gift for life. wmda handbook for blood stem cell donations /23/ec/2006 for setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells human tissue authority obtaining an accepted investigational new drug application to operate an umbilical cord blood bank fda guidance for industry and fda staff investigational new drug applications (inds) for minimally manipulated, unrelated allogeneic placental/umbilical cord blood intended for hematopoietic reconstitution for specified indications probability of finding hla mismatched related or unrelated marrow or cord blood donors cord blood stem cells for hematopoietic stem cell transplantation in the uk: how big should the bank be? quality rather than quantity: the cord blood bank dilemma use of cost-effectiveness analysis to determine inventory size for a national cord blood bank cord blood transplantation and cord blood bank in japan the minimum number of cord blood units needed for koreans is 51 -dq in 122 unrelated cord blood/patient pair transplants hardly improves long-term clinical outcome effect of donor-recipient hla matching at hla a, b, c and drb1 on outcomes after umbilical-cord blood transplantation for leukaemia and myelodysplastic syndrome: a retrospective analysis estimation of size of cord blood inventory based on high-resolution typing of hlas creation of a double chimera after the transplantation of umbilical-cord blood from two partially matched unrelated donors transplantation of 2 partially hla-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy single versus double unrelated umbilical cord blood units for allogeneic transplantation in adults with advanced hematological malignancies: a retrospective comparison of outcomes ex vivo expansion of umbilical cord blood notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution direct intrabone transplant of unrelated cord-blood cells in acute leukaemia: a phase i/ii study results of a pilot study on the use of thirdparty donor mesenchymal stromal cells in cord blood transplantation in adults unrelated umbilical cord blood transplants in adults: early recovery of neutrophils by supportive co-transplantation of a low number of highly purified peripheral blood cd34+ cells from an hla-haploidentical donor infectious complications following unrelated cord blood transplantation key: cord-316330-55nd3pwe authors: ramos-lopez, omar; daimiel, lidia; ramírez de molina, ana; martínez-urbistondo, diego; vargas, juan a.; martínez, j. alfredo title: exploring host genetic polymorphisms involved in sars-cov infection outcomes: implications for personalized medicine in covid-19 date: 2020-10-19 journal: int j genomics doi: 10.1155/2020/6901217 sha: doc_id: 316330 cord_uid: 55nd3pwe objective: to systematically explore genetic polymorphisms associated with the clinical outcomes in sars-cov infection in humans. methods: this comprehensive literature search comprised available english papers published in pubmed/medline and scopus databases following the prisma-p guidelines and pico/axis criteria. results: twenty-nine polymorphisms located in 21 genes were identified as associated with sars-cov susceptibility/resistance, disease severity, and clinical outcomes predominantly in asian populations. thus, genes implicated in key pathophysiological processes such as the mechanisms related to the entry of the virus into the cell and the antiviral immune/inflammatory responses were identified. conclusions: although caution must be taken, the results of this systematic review suggest that multiple genetic polymorphisms are associated with sars-cov infection features by affecting virus pathogenesis and host immune response, which could have important applications for the study and understanding of genetics in sars-cov-2/covid-19 and for personalized translational clinical practice depending on the population studied and associated environments. coronaviruses (covs) have become one of the leading pathogens of the latest emerging outbreaks of respiratory disease, representing a serious public health burden worldwide [1] . a novel coronavirus was identified to play a crucial role in the severe acute respiratory syndrome (sars) in 2003 [2] . later, the severe acute respiratory syndrome coronavirus-2 (sars-cov-2, are related to an exacerbated immunological response that the viral infection produces in the host [5] . in some cases, this reaction is excessive (inflammatory cytokine storm), triggering an extensive tissue damage and body dysfunction [6] . the clinical manifestations of this infection include typical symptoms such as cough, fever, asthenia, and mild respiratory distress. however, more serious respiratory injuries include pneumonia, acute respiratory distress syndrome, and respiratory failure, accompanied by inflammatory outcomes affecting adipokines and other mediators [6] . furthermore, recent investigations have reported the presence of neurological, renal, hepatic, and cardiac complications in some patients [7] . on the other hand, a group of patients are asymptomatic or with very mild manifestations during the course of the infection, with a relatively short recovery time [8] . certain risk factors for the chronicity and severity of covid-19 infection have been reported, including age (over 60 years), male gender, and the presence of concomitant metabolic conditions such as obesity, diabetes, and hypertension [9, 10] . nevertheless, there is a constant search for biological factors associated with the evolution of covid-19. evidence suggests that genetic factors may influence the onset and progression of infectious diseases [11] . in this context, a number of genetic variants, mainly single nucleotide polymorphisms (snps), have been associated with the susceptibility/resistance to viral respiratory infections [12] . until now, the specific role of the genetic make-up in sars-cov-2/covid-19 disease has been insufficiently analyzed, although proinflammatory-related pathways have been involved [6] . understanding the host genetic factors involved in sars-cov infection could contribute to the identification of new therapeutic tools for advancing in the prevention and clinical management of this disease through a personalized medicine approach [13] . due to the lack of information concerning genetics and sars-cov-2/covid-19 infection, the aim of the present review was to systematically explore the genetic polymorphisms associated with the clinical outcomes in sars-cov infection in humans in order to translate this information to sars-cov-2/covid-19 research and clinical implementation. a systematic review was performed in march 2020 to analyze the association of genetic polymorphisms with sars-cov infection outcomes. the methodological procedures of this systematic review were performed according to the preferred reporting items for systematic reviews and meta-analyses protocols (prisma-p) guidelines [14] . observational (cross-sectional, cohort, or case-control) studies exploring the role of host genetic polymorphisms in sars-cov disease (infection susceptibility, disease progression, and clinical outcomes) in adult subjects were included. articles written only in the english language were selected. duplicates, reviews, clinical trials, in vitro assays, animal experiments, and human studies reporting negative results or focused on other infectious diseases or genetic factors were excluded. the comprehensive literature search encompassed available papers compiled in pubmed/medline and scopus databases without a period of time or study population restrictions. advanced search techniques for each database were used to make the search more efficient (i.e., "mesh" and "tiab" terms). the keywords used were as follows: ("genetic polymorphisms") or ("genetic variations") or ("gene polymorphisms") or ("genetic variations") and ("sars-cov") or ("sars cov") ("sars coronavirus") or ("sars virus"). the reference sections of the included articles were also scrutinized. this search strategy was replicated at different times to guarantee reproducibility. moreover, two researchers performed independently the research. using these terms, a total of 202 articles were identified. five additional articles were added from reviews, which did not appear in this first stage ( figure 1 ). first, duplicates were eliminated (n = 132), obtaining 75 records for the eligibility criteria assessment. after the duplicates were removed, the titles, abstracts, and keywords of 75 manuscripts were screened, excluding those that did not meet the eligibility criteria (n = 41) or reported negative/inconsistent findings (n = 8). finally, 26 articles were included in the current analyses ( figure 1 ). the most relevant information about each of the 26 selected studies included in the present review was analyzed using the population, intervention, comparison, outcome (pico) criteria [15] . the selected articles met at least 3 inclusion criteria (supplementary table 1 ). the quality assessment of the analyzed studies was performed using the appraisal tool for cross-sectional studies (axis tool), a validated 20-point questionnaire with 6 subdomains (introduction, methods, results, discussion, and others) addressing study quality and reporting encompassing study design, sample size justification, target population, sampling frame, sample selection, measurement of validity/reliability, and overall methods [16] . because the axis tool does not provide a numerical scale for assessing the quality of the study, subjective assessments by the authors are required [16] . thus, the authors of this study performed a consensus procedure to evaluate the general and overall quality of records in order to select those to be included in the systematic review. the screening was completed with references coming from citations of the 26 selected studies. the results of the axis tool are summarized (supplementary table 2 ). the present systematic review revealed that 29 polymorphisms located in 21 genes were associated with sars-cov susceptibility/resistance, disease severity, and clinical manifestations/outcomes (table 1) . available studies used mainly a gene-candidate approach to select those genes involved in disease pathogenesis and phenotypes based on a priori knowledge. thus, genes implicated in key physiological processes such as the mechanisms related to the entry of the virus into the cell and the antiviral immune/inflammatory response were found (table 1) . 2 international journal of genomics of note, research was predominantly performed in asian populations, including chinese, vietnamese, and taiwanese populations. moreover, genetic polymorphisms were mainly snps in coding and promoter regions, although insertion/deletion and tandem repeats were also described. for instance, chinese individuals homozygous for the tandem repeat domain in exon 4 of the clec4m gene were less susceptible to sars infection [17] . also, the insertion/deletion (i/d) polymorphism in the ace1 gene was associated with the progression of pneumonia in sars patients [18] . both genes are involved in virus entry. the rest of the genetic polymorphisms were located in cytokine and chemokine genes implicated in the immune response and inflammatory processes. for example, the ifn-γ +874a allele was associated with the susceptibility to develop sars in two independent populations [19, 20] . also, the minor alleles of the -88g>t and -123c>a mxa promoter snps were significantly associated with a lower risk of sars-cov infection in chinese [21] . nevertheless, the g/t snp at position −88 in the promoter region of this gene was associated with sars-cov disease progression [22] . genetic variations in the inflammatory tnf-α gene were also screened. thus, whereas the ct genotype at the tnf-α -204 locus of this gene was found to be associated with a pro-tective effect on sars, a higher a allele frequency of the tnf-α -308g/a polymorphism was found in the sars population when compared with healthy controls [23] . moreover, the prevalence of the cd14-159cc polymorphism was significantly higher in patients with severe sars than in those with mild stages of the disease or controls [24] . the distribution of hla alleles has been widely used as a strategy in the search for the etiology of infectious diseases. in this sense, three genetic polymorphisms in the hla-b gene were related to different sars-cov outcomes, including infection predisposition, a protective phenotype [25] , and severe forms of the disease [26] . in addition, the cw * 0801 and cw * 1502 variants in the hla-cw gene were associated with sars risk [27] and sars resistance [28] in taiwanese, respectively. personalized medicine involves a balanced knowledge of genotype, phenotype, and clinical features of the patient [13] , where susceptibility to exacerbated infection is often dependent. similar to other viral diseases, the first step in sars-cov infection includes the attachment of the virus to the host cell receptors [29] . in the present review, genetic international journal of genomics variants in ace1, clec4m, and cd209 genes were associated with sars-cov-related disease pathogenesis. particularly, the angiotensin-converting enzyme 2 (ace2, a homologue of ace1) has been identified as a functional receptor for sars-cov [30] . also, the c-type lectin domain family 4 member m (clec4m, also known as l-sign) was recognized as a binding receptor for this virus [31] . cd209 (also known as dc-sign) shares amino acid identity with clec4m, which was involved in facilitating sars-cov viral transmission to susceptible cells [32] . these findings demonstrate the important functions of these molecules in the virus entry. nevertheless, two additional studies failed to demonstrate a relationship between ace2 polymorphisms and sars-cov outcome in asians so far [33, 34] , emphasizing the need for future research in other populations. virus survival and virus-induced disease outcomes (including sars-cov) are commonly linked to the modulation of the immune response in the host [35] . as expected, in this review, genetic polymorphisms in genes involved in immunocompetence/inflammatory pathways were found (icam-3, ifn-γ, rantes, oas-1, mxa, il-12 rb1, fcgriia, mlb, ccl2, ahsg, tnf-α, cd14). most of them are cytokines or chemokines that are known to be important mediators of early defense against infections, activating protective mechanisms in infected cells [36] . these processes occur during the immune response driven by the sars-cov infection and comprise t cell stimulation (icam-3, il-12 rb1), macrophage activation/deactivation (ifn-γ, ahsg), cell trafficking (rantes, ccl2), rna degradation and the inhibition of viral replication (oas-1, mxa), link between humoral and cell-mediated immune reaction (fcgriia), virus binding (mlb), and inflammation (tnf-α, cd14) (http://www.genecards.org). genetic variations in hla class i or ii genes have been often associated with susceptibility/resistance to a wide range of infections, including sars-cov [37] . accordingly, the genes of the hla complex were associated with sars-cov outcomes in this study (hla-b, hla-drb1, hla-drb4, hla-drb3, hla-cw). hla play a critical role in the presentation of antigenic peptides to t cells, orchestrating an immune response aimed at removing nonself material via neutralization of antibodies, cytokines, and activated cytotoxic t cells [38] . robust evidence support that genetic variation in human populations contributes to the onset and development of several chronic diseases, including those of an infectious nature [39] . the results of this systematic review show that research exploring the genetic contribution to sars-cov infection was predominantly performed in asian populations, which may be related to its epidemiological origin in china in 2002 [40] . however, allele and genotype frequencies of genetic variants may vary depending on region; therefore, the results should not be extrapolated to other populations without prior exploration. therefore, further investigation is required to determine the pattern of distribution of these and other candidate polymorphisms in other groups as well as confirm associations with sars-cov outcomes. this knowledge is particularly important in populations with heterogenic heritages exposed to absolutely different environmental factors. an example could be latin american countries, including mexico, which have an admixture genome with amerindian, european, and african ancestries; a high prevalence of obesity; and associated comorbidities as well as the adoption of an unhealthy lifestyle (high-fat/sugar diet and physical inactivity) that could exacerbate the outcome of viral infections [41] . or even some snps that have not been associated with sars-cov disease outcomes in the asian population could become a significant association in other populations because of a strong environmental pressure. thus, we cannot rule out the possibility that other snps in other genes could play a role in susceptibility to sars-cov infection in these populations. in this context, personalized medicine is an integrative therapeutic approach that considers conventional factors (age, gender, clinical phenotype), as well as emerging genetics and interactions with environmental factors to individualize prevention, diagnosis, treatments, and prognosis [42] . applying the principles of personalized medicine in the current care schemes for the control of infectious diseases (i.e., sars-cov) could allow the identification of genetically susceptible groups, disease risk prediction, and personalized therapies to reduce infection-related complications, high mortality rates, and the optimization of economic resources for health care and individualized management of inflammation ( figure 2) . currently, the high incidence of sars-cov-2/covid-19 around the world, the speed of its spread, and the absence of a specific drug for its pharmacological management emphasize the need to identify risk factors associated with the dynamics of infection and disease progression in order to mitigate its negative impact on the society. a better understanding of the relationship between the genetic make-up and sars-cov-2/covid-19 will provide new insights into the disease pathogenesis by explaining particular phenotypes and clinical responses. moreover, this knowledge will also aid in identifying biomarkers as potential therapeutic targets for evaluating the efficacy of genome-based interventions and other personalized treatments within the new era of precision medicine. hla-dr dr * 0301 dr * 0301 lower susceptibility to sars-cov infection incidence 0.11 taiwanese [28] 5 international journal of genomics in addition to the polymorphisms analyzed in this review, new genetic variants affecting sars-cov and sars-cov-2/covid-19 susceptibility need to be further explored as well as the interplay of other emerging parameters of individualization such as epigenetic signatures, metabolomic fingerprints, metagenomic data, and lifestyle factors through an integrative holistic approach [43] . potential gen-e×drug or gene×inflammation interactions also deserve further investigation. in this regard, drugs targeting the inflammatory cytokines il-1 and il-6 have been proposed to reduce extreme immune reaction to the virus and extensive tissue damage [44, 45] , where genetics may play a pivotal role. additionally, genotyping of virus isolates to detect specific multiple mutations is of great importance for the understanding of the evolution and transmission of sars-cov infections as well as for vaccine development and disease control [46] . although caution must be taken, the results of this systematic review suggest that multiple genetic polymorphisms are associated with sars-cov infection outcomes (susceptibility and severity, hospitalization, icu stays, and medications) by affecting virus pathogenesis and host immune response, which could have important applications for the study and understanding of genetics in sars-cov-2/covid-19 and for personalized translational clinical practice depending on the population studied and associated environments. all data are available in the article. the authors declare that there is no conflict of interest regarding the publication of this paper. orl and jam performed the search of articles and wrote the draft of the manuscript. ld, arm, dmu, and jav contributed to the analysis and critical interpretation of the data. all authors read and approved the final manuscript. international journal of genomics sars and mers: recent insights into emerging coronaviruses a novel coronavirus associated with severe acute respiratory syndrome novel coronavirus disease (covid-19): a pandemic (epidemiology, pathogenesis and potential therapeutics) coronavirus cis-acting rna elements pathological 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inflammation mediated by il-1 anti-il6r role in treatment of covid-19-related ards genotyping coronavirus sars-cov-2: methods and implications cd209 (dc-sign) -336a>g promoter polymorphism and severe acute respiratory syndrome in hong kong chinese association of a single nucleotide polymorphism in the cd209 (dc-sign) promoter with sars severity functional role of icam-3 polymorphism in genetic susceptibility to sars infection association of icam3 genetic variant with severe acute respiratory syndrome the association of rantes polymorphism with severe acute respiratory syndrome in hong kong and beijing chinese association of sars susceptibility with single nucleic acid polymorphisms of oas1 and mxa genes: a case-control study a case-control study on the mxa polymorphisms and susceptibility to severe acute respiratory syndromes il-12 rb1 genetic variants contribute to human susceptibility to severe acute respiratory syndrome infection among chinese influence of fcgam-mariia and mbl polymorphisms on severe acute respiratory syndrome association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection functional polymorphisms of the ccl2 and mbl genes cumulatively increase susceptibility to severe acute respiratory syndrome coronavirus infection genetic variation of the human α-2-heremans-schmid glycoprotein (ahsg) gene associated with the risk of sars-cov infection immunogenetics in sars: a case-control study association of human leukocyte antigen class ii alleles with severe acute respiratory syndrome in the vietnamese population the support from ciberobn, imdea-food institute, and hm hospitals is gratefully acknowledged. supplementary key: cord-284944-hcgfe9wv authors: silvin, aymeric; chapuis, nicolas; dunsmore, garett; goubet, anne-gaëlle; dubuisson, agathe; derosa, lisa; almire, carole; hénon, clémence; kosmider, olivier; droin, nathalie; rameau, philippe; catelain, cyril; alfaro, alexia; dussiau, charles; friedrich, chloé; sourdeau, elise; marin, nathalie; szwebel, tali-anne; cantin, delphine; mouthon, luc; borderie, didier; deloger, marc; bredel, delphine; mouraud, severine; drubay, damien; andrieu, muriel; lhonneur, anne-sophie; saada, véronique; stoclin, annabelle; willekens, christophe; pommeret, fanny; griscelli, frank; ng, lai guan; zhang, zheng; bost, pierre; amit, ido; barlesi, fabrice; marabelle, aurélien; pène, frédéric; gachot, bertrand; andré, fabrice; zitvogel, laurence; ginhoux, florent; fontenay, michaela; solary, eric title: elevated calprotectin and abnormal myeloid cell subsets discriminate severe from mild covid-19 date: 2020-08-05 journal: cell doi: 10.1016/j.cell.2020.08.002 sha: doc_id: 284944 cord_uid: hcgfe9wv summary blood myeloid cells are known to be dysregulated in the coronavirus disease 2019 (covid-19) caused by sars-cov-2. it is unknown whether the innate myeloid response differs with disease severity, and whether markers of innate immunity discriminate high risk patients. thus, we performed high dimensional flow cytometry and single cell rna sequencing of covid-19 patient peripheral blood cells and detected the disappearance of non-classical cd14lowcd16high monocytes, the accumulation of hla-drlow classical monocytes, and the release of massive amounts of calprotectin (s100a8/s100a9) in severe cases. immature cd10lowcd101-cxcr4+/neutrophils with an immuno-suppressive profile accumulated as well in blood and lungs, suggesting emergency myelopoiesis. we finally showed that calprotectin plasma level and a routine flow cytometry assay detecting decreased frequencies of non-classical monocytes could discriminate patients who develop a severe covid-19 form, suggesting a predictive value that deserves prospective evaluation. coronavirus disease 2019 is caused by severe acute respiratory syndromecoronavirus 2 (sars-cov-2), which infects the lungs leading to fever, cough and dyspnea (guan and zhong, 2020) . most patients presenting with mild disease develop an efficient immune response (thevarajan et al., 2020) , but some go on to develop acute respiratory distress syndrome leading to admission in intensive care unit (icu), often culminating in multi-organ dysfunction and death . in addition to cell autonomous effects of the viral infection, a dysregulated immune response participates in the sudden deterioration of covid-19 patients, ultimately overwhelming infected and non-infected tissues (vabret et al., 2020) . this overt inflammatory response centers around a cytokine storm (chen et al., 2020a) with elevated blood concentrations of interleukin-6 (il-6). accordingly, therapeutic agents targeting the il-6/il-6r-gp130 axis can alleviate the inflammatory response (michot et al., 2020) and ameliorate immune dysregulation (giamarellos-bourboulis et al., 2020) , emphasizing the clinical significance of this cytokine. a marked lymphocytopenia is also associated with covid-19 severity (chen et al., 2020a) ; however, the primary source both of the cytokine storm and the mechanisms behind the lymphocytopenia remains elusive . a growing body of evidence points to dysregulation of innate immune cells of the granulomonocytic lineage during lung viral infections. a variety of human viruses infects monocytes and macrophages to spread through the tissues (al-qahtani et al., 2017; desforges et al., 2007; nottet et al., 1996; smith et al., 2004; yilla et al., 2005) . sars-cov-2 mrna is detectable in lung monocytes/macrophages of severe covid-19 patients (bost et al., 2020) , though its ability to enter these cells in the peripheral blood and activate them directly remains unclear. also, tissue damage induced by sars-cov-2 infection may lead to the release of pathogen-and damage-associated molecular patterns that, in turn induce the activation and recruitment of inflammatory cytokine-and chemokine-producing innate immune cells in an amplifying loop (liao et al., 2020) . it remains unclear to what extent immune patterns associated with covid-19 pathophysiology are causative and exacerbating the disease and/or could be used for accurate patient stratification. here, using high dimensional single cell approaches including single cell rna sequencing, mass cytometry and 25-parameter spectral flow cytometry, we show that patients doomed to develop a severe disease exhibit a massive release of s100a8/s100a9 calprotectin accompanied by changes in monocyte and neutrophil subsets. we further discover that this pathological immune system reorganization is initiated by the onset of an emergency myelopoiesis that release immature myeloid cells with an immunosuppressive phenotype into peripheral blood and lungs. together, our study integrates frequencies of non-classical monocytes and immature neutrophils with calprotectin plasma level as robust biomarkers of covid-19 severity, suggesting potential therapeutic strategies targeting calprotectin to alleviate severe covid-19. this non-interventional study enrolled 158 patients (table s1) , including 86 and 72 subjects referred to the hospital with various flu-like symptoms who were diagnosed or not with covid-19, based on positive and negative rt-pcr on pharyngeal swabs respectively. patients were stratified according to disease severity: mild disease (n=27) was defined as having no or limited clinical symptoms and not requiring ct-scan or hospitalization; moderate disease (n=16) was defined as being symptomatic, with dyspnea and radiological findings of pneumonia on thoracic ct scan, requiring hospitalization but with a maximum of 9 l/min of oxygen; and severe disease (n=43) was defined as respiratory distress requiring admission into the intensive care unit (icu). mild and moderate cases were mixed in the discovery part of the study and considered separately to explore the ability of a routine flow assay to discriminate patients that require hospitalization. in order to explore changes induced by sars-cov-2 infection in circulating immune cell phenotype, we first collected peripheral blood samples from a discovery cohort of 13 patients positive for sars-cov-2 (hereafter "covid-19 patients") by rt-pcr and 12 patients suffering from flu-like symptoms but negative for sars-cov-2. the former group included 5 patients with mild disease and 8 patients with severe covid-19 (table s2) . after red blood cell lysis, we labelled peripheral blood cells with a panel of 25 antibodies recognizing immune cell surface markers (key resource table) and analyzed them by spectral flow cytometry ( figure 1a , s1a and s1b). by pooling the data from the 25 control and covid-19 patients and subjecting them to dimensionality reduction using the non-supervised umap algorithm , we identified populations of cd4 + t cells, cd8 + t cells, cd19 + b cells, cd14 high monocytes and cd15 + cd66b + neutrophils (figure 1b and 1c) . we also identified hla-dr high cd11b + and cd16 high monocytes as well as neutrophils expressing cd11b, cd15, cd16 and cd64 (figure 1b and 1c) . analysis and visualization, using umap dimensionality reduction to the cell surface marker expression datasets from control, and mild and severe covid-19 groups, suggested differences in the repartition of cell populations ( figure 1d ). severe patients exhibited an expansion in the proportion of circulating neutrophils within the peripheral blood cell population (figure 1e) , which was associated with an increase in their absolute number (table s2) , as already reported . focusing on neutrophil subsets, we noticed a slight increase in the fraction of cd10 low cd101 + neutrophils in mild covid-19 patients (figure 1f) , whereas the fraction of cd10 low cd101neutrophils was remarkably amplified in severe patients, suggesting an accumulation of immature subsets of neutrophils (ng et al., 2019) in the peripheral blood of these patients ( figure 1g and s1c-e). in severe patients, the absolute number of circulating monocytes (table s2 ) and the proportion of total monocytes among peripheral blood leucocytes (figure 1h and s1f) were similar to controls, but we noticed changes in monocyte subset repartition. the fraction of cd14 high cd16 high intermediate monocytes was significantly greater in mild covid-19 patients (16.95+/-6.75%) than in control (5.84+/-1.02%) or severe (6.77+/-1.10%) groups, while the non-classical cd14 low cd16 high monocyte fraction was lower in severe covid-19 patients (1.31+/-0.35%) than in mild (5.46+/-1.57%) or control groups (6.68+/-1.14%) (figure 1i and 1j). within the cd14 high cd16 low classical monocyte subset (figure s1g), we detected higher frequencies of cd11b high monocytes with increased disease severity (figure s1h) , while the intensity of hla-dr expression was lower across both cd11b + and cd11bmonocyte populations of severe covid-19 patients ( figure 1k and 1l) . changes in myeloid cell repartition observed in severe patients were associated with lower frequencies of b cells compared to controls (p<0.001), and of cd4 + (p<0.001) and cd8 + t cells (p<0.01) relative to both controls and mild patients, while cd56 + nk cell frequencies remained comparable across all groups ( figure 1m ). altogether, these data suggested sars-cov-2-induced changes in the relative abundance of monocyte and neutrophil subsets within the peripheral blood cell population, with loss of non-classical cd14 low cd16 high monocytes, reduced expression of hla-dr on classical monocytes and drop in cd101 and cd10 expression on neutrophils, characterizing severe cases. as a second step in our discovery process, we collected peripheral blood samples from three control patients with flu-like symptoms tested negative for sars-cov-2, and three sars2-cov-2 positive patients, one outpatient with mild disease and two patients with severe disease admitted to icu (figure 2a and 2b ; table s3 ). using the 10x chromium dropletbased platform, these samples were subjected to single cell rna sequencing (scrnaseq) immediately after collection and red blood cell lysis, without additional sorting or freezing, in an attempt to preserve fragile cell populations, mainly neutrophils. unsupervised clustering based on gene expression identified b and t cells as well as neutrophils, monocytes, erythroid cells and platelets ( figure 2c and figure s2a and s2b). samples analyzed by scrnaseq were simultaneously analyzed by spectral flow cytometry for comparison ( figure 2d ). umap analysis of spectral flow cytometry data suggested lower proportions of cd4 + and cd8 + t cells while the neutrophil fraction was greater in severe patients compared to controls and to the unique mild patient (figure 2e and s2c) . the three sars2-cov-2-infected patients were sampled again 10 days later to monitor progression of the immune response in relation to clinical status (figure 2a to 2e) . umap visualization of monocytes analyzed by scrnaseq identified three clusters (figure 3a) , that may correspond to well-defined monocyte subsets (guillams et al., 2018) . cells of cluster 1 expressed cd14, itgam (encoding cd11b) and klf4 while poorly expressing fcgr3a (encoding cd16), suggesting classical monocytes. cells of cluster 3, which expressed high levels of fcgr3a and low levels of cd14, may correspond to non-classical monocytes, and cluster 2, in which cells expressed both cd14 and fcgr3a, evoked intermediate monocytes ( figure 3a ). differential expressed genes (degs) and pathway analyses delineated a type i interferon signature in the mild covid-19 patient monocytes (figure 3b, s3a and s3b; table s4 ). this signature was less pronounced in the two severe covid-19 samples, contrasting with the elevated expression of genes involved in the production of reactive oxygen species (ros) and nitric oxygen species (nos) (figure s3a and s3b). a non-supervised umap analysis of the data collected by spectral flow cytometry of the same samples detected variations in monocyte subset repartition among patients: compared to controls and the mild patient, severe patient #1 showed a lower fraction of cd14 low cd16 high non-classical monocytes at day 0 while the other severe patient showed a high level of this monocyte fraction ( figure 3c) . additionally, the two severe patients showed markedly higher levels of classical cd14 high cd16 low monocytes, expressing more cd141 (thbd) at their surface ( figure 3d ), in accordance with the scrnaseq analysis (table s4 ). in the mild patient, one of the most highly expressed genes in classical monocytes was the interferon stimulated gene (sevelsted et al., 2015) siglec-1, consistent with the high level of expression of cd169, the corresponding protein, at the surface of classical monocytes at day 0 ( figure 3e ). ten days later, siglec-1 gene expression was down-regulated and cd169 expression was undetectable at the surface of hla-dr high classical monocytes ( figure 3b and 3e). the two severe patients exhibited low expression of hla-dr protein on their monocyte surface at day 0, without significant change at day 10 ( figure 3e ). validating these discovery experiments, we performed mass cytometry analysis of an independent cohort of 12 patients (four in each group; control, mild and severe) ( table s5) , which showed a lower fraction of cd14 low cd16 high non-classical monocytes in severe compared to mild patients ( figure 3f and 3g ). in accordance with pathway analysis of scrnaseq data highlighting nf-ĸb activation as a prominent feature in monocytes of severe patients ( figure 3b and s3b), we observed significantly higher levels of the phosphorylated transcription factor rela/p65 (p-p65), a critical effector of the canonical nf-ĸb pathway, in hla-dr low cd14 high classical monocytes from severe patients compared to controls ( figure 3h and 3i). we also measured p-p65 expression in circulating cd34 + cells, identifying increased expression in severe disease ( figure s3c ). umap analysis of neutrophils identified two clusters ( figure 4a ). we observed an increase of cluster 2 cells in severe covid-19 patients ( figure 4b ). cluster 1 expressed il1r2 gene whereas cluster 2 expressed also high levels of s100a8 and s100a9, cxcr4, sell and spi1 (figure 4c and s4a) . degs and pathway analyses in mild patient neutrophils informed of a type i interferon response at day 0 that was lost by day 10 (figure 4d, s4b and s4c ). this signature was absent in controls and also in the two severe patient samples collected at later time-points ( figure 4d ), demonstrating high expression of genes involved in the production of ros, the inducible nos pathway, il-1 signaling and nf-κb activation pathways ( figure s4b and s4c). analysis of the data collected by spectral flow cytometry of the same samples distinguished cd10 + cd101 + mature neutrophils from cd10 low cd101immature neutrophils. at day 0, the two severe patients had more circulating cd10 low cd101immature neutrophils compared to controls or the mild patient ( figure 4e ). severe patient #1 neutrophils had increased the expression of cd101 on their surface at day 10 while severe patient #2 neutrophils retained their immature phenotype at day 10. focusing on the expression of a pre-neutrophil hallmark cxcr4 at the surface of cd10 low cd101immature neutrophils (ng et al., 2019) , we observed an increase in the proportion of neutrophils with a cd10 low cd101 -cxcr4 + phenotype, which presumably are pre-neutrophils ( figure 4f ). mass cytometry analysis of an independent cohort of 12 patients (four controls, four mild and four severe covid patients, table s5 ) again suggested a higher fraction of cd10 low cd101immature neutrophils in severe patients compared to control patients ( figure 4g and 4h) . altogether, results of these exploratory scrnaseq experiments identified a transient type i interferon response in cells of a patient with a mild disease and the presence of phenotypically immature subsets of monocytes and neutrophils in two patients with a severe disease, which were further identified by mass cytometry. calprotectin plasma levels distinguish mild from severe covid-19 patients s100a8 and s100a9 alarmins, representing ~45% of the cytoplasmic proteins in neutrophils, are released under inflammatory conditions and form a stable heterodimer known as "calprotectin" (wang s et al., 2018) . in accordance with preliminary results generated by scrnaseq ( figure s4a) , rt-qpcr analysis detected higher expression of s100a8 and s100a9 genes in peripheral blood nucleated cells of patients with severe covid-19 (n=8) compared to controls (n=8) and patients with a mild disease (n=16) ( figure s5a and table s5 ). this led us to measure the plasma level of calprotectin, together with type i interferon (ifnα) and 40 other cytokines and chemokines in samples from a cohort of 84 patients ( table s6) . as observed in figure 5a , patients with mild disease showed significantly less cxcl8 (figure 5c and s5b) and significantly more type i ifnα (figure 5a and 5c) compared to controls. severe j o u r n a l p r e -p r o o f patients exhibited dramatically higher calprotectin levels compared to mild covid-19 or controls, without further increase in ifnα plasma level above mild disease levels ( figure 5b , 5c and s5c). calprotectin was the most significantly increased circulating biomarker in severe patients, accompanied by a rise in 23 other tested chemokines and cytokines, including cxcl-8, cxcl-12 and il-6 ( figure 5b, 5c and s5b ). age and comorbidities (including obesity, diabetes mellitus, cardiovascular and respiratory diseases and cancer) are predictors of severe covid-19 disease (richardson et al., 2020) . we found that plasma calprotectin levels were significantly higher in control patients with comorbidities, as well as in mild or severe covid-19 patients with comorbidities ( figure 5d) . nevertheless, the increase in calprotectin in severe covid-19 far exceeds that correlations associated with comorbidities. none of the other measured circulating proteins were significantly higher in patients with comorbidities ( figure 5e ). bacterial infections can occur in severe covid-19 (chen et al., 2020c; llitjos et al., 2020) and were present in some of our severe patients but did not significantly modify the profile of released proteins ( figure s5b and s5c), including calprotectin ( figure 5f ). no correlation between calprotectin and age was observed in each group of patients ( figure s5d ). calprotectin concentration correlated with neutrophil counts (figure 5g ), fibrinogen plasma levels ( figure 5h ) and d dimers (figure 5i) , the latter being fibrin degradation products reflecting a hypercoagulability state. modeling calprotectin plasma level using multivariable linear regression to take into account potential confounding factors (age, sex, comorbidities) and the correlation with neutrophil count, fibrinogen et d-dimers, these associations were still statistically significant (neutrophils p-value=1.154e-04; fibrinogen p-value=5.688e-05; d-dimers p-value=2.099e-03). we also uncovered a weak correlation between il-6 plasma concentration and levels of calprotectin ( figure s5e ), blood neutrophil count ( figure 5j ), fibrinogen ( figure 5k ) and d dimers (figure 5l ), which disappeared after adjusted multilinear regression. finally, a logistic regression including age, sex and comorbidities together with biological parameters identified plasma levels of calprotectin, cx3cl1, cxcl11 and cxcl13 as the parameters that best discriminate controls / mild covid-19 patients from severe patients. these results indicate that high plasma levels of calprotectin are seen in severe covid-19 patient, not in those with a mild disease. importantly, this increase is independent from confounding factors for prognosis such as advanced age, co-morbidities or concurrent bacterial infection which have only minor effects on plasma calprotectin levels. hypothesis from the scrnaseq-based identification of cd37, cd63 (lamp3), cd169 (siglec-1) and cd184 (cxcr4) biomarkers of blood cell subsets whose relative proportions differ in mild and severe covid-19 patients, prompted us to add additional antibodies targeting these proteins to the spectral flow cytometry panel. we applied this new panel to samples from an independent validation cohort of 90 patients. this cohort included 48 control patients and 42 covid-19 positive, of which 16 had mild disease and 26 had severe disease ( figure 6a and table s6 ). non-supervised analysis and umap visualization identified the main cell populations in the three categories of patients combined ( figure s6a and s6b) . analyzing patients individually confirmed the significant decrease in b cell, cd4 + t cell and cd8 + t cell fractions in severe patients compared to control and mild groups ( figure 6b) , which may be a consequence of the increased neutrophil fraction ( figure 6c ) and absolute number (table s6) . within neutrophils, we observed more specifically a shift in cd10 low cd101neutrophils ( figure 6d and 6e ) and the subset of cd10 low cd101neutrophils that express cxcr4 (cd10 low cd101 -cxcr4 + cells) ( figure 6f ) that we previously observed in severe patients. finally, the fraction of cd10 low cd16 low neutrophils was also higher in severe patients ( figure s6c ), further suggesting for the accumulation of immature neutrophils in the blood of severe covid patients. scrna-seq analyses of monocyte subsets had indicated differential changes in the distribution of non-classical cd14 low cd16 high monocyte fractions between the two patients with severe disease (see figure 3c ). since samples were collected from patients at various time points after admission in icu, we asked if the duration of icu stay affects the monocyte subset distribution. in the 26 severe patients of this cohort (table s6) , we observed a significant correlation between the time spent in icu and the fraction of non-classical monocyte subset, irrespective of the presence or absence of concurrent bacterial infection ( figure 6g ). mean time spent in icu was 5.46 days for patients with less than 5% nonclassical monocytes compared to 8.83 days for those with 5% and more non-classical monocytes ( figure 6h and 6i) . then, we examined other monocyte subsets: in the majority of mild patients, we observed a fraction of classical monocytes that express cd169, which was decreasing in severe patients ( figure 6j , 6k and s6d). cd169 expression correlated with plasma levels of ifnα ( figure s6e ). independent of the time they spent in icu, severe patients also showed a larger fraction of classical monocytes expressing high levels of cd141 compared to controls ( figure 6j and 6l) and of monocytes expressing low levels of hla-dr compared to controls and mild patients ( figure s6f) . finally, the time spent in icu did not significantly affect the repartition of lymphocyte populations or neutrophil subsets ( figure s6f and s6g). thus, severe covid-19 patients exhibited a transient decrease in non-classical monocyte frequencies, a stable decrease in hla-dr low cd141 + classical monocytes and a major increase in cd10 low cd101 -cxcr4 +/immature neutrophils. we next investigated whether changes in circulating myeloid cell phenotypes could be used to discriminate patients who develop severe covid-19. within our previous cohort, we separated mild (n=12) from moderate (n=6) and severe (n=27) patients using clinical criteria. patients classified as "moderate" demonstrated intermediate changes between those of mild outpatients and severe patients in icu ( figure s7a ). the fraction of cd10 low cd101neutrophils in moderate patients was intermediate but not significantly different to any group ( figure 7a) . however, the amount of calprotectin measured in moderate covid-19 patients was significantly higher than mild outpatients but still significantly lower than severe covid-19 patients ( figure 7b ). in comparison, ifnα levels were not significantly different between moderate and mild or severe patients ( figure s7b) . the difference in nonclassical monocyte fraction was significant between mild and moderate patients, dropping down to levels comparable to severe cases ( figure 7c ). thus, we hypothesized that the decreased non-classical monocyte fraction could be used as a fast and simple diagnostic test to distinguish moderate from mild covid-19 cases, especially in cases were clinical symptoms may be substantially overlapping. this would facilitate rapid and accurate identification of currently classified as "mild" patients at the cusp of potentially progressing to more severe disease. we therefore employed a low dimensional flow cytometry approach that measures the fraction of classical (cd14 high cd16 low ), intermediate (cd14 high cd16 high ) and non-classical (cd14 low cd16 high ) monocyte subsets among total peripheral blood monocytes, and applied it initially to a learning cohort of 98 patients, consisting of 16 mild, 10 moderate and 16 severe covid-19 patients, along with 56 controls ( table s7 ). all hospitalized patients were sampled within ten days of admission to limit the potential impact of time in icu (see figure 6g ); the mean time spent in icu was 5.5 days at the point of sampling. the cohort also included 56 controls. patients with mild disease showed a fraction of non-classical monocytes similar to that observed in controls. in contrast, moderate patients showed lower levels of non-classical monocytes, as observed in severe patients ( figure 7d) . to measure the global performance of this test, we used a receiver operating characteristic (roc) curve (hajian-tilaki, 2013) . the point of the roc corresponding to the best sensitivity/specificity compromise indicated that a non-classical monocyte fraction below 4% separated patients with moderate or severe covid-19 from those with mild or no disease with 76.9% sensitivity (95% bootstrap confidence interval (bci) [61.5%; 92.3%]) and 89% specificity (95% bci [80.6%; 95.8%]) ( figure s7c ). we then applied these analyses to blood samples from an independent validation cohort of 24 hospitalized patients from a different clinical center (10 controls, 3 mild, 4 moderate and further confirming these observations, serial sampling of two severe patients who responded to anti-il-6r antibodies documented that their clinical recovery was associated with the reappearance of non-classical monocytes in the blood ( figure s7d) . alongside, one patient who was referred initially with limited symptoms (atypical thoracic pain) and was sars-cov-2 pcr negative unexpectedly exhibited a low fraction of non-classical monocytes (3.4%), accompanied by 10% hla-dr low classical monocytes. the following day, pulmonary symptoms appeared, the patient was hospitalized requiring oxygen therapy, and a lung ctscan revealed characteristic covid-associated injury. such cases suggest that the loss of nonclassical monocyte fraction could be a strong indicator of existing or impending severe covid-19. additional informative parameters could be added to this flow assay to increase its specificity to identify a transition to severe covid-19, including a decreased expression of hla-dr at the surface of classical monocytes ( figure s7e ) that is associated with a decrease in non-classical monocyte fraction below 4% (figure s7f) , and an increase in the fraction of cd16 low neutrophils ( figure s7g ). comparison of roc curves indicated that calprotectin plasma level and monocyte or neutrophil subset analyses distinguished mild covid-19 in outpatients from moderate or severe disease in hospitalized patients, while ifnα2a plasma level did not ( figure s7h ). together with calprotectin plasma level, flow identification of a decrease in non-classical monocyte fraction below 4% of total monocytes could provide improved resolution to clinical observations when categorizing patients at the borders of mild and moderate/severe covid-19. this would potentially identify those individuals at greatest risk of rapid decline and highlight the need for pro-active management/intervention and intensive monitoring. this assay could be reinforced by analysis of hla-dr low classical monocyte and cd16 low neutrophil fractions. the lungs are a major organ affected in severe covid-19 patients. to better understand how the distinctive cell signatures found in the blood of severe covid-19 patients, particularly the presence of immature neutrophils and hla-dr low monocytes, affected immune cell compartments in the lungs, we integrated our dataset using the seurat v3 pipeline (stuart et al., 2019) with the published scrnaseq dataset of cells from 12 bronchoalveolar lavage fluids (balf) of control (n=3), mild (n=3) and severe (n=6) covid-19 patients (liao et al., 2020; gse145926) . this analysis provided an unbiased global map of immune cells in the blood and balf of control, mild and severe covid-19 patients. using dimensional reduction, we identified 5 regions based on degs across pooled data from all samples (figure 7f and s7i), including t cells (characterized by the expression of genes including nkg7, cd8a, cst7, gzmb and gzma), b cells (iglv3-19, ighv4-34, ighg1, igha1 and jchain), neutrophils (g0s2, rsad2, il1r2 and il1rn), alveolar macrophages (apoe, msr1, marco and fbp1) and monocytes/macrophages (fn1, cxcl10, cd68 and nupr1). validating this approach, the alveolar macrophage region was mainly present in balf of control patients but was dramatically decreased in mild and severe covid-19 patients and only one cell from our blood scrnaseq matched in this region (figure 7f and 7g) . we also observed changes in the monocyte/macrophage region of the balf from mild or severe patients versus controls and a dramatic neutrophil accumulation in severe disease ( figure 7g ). monocytes/macrophages were increased in balf of mild compared to control and severe groups (figure 7h and 7i) and these cells were characterized by the expression of the isgs (siglec-1, ifi44 and ifitm3) ( figure 7j ) with pathway analyses indicating the upregulation of viral replication and interferon type i signaling pathways. in contrast, nos biosynthetic process and monocyte chemotaxis were upregulated in balf monocytes/macrophages of severe patients ( figure s7j ) that, similar to blood monocytes, expressed lower levels of hla-dra and hla-drb1 and higher levels of nfkbia mrna compared to controls or mild covid-19 patients ( figure 7j) . finally, neutrophils were present at high frequencies in balf from severe covid-19 patients but not in balf from controls or mild patients ( figure 7k and 7l) . umap integration of severe patient samples indicated that balf neutrophils, similar to blood neutrophils (figure 7l) , were characterized by high expression of s100a8, s100a9 as well as cxcr4, indicating an immature state, (figure 7m, 7n and s7k) . altogether, integration of blood and balf myeloid cells identified in severe covid-19 patients the loss of hla-dra and hla-drb1 and high nfkbia expression in monocytes/macrophages (not including alveolar macrophages), together with an accumulation of neutrophils expressing high levels of s100a8/a9 and cxcr4. this study presents evidence that patients who develop a severe covid-19 exhibit high levels of calprotectin and inflammatory cytokines and chemokines correlating with an emergency myelopoiesis generating ros-and nos-expressing immunosuppressive myeloid cells (hla-dr low monocytes and immature subsets of neutrophils). the first line of defense in viral-infected patients typically involves a protective innate response incorporating the transient and strong production of type i ifns. through inducing expression of isgs, type i ifns inhibits virus replication and promotes an effective innate and adaptive immune response (thevarajan et al., 2020; totura and baric, 2012) . this antiviral response may be overflowed in covid-19 patients who suddenly evolve into clinically threatening disease (hadjadj et al., 2020) . severe covid-19 frequently develops in the context of advanced age and comorbidities that provide a degree of underlying systemic chronic inflammation (furman et al., 2019) . such inflammation could disrupt the timing of type i ifn response relative to the kinetics of virus replication (teran-cabanillas and hernandez, 2017), which was shown to be critical in mouse models of coronavirus infection (channappanavar et al., 2019) . an imbalance between the type i ifn and inflammatory responses could also be favored by the highly efficient replication of sars-cov-2 in human tissues (chu et al., 2020) , and by the ifn-neutralizing effects of structural and non-structural viral components shared between sars-cov-2 and other virulent human coronaviruses (chen et al., 2014; yang et al., 2015) . severe covid-19 patients exhibit abnormal partition of circulating monocytes and of neutrophils expressing s100a8 (calgranulin a / myeloid-related protein 8) and s100a9 (calgranulin b / myeloid-related protein 14) alarmin genes. importantly, an accumulation of neutrophils expressing high levels of s100a8/a9 genes was also observed in the balf of these patients. the release of massive amounts of calprotectin, the heterodimer formed by s100a8 and s100a9 proteins, is a striking event associated with severe covid-19. this heterodimer promotes cell migration and boosts nadph (nicotinamide adenine dinucleotide phosphate) oxidase activity. calprotectin is a tlr4 and rage (receptor for advanced glycation end products) ligand that, upstream of tnfα (vogl et al., 2018) and cxcl8 (simard et al., 2014) synthesis and secretion, promotes nf-ĸb activation (riva et al., 2012) and the secretion of multiple inflammatory proteins as il-6 (wang et al., 2018) . thus, we propose that calprotectin may account for, and possibly trigger the cytokine release syndrome that characterizes severe covid-19. its production may be amplified by tissue damage, generating a harmful hyper-inflammation loop (kuipers et al., 2013) that precludes these peptides from exerting more protective functions (austermann et al., 2014; freise et al., 2019; ulas et al., 2017; vogl et al., 2018) . chronic inflammation from comorbidities may synergize with sars-cov-2 viral infection to induce a systemic release of calprotectin, which translate by the up-regulation of nf-kb and the loss of hla-dr on classical monocytes and the presence of immature neutrophils, altogether converging to a state of chronic inflammatory induced immunosuppression. abnormal neutrophils were observed previously in severe covid-19 patients (wilk et al., 2020) . however, authors concluded that these neutrophils transdifferentiate from b cells. we have no supporting results suggesting that it could be the case. in healthy conditions, roughly 85% of total circulating monocytes are cd14 high cd16 low hla-dr high cells that are rapidly recruited to inflamed tissues (guilliams et al., 2018) . as in other severe illnesses (lukaszewicz et al., 2009) , the expression of hla-dr on cd14 high circulating monocytes is low in severe covid-19, which correlates with, and could be mediated by, il-6 overproduction (giamarellos-bourboulis et al., 2020). a more specific feature of covid-19 is the low fraction of cd14 low cd16 high non-classical monocytes. this fraction commonly increases in patients with sepsis and inflammatory diseases, including viral infections (kratofil et al., 2017) . the decrease in non-classical monocyte fraction could involve the ability of calprotectin to fasten the trans-endothelial migration of leucocytes (fassl et al., 2015) , unless these cells strongly adhere to the endothelium, or the conversion of classical into non-classical monocytes is stuck (hanna et al., 2011; hofer et al., 2015 ) (selimoglu-buet et al., 2018 . whatever the mechanism, the lower than normal frequencies of non-classical monocytes (thevarajan et al., 2020; hadjadj j et al., 2020) suggest a sars-cov-2 characteristic effect that is not observed in other viral infections. most importantly, this decrease generates a highly characteristic biological signature of covid-19's aggressive form, with the potential to be easily measured using standard diagnostic flow cytometry and to provide information on the real-time immunological severity of the infection. the burst of calprotectin detected in covid-19 patients may trigger nf-ĸb-driven emergency myelopoiesis, generating immature and dysplastic cells (basiorka et al., 2016; chen et al., 2013) . given the considerable hematopoietic potential of the lung (lefrancais et al., 2017) , the burst of calprotectin could also promote the contribution of lung megakaryocytes to disease pathogenesis in this organ. whatever the mechanism, the immature and mature cells released into the peripheral blood by emergency myelopoiesis may be endowed with immunosuppressive functions, suggesting that myeloid derived suppressive cells (mdscs) as detected in cancer, inflammation and other diseases (veglia et al., 2018) might be important in covid-19. in addition to hla-dr low monocytes whose phenotype is that of monocytic mdscs (m-mdscs), cd10 low cd101 -cxcr1 + immature cells are reminiscent of granulocytic mdscs (g-mdscs) (aarts et al., 2019; mastio et al., 2019; veglia et al., 2018) . thus, neutrophil precursors such as the pre-neutrophil (preneu) population that are cxcr4 positive (evrard et al., 2018) , may be released into the blood from the bone marrow prematurely and infiltrate the lung tissue in severe patients. the emergence of these populations could be a predictor of switch to a severe disease. further research will be required to determine their specific role in disease development. altogether, we observed that severe covid-19 is specifically associated with 1) a burst of circulating calprotectin that precedes cytokine release syndrome, 2) low levels of nonclassical monocytes in the peripheral blood, and 3) an emergency myelopoiesis that releases immature and dysplastic myeloid cells with an immune suppressive phenotype. calprotectin j o u r n a l p r e -p r o o f plasma level and non-classical monocytes monitoring in the blood of patients could be implemented in a routine lab to discriminate patients with early immunological signs consistent with developing more severe disease, as recently suggested (chen et al., 2020b) . finally, in addition to the network of potential drug targets recently depicted by analysis of sars-cov-2 interactions (gordon et al., 2020) , our work provides further rationale for the testing of several clinical strategies, including: blocking emergency myelopoiesis with lenzilumab (nct04351152), a recombinant anti-human gm-csf antibody (patnaik et al., 2020) ; testing the oral quinoline-3-carboxamide tasquinimod (fizazi et al., 2017) and related molecules such as abr-215757 (paquinimod) which blocks the binding of s100a9 to tlr4 and rage (kraakman et al., 2017; raquil et al., 2008) and the preclinical anti-cd33 monoclonal antibodies (walter, 2018) which may prevent the interaction of s100a9 with myeloid progenitors (eksioglu et al., 2017) . these analysis provide snapshots of the differences in innate immune cell phenotype and calprotectin plasma level between outpatients with a mild disease at the time of sampling, having no or limited clinical symptoms and not requiring ct-scan or hospitalization, and moderate to severe patients whose clinical situation requires hospitalization and, in most cases, oxygen supply. although all the statistical analyses indicate that these biomarkers efficiently discriminate these two clinical situations and may help for urgent patient triage, a prospective serial analysis is now required to evaluate how these biomarkers can predict the switch from a mild to a moderate or severe covid-19 and inform on the mechanisms involved in this switch. non-supervised umap analysis of data from 25 patients (controls=12; mild=5; critical=8); c. cell surface marker expression within umap analysis shown in panel b; d. non-supervised umap analysis of patient blood samples in control, mild and severe groups; e. percentage of neutrophils within total cells in each individual sample within indicated patient groups; f. partition of neutrophil subsets, based on cd101 and cd10 expression, within each patient group (data pooled per group); g. percentage of cd10 low cd101 +/neutrophils among total neutrophils, as in e; h. percentage of monocytes within total cells, as in e . i. partition of monocyte subsets in in each individual sample within patient groups, based on cd14 and cd16 expression (left panels) or cd11b and hla-dr expression (right panels); j. fractions of non-classical monocytes within total monocytes, as in e ; k. cd11b and hla-dr expression on classical monocytes within each patient group (data pooled per group); l. percentage of hla-dr low classical monocytes within classical monocytes, as in e; m. percentage of b, cd4 + t, cd8 + t, and nk cells within total cells, as in e; kruskal-wallis test, * p<0.05; ** p <0.01; *** p<0.001; ns, non-significant. two blood samples were collected ten days apart from 3 covid-19 patients. blood was also collected once from 3 outpatient controls whose sars-cov-2 rt-pcr was negative. individual cell mrnas were sequenced using chromium 10x technology; b. time line of sample collection in the three patients; further details in table s4 figure 2a , and violin plots of gene expression in three statistically defined clusters; b. heatmap of differentially expressed genes (degs; logfc ± 0.25; fdr<0.05) in total monocytes; columns labelled "0" identifies degs generated by comparing each patient sample at day 0 to the pool of the three controls and the two other patient samples at day 0; columns labelled "10" identifies the expression of these genes in each patient sample at day 10 compared to day 0; genes shown in table s4 . c-e. spectral flow analysis in pooled controls and each individual patient sample at day 0 and day 10 of monocyte subset partition in samples analyzed by scrnaseq (c), cd11b and cd141 expression among classical monocytes (d), and cd169 and hla-dr expression among classical monocytes (e); f-i. mass cytometry analysis of monocyte subsets in 4 patients within each group (pooled data) (f); non-classical monocyte fraction among total monocytes in each individual sample within the 3 groups (g); p65/nf-κb expression in hla-dr low classical monocyte subset as in f (h); fraction of p65/nf-κb high hla-dr low classical monocytes among classical monocytes as in g (i). figure 2a ; b. umap profile of neutrophils within the 3 controls, the mild and the two severe cases with the cluster gates overlaid; c. violin plots of indicated gene expression in two statistically defined neutrophil clusters; d. heatmap of degs in total neutrophils generated as described in figure 3b ; e-f. spectral flow analysis in pooled controls and each individual patient sample at day 0 and day 10 of neutrophil subsets, based on cd10 and cd101 expression (e), and cxcr4 and cd11b expression among cd10 low cd101neutrophils (f) in indicated samples (pooled controls). g-h. mass cytometry analysis of neutrophil subsets in 4 patients within each group (pooled data) as in figure 3f -i, based on cd10 and cd101 expression (g) and the fraction of cd10 low cd101neutrophils among total neutrophils in each sample within the 3 groups (h). plasma levels of calprotectin (s100a8/s100a9), interferon alpha (ifnα2a) and 40 cytokines and chemokines in blood samples collected from 84 patients (controls, 40; mild disease, 18; moderate or severe disease, 25). a. volcanoplot representation of cytokine levels in mild covid-19 compared to controls; ifnα2a shown in orange, b. volcanoplot representation of cytokine levels in severe covid-19 compared to control patients; ifnα2a shown in orange, calprotectin, cxcl11, cxcl13 and cx3cl1 in red being are the most significantly associated with severe forms; c. circulating levels of cxcl8, ifnα2a, calprotectin and il-6 in individual samples within each group; d. impact of comorbidities (see table s6 ) on calprotectin plasma level within each group; e. volcanoplot representation of cytokine levels in severe patients with and without comorbidities; f. impact of bacterial infections on calprotectin plasma level within each group; g-i. spearman correlations between calprotectin plasma level and neutrophil count (g), fibrinogen (h), and d-dimers (i); j-l. spearman correlations between il-6 plasma level and neutrophil count (j), fibrinogen (k), and d-dimers (l). wilcoxon rank-sum test, * p<0.05; ** p <0.01; *** p<0.001; **** p<0.0001; ns, non-significant. table s3 -s5. monocyte analysis by single cell rna sequencing, spectral flow cytometry and mass cytometry. a. pathway analysis generated by comparing degs in monocytes of each sars-cov-2 positive patient to the same population in the three control patients considered together using ipa software (mild patient in blue, severe #1 in red, severe # 2 in orange); b. the same degs were used to perform a gene ontology network analysis using cluego software, considering the two severe patients together; c. combined (left panel) and individual (right panel) mass cytometry analysis of p65/nf-κb expression in circulating cd34 + cells in each group. figure 4 ; table s3 -s5. neutrophil analysis by single cell rna sequencing, spectral flow cytometry and mass cytometry. a. heatmap of the top 20 degs defining two neutrophil clusters. b. pathway analysis generated by comparing degs in neutrophils of each sars-cov-2 patient to the same population in the three control patients considered together using ipa software (mild patient in blue, severe #1 in red, severe # 2 in orange); c. the same degs identified in neutrophils were used to perform a gene ontology network analysis using cluego software, considering the two severe patients together. rt-qpcr analysis of s100a8 and s100a9 gene expression in the three groups of patients, using hprt as a control gene; b. heatmap of cytokines, chemokines, ifnα2a and calprotectin plasma levels in 37 covid-19 patients compared to 40 controls. sars-cov-2-positive patients included 14 mild and 23 severe patients. associated bacterial infection at sample collection is indicated in purple. the heatmap shows z score-normalized concentrations, each column represents one patient and each row one protein; the color gradient from blue to red indicates increasing concentrations. rows and columns are clustered using correlation distance and average linkage; c. volcano-plot representation of cytokine levels in severe sars-cov-2 patients with (n=11) or without (n=14) bacterial infection at the time of sample collection; d. spearman correlation between calprotectin concentration and age showing control patients in green, mild in orange and severe in red; e. spearman correlation between il-6 and calprotectin concentrations (color code as in d). figure 6 . related to figure 6 ; table s6 . validation of innate immune signature of severe covid-19. a-b non-supervised umap representation generated by pooling data from all the patient samples; cell identification (a) surface marker expression (b); c. bar plots representing the percentage of cd10 low cd16 low neutrophils among all neutrophils in individual patients from each group in the validation cohort (n=90). d. spearman correlation between cd169 (siglec-1) mean fluorescence intensity (mfi) and days spent by severe patients in icu. e. spearman correlation between cd169 (siglec-1) mean fluorescence intensity (mfi) and plasma ifnα concentration; yellow, mild covid-19 patients; red, severe covid-19 patients. f.g. bar plots representing the percentage of hla-dr low classical monocytes, b cells, cd4 + and cd8 + t cells and nk cells (f) and neutrophils among cd45 + cells, cd10 low cd101neutrophils among all neutrophils and cd10 low cxcr4 + neutrophils among cd10 low cd101neutrophils (g) in individual patients from each group in the validation cohort (n=90). figure 7 ; table s6-s7. changes in innate immune cell phenotype are detected in moderate covid-19 patients. a. bar plots representing the percentage of b cells, cd4 + t cells, cd8 + t cells, nk cells, total monocytes, cd169 + , hla-dr low and cd141 + classical monocyte subsets, total neutrophils among cd45 + cells, and cd10 low cd101and cd10 low cd16 low neutrophil subset among all neutrophils in individual patients from each group, with the moderate category (6 patients) highlighted. b. plasma concentration of ifnα in moderate covid-19 patients compared to the three other groups. c. roc analysis showing performance of a diagnostic test using percentage of non-classical monocytes among total monocytes to distinguish controls and mild covid patients from moderate and severe covid patients; d. monocyte subset analysis in the peripheral blood of 2 severe patients, before (left panels) and after (right panels) treatment with the indicated anti-il-6 antibodies; e. percentage of hla-dr low classical monocytes among classical monocytes in a cohort of 22 patients and 17 controls grouped into 4 clinical categories; f. correlation between the percentage of hla-dr low classical monocytes and non-classical monocytes; g. percentage of cd16 low neutrophils among neutrophils in control and covid-19 patients of the learning cohort described in figure 7 . h. roc curves evaluating the discriminating significance of calprotectin plasma level (yellow), nonclassical monocyte fraction (red), cd16 low circulating neutrophils (blue) and ifnα2a plasma level (white) between controls/mild patients and moderate/severe patients. auc, area under the curve; mann whitney test; i. heatmap of blood and bronchoalveolar lavage fluid scrnaseq cells integrated defining the 5 regions of cell populations; j. pathway analysis (cytoscape and cluego) of degs expressed at a higher level in bronchoalveolar monocytes/macrophages from mild versus severe patients. k. umap analysis of neutrophils with s100a8 (left panel) and s100a9 (right panel) gene expression level projection (low expression = grey dots; high expression = dark blue dots). * p<0.05; ** p <0.01; *** p<0.001. lead contact. further information and request for resources and reagents should be directed to and will be fulfilled by the lead contact: florent_ginhoux@immunol.astar.edu.sg (f.gi.) patients. this non-interventional study was approved by institutional review boards of cochin-port royal (paris, france) and gustave roussy (villejuif, france) hospitals and the ethical committee of cochin-port royal hospital (clep decision n°: aaa-2020-08023), and conformed to the principles outlined in the declaration of helsinki. controls (n = 72) were symptomatic patients who were seen at hôtel-dieu or gustave roussy covid-19 screening unit and were negative for sars-cov-2 rt-pcr on pharyngeal swab. mild covid-19 patients (n = 27) were defined by having limited clinical symptoms (fever, cough, diarrhea, myalgia, anosmia/ageusia) that did not require ct-scan or hospitalization. moderate cases (n = 16) were defined as symptomatic patients with dyspnea and radiological findings of pneumonia on thoracic ct scan, requiring hospitalization and a maximum of 9 l/min of oxygen. in the larger part of this study, mild and moderate cases were analyzed together and grouped under "mild category". severe patients (n = 43) were those hospitalized in the icu with respiratory distress requiring 10l/min of oxygen or more, without or with endotracheal intubation and mechanical ventilation. sampling. whole human peripheral blood was collected into sterile vacutainer tubes containing edta or heparin. except for single cell rna sequencing, tubes were centrifuged at 300 g for 5 min at room temperature and plasma was collected. whole blood was mixed at a 1:1 ratio with whole blood cell stabilizer (cytodelics), incubated at room temperature for 10 min and transferred to -80°c freezer to await analysis. these samples were secondarily thawed in a water bath set to +37°c. cells were fixed at a ratio 1:1 with fixation buffer (cytodelics, ratio 1:1) and incubated for 10 min at room temperature. red blood cells were lysed by addition of 2 ml of lysis buffer (cytodelics, ratio 1:4) at room temperature for 10 min. white blood cells were washed with 2 ml of wash buffer (cytodelics, ratio 1:5). spectral flow cytometry. cells were resuspended in 100 µl extra-cellular antibody cocktail and incubated at room temperature for 15 min. for intra-cellular labelling, a step of permeabilization was performed using 200 µl of bd cytofix/cytoperm kit (bd); cells were then incubated for 40 min at +4°c, washed in perm buffer (bd) and resuspended in intra-cellular antibody cocktail. after incubation, cells were washed in flow cytometry buffer (1% bsa, 0.5% na-azide and 0.5m edta in pbs) and resuspended to proceed to the acquisition. all antibodies are listed in the key resource table. samples were acquired on cytek aurora flow cytometer (cytek biosciences). fcs files were exported and analyzed using flowjo software. to fully capture peripheral blood cell heterogeneity, we analyzed fresh samples without cell sorting or freezing and without ficoll enrichment, minimizing time of incubation and processing. sample preparation was done at room temperature. after red cell lysis, single-cell suspensions were loaded onto a chromium single cell chip (10x genomics) according to the manufacturer's instructions for coencapsulation with barcoded gel beads at a target capture rate of ~7000 individual cells per sample. to analyze neutrophils, we added rnase inhibitor (rnase out recombinant ribonuclease inhibitor invitrogen, 40u/ml) into the loading buffer. captured mrnas were barcoded during cdna synthesis using the chromium single cell 3' solution v3 (10x genomics) according to the manufacturer's instructions. of note, we increased the pcr cycles by two during cdna amplification. all samples (at day 0 and day 10) were processed simultaneously with the chromium controller (10x genomics) and the resulting libraries were prepared in parallel in a single batch. we pooled all of the libraries for sequencing in a single sp illumina flow cell. all of the libraries were sequenced with an 8-base index read, a 28-base read1 containing cell-identifying barcodes and unique molecular identifiers (umis), and a 91-base read2 containing transcript sequences on an illumina novaseq 6000. reads were aligned to the hg19 genome and were used for subsequent analysis. using the package seurat v3 (stuart et al., 2019) , we normalized and scaled scrna sequencing data. we next applied a principle component analysis to the scrna sequencing results yielding a number of significant pcs (using jackstraw plot analysis). in addition, the standard deviation differences from one pc to another was taken into account as described by the seurat v3 manual (struart et al., 2019) . to generate umap plots, min_distance was set as 0.3 and n_neighbors was set to 30. by dimensionality reduction, distinct clusters were identified and described by performing the findclusters feature. the resolution of this feature was reduced to 0.3 to identify main cellular population only. following this, differential genes were identified by performing the findallmarkers function and selecting genes that were differentially expressed (logfc >/= +/-0.25 and fdr < 0.05). this approach identified a number of well characterized blood cell populations. clustering and analysis of specific cell populations were performed in a similar manner to as previously stated. cells were clustered and separated based on well described markers (cd14/cd16 as describing monocyte populations). the bronchoalveolar dataset was downloaded from the nih geo database (liao et al., dataset gse145926) and integrated with our own blood scrnaseq data using the seurat v3 anchoring method (stuart et al., 2019) . briefly, the datasets were normalized independently and the highly variable genes were identified for each dataset using the seurat pipeline. a corrected data matrix with both datasets was then generated using the seurat v3 anchoring procedure to allow for joint analysis. the matrix was scaled and a principal component analysis (pca) was performed using the seurat v3 pipeline. a umap was performed on the 30 first principal components (pcs) . these principle components and subsequent clustering and analysis of scrna sequencing data was performed as previously described. comparisons between patient samples were performed by a variation of the findmarkers function that compared the differentially expressed genes from different samples, patient groups, and organs. cutoff values were determined as previously described. rt-qpcr analysis. total rna was extracted with rneasy mini kit (qiagen) and reverse transcribed with superscript™ iv vilo™ master mix with ezdnase™ enzyme (invitrogen). real-time quantitative polymerase chain reaction (rt-qpcr) was performed using power sybr™ green pcr master mix in a biorad cfx96 thermocycler using the standard sybr green detection protocol as outlined by the manufacturer (applied biosystems). briefly, 12 ng of total cdna, 50nm (each) primers and 1× sybr green mixture were used in a total volume of 20 μl. human primer sequences are the following: gus (f: gaaaatatgtggttggagagctcatt; r: ccgagtgaagatcccct tttta); hprt (f: ggacaggactgaacgtcttgc; r: cttgagcacacagagggctaca); s100a8 (f: caacactg atggtgcagttaacttc; r: ctgccacgcccatctttatc); s100a9 (f: ctgagcttcgagg agttcatca; r: cgtcaccctcgtgcatcttc). table 6 ) were centrifuged for 15 min at 1,000 g, diluted 1:4, then monitored using the bio-plex pro tm human chemokine panel 40-plex assay (bio-rad, ref: 171ak99mr2) according to the manufacturer's instructions. 40-plex cytokines and chemokines provided are: ccl1, ccl11, ccl13, ccl15, ccl17, ccl19, ccl2, ccl20, ccl21, ccl22, ccl23, ccl24, ccl25, ccl26, ccl27, ccl3, ccl7, ccl8, cx3cl1, cxcl1, cxcl10, cxcl11, cxcl12, cxcl13, cxcl16, cxcl2, cxcl5, cxcl6, cxcl8, cxcl9, gm-csf, ifnα, il-10, il-16, il-1b, il-2, il-4, il-6, mif, tnfa. acquisitions and analyses were performed on a bio-plex 200 system (bio-rad) and a bio-plex manager 6.1 software (bio-rad), respectively. soluble calprotectin (diluted 1:100) and ifnα2a were analyzed using a r-plex human calprotectin antibody set (meso scale discovery, ref: f21yb-3) and the ultra-sensitive assay s-plex human ifnα2a kit (meso scale discovery, ref: k151p3s-1), respectively, following manufacturer's instructions. acquisitions and analyses of soluble calprotectin and ifnα were performed on a meso™ quickplex sq120 reader and the msd's discovery workbench 4.0. each plasma sample was assayed twice with the average value taken as the final result. data representation was performed with software r v3.3.3 using tidyverse, dplyr, ggplot2, ggpubr, pheatmap, corrplot or hmisc packages. mass cytometry. cells were barcoded using the 20-plex pd barcoding kit (fluidigm). briefly, they were washed in barcode perm buffer, resuspended in 800 µl of barcode perm buffer and 100 µl of each barcode were transferred to the appropriate sample. cell suspensions were incubated for 30 min at room temperature, washed twice with cell staining buffer (fluidigm) and pooled, suspended in 100 µl filtered antibody cocktail, and incubated for 30 min at +4°c. all antibodies used are listed in key resource table. after staining, cells were washed with cell staining buffer and permeabilized with 200 µl of fix/perm from foxp3/transcription factor staining buffer kit (ebiosciences), 40 min at +4°c. after incubation, cells were washed in perm buffer from foxp3/transcription factor staining buffer kit (ebiosciences), resuspended in 100 µl filtered antibody cocktail, incubated for 30 min at +4°c, washed in cell staining buffer and resuspended in 50 µl of cytofix/perm for 5 min at room temperature. then, 400 µl of pbs containing 1.6% pfa + iridium (1:4000) were added for 35 min at room temperature. finally, cells were washed in cell staining buffer, resuspended in 50 µl and stored at +4°c until acquisition. cells were counted, washed and resuspended in maxpar cell acquisition solution at 0.5x 10 6 / ml and mixed with 10% eq beads immediately before acquisition on helios mass cytometer using noise reduction, event length limits of 10-150 pushes. an average of 500,000 events were acquired per sample at a flow rate of 0.03ml/min. mass cytometry standard files were normalized to a global standard determined for each log of eq beads using cytof software v. 6.7.1014 (fluidigm) . fcs files were exported and analysed using flowjo software. umap was performed with n_neighbours of 15 and a min_distance of 0.2. clusters were identified by the detection of commonly used cell markers (t cells expressing cd4 or cd8, neutrophils expressing cd15, and monocytes expressing cd14 and or cd16). whole-blood samples (200µl) were labelled with anti-cd14-pc7 (clone rmo52); cd16-pb (clone 3g8); cd2-fitc (clone 39c1.5); cd56-pc5.5 (clone n901); cd24-pe (clone alb9); cd45-ko (clone j33) and hla-dr-apc (clone immu-357) antibodies, all purchased from beckman-coulter. red blood cells were lysed with 1 ml versalysetm (beckman coulter) before sample analysis with a navios cytometer (beckman coulter) as described (tarfi et al., 2019) . monocytes were selected as cd45 high /ssc int cells among living cells and singlets before excluding t cells as cd2 + /ssc low , nk cells as cd56 + /ssc low/int , b cells as cd24 + /ssc low , immature and mature granulocytes as cd24 + /ssc int/high , cd16 bright residual granulocytes, and remaining cd14 − cd16 − cells corresponding mainly to basophils and nk cells not previously excluded. monocyte subsets were detected on a cd45/scc dot plot, using a cd14/cd16 scattergram that separates cd14 high cd16 low (classical), cd14 high /cd16 high (intermediate) and cd14 low cd16 high (nonclassical) subsets. finally, the proportion of monocytes hla-dr low was evaluated on a hla-dr/cd14 scattergram. data analysis. calculations and statistical tests were performed using r v3.3.3. unless stated, p-values are two-sided with 95% confidence intervals for the reported statistic of interest. individual data points representing the measurement from one patient are systematically calculated from the corresponding distribution. wilcoxon rank-sum test was applied to assess differences in concentration between two different groups. when indicated, the false discovery rate (fdr, p > 0.05) was controlled using the benjamini-hochberg procedure. spearman correlations were computed using hmisc r package and cytokine results were shown using r package pheatmap. soluble factor fold ratios were calculated as log2 transformation of values of mild and severe patients versus median value of all control patients, and were converted to z scores. hierarchical clustering of the patients based on the z score of 42 soluble factors was performed using euclidean distance and ward.d clustering. gene ontology networks were made by subjecting the degs from previous scrna sequencing analysis to the cytoscape addon cluego. the selected degs were specific to those with increased expression by monocytes and neutrophils from mild or severe sars-cov-2 positive patients. biological process gene ontologies selected had an fdr < 0.05. other statistical analyses were performed using graphpad prism 7. a generalized linear model was also used to analyze interactions between biological parameters. first, neutrophil count, calprotectin, fibrinogen, il-6 and d-dimers were normalized using log transformation. then, calprotectin plasma level was modeled using multivariable linear regression adjusted for the other parameters, and their interaction with the groups. similar approach was performed to model il-6. backward selection was applied to obtain a parsimonious model. to identify the most discriminant markers, we used a logistic regression adjusted for the scaled log2-transformed markers. parameters were penalized using the least absolute shrinkage and selection operator (lasso) to limit overfitting due to the high number of markers. the regularization parameter was selected from 10 folds cross-validation using the glmnet r package (friedman et al., 2010) . the final auc estimate was corrected for optimism using the harrell's method (harrell et al., 1996) , and its confidence interval was computed using the two-stage approach proposed by noma et al (noma et al., 2020) with 2000 bootstrap samples for each stage. in this analysis, we included age, sex and comorbidities together with biological parameters. given the absence of validation cohort, auc was corrected to limit overfitting bias. this correction indicated an auc at 99.7% (95% confidence interval [98.8%; 100.0%]). the final score corresponds to the following equation: gustave roussy cancer campus gustave roussy cancer campus gustave roussy cancer campus gustave roussy cancer campus gustave roussy cancer campus hôpital hôtel-dieu, 75014 service de médecine intensive et réanimation gustave roussy cancer campus gustave roussy cancer campus service de réanimation médicale, gustave roussy cancer campus activated neutrophils exert myeloid-derived suppressor cell activity damaging t cells beyond repair middle east respiratory syndrome corona virus spike glycoprotein suppresses macrophage responses via dpp4-mediated induction of irak-m and ppargamma alarmins mrp8 and mrp14 induce stress tolerance in phagocytes under sterile inflammatory conditions the nlrp3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype dimensionality reduction for visualizing single-cell data using umap host-viral infection maps reveal signatures of severe covid-19 patients ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes clinical and immunological features of severe and moderate coronavirus disease 2019 elevated serum levels of s100a8/a9 and hmgb1 at hospital admission are correlated with inferior clinical outcomes in covid-19 patients epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study the architecture of a scrambled genome reveals massive levels of genomic rearrangement during development induction of myelodysplasia by myeloid-derived suppressor cells comparative replication and immune activation profiles of sars-cov-2 and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid-19 activation of human monocytes after infection by human coronavirus 229e star: ultrafast universal rna-seq aligner novel therapeutic approach to improve hematopoiesis in low risk mds by targeting mdscs with the fc-engineered cd33 antibody bi 836858 developmental analysis of bone marrow neutrophils reveals populations specialized in expansion, trafficking, and effector functions transcriptome assessment reveals a dominant role for tlr4 in the activation of human monocytes by the alarmin mrp8 a randomized, double-blind, placebo-controlled phase ii study of maintenance therapy with tasquinimod in patients with metastatic castration-resistant prostate cancer responsive to or stabilized during first-line docetaxel chemotherapy signaling mechanisms inducing hyporesponsiveness of phagocytes during systemic inflammation chronic inflammation in the etiology of disease across the life span complex immune dysregulation in covid-19 patients with severe respiratory failure a sars-cov-2 protein interaction map reveals targets for drug repurposing clinical characteristics of covid-19 in china. reply developmental and functional heterogeneity of monocytes impaired type i interferon activity and exacerbated inflammatory responses in severe covid-19 patients. medrxiv receiver operating characteristic (roc) curve analysis for medical diagnostic test evaluation the transcription factor nr4a1 (nur77) controls bone marrow differentiation and the survival of ly6c-monocytes slan-defined subsets of cd16-positive monocytes: impact of granulomatous inflammation and m-csf receptor mutation clinical features of patients infected with 2019 novel coronavirus in wuhan neutrophil-derived s100 calcium-binding proteins a8/a9 promote reticulated thrombocytosis and atherogenesis in diabetes causal analysis approaches in ingenuity pathway analysis monocyte conversion during inflammation and injury high levels of s100a8/a9 proteins aggravate ventilator-induced lung injury via tlr4 signaling the lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors sars-cov-2 and viral sepsis: observations and hypotheses single-cell landscape of bronchoalveolar immune cells in patients with covid-19 high incidence of venous thromboembolic events in anticoagulated severe covid-19 patients monocytic hla-dr expression in intensive care patients: interest for prognosis and secondary infection prediction identification of monocyte-like precursors of granulocytes in cancer as a mechanism for accumulation of pmn-mdscs tocilizumab, an anti-il6 receptor antibody heterogeneity of neutrophils mechanisms for the transendothelial migration of hiv-1-infected monocytes into brain phase 1 study of lenzilumab, a recombinant anti-human gm-csf antibody, for chronic myelomonocytic leukemia (cmml) blockade of antimicrobial proteins s100a8 and s100a9 inhibits phagocyte migration to the alveoli in streptococcal pneumonia presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area induction of nuclear factor-kappab responses by the s100a9 protein is toll-like receptor-4-dependent a mir-150/tet3 pathway regulates the generation of mouse and human non-classical monocyte subset cesarean section and chronic immune disorders cytoscape: a software environment for integrated models of biomolecular interaction networks human s100a9 potentiates il-8 production in response to gm-csf or fmlp via activation of a different set of transcription factors in neutrophils human cytomegalovirus induces monocyte differentiation and migration as a strategy for dissemination and persistence comprehensive integration of single-cell data disappearance of slan-positive non-classical monocytes for diagnosis of chronic myelomonocytic leukemia with associated inflammatory state role of leptin and socs3 in inhibiting the type i interferon response during obesity breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon s100-alarmin-induced innate immune programming protects newborn infants from sepsis advancing scientific knowledge in times of pandemics myeloid-derived suppressor cells coming of age autoinhibitory regulation of s100a8/s100a9 alarmin activity locally restricts sterile inflammation investigational cd33-targeted therapeutics for acute myeloid leukemia s100a8/a9 in inflammation. front immunol 9 comorbidities and multi-organ injuries in the treatment of covid-19 a single-cell atlas of the peripheral immune response in patients with severe covid-19 middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets sars-coronavirus replication in human peripheral monocytes/macrophages our teams are supported by grants from ligue nationale contre le key: cord-288916-i8ukefp8 authors: gómez-herranz, maria; nekulova, marta; faktor, jakub; hernychova, lenka; kote, sachin; sinclair, elizabeth h.; nenutil, rudolf; vojtesek, borivoj; ball, kathryn l.; hupp, ted r. title: the effects of ifitm1 and ifitm3 gene deletion on ifnγ stimulated protein synthesis date: 2019-04-02 journal: cell signal doi: 10.1016/j.cellsig.2019.03.024 sha: doc_id: 288916 cord_uid: i8ukefp8 interferon-induced transmembrane proteins ifitm1 and ifitm3 (ifitm1/3) play a role in both rna viral restriction and in human cancer progression. using immunohistochemical staining of ffpe tissue, we identified subgroups of cervical cancer patients where ifitm1/3 protein expression is inversely related to metastasis. guide rna-cas9 methods were used to develop an isogenic ifitm1/ifitm3 double null cervical cancer model in order to define dominant pathways triggered by presence or absence of ifitm1/3 signalling. a pulse silac methodology identified irf1, hla-b, and isg15 as the most dominating ifnγ inducible proteins whose synthesis was attenuated in the ifitm1/ifitm3 double-null cells. conversely, swath-ip mass spectrometry of ectopically expressed sbp-tagged ifitm1 identified isg15 and hla-b as dominant co-associated proteins. isg15ylation was attenuated in ifnγ treated ifitm1/ifitm3 double-null cells. proximity ligation assays indicated that hla-b can interact with ifitm1/3 proteins in parental siha cells. cell surface expression of hla-b was attenuated in ifnγ treated ifitm1/ifitm3 double-null cells. swath-ms proteomic screens in cells treated with ifitm1-targeted sirna cells resulted in the attenuation of an interferon regulated protein subpopulation including mhc class i molecules as well as ifitm3, stat1, b2m, and isg15. these data have implications for the function of ifitm1/3 in mediating ifnγ stimulated protein synthesis including isg15ylation and mhc class i production in cancer cells. the data together suggest that pro-metastatic growth associated with ifitm1/3 negative cervical cancers relates to attenuated expression of mhc class i molecules that would support tumor immune escape. interferons (ifns) are pleiotropic cytokines produced by the innate immune system as a defensive response [1] . many biological functions have been described for the interferon pathway. among them, the best characterized are anti-tumor activity, immunomodulatory effects, antipathogen, and anti-viral activity [2] . ifn effects rely on three different types of receptors: type i (ifnα, ifnβ and ifnω), type ii (ifnγ) and type iii (ifnλ) [3, 4] . ifns increase in response to a broad range of factors such as persistent viral infection or dna damaging agents which activate the jak kinase-stat pathway. ultimately, this signalling cascade will regulate the transcriptional synthesis of interferon stimulated genes (isgs) [5, 6] . type i ifns facilitate anti-proliferative and pro-apoptotic pathways in a wide range of cell types and it has been extensively used as antitumor therapeutic agent. high doses of ifns are used for cancer therapy and can activate anti-tumor immunity as well as pro-apoptotic and antiproliferative programs. by contrast, it has been demonstrated that sustained low level of ifn causes a steady-state expression of the interferon resistance dna damage signature (irds) which is comprised of a subset of interferon-stimulated genes [7] . irds proteins promote phenotypes that contribute to the tumor development such as resistance to dna damage, suppression of t cell toxicity, metastasis, and facilitation of epithelial-mesenchymal transition [8] . ifitm1 is a pro-oncogenic receptor which is a component of the irds pathway. basal expression of ifitm proteins is observed in some cells and expression can also be induced by type i and type ii interferons. ifitm1 is up-regulated during development of radiation resistance, escaping from pro-apoptotic and anti-proliferative effects [9, 10] . ifitm1 expression has been extensively reported in many types of cancer; breast, cervix, colon, ovary, brain and oesophagus cancer and its high expression correlates with tumor progression and can lead to a poor outcome [10] [11] [12] [13] [14] [15] [16] . ifitm1 was previously known as ifi 17 (interferon induced protein 17), ifi 9-27 (interferon inducible protein 9-27), cd225 (cluster of differentiation) and leu-13 (leucocyte surface protein). it is a membrane protein that plays a key role in restriction of anti-viral immune response and belongs to the interferon induced transmembrane family (ifitm) [17] . ifitm1 is coded by the ifitm1 gene located on chromosome 11p15.5 and flanked by ifitm2 and ifitm3 genes. the ifitm immunity-related protein family are composed of short amino-terminal and carboxy-terminal domains, two transmembrane domains, and a cytoplasmic domain. ifitm1 is slightly different from ifitm2 and ifitm3, with some studies demonstrating that ifitm1 is uniquely expressed at the cell surface [18, 19] . ifitm family members are capable of attenuating the entrance of many human and animal viruses. they suppress the entry of viruses such as influenza a virus (iav), west nile virus, dengue virus, sars coronavirus, filoviruses, vsv, and hcv among others [20] . ifitm family proteins inhibit cytosolic entry of viruses by preventing fusion of viral and host membranes. the protein-protein interaction networks by which ifitm proteins inhibit viral propagation are just emerging. one study using yeast two-hybrid methodology has identified an interaction of ifitm3, ifitm2, and ifitm1 with vapa that in turn mediates an accumulation of cholesterol in multivesicular structures [21] . this reduces the fusion of intraluminal virion-containing vesicles with endosomal membranes and thereby inhibits virus release into the cytosol. in addition, different family members exhibit specific viral preferences. in the case of ifitm1, it is more active in controlling filoviruses, influenza a, and sars [17, 20, 22] . in this report, we focus on applying methodologies in interactomics, gene editing, swath-immunoprecipitation mass spectrometry, and pulse-silac mass spectrometry to propose an ifnγ responsive biochemical function for the ifitm1/3 proteins. all chemicals and reagents were obtained from sigma unless indicated otherwise. all antibodies from thermofisher unless indicated otherwise. siha, ifitm1 null-siha, and ifitm1/ifitm3 null-siha cell lines were grown in rpmi 1640 medium (invitrogen) supplemented with 10% (v/v) fetal bovine serum (labtech) and 1% penicillin/streptomycin (invitrogen) and incubated at 37°c with 5% co 2. cervical cancer samples were obtained at the masaryk memorial cancer institute where patients gave written consent for tissue use according to local ethical regulations. tissues were fixed in 4% formaldehyde for approximately 24 h before processing into paraffin wax and sectioned (4 μm). after sections were cut, the antigens were retrieved, and samples were probed with primary antibodies (including those to p16 protein (data not shown), and ifitm1/3 proteins (using the mab-mhk); supplementary fig. 1 ). following this stage, the tissue sections were incubated with secondary antibodies conjugated to streptavidin-hrp. sections were incubated with dab (dako), counterstained with haematoxylin and mounted for visualization, as previously described [23] . 2.3. generation of ifitm1 null and ifitm1/ifitm3 double null cell line using crispr/cas9 technology guide rna sequences were designed for ifitm1 (5′ tccaaggtcc accgtgatca 3′) and for ifitm3 (5′ gtcaacagtggccagccccc 3′). the guide rna was cloned into the lenticrisprv2 expression vector [24] . after transfection, cells were selected with puromycin and sorted in a 96-well plate using facs. after 3 weeks, individual clones were screened for absence of ifitm1 and/or ifitm3 protein expression by western blot using the mab-mhk ( supplementary fig. 1 ). chromosomal dna from the positive clones were sequenced for a final validation to define the precise gene edit (as in figs. 2 and 3 ). chromosomal dna was extracted from frozen cell pellets following the instruction manual (gentra puregene cell kit, qiagen). validation of the edited dna sequence was confirmed by cloning the genomic ifitm1 and ifitm3 pcr products into a holding vector and by amplifying the entire gene and 500 bp surrounding the guide rna targeting site followed by sanger sequencing at source bioscience service (scotland). 2.5. cloning, transfection and affinity purification of sbp-ifitm1 protein ifitm1 cdna was cloned by pcr into pexpr-iba105 expression vector containing sbp tag at the n-terminus of the coding region (sbp vector (iba)). siha cells were grown in rpmi as a duplicate. for transfection, cells were grown to approximately 80% confluency and transfected using attractene (qiagen). cells were transfected with sbpempty vector (control cells) and sbp-ifitm1 for 48 h. 48 h after transfection, cells were treated with carrier or with 100 ng/ml ifnγ (invitrogen) for 24 h in order to "stabilize" potential interferon-activated sbp-ifitm1 interacting proteins. cells were washed twice in ice cold pbs and scraped into buffer containing 100 mm kcl, 20 mm hepes ph 7.5, 1 mm edta, 1 mm egta, 0.5 mm na 3 vo 4 , 10 mm naf, 10% (v/v) glycerol, protease inhibitor mix, and 0.1% triton x-100, then incubated for 30 min on ice and centrifuged at 13,000 rpm for 15 min at 4°c. equal amounts of protein were used for performing the affinity capture. streptavidin agarose conjugated beads (millipore) were prewashed with in pbs. then cell lysate was added and incubated for 2 h at rt with gentle rotation. binding proteins were eluted with a buffer containing 20 mm hepes ph 8, 2 mm dtt, and 8 m urea. small interfering rna directed against the human ifitm1 (qiagen) and an allstars negative controls flexitube sirna (qiagen) were used to transfect siha cells for 48 and 72 h. cells were transfected using hiperfect (qiagen) following the manufacturer's instructions. for performing "pulse" silac, parental siha, ifitm1/ifitm3 double null cells, and ifitm1 single null cells were grown as biological triplicate and incubated with silac heavy media for 6 and 24 h with or without 100 ng/ml ifnγ before harvesting [25] [26] [27] . cells were isotopically pulse-labeled using silac rpmi-heavy media (dundee cell products, uk); l-[ 13 c6 14 n4] arginine (r6) and l-[ 13 c6 14 n2] lysine (k6). cells were harvested in a buffer containing 8 m urea, 0.1 m tris ph 8.5. total protein extracts were measured by the bradford assay [28] . proteins were detected using the following primary antibodies: mouse monoclonal anti-bodies generated to a peptide that is identical in ifitm1 and ifitm3 (mab-mhk) (moravian biotechnology, this study is their first description). the antibody we name mab-mhk can therefore detect co-expression of both ifitm1 and ifitm3 proteins ( supplementary fig. 1 ). other sources include, rabbit monoclonal anti-stat1 (cell signalling), mouse monoclonal anti-irf1 (bd transduction laboratories), rabbit polyclonal anti-hla-b (thermo fisher), rabbit polyclonal antibody anti-isg15 (cell signalling), mouse monoclonal anti-sbp-tag (sigma), mouse monoclonal anti-β-actin (sigma), and the mouse monoclonal anti-gapdh (abcam). protein from lysed samples was quantified using protein assay dye reagent (bio-rad). proteins were resolved by sds-page using 15% gels [29] and transferred onto nitrocellulose membranes (amersham protran, ge healthcare). immunoblots were processed by enhanced chemiluminescence (ecl). parental siha and ifitm1/ifitm3 double null cells were grown over 16 mm diameter glass coverslips. stimulated cells were treated with 100 ng/ml ifnγ for 24 h. cells were fixed with 4% (v/v) paraformaldehyde in pbs for 15 min at room temperature and permeabilized using 0.25% triton x-100 in pbs for 10 min. then, the cells were blocked with 3% bsa in pbs for 30 min. the primary antibody was incubated at a 1:1000 dilution overnight. alexa fluor 488 goat antirabbit igg (h + l) (invitrogen) was incubated as a secondary antibody at 1:2000 dilution for 1 h. the fluorescent signal was detected using a zeiss axioplan 2 microscope at 63×. replicates are described in the fig. 6 legend. fluorescence was measured using imagej software; cells were selected, and information was extracted on the area, integrated density, and mean gray values by selecting set measurements in the analyse menu. a region with no fluorescence was selected as background for each image. the following formula was applied for each 50 cells analyzed: ctcf = integrated density -(area of selected cell × mean fluorescence of background readings); *ctcf = corrected total cell fluorescence. parental siha and ifitm1/ifitm3 double null cells were grown and processed as described in the immunofluorescence method (above). primary antibodies from different species were incubated with the fixed and permeabilized cells: mhk mouse mab with rabbit polyclonal anti-hla-b, at 1:250 dilution overnight. duolink® assays (sigma) were carried out following supplier's instructions. the fluorescent signal was detected using a zeiss axioplan 2 microscope at 63×. fluorescence was measured using imagej software, as above reviewed for standard immunofluorescence. 2.12. mass spectrometric experimental screens 2.12.1. peptide generation using fasp cell lysates, immunoprecipitates, or gradient fractions were processed using filter-aided sample preparation protocol (fasp) [30, 31] . urea buffer (8 m urea in 0.1 m tris ph 8.5) was added to a 30 kda spin filter column (mrcprt010, microcon). protein concentration was determined using the rc-dc method (bio-rad). normalized sample was added into the spin filter column and was centrifuged at 14000 g for 15 min at 20°c. urea buffer was added again with 100 mm tris (2carboxyethyl) phosphine hydrochloride (aldrich) and mixed. the column was left on a thermo-block set at 37°c shaking at 600 rpm and centrifuged at 12,210 rpm at 20°c for 15 min. urea buffer and 300 mm of iodoacetamide (sigma) were mixed using a thermo-mixer at 600 rpm in the dark for 1 min, then was maintained statically for a further 20 min at room temperature in the dark. the sample was centrifuged at 12,210 rpm at 20°c for 15 min and the supernatant was discarded. a solution containing 100 mm ammonium bicarbonate were added to the column and then it was centrifuged at 12,210 rpm at 20°c for 20 min. this step was repeated one more time. the column was placed in a new collecting tube (low binding affinity) and 50 mm ammonium bicarbonate was added along with trypsin diluted in trypsin buffer (promega) at a 1:100 ratio. the column was incubated at 37°c overnight. the following day the column was centrifuged at 12,210 rpm at 20°c for 15 min. determination of the peptide concentration was performed using the quantitative colorimetric peptide assay (pierce, thermo-scientific). peptides were desalted on micro spin columns c-18 (harvard apparatus, usa). c-18 columns were conditioned three times with 100% acetonitrile (acn) and 0.1% formic acid (fa) and centrifuged at 1200 rpm at room temperature for 2 min. the column was washed with 0.1% fa and centrifuged at 1200 rpm at room temperature for 2 min. the column was hydrated in 0.1% fa for 15 min following centrifugation at 1800 rpm at room temperature for 3 min. the sample was loaded into the column and centrifuged at 2300 rpm for 3 min. after washing the column three times with 0.1% fa, the peptides were eluted in three consecutive centrifugations at 2300 rpm for 3 min using 50%-80% and 100% acn with 0.1% fa. subsequently, peptide eluates were evaporated and dissolved in 5% acn with 0.05% fa. there were three distinct mass spectrometric screens used in the manuscript. the rationale for replicates in each distinct approach is as follows; (i) the swath-ip (immunoprecipitation) to identify ifitm1enriched associated proteins (fig. 8) . the immunoprecipitation and immunoblots of sbp-ifitm1 vs sbp control is representative of experiments performed at least three times. the representative enrichment of ifitm1-associated proteins (in fig. 8 from supplementary table 2 ) was processed by label free (swath) mass-spectrometry in technical triplicates. the measured fold-changes and p-values for quantified proteins are listed in supplementary table 2 and fig. 8 ; (ii) the sirna swath-ms ( fig. 9a and b). targeted sirna to deplete ifitm1 in siha cells was performed in at least three separate experiments. a representative depletion of ifitm1 protein (fig. 9a ) was performed in technical triplicates using two different biological states (48 and 72 h). the statistical rationale for performing two biological states (equivalent plating of cell number and differing by time of interferon exposure) rather than two biological replicates at the same time point, was due to the variable induction and suppression of the interferon cascade over this time frame. thus, any proteins that are observed in two biological states as a function of time are thought to have higher significance than an analysis performed in duplicates at one time point. the samples in the two biological states were processed in technical triplicate for label-free (swath) analysis ( fig. 8 ; supplementary table 2 ); (iii) pulse silac to measure protein synthesis as a function of genotype. twelve enzymatically digested samples (four samples of parental siha, four samples of ifitm1 null, and four samples of ifitm1/ifitm3 double null), each of them as independent biological triplicates, were processed using isotopically labeled amino acids and were separated using lc-ms/ms analysis ( table 1 ). the statistical rationale for using three biological replicates with one injection per replicate, rather three technical replicates from one biological sample, relates to the dynamics and variability in the interferon dynamics. by using biological triplicates, any common overlaps are deemed to be more significant because of the possible variability in the cell plating and interferon cascade. peptides were loaded on a pre-column (μ-precolumn, 30 mm i.d., 5 mm length, c18 pepmap 100, 5 μm particle size, 100 å pore size) and further separated on an acclaim pepmap rslc column (75 mm i.d., length 500 mm, c18, particle size 2 mm, pore size 100 å) with a 300 nl/min flow rate using a linear gradient: 2% b over 4 min, 2-40% b over 64 min, 40-98% b over 2 min, with a = 0.1% aq. formic acid and b = 80% acn in 0.08% aq. formic acid. peptides eluting from the column were introduced into an orbitrap elite (thermo fisher scientific, massachusetts, usa) operating in top20 data dependent acquisition mode. a survey scan of 400-2000 m/z was performed in the orbitrap at 120000 resolution with an agc target of 1 × 106 and 200 ms injection time followed by twenty data-dependent ms2 scans performed in the ltq linear ion trap with 1 microscan, 10 ms injection time and 10,000 agc. the data from mass spectrometer were processed either using proteome discoverer 1.4 or proteome discoverer 2.2 that is employed with imbedded statistical tools (both programs were from thermo fisher scientific, massachusetts, usa). proteome discoverer 1.4 processed the data using mascot engine with the following search settings: database swiss-prot human (april 2016); enzyme trypsin; 2 missed cleavage sites; precursor mass tolerance 10 ppm; fragment mass tolerance 0.6 da; dynamic modifications: carbamidomethyl [c], oxidation [m], acetyl [protein n-terminus]. the results of the search were further submitted to generate the final report considering 1% fdr on both psm and peptide group levels. only unique peptides were used for the protein quantification. silac labels of r6 and k6 were chosen for heavy and r0 and k0 for light. the relative quantification value was represented as heavy/light ratio (supplementary table 1 ). in the processing and consensus workflows subsequent nodes were used: the minora feature detector, the precursor ion quantifier, and the feature mapper. the data were processed using sequest ht engine with the following search settings: database swiss-prot 2017-10-25, # sequences 42,252, taxonomy: homo sapiens (updated february 2018); enzyme trypsin; 2 missed cleavage sites; precursor mass tolerance 10 ppm; fragment mass tolerance 0.6 da; static modification carbamidomethyl [+57.021 da, (c)], label 13c(6) [+6.020 da (k, r)]; dynamic modifications oxidation [peptide terminus, +15.995 da (m)], met-loss +acetyl [protein terminus, −89.030 da (m)]. the results of the search were further submitted to generate the final report with a 1% fdr using percolator. for the protein quantification and statistical assessment of the biological triplicates, only unique peptides and razor peptides were used. the relative quantification value is represented as the relative peak area of the peptides with the heavy isotope labels with ifnγ treated cells/ifnγ untreated cells ratios (supplementary table 3b ). label free quantitation was performed using fasp-processed tryptic digests with liquid chromatography coupled to tandem mass spectrometry on an eksigent ekspert nanolc 400 (sciex, california, usa) online connected to a tripletof 5600+ (sciex, toronto, canada) mass spectrometer. cells lysates were processed in technical triplicates. prior to the separation the peptides were concentrated and desalted on a cartridge trap column (300 μm i.d. × 5 mm) packed with a c18 pepmap100 sorbent with a 5 μm particle size (thermo fisher scientific, waltham, ma, usa). after a 10 washing using 0.05% trifluoroacetic acid in 5% acetonitrile and 95% water, the peptides were eluted using a gradient of acetonitrile/water (300 nl/min) using a capillary emitter column picofrit® nanospray columns 75 μm × 210 mm (new objective, massachusetts, usa) self-packed with prontosil 120-3-c18 aq sorbent with 3 μm particles (bischoff, leonberg, germany). mobile phase a was composed from 0.1% (v/v) formic acid in water, and mobile phase b was composed of 0.1% (v/v) formic acid in acetonitrile. gradient elution started at 5% mobile phase b for the first 30 min and then the proportion of mobile phase b increased linearly up to 40%b for the following 120 min. output from the separation column was directly coupled to an ion source (nano-electrospray). nitrogen was used as a drying and nebulizing gas. temperature and flow of the drying gas was set to 150°c and 12 psi. voltage at the capillary was 2.65 kv. pooled sample for the spectral library was measured in data-dependent positive mode (ida). the ms precursor mass range was set from m/z 400 up to m/z 1250 and from m/z 200 up to m/z 1600 in ms/ ms. cycle time was 2.3 s and in each cycle 20 most intensive precursor ions were fragmented. subsequently, their corresponding ms/ms spectra were measured. precursor exclusion time was set to 12 s. precursor ions with intensity below 50cps were suspended from the ida experiment. the extraction of mass spectra from chromatograms, their annotation and deconvolution were performed using protein pilot 4.5 (sciex, toronto, canada). ms and ms/ms data were searched using the uniprot+swissprot database (02. 2016, 69,987 entries) restricted to homo sapiens taxonomy. fixed modificationalkylation on cysteine using iodoacetamide and digestion using trypsin was set for all searches. fdr analysis was performed by searching the shotgun data against the decoy search database. the resulting group file was imported into peakview 1.2.0.3 (sciex, toronto, canada), where only proteins with fdr below 1% were imported into the spectral library (465 proteins for sbp-ifitm1 pull down swath). in swath mode, the mass spectrometer operated in high sensitivity positive mode. precursor range was set from m/z 400 up to m/z 1200. it was divided into 67 precursor ion windows with the width of 12 da and 1 da overlap. accumulation time was 50 ms per swath window and the duty cycle was 3.0 s, which enabled acquisition of at least 10 data points across a chromatographic peak. product ions were scanned from m/z 400 up to m/z 1600. data extraction was performed in peakview 1.2.0.3 (sciex, toronto, canada) with the spectral library. the retention time window for extraction was manually set to 4 min for sbp-ifitm1 pull down swath and for the sirna swath ( fig. 9 ). protein quantification was performed using up to 4 peptides and 6 transitions per protein to define the statistically significant proteins. scope of peptides used for quantification was narrowed to only those with peptide confidence higher than 99% and without any variable modification. protein summed peak areas were normalized using total area sums option in markerview 1.2.1.1 (sciex, toronto, canada). samples were compared pairwise using paired t-test. the mass spectrometric data have been deposited to the proteomexchange consortium via the pride partner repository with the dataset identifier pxd007562". for reviewers, the details include; username: reviewer17106@ebi.ac.uk and password: oftomsm7. the parental siha and ifitm1/ifitm3 double null cells were grown in rpmi (supplemented with 10% fbs, pen-strep and pyruvate) to 50% confluence in 6-well plates and treated with 100 ng/ml ifnγ for 24 h. cells were harvested using accutase (sigma-aldrich, a6964), centrifuged 1000 rpm for 5 min with rpmi and kept on ice in 1% bsa in pbs for 20 min. primary antibodies (hla-b: pa5-35345) were diluted in 1% bsa/pbs to 1:100. cells were centrifuged as before, and cell pellets were resuspended in 100 μl of diluted primary antibodies or in the same amount of 1% bsa/pbs (for control samples) and incubated 45 min a room temperature on a tube rotator. after a triple wash in icecold 1% bsa/pbs, cells were incubated with 100 μl of secondary antibody (abcam, goat anti-rabbit igg h&l dylight 488), diluted 1:200, on a tube rotator for 30 min at room temperature. after a triple wash in ice-cold 1% bsa/pbs, cells were resuspended in 500 μl of 1% bsa/pbs and kept on ice before measurement. samples were measured on facsverse (bd biosciences) and data were analyzed using facsuite software (bd biosciences). a negative control without primary antibody was prepared for each sample. hla expression on the cell surface was counted as fitc median fluorescence intensity (mfi) divided by the mfi of the negative control. two independent experiments were performed, each with two independently isolated ifitm1/ifitm3 double null cells and two different hla antibodies (hla-a (data not shown) and hla-b (fig. 6) ). identifying a clinically relevant model to dissect ifitm1 and ifitm3 (ifitm1/3) signalling ifitm1 and ifitm3 (ifitm1/3) can function as oncogenic factors in several cancer cells [32, 33] . attenuation of ifitm1 protein expression can inhibit growth, invasion, and/or migration of cancer cells [34] . patient subgroups with clinically relevant expression data are not welldefined. we developed a panel of monoclonal antibodies to a n-terminal peptide with a high homology between ifitm1 and ifitm3 (supplementary fig. 1a , b). this would allow the development of monoclonal antibodies that detect the co-expression of both ifitm1 and ifitm3 proteins. we aimed to use such tools to screen a large panel of human cancers for those that express ifitm1/3 proteins. this would identify clinically relevant models for a focus to dissect ifitm1/3mediated signalling pathways. the monoclonal antibody chosen (named mab-mhk; supplementary fig. 1b) can bind to ifitm1/3 antigens in a range of human cancer cells ( supplementary fig. 1c ). some cancer cells exhibit no expression of ifitm1/3 such as the lymphoma cell line whu-nhl ( supplementary fig. 1c ). further studies confirmed that mab-mhk can bind to both ifitm1 and ifitm3 proteins, as defined using single ifitm1 single null and ifitm1/ifitm3 double null cells (see below). mab-mhk was used to screen large panels of archival formalin-fixed human cancer tissues to identify potential clinically relevant models. we could detect differential expression of ifitm1/3 in breast cancer, colon cancer, and oesophagus cancer (data not shown). we could also detect differential expression of ifitm1/3 protein in a cervical cancer array ( fig. 1a-f) . squamous cervical cancer samples expressed either high levels of ifitm1/3 (fig. 1a) , lower levels of ifitm1/3 (fig. 1b) , or undetectable levels of the antigens (fig. 1c ). cervical adenocarcinoma often exhibited high expression (fig. 1d) . interestingly, some normal squamous cervical epithelium exhibited high expression in the basal 'stem' cell or pluripotent layer only (fig. 1e ), but not the differentiating cell layers. we can conclude that 64 out of 74 cervical cancer specimens are positive for ifitm1/3 proteins using the mab-mhk (fig. 1f, top panel) . what is also interesting is that there is a statistically significant inverse association between ifitm1/3 protein expression and the number of lymph node metastases in patients (fig. 1f, bottom panel) . this will be rationalized in the discussion based on data that emerges below. developing an ifitm1 and ifitm3 double null cell line using a crispr-guide rna methodology ifitm1 is implicated in ifnγ mediated growth control in some cancer cells with an active p53 pathway [35] . ifitm1 is also implicated in a growth stimulatory role in cervical squamous cancers [36] [37] . the hpv16+ and ifitm1/3 positive cervical cancer cell line siha [38] [39] exhibit ifnγ inducible stat1, irf1, and ifitm1/3 proteins (fig. 1g ). as such, we focused on using this cervical cancer cell line (siha) as a model to identify ifitm1/3 dependent signalling pathways. in order to continue to develop a cervical cancer cell model that reflects the clinical data (ifitm1/3 positive or ifitm1/3 negative cancers; fig. 1f ), we set out to develop a double null ifitm1 and ifitm3 cell line model. we first generated an isogenic ifitm1 null cell panel through gene editing to validate the guide rna. ifitm1 knockout mice are viable [40] so it was likely we would be able to generate ifitm1 null cells. guide rnas targeting exon 1 in the ifitm1 gene ( figure 2a) were cloned into plentiv2. cells were transfected and selected for resistance to puromycin to allow stable integration of the ifitm1 targeting guide rna cassette. cell clones were chosen for sequencing across the guide rna targeting site ( fig. 2a) using pcr (fig. 2b ). both ifitm1 alleles were gene edited in a representative ifitm1-null clone that creates two distinct frameshifts ( fig. 2c and d) . we examined four representative ifitm1 null cells in dna damage response assays and all lines were shown to be either chemosensitive or x-irradiation sensitive (data not shown). since all single knock-out clones behaved similarly, we chose one representative ifitm1 null cell line for continued study. the ifitm3 gene was next targeted using guide rna methodologies (fig. 3a) to to create a ifitm1/ifitm3 double null cell ( fig. 3a-c) . ifitm3 null mice, and mice with the entire ifitm chromosomal locus deleted, are also viable [40, 41] . this created an isogenic cell model that removed any redundancy of ifitm3 in the ifitm1 interaction landscape, especially as they both are reported to interact with vapa [21] . immunoblotting confirmed that ifitm1 and ifitm3 proteins are not detected in the ifitm1/ifitm3 double null cell, respectively ( fig. 3d ; lanes 5 and 6). we chose a representative doublenull cell line for subsequent studies. given that one established effector of ifitm1 is ifnγ [35] , we evaluated ifnγ responsive protein synthesis in ifnγ stimulated parental siha, ifitm1 single null, and ifitm1/ifitm3 double null cells. the parental siha, ifitm1 single null, and ifitm1/ifitm3 double null cells were treated with heavy isotopic amino acid labeling media (the silac method) for 6 or 24 h, in the absence or presence of ifnγ (fig. 4) . cell lysates were then processed by fasp [30, 31] and then analyzed for ifitm1/3-dependent protein synthesis using mass spectrometry. the silac methodology has been subjected to an analysis of random error associated with the multiple steps in this approach including; cell plating in biological replicates, switch to heavy isotopic media, cell recovery from plastic plates, cell lysis, centrifugation, filter assisted trypsinization, and tryptic peptide recovery and processing. this error can be reduced by employing multiple replicates (n = 3) as highlighted previously [42] . to highlight the importance of biological replicates and the inherent error in this multiple step process, we plot the data not as an average of three replicates, but as individual points from all three replicates (as in supplementary fig. 2 and fig. 5) . the dominant ifnγ responsive protein to be identified at 6-h post labelling is irf1 protein (supplementary fig. 2b vs 2a) with an attenuation of isotopically labeled irf1 peptides recovered in the biological triplicates from ifitm1/ifitm3 double null cells ( supplementary fig. 2h ). this suggests that irf1 is partially dependent upon ifitm1/3 signalling and this was confirmed using irf1 transcriptional reporter assays (data not shown). this data provides some degree of confidence that the methodology is able to identify a known ifnγ responsive target (irf1). there are other proteins whose synthesis was detected at 6 hours post-isotopic labelling including stat1, eif1, and b2m ( supplementary fig. 2i-k) . eif1 is not known to be linked to interferon signalling, but it is known to regulate the accuracy of aug codon selection by the scanning pre-initiation complexes [43] . eif1 might prove to be involved in regulating interferon dependent anti-viral mrna selection. nevertheless, all three proteins are also iftm1/3-independent ( supplementary fig. 2i-k) . stat1 and b2m are also both known ifnγ responsive proteins further validating the methodology used to measure quantitative changes in protein synthesis. that all three proteins (stat1, eif1, and b2m) exhibit equivalent protein synthesis rates in the parental and double-null cell model indicates that the double null has retained many key regulatory features of the parental cell. this suggests that many ifn regulatory features of the double null cell have been retained despite the selection process creating the cell model. by 24-h post ifnγ treatment of siha cells, hla family members, b2m, and stat1 proteins were detected (fig. 5b vs 5a) , again indicating that the methodology can detected known ifnγ inducible proteins. by 24-h post ifnγ treatment, isotopically labeled irf1 fig. 1 . immunohistochemical analysis of ifitm1/3 protein expression in cervical cancers using the mab-mhk. formalinfixed, paraffin-embedded cervical carcinoma tissue was processed as stated in the experimental procedures using the mab-mhk that binds to shared epitopes in the n-terminal domains of ifitm1 and ifitm3 ( supplementary fig. 1a, b) . peptides are attenuated (fig. 5h) , which is consistent with the early and transient induction of irf1 by ifnγ. the synthesis of mhc class i molecules was ifitm1/3-dependent (fig. 5b vs 5f ; quantified in biological triplicates in fig. 5l ). all three, major hla alleles exhibited attenuated synthesis in the double null cell, as defined using the tryptic peptide coverage (supplementary fig. 3 ). isotopically labeled isg15 tryptic peptides are also not observed in the early interferon response ( supplementary fig. 2) , and the isotopically labeled isg15 peptide recovery after 24 h is attenuated in the ifitm1/ifitm3 double null cells (quantified in biological triplicates in fig. 5g ) suggesting that isg15 protein synthesis is largely ifitm1/3 dependent. by contrast, stat1 protein synthesis at 24 h appears largely ifitm1/3-independent (quantified in biological triplicates in fig. 5i ). providing another internal control, another well-known inducible ifnγ protein, b2m, exhibits equivalent synthesis in the ifitm1/ifitm3 double null cell (quantified in biological triplicates in fig. 5k ). this indicates that one key regulatory feature of the double null cell, stat1 production, has remained intact. these data first demonstrate that using the pulse-silac methodology, the siha cell model reflects the classic ifnγ responsive induction of stat1, irf1, b2m, isg15, and mhc class i molecules (fig. 5b vs 5a and supplementary fig. 2) . also of note is attenuation of hla-a, hla-b, hla-c, and isg15 protein synthesis 24 h post-ifnγ treatment in the ifitm1/ifitm3 double null cells compared to parental siha (fig. 5f vs 5b). in order to determine if ifitm1 deletion alone impacted on this set of gene products, the parental siha and ifitm1-null cells were in parallel treated with silac heavy-labeling media for 6 or 24 h. as with the double ifitm1/ifitm3 double null cells, ifnγ dependent induction of irf1 protein synthesis is attenuated 6 hours post labelling in the ifitm1-single null cells ( supplementary fig. 2d vs 2b ). this suggests that irf1 is dependent upon ifitm1. elevation of stat1 protein synthesis are ifitm1-independent based on the equivalent induction of stat1 in the ifitm1-single null cells (fig. 5i) . hla-b protein synthesis is attenuated 24 hours post-ifnγ treatment in the ifitm1 single null cells (fig. 5l) . isg15 synthesis is also attenuated in the ifitm1 single null cell (fig. 5g) . together, these data suggest that mhc class i family members and isg15 require at least ifitm1 for maximal ifnγ stimulated protein synthesis. the software used to identify hla orthologues in the pulse-silac screen (fig. 4) can discriminate between hla-a, hla-b, and hla-c based on tryptic peptide sequences (supplementary fig. 3) . we focus here on hla-b, which shows accurate identification of hla-b specific peptides and it is also a member of the irds pathway [7] . we thus validated hla-b protein expression in orthogonal assays to determine whether apparent reductions in hla-b protein synthesis reduction in the ifitm1/ifitm3 double null cells was reflected in total steady state protein levels and subcellular localization on the plasma membrane. first, immunofluorescence of hla-b was defined in parental siha and ifitm1/ifitm3 double null cells. parental siha cells revealed significant induction of hla-b immunoreactivity 24 h after ifnγ treatment (fig. 6c vs 6b and quantified in 6g) . by contrast, basal hla-b protein expression was attenuated in the ifitm1/ifitm3 double null cells after ifnγ treatment (fig. 6f vs 6e) . quantitation of the total immunofluorescence in the absence and presence of ifnγ, in the parental siha and ifitm1/ifitm3 double null cells, also confirms attenuated hla-b induction in the null cell panel (fig. 6h vs 6g ). this is consistent with the reduced protein synthesis observed for hla-b in the pulse silac quantitation in the ifitm1/ifitm3 double null cells. the dominant subcellular localization of hla-b is thought to reside on the cell surface as an antigen presentation carrier. we therefore evaluated whether hla-b expression on the plasma membrane was altered in the ifitm1/ifitm3 double null cell using facs analysis with non-permeabilized cells. two independent ifitm1/ifitm3 double null cell clones were used as a form of biological replicate in comparison to the parental siha cell line. twenty-four hours post treatment, hla-b was elevated on the plasma membrane in the parental siha cell line (data not shown). quantitation revealed reduced levels of hla-b in both independent ifitm1/ifitm3 double null biological replicates in the absence and presence of ifnγ (fig. 6i) . these data indicate that the fig. 4 . methodological approach to identify signalling pathways altered in ifitm1 and ifitm3 double null cells. the indicated cells (parental siha, ifitm1 single null, or ifitm1/ifitm3 double null) were pre-treated with carrier or ifnγ for 24 h. the media was replaced with r6k6 isotopically labeled media with carrier or ifnγ for 6 or 24 h. cells were harvested, lysed, and tryptic peptides processed for analysis by mass spectrometry (ms) as indicated in the experimental procedures. (caption on next page) reduced synthesis of hla-b in the ifitm1/ifitm3 double null cell (fig. 5) has an impact on its subcellular localization at the plasma membrane. we next also examine whether there is any direct protein-protein interaction between ifitm1 and hla-b since they are both co-synthesized, have transmembrane localizations on the cell surface, and are both irds components. in vivo proximity ligation assays are emerging methodologies that have been shown to demonstrate the "association" of two endogenously expressed proteins in fixed cells without the need for harsh lysis [44] . the method can be considered as an in situ mimic of an "immunoprecipitation assay". proximity ligation assays can identify a protein-protein interaction/association with a distance of 10-30 nm that is in the upper range of that observed using fret (5-20 nm) and this methodology can detect authentic endogenous proteins in situ that does not rely on transfected or artificially gfptagged protein vectors [45] . we evaluated whether ifitm1/3 and hla-b co-associate using this methodology using antibodies to hla-b and mab-mhk (that can bind to both ifitm1 and ifitm3 proteins; supplementary fig. 1) . a significant protein-protein interaction was observed in the ifnγ treated cells (representative images; fig. 7a-d) . these foci were absent in the ifitm1/ifitm3 double null cells (fig. 7e-h) . together, these data validate the pulse-silac data that identified hla-b as a downstream effector of ifitm1/3. isg15 was not easily visualized using immunofluorescence in situ table 1 ). cells were incubated with ifn-γ for 24 h in b, d and f. data were plotted as a function of log10 fold change of heavy/light peptide intensities. triplicates were represented in the x, y, and z-axis. in samples (g-l), representative peptides used for quantification in biological triplicates are highlighted to demonstrate a protein that is induced independent of ifitm1/ifitm3 (stat1, i and eif1, j) and proteins that are ifitm1/3 dependent (isg15; g, hla-b, l). (data not shown) nor could we identify a protein-protein association between ifitm1/3 proteins and isg15 using proximity ligations (data not shown). we thus used an independent assay for orthogonal validation of isg15 induction. we developed a sbp-tagged ifitm1 expression construct in order to ectopically express the protein in the parental siha cells and design methodologies for capturing ifitm1 associated proteins. the transfection of the sbp-ifitm1 expression could be detected as migrating at a higher mass (due to the sbp tag) than endogenous ifitm1/3 proteins in the parental siha cells (fig. 8a , lane 3 vs lane 1) and specifically captured after affinity purification following expression in the ifitm1/ifitm3 double null cell (fig. 8a table 2 ). the data first highlight the detection of peptides belonging to a homologous sequence for ifitm1/2/3 proteins (fig. 8b, fig. 8c ). since ifitm1 was our bait protein we presume that it was the detected isoform, serving as an internal positive control (fig. 8a) . nevertheless, ifitm1 may also be interacting with ifitm2 or ifitm3. the previously identified ifitm1/2/3 interacting protein vapa was also detected (fig. 8b and c) , supplementary table 2. the proteins with the most significant p-values were enriched in ifn-γ treated cells, relative to non-treated cells ( fig. 8b and c, supplementary table 2 ). these included several proteins involved in cell-cell or cell matrix interactions including cornifin, galectin-7, desmocolin, jup, hornerin, and desmoglein ( fig. 8c; supplementary table 3 ). this is suggestive of a pathway interaction of ifitm1 with membrane-dependent cell-cell communications. the enrichment of higher confident targets in ifn-γ treated cells also suggests that ifn-γ treatment may be required to fully 'activate' the ifitm1 protein interaction landscape. interestingly, proteins related to ifnγ signalling were also detected; isg15 and hla-b ( fig. 8b and c) . immunoblotting also confirmed that isg15ylation was for quantitation, three independent assays were performed, and each assay had two independent biological replicates. for each assay, fluorescence was measured in at least 50 cells per condition. fluorescence was measured using imagej software. statistical study was performed with 1-way anova and bonferroni correction (p-value < .0001). (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) attenuated in ifitm1/ifitm3 double null cells after ifnγ stimulation (fig. 8d) . by contrast free isg15 protein remained equivalent in both cells (fig. 8d) , suggesting that the isg15 synthesis detected using pulse silac is directly linked to ifn stimulated protein conjugation. the ratio changes of conjugated to free isg15 is summarized in fig. 8e . together, the orthogonal validation of hla-b and isg15 is consistent with the pulse silac data; ifitm1/3 proteins are required for maximal induction of hla-b and isg15ylation in ifnγ treated cells. we were unable to complement the ifitm1 and ifitm3 gene back into the double-null siha cells to stimulate isg15ylation and hla-b protein levels in ifn-γ treated cells (data not shown). as such, we took an independent approach to define dominating ifitm1-dependent signalling pathways and whether these overlap with the signalling proteins identified using the pulse-silac experimental methods. although the majority of analyses identified hla-b and isg15 protein expression changes using ifitm1/ifitm3 double null cells, we did create an ifitm1 single null cell that revealed similar reductions in isg15 and hla-b after ifnγ treatment (fig. 5d, g, and l) . as such, we examined ifitm1 dependence in the steady-state proteome levels using ifitm1 targeted sirna treatment of parental siha cells (fig. 9a, lanes 4 and 2 vs 3 and 1) to determine if attenuation of ifitm1 by this method gave rise to similar proteome changes. however, there is a caveat to this method. similar to plasmid transfection [46] , double stranded rna can also induce an irf-1 dependent transcriptional interferon response [47] . accordingly, we have also found that sirna transfection methodologies can induce an irf1 transcriptional response (data not shown). nevertheless, sirna is a powerful tool to determine gene dependencies in cell models. when parental siha cells, treated from 48 and 72 h with control sirna or ifitm1-targeted sirna, there was table with selected binding proteins detected by mass spectrometry for sbp-ifitm1 enrichment without or with ifn-γ stimulation (from supplementary table 3 ). (d). cells (parental siha; lane 1; or ifitm1/ifitm3 double null (lane 2) were transfected with empty vector (as in 8a) and treated with ifnγ. samples were processed by immunoblotting with antibodies to isg15. free, monomeric isg15 protein is highlighted, as well as conjugated isg15. free isg15 protein is expressed at similar levels in both cells whilst conjugated isg15 is attenuated in the ifitm1/ifitm3 double null cell (lane 2 vs 1). this suggests that isg15ylation protein synthesis is linked to covalent conjugation under these conditions, under conditions where attenuated isg15 protein synthesis was observed using pulse silac (fig. 5 ). antibodies to β-actin and ifitm1/3 proteins were used as loading controls, as indicated. (e). free and conjugated isg15 were quantified using imagej software. the relative units (in a.u.) define expression as a function of free or conjugated isg15 in parental and ifitm1/ifitm3 double null cells. the relative change in isg15 conjugation over free isg15 was 5.58 a.u. in parental cells. the relative change in isg15 conjugation over free isg15 was 3.63 a.u. in ifitm1/3 double null cells. striking and selective reduction in interferon-responsive proteins using label free quantitative mass spectrometry at both time points (fig. 9b and supplementary table 3 ). some of these proteins, including ifitm1 itself, are also components of the interferon-stimulated dna damage resistant signature of proteins [9, 10] . interestingly, isg15 and hla-b are also lowered after the treatment of siha cells with ifitm1 targeted sirna. however, there is one notable difference in the pulse-silac (fig. 5 ) vs the sirna methodology (fig. 9b) ; stat1 and b2m are ifitm1/3-independent as defined using pulse silac (fig. 5) ; whilst stat1 and b2m are ifitm1-dependent using the sirna methodology fig. 9 . the impact of ifitm1 targeted sirna on the steady-state proteome. (a). immunoblotting to demonstrate that ifitm1-targeted sirna can attenuate ifitm1/3 protein levels. cells were treated with the indicated control or targeted sirna for two time points to capture the overlap in the transient dynamics of the interferon signalling cascade. lysates were immunoblotted with the mab-mhk antibody (the mhk monoclonal antibody cross reacts to a common n-terminal epitope in ifitm1 and ifitm3, see supplementary fig. 1 ) to quantify ifitm1/3 protein and the loading control (gapdh), as highlighted. (b). evaluation of the total steady-state proteome in response to sirna targeting of ifitm1 in siha cells using swath-ms (data from supplementary table 3 ). the impact of ifitm1-targeted sirna treatment for 48 and 72 h. these time points were a point of focus since the sirna treatment activates the irf1 transcriptional response over these two-time frames (data not shown). as such, the screen is conducted under experimental conditions in which we consider that the irf1 response is activated by rna treatment. the data from these two biological states are plotted as log2 fold change in protein levels (using swath-ms) as a function of either the 48 or 72-hour time point. the key proteins whose steady-state levels were suppressed after ifitm1-targeted sirna treatment are highlighted in red, in the lower left quadrant. (c) the ifitm1 signalling model. pulse labelling using silac methodologies identified stat1 as a dominant protein synthesized after ifnγ treatment. this forms an internal positive control and is consistent with the classic jak-stat response to ifnγ treatment. (i). stat1 can produce mrnas that are translated in response to ifnγ treatment including irf1 and other interferon effectors such as b2m. (ii). by contrast to stat1 and b2m, some of the ifnγ stimulated factors are ifitm1 dependent including mhc class i molecules and isg15 (fig. 5g and l) . the sirna-mediated depletion of ifitm1 represents an orthogonal assay that identified reductions in isg15 and mhc class i molecules (fig. 9a) . stat1 protein reflects a distinct mechanism of control by ifitm1/3. although pulse silac revealed that stat1 synthesis is ifitm1/3-independent (fig. 5) , the steady state levels of stat1 protein are reduced after targeted depletion of ifitm1 in siha cells (fig. 9a) . these data suggest that turnover of stat1 protein might be dependent on ifitm1/3, but its synthesis is independent of ifitm1/3. however, these methodologies are complicated to compare directly, since the sirna methodology uses an intrinsic rna signal (double stranded rna) that stimulates irf1 but without exogenously added ifnγ, whilst the pulse silac used ifnγ without rna ligands. altogether, these data place ifitm1/3 proteins as a coordinator of the synthesis and/or steady state levels of a subset of key players in the ifnγ response. the notable induction of mhc class i molecules and isg15 in an ifitm1/3 dependent manner identifies a coordinated signalling pathway with potential clinical relevance (fig. 1) . the recent observation that lowered hla-a, hla-b, and hla-c alleles correlates with poor prognosis and enhanced metastatic growth in cervical cancers [54] is further consistent with the existence of an ifitm1/3:hla signalling pathway regulating cervical cancer outcomes. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) (fig. 9b, supplementary table 3 ). this might reflect that fact that the rna ligand (sirna) induces additional rna-activated pathways in the targeted sirna proteome screen than ifnγ alone. in conclusion, the pulse silac methodology in the ifitm1 single null (fig. 5 ) and the sirna treatment using ifitm1 targeted sirna in parental siha cells (fig. 9 ) both gave rise to overlapping proteome changes; mainly isg15 and hla-b. these data suggest that ifitm1 might alone function as an effector to these two proteins. however, we cannot rule out a role for ifitm3 in the ifitm1 single null cell, since ifitm3 is also reduced by ifitm1 targeted sirna treatment of siha cells (fig. 9b) . thus, ifitm1 and ifitm3 might cooperate to mediate these effects. the ifitm protein family was identified in whole genome sirna screens as rna virus restriction factors [20] . the molecular functions of the ifitm family in the anti-viral response are just beginning to be defined. yeast-two hybrid, with ifitm3 as a bait, was used as a methodology to discover new protein-protein interactions within the ifitm family that mediate viral restriction [21] ; a protein-protein interaction was identified for ifitm3 and the vesicle-membrane-protein-associated protein a (vapa). the vapa interaction occurred with ifitm1, ifitm2, or ifitm3 and these three were not distinguished from each other in this assay. nevertheless, the study focused on defining how vapa interaction with ifitm3 results in reduction of vapa binding to oxysterol-binding protein (osbp), disrupting cholesterol homeostasis, and preventing viral maturation. as ifitm1 and ifitm2 are also reported to bind to vapa, it is not yet clear whether they play minor and/ or redundant roles in controlling cholesterol-mediated viral maturation [21] . interestingly, vapa was also identified in our swath-ip using sbp-tagged ifitm1 (fig. 8) , which is consistent with vapa being a dominant interactor for the ifitm family. in addition to vapa, palmitoylation of ifitm1/2/3 family members has been reported to be required for some anti-viral functions [48] . whether the anti-viral associations with vapa or palmitoylation by the ifitm family impact on cancer growth control is not yet known. the ifitm family also have reported roles in oncogenic and/or prometastatic cancer cell growth. it is not known if the anti-viral and prooncogenic functions of the ifitm family overlap. within the ifitm1 family, iftm1 is the most well documented to have pro-metastatic roles and it is the only ifitm family member with a partial residence in the plasma membrane [19] . ifitm1 is also the only ifitm family member that is a component of the interferon-regulated dna damage resistance (irds) pathway [10] . the irds contains a subset of approximately 30 interferon responsive genes that are up-regulated late in the viral response, upregulated during development of radiation resistance, and upregulated as a result of chronic exposure to lower levels of type i or type ii interferons. irds pathway expression is mediated by 'unphosphorylated' stat1 [49] and can be stimulated by irf9 [50] . interestingly, two irds genes (isg15 and hla-b) are induced after ifnγ exposure in a 'ifitm1-dependent' manner (fig. 9c ). our unpublished data also indicate that ifitm1 single null cells are x-ray sensitive or cisplatin-sensitive, consistent with the hypothesis that ifitm1 expression is linked to chemo or irradiation resistance. the mechanisms whereby ifitm1 itself regulates such chemoresistant or pro-metastatic cell signalling events have not been mechanistically defined. in this report, we begin to set up biochemical assays that could shed light on dominant cancer-associated functions of ifitm1. we first aimed to identify a clinically relevant human cancer model to study ifitm family of proteins. this required us to develop a pan-specific antibody that binds to both ifitm1 and ifitm3 proteins ( supplementary fig. 1) . this resulted in a focus on cervical cancer (fig. 1 ) that exhibited high, medium or no expression of ifitm1/3 proteins (fig. 1f) . of particular interest was the inverse correlation between ifitm1/3 protein expression and lymph node metastasis ( fig. 1f) suggesting that loss of ifitm1/3 protein expression correlates with evasion of the immune system. if ifitm1/3 are 'pro-oncogenic', why would cervical cancer panels reveal an inverse correlation between ifitm1/3 expression and cancer-positive lymph nodes in patients? it is now becoming apparent that there are two distinct modes of metastatic cell growth. the first is the more classically defined metastasis due to enhanced 'invasion and migration" to secondary tissue niches. the second represents metastasis due to cancer cell escape from immune surveillance [51] . ifitm1/3-positive cancers might indeed be pro-invasive, depending on the microenvironment and tissue type, leading to poor clinical prognosis. the methodologies that highlighted such classic pro-metastatic roles for ifitm1 include over-expression by ectopic transfection of plasmid encoded genes or attenuation of gene expression using targeted sirna [14] . by such experimental approaches, ifitm1 does indeed promote cancer cell growth and/or 'cell invasion' (i.e. a model of metastatic growth) [16] . consistent with this, using clinical material ifitm1 protein has often been shown in literature to be over-produced in cancers using immunohistochemistry and this often correlates with poor prognosis [12, 52] . however, our data reviewed using cervical cancer (fig. 1) indicate that there can be two distinct states of cervical cancer with respect to ifitm1/3 expression. ifitm1/3 negative cervical cancers might also be more pro-invasive due to immune escape. this is based on the data indicating that the most dominant proteins whose synthesis depends on ifitm1/3 using the pulse silac methodology are in fact hla family members (figs. 4 and 5). hla family members are components of the irds, they play a role in anti-viral immunity through the presentation of viral peptides through the mhc class i system, and hla expression is linked to immune rejection of cancer cells [53] . this suggests that although ifitm1/3 might be pro-oncogenic under some conditions, ifitm1/3-hla signalling would presumably function as a 'tumor suppressor' signal via engagement of cd8+ t-cells. such ifitm1/3 and hla positive cancers might not metastasize because they produce neoantigen presenting mhc class i molecules that keep the primary tumor in a local chronic state of equilibrium with the immune system. ifitm1/3-negative cancers by contrast might be expected to produce lower amounts of mhc class i molecules following ifnγ stimulation ( fig. 9 ) resulting in lowered neoantigen expression, immune escape, and metastasis. such hypotheses are consistent with two clinical observations in cervical cancers. first, an inverse correlation exists between ifitm1/3 expression and lymph-node positive cancers (fig. 1) . second, a recent study has highlighted that lowered mhc class i expression also predicts poor prognosis in cervical cancers [54] . this data is also consistent with several studies that highlight elevated rates of metastatic cancer cell growth in vivo are inversely correlated to mhc class i expression including deletion of the mhc class i locus [53] . although expression of ifitm1/3 was detected in cancer cells (fig. 1) , it is interesting that ifitm1/3 protein expression was observed and confined to the basal 'stem cell' layer in representative normal tissue controls using immunohistochemistry (fig. 1 e) . expression was not observed even on one cell layer up from the basal layer. in squamous human skin, this basal layer is reflective of p63 (a squamous stem cell transcription factor) and phosphorylated p53 positive stem cell populations after uv irradiation [55] . thus, this expression might reflect a role for ifitm1 in squamous stem cell pluripotency. we do not have very many samples reflecting a 'normal' human cervical epithelium, but the few cases we have exhibit very strong staining in the basal layer (data not shown). during the course of these studies the human protein atlas has also populated their immunohistochemical library with expression patterns of ifitm1 protein in normal tissues. we can see in this library that ifitm1 protein is also confined to the "basal stem cell" layer in normal squamous oesophagus, cervix, and oral mucosa ( supplementary fig. 4) . together, these data would suggest that ifitm1 might play a role in squamous stem cell physiology as its expression appears specifically confined to the basal layer and is not expressed in suprabasal normal squamous cells. as the 'normal' squamous cervical epithelium we have used is from patients undergoing screening for cervical cancer or dysplasia, we cannot rule out a role for hpv in this expression in normal cervical cells. however, as the oesophagus squamous epithelium also exhibits ifitm1 protein expression in the basal cells of normal squamous epithelium (protein atlas, supplementary fig. 4) , and the normal oesophagus is not noted for hpv infection, we would suggest that the expression of ifitm1 protein in basal cells is not related to hpv status. the signalling pathways that might trigger basal ifitm1 protein expression in squamous stem cell populations are not precisely defined. however, at the mrna level, differential expression patterns of ifitm1 gene expression were identified in the uteri of mice and there were correlations between the patterns of ifitm1 gene expression and wnt/β-catenin expression [56] . these data might suggest a role for wnt signalling as an upstream regulator of ifitm1 in stem cells. our focus on cervical cancer as a model to understand the cancerassociated role of ifitm1/3 is interesting considering ifitm1/ifitm3 are themselves rna viral restriction factors and hpv infection is a risk factor in cervical cancer progression. there is evidence that ifitm1 and ifitm3 might be a positive cofactor for a dna virus; hpv16 viral propagation [57] . we do not have any data that defines the hpv status of the cervical cancers we have analyzed (fig. 1) , as the main clinically approved assay for diagnostics is p16 positive cancer cells [58] . there is a close relation between persistent viral infection, development of cancer and failure in immune response. as an example, cervical as well as vulvar intraepithelial neoplasia are pre-cancerous conditions characterized by sustained hpv-16 infection. a clinical study shows favorable prognosis related to an increase in ifnγ-producing cd8+ cytotoxic t following to robust cell response induced by vaccination [59] . vaccination delivers a high dose of specific antigen against hpv-16 oncoproteins e6 and e7 and mediates mhc-binding peptide complex presentation [60] . a similar outcome was also observed after vaccination of a preclinical mouse model of hpv positive cervical cancer [61] . there is a significant correlation between mhc class i (not found for mhc class ii) expression on malignant cells and t-cell infiltration (til) in human ovarian cancer [62] , being a positive prognostic factor. thus, ifitm1 and ifitm3 might have dual roles in stimulating hpv propagation, but also in suppressing cancer escape from immune surveillance. one of the key approaches we used to evaluate the role of ifitm1 in ifnγ dependent protein production was the utilization of crispr-cas9 guide rnas to create isogenic knock-out cells. at the outset, we relied less on the use of sirna to deplete ifitm1 since this would only give a transient reduction in a target protein but also sirna itself can induce an interferon response (data not shown; [46] ). this would have complicated our analysis of the interferon-responsive nature of any ifitm1 protein interactions we visualize and measure. nevertheless, sirna was used as a final orthogonal approach to define ifitm1 signalling events (fig. 9a, b) . by contrast, the limitation of using gene knockout tools to reduce expression of a protein requires that loss of the protein is not a lethal event. in the case of ifitm1 and ifitm3 knock-out mice are reported as viable [41] , thus it was not unexpected that we were able to generate single ifitm1 null and double ifitm1/ifitm3 double null cell panels (figs. 2 and 3) . however, we were unable to generate single ifitm3 null cells under experiments carried out in parallel to those reported in this manuscript (data not shown). using these isogenic cell panels, we were able to determine whether there were defects in ifnγ dependent protein synthesis using pulse silac methodologies. we chose to use a pulse-silac approach to identify the isotopically labeled tryptic peptides with the most significant fold change after ifnγ treatment and which are altered in the double ifitm1/ifitm3 or ifitm1 single null cells. the most significantly suppressed proteins in the double ifitm1/ifitm3 or ifitm1 single null cells after ifnγ treatment were mhc class i orthologues encoded by the hla-a, hla-b, and hla-c genes (fig. 5) . as rationalized above, the data suggested an inverse correlation between ifitm1/ 3 protein and hla expression with metastatic growth in cervical cancer. the interferon-responsive protein isg15 was also attenuated in the double ifitm1/ifitm3 null cells or single ifitm1 single null cells after ifnγ treatment (fig. 5g) . the fact that isg15 and hla-b are enriched in the sbp-ifitm1 protein affinity purification after ifnγ treatment (fig. 8) suggests a co-operative activity exists between the two proteins. indeed, as hla-b can interact in situ with ifitm1/3 after ifnγ treatment (fig. 7) , and as isg15 is a high-confident ifitm1-associated protein using swath-ip mass spectrometry (fig. 8) , these data suggest that the two proteins are directly involved in the ifitm1/3 dependent ifnγ response. isg15 is also a component of an interferon and immune responsive gene cluster that are suppressed by stem cell pluripotent gene product expression [63] , suggesting that, like hla suppression, isg15 suppression might be co-incident with immune escape. in the case of ifitm1/3 signalling, we do not see defects in 'free' monomeric isg15 in the double ifitm1/ifitm3 null cells, but reductions in the conjugation of higher molecular mass isg15ylated adducts (fig. 8d) . these data suggest that conjugation of proteins to isg15 during interferon stimulation might play a coordinated role in the ifitm1/3 dependent immune-tumor cell interactions. overproduction of isg15 has been reported previously to stabilize ifitm3 [64] , consistent with our data that isg15 is detected in the sbp-ifitm1 complex (fig. 8) . ubiquitination of ifitm3 might counteract the stimulatory effect of isg15 on the anti-viral functions of the protein [65] . how ubiquitination and isg15ylation regulate ifitm1 and/or ifitm3 in a coordinated fashion is not defined. these data together provide a novel biochemical pathway relevant for cancer associated functions of ifitm1/3 that correlates with the interferon-responsive nature of ifitm1/3 signalling; they can mediate ifnγ dependent protein production of mhc class i proteins and isg15 (fig. 5) , whilst the maintenance of stat1 protein in response to ifnγ involves by a different signalling mechanism that is ifitm1/3-independent (fig. 9b ). both antigen presentation and isg15ylation signalling events are important for anti-viral signalling as well as immune regulation of cancer cells at the immune-cancer synapse [66] [67] [68] [69] [70] [71] . further research will shed light on how reductions in hla and isg15ylation can impact on both oncogenic signalling and/or anti-viral activity in response to ifitm1/3 expression. supplementary data to this article can be found online at https:// doi.org/10.1016/j.cellsig.2019.03.024. interferon signalling network in innate defence mechanisms of type-i-and type-ii-interferon-mediated signalling 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present internal influenza epitopes regulation of the e3 ubiquitin ligase activity of mdm2 by an n-terminal pseudosubstrate motif key: cord-304635-z5vmhopa authors: ji, wei; niu, ling; peng, weiyu; zhang, yongli; cheng, hao; gao, feng; shi, yi; qi, jianxun; gao, george f.; liu, william j. title: salt bridge-forming residues positioned over viral peptides presented by mhc class i impacts t-cell recognition in a binding-dependent manner date: 2019-06-18 journal: mol immunol doi: 10.1016/j.molimm.2019.06.005 sha: doc_id: 304635 cord_uid: z5vmhopa the viral peptides presentation by major histocompatibility complex class i (mhc i) molecules play a pivotal role in t-cell recognition and the subsequent virus clearance. this process is delicately adjusted by the variant residues of mhc i, especially the residues in the peptide binding groove (pbg). in a series of mhc i molecules, a salt bridge is formed above the n-terminus of the peptides. however, the potential impact of the salt bridge on peptide binding and t-cell receptor (tcr) recognition of mhc i, as well as the corresponding molecular basis, are still largely unknown. herein, we determined the structures of hla-b*4001 and h-2k(d) in which two different types of salt bridges (arg62-glu163 or arg66-glu163) across the pbg were observed. although the two salt bridges led to different conformation shifts of both the mhc i α helix and the peptides, binding of the peptides with the salt bridge residues was relatively conserved. furthermore, through a series of in vitro and in vivo investigations, we found that mhc i mutations that disrupt the salt bridge alleviate peptide binding and can weaken the tcr recognition of mhc i-peptide complexes. our study may provide key references for understanding mhc i-restricted peptide recognition by t-cells. the presentation of viral peptides to host t lymphocytes is crucial for adaptive cellular immunity to clear viruses during the infection. in the structures of mhc i presenting these short peptides, the α1 and α2 domains of mhc i form a bed-like groove to comfortably accommodate the peptides: two α-helices as the bedrails and the β-sheets beneath as the mattress (bjorkman et al., 1987; niu et al., 2013) . the short peptides interact with the amino acids within the peptide binding groove (pbg) through different binding modes. first, the featured residues at the second position (p2) from the n-terminus and the last residue (pω) at the c-terminus of the peptides, termed primary anchor residues, protrude into the specific pockets in the pbg of the mhc i. though the positions of the primary anchors on the peptides are relatively conserved, the binding modes of the anchoring residues of the peptides to different mhc i molecules are diversified due to polymorphisms in the pocket-forming residues. second, the peptides also interact with the pbg via contacts between the backbone atoms of the peptides and the pbg residues (mitaksov and fremont, 2006) . third, a hydrogen bond network is formed between the two termini of the peptides and the conserved residues in the pbg. the latter two interaction modes are relatively conserved between different mhc i molecules (fremont et al., 1992; madden et al., 1993) . generally, mhc i binding peptides are located in the bottom of a ringent pbg, which leaves a solvent-exposed top surface of the entire peptide from the n-to the c-terminus, ready for the recognition of the t-cell receptors (tcr) (bjorkman et al., 1987) . however, based on the structures of a series of mhc i molecules, such as human hla-b*2705 (madden et al., 1991) , rhesus macaque mamu-a*02 , and mouse h-2k d (mitaksov and fremont, 2006; zhou et al., 2004) , there is a salt bridge positioned over the peptides formed by opposite charged residues from the α1 and α2 helices of mhc i, respectively. in humans and macaques, the salt bridge is formed by the positively charged arg62 on the α1 helix and the negatively charged glu163 on the α2 helix. meanwhile, in mice, the involved residues are arg66 on α1 helix and glu163 on α2 helix. both of these types of salt bridges overhang the n-terminal portion of the t-cell epitopes in the groove. obviously, the salt bridge forms a ligature to tightly fix the peptides into the pbg. however, thus far, it is still largely unknown how the formation of these salt bridges in mhc i molecules impacts peptide binding. further, considering that these salt bridges are also solvent-exposed, they are in the position to be recognized by tcrs. generally, t-cell activation by the mhc-peptide complex may occur in three correlated ways: 1) direct interaction of the tcr with the mhc, termed mhc restriction; 2) direct interaction of the tcr with the solvent-accessible peptide main chain and side chains; and 3) interaction of the peptide with the tcr mediated by conformational perturbations in the mhcpeptide complex (baker et al., 2012; gras et al., 2012) . crystallographic studies clearly demonstrate that the polymorphic amino acids in the grooves of mhc i molecules from different mammals may influence the peptide conformation, subsequently impacting t-cell recognition (borbulevych et al., 2009; hulsmeyer et al., 2004; insaidoo et al., 2011) . nevertheless, to our knowledge, it has not been determined if the salt bridges in some mhc i molecules impact tcr recognition through a direct interaction with the tcr or indirectly through modulation of the peptide conformation. herein, by determining the crystal structures of human mhc i hla-b*4001 complexed with a severe acute respiratory syndrome coronavirus (sars-cov) nucleocapsid (n)-derived t-cell epitope (oh et al., 2011) and mouse mhc i h-2k d bound to an immunodominant t-cell epitope from human hepatitis b virus (hbv) core antigen (hbc) (li et al., 2005) , we clearly demonstrated the molecular features of mhc i molecules with two different salt bridges formed by the residues pairs arg62-glu163 and arg66-glu163, respectively. we further investigated the impacts of the salt bridges on peptide binding and t-cell recognition by constructing a series of mhc i mutants in vitro and in vivo. our results elucidated the key features of mammalian mhc i molecules with salt bridges in the pbg and will benefit t-cell based diagnosis and vaccine development. the human hbv hbc protein-derived peptide hbc87-95 (syvnt-nmgl) (li et al., 2005) and sars-cov n protein-derived peptide n216-225 (getalallll) (oh et al., 2011) were synthesized with 95% purity by reverse-phase high performance liquid chromatography (scilight biotechnology, beijing, china). the peptides were stored at −80°c as freeze-dried powders and were dissolved in dimethyl sulfoxide before use. the expression plasmid for mouse mhc i h-2k d was constructed previously in our lab (zhou et al., 2004) . we also constructed three h-2k d salt bridge mutants (r66a, e163a, and r66a&e163a) and three control mutants (s69a, r157a, a158 g) based on the wt plasmid (genewiz). for the hla-b*4001 expression construct, the gene (genbank q04826) was synthesized and ligated into the pet21a vector (shanghai generay). the three hla-b*4001 salt bridge mutants (r62a, e163a, r62a&e163a) and three control mutants (i66a, r157a, a158 g) were also constructed on the basis of the wt construct. six-to eight-week-old female balb/c mice were purchased from vital river laboratory animal technology company (beijing, china) and raised under specific pathogen-free conditions. all experiments were performed in strict compliance with the guide for the care and use of laboratory animals of the people's republic of china and approved by the committee on the ethics of animal experiments of national institute for viral disease control and prevention, chinese center for disease control and prevention. mice in the experimental group were subcutaneously injected at multiple sites with hbv peptide hbc87-95 and the n-terminal fragment n333 (23-355aa) of murine gp96 emulsified in complete freund's adjuvant for the first dose. mice were administered the mixture of peptide, gp96 fragment, and incomplete freund's adjuvant twice in 2-week intervals for the next two doses. mice were sacrificed to harvest splenocytes on day 7 after the final immunization. mice in the control group were immunized with a mixture of phosphate buffered saline (pbs), gp96 fragment, and freund's adjuvant. antigen-specific t lymphocyte responses were detected with an ifnγ-secreting elispot assay by using splenocytes as previously described (tan et al., 2017) . briefly, 96-well plates were coated with 100 μl/well of 5 mg/ml anti-mouse ifn-γ antibody (bd pharmingen, san diego, ca) overnight at 4°c. after washing with pbs twice, the plates were blocked with culture medium for 2 h at room temperature. mouse spleens were ground and filtered through cell strainers. cells were processed with red blood cell lysis buffer (0.144 m nh 4 cl and 17 μm tris, ph 7.2). we washed cells with pbs twice and centrifuged them to harvest lymphocytes. fresh mouse splenocytes (2.5 × 10 5 ) in 100 μl roswell park memorial institute (rpmi) 1640 medium with 10% fetal bovine serum (fbs) were seeded in each well. then, the peptides (10 μg/ml) were added to the wells and incubated at 37°c in 5% co 2 for 18 h. phytohaemagglutinin (pha) was added as the positive control for nonspecific stimulation. cells incubated with medium alone were employed as a negative control that produced less than five spots in 90% of the experiments. finally, the cells were removed, and the plates were processed according to the manufacturer's instructions (bd). the colored spots, which represent epitope-specific t-cells, were counted and analyzed using an automatic elispot reader (ctl corp.). murine mhc class i h-2k d and β 2 m or human mhc class i hla-b*4001 heavy chain and human β 2 m were overexpressed in escherichia coli as inclusion bodies and subsequently refolded in vitro in the presence of a high concentration of peptide, as described previously (xiao et al., 2016) . briefly, the dissolved mhc i heavy chain and β 2 m inclusion body and peptides were diluted at a molar ratio of 1:1:3, respectively, in refolding buffer (100 mm tris−hcl, 400 mm l-arginine, 2 mm edta-na, 5 mm glutathione [gsh], and 0.5 mm l-glutathione oxidized [gssg]). after 12 h of slow stirring at 4°c, the mhc i/peptide complex was then concentrated and purified via superdex 200 10/ 300 g l (ge healthcare) chromatography. wt h-2k d and the three h-2k d mutant-restricted tetramers of hbvpeptide were prepared as previously described (liu et al., 2012a; zhang et al., 2018; zhou et al., 2006) . briefly, recombinant h-2k d /peptide complexes were purified and then biotinylated by incubation with dbiotin, atp, and the biotin protein ligase bira (avidity) at 4°c for 12 h. the biotinylated h-2k d was further purified by gel filtration to remove free biotin, and then the multimers were produced by using pe-streptavidin (sigma). cells from the subjects were stained with pe-tetramer and fitc-conjugated anti-cd8 antibody. all samples were analyzed with a facscalibur flow cytometer (bd biosciences) after staining. the conditions for the protein purification and the crystal growth of the complex formed by h-2k d with hbv peptide hbc87-95 was described previously (zhou et al., 2004) . human β 2 m was used to generate the h-2k d /hbc87-95 complex. the renatured hla-b*4001/n216-225 complex was further purified by resource q anion-exchange chromatography before crystallization. crystallization was performed using the hanging drop vapor diffusion technique. the concentration of protein was 12 mg/ml, and the crystals grew in 0.1 m sodium citrate tribasic dehydrate (ph 6.5) and 18% (w/v) polyethylene glycol 3350 at 4°c. for cryoprotection, crystals were transferred to reservoir solutions containing 15% glycerol, flash-cooled, and maintained at 100 k in a stream. x-ray diffraction data were collected at 100 k at the ssrf beamline bl17u (shanghai, china) at a wavelength of 0.97907 å ( table 1 ). the structure of h-2k d /hbc87-95 and hla-b*4001/n216-225 were resolved through the molecular replacement method using the mhc i structures with pdb codes 1fg2 and 4f7m, respectively (liu et al., 2012b) as the model in the crystallography and nmr system (cns) program. detailed model building was performed by hand using coot, and restrained refinement was performed using refmac5. the stereochemical quality of the final model was assessed with the program procheck. structure-related figures were processed by pymol. to evaluate the thermostability of different mhc i complexes and also their mutants, we used cd spectroscopy as previously described (n. zhang et al., 2011) . we repeated at least three times for each protein. all complexes were prepared as described above and diluted to 0.2 mg/ ml in 20 mm tris−hcl (ph 8.0) and 50 mm nacl. thermal denaturation curves were determined by monitoring the cd value at 218 nm using a 1-mm optical path-length cell as the temperature was raised from 20 to 100°c at a rate of 1°c/min. the temperature of the sample solution was directly measured with a thermistor. the fraction of unfolded protein was calculated from the mean residue ellipticity (θ) by the standard method. the unfolded fraction (%) is expressed as (θ -θ n ) / (θ u -θ n ), where θ n and θ u are the mean residue ellipticity values in the fully folded and fully unfolded states, respectively. the midpoint transition temperature (t m ) was determined by fitting the data to the denaturation curves using the origin 8.0 program (originlab). for comparisons between multiple groups, two-way anova with bonferroni post-tests was performed. one-way anova analysis with bonferroni post-tests was used for comparison between multiple columns. statistical significance of differences between two columns was determined by student's t-test all tests were two-tailed with a significance level of 0.05. all data analyses were performed with graphpad prism. the coordinates and structure factors of sars-cov peptide n216-225 complexed to hla-b*4001 and hbv peptide hbc87-95 complexed to h-2k d have been deposited in the pdb under accession numbers 6iex and 1vgk, respectively. the retrievement of the mhc i protein sequences available in the ipd-mhc database (https://www.ebi.ac.uk/ipd/mhc/) showed that 12%-26% of the mhc i alleles available possess the paired residues for the potential salt bridge formation ( table 2 ). the type 1 salt bridge is formed by r66 on the α1-helix and e163 on the α2-helix, such as in h-2k d , while the type 2 salt bridge is constructed by r62 and e163 on the α1-and α2-helices, respectively, such as in hla-b*4001. moreover, we retrieved the allele frequency of mhc i molecules which are confirmed to possess salt bridge from the crystal structures in . the allele h-2k d with type 1 salt bridge is also common in mouse mhc alleles. thus, the impacts of the salt bridge formation overhead the pbg on the peptide binding and recognition by tcr would be a substantial phenomenon for different vertebrates, which has emerged in fish. to determine the potential role of the mhc i salt bridge in peptide binding, we utilized the peptide hbc87-95 to facilitate the in vitro renaturation of h-2k d and its salt bridge mutants h-2kd-m1(mutant r66a), h-2kd-m2 (mutant e163a), and h-2kd-m3 (mutant r66a& e163a) followed by size exclusion chromatography (gel filtration) analyses. the control mutants h-2kd-c1 (mutant r157a), h-2kd-c2 (mutant a158 g), h-2kd-c3 (mutant s69a), which preserve the salt bridge were proceeded the same experiment as control mutants. compared to wild type (wt) of h-2k d , all the three salt bridge mutants generated relatively lower yields of refolded products at the size expected for an mhc i monomer. however, the control mutants showed similar yields of renatured products as wt h-2k d (fig. 1a and table s2 ). the binding stabilities of the peptide hbc87-95 with h-2k d and all the mutants were further analyzed by circular dichroism (cd) spectroscopy (fig. 1b) , with the t m s determined from melting curves. wt h-2k d complexed with the hbc87-95 peptide was stable, with a t m of 87.3 ± 2.3°c. as expected, the h-2k d salt bridge mutants h-2kd-m1, h-2kd-m2, and h-2kd-m3 displayed significantly decreased stability with lower t m s (81.3 ± 0.5°c for h-2kd-m1, 78.5 ± 3.8°c for h-2kd-m2, and 80.4 ± 0.5°c for h-2kd-m3). moreover, the t m s of the control mutants have no statistically differences with wt h-2k d (87.8 ± 0.4°c for h-2kd-c1, 88.2 ± 0.3°c for h-2kd-c2, 87.8 ± 0.3°c for h-2kd-c3). we also determined the binding capacity of the sars-cov-derived peptide n216-225 with the human mhc i hla-b*4001, and its salt bridge mutants b4001-m1 (mutant r62a), b4001-m2 (mutant e163a), and b4001-m3 (mutant r62a&e163a) and its control mutants b4001-c1 (mutant a158 g), b4001-c2 (mutant r157a), b4001-c3 (mutant i66a). we found that three salt bridge mutants b4001-m1 (84.2 ± 0.4°c), b4001-m2 (82.8 ± 0.7°c), and b4001-m3 (82.9 ± 0.7°c) displayed significantly lower binding capacity for the peptide compared to wt hla-b*4001(85.9 ± 0.6°c) ( fig. 1c and 1d ), while the control mutants showed the identical binding capacity with the peptides as wt hla-b*4001 ( fig.1c and 1d) . thus, the salt bridge formation in both pbgs of h-2k d and hla-b*4001 contributed to the binding of the peptides, though breaking the salt bridge did not abolish peptide binding. to further investigate the role of the mhc i salt bridge above the pbg in t-cell recognition, we established a mouse model via three rounds of fortnightly in vivo injection of the hbv-derived peptide hbc87-95. elispot assays were then performed using freshly isolated splenocytes to assess the t-cell immune responses induced by the peptide. no specific reactivity of ifn-γ secretion could be detected in splenocytes separated from placebo-immunized mice (< 10 sfcs/10 5 splenocytes). in contrast, cd8 + t-cells from splenocytes of the mice immunized with peptide hbc87-95 presented strong ifn-γ production ( fig. 2a) . the splenocytes from the peptide-inoculated mice were also stained with the h-2k d tetramers prepared in the presence of peptide hbc87-95. the splenocytes from hbc87-95-inoculated mice contained 0.75 ± 0.36% of peptide-specific cd8 + t-cells ( fig. 2b and c) . no peptide-specific cd8 + t-cells were identified from splenocytes of mice inoculated with placebo, as judged by hbc87-95 tetramer staining. we also constructed tetramers based on a series of mutated h-2k d mhc i heavy chains, including h-2kd-m1, h-2kd-m2, and the dual mutant h-2kd-m3. when compared to the wt tetramer, the three mutant tetramers stained a lower proportion of hbc87-95-specific cd8 + t-cells within splenocytes from mice inoculated with peptide hbc87-95 (0.31 ± 0.06%, 0.30 ± 0.05%, and 0.29 ± 0.09%, respectively). these results indicated that mutations of the residues forming the salt bridge positioned over the peptide may impact specific-t-cell recognition. though both h-2k d and hla-b*4001 contain the conserved negatively charged residue e163, the structures of these two mhc i molecules clearly display two different types of salt bridges over the n-terminus of their complexed peptides (fig. 3) . compared to h-2k d , the salt bridge of hla-b*4001 is located closer to the n-terminus of the pbg, and an angle of˜30°can be observed between the positions of the two salt bridges. this is due to the different positively charged residues in the salt bridges of the two mhc i molecules. however, in the structures of some mhc i available online thus far (e.g., h-2l d , h-2d b , and h-2d d ), though they possess the same salt bridge-forming residues (r62 and e163) as hla-b*4001, no salt bridge is formed (pdb codes: 1ld9, 1ce6, 1bii) (supplemental fig. s1 ). the "broken bridge" is also found in several hla-b*2705 structures (e.g. pbd codes: 2bst and 1jge) (supplemental fig. s1 ). particularly, h-2d d possesses both r62 and r66 in its α1 helix, but no salt bridge is formed the balb/c mice were immunized with hbc87-95 peptide (peptide group) or pbs (adjuvant group) together with adjuvants. elispot assays were performed using freshly isolated mouse splenocytes. the non-specific stimulant pha was used as a positive control, and mock indicates the negative control without any stimulant. (b) peptide-specific cd8 + t-cells stained by tetramers of h-2k d and mutants. the hbc87-95 peptide-specific cd8 + t-cells in the freshly isolated splenocytes from vaccinated mice were stained by h-2k d tetramer, kd-m1 (mutant r66a) tetramer, kd-m2 (mutant e163a) tetramer, and kd-m3 (mutant r66a&e163a)tetramer, respectively. the hollow dots represent the peptide-immunized group, and the black dots represent the adjuvant group. the control was staining with unrelated tetramer (hla-a2/influenza peptide gl9). error bars represent means ± sd. n = 5 mice for per peptide group, n = 3 mice for per adjuvant group. (c) the representative peptide-specific cd8 + t-cells stained by tetramers of h-2k d and mutants. the statistical analysis between two groups used two-way anova with bonferroni post-tests. ** p < 0.01, *** p < 0.001. between e163 and either of these positively charged residues. next, we analyzed the hydrogen bonds within the two residues forming the salt bridge and the adjacent residues of both mhc i and the peptides (fig. 4) . three different interactions contribute to the formation of the salt bridges: 1) the electrostatic interaction between r62 or r66 in the α1 helix and the e163 in the α2 helix. the hydrogen bonds constructed between residues r66 and e163 in h-2k d and between r62 and e163 in hla-b*4001 are 2.59 å and 2.75 å, respectively. 2) r62 or r66 in the salt bridge can form a hydrogen bond with the carbonyl group of the p2 residue of the bound peptide in the pbg (as in both the h-2k d and hla-b*4001 structures, and through a water molecule in mamu-a*02). 3) for the mhc i-binding peptides with a hydrophilic residue at the p1 position, hydrogen bonds can be found between e163 and the side chains of the p1 residue of the peptide (as in the h-2k d structure but not hla-b*4001) (fig. 4) . in hla-b * 4002 and hla-b*0702, a water molecule links the interaction between r66 and e163 (fig. 4) . further analysis indicated that this water molecule is conserved in a series of hla-b*2705 structures (1uxs, 2a83, 2bsr, 3b6s, 3bp4, 1of2, 1uxw, and 1w0w) (supplemental fig. s2) . we further analyzed the structures of h-2l d , h-2d b , and h-2d d , which contain the same residues for salt bridge forming (r62 and e163) as hla-b*4001 but do not contain salt bridges. in the mhc i h-2l d , h-2d b , and h-2d d structures, r62 is pulled away by e56 on the α1 helix, and r66 or k66 forms hydrogen bonds with e63 of the α1 helix. thus, no salt bridge is formed in these three mhc i structures despite the fact that they contain the corresponding residues (supplemental fig. s3 ). to investigate the potential influence of salt bridge formation on the structural conformation of the mhc/peptide complex, we superimposed the structures of the mhc i molecules with the two different salt bridges: r66-e163 (h-2k d , mamu-b*17, and mamu-b*098) and r62-e163 (hla-b*4001, mamu-a*02, rt1-a1c, hla-b*4002, hla-b*0702, and hla-b*2705) (fig. 5a) . compared to the structure of mhc i with the r62-e163 salt bridge, the α2-helix of mhc i molecules with the r66-e163 salt bridge has an conformational shift (approximately 1.65 å between h-2k d and hla-b*4001), using the α1-helix of mhc i as the benchmark during the alignment (fig. 5b) . the peptides in the structures of mhc i complexes with r66-e163 salt bridges also display conformational differences for the two residues at the p1 and p2 positions compared to the peptides under the r62-e163 salt bridges (fig. 5c ). the cα of the p2 residues in the h-2k d -binding peptides are closer to the c-terminus of the pbg and located lower and closer to the bottom of the groove (fig. 5d and 5e ). the r66-e163 salt bridge acts as a "seatbelt" for the peptide, pushing both the conformational shift of the peptides and the α2-helices. herein, we determined the crystal structures of hla-b*4001 and h-2k d molecules with two representative different salt bridges spanning the presented peptides. compared to wt, the peptide binding capacity of the salt bridge mutants was significantly lower. meanwhile, the control mutants showed no statistical difference. though the two salt bridges had distinct impacts on the conformational changes of the mhc i structures, both enhanced peptide binding and potentially contributed to tcr recognition. though the mhc i salt bridge-breaking mutants still bound to the peptides, their binding capacity were lower than wt. the impact on the thermostability of hla-b*4001 by the mutants was not as much as that observed in the h-2k d mutants. this is may be due to the intrinsic features of different salt bridges in the two molecules. using structural analysis, we found that the enhancement effect of peptide binding to mhc i was not only contributed by the direct formation of the salt bridge above the peptide main chain but also contributed by the conserved hydrogen bonds between the salt bridge-forming residues with the atoms on the main chain and the p1 residues of the peptides. a previous study indicates that a salt bridge (also called an ion pair in the study) formed by argα80 and aspβ57 in mhc class ii heterodimers is also critical for surface expression and peptide presentation (nalefski et al., 1995) . however, unlike the salt bridge overhanging the mhc i peptide, this argα80-aspβ57 salt bridge is located under the main chain 4 . the interactions between the two residues forming the salt bridge and the adjacent residues of both mhc i and the peptides. the interactions between r66/r62 and e163 that form the salt bridge are denoted as red arrows. the cyan arrows represent the hydrogen bond between e163 and the side chains of the p1 residues of the peptides. the dark blue arrow marks the hydrogen bond between r62 or r66 and the carbonyl group of the p2 residue of the bound peptide in the pbg. in hla-b*4002, hla-b*0702 and mamu-a*02, the cyan dots represent water molecules. the black dashed lines in the structures represent the hydrogen bonds. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). of the mhc ii-presented peptide and may contribute to p9 binding and the c-terminal hanging conformation of the mhc ii-bound peptides. the salt bridge above the peptide both enhances mhc i-peptide binding and contributes to tcr recognition of mhc i-peptide complexes. birkinshaw et al. studied that a prototypical autoreactive bk6 tcr bound cd1a when the presented ligands were permissive. tcr docked over the a' roof of cd1a and a central cd1a salt-bridge network (arg76-glu154 salt-bridge) prevented the interaction between bk6 tcr and permissive ligands, and its autoreactivity was determined by contacts with cd1a. nonpermissive ligands disrupted salt bridge interaction of cd1a, disrupt the properties of a' roof and could hinder engagement of hydrophobic cdr3β loop of the bk6 tcr with cd1a (birkinshaw et al., 2015) . nurzia et al. indicate that the positive charging of arg62 is preferred for the tcr recognition of ankylosing spondylitis-associated b*2705 complexes, as revealed by the r62 k and r62a mutants with an overall reduced capability to present peptides to cd8 + t-cells (nurzia et al., 2012) . our data also demonstrate that tcell recognition was significantly alleviated when we broke the mhc i salt bridge with r66a and e163a mutations. previously determined complex structure between hla-b*07:02 and kfj37 tcr showed that both the r62 and e163 of hla-b*07:02 can form hydrogen bonds with the residues in the cdr3α and cdr1α loops of the tcr, respectively (chan et al., 2018) . this indicated that the residues in the salt bridge can be directly-recognized by the tcr (tynan et al., 2007) . meanwhile, our analysis also demonstrated that the salt bridge can lead to a conformational shift of the presented peptide and the relative locations of the α1 or α2 helices, which may subsequently and indirectly impact tcr recognition. further structural studies are required to investigate the accurate and detailed recognition of mhc i/peptide complex containing the salt bridge by the tcr. nevertheless, our current study still has limitations. the mutant mhc i tetramers used in this study including the h-2k d mutants at r66 and/or e163a can dismantle the salt breakage and then may indirectly alter t cell response. however, these residues can also be recognized by tcr, thus, the mutation of these residues may also directly influence the tcr response. we cannot exclude the possibility that the altered t cell response was due to altered contacts with the mutated residues, rather than the loss of the salt bridge interaction. in summary, our study indicates that the two different salt bridges in mammalian mhc i molecules, represented by hla-b*4001 and h-2k d , respectively, have different structural characteristics. and the mutations of the salt bridge-forming residues may impact both peptide binding and tcr recognition, directly or indirectly. our data is helpful for the understanding of the cd8 + t-cell recognition of mhc i-presented peptides. structural and dynamic control of t-cell receptor specificity, cross-reactivity, and binding mechanism alphabeta t cell antigen receptor recognition of cd1a presenting self lipid ligands structure of the human class i histocompatibility antigen, hla-a2 t cell receptor cross-reactivity directed by antigen-dependent tuning of peptide-mhc molecular flexibility divergent t-cell receptor recognition modes of a hla-i restricted extended tumourassociated peptide crystal structures of two viral peptides in complex with murine mhc class i h-2kb a structural voyage toward an understanding of the mhc-i-restricted immune response: lessons learned and much to be learned loss of t cell antigen recognition arising from changes in peptide and major histocompatibility complex protein flexibility: implications for vaccine design generation of murine ctl by a hepatitis b virus-specific peptide and evaluation of the adjuvant effect of heat shock protein glycoprotein 96 and its terminal fragments diverse peptide presentation of rhesus macaque major histocompatibility complex class i mamu-a 02 revealed by two peptide complex structures and insights into immune escape of simian immunodeficiency virus crystal structure of cell adhesion molecule nectin-2/cd112 and its binding to immune receptor dnam-1/ cd226 cross-allele cytotoxic t lymphocyte responses against 2009 pandemic h1n1 influenza a virus among hla-a24 and hla-a3 supertype-positive individuals the antigenic identity of peptide-mhc complexes: a comparison of the conformations of five viral peptides presented by hla-a2 the structure of hla-b27 reveals nonamer self-peptides bound in an extended conformation structural definition of the h-2kd peptide-binding motif an ion pair in class ii major histocompatibility complex heterodimers critical for surface expression and peptide presentation structural basis for the differential classification of hla-a*6802 and hla-a*6801 into the a2 and a3 supertypes interaction pattern of arg 62 in the a-pocket of differentially disease-associated hla-b27 subtypes suggests distinct tcr binding modes engineering t cells specific for a dominant severe acute respiratory syndrome coronavirus cd8 t cell epitope hemagglutininspecific cd4(+) t-cell responses following 2009-ph1n1 inactivated split-vaccine inoculation in humans a t cell receptor flattens a bulged antigenic peptide presented by a major histocompatibility complex class i molecule diversified anchoring features the peptide presentation of dla-88*50801: first structural insight into domestic dog mhc class i crystal structure of swine major histocompatibility complex class i sla-1 0401 and identification of 2009 pandemic swine-origin influenza a h1n1 virus cytotoxic t lymphocyte epitope peptides evaluation of zika virus-specific t-cell responses in immunoprivileged organs of infected ifnar1-/-mice screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes complex assembly, crystallization and preliminary x-ray crystallographic studies of mhc h-2kd complexed with an hbv-core nonapeptide we thank prof. jianfang zhou (national institute for viral disease control and prevention, chinese center for disease control and prevention) for excellent assistance with flow cytometry. we thank dr. minghai zhou (the scripps research institute, scripps florida, usa) for his excellent work in crystallization of the proteins. we also thank dr. jianhui li (institute of biophysics, chinese academy of sciences) for instruction in circular dichroism spectroscopy. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.molimm.2019.06.005. key: cord-299279-v0vznri2 authors: røder, gustav; kristensen, ole; kastrup, jette s.; buus, søren; gajhede, michael title: structure of a sars coronavirus-derived peptide bound to the human major histocompatibility complex class i molecule hla-b*1501 date: 2008-05-17 journal: acta crystallographica section f structural biology and crystallization communications doi: 10.1107/s1744309108012396 sha: doc_id: 299279 cord_uid: v0vznri2 the human leukocyte antigen (hla) class i system comprises a highly polymorphic set of molecules that specifically bind and present peptides to cytotoxic t cells. hla-b*1501 is a prototypical member of the hla-b62 supertype and only two peptide–hla-b*1501 structures have been determined. here, the crystal structure of hla-b*1501 in complex with a sars coronavirus-derived nonapeptide (vqqessfvm) has been determined at high resolution (1.87 å). the peptide is deeply anchored in the b and f pockets, but with the glu4 residue pointing away from the floor in the peptide-binding groove, making it available for interactions with a potential t-cell receptor. major histocompatibility complex class i (mhc-i, or hla-i in humans) molecules are cell-surface glycoproteins. their function is to select peptides derived from the intracellular protein metabolism and present them to cytotoxic t cells of the adaptive immune system, thereby allowing the protein content of cells to be surveyed and controlled (reviewed in guermonprez et al., 2002) . the specificity of mhc-i molecules determines which peptides will be presented and potentially recognized by the immune system. the hla system is highly polymorphic in a manner that effectively diversifies immune responsiveness. almost 1600 different hla-i alleles have been identified in human populations, making mhc-i the most polymorphic gene system known. in an attempt to reduce this complexity, it has been suggested that the majority of hla-i molecules can be clustered into one of 12 currently defined hla supertypes. more than 120 different hla-i crystal structures have been reported and the majority of these belong to the hla-a2 supertype; other supertypes are sparsely populated or not at all. only a few structures of hla-i alleles belonging to the hla-b62 supertype have been reported, including two structures of hla-b*1501 (røder et al., 2006) . hla-b*1501 is the prototypical member of the hla-b62 supertype. hla-b*1501 is frequent in southeast asian and in european populations, where it often accounts for more than 10% of the hla-i phenotype. we are involved in the human mhc project, which aims to describe and predict the peptide-binding specificity of one or more members of each hla supertype (lauemøller et al., 2000) . to this end, we are generating structural (blicher et al., 2005 røder et al., 2006) and biochemical (buus et al., 1995; sylvester-hvid et al., 2002; lauemøller et al., 2000) information on peptide-hla interactions and using this information to generate fast computational predictions of peptide binding (stryhn et al., 1996; buus et al., 2003; peters et al., 2006) . a detailed understanding of peptide-mhc interactions will support a general understanding of the nature of t-cell recognition and a rational exploitation of human immune responses (lauemøller et al., 2000) . thus, predictive algorithms allow entire genomes to be rapidly and accurately screened for potential immunogenic epitopes (wang et al., 2007) . one approach, which might be independent of large-scale biochemical experimentation, would be to use structural information to predict peptide binders (rognan et al., 1999) . to populate the 'structure space' of hla molecules, we have previously reported two hla-a*1101 (blicher et al., 2005 and two hla-b*1501 structures (røder et al., 2006) . here, we report a crystal structure of hla-b*1501 in complex with a sars coronavirus-derived nonapeptide (vqqessfvm). a genetic construct representing the hla-b*1501 protein, positions 1-276, was produced by site-directed mutagenesis from an hla-a*0201 gene using the quikchange multi site-directed mutagenesis kit (stratagene). this procedure involved 81 nucleotide mutations, which could be accomplished using 13 dna primers. the final hla-b*1501 sequence was inserted into the pet28a vector (novagen). the sequence was verified by dna sequencing (3100 avant, abi). escherichia coli bl21 (de3) cells were transformed with the hla-b*1501-pet28 vector and the protein was produced in a 2 l fermentor (infors) by induction with iptg as described previously (ferré et al., 2003) . inclusion bodies were obtained by cell disruption (constant cell disruption systems), washed twice with pbs containing 0.5%(v/v) np40 and 0.5%(w/v) deoxycholate and dissolved in 8 m urea and 25 mm tris-hcl ph 8.0. the dissolved hla-b*1501 protein was purified in 8 m urea at 285 k by ionexchange chromatography (q-sepharose fast flow). buffer a contained 8 m urea and 25 mm tris-hcl ph 8.0 and buffer b was the same but included 1 m nacl. a 0-50% gradient in buffer b spanning four column volumes was applied. the eluted protein was further purified by hydrophobic interaction chromatography (phenyl sepharose hp) to separate correctly disulfide-bonded hla-b*1501 (buffer a, 8 m urea, 25 mm tris-hcl ph 8.0 and 100 g l à1 ammonium sulfate; buffer b, 8 m urea and 25 mm tris-hcl ph 8.0; gradient, 0-40% in buffer b spanning six column volumes). finally, the hla-b*1501 sample was purified by sephacryl-s200 chromatography in 8 m urea, 25 mm tris-hcl ph 8.0 and 150 mm nacl, concentrated by ultrafiltration and subsequently stored at 253 k. the vqqessfvm peptide was synthesized by conventional fmoc chemistry and subsequently purified by reversed-phase hplc (schafer-n, copenhagen). the peptide identity was verified by reversed-phase hplc followed by ion-trap mass spectrometry (bruker daltonics). the purity was determined to be higher than 99%. for complex assembly, 3 mg denatured hla-b*1501 heavy chain was rapidly diluted in 1 l 50 mm tris-hcl ph 7.5, 3 mm edta and 150 mm nacl containing 2 mg 2 -microglobulin ( 2 m) and 1 mg peptide (the final heavy chain, 2 m and peptide concentrations were 100, 170 and 1000 nm, respectively). the folding reaction was incubated at 291 k for 48 h and then concentrated to 10 ml using a pressure cell (amicon) equipped with a 10 kda molecular-weight cutoff filter. the highly concentrated folding reaction was allowed to settle overnight and was subsequently concentrated to 0.5 ml on a 10 kda spin filter (amicon). folded mhc-i complex was separated from aggregated hla-b*1501 heavy chain, free 2 m and peptide on a superdex-200 column (amersham biosciences). the fractions were analyzed (sds-page and ion-trap mass spectrometry) and those containing the two protein components, hla-b*1501 heavy chain and 2 m, were pooled and concentrated on a 10 kda spin filter to 4.2 mg ml à1 (96 mm) in 50 mm tris-hcl ph 7.5, 3 mm edta and 150 mm nacl. crystals of the hla-b*1501 complex were grown using the hanging-drop technique. buffers from crystal screens 1 and 2 (hampton research) were used in screening (reservoir volume, 500 ml; drop volume, 1 ml reservoir solution and 1 ml protein solution). the experiments were set up at 277 k. no crystals appeared within the first two months, after which the crystallization trials were left at 277 k for 2 y. single crystals appeared under initial conditions consisting of 0.2 m magnesium chloride hexahydrate, 0.1 m hepes, 30%(v/v) peg 400 ph 7.5. diffraction data were collected at cryogenic temperature on the i911-5 beamline at max-lab, lund, sweden. the hla-b*1501 complex crystallized in space group p2 1 2 1 2 1 , with one protein-peptide complex per asymmetric unit (see table 1 ). intensities were integrated with mosflm (powell, 1999) and scaled with scala (evans, 2006) . the structure was solved by molecular replacement using the program phaser (mccoy et al., 2005) with hla-b*1501 (pdb code 1xr9) as the search model (røder et al., 2006 ). an initial model consisting of the hla-b*1501 heavy chain and 2 m was automatically built with arp/warp (cohen et al., 2004) and residue side chains were substituted using guiside (cohen et al., 2004) . multiple rounds of manual inspection and model building in coot (emsley & cowtan, 2004) were performed, followed by restrained refinements in refmac5 (murshudov et al., 1997) . this included manual building jf o j. § as r, but calculated on 5% of the data excluded from the refinement. } calculated with procheck (laskowski et al., 1993). of the vqqessfvm peptide. the final structure contains hla-b*1501 (residues 1-275), 2 m (residues 1-99), the vqqessfvm peptide, one putative peg fragment, a putative hepes molecule and 357 waters ( table 1) . the mhc-i -chain, the 2 m chain and the bound peptide will be referred to in the following as 'a', 'b' and 'p', respectively. the structure has been deposited in the rcsb protein data bank with code 3c9n. all figures were prepared with pymol (delano, 2002). this is the third crystal structure of hla-b*1501 in complex with a peptide. the two previous structures either had leu or glu in position 2 of the peptide (røder et al., 2006) . the structure reported here has gln at this position. furthermore, the two reported structures both have tyr as the c-terminal peptide residue, whereas this structure has a met, which slightly alters the position of the peptide c-terminus. the overall structure of the present hla-b*1501 peptide-binding groove is similar to the previously two determined hla-b*1501 structures (røder et al., 2006) and to other mhc-i molecules (see fig. 1 ). structural superpositioning of this and a previously published hla-b*1501 structure (pdb code 1xr9) using the first 181 ca atoms in the -chain yields a root-mean-square deviation of 0.34 å . the structure consists of all of the residues of the heavy (a) and light (b) chains and the bound peptide (p) (see table 1 for details). the model was refined to an r value of 18.8% and an r free value of 22.7%. generally, the electron density is clearly defined and all the residues lining the peptide-binding groove and the peptide residues are completely resolved (see fig. 2 ). furthermore, the disulfide bond in the 2 domain of the peptide-binding groove is clearly defined, as depicted in the inset in fig. 1 . it has been speculated that the oxidation state of this disulfide bond is important for the loading of the peptide-binding groove with a peptide (park et al., 2006) . when this disulfide bond is oxidized and a peptide is bound, the 1 and 2 domains are thought to be more stable. some solvent-exposed residues (including pser5 of the peptide) are found in multiple conformations. a ramachandran analysis estimated that 91.4% of the residues are found in the favoured regions and no residues are found in disallowed regions. the electron density of the peptide bound in the hla-b*1501 binding groove is well defined except for the pglu4 side chain, which is solvent-exposed and thus only partially defined (see fig. 2 ). 3.2.1. peptide residue 1. the c atoms of the pval1 side chain in the a pocket are positioned towards the aromatic ring system of atrp167, thereby engaging in hydrophobic interactions. furthermore, this hydrophobic p1 residue also interacts with the atyr59 and aleu163 residues in hla-b*1501 as described previously (røder et al., 2006) . the n-terminal n atom is oriented towards the 2 helix. this is unusual as a water-mediated hydrogen bond is generally formed to protein residues (ogata & wodak, 2002) . in the present structure, the corresponding water molecule (hoh287) makes hydrogen bonds to atyr7, atyr59 and potentially aglu63. interactions in the hla-b*1501 peptide-binding groove. the hla-i complex is visualized from above, looking directly towards the floor of the peptide-binding groove. the peptide (vqqessfvm) is depicted in yellow and the peptide n-terminal region is located to the left. peptide-binding groove residues interacting with the peptide are coloured black and labelled. the insert shows the electron density (at 1, to a distance of 1 å ) of the characteristic disulfide bond in the 2 helix. 3.2.2. peptide residue 2. the pgln2 peptide residue is located in the b pocket in the same orientation as the pglu2 in the previously described hla-b*1501-lekargsty structure (røder et al., 2006) . this residue is completely shielded from the surroundings by aarg62, thereby prohibiting any interaction with a potential t-cell receptor. the pgln2 residue makes hydrophobic interactions with atyr7 and aile66 that make up the bottom and top side wall of the b pocket, respectively. the n and o atoms of pgln2 interact with atyr9, aglu63, aser67 and a bridging water molecule (hoh57) located at the apex of the b pocket. 3.2.3. peptide residue 3. the pgln3 peptide residue is located in the d pocket and points towards the 2 helix. this peptide residue forms hydrophobic interactions with atyr99, atrp156 and atyr159. in addition, weak hydrogen bonds to two water molecules (hoh341 and hoh539) are observed, thus bridging the peptide to the hla-b*1501 side chains, which results in further stabilization of the bound peptide residue in this pocket. 3.2.4. peptide residue 4. the pglu4 peptide residue points away from the peptide-binding groove and the side chain does not interact with any hla-b*1501 residues. this orientation of pglu4 makes it available for potential interactions with t-cell receptors. 3.2.5. peptide residue 5. the pser5 peptide residue is found in two conformations, both of which point towards the 1 helix of the c pocket. the o atom in the a conformation makes a hydrogen bond to the n 2 atom of aasn70 and the backbone o atom of aile66, both of which are located in the 1 helix, and forms a weak hydrogen bond to a water molecule located at the surface (hoh327). in the b conformation, a hydrogen bond is formed to the n 2 atom of aasn70 and two water molecules (hoh79 and hoh364). 3.2.6. peptide residue 6. the pser6 peptide residue points down towards the -sheet floor of the peptide-binding groove. the conformation observed does not interact with any hla-b*1501 residues, but makes a hydrogen bond to two water molecules (hoh67 and hoh348) present between the peptide and the floor of the peptide-binding groove, thereby forming water-mediated contacts with the hla-b*1501 protein. therefore, only larger peptide residues at this position are likely to contribute significantly to the overall mhc-i binding. 3.2.7. peptide residue 7. the pphe7 peptide residue is solventexposed and points directly away from the floor of the peptidebinding groove. the electron density of this residue is clearly defined, revealing that pphe7 is tilted slightly towards the 2 helix. the conformation of pphe7 allows contacts with aala150 located on the 2 helix segment. the backbone n atom of this residue also makes a hydrogen bond to the o "1 atom on the aglu152 residue. the large atrp147 also excludes the possibility of the pphe7 residue side chain packing into the peptide-binding groove, which forces the pphe7 residue to be solvent-exposed. the orientation of pphe7 makes it available for interactions with a potential t-cell receptor. 3.2.8. peptide residue 8. the side chain of this peptide residue does not contact any hla-b*1501 residues. the pval8 residue protrudes from the binding pocket. however, owing to the small size of pval8, it is unlikely to be a dominating peptide residue that plays any significant role in the t-cell receptor docking and recognition. 3.2.9. peptide residue 9. the last pocket of the peptide-binding groove of hla-i is the f pocket. as previously described for hla-b*1501, this pocket is lined by hydrophobic residues and can accommodate large aromatic residues such as tyr and phe (røder et al., 2006) . the present hla-b*1501-peptide structure contains a c-terminal methionine residue, pmet9, which is found in an extended conformation reaching the apex of the f pocket. the f pocket is lined by aleu81, aleu95, atyr123 and atrp147, rendering this environment mostly hydrophobic. aser116 is located at the bottom of the f pocket and points towards the 1 domain of the peptide-binding groove. hence, aser116 points away from the docked pmet9 side chain and is unable to interact with this c-terminal peptide residue. pmet9 along with a114 have been shown to not only have a direct influence on the f-pocket specificity but also to influence the interaction of hla-i with tapasin (park et al., 2003; thammavongsa et al., 2006) . the peptide c-terminal o atoms make one hydrogen bond to the n 2 atom of aasn80 and one to the n atom of alys146, which stabilizes peptide binding and at the same time shields the peptide c-terminus from interactions with a t-cell receptor. the pymol molecular graphics system this work was funded by the danish centre for synchrotron radiation (dansync), eu 6fp 503231 and nih hhsn2662004-00025c. key: cord-329825-e9mepqvn authors: giamarellos-bourboulis, evangelos j.; netea, mihai g.; rovina, nikoletta; akinosoglou, karolina; antoniadou, anastasia; antonakos, nikolaos; damoraki, georgia; gkavogianni, theologia; adami, maria-evangelia; katsaounou, paraskevi; ntaganou, maria; kyriakopoulou, magdalini; dimopoulos, george; koutsodimitropoulos, ioannis; velissaris, dimitrios; koufargyris, panagiotis; karageorgos, athanassios; katrini, konstantina; lekakis, vasileios; lupse, mihaela; kotsaki, antigone; renieris, george; theodoulou, danai; panou, vassiliki; koukaki, evangelia; koulouris, nikolaos; gogos, charalambos; koutsoukou, antonia title: complex immune dysregulation in covid-19 patients with severe respiratory failure date: 2020-04-21 journal: cell host microbe doi: 10.1016/j.chom.2020.04.009 sha: doc_id: 329825 cord_uid: e9mepqvn proper management of covid-19 mandates better understanding of disease pathogenesis. the sudden clinical deterioration 7–8 days after initial symptom onset suggests that severe respiratory failure (srf) in covid-19 is driven by a unique pattern of immune dysfunction. we studied immune responses of 54 covid-19 patients, 28 of whom had srf. all patients with srf displayed either macrophage activation syndrome (mas) or very low human leukocyte antigen d related (hla-dr) expression accompanied by profound depletion of cd4 lymphocytes, cd19 lymphocytes, and natural killer (nk) cells. tumor necrosis factor-α (tnf-α) and interleukin-6 (il-6) production by circulating monocytes was sustained, a pattern distinct from bacterial sepsis or influenza. sars-cov-2 patient plasma inhibited hla-dr expression, and this was partially restored by the il-6 blocker tocilizumab; off-label tocilizumab treatment of patients was accompanied by increase in circulating lymphocytes. thus, the unique pattern of immune dysregulation in severe covid-19 is characterized by il-6-mediated low hla-dr expression and lymphopenia, associated with sustained cytokine production and hyper-inflammation. proper management of covid-19 mandates better understanding of disease pathogenesis. giamarellos-bourboulis et al. describe two main features preceding severe respiratory failure associated with covid-19: the first is macrophage activation syndrome; the second is defective antigen-presentation driven by interleukin-6. an il-6 blocker partially rescues immune dysregulation in vitro and in patients. in december 2019, authorities in wuhan, china reported a cluster of pneumonia cases caused by an unknown etiologic agent. the pathogen was soon identified and sequenced as a novel coronavirus related to the agent of severe acute respiratory syndrome (sars) and was subsequently termed sars coronavirus-19 (sars-cov-2). the infection spread in the subsequent 3 months on all continents and was declared a pandemic by the world health organization. as of april 2, 2020, 961,818 documented cases were reported worldwide, and 49,165 patients had died (https://www.who.int/emergencies/diseases/ novel-coronavirus-2019). this novel coronavirus has a tropism for the lung, causing community-acquired pneumonia (cap). some patients with pneumonia suddenly deteriorate into severe respiratory failure (srf) and require intubation and mechanical ventilation (mv). the risk of death of these patients is high, reaching even 60% (arabi et al., 2020) . proper management mandates better understanding of disease pathogenesis. the majority of physicians use sepsis as a prototype of critical illness for the understanding of severe coronavirus disease 2019 pathogenesis. this is mostly because severe covid-19 is associated with hyper-cytokinemia (guan et al., 2020; huang et al., 2020) . lethal sepsis is commonly arising from bacterial cap, often leading to srf and the need for mv. the peculiar clinical course of cap caused by sars-cov-2, including the sudden deterioration of the clinical condition 7-8 days after the first symptoms, generates the hypothesis that this illness is driven by a unique pattern of immune dysfunction that is likely different from sepsis. the features of lymphopenia with hepatic dysfunction and increase of d-dimers (qin et al., 2020) in these patients with severe disease further support this hypothesis. immune responses of critically ill patients with sepsis can be classified into three patterns: macrophage-activation syndrome (mas) (kyriazopoulou et al., 2017) , sepsis-induced immunoparalysis characterized by low expression of the human leukocyte antigen d related (hla-dr) on cd14 monocytes (lukaszewicz et al., 2009) , and an intermediate functional state of the immune system lacking obvious dysregulation. we investigated whether this classification might apply to patients with srf caused by sars-cov-2. results revealed that approximately one fourth of patients with srf have mas and that most patients suffer from immune dysregulation dominated by low expression of hla-dr on cd14 monocytes, which is triggered by monocyte hyperactivation, excessive release of interleukin-6 (il-6), and profound lymphopenia. this pattern is distinct from the immunoparalysis state reported in either bacterial sepsis or srf caused by 2009 h1n1 influenza. all patients with severe respiratory failure caused by sars-cov-2 have immune dysregulation or mas we assessed the differences of immune activation and dysregulation between sars-cov-2 and other known severe infections in three patient cohorts: 104 patients with sepsis caused by bacterial cap; 21 historical patients with 2009 h1n1 influenza; and 54 patients with cap caused by sars-cov-2. patients with bacterial cap were screened for participation in a large-scale randomized clinical trial with the acronym provide (clinicaltrials.gov nct03332225). patients with 2009 h1n1 influenza have been described in previous publications of our group (giamarellos-bourboulis et al., 2009; raftogiannis et al., 2010) . the clinical characteristics of patients with bacterial cap and cap caused by covid-19 are described in table 1 . each cohort (bacterial sepsis and covid-19) is split into patients who developed srf and required mv and those who did not. three main features need to be outlined: (1) patients with covid-19 and srf are less severe than those with severe bacterial cap, on the basis of the traditional severity scores of sequential organ failure assessment (sofa) and acute physiology and chronic health evaluation (apache) ii; (2) this leads to the conclusion that covid-19 patients undergo an acute immune dysregulation with deterioration into srf before the overall state of severity is advanced; and (3) although the burden of co-morbidities of patients with covid-19, as expressed by the charlson's co-morbidity index, is higher among patients with srf than among patients without srf, it remains remarkably lower that traditional bacterial cap and sepsis. it was also notable that the admission values of glasgow coma scale scores of patients with bacterial cap were 8.80 ± 4.76, and that of patients with covid-19 was 14.71 ± 0.20 (p < 0.0001). this finding is fully compatible with clinical descriptions of severe covid-19: patients are admitted at a relatively good clinical state and suddenly deteriorate. coronary heart disease 7 (14.6) 2 (7.7) 0.479 10 (17.9) 5 (17.9)* 1.0 comparisons with the respective groups without respiratory failure by the student's t test: *p-non-significant; **p < 0.05; # p < 0.001; ## p < 0.0001. abbreviations are as follows: alt, alanine aminotransferase; aptt, activated partial thromboplastin time; ast, aspartate aminotransferase; apache, acute physiology and chronic health evaluation; cci, charlson's comorbidity index; inr, international normalized ratio; psi, pneumonia severity index; sd, standard deviation; sofa, sequential organ failure assessment immune classification of patients with sars-cov-2 was performed by using the tools suggested for bacterial sepsis, i.e., ferritin more than 4,420 ng/ml for mas (kyriazopoulou et al., 2017) , and hla-dr molecules on cd14 monocytes lower than 5,000, in the absence of elevated ferritin, for the immune dysregulation phenotype (lukaszewicz et al., 2009) . it was found that contrary to the patients with bacterial cap and srf, all patients with srf and sars-cov-2 had either immune dysregulation or mas (table 2) . major decrease of hla-dr on cd14 monocytes is associated with srf immunoparalysis of sepsis is characterized by significant decrease of the number of hla-dr molecules on cd14 monocytes. this also happens in immune dysregulation caused by sars-cov-2 ( figures 1a and 1b) . although patients with bacterial-cap-associated mas also have decreased hla-dr molecules on cd14 monocytes, their circulating ferritin is significantly higher than normal. this is a feature found only in a few patients with sars-cov-2 and mas ( figure 1c ). the absence of traits of mas among cases of sars-cov-2 with immune dysregulation is further proven by the low scores of hemophagocytosis (hs). hscore is proposed as a classification tool for secondary mas, and values more than 169 are highly diagnostic (fardet et al., 2014) . seven patients with sars-cov-2 had hs above this cut-off, and all were properly classified by using ferritin (figure 1d ). among patients with bacterial cap at an intermediate immune state, the number of molecules of hla-dr on their cd14 monocytes was lower than in healthy patients. however, patients with pneumonia caused by sars-cov-2 at an intermediate immune state maintained their number of molecules of hla-dr on cd14 monocytes much closer to the healthy condition. when this number suddenly dropped, srf supervened ( figures 1a and 1b) . moreover, the absolute counts of neutrophils and monocytes were higher among patients with immune dysregulation than patients with mas ( figures 1e and 1f ). the absolute counts of cd3 + cd4 + cd45 + lymphocytes, cd3 + cd8 + cd45 + lymphocytes, cd3 à cd16 + cd56 + cd45 + cells, and cd19 + cd45 + lymphocytes were lower among patients with covid-19 compared with those in 10 healthy subjects adjusted for age and gender. compared with patients with cap caused by 2009h1n1, patients with covid-19 had lower cd3 + cd4 + cd45 + lymphocytes but higher cd3 + cd16 + cd45 + cells and cd19 + cd45 + lymphocytes (figures 2a-2e ). those patients with immune dysregulation caused by covid-19 had lower counts of cd3 + cd4 + cd45 + lymphocytes, cd3 + cd8 + cd45 + lymphocytes, and cd3 à cd16 + cd56 + cd45 + cells than those at an intermediate immune state. when comparisons were limited to patients with srf and infection by one of the two viruses, it was found that infection by sars-cov-2 was accompanied by lower cd3 + cd4 + cd45 + lymphocytes but with higher cd3 à cd16 + cd56 + cd45 + cells and cd19 + cd45 + lymphocytes than 2009h1n1 (figures 2f-2j ). the th17 function as assessed by il-17 production capacity was downregulated among patients with immune dysregulation ( figure 2k ). the next question was how this change of cd19 + cd45 + lymphocytes is translated to serum immunoglobulins (igs). concentration of iggs and their subclasses in the plasma of covid-19 patients was low, as it was in bacterial cap ( figures 3a-3d) . however, igm and iga were higher than in bacterial cap ( figures 3e and 3f) . overall, patients at mas had lower igg2, igm, and iga than those at an intermediate immune state, and patients at immune dysregulation had lower igm than those at an intermediate immune state. the il-6 blocker tocilizumab partially rescues the immune dysregulation driven by sars-cov-2 sepsis-induced immunoparalysis is characterized by profound deficiency of monocytes for cytokine production upon ex vivo stimulation (giamarellos-bourboulis et al., 2011) . indeed, production of tumor necrosis factor-a (tnf-a) by lps-stimulated peripheral blood mononuclear cells (pbmcs) of patients with bacterial cap classified for immunoparalysis was significantly lower than in patients at an intermediate state ( figure 4a ). that was not the case for patients with pneumonia caused by sars-cov-2, in whom pbmcs showed sustained tnf-a production after stimulation with lps ( figure 4b ). the function of pbmcs in patients with srf caused by 2009h1n1 was also impaired, and there was lower tnf-a production, a pattern different from covid-19 patients ( figure 4c) . surprisingly, stimulation of il-1b was lower among patients with immune dysregulation than among patients with an intermediate immune state (figures 4d-4f) . il-6, however, followed the stimulation pattern of tnf-a ( figures 4g-4i) . this generated the hypothesis that in the case of srf-aggravated pneumonia caused by sars-cov-2, there is a unique combination of defective antigen presentation and lymphopenia that leads to defective function of lymphoid cells, whereas monocytes remain potent for the production of tnf-a and il-6. as a next step, we measured circulating concentrations of tnf-a, interferon-g (ifn-g), il-6, and c-reactive protein (crp) in patients infected by sars-cov-2. ifn-g was below the limit of detection in all patients (data not shown), indicating that th1 responses do not contribute to over-inflammation. no differences of circulating tnf-a concentrations were found between covid-19 patients at the three states of immune classification (figure 4j ). in contrast, il-6 and crp concentrations were significantly higher among patients with immune dysregulation than among patients at an intermediate state of immune activation ( figures 4k and 4l ). given that il-6 was below the limit of detection in some patients with immune dysregulation, we divided them into two groups as follows: seven patients with il-6 below the limit of detection and 14 patients with detectable il-6. their severity was similar given that sofa score and pneumonia severity indexes were similar (p values of comparisons were 0.937 and 0.877, respectively; data not shown). il-6 is known to inhibit hla-dr expression (ohno et al., 2016) , leading to the hypothesis that il-6 over-production mediates the low hla-dr expression on cd14 monocytes of covid-19 patients. in agreement with this, negative correlation was found between serum amounts of il-6 and the absolute number of hla-dr molecules on cd14 monocytes of patients with covid-19 but also between the absolute lymphocyte count and the absolute number of mhla-dr on cd14 monocytes of patients with covid-19 ( figures 5a and 5b) . furthermore, pbmcs from patients with immune dysregulation were cultured overnight in the presence of plasma of the covid-19 patients, which was already shown to be rich in il-6. the expression of hla-dr on cd14 monocytes was strongly inhibited by covid-19 plasma from patients with immune dysregulation but not by plasma from patients with an intermediate immune state of activation ( figures 5c-5f ); the addition of the specific blocker of the il-6 pathway tocilizumab partially restored the expression of hla-dr on monocytes of all patients with immune dysregulation (figures 5e and 5f ). treatment with tocilizumab in six patients was accompanied by increase of the absolute lymphocyte blood count within the first 24 h ( figure 5g ). il-6 was produced partly by cd14 monocytes and partly by cd4 cells (figure 5h ). (f-j) absolute counts of cd3 + cd4 + cd45 + lymphocytes (f), cd3 + cd8 + cd45 + lymphocytes (g), cd3 + cd16 + cd56 + cd45 + lymphocytes (h), cd3 à cd16 + cd56 +-cd45 + cells (i), and cd19 + cd45 + lymphocytes (j) among patients with severe respiratory failure developing in the field of cap caused by the 2009h1n1 influenza virus and covid-19. all patients with pneumonia caused by sars-cov-2 who develop srf display hyper-inflammatory responses with features of either immune dysregulation or mas, both of which are characterized by pro-inflammatory cytokines: the immune dysregulation described here, which is driven by il-6 and not by il-1b, and mas, which is driven by il-1b. there are two key features of this immune dysregulation: over-production of proinflammatory cytokines by monocytes and dysregulation of lymphocytes characterized by cd4 lymphopenia and subsequently b cell lymphopenia. in parallel, the absolute natural killer (nk) cell count is depleted, probably as result of the rapidly propagating virus. it was previously shown that the addition of il-6 in the growth medium of healthy dendritic cells attenuated hla-dr membrane expression and decreased the production of ifn-g by cd4 cells (ohno et al., 2016) . three findings of our study are compatible with the proposal that il-6 is one of the drivers of decreased hla-dr on the cd14 monocytes: (1) il-6 concentrations in the blood is inversely associated with hla-dr expression ( figure 5a ), (2) the addition of tocilizumab in the plasma-enriched medium of cells partially restored the expression of hla-dr on cell membranes ( figures 5e and 5f ), and (3) absolute lymphocyte counts of six patients increased after tocilizumab treatment ( figure 5g ). our findings are indirectly supported by the increase of circulating hla-dr + cells during convalescence of one case of covid-19 of moderate severity (thevarajan et al., 2020) . in conclusion, we identified a unique signature of immune dysregulation in the patients with sars-cov-2, characterized on the one hand by normal or high cytokine production capacity and bars in each graphic represent mean values and standard errors. only statistically significant comparisons are indicated by the arrows; *p < 0.05; **p < 0.0001; ***p < 0.0001. comparisons were done by the mann-whitney u test followed by correction for multiple comparisons. (k) il-17 production by pbmcs after stimulation with heat-killed candida albicans. bars in each graphic represent mean values and standard errors. statistical comparisons are indicated by the arrows; ns: non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. comparisons were done by the mann-whitney u test followed by correction for multiple comparisons. (legend continued on next page) increased circulating cytokines (especially il-6) and on the other hand by defects in the lymphoid function associated with il-6mediated decrease in hla-dr expression. these findings support the rationale of launched clinical trials on the efficacy of ana-kinra, sarilumab, siltuximab, and tocilizumab (clinicaltrials.gov nct04330638, nct04317092, and nct04315298 and eudract number 2020-001039-29) to elaborate anti-inflammatory responses in these patients. bars in each graphic represent mean values and standard errors. statistical comparisons are indicated by the arrows; ns: non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. comparisons were done by the mann-whitney u test followed by correction for multiple comparisons. six patients with immune dysregulation were treated with single intravenous infusion of 8 mg/kg of tocilizumab (roacremra, roche) based on the national health care off-label provision for covid-19. absolute lymphocyte blood counts were compared before and after tocilizumab infusion. qualitative data were presented as percentages and confidence intervals (cis) and compared by the fisher exact test. qualitative data were presented as mean and standard deviation and compared by the student' s ''t-test''; they were presented as mean and standard error and compared by the mann-whitney u test for small groups. paired comparisons were done by the wilcoxon's rank-signed test. non-parametric correlations were done according to spearman. any value of p below 0.05 was considered significant. covid-19: a novel coronavirus and a novel challenge for critical care development and validation of the hscore, a score for the diagnosis of reactive hemophagocytic syndrome effect of the novel influenza a (h1n1) virus in the human immune system inhibition of caspase-1 activation in gram-negative sepsis and experimental endotoxemia clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan macrophage activation-like syndrome: an immunological entity associated with rapid progression to death in sepsis monocytic hla-dr expression in intensive care patients: interest for prognosis and secondary infection prediction il-6 down-regulates hla class ii expression and il-12 production of human dendritic cells to impair activation of antigen-specific cd4 (+) t cells dysregulation of immune response in patients with covid-19 in wuhan indication for a role of regulatory t cells for the advent of influenza a (h1n1)-related pneumonia the third international consensus definitions for sepsis and septic shock (sepsis-3) resource availability lead contact further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact covid was diagnosed for patients with cap confirmed by chest x-ray or chest computed tomography and positive molecular testing of respiratory secretions. for patients who required mv, blood sampling was performed within the first 24 h from mv and results were used for this analysis. exclusion criteria were infection by the human immunodeficiency virus; neutropenia; and any previous intake of immunosuppressive medication (corticosteroids, anti-cytokine biologicals, and biological response modifiers). srf was defined as severe decrease of the respiratory ratio necessitating intubation and mechanical ventilation written informed consent was provided by patients or by first-degree relatives in case of patients unable to consent. all data of patients with bacterial cap screened for participation in the randomized clinical trial with the acronym provide (clinicaltrials.gov nct03332225) were used. all patients admitted for cap by sars-cov-2 from march 3 until march 30, 2020 participated in the study white blood cells were incubated for 15 min in the dark with the monoclonal antibodies anti-cd14 fitc, anti-hla-dr-pe ) and quantibrite hla-dr/anti-monocyte percp-cy5 ) for 60 min at 37 c in 5% co 2 followed by incubation in the dark for 15 min at room temperature with anti-cd4 fitc, anti-cd14 fitc (beckman coulter) and anti-cd45 pc5 followed by red blood cells lysis. stained cells were then washed with dulbecco's phosphate buffered saline and permeabilized with the intraprep parmeabilization reagent kit they were then diluted in rpmi 1640 enriched with 2mm of l-glutamine, 500 mg/ml of gentamicin, 100 u/ml of penicillin g, 10 mm of pyruvate, 10% fetal bovine serum (biochrom) and suspended in wells of a 96-well plate. the final volume per well was 200 ml with a density of 2 3 10 6 cells/ml. pbmcs were exposed in duplicate for 24 h or 5 days at 37 c in 5% co 2 to different stimuli: 10 ng/ml of escherichia coli o55:b5 lipopolysaccharide (lps detailed methods are provided in the online version of this paper and include the following: e.j.g.-b. conceptualized and designed the study, analyzed the data, wrote the manuscript, and gave approval of the final version to be submitted. m.g.n. designed lab experiments, analyzed the data, drafted the manuscript, and gave approval of the final version to be submitted. n.r., k.a., a.a., n.a., m.e.a., p.k., m.n., m.k., g.d., i.k., d.v., v.l., m.l., a.k., d.t., v.p., e.k., n.k., and a.k. collected blood samples, collected clinical information, reviewed the manuscript, and gave approval of the final version to be submitted. g.d., t.g., p.k., a.k., k.k., and g.r. performed lab experiments, reviewed the manuscript, and gave approval of the final version to be submitted. c.g. and a.k. contributed in data analysis, drafted the manuscript, and gave approval of the final version to be submitted. e.j.g.-b. has received honoraria from abbvie usa, abbott ch, inflarx gmbh, msd greece, xbiotech inc. and angelini italy; independent educational grants from abbvie, abbott, astellas pharma europe, axisshield, biomé rieux inc, in-flarx gmbh, and xbiotech inc; and funding from the framework 7 program hemospec (granted to the national and kapodistrian university of athens), the horizon2020 marie-curie project european sepsis academy (granted to the national and kapodistrian university of athens), and the horizon 2020 european grant immunosep (granted to the hellenic institute for the study of sepsis). a.a. has received honoraria and independent educational grants from gilead, pfizer, msd, viiv, astellas, and biotest. key: cord-306308-zjq6cscm authors: de moura, ronald rodrigues; agrelli, almerinda; santos-silva, carlos andré; silva, natália; assunção, bruno rodrigo; brandão, lucas; benko-iseppon, ana maria; crovella, sergio title: immunoinformatic approach to assess sars-cov-2 protein s epitopes recognised by the most frequent mhc-i alleles in the brazilian population date: 2020-08-05 journal: j clin pathol doi: 10.1136/jclinpath-2020-206946 sha: doc_id: 306308 cord_uid: zjq6cscm aims: brazil is nowadays one of the epicentres of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic and new therapies are needed to face it. in the context of specific immune response against the virus, a correlation between major histocompatibility complex class i (mhc-i) and the severity of the disease in patients with covid-19 has been suggested. aiming at better understanding the biology of the infection and the immune response against the virus in the brazilian population, we analysed sars-cov-2 protein s peptides in order to identify epitopes able to elicit an immune response mediated by the most frequent mhc-i alleles using in silico methods. methods: our analyses consisted in searching for the most frequent human leukocyte antigen (hla)-a, hla-b and hla-c alleles in the brazilian population, excluding the genetic isolates; then, we performed: molecular modelling for unsolved structures, mhc-i binding affinity and antigenicity prediction, peptide docking and molecular dynamics of the best fitted mhc-i/protein s complexes. results: we identified 24 immunogenic epitopes in the sars-cov-2 protein s that could interact with 17 different mhc-i alleles (namely, hla-a*01:01; hla-a*02:01; hla-a*11:01; hla-a*24:02; hla-a*68:01; hla-a*23:01; hla-a*26:01; hla-a*30:02; hla-a*31:01; hla-b*07:02; hla-b*51:01; hla-b*35:01; hla-b*44:02; hla-b*35:03; hla-c*05:01; hla-c*07:01 and hla-c*15:02) in the brazilian population. conclusions: being aware of the intrinsic limitations of in silico analysis (mainly the differences between the real and the protein data bank (pdb) structure; and accuracy of the methods for simulate proteasome cleavage), we identified 24 epitopes able to interact with 17 mhc-i more frequent alleles in the brazilian population that could be useful for the development of strategic methods for vaccines against sars-cov-2. immunoinformatic approach to assess sars-cov-2 protein s epitopes recognised by the most frequent mhc-i alleles in the brazilian population in december 2019, a novel coronavirus strain was detected and isolated in the city of wuhan, hubei province, china. 1 this emerging viral infection was associated with severe human respiratory disease with a fatality rate of ~2%-3%. 2 despite the strict containment measures, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has spread rapidly worldwide, with cases now confirmed in multiple countries and propagation still ongoing. on 30 january 2020, the who declared covid-19 a public health emergency of international concern. 3 brazil is actually one of the epicentres of the pandemic. 4 day by day, the epidemic advances with a high daily rate of cases per million people. nonetheless, these data may be underestimated because of the reduced number of sars-cov-2 molecular detection tests being made. 5 the sars-cov-2 is an enveloped positivestrand rna virus belonging to the betacoronavirus genus of the coronaviridae family of the nidovirales order. 6 sars-cov-2 genome has ~30 kilobases that encode structural and nonstructural proteins. 7 the 5′ encodes two large genes (orf1a and orf1b), which are translated into two polyproteins (pp1a and pp1ab), which are cleaved in a set of non-structural proteins essential for virus replication. 8 the 3′-terminal region of the genome encodes four structural proteins, namely spike (s), nucleocapsid (n) envelope (e) and membrane (m). 9 the protein s on the virion surface mediates receptor binding and membrane fusion. 6 for sars-cov-2 to infect a host cell, it is mandatory that protein s is cleaved (s1/s2 cleavage) by host cell proteases into two units: the n-terminal ectodomain (s1) subunit and the c-terminal membrane-anchored (s2) subunit. 10 during the receptor binding process, s1 binds via its receptor binding domain to the extracellular peptidase domain of the host receptor ace-2, which is mainly expressed in the lung, gastrointestinal tract, kidney and heart tissues, although it is also present in other tissues, dictating viral tropism. 10 11 after that, s2 subunit mediates membrane fusion. although cell entry is not yet fully understood, it is likely that a second proteolytic site (s2′) at subunit s2 is required for viral entry. 12 viral entry triggers the host's immune system and initiates an inflammatory cascade that starts with the mechanism of antigen presentation. 6 during the process of presentation of antigenic peptides to cd8 + (cytotoxic) t cells by class i major histocompatibility complex (mhc-i) molecules, peptides are generated by proteasomal cleavage in the cytosol and transported to the lumen of the endoplasmic reticulum by original research a transporter associated with antigen processing protein before they can bind mhc groove and trigger an immune response. 13 the correlation between mhc-i and the severity of the disease in patients with covid-19 has been previously hypothesised. 14 the understanding of which sars-cov-2 epitopes are immunogenic may help advances in the development of diagnostic kits and prophylactic vaccines. an immunoinformatic approach is suitable for an initial screening, since it may predict algorithms and test for the immunogenicity of a vast quantity of peptides and alleles in a cost-effective manner, reducing the costs and time on workbench. here, we analysed sars-cov-2 protein s peptides aiming to prospect epitopes and their ability to elicit an immune response mediated by the most frequent mhc-i alleles in the brazilian population. we searched for the most frequent mhc-i alleles in the brazilian population through the human leukocyte antigen (hla) allele frequency database. 15 we selected allele frequency data from overall brazilian populations, excluding genetic isolates, such as indigenous tribes or quilombos. after that, we calculated the average allelic frequency for each hla-a, hla-b and hla-c allele with data from more than one population data. finally, we chose the 10 most frequent class i alleles. the protein data bank (pdb) structure of some alleles was not available (table 1) . therefore, we used swiss-model 16 to perform modelling by homology. the best model was chosen using the default parameters. model refinement was made using 3drefine software. 17 the quality of the refinement was evaluated by the molprobity score. for model validation, errat 18 and rampage 19 softwares were used with their default parameters. for immunogenicity inference, we used the netmhcpan v.4.0 20 server, by importing the fasta sequence of the sars-cov-2 spike protein retrieved from pdb (pdb id: 6vsb) that will be subsequently cleaved in peptides with a length of 10-mer. the interrogation of each 10-mer peptide was made querying the selected alleles ranked in the previous section. we accepted as possible candidates, those allele:peptide complexes with strong binding affinity, that is, those with %rank score <0.50. after that, we tested the strong binding peptides for probable antigenicity using vaxijen v.2.0, 21 using a threshold of score <0.5 to filter out possible non-antigenic peptides. peptide docking was carried out using the cabs-dock server. 22 we submitted to a docking simulation, those peptides that showed strong binding affinity and probable antigenicity to their specific mhc-i allele using the default parameters. after the docking, we excluded the complexes that showed root mean squared deviation (rmsd) values above 3 å to follow the molecular dynamics (md). 23 the complexes from the docking simulations (rmsd values below 3 å) were further investigated by md simulations using gromacs 2020.1. 24 the complex was inserted in a 0.7 nm cubic box, filling the voided space with spc-e water molecules. calcium and sodium ions were added for reaching the system electronic equilibrium. energy minimisation was carried out using default parameters. the equilibrium phase was made during 10 ns (for temperature and pressure), while the production phase lasted 100 ns. these steps were also performed using default parameters. in order to investigate the stability of the complexes, we calculated rmsd, root mean squared fluctuation, radius of gyration (rg), solvent accessible surface area and the determination of hydrogen bonds. table 1 shows the most frequent hla-a, hla-b and hla-c alleles, according to the hla allele frequency database. the top 10 hla-a alleles correspond to 68.7% of the a* alleles, whereas the top 10 hla-b alleles represent 53% and the hla-c alleles 68.8%. these values may not be sufficient to depict the mch-i allelic variation, but it can give an estimation of the major diversity of the class i genes. we modelled the mhc-i alleles, whose pdb structures were not available. the templates used for model prediction, refinement and validations are reported in online supplementary table 1. overall, the models were considered suitable for docking analysis since they presented significant amino acid sequence similarity to the templates and were above rampage and errat thresholds (98% and 80%, respectively). two hundred twenty-eight peptides were considered with strong binding affinity, according to netmhcpan v.4.0 results (online supplementary table 2); of these, 73 were also considered probably antigenic based on the vaxijen v.2.0 results. we carried out peptide docking simulations for all of them except for two (pep88 and pep111), which could not be docked by cabs-dock server, possibly due to some issues with the mhc-i structure. as result, 61 peptides had good docking predictions, as they showed average rmsd <3 å (online supplementary table 3 analysing the trajectories for hla-c, it was possible to observe that the most stable complexes were hla-c*05:01, with pep103 (online supplementary figure 19) ; hla-c*07:01, with pep105 (online supplementary figure 20) and hla-c*15:02, with pep109 (online supplementary figure 23 ). the latter complex, though, appeared to be stable only by the end of the simulation. there are a few works in the literature using a similar approach to evaluate possible immunogenic peptides in the sars-cov-2 protein s. one study identified five cd8 + t cells epitopes (ylqprtfll, gvyfastek, epvlkgvkl, vvnqnaqal and wtagaaayy) and eight b cell epitopes that bind the mhc class i and ii alleles more frequent in china. 25 a second study found 13 mhc-i (sqcvnlttr, gvyyhknnk, gkqgnfknl, giyqtsnfr, vsptklndl, kiadynykl, kvggnynyl, egfncyfpl, gpkkstnlv, sprrarsva, lgaensvay, fknhtspdv and deddsepvl) and three mhc-ii possible protein s antigenic peptides. 26 joshi et al 27 reported the mhc-i itlcftlkr as a possible candidate for vaccine development. comparing these previous results with our simulations, only gwtagaaayy complexed with hla-a*01:01 and hla-a*26:01 presented good netmhc-pan and vaxijen scores, as well as good docking stability and rmsd during the md. in conclusion, being aware of the intrinsic limitations of in silico analysis (differences between the real and the pdb structure, accuracy of the methods for simulate proteasome cleavage, as well as molecular modelling, docking and dynamics' shortcomings), we described 24 epitopes present in the sars-cov-2 protein s that could interact with 17 different mhc-i alleles in the brazilian population. these epitopes can elicit an effective cd8 + t cells immune response and could be useful to develop strategic methods for vaccines against covid-19. finally, our immunoinformatic approach could be a useful tool to determine a guided starting point to design and develop epitope-based vaccines. take home messages ► the understanding of which severe acute respiratory syndrome coronavirus 2 (sars-cov-2) epitopes are immunogenic may help in the development of diagnostic kits and prophylactic vaccines. ► an immunoinformatic approach is suitable for major histocompatibility complex class i (mhc-i) screening, to predict the immunogenicity of a vast quantity of peptides and alleles in a cost-effective manner. ► sars-cov-2 protein s peptides have been analysed in silico aiming to prospect epitopes and their ability to elicit an immune response mediated by the most frequent mhc-i alleles in the brazilian population. ► twenty-four epitopes present in the sars-cov-2 protein s have been observed, which could interact with 17 different mhc-i alleles identified in the brazilian population. handling editor runjan chetty. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia real estimates of mortality following covid-19 infection covid 19 public health emergency of international concern (pheic) who. coronavirus disease (covid-2019) situation reports coronavirus pandemic (covid-19) -the data sars-cov-2 and coronavirus disease 2019: what we know so far covid-19, sars and mers: are they closely related? emerging novel coronavirus (2019-ncov)-current scenario, evolutionary perspective based on genome analysis and recent developments preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies structural basis for the recognition of sars-cov-2 by fulllength human ace2 a novel coronavirus associated with severe acute respiratory syndrome the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade characterization of the binding profile of peptide to transporter associated with antigen processing (tap) using gaussian process regression total predicted mhc-i epitope load is inversely associated with mortality from sars-cov-2 allele frequency net database (afnd) 2020 update: gold-standard data classification, open access genotype data and new query tools swiss-model: homology modelling of protein structures and complexes 3drefine: an interactive web server for efficient protein structure refinement verification of protein structures: patterns of nonbonded atomic interactions structure validation by cα geometry: ϕ,ψ and cβ deviation netmhcpan-4.0: improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site heterologous expression of plasmodium vivax apical membrane antigen 1 (pvama1) for binding peptide selection gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of 2019-ncov development of epitope-based peptide vaccine against novel coronavirus 2019 (sars-cov-2): immunoinformatics approach epitope based vaccine prediction for sars-cov-2 by deploying immuno-informatics approach acknowledgements cas-s and amb-i thank capes (coordination for the improvement of higher education personnel, brazil) and cnpq (brazilian national council for scientific and technological development) for financial support and scholarships. patient consent for publication not required.provenance and peer review not commissioned; internally peer reviewed.data availability statement data are available upon reasonable request. all data relevant to the study are included in the article or uploaded as supplementary information. raw data generated in this study may be requested by sending an email to lpm. ccm@ ufpe. br.this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid-19 pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. ronald rodrigues de moura http:// orcid. org/ 0000-0002-9112-7167 key: cord-334603-yt2pmxi3 authors: de sousa, eric; ligeiro, dário; lérias, joana r.; zhang, chao; agrati, chiara; osman, mohamed; el-kafrawy, sherif a; azhar, esam i; ippolito, giuseppe; wang, fu-sheng; zumla, alimudin; maeurer, markus title: mortality in covid-19 disease patients: correlating association of major histocompatibility complex (mhc) with severe acute respiratory syndrome 2 (sars-cov-2) variants date: 2020-07-18 journal: int j infect dis doi: 10.1016/j.ijid.2020.07.016 sha: doc_id: 334603 cord_uid: yt2pmxi3 abstract as the 2019 (covid-19) pandemic caused by the novel coronavirus, sars-cov-2 spreads globally, differences in adverse clinical management outcomes have been associated with associated with age >65years, male gender, and co-morbidities such as smoking, diabetes, hypertension, cardiovascular comorbidity and immunosuppression. ethnicity has been the focus of attention after data from the united kingdom showed a disproportionate number of deaths among healthcare workers from black, asian and other ethnic minority backgrounds (1). in addition to ethnicity, socio-economic factors, prior vaccinations and exposure to other coronaviruses, other factors need to be considered to explain geographical and regional variations in susceptibility, severity of clinical expression of covid-19 disease and outcomes. in the united states there have been disproportionate covid-19 death rates among african americans at around 2.6 times higher than that of other groups. although these data could be due to multiple cultural and socioeconomic factors an underlying genetic susceptibility to sars-cov-2 infection may be a factor. as the 2019 (covid-19) pandemic caused by the novel coronavirus, sars-cov-2 spreads globally, differences in adverse clinical management outcomes have been associated with associated with age >65years, male gender, and co-morbidities such as smoking, diabetes, hypertension, cardiovascular comorbidity and immunosuppression. ethnicity has been the focus of attention after data from the united kingdom showed a disproportionate number of deaths among healthcare workers from black, asian and other ethnic minority backgrounds (1). in addition to ethnicity, socio-economic factors, prior vaccinations and exposure to other coronaviruses, other factors need to be considered to explain geographical and regional variations in susceptibility, severity of clinical expression of covid-19 disease and outcomes. in the united states there have been disproportionate covid-19 death rates among african americans at around 2.6 times higher than that of other groups. although these data could be due to multiple cultural and socioeconomic factors an underlying genetic susceptibility to sars-cov-2 infection may be a factor. genetic factors were thought to play a causative role in the pathogenesis of the sars outbreak in 2003 in a group of taiwanese patients, where the hla-b*4601 haplotype was associated with severity of sars infection (2) . in hong kong chinese patients a strong association was shown j o u r n a l p r e -p r o o f between hlab* 0703 and hla-drb1*0301 alleles and an increased susceptibility to sars infection (3) . in contrast, l-sign homozygote individuals seem to have a significantly lower risk of sars infection (4) . generally, peripheral blood lymphocytes counts of black americans show lower neutrophil counts and proportionally higher frequency of lymphocytes compared to the rest of the population (5). hla-association studies of sars-cov-1 with hla-ligands for sars-cov-2 have been compiled (6) . the biological and clinical relevance of immune responses to sars-cov-2 requires further discussion: 1. autoimmune associations with covid-19. some individuals with covid-19 experienced neurological symptoms, e.g. guillain barre syndrome (7), suggesting an autoimmune background, which has been associated with mhc alleles (8) . the role of mhc variants in increased susceptibility to infections or, vice versa, immune protection is well known for a number of viral diseases, e.g. the role of mhc alleles in hiv-control, or increased risk for chronic hepatitis b (9). dampens autoimmune responses and confers protection from type i diabetes (10) (11) associated with strong ifn-production. hla-dqb1*06:02 has been selected for increased resistance to yersinia pestis in immigrants from africa to europe, engagement of cd4+ t-cells to hla-dqb1*06:02 leads to increased, pro-inflammatory il-17 production, independent of the mhc class ii presented peptides (12) and confers increased risk to the development of anti-myelin directed autoimmune responses (13) . the haplotypes hla-dr2-dq6, dr4-dq8, and dr3-dq2 accommodate peptides from infectious pathogens to cd4+ t-cells from europeans who survived the bottleneck of different, life-threatening infections prevalent in europe (9). these alleles have also shown to be associated with increased risk for autoimmune diseases, for instance to dietary antigens (celiac disease) (14) in part due to their intrinsic capacity to stimulate stronger il-17 production, that facilitates central nervous system (cns)associated disease manifestations (15) . (33) . we used for this prediction a 33 residue peptide with the mutation site in the middle (vnkitygacpkyvkqntlklatgmrnvpekqtr) and thebest fitting peptide with 15 residues that was predicted to binds to hla-drb1*04:01, is exactly the peptide reported earlier (33) recognized by a flu epitope specific t-cell clone. in contrast, the variant (t314k) peptide does not bind ( table 1) . (40), type 1 diabetes (41, 42) , and lyme disease induced arthritis (43) . drb1*09:01 is associated with early childhood myasthenia gravis (44) . drb3*02:02 is linked to grave's disease (44) , serum igg antibodies to chlamydia pneumoniae with essential hypertension (45) and acute necrotizing encephalopathy (46) in conclusion, there appears to be no selective pressure from mhc class i alleles for sars-cov-2 variants tested. most likely there is selective pressure from mhc class ii alleles in regard to binding of the orf8 (l84s) variants assuming that this mutation may be biologically relevant (25, 26) . this data underlines the need to examine sars-cov-2 variants and mhc-associations along with clinical outcomes, a detailed longtime observation with a particular focus on cns-associated symptoms, particularly in individuals with increased risk to develop autoimmune responses. is ethnicity linked to incidence or outcomes of covid-19? association of hla class i with severe acute respiratory syndrome coronavirus infection association of human-leukocyteantigen class i (b*0703) and class ii (drb1*0301) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome homozygous l-sign (clec4m) plays a protective role in sars coronavirus infection black/white differences in leukocyte subpopulations in men hla studies in the context of coronavirus outbreaks guillain-barre syndrome associated with sars-cov-2 infection: causality or coincidence? association between human leukocyte antigen-dr and demylinating guillain-barre syndrome the mhc locus and genetic susceptibility to autoimmune and infectious diseases genetics of the hla region in the prediction of type 1 diabetes crystal structure of hla-dq0602 that protects against type 1 diabetes and confers strong susceptibility to narcolepsy hla class ii molecules influence susceptibility versus protection in inflammatory diseases by determining the cytokine profile dqb1*06:02-associated pathogenic anti-myelin autoimmunity in multiple sclerosis-like disease: potential function of dqb1*06:02 as a disease-predisposing allele the prevalence of hla dq2 and dq8 in patients with celiac disease, in family and in general population mechanisms regulating regional localization of inflammation during cns autoimmunity the autoimmune basis of narcolepsy hpv variants and hla polymorphisms: the role of variability on the risk of cervical cancer association between human papillomavirus 16 e6 variants and human leukocyte antigen class i polymorphism in cervical cancer of swedish women structural analysis of a peptide--hla class ii complex: identification of critical interactions for its formation and recognition by t cell receptor cytotoxic tcell activity antagonized by naturally occurring hiv-1 gag variants natural variants of cytotoxic epitopes are t-cell receptor antagonists for antiviral cytotoxic t cells separation of il-4 production from th cell proliferation by an altered t cell receptor ligand longitudinal analysis of mycobacterium tuberculosis 19-kda antigen-specific t cells in patients with pulmonary tuberculosis: association with disease activity and cross-reactivity to a peptide from hivenv gp120 human papillomavirus type 16 e7 peptide-directed cd8+ t cells from patients with cervical cancer are cross-reactive with the coronavirus ns2 protein phylogenetic network analysis of sars-cov-2 genomes on the origin and continuing evolution of sars-cov-2. national science review tepitool: a pipeline for computational prediction of t cell epitope candidates peptide binding predictions for hla dr, dp and dq molecules netmhciipan-3.0, a common pan-specific mhc class ii prediction method including all three human mhc class ii isotypes quantitative predictions of peptide binding to any hla-dr molecule of known sequence: netmhciipan a review of hla allele and snp associations with highly prevalent infectious diseases in human populations chemistry of peptides associated with mhc class i and class ii molecules structure of a complex of the human alpha/beta t cell receptor (tcr) ha1.7, influenza hemagglutinin peptide, and major histocompatibility complex class ii molecule, hla-dr4 (dra*0101 and drb1*0401): insight into tcr cross-restriction and alloreactivity balancing selection and heterogeneity across the classical human leukocyte antigen loci: a meta-analytic review of 497 population studies association of hla-drb1/dqb1 polymorphism with high-risk hpv infection and cervical intraepithelial neoplasia women from shanghai impact of host molecular genetic variations and hiv/hpv co-infection on cervical cancer progression: a systematic review drb1*03/drb3*0101, and drb3*0202 are susceptibility genes for graves' disease in north american caucasians, whereas drb1*07 is protective e6 and e7 gene polymorphisms in human papillomavirus types-58 and 33 identified in southwest china multiple sclerosis and autoimmune diseases: epidemiology and hla-dr association in north-east italy analysis of hla dp, dq, and dr allesles in adult italian rheumatoid arthritis patients genes for insulin-dependent diabetes mellitus (iddm) in the major histocompatibility complex (mhc) of african-americans hla-encoded genetic predisposition in iddm: dr4 subtypes may be associated with different degrees of protection identification of lfa-1 as a candidate autoantigen in treatment-resistant lyme arthritis a variant of childhood-onset myasthenia gravis: hla typing and clinical characteristics in japan association of hla-drb3*0202 and serum igg antibodies to chlamydia pneumoniae with essential hypertension in a highly homogeneous population from majorca molecular analysis of hla class iiassociated susceptibility to neuroinflammatory diseases in korean children hla-dpb1 and hla class i confer risk of and protection from narcolepsy dna methylation as a mediator of hla-drb1*15:01 and a protective variant in multiple sclerosis professor ippolito, sir zumla and prof mohamed osman are co-investigators investigators of the pan-african network on emerging and re-emerging infections (pandora-id-net) funded by the european and developing countries clinical trials partnership the eu horizon 2020 framework programme for research and innovation. sir zumla is in receipt of as uk-nihr senior investigatorship. professor maeurer is a member of the innate immunity advisory group of the gates foundation and his work is funded by the champalimaud foundation. all authors are members of the global cancer and infectious diseases consortium for host-directed therapies: weblink: https://fchampalimaud.org/covid19/aci key: cord-018714-i291z2ju authors: criado, paulo ricardo title: adverse drug reactions date: 2016-12-31 journal: dermatology in public health environments doi: 10.1007/978-3-319-33919-1_26 sha: doc_id: 18714 cord_uid: i291z2ju adverse events and adverse drug reactions are common in clinical practice. side effects range from the common to the rare and may be confused with other mucocutaneous manifestations resulting from several medications to treat infections, other medical conditions, and in the clinical setting of oncologic treatment. the objective of this chapter to review current data on adverse drug reactions, here classified as (i) severe adverse drug reactions, (ii) uncomplicated cutaneous adverse drug reactions, and (iii) adverse drug reactions caused by chemotherapy drugs, particularly those cases whereby the dermatologist is requested to issue a report and asked to comment on the safety and viability of readministration of a specific drug. we describe aspects associated with these events, presenting a detailed analysis of each of them. • if possible identify the physiopathologic mechanism involved in the reaction; • identify as rapidly as possible the drug inducing the reaction and always opt for its withdrawal; in some circumstances the choice is difficult as there is no alternative drug and its use is essential for the maintenance of life; • a careful and intensive observation is recommended for the occurrence of warning signs regarding the appearance of a potentially severe adverse drug reaction, especially in relation to mucous, oral, ocular, and genital involvement and progression of any present cutaneous eruption; • it is imperative that the drug responsible may be withdrawn on a permanent basis together with chemically related com-pounds, and this advice is also valid for first-degree relatives who can present the same type of reaction. function. the aim of this study is to describe these reactions in order to facilitate recognition and treatment. this group of drug reactions includes anaphylaxis, stevens-johnson syndrome (sjs), toxic epidermal necrolysis (ten), and, depending on the systemic involvement, erythroderma. in this chapter we approach the characteristics and treatment of some adverse reactions to drugs, including anaphylaxis, erythroderma, sjs, and ten. the prevalence of scards is estimated at 1 in 1,000 hospitalized patients. sjs and ten are particularly severe [4] . in general, fatal cutaneous drug-induced reactions occur in 0.1% of clinical patients and 0.01% of surgery patients [1] . scards may be defined as usually requiring hospitalization, at times in intensive therapy or burn care units for close observation of vital signs and visceral function. this group of drug reactions includes anaphylaxis, sjs, ten, drug hypersensitivity, and, depending on the systemic involvement, erythroderma, acute generalized exanthematous pustulosis (agep), cutaneous necrosis induced by anticoagulants, druginduced vasculitis, and reactions such as serum disease [4] . quick differentiation between a scard and a less severe eruption may be difficult, although essential. withdrawal of the suspected drug is the surest way of intervening to reduce mortality [4] . most cutaneous reactions to drugs are usually observed as a morbilliform or maculopapulous exanthema [2, 5, 6] . unfortunately, erythema morbilliform ( fig. 26 .1) most often characterizes the appearance at onset in the severest of cases, including ten, serum disease, and drug hypersensitivity syndrome [4] . djien et al. [3] , studying 133 patients with reactions to drugs clinically presenting with erythematous cutaneous eruptions (morbilliform and scarlatiniform exanthema, maculopapulous, and small isolated papules), concluded that three types of severe clinical markers exist with respect to this kind of reaction: fever, lymphadenopathy, and extensive cutaneous affection. the authors excluded specific forms from the study, such as sjs, ten, fixed drug eruption (fde), agep, phototoxicity, and vasculitis. this suggests that in cases of drug-induced reactions with extensive cutaneous affection, with or without lymphadenopathy, a laboratory investigation is required with a complete hemogram and hepatic function test. in 1994, roujeau and stern [4] put forth clinical and laboratory criteria leading to the suspicion that a reaction to drugs could develop into more severe behavior (chart 26.1). we next discuss the following reactions: anaphylaxis and anaphylactoid reactions, erythroderma, and the clinical spectrum of sjs and ten (lyell's disease). anaphylaxis is a quick systemic reaction usually presenting a risk to life and resulting in immediate hypersensibility mediated by immunoglobulin e (ige). anaphylactoid reactions mimic anaphylaxis, although they are not related to immunologic mechanisms [4, 7] . these reactions lead to a powerful activation of mastocytes, with a massive release of mediators [7, 8] . drugs are not the more important cause of anaphylaxis, as they are responsible for merely 13-20% of cases [8] .drugs that do cause anaphylactic reactions include β-lactam antibiotics (responsible for 75% of fatal anaphylactic reactions in the united states), cephalosporin, sulfonamides, hemoderivatives, enzymes (trypsin, chymopapain, and streptokinase), insulin (very rare nowadays, owing to use of recombinant human insulin), vaccines (due to preservatives, proteic components, and gelatin; some reports of patients show sensitivity to eggs and allergic reactions to vaccines), allergenic extracts, protamine, and progesterone [7, 8] . anaphylactoid reactions may occur with acetylsalicylic acid, nonhormonal anti-inflammatory drugs, iodide contrasts, angiotensin-converting enzyme (ace) inhibitors, and fluorescein [7] . during general anesthesia, anaphylactic and anaphylactoid reactions may occur. these are difficult to differentiate because of the large amount of medications used, such as anesthetics, muscular relaxants, analgesics, nonhormonal anti-inflammatories, and antibiotics [7] . their clinical emergence tends to occur suddenly, within 30-min to 1-h intervals after contact with the precipitating factor, although delayed reactions are rarer. they show an appearance of pruritus, urticaria ( fig. 26. 2), rhi3 lymphocytosis with atypia abnormalities of liver enzymes or function noconjunctival symptoms, angioedema symptoms (especially laryngitis), hypotension, and lung sounds [7] . the following ailments may be observed: abdominal pain, diarrhea, vomiting, uterine contraction, and cardiac arrhythmia. after a few hours symptoms may reappear during a late phase, although this is by no means automatic [4, 7] . patients with anaphylaxis must be identified as fast as possible, and treatment must be initiated immediately [8] . this reduces the risk of fatal reactions [8] . the following are signs of anaphylaxis that pose a risk to life: presence of stridor, edema of the glottis, intense dyspnea, lung sounds, hypotension, cardiac arrhythmia, shock, convulsions, and loss of consciousness [7, 8] . in patients using β-blockers, anaphylaxis is often severe and may be resistant to conventional treatment [8] . various conditions must be considered in the differential diagnosis when suspecting anaphylaxis [8] : cardiac arrhythmias, acute myocardial infarction, food aspiration, convulsive disease, reaction to insulin, pulmonary embolism, syndrome etiology (e.g., the presence of carcinoid tumors or reaction to alcohol and chlorpropamide), hysterical behavior, vasovagal reactions, and fictitious allergic reactions. vasovagal reactions are most often confused with anaphylaxis [8] . in general they are consequences of procedures such as injections, which present as a clinical condition consisting of facial paleness, nausea, profuse sweating, and syncope, with symptoms improving without treatment 20 to 30 min later [8] . absence of pruritus in the presence of a slow pulse and normal blood pressure distinguish vasovagal reactions from anaphylaxis [8] . the treatment of anaphylaxis consists of short-and long-term measures [8] . the immediate goal is to maintain the permeability of the airways and blood pressure, in addition to administering oxygen in more severe cases [8] . epinephrine must be administered as soon as possible, with a standard dose of 0.01 mg/kg of a 1:1,000 solution, up to a maximum of 0.3-0.5 ml, subcutaneously every 10-20 min until the patient is stabilized. this is a condition characterized by a state of generalized erythema and scaling (exfoliative dermatitis) of the skin. it has the morphologic appearance of various cutaneous diseases such as psoriasis, atopic dermatitis, t-cell cutaneous lymphoma, and reactions to drugs [9] . the dissemination of a maculopapular condition caused by medication may lead to the emergence of an erythrodermic syndrome. various types of drug-induced cutaneous reactions (including contact dermatitis, photosensitivity, and maculopapulous reactions) would be responsible for roughly 7.3% of erythroderma cases [10] .the secondary drug-induced erythroderma conditions, as opposed to erythrodermas resulting from other etiologies, most often set in quickly and also tend to regress quickly after withdrawal of the medication being used [10] . pruritus arises 1-4 weeks after starting drug use, in association with diffuse erythema covering roughly 90% of the body surface, followed by lymphadenopathy and scaling. when acute, large amounts of epidermis are exfoliated; when chronic, it produces small elements [9] ( fig. 26.3 ). pruritus and a sensation of diffuse burning occur [9] . exfoliative dermatitis leads to systemic complications such as hydroelectrolytic and thermoregulatory disturbances, high cardiac insufficiency, tachycardia, capillary leak syndrome, and infection [11] [12] [13] . the effect of exfoliative dermatitis on the organism depends on the intensity and duration of the process [13] . common laboratory findings in the erythrodermic state include light anemia, leukocytosis with eosinophil, high ige, an increase of the hemosedimentation process, a drop in serum albumin, and a rise in uric acid [9, 13] . increased ige and eosinophil is a nonspecific finding and is found only in secondary drug-induced erythrodermas, although it might also be due to atopic dermatitis [9, 13] . multiple cutaneous biopsies performed simultaneously on distinct points of the skin might increase the accuracy of the diagnosis of the base disease [9] . in drug reactions vacuolar alterations may be observed on the epidermis, as well as necrotic keratinocytes [9] . the initial treatment of drug reaction in an erythrodermic patient is identical to that for erythrodermas of other causes [9, 13] . suspending the drug is the quickest way to improve the patient's condition. one ought to consider the nutritional state and hydroelectrolytic replacement, as well as administering local measures such as antiseptic baths, humid compresses on the crusts, application of soft emollients, and low-strength corticosteroids [9] . classic oral antihistamines may be prescribed to alleviate the pruritus and anxiety. they provide the patient with a warm and humid environment so as to prevent hypothermia and improve cutaneous hydration [9, 13] . symptoms and signs of cardiac and respiratory insufficiency may require emergency assistance and hospitalization [9] . the most aggressive and debilitating erythrodermic states may require care similar to that offered to sjs or ten patients. the differential diagnosis must be performed with other types of secondary erythrodermas to cutaneous diseases, such as psoriasis, contact dermatitis, seborrheic dermatitis, lichen planus, bullous pemphigoid, and pemphigus foliaceus, as well as systemic diseases such as leukemias, t-cell cutaneous lymphoma, and hodgkin's lymphoma, in addition to erythrodermic states secondary to internal cancer [9, 13] . what currently exists is a combination of concepts according to which spectrum of erythema multiforme (em), including em minor and em major (emm), is separated from another spectrum of reactions, which includes sjs and ten (lyell's syndrome), referred to here as the sjs/ ten spectrum [14] [15] [16] [17] . however, according to assier et al. [18] , it seems possible to separate emm patients from true sjs patients based on clinical symptoms and disease origin. these authors define the emm pattern as consisting of characteristic mucous erosions and cutaneous lesions (typical targets, with or without blisters), symmetrically distributed and commonly acral. sjs would be represented by mucous erosions and disseminated cutaneous purpuric macules that are frequently confluent, with a positive nikolsky sign and epidermal scaling limited to less than 10% of the body surface [14, 18] . em would include recurrent, postinfectious cases (especially related to herpes simplex and mycoplasma), or eventually related to exposure to medication, with a low mortality rate and without lethality. on the other hand, sjs would comprise a severe adr with high mortality rates and a reserved prognosis for many cases [4, 14, 19] . in 1993, bastuji-garin et al. [19] put forward a clinical classification of the spectrum that included me bullosa up to ten. to better understand this classification [19] , we note the characteristics of the dermatologic lesions of which the group consists, defined as follows: • epidermal detachment: refers to epidermal loss, which at times occurs in flaps ( fig. 26.4 ). • typical targets: lesions less than 3 cm in diameter, in disc shape, with well-defined borders, and exhibiting at least three distinct zones, namely two concentric halos around a central disc ( fig. 26 .5). • atypical flat targets: lesions that are not raised, but are round or disc shaped, with two zones and/or borders that are not well defined. • atypical raised targets: round or disc-shaped lesions, palpable or raised, but without the two zones and/or well-defined borders. • macules/spots: erythematous or purpuric stains, irregularly shaped or confluent, with or without blisters (fig. 26.6 ). insofar as the area of epidermal necrolysis makes up one of the two main factors of prognosis, a consensus was reached on classifying the spectrum [14, 19] : (i) sjs in cases with mucous erosions and disseminated purpuric macules and scaling of the epidermis below 10%; (ii) sjs/ten superposition or transition in cases with epidermal scaling between 10% and 30% of the body surface; and (iii) ten in cases with disseminated purpuric macules and epidermal scaling above 30%; or (iv) in rare cases with disseminated necrolysis (over 10% scaling) without any of the lesions described above. sjs is an entity characterized by the presence of lesions similar to those of em, but with purpuric macula and widely distributed blisters or even lesions in atypical targets dispersed over the dorsal aspect of the hands, palms, soles of the feet, extensor region of the extremities, neck, face, ears, and perineum; the face ( fig. 26 .6) and trunk ( fig. 26 .7) are prominently involved [4] . incidence of sjs is estimated at roughly one in three cases per million residents yearly [20] [21] [22] . sjs may be preceded by a discrete maculopapulous eruption similar to exanthema morbilliform [19] . blister formations are possible, though usually not determined by an epidermal detachment of over 10% of the body surface [4, 14, 19] . mucous involvement occurs in roughly 90% of cases, generally on two distinct mucous surfaces; this may precede or follow cutaneous involvement [4, 14, 19] . onset begins with enanthema and edema, which give rise to erosions and pseudomembranous formations on the eyes, mouth, genitals, pharynx, and upper airways [19] . some 10-30% of cases occur with fever and lesions in the gastrointestinal and respiratory tracts [4] . its prognosis seems to not be affected by the type and dose of the drug responsible, nor by human immunodeficiency virus (hiv) infection [4] . the therapeutic options for sjs are limited and controversial [4, 23, 24] . corticosteroids are frequently used [25] , although some cases have not shown a satisfactory response [24] . in agreement with most authors, the use of systemic corticosteroids on the initial sjs and ten forms do not currently demonstrate any proven benefits. the advanced forms of this spectrum of relations have clearly deleterious effects on the patient [26] . the treatment and prognosis of sjs are tackled in combination with that of ten. ten is an entity characterized by extensive scaling of the epidermis in the wake of necrosis (epidermal necrosis) [4, 14, 15] . the term "toxic epidermal necrosis" was introduced by lyell in 1956 [14] . fortunately, it consists of a very rare adverse reaction to drugs. in europe, its incidence is estimated to be at 1.1 [4] cases per million residents yearly [26] . in aids patients, however, the risk of this reaction does rise, estimated at one case in every 1,000 patients yearly [14] . in general, there is a slight predominance among women (1.5-2 cases in females for every male case). indeed, the disease's occurrence in aids patients ends up balancing out the incidence rate between the sexes [14] . the initial characteristics of ten are nonspecific influenza-like symptoms, such as fever, sore throat, coughing, and burning eyes. these are considered prodromic manifestations preceding a cutaneous and mucous affection by 1-3 days [4] . an erythematous eruption emerges symmetrically on the face ( fig. 26 .8) and the upper part of the trunk, extending to the craniocaudal region and provoking symptoms of burning or painful skin [4, 14] . the individual cutaneous lesions are, for the most part, characterized by erythematous macules with poorly defined contours and a purple center. they progressively spread over the anterior thorax and back [4, 14] . less commonly, the initial eruption may consist of an extended scarlatiniform exanthema. in roughly 2-5 days or, at times, within a few hours, or more seldom in about a week, complete extension of the cutaneous condition occurs [14] . at first, some cases may present lesions persisting in sun-exposed areas of the skin [14] . the apex of the process consists of characteristic denuding of the necrotic epidermis, standing out as veritable red strips or flaps on the areas affected by the base erythema ( fig. 26.9 ) [4, 14] . the epidermis is raised by the serum content of flaccid blisters, which are progressively confluent and provoke rupture of the blisters and detachment of the skin. this causes an aspect of severe burns on the patient's skin, with the skin denuded, bleeding, and with an erythematouspurple color, as well as continued elimination of serosity, which contributes to hydroelectrolytic unbalance and accentuated protein loss [4, 14] . the nikolsky sign is positive over widespread areas of the skin [4, 14] . the areas of the skin subjected to pressure, such as the lower shoulders, back, and buttocks, are the first to release epidermal flaps [4, 14] . cutaneous extensor affection might determine a state of acute cutaneous failure [15, 27] . the cutaneous surface can virtually be 100% affected, although scalp affection is exceptional [14] . the mucous membranes are affected in 85-95% of patients, commonly preceding skin involvement by a day or two [14] . in the order of frequency, the disease afflicts the oropharynx, eyes, genitalia, and anus [14] . extensive and painful erosions lead to labial crusts, salivation, feeding obstruction, photophobia, and painful urination and evacuation [14] . severe eye sequelae, with the formation of synechiae between the eyelids and conjunctiva by pseudomembranous conjunctival erosions, and blindness may occur [4, 14] . ceratitis and corneal erosions have been reported, as well as a secondary sicca-like syndrome [14] . high fever or hypothermia may occur because of a thermoregulatory imbalance until complete healing, even in the absence of concomitant infections [14] . the abrupt drop in temperature is more indicative of sepsis than of fever itself [14] . psychomotor agitation and mental confusion are not uncommon, and usually indicative of hemodynamic complications and sepsis [14] . many internal organs are affected by the same pathologic process that involves the skin and determines a spectrum of systemic manifestations [4, 14] . systemic involvement occurs, causing erosion in the esophagus and gastrointestinal tract, which may progress to esophageal constrictions, transaminase increases in 50% of cases (hepatitis in 10%), pseudomembranous colitis, and pancreatitis [23] . in the respiratory tract tracheobronchial erosions and secondary pulmonary interstitial edema, with the correction of hypovolemia, can be found [15] . anemia can be constantly observed, as well as lymphopenia in up to 90% of patients [15] . thrombocytopenia is found in 15% of patients; neutropenia occurs in 30% of cases, and when present indicates a worse prognosis [15, 23] . the medications most commonly causing ten are sulfas, phenobarbital, carbamazepine, dipyrone, piroxicam, phenylbutazone, aminopenicillin, allopurinol, and nevirapine. however, it is necessary to consider that new drugs are continually being reported as triggering ten [4, 14, 15, 23] . the exact mechanism by which sjs and ten develop is not well defined. some authors have suggested the participation of the altered metabolism of drugs with the predominance of a slow acetylator genotype in sjs and ten patients, and a deficiency in the mechanisms involved in detoxification of reactive intermediary metabolites [28] [29] [30] . in addition to the metabolic mechanisms, there is evidence to suggest that, especially in ten, the epidermal necrosis is mediated immunologically [4, 14, 30] . it is known today that sjs and ten are disturbances mediated by t cells, similarly to acute graft-versus-host disease (gvhd), with cytotoxic t cells being responsible for the epidermal necrosis through an apoptosis in keratinocytes [14, 30] . posadas et al. [31] have shown the association of high tumor necrosis factor α (tnf-α) levels with the severity of the reaction. this cytokine has been related to an induction in the adhesion and activation of t cells and monocytes. it also participates in apoptosis, irrespective of the action of perforins [31] . it has been demonstrated also that apart from tnf-α, the perforins granzyme b (grb) and fas ligand (fasl) are found to be high in the initial stages of a drug reaction, particularly in sjs and ten. this reinforces the hypothesis of the participation of cytotoxic mechanisms [31] . nowadays, cytotoxic reaction caused by granulosin liberation from t cells is the major mediator involved in cell apoptosis, and the spectrum of sjs/ten is grouped in type ivc of immune reactions, as classified by pichler. correia et al. [32] have observed a similar seric cytokine profile between ten and acute gvhd. these authors showed a significantly high serum level of interleukin (il)-6 and il-10 in ten and acute gvhd patients as opposed to normal blood donors [32] . il-6 is a multifunctional proinflammatory cytokine produced by various cells, including keratinocytes. it consists of a main circulating endogenous pyrogen [32] . this explains the presence of fever that is unrelated to the infection in the first days of ten and gvhd [32] . in turn, il-10 is an endogenous antipyrogen agent. it is produced by keratinocytes with the purpose of blocking inflammatory cytokines such as il-1, il-6, and tnf-α, in addition to being a powerful suppressant of macrophage, t-cell, and natural killer cell functions [32] . by contrast, as il-10 recruits cd8 + lymphocytes from the peripheral blood, its increase in blister fluid explains the high number of these cells in patients' epidermis [32] .the elevation of il-10 creates a natural mechanism against excessive tissue inflammatory reaction [32] . chosidow et al. [33] have suggested that the cellular cytotoxic targets are viral antigens with a potential to alter immune responses resulting from exposure to medications. treatment for sjs and ten patients is similar to that for patients who have suffered extensive burns, with a number of rare exceptions [23] . all patients have to submit to cutaneous biopsy to confirm the diagnosis [23] . the patient must be observed in an intensive therapy unit, in an isolated and heated environment so as to avoid any cutaneous trauma [4, 14, 23] . the treatment must proceed by suspending any drug that is not essential to the patient's life and begin replacement of intravenous fluid, mainly when an oral mucous lesion obstructs liquids from being ingested [4, 14, 23] . isolation and feeding must be carried out through the nasogastric probe because the patient shows calorie and protein loss [4, 14, 23] . corticosteroids should only be administered within 48 h of the condition's onset. it has not proved to be beneficial after this period because of its delaying epithelialization and increasing protein catabolism, in addition to increasing the risk of infection [23, 26] . antibiotic therapy has to be administered to cases whereby a sudden drop in temperature occurs and with a concomitant drop in the general status or increase of cultivated bacteria on the skin with a predominance of a single strain [23, 26] . it must be emphasized that during the first days, the most common infections are by staphylococcus aureus and later by gramnegatives (pseudomonas aeruginosa) or candida albicans [23] . noncontrolled reports and studies on the treatment of ten exist, reporting the use of intravenous immunoglobulin, cyclosporine, cyclophosphamide, plasmapheresis, and anticytokine monoclonal antibodies, among others, in an attempt to curb the process of epidermal necrosis. the value of these studies has been questioned, however, particularly owing to the fact that in most patients who are hospitalized the phenomenon of necrosis virtually comes to a halt [15] . prins et al. [34] published a multicenter, retrospective study on intravenous immunoglobulin use in treating ten patients, which obtained excellent results. a 48-patient cohort, average age 43 years (±24) and consisting of 24 women and 24 men, with a 10-95% variation of epidermal detachment of the total body surface area, was treated. mucous membrane was affected in 91.7% of these patients. the patients received intravenous infusion of gammaglobulins begun on average 7 days after onset of ten (with a variation of 2-30 days). it was administered over a period of 1-5 days, in doses varying from 0.65 to 5.8 g/kg (mean total dose of 2.7 g/kg). an objective positive response to treatment occurred with a break in the progression of ten, observed in 43 (90%) of the 48 patients. in all there were six deaths. the authors concluded that early use of intravenous gammaglobulin is safe, with a recommended dose of 1 g/kg daily for 3 days in a row. in contrast to the studies of prins et al. [34] , a french group (bachot, revuz, and roujeau) led a noncomparative prospective study of 34 patients diagnosed with sjs (nine patients), sjs/ten overlapping (five patients), and ten (20 patients). they concluded that intravenous gammaglobulin in a 2-g/kg daily dose, administered for 2 days in a row, did not reduce patient mortality [35] . until such discrepancies in the results have been cleared up, intravenous gammaglobulin use in treating ten will remain controversial [36] . however, as the volume of data encourages its application and effective alternative therapies remain lacking, it seems difficult to not suggest a high dose of intravenous gammaglobulin, especially as a way of intervening early in quickly progressing ten cases. whereas mortality rate is low for emm (<1%) and sjs (roughly 5%), it is above 40% for ten patients with macules [37] . the mortality rate rises with age range and increased surface area of the epidermal scaling [37] . we reiterate the classification methodology adopted by multicenter studies, prospectively named scard (severe cutaneous adverse reactions). the results of the latter were recently published based on the analysis of 552 patients and 1,720 controls [38] . this classification system (named scorten) is summarized in chart 26.2. despite the large range and amount of drugs that may pose a great risk of contracting sjs and ten, an annual risk rate of five cases per year among medication users has not been exceeded [39, 40] . [41] [42] [43] dress syndrome is an acronym derived from the term "drug rash with eosinophilia and systemic symptoms" coined by bocquet et al. also known as drug-induced hypersensitivity syndrome (dihs), it was first recognized in 1950 by chaiken in a patient using an anticonvulsant. there are many synonyms used, most of them referring to the origin of the drugs involved in the drug reaction, such as dapsone syndrome, allopurinol hypersensitivity syndrome or the anticonvulsant hypersensitivity syndrome. although a dermatosis is usual in dress, the extent of skin involvement is variable and therefore the "r" in dress was subsequently changed from "rash" to "reaction." clinically, in its complete form, this syndrome includes an extensive mucocutaneous rash, fever, lymphadenopathy, hepatitis, and hematologic abnormalities with eosinophilia and atypical lymphocytes, and may involve other organs with eosinophilic infiltration, producing damage in several systems, especially in kidney, heart, lungs, and pancreas. this multivisceral involvement differentiates dress from other common skin reactions to drugs. another unique feature of this syndrome is its late onset in relation to the period of introduction of the causative drug, i.e., at around 3 weeks to 3 months, and its possible persistence or worsening despite the withdrawal of the offending drug. the incidence of this syndrome is estimated to vary from one case among 1,000-10,000 drug exposures. adults are more affected than children, and although the precise incidence of drug reaction has not yet been determined, it is much more common than sjs, which has an incidence of 1.2-6 cases per million person-years, and most cases are sporadic, with no gender predilection. recognition of this syndrome is of paramount importance, since the mortality rate is about 10-20% and a specific therapy may be necessary. the exact mechanism of dress/dihs remains to be determined but, in cases related to anticonvulsant drugs, three components are considered: (i) deficiency or abnormality of the epoxide hydroxylase enzyme that detoxifies the metabolites of aromatic amine anticonvulsants (metabolic pathway); (ii) associated sequential reactivation of herpesvirus family; and (iii) ethnic predisposition with certain human leukocyte antigen (hla) alleles (immune response). this type of reaction is most commonly seen using seven different drug groups: (i) anticonvulsants, such as the aromatic anticonvulsants (phenytoin, carbamazepine, phenobarbital, primidone), mexiletine, lamotrigine, valproate, ethosuximide, and zonisamide; (ii) antidepressants (desipramine, amitriptyline, fluoxetine); (iii) sulfonamides and sulfones (dapsone, sulfasalazine, trimethoprim-sulfamethoxazole, salazosulfopyridine); (iv) anti-inflammatory drugs (piroxicam, naproxen, diclofenac, sundilac, phenylbutazone, ibuprofen); (v) anti-infectives (abacavir, cidofovir, terbinafine, nevirapine, minocycline, linezolid, doxycycline, telaprevir, nitrofurantoin, zalcitabine, spiramycin, metronidazole, piperacillin-tazobactam, ceftriaxone); (vi) angiotensin-converting enzyme inhibitors (captopril, enalapril); and (vii) β-blockers (atenolol, celiprolol). cases have been reported with allopurinol, gold salts, thalidomide, calcium channel blockers (diltiazem), ranitidine, sorbinil, azathioprine, dobutamine, methimazole, propylthiouracil, and efamizulab. the cases more consistent with dress/dihs were caused by aromatic anticonvulsants, dapsone, salazosulfopyridine, allopurinol, and minocycline. other drugs causing less typical cases are reported in the literature, but less frequently. aromatic anticonvulsants have an estimated occurrence of dress/dihs of one case for every 5,000 people exposed to the drug, and the reaction is especially common among black patients. the aromatic anticonvulsant drugs that have been associated most frequently with drss/dihs are phenytoin, phenobarbital, and carbamazepine. however, newer anticonvulsant medications also containing aromatic structure (felbamate, oxcarbazepine, zonisamide, and lamotrigine) can also be involved, and the cross-reactivity between the various aromatic anticonvulsant drugs is well documented, varying between 40% and 80%. the pathogenic mechanism of idiosyncratic reactions to drugs, such as dress/dihs, has not been fully elucidated. sullivan and shear proposed a multifactorial model for the pathogenesis of dress/dihs. its occurrence would be determined by the combination of exposure to a drug capable of causing adverse reaction given in sufficient dosage and period of use to a susceptible patient. a certain group of drugs associated with dress/dihs, including the aromatic anticonvulsants, is metabolized to reactive oxygen intermediates that appear to be inefficiently detoxified in patients with acquired or pharmacogenetic variations in the metabolism of these drugs. aromatic anticonvulsants such as carbamazepine, phenytoin, and phenobarbital are metabolized by the hepatic cytochrome p450 enzymes and undergo oxidation by aromatic hydroxylation, with subsequent formation of arene oxides. arene oxides are toxic reactive intermediates that are normally enzymatically converted to nontoxic metabolites by epoxide hydroxylase or glutathione transferase. in addition, spontaneous conversion to nontoxic phenol derivatives can occur. in cases of defective or deficient epoxide hydroxylase, arene oxides can accumulate and cause direct cellular toxicity or immune response ( fig. 26.10 ). drug interactions can be important in this syndrome. concomitant use of lamotrigine and valproic acid increases the occurrence of the syndrome. it is thought that the mechanism for this drug interaction is the competition between valproic acid and lamotrigine for hepatic metabolism by glucuronidation, which doubles the halflife of lamotrigine and predictably would increase the possibility of adverse effects. positive patch tests and testing of blast transformation of lymphocytes indicate the presence of an immune reaction in which t cells participate in specific core function. clones of drugspecific t cells have been isolated from patients sensitive to carbamazepine and lamotrigine. several clinical similarities that could be observed between dress/dihs and infectious mononucleosis (im) have led researchers to implicate a possible range of viruses as triggers for this syndrome. in addition, unique features of this syndrome are its late onset in relation to the period of introduction of the causative medication and frequent clinical and laboratory deterioration, as well as episodes of exacerbation despite the withdrawal of the offending drug, so that these characteristics are not necessarily typical of a reaction of specific drug etiology. although there are conflicting views on the pathogenesis of dress/dihs in different parts of the world, recent studies have suggested a close relationship between human herpesvirus 6 (hhv-6) and the development of dress/dihs. sporadic reports have shown that not only hhv-6, but also other herpesviruses such as hhv-7, epstein-barr virus (ebv), and cytomegalovirus (cmv), can be reactivated during the course of the dress/dihs. results obtained with analysis by polymerase chain reaction showed that various herpesviruses are sequentially reactivated during the course of dress/dihs in most patients. the cascade of viral reactivation is initiated by ebv or hhv-6 and extends over a period to hhv-7 and eventually to cmv [1] . in some patients, the clinical manifestations of this syndrome persist despite discontinuation of the drug involved, coinciding with the reactivation of herpesvirus, as shown in fig. 26 .11. the reactivation of hhv-6 is evidenced by increases in the titers of igg anti-hhv-6 dna levels, and hhv-6 is commonly found in the second or third week after the onset of rash, despite the high variability of clinical manifestations among patients with this drug reaction. since the reactivation of hhv-6 can be detected only in patients with dress/dihs, but not other adrs, in japan this diagnostic test has become sensitive and specific for the diagnosis of all patients with dress/dihs [7] . the detection of hhv-6 reactivation seems to be the gold-standard diagnostic test for dress/dihs in japan, with other asian countries and europe helping to confirm the identification of this condition. however, it is still unknown how detection of the viral genome in peripheral blood reflects the true status of viral reactivation in progress in many different organs and systems. specifically, it is possible that in different compartments and organs such as spleen and lymph nodes, different herpesviruses can reactivate in sequential order completely independent of what occurs in the blood, which would explain why blood samples negative for the viral genome are obtained during the clinical activity of dress/dihs. what remains unclear is the role of herpesvirus in early dress/dihs. there are two possibilities: (i) dress/dihs began as an "allergic" immune reaction to a particular drug, which seems to possess an innate ability to stimulate t cells. (2) in genetically predisposed individuals or by additional factors, an impaired detoxification and accumulation of these metabolites occur (3), which can cause cellular damage generating danger signs that can stimulate resting t cells, inducing costimulatory pathways (4). in addition, ethnic predisposition to certain hla types may contribute to the formation of neoantigens from the combination of these intermediary reactive metabolites with tissue macromolecules and formation of haptens (5a), which can be presented via the human histocompatibility complex class i (hla-dr) or class ii (hla-a, -b or -c), to cd4 or cd8 t cells (6) . it was demonstrated that carbamazepine, valproic acid, and amoxicillin are able to exert immunomodulatory actions by inhibiting histone decarboxylase on b lymphocytes, producing a hypogammaglobulinemia that precedes the clinical onset of dress/dihs. the clonal expansion of t cells requires sequential reactivation of latent herpesvirus, and at the same time cd8 + cla + t cells are produced, which are directed toward skin, cd8 + ccr4 + t cells addressed to the lungs (7b), and cd4 + il-4, il-5 producer and il-17 cd4 th17 + producer that cause tissue and peripheral eosinophilia in the context of t-cell activation is a massive activation of herpesvirus housed in these cells, since the stimulation of t cells by the drug may reactivate the viral genome into the cell. thus, the drug in turn can activate a specific cellular and humoral immune response to herpesvirus. this could explain why different herpesviruses are activated and because in another intense immune process, so-called gvhd, a similar reactivation can be observed. (ii) the viral reactivation can occur but is initially clinically unapparent. however, t cells stimulated by virus present significant crossreactivity with certain drugs, and exposure to these drugs leads to an expansion of t cells specific to the drug (and viruses), which persists even after drug withdrawal due to persistence of viral antigens. the simultaneous appearance of multiple concurrent viral reactivation could be explained by the ability of hhv-6 and hhv-7 counterparts to reactivate virus. thus, if the symptoms of dress/ dihs are mediated by both the various gene products and herpesvirus immune responses to viral replication, the frequent deterioration or the several exacerbations that occur despite withdrawal of the offending drug could derive, at least in part, from the sequential activation of this herpesvirus. the viral reactivation may provide a "danger signal" (danger sign) that stimulates massive clonal expansion of both cd8 + and cd4 + nonspecific t cells and causes the complete development of the syndrome. shiohara et al. proposed the possibility that the clinical symptoms during the course of evolutionary dress/dihs do not seem to be only mediated by oligoclonal expansion of drug-specific t cells, but also by antiviral t cells that cross-react with drugs. how is hhv-6 acquired? hhv-6 infects almost all humans around 2 years of age. most infections arise through the exchange of infected saliva during the first year of life, although perinatal transmission can occur. it was demonstrated that the dna of hhv-6 can be integrated into the host dna, and once part of the human dna, congenital transmission can occur [7] . this was also demonstrated in the course of the dress/dihs. the temporal relationship between onset of drug use and the onset of dress/dihs (3 weeks to 3 months) suggests that viruses have no primary function in the syndrome, favoring primary pathogenesis related to drug allergy. patients with dress/dihs have decreased total serum igg, iga, igm, and b-lymphocyte count at onset, while there is an expansion of memory t cells that cross-react with both drug and virus. it is noteworthy that the lymphocyte transformation test is negative in the first week of illness and remains negative in 90% of patients 2 weeks after the onset of symptoms, becoming positive only 5-7 weeks after the initial drug reaction. this could be due to the expansion of regulatory t cells (which suppress the proliferation of memory t cells) in the early stages of the disease and its subsequent reduction by apoptosis. several cytokines are increased during dress/dihs. in particular, levels of tnf-α and il-6, which are typically proinflammatory cytokines, are elevated in this syndrome before the reactivation of hhv-6. interestingly, il-6 becomes undetectable during viral replication and increases again after the infection in most patients. dress/dihs is an entity distinct from other serious adrs because of the dynamic changes in the immune response observed during the course of the disease. the phenotype of circulating cd4 + t cells is changed to cd8 + phenotype at the time of viral reactivation. regulatory t cells are initially increased in number in the circulation and skin, but decrease in parallel the function of the different organs or systems. the reactivation of hhv-6 is considered a condition requiring immunosuppression, demonstrated on several immune abnormalities in the early syndrome: marked decrease of serum immunoglobulins, the number of circulating b cells, and regulatory t-cell dysfunction. moreover, the participation of skin inflammation may be involved in the induction of immunosuppressive conditions. sugita et al. demonstrated a reduction in the number of plasmacytoid dendritic cells (pdc) in peripheral blood of patients, but an increase in the expression of these cells in skin affected by the rash. the pdc human leukocyte subtypes are capable of producing large amounts of interferon-α (ifn-α), which induces the maturation of b cells in order to produce igg and plays a critical role in antiviral defense. the pdc from circulation may accumulate in the skin and thus reduce the number of pdc in the circulation. therefore, antiviral responses may be reduced, facilitating viral reactivation in peripheral blood and tissues other than the skin. although the terms dress and dihs are often and mistakenly used interchangeably, there is currently a tendency to believe that the dihs represents the most severe cases of dress, with reactivation of hhv-6 detected in a large majority of patients and only in a limited number of patients with dress [6] . the most popular hypothesis to explain the immunoallergic reactions to drugs is the theory of hapten/pro-hapten: according to this hypothesis, the drug (or metabolite) is processed by antigen-presenting cells (apcs) and expressed in the cell membrane in the context of hla-a, -b, or -c type i (mhci) or hla-d type ii (mhcii). the complex hla drug (hapten) is presented to native t cells (naive) via their t-cell receptor (tcr), which initiates different types of immune responses, depending on the hla expressed on the apc and the cytokine environment. the story of "hla-drug" correlation truly began in the twenty-first century with abacavir. in 2002, two independent groups observed the abacavir hypersensitivity syndrome and that this was restricted to the allele hla-b*5701, which conferred an elevated odds ratio (>100). glaxosmithkline (london, uk) led the largest international randomized pharmacogenetic clinical trial to date, which demonstrated the correlation between abacavir hypersensitivity reactions and patients with this allele, and proved that the exclusion of abacavir introduction to the patients with this allele resulted in the disappearance of the syndrome, which was first seen in 5% of patients overall who received the drug during the first weeks of antiretroviral treatment. this allele test is now routinely used before the introduction of abacavir in several countries. the hla alleles have a high negative predictive value but low positive predictive value in relation to adrs, indicating that these biogenetic markers are necessary but not sufficient to trigger the allergic immune reactions. according to the theory of hla-drug (hapten), the complex hapten only triggers an immune-allergic reaction in the presence of a specific hla allele. thus, prospective hla screening should prevent some patients from having serious idiosyncratic reactions such as dress/dihs, sjs, and ten if they have a specific risk allele by not receiving the drug related to it. hla pharmacogenomics is a recent field of study that has been rapidly developed and implemented into clinical practice and has improved drug prescription, which is likely to become more and more important in coming years. besides causing sjs and ten, carbamazepine also induces other types of adrs, including maculopapular exanthema (mpe) and dress/ dihs. the association between hla-b*1502 and carbamazepine-induced mpe was not detected in populations of ethnic han chinese and hong kong or thai populations. studies involving 18 han chinese residents in taiwan and 56 caucasians showed no association between cases of dress/dihs caused by carbamazepine and hla-b*1502. these data indicate that the association between hla-b*1502 and cutaneous adrs induced by carbamazepine are specific to sjs/ten. kano et al. showed that in four of their 13 japanese patients (30.8%) with dress/dihs in whom reactivation of hhv-6 was proved, the syndrome was triggered by aromatic anticonvulsants (carbamazepine in ten, phenobarbital in two, and phenytoin in one) had hla-b*1301. the frequency of this allele was much higher than in the japanese population (1.3%). although this difference was not statistically significant after correction for multiple comparisons, the authors proposed that the presence of certain alleles of hla-b on the reactivation of the virus contributed, at least in part, to the association of hla-b allele with dress/dihs. kashiwagi et al. demonstrated a significant association between adverse skin reactions to carbamazepine and hla-a*3101 among 22 japanese patients, including erythema multiforme, erythroderma, dress/dihs, ssj, and other drug reactions. eleven of these patients (50%), including two patients with sjs and others, were carriers of hla-a*3101 and allele frequency was much higher in these patients (25%) than in the japanese population (7.1%) (p = 4 × 10 -4 , odds ratio (or) = 4.33). in a case-control study in a han-chinese population a strong association between the presence of hla-b*5801 and sjs/ten, or dress/dihs triggered by allopurinol among 51 patients (100%) was found, compared with 20 out of 135 (15%) allopurinol-tolerant patients and 19 out of 93 controls (20%) (p (pc value 4.7 × 10(-24), or = 580). japanese patients with different clinical types of cutaneous adrs caused by allopurinol, including sjs, ten, and dress/dihs, had the same hla-b*5801 allele. pirmohamed et al. found an increased frequency of hla-dr3 and hla-dq2 in a group of patients with carbamazepine-induced dress/ dihs (respectively p = 0.01, or = 3.3; p = 0.04, or = 2.7). it was demonstrated that activation of cd4 + t cells with il-2 is essential for the spread of hhv-6 in vitro. genotyping of patients revealed that they had positive hla-dr3 (drb1*0301) and hla-dq2 (dqb1*0201). thus, in recent years increased attention has been given to genetic factors as a cause of variation in both the interpersonal effectiveness and adverse effects of medicines. idiosyncratic reactions are often mediated through immune, usually severe, and unpredictable course. the main region of human dna with genetic variations that predispose to drug hypersensitivity reactions is the region hla. this region harbors the gene locus of most diseases and contains many genes associated with immune functions. although strong associations have been demonstrated between certain hla alleles and some types of adverse skin reaction to drugs, there is no definitive evidence or published data concerning the functions involved in these alleles. the activation of t cells restricted to hla is necessary for the induction of immune reactions and, moreover, there is the possibility that some hla proteins have high binding affinity combined with other drugs or a metabolite of the drug through covalent and noncovalent mechanisms. on the other hand, a protective effect of hla has also been suggested. alfirevic et al. reported a potential protective effect of hla-b*0702 against severe adverse skin reactions induced by carbamazepine in caucasian patients. the implications of pharmacogenomics are varied; one example is the recommendation of the us food and drug administration (fda), which currently recommends genetic testing for users of more than ten drugs currently marketed in that country. histopathology of the skin shows a diffuse, dense superficial and/or perivascular lymphocytic infiltrate. eosinophils in the dermis or swelling may or may not be present ( fig. 26.12a ). on some occasions there is a band-like infiltrate with atypical lymphocytes simulating epidermotropism such as mycosis fungoides. fernando et al. described a patient with dress/dihs triggered by carbamazepine whose rash biopsy presented an unusual form of superficial perivascular inflammatory infiltrate, in which tiny granulomas along with a moderate number of lymphocytes were found. the authors speculated that granuloma formation may be due to a sustained exposure to the drug, even after the onset of dress/dihs. the expansion of cd4 + t cells producing ifn and other cytokines results in recruitment of macrophages which, as a result of maintained exposure to the drug and persistence of cytokine release, promote differentiation into epithelioid cells, which then secrete tnf to promote fusion of these cells into multinucleated giant cells. thus, biopsies of organs involved in dress/ dihs, such as skin and liver, on a significant number of patients may demonstrate the true frequency of granulomatous infiltration in the disease and assist in understanding the pathogenesis of the reaction. the syndrome usually develops within 2 months after drug introduction, more often in 3 weeks to 3 months of the introduction of the drug, or earlier if constituting readministration. fever, often high (38°-40 °c), which is the most common symptom (seen in 90-100% of cases), and rash (87% of cases) are the first signs, especially when related to antiepileptic drugs. the cutaneous eruption consists of a morbilliform rash, which is indistinguishable from the rash of other less severe reactions ( fig. 26.12b, c) . the face, upper trunk, and upper extremities are initially affected, with subsequent progression to the lower extremities occurring in about 90% of cases, which later spreads to the legs and the development of erythrodermic rash. the maculopapular eruption later becomes infiltrated with edematous follicular accentuation. swelling of the face, with marked periorbital involvement, is a warning for the diagnosis, occurring in about 25% of patients, and can be so intense that the patient becomes disfigured. vesicles may arise, and fine bubbles caused by edema of the dermis can be present. no necrosis of the epidermis such as ten occurs, except in rare cases of overlapping dress/dihs and ten. small sterile perifollicular pustules and nonfollicular pustules may appear, which are different from acute generalized exanthematous pustulosis and do not predominate on the main ridges of the skin. often atypical targets may arise. over time the rash becomes purplish, with sharp definition on lower limbs and the resolution of scaling another form of presentation is a picture of exfoliative dermatitis, which may be associated with mucosal involvement, such as cheilitis, erosions, pharyngitis, and enanthematous enlarged tonsils. bilateral edema and infiltration of the salivary glands with xerostomia has been frequently reported. lymphadenopathy is common (70-75% of cases), limited to the lymph nodes or generalized, and painful, gradually resolving with the withdrawal of the drug. the lymph nodes may reveal two distinct types of involvement: a benign pattern of lymphoid hyperplasia with maintenance of normal lymph node architecture, and another a b c various hematologic abnormalities are observed, which consist of marked leukocytosis, eosinophilia (30% of cases), and atypical lymphocytes similar to mononucleosis. these findings guide the diagnosis toward dress, but can sometimes be difficult to distinguish from viral infections such as infection by ebv or hematologic diseases. lymphopenia, leukopenia, or leukocytosis usually precedes it, although they often are not detected because they occur several days before establishment of the clinical syndrome. leukocytosis may be high, up to 50,000 leukocytes/mm 3 , and eosinophilia reaches values higher than 20,000/mm 3 . the eosinophilia may determine the involvement of internal organs with pulmonary infiltrates. in general, eosinophilia may be observed about 1-2 weeks after the onset of the syndrome, or may even occur after the increase in liver enzymes has normalized. hemophagocytic syndrome (hps) can rarely be observed in the course of dress/dihs. hps is associated with and triggered by various conditions, including viral infections, particularly ebv, malignant tumors, or autoimmune diseases. when involved with the course of dress/dihs, hps usually occurs 2 weeks after the onset of drug eruption. there is a decrease in white blood cells and platelets that are detected simultaneously with elevation of lactate dehydrogenase (ldh). bone marrow aspirate reveals hemophagocytosis in an increased number of macrophages. multiorgan involvement may include a wide variety of organs and systems with myocarditis/ myositis, pericarditis, interstitial nephritis (11% of cases), necrotizing granulomatous vasculitis in kidney, brain involvement (encephalitis or meningitis), colitis, and thyroiditis. this potentially fatal visceral involvement form may be symptomatic or not, and begins 1-2 weeks after the onset of rash. we observed a patient who developed acute pancreatitis that evolved into a lethal course. there are reports of shock and respiratory distress syndrome with hypotension, pyrexia, hepatitis, and renal failure related to a hydantoin reaction. arthritis or arthralgia may occur in the context of this syndrome, including myositis. liver involvement is the most common visceral manifestation (50-60% of patients) after lymphadenopathy. hepatomegaly may constitute a finding on physical examination. hepatitis with isolated elevation of liver transaminases is common (51% of cases), usually anicteric, but liver failure is a leading contributory factor to mortality. liver biopsy shows central lobular necrosis and dense inflammatory infiltrate of lymphocytes and eosinophils or granulomas. the reaction is accompanied by cholestasis and hepatocyte necrosis. in more severe cases, widespread or focal hepatic necrosis may be present. the presence of an active coinfection with hepatitis viruses b and/or c often determines deterioration in liver function and prolonged liver dysfunction. there are few cases reported in the literature of dress/dihs with severe acute hepatitis (defined by the presence of alanine aminotransferase (alt) to more than 10× upper limit of normal and/or acute liver failure, such as coagulopathy and encephalopathy), mostly observed in women between the second and fourth decade of life, especially in relation to the use of sulfasalazine. about 15% result in death or liver transplantation, and the course of the disease is apparently unchanged by the use of immunosuppressants. the rapid recognition of the syndrome and prompt withdrawal of the drug can limit the liver damage, although this may be possibly even worse for several weeks and take months to resolve. renal involvement occurs in about 11% of cases, being particularly evident in cases arising from the use of allopurinol, whereby there was an increase in serum creatinine and urea and decreased creatinine clearance. in urine tests, increased content of eosinophils can be observed. although pulmonary involvement is rarely reported in dress/dihs, interstitial pneumonia with eosinophilia is often observed among patients whose syndrome was triggered by minocycline. possibly the cases with lower intensity of pulmonary manifestations are less reported, leading to a bias in the published literature. pulmonary complications include acute interstitial pneumonitis, lymphocytic interstitial pneumonia, and adult respiratory distress syndrome (ards). myocarditis may develop at the beginning of the syndrome or up to 40 days after establishment. symptoms include heart failure, chest pain, sudden tachycardia, dyspnea, and hypotension in early dress/dihs, but some patients are asymptomatic. the echocardiogram shows a reduction in ejection fraction, chest x-ray demonstrates cardiomegaly, and the electrocardiogram shows nonspecific changes in the st-t segment. there is an increase in enzymes such as cpk and ck-mb, but no apparent changes in levels of troponin-1. neurologic complications include meningitis and encephalitis. meningoencephalitis occurs about 2-4 weeks after initiation of drug reaction, and may lead to coma, seizures, headaches, disorders of speech, and paresis and paralysis of the cranial nerve. gastrointestinal bleeding may be an abrupt complication caused by ulcers derived from cmv. endoscopic examination reveals arterial bleeding from punched-out gastric ulcerations. kennebeck compiled the frequency of clinical manifestations and laboratory data of the anticonvulsant hypersensitivity syndrome: fever (90-100%), cutaneous eruption (87-90%), lymphadenopathy (70%), hepatitis (50-60%), hematologic abnormalities (23-50%), periorbital and orofacial edema (25%), myalgia and arthritis (20%), nephritis (11%), pharyngitis (10%), and pulmonary manifestation (9%). the visceral involvement in acute dress/ dihs until resolution of clinical disease is, therefore, extensive and varied, some of these events being closely related to hhv reactivation: enterocolitis and intestinal bleeding, hemophagocytic syndrome (hps), hepatitis, limbic encephalitis, myocarditis, nephritis, mumps, pneumonia, pleurisy, and the syndrome of inappropriate antidiuretic hormone (siadh). the exclusion of other serious infections, particularly bacteremia, neoplastic diseases (lymphoma, leukemia, hypereosinophilic syndrome, paraneoplastic syndrome), and autoimmune or connective tissue conditions (adult-onset still's disease, lupus erythematosus, vasculitis) is necessary for an accurate diagnosis of dress/ dihs. complications are rare and include limbic encephalitis, thyroid disease, renal failure, splenic rupture, eosinophilic colitis, eosinophilic esophagitis, enterocolitis, and fatal cmv. the mortality rate can reach 20%, especially in cases related to advanced age, renal impairment, jaundice, and hepatitis with reactivation of cmv. by contrast, cases where there is a reactivation of ebv seem to have a less severe course, but are more likely to later (usually after several years) develop autoimmune diseases such as diabetes mellitus type 1 and autoimmune hypothyroidism. several authors have reported the occurrence of autoimmune diseases and/or the production of autoantibodies after the resolution of dress/ dihs, in a period ranging from several months or years after the resolution of the syndrome, and some are similar to those seen after bone marrow transplant. the related conditions include diabetes mellitus type 1, lupus erythematosus, hashimoto's thyroiditis, enteropathy, sclerodermiform lesions, gvhd, and bullous pemphigoid. the diagnosis is difficult since there are incomplete or less characteristic clinical features, for example, hepatitis without rash, or merely pulmonary infiltrate with eosinophilia. bocquet, bagot, and roujeau were the first authors who proposed criteria for dress diagnosis. according to these authors the diagnosis is established if there are at least three criteria present: there is still no international consensus on the best criteria for the definition of dress/dihs diagnosis. bocquet et al. and southeimer and houpt have proposed different definitions and nosology for dress/dihs in order to clarify clinical and pathologic characteristics of this syndrome. the japanese study group for severe cutaneous adverse reactions to drugs (scar-j) has adopted other criteria, as presented on chart 26.4. however, the universal adoption of these criteria may be impaired, because one of the criteria is viral replication during the course of infection, and some tests, such as measurement of igg titer anti-hhv-6, are not yet routinely available in all hospitals or laboratories. in our view, the criteria adopted by the european group regiscar, published by kardaun et al. in 2007, is the best to meet the needs in the diagnosis of dress/dihs. here the use of a system score for the diagnosis of dress/ dihs was suggested, based on the presence of symptoms and clinical and laboratory signs, as displayed in table 26 .1. given the suspicion of the syndrome relevant examinations should be performed, keeping in mind that this syndrome has evolutionary behavior. the initial tests are oriented to verify the data and research into hematological visceral involvement, as proposed by descamps et al. at admission: complete blood count, alt, aspartate aminotransferase (ast), total bilirubin, γ-glutamyl transferase, alkaline phosphatase, sodium, potassium, creatinine and creatinine clearance, 24-h urine protein and urinary eosinophil count, cpk, ldh, ferritin, triglycerides, calcium and parathyroid hormone, blood glucose, prothrombin time and activated partial thromboplastin time, lipase, protein electrophoresis, c-reactive protein, quantitative pcr for hhv-6, -7, ebv, and cmv, blood culture, and antinuclear factor. follow-up (two times per week): complete blood count, alt, ast, creatinine, ldh, and other laboratory tests according to changes found on admission tests. evolutive follow-up: quantitative pcr for hhv-6, -7, ebv, and cmv, complete blood count, alt, ast, alkaline phosphatase, creatinine, ldh, ferritin, and triglycerides. the early recognition of adrs and withdrawal of the offending drug is the most important and essential steps toward clinical improvement. empiric treatment with antibiotics or antiinflammatory drugs should not be administered during the acute disease, since they may confuse or worsen the clinical picture of patients because of an unexplained cross-reactivity between drugs. prognosis is generally worse in the elderly while the recovery is usually faster and usually complete in children. for many years, the treatment of dress has been based on the use of systemic corticostethe diagnosis is confirmed by the presence of the seven criteria (typical dihs) or of the first five criteria (atypical dihs) a this can be replaced by other organ involvement such as renal involvement b reactivation is detected from the second to third week after symptom onset, through igg anti-hhv-6 titer elevation roids (dose equal to or greater than 1-1.5 mg/ kg/day of prednisone or equivalent) with marked improvement of symptoms and laboratory parameters only several days after the start of treatment. systemic corticosteroids should have their dose reduced, after clinical and laboratory control of the disease, slowly over 6-8 weeks to prevent recurrence of the symptoms of disease. abrupt deterioration of various symptoms is observed when the withdrawal is accidental or by rapid reduction of the dose of corticosteroids. shiohara et al. recommend that all patients should be hospitalized even when the initial presentation is mild. if symptoms worsen despite the use of oral corticosteroids, other options used in case series are the use of pulsed methylprednisolone (30 mg/kg intravenously for 3 days), intravenous immunoglobulin (ivig), and plasmapheresis, or a combination of these therapies. it should be remembered that the immunosuppressive therapies may increase the risk of infectious complications and sepsis. mild cases can recover simply by drug withdrawal and supportive treatment after a few weeks, even without the use of corticosteroids. however, even in mild cases, the monitoring of liver function tests should be conducted and appropriate tests ordered to rule out the involvement of other organs such as lungs, thyroid, and heart. special attention should be given to possible reactivation of cmv, especially in patients with severe dress/dihs. physicians should also pay attention to a proper balance between the needs of corticosteroids for relief of symptoms and been shown that dihs/dress is a manifestation of newly observed immune reconstitution syndrome (irs), and herpes zoster is observed as the most common manifestation of irs after highly active antiretroviral therapy for aids. relevant clues related to dress syndrome include: • dress/dihs is an adr caused by an apparent group of drugs, and one-third of cases are related to anticonvulsants, in addition to sulfonamides and allopurinol, which can cause 10-20% mortality. • the syndrome is characterized by a latency period ranging between 3 weeks and 3 months after the introduction of the offending drug, and its course is marked by apparent sequential reactivation of hhv and subsequent development of autoimmune diseases, providing an opportunity to establish a connection between viral infections and the emergence of autoimmune diseases. • in early dress/dihs hypogammaglobulinemia and reduced peripheral b cells are found, and cd4 + cd25 + foxp3 + (regulatory t cells) levels are high at the beginning of the syndrome, regardless of whether or not patients are treated with corticosteroids. this clonal expansion of regulatory t cells appears to prevent activation of antiviral t cells in an appropriate manner and sequential reactivation of virus is presented in the syndrome. these regulatory t cells have the phenotype ccr4 + and cla + , which address the skin. in the last stage of the syndrome's activity, phenotype of cytotoxic t cells becomes prominent and cd4 + lymphocytes are intensely diminished. these cells are depleted over time, suffering apoptosis and becoming reduced after the resolution of the syndrome, which could be a predisposing factor for the development of autoimmunity. pustulosis [45] agep is a clinical entity that appears in the intertriginous areas or on the face as a diffuse erythema (scarlatiniform) with acute presentation. patients report pruritus or local burning sensation. after this appearance, the erythema is replaced by hundreds of nonfollicular sterile small pustules (<5 mm in diameter) (figs. 26.13 and 26.14). these pustules may sometimes converge and mimic nikolsky's sign, leading to misdiagnosis as ten. intense edema of the face may occur, with purpuric lesions mainly on the legs and the onset of lesions similar to em of the legs. there may be mucous involvement in about 20% of the patients, although it is usually mild and self-limited, occurring in just one location. the cutaneous symptoms are almost always accompanied by fever of >38 °c. frequently there is leukocytosis in the blood count, and eosinophilia may also occur in one-third of the patients. usually this eruption regresses within 4-10 days after withdrawal of the drug and in typical cases leaves a lamellar or punctiform desquamation. disease prognosis worsens when there is hyperthermia or infection of the lesions, and when it affects elderly individuals, who should be hospitalized. the drugs described as a cause of agep are most frequently β-lactams (penicillin, cephalosporins), macrolides (azithromycin, erythromycin), cyclines (doxycycline), sulfonamides (trimethoprim, sulfasalazine), chloramphenicol, isoniazid, streptomycin, vancomycin, quinolones (ciprofloxacin, norfloxacin), itraconazole, terbinafine, allopurinol, carbamazepine, phenytoin, diltiazem, nifedipine, chromium picolinate, diclofenac, enalapril, disulfiram, furosemide, hydroxychloroquine, paracetamol, mercury, thalidomide, protease inhibitors, and bamifylline. sidoroff and et al. proposed some characteristics that might aid in the differentiation between pustular psoriasis and agep. in the latter, a history of psoriasis is rare, the lesions are most frequent in the cutaneous folds, the duration of the fever and the pustules is short, and there is usually a history of recent exposure to the drug; arthritis is rare. histopathology may show subcorneal and/or intraepidermal spongiform pustules, edema of the papillary dermis, vasculitis, exocytosis of eosinophils, and focal necrosis of keratinocytes (fig. 26.15 ). on the other hand, in pustular psoriasis a history of psoriasis is common, the involvement is generalized, the duration of the fever and the pustules is longer, history of drug exposure is less frequent, arthritis occurs in about 30% of the patients, and histopathologic recently, britschgi and colleagues demonstrated high expression of il-8 in these patients. it is known that il-8 is a chemokine with potent activity in the recruitment of neutrophils, which is produced by the keratinocytes and mononuclear cells of the cutaneous inflammatory infiltration. these authors concluded that agep might be the expression of a reaction whereby a cell bound to the drug triggers a drug-specific cd4 + and cd8 + immune response, which results in high expression of il-8 (type vid in the pichler classification). [45] in 1905, von piquet and shick described serum sickness in children treated with horse serum containing diphtheria antitoxin. more recently serum sickness has been observed in patients treated with horse antithymocyte globulin or vaccines of rabbit antihuman diploid cells. this constitutes a type iii hypersensitivity reaction, mediated by immunocomplexes deposited on the walls of the vessels, activation of the complement, and recruitment of granulocytes. it presents particular cutaneous manifestations: typically there is erythema in the lateral portion of the fingers and toes that precedes a more disseminated eruption (occurring in 90% of cases), which frequently is morbilliform (twothirds of the patients) and sometimes urticariform. the presence of urticaria, leukocytoclastic vasculitis, and multiform erythema is rarely observed. in half of the cases there is visceral involvement [1] . the following clinical findings are common: fever, cutaneous eruption, constitutional symptoms, arthritis, and arthralgia. the disease begins about 8-14 days after the initial exposure to the foreign protein. the drugs related with this type of manifestation are the heterologous sera and vaccines. serum sicknesslike reactions can also be caused by penicillin, cephalosporin, minocycline, propranolol, streptokinase, and nonhormonal anti-inflammatories. there are no data on the prevalence of this disease in brazil, although reports of cases of this disease are not infrequent in the medical literature. fractions c3 and c4 of the complement are strongly decreased in serum sickness while they are usually normal in serum sickness-like reactions. treatment of the disease constitutes withdrawal of the drug allied to the use of systemic corticosteroids, in addition to antihistamines for symptomatic relief of pruritus when present. careful observation of the clinical course of the patient's systemic involvement is imperative. [45, 46] several medications can induce a cutaneous vasculitis-type response, the histopathologic definition of which is the presence of inflammation and necrosis in the wall of the cutaneous blood vessels. clinically it presents as tangible purpura or maculopapular purpuric eruption. this disease can also occur in the form of hemorrhagic blisters, urticaria, ulceration, nodules, raynaud's disease, and digital necrosis. the same vasculitis the disease develops about 7-21 days after initiating the drug; however there can be a longer time interval, and any medication instituted within the 2 months prior to the presentation should be considered suspect. given the absence of confirmatory tests for this entity, one should value anamnesis and the correlation with drug exposure, which in general occurs 1-3 weeks before onset of the cutaneous picture. however, the exposure can have occurred in periods as disparate as 2 days to 9 years. withdrawal of the drug leads to a rapid resolution of the picture, and systemic corticosteroids can benefit some patients. the process is usually solved without sequels. the clinical, epidemic, and pathologic characteristics of drug-induced vasculitis have been little reported in the medical literature, since there is no consensus in the definition of this disease, with various revisions using different criteria for inclusion of cases. vasculitis attributed to exposure to medicines is rare, but seemingly account for about 10-20% of dermal vasculitis cases. it is difficult to quantify the frequency with which drug-induced vasculitis is strictly cutaneous. clinical experience suggests that most of the cases are confined to the skin and have a selflimited course, although it can be associated with varied degrees of systemic symptoms including arthralgia, indisposition, and fever. visceral involvement is well described and pathologically heterogeneous. glomerulonephritis and interstitial renal disease, varied degrees of hepatocellular damage, and formation of granulomas in the liver have been described, besides involvement of the heart, lungs, and central nervous system. furthermore, there are rare cases of drug-induced vasculitis with renal and hepatic involvement in the absence of cutaneous disease. the drugs most frequently referred to in the literature, in the form of case reports or series studies, as causative of vasculitis are propylthiouracil, hydralazine, granulocyte colony-stimulating factor (g-csf), cefaclor, minocycline, allopurinol, d-penicillamine, phenytoin, isotretinoin, and methotrexate. as many of the cases of drug-induced vasculitis are not reported in the literature, other drugs are also possible important causative agents of this reaction type. other drugs have been reported less often as causal agents of vasculitis: several antibiotics, etretinate, didanosine, zidovudine, acebutolol, atenolol, sotalol, propranolol, chlorothiazide, furosemide, diltiazem, nifedipine, methyldopa, captopril, enalapril, lisinopril, losartan, procainamide, quinidine, antithyroid medications, painkillers and antipyretics, levamisole, tamoxifen, arabinoside c, interferon, interleukin-2, sulfasalazine, etanercept, gold, carbamazepine, antidepressants, zafirlukast, chromalin, cimetidine, ranitidine, l-tryptophan, radiocontrast, streptokinase, heparin, coumarin, chlorpromazine, metformin, pimagedine, and diphenhydramine. drugs that induce vasculitis associated with antineutrophil cytoplasmic antibodies (anca) include hydralazine, propylthiouracil, minocycline, and anti-tnfα biological agents. about 20% of the patients who use propylthiouracil develop anca, a fact that is related to a higher risk of glomerulonephritis. a particularly relevant form among the drug-induced vasculites is propylthiouracil hypersensitivity vasculitis. there are cases with other antithyroid compounds, such as methimazole, thiamazole/ methylthiouracil, and carbimazole, which, similarly to propylthiouracil, contain a thioamide group and cause allergic cross-reactions. although uncommon, nowadays a larger number of case reports of this entity are observed, suggesting that cases were previously not reported or were included among other nosologic entities, since propylthiouracil is a drug classically dedicated to the treatment of hyperthyroidism. the clinical symptoms and signs begin after initiating propylthiouracil. although the duration of drug use is extremely variable, from 1 week to 13 years, it appears under a classic tetrad of symptoms that include fever, sore throat, arthralgia and cutaneous eruption; there can also be myalgia, fatigue, weight loss, conjunctivitis, rhinitis, and hemoptysis. the disease course is that of a systemic vasculitis. there can be a lupus-like syndrome, wegener-like granulomatosis, or nodular-like polyarthritis with multiple involvement of organs, such as kidneys, joints, lungs, and others associated with cutaneous lesions. the cutaneous lesions usually consist of plaques or acral purpuric nodules arranged in a livedoid pattern, with a preference for the extremities (fig. 26.16) , face, breasts, and characteristically the lobes and helices of the ears, mimicking the leprosy type reaction of lucio's phenomenon. hemorrhagic blisters appear on these lesions that progress to central necrosis of the skin, which can be so extensive that it simulates the clinical presentation of purpura fulminans observed in septic infectious states with disseminated intravascular coagulation. laboratory tests reveal anemia, leukopenia, and platelet depletion in the blood count; increased erythrocyte sedimentation rate, urea, creatinine, transaminases, and bilirubin; hypoalbuminemia; alterations in the coagulation time, prothrombin time, and partial activated thromboplastin time; and immunological abnormalities such as positive anca, rheumatoid factor, and hypergammaglobulinemia can be found. positivity can also be present in anti-ssa, antidouble-stranded dna, anticardiolipin, antismooth muscle antibodies, antimitochondrial, parietal, and antiadrenergic antibodies, besides hypocomplementemia, cryoglobulinemia, and elevation of c-reactive protein. histopathologic study demonstrates a leukocytoclastic vasculitis of the superficial and profound vessels of the dermis. the finding of immunocomplexes deposited in the vascular wall is uncommon, such that some authors have named them pauci-immune anca-positive vasculitis. most of the patients recover completely following withdrawal of propylthiouracil, although some develop impairment of the kidneys or other internal organs, or skin, requiring high doses of prednisone for several months. the dermatologic findings in patients with drug-induced vasculitis associated with anca include plaques and purpuric acral nodules, which appear more commonly on the extremities, face, breasts, and ears. in addition, the patients report the same signs and symptoms as found in other small-vessel vasculites associated with anca (wegener's granulomatosis, churg-strauss syndrome), including glomerulonephritis, pulmonary hemorrhage, and digital gangrene. besides withdrawal of the offending drug, it is generally necessary to use corticosteroids in high doses or in pulse therapy, plasmapheresis, and immunosuppressants for several months. the mortality rate is approximately 10%. necrosis [45] this is a rare and severe adverse effect from treatment with warfarin (anti-vitamin k agents), occurring with cutaneous necrosis secondary to occlusive thrombosis in the vessels of the skin and subcutaneous cellular tissue. it usually presents 3-5 days after use of the drug, as painful (fig. 26.17) , with hemorrhagic blisters or necrotic scars in the areas rich in subcutaneous tissue, such as buttocks, breasts, and hip. the risk of this disease increases in patients who are female, obese, and users of high doses of the medication [1] . the necrotic tissue requires debridement and grafts. this type of reaction has also been described with the use of heparin. this kind of adr is represented by several conditions related to drug exposure, which do not represent life-threatening conditions to the patients except discomfort. there is no severe temporary or permanent remaining lesions or long-term internal organ sequelae and, in most of cases, no mortality or severe impact on patients' health. in contrast to severe adrs, in the clinical setting of uncomplicated cadrs admission to the intensive care or burn unit is usually not necessary for the majority of patients. for this reason, the physician must be able to identify the signs and symptoms that indicate severe cadr [47] . in particular, dermatologists must pay attention to identifying these reactions, since the skin is among the most common organs or systems of clinical manifestation of adrs and concurs with at least 15% of adrs [47] . the most common forms of cadrs are urticarial and exanthematous eruptions, which together constitute 90-95% of all cards [47] . these two types carry few to no long-term consequences [47] . the severe adrs (sadrs), described earlier in this chapter, probably represent around 2% of all adrs [47] . sadrs often are associated with high levels of morbidity and mortality, and therefore a prompt recognition of the reaction, withdrawal of all possible offending agents, and appropriate triage, hospital admittance, workup, and specific treatment are critical [47] . regarding studies of severe versus uncomplicated adrs, swanson and colven [47] proposed a staged patient evaluation as shown in fig. 26 .17. signs and symptoms severe adr are listed in fig. 26 .18, and in this clinical scenario the physician should have a low threshold to admit the patient to hospital, perform a complete workup including evaluation of other medical specialties, withdraw suspected medications, and initiate adequate therapy when indicated [47] . another relevant aspect in recognizing the type of adr is the time from medication introduction to the onset of a cutaneous reaction, since this is related to the subtype of adr, as proposed in fig. 26 .19 [47] . often patients have been exposed to several medications in the same period, and creating a "drug list" that details the dates of all medications taken is helpful in narrowing down the most probable culprits [47] . physicians should pay attention to prodromal symptoms (skin pain, fever, malaise, throat pain or discomfort, arthralgia, etc.) that can to precede the cutaneous eruption, and associated internal symptoms (abdominal pain, ocular discomfort, dysuria, respiratory distress, etc.), and proceed to complete physical examination including full skin examination of groin, genitalia, eyes, oropharynx, thorax auscultation, abdomen, and lymph node palpation [47] . the physician needs remember that several risk factors for the development of more severe cutaneous adrs have been identified, including female gender, older age, viral infections (herpesvirus family or hiv), genetic susceptibility (specific single-nucleotide polymorphisms in the hla region), iatrogenic immunosuppression, underlying immune-mediated diseases, and cancer [48] . [48, 49] exanthematous or maculopapular drug eruptions, sometimes inappropriately designated "drug rashes" or "drug eruptions" by some generalists, are the most common adrs in the skin. the eruption usually occurs between 4 and 14 days after the initiation of a new medication or chemical substance, although it can develop sooner, exanthematous eruptions generally are composed of erythematous macules and/or papules and more rarely by vesicles or pustules, usually with a pattern of symmetric distribution on skin. the eruption often begins on the trunk followed by centrifugal dissemination to the proximal limbs. skin lesions progressively become confluent and may cover large areas of the body (fig. 26.20 ). pruritus and/or low-grade fever are often associated with the exanthema. in some patients the exanthema may progress to erythroderma or more severe reactions such as sjs/ten or dress syndrome after some days or weeks. under histopathology examination this type of adr demonstrates interface dermatitis with vacuolar changes in keratinocytes at the basal layer of the epidermis, and upper dermal mononuclear cells infiltrate with some eosinophils. the pathogenesis involves the overexpression of several cytokines of th2 pattern, such as il-5 and il-13, causing epidermal damage by uncomplicated exanthematous drug eruptions can occur with almost any medication, but the following drugs have higher risks (more than 3% of patients): allopurinol, aminopenicillins, cephalosporins, antiepileptic drugs, and antibacterial sulfonamides. viral infections may increase the incidence of morbilliform drug eruptions, as seen in the setting of mononucleosis infection under treatment with ampicillin, or in severe exanthema with internal damage as in dress syndrome related to the hhv family (ebv, cytomegalovirus, hhv-6 and -7). morbilliform reaction is the most common presentation of exanthematous drug eruption. morbilliform is defined as a rash resembling measles and is clinically depicted by erythematous macules and/or papules, often coalescing into larger plaques. many studies have shown that cutaneous biopsy alone cannot distinguish with certainly that a reaction is due to a drug. there are some clues that suggest the diagnosis: (i) epidermis (mild spongiosis is the most consistent feature, with occasional hyperplasia of the epidermis. few lymphocytes are commonly present in the epidermis. in 97% of biopsies, vacuolization was found in the dermoepidermal junction); (ii) dermis (perivascular infiltrate is virtually always present, composed of lymphocytes and in 60% of cases scattered eosinophils); (iii) papillary dermal edema; (iv) dilated lymph and blood vessels. the primary differential diagnosis for morbilliform eruptions includes viral exanthemas (e.g., ebv, hhv-6, and cmv), bacterial toxin resection (streptococcal or staphylococcal), kawasaki syndrome, and others such as secondary syphilis, scarlet fever, acute hiv, or acute gvhd. the treatment is supportive. the first measure is the withdrawal the causative agent. topical corticosteroids and systemic antihistamines can be administered in the first step. if necessary, this can be combined with a short cycle of systemic corticosteroids (oral prednisone, 0.5 mg/kg/day, with progressively tapering dosages over several days). antihistamines are indicated as adjuvant therapy in cases of itching. [48] [49] [50] drug-induced urticaria is the second most common form of cutaneous drug reaction after exanthematous reactions. urticarial eruption can be broken down into simple acute urticarial eruptions, those involving angioedema or anaphylaxis, and serum sicknesslike reactions as previously described in this chapter. simple urticarial reactions caused by drugs consist of erythematous and edematous lesions, which have central clearing with a red border. the lesions can be located anywhere on the body and wax and wane over hours to days. pruritus is an associated symptom. this type of drug reaction takes place minutes to days after exposure to the offending drug. common drugs responsible for urticarial reactions include antibiotics, such as penicillins, cephalosporins, sulfonamides, and tetracyclines, generally due to ige-mediated hypersensitivity reaction. another common class of drugs related to urticarial eruptions is nonsteroidal anti-inflammatory drugs (nsaids). nsaids cause most frequently non-ige-mediated urticaria and angioedema because of their pharmacologic activity of cyclooxygenase-1 enzyme inhibition, particularly of prostaglandin e2, and results in the generation of leukotriene c4 and activation of inflammatory cells. urticaria and angioedema are associated in about 50% of cases. regarding ace inhibitors, angioedema is described in 0.5% of patients treated with this class of drugs, and often without urticarial lesions. rarely, angiotensin ii receptor blockers result in the same complication. oral or injectable antihistamines and systemic corticosteroids are sometimes needed for severe acute urticaria and intramuscular epinephrine for angioedema. in cases of isolated angioedema caused by ace inhibitors, epinephrine will not control the symptoms and it is necessary to use the selective bradykinin b2 receptor antagonist icatibant in this clinical setting. [49, 51] this entity is defined as recurrent lesions that, upon repeated uptake of the causative drug, always appear at the same skin or mucosal sites. fdes present as well as circumscribed, single or multiple, often pruritic or burning erythematous, dusky patches (fig. 26.21) , ranging from several millimeters to over 10 cm in diameter. vesicles or even blisters can develop. as the lesions resolve, they leave residual hyperpigmentation. the hallmark of fdes is geographic memory. if a reaction recurs, it tends to recur in the same location as previously (although a new location can also be involved). lips, hands, genitalia (especially male genitalia), and occasionally oral mucosa are favored sites of fde occurrence, although the lesions can be found anywhere on the skin and mucous membranes. after intake of the offending drug, fde appears within minutes up to several hours (about 30 min to 8 h). the cutaneous lesions can be accompanied by general symptoms such as fever, nausea, dysuria, abdominal cramps, and diarrhea, though rarely. on occasion the disease presents in an atypical form with blunt-margined, non-pigmented, giant (>20 cm in diameter), urticarial, purpuric, targetoid, linear, reticular, and butterflylike lesions. the histopathologic hallmark is brisk interface dermatitis with varying amounts of epidermal necrosis as well as melanophages and eosinophils in the upper dermis. fdes reveal a reaction pattern with lichenoid or erythema multiforme-like changes. atypical histopathologic reaction patterns such as leukocytoclastic vasculitis, neutrophilic reaction, and a predominantly dermal reaction without pigment incontinence in what is termed nonpigmented fde have been reported. the pathogenesis of fde is based on the new subclassification of delayed type iv immune reactions (werner pichler), a type ivc reaction. in this kind of immune response cytotoxic t cells play a predominant function, whereby autoaggressive αβ + cd8 + memory t cells persist intraepidermally in previous fde sites and play a central role in new flare-ups during drug recall. under drug exposition, keratinocytes are stimulated to participate in immune response through tnf-α and a rapid expression of icam-1 molecule, and then stimulate cd8 + t cells to produce ifn-γ and many drugs have been found to cause fde, with common offenders including sulfonamides, nsaids (e.g., ibuprofen), allopurinol, barbiturates, hydroxyzine, laxatives, tetracycline, phenolphthalein, and feprazone. usually there is only one causative drug (monosensitivity), although sometimes several drugs can induce fdes in the same patient (multisensitivity). the most common multisensitivity is the cross-reaction between chemically related drugs such as tetracyclines. less frequently, multisensitivity can occur because of polysensitivity, whereby two or more chemically unrelated drugs either induce the identical fde lesion or each drug determine flare-ups in separate lesions. treatment is mainly symptomatic with discontinuation of offending agent, topical corticosteroids, and antihistamines. [49, 52] this kind of adr produces lesions resembling acne vulgaris, but unlike acne vulgaris, druginduced acneiform eruptions typically are not associated with the presence of comedones (blackheads and whiteheads). acneiform drug eruptions appear as erythematous papules or erythematous pustules on the face and trunk (fig. 26.22 ) and proximal extremities, but sometimes can be present on the forearms and legs, an unusual site in acne vulgaris. the most relevant hallmark is the monomorphous pattern of this eruption and the resolution without scarring. drug-induced acneiform eruptions represent only 1% of drug eruptions. several medications are related to flare-ups of drug-induced acneiform eruptions, the most strongly associated being lithium, androgens, oral contraceptives, corticosteroids, vitamin b complex, and nowadays epidermal growth receptor (egfr) inhibitors for chemotherapy. iodine, bromide, isoniazid, actinomycin d, and phenytoin have also been associated. in the last decade, the use of supplementary complexes by bodybuilders, such as milk and whey proteinbased products, have been reported as being involved in acneiform eruptions. this is an effect caused by elevations of postprandial insulin and basal insulin-like growth factor i plasma levels. treatment involves discontinuing the use of the offending drug, except in the case of egfr inhibitors, when discontinuation may not be possible. benzoyl peroxide, topical retinoids, and topical or oral antibiotics, such as doxycycline, can be used to treat the reaction, similar to the treatment of acne vulgaris. lichenoid eruptions [49, 53] drug-induced lichenoid eruptions are uncommon adrs that appear similar or even identical to lichen planus, with shiny violaceous polygonal papules and plaques (fig. 26.23) . drug-induced lichenoid eruptions can present virtually anywhere on the body surface, but certain clues in the distribution can help suggest drug eruption over lichen planus. drug-induced lichenoid eruptions tend to be absent from the flexor surface of the wrists, genitals, and mucous membranes, whereas these locations are often involved in common lichen planus. lichen planus drug eruptions also often favor sun-exposed areas of the body. several drugs have been reported to be related to drug-induced lichenoid eruptions: gold salts, antimalarials, methyldopa, nsaids, penicillamines, lithium, sulfonylureas, phenylenediamine derivatives, thiazide diuretics, β-blockers, omeprazole, and pantoprazole. the time from initiation of the drug to onset of lichenoid drug eruption varies greatly depen ding on the causative medication. reactions caused by naproxen, for example, tend to occur approximately 10 days after administration. by contrast, certain drugs such as lithium, methyldopa, and acebutolol can develop lichenoid eruptions several years later. hiv infection can contribute to lichenoid drug eruptions on photoexposed areas of the body. treatment typically is symptomatic, with topical corticosteroids a mainstay. once discontinuation of the medication has been accomplished, the eruption resolves spontaneously after a period of a few weeks or months. [54] [55] [56] acute photosensitivity ranges from common polymorphous light eruptions to phototoxicity, or rare photoallergies. photosensitivity refers to reactions that occur when a photosensitizing agent (chromophore substance) in or on the skin reacts with ultraviolet (uv) radiation, often in doses smaller than those associated with sunburn. up to 8% of cutaneous drug reactions are photosensitivity eruptions. typically, a photosensitivity reaction occurs within hours to days of exposure to sunlight and may last for up to 1 week or more. more frequent reactions are named "phototoxic reactions," in which skin signs resemble moderate to severe sunburn, with erythema, blistering, weeping, and desquamation. photoallergic reactions resemble eczematous lesions, often in subacute or chronic presentation. phototoxic and photoallergic reactions occur in sun-exposed areas of the skin; however, widespread eruptions can occur, which may suggest a systemic photosensitizing agent (photoallergy). these reactions are dose related and are most commonly seen in patients who have been exposed to high doses of both the drug and uv radiation. one's susceptibility to this type of syndrome is variable and likely based on drug absorption and metabolism, as well as the amount of melanin in the skin. piroxicam, fluoroquinolone antibiotics, tricyclic antidepressants, and nsaids are classes of drugs that have been reported to be frequent photosensitizers, with fluoroquinolones being the most potent. other antibiotics, such as tmp-smx and tetracyclines, have also been implicated. recently voriconazole, a third generation of azole antifungal agents, has been reported as a photosensitivity agent, especially in phototoxic reactions (fig. 26.24 ), in 8% of outpatients treated, besides increasing the potential of nonmelanoma skin cancer (particularly squamous cell carcinomas) arising from potential photocarcinogenesis related to voriconazole. phototoxic reactions are the most common dermatologic adverse effect of amiodarone therapy, affecting 25-75% of patients on long-term treatment. photoallergy is considerably less likely to occur, but the risk also increases with prolongation of the therapy. skin changes usually occur after at least 4 months of therapy and with the minimal cumulative dose, which is 40 g. management of photosensitivity reactions includes limiting exposure to sunlight, using potent sunscreen, and wearing protective clothing. oral and topical corticosteroids agents may be employed in the treatment. [57, 58] coma blisters are uncommon skin eruptions seen in patients with impaired consciousness. the original case was described in a patient who was heavily sedated because of barbiturate intoxication. subsequently anticonvulsants have been reported, including certain benzodiazepines such as clobazam (fig. 26.25) , and valproic acid and amitriptyline overdose. there were a few reports of coma blisters and peripheral neuropathy caused by amitriptyline overdose. these blisters are most often seen in pressure areas, particularly over bony prominences in contact with hospital beds. the hallmark histologic feature that defines coma blisters is eccrine gland necrosis in the skin. differential diagnosis with bullous pemphigoid is obtained with a negative direct immunofluorescence biopsy of the skin. until recently, coma blisters were thought to be a self-limiting process that did not require withdrawal of the offending agent. however, in some patients the eruption resolves only upon withdrawal of the drugs. nodosum [59] erythema nodosum is a skin reaction manifested by tender or painful erythematous subcutaneous nodules, located usually on the extensor aspects of the lower extremities. histologically it is a septal panniculitis without vasculitis. several conditions can be induce and act as an antigenic stimuli, including drugs, benign and malignant systemic diseases, leprosy, and bacterial (e.g., tuberculosis) and fungal infections. frequently the cause is unknown. drugs that may cause erythema nodosum are antimicrobial agents (amoxicillin, penicillin, sulfonamides), bromide, iodine, gold salts, analgesics and antipyretics (including paracetamol), carbimazole, isotretinoin, azathioprine, vemurafenib, gm-csf, oral contraceptives (estrogens/ progesterones), and estrogens. erythema nodosum disappears within a couple of weeks after withdrawal of the causative drug. psoriasis [57, 60] drug-induced psoriasis is well documented. such eruptions may occur in patients with pre-existing psoriasis (exacerbation phenomenon) or those without a personal or family history. lesions typically improve with drug withdrawal, although persistent disease is possible. more frequent drugs involved are β-blockers, lithium, antimalarials, nsaids, anti-tnfα agents (fig. 26.26) , and bupropion. exacerbation of psoriasis caused by the following medications has also been observed: adrenergic antagonists, ifn, gemfibrozil, iodine, digoxin, and clonidine. exanthema [61] in individuals previously sensitized to an allergen through contact, systemic exposure results in the development of a condition classically termed systemic contact dermatitis. one of the most common manifestations of this condition is socalled baboon syndrome (bs). a subsequent study by hausermann et al. [62] examined a series of 100 cases of bs and found that about half of the patients exhibited no evidence of prior skin sensitization. for that group the authors proposed the term "symmetric drugrelated intertriginous and flexural exanthema" (sdrife) to describe a peculiar form of drug rash with symptoms similar to those of true bs. bs is historically often equated with a mercuryinduced exanthem in patients with previous contact sensitization. sdrife specifically refers to the typical clinical pattern of this drug eruption, and the following diagnostic criteria are proposed [62] : (1) exposure to a systemically administered drug either at the first or repeated dose (excluding contact allergens); (2) sharply demarcated erythema of the gluteal/perianal area and/or v-shaped erythema of the inguinal/perigenital area; (3) involvement of at least one other intertriginous/flexural localization (fig. 26.27) ; (4) symmetry of affected areas; and (5) absence of systemic symptoms and signs. several drugs are reported to induce sdrife [63] : (i) β-lactam antibiotics (amoxicillin, ampicillin, amoxicillin/clavulanic acid, pivampicillin, penicillin v, ceftriaxone, cefuroxime, cephalexin) and non-β-lactam antibiotics (including clindamycin, roxithromycin); (ii) corticosteroids: deflazacort; (iii) radiocontrast barium; (iv) other drugs: sulfate iomeprol, iopromide, monoclonal antibodies (cetuximab, glembatumumab), vedotin (cr011, vcmmae), psychopharmaceuticals (risperidone loflazepate ethyl), allopurinol, cimetidine hydroxyurea, heparin (intravenous), ivig, mitomycin c, naproxen, oxycodone, pseudoephedrine, salsalate, terbinafine, and valacyclovir. [64, 65] in 1924, freudenthal described full-thickness dermal necrosis associated with intramuscular injection of oily bismuth suspension, which was used to treat syphilis at that time. he described the histologic appearance of these suspended particles deep within the cutaneous arteries, distant from the injection site. this condition was also described by nicolau the following year, and the syndrome more often bears his name despite freudenthal's precedence in the literature. nicolau syndrome is an iatrogenic syndrome caused by intramuscular injection leading to variable degrees of tissue necrosis, with variable severity, including the skin and deeper tissues. intense pain in the immediate postinjection period and purplish discoloration of the overlying skin, with or without a reticulate pattern (livedo racemosa-like pattern), is highly characteristic of this syndrome. intramuscular, subcutaneous, intravenous, and intra-articular injections have been reported to produce this syndrome. the skin necrosis resolves with severe and disfiguring scarring. it is therefore important that dermatologists and cutaneous surgeons are aware of this agonizing and deforming iatrogenic complication of injections. discoloration of the skin may result in necrosis and ulceration, which might involve the subcutaneous tissue and the muscular layer. paralysis of the lower extremities has been reported and attributed to embolization of the medication, mainly resulting from the force of injection from the gluteal vessels into the internal iliac arteries, and ischemia of sciatic nerve. application of cold devices or compress tends to aggravate the tissue injury and necrosis. several drugs are related to ecm: (i) intramuscular injections (vitamin k, nsaids, hydroxyzine, vaccination, bismuth, benzathine penicillin, penicillin g); (ii) intravenous injections (polidocanol 1%); (iii) subacromial injection (triamcinolone acetate); (iv) subcutaneous injection: pegylated ifn-α, glatiramer acetate; and (v) intra-articular: glucocorticoid. aspirating just before injecting has been suggested as a method of preventing nicolau syndrome, as it is thought to help prevent embolism caused by intra-arterial deposition of medication. however, it is doubtful as to whether nicolau syndrome can be prevented by this method, as the spasm of the vessel or vasocompressive effect in nicolau syndrome is usually difficult to recognize. the essential difference between those cases of ecm and the pathophysiology seen with vascular obstruction by dermal fillers (hyaluronic acid, polymethylmethacrylate microspheres) is that the former often involves inflammatory pathways being activated by the injected material, whereas the latter typically involves a more purely mechanical vascular obstruction (although some dermal fillers may promote blood clotting, hyaluronic acid-based dermal fillers by design are minimally reactive in tissues). the phenomena are similar in that the inciting event is accidental intravascular injection, followed by some degree of intravascular transport, finally resulting in distal vascular obstruction, ischemia, and so forth, such that the ultimate clinical presentation is the same. diagnosis is mainly clinical; cutaneous biopsy reveals necrotic changes caused by ischemia. ultrasonography study of the skin and magnetic resonance imaging help in delineating the extent of damage. prompt treatment has been reported to avert necrosis of the skin. in the immediate post-event period, treatment is based on various approaches to improve blood supply such as pentoxyphylline, hyperbaric oxygen, intravenous alprostadil, and thrombolysis with heparin. intralesional corticosteroid has also been used to reduce inflammation. surgical debridement of the necrotic scar is of utmost importance as it reduces infection and enhances wound healing. [49, 66] the autoimmune form. the most commonly used medication associated with this kind of adr is vancomycin; however, other drugs include amiodarone, atorvastatin, captopril, ceftriaxone, diclofenac, furosemide, lithium, metronidazole, penicillin, phenytoin, piroxicam, rifampin, and trimethoprim-sulfamethoxazole. treatment of drug-induced labd includes discontinuation of the causative agent and treatment with topical or systemic steroids, dapsone, and/or nonsteroidal systemic immunosuppressive agents. pemphigoid [49] this entity is very similar to the autoimmune form. multiple tense bullous lesions appear on the skin and pruritus is a common symptom. often the medications associated with druginduced bullous pemphigoid include furosemide, ace inhibitors (especially captopril and enalapril), penicillin, ampicillin, chloroquine, psoralen-uva treatment, and sulfasalazine. treatment is aimed at discontinuation of the offending drug as well as topical or systemic corticosteroids and steroid-sparing immunosuppressive drugs as indicated. [49] similar other drug-induced reactions related to counterpart autoimmune conditions, druginduced pemphigus most closely resembles pemphigus foliaceus, with flaccid vesicles or bullae that rupture, creating crusted or desquamated erosions, with mucous membranes often spared. the histologic hallmark of this drug-induced eruption is the acantholysis of epidermal cells, but this phenomenon is not a pathognomonic sign of this type of adr. both autoimmune (idiopathic) and drug-induced pemphigus have a positive nikolsky sign, as observed in the sjs/ten spectrum, although sjs/ten does not demonstrate acantholysis in the skin biopsy. drugs containing thiol molecules (penicillamine, thiopurine, pyritinol, gold sodium thiomalate, captopril) are responsible for 80% of the cases, and other drugs implicated include levodopa, penicillin, phenobarbital, piroxicam, propranolol, and rifampicin. drug-induced pemphigus can occur any time within the first year of initiation of one of the offending drugs. treatment generally consists of withdrawal of the drug and use of systemic corticosteroids. [44, [67] [68] [69] [70] the skin, mucous membranes, annexes (sebaceous and sudoriferous glands), and the phaneros (hair and nails) are tissues with rapid cellular proliferation and are thus susceptible to adverse reactions (toxic or hypersensitive) resulting from systemic chemotherapeutic treatment. antineoplastic agents are defined as substances that inhibit or prevent the proliferation of neoplasms. because of their high metabolic rate, the skin, mucous membranes, and annexes are the most important target organs of the toxicity associated with chemotherapy. reactions can present with disseminated exanthematous eruptions, nonspecifically, or as distinct cutaneous lesions. some drugs can trigger localized reactions caused by extravasation to tissues adjacent to the areas of application. exanthematous reactions, such as nonspecific erythema multiforme, are more common, and many of them are attributed to hypersensitivity mechanisms. certain local toxicity, such as alopecia, mucositis, nail alterations, or hand-foot syndrome, is more specific and less common, frequently associated with particular drugs or groups of drugs. the identification of the reaction pattern associated with the trigger drug and of the possible dose-limiting toxicity is of extreme importance to the physician, as is the differential diagnosis with infectious processes and specific manifestations of the neoplasm. [44, [67] [68] [69] [70] alopecia [44, [67] [68] [69] [70] alopecia is the most common adverse skin manifestation of chemotherapeutic treatment. there are two types of drug-induced alopecia: the anagen effluvium and the telogen effluvium. in the anagen effluvium hair loss occurs because of the sudden interruption of the mitotic activity of the hair matrix, 1-2 weeks after the start of chemotherapy, leading to lack of hair production or its thinning (pohl-pinkus constrictions). the weakening of the hair shaft in this context predisposes the hair to breakage and shedding during the act of combing. they involve the hair, eyebrows, beard, axillary hair, and pubic hair. it is dose-dependent and reversible. new hairs often grow back with a different color and texture. in the telogen effluvium, hairs move prematurely to a resting phase with subsequent loss of normal hair. the antineoplastic agents that most frequently cause the anagen effluvium lead to diffuse hair loss, of sudden onset, from 7 to 10 days after the start of chemotherapy. hair loss becomes more pronounced about 1-2 months after the start of treatment. even though hair loss is intense, about 10% of the pilous follicles are usually in a resting phase at the time of the administration of the drug, and this determines incomplete hair loss. with repeated treatment cycles, alopecia totalis may occur. this type of effluvium is generally reversible when treatment is suspended, and occasionally permanent with the use of cyclophosphamide and busulfan. hair grows around 1 cm per month, possibly showing new texture and color. the chemotherapeutic drugs more often associated with alopecia when used in isolation are: (i) complete alopecia (cyclophosphamide at high dose, doxorubicin, docetaxel, dactinomycin, irinotecan, topotecan, bleomycin, paclitaxel); (ii) incomplete alopecia (etoposide, ifosfamide, mitomycin c, 5-fluorouracil, melphalan, mitoxantrone, gemcitabine, vinca alkaloids). most reactions can be reversed by dose reduction or by increasing the interval between doses. some toxic effects can be successfully treated or prevented. medication administered before the chemotherapeutic treatment can prevent hypersensitivity reactions. the use of oral antiseptic solutions is useful in the control of mucositis. some dermatologic reactions to new antineoplastic agents, such as egfr inhibitors, have been associated with anticancer efficacy. other adverse effects may be mistaken for reactions to chemotherapeutic drugs and include infections resulting from immunosuppression, paraneoplastic syndromes, gvhd, nutritional deficiencies, development of skin malignancies, and metastatic primitive tumor. there are several classifications of reactions to antineoplastic drugs. the lack of a systematized multidisciplinary approach does not provide all the microscopic data and physiopathogenic mechanisms that delineate the lesions. therefore, the classification adopted didactically groups with the eruptions based on the target cells and mechanism of action of the drugs. preventive measures to limit hair loss have had limited success. hypothermia of the hair scalp or tourniquets applied in this region may reduce the perfusion of the drug in the pilous follicles and delay the start of or minimize hair loss. this procedure is contraindicated for patients with hematologic neoplasms such as leukemias, lymphomas, and other potentially metastatic tumors of the hair scalp. topical minoxidil is not effective in the prevention of drug-induced alopecia, but may shorten its duration. [44, [67] [68] [69] [70] hair alterations with acceleration of growth and shaft changes are observed with the use egfr inhibitors (fig. 26.28) . alterations [44, [67] [68] [69] [70] nail alterations can present with a reduction of the nail growth speed, fragility, lines of discoloration (mees' lines), transversal depressions (beau's lines), hyperpigmentation, onycholysis with subungual aseptic abscesses, photo-onycholysis, paronychia, and pyogenic granulomas of the periungual folds. nearly all antineoplastic agents can lead to reduction of growth speed, nail fragility, mees' lines, and beau's lines. hyperpigmentation can occur after the use of cyclophosphamide, hydroxyurea, fluoropyrimidines such as 5-fluorouracil, and especially anthracyclines such as doxorubicin and daunorubicin. painful onychomycosis and subungual abscesses are due to the use of taxanes (docetaxel/ paclitaxel) and anthracyclines (doxorubicin). ingrown nails, paronychia, and pyogenic granuloma are associated with the use of tyrosine kinase inhibitors of egfr (fig. 26.29 ), such as erlotinib and gefitinib. the fenestration or avulsion of the lamina should be considered when abscesses that involve more than 50% of the nail bed are present. in these more severe cases, the temporary suspension of treatment, longer intervals between cycles, and dose reduction should be considered. [44, [67] [68] [69] [70] this rare, nonspecific disease often occurs when chemotherapeutic drugs are used in combination, making it difficult to know which drugs are responsible for causing the disease. cytarabine is the most commonly cited drug; however, others are also implicated, such as bleomycin, chlorambucil, cyclophosphamide, cytarabine, doxorubicin, lomustine, mitoxantrone, busulfan, carmustine, cisplatin, cyclophosphamide, etoposide, 5-fluorouracil, methotrexate, and thiotepa. some authors consider neutrophilic eccrine hidradenitis (neh) as a paraneoplastic phenomenon, since it has been found in an early case of acute myeloid leukemia not yet treated. it has been associated with hiv infection, nocardia, serratia, enterobacter, staphylococcus, and with patients receiving gm-csf. the mechanism is unknown, but may be due to the excretion of the chemotherapeutic drug by the eccrine glands and its direct toxic effect on the eccrine epithelium. the clinical condition may be preceded by fever and unspecific clinical signs. skin eruptions are distributed in the head, neck, trunk, and extremities, with lesions that vary from erythema, papules, nodules, and pustules to papular plaques. lesions may be purpuric or hyperchromic, single or multiple. they appear between 2 days and 3 weeks from the start of treatment, regressing spontaneously without scarring or sequelae 1-4 weeks after the suspension of the drug. the differential diagnosis is vast and includes sepsis, septic embolism in a postchemotherapeutic neutropenic patient, vasculitis, leukemia cutis, hypersensitivity reaction, urticaria, polymorphous erythema, and neutrophilic dermatoses such as sweet's syndrome, bullous pyoderma gangrenosum, and atypical pyoderma gangrenosum. owing to the unspecific clinical presentation of the disease and the great number of differential diagnoses, some authors suggest that neh be included in the diagnostic hypotheses of any ingrown nail and pyogenic granuloma in a patient using erlotinib eruption that may occur in patients undergoing chemotherapy, and its final diagnosis is established by histopathology. therefore, histopathology is essential for conclusive diagnosis. it is constituted by a dense neutrophilic infiltrate, inside and around the eccrine glands, with necrosis of the eccrine epithelium cells. involvement of the apocrine glands has been reported. occasionally, squamous syringometaplasia, hemorrhage and edema of the dermis, spongiosis and/or vacuolization of the basal layer of the epidermis, necrosis of keratinocytes, and mucin deposits inside and around the eccrine glands may occur. in patients with severe neutropenia, the neutrophilic infiltrate may be absent; however, necrosis of the eccrine epithelium is typical. neh is a self-limiting adverse reaction. frequently the process resolves within a month, without treatment. in other chemotherapy cycles, 60% of the patients may relapse. the efficacy of the prophylactic or therapeutic use of systemic corticosteroids, dapsone, or nonhormonal antiinflammatories is still questionable. [44, [67] [68] [69] [70] eccrine squamous syringometaplasia is an unusual adverse reaction to chemotherapeutic drugs. it can also be found in association with chronic ulcerations, skin tumors, exposure to toxic agents, and several inflammatory processes. therefore, it is not a histopathologic reaction exclusive to the use of chemotherapeutic drugs. the mechanism of neutrophilic eccrine hidradenitis is unknown, but it can be the result of the excretion of the drug by the eccrine glands and its direct toxic effect on the eccrine epithelium. it is postulated that eccrine squamous syringometaplasia represents the final noninflammatory spectrum of adverse reactions to chemotherapeutic drugs in the eccrine glands. similarly to neh, eccrine squamous syringometaplasia also has an unspecific clinical presentation, constituted by erythematous maculae, papules, and papular plaques or vesicles, localized or disseminated. lesions develop between 2 and 39 days after the start of chemotherapy and improve spontaneously after 4 weeks. the diagnosis is histopathologic, characterized by the presence of squamous metaplasia of the eccrine glands in the papillary dermis. minimal and focal necrosis of the eccrine gland epithelium, fibroblastic proliferation, and edema of the periductal stroma may occur. contrary to neh, the neutrophilic infiltrate is minimal or absent. squamous eccrine syringometaplasia has been described as an accidental histologic finding in other conditions not associated with chemotherapy. eccrine squamous syringometaplasia does not appear to be associated with a specific chemotherapy agent or malignancy. numerous drugs have been related such as cytarabine, mitoxantrone, daunorubicin, cisplatin, 5-fluorouracil, doxorubicin, cyclophosphamide, etoposide, methotrexate, busulfan, melphalan, and carmustine. eccrine squamous syringometaplasia has been observed in association with palmoplantar erythrodysesthesia syndrome, in radiation-induced memory reactions, and in patients who underwent bone marrow transplantation and received high doses of chemotherapeutic drugs. the condition often spontaneously resolves. [44, [67] [68] [69] [70] first described in 1974, this syndrome is also known as burgdorf's syndrome, palmoplantar erythema, hand-foot syndrome, and toxic erythema of the palms and soles. it occurs more frequently in patients treated with cytarabine and, fluoropyrimidines, especially capecitabine, which is the oral 5-fluorouracil prodrug. after alopecia and mucositis, it is the most common adverse reaction to chemotherapy. other agents less frequently associated with palmoplantar erythrodysesthesia syndrome are cisplatin, cyclophosphamide, cytarabine, doxorubicin, daunorubicin, doxifluridine, etoposide, floxuridine, hydroxyurea, mercaptopurine, methotrexate, mitotane, paclitaxel, docetaxel, and vinorelbine. it is estimated that this adverse reaction occurs in 6-64% of the patients treated with different chemotherapeutic regimens. most patients show a prodrome of dysesthesia, with a tingling (pins and needles) sensation on the palms and soles. within a few days the reaction evolves to a feeling of pain and burning with a well-demarcated edema and erythema. the erythema is symmetric and sometimes more pronounced on the soft parts of the distal phalanges. hands are often more affected than feet (fig. 26.30a) . some patients show light scaling with or without erythema. a bullous variant has been described (fig. 26.30b) , representing a more severe form of the reaction, specifically associated with cytarabine and methotrexate. lesions are aggravated if the treatment is not suspended, and the associated pain and edema may limit the movement of fingers. when the drug is suspended, the reaction progressively improves within 2 weeks. in some patients, when treatment is maintained despite the development of erythrodysesthesia syndrome, palmoplantar keratodermia may occur. the reaction occurs more frequently in patients who undergo oral or continuous infusional therapy with fluoropyrimidines (2-18%), as compared with those submitted to bolus therapy (0.4-3%). it is thought that in the pathogenesis of the process the local accumulation of the drug leads to degeneration with necrosis of the sweat glands, because its microscopic aspects are similar to those of eccrine squamous syringometaplasia and neutrophilic eccrine hidradenitis. in the differential diagnosis the following should be considered: polymorphous erythema, erythromelalgia, eccrine squamous syringometaplasia, and neutrophilic eccrine hidradenitis. the most relevant differential diagnosis is acute gvhd. the fundamental difference is that acute gvhd occurs in patients who have received a bone marrow transplant, in addition to extracutaneous involvement with gastrointestinal alterations (abdominal pain and diarrhea, elevation of hepatic enzymes). in cases of acute gvhd without extracutaneous manifestations, differentiation may be difficult. nevertheless, acute gvhd presents with diffuse erythema and can form papules, whereas palmoplantar erythrodysesthesia syndrome shows a well-demarcated erythema and edema. there are no relevant histopathologic differences between them, except for necrosis of the satellite cell in all layers of the epidermis (apoptotic keratinocytes adjacent to lymphocytes) in acute gvhd and sometimes presence of squamous syringometaplasia in palmoplantar erythrodysesthesia syndrome. the differentiation between these two disorders is essential because the use of cyclosporine is necessary to treat acute gvhd, but worsens the patient's pain if used in the treatment of palmoplantar erythrodysesthesia. apart from dose reduction, longer intervals between the cycles of chemotherapy and, as a last resort, the suspension of the drug, there is no specific treatment for palmoplantar erythrodysesthesia syndrome that has proved to be effective in a large series of cases. some treatments have been suggested for small series of patients or case reports. general measures should be taken, such as reduction or suspension of the drug, longer intervals between chemotherapy cycles, dressings, elevation of the extremity, cold compresses, analgesic medication, and emollients. as a specific treatment, pyridoxine can be used if 5-fluorouracil, liposomal doxorubicin, doxorubicin, docetaxel, and etoposide have been administered; hand cooling (docetaxel); oral corticosteroids (doxorubicin, 5-fluorouracil); strong topical corticosteroids (liposomal doxorubicin, cisplatin, and 5-fluorouracil); and topical dimethyl sulfoxide (dmso) at 99% (liposomal doxorubicin). symptoms can be relieved with lesion care to prevent infection and elevation of the limb to reduce the edema. cooling of hands and feet during treatment reduces the blood flow in these areas and may decrease the severity of the reaction. strong topical corticosteroids have been used with mixed results when associated with emollients. systemic corticosteroids are useful in some situations. pyridoxine (vitamin b6) in doses of 200-300 mg/ day can be useful to treat and prevent this reaction, except when cytarabine or vincristine is used. topical dmso at 99% four times a day for 14 days has cured some cases of palmoplantar erythrodysesthesia syndrome induced by pegylated liposomal doxorubicin. [44, [67] [68] [69] [70] some authors prefer to associate toxic erythema caused by chemotherapy with clinical lesions that present with painful erythema, with or without edema, often affecting the hands and feet, intertriginous areas such as the axillary and inguinal regions, and less frequently the elbows, knees, and auricular pavilion. these eruptions may have a bullous component, are self-limited, and generally evolve with resolution and scaling associated with postinflammatory hyperpigmentation. many denominations used refer to histopathologic findings or those given by various authors on different occasions. disorders such as eccrine squamous syringometaplasia, neh, acral erythema, and palmoplantar erythrodysesthesia syndrome would be, according to these authors, grouped under toxic erythema caused by chemotherapeutic drugs. the objective to group many disorders under the same denomination seeks to emphasize the superposition of clinical characteristics and promote an easy dialogue between medical specialties and with the patient. the clinical characteristics of the toxic erythema associated with chemotherapy are: (1) maculae or erythematous and/or edematous plaques on the hands and feet, intertriginous areas, and less frequently on the elbows, knees, and auricular pavilions, often appearing 2-3 days after the administration of the drug; (2) associated symptoms of pain (that may be debilitating), burning, paresthesia, pruritus, and/or hypersensitivity; (3) pale color, petechiae, and/or sterile blisters, followed by erosion in areas of intense erythema; (4) scaling and spontaneous resolution without specific treatment; and (5) chance of relapse if an equal or higher dose is administered. isolated papules may be found in the periphery of plaques. papules and plaques may also be found in the head, cervical region, trunk, and extremities. onset of lesions after 2-10 months can be observed. the histologic characteristics observed are atypia (larger cells and nuclei and nuclear pleomorphism), apoptosis of keratinocytes, mitotic figures and bizarre mitotic configurations (astral mitosis), loss of polarity of the epidermal cells and apoptosis of keratinocytes, vacuolar degeneration of the basal layer of the epidermis, dermal edema, and eccrine squamous syringometaplasia. moreover, necrosis of the upper epidermis, similar to the alterations observed in pellagra, may also occur. the inflammatory infiltrates are usually minimal despite their abundant clinical profile. from these observations, it has been suggested that erythema is secondary and results from damage to keratinocytes, leading to the release of cytokines and vasodilation. [44, [67] [68] [69] [70] acneiform eruption is the adverse effect more often associated with the use of egfr inhibitors. onset occurs 1 week after the start of treatment with the egfr inhibitor as a self-limiting eruption, dose-related, that affects the face, central region of the thorax, upper dorsum and, more rarely, limbs. it presents with follicular erythematous papules, pustules with or without comedones, and scaling of the interfollicular skin (fig. 26.31) . often an association with the following conditions is observed: acral asteatosis, paronychia with pyogenic granuloma, oral and nasal aphthous ulcerations, and hair alterations. palms and soles are often free of lesions. excessive follicular hyperkeratosis leading to the obstruction of the ostium with formation of a follicular corneal plug, rupture of the glandular wall, and consequent inflammatory process are suggested as pathogenic mechanisms. in the histopathologic examination a prominent corneal plug, with dilated infundibulum, with or without neutrophilic folliculitis, is observed. there is a positive correlation between the severity of the eruption and the tumoral response and survival. we emphasize the need for attention to the eruption to improve adherence to the chemotherapeutic treatment. the use of topical anti-acne agents and oral tetracyclines improve the condition. topical emollients are indicated to treat xerosis. [44, [67] [68] [69] [70] stomatitis [44, [67] [68] [69] [70] oral mucositis is the main dose-limiting reaction of most chemotherapeutic drugs. about 40% of the patients being treated show some type of oral complication. these complications are often associated with drugs that affect the synthesis of dna. the main causative agents are antimetabolic drugs and antitumoral antibiotics. the drugs more frequently associated with stomatitis are bleomycin, dactinomycin, methotrexate, topotecan, and 5-fluorouracil. unusually, the stomatitis caused by 5-fluorouracil is related to its continuous infusional administration or to the use of its oral prodrug, capecitabine, and is less frequently observed when 5-fluorouracil is administrated in bolus. the main mechanism is the direct toxicity of the drug, but it can result secondarily from the indirect effects of the drug on the bone marrow. in patients with head and neck tumors, cisplatin used during radiotherapy acts as a strong radiosensitizer. in these cases there is more tumoral control but also greater severity of stomatitis caused by a boost in the direct effect of radiotherapy. since oral epithelium cells have a high mitotic index (renewal every 7-14 days), they become susceptible to the toxic effects of chemotherapeutic drugs. moreover, there is atrophy of the oral mucosa, causing odynophagia, burning, xerostomia, and mucous membrane ulcerations. ulcerations may be initially focal and then become diffuse and confluent, with occasional vesicles and blisters. these alterations are more common in the nonkeratinized mucosa and appear 4-7 days after use of the drug. resolution of lesions may occur after treatment is suspended, often within 3-4 weeks. spontaneous or induced hemorrhage, especially gingival, may occur when the platelet count is below 10,000/mm 3 . patients at a higher risk of developing stomatitis are those with hematologic neoplasms, those who are under 20 years old (high mitotic activity of the epithelium), and patients with pre-existing oral disease and poor mouth hygiene. preventive measures include proper maintenance of oral hygiene by washing the mouth with water, saline solution, sodium bicarbonate, or hydrogen peroxide. the use of cold water to prevent mucositis induced by 5-fluorouracil and melphalan in high doses appears to be helpful. other alternative clinical procedures, still not fully proven, consist in the use of chlorhexidine gluconate, β-carotene, and benzydamine chlorhydrate or sucralfate. treatment essentially consists of support with oral care, using agents such as magnesium or aluminum hydroxide and vitamin e. in addition, pain-relief drugs such as paracetamol and opioids (codeine and morphine) may be necessary when the use of topical anesthetics such as benzocaine and lidocaine are not effective. additional complications occur because of secondary bacterial, viral, or fungal infections that may become systemic. palifermin, when used prophylactically, reduces the occurrence and duration of severe stomatitis in patients with hematologic tumors and submitted to bone marrow transplantation. palifermin is a human recombinant factor of keratinocyte growth and protects various epithelial tissues. it acts not only on stomatitis but also on mucositis in general. a possible tumoral stimulating factor still limits its use in patients with epithelial tumors. hyperthermic chemotherapy with mitomycin c [71] [72] [73] peritoneal carcinomatosis frequently signals the terminal stage in some cancers of gastrointestinal and gynecologic origins. cytoreductive surgery and intraperitoneal hyperthermic chemotherapy was introduced in the early 1990s and has become the mainstay of treatment in particular clinical settings for patients with peritoneal carcinomatoses, especially in pseudomyxoma peritonei, significantly improving overall survival rates. there are four case reports of this recently described entity using a new procedure to treat peritoneal carcinomatosis after intraperitoneal hyperthermic chemotherapy with mitomycin c. one of them was described [71] in a male patient after 9 days of the procedure. the patient developed pain and scrotal necrosis on the anterior aspect of the scrotum (fig. 26.32) . two possible causes of the scrotal ulcers were proposed. (i) a patent processus vaginalis, allowing mitomycin c to become sequestered in the scrotum, inducing an inflammatory reaction, resulting in scrotal wall inflammation and subsequent ulceration (fig. 26.33 ). this was proposed since previous studies have shown that intradermal administration of mitomycin c inhibits wound healing and induces skin necrosis. (ii) local spillage of mitomycin c onto the scrotal skin, with resulting inflammation and ulceration. previous studies with patients on continuous ambulatory peritoneal dialysis (capd) have shown that 10% of capd patients developed genital swelling [3] . a possible cause of the scrotal swelling is a patent processus vaginalis causing a communicating hydrocele, which can be found in 15-37% of adult men and may not be clinically evident until capd has begun. patients may develop this complication months after the surgery; hence, any complaint of scrotal pain or discomfort in these patients should warrant immediate investigation and attention, even if the complaints present much later. our group suggests that an image investigation, such as computed tomographic peritoneography, should be considered for male patients prior to intraperitoneal chemotherapy, since this complication is potentially serious for the patient. vasomotor changes [74] various vascular alterations have been described, probably as a result of a direct effect on arterial smooth muscle fibers or by acting on the autonomic nervous system. manifestations may include blood vessel spasms with livedo, raynaud's phenomenon, and distal necroses, which may be triggered by bleomycin and cisplatin. vasodilatation with erythema and flushing may result from the use of bleomycin, cisplatin, asparaginase, dacarbazine, taxanes, 5-fluorouracil, doxorubicin, cyclophosphamide, gefitinib, and carmustine. flushing [74] flushing consists of a temporary erythema of the face, neck, upper chest, ears, or upper abdomen. the mechanism responsible for flushing is a transitory vasodilation mediated by the autonomic nervous system or by the direct effect of circulating substances that act on the musculature of the vessel walls. the nerves of the autonomic nervous system also control the sweat glands so that flushing mediated by these nerves is known as "wet flushing," whereas when the substance acts directly on the vascular wall muscles it is known as "dry flushing." derivatives of biological agents probable mechanism of mitomycin c deposition in a patent processus vaginalis into the scrotum such as l-asparaginase and bleomycin are notorious for causing flushing, which occurs soon after infusion. irinotecan, a topoisomerase i inhibitor, causes dysautonomia, the symptoms of which include diarrhea, bradycardia, and flushing. hormonal agents such as antiestrogens (tamoxifen, anastrozole), lhrh analogs (leuprolide), and antiandrogens (flutamide and diethylstilbestrol) may result in flushing. other agents that also deserve mention include 5-fluorouracil, carboplatin, cisplatin, cyclophosphamide, dacarbazine, doxorubicin, etoposide, and methotrexate. interaction with uv light [74] eruptions resulting from photosensitivity are caused by various agents, principally following exposure to uv radiation. phototoxicity caused by dacarbazine, fluoropyrimidines (systemic 5-fluorouracil, topical 5-fluorouracil, tegafur, and capecitabine) and vinblastine has been well documented. phototoxicity caused by dactinomycin, doxorubicin, hydroxyurea, procarbazine, brequinar sodium, mitomycin, 6-thioguanine, and flutamide, as well as by the porphyrin compounds that are used in photodynamic therapy, is uncommon. reactivation of sunburn is a well-documented adverse effect following the use of methotrexate. it occurs when the drug is administered 1-3 days after exposure to uv radiation, when the erythema from the previous exposure has been in the process of disappearing. leucovorin does not prevent this reaction. phototoxic reactions resemble intense sunburn in areas of the skin that are exposed to light, with erythema, edema, pain, or pruritus. blisters may be present and desquamation may occur in severe cases. residual hyperpigmentation may persist for months. hydroxyurea has been described as being associated with development of dermatomyositislike eruption due to photosensitivity (fig. 26 .34) [74, 75] . hydroxyurea is an anticancer agent that inhibits dna synthesis through its action on the enzyme ribonucleotide reductase [75] . it is used in chronic myeloproliferative diseases such as polycythemia vera, chronic myeloid leukemia, and essential thrombocytosis, although it has also been prescribed to patients with refractory psoriasis [75] . patients on long-term therapy with hydroxyurea can develop various side effects, including a wide variety of mucocutaneous manifestations, which appear in 10-35% of patients [75] . the most common skin changes are facial erythema, hyperpigmentation, xerosis, alopecia, skin atrophy, melanonychia, and ulcers on the [75] . other less frequent adverse effects are dermatomyositis-like rash and nonmelanoma skin cancer (fig. 26.35 ) [75] . dermatomyositis-like rash resembles true dermatomyositis both clinically and histologically [75] . it presents as desquamating erythematous papules or plaques on the dorsum of the hands, typically associated with facial erythema and pronounced xerosis of the skin. patients rarely report other accompanying symptoms and there are usually no significant alterations of laboratory tests [75] . histologically, a lichenoid inflammatory infiltrate is found at the dermoepidermal interface, with vacuolization of the basal layer, dyskeratosis, and, rarely, mucin deposition ( fig. 26.36 ) [75] . diagnosis is made according to the distribution of the lesions and by the temporal relationship between chemotherapy and light exposure. treatment includes discontinuation of the agent and protection from the sun for at least 2 weeks. physical sunscreens are recommended. cold compresses, systemic antihistamines, and topical or oral corticosteroids are used as associated symptomatic treatment. [74] this is a phenomenon whereby the chemotherapeutic agent induces an inflammatory reaction in an area previously exposed to radiation. these reactions are predominantly cutaneous; however, they may affect internal organs such as the lungs, heart, bladder mucosa, esophagus, oral or bowel mucosa, and supraglottic larynx. it occurs more often with the use of doxorubicin, dactinomycin, and gemcitabine and is less common with bleomycin, etoposide, hydroxyurea, methotrexate, trimetrexate, vinblastine, 5-fluorouracil, lomustine, daunorubicin, melphalan, cyclophosphamide, cytarabine, docetaxel, edatrexate, idarubicin, paclitaxel, tamoxifen, and vinblastine. the mechanism of radiation recall is unknown but it is probably related to dna repair. relapsing dermatitis or radiation recall may occur between 8 and 15 days following radiotherapy and generally appears hours to days after administration of the chemotherapeutic agent. clinically, the patient may or may not experience a painful erythema with or without vesiculation, edema, desquamation, and pruritus. the borders of the lesion are well defined and correspond to the exact site at which the radiation was applied. in severe cases, necrosis and ulceration may occur. the severity appears to directly reflect the brevity between radiation and chemotherapy as well as the doses of both radiation and chemotherapy. the reaction improves spontaneously within hours or weeks following cessation of chemotherapy, treatment being symptomatic. the use of systemic corticosteroids associated with the discontinuation of chemotherapy generally results in a marked improvement and may permit reintroduction of the treatment. [74] this occurs when a chemotherapeutic agent increases the toxicity of radiotherapy. this phenomenon may occur in virtually all the organs of the body including the skin, mucosa, esophagus, lungs, heart, digestive tract, kidneys, liver, brain, bladder, and eyes. the agents most associated with exacerbation of radiation are bleomycin, gemcitabine, dactinomycin, doxorubicin, 5-fluorouracil, hydroxyurea, 6-mercaptopurine, oxaliplatin, and methotrexate. clinically, the reaction resembles residual dermatitis secondary to acute dermatitis from radiation, with erythema, edema, vesiculation, blisters, or erosions. the reaction generally appears at the site of radiation; however, the area affected may be more extensive. severe mucositis may occur. the reaction is associated with the dose, the type of drug used, and the sequence of time between radiation and the use of chemotherapy. toxicity may be additive or supra-additive (synergic). in supra-additive toxicity, the reaction is greater than that of the sum of each one of the types of treatment alone. treatment is symptomatic: applying cold compresses, taking precautions at the site to prevent infection and avoiding trauma, heat, and uv light. sequelae such as fibrosis, skin atrophy, and telangiectasia-related disorders may occur. [74] in theory, all chemotherapeutic agents may trigger hypersensitivity eruptions. with certain drugs derived from biological agents such as l-asparaginase, mitomycin c, and bleomycin in addition to paclitaxel, the incidence of hypersensitivity reactions is high. in the case of paclitaxel, this is due to the fact that it is dissolved in cremophor el castor oil. according to the classification system defined by gell and coombs, the majority of hypersensitivity reactions are type i, i.e., ige-mediated. they present as urticaria, pruritus, angioedema, and anaphylaxis. they generally occur within the first hour after use of the drug, but onset may be delayed until up to 24 h after using the medication. type iii reactions occur because of formation of circulating immunocomplexes, and cause eruptions such as polymorphous erythema and vasculitis. nonetheless, l-asparaginase and procarbazine cause urticarial reactions via type iii reactions. allergic contact dermatitis, a type iv reaction, may occur, principally as a consequence of the topical use of nitrogen mustard (mechlorethamine). other severe reactions may occur, such as sjs and ten, as well as exanthematous eruptions, all currently classified as type iv reactions according to the extended gell and coombs classification, i.e., sjs and ten, respectively (type ivc, mediated by fas, granzymes, and perforin) and exanthematous eruptions (type ivb, mediated by t cells with il-5 production, with chemotaxis of eosinophils). agents [74] local toxicity [74] antineoplastic drugs may be classified according to their potential aggressiveness toward blood vessels and adjacent tissues. they may be nonirritating, irritating, or vesicant, causing effects that range from mere local discomfort to tissue necrosis. nonirritating drugs (thioguanine, asparaginase, bleomycin, cyclophosphamide, chlorambucil, methotrexate, hydroxyurea) provoke an edema that is indicative of a site of extravasation; however, they do not cause necrosis or tissue irritation. irritating drugs (5-fluorouracil, carmustine, docetaxel, and etoposide) cause tissue damage that does not progress to necrosis. they trigger erythema, pain, inflammation at the puncture site and along the venous pathway, burning, and local edema, without blistering. the vesicant drugs (dactinomycin, doxorubicin, melphalan, vincristine, vinblastine, and dacarbazine) cause severe skin irritation with pain, erythema, edema, blistering, and necrosis with functional and aesthetic damage. [74] this is defined as the leakage of a chemotherapeutic drug from the vessel bed to the surrounding tissues, either as a result of vascular rupture or by direct infiltration. the frequency of this event in adults is estimated at 0.1-6% and it is more common among children. severe sequelae are rare. the severity of tissue damage is related to the type of chemotherapeutic agent used and the quantity and concentration of the drug administered. cytotoxic agents are classified as irritants or vesicants as a function of their potential for local toxicity. an irritant is defined as an agent that causes an inflammatory reaction, paresthesia, pain, or phlebitis at the puncture site or along the venous pathway. clinical signs include sclerosis and hyperchromia along the passage, as well as burning, increased temperature at the site, discomfort, erythema, and pain at the area of extravasation. necrosis does not occur with this condition. the symptoms are generally short-lived and leave no sequelae. the drugs most associated with this complication are 5-fluorouracil, carboplatin, cisplatin, bleomycin, mitomycin, dactinomycin, idarubicin, daunorubicin, dacarbazine, ifosfamide, cyclophosphamide, mechlorethamine, carmustine, mitoxantrone, paclitaxel, docetaxel, streptozotocin, vinblastine, vinorelbine, and etoposide. the vesicant agents (melphalan, bleomycin, mechlorethamine, carmustine, mitomycin, mitoxantrone, cisplatin, paclitaxel, dacarbazine, dactinomycin, daunorubicin, streptozotocin, doxorubicin, epirubicin, vinblastine, vincristine, etoposide, vindesine, and vinorelbine) have the potential to cause more severe and long-lasting tissue damage, including necrosis of the affected area. the initial manifestations are often subclinical and may appear immediately following extravasation or after several days or weeks. the initial signs include local burning or paresthesia at the site of infusion, mild erythema, pruritus, and edema. a change in the infusion rate or the absence of venous return in the aspirate may indicate the occurrence of extravasation. after 2-3 days, erythema increases and there is pain, a brownish discoloration, induration, dry desquamation, or the appearance of blisters. if the amount extravasated was small, the signs and symptoms may disappear in the following weeks. if a significant amount was extravasated, the following symptoms may appear in the coming weeks: necrosis, formation of eschar and painful, necrotic ulceration with raised, erythematous borders and a yellowish base. there is generally no granulation tissue with these ulcerations. they may resolve slowly or persist, increasing gradually in area. involvement of the tendons, nerves, and vessels may occur if appropriate treatment is not given, leading to severe sequelae such as nerve compression syndrome, a reduction in joint mobility, contractures, neural deficits, and reflex sympathetic dystrophy. cellulitis and the formation of abscesses are rare events. the interval between detecting the condition and adopting the appropriate measures should be short as possible. the nursing team should be trained in this respect. preventive measures should be adopted such as avoiding puncturing emaciated limbs, lower limbs, limbs with multiple punctures, limbs with phlebitis or those that have been subjected to radiation, the ipsilateral limb to a mastectomy, in vena cava syndrome, and in veins that protect joints, nerves, and tendons. it is important to evaluate the venous conditions of the patient and, if necessary, to use an indwelling catheter. the use of common needles for venous access should be avoided. adequate fixation should be performed and blood reflux should be tested, with an infusion of 0.9% saline solution or 5% glucose-saline solution used for every 2 ml of the chemotherapeutic agent. after administration of all the drugs, 20 ml of saline or glucose-saline solution should be infused to reduce any possibly toxic residues. vesicant drugs should always be given first. in prolonged sessions of chemotherapy (those lasting over an hour) with vesicant drugs, central venous access should be used. always listen to the patient's complaints. if extravasation occurs, stop the infusion immediately. remove the puncture device and elevate the affected limb. in the case of extravasation of drugs such as etoposide, paclitaxel, vinblastine, vincristine, and vinorelbine, apply local heat (leading to vasodilation and dilution of the drug) for 30 min and ice (venous constriction and greater degradation of the toxic metabolites in addition to alleviating pain and inflammation) every 30 min, six times a day in the first 48 h. for the other drugs, apply ice every 30 min, six times a day. when indicated, the specific antidote for the drug in question should be used. the use of intralesional corticosteroid and sodium bicarbonate should be avoided. ulcers that fail to heal may require debridement and grafting. in case of persistent edema and erythema and pain without ulceration that persists despite conservative therapy or in the presence of extensive areas of necrotic tissue or skin ulceration, surgery may be indicated. periorbital edema [74] edema of the eyelids has been described with the use of gemcitabine. recovery [74] cutaneous eruption of lymphocyte recovery (elr) is observed in leukemia patients who receive bone marrow ablation. in general, it appears between the 6th and 21st days after chemotherapy. this point corresponds to the beginning of the recovery of peripheral lymphocytes following the nadir of leukocyte count induced by chemotherapy. although the exact mechanism has yet to be clarified, it is believed that the eruption is caused by the return of immunocompetent lymphocytes to peripheral circulation with cutaneous cytotoxicity. t lymphocytes and langerhans cells are found on histopathologic evaluation of these reaction sites. clinically, the condition consists of pruriginous, erythematous macules, papules, or papulous plaques that become confluent. erythrodermia may occur. in addition, this condition is associated with an elevation in body temperature that occurs together with the appearance of the eruption. the temperature falls in the following 2-3 days and the skin eruption tends to diminish after several days, progressing with desquamation and mild residual hyperchromia. the drugs most associated with these reactions are cytarabine, daunorubicin, amsacrine, etoposide, cyclophosphamide, and vincristine. differential diagnosis should be made with sepsis, viral exanthems, gvdh, leukemia or lymphoma cutis, and drug hypersensitivity or toxicity. histopathology is nonspecific. the most characteristic findings are superficial perivascular mononuclear cell infiltrate, mild epidermal alterations such as spongiosis, vacuolar alteration of the basal cell layer, and loss of keratinocyte maturation secondary to chemotherapy. dyskeratotic keratinocytes are rare and eosinophils are absent. on occasions when the patient was treated with gm-csf associated with il-3, atypical lymphocytes with large pleomorphic and hyperchromatic nuclei were found at histopathology. differentiation may be difficult between elr and gvhd. receptor tyrosine kinase inhibitors [74] anti-egfrs currently consist of panitumumab, cetuximab, erlotinib, and gefitinib. skin toxicity with anti-egfrs is actually more of a pharmacologic effect than a hypersensitivity reaction, since this is a clinical marker of the efficacy of the inhibiting effect of these drugs on the tumor, with the severity of the eruption corresponding to tumor response. the skin effects observed with the anti-egfr are alterations in capillary growth and in the texture of the hair, paronychia with or without secondary infection, or the formation of pyogenic granuloma, generalized asteatosis, skin desquamation, and blepharitis. the most characteristic and intense manifestation is a papulopustular, follicular, comedone, or non-comedo (acneiform eruption) that occurs on the head, neck, and the central portion of the chest and back, which later disseminates. there may be pruritus, which differentiates this reaction from the acneiform eruptions caused by corticosteroids, antiepileptic drugs, and vitamins b6 and b12. acneiform eruptions occur in more than 50% of patients with use of cetuximab, and this percentage may reach as high as 75-100%. the manifestations generally occur in the first weeks (2 days to 6 weeks) after the beginning of treatment (cetuximab and panitumumab). the eruption is dose dependent; however, the duration of the condition does not correlate with the duration of treatment. the acneiform eruptions induced by monoclonal antibodies are more severe and extensive than those resulting from the use of tyrosine kinase inhibitors. blepharitis caused by anti-egfrs may range from mild to intense. histopathology of the papulopustular lesions shows no increase in sebaceous gland activity, comedones, or follicular rupture that would explain the inflammation, differentiating it from acne vulgaris. the follicles are rather wide and at times obstructed by an excess of keratinocytes. in the dermis, neutrophilic infiltrate may be found, particularly involving the follicular infundibulum. intraepidermal acantholysis may be present in association with the eccrine gland ducts. in the lesions of patients using gefitinib, there is an expressive thinning of the stratum corneum layer with loss of the normal basket-weave pattern. paronychia occurs in around 10-15% of patients who are using cetuximab and gefitinib, appearing at 6-8 weeks of treatment or sometimes after 6 months. it affects various fingers and the first toes. treatment consists of potent topical corticosteroids. in case of onychocryptosis, anti-egfr may be temporarily interrupted and canthotomy performed. asteatosis occurs in around 35% of patients, particularly with the use of gefitinib. there is a predilection for the areas previously or simultaneously affected by acneiform eruption. some patients have xerosis of the vaginal mucosa, with dysuria. xerosis may progress to chronic asteatotic eczema (fig. 26.37) , with a greater susceptibility to staphylococcus aureus infection or hhv-1. emollients and topical corticosteroids should be used for the eczema. fissures can be treated with a solution of 50% propylene glycol under plastic occlusion or a hydrocolloid dressing. adverse drug reactions (adrs) include all unintended pharmacologic effects of a drug except therapeutic failures, intentional overdosage, abuse of the drug, or errors in administration. they can be classified as predictable (type a -80% of the adrs) or unpredictable (type b). anaphylaxis an immediate systemic reaction that occurs when a previously sensitized individual is re-exposed to an allergen. it is caused by rapid ige-mediated immune release of vasoactive mediators from tissue mast cells and blood basophils with a potential late component. this is a systemic severe adr affecting skin, mucous membranes, gastrointestinal tract, respiratory tract, and cardiovascular system. drug allergy an immunologically mediated response to a pharmaceutical and/or formulation (excipient) agent in a previous sensitized patient. drug idiosyncrasy an abnormal and unexpected effect that is unrelated to the intended pharmacologic action of a drug and has an unknown mechanism. it is not mediated by a humoral or cellular immune response but is reproducible on readministration. it may be due to underlying abnormalities of metabolism, excretion, or bioavailability. drug intolerance an undesirable pharmacologic effect that may occur at low or conventional doses of the drug without underlying abnormalities of metabolism, excretion, or bioavailability of the drug. humoral or cellular immune mechanisms are not thought to be involved, and a definitive mechanism for such exaggerated responses has not been established (e.g., acetylsalicylic acid-induced tinnitus at low doses). immediate systemic reactions that mimic anaphylaxis but are caused by non-ige-mediated release of mediators from mast cells and basophils. often caused by radiocontrast agents. severe adrs include all adverse effects which are unpredictable life-threatening adrs and need prompt recognition to reduce integumentary and internal organ damage and, thus, morbidity and mortality. uncomplicated adrs include mild or moderate adverse drug effects on healthy patients, not involving life-threatening situations. diagnostic testing for drug hypersensitivity drug-induced reactions: a report from the boston collaborative drug surveillance program on 15.438 consecutive inpatients sémilogie et marqueurs de sévérité des toxidermies érythémateuses severe adverse cutaneous reaction to drugs the nature of adverse events in hospitalized patients: results of the harvard medical practice study ii cutaneous drug reactions: clinical types and causative agents: a five-fig. 26.37 intense xerosis caused by erlotinib year survey of in-patients (1981-1985) joint task force on practice parameters of american academy of allergy, asthma and immunology, american college of allergy, asthma and immunology, joint concil of allergy, asthma and immunology anaphylaxis and anaphylactoid reactions. a guide to prevention, recognition and emergent treatment erythroderma: analysis of 247 cases dermatitis exfoliativa: cases admitted in the decade 1948-1957 to the dermatological clinic, karolinska sjukhuset exfoliative dermatitis: a prospective study of 80 patients dermatology in general medicine advances in toxic epidermal necrolysis toxic epidermal necrolysis erythema multiforme: a critical review of characteristics, diagnostic criteria, and causes acute disseminated epidermal necrosis types 1, 2, and 3: study of sixty cases erythema multiforme major and stevens-johnson syndrome are clinically different disorders with distinct etiologies clinical classification of cases of toxic epidermal necrolysis, stevens-johnson syndrome and erythema multiforme epidemiology of erythema exsudativum multiforme majus, stevens-jonhson syndrome, and toxic epidermal necrolysis in germany (1990-1992): structure and results of a populationbased registry toxic epidermal necrolysis (lyell syndrome). incidence and drug etiology in france the incidence of erythema multiforme, stevens-johnson syndrome, and toxic epidermal necrolysis. a population-based study with particular reference to reactions caused by drugs among outpatients toxic epidermal necrolysis (lyell syndrome) intravenous immunoglobulin therapy for stevens-johnson syndrome effectiveness of early therapy with corticosteroids in stevens-johnson syndrome: experience with 41 cases and a hypothesis regarding pathogenesis treatment of severe drug reactions: stevens-johnson syndrome, toxic epidermal necrolysis and hypersensitivity syndrome o espectro do eritema multiforme (eritema multiforme minor e major) e o espectro da síndrome de stevens-johnson e da necrólise epidérmica tóxica (síndrome de lyell) low n-acetylating capacity in patients with stevens-johnson syndrome and toxic epidermal necrolysis a slow acetylator genotype is a risk factor for sulphonamide-indiced toxic epidermal necrolysis and stevens-johnson syndrome cutaneous adverse drug reactions. stevens-johnson syndrome and toxic epidermal necrolysis delayed reactions to drugs show levels of perforin, granzyme b, and fas-l to be related to disease severity increased interleukin 10, tumor necrosis factor a, and interleukin 6 levels in blister fluid of toxic epidermal necrolysis drug rashes. what are the targets of cell-mediated cytotoxicity? treatment of toxic epidermal necrolysis with high-dose intravenous immunoglobulins intravenous immunoglobulin treatment for stevens-johnson syndrome and toxic epidermal necrolysis treatment of toxic epidermal necrolysis epidemiology of druginduced severe skin reactions correlations between clinical patterns and causes of erythema multiforme majus, stevens-johnson syndrome, and toxic epidermal necrolysis medication use and the risk of stevens-johnson syndrome or toxic epidermal necrolysis severe cutaneous adverse reactions to drugs -relevant aspects to diagnosis and treatment -part i: anaphylaxis and anaphylactoid reactions, erythroderma and the clinical spectrum of stevens-johnson syndrome and toxic epidermal necrolysis (lyell's disease) drug reaction with eosinophilia and systemic symptoms (dress): a complex interaction of drugs, viruses and the immune system drug reaction with eosinophilia and systemic symptoms (dress)/drug-induced hypersensitivity syndrome (dihs): a review of current concepts drug reaction with eosinophilia and systemic symptoms/drug-inducedhypersensitivity syndrome: clinical features of 27 patients adverse mucocutaneous reactions to chemotherapeutic agents: part i severe cutaneous adverse drug reactions: relevant aspects to diagnosis and treatmentpart ii propylthiouracil-induced vasculitis with antineutrophil cytoplasmic antibody approach to the patient with a suspected cutaneous adverse drug reaction adverse drug reactions and organ damage: the skin a review of cutaneous drug eruptions dermal dendrocytes fxiiia+ phagocytizing extruded mast cell granules in drug-induced acute urticaria fixed drug eruption: state of the art evidence for acne-promoting effects of milk and other insulinotropic dairy products lichenoid drug eruption with proton pump inhibitors getting under the skin of adverse drug reactions cutaneous adverse reactions of amiodarone phototoxicity and photocarcinogenesis associated with voriconazole an update on pediatric cutaneous drug eruptions coma blisters, peripheral neuropathy, amitriptyline overdose: a brief report in vitro interferon-gamma release test in the diagnosis of druginduced erythema nodosum drug induced psoriasis advances in the diagnosis of drug eruptions baboon syndrome resulting from systemic drugs: is there strife between sdrife and allergic contact dermatitis syndrome? contact dermatitis a new proposal for a clinical-oriented subclassification of baboon syndrome and a review of baboon syndrome nicolau syndrome: an iatrogenic cutaneous necrosis complications of the injectable fillers, part 2: vascular complications cutaneous drug reactions in the pediatric population chemotherapeutic agents and the skin: an update mucocutaneous reactions to chemotherapy cutaneous reactionsto chemotherapy drugs: the art of consultation reações cutâneas desencadeadas por drogas scrotal ulcer developed after intraperitoneal hyperthermic chemotherapy with mitomycin-c scrotal ulcer after intraperitoneal hyperthermic chemotherapy scrotal pain and ulceration post hipec: a case report adverse mucocutaneous reactions related to chemotherapeutic agents: part ii dermatomyositis-like eruption associated with hydroxyurea therapy: a premalignant condition key: cord-294212-nlekz39f authors: wang, dongliang; mai, jinhui; zhou, wenfeng; yu, wanting; zhan, yang; wang, naidong; epstein, neal d.; yang, yi title: immunoinformatic analysis of tand b-cell epitopes for sars-cov-2 vaccine design date: 2020-07-03 journal: vaccines (basel) doi: 10.3390/vaccines8030355 sha: doc_id: 294212 cord_uid: nlekz39f currently, there is limited knowledge about the immunological profiles of severe acute respiratory syndrome coronavirus 2 (sars-cov-2). we used computer-based immunoinformatic analysis and the newly resolved 3-dimensional (3d) structures of the sars-cov-2 s trimeric protein, together with analyses of the immunogenic profiles of sars-cov, to anticipate potential b-cell and t-cell epitopes of the sars-cov-2 s protein for vaccine design, particularly for peptide-driven vaccine design and serological diagnosis. nine conserved linear b-cell epitopes and multiple discontinuous b-cell epitopes composed of 69 residues on the surface of the sars-cov-2 trimeric s protein were predicted to be highly antigenic. we found that the sars-cov-2 s protein has a different antigenic profile than that of the sars-cov s protein due to the variations in their primary and 3d structures. importantly, sars-cov-2 may exploit an immune evasion mechanism through two point mutations in the critical and conserved linear neutralization epitope (overlap with fusion peptide) around a sparsely glycosylated area. these mutations lead to a significant decrease in the antigenicity of this epitope in the sars-cov-2 s protein. in addition, 62 t-cell epitopes in the sars-cov-2 s protein were predicted in our study. the structure-based immunoinformatic analysis for the sars-cov-2 s protein in this study may improve vaccine design, diagnosis, and immunotherapy against the pandemic of covid-19. the outbreak of the coronavirus disease 2019 (covid-19) is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1] . by 16 june 2020, sars-cov-2 has been reported in 216 nations and has resulted in 7,941,791 confirmed cases (https: //www.who.int/emergencies/diseases/novel-coronavirus-2019). coronavirus (cov) belongs to the family of coronaviridae, and it is an enveloped, positive-sense single-stranded rna virus. both sars-cov-2 and sars-cov fit into the subgenus of sarbecovirus within the genus of betacoronavirus (beta-cov), based on phylogenetic tree analysis [1] [2] [3] . the viral genome, approximately 30 kb in size, encodes four structural proteins including the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. the s protein is composed of an ectodomain, sars-cov infection triggers a series of humoral and cellular immune responses, including the production of high titers of specific neutralizing antibodies and specific cytotoxic t lymphocyte responses to sars-cov [12, 13] . the s protein is the major structural antigenic component through which effective protective immunity is raised against virus infection. a vaccine based on the s protein could elicit antibodies to neutralize virus infection by blocking virus fusion and entry. the sars-cov-2 s protein shares a high degree of similarity to the sars-cov s protein [14, 15] , and it also binds in similar fashion to the human ace2 receptor and thus is likely to employ a similar cell entry mechanism [4, 16] . as such, the s protein is an effective antigenic component for sars-cov-2 vaccine design and development. however, currently, there is little or limited information about the immunogenic profiles of sars-cov-2 and the immune responses against sars-cov-2. despite this, computer-based immunoinformatics [17] , together with the recent progress on the 3-dimensional (3d) sars-cov-2 s protein [14, 15, [18] [19] [20] [21] , offers a powerful strategy providing rational and rapid guidelines for the design and development of effective vaccines against this emerging infectious disease. in this study, the close genetic relationship of sars-cov-2 with other members of the genus of beta-cov, especially with sars-cov, prompted us to explore the potential immunogenic profiles of sars-cov-2 for vaccine design and development. we used computer-based immunoinformatic analysis, together with analyses of the immunogenic profiles of sars-cov, to anticipate potential b-cell and t-cell epitopes of the s protein of sars-cov-2 for vaccine design, particularly peptide-driven vaccine design, immunotherapy, and serological diagnosis. linear b-cell epitopes of the sars-cov-2 s protein were predicted by bepipred 2.0 in iedb (bepipred 2.0., immune epitope database and analysis resource, national institute of allergy and infectious diseases, bethesda, md, usa) with a threshold of 0.55 (corresponding specificity > 0.817 and sensitivity < 0.292), and only the epitopes with more than 8 residues were considered for subsequent antigenicity analysis. antigenicity was evaluated via the vaxijen v2.0 server online tool (vaxijen v2.0., the jenner institute, oxford, uk) [22] . discontinuous b-cell epitopes were predicted via the discotope 2.0 server tool in iedb with a default threshold of −3.7 (corresponding specificity > 0.75 and sensitivity < 0.47), based on the 3-dimensional (3d) structures of the sars-cov-2 s protein (pdb id: 6vyb, b chain) and the sars-cov-2 s protein rbd (pdb id: 6m0j, b chain). cd8 t-cell epitopes were predicted based on the net mhc pan 4.0 algorithm in iedb with a peptide size of 9 residues, and the 8 most frequent hla class i alleles (hla-a*01:01, hla-a*02:01, hla-a*03:01, hla-a*11:01, hla-a*24:02, hla-b*07:02, hla-b*08:01, and hla-b*40:01) in the worldwide population (phenotypic frequency > 10%) were selected [23] . the top 1% of peptides with high scores were chosen for subsequent immunogenicity evaluation, which was analyzed by the vaxijen v2.0 server. for cd4 t-cell epitope prediction, an iedb-recommended 2.22 algorithm based on 7 alleles (drb1*0301, drb1*0701, drb1*0501, drb3*0101, drb3*0202, drb4*0101, and drb5*0101) [24] at a default 15-aa peptide was used with a median consensus percentile of prediction threshold ≤ 20, as recommended. b-cell and t-cell epitopes of the sars-cov s protein were searched in iedb by using iedb's immunobrowser tool. to identify b-and t-cell epitopes tested by experiments, only the epitopes with the response frequency (rf) values more than 0.5 were considered as positive. 3d structures of all peptides were modelled via the pep-fold3 online server [25] . all the peptides were docked to the mhc i molecules hla-b7 (pdb id: 3vcl) and hla-a*01:01 (pdb id: 4nqv) via the patchdock rigid-body docking server based on the defined threshold [26] . the docking transformation with good molecular shape complementarity was selected based on the geometry docking algorithm in patchdock, and then scoring and refining of the docked complexes were performed using the firedock server [27, 28] . the docking complexes with high global energy, attractive van der waals (vdw) energy, and hydrogen-bonding energy were used for subsequent analysis. protein-peptide connection was examined via ligplot+ v.2.2, and pymol (version 1.8.4.0, schrödinger, inc, new york, nj, usa) was used to analyze docked complexes. in all, 17 potential linear b-cell epitopes were predicted by the bepipred 2.0 program (table s1 , supplementary materials), and nine linear b-cell epitopes were chosen for further analysis after their antigenicity was evaluated via the vaxijen v2.0 program, based on the scores (table 1) . all the predicted b-cell epitopes were localized to a strictly conserved region and shared 100% identity throughout the 138 sars-cov-2 isolates. structure simulations demonstrated that all the nine epitopes were located on the surfaces of either the monomer or the trimer of the s protein ( figure 2a , top panel). of note, of the nine epitopes, epitope 5 ( 405 devrqiapgqtgki 418 ) was localized to the rbd and (table 1) . we also reviewed seven dominant linear b-cell epitopes of the sars-cov s proteins based on previous experimental tests and response frequency (see methods). of the seven epitopes, two epitopes were identical throughout all the 87 sars-cov isolates and four were highly conserved (≥93.1% sars-cov isolates had identical epitopes) (table s2 , supplementary materials). these results suggested that the majority of linear b-cell epitopes of the s protein were highly conserved in sars-cov and sars-cov-2 isolates, respectively ( table 1 and table s2 ). it is worth noting that one epitope ( 786 qilpdplkptkrsfiedllfnkvtla 811 ) located in the s2 subunit of the sars-cov s protein is an important linear b-cell epitope capable of eliciting the production of a neutralizing antibody (nab) identified in patients who recovered from sars-cov infection (table s2 ) [13] . in addition, we also predicted linear b-cell epitopes of the sars-cov s protein, and six of the seven dominant linear b-cell epitopes were predicted by bepipred 2.0, since the six dominant b-cell epitopes had overlapping sequences with their counterparts in the predicted epitope pool, thereby supporting bepipred 2.0 as a reliable and powerful tool for predicting linear b-cell epitopes. finally, the comparison of the epitope sequences revealed that there were no overlapping sequences between the nine potential linear b-cell epitopes of sars-cov-2 and the seven dominant linear b-cell epitopes of sars-cov (table 1 and table s2 ), suggesting that the immunogenetic profile of the sars-cov-2 s protein may be different from that of sars-cov. besides the linear b-cell epitopes, 69 residues on the surface of the s protein of the sars-cov-2 were predicted to form the multiple discontinuous b-cell epitopes ( table 2) . furthermore, based on the primary structure and 3d structure of the trimeric s protein, these residues were mainly distributed within eight regions ( table 2 and figure 2b ). notably, region s1-2 containing 35 residues accounted for more than half of the residues (35/69) comprising the discontinuous b-cell epitopes, and these 35 residues of region s1-2 were all located in the rbd. furthermore, 31 of the 35 residues were in the rbm (region s1-2 in table 2 ). this result suggested that the rbd, particularly the rbm, was highly antigenic. in addition, among the discontinuous epitope(s) of region s1-2, 10 residues (g417, g446, y449, q493, g496, q498, t500, n501, g502, and y505) were identified as the key residues contributing to the binding to the host receptor ace2 [19] (table 2 ). in region s2-2, two residues (p793 and i794) were located in the fusion peptide (fp) and exposed on the surface of the s2 subunit (table 2 and figure 2b , top panel). therefore, antibodies targeting these two regions may block the virus binding to the host cell receptor and the subsequent membrane fusion between virus and host cell. in addition, sequence alignments revealed that these 69 residues were strictly conserved among the s proteins of the 138 sars-cov-2 isolates, except that a point mutation (p1143l, region s2-5, table 2 ) occurred in the australia/qld02/2020 strain. although this mutation did not change the secondary structure ( figure s1a , supplementary materials), it caused a slight increase in the antigenicity (the antigenic scores increasing from 0.558 to 0.565). indeed, as shown in figure s1b , the longer side chain of 1143 l caused an apparent alteration of the surface structure. note: residues in the epitopes that are present in the crystal structure of the sars-cov-2 trimeric s protein are underlined; otherwise, they were absent in the crystal structure. table 2 . predicted discontinuous b-cell epitopes of the sars-cov-2 s protein. sequences domain/motif s1-1 k97, s98, k187, p209, n211, e281, n282 ntd s1-2 t415, k417, d420, y421, n439, n440, s443, k444, v445, g446, g447, n448, * y449, * n450, l452, r454, k458, s459, n460, k462, s477, p491, * l492, q493, s494, * g496, f497, q498, p499, * t500, n501, * g502, v503, g504, * y505 rbd between fp and hr1 s2-4 y917, e918 hr1 s2-5 q1071, n1074, t1100, l1141, q1142, p1143, e1144, l1145, d1146, s1147 between hr1 and hr2 residues in the epitopes that are involved in binding of the sars-cov-2 rbd to hace2 are underlined. * indicates the residues that are present at the identical positions of both sars-cov and sars-cov-2 s proteins. cell. in addition, sequence alignments revealed that these 69 residues were strictly conserved among the s proteins of the 138 sars-cov-2 isolates, except that a point mutation (p1143l, region s2-5, table 2 ) occurred in the australia/qld02/2020 strain. although this mutation did not change the secondary structure ( figure s1a , supplementary materials), it caused a slight increase in the antigenicity (the antigenic scores increasing from 0.558 to 0.565). indeed, as shown in figure s1b , the longer side chain of 1143 l caused an apparent alteration of the surface structure. next, we examined all the discontinuous b-cell epitopes of the sars-cov s protein deposited in the iedb database, and three main epitopes (epitope id: 77442, 77444, and 910052) were obtained from the database (table s3 , supplementary materials). furthermore, these conformational epitopes could be recognized by a variety of neutralizing mabs (80r, m396 and s230) in previous studies [29, 30] . 3d structure analyses revealed that the residues among the three discontinuous b-cell epitopes were exclusively mapped onto the rbd surface of the s1 subunit, suggesting that the rbd of the sars-cov s protein is also highly antigenic. we also compared the common residues comprising the discontinuous epitopes within the rbds of both the rbds of sars-cov and sars-cov-2 s proteins. only seven residues (y449, n450, l492, g496, t500, g502, and y505 of the sars-cov-2 s protein) were present in the identical positions of both s proteins (table 2) . therefore, the three mabs (80r, m396, and s230) recognizing the rbd of the sars-cov s protein hardly bound the sars-cov-2 rbd, although the rbds of both sars-cov and sars-cov-2 exhibit a high degree of 3d structural homology [14] . altogether, compared to the sars-cov s protein, the sars-cov-2 s protein may have a distinct antigenic profile, although both viruses are closely related by phylogenetic analysis (figures s2 and s3 , supplementary materials). in all, 40 peptides were predicted as the potential cd8 t-cell epitopes following analysis of peptide-mhc-i binding of the sars-cov-2 s protein using the net mhc pan 4.0 server and their subsequent evaluation of antigenicity using vaxijen v2.0 ( 22 potential cd4 t-cell epitopes were predicted to be present in the sars-cov-2 s protein ( table 3) . three of the 62 predicted t-cell epitopes (cd8 and cd4 t-cell epitopes above) of the sars-cov-2 s protein have been reported as t-cell epitopes of the sars-cov s protein in previous studies [31] [32] [33] [34] (table s5 , supplementary materials). the first and second were cd8 t-cell epitopes ( 493 pyrvvvlsf 501 , epitope id: 50166; 1174 nlneslidl 1182 , epitope id: 44814), while the first one was located in the rbd of the sars-cov s protein, which is known to be important for receptor binding and virus entry [35] . the third one was encompassed in one of the cd4 t-cell epitopes ( 993 qliraaeirasanlaatk 1010 , epitope id: 100428) of the sars-cov s protein (the epitope highlighted with underline showed the predicted cd4 t-cell epitope derived from the sars-cov-2 s protein). the underlined epitope is also identified as a t-cell epitope of the sars-cov s protein. before molecular docking with hla molecules, the 3d structures of the 40 potential cd8 t-cell epitopes were modelled via pep-fold3. only the best 3d model of each epitope was chosen for the subsequent molecular docking with hla molecules. among the 40 epitopes, four were docked into hla-b7 and nine were docked into hla-a*01:01. for the four peptide-hla-b7 molecular docking, the binding efficiency of each epitope was evaluated by the global and vdw energies, which were computed ranging from −11.55 to −26.14 kcal/mol and −18.15 to −25.38 kcal/mol, respectively (table 4 ). all the four peptides were predicted to be well docked into the groove of the hla-b7 molecule and formed stable hydrogen bonds with the residues in the groove of the hla ( figure 3a) . notably, both t73 and e152 in the hla-b7 groove frequently interacted with the epitopes via hydrogen bonding within 3.1å ( figure s4a, supplementary materials) . furthermore, the global and vdw energies of the nine peptide-hla-a*01:01 dockings ranged from −12.90 to −46.66 kcal/mol and −12.10 to −25.71 kcal/mol, respectively (table 4 ). of these nine peptides, five peptides showed high binding affinities and the other four peptides showed even higher binding affinities with hla-a*01:01. hydrogen bonds less than 3.1å were frequently observed in docking complexes. t73, n77, t143, and r156 within the groove were the major residues interacting with these peptides and formed stable complexes ( figure 3b and figure s4b ). the underlined epitopes are also identified as t-cell epitopes of the sars-cov s protein. (table 4 ). of these nine peptides, five peptides showed high binding affinities and the other four peptides showed even higher binding affinities with hla-a*01:01. hydrogen bonds less than 3.1å were frequently observed in docking complexes. t73, n77, t143, and r156 within the groove were the major residues interacting with these peptides and formed stable complexes ( figure 3b and figure s4b ). table 4 . table 4 . vaccination is the most effective medical strategy against a variety of infectious diseases. unfortunately, to date, no vaccine against coronavirus-associated diseases has been approved by the fda for use in humans. therefore, a vaccine against covid-19 is urgently needed to control the pandemic caused by the highly contagious sars-cov-2. the lack of knowledge about sars-cov-2 immunogenic profiles and immune responses is a challenge to vaccine design and development. the s protein is a leading potential target for vaccine design for either sars-cov or sars-cov-2 infection because of its strong immunogenicity and its roles in virus attachment and cell entry [16, 36] . importantly, the s protein of sars-cov is capable of inducing the production of neutralizing antibodies (nabs), which have been found in convalescent plasma samples from sars patients [13] and in animal models [37, 38] . therefore, antibodies targeting the s protein, particularly the rbd/rbm or the s2 fusion machinery, may exhibit neutralizing activity against sars-cov-2 infections. sars-cov-2 and sars-cov belong to the same genus of the betacoronavirus and both of them, together with the three coronaviruses from bat, show a very close genetic relationship in the evolution of the virus ( figure s2 , supplementary materials). however, the similarity of the immunogenic properties of these viruses remains to be determined. several immunological questions are critical: "are the immunogenic profiles of sars-cov-2 and sars-cov as similar as the genetic relationship shown in the phylogenetic trees?"; "do the immunogenic properties of both viruses differ significantly from each other?"; and "can the nabs raised against sars-cov provide effective protection against the infection of sars-cov-2?". we sought to identify the potential b-cell and t-cell epitopes of the sars-cov-2 s protein by using various state-of-the-art tools. the results improve our understanding of s protein immunogenesis and vaccine design. nine linear b-cell epitopes were predicted and localized to the surface of the sars-cov-2 s protein (table 1) , while seven linear b-cell epitopes of the sars-cov s protein have been confirmed by previous investigations [13, [39] [40] [41] [42] . the two groups of epitopes do not share any similarities, even though the s proteins from both viruses are close to each other in their primary structure. one critical linear b-cell epitope ( 786 qilpdplkptkrsfiedllfnkvtla 811 ) of the sars-cov s protein was reported to be recognized by nabs obtained from convalescent sars patients in a previous report [13] . another group also reported that an epitope ( 803 llfnkvtladagfmkqygeclgdina 828 ) was able to induce the production of nabs in animal models [41] . both epitopes localize to the s2 subunit and have a nine-residue overlap from position 803 to 811. furthermore, two of our predicted linear b-cell epitopes (predicted via bepipred 2.0 in iedb) were also found to map to the region between 786q-828a (data not shown), suggesting that the region (786q-828a) is an epitope-rich region of the sars-cov s protein. the function of this region in the s2 subunit is still unknown, but we note that the region has a three-residue overlap with the fusion peptide (fp) from 770 to 788 (figure 1) , and a proteolytic cleavage site (s2 ) upstream of the fusion peptide is conserved in all known coronaviruses [43] . therefore, antibodies targeting this epitope-rich site could potentially block fp function in membrane fusion during virus cell entry. comparison of the sars-cov and sars-cov-2 s proteins reveals that the s2 subunit is structurally conserved and shares higher aa identity (~90%), than does the s1 subunit (~68%). likewise, we also identified a homologous peptide ( 804 qilpdpskpskrsfiedllfnkvtla 829 ) in the s2 subunit of the sars-cov-2 s protein, which differs by only two residues from the corresponding region in sars-cov (see the residues indicated by the underlines). however, this homologous peptide in the sars-cov-2 s protein does not qualify as a predicted linear b-cell epitope. we noticed that the two residues in sars-cov (792l and 795t) are replaced by residues with less bulky side chains in sars-cov-2 (810s and 813s), which may decrease the antigenicity of the peptides and support sars-cov-2 countering host immune surveillance and clearance. indeed, we evaluated the antigenicity of both peptides using vaxijen v2.0, and found that the antigenicity score of the linear b-cell epitope in the sars-cov s protein was 0.4121, almost double the score of the peptide in the sars-cov-2 s protein (0.2114). although the antigenicity of this peptide in sars-cov-2 remains to be experimentally determined, and currently, most vaccine designs are focusing on the rbd of the s1 subunit, we rspeculate that a vaccine based on this epitope-rich region (786q-828a) in the s2 subunit of the sars-cov s protein may also elicit broad nabs that can cross-react with other virus members among this coronavirus family to provide broader protections (i.e., against simultaneous infections of both sars-cov and sars-cov-2). very recently, poh et al. used sera from covid-19 convalescent patients to identify peptides eliciting nabs from a pool constructed from overlapping sequences of the sars-cov-2 s protein [44] . one of the identified peptides ( 809 pskpskrsfiedllfnkv 826 ) is also from the s2 subunit, located near the fusion peptide of the sars-cov-2 s protein. however, as discussed above, the antigenicity of this peptide and its effect across human populations of different ages, genetic backgrounds, and immune status requires further evaluation before being used for vaccine design, due to its lower antigenicity score compared to the concomitant sars-cov s protein. in addition, s proteins of coronaviruses are decorated with an extensive glycan shield, which blocks neutralizing antibody recognition and presents a challenge for vaccine development. walls et al. recently characterized the s glycan shield of the sars-cov s protein [43] . according to their result, this epitope-rich region (786q-828a) located in a glycan hole that is sparsely glycosylated provides access to host protease for further proteolysis and subsequent induction of membrane fusion. taken together, this epitope-rich region is an ideal target for sars-cov-2 vaccine design. besides these critical linear b-cell epitopes, multiple discontinuous conformational b-cell epitopes distributed throughout eight regions were located on the surface of the trimeric s protein (table 2 and figure 2b ). region s1-2 is one of the critical sites that could be targeted by nabs since it is located in the rbd, and specifically on the surface of the rbm. ten of the 35 residues among the conformational epitope(s)/regions are potentially involved in binding of the sars-cov-2 rbd to hace2: (g417, g446, y449, q493, g496, q498, t500, n501, g502, and y505) ( table 2 ) [19] . it is possible that antibodies compete with hace2 to bind the sars-cov-2 rbd, and thereafter block the interaction of the virus with the receptor and the subsequent virus cell entry. in addition, seven of the 35 residues (y449, n450, l492, g496, t500, g502, and y505) of the sars-cov-2 rbd are also identified in the sars-cov rbd. these are the key residues forming a conformational epitope recognized by nabs in previous studies [45, 46] . cell-mediated immunity plays crucial roles in the response to virus infection as well as cancer therapy. cd8 cytotoxic t cells kill cells via t-cell receptor (tcr) recognition of the cognate peptide presented by mhc class i. two critical cd8 t-cell epitopes ( table 4 , a5 and a9 in figure 3 ), previously reported in sars-cov studies, were also predicted as cd8 t-cell epitopes of the sars-cov-2 s protein in our study. importantly, both the epitopes can be docked onto the hla-a*01:01 allele in an energetically favorable manner (table 4 ). these results strongly suggest that both of the cd8 t-cell epitopes are authentic epitopes of the sars-cov-2 s protein and are possibly involved in cell-mediated immune responses against sars-cov-2 infection. currently, most vaccine designs of the virus focus on the nab production elicited by the s protein, but t cell-mediated immunity (both cd8+ and cd4+ helper cells) against the viral infection deserves more attention. furthermore, "human-like" t-cell epitopes in sars-cov-2 should be removed from the vaccine since these epitopes are able to activate treg cells and suppress the immune response [47] . epitope prediction via immunoinformatics has accelerated the identification of antigens capable of eliciting a strong immune protective response against pathogen infections. likewise, it can remove deleterious epitopes from the antigen pool, which may cause antibody-dependent enhancement (ade), cytokine storm, autoimmune responses, and pathological lesions. the authenticity and effectiveness of these predicted epitopes may be improved through the use of threshold scoring and further confirmed by in vitro experiments and animal models. since epitope mapping of a new pathogen is time-consuming and laborious work, epitope prediction by immunoinformatics improves the efficiency of vaccine design and development. for instance, gutiérrez et al. predicted cross-conserved t-cell epitopes of seven representative strains of influenza a virus (iav) in us swine herds [48] . following the prediction, researchers in tanja opriessnig's group designed and tested a dna vaccine containing these predicted cross-conserved t-cell epitopes followed by an inactivated vaccine for boost. the new designed vaccine (prime-boosting regimen) exhibits an additive increase in cell-mediated immunity and an excellent clinical protection [49] . a pool of epitopes may be chosen as the core immunogen to develop various peptide-driven vaccines, such as a peptide vaccine, a dna/rna vaccine encoding these tandem epitopes, or a subunit vaccine via grafting these epitopes onto a defined nanoparticle, i.e., virus-like particles (vlps). in contrast to the whole pathogen-based vaccine, the injurious side effects (i.e., ade, cytokine storm, autoimmune responses, and pathological lesions) of these isolated epitopes can be evaluated in vitro or in animal models. thereafter, the peptide-driven vaccine with the optimized epitope combination will be safer and more effective as a result of its more precise design guided by a variety of versatile immunoinformatic tools. cytokine storm is one of the most dangerous and potentially lethal sequelae of covid-19 infection but the details of its onset and why it affects one patient rather than another remain unknown. several possible pathways may be responsible for sars-cov-2-associated cytokine storm, but it is likely that a failure to initially suppress viral replication leads to severe tissue damage from an overwhelming infection and a subsequent uncontrolled immune response. the initial cytokine wave following sars-cov-2 infection comes from the innate immune response. pattern recognition receptors (prrs), such as toll-like receptors (tlr-3, -7, and -8), rig-i-like receptors (rlrs), and nod-like receptors (nlrs) recognize the viral rna genome or its intermediates during replication. this recognition causes substantial releases of proinflammatory cytokines, such as tnf-α, il-1β, and il-6. second, the proliferation of sars-cov-2 in host cells leads to a large amount of cell death, and the content of the dead cells release damage signals, which further amplify cytokine release leading to cytokine storm [50] . lastly, cytokine storm may be exacerbated through a complement cascade after infection. the large number and combinatorial diversity of n-linked glycans on the surface of the sars-cov-2 s protein can be recognized by mannose-binding lectin (mbl), which is able to initiate the complement cascade and activate macrophages. subsequently, these activated macrophages also release a large amount of cytokine, i.e., il-1, il-6, and tnf-α. thus, a peptide-driven vaccine in the absence of viral genome and various glycan-conjugated antigens, theoretically, promises to limit inappropriate cytokine release. an autoimmune response induced by foreign antigens presents another safety issue for vaccine design. generally, pathogens may evade host immunosurveillance through producing "host-like" b-cell or t-cell epitopes and activating self-reactive t-regulatory cells that suppress the immune response or induce tolerance to the pathogens. however, in the context of a vaccine formula (combinations of antigens and adjuvants), these "host-like" epitopes may reversibly activate to induce an autoimmune response. recently, epivax, inc., has launched a new immunoinformatic tool called janusmatrix that helps to identify "human-like" epitopes from pathogens [47] . removing these "human-like" epitopes from a vaccine formula further enhances the safety and efficacy of such vaccines. compared to the use of the whole virus proteome, the epitopes predicted in our study are more easily evaluated by these newly developed tools such as janusmatrix, which removes the "human-like" epitopes from the vaccine. finally, ade is a critical safety concern for vaccine design and development. in general, ade is mediated by non-neutralizing antibodies binding to virus and then promoting virus cell entry via fcγ receptors (fcγrs). actually, ade was discovered in the course of vaccinations (i.e., rsv and dengue virus) and is difficult to predict. ade has been reported in animal experiments during vaccine trials of sars-cov and mers-cov [51, 52] . however, ade during vaccination for sars-cov or mers-cov, let alone sars-cov-2, remains to be determined in human. epitope prediction and in vitro immunological assays can aid scientists in the identification and removal of potential ade-promoting b-cell epitopes. the refined pool of candidate vaccine antigens can then be more exhaustively tested in animal models for evidence of ade. the results presented in this study highlight sars-cov-2 evolution and the structure-relevant immune profiles of both s proteins (sars-cov-2 and sars-cov). this perspective improves vaccine design and immunotherapy and works to minimize the side effects of vaccination for sars-cov-2. supplementary materials: the following are available online at http://www.mdpi.com/2076-393x/8/3/355/s1, figure s1 : the effect of p1143l mutation on the secondary structure and predicted 3d structure, figure s2 : neighbor-joining tree analysis of sars-cov-2, figure s3 : maximum-likelihood tree analysis of sars-cov-2, figure s4 : (a) 2d graphical representation of interaction analyses between human hla-b7 protein and mhc-i binding peptides, (b) 2d graphical representation of interaction analyses between human hla-a*01:01 protein and mhc-i binding peptides, figure s5 : (a) sequence variability of the s protein between ratg13 and sars-cov-2, (b) sequence variability of the s protein among 138 sars-cov-2 clinical isolates, table s1 : 17 predicted linear b-cell epitopes of the sars-cov-2 s protein using bepipred 2.0, table s2 : linear b-cell epitopes of the sars-cov s protein, table s3 : discontinuous b-cell epitopes of the sars-cov s protein, table s4 : predicted mhc class-i epitopes in the sars-cov-2 s protein, table s5 : mhc class i and class ii epitopes identified in the sars-cov s protein, table s6 : detailed information of 138 sars-cov-2 sequences, table s7 : detailed information of reference coronavirus strains in 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followed by a whole virus vaccine effectively protected pigs in the pandemic h1n1 pig challenge model cytokine storm induced by sars-cov-2 a decade after sars: strategies for controlling emerging coronaviruses recent advances in the vaccine development against middle east respiratory syndrome-coronavirus we thank all the researchers who submitted their sequencing data from gisaid on which this study is based. the authors declare no conflict of interest. key: cord-329669-z3t7plvh authors: poulton, kay; wright, paul; hughes, pamela; savic, sinisa; welberry smith, matthew; guiver, malcolm; morton, muir; van dellen, david; tholouli, eleni; wynn, robert; clark, brendan title: a role for human leucocyte antigens in the susceptibility to sars‐cov‐2 infection observed in transplant patients date: 2020-07-05 journal: int j immunogenet doi: 10.1111/iji.12505 sha: doc_id: 329669 cord_uid: z3t7plvh we analysed data from 80 patients who tested positive for sars‐cov‐2 rna who had previously been hla typed to support transplantation. data were combined from two adjacent centres in manchester and leeds to achieve a sufficient number for early analysis. hla frequencies observed were compared against two control populations: first, against published frequencies in a uk deceased donor population (n = 10,000) representing the target population of the virus, and second, using a cohort of individuals from the combined transplant waiting lists of both centres (n = 308), representing a comparator group of unaffected individuals of the same demographic. we report a significant hla association with hla‐ dqb1*06 (53% vs. 36%; p < .012; or 1.96; 95% ci 1.94–3.22) and infection. a bias towards an increased representation of hla‐a*26, hla‐drb1*15, hla‐drb1*10 and drb1*11 was also noted but these were either only significant using the uk donor controls, or did not remain significant after correction for multiple tests. likewise, hla‐a*02, hla‐b*44 and hla‐c*05 may exert a protective effect, but these associations did not remain significant after correction for multiple tests. this is relevant information for the clinical management of patients in the setting of the current sars‐cov‐2 pandemic and potentially in risk‐assessing staff interactions with infected patients. factor in an infected individual's ability to produce an effective immune response (ishibashi et al., 2009) . a number of in silico studies have defined the potential for immune responsiveness based on differential viral peptide binding to hla alleles (fast, altman, & chen, 2020; nguyen et al., 2020) . these are useful in vaccine design but do not represent the situation in natural infection where the full hla type of the individual, comprising up to twelve alleles across six loci, informs the capacity for immune response. this study investigated hla profiles of patients admitted with pcr-confirmed sars-cov-2 infection to identify any potential hla bias which might indicate an impaired capacity to mount an effective immune response to the infection. the information is of value towards risk stratification and clinical management of patients and potentially in risk-assessing staff interactions with infected patients. the aim of this study was to analyse the hla types of individuals receiving hospital care for covid-19 symptoms, where the hla type was already known. the inclusion criteria for this study were (a) that the patient must have been previously hla typed at least at hla-a, b, c, drb1 and dqb1 and (b) must have a positive test for the presence of sars-cov-2 rna. a total of 80 patients were included in the study, 41 from manchester and 39 from the leeds transplant centre. data were combined from both units to achieve a sufficient number for early analysis. all patients included in the study had previously been hla typed to support transplantation and required hospital treatment for covid-19 disease, indicating that their symptoms were severe, requiring clinical support or intervention. patients included in the study were either kidney transplant patients (n = 33), individuals on the solid organ transplant waiting lists (n = 40), or patients who had received haematopoietic stem cell transplantation (n = 7). for the leeds patients, the male:female ratio was 32:8, and the median age was 60.6 years (range 27-87 years). for the manchester patients, the male:female ratio was 20:20, and the median age was 58.9 years (range 3-80 years). hla typing had been performed using either of two methods: (a) labtype ® sso hla class i (hla-a, b and c) and hla class ii (drb1/3/4/5 and hla-dq) loci (onelambda inc), or (b) next-generation sequencing (holotype hla™; omixon or trusight hla, illumina). all tests were performed according to manufacturers' instructions. hla frequencies were analysed at first field resolution, and where second field typing data were available, types were converted into a serological equivalent specificity for analysis. we have performed analyses against two control populations group a: a uk (n = 10,000) deceased donor pool13 (https://nhsbtdbe.blob. core.windows.net/umbrarco-assets-corp/2925/antigen.pdf)and group b: a local (n = 308) wait-listed renal patient cohort comprising the active wait list from leeds (n = 150) and a randomly selected cohort (n = 158) from the manchester centre. the rationale behind the use of two separate control groups was an acknowledgement that problems exist in respect of both. control group a is representative of the uk population and the infection target of the virus, but it lacks proportionate representation of black, asian and minority ethnic (bame) groups. comparisons made with transplant patient groups may be confounded by this variance. the advantage of using the control group a in the context of this study is, however, that it represents a population assembled prior to the sars-cov-2 pandemic. conversely, the n = 308 cohort (group b) is derived by combining unselected individuals on the renal transplant waiting list from each centre, but the sars-cov-2 infection/exposure history of the group is uncertain and incorrect inferences may then be drawn from its use. alternative control groups were considered but precluded on the basis of the problems in their confident construction at this time. statistical analysis was performed using medcalc v19.3 (medcalc software) to compare frequencies of allele group carriage between the patient and control populations using fisher's exact test. odds ratios were calculated at 95% confidence intervals (ci) using medcalc software. each allele group was compared independently, and p ≤ .05 was considered to be significant. bonferroni correction was applied to accommodate multiple testing within each locus tested (gaetano, 2018) . the allelic frequencies were assessed in all populations. allele group carriage is defined as the number of individuals carrying the respective allele group. allelic frequencies are defined as the number of occurrences of the respective allele group divided by the total number of allele groups in the cohort. an allele group was considered to raise the risk of viral susceptibility when the or was above 1 and considered protective against viral susceptibility when or was below 1. allele group carriage frequencies for all specificities where associations which were significant, or approaching significance are included in table 1 . seventeen of the top twenty most frequent hla haplotypes in the uk population (allelefrequencies.net) were represented in this cohort revealing a broad susceptibility to infection. when comparing our patients with control group a, there were more significant associations observed than when compared against control group b. the strongest associations observed in risk of viral susceptibility observed were with hla class ii specificities. after bonferroni correction, hla-dqb1*06 remains a significantly increased risk when compared with group b controls, but not group group b: 72.5% vs. 60.1%; p = .0513, p c = not significant; or = 1.96 ci 1.19-3.22, p = .0421, p c = not significant). when the broad specificities were considered independently, hla-dqb1*05 was not significant in its correlation with viral infection, suggesting that the primary association lies with dqb1*06, rather than dq1. with such a small cohort, it is not possible to ascertain whether these observations are due to linkage disequilibrium between hla-dr and dq, or whether this is an independent observation. for hla class i, hla-a*26 was also significantly increased in the patients compared with group a controls (11.3% vs. 4.0%; p = .0049, p c = .0198; or = 3.04 cd8 t-cell response (dipiazza, richards, knowlden, nayak, & sant, 2016) . cal manifestations of the disease described as covid-19 . while almost all patients develop covid pneumonia, approximately 29% cases progress to acute respiratory distress syndrome which occurs 10-12 days after the onset of symptoms . furthermore, individuals who develop respiratory distress have often been responding well during the first phase of the disease, but their symptoms return aggressively, requiring clinical support within a hospital environment. this phenomenon is suggestive of an immunological hiatus, in which these individuals have been unable to effectively mature their immune response to generate the production of potent neutralizing antibody. in this series of individuals requiring admission following inin this regard, hla-a*26 has previously been identified as a predisposing type to infection with visceral leishmaniasis (kala-azar; singh, agrawal, & rastogi, 1997) and has been associated with the development of epstein-barr virus-driven post-transplant lymphoproliferative disorder in solid organ transplantation (reshef et al., 2011) . the occurrence of other specificities potentially within the hla-a*26 superset is also noteworthy and is the subject of further work in our laboratories. we also note the prediction, based on an in silico analysis, for poor ability of the closely related antigen hla-a*25 to present a repertoire of sars-cov-2 epitopes in the current paper from nguyen et al. (2020) . this paucity would be expected to impact on the quality of the linked immune response to the virus. utilizing the immune epitope database analysis resource, tepitool (tepitool iedb analysis resource), we therefore examined the top potential epitopes (fast et al., 2020) for key sars-cov-2 proteins in regard to their binding affinity to hla-a*26:01. based on ic50 < 500 nm, only one epitope (ftisvtte) was identified that would be predicted to support a t-cell-based immune response. this link between a real-world observation and an in silico predication provides validation of both findings. in this study, the individuals who were positive for hla-a*26 were not also positive for hla-drb1*10, although these have been documented to be inherited on the same haplotype (allelefrequencies.net). a study of sars-cov-2-positive individuals in a uk biobank population has also identified drb1*15:01 (or = 1.33, ci 1.01-1.74) and dqb1*06:02 (or = 1.32, ci 1.01-1.74) to be associated with an increased likelihood of testing positive for the virus (kachuri et al., 2020) . this is an in silico study in which hla types have been imputed, but these observations add support to our own observation in regard to hla-dr15 and dq6 in this early study of individuals with known hla types. the upregulation of hla-dr expression in lung tissue of patients affected with covid pneumonia suggests that this process is instrumental in the immunological control of the disease process and therefore must be considered to be biologically relevant. hla-dr10 and dr11(5) have previously been implicated in patients with impaired response to cytomegalovirus infection (ishibashi et al., 2009) , while hla-dr11 (5) has also been documented to be associated with severe response to infection with mers-cov (hajeer, balkhy, johani, yousef, & arabi, 2016) . hla-dr10 has also been proposed to be the vector mechanism for viral entry of epstein-barr virus into b cells, reinforcing the concept that the molecule itself may be a mechanism for driving viral infection li and cohen (2019) . a primary inefficiency of the alleles hla-dqb1*05 and *06 cannot be discounted in this very early uk study, due to linkage disequilibrium. this study has been deliberately independent of ethnicity as the uk population in this region of england is genetically heterogeneous with multiple populations of different heritages co-existing. it could be argued that the use of the uk deceased donors as a control population in this study has simply highlighted more frequent occurrence of antigens common to bame communities in our covid-19 population requiring hospital support for their disease. conversely, it may be legitimately argued that the bame link with susceptibility is explained through possession of these antigens. while either point remains valid, other specificities common to bame groups such as hla-a*34 and drb1*16 were not implithese are obviously tentative findings based on a limited number of cases in two centres. an unbiased surveillance of the transplant population to include those with mild symptoms or no symptoms would help to clarify this observation. we are now seeking to accrue additional data in substantiation of the association and to extend the scope of our study to in order to establish its basis. https://orcid.org/0000-0002-5608-8107 paul wright https://orcid.org/0000-0002-0516-3218 brendan clark https://orcid.org/0000-0001-7243-1916 allele frequency net database identification of major histocompatibility complex class ic molecule as an attachment factor that facilitates coronavirus hku1 spike-mediated infection the role of cd4 t cell memory in generating protective immunity to novel and potentially pandemic strains of influenza potential t-cell and b-cell epitopes of 2019-ncov. biorxiv, 1-13 holm-bonferroni sequential correction: an excel calculator (1.3) association of human leukocyte antigen class ii alleles with severe middle east respiratory syndrome-coronavirus infection clinical features of patients infected with 2019 novel coronavirus in wuhan association between antibody response against cytomegalovirus strain-specific glycoprotein h epitopes and hla-dr the landscape of host genetic factors involved in infection to common viruses and sars-cov-2 epstein-barr virus and the human leukocyte antigen complex human leukocyte antigen susceptibility map for sars-cov-2 association of hla polymorphisms with post-transplant lymphoproliferative disorder in solid-organ transplant recipients common and well-documented hla alleles over all of europe and within european sub-regions: a catalogue from the european federation for immunogenetics infectious diseases and immunity: special reference to major histocompatibility complex the laboratory tests and host immunity of covid-19 patients with different severity of illness self-reported symptoms of covid-19 including symptoms most predictive of sars-cov-2 infection are heritable a role for human leucocyte antigens in the susceptibility to sars-cov-2 infection observed in transplant patients key: cord-343262-zopxdw1d authors: srinivasan, raghuraman c.; strom, stephen c.; gramignoli, roberto title: effects of cryogenic storage on human amnion epithelial cells date: 2020-07-15 journal: cells doi: 10.3390/cells9071696 sha: doc_id: 343262 cord_uid: zopxdw1d perinatal stem cells and epithelial cells isolated from full term amnion membrane, in particular, have attracted interest over the last decade, as a promising source of multipotent cells for cellular therapies. human amnion epithelial cells (haec) have been used to treat monogenetic liver disease such as maple syrup urine disease or fibrosis of the liver in preclinical studies. in most studies xeno-transplants of haec were conducted without providing immunosuppression to recipients, reflecting the tolerogenic properties of haec. for many cell types, successful cryopreservation is critical for providing a readily available, off-the-shelf product. in this study, haec were isolated from full-term human placenta from 14 different donors, cryopreserved using a protocol and reagents commonly adopted for epithelial cell preservation. the cells were analyzed in terms of survival, recovery, and homogeneity, profiled for surface markers characteristic of epithelial, mesenchymal, endothelial, or hematopoietic cells. there were no significant differences observed in the percentage of cells with epithelial cell markers before and after cryopreservation. the relative proportion of stromal and hematopoietic cells was significantly reduced in haec preparations after cryopreservation. the expression of stem cell and immunomodulatory molecules were confirmed in the final product. since multipotent cells are readily available from full-term placenta, this novel cell source might significantly increase the number of patients eligible to receive cellular therapies for liver and other diseases. cellular therapeutics have potential utility for a large number of unmet medical needs. large-scale clinical-grade isolation and subsequent banking of stem cells has been the backbone of the field of regenerative medicine. successful translation of cell-based therapies frequently requires successful long-term storage under conditions that maintain the safety and efficacy of the final cell product. cell cryopreservation is an established procedure for several clinical therapies, as decades of bone marrow and cord blood hematopoietic stem cell transplants have shown. nevertheless, epithelial cell-based treatments, including hepatocytes, have suffered from limited reanimation after cryogenic procedure, with considerable loss of viability and function. human amnion epithelial cells (haec) are unique stem cells isolated from perinatal tissues. they form a thin epithelial layer lined up in the amniotic cavity surrounding the fetus during gestation. it is perhaps haec origin that makes these cells unique and attractive for interventional treatments [1] . the generation of the amnion epithelial lineage occurs prior to the specification of the three germ layers. this unique place in development, between the pluripotent cells of the epiblast and the specification of the three germ layers, may explain the lineage plasticity to haec, a property not shared with other perinatal cells. it has been reported that haec differentiate into different lineages and cell types, including ectoderm (neural cells) [2] , endoderm (hepatocyte-like cells [3] [4] [5] , and insulin producing islet-like cells) [4, 6] , as well as mesoderm lineage [7] . in preclinical studies, haec have been used to treat stroke, multiple sclerosis [8] , cardiac degenerative diseases [7] , type 1 diabetes [9] , pulmonary [10] [11] [12] , and metabolic liver diseases [13] [14] [15] . amnion products have been shown effective in reversing fibrosis in models of cirrhosis in mice [16] and rats [17] . recently, a 2-year report described safety and efficacy of haec clinical infusions in preterm babies with bronchopulmonary dysplasia [18] . in all xeno-transplants and clinical infusion, allogenic human cells have been used with no sign of rejection. in addition to their plasticity and tissue remodeling, haec have been reported to possess immune-modulatory properties that makes them appealing for cellular therapy. human amnion epithelial cells are poorly recognized by the immune system, following allogenic [18, 19] , and even xeno-transplants [9] [10] [11] [12] [13] [14] [15] [16] . this immune privilege is partly due to their uniquely regulated expression of human leukocyte antigens (hla): with the absence of hla class ii antigens or costimulatory molecules [20] , low levels of the polymorphic hla class 1 [20] , and constitutive expression of non-polymorphic hla-g [21, 22] . amnion-derived cells have been shown to have powerful immunomodulatory properties, crucial for evading an immune mediated rejection of the developing embryo by the maternal system, and in support of allogenic transplantation of haec in immune competent animals without immunosuppression [9] [10] [11] [12] [13] [14] [15] [16] 21] . preclinical studies report a re-education of the host immune-system into a more tolerogenic and regenerative setting [23] [24] [25] . finally, haec are not immortal [4] , and not tumorigenic when transplanted [4, 14, 26] . the current study is part of a multi-step investigation of safety and efficacy in consideration of moving the haec therapy into clinical practice. recently, standardized reagents and reproducible procedures, in accordance with current good manufacturing procedure (gmp) requirements, have been standardized [27] . criteria for characterization and release of the final product [27] , in addition to biodistribution analysis, have validated an efficient and safe route of administration for qualified haec batches into the hepatic parenchyma [28] . in anticipation that the transplant product used in any future clinical trial will be previously cryopreserved cells, the current study was undertaken to determine if there are any significant differences between the populations initially isolated from the amnion membranes, and the cells recovered following the cryopreservation procedure. cell surface markers characteristic of different cell types were investigated both before and after cryopreservation. additional pathways thought to be critical to haec engraftment and long-term function following transplantation were investigated in cryopreserved lots of haec to determine if differences could be identified that might provide insight into cases that might be more useful for transplantation as opposed to others. the human placenta was procured from uncomplicated full-term cesarean resection from healthy mothers. the placenta was received from karolinska institute hospital, stockholm under ethical permit number: 2015/419-34/4. signed, informed consent was provided by the mother to use the sample for research purposes. all the pathogen-positive deliveries (including hbv, hcv, syphilis, and hiv) were excluded, and current revised exclusion criteria includes positiveness for covid-19. no information regarding human donors (mother and newborn child) was transmitted or diffused, and the details were de-personalized, in order to respect privacy. placentae were delivered and processed within 3 h post-partum. a protocol for haec isolation was previously described [27] . briefly, the amnion membrane was removed from the inner surface of the placenta and washed multiple times with ringers solution (baxter, sweden) and plasmalyte solution (baxter, norfolk, uk) to remove blood. the amnion membrane was digested using tryple 10× (gibco, grand land, ny, usa) for 30 min at 37 • c to primarily release epithelial cells from the amnion membrane. the dispersed haec were cells 2020, 9, 1696 3 of 13 collected by centrifugation and filtered through a 100 µm cell strainer. cell viability was determined by the trypan blue exclusion (tbe) method (thermofisher, waltham, ma, usa). haec were not maintained in culture or selected ex vivo prior to freezing. following cell isolation, haec were immediately re-suspended in university of wisconsin solution (uw; bel gen 1000, lissieu, france) supplemented with 10% dimethyl sulfoxide (dmso; sigma-aldrich, mo, usa), at a cell density of 10 million viable cells/ml. every cell batch was aliquoted into 1.5 ml cryotubes (corning, ny, usa) and transferred in a controlled freezing container that lowered the temperature by 1 • c per minute when stationed in a −80 • c freezer (biocision, larkspur, ca, usa). the cells were stored in the vapor phase of a liquid nitrogen storage tank for a minimum of 30 days and a maximum of 36 months before analysis. the cryovials were removed from the liquid nitrogen tank and rapidly thawed by partial emersion in a water bath maintained at 37 • c, until small ice crystals remain (commonly for 80-120 s). cells were diluted into 10 volumes of ice cold plasmalyte solution (baxter), supplemented with 10% bovine calf serum, and centrifuged at 250 × g for 5 min. cell pellet was resuspended in cold plasmalyte and filtered through a 100 µm cell strainer. cell viability and recovery were determined by tbe method. the heterogeneity of the cell suspension was evaluated based on surface markers quantified by fluorescence-activated cell sorting (facs). both freshly isolated and cryopreserved haec were incubated with monoclonal antibodies directed against cell-specific surface protein, properly diluted in pbs solution and incubated for 30 min at 4 • c. the human-specific antibodies included in the study were cd326 (epithelial cell adhesion molecule, epcam; clone-hea-125; miltenyi biotech); cd31 (pecam1; clone wm59), cd44 (hcam; clone g44-26), cd45 (clone-t29/33), cd49f (alpha 6 integrin subunit; clone-goh3), cd105 (endoglin; clone-sn6; all from bd biosciences, san jose, ca, usa). all six monoclonal antibodies were directly conjugated with one of three specific dyes, fluorescein isothiocyanate (fitc) or phycoerythrin (pe) or allophycocyanin (apc) to perform multilineage evaluation on the same suspension. corresponding isotype controls were also analyzed. the cells were washed and fixed with 2% bd™ stabilizing fixative (bd biosciences) for 10 min at room temperature. cells were washed and re-suspended in ice cold pbs, and analyzed on a facscanto (bd biosciences) using flowjo™_v10 software. statistical differences were determined by paired t-test; p < 0.05 was chosen as the minimum level of significance. results are presented as histograms showing data plots, mean ± standard deviation. all data were analyzed by graphpad prism software (version 6.0, graphpad software inc., san diego, ca, usa). fourteen human placentae were collected and generated cell suspensions characterized by limited variability (16 ± 7 million viable cells/gr of processed tissue). cell viability measured immediately after isolation was 90% ± 4% (n = 14). when cells from the same 14 cases were thawed months to years later, the average cell viability was significantly lower (78% ± 5%; p < 0.0001; figure 1 ). ten million viable haec were initially cryopreserved. on average, a lower number of cells were recovered (6.5 ± 1.1 million/ml), corresponding to 55-95% of the initially cryopreserved cells. cases characterized with the highest viability post-cryogenic procedure did not always result in highest cell recovery ( figure 1 ). statistical differences were determined by paired t-test; p < 0.05 was chosen as the minimum level of significance. results are presented as histograms showing data plots, mean ± standard deviation. all data were analyzed by graphpad prism software (version 6.0, graphpad software inc., san diego, ca, usa) fourteen human placentae were collected and generated cell suspensions characterized by limited variability (16 ± 7 million viable cells/gr of processed tissue). cell viability measured immediately after isolation was 90% ± 4% (n = 14). when cells from the same 14 cases were thawed months to years later, the average cell viability was significantly lower (78% ± 5%; p < 0.0001; figure 1 ). ten million viable haec were initially cryopreserved. on average, a lower number of cells were recovered (6.5 ± 1.1 million/ml), corresponding to 55-95% of the initially cryopreserved cells. cases characterized with the highest viability post-cryogenic procedure did not always result in highest cell recovery ( figure 1 ). there is some heterogeneity in the types of cells recovered from amnion membrane depending on the isolation procedure and the reagents used. preparations of haec are generally characterized for the presence of static surface markers to identity different cell types in amnion-derived cell product [27] . facs analysis was performed on each haec case, before and after cryopreservation ( figure 2 ). the presence of specific epithelial cell markers (cd49f and cd326) was confirmed on the majority of isolated cells (figure 2b ). the average expression of cd49f on haec was unaffected by cryopreservation procedure and it was present on 99% ± 1% of freshly isolated and cryopreserved cells (p = 0.1386) (figure 2b) . similarly, the expression of another epithelial marker cd326 was nonsignificantly different between fresh (88% ± 8%) and cryopreserved cells (91% ± 6%; p = 0.0939) ( figure 2b ). similarly, haec viability after cryopreservation is represented as black bars. the first bars on the left represents average viability ± standard deviation (avg). cell recovery for every case and average are shown as black and white, checkered bars. * p < 0.001, t-test on paired values. there is some heterogeneity in the types of cells recovered from amnion membrane depending on the isolation procedure and the reagents used. preparations of haec are generally characterized for the presence of static surface markers to identity different cell types in amnion-derived cell product [27] . facs analysis was performed on each haec case, before and after cryopreservation ( figure 2 ). the presence of specific epithelial cell markers (cd49f and cd326) was confirmed on the majority of isolated cells ( figure 2b ). the average expression of cd49f on haec was unaffected by cryopreservation procedure and it was present on 99% ± 1% of freshly isolated and cryopreserved cells (p = 0.1386) ( figure 2b) . similarly, the expression of another epithelial marker cd326 was non-significantly different between fresh (88% ± 8%) and cryopreserved cells (91% ± 6%; p = 0.0939) ( figure 2b ). digestion of amnion membrane to deliver epithelial cells could result in stromal cell release from the inner layer of the amnion membrane. the presence of amnion-derived mesenchymal stromal cells (msc) was investigated with common stromal markers, cd105 and cd44 [29] . approximately 50% of the haec preparations contained a small amount of cells with msc surface markers. cells positive for cells 2020, 9, 1696 5 of 13 cd44 were found on 3/14 preparations, on an average of 0.5% ± 0.8% of the total cell number ( figure 2c ), while cd105 was expressed in 9/14 preparations on 1.25 ± 1.6% of the cells. after cryopreservation, stromal marker positive cells were below the limits of detection on 9/14 haec ( figure 2c ): with 0.1% ± 0.4% (p = 0.173) and 0.6% ± 1.4% (p = 0.0128) of the total cells remaining positive for cd44 and cd105, respectively. digestion of amnion membrane to deliver epithelial cells could result in stromal cell release from the inner layer of the amnion membrane. the presence of amnion-derived mesenchymal stromal cells (msc) was investigated with common stromal markers, cd105 and cd44 [29] . approximately 50% of the haec preparations contained a small amount of cells with msc surface markers. cells positive for cd44 were found on 3/14 preparations, on an average of 0.5% ± 0.8% of the total cell number hematopoietic or endothelial cells may be present in the final cell suspension from residual contamination with blood or placental vascular tissue. the presence of cd45, a receptor linked to protein tyrosine phosphatase present in cells of the hematopoietic lineage, was detected in 9/12 of haec preparations immediately after isolation at an average level of 1.2% ± 1.4% cells, but greatly reduced after cryopreservation and washing steps, where only four preparations still contained an extremely low number of cd45 positive cells (0.3% ± 0.4%; p = 0.0146) ( figure 2d ). the endothelial cell marker (cd31) was undetectable in all 14 cases, after isolation or cryopreservation (data not shown). mismatches in human leukocyte antigens (hla) expression are recognized by immune cells and generally induce rejection. the expression of classical hla-ia antigens (hla-a, -b, -c) and the hla-ii were quantified ( figure 3a ). expression of hla-ia was detected on an average of 39% ± 18% of the cells, with a variable range of expression in different cryopreserved haec cases (10-55%). as previously reported [20] , human leukocyte antigens class ii was negative on all haec, before and after cryopreservation. amnion epithelial cells have been reported to express characteristic immunomodulatory molecules, such as hla class ib [21, 22] . these and other surface proteins have been ascribed as mediators in immune-recognition and reactions against innate or adaptive immune cells. the presence of hla-g on haec after isolation was confirmed using a specific antibody, and hla-g amnion epithelial cells have been reported to express characteristic immunomodulatory molecules, such as hla class ib [21, 22] . these and other surface proteins have been ascribed as mediators in immune-recognition and reactions against innate or adaptive immune cells. the presence of hla-g on haec after isolation was confirmed using a specific antibody, and hla-g expression was maintained after cryopreservation on 79% ± 9% of the cells (range, 66-91%) ( figure 3a ). hla-ib expression by haec was investigated by transcriptome analysis: hla-g, -e, and -f forms were detected in all the haec cases analyzed, with hla-e expression approximately 100-fold higher than hla-g and hla-f ( figure 3b ). recent studies indicate that ecto-enzymes, such as adenosinergic enzymes cd39 and cd73 play an activate role in modulation of the immune system [25] . both hla molecules and ecto-enzymes cd39 and cd73 (30% ± 7% and 88% ± 7%, respectively) were expressed on all 14 cases of haec after thawing ( figure 3a) . as with hla-ib, cryogenic procedure does not significantly affect expression of ecto-nucleotidase enzymes on haec ( figure 3b) . finally, indoleamine-pyrrole 2,3-dioxygenase (ido), another enzyme involved in immune modulation and immune tolerance by limiting t-cell function and described in immunomodulatory cells, was analyzed but undetectable in all haec cases (data not shown). multipotency is another important characteristic ascribed to haec [2, 17] . the expression of the transcription factors oct-4, nanog, and sox2 are critical in the maintenance of pluripotency, and all three transcription factors were expressed in all haec cases ( figure 4a ). another transmembrane protein evolutionarily conserved and responsible for several developmental processes such as cellular fate determination and terminal differentiation, delta-like 1 homolog (dlk-1), was found to be expressed in all preparations ( figure 4a ). finally, telomerase reverse transcriptase (tert), a component of telomerase complex responsible for long-term survival and repetitive cycles of cell replication was undetectable in all haec cases (data not shown). transplanted epithelial cells require integration into parenchyma to establish cell-to-cell and cell-to-ecm interaction supporting their survival and growth. integration and nidation is in part facilitated by the secretion of matrix metalloproteinases (mmp), enzymes that degrade ecm and enhance cell adhesion, migration, and proliferation. several mmps were analyzed in the haec cases. mmp2, mmp3, and mmp9 were expressed in all cases ( figure 4b ). additional isoforms, mmp7, mmp8, and mmp13, were also analyzed but were undetectable in all haec cases ( figure 4b ). low level expression of mmp12 was detected in one case, however, it was undetectable in the other 13 cases. finally, the tissue inhibitor of metalloproteinases timp1 was highly expressed in all preparations ( figure 4b ). all three transcription factors were expressed in all haec cases ( figure 4a ). another transmembrane protein evolutionarily conserved and responsible for several developmental processes such as cellular fate determination and terminal differentiation, delta-like 1 homolog (dlk-1), was found to be expressed in all preparations ( figure 4a ). finally, telomerase reverse transcriptase (tert), a component of telomerase complex responsible for long-term survival and repetitive cycles of cell replication was undetectable in all haec cases (data not shown). several liver diseases have been treated with liver cell transplants [30] [31] [32] , although human hepatocyte transplants are limited in part by the paucity of organs for cell isolation [31] . recent studies support the use of haec transplants as an alternative for hepatocytes [5, [13] [14] [15] 23] . transplantation of haec has been shown to correct amino acids and neural transmitter abnormalities in mouse models of pku and msud [13] [14] [15] , and to reverse the lethal effects of acute liver failure [15, 16, 33] . the studies with mice, like the human patient transplants, were conducted on subjects that have normal, active immune systems and haec were transplanted without providing immunosuppression. yet, haec engrafted without evidence of immune recognition and rejection [13, 16, 33] . as cell therapies become more feasible, the demand for a constant supply of quality cells becomes increasingly important. an optimized enzymatic isolation method has been proved to generate a large amount of haec from full-term placentae, and the efficiency in cryopreservation with simple, controlled conditions may fulfill the growing demand for haec transplantation. in this study, haec were harvested from 14 different donors based on a protocol using entirely clinical grade reagents [27] . the formulation of reagent to thaw haec has originally included additive of animal origin (fbs). such supplement has been replaced with human albumin in revised procedure, and matched reliability and efficiency (as validated in several haec batches, including cases here analyzed, data not shown). the quality of cell product is regularly characterized based on the presence of static cell markers (as regularly performed with other cellular therapies). the lack of need for a pre-step based on in vitro selection allowed to immediately direct haec to cryogenic preservation, where they were banked for several weeks to years (as commonly performed in perinatal cell banks for clinical use). upon thawing, the cryopreserved cells were analyzed for surface markers and transcriptome analysis. the present data support the consistency of the cellular haec product, before and after cryopreservation. while there was approximately a 12% loss of viability upon thawing, the cells recovered after cryopreservation maintain the same surface markers as the cells prior to cryopreservation. constitutive presence of epithelial markers, such as cd49f and cd326, showed that the isolation procedure is highly selective for epithelial cells, and that they nicely tolerate the cryogenic procedure. thawing and subsequent wash steps resulted in an average loss of 35% of the cells initially frozen. as expected, the endothelial marker, cd31, was absent from all preparations examined. since the amnion is an avascular membrane, vascular endothelial cells would not be expected to be present in isolates of haec cells. the presence of passenger (maternal) hematopoietic cells in a haec preparation could elicit an allogeneic immune reaction if transplanted, and sporadic preparations of haec showed up to 4% cd45 positive cells before cryopreservation. however, only five of 14 preparations contained remaining cd45 cells upon thawing, and in an extremely low amount (1% or less). other surface markers present in some preparations were the mesenchymal markers: cd105 and cd44, which represented up to 5% (cd105) or 3% (cd44) in different preparations, before the cryogenic procedure. after cryopreservation, cd44 positive cells were absent in 12/14 cases and cd105 positive cells were only detectable in four of 14 cases, and sporadically (one case only) had more than 1% cd105 positive cells. it is not even clear that residual msc in an haec preparation would be detrimental since the immunomodulatory properties of msc are similar to haec. a recent report of the transplantation of haec into pre-term babies with established bronchopulmonary dysplasia reported release criteria as cells with >96% epithelial markers and less than 1% of cells positive for cd45 or mesenchymal markers [19] . the present studies indicate that those product release criteria would have been met in 13 of 14 of the cases reported here. once it was determined that the haec recovery after cryopreservation was acceptable, attention was focused on the examination of the properties that are thought to be critical for haec engraftment and long-term function after transplant. in clinical studies, allogeneic haec were transplanted with no signs of rejection [18, 19] . in immune-competent models and patients, haec cells engrafted and survived, without administration of immunosuppressive drugs [13, 16, 33] . the immunomodulatory properties of haec are a key feature that makes them attractive candidates for cell transplantation. the haec are often referred as 'immuneprivileged' cells, since they lack the expression of hla class ii antigens (as depicted by hla-dr staining in figure 3 ) as well as co-stimulatory molecules [19, 20] . polymorphic histocompatibility complexes have been shown to be expressed at different levels on haec: a low-density expression of hla-ia molecules is commonly detected on haec [20] . recently, a constitutive expression of non-canonical, oligomorphic hla class ib molecules on haec was reported [21] . hla-g molecules play a vital role in protecting the fetus, a 'semi-allograft' tissue from the mother's immune system and can induce apoptosis in activated t cells and also modulates the activity of the natural killer cells (nk) by binding to specific receptors cd85d, cd158d, and cd85j, respectively [34, 35] . like hla-g, hla-e and hla-f have potent immunomodulatory properties, although less is known about their mode of action. transcriptome analysis showed expression of all non-polymorphic molecules (hla-e, -f, and -g) by haec. in addition to the hla class ib molecules, other immunomodulatory functional enzymes, previously described on msc, have been recently identified on haec [25] . plasma membrane nucleotidases are cell surface enzymes, which represent functional bridges between cell cytoplasm and the environment. purinergic mediators, such atp and adenosine released into the extracellular space, have a role in proliferation and angiogenesis, and contribute to immune cell function by the inhibition of dendritic, t, and nk cells, and the promotion of t regulatory cells and m2 macrophages. once in the extracellular environment, atp is hydrolyzed into adenosine, by ecto-nucleotidase cd39 and cd73 in a tightly regulated process [36] . alternatively, adenosine is produced by metabolizing nad + , and this alternative pathway is initiated by cd38 and cd203a and culminates in cd73 [37] . all of these ectoenzymes play a role in modulating immune effector cells [25] . previous reports have suggested that the ecto-nucleotidases operate in a discontinuous fashion, where different receptors are expressed on different cell types, resulting in a synergic effect based on cell-to-cell interactions. human amnion epithelial cells were the first cell population reported to express all of these functional ectoenzymes on a single cell type, to drive the production of adenosine via canonical and alternative pathways [25] . additionally, all these surface enzymes were maintained after cryogenic procedure. stem cell marker genes were investigated, since expression of these pathways will likely contribute to proliferation and self-renewal of haec as well as determine their ability to differentiate to different cell types [4] . the expression of sox2, oct4, and nanog was quite consistent in the 14 cases examined. high levels of dlk1 were observed in all cases. it was reported that when transplanted into the liver, dlk positive precursor cells differentiate into hepatocytes [38] and hepatic differentiation of haec has been reported by us and others [5, [13] [14] [15] [16] 23] . while embryonic stem cells express many properties of multipotent or pluripotent stem cells, they are different in important aspects from haec. telomerase activity contributes to the regulation of proliferation, however telomerase reverse transcriptase (tert) was not expressed by haec, suggesting a cell type with more limited proliferation capacity than other stem cells [39] . this may be an important safety issue by reducing the possibility of unlimited growth and possible tumor formation. the matrix metalloprotease (mmp) and tissue inhibitors of matrix metalloprotease (timp) play important roles in cell migration, invasion, tissue remodeling, and proliferation [40, 41] . cell migration and remodeling of matrix is also a necessary step in engraftment of cells following transplantation [42] . breakdown of extracellular matrix results in the release of growth factors that were bound to ecm and makes them available to cellular receptors [40] . the haec predominantly express mmp-2, -3, and -9 in all cases analyzed. both mmp-2 and -9 degrade collagens and play a pivotal role in mobilization, homing, and engraftment of hematopoietic stem/progenitor cells as well as other cell types [41] . substrates for mmp-3 include collagens, fibronectin, laminin, and elastin and mmp-3 can also activate mmp-9. active mmps are inhibited by the tissue inhibitors of metalloproteinases (timps) [41, 43] . the balance between mmps and timps such as timp-1 are important in regulating mmp activities [40] . further studies will evaluate and determine the functional activity and ecm remodeling activity performed by different batches of primary haec. there are now two reports describing the transplantation of amnion-derived cells in patients affected by life-threating disorders. five patients with niemann-pick disease type b were treated with amnion-derived cells [44] , and recently six premature babies with bronchopulmonary dysplasia received haec treatment [19] , confirming the strong immunomodulatory and regenerative capacity of haec. our group has extensive experience with liver cell-based therapy of acute, chronic, and congenital liver disorders in patients [30, 31] . encouraging evidence of hepatic maturation by implanted haec in immune-proficient animals led our group to obtain approval to transplant allogenic haec into patients without the administration of immunosuppressive drugs. the recipient candidate are patients otherwise qualified for hepatocyte transplant. data contained in this report are an important step in the collection of preclinical data necessary to proceed with manufacturing and banking of haec for clinical use. surface marker expression analysis indicates that the population of haec recovered after cryopreservation is highly enriched for haec with minimal inclusion of other cell types (less than 1%). the expression of many pathways necessary for engraftment and long-term survival of haec following transplant procedures, including stem cell, immunomodulatory, and matrix remodeling pathways are maintained in cryopreserved haec. author contributions: conception and design (s.c.s., r.g.); collection and assembly of data (r.c.s., s.c.s., r.g.); data analysis and interpretation (r.c.s., r.g.); manuscript writing (r.c.s., s.c.s., r.g.). all authors have read and agreed to the published version of the manuscript. a rational strategy for the use of amniotic epithelial stem cell therapy for liver diseases stem cells derived from human fetal membranes display multilineage differentiation potential human amniotic epithelial cells possess hepatocyte-like characteristics and functions hepatic differentiation of amniotic epithelial cells human amnion-isolated cells normalize blood glucose in streptozotocin-induced diabetic mice in vivo differentiation of human amniotic epithelial cells into cardiomyocyte-like cells and cell transplantation effect on myocardial infarction in rats: comparison with cord blood and adipose tissue-derived mesenchymal stem cells the emergence of amnion epithelial stem cells for the treatment of multiple sclerosis shielding islets with human amniotic epithelial cells enhances islet engraftment and revascularization in a murine diabetes model human amnion epithelial cell transplantation abrogates lung fibrosis and augments repair human amnion epithelial cells prevent bleomycin-induced lung injury and preserve lung function improved amino acid, bioenergetic metabolite and neurotransmitter profiles following human amnion epithelial cell transplant in intermediate maple syrup urine disease mice placental stem cell correction of murine intermediate maple syrup urine disease therapeutic use of human amnion-derived products: cell-based therapy for liver disease transplantation of human amnion epithelial cells reduces hepatic fibrosis in immunocompetent ccl4-treated mice anti-fibrotic effects of fresh and cryopreserved human amniotic membrane in a rat liver fibrosis model two-year outcomes of infants enrolled in the first-in-human study of amnion cells for bronchopulmonary dysplasia first-in-human administration of allogeneic amnion cells in premature infants with bronchopulmonary dysplasia: a safety study concise review: isolation and characterization of cells from human term placenta: outcome of the first international workshop on placenta derived stem cells human amnion epithelial cells expressing hla-g as novel cell-based treatment for liver disease increased immunomodulatory capacity of human amniotic cells after activation by pro-inflammatory chemokines immunogenicity and immunomodulatory properties of hepatocyte-like cells derived from human amniotic epithelial cells immunogenicity and immunomodulatory effects of amnion-derived multipotent progenitor cells ectonucleotidase expression on human amnion epithelial cells: adenosinergic pathways and dichotomic effects on immune effector cell populations amnion-derived pluripotent/multipotent stem cells isolation of human amnion epithelial cells according to current good manufacturing procedures evaluation of different routes of administration and biodistribution of human amnion epithelial cells in mice minimal criteria for defining multipotent mesenchymal stromal cells. the international society for cellular therapy position statement human hepatocyte transplantation: worldwide results clinical hepatocyte transplantation: practical limits and possible solutions human hepatocyte transplantation for liver disease: current status and future perspectives therapeutic efficiency of human amniotic epithelial stem cell-derived functional hepatocyte-like cells in mice with acute hepatic failure kir2dl4 (cd158d): an activation receptor for hla-g. front a human histocompatibility leukocyte antigen (hla)-g-specific receptor expressed on all natural killer cells purinergic signalling and immune cells a non-canonical adenosinergic pathway led by cd38 in human melanoma cells induces suppression of t cell proliferation isolation of hepatoblasts based on the expression of dlk/pref-1 prevalence of telomerase activity in human cancer structure and function of matrix metalloproteinases and timps hematopoietic stem cell mobilization and homing after transplantation: the role of mmp-2, mmp-9, and mt1-mmp the tissue inhibitors of metalloproteinases (timps): an ancient family with structural and functional diversity treatment of sphingomyelinase deficiency by repeated implantations of amniotic epithelial cells acknowledgments: this research was supported by the national pku alliance (rg).conflicts of interest: scs has stock in noveome biotherapeutics, inc., although no noveome products or services were used in the present study. key: cord-347714-vxxhglx7 authors: abitogun, folagbade; srivastava, r.; sharma, s.; komarysta, v.; akurut, e.; munir, n.; macalalad, l.; olawale, o.; owolabi, o.; abayomi, g.; debnath, s. title: covid19: exploring uncommon epitopes for a stable immune response through mhc1 binding date: 2020-10-14 journal: biorxiv doi: 10.1101/2020.10.14.339689 sha: doc_id: 347714 cord_uid: vxxhglx7 the covid19 pandemic has resulted in 1,092,342 deaths as of 14th october 2020, indicating the urgent need for a vaccine. this study highlights novel protein sequences generated by shot gun sequencing protocols that could serve as potential antigens in the development of novel subunit vaccines and through a stringent inclusion criterion, we characterized these protein sequences and predicted their 3d structures. we found distinctly antigenic sequences from the sars-cov-2 that have led to identification of 4 proteins that demonstrate an advantageous binding with human leukocyte antigen-1 molecules. results show how previously unexplored proteins may serve as better candidates for subunit vaccine development due to their high stability and immunogenicity, reinforce by their hla-1 binding propensities and low global binding energies. this study thus takes a unique approach towards furthering the development of vaccines by employing multiple consensus strategies involved in immuno-informatics technique. coronaviruses are a large group of viruses that are rather common throughout the community, causing illness ranging from the common cold to more severe diseases such as severe acute respiratory syndrome (sars-cov) and middle east respiratory syndrome (mers-cov) (1) . the previous outbreaks of these coronaviruses in 2003 and 2015 respectively show similarities to the novel coronavirus (2) , which was first reported in december 2019 in wuhan (3) . it was later declared as a pandemic on 11th march, 2020 by the world health organization (who) having caused a global emergency across many countries and territories around the world. the causative agent of the covid-19 disease is the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which was initially tagged as 2019 novel coronavirus (2019-ncov) by the world health organization (4) before genomic studies revealed a 79.5% and 96% nucleotide level similarity with sars-cov and bat coronavirus respectively (5, 6) , hence necessitating its new identity. further analysis on its similarity with sars-cov and mers-cov including phylogeny revealed that it is categorized under the family coronaviridae, order nidoviralae and genus betacoronavirus alongside the other two viruses which have also caused pandemic situations in the past, causing thousands of death globally. (7, 8) the structural architecture of sars-cov-2 is an elaborate one, containing a positive single stranded rna as its genetic element with genomic length of about 29-30 kilobases, (9) which encodes the major structural proteins being the spike (s) glycoprotein, membrane (m) protein, envelope (e) protein, and nucleocapsid (n) protein. (10, 11) the structure of the spike glycoprotein of the virus is also an extended similarity with sars-cov, (4) which together with covid19: exploring uncommon epitopes for a stable immune response through mhc1 binding other proteins of the virus are candidates for vaccine development and are being explored in different settings due to the active roles of the proteins in the infectivity of the virus. (12) (13) (14) (15) developing an effective treatment for sars-cov-2 is a research priority. currently, different therapeutic strategies are being utilized by researchers to combat this dangerous covid-19, with much of the focus being on developing novel drugs or vaccines. however, in recent years, the development of vaccine design has been revolutionized by the reverse vaccinology (rv), which aims to first identify promising vaccine candidate through bioinformatics analysis of the pathogen genome. one of the successes of this method is the bexsero vaccine, discovered for group b meningococcus. (16) this immuno-informatics method includes the identification of peptide epitopes in the virus genome which can then be prepared by chemical synthesis techniques. these epitopes are easier in quality control; however, there is the need for structural modifications as well as the inclusion of delivery systems, and adjuvants in their formulation due to the low immunogenicity of the epitopes which is a result of their structural complexity and low molecular weight. (17) recently, a set of b and t cell epitopes highly conserved in sars-cov-2 were identified from the s and n proteins of sars-cov. (18) however studies have shown that full length spike protein vaccines for sars-cov may lead to antibody mediated disease enhancement causing inflammatory and liver damage in animal models (19, 20) which is why in this manuscript, we applied immuno-informatics "in silico" approaches to identify potential cd8+ cytotoxic t cell epitopes from proteins of sars-cov-2, sars-cov and mers-cov. in achieving this, the ~29.8kb genome of the sars-cov-2 which encodes for 28 proteins comprising 5 structural proteins, 8 accessory proteins and 15 non-structural proteins as well as the ~30kb genome of sars-cov-1 comprising 14 open reading frames (orf) were explored for some uncommon proteins of the viruses with therapeutic potentials. identified proteins were thus considered for highly antigenic epitope prediction and evaluation. the antigenicity and immunogenicity of all identified epitopes was estimated and their interactions with the human leukocyte antigen (hla) class i system were evaluated, owing to the wide usage of the hla system as a strategy in the search for the etiology of infectious diseases and autoimmune disorders, (21) as was explored in the case of sars-cov-1 after the first epidemic that broke out in east asia in 2002−2003. this is linked to the fact that hla genes are known to display the highest level of diversity in the genome, with thousands of different alleles now reported. each of these alleles is also a combination of multiple single nucleotide polymorphisms (snps). this, we also investigated the hla-class i system for evidence of disease association, with the hope that the discovery of disease susceptibility will help in developing protection such as vaccines against the virus. the uniprot (22) database was searched for the protein sequences inferred from the genome sequence analysis of different isolates of highly pathogenic human coronaviruses. the sars cov, mers-cov, and sars-cov-2 proteins without experimentally defined 3d structure or functional annotation were chosen for further analysis and their sequence files in the fasta format were downloaded. the fasta sequences were used to infer the physicochemical properties of the chosen each model was refined using a molecular dynamics simulation approach implemented by galaxy refine (36) tool or rapid energy minimization using discrete molecular dynamics with an all-atom representation for each residue implemented by chiron server. antigenicity prediction of the top eight uncharacterized coronavirus proteins selected after 3d molecular models' validation was performed using vaxijen tool. (44) a threshold value of 0.4 was taken into account. non-antigenic peptides having vaxijen scores less than 0.4 were discarded, while antigenic epitopes with scores higher than 0.4 were further prioritized for their immunogenicity. the proteins predicted to be non-antigens were discarded from the further analysis. antiviral immunity relies on the ability of major histocompatibility complex (mhc) class i molecules to bind antigen molecules and display them to t cells. each candidate is subsequently refined by restricted interface side-chain rearrangement and by soft rigid-body optimization. the side-chain flexibility is modeled by rotamers and the obtained combinatorial optimization problem is solved by integer linear programming. following rearrangement of the side-chains, the relative position of the docking partners is refined by monte carlo minimization of the binding score function. the refined candidates are ranked by the binding score which is a combination of atomic contact energy, softened van der waals interactions, partial electrostatics and additional estimations of the binding free energy. in order to identify functionally important regions in the selected proteins and the selection of epitopes with the desired degree of conservation, consurf (54) a total of 8 non-structural sequences were identified and retrieved from uniprotkb database. the mass, isoelectric-ph, basic amino acid %, cysteine %, polarity and hydrophobicity of the protein sequences were computed and outlined in table 1. the lengths of these protein sequences varied from 39 to 275 amino acid residues. cysteine was found to have the highest presence in the selected proteins with an average of 5.56% and its highest presence was observed in proteinq7tfa0 with 15.40%. arginine, lysine and histidine have an average presence of 2.35%, 3.69% and 4.36% respectively. the polarity and non polarity of the final selected proteins ranged from 27.907%to 63.839 % and from 36.161%to 72.093% respectively with m4svf1 having the highest percentage of polar amino acids (63.839%) while p0dtd8 has the highest percentage of non-polar amino acids (72.093%). likewise, the highest hydrophobicity index was recorded for p0dtd8. after obtaining the 3d structures of the proteins from modeling servers, the models were refined using galaxyrefine. upon refinement, the models were subjected to stereochemical and thermodynamic stability analysis. particularly, the models with highest ramachandran favored residues and lowest clash scores were used for energy analysis using chiron, scoop and errat servers as highlighted in table 2. all the protein structures had a ramachandran score of over 89% and an errat value of over 60, with q7tfa0, p0dtd8 and p0dtd3 all having a perfect 100% ramachandran scores. the free energy of the structures ranged from -28.7kcal/mol (q7tfa0) to -2.2 kcal/mol (a0a6h2lbe3). force field values ranging from -5955.989 (a0a6h2lbe3) to -435.783 (p0dtd8) were observed using gromos96 implementation of the swiss-pdb viewer. the models with lowest overall energies were determined to be stable. vaxijen antigenicity prediction was used to predict the overall antigenicity of the 8 proteins as outlined in table 3. two proteins: m4svf1_mers and q7tfa0 were predicted to be non-antigens with their antigenicity scores of 0.3373 and 0.1251 respectively while p0dtd8 and p0dtc8 showed very high antigenicity owing to their high scores of 0.8462 and 0.6502 respectively. t-cell epitope prediction was carried out using iedb, epijen, pepvac, rankpep, and netmhc. initially, 15,181 hla class i binding epitopes were predicted from 6 out of 8 stable proteins which were predicted to be antigenic. scrutiny on the basis of percentile rank filtered 112 peptide epitopes. considerable binding affinity for 5 hla-1 subtypes with high frequencies in asia and africa (hla-a*02:01, hla-a*11:01, hla-b*08:01, hla-b*27:05 and hla b*58:01) was observed from netmhcpan server which predicted high affinity binders (<0.5 percentile rank) using el-ranks. among predicted epitopes of sars-cov-2 virus, ten (10) epitopes showed considerably high immunogenicity towards the mhc-class 1 molecules which were then selected for further analysis (table 4 ). all the predicted epitopes were further subjected to allergenic and toxicity analysis, with all 10 coming out as non-allergens and non toxins (table 4) . epitopes including hlvdfqvti, rksapliel, and ylcflafll showed remarkably high antigenic tendencies, scoring 1.4119, 1.1591 and 0.9143 respectively. all of these epitopes, along with their features are reported in table 4 (table 5) . further docking analysis resulted in a total of 10 global binding energies predictions which were refined with firedock ( table 6 ). the global binding energies range from -68.71 to -11.19 indicating that all the proposed epitope sites from the modelled proteins can serve as good antigens. the conservation status of each residue in the selected t-cell epitopes in four (4) sars cov-2 proteins (q7tlc7, p0dtc6, p0dtd8, p0dtc8) were examined using consurf. results revealed that the highly conserved and exposed residues are found in all 4 proteins, with p0tdc6 and p0dtc8 having the larger amount of these residues. particularly, the epitopes without allergenicity and toxicity containing at least one predicted functional residue (highly conserved and exposed) included epitopes 'rksapliel' and 'hlvdfqvti' while t-cell epitopes 'rksapliel', 'flafllflv', and 'hlvdfqvti' contained at least one predicted structural residue (highly conserved and buried). interestingly, none of the epitopes from p0dtd8 contained any functional residues (highly conserved and exposed) while the highest concentration of predicted structural and functional residues was found in two epitopes hlvdfqvti (1 functional residue and 3 structural residues) and rksapliel (3 functional residues and 1 structural residue)-from the protein p0dtc6 . despite the continuous unrest caused by the covid19 pandemic, considering the 1,886,643 new cases recorded as of 7th september, 2020 which has spiked up the total reported cases to over 26.7 million and a total of 876,616 deaths globally, there is currently no available fda-approved vaccine against covid-19 (57). if a vaccine is successfully developed against covid-19, this will improve global human health. advancement in technology has led to various bioinformatic approaches such as reverse vaccinonology (rv) and immune-informatics being applied to revolutionize vaccine production in terms of production cost and time which has been a major setback in vaccine development. antibody response as well as cell mediated immunity can be established by using proper protein antigen. reverse vaccinology (rv) has been useful in identification of potential vaccine candidates against pathogens and the world is in a race to get one against sars-cov2. this approach presents an advantage over other vaccine approaches because it can identify all potential antigens coded by a genome irrespective of their abundance, phase of expression and immunogenecity. while most of the previous protein vaccines against sars-cov have focused on the full length spike protein of the virus, the vaccine development race against the covid19 virus have followed the same path, with a larger percentage of the 69 of the 210 vaccines currently in development utilizing the spike protein of the sars-cov-2 (58) not minding the reported negative side effects, including liver damage and antibody mediated inflammatory diseases, of the full length spike protein vaccines and other whole organism vaccines in the host. additionally, while novel technologies and approaches such as next-generation vaccines enabled through advances in nanotechnology which relies on the principle that nanoparticles and viruses operate at the same length scale are poised to make a clinical impact for the first time, many of these platforms may be several years away from deployment and therefore may not have an impact on the current sars-cov-2 pandemic, further necessitating the priority given to the quicker and safer vaccine development platforms. the current study focused on the hla-1 binding propensities of some uncommon proteins from covid-19 virus. obtaining these antigenic epitopes using the immune-informatics approach will help inform which epitopes to use in the construct of protein vaccines against sars-cov-2 using proteins other than the spike protein. from the 8 selected proteins obtained all the models with good stability analysis including the ramachandran analysis, which is a two dimensional plot drawn in the space of angles ϕ and ψ that quantify the rotation of the protein backbone and thus play a crucial role in the secondary structure of proteins and later the tertiary structure, were further analyzed for antigenicity and only highly antigenic proteins were selected for further analysis since the highly antigenic proteins will induce better immune response in the host. five (5) of the 35 most common hla-1 subtypes in africa and asia that were identified in this study were included in the analyses and their potential interactions between identified epitopes from the proteins were predicted using the netmhcpan 4.1 server. the binding affinities between these hla subtypes and the epitopes were indicated by the server which allowed the selection of epitopes that will have strong interactions with the hla alleles. the analysis of this interaction is important because vaccination is a proven strategy for protection from disease and an ideal vaccine would include antigens that elicit a safe and effective protective immune response. hla-restricted epitope vaccines, including t-lymphocyte epitopes restricted by hla alleles, embodies a newly developing and favorable immunization approach. research in hla restricted epitope vaccines to be used for the treatment of tumors as well as for the prevention of viral, bacterial, and parasitic infections have, in recent years, achieved substantial progress and this further strengthens the resolve of the hla-binding propensities analysis done in this study. the predicted strongest binding epitopes were thus further subjected to immunogencity evaluation which checked the ability of the epitope to induce an immune response and also predicted how strongly they will induce an immune response; allergenicity which verified the potential of our epitopes to cause sensitization or allergic reactions related to ige antibody responses; and toxicity which is its ability to cause bodily harm. using these criteria, 10 epitopes were identified and these could be potential epitopes for vaccine constructs against sars-cov-2. this was also evinced by the lower global binding energies between our identified epitopes and the hla molecules, ranging from -68.71 to -11.19, indicating stable complexes formed between them. our extensive approach towards the prediction of stable protein structures from previously uncharacterized proteins and the subsequent application of immune-informatics for the identification of immunogens has resulted in the classification of 10 different epitope sites from 8 different proteins that have high antigenicity and low binding energies with hla-1 alleles that are most commonly present in the continent of asia and africa. these epitopes have high tendencies to provide effective and strong protective cytotoxic immunity against the sars cov-2 if they are adequately exploited for vaccine production. a multiple consensus approach ensures the reliability and reproducibility of these results. this study thus provides further knowledge on the association of these hla-1 alleles with covid19, suggesting that good peptide coverage can be achieved by many different combinations of hla-1 alleles. multi-epitope based peptide vaccine design using three structural proteins (s, e, and m) of sars-cov-2: an in silico approach knowledge, 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knowledge-based force field protein structure refinement driven by side-chain repacking automated minimization of steric clashes in protein structures procheck: a program to check the stereochemical quality of protein structures verify3d: assessment of protein models with three dimensional profiles verification of protein structures: patterns of nonbonded atomic interactions deviations from standard atomic volumes as a quality measure for protein crystal structures scoop: an accurate and fast predictor of protein stability curves as a function of temperature the gromos biomolecular simulation program package a server for prediction of protective antigens, tumour antigens and subunit vaccines gold-standard data classification, open access genotype data and new query tools netmhcpan-4.1 and netmhciipan-4.0: improved predictions of mhc antigen presentation by concurrent motif deconvolution and integration of ms mhc eluted ligand data the immune epitope database (iedb): 2018 update silico approach for predicting toxicity of peptides and proteins pep-fold3: faster de novo structure prediction for linear peptides in solution and in complex ucsf chimera -a visualization system for exploratory research and analysis patchdock and symmdock: servers for rigid and symmetric docking firedock: a web server for fast interaction refinement in molecular docking using evolutionary data to raise testable hypotheses about protein function consurf: identification of functional regions in proteins by surface-mapping of phylogenetic information development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines coronavirus disease (covid-19) situation reports milken institute covid-19vaccinetracker table 3. immunogenicity analysis of predicted mhc class 1 epitopes we acknowledge the support of dnacompass, cmesbahf nigeria, biotrust scientific, galaxy project and neliref for this project. we also acknowledge the provision of figure images generated by rajavee srivastava and folagbade abitogun from ucsf chimera, discovery studio, pymol, mhc cluster server v 2.0 and consurf. we acknowledge t. thao, h. aminu, and v. ekundayo for their contribution to this project. folagbade abitogun is a research associate at the university college hospital, ibadan, nigeria.he is currently actively involved in antimicrobial resistant research and he has deep interest in the interplay between the human immune system and the clearance of infectious diseases, using molecular and bioinformatics approaches. coloured 'e' represents exposed residues, the green colored 'b' represents buried residues, the red colored 'f' represents functional residues (highly conserved and exposed) and the dark blue colored 's' represents structural residues (highly conserved and buried). the conservation scale represents the status of conservation from variable, average to conserved. key: cord-310252-0cdqhrcw authors: seliger, barbara; ruiz‐cabello, francisco; garrido, federico title: chapter 7 ifn inducibility of major histocompatibility antigens in tumors date: 2008-12-03 journal: adv cancer res doi: 10.1016/s0065-230x(08)00407-7 sha: doc_id: 310252 cord_uid: 0cdqhrcw interferons represent a protein family with pleiotropic functions including immunomodulatory, cytostatic, and cytotoxic activities. based on these effects, interferons are involved in innate as well as adaptive immunity, thereby shaping the tumor host immune responses. these cytokines, alone or in combination, have been successfully implemented for the treatment of some malignancies. however, it has been recently demonstrated that tumor cells could be resistant to interferon treatment, which may be associated with an escape of tumor cells from immune surveillance. therefore, the aim of this chapter is to summarize the frequency of impaired interferon signal transduction, their underlying molecular mechanisms, and their clinical relevance. ag, antigen; apc, antigen presenting cells; apm, antigen-processing machinery; bh, bleomycin hydrolase; bp, base pairs; ciita, class ii transactivator protein; clip, class ii invariant chain peptide; ctl, cytotoxic t lymphocyte; dc, dendritic cell; er, endoplasmic reticulum; gas, gammainterferon-activated site; ifn, interferon; ifn-r1, interferon-receptor-1; il, interleukin; irf, interferon regulatory factor; isg, interferon-stimulated genes; isgf3, ifn-stimulated gene factor 3; isre, interferon-stimulated response element; jak, janus kinase; lps, lipopolysaccharide; mapk, mitogenactivated protein kinase; mca, methylcholanthrene; mhc, major histocompatibility complex; nf, nuclear factor; nk, natural killer; pkc, protein kinase c; rcc, renal cell carcinoma; sclc, small-cell lung carcinoma; socs, suppressor of cytokine signaling; stat, signal transducer and activator of transcription; ta, tumor antigen; tap, transporter associated with antigen processing; tcr, t cell receptor; tfbs, transcription factor-binding sites; tnf, tumor necrosis factor; tpn, tapasin; tppii, tripeptidyl peptidase ii; tsa, trichostatin a; tyk, tyrosine kinase; uirr, upstream interferon response region; usf1, upstream stimulatory factor 1; wt, wild type. interferons (ifns) represent proteins that are secreted from cells in response to various stimuli and provide the basis for the understanding of the evolution, structure, and function as well as the pathways of other cytokines and their receptors (pestka, 2000; pestka et al., 2004) . they exert pleiotropic effects and are involved in host responses to bacterial and viral infection, in tumor surveillance mechanisms as well as in innate and adaptive immune responses (decker et al., 2005; pestka et al., 2004; stetson and medzhitov, 2006; takaoka and yanai, 2006) . in addition, ifns were the first cytokines used for the treatment of tumor patients. however, it has been suggested that tumor cells might develop either a transient or a permanent ifn insensitivity. this phenotype is linked to cytotoxicity resistance and might lead to escape of tumor cells from immune surveillance. we here summarize the current knowledge about (i) pleiotropic functions of ifns that mediate various biological responses, (ii) mechanisms of action and transduction pathways, (iii) the effect of type i and type ii ifns on the expression levels of molecules involved in proper major histocompatibility complex (mhc) class i and class ii antigen processing and presentation of tumor cells, (iv) the frequencies and the underlying molecular mechanisms of ifn resistance in tumors in association with alterations of the mhc class i and ii antigen-processing machinery, and (v) the clinical relevance of aberrant ifn signaling. the elucidation of the mechanisms leading to dysregulation of ifn signal transduction cascades triggering immune dysfunction and to tumor immune escape will benefit the design of strategies reversing these deficiencies, which could be of clinical relevance. interferons (ifns) are a family of multifunctional cytokines, which were originally described as antiviral cytokines, thereby protecting cells from viral infection (isaacs and lindenmann, 1957) . however, based on the current knowledge they exhibit a broad spectrum of activities including antiproliferative, immunomodulatory, anti-inflammatory, apoptosis-inducing, stress-mediated effects as well as regulation of cell differentiation steps and angiogenesis (amadori, 2007; baccala et al., 2005; theofilopoulos et al., 2005) . the ifn family is divided into type i, type ii, and type iii ifns. type i ifns consist of 13 ifn-members and single members of ifn-, ifn-, ifn-, and ifn-e, respectively, which are all clustered on chromosome 9. in contrast, type ii ifn is represented only by a single gene, ifn-, encoded by chromosome 12 (decker et al., 2005) . recently, type iii ifns have been discovered as a novel class of antiviral cytokines which are classified into ifn-1, -2, and -3 (oesterlund et al., 2007; sheppard et al., 2003; uze and monneron, 2007) . ifns bind to two distinct cell surface receptors. type i and ii ifn signal through a common -chain, thereby activating discrete, but related pathways leading to the transcriptional activation of the so-called interferonstimulated genes (isgs) ( table i; fig. 1 ). isgs represent a functionally diverse group of genes involved in many cellular activities such as transcription, translation, regulation of cell cycle and apoptosis, intracellular communication as well as the processing and presentation of antigens. the transcriptional activity of isgs is necessary to mediate the effect of ifns. because of their diverse activities, ifns have been used for the treatment of various diseases such as chronic viral infections, like hepatitis c, multiple sclerosis, hematopoietic malignancies as well as solid tumors including renal cell carcinoma (rcc) and melanoma. the ifn therapy has been shown to reduce the rates of relapses and mortality by between 12 and 30% in tumor patients (kirkwood et al., 2004) . however, during the last decade no further progress concerning the adjuvant therapy of tumor patients has been achieved. therefore, a better knowledge of the underlying molecular mechanisms of ifn action may lead to improved and more effective applications and the design of innovative, intelligent treatment strategies using ifns alone or in combination with other therapeutics. a wealth of information is available on the molecular processes underlying some of the ifn-induced signaling cascades. binding of ifns to their specific receptors lacking intrinsic kinase activity induces oligomerization of receptor subunits triggering diverse signaling pathways ( fig. 1) , thereby leading to the transcriptional regulation of a plethora of target genes (kaur et al., 2005; li et al., 2004; schindler et al., 2007) . the physiologic relevance of ifn-dependent signal transduction cascades including the stat/jak pathway was established by generating and characterizing mice with targeted disruption of genes encoding stat1/stat2 or jak1, respectively (platanias, 2005; ramana et al., 2002) . both type i and type ii ifn receptors (ifn-r) initiate the activation of the jak/stat cascade, which consists of four janus kinases (jak1, jak2, jak3, and jak4) and seven signal transducers and activators of transcription (stat1, stat2, stat3, stat4, stat5a, stat5b, stat5c; fig. 1 ifn signal transduction cascade and defects in this pathway. the type i and type ii receptors are transmembrane glycoproteins whose extracellular domains serve as ifn-binding sites, whereas their cytoplasmic domains associate with members of the jak kinase family and initiate signal transmission (dunn et al., 2006) . upon binding to their specific receptors both type i and type ii ifns induce a number of signal transduction cascades, which involve the phosphorylation of various components such as tyk2, jaks, and stats. after recruitment to the receptor, stats become phosphorylated, form homo-or heterodimers, and migrate to the nucleus to bind to specific sequences in the promoter of target genes. type i ifn-induced signaling then induces homodimerization of stat1 and heterodimerization of stat1 and stat2. stat1 and stat2 associate with the cytosolic transcription factor ifn-regulatory factor 9 (irf9), forming a trimeric complex known as ifn-stimulated gene factor 3 (isgf3) and activates transcription by binding to the isres. type ii ifn associates kinases, jak1 and jak 2 phosphorylate stat1, which then forms homodimers, translocates to the nucleus, and activates transcription by binding to the gas sequences. ifn-mediated signaling is controlled by several mechanisms including dephosphorylation of ifn-r1, jak1, and stat1 (mediated by sh2-domain-containing protein tyrosine phosphatase 2, shp2), inhibition of the jaks (mediated by suppressor of cytokine signaling 1, socs1), proteasomal degradation of the jaks, and inhibition of stat1 (mediated by protein inhibitor of activated stat1, pias1). shin-ya et al., 2005; yu and jove, 2004) . stats, sh2-containing transcription factors, represent cytosolic proteins of 750-800 amino acids and are composed of (i) an extracellular domain that plays an important role in the association of stat with receptor molecules, (ii) a ligand-binding domain, and (iii) an intracellular domain that is responsible for the stat dimer formation. stat1 induces the expression of ifn-responsive genes through the activation of ifn-stimulated response element (isre)-containing promoters (yu and jove, 2004) . however, it has now become apparent that the activation of jak-stat pathways alone is not sufficient for the generation of all biological activities of ifns. there exists accumulating evidence that several other ifn-regulated signaling elements and cascades are required for the generation of many ifn responses. some of them operate independently of the jak-stat pathway, whereas others cooperate with stats to optimize the transcriptional regulation of target genes. these include in particular pathways linked to cellular stress and cell death like the mitogenactivated protein kinase (mapk), the stress-induced kinase p38, and protein kinase c (pkc) signaling cascades. pkcs are known to be involved in both ifn-andsignaling pathways (kwon et al., 2007) . in this context, it is noteworthy that the ifn-, ifn-, and ifn-cascades exhibit overlapping activities, but also clearly different features ( fig. 1 ; levy et al., 1990) . after the engagement with the type i ifn receptors (ifn-r), ifnbinding stimulates the cross-linking between the ifn-r chain 1 (ifn-r1) and 2 (ifn-r2), thereby bringing the receptor-associated kinases tyk2 and jak1 into close proximity. this triggers the activation of jak1 and tyk2 leading to the phosphorylation of tyr-466 of the ifn-r1, which serves as a docking site for stat2. the activated kinase subsequently phoshorylates stat2 and stat1 on tyr-690 and tyr-701, respectively. both phosphorylated stats form a heterodimer and associate with the interferon regulatory factor (irf)9, which does not undergo tyrosine phosphorylation to form the ifn-stimulated gene factor 3 (isgf3), which, in turn, translocates to the nucleus and binds specific elements known as isres that are present in the promoters of certain isgs initiating the transcription of a broad variety of genes. in addition, phosphorylated stat1, other stat complexes, and combinations of different stat-containing complexes can be formed which translocate to the nucleus and bind to the ifn--activated site (gas) leading to the transcription of further genes (caraglia et al., 2005) . it is noteworthy that ifn-can also activate stat3 and stat5, but the role of stat5 in the ifn--mediated activity has still to be elucidated (uddin et al., 2003) . in contrast, ifn-mainly activates stat5b. however, one can speculate that a fine balance between different stat complexes might account for specific responses and represent a key mechanism for ifn--induced activities. ifn-acts through a heterodimer consisting of the ifn-receptor-1 (ifn-r1) and ifn-r2 expressed on most cells, thereby upregulating specific genes. binding of ifn-initially leads to the formation of an ifn-r1 homodimer, which consecutively attracts the ifn-r2 chains. the ifn-r1 and -r2 homodimer is constitutively associated with jak1 and jak2, which phosphorylate the tyrosine 440 at the intracellular domain of the ifn-r1 serving as a docking site for the latent cytosolic transcription factor stat1. stat1 is subsequently phosphorylated on tyrosine 701 and serine 727 leading to the homodimerization of phospho-stat1 molecules. these form a complex named the -activating factor (gaf) that translocates into the nucleus and upregulates the transcription of ifn--regulated genes including in particular the interferon-regulated factors (irf)1 and irf7 which represent transcriptional activators, whereas the constitutively expressed irf2 generally acts as a transcriptional repressor (harada et al., 1989) . irf1 subsequently activates the transcription of caspase genes involved in apoptosis next to genes encoded in the major histocompatibility complex (mhc) in particular components of the mhc class i and class ii antigen-processing machinery (apm) as well as 2 -microglobulin ( 2 -m) located on chromosome 15. the molecules of the antigen-processing pathway are required for the initiation and triggering of proper cd4 þ or cd8 þ t-cell responses, respectively. in addition, stat1 and irf1 cooperate with the ubiquitously expressed transactivating factor upstream stimulatory factor (usf)1 to activate the transcription of the class ii transactivator protein promoter iv (ciita-piv) that controls the expression of mhc class ii molecules (chen et al., 2007) . the expression of mhc class i and class ii molecules is critical for the presentation of antigens and essential for the generation of an adaptive immune response (cresswell et al., 2005; jensen, 2007) . in the last decades, cd8 þ cytotoxic t lymphocytes (ctl) have been implicated as main effector cells in antitumor responses. they recognize and attack tumor cells presenting intracellular antigens derived from different nonself peptides on their surface through the interaction of the t-cell receptor (tcr) with mhc class i peptide complexes. the generation and presentation of these antigens (ag) requires a coordinated expression of several genes ( fig. 2a) . briefly, endogenously synthesized proteins are cleaved by the multicatalytic proteasome complex, in particular the ifn--regulated proteasome subunits, such as the low molecular weight proteins (lmp)2, lmp7, and lmp10. these peptides are further trimmed by cytosolic enzymes such as, for example, the tripeptidyl peptidase (tpp)ii and the bleomycin hydrolase (bh) generating the correct n-terminus (kloetzel, 2004; rock et al., 2004) . then the peptides are transported from the cytosol into the endoplasmic reticulum (er) via the transporter associated with antigen processing (tap), a heterodimer consisting of the tap1 and tap2 subunits. in the lumen of the er the mhc class i assembly occurs, which is assisted by various chaperones such as calnexin, calreticulin, the oxido thiol reductase erp57, and tapasin (tpn). tpn facilitates the peptide loading onto mhc class i molecules. after successful peptide loading, mhc molecules are released from the peptide loading complex and the trimer consisting of mhc class i heavy chain (hc)/ 2 -m/peptide is then transported through the trans-golgi apparatus to the cell surface and presented to cd8 þ ctl. thus, proper expression of the major components of the complex mhc class i apm components is obligatory for effective t-cell recognition of tumors (groettrup et al., 1996; jensen, 2007; seliger et al., 2006) . recently, it has been demonstrated that cd4 þ t cells are also important for proper antitumor immune responses (drozina et al., 2005; jensen, 2007) . these t cells recognize via their tcr antigens presented on mhc class ii molecules. in contrast to mhc class i antigens which are expressed on all nucleated adult cells, the expression of the heterodimeric mhc class ii molecules also representing transmembrane glycoproteins is highly restricted and preferentially found on the cell surface of professional antigen presenting cells (apcs). however, mhc class ii antigen expression can be induced in other cell types by various cytokines, in particular ifn-. mhc class ii expression is mainly controlled by the class ii transactivator protein (ciita), which acts as a master regulator for its coordinated constitutive and ifn--induced expression which also involves pkc delta (kwon et al., 2007; giroux et al., 2003) . ciita interacts with the transcription factors rfx, nfy, and creb (van den in the cytosol, endogenous peptides are generated by the proteasome, which were further trimmed by other peptidases and then transported into the er via the heterodimeric tap. erap is involved in the final aminoterminal trimming of peptides. the loading of mhc class i molecules with peptides is further assisted by the chaperone tapasin which is also a component of the plc. upon peptide loading, the plc dissociates and then transported via the trans golgi to the cell surface and there exposed to cd8 þ cytotoxic t lymphocytes. (b) mhc class ii pathway. mhc class ii molecules assemble in the er with the invariant chain (li), which contains an endosomal targeting signal. this complex is then transported to the endosomal compartment and there the ii is cleaved by a number of proteases leaving only the clip fragment, which occupies the peptide-binding groove. hla-dm and -do catalyze the release of clip, which is exchanged by antigenic peptides. hla-dm edit the repertoire of the mhc class ii-peptide complexes, which are then transported to the cell surface for recognition by cd4 þ t lymphocytes. exogenous proteins are internalized into the endosomal pathway by different mechanisms then unfolded and cleaved which is catalyzed by different proteases. in addition, the yielded peptides are further trimmed after binding to mhc class ii molecules. elsen et al., 2004) , thereby forming an enhanceosome governing the mhc class ii transcription. in addition, a coordinated expression of various mhc class ii apm components exists. mainly exogenous antigens are phagocytosed by apcs, directed then to lysosomes where they are cleaved into small peptide fragments (fig. 2b) . mhc class ii antigens are assembled in the er. the peptide-binding groove of these molecules is initially occupied by the invariant chain which is degraded into the class ii invariant chain peptide (clip) fragment by a series of key cleavage events, thereby protecting the mhc class ii-binding groove. the loading of mhc class ii molecules with exogenously derived peptides is assisted by the chaperone-like components hla-dm and -do, which results in an exchange of the clip fragment by these antigens. hla-dm is editing the peptides presented to cd4 þ t cells by catalyzing multiple rounds of peptide exchanges possibly favoring the most stable complexes. the peptide-loaded mhc class ii molecules are then transported to the cell surface and presented to cd4 þ t lymphocytes. in professional apc, exogenous antigens can gain access to the mhc class i pathway through distinct cross-presentation mechanisms. furthermore, the endosomal mhc class ii loading pathway could also receive peptides derived from endogenous antigens through autophagy and other mechanisms (dengjel et al., 2005; schmid et al., 2007) . the promoters of the mhc class i and class ii apm components have been intensely characterized and exert some similarities, but also unique properties. concerning the promoter of mhc class i apm components, some of them contain tata and caat boxes, whereas others completely lack these regulatory domains in the promoters). in addition, it is noteworthy that both tap1 and lmp2 are transcribed from a shared bidirectional promoter of only 596 base pairs (bp) separating their atg translation initiation codon (wright et al., 1995) . the promoter of the major mhc class i apm components contain a combination of distinct transcription factor-binding sites (tfbs), like sp1, creb, the nuclear factor (nf)-b, e2f, and p300, but all exhibit ifn-response elements, which hint toward their regulation by irfs chatterjee-kishore et al., 1998 , 2000 fig. 3) . in terms of the mhc class ii pathway, the promoters of the invariant chain, hla-dm/-do and the mhc class ii hc, respectively, contain similar, but also distinct transcription factorbinding sites, whereas all of them contain an ifn-response element in their promoter. an exception is represented by ciita, which is regulated by multiple promoters differing in their tfbs composition. there exist three tissue-specific promoters for ciita, pi, pii, and piii. one promoter controls the constitutive ciita expression in dendritic cells (dc), whereas another is specific for the constitutive expression in b cells. the ciita-piv regulates the induction of ciita expression in different cell types. it contains several cis elements including a putative nf-b site overlapping with an ap2 site, the ifn-activating sequence (gas), the e box, and an irf element (dong et al., 1999; muhlethaler-mottet et al., 1998) . thus, the activity of the different mhc class i and ii apm component promoters can be induced, but to a different extent, by type i and type ii ifns, respectively. ifn-is a stronger inducer when compared to type i ifns, whereas a combination of both substances exerts additive or even synergistic effects on mhc class i and ii apm components. activation of adaptive immune responses by ifn, in particular ifn-, is partially due to transcriptional activation of genes encoding the mhc class i and class ii antigens and respective apm components such as the invariant chain, hla-dm/-do, ciita, tap, tpn, the lmps, and erap1/2. fig. 3 promoter structure of major apm components. the structure of representative promoters of the major apm components is schematically illustrated, demonstrating a number of transcription factor-binding sites such as nf-b, ap1, sp1, and creb as well as interferon regulatory response elements (isre), which are involved in the inducibility by this cytokines. decrease in or absence of mhc class i molecules has been observed in a diversity of human tumor types (garrido and algarra, 2001; garrido et al., 1993 garrido et al., , 1997 ). an increasing proportion of tumors were found with total or selective hla allelic losses supporting the theory that altered hla expression phenotypes represent a major mechanism of tumor escape from t-cell recognition due to downmodulation of presentation of immunodominant tumor antigens. distinct hla class i abnormalities, including total loss or downregulation of hla class i antigens (paschen et al., 2003) , hla haplotype loss (ramal et al., 2000) , hla locus or allele loss (jimenez et al., 2001) has been described in tumors originating from different tissues and multiple molecular mechanisms have been identified as responsible for these changes (garrido and algarra 2001) . the mechanisms that underlie total or partial loss of hla class i antigens (table ii) include mutations of the 2 -microglobulin ( 2 -m) gene (perez et al., 1999) and loss of heterozygosity (loh) of mhc genes (maleno et al., 2004) . other causes of total hla class i downregulation comprise defects in the regulation of different components of the mhc class i antigen processing. structural defects of apm components cannot be corrected by cytokine treatment; therefore, it does not restore hla class i surface antigen expression. t-cell-based therapy may not be effective due to the irreversible loss of hla class i molecules. this is important when selecting the appropriate immunotherapy for a given cancer patient. abnormalities in the expression of various mhc class i apm components occur at a high frequency in human tumors of distinct origin like small-cell lung carcinoma (sclc), melanoma, colon carcinoma, breast carcinoma, renal cell carcinoma, and hematological malignancies and are frequently associated with malignant transformation (table ii) . this phenotype allows the tumor cells to evade recognition by mhc class i-restricted, tumor antigen (ta)-specific ctl. mutations in different apm components appear to be a rare event postulating that dysregulation rather than structural alterations is the major cause for aberrant apm component expression (fernandez et al., 2000; ramal et al., 2000; seliger et al., 2006; table ii) . this hypothesis is supported by experiments (i) identifying only few mutations in these molecules, (ii) characterizing the apm promoter activity in tumors, (iii) determining posttranscriptional regulatory mechanisms, and (iv) treating tumor cells with ifns to analyze whether deficiencies of apm component expression could be overcome by cytokines. indeed, impaired apm component expression of tumor cells could be often restored by ifn-/ and/or ifn-treatment. the ifn-mediated upregulation of apm components often results in enhanced mhc class i surface expression, which is required for the generation of an effective antitumor-specific immune response. indeed, the ifn-induced upregulation of apm components improves antitumor-specific ctl responses (seliger et al., 1997; tajima et al., 2004) and therefore represent a valuable strategy for the treatment of patients with apm component deficiencies. however, in some cases, tumors remain insensitive to ifn treatment despite the lack of structural alterations in apm components, rather suggesting an impaired ifn signal transduction. the unresponsiveness to ifn treatment was analyzed in a number of different tumor types and according to kaplan et al. (1998) can be frequently found in human cancers. approximately 33% of 33 melanoma and 17 non-adenocarcinoma lung tumor cell lines analyzed exhibit a quantitative reduction in ifn-sensitivity, while 2 out of 17 lung adenocarcinoma cell lines were totally unresponsive to ifn-. these data were extended in a recent study in which 57 melanoma cell lines were analyzed for the ability to upregulate mhc class i surface antigens in response to stimulation with ifn-. a total unresponsiveness to ifn-was found in 2 out of 57 melanoma cell lines (rodriguez et al., 2007b) . however, the number of tumor types and tumor samples analyzed for ifn resistance is still limited and requires further studies in order to determine the frequency, relevance, and molecular mechanisms of these deficiencies. it is noteworthy that an impaired ifnresponse despite a functional ifn-induction may exist. on the other hand, a lack of ifn-responsiveness can also be found in the presence of ifnsensitivity, suggesting that the ifn signal transduction cascades are not coordinately regulated in tumor cells. the importance and involvement of ifn signal transduction pathways in the transcriptional regulation of apm promoters have been established, but there exists only limited information about the underlying molecular mechanisms of defective ifn-inducible apm component expression. the impairment could occur at different steps along the ifn signal transduction pathways and might involve sequence abnormalities and/or different regulatory processes such as transcriptional, posttranscriptional, and epigenetic control ( fig. 1 ; table iii ). the physiological relevance of the stat/jak and pi3k pathway has been established in mice with a targeted disruption of these genes. the lack of jak1 activity was associated with a loss of ifn-to induce growth arrest and apoptosis as well as an increased tumorgenicity (sexl et al., 2003) . however, the observed ifn-response with respect to growth inhibition might also be attributable to the ifn-inducibility of lmp2 (hayashi et al., 2006) . so far, there exists only limited information regarding the molecular mechanisms of ifn resistance in tumors (huang et al., 2002; lesinski et al., 2007; wellbrock et al., 2005; wong et al., 1997) . based on the current knowledge that stat1 and irf1 are involved in the transcriptional regulation of the dual tap1 and lmp2 promoter, the loss of tap1 and lmp2 expression may be attributable to deficiencies of these regulatory factors. regarding the ifn-resistance of rcc cell lines, it is associated with a defective induction of stat1 that could be restored by the addition of a supernatant from pma-stimulated peripheral mononuclear cells (brinckmann et al., 2002) . this effect appears to be mediated by ifn-although other cytokines might also be involved in this process. in addition, the loss of the ifn--mediated upregulation of mhc class i apm components in some rcc cell lines appears to be due to the lack of irf1-and stat1-binding activities upon ifn-stimulation. the stat1, jak1, and jak2 proteins were expressed but not phosphorylated in the presence of ifn-. the ifn--mediated inducibility was not restored by gene transfer of jak1 and/or jak2 into rcc cells, whereas jak1 overexpression increased both tap1 and lmp2 expression independent of ifn-. therefore, the loss of tap1 and lmp2 induction was associated with a defect of an early step in the ifn-signal transduction pathway (dovhey et al., 2000) . furthermore, an association of impaired stat1 phosphorylation with the loss of ifn-mediated hla class i induction was also found in melanoma cell lines (rodriguez et al., 2007b) . the absence of stat1 phosphorylation was at least partially due to the constitutive expression of the suppressor of cytokine signaling (socs)-1 protein, which could be mediated by the jak2 kinase inhibition via the socs phosphatase. socs-1 modulates the ifn-mediated signaling by binding to the autophosphorylation site of jak2 and by targeting bound jak2 to the proteasome for degradation (waiboci et al., 2007) . in addition, socs-1 expression correlates with melanoma progression and confers growth advantage (komyod et al., 2007; li et al., 2004) . in another study, the ifn-resistance was associated with socs3 expression. the resistant cell lines differed from the sensitive cells by a constitutive expression of socs3, by the absence or a low degree of socs1-3 activation following ifn-treatment, and by a short duration of the cytokine activatory signal (fojtova et al., 2007) . the expression of ifn--responsive genes is also reduced in the choriocarcinoma cells jeg3 and jar in comparison to the epithelial cell line hela (choi et al., 2007) . this is mediated by a compromised tyrosine phosphorylation of jak2 and stat1 at tyrosine 701 and the reduced expression of irf1. in addition, inhibition of the tyrosine phosphatases results in increased jak1 and stat1 phosphorylation and ifn--induced gene expression in these cells (choi et al., 2007) . the impaired expression of irf1 and deficient phosphorylation of stat1 were also observed in primary trophoblast cell lines suggesting that these defects are of clinical relevance. besides the posttranslational regulation of components of the ifn signal cascades, the absence of the ifn--mediated mhc class i expression can be controlled by epigenetic alterations in this pathway. indeed, methylation affects the binding of irf1 leading to an abrogation of the irf1 transactivation (rodriguez et al., 2007b) . treatment with the demethylating agent 2 0 5 0deoxyazacytidine (dac) restored the irf1 expression and consecutively led to the reconstitution of the ifn--mediated mhc class i inducibility. other studies have identified that the ifn unresponsiveness is attributed to low expression of stat1 rather than to an absence of its phosphorylation (abril et al., 1998; xi et al., 2006) . the absence of stat1 expression has been correlated with the methylation of its promoter (xi et al., 2006) . finally, there exists evidence that genetic instability in tumor cells may lead to modulation of the expression of the ifn-r, which in some cases has been reported to be associated with cancer prognosis. for instance, the loss of ifn-r independently predicts poor prognosis in ovarian cancer and may be responsible for the limited success in the outcome of treatment of ovarian cancer with ifn(duncan et al., 2007) . the multiple activities of ifns on tumor cells might coordinate the antitumor immune responses so that the early recognition and/or elimination of cancer cells by the innate immune system transitions to immune attack by the adaptive immune system (dunn et al., 2006) . the ifn-on the tumor cell immunogenicity mediate the immune response directed against tumor cells through distinct mechanisms. ifn-can downregulate the expression of the nkg2d ligands and at the same time increase the expression of mhc class i molecules (bui et al., 2006) . in vitro treatment with ifn-decreased the death by nk cells independently from the expression of hla class i molecules, whereas an increased mhc class i expression increased the sensibility ctl-mediated lysis. besides these in vitro results, there also exist information that abnormalities in the ifn signaling occurs in vivo. lmp2-/-mice exhibit an impaired proteasome function and 36% of female lmp2-/-mice develop uterine leiomyosarcomas by 12 months of age. thus, the development of spontaneous human uterine leiomyosarcomas might be probably due to defects in early steps of the ifn signal cascade. indeed, the defective tap1 and lmp2 expression in these tumors is associated with a g871e mutation in the atp-binding region of the jak1 kinase domain, thereby affecting jak1 kinase activity, but neither jak1 expression and production nor its degradation (hayashi et al., 2006) . this allows the tumor cells to evade antitumor-specific immunity. in different tumor types, immunosuppression associated with stat3 activation and stat3-mediated inhibition of dc function has been reported (yu and jove, 2004) . the biological function of stat1 and stat3 differs in terms of cell growth and induction of an antitumor immune response. whereas stat1 abrogates growth and mediates antitumor effects, stat3 promotes cell proliferation and tumorigenicity as it has been shown in melanoma and head neck squamous carcinoma. in both tumor entities, stat3 expression is associated with tumor progression and mediates immune suppression. in addition, unphosphorylated or phosphorylated stat1 and stat3 are coordinately upregulated by both ifn-and ifn-and may represent a marker for the dynamic mechanism of melanoma progression and host response. using methylcholanthrene (mca) and untreated ifn-r -/-, a significant tumor development was observed in the ifn-r control mice. the crossing of ifn-r1 and stat1 -/mice with p53 -/mice resulted in a spontaneous and more rapid tumor development in particular teratomas, hemangiomas, and chondrocytomas, whereas lymphoid tumors generally develop in ifn--sensitive p53 -/mice. interestingly, the ifn-sensitive tumor cells transfected with the dominant negative ifn-r mutant grew faster than untransfected tumors and were not rejected upon their treatment with lipopolysaccharide (lps) effectively eliminating control tumors . furthermore, downregulation of the ifn-r in association with loss of fas function is linked to tumor progression (yang et al., 2008) . thus, the ifn-responsiveness is an important mechanism in the control of tumor growth. an increased responsiveness to metastasespromoting agents might be induced by many mediators in the microenvironment of melanoma including type i and type ii ifns. both cytokines cooperate with tnf-, which involves a positive interplay between jak1 and pkc signal transduction (bianchini et al., 2006) . these data suggest that multiple signals were generated by the host inflammatory cells, which are accompanied by cooperate with the invasive properties of tumor cells. therefore, strategies targeting this cross-talk among tumor and host cells in the microenvironment are needed to prevent tumor growth. the chimeric ret/ptc (rearranged in transformation/papillary thyroid carcinoma) oncoproteins were constitutively expressed in papillary thyroid cancer and are able to phosphorylate the y107 of stat1, which is accompanied by irf1 expression (hwang et al., 2004) . this is associated with an enhanced transcription of ciita and consequently with mhc class ii expression of papillary thyroid carcinoma cells and explain the immune cell infiltration of ret/ptc-positive cancers. furthermore, a synergistic activity of tnf-and ifn-on ciita was found in thyroid carcinoma (rahat et al., 2001) the ciita-independent mhc class i expression could be upregulated by histone deacetylases like trichostatin a (tsa) (chou et al., 2005; gialitakis et al., 2006) . ciita was refractory to ifn induction in many tumors. in colorectal and gastric carcinoma cells, ciita is silenced by epigenetic mechanisms resulting in the lack of ifn--induced mhc class ii expression (satoh et al., 2004) . in order to correlate the ifn unresponsiveness with the expression profile of isgs, cdna microarray analyses were employed using a customized microarray consisting of 850 isg (holko and williams, 2006) . expression of genes associated with transcription precedes the expression of genes involved in signal transduction, whereas no differences in the stat1 induction were observed. however, subtle alterations in the expression profile might be responsible for the insensitivity to this cytokine. the maintenance of transcriptional activation following ifn treatment appeared to enhance ifn sensitivity. ifns have been used in various clinical settings, since they are potent negative regulators of cell growth either by modulating the cell cycle or by inducing pro-apoptotic genes. ifn-has been extensively studied in the treatment of various malignancies during the last two decades demonstrating improved clinical outcome of hematological malignancies (chronic myeloid leukemia, cutaneous t-cell lymphoma, hairy-cell leukemia, multiple myeloma), solid tumors including malignant melanoma, renal-cell carcinoma (rcc), aids-related kaposi's sarcoma, and viral syndromes (hepatitis c, hepatitis b, severe acute respiratory syndrome). ifn-has shown positive results in the treatment of chronic granulomatous disease, multiple sclerosis, and severe malignant osteopetrosis (parmar and platanias, 2003, for review). however, the resistance to ifns has been described, which limits their anticancer activity. the impaired expression of ifn-responsive genes might have important implications not only in immunotherapy but also in transplantation, pregnancy, and the development of tumors such as choriocarcinoma. despite proven clinical efficacy in malignancies, viral infections, and multiple sclerosis, a substantial number of patients fail to develop positive clinical response to ifn therapy. although ifn-2b is a clinically active therapeutic agent for malignant melanoma and rcc, only 15-20% patients with metastatic melanoma respond to ifn therapy (marincola et al., 1995) . other reviews report even lower response rates of only 6% of treated melanoma patients (quesada et al., 1985; umeda and niijima, 1986) . in rcc, the best results of ifn treatment as determined by the response rate and the duration of the effect were obtained in patients with a previous nephrectomy without chemotherapy, in a good functional state, and with preferentially lung metastasis. in these patients the survival rate increased from 49 to 115 weeks upon ifn-administration (logothetis, 1992) . despite these positive results, there exist many aspects of these response factors which are not well understood. actually, none of these factors has been proved to be associated in an unambiguous way with the cytokine response and the patients' survival. the key aspect may be the right selection of patients, since currently all of them independent of previous nephrectomy and the presence of metastasis are enrolled into the treatment with poor clinical outcome. unlike type i ifns, ifn-has not been approved for cancer treatment by the fda. ifnproduces numerous antitumor effects and plays a central role in promoting natural immune responses directed against developing tumors. however, its practical application in immunotherapeutic protocols has been very limited. in clinical trials, an improved survival was observed in patients with ovarian cancer of stage ic-iiic treated with ifn(windbichler et al., 2000) , when ifn was intravesically administered to patients with transitional-cell bladder carcinoma (giannopoulos et al., 2003) or when ifn was used in isolatedlimb perfusion of individuals with non-melanoma cancers of the extremities (lienard et al., 1998) . however, no effect was detected upon ifn-treatment of patients with metastatic rcc (gleave et al., 1998) , advanced colon cancer (wiesenfeld et al., 1995) , or small-cell lung cancer (jett et al., 1994) . the limited success of the therapeutic use of ifn-might reflect the inability to target ifns in the right place with an efficient concentration (dunn et al., 2006) . despite the proven pivotal role of endogenously produced antitumor immunity of ifn-in animal models, the limited success of this cytokine in cancer immunotherapy trials in humans might be explained by the resistance of tumor cells to ifn(kaplan et al., 1998; rodriguez et al., 2007a; wong et al., 1997) . in this context, it is important to note that unlike type i ifns, ifn-has a direct effect on tumor cells during the antitumor immune response supporting the relevance of ifn-in the cancer immunoediting process (dunn et al., 2004) . the targets of the immunologic unresponsiveness represent genes encoding components of the mhc apm components or the constituents of the ifn-r signaling pathway. in this context, in two recent studies from our laboratory, the physiological relevance of hla class i surface expression during the tumor rejection process in patients receiving different protocols of immunotherapy was assessed (cabrera et al., 2007; carretero et al., submitted) . in the first study, a significant difference in the immunotherapeutic response of patients exhibiting metastases with low levels of mhc class i surface antigens and those with high levels of mhc class i expression was detected. in a second trial, the impact of cytokine unresponsiveness was demonstrated by determination of hla class i antigen expression levels on metastatic melanoma lesions during the course of the disease in one patient undergoing ifn-2b and autologous vaccination plus bcg (m-vax). bcg triggers the il-12/ifn-axis and induces upregulation of genes associated with antigen presentation (feinberg et al., 2004; saban et al., 2007) . the level of the mhc class i antigen expression was dependent on the ifn response since neither of the progressor metastases increased the expression of hla class i antigens after vaccination. however, a significant increase in the hla class i surface expression was detected in the regressor metastases. therefore, the hla class i surface antigen on tumor cells significantly contributed to the therapeutic effect of bcg. in connection with these findings, downregulation of hla class i surface antigens in cancer cells has been considered a significant risk factor for recurrence in patients with intravesical bcg immunotherapy for bladder cancer (kitamura et al., 2006) . based on these results, a better understanding of the molecular mechanisms by which tumors modulate the cytokine signaling may be essential for the development of immunotherapeutic strategies with the aim to enhance mhc class i surface antigen expression in tumor cells. the balance of stat phosphorylation versus socs expression might be crucial in the activation of immunologic response through apm and mhc class i transactivation (wang et al., 2007) . for instance, the effects of high-dose ifn are associated with immunologic processes such as an upregulation of tap1, tap2, tpn, and lmp2. the stat1 and stat2 pathways in melanoma cells are sensitized to ifn-by pretreatment of the cells with ifn-. thus, the biological response to ifn-might be mediated by a direct effect on melanoma cells and suggests also a potential role for ifn-in the treatment of this disease (carson, 1998) . in addition, it has recently been demonstrated that ifntreatment of patients with cutaneous melanoma significantly modulates the balance of stat1/stat3 in tumor cells and host lymphocytes. this results in an upregulation of tap2 and an increased immune response (wang et al., 2007) . an increased knowledge of the factors responsible for the resistance to ifns might lead to an improved use of these cytokines in malignant diseases. the application of the molecular analysis of tumor tissues has now advanced to the point where better classification schemes and prognostic variables are used leading to an optimization of specific treatment programs and patients' selection. the identification of tumor lesions with the capacity to upregulate mhc gene expression will determine the ability to present new antigenic peptides to t lymphocytes favoring regression of primary or metastatic tumor lesions. in contrast, the identification of tumors with mhc irreversible genetic lesions will maintain an unaltered mhc expression, thereby not exposing new antigenic peptides to t cells, which subsequently favors tumor and/or metastases progression. we propose that suppression of ifn signaling in tumors contributes to tolerance by inhibiting expression of genes encoding subunits of hla class i/ii antigens and/or components of the mhc class i/ii apm that could be detrimental to successful antitumor responses. unresponsiveness to interferon associated with stat1 protein deficiency 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infection autophagy promotes mhc class ii presentation of peptides from intracellular source proteins ifn-gamma regulation of the type iv class ii transactivator promoter in astrocytes loss of interferon-gamma inducibility of tap1 and lmp2 in a renal cell carcinoma cell line expression of mhc ii genes loss of ifn gamma receptor is an independent prognostic factor in ovarian cancer the immunobiology of cancer immunosurveillance and immunoediting interferons, immunity and cancer immunoediting bacillus calmette guerin triggers the il-12/ifn-gamma axis by an irak-4-and nemodependent, non-cognate interaction between monocytes, nk, and t lymphocytes beta2-microglobulin gene mutation is not a common mechanism of hla class i total loss in human tumors development of ifn-gamma resistance is associated with attenuation of socs genes induction and constitutive expression of socs 3 in melanoma cells mhc antigens and tumor escape from immune surveillance natural history of hla expression during tumour development implications for immunosurveillance of altered hla class i phenotypes in human tumours coordinated changes of histone modifications and hdac mobilization regulate the induction of mhc class ii genes by trichostatin a the immunomodulating effect of interferon-gamma intravesical instillations in preventing bladder cancer recurrence ifn-gamma-induced mhc class ii expression: transactivation of class ii transactivator promoter iv by ifn regulatory factor-1 is regulated by protein kinase c-alpha interferon gamma-1b compared with placebo in metastatic renal-cell carcinoma peptide antigen production by the proteasome: complexity provides efficiency structurally similar but functionally distinct factors, irf-1 and irf-2, bind to the same regulatory elements of ifn and ifn-inducible genes the mutation in the atp-binding region of jak1, identified in human uterine leiomyosarcomas, results in defective interferongamma inducibility of tap1 and lmp2 functional annotation of ifn-alpha-stimulated gene expression profiles from sensitive and resistant renal cell carcinoma cell lines stat1 negatively regulates angiogenesis, tumorigenicity and metastasis of tumor cells regulation of signal transducer and activator of transcription 1 (stat1) and stat1-dependent genes by ret/ ptc (rearranged in transformation/papillary thyroid carcinoma) oncogenic tyrosine kinases virus interference. i. the interferon. by a. isaacs and j. lindenmann, 1957 recent advances in antigen processing and presentation phase iii trial of recombinant interferon gamma in complete responders with small-cell lung cancer a nucleotide insertion in exon 4 is responsible for the absence of expression of an hla-a*0301 allele in a prostate carcinoma cell line demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice the pi3 0 kinase pathway in interferon signaling a pooled analysis of eastern cooperative oncology group and intergroup trials of adjuvant highdose 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metastatic urothelial tumors combination therapy with interferon alfa-2a and interleukin-2 for the treatment of metastatic cancer distribution of hla class i altered phenotypes in colorectal carcinomas: high frequency of hla haplotype loss associated with loss of heterozygosity in chromosome region 6p21 stat1 regulates lipopolysaccharide-and tnf-alpha-dependent expression of transporter associated with antigen processing 1 and low molecular mass polypeptide 2 genes in macrophages by distinct mechanisms activation of the mhc class ii transactivator ciita by interferon-gamma requires cooperative interaction between stat1 and usf-1 ifn regulatory factor family members differentially regulate the expression of type iii ifn (ifn-) genes complete loss of hla class i antigen expression on melanoma cells: a result of successive mutational events a new beta 2 microglobulin mutation found in a melanoma tumor cell line the human interferon alpha species and receptors interferons, interferon-like 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interferons (alpha/beta) in immunity and autoimmunity role of stat5 in type i interferon-signaling and transcriptional regulation phase ii study of alpha interferon on renal cell carcinoma. summary of three collaborative trials il-28 and il-29: newcomers to the interferon family transcriptional regulation of antigen presentation both the suppressor of cytokine signaling 1 (socs-1) kinase inhibitory region and socs-1 mimetic bind to jak2 autophosphorylation site: implications for the development of a socs-1 antagonist modulation of signal transducers and activators of transcription 1 and 3 signaling in melanoma by high-dose ifnalpha2b stat5 contributes to interferon resistance of melanoma cells controlled clinical trial of interferon-gamma as postoperative surgical adjuvant therapy for colon cancer interferon-gamma in the first-line therapy of ovarian cancer: a randomized phase iii trial interferon-resistant human melanoma cells are deficient in isgf3 components, stat1, stat2, and p48-isgf3gamma coordinate regulation of the human tap1 and lmp2 genes from a shared bidirectional promoter decreased stat1 expression by promoter methylation in squamous cell carcinogenesis downregulation of ifn-gammar in association with loss of fas function is linked to tumor progression the stats of cancer -new molecular targets come of age this work was supported by grants from the fondo de investigaciones sanitarias (fis), red genomica del cancer (retic rd 06/0020), plan andaluz de investigacion (group cts 143), consejeria andaluz de salud (sas), proyecto de excelencia de consejeria de innovacion (cts 695), proyecto de investigacion iþd (saf 2007-63262) in spain; and from the integrated european cancer immunotherapy project (oj2004/c158, 518234) and by grants from the deutsche forschungsgemeinschaft dfg se581 9-1/2 and 11-1 (b.s). in addition, we thank tarish abbas for providing figure 3 and anne wasilewski for excellent secretarial help. key: cord-333670-qv1orlv5 authors: mutti, luciano; pentimalli, francesca; baglio, giovanni; maiorano, patrizia; saladino, rita emilena; correale, pierpaolo; giordano, antonio title: coronavirus disease (covid-19): what are we learning in a country with high mortality rate? date: 2020-05-28 journal: front immunol doi: 10.3389/fimmu.2020.01208 sha: doc_id: 333670 cord_uid: qv1orlv5 nan covid-19 has been declared a pandemic by the who (1) . following the outbreak of the disease in china, italy was the first european country to be heavily struck (2, 3) . initially, three covid-19 cases were reported in early february, which were all related to individuals who had traveled to china; then, on the 20th, a young man who had not traveled abroad presented with severe sars-cov-2-induced pneumonia in lombardy, a region in the north of the country (2) . over the next 2 weeks, many patients in the surrounding areas were diagnosed with covid-19, which was often severe, and another cluster was identified in the nearby region of veneto (2) . there then followed an exponential increase in cases, mostly in the north, although the disease spread throughout the whole country, leading to the hypothesis that the virus had been circulating since january (2, 4) . at that point, italy reached incidence and mortality rates that were amongst the highest in the world (2) (3) (4) . many factors explain differences from other countries, including different application of detection tests, a larger elderly population, and different prevention policies and capacity to provide intensive care (2) . while it is paramount to conceive preventive strategies and apply more effective early treatments, it is also crucial to understand the biological mechanisms underlying these fatal outcomes. in italy, the possibility of performing autopsies or post-mortem diagnostic studies on suspect, probable, or confirmed covid-19 cases has been intensively debated (5, 6) ; however, postmortem pathological analysis of covid-19 patients in china has shown findings consistent with acute respiratory distress syndrome (ards) (7-9) (figure 1 ). at present, the exact nature of the acute lung injury trigger is not yet fully clarified; however, it could be ignited by t cells overreacting to virus-specific epitopes, thus recruiting multiple cytokineactivated inflammatory cell lineages (10) (11) (12) . other possibilities that deserve further experimental evidence include an exaggerated antibody-mediated response with complement activation and/or fcγ1 receptor-mediated leukocyte engagement and/or a hypothetical cytopathic effect of the virus (13, 14) . the latter could explain the recently described microvascular damage leading to disseminated intravascular coagulation (manifested as thrombosis, thrombocytopenia, and gangrene of extremities), anti-phospholipid syndrome, and mimicry of vasculitis, which are described in both chinese cohorts (15) and italian patients (16) (17) (18) . in our experience, ≈18% of patients develop interstitial pneumonia, and a subset of these (≈5%) develop ards that, especially when so serious as to require invasive ventilation, is mostly fatal. the risk of ards rises with age, and almost all deaths regard patients with pre-existing chronic conditions (19, 20) . pre-admission hypertension, in particular, has been reported as a key mortality risk factor (19) . the risk of death further rises where there is a lack of ventilators or ventilation is refused, as described in xu et al. (9) . moreover, an increasing number of clinical reports describe a biphasic behavior: a first phase where covid-19-infected patients are completely asymptomatic, which lasts on average seven days, and a second phase where the patients present mild to moderate flu-like symptoms, anosmia, ageusia, and blind conjunctivitis, which may last 10-15 days (21, 22) . a minority of patients who are unable to achieve complete virus coverage develop severe cardio-respiratory symptoms with radiological signs of pneumonitis, ards, and then multiorgan failure (23) . the last phase occurs, on average, 15-30 days after infection. in the latter case, patients may test negative for covid genome research standard molecular tests. altogether, these clinical findings, as well as the available pathology studies, support the hypothesis of an inappropriate immunerelated inflammatory response to covid19 epitopes and consequent auto-antigen release and t-cell cross-presentation in the damaged alveolar tissue. consistently, recent results indicate that a systemic immune dysregulation that triggers auto-sustaining inflammatory lung damage, causing fatal respiratory-failure and consequent multiorgan-failure, is the main virus-related-death cause in patients who develop sars-cov-2 (10). the culprit is the cytokine storm unleashed in this context by the infection and already described in cancer patients treated with cart or immunotherapy, including the "old" treatments with interleukins (il2 and il12, in particular) and the newest anti-ctla-4 and or anti-pd-1/pdl1 immunecheckpoint inhibitors. a greater risk of pneumonitis has already been recorded in chinese patients bearing a highfrequency of specific class-i and ii hla alleles associated with poor virus clearance and development of immune-related pneumonitis and other inflammatory-related autoimmune diseases (24) . this viral-load-independent different response to the infection might depend on a genetic predisposition causing extreme and often lethal inflammatory reactions. given the inefficacy of steroids (9), understanding the molecular features underlying such threatening immune-related events provides a strong rationale for using biological drugs for the early treatment of symptomatic patients, aimed at hampering the effects of the most relevant cytokines able to trigger an antibody response and acute inflammatory reaction, such as il6 and il1α. to this purpose, abs against the il6 receptor, or drugs able to disrupt its downstream signals, can inhibit its function on specific inflammatory cell subsets. these agents have so far been promising in the clinical setting for curbing the inflammatory response to control the severe immune-related adverse events related to cart-therapy and immune-checkpoint blockade and autoimmune diseases, including juvenile rheumatoid arthritis, psoriatic arthritis, and ulcerative colitis, all related to particular hla class i and ii alleles, some of which, like class i b * 27 and b * 35, might sustain both mitochondrial stress and cross-reactivity with several pathogens (25) . therefore, while antiviral drugs help to contain viral replication, moabs to il6 in the early phase of respiratory involvement could control the risk of a fatal virus-inducedcytokine storm. a great effort should be made to recognize lung involvement as, at least theoretically, the earlier the treatment, the better the outcome will be, with il6 inhibitors being able to "nip in the bud" the inflammatory cascade and prevent the fatal permanent damage to the alveolar pneumocytes. on this basis, il6 inhibitors are currently being tested in china and italy in patients with respiratory failure, and other il6 inhibitors are also being considered. iatrogenic cues might also contribute to exacerbating the acute inflammatory lung injury triggered by the virus. most hospitalized patients in fact received oxygen either through intubation or mechanical or non-invasive ventilation (20) ; however, oxygenation in ards patients with acute lung inflammation has been previously shown to interfere with the anti-inflammatory response induced locally by hypoxia through the activation of the adenosine a2a receptor (26) . similarly, in covid-19, patients, oxygen therapy could worsen lung injury by weakening such anti-inflammatory pathways. consistently with this hypothesis, in a cohort of 5,700 patients hospitalized with covid-19 in the new york city area, mortality reached 88.1% for those requiring mechanical ventilation (27) . in lombardy, the intensive care unit mortality was 26%, and indeed, a large proportion of admitted patients required mechanical ventilation (20) . these data support the possible use of adenosine agonists in patients presenting with ards (figure 1) . identifying infected patients at higher risk of poor prognosis even without evident risk factors could represent an important step forward. in this direction, zhou et al. reported some predictive biomarkers of the severity of the infection (23). nguyen and colleagues, in a preprint article, analyzed the sars-cov-2 proteome and identified a range of hla alleles potentially able to present (or not) viral epitopes. they suggest that individuals bearing hla-b * 46 (which has the fewest predicted binding peptides for sars-cov-2) may be particularly vulnerable to covid-19, whereas individuals bearing hla-b * 15 (which has the greatest predicted capacity to present sars-cov-2 peptides) could exhibit cross-protective t-cell based immunity. the authors highlight that a thorough understanding of how hla variation correlates with covid-19 onset and outcome could help identify high-risk subjects (28) . indeed, we have preliminary evidence that the prevalence of specific hla class i alleles across italian regions/provinces correlates with increased covid-19 incidence (correale p., mutti l., submitted for publication). if confirmed in wide case-control studies, the identification of hla alleles that are more permissive to viral infection would provide the first genetic explanation for the wide differences in covid-19 incidence rates among italian regions and also among nearby provinces with similar environmental factors. figure 1 | host response and possible outcomes of sars-cov-2 infection. viral infection seems to occur mainly upon sars-cov2 engagement of angiotensin i converting enzyme 2 (ace2), which acts as a functional receptor for the spike glycoprotein of the coronavirus. the hla genetic system acts as a key player in determining the anti-viral immune response. in particular, the ability of hla to trigger an adequate cytotoxic t-lymphocyte (ctl) response will result in viral clearance and host healing, along with the development of the igm, iga, and igg humoral response. conversely, an inadequate hla asset will result in an inefficient ctl response and, consequently, incomplete viral clearance. in this context, various factors underlie increased covid-19 severity, including an exaggerated ab response, complement activation, leukocyte-mediated antibody-dependent cell-mediated cytotoxicity (adcc), and t-cell-mediated inflammation, as discussed in the text. without a protective immune response, the virus is able to migrate, propagating into other ace2-expressing tissues, while the damaged lung cells induce high inflammation, triggering the cytokine storm that represents the main cause of the acute respiratory distress syndrome (ards) and subsequent multiorgan failure. incomplete viral clearance can also lead to virus hiding in sanctuary sites and patient relapse with symptoms arising in new districts. in the purple boxes, different therapeutic approaches aimed at targeting either the virus or endogenous host players are represented. overall, understanding the role of pro-inflammatory cytokines certainly unravels a new battleground against the lethal clinical effect of codiv-19 infection; this, along with the identification of a high-risk autoimmune profile, including the genotyping of class i and ii hla, which have a key role in shaping the anti-viral immune response and th1/th2 lymphocyte subset response (figure 1) , and immune-profiling, could also help to prevent these dangerous evolutions of the disease (29) . in particular, the isolation of genetically at-risk individuals, including healthcare workers, will inform future vaccination campaign priorities and clinical management strategies. the finding of healed patients retesting positive after an apparent complete virus clearance is a matter of intense debate in italy and worldwide. assuming that testing was reliable, various hypotheses are being considered, including viral mutation, although variation among sequences seems very low at present (30) . a preprint study in rhesus macaques argues against a risk of re-infection (31) . host inability to develop immunological memory with subsequent longterm protection is also being evaluated. interestingly, another preprint study identified specific sars-cov-2 neutralizing antibodies (nabs) in the plasma of patients who had recovered from infection and recorded that 30% of patients failed to develop high titers of nabs after covid-19 infection (32) . another possibility is that newborn sars-cov-2 might hide in sanctuary sites, such as the ncs and/or testis, which are protected from both antiviral drugs and proficient immuno-effectors; this hypothesis is supported by the recent description of viral detection in the cerebrospinal fluid but not in the nasopharyngeal swab in a case report (33) . overall, these distinct biological patterns of response to the virus should be taken into account for the design of new preventive and therapeutic strategies. lm, pc, and ag conceived the study. fp and gb evaluated current data. rs and pm studied hla involvement. pc conceived and fp sketched the figure. all authors contributed to manuscript writing and agreed with content. this work was supported by the sbarro health research organization (www.shro.org) and the commonwealth of pennsylvania. available online at case-fatality rate and characteristics of patients dying in relation to covid-19 in italy covid-19) situation report -57 coronavirus disease 2019 (covid-19) in italy the autopsy debate during the covid-19 emergency: the italian experience covid-19 deaths: are we sure it is pneumonia? please, autopsy, autopsy, autopsy! a new coronavirus associated with human respiratory disease in china clinical features of patients infected with 2019 novel coronavirus in wuhan pathological findings of covid-19 associated with acute respiratory distress syndrome molecular immune pathogenesis and diagnosis of covid-19 immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of 2019-ncov covid-19 infection and rheumatoid arthritis: faraway, so close monitoring kinetics reveals critical parameters of iga-dependent granulocyte-mediated anti-tumor cell cytotoxicity identification of coronavirus isolated from a patient in korea with covid-19. osong public the use of antiinflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the experience of clinical immunologists from china is covid evolution due to occurrence of pulmonary vascular thrombosis? covid-19 pulmonary involvement: is really an interstitial pneumonia? pulmonary embolism or pulmonary thrombosis in covid-19? is the recommendation to use high-dose heparin for thromboprophylaxis justified? characteristics, treatment, outcomes and cause of death of invasively ventilated patients with covid-19 ards in baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region objective evaluation of anosmia and ageusia in covid-19 patients: a single-center experience on 72 cases conjunctivitis and covid-19: a meta-analysis clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study werner syndrome: clinical features, pathogenesis and potential therapeutic interventions major histocompatibility complex genomics and human disease oxygenation inhibits the physiological tissue-protecting mechanism and thereby exacerbates acute inflammatory lung injury presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area human leukocyte antigen susceptibility map for sars-cov-2 could pd-1/pdl1 immune checkpoints be linked to hla signature? the establishment of reference sequence for sars-cov-2 and variation analysis lack of reinfection in rhesus macaques infected with sars-cov-2. biorxiv neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications. medrxiv a first case of meningitis/encephalitis associated with sars-coronavirus-2 we are grateful to barbara colombo for the figure graphics. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 mutti, pentimalli, baglio, maiorano, saladino, correale and giordano. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-344364-vu389d88 authors: wang, wei; zhang, wei; zhang, jingjing; he, ji; zhu, faming title: distribution of hla allele frequencies in 82 chinese individuals with coronavirus disease‐2019 (covid‐19) date: 2020-06-02 journal: hla doi: 10.1111/tan.13941 sha: doc_id: 344364 cord_uid: vu389d88 covid‐19 is a respiratory disease caused by a novel coronavirus and is currently a global pandemic. hla variation is associated with covid‐19 because hla plays a pivotal role in the immune response to pathogens. here, 82 individuals with covid‐19 were genotyped for hla‐a, ‐b, ‐c, ‐drb1, ‐drb3/4/5, ‐dqa1, ‐dqb1, ‐dpa1, and ‐dpb1 loci using next‐generation sequencing (ngs). frequencies of the hla‐c*07:29, c*08:01g, b*15:27, b*40:06, drb1*04:06, and dpb1*36:01 alleles were higher, while the frequencies of the drb1*12:02 and dpb1*04:01 alleles were lower in covid‐19 patients than in the control population, with uncorrected statistical significance. only hla‐c*07:29 and b*15:27 were significant when the corrected p‐value was considered. these data suggested that some hla alleles may be associated with the occurrence of covid‐19. covid-19 is a respiratory disease caused by a novel coronavirus and is currently a global pandemic. hla variation is associated with covid-19 because hla plays a pivotal role in the immune response to pathogens. here, 82 individuals with covid-19 were genotyped for hla-a, -b, -c, -drb1, -drb3/4/5, -dqa1, -dqb1, -dpa1, and -dpb1 loci using next-generation sequencing (ngs). frequencies of the hla-c*07:29, c*08:01g, b*15:27, b*40:06, drb1*04:06, and dpb1*36:01 alleles were higher, while the frequencies of the drb1*12:02 and dpb1*04:01 alleles were lower in covid-19 patients than in the control population, with uncorrected statistical significance. only hla-c*07:29 and b*15:27 were significant when the corrected p-value was considered. these data suggested that some hla alleles may be associated with the occurrence of covid-19. a new type of pneumonia with an unknown causative agent broke out in wuhan, hubei province, china in late december 1, 2019. 1,2 a novel coronavirus was subsequently confirmed as the agent causing the disease. 1, 2 the novel coronavirus was named 2019-ncov by the world health organization (who) on january 12, 2020, and was later named severe acute respiratory syndrome coronavirus-2 (sars-cov-2) by the international classification committee of viruses on february 11, 2020. 3, 4 now, the associated disease has been named coronavirus disease 2019 (covid-19) by the who. 3, 4 the transmission mode for covid-19 has been shown to be human to human. 3 covid-19 is currently a global pandemic, and over 2 740 000 cases of covid-2019 and 191 000 deaths have been reported globally as of april 24, 2020. the hla system participates in immune regulation in humans and it plays an important role in the occurrence and development of infectious diseases. [5] [6] [7] chen et al reported an odds ratio of 4.4 for sars-cov infection in individuals homozygous or heterozygous for hla-c*08:01. 7 we hypothesized that hla variation in the population may be associated with the occurrence of covid-19, because hla plays a pivotal role in the immune response to pathogens. here, we report hla allele frequencies in chinese han individuals with covid-19 and a comparison between hla allele distribution in covid-19 patients and healthy individuals. eighty-two han individuals from zhejiang with confirmed covid-19 were tested. all patients had mild or severe covid-19, with none in a critical condition. individuals were recruited for plasma donation after recovery and the plasma was used for treatment of other covid-19 patients with severe or critical symptoms. the ages of the individuals ranged from 20 to 54. all samples were collected during the plasma donation process and informed consent was obtained from all individuals. the samples were genotyped at the hla-a, -b, -c, -drb1, -drb3/4/5, -dqa1, -dqb1, -dpa1, and -dpb1 loci by ngs-based typing, using an alltype ngs 9-loci amplification kit (one lambda inc., canoga park, california), according to the manufacturer's instructions. hla genotypes were assigned using typestream visual software version 2.0 (one lambda inc.). the chinese hla common and well-documented principle was used to solve ambiguous hla allele combination assignments. 8 hardy-weinberg equilibrium (hwe) was assessed by maximum likelihood, using the method of guo and thompson, which has been implemented in arlequin version 3.5. 9 odds ratios (95% confidence interval [ci]) and p-values were calculated using fisher's exact test or yates continuity-corrected χ 2 test in prism 5.0 software (graphpad, san diego, california). corrected p-values (pc) were obtained by multiplying the number of alleles at each locus using the benjamini-hochberg method. the significance of the pc-value was set at a level of .05. the results of hla-a, -b, -c, -drb1, -drb3/4/5, -dqa1, -dqb1, -dpa1, and -dpb1 genotyping were fitted for hwe after considering the pc-value (table s1) (table s2 ). the allele distributions of hla-a, -c, -b, -drb1, -dqb1, and -dpb1 loci were compared between covid-19 patients and control individuals. the resulting ors (95% ci) and p-values are presented in table s2 . data for control individuals were obtained from our previous studies of bone marrow donors in the zhejiang han population. 10, 11 there were 3548 individuals in the control group with genotyping data for the hla-a, -b, -c, -drb1,-dqb1 loci, and 242 with data for hla-dpb1 locus. 10, 11 hla genotyping of the control individuals was performed using polymerase chain reaction at two-field resolution, as in our previous reports. 10, 11 the frequencies and ors of the hla alleles, with uncorrected significance, for covid-19 patients are listed in table 1 . hla-c*07:29, c*08:01g (including c*08:01 and c*08:22), b*15:27, b*40:06, drb1*04:06, and dpb1*36:01 frequencies were higher in covid-19 patients than in the control population, with uncorrected statistical significance (p < .05). the ors were 130.20, 1.65, 3.59, 2.43, 2.39, and 12.08, respectively. meanwhile, the frequencies of the drb1*12:02 and dpb1*04:01 alleles were significantly lower in covid-19 patients (pc < .05), with ors of 0.44 and 0.40, respectively. however, only the frequencies of hla-c*07:29 and b*15:27 remained significantly different after p-value correction. the hla-c*07:29 allele is a well-documented allele in the chinese population, with five cases in the 816 486 bone marrow donors. 8 in the present study, hla-c*07:29 was found in one covid-19 patient, but in no individuals in the control group. therefore, the significance of c*07:29 should be interpreted with caution and this result needs to be confirmed in further studies with larger sample sizes. two alleles, b*07:02 (1.38%) and b*27:04 (1.92%), with frequencies greater than 1% in the control group, were not found in covid-19 patients. the odds ratios for b*07:02 and b*27:04 were 0.216 (95% ci: 0.01335-3.494, p = .2408) and 0.155 (95% ci: 0.009598-2.503, p = .134). because there were no data for hla-drb3/4/5, -dqa1, or -dpa1 loci in the control group, 10,11 the allele frequencies of these loci could not be compared between the two groups. the allele frequency data for the hla-drb3/4/5, -dqa1, and -dpa1 loci are summarized in table s3 . the hla system is an important host genetic factor that plays an important role in determining the outcome of many infectious diseases, including human immunodeficiency virus (hiv) and severe acute respiratory syndrome (sars). 6, 12, 13 associations between hlas and the development and/or severity of sars have been found in some populations. 7,13,14 hla-b*07:03, b*46:01, drb1* 03:01, drb1*12:02 alleles have been reported to be associated with susceptibility to sars, but some studies have note: #c*08:01g ( 9.02%) was included c*08:01 and c*08:22. the number of the individuals in the control group were 3548 for hla-b, -c, -drb1 loci, and 242 for hla-dpb1 locus. not confirmed these results. 15 the sequence of sars-cov-2 shows some homology with sars-cov, but there are distinct differences between the two viruses. 4 therefore, the association of hla alleles with covid-19 warrants further research. 15, 16 in the present study, these sars-susceptibility alleles were not found to occur at a significantly different frequency in covid-19 patients after p-value correction. nguyen et al 16 predicted the binding affinity of sars-cov-2 to 145 hla class i alleles, and hla-a*02:02, hla-b*15:03, and hla-c*12:03 were found to be the top presenters of conserved peptides. we found that b*15:27 alleles may be associated with the occurrence of covid-19. hla-b*15:03 and b*15:27 belong to the b*15 group and have 10 nucleotide differences. prediction of the peptide-binding groove of these alleles may help to explain their association with covid-19. although the number of samples in the present study was small, these data will still be useful for exploring the influence of hla gene polymorphisms on susceptibility to covid-19 and patient outcomes. additional supporting information may be found online in the supporting information section at the end of this article. estimating clinical severity of covid-19 from the transmission dynamics in wuhan the outbreak of covid-19: an overview spread of sars-cov-2 systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov hla variation and disease hiv and host immunogenetics: unraveling the role of hla-c epidemiological and genetic correlates of severe acute respiratory syndrome coronavirus infection in the hospital with the highest nosocomial infection rate in taiwan in 2003 hla common and well-documented alleles in china. hla arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under linux and windows the distributions of hla-a, hla-b, hla-c, hla-drb1 and hla-dqb1 allele and haplotype at high-resolution level in zhejiang han population of china establishment of a polymerase chain reaction sequencing based typing method for hla-dpb1 exons 2 and 3 and investigation of their polymorphisms lack of association between hla-a, -b and -drb1 alleles and the development of sars: a cohort of 95 sars-recovered individuals in a population of guangdong, southern china association of hla class i with severe acute respiratory syndrome coronavirus infection human-leukocyte antigen class i cw 1502 and class ii dr 0301 genotypes are associated with resistance to severe acute respiratory syndrome (sars) infection hla studies in the context of coronavirus outbreaks human leukocyte antigen susceptibility map for sars-cov-2 this work was sponsored by the science research foundation of zhejiang province (lgf19h100005) and the science research foundation of zhejiang healthy bureau (2020rc053). the authors have declared no conflicting interests. data sharing is not applicable to this article as no new data were created or analyzed in this study. faming zhu https://orcid.org/0000-0002-1963-9176 key: cord-342942-1s32o9m8 authors: stamatakis, george; samiotaki, martina; mpakali, anastasia; panayotou, george; stratikos, efstratios title: generation of sars-cov-2 s1 spike glycoprotein putative antigenic epitopes in vitro by intracellular aminopeptidases date: 2020-06-22 journal: biorxiv doi: 10.1101/2020.06.22.164681 sha: doc_id: 342942 cord_uid: 1s32o9m8 presentation of antigenic peptides by mhci is central to cellular immune responses against viral pathogens. while adaptive immune responses versus sars-cov-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the s1 spike glycoprotein of sars-cov-2, by three key enzymes that generate antigenic peptides, aminopeptidases erap1, erap2 and irap. all enzymes generated shorter peptides with sequences suitable for binding onto hla alleles, but with distinct specificity fingerprints. erap1 was the most efficient in generating peptides 8-11 residues long, the optimal length for hla binding, while irap was the least efficient. the combination of erap1 with erap2 greatly limited the variability of peptide sequences produced. less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. these experimentally generated putative epitopes could be prioritized for sars-cov-2 immunogenicity studies and vaccine design. we furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the pathogen responsible for coronavirus disease that is behind a major ongoing pandemic (1) (2) (3) . virus entry into host cells is dependent on the s1 spike glycoprotein that forms homotrimers on the surface of the virion and interacts with the ace2 receptor in susceptible cells (4) (5) (6) . many studies on clinical characteristics and mortality resulting from sars-cov-2 infection have highlighted the need for a detailed understanding of immune responses against this pathogen (7) . while appropriate innate and adaptive immune responses are necessary for recovery from infection, aberrant immune responses can be a major contributing factor to mortality (8, 9) . in parallel, understanding immune recognition of sars-cov-2 is crucial to the ongoing massive global effort into developing an effecting vaccine against this pathogen (10) . although early analyses have focused on the development of neutralizing antibodies, cellular immune responses are emerging of vital importance (11) both for understanding normal immune response against this pathogen and for designing and optimizing vaccines (12) . in particular t-cell mediated immunity appears to be important for both viral clearance and for long-term immunity (11) . thus, analysis of antigenic epitopes from sars-cov-2 should be a priority for the design of vaccines that induce effective and longlasting cellular immune responses (13) . cytotoxic t-cell responses against virus-infected cells hinge on the presentation of small peptidic fragments of viral proteins, called antigenic peptides, by specialized proteins on the cell surface that belong to the major histocompatibility class i complex (mhci, also called human leukocyte antigens, hla, in humans). antigenic peptides are derived from viral proteins that are proteolytically degraded by complex proteolytic cascades (14) . intracellular aminopeptidases, er aminopeptidase 1 (erap1), er aminopeptidase 2 (erap2) and insulinregulated aminopeptidase (irap) play important roles in producing antigenic peptides, by down-sizing longer peptides to the correct length for binding onto mhci (15) . appropriate processing of pathogen antigens by these enzymes can determine the generation of cytotoxic immune responses and aberrant processing can lead to immune evasion (16) . thus, it is important to understand how these enzymes process sars-cov-2 antigens, so as to gain insight into the efficacy of antiviral cytotoxic responses and reveal possible avenues to enhance them. in this study, we utilized a novel approach to analyze antigen trimming by intracellular aminopeptidases erap1, erap2 and irap, focusing on the largest antigen of sars-cov-2, namely s1 spike glycoprotein. by using tandem lc-ms/ms analysis, we were able to follow trimming in parallel of a large ensemble of peptides derived from the full length of s1 protein. this approach was inspired by two established observations: i) that these enzymes are expected to normally encounter a very large number of potential substrates concurrently in the cell and ii) accommodation of peptides inside a large cavity of each enzyme can lead to complex interactions between substrates that have to compete for the same space in the cavity (17) (18) (19) . our analysis provides novel insight into the differences in specificity between the three enzymes and provides a potential filter of traditional bioinformatic approaches that aim to predict antigenic epitopes. finally, we propose a limited list of peptides that are potential ligands for common hla alleles and could be prioritized for further immunological analyses and vaccine design efforts. recombinant erap1, erap2 and irap were expressed and purified as described previously. briefly, erap1 and erap2 were expressed by hi5 insect cells in culture after infection with baculovirus carrying the appropriate gene and purified by affinity chromatography using a c-terminal his tag (20, 21) . the enzymatic extracellular domain of irap was expressed by stably transfected hek 293s gnti (-) cells and purified by affinity chromatography using a c-terminal rhodopsin 1d4 tag (22) . enzymes were stored with 10% glycerol in aliquots at -80c until needed. the pepmix sars-cov-2 peptide mixture was purchased by jbt peptide technologies gmbh. peptide pools were dissolved in dmso. prior to reactions the two peptide collections (158 and 157 peptides respectively) were mixed at equimolar concentrations and diluted in buffer containing 10mm hepes ph 7, 100mm nacl to a final concentration of 48μm. enzymatic reactions were performed in triplicate in a total volume of 50μl in 10mm hepes ph 7, 100mm nacl. freshly thawed enzyme stocks were added to each reaction to a final concentration of 50nm. reactions were incubated at 37c for 2 hours, stopped by the addition of 7.5μl of a 10% tfa solution, flash frozen in liquid nitrogen and stored at -80c until analysis. the sample was preconcentrated on a pepmap lc trapping column (0.3x5mm) at a rate of 30ul of buffer a (0.1% formic acid in water) in 5min. the lc gradient used was 5% buffer b (0.1% formic acid in acetonitrile) to 25% in 36 min followed by an increase to 36% in 5 min and a second increase to 80% in 0.5min and then was kept constant for 2min. the column was equilibrated for 15 min prior to the next injection. a full ms was acquired with a q exactive hf-x hybrid quadropole-orbitrap mass spectrometer, in the scan range of 350-1400m/z using 60k resolving power with an agc of 3x 10 6 and max it of 45ms, followed by ms/ms scans of the 12 most abundant ions, using 15k resolving power with an agc of 1x 10 5 and max it of 22ms and an nce of 28. the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride (23) partner repository with the dataset identifier pxd019901. we employed the maxquant computational proteomics platform version 1.6.14.0 to search the peak lists against the spike glycoprotein sars2 fasta file (swissprot accession number p0dtc2) and a file containing 247 frequently observed contaminants. n-terminal acetylation (42.010565 da) and methionine oxidation (15. 994915 da) were set as variable modifications. the second peptide identification option in andromeda was enabled. the false discovery rate (fdr) were set to of 0.01 both for peptide. the enzyme specificity was set as unspecific. the minimum peptide length was set to 6 amino acids. the initial allowed mass deviation of the precursor ion was set to 4.5 ppm and the maximum fragment mass deviation was set to 20 ppm. to investigate the trimming of antigenic epitope precursors by intracellular aminopeptidases that generate antigenic peptides, we used a mixture of 315 synthetic peptides derived from the sequence of the sars-cov-2 s1 spike glycoprotein. all peptides were 15 residues long and spanned the entire sequence of the protein with an 11 residue overlap between adjacent peptides. this mixture allows the systematic sampling of the entire sequence of the protein. the peptide mixture was incubated with either erap1, erap2, irap or an equimolar mixture of erap1 and erap2 at a substrate to enzyme ratio of 1000:1 and the digestion products analyzed by lc-ms/ms using a custom search database generated by in silico digestions of the full s1 protein sequence (uniprot id: p0dtc2). all analyses were performed on three biological replicates for each reaction, as well as a control reaction that was performed in the absence of enzyme. an additional technical replicate for the control sample was also analyzed. for statistical robustness, we performed a t-test between the control sample and each reaction and selected for further analysis only the peptides for which the quantification value changed by a statistically significant degree (p-value<0.05). the relative abundance of each peptide before and after the reaction was compared by label-free-quantification. analysis identified 263 unique 15mers in the samples out of the total 315 in the mixture. this represents an 83% coverage of included peptides which may be due to poor ionization and detection for some peptide sequences. as a result, further analysis was limited to the peptides detectable by our experimental setup. on average, incubation with enzyme reduced the relative abundance of the 15mer peptides indicating successful digestion ( figure 1a , b). this reduction was much more evident for erap1 and erap2 (and their mixture) than for irap. each enzyme featured a unique digestion fingerprint, suggesting different selectivity, as suggested in previous studies (24) . since the majority of peptides presented by hla are 8-11 residues long, we analyzed the comparative abundance of 8-11mers generated from each reaction ( figure 1c, d) . of the three enzymes, erap1 was the most efficient in generating peptides within this length range, consistent with previous reports on the mechanism of action of this enzyme (17, 25) . erap2 and the erap1/erap2 mixture followed, while irap was the least efficient. similar to the trimming of 15mers, the generation of 8-11mers followed a unique fingerprint for each enzyme. this is consistent with the previous hypotheses that each of these enzymes accommodate peptides in a large internal cavity and selectivity is driven by interactions with the whole sequence of the peptide (17, 21, 22) . indeed, comparing the peptide sequences generated by each enzyme, out of 1184 peptides identified, 142 were common between all three enzymes, 244 between erap1 and erap2 and 220 between erap1 and irap ( figure 2a) . furthermore, 169 peptides were unique for erap1, 234 for erap2 and 303 for irap. a similar situation was evident for 8-11mer peptides ( figure 2b) . strikingly, the mixture of erap1 with erap2 generated the fewest number of distinct sequences of 8-11mers ( figure 2c ). this was in contrast to the finding that the erap1/erap2 mixture generated about the same average signal intensity as erap2 ( figure 1d ). this was due to erap1/erap2 mixture generating fewer, in terms of sequence, distinct peptides, which were however relatively abundant. this finding is consistent with the proposed synergism of erap1 and erap2 (26) and suggests that the combination of these two enzymes is more efficient in trimming variable sequences and can thus over-trim peptides to lengths below 7 residues that are not detectable in our experimental setup and should not be able to stably bind onto mhci ( figure 2c ). as a result, incubation with erap1/erap2 mixture, accumulates only peptides that are resistant to degradation by both enzymes. since epitope length is a key parameter for binding onto mhci (the majority of presented peptides are 9mers) we analyzed the distribution of lengths of peptides generated by each enzymatic reaction ( figure 3 ). erap1 was very efficient in trimming the 15mer substrates and generated primarily 9mer peptides, consistent with its proposed property as a "molecular ruler" (25) . neither erap2 nor irap were able to accumulate 9mers preferably, but still generated significant numbers. the mixture of erap1 and erap2 showed a similar fractional distribution of peptide lengths ( figure 3b ), but produced a much lower number of distinct peptide sequences ( figure 3a ), presumably due to over-trimming to smaller lengths or even single amino acids. table 1 ). for each peptide we selected the best scoring hla-allele and plotted the calculated percentile rank of the predicted score for each enzymatic reaction ( figure 4a ). the geometric mean of the predicted affinity was lowest for erap1 (indicating that the erap1 generated peptides had the highest average affinity for hla), followed by irap and then erap2. only a subset of generated peptides was predicted to bind with sufficient affinity onto at least one hla: 23% for erap1, 22% for erap2, 6% for erap1/erap2 mixture and 21% for irap (peptide sequences are listed in table 1a ). these peptides spanned the whole sequence of the s1 spike glycoprotein, although each enzyme presented a unique signature onto this sequence ( figure 4b ). showing the predicted affinity of produced peptides for common hla alleles as calculated by hlathena. color region encompasses peptides that are predicted to bind to at least one of the common hla alleles used in the analysis. panel b, schematic representation of relative locations in the s1 protein sequence where the generated peptides are found. panel c, venn diagrams depicting overlap between peptides of s1 protein predicted to bind to common hla alleles and peptides produced experimentally by erap1, erap2, irap or erap1/erap2 mixture. numerals indicate number of peptides in each separate segment. in a recent publication, the authors proposed that different hla alleles can have significant variability in their ability to present sars-cov-2 epitopes, with hla-b46:01 having the capability to present the fewest and hla-b15:03 being able to present the most (29) . we thus used hlathena to analyze the propensity of the experimentally produced peptides to bind onto these two alleles (supplemental table 2 ). erap1 was found to produce 15 potential ligands for hla-b15:03 but only 6 for hla-b46:01, consistent with the proposed trend. in contrast, erap2 produced 8 potential ligands for both alleles and irap produced 6 for hla-b15:03 and 4 for hla-b46:01. strikingly, the mixture of erap1 with erap2 produced 4 peptides that could bind onto hla-b15:03, but no peptides predicted to bind onto hla-b46:01 (table 1b) . thus, our findings appear to validate the hypothesis that hla-b15:03 is likely to present more sars-cov-2 epitopes than hla-b46:01, but only for erap1, which however is considered the dominant aminopeptidase activity in the cell for generating antigenic peptides. bioinformatic epitope predictions based on antigen sequence are often used as a tool to study the potential antigenicity of a particular epitope or pathogen. the power of those predictions is constantly evolving and primarily relies on predictions of binding affinity on hla. to compare such predictions to our experimentally generated peptides, we used the full sequence of the s1 spike glycoprotein and the netmhcpan 4.1 server (30) to predict potential epitopes that could be presented by the common hla alleles indicated in the previous paragraph. the server predicted 929 potential epitopes with lengths of 8-12 residues (supplemental table 3 ). of those potential epitopes however, less than 7% were found to be produced experimentally by one of the enzymes tested and more specifically 58 by erap1, 52 by erap2, 4 for the erap1/2 mixture and 53 by irap ( figure 4c ). while our experimental approach has limitations as discussed below, this finding suggests that intracellular antigen processing by aminopeptidases may constitute a major filter in determining which peptides will be presented by mhci. indeed, it has been recently proposed that the main function of erap1 is to limit the peptide pool available for mhci (31) . in this context, this experimental approach could be useful in optimizing bioinformatic predictions. our findings provide new information on both the general biological functions of intracellular aminopeptidases that generate antigenic peptides as well as on specific processing of a key antigen from sars-cov-2. specifically, our results highlight that each enzyme bears a unique trimming fingerprint to antigen processing. although this has been suggested before based on differences in specificity towards specific peptide substrates (26, (32) (33) (34) , it has not been observed in the context of peptide ensembles. this is potentially important since competition of different peptides for the cavity in these enzymes could result in complex substrate interactions. at first glance, major differences in trimming fingerprints between each or the three enzymes, may appear to impose an unnecessary complication to antigenic peptide generation. it is conceivable, however, that this trimming variability is desirable for the immune system, since it can expand the breadth of possible antigenic peptides detected in different immunological settings and cell types. our results also highlight a previously proposed property of erap1: the specialization in trimming large peptides and producing peptides that have the ideal length for mhci binding -most of the erap1 products fall well within that range (25) . in contrast, both erap2 and irap appear to be less optimized for length selection. however, they are still able to produce many peptides that are potential cargo for mhci, casting some doubt on whether the unique trimming properties of erap1 are absolutely necessary for this basic function. furthermore, the combination of erap1 with erap2 appears to provide significant synergism in trimming, to the point of over-trimming peptides and limiting available sequences. synergism between erap1 and erap2 has been demonstrated before in trimming isolated peptides and these two enzymes have been proposed to also form functional dimers (33, 35) . according to our observations, their combination is especially efficient in trimming. while the biological repercussions of this are not fully clear yet, it is conceivable that the strong associations between erap2 activity and predisposition to autoimmunity may be related to this effect (36) . despite the current importance in understanding immune reactions in covid19, very little is known about the cellular adaptive immune responses against sars-cov-2. cellular immune responses are emerging as a central player in clearing the infection and as targets for vaccine efforts (37, 38) . furthermore, hla polymorphic variation has been suggested to underlie the large variability in virus clearance that has been observed amongst individuals (39) . our analysis of the largest antigen of sars-cov-2, s1 spike glycoprotein, suggests that aminopeptidase trimming can be a significant filter that helps shape which peptides will be presented by hla. thus, we propose a short list of candidate peptides that could be prioritized in downstream antigenic analysis as well as in vaccine design and efficacy studies. while the functions of erap1, erap2 and irap have been studied in both in vitro and in vivo contexts during the last decade, their relative functional differences have only been compared in processing specific substrates at a time. however, all these enzymes have a broad substrate specificity and can normally encounter thousands of different peptides in the er or endosomal compartments. on the other hand, studies focusing on the presented immunopeptidome have revealed effects attributed to erap1 and erap2 trimming, but direct comparisons have been difficult because of the dominant effect of mhci affinity on presentation (40). our approach stands in-between these two types of studies. it mimics the multiple-substrate situation that is likely normal in vivo but focuses on antigenic peptide precursor trimming. in this context, our approach may have broader application for the quick prediction of potential antigenic epitopes as an additional filter on bioinformatic predictions. indeed, bioinformatic predictions result in many candidate peptides, very few of which will provoke an immune response; adding more filters can increase the usefulness of these rapid approaches. however, our approach also has limitations that need to be taken into account when interpreting results. due to differences in ionization and detection by the lc-ms/ms some peptides may not be detected or may be under-represented compared to other sequences, making comparisons between different peptides less reliable. furthermore, it is an in vitro approach that is limited to the peptide pool used and cannot take into account the dynamics of mhci binding that can protect peptides from further aminopeptidase degradation (41) or peptide proofreading by chaperone components or the peptide loading complex (42) . due to those limitations, we restricted our analysis to statistical comparisons and avoided drawing conclusions regarding particular peptide sequences. in summary, we analyzed the trimming of a peptide ensemble spanning the sequence of the s1 spike glycoprotein of sars-cov-2, the pathogen responsible for the recent covid-19 pandemic. our analysis provided novel insight into the function of antigen trimming enzymes and suggested that aminopeptidase trimming may be a significant filter in determining which peptides can be presented by mhci. furthermore, we have identified a limited set of peptides that were experimentally produced by elongated precursors which could be prioritized in future studies aiming to investigate the antigenicity of sars-cov-2 infected cells and assist in the design of highly effective vaccines that aim to produce adaptive cytotoxic responses. we propose that our experimental approach may also be useful as a general tool for enhancing bioinformatic predictions of antigenic epitopes. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 a new coronavirus associated with human respiratory disease in china structural insights into coronavirus entry structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structural basis of receptor recognition by sars-cov-2 early insights into immune responses during covid cytokine storm induced by sars-cov-2 sars-cov-2 infection-induced immune responses: friends or foes? avoiding pitfalls in the pursuit of a covid-19 vaccine t cells found in coronavirus patients 'bode well' for long-term immunity protective adaptive immunity against severe acute respiratory syndrome coronaviruses 2 (sars-cov-2) and implications for vaccines immunoinformatics and structural analysis for identification of immunodominant epitopes in sars-cov-2 as potential vaccine targets. vaccines (basel) 2020 degradation of cell proteins and the generation of mhc class i-presented peptides peptidases trimming mhc class i ligands the final touches make perfect the peptide-mhc class i repertoire mechanism for antigenic peptide selection by endoplasmic reticulum aminopeptidase 1 antigenic peptide trimming by er aminopeptidases--insights from structural studies coding single nucleotide polymorphisms of endoplasmic reticulum aminopeptidase 1 can affect antigenic peptide generation in vitro by influencing basic enzymatic properties of the enzyme critical role of interdomain interactions in the conformational change and catalytic mechanism of endoplasmic reticulum aminopeptidase 1 structural basis for antigenic peptide recognition and processing by endoplasmic reticulum (er) aminopeptidase 2 crystal structure of insulin-regulated aminopeptidase with bound substrate analogue provides insight on antigenic epitope precursor recognition and processing the pride database and related tools and resources in 2019: improving support for quantification data probing the s1 specificity pocket of the aminopeptidases that generate antigenic peptides the er aminopeptidase, erap1, trims precursors to lengths of mhc class i peptides by a "molecular ruler" mechanism concerted in vitro trimming of viral hla-b27-restricted ligands by human erap1 and erap2 aminopeptidases biovenn -a web application for the comparison and visualization of biological lists using area-proportional venn diagrams a large peptidome dataset improves hla class i epitope prediction across most of the human population netmhcpan-4.1 and netmhciipan-4.0: improved predictions of mhc antigen presentation by concurrent motif deconvolution and integration of ms mhc eluted ligand data cell surface mhc class i expression is limited by the availability of peptide-receptive "empty" molecules rather than by the supply of peptide ligands the specificity of trimming of mhc class i-presented peptides in the endoplasmic reticulum concerted peptide trimming by human erap1 and erap2 aminopeptidase complexes in the endoplasmic reticulum placental leucine aminopeptidase efficiently generates mature antigenic peptides in vitro but in patterns distinct from endoplasmic reticulum aminopeptidase 1 erap1-erap2 dimerization increases peptide-trimming efficiency a genome-wide association study identifies a functional erap2 haplotype associated with birdshot chorioretinopathy targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals a single-cell atlas of the peripheral immune response in patients with severe covid-19 distribution of hla allele frequencies in 82 chinese individuals with coronavirus disease-2019 (covid-19). hla 2020. 40. de castro, j. a. l., how erap1 and erap2 shape the peptidomes of disease-associated mhc-i proteins cutting edge: h-2l(d) class i molecule protects an hiv n-extended epitope from in vitro trimming by endoplasmic reticulum aminopeptidase associated with antigen processing mhc i chaperone complexes shaping immunity key: cord-346032-188gnf8j authors: cheung, ying-kit; cheng, samuel chak-sum; sin, fion wan-yee; chan, kin-tak; xie, yong title: induction of t-cell response by a dna vaccine encoding a novel hla-a*0201 severe acute respiratory syndrome coronavirus epitope date: 2007-08-10 journal: vaccine doi: 10.1016/j.vaccine.2007.05.025 sha: doc_id: 346032 cord_uid: 188gnf8j the severe acute respiratory syndrome coronavirus nucleocapsid protein (sars-cov n) is one of the major targets for sars vaccine due to its high potency in triggering immune responses. in this study, we have identified a novel hla-a*0201 restricted epitope, n220 (lalllldrl), of the sars-cov n-protein through bioinformatics analysis. the n-protein peptide n220 shows a high binding affinity towards human mhc class i in t2-cells, and is capable of activating cytotoxic t-cells in human peripheral blood mononuclear cells (pbmcs). the application of using the n220 peptide sequence with a single-chain-trimer (sct) approach to produce a potential dna vaccine candidate was investigated in hla-a2.1k(b) transgenic mice. cytotoxicity assay clearly showed that the t-cells obtained from the vaccinated animals were able to kill the n-protein expressing cells with a cytotoxicity level of 86% in an effector cells/target cells ratio of 81:1 one week after the last vaccination, which is significantly higher than other n-protein peptides previously described. the novel immunogenic n-protein peptide revealed in the present study provides valuable information for therapeutic sars vaccine design. severe acute respiratory syndrome (sars) is caused by a novel coronavirus known as sars-associated coronavirus (sars-cov) and the investigation of the sars-cov immunity has garnered significant attention since the worldwide outbreak spreaded to 30 countries in 2003 [1] [2] [3] . the sars-cov is an enveloped positive-stranded rna virus encoding four major structural proteins, known as a spike glycoprotein (s), a small envelope protein (e), an integral membrane glycoprotein (m), and a nucleocapsid rna-binding protein (n) [4] . upon viral infection, the viral proteins expressed within the infected cells are degraded in the cytosol by proteasomes. peptides generated by the proteasomes are transported by the transporter associated with antigen processing (tap) into the lumen of endoplasmic reticulum (er), where an er aminopeptidase produces the mature mhc class i-peptide complex by trimming the peptide to eight or nine amino acids [5] . the resulting mhc class i-peptide complex expressed on the surface of professional antigen presenting cells, such as dendritic cells, plays important roles in the activation of specific cytotoxic t-cells for infected cell killing. on the other hand, the mhc class i-peptide complexes expressed on the virus infected cells is a "marker" for being killed. therefore, formation of stable mhc class i-peptide complexes is critical to elicit cytotoxic t-cell response to eliminate the infected cells. immunogenicity of a peptide sequence for the cytotoxic t-cell is determined by its binding affinity towards the human mhc class i molecule and its availability upon proteasome digestion of the parental protein. subunit vaccines containing hla-a*0201 restricted peptides is highly effective for the induction of strong cytotoxic t-cell response against infectious virus [6] . among the four structure proteins of the sars-cov, the s and n proteins are the major targets for vaccine research studies due to their potency in triggering immune responses [7] [8] [9] [10] [11] [12] [13] [14] . the s protein contains 1256 amino acids and is involved in viral entry through 0264-410x/$ -see front matter © 2007 elsevier ltd. all angiotensin-converting enzyme 2 receptor on host cell surface [15, 16] and the n protein contains 422 amino acids and is involved in the viral rna packaging [17, 18] . previous studies suggested that hla-a*0201 restricted sars s and n protein peptides can trigger specific human cytotoxic tcell response against sars in vitro [12, 14] . in the present study, we have identified a noval hla-a*0201 restricted nprotein peptide (n220: lalllldrl). the binding affinity of the n220 peptide towards human mhc class i molecule is comparable to the n223 peptide and is higher than the n227 and n317 peptides previously described [12] . moreover, in order to stablize the mhc class i-peptide complex for antigen presentation, the n220 sequence was genetically linked to the cdna of human ␤ 2 -microglobulin and the alpha-1 and alpha-2 domains of the human mhc class i heavy chain to form a single-chain-trimer (sct) in our dna vaccine design. the sct approach makes the mhc class i molecules less dependent on chaperone assistance for peptide loading and hence a stable mhc class i-peptide complex for antigen presentation is produced [19] [20] [21] . the potency of the n220 dna vaccine to trigger cytotoxic t-cell response against n-protein expressing cells was demonstrated in a hla-a2.1k b transgenic mouse model and our results indicated that the sars protein n220 peptide is a good vaccine candidate for sars vaccine development. the potential sars n-protein peptide sequence for human mhc class i binding was searched by a hla peptide binding prediction program, syfpeithi (http://www.syfpeithi.de) [22] . seven nine-amino acid peptides with high scores for human mhc class i binding (n139-147 alntpkdhi, n160-168 lqlpqgttl, n220-228 lalllldrl, n223-231 llldrlnql, n227-235 rlnqlesky, n317-325 gmsrigmev, n352-360 illnkhid) were synthesized by a solid-phase strategy using the fmoc-based protocol on an automated peptide synthesizer. the crude products were purified on a reverse-phase preparative high performance liquid chromatography (hplc) column and the homogeneity of the final products was analyzed by reverse-phase hplc of purity of 95%. the peptides were stored in dimethyl sulfoxide (dmso, sigma) at −80 • c until use. the dna sequence encoding the n-protein was cloned into a expression vector pet22b (novagen) and the recombinant n-protein is expressed in a escherichia coli system bl21-condonplus (de3)-ril (stratagene) followed by purification with ni-nta his-bind resin (novagen). after protein purification, the purity and the quantity of the recombinant protein was analyzed by sds-page analysis. to establish a n-protein expressing cell-line for the cytotoxicity assay, lung tissues from the hla-a2.1k b transgenic mice were isolated, minced and treated with collagenase (gibco) at 100 u/ml for 30 min. tissue samples were seeded on a plate and cultured for 1 week, fibroblasts were then collected and immortalized by transduction of a recombinant retrovirus containing the sars n gene and the hpv16 e6-e7 genes [23] . expression of the nprotein in the lung fibroblast cell-line was confirmed by rt-pcr (with primers: n: sense: 5 -atgtctgataatgg-3 , antisense: 5 -ttatgcctgagttg-3 ; e6e7: sense: 5 -atgtataaaactaaggg cgtaacc-3 , antisense: 5 -ttatggtttctgagaacagatgg-3 ), and the cell-line is named as n/e6e7/a2.1k b . the t2-cell (174 × cem.t2), which are hla-a2 positive, but deficient for the tap protein for endogenous antigen presentation, were cultured according to the procedure stated in the atcc manual. to determine whether the selected n-protein peptides could bind to the human mhc class i molecule, the t2-cells (1 × 10 5 ) were pulsed with each of the n-protein peptides (10 g/ml) in the presence of 5 g/ml human ␤ 2 -microglobulin (sigma) for 4 h. after peptide pulsing, the t2-cells were washed twice with cold pbs containing 2% fbs and then stained with a mouse anti-human hla-a2 antibody (bb7.2) followed by a goat anti-mouse igg fitc antibody (zymed). the peptide bound t2-cells were fixed with 1% paraformaldehyde (sigma) and subjected to flow cytometry to measure the relative amount of mhc class i/peptide complexes formed. the results were presented as the fluorescent index (fi) calculated by the mean fluorescence intensity (mfi) of t2 cells using the formula: fresh human hla-a*0201 positive peripheral blood mononuclear cells (pbmcs) were isolated by ficoll-hypaque density gradients (amersham), followed by enrichment of cd8+ t-cells by human cd8 microbeads (macs). the purified human cd8+ t cells were then subjected to autologous dendritic cells mediated t-cell activation in vitro. in brief, the pbmcs were seeded on dishes for 2 h and the non-adherent cells were removed. the adherent monocytes were then cultured in aim-v medium (invitrogen) supplemented with 5% human ab serum, 800 u/ml recombinant granulocyte-macrophage colony-stimulating factor (gm-cff) and 500 u/ml recombinant human interleukin 4 (il-4) to obtain dendritic cells. on day 6, the dendritic cells were matured by addition of 100 u/ml recombinant human interleukin 1 beta (il-1␤), 1000 u/ml recombinant human interleukin 6 (il-6), 10 ng/ml recombinant human tumor necrosis factor-alpha (tnf-␣) and 1 g/ml prostaglandin e2 (pge2). on day 7, the mature dendritic cells were loaded with the selected n-protein peptides in serum free medium for 4 h. the peptide-loaded dendritic cells were then cocultured with the purified cd8+ t cells in the presence of 20 u/ml recombinant human interleukin 2 (il-2) and 10 ng/ml recombinant human interleukin 7 (il-7) for t-cell stimulation. the stimulation procedure was repeated once a week and totally three times of t-cell stimulation by the peptide-loaded dendritic cells was conducted. activation of t-cell was investigated by an ifn-␥ elispot assay as previously described with minor modifications [24] . briefly, a 96-well elispot plate (multiscreen-ha, millipore) coated with anti-human ifn-␥ antibodies (5 g/ml, ebioscience) was blocked with aim-v medium containing 5% human ab serum. the stimulated cd8+ t cells (1 × 10 5 ) were then added into the wells together with autologous n-protein loaded b-cells (1 × 10 4 ) and incubated for 24 h. subsequently, the plate was washed and followed by incubation with 0.5 g/ml biotinylated ifn-␥ antibody (ebioscience) at 4 • c for another 24 h. after washing, 1 g/ml streptavidin-ap (zymed) was added and incubated at room temperature for 45 min. spots were developed by adding a bcip/nbt solution (invitrogen) and digitally recorded with an elispot plate reader (service provided by beijing swan tech co., china). the dna sequence containing the human ␤ 2 -microglobulin and the chimeric mhc heavy chain, was synthesized by connecting the mouse ␤ 2 -microglobulin cdna, human hla-a*0201 ␣-1 cdna, hla-a*0201 ␣-2 cdna and mouse h2-k b ␣-3 domain cdna together by overlapping pcr to construct an mhk gene (m = mouse ␤ 2 -microglobulin, h = human hla-a*0201 ␣-1 and ␣-2, k = mouse h2-k b ␣-3) using two templates. the ova2␤ 2 mk b sct gene which was a generous gift from prof. hansen was the template for mouse ␤ 2 -microglobulin and mouse h2-k b ␣-3 domain [25] . the hhd gene, which was provided by prof. lemonnier, was another template for the human hla-a*0201 ␣-1 and hla-a*0201 ␣-2 [26] . the dna fragments encoding the selected n-protein peptides were then linked to the n-terminal of the mhk gene by pcr using the primers with the corresponding dna sequence at the 5 region to generate single-chain-trimers. the constructed single-chain-trimer dna fragments were cloned into agei and xhoi sites of pvax1 (invitrogen) to construct n-protein peptide-expressing plasmids, n220mhkpvax1, n223mhkpvax1, n227mhkpvax1, and n317mhkpvax1 for vaccination purpose. ovamhkpvax1 was also constructed as a control plasmid expressing a siinfekl peptide of the ovalbumin (ova257). [27] were bred and maintained under pathogen-free conditions (animal care center, hkust, hong kong). the dna vaccine was delivered into 6-8 weeks old transgenic mice with a gene gun device as previously described with some minor modifications [21] . briefly, cartridges were prepared by precipitating the plasmid dna on 1 m gold particles dissolved in 0.05 m spermidine and 1 m cacl 2 . the microcarrier/dna suspension was coated on plastic tubing in the presence of 0.5 mg/ml polyvinylpyrrolidone (sigma). the coated tubing was cut into 0.5 inch cartridges so that each cartridge contained 1 g of dna. dna-coated gold particles were delivered to the shaved abdominal region of mice using a helios gene gun (bio-rad) with a discharge pressure of 400 psi. each mouse was administrated with 1 g dna per injection for a total of three vaccinations within a 3-week interval. to investigate the cell mediated cytotoxic response triggered by the sct-dna vaccine, mice were sacrificed 1 week after the last vaccination. splenocytes were obtained and cultured in imdm medium (gibco) containing 2 g/ml of the corresponding target peptides and 20 units of interleukin-2 (peprotech) in a 12-well plate (nunc) at 37 • c for 3 days. t-cell cells from spleen were harvested by ficoll-hypaque (amersham) density gradient centrifugation and used as effector cells in a cytotoxicity assay. the n-protein transduced n/e6e7/2.1k b cells were used as target cells. in the cytotoxicity assay, the effector cells and the target cells were co-cultured in the ratios of 81:1, 27:1, 9:1, 3:1 and 1:1. after 5 h incubation, the culture plates were centrifuged and the medium was collected for further analysis using a lactate dehydrogenase (ldh) cytotoxicity detection kit (roche) according to the procedures stated by the manufacturer. the absorbance of the samples was measured by elisa reader at 490 nm with 630 nm as reference wavelength. the spontaneous release of ldh by target cells or effector cells the full-length amino acid sequence of the sars n-protein was subjected to bioinformatic analysis to search for hla-a*0201 restricted nine-amino acid peptides. the scores of the peptides are within the range from −4 to 30, and the amino acid sequence of the seven peptides selected that have the high scores are listed in the middle column. a flu m1 peptide (gilgfvftl) (which is used as a positive control) shows a score of 30 is listed at the bottom. was assessed by incubation of target cells in the absence of effector cells and vice versa. the maximum release of ldh was determined by incubation of the target cells in 1% triton x-100 in assay medium. the percentage of specific cell mediated cytotoxicity was determined by the following equation: specific cytotoxicity (%) = experimental value − effector cell spontaneous release − target cell spontaneous release maximum target cell release × 100 in order to search for the hla-a*0201 restricted peptide sequence of the sars n-protein, the syfpetithi program was employed to compare the binding affinity of the nprotein peptides towards the human mhc class i molecule through a bioinformatic analysis. seven high score peptides were selected (table 1 ) and the binding activity of these peptides towards the human mhc class i molecules was investigated by comparison of their ability to stabilize the empty mhc class i molecules expressed on the cell surface in the presence of ␤ 2 -microglobulin. the presence of the stable mhc class i-peptide complex was then detected by the antibody (bb7.2) through facs. the results clearly show that among the seven n-protein peptides, n220 and n223 display the highest binding affinity towards the human mhc class i and the resulting fluorescence intensities are higher than the positive control flu m1 peptide (58-66, gilgfvftl) [28] ( fig. 1) , suggesting a high binding affinity of the n220 and n223 peptides towards the human mhc class i. the t-cell immunogenicity of the seven selected peptides was further tested by their ability to stimulate human cd8+ t cells isolated from healthy donor pbmcs. the purified cd8+ t-cells were primed three times with autologous mature peptide-loaded dendritic cells and the level of tcell activation was then investigated by the release of ifn-␥ through ifn-␥-elispot in the presence of autologous bcells loaded with the recombinant n-protein. if the target peptide of the n-protein can be generated and presented by the target cells, the primed cd8+ t-cells could specifically recognize the peptide-mhc complex and release ifn-␥. the elispot results show that t-cells primed with n223 (lll-drlnql) and n220 (lalllldrl) produce the highest number of spots that are six to seven times higher than that found in the irrelevant flu peptide primed t-cells and is significantly higher than that of the other selected n-protein peptides (fig. 2) . the results of the t-cell stimulation assay demonstrated that the novel n-protein peptide revealed in the present study is able to trigger specific cytotoxic t-cell response in human pbmcs. the four most immunogenic peptides (n220, n223, n227 and n317) selected in the t2-cell binding assay and the human t-cell stimulation assay were further tested for their potency in triggering immune response against the sars n-protein expressing cells in an animal model. to facilitate antigen presentation, the dna sequence of the target peptides was genetically linked to the cdna sequence of the mouse ␤ 2 -microglobulin and the chimeric mhc class i heavy chain domains (fig. 3 ) that the peptide can be translated as a peptide-spacer-␤ 2 -microglobulin-space-h-chain complex for t-cell activation. under this covalently linked mhc class i-peptide construction, the linked mhc molecule is at least 1000-fold less accessible to exogenous peptides than normal mhc loaded with endogenous peptide and is more potent in the stimulation of cytotoxic t-cells [25] . the cytotoxic tcell response triggered by the dna vaccine was investigated by the activity of the spleen t-cells to kill the target cells n/e6e7/a2.1k b , which are immortalized primary a2.1k b fibroblasts expressing the sars n-gene (fig. 4), 1 week after the last vaccination. the cytotoxicity level was measured by the amount of lactate dehydrogenase (ldh) released from the target cells. the results demonstrated that the dna vaccines encoding the n-protein peptides n220 and n223 trigger the highest t-cell cytotoxicity towards the n-protein expressing cells with 86 and 61% cytotoxicity level, respectively, in effector cells/target cells ratio of 81:1 compared to the cytotoxicity level of the previously described peptide n317, which shows only 42% cytotoxicity, and n227, which is similar to the negative control using an irrelevant ova257 peptide for vaccination (fig. 5 ). the worldwide outbreak of sars in 2003 caused a severe economical loss and the development of sars vaccine is one of the most effective ways to prevent the outbreak in the future. an ideal vaccine against infectious virus should be able to trigger both the neutralizing antibody production and the cytotoxic t-cell responses that play a critical role in the elimination of virus infected cells in controlling viral pathogensis. during sars-cov infection, the n-protein is reported to be highly immunogenic [8] and it has been the y-axis indicates the percentage of cytotoxicity. four groups of mice were vaccinated with the n-protein peptide plasmids, n220mhkpvax1 ( ); n223mhkpvax1 ( ); n227mhkpvax1 ( ); and n317mhkpvax1 ( ). mice vaccinated with an irrelevant plasmid, ovamhkpvax1 ( ), was used as a negative control. the cytotoxicity level was calculated as previously described. the differences in cytotoxicity level between all peptides with various effector cells: target cells ratios were calculated with t-test (p < 0.05). shown that n vaccine can trigger specific t-cell response in mice [29] . however, only six t-cell specific epitopes of the n-protein have been reported up-to-date [12, 14, 30] . the objective of this study is to identify immunogenic n-protein peptides that can serve as cytotoxic t-cell epitope in sars vaccine. a peptide sequence useful for inducing the cytotoxic t-cell response should be presented as endogenous peptide epitope through proteasome digestion and have a high binding affinity towards the human mhc class i molecules. in contrast to the conventional method of screening numerous amino acid sequences manually from synthetic peptide library that is costly and time consuming, bioinformatics analysis was employed to search for the most immunogenic peptide sequences of the sars n-protein. although the efficiency of the bioinformatics analysis in searching the immunogenic peptide is high, discrepant results could also be obtained from different programs online. for instance, although the novel immunogenic n220 peptide revealed in the present study is the third potent peptide found in the syfpetithi program, it is ranked 13 according to the hla peptide motif search provided by the national institutes of health and was totally ignored in the previous study about searching the t-cell epitopes in the n-protein [12] . to investigate whether the peptide predicted from the computer program has a high binding affinity to human mhc, a t2-cell binding assay was conduced that is based on the binding affinity of the peptide to the empty mhc class i molecules on the cell surface and to stabilize the mhc class i-peptide complex formed. the results clearly show that the novel peptide, n220, identified in the present study has a high binding affinity towards the human mhc class i molecule hla-a2.1 that is comparable to the n223 peptide and is significantly higher than that of the n227 and n317 peptides previously described [12] . previous studies suggested that a peptide sequence with a high binding affinity to hla-a2.1 is not able to trigger t-cell response if the peptide sequence is not generated from endogenous process through the proteasome system. for instance, although the ny-eso-1 peptide 159-167 has a high affinity towards the hla-a2 molecules, it is not able to elicit cytotoxic t-cell response since the peptide is not processed naturally from the parental ny-eso-1 protein by the proteasome [21, 31, 32] . to address the question concerning with intracellular protein processing, a t-cell activation assay with n-protein loaded b-cells, which is able to cross-present protein antigen through the proteasome system was preformed [33, 34] . the t-cell activation assay demonstrated that when the n-protein loaded b-cells were used as target cells, the n220 primed t-cells were induced for ifn-␥ production and the level of ifn-␥ produced is comparable to the n223 peptide and is significantly higher than that of the n227 and n317 peptides previously described [12] , that is consistent to the results obtained in the t2-cell binding assay. although a study has previously mapped the three hla-a*0201 restricted n-protein peptides (n223, n227 and n317) in vaccinated transgenic mice [12] , and the vaccine potency has not been addressed. in the present study, the potency of the novel n220 peptide together with the three previously described peptides [12] , vaccine was investigated in a transgenic mouse model expressing the human hla-a2.1. in contrast to the conventional method using expensive synthetic peptides for injection [14] that is infeasible for practical used in a large vaccination program, a sct display dna vaccine mechanism was employed. a dna vaccine is much less costly compared to the synthetic peptides and when couple with the sct display system, the immunogenic peptide is translated together with the ␤ 2 -microglobulin and the mhc class i heavy chain molecule as a complex. after translation, the whole covalently linked mhc class i-peptide complex can be transferred from the er to the cell surface for antigen presentation. the high stability of the covalently linked mhc class i-peptide complex produced the sct system excludes competing peptides and is a potent stimulator for t-cells [25] . thus, it eliminates the uncertainty of the antigen processing in the professional antigen presenting cells and the cytotoxic t-cells can be primed more directly to ensure that the peptide encoded in the vaccine can be presented for vaccination. in our study, the dna vaccine was injected into the transgenic mice for vaccination and the cytotoxicity assay demonstrated that vaccination of the n220 peptide is able to trigger the cytotoxic t-cell response against the n-protein expressing cells as early as 1 week after the last vaccination even in the absence of any adjuvants. comparative results obtained from cytotoxicity assay among the tested peptides indicated that the n220 peptide represents as one of the most potent amino acid sequences of the n-protein that is able to trigger the cytotoxic t-cell response. interestingly, all the reported immunogenic t-cell epitopes of n-protein, including the n220 revealed in the present study, are mostly clustered between the amino acid residues 220 and 362. therefore it will be interested to investigate whether a vaccine composed of this 140 amino acid peptide (n220 to n362) coupled with the known sars b-cell epitopes previously described [9, 35] is sufficient to trigger a protective immune response against the sars-cov in human. in summary, we have identified a novel hla-a2.1 specific sars-cov n protein epitope (n220-n228 lalllldrl) which can activate cytotoxic t-cells in vitro and when used with the sct system, it is sufficient to elicit cytotoxic tcell response against n-protein expressing cells in the hla-a2.1k b transgenic mouse model. the findings of the novel cytotoxic t-cell epitope presented in this study provide worth information in sars vaccine design that may contribute to the sars controlling program in the future. sars: clinical presentation, transmission, pathogenesis and treatment options the comparative pathology of severe acute respiratory syndrome and avian influenza a subtype h5n1-a review the aetiology, origins, and diagnosis of severe acute respiratory syndrome characterization of viral proteins encoded by the sars-coronavirus genome cellular mechanisms governing crosspresentation of exogenous antigen the carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic t lymphocytes and protects chickens from acute infection enhanced induction of sars-cov nucleocapsid protein-specific immune response using dna vaccination followed by adenovirus boosting in balb/c mice antibody response of patients with severe acute respiratory syndrome (sars) targets the viral nucleocapsid the epitope study on sars-cov nucleocapsid protein antibody responses against sars-coronavirus and its nucleocaspid in sars patients preparation and characterization of a novel monoclonal antibody specific to severe acute respiratory syndrome-coronavirus nucleocapsid protein hla-a*0201 t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus nucleocapsid and spike proteins tcell epitopes in severe acute respiratory syndrome (sars) coronavirus spike protein elicit a specific t-cell immune response in patients who recover from sars screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a model of the ace2 structure and function as a sars-cov receptor sars coronavirus: a new challenge for prevention and therapy comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection cancer immunotherapy using a dna vaccine encoding a single-chain trimer of mhc class i linked to an hpv-16 e6 immunodominant ctl epitope cells expressing a major histocompatibility complex class i molecule with a single covalently bound peptide are highly immunogenic plasmids encoding foot-and-mouth disease virus vp1 epitopes elicited immune response in mice and protected swine against viral infection genomic characterization of the severe acute respiratory syndrome coronavirus of amoy gardens outbreak in hong kong treatment of established tumors with a novel vaccine that enhances major histocompatibility class ii presentation of tumor antigen transduction of dendritic cells with recombinant adenovirus encoding hca661 activates autologous cytotoxic t lymphocytes to target hepatoma cells cutting edge: single-chain trimers of mhc class i molecules form stable structures that potently stimulate antigen-specific t cells and b cells perarnau b. hla-a2.1-restricted education and cytolytic activity of cd8(+) t lymphocytes from ␤2-microglobulin (␤2m) hla-a2.1 monochain transgenic h-2db ␤2m double knockout mice analysis of the hla-restricted influenza-specific cytotoxic t lymphocyte response in transgenic mice carrying a chimeric human-mouse class i major histocompatibility complex the minimum peptide epitope from the influenza virus matrix protein. extra and intracellular loading of hla-a2 induction of th1 type response by dna vaccinations with n, m, and e genes against sars-cov in mice long-lived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sars-recovered patients multiepitope cd8(+) t cell response to a ny-eso-1 peptide vaccine results in imprecise tumor targeting cross-presentation of hla class iu epitopes from exogenous ny-eso-1 polypeptides by nonprofessional apc cpg-dna aided cross-priming by cross-presenting b cells b lymphocytes participate in crosspresentation of antigen following gene gun vaccination antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein we thank the hong kong research fund for the control of infectious diseases for funding this project. key: cord-346957-bmajkabp authors: lv, yanbo; ruan, zhihua; wang, li; ni, bing; wu, yuzhang title: identification of a novel conserved hla-a*0201-restricted epitope from the spike protein of sars-cov date: 2009-12-03 journal: bmc immunol doi: 10.1186/1471-2172-10-61 sha: doc_id: 346957 cord_uid: bmajkabp background: the spike (s) protein is a major structural glycoprotein of coronavirus (cov), the causal agent of severe acute respiratory syndrome (sars). the s protein is a potent target for sars-specific cell-mediated immune responses. however, the mechanism cov pathogenesis in sars and the role of special ctls in virus clearance are still largely uncharacterized. here, we describe a study that leads to the identification of a novel hla-a*0201-restricted epitope from conserved regions of s protein. results: first, different sars-cov sequences were analyzed to predict eight candidate peptides from conserved regions of the s protein based upon hla-a*0201 binding and proteosomal cleavage. four of eight candidate peptides were tested by hla-a*0201 binding assays. among the four candidate peptides, sp8 (s(958-966), vlndilsrl) induced specific ctls both ex vivo in pbls of healthy hla-a2(+ )donors and in hla-a2.1/k(b )transgenic mice immunized with a plasmid encoding full-length s protein. the immunized mice released ifn-γ and lysed target cells upon stimulation with sp8 peptide-pulsed autologous dendritic cells in comparison to other candidates. conclusion: these results suggest that sp8 is a naturally processed epitope. we propose that sp8 epitope should help in the characterization of mechanisms of virus control and immunopathology in sars-cov infection. severe acute respiratory syndrome (sars), a newly emerging infectious disease, is caused by a sars-associated coronavirus (sars-cov) [1] [2] [3] , which may originate from some wild animals [4] . after its first occurrence, sars rapidly spread around the world along international airtravel routes, reaching all five continents and resulting in several hundreds of deaths [5] . the most recent epidemic of sars occurred in beijing and anhui, china in april 2004 and originated from laboratory contamination (who update 7; see further information). although the outbreaks seem to be over, sars remains a safety concern because of the possible reintroduction of a sars-like coronavirus (sl-cov) into humans and the risk of an escape of sars-cov from laboratories [6] [7] [8] . more importantly, a new recombinant virus derived from human [9], swine and/or avian influenza virus, might re-emerge as a new sars-cov type, much like the recent emergence of a novel swine-origin influenza a (h1n1) in humans. thus, it is essential to develop various and distinct strategies to combat this highly contagious disease. the published literature reports that high titres of neutralizing antibodies and sars-cov-specific cytotoxic t lymphocyte (ctl) responses were detected in patients who had recovered from sars [10, 11] , and the levels of those responses correlated well with disease outcome [12] . hence, both humoral and cellular immune responses appear to be crucial for the clearance of sars-cov infection. immune responses can be raised directly against several of the sars-cov proteins [13] [14] [15] . targeting the spike (s) structural glycoprotein [12, [16] [17] [18] in particular induces a robust immune response, suggesting it plays an important role in the systemic clearance of sars-cov [10] . the viral surface s protein is involved in host cell receptor recognition, virus attachment and entry [19] ; adaptive evolution of s protein, thus, contributes to sars-cov overcoming the species barrier [20] . hence, many vaccines and therapeutics against sars-cov target the s protein [19] . considering that cytotoxic t-cell responses participate in the clearance of virus from recovered sars patients and contribute to immunopathology in early stages of the disease [21] , one of the most attractive s protein-based strategies proposes eliciting a sars-cov ctl response to clear the infection. to this end, a detailed understanding of the s protein-mediated ctl response is essential. development of effective treatments and vaccines against sars-cov depends upon the underlying mechanisms of various immune effectors in protective immunity and identification of the protective antigens recognized by each. epitopes are the basic antigenic elements of virus structural proteins, which functionally induce the host cell-mediated immune response. identification of the ctl-specific epitopes of sars-cov proteins could provide the basis for the development of sars immunity-based treatments and aid in the understanding of mechanisms underlying sars-cov pathogenesis. hla-a*0201 is expressed 39-46% of all major ethnicities [22] . the identification of hla-a*0201-restricted sars-cov/s ctl epitopes is an important contribution towards understanding the role of ctls in sars-cov pathogenesis and protection. currently, several ctl-specific sars-cov s protein epitopes have been identified in the context of hla-a*0201, including s 411-420 , s 787-795 , s 978-986 , s 1042-1050 , s 1167-1175 , s 1203-1211 and ssp-1 [23-26], and the h2 complex, including s 366-374 , s 436-443 , s 525-532 and s 1031-1047 [18, 27] . it is likely that additional s protein ctl epitopes exist. s protein is relatively large in size and usage of different detection methods may result in the identification of novel s protein-derived epitopes. in turn, this data will provide advances towards understanding the mechanisms of sars-cov infection, and contribute to the development of future sars-cov infection intervention strategies. in this study, we identified a novel sars/s-specific, hla-a*0201-restricted epitope that was conserved among sars-cov strains. based on a binding affinity-based prediction and a proteosomal cleavage site prediction, we constructed a panel of potential hla-a*0201-restricted ctl peptides from the s protein. each candidate peptide was evaluated for its binding affinity to hla-a*0201 molecules using the t2 cell-peptide binding test. we then evaluated the ability of hla-a*0201 binding peptides to provoke ctl responses in peripheral blood lymphocytes (pbl). pbl preparations from major histocompatibility complex (mhc)-matched healthy donors or hla-a2.1/ k b transgenic (tg) mice, were incubated with dendritic cells (dcs) that had been pre-pulsed with the peptides of interest. we identified a novel sars-cov s proteinderived ctl epitope s (958-966) (vlndilsrl) that was capable of priming the s protein-specific hla-a2.1-restricted ctl response. the effective ctl response was evidenced by cell death of peptide-pulsed t2 and peptide-pulsed jurkat-a2/k b cells. the findings of this study should provide insight into the immunological characteristics of spike protein and provide an alternative strategy for the future development of sars-cov s protein ctl epitope-based vaccines. we selected candidate ctl epitopes derived from sars-cov s protein by two criteria: (i) conservation between different strains of sars-cov to encompass as many sars-cov strains as possible, and (ii) high representation in the general population, i.e., hla-a*0201-restricted. after alignment of the amino acid residues of s protein with eighteen sars-cov strains (figure 1 ), the bj01 strain s protein was selected to predict the s protein specific, hla-a*0201-restricted ctl epitopes. based on the presence of hla-a*0201 binding motifs and the cleavage sites for proteasomes and immunoproteasomes, eight candidate peptides were predicted and synthesized ( figure 1 and table 1 ), termed sp1-8. these peptides were further verified as having an absence of shared sequence homology with the human or murine proteins using the blast search engine http://www.ncbi.nlm.nih.gov/blast/, to avoid any autoreactive response. to evaluate the binding affinity of these peptides to hla-a*0201 molecules, a t2 cell-peptide binding test was used [28] . t2 cells lack the transporter associated with antigen processing (tap), a key factor involved in endogenous antigen processing and presentation, causing the empty hla-i molecules on the t2 cell surface to be very unstable and to degrade rapidly after cell surface presentation. however, when exogenous epitope peptides bind to the hla-i molecules on the cell surface, they become stable [28, 29] . accordingly, the peptide-induced upregulation of hla-i on tap-deficient t2 cells is used to monitor peptide binding to class i molecules, which then indicates the binding affinity of peptides to hla-i molecules. higheraffinity peptides will induce more hla-a*0201 expression on the cell surface than will lower-affinity peptides. as shown in table 1 , of the eight candidate peptides only sp5, sp6, sp7 and sp8 were high-affinity epitopes (fi = 1.1, 1.1, 1.2 and 1.5, respectively). the positive control peptide, s 411-420 , bound hla-a*0201 strongly (fi = 1.5), whereas no binding was observed with the negative control hbcag (131-140) peptide (fi = 0.1). to investigate the capacity of candidate peptides to mobilize a human ctl repertoire, hla-a2 + pbls from ten hla-a2 + donors were stimulated in vitro by dcs loaded with alignment of the putative amino acid sequences of s proteins from eighteen sars-cov strains a dot among the individual sequences denoted nucleotides that are the same as the consensus. the candidate epitope peptides were shown in bold text. * hla-a*0201-binding motif score from algorithm syfpeithi ≥ 24. the eight peptides were also predicted and selected using propred1, in which the threshold of hla-a2-binding motif is 4% and the threshold of proteasomal and immunoproteasomal cleavage site is 8%. † increase of hla-a*0201 molecules on t2 cell surface. fi = [(mean fitc fluorescence with the given peptide -mean fitc fluorescence without peptide)/(mean fitc fluorescence without peptide)]. fi > 1.0 indicates high-affinity peptides; fi ≤ 1.0, low-affinity peptides. hla-a*0201-restricted peptide s 411-420 was used as a positive control for hla-a*0201-binding ability, while the h-2 b -restricted peptide hbcag (131-140) was used as a negative control. the eight candidate peptides, positive control peptides s 411-420 , or negative control peptides hbcag (131) (132) (133) (134) (135) (136) (137) (138) (139) (140) . t2 cells loaded with each of peptides were used as target cells in cytotoxicity assays. of the eight peptides tested, sp6, sp7 and sp8 induced more cd8 + t-cells that specifically produced ifn-γ in response to dcs pulsed with the relevant peptides or positive control peptide in comparison to other groups ( figure 2a ). furthermore, these cd8 + t cells could lyse the t2 cells loaded with relevant peptides or positive control peptide more efficiently than other groups ( figure 2b ). the cytolysis observed were specific because the ctls could not lyse t2 cells loaded with irrel-evant peptides ( figure 2b ). due to the limited induction of ifn-γ secreting t cell frequency and the low ctl cytolysis ability, groups other than sp6, sp7 and sp8 were excluded from further study. the ex vivo results showed the existence of functional anti-sars-cov/s ctl precursors in the peripheral t cell repertoire of healthy donors. furthermore, sars-cov/s-derived peptides sp6, sp7 and sp8 could not only induce the increased s protein specific ifn-γ secreting t cell frequency but also the enhanced cytolytic capacity of these ctls. to further address whether the immunogenic candidate peptide is naturally processed and presented, hla-a2.1/ k b transgenic mice were immunized with s/pvax1 plasmid containing a full-length cdna encoding the sars-cov/s protein. splenocytes were collected from mice seven days after four weekly injections with s/pvax1, and re-stimulated ex vivo by mouse bone marrow-derived dcs loaded with the candidate peptides, the positive peptide s 411-420 , the irrelevant peptides hbcag (131-140) , or dcs alone, for an additional 6 days. investigation of ifn-γ production and the cytolytic ability of the effector ctl cells were carried out following the re-stimulation. the j(a2/ k b ) cells loaded with the corresponding peptides were used as targets in cytotoxicity assays. as shown in figure 3a , ctls from s/pvax1-immunized mice were able to lyse three candidate peptides-pulsed j(a2/k b ) cells but did not lyse j(a2/k b ) cells alone or j(a2/k b ) cells loaded with irrelevant peptide hbcag (131-140) at any e/t ratio. of the three candidate peptides tested, sp8 exhibited the most lytic capacity at each e/t ratio, which was comparable to the positive control peptide ( figure 3a ). in accordance with results from the cytolytic assays, bulk ctls released ifn-γ only in response to dcs pulsed with sp6, sp7, sp8 and the positive control peptide, but not to those pulsed with irrelevant peptide hbcag (131-140) or dcs alone ( figure 3b ). again, among the three tested candidate peptides, sp8 released the most ifn-γ following peptide stimulation ( figure 3b ). it is known that sars-cov can induce a strong specific ctl response in infected patients, besides high titres of neutralizing antibodies [10, 11] . furthermore, the ctl response levels correlate with disease outcome [12] , suggesting ctl response is crucial for the clearance of sars-cov. among all the encoded proteins in the sars-cov genome, s protein is currently considered the most important target to prime the host immune response [12, [16] [17] [18] . it has been reported that an inflammatory cell influx of airway macrophages and a massive release of cytokines occur during the peak of sars infection [30] . thus, it is in this study, we predicted and validated a novel ctl epitope of sars-cov s protein. we used two prediction systems to identify candidate ctl epitopes of s protein (i.e., hla-a2-binding peptide prediction method combined with a proteosomal cleavage site prediction system) to improve prediction accuracy. the eight predicted peptides were then verified via mhc peptide binding assay (table 1) . among the eight candidate peptides, sp5, sp6, sp7 and sp8 exhibited the highest capacity to induce more potent ctls secreting ifn-γ and to lyse target cells from hla-a*0201-matched healthy donor pbls ( figure 2 ). further in vivo investigation showed that plasmid encoding the full-length sars-cov s gene elicited strong ctl response in hla-a2.1/k b transgenic mice. these ctls could produce substantial amounts of ifn-γ and kill target cells in a peptide-specific and hla-a*0201-restricted manner (figure 3 ), suggesting the predicted candidate peptides were native epitopes, capable of priming ctl responses in vivo. we found that candidate peptide sp8 held the greatest ability to secrete ifn-γ and kill target cells in vivo (figure 3 ). another candidate peptide, sp7, failed to induce the most potent peptide specific ctls in tg mice (figure 3 ), despite it having had the highest such ability in comparison to the rest of the in vitro stimulation set (figure 2 [18, 27] . in this study, we predicted and validated a novel ctl epitope of sars-cov s protein, sp8 (s 958 , vlndilsrl). this may be due to the unique predictive methods used in our study. we combined strategies for prediction (i.e., hla-a2-binding peptide prediction method combined with a proteosomal cleavage site prediction system). previous studies used single methods, such as hla peptide binding prediction or overlapping peptide strategy [18, [23] [24] [25] [26] [27] , suggesting different prediction strategies might lead to different results. in any case, the predicted candidate peptides require additional validation methods to ensure accuracy. in our study, we also determined that among the eight peptides we predicted, four could potent prime ctls to produce significant ifn-γ and lyse target cells; although, sp8 peptide exhibited the most potency for ctl priming. however, zhou et al. reported that they only found one predicted peptide that could stimulate ifn-γ secretion and target cell lysis [26] . this may reflect the different stimulators used in these studies; zhou used peptides to stimulate the effector cells directly while we used dcs loaded with the candidate peptides. we argue for the use of dcs as stimulator cells in ex vivo study because dcs are the most potent apcs for priming t cells, and they not only present peptides to t cells but sars-cov s protein specific ctls in dna vaccine-immu-nized hla-a2.1/k b -tg mice our study has identified a novel conserved hla-a*0201restricted epitope from the spike protein of sars-cov. we propose that the newly identified epitope could be used for evaluation of sars-cov-specific cd8 + t-cell responses during the course of sars infection and treatment. this epitope should also aid in the characterization of virus control mechanisms and immunopathology of sars-cov infection. ultimately, our findings may be relevant to the development of ethnically unbiased, widely applicable immunotherapeutic approaches for sars disease. to identify potential hla-a*0201-binding peptides within the s protein of the sars-cov (bj01) strain, a combination of two computer algorithms was utilized. the predictive algorithm, "propred1"[31], is a matrix-based method that allows the prediction of mhc binding sites in an antigenic sequence for 47 mhc class-i alleles. we restricted our analysis to the hla-a2 allele, since it is prevalent in a large percentage of all major ethnicities and it is the most extensively studied hla class-i antigen [22] . propred1 also allows the prediction of the standard proteasomal and immunoproteasomales cleavage sites in an antigenic sequence. the simultaneous prediction of mhc binding and proteasomal cleavage sites in an antigenic sequence leads to the identification of potential t-cell epitopes. the second algorithm, "syfpeithi", was developed by h. g. rammensee et al [32] , and ranks peptides according to a score that takes into account the presence of primary and secondary mhc-binding anchor residues. the 9 mer peptides with a score exceeding 24 were selected in "syfpeithi". the amino acid sequence of sars-cov/s (bj01) was analyzed on both of the computer programs for the existence of 9-amino acid peptides predicted to bind to hla-a2. the candidates peptides were synthesized at shenyou biotech (shanghai, china) and purified by reverse phase hplc to > 95%, as confirmed by mass spectrometry. the published hla-a*0201-restricted peptide s 411-420 (klp-ddfmgcv) derived from the s protein of sars-cov [26] was used as a positive control for hla-a*0201-binding ability, and the hbcag-derived h-2 b -restricted peptide hbcag (131-140) (ayrppnapil) was used as a negative control. lyophilized peptides were dissolved in pbs at a concentration of 1 mg/ml and stored in aliquots at -20°c. hla-a2 + individuals were selected by flow cytometry screening using the anti-hla-a2 monoclonal antibody bb7.2. buffy coats from hla-a2 + normal donors were purchased from southwest hospital (third military medical university, chongqing, china). pbl from an hla-a2 + healthy donor were separated on ficoll-hypaque density gradients (tbd, inc, tianjin, china), washed three times in phosphate-buffered saline (pbs), resuspended in rpmi1640 medium (gibco, brl) supplemented with lglutamine (10 mg/ml), penicillin (5 × 10 4 u/l), streptomycin (50 mg/l) and 10% fetal calf serum (fcs), and plated in 6-well plates at 4 × 10 6 cells per well. human tap-deficient t2 cell line and bb7.2 cell line producing mab against hla-a2 were purchased from american type culture collection. t2 cell line was maintained in rpmi1640 medium supplemented with 20% fetal bovine serum and 100 μg/ml penicillin/streptomycin. bb7.2 cell line was maintained in dmem containing 10% fcs, 4 μg/l glucose, penicillin (5 × 10 4 u/l) and streptomycin (50 mg/l). jurkat-a2/k b cells, a generous gift from dr. w. martin kast (the norris comprehensive cancer center, los angeles, ca) and dr. jehad charo (the max delbruck center for medicine, berlin, germany), were transfected with the hla chimeric molecule containing the α1 and α2 domains from human hla-a*0201 and α3 from mouse h-2k b , to serve as a model system of hla restricted responses [33] . the jurkat-a2/k b (j(a2/kb)) cell line was maintained in rpmi1640 medium (gibco, brl) plus 10% calf serum and supplemented with 4 μg/l glucose, penicillin (5 × 10 4 u/l) and streptomycin (50 mg/ l). all cell lines mentioned above were kept at 37°c in a humidified atmosphere of 5% co 2 in air. hla-a2.1/k b transgenic (tg) mice were purchased from the jackson laboratory (bar harbor, me). for experimen-tal purposes, six to eight week-old mice were used. cell surface hla-a*0201 expression was assessed by flow cytometry using fluorescein isothiocyanate (fitc)labeled hla-a2-specific mab bb7.2 (sterotec ltd, oxford, uk). mice were kept in spf animal care facilities and all experiments were performed according to the guidelines in the institutional animal committee of tmmu. binding assay of candidate peptides to hla-a2 all candidate peptides were tested individually for their capacity to bind to hla-a2 molecules on the surface of human tap-deficient t2 cells [28] . briefly, t2 cells were incubated with 20 μg/ml candidate peptides and 3 μg/ml human β2-microglobulin (sigma, st louis, mo) in serum-free rpmi1640 for 18 hours at 37°c in a 5% co 2 atmosphere. expression of hla-a*0201 on t2 cells was then determined by staining with fitc-conjugated anti-hla-a2 mab bb7.2 and data analyzed using a facscalibur flow cytometer (becton dickinson, mountain view, ca) and cellquest software (becton dickinson). the published peptide s 411-420 and hbcag (131-140) served as positive and negative control, respectively. the former is known to bind to hla-a2 molecule with high affinity, the latter has been identified as mouse h2k d epitope that has little binding affinity with hla-a2 molecule. the fluorescence index (fi) was calculated as follows: fi = [(mean fitc fluorescence with the given peptide -mean fitc fluorescence without peptide)/(mean fitc fluorescence without peptide)]. peptides with an fi more than 1 were regarded as high-affinity epitopes. human peripheral blood monocyte-derived dcs were generated as described previously [28] with minor modifications. briefly, human pbls were suspended in serumfree rpmi1640 and allowed to adhere to 6-well plates at a final concentration of 1 × 10 7 cells/3 ml/well and cultured in 5% co2 at 37°c. after 2 hours, non-adherent cells were gently removed with warm medium. the resulting adherent cells were cultured in rpmi1640 medium supplemented with 10% fcs, 20 ng/ml recombinant human interleukin-4 (il-4) (r&d systems, minneapolis, mn) and 800 u/ml recombinant human granulocyte-macrophage colony stimulating factor (gm-csf; sandoz, basel, switzerland) in 5% co 2 at 37°c. every two days, one-half of the medium was replaced by fresh medium containing double concentration of gm-csf and il-4 as indicated above. cell suspensions were collected for analysis of surface phenotype at different stages of development. after five days of culture, dcs were harvested for subsequent experiments (90% pure as confirmed by analysis of relatively dc-specific phenotype and with a typical dc morphology). 10 ng/ml recombinant human tumor necrosis factor (tnf-α, peprotech, rocky hill, nj) was added to the medium to induce phenotypic and functional matura-tion. then, 48 hours later, dcs were used to prime autologous pbls as follows, dcs were pulsed with 20 μg/ml peptide in the presence of 3 μg/ml β2-microglobulin at 37°c for 5 hours and irradiated at 30 gy before use. pbls (2 × 10 6 cells/3 ml culture medium) were co-cultured with 2 × 10 5 peptide-pulsed irradiated autologous dcs in a 6well plate in the presence of 10 ng/ml recombinant human interleukin-7 (il-7; peprotech). after 24 to 48 hours 20 iu/ml human interleukin-2 (il-2, sigma) was added to the culture medium. lymphocytes were re-stimulated each week in the same manner. three days after the second round of re-stimulation, induced cells were harvested and tested by cytokine determination and cytotoxicity assays. a plasmid s/pvax1 encoding sars-cov s protein was constructed and used to immunize the hla-a2.1/k b transgenic mice at a dose of 100 μg (in 100 μl of pbs) of plasmid s/pvax1 by injection into tibialis anterior muscles. mice were re-inoculated four times every seven days under the same conditions. in this study, bone marrowderived dcs were generated from transgenic mice as previously described [34,35] with some modification. dcs were pulsed with 20 μg/ml peptide in the presence of 3 μg/ml β2-microglobulin at 37°c for 5 hours and irradiated at 30 gy before use. spleens were aseptically removed after the final scheduled immunization. splenic singlecell suspensions were then harvested and cultured in 6well plates at a density of 1 × 10 7 cells/3 ml/well, in the presence of 1 × 10 5 peptide-loaded irradiated syngeneic dcs. on day six of culture, induced cells were harvested and tested by cytokine determination and cytotoxicity assays. cytotoxic activity of ctls was determined in a standard 4hour 51 cr release assay as previously described [36] with some modification. in human cytotoxicity assays, dcs derived from a healthy hla-a2 + donor were incubated with each of candidate peptides and used to stimulate autologous healthy hla-a2 + donor pbls. t2 cells loaded with the relevant peptides were used as target cells in cytotoxicity assays. as a positive control group, hla-a2 + pbls were stimulated with s 411-420 -pulsed autologous dcs. hla-a2 + pbls stimulated with hbcag (131-140) -pulsed autologous dcs served as the negative control group. in dna-immunized mice cytotoxicity assays, the target cells were the j(a2/kb) cells loaded with the candidate peptides, the positive control peptide s 411-420 , the irrelevant peptides hbcag (131) (132) (133) (134) (135) (136) (137) (138) (139) (140) , and j(a2/kb) cells alone. first, t2 cells and/or j(a2/kb) cells were loaded with 20 μg/ml peptides and 3 μg/ml human β2-microglobulins and incubated at 37°c for 2 hours. then peptide-pulsed t2 cells and/or j(a2/kb) cells were labeled with 51 cr sodium chromate (na 51 cro 4 , perkin-elmer life science, boston, ma) for 90 minutes at 37°c. 51 cr-labeled target cells were washed three times and mixed with graded doses of effectors in 96-well plates. after incubation at 37°c for 4 hours, a total of 100 μl supernatant was collected from each well and radioactivity was counted with a gamma counter. each assay was performed in triplicate. percent specific lysis was determined according to the following formula: percent specific lysis = [(mean experimental cpm -mean spontaneous cpm)/(mean maximum cpm -mean spontaneous cpm)] × 100%. spontaneous and maximum releases were determined by incubating the labeled targets with medium alone or 1 m hcl, respectively. spontaneous release was always < 15% of maximum release. elispot assay was performed using a commercially available kit (u-cytech, netherlands) according to the manufacturer's instructions and published literature [37] with some modification. autologous dcs were pulsed with 20 μg/ml candidate peptides and used as stimulators for hla-a2.1 + pbls from the immunized mice. effector cells (1 × 10 5 ) and stimulator cells (1 × 10 5 ) were seeded into 96-well polyvinylidene fluoride (pvdf)-backed microplates pre-coated with anti-ifn-γ mab. after incubation at 37°c for 48 hours, cells were removed and plates processed as described in the instruction. resulting spots were counted with a stereomicroscope (carl zeiss, thornwood, ny) under magnifications of ×20 to ×40. only brown and/or blue colored spots with fuzzy borders were scored as spot-forming cells (sfcs). as a positive control, s 411-420 -loaded dcs were used as stimulator cells. the hbcag (131-140) -loaded dcs, dcs alone, and medium were used as negative controls. negative control values were always < 20 sfc per 10 6 input cells. results were considered positive when at least 120 sfc/10 6 pbl were detected. each assay was run in triplicate and results were representative of three experiments. identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome (sars): a year in review infectious diseases. mounting lab accidents raise sars fears laboratory-acquired sars raises worries on biosafety emergence of a novel swine-origin influenza a (h1n1) virus in humans immunological responses against sars-coronavirus infection in humans b-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the s2 domain of the surface spike glycoprotein long-term persistence of robust antibody and cytotoxic t cell responses in recovered patients infected with sars coronavirus search for potential target site of nucleocapsid gene for the design of an epitope-based sars dna vaccine induction of th1 type response by dna vaccinations with n, m, and e genes against sars-cov in mice severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity immunogenicity of a receptor-binding domain of sars coronavirus spike protein in mice: implications for a subunit vaccine identification of murine cd8 t cell epitopes in codon-optimized sars-associated coronavirus spike protein the spike protein of sars-cov--a target for vaccine and therapeutic development adaptive evolution of the spike gene of sars coronavirus: changes in positively selected sites in different epidemic groups measurement of subgroups of peripheral blood t lym yl carried out the cell and animal manipulation, the ctl cytolysis assay. zr carried out the immunoassays. lw participated in the design of the study and performed the statistical analysis. bn and yw conceived of the study, and participated in its design and coordination and helped to draft the manuscript. all authors read and approved the final manuscript. key: cord-346445-hgqohdct authors: toyoshima, yujiro; nemoto, kensaku; matsumoto, saki; nakamura, yusuke; kiyotani, kazuma title: sars-cov-2 genomic variations associated with mortality rate of covid-19 date: 2020-07-22 journal: j hum genet doi: 10.1038/s10038-020-0808-9 sha: doc_id: 346445 cord_uid: hgqohdct the coronavirus disease 2019 (covid-19) outbreak, caused by sars-cov-2, has rapidly expanded to a global pandemic. however, numbers of infected cases, deaths, and mortality rates related to covid-19 vary from country to country. although many studies were conducted, the reasons of these differences have not been clarified. in this study, we comprehensively investigated 12,343 sars-cov-2 genome sequences isolated from patients/individuals in six geographic areas and identified a total of 1234 mutations by comparing with the reference sars-cov-2 sequence. through a hierarchical clustering based on the mutant frequencies, we classified the 28 countries into three clusters showing different fatality rates of covid-19. in correlation analyses, we identified that orf1ab 4715l and s protein 614g variants, which are in a strong linkage disequilibrium, showed significant positive correlations with fatality rates (r = 0.41, p = 0.029 and r = 0.43, p = 0.022, respectively). we found that bcg-vaccination status significantly associated with the fatality rates as well as number of infected cases. in bcg-vaccinated countries, the frequency of the s 614g variant had a trend of association with the higher fatality rate. we also found that the frequency of several hla alleles, including hla-a*11:01, were significantly associated with the fatality rates, although these factors were associated with number of infected cases and not an independent factor to affect fatality rate in each country. our findings suggest that sars-cov-2 mutations as well as bcg-vaccination status and a host genetic factor, hla genotypes might affect the susceptibility to sars-cov-2 infection or severity of covid-19. the novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which causes coronavirus disease 2019 (covid19) , was first reported in wuhan, china in december 2019 [1, 2] . soon after, the virus caused an outbreak in china and has spread to the world. according to the world health organization, the current outbreak of covid-19 has nearly 11.5 million confirmed cases worldwide with more than 530,000 deaths, as of july 6, 2020. the sars-cov-2 genome comprises of around 30,000 nucleotides organized into specific genes encoding structural proteins and nonstructural proteins (nsps) [1, 2] . structural proteins include spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. surface s glycoprotein is involved in the interaction with the host's angiotensin-converting enzyme 2 (ace2) receptor and plays an important role in rapid human to human transmission. nsps, which are generated as cleavage products of the open reading frame 1ab (orf1ab) viral polyproteins, assemble to facilitate viral replication and transcription. rna-dependent rna polymerase, also known as nsp12, is the key component that regulates viral rna synthesis with the assistance of nsp7 and nsp8 [3] . in addition, five accessory proteins are encoded by orf3a, orf6, orf7a orf8, and orf10 genes. sars-cov-2 has rapidly spread around the world compared with sars-cov appeared in 2002 and middle east respiratory syndrome coronavirus (mers-cov) in 2012. although the estimated fatality rate in the confirmed cases is 6.6% in sars-cov-2, which is lower than those of sars-cov and mers-cov, 9 .6% and 34.3%, respectively [4] , there is an urgent need for its effective treatment based on antivirals and vaccines that reduce the mortality and morbidity rates of covid-19. however, up to now, the causes of the large country-by-country difference of the mortality rates related to covid19 have not been clearly understood. although many studies were conducted, the effects of sars-cov-2 genetic variations and host genetic factors remain elusive. in this study, we comprehensively analyzed 12,343 sars-cov-2 genome sequences isolated from patients/ individuals in six geographic areas, including asia, north america, south america, europe, oceania, and africa, and investigated their correlations to the fatality rates in 28 different countries. we also investigated the associations with bcg-vaccination status as well as human leukocyte antigen (hla), which is an important molecule to recognize virus by our host immune system. full-length viral nucleotide sequence of the reference sars-cov-2 (accession number mn908947) [1] was downloaded from the ncbi genbank. we used a total of 12,343 sars-cov-2 sequences isolated in 50 different countries of six geographic areas, including 1062 sequences from asia, 4060 from north america, 99 from south america, 6012 from europe, 1028 from oceania, and 82 from africa regions, which were deposited in the global initiative on sharing avian influenza data as of 7 may 2020 [5] . to analyze mutations based on countries, we used the data of 28 countries in which more than 30 sars-cov-2 sequences are available, among the 50 countries. we analyzed mutations of sars-cov-2 as described previously [6] . briefly, we first aligned each of the sars-cov-2 sequences to the reference sequence sars-cov-2_wuhan-hu-1 (accession number mn908947) using blat software [7] . after the alignment, we extracted nucleotide sequences corresponding to individual proteins of sars-cov-2, translated them into amino acid sequences, and then compared them to reference amino acid sequences of sars-cov-2_wuhan-hu-1 (accession numbers qhd43415-qhd43423, qhi42199). data on numbers of confirmed cases and deaths related to covid-19 were obtained from the worldometer (https://www.worldometers.info/coronavirus/) on 7 may 2020 (supplementary table 1 ). data of confirmed cases and deaths in each state in the united states were obtained on 3 july 2020. fatality rate in infected individuals was calculated from total infected cases and total deaths in each country. the allelic frequencies of hla genes were obtained from the allele frequency net database [8] . data on bcg-vaccination status in each country were obtained from the previous reports [9] [10] [11] . continuous variables were compared using the student's t test. fisher's exact test was used to analyze differences of mutation rates of sars-cov-2 among the different geographic areas. a hierarchical clustering was performed to identify clusters corresponding to distinct subgroups with the selected mutations using r package stats. global maps of clusters or mutations were drawn using r package rworldmap. pearson's correlation was used to evaluate correlations among mutant frequencies, hla allele frequencies and fatality rates. haploview software was used to analyze and visualize the haplotypes of sars-cov-2 mutations [12] . multiple regression analysis was used to test for an independent contribution of identified factors to fatality rates of covid-19. all statistical analyses were carried out using the r statistical environment version 3.6.1. all replicating viruses, including coronavirus, continuously accumulate genomic mutations that persist due to natural selections. these mutations contribute to enhancement of ability of viral proliferation and infection as well as an escape from host immune attack. we firstly investigated mutations in 12,343 sars-cov-2 genome sequences isolated from patients/individuals in six different regions, including asia, north america, south america, europe, oceania, and africa. we identified a total of 1234 mutations detected in at least two independent samples, including 131 mutations found at a frequency of more than 10% (supplementary table 2 ). a hierarchical clustering using 16 common amino acid mutations classified 28 countries into three clusters (fig. 1a) . the cluster 1 includes most of the asian countries we analyzed, whereas the cluster 2 includes european and south american countries, and the cluster 3 includes european, north american, oceania, african and a few asian countries (fig. 1b) . comparing the mutations among the three clusters, the average frequency of an l variant of an orf1ab p4715l in the countries classified as the cluster 1 was 14.7%, which is significantly lower than 81.3% and 73.2%, respectively, in the countries classified as the clusters 2 and 3 (p = 1.3 × 10 −6 and p = 2.5 × 10 −5 , respectively; supplementary fig. 1a ). the orf1ab 4715l variant was detected at the significantly low frequency in asian countries compared with the other areas (20.8% vs. others 54.9-86.8%, p = 1.1 × 10 −118 ; supplementary fig. 2) . similarly, the frequency of a g variant of s protein d614g was significantly lower in the cluster 1 than the other two clusters (p = 1.2 × 10 −6 and p = 1.7 × 10 −5 , respectively, for the clusters 2 and 3; supplementary fig. 1b ). in the cluster 2, k/r variants of n protein r203k/ g204r mutations were significantly enriched at 43.1%, compared with the other clusters (5.2%, p = 0.00011 for the cluster 1 and 11.8%, p = 5.6 × 10 −7 for the cluster 3; supplementary fig. 1c ). in addition, in the cluster 1, l and f variants of n p13l and orf1ab l3606f were predominantly enriched. the l variant of n p13l was found at 17.8%, which was significantly higher than 0.2% and 1.4%, respectively, in the clusters 2 and 3 (p = 0.012 and p = 0.0079; supplementary fig. 1d ). the f variant of orf1ab l3606f was detected at a higher frequency of 40.1% than 10.0% and 7.9% in the clusters 2 and 3, respectively (p = 0.0035 and p = 0.00050; supplementary fig. 1e ). to further analyze the mutational profile, we performed a haplotype analysis by drawing a linkage disequilibrium (ld) map for sars-cov-2 viral genomes ( supplementary fig. 3 ). we found that orf1ab 4715l and s protein 614g variants were in a nearly complete ld (r 2 of ld = 0.98 and d' = 1.00). n protein 203k/204r variants were additionally acquired in the s protein 614g type of virus genome as indicated as r 2 of ld = 0.11 and d' = 0.99. these results indicate that s protein 614g-n protein 203k/204r haplotype characterizes the cluster 2. we then investigated the association with the fatality rates among confirmed cases in the 28 countries. in the analysis comparing the fatality rates in the countries classified as either of the three clusters, average fatality rate of the countries belonging to the cluster 2 was 9.3%, which was higher than 3.0% and 5.8% of averages of the countries fig. 1c ). among the mutations we analyzed, the frequencies of orf1ab 4715l-type and s 614g-type viruses showed significant positive correlations with fatality rates (pearson's correlation coefficient (r) = 0.41, p = 0.029 and r = 0.43, p = 0.022, respectively; fig. 2a, b) . since the clusters 2 and 3 were separated mainly by the frequency of n 203k/204r, we also examined the correlations of this variant or s 614g-n 203r/204g haplotype with fatality rates; however, the correlations were not statistically significant (r = 0.31, p = 0.11; r = 0.27, p = 0.17, respectively; supplementary fig. 4a, b) . it is reported that fatality rates are different among the areas or states in the united states [13] . when we compared fatality rates among the three different areas, western, central and eastern, in the united states, an eastern area showed a higher fatality rate of 6.5% than that of 2.2% in a western area (p = 0.010) and that of 3.9% in a central area (p = 0.10; fig. 3a ). therefore, we further investigated the correlations of the variants with fatality rates in the 17 states. the frequencies of orf1ab 4715l-and s protein 614g-types tended to show positive correlations with the fatality rates (r = 0.49, p = 0.047; r = 0.45, p = 0.070, respectively; fig. 3b, c) . even when integrating the data of 17 states and the remaining 27 countries, the significant correlations kept significant (r = 0.38, p = 0.014; r = 0.39, p = 0.011, respectively; supplementary fig. 5a, b) . several other factors are investigated in association with mortality related to covid-19. ecological studies have suggested that countries that mandate bcg vaccination for the population have a lower number of infections and a reduced mortality from covid-19, although the association is still controversial and the underlying mechanism has not been clarified [9, 14, 15] . we classified 28 countries into two groups according to the bcg-vaccination status as the routine vaccine schedules. as a result, the mean of fatality rates was significantly lower in 11 bcg-vaccinated countries than in 17 bcg-non-vaccinated countries (4.1% vs. 8.1%, p = 0.031; fig. 4a ). when we divided bcgvaccinated countries into subgroups according to the strains of bcg vaccine, we observed some differences in the fatality rates among the countries by different strains of bcg vaccine, but sample sizes of subgroups are too small to evaluate statistical significance ( supplementary fig. 6 ). we also found the frequencies of s 614g variant showed a trend of positive correlation with fatality rates (r = 0.54, p = 0.090; fig. 4b ) in bcg-vaccinated countries, but such correlation was not observed in bcg-non-vaccinated countries (r = 0.19, p = 0.47; fig. 4b ). in addition, the number of confirmed cases per million population was significantly lower in bcg-vaccinated countries than in bcg-non-vaccinated countries (710 vs. 2912, p = 0.0012; fig. 4c ). these results suggest that bcg-vaccination may protect from sars-cov-2 infection by potentiation of innate immune response; however, orf1ab 4715l-type and s protein 614g-type sars-cov-2 variants may escape from the immune response. host genetic differences, especially in hla loci, are well-known to contribute to individual variations in the immune responses to pathogens. we finally searched peptide epitopes with a high binding affinity to hla molecules, which we previously reported [6] , involving the two sars-cov-2 mutations, orf1ab p4715l and s d614g, to investigate the association with host immune responses. we found that several epitopes, which include the position of orf1ab p4715l or s protein d614g, are possibly bind to hla molecules, including hla-a*02:06, hla-a*11:01, hla-b*07:02, and hla-b*54:01, although the mutated epitopes from variant sars-cov-2 also predicted to bind to hla molecules at similar affinities (supplementary table 3 ). using the information of 21 countries in which allele frequency data are available, we examined a fig. 1a relationship between allele frequency of hla-a*11:01 and the fatality rates. consequently, we found a significant negative correlation (r = −0.61, p = 0.0031; fig. 5a ). similarly, a trend of negative correlations was observed between allele frequencies of hla-a*02:06 or hla-b*54:01 and the fatality rates (r = −0.39, p = 0.14, n = 16 and r = −0.60, p = 0.017, n = 15; fig. 5b, c) . however, the significant correlations became not statistically significant after adjusted by the frequency of s 614g variant in multiple regression (p = 0.13 for hla-a*11:01, p = 0.73 for hla-a*02:06 and p = 0.45 for hla-b*54:01). we also found negative correlations between allele frequencies of the hlas and the number of confirmed cases per million population (r = −0.43, p = 0.054 for hla-a*11:01, r = −0.44, p = 0.086 for hla-a*02:06 and r = −0.52, p = 0.047 for hla-b*54:01; fig. 5d-f ). together, these results suggest that differences in hla allele frequencies may explain different susceptibilities to sars-cov-2 infection among the countries, although there are many other potential confounding factors needed to be considered. the current outbreak of covid-19 has rapidly spread worldwide. most patients with covid-19 exhibit no or mild to moderate symptoms, but~15% progress to severe pneumonia and about 5% eventually develop acute respiratory distress syndrome, septic shock, and multiple organ failures. the mortality rates related to covid-19 vary among countries, generally known to be significantly higher in european and north american countries than those of asian countries. although several possibilities to explain the differences in the mortality rates are demonstrated, including the difference of age distribution, bcgvaccination status, virus genomic types, and genetic backgrounds, nothing is clear at this moment. in this study, we investigated the sars-cov-2 virus mutations and found that the frequencies of s protein 614g variant and its highly linked variant, orf1ab 4715l, were significantly correlated with fatality rates in the 28 countries and 17 states of the united states. the d614g spike mutation is the mutation detected in europe in the early phase and has widely spread around the globe, especially to european and north american countries [16] [17] [18] [19] . spike glycoprotein is essential for interaction with ace2 expressed in host cells and is important for viral transmission [20, 21] . therefore, spike glycoprotein is most vital hotspot of amino acid mutations when viruses acquire mutations to enhance the virus-cell entry to adapt environments. structural analyses indicated that s protein having a d614g substitution is located on the surface of the virus and interacts with ace2. concordant to our results, a few reports demonstrated that s 614g variant was associated with the mortality related to covid-19 [13, 22] . orf1ab p4715l is located in nsp12, which is important for viral rna replication. we found significant associations between these mutations and the fatality rates; however, the functional significance of these mutations has not clarified yet. since immune responses through hla and t cells are important to protect from virus infections and also known to be involved in the progression of covid-19, we screened epitopes around the mutations associated with fatality rates (supplementary table 3 [23] [24] [25] [26] . although further studies are required to elucidate whether such cytotoxic t lymphocytes targeting the epitopes are present in peripheral blood in patients, especially in severe patients, and also large scale casecontrol association studies are needed to confirm the association of hla genotype with susceptibility or disease progression of sars-cov-2 infection, these findings in the current study provide an important insight into treatment of the current sars-cov-2 and prevention of the second sars-cov-2 pandemic. in summary, we comprehensively investigated sars-cov-2 genome mutations, bcg-vaccination status, and hla genotypes in the 28 different countries and identified significant associations of some virus genome variants with the fatality rates. these results may explain, at least a part of the differences of the sars-cov-2 infection or the mortality rates related to covid-19 among various countries. acknowledgements the super-computing resource was provided by human genome center, the institute of medical science, the university of tokyo (http://sc.hgc.jp/shirokane.html). conflict of interest yn is a stockholder and a scientific advisor of oncotherapy science, inc. kk is a scientific advisor of cancer precision medicine, inc. this study is unrelated to the activity in these companies. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin one severe acute respiratory syndrome coronavirus protein complex integrates processive rna polymerase and exonuclease activities a novel coronavirus outbreak of global health concern global initiative on sharing all influenza data-from vision to reality bioinformatic prediction of potential t cell epitopes for sars-cov-2 blat-the blast-like alignment tool allele frequency net database (afnd) 2020 update: gold-standard data classification, open access genotype data and new query tools is bcg vaccination affecting the spread and severity of covid-19? mapping the global use of different bcg vaccine strains the bcg world atlas: a database of global bcg vaccination policies and practices haploview: analysis and visualization of ld and haplotype maps sars-cov-2 viral spike g614 mutation exhibits higher case fatality rate is global bcg vaccination-induced trained immunity relevant to the progression of sars-cov-2 pandemic? sars-cov-2 rates in bcgvaccinated and unvaccinated young adults phylogenetic network analysis of sars-cov-2 genomes emergence of drift variants that may affect covid-19 vaccine development and antibody treatment introductions and early spread of sars-cov-2 in the new york city area genomic surveillance reveals multiple introductions of sars-cov-2 into northern california functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor could the d614g substitution in the sars-cov-2 spike (s) protein be associated with higher covid-19 mortality? association of hla class i with severe acute respiratory syndrome coronavirus infection association of human-leukocyte-antigen class i (b*0703) and class ii (drb1*0301) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome epidemiological and genetic correlates of severe acute respiratory syndrome coronavirus infection in the hospital with the highest nosocomial infection rate in taiwan in 2003 association of human leukocyte antigen class ii alleles with severe middle east respiratory syndrome-coronavirus infection key: cord-288496-7rrh2gg6 authors: stryhn, anette; kongsgaard, michael; rasmussen, michael; harndahl, mikkel nors; østerbye, thomas; bassi, maria rosaria; thybo, søren; gabriel, mette; hansen, morten bagge; nielsen, morten; christensen, jan pravsgaard; randrup thomsen, allan; buus, soren title: a systematic, unbiased mapping of cd8(+) and cd4(+) t cell epitopes in yellow fever vaccinees date: 2020-08-31 journal: front immunol doi: 10.3389/fimmu.2020.01836 sha: doc_id: 288496 cord_uid: 7rrh2gg6 examining cd8(+) and cd4(+) t cell responses after primary yellow fever vaccination in a cohort of 210 volunteers, we have identified and tetramer-validated 92 cd8(+) and 50 cd4(+) t cell epitopes, many inducing strong and prevalent (i.e., immunodominant) t cell responses. restricted by 40 and 14 hla-class i and ii allotypes, respectively, these responses have wide population coverage and might be of considerable academic, diagnostic and therapeutic interest. the broad coverage of epitopes and hla overcame the otherwise confounding effects of hla diversity and non-hla background providing the first evidence of t cell immunodomination in humans. also, double-staining of cd4(+) t cells with tetramers representing the same hla-binding core, albeit with different flanking regions, demonstrated an extensive diversification of the specificities of many cd4(+) t cell responses. we suggest that this could reduce the risk of pathogen escape, and that multi-tetramer staining is required to reveal the true magnitude and diversity of cd4(+) t cell responses. our t cell epitope discovery approach uses a combination of (1) overlapping peptides representing the entire yellow fever virus proteome to search for peptides containing cd4(+) and/or cd8(+) t cell epitopes, (2) predictors of peptide-hla binding to suggest epitopes and their restricting hla allotypes, (3) generation of peptide-hla tetramers to identify t cell epitopes, and (4) analysis of ex vivo t cell responses to validate the same. this approach is systematic, exhaustive, and can be done in any individual of any hla haplotype. it is all-inclusive in the sense that it includes all protein antigens and peptide epitopes, and encompasses both cd4(+) and cd8(+) t cell epitopes. it is efficient and, importantly, reduces the false discovery rate. the unbiased nature of the t cell epitope discovery approach presented here should support the refinement of future peptide-hla class i and ii predictors and tetramer technologies, which eventually should cover all hla class i and ii isotypes. we believe that future investigations of emerging pathogens (e.g., sars-cov-2) should include population-wide t cell epitope discovery using blood samples from patients, convalescents and/or long-term survivors, who might all hold important information on t cell epitopes and responses. examining cd8 + and cd4 + t cell responses after primary yellow fever vaccination in a cohort of 210 volunteers, we have identified and tetramer-validated 92 cd8 + and 50 cd4 + t cell epitopes, many inducing strong and prevalent (i.e., immunodominant) t cell responses. restricted by 40 and 14 hla-class i and ii allotypes, respectively, these responses have wide population coverage and might be of considerable academic, diagnostic and therapeutic interest. the broad coverage of epitopes and hla overcame the otherwise confounding effects of hla diversity and non-hla background providing the first evidence of t cell immunodomination in humans. also, double-staining of cd4 + t cells with tetramers representing the same hla-binding core, albeit with different flanking regions, demonstrated an extensive diversification of the specificities of many cd4 + t cell responses. we suggest that this could reduce the risk of pathogen escape, and that multi-tetramer staining is required to reveal the true magnitude and diversity of cd4 + t cell responses. our t cell epitope discovery approach uses a combination of (1) overlapping peptides representing the entire yellow fever virus proteome to search for peptides containing cd4 + and/or cd8 + t cell epitopes, (2) predictors of peptide-hla binding to suggest epitopes and their restricting hla allotypes, (3) generation of peptide-hla tetramers to identify t cell epitopes, and (4) analysis of ex vivo t cell responses to validate the same. this approach is systematic, exhaustive, and can be done in any individual of any hla haplotype. it is all-inclusive in the sense that it includes all protein antigens and peptide epitopes, and encompasses both cd4 + and cd8 + t cell epitopes. it is efficient and, importantly, reduces the false discovery rate. the unbiased nature of the t cell epitope discovery approach presented here should support the refinement of future peptide-hla class i and ii predictors and tetramer technologies, which eventually should cover all hla class i and ii isotypes. we believe that future investigations of emerging the immune system attempts to protect its host against invading pathogens; yet, it can also cause serious pathology. the ability to discriminate between foreign and self is key to exerting immune protection without inflicting immune pathology. immune recognition is therefore of immense interest and efficient methods to identify and validate immune epitopes are a high priority. in this context, t cells, which effectively orchestrate the overall immune response, are of particular interest. t cells are specific for compound ligands consisting of peptides, generated intracellularly by proteolytic degradation of protein antigens, which are presented in the context of major histocompatibility complex (mhc) [or human leucocyte antigens (hla)] molecules on the surface of antigen presenting cells (apc) (1) . the interaction between peptide and hla is specific; the resulting hla-mediated t cell epitope selection process being greatly diversified by the polygenic and polymorphic nature of the hla. this significantly affects the peptide-binding specificity of the set of hla molecules that are available to any given host; something that effectively individualizes our immune responses. although other events are also involved in antigen processing and presentation, the single most selective event is that of peptide-hla binding. it is estimated that ca. 0.5% of all possible peptide-hla combinations are of a sufficiently high affinity that they potentially, but not necessarily, could be immunogenic (2) . major efforts have been devoted to understand, quantitate and preferably predict peptide-hla binding as a means to identify t cell epitopes. proposed in 1999, the "human mhc project" aims at mapping all human mhc (or hla) specificities (3, 4) . established in 2004, the "immune epitope database" (iedb) has become an authoritative repository of hla binding peptides and t cell epitopes, and of methods to predict these (5) . the recent breakthrough in cancer immunotherapy has reinforced the interest in fast and efficient methods to identify t cell epitopes with special emphasis on identifying immunogenic neoepitopes for personalized cancer immunotherapy. thus, several recent international research efforts, such as the "human immunopeptidome project and consortium, " "tumor neoantigen selection alliance" and others, have focused on t cell epitope discovery. employing recent advances in mass spectrometry to perform large-scale identification of peptides eluted of hla molecules, these efforts promise to identify natural ligands thereby capturing information on both antigen processing and hla binding (6) . over the past decades, substantial progress has been made on predicting peptide-hla interactions, particularly for hla class i (hla-i), which restricts cd8 + cytotoxic t cells (ctl's), and to a lesser degree on predictions for hla class ii (hla-ii), which restricts cd4 + helper t cells (th) (7) (8) (9) (10) (11) (12) (13) (14) (15) . state-of-theart predictors such as netmhcpan, an artificial neural network method based on a large collection of experimental peptide-hla-i binding data, can successfully identify 96.5% of cd8 + t cell epitopes, while rejecting 98.5% of non-epitopes (16) . however, considering that only 1 of 2,000 (2) to 8,000 (17) random peptides is a t cell immunogen in the context of a given hla molecule, even a rejection rate as high as 98.5% translates into a high false discovery rate (fdr) (8, 10, 11, 18) . this is a general problem of current peptide-hla binding predictors (10, 11) , and it is particularly problematic when trying to develop a neoepitope-specific, personalized cancer immunotherapy where timely delivery of a few unique cancer neoepitopes is of paramount importance; something that potentially could be achieved with even better predictors (8, (19) (20) (21) . yellow fever virus (yfv) is a mosquito-borne flavivirus (i.e., a ssrna virus) (22, 23) . it remains an important human pathogen despite the existence of an effective live attenuated vaccine (24) . particularly relevant to this study, previous analyses of the cd8 + t cell response against a limited number of epitopes have revealed that vaccination with this live vaccine represents an excellent model for studying the host response to a viral infection (25, 26) . the main advantages are that the precise time and the exact identity of the immune challenge are both known [note that the vaccine strain used here is known to be stable (27) ]; issues that otherwise might complicate the interpretation of immune responses observed in patients that are naturally infected with a variable pathogen. here, we have generated a comprehensive, population-wide t cell epitope discovery approach with a much-reduced fdr, and used it to identify and validate immunodominant cd8 + and cd4 + t cell epitopes in a cohort of 210 hla-typed, primary yfv vaccinees. this involves using a "forward (or direct) immunology" approach, where you start with a specific t cell response of interest and then search for the epitope(s) being recognized (28, 29) , to perform an initial identification of t cell stimulatory peptides. subsequently, a "reverse immunology" approach, where you start by predicting possible t cell epitopes and then search for a t cell response of the corresponding specificity (30, 31) was used, to perform a final identification and validation of the underlying specific t cell epitopes and their hla restriction elements. from here on, this approach is denoted as a "hybrid forward-reverse immunology" (hfri) approach. briefly, in the "forward immunology" step, pbmc's obtained 2-3 weeks after primary yfv vaccination were ex vivo stimulated with an overlapping peptide library representing the entire 3,411 amino acid yfv proteome and tested by an ifnγ-specific intracellular cytokine secretion (ics) assay thereby identifying cd8 + and cd4 + t cell stimulatory yfv-derived peptides. in the subsequent "reverse immunology" step, predictors were used to select appropriate peptide-hla combinations for the generation of peptide-hla tetramers, which then were used to identify and validate the underlying t cell epitopes and their hla restriction elements. applying this hfri approach to t cell epitope discovery in 50 yfv vaccinees, we identified and tetramervalidated 92 cd8 + and 50 cd4 + t cell epitopes covering 40 hla-i and 14 hla-ii allotypes, respectively (note that he tetramer-validation step could not be performed exhaustively for the cd4 + t cell epitope discovery process and that the true number of cd4 + t cell epitopes probably was many times larger than the 50 validated cd4 + t cell epitopes reported here). with a cohort of 210 yfv vaccinees, the prevalence of responses against the cd8 + t cell epitopes could be examined. about a third (31%) of these epitopes were recognized in >90% of the individuals expressing the hla-i in question. by this token, they could be considered strongly immunodominant. we conclude that t cell epitope discovery using this hfri approach is highly efficient, in particular when examining larger populations responding to the same pathogen (e.g., an infectious pathogen e.g., sars, ebola, zika, sars-cov-2). furthermore, we suggest that the hfri approach is unbiased and that the resulting t cell epitopes should serve as a valuable benchmark for future improvements of predictive algorithms of immunogenicity. primary vaccination with the attenuated yfv vaccine, 17d-204, is known to trigger a prompt and vigorous cellular immune reaction (25, 26) . here, 210 vaccinees were recruited, and peripheral blood mononuclear cells (pbmc) were prepared from 50-to 200-ml blood samples obtained before and ca. 2 weeks after primary vaccination, respectively (26) . the typical yield from the latter was ca. 450 million pbmc. all vaccinees were hla typed at high-resolution (i.e., 4 digit) including all nine classical, polymorphic hla loci (i.e., hla-a, b, c, drb1, drb3/4/5, dqa1, dqb1, dpa1, and dpb1) (26) . the 17d-204 vaccine encodes a single polyprotein precursor of 3,411 amino acids (aa), which is processed into 15 proteins. the full genome (genbank accession# x15062) and proteome (swiss-prot accession# p03314) sequences of the 17d-204 have been determined (32) . a library of 850 overlapping 15 mer peptides overlapping by 11 aa, spanning the entire yfv precursor protein (essentially the yfv proteome), was generated. additionally, 50 peptides representing potentially aberrant yfv translation products were selected. of the resulting 900 peptides, synthesis failed for 30 peptides (3%) leaving 870 peptides for analysis. since testing each of these peptides individually would exhaust the available pbmc's, the peptides were tested in pools. initially, the peptides were organized into a single 30×30 matrix from which 30 "column pools" and 30 "row pools" were generated leading to a total of 60 pools each containing ca. 30 different peptides. each peptide would be present in two pools: one column and one row pool (supplementary figure s1a) . the intersections of stimulatory column and row pools should ideally identify which peptide might be immunogenic and therefore should be further investigated on an individual basis. this 30×30 matrix strategy was initially tested using an exvivo ifnγ elispot assay as readout. after the first 94 primary vaccinated donors had been recruited, the average number of positive column/row intersections was found to be 418 (range 26-870) ( figure 1a) suggesting that the hit rate from pools containing 30 peptides was too high, at least in the setting of this acute viral response, to be effective in eliminating nonstimulatory peptides from further consideration. to reduce the hit rate per peptide pool, the peptides were re-organized into four smaller matrices, three 15×15 matrices and one 14×15. for each matrix, 14 to 15 column pools and 15 row pools were generated leading to a total of 119 pools, which each contained 14 to 15 different peptides (supplementary figure s1b) . to further reduce the number of relevant intersections, the ifnγ elispot assay was replaced by an ifnγ intracellular cytokine staining (ics) assay, which can discriminate between cd8 + and cd4 + t cells and therefore eliminate intersections with mismatched cd8 + and cd4 + t cell responses. furthermore, to increase the number of t cells available for the ics assay, pbmc were expanded in four separate in vitro cultures containing a pool of ca. 225 peptides corresponding to each of the four matrices, respectively (the potential bias introduced by this in vitro culture is discussed in supplementary results and discussion). after 8 days, each matrix-expanded pbmc culture was tested against the appropriate row and column pools using ifnγ ics as readout. for comparison, 36 donors, which had already been analyzed using the 30×30-matrix, elispot-based screening strategy, were re-screened using the 4×(15×15)-matrix, icsbased screening strategy (figures 1b-d) . the aggregated cd4 + and cd8 + t cell responses were calculated for ics responses (denoted "all t cells" in figure 1 ) and compared to those from elispot responses. the total number of intersections needing deconvolution was significantly lower for the 4×(15×15 ics strategy [average intersections 253 (range 20-589)] than for the 30×30 elispot strategy [average 418 (range 26-870), (p < 0.0001, n = 36, mann-whitney u-test), figures 1a,b] . the 253 intersections, which on average were detected by the ics-based screening strategy, could further be broken down into an average of 80 (range 2-374) ( figure 1c ) and 197 (range 7-514) ( figure 1d ) intersections representing cd8 + and cd4 + t cell responses, respectively (30 of these intersections were shared). the peptides corresponding to these intersections were subsequently tested individually to identify which of the intersections truly represented cd8 + and/or cd4 + t responses. figure 1 | comparing the number of peptide-containing peptides identified by the two different approaches. t cells obtained ex vivo from primary yfv vaccinees were stimulated with matrix-derived pools of yfv peptides and responses were read by ifnγ-specific elispot or ics. the peptides were distributed into matrixes, column and row pools of peptides were generated, and used to test t cell stimulation. intersections of stimulatory column and row pools putatively identified single stimulatory peptides for further analysis (supplementary figure s3) . (a) peptides were distributed into one 30×30 matrix generating 30 + 30 = 60 pools, which were used to stimulate t cell responses in 94 donors using an ifnγ-specific elispot assay as readout of all (i.e., cd4 + and cd8 + ) t cell responses [average 418 positive intersections (range ]. (b,c) peptides were distributed into four ca. 15×15 matrices generating 4x(ca. 15 + 15) ca. 120 pools, which were used to stimulate t cell responses in 36 donors using an ifnγ-specific ics assay as readout of (b) all t cell responses [average 253 intersections (range 20-589)], (c) cd8 + t cell responses [average 80 intersections (range 2-374), and (d] cd4 + t cell responses average 197 intersections (range 7-514). the symbols representing the 36 donors that were examined by both elispot and ics have been shaded. mann whitney u test was used to determine the significance of the difference between the indicated groups (***p < 0.0001). the complete screening and validation procedure is illustrated using donor yf1067. the blood sample for donor yf1067 was collected at day 16 post vaccination, which is within the time span of optimal post vaccination yfv responses (25, 26) . it gave a relatively high yield of 700 × 10 6 pbmc's for the subsequent epitope discovery effort. donor yf1067 was initially analyzed by the 30×30-matrix, ifnγ elispot-based screening strategy where 690 positive intersections were identified. a total (i.e., cumulative) yfv-specific response of 8000 sfu were obtained suggesting a t cell response of considerable breath and magnitude. re-analyzing this donor using the four-matrix, ics-based screening strategy, the number of intersections for figure 2 | t cell epitope screening strategy. (a) identification of stimulatory 15mer peptides: pbmc's from yfv vaccinated donors were divided into four cultures and in vitro stimulated with peptide sublibraries corresponding to each of the four 15×15 peptide matrices. after 8 days, each sublibrary expanded pbmc culture was tested by ics against the matrix-specific row and column peptide pools. subsequently, individual peptides representing stimulatory matrix intersections were analyzed to identify single t cell stimulatory 15-mer peptides. (b) identification of t cell epitopes and their hla restriction elements: cd4 + t cell epitope deconvolution: single cd4 + t cell stimulatory 15mer peptides were tested for binding to the donor's hla-dr molecules using a biochemical hla class ii binding assay, positive interactions were used to generate peptide-hla class ii tetramers, and these tetramers were used to stain expanded t cells, and the resulting epitopes were eventually validated by ex vivo elispot analysis. cd8 + t cell epitope deconvolution: single cd8 + t cell stimulatory 15mer peptides were submitted to the netmhcpan 2.4 predictor together with the donor's hla class i haplotype to identify optimal epitopes and their hla-restriction elements. these optimal epitopes were subsequently synthesized and validated by ex vivo peptide-hla class i tetramer staining. follow-up analysis could be reduced to 253; 78 representing cd8 + t cell responses and 218 representing cd4 + t cell responses (43 of the intersections contained both cd4 + and cd8 + t cell responses). peptides corresponding to the 253 intersections were tested individually by ics; this identified 27 and 31 cd8 + and cd4 + t cell stimulatory 15mer peptides, respectively (for a detailed listing of these peptides, see tables 1,2 below). the next steps aimed at identifying the underlying cd8 + and cd4 + t cell epitope(s) and their hla restriction element(s), preferably by generating the corresponding tetramer(s), and validate the epitope(s). for a general outlined of this epitope discovery scheme, see figure 2 . identification and validation of cd8 + t cell epitopes exemplified by donor yf1067 the sequences of the 27 15mer peptides, which stimulated cd8 + t cell responses in donor yf1067, were submitted, along with the donor's hla-i allotypes (in casu hla-a * 02:01, -a * 32:01, -b * 07:02, -b * 40:01, -c * 03:04 and -c * 07:02), to our webserver netmhcpan (version 2.4 at http://www.cbs.dtu.dk/ services/netmhcpan-2.4 was available at the time of this analysis). in silico, this predictor considered all 26 submer peptides of 8-11mer length, which could possibly be generated from a 15mer peptide, and predicted their binding to all six hla-i allotypes of donor yf1067, a total of 6 × 26 = 156 submer-hla-i combinations per 15mer peptide, and returned a ranked list across all six hla-i allotypes of the most likely epitope(s) and their hla-i restriction element(s). for all 27 cd8 + t cell stimulatory peptides, this amounted to predicting the binding affinities of 27 × 156 = 4,212 submer-hla-i combinations. for each 15mer peptide, submers representing the top one to three predicted affinities were synthesized and the stabilities of the corresponding peptide-hla-i interactions were measured experimentally ( table 1) . fluorochrome-labeled tetramers corresponding to the most stable peptide-hla-i interactions were generated and used to label relevant cd8 + t cells. when available, surplus t cells from the initial expansion cultures were used as a first line of identification of cd8 + t cell epitopes and their restriction elements, however, ex vivo tests were always used for the final cd8 + t cell epitope validation, and for enumerating and characterizing epitope-specific cd8 + t cells. the matrix-identified cd8 + t cell stimulatory 15mer peptides and the corresponding tetramer-validated optimal the 27 cd8+ t cell stimulatory 15mer peptides are given including their sequences and ics stimulation values. the epitopes eventually identified are given in red. as shown, many epitopes were found in consecutive 15mer peptides. these 15mer peptides were submitted along with the donors hla-i haplotype to netmhcpan 2.4, which returned suggested epitopes and restriction elements. in most cases, the epitope was found as a top ranking prediction. the stabilities of the suggested peptide-hla-i complexes were measured and the corresponding tetramers generated. finally, these tetramers were used to validate the cd8+ t cell epitopes and to enumerate the responding cd8+ t cells ex vivo. cd8 + t cell epitopes and their restriction elements are listed ( table 1) . for each of the 27 cd8 + t cell stimulatory 15mer peptides identified in donor yf1067, one or more cd8 + t cell epitopes and their hla-i restriction elements were identified. some of the epitopes were present in two consecutive overlapping 15mer peptides and should therefore only be counted as epitopes once. with this in mind, 19 unique cd8 + t cell epitopes were recognized by donor yf1067 (7 epitopes restricted by hla-a * 02:01, 2 by hla-a * 32:01, 4 by hla-b * 07:02, 6 by hla-b * 40:01, and none by hla-c * 03:04 or -c * 07:02) ( table 1) . cd8 + t cell specific for the 19 unique yfv-derived epitopes were readily detectable and enumerable ex vivo during the acute primary response of donor yf1067. the frequencies of total, as well as activated, tetramer-positive cd8 + t cells were determined ( table 1) . the most frequently and immunodominant epitope of all, the hla-a * 02:01-restricted ns4b 214−222 epitope, was recognized by 4.6% of cd8 + t cells in donor yf1067. the frequencies of cd8 t cells recognizing each of the other 18 epitopes ranged from 0.03 to 0.9%; the total frequency of cd8 + t cells recognizing the 19 yfv epitopes was ca. 8%. the yfv vaccine induced a measurable increase in the overall frequency of activated cd8 + t cells (i.e., cd38 + hla-dr + cd8 + t cells) (26) . in donor yf1067, the yfv vaccine induced an increase in activated cd8 + t cell from 0.6% preto 7% post-vaccination. notably, the hla-a * 02:01-restricted ns4b 214−222 -epitope was recognized by 41% of the activated cd8 + t cells in donor yf1067. the frequencies of activated cd8 + t cells recognizing each of the other 18 epitopes ranged from 0.1 to 4.1%. in total, the 19 identified yfv-specific cd8 + t cell epitopes accounted for the majority (ca. 60%) of activated cd8 + t cells observed during the acute response following primary yfv vaccination. the cd8 + t cell epitope discovery strategy described above for donor yf1067 was extended to 50 randomly selected donors, who were sampled at day 12-21 after vaccination i.e., at the peak of a primary anti-yf vaccine response (25, 26) . cd8 + t cell responses specific for 120 different peptide hla-i combinations were identified and validated by ex vivo tetramer staining (for an overview, see figure 3 , and for details, see supplementary table si) . this represented 92 different cd8 + t cell epitopes restricted by 40 different hla-i molecules; 68, 20 and 4 epitopes were restricted by 1, 2, and 3 different hla-i molecules, respectively. the hla-a, -b and -c allotypes covered by the 50 donors were, respectively 19, 30, and 20 of which the majority, 15, 27, and 16, were available to us for tetramer validation. thirteen of the 15 different hla-a allotypes tested served as restriction elements of 38 different cd8 + t cell peptide epitopes leading to the presentation of 44 immunogenic peptide-hla-a combinations; 26 of the 27 different hla-b allotypes tested served as restriction elements of 56 different epitopes leading to the presentation of 74 immunogenic peptide-hla-b combinations; whereas only one of the 16 different hla-c allotypes tested served as restriction elements of 2 different epitopes leading to the presentation of 2 immunogenic peptide-hla-c combinations. the average number of cd8 + t cell epitopes identified per hla-a and -b allotype, 3.4 and 2.8, respectively, were not significantly different [p > 50%, fishers exact test, two-tailed (graphpad)]. to the best of our knowledge, 84 of the 92 yf-specific cd8 + t cell epitopes, and 110 of the 120 epitope-hla-i combinations reported here and in previous publications (26, 33) , were first identified as a result of this hfri project. for the previously reported epitopes or epitope-hla-i combinations, minor adjustments of the already available information could be made: some had not been tetramer validated before, and others were also found to be restricted by other, albeit closely related, hla-i allotypes than those previously reported. in a few cases, tetramers representing the exact epitope-hla-i combinations previously reported failed to label cd8 + t cells in our donors despite expressing the appropriate hla-i allotype (for details see supplementary table si) . our in-house peptide repository included 533 yfv-derived peptides from previous hla mapping efforts (34) . using the contemporary netmhcpan2.4 at %rank cut-off of 0.5% to select putative binders from this repository, we generated 90 additional peptide-hla-i tetramers (i.e., tetramers that had not already been prepared in the course of the present hfri approach). we included these tetramers in the immunodominance analysis described below. nine additional peptide-hla-i combinations, which had not been observed previously, were identified; four representing previously identified epitope presented by an alternative hla-i restriction element, and five representing new yfv-specific cd8 + t cell epitopes (supplementary table sii) . thus, the total number of cd8 + t cell epitopes discovered and tetramer validated here was 97 of which 92 (or 95%) were identified by the hfri approach. we systematically extended the analysis of ex vivo responses to additional donors expressing relevant hla-i restriction elements and evaluated them in terms of prevalence (the frequency of responders in donors with the hla-i restriction element in question) and response magnitude (the average ex vivo frequency of tetramer positive, activated cd8 + t cells of the responding donors) supplementary tables si, sii. to allow for a reasonable assessment of prevalence, the final analysis included epitopes restricted by hla-i molecules represented by at least 5 donors, who had donated blood samples 12-21 days after vaccination. this involved a total of 98 peptide-hla combination representing 81 epitopes presented by 24 hla-i allotypes (figure 4) . immunodominance was frequently observed. from an epitope point of view, 25 (or 31%) of the 81 epitopes had a prevalence of ≥90% and a median magnitude >0.03%, and 50 (or 62%) had a prevalence of ≥50% and a median magnitude >0.02%. from an hla point of view, 16 (or 67%) of the 24 hla-i molecules presented at least one epitope with ≥90% prevalence, and all 24 hla-i molecules presented at least one epitope with at least 50% prevalence. in terms of hla-i coverage and immunodominance, the vast majority of our cohort, 97, 79, and 43%, carried at least one, two or three hla-i allotypes, respectively, which presented at least one epitope with ≥90% prevalence. a selection of 10 immunodominant epitopes representing the most frequent hla-a and -b allotypes would cover 95% of the caucasian population. the "all cd8" bar indicates the positions of each cd8 + t cell stimulatory 15mer peptide relative to the yfv polyprotein. each 15mer is shown as a frame that horizontally indicates the starting and ending positions of the 15mer peptide, vertically indicates the number of donors, of the 50 donors tested, who responded to the peptide, and the color-coded is according to the polyprotein coloring scheme (to enhance the visualization of overlapping peptide sequences, this coloring is translucent). the lower hla-i allotype-designated bars indicate the tetramer-validated epitopes and their hla-i restriction elements (e.g., a*01:01 is shorthand for hla-a*01:01). again, the frame horizontally indicates the starting and ending positions of the epitopes; however, for visual clarity, all frames have the same vertical dimension. the details of each epitop (epitope sequence, cd8+ t cell stimulation, ex vivo tetramer staining frequency, and response prevalence is given in supplementary table si) . figure 4 | prevalence and magnitude of cd8 + t cell responses. to determine the prevalence and the median magnitude of the cd8 + t cell responses toward the epitopes discovered by the hfri approach in 50 donors were extended to additional donors expressing the relevant hla-i restriction elements. only donors sampled 12-21 days after vaccination were included. the prevalence (gray columns) and the median magnitude of the responses (black diamond) were determined for each epitope-hla combination. only epitope-hla combinations analyzed in 5 or more donors were included. the epitopes are organized according to restriction elements. the top figure shows the hla-a restriction elements; the bottom figure shows the hla-b and -c restriction elements. it has been suggested that immunodominant epitopes can curtail responses to other epitopes (reviewed in 35). the hla-a * 02:01 restricted, yfv ns4b 214−222 -epitope may represent a unique opportunity to address this in an outbred human population: it represents an exquisitely dominant cd8 + t cell response as all 93 hla-a * 02:01-positive donors examined here responded to this epitope and an average of 29% of all activated cd8 + t cells from ex vivo blood samples obtained 2-3 weeks after yfv vaccination were specific for this epitope. it has recently been suggested that this massive response can be explained by the invariant cdr1α loop of trav12-2 taking part in the recognition of this epitope (35) . in donors, who had donated blood samples at the peak of the response (12-21 days after vaccination), we examined whether the presence of hla-a * 02:01, -a * 01:01, or -a * 03:01, could be correlated to the strength of cd8 + t cell responses restricted by other restriction elements, in casu all available hla-b allotypes. we included 142 donors, which, respectively, could be split into 71 and 71 hla-a * 02:01 positives and negatives, 39 and 103 hla-a * 01:01 positives and negatives, or 30 and 112 hla-a * 03:01 positives and negatives; and used tetramers to examine the ex vivo frequencies of up to 43 different hla-b-restricted responses. in the presence or absence of each of the three hla-a restriction elements, the average frequencies of each of the hla-b-restricted responses were determined leading to the generation of up to 43 matched-pairs per hla-a. the frequencies, or magnitude, of the hla-b-restricted responses were significantly reduced in the presence vs. absence of hla-a * 02:01 (median reduction of 0.0432%, p < 0.0001). in contrast, in the presence vs. absence of hla-a * 01:01, which have lesser immunodominant cd8 + t cell responses, there was a smaller and not significant reduction (median reduction of 0.0165%, p = 0.1353); in the presence vs. absence of hla-a * 03:01, which have even fewer immunodominant cd8 + t cell responses, there was a very small and non-significant increase (median increase of 0.0041%, p = 0.51) (wilcoxon signed-rank test, figure 5 ). we suggest that this may be the first demonstration of immunodomination in an outbred human population. the length of the 97 discovered cd8 + t cell epitopes ranged from 8 to 11mers with a predominance of 9mers (67 (69%) 9mers, 18 (19%) 10mers, 5 (5%) 11mers, and 7 (7%) 8mers) (supplementary figure s2) . this matches well with available data for peptides eluted of hla-a and -b molecules (33) . some of the epitopes were size variants of the same peptide sequence. in six cases, such size variants were presented by the same hla-i restriction element (four cases involving two size variants each and two cases involving three size variants each, supplementary table si). we reasoned that cd8 + t cell recognition of these identically restricted size variants could either involve cross-recognition of shared epitope structure(s) by the same tcr(s), or involve recognition of genuinely different epitope structures by different tcr(s). to evaluate this, tetramers of the different epitope size variants and the relevant hla restriction elements were produced with unique fluorochrome labels and used to determine whether the epitope size variants were recognized by the same or different t cell populations. in some cases, distinctly defined and shared subpopulations were observed (figures 6a,d,i,j) indicating that one or more unique shared epitope structures were presented and recognized; in other cases, we observed shared subpopulations merging with populations that were single-stained with one of the lengthvariant tetramers (figures 6c,g,h,i) ; and finally, in some cases, no shared subpopulations were observed suggesting that the corresponding length-variants were presented and recognized as being distinctly different (figures 6b,e,f) . accommodating length-variants by extending the peptide-binding groove or by one or more aa's bulging out of the groove (34) could affect the presented epitopes dramatically, whereas accommodating length-variants by protruding out of the n-or c-terminal ends of the groove could leave the non-protruding end of the epitope unaltered. elucidating the structural basis of these various recognition modes is beyond the scope of this paper. one of the more frequent hla allotypes, hla-b * 07:02, offered an opportunity to compare the hfri approach with a strictly reverse immunology approach. theoretically, a total of 13,610 peptides of 8-11mer size could be generated from the yfv proteome. netmhcpan 2.4 predicted 54 of these as being strong hla-b * 07:02 binders at a %rank of <0.5%. we selected 40 of those for further examination (supplementary table siii) . with one exception, all of these predicted binders supported hla-b * 07:02 tetramer generation, which subsequently were used to examine ex vivo obtained pbmc's from at least 16 hla-b * 07:02 + donors. apart from the epitopes that had already been described (supplementary tables si, sii) , no additional hla-b * 07:02-restricted cd8 + t cell epitopes were identified. thus, a final count can be made: combining the hfri and a strictly reverse immunology approach, a total of 10 unique hla-b * 07:02-restricted cd8 + t cell epitopes were found; the hfri strategy identified nine of these, whereas the reverse immunology strategy identified eight; seven (70%) of these epitopes were shared. assuming that the number of true positive hla-b * 07:02 epitopes is ten, then both strategies were sensitive (correctly identifying 80-90% of the 10 epitopes) and at the same time very specific (correctly rejecting 99.6% of the 13,600 non-epitopes); the hfri approach being slightly more sensitive and specific than the reverse immunology approach. the major performance difference between the two strategies arose from the lower false discovery rate (fdr) where the hfri screening strategy required 13 peptides to identify nine of the ten epitopes found (a fdr of 17%; albeit some of these apparently false positive peptides were eventually identified as epitopes restricted by other hla-i restricting elements expressed by the donors suggesting that the true false discovery rate of the hfri approach was even smaller), whereas the reverse immunology approach required 54 peptides to identify eight of the ten epitopes suggesting a false discovery rate of 85%. in conclusion, the present hfri approach ranks epitope at the very top of the list of candidates while decimating the false discovery rate (further comparisons of hfri vs. reverse immunology is described in the discussion and detailed in supplementary results and discussion). the unbiased nature of our cohort of 120 different hfriidentified peptide hla-i combinations covering 40 hla-i restriction elements provided an opportunity to evaluate the performance and discriminatory power of various prediction methods such as the authoritative netmhcpan [both the contemporary version 2.4 (36) and the most recent version 4.0 (37) trained on both eluted ligands (el) and peptide binding affinity (ba)], and the recent mhcflurry (38) (trained either only on ba data or on both el and ba data) (38) and mixmhcpred (trained only on el data) (39) . in addition to these peptide-hla-i affinity predictors, we also included a stability predictor, netmhcstabpan 1.0 (40) . for each of these methods, predictions scores of all 13,610 peptides of length 8-11 aa that could be generated from the 3,411 aa yfv proteome were predicted for the relevant hlas (using %rank scores allowing comparisons across hla allotypes and predictors as read-outs). subsequently, a receiver operating characteristics (roc) analysis was performed and the area under the curve (auc) was determined. a non-discriminatory predictor has an auc of 0.5, whereas a perfectly discriminating predictor has an auc of 1.0. applied to this unbiased and validated set of epitopes, all of these predictors gave highly discriminatory auc's of 0.98743 to 0.99797 (supplementary figure s3) . these impressive auc's are heavily influenced by the many nonimmunogenic peptides being correctly rejected; however, this may still leave considerable room for false positive discovery rates (fdr). in this case, a more fdr-averse way to visualize the performance is to use the frank score, which is the number of false positive predictions (fp) relative to the total number of peptides (n) that can be generated from the source protein (i.e., frank = fp/n). a frank score of 0 indicates a "perfect prediction" where a true epitope receives the highest prediction value of all peptides within the source protein and avoids any false positive predictions, whereas a frank score of 0.5 indicates a random prediction where half of the predictions are false positives. frank values were calculated for each epitope-hla pair and predictor (supplementary figure s2) . the best predictors were netmhcpan 4.0 el and mixmhcpred, which, respectively, scored 21 and 20 "perfect" predictions, obtained an average frank score of 0.001875 and 0.003809, and a median frank score of 0.000405 and 0.000588, respectively. the median, being a more "outlier-resistant" measure, would, respectively, indicate that the netmhcpan 4.0 el and mixmhcpred methods would place 6 and 8 false-positive non-epitopes ahead of each epitope, corresponding to a false discovery rate of 85 and 89%. these numbers should be appreciated in the context of a random predictor, which would yield a fdr of 99%, and a perfect predictor which would yield an fdr of ca. 50% [assuming that only 50% of hla-presented peptides are immunogenic (2)]. in line with earlier work (41) , comparing the predictive power of the various predictors in terms of the frank values, netmhcpan 4.0 el was found to significantly outperform all other predictors (p < 0.02 in all cases, wilcoxon matched-pairs signed rank test) (supplementary figure s2) . identification and validation of cd4 + t cell epitopes (exemplified by donor yf1067) in donor yf1067, the ics-based screening analysis identified 31 15mer peptides as stimulating cd4 + t cell responses. at face value, these 15mer peptide sequences qualified as cd4 + t cell epitopes (the iedb epitope curation manual 2.0 defines a cd4 + t cell epitope of 15 residues or less in length as an "exact epitope"). to identify the underlying hla class ii restriction elements, the binding of each of the 31 15mer peptides to each of the hla-dr molecules of donor yf1067 (in casu hla-drb1 * 13:02, -drb1 * 15:01, -drb3 * 03:01 and -drb5 * 01:01) was tested in a biochemical binding affinity assay (42) . nine (28%), eleven (34%), four (13%) and four (13%) of the epitopes bound with an affinity better than 50 nm to one, two, three and four of the donor's hla-dr molecules, respectively ( table 2) , whereas three (9%) bound to none of them. secondly, we generated tetramers for 50 of the (9×1 + 11×2 + 4×3 + 4×4) = 59 strongly interacting peptide-hla combinations and used these to label in vitro expanded cd4 + t cells from donor yf1067. twenty-two of the 50 tetramers successfully identified cd4 + t cell epitopes and their hla-dr-restriction elements (figure 7) . the final validation and enumeration of specific cd4 + t cell was performed by an ex vivo ifnγ elispot analysis ( table 2) . in one case, the same epitope was presented by two different hla-drb allotypes and should therefore only be counted as epitope once. thus, 21 of the 31 different hla-dr-restricted cd4 + t cell epitopes observed in donor yf1067 were identified at the tetramer level; the remaining eleven epitopes were not resolved. the latter could potentially be explained as being restricted by hla-dq or dp molecules; something that could not be readily addressed by our tetramer capabilities at the time; albeit, in one case, we successfully generated a ns4b 233−247 -dpa1 * 01:03-dpb1 * 04:01 tetramer and identified an hla-dprestricted epitope. although 19 of the 32 cd4 + t cell stimulatory peptides bound to more than one of the four hla-dr allotypes of donor yf1067, there was only one epitope that exploited more than one of the available hla-dr allotypes as restriction element: the ns5 551−565 epitope, which was recognized by cd4 + t cells in the context of both hla-drb1 * 15:01 and hla-drb5 * 01:01. that this was not a case of tcr cross-recognition was shown by double staining with the two tetramers showing two distinctly different cd4 + t cell populations recognizing the ns5 551−565 -epitope presented by either hla-drb1 * 15:01 or hla-drb5 * 01:01 (see section recognizing the same cd4+ t cell epitope presented by two to three different hla-dr allotypes below). thus, in donor yf1067, a total of 31 cd4 + t cell epitopes were identified; 22 of these could be hla-dr or -dp tetramer validated. the cd4 + t cell epitope discovery strategy was extended to the same 50 donors used for cd8 + t cell epitope discovery. a total of 192 cd4 + t cell stimulatory 15mer epitopes were identified (for an overview, see figure 8 "all cd4", and for details, see supplementary table siv) . some of these epitopes were frequently recognized. thus, the single most recognized cd4 + t cell epitope, enve 44−58 , was recognized in 22 (71%) of 31 tetramer-tested donors tested, and another 12 epitopes were recognized in 10-16 (32-52%) of 31 donors. however, most of the 192 epitopes were much less frequently recognized; in fact, 76 of the peptides were recognized in only one (3%) of the 31 donors. we suggest that the strongest and most immunodominant cd4 + t cell epitopes have been found. an important objective was to identify and validate the hla-dr restriction element(s) used to present these epitopes (for an overview, see figure 8 , and for details, see supplementary table sv) . we have evaluated the restriction elements for 74 of the 192 epitopes. for each epitope, the most likely hla-dr restricting element was selected based on its affinity to one or more of the hla-dr allotypes available to the donor. guidance was also obtained from which hla-dr allotypes were shared amongst the epitope-responding donors. in some cases, more than one strong binding hla-dr allotype and/or more than one shared hla-dr allotype were found highlighting that multiple hla-dr allotypes would have to be considered as potential restriction elements. in total, 152 peptide-hla-dr tetramers were generated and used to validate the cd4 + t cell epitopes. of these, 64 the 31 cd4 + t cell stimulatory 15mer peptides are given including their sequences and ics stimulation values. overlaps between two consecutive peptides are given in red. the binding affinity of the 15mer peptides to the four hla-drb1 allotypes of donor yf1067 were measured. tetramers corresponding to the strongest binders were generated. the resulting tetramers were used to stain and analyze expanded cd4 + t cells by flow cytometry gating on cd3 + cd4 + t cells. in 21 cases, staining with a hla-dr tetramer was demonstrated ( figure 7) . note, that no hla-dr-restricted cd4 + t cell responses were found for the ns4b (233-247) epitope, yafvgvmynlwkmkt. eventually, it was found to be a dpa1 * 01:03-dpb1 * 04:01 binder, the corresponding tetramer was generated, and cd4 + t cell staining could be demonstrated (figure 7) . finally, an ex vivo elispot assay was performed to validate the cd4+ t cell epitopes. tetramers were tested positive for cd4 + t cell staining in one or more donors. this covered 50 cd4 + t cell peptide epitopes restricted by 13 different hla-dr molecules (some epitopes were presented by more than one hla-dr allotype) and one hla-dp molecule. for 17 of the 50 epitopes, the hla-dr molecules available to us for tetramer generation did only partially cover the hla-dr molecules observed in one or more of the responding donors. as an example, the most frequently recognized epitope, enve 44−58 , was found in 23 donors (supplementary tables siv, sv) . using appropriate tetramers, two restriction elements, hla-drb1 * 03:01 and -drb3 * 03:01, were identified, however, four of the enve 44−58 responding donors expressed neither the drb1 * 03:01 nor the drb3 * 03:01. this suggested that one or more additional, not yet identified, restriction element(s) existed for this epitope; something that could apply to more of the 17 epitopes. for each peptide, a biochemical hla class ii binding assay was used to identify which of donor yf1067's hla-ii molecules could bind the peptide and therefore could serve as restriction elements. productively interacting peptide-hla-ii combinations were used to design and generate peptide-hla class ii tetramers. the resulting tetramers were used to stain and analyze expanded cd4 + t cells by flow cytometry gating on cd3 + t cells. note, that the selective hla class ii tetramer staining of cd4 + , not cd8 + , t cells is a demonstration of the specificity of the tetramer staining. the identities of the epitopes and their restricting hla-ii elements are indicated. some of the 15mer peptides could stimulate cd4 + t cell responses restricted by two or three different hla-dr restriction elements (supplementary table sv) . no donor happened to possess three appropriate hla-dr molecules, but some did possess two and could generate appropriate cd4 + t cell response restricted by both of these restriction elements. in these cases, staining cd4 + t cells with two uniquely labeled tetramers, representing either of the two restriction elements, allowed us to address whether the same epitope presented by two different restriction elements were recognized by the same, or by distinctly different, cd4 + t cells. (figures 9a-e) . the remaining three epitopes were presented by the closely related hla-dr allotypes (hla-drb1 * 13:01 and -drb1 * 13:02 (one amino acid difference, a v86g, a part of the peptide binding site interacting with p1 of the core sequence), ns3 57−71 , ns5 59−73 , and ns1 111−125 , showed various degrees of cross-recognition. the ns3 57−71 peptide presented by hla-drb1 * 13:01 and -drb1 * 13:02 is mostly recognized by separate t cell populations; only a small population recognized the peptide presented by both molecules (figure 9f ). for peptides, ns5 59−73 and ns1 111−125 about half of the t cells recognizing the peptides presented by hla-drb1 * 13:01 cross-recognized the peptides presented by hla-drb1 * 13:02, with none or a very small t cell population recognizing the peptides presented only by hla-drb1 * 13:02 (figures 9g,h) . we speculate that a peptide presented by two restricting hla-dr molecules with only a few polymorphic amino acid differences may be cross-recognized by some, but not necessarily all, cd4 + t cells of appropriate specificity, whereas, presentation by two restricting hla-dr molecules with many polymorphic amino acid differences are more likely to be recognized as being distinctly different. in 16 cases, two consecutive overlapping 15mer peptides stimulated cd4 + t cell responses restricted by the same hla-dr restriction element. if the two peptides of such an overlapping 15mer peptide pair were presented through two different core regions, one for each, then the two neighboring epitopes should be perceived as being distinctly different and should be recognized by two disparate cd4 + t cell populations. alternatively, if the two peptides were presented through the exact same core region, then the two neighboring epitopes could potentially be perceived as being identical and be recognized by the same cd4 + t cell populations. to examine this, cd4 + t cells were double-stained with hla-dr tetramers, which had been prepared with each of the overlapping peptides of a 15mer pair and labeled with a unique fluorochrome. we analyzed 10 such pairs and found a wide variety of staining patterns. in no case did two peptides of an overlapping pair engage two distinctly different cd4 + t cell populations; rather, in all cases observed, the two peptides engaged at least some shared cd4 + t cell populations suggesting usage of shared core regions. in most cases, a plethora of shared, yet subtly different, cd4 + t cell populations were observed (figure 10) . by way of examples, tetramers representing the overlapping hla-drb1 * 01:01-restricted 15mer peptides, capc 49−63 and capc 53−67 , revealed multiple distinct cd4 + t cell subpopulations, which recognized one, the other, or both tetramers at various efficiencies ( figure 10a) ; whereas tetramers representing the overlapping hla-drb1 * 01:01-restricted 15mer peptides, ns5 471−485 and ns5 475−489 , revealed almost exclusively cd4 + t cell subpopulations recognizing both tetramers, albeit clearly comprising multiple distinct subpopulations ( figure 10b) . we argue that this phenomenon increases and diversifies cd4 + t cell responses. apart from two small proteins, the 20 aa er anchor and the 164 aa prm proteins, all yfv proteins contained both cd8 + and cd4 + t cell epitopes. on average, the frequencies of cd8 + and cd4 + t cell epitopes were ca. 3 and 6 per 100 aa, respectively (table 3) . notably, the cd8 t cell epitopes, which have been tetramer mapped exhaustively, exhibited stretches of overlapping epitopes restricted by several different hla class i molecules: twelve stretches encompassing two epitopes, five stretches encompassing three epitopes, and three larger hot-spots areas encompassing four to six epitopes, many of which were presented by several different hla molecules. thus, the frequently recognized enve 200−240 sequence comprised six peptide epitopes and ten hla-restriction elements giving a total of twelve epitope-hla combinations (figure 11) . these three hot-spot regions accounted for about 15 of the 97 (15%) cd8 + t cell epitopes identified, and encompassed 15 hla-i restriction elements covering ca. 77% of the caucasian race. although the yf protein was generated as one long precursor polyprotein, no epitopes were found in any of the overlaps between the different processed proteins. no epitopes were found in any of the peptides representing products of alternative translation initiation codons. in order to understand the complexity of the human t-cell response to a circulating pathogen, and its potential impact on population dynamics of both pathogen and host, knowing a wide range of epitopes relevant for t-cell/pathogen interplay is essential. however, identifying the exact epitope sequence and the exact hla allotype involved in t cell recognition of a specific pathogen is a demanding challenge. over the years, a plethora of methods have been used to identify t cell epitopes. there are two major and principally different approaches of t cell epitope discovery. the "forward immunology" approach (28, 29) uses specific t cell responses as a starting point to search for the underlying t cell epitope and its mhc restriction, whereas the "reverse immunology" approach (30, 31) uses predictions (e.g., of peptide-mhc interactions) to suggest possible t cell epitopes and then screen them for their ability to stimulate specific t cell responses [reviewed in (9, 43, the frequencies of epitopes per 100 aa are also given. ]. the experimental procedures involved in both of these epitope discovery modes tend to involve slow, low throughput, cumbersome and expensive processes [e.g., expression cloning of antigen libraries and/or hla genes (28, [45] [46] [47] [48] , synthesis of peptide libraries etc.]. in contrast, the bioinformatics component of a reverse immunology approach offers a process that is fast, of high capacity and throughput, yet very easy and inexpensive; a process, which is well-suited to support systematic analyses of genomic and proteomic information (3, 4, 9, 30) . it is not surprising that reverse immunology has become the preferred approach to t cell discovery. the need for high speed and capacity is of obvious importance in emerging infectious diseases (including bioterrorism), and even more so in personalized cancer immunotherapy where fast and high-throughput methods are essential for the selection of relevant and safe cancer neoepitopes in real time. current peptide-mhc predictors are highly sensitive and specific [96.5 and 98.5%, respectively (16) ]. however, despite continued improvements of these predictors, the false discovery rate (fdr) is very high (8, 10, 18) ; something that compromises the successful inclusion of one, or preferably more, t cell epitopes in cancer immunotherapy even if these encompass up to 10-20 predicted epitopes (20, 21) . reducing the fdr while maintaining the sensitivity will be needed if reverse immunology in the future should fully support neoantigen discovery and secure timely, personalized immunotherapy of cancer (19) . indeed, most of the larger cd8 + and cd4 + t cell epitope submissions to the iedb have been identified by "reverse immunology." thus, sette and coworkers used "reverse immunology" to identify dengue virus-specific t cell epitopes and have, as of july 2019, contributed with the single largest submissions of cd8 + and cd4 + t cell epitopes (iedb reference id 1027503, 1031475, and 1031301). in contrast, the "forward immunology" approach has fallen relatively into disuse. an innovative approach pioneered by koelle and co-workers, which has resulted in larger iedb submissions of cd8 + and cd4 + t cell epitopes (e.g., iedb reference id 1021375), have used a "forward" component where co-transfecting panels of apc with cdna encoding antigen and hla class i or ii, each apc representing a single antigen and a single hla restriction element, were used to interrogate cd4 + and cd4 + t cell responses of virus infected donors (48) . the "forward" component of this approach identified intact immunogenic protein antigens and their restriction element(s); however, for the epitope discovery part of this work, the entire antigen was subjected to a "reverse" component predicting the epitope(s) and its hla restriction element(s). another innovative approach, tetramer guided epitope mapping (tgem), pioneered by kwok and james, which has resulted in large cd4 + t cell epitope submissions [iedb references id 1026930 (49) , 1013360, 1016040, and 1020783], have also used a "forward" component. longer overlapping peptides representing entire antigens were offered to single hla class ii molecules and the resulting peptide-hla class ii complexes were multimerized and the ensuing tetramers used to interrogate cd4 + t cell responses of appropriate donors. using shorter overlapping peptides suitable as cd8 + t cell epitopes, maeurer and coworkers established a tetramer-based approach for cd8 + t cell epitope discovery, which also resulted in larger iedb submissions [iedb id 1026840 (50) ]. this latter approach would obviously be very peptide intensive if every relevant peptide was to be tested in that way (e.g., the yellow fever proteome would require 13610 peptides to represent all possible 8-11mer peptides). here, we have generated a "hybrid forward-reverse immunology" (hfri) approach capable of doing concurrent cd8 + and cd4 + t cell epitope discovery and demonstrated that it can perform large-scale epitope discovery and at the same time decimate the false positive discovery rate. for the initial "forward immunology" screen, we used an overlapping peptide library of 850 15mer peptides overlapping by 11 aa, which represented the entire 3,411 aa yellow fever virus proteome, to stimulate pbmc's obtained ex vivo from primary yellow fever virus vaccinees at the peak of the resulting t cell response. since 15mer peptides are further processed during in vitro ics and/or elispot assays, this peptide library represented all possible yfv-specific cd4 + and cd8 + t cell epitopes of up to 12 aa in length; some, but not all, epitopes of 13-15 aa in length; and no epitopes of a length longer than 15 aa. distributing this peptide library into 4 matrices, the initial screening effort could be reduced to testing ca. 120 peptide pools for their ability to stimulate t cell responses preferably by ics analysis. the matrix design subsequently allowed us to home in on the individual t cell stimulatory peptides. following the "forward immunology" screening, a "reverse immunology" approach was applied to all the 15mer peptides containing cd8 + t cell epitopes. in silico, the affinities of all possible 8-11mer peptides that could be generated from the 15mer were predicted in the context of up to 6 different hla-a, -b and -c allotypes per individual. this reduced the number of potential peptide-hla-a, -b or -c combinations from 156 per stimulatory 15mer peptide to typically one to three combinations. the most likely peptide binders were synthesized and used to generate appropriate peptide-hla-i tetramer(s), which subsequently were used to validate cd8 + t cell epitope(s). for the vast majority of t cell stimulatory 15mer peptides, at least one epitope was identified per 15mer peptide. once the stimulatory 15mer peptides had been identified, predicting the exact epitope and its restriction element was a highly efficient process; typically, the epitopes ranked first, second or third amongst the many potential epitope-hla combinations. as a cost-saving measure, if the predictions clearly discriminated between the candidates, a consecutive process was applied whereby the top peptide(s) were synthesized and tested before any next tier peptides were synthesized and tested. this hfri approach was extended to 50 primary yfv vaccinees, where it identified and tetramer-validated 92 cd8 + t cell epitopes (predominantly of size 9-10mer, range 8-11mer) covering 40 hla-i allotypes (representing a total of 120 peptide-hla-i combinations). before this work, the iedb had registered ten yfv-specific cd8 + t cell epitopes as being "exact epitopes" (i.e., length from 7 to 11 aa) and restricted by an hla allotype defined at high (4-digit) resolution; however, none of them were tetramer validated. four of the ten already registered yfv-specific, cd8 + t cell epitopes were included in the 92 epitopes identified here. thus, the present approach identified and validated 92 -4 = 88 new, or ca. 90% of all currently known, yfv-specific, cd8 + t cell epitopes. the total number of exact cd8 + t cell epitopes with high resolution hla-i restriction, which are currently registered in the iedb is 2,612 of which 1,101 have been tetramer validated (extracted from the iedb, july 2019). thus, this study accounts for >8% of these tetramer-validated human cd8 + t cell epitopes. to evaluate the prevalence of the different yfv-specific cd8 + t cell immune responses, the tetramer analysis was extended to additional vaccinees with the appropriate hla-i allotypes. many epitopes were frequently observed (i.e., were highly prevalent) in vaccinees with the appropriate hla allotype. thus; 25 (ca. 31%) and 50 (ca. 62%) of 81 cd8 + t cell epitopes were observed in ≥90 and ≥50%, respectively, of vaccinees with the appropriate hla-i allele. conversely, 18 (ca. 75%) of 24 hla-i allotypes presented at least one cd8 + t cell epitope with a prevalence of ≥90%. thus, the hfri approach identified a cohort of immunodominant yellow fever-derived peptides, which could be of broad diagnostic and therapeutic interest. large-scale t cell epitope discovery could also address more fundamental issues in immunobiology. pertinent examples of phenomena that are poorly understood include the closely related immunodominance (that the immune response is focused on just a few of the many available determinants expressed by a pathogen) and immunodomination (that the immune response of one specificity can suppress the response of another specificity). not surprising, these phenomena are closely related to antigen processing and presentation including mhc and t cell repertoire (51) . the vast majority of experimental data on immunodominance and immunodomination emanates from studies involving inbreed mice. few studies in humans address immunodominance [e.g., (52) ]; to the best of our knowledge none involve immunodomination. the latter is particularly difficult to address in an outbreed system like the human where the extremely diverse hla creates context dependent effects that confounds attempts to address immunodomination. assuming that the context-dependent effects hla could even out in larger donor cohorts, we exploited the size of our study to ask whether the presence of hla-a * 02:01, which restricts a strongly immunodominant, ns4b 214−222 -specific t cell response, would correlate with a reduction of responses restricted by other hla allotypes. indeed, under these conditions, we could demonstrate such a correlation in the presence of hla-a * 02:01, but not in the presence of hla-a * 01:01 or -a * 03:01. note that the hla-a * 01:01 or -a * 03:01 allotypes themselves featured a hierarchy of immunodominant t cell responses i.e., they are valid hla restricting elements. this may be the first demonstration of primary anti-virus responses being subjected to immunodomination in humans. a further analysis of the mechanism of behind these phenomena is beyond the scope of this paper. hla-c restricted, cd8 + t cell epitopes were scarcely represented (<3%) in the iedb; something that potentially could be explained by hla-c being insufficiently investigated. a priori, we expected that the unbiased nature of our approach would reveal several hla-c restricted cd8 + t cell epitopes, however, we only found one case of a strong and highly prevalent cd8 + t cell response, which could not be explained by any of the hla-a or -b allotypes available to the responding donors. instead, a strongly predicted binding to a shared hla-c allotype amongst the responding donors suggested an hla-c * 06:02 restricted response. eventually, two hla-c * 06:02restricted epitope length variants; ns3 207−213 (trrflpqil) and ns3 208−213 (rrflpqil), were tetramer validated. these were the only hla-c restricted cd8 + t cell epitope identified; all other identified cd8 + t cell epitopes were validated as being either hla-a or -b restricted. hla-c is less polymorphic and is known to be expressed at a lower level than hla-a and -b (53) (54) (55) (56) ; something that has been correlated with reduced cytotoxic t lymphocyte responses (57, 58) . in the case of the hla-c * 06:02-restricted ns3 207−213 (trrflpqil) epitope identified here, any reduced expression level of hla-c * 06:02 might have been compensated by the very strong predicted binding affinity for ns3 207−213 . although weaker hla-c-restricted cd8 + t cell responses may have been missed, we would argue that it is unlikely that we have missed strong and prevalent hla-c restricted cd8 + t cell epitopes. thus, we suggest that the paucity of strong hla-c restricted cd8 + t cell responses, at least in an acute viral infection like yellow fever virus, is not due to hla-c having been neglected in the scientific literature, but rather reflects a true biological phenomenon. notwithstanding, future cd8 + t cell discovery efforts should include hla-c, in particular if one or more hla-c restricted epitopes can be suggested in a situation where there are no obvious hla-a or -b restricted candidates. concurrent with cd8 + t cell discovery, the "forward-reverse immunology" approach also allowed hla-ii-restricted cd4 + t cell epitope discovery. the initial matrix-driven "forward" analysis of 50 donors identified 192 cd4 + t cell stimulatory yfv-derived 15mer peptides. this suggests that cd4 + t cell epitopes are more numerous than cd8 + t cell epitopes, perhaps as much as 2-3 times greater. if generalizable, this would have important implications for cd4 + t cell immunity since, everything else being equal, it would be more difficult for a microorganism to escape many cd4 + t cell epitopes than fewer cd8 + t cell epitopes. addressing the number of immunogenic open reading frames, other have also hinted at a greater preponderance of cd4 + than cd8 + t cell epitopes (59, 60) ; to the best of our knowledge, ours is the first proteome-wide study that have made this observation at the epitope level. the identification of the restricting hla class ii element(s) is a serious challenge in part due to different hla-ii allotypes having overlapping peptide binding repertoires (61) . in fact, this problem is so manifest that sette et al. have developed a panel of 46 different single hla-ii transfected cell lines to identify hla-ii restriction elements (62) . it would be ideal if hla-ii restrictions could be identified by predictors and then validated by tetramer analysis. unfortunately, the contemporary cd4 + t cell epitope discovery tools were immature (e.g., the early netmhciipan predictors were relatively inefficient and focused solely on the hla-dr isotypes), and access to peptide-mhc class ii tetramers was very limited. moreover, ex vivo frequencies of tetramer-positive cd4 + t cells tend to be <0.01%, which make them difficult to detect. thus, our cd4 + t cell epitope discovery process was not exhaustive; however, as cd4 + t cell discovery tools mature, we believe that the efficiency of cd4 + t cell epitope discovery eventually should approach that of cd8 + t cell epitope discovery. here, using a panel of recombinant hla-dr molecules, we measured the binding affinity of the overlapping 15mer peptides to the most common hla-dr allotypes. for each stimulatory 15mer peptide, this suggested which of the donor's hla-dr molecules should be used to generate peptide-hla-dr tetramers for validation of cd4 + t cell epitopes. this "brute force" approach was extended to 31 donors, where we tetramervalidated 50 cd4 + t cell epitopes covering 13 different hla-dr allotypes (and one hla-dp allotype). as of july 2019, the iedb has registered a total of 1,915 yfv-specific cd4 + t cell epitopes as being "exact cd4 + t cell epitopes" (i.e., length 15 aa, or less) and restricted by an hla-ii defined at high (i.e., 4-digit) resolution; 368 of which have been tetramer-validated. thus, the tetramer-validated yfv-specific cd4 + t cell epitopes reported here represents a significant increase in the number of tetramer-validated cd4 + t cell epitopes. it should be noted that james and coworkers have identified and tetramer-validated 94 different yfv-specific cd4 + t cell epitopes [iedb reference id 1026930 (49) ] that are 17 aa long and therefore fall just outside the definition of an exact cd4 + t cell epitope. a detailed examination of cd4 + t cell responses revealed a phenomenon that could have profound biological and practical implications for cd4 + t cell recognition. in many cases, two consecutive overlapping 15mer peptides stimulated cd4 + t cell responses, which were restricted by the same hla-dr restriction element. when the responding cd4 + t cells were double-stained with hla-dr tetramers, which had been prepared with each of the overlapping peptides of a 15mer pair and labeled with a unique fluorochrome, we observed a plethora of different, yet partially shared, cd4 + t cell specificities. situations where overlapping peptides are presented must occur regularly in vivo since experiments sequencing natural peptides eluted of hla-ii molecules frequently find large series of staggered peptides surrounding each core region (63) . exploiting this wealth of closely related peptides to engage a large number of different cd4 + t cell specificities recognizing the same core region in slightly different ways [something that actually was noted years ago (64) ], may represent a biologically significant diversification mechanism of cd4 + t cell responses reducing the risk of pathogen escape and increasing the chances of recognizing a given target. this phenomenon is also important for how cd4 + t cell responses should be analyzed. a single peptide-hla-ii tetramer is likely to engage a range of t cells of various avidities for the tetramer; something that might explain why single hla-ii tetramers often appear to label a poorly defined, non-base line separated, mono-specific cd4 + t cell population of low frequency. if examined with two "overlapping" tetramer, this could in reality turn out to be a heterogeneous collection of better defined and separated cd4 + t cell populations of a higher accumulated frequency. from a practical perspective, this implies that double staining involving two overlapping peptide-hla-ii tetramers will be needed to faithfully enumerate and monitor any cd4 + t cell response. from a technical perspective, this will increase the observed frequencies of specific cd4 + t cells for a given specificity (i.e., increase the sensitivity of the analysis), and it will increase the resolution of the flow cytometric analysis since it may separate various positively staining subpopulations and provide a better discrimination against negatively staining cd4 + t cells. the t cell epitope discovery approach described here has several advantages. in the forward immunology component, an overlapping peptide library is used to search for peptides containing cd4 + or cd8 + t cell epitopes. in the subsequent reverse immunology component, pan-specific predictors are used to identify the underlying epitope and its hla restriction element. these steps can be done in any (obviously outbred) individual of any hla haplotype using simple and standardized conditions. this reduces the number and combinations of peptides and hla allotypes that should be considered for peptide-hla tetramer generation and used in the final validation of the discovered t cell epitopes. as shown here, this approach is efficient and, not surprisingly, it reduces the false discovery rate. as peptide-hla class i and ii predictors and tetramer technologies mature, this approach will eventually be able to cover all frequently found hla class i and ii iso-and allotypes. this approach is systematic, all-inclusive, complete, and global in the sense that it includes all protein antigens and peptide epitopes, encompasses both cd4 + and cd8 + t cell epitopes, and can be extended to all individuals of all populations. this approach could be extended to other attenuated live virus vaccines (e.g., those targeting measles, mumps, rubella, polio, and hpv). compared to a strictly reverse approach, significant disadvantages of the hfri approach include the time and cost associated with establishing a complete overlapping peptide library as well as using a cellular readout as an initial selection step. therefore, this will probably not be justified if the aim is to identify epitopes in an urgent effort involving one donor (e.g., for cancer immunotherapy purposes); rather, it would be appropriate if the aim is to examine a large panel of donors in order to get population-wide data including immunodominance, candidates for diagnostics and vaccine development for infectious disease purposes (examples include a range of emerging and re-emerging infectious diseases like hiv, sars, mers, chikungunya, dengue, west nile, zika, ebola and sars-cov-2). for all of the above examples, the proteomes are small enough that their entire proteomes could be addressed by an overlapping peptide strategy using the number of pbmc's that could be obtained from a donor. addressing larger pathogen proteomes (e.g., herpes virus, bacteria or parasites; or smaller highly variable virus like hiv and dengue) in their entirety would require either a selection process downsizing the source target protein antigens, or the development of novel miniaturized, yet high-capacity, technologies. one could envision that future investigations of emerging diseases would include population-wide t cell epitope discovery efforts using blood samples from patients, convalescents and/or longterm survivors, which all possess important information on t cell epitopes and responses. similarly, one could envision that approval and registration of new vaccines could include population-wide analysis of t cell epitopes and responses. one could also envision that population-wide information on t cell epitopes and immunodominance could be used to design subunit vaccines. another important advantage of the forward-reverse approach presented here is the unbiased nature of the t cell epitope discovery process. whereas, current data-driven bioinformatics peptide-mhc predictors are quite accurate, the need for even better predictors stresses not only the need for high-quality training data, but also the need for high-quality validation data. in this context, there is an inherent problem in most epitope discovery efforts being dependent on peptide-mhc predictors since this effectively means that current t cell epitope discovery submissions tend to be biased by current predictors; something that might compromise the validation and benchmarking of predictors. having reasoned that our forward-reverse approach captures about 90% of the true t cell epitopes, we would like to propose that the resulting data is largely unbiased and should serve as an appropriate benchmark [others have reached similar conclusions (47)]. as an example, we used the cd8 + t cell epitopes identified here to benchmark current predictors. all current predictors were quite efficient and accurate. the newer predictors, some of which included immunopeptidomics and therefore may also reflect antigen processing, were better than the older predictors [as also noted by others (41) ]. however, these improvements are incremental and even the newest predictors were afflicted by high fdr's. taken together, this could be interpreted as a need for a change in how we predict t cell epitopes that is more fundamental than merely acquiring more peptide-mhc affinity and/or stability data e.g., by including t cell receptor specificities and repertoire propensities. a source of unbiased t cell epitope data would be instrumental in improving predicting tools. in conclusion, for smaller proteomes, it is possible to design a limited set of overlapping peptides spanning the entire proteome and use these to reveal the vast majority, if not all, specific cd4 + and cd8 + t cell responses concurrently; use predictors to identify the underlying combination of peptide epitopes and mhc restriction elements; and finally use this information to construct suitable peptide-mhc multimers and validate the t cell epitopes discovered. performing this in cohorts of patients or vaccinees allows for a systematic, global and cost-efficient analysis of cd4 + and cd8 + t cell epitopes, and evaluation of their immunodominance. as previously described (26) , this study was approved by the danish national committee on health research ethics (protocol # h-1-2009-095), and the collection of data and cells was approved by the danish data protection agency (permission 2008-41-2732). all volunteers gave written informed consent prior to participation. based on previous yfv vaccination history and their international card of vaccination, healthy volunteers, who for traveling purposes were about to receive a primary yfv vaccination, were recruited. the attenuated yfv vaccine, 17d-204 (sanofi pasteur; marketed as stamaril in more than 70 countries globally and as yf-vax in the usa) was administered intramuscularly. about 42% of the volunteers received an yfv vaccination only, whereas the remaining 58% received additional vaccines, typically killed, inactivated or subunit vaccines; in no case was the yfv vaccine co-administered with another live attenuated vaccine. as previously described (26) , blood samples were obtained just prior to and after the yfv vaccination (typically day 10-20 post vaccination, range 9-41 days). peripheral blood mononuclear cells (pbmc) were isolated by density gradient centrifugation using ficoll-paque tm plus (ge healthcare europe, brøndby, denmark). they were either examined directly ex vivo or cryopreserved in 10% dmso and 90% fcs at −150 • c for later in vitro analysis. chromosomal dna was prepared from the pbmc's and sequence-based typing (sbt) was used to perform highresolution (i.e., 4 digit) hla-typing (genome diagnostics, utrecht, the netherlands). all loci encoding classical hla molecules were typed i.e., the three class i ioci, hla-a, -b, -c and the six class ii loci, hla-drb1, -drb3/4/5, -dqa1, -dqb1, -dpa1, and -dpb1. the pbmcs were analyzed ex vivo for the t cell markers, cd3, cd4, and cd8, and the extracellular t cell activation markers, cd38 and hla-dr as previously described (26) . briefly, pbmcs were incubated with fluorochrome-conjugated anti-cd3, -cd4, -cd8, -cd38, and -hla-dr antibodies for 30 min at room temperature, washed, fixed with 1% formaldehyde, and analyzed by flow cytometry (lsr-ii, bd biosciences) using diva software. all antibodies were obtained from biolegend (san diego, ca, usa). all peptides were synthesized by standard 9fluorenylmethyloxycarbonyl (fmoc) chemistry and purified by reversed-phase high-performance liquid chromatography (purity at least 80%, usually >95%), mass spectrometry validated and lyophilized (schafer-n, copenhagen, denmark). an overlapping peptide library systematically covering the entire proteome of the vaccine strain, yf-17d-204 (uniprot# p03314), was synthesized. this encompassed the entire yf precursor protein of 3411 aa, which could be represented by 850 peptides, each 15 aa long and overlapping its neighboring peptides by 11 aa. in addition, 50 peptides, which were predicted to be binders to hla-a or -b supertype representatives by our netmhcpan predictor, were selected from putative products of alternative translation initiation codons (65) . one hundred and seven (107, or 11.9%) of the total of 900 selected peptides were difficult to synthesize and/or purify; many of which had long stretches of hydrophobic aa. adding one or two lysine to their n-or c-termini allowed the successful synthesis and purification of 77 of these "difficult to synthesize" peptides leaving 30 peptides that could not be synthesized and/or purified. thus, 870 (97%) of the selected peptides could be included in this epitope screening effort. these peptides were initially organized in a 30×30 matrix, and eventually in four 15×15 matrices (supplementary figure s1 ). fresh or thawed pbmcs were tested using an interferon-γ (ifnγ) specific eli spot assay as previously described (66) . briefly, 2-3 × 10 5 cells/well were plated in an eli spot plate (mahas4510, merck millipore, usa) and in vitro cultured for 18-24 h in media supplemented with or without peptide at 0.5 µm [or, as positive control, with 1 µg/ml staphylococcal enterotoxin b (seb, sigma aldrich, st. louis, usa)]. an ap conjugate substrate kit (bio-rad) was used for visualization of spots. eli spots were counted using a ctl immunospot series 5 uv analyzer. immunospot 5.0.9 software (c.t.l., shaker heights, usa) was used for analysis. wells with spotforming units spu > 2 times the background wells were considered positive. pbmcs were incubated overnight (37 • c, 5% co 2 ) in x-vivo 15 media (fisher scientific) supplemented with 5% ab serum (invitrogen) and a mixture of relevant peptides at a concentration of 0.5 µm of each peptide. the cells were harvested and washed, and subsequently plated in 24-well plates at a concentration of 5 × 10 6 /ml supplemented with 50 u/ml il-2 for expansion. fresh media and il-2 were supplemented every second day until the cells were harvested at day 8, and il-15 (15 ng/ml) was added the last 4 days. in vitro cultured pbmc's were harvested, washed, and aliquoted at 2-4 × 10 5 cells/well in a round bottom 96 well-plate. the in vitro cultured pbmc's were stimulated for 4 h at 37 • c, 5% co 2 with, for the matrix analysis, each of the 15 column and 15 row mixes (1 µm/peptide), and for the epitope identification with a single peptide (0.8 µm). after 1 h of stimulation 1 µg/ml brefeldin a (bd biosciences) was added. the cells were subsequently permeabilized (becton dickinson permeabilizing solution 2) and stained with anti-cd3-pacific blue, anti-cd4-percp, anti-cd8-apc, anti-cd69-pe, and anti-ifnγ-fitc, according to the "fastimmune" protocol (becton dickinson). the cells were subsequently analyzed by flow cytometry using a lsrii (bd biosciences). the gating strategy is illustrated in supplementary figure s4 . staining of more than 0.8% of cd8 + or cd4 + t cells was considered positive. hla class i tetramers were produced as previously described (67) . briefly, recombinant, biotinylated hla class i heavy chain, human β 2 -microglobulin and peptide were incubated in 50 mm tris-maleate ph 6. hla-dra and hla-drb chains were produced as previously described (42) . for tetramer production, hla-dra and hla-drb chains were mixed in a 1:1.5 ratio and incubated in 3 µm peptides in pbs (ph 7.4) with 20% glycerol and 0.1% pluronic f68 for 96 h at 18 • c. the resulting monomers were buffer changed into pbs with 5% glycerol and concentrated on 10kd vivaspin (satorius) and quantitated by luminescent oxygen channeling immunoassay (loci)-driven assay (42) . the resulting monomers were tetramerized by addition of fluorochrome labeled streptavidin (streptavidin-phycoerythrin (sa-pe) or streptavidin-allophycocyanin (sa-apc); both from biolegend) sequentially over 60 min at a 1:4 molar ratio of streptavidin to monomer. for t cell analysis, pellets of 4 × 10 5 cells obtained from in vitro peptide stimulated cell cultures, were re-suspended in a 40 µl tetramer diluted in media to a final concentration of ca. 30 nm, and incubated for 1 h at 37 • c, followed by 30 min incubation with fluorochrome conjugated anti-cd3, -cd8, -cd38, and -hla-dr antibodies. the cells were analyzed by flowcytometry (fortessa or lsr-ii, bd biosciences) using diva software. for each donor, all 15mer peptides eliciting a cd8 + t cell response were submitted to our bioinformatics predictor, netmhcpan (36) , which returned a prioritized list of predicted optimal epitopes, which could bind to any of the up to six hla-a, -b, or-c molecules of the donor in question. for each donor, all 15mer peptides eliciting a cd4 + t cell response were submitted to our bioinformatics predictor, netmhciipan (68) , which returned a prioritized list of predicted epitopes including a predicted core-region, which could bind to any of the up to four hla-drb1, or-drb3/4/5 molecules of the donor in question. the stability of peptide-hla class i complexes was measured using dissociation of 125 i radiolabelled β 2 m in a scintillation proximity assay (spa) as previously described (69) . briefly, recombinant, biotinylated hla class i heavy chains were diluted into a refolding buffer containing the test peptide and trace amounts of 125 i radiolabeled β 2 m, and allowed to refold at 18 • c for 24 h in a streptavidin-coated scintillation microplate (flashplate plus, perkin elmer, boston, ma). dissociation was initiated by adding excess unlabeled β 2 m and placing the microplate in a scintillation counter (topcount nxt, packard) adjusted to 37 • c. reading the microplate continuously for 24 h allowed determination of the dissociation of radiolabeled β 2 m. peptide-hla-ii binding affinities were determined using a previously described luminescent oxygen channeling immunoassay (loci)-driven assay (42) . briefly, denatured and purified recombinant hla-ii alpha and beta chains were diluted into a refolding buffer (tris-maleate buffer, ph 6.6) with graded concentrations of the test peptide, and incubated for 48 h at 18 • c to allow for equilibrium to be reached. the peptide concentration leading to half-saturation (ed 50 ) was determined as previously described (42) . under the limited receptor concentrations used here, the ed 50 reflects the affinity of the interaction. graphpad prism 8 was used for statistical analyses (unpaired and paired mann-whitney-wilcoxon tests, unpaired and paired t-tests, fishers exact test, and roc analysis). all datasets presented in this study are included in the article/supplementary material. towards a systems understanding of mhc class i and mhc class ii antigen presentation immunodominance in major histocompatibility complex class i-restricted t lymphocyte responses description and prediction of peptide-mhc binding: the human mhc project identifying cytotoxic t cell epitopes from genomic and proteomic information: the human mhc project the immune epitope database and analysis resource program 2003-2018: reflections and outlook a case for a human immuno-peptidome project consortium computational tools for the 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quantitative predictions of peptide binding to any hla-dr molecule of known sequence: netmhciipan real-time, high-throughput measurements of peptide-mhc-i dissociation using a scintillation proximity assay the authors thank doctors and nurses at the two recruiting centers and the blood bank of the university hospital, and members of the buus laboratory for expert technical assistance. the studies involving human participants were reviewed and approved by danish national committee on health research ethics (protocol # h-1-2009-095). the patients/participants provided their written informed consent to participate in this study. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.01836/full#supplementary-material key: cord-326983-h6gdck2u authors: ferretti, andrew p.; kula, tomasz; wang, yifan; nguyen, dalena m.v.; weinheimer, adam; dunlap, garrett s.; xu, qikai; nabilsi, nancy; perullo, candace r.; cristofaro, alexander w.; whitton, holly j.; virbasius, amy; olivier, kenneth j.; buckner, lyndsey r.; alistar, angela t.; whitman, eric d.; bertino, sarah a.; chattopadhyay, shrikanta; macbeath, gavin title: unbiased screens show cd8+ t cells of covid-19 patients recognize shared epitopes in sars-cov-2, most of which are not located in the spike protein date: 2020-10-20 journal: immunity doi: 10.1016/j.immuni.2020.10.006 sha: doc_id: 326983 cord_uid: h6gdck2u developing effective strategies to prevent or treat covid-19 requires understanding the natural immune response to sars-cov-2. we used an unbiased, genome-wide screening technology to determine the precise peptide sequences in sars-cov-2 that are recognized by the memory cd8+ t cells of covid-19 patients. in total, we identified 3–8 epitopes for each of the six most prevalent human leukocyte antigen (hla) types. these epitopes were broadly shared across patients and located in regions of the virus that are not subject to mutational variation. notably, only 3 of the 29 shared epitopes were located in the spike protein, whereas most epitopes were located in orf1ab or the nucleocapsid protein. we also found that cd8+ t cells generally do not cross-react with epitopes in the four seasonal coronaviruses that cause the common cold. overall, these findings can inform development of next-generation vaccines that better recapitulate natural cd8+ t cell immunity to sars-cov-2. identification of cd8 + t cell epitopes in five hla-a*01:01 patients coronavirus disease 2019, or covid-19, is a global pandemic that has claimed over a million lives world-wide and has affected countless more. developing effective vaccines and therapies requires understanding how the virus and the immune response affect disease pathology and how the adaptive immune system recognizes and ultimately clears the virus. to date, most efforts have focused on the b cell-mediated antibody response to the virus. notably, the vast majority of current vaccine development efforts are focused on eliciting neutralizing antibodies to the virus, most frequently by immunizing with the spike (s) protein of sars-cov-2, or even with just the receptor binding domain (rbd) of the s protein (vabret et al., 2020) . how cytotoxic cd8 + t cells recognize and clear infected cells is less understood. in individuals who recovered from the closely related coronavirus, sars-cov, virus-specific memory cd8 + t cells persist for at least six to eleven years, whereas memory b cells and anti-viral antibodies are largely undetectable at these later timepoints (peng et al., 2006; tang et al., 2011) . similarly, antibody responses to sars-cov-2 can be detected in most covid-19 patients 10 -15 days following symptom onset, but responses decline to baseline in many patients within 3 months (seow et al., 2020) . these findings suggest that vaccines focused solely on eliciting neutralizing antibodies to the s protein may be insufficient to elicit long-term immunity to coronaviruses. in mice infected with sars-cov, virus-specific cd8 + t cells are sufficient to enhance survival and diminish clinical disease (zhao et al., 2010) and immunization with a single immunodominant cd8 + t cell epitope confers protection from lethal viral infection (channappanavar et al., 2014) . these studies highlight the importance of understanding the natural cd8 + t cell response to sars-cov-2 as a route to designing more durable vaccines. sars-cov-2-specific cd8 + t cells can be detected both in convalescent patients (braun et al., 2020; grifoni et al., 2020; le bert et al., 2020; peng et al., 2006; sekine et al., 2020; thieme et al., 2020) and in subjects participating in vaccine trials (folegatti et al., 2020; jackson et al., 2020; sahin et al., 2020) . however, these studies used complex pools of predicted epitopes, and it is therefore not clear what specific epitopes are being recognized and, in the case of vaccine trials, if the epitopes being recognized are the ones driving the natural cd8 + t cell response to viral infection. to circumvent potential bias introduced by epitope prediction algorithms, we built upon an unbiased, genome-wide screening technology (kula et al., 2019) to simultaneously screen all of the memory cd8 + t cells in convalescent patients against every possible epitope in sars-cov-2. we focused on memory cells to identify epitopes that are functionally recognized during the course of sars-cov-2 infection and included patients with a j o u r n a l p r e -p r o o f range of symptoms to determine if any obvious associations are observed between cd8 + t cell response and disease severity. cd8 + t cells in these patients responded to a few highly antigenic epitopes in sars-cov-2 that were shared among patients with the same hla type. these epitopes were largely unique to sars-cov-2, were invariant among viral isolates, were frequently targeted by multiple clonotypes within each patient, and did not occur in "common cold" coronaviruses. only ~10% of the epitopes were found in the s protein, with ~50% located in orf1ab and the highest density of epitopes located in the nucleocapsid (n) protein. these results provide the necessary tools to better understand the cd8 + t cell response in and provide a path to the design and development of next-generation vaccines. to determine the global landscape of cd8 + t cell recognition in an unbiased fashion, we built upon a genome-wide screening technology, termed t-scan (kula et al., 2019) , that enabled us to simultaneously screen all the memory cd8 + t cells in a patient, one hla allele at a time, against every possible viral epitope in sars-cov-2, as well as the four seasonal coronaviruses that cause the common cold ( figure 1a ). briefly, cd8 + t cells were co-cultured with a genomewide library of target cells (modified hek 293 cells), engineered to express a single hla allele. each target cell in the library also expressed a different 61-amino acid (61-aa) protein fragment. these fragments were processed naturally by the target cells and the appropriate peptide epitopes were displayed on class i mhc molecules on the cell surface. if a cd8 + t cell encountered its target in the co-culture, it secreted cytotoxic granules into the target cell, inducing apoptosis. early apoptotic cells were then isolated from the co-culture and the expression cassettes sequenced, revealing the identity of the protein fragment. because the assay is non-competitive, hundreds to thousands of t cells were screened against tens of thousands of targets simultaneously. to address the bottleneck of extensive sorting needed to isolate rare recognized target cells in high complexity libraries (kula et al., 2019) , we engineered the target cells to express both a granzyme b (gzb)-activated fluorescent reporter, as previously described, as well as a gzb-activated version of the scramblase enzyme xkr8, which drives the rapid and efficient transfer of phosphatidylserine to the outer membrane of early apoptotic cells (supplemental methods and figure 1a ). early apoptotic cells were then enriched by magnetic-activated cell sorting with annexin v, followed by fluorescence-activated j o u r n a l p r e -p r o o f sorting with the fluorescent reporter. this modification increased the throughput of the t-scan assay 20-fold, enabling the rapid processing of a large number of patient samples. to comprehensively map responses to sars-cov-2, we generated a library of 61-aa protein fragments that tiled across all 11 open reading frames (orfs) of sars-cov-2 in 20-aa steps ( figure 1b) . to capture the known genetic diversity of sars-cov-2, we included all protein-coding variants from the 104 isolates that had been reported as of march 15, 2020. we also included the complete set of orfs (orfeome) of sars-cov and the four endemic coronaviruses that cause the common cold (betacoronaviruses hku1 and oc43, and alphacoronaviruses nl63 and 229e). as positive controls, we included known immunodominant antigens from cytomegalovirus, epstein-barr virus, and influenza virus (currier et al., 2002) . finally, each protein fragment was represented ten times, each encoded with a unique nucleotide barcode to provide internal replicates in our screens, for a final library size of 43,420 clones. to identify the epitopes functionally recognized during the course of sars-cov-2 infection, we collected peripheral blood mononuclear cells from 78 adult patients who had tested positive by viral pcr (swab test), had recovered from their disease, and had been out of quarantine according to centers for disease control and prevention guidelines for at least two weeks (supplemental methods). patients were recruited at either of two centers: atlantic heath system in morristown, nj and ochsner medical center in new orleans, la. all patients were hlatyped, and a summary of their characteristics and hla types are provided in tables s1 and s2. as hla a*02:01 is the most common mhc allele world-wide (gonzalez-galarza et al., 2020; maiers et al., 2007) , we started by selecting nine hla-a*02:01 patients with a broad range of clinical presentations: six had mild symptoms and were not hospitalized, two required supplemental oxygen, and one required invasive ventilation. in each case, we purified bulk memory cd8 + t cells (cd8+, cd45ro+, cd45ra-, cd57-) by negative selection, expanded the cells with antigen-independent stimulation (anti-cd3), and screened them against the sars-cov-2 library. target cells expressing only hla-a*02:01 were used to provide unambiguous mhc restriction of discovered antigens. the sars-cov-2 screening results for one representative patient and one covid-19-negative healthy control are shown in figure 1c . we found reactivity to at least eight regions of sars-cov-2 proteins in the convalescent patient and none in the control. importantly, we observed reproducible performance of four technical screen replicates, internal nucleic acid barcodes, and overlapping protein fragments, collectively j o u r n a l p r e -p r o o f suggesting robust screen performance. additionally, we detected reactivity to the control cmv epitope (nlvpmvatv) in the healthy control, who was known to be cmv-positive, and reactivity to two ebv epitopes in both the covid-19 patient and the healthy control ( figure 1c ). next, we examined the screen results for the full set of nine hla-a*02:01 patients and detected reactivity to specific segments of sars-cov-2 orfs in 8/9 patients ( figure 2a ). in keeping with what has been observed for other viruses (yewdell, 2006) , we found that specific fragments of sars-cov-2 were recurrently recognized by the t cells of multiple patients (i.e., are immunodominant). for example, orf1ab aa 3881-3900 and s aa 261-280 were each recognized by 7/9 patients ( figure 2a ). overall, we identified six regions that were targeted by cd8 + t cells from at least three different patients. in addition to being shared across patients, these regions were among the strongest responses observed in each patient. we next sought to identify the precise peptide epitopes underlying the shared t cell reactivities detected in our screens. the overlapping design of our antigen library allowed us to map the t cell reactivities to specific 20-aa segments. we then used netmhc4.0 (andreatta and nielsen, 2016; nielsen et al., 2003) to identify specific, high-affinity hla-a*02:01 peptides in each pre-identified 20-aa stretch. an example of a predicted epitope and the corresponding screen data is shown in figure 2b . notably, the fragments scoring in our screens were enriched for high-affinity hla-binding peptides compared to the library as a whole ( figure s1 ). to visualize the results across all nine patients, we collapsed the screening data into a single value (mean of screen replicates and redundant tiles), revealing a set of six predicted epitopes that were recurrently recognized by three or more patients ( figure 2c , table 1 ). we then synthesized peptides corresponding to each epitope to validate our findings. all six epitopes induced peptide-dependent t-cell activation as determined by interferon-gamma (ifnγ) secretion ( figure 2d ) and cd137 upregulation ( figure s2 ). both ifnγ secretion and cd137 upregulation correlated with the fold enrichment in the t-scan screen ( figures s2 and s3 ). as further validation, we constructed mhc tetramers with the six peptides and used them to stain the memory cd8 + t cells of all nine a*02:01 patients, as well as an additional test-set of 18 a*02:01 patients that had not been screened. positive tetramer staining was observed in a subset of patients for all six peptides, including patients in the independent test-set ( figure 2e ). additionally, the magnitude of enrichment in the screens correlated well with the frequency of cognate t cells in the patient samples (r = 0.73, p < 0.0001; figure 2f ), allowing us to determine that our screens detected the targets of t cells that were present at ≥0.1% frequency j o u r n a l p r e -p r o o f in the memory cd8 + t cell pool. notably, the three most commonly recognized epitopes we discovered -klw, ylq, and lly -were each recognized by 67% of the patients we screened, and all nine patients had a detectable response to at least one of the top three epitopes ( figure 2g ). a similar analysis of the tetramer staining data in all 27 a*02:01 patients showed recognition of at least one of these epitopes in 23/27 patients (85%; figure 2h ). taken together, these analyses revealed the limited set of a*02:01-restricted shared epitopes recognized by patient t cells. as cd8 + t cell responses are profoundly shaped by host mhc alleles, we next mapped memory cd8 + t cell reactivities for five additional mhc alleles: hla-a*01:01, hla-a*03:01, hla-a*11:01, hla-a*24:02, and hla-b*07:02. along with hla-a*02:01, these alleles provide a broad perspective on the nature of anti-sars-cov-2 cd8 + t cell immunity, as ~90% of the u.s. population and ~85% of the world population is positive for at least one of the six alleles we examined (gonzalez-galarza et al., 2020; maiers et al., 2007) . for each allele, we selected five hla + convalescent covid-19 patients and screened their memory cd8 + t cells against the sars-cov-2 library in target cells expressing only the single hla of interest. as some patients were positive for more than one allele, their t cells were used in more than one hla-specific screen. a total of 25 distinct patients were needed for the 34 hla-specific screens. as with a*02:01 patients, we found robust t cell recognition of multiple regions in the sars-cov-2 orfeome for patients with each hla allele ( figure s4 ) and confirmed that the scoring fragments were enriched for predicted high-affinity mhc binders for each respective allele ( figure s1 ). we again observed recurrent recognition of specific protein fragments by most or all patients for each allele ( figure 3a ), indicating a narrow set of shared responses. as before, we used netmhc4.0 to identify the precise epitopes underlying the top hits from our screens, and validated these peptides using ifnγ secretion ( figure 3b ) and cd137 upregulation ( figure s2 ). we identified three or more recurrently recognized epitopes for each screened mhc allele and found that 92% of patients recognized at least one of the top three allele-specific epitopes for these five additional hla types ( figure 3c ). collectively, we mapped and validated 29 cd8 + t cell epitopes that were shared among covid-19 patients with the same hla type (table 1) . these epitopes represent the global landscape of mhc class i immunodominance in sars-cov-2 across the six most prevalent hla types. the unbiased antigen mapping we performed enabled us to interrogate various features of cd8 + t cell immunity to sars-cov-2. first, we examined the scope of recognized viral j o u r n a l p r e -p r o o f proteins. we observed broad reactivity to many sars-cov-2 proteins, including orf1ab, s, n, m, and orf3a ( figure 4a ). notably, only three of the 29 epitopes were located in the s protein, with most (15 of 29) located in orf1ab and the highest density of epitopes located in the n protein. when taken in aggregate, our results are consistent with previous orf-level analyses using peptide pools (altmann and boyton, 2020; braun et al., 2020; grifoni et al., 2020; le bert et al., 2020; thieme et al., 2020) . however, our approach provided an increased level of granularity that enabled identification of specific epitope sequences and highlighted hla allelespecific differences. for example, we observed shared epitopes in the s protein for hla-a*02:01, hla-a*03:01, and hla-a*24:02, but not for hla-a*01:01, hla-a*11:01, or hla-b*07:02. notably, we detected only one recurrent response in the receptor-binding domain (rbd) of the s protein (kcy on hla-a*03:01). next, we asked how the cd8 + t cell response to sars-cov-2 intersects with the emerging genetic diversity of the virus. recent analyses, which examined the genome sequences of over 10,000 isolates of sars-cov-2 sampled from 68 different countries, identified a set of 28 non-synonymous coding mutations detected in at least 1% of strains (koyama et al., 2020) . only one of these mutations (m protein t175m, detected in 2% of strains) was found in the shared epitopes we identified (hla-a*01:01 ats and hla-a*11:01 ats). this suggests that the recognition of the epitopes we identified is unlikely to be significantly influenced by the sars-cov-2 genetic diversity observed thus far. identifying specific sars-cov-2 epitopes allowed us to examine the features of the t cell receptors (tcrs) recognizing these shared epitopes. we used tetramers loaded with three hla-a*02:01 epitopes (klw, ylq, and lly) to stain and sort antigen-specific memory cd8 + t cells from the initial nine hla-a*02:01-positive convalescent covid-19 patients. for each of the other five hla alleles, we used tetramer or cd137 staining to sort cd8 + t cells reactive to the 3-4 most frequently shared epitopes in two patients each. we then used 10x genomics single-cell sequencing to identify the paired tcr α and tcr β chains expressed by these t cells. collectively, we found tcrs recognizing 17 shared epitopes for a total of 421 sars-cov-2-reactive tcrs. next, we examined the tcr sequences themselves, focusing on the three hla-a*02:01 antigens that were explored across a larger set of patients. we identified paired clonotypes reactive to each antigen in 5/9 (klw, alw) or 6/9 (ylq) patients. for a majority of responses (9/16), we detected oligoclonal recognition by five or more distinct clonotypes. striking similarity was observed among the tcrs recognizing each antigen in terms of vα gene j o u r n a l p r e -p r o o f segment usage and, to a lesser extent, vβ usage ( figure 4b ). specifically, 26/61 klw-reactive clonotypes used trav38-2/dv8, 24/31 ylq-reactive clonotypes used trav12-1, and 14/29 lly-reactive clonotypes used trav8-1. notably, these dominant vα genes were used across all of the patients for whom we identified reactive clonotypes. taken together, these data suggest that certain tcr vα regions provide the structural features necessary for high-affinity binding to peptide-mhc, and that these features may explain the recurrent recognition of these epitopes among patients with the same hla type. although our study was not designed to test specific clinical hypotheses, we looked for potential associations between virus-specific t cell responses and clinical characteristics. we focused on the 27 a*02:01 patients for which tetramer staining data were available, as this represents the most uniform set of t cell data in our study. no obvious association was observed between t cell response and sex ( figure s5 ), but a significant negative correlation was observed with time from diagnosis to blood draw (p=0.0012; figure 4c and figure s5 ). this is expected, as antiviral t cells, including effector memory cells, naturally contract following an acute infection (badovinac et al., 2002; wherry and ahmed, 2004) . this observation is important, however, as future epidemiological studies should control for this variable. we also observed a trend in which patients with severe disease exhibited fewer virus-specific t cells than those with mild disease (p=0.041; figure 4d and figure s5 ). additionally, older patients had lower t cell responses than younger patients ( figure s5 ). these observations should be treated with caution as the number of patients in these studies is small, particularly those requiring invasive ventilation. appropriately powered studies to address this question are warranted, as they could shed light on whether these shared epitopes are potentially protective against severe disease. another key question is how pre-existing immunity to other coronaviruses shapes the cd8 + t cell response to sars-cov-2. there are four commonly circulating coronaviruses, oc43, hku1, nl63, and 229e, and cross-reactive responses to these viruses have been theorized as a potential protective factor during sars-cov-2 infection (sette and crotty, 2020). moreover, understanding the extent of cross-reactivity has implications for accurately monitoring t cell responses to sars-cov-2 and for optimizing vaccine design. if the immune response to sars-cov-2 is shaped by pre-existing cd8 + t cells that recognize other coronaviruses, we reasoned that covid-19 patients should have reactivity to the regions of the other coronaviruses that j o u r n a l p r e -p r o o f correspond to the sars-cov-2 epitopes we identified. we therefore examined t-cell reactivity to sars-cov-2, sars-cov, and all four endemic coronaviruses in the 34 genome-wide screens that we conducted across all patients and mhc alleles ( figure 5a ). we observed broad reactivity to the corresponding epitopes in sars-cov in over half of cases, consistent with a recent study reporting the existence of long-lasting memory t cells cross-reactive to sars-cov-2 in patients that had been infected in sars-cov during the 2002/2003 sars outbreak (le bert et al., 2020) . in contrast, we detected almost no reactivity to oc43 and hku1 (2/29 dominant epitopes) and none to nl63 and 229e. beyond the 29 epitopes, we observed no reproducible cross-reactivity to any other regions of the four endemic coronaviruses, again suggesting that prior exposure to these viruses is unlikely to provide cd8 + t cell-based protection from sars-cov-2. mapping the specific shared epitopes in sars-cov-2 enabled us to determine the molecular basis for this lack of cross-reactivity. in some cases, the corresponding region is poorly conserved in the other coronaviruses and high-affinity binding to mhc is lost (see, for example, the corresponding regions of the klw epitope in nl63 and 229e; figure 5b ). in other cases, the corresponding epitopes are still predicted to bind with high affinity to mhc, but sars-cov-2-reactive t cells did not recognize them (see, for example, the corresponding regions of the klw epitope in oc43 and hku1; figure 5b ). in one notable case, we did identify a strong cross-reactive response. the hla b*07:02 epitope spr, which lies in the n protein, is highly conserved across betacoronaviruses and all four of the patients that demonstrated reactivity to spr also exhibited reactivity to the corresponding epitopes in oc43 and hku1 ( figure 5c ). overall, however, we conclude that the cd8 + t cell response to sars-cov-2 is not significantly shaped by pre-existing immunity to endemic coronaviruses. in contrast to reports that unexposed individuals have t cells that cross-react with sars-cov-2 le bert et al., 2020; mateus et al., 2020; weiskopf et al., 2020) , our data showed that the most expanded memory t cells in convalescent patients did not cross-react with endemic coronaviruses. these discordant results may be explained by our focus on cd8 + t cell responses, whereas most cross-reactivity is detected in cd4 + t cells. additionally, it is possible that weakly cross-reactive t cells exist in unexposed individuals, but these t cells are overtaken by de novo responses during sars-cov-2 infection. if pre-existing memory responses to other coronaviruses efficiently recognize sars-cov-2, the reacting t cells should expand, and their targets would likely have been detected in our screens. as a result, the paucity of identified cross-reactive responses argues against substantial protection against sars-cov-2 stemming from cd8 + t cell immunity to the four coronaviruses that cause the common cold. we did identify two epitopes that were shared with oc43 and hku1, however, which could be of interest in the design of vaccines intended to boost pre-existing t cell immunity. our findings have broader implications for sars-cov-2 vaccine design. the vast majority of shared epitopes we uncovered (26/29) were located in orf1ab, n, m, and orf3a; only three were in s and only one was in the receptor-binding domain of s. these findings j o u r n a l p r e -p r o o f provide high-resolution insight into peptide pool studies observing responses outside the s protein, and are consistent with the detectable but modest cd8 + t cell responses generated by vaccines targeting the s protein le bert et al., 2020; mulligan et al., 2020) . importantly, the protective or pathogenic role of cd8 + t cell responses to specific proteins, individual shared epitopes, or epitopes that are only recognized following vaccination remains to be determined. the epitopes we identified can serve as the basis of experimental and correlational studies to address this critical question. moreover, our findings enable the design and evaluation of next-generation vaccines that more fully recapitulate the scope of natural cd8 + t cell responses to sars-cov-2 infection. while our screening approach assayed all patient memory cd8 + t cells as a pool, it is best suited for the discovery of targets recognized by the most abundant t cell specificities (≥0.1% based on our estimates). additional specificities recognized by less frequent t cell clonotypes may have been missed. in addition, sample limitations necessitated polyclonal expansion of the memory cd8 + t cells ex vivo that may have altered the relative abundance of some clonotypes. finally, our study was underpowered to evaluate the clinical impact of cd8 + t cells recognizing specific epitopes. additional studies are needed to determine whether cd8 + t cell responses to individual proteins or epitopes are associated with protection from the virus or specific clinical outcomes. all donors provided written consent. the study was conducted in accordance with the declaration of helsinki (1996) we thank the patients and their families who participated in these studies. we also that stephen elledge and kai wucherpfennig for helpful discussions. this work was supported by tscan therapeutics, a privately-owned biotechnology company. peptides generated in this study are available for research purposes upon signing a materials transfer agreement. peptides and peptide sequences for commercial purposes (e.g., diagnostics or vaccine development) are available through license agreement. t-scan screening data are available upon request. amino acid sequences for the coronavirus peptidome library (43,420 sequences) are available upon request. t cell receptor sequences for the purposes of therapeutic development are available through license agreement. all donors provided written consent. the study was conducted in accordance with the declaration of helsinki (1996) , approved by the atlantic health system institutional review board (irb) and the ochsner clinic foundation irb and registered at clinicaltrials.gov (# nct04397900). patients who had recovered from covid-19 were eligible for this study. they were required to be >18 years of age and have laboratory-confirmed diagnosis of covid-19 using cdc or state health labs or at hospitals using an fda emergency use authorized molecular assay. time since discontinuation of isolation was required to be >14 days and discontinuation of isolation followed cdc guidelines (accessed on march 19, 2020) using either test-based or non-test-j o u r n a l p r e -p r o o f based criteria for patients either in home isolation or in isolation at hospitals. patients were also required to have no anti-pyretic use for >17 days and be able to sign informed consent for blood draws for 4 tubes of whole blood with approximately 7.5 ml of blood per tube. eligible patients were identified by the participating sites through advertising and direct contact. case report forms did not contain identifying information. samples were de-identified at the participating sites with an anonymous code assigned to each sample. anonymized blood samples were sent to tscan laboratories with limited demographic and clinical data. demographics included age, sex, and ethnicity. clinical data included date of diagnosis, specifics of diagnostic testing, duration of symptoms, and whether the patient required hospitalization, supplemental oxygen, or icu care/ ventilator support. comorbidities and current medications were also recorded. convalescents who met eligibility criteria and consented to described procedures were enrolled and sampled from two sites: atlantic health (new jersey, 51 samples) and ochsner (new orleans, 27 samples). these sites played a critical role in treating patients from early epicenters of sars-cov-2 outbreaks. recruitment materials clearly requested patients that had recovered from covid-19 with the goal of designing effective vaccines and treatments for this indication. average self-reported duration of symptoms was 18 days (1-80 days range) in females and 21 days (0-76 days range) in males. hospitalizations made up ~32% of total convalescent samples received, with 31% requiring oxygen and 5% placed on a ventilator. blood samples were collected in four 10-ml k2 edta vacutainer tubes (becton dickinson) and processed within 24-30 h to pbmcs or memory cd8+ t cells. a 1-ml sample was removed and centrifuged at 500xg for 10 min to obtain plasma. to isolate pbmcs, blood samples were diluted with an equal volume of macs separation buffer (phosphate buffered saline, 0.5% bovine serum albumin, 2 mm edta), then layered onto lymphocyte separation media (corning) and centrifuged at 1200xg for 20 min. the interface was removed and washed once with macs buffer before further processing or cryopreservation. memory cd8+ t cells were isolated from pbmcs using macs microbead kits according to the manufacturer's instructions (miltenyi). following separation, purity was confirmed using antibodies to cd3, cd8, cd45ra, cd45ro and cd57 (biolegend). immediately following isolation, memory cd8+ t cells were expanded j o u r n a l p r e -p r o o f by co-culturing with 2x10 7 mitomycin c treated (50 µg/ml, 30 min) allogenic pbmcs in the presence of 0.1 µg/ml anti-cd3 (okt3, ebioscience), 50 u/ml recombinant il-2 (peprotech), 5 ng/ml il-7 and 5 ng/ml il-15 (r&d systems). after 10 days of expansion, the cells were collected and cryopreserved. coding sequences of all deposited sars-cov-2 strains were downloaded from ncbi on march and incubating for an additional 15 min at room temperature. the stained cells were pelleted and washed three times before resuspending in a 5 µg/ml dapi solution and analyzed by flow cytometry (cytoflex s, beckman coulter). the limit of detection was defined as the mean + 2 sd of the frequency of three mhc-mismatched controls. single-cell tcr-seq (sctcr-seq) libraries were prepared following the 10x genomics single cell v(d)j reagent kit (v1) all statistical analyses were performed using graphpad prism, excel, or python. the details of the statistical tests are displayed in the figure legends. immune functional assays, from custom to standardized tests for precision medicine sars-cov-2 t cell immunity: specificity, function, durability, and role in protection gapped sequence alignment using artificial neural networks: application to the mhc class i system programmed contraction of cd8(+) t cells after infection sars-cov-2-reactive t cells in healthy donors and patients with covid-19 virus-specific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection a panel of mhc class i restricted viral peptides for use as a quality control for vaccine trial elispot assays safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, singleblind, randomised controlled trial allele frequency net database (afnd) 2020 update: gold-standard data classification, open access genotype data and new query tools targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals an mrna vaccine against sars-cov-2-preliminary report variant analysis of sars-cov-2 genomes t-scan: a genome-wide method for the systematic discovery of t cell epitopes sars-cov-2-specific t cell immunity in cases of covid-19 and sars, and uninfected controls clinical and immunological assessment of asymptomatic sars-cov-2 infections high-resolution hla alleles and haplotypes in the united states population selective and cross-reactive sars-cov-2 t cell epitopes in unexposed humans phase 1/2 study to describe the safety and immunogenicity of a covid-19 rna vaccine candidate (bnt162b1) in adults 18 to 55 years of age: interim report memory t cell responses targeting the sars coronavirus persist up to 11 years post-infection reliable prediction of t-cell epitopes using neural networks with novel sequence representations long-lived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sars-recovered patients concurrent human antibody and th1 type t-cell responses elicited by a covid-19 rna vaccine robust t cell immunity in convalescent individuals with asymptomatic or mild covid-19 longitudinal evaluation and decline of antibody responses in sars-cov-2 infection recognize 3-8 shared epitopes for each hla type studied • ~90% of shared epitopes are not located in the spike protein • cd8 + t cells show almost no cross-reactivity with epitopes in seasonal coronaviruses etoc blurb ferretti et al. reveal specific sars-cov-2 epitopes that are broadly shared by cd8+ t cells of covid-19 patients but exhibit limited cross-reactivity with seasonal coronaviruses j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f key: cord-330615-h8sktgo6 authors: jabri, ‡ § bana; de serre∥, natacha patey–mariaud; cellier¶, christophe; evans, kelly; gache, cécile; carvalho, carla; mougenot‡, jean–françois; allez, matthieu; jian, raymond; desreumaux‡‡, pierre; colombel‡‡, jean–fréderic; matuchansky§§, claude; cugnenc¶, henri; lopez–botet∥∥, miguel; vivier¶¶, eric; moretta, alessandro; roberts, arthur i.; ebert, ellen c.; guy–grand‡‡‡, delphine; brousse∥, nicole; schmitz‡, jacques; cerf–bensussan, nadine title: selective expansion of intraepithelial lymphocytes expressing the hla-e–specific natural killer receptor cd94 in celiac disease date: 2000-05-31 journal: gastroenterology doi: 10.1016/s0016-5085(00)70173-9 sha: doc_id: 330615 cord_uid: h8sktgo6 abstract background & aims: celiac disease is a gluten-induced enteropathy characterized by the presence of gliadin-specific cd4+ t cells in the lamina propria and by a prominent intraepithelial t-cell infiltration of unknown mechanism. the aim of this study was to characterize the subset(s) of intraepithelial lymphocytes (iels) expanding during active celiac disease to provide insights into the mechanisms involved in their expansion. methods: flow-cytometric analysis of isolated iels and/or immunohistochemical staining of frozen sections were performed in 51 celiac patients and 50 controls with a panel of monoclonal antibodies against t-cell and natural killer (nk) receptors. in addition, in vitro studies were performed to identify candidate stimuli for nk receptor expression. results: in normal intestine, different proportions of iels, which were mainly t cells, expressed the nk receptors cd94/nkg2, nkr-p1a, kir2d/3d, nkp46, pen5, or cd56. during the active phase of celiac disease, the frequency of cd94+ iels, which were mostly αβ t cells, was conspicuously increased over controls. in contrast, the expression of other nk markers was not modified. furthermore, expression of cd94 could be selectively induced in vitro by t-cell receptor activation and/or interleukin 15, a cytokine produced by intestinal epithelial cells. conclusions: the gut epithelium favors the development of t cells that express nk receptors. in active celiac disease, there is a specific and selective increase of iels expressing cd94, the hla-e–specific nk receptor that may be related to t-cell receptor activation and/or interleukin 15 secretion. gastroenterology 2000;118:867-879 background & aims: celiac disease is a gluten-induced enteropathy characterized by the presence of gliadinspecific cd4 ؉ t cells in the lamina propria and by a prominent intraepithelial t-cell infiltration of unknown mechanism. the aim of this study was to characterize the subset(s) of intraepithelial lymphocytes (iels) expanding during active celiac disease to provide insights into the mechanisms involved in their expansion. methods: flow-cytometric analysis of isolated iels and/or immunohistochemical staining of frozen sections were performed in 51 celiac patients and 50 controls with a panel of monoclonal antibodies against t-cell and natural killer (nk) receptors. in addition, in vitro studies were performed to identify candidate stimuli for nk receptor expression. results: in normal intestine, different proportions of iels, which were mainly t cells, expressed the nk receptors cd94/ nkg2, nkr-p1a, kir2d/3d, nkp46, pen5, or cd56. during the active phase of celiac disease, the frequency of cd94 ؉ iels, which were mostly ␣␤ t cells, was conspicuously increased over controls. in contrast, the expression of other nk markers was not modified. furthermore, expression of cd94 could be selectively induced in vitro by t-cell receptor activation and/or interleukin 15, a cytokine produced by intestinal epithelial cells. conclusions: the gut epithelium favors the development of t cells that express nk receptors. in active celiac disease, there is a specific and selective increase of iels expressing cd94, the hla-e-specific nk receptor that may be related to t-cell receptor activation and/or interleukin 15 secretion. c eliac disease is an enteropathy that is induced by gliadin in genetically susceptible individuals expressing hla-dq2 (dqa1*0501, b1*201) or hla-dq8 (dqa1*031, b1*302). 1, 2 the nature of its pathogenesis remains unclear and may involve both direct toxic and immune-mediated effects of gliadin. 1 active celiac disease is associated with the presence of serum autoantibodies against a tissue transglutaminase, 3 with activated cd4 ϩ cd25 ϩ t cells in the lamina propria and with massive intraepithelial infiltration by proliferating t lymphocytes. 4, 5 interestingly, transglutaminase, the target of celiac disease autoantibodies, is an enzyme that enhances the binding of gliadin peptides to dq2 and dq8 molecules through deamidation of glutamine residues. 6, 7 furthermore, gliadin-specific dq2-or dq8restricted t helper (th) 1 cd4 ϩ t cells have been derived from the lamina propria of celiac patients. 6, 8 these findings suggest a model whereby enhanced presentation of gliadin peptides by dq2 or dq8 molecules to cd4 ϩ cells in the lamina propria results in secretion of interferon gamma (ifn-␥) and other cytokines that may be deleterious to gut epithelial cells (ecs). 9 in contrast, the role and mechanism of the intraepithelial t-cell infiltration, a hallmark of the disease, are debated. it does not appear, as previously suggested, 4 that the expansion of the intraepithelial lymphocyte (iel) population is a nonspecific reaction to the underlying inflammatory process, because iels are not significantly increased in crohn's disease 10 and they are moderately expanded in autoimmune enteropathy of childhood, a disease with a strong inflammatory reaction in the lamina propria and a subtotal to total villous atrophy. 11 in contrast, celiac disease-associated iel infiltration seems to be of particular significance. it is detected as early as 12 hours after oral or rectal gluten challenge in patients receiving a long-term gluten-free diet (gfd) and precedes the onset of epithelial lesions. 12, 13 furthermore, iels can undergo malignant transformation during the course of 2 rare but severe complications of celiac disease: enteropathyassociated t-cell lymphomas 14 and refractory sprue. 15 these complications, which are not observed in other inflammatory bowel diseases, support the idea that celiac disease iels are permanently submitted to stimuli that promote their expansion and ultimately may favor their transformation. altogether, these observations underline the importance of understanding the mechanism(s) that drives the expansion of iels and their role in the pathogenesis of the epithelial lesions. iels represent a heterogeneous population of t lymphocytes with distinct ontogenic, phenotypic, and functional properties shown in mice. 16 in humans, the main subset expresses ␣␤ t-cell receptor (tcr), more often than cd4. the other subset expresses ␥␦ tcr and is cd4 ϫ cd8 ϫ or cd8 ϩ . a variable albeit significant increase in the numbers of tcr␥␦ iels, mainly using the v␦1 chain, has been observed in treated as well as in untreated celiac disease. 5, 17, 18 these ␥␦ t cells, which recognize the mica and micb antigens, 2 closely related hla molecules that are induced by stress on intestinal ecs, may therefore be part of a stress response to celiac disease-associated ec injury. 19 in addition, a prominent increase in tcr␣␤ ϩ cd8 ϩ iels has been observed in active celiac disease and reported to disappear after gluten avoidance and recovery of a normal villous architecture. 5, 20 because there is no evidence for gliadin-specific major histocompatibility complex (mhc)-restricted ␣␤ t cells among cd8 ϩ iels, the mechanism driving their expansion remains to be elucidated. recent studies suggest that a subset of iels express surface receptors and functional properties that are normally associated with the natural killer (nk) lineage. [21] [22] [23] [24] these iels may respond to modifications of ecs, as suggested by their natural cytotoxicity against corona virus-infected cells in mice 25, 26 or against ec tumors in humans. 27, 28 in mice, in which they have been extensively characterized, they constitute a fraction of both tcr␣␤ ϩ and tcr␥␦ ϩ iels and express a cd8␣ ϩ ␤ ϩ cd4 ϫ or cd8␣ ϫ ␤ ϫ cd4 ϫ phenotype (reviewed by rocha et al. 29 ). studies in humans have also identified the presence of iels expressing nk receptors. cd56 and cd94 were found on both cd3-positive and -negative cells, whereas cd16 expression was mainly restricted to cd3-negative cells. 21, 22, 24 the proportion of nk-like cells decreased in one study of active celiac disease, 24 whereas another study suggested that the disappearance of cd16 ϩ granular iels was associated with a more severe and persistent disease. 21 with the idea in mind that nk receptors may play an important role in the interactions between iels and ecs in the normal or diseased human intestine, we used a panel of antibodies against nk receptors to characterize the surface phenotype of iels in normal subjects and patients with celiac disease. we found that a significant proportion of normal human t-iels (iels expressing tcr) expressed one or several nk receptors. remarkably, most of the tcr␣␤ ϩ iels that expanded in active celiac disease consistently and selectively expressed cd94, the hla-e-specific nk receptor. [30] [31] [32] in contrast, there was no change in the proportion of iels expressing nk receptors that bind classical mhc class i molecules or other ligands unrelated to mhc molecules. furthermore, the increase in cd94 ϩ iels was specific of active celiac disease because it was not observed in various other inflammatory diseases and it subsided under gfd. finally, in vitro studies with isolated iels identified interleukin (il)-15, a cytokine secreted by intestinal ecs, 33 as a likely candidate for inducing cd94 expression. altogether these findings suggest that the cd94/ hla-e and il-15r/il-15 receptor/ligand pairs may control the expansion and/or function of iels in celiac disease. twenty-four children (age, 4 ϯ 3 years) had celiac disease according to epsgan criteria. 34 at the time of the study, 10 had untreated celiac disease and total villous atrophy, 10 had been on a gfd for 1-3 years and had recovered a normal mucosa or showed a moderate villous atrophy, and 4 additional patients were studied before and after gfd. twenty-seven adults, showing symptoms of malabsorption and severe to subtotal villous atrophy, were diagnosed with celiac disease based on the presence of high titers of serum antiendomysium antibodies. all had the dqa1*0501/ dqb1*0201 genotype. at the time of study, 16 patients (age, 43 ϯ 13 years) had active celiac disease with severe to subtotal villous atrophy and 11 (age, 57 ϯ 16 years) were on gfd-induced remission with normal mucosa or moderate villous atrophy. isolation of lymphocytes from biopsy specimens was performed in 15 of the adult celiac patients, 8 with active celiac disease and 7 on gfd with normal intestinal histology, and in 6 controls upon informed consent and approval by the institutional ethics committee of hôpital necker-enfants malades. histologically normal intestinal biopsy specimens were obtained from 12 children (age, 6 ϯ 5 years), 2 with chronic colitis and 10 investigated for short stature syndrome without digestive symptoms, and from 11 adults undergoing duodenal biopsies for diagnostic purposes (age, 35 ϯ 20 years) but without small intestinal disease. for 3 of the adult patients, intestinal lymphocytes were isolated from the biopsy specimens. control ileal surgical samples with an important inflammatory infiltrate in the lamina propria were obtained from 2 children (aged 12 and 14 years) with active ileocolic crohn's disease undergoing surgery for occlusive symptoms. control intestinal biopsy specimens from patients with villous atrophy distinct from celiac disease were obtained from 9 children and 3 adults. four children with moderate villous atrophy had cow's milk intolerance (n ϭ 1), intestinal giardiasis (n ϭ 1), and intestinal graft rejection (n ϭ 2). 35 five children (aged 1-8 years) with severe to total villous atrophy had autoimmune enteropathy (n ϭ 2), 11 epithelial dysplasia (n ϭ 1), 36 or graft-versus-host disease after bone marrow graft for severe combined immunodeficiency (n ϭ 2). three adult patients (aged 41-60 years) were included as controls for villous atrophy. in these patients, malabsorption and subtotal villous atrophy were not caused by celiac disease, as indicated by the absence of antiendomysium antibodies, lack of dq2 or dq8 haplotype, and lack of clinical or histological improvement after gfd for over 6 months. all 3 had severe infiltration of the lamina propria and the epithelium by cd3 ϩ cd4 ϩ (n ϭ 1), cd3 ϩ cd8 ϩ (n ϭ 1), or cd3 ϫ cd56 ϩ (n ϭ 1) small lymphocytes, suggesting low-grade lymphoproliferative disease without lymphoma. surgical samples of histologically normal proximal small intestine, 3-6 cm long, were obtained from 15 adult patients (aged 60 ϯ 12 years) undergoing intestinal surgery for gastric or pancreatic cancers or for chronic pancreatitis. peripheral blood lymphocytes (pbls) were obtained from 17 healthy adult volunteers. fluorescein isothiocyanate (fitc)-or phycoerythrin (pe)-conjugated antibodies against cd3, cd8, cd4, cd16, cd45ro, cd56, and cd57 were obtained from becton dickinson (le pont de claix, france; and mountain view, ca), and those against cd103, tcr␣␤, and tcr␥␦ were from coulter-immunotech (marseille, france). purified hp-3b1 anti-cd94 was biotinylated according to standard procedures. this antibody recognizes cd94 alone or associated with members of the nkg2 family. 30 the following anti-nk antibodies were used as supernatants (table 1) for histological studies, endoscopic duodenal biopsy specimens were fixed in 10% formalin. villous atrophy was graded as moderate (crypt-to-villus ratio ͼ 1) or severe (crypt-to-villus ratio յ1) and subtotal or total. for immunohistochemical staining with anti-cd94 antibody, endoscopic biopsy specimens were embedded in o.c.t. compound (miles inc., elkhart, in) and snap-frozen in liquid nitrogen. serial cryostat sections, cut at 4 µm, were labeled with purified anti-cd94 antibody or with isotype-matched control immunoglobulin (ig) g2a at 10 µg/ml (coulterimmunotech) and revealed using an indirect immunoperoxidase technique. 44 the number of labeled iels was estimated on serial sections by counting peroxidase-stained cells per 100 ecs. five hundred to 1000 ecs were counted in each section. comparable results were obtained by 2 different investigators (n.p., b.j.). numbers of iels in the different groups were compared using the kruskal-wallis nonparametric test. for immunohistochemical staining with anti-il-15 antibody, samples were fixed in formalin and embedded in paraffin. sections were dewaxed, microwave-heated (2 ϫ 5 minutes at 750 w in citrate buffer, ph 6), rinsed in phosphate-buffered saline, and then incubated with anti-il-15 antibody (2 µg/ml) overnight at 4°c. staining was revealed using cy3-labeled goat anti-mouse igg (amersham, buckinghamshire, england). for control staining, a saturating concentration of recombinant il-15 (r&d systems) was added before staining with anti-il-15 antibody. iels were isolated from surgical intestinal samples, as described previously, 45 or from biopsy specimens. five to 6 endoscopic biopsy specimens were pooled and incubated under constant shaking for 30 minutes at 37°c in rpmi 1640 (gibco-brl, life technology, cergy-pontoise, france) containing 1% dialyzed fetal calf serum (gibco-brl), 1.5 mmol/l mgcl 2 , and 1 mmol/l ethylene glycol-bis(␤aminoethyl ether)-n,n,nј,nј-tetraacetic acid. the supernatant containing the iels was passed through a nylon filter (falcon 2360; becton dickinson), and iels were washed twice in phosphate-buffered saline supplemented with 5% human ab serum. pbls were isolated on ficoll-hypaque (lymphoprep; nycomed pharma, oslo, norway) according to standard procedures. iels isolated from 6 normal surgical intestinal samples and pbls from 3 normal individuals were labeled with anti-cd94 and anti-cd8 and sorted with a fluorescenceactivated cell sorter (facs) vantage flow cytometer (becton dickinson). next, 1 ϫ 10 5 to 2 ϫ 10 5 cd94-negative iels (90%-95% tcr␣␤ ϩ ) and cd94-negative cd8-high pbls (98% tcr␣␤ ϩ ) were cultured in rpmi plus 10% fetal calf serum for 18-48 hours in round-bottom microwells, alone or in the presence of 10 ng/ml il-15. for tcr-mediated stimulation, cells were cultured in round-bottom microwells precoated overnight with 10 µg/ml anti-cd3 antibody. kato, a human ec line of gastric origin, was cultured in rpmi with 10% fetal calf serum and antibiotics. after isolation, lymphocytes were first incubated for 10 minutes in phosphate-buffered saline with 10% human ab serum to block fc receptors. for each staining, 5 ϫ 10 4 to 1 ϫ 10 5 cells were used. for triple membrane staining with directly conjugated antibodies, cells were labeled with a combination of antibodies conjugated to red (pe) or green (fitc) fluorescent dyes or to biotin according to standard procedures. biotinylated antibodies were revealed with streptavidin tricolor (caltag laboratories, san francisco, ca). for single staining, supernatants containing antibodies were used at a 1:4 dilution and revealed using fitc-conjugated fabј2 rat anti-mouse igs at a 1:200 dilution (jackson immunoresearch, west grove, pa). for multicolor analysis with antibody-containing supernatants, cells were first incubated with a 1:4 dilution of antibodycontaining supernatant, washed twice, and stained by an fitc-conjugated rat fabј2 anti-mouse ig. free antibody sites were then blocked with a 50 µg/ml solution of purified mouse igs (sigma chemical co., st. louis, mo) for 10 minutes before adding pe and biotin-conjugated antibodies for 15 additional minutes. biotinylated antibodies were revealed by a final incubation in streptavidin tricolor. fluorescence was analyzed on a facscan (becton dickinson). for analysis of cd94-positive lymphocytes, statistical quadrants were set so as to score as negative more than 99% of control-stained cells (cells stained with pe-conjugated igg2a control isotype; coulter immunotech). cd94-positive cells include cd94-low and cd94-high cells, as previously reported for nk cells stained with either anti-cd94 or hla-e tetramers. 29, 30 the cd94-low cells, which include cells expressing cd94/nkg2c complexes, partially overlap with cd94negative cells, and thus their frequency might have been underestimated. the cd94-high cells include cells expressing cd94/nkg2a complexes and are brightly stained. 30, 31 results expression of nk receptors was compared using flow cytometry on iels isolated from 15 surgical samples of histologically normal small intestine and on pbls obtained from 14 healthy volunteers. as shown in table 1 , a substantial fraction of iels, mostly cd3 ϩ t cells, expressed nk receptors. two nk receptors of the c-lectin-like family were expressed by a large fraction of normal human iels. nkr-p1a (cd161), a receptor with no defined ligand that is expressed by nk cells and a subset of peripheral t cells in humans, 46 was present on 58% of intraepithelial t lymphocytes but only on 14% of blood t lymphocytes. this receptor can costimulate tcr-mediated signaling. 47 another c-lectin-like nk receptor is cd94, a glycoprotein that associates with molecules of the nkg2 family to form heterodimers that recognize the nonpolymorphic mhc class i molecule hla-e and transduce either inhibitory or activating signals. 31, 48 cd94 was expressed by 27% of t-iels, a result comparable to that obtained by eiras et al., 24 and by 11% of blood t lymphocytes. two nk receptors expressed by pbls were virtually absent among iels: cd57, a marker of unknown function, and cd16, an fc receptor involved in antibodydependent cytotoxicity. on the other hand, 2 nk receptors not found on blood t cells, pen5, a receptor of unknown function, 38 and nkp46, an activatory receptor of the ig-like family, 39, 49 were expressed by 13% and 7% t-iels, respectively. other nk receptors were expressed at a similar frequency by both iels and pbls. a small subset of iels (2%-10%) expressed nk receptors of the ig-like kir family, mainly kir2dl/s receptors stained by the nkvsf1 antibody. these kir receptors are biased toward recognition of hla-cw3 and hla-bw4 class i molecules and transduce either inhibitory (kir2dl) or activating (kir2ds) signals to nk cells. 46 the frequency of kir ϩ iels was comparable to that of kir ϩ pbls, although the latter were mainly cd3-negative nk cells and the former included up to 50% cd3-positive t lymphocytes. in addition, cd56, an nk marker of unknown function, was expressed by a fraction of iels, as reported previously. 22, 24 the frequencies of cd56positive cells among t-iels and t-pbls (pbls expressing the tcr) were similar, approximately 7%. using multicolor analysis we determined the pattern of coexpression of several nk receptors with cd94 on iels of 4 different individuals. the results indicated that, with the exception of cd161, which is present on most iels, only a minor fraction of cd94 ϩ iels expressed the various other nk receptors ( figure 1) . conversely, cd94, which was only expressed by 27% of t-iels, was present on most iels expressing kir2dl/s, cd161, cd56, pen5, and nkp46 ϩ iels, indicating that these nk receptors tended to be coexpressed with cd94 (data not shown). we first studied infiltration of the epithelium by cd3 ϩ intraepithelial t cells in active celiac disease. as expected, the surface epithelium was massively infiltrated by cd3 ϩ iels in both children (91 ϯ 31 iels/100 ecs vs. 20 ϯ 13 iels/100 ecs in normal control biopsy specimens and 23 ϯ 15 iels/100 ecs in control biopsy specimens with villous atrophy of other origin) and adults (100 ϯ 13 iels/100 ecs vs. 34 ϯ 20 iels/100 ecs in normal control biopsy specimens). in preliminary immunohistochemical studies of intestinal frozen tissue sections, the pattern of staining of iels with anti-cd56, -pen5, -p46, -p58, and -cd94 antibodies was compared in 4 adult patients with active celiac disease and in 4 normal controls. cd56, p58, pen5, and nkp46 were expressed by less than 5% of iels in either group, and nkr-p1a was expressed by 70%-80% of iels in both groups (not shown). strikingly, cd94 was the only receptor whose frequency was markedly increased. to confirm the increase in cd94 ϩ iels on a larger population of celiac patients and to study its specificity and its relationship with disease activity, we examined frozen sections from 24 children and 27 adults at various stages of celiac disease and from 32 controls (21 children, 12 with normal villi and 9 with villous atrophy not related to celiac disease, and 11 adults with normal villi). counts of cd94 ϩ iels/100 ecs were determined. as shown in figure 2 , the number of cd94 ϩ iels/100 ecs was considerably increased over normal age-matched controls during active celiac disease in children (40 ϯ 11 vs. 5 ϯ 2; p ͻ 0.001) as well as in adults (40 ϯ 16 vs. 10 ϯ 7; p ͻ 0.001). in contrast, the numbers of cd94 ϩ iels/100 ecs in celiac patients on gfd (children, 13 ϯ 8; adults, 15 ϯ 7) were not significantly different from those in normal controls (children, 5 ϯ 2; adults, 10 ϯ 7) (figure 2a and c) . furthermore, a longitudinal follow-up of 4 children showed that high counts of cd94 ϩ iels before gfd returned to normal after gfd and recovery of a normal villous architecture ( figure 2b ). altogether, these results indicate that there is a selective increase in the number of cd94 ϩ iels that is dependent on the activity of the disease. we next asked whether the increase in the number of cd94 ϩ iels was specific to celiac disease by examining control patients with villous atrophy or patients with inflammatory bowel disease unrelated to celiac disease. no increase in the number of cd94 ϩ iels/100 ecs was observed in 4 children with moderate villous atrophy because of giardiasis (n ϭ 1), cow's milk intolerance (n ϭ 1), and intestinal graft rejection (n ϭ 2), or in 5 children with total or subtotal villous atrophies because of epithelial dysplasia (n ϭ 1), autoimmune enteropathy (n ϭ 2), and graft-versus-host disease (nϭ2). only one 2-year-old child with graft-versus-host disease and subtotal villous atrophy had a moderately increased number of cd94 ϩ iels (28 iels/100 ecs) (figure 2a) . finally, no increase in the number of cd94 ϩ iels/100 ecs was observed in 2 pediatric biopsy samples with severe crohn's disease showing an important inflammatory infiltrate in the lamina propria but no villous atrophy (4 iels/100 ecs and 8 iels/100 ecs). to determine whether the increase in cd94 ϩ iels simply reflected the overall increase of iels or whether it was selective, flow-cytometric experiments were performed to precisely measure the proportion of cd94 ϩ iels among total iels. iels were isolated from biopsy specimens of 15 adult celiac patients and compared with those isolated from 6 adult controls (figure 3 ). the percentage of cd94 ϩ lymphocytes among total iels was markedly increased in patients with active celiac disease (74% ϯ 13%; n ϭ 8) over controls without villous atrophy (23% ϯ 9%; n ϭ 3), controls with villous atrophy unrelated to celiac disease (adults 9% ϯ 12%; n ϭ 3), or celiac patients receiving gfd (36% ϯ 13%; n ϭ 7) (p ͻ 0.001) ( figure 3a and b) . additional controls included biopsy specimens from 2 children, 1 with crohn's disease and 1 with autoimmune enteropathy, that also showed normal frequencies of cd94 ϩ iels (22% and 18%, respectively; data not shown). the frequencies of cd94 ϩ iels in control biopsy specimens without villous atrophy and in biopsy specimens from patients on gfd were similar to that observed in iels isolated from surgical samples (23% ϯ 9% [n ϭ 3] vs. table 1 and figure 3b ). despite a marked relative increase of cd94 ϩ iels in active celiac disease, the percentage of iels expressing other nk receptors was within the normal range (table 2 ). these results show that untreated celiac disease is associated with a marked selective increase in cd94 ϩ iels. in addition, flow-cytometric analysis showed that the majority of cd94 ϩ iels in active celiac disease, as well as in controls, expressed the ␣␤ tcr (celiac disease: 75% ϯ 15%, n ϭ 6; control biopsy specimens: 83% ϯ 15%, n ϭ 3; surgical controls: 81% ϯ 13%, n ϭ 15) (figure 4) . only a small and variable fraction of cd94 ϩ iels expressed the ␥␦ tcr in active celiac disease (12% ϯ 10%, n ϭ 6), a proportion comparable to that found in controls (control biopsy specimens: 19% ϯ 15%, n ϭ 3; surgical controls: 19% ϯ 24%, n ϭ 15) ( figure 4) . cd94 is usually expressed as a heterodimer associated with various members of the nkg2 family, some of which inhibit and others enhance cell activation. 30 flowcytometric analysis using an antibody directed against the inhibitory isoform nkg2a 37 showed that, in active celiac disease, 5% ϯ 5% of cd94 ϩ iels expressed nkg2a (n ϭ 4), whereas the frequency in normal controls was 29% ϯ 18% (n ϭ 5). thus, the increase in cd94 ϩ iels is not related to an expansion of cells expressing the cd94 ϩ nkg2a ϩ inhibitory receptor. finally, to investigate whether the cd94 receptor might participate to the interactions between iels and ecs, we examined the expression of its ligand, the nonclassical mhc molecule hla-e, on the surface of ecs. hla-e was constitutively expressed on the surface of the kato ec line and upon ifn-␥ stimulation in kato as well as in ht29 intestinal cell lines ( figure 5 and data not shown, respectively). previous studies indicated that cd94 could be induced on pbls, but that induction required the prolonged combination of tcr engagement and exposure to il-15. 50 this led us to examine the effect of tcr stimulation and il-15, alone or in combination, on cd94 expression by iels. iels were isolated from histologically normal surgical samples, and cd94-negative cells were further purified by cell sorting. cd94-negative iels, which included 90%-95% tcr␣␤ ϩ cd8 ϩ cells, were stimulated during a short period of time, 18-36 hours, with anti-cd3 antibodies and/or il-15 before multicolor flow-cytometric analysis using anti-cd8 and cd94. figure 6 shows that il-15 alone, or tcr stimulation alone, rapidly and selectively induced cd94 on up to 40%-50% of iels, whereas less than 10% of unstimulated cells induced cd94. induction of cd94 was detected as early as 18 hours after culture and is shown at 24 hours. the prompt and important increase in the frequency of cd94 expression, in the absence of changes in cell recovery (data not shown), rules out the possibility of selective expansion or survival of a minor subset of cd94 ϩ contaminants among the facs-purified cells. adding anti-cd3 antibody did not significantly enhance the effect of il-15 in 6 separate experiments. in contrast, expression of cd56 and cd16 was consistently unaffected (data not shown). in addition, within a time frame ranging from 18 to 36 hours in 3 different experiments, ifn-␥ had little effect on cd94 expression, and other cytokines that may be present in normal and/ or inflammatory intestine, il-12, tnf-␣, and transforming growth factor ␤, had no detectable effect alone or in combination with tcr stimulation. we confirmed in 3 separate experiments that, within this time frame, neither il-15 alone nor the combination of il-15 and tcr stimulation induced cd94 on cd8-high pbls (which include 98% tcr␣␤ ϩ ), as previously reported. 50 furthermore, we showed that even the minor subset of cd45ro ϩ cd8 ϩ pbls that may be functionally closer to iels than cd45ro ϫ cd8 ϩ pbls displayed little induction of cd94 (5%-7%) ( figure 6 ). thus, in marked contrast with both ro ϩ and ro ϫ cd8 ϩ pbls, a large fraction of iels promptly expressed cd94 in response to il-15 alone and, to a lesser degree, to tcr stimulation alone. finally, to examine whether il-15 might play a role in the expression of cd94 by iels in celiac disease, we examined the presence of il-15 in intestinal tissue sections. the il-15 protein was mainly detected in ecs of the villi in the normal intestine and was strongly expressed by crypt and villi ecs as well as by numerous lamina propria cells in active celiac disease (figure 7 ). we show in this study that most normal human t-iels express at least 1 nk receptor and that the ␣␤ tcr ϩ iels that expand in untreated celiac disease specifically and selectively express the nk receptor cd94. we also provide functional evidence that increased expression of cd94 in active celiac disease may be the consequence of local release of il-15, a potent cytokine produced in situ by intestinal ecs and activated macrophages (reviewed by waldmann and tagaya 51 ) and/or direct tcr activation. t cells that express various receptors of the nk lineage have been reported in mice and humans, in normal as well as in pathological states. 23,52-59 nk receptor expression may be part of a genetic program of differentiation, 52 or alternatively it may be imparted on activation of mature t cells. 50, 60 in the present study we confirmed the expression of cd56 and cd94 by 7% and 27% t-iels, figure 6 . il-15 alone induces the expression of cd94 by iels but not by pbls. altogether, 1 ϫ 10 5 to 2 ϫ 10 5 facs-sorted cd94 ϫ cd8 ϩ iels and pbls were cultured in rpmi plus 10% fetal calf serum alone or in the presence of saturating amounts of il-15 in round-bottom microwells. for tcr-mediated stimulation, cells were cultured in round-bottom microwells precoated overnight with 10 µg/ml anti-cd3. cells were analyzed by flow cytometry after 24 hours (iels) or 48 hours (pbls) in culture. pbls are gated on cd8 ϩ cd45ro ϫ (top row) and cd8 ϩ cd45ro ϩ (middle row), and iels are gated on cd8 ϩ (bottom row). quadrants for statistical analysis were set so as to score as negative more than 99% of control-stained cells. respectively, and the paucity of cd16 expression, as previously reported on t-iels. 22, 24 we determined for the first time the pattern of expression of an extended set of nk receptors by t-iels and compared it with that of t-pbls. the results showed that iels and pbls markedly differed in the frequency of t cells expressing nk receptors as well as in the nature of the nk receptors expressed. thus, more than 60% of t-iels vs. only 20%-30% of t-pbls expressed at least 1 nk receptor. nkr-p1a was expressed on a much larger subset of t-iels than t-pbls, and pen5 and nkp46 were expressed by a subset of t-iels but not by t-pbls. conversely, cd57 was expressed by a subset of t-pbls but not by t-iels. these nk receptor-expressing t-iels, or a subset of them, might be the counterpart to the mouse nk receptor-expressing t-iels, which are thought to differentiate in the gut wall and expand in response to self antigens. 29, 61, 62 alternatively, the expression of nk receptors on human t-iels could be the consequence of particular activation events. in any case, our data suggest that the gut epithelium is a unique environment that favors the differentiation and/or expansion of particular subsets of nk receptor-expressing t cells in humans as well as in mice. some nk receptors, such as cd16, endow t-iels with nk-like properties 23 that may contribute to the innate defense of the intestinal epithelium. [25] [26] [27] [28] other nk receptors, such as cd94, [63] [64] [65] function as regulators of tcr-mediated signaling by modulating the activation threshold of the tcr. accordingly, our recent studies of t-cell clones derived from cd8 ϩ tcr␣␤ ϩ iels indicate that cd94 can modulate tcr-mediated cytotoxicity (unpublished data, bana jabri and nadine cerf-bensussan, january 1999). these functional properties may profoundly influence the outcome of the local immune responses, and it is therefore of particular significance that distinct patterns of nk receptors may be expressed in particular types of inflammatory processes of the intestine. a massive increase in the frequency of cd8 ϩ tcr␣␤ ϩ iels was previously reported in active celiac disease and found to subside after gluten eviction. 5, 20 this iel expansion does not seem to be an obligatory consequence of the underlying th1 response. 17 in fact, iel expansion in celiac disease could be dissociated from the th1 response of the lamina propria because treatment with ctl4-ig could block ifn-␥ production without inhibiting the migration and/or expansion of iels that occurred within 12 hours of gluten challenge. 66 these observations suggested that gluten itself might, directly or indirectly, cause injury to the gut epithelial microenvironment, which in turn would induce the expansion and/or migration of iels. in this context, it was important to further characterize the nature of the expanding cells, particularly with respect to their pattern of nk receptor expression, because nk receptors may regulate the interactions of iels with the altered epithelium of celiac patients. our results indicated that active celiac disease is associated with a striking increase in cd8 ϩ tcr␣␤ ϩ iels expressing the cd94 nk receptor. this increase was specific of active celiac disease and was not observed in various other types of villous atrophies, including autoimmune enteropathy, or in crohn's disease. furthermore, it was tightly associated with disease activity because it subsided after gluten eviction. a remarkable finding of this study was that the induction of nk receptors seemed to be a selective process because cd94 was the only nk receptor tested whose expression was markedly increased in active celiac disease. this observation suggested that induction of cd94 could be dissociated from that of other nk receptors in vivo. our in vitro studies further confirmed that cd94 induction was selective because cd94, but not the other nk receptors tested (such as cd16 and cd56, data not shown), could be induced on cd8 ϩ t-iels upon tcr and il-15 stimulation. a selective induction of cd94 upon treatment with anti-tcr and il-15 was also recently reported for pbls. 50 however, there seem to be some interesting differences between the response of pbls and iels to these stimuli. cd94 expression by pbls required the combined stimulation by anti-tcr and il-15 over a period of 4-6 days, 50 but iels expressed cd94 as early as 18 hours after stimulation with il-15 alone, and, to a lesser degree, with tcr stimulation alone. this unusual property of iels was not shared by the minor subset of cd45ro ϩ cd8 ϩ pbls, which is functionally closer to iels than the bulk of pbls, suggesting that the ability to rapidly induce cd94 upon exposure to il-15 is not a common feature of memory cd8 ϩ t cells. a possible explanation for these differences is that, by prior exposure to antigens in the gut microenvironment, t-iels were ''primed'' for cd94 induction. our in vitro studies further indicated that cytokine-mediated induction of cd94 was the restricted property of il-15 because other inflammatory cytokines such as il-12, ifn-␥, and tnf-␣ consistently failed to induce high levels of cd94. il-15 can be secreted by the intestinal epithelium 33 and is a potent activator of human iels, stimulating their proliferation, cytotoxicity, and ifn-␥ secretion. 67 in addition, il-15 favors migration of lymphocytes 68 and induces expression of several chemokines and chemokine receptors on lymphocytes. 69 altogether, these findings suggest a scenario whereby after tcr-mediated activation and exposure to the cytokines of the epithelial microenvironment, including il-15, celiac disease iels not only up-regulate cd94 expression, but also increase their cytotoxic properties through the induction of perforin and granzyme b. 70 our finding that il-15 is abundant in the intestinal epithelium of celiac disease is consistent with this scenario. the local events leading to tcr engagement remain to be elucidated. one could speculate that celiac disease iels recognize as yet undefined antigens induced on the intestinal epithelium in response to gliadin. such a mode of response to stress has recently been suggested for tcr␥␦ ϩ iels using the variable v␦1 region, a subset of iels known to be increased in celiac disease 5, 17, 18 and that respond to the stress-induced mica/micb hla molecules. 19 the selective induction of cd94 expression by celiac disease iels also points to a specific function of the cd94 receptor and its ligand, the nonpolymorphic mhc class i molecule hla-e, 31, 32, 48 in the immune processes associated with celiac disease. although the surface expression of hla-e on intestinal cells has not yet been shown with the available reagents, significant levels of messenger rna have been reported. 71 furthermore, we found constitutive membrane expression of hla-e and marked up-regulation by various cytokines, including ifn-␥, on intestinal ec lines such as ht29 colonic cells ( figure 5 and data not shown). depending on the associated nkg2 isotype, the engagement of cd94 induces a range of activating or inhibitory effects on proliferation, target killing, and cytokine secretion by nk cells. 71 in cd94 ϩ t cells, signaling via cd94 can modulate, positively or negatively, activation through the tcr, depending on the associated nkg2 molecule. 54, 57, 59, 72 because cd94 ϩ iels display cytotoxic activity and ifn-␥ secretion after tcr stimulation (unpublished data, bana jabri and nadine cerf-bensussan, january 1999), engagement of cd94 by hla-e may modulate the consequences of tcr engagement. interestingly, we found that the proportion of cd94 ϩ t-iels expressing the cd94/nkg2a inhibitory receptor was low in celiac disease. further studies are in progress to define whether the nkg2 isotypes expressed by cd94 ϩ iels in celiac disease belong to the activating group and whether they modulate the cytotoxicity of iels. in conclusion, we report a selective and specific increase in the number of tcr␣␤ ϩ iels that express the hla-e-specific nk receptor cd94 during the active phase of celiac disease. we show that il-15, which is expressed by intestinal ecs, or tcr engagement can selectively induce cd94 on iels. these findings suggest a scenario whereby iels primed in the celiac disease microenvironment may be locally regulated positively or negatively by il-15 and hla-e. thus, ec injury and the resulting intestinal dysfunction in celiac disease may not only depend on the activation of gliadin-specific, dq2-, the pathogenesis of celiac disease evidence for a primary association of celiac disease to a particular hla-dq ␣/␤ heterodimer identification of tissue transglutaminase as the autoantigen of celiac disease activated t lymphocytes in the celiac lesion: non-proliferative activation (cd25) of cd4 ϩ ␣/␤ cells in the lamina propria but proliferation (ki-67) of ␣/␤ and ␥/␦ cells in the epithelium numbers of t cell receptor (tcr) ␣␤ ϩ but not of tcr ␥␦ ϩ intraepithelial lymphocytes correlate with the grade of villous atrophy in coeliac patients on a long term normal diet tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived t cells in celiac disease selective deamidation by tissue transglutaminase strongly enhances gliadin-specific t cell reactivity heterogeneous reactivity patterns of hla-dqrestricted, small intestinal t-cell clones from patients with celiac disease gluten specific, hla-dq restricted t cells from coeliac mucosa produce cytokines with th1 or th0 profile dominated by interferon ␥ quantitation of intraepithelial lymphocytes in human jejunum classification of intractable diarrhea in infancy using clinical and immunohistological criteria time dose responses of celiac mucosae to graded oral challenges with frazer fraction-iii of gliadin gluten, major histocompatibility complex, and the small intestine. a molecular and immunobiologic approach to the spectrum of gluten sensitivity ('celiac sprue') intestinal lymphoma and enteropathy abnormal intestinal intraepithelial lymphocytes in refractory sprue subsets of intraepithelial lymphocytes in normal intestine and in coeliac disease intraepithelial t cells of the tcr ␥/␦ ϩ cd8 ϫ and v ␦ 1/j ␦ 1 ϩ phenotypes are increased in coeliac disease expression of disulfide-linked and non-disulfide-linked forms of the t cell receptor ␥/␦ heterodimer in human intestinal intraepithelial lymphocytes recognition of stress-induced mhc molecules by intestinal epithelial ␥␦ t cells effect of diet and age on jejunal and circulating lymphocyte subsets in children with coeliac disease: persistence of cd4 ϫ 8 ϫ intraepithelial t cells through treatment occurrence of large granular lymphocytes and natural killer cells in the epithelium of the gut distinguishes two different coeliac diseases intra-epithelial lymphocytes. evidence for regional specialization and extrathymic t cell maturation in the human gut epithelium complexity of the mouse gut t cell immune system: identification of two distinct natural killer t cell intraepithelial lineages flow cytometry description of a novel cd3 ϫ /cd7 ϩ intraepithelial lymphocyte subset in human duodenal biopsies: potential diagnostic value in coeliac disease characteristics of natural killer cells in the murine intestinal epithelium and lamina propria intraepithelial leukocytes contain a unique subpopulation of nk-like cytotoxic cells active in the defense of gut epithelium to enteric murine coronavirus spontaneous cytotoxicity of human intraepithelial lymphocytes against ec tumors spontaneous cytotoxicity of intestinal intraepithelial lymphocytes: clues to the mechanism extrathymic t cell differentiation structure and function of the cd94 c-type lectin receptor complex involved in recognition of hla class i molecules hla-e binds to natural killer cell receptors cd94/ nkg2a, b, and c hla-e is a major ligand for the natural killer inhibitory receptor cd94/nkg2a intestinal ecs both express and respond to interleukin 15 management of infantile gastroenteritis small bowel transplantation in children: an immunohistochemical study of intestinal grafts intractable diarrhea of infancy with epithelial and basement membrane abnormalities the cd94 and nkg2-a c-type lectins covalently assemble to form a natural killer cell inhibitory receptor for hla class i molecules developmental regulation of a mucin-like glycoprotein selectively expressed on natural killer cells molecular cloning of nkp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity existence of both inhibitory (p58) and activatory (p50) receptors for hla-c molecules in human natural killer cells physical and functional independency of p70 and p58 natural killer (nk) cell receptors for hla class i: their role in the definition of different groups of alloreactive nk cell clones the natural killer cell receptor specific for hla-a allotypes: a novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains and is expressed as a 140-kd disulphide-linked dimer il-12-induced upregulation of nkrp1a expression in human nk cells and consequent nkrp1a-mediated down-regulation of nk cell activation same peculiar subset of hml1 ϩ lymphocytes present within normal intestinal epithelium is associated with tumoral epithelium of gastrointestinal carcinomas intraepithelial lymphocytes of human gut: isolation, characterisation and study of natural killer activity nk cell receptors nkr-p1a) costimulation of cd1d-dependent activation of human t cells expressing invariant v ␣ 24 j ␣ q t cell receptor ␣ chains hla-e-bound peptides influence recognition by inhibitory and triggering cd94/nkg2 receptors: preferential response to an hla-g-derived nonamer p46, a novel natural killer cell-specific surface molecule that mediates cell activation hla class i-specific inhibitory receptors in human t lymphocytes: interleukin 15-induced expression of cd94/nkg2a in superantigen-or alloantigen-activated cd8 ϩ t cells the multifaceted regulation of interleukin-15 expression and the role of this cytokine in nk cell differentiation and host response to intracellular pathogens mouse cd1-specific nk1 t cells: development, specificity, and function t cell clones expressing the natural killer cell-related p58 receptor molecule display heterogeneity in phenotypic properties and p58 function cytolytic t lymphocytes displaying natural killer (nk)-like activity: expression of nk-related functional receptors for hla class i molecules (p58 and cd94) and inhibitory effect on the tcr-mediated target cell lysis or lymphokine production superantigendependent, cell-mediated cytotoxicity inhibited by mhc class i receptors on t lymphocytes abnormal development of intestinal intraepithelial lymphocytes and peripheral natural killer cells in mice lacking the il-2 receptor ␤ chain major histocompatibility complex class i molecules modulate activation threshold and early signaling of t cell antigen receptor-␥/␦ stimulated by nonpeptidic ligands expression of hla class i-specific inhibitory natural killer cell receptors in hivspecific cytolytic t lymphocytes: impairment of specific cytolytic functions control of self-reactive cytotoxic t lymphocytes expressing ␥ ␦ t cell receptors by natural killer inhibitory receptors inhibitory mhc class i receptors on nk cells and t cells selection of intraepithelial lymphocytes with cd8 ␣/␣ co-receptors by self-antigen in the murine gut immune deviation of 2c transgenic intraepithelial lymphocytes in antigen-bearing hosts cd94/nkg2 inhibitory receptor complex modulates both anti-viral and anti-tumoral responses of polyclonal phosphoantigen-reactive v␥9v␦2 t lymphocytes c-type lectin-like receptors in peptide-specific hla class i-restricted cytotoxic t lymphocytes: differential expression and modulation of effector functions in clones sharing identical tcr structure and epitope specificity triggering of effector functions on a cd8 ϩ t cell clone upon the aggregation of an activatory cd94/kp39 heterodimer blockage of t-cell costimulation inhibits t-cell action in celiac disease interleukin 15 is a potent stimulant of intraepithelial lymphocytes activation of peripheral blood t cells by interaction and migration through endothelium: role of lymphocyte function antigen-1/intercellular adhesion molecule-1 and interleukin-15 il-15 induces the expression of chemokines and their receptors in t lymphocytes evidence that intestinal intraepithelial lymphocytes are activated cytotoxic t cells in celiac disease but not in giardiasis differential expression of hla-e, hla-f, and hla-g transcripts in human tissue association of dap12 with activating cd94/nkg2c nk cell receptors inhibitory mhc class i receptors on nk and t cells: a standard nomenclature (letter) faculté necker, 156, rue de vangirard, 75730 paris cedex 15, france. e-mail: cerf@necker.fr. supported by grants from l'association pour la recherche contre le cancer (contrats arc 9216, 9962), assistance publique hôpitaux de paris (phrc aom96082), and la société nationale française de gastroentérologie. the authors thank a. beavis for help with cell sorting, m. leborgne for immunohistochemical staining or dq8-restricted lamina propria cd4 ϩ cells, but may also be regulated or mediated by cd94 ϩ tcr␣␤ ϩ iels. key: cord-348283-7xorq5ce authors: naz, anam; shahid, fatima; butt, tariq tahir; awan, faryal mehwish; ali, amjad; malik, arif title: designing multi-epitope vaccines to combat emerging coronavirus disease 2019 (covid-19) by employing immuno-informatics approach date: 2020-07-10 journal: front immunol doi: 10.3389/fimmu.2020.01663 sha: doc_id: 348283 cord_uid: 7xorq5ce a recent pandemic caused by a single-stranded rna virus, covid-19, initially discovered in china, is now spreading globally. this poses a serious threat that needs to be addressed immediately. genome analysis of sars-cov-2 has revealed its close relation to sars-coronavirus along with few changes in its spike protein. the spike protein aids in receptor binding and viral entry within the host and therefore represents a potential target for vaccine and therapeutic development. in the current study, the spike protein of sars-cov-2 was explored for potential immunogenic epitopes to design multi-epitope vaccine constructs. the s1 and s2 domains of spike proteins were analyzed, and two vaccine constructs were prioritized with t-cell and b-cell epitopes. we adapted a comprehensive predictive framework to provide novel insights into immunogenic epitopes of spike proteins, which can further be evaluated as potential vaccine candidates against covid-19. prioritized epitopes were then modeled using linkers and adjuvants, and respective 3d models were constructed to evaluate their physiochemical properties and their possible interactions with ace2, hla superfamily alleles, tlr2, and tlr4. a rapid increase in the human population and its mobility has led to urbanization and subsequent climate and ecological changes, catering to emerging infectious diseases that galvanize an implacable threat to human health around the world (1) . the human race has encountered multiple bacterial and viral pathogens, some being inconsequential while others causing global chaos. interestingly, before the twenty-first century, human coronaviruses were thought to be trivially harmful, causing only common cold in healthy individuals (2) . coronaviruses have an enveloped positive-sense rna genome comprising about 25-32 kilobases. they have been identified in multiple mammalian hosts, including dogs, cats, bats, camels, pigs, and civets (3) . according to centers for disease control and prevention (cdc), common human infecting coronaviruses include 229e coronavirus, nl63 coronavirus, oc43 beta coronavirus, hku1 coronavirus, mers-cov, sars-cov, and the recently emerged deadly coronavirus disease 2019 . the first four account for 10-30% of upper respiratory tract infections in human adults. while the latter three have emerged as perpetual challenge for the scientific community. in november 2002, an outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in guangdong, china, led to the deaths of around 774 out of ∼8,000 infected individuals from 37 different countries (4) . common symptoms in sarsinfected individuals were documented as cough, fever, dyspnea, and occasional diarrhea. although sequence analysis of the virus depicted that bats were its hosts, human-to-human transmission was also observed (5, 6) . likewise, in 2012, the emergence of middle east respiratory syndrome coronavirus (mers-cov) was reported in saudi arabia (7) . the symptoms included atypical pneumonia along with gastrointestinal problems and kidney failure. as a result, out of 2,494 reported cases, 858 patients have died to date as of november 2019 (world health organization report). in december 2019, covid-19 was initially encountered in wuhan, china, and has now rapidly spread to multiple countries. the affected individuals exhibit mild symptoms that turn into pneumonia as the illness progresses (8) . according to nature news, as of february 7th, this virus is responsible for infecting about 31,161 humans in china, leading to the death of 630 patients. the majority of the cases tend to have some connection to the seafood and animal market, which indicates the virus is zoologically transmitted. this situation has gained the attention of authorities at both a local and state level and has highlighted an urgent need to devise a method for rapid treatment of the deadly pathogen (9, 10) . recent research has established that the rna genome of recently discovered sars-cov2 comprises of 9,860 amino acids. it features two untranslated regions at both flanking ends while only a single polyprotein encoding open reading frame is present between them. the genome is organized in a sequential manner starting from 5' replicase, and it is followed by structural proteins: the spike, envelope, and nucleocapsid at the n terminal (11) . reportedly, the spike protein acts as multifunctional molecular machinery to mediate viral entry into host cells and is involved in viral transmission. initially, it binds the host cell-surface receptor via the s1 subunit domain and afterwards carries out the fusion of host and viral cell membranes with the help of the s2 domain. a wide variety of host receptors can be recognized by two subsequent domains in s1 region of sars-cov-2, leading to viral attachment. the n-terminal peptide domain (ranges from amino acid 14-305 in the sequence) as well as the c-terminal peptide domain (the receptor binding domain ranging from amino acid number 319 to 541) of the s1 zone have the ability to bind host cell receptors. it has been suggested that sars-cov-2 exploits angiotensin-converting enzyme 2 (ace2) as a cell receptor (10, 12, 13) . outbreaks of infectious disease like covid-19 poses a serious challenge to the scientific community since they usually arise from unrecognized zoonotic sources or due to scarcity data. viruses can emerge by evolving from their animal-restricted form to another form that can infect humans by attainment of their receptors and biosynthetic machinery. a majority of the recently emerging pathogens are difficult to treat due to the lack of specific therapeutic options (14) . so far, no therapeutic vaccine for either sars-cov, mers-cov, or sars-cov-2 currently exists in the market, although some clinical trials are in progress (15) . innovative computational biology approaches have enabled us to obtain immunogenic and highly conserved epitopes from bacterial and viral antigens (16) (17) (18) (19) . both cd4 + and cd8 + epitopes can be used separately or in combination to construct broad spectrum vaccine candidates. the proposed vaccines can combat a wide variety of pathogens and possess the ability to elicit cellular and humoral responses in human hosts. once administered, the mock epitopes from the vaccine are presented by mhc. the presented epitopes are recognized by their corresponding t-cell receptors that proliferates and generates suitable immune responses. considering this, tcell epitopes from deadly pathogens can facilitate t-cell-based vaccine development (cd4 + and cd8 +) . more precisely, a cd4 + -based subunit vaccine usually deals with exogenous antigens that are phagocytosed by apcs and subsequently bind to mhc-ii, which presents them to cd4 + t cells. accordingly, a cd8 + -based t-cell vaccine encompasses endogenous antigens that are degraded by apcs and later presented via mhc-i to cd8 + t cells (17, 19, 20) . epitope-based chimeric/subunit vaccines have many advantages when compared to vaccines produced via conventional vaccinology. for instance, they are cheaper to develop, do not require microbial culturing, and can surpass many wet lab experiments, saving time. they are a safer option, as they do not contain the entire pathogen and are highly specific and stable (21) . nevertheless, due to the presence of mutable hla variants, epitope-based vaccines targeting limited hla alleles usually do not produce the required/equal effect among the human population. hence highly promiscuous epitopes can bind multiple alleles at a time and can ensure the desired immune response among a heterogeneous human population (18) . the current study focuses on finding promiscuous cd4 + and cd8 t + cell epitopes for chimeric covid-19 vaccine development using a variety of web-based tools. the proposed potential vaccine is then checked for its binding affinity with suitable receptors. the surface glycoprotein sequence of the pneumonia virus discovered at the wuhan seafood market (qhd43416.1 from mn908947.3 reference genome) was retrieved from ncbi (22) . to scrutinize required hla binding epitopes, a tepitool from iedb was used (23) . a set of 12 mhc class i super-types (a * 01:01, a * 02:01,a * 03:01, a * 24:02,a * 26:01, b * 07:02, b * 08:01, b * 27:05, b * 39:01, b * 40:01, b * 58:01, and b * 15:01) were used, and the two highest-scoring epitopes (based on percentile rank and ic50 values) for each allele were selected. a percentile rank is calculated by the comparison of the peptide's predicted bindingaffinity against a panel of a variety of peptides randomly selected from the swiss-prot. hence, a lower percentile rank numerical value depicts better binders. additionally, all the predicted peptides were checked for their ic50 value, and those with ic50 ≤ 500 nm were taken into account. specific immune responses are based on cd4 + and cd8 + t cells, and protective vaccines should thus induce specific t-cell responses based on peptides represented by mhc-i and mhc-ii alleles. the rationale behind prioritizing hla binding epitopes is to ensure the specific immune response in infected macrophages. for mhc-ii-binding peptide epitopes, the sevenallele method was used. this selection is based on the median of consensus percentile ranks among the seven commonly encountered dr alleles, namely, hla-drb1 * 03:01, hla-drb1 * 07:01, hla-drb1 * 15:01, hla-drb3 * 01:01, hla-drb3 * 02:02, hla-drb4 * 01:01, and hla-drb5 * 01:01. epitopes with a median consensus percentile rank ≤20.0 were designated as good binders. the scrutiny of promiscuous peptide epitopes was established based on the median of the consensus percentile rank of the seven preselected alleles. for b-cell epitope prediction, bepipred 2.0 from immune epitope database analysis resource (iedb-ar) was used (24) . iedb-ar is linked to iedb and offers computational analysis regarding both b and t cell epitope prediction and their subsequent analysis. bepipred 2.0 works on the basis of a randomly chosen forest algorithm that has been trained on epitopes acquired from antibody-antigen models obtained from interactive protein structures (25) . owing to the significance of spike protein, the selected epitopes were manually screened for their presence in this zone. the epitopes were further examined for antigenic potential via vaxijen version 2.0 (26) . a threshold value of 0.5 was taken into account. non-antigenic peptides (having vaxijen score < 0.5) were discarded, while antigenic epitopes (with threshold value > 0.5) were further prioritized for their immunogenicity. the immune epitope database (iedb) tool for immunogenicity score calculation was used to predict immunogenicity scores for all mhc-i predicted epitopes (27) . this tool is designed to predict immunogenicity of the peptide based on amino-acid position and properties. immunogenic epitopes were then verified for their presence in iedb database. shortlisted top-scoring epitopes were checked for their binding affinity with each other for determining the final sequence of the chimeric vaccine. the epitopes were analyzed using a haddock web server (guru interface) (28) . clusters representing two epitopes, which possessed the highest interaction scores, depicting their maximum interaction, were refined by removing the water molecules, which may hinder their interaction, and then having them dock to the third epitope. likewise, evaluation of clusters with three epitopes was done. the refined and the highest-scoring cluster was docked to the fourth epitope to obtain the final sequence. to facilitate the process of vaccine development, a flexible linker ggggs was added between each epitope. this helps to restore protein folding by allowing interaction between different domains (16, 29) . additionally, another linker eaaak was added at the n terminal to separate bi-functional domains. designed vaccines were then tested with different epitopes, including truncated ov-asp-1 protein (residues 10-153) and beta defensin (45 residues long), and constructs having higher antigenicity and that are predicted to produce high antibody titers were added with the multi epitope vaccine construct to the enhance immune response (30) . three different constructs were designed in this study, one comprising the top-scoring cd4 and cd8 epitopes lying in the s1 domain, while another is formed by taking two epitopes from the s1 domain and two from the s2 domain, representing mhc-i and mhc-ii binders. finally, the third one is formed by adding a b-cell epitope to the second one but with a different adjuvant. the final sequences of the chimeric vaccine constructs were screened for its antigenic potential and solubility using antigenpro and solpro (31) . allertop version 2.0 was used to check the probability of the construct to cause an allergic reaction (32) . sequence of the finalized vaccine candidate in fasta format was given as an input to expasy server, in order to calculate various parameters like molecular weight, theoretical pi, half-life of the protein, instability index, amino acid composition, aliphatic index, and gravy (33). secondary structures of the vaccine constructs were predicted using pdbsum (34) . this step was executed to better understand the structures of predicted vaccines. pdbsum is a database that is exclusively designed to show the molecules that build dna or proteins, ligands, and metal ions along with the illustration of graphical representation of their interactions with each other. to generate 3d structures of the vaccine candidates, 3dpro was used (31) . the predicted models were then refined using galaxy refine server (35) . this server is responsible for subjecting the predicted 3d model to structural perturbations and subsequent structural relaxations. it generates five different models. all five models for each vaccine construct were screened for gdt-ha, rmsd, and poor rotamers, and the finest predicted models were taken to the next step. the finalized models were further evaluated using errat scores and ramachandran plot analysis for verification. in order to obtain stabilized vaccine constructs, energy minimization was carried out using online yasara server. yasara deals with molecular-dynamics simulations of the given models in solvent, using an exclusive forcefield that has been derived from amber, whose constraints have been improved to minimalize the impairment done to protein structure during the process of energy minimization (36) . in order to study the binding affinity of the putative vaccine candidates with immune receptors, molecular docking technique was adopted. prioritized vaccine constructs were docked to ace2 receptor (pdb id: 3sci), tlr2 (pdb id: 2z7x), and tlr4 (pdb id: 4g8a). vaccine 3, having a b-cell epitope, was also checked for its interaction with a b-cell receptor (bcr) cd79 (pdb id: 3kg5). for this protein-protein docking validation process, the haadock server (guru interface and refinement interface) was used (28) . additionally, to obtain a graphical illustration of the interactions between vaccine and receptor, pdbsum was used (34) . moreover, in order to verify the binding affinity of our multiepitope peptide vaccines with hla alleles, all our vaccine constructs were docked with class i and class ii superfamily alleles to reveal the interaction of epitopes with mhc alleles when combined as well. hence, for this purpose, class i [hla a * 02 01 (pdb id 4u6y), hla b * 51 01 (pdb id 4mji)] and class ii [hla-drb1 * 1402 (pdb id 6atf)] were used; they represent broad-spectrum peptide-binding repertoires. population coverage of epitopes was determined using iedb for prioritized epitopes, as it helps to determine the percentage population that can respond to the particular epitope and can elicit an immune response against it. initially, 15,181 hla class i epitopes have been predicted within spike glycoprotein of covid-19. scrutiny on the basis of percentile rank filtered 24 peptide epitopes. each of them had a considerable binding affinity for the 12 superfamily alleles. all of these epitopes, along with their features and respective binding alleles, are reported in table s1 . further analysis revealed that 11 predicted epitopes lie within the s1 domain of the spike protein, eight epitopes lie in the n terminal domain (13-317 aa), and three epitopes are in the receptor-binding domain (347-520aa). vaxijen antigenic score prediction at a threshold of 0.5 was used to detect the antigenicity of peptide epitopes. antigenic epitopes tend to trigger a large number of antibody titers to fight the infection. among predicted epitopes of covid-19 virus, six epitopes showed considerable antigenic potential, including five from the n-terminal domain and one from the receptor binding domain. an immunogenicity analysis was then carried out for further filtration, and, consequently, five epitopes were screened out; one of the epitopes lying within n-terminal domain showed relatively less immunogenicity value. out of these five mhc-i epitopes, two epitopes from s1 domain with high antigenic and immunogenicity score were further selected for multiepitope vaccine construction. these were 89 gvyfastek 97 and 50 stqdlflpf 58 . 89 gvyfastek 97 is a part of the n-terminal binding domain with antigenicity and immunogenicity scores of 0.7112 and 0.09023, respectively. epitope 50 stqdlflpf 58 also lies within the n-terminal domain and has an antigenicity and immunogenicity score of 0.6619 and 0.06828, respectively. moreover, another hla class i epitope 733 ktsvdctmy 741 from the s2 domain of the spike proteins was also screened to be potential candidates for multi-epitope vaccine construction. a total of 1,772 unique epitopes against seven drb alleles were identified. twenty (15-mer epitopes) epitopes were screened out via filtration on the basis of median percentile rank <20 ( table s2 ). the major portion of binding energy between a peptide epitope and mhc class ii receptor molecule is delivered through the basic peptide core, comprising ∼9 amino acids in length. nevertheless, the existence of extra amino acids around the basic binding core seems to play a significant role in stable binding even if they do not precisely bind the peptide-bindinggroove of the mhc receptor. the 15-mer epitopes for binding with mhc-ii are thus usually recommended (23) . ten of mhc class ii epitopes (three in the receptorbinding area and seven in the n-terminal domain) were found to be a part of the s1 domain. while considering a total of 10 s1 epitopes, four were found to be highly antigenic (threshold > 0.5). among these, three belonged to the nterminal domain of s1 while 1 was a part of the receptorbinding domain. two epitopes 191 efvfknidgyfkiys 205 and 506 qpyrvvvlsfellha 520 were selected for vaccine 1 construction on the basis of their high antigenic potential. the former belonged to the n-terminal domain with an antigenicity score of 1.0339, while the later was a part of the receptorbinding domain and had an antigenicity score of 0.9109. an epitope 731 mtktsvdctmyicgd 745 from the s2 domain was also prioritized and was used along with the s1 epitope 506 qpyrvvvlsfellha 520 for vaccine 2 construction. to the best of our knowledge, none of the epitopes reported in this study have been previously added to the iedb database. table 1 shows the final epitopes picked for vaccine development. an iedb server was used to identify 34 b cell epitopes. out of these, 11 were found to be antigenic in nature (threshold > 0.5). they were further checked for their allergenicity, and the highly antigenic epitope, found to be non-allergenic in nature ( 369 ynsasfstfkcygvsptklndlcft 393 ), was picked. this epitope was conjugated with the s1 and s2 epitopes along with a beta defensin adjuvant to design the vaccine 3 construct. envelope-affixed spike protein of coronaviruses plays an important role in receptor recognition. several virology studies have been carried out to discover the exact mechanism of receptor binding and subsequent entry into the host cells. the sars-cov-2 spike protein has been found to be 76% identical to the sars-cov urbani stains' spike protein and 80% identical to the bat sarsr-cov zxc21 and zc45 spike protein (37) . the shortlisted epitopes have also been subjected to conservation analysis, hence manifesting cross protection against other species. conservation analysis revealed the high similarity between the prioritized epitopes of the sars-cov-2 spike protein with mers and sars spike protein epitopes ( table 2 ). all our seven epitopes were found to be a part of at least eight viral sequences present on ncbi, while one of the prioritized epitopes, ktsvdctmy, was found to be 100% identical in 43 available coronavirus sequences (table s3) . the finalized epitopes in table 1 were examined for their interactive ability with one another using haddock. all possible combinations of epitopes along with a flexible linker ggggs between them were explored. for vaccine 1, the e1s1-e4s1 combination had the highest haddock refinement score. the binding affinity of the e1s1-e4 s1 combination with the other two epitopes was determined to find the best combination of three epitopes. e1 s1 -e4 s1 -e3 s1 was thus formed. finally, the vaccine construct obtained after combination analysis was e1 s1-e4 s1-e3 s1-e2 s1 (table 3) . similarly, for vaccine 2, e1-s1 -e4 s1 was the first combination, and it was followed by e1s1 -e4 s1-e2 s2 and e1 s1-e4 s1-e2 s2 -e1 s2. each best combinations haddock refinement score vaccine 1 e1 s1-e4 s1 −79.1 +/-2.3 e1 s1 -e4 s1 -e3 s1 −113.2 +/-2.5 e1 s1-e4 s1-e3 s1-e2 s1 −123.7 +/-1.3 vaccine 2 e1 s1-e4 s1 −79.1 +/-2.3 e1 s1-e4 s1-e2 s2 −100.4 +/-1.2 e1 s1-e4 s1-e2 s2-e1 s2 −92.7 +/-2.6 vaccine 3 e4 s1-e5 s1 −128.4 +/-1.7 e4 s1-e5 s1-e2 s2 −96.6 +/-0.4 e4 s1-e5 s1-e2 s2-e1 s2 −96.6 +/-0.4 e4 s1-e5 s1-e2 s2-e1 s2-e1 s1 −77.2 +/-1.3 probable combination lined up for putative vaccine design along with their corresponding haddock scores is present in table 3 . moreover, truncated ov-asp1 (ivvavtgyncpgg kltalerkkivgqnnkyrsdlingklknrngtymprgk nmleltwdcklessaqrwanqcifghsprqqregvgen vyaywssvsveglkktagtdagkswwsklpklyennpsn nmtwkvagqgvlhftq) was attached to the n terminal of both the putative vaccines using another linker, eaaak. the finalized vaccines together with the linkers and adjuvant were 212 amino acids long. ov-asp-1 reportedly has ability to activate antigen-processing cells (apcs) which define its good adjuvanticity for a number of vaccines and antigens (30) . they are thus added in vaccine constructs to improve the efficacy of these new generation subunit vaccines. in order to ensure both cell and humoral mediated responses, a potent b-cell epitope was added to vaccine 2 based on the best docking scores predicting the combination pattern of epitopes. vaccine 3 was created with an order; e4 s1-e5 s1-e2 s2-e1 s2-e1 s1 and the corresponding docking score are enlisted in table 3 . for comparison purposes, another adjuvant betadefensin (giintlqkyycrvrggrcavlsclpkeeqigkcstr grkccrrkk) was added to this combination. beta defensin has previously been reported as a potent adjuvant when conjugated with mers-cov antigens (38) . vaccines containing defensins as adjuvants have been shown, both in vivo and in vitro, to activate the primary innate antiviral immune response and mediate other immunomodulatory activities against a number of viruses, including coronaviruses (38, 39) . vaccine 3, after addition of this adjuvant at the n terminal along with eaaaak and ggggs linkers, consisted of 143 residues. the final combination of epitopes of all three vaccine constructs have been shown in figure 1 . various physiochemical properties were examined for both the constructs. the molecular weight of vaccine 1 is 23235.26 g/mol while the theoretical pi is 9.50, depicting the basic nature of the peptide construct. the instability index ii showed that the construct is stable with a score of 24.79. the gravy (grand average of hydropathy) index was calculated to be −0.479, validating the hydrophilic nature of the construct that can form interactions with surrounding water molecules. the aliphatic index 67.12 illustrated that the construct is thermostable in nature. vaccine 2 has a molecular weight of 23013.07 g/mol, and its theoretical pi is 9.33. hence, this construct was also found to be basic in nature. likewise, instability analysis showed that the protein is stable with a score of 24.50. the gravy index testified the hydrophilic nature of this construct as well (−0.492). the thermostable nature of the construct was established by the value of aliphatic index, 62.97. the predicted values of antigenicity for both the vaccines were found to be 0.883591 and 0.946425, respectively. this ensured highly antigenic nature of the constructs. similarly, the solubility upon overexpression was predicted to be 0.864955 and 0.951926. furthermore, both vaccine constructs designed in this study were designated as non-allergenic by allergenpro. vaccine 3 has a molecular weight of 15084.28 g/mol and its theoretical pi is 9.25. therefore, this vaccine construct was also found to be basic in nature. the gravy index testified the hydrophilic nature of this construct as well (−0.253). the thermostable nature of the construct was established by the value of aliphatic index, 55.87. the predicted values of antigenicity for this particular the vaccine was 0.883570. this ensured highly antigenic nature of the construct. similarly, the solubility upon overexpression was predicted to be 0.806206. furthermore, like both the previous vaccine constructs designed in this study, this vaccine was also found to be non-allergenic by allergenpro. the secondary structure of vaccine 1 includes six helices, 35 beta turns, seven gamma turns, and nine helix-helix interactions. the secondary structure of vaccine 2 has eight helices, 22 beta turns, 12 gamma turns, and nine helix-helix interactions. for vaccine 3, secondary structure consisted of two beta strand, one hairpin, one sheet, four helices, 23 beta turns, 23 gamma turns, and one helix-helix interaction. helix-helix interaction presents facts about different pairs of helices, interacting with each other with the vicinity of the protein structure, whereas beta turns depict frontiers in immunology | www.frontiersin.org four consecutive residues. these four residues are represented by i, i + 1, i + 2, and i + 3. this is possible when the measured distance between the alpha carbon atom of the first residue (i) and alpha carbon atom of the fourth residue (i + 3) is <7 å plus the two residues between them are not helical. a gamma turn comprises of three residues i, i + 1, and i + 2. this is possible when a hydrogen bond is present between the two residues (i.e., i and i + 2). moreover, the phi angle and the psi angle of the second residue i.e., i + 1 lies within a range of 40 degrees in one of the next two cases: (1) classic [phi i + 1(75), psi i + 1(−64)] or (2) inverse [phi i + 1(−79), psi i + 1(−69)]. the 3dpro tool, which works on the basis of ab initio method for predicting tertiary structure, was used to predict three dimensional structures of proposed vaccine constructs. this strategy was adopted due to the lack of fine homolog proteins that could be exploited for homology modeling. the obtained models were then refined via several structure perturbations and subsequent structure relaxations using glaxyrefine server. the obtained best models are shown in figure 2 . the errat score for 3d models of three vaccines were calculated as 74.1379, 67.5676, and 74.2574, respectively. while ramachandran plot analysis showed 97.1% residues in favored region for vaccine 1, 98.1% residues in the favored region for vaccine 2 and 86.5% for vaccine 3 (figure 2) . these analyses authenticated the reliability and stability of the predicted structures. energy minimization by a yasara server was performed. for vaccine 1, the yasara force field was applied to 2,032 atoms. a total of 5,282 water molecules were found. the initial energy was −68794.3kj/mol (z score −1.90), which was minimized to −97974.1 kj/mol (−1.93). for vaccine 2, the yasara force field was applied to 2,006 atoms while the water molecules were 5,208. initial energy was −66687.0 kj/mol (z score −2.08); however, the final energy was 101214.7 kj/mol (z score −1.47). for vaccine 3, the yasara force field was applied to 2,093 atoms while the water molecules were 4,185. initial energy was −53609.9 kj/mol (z score −3.35); however, the final energy was −60374.6 kj/mol (z score −3.16). sars-cov spike protein has been studied previously for its exceptional binding affinity with human ace-2. it should be noted that, structurally, sars-cov-2 and sars-cov spike proteins are highly homologous in nature, sharing 76.5% identical amino acids. atomic level studies between sars-cov and ace-2 show promising interactions between the two, and therefore, owing to the structural and sequence similarity, it is anticipated that an ace-2 blocker might be handy in curbing sar-cov-2 (40) . for vaccine 1 and the ace-2 receptor, therefore, docking was carried out. a haddock server clustered 36 probable structures into seven different clusters, which represented a total of 18.0 % of the water-refined models. the top-scoring cluster had a score of 39.8 +/-29.1 and a z score of −1.6. similarly, for vaccine 2 and the ace-2 receptor, haddock clustered 18 structures in three clusters, which represented 9.0% of the water-refined models. the topscoring cluster had a value of 0.3 +/-9.8 and a z score of −1.3. likewise, for vaccine 3, haddock clustered 22 structures into five clusters, which depicted 11% of the water refined models generated by haddock. here, the best cluster had a score of 147.5 +/-15.0 and a z score of −1.2 (figure 3) . tlr2 and tlr4 are well-studied toll-like receptors that identify both structural and non-structural proteins of the virus and subsequent cytokine production and inflammation. they are present on the surface of cells and are triggered by viral glycoproteins. tlr agonists have the potential to initiate an immune response and actively participate in viral clearance (41) . the prioritized vaccine constructs were therefore also explored for their interaction with toll-like receptors tlr2 and tlr4. vaccine 1 and tlr2 interaction revealed 40 structures in a total of six clusters that represented 20.0% of the water-refined models. the model with the highest score, −4.2 +/-20.8 had a z value of −1.2. likewise, for vaccine 2 and tlr2, haddock clustered 80 structures in 12 clusters, which represented 40.0% of the water-refined models. here, the highest-scoring model had a score of −23.7 +/-12.1 with a z-value of −1.3. for vaccine 3 and tlr2, haddock clustered 136 structures in 10 clusters, which represented 68.0% of the water-refined models. the highest scoring model had a score of −16.7 +/-14.0 with a z-value of −1.8. moreover, haddock clustered 157 structures in 13 clusters to determine vaccine 1 and tlr4 interaction, which represented 78.5% of the water-refined models. the top-scoring model had a score of 37.9 +/-7.8 and a z-value of −2.2, whereas the interaction of vaccine 2 and tlr4 is determined by 47 structures in nine cluster(s), which represents 23.5% of the water-refined models. the top-scoring model had a score of −16.8 +/-23.4 (z-value −1.6). similarly, haddock clustered 93 structures in eight clusters to determine vaccine 3 and tlr4 interaction, which represented 46.5 % of the water-refined models. the top-scoring model had a score of 23.3 +/-5.7 and a z-value of −1.3. models from top clusters were refined using haddock refinement interface. this server was used to cluster 20 structures, obtained via haddock, into one cluster. this final cluster symbolized 100% of water-refined models that were generated by haddock. the statistics observed in interactions of vaccine 1, vaccine 2, and vaccine 3 from their refined clusters can be seen in table 4 , and complexes are shown in figure 4 . the pdbsum analysis of vaccine 1 with ace2 showed 18 hydrogen bonds and one salt bridge. additionally, 42 interface residues of vaccine 1, representing an interface area of 2,502 (a 2 ), were found while the corresponding ace2 had 45 interface residues covering an area of 2,319 (a 2 ). for vaccine 2 and ace2, there were two salt bridges and seven hydrogen bonds predicted by pdbsum. additionally, 28 and 38 residues from vaccine 2 and ace2 interacted with each other covering an area of 1,997 and 1,832, respectively. likewise, for vaccine 3 there was one salt bridge and 13 hydrogen bonds predicted by pdbsum. additionally, 27 and 22 residues from vaccine 3 and ace2 interacted with each other, covering an area of 1,228 and 1,271, respectively. an interaction analysis of vaccine 1 with the tlr2 interacting complex via pdbsum exhibited 19 hydrogen bonds and one salt bridge. furthermore, 35 interface residues of vaccine 1, representing an interface area of 1,795 (a 2 ), were found while a corresponding tlr2 had 36 interface residues encompassing an area of 1,880 (a 2 ). for vaccine 2 and tlr2, there were two salt bridges and 14 hydrogen bonds predicted by pdbsum. additionally, 23 and 25 residues from vaccine 2 and tlr2 interacted with each other, covering an area of 1,362 and 1,443, respectively. lastly, for vaccine 3 and tlr2 pdbsum, 17 hydrogen bonds and five salt bridges were found. furthermore, 25 interface residues of vaccine 3, representing an interface area of 1,104 (a 2 ), were found while corresponding a tlr2 had 21 interface residues, encompassing an area of 1,194 (a 2 ). similarly, the interaction of vaccine 1 with tlr4 exhibited eight hydrogen bonds and 18 interface residues of vaccine 1, representing an interface area of 1,171 (a 2 ) while a corresponding tlr4 had 19 interface residues, encompassing an area of 1,146 (a 2 ). for vaccine 2 and tlr4, there were three salt bridges and 20 hydrogen bonds predicted by pdbsum. additionally, 33 and 34 residues from vaccine 2 and tlr4 here, a lower haddock score indicates the higher strength of interaction between the proteins. z-score of all docking complexes came out to be 0. interacted with each other, covering an area of 1,763 and 1,745, respectively. in case of vaccine 3 and tlr4, 15 hydrogen bonds and three salt bridges were found, while 34 interface residues of vaccine 3 represented an interface area of 1,397 (a 2 ) and a corresponding tlr4 had 27 interface residues, encompassing an area of 1,396 (a 2 ). for the interaction analysis of vaccine 3 and bcr (cd79), the haddock server clustered 140 probable structures into 13 different clusters, which represented a total of 70% of the water-refined models. the top scoring cluster had a score of −43.6 +/-16.0 and a z score of −1.7. models from top clusters were refined using haddock refinement interface. this server was used to cluster 20 structures, obtained via haddock, into one cluster. this final cluster symbolized 100% of waterrefined models that were generated by haddock. the statistics observed in interactions of vaccine 3 and bcr from its particular refined clusters can be seen in table 4 . pdbsum analysis showed that 26 and 18 residues from vaccine 3 and bcr interacted with each other covering an interface area (a2) of 1,181 and 1,205, respectively. they formed one salt bridge and 10 hydrogen bonds. for interaction analysis of vaccine 1 and hla a allele, the haddock server clustered 118 probable structures into 17 different clusters, which represented a total of 59.0 % of the waterrefined models. the top scoring cluster had a score of −26.5 +/-2.7 and a z score of −2.5. similarly, for vaccine 2 and the hla a allele, haddock clustered 97 structures in 17 clusters, which represented 48.5 % of the water-refined models. the top scoring cluster had a value −57.5 +/-12.8 and a z score of −2.3. likewise, for vaccine 3 haddock clustered 187 structures into three clusters, which depicted 93.5% of the water refined models generated by haddock. here the best cluster had a score of −34.7 +/-1.9 and a z score of −1.1. for vaccine 1 and hla b allele, 115 probable structures were clustered by haddock into 15 different clusters, which represented a total of 57.5 % of the water-refined models. the top scoring cluster had a score of −57.5 +/-12.8 and a z score of −2.3. similarly, for vaccine 2 and the hla b allele, haddock clustered 84 structures into nine clusters, which represented 42% of the water-refined models. the top scoring cluster had score of −18.7 +/-8.7 and z score of −1.6. likewise, for vaccine 3 haddock clustered 168 structures into 10 clusters, which depicted 84% of the water refined models were generated. here, the best cluster had a score of −41.2 +/-18.7 and a z score of −2.1. furthermore, for vaccine 1 and the hla drb1 allele docking, the haddock server clustered 67 probable structures into 10 different clusters, which represented a total of 33.5 % of the waterrefined models. the top-scoring cluster had a score of −27.8 +/-6.0 and a z score of −2.3. similarly, for vaccine 2 and hla drb1 allele, haddock clustered 64 structures in 11 clusters, which represented 32% of the water-refined models. the topscoring cluster had score of −24.8 +/-25.6 and z score of −1.7. likewise, for vaccine 3, haddock clustered 93 structures into 13 clusters, which depicted 46.5% of the water refined models generated by haddock. here the best cluster had a score of −37.1 +/-11.8 and a z score of −1.5. models from top clusters were refined using haddock refinement interface. this server was used to cluster 20 structures, obtained via haddock, into one cluster. this final cluster symbolized 100% of waterrefined models that were generated by haddock. the statistics observed in interactions of vaccine 1, vaccine 2, and vaccine 3 from their particular refined clusters can be seen in table s4 . epitope population coverage was checked by iedb population coverage tool. resultantly, all epitopes had a combined class i and class two average coverage score of 94%. this step was performed by using the entire world population datasets and the mhc restricted alleles used in this case were (a * 01:01, coronavirus can reportedly spread from person to person via droplet transmission. however, there is currently no available fda-approved vaccine against covid-19 (42, 43) . a vaccination regime, if successfully developed against covid-19, has the ability to improve global human health statistics. the advent of immuno-informatics approaches has revolutionized the area of vaccine development. antibody response as well as cell mediated immunity can be established by using proper protein antigens (44) . notably, the natural infections elicit a minimal immune response that can be enhanced by developing epitope-based vaccines. therefore, rational selections are done to separate the constituents required for the desired immune response. efforts to identify suitable tcell epitopes as well as the design of effective strategies in order to deliver those epitopes are under consideration. the benefits of epitope-based vaccine construction includes improved safety levels, time saving, and, additionally it can provide the opportunity to specifically attach/engineer combinations of epitopes for augmented potency. this also facilitates to emphasize the required immune responses on antigenic/ conserved epitopes (45) . spike proteins of coronaviruses are responsible for selection and entry into the target cells. any therapeutic approach to target the spike protein can prove to be fruitful to curb the deadly pathogen. moreover, it has been reported that like sars-cov, sars-cov-2 uses the ace2 human receptor to bind and enter the cells (12) . peptides that potentially interact with the functional domain of the coronavirus spike protein, can be designated as viral entry inhibitors. in our study, the chosen cd4+ and cd8+ t cell epitopes are predicted to be antigenic and immunogenic, and they can thus play a vital role in viral clearance mechanisms. to further validate the authenticity of our proposed vaccines, more detailed docking analysis and experimentation has to be performed. nevertheless, it might take months to years to actually derive a vaccine against covid-19, we believe that our contribution in this case might be a useful to initiate the process. for vaccine 1, four epitopes from the s1 domain were picked. the s1 domain, which comprises of amino acids from 14 to 685, is further divided into the n-terminal domain and receptor-binding domain. analysis showed that three of our chosen epitopes lied in the n-terminal domain of the s1 protein while one " 506 qpyrvvvlsfellha 520 " was a part of receptorbinding domain (319-541). viral infections are prompted by the interaction of the spike-protein with the receptor, present on the surface of the target cell. this process is mediated by the receptor binding portion of the s1 domain. hence, it plays a significant role in the attachment, and subsequent fusion and entry of the virus into the host cell. hence this particular portion can be targeted for designing antiviral agents (46) . vaccine 2 is comprised of a combination of strong and weak epitopes. it had two epitopes (mhc-i and mhc-ii) that were found to be the best epitopes for s1 domain. regardless of the fact that 91 efvfknidgyfkiys 205 had a higher antigenicity score compared to 506 qpyrvvvlsfellha 520 (1.0339 and 0.9109), the latter was used in vaccine construction due to its presence in receptor binding domain. additionally, another experimental strategy was applied; comparatively weak epitopes from s2 were selected and their binding affinity was checked. docking with tlrs and ace2 showed that they bind effectively; from 731 mtktsvdctmyicgd 745 , thr 192 , val 197 , lys 186 , thr 187 , and ser 186 bound to tlr4; lys 186 had affinity for ace2 receptor. the other s2 epitope 733 ktsvdctmy 741 completely overlapped with 731 mtktsvdctmyicgd 745 . vaccine 3 is a modified form of vaccine 2 with an additional 25mer b-cell epitope 369 ynsasfstfkcygvsptklndlcft 393 integrated. the whole idea to include b-cell epitopes along with was to ensure both cellular and humoral defense responses (47) . b-cell epitopes are precisely amino acids clusters present at the cell surfaces that are identified by certain antibodies or bcell receptors, that in turn elicit cellular or hormonal immune response (48) . antibodies released by b-cells can neutralize toxins and thus label them for destruction (49, 50) . in this case, in addition to considerable interactions with tlrs and hla superfamily alleles, notable interactions were observed between cys 93 and phe 94 from b cell epitope and arg 8 and glu 96 from bcr, respectively. designed vaccines have been tested against different receptors to identify their potential to induce immune response within the host. results revealed that proposed vaccines are likely to be presented by mhc-i and mhc-ii, as that was the prime objective of this study. also, they may interact with human tlr2 and tlr4 to induce innate immune response, as these receptors have been revealed to play a key role in the induction of immune responses (51) . moreover, the spike protein of sars-cov has been reported to play a significant role in the induction of neutralizing-antibodies and t-cell responses as well as protective immunity during the infection (52) . therefore, keeping in view the importance of spike proteins in immunity, we applied this predictive framework to identify potential vaccine candidates in spike protein of sars-cov-2 against its potential host receptor ace2 as well as against tlr4 and tlr2. recent studies have strongly suggested that covid-19 uses angiotensin-converting enzyme 2 (ace2) as its potential receptor. several critical residues in covid-19 receptor binding motif (rbm) of s1 domain particularly gln493 provide favorable interactions with human ace2 (53) . thus, it has been proposed in several studies that spike-protein-based vaccines can be potential therapeutic targets against sars-cov-2, as they may block the viral interaction with ace2 and may thus prevent the downregulation of ace2 and ultimately the pulmonary vascular permeability (54) . vaccines designed in this study may also interact with ace2 resulting interrupted interaction of the receptor with the viral spike protein and thus can be a potential therapeutic target against covid-19. the overall effect of all these interactions within the host is still unknown and requires further experimental studies for their clear role in the immune regulation and virus clearance. concisely, we have combined several immuno-informatics tools to propose a set of potentially antigenic and immunogenic peptide epitopes that can facilitate vaccine design. the predicted vaccine constructs consist of distant epitopes. the authenticity of these constructs must be validated via further experimentation. however, further experimental authentication is required to verify this study. we anticipate promising outcomes from the predicted peptide epitopes to curb the deadly covid-19 pandemic. all datasets presented in this study are included in the article/supplementary material. an, fs, and fa conceptualized the study, validated the study, and wrote the original draft. fs performed the data curation and developed the software. an and fs performed the formal analysis, methodology, and visualized the study. tb, aa, and am performed the funding acquisition. an performed the investigation and supervised the study. an and am performed the project administration. an, tb, fa, aa, and am wrote, reviewed, and edited the manuscript. all authors contributed to the article and approved the submitted version. does urbanization make emergence of zoonosis more likely? evidence, myths and gaps coronavirus infections-more than just the common cold genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding can we contain the covid-19 outbreak with the same measures as for sars? severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia first case of 2019 novel coronavirus in the united states return of the coronavirus: 2019-ncov a novel coronavirus from patients with pneumonia in china genomic 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server for in silico prediction of allergens protein identification and analysis tools on the expasy server. the proteomics protocols handbook pdbsum: a web-based database of summaries and analyses of all pdb structures galaxy: a platform for interactive large-scale genome analysis yasara: a tool to obtain structural guidance in biocatalytic investigations structure, function, and antigenicity of the sars-cov-2 spike glycoprotein human β-defensin 2 plays a regulatory role in innate antiviral immunity and is capable of potentiating the induction of antigen-specific immunity towards the application of human defensins as antivirals angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target toll-like receptors in antiviral innate immunity clinical features of patients infected with 2019 novel coronavirus in wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia genome-based approaches to develop vaccines against bacterial pathogens epitope-based vaccines: an update on epitope identification, vaccine design and delivery receptor-binding domains of spike proteins of emerging or re-emerging viruses as targets for development of antiviral vaccines in silico screening of antigenic bcell derived t-cell epitopes and designing of a multi-epitope peptide vaccine for acinetobacter nosocomialis current progress of immunoinformatics approach harnessed for cellular-and antibody-dependent vaccine design fundamentals and methods for t-and b-cell epitope prediction toll-like receptors: the swiss army knife of immunity and vaccine development the spike protein of sars-cov-a target for vaccine and therapeutic development receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars a review of sars-cov-2 and the ongoing clinical trials we want to acknowledge institute of molecular biology and biotechnology (imbb) at the university of lahore for providing the facilities and confidence to publish this article. we also want to acknowledge atta-ur-rehman school of applied biosciences (asab) at the national university of sciences and technology for collaboration and support. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.01663/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 naz, shahid, butt, awan, ali and malik. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-319993-er3sm4u8 authors: terry, frances e; moise, leonard; martin, rebecca f; torres, melissa; pilotte, nils; williams, steven a; de groot, anne s title: time for t? immunoinformatics addresses vaccine design for neglected tropical and emerging infectious diseases date: 2015-01-02 journal: expert rev vaccines doi: 10.1586/14760584.2015.955478 sha: doc_id: 319993 cord_uid: er3sm4u8 vaccines have been invaluable for global health, saving lives and reducing healthcare costs, while also raising the quality of human life. however, newly emerging infectious diseases (eid) and more well-established tropical disease pathogens present complex challenges to vaccine developers; in particular, neglected tropical diseases, which are most prevalent among the world’s poorest, include many pathogens with large sizes, multistage life cycles and a variety of nonhuman vectors. eid such as mers-cov and h7n9 are highly pathogenic for humans. for many of these pathogens, while their genomes are available, immune correlates of protection are currently unknown. these complexities make developing vaccines for eid and neglected tropical diseases all the more difficult. in this review, we describe the implementation of an immunoinformatics-driven approach to systematically search for key determinants of immunity in newly available genome sequence data and design vaccines. this approach holds promise for the development of 21st century vaccines, improving human health everywhere. vaccines have been invaluable for global health, saving lives and reducing healthcare costs, while also raising the quality of human life. however, newly emerging infectious diseases (eid) and more well-established tropical disease pathogens present complex challenges to vaccine developers; in particular, neglected tropical diseases, which are most prevalent among the world's poorest, include many pathogens with large sizes, multistage life cycles and a variety of nonhuman vectors. eid such as mers-cov and h7n9 are highly pathogenic for humans. for many of these pathogens, while their genomes are available, immune correlates of protection are currently unknown. these complexities make developing vaccines for eid and neglected tropical diseases all the more difficult. in this review, we describe the implementation of an immunoinformatics-driven approach to systematically search for key determinants of immunity in newly available genome sequence data and design vaccines. this approach holds promise for the development of 21st century vaccines, improving human health everywhere. neglected tropical & emerging infectious diseases: new challenges climate change and international travel have had a dramatic impact on the geographic distribution of pathogens infecting humans and animals. old world pathogens such as dengue and chikungunya virus, previously restricted to the middle east, africa and asia have now appeared in the americas [1] . newer pathogens such as middle east respiratory syndrome coronavirus (mers-cov), an entirely new coronavirus affecting humans, have been spreading beyond the region of the world from which they derive their names [2] . meanwhile, human populations in developing areas of the world continue to be threatened by neglected tropical diseases (ntd). more than two billion people -nearly 30% of the world's population -suffer from one or more ntd [3] , which include leishmaniasis, lymphatic filariasis, onchocerciasis, schistosomiasis and soil-transmitted helminthiasis, among others (table 1 ). in addition to climate change and airline travel, economic conditions leading to transmigration contribute to the spread of ntd. recent examples include the reemergence of leishmania in spain [4] and chagas disease in texas [5] . even while ntd expand their reach, vaccine development for these diseases lags behind. in contrast, vaccines for important emerging infectious diseases (eid, table 2) are being developed, to a certain extent, by large biotechnology companies, particularly when these companies receive guaranteed purchase agreements or other incentives to accelerate vaccine development. examples include the development of a vaccine for h7n9 (an emerging avian influenza) by novartis and novavax in 2013 [6, 7] and work toward the development of a new mers-cov vaccine in 2014 [8] . however, the standard approach to develop new vaccines for emerging (and reemerging) infectious disease threats, which is to implement previously existing vaccine design methodologies such as cloning and expressing the dominant surface antigen [9] , frequently results in the development of vaccines that are only effective when given with strong adjuvants [10] . this approach is particularly unlikely to work for pathogens that have complex lifecycles (such as parasites) or are highly mutable (such as rna viruses). this article will discuss new, computational approaches that may accelerate and improve the design of vaccines for ntd and eid. truly effective vaccines do not exist for the majority of ntd. although vaccines are in development for several ntd pathogens [11] , lack of financial incentive to invest in research and development programs for diseases concentrated in lowincome countries has mired the progress of vaccine efforts among large pharmaceutical companies [3] . preventative chemotherapy mass drug administration (mda) programs employing donated or extremely low-cost generic drugs are currently in progress to control lymphatic filariasis, onchocerciasis, leprosy, trachoma and helminthiases in many areas of the world [12] . a complication inherent in this strategy is that because the regions affected by different ntd overlap, coinfections can be difficult to manage with antiparasitic agents [13] . the success of these long-term efforts and off-target effects of mass-eradication campaigns at the individual and population levels remain to be determined [14] . as has been observed for polio, geopolitical upheaval may hamper global efforts to eradicate ntd. thus, effective ntd vaccines are still needed [15] . new cost-effective design methodologies can help to bridge the gap between the great need for these vaccines and return on investment for pharmaceutical companies. fortunately, genomes for many newly emerging pathogens and neglected tropical disease-associated pathogens are becoming available due to research efforts worldwide [16] . the availability of these genomes now makes it possible to apply computational vaccinology tools to these diseases of global health importance. host immune response to pathogens is mediated by the innate and adaptive arms of the immune system. innate immune cells such as macrophages, neutrophils and natural killer cells are responsible for the first line of defense, while adaptive immunity provides a more targeted response to pathogens that establishes immune memory for more rapid responses upon repeated exposures. b cells produce antibodies which are capable of recognizing and neutralizing pathogenic antigens. t cells support antibody production, activation and memory development, and are capable of lysing infected cells. the potent response from t and b cells, however, comes at a cost. whereas innate immune cells can respond within 24-72 h, the primary adaptive response normally takes 7-14 days to mature. secondary adaptive responses driven by immune memory are much faster and much stronger. this is the principle behind vaccination: pre-exposure to pathogen-derived antigens can induce pathogen-specific immune memory. the discovery of critical antigens that drive protective memory is facilitated by new computational tools. indeed, the general principle that immune cells develop memory to specific pathogen components -has driven the development of genome-derived vaccines over the past two decades. since t cells play a critical role in adaptive immunity and the development of immune memory required for an efficacious vaccine, computational tools have been used to search for small linear peptides (t-cell epitopes) derived from protein antigens that drive class i and class ii t-cell responses. these peptides are displayed on the surface of apc by multiple alleles of the mhc. as human beings express multiple alleles of class i and class ii mhc molecules, called human leukocyte antigens (hla), computational vaccinologists now search for t-cell epitopes that can bind to the most common hla alleles in the human population, reasoning that broad hla coverage will contribute to the development of effective genome-derived vaccines. computational tools can also be used to select epitoperich surface proteins that are better immunogens to drive b-cell response. fortunately, while b cells and antibodies generally recognize surface proteins, t cells recognize epitopes derived from a broader range of proteins, giving the computational vaccinologist many possible sources (internal and external proteins as well as secreted proteins) for the selection of t-cell epitopes for vaccines. exposure to a given pathogen generates memory t-cell clones capable of rapid and efficient response upon subsequent reinfection [17] . this response may include t cell help for induction of higher antibody titers, t-cell-mediated lysis of infected cells and the expression of cytokines to coordinate other cell-mediated immune processes such as activation of apc. breadth of t-cell response (responding to many different epitopes) appears to be correlated with protection from severe disease for many pathogens that affect humans. more specifically, for hiv, hbv, hcv, lymphocytic choriomeningitis virus and malaria, protection from disease has been correlated with broad t-cell epitope response to both 'immunodominant' and subdominant t-cell epitopes [18] [19] [20] [21] [22] . these discoveries have contributed to the concept that vaccines can be made directly from genomes by selecting sets of epitopes that will stimulate immune responses and protect against diseases. based on the observation that broad t-cell response may be protective, computational vaccinologists have worked to define collections of t-cell epitopes that can recreate the requisite features of this response. t-cell-driven, epitope-based strategies for developing vaccines against eid and ntd are currently the focus of several ntd research laboratories. proof of principle exists for a number of disease models: cellular immunity elicited by epitope immunization provided complete protection against respiratory syncytial virus challenge, partial protection of balb/c mice against sporozoite challenge, elimination of malaria-infected hepatocytes in vitro, partial protection of balb/c and cba against encephalitis following intracerebral challenge with a lethal dose of measles virus, complete protection from intraperitoneal hsv challenge, protection against infection with malaria or influenza a virus and full protection of sheep against bovine leukemia virus (these examples are reviewed in [23] ). we have demonstrated complete protection against lethal vaccinia challenge [24] and successful clearance of a chronic bacterial infection (helicobacter pylori) following t-cell epitope-driven vaccination [25] . in earlier studies, we achieved partial protection against an aerosolized bacterial pathogen (tularemia [26]) using a vaccine that contained only 14 epitopes. while mice are not humans, growing evidence that t-cell epitope-driven vaccines can be effective in humans has led to the establishment of a number of biotech startups and venture-backed companies focused entirely on t-cell epitopebased vaccines. t-cell epitopes as ' payload' as described in the following sections, computational tools are being used to identify proteins or antigens of interest directly from the genomes of pathogens. in theory, a minimal set of antigens or epitopes that induce a competent immune response to a pathogen can be discovered using the new tools. adjuvant triggers innate immunity, which is an essential component of the protective immune response, directing it toward inflammation rather than tolerance. when combined with the minimum antigenic components that comprise the 'payload' of a genomederived vaccine, delivered in the right vehicle, may trigger protective immune response. the fundamental principle of the genome-derived epitope-driven vaccine approach is illustrated tthus: the importance of epitopes as key determinants of protective immune responses is reflected by the flurry of immunoinformatics activity over the past decades. a number of t-cell epitope mapping tools have been developed to accelerate the identification of these critical components of the immune response. using methods such as frequency analysis, support vector machines, hidden markov models and neural networks, researchers have developed highly accurate tools for modeling the mhc-peptide interface and predicting t-cell epitopes. computational vaccinologists have been unable to successfully develop accurate tools for b-cell epitope prediction, even though one of the most commonly measured outcomes of vaccination and accepted determinant of protection is antibody generation [35, 36] . thus, current computational vaccinology approaches to vaccine development must take b-cell response into consideration and develop approaches that include means of stimulating effective humoral immunity where it is required for protection against challenge. given t-cell dependence for essential features of an effective antibody response, including bcell affinity maturation, class switch recombination, plasma cell differentiation and memory b-cell differentiation [37] , t-cell epitope analysis and quantification have been used by our group as a proxy for identifying good b-cell immunogens, linking in silico sequence analysis to desired putative b-cell responses [28] . the ivax approach to design genome-derived epitope-driven vaccines de groot and colleagues have integrated epitope-mapping tools with a wider array of vaccine design algorithms into the webbased ivax toolkit, which will be described in some detail in the following sections. the tools were initially used by epivax and collaborators [23, [38] [39] [40] [41] [42] , and then expanded and refined for projects that have been in progress at the institute of immunology and informatics (icubed) [43] [44] [45] . the ivax toolkit is currently in use for ntd research at the icubed and with academic collaborators under an agreement established between epivax and uri in 2009. ivax tools are being used to evaluate the protective potential of existing ntd and eid vaccines [46, 47] , to predict immune response to newly emerging pathogens [9, 10] and to design novel ntd vaccines composed of t-cell epitopes (for chagas disease, brugia malayi and several different species of leishmania [48] ). in the following few sections, we describe the ivax approach to design genomederived epitope-driven vaccines for ntd and eid. one of the first questions facing computational vaccinologists is how to prioritize their search for antigenic proteins and epitope subunits. the entire set of proteins derived from a pathogen's genome is an unlikely point of departure for epitope mapping, since many of these proteins may not be part of the 'core genome' for a set of bacterial or viral strains of the same pathogen. others may be proteins that serve as 'housekeeping' genes that are also well conserved in harmless commensal organisms. on the other hand, proteins that are highly conserved across variant strains, that are pathogen-specific and those that are upregulated during interactions with the host, particularly those that are secreted by a pathogen (presumably in an attempt to alter the host environment), are excellent targets for vaccine development. in addition to targeting upregulated, secreted and pathogen-specific antigens, other means of selecting antigens for epitope screening include identifying proteins that are more common in virulent as compared to avirulent strains, selection of genes differentially expressed in immunopathogenesis, prioritizing proteins exposed on the surface of the pathogen and focusing on proteins that are expressed early in the course of natural infection. the expert protein analysis system (expasy) proteomics server of the swiss institute of bioinformatics offers a wide variety of proteomics tools that can be used for this purpose, including tools related to protein identification and characterization. our groups have adapted an approach first described by gennaro et al. for mycobacterium tuberculosis (mtb) [49] , employing a series of expasy tools (signalp, tmpred and prosite scan [50] ) to triage pathogen genomes, reducing the number of potential targets from thousands of proteins to several dozen candidate antigens. in our first test of this approach, we found that a subset of epitopes derived from the mtb genome elicited ifn-g response from mtb-exposed human samples, and prototype epitope-based tb vaccines were shown to be robustly immunogenic in murine studies [51] . reflect more epitope content than expected, while negative scores reflect less epitope content than expected [52] . large numbers of protein sequences derived directly from the genome of selected pathogens can be ordered by potential class i (ctl), class ii (t helper) or both class i and class ii epitope content and placed on an immunogenicity scale (figure 1). this tool allows researchers to quickly rank a given set of proteins both in relative (i.e., relative to each other) and absolute (i.e., relative to a panel of known immunogens and nonimmunogenic proteins) terms [53] . in our experience, epitope-rich proteins are good vaccine targets and elicit strong antibody responses -thus, as previously stated, t-cell epitope content is a useful proxy for overall immunogenic potential. antigen selection is particularly complicated when targeting parasitic organisms due to their comparatively massive genomes and multistaged life cycles, and ranking of these antigens may assist with the selection of better targets. for example, in figure 1, we show two candidate antigens derived from b. malayi, a causative agent of lymphatic filariasis, whose life cycle is divided into multiple larval stages including a microfilarial stage [54] . in this case, tpx-2, a protein that has been identified as a potential vaccine target [55] , is shown to contain minimal t-cell epitope content, with an immunogenicity score of -27.61, and thus it may be less successful as a vaccine candidate. in contrast, juv-p120, a b. malayi ortholog of a litomosoides sigmodontis antigen implicated in conferring protection against microfilarial infection [56] carries substantially more t-cell epitope content, scoring +94.14 on the immunogenicity scale, in the same range as other well-known immunogens. furthermore, as is illustrated here in the case of b. malayi, we frequently find evidence that pathogens appear to reduce t-cell epitope content in key proteins to avoid human immune responses. epitope deletion is an established means of immune evasion in hiv and hcv [57, 58] ; thus, the mechanism may also be relevant in the context of infections that are associated with chronic infection caused by filaria, leishmania and other chronic ntd, particularly in stages associated with chronic parasitism and parasite persistence in the face of immune pressure. we will discuss additional means of immune evasion that can be uncovered by computational tools below. epimatrix: t-cell epitope mapping of selected antigens t-cell epitopes are short linear peptides that can bind to mhc molecules and engage t cells through their receptors (tcr), activating specific populations of cd8+ and/or cd4+ lymphocytes. these epitopes are key to forming the immunological synapse between antigen-presenting cells and t cells. because tcrs are produced in a myriad of possible conformations (much like antibodies, to which they are related), mhc binding is the dominant event in immune recognition. in other words, most mhc ligands are also t-cell epitopes, and t-cell epitopes are by definition, mhc ligands. the mhc-peptide interaction is well characterized [59, 60] . based on these characterizations, pattern-matching algorithms such as epimatrix have been developed to screen protein sequences for peptides that will bind mhc. the human mhc molecules, or hla, are among the most variable proteins in the human genome. this variation ensures that the surveillance capabilities of the human immune system are both broad and deeply redundant, making immune escape through mutation more difficult for pathogenic organisms. fortunately, some alleles are much more common than others in the human population and the binding repertoire of many alleles significantly overlap. by focusing on alleles that are both common (in the human population) and significantly different from each other (representative of human diversity), hla alleles can be grouped into all other factors being equal, the more hla ligands (i.e., putative t-cell epitopes) contained in a given protein, the more likely that protein is to induce an immune response. to capture this concept, the epimatrix immunogenicity scale presents proteins by the epimatrix protein score, and compares them to other known immunogens. the epimatrix protein score is the difference between the number of predicted t-cell epitopes expected in a protein of a given size and the number of putative epitopes predicted by the epimatrix. the epimatrix protein scores are 'normalized' and can be plotted on a standardized scale. 'average' proteins score near zero. protein scores above zero indicate the presence of excess mhc ligands and denote a higher potential for immunogenicity, while scores below zero indicate the presence of fewer potential mhc ligands than expected and a lower potential for immunogenicity. the epimatrix protein score is correlated with observed immunogenicity in vitro and in vivo. as shown here, proteins scoring above +20, such as brugia malayi antigen juv-p120, are considered to have a significant immunogenic potential. proteins scoring below -20, such as tpx-2 above, are less likely to be immunogenic in vivo. 'supertypes,' which can reduce the search space to a manageable number of evaluations. six of these class i super-type alleles that 'cover' the genetic backgrounds of most humans worldwide have been used to define ctl epitopes: a*0101, a*0201, a*0301, a*2402, b*0702 and b*4403 [61] . for class ii t helper epitopes, mapping for a panel of eight common alleles: drb1*0101, *0301, *0401, *0701, *0801, *1101, *1301 and *1501, gives broad t helper epitope coverage [62] . the concept of supertype alleles is generally accepted and widely applied to vaccine design in the field of computational vaccinology [61, 62] . using the set of selected protein antigens as a starting point, ivax uses epimatrix to parse each into overlapping 9-mer frames where each 9-mer overlaps the last by eight amino acids. each 9-mer is then scored for predicted binding affinity to a panel of class i or class ii hla alleles. the epimatrix algorithm compares the amino acid sequence of each given 9-mer peptide to the coefficients contained in stored probability matrices and produces a raw score. in order to compare potential epitopes across multiple hla alleles, epimatrix raw scores are converted to a normalized 'z' scale. peptides scoring above 1.64 on the epimatrix 'z' scale (typically the top 5% of any given sample) are likely to be mhc ligands [63] . evidence from animal studies suggests that the number of epitopes required for full protection is a small and definable subset (~50) [64, 65] ; thus, epitope-driven vaccines developed by our group generally contain a payload of 50-100 epitopes that provide broad coverage of human genetic backgrounds. with a combination of promiscuous class ii epitopes and class i supertype epitopes, it is possible to attain >99% coverage of the hla of most human populations [61, 62] . eliminating regulatory or suppressor epitopes using janusmatrix a recent development in vaccine design includes the consideration of epitopes that induce regulatory or suppressive immune responses [66] . our group has been investigating epitope crossconservation with the human genome and its association with diminished or regulatory immune responses. using a recently developed tool called janusmatrix we first determined that published effector t-cell epitopes can be distinguished from reported regulatory t-cell epitopes on the basis of tcr-specific cross-reactive potential with the human genome and human microbiome [67] . janusmatrix differs from whole-sequence alignment tools such as blast [68] in its basis upon t-cell receptor homology. pathogenic peptides whose tcr-facing residues are identical to the epitopes contained in multiple self may be recognized by t cells specific to those human proteins. of course, even though the mhc-facing residues may differ, these peptides must still have the capacity to bind to the same mhc as the pathogen sequence, provided that binding is preserved. taking this into account, janusmatrix compares the tcr-facing contour of pathogen ligands to other genomes of interest, identifying matches therein that are predicted to bind the same mhc. tcr-homologous epitopes shared between pathogens and humans, or pathogens and other microbes, can be uncovered with remarkable speed using the janusmatrix tool. exploring further, we have uncovered a high degree of host (human) homology in viruses that tend to establish chronic infections in humans such as ebv and cmv [69] . furthermore, 'commensal' viruses can be shown to contain significantly more human genome-homologous epitopes relative to those causing acute infection (e.g., ebola, marburg) [69] . the limited clinical efficacy of some vaccines against selected microbial pathogens may, in fact, have been due to their extensive crossconservation with the human genome [10] . the janusmatrix tool is currently being used by our team and collaborators to identify significant homology between candidate payload epitopes and proteins contained within the human genome and the human microbiome. using the tool, we find that not only viruses but also bacteria that establish chronic infections in humans 'deimmunize' (remove t-cell epitopes) and 'tolerize' (modify epitopes to be more cross-reactive to human t-cell epitopes). comprehensive studies of ntd genomes (and stageby-stage analysis of parasite antigens) will be performed using the janusmatrix tool in the near future. it follows that careful selection of t-cell epitopes, and redesign of whole antigens, to avoid the inclusion of t-cell epitopes that may be highly cross-reactive with the human genome could improve the efficacy of whole-antigen and epitope-based vaccines. janusmatrix complements recent research [70] on the development of adaptive immunity and supports the hypothesis that adaptive t-cell responses are reinforced by cross-reactivity with the human microbiome [71] [72] [73] . cytoscape is an online tool that is usually used by bioinformaticians to illustrate the relatedness between proteins, for example, all of the intracellular proteins that might be involved in the stimulation of a cell through toll-like receptors. we have repurposed cytoscape to describe the relationship between epitopes across proteins in groups of sequences (the human genome, the human microbiome, pathogen genomes [67] ). using cytoscape [74] , the results of janusmatrix analysis (e.g., comparing a pathogen epitope to the human genome) can be visualized as networks where each epitope derived from a pathogen is linked to its tcr-matched counterparts in the search database, which themselves are linked to their source proteins. for example, an influenza t-cell epitope previously identified by mark davis and colleagues [70] that stimulates t cells in subjects never exposed to influenza can be shown to have an extensive network of cross-reactive tcr-facing epitopes in the human microbiome. in contrast, an epitope from vaccinia virus synthesized and tested by larry stern's group is shown to have extensive cross-reactivity with the human genome by janusmatrix. this epitope was nonimmunogenic in vitro (by ifn-g elispot) even though it was shown to bind to the correct class ii mhc [75] . this epitope fits the emerging in silico definition of a treg epitope. due to their commensal nature and need to avoid human immune responses over many years of coexistence, it is even ntd/eid: time for t? review informahealthcare.com more likely for selected human parasites to share putative t-cell epitope content with their human hosts. in figure 2, we offer two example peptides from b. malayi antigens tpx-2 and juv-p120, compared to published treg epitopes from human immunoglobulin (tregitopes) and effector epitopes, the ceft pool (a set of peptides used as 'control positive' peptides in eli-spots [67] ). the potential cross-reactivity network differential is evident between the tpx-2 epitope, with many related epitopes derived from human sequences, and the juv-p120 sequence, whose related human epitopes are few. this finding underscores the importance of validating the response phenotype of t cells stimulated by epitopes identified in silico prior to their inclusion in vaccine constructs, and also illustrates the importance of this type of analysis for the selection of candidate epitopes for ntd. promiscuous hla binding potential is a feature of class ii-restricted t-cell epitopes particularly exploitable for vaccine design purposes. it has been shown that putative epitopes for hla class ii are not often distributed evenly across protein sequences, but instead tend to cluster in specific regions, where it is not uncommon to observe several reactive 9-mer frames in close proximity [76] . these 'clusters' of unusually high predicted epitope density can be identified in silico using the clustimer algorithm. in general, t-cell epitope clusters identified by the clustimer algorithm tend to be promiscuous mhc binders and are frequently t-cell epitopes [52] . due to overlapping peptide-binding preferences among hla-dr alleles, it is also possible to identify single 9-mers capable of binding four or more hla alleles [76] . these sequences have been dubbed 'epibars' due to their horizontal, band-like signature in readout from epimatrix (figure 3) . t-cell epitope clusters can be very powerful, and epibars may be a characteristic feature of highly immunogenic, promiscuous class ii epitopes. these compact, highly reactive peptides are relatively easy to deliver and show great promise as vaccine components when cross-reactivity with the human genome is limited (see above). we have used these clusters extensively in our own work [24, 43, 51] . promiscuous t-cell epitopes also exist, to a certain degree, for class i alleles; however, this is much less common than for class ii. some laboratories have demonstrated cross-presentation of peptides within hla 'superfamilies,' such as the a3 superfamily: a3, a11, a31, a33 and a68 [77] . cross-mhc binding and presentation to t cells has been confirmed in hiv vaccine studies [78] . however, we have found that weighting toward the selection of highly promiscuous class i epitopes may lead to identification of candidate epitopes that have lower binding affinities overall. higher binding affinities appear to be a critical aspect of ctl epitope efficacy [79] , thus our group prefers to select a small set of the best-scoring putative epitopes for each of the six class i hla superfamilies from a given protein or set of conserved peptides (figure 4) . potential lf t eff epitope (juv-p120) potential t eff epitope ceft pool published t reg epitope human igg a b c d figure 2 . janusmatrix analysis. this tool considers identity of tcr-facing residues to target proteins or genomes independently from residues that contribute to mhc binding. peptides that have similar tcr-facing residues and are presented in the context of the same hla can be identified. extensive homology is easy to identify using the cytoscape network visualization tool. the extent of the network can be used to distinguish potential regulatory t-cell epitopes (a) from potential effector t-cell epitopes (b). a published treg epitope example is shown in (c), and several published teff epitope examples are shown in (d). for these illustrations, yellow hexagons identify the source antigens, turquoise diamonds identify the source t-cell epitope clusters, gray squares indicate the source 9-mers, dark blue triangles indicate matched human 9-mers and light blue circles indicate human antigens in which matched 9-mers are found. data for c, d taken from [67] . selecting epitopes that are broadly reactive across circulating strains can enhance broad applicability of new vaccines. the problem of pathogen variability significantly complicates the selection of epitopes for vaccine design. to address this problem, epivax has developed epiassembler [80] to identify sets of overlapping, conserved and immunogenic epitopes and to assemble them into extended immunogenic consensus sequences (ics, figure 5 ). the theory behind developing ics is that processing and presentation of these sequences would allow for presentation of the highly conserved class ii-restricted epitopes contained in the ics in the context of more than one mhc. the resulting peptide is not a 'pseudo-sequence' as such, since each constituent epitope occurs in its corresponding position in the native protein; adjacent epitopes may be similarly conserved but not in the same variant of the pathogen. the ics approach has been useful for identifying highly immunogenic epitopes for hiv vaccine design [38] . using hiv as an example, while the full composite ics peptides happen to be exactly conserved in a few individual strains of hiv, each peptide represents a significant percentage of circulating strains because every constituent overlapping epitope is conserved in a large number (range 893-2254) of individual hiv-1 strains [38] . by extending the approach described above, it is possible to develop completely synthetic antigens whose sequences are optimized for t helper potential. with an eye to structural considerations, even recombinant protein-only vaccines could be optimized in this way, enabling primary cognate t help to be maximized and b-cell memory to be elicited. an ideal vaccine might include whole proteins in addition to some epitopes; some or all of these antigens could be optimized using the ics approach. linking ics epitopes to a carrier protein (such as a surface protein target of b-cell response) would further maximize primary cognate t help, since b cells that capture the recombinant proteins would be able to process and present t helper epitopes derived from more variable proteins. as compared with ics, randomly selected counterparts, on average, contain half as many binding motifs and cover one-third fewer isolates [40] . to develop vaccines of equivalent antigenic 'payload' using conventional methods would be prohibitively expensive, as it would require use of multiple variants of each antigen. we believe that this and similar approaches that harness conserved t help have tremendous potential and deserves careful consideration in vaccine design. after generating a preliminary list of candidate vaccine components, the next step in the ivax approach is to review the putative epitopes produced by the epimatrix system, adding qualitative and quantitative annotations wherever possible, leading to an investigator-driven down-selection process. putative epitopes derived from known antigens or from proteins overexpressed during early stages of infection or proteins known to be exposed to immune surveillance as reported in the literature may be prioritized. furthermore, putative epitopes with the in silico profile of potential regulatory t-cell epitopes (based on janusmatrix analysis) are removed from further consideration. figure 3 . example of an epibar: epimatrix analysis of candidate lymphatic filariasis epitope. in addition to providing an overall immunogenicity score, epimatrix can be used to analyze epitopes at the local level. a brugia malayi juv-p120 peptide is shown above, parsed into 9-mer frames and analyzed for predicted immunogenicity. epimatrix assessments above 1.64 constitute the top 5% of predicted hla binders and are shaded medium blue, while scores above 2.32 fall in the top 1% and are shaded dark blue. this juv-p120 peptide registers significant scores for all eight alleles in epimatrix in a single 9-mer frame, and based on the epimatrix method, has a cluster score of 16.81 (reflecting the number of predicted binders per amino acid length). cluster scores higher than 10 are considered to be significant based on retrospective and prospective studies carried out by the epivax group. the band-like pattern illustrated in frame 35 is called an epibar and is characteristic of promiscuous epitopes. algorithms can also be helpful to interpret vaccine component responses in preclinical and clinical studies. in studies of immune response to therapeutic proteins and vaccines, the authors have observed that subject-to-subject variation in t-cell response closely relates to subject hla type and the number of motifs or peptides that match the subject's hla haplotype. to describe this relationship, epivax researchers have developed a metric that may be useful in clinical assessment of immune response to vaccines, called the 'individualized t-cell epitope measure' or item. for a given t-cell epitope, an individual's item score can be calculated by weighting and summing the epitope's epimatrix z-scores for each hla allele in a given subject's haplotype. this calculated score allows for individualized immunogenic potential to be predicted based on the number of putative epitopes contained in a protein and a given individual's hla haplotype. using this score, it is possible to analyze the contribution of haplotype to the corresponding t-cell response. in prospective and retrospective evaluations, significant correlations were found between the ifn-g response to a given antigen and the item scores for individual subjects [42] . in addition, correlations between the item score and patient hla have been observed for antibody titers [40, 81, 82] , reflecting the importance of hla-restricted t-cell responses to the genesis of a robust antidrug antibody response. a number of methods for enhancing epitope-based vaccines have been described and implemented [83, 84] . one approach is to align the individual epitopes in a protein or dna vaccine construct as a 'string of beads' without any intervening figure 4 . class i epitope 'staircase' ranking. in the process of generating a selection of predicted high-affinity class i epitopes for inclusion in t-cell-driven vaccines, parsed 9-mers from any antigen are ranked by potential to bind supertype hla alleles and collated in a 'staircase' report. in this example, the top five highest-scoring peptides from a given antigen are shown. in general, prioritizing class i epitopes by score for each of the individual alleles is preferred to define epitopes that bind across alleles. sequences or spacers between the payload epitopes [85] . however, the lack of spacers between the payload epitopes has raised concern that these sequences may contain junctional epitopes. vaccinecad, an algorithm that iteratively analyzes epitope assemblies and minimizes the potential for junctional immunogenicity in any string-of-beads construct, has been developed to address this concern [40] . peptide sequences contained in the junctional regions between the target epitopes are evaluated for potential immunogenicity. the highest scoring junction is identified and the algorithm optimizes the order of epitopes by evaluating potential alternative sequences. the process is repeated until no additional reductions in junctional immunogenicity can be achieved or until all junctional immunogenic potential has been eliminated. when the potential for junctional immunogenicity cannot be sufficiently reduced, a cleavage promoting spacer sequence, typically 'aay' for class i restricted constructs [86] or a binding inhibiting 'breaker' sequence such as 'gpgpg' for class ii restricted constructs [87] is placed between the two offending epitopes. the ability to minimize junctional immunogenicity while simultaneously minimizing the presence of transmembrane domains or highly hydrophobic peptide segments which may be difficult to express would be a logical extension of this tool's capabilities. the integration of computational tools for epitope discovery has enabled the development of genome-derived vaccines [41, 45, 44] . compared to conventional strategies, this approach has the potential to create more effective and safer next-generation vaccines, as carefully selected epitopes focus immune responses on the minimal, essential pathogen-specific antigenic elements; epitopes directed against conserved 'self' (host) antigens are eliminated. this approach is also well suited for highly variable pathogens, as selection of epitopes that are conserved across multiple strains or subtypes enables the development of a broadly applicable, multipathogen vaccine. the genome-derived vaccine strategy has been applied by our team to a wide range of pathogens, including f. tularensis, variola, hiv, mtb, h. pylori and influenza. these studies demonstrate that immunoinformatic-predicted epitopes are immunoreactive in vaccinees and survivors of infection, and stimulate de novo, protective immune responses in vivo in hla transgenic mice (e.g., [24] [25] [26] 51] ). epitope-driven vaccines offer distinct advantages over traditional subunit vaccines. multiple epitopes derived from several antigens can be packaged together. thus, a broad-based immune response directed against several different antigenic proteins can be elicited without manufacturing and administering the entire protein, much of which will be immunologically irrelevant. this may reduce formulation challenges, cost and safety risk. the use of epitopes also mitigates safety concerns arising from the use of intact recombinant proteins that may have undesired biological activity. this review of vaccine design tools developed by the epivax team is by no means comprehensive, and has mainly focused on antigen selection and design. topics not covered in this review include formulation of epitope-driven vaccines, route of delivery (mucosal, intradermal, etc.), adjuvanting, selection of delivery vehicles and preclinical and clinical testing. a major caveat concerning the use of the ivax toolkit is that none of the vaccines designed using these tools have advanced to the clinic. given the cycle of vaccine development, this is not surprising (it may take up to 20 years to develop a vaccine with full industry support). retrospective and prospective studies have provided extensive validation of the tools described here [10, 28, 46, 47] . nonetheless, algorithms developed and applied by this group to a wide range of pathogens have met with significant preclinical success and are currently in use for the development of vaccines against ntd parasites, eid viruses and bioterror pathogens. access to nearly all of the tools described in this article is freely available to trained users through the ivax toolkit [88] . the website was developed with funding from the national institutes of health in 2010. access to the ivax toolkit and training on the tools is available for interested researchers under collaborative agreements with the university of rhode island (primarily for ntd, but other arrangements are possible). commercial users are directed to epivax [89] , which provides a secure-access version of the ivax website for commercial users. in general, the field of vaccine research has been slow to adopt new vaccine design tools, and even fewer ntd researchers are familiar with the use of the tools, despite proof of principle for the genome-derived vaccine approach and the fact that it significantly reduces time and effort to make vaccines. for eid, 'tried and true' approaches often win out over newer strain: a b c d e f g h conserved epitope figure 5 . epiassembler construction of immunogenic consensus sequences. this figure illustrates the process of assembling highly conserved t-cell epitopes into a single molecule. first, a highly conserved, promiscuous epitope is identified to form the 9-mer core of the ics peptide (red bar). overlapping conserved epitopes (pink, orange, green and blue bars) are then added to the n-and c-termini of the peptide until a suitable length is reached for binding in the class ii hla binding groove. this economical approach allows for targeting of multiple strains of a given pathogen using a single peptide, as illustrated by the blended bar at the bottom of the figure. ics: immunogenic consensus sequences. a similar approach was used during the emergence of sars and completely failed to protect against rapidly evolving sars viruses in animal challenge models [9, 90] . application of advanced immunoinformatics tools to ntd vaccines has also lagged for a number of reasons. ntd researchers do not use the tools because they lack access to and familiarity with them, and there are no widely publicized examples focusing on diseases that impact the developing world. a series of technical challenges for ntd vaccines have been described recently, including antigen discovery, process development, preclinical development, clinical trials in resource-poor settings and the immune response to ntd infection, including what is commonly referred to as the ige trap, through which certain individuals, perhaps especially those in endemic regions, may have elevated preexisting ige antibodies for potential ntd vaccine antigens, leading to increased risk with vaccination [91] . computational vaccinology cannot currently address all of these challenges; however, the approach described here offers a unique opportunity to address certain hurdles early in the developmental process. early in the pipeline, antigen discovery using t-cell epitope prediction and ranking, along with candidate epitope triage using cluster analysis and crossreactivity prediction provide valuable leads. selected peptide candidates can be screened ex vivo in order to verify the phenotype of the immune response prior to inclusion in a final vaccine product. finally, t-cell epitope-based strategies are exceptionally platform-flexible, adaptable to synthetic peptide formulations deliverable in saline, emulsion or microparticle, or encoding into plasmid vectors for dna vaccination or recombinant protein production, thus allowing for novel distribution strategies necessary to reach the world's poorest. this flexibility extends to the antigen discovery approach as well, in that many kinds of targets may be explored using immunoinformatics tools. a pertinent example for ntd and eid applies to vector-based targets. vaccine components based upon the salivary proteins of arthropod vectors are already under investigation [92] . however, vector salivary antigens also have known immunomodulatory properties allowing for extended host tolerance [93, 94] . the same discovery and evaluation strategy described for pathogenic antigens could be applied to such proteins, potentially providing a mechanism through which to stimulate robust immune response in the absence of the immunomodulatory properties of the complete salivary antigens. the amount of data generated through new technologies, such as next-generation sequencing, continues to expand exponentially. by applying these technologies to the study of eid and ntd causative agents, and expanding our genomic knowledge of these organisms, the feasibility of using high-throughput, informatics-based tools for the identification of putative protein and peptide targets increases. vaccine efficacy may also improve, as the selection of targets can be refined by comparing the antigen to other genome sequences, such as the human genome and the human microbiome. the in silico-based approach to vaccine design may also alleviate many of the funding-associated challenges common to traditional vaccine design, by reducing the number of assays that need to be performed to select vaccine targets. reduced cost should allow for the reallocation of critical funding to the testing of in silico-predicted targets and constructs. and finally, improved safety, by eliminating human genome cross-conserved epitopes, may reduce unwanted adverse effects. looking further into the future, we are confident that the evolution of the tools described here will eventually contribute to the development of personalized, on-demand vaccines [95] . considering the importance of controlling infectious diseases to global economic stability, the integration of computational vaccinology tools and their application to the design of vaccines for ntd and eid is of paramount importance. delay is no longer acceptable. vaccine developers must implement computational vaccinology tools if they wish to contribute to improve world health in the 21st century. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no writing assistance was utilized in the production of this manuscript. • new immunoinformatics tools have been developed that address critical problems in vaccine design. • these tools have been extensively validated in preclinical models. • the design of vaccines for neglected tropical diseases would benefit from expanded use of these tools. chikungunya fever diagnosed among international travelers-united states the emergence of the middle east respiratory syndrome coronavirus control of neglected tropical diseases needs a long-term commitment re-emergence of leishmaniasis in spain neglected parasitic infections in the united states: chagas disease bringing influenza vaccines into the 21st century a recombinant viruslike particle influenza a (h7n9) vaccine current advancements and potential strategies in the development of mers-cov vaccines how the sars vaccine effort can learn from hiv-speeding towards the future, learning from the past cross-conservation of t-cell epitopes: now even more relevant to (h7n9) influenza vaccine design this study connects the dots between low t-cell epitope content and low immunogenicity of h7n9 vaccines in humans, and points out important changes to t-cell epitopes that might further reduce immune response through the induction of tregs innovation for the 'bottom 100 million': eliminating neglected tropical diseases in the americas preventive chemotherapy as a strategy for elimination of neglected tropical parasitic diseases: endgame challenges neglected tropical diseases and the millennium development goals: why the "other diseases" matter: reality versus rhetoric the contribution of mass drug administration to global health: past, present and future the human hookworm vaccine genomics of emerging infectious disease: a plos collection evolution of the t-cell repertoire during primary, memory, and recall responses to viral infection cytotoxic t-lymphocytes in asymptomatic long term nonprogressing hiv-1 infection. breadth and specificity of the response and relation to in vivo viral quasispecies in a person with prolonged infection and low viral load degenerate cytotoxic t-cell epitopes from p. falciparum restricted by multiple hla-a and hla-b supertype alleles human memory ctl response specific for influenza a virus is broad and multispecific vaccines: correlates of vaccine-induced immunity conformational b-cell epitope prediction on antigen protein structures: a review of current algorithms and comparison with common binding site prediction methods follicular helper cd4 t cells (tfh) • a study that links innate, adaptive and humoral immunity at the follicular dendritic cell level confirmation of immunogenic consensus sequence hiv-1 t-cell epitopes in bamako diversity of francisella tularensis schu4 antigens recognized by t lymphocytes after natural infections in humans: identification of candidate epitopes for inclusion in a rationally designed tularemia vaccine hiv vaccine development by computer assisted design: the gaia vaccine developing an epitope-driven tuberculosis (tb) vaccine coupling sensitive in vitro and in silico techniques to assess cross-reactive cd4(+) t cells against the swine-origin h1n1 influenza virus careful prospective validation of epitope predictions demonstrating that h1n1 'seasonal' vaccination or exposure might protect against h1n1 'pandemic' disease at the t-cell level. it also validates the individualized t-cell epitope measure tool for hla-specific immunogenicity prediction human immune responses to h. pylori hla class ii epitopes identified by immunoinformatic methods peptide-pulsed dendritic cells induce the hepatitis c viral epitope-specific responses of naïve human t cells immunogenic consensus sequence t helper epitopes for a pan-burkholderia biodefense vaccine analysis of chimerivax japanese encephalitis (je) virus sequence for t cell epitopes and comparison to circulating wild type je virus strains immunoinformatic comparison of t-cell epitopes contained in novel swine-origin influenza a (h1n1) virus with epitopes in 2008-09 conventional influenza vaccine a study demonstrating that analysis of hemagglutinin prior to production of vaccine might reveal important insights: in this case, that seasonal influenza might protect against pandemic influenza, a prediction that was later corroborated by other researchers in vitro and in vivo. prospective use of this method for evaluating influenza antigens might be cost-saving in the context of vaccine development and world health programs immunogenicity and immune modulatory effects of in silico predicted l. donovani candidate peptide vaccines identification of secreted proteins of mycobacterium tuberculosis by a bioinformatic approach expasy bioinformatics resource portal epitope-driven tb vaccine development: a streamlined approach using immuno-informatics, elispot assays, and hla transgenic mice immunomics: discovering new targets for vaccines and therapeutics low immunogenicity predicted for emerging avian-origin h7n9: implication for influenza vaccine design a fearless prediction of h7n9 immunogenicity that was later validated (see cross-conservation publication in the same journal apple trees productions, llc novel phage display-based subtractive screening to identify vaccine candidates of brugia malayi juvenile female litomosoides sigmodontis produce an excretory/secretory antigen (juv-p120) highly modified with dimethylaminoethanol mutational escape from cd8+ t cell immunity: hcv evolution, from chimpanzees to man mechanisms of hiv-1 escape from immune responses and antiretroviral drugs allele-specific motifs revealed by sequencing of self-peptides eluted from mhc molecules • one of the first mhc-binding motif descriptions exact prediction of natural t cell epitope nine major hla class i supertypes account for the vast preponderance of hla-a and -b polymorphism one of the several studies describing hla class i 'supertypes', that is, families of hlas that group together based on their hla binding preferences. this one describes supertypes for hla-a and -b (class i). these papers made it possible to design vaccines in silico several common hla-dr types share largely overlapping peptide binding repertoires hlas that group together based on their hla binding preferences. this one describes supertypes for hla-dr (class ii). these papers made it possible to design vaccines in silico a comparison of two methods for t cell epitope mapping: "cell free" in vitro versus immunoinformatics a consensus epitope prediction approach identifies the breadth of murine t(cd8+)-cell responses to vaccinia virus putting immunoinformatics to the test elimination of il-10-inducing t-helper epitopes from an igfbp-2 vaccine ensures potent antitumor activity the two-faced t cell epitope: examining the host-microbe interface with janusmatrix basic local alignment search tool integrated assessment of predicted mhc binding and cross-conservation with self reveals patterns of viral camouflage a mathematical exploration of the cross-conservation between human pathogen (viruses) and human host that demonstrated a significant increase in the number of cross-conserved epitopes in viruses that 'hit and stay' (commensals) as compared to 'hit and run' viruses such as ebola virus-specific cd4(+) memory-phenotype t cells are abundant in unexposed adults further evidence that cross-reactive t-cell recognition is discernable gut immune maturation depends on colonization with a host-specific microbiota 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of immunogenicity of protein vaccines and biologic therapeutics: individualized t cell epitope measure (item) targeting a polyepitope protein incorporating multiple class ii-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by cd4+ cytotoxic t lymphocytes: a novel approach to vaccine design enhancing dna immunization defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string dna vaccine optimization of epitope processing enhances immunogenicity of multiepitope dna vaccines evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses vaccines to combat the neglected tropical diseases a listeria-based vaccine that secretes the sand fly salivary protein ljm11 confers long-term protection against vector-transmitted leishmania major sand-fly saliva-leishmania-man: the trigger trio tick salivary compounds: their role in modulation of host defences and pathogen transmission making vaccines "on demand": a potential solution for emerging pathogens and biodefense? disability-adjusted life years (dalys) for 291 diseases and injuries in 21 regions, 1990-2010: a systematic analysis for the global burden of disease study key: cord-320490-3jmo35jc authors: ismail, saba; ahmad, sajjad; azam, syed sikander title: immuno-informatics characterization sars-cov-2 spike glycoprotein for prioritization of epitope based multivalent peptide vaccine date: 2020-04-12 journal: biorxiv doi: 10.1101/2020.04.05.026005 sha: doc_id: 320490 cord_uid: 3jmo35jc the covid-19 pandemic caused by sars-cov-2 is a public-health emergency of international concern and thus calling for the development of safe and effective therapeutics and prophylactics particularly a vaccine to protect against the infection. sars-cov-2 spike glycoprotein is an attractive candidate for vaccine, antibodies and inhibitor development because of many roles it plays in attachment, fusion and entry into the host cell. in this study, we characterized the sars-cov-2 spike glycoprotein by immune-informatics techniques to put forward potential b and t cell epitopes, followed by the use of epitopes in construction of a multi-epitope peptide vaccine construct (mepvc). the mepvc revealed robust host immune system simulation with high production of immunoglobulins, cytokines and interleukins. stable conformation of the mepvc with a representative innate immune tlr3 receptor was observed involving strong hydrophobic and hydrophilic chemical interactions, along with enhanced contribution from salt-bridges towards inter-molecular stability. molecular dynamics simulation in solution aided further in interpreting strong affinity of the mepvc for tlr3. this stability is the attribute of several vital residues from both tlr3 and mepvc as shown by radial distribution function (rdf) and a novel analytical tool axial frequency distribution (afd). comprehensive binding free energies estimation was provided at the end that concluded major domination by electrostatic and minor from van der waals. summing all, the designed mepvc has tremendous potential of providing protective immunity against covid-19 and thus has the potential to be considered in experimental studies. in december 2019, a new strain of coronavirus emerged in wuhan city of hubei province in china and has since spread globally. the virus belongs to clade b of family coronaviridae in the order nidovirales, and genera betacoronavirus and caused pulmonary disease outbreak [1, 2] . it is positive-sense rna, enveloped and non-segmented virus and named as sars-cov-2 as it share 82% sequence identity with sars coronavirus (sars-cov) [3, 4] . sars-cov-2 caused coronavirus disease-19 and evidence suggest a zoonotic origin of this disease [5] . though the zoonotic transmission is not completely understood but facts provide the ground that it proliferates from the seafood market huanan in wuhan and human-to-human transmission resultant into the exponential increase in number of cases [6, 7] . as of march 24, 386,332 cases are reported worldwide with 16,747 deaths and 102,333 recovered patients. among the active cases, 267,252 are currently infected, 255,166 (95%) are in mild conditions and 12,086 (5%) are seriously ill. serisouly illed. among the 119,080 closed cases, 102,333 (86%) are recovered whereas 16,747 (14%) die. on march 11, the world health organization (who) affirmed covid-19 as a pandemic (https://www.worldometers.info/coronavirus/). sars-cov-2 utilizes a highly glycosylated, homotrimeric class i viral fusion spike protein to enter into host cells [8] . this protein is found in a metastable pre-fusion state which undergoes a structural rearrangement facilitating viral membrane fusion with the host cell [9] [10] [11] . the binding of s1 subunit to a host-angiotensin-converting enzyme initiates this process and disrupts the prefusion trimeric structure resulting into s1 subunit dispersion and stabilizes the s2 subunit to a post-fusion conformation [12] . the receptor-binding domain (rbd) of s1 goes through a hingelike conformational change that temporarily hides or exposes the determinants of receptor binding in order to occupy a host-cell receptor [11] . down and up conformation states are recognized where former is related to the receptor-inaccessible state and the later one explains receptor-accessible state and considered as less stable [13] [14] [15] [16] . this critical role of the spike protein makes it an important target for antibody-mediated neutralization, and detailed study of the pre-fusion s structure would provide information at atomic-level helping in the design and development of a vaccine [17] [18] [19] [20] [21] . current data indicates that sars-cov-2 spike and sars-cov spike both share the same functional receptor (host cell) -angiotensin-converting enzyme 2 (ace2) [22, 23] . interestingly, ace2 binds to sars-cov-2 spike ectodomain with ∼15 nm affinity, about 10-20 folds higher than ace2 binding to sars-cov spike [24] . one possible reason for sars-cov-2 capability of spreading infection from human-to-human is sars-cov-2 spike's high affinity for human ace2 [25] . series of cellular immune and humoral responses can be triggered by sars-cov-2 infections [26] . immunoglobulin g (igg) and igm were noticeable after the 2 weeks of onset of infection which are specific antibodies to sars-cov-2. high titers of neutralizing antibodies and sars-cov-2 specific cytotoxic t lymphocyte responses have been identified in the patients who have improved from sars-cov-2. this phenomenon clearly suggest that both cellular and humoral immune reactions are vital to clearing the sars-cov-2 infection [26] [27] [28] [29] [30] . the study presented, herein, is an attempt to get insights about antigenic determinants of sars-cov-2 spike glycoprotein and highlight all antigenic epitopes [31] of the spike that can be used specifically for the design of a multi-epitope peptide vaccine construct (mepvc) [32] to counter covid-19 infections. the epitopes predicted by immunoinformatics techniques were fused together as well as to β-defensin adjuvant [33, 34] to boost the antibody production and longthe mepvc affinity for an appropriate immune receptor as an agonist was checked in the step of molecular docking [60] . tlr3 available under pdb id of 1ziw was retrieved and used as a receptor molecule. tlr3 also named as cd283 is a transmembrane protein belongs to the family of pattern recognition receptor [61] . it detects viral infection-associated dsrna and evoke the activation of interferon regulatory transcription factor (irf3) and (nuclear factor kappa-lightchain-enhancer) nf-kb [62] . unlike other tlrs, tlr3 uses tir-domain-containing adapterinducing interferon-β (trif) as a primary adapter [63] . irf3 eventually induces the development of type i interferons leading to the activation of innate immune system and eventually to long lasting adaptive immunity [64] . the tlr3 receptor and mepvc were used in a blind docking approach through an online patchdock server interface [65] . the interacting molecules were docked according to the shape complementarity principle. the clustering rmsd is allowed to default 4.0 å. the output docked solutions were immediately refined with fast interaction refinement in molecular docking (firedock) server [66] which provides an efficient framework for refining patchdock complexes. the refined complexes were examined and one with lowest global energy was considered as top ranked. the opted complex was subjected to in-depth mepvc conformation with respect to the tlr3 using ucsf chimera 1.13.1 [67] . md simulation was applied on the selected top complex for 50-ns to understand complex dynamics and stability for practical applications. this assay was categorized into three phases: (i) parameters file preparation (ii) pre-processing, and (iii) simulation production [68] . in first phase using an antechamber module of amber16 [69] , complex libraries and set of parameters for tlr3 and mepvc were generated. the complex system was solvated into 12 å tip3p solvation box achieved through leap module of amber. the intermolecular and intramolecular interactions of the system were determined by ff14sb force [70] field. counter ions in the form of na + were added to the system for charge neutralization. in the system pre-processing stage, complex energy was optimized through several rounds: minimization of complete set of 6 hydrogen atoms for 500 steps, minimization of system solvation box energy for 1000 steps with restraint of 200 kcal/mol -å 2 on the remaining system, minimization of complete set of system atoms again for 1000 steps with applied restraint of 5 kcal/mol -å 2 applied on system carbon alpha atoms, and 300 steps of minimization on system non-heavy atoms with restraint of 100 kcal/mol -å 2 on other system components. the complex system then underwent a heating step where the complex was heated gradually to 300k through nvt ensemble, maintained through langevin dynamics [71] and shake algorithm [72] to restrain hydrogen bonds. complex equilibration was achieved for 100-ps. pressure on the system was maintained using npt ensemble allowing restraint on cα atoms of 5 kcal/mol -å 2 . in the simulation production, trajectories of 50-ns were produced on time scale of 2-fs. non-bounded interactions were differentiated by describing cut-off distance of 8.0 å.cpptraj module [73] was lastly used for statistical computation of different structure parameters to probe complex stability. the md simulation trajectories were visualized and analyzed in visual molecular dynamics (vmd) 1.9.3 [74] . axial frequency distribution or simply afd [75] is a novel analytical technique run on simulation trajectories to access ligand 3d conformation with respect to reference receptor atom. such local structural movements are not captured by any other available technique. afd can be mathematically presented by eq.i, where, i and j are ligand atom coordinates on x and y axis with cut-off value k and l, respectively. the mi,j sums interactions frequency that fall in the coordinate (i,j). the interaction energy and solvation free energy for tlr3 receptor, mepvc, tlr3-mepvc complex were calculated utilizing the mmpbsa.py module [76] of amber16. also, an average of the above was estimated as a net binding free energy of the system. the binding free energy was computed through mm-pbsa method and its counterpart mm-gbsa of amber with objective to derive the difference between bound and unbound states of solvated conformations of the same molecule [77] . 1 . computational approach adopted for the design of a sars-cov-2 spike protein based mepvc. the sars-cov-2 spike protein was targeted for mepvc designing because of many filters it fulfilled required for a potential vaccine candidate. first, it does not share any significant homology to the human host and as such chances of autoimmune responses are negligible [78] . second, the protein is also not found to have any sequence identity to the mouse proteome and thus accurate immunological findings can be deciphered from in vivo mice experimentations [79] . this spike protein only harbored one transmembrane helix ensuring the wet lab protein cloning and expression for antigen analysis easy [80] . antigenicity is another factor that make this candidate highly suitable for vaccine designing as this allows efficient binding to the products of host immune system [81] . further, this protein is strongly adhesive which makes it an excellent target for creation of adhesion based vaccine [82] . lastly, all the sequences of sars-cov-2 spike protein are highly conserved thus a vaccine based on its sequence will be highly likely to have broad spectrum immunological implications [83] . prioritization of potential epitopes for the sars-cov-2 spike protein commenced with the mapping of b-cell epitopes that predicted total of 34 epitopes of vary length ranging from one to 62 (s table 1 ). the average score predicted for these b cell epitopes is 0.470, maximum (max) of 0.696 and minimum (min) of 0.188 (fig.2) . each bcell epitope was then analyzed in mhc-1 alleles binding regions prediction [84] . the predicted epitopes were then screened and stringent criteria of lowest percentile score was used to choose the excellent binders. afterward, the b cell epitopes were simultaneously run in mhc-ii alleles binding [85] . likewise mhc-i, reference set of mhc-ii binding were: hla-drb4*01:01, hla-drb1*04:01, hla-drb1*04:05, hla-drb1*07:01, hla-drb1*09:01, hla-drb1*11:01, hla-drb1*03:01, hla-drb1*13:02, hla-drb1*15:01, hla-drb3*01:01, hla-drb1*12:01, hla-drb3*02:02, hla-drb1*08:02, hla-drb1*01:01, and hla-drb5*01:01. the mhc-ii predicted epitopes were also filtered on basis of percentile score and then cross checked with the selected mhc-i allele and those common in both classes were considered only which were 50 in numbers. the shortlisted common mhc-i and mhc-ii epitopes then subjected to antigenicity check. in this check, ability of the filtered b-cell derived t-cell epitopes ability to evoke and bind to products of adaptive immunity. this yielded 38 epitopes all of which have strong ability to bind to the most prevalent drb*0101 with average ic50 score of 35.6552, max of 98 and min of 0.89.the antigenic epitopes then underwent allergenicity check to discard allergic peptides that may cause allergic reactions [86] . this resulted into 31 epitopes. non-toxic epitopes were 7 whereas 6 were ifn-gamma producer (fig.3) . the set of epitopes obtained at different stages of epitope mapping phase is tabulated in table 1 . these epitopes appear to provide coverage to 98% of the world population (fig.4) and green peaks are those predicted as epitopes and non-epitopes, respectively. prioritized t cell epitopes derived from b cells were fused together tandemly by aay linkers to make a multi-epitope peptide (mep). aay linker avoid formation of junctional epitopes and as such enhance epitope presentation [87] . to the n-terminal of mep, eaaak linker was added to attach β-defensin as an adjuvant leading to the design of a mepvc. the mepvc is schematically shown in fig.5a . mepvc offers many advantages compared to a separate antigenic peptide. such vaccines induce both cd4+ and cd8+ responses and the antigens optimization are optimal. eaaak is a rigid spacer and allow separation of the attached domain and promoting efficient immune processing of the epitopes [88] . β-defensins are potent immune adjuvants as they are capable of significantly enhancing production of lymphokines resulting into antigen-specific ig production and t cell-dependent cellular immunity. the sequence of mepvc is: giintlckyycrvrggrccvcsccpkeeqigkcstrgrkccrrkkecaakyawnrk cisacyiapgqtgkiccyprrarsvcsacytvydpcqpcaayvydplcpelcayckn htscdv. physicochemical properties of the mepvc were evaluated in order to assist experimentalists in the field to setup experiments accordingly in vitro and in vivo. the length of mepvc is spanned across 110 amino acids and has molecular weight of 13.30 kda. vaccine construct with weight less than 110 kda is generally believed to effective vaccine target because of its easier purification. theoretical pi of mepvc is 9.8 and aids in locating mepvc on 2d gel. mepvc aliphatic index is 69.08 projecting the vaccine thermostable at different temperatures. the total number of negatively charged and positively charges residues are 8 and 22, respectively. the grand average of hydropathicity (gravy) score computed for the mepvc is -0.545, illustrating hydrophilic nature of the protein and is likely to interact with water molecules. the estimated half-life of mepvc in mammalian reticulocytes, in vitro is 30 hours, yeast, in vivo is greater than 20 hours, and escherichia coli, in vivo higher than 10 hours. the antigenicity of the mepvc was cross-checked and predicted highly antigenic with value of 0.69. total entropy of the protein is 17.0170 which is considered ideal and also the vaccine has no transmembrane helices (alpha helical transmembrane protein, 0.0474783 and beta barrel transmembrane protein, 0.0060384) hence no difficulties can be anticipated in cloning and expression analysis. the predicted solubility upon overexpression of mepvc is 0.965751 reflecting higher solubility of mepvc. the 3d model of the mepvc was constructed using ab initio 3dpro predictor as no appropriate template was available for homology modeling and threading methods. the 3d structure of mepvc is shown in fig.5b . the structure secured 85.4% of residues in the ramachandran favored, 12.6%, 1.9% and 0% residues in additionally allowed, generously allowed and disallowed regions, respectively. as the predicted mepvc unit has number of loop regions that need to be modeled proper before moving forward. in total, five sets of residues: alas7-lys32, ile63-gly69, cys73-arg77, thr87-pro102, and asn113-val119 were loop modeled. the loop modeled structure increased the ramachandran favored residues percentage to 92.3%, residues in allowed region reduced to 6.7%, residues of generously allowed region to 1.0% and disallowed remained to 0%. the structure was subjected to structure perturbations and relaxations to obtain a refined model. among the generated structures (stable 2 ), the first model was selected as it has improved rama favored score, lowest stable galaxy energy of 0.96, improved clash score of 23.1 and good molprobity value. similarly, the structure lacks poor rotamers in contrast to the original structure. the ramachandran statistics for the refined 3d structure are in following order: ramachandran favored residues (93.2%), additionally allowed region (5.8%), generously allowed region (1.0%) and disallowed region (0 %). the z-score of the refined mepvc is -4.3 and within the score range of same size protein in structure data bases (sfig.1). fig.5 . a. schematic depiction of the mepvc. b. the original predicted 3d mepvc structure and refined along with respective ramachandran plots. aay linkers are shown in red while epitopes are in coal and yellow is for eaaak linker. cyan color represents the β-defensin adjuvant. in the ramachandran plot, the torsion angles are shown by black squares dispersed across the core secondary structures (colored as red). the allowed regions can be understand by yellow, generously allowed by pale yellow and disallowed by white region. the top right, top left, bottom right and bottom left represent quadrants for left handed alpha helices, beta sheets, right handed alpha helix, and no elements, respectively. further, disulfide engineering of the mepvc was performed in order to optimize molecular interactions and confer considerable stability by attaining precise geometric conformation [89, 90] . eight pairs of residues were selected to be replaced with cysteine amino acid. these pairs are: gln7-ala19 (χ3 angle,+118, energy value, 4.20 kcal/mol), cys18-leu21 (χ3 angle,+84.35, energy value, 3.69 kcal/mol), lys44-ala47 (χ3 angle,+74.17, energy value,5.59 kcal/mol), arg57-ala61 (χ3 angle,+122.71 , energy value,6.14 kcal/mol), ala72-ala85 (χ3 angle,-62.92, energy value,4.40 kcal/mol), ala73-ala82 (χ3 angle, , energy value, kcal/mol), leu92-glu95 (χ3 angle, -102.37 , energy value, 3.86 kcal/mol), and phe111-pro117 (χ3 angle,-96.20 , energy value,4.14 kcal/mol). these residues have either higher energy level i.e. > 2 kcal/mol and χ3 angle out of range (< −87 and > + 97) were selected on purpose to make them stable. the original and disulfide mutant mepvc structures are shown in fig.6 . the primary purpose of in silico cloning of the mepvc was guide molecular biologist and genetic engineers about the possible cloning sites and predicted level of expression in a specific expression system for instance here in this study we used e. coli k12 system. prior to cloning, reverse translation of the mepvc sequence was conducted to have an optimized codon usage as per e. coli k12 to yield its max expression. the cai value of the improved mepvc sequence is 1 indicating ideal expression of the vaccine [91] . the gc content whereas is 53.2 % nearly to the e. coli k12 and range within the optimum ranged between 30 % and 70%. the cloned mepvc is shown in fig.7 . both primary and secondary immune responses seem to play a significant contribution against the pathogen and may be compatible to the actual immune response. the in silico host immune system response to the antigen is shown in fig.8 . high concentration of igg +igg and igm was characterized at the primary response, followed by igm, igg1+ igg2 and igg1 at both primary and secondary stages with concomitant of antigen reduction. additionally, robust response of interleukins and cytokines were observed. all this suggest the efficient immune response and clearance of the pathogen upon subsequent encounters. elevated b cell population including memory cells and different isotypes in response to the antigen, points to the long lasting formation of memory and isotype switching. the t helper cell population additionally with the cytotoxic t cell and their respective memory development are in strong agreement of strong response to the antigen. bioinformatic modelling driven molecular docking of the desingned mepvc to one representative innate immune response receptor tlr3 was carried out in order to decode mepvc potential of binding to the innate immune receptors. this was fundamental to understand as tlr3 is significant in recognition of virus associated molecular patterns and of activaiton of type i interferons and nf-kappa b. the docking assesment predicted top 20 complexes sorted mainlny on scoring functions along with interacting molecules area size, desolvation energy, and complexes actual rigid transformation. following, the complexes were subjected to firedock web server for refinement assay. this ease in discarding flexibility errors of the docking procedure and provide a deep refinement of the predictions thus limiting the chances of false positive docking calculations. according to the global energy, solution 8 was considered as a best complex with net global energy of -20.78 kj/mol ( table 2 ). this energy is the output of -16.88 kj/mol attractive van der waals (vdw), 3.81 kj/mol repulsive vdw, 8.14 kj/mol atomic contact energy (ace), and -0.93 kj/mol hydrogen bond energy. the docked conformation and chemical interacting residues of the mepvc with tlr3 is shown in fig.9 . visual inspection of the complex leads to observation of deep binding of the mepvc at the center of tlr3 and favor rigorously rigoursly hydrogen and weak van dar waals interactions with various residues of tlr3. within 3 å, the mepvc was noticed to formed interactions with his39,val55,asn57,asp81,phe84,val103,asn105,gln107,his108,thr126,glu127,ser132,hi s129,thr151,his156,gln174,glu175,lys200,lys201,glu203,asn229,ser256,asp280,ser28 2,tyr283,tyr302,phe304,tyr307,lys330,tyr383,tyr326,asn328,his359,asn361, and glu363. the stability of mepvc with tlr3 was further investigated through md simulations. the trajectories of md simulations were used in vital statistical analysis to decode backbone stability and residual flexibility. root mean square deviation (rmsd) [92, 93] was performed first that compute average distance of backbone carbon alpha atoms of superimposed frames (fig.10a) . the average rmsd for the system is 3.23 å with max of 4.4 å at 24-ns. an initial sudden change in rmsd can be seen up to 2.7-ns that may be due to adjustments adopted by the complex when exposed to dynamics forces and milieu. the second minor rmsd shift can be noticed between 22-ns to 26-ns. afterward, the system is quite stable with not global and local conformational changes specified. next, root mean square fluctuations (rmsf) [94] was applied on the system trajectories (fig.10b) . rmsf is the average residual mobility of complex residues from its mean position. mean rmsf for the mepvc-tlr3 complex calculated is 1.60 å with max of 8.6 å pointed at the n-terminal of the mepvc. most of the interacting residues of the mepvc with tlr3 are subject to minor fluctuations, a fact in analogy to complex high stability. the thermal residual deviation was assessed afterward by beta-factor (β-factor) [95] , the outcomes of which is strongly correlated to the rmsf and hence further affirming system stability (fig.10c) . the average β-factor of the system analyzed is 88.64 å² with max of 1956.23 å². lastly, we evaluated the compactness of the system by means of radius of gyration (rg) [96] analysis (fig.10d) . high rg and low rg illustrate the magnitude of system compactness and system less tight packing. it further tell us the whether the system of interest in order or not. highly compact system is an indication of system stability and vice versa. the mean rg for our system is 55.8411 å with max score of 74.884 reflecting higher ordered and compact nature of the system. hydrogen bonds are dipole-dipole attractive forces and formed when a hydrogen atom bounded to a highly electronegative atom such as f, n, and o is attracted by another electronegative atom [97] . the strength of a hydrogen bond vary from 4 kj to 50 kj per mole. hydrogen bonds are deemed vital in molecular recognition and provide rigidity in achieving stable conformation [98] . the frequency of hydrogen bonds in each frame of the md simulation trajectories can be visualized in fig.11a . these hydrogen bonds are extracted by mean of vmd hbonds plugin and are 104 in number as tabulated in table 3 . the cut-off distance set is 3.0 å and cut-off angle 20 degrees. each residue pair may for one, two or more each of which is counted separately. the min, mean and max number of hydrogen bonds between mepvc and tlr3 are 1, 5, and 12, respectively. the distribution and bonding pattern of intermolecular interactions of the mepvc residues atom(s) with respect to the tlr3 were studied through radial distribution function (rdf) (abbasi et al., 2016; donohue, 1954; kouetcha et al., 2017) . rdf mainly describes distance 'r' between two entities and is represented by g(r). the factor 'r' is extracted from simulation trajectories and range from o to ∞ [75] . the hydrogen bonds predicted by vmd were utilized in rdf that shown only 8 interactions between mepvc and tlr3 with good affinity for each other. in these interactions, tlr3 residues (atoms) are: asp52:od1, gln78:he21, glu98:oe1, asp124:od2, lys171:hz3, asp251:od2, glu323:oe2, and glu328:oe1 are found to have strong radii distribution to their counterpart mepvc residues (atoms): arg705:hh11, arg705: o, lys679:hz3, arg706:hh12, glu675:oe1, asn633:hd22, arg643:hh22, and tyr638: hh, respectively. the rdf plots for the above said interactions are illustrated in fig.11b . the interaction between asp124-od2 and arg706-hh12 has a refined distribution pattern and highest density distribution among all. the max g(r) value for this interaction is 3.51 observed at distance range of 1 å. this is followed by glu328-oe1-tyr638-hh with max g(r) value of 3.26 mostly interaction at distance range of 0.6 å. the glu323-oe2-arg643-hh22 is also much refined having g(r) value of 1.98 and mostly interacts within distance range of 0.6 å. the remaining interactions density distribution is not confined and vary considerably but important from mepvc and tlr3 interaction point of view. salt bridges are non-covalent in nature and the outcome of interactions between two ionized states [101] . these interactions comprised two parts: an electrostatic interaction and a hydrogen bond. in salt bridges, lysine or arginine typically behave as base where glutamine or aspartate as acid and the bridge is created when carboxylic acid group allows a proton migration to guanidine and amine group in arginine. salt bridges are the strongest among all non-covalent interactions and contribute to a major extent in biomolecular stability [102] [103] [104] . in total, 17 salt bridges were identified between tlr3 and mepvc within the cut-off distance of 3.2 å as can be depicted from fig.11c . the higher numbers of salt bridges were recorded for tlr3-glu628 and mepvc-lys685. the mean number of salt bridges for this interaction is 18, max, 35 and min, 3. the count for other salt bridges from tlr3 to mepvc is in following order: asp124-arg706 (mean, 3, max,7 and min,3), glu98-lys679 (mean, 5, max,10 and min,4), asp402-arg641 (mean, 12, max, 18 and min, 4), glu146-arg706 (mean,7 max,11 and min,3), glu146-lys679 (mean,5 max,13 and min,2), glu272-lys655 gln722-side 0.02% the vital hydrogen bond interactions involved between tlr3 receptor and mepvc shortlisted by vmd were subjected to a novel afd analysis to elucidate 3d movements of mepvc atoms with respect to a reference tlr3 residues atom in simulation time. to this objective, interactions mentioned in the rdf were used in afd. preliminary investigation suggested that only three interactions: tlr3-asp52-mepvc-arg705, tlr3-glu328-mepvc-tyr638, and tlr3-glu323-mepvc-arg643 are mainly represented frequently and found in most of the simulation frames. the tlr3-asp52-mepvc-arg705 is uncovered in 4997 frames, tlr3-glu328-mepvc-tyr638 in 4988, and tlr3-glu323-mepvc-arg643 in 4985 making these interactions ideal for interpreting density distribution of the interactions on xyz planes and also appropriate for gaining ideas about conformational changes of the interacting atoms with respect to each other. as the local structure movements and rotations are responsible for functional shifts, their understanding in our system is important to be unveiled. for tlr3-asp52-mepvc-arg705 (fig.12) , the density distribution is not uniform, dispersed and behave flexibility in affinity on all three axis for the receptor atom. parallel, the strength of interaction is also observed affected due to these minor structural movements of the mepvc residue atom. though, the mentioned interaction depicts mepvc is still within the vicinity of the tlr3 reference residue and enjoys this interaction flexibility with the said mepvc residue during simulation. tlr3-glu328-mepvc-tyr638 interaction (fig.13 ) has less distribution area and has much higher intensity illustrating strong affinity of the interacting atoms for each other. it also gives an idea of the lesser movements of the atoms with respect to each other, an indication of a correct system conformation. the distribution area tlr3-glu323-mepvc-arg643 is much dispersed though high intensity of the interaction can be seen in close vicinity (fig.14) . the net free energy of binding (δtotal) in both gb and pb models are revealed favorable mepvc-tlr3 complex in pure water. the net gb and pb energy for the mepvc-tlr3 complex is -53.81 kcal/mol and -89.02 kcal/mol, respectively. to this energy, high contribution was noticed from gas phase energy (δg gas) compared to highly insignificant contributions from solvation energy (δg solv). in gb model, the δg gas energy for the system is -1889. 76 the net free energy of the simulated system was subjected to per residues and pairwise residues decomposition to point residues that contribute majorly in system stabilization and lower energy. molecular docking simulation studies demonstrated 64 residues from the tlr3 receptor that are in direct contact with the mepvc but per residue decomposition assay illustrated that among the residues only hie31, phe55, glu98, hie100, met102, ile121, thr122, asp124, glu146, glu146, glu174, ser176, phe198, asn200, ser225, met249, asp251, tyr254, tyr273, phe275, tyr278, tyr297, glu323, hie324, and tyr348 are hotspot as they contribute rigoursly in binding interaction with mepvc at the docked side. the side chain of hie100, thr122, asp124, glu174, ser176, tyr254, and glu323 contribute significantly in chemical interactions and have energy value in following order: -2.86602 kcal/mol, -3.71782 kcal/mol, -3.77019 kcal/mol, -3.80724 kcal/mol, -2.71475 kcal/mol, -2.40187 kcal/mol, and -3.54158 kcal/mol, respectively. to these tlr3 hotspot residues, the mepvc interacting residues were also observed in quite lower energies illustrating high affinity for the receptor residues for chemical interactions. the binding free energy of the tlr3-mepvc complex, tlr3 receptor, mepvc and the net system energy is further decomposed into 100 frames extracted from simulation trajectories (sfig.3 ). this information deemed vital in predicting the simulation time where higher intermolecular affinity was observed and can guide about the most suitable docked conformation. in general the complex, receptor and construct energies are higher in pb compared to gb but for the total energy, the pb energies are quite lower for frames in contrast to gb. for the complex, the min, max and average binding energy reported are -67264.7 kcal/mol, -66901.5 kcal/mol, and -67069.5 kcal/mol, respectively in gb. the pb max frame energy is -66751.4 kcal/mol, min is -67120.2 kcal/mol and average is -66921.6 kcal/mol. the gb receptor max is -57111.5 kcal/mol whereas the min is -57381. 8 pair-wise energy contribution to the net energy of the system was accomplished in order understand pair residues role from both tlr3 and mepvc in complex stability. we found that the thr122 and asp124 (-4.56 kcal/mol in gb and -5.45 kcal/mol in pb), glu174 and ser176 (-3.45 kcal/mol in gb and -3.77 kcal/mol in pb), glu323 and hie324 (-2.86 kcal/mol in gb and -3.99 kcal/mol in pb) of tlr3 receptor have high combine contribution to the net energy. in case of mepvc, asn633 and thr634 (-3.21 kcal/mol in both gb and pb), val642 and arg643 (-5,87 kcal/mol in gb and -3.27 kcal/mol in pb) and arg705 and arg708 (-2.74 kcal/mol and 2.04 kcal/mol). taken together, we characterized sars-cov-2 spike glycoprotein for antigenic peptides and proposed a mepvc by means of several computational immunological methods and biophysical calculations. the outcomes of this study could save time and associated cost that go into experimental epitope targets study. the mepvc is capable of activating all components of the host immune system, have suitable structural and physicochemical properties. also, it seems to have very stable dynamics with tlr3 innate immune receptor and thus has higher chances of presentation to the host immune system. however, additional in vivo and in vitro experimentations are needed to disclose its potential in fight against covid-19. no conflict of interest was reported by the authors. authors are highly grateful to the pakistan-united states science and technology cooperation program (grant no. pak-us/2017/360) for granting the financial assistance. fig.1 . prosa-z energy plot for the mepvc. fig.2 . residue wise decomposition of net binding energy into tlr3 receptor and mepvc interacting residues. top (gb) and bottom (pb). fig.3 . binding energy decomposition per frame for tlr3-mepvc complex (a), tlr3 receptor (b), mepvc (c) and net energy (d). stable 1 . b cell epitopes predicted for the sars-cov-2 spike glycoprotein. stable 2 . top 5 refined model of the mepvc. the input mepvc is also provided. glu323-arg646 (mean,5 max,10 and min,3), glu323-lys655 (mean,13 max,25 and min,3), glu328-arg641 (mean,10 max,17 and min,4), glu628-lys699 (mean,9 max,28 and min,2), glu675-lys171 (mean,4 max,13 and min,2), glu675-lys172 (mean,10 max,16 and min,2), glu98-arg705 (mean,9 max,14 and min, 5), glu98-arg706 (mean covid-19, an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics a review of the 2019 novel coronavirus (covid-19) based on current evidence emerging coronaviruses: genome structure, replication, and pathogenesis the deadly coronaviruses: the 2003 sars pandemic and the 2020 novel coronavirus epidemic in china 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an indicator of protein structure compactness the hydrogen bond in molecular recognition hydrogen bonds in proteins: role and strength ultrafast scalable parallel algorithm for the radial distribution function histogramming using mpi maps radial distribution functions of some structures of the polypeptide chain defining the role of salt bridges in protein stability do salt bridges stabilize proteins? a continuum electrostatic analysis protein stabilization by salt bridges: concepts, experimental approaches and clarification of some misunderstandings contribution of surface salt bridges to protein stability: guidelines for protein engineering key: cord-347647-m9vk9m7h authors: eapen, mary; zhang, mei-jie; tang, xiao-ying; lee, stephanie j.; fei, ming-wei; wang, hai-lin; hebert, kyle m.; arora, mukta; chhabra, saurabh; devine, steven m.; hamadani, mehdi; d'souza, anita; pasquini, marcelo c.; phelan, rachel; rizzo, j. douglas; saber, wael; shaw, bronwen e.; weisdorf, daniel j; horowitz, mary m. title: hematopoietic cell transplantation with cryopreserved grafts for severe aplastic anemia date: 2020-05-08 journal: biol blood marrow transplant doi: 10.1016/j.bbmt.2020.04.027 sha: doc_id: 347647 cord_uid: m9vk9m7h the covid-19 pandemic and the ensuing barriers to the collection and transport of donor cells, it is often necessary to collect and cryopreserve grafts before initiation of transplant conditioning. the effect on transplant outcomes in non-malignant disease is unknown. this analysis examined the effect of cryopreservation of related and unrelated donor grafts for transplantation for severe aplastic anemia in the us during 2013-2019. included are 52 recipients of cryopreserved grafts who were matched for age, donor type, and graft type to 194 recipients who received non-cryopreserved grafts. marginal cox regression models were built to study the effect of cryopreservation and other risk factors associated with outcomes. we recorded higher 1-year rates of graft failure (hr 2.26, 95% ci 1.17 – 4.35, p=0.01) and of 1-year overall mortality (hr 3.13, 95% ci 1.60 – 6.11, p=0.0008) after transplantation of cryopreserved compared to non-cryopreserved grafts with adjustment for sex, performance score, comorbidity, cytomegalovirus serostatus, and abo blood group match). acute and chronic gvhd did not differ between groups. adjusted probabilities of 1-year survival were 73% (95% 60 – 84) and 91% (95% ci 86 – 94) with cryopreserved and non-cryopreserved grafts, respectively. these data support the use of non-cryopreserved grafts, when possible, for severe aplastic anemia. the included are 52 recipients of cryopreserved grafts who were matched for age, donor type, and graft type to 194 recipients who received non-cryopreserved grafts. marginal cox regression models were built to study the effect of cryopreservation and other risk factors associated with outcomes. we recorded higher 1-year rates of graft failure (hr 2.26, 95% ci 1.17 -4.35, p=0.01) and of 1-year overall mortality (hr 3.13, 95% ci 1.60 -6.11, p=0.0008) after transplantation of cryopreserved compared to noncryopreserved grafts with adjustment for sex, performance score, comorbidity, cytomegalovirus serostatus, and abo blood group match). acute and chronic gvhd did not differ between groups. adjusted probabilities of 1-year survival were 73% (95% 60 -84) and 91% (95% ci 86 -94) with cryopreserved and non-cryopreserved grafts, respectively. these data support the use of non-cryopreserved grafts, when possible, for severe aplastic anemia. the emergence of coronavirus disease 2019 (covid-19) as a global pandemic triggered an unprecedented worldwide health-care crisis. it also impacted the world economy and disrupted travel across international borders and within countries. these travel restrictions combined with potentially reduced hct donor availability (due to infection, quarantine, and constraints on travel to collection centers) and complex allograft processing logistics (donor assessment, collection, on-schedule delivery for fresh infusion) directly impacted the ability to infuse fresh donor-cells into intended recipients on the scheduled day of transplantation. consequently, the american society for transplantation and cellular therapy (astct) 1 thus, the current analysis was undertaken to inform clinical practice for transplantation for severe aplastic anemia, a common non-malignant indication for hct. patients with severe aplastic anemia were identified from the database of the cibmtr the primary outcome was 1-year survival. death from any cause was considered an event and surviving patients were censored at 1-year or earlier for follow-up less than 1 year. neutrophil recovery was defined as the first of 3 consecutive days with absolute neutrophil count (anc) ≥ 0.5 x 10 9 /l, and platelet recovery, ≥ 20 x 10 9 /l without transfusion for 7 days. graft failure was defined as a failure to achieve anc ≥0·5 x 10 9 /l or anc decline to <0·5 x 10 9 /l without recovery after having achieved anc ≥0·5 x 10 9 /l, or myeloid donor chimerism (<5%), or second transplant. 8 other outcomes studied were grade ii-iv acute and chronic gvhd, graded using standard criteria. 9,10 fifty-two patients (cases) who were transplanted with a cryopreserved graft were matched on age (≤17, 18 -39 and ≥40 years), 11, 12 donor type (hla-matched sibling, haploidentical relative, hla-matched or hla-mismatched unrelated donor) 12, 13 and graft type (bone marrow or peripheral blood) 14, 15 to 195 controls from a pool of 979 patients transplanted during the same period with non-cryopreserved grafts. forty-five cases were matched to 4 controls, 2 were matched to 3 controls, 4 were matched to 2 controls and 1 was matched to 1 control. to study the effect of cryopreserved compared to non-cryopreserved grafts, (matched-pairs) marginal cox regression models were built and adjusted for sex, cytomegalovirus serostatus, performance score, comorbidity score, and donor-recipient abo blood group match. 16 all variables met the assumptions for proportional hazards. results are expressed as hazard ratio (hr) with 95% confidence interval (ci). adjusted probabilities for outcomes of interest were generated from the marginal cox model. 17, 18 the level of significance was p-value ≤0.01 (two-sided), in consideration of the multiple comparisons. analyses were done using sas version 9.4 (cary, nc). the characteristics of the treatment groups matched for age, donor type and graft type, are shown in table 1 . [11] [12] [13] [14] [15] females were more likely to receive cryopreserved grafts but other characteristics such as recipient cytomegalovirus serostatus, performance score, comorbidity index and donor-recipient abo blood group match, transplant conditioning regimen and gvhd prophylaxis were similar between treatment groups. although the total nucleated cell doses (tnc) of harvested bone marrow were similar between the groups, the tnc dose infused differed with recipients of cryopreserved bone marrow grafts receiving significantly lower cell doses ( table 1) . the difference between cell dose at harvest and infusion was statistically significant (paired t-test, p=0.0008). cd34 doses for peripheral blood grafts were not significantly different between cryopreserved and non-cryopreserved grafts ( table 1 ). the median follow-up of surviving cases and controls was 35 months (range 6 -74) and 26 months (range 5 -76), respectively. we did not record statistically significant differences in day-28 neutrophil recovery between those who received cryopreserved and fresh grafts, 83% (95% ci 71 -92), and 91% (95% ci 86 -94), p=0.17, respectively. the corresponding incidences of day-100 platelet recovery were 91% (95% ci 79 -98) and 90% (95% ci 86 -94), p=0.89. in multivariate analysis, the rate of neutrophil (hr 0.76, 95% ci 0.54 -1.08, p=0.13) and platelet (hr 0.77, 95% ci 0.57 -1.04, p=0.08) recovery was lower with transplantation of cryopreserved grafts but the difference did not reach statistical significance. however, the risk of 1-year graft failure was higher after transplantation of cryopreserved compared to non-cryopreserved graft (hr 2.26, 95% ci 1.17 -4.35, p=0.01), figure 1a . graft failure was primary for 7 patients who received cryopreserved and for 8 patients who received non-cryopreserved grafts. three patients who received cryopreserved and for 11 patients who received non-cryopreserved grafts were reported to have developed secondary graft failure. the likelihood of hematopoietic recovery and risk for graft failure were adjusted for sex, recipient cytomegalovirus serostatus, performance score, comorbidity index, and blood group abo match. we did not observe differences in grade ii-iv acute gvhd (hr 0.93, 95% ci 0.41 -2.13, p=0.87) and chronic gvhd (hr 0.79, 95% ci 0.41 -1.50, p=0.46) by treatment group. the day-100 incidence of grade ii-iv acute gvhd after transplantation of cryopreserved and non-cryopreserved grafts were 12% (95% ci 5 -22) and 13% (95% ci 8 -18), p=0.94, respectively. the corresponding incidence of 1-year chronic gvhd was 23% (95% ci 12 -37) and 28% (95% ci 21 -35), p=0.49. one-year mortality was higher after transplantation of cryopreserved compared to noncryopreserved grafts (hr 3.31, 95% ci 1.60 -6.11, p=0.0008) after adjusting for sex, recipient cytomegalovirus serostatus, performance score, comorbidity index and blood group abo match. the adjusted 1-year probabilities of overall survival were 73% (95% 60 -84) with cryopreserved and 91% (95% ci 86 -94) with non-cryo-reserved grafts, the current analysis was undertaken to examine whether there are differences in survival or other transplant outcomes after transplantation of cryopreserved bone marrow or peripheral blood for severe aplastic anemia. recipients of cryopreserved grafts were matched to recipients of non-cryopreserved grafts for age at transplantation, donor type/donor-recipient hla-match and graft type, factors that are consistently associated with transplant outcomes for this disease. [11] [12] [13] [14] [15] the analyses also considered the effect of other potential risk factors on transplant-outcomes. after carefully controlled analyses, we observed higher graft failure and mortality rates after transplantation of cryopreserved compared to non-cryopreserved grafts. thus, our findings favor the transplantation of non-cryopreserved grafts for severe aplastic anemia. transplant conditioning-regimen for severe aplastic anemia varies by type of donor. 19 other reports have shown an effect of conditioning-regimen for survival after hlamatched sibling transplants. 19 none of the patients in the current analysis received cy alone or cy with flu, the conditioning regimens that are associated with higher graft failure and mortality rates. 19 the cell dose of the graft has also been associated with graft failure. 20 it is recommended that bone marrow grafts contain a minimum of 3 x 10 8 /kg tnc to avoid graft failure. 20 these data derive from an analysis of noncryopreserved bone marrow grafts. data on infused bone marrow tnc was available for only 70% (23 of 33) cryopreserved and 83% (109 of 132) and non-cryopreserved grafts. despite this limitation, we observed significantly lower tnc infused after cryopreservation of bone marrow grafts. although 68% of patients receiving cryopreserved bone marrow grafts had ≥3 x 10 8 /kg tnc harvested, only 26% had that number infused. this loss of cells may have resulted in the observed differences in outcomes. the difference between tnc at harvest and infusion implies cryopreservation/thaw process is associated with cell loss. however, there may be other unmeasured or unknown factors that also resulted in the observed differences in outcome. we do not have data on cell function at any time point. an earlier report on the functional assay of cryopreserved bone marrow suggests preservation of cell function although that report included only 7 grafts. 21 an analysis of non-cryopreserved bone marrow cellular subsets for unrelated donor transplantations failed to show an effect of graft composition on hematopoietic recovery or survival although that report included only 7 patients with aplastic anemia. 22 all cryopreserved peripheral blood grafts in the current analysis had cd34+ doses greater than 2 x 10 6 /kg, the recommended minimum dose for severe aplastic anemia. 23 a subset analysis limited to recipients of peripheral blood was consistent with the findings of the main analysis. cryopreserved cd34+ cells from peripheral blood are reported to have significant loss of membrane integrity, viability, and cfu potential that collectively could contribute to the adverse effect of transplantation of cryopreserved peripheral blood in our study. 23 we hypothesize that several factors led to the poor outcomes after transplantation of cryopreserved grafts. optimizing cell dose is desirable but a controlled study that examines for changes in graft composition with cryopreservation/thaw that is specific for aplastic anemia is needed. a detailed analysis of the composition and function of cryopreserved grafts is beyond the scope of this study. we did not observe statistically significant differences in neutrophil and platelet recovery despite lower rates of recovery after transplantation of cryopreserved grafts. we hypothesize the absence of significant differences is attributed the modest number of patients in the current analysis. we do not know the indication for using a cryopreserved graft for the patients included in this analysis. the data on the interval between diagnosis and transplant was not different between the treatment groups. further, the timing of transplantation by donor type is also consistent with accepted clinical practice guidelines. hla-matched sibling transplants were mostly offered within 6 months from diagnosis and alternative donor transplants later after failure of at least one course of immunosuppressive therapy. 24 recipients of cryopreserved and noncryopreserved grafts were matched for graft type (bone marrow or peripheral blood). subset analyses limited to peripheral blood transplants confirmed higher graft failure and mortality consistent with the main analysis and suggest a greater effect than with bone marrow grafts. these findings differ from findings in studies of patients receiving cryopreserved grafts for hematologic malignancies. patients with malignancy often come to transplant after multiple chemotherapy and immune-suppressive therapies and also usually receive more intensive pretransplant conditioning than patients with aplastic anemia. for these reasons, and perhaps because of difference in the nature of the underlying diseases, the risk of graft failure is in general lower after hct for malignant disease than the risk after hct aplastic anemia and may be less affected by any alterations in cell dose or function induced by cryopreservation. in summary, the data support the use of non-cryopreserved bone marrow or peripheral blood for transplantation for severe aplastic anemia. if this is not possible, it may be prudent to delay transplantation until it is. these transplants are often not deemed urgent and every effort must be made to provide the best available supportive care for the patient until the transplant center can ensure the availability of a noncryopreserved graft. if a delay is not possible, careful assessment of the risk of cryopreservation versus the risk of not receiving the graft when it is needed is necessary. the nmdp/be the match considers the diagnosis of aplastic anemia a valid reason to try to deliver a fresh graft for unrelated donor transplants for severe aplastic anemia. figure 1a . the 1-year graft failure for cryopreserved and non-cryopreserved transplants was 19% (95% ci 10 -31) and 10% (95% ci 6 -14), respectively figure 1b . the 1-year overall survival for cryopreserved and non-cryopreserved transplants was 73% (95% ci 60 -84) and 91% (95% ci 86 -94), respectively national marrow donor program (be the match) has allogeneic stem cell cryopreservation been given the 'cold shoulder'? an analysis of the pros and cons of using frozen versus fresh stem cell products in allogeneic stem cell transplantation long-term follow-up of leukaemia patients after related cryopreserved allogeneic bone marrow transplantation similar outcomes of cryopreserved allogeneic peripheral stem cell transplants (pbsct) compared to fresh allografts cryopreservation of allogeneic pbsc from related and unrelated donors is associated with delayed platelet engraftment but has no impact on survival graft cryopreservation does not impact overall survival allogeneic hematopoietic cell transplantation using posttransplant cyclophosphamide for gvhd prophylaxis graft failure in the modern era of allogeneic hematopoietic sct consensus conference on acute gvhd grading committee of the international bone marrow transplant registry. consensus among bone marrow transplanters for diagnosis, grading and treatment of chronic graft versus host disease impact of age on outcomes after bone marrow transplantation for acquired aplastic anemia using hla-matched sibling donors how i treat acquired aplastic anemia evaluation of hla matching in unrelated hematopoietic cell transplantation for nonmalignant disorders worse outcome and more chronic gvhd with peripheral blood progenitor cells than bone marrow in hlamatched sibling donor transplants for young patients with severe acquired aplastic anemia effect of stem cell source on outcomes after unrelated donor transplantation in severe aplastic anemia regression models and life tables a sas macro for estimation of direct adjusted survival curves based on a stratified cox regression model sas macros for estimation of direct adjusted cumulative incidence curves under proportional subdistribution hazards models choice of conditioning regimens for bone marrow transplantation in severe aplastic anemia guidelines for the diagnosis and management of aplastic anemia implications of cd34+ cell dose on clinical and hematological outcome of allo-sct for acquired aplastic anemia effect of the composition of unrelated donor bone marrow and peripheral blood progenitor cell grafts on transplantation outcomes transportation and cryopreservation may impact hematopoietic stem cell function and engraftment of allogeneic peripheral blood stem cells and not bone marrow aplastic anemia cy dose=120 mg/kg (n=3) flu + cy + atg cases: cy dose=120 mg/kg (n=5), cy dose 60 mg/kg (n=1) controls cy dose= 120 mg/kg (n=15) cy dose=120 mg/kg (n=2) controls cy dose=200 mg/kg (n=2 cy dose=50 mg/kg (n=15), cy dose=29 mg/kg (n=19) interval between diagnosis and transplant ♯77% were hla-matched sibling and 23% were hla-matched or mismatched unrelated donor transplants ╪55% were hla-matched sibling, 30% were hla-matched or mismatched unrelated donor and 14% were hla-haploidentical transplants ║23% were hla-matched sibling, 59% were hla-matched or mismatched unrelated donor and 19% were hla-haploidentical transplants ♯20% were hla-matched sibling key: cord-344691-vmc0rrrk authors: srinivasan, k.n.; zhang, g.l.; khan, a.m.; august, j.t.; brusic, v. title: prediction of class i t-cell epitopes: evidence of presence of immunological hot spots inside antigens date: 2004-08-04 journal: bioinformatics doi: 10.1093/bioinformatics/bth943 sha: doc_id: 344691 cord_uid: vmc0rrrk motivation: processing and presentation of major histocompatibility complex class i antigens to cytotoxic t-lymphocytes is crucial for immune surveillance against intracellular bacteria, parasites, viruses and tumors. identification of antigenic regions on pathogen proteins will play a pivotal role in designer vaccine immunotherapy. we have developed a system that not only identifies high binding t-cell antigenic epitopes, but also class i t-cell antigenic clusters termed immunological hot spots. methods: multipred, a computational system for promiscuous prediction of hla class i binders, uses artificial neural networks (ann) and hidden markov models (hmm) as predictive engines. the models were rigorously trained, tested and validated using experimentally identified hla class i t-cell epitopes from human melanoma related proteins and human papillomavirus proteins e6 and e7. we have developed a scoring scheme for identification of immunological hot spots for hla class i molecules, which is the sum of the highest four predictions within a window of 30 amino acids. results: our predictions against experimental data from four melanoma-related proteins showed that multipred ann and hmm models could predict t-cell epitopes with high accuracy. the analysis of proteins e6 and e7 showed that ann models appear to be more accurate for prediction of hla-a3 hot spots and hmm models for hla-a2 predictions. for illustration of its utility we applied multipred for prediction of promiscuous t-cell epitopes in all four sars coronavirus structural proteins. multipred predicted hla-a2 and hla-a3 hot spots in each of these proteins. molecules of adaptive immune responses diversified very rapidly in early vertebrates. major histocompatibility complex (mhc) molecules play a vital role in the regulation of immune responses (hudson and ploegh, 2002; watts and amigorena, 2001) . foreign and host proteins are degraded by specialized intracellular mechanisms to short antigenic peptides. the primary function of mhc molecules is to bind and present antigenic peptides on the cell surface for recognition by antigen-specific t-cell receptors (tcrs) of lymphocytes. these processing and presentation mechanisms are essential processes for cellular immune recognition of antigens. mhc class i peptides are primarily generated by the proteasome complex, and are translocated from the cytosol into the lumen of the endoplasmic reticulum (er) by a transporter associated with antigen processing. in the er, peptides are loaded onto the mhc class i molecules and are exported to the cell surface for presentation to tcrs. short peptides presented to tcrs, termed t-cell epitopes, are critical elements for understanding the basis of immunity (parker et al., 1994; van kaer, 2002; britschgi et al., 2003) . precise identification of t-cell epitopes is a prerequisite for accurate epitope mapping and for design of vaccines and immunotherapies. peptides that bind to more than one mhc allelic variant ('promiscuous peptides') are important because they are relevant to higher proportions of the human populations and are targets for vaccine and immunotherapy development. computational methods have been used for the prediction of t-cell epitopes and are now a standard methodology (schirle et al., 2001; yu et al., 2002) . in silico, t-cell epitope mapping using computational models is emerging as a new approach for the study of peptide vaccines (de groot et al., 2001) . a number of predictive methods for mhc classes i and ii binding peptides are available, including those based on binding motifs (rammensee et al., 1995) , quantitative matrices (parker et al., 1994) , artificial neural networks (anns) , hidden markov models (hmms) (mamitsuka, 1998) , multivariate statistical approaches (guan et al., 2003) , support vector machines (zhao et al., 2003) and decision trees (savoie et al., 1999) . computational strategies for promiscuous class ii binding peptides using multiple quantitative matrices (sturniolo et al., 1999) have been used for vaccine development in cancer (kobayashi et al., 2001) and infectious disease (panigada et al., 2002) . in our prediction of promiscuous class i t-cell epitopes, we made predictions of t-cell epitope hot spots in nucleocapsid protein of the severe acute respiratory syndrome coronavirus (sars-cov). multipred, a computational system developed for human leukocyte antigen (hla) classes i-a2 and i-a3 binding, predicts individual 9-mer t-cell epitopes and also promiscuous class i regions as immunological hot spots, based on hmm and ann models (zhang et al., 2003) . severe acute respiratory syndrome, an outbreak of atypical pneumonia was first reported in guangdong province, china in november 2002 and spread to other parts of the world (rota et al., 2003; booth et al., 2003) . genome analysis of sars-cov revealed the virus to be of completely new pathogenic strain and distantly related to other cov members (ruan et al., 2003; holmes and enjuanes, 2003; holmes, 2003) . the four major structural proteins of sars-cov are: surface spike (s), nucleocapsid (n), envelope (e) and membrane (m) (marra, 2003; holmes, 2003) . the packaging of the genome to form the viral nucleocapsid is by the n protein, which is incorporated into virions by intracellular budding through a membrane containing three proteins: the s glycoprotein, the m glycoprotein and the small e protein (kuo and masters, 2002) . it has been demonstrated that antibodies to sars n proteins are predominant among the early responses to infection (shi et al., 2003; liu et al., 2004) . we used ann and hmm as the prediction engines. the ann learning algorithm in multipred is the error backpropagation with sigmoid activation function. the ann is a three-layer network with structure 267-4-1. the inputs to the ann are binary strings representing the virtual peptides; the outputs are the binding scores ranged from 1 to 9. in the training dataset, scores 8 and 9 denote high binding affinity; 6 and 7 moderate binding affinity; 4 and 5 low binding affinity. scores less than 4 denote non-binding. the maximum number of the ann training cycles is set to 300. the training was repeated four times, and four sets of weights were obtained. the value of learning momentum was 0.5 and of learning rate was 0.001. algorithm details of neural network can be found in brusic et al. (1998) . the hmm algorithm, training and testing were described earlier . peptide data containing both binding and non-binding 9-mer peptides were extracted from literature sources, mhcpep (brusic et al., 1994) , and a set of hla non-binding peptides (brusic, unpublished data) for hla-a2 and hla-a3 alleles. the dataset had a total of 2962 (604 binders and 2358 nonbinders) 9-mer peptides representing 15 different hla-a2 alleles and 2216 (680 binders and 1536 non-binders) 9-mer peptides for eight different hla-a3 alleles. the available dataset was divided into training and testing datasets. the training set for a given allele contained virtual peptides that are known to bind other alleles and the test set included all peptides with known binding affinity for the allele to be tested, as described earlier . the performances of multipred ann and hmm in predicting promiscuous binders to different hla alleles were tested by a number of trained ann and hmm models, one model for the prediction of peptide binding to each selected hla allele. models for the prediction of alleles with small number of peptides in the dataset could not be tested reliably and were excluded. the percentage of binders represents ∼25% of the training dataset, while non-binders represent the remaining 75%. to optimize the disproportionate numbers of binders and nonbinders in the training dataset, new training datasets were constructed using a novel approach in which one or more copies of binders (up to 10 copies) were used in the training datasets (zhang et al., manuscript in preparation) . we trained ann models to each of the hla-a2 and hla-a3 alleles using 10 sets of data to find the composition of training data that result in best predictive performance of the training system. the predictive performance was assessed by the sensitivity (se), specificity (sp) and receiver operating characteristic (roc) analysis as described previously . multipred can perform a 10-fold internal crossvalidation and calculate a roc values (measure of overall prediction accuracy) of high, moderate and low binders. the accuracy of prediction is poor for values of a roc < 70%, good for values of a roc > 80%, and excellent for values of a roc > 90% (described in brusic et al., 2002) . peptides that are predicted to bind to multiple hla alleles are considered promiscuous t-cell epitope candidates. the performances of the different multipred ann models containing 1-10 copies of binders (hla-a2 and hla-a3) were compared. the results showed that the binder/non-binder composition of a dataset influences predictive performance of the model. ann models trained on the raw dataset containing a single copy of both binders and non-binders produced inferior prediction results. ann models trained using datasets when top 10% of the predicted peptides were considered as potential t-cell epitopes, multipred could predict all the experimental hla-a3 restricted peptides. *numbers in the parentheses indicate the total number of 9mer peptides predicted for that protein. containing four and six copies of binders provided higher a roc values. because the performance of ann models with four and six copies of binders were comparable, the simpler ann model with four copies was chosen for the prediction of hla-a2 promiscuous peptides. for hla-a3, the ann model with six copies was found to be more accurate in predicting low (l), moderate (m) and high (h) binding peptides. the prediction performance of multipred for hla-a2 and hla-a3 binding was assessed using experimentally known binders. hla-a2 and hla-a3 restricted peptides from four melanoma associated proteins, gp100, tyrosinase, tyrosinase-related protein 2 and melanocortin-1 receptor, (reynolds et al., 1998) were used for validation of multipred. all duplicate 9-mer peptides in the training dataset were removed and the models were re-trained for prediction of promiscuous peptides to hla-a2 and hla-a3. when top 10% of the predicted peptides were considered as potential t-cell epitopes, multipred could predict most of the hla-a2 and all the hla-a3 restricted peptides of the four proteins tested, suggesting that the performance and accuracy of multipred is reliable. of 28 known binders tested for hla-a2, both multipred ann and hmm predicted 27 peptides within top 10% of the scores. within top 5%, ann predicted 22 peptides and hmm 24 peptides. hence the prediction accuracy was 96% for top 10% prediction (both methods) and 78.5% and 85.7% for ann and hmm top 5% prediction, respectively. the prediction accuracy of hla-a3 peptides tested was 100% for both top 10% and 5% of the predicted peptides (table 1) . to assess the accuracy of individual 9-mer predictions, we compared predictions of hpv e7 hla-a2 binding peptides with experimental binding measured by kast et al. (1994) . hpv e7 is 98 amino acids long and contains six 9-mer hla-a2 binders (7-15, 11-19, 12-20, 82-90, 84-92 and 85-93) . of these, four peptides were within top 5% and five were within top 10% of predictions. top 5% predictions contained one false positive and top 10% predictions contained five false positives. we have developed a scoring scheme to identify class i regions termed 'immunological hot spots' within antigens that are based on high scoring individual 9-mers within a window of 30 amino acids. immunological hot spots are thus defined as antigenic regions of up to 30 amino acids that are predicted to bind multiple hla alleles. for validation of hot spot predictions, a test dataset for peptides to hlaclass i alleles were taken from a set of 240 9-mer peptides of human papillomavirus type 16 e6 and e7 proteins reported by kast et al. (1994) . all duplicate 9-mers peptides pertaining to e6 and e7 proteins were removed from the training dataset. the class i epitopes were predicted by the use of both multipred models. the results were sorted to the average score of top four 9-mers within the region (across all the alleles studied for promiscuous prediction) and regions of immunological hot spots were identified. by use of this strategy, our models were successful in identifying class i hot spots for e6 and e7 proteins. multipred ann and hmm hla-a3 output against experimental data from human papillomavirus protein e6 (kast et al., 1994) is shown in figure 1 . hpv protein e7 and e6 hla-a2 hot spots were predicted for validation of multipred ann and hmm models. the known hla-a2 hot spots for protein e7 (e7:7-20 and e7:82-94) that were previously demonstrated (kast et al., 1994) , were predicted by multipred ann and hmm models, with a false negative prediction for e7:7-20 by hla-a2 ann prediction at threshold >60. similarly, the known hla-a2 hot spot for protein e6 (e6 7-34) was predicted with multi-pred hmm and ann models. ann and hmm predictions produced similar results, with a false positive at position 85-105. the results of validation of hot spot predictions by multipred models suggests that the overall performances of were reliable and a single multipred model could make reasonably accurate predictions of peptides binding to multiple alleles of hla molecules, and also for variants of hla supertypes that lack experimental binding data. these predictions will be further improved by increased number of training datasets and additional rigorous testing strategies. the testing results on e6 and e7 proteins indicate that with available datasets ann models appear more accurate for prediction of hla-a3 hot spots, while hmm models appear more accurate for hla-a2 predictions. therefore we selected hmm as a method of choice for prediction of hla-a2 t-cell hot spots and ann for prediction of hla-a3 hot spots. testing results provided a basis for determination of prediction thresholds: a2 scores were calculated as sum of individual predictions for eight hla-a2 variants and a3 scores as sum of individual predictions for six hla-a3 variants. the suitable thresholds for both ann a2 and hmm a2 hot spot (kast et al., 1994) . ann prediction is shown at the top and hmm at the bottom. all three hot spots have been captured with ann a3 predictions with the threshold >35 with a false positive shown inside the square. ann predictions appear to be more accurate than hmm predictions for hla-a3 hot-spots. prediction thresholds were set to >60, while ann a3 and hmm a3 thresholds were set to >35. to illustrate the utility of multipred for prediction of immunological hot spots, we analyzed four structural proteins from the sars-cov. the sars-cov protein sequences were retrieved from ncbi genbank database (ay283798). the four proteins were submitted to multipred for prediction of class i (hla-a2, a3) t-cell epitope hot spots. the hot spots were derived from consensus predictions of both (ann and hmm) models in multipred. the results of the analysis showed that sars-cov e and m proteins were predicted to possess one hot spot each to hla-a2 (e 1-52 and m 4-83) and hla-a3 (e 1-76 and m 74-220) supertypes. further, we have identified two and four immunological hot spots in sars n protein. as for sars s protein, the system predicted three hot spots to hla-a2 (s 848-880, s 937-994 and s 1181-1231) and eight hot spots to hla-a3 (s 270-411, s 1026-1147, s 743-828, s 134-163, s 76-115, s 9-53, s 418-465 and s 886-950) . these results indicate the presence of immunological hot spots of both hla-a2 and hla-a3 molecules in all sars-cov structural proteins. similar patterns have been observed in 10 dengue virus proteins (data not shown). we propose that t-cell epitopes tend to cluster in certain regions of protein antigens in a hla supertype-dependent manner. these regions therefore represent immunological hot spots containing multiple t-cell epitopes. our strategy of peptide-based vaccines is to identify promiscuous t-cell epitopes that are representative of large proportion of the human population. the majority of publicly available methods has not been properly assessed for predictive accuracy and do not predict promiscuous peptides for a broad range of hla alleles. in this context, we have developed a computational system multipred that identifies promiscuous peptides across hla-a2 and hla-a3 alleles, and also a scoring scheme for prediction of immunological hot spots of class i molecules, using multipred ann and hmm models. the system was trained and rigorously tested using experimentally known peptides, human melanoma-related proteins and human papillomavirus type 16 proteins e6 and e7. it was found that ann model could predict hla-a3 with more accuracy than the hmm model, while hmm appeared to be more accurate for hla-a2 predictions. severe acute respiratory syndrome was a great threat both to public health and economy affecting more than 30 countries around the globe and was of great concern due to formidable morbidity and mortality. although sars looked a devastating pandemic and the outbreak was deemed to be under control, the world health organization (2003, http:// www.who.int/csr/sars/country/table2003_09_23/en/) has urged health authorities not to be contented. hence there is a need to design a more efficient vaccine to combat the deadly sars. current therapeutic strategies to sars involve the use of convalescent plasma (burnouf and radosevich, 2003) , glucocorticoids , interferons (cinatl et al., 2003) , but still remains empirical. peptide-based vaccines offer several potential advantages over the conventional whole proteins in terms of high specificity in eliciting immune responses, ease of manufacturing and quality control and proven successful against specific allergy (alexander et al., 2002) , malaria (lopez et 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in the treatment of severe acute respiratory syndrome patients: a preliminary report profile of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (sars)-associated coronavirus in probable sars patients a synthetic malaria vaccine elicits a potent cd8(+) and cd4(+) t lymphocyte immune response in humans. implications for vaccination strategies predicting peptides that bind to mhc molecules using supervised learning of hidden markov models the genome sequence of the sars-associated coronavirus scheme for ranking potential hla-a2 binding peptides based on independent binding of individual peptide side-chains identification of a promiscuous t-cell epitope in mycobacterium tuberculosis mce proteins mhc ligands and peptide motifs: first listing hlaindependent heterogeneity of cd8+ t cell responses to mage-3, melan-a/mart-1, gp100, tyrosinase, mc1r, and trp-2 in vaccine-treated melanoma patients characterization of a novel coronavirus associated with severe acute 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world health organization. cumulative number of reported probable cases of sars: 1 methods for prediction of peptide binding to mhc molecules: a comparative study neural models for predicting viral vaccine targets application of support vector machines for t-cell epitopes prediction this research was supported by the national institutes of health grants, niaid r37 ai 41908 (jta), and u19 ai 56541 (kns, gz, jta, vb); biomedical research council grant, singapore 03/1/55/20/282 (kns, gz, jta, vb); and the national university of singapore graduate scholarship (amk). the authors are also grateful to the agency for science, technology and research, singapore. key: cord-333966-st6gyozv authors: taherkhani, reza; farshadpour, fatemeh; makvandi, manoochehr title: design and production of a multiepitope construct derived from hepatitis e virus capsid protein date: 2015-03-17 journal: j med virol doi: 10.1002/jmv.24171 sha: doc_id: 333966 cord_uid: st6gyozv the aim of this study was to design a high density multiepitope protein, which can be a promising multiepitope vaccine candidate against hepatitis e virus (hev). initially, conserved and antigenic helper t‐lymphocyte (htl) epitopes in the hev capsid protein were predicted by in silico analysis. subsequently, a multiepitope comprising four htl epitopes with high‐affinity binding to the hla molecules was designed, and repeated four times as high density multiepitope construct. this construct was synthesized and cloned into pet‐30a (+) vector. then, it was transformed and expressed in escherichia coli bl21 cells. the high density multiepitope protein was purified by ni‐nta agarose and concentrated using amicon filters. finally, the immunological properties of this high density multiepitope protein were evaluated in vitro. the results showed that the high density multiepitope construct was successfully expressed and purified. sds‐page and western blot analyses showed the presence of a high density multiepitope protein band of approximately 33 kda. approximately 1 mg of the purified protein was obtained from each liter of the culture media. moreover, the purified multiepitope protein was capable of induction of proliferation responses, ifn‐γ elispot responses and ifn‐γ and il‐12 cytokines production in a significant level in peripheral blood mononuclear cells (pbmcs) isolated from hev‐recovered individuals compared to the control group. in conclusion, the newly produced multiepitope protein can induce significant t helper type 1 responses in vitro, and can be considered as a novel strategy for the development of hev vaccines in the future. j. med. virol. 87:1225–1234, 2015. © 2015 wiley periodicals, inc. hepatitis e virus (hev) is a positive-stranded rna virus which has been classified as the member of the genus hepevirus in the family hepeviridae [yamashita et al., 2009 ]. at least four major hev genotypes have been identified, which have different geographical distributions and host ranges [xing et al., 2011] . despite classification into four geographically distinct genotypes, the hev strains antigenically belong to a single serotype and share cross-reacting epitopes due to the high degree of sequence conservation [wang and zhuang, 2004; xing et al., 2011] . genotype 1 includes most of human hev strains which is endemic in asia and north africa, genotype 2 is identified in human and includes the mexican strain and few strains of african countries. genotypes 3 and 4 have been identified in pigs and other animals; however, humans can be infected with these two genotypes too. genotype 3 predominates in industrialized countries, and genotype 4 is endemic in asia, particularly china, taiwan, and japan yamashita et al., 2009] . hev causes acute hepatitis in one-third of the world's population [zhu et al., 2010] , with mortality rate of 1-15% in the general population [xing et al., 2011] . however, sporadic and epidemic outbreaks of the acute hepatitis usually lead to a self-limited disease, but the infection is potentially fatal and causes fulminant hepatitis in susceptible individuals such as pregnant women and patients with underlying liver disease [zhu et al., 2010; xing et al., 2011] , and has high mortality rate of up to 30% among pregnant women [xing et al., 2011] . therefore hev is a significant public health problem worldwide especially in endemic areas and primary prevention of the hev infection seems essential. currently, no protective vaccine against hev infection has been licensed and primary prevention remains limited [wang and zhuang, 2004] . development of an effective vaccine, which can significantly reduce the prevalence of this disease, is a desirable goal. killed and live attenuated vaccines are not available due to the lack of a permissive cell culture system for hev replication . therefore the main focus for vaccine design is on molecular approaches, especially expression of viral proteins in different expression systems jimenez de oya et al., 2012] . the hev genome ($7.2 kb) contains three partially overlapping open reading frames (orfs), including orf1, orf2 and orf3 [zhou et al., 2004; jimenez de oya et al., 2012] . orf2, which encodes capsid protein, is highly conserved and immunogenic among hev species . due to hydrophobicity and insolubility of full-length capsid protein (72 kda) [tsarev et al., 1994; zhang et al., 2001] , new generation vaccines such as multiepitope vaccines are of particular interest. therefore, the present study was undertaken to design a high density multiepitope protein compromising four htl epitopes with high-affinity binding to the hla molecules using the in silico analysis, and to evaluate the immunological properties of this protein in vitro. overall, this study represents a novel strategy for the development of hev vaccines in the future. construct from hev orf2 protein the amino acid sequences of the orf2 from standard strains of hev genotype 1 including pakistan/ sar-55 strain (accession number p33426), china strain (accession number q81871), burma strain (accession number p29326), myanmar strain (accession number q04611) and india strain (accession number q68985) were obtained from uniprot knowledgebase (www. uniprot.org). the sequences were aligned by mega software version 4.0 (biodesign institute, tempe, az) [tamura et al., 2007] , and then the conserved sequences among these strandard strains were determined. the highly conserved hev-derived hla-dr-restricted htl epitopes of these strains were predicted by syfpeithi online software (http://www.syfpeithi.de/ home.htm). hla-dr binding affinity of the predicted epitopes was determined by iedb analysis resource online program (http://tools.immuneepitope.org/analyze/html/mhc_ii_binding.html). genetic diversity of mhc molecules is very high in human population; therefore htl epitopes with high population coverage are needed for this study. four epitopes with highaffinity to the most common hla-dr alleles in iran and world population were selected. therefore it is predicted that this construct could cover the most world population. the four htl epitopes (15 amino acids) derived from hev capsid protein including hla-drb1 ã 1501 (a150-164), hla-drb1 ã 0701 (b320-334), hla-drb1 ã 1101 (c370-384), hla-drb1 ã 0401 and hla-drb3 ã 0101 (dr52) (d491-505)restricted epitopes were binded to each other as a linear multiepitope (abcd). to avoid creation of junctional epitopes, the construct was designed with amino acid spacer sequences (gly-pro-gly-pro-gly) between sequential htl epitopes [livingston et al., 2002; depla et al., 2008] . the four times repeated multiepitope was constructed as high density multiepitope (fig. 1a) . based on e. coli codon usage, the nucleotide sequence of the high density multiepitope gene was optimized using genscript rare codon analysis tool (genscript, piscataway, nj) for efficient expression. in order to simplify the purification of the target protein, a sequence encoding 8-his tag was added at the c-terminal of the optimized gene followed by adding two stop codons (taa and tag) to terminate protein synthesis. two restriction-enzyme digestion sites, ndei and xhoi, were placed in the both ends of the codon-optimized gene to subclone it into the pet30a (þ) vector. ndei digestion site (catatg) with starting codon (atg) was inserted at the n-terminal of the construct. the xhoi digestion site (ctcgag) was inserted at the c-terminal of the construct after the second termination codon. in silico digestion was performed using clone manager basic software version 9 (sci-ed software, cary, nc), and was checked by vector nti software (invitrogen, carlsbad, ca). finally, the optimized high density multiepitope sequence was synthesized and cloned into the commercial cloning vector, pbluescript ii sk (þ) by biomatik company (biomatik corporation, cambridge, canada). both pbluescript ii sk (þ) vector carrying the high density multiepitope gene (pbluescript ii sk-high density multiepitope) and the pet-30a (þ) plasmid (novagen, madison, wi) were digested by ndei and xhoi restriction enzymes (new england biolab, ipswich, ma). after ligation by t4 dna ligase (new england biolab, ipswich, ma), the recombinant plasmid pet30a-high density multiepitope was generated and transformed into e. coli dh5a competent cells (novagen) by electroporation [chassy et al., 1988] . the subcloning was confirmed by colony pcr, restriction-enzyme digestion, and dna sequencing. the recombinant plasmid was then transformed into competent e. coli bl21 (de3) cells to enhance protein expression (novagen). e. coli bl21 (de3) cells containing the recombinant plasmid were cultured overnight, then inoculated in terrific broth (himedia, mumbai, india) supplemented with kanamycin (50 mg/l) in a 1:100 volumetric ratio, and grown with shaking at 250 rpm at 37˚c until the optical density at 600 nm (od 600) reached 0.5. the bacterial culture was induced by adding various concentrations of iptg (0.1-1 mm) (sigma-aldrich, st. louis, mo), and grown with shaking for different induction times (2, 4, 6, 8, and 16 hr) at different induction temperatures (37˚c, 30˚c, and 25˚c) to determine the optimal conditions of protein expression. after the large-scale protein expression, the target protein was purified by ni-nta (qiagen, hilden, germany) under denaturing conditions using 8 m urea . the purified protein was dialyzed in pbs containing 10% glycerol with a linear urea gradient of 6, 4, and 2 m at 4˚c for 8 hr in order to decrease the amount of urea, and concentrated using amicon ultra-4 centrifugal filter unit (emd millipore, billerica, ma). sds-page and western blot were performed to confirm the presence of the target protein [laemmli, 1970; hao et al., 2013; taherkhani et al., 2014] . concentration of the protein was measured by the bradford method [bradford, 1976] . to remove bacterial endotoxin contamination in the purified protein, toxin eraser endotoxin removal kit was used according to the manufacturer procedure (genscript). the truncated orf2 protein (aa 112-608) of hev strain sar-55 ( fig. 1b) was expressed in e. coli bl21 host cells using the recombinant plasmid pet-30a-orf2 (aa 112-608) and purified by ni-nta (qiagen) as described previously . this protocol was approved by the ethical committee of ahvaz jundishapur university of medical science. a statement on ethical committee approval and the informed consent was taken from all participants. ten recovered individuals from acute hepatitis e infection (6 males and 4 females; mean age ae sd, 40.30 ae 13.76 years) with positive anti-hev igg antibody were enrolled in this study. the control group consisted of 12 healthy individuals (7 males and 5 females; mean age ae sd, 38.08 ae 15.35 years) with no history of acute hepatitis, and with negative anti-hev igm/igg antibodies. the commercial elisa kits (hev igg and hev igm elisa kits, dia.pro, milan, italy) were used for all the participants. all the participants were negative for hepatitis b surface antigen, anti-hav igm antibody and anti-hcv antibodies (hbs ag elisa kit, hav igm elisa kit, hcv ab elisa kit, dia.pro) with normal alt level. peripheral blood mononuclear cells (pbmcs) of the each individual were isolated from 10 ml heparinized venous blood by density gradient centrifugation using ficoll-hypaque (lymphoflot, biotest, dreieich, germany). the isolated pbmcs were washed and resuspended in complete rpmi 1640 medium (invitrogen). the viability of the cells was assessed by trypan blue staining. the cell proliferation assay was performed using non-radioactive mtt (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) assay [majumdar et al., 2013] . in brief, approximately 1 â 10 5 cells/well of pbmcs of each sample in rpmi 1640 and 10% fcs were added to four wells of round-bottom 96-well plates in total volume of 180 ml/well, stimulated with 20 ml/well of truncated orf2 protein (10 mg/ml), high density multiepitope (10 mg/ml) and phytohemagglutinin (pha) (5 mg/ml) (sigma-aldrich) separately, and incubated at 37˚c for 4 days. the unstimulated well of each sample was used as negative control. rpmi 1640 (20 ml) was used as blank. after 4 days, 10 ml of mtt solution (5 mg/ml) was added to each well and incubated at 37˚c for 4 hr. then, 100 ml of isopropanol containing 0.04 n hcl was added to each well and mixed thoroughly. finally, the absorbance of the wells was read at 570 nm with a reference wavelength of 630 nm by an elisa reader (tecan sunrise elisa reader; tecan trading ag, mannedorf, switzerland). the results were showed as proliferation index (pi), calculated as od570 stimulated sample/od570 unstimulated sample. the elispot assay for hev-specific ifn-g-secreting cells was carried out by human ifn-g elispot ready-set-go kit (ebioscience, san diego, ca) according to manufacturer's protocol. briefly, a 96-well pvdf bottomed elispot plate (millipore, bedford, ma) was coated with 100 ml/well of capture antibody solution and incubated overnight at 4˚c. approximately 2 â 10 5 cells/well of pbmcs of each sample suspended in rpmi 1640 and 10% fcs along with 10 mg/ml high density multiepitope protein and 10 mg/ ml truncated orf-2 protein separately were added to 2 wells in total volume of 150 ml/well. for negative control unstimulated pbmcs was used. for positive control, 5 mg/ml of pha (sigma-aldrich) was used. after 24-hr incubation at 37˚c, cells and medium were decanted from the plate and the plate was washed three times with elispot wash buffer. 100 ml of biotinylated detection antibody was added to each well and incubated overnight at 4˚c. after incubation, the plate was emptied and washed as described above. 100 ml/well of avidin-horseradish peroxidase reagent was added, and the plates were incubated at room temperature for 45 min. after incubation, the plates were washed three times with elispot wash buffer and two times with 1x pbs; then, 100 ml/well of freshly prepared aec (3-amino-9ethyl carbazole) substrate solution (sigma-aldrich) was added to the wells and incubated at room temperature until dark purple spots appears. the reaction was stopped by washing wells three times with 200 ml/well distilled water. plate was air-dried and the number of ifn-g-producing cells in each well was counted by a dissection stereoscope (nikon, tokyo, japan) and expressed as spot-forming cells (sfcs) per 10 5 cells. at a concentration of 1 â 10 5 cells/well, pbmcs of each sample in rpmi 1640 and 10% fcs were added to the four wells of 96-well plates, and stimulated with truncated orf2 protein (10 mg/ml), high density multiepitope protein (10 mg/ml), and pha (5 mg/ml) separately at 37˚c. the unstimulated pbmcs of each sample were used to evaluate spontaneous production of cytokines. culture supernatants were collected after 48 hr (24 hr for il-12). levels of four cytokines (il-12 p70, ifn-g, il-4 and il-10) were measured in duplicate by commercial elisa kits (ebioscience) according to the manufacturer's instructions. the elisa results were expressed as picograms per milliliter (pg/ml). statistical analysis was performed using spss 17 software (spss, chicago, il) and p values of less than 0.05 were considered statistically significant. discrete variables were compared using the pearson's chi-square test or fisher's exact test. intergroup comparisons (recovereds vs. controls) were performed using independent t test or mann-whitney u test. intragroup comparisons (high density multiepitope versus orf2) were made by paired t test or wilcoxon test. all data were presented as mean ae se. the high expression of the protein was induced by iptg at a final concentration of 1 mm at 30˚c for 4 hr. sds-page and western blotting analysis of the resultant protein showed a protein band of approximately 33 kda corresponding to high density multiepitope (fig. 2) . approximately 1 mg of purified protein was obtained from each liter of culture media. the cell proliferative responses in hev-recovered group following stimulation with high density multiepitope protein and truncated orf2 protein were found higher than control group (p < 0.001); and these responses were observed higher with high density multiepitope protein than truncated orf2 protein in hev-recovered group (p ¼ 0.013) ( table i, fig. 3 ). ifn-g elispot responses to high density multiepitope protein and truncated orf2 protein were found significantly higher in hev-recovered individuals than control group (p < 0.001). these responses to high density multiepitope protein were observed slightly higher than truncated orf2 protein in hevrecovered group, with no significant observation (p ¼ 0.220). table i and figure 4 show the frequency of ifn-g-secreting cells following stimulation with high density multiepitope protein and truncated fig. 2 . a: sds-page analysis of the expressed and purified high density multiepitope protein in e. coli. the high density multiepitope protein band is visualized at approximately 33 kda. lane 1, uninduced control; lane 2, induction with 1 mm iptg for 4 hr; lane 3, induction with 1 mm iptg for 8 hr; lane 4,5, flow-through; lane 6-8, washing steps of the ni column; lane 9-11, the first, the second, and the third purified eluates of high density multiepitope protein from the ni column; lane 12, prestained protein ladder (cinagen, tehran, iran). b: western blot analysis of the expressed and purified high density multiepitope protein in e. coli. the high density multiepitope protein band is visualized at approximately 33 kda. lane 1, prestained protein ladder (cinagen); lane 2, flow-through; lane 3, wash; lane 4-7, the first, the second, the third, and the fourth eluates. orf2 protein in hev-recovered individuals and control group. the levels of ifn-g and il-12 p70 responses to truncated orf2 protein and high density multiepitope in the recovered group were found significantly higher compared to control group (p < 0.001). however, the levels of these stimulated cytokines with high density multiepitope protein were found higher than truncated orf2 protein with no significant observation. the levels of specific il-10 for high density multiepitope protein and truncared orf2 protein were found moderate among the hev-recovered group, while the levels of specific il-4 for the both proteins were low (table i, fig. 5 ). a better understanding of the hev-specific cellular immune responses may help us in designing and development of vaccine for the prevention and control of the hev infection. although some progress has been achieved in developing hev vaccine, but due to some limitations in current approach for vaccine preparation such as the duration of the response and cost effectiveness of vaccine, the attempt was made to develop an alternative hev vaccine [jimenez de oya et al., 2012] . the virus-specific t-helper cell immune responses have been shown to be essential for controlling and clearing viral infection by activating cytotoxic t cells and producing cytokines especially inf-g which can suppress viral replication. thus, viral proteins comprising helper t-lymphocyte (htl) epitopes may be useful as protective vaccines [aggarwal et al., 2007] . it has been shown that hev capsid protein has several immunogenic epitopes [gu et al., 2004] . shata et al. identified dominant and subdominant epitopes of hev capsid protein such as amino acids 271-286, 199-214, 463-478, 583-598, and 631-646 [shata et al., 2007] . aggarwal et al. mapped cd4 t-cell epitopes in hev orf2 protein, and found the immunodominant t-cell epitopes of capside protein are encompassed amino acids 73-156, 289-444, and 505-588 [aggarwal et al., 2007] . khudyakov et al. reported the antigenic regions in hev orf2 protein are encompassed amino acids 319-340, 631-648, and 641-660 [khudyakov et al., 1993] . selection of the epitopes is the most important part of the design of the epitope-based vaccines that can guarantee the effectiveness and extent of the desired immune responses. this study focused on the consereved epitopes with high-affinity binding to the different hla molecules to cover the majority of the human population, on this basis the multiepitope protein comprising these epitopes was constructed. this multiepitope design is a new strategy and may counter the limitations present in current vaccine against hev, but it requires further investigation. to date, no application of multiple hla-dr-restricted epitopes was reported to induse htl responses against hev infection. oany et al. designed a multiepitope vaccine candidate compromising b cell and t cell epitopes from spike protein of human coronavirus by in silico analysis. in their study, sequences of the spike proteins were collected from the uniprotkb database and the consereved epitopes with high-affinity binding to the different hla molecules were predicted using in silico tools to cover the majority of the world population. this computational design was a novel study to determine antigenic epitopes in spike protein and to design an epitope-based vaccine against human coronavirus [oany et al., 2014] . these type of vaccines were reported to have many advantages including the stability, lack of carcinogenic, large scale production with the least equipments, no need for cold chain storage, the ability to stimulate the immune system against a wide variety of strains and genotypes of a microorganism, and use in different races or high population coverage [elliott et al., 1999; livingston et al., 2002; sette and fikes, 2003; jackson et al., 2006] . meanwhile, some disadvantages like the creation of junctional epitopes in the sequential arrangement of the epitopes have limited using these constructs [livingston et al., 2002; depla et al., 2008] . junctional epitopes is created by the juxtaposition of two epitopes, and can suppress the immune responses to the main epitopes [livingston et al., 2002] . this problem was overcome through application of the spacer residues between the epitopes to improve appropriate epitope processing. furthermore, it has been reported that if the epitope is expressed with high density will enhance protective immunity [liu et al., 2004; lu et al., 2008] . lu et al. designed constructs consisting of various v3 epitope densities from hiv-1 gp120, and found that high epitope density in one single protein molecule could enhance protective immunity [lu et al., 2008] . liu et al. reported that high epitope density from m2 protein of influenza virus enhanced specific humoral immunity against flu virus [liu et al., 2004] . vaccine development is dependent not only on selecting antigens and epitopes but also on defining the immune responses against target epitopes. by evaluating the antigenicity and immunogenicity of the high density multiepitope protein, it was indicated that the high density multiepitope protein stimulates slightly stronger t helper cell responses compared to truncated orf2 protein. it has been shown that during acute infection, virus-specific t helper cell responses can clear viral infection but lack of its activity leads to chronic infection [macdonald et al., 2002] . in this study, the hla typing was not carried out and the frequencies of various hla alleles among the participants were not determined. so it is not clear which type of hla alleles among the hev-recovered individuals had a low or highaffinity hla binding epitopes against the multiepitope protein. li et al. designed a multiepitope construct comprising six low-affinity hiv gag and pol ctl epitopes restricted to hla-a ã 0201 and the hiv p24 antigen by bioinformatics analysis. they used cryptic epitope modification to enhance the immunogenicity of lowaffinity hla-a2.1-binding epitpes, and found the multiepitope can induce a strong cd8þ t cell immune responses in mice [li et al., 2013b] . jafarpour et al. designed and synthesized a multiepitope construct encoding conserved and immunogenic epitopes from hiv-1 tat, pol, gag, and env proteins by bioinformatics analyses as a new dna vaccine candidate. the designed multiepitope plasmid was able to induce proper immune responces in balb/c mice [jafarpour et al., 2014] . li et al. designed a multiepitope construct encoding t and b lymphocyte epitopes from latent membrane protein 2 (lmp2) of epstein-barr virus (ebv) based on mammalian codon usage. the recombinant plasmid pcdna3.1þ/ ebv-lmp2 multiepitope was constructed and transfected into cos-7 cells. this multiepitope plasmid dna was able to stimulate strong cellular and humoral immunity in mice [li et al., 2013a] . in the present study, high level of specific ifn-g and il-12 responses (t helper type 1 cytokines) and low level of il-10 and very low level of il-4 responses (t helper type 2 cytokines) against high density multiepitope protein and truncated orf2 protein were observed among the hev-recovered individuals. thus, both proteins can be considered as promising vaccine candidate against hev infection. hashish et al. designed a multiepitope fusion protein compromising two b cell and t cell epitopes from the e2 glycoprotein of bovine viral diarrhea virus (bvdv), and also k99 major subunit fanc and sta toxoid of enterotoxigenic escherichia coli (etec). in their study, fanc-sta-e2 gene construction was cloned into the expression vector pet28 and transformed into e. coli bl21 cells. fanc-sta-e2 fusion protein was well expressed by iptg-induction (1 mm) at 37˚c for 4 hr and purified by ni-nta agarose using his-tag. this multiepitope fusion protein could develop neutralizing antibodies against etec and bvdv in vitro [hashish et al., 2013] . beside vaccine development, multiepitope technology can be used for diagnosis. several studies have been performed on multiepitopes proteins for development of diagnostic kit. the results of these studies showed that the use of multiepitope proteins can increase the sensitivity and specificity of the tests, since this kind of proteins has great efficiency to expose conserved and immunodominant epitopes [dipti et al., 2006; de souza et al., 2013] . de souza et al. designed a high density multiepitope protein containing three copies of four conserved epitopes from hepatitis b core protein (hbcag), and developed an elisa using this recombinant protein. the developed elisa was useful for hepatitis b diagnosis [de souza et al., 2013] . dipti et al. designed a novel recombinant multiepitope protein comprising six conserved and immunodominant epitopes from hepatitis c virus, and developed an anti-hcv diagnostic kit using this protein. the multiepitope protein was able to detect anti-hcv antibodies in human plasma with high degree of sensitivity and specificity [dipti et al., 2006] . although several microorganisms and cell lines are currently used for expression of foreign genes, but still e. coli are routinely used for expression of target proteins [vincentelli and romier, 2013] . fast cultivation, well-known genetics and high-level expression capabilities with high purity are some other advantages of e. coli expression system [fakruddin et al., 2013] . despite all these advantages, expression of a foreign gene in e. coli may be hampered by the presence of rare codons and high gc contents within the gene-coding region which result in decrease of the expression level or production of insoluble and nonfunctional proteins [mondal et al., 2013; elena et al., 2014] . in the present study, e. coli bl21 cell was used for expression of the high density multiepitope protein. the target genes were cloned in to the pet30aþ plasmid and expressed by iptg-induction under control of strong bacteriophage t7 promoter and t7 rna polymerase of the e. coli bl21 cell. the iptg-induction is economically desirable method and increases protein production [mondal et al., 2013] . previous studies have reported high yield protein purification by ni2þ chelate affinity chromatography using his-tag [glynou et al., 2003; farshadpour et al., 2014] . the application of affinity tags is simple, rapid, cost effective and also efficient for purification of proteins, and also has no effect on the structure, folding and function of the protein [glynou et al., 2003; carson et al., 2007] . in the present study, histag was used for the purification. the results of this study show that e. coli is a good system for expression of recombinant proteins with high yield. in conclusion, a high density multiepitope protein capable of induction of t helper type 1 responses was successfully produced and evaluated in vitro. the results of this study provide much information for the design of hev multiepitope vaccine. however, further works will be needed to evaluate protective immune response of the high density multiepitope protein in vivo. t-cell epitope mapping of orf2 and orf3 proteins of human hepatitis e virus a rapid and sensitive method for the quantitation of microgram quantities of 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of a recombinant hepatitis e vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled key: cord-353877-wzndpcq3 authors: albagi, sahar obi abd; al-nour, mosab yahya; elhag, mustafa; abdelihalim, asaad tageldein idris; haroun, esraa musa; essa, mohammed elmujtba adam; abubaker, mustafa; hassan, mohammed a. title: a multiple peptides vaccine against ncovid-19 designed from the nucleocapsid phosphoprotein (n) and spike glycoprotein (s) via the immunoinformatics approach date: 2020-05-20 journal: biorxiv doi: 10.1101/2020.05.20.106351 sha: doc_id: 353877 cord_uid: wzndpcq3 due to the current covid-19 pandemic, the rapid discovery of a safe and effective vaccine is an essential issue, consequently, this study aims to predict potential covid-19 peptide-based vaccine utilizing the nucleocapsid phosphoprotein (n) and spike glycoprotein (s) via the immunoinformatics approach. to achieve this goal, several immune epitope database (iedb) tools, molecular docking, and safety prediction servers were used. according to the results, the spike peptide peptides sqcvnlttrtqlppaytnsftrgvy is predicted to have the highest binding affinity to the b-cells. the spike peptide ftisvttei has the highest binding affinity to the mhc i hla-b1503 allele. the nucleocapsid peptides ktfpptepk and rwyfyylgtgpeagl have the highest binding affinity to the mhc i hla-a0202 allele and the three mhc ii alleles hla-dpa1*01:03/dpb1*02:01, hla-dqa1*01:02/dqb1*06:02, hla-drb1, respectively. furthermore, those peptides were predicted as non-toxic and non-allergen. therefore, the combination of those peptides is predicted to stimulate better immunological responses with respectable safety. human coronaviruses (hcovs) including the severe acute respiratory syndrome coronavirus 2 (sars-cov-2, covid-19) are enveloped, positive-sense, single-stranded polyadenylated rna viruses belong to the coronaviridae family. they cause systemic and respiratory zoonotic diseases [1] . the sars-cov-2 is a novel strain detected firstly in the city of wuhan, the republic of china in december 2019 [2] . it causes fever, cough, dyspnea, bilateral infiltrates on chest imaging and may progress to pneumonia [3] . the covid-19 is characterized by rapid spreading; "as the 27 feb, it is reported in 47 countries, causing over 82,294 infections with 2,804 deaths" [4] and till the 5th may, more than 3. 5 million positive cases and 0.25 million deaths have been identified globally [5] . unfortunately, until now covid-19 has no effective antiviral drug for the treatment or vaccine for the prevention, hence extensive researches should be conducted on the development of safe and effective vaccines and antiviral drugs [4] . to develop a safe and effective covid-19 vaccine rapidly, the who recommended that "we must test all candidate vaccines until they fail to ensure that all of them have the chance of being tested at the initial stage of development". ensuing this point, recently, there are over 120 proposed vaccines. six of them in the clinical evaluation and 70 in pre-clinical evaluation [6] . the vaccine development is achieved by multiple approaches including the inactivated, live-attenuated, non-replicating viral vector, dna, rna, recombinant proteins, and peptide-based vaccines. "as of 8 april 2020, the global covid-19 vaccine r&d landscape confirmed 78 active candidate vaccine" [7] . consistent with global efforts, this study aims to predict potential covid-19 peptide-based vaccine utilizing the nucleocapsid phosphoprotein (n) and spike glycoprotein (s) via the immunoinformatics approach. due to the respectable antigenicity of the nucleocapsid and spike glycoprotein, they are appropriate targets for vaccine design [8] . the peptide vaccines are sufficient to stimulate cellular and humoral immunity without allergic responses [9] . they are" safe, simply produced, stable, reproducible, cost-effective" [10] , and permits a broad spectrum of immunity [9] , consequently, they are the targets for this study as well as they utilized in multiple studies concerning covid-19 vaccine [11] [12] [13] [14] . a total of 100 covid-19 nucleocapsid phosphoprotein (n) and spike glycoprotein (s) were retrieved from the national center for biotechnology information (ncbi) database [15] as fasta format in march 2020. the sequences with their accession number are listed in the supplementary file s1. the protein sequences of mhc i alleles hla-a*02:01, hla-b15:03, hla-c*12:03, and mhc ii alleles hla-dpa1*01:03/dpb1*02:01, hla-dqa1*01:02/dqb1*06:02, hla-drb1 were obtained from the immuno polymorphism database (ipd-imgt/hla) [16] . the retrieved covid-19 nucleocapsid phosphoprotein (n) and spike glycoprotein (s) sequences were aligned using the clustalw algorithm [17] on the bioedit software version 7.2.5 [18] to identify the conserved regions between sequences. the b-cells peptides were predicted from the conserved regions using the linear epitope prediction tool "bepipred-test" on the immune epitope database (iedb) [19] at the default threshold value -0.500. to predict the epitopes accurately, a combination between the hidden (parker and levitt) method and the markov model (hmm) [20] was used. the surface accessibility of b-cells peptides was predicted via the emini surface accessibility tool [21] on the iedb [19] at the default threshold holding value. to identify the antigenic sites within the nucleocapsid phosphoprotein and spike glycoprotein, the kolaskar and tongaonker method's on the iedb [19] at the default threshold value. to predict the interaction with different mhc i alleles, the major histocompatibility complex class i (mhc i) binding prediction tool on the iedb [19] was used. all peptide length was set as 9amino acid. to predict the binding affinity, the artificial neural network (ann) prediction method was selected with a half-maximal inhibitory concentration (ic50) value of less than 100. in contrast, to predict the interaction with different mhc ii alleles, the major histocompatibility complex class ii (mhc ii) binding prediction was used. to predict the binding affinity, the nn align algorithm was selected with an ic50 value of less than 500. the human allele reference sets (hla dr, dp, and dq) were included in the prediction. to predict the percentage of peptides binding with various mhc i and mhc ii alleles that cover the world population, the population coverage tool on the iedb [19] was used. to predict the peptides' allergicity, the allergenfp v.1.0 [22] and allercatpro v. 1.7 [23] servers were used. in contrast, to predict the peptides' toxicity, the toxinpred server [24] was used. to model the 3d structure of the nucleocapsid, spike, and mhc molecules, the swiss-model server [25] , and the phyre2 web portal [26] were used. to visualize the modeled structures, the uscf chimera 1.8 software [27] was used. the predicted peptides were docked with mhc i alleles hla-a*02:01, hla-b15:03, hla-c*12:03, and mhc ii alleles hla-dpa1*01:03/dpb1*02:01, hla-dqa1*01:02/dqb1*06:02, hla-drb1. the modeled mhc structures were prepared for the docking via cresset flare software [28] at the normal type calculation method. to dock the predicted peptides, the mdockpep [29] and hpepdock [30] [31] [32] [33] [34] servers were used. the predicted peptides were submitted as amino acid sequences. the 2d and 3d interactions were visualized using the poseview [35] at the proteinplus web portal [36] and cresset flare viewer [28] , respectively. according to the iedb [19] prediction, the average binding score for the nucleocapsid phosphoprotein and spike glycoprotein were 0.558, 0.470, respectively. all values equal to or greater than the default threshold were predicted as potential b-cell binders. regarding the cytotoxic t-lymphocyte peptides, the mhc i binding prediction tool predicts 46 peptides from the nucleocapsid and 192 peptides from the spike glycoprotein could interact with the different mhc i alleles. the most promising peptides were listed in table 3 . in contrast, the mhc ii binding prediction tool predicts 774 peptides from the nucleocapsid and 1111 peptides from the spike glycoprotein could interact with the different mhc ii alleles. the most promising peptides were listed in table 4 according to the mdockpep [29] and hpepdock [30] servers prediction, the spike peptide (ftisvttei) has the highest binding affinity to the mhc i hla-b1503 allele and the spike peptide (miaqytsal) has the highest binding affinity to the mhc i hla-c1203 allele. the nucleocapsid peptide (ktfpptepk) has the highest binding affinity to the mhc i hla-a0202 allele ( table 6) according to the allergenfp v.1.0 [12] , allercatpro v. 1.7 [13] , and toxinpred servers [14] , all the predicted peptides except the spike peptide (evfnatrfasvyawn) were nonallergen and non-toxin (tables 8and 9) . the hydrophobicity scores of the spike glycoprotein peptides were not available. due to the current covid-19 pandemic, the rapid discovery of a safe and effective vaccine is an essential issue [37] . since the successful vaccine relies on the selection of the most antigenic parts and the best approaches [38] , covid-19 nucleocapsid phosphoprotein (n) and spike glycoprotein (s) were selected to design a peptide vaccine. the antigenicity of the nucleocapsid and spike is well predicted [8] , and the advantages of the peptide vaccines are well established [9, 10] . the peptide design via the immunoinformatics approach is achieved through multiple steps including the prediction of; b-cells and t-cell peptides, the surface accessibility, antigenic sites, and the population coverage. after the selection of the candidate peptides, their interaction with the mhc molecules is simulated and their safety is predicted [39] . regarding the b-cells peptides prediction, the successful candidates must pass the threshold scores in the bepipred, parker hydrophilicity, kolaskar and tongaonkar antigenicity, as well as emini surface accessibility tests [40] . the iedb bepipred test [19] on the nucleocapsid showed that eleven peptides were predicted, however, the peptide dayktfpptepkkdk-kkkadetqalpqrqkkqqtvtllpaadldd was the only one that passed all the tests. in contrast, the iedb bepipred test [18] on the spike showed that forty-two peptides were predicted, but the peptides sqcvnlttrtqlppaytnsftrgvy and lgky the only two that passed all the tests. as the length of effective b-cell peptides varies from 5-30 amino acids [41] , the peptide lgky is too short and the peptide dayktfpptepkkdk-kkkadetqalpqrqkkqqtvtllpaadldd is too long. consequently, the spike peptide peptides sqcvnlttrtqlppaytnsftrgvy is predicted to have the highest binding affinity to the b-cells (table 2) . concerning the t-cells peptides prediction, the test measures the peptides' binding affinity to the mhc molecules [19] . the available mhc i alleles hla a, hla b, hla c, hla e, and mhc ii alleles hla-dr, hla-dq, and hla-dp were used. the mhc i iedb tests [19] the results of collective the iedb tests [19] revealed that the spike glycoprotein peptides ftisvttei, miaqytsal, and the nucleocapsid peptide ktfpptepk are the most promised mhc1 peptides. on the other hand, the spike peptides evfnatrfasvyawn, vfrssvlhstqdlfl, and the nucleocapsid peptides aalalllldrlnqle, alall-lldrlnqles, prwyfyylgtgpeag, rwyfyylgtgpeagl are the most promised mhc ii peptides. to stimulate better immunological responses by the predicted peptides, they must interact and bind effectively with the mhc1 and mhc ii molecules [42] , therefore we must study their interaction with the mhc molecules. the simulation and prediction of the interaction between the predicted peptides and the mhc molecules are conducted using molecular docking studies that rely on the calculation of the binding free energy. the lowest binding energy scores of the mhc-peptide complex will indicate the best interaction and the highest stability [43] . to validate the results of molecular docking, mdockpep [29] and hpepdock [30] [31] [32] [33] [34] servers were used. the mdockpep server predicts the mhc-peptides interaction by "docking the peptides onto the whole surface of protein independently and flexibly using a novel the conformation restriction in its novel iterative approach. it ranks the docked peptides via the itscorepep scoring function that uses the known protein-peptide complex structures in the calculations" [29] . in contrast, hpepdock uses "a hierarchical flexible peptide docking approach" to predict the mhc-peptides interaction [30] . the mhc i, hla-a0202, hla-b1503, hla-c1203 was predicted to present the highly conserved sars-cov-2 peptides more effectively [44] , hence, they were used in molecular docking study. the molecular docking results showed that the spike peptide ftisvttei has the lowest docking energy score with the mhc i hla-b1503 allele, hence it is predicted to have the highest binding affinity. the spike peptide miaqytsal showed the lowest docking energy score with the mhc i hla-c1203, consequently, it is predicted to have the highest binding affinity to the mhc i hla-c1203 allele. in contrast, regarding the mhc i hla-a0202; the results of the mdockpep [29] server showed that the nucleocapsid peptide ktfpptepk has the lowest docking energy score, but the results of hpepdock [30] server showed the spike peptide miaqytsal has the lowest docking energy score ( table 6 ). to illustrate the mhc-peptide interaction, the poseview [35] at the proteinplus web portal [36] that illustrate the 2d interactions and cresset flare viewer [8] that illustrate the 3d interaction were used. the spike peptide ftisvttei interacts with the mhc i hla-b1503 allele by forming hydrogen bonds with the amino acids thr2, ser4, ser101a, asn104a, tyr123a, thr167a, lys170a, trp171a, glu176a and hydrophobic bonds with the amino acids thr2, thr6, trp98a, ser101a, trp171a, ala174a, leu180a. the spike peptide miaqytsal interacts with the mhc i hla-c1203 allele by forming hydrogen bonds with the amino acids tyr33a, arg86a, lys90a, gln179a, thr187a and hydrophobic bonds with the amino acids ile2, gln4, gln94a, ala8, arg86a, tyr183a, trp191a. in comparison, it interacts with the mhc i hla-a0202 allele by forming hydrogen bonds with the amino acids thr6, glu87a, arg121a, trp180a, and hydrophobic bonds with the amino acids ile2, trp171a, tyr183a, trp191a. it forms six hydrogen bonds and seven hydrophobic bonds with the mhc i hla-c1203 allele that is more than its bonds with the mhc i hla-a0202 allele. this finding indicates the higher binding affinity of spike peptide miaqytsal to the mhc i hla-c1203 allele and supports the mdockpep [3] server score (table 6 and figures 5, 6 ). the nucleocapsid peptide ktfpptepk interacts with the mhc i hla-a0202 allele by forming hydrogen bonds with the amino acids pro4, glu82a, lys90a, asp101a, trp171a and hydrophobic bonds with the amino acids pro4, pro8, leu105a, thr167a, trp171a, ala174a, val176a (figures 5 and 6 ). among the reported mhc i alleles, the hla-b1503 allele was predicted to have "the greatest ability to present the highly conserved sars-cov-2 peptides" [44] , therefore, the spike peptide ftisvttei is predicted to make the highest response, since the binding with the mhc i stimulates the natural killer and the cytotoxic t cells [45] . regarding the interaction with the mhc ii molecule, the spike peptide evfnatrfasvyawn showed the lowest docking energy score with the three mhc ii alleles hla-dpa1*01:03/dpb1*02:01, hla-dqa1*01:02/dqb1*06:02, and hla-drb1, hence it is predicted to have the highest binding affinity to the three alleles. hence, it predicted to stimulate the cd4+ (helper) t cells more effectively, since the mhc ii molecule presents the antigenic peptides to the cd4+ (helper) t cells [46] . on the contrary, the nucleocapsid peptide (rwyfyylgtgpeagl) showed lower docking energy scores with the mhc ii allele hla-dqa1*01:02/dqb1*06:02. the results of the mdockpep [29] server showed that the peptide (prwyfyylgtgpeag) has the lowest docking energy score, however, the results of hpepdock [30] server differed from it in the mhc ii alleles hla-dpa1*01:03/dpb1*02:01 and hla-drb1 (table 7) . spike peptide (evfnatrfasvyawn) interacts with hla-dpa1*01:03/dpb1*02:01 allele by forming hydrogen bonds with the amino acids val2, ser10, tyr12, tyr40a, glu59a, hydrophobic bonds with the amino acids val2, phe8, tyr40a, ala41a, ala42a, asn93a, leu101a, leu104a, trp14, tyr174a, and pi-pi bonds with the aromatic amino acid phe8, ser10, tyr12, trp14, tyr174a. it interacts with the hla-drb1 allele by forming hydrogen bonds with the amino acids val2, thr6, ser10, phe46b, tyr107b, tyr152b, gly154b, hydrophobic bonds with the amino acids val2, asn4, thr6, phe8, tyr12, trp14, glu43b, cys44b, phe46b, asn111b, tyr112b, pro153b, trp182b, and pi-pi bonds with the aromatic amino acid phe8, tyr12, trp14. its interaction with the hla-dqa1*01:02/dqb1*06:02 allele was not obtained from the server. the nucleocapsid peptide (rwyfyylgtgpeagl) interacts with hla-dpa1*01:03/dpb1*02:01 allele by forming hydrogen bonds with the amino acids glu12, leu101a, hydrophobic bonds with the amino acids trp2, phe4, tyr6, gly10, glu12, gln45a, leu97a, leu101a, leu104a, phe144a, pro146a, pro170a, tyr174a, and pi-pi bonds with the aromatic amino acid trp2, phe4, tyr6, phe144a, tyr174a. it interacts with the hla-dqa1*01:02/dqb1*06:02 allele by forming hydrogen bonds with the amino acids glu12, asn8a, asn95a, leu96a, thr109a, asn110a, hydrophobic bonds with the amino acids trp2, phe4, tyr6, gly10, glu12, gly14, gln40a, phe41a, ala92a, tyr103a, thr106a, ala107a, ala108a, and pi-pi bonds with the aromatic amino acid trp2, phe4, tyr6, tyr103a. it interacts with the hla-drb1 allele by forming hydrogen bonds with the amino acids glu12, val40b, cys44b, tyr59b, tyr107b, asn111b, gln178b, hydrophobic bonds with the amino acids trp2, phe4, tyr6, gly10, glu12, lys41b, his42b, tyr107b, asn111b, tyr112b, trp182b, and pi-pi bonds with the aromatic amino acid trp2, phe4, tyr6. the nucleocapsid peptide (rwyfyylgtgpeagl) interacts most effectively with the hla-dqa1*01:02/dqb1*06:02 allele, hence it showed the highest binding affinity to it (figures 7 and 8) . concerning the two nucleocapsid peptides (rwyfyylgtgpeagl) and (prwyfyylgtgpeag) in the interaction with the mhc ii alleles hla-dpa1*01:03/dpb1*02:01 and hla-drb1, the 2d and 3d interaction results showed that the peptide (prwyfyylgtgpeag) more effectively with the hla-dpa1*01:03/dpb1*02:01 allele and the peptide (rwyfyylgtgpeagl) more effectively with the hla-drb1 allele, however, they differ only in the first and last amino acid (figures 9 and 10 ). in comparison between the spike and nucleocapsid peptides, the spike peptide (ftisvttei) showed a higher binding affinity to the mhc i hla-b1503 allele. the nucleocapsid peptides (ktfpptepk) and (rwyfyylgtgpeagl) showed a higher binding affinity to the mhc i hla-a0202 allele and the three mhc ii alleles hla-dpa1*01:03/dpb1*02:01, hla-dqa1*01:02/dqb1*06:02, hla-drb1, respectively, however, the total population coverage of the peptides ftisvttei and ktfpptepk is not high (table 10) . joshi a, et al. in their predictive covid-19 peptide-based vaccine study found that the orf-7a protein's peptide (itlcftlkr) binds most effectively with the mhc i hla alleles hla-a*11:01, hla-a*68:01 [13] . enayatkhani m, et al. in their predictive study included nucleocapsid n, but they studied its interaction with the mhc i hla-a*11:01 allele [11] . kalita p, et al. included the nucleocapsid protein and spike glycoprotein. they used the predicted peptides from nucleocapsid, spike, and membrane glycoprotein to design a subunit vaccine [12] . singh a, et al. used the nucleocapsid protein to design multi-peptides vaccine [14] . since we didn't include the two alleles hla-a*11:01, hla-a*68:01 in our study and didn't design subunit or multi-peptides vaccine, the logical comparison will not be applied. besides the binding with the mhc molecules, the predicted peptide must be non-toxic and non-allergen, hence, their safety was predicted using the allergenfp v.1.0 [22] , allercatpro [23] v. 1.7, toxinpred [24] servers. the result showed that all the peptides were non-toxic. the allercatpro v. 1.7 [23] server results showed there is no evidence about the allergicity of all peptides, however, the allergenfp v.1.0 [22] server predicts spike peptide (evfnatrfasvyawn) as an allergen (tables 8 and 9 ). a potential covid-19 peptide-based vaccine was predicted from the nucleocapsid phosphoprotein (n) and spike glycoprotein (s) via the immunoinformatics approach. the spike peptide peptides sqcvnlttrtqlppaytnsftrgvy is predicted to have the highest binding affinity to the b-cells. the spike peptide ftisvttei has the highest binding affinity to the mhc i hla-b1503 allele. the nucleocapsid peptides ktfpptepk and rwyfyylgtgpeagl have the highest binding affinity to the mhc i hla-a0202 allele and the three mhc ii alleles hla-dpa1*01:03/dpb1*02:01, hla-dqa1*01:02/dqb1-*06:02, hla-drb1, respectively. furthermore, those peptides were predicted as non-toxic and non-allergen. therefore, the combination of those peptides is predicted to stimulate better immunological responses. since the study is an in silico predictive work, further experimental studies are recommended to validate the obtained results. a comparative sequence analysis to revise the current taxonomy of the family coronaviridae novel coronavirus ( 2019-ncov) : situation report, 3 coronavirus disease 2019 (covid-19): epidemiology, virology, clinical features, diagnosis, and prevention sars-cov-2: an emerging coronavirus that causes a global threat covid-19 situation update worldwide who solidarity trial -accelerating a safe and effective covid-19 vaccine the covid-19 vaccine development landscape genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan peptide vaccine: progress and challenges peptide-based vaccine successfully induces protective immunity against canine visceral leishmaniasis reverse vaccinology approach to design a novel multi-epitope vaccine candidate against covid-19: an in silico study design of a peptide-based subunit vaccine against novel coronavirus sars-cov-2 epitope based vaccine prediction for sars-cov-2 by deploying immuno-informatics approach designing a multi-epitope peptide-based vaccine against sars-cov-2 database resources of the national center for biotechnology information ipd-imgt/hla database clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties, and weight matrix choice bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt the immune epitope database (iedb) 3.0 improved method for predicting linear bcell epitopes induction of hepatitis a virusneutralizing antibody by a virus-specific synthetic peptide allergenfp: allergenicity prediction y descriptor fingerprints allercatpro-prediction of protein allergenicity potential from the protein sequence in silico approach for predicting toxicity of peptides and proteins swiss-model: an automated protein homology-modeling server the phyre2 web portal for protein modeling, prediction, and analysis ucsf chimera--a visualization system for exploratory research and analysis a. molecular field extrema as descriptors of biological activity: definition and validation mdockpep: an ab-initio protein-peptide docking server hpepdock: a web server for blind peptide-protein docking based on a hierarchical algorithm hhblits: lightning-fast iterative protein sequence searching by hmm-hmm alignment improved tools for biological sequence comparison comparative protein structure modeling of genes and genomes the protein data bank poseview--molecular interaction patterns at galance proteinsplus: a web portal for structure analysis of macromolecules developing covid-19 vaccines at pandemic speed vaccines: from empirical development to rational design immunoinformatics approach for epitope-based peptide vaccine design and active site prediction against polyprotein of emerging oropouche virus antibody epitope prediction -tutorial present yourself! by mhc class i and mhc class ii molecules relative binding free energy calculations in drug discovery: recent advances and practical considerations human leukocyte antigen susceptibility map for sars-cov-2," medrxiv major histocompatibility complex (mhc) class i and mhc class ii proteins: conformational plasticity in antigen presentation function and regulation of mhc class ii molecules in t-lymphocytes: of mice and men the authors declare that have no conflict of interest. key: cord-010088-s9tfvtao authors: nan title: oral abstracts date: 2013-11-01 journal: vox sang doi: 10.1111/vox.12100_1 sha: doc_id: 10088 cord_uid: s9tfvtao nan tadokoro k and satake m japanese red cross society, tokyo, japan it is estimated that there are 2 billion hbv-infected people including 350 million hbv-carriers in the world. it is highly endemic in south africa, amazon, and southeast-central asia. the genotype of hbv is geographically characteristic, e.g. genotype b and c are the major in east asia. hbv transmission remains the most frequent transfusion-transmitted viral infection despite the implementation of various screening tests applied in different settings. the residual risk is mainly related to donations either in the pre-sero (or pre-dna)-conversion window period or occult hbv infection (obi) where blood test is hbv-dna-positive and hbs-ag-negative. infectivity of hbv depends on the transfused blood (viral load, phase of infection, genotype, and anti-hbs in the concurrent blood) and immune status of the recipients (anti-hbs, immunocompetence). it was shown that infectivity is dependent on viral load. allain et al reported that ffps is more infectious than pcs or rbcs. the minimal infectious dose of blood in late acute infection phase in chimpanzee and chimeric mice is approximately 10 times higher than that of pre-acute phase. satake et al reported that transmission rate of obi-derived components with low titer anti-hbc was 1/33(3%), whereas that of anti-hbc-negative components was 11/22(50%), which was verified in the lookback programme conducted in japan. allain et al showed in the study conducted in europe that adjusted transmission rate of obi blood was 28%, and the rate was higher without anti-hbs(63.8%) and lower with anti-hbs(15.4%). discrepancy of transmission rate of obi-derived blood between above two reports might be related to the different cutoff levels of anti-hbc and presence or absence of anti-hbs. dna-positivity rate among obi-derived components is higher in those with the higher levels of anti-hbc and lower in those with the presence of anti-hbs. there has been no report of transmission by obi-derived blood with anti-hbs of 200miu/ml or more. screening test for hbv is different between countries. low endemic countries screen blood for hbs-ag, anti-hbc and mini-pooled nat, while highly endemic countries test for hbs-ag without anti-hbc, because high prevalence of anti-hbc-positive donation hamper securing necessary blood. japan as a moderately endemic country had tested for hbs-ag, mini-pool nat and anti-hbc/anti-hbs where anti-hbs of more than 200 miu/ml irrespective of anti-hbc and low anti-hbc with agglutination-inhibition titer of no more than 2 5 is qualified. transfusion-transmitted hbv cases related to window period donations declined by increasing the sensitivity of mini pool nat, whereas those related to blood with low titer anti-hbc remained stable with around 10 cases annually. in order to decrease such transmission japanese red cross implemented a novel strategy to eliminate all anti-hbc-positive donations with anti-hbs <200 miu/ml. considering the frequency of donations with low titer anti-hbc has decreased to 1.3%, loss of those donations was estimated to be covered by promoting donations, each country should establish its own hbv screening strategy considering the prevalence of hbv, residual risk of transmission, balance between safety and securing blood, and cost-effectiveness. implementation of individual donation nat and universal vaccination could further reduce further the risk of hbv transmission. our blood service is to motivate other population groups to diversify the age and gender composition of our donors. forty-two percent of the whole blood donors participate in blood donation only once a year, so we need develop programs to motivate them to re-join blood donation. also, we have to solve the annually recurring problem of blood shortages for transfusion during the winter and summer season. in the long term, our donor base is going to decrease gradually because of the low birth rate in korea and our rapidly aging population. therefore we should prepare a sustainable solution for a stable blood supply. aims: present methods to recruit blood donors based on age, gender and occupation groups for a stable blood supply. methods: to make blood donation more accessible to individual donors, fixed donation sites are continuously developed. facilities of our fixed and mobile sites are improved to provide safe and comfortable environment to donors. since 1999, we have been operating the 'registered donor system' for registered donors who agreed to donate their blood on a regular basis. to raise awareness of the importance of blood donation among youth, high-school students blood donor groups called 'red campaigners' and groups of university students called 'blood donation supporters' are actively participating in blood donation campaigns. to increase participation of the middle-aged group, agreements were made not only with enterprises and organizations but also with the government and public institutions. we have developed a computerized system for scheduling group donations according to demand and supply and for performance management. to recognize the necessity of blood donation, every 13th was designated as 'blood donation day' in 2012. to deal with donor complaints and requests, a customer relationship management center has been established. results: by increasing the number of fixed donation sites and making donation more accessible, rate of individual donations is getting increased. as of the end of june 2013, 250 agreements for blood donation had been signed with enterprises and organizations. every year more than 100 thousand donors agree to donate on a regular basis, and so far about 600 thousand donors have been registered in the 'registered donor system'. the campaign 'every 13th is blood donation day' has contributed to spread a positive perception about blood donation. conclusions: we have been very successful in engaging youth in blood donation. diversification of the donor group will take time and constant effort. however, with more participation of female donors and retention of our first-time donors, it will be possible to be self-sufficient in blood supply for transfusion and plasma fractionation. the provision of sufficient safe blood products to patients requiring transfusion is the common goal of blood transfusion services and the public expectation of absolute safety continues to be a challenge. although the focus of blood safety falls on laboratory testing, the role of pre-donation donor selection cannot be underestimated. transfusion-transmitted infections (ttis) are blood-borne microbes that can be spread from blood donor to recipient via transfusion. to prevent tti, donated blood must go through validated laboratory testing. in most countries, blood is tested for hiv, hbv, hcv, and syphilis. screening for other ttis may also be implemented in some countries after individual assessment of the prevalence of the infection in their general populations, for example, west nile virus in the united states. with the advance in testing technology, in particular the nucleic acid test, the window periods for the detection of various ttis have been significantly shortened. however, the risk of window donation still exists. furthermore, there are known or emerging ttis such as the variant creutzfeldtjakcob disease (vcjd) where there is still no suitable testing system for routine screening of donated blood. therefore, blood transfusion services have to continue practicing effective pre-donation donor selection to mitigate the tti risks. who recommends selecting voluntary, non-remunerated donors from low-risk populations for blood collection as the first step in reducing the risk of ttis. donor selection is usually conducted by a health history questionnaire to be completed by potential donors and a confidential interview. the questions asked should be effective in assessing whether the respondent's health status is suitable to donate and there is no tti risk factor. people who are deferred should be counseled and given the reason for and duration of the deferral. who has published a document titled 'blood donor selection -guidelines on assessing donor suitability for blood donation'. it recommends the following: national donor selection guidelines and criteria should be based on epidemiological and/or scientific evidence or, where evidence is limited or lacking, on best practice. donor acceptance and deferral policies for the prevention of tti should be based on up-to-date information on the local epidemiology of infections, the markers screened for, the availability of suitable blood screening and confirmatory assays, and the technologies in use. national donor selection criteria should define conditions of acceptance and deferral for each criterion. adequate resources, including a sufficient number of qualified and trained staff, should be made available for the consistent and reliable assessment of donor suitability for blood donation. quality systems should be in place for donor selection, including selection criteria, staff training and documentation. blood transfusion services should establish mechanisms for monitoring and evaluation to assess the implementation and effectiveness of donor selection criteria. in conclusion, pre-donation selection is essential to protect the safety and sufficiency of blood supply, and safeguard the health of recipients and donors. background: blood components may be contaminated by a variety of commensal, pathogenic or environmental bacteria during collection, manufacture or storage. the outcome of transfusion is dependent on the ability of the specific strain to multiply to clinically-relevant titers during storage, the pathogenicity of the strain and the patients' situation. platelets in particular are stored in conditions conducive to bacterial growth and septic reactions to these products are the most frequently documented infectious risk of transfusion. aim: the objective of this update is to review estimates of risk of bacterial sepsis and contamination of platelets, and recent findings describing interventions designed to safeguard patients. methods: this update will use recent reports and unpublished data to describe our current understanding of the role of bacteria in transfusion safety. results: historical data suggests that~1:1-3000 platelet products are contaminated with bacteria, and septic transfusion reactions occurred in 1:15-100,000 transfusions. clinical awareness of the danger and a 2004 aabb standard to 'limit and detect bacteria' in platelet products have driven the development of safety systems. the last decade has seen substantial progress in the implementation of optimal skin disinfection techniques and sample diversion strategies to reduce contamination, and many centers implemented either bacterial culture testing or pathogen inactivation processes to reduce the risk. culture testing can reduce but cannot eliminate the risk of exposure to contaminated components and sepsis. the majority of contaminated products do not cause adverse reactions, however, at the time of collection and manufacture the only means to prevent serious and fatal reactions is to ensure that that the component is functionally sterile. pathogen inactivation technologies show variable efficacy at killing bacteria, suggesting a need for strict adherence to the manufacturers suggested protocols to ensure optimum performance. alternatively, assays performed on the day of transfusion prevent the transfusion of high concentrations of bacteria that are associated with the most severe adverse reactions. conclusions: bacterial contamination and sepsis remain the greatest infectious risks of transfusion. enhanced testing or pathogen inactivation should be implemented to ensure patient safety. the australian red cross blood service, sydney, australia background: pathogen reduction technologies have been developed as a means of reducing the risk of transfusion transmission of blood-borne pathogens. published literature indicates that systems currently in use or under development effectively inactivate a range of pathogens in platelets and plasma, with the exception of some non-lipid enveloped viruses, bacterial spores and prions. development of pathogen reduction technology (prt) is ongoing, and many countries have adopted prt as part of their routine blood component processing. the aim of this update will be to review technologies currently in use or under development for treatment of labile blood components during processing. the impact of prt on blood component quality as well as ongoing challenges will be reviewed. platelets: there are currently three systems for prt treatment of platelets. these are the mirasol tm system (terumobct), the intercept blood system tm (cerus corporation) and the theraflex uvc tm system (macopharma). the intercept and mirasol systems are used widely in blood centres in europe, asia and the middle east for the treatment of both platelets and plasma, whereas clinical trials of platelets treated using the theraflex uvc system are ongoing. in vitro data suggests prt treatment leads to some loss of platelet function. however, haemovigilance reports from sev-eral countries where intercept treated platelets are transfused indicate no change in component usage, and indeed a reduction in transfusion related adverse events. similarly, there have been no reports to date of serious adverse events relating to clinical use of mirasol-treated platelets. plasma: the intercept and mirasol systems can be used to treat both plasma and platelets, and the theraflex system utilises methylene blue (mb) with visible light for prt treatment of plasma. there are some losses of coagulation factors following treatment with each of these systems. theraflex mb plasma has been transfused world-wide since 1999, and haemovigilance data indicates there is not a higher incidence of allergic reactions or other adverse events with mb-treated plasma. red cells/whole blood: commercial prt systems for red cell or whole blood components are under development. cerus corporation has continued development of a second-generation s-303 system for red cells, demonstrating in vivo recovery after 35 days storage. a mirasol prt system for treatment of whole blood is also being developed by terumobct. preliminary in vitro quality and in vivo recovery and survival data indicate that this technology may eventually become available, but further development and clinical trials are still required. challenges: concerns still exist regarding long term effects on patients receiving prt treated blood components, particularly the potential toxic effects of residual products following photochemical treatment. ongoing post-marketing surveillance and clinical trials are required to address these concerns. ideally, prt should provide proactive protection against emerging pathogens and reduce the need to introduce additional pathogen testing, minimise bacterial contamination and potentially replace processing steps such as gamma irradiation. the challenge for blood services is to understand and rationalise the costs, risks and benefits of prt compared to removing any of these tests or processes, whilst aiming to provide the highest level of blood safety. steps in getting a paper published/abstract accepted 1b-h4-01 no abstract available. 1b-h4-02 how to get your abstract accepted and how to present it daniels g many abstracts are submitted to the isbt for presentation at international and regional congresses. all abstracts are refereed by a large panel of international experts, who decide which abstracts should be accepted and which ones should be rejected. they also decide which of the accepted should be presented orally or as a poster. most of the submitted abstracts are accepted, though there is plenty of room for improvement in the quality of many of those abstracts. in this session i will describe why some abstracts are rejected and discuss how you might avoid this pitfall. i will also discuss some methods for improving your abstracts so that they represent the quality of the work you are describing and enhance the reputation of yourself and your institution. once your abstract has been accepted you will have to present it at the congress. if it is accepted for poster presentation, you will have to make a poster. the easiest way to produce a poster is to design it on a single powerpoint slide and then send the file to a company that will turn it into a poster on paper, or even on cloth for easier transportation. if your budget does not run to that, there are cheaper ways, such as printing it on single pages of a4 with the text printed in a very large font. the best posters do not have too much written information (bullet points are often best), contain diagrams and/or pictures, and are colourful and visually attractive. if, however, your abstract is accepted for oral presentation, then you will have to give a talk, usually of 10 min, and then be prepared to answer questions. this may be a daunting prospect, especially if you are not experienced in public speaking, and particularly if english is not your first language. i will discuss techniques for improving your presentation skills, both from the points of your spoken presentation and your visual aids, which will usually be a powerpoint presentation. if you are thoroughly prepared before you stand up in front of an audience, you will feel more confident and, consequently, less nervous. devine d publication of research findings and novel concepts in the biomedical literature is the mainstay of knowledge mobilization. the communication of scientific work as papers follows a well-established framework. a clear understanding of the nature of this framework and how to assess one's own work against it is critical to successful acceptance and subsequent publication of manuscripts. in this session, we will review the standard framework for scientific communication. communicating your findings is a form of scientific story telling. one must clearly explain why it was important to write the paper so that the reader will be interested and want to keep reading it. generally, an author must capture the interest of a potential reader right at the abstract which is sometimes the only way a reader sees the paper if they have discovered the work using a search engine such as medline. in the main body of the paper, the author must clearly explain how the study was designed and carried out with careful attention paid to any important details and to the statistical analysis, if appropriate. results must be presented in a manner that is clear and easily interpreted by the reader. then the work should be discussed in the context of other work in the area, emphasizing the novel findings. in this session, we will also address the following questions: how do i know if i have enough information to write a paper? where should i try to publish the paper? how should my paper be put together to give it the best chance of being accepted? what happens to my paper after i submit it? if it is returned to me with comments from the reviewers, what should i do? what is my next step if my paper is rejected, either without review or with review? we will also consider how to prepare papers in a language other than one's native language. the various sorts of scientific communications (original papers, review articles, reports, letters to the editor) will be discussed. the session is intended to provide general guidance for the publication of papers in the biomedical literature rather than be specifically focused on publication in the society's journal vox sanguinis. the membership survey commissioned by the isbt central office in 2012 gave a good insight into what members and non-members expect from the society. the main message was that members and potential members would like more educational and training resources to be available. many respondents requested that isbt should write international guidelines and develop standards. developing international guidelines when so many are already available and when countries and regions have different requirements would be a titanic task. the isbt board decided that the society should put together a repository (library) of guidelines, standards and regulatory documents that are already currently available. the repository is now ready and launched during the 24th regional congress of the isbt in kuala lumpur. for practical purposes the repository contains recent guidelines in the english language that are freely available, however following consultation with experts some manuals are included that are either relatively old or are not yet freely available on line. approximately 250 documents are in the repository from around 25 countries. the repository is a work in progress and further documents will be added in other languages. the repository is available via a link on the isbt home page www.isbtweb.org to the isbt academy e-learning portal. it can be accessed by country and by subject and a search facility is available. the guidelines are organised by six main subjects; donor, clinical, laboratory, quality/haemovigilance, processing and regulatory. there are sub topics for each main subject. isbt anticipates that you will find the repository a useful resource. background: blood services in africa operate at different levels of development from those comparable to the first world to those operating at a basic level with just hospital based blood banks and no national coordination. in recent years, countries have made efforts to improve their blood services based on voluntary non-remunerated blood donation. major challenges include low knowledge levels, inadequate funding, weak regulatory framework and poor quality systems. many international standards are too stringent for most economies in sub-saharan africa. to address these challenges, the africa society for blood transfusion (afsbt) started a program to develop blood transfusion standards relevant to africa. the afsbt step-wise accreditation standards: these standards were drafted by an afsbt task team for accreditation with guidance from the aabb and input from a team of experts. they were initially based on the who aide memoire for blood safety. an evidence-based decision-making process, where possible, was used to modify existing requirements or create new specific requirements. the goal of the standards is to provide a benchmark for accreditation of facilities and to maintain and enhance the quality and safety of blood transfusion in africa. the development process started in 2009. there is one standard with 3 progressively more rigorous steps of achievement as follows: level 1: minimum quality and operational requirements level 2: intermediate quality and operational requirements level 3: full accreditation at international standard a facility chooses the level to be assessed at. however accreditation at any grade is attained if facilities meet standards of the grade but also comply with specific requirements in section 11 which deals with legal and regulatory requirements, blood supply, equipment and supplies and clinical use of blood and blood products. application of the standards: they apply to facilities that perform any or all of the following functions: mobilization, recruitment and selection of blood donors; blood collection; components preparation; blood group and serological testing; compatibility testing; storage, handling, transportation and distribution of blood products. requirements do not apply in cases where the blood transfusion service is not responsible for an activity e.g compatibility testing. guidance document: there is a guidance document which clarifies and enhances understanding of the requirements of some of the standards. other standards are straight forward and do not need guidance. the guidance document is being updated as queries are received from the field. training: to facilitate meeting the requirements of the standards, there is a training committee, tasked with supporting training for blood services. a full training program is currently being developed. piloting: the standards were piloted in namibia and malawi. the namibia piloting was completed in 2012 while the malawi piloting will be completed in 2013. comments from these pilot sites have provided valuable input into the standards development process. conclusion: stepwise accreditation program is relevant in encouraging improvements for developing blood transfusion services. accreditation standards need to be commensurate with local needs. training is very important in helping developing blood transfusion services attain accreditation status. blood transfusion has become an integral part of modern healthcare. when it is required, it is an essential element of therapy, helps safe lives and improves quality of life. however, it is not without risk. being of human tissue origin, this risk is related to its source as well as the process involved in its provision. to ensure that blood transfusion is safe and does not cause harm to patients, these risks have to be monitored, evaluated and managed appropriately. therefore it is essential to develop system of ensuring safe blood supply and safe transfusion. quality systems should be developed covering the whole transfusion chain. policies, standards and guidelines are important tools. to ensure that the system is effective, there are various mechanisms to monitor, evaluate and analyse in order to bring about improvement. these include developing indicators, quality and clinical or transfusion audits, quality assessment programmes and haemovigilance programme. indicators can be used to monitor transfusion practice such as prevalence of transfusion transmitted disease among blood donors, crossmatch transfusion ratio, expiry fate of blood components and transfusion error. these indicators not only measure safety but also efficiency of the blood service. in countries where the blood supply is not consistent, the rate of blood requirement not met is an important indicator to monitor. performance of laboratories providing blood can be measured using quality assessment programmes. this is an important element that links the source of blood to the patient. audits covering all processes and procedures ensure that the quality and safety of blood is maintained. while these processes and procedures are controlled in ensuring safety and quality of the blood supply, the process involved in the transfusion process is sometimes not controlled by documented procedures and poorly supervised. auditing blood usage which is more complex and laborious provides an important mechanism for ensuring that this precious national resource is utilised efficiently. guidelines whether national or international are required against which these audits are performed. transfusion audits looks at the quality of care of patients besides the use of blood. it analyses the process of diagnosis, the decision making in treatment and the use of available resources. over the last two decades, haemovigilance has evolved and expanded worldwide. it focuses on adverse events that occurred throughout the transfusion chain, analysis of the facts and provide an avenue for corrective actions to be taken to prevent further recurrence. initially, its focus was more on adverse events that occurred to patients, it is now used to monitor adverse events relating to blood donors and blood donating process. in resource limited economies these tools can be used effectively when a stepwise approach is adopted. the aim is to create a culture of professionalism in delivering quality of care to patient with efficient use of available resources. however, its benefit can be extended to influence change and improvement in healthcare in general. only when deficiencies are demonstrated that request for resources can be shown to be justified. serological methods have the advantage of being fast, simple, and determining the expression of antigens directly. however, genotyping and molecular diagnostic methods have their advantages in determining subtypes and variants, as well as in detecting other rare blood groups. commercial blood group genotyping kits have been widely used, not only in clinical blood banks but also in transfusion services. these commercial kits mainly aim to analyze coding genes of abo and rh blood group systems. common alleles found in local populations are included in these kits. at present, many kinds of molecular diagnostic methods are employed in detecting blood group alleles by transfusion laboratories in china. these methods include multiplex pcr, multiplex pcr combined with pool system, sequencing, and gene chip technology. considering the genetic background of several rare phenotypes with clinical importance in chinese persons, one multiplex pcr system was developed to detect fy a , s, and ok a antigens, and the other system was developed to detect di b , k, and js b antigens. using the existing multiplex pcr-ssp assays, only one sample can be detected in a single pcr tube because the primers used in these assays are specifically devised for corresponding high-frequency alleles, and aim to screen for the negative results. therefore, the positive results would mask the negative results by adding more samples in one pcr tube. to improve the efficiency of screening, recently we established a novel method combining the multiplex pcr-ssp assays with the dna pooling strategy. the primers were designed to amplify the corresponding low-frequency snp sites. in each multiplex pcr-ssp assay, every dna pool is tested for the presence of the low-frequency snp sites. a pool is released for further processing when the positive results were found in any site. after then, each individual dna sample of the positive pool was detected respectively to determine the positive sample. finally, pcr-ssp methods were used to determine whether the positive result was caused by homozygous or heterozygous alleles. normally, the homozygous low-frequency alleles would lead to rare phenotypes. at the same time, the control system based on site-directed mutagenesis solves the problem of the lack of experiment controls due to unavailable negative or positive dna control samples. with large scale screening of population samples and diagnosis of various clinical special samples, the relationship between blood group coding genes and the expression of proteins is being clear. the frequency of multiple blood group alleles has also been investigated. the specific molecular events found in asians and chinese populations are revealing the difference among races and regions, as well as the expression diversity of blood group alleles. 1c-h6-02 diagnosis and treatment of autoimmune haemolytic anaemia autoimmune haemolytic anaemia (aiha) occurs as a result of antibodies directed against self-red blood cell (rbc) antigens, leading to the premature destruction of rbc. rbc destruction may occur within the vasculature, often mediated by the membrane-lysing complex, which is generated upon complement activation or occur extravascularly, within the reticulo-endothelial system that expresses fcî¥r and c3 receptors, that bind antibody and complement coated rbc. the laboratory hallmark of aiha is a positive direct antiglobulin test (dat) although it has to be recognized that occasional cases of dat-negative aiha may occur. secondary causes for aiha such as an underlying autoimmune disorder, infections or malignancies should be considered as part of the investigation process for aiha. immunohaematology investigations for aiha should always include a dat using monospecific anti-igg and anti-c3d. on occasions, further testing with anti-iga or -igg subtypes may be necessary. while the dat provides information on bound rbc antibodies, the indirect antiglobulin test (iat) using screening and antibody identification cells will provide information on the specificity of circulating antibodies. often this will give a pan-reactive pattern although it is not unusual to identify coincident autoantibodies with defined red cell specificity. negative iat with screening and identification cells in the presence of a positive dat should raise suspicion of drug-induced aiha. in patients who may have been potentially alloimmunized, further procedures should be performed to exclude the coincident presence of alloantibodies as failure to recognize alloantibodies may result in an immediate or delayed haemolytic transfusion reaction if red cell transfusions are initiated. elution and auto-or alloadsorbtion techniques are useful to separate allo-and autoantibodies. complete red cell phenotyping should be concurrently performed to aid in identifying antibody specificities. heavily antibody-coated red cells may be difficult to phenotype with some anti-sera, in which case, molecular based red cell genotyping should be initiated. molecular based red cell typing is also particularly useful where the patient has recently been transfused and the initial red cell phenotype of the patient is not known. the primary management of the patient with aiha is amelioration of the underlying condition and suppression of immune mediated haemolysis. this is usually achieved with administration of steroids or immunosuppressive agents. splenectomy may be an effective second line treatment. rituximab is increasingly becoming a promising treatment option in patients who are steroid-refractory and not suitable for splenectomy. other treatment modalities for the refractory patient include intravenous immunoglobulin and danazol. red cell transfusions in patients with aiha should be undertaken carefully although they should never be denied blood transfusions because of inability to find compatible units. as far as possible, phenotype matched red cells, cross-match compatible with the patient's autoadsorbed serum should be transfused. if coincident alloantibodies are identified, antigen-negative red cells will need to be selected. clinical immunology and transfusion medicine, university & regional laboratories, lund, sweden what is the value of discussing case studies? they provide us with a forum for sharing our combined experience and permit the development of ideas and techniques as well as the possibility to see a given situation from another perspective. cases that illustrate the following will be discussed: (1) the presence of polyagglutination in a patient with a bacterial infection in europe and the usa, polyagglutination is rarely seen since monoclonal blood grouping reagents are the norm; however across asia, a broad spectrum of blood typing reagents are used and polyagglutination maybe encountered. (2) investigation of an antibody to a low-prevalence antigen in a case of haemolytic disease of the foetus and newborn antibodies to low-prevalence antigens occur not infrequently. how can they be investigated and what should be considered? (3) antibodies to a high-prevalence antigen the discussion will consider how to resolve such a case in laboratories with different levels of resources. what should be considered in different clinical situations? the goal is provide useful tips for investigation of difficult serological cases and information on how to proceed when the investigation is beyond the resources of the laboratory. haemovigilance is an important element of blood safety. it aims to identify, monitor and prevent adverse reactions, incidents and adverse events related to transfusion for both donors and patients (from 'vein to vein'). various local, regional and national haemovigilance models exist, which reflect the range of health systems and blood systems in different countries; for example, some haemovigilance systems are coordinated by professional bodies, some by blood suppliers, and some by health authorities (health departments or regulators). participation may be voluntary or mandatory, and may differ depending on whether all events or only serious ones are reportable. some systems capture events with all levels of imputability, whereas others record only those which are confirmed or highly probable. 'near miss' events are captured by some systems, and many valuable lessons can be learned from these cases. from an early stage of haemovigilance reporting it has been identified that processrelated problems are a major cause of serious transfusion complications. these include 'incorrect blood component transfused' events, where the blood component was intended for another recipient (frequently due to errors in patient identification at the time of collection of the pre-transfusion sample, or at the time of bedside administration), or did not meet the patient's special needs (such as a patient with a red cell antibody who did not receive the required antigen-negative unit). human and system factors, such as lack of awareness or training, working environment, interruptions and inadequate communications between clinical teams, or between the clinical teams and the transfusion laboratory, are very important contributors to these events. investigation of transfusion reactions, incidents and events at the hospital level is essential to identify clinical consequences, contributing factors and to develop and implement plans to prevent recurrence. reportable incidents should be notified to the haemovigilance programme. transfusion safety officers, transfusion nurses and similar roles have been introduced in many countries and they play important roles in haemovigilance, especially at the hospital level. adequate medical and transfusion laboratory support for hospital haemovigilance activities is also essential for success. hospital transfusion committees should oversee haemovigilance activities and reporting, and should ensure that hospital senior management is aware of and responds to serious reactions and events, especially where systems issues are identified to be contributory. at an international level, isbt's working party on haemovigilance brings together isbt members with an interest in haemovigilance. isbt works closely with ihn, an international collaboration of regional or national haemovigilance programmes, and other partners. ihn operates the istare database for international data sharing and benchmarking. haemovigilance reporting can identify priority areas for action (either where the events have serious clinical consequences, and/or occur frequently) and can help identify and monitor the implementation of solutions. an important feature of haemovigilance programs is the sharing of experiences and results. haemovigilance reports can both provide valuable feedback to clinical teams and hospitals locally, as well as share experiences nationally and internationally to improve patient outcomes. wiersum-osselton jc trip national hemo-and biovigilance office, the hague, the netherlands background: haemovigilance systems capture data on adverse reactions and infections in recipients of blood transfusions as well as on errors and incidents in the transfusion chain. the objective is to analyse them and make recommendations for improving transfusion safety. many haemovigilance systems also collect data on complications in blood donors with a view to monitoring and improving blood donor safety. standardised definitions are necessary for classifying and comparing data in all these domains and at all levels. method: since 2004, at international meetings of blood transfusion professionals, members the international haemovigilance network (ihn) and the haemovigilance working party of the international society for blood transfusion (isbt) have collaborated in developing and validating definitions for non-infectious transfusion complications, errors and incidents in the transfusion chain as well as adverse reactions in blood donors. from 2012 contacts with other groups including the who have been put in place to ensure wide consultation as well as awareness and use of the definitions. results: standardised definitions have been published for recipient adverse reactions, for complications of blood donation and for a limited number of types of incident in the transfusion chain. the isbt haemovigilance working party and ihn are committed to ensuring that the definitions remain up-to-date and that revisions and improvements are conducted with wide consultation of professionals in relevant organisations worldwide. the haemovigilance working party and working party on transfusion-transmitted infections are collaborating on the development of definitions and criteria for assessing suspected transfusion-transmitted infections. conclusion: internationally agreed definitions are available for registration and surveillance of complications of blood donation and most types of adverse reaction in patients receiving blood transfusions. for errors and incidents in the transfusion chain, further work is necessary to improve comparability of data between hemovigilance systems. 1d-h7-03 distler pb and ashford p critical to patient safety is the capability of rapidly tracing a medical product of human origin (mpho) from donor to recipient and vice versa. traceability requires that each product be uniquely identified in order to provide a clear, unambiguous path. historically, uniqueness was defined only in the context of a single organization. for example, a blood product identifier was unique only to the blood bank that assigned it. because some medical products of human origin (mpho), especially cells and tissues, are frequently distributed across international borders, it is becoming increasingly important that identifiers of mpho need to be unique not only within an organization, but globally as well. in 2010, the world health assembly urged member states 'to encourage the implementation of globally consistent coding systems for human cells, tissues, and organs as such in order to facilitate national and international traceability of materials of human origin for transplantation.' more recently who has recognized the need for common strategies for global governance of all mhpo, including the global use of isbt 128, to ensure unique identification, optimal traceability, and interoperability between countries and across all mpho for both routine and emergency use. this requires a globally consistent coding system that can provide: a mechanism to allow distinct items to be uniquely identified and consistently characterized to all participants within the system, the means to allocate identifiers in a manner that avoids duplication, and the information infrastructure on which effective traceability can be built. isbt 128 is an international terminology, coding, and labelling system that supports the assignment of unique identifiers to support global traceability of mpho. it is currently in use by many blood banks, tissue banks, and cellular therapy facilities around the world and the who global forum on blood safety has recognized promotion of the use of isbt 128 as a priority for action in improving quality management and haemovigilance. 1d-h8-01 the university of tokyo, tokyo, japan antibodies directed to human platelet antigens (hpa) play important roles in the pathogenesis of neonatal alloimmune thrombocytopenia (nait), platelet transfusion refractoriness (ptr) and post-transfusion purpura (ptp). presently, six hpa biallelic systems, namely hpa-1 to -5 and -15, which are involved in immune mediated thrombocytopenia, are characterized. there are important ethnic differences in the frequency distribution of these hpa systems, the incompatibility of the hpa-1 system being the mostly involved in thrombocytopenic conditions in caucasian, whereas in japanese, the hpa-4 system is the mostly involved. the frequency distribution of hpa systems reported in other parts of asia seems to be different from caucasian as well as japanese, especially related to hpa-1 and -4, respectively. in addition to hpa, antibodies to human leukocyte antigen (hla), blood group abo, and human neutrophil antigens (hna) have also been shown to be involved in immune mediated thrombocytopenia. in fact, the majority of the cases of ptr are dependent on anti-hla antibodies, and anti-hpa antibodies comprise only a small proportion. the identification of the causative antibody is very important for the implementation of preventive/therapeutic measures for ptr, such as the selection of hla-and/or hpa-compatible platelets. on the other hand, the involvement of anti-hla antibodies in the pathogenesis of nait is questioned, but cases in which the causative antibody cannot be determined still remain relatively high. thus, for the implementation of preventive and therapeutic measures for the immune mediated thrombocytopenia, the detection and identification of the causative antibody is essential. the standard methods applied varies among the regions, the monoclonal antibody-specific immobilization of platelet antigens (maipa) and the platelet immunofluorescence test (pfit) being the preferred methods in the us and europe, whereas in japan, the mixed-passive hemagglutination is the most applied one. neither of the methods alone, however, is able to detect all the clinically significant antibodies, thus, improvement of the available methods as well as the development of new technologies is required. considering the ethnical differences of the hpa frequency distribution, we considered important to develop the research of this field also in asia, and for this purpose, the isbt platelet immunobiology working party, asia regional (isbt piwp-ar) was launched in 2010, during the xxxist international congress of the isbt in berlin, which aims the sharing of knowledge and improvement of technology in asia. a training course on platelet immunology methods and genotyping was provided in tokyo in 2010, and the first workshop of the piwp-ar was organized in taipei in 2011. in may 2013, the second training course on platelet immunology methods and genotyping was organized in guangzhou, china, and the 2nd workshop of the piwp-ar is going to be held in kuala lumpur, malaysia, during the 24th regional congress of the isbt. the presently available methods for the antigen/antibody testing, the problems related with antibody detection, and the activities of the platelet working party will be presented. nanning institute of transfusion medicine, nanning, china background: immunization against human platelet antigens (hpa) is associated with a number of clinical syndromes, including neonatal alloimmune thrombocytopenia (nait), platelet transfusion refractoriness (ptr), post-transfusion purpura (ptp), and other platelet immune disorders. the detection and identification of the clinical relevant platelet antibodies are important for the diagnosis and management of the affected patients with immune thrombocytopenia. aims: the aims of this study is to investigate the characteristic of the detection of clinical relevant platelet antibodies in the asian population, and to evaluate the ability for the detection and identification of platelet antibodies in the platelet immunology laboratories in asian countries. methods: total of 378 cases that were diagnosed and studied as naitp (70), ptr (305) and ptp (3) in asian platelet immunology laboratories were reviewed. of the 378 cases, 167 were japanese in japan, 142 were chinese in china, 30 were in india, 24 were chinese in taiwan, china, 5 were in south korea, 4 were in thailand, 3 were in kuwait, and 1 case each for oman, lebanese and palestinian. the specificities of platelet antibodies in these cases were investigated and compared with the data from western country's laboratories. the methodology of detection and identification of antiplatelet antibodies in asian labs were also reviewed. results: among 378 cases, the immune thrombocytopenia associated antiplatelet antibodies were two cases of anti-hpa-1(in kuwait), 2 of anti-hpa-2 (1 in china and 1 in japan), 5 of anti-hpa-3 (three in japan, one in china and one in taiwan, china), 5 of anti-hpa-4 (all in japan), 4 of anti-hpa-5 (1 in china and 3 in japan), 1 of anti-hpa-7new(antihit a )(in japan), 1 of anti-hpa-15 (in japan), 2 of anti-hpa-21bw (in japan), 309 of anti-hla(101 in china, 147 in japan, 5 in korea,3 in thailand, 23 in taiwan, china and 30 in india), 1 of anti-a (in japan) as well as 15 cases of anti-cd36(nak a ) isoantibody (eight in china, three in japan and one case each in thailand, oman, lebanese and palestinian). thirty one cases of antibodies could not find the specificities (30 in china and 1 in kuwait). the methods of detection and identification of antiplatelet antibody, such as monoclonal antibody immobilization of platelet antigens (maipa), mixed passive hemagglutination (mpha) assay, platelet immunofluorescence test (pift), modified antigen capture elisa (mace), and solid phase red cell adherence (spaa) have been used in asian laboratories summary/conclusions: platelet alloantibodies are found with variable frequencies in different ethnic groups in asian population. anti-hpa-4 is mainly found in japanese individuals, while anti-cd36 (nak a ) isoantibody that occurred in cd36 type i deficient individuals is most frequent found in asian population especially in chinese population. only two cases of anti-hpa-1 antibodies were found in asian population (all in kuwait), while anti-hpa-1 is the most frequent antibody associated with severe complications in caucasian populations. however, anti-cd36 isoantibody is of a risk factor of immune thrombocytopenia in asian population, as the incidence of cd36 deficiency is 3-11%. the human platelet antigens (hpa) are a group of polymorphic antigens, expressed relatively specific on platelets, capable of eliciting an immunological response with development of alloantibodies. hpa directed alloantibodies have been implicated in neonatal alloimmune thrombocytopenia (nait), post-transfusion purpura and refractoriness to platelet transfusions. twenty-seven hpa are recognized, of which antibodies to both the given alloantigen and the antithetical alloantigen has been reported for hpa-1 to 5 and hpa-15. the remaining hpa are designated with a 'w' as an alloantibody to the antithetical antigen has yet to be reported. most hpa polymorphisms are a result of a mis-sense single nucleotide alteration and are readily detectable using standard molecular typing methods. dna based population wide genotyping have revealed considerable variation in hpa allele frequencies among different ethnic groups. the 'b' forms of hpa-1, -2, -3, and -5 are common among caucasians while hpa-4b is extremely rare. in the chinese however, hpa-1b is extremely rare while uncommon in malays. hpa-4b and hpa-21bw meanwhile are more commonly seen among asians compared to caucasians. consequent to this, alloanti-hpa-4b is the most common cause for nait in the asian population as compared to alloanti-hpa-1a among caucasians. several cases of nait due to anti-hpa-21bw have also been reported in asia. in contrast, nait due to anti-hpa-6b is rare despite the alloantigen being more common among asians. although platelet transfusion refractoriness is more commonly associated with hla antibodies, anti-hpa antibodies have been implicated in some cases. management of nait and platelet transfusion refractoriness include the supply of antigen negative platelet units. a platelet donor registry with a critical mass of hla and hpa typed blood donors is therefore necessary for effective management of these conditions. ready availability of low-cost high-throughput snp genotyping platforms allow for establishment of large hpa genotyped donor pools. knowledge of hpa genotype prevalence in the local population as well as its implications on nait and platelet refractoriness is however crucial before deciding on donor screening strategies, careful considerations would also need to be made with regards to the cost-effectiveness of such ventures in light of alternative management strategies and local incidence rates of nait. clinical -improving patient outcomes 2a-s01-01 setting up a patient blood management programme wood e 1 , engelbrecht s 1,2 and robinson k 2 1 transfusion research unit, monash university, melbourne, australia 2 australian red cross blood service, adelaide, australia patient blood management (pbm) aims to minimise unnecessary transfusions, and also to ensure that if transfusion is required it is managed appropriatelyby individualising care so that patients receive what they need when they need it. pbm is comprehensive and patient-centred, with active participation by patients and a multidisciplinary approach from the hospital team to achieving these aims. 'three pillars' of pbm have been suggested, to optimise a patient's red cell mass, reduce bleeding and improve tolerance of anaemia. in the perioperative setting, important elements of pbm include attention to medical, surgical and anaesthetic interventions and techniques to improve haemostasis and reduce blood loss. where significant intraoperative blood loss is anticipated, use of cell salvage techniques can be very valuable. pbm concepts also apply outside the perioperative setting, and the broad principles can be applied to a wide range of clinical settings, including obstetrics, trauma, critical care and haematology/oncology and other medical settings (e.g. gastroenterology). an effective hospital pbm programme requires planning and communication, with a stepby-step approach to implementation, identifying priority areas for action, engagement, education and training of staff, and on-going monitoring against plans to demonstrate progress and identify areas for improvement. feedback to staff on progress provides a sense of achievement and helps engagement. adequate resources are required, including from medical, nursing and laboratory staff from a range of disciplines who have important contributions to make in clinical practice, education and training, and audit and review. minimisation of unnecessary transfusions saves money for hospitals and the community, and other resources such as staff time, and therefore offsets the costs of establishing and maintaining a pbm programme. effective implementation requires change at individual and organizational levels, and therefore support of hospital executive management, local health authorities and the blood supplier are also very valuable. oversight of the program can be by the hospital transfusion committee or a pbm programme committee, but the particular structure and governance arrangements should be developed to suit local needs. general practitioners can play key roles in preparing patients for surgery by identification and management of anaemia, as well as other pbm activities. ultimately an effective pbm programme can optimise care and outcomes for patients, make better use of limited and precious blood supplies, and reduce risks and costs. trauma is a leading cause of death around the world, with haemorrhage accounting for more than a third of the preventable mortality, with the majority of these deaths occurring within the initial 24 h. massive blood transfusion is generally defined as the replacement bytransfusion of more than 10 units of red cells over 24 h. massive transfusionprotocols (mtps) have evolved over the past decade and are especially importantin facilitating the early delivery of copious amounts of blood products topatients who have major injuries and severe haemorrhage. various studies havealso demonstrated that with mtps, there is a more efficient use and lesswastage of blood products. however, for trauma patients, stopping thehaemorrhage and resuscitating the patient does not only involve the expedientdelivery of red cells to the injured patient. mtps have also emphasized theneed for a more balanced ratio of delivery of blood products, includingplatelets and plasma, to patients who sustain massive blood loss and havedeveloped acute traumatic coagulopathy (atc). often times, mtps also stress theimportance of the consideration of use of haemostatic adjuncts, such astranexamic acid, activated factor vii, level one transfusion units and the useof blood warmers to reverse the potential effects of hypothermia with sustainedtransfusions of large amounts of blood products. ultimately, in tandem withdamage control resuscitation, which allows permissive hypotension whilstsecuring haemostasis, mtps have been shown to also improve survival of theseseverely injured patients. a more recent paper describes the development of amassive haemorrhage protocol to aid in the identification of patients who wouldbenefit from the mtp and this may be the next step in the evolution of a workprocess by which resuscitation for severely injured patients may be optimized. background: rh blood group antigens are highly immunogenic and transfusion of rh d positive blood in rh d negative recipients is avoided. platelets do not express rh antigens, however they may contain significant amount of contaminating red cells to illicit an immune response in the patient. due to limited shelf life of platelets and inventory issues, rh d positive platelets which are not visibly contaminated with red blood cells maybe transfused to rh d negative patients including children and females of child bearing age. there has been increased focus on whether rh immunoglobulin should be routinely administered after such transfusions. in saudi arabia no clear guidelines exist on the administration of rh immunoglobulin after d positive transfusions in d negative patients. aim: the purpose of this study was to determine whether the transfusion of rh d positive platelets to patients who are rh d negative, results in d alloimmunization and whether rh immunoglobulin should be routinely administered in such patients. methods: eligibility criteria for inclusion in the study included the following: transfusion of rh d positive platelets, no anti d detectable before transfusion, no previous exposure to rh d positive blood components, and results of follow-up testing of anti-d in patients serum available. the patients blood group and antibody screening was done using liss-iat gel technology (diamed). results: one hundred rh d negative patients who received rh d positive blood components were identified. out of this 63 (63%) patients received rh d positive platelet transfusions. in 47 (74%) patients out of this, the results for post transfusion antibody screening were available. the mean age of the patients was 46.5 years and included 30 males and 17 females. average number of rh d positive platelets transfused per patient was 11.3 units and total number of platelet units transfused was 523. anti d was detectable in 4 (8.5%) patients post transfusion and included one male and three female patients. of the female patients one was a 3 year old child who received 50 units of random donor platelets and second a 21 year old women who presented in the emergency department as a case of trauma. the third female was 54 year old post-menopausal woman. conclusion: we conclude that the chances of developing d alloimmunization after receiving rh d positive platelets is generally low. however keeping in view the antenatal complications which can arise in the future in females due to this alloimmunization, it is highly recommended that rh immunoglobulin be administered to all rh d negative women in the reproductive age group and female children who receive rh d positive platelets. background: hemovigilance is a quality process which takes into account all the activities of a blood transfusion chain with the aim to improve quality and enhance safety of blood transfusion. we have implemented a transfusion feedback reporting mechanism in our hospital as a part of hemovigilance. the current study aims to collect and analyze this data to improve our transfusion system. aim: to systematically analyze the transfusion process from issue of blood components to completion of the transfusion material and methods: the transfusion feedback forms received back at the transfusion medicine department during a 3 months period were systematically analyzed for documentation related to patient identification, product identification, documentation, completeness, etc. results were analyzed statistically for specific co-relation with patient's location, time of transfusion and type of component transfused. results: of 3474 blood components were issued during the study period. transfusion feedback form were received for 2000 (57.5%) blood components, as follows: prbc-800 (40%), platelets-651 (32.5%), ffp -441 (20.7%). patient identification number /wristband check was not done in 25 (1.25%) cases. pre-transfusion verification of blood group, patients name and patient's identification number was done in 1963 (98.15%) cases. cross checking of component unit with request form was missed in 16 (0.8) cases. pre-transfusion and post-transfusion monitoring of blood pressure was documented in 698 (34.9%) episodes, monitoring of pulse in 696 (34.8%) episodes and patient's temperature was monitored in 681 (34%) episodes. signature of nurse was missing in 86 (4.3%) and that of medical officer in 11 (05%) of the form. adverse transfusion reaction was documented in 1/2000 forms, whereas the transfusion reactions notified at the blood bank during the same period were 2. of 553 (27.65%) transfusions were carried out during non routine work hours. the mean time between the issue of the components and start of transfusion was 28 min for rbc components, 21 min for platelets and 16 min for ffp. patient identification and monitoring and product identification related non-compliances significantly correlated with out of routine transfusions (p = 0.002). conclusion: though overall compliance with established procedure for transfusion was evident, activities related to unit identification, patient monitoring and identification and reporting of adverse reactions were not well documented. this emphasizes the need for ongoing training of nursing staff and medical doctors in safe blood administration practices. introduction and method: matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (maldi-tof ms) is an ideal tool for high-throughput blood group genotyping. using this technique, this swiss (bts zurich) -german (sequenom gmbh, hamburg) cooperation aims to genotype swiss blood donors for blood groups rh, kell, kidd, duffy, mnss (n = 6000), hpa and hna (n = 3000) and high frequency antigens (hfa, n = 36,000) within 3 years. specificities are grouped into single multiplex reactions (mpx, n = 10) with up to 17 snps tested simultaneously in one tube. results: using the kel-jk-fy mpx, more than 4000 donor dnas have been tested and compared to serological pre-values. concordance between geno-and phenotypes reached 100% for k/k, 99.98 for kp a 99.93% for jkand 99.23% for fy a/b/x/0 . only one discrepancy each for kp, jk and fy could be attributed to genetics, the others were erroneous serotypes revealed by genotyping. genetic discrepancies were three new variants: kel*02.03(r700g)nulla kp a relative, jk*b(mutation n.d.) and fy*b (g261r)null. serology for js a/b of some few kel*02.06 positives confirmed validity of genotyping. numbers of detected known kel*mod and null, jk*null and fy*null (gata) alleles were 3, 2 and 51, respectively. call failures (no result) were observed in less than 2% of all mpxs. genotyping for rhd, rhce (5 mpx), gypa and b (mnss, 1 mpx) on more than 4000 samples, delivered results with discrepancy rates for mnss comparable to above, and better rates for rhdce. call failure were at approximately 2% again. reproducibility, robustness and analytical accuracy of the technique allowed measurement of gene copy numbers with relevance for rhd zygosity estimation and detection of the gypb deletion in u negatives. rhd category, partial, weak, del and null alleles and the genetic correspondents of vw, mg, mi(a), he, and uvar were observed. genotyping for hpa and hna (1 mpx) showed expected results among 2300 samples with the exception of hna-1a, b and c, where frequent duplications and deletions of fcgriiib pose difficulties for all genotyping approaches in general. in hfa ('rare') genotyping (2 mpx), currently, more than 13,000 blood donors were analyzed, and delivered: 29 kk, 2 kp(a+bã�), 48 js(a+bã�), 41 kel11+kel17+, 20 lu(a+bã�), 3 lu14+lu08ã�, 52 yt(aã�b+), 17 co(aã�b+), 11 kn (aã�b+), 98 lw(a+b+), and >10 others. specificities for vel negativity and scianna have been included into hfa typing, recently. conclusion: analysis for kell, kidd and duffy showed that genotyping worked qualitatively better and to costs comparable to serology. consequently, genotyping kell, kidd and duffy instead of routinely performing a second round of serotyping as mandatory for donors in switzerland, is recommended. ahead of comparable suggestions with regard to rh and mnss, a more detailed statistical analysis of existing raw data is needed. however, genetically identified donors with rare blood phenotypes, e.g. such as yt(aã�b+), are already selected for respective transfusions and are a strong indicator for the value of the presented project. the serological data suggest that retention of the 208-210 tcc (51s in glycophorin bs) codon from the gypb pseudoexon, prior to the gene conversion insertion of gypa sequence and coding for serine in the hybrid glycophorin, is the basis of 'anek-like' activity for both hybrid glycophorins. it should be considered that these antisera react with a new kipp-related mns system antigen. genetic studies revealed the same crossover for gp.kipp and gp.yak that have been independently reported in conference abstracts, suggesting that these are in fact the same hybrid glycophorin. background: with the increasing knowledge of the genetics of blood group antigens, molecular immunohaematology is gaining popularity. molecular immunohaematology refers to the use of genotyping to encode red blood cell antigens, representing an indirect method used to predict one's phenotype. there are certain advantages of genotyping, namely, typing of red blood cells with autoantibodies, prenatal testing and patients with multiple transfusions. molecular methods are also useful in phenotyping donors as it enables the prediction of numerous phenotypes in one single test. however, there are several misgivings about the cost of molecular methods used to genotype antigens. aim: to evaluate and compare the cost of conventional serological phenotyping and molecular genotyping. methods: a batch of 30 donor samples was typed using both serological and molecular methods. the test kit used for molecular methods, from gen-probe, included the typing for the following antigens -kell (k, k, kpa, kpb, kpc, jsa and jsb), kidd -(jka, jkb, jk), duffy (fya, fyb, fyx, fygatasil) , mns (m, n, s, s, s-s-uvar), rh (c, c, e, e) and dombrock (doa, dob). negatives that are obtained using this method were confirmed using serology. serological typing was performed with available antisera according to the various manufacturers' instructions. this includes kell (k, k), kidd (jka, jkb), duffy (fya, fyb), mns (m, n, s, s) and rh (c, c, e, e). the cost for the different tests were tabulated and compared. the cost includes labour, consumables and equipments. results: the results show that molecular method of typing red blood cells is more expensive than the traditional serological method. the cost of consumables is comparable for both methods. the consumables make up about 80% of the total cost for serological methods, and about 82% for molecular methods. the cost of labour is about 9% for serological methods and <1% for molecular. equipment cost contributes to <1% of the cost using serological methods and about 14% using molecular methods. conclusion: molecular methods may seem more expensive, about two times the cost of serology but results are obtained faster and are less labour intensive which could prove to be an advantage when phenotyping samples in large quantities, especially for donor phenotyping. molecular immunohaematology is a relatively new process, thus kits for these methods are expensive at the moment. as development and adoption of genotyping progresses, consumables and equipment costs are expected to become more affordable. serological methods do have their limitations even for patient testing, especially for patients with autoantibodies and patients who have had multiple transfusions. molecular methods may serve useful in overcoming these limitations. background: transfusion dependant patients often develop multiple antibodies to red cell antigens on exposure to red cells and require antigen-negative red blood cells for further transfusions. finding suitable red cell units for such patients is often difficult and time consuming, requiring phenotyping of large numbers of red cell units in inventory. the increasing cost and scarcity of anti-sera also makes this an expensive exercise. aim: in order to improve provision of phenotype-matched blood for such patients, we studied the feasibility of large-scale snp-based genotyping of common blood groups for establishment of an antigen-negative red blood cell inventory. methods: genomic dna was extracted from donor blood samples using an automated platform. samples were subjected to snp-typing for common local polymorphisms of rhce (ccee), fy (fy a /fy b ), jk (jk a /jk b ), mns (s/s) and co (co a /co b ) using taqman 㢠snp genotyping assays in 384-well plates at 5 ll final reaction volume, on a lightcy-clerii 480 real-time instrument. identification of the cc polymorphism was by probes targeted to the 48c>g and 307t>c of the rhce allele while probes targeted to 676c>g was utilised for ee detection. for the remaining alleles, probes were designed only to detect the most common mutation for the polymorphism within an east-asian population. results were analysed and integrated into the blood donor software system. results: the 48c>g probe designed for identification of c was unsuccessful with poor discrimination between genotype calls. the remaining probes showed satisfactory discrimination between genotypes, with 462 of the 505 (91%) samples analysed, fully genotyped for the five alleles studied. relatively rare genotypes were successfully identified using this strategy: ccee (15/505, 3.0%), fya-/fyb+ (19/505, 3.8%), ss (5/505, 1.0%). the co2 allele responsible for co b was identified in the heterozygous state in four of 494 evaluable samples giving an allele frequency of 0.4% in our population. the estimated reagent and consumables cost for sample dna preparation and snp testing was usd12.00 compared to usd27.15 for phenotyping using commercial anti-sera. we did not factor in the cost of capital acquisition of instruments for genotyping as we used facilities which were common for other molecular tests in the hospital. conclusions: results of this study indicate that snp genotyping would be a costeffective strategy for screening and establishment of an antigen-negative red cell inventory and genotyped whole blood donor pool. the cost of genotype screening was effectively reduced by use of small final reaction volume and extensive use of automation. in addition, genotyping allowed us to identify local prevalence of blood groups which were previously unidentifiable due to lack of commercially available anti-sera. background: over the past 20 years the molecular, biochemical and serological basis of almost all blood groups have been determined, highlighting the different frequencies of antigen, related to different ethnic groups. since october 2012, in our transfusion center (asl caserta) in order to have a blood bank that ensures the different transfusion needs, extended erythrocyte typing is practiced on periodic donors with specific features such as age, group and rh phenotype. aims: the aim of our study was to test the validity of the molecular method of erthrocyte phenotype typing with the serological technique by comparing the results obtained by each procedure. we also compared each method in regards to reliability, ease of use and reproducibility of results. methods: we selected 250 donors aged <45 years old, group o and a, rh homozygous phenotype. samples from each patient were tested by serological typing in solid phase (capture r immucor) and then dna was extracted and each sample was tested by molecular typing in microarray (bioarray solutions immucor) results: see table 1 . table 1 : summary/conclusions: the results show that the frequencies of more immunogenic antigens (k, fya, jka) reflect the specific and ethnic frequencies of the donors. we emphasise the absence of donors having an antigenic structure that is defined as rare (that is present with a frequency of 1:1000 according to american rare donor program (ardp), council of europe, international donor panel (idp), international society of blood transfusion (isbt), council of europe, japanese red cross). we only found one case of a donor expressing weak fyb which is due to mutation in fy 265c> t. the results were confirmed in 99.6% of cases by serological technique. however for the phenotype fyb weak, the result was discordant in serology, where the result was fyb-. in conclusion we can confirm the validity and the need to use both techniques in order to obtain a reliable and reproducible result. the molecular technique is able to identify mutations in particular genes, especially in specificity whilst the serological technique excels in sensitivity and speed but fails to confirm the data obtained. background: pathogen inactivation of blood components promises a new layer of transfusion safety, enhancing existing strategies and providing proactive protection against emerging infectious risks. processes for plasma and platelets are well established in many blood centers and those for red cells and whole blood are in development. clinical acceptance of new technologies depends on the demonstration of product safety, a minimal effect on product efficacy both in vitro and in vivo, and the range and degree of pathogens inactivated. aims: to review the major processes of pathogen inactivation of plasma and cellular blood components with a focus on the range and degree of inactivation of relevant pathogens, especially with regards to platelet pathogen inactivation. methods: this review will use recent reports and unpublished data to describe our current understanding of the potential efficacy of pathogen inactivation in improving transfusion safety. results: the major indication for platelet pathogen inactivation is bacterial contamination, a persistent problem despite multiple innovations to minimize and detect contamination. pathogen inactivation processes need to cover a wide range of possible bacterial concentrations and species. there have been few published reports using the current commercially available systems: optimized in vitro testing documents the ability of the terumo bct mirasol tm , cerus intercept tm and macopharma theraflex tm uv systems to effect a 10 2 ->10 6 log reduction of various bacterial species. most systems are less effective at inactivating bacterial spores, a particular problem as bacillus spp. is common platelet contaminant. testing the level of pathogen inactivation under clinical conditions at very low and high concentrations of bacteria reveals further weaknesses in pathogen inactivation strategies. conclusions: clinical trials of pathogen inactivated platelets have focused on the safety and clinical efficacy of pathogen inactivated treated platelets, but little has been reported on the efficacy of pathogen inactivation to reduce the risk of infection transmission. blood centers should focus on this aspect of efficacy as they decide whether to implement, and to favor those commercially available systems that best meets the clinical need for pathogen protection. for pathogen inactivation (pi) using amotosalen and uva light induces the formation of covalent adducts and interstrand crosslinks between amotosalen and nucleic acids, thus preventing dna replication and rna translation. pi technology has been adopted into routine use in some european countries and is under fda review for licensure in the us. current documentation of pi efficacy relies on illuminator sensors that measure the uva light dose delivered. an indirect methodology is utilized for the validation and qc of the process in some centers that measures the amount of amotosalen consumed during illumination in platelets and before removal with the compound removal device. the% amotosalen remaining is a direct measurement of the uva light delivered and photochemical conversion of amotosalen. however, a functional qc method that measures a direct target within the treated blood product has not been introduced into clinical use. residual leukocytes, platelets and potentially plasma contain mitochondrial dna (mtdna) which is a collateral target of the pi process. aims: the goal of this study was to quantify the impact of intercept treatment on platelet-derived mtdna by real-time pcr. methods: to evaluate the feasibility of detecting pi-induced mtdna modifications by real-time pcr, we spiked purified human leukocyte dna into human plasma, 35% plasma/65% intersol, or pbs. each sample (3.6 ml) was treated with 150 lm amotosalen and 3 j/cm 2 uva (n = 2). control samples were either untreated or treated in the absence of amotosalen or uva. dna was extracted from each sample (0.2 ml) in duplicate and assessed by measuring the inhibition of real-time pcr amplification in duplicate wells using mtdna-specific primers and sybr greenbased detection. amplification of sequences ranging in size from 73 to 1065 bp was evaluated over 45 cycles of amplification. subsequent to the initial feasibility tests, a pilot validation was performed by treatment of platelets in 35% plasma/65% inter-sol (30 ml). six different platelet units were tested as outlined above for inhibition of amplification of endogenous mtdna sequences. results: treatment of purified dna with amotosalen plus uva resulted in 1.3 log to >6.0 log inhibition of pcr amplification (results shown in table) . the extent of pcr reduction roughly correlated with the size of the amplicon, as well as with the type of diluent in the following order: pbs > 35% plasma > 100% plasma. exposure of platelets to amotosalen and uva showed an average of 2.5 log to 3.6 log reduction in pcr signal, with increasing inhibition observed for larger amplicons. in all cases, no pcr inhibition was observed in the absence of amotosalen and/or uva. conclusions: a quantitative real-time pcr assay specific for mtdna is capable of documenting pi-induced collateral nucleic acid modification in platelets. based on this work, this assay can be developed further for use as a quality control method for pi efficacy. background and aims: pathogen reduction technology (prt) provides a proactive approach to improving transfusion safety for platelet concentrates (pcs). however, prt treatment is known to exacerbate the effects of the platelet storage lesion, leading to increased platelet activation and secretion of immunomodulatory factors. little is known regarding how prt-treated platelets may affect the cells of the recipient's immune system once transfused; therefore the aim of this study was to examine the cytokine responses of a recipient's inflammatory cells after exposure to prt-treated platelets using an in vitro whole blood model of transfusion. methods: two abo/rhd matched buffy coat derived pcs were pooled and split to form matched pairs on day-1 post-collection (n = 11). one unit was treated with the mirasol prt tm system (terumo bct), while the other unit remained as an untreated control. all units were stored at 22â°c with agitation and samples were taken on day 2 and 7 post-collection for 'in vitro transfusion' experiments. to represent a transfusion in vitro, 10% v/v platelets were incubated with abo/rhd-matched fresh whole blood, with or without lipopolysaccharide (lps; 1 mg/ml) for 6 h at 37â°c/5% co 2 . protein secretion was inhibited using brefeldin (2 lg/ml) to allow detection of intra-cellular cytokine production in monocytes (cd45 + /cd14 + ) and neutrophils (cd45 + / cd16 + ), using multi-colour flow cytometry. the fold change of cytokine production was calculated by comparison to a 'no-transfusion control' (whole blood without addition of platelets). data was analysed using a one way anova with post-hoc tests for pair-wise comparisons. results: in the absence of lps, both prt-treated and untreated platelets stored for 7 days significantly increased monocyte mip-1b expression (1.5-fold; p = 0.047), whereas exposure to day 2 platelets did not result in any significant differences in mip-1b expression. as expected, lps stimulation significantly increased monocyte production of both il-12 (5.0-to 7.4-fold) and mip-1b (1.8-to 2.4-fold). however, lps-induced monocyte il-12 production was significantly reduced by exposure to prt-treated or untreated platelets stored for 2 days (prt-treated: 3.0-fold, untreated: 2.5-fold; p < 0.0001) and 7 days (prt-treated: 4-fold, untreated: 2.1-fold; p < 0.0001). furthermore, il-12 production was significantly lower following exposure to day 7 prt-treated platelets compared to untreated platelets (p = 0.006); however there was no significant difference following exposure to day 2 platelets. lps-induced mip-1b production was not significantly differentfollowing exposure to either day 2 or day 7prt-treated or untreated platelets. exposure to platelets (untreated or prt-treated) did not significantly modulate monocyte production of ip-10, mcp-1, mip-1a, il-1a, il-1b, il-6, il-8, il-10, or tnf-a. co-culture of platelets and whole blood did not result in any significant changes to cytokine expression in neutrophils. conclusion: using an in vitro whole blood transfusion model, we have demonstrated that exposure to prt-treated platelets stored for 7 days results in significant changes in the il-12 production by monocytes. these changes may reflect the way prt-treated platelets interact with immune cells upon transfusion. therefore, the effect of stored prt-treated platelets, especially in recipients with underlying inflammation, should be further examined. backgrounds: hepatitis c virus (hcv) infection is one of the major causes of chronic hepatitic disease. hcv has six genotypes and more than 80 subtypes. the epidemiology of hcv subtypes vary with different geographic distribution. understand the subtypes prevalent in certain area will help to understand the transmission modes and the spreading trend of hcv and thus help to make effective precautionary measures. hcv subtypes are also closely related with clinical therapeutic effect. it is important for guiding clinical therapy and prognosis and predicting the possible burden of hcv infection and treatment in the future. aims: to investigate and compare the prevalence of hcv subtypes in clinical patients and blood donors in guangdong china. methods: of 191 samples ofclinical patients and 222 samples of blood donors whose hcv rna were positive were collected from guangdong province. hcv ns5b gene was amplified by rt-nested pcr and then sequenced. hcv subtypes were assigned by constructing phylogenetic trees with mega5 software. moreover, spss16.0 software was applied to compare the difference between these two groups. results: of 191 clinical patients, hcv genotype 1a, 1b, 2a, 3a,3b, 6a and 6n was 2 (1.05%), 127 (66.49%), 17 (8.90%), 5 (2.62%), 5 (2.62%), 34 (17.80%) and 1 (0.52%), respectively. of 222 blood donors, hcv genotype 1a, 1b, 2a, 3a,3b, 6a and 6n was 1 (0.45%), 92 (41.44%), 1 (6.76%), 19 (8.56%), 11 (4.95%), 83 (37.39%) and 1 (0.45%), respectively. the proportion of hcv 1b was higher in clinical patients than in blood donors(v 2 = 25.866, p = 3.66e-07), while the proportion of 3a and 6a subtypes were higher in blood donors than in clinical patients(v 2 = 6.602, p = 0.010; v 2 = 19.398, p = 1.06e-05). one possible cause was the transmission modes varied with different hcv subtypes. hcv 1b is more related with blood transfusion while 3a and 6a are more relevant to intravenous drug abuse and sexual behavior. with the anti-hcv screen implemented from 1993, the risk of hcv infection by transfusion is diminishing. the other reason was the average time from hcv infection to serious pathological lesions is about 20 years and hcv 6a was transmitted into guangdong later than 1b. conclusions: in guangdong province, hcv 1b and 6a were the predominant subtypes in clinical patients and blood donors. the proportion of hcv 1b, 3a and 6a subtypes were significantly different between clinical patients and blood donors. the reason may relate with the time of hcv transmission into guangdong area and the hcv propagation modes. blood safety and increased public health initiatives to reduce hcv infection from those in these high risk groups to the general population remain a priority. background: in japan, we routinely evaluate the presence of transmission of hbv, hcv and hiv in all transfused patients at three months after the last transfusion. although the sensitivity of detection of hepatitis b virus (hbv) in blood donation improved during recent years, transfusion transmitted hbv infection is left as a serious problem. the japanese society of transfusion medicine and cellular therapy (jstmct) conducts the nationwide questionnaire survey about the clinical blood transfusion activities, supported by ministry of health, labour, and welfare, every year. in this survey, we can collect detailed characteristics of patients, who showed a positive result of post-transfusion hbv-marker-test. aims: the aim of this study is to elucidate the cause of hbv positive in transfused patients. materials and methods: data concerning to transfused patients with positive hbvmarker were collected using results of the nationwide questionnaire survey in 2007-2011. in this questionnaire, if there is a patient showing a positive result of hbsag and/or hbvdna evaluated by post-transfusion-test, it is requested that detailed patient's characteristics, including a total amount of transfusion, disease (hematologic or non-hematologic), therapeutic methods (surgery, use of anticancer drugs, use of immunosuppressive drugs, use of molecular target drugs, blood stem cell transplantation), and results of hbv-marker-test prior to the first transfusion, should report to the jstmct office. a number of hbsag and/or hbvdna positive patients were 234 cases in 2007-2011. among them, 19 patients were not eligible because of the absence of results of hbv-related markers. finally, a total number of 215 patients (36, 37, 41, 43, 45 patients in 2007, 2008, 2009, 2010, 2011, respectively) were enrolled for the present study. results: all the eligible patients showed positive results of hbsag and/or hbvdna in samples obtained from three months after the last transfusion. we classified the cause of these results into five categories according to results of hbv-markers tests prior to the first transfusion. (i) if a result of hbsag and/or hbvdna performed prior to transfusion is positive, a patient is categorized as the hbv carrier group. (ii) a patient showing both a negative result of hbsag and positive results of anti-hbs and/or anti-hbc are categorized as the hbv past-infection group. (iii) if a patient shows no hbv-related marker prior to the first transfusion and patient's hbvdna is identical to donor's hbvdna, we categorized as the transfusion transmitted infections (tti) group. (iv) if a patient does not show any hbv-related marker prior to the first transfusion and hbvdna is not detected in donor's blood by single nat, we categorized as the unknown cause group. results were summarized in table 1 . conclusion: although the positive result of hbsag and/or hbvdna of transfused patients has been considered the sequel of blood transfusion, we showed that hbv reactivation was also an important cause of it. to distinguish hbv reactivation from transfusion transmitted infection, we have to perform hbv-marker-test prior to the first transfusion, or we should freeze pre-transfusion patient's serum. plenary session: it's all about red cells 2b-pl1 the english dictionary provides several definitions for the word 'myth'. one definition is, 'a widely held but false belief'. this seems suitable for the purposes of this presentation. but who decides what is false? as we shall see in the course of this presentation, some 'myths' of blood groups may not be myths at all, and some established facts may indeed be myths. perhaps a more scientific definition would be, 'a widely held but unproven belief'. abo, the original and most important blood group in transfusion and transplantation medicine, has engendered many 'myths'. these have mostly arisen through associations between abo groups and other characteristics such as personality, dis-ease, psychological traits, and ideal diet. although many may indeed be myths, or even fabrications advanced for political reasons or financial gain, some are not. for example, the statistical associations between abo type and thrombosis, where a biochemical basis involving clearance of von willebrand factor from the blood by enzymatic cleavage appears to be affected by abh glycosylation. in rh, the first myth was that the human antibody, now called anti-d, was the same as the antibodies made by immunising rabbits with rhesus monkey red cells. hence the name rhesus, now rh, for the blood group system. in the 1940s, early serological work with rh antibodies led to two genetic theories, involving either one or three rh genes. both theories have now been rejected because molecular genetics revealed two rh genes. but was the three-gene theory really wrong, when the boundaries of genes were not understood at the time? since the 1960s it has been commonly understood that there are two types of variant d antigens: weak d and partial d. policies for transfusing patients with these variants have often been based on this dichotomy. but is this a myth? the difficulty we have in defining the terms weak d and partial d suggests that it might be. it is always tempting to dismiss anything that we don't agree with as a myth. as wiener, one of the discoverers of the 'rhesus' antigen, wrote several papers on 'blood group mythology', doing just that. scientists should beware of falling into this trap. perhaps 'myth' is a term best avoided in science. five new blood groups -what next? the past 2 years has seen the discovery of five new blood groups. at the isbt meeting in 2012, fors, jr and lan were ratified as blood group systems and since then, the molecular basis of the vel blood group antigen has been elucidated, and the complement regulator protein, cd59 has been shown itself to be a blood group antigen. these last two discoveries will no doubt lead to their elevation to blood group systems. how has this happened? it turns out to be a mixture of old and new techniques. rapid advances in molecular biology and in our understanding of the human genome have opened new fields of discovery within human blood groups. the development of comprehensive snp arrays, exome sequencing and rapid sequencing techniques, e.g. next-generation sequencing, has provide us with tools for rapid discovery. combining these with sophisticated algorithms for database mining has resulted in the identification of the molecular bases behind the hitherto unresolved, clinically relevant blood group antigens jr a and vel. however classic biochemistry and subsequent peptide and dnasequencing still play an important role and lie behind the (simultaneous) discoveries of jr a and vel, but also of lan and fors. a rare cd59-deficient patient produced an alloantibody to a high-prevalence antigen that was shown to be targeted at the cd59 protein, which was confirmed by routine dna-sequencing. the jr a and lan antigens were assigned to already well-investigated abc-transporter proteins (abcg2 and abcb6 respectively) whose presence on the red blood cell (rbc) had not been established previously and for which the function on rbcs is still not known. fors antigen was shown to result from the reactivation of the human pseudogene gbgt1. this enzyme builds the carbohydrate forssman antigen on sheep and dog rbcs but is normally inactive in humans. a mutation that reactivated the enzyme explained the unusual a pae phenotype in two families. vel was shown to be carried on a hitherto unknown protein on the rbc, smim1. the function of the protein remains unknown although the protein is highly conserved across all species, which is both intriguing and hints at a fundamental function. absence of cd59 whose function in complement regulation is well-understood, resulted in production of an alloanti-cd59, demonstrating immunogenicity of this protein for the first time. these simultaneous discoveries have shown that regardless of the route of identification, assignation of orphan antigens to their blood group 'home' continues at a rate unparalleled since the 1990s. as the '-omics' fields identify new erythroid genes and proteins, and next generation sequencing permits rapid genome sequencing of individuals, we can anticipate that many more currently unassigned antigens will find their genetic and molecular home. 2c-s04-01 teo d blood services group, health sciences authority, singapore, singapore an emerging infectious disease (eid) is defined as one that has appeared in a population for the first time, or that may have existed previously but is rapidly increasing in incidence or geographic range. in 1992, an institute of medicine report predicted continued emergence and re-emergence of microbial pathogens, facilitated by changes in human populations, environment and infectious agents. east and southeast asia, with 30% of global population, has a reputation as a hot spot for eid. within the region, the forces of rapid social, economic and environmental change have resulted in factors such as urbanization, deforestation, agricultural intensification, rapid population growth and mobility which contribute to the increased exposure and efficient transmission of new pathogens. the emergence of severe acute respiratory syndrome (sars) exactly 10 years ago has dramatically transformed individual and national awareness and capabilities for identifying and responding to regional eid threats. the re-emergence of highly pathogenic avian influenza a(h5n1) virus in 2004, isolation of novel bat-associated reoviruses in 2006, emergence of artemisinin-resistant malaria and discovery of a tick-borne bunyavirus associated with fever and thrombocytopenia in 2009 are some examples of eid emerging within the region since sars. at the time of writing, the situation involving human cases infected with avian influenza a(h7n9) virus is evolving, and there has been a recent report of human infection with avian influenza a(h6n1) virus as well. additionally, there are imported eids such as influenza a(h1n1) virus, west nile virus, and the present cause for concern in middle east respiratory syndrome coronavirus. climate changes in recent years have also accelerated the increase in incidence and range of existing diseases such as dengue and chikungunya. 40% of the world's population is now at risk of dengue, with the majority living in the asia-pacific region. the scourge of dengue is sufficiently high in the region for the association of southeast asian nations (asean) in 2011 to designate 15 june as asean dengue day. hepatitis e is another infection that is widespread in some parts of the region, and reports of silent infection in blood donors are cause for further study. the impact of eids on the blood supply may be direct through the potential for transmission through transfusion, the effect on blood donor attendance and eligibility and the effect on blood demand. blood products such as hyper-immune plasma preparations may be useful treatment options in some eids. there is a need for constant surveillance and the capacity to identify, assess and manage eid risks to the blood supply. in recent years, the strengthening of regional and international partnerships and the availability of new diagnostic tools has improved our ability to respond to infectious threats. nonetheless, the volatile and ever-changing nature of eids will remain a constant challenge to the vigilance and response capabilities of the transfusion medicine community. summary: the residual transmission risk is relatively high in hbv followed by hcv and hiv-1. this finding is not new as malaysia is a country of medium seroprevalence for hbv which ranges from 1.5% to 9.8% in the general population, but is relatively low in blood donor population (0.04%). the obi nat yield was higher than acute phase wp in hbv-nat yield. these obi positivity have shown inconsistent nat results on repeat testing and also low viral loads ranging from <12 to 317 iu/ml. in contrast for hiv and hcv nat yields, their viral loads were consistently high (34,220-424,310 cp/ml and 42,780-5,980 ,350 iu/ml respectively). implementation of id-nat is probably the best option to improve blood safety especially for the detection of low viral load such as in obi and sero-negative wp donation in malaysia scenario. blood centers reported to kcdc. repository samples of these donors were tested for anti-hav igm/igg and hav-rna. if any of these test result was positive, the recipients of the blood components generated from these donations were traced. transfusion records of hospital were reviewed to identify recipients of blood suspected to be contaminated with hav. recipients were contacted by telephone. if the recipients agreed participating in the investigation, laboratory test was performed. results: from may 2007 to december 2011, 15 donors notified the blood center of having been diagnosed with hepatitis a. the median interval from donation to diagnosis in donors was 18.2 days (table) . eleven (73%) of these 15 were male donors, and the median age was 28 years (range 19-42 years). the pcr for hav rna was positive in all of 15 depository samples of these donors. none of the repository samples showed positive result of anti-hav igm. a total of 44 blood components (rbc 15 units, pc 14 units and ffp 15 units) were delivered to hospitals. twenty six products were transfused to 26 patients, and the rest 18 blood components (4 rbcs, 1 pc, and 13 ffps) were recalled immediately and discarded. twelve recipients (46%) were already expired. fourteen recipients agreed to participate in the lookback procedure through testing. twelve recipients did not showed viremic for hav, however two recipients showed positive either on the test for anti-hav igm or hav-rna. these two recipients who were 35 and 37 years old developed symptoms on 46 and 48 days after blood transfusion respectively. they were treated for hepatitis a successfully. summary/conclusions: recipients who had anti-hav igg were not infected with hav, even though they were transfused with blood suspected to be contaminated with hav. however ttha cases were those who did not have anti-hav igg and all of them were 30s. in korea, most people under 40 years old are susceptible to hav because of low immunity. there is a risk of transfusion transmitted infection, if recipients received blood contaminated with hav. 2c-s04-04 cable rt 1 , pistorius c 1 , andersson m 2 , maponga t 2 , lopez t 2 , preiser w 2 and tedder r 3 1 hav seroprevalence was highest in the black donors (86%), lower in coloured donors (62%) and lowest white donors (36%). all were hav igm negative. there was an age-related increase from 44.8% (52/116) in those 16-25 years old to 81% (47/58) in those >46 years. although no active hev infection was identified by pcr on pooled samples, 25% of donors were hev igg positive. rates were highest in whites at 33%, followed by coloured at 23% and lowest in black at 19%. again prevalence increased with age from 12.1% in those 16-25 years to 48.3% in those >46 years. discussion: the results show a lower hav seroprevalence in the white population compared to the black and coloured population groups, likely to be due to socioeconomic living conditions such a contrast is striking given the introduction of democracy in south africa almost 20 years ago. the reduction in anti-hbc (as a marker of past hbv infection) with age is reassuring, suggesting that hbv vaccination is impacting hbv population prevalence. the pattern of hev exposure appears to implicate a zoonotic transmission route rather than being related to socio-economic circumstances. given the subclinical nature of hev infection in healthy donors, larger studies are urgently needed to establish the prevalence of active infection in blood donors. background: hbv demonstrates remarkable genetic variability, with eight genotypes and more subgenotypes. in addition, mutations in the polymerase region may lead to drug resistance and changes in the pres region (including deletions and mutations such as t31c and t53c) and prec/bcp region (mutations including a1762t/g1764a, g1896a, t1896a, c1766t, t1768a) are associated with higher risk of hepatocellular carcinoma (hcc). aims: to study the hbv subgenotype distribution and analyze the changes in pres region, prec/bcp and the polymerase region of hbv in chinese blood donors. methods: of 245 blood samples were selected randomly from hbsag positive blood donors from five blood centers in china. the pres plus s region or the whole genome of hbv was amplified and sequenced and hbv subgenotype was determined. the amino acid sequences of the polymerase region were aligned and the mutations related to drug resistance were determined. the nucleotide sequences of pres region and prec/bcp region were aligned and the mutations related to hcc were determined. distribution of genotype, subgenotype, and mutations by different regions were examined using chi-square statistics. results: of 200 samples (81.6%)were subgenotyped successfully. the predominant subgenotypes were b2, c2, d1 and a1 which accounted for 50.5%, 19.2%, 5.3%, and 3.4% respectively. deletions and mutations (t31c and t53c) in pres region were found in 28 (28/200, 14.0%) samples. five of these 28 samples (2.5% of all samples) have deletions and no deletions specific to genotype d was detected. the prevalence of mutations in pres region was significantly higher in genotype c than in genotype b (p < 0.001). mutations in polymerase region were found in 14 samples (7%, 14/ 200), most of which were related to resistance to adefovir and lamivudine. mutations in prec/bcp region were found in 68 samples (29.8%, 68/228). the prevalence of hbeag was significantly lower in samples with mutations in prec/bcp region than that in the samples with no mutation (p = 0.02). more a1762t/g1764a mutations were found in c than b genotype while the opposite was observed for g1896a mutation (p's < 0.01). conclusions: subgenotype b2 was the most frequent strain circulating in hbv infected chinese blood donors, followed by c2. hcc related mutations were found less in pres region but more in prec/bcp region in blood donors. the prevalence of mutations in pres region and a1762t/g1764a mutations was higher in genotype c than in genotype b while the opposite is the case for g1896a mutations. this is consistent with the distribution of hcc related mutations in general hbv carriers in china. since all donors in this study reported not having received hbv treatment, it is not clear whether drug resistance mutations occurred spontaneously in the hbv-infected blood donors or were acquired by the donors from hbv infected patients who underwent antiviral therapy. 2c-s05-01 a fresh look at measuring quality in blood components devine d confidence in the quality of blood components produced for transfusion, particularly with respect to their safety and efficacy, is a necessity for clinicians and patients alike. the assurance of blood product quality is dependent upon the collection of data that can demonstrate products are within specification. however, the linkage between confidence that an individual blood component unit will perform as expected and the conduct of quality testing is imperfect. this begins with the manner in which we conduct these tests. although we describe our practice as 'quality control', it is, in fact, a type of process control testing in which we test a small proportion of our production inventory to ensure that the process was conducted properly. this testing is often conducted at product outdate, long after problem products have been issued and used in hospitals. in addition, the standards used to assess blood components often have 'wiggle room'. for example, the north american standards for the number of platelets in a whole blood-derived platelet concentrate require that at least 75% of tested products meet or exceed the required platelet count. in practice, this means that up to 25% of individual platelet components issued to hospitals may have fewer platelets than the user specification requires. there are other examples in component quality standards of this same phenomenon. in an utopian blood production laboratory, there would be real time quality control measures that would be made prior to release of a unit to the hospital blood bank or transfusion service, and these measures would be highly predictive of product efficacy. as a community, we have considerable work to do to get to this utopian ideal. first we must identify better product characteristics to use as standards for blood component production. modern science, especially in the field of cell biology, has made huge strides since quality standards were first introduced some 50 years ago, yet we have not applied these advances to quality assessment for blood products. those studying blood component quality have begun to collect data sets that will help to inform this change over to new standards. production methods are only one way to impact component quality; another is the actual features of the donors themselves. this biological variation includes not only well known phenomena such as the range of platelet counts in normal healthy humans or the distribution of plasma factor viii or fibrinogen levels, but also appears to extend to storage characteristics of components made from individual donations. this presentation will review the state of the science of product quality and the regulation of blood products, including new information arising from clinical trials, and the application of modern scientific methods such as proteomics and metabolomics to the broad question of blood component quality. background: blood transfusion has been shown to be associated with poorer surgical outcomes such as higher incidence of infection, higher mortality, and increased number of serious adverse events. microparticles (mp) released in packed red cells (pc) in storage have been suggested to be mediators for transfusion-related complications. however, the underlying mechanism for mp release during storage is mostly unresolved. aims: to examine mp released in pc in storage for procoagulant and proinflammatory activity and define the role of residual platelets in pc in generation of mp during storage. methods: (i) leukoreduced (lr) and non-lr (nlr) packed cells (pc) were stored according to blood bank standards and sampled at day 0, 10, 20, 30, and 40. assay of mp was by flow cytometry using mab to label cd235a, cd45, cd41, and cd62e. thrombin generation (tg) and cd11b expression were used as measures of procoagulance and proinflammation, respectively. (ii) the impact of platelets was further evaluated by reconstituting lr pc with increasing concentrations of platelets at day 0. results: (i) multiple species of mp were released in a time-dependent manner. using nlr pc, we found that, relative to day 0, red cell mp (rmp) were increased by 2.59 at day 20, and by 8.59 by day 40. small amounts of mp from leukocytes (lmp), platelets (pmp), and endothelia (emp) were detected, generally <20% as many as rmp. levels of pmp rose rapidly from day 0 and peaked at day 20. lmp began rising at 20 days, increasing to 1.59 at day 30 and 2.49 at day 40. emp changed little over 40 days. (ii) comparing mp in nlr vs lr pc. as expected, pmp and lmp were higher in nlr pc. unexpectedly, however, the rate of rmp production was <50% as fast in lr vs nlr pc. the levels of rmp were found to be significantly associated with residual platelet counts. therefore, we investigated the possible role of platelets in rmp production. (iii) effects of residual platelets on rmp release. when lr pc were incubated with increasing numbers of platelets (0-200,000 per ll), rmp as well as pmp generation increased. rate of increase of rmp was closely associated with platelet count and storage time. this shows that residual platelets catalyze rmp generation. (iv) procoagulant and proinflammatory activities. mp-mediated thrombin generation and cd11b expression increased from day 0 to day 40 in both lr and nlr pc, but in lr pc, it was only 30-50% magnitude of nlr pc. the time course curves did not match any specific mp species. conclusions: procoagulant and pro-inflammatory mp were generated in a timedependent manner. the new finding is that residual platelets markedly augment release of rmp, which is a known indicator for storage lesion. the benefits of leukoreduction may be due to reduction of platelets and mp production in addition to reduction of wbc. reducing platelet count in pc may be beneficial in reducing storage lesion and transfusion related complications. background: it has been reported the soluble cd40 ligand (scd40l, scd154) that was accumulated during platelet storage induce polymorphonuclear leukocyte (pmn) mediated damage of human pulmonary microvascular endothelial cells(hmvecs), was a potential cofactor in developing the transfusion-related acute lung injury (trali). however the amount of scd40l was only slightly elevated in the recipient by transfused blood components as it was fully diluted in the recipient's blood circulation. the mechanism by which cd40l exert its effect is still needs to be elucidated. aim: to determine the effect of platelet derived microparticles (pmp) on pmn mediated hmvecs damage, and its correlation with pmp bounded cd40l. method: the pmps were isolated by centrifugation of the platelet-free plasma from 10 apheresis platelet concentrates (a-plts) at 20,000 g for 1 h. the pmps were counted by flow cytometric analysis, followed by western blotting that was performed on isolated pmps. the scd40l was assayed with elisa. the priming of the formyl-met-leu-phe (fmlp) activated pmn respiratory burst was measured with the hydrogen-peroxide production. a two-insult in vitro model of pmn-mediated hmvecs damage was used to investigate the effect of pmp. result: the pmp priming activity to pmn are correlative with pmp accumulations and their level of scd40l during 5 days storage (correlation was significant at the 0.05 level); pmn respiratory burst are declined by removing pmp with 0.1 lm pvdf membrane filtration or depletion pmp with cd154 monoclonal antibody combining magnetic dynabeads pan mouse igg; the lipopolysaccharides(lps) activated hmvecs were damaged more significantly by pmp isolated from 5-day stored a-plts compared with 1-day stored a-plts (p < 0.05). conclusion: platelet-derived microparticles carry concentrated cd40l signal, promote pmn mediated hmvecs damage, may be relative to developing of trali. background: continuous efforts has spared on improving the quality of platelets harvested from plateletpheresis. little is known about the contribution of donors on the variation of platelet quality, particular the effect of frequent platelet donation on donor's platelet function. aims: aim of this study is to investigate the effect of frequent platelet donation on the state of in vivo platelet activation in platelet donors. material and methods: of 107 whole blood donors and 335 platelet donors with vary donation history from 1 to 74 times (mean at 11.5 ae 12.7) were recruited. they were stratified into three subgroups according to their previous plateletpheresis history (g1: 0-1 time, g2: 2-10 times and g3: >10 times). blood sample were collected from each participant for the determination of plasma levels of soluble p-selectin (sp-selectin), marker of platelet activation and total platelet p-selectin (tp-selectin), as well as platelet count and platelet indices. results: following the increase of donation times, sp-selectin levels were steady increase (g1: 20.50 ae 5.76, g2: 23.21 ae 6.70 and g3: 25.33ae7.67 ng/ml respectly, p = 0.001) and mean platelet volume (mpv) was decrease (g1: 9.29 ae 0.89, g2: 9.17 ae 0.84 and g3: 9.02 ae 0.91 fl respectly, p = 0.039) as estimated by the analysis of covariance adjusted for sex and gender. no significant changes in tp-selectin, platelet count, platelet distribution width (pdw) were observed. further multivariate regression analysis including variables of abo blood groups as well as donation history, sex, age indicated that increased plateletpheresis donations are positively associated with the elevated sp-selectin levels in blood donors (t = 4.16, p < 0.0001). conclusion: our data suggested that frequent plateletpheresis result in the increased level of sp-selectin in platelet donors, implicating a higher state of platelet activation in vivo in frequent platelet donors. the potential effects of frequent plateletpheresis on the quality of platelet harvested and the donor complication are worthy of attention. background/aims: structural and functional changes in erythrocytes occur during ex vivo storage, including the accumulation of bioactive substances in the supernatant of red cell concentrates (rccs). many of the constituents of the supernatant fraction, which are potential mediators of transfusion-related complications, may be reduced by washing of rccs. with emerging paediatric clinical data supporting a beneficial effect of rcc washing prior to transfusion, the aim of the current study was to characterise the effects of rcc age and post-washing storage on erythrocyte structure, function and the accumulation of bioactive substances in paediatric-sized washed rccs. methods: two units of abo/rhd-and age-matched rccs (either 1-or 4-days old; n = 11 each) were pooled and equally split to obtain matched pairs (day 0). one unit was washed with 0.9% saline by repeated centrifugation then resuspended in 100 ml sag-m, while the other remained unwashed. subsequently, both rcc units were divided equally to produce 4 units of paediatric-sized washed or unwashed rccs. all units were stored at 2-8â°c and samples were taken on days 0, 1, 2, 7, and 14 of storage to measure metabolic activity and quality of rccs, as well as the concentration and activity of bioactive substances in the rcc supernatant. the overall effects of washing and storage were compared using repeated measures anova with posthoc paired t-tests as required, with p < 0.05 considered significant. results: the washing process resulted in reductions in red cell count (9.3%), haemoglobin (9.2%) and haematocrit (5.9%) compared to unwashed rccs. overall, washing and subsequent storage of 1-and 4-day old rccs significantly reduced the ph (p < 0.0001), lactate production rate (p < 0.0001), and 2,3-diphosphoglycerate concentration (p = 0.046). although the atp content of the rcc decreased during storage, it was not changed by washing (p = 0.570). haemolysis in the rccs was increased by the washing process, but remained <0.15% on day 14 for all products. extracellular potassium was significantly reduced by washing (p < 0.0001), but increased during storage in both washed and unwashed red cells (p < 0.0001 for both). washing significantly reduced the number of microparticles in the supernatant of both 1-and 4-day-old rcc compared to unwashed rccs (p = 0.01 and 0.012 respectively). however, the microparticle number in the supernatant of all rccs increased during storage. washing of both 1-and 4-day old rcc also markedly reduced the supernatant concentration of monocyte chemoattractant protein-1, scd62p, rantes, anaphylatoxins (c3a, c4a, and c5a) and iga to levels below or near the limit of detection. incubation of cultured human umbilical vein endothelial cells (huvecs) with supernatant from unwashed rcc led to endothelial cell activation, with increased cell-surface expression of e-selectin and vcam (p < 0.0001 for both). however, little or no activation was observed when huvecs were incubated with supernatant from washed rcc. conclusion: although washing affected some aspects of the in vitro quality of rccs, it effectively reduced the concentration and activity of bioactive substances in the supernatant of rccs, leading to reduced endothelial cell activation. such a reduction may be clinically beneficial in selected patient groups. blood services group, health sciences authority, singapore, singapore many of the critical issues associated withbiobanking have been effectively addressed in blood banking. blood transfusion therapy with its emphasison traceability has developed robust systems for inventory and product release. the ethos of proper quality management hasalso been an integral part of blood banking. the lessons learnt have been applied into the biobanking of cord blood, stem cells, tissue and organs. biobanking includes both banking of tissuefor research only as well as public cord blood banks that play a vital rolesupporting clinical stem cell transplantation. the growth of regenerative medicine will only increase the scope, variety and numbers in biobanking. similarly, the discovery of induced pluripotent stem cells (ips) and itspotential myriad applications has highlighted the central role of biobanking inboth diagnostics, research and therapeutics. principles and key considerations in biobanking including biospecimenprocurement, consent, processing, preservation and traceability will beaddressed. introduction: cell therapy generally includes the extraction, processing, manipulation and implantation of characterized cells effectuating specific functions in a patient. however, adjacent fields like tissue banking or tissue engineering should be incorporated. all together have donor selection and validated core procedures in common. production cycles are carried out in gmp clean rooms. furthermore, quality control includes assays which are common in transfusion medicine. it might be tempting to speculate, that cell therapy is closely related with transfusion medicine and requires minor adaptions. the big moat surrounding cell therapy: but there are huge differences: cell therapy is a domain of specialists rooted in patient care with profound knowledge about specific pathologies and how to target them. cell therapy derives from individual clinical needs and rarely is a prefabricated procedure. this is in stark contrast to transfusion medicine, which focuses on standardized products for any patient in need. today, complex regulations for cell therapy surpass those in transfusion medicine. this distracts clinicians in a cell therapy program. this may aspire transfusion medicine specialists to engage in cell therapies. however, only a small niche is left for blood centers, as two major trends arise: one is the more or less 'academic gmp' setting, utilizing the hospital exemption status. the other is the commercial arena, where companies produce standardized cell therapies to achieve maximized market shares. 'academic gmp' entities promote individualizedmainly autologoustherapies, which require a constant flow of financial assets to keep underused clean rooms running. therefore it is serious to ask why and where blood centers should engage in cell therapies. strategy first: transfusion medicine has been heavily influenced by external factors such as viral safety, blood usage, cost pressures and low resources. the term 'transfusion medicine' misleads outsiders to suppose, that it deals with transfusions, nothing else. a wise strategy must include a change in the mind-set of all. this is the most crucial issue as many clinicians are needed to collaborate and co-develop cell therapies. without deeply rooted partnerships it is impossible to establish sustainable cell therapy programs in a blood center. furthermore, a thorough evaluation includes possible products, quantities, as well reimbursement schemes. cell therapy is one of the most expensive treatments and financial assets must be secured first. second: establishing a cell therapy facility: a mock-up facility, without clean room status is highly recommended. processes will be developed, staff is enabled to learn and define procedures, before a cell therapy unit is planned. planning and establishing a cell therapy unit is very complex and specific expertise is scarce. a basic prerequisite is a project manager carrying out final decisions with a profound knowledge about processes interacting with different technologies (hvac, controls, microbiology). cell therapy units fail if project management has flaws and deep involvement of engineering with medical expertise is ignored. a cell therapy unit is an endless, stressful, path riddled with expensive failures, but rewards in the long run a blood center with exciting future prospects integrating grateful clinicians and patients. any kind of accreditation, either by regulatory authorities or any professional entity, like jacie/fact, is an important milestone in the time line of a new cell therapy and a possible showstopper of a long, expensive and enduring project. honest and thorough preparations before accreditation should start before an application may be sent. three phases are distinguishable: keeping the basics on track: running a cell therapy unit is a high wire task. fundamental knowledge about the medical background, processes, quality control, specifications is interwoven with engineering and controlling tasks. it is inevitable for anyone working in this environment to know about air quality, hygiene, staff education and additional technical features. especially controls and the design of engineering and its qualification should be documented continuously. maintenance, re-qualifications, adjustments in the control-system of a clean room facility offer chances to learn the interplay of systems. build a sufficient knowledge base and freeze the process: protocols for cell therapy are often introduced on primary events and work well in first shot experiments. as further patients are included it is very probable, that the whole process and specifications will be modified and sometimes fundamentals and documentation are out of focus. altering and scaling methods, processes and assays may or may not change the product or its intended use. slippage is often not detected and therefore first steps aim at the build-up of as much information as possible. firstly, current literature has to be collected, reanalyzed and mirrored onto the own processes. then the regulatory framework has to be analyzed. the main question is, how the product fits into the regulatory system. is it an atmp or non-manipulated cell product, is it a blood product or a pharmaceutical? pros and cons about alterations should be meticulously discussed and alternative procedures highlighted in this phaseespecially those which are already accredited. it will be very probable that the same inspectors, who have inspected similar cell therapy units, know about alternative procedures and will raise comparative questions. after building up the knowledge base it is advisable to finish a risk assessment focused on the intended use in patients and to revamp the process. after adjusting all methods, processes and assays the whole production has to be frozen. further changes are prohibited and the documentation has to be refreshed. the last test trial: in the last phase before accreditation all aspects of quality management have to be finished. risk assessments should focus on the safety and effectivity of the cell therapy. donor/patient eligibility criteria, quality control of incoming cells and tissues, production processes and their internal quality control criteria as well storage conditions have to be well documented and validated. under certain circumstances a file of clinical studies has to be prepared. all documents and clinical studies should be reviewed, regulatory aspects should be clear. a preparation project gives better chances to pass the last milestone before patients can be treated on a regular cell therapy. 2d-s07-01 burnouf t plasma fractionation is a complex biotechnological process exhibiting unique features compared to downstream technologies used for recombinant proteins, vaccines, and animal-derived antisera. in human plasma fractionation, by contrast to other biological products, multiple end-products (typically 5-10 or more) are obtained from the same manufacturing pool. some of the targeted proteins are present in plasma in large amounts, as is the case for albumin and immunoglobulin, whereas, by contrast, others, such as coagulation factors and anticoagulant proteins are in trace amounts. with the emergence of selective hemotherapy, plasma fractionation has, over the years, turned into a highly integrated protein separation process carefully designed to isolate various proteins under optimized conditions of yield and purity. current plasma fractionation methodologies combine diverse protein purification tools based on 'crude' precipitation techniques and refined chromatographic procedures. some proteins are stable and not prone to degradation, while others, with specialized functional activity, are fragile and prone to enzymatic degradation, activation, or aggregation. contamination of plasma products with harmful residual plasma protein impurities (such as activated coagulation factors or proteases) can lead to serious adverse events in some patients. in addition, while a few plasma products, like albumin and immunoglobulin, can be formulated as liquid preparations, all others products are freeze-dried to ensure long-term stability, which increases cost and technical difficulties. the diversity of protein products made from plasma explains why plasma fractionation facilities have complex design. the manufacturing lines of the various fractions should be strictly segregated from one another. in addition, the risk of viral contamination of plasma pools requires that each product be subjected to several (typically two or more) dedicated viral reduction treatments, the goal being to gradually increase the degree of viral safety along the downstream process. production zones should therefore be physically segregated to limit risks of cross-or downstream contaminations, adding to the complexity of the plant design, flows of product, personnel and wastes, and working procedures. the plasma fractionation industry has a long history from the years 1940's, when cohn and co-workers developed a sequential cold ethanol precipitation process. this method which evolved over the years, remains the core fractionation process, albeit integrated with cryoprecipitation and multiple chromatographic steps. combined with modern viral removal treatments, the current fractionation process ensures therapeutic protein products of established quality and safety, at more or less acceptable yields. the current safety record of plasma products, which contrasts with that of earlier product generations, somehow represent an impediment to the emergence of new fractionation technologies. novel plasma fractionation processes based on integrated chromatographic steps, membrane electrophoresis, aqueous two-phase system, mini-pool fractionation in disposable equipment, are being developed at pilot-scale and represent interesting alternatives. to reach the market, such technologies should be integrated with robust viral reduction steps and proven to achieve at least equal, if not superior, products yield, quality, safety, and productivity to justify the regulatory load, clinical trials, and licensing of what would be regarded by most regulatory agencies as new plasma products requiring full validation. effective specific antiviral agent is generally not available for emerging infectious disease agents such as sars-coronavirus and middle-east-respiratory-syndromecoronavirus. passive immunotherapy with plasma or plasma derived hyperimmune globulins have been used for treatment or prophylaxis against many exotoxin mediated bacterial or viral diseases such as viral hemorrhagic fever and 1918 influenza despite the lack of data from randomized control trial. antigenically shifted influ-enza a virus causes pandemics and antigenically drifted viruses are associated with seasonal outbreaks. poultry to human transmission of avian influenza a h7n9 and h5n1 virus can cause acute community acquired pneumonia with 25 to 50% mortality. risk factors including extremes of age, pregnancy, underlying medical illness and low serum igg2 are associated with severe pneumonia with delayed clearance of viral load and excessive proinflammatory response. though treatment with neuraminidase inhibitors within 48 hours after onset of symptoms should be effective, those with severe disease and respiratory failure usually present later than 5 days after symptom onset. during the 2009 pandemic of h1n1 influenza, we harvested convalescent plasma from a small percentage of recovered adults sufficient for a case control study for treating severe cases during the pandemic in hong kong. plasma supply is constrained by plasmapheresis capacity during most stages of the epidemic. between august 26 to october 31, 2009, a total of 9101 persons were successfully contacted. a total of 1309 screening and 619 whole blood donation appointments were made. in the former 786 (60.0%) attended screening but only 301 could donate plasma by apheresis because of failure to meet blood donation eligibility criteria, failed laboratory tests, insufficient neutralization antibody titers, and inability to make the apheresis appointment. for those who opted for whole blood donation, 379 (61.2%) had attended and donated. 10.5 l (21 units) and 276 l of convalescent plasma with sufficient neutralization antibodies titers was collected for passive immunotherapy as convalescent plasma or h-ivig production respectively. we recruited 93 patients with severe h1n1 2009 infection already put on neuraminidase inhibitors and requiring intensive care. twenty patients (21.5%) received convalescent plasma. mortality in the treatment group was significantly lower than the demographically matched control nontreatment group (20.0% vs 54.8%; p = 0.01). convalescent plasma treatment was associated with significantly lower day 3, 5, and 7 viral load, compared with the control group (p < 0.05) and corresponding lower serum levels of il6, il10, and tnf (p < 0.05). between 2010 and 2011, 35 patients were randomized to receive h-ivig (17 patients) or ivig (18 patients). no adverse event related to treatment was reported. serial respiratory viral load demonstrated that h-ivig treatment was associated with significantly lower day 5 and 7 posttreatment viral load when compared to the control (p = 0.04 and p = 0.02 respectively). the initial serum cytokine level was significantly higher in the h-ivig group but fell to similar level 3 days after treatment. subgroup multivariate analysis of the 22 patients who received treatment within 5 days of symptom onset demonstrated that h-ivig treatment was the only factor which independently reduced mortality [or:0.14, 95% ci, 0.02-0.92; p = 0.04]. background: in recent years, with the global spread of the west nile virus (wnv), dengue virus (denv) and chikungunya virus (chikv) that are transmissible by mosquitoes, concern has arisen regarding their entry to japan. furthermore, chikv as well as wnv and denv are potentially transfusion-transmissible, posing a serious risk for transfusion medicine. of these, wnv and denv belong to the flavivirus genus, as does the japanese encephalitis virus (jev), and they have similar antigenicity. since most japanese people are periodically vaccinated against jev, there is a possibility that anti-jev antibodies cross-react with wnv and denv and induce a protective immune response. however, because wnv and denv have similar antigenicity to jev, there is concern that the anti-jev antibodies are present in japanese plasma against wnv and denv owing to antibody-dependent enhancement (ade) in fccr-expressing cells. furthermore, if the anti-jev antibodies present in japanese plasma have high ade activity, these antibodies may act in an infection-enhancing manner rather than an infection-neutralizing manner against wnv and denv in vivo. aims: using intravenous immunoglobulin (ivig) prepared from pooled plasma from japanese donors, we evaluated its neutralizing activity and ade activity against these viruses for the purpose of estimating the potency of the japanese individuals to protect themselves against these viruses. methods: neutralizing activity (tcid 50 ) and ade activity were compared among three types of ivig, nisseki polyglobin n (made in japan), gammagard (made in germany) and sanglopor (made in the usa). tcid 50 was calculated from the results of cytopathic effect (cpe) assay using vero cells as target cells. ade activity was measured by plaque assay using bhk cells and fccr-expressing bhk cells as target cells. results: nisseki polyglobin n showed a significantly higher neutralizing activity against jev than gammagard and sanglopor. the neutralizing activity of nisseki polyglobin n against wnv was approximately a log reduction factor of 2.3 higher than that of sanglopor. furthermore, the neutralizing activity of nisseki polyglobin n showed approximately the same neutralizing activity as gammagard against wnv. none of the ivig preparations showed significant neutralizing activity against denv or chikv. nisseki polyglobin n showed only marginal ade activity against any of the viruses. conclusion: although the neutralizing activity of plasma from japanese individuals is not known, it is suggested that the japanese population as a whole has a potency to protect themselves from infection by wnv to some extent, probably due to the cross-reaction of anti-jev antibodies to wnv as a result of jev vaccination and natural infection. therefore, if wnv invades japan, a pandemic may not occur and the risk of wnv infection by blood transfusion may be low. it was suggested that plasma from the japanese individuals has almost no neutralizing activity against denv and chikv. nisseki polyglobin n showed only marginal ade activity against wnv and denv, suggesting the low possibility of the anti-jev antibodies present in japanese plasma acting as infection-enhancing agents against wnv and denv as a function of ade. background: platelet-rich-plasma (prp), platelet gel (pg), and platelet lysate (pl), are used in regenerative medicine to stimulate the healing of soft and hard tissues. in addition to their tissue regenerative properties, platelet materials are claimed, mostly through anecdotal observations, to limit post-surgical inflammation and decrease pain. the anti-inflammatory properties are thought to be due to the release of platelet components, including transforming growth factor-b1 (tgf-b1) and hepatocyte growth factor (hgf), which are known to inhibit some inflammatory pathways in vitro. however, there is a large diversity in the type of platelet fractions used in clinics. they differ significantly in protein composition and content of proinflammatory and anti-inflammatory molecules and may therefore not be equally effective in controlling inflammation. one needs to elucidate the factors responsible for the possible anti-inflammatory properties of platelet materials to standardize the preparation and clinical use of these products when an anti-inflammation effect is clinically beneficial. aims: to investigate the potential anti-inflammatory effect of various plasma/ platelet fractions using an established in vitro model of raw 264.7 mice macrophages stimulated by lipopolysaccharide (lps), and studying the production of nitric oxide (no), inducible nitric oxide synthase (inos), and cyclooxygenase-2 (cox-2). methods: apheresis platelet concentrates (pc) were obtained from three donors and separated within 3 days into pc, platelet poor plasma (ppp), platelet gel releasate (pg), frozen-thawed platelet lysate (pl), and solvent-treated platelet lysate (s/d-pl). proteins were determined, sds-page patterns obtained, and growth factors quantified by elisa. raw 264.7 cells were grown in medium supplemented with 10% of fetal bovine serum (fbs) or plasma/platelet fractions, with or without lps (500 ng/ ml added after 24 h of culture). cell growth and cytotoxicity were checked by cell count determination and mtt assay. no was determined in cell culture supernatants by colorimetric assay and inos and cox-2 in cell extracts by western blot. prp from mice and quercetin, a known anti-inflammatory compound, were used as controls. results: pc and s/d-pl had the highest mean tgf-b1 content ranging from approximately 100-200 ng/ml, and ppp the lowest (5-10 ng/ml). cell count analysis and mtt assays showed consistent cell growth and viability in all conditions evaluated but were slightly lower in the presence of lps and quercetin, as expected. there was no no, inos, cox-2 detected in absence of lps stimulation. the plasma and platelet fractions were all found to reduce no production and inos expression, when compared to fbs, after lps stimulation. interestingly, inhibition of no production and inos was generally more pronounced with s/d-pl. cox-2 synthesis was lower in the presence of s/d-pl compared to other plasma/platelet fractions and higher with pg. the mice prp did not exert any stronger anti-inflammatory action in this mice cellular model suggesting absence of species specificity. conclusions: the plasma and platelet fractions evaluated exert, to various degrees, an anti-inflammatory effect mostly revealed by inhibition of no production and inos. impact on cox-2 synthesis is less obvious. the fact that s/d-pl exhibits stronger global anti-inflammatory activity requires further studies. 2d-s08-01 tani y japanese red cross kinki block blood center, ibaraki, japan rare blood is generally defined as one that occurs at a frequency of 1:100~1000 individuals or less, and it is sometimes difficult to provide such blood types to patients because of their rarities. the japanese red cross (jrc) society lists 46 rare blood phenotypes that are divided in two categories as shown in table 1 . the rare blood types listed in category i occur much less frequently than those listed in category ii. we screen for rare blood cells using monoclonal antibodies (moabs). since 1987, our blood center has established 93 moabs (32 human and 61murine), and has provided them to the other blood centers. many of igg moabs are available on the machine such as beckman coulter pk-7300 blood grouping analyzer by saline or bromelin method by cross-linking with anti-human or anti-mouse igg. in addition, we routinely screen for antigen negative-cells (rhc, c, e, e, jk a , jk b , fy b , di a , m, le a , s to which antibodies are believed to be clinically significant). thus, more than 10,000 donors with rare blood phenotypes (category i, 748; category ii, 9314) are registered in japan, but the number of donors with some category i blood types are insufficient. we freeze rare blood, particularly category i types, and domestically and sometimes internationally supply these units of blood. since 1977, a total of 576 units of rare blood with phenotypes di(b-), d-, jr(a-), ko, jk(a-b-), (para-)bombay, p, en(a-), m k m k , lan-and rhnull have been supplied to 23 international countries. thus, the jrc contributes to the international panel of donors of rare blood type (idp) which is maintained by the international blood group reference laboratory (ibgrl) in bristol, uk. the idp provides information on the location of rare blood donors when they cannot be found in their respective countries. the jrc has encountered difficulties in finding rare donors with rhnull, p k , m k m k , en(a-), u-and ge-phenotypes. we also joined the isbt working party on rare donors which handles all matters related to rare blood. our rare blood donor program is successful because of international cooperation. 2d-s08-02 the china national rare blood group screening program started in 2008. the program has screened more than 1,500,000 donors in thirteen regional blood centers by using large-scale serological tests, gene diagnosis, and other different specific screening technique. now, more than 1300 donors with rare blood phenotypes have been found. including rh null, d --, wrb-, lu(a-b-), jk(a-b-), vel-, lan-, coa-, k 0 , dib-, s-as well as yta-. the primary target for the project is to screen the negative blood antigens whose conjugational antigens have high frequency, and the blood groups which is easy to produce antibodies and the negative antigen in the system is very rare in chinese population (for example, fya-, whose frequency was 1/400 in chinese han population). a professional website for rare blood groups (http://www.chinarareblood.cn)was set up and serviced in jan, 2010. the information of blood donors with rare blood types is registered into the national registering system for blood donors with rare blood types by professional technician from organizations join the program. the information includes the specificity of the rare blood types, other common blood groups, personal data of donors, and the information of contacts. except the network administrator, other visitors could only see the specificity of the rare blood type and the number of the rare blood in rare blood bank storage. application for the rare blood products should communicate with contacts through administrator. according to the standard of the pretransfusion test in china, all the donors and recipients must be identified the abo and rh systems and done the cross-match test, in addition, all the donors must pass the test for contagious marks. nowadays, more and more chinese blood centers use nucleic acid technique to detect hiv and hcv. the results of infection test must be added to the information of the blood products at every time for collecting the rare blood. in recent 2 years, 21 units (1 unit = 200 ml) blood products with different rare blood types have been used in clinical treatment. background: anti-emm is a rare specificity detecting the high-prevalence red blood cell (rbc) antigen emm (isbt 901008). five cases were reported by daniels et al, (transfusion, 1987; 27:319) and two by reid et al, (transfusion 1998; 38 (suppl) :101s). six of these were in untransfused males and anti-emm had not been implicated in a transfusion reaction, most likely because the patients were not transfused. case study: a 58-year-old, untransfused, japanese man with group a, d+, datnegative rbcs, urgently needed transfusion due to an abdominal stab wound. pretransfusion testing using gel column agglutination and peg-iat, demonstrated an antibody reactive with all panel cells, but non-reactive with autologous rbcs; anti-le a was detected by papain methods using gel column. unavoidably, one bag of crossmatch-incompatible le(a-) rbcs had to be transfused. thirty minutes after transfusion, he experienced a drop in blood pressure and hematuria. however, as his hb level was 5.5 g/dl, another two rbc bags were transfused, and his vital signs became stable. on day 3, he was transfused one rbc bag without a hemolytic transfusion reaction. on day 6, after receiving 30ml of rbcs, the patient vomited and had cola-colored urine (t-bil 6.1 mg/dl, ldh 912 u/l) and the transfusion was stopped. following transfusion, his rbcs reacted in the dat: 1+ on day 1, 2+ on day 3 and negative on day 7. thereafter his anemia improved gradually by iron medication and he was discharged on day 24. aims: to identify the antibody in the patient's plasma that caused the transfusion reaction. methods: serological testing was performed by standard methods. result: the patient's plasma reacted in saline at 4c (2+), by albumin iat (2+), peg-iat (2+), and papain-iat (3+) with all panel cells, and by peg-iat with 30 samples lacking high-prevalence antigens; the autologous rbcs were non-reactive. testing his plasma against phenotypically-similar enzyme or chemically treated rbcs showed that the antigen detected was resistant to bromelin, papain, ficin, trypsin, a-chymotrypsin, pronase, dtt, aet, and acid. two examples of emm-rbcs were non-reactive and the antibody was identified as anti-emm. the reactivity of enzyme and chemically treated rbcs is consistent with anti-emm. his rbcs reacted with antibodies to 17 high-prevalence antigens, but could not be confirmed as emmbecause anti-emm was not available. the anti-emm was igg1 and igg3 with a titer of 16 by peg-iat before transfusion rising to 128 on day 10; his serum demonstrated hemolysis after day 6. no other underlying antibodies were detected in the patient's plasma alloadsorbed to remove the anti-emm. conclusions: we report the first japanese case of anti-emm and the first to cause an acute hemolytic transfusion reaction (ahtr). in previous reports, six people with anti-emm were untransfused men and one was in a woman whose transfusion history was unknown, suggesting that the antibodies may be 'naturally-occurring'. the anti-emm reported here, also in an untransfused man, also may be considered a 'naturally-occurring' antibody. similar to the first reported example of anti-emm, the antibody reacted, albeit weakly, at 4c. our case suggests that anti-emm is clinically-significant as the patient experienced an ahtr. 2d-s09-01 background: to recruit blood donation volunteers and provide stable blood supply according to demand, it is more important to change the overall social perception than to carry out one-time event or short-term campaign. the social understanding of blood donation is formed as valuable and honorable service over certain level in korean society. nevertheless, there still are many people who don't even try to participate in blood donation because of fear, health concern, and expectation for reward. to change this culture and social awareness, it is important for the present and future blood donors to have a perception that the blood donation is the sharing one's life and a genuine service which helps other people for nothing. aim: the main purpose of this article is to introduce korean red cross' recent efforts (operation of the red campaigner and blood donation supporters, the construction of virtual blood donation experience center, the blood donation promotion program) to change the blood donation culture and widen the donor base in korea and to present their effect and improvement direction. (1) this article is comprised of the case study and analysis on the operation of red campaigner and blood donation supporters, the construction of virtual blood donation experience center and blood donation promotion program (2) this article outlines the red campaigner and the blood donation supporters and the related programs and addresses their significance in addition, describes the effect and direction for improvement, presenting research related data. (3) this article outlines virtual blood donation experience center construction and presents the exhibition in it and it suggests the effect and possibility on the authority of the research case that the education with fun has more considerable impact than learning by rote. result: to change current culture and increase positive understanding about blood donation, korean red cross is making various efforts. the red campaigner, consisting of high school students and the blood donation supporters, consisting of college students, aim to influence the youth, the potential blood donors, to have a positive understanding of blood donation and to carry out continuous and organized publicity of its importance and safety. in addition, korean red cross is making a progress in the construction of the virtual blood donation experience center which is going to be completed by the end of 2013. we expect that we can achieve the educational purpose that sends a message that the blood donation is a volunteer work to save life and make future donors to recognize the blood donation as an object of not fear, but interest. finally, 'the blood donation promotion program' which began in 2009 is designed to encourage for general groups or organizations to participate in the blood donation campaign and to create the voluntary blood donation culture. conclusion: various projects operated by korean red cross contribute to widen blood donor based by changing blood donation culture in korea and are expected to make continuous contribution. but these projects have a limitation that the partici-pant is restricted and continuous participation isn't progressing in terms of national participation. continuous and positive endeavor to foster these projects as a national campaign should be encouraged. although it is possible to increase the blood donor temporarily through one-time event when blood shortage recurs, widening the donor base by changing blood donation culture should be the fundamental solution. the changing blood donation culture and donor understanding may not be optimistic for a short-term blood shortage problem but will be useful to make conditions that expand the donor base and increase voluntary donors in the medium to longer term. understanding our future donors is of critical importance to blood collection agencies worldwide. not only do we need to know who they are, but also why they do or do not engage in the required behaviour of blood (product) donation. the surge in psychological research into blood donation in the last decade has provided significant insight into understanding the psychological factors underpinning the commencement and continuation of blood donation. this review will summarise the state of our current understanding of knowing how to effectively recruit the non-donor and the complexities that lie ahead for all involved. at the descriptive level, and stemming from the systematic application of various psychological theories and models within blood donation contexts, we now know more than ever about the key factors that non-donors report impact on their blood donation intentions and behaviour. from this, certain psychological elements such as perceived control or self-efficacythe individuals' self perception that they can cope with donationhave emerged as key determinants of donor recruitment. drawing on these results, research psychologists have worked collaboratively with blood collection agencies to develop and evaluate recruitment materials designed to target these central constructs. both laboratory and in field trials have been undertaken which have consistently shown positive effects. for example, participants receiving these materials are more likely to volunteer to donate blood than those receiving standard recruitment materials. the collaboration of researchers and blood collection agencies has furthered understanding and recruitment efficacy generating measurable, operational deliverables. however, this collaborative research has also served to highlight the challenges that lie ahead both in terms of the diversity of our non-donor population as well as the limitations of our current theoretical models. further, there is an increasing need for large scale randomized controlled field trials to systematically evaluate interventions designed on the basis of psychological research. while these needs may provide substantial challenges for researchers and blood collection agencies alike, the promise of psychology in providing the 'who' and 'how' to effectively recruit remains. background: developed countries rely solely on voluntary non-remunerated donors to ensure adequate blood supply but ageing has brought in significant pressure on the health care system including blood supply. in hong kong, blood demand has recorded a 17% increase in blood demand from year 2008 to 2012 with almost 60% blood being transfused to patients aged >60. therefore, sourcing for more blood to meet demand is one of the most urgent issues in blood service. in this report, we report how we successfully modeled donation preference into the development of a new collection site to leverage the blood demand. material and methods: demographic information of all donors with successful donations was retrieved from blood bank computer system for the years 2004-2007. statistical analyses were performed to determine how frequent they came back to donate, whether residential location affected their donation frequencies and lastly identify potential district in hong kong to build a new donor center against the donor and general population distribution. results: of 775,690 donations made by 191,302 male and 201,446 female donors were analyzed. on average 75.8% of donors donated only once during the calendar year but donors were more likely to make repeated donations at donor centers than mobile venues. significantly more donors would come back for second or more donations at donor center (36.1% vs 20.1%, p < 0.05). male were more likely to come back for repeated donations than female (43.3% vs 30.9%, p < 0.05). a total of 466,121 donations made at donor centers were further studied. upon matching with their residential address, distance from donor centers was shown to be a determining factor on their choice of donation through linear regression analysis. reduced donation frequencies were observed with increasing distance from donor centers. regression analysis then identified several districts where many donors had to travel a long distance to the nearest donor center. the district with the highest expected increase in donations, adjusted by the expected growth rate, was then chosen as the site for building the new donor center. a fixed donor center was so selected at yuen long district and open to donors by 29 july 2011. by the end of year 2011, 6160 donations (consisting of 3047 males and 3113 females) were made with more than 88.1% collection given by donors residing at yuen long and the nearby district, tuen mun. when the whole year figure was reviewed in 2012, 16,990 donations were collected which already exceeded the planned collection target of 15,000 by year 3. interestingly, same 88.1% donations were given by donors residing at these two districts. conclusion: despite being a small city, the retrospective analysis of donation behavior has provided valuable information in the service planning in hong kong. the new donor center was able to reach the planned third-year collection target by 15 months earlier. further work is being done by using the more recent data to identify the next optimal collection sites for future expansion under most best cost effective way. singapore red cross, singapore, singapore background: singapore is moving towards providing more fixed blood donation sites with the aim of enhancing donation experience and encouraging repeat donations. it was recognized that the choice of location and an understanding of the human traffic in the vicinity are elements of success. a 2-prong approach was undertaken to: collect information on the footfalls, the social profile and demographics in the designated location. collect information from potential donors on their preferences and sentiments in relation to operating hours and outreach channels for marketing communications. method: a month-long study was conducted in the vicinity by observing and counting the human traffic, the crowd density at various exit/entry points at various timings, the flow and direction of the general foot traffic, overall make up of the surrounding district such as types of corporate, civic & religious organizations operating in the vicinity and number of educational institutions. in addition, an on-site survey to determine potential donor preferences and sentiments in relation to operating hours, tactical outreach and design mechanism of the blood centre was also conducted to help develop a tactical marketing communications strategy. the results indicated that during week days, about 85% of people visiting that vicinity were youths aged 16 to 25 (50.5%) and young adults aged 26 to 39 (33.4%). the main purposes of the visit were for work related activities (22%), attending school (19.1%) and shopping (38.1%). on weekends, 76% of these age groups visited the vicinity for leisure activities or to church. another 24% who visited there were adults age 40-60 years old. most of the respondents had a specific destinations, and most of them indicated lunch time and after office hours as their preferred donation periods. a tactical marketing communications strategy targeting youth and young adults was developed to meet the behavior and preferences of the target group. social media platforms such as online mobile app advertising and locational media buys were employed. in addition, partnerships were developed with nearby educational institutions and churches to host road shows and blood donation awareness activities to engage youths and to foster fun and excitement in the social media atmosphere for the blood collection center. the strategies undertaken proofed favorable as the daily attendance at the new blood collection center has surpassed its baseline target collection of 50 units of blood a day within the 3 months of its operation (jan 2013); and now, has a daily average collection of 60 units of blood. 3a-s10-01 setting up haemovigilance programme from the very first step background: national haemovigilance programmes wherever established have yielded significant data to implement blood safety initiatives. settting up a national haemovigilance programme requires meticulous planning and the following issues need to be addressed; whether reporting will be voluntary or mandatory, what is to be reported and by whom, reporting formats and data submission, resources to sustain the programme, staff training and responsibilities. india is a country of 1.18 billion people, 2700 blood centres, one-third each in public, charitable and private healthcare sectors and annual blood collection of 9 million. given the diversity of blood centre management, setting up such a national programme is a complex task. aim: to set up a national haemovigilance programme in india. method: the ministry of health and family welfare (mohfw), govt. of india had launched the pharmacovigilance programme of india (pvpi) in july 2010, with oversight by the indian pharmacopoeia commission (ipc). adverse drug reaction (adr) monitoring centres were setup in 90 medical institutions in the country and trained staff was recruited, for data collection and submission. after the successful launch of pvpi, the mohfw, initiated the haemovigilance programme, distinct from pvpi with the co-ordinating centre at national institute of biologicals (nib), but in close collaboration with ipc. the broad organizational structure of the programme is as follows; reports will be generated in the medical institution based blood centressubmitted online to the haemovigilance national co-ordinating centre at nibreports will be reviewed by the national advisory committee which will make recommendations to the national co-ordinating centre, ipc for onward transmission to the regulatory authoritythe central drugs standards control organisation to formulate safety related regulatory changes and communicate to blood centres. the programme was formally launched in december 2012. terms of reference of the national advisory committee are: to finalize transfusion reaction reporting form (trrf) for the country. to give expert opinion for collection, collation and analysis of data and development of software for the same. to monitor the quality of data collected. to develop training modules and guidelines for implementation of haemovigilance programme. results: based on recommendations of the committee, the initial focus is on reporting serious adverse reactions as defined by the working party of the international society of blood transfusion, reporting is voluntary and a guidance document and trrf have been made available to the medical institution blood centres, which are the designated reporting centres. the reporting is online through a software -haemo-vigil accessible on the nib website. each centre has been given a unique username and password for login. the security of data submitted through the software has been validated. reporting commenced from february 2013 and till date 434 reports of adverse reactions have been submitted. the data is yet to be analysed. a series of awareness workshops are planned countrywide, five have already been organized. specific funds have been allocated by the mohfw for this programme. conclusion: a well structured programme of haemovigilance has been initiated in india and is now in a phase of development. 3a-s10-02 background: congenital haptoglobin (hp) deficiency being homozygous for a deleted allele of the hp gene, hpdel, was identified in a japanese pregnant woman who had experienced severe anaphylactic transfusion reactions (trs) after infusion of red blood cells (rbcs) and human serum albumin in 1998. in addition, the allelic frequency of hpdel was calculated to be 0.015 by a genetic study of a limited number of the japanese individuals, suggesting that hp deficiency might distribute among the japanese population as a phenotype of serum hp. aims: in this report, we present the results obtained from a hemovigilance survey carried out between 1998 and 2012, in which hp deficiency was identified among japanese patients who had experienced nonhemolytic trs (nhtrs), and those obtained from a screening of hp-deficient japanese healthy blood donors. materials and methods: patients with nhtrs: a total of 19,675 patients who had experienced nhtrs, reported by hospitals to the japanese red cross society between january 1998 and december 2012, were examined. healthy blood donors: a total of 272,068 blood donors who visited the japanese red cross blood centers in the tokyo area between june and august 2010 were examined. testing for identification of hp deficiency: (i) serum hp concentration was determined using peak-rate nephelometry with the detection limit of 6 mg/dl followed by simplified sandwich elisa with the detection limit of 3 lg/dl. individuals who showed a negative result of elisa were assessed as hp-deficient. (ii) the presence of the hpdel allele was analyzed using an allele-specific pcr method. testing for anti-hp antibody: the anti-hp antibody produced in all the hp-deficient individuals was measured by elisa and western blot analysis. it was further analyzed using ouchterlony or surface plasmon resonance technology in some cases. results: thirty-one individuals were identified as hp-deficient among the 19,675 patients who had experienced nhtrs (0.16%). all the patients, except three who could not be tested, were homozygous for the hpdel allele. they were transfused blood products [pc, 17; ffp, 1; rbcs, 9; mixed, 4] . nineteen of them (61%) experienced anaphylactic trs accompanied by severe hypotension and the other patients (39%) experienced milder nhtrs. all the patients except one had a history of transfusion. the anti-hp igg antibody was detected in 28 patients (90%). in addition to the igg antibody, the anti-hp ige antibody was detected in 20 patients (64%). hpdeficient individuals were identified among japanese blood donors with a prevalence of 105/272,068 (0.039%). all the donors were homozygous for hpdel, except one who was heterozygous for hpdel, hpdel/hp2. the anti-hp antibody was not detected in 104/105 (99%) of the hp-deficient donors. conclusions: the higher prevalence of hp deficiency caused by hpdel -homozygosity producing the anti-hp antibody among the patients with nhtrs than in the normal donors (p < 0.001), suggests a higher risk of such trs in hp-deficient patients. hp-deficient individuals are present among normal healthy donors with a prevalence that is expected from its allele frequency. they might be expected as suitable donors for hp-deficient patients to prevent hp-related anaphylaxis. hlaing aa background and objective: collection of the correct blood sample from the correct patient is a vital step in the process of safe blood transfusion. transfusion laboramethods: a study on all reports of mislabeled and miscollected specimens from january 2010 to december 2012 was undertaken and the results were analyzed. mislabeled samples were defined as samples that were incorrectly labeled at the time of collection and miscollected samples were 'wrong blood in tube' samples due to patient misidentification. errors resulting in discrepancies in blood group between the current blood sample and historical records were identified by program flagging during validation of blood group results. these discrepancies were resolved by requesting a second sample from the patient collected by another person. some errors detected at the ward level, were reported by the staff member who had sent the blood sample. workload data for group and screen samples received during 2010-2012 was collected from the annual transfusion laboratory records. using these data, ratio of errors from mislabeled and miscollected samples, to number of group and screen samples received was calculated. results: between january 2010 and december 2012 a total of 162,999 samples were received for abo grouping. of 77 incidents were recorded relating to errors in either sampling or labeling. the overall sample error rate was 0.047% or 1 in 2116 samples. of 52 cases resulted from wrong labeling during collection, 17 cases were due to patient misidentification, five were errors that had occurred during the initial request, in two incidents the cause could not be identified and one labeling error occurred in the laboratory. all errors in labeling resulted from failure to check the pre printed name label with the label on the patient's identity band. in 14 incidents, labeling was performed away from the bedside, in 11 cases name labels of a different patient were found in the correct patient's medical file, in 25 cases labels were taken from wrong patient's file and two errors were due to using prelabeled tubes. of 17 patients had been misidentified and blood taken from the wrong person. root cause for these errors was not following hospital polices in patient identification and sample collection. sixty-four percent of the errors occurred out of normal working hours, mainly during the night while the rest 36% had occurred during normal hours. conclusion: we conclude that mislabeling and miscollected sample errors represent a potential for mistransfusion in our institution. the rates of mislabeled and miscollected samples can be used as key performance indicators in this important step in the clinical transfusion process. this baseline data will be used in formulating standards of performance for sample collection and patient identification and, for implementing risk -reducing strategies. background: haemovigilance is a surveillance programme dedicated for the practice of blood transfusion. it is an important part of the quality system of the blood programme. haemovigilance programme in malaysia was initiated as a national programme in 2003 under the ministry of health (moh). since its inception in 2003 the programme has evolved and has become an integral part of our transfusion service. all adverse events and near misses were reported to the national coordinating haemovigilance unit at the national blood centre (nbc) using a standardised form and predefined criteria. these were compiled and analysed into an annual report for the national background: the process of blood transfusion from blood collection and processing to issue and bedside transfusion of blood components involves several areas of human participation. human error is therefore inevitable in this chain of events. transfusion laboratories have long focused their attention on quality control methods and quality assessment programmes dealing with analytical aspects of blood testing. however, there is enough evidence to suggest that the steps most prone to error are in fact in the pre and post analytical phases. various international accreditation bodies now require clear and effective incident reporting protocols to enhance measures for error trapping and error avoidance. aims: this study aims to quantify and characterize transfusion errors in a joint commission international (jci) accredited tertiary care centre in india. methods: all reports of transfusion related errors, registered in the blood bank or outside, between january 2008 till december 2012 were reviewed and categorised into pre-analytical, analytical and post-analytical events. the process adopted at our centre for assessing transfusion related events at the patient's side uses widely tested criteria of: (1) incident reporting (2) root cause analysis (3) identification of corrective actions results: during the study period 89,156 requests for blood and blood components were received and total of 1,98,505 blood components were issued within the hospital. a total of 16,834 reported errors were analysed. pre-analytical errors comprised a large majority (13,676; 81.24%), most of them being errors of inadequate patient information on request forms (10976; 65.2%) followed by sampling errors (1756; 10.4%). analytical errors comprised (563; 3.3%) and post analytical errors accounted for (2595; 15.4%) of the total errors reported. there was no incidence of acute haemolytic transfusion reaction or direct patient harm during the study period but on several occasions near miss events were reported which, if missed could have background: in australia, we rely on non-remunerated, voluntary donors to provide sufficient blood to meet patients' needs. for fresh components, the australian red cross blood service (blood service) is unable to import components for routine use, so is 100% self-sufficient. hospitals and pathology laboratories are under increasing pressure from government/s to improve value for money for blood and blood products, which is resulting in extra demands being placed on the blood service, especially in relation to lower age at issue and a continuing trend to hold more group o stock and less of the other groups, especially ab. with a typically seasonal inventory pattern for red cells, the blood service has focussed on closer management of blood inventory. aim: the aim of the inventory program was to improve reliability of blood coming into the supply chain and therefore improved reliability in delivery to customers. this is measured by average and variance in the number of whole blood collection packs being receipted at the processing centre. the aim was to reduce the variability in this metric, which would naturally lead to decreased inventory holding requirements, greater control and efficiency, and increased reliability and service to the customers. order fulfilment is another measure used to demonstrate improvement. methods: in order to manage blood inventory effectively, the first step was to introduce a minimum and maximum inventory level, by blood type, by state. this provided a transparent target to aim for. the bands were calculated by firstly setting a minimum inventory level using traditional supply chain safety stock calculations. the next step was to develop a 12 week inventory forecast, using a number of planning assumptions. one of the core assumptions is the number of appointments booked in the lead up to a donation. in order to improve reliability, minimum tar-gets were agreed at 3 months out (re-booking time) through to one week out. a 'traffic light' style appointments portal was developed to provide improved visibility of appointment levels for each collection mode and by state. results: results show that the quarterly standard deviation of blood coming in to inventory has improved from 1711 to 1285 in the last four financial yearsa 25% reduction. in addition, order fulfilment has improved from 82% to 95%, demonstrating that, with improved planning systems and processes, it is possible to manage inventory effectively. the results are demonstrated in the two graphs attached. summary/conclusion: the blood service in australia set a goal to improve reliability of fresh components, in particular, red cells entering finished goods inventory, to improve order fulfilment and provide service excellence to customers. by implementing robust and disciplined planning and reporting systems, reliability has improved which shows that there are methods available to improve the effectiveness of inventory management for blood components. 3a-s11-02 wooi-seong k one of the fundamentals of a blood collection center that procures, processes, stores and supplies blood and blood components to hospitals or other blood banks is an effective and sound management of blood inventory. as blood supply is dynamic, blood supply management requires continuous monitoring and interfacing between blood procurement and inventory management and with hospitals. in an effort to provide adequate, safe and equitable blood supply from voluntary non-remunerated blood donors in the face of increasing demand and decreasing donor population, blood collection centers are also challenged with blood shortages, which unless managed, could impact the healthcare delivery and negatively affect patient care. in order to provide a consistent and reliable blood supply blood centres will have to resort to creative and innovative measure. malaysia, a unique multicultural and multiethnic country celebrates significant religious and historic events as well as commemorations. as such, malaysia observes numerous national and state holidays. in fact, malaysia is ranked as the seventh country in the world in the number of observed holidays. by virtue of its geographical location, malaysia is not exempt from natural and man-made disasters, the most severe being seasonal monsoon floods and flash floods. these and the poor response to blood donation campaigns as a result of 'balik kampung' phenomenon during major holidays due to mass exodus of malaysians to their hometowns, contribute to acute seasonal blood shortages in blood collection centers around the country as well as within the region. adopting a proactive approach to blood shortages includes embracing new measures to recruit and retain blood donors, establishing a blood forecast system, developing a strong network among blood collection centers, being transparent with the blood inventory levels which will lead to greater trust and increased confidence in bts and having a contingency plan. at national blood centre (nbc), the blood action team was formed in 2010. it is a multidisciplinary team comprising of members from the donor education and publicity, donor recruitment, blood procurement, component and processing and inventory divisions as well as medical officers, transfusion medicine specialists and consultant. meetings are held regularly and this has greatly improved the communication interdepartmentally, and has fostered a team whose members are committed to improving blood supply management and preventing blood shortages through discussion and brainstorming sessions. also, blood forecasting is carried out as far ahead as months in advance. the blood stock forecasts are also communicated to blood banks from public and private hospitals which are supplied by nbc, a measure to increase transparency. since the implementation of these measures, nbc has successfully and effectively overcome the annual seasonal blood shortages for the last 3 years. evidently, blood shortages are largely preventable by adopting a proactive approach. 3a-s11-03 . the c/t ratios were calculated and analyzed for each major department. nbc and hkl had continuously introduced several interventions to reduce c/t ratios during the period of this study. results: the overall c/t ratio for hkl had been reduced from 2.2 in year 2005 to 1.9 in year 2012. the four major departments in hkl that showed high reductions in c/t ratio for the same period were obstetrics and gynecology (6.4 reduced to 2.5), surgical (2.6 reduced to 2.1), orthopedic (2.6 reduced to 2.1) and neurology (3.8 reduced to 2.2). in this study, interventions that had contributed to the drastic reduction in c/t ratio were compliance to the maximum surgical blood order schedule (msbos) which was periodically updated within each department, effective communication between clinician and blood bank staff, and continuous medical education (cme) for house officers and clinicians. the active hospital transfusion committee (htc) and hospital transfusion team (htt) also played an important role in reducing the c/t ratio by creating awareness among the paramedics and medical officers regarding the judicious use of blood and blood products, and regular monitoring and audits of the whole transfusion process starting from blood sampling to monitoring of patients during and after transfusion. summary/conclusions: this study showed that several interventions that have been introduced by hkl and nbc such as continuous medical education, compliance with updated msbos, active role of htc and htt, and effective communication between clinician and blood bank staff have successfully reduced c/t ratio in four major departments in hkl. this successful achievement needs continuous monitoring and evaluation to ensure consistency. this can also be a role model that is shared with other hospitals to ensure that the c/t ratio is within the set target. 3a-s11-04 fusion) were collected. during second step, a modeling and simulation were used to define the optimal rcl inventory for the metropolitan area. average rc cell shelflife of regional inventory as well as the number of transfused rcs were calculated. in addition it was hypothesized that an efficient turnover of rc inventory will result in inventory reduction and relatively fresh blood for the transfusion reducing the blood utilization and frequency of transfusion among non-surgical patients especially those with chronic transfusion. results: dynamic inventory management and application of inventory index at regional level (four referral hospitals providing direct health care services to 1.4 and specialized services to 2.0 million population) reduced the regional rc inventory by 32% (1100 rcs to 750 rcs; optimal hospital inventory index of 7.5). this change in inventory was accompanied by a reduction in shelf-life of transfused red cells at 36% (average shelf-life of transfused rcs reduced from 3.45 to 2.21 weeks). the total annual rc utilization and specific categorical data of patients prior to and after the implementation of bump (2010 and 2012) included in table 1 . conclusion: optimization of rc inventory by application of inventory index improved the pattern of regional blood utilization. red cell utilization among chronically transfused patients was decreased by 20% (average). chronically transfused hematology and renal patients showed the highest reduction on rcu (21-26%, p value < 0.0001). that was no change in amount of surgical transfusions. non-surgical (medical) category showed a mild reduction (4%) but statistically was not significant. the results indicate that implementation of bump could significantly improve red cell utilization among chronically transfused patients. this change may also result in reduction of transfusion associated adverse reactions and long term complications like as iron overload. 3a-s12-01 no abstract available. 3a-s12-02 burnouf t 1 , tzeng ys 2 , deng sc 2 , wang ch 2 , tsai jc 3 and chen tm 2 1 taipei medical university, taipei, taiwan 2 tri-service general hospital, taipei, taiwan 3 tatung university, taipei, taiwan background: approximately 15% of diabetic patients develop chronic ulcers, and 25% of those may undergo foot amputation. activated platelet gel, which contains growth factors, has been proposed as an adjunct to promote healing of small diabetic foot ulcers. for large un-healing diabetic ulcers, skin graft is usually needed. we have demonstrated that single-donor allogeneic platelet gel and fibrin glue improve skin grafting to achieve successful persistent healing of large ulcers [1]. however, it is not known whether autologous platelet gel can be beneficial in this application. aims: to evaluate in a prospective study the safety and efficacy of using autologous platelet gel to enhance skin graft take in non-healing diabetic lower extremity ulcers. methods: approval was obtained from the institutional review board of our hospital. eight consecutive diabetic patients aged 25-82 with nine non-healing lower extremity ulcers (median size of 50 cm 2 ; range 15-150 cm 2 ) were enrolled. their median duration of diabetes and ulcer was 10.6 years (range 5-25 years) and 6.5 months (range 3-24 months). none of the patients had received conventional skin grafting in the past. prp was prepared from 100 ml of venous blood using sep-ax-vgr protocol (biosafe sa, switzerland). autologous thrombin was prepared by activating 10 ml of plasma (tgd-001; merries international inc., taiwan). skin ulcer was debrided to remove the infected and necrotic tissues and covered with moist saline dressing. daily dressing change without additional treatment was performed. the wound was sprayed after 7 to 10 days with equal volumes (5 to 7 ml) of autologous prp and autologous thrombin to form the platelet gel within 5-10 s. thin-splitthickness skin graft with multiple slits was then applied on the wound bed and fixed with staples or cat-gut sutures. each patient was placed on antibiotics during the course according to wound cultural results. bolster dressing with sofa-tulle were used to avoid post-graft hematoma formation. negative pressure wound therapy (vac) was not used in this study. results: there was no adverse reaction during the study. eight out of nine skin grafts took well (88% healing rate). the interval between skin graft and complete wound healing ranged from 2 to 3 weeks in the eight successful cases. no ulcer recurrence was noted during the 2-19 months follow-up period. the non-successful case was an attempt to treat an ulcer that was deep to the periosteum of calcaneus bone. free tissue transfer would have been required, but the patient refused the microsurgery, due to age and medical condition, which led to skin graft loss. conclusion: this study shows for the first time, to our knowledge, the possibility to use platelet materials in combination with skin graft procedures to treat large nonhealing diabetic ulcers of lower extremity recently, human platelet lysates (pl) rich in growth factors were shown to replace fbs for ex vivo expansion of various cells, but whether they can be used for cec expansion is unknown. aims: to evaluate the possibility to isolate and propagate cecs ex vivo using a xenogeneic-free, recombinant growth factor-free medium supplemented exclusively with human pl. methods: pl was prepared by cacl 2 activation of apheresis platelet concentrates from three volunteer donors, and centrifuged to obtain a fibrin-free supernatant that was heat-treated (56â°c/30 min; hpl) or not. pl was characterized for proteins, platelet growth factors (pdgf-ab, bdnf, egf and vegf) and chemical composition. cecs were obtained from over 10 bovine corneas (bcecs) using standard procedures and grown in a dmem-f12 medium (containing sodium bicarbonate, selenium, and antibiotics) supplemented either with (i) 5% fbs, 0.5% dmso, 2 ng/ml rhu-egf, 5 lg/ml insulin, 5 lg/ml transferrin, and 1 nm cholera toxin (termed '5% fbs medium'), or with (ii) 2.5%, 5%, 7.5%, or 10% pl or hpl as the only source of protein nutrients and growth factors. cells were grown in duplicates in 6, 24 or 96-well plates at 37â°c in a controlled atmosphere containing 5% co 2 , with medium changes every two days. viable cells were counted for 7 days and cell viability was determined by mtt assay. bcec phenotype was determined by immunostaining using anti-phospho-connexin 43, anti-na/k atpase alpha-1 subunit, anti-zo-1 and purified anti-n-cadherin. anti-mouse and anti-rabbit igg fitc were used as the second fluorescent antibodies. results: pl or hpl contained 55-60 mg/ml total proteins, and a range of approximately 30-40.5, 23.1-32, 0.44-1.82, and 0.24-0.39 ng/ml of pdgf-ab, bdnf, egf, and vegf platelet growth factors, respectively. cecs could be expanded in a med-ium supplemented with 2.5-10% pl or hpl. interestingly, better cell bcec morphology and adherence was found when using hpl compared to pl. cell growth and mtt equivalent to that of the '5% fbs medium' could be achieved only using 10% hpl. in addition, bcecs could be isolated from bovine corneas and subsequently expanded using the dmem/f12 medium supplemented with 10% hpl. bcecs expanded in the hpl-medium maintained their typical morphology, adherence, transparency and phenotype. conclusion: bcecs can be isolated and expanded ex vivo in a growth medium supplemented solely with human platelet lysate material. although further studies using cec from human origin are mandatory to confirm these conclusions, such findings open a possible new paradigm for gmp-compliant, clinical-grade ex vivo propagation of cec and regenerative therapy protocols of human corneal endothelium. platelets are the smallest and second most abundant circulating cells in the blood and their primary role is to maintain the integrity of the vasculature. when blood vessel injury occurs, platelet adhesion and activation receptors recognize subendothelial matrix proteins such as collagen and this can initiate a coordinated series of reactions leading to the formation of a fibrin clot to arrest bleeding. it appears, however, that in addition to hemostasis, platelets also have important inflammatory and immunological functions. as early as the 1960's, reports began to demonstrate that platelets may play an active role in the stimulation and regulation of immune responses. for example, platelets can store and secrete several pro-and anti-inflammatory chemokines (e.g. platelet factor 4 and rantes) and cytokines (e.g. interleukin-1b and transforming growth factor-b) that can affect local immune responses such as chemoattracting neutrophils to sites of tissue damage. on the other hand, platelets may be able to directly regulate adaptive immune responses via their ability to express and secrete cd40/ cd40l co-stimulatory molecules. more recent reports have also suggested that depending on their activation state, platelets may be able to either suppress cd8+ t cell responses or under certain circumstances, present mhc class i associated peptides to activate cd8+ t cells. these studies have suggested that platelets represent a critical link between innate and adaptive immunity. platelet mhc class i expression may also have a detrimental role by conferring tumor cell resistance against immune attack. of perhaps greater interest, platelets have been shown to express the entire family of tolllike receptors (tlr) and this may allow them to act as circulating sentinel cells that first encounter bacterial products for presentation to the innate immune system. in particular, surface expression of platelet tlr4 enables platelets to present lipopolysaccharide to mononuclear cells and neutrophils which modulates their phagocytic capabilities and this has implications for the development of immune platelet disorders. furthermore, tlr9 appears to be contained within a unique platelet granule underneath the cell surface that can be expressed by platelet activation. thus, elucidating the role of platelets in sepsis and a better understanding of the apparent central role that they play as immune cells may be important for the potential development of efficient therapeutic modalities against infections. this lecture will highlight the many characteristics of platelets as immune-like cells will discuss how platelets may be the major controllers of immune responses. macquarie university, sydney, australia malaria remains a major health problem in most of the tropics, and is especially burdensome in economically underprivileged areas. our ability to reduce the high rates of morbidity and mortality due to malaria are hampered by wanning efficacy of current antimalarial drugs and the spread of insecticide-resistant mosquitoes. we desperately need a greater understanding of how the plasmodium parasite succeeds in invading and growing within red cells, how the host responds to an infection, and importantly, the protective mechanisms employed by the host to combat the infection. platelets regulate blood haemostasis, but are now also regarded as an important component of the body's early innate defense against invading microbial pathogens. recently, my laboratory discovered that platelets are able to protect against a malaria infection. in mouse models of malaria, survival to a chronic infection is reduced when platelet levels are artificially depleted. purified human platelets directly bind to p. falciparum-infected red cells in culture and kill parasite within. our current work is exploring how platelets can kill intrerythrocytic malaria parasites. i will present our current understanding of the platelet and red cell molecules involved in the killing mechanism. these include the platelet cytocidal molecule, platelet factor 4 (pf4) and the erythrocyte duffy-antigen molecule, which binds pf4 and mediates the platelet killing effect. the critical requirement of duffy has lead us to propose that platelet-mediated protection against p. falciparum infection is compromised in individuals homozygous for the common duffy-antigen negative allele. 3c-s13-01 graduate school of medicine, the university of tokyo, tokyo, japan although bleeding is a major side effect of heparin, which is used for treatment of thrombosis, heparin also causes a prothrombotic adverse drug reaction called heparininduced thrombocytopenia (hit). hit is caused by the development of platelet-activating antibodies (hit antibody), which is directed against the heparin and platelet factor 4 (pf4) complex. these reactions accelerate platelet activation and coagulation, leading to thrombosis. thus, if hit is strongly suspected clinically in cases of thrombocytopenia and thromboembolism that occur during or after heparin therapy, it is vital to stop all heparins and start administering an alternative antithrombotic drug immediately. in japan, a test to screen for hit antibody (the automated immunoassays based on two types of chemiluminescent immunoassay and a latex-enhanced immunoturbidimetric assay) was approved as a clinical laboratory test in the medical insurance system, in september 2012. only the latex agglutination test is now widely used clinically because of its simplicity, convenience and cost-effectiveness. however, these immunological methods, including enzyme immunoassays (eias), which detect binding of antibodies to immobilized pf4/heparin complexes, may not be employed suitably. the immunological hit tests are useful in diagnosing hit because of their high sensitivity; however, they also often cause overdiagnosis of hit. the value of the selected cut-offs is the key element in ruling out hit. consequently, hit should be confirmed through laboratory detection of platelet-activating antibodies by using functional assays for the hit antibody; it must also be diagnosed based on careful consideration of the clinical picture. in order to diagnose hit properly, our laboratory asks clinicians to assess the pretest probability of hit by using the scoring system (the '4t' scoring: thrombocytopenia, timing, thrombosis, and other explanations). furthermore, our expert staff ensures that the diagnosis is correctly performed, since hit has not been fully recognized in clinical practice in japan as compared to in western countries. the functional assay for hit antibody has been regarded as the gold standard for diagnosing hit in patients in spite of its disadvantages. the platelet activation test procedure is cumbersome, the tests are technically challenging, and limited to specialized laboratories. additionally, the most important requirement for the test is the selection of platelet donors with high reactivity to the platelet activation antibodies. accordingly, in japan, there are very few places where the functional assay is conducted, whereas many institutions still assess patients with only the immunological assay. in our laboratory, the heparin-induced platelet aggregation method is performed, as isotopes such as radiolabeled serotonin release assay should not be commonly used in routine laboratories. in an attempt to improve the sensitivity of the functional assay, we developed two methods for increasing hit antibody reactivity in donor platelets. one is the cooling donor platelet method, used for improving reactivity, and the other a way of donor selection by using monoclonal hit antibody. further studies are necessary to introduce a simple assay method in ordinary laboratory testing to detect platelet-activating antibodies. 3c-s13-02 autoimmune or immune thrombocytopenia (itp) is an acquired bleeding disorder with a low platelet count mediated by immune-mediated mechanisms. this condition is seen in patients with various associated diseases, such as systemic lupus erythematosus, and can also occur without an underlying disease. production of igg autoantibodies to platelet surface glycoproteins, such as gpiib/iiia and gpib, is the hallmark of the disease. it has been thought that anti-platelet autoantibodies promote platelet clearance in the reticuloendothelial system, but recent findings indicate that anti-platelet antibodies also suppress megakaryogenesis, resulting in impaired platelet production. the diagnosis of itp continues to be one of exclusion. several antigen-specific assays for detection of anti-gpiib/iiia and anti-gpib antibodies are reported to be useful in identifying itp patients, but these assays require complicated procedures such as platelet solubilization, and use of commercially unavailable monoclonal antibodies. to solve these problems, we have developed an enzyme-linked immunospot (elispot) assay for detection of circulating b cells secreting igg anti-gpiib/iiia and gpib antibodies, which is a sensitive, specific, and convenient method for evaluating the anti-platelet autoantibody response. in addition, reticulated platelets and circulating thrombopoietin (tpo) are useful in evaluating platelet production status. these findings led us to propose preliminary diagnostic criteria for itp based on a combination of itp-associated laboratory findings, including erythrocyte and leukocyte counts, anti-gpiib/iiia antibody-producing b cells, platelet-associated anti-gpiib/iiia antibodies, percentage of reticulated platelets, and plasma tpo. although the etiology of itp remains unknown, complex dysregulation of the immune system is observed in itp patients. based on a series of experiments using cd4+ t cells reactive with gpiib/iiia derived from itp patients, we have proposed a 'continuous pathogenic loop' model as a mechanism that explains ongoing antiplatelet antibody response in itp patients. this model includes b cells that produce anti-platelet antibodies, reticuloendothelial macrophages that phagocytose opsonized platelets via fcc receptors and present platelet-derived antigenic peptides, and platelet-reactive cd4+ t cells that exert their helper activity upon recognition of the antigenic peptides. once this pathogenic loop is established, anti-platelet antibody production would go on endlessly. recently, regulatory systems that control this pathogenic loop are attracting a great deal of attention. a series of studies in itp patients have found that foxp3+ regulatory t cells are reduced in circulation, bone marrow, and spleen, and are deficient in their suppressive function. in addition, a critical role of regulatory t cells in preventing the anti-platelet autoimmune response has been demonstrated in mice deficient in regulatory t cells, which spontaneously develop anti-platelet autoantibody-mediated thrombocytopenia. in addition, our recent analysis indicates that the eradication of helicobacter pylori leads to up-regulated expression of inhibitory fccgriib on macrophages, resulting in the attenuation of the pathogenic loop. therefore, therapeutic strategies aimed at interrupting this pathogenic loop would inhibit anti-platelet autoantibody production and subsequent increase in platelet count. in fact, current treatment regimens for itp, including corticosteroids, splenectomy, and rituximab, are able to suppress the pathogenic loop. interestingly, tpo mimietics have a potential to induce peripherally induced regulatory t cells, resulting in suppression of the pathogenic loop. 3c-s13-03 seguchi s 1 , maeda t 1 , kanaumi y 1 , kawamura s 1 , kodama m 1 , kawai t 1 , okazaki h 2 and miyata s 1 1 national cerebral and cardiovascular center, osaka, japan 2 the university of tokyo hospital, tokyo, japan background: heparin-induced thrombocytopenia (hit) is a devastating immunemediated thromboembolic complication of heparin therapy. heparin administration can cause conformational changes in platelet factor 4 (pf4), resulting in the production of anti-pf4/heparin antibodies. a subset of these antibodies can activate platelets and monocytes (hit antibodies), leading to thrombocytopenia and a thrombininduced hypercoagulable state. up to half of hit patients suffer from arterial or venous thrombosis. if platelet concentrates are transfused into hit patients, it is conceivable that the transfused platelets can be activated by the same mechanisms that affect the patient's own platelets and trigger the onset of new thromboembolism or exacerbate hit-associated thromboembolism. thus, platelet transfusion is thought to be contraindicated in acute hit patients. however, it remains uncertain whether platelet transfusion is a risk factor for thrombosis in hit patients since only a few studies have investigated this issue systematically. aim: the goal is to clarify whether platelet transfusion increases the risk of thrombosis in hit patients. methods: we constructed a nationwide registry for hit with the approval of the ethical review committee. between august 2008 and may 2013, 329 patients from 185 hospitals clinically suspected of having hit were retrospectively included in the registry with clinical information such as changes in platelet count, timing of heparin administration, episodes of transfusion, thromboembolic events, and the results of serological assays for hit antibodies. hit was definitely diagnosed by the detection of anti-pf4/heparin igg with platelet-activating properties at a therapeutic heparin concentration, but not at a high heparin concentration or with anti-fccriia antibodies. the assay was performed using washed platelets prepared from hit antibody-sensitive healthy volunteers at a reference laboratory. we examined patients who received transfusions of platelet concentrates after hit was suspected. results: of the 329 patients, 85 patients were ultimately diagnosed with hit (25.8%). optical density values of anti-pf4/heparin antibodies detected by elisa were significantly higher in hit patients than in non-hit patients (2.3 ae 0.95 vs 0.36 ae 0.53 for igg/a/m, p < 0.001; 1.68 ae 0.80 vs 0.25 ae 0.37 for igg, p < 0.001). the incidence of thromboembolic events was significantly higher in hit patients (51.8%) than that in non-hit patients (28.3%; p < 0.001). among the 85 hit patients, 20 patients received platelet transfusions after the onset of hit. only two of them experienced a thromboembolic event after platelet transfusion, one within 24 h and the other after 9 days. notably, neither patient was being treated with a thrombin inhibitor at the time. the incidence of thromboembolic events in hit patients who received platelet transfusions was not significantly higher compared to hit patients who did not receive platelet transfusion or non-hit patients who received platelet transfusions after the suspicion of hit arose, respectively. conclusions: to our best knowledge, this is the first systematic report that clarifies the clinical impact of platelet transfusion on the occurrence of thromboembolic events in acute hit patients whose diagnosis was confirmed by a washed plateletactivating assay. even in acute hit patients who possess platelet-activating antibodies, the transfusion of platelet concentrates does not appear to increase the risk of thromboembolism, especially while on thrombin inhibitor therapy. 3c-s13-04 lu p, ling b and li rs background: transfusion platelet matches with antigenic similarity would evoke less allorecognition and immune activation. strategies have been based on the theory that selection for hla-a and hla-b cross-reactive groups (cregs) compatible donor as well as abo/hpa-matched donor will predict good increment in platelet corrected count after platelet transfusion. aim: establish large-sized platelet donor registry with hla class i,hpa,abo-typed to meet the needs of immunized patients with platelet transfusion refractoriness. evaluate the effectiveness of platelet transfusion therapy in ptr patients. progressive management to ptr patients maintain a long-term platelet transfusion strategy. methods: to establish platelet aphaeresis donor registry in shanghai, 1931 repeat donors were typed for hla-a, -b and hpa-1,-2, -3, -4, -5, -6 and -15 using standard pcr-ssp method. eighteen patients with hematologic or oncologic diseases which refractoriness to platelet transfusions from random donors who are receiving 36 units of apheresis platelet products transfusion were studied. results: eighteen patients(eight male, 10 female)showing platelet refractoriness to random donor platelets [1 h corrected count increment (cci) <7500 ml/m 2 , percentage of platelet recovery (ppr) <20%] before. patients phenotyped for both hla-a,b and hpa-1,-2, -3, -4, -5, -6 and -15. apheresis platelets from donor registry in shanghai matched to patients abo, hpa and hla-a and hla-b cross-reactive groups (cregs) are transfused. ten patients ( show 24 h ppr >20%. the mean 1, 24 h cci and ppr values from the best donors were significantly higher than those from random donors they transfused before. conclusion: the use of hla-a,-b and hpa,abo-compatible aphaeresis platelet improves posttransfusion 1, 24 h cci values and percentage of platelet recovery in refractory patients. transfusion with hla-a,-b and hpa,abo-matched platelets is mandatory to reduce the risk of bleeding in ptr patients. refractoriness to platelet transfusions developed at least in 50% of the patients we observed and to maintain a long-term platelet transfusion strategy. establish large-sized platelet donor registry with hla class i, hpa, abo-typed may be needed to circumvent platelet-specific antibodies of unknown specificity in all chronically transfused patients. the optimal strategy for platelet substitution in immunized patients remains a challenge. 3c-s13-05 xia w 1 , xu x 1 , ye x 1 , fu y 1 , deng j 1 , liu j 1 , ding h 1 , chen y 1 , shao y 1 , wang j 1 , li h 2 and santoso s 3 1 guangzhou blood center, guangzhou, china 2 department of biotechnology, guangdong food and drug vocational college, guangzhou, china 3 he institute for clinical immunology & transfusion medicine, justus-liebig univ., giessen, germany background: immunization against cd36 leads to the production of anti-nak a antibodies associated with fetal/neonatal alloimmune thrombocytopenia (fnait), platelet transfusion refractoriness (ptr) and post-transfusion purpura (ptp). however, no data regarding the clinical relevance of cd36 immunization is currently available for chinese population. study design and methods: platelets and monocytes derived from 998 healthy blood donors were typed for cd36 deficiency using flow cytometry. in addition, four patients with suspected fnait (one case) and ptr (three cases) were investigated. nucleotide sequencing was performed to identify the mutations underlying the cd36 deficiency. transfection in mammalian cells (hek-293t) with cd36 mutated constructs was conducted to confirm these results. anti-nak a antibodies were screened by the use of platelet solid-phase kit (pak-plus, gti diagnostics). results: of 18/998 blood donors failed to express cd36 on their platelets surface. in 5/12 individuals no cd36 expression was detected both on platelets and monocytes, suggesting that the frequencies of type i cd36 deficiency (platelets and monocytes) and type ii cd36 deficiency (platelets only) were approximately 0.5% and 1.3%, respectively. nucleotide sequencing analysis of type i cd36 deficient individuals revealed eight different mutations; four of them were not described so far. however, 1228-1239del attgtgcctatt and 329-330delac appeared to be the most common mutations related to type i cd36 deficiency in south chinese population. further analysis showed that the presence of anti-nak a antibodies in one healthy donor (donor 1) as well as in three cases of ptr (patients 2-4) and one case of fnait (patient 1). these results could be confirmed by immunoprecipitation using biotinylated platelets and by antigen capture assay with stable transfected cd36 cell lines. in all ptr patients, transfusion with platelets derived from cd36 negative donors resulted in good increment (24 h, ppr >20%). table 1 shows the mutations found in these five gpiv defective individuals. conclusions: more than 0.5% of cd36 type i deficient individuals are at risk to be immunized through blood transfusion or pregnancy in china. in this study, we could demonstrate that this immunization is of clinical relevance for the development of ptr and fnait. therefore, testing of anti-nak a antibodies should be considered in suspected immune mediated thrombocytopenia. a national registry of cd36 deficient blood donors should be established to maintain bleeding disorders associated with anti-nak a antibodies. since immunization against cd36 is conceivable for other asian populations an international network within laboratories in south asian region should be established in the future. 3c-s14-01 do we really need ffp? the evolving role of pf24 and pre-thawed plasma devine d fresh frozen plasma (ffp) is defined as plasma frozen within 8 hours of collection. while this product maintains a high functional activity of both coagulation factors and anticoagulant proteins, there has been recent movement in some jurisdictions away from reliance on ffp. in many blood systems, an increasing role for plasma frozen within 24 h of collection (fp24) is seen. such plasma shows little difference in functional protein levels when compared to ffp, with the exception of fviii levels which a show time related decay of activity. even factor viii loss can be consistently minimized if whole blood is held on controlled rate cooling plates prior to preparation of fp24. in addition, the activity profiles of coagulation proteins in fp24 prepared in routine production closely resemble those of commercial pooled plasma products. taken together, these observations have led many blood systems to move from the exclusive use of ffp to a mix of inventory of ffp and fp24, if not to the complete removal of ffp from their menu of offerings. the preparation of cryoprecipitate has also been a driver for the retention of plasma frozen within 8 h of collection. since the most common labeling of cryoprecipitate has focused on the content of both fibrinogen and factor viii, in part owing to original role of the latter in the treatment of hemophilia a, collection of ffp has persisted as the starting material for cryoprecipitate production. in jurisdictions where hemophilia or other factor viii deficiencies are treated with factor concentrates, the labeling of cryoprecipitate to emphasize its antihemophilic factor activity is no longer warranted. as data began to accumulate on fp24, similar studies began to appear that investigated the effect of prolonged cold storage of plasma that had been thawed. this led to the introduction in some jurisdictions of the extension of the allowable period of use for thawed plasma from 24 h to up to 5 days, if stored at 4â°c. such practice is increasingly widespread and there is no evidence that patients receiving such products are compromised. from the perspective of health resources management, the use of both fp24 and pre-thawed plasma reduces discard of products or prevents the use of these products in non-group specific recipients. with the advent of massive transfusion protocols which may require pre-thawed plasma at the ready, it also allows better use of relatively scarce but high demand products such as ab plasma. this presentation will focus on a review of the relevant studies of plasma quality for ffp, fp24 and pre-thawed plasma. we will review the appropriate uses of these different components as well as groups of patients for whom specific products should be restricted or supplemented. 3c-s14-02 background: in 2008, the australian red cross blood service (blood service) began a programme of process improvement aimed at maximising the manufacture of clinical plasma components from male donors, which is a key mitigation to the risk of trali (transfusion related acute lung injury). the challenge of sourcing all clinical plasma from male donors is exacerbated in australia due to its adherence to the council of europe guidelines that stipulate a maximum allowable time between collection and freeze of apheresis-derived clinical plasma of six hours, which is considerably more stringent than for most other blood services despite the greater tyranny of distance that exists in australia. aim: the aim of the programme was to achieve a result of 100% of clinical plasma sourced from male only donors. methods: work began in early 2008 to gather detailed quantitative data that linked information on donor panel to collection centre to production facility, and that could be broken down by blood group and by day of the week. this was then formulated into a suite of reports, highlighting opportunities and variance in performance. based on those reports, a cross-functional team designed a range of improvement initiatives across disciplines such as transport, systems enhancements, donor acquisition and processing, such as: incoming blood donation shippers were marked with colour coded labels that notified the receiving production facility of clinical suitability. this assisted with prioritisation and workflow management. additional deliveries of blood from collection centres to processing centres. a range of targeted campaigns and marketing collateral were produced to attract male donors to apheresis plasma donation donor centre collection staff were trained to convert male ab donors over to plasmapheresis donation activity. changes to progesa to prevent manufacture of female plasma were made (after a time). results: the first report in july 2008 showed that the blood service were issuing 87.71% male clinical plasma; the group ab rate was 57.86%. the results improved dramatically by november 2010 in groups o, a and b (99.64%, 99.72% and 99.62% respectively), allowing for progesa to be configured to prevent the routine manufacture of female plasma from those groups, whilst still allowing supervisor over-ride. by march 2012, the results in group ab had improved to 97.77% (chart below) and the directive was given to manufacture male clinical plasma only as routine. january 2013 was the first month ever where 100% of all clinical plasma was sourced from male donors only. in 2012/13, 99.98% of clinical plasma was male onlythe 0.02% constituted short lead time requests for iga deficient plasma. subsequent to a system change that allowed for a donor identification marker for iga deficient donors, inventory levels of this sub-product have increased by 34%, negating the need to turn female production on to accommodate sporadic demand. summary/conclusion: the multi-disciplinary efforts over an extended period of time have resulted in the practical removal of the risk of trali in australia. this is an achievement that many thought impossible and one that many other blood services have been unable to attain. results: quality control (qc) parameters were measured in the prepared blood components and were listed in table 1 . by standardising bc volume and haematocrit in the primary separation, recovery of red cells and plasma was optimised in both the red cells in sagm and plasma units while wbc and rbc contamination levels in pc and plasma were maintained low. all parameters were well within the blood component specifications set out in the council of europe guide (16th edition), the standard adopted by hkrcbts. qc parameters of the pathogen reduction-treated platelet concentrates in pas so produced were also within the hkrcbts blood component specifications. low contamination with red cells and white cells were demonstrated and the ph range was acceptable after 5 days of storage at 20-24â°c. the new t&b production method for the separation of 450-ml quadruple wb and the preparation of intercept platelet concentrates in ssp+ pas was successfully developed and can be applied to the production of high quality blood components in preparation for clinical evaluation of the pathogen reduction-treated platelet concentrates. 3c-s15-01 blood donors are healthy volunteers who give whole blood or blood components by apheresis for altruistic motives. they should be managed in a way that ensures high standards of care. nevertheless, there are recognized adverse reactions that can occur during blood donation. the overall incidence of complications directly related to blood donation is 1%. they are generally more common in women, in younger and in first-time donors. although the incidence seems to be small, it is of great importance considering the large number of people giving blood each day worldwide. adverse blood donation reactions can generally be minimized or avoided by appropriate donor selection and care, and appropriately trained staff. vasovagal episodes and soft tissue injuries (bruises/haematomas at the venepuncture site) are the most common donor reactions. the majority of these are minor and donors usually recover quickly; however, these reactions can be of concern to donors and reassurance should be provided. other reactions include nerve injury and arterial puncture which, although less frequent, may require medical care outside the blood service and may lead to prolonged symptoms or incapacity. staff should be trained to recognize and manage such adverse reactions, including the provision of first aid. the incidence of bruising should be monitored so that further venepuncture training may be provided to staff as necessary. iron deficiency in regular blood donors has been a top donor health and safety concern in many countries. as each donation causes a loss of 213-236 mg of iron, repeat donation can lead to a continuous depletion of body iron stores. studies have shown that donation frequency had the greatest impact on iron deficiency and further risk factors were lower weight and female gender. to address this issue, many blood services encourage donors to take iron-rich food and/or give them iron supplements. adverse reactions such as delayed faint may occur after the donor leaves the donation venue. donors should be advised to inform the donor centre staff of any ill-effects they suffer after donating. a system for the reporting and investigation of adverse donor events and reactions should be in place as part of the donor haemovigilance system. all adverse events and reactions in donors should be identified, documented and reported. these data should be regularly analysed for possible corrective and preventive actions. the goal of donor haemovigilance is to reduce the occurrence of adverse events and reactions and improve the outcomes both for donors and patients. for various reasons, even today, donors often do not receive detailed information on the blood donation procedure and possible complications. not only do blood services have the ethical duty to inform donors of possible adverse events to enable them to give informed consent and take action for preventing adverse effects, protecting the safety of donors is also important for donor retention because a safe and good donation experience ensures donors will return regularly. 3c-s15-02 wiersum-osselton jc trip national hemo-and biovigilance office, the hague, the netherlands background: without blood donations and the availability of blood transfusion, many important therapeutic advances could not have been achieved. donor hemovigilance is the systematic monitoring of adverse reactions and incidents in the whole chain of blood donor care, with a view to improving quality and safety for blood donors. method: this 'global update' draws on work by the international haemovigilance network and international society for blood transfusion haemovigilance working party, experience in the netherlands, as well as a pubmed search using terms blood donor and adverse reaction. results are discussed for vasovagal reactions, needlerelated complications, long-term morbidity, donor iron status and frequent apheresis. results and discussion: the occurrence of vasovagal reactions is associated with young, female donors, lower body weight and estimated blood volume, first-time donor status. a reduction of vasovagal reactions has been documented with use of a water drink before donation, muscle tensing, social distraction and lower collection volume for donors with small estimated blood volume. needle injury is relatively frequent as a cause in cases of long-term morbidity; needle injury is associated with traumatic phlebotomy and in some cases nerve damage is documented. repeated whole blood donations lead to reduction of body iron stores and in some cases anaemia. some blood services adjust donation intervals to avoid or reduce this, while others have or are considering a policy of iron replacement therapy. fewer studies on acute complications in plasma and other types of apheresis have been published. preliminary studies of bone density and protein levels in non-commercial frequent plasma donors have not substantiated any specific hazard despite theoretical concerns of calcium or protein depletion. international collaboration in strengthening donor vigilance definitions and data analysis may in future increase potential for study of risk factors and measures to improve donor care worldwide. conclusion: donor vigilance is gaining international interest and has increased knowledge of risk factors for vasovagal reactions associated with blood donation. there remains a need of research and of developing preventive measures, including prevention and treatment of needle injury as well as possible long-term effects of frequent donation. assuming that these donors were newly infected, it is crucial for bts to monitor the prevalence of this category of donors in order to strategize specific measures to these targeted groups to improve blood safety. aims: this study aimed to profile blood donors who donated during the hiv serological window period and to identify the risk factors of these blood donors. methods: past donor records of blood donors who had donated blood during the hiv serological window period (nat ultrio and discriminatory detected and negative for both anti-hiv and p24 antigen) at nbc or at blood mobiles organized by nbc from november 2007 until july 2013 were retrieved and analyzed using spss 15.0. results: a total of 18 donors were nat detected and negative for anti-hiv and p24 antigen (none in 2007, 2 in 2008, 2 in 2009, 1 in 2010, 6 in 2011, 4 in 2012 and 3 in 2013 introduction: in pakistan, the predominant reliance for blood supply is on the replacement donors, as sufficient numbers of voluntary blood donors are not available. an increase in the proportion of voluntary donors following the promotion of the concept of voluntary non-remunerated blood donation (vnrbd) will enhance safety and will also help to shift the responsibility for arranging blood availability from the patients to the health care system. objective: the objective of the current study was to promote vnrbd through a public awareness campaign (pac) based on a thorough analysis of the knowledge, attitudes and practices (kap) of a key segment of the society, i.e. 18-25 years old college and university students. material and methods: a cross-sectional, descriptive study was conducted over a period of three months (jan-mar 2012). multi-stage random cluster sampling approach was followed and college and university students were targeted through 20 university based blood donor organisations (bdos) out of a total 56 bdos identified. all the participants voluntarily participated in the study and informed consent was obtained orally. a pre-tested questionnaire comprising of 28 questions related to knowledge, attitudes and practices was applied. the questionnaire was kept anonymous and each question included multiple options or statements. statistical analysis was conducted by the assistance of statistical package for social sciences (spss) software version 17. results: a majority (65%) of the students had heard about blood donation through family/friends and a minority (9%) through the internet, although 45% preferred internet as spare time activity. majority (+85%) of the students had access to the internet and mobile phone. more than 50% of the respondents had donated blood: 22% donated for family, relatives or friends, 4% donated voluntarily as an act of altruism, and 25% donated voluntarily once and then stopped donating, but 55% of these respondents still considered themselves as volunteer blood donors. 35% indicated that important people in their environment had an influence on crucial decisions that they made. motivation for blood donation was a desire to help other people (73%), 28% followed friends invitations, in 21% cases respondents family or friends had received blood transfusions, 16% followed the example of fellow students. restriction for blood donation: 40% generally feared donation, 25% had a specific fear of the needle, 20% had no confidence in the public (health) sector, 25% condemned blood selling practice, 14% had no confidence in the donation procedure (hygiene), 13% experienced parental discouragement. conclusion: to overcome the apprehensions and fears of the donors it is important to provide adequate information about donation to potential donors. this strategy will help convince family replacement donors to become vnrbd and also recruit healthy individuals to become a vnrbd. the approaches and strategies for this transition can be based on the findings of the study. the reported preference for internet as leisure time activity suggests that internet can be utilized as an important tool for information dissemination in a pac, for which detailed study is required. 3d-s16-01 murphy mf platelet refractoriness is the repeated failure to obtain satisfactory responses to platelet transfusions. there are immune and non-immune causes of platelet refractoriness. the main immune cause is hla alloimmunisation which occurs predominantly in females with a history of pregnancy. other immune causes include hpa alloimmunisation, abo incompatibility, platelet autoantibodies and drug-related platelet antibodies. the incidence of alloimmune platelet refractoriness due to hla antibodies has declined due to leucocyte-reduction of blood components and more aggressive treatment for patients with haematological malignancies and other cancers. in current practice, platelet refractoriness is mainly due to shortened platelet survival associated with non-immune clinical factors, such as infection and its treatment with antibiotics and antifungal drugs, dic and splenomegaly. if there are poor responses to hla-matched platelet transfusions, the reasons should be sought including hla incompatibility which is most likely to occur in patients with unusual hla types with few well-matched donors, non-immune platelet consumption, and hpa and abo incompatibility. further serological investigations including testing for hpa antibodies may be used to differentiate between these possibilities. depending on the results, the appropriate management could be the use of abo-identical or hpa-matched platelet concentrates if the specificity of the hpa antibodies can be identified. platelet crossmatching may be helpful in some patients with non-specific hpa antibodies. the management of patients with hla and/or hpa alloimmunisation and no compatible donors may be very difficult. there is no evidence that alloimmunised patients benefit from incompatible platelet transfusions which do not produce an increase in the platelet count, and prophylactic platelet support should be discontinued. if bleeding occurs, platelet transfusions from random donors or the bestmatched donors, despite being incompatible, may reduce the severity of haemorrhage although increased doses of platelets may be required. other management approaches such as the use of high-dose intravenous immunoglobulin, splenectomy, and plasma exchange have not been shown to be effective. the management of patients with non-immune platelet consumption is similarly problematic. the usual practice is to continue with daily platelet transfusions as prophylactic platelet support, but it is not known whether this approach is effective, or whether platelet transfusions should be discontinued or the dose of platelets increased. 3d-s16-02 managing bleeding in cardiac surgery: despite major advances in the management of perioperative blood conservation, transfusion rates in cardiac surgery remain very high, with large variations among individual centres. among all major surgical procedures, cardiac surgery with cpb still consumes a large part of the available blood supply. in england indicated that 10-15% of the blood units supplied by the national blood service is used in cardiac surgery units. in the usa, nearly 20% of blood transfusions are associated with cardiac surgery. during the early history of cardiac surgery, patients received large amounts of allogeneic blood. in the 50's, most operations were performed to correct congenital heart disease. during the 60's and 70's, the introduction of satisfactory valve prosthesis and direct grafting for atherosclerotic coronary artery disease led to rapid growth in the scope and number of patients having open heart surgery. in the 80's pharmacological methods to reduce bleeding were introduced and the focus of blood conservation was expanded to include blood components as well as red cells. with increasing application of cardiac surgery in acutely ill older patients with more comorbidities as well as the increasing safety of blood supply have contributed to an increasing incidence of allogeneic transfusions. not surprisingly, physicians, surgeons and anaesthesiologists have shown a great interest in the promotion of safe and effective alternatives to the transfusion of allogeneic blood in cardiac surgery. perioperative risk factors for allogeneic transfusion can be regrouped in three main categories: factors affecting the patient's preoperative rbc mass, factors affecting the perioperative blood losses, and factors affecting the transfusion practice. the ability to predict a patients risk for transfusion allows modification of patient management with the goal of decreasing allogeneic transfusions. using the trac and trust scoring system predicts candidates likely for transfusion. diminished rbc mass appears to be one of the strongest predictors of transfusion. the acceptance of a lower postoperative haematocrit (in ijn the hct on bypass is 20-25% and post bypass is >25%) or haemoglobin concentration represent an important element in current blood conservation practice. the decision to transfuse a patient cannot be based only on haematocrit concentration. optimizing preoperative rbc mass involves the early detection of anaemia and its correction before surgery. preoperative autologous blood donation can be used to conserve allogeneic blood. besides economic concerns, one essential argument against pad is the lack of sufficient time because of the uncertainty of waiting list. erythropoietin has also been used to augment pad in elective cardiac surgery. acute normovolaemic haemodilution (anh) aims at reducing allogeneic blood exposure through a reduction in the net red blood cell mass lost during or just after surgery. perioperative cell salvage (cs) also aims at reducing allogeneic blood exposure through a reduction in perioperative blood loss. antifibrinolytics (ltranexamic acid or epsilon aminoaproic acid) or serine protease inhibitors (aprotininnow unavailable) may reduce excessive fibrinolysis and platelet dysfunction. the use of activated f vii has been reported in intractable bleeding post cardiac surgery. 3d-s17-01 the university of tokyo, tokyo, japan antibodies against human neutrophil antigens (hna) are involved in the pathogenesis of immune neutropenia, such as neonatal alloimmune neutropenia (nan), refractoriness to granulocyte transfusions, and transfusion reactions, such as febrile non-hemolytic transfusion reactions, and transfusion-related acute lung injury (trali). the hna systems are assigned to five antigen groups, namely hna-1 to 5. hna-1, -2, and -3a alloantibodies have been implicated in the pathogenesis of trali, and especially hna-3a alloantibody has been found in the severe cases requiring artificial ventilation or with fatal reactions. besides alloantibodies to hna-1, -2 and -3, those against hna-4a and hna-5a have been implicated in nan. the identification of the causative antibodies is essential for the diagnosis as well as for the prevention of these disorders. the detection of hna antibodies has been mainly dependent on cell-based assays so far. among them, the granulocyte agglutination test (gat), the granulocyte immunofluorescence test (gift) and the monoclonal antibody immobilization of granulocyte antigens (maiga) are the most commonly applied. according to the isbt working party on granulocyte immunobiology, the combination of gat and gift is presently the best means of hna antibody detection. gift is usually more sensitive than gat, however, hna-3a antibodies associated with severe cases of trali are better detectable by gat. in gat and gift, the presence of hla antibodies with broad specificities may affect the detection of hna antibodies. on the other hand, the maiga assay allows the differentiation between hna and hla antibodies. these classical methods, however, require fresh neutrophils from hna-typed donors. also, these assays are time-consuming, which makes them not appropriate for the large-scale antibody screening. in our lab, we modified the mixed-passive hemagglutination (mpha) assay, the method largely applied in japan for platelet antigen/antibody detection, for the detection of hna antibodies. recently, alternative assays have been developed, including elisa with recombinant hna (rhna), and immunofluorescence tests with transfectant cells of hna (hna-1, -2, -4, and -5). more recently, the molecular basis of hna-3 antigen has been elucidated, and stable cell lines expressing hna-3 antigens became available. these cell lines seem to have low background, and do not express detectable levels of hla antigens, which make the identification of hna antibodies easier. additionally, kits that use luminex microbeads coated with hna antigen are being developed. these kits, however, do not include hna-3 antigens. these new technologies significantly help improving the detection and identification of hna antibodies, and allow the large scale screening of hna antibodies, contributing for the reduction of the risk of the pathological conditions associated with hna antibodies, especially trali. however, these new technologies significantly increase the cost of the tests. presently, although many assays have been developed, the standard hna antisera are not necessarily available in every lab, which makes their validation difficult to be conducted. thus, the collaborative study among the various labs, by exchanging the available antisera, and comparing the test results, is essential for the improvement of this field. 3d-s17-02 one of the main sites where pmns carry out vital to surveillance functions is in the lungs. the large surface area of the lung is needed for gaseous exchange but lungs also present a vital direct mechanical barrier to the external environment. to patrol and protect this interface, about 28% of the body's total pmns are located in the pulmonary microvasculature. illness may increase the number of lung pmns as well as change their phenotype from quiescent to primed. in trali, the transfusion of blood products with either pmn reactive antibodies or biological response modifiers can activate this concentration of primed pmns to produce an augmented respiratory burst. this causes injury to the pulmonary microvasculature and consequently the symptoms of trali. circulating antibodies to pmns also can compromise their numbers and function. pmns carry human neutrophil antigens (hna) as well as class i hla, which can become targets for pmn reactive antibodies. the granulocyte immunofluorescence test (gift) and granulocyte agglutination test (gat) are primary tools for investigating these pmn reactions, as they are able to detect reaction of hna as well as some hla class i antibodies. immune neutropenias: alloimmune neonatal neutropenia (ann) occurs when a neonate's pmns are destroyed by transferred maternal antibodies developed against an inherited paternal neutrophil antigen. this is similar to haemolytic disease of the newborn, but importantly can occur with the first pregnancy. in early childhood, some children develop severe neutropenia as a result of pmn auto-antibodies. although the pathogenesis of such chronic benign autoimmune neutropenia is still not understood most of these autoantibodies demonstrate specificity for the hna system. passively acquired autoimmune neutropenia, wherein pmns are destroyed by maternal pmn auto-antibodies crossing the placenta are a rare finding. hna specificity is unlikely. autoimmune neutropenia in adults is either primary, secondary to another autoimmune disease or drug related. it can present a clinical and diagnostic challenge as many adults invariably have alloantibodies to neutrophils and the patient's neutrophil count is too low to make a definitive identification of a self reactive autoantibody. hna specificity is extremely rare. the severity of trali and immune neutropenias demand rapid and precise diagnosis with reliable neutrophil serology. the isbt granulocyte immunobiology working party maintains a list of granulocyte immunobiology reference laboratories around the world. 3d-s17-03 when seven sera from 778 donors were screened for neutrophil specific antibodies, 9% samples showed positive reaction. these results, however, could not be confirmed by gift and gat. conclusions: in this study, we found alloimmunization against hla class i and ii in 4.6% male,~4.3% nulliparous and~13.9% parous females. in contrast, alloimmunization against hna was not detectable in this cohort. these results indicate that the use of plasma containing blood products from parous females without hla antibodies pre-testing may increase the risk of trali reaction. although alloimmunization against hna seems to be a rare event in china, further observation is necessary to exclude the necessity of hna antibodies screening in our blood products. it is becoming clear that the ccn family of extracellular proteins play an important role in the health and function of several cells of the hematopoietic lineage. ccn2, also known as connective tissue growth factor, ctgf, has recently been found to be in high abundance in platelets and released upon activation, an effect inhibited by aspirin, suggesting a role in blood clotting and/or wound healing. on the other hand, ccn3, also known as nephroblastoma-overexpressed, nov, has been found to play an important role in hematopoietic stem cell health and function. in fact, treatment with ccn3 has recently been shown to promote hematopoietic potential, a discovery with dramatic clinical potential. initially using bioinformatics, our laboratory has discovered a signaling pathway that connects these two discoveries and appears to be a key functional node within the development of blood cells. specifically, we have found that the myeloid zinc finger protein 1, mzf1, is a transcription factor that trans-activates both ccn2 and ccn3, in distinct cell types. for example, we have shown that mzf1 can stimulate ccn2 production and secretion in stromal fibroblasts, which is then taken up by megakaryocytes and loaded into platelets. this is the first time that ccn2 loading into developing platelets has been directly achieved and observed in vitro. secondly, we have discovered that mzf1 also regulates the synthesis of ccn3 in several hematopoietic cell types. putting these results together with previous data suggests a new and immediately testable clinical treatment. it is known that both vitamin d (calcitriol) and vitamin a (all-trans retinoic acid) stimulate transcription of the mzf1 gene. (we also have new data exploring the mechanism and suggesting other pharmacological ligands). we have confirmed that treatments with either vitamin a or d activate this pathway and results in increased production of ccn2 in stromal fibroblasts, which in turn results in enhanced loading of ccn2 into developing platelets in vitro. similarly, we have observed that both vitamin a and vitamin d induce ccn3 expression, through mzf-1, and we are currently testing if this will lead to enhanced hematopoietic potential. this work could impact the efficacy of blood donation and transfusion, bone marrow transplants, and the treatment of bleeding and clotting diseases as well as lymphomas and leukemias. 3d-s18-05 background: foxp3 + t regulatory cells (tregs) consisting of natural and induced treg subsets play a crucial role in the maintenance of immune homeostasis against self-antigen. while recent studies demonstrated that natural tregs are instable and dysfunctional in the inflammatory condition, induced tregs (itregs) may have a different feature. furthermore, it was reported that tolerogenic dendritic cells (tdcs) could expand itregs in vitro and this action designed to correct defects in numbers or functions of itregs may be therapeutic in the treatment of autoimmune diseases. in this study, the suppression efficacy of tgf-beta-induced tregs expanded by tdcs in vitro and in mouse model of autoimmune arthritis was determined. method: in vitro, first, cd4+ cd25ã� t cells were purified from splenocytes of d1 mice and stimulated by anti-cd3/cd28 in the presence of tgf-b1 for 5 days, which were termed 'itreg'. and tdcs derived from bone marrow of d1 mice were induced by gm-csf, il-10 and tgf-b1 and harvested after 10-day cultivation. then, itregs were expanded by tdcs at the ratio 5:1 and collected after 4 days (termed 'itreg tdc '). the phenotype, proliferation, suppression of cd4t proliferation, induction of foxp3 + tregs from foxp3ã� t cells and suppression of th17 cell differentiation were assessed. for in vivo experiments, the animal models of ra were established. in this model, arthritis was induced in d1 mice after immunized with bovine type ii collagen (cii) on day 0 and day 21, termed collagen-induced arthritis (cia). and 1 9 10 6 itregs or itreg tdc cells were transferred <iv. into cia recipient mice when cia onset. control mice were treated with pbs. clinical and histopathologic scores, cytokine and anti-cii antibody secretion in serum and cd4 + th subsets were analyzed. results: after 5-day induction in vitro, the percent of foxp3 + itregs increased from 9.8 ae 1.5% to 54.9 ae 2.5%. and these itregs could be expanded effectively by tdc in additional 5-day co-incubation, which continued to express higher levers of foxp3 (82 ae 3.6%) and increased 4.17 ae 0.17 fold. compared with itregs, the itreg tdc cells showed stronger inhibitory effect, and could prompt cd4t cells converting to foxp3 + itreg and resistant to th17 cell differentiation more effectively. during the 4-weekobservation-period, a more remarkable anti-arthritic activity, improved clinical scores and histological end-points were found in the itreg tdc -treated group than those in itreg-treated group. serological levels of tnf-a, il-6, il-17 and anti-cii antibodies were also significantly lower and tgf-b production were higher in cia mice following adoptive transfer of itreg tdc cells as compared with itregs. moreover, the itreg tdc treatment resulted in an increase in the number of tregs (cd4+ foxp3+) and a decrease in the percentage of th17 cells (cd4+ il-17+) more obviously. conclusion: these results indicated that itregs expanded by tdcs showed more effective suppression in vitro, and reduced more markedly the severity and progression of cia than itregs. this reduction was associated with modulating inflammatory cytokine and anti-cii igg secretions to lower levels and polarizing the treg/ th17 balance to treg more obviously. this study highlights the potential therapeutic utility of itregs expanded by tdcs in autoimmune arthritis and should facilitate the future design of itreg tdc immunotherapeutic strategies against ra. in may 2010 the world health assembly passed a resolution, wha 63.12, on the availability, safety and quality of blood products. this urges member states to 'take all necessary steps to establish, implement and support nationally co-ordinated bts with the aim of achieving self-sufficiency'. the resolution applies to both fresh blood components and plasma derived medicinal products (pdmps). who defines self-sufficiency as meeting national needs for six products from blood and plasma collected locally. these comprise red cells, platelet concentrates, fresh frozen plasma, and factor viii, immunoglobulin and albumin solution. the definition and overall goals were established by a who expert consensus statement on 'achieving self-sufficiency based on vnrbd' developed at a meeting of who experts in geneva switzerland in 2011. blood services develop in line with the health care system that they support. typically this involves a transition from a supply for whole blood mainly in the context of emergency management of trauma and child birth to the implementation of blood component therapy. blood component production will likely lead to an excess of recovered plasma. effective utilisation of this requires access to plasma fractionation facilities. if this can be achieved then this will reduce reliance on commercial pdmps and ensure maximal utilisation of the donated whole blood. increasing demand for pdmps will potentially lead to a requirement for introduction of plasmapheresis programmes if the goal of self-sufficiency is to be maintained. there are a number of significant barriers to both utilisation of recovered plasma and the implementation of effective plasmapheresis programmes. individual countries will need to consider these as part of their long term strategic planning process. the main barriers for low and medium hdi index countries are access to suitable plasma fractionation facilities. few countries will be able to justify creation of a national fractionation plant. regional co-operation and development of facilities will be needed. secondly plasma for fractionation is seen as a critical material in the manufacture of a medicine. quality system requirements, including full compliance with a code of good manufacturing practice will be a pre-requisite for acceptance of the plasma as suitable for fractionation. there is an urgent need to develop support programmes to raise standards to achieve this. in contrast the main barrier for most high hdi countries is cost and the easy availability of pdmps produced by commercial countries from paid plasma. the safety profile of pdmps is currently excellent since the integration of a number of specific viral inactivation steps in the manufacturing process overcomes any infectious burden associated with payment for plasma. the critical question is then how a country can assure ongoing supply at a reasonable cost. ethical principles will play an important role in defining the strategy and individual countries should be able to define their own approach without unnecessary intervention and competition from the commercial plasma industry. 4a-s19-02 background: blood transfusion service of nepal red cross society was established in the year 1966. since the beginning nrcs has been running the service exclusively with assurance of government of nepal. in 1991 the government of nepal elaborated the mandate with clear policies and declared nepal red cross society as the sole authority to conduct the national blood program through out the country. at present it consists of 86 centers in 62 districts. national leadership and oversight of management and quality standards is the responsibility of the central blood transfusion service, who in partnership with regional and district centers and aims to provide a safe and secure supply of blood/blood products to all needy in the country. in the initial years the service was made possible through collection of blood from professional donors but since 1982 collection of blood was emphasized from voluntary non-remunerated donors only. aims: blood service is provided according to the need base and with the expansion of health services, establishments of medical colleges, government hospitals, private hospitals and nursing homes the demand of blood is rapidly going up through out the country. nationwide more than 250,000 units of blood were served to the needy and around 60% of it is served through the central blood transfusion service. results: with the view of providing safe blood and bloodproducts to the needy patients all collected blood is routinely tested for hiv, hbsag, hcv, syphilis besides grouping, cross-matching and anti-d antibody identification. blood components service is limited in few centers. conclusion: blood program is run through cost recovery basis with material and nominal service charge nationwide. no defined budget is allocated for blood program. there is on and off support provided from nepal government, un agencies, national societies and international non government organizations. currently, the total revenue raised for the blood program is insufficient to meet the total cost of production of blood and blood products. 4a-s19-03 background: blood transfusion services in cambodia face a number of significant challenges in providing a supply of safe blood and blood products sufficient to meet the clinical needs of patients. these challenges include access to adequate and sustainable financing, trained staff, sufficient voluntary non-remunerated blood donors (vnrbd) and appropriate equipment and facilities. aims: a 2011 programme funded by the us presidents emergency plan for aids relief (pepfar) and managed by the us centres for disease control (cdc) was initiated by the cambodian based us cdc office, in a collaborative partnership with the nbts and ministry of health (moh). specific blood program needs were identified and presented in a logical task order framework. the task order required support at both the national and provincial level across the whole blood sector; and priorities three key deliverables: comprehensive assessment of the country's current practices and infrastructure including national policy, legislation, collection, testing and the clinical use of blood. vnrbd recruitment strategies/protocols. a new 5 year national strategic blood plan. methods: the selected technical assistance partner (ta) developed a proposal that addressed the areas of the task order, and utilised a number of strategies to deliver the technical program; including: a critical initial design phase with an introductory visit to map prior local and development partner work and draw on in-country. seeking ongoing engagement and advocacy from local thought leaders. tailoring assessment and strategy planning tools to local requirements. use of inclusive communications with all stakeholders. strengthening of local governance infrastructure (blood safety working group) . the 1 year program resulted in development and delivery of an assessment report on cambodia's blood transfusion services, a knowledge, attitudes and practices survey and a five year national strategic blood plan (nsp background: indonesia has recognized that blood transfusion plays a key role in their modern health care; yet ensuring the availability of safe blood is a challenge for a population of over 250 million living on 6000 islands with limited health care resources with a high background prevalence of hepatitis b virus (hbv) of 9.4%, human immunodeficiency virus (hiv) of 0.2%, and hepatitis c virus (hcv) 0.4%. aim: a health economic study was performed by the irc to evaluate the economic impact of individual donor (id) nat on the burden of disease (hbv, hcv, hiv) associated with transfusion transmitted infections (ttis) and understand the comprehensive costs to the ministry of health (moh) of providing id-nat-tested blood. method: a health economic analysis was performed using a markov method, which predicts the probability of an outcome based on a chain of outcomes and defined transition probabilities. the two arms of the analysis compared the testing costs and treatment costs of: (i) serology alone, and (ii) serology and nat. testing costs were defined as the total costs to the indonesian moh of purchasing the reagents used in conducting either serological testing alone or serological and id-nat testing on donor blood units. the treatment costs were defined by the outcomes associated with each of the three viruses (hbv, hiv, hcv) and the likelihood of various clinical outcomes for patients infected by transfusion. indonesia-specific data drawn from peerreviewed published sources was used to populate the model. in cases where published local data was not available, proxy published data from other geographies and data collected from interviews with local transfusion experts were used. cost information for id-nat and serology testing was sourced from test manufacturers. results: previous effectiveness of nat testingin annual collection of 2.1 million donations determined that id-nat could prevent 3187 infections missed by serology screening. if id-nat assays were not available ttis represents a calculated usd $73 million in treatment costs for the indonesian healthcare system. the introduction of id-nat could saveusd $27 million per year (or~20% savings) due to reduced treatment costs by preventing ttis. the estimated savings exclude costs associated with lost productivity and litigation. conclusion: sincethe initial analysis, additional testing has been performed in variousdemographic locations and it is now estimated that 8500 whole blood units andup to 15,000 blood components could be interdicted with id-nat. these new datarepresent even more favorable economics to the moh. while id-nat represents an additional cost tokeep blood as safe as possible, the incremental cost comparison of serology andid-nat compared to serology testing alone shows a minimum estimated savings ofusd $27 million. based on these results, id-nat blood screening appears to be aclinically and cost-effective way to prevent transmission of infection to patients. 4a-s20-01 yee-sin l tan tock seng hospital, singapore, singapore arbovirus is an acronym for a group of viruses that are transmitted by arthropod vectors, commonly mosquitoes and ticks. person-to-person transmission of arboviruses is not common. however, transmission can occur through transfusion of blood and blood products, and organ transplantation, if the virus is present in the donor's blood or organ. the majority of arboviral infections in humans are asymptomatic. if symptomatic, the symptoms generally occur 3-15 days after exposure. clinical features of arboviral infections can be classified into four groups: acute central nervous system illness, acute benign fever with or without exanthem, hemorrhagic fever, and polyarthritis. there have been published reports of transfusion-transmitted infections of a number of arboviruses, including west nile and dengue viruses, as well as colorado tick fever and tick-borne encephalitis viruses. the risk of transmission depends largely on the burden of the disease in a particular geographical location, the proportion of asymptomatic infections in which clinical diagnosis is not possible, and the availability of diagnostics to detect low levels of viruses in the blood. west nile virus (wnv) entered the united states (us) in 1999 and has spread across the entire north american continent. wnv is now the most significant arboviral infection in the us. one out of every five exposed individuals will develop illness and 1 out of 150 may suffer from severe infection with significant neurological sequelae. in 2002, 23 transfusion-transmitted wnv infections were described. since 2003, all blood donations in the us have been routinely screened for wnv rna using wnv nucleic acid test (nat). since the implementation of screening, 13 transfusion-associated transmissions of wnv have been documented (mmwr 2012) . dengue is the most common arboviral infection in the world. the world health organisation (who) estimates that 50 million dengue infections occur annually and approximately 2.5 billion people live in dengue endemic countries (who 2009 ). the ratio of asymptomatic to symptomatic infections vary widely. singapore reported three cases of transfusion-associated dengue infections involving one blood donation episode. an asymptomatic repeat blood donor informed the national blood bank that he had fever the day after blood donation. by then, packed red cells and other blood products from his donation had already been transfused to three recipients. all three recipients were infected; two of the recipients' blood and the donor's stored serum sample were tested positive for dengue serotype 2 virus by pcr. wilder-smith et al. estimated the risk for transfusion-associated dengue transmission in singapore to be 1.6-6/10,000 blood transfusions in the year 2005, assuming a ration of asymptomatic-to-symptomatic infections of 2:1 to 10:1. subsequently, teo et al. reported the practice of deferring blood donation from recently returned travellers from dengue endemic areas. the massive outbreaks of chikungunya fever in the indian ocean and southeast asia again raise concerns about blood safety. although there has yet to be a reported case of transfusion-associated chikungunya virus infection, such a transmission route remains a distinct possibility. liumbruno gm et al. reported an economic loss of approximately 2 million euros, resulting from the suspension of blood donations at the peak of the chikungunya outbreak in italy in 2008. 4a-s20-02 australian red cross blood service, perth, australia background: arthropod-borne viruses (arboviruses) are an expanding global threat to human health. among them dengue (denv) and west nile virus (wnv) have both been documented to be transfusion-transmissible and transfusion is a plausible route of infection for several others including chikungunya virus (chikv) and ross river virus (rrv). addressing the transfusion risk associated with arboviral outbreaks poses new challenges. unlike established viral threats to the blood supply (e.g. hiv, hcv and hbv) in which the risk is generalized to the population-at-large and chronic infection is common, arboviruses are characterised by focal and often seasonal outbreaks of acute, predominantly self-limiting infection. thus, unlike traditional viral threats the risk is not 'continuous' in nature complicating risk mitigation strategies. aims: to describe possible risk management strategies for arboviral transmission including; geographical donor deferral/restrictions on labile component manufacture, donation testing and pathogen inactivation (pi) which can be applied individually or in combination. methods: how strategies are applied depends on the nature of the risk i.e. whether internal (i.e. local outbreak) or an external (i.e. potentially viraemic donors returning from an outbreak affected country). geographical donor deferral can take several forms ranging from deferral of donors returning from precisely defined 'outbreak risk' areas (e.g. wnv deferral for travel to n. america) to a 'blanket' deferral of all donors returning for a defined period after return from overseas (e.g. the netherlands). while this can be an effective risk mitigation strategy it can lead to the deferral of significant numbers of donors and the impact on sufficiency needs careful consideration. in some cases (e.g. for local dengue outbreaks in australia) this can be partially addressed by restricting manufacture to 'plasma for fractionation' since pi within the fractionation process satisfactorily addresses the transmission risk. triggering implementation of deferrals for specific outbreaks is problematic because a high proportion of arboviral infections are asymptomatic so asymptomatic donor viraemia poses a risk before the first symptomatic case is apparent. instituting a deferral only during the 'transmission season' is complicated because of the difficulty in precisely defining the beginning and end donors for major transfusion-transmitted infectious diseases (ttids; hiv, hbv and hcv) has evolved from performance of serologic assays only to include nucleic acid testing (nat) for detection of window phase and occult infections in most developed and some developing countries. nat screening has also been implemented for other viruses known to be transmitted by blood components and/or plasma derivatives (parvovirus b19, hav), and for several emerging/expanding infectious disease (eid) agents that threaten blood safety in some regions (e.g., wnv in the us, hev in japan, and dengue virus in puerto rico). despite this dramatic progress, eid threats continue to be identified and cause pressure to further enhance the safety of transfusions. the concept of a 'global village' has emerged, which reflects the fact that a potential blood-borne infectious agent present in any region of the world could 'traffic' to other regions overnight. there are increasing examples of zoonotic transmissions of infectious agents, with the potential for rapid adaptation to humans and subsequent spread to blood donors and recipients. there is also intense research to identify new blood born infectious agents using novel molecular discovery strategies. although recent examples of putative new agents have proven to be non-pathogenic (hgv/gbvc, ttv and sen-v), not transmitted at significant rates by transfusions (hhv-8), or contaminants (xmrv), every newly discovered agent requires serious investigation to assess its relevance to transfusion safety. this presentation will highlight recent progress in developing approaches for systematic assessment of eid risks and for applying rational approaches to respond to new pathogens. in an effort to proactively assess transfusion safety threats in the united states and canada, the transfusion-transmitted diseases committee of the aabb has systematically reviewed relevant data and prioritized the risks of individual established and emerging disease agents; this program includes aabb website accessible fact sheets for~100 agents which are updated as new information becomes available or new agents of concern are identified. recently a research program has been started using data of id-nat users for analyses of the efficacy of various testing strategies this allows comparison of the yield and risk reduction obtained by nat and serology assays in countries with differing incidence and prevalence rates of the three major viral infections. furthermore the aabb eid sub-group and the european cdc have developed 'toolkits' for assessing the risk of eids with respect to potential impact on blood safety and availability, and for evaluating and validating the effectiveness of proposed interventions. the toolkit process, including methods to monitor eid agent emergence, identify and recognize a transfusion-transmission threat, processes to quantify risk, and the appropriate management of such threats using formal decision analysis methodologies, should be considered for implementation by all blood systems. the main challenges for hospital transfusion are patient safety, the effective use of blood, robust methods for documentation and monitoring practice, good blood stock management and low wastage, good staff training, and the rapid availability of blood for those patients who need it urgently. patient safety is paramount, but haemovigilance schemes worldwide continue to demonstrate that errors in the transfusion process result in patient morbidity and mortality. this talk will indicate where progress has been made in improving transfusion safety in hospitals in the uk, and use the implementation of an electronic transfusion management system in oxford as an example of improving patient safety. the project involved the development of an end-to-end electronic transfusion process involving barcode patient identification, handheld computers at the bedside and electronically controlled blood fridges linked to the blood transfusion laboratory information system. it was taken through pilot stages between 2001 and 2006 through to its full implementation across the acute hospitals in oxfordshire in 2006/07, and its maintenance and further development since then. there was an improvement from 11.8% to 100% of staff following the process for correct bedside patient identification; no abo incompatible red cell transfusions or serious wrong blood events in over 10years; a reduction in wrong blood in tube events; reduced nursing (one nurse rather than 2 and half the time to administer blood) and laboratory workload; and more rapid delivery of urgently required red cell units (from a median of 18 min to 45 s). the electronic system provided a simple mechanism for compliance with uk/eu regulatory requirements for the traceability of blood, and the documentation of transfusion and training. feedback from both staff and patients was positive. our group wrote a national specification and provided the material for a national recommendation for hospitals to adopt the electronic transfusion process, but wider implementation by other hospitals has been slow. an additional module into the electronic transfusion process is being implemented to provide decision support for doctors ordering blood to minimise inappropriate use of blood as part of a patient blood management programme. there are considerable barriers to improving the safety of transfusion practice in hospitals, but they can be overcome by 're-engineering' the process with the support of a multidisciplinary team with shared, achievable and measurable objectives, engaging with senior hospital management, and having a determination to see the development of a new process through to completion and its implementation as routine practice. whats new with trali? in the last 20 years there have been substantial efforts to minimise or eliminate trali. despite these activities, in 2012 the fda still reported trali as the top cause of transfusion-associated mortality. the pathophysiology of trali follows a two event mechanism. the patient's illness leads to activation of the pulmonary endothelium with the sequestration of primed neutrophilsthe first event. the transfusion is the second event, and leucocyte antibodies or biological responsive modifiers (brms), present in the blood product, activate the microbicidal arsenal of the primed neutrophils to cause injury to the pulmonary microvasculature and consequently acute lung injury (ali). hence, trali may be antibody-mediated or brm-mediated. antibodies to human neutrophil antigens (hna), hla class i and class ii have been associated and implicated with trali. interestingly, a large prospective case-controlled study of over 400,000 transfusions reported that recipients with cognate antigens to hna and hla class ii antibodies had increased risk of trali but found no statistical significance with hla class i (toy et al. 2012 blood) . that hna-3a antibodies are associated with more severe trali indicates that antibody specificity is another determinant of trali injury (reil et al. 2008 vox sang) . additionally, murine models demonstrated that antibody titre also determined the severity of injury (fung et al. 2010 blood) . while the use of male predominant plasma has significantly reduced the incidence of antibody-mediated cases, trali still occurs likely due to brms. brms are generated during the routine storage of blood products. a meta-analysis of cardiac and trauma patients concluded that older blood is associated with a significant risk of death (wang et al. et al. 2012 blood) . given the success of male predominant plasma in managing the risk of antibody-mediated trali, ongoing efforts to reduce the risk of trali are likely to focus on brm-mediated trali. developing a better understanding of the how specific brms may contribute, either individually or in combination, to the development of trali, and how brm concentrations vary between donors, will be crucial to the future development of strategies to manage the risk of brm-mediated trali. they were all thais and their forebears have lived in northeast thailand for at least two generations. hna-1,-3, and -4 were typed by the pcr-sequence specific primer (pcr-ssp). results: the gene frequencies of hna-1a, -1b, -1c were 0.697, 0.302 and 0.001, respectively. hna-1 null allele was found in one sample. the gene frequencies of hna-3a and -3b were 0.780 and 0.220, respectively. hna-4a and -4b gene frequenfrom asian populations aims: to assess whether symptoms consistent with dengue are more common in transfused patients who test positive for denv rna compared to patients who do not. methods: in 2012 and rio de janeiro, patients who were transfused were enrolled and interviews inquiring about symptoms (fever, headache, bone or joint pain, body or muscle pain, vomiting, rash, painful eyes, and bruising) were conducted between 3 to 7 days, and about 30 days following transfusion. inpatients and outpatients were included. blood samples were collected at the time of first interview. subsequently, coded samples were tested for denv rna using the hologic/novartis tma assay. we used two definitions for denv disease: the who clinical definition consisting of fever with at least two contemporaneous symptoms, and a more general definition of having â�¥3 symptoms of any kind. we calculated odds ratios (or) and 95% confidence intervals (ci) for symptomatic infection comparing transfused denv rna+ to rnaã� patients. results: of 595 patients with samples and interviews 3-7 days following transfusion 54% were inpatients while it is possible that viremia was missed in some recipients who had symptoms consistent with dengue, we did not find evidence of significantly increased rates of symptomatic infection in denv rna+ compared to rnaã� recipients. investigation into whether any of these patients acquired denv through transfusion and mie prefectures) where there are many immigrants from latin america including japanese-latin americans. methods: we conducted a questionnaire survey on chagas disease for the subjects who were borne or grew up in latin america. since jan. 8th 2013 we have performed testing for anti-t. cruzi for donors from whom informed consent for epidemiological study was obtained. tests used were ortho t. cruzi elisa assay and an immunochromatographic assay donors who were aware of chagas disease and kissing bug were 67% and 56%, respectively. nobody has ever been bitten by kissing bug. 14% of the donors had been tested for anti-t. cruzi in their country. four donors had a family member with chagas disease and they were all brazilian. four donors had been diagnosed as having a cardiac or digestive system disease before. all of the 287 blood samples obtained from the subjects were anti-t. cruzi-negative except for one false positive result. conclusions: none of the subjects surveyed were anti-t. cruzi-positive. most of latin american blood donors in tokai region of japan are brazilian and about twothirds of them were younger than 40 years. we consider that the risk of transfusion background: leukocyte antibodies presence in the blood product(s) was responsible for the development of immune mediated transfusion related acute lung injury (trali) in recipients. several studies had demonstrated that hla antibodies (class i and class ii) as well as hna antibodies (hna-1, -2, and -3) were capable to induce trali reaction. among them, hla class ii and hna-3a antibodies seemed to be the most frequent antibodies responsible for severe trali reaction. therefore, screening of hla and hna antibodies in our female blood donors may reduce the risk of trali. however, the prevalence of leukocyte antibodies among parous female blood donors in china is currently not available. study design and methods: blood samples were collected from 142 nulliparous females, 343 parous females and 453 male donors. pregnancy history was obtained from consenting donors. hla class i and ii antibodies were screened by the use of lifecodes lifescreen deluxe (lmx), and antibody specificities were determined with lifecodes lsatmclass i & ii (single antigen). antibodies against neutrophil antigens hna-1 and -2 were screened by the labscreen assay (one lambda, inc) and rapid elisa detection (bayat et al, 2009; werth et al, 2013). hna-3 antibodies were analyzed by antigen capture assay (bayat et al, 2012). the specificity of positive samples was reanalyzed by the granulocyte immunofluorescence test (gift), the granulocyte agglutination test (gat). results: screening of the total cohort of 938 donors (453 males and 485 females) showed higher prevalence of hla antibodies production in female compared with male donors (18.9% vs 4.63%). we found in 61 out of 485 females donors alloantibodies against hla class i (12.58%) and hla class ii (10.10%). analysis among female additional immunosuppression may be used in patients with low adamts13 due to autoantibodies. aims: to investigate differences between idiopathic and secondary ttp with regard to clinical presentation, management and outcomes. methods: the australian ttp registry was established in 2009, and expanded in 2012 to include all tma. of 34 hospitals currently participate in the registry. of 103 cases in 94 patients were registered from 18 sites; eight were excluded for incomplete data, leaving 95 episodes in 86 patients. results: median age was 46 years (15-83 years), 53% were female, 16% were relapses. of 51% were idiopathic and 49% had â�¥1 identified precipitant, including infection 23%, autoimmune 18%, malignancy 12%, medication 14%, pregnant/postpartum 4%, solid organ transplant 3%, and allograft 3%. adamts13 levels were available in 66 cases (37 idiopathic and 29 secondary). adamts13 was <10% in 22/37 (59%) idiopathic and 9/29 (31%) secondary cases. precipitants in cases with adamts13 <10% included infection (n = 5), autoimmune (n = 5), medication (n = 1), and pregnancy (n = 1). neurological, gastro-renal, bleeding and thrombotic presentations were similar in idiopathic and secondary cases. data on plasma therapy were available in 88 cases (table 1) . of 84/88 (95%) received pex, by centrifugation in 28/84, membrane filtration in 25/84 (30%), and unknown method in 29/84 (35%). plasma infusions were used in 11/88 cases (without pex in 4). data on additional therapy was available for 89 cases (table 2) . rituximab was given in 12/45 cases with â�¥1 precipitant, including infection (n = 5), non-haematological malignancy (n = 1), haematological malignancy (n = 3), autoimmune (n = 5), medication (n = 2), and pregnancy (n = 1). death rates were lower in idiopathic than in secondary cases (8% vs 25%, p = 0.05). conclusion: our data confirm that very low adamts13 does occur in cases with secondary causes identified. clinical presentation did not differ between idiopathic and secondary cases. plasma therapy was similar in both groups and additional immunosuppression was not only used in idiopathic cases, but also in secondary cases. mortality remains high, and more data to support improved diagnostic and therapeutic approaches are needed. background: living donor liver transplantation (ldlt) is a curative treatment for patients with end-stage liver disease. ldlt is associated with major blood loss and allogenic blood transfusions. although transfusion practices in adult liver transplant (lt) has been widely studied, the same in pediatric lt has not been well documented in india. aims: the purpose of the study was to evaluate the intraoperative transfusion requirements in pediatric ldlt recipients (<192 months) against the diagnosis, preoperative clinical and laboratory variables. methods: the data was collected retrospectively from blood bank and patient records. demographic and important preoperative variables namely clinical diagnosis, peld score, hemoglobin (hb), platelet count, inr, albumin, and fibrinogen levels were reviewed. data on intraoperative transfusion of packed red cells (prc), fresh frozen plasma (ffp), platelet concentrates (plt), cryoprecipitate was computed as ml/kg bw. massive transfusion (mt) was defined as >80 ml/kg of blood components during the surgery. analysis was done using spss software version 16. median levels were compared between mt and non-mt group using mann whitney test. results: between august 2010 to july 2013, 60 pediatric ldlt were performed in a single center in south india. thirty five (58.3%) of them were females and 25 (41.7%) males; 32 (54%) of the recipients blood group were o positive, 14 (23%) b, 9 (15%) a and 5 (8%) ab. recipient characteristics are given in table. transplant indications were biliary atresia and cirrhosis in 28, metabolic /hereditary liver disease in 24, hepatic tumors in 5 and acute liver failure in 3 recipients. of 56 (93.3%) patients received prc, 40 (70%) ffp, 17 (28.3%) plt and 34 (56.4%) cryoprecipitate. sixteen (26.7%) patients received massive transfusion (mt) with a median peld score of 22 (ã�7 to 43) compared to 9 (ã�9 to 46) recipients without mt (p < 0.005). also, mt group had significantly lowered median levels (preoperative) compared to non-mt group, viz. hb (8.6 vs 10.3 mg/dl), platelet count (96 vs 186 9 10 3 per mm 3 ), fibrinogen (137 vs 248 mg/dl), and a higher bilirubin (16.5 vs 3.7 g/dl). transfusion requirements of ffp was higher in acute liver failure (53 ae 17.3 ml/kg) compared to metabolic liver disease (11.1 ae 2.2 ml/kg) and biliary atresia (19.5 ae 4.2 ml/kg); (p = 0.001). conclusion: to conclude, massive blood transfusion requirement in pediatric recipients during ldlt was associated with higher peld score, and more deranged preoperative hematological and coagulation status. in depth analysis of recipient disease status, controlling for the effect of surgical interventional variables on larger samples are recommended to develop predictive models of transfusion therapy. conclusions: this study is the first report of hna gene frequencies in ethnic northeast thais. it could be used for the risk prediction of alloimmunization to hna and estimation of alloimmune neutropenia and trali in the ethnic northeast thai population. 3d-s18-01 distler pb 1 , slaper-cortenbach i 2 and ashford p 1 1 iccbba, san bernardino, united states of america 2 university medical center utrecht, utrecht, the netherlandsbackground: standardized isbt 128 terminology is used by cellular therapy organizations in many countries. as products evolve and new products are created, terminology is required to support the new products. changes to the terminology are managed by the cellular therapy coding and labeling advisory group (ctclag), a committee of experts representing international professional cell therapy societies, technical experts, and regulatory liaisons. since the early nomenclature was devel-oped, the ctclag has approved classes and terminology for very innovative products, including some for which therapeutic benefit has yet to be clearly demonstrated. because the term 'therapeutic cell' was used in the terminology, this became a great concern to the us fda, even to such an extent that the use of isbt 128 for cellular therapy products in the usa could be problematic. aims: the ctclag recognized the concerns raised by regulators and determined it needed to revise nomenclature to address these concerns and to be consistent with isbt 128 nomenclature used in related fields. methods: the ctclag held a face-to-face meeting and reconsidered the use of tc (therapeutic cells) terminology for products and proposed new nomenclature for the problematic terms. a draft of new nomenclature was developed and made available for public comment as well as review by the boards of ctclag sponsoring organizations (aabb, apbmt, asbmt, asfa, ebmt, fact, isbt, isct, jacie, nmdp and wmda). following this review, terminology was updated. results: major changes are:(1) the class name will comprise the type of cells and, where appropriate, the source (eg. 't cells, cord blood').(2) hyphenated class names will be replaced (3) tc and therapeutic terminology will be replaced (4) modifiers will be replaced with attributes providing the same information (5) new attributes will be added the terminology remains compatible with the single european coding system. summary/conclusions: changing terminology will create rework for facilities that have implemented isbt 128 and delay for those in the process of implementation. however, it was felt the revised terminology will provide a strong foundation for consistent nomenclature as new products are developed and address regulatory concerns. an appropriate timescale for implementation of the revised terminology in facilities already using isbt 128 will be developed. this presentation will describe the revised terminology and explain the reasoning supporting the changes.3d-s18-02 kordelas l 1 , rebmann v 2 , ludwig a-k 2 , radtke s 2 , beelen dw 1 , giebel b 2 and horn p 3 1 department of bone marrow transplantation, university hospital essen, essen, germany 2 institute for transfusion medicine, university hospital essen, essen, germany 3 university hospital essen, essen, germanybackground: graft-versus-host disease (gvhd) is a major cause of morbidity and mortality after allogeneic stem cell transplantation. a number of studies reported positive impacts of systemically applied mesenchymal stem cells (mscs) for preventing or treating acute gvhd. in contrast to the initial paradigm that mscs intercalate into injured tissues and thus reduce tissue damage, it is now widely assumed that mscs secrete a number of immune-modulatory factors, which impair inflammation and thus help to suppress gvhd. exosomes are secreted cell organelles, which exert immune-modulatory properties. these small membrane vesicles are released by a huge variety of different cell species, including mscs. methods: here, we enriched exosomes from bone-marrow derived mscs of four different unrelated stem cell donors and compared their immune modulatory properties in vitro. next, we administered immunosuppressive msc-derived exosomes in escalating doses into a 22-years female gvhd patient. this patient suffered a severe and therapy-refractory cutaneous and intestinal gvhd grade iv. we monitored the clinical effects on an in-hospital basis and correlated this with the levels of inflammatory cytokines measured in the patient's plasma. results: we show that even though all propagated msc lines released exosomes, exosome-enriched fractions differed in their potential to modulate immune responses in vitro. administration of the exosome-enriched fraction with the strongest immune suppressive in vitro effect into the gvhd patient was well tolerated and appeared to be safe. during the course of the exosome therapy a clear reduction of the proinflammatory cytokines il-6, il-8 and il-17 was observed in the patient's plasma. in line with that, the clinical cutaneous and intestinal gvhd symptoms improved significantly and the dosage of the immunosuppressive agentsparticularly of steroidscould be reduced. in total the patient was stable for 5 months. interpretation: msc exosome-enriched fractions exert immune suppressive functions in vitro and in vivo. since the in vivo administration seems to be safe, msc exosome administration appears as a promising new treatment option for steroid refractory gvhd patients. key: cord-333391-6l0cpvgr authors: bortolotti, daria; gentili, valentina; rizzo, sabrina; rotola, antonella; rizzo, roberta title: sars-cov-2 spike 1 protein controls natural killer cell activation via the hla-e/nkg2a pathway date: 2020-08-26 journal: cells doi: 10.3390/cells9091975 sha: doc_id: 333391 cord_uid: 6l0cpvgr natural killer cells are important in the control of viral infections. however, the role of nk cells during severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection has previously not been identified. peripheral blood nk cells from sars-cov and sars-cov-2 naïve subjects were evaluated for their activation, degranulation, and interferon-gamma expression in the presence of sars-cov and sars-cov-2 spike proteins. k562 and lung epithelial cells were transfected with spike proteins and co-cultured with nk cells. the analysis was performed by flow cytometry and immune fluorescence. sars-cov and sars-cov-2 spike proteins did not alter nk cell activation in a k562 in vitro model. on the contrary, sars-cov-2 spike 1 protein (sp1) intracellular expression by lung epithelial cells resulted in nk cell-reduced degranulation. further experiments revealed a concomitant induction of hla-e expression on the surface of lung epithelial cells and the recognition of an sp1-derived hla-e-binding peptide. simultaneously, there was increased modulation of the inhibitory receptor nkg2a/cd94 on nk cells when sp1 was expressed in lung epithelial cells. we ruled out the gata3 transcription factor as being responsible for hla-e increased levels and hla-e/nkg2a interaction as implicated in nk cell exhaustion. we show for the first time that nk cells are affected by sp1 expression in lung epithelial cells via hla-e/nkg2a interaction. the resulting nk cells’ exhaustion might contribute to immunopathogenesis in sars-cov-2 infection. in december 2019, a novel coronavirus was isolated in wuhan, china [1] . the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was the causative agent for coronavirus disease 2019 . sars-cov-2 is a β-coronavirus with a 79.5% sequence homology with sars-cov [2, 3] . the covs have been demonstrated to be able to adapt quickly and cross the species barrier, as happened with sars-cov and middle east respiratory syndrome cov (mers-cov), with resulting epidemics or pandemics. the effect of these infections on humans often leads to severe clinical symptoms and high mortality [3] . the number of sars-cov-2-infected cases is more than 22 million, with typical clinical manifestations including fever, cough, diarrhea, and fatigue [4] . several studies are currently investigating the potential response of the immune system during sars-cov-2 infection [5] . it has already been shown that, during the infection, patients develop an uncontrolled immune response and hyperactivation of macrophages and monocytes [6] . this immune dysregulation is associated with an increase in interleukin-6 (il-6), neutrophils, natural killer (nk) [7] cells, and reactive protein c (pcr) and in a decrease in the total number of lymphocytes [5, 8] . interestingly, nk cells showed a functional exhaustion with increased nkg2a expression [8] . nk cells are important effectors in antitumor and anti-infection immunity [9] . the activity of nk cells is controlled by activating and inhibitory receptors [10] . the cd94/nk group 2 member a (nkg2a) is a heterodimeric inhibitory receptor expressed by nk cells [11] . it binds to the nonclassical hla class i molecule (hla-e), which presents peptides derived from leader peptide sequences of other hla class i molecules, including hla-g [12] [13] [14] . the ligation of the peptide-loaded hla-e with nkg2a transduces inhibitory signaling through two inhibitory immune-receptor tyrosine-based inhibition motifs, which suppress nk cytokine cytotoxicity and secretion [11, 12, 15, 16] . by now, no data are available on how sars-cov-2 might control nk cells. we evaluated the possible role of sars-cov-2 spike proteins in modifying nk cell functions. human primary nk cells were obtained from the peripheral blood of four healthy blood donors. this study was approved by the "ferrara ethics committee". all subjects gave written informed consent in accordance with the declaration of helsinki. primary nk cells were separated from peripheral blood samples using the negative magnetic cell separation (macs) system (miltenyi biotech, gladbach, germany) [17] . the analysis of purified cell fraction by flow cytometry with cd3-percp-cy5.5, cd56-fitc moabs (e-bioscience, frankfurt, germany), demonstrated that the nk cell content was > 90% ( figure s1a ). rna extraction was performed by using the magmax viral/pathigen nuclei acid isolation kit (thermofisher, milano, italy) according to the manufacturer's instructions. the rt-pcr was performed with the taqman 2019ncov assay kit v1 (thermofisher, milano, italy). nk cell were supplemented with il-12 (10 ng/ml) (becton dickinson, san jose, ca, usa) to have a positive control for ifn-gamma secretion [18] . the beas-2b (atcc crl-9609) bronchial epithelial cell line was grown in begm culture medium (begm kit catalog no. cc-3170; lonza/clonetics corporation, usa). the k562 (atc ccl-243) lymphoblastoid cell line was cultured in roswell park memorial institute medium (rpmi) (gibco, milano, italy) supplemented with 10% of fcs (fetal calf serum, euroclone, pero, mi, italy) and 100 u/ml penicillin and 100 µg/ml streptomycin. cell cultures were maintained at 37 • c in a humidified atmosphere of 5% co 2 in air. the role of hla-e and nkg2a was evaluated by incubating the cells with anti-hla-e (clone mem-e/08, exbio, praha, cz) or anti-cd94/nkg2a (clone 131411, bd, italy) antibodies (7.5 ng/ml). gata3 dna-binding activity was inhibited by adding pyrrothiogatain (cat#sc-352288a, santa cruz, ca, usa) to cell cultures (10 µm) [19] . in total, 1 × 10 5 enk cells were labeled with fluorophore-conjugated antibodies: cd3-pe (clone sp34-2, cd16-apc (clone b73.1), cd56-pe-cy7 (clone b159), nkg2a-apc (clone 131411), and matched isotype controls. in total, 5 × 10 5 bronchial epithelial cells were stained specific ab hla-i (hla-a, -b, -c)-pe (becton dickinson, san jose, ca, usa), hla-e (clone mem-e/08)-fitc, and matched isotype controls. gata3 expression was evaluated in fixed and permeabilized cells with intraprep reagent (beckman coulter; brea, ca, usa) and stained with anti-gata3 (bv421; bd)-pe moab. ck8 expression was evaluated with anti-ck8 (ts1, novus biologicals, milano, italy)-pe moab. sp1, sp2, and s proteins expression in k562 and beas-2b transfected cells were evaluated with anti-spike 1 (clone 2c1), anti-spike 2, and anti sars-cov spike antibodies (mybiosource, san diego, ca, usa). data were analyzed using facs cantoii flow cytometer (bd, milan, italy) and flowjo llc analysis software (tree star inc, ashland, or, usa). in total, 10,000 events were acquired. hla-e expression was analyzed by immunofluorescence with anti-hla-e-pe antibody (mem-e/08-pe), as previously described [20, 21] . in particular, 10 5 cells were spotted onto glass slides and fixed by cold acetone for 30 min at 20 • c. slides were air-dried and kept at 20 • c until use. for assay, slides were rehydrated by washings in pbs, and incubated with anti hla-e-pe-labelled antibody diluted 1:100 in pbs for 35 min at 37 • c in a humidified chamber. slides were washed twice with pbs for 10 min, once for 1 min with tap water, and once for 1 min with distilled water. after washings as described, slides were stained with 1:2000 in pbs diluted hoechst stock solution (thermofisher, milano, italy) for 5-10 min, washed in distilled water, and finally mounted with 50% glycerol in pbs for fluorescence microscope observation. all samples were observed under a uv light microscope (nikon eclipse e600) equipped with a digital camera (dmx 1200). the assay was performed using the cornig transwell system, with inserts with 5-µm pore polycarbonate membrane. briefly, 1 × 10 6 cells were seeded in the upper chamber in 150 µl of rpmi with the 0.5% of fbs and il-2 (100 iu/ml; sigma biosciences, mo, usa). the inserts containing cells were positioned into a 24-well plate, which provides the lower reservoirs for the migration system. each reservoir was filled with 650 µl of medium (rpmi) containing sars-cov-2 proteins (spike protein s1 subunit, spike protein s2 subunit) (raybiotech, peachtree corners, ga, usa) or control sars-cov spike protein (mybiosource, san diego, ca, usa) at the final concentration of 100 ng/ml. medium with cxcl12 (100 ng/ml; biolegend, san diego, ca, usa) [22] was used as the positive. migration was performed for 3 h at 37 • c and then the plate was briefly centrifuged at 300× g for 5 min in order to collect migrated cells in the lower reservoir for the cell count. every condition was tested in triplicate and results were reported as the number of migrated cells compared to untreated nk cells. k562 or beas-2b cell lines were transfected using the pierce protein transfection kit (thermofisher, milano, italy) following the product instructions. a total of 4 × 10 5 cells were transfected with 1 µg of protein (spike protein s1 subunit, spike protein s2 subunit) of sars-cov-2 or sars-cov spike protein. transfection was performed for 3-4 h at 37 • c in 1 ml of medium without fbs. after transfection, a volume of complete medium with 20% fbs was added to each well. k562 or beas-2b cells treated with transfection reagent alone or transfected with 0.5 µg of control fluorescent antibody (provided in the kit) were used as the negative and efficiency control, respectively. the ldh assay was performed to evaluate the effect of the transfection with sars-cov-2 and sars-cov proteins in k562 or beas-2b cells on cell viability. transfected k562 or beas-2b cells were suspended at 5 × 10 4 cells/ml and cultured for 4 h on a 96-well microplate at 37 • c with 5% co 2 . a colorimetric-based lactate dehydrogenase (ldh) assay (cytotoxicity detection kitplus; switzerland) was used, according to the manufacturer's instructions. in vitro cytotoxicity experiments were performed using k562 or beas-2b cells as the target and nk cells as effector cells. nk cells were added to k562 or beas-2b cells with a 5:1 effector:target ratio [23] . nk cell degranulation was evaluated by cd107a staining (anti-cd107a-pe; clone h4a3; bd biosciences) after 3 h of treatment with golgi stop solution (bd). labeled cells were analyzed with a facscantoii flow cytometer (bd, milano, italy) and flowjo software (tree star inc., ashland, or, usa). k562 or beas-2b cells were labeled with carboxyfluorescein diacetate succinimidyl ester (cfse) to assess cell-mediated cytotoxicity, using a 7aad/cfse cell-mediated cytotoxicity assay kit (ann arbor, mi, usa). in total, 10 7 cells/ml were resuspended in cfse staining solution and incubated for 15 min at 37 • c. control target cells were resuspended in 0.1% bsa. then, cells were washed two times with culture medium and incubated for 30 min at 37 • c. nk cells were put in co-culture with cfse-labeled infected cells at a 1:5 ratio. the cell mixture was incubated for 4 h, centrifuged, and resuspended in 7-aad staining solution. control target cells were resuspended in assay buffer. cells were incubated for 15 min in the dark at 4 • c. then cells were centrifuged and resuspended in assay buffer. cells were analyzed with a facscantoii flow cytometer and flowjo software. ifn-gamma levels were detected by an ifn-gamma elisa kit (mybiosource, san diego, ca, usa) following the instructions. in particular, standards and samples were pipetted into the wells and ifn gamma present in a sample was bound to the wells by the immobilized antibody. the wells were washed and biotinylated anti-human ifn gamma antibody was added. after washing away unbound biotinylated antibody, hrp-conjugated streptavidin was pipetted to the wells. the wells were again washed, a tmb substrate solution was added to the wells, and color developed in proportion to the amount of ifn gamma bound. the stop solution changed the color from blue to yellow, and the intensity of the color was measured at 450 nm (thermofisher elisa reader). rna extraction was performed by using the rneasy kit (qiagen, hilden, germany) according to the manufacturer's instructions. the real-time pcr was performed with the taqman 2019ncov assay taqman gene expression assays for hla-e (hs00428366_m1), gata3 (hs00231122_m1), eotaxin3 (hs00171146_m1), and nkg2a (hs00970273_g1) (thermofisher). the hla-e binding prediction was made using the iedb analysis resource ann aka netmhc (ver. 4.0) tool at the http://tools.iedb.org/mhci/help/, using the viral spike 1 protein from the sars-cov-2 sequence (qho62112.1). since the biological variables presented a normal distribution (kruskal-wallis test, p > 0.05), they were evaluated by student t test by graph pad prism 6 software (https://www.graphpad.com/ scientific-software/prism/). a p-value < 0.05 was defined statistically significant. all relevant data are within the manuscript. although the role of nk cells in the immune response towards viral infections is generally accepted, there are few data on the early nk cell trafficking in response to coronavirus infections [5] . the evaluation of nk response to sars-cov-2 infection is important to determining the innate immune response per se and for the cross-talk with adaptive immune cells [24] . we explored the migration, interferon-gamma (ifn-gamma) secretion, and degranulation of nk cells in the presence of spike 1 (sp1) and spike 2 (sp2) sars-cov-2 proteins and sars-cov spike proteins (s). we used a cell migration system in a 5-µm transwells w/polycarbonate filter, without a cell barrier. we purified nk cells from the peripheral blood of control subjects ( figure s1a ) negative for both sars-cov-2 and sars-cov viremia (data not shown). this condition ensured that nk cells were naïve for spike proteins. we cultured nk cells in the presence of sp1, sp2 from sars-cov-2, or spike protein (s) from sars-cov and we observed no effect on cell viability ( figure s1b ). we used cxcl12 as a positive control for nk cell migration [22] . we observed an increase in nk cell migration in the presence of both sp1 and sp2 (sp1 p = 0.021; sp2 p = 0.013 student t test) ( figure 1a) . similarly, nk cell migration was induced by the sars-cov s protein (p < 0.01; student t test) ( figure 1a) . no modifications in cell viability were observed ( figure s1b ). these data suggest that both sars-cov-2 and sars-cov spike proteins are able to chemo-attract nk cells. to evaluate if spike proteins are able to also induce nk cells' activation, we analyzed the secretion of ifn-gamma. we observed an increase in ifn-gamma secretion in the presence of sp1, sp2, and s-protein (p < 0.001; student t test) ( figure 1b) . the basal ifn-gamma secretion might be enhanced by the il-2 treatment of nk cells, which is necessary for the in vitro maintenance of primary nk cells. then, we evaluated the cytotoxicity of nk cells in the presence of spike proteins, using cd107a staining, a marker of degranulation [23] . we mimicked the expression of spike proteins inside nk target cells, the k562 cell line, transfecting directly the proteins. we obtained a mean transfection efficiency of 95% for sp1, sp2, and s proteins ( figure 1c ). the k562 cell viability, evaluated by the ldh assay, was not affected by protein transfection ( figure 1d ). we incubated nk cells for four hours with k562 and evaluated the expression of cd107a on cd56-gated nk cells ( figure s1c ). we observed an increase in cd107a staining in all the culture conditions, with no difference in the presence of sp1, sp2, and s proteins in comparison with the untreated co-culture (s1 p = 0.078; s2 p = 0.087; s p = 0.081; student t test) ( figure 1e ,f). to be sure that nk cells expressing cd107a were able to kill k562 cells, cfse (carboxyfluorescein diacetate succinimidyl ester), the staining of target cells was detected by flow cytometry [20] . we observed an increase in the k562 cfse+/7-aad+ cell percentage in all the co-culture conditions comparable with the killing observed in the untreated co-culture (p < 0.001; student t test) ( figure 1g ), confirming the results observed with cd107a staining (figure 1e ,f). since the target cells for sars-cov-2 replication are lung epithelial cells [4] , we considered the activation status of nk cells in this context. we mimicked the expression of sp1, sp2, and s proteins inside beas-2b lung epithelial cells, transfecting directly the proteins. we obtained an average 95% efficiency of transfection with all the proteins (figure 2a ). the transfection did not affect cell viability ( figure 2b ) and the expression of ck8 epithelial cell markers (figure 2a ). we incubated nk cells for four hours with lung epithelial cells and evaluated the expression of the cd107a degranulation marker. we observed a decrease in cd107a staining in the culture condition with sp1-transfected beas-2b cells (p < 0.001; student t test) ( figure 3a,b) . on the contrary, we observed an increase in cd107a expression in the co-culture conditions with sp2 and s protein-transfected beas-2b cells, which is similar to that observed with the control of the transfection condition (null-transfected) and untreated co-culture (p = 0.079; p = 0.085, respectively; student t test) ( figure 3a,b) . to be sure that nk cells expressing cd107a were able to kill beas2b cells, cfse staining of target cells was detected by flow cytometry. we observed an increase in the beas-2b cfse+/7-aad+ cell percentage in the co-culture with nk cells in sp2 and s protein-transfected beas2b cells ( figure 3c) , while there was a decrease in the beas-2b cfse+/7-aad+ cell percentage in the co-culture with nk cells in sp1-transfected beas-2b cells (p < 0.001; student t test) ( figure 3c ), confirming the results observed with cd107a staining (figure 3a,b) . since the presence of intracellular sp1 protein in lung epithelial cells induced a decrease in the cytotoxic activity of nk cells, we analyzed the possible factors involved in the modification of nk cell status. since viral proteins are commonly recognized and degraded by the proteasome inside the infected cells [25] , we hypothesized that sp1 peptides might be presented to nk cells. intracellular peptide presentation is performed by human leukocyte antigen (hla) class i molecules, which are expressed by all nucleated cells. firstly, we evaluated the expression of hla class i molecules in beas-2b cells transfected with sp1, sp2, and s proteins. when we stained the cells with anticlassical hla class i molecules (hla-a, hla-b, hla-c) antibody, we recognized a significant decrease in their membrane expression when lung epithelial cells were transfected with sp1 protein (p < 0.001; student t test) ( figure 3d ,e). sp2 and s protein slightly inhibited hla-i expression (p = 0.039; p = 0.041, respectively; student t test). epithelial lung cells can express also non-classical hla-e molecule [7] . hla-e binds peptides primarily derived from specific signal sequences [26] and interacts with nkg2a/cd94 nk cell inhibitory receptors [15] . when we performed the lung epithelial cell staining with mem-e/06 anti-hla-e antibody, we observed no expression in basal conditions, which was not affected by sp2 and s transfection ( figure 3f,h) . on the contrary, there was a significant increase in hla-e protein (p = 0.011; student t test) (figure 3f ,g) and mrna expression (p < 0.001; student t test) ( figure 3h ), in the presence of sp1 protein. hla-e expression is controlled by the gata3 transcription factor [12] , which is known to also be expressed by lung epithelial cells [27, 28] . we observed an increase in gata3 protein (p < 0.001; student t test) ( figure 4a ,b) and mrna (p < 0.01; student t test) ( figure 4c ) expression only in the presence of sp1. to confirm the transcriptional activity of gata3 induced by sp1, we analyzed the expression levels of a target gene for gata3, the eotaxin3/ccl26 [29] . we observed the induction of ccl26 transcription only in the presence of sp1 (p < 0.001; student t test) ( figure 4d ). the treatment with a gata inhibitor, the pyrrothiogatain [19] , reduced both gata3 protein ( figure 4a ,b) and mrna ( figure 4c ) expression. to be sure that the increase in hla-e expression in lung epithelial cells transfected with sp1 protein is controlled by gata3 transcription factor, we treated the cells with pyrrothiogatain. this inhibitory molecule reduced the expression and transcription of the hla-e gene in beas-2b cells ( figure 3f ,h). the immune-fluorescence staining showed increased modulation of the expression of hla-e molecules in the presence of intracellular sp1, which was reduced with the addition of gata inhibitor ( figure 5a,b) . to evaluate if sp1 peptides might be presented by hla-e molecules, we performed mhci (major histocompatibility class i) binding predictions, made on 26 april 2020 using the iedb analysis resource ann aka netmhc (ver. 4.0) tool. we observed that an 8mer peptide in the sp1 domain (270-277, lqprtfll) showed a high affinity mainly for the hla-e*0101 binding groove (ic50: 0.02) and in a lower extent for the hla-e*0301 allele (ic50: 0.76), with a similar consensus motif with hla-e binding (vmaprtvll) [14] . we might speculate that the induction of gata3 and the binding of sp1 peptide might induce hla-e membrane expression on lung epithelial cells. since the functional control of nk cells by hla-e is possible in the presence of nkg2a/cd94 on nk cells, we evaluated the expression of this receptor on the surface of nk cells. when nk cells were co-cultured with sp1-transfected beas-2b cells, we observed an increase in the protein (p < 0.001; student t test) ( figure 6a ,b) and mrna (p < 0.01; student t test) ( figure 6c ) expression of the inhibitory receptor nkg2a/cd94. interestingly, the percentage of nkg2a-positive nk cells increased from an average of 16% to 80%. interestingly, a high percentage of nkg2a-positive nk cells were also cd107a negative ( figure 6a ). to be sure that the resulting inactivity of nk cells towards lung epithelial cells expressing sp1 was determined by hla-e/nkg2a interaction, we treated the cell culture with anti-hla-e or anti-nkg2a/cd94 antibodies. we incubated nk cells for four hours with the sp1-transfected beas-2b cell line and evaluated the expression of cd107a in the presence or absence of anti-hla-e or anti-nkg2a antibodies. we observed a decrease in cd107a-positive nk cells in the culture condition with sp1-transfected beas-2b cells (p < 0.001; student t test) ( figure 7a,b) , which was recovered by the treatment with anti-hla-e and anti-nkg2a/cd94 antibodies ( figure 7a,b) . as a proof of concept, the secretion of ifn-gamma was reduced by sp1 treatment (p < 0.01; student t test) ( figure 7c ), while it was enhanced after the treatment with anti-hla-e, anti-nkg2a/cd94 antibodies, and gata inhibitor ( figure 7c ). the gaps in the activation of the immune system during sars-cov-2 infection translate into the severity of the covid19 disease. recent studies have documented a modification in the nk cell number and phenotype [5, 8, 30] . the total number of nk cells decreased in patients with sars-cov-2 infection and the expression of nkg2a on the surface of nk cells was increased, suggesting an exhausted phenotype [31, 32] . interestingly, when the patients were rescued after the infection, nkg2a expression was decreased simultaneously with the increase in the number of nk cells [30] . these results suggest that sars-cov-2 infection might compromise the innate antiviral immunity exhausting nk cells' functions [33, 34] . we evaluated the effect of sars-cov-2 spike proteins in the control of nk cell activation. we considered spike 1 protein, which is involved in the attachment of the virion to the cell membrane by interacting with ace2 receptor [35] , and spike 2 protein that mediates the fusion of the virion. we observed that the extracellular spike proteins from sars-cov-2 and sars-cov are able to induce nk cell chemotaxis and activation, via the induction of ifn-gamma secretion in the k562 cell model. these results are interesting considering the efficacy of ifn-gamma in inhibiting sars-cov replication partly through the downregulation of ace2 [36] . our data sustain the role of the immune response of nk cells during sars-cov-2 infection [37] . they would migrate to the infected sites and respond to viruses producing ifn-gamma, killing virus-infected cells, and boosting the adaptive immune response with the production of innate proinflammatory cytokine and type i ifns [5] . the spike proteins, per se, are not able to affect nk cell activation and ifn-gamma secretion. since sars-cov-2 infection is mainly localized to lung epithelial cells, where the detrimental effects of this infection are more evident [4] , we evaluated the effect of sars-cov proteins on this cell type. surprisingly, we showed that the intracellular expression of s1 protein in lung epithelial cells reduces the activation of nk cells and their ability to degranulate, which is the opposite pattern to that observed in k562 cells. these results account for the in vivo observation of a break in the interplay of lung epithelial cells and immune cells during sars-cov-2 infection [37] , with a consequent exhausted immune response [37] . to evaluate the possible mechanisms used by sars-cov proteins to control nk cells' activation, we considered that the activation of nk cells is partly controlled by the expression of hla class molecules, via the interaction with specific nk cell receptors [38] . we evaluated the possible modification of surface hla class i molecules on lung epithelial cells. the presentation of pathogen-derived peptides by hla molecules and the genetic variability of hla alleles in human populations account for their role in the individual responses to sars-cov-2 infection and/or progression. we showed that s1 protein on one side diminished classical hla class i molecule expression but on the other side upregulated hla-e expression. the protein hla-e is a non-classical major histocompatibility complex molecule that binds peptides derived from a specific signal sequence [14] . we recognized an sp1-derived hla-e binding peptide that might stabilize hla-e expression on the surface of lung epithelial cells during sars-cov-2 infection. interestingly, the highest affinity is demonstrated for the hla-e*0101 allele. hla-e surface expression conferred cell resistance to nk cell lysis, interacting with the nk cell inhibitory receptor cd94/nkg2a [14, 38] . the involvement of hla-e in the control of nk cell activation is confirmed also by the different results observed in k562 and lung cell models. k562 cells express no hla molecules, providing no effective molecules to sars-cov spike 1 protein in order to control nk cells. for this, we observed ifn-gamma secretion and nk cell activation also in the presence of sars-cov spike 1 protein in the k562 cell model. on the contrary, lung cells, which express hla-e molecules in the presence of sars-cov spike 1 protein, are able to inhibit ifn-gamma secretion and nk cell activation. since, it was shown earlier that hla-e is tightly upregulated through gata3 response elements [12] , we evaluated the role of this transcription factor. we observed that hla-e up-modulation by sp1 is controlled by gata3 transcription factor. in fact, the treatment with gata inhibitor reduced the expression of hla-e even in the presence of sp1. gata3 is a transcription factor that drives the differentiation of t helper (th) 2 cells [28] , immune regulation [27] , and embryonic and adult non-hematopoietic cells' differentiation, including the lung [39] . the upregulation of gata3 mrna and protein by sp1 protein might have other important effects on lung epithelial cells that surely deserve to be evaluated. more interestingly, gata3 is not only up-modulated, but it is transcriptional active, as showed by the induction of mrna transcription of a gata3 target gene, as ccl26. the induction of ccl26 might modify nk cell status. in particular, it has been shown that ccl26 is able induce the migration of nk cells infiltrated in the epithelial layers of nasal tissue [40] . we hypothesize that sp1 induction of gata3 and the consequent secretion of ccl26 might induce nk cell migration, as we previously observed ( figure 1a) . further experiments are necessary to prove this hypothesis. simultaneously, nk cells showed increased levels of nkg2a/cd94 inhibitory receptor in the presence of sp1 intracellular expression in lung epithelial cells. the percentage of nkg2a-positive cells increased from the 16% to 80%. these nkg2a-positive nk cells were also cd107a negative, supporting the role of this inhibitory receptor in the control of nk cell activation in the presence of sp1. these data are in agreement with the recognized crosstalk between hla-e and nkg2a/cd94, that induces a higher surface level of hla-e molecules concurrently with a prevalent expression of nkg2a receptor on the surface of nk cells [15] . moreover, the maintenance of nk cell activation towards k562 cells, even in the presence of sp1, might be associated to the inability of k562 to express hla-e molecules [41] and consequently to interact with nkg2a. the internalization of viral sp1 might induce a cellular stress condition in lung epithelial cells [42] , which can result in endoplasmic reticulum stress and consequent down-modulation of classical hla class i molecules and upregulation of the gata3 transcription factor. the processing of sp1 by proteasome might create hla-e-specific peptides that enhance hla-e surface expression and consequently stimulate nkg2a/cd94 receptors on the surface of nk cells. these aspects deserve further evaluation and more accurate analysis. further experiments might also elucidate the possible interaction of nk cells with the regulation of adaptative immunity, in particular t cell activation. these new aspects of interaction between sars-cov-2 s1 protein and the host cells might have important implications in the pathogenesis of covid19, providing opportunities for developing new therapies against sars-cov-2. in particular, counteracting the cellular stress, targeting the s1 protein or using the anti-nkg2a monoclonal antibody monalizumab, currently in use for management of rheumatoid arthritis and several neoplastic disorders [43] , might represent new anti-sars-cov-2 strategies to enhance the innate immune response at the early stage of the disease, inducing mucosal immunity that might lead to a long-term protection against sars-cov-2 infection [44] . funding: this research was funded by university of ferrara covid19 grant (rr), university of ferrara far 2018 (rr), 2019 (rr). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. a novel coronavirus from patients with pneumonia in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical characteristics of coronavirus disease 2019 in china dysregulation of immune response in patients with covid-19 in wuhan sars-cov-2: a storm is raging hla-e-expressing pluripotent stem cells escape 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entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor interferon-gamma and interleukin-4 downregulate expression of the sars coronavirus receptor ace2 in vero e6 cells sars coronavirus and innate immunity human nk cell education by inhibitory receptors for mhc class i gata3 acts downstream of beta-catenin signaling to prevent ectopic metanephric kidney induction novel cooperation between cx3cl1 and ccl26 inducing nk cell chemotaxis via cx3cr1: a possible mechanism for nk cell infiltration of the allergic nasal tissue engagement of cd83 ligand induces prolonged expansion of cd8+ t cells and preferential enrichment for antigen specificity er stress impairs mhc class i surface expression and increases susceptibility of thyroid cells to nk-mediated cytotoxicity monalizumab: inhibiting the novel immune checkpoint nkg2a nk cells: a double edge sword against sars-cov-2 we thank linda sartor for writing assistance. the authors declare no conflict of interest. key: cord-344933-k23kzqxl authors: flahou, charlotte; sugimoto, naoshi; eto, koji title: innovations dans la culture de plaquettes à partir de cellules souches pluripotentes induites* date: 2020-09-28 journal: bull acad natl med doi: 10.1016/j.banm.2020.09.040 sha: doc_id: 344933 cord_uid: k23kzqxl la production in vitro de plaquettes offre une opportunité de résoudre les problèmes liés aux limitations d’approvisionnement et à la sécurité des dons de produits dérivés du sang. les cellules souches pluripotentes induites – ou ipsc – sont une source idéale pour la production de cellules à des fins de thérapies régénératives. nous avons précédemment établi avec succès une lignée mégacaryocytaire immortalisée à partir d’ipsc. celle-ci possède une capacité de prolifération fiable. par ailleurs, il est possible de les cryoconserver. elle est donc une source adaptée de cellules primaires pour la production de plaquettes suivant les bonnes pratiques de fabrication (bpf). dans le même temps, la capacité améliorée des bioréacteurs à reproduire certaines conditions physiologiques, telle que la turbulence, de pair avec la découverte de molécules favorisant la thrombopoïèse, a contribué à l’accomplissement de la production de plaquettes en quantité et qualité suffisantes pour répondre aux besoins cliniques. la production de plaquettes à partir de cellules ips s’étend aussi aux patients en état de réfraction allo-immune, par la production de plaquettes autologues ou dont on a génétiquement manipulé l’expression des antigènes des leucocytes humains (hla) et des antigènes plaquettaires humain (hpa). considérant ces avancées fondamentales, les plaquettes ipsc avec expression des hla modifiées se présentent comme un potentiel produit de transfusion universel. dans cette revue, nous souhaitons apporter une vue d’ensemble de la production in vitro de plaquettes à partir de cellules ips, et de son possible potentiel transformatif, d’importance capitale dans le domaine de la transfusion des produits sanguins. ex vivo production of human platelets have been pursued as an alternative measure to resolve limitations in the supply and safety of current platelet transfusion products. to this end, induced pluripotent stem cells (ipscs) are considered an ideal global source, since they are not only pluripotent and self-renewing, but also are available from basically any person, have relatively few ethical issues, and are easy to manipulate. from human ipscs, megakaryocyte (mk) lines with robust proliferation capacity have been established by the introduction of specified sets of genes. these expandable mks are also cryopreservable and thus would be suitable as master cells for good manufacturing practice (gmp) grade production of platelets, assuring availability on demand and safety against blood-borne infections. meanwhile, developments in bioreactors that physically mimic the in vivo environment and discovery of substances that promote thrombopoiesis have yielded competent platelets with improved efficiency. the derivation of platelets from ipscs could further resolve transfusion-related alloimmune complications through the manufacturing of autologous products and human leukocyte antigen (hla)-compatible platelets by manipulation of hlas and human platelet antigens (hpas). considering these key advances in the field, hla-deleted platelets could become a universal product that is manufactured at industrial level to safely fulfill almost all demands. in this review, we overview the ex vivo production of ipsc-derived platelets towards clinical applications, a production that would revolutionize the blood transfusion system. les plaquettes sont des cellules sanguines anucléées de petite taille (2-3µm de diamètre), et fragmentées dans le système vasculaire à partir de la membrane plasmique de mégacaryocytes (mk) résidants dans la moelle osseuse. elles sont indispensables dans l'équilibre homéostatique, et leur dysfonctionnement induit diverses complications hémorragiques. certaines maladies hématologiques, lourdes pertes de sang, infections sévères, syndromes d'hyper-coagulation, ainsi que des interventions thérapeutiques telles que certaines chirurgies cardiaques, chimiothérapie, radiothérapie et conditionnement des cellules souches hématopoïétiques, provoquent une thrombocytopénie, c'est-à-dire une diminution du nombre de plaquettes au point qu'il en devient insuffisant [1] . depuis des dizaines d'années, le traitement standard pour palier une thrombocytopénie reste l'allo-transfusion [2, 3] . par le passé, les dons de sang ont assuré l'apport en produits plaquettaires pour la transfusion et ont sauvé de nombreuses vies. en dépit de l'adroite gestion actuelle des incommodités liées à l'apport et de la sécurité des dons de plaquettes, il est fondamental de répondre à ses fragilités [4, 5] . en effet, pour maintenir leur viabilité de manière optimale, les produits plaquettaires sont gardés à température ambiante sous agitation douce. ceci restreint leur fenêtre d'utilisation déjà limitée à 5 jours au japon (7 jours en france, si elles sont inactivées pour les pathogènes), donc leur ré-apport doit rester constant. par nature, l'allo-transfusion induit inévitablement des risques de transmission d'infections du sang, d'allergies ; des risques liés à l'allo-immunisation [2] . les produits contaminés par des agents infectieux tels que les virus de l'hépatite, vih, virus tlymphotrope humain (htlv) etc., sont exclus de manière fiable grâce à des tests hautement sensibles, mais le risque ne peut être complètement éliminé en raison de la période de détection. les plaquettes étant gardées à température ambiante, elles sont par ailleurs sensibles au risque de contamination bactérienne. de plus, contrairement aux réponses allo-immunitaires (par exemple la maladie du greffon contre l'hôte), qui sont évitables par la déplétion des leucocytes ou l'irradiation des produits dérivés du sang, être les cas réfractaires aux transfusions de plaquettes nonconcordantes pour les antigènes des leucocytes humains (hla) et des antigènes plaquettaires humains (hpa) n'ont pour l'instant pas de solution [6] . d'autre part, certaines molécules bio-réactives incluses dans le sérum des produits plaquettaires, en particulier les cytokines, participent probablement à des complications comme la fièvre, les symptômes allergiques et les oedèmes pulmonaires lésionnels (trali). la production de plaquettes ex-vivo à partir de cellules souches pluripotentes induites (ipsc) est une solution pour résoudre les problèmes liés à la transfusion allogénique. en médecine régénérative, le risque de tumorigenèse liées aux plaquettes provenant d'ipsc est faible. cependant, le nombre de cellules requises dépasse de plusieurs ordres de grandeur celui demandé par d'autres thérapies cellulaires. motivées par ces problématiques, plusieurs avancées fondamentales ont été réalisées dans la production de plaquettes à partir d'ipsc, de telle sorte que les applications cliniques sont attendues dans les prochaines années. cette revue a pour objectif de résumer les avancées technologiques récentes dans ce domaine. nous discuterons des lignées mégacaryocytaires, des systèmes à bioréacteurs, et des nouveaux composés découverts. ces avancées seront également mentionnées dans le cadre des complications liées à l'allo-immunisation, en tant qu'importante direction thérapeutique à explorer. la production ex-vivo de plaquettes est indépendante des dons du sang et représente donc un apport stable et facilement contrôlable. elles peuvent être préparées à partir d'une source de cellule exempte de pathogène produite dans des conditions strictes avec peu de problèmes liés à la disponibilité de dons. de fait, le principal défi réside dans la production suffisante de plaquettes. en pratique, chaque dose transfusée demande un nombre élevé de plaquettes, de l'ordre de 200 ou 300 milliards. ces dernières étant nonprolifératives, anucléées, et restant en circulation seulement 3 à 10 jours, les transfusions doivent être constantes jusqu'à ce que le patient en recouvre un nombre adéquat. pour atteindre un nombre suffisant de plaquettes ipsc produites, nous avons développé une source cellulaire pouvant être amplifiée de manière fiable. les mégacaryocytes (mk), progéniteurs unipotents des plaquettes, résident en faible quantité dans la moelle osseuse. ces cellules sont donc une source peu fiable. en revanche, en amont de la lignée développementale, les cellules souches hématopoïétiques (csh) peuvent être obtenues sans procédure invasive à partir du sang de cordons ombilicaux. cependant, une unité contient seulement environ 1 million de csh [7] . cela signifie qu'une stratégie hautement efficace d'amplification, de différenciation et de production de plaquettes est nécessaire [8] . seulement, l'amplification des csh n'est pas optimale pour la production de mk in vitro, et cette option est donc aussi peu réaliste (tableau 1). encore plus en amont dans la lignée développementale se trouvent les cellules souches pluripotentes : les cellules souches embryoniques (cse) et les ipsc [9, 10] . contrairement aux csh, les cellules souches pluripotentes peuvent être amplifiées indéfiniment in vitro, puis différenciées en mk et en plaquettes [11] [12] [13] [14] [15] [16] . à noter : il n'y a pas de détection de csh dans ce procédé de différenciation, bien que ces cellules soient considérées comme un intermédiaire obligatoire pour les cellules souches pluripotentes lorsque qu'elles suivent la trajectoire développementale mégacaryocytaire. la raison de leur absence n'a pas encore été élucidée. néanmoins, les plaquettes produites de cette manière ont des propriétés comparables aux plaquettes humaines. alors que les cse sont obtenues à partir d'embryons, un équivalent fonctionnelles ipsc -peut être produit à partir de cellules somatiques par l'introduction de gènes de reprogrammation. ces cellules présentent donc moins de dilemmes éthiques et leur acquisition est peu invasive [9, 10] . malheureusement, nous ne disposons pas à l'heure actuelle de protocoles amplifiant les ipsc de sorte à atteindre les niveaux de plaquettes 6 requis pour une application clinique. ceci est sans doute dû à la longueur, la complexité, le coût, et la faible efficacité de leur différenciation en mk, résultant en une variabilité significative entre les lots de culture (tableau 1). atteindre une échelle de production applicable en clinique est donc irréalisable dans ces conditions. toutefois, les ipsc étant facilement modifiables génétiquement, il a été possible d'induire une surexpression de gènes spécifiques pour établir une lignée mégacaryocytaire amplifiable. c'est cette source cellulaire qui est actuellement considérée comme la plus adaptée pour produire des plaquettes [17, 18] . les ipsc, contrairement aux csh et aux cse, peuvent être obtenues à partir d'un petit fragment de peau, de sang périphérique ou ombilical, voire d'urine [19] de n'importe quel donneur, même ceux ayant une maladie hématologique. à l'heure actuelle, une banque d'ipsc est maintenue pour fournir des cellules autologues à partir de donneurs pouvant être compatibles au regard des hla. celle-ci est une source d'allogreffe possible (tableau 1) [20] [21] [22] . l'autre possibilité consiste à manipuler l'expression des hla and hpa pour permettre aux plaquettes produites in vitro d'échapper à la réponse immunitaire [12, 23, 24] . l'hypothèse concernant la trajectoire développementale des érythroblastes et des mk leur attribue un progéniteur commun, les mk-érythroïdes (mep) [25] . dans ce système, la trombopoïétine (tpo) et son récepteur associé (mpl) jouent un rôle majeur dans l'amplification, la différenciation et la maturation des mk [26] . de fait, la combinaison de tpo, d'autres cytokines et de divers facteurs participe à la différenciation des mk in vitro [12, [27] [28] [29] . des études récentes suggèrent qu'il existerait progéniteur spécifique de la trajectoire développementale des mk dans l'hématopoïèse adulte -mkp -qui proviendrait directement des csh ou de progéniteurs sanguins multipotents [30] , en plus de l'existence de mk ayant des propriétés d'auto-renouvèlement ( figure 1 ) [31, 32] . dans le but d'obtenir des mkp, nakamura et al., ont établi une lignée cellulaire immortelle de progéniteurs mk (immkcl) à partir de progéniteurs hématopoïétiques dérivés d'ipsc, par l'introduction séquentielle des gènes c-myc et bmi1 suivis par le gène bcl-xl (table2). ces transgènes sont sous le contrôle du système tet-on qui permet de faire passer les immkcl d'un état prolifératif et d'auto-renouvèlement à un état de maturation et de production de plaquettes par l'utilisation de doxicycline (dox, dox-on : prolifération, dox-off : maturation) [18] . la surexpression de c-myc favorise l'amplification des mk sans induire leur maturation [33, 34] , alors que le complexe du groupe polycomb bm1 et le facteur anti-apoptotique bcl-xl suppriment respectivement la sénescence et l'apoptose via la régulation d'arf et de ink41, et de caspases [35] . la culture de ces des cellules en condition dox-off bloque la surexpression de ces transgènes et augmente l'expression de gata1 et de nf-e2, des facteurs cruciaux pour la différenciation et la maturation des mk [36, 37] . la génération de plaquettes à partir d'immkcl est plus rapide et possède une efficacité supérieure à leur production à partir d'ipsc, puisque ce système souffre d'une 7 faible efficacité de différenciation en mk. pour illustrer ce fait, plus de vingt jours ont été nécessaires pour différencier des mk à partir d'ipsc, avec une efficacité d'environ 3 mk par ipsc [33] . de plus, les lignées mégacaryocytaires peuvent être cryoconservées puis amplifiées à la demande. des produits plaquettaires respectant les règles de bpf peuvent être produits à partir de cellules sources libres de pathogènes en conditions stériles ( figure 2 ). un obstacle majeur à l'utilisation des cellules dérivées d'ipsc ou de matériel biologique génétiquement modifié concerne le risque de tumorigénicité. nous discuterons de la mitigation de ce problème par un procédé de purification amélioré et l'irradiation des produits finaux dans la section iv. l'établissement d'une lignée amplifiable mégacaryocytaire ne correspond qu'à la moitié du travail : les mk doivent ensuite être induits à maturation pour produire efficacement des plaquettes fonctionnelles. pendant leur maturation, les mk passent par plusieurs endomitoses et deviennent des cellules géantes polyploïdes, pouvant atteindre 50-100µm de diamètre et une quantité d'adn jusqu'à 128n. suivant cette augmentation en volume, les mk étendent et invaginent leurs membranes cellulaires pour former un système de membranes de démarcation (dms). le procédé est accompagné par le bourgeonnement de nombreuses pro-plaquettes à partir du dms dans les vaisseaux sinusoïdaux de la moelle osseuse. les pro-plaquettes contiennent les organelles des plaquettes et possèdent des gonflements correspondants à la taille de ces dernières, arrangées en tandem, qui dépendent du réarrangement de leurs microtubules. dans les dernières étapes de la thrombopoïèse, des particules anucléées se détachent de la partie distale des protrusions pro-plaquettaires et entrent en circulation, où elles sont de nouveau fragmentées en plus petites particules : les plaquettes [38] [39] [40] [41] . à la différence du mécanisme stationnaire de la thrombopoïèse, qui est régulé par la tpo, un nouveau mécanisme de production de plaquettes qui suit une infection grave ou d'importants saignements a été découvert. ce mécanisme induit la libération rapide de plaquettes selon un mode de rupture sous l'effet de la cytokine il-1α ( figure 3 ) [42] . les plaquettes produites de cette façon sont plus larges et montrent des détériorations mineures, mais leur fonction thrombotique est intacte. alors que le mécanisme stationnaire est le modèle principal de la production de plaquettes ex-vivo, le mécanisme de production d'urgence de plaquettes, basé sur des signaux inflammatoires, offre des possibilités d'exploration de stratégies alternatives pour augmenter l'efficacité de la production de plaquettes ex-vivo. dans cette optique, l'utilisation d'il-1β, qui partage le même récepteur que l'il-1α, a été examinée [17, 28, 43] . un seul mk dans la moelle épinière peut potentiellement produire 1000 à 2000 plaquettes, mais dans des conditions statiques en culture in vitro, ce nombre est comparativement faible, et se réduit à 3 ou 10 [18, 44] . de plus, les plaquettes in vitro sont typiquement plus larges, moins réactives et ont une viabilité inférieure [18, 44, 45] . cette 8 importante différence a motivé de plus amples études pour optimiser la maturation des mk et la qualité des plaquettes produites. dans le but d'imiter au mieux les conditions physiologiques retrouvées in vivo, plusieurs types de bioréacteurs ont été conçus (table 3 ) [46] . les bioréacteurs permettent une culture cellulaire en 3d, ce qui augmente la surface disponible aux mk pour produire des pro-plaquettes. ils permettent aussi de récapituler les forces de cisaillement qui existent dans la circulation et de libérer des plaquettes à partir de la partie distale des proplaquettes [38, 47] . à noter qu'un mécanisme similaire de production de plaquettes a été décrit dans les poumons [48, 49] . les bioréacteurs récapitulant de manière optimale les forces de cisaillement améliorent significativement le nombre et le ratio de plaquettes produites [50] . ont suivi d'autres développements de systèmes plus performants permettant d'accroître encore le nombre de plaquettes produites ainsi que leur production dans un état quiescent, leur activation réduisant leur fonction (tableau 3). par exemple, thon et al., ont conçu un bioréacteur sur puce qui reproduit les composants physiques et chimiques de la moelle osseuse telle que la rigidité, la composition de la matrice extracellulaire, la taille des pores, le contact avec les cellules endothéliales, et les forces de cisaillement [51] . une particularité de leur bioréacteur est de pouvoir incorporer de la microscopie en temps réel. notre équipe a suggéré que l'angle du flux pourrait être un paramètre important, puisque des expériences avec des angles divers ont montré une corrélation avec une variation du nombre de plaquettes, le meilleur étant de 60 degrés [44] . dans une tentative de récapituler la niche environnementale des mk, balduini et al., ont utilisé un support unique fait de protéines de soie contenant des cytokines, des composés de la matrice extracellulaire, et des protéines dérivées de cellules endothéliales [43, 52] . blin et al., ont construit un dispositif microfluidique qui capture les mk dans des micro-piliers enduits de vwf et libère les plaquettes selon différents taux de cisaillement [53] . utilisant une technologie permettant des forces de cisaillement basée sur des membranes en nanofibres, avanzi et al., ont assemblé un bioréacteur capable de produire étape par étape des plaquettes à partir de cellules cd43+ provenant de sang de cordon ombilical [54] . cependant, en considérant le potentiel physiologique des mk et le niveau de production requis, les bioréacteurs ont encore une marge de progression importante quant à l'optimisation de la production de plaquettes [46] . de plus, les bioréacteurs qui exploitent les forces de cisaillement n'ont pas réussi à obtenir des plaquettes ayant des propriétés hémostatiques satisfaisantes. une approche alternative pour la production de plaquettes est d'injecter en intraveineuse des mk pour utiliser la caractéristique des poumons à être un site de thrombopoïèse [48, 49] . dans ce cas, les poumons retiennent les mk, qui libèrent ensuite les plaquettes dans les vaisseaux pulmonaires et convertissent les poumons en sorte de bioréacteurs in vivo [55, 56] . en combinant les bioréacteurs à des molécules stimulant la thrombopoïèse, il est possible d'améliorer la production de plaquette ex vivo. parmi les composés candidats on 9 retrouve des inhibiteurs de la polymérisation d'actine et de la kinase rho. ils améliorent la polyploïdisation des mk et la formation de pro-plaquettes [54, 57, 58] . d'autre part des études ont montré que l'hypercholestérolémie entraîne la production de plaquettes. on pourrait donc l'optimiser en supplémentant le milieu de culture en cholestérol [59, 60] . stemregenin 1 (sr1), antagoniste du récepteur aryl-hydrocarbone (ahr), non seulement améliore l'amplification des csh [61] [62] [63] , mais augmente également la polyploïdie des mk et favorise l'amplification in vitro de leur précurseurs [62, 64] . parmi les complications immunitaires observées après une transfusion de plaquettes, on retrouve des réactions aigües telles que de la fièvre, des réactions allergiques, le trali, mais aussi des réactions plus tardives telles que la gvhd et une réfraction allo-immune [2, 6, 65] . les causes potentielles peuvent être attribuées aux cytokines, allergènes, ou autres molécules encore non-identifiés. des étapes de lavages de plaquettes (dites transformation « déplasmatisée ») préviennent les réactions aiguës [2] . de la même manière, le lavage des plaquettes ipsc devrait réduire leur risque de provoquer ce type de réaction. dans le cas de la gvhd, elle est provoquée par la contamination lymphocytaire du concentré de plaquettes et celle-ci peut être prévenue par leuco-réduction ou irradiation [65] . ce problème ne se pose pas pour les plaquettes ipsc puisque les lymphocytes ne se différencient pas à partir de mk [18, 66] . la réfraction immunitaire à la transfusion de plaquettes est basée sur l'alloimmunité liée à l'expression des antigènes sanguins abo, mais aussi hla classe i et hpa. suivant la transfusion, une réponse immunitaire contre ces antigènes provoque un rejet dans 5 à 15% des cas [2, 6] . une non-concordance majeure des antigènes abo réduit la durée entre les transfusions, mais crée peu de conséquences cliniques [67] . ce n'est pas le cas pour la non-concordance sur les hla et hpa, qui entraîne de sérieuses complications et demandent une réponse immédiate pour éviter de sérieux problèmes de saignement [2, 6] . les anticorps contre les hla classe i, et dans une moindre mesure les hpa, sont la cause principale d'allo-réfraction. il est donc nécessaire de vérifier leur compatibilité avant transfusion [6] . certains patients deviennent sensibilisés et produisent des anticorps contre le hla et/ou hpa après plusieurs transfusions, ou après une grossesse. actuellement, la transfusion de plaquettes concordantes pour les hla ou hpa est la seule mesure pour éviter ce type de réaction, mais leur disponibilité est limitée, demande une logistique au coût plus important, et peut manquer en situation d'urgence. plusieurs approches utilisant les cellules dérivées d'ipsc peuvent être mises en oeuvre pour assurer une solution à ces problèmes. d'une part, il est possible de produire des plaquettes dérivées d'ipsc produite à partir des cellules du patient, qui dans ce cas n'éliciteront pas de réponse immunitaire. à l'heure actuelle, un essai clinique utilise des plaquettes autologues suivant cette stratégie [68] . cependant, établir des ipsc autologues, produire la lignée cellulaire mk, puis finalement produire des plaquettes est un processus long et complexe à la qualité variable. une approche alternative est de préparer une banque de cellules ayant des propriétés immunogènes amoindries. des ipsc de donneurs homologues pour les hla classe i ont déjà été collectés dans cette optique [20] [21] [22] . les produits homologues pour les hla classe i ne doivent être compatibles qu'avec un seul des deux loci du gène hla classe i du receveur, et peuvent donc être utilisés pour bien plus de patients que les produits hétérologues. conformément aux estimations, la collection de 100 des types de hla les plus fréquents pourrait couvrir plus de 80% de la population japonaise [69] . cette stratégie permet aux cliniciens de s'appuyer sur des stocks stables, à la place de donneurs compatibles à la disponibilité incertaine. les hla sont toutefois très variables dans la population mondiale. en conséquence, plus la couverture est grande et plus la banque cellulaire doit être large. enfin, une future stratégie pour minimiser le risque d'allo-réfraction consiste à modifier génétiquement les ipsc pour en supprimer l'expression des hla classe i et en dériver des plaquettes hla-nul. en utilisant des technologies telles que shrna, talen ou bien crispr/cas9, la diminution de l'expression des hla est possible soit par l'extinction du gène de la microglobuline beta-2 (2m), soit par sa délétion [12, 70] . avec cette stratégie, il faut néanmoins prendre en compte le risque posé par cellules natural killer (nk), qui ciblent et éliminent les cellules qui ont une expression réduite des hla [71] . bien que l'allo-réactivité des nk n'ait pas été démontrée contre les plaquettes, une réaction pourrait apparaître après transfusion de cellules n'ayant aucune expression des hla. l'utilisation des shrna pourrait être une solution pour laisser une expression minimale mais suffisante pour prévenir l'activation des nk [72, 73] . des études ont été menées sur l'expression forcée de hla classe i non classiques (hla-e, hla-g) fusionnés avec la 2m dans le but d'inhiber la réponse des nk par leur récepteur cd94, ainsi que sur la suppression sélective de l'expression des hla-a/b en maintenant l'expression des hla-c [45, [73] [74] [75] . au-delà de ces considérations, nous avons mis en évidence que les plaquettes ipsc génétiquement modifiées afin qu'elles n'expriment pas les hla classe i ne provoquent pas de réponse immunitaire des nk, et peuvent circuler adéquatement dans un modèle de souris reconstituées avec des cellules nk humaines et des anticorps anti-hla classe i [76] . l'allo-réfraction provoquée par les hpa est moins fréquente, mais les patients peuvent aussi manifester des réponses cliniques graves telles que des cas de purpura posttransfusionnel (ptp) ou thrombopénie foetale/néonatale allo-immune (fnait) [77] . dans le cas du ptp, quelques semaines après la transfusion de plaquettes non concordantes pour les hpa, le nombre de plaquettes est plus bas qu'avant transfusion. ceci suggère que l'élimination des plaquettes inclue aussi celle du patient [77] . la fnait apparaît lors de la grossesse, lorsque le système immunitaire de la mère réagit contre les plaquettes foetales ayant des hpa non concordants. ces anticorps traversent le placenta et causent une thrombopénie chez le nouveau-né, induisant des risques de complications sévères potentiellement à long terme [77, 78] . l'antigénicité provient principalement de polymorphismes singuliers de nucléotides (snp) dans les molécules de hpa [77] [78] [79] . étant donné que leur expression est nécessaire pour les plaquettes, une stratégie d'élimination similaire à celle proposée pour les hla n'est pas réalisable. il est cependant possible de convertir les snp et de changer l'allo-type des hpa en utilisant le système crispr/cas9 [24] . ensembles, les technologies pouvant manipuler les antigènes des plaquettes sont capables d'apporter jusqu'en clinique des plaquettes allo-antigèniques concordantes pour les antigènes impliqués dans les effets secondaires immunitaires des transfusions. une seule lignée cellulaire mk hla-nul peut servir de cellules primaires pour une production de masse de cellules universelles pour les hla, et servir de base pour de futurs développements dans la modification des hpa. la production de plaquettes ex vivo a grandement progressé ces dernières années. la mise en place d'une lignée mégacaryocytaire, associée au développement de bioréacteurs et la découverte de nouveaux agents, a drastiquement amélioré l'efficacité et la quantité de plaquettes produites et a permis de se rapprocher d'applications en clinique. en raison de la sécurité offerte par le fait que les plaquettes soient anucléées et non prolifératives, des tests cliniques sont attendus dans un avenir proche. les plaquettes produites ex vivo sont à l'avant-garde de la médecine régénérative utilisant les ipsc, au regard du nombre considérable de personnes nécessitant des transfusions de plaquettes. les obstacles à surmonter restent la génération de plaquettes suivant les principes de bpf avec un haut rendement et qui ont des qualités fonctionnelles au plus proche des plaquettes naturelles, ainsi que le développement de procédés de purification pour éliminer les contaminations cellulaires et chimiques, ou encore des méthodes confirmant la sécurité des produits finis et leur préservation optimale [45] . à travers le monde, de nombreuses équipes de recherche travaillent à faire avancer le domaine de la production de plaquettes ex vivo. d'ici quelques années, on peut s'attendre à voir publié les premiers résultats d'essais cliniques dans plusieurs pays, et qu'un accès facile, sûr et abordable à des transfusions plaquettaires soit assuré par leur production ex vivo à partir d'ipsc. les auteurs remercient dr peter karagiannis et colin deschamps pour leur relecture critique du manuscrit. k. e. a soumis des brevets liés aux travaux des références [16, 18, 33, 42, 44, 69] . k. e. est un co-fondateur de megakaryon corporation. les intérêts de k.e. ont été investigués et sont gérés par l'université de kyoto en accord avec ses règles de conflits d'intérêts. en raison des limitations de texte, plusieurs études ayant néanmoins grandement participé à l'avancement de la production de plaquette ex vivo n'ont pas pu être mentionnées. les auteurs s'excusent de ces omissions. lignée mégacaryocytaire amplifiable, établie à partir d'ips par transduction de gènes particuliers. ces cellules peuvent être préservées et servir de cellules primaires qu'il est facile de mettre en culture, d'amplifier puis d'en 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human platelet antigens, antibody detection strategies, and genotyping induction of functional platelets from mouse and human fibroblasts by p45nf-e2/maf direct conversion of fibroblasts to megakaryocyte progenitors gènes introduits référence immkcl ips c-myc, bmi1 et bcl-xl [18] fopmk ips gata1, fli1 et tal1 [17] converted mk fibroblaste nf-e2, mafg et mafk [80] gata2, runx1, gata1, tal-1, lmo2 et c-myc [81] key: cord-009571-mygj2nd4 authors: nan title: proceedings of the 42nd annual meeting of the american rheumatism association a section of the arthritis foundation june 1 & 2, 1978 new york city abstracts of papers presented date: 2005-11-23 journal: arthritis rheum doi: 10.1002/art.1780210508 sha: doc_id: 9571 cord_uid: mygj2nd4 nan azathioprine has been shown t o be an effective agent in the treatment of active rheumatoid arthritis (ra); however, potential long-term neoplastic effects remain a serious concern. to clarify this relationship, 36 patients with classic or definite ra being treated with azathioprine (group 1) were chosen at random and studied by means of clinical, laboratory, and radiologic measures to detect potential early markers of neoplastic disease. nineteen age matched patients with ra were concurrently similarly studied as controls (group 2). group 1 had a longer disease duration (15.3 versus 9.4 years). the mean duration of treatment with azathioprine was 38.3 months, with a mean dose of 7 1.2 mg/day. histories of cigarette smoking, hormone treatment, and fertility were comparable. chest x-rays revealed comparable numbers of inflammatory and fibrotic abnormalities. there was an increased frequency of tumors in first degree relatives in group i (15 versus 2). hematologic studies revealed a trend to lower cell counts in group 1 with a striking increase of marrow megaloblastosis in this group (28 versus i ) . there were no differences in iron saturation or b12 levels. serum folate was decreased in group i (14 versus 2). there were neither liver function test abnormalities in group 1, nor evidence of malignancy in gastrointestinal x-rays when clinically indicated. no neoplastic lesions were identified on mammography in either group. four patients in group 1 and none in group 2 had atypical or dyskaryotic cells on urine cytology. one patient in group 1 had an abnormal pap smear and was subsequently shown to have endometrial carcinoma, on chromosomal analysis, group i showed a greater percent of cells with aberrations, a higher aberration incidence and index, and more patients with hyperdiploidy. patients in group 2 had a history of 3 benign neoplasms. patients in group i had a history of i benign neoplasm prior to the initiation of azathioprine; subsequently 7 neoplasms were diagnosed, 4 benign and 3 malignant (2 endometrial carcinomas and carcinoma-in-situ of the cervix). in summary, group i had an increased number of cellular abnormalities, with cytologic atypia, megaloblastic bone marrows, and increased chromosomal aberrations. clinically this group had more tumors diagnosed while on treatment, particularly in females, and arising from the genitourinary tract. more long-term studies of patients on cytotoxic agents are clearly warranted. solid phase clq (sclq) radioimmunoassays. c3 was determined by radial immunodiffusion and antibodies to dna by binding of radiolabeled dna by the millipore filter technique. these assays were correlated with each other, general presence or absence of symptomatic disease, active renal disease, and active joint disease. to evaluate predictive capabilities, a change in cic result was correlated with a change in disease activity. change in disease activity was defined as an sle related event requiring hospitalization, a change in symptoms requiring an increase in or addition of steroids or immunosuppressive agents, or a marked improvement in symptoms such that drugs were tapered or discontinued. the fclq results did not correlate with any parameters of disease activity. a fall in c3 or an increase in antibodies to dna usually signaled a change in disease activity; however, many patients experienced a change in disease activity without any change in c3 or dna binding. the sclq assay correlated with symptoms of sle in general (p < 0.001), with active renal disease (p < 0.025), and with active arthritis (p < 0.001). an appropriate change in sclq determination correlated best with a significant change in disease activity (p < 0.001). we conclude that the sclq method of determining cic correlates best with active clinical manifestations of sle and is a useful predictor of disease activity. we have recently found that certain rheumatoid factors (rfs) cross react with dna-protein (dnp) . this type of cross reacting r f is frequently present in seropositive rheumatoid arthritis (ra), 21 of 58 patients, and also in order r f positive patients: rheumatoid overlap syndrome 4/10, mixed connective tissue disease (mctd) 5/7, other diseases 1/10, thus far rfs reactive with dnp have not been found in systemic lupus erythernatosus (sle) 0/8 or essential mixed cryoglobulinemia 0/7. rfs which react with dnp can give positive antinuclear antibody (ana) and le tests which can be blocked by aggregated igg or dnp. in contrast, positive ana and le tests in sle are not blocked by aggregated igg. hence rf induced ana and le tests may be considered "false" positives when these tests are used in the diagnosis of sle. in some sera these rfs could be detected only following isolation with insoluble igg or dnp, suggesting the presence of rfs complexed with antigen. by use of a monoclonal r f which cross reacts with dnf in a sensitive competitive inhibition radioimmunoassay designed for detection of immune complexes (jci 59:990, 1977) , complexes larger than 19s by ultracentrifugation studies could be detected in several ra sera and synovial fluid and mctd sera. in some studies dnase treatment of these complexes resulted in a decrease in size. these studies suggest that complexes consisting of dnp antigen and r f may be present in the circulation in ra and mctd. the relationship, if any, of these complexes to the immunopathology in these diseases is currently under study. these studies also suggest that not all rfs may be induced by immune aggregated igg and that a re-exploration of rfs in various diseases for cross reactions with non-igc antigens is indicated, such antigen may provide clues to etiologic factors in these diseases. previous studies demonstrated that patients with rheumatoid arthritis (ra) frequently have an antibody referred to as rheumatoid arthritis precipitin (rap). this antibody was shown by the indirect immunofluorescent technique to react with a nuclear protein in human, b lymphocytes from continuous cell culture (wil,) and the antigen was termed ra associated nuclear antigen (rana) . infectivity experiments with epstein-barr virus (ebv) or other herpes viruses have suggested that rana may be associated with ebv infection; although rana shares some characteristics with eb nuclear antigen, they are probably two different antigens. in this study we have expanded our work on rana. in addition to its presence in wil, cells, it has now been found in 6 other human, b lvmphocyte lines but was not present in 2 t lymphocyte lines. rana was not found in extracts from normal spleen, kidney, heart, or lungs, or a spleen extract from a patient with felty's ra. this antigen was not present in normal lymphocytes stimulated with b or t cell mitogens. rana was not found in peripheral leukocytes from patients with ra. however, it was found in 2 of 3 extracts made from pannus layers and an extract made from nodules taken from patients with seropositive ra but was not present in 2 extracts made from pannus layers taken from seronegative ra patients and synovium from a traumatized joint. initial physicobiochemical characterization of rana demonstrated that it was stable to heat (37oc or 56oc) or cold ( 4 o to -7ooc) treatments or at ph values of ph 6.2-8.6. studies performed by immunoelectrophoresis indicated that rana was an anion at ph 7.4-8.6. will extracts were separated on a calibrated 10% agarose column and the molecular weight estimated at 120,000 d. in other studies the antigen could not be detected on the surface of wil, cells. immunodiffusion studies using burkitt's lymphoma sera (12) or nasopharyngeal carcinoma sera (12) containing high titers of ebv antibodies to: viral capsid antigen or early antigens d or r do not react with wil, extract, suggesting these ebv antigens are not present in the extract and therefore cannot be rana. the data suggest that rana may be a new, heat stable, acidic, nuclear protein associated with ebv infection which could be important in the pathogenesis of ra. after medical workup, arthroscopy was performed on 100 patients (5: 2, men: women; ages 14-74) with painful knees of unknown cause. arthroscopy of the knee was achieved on ambulant patients under sterile technique in an outpatient minor surgery suite utilizing a needlescope or watanabe 521 arthroscope. biopsies were guided by visualization. postarthroscopy diagnoses are reflected in the table. in most cases the procedure allowed adequate visualization of the suprapatellar sac, articular cartilage, menisci, anterior cruciate, and synovial lining. no arthroscopic abnormality was visualized in 12%. nearly half of the patients had osteoarthritis including 12 patients with chondromalacia patella. the group with osteoarthritis also included 9 patients who had at least one torn meniscus. septic arthritis was confirmed in 4% by synovial biopsy including one case of tuberculosis. previous undiagnosed synovitis was confirmed but not further defined in 4 patients. four biopsies suggested rheumatoid synovitis; the diagnosis was confirmed by subsequent clinical and laboratory course. results of arthrography (46) were confirmed in 26, contradicted in 12, and provided additional findings in 2 i. patients were ambulant after the procedure with minimal morbidity-6 hemarthroses and an acute pseudogout, all treated by arthrocentesis. the gainful knee often presents a diagnostic enigma particularly when degree of disability is a question. ar-throscopy is a safe minor surgery procedure that may reveal the underlying cause of pain and preclude the need for arthrotomy. diagnosis computer assisted tomography of the spine and/or roentgenographic transaxial lumbar tomography were performed on 25 patients (age 69 iz 12 sd years) with back pain and concomitant paget's disease. clinical assessment with routine lumbosacral x-rays was performed by a rheumatologist and an orthopedic surgeon. both tomographic methods are xray techniques that transect the spinal canal and the intervertebral foramina producing a computer printout or laminogram along the axial plane of the spine. the following was found: 1. spinal stenosis: spondylotic osteophytes encroaching on the spinal canal in 14 patients. three had a history of pseudointermittent claudication. lumbar x-rays supported a diagnosis of paget's disease at the involved site in 6. 2. lateral recess syndrome: spondylotic osteophytes encroaching on the intervertebral foramina in 9 patients. all had sclerotogenous pain (nonspecific pain referred to the buttocks, groin, and/or thighs). lumbar x-rays supported a diagnosis of paget's disease at the involved site in 4. 3. pagetic pain: increased thickness of the laminae, pedicles, or bodies of the vertebrae secondary to paget's disease in 3 subjects without alteration of the spinal canal. 4. spondylotic changes of articular facets without stenosis or lateral recess in 6; 5 had adjacent pagetic changes. in the assessment of back pain, axial tomography proved to be a useful tool in defining anatomic spinal lesions that were not appreciated by routine x-ray techniques. the methods were particularly helpful in outlining the zygapophyseal articulation for definition of radiographic joint space and hypertrophic changes. neural entrapment by bone due to hy-pertrophic changes was similarly defined. in several cases the procedure obviated the need for myelography. in paget's disease, determining the degree of spondj4osis could guide therapy since it would predict those that might be expected to respond to suppressive therapy of their paget's disease. hla-b27 is a potent genetic marker for ankylosing spondylitis (as), reiter's syndrome (rs), and other spondylitic variants. this cell surface antigen is determined by the hla-b locus, one of a linear array of genes clustered on the sixth chromosome. whether hla-b is the disease-conferring gene or only linked to another nearby gene remains unclear. studies of hla-d on one side of hla-b (1 map unit) have shown no associations with as or b27. the hla-c locus lines 0.2 map units to the opposite side of hla-b. it was our purpose to study c locus antigens in b27 positive and negative patients in order to more precisely localize the disease promoting gene. eighty-eight patients (30 as, 37 rs, 9 sacroiliitis, 9 psoriatic spondylitis, and 3 colitic spondylitis) were hla cwl, p < 0.025 for cw2, and p < 0.005 for either cwl and cw2. similar significant differences were found when both the b27+ patients and the b27+ controls were compared with the matched controls. there were no significant differences noted between b27+ patients and b27+controls or between b27patients and matched controls. the occurrence of cw3 and cw4 was similar within all groups. thus, these data confirm strong linkage disequilibrium between b27, cwl, and cw2 which does not relate to disease. most importantly, the absence of cwl and cw2 in b27patients provides further evidence that recombination between b and c loci is unlikely, and that disease susceptibility is intimately related to or inseparable from the b locus. control groups (n=68), % (n=20), % (n=64), % (n=88),% typed for a, b, and the c locus antigens, cwl, cw2, cw3, and cw4. in addition, 88 age, sex, and race matched normal controls and 64 b27 positive normal controls were similarly studied and compared to those with disease. hla-b27 was present results of c locus typing are shown in the table. an immunofluorescent complement fixation (ifcf) test was used to determine if nuclear antigen-antibody complexes of different specificities possessed different capacities to fix complement. sera from patients with rheumatic diseases were specially selected and demonstrated to contain only antibodies to sm antigen (5 patients), nuclear rnp (7), ss-b antigen (3), or nuclear histones (7). column chromatography isolated-igg from these sera were reacted with sections of mouse liver to form nuclear ag-ab complexes of the different specificities described above. binding of complement components to the nuclear ag-ab complexes was determined after washing away excess serum and incubating with 3 different sources of complement: 1) normal human serum (nhs); 2) nhs-egta mg, conditions which are known to block activation of the classic pathway; 3) immunologically clq-depleted sera in which other complement proteins were demonstrated to be 90% hemolytically active. fixation of complement was determined by using fluorescein-conjugated antiserum to clq, c4, properdin, factor b, c3, c6, and c9. all sera with antibodies to sm antigen, nuclear rnp, and ss-b nuclear antigens showed fixation of all complement proteins when nhs was used as the source of complement. when the source of complement was egta-mg or clq-depleted serum, clq and c4 were negative by ifcf. however, complement proteins properdin, factor b, c3, c6, and c9 were positive, showing that these nuclear antigen-antibody complexes were able to fix complement proteins of the alternative pathway, without concomitant fixation of the classic pathway. the 7 sera with antibodies to nuclear histones did not fix any complement component of either the classic or alternative pathways. these studies demonstrate two important findings. bodies of certain specificities to activate complement may have certain nuclear ag-ab complexes are capable of activating the relationship to their capacities to cause tissue damage by realternative complement pathway, independent of classic path-lease of inflammatory peptides mediated by complement actiway activation. reactions between nuclear histones and anti-vation. our results may explain why drug-induced lupus erbody, as demonstrated by the immunofluorescent technique, ythematosus, characterized by anti-histone ab, is rarely appear not to be complement-fixing reactions or at least not associated with inflammatory kidney disease. detectable by this technique. the capacity of antinuclear antiit has been suggested that renal involvement in sle patients may depend upon qualitative immunochemical characteristics of antibodies to dna, e.g., precipitating ability, affinity, ig class or subclass, and complement fixing (cf) capacity. we used the immunofluorescent crithidia luciliae method [a-dna(cl)] to delineate some of these characteristics in sera from 35 patients with active sle. using fitclabeled polyspecific antiserum to igg, iga, and igm, a-dna(cl) was found in 17 of 18 patients with active lupus nephritis (gp-i) and 11 of 17 patients with lupus activity without nephritis . median titer in gp-i was 1 : 160 and in gp-i1 1 : 40; the difference in titers between these groups was significant (p < 0.005). c f antibodies to dna were found in 13 patients in g p i and 5 patients in gp-i1 using a double sandwich technique to detect c3 binding. when titer of c f activity was compared with a-dna(cl) titer, a strong correlation was found (r = 0.73; p < 0.001). igg, iga, and igm a-dna(cl) were detected with high frequency in both groups. in addition, we compared titers of a-dna(cl), which detects antibodies primarily directed against dsdna determi-nants, with dna binding capacity detected by the millipore filter (mf) method of ginsberg and keiser, in each of these groups. as antigen we employed 1*61-labeled calf thymus dna (type i, sigma) passed through an 0.45 p, type ha millipore filter. in contrast to the cl method, levels of a-dna(mf) did not differ significantly in gps i and 11. when we compared a-dna(cl) titer in gp-i sera with quantitative a-dna(mf), a highly significant correlation was found (r = 0.89, p < 0.001), while no correlation between these methods was evident in sera from gp-i1 patients. these findings suggest that: 1) the presence of high titered a-dna(cl) is strongly correlated with active renal lupus; 2) detection of c-fixing a-dna(cl) may reflect total amount of antibody; demonstration of c-fixing ability may generally be expected in high titered sera; 3) antibodies to at least 2 sets of constituents of the dna preparation employed in the mf method can be detected, one occurring in active lupus nephritis and correlated with antibodies detected by cl, and one in patients with active lupus without nephritis, detected minimally or not at all by the cl method. cell mediated cytotoxicity is an important pathogenic mechanism in certain rheumatic diseases. recent studies from this laboratory have demonstrated that adherent monocytes are potent killers of antibody coated (adcc) nonerythroid target cells and also kill uncoated target cells to a lesser extent. although immune complexes can inhibit lymphocyte cytotoxicity, their effect on monocyte cytotoxicity is unknown. this study compares human monocytes adcc and natural killer (nk) activities with that of lymphocytes. the effects of heat aggregated igg (0.02-2 mg per ml) and preformed soluble keyhole limpet hemocyanin (klh)/anti-klh complexes were evaluated. monocytes and lymphocytes were separated by their differential adherence properties in plastic microtiter wells and were at least 95% pure. cytotoxicity was measured by "chromium released from antibody coated (adcc) and nonsensitized (nk) k 562 cells, an established myeloid cell line. precipitated klh/anti-klh complexes formed at equivalence were the strongest inhibitors of cell mediated cytotoxicity (suppression: monocyte nk 900/0, monocyte adcc 60%, lymphocyte nk 8o%, lymphocyte adcc 30%). aggregated igg also strongly inhibited both monocyte and lymphocyte nk (50% and 75% respectively) and also suppressed adcc to a lesser extent (monocyte 25%, lymphocyte 20%). klh/anti-klh in antigen excess had no effect on monocyte nk and suppressed lymphocyte nk by only 25%. although soluble complexes did not block monocyte nk, they did significantly block monocyte adcc (45%). by use of serial dilutions of complexes in aggregated igg, a dose dependent relationship was established between complexes and percent suppression of cytotoxicity. these studies reveal that both klh/anti-klh immune complexes and aggregated igg can inhibit monocyte cytotoxic activity and that, in general, these effects correlated with lymphocyte nk and adcc. significant differences between soluble immune complexes and aggregated igg were demonstrated. finally, the finding that certain immune complexes inhibit both nk and adcc would imply that fc receptors are required for both cytotoxic responses. the relatively greater inhibitory effect on nk would imply that antibody fixed to target cells potentiates attachment by the effector cell and renders it less susceptible to inhibition by immune complexes. gout is easily diagnosed from a classic clinical history and the demonstration of urate crystals in synovial fluid and/ or tophi; however the diagnosis may not be suspected in patients with an atypical history or physical examination. to ascertain the prevalence of atypical findings we are conducting a prospective study in our gouty population randomly admitted to the clinical research unit. we report the following findings in the first 30 patients with crystal proved gout: 1) 30% had saturnine gout 2" to moonshine consumption, 37% gave a history of heavy ethanol consumption but did not have lead intoxication, and 53% were obese. all patients were male, 53% black, 40% white and 7% american indian. the average age at the time of study was 53.2 years with a mean duration of gout of 14 years. seventythree percent had evidence of liver disease (elevated sgot or hepatomegaly), 77% were hypertensive, 47% had elevated triglycerides, and 47% had abnormal glucose tolerance. 2) twenty-seven percent had a polyarticular onset of gout (> 1 joint); 60% subsequently had polyarticular attacks. seventythree percent had tophi at the time of the study. chronic synovitis was present in 63% of patients. in the 30 patients examined the following joints of the upper extremity were involved; 40% m p and/or ip, 20% wrist, 209'0 elbow, 10% shoulder. in the lower extremity the knee was involved in 40%, m p joints in 27%, and ankles in 13%. 3) a positive latex fixation test was observed in 31% of patients; 6 being > 1 : 40 and 4> 1 : 320. ra latex was present in 36% of patients with tophi and 38% of patients without tophaceous deposits. in the patients with chronic synovitis 37% had a +ra latex, and 36% free of chronic synovitis were also positive. the mean age in patients with a positive test was 53.1 years and 53.3 years for those who were negative. in contrast to these negative associations, evidence of liver disease was correlated with the finding of a +ra latex. fifty percent of patients with liver disease had a +ra latex whereas it was absent in all 8 without liver disease. we conclude that chronic synovitis and polyarticular arthritis are common in our gouty patients. in addition, rheumatoid factor is found in a significant proportion of gouty patients and it does not seem to correlate with age, tophi, or chronic synovitis. ra latex seems to correlate best with the presence of liver disease which in most instances was a concomitant of chronic ethanol abuse. a polysaccharide iden tical to a proprionibacterium acnes polysaccharide (pps) has been isolated from hyaluronidase treated rheumatoid synovial fluids and synovial leukocyte pellets using a phenol-water (westphal) extraction and sepharose 4b column chromatography. pps was identified by counterimmunoelectrophoresis (cie) by rabbit antibody to sonicated p acnes organisms. sensitivity by this method is approximately 3.5 ng of pps and identity of synovial and bacterial pps was shown by cie. of common aerobic and anaerobic bacteria tested, only ps aeruginosa contained a similar antigen. pps is not present in e coli or s typhosa lipopolysaccharide. pps could not be identified in synovial fluid without extraction procedures even when a more sensitive radioimmunoassay with lzsi labeled rabbit antibody was used. sixty percent of 35 rheumatoid fluids and 70% of 10 rheuma-toid synovial leukocyte pellets contained pps. only 1 of 16 (6%) nonrheumatoid fluids was positive. two of 9 (23%) nonrheumatoid inflammatory synovial leukocyte pellets contained pps. antibody to pps by cie was found in 33% of rheumatoid sera ( p < 0.01), 45% of nonrheumatoid inflammatory arthritic sera ( p < 0.05), and 67% of normal control sera. characteristics of pps (bacterial and synovial) include sensitivity to 50 mm periodate oxidation, resistance to rnase, dnase, pronase, and boiling. uronic acid and hexosamine account for approximately 50% by weight. less than 0.5% protein is present. it appears polydisperse on sephadex g200 with a m w of approximately 5 x 1 0 ' to >lp. electrophoresis in 3.5% polyacrylamide showed a single pas positive band which formed a preciptin line with anti-p acnes antibody by crossed cie. no lipid staining bands were noted. these findings are consistent with a polysaccharide antigen. the isolation of a bound antigenic bacterial polysaccharide primarily from rheumatoid effusions associated with diminished antibody suggests a pathogenetic role for pps in rheumatoid arthritis possibly as an immune complex type. an in vitro system for examining the cellular requirements necessary for initiating and regulating antinucleic acid antibody responses has been developed to facilitate a better understanding of the mechanisms responsible for triggering antibody responses to this group of antigens in murine and human sle. it has been found that spontaneously appearing anti-ssdna hemolytic plaque forming cells (pfc) can be detected when spleen cells from normal mice of different hz types (cba, dba, bdf1, cfw, c57/b16) or young (4 weeks) autoimmune (nzb x nzw f1 (b/w), nzb, nzw) strains of mice are cultured under standard mishell dutton conditions in the absence of added antigen. such pfc(200-2600/culture) which peak on day 5 and cannot be detected prior to culture consist entirely of specific igm anti-ssdna antibody. the response, which appears to be dependent on active antibody synthesis, can be selectively abolished by specific removal of ssdna antigen binding cells, by rosette depletion. while low responses can be augmented by pokeweed mitogen, there is no obligatory requirement of serum, thymic (t) cells, or macrophages in the culture system. this in vitro response can blocked by: 1) adding small quantities of ssdna (lo-5opg) but not similar concentrations of dna or rna directly to culture on day 1; 2) pre-incubation of spleen cells with ssdna (1 hour) and washing prior to culture. unblocking can be demonstrated by subsequently treating these cells with trypsin-dnase, suggesting that this inhibition may be due to a membrane receptor blockade mechanism; 3) the addition to young b/w spleen cells of t cells from young (4 weeks) or old (> 6 months) syngeneic mice similarly abrogates this in vitro anti-ssdna response without effecting cell viability or the anti-srbc antibody response. on the other hand syngeneic bone marrow cells which markedly suppress the anti-srbc antibody response have no effect on the anti-ssdna response of these spleen cells. these in vitro studies suggest mechanisms that may be relevant to the normal regulation of anti-nucleic acid antibody and other autoantibody responses in vivo. the ability of old b/w t cells to exhibit inhibitory effects similar to those of young preautoimmune b/w mice on the in vitro antinucleic acid antibody response suggests that the suppressor potential of these old b/w t cells may be blocked in vivo. the ehlers-danlos syndrome (ed-s) is an uncommon, genetically determined disorder of connective tissue. of the seven clinically distinct types, a basic molecular defect has been reported in types iv, vi, and vii, all of which are inherited by a recessive mode, and appear to be related to deficiencies in the activity of specific enzymes involved in postribosomal changes. types i to i11 are inherited by a dominant mode and might be expected to have defects at the transcriptional level. type v is sex-linked and has been reported to result from a cross-link deficiency. we have studied 8 patients with ed-s type i, 3 patients with type 11, and 3 with the x-linked type v. the results show that the reducible crosslinks are present and undergo the same maturation process to nonreducible cross-links as in normal skin. we were unable to confirm the absence of reducible cross-links in the x-linked type ed-s. the ability of the ed-s skin to produce apparently normal cross-linked collagen was supported by cell and tissue culture studies. transmission electron micr~scopy and optical birefringence revealed a normal ultrastructure of the collagen fibrils. at a higher morphological level of organization scanning electron microscopy demonstrated a gradual increase in fiber bundle disorder from the x-linked to the myasthenia gravis, where the fibers making up the large fiber bundles demonstrated a considerable inability to aggregate. this disorder could account for the prolonged lag phase in the stressstrain curves, while the presence of cross-links ensures that once stress is on the individual fibers the elasting modulus is normal and the skin can return to normal after hyperextension. the absence of cross-links would lead to an irreversible elongation of the fiber. the low breaking point of the fiber is therefore due to the lack of integrity of the fiber bundles and suggests that the defect lies at a higher order of organization. the cryoprecipitates (cryos) from many sle sera contain lymphocytotoxic antibody and, when used to immunize rabbits, induce antibodies to human lymphocytes, suggesting that they contain membrane fragments complexed to antibody. lymphocyte-reactive antibodies in sle sera also recognized antigens on neuronal cells. to determine whether the membrane fragments in the cryos have the antigenic determinants shared with neurons, antisera generated against 12 sle cryos were tested in an antibody-dependent, cell-mediated cytotoxicity assay by use of 61cr-labeled human neuronal cells, sk-n-sh, as targets and normal human peripheral blood lymphocytes (pbl) as effectors. the anti-cryos produced 9.7 f 3.8 mean % "cr-release. two of the antisera were strongly positive at 35 and 38% cytotoxicity, and 3 others were modestly cytotoxic in the range of 8.3 to 12.4%. the remaining antisera were not significantly different from the controls; 1.3 f 0.3% '0-release from 12 normal rabbit sera (nrs), and 1.7 f 0.5% cytotoxicity from a control group consisting of 4 non-sle anti-cryos. the antibody activity was not linked to sk-n-s h cells. the same sera reacted with human neuronal line, la-n-1, and 2 human glial lines, a-i72 and u-l18mg. unexpectedly there was no correlation between reactivity against the neuronal cells and antibody activity against normal human pbl. antibody t o pbl was not detected in 4/5 anti-cryos with neuronal antibody, and 2 of the antisera unreactive with sk-n-sh had anti-pbl activity. absorption of the anti-cryo reactive against both sk-n-sh and pbl with the human lymphoblast line wll2 removed all anti-pbl activity but not the neuronal reactivity. absorption with sk-n-sh eliminated the neuronal reactivity and also diminished the antibody to pbl. the antineuronal activity was not removed by absorptions with human platelets and human rbc. passage of the anticryos over immunoglobulin containing immunoabsorbents removed the detectable anti-immunoglobulin activity without diminishing the neuronal reactivity. thus, sle cryoproteins contain antigenic determinants shared by neuronal and glial cells but not by the major cellular elements in blood. it is not yet clear whether those antigens are present on fragments of neuronal membranes or on antigenic structures eliciting cross reactive antibodies. it is generally accepted that antibodies to double stranded (ds) dna are a feature of active sle, while antibodies to single stranded (ss) dna occur in many rheumatic diseases. the natural occurrence of antibodies to dsdna presumes an interaction with the sugar-phosphate backbone of dna, the immunogenicity of which is unproved. we have attempted t o define the preferential reaction site of dsdna antibodies on a molecular species of dsdna containing the multiple ss regions, herewith designated ds/ssdna. calf thymus dna was sheared to a molecular weight of 1 x iw daltons. characterization by hydroxyapatite and benzyolated naphthoylated deae cellulose chromatography (j immunol 118:694, 1977) indicated the dna was predominantly ds with numerous ss regions. antibodies to chromatographically pure dsdna (> 80% binding in a standard farr assay) were partially purified by ammonium sulfate precipitation, iodinated with lz6i, and reacted with ds/ssdna. the resulting complexes were precipitated with 4.5% polyethylene glycol (mw, 6000) and redissolved in 0.1 m tris-hci, ph 8.0, 0.01 m mgci2, 0.15 m nacl (the endonuclease buffer). the solubilized complexes were reacted with ss specific endonuclease from neurospora crassa (0.5pg enzyme/700pg dna) and compared t o undigested complexes by velocity sedimentation in a 5% to 40% sucrose gradient run at 135,ooog for i5 hours. undigested complexes sedimented at a position of 19s; after endonuclease digestion there was a dramatic decrease in the sedimentation velocity to a 5 7 s position. these findings could be explained by: 1) a disintegration of the dna into many smaller ds molecules still attached to labeled antibodies, 2) a liberation of antibodies formerly attached to ss regions of the ds/ssdna, 3) a displacement, by the endonuclease, of antibodies reactive with ss regions. to test these possibilities the procedure was repeated without labeling of the immunoglobulins, the resulting complexes were reacted with lz5l dsdna in a standard farr assay. after endonuclease digestion free dna antibodies were readily detected, as shown by a 20-fold increase in dna binding, 3% to 63%. these findings indicate that dsdna antibodies preferentially react with ss portions of dsdna molecules. whether dsdna antibodies ever react with determinants unique to dsdna, or are merely a manifestation of high avidity binding to a very short ss region of the dsdna molecule, remains to be determined. t cell cytotoxic function in nzb mice has previously been tested utilizing target cells differing from nzb at the major histocompatibility complex (mhc) of mice, h-2. in the experiments reported here, t cell cytotoxic functions of nzb mice against targets identical to nzb at the mhc were investigated. normal mice do not generate cytotoxicity under these circumstances. spleen cell suspensions of nzb mice (h-2d) were cultivated for 5 days in the presence of irradiated stimulator cells of other h-2d strains (b10.d2, balb/c, dba/2, hw19). on the fifth day labeled target cells identical to the stimulating cells were added and the6'cr release from these targets was measured after 4 hours. nzb effector cells exerted a highly significant specific lysis in all four of the h-2d strains tested; none of these reacted against nzb. in experiments in which nzb cells were sensitized against one h-2d strain (balb/c) target cells from all four h-2d strains were lysed, demonstrating crossreactivity of the lytic reaction. the amount of lysis observed was dependent on the ratio of effector to target cells. the action of the effector cells was abolished by treatment with anti-thy-1 serum, indicating their t cell character. the capacity to respond against balb/c could be demonstrated in nzb mice as early as 4 weeks and as late as 16 months of age. the capacity of nzb mice to generate cytotoxic t-cells following primary in vitro sensitization against targets carrying the same major transplantation antigens as nzb establishes a qualitative difference between nzb mice and all normal strains since generation of cytotoxicity is restricted under the applied conditions to targets differing at the h-2 in all normal strains heretofore tested. it is suggested that the observed lack of restrictioq of the cytotoxic reaction in nzb mice is related to the autoimmune reactivity of this strain. immune deposits in the choroid plexus of patients with neuropsychiatric manifestations of systemic lupus erythematosus (sle) and nzb/nzw f, hybrid mice are thought to relate to the pathogenesis of central nervous system (cns) lupus. since previous studies did not include in their control groups patients with sle who had no neuropsychiatric manifestations, we looked for immune deposits in the choroid tissue of patients with and without cns lupus. fixed brain tissue containing choroid plexus was retrieved from 6 sle patients and 3 cont.rols. ependymal lining cells, choroid stroma, and cortical vessels were reviewed for deposits of igg, igm, iga, igd, ige, and kappa and lambda light chains by an immunoperoxidase technique (rabbit antihuman immunoglobulin and peroxidase-antiperoxidase). complement components were destroyed by fixation. immunoglobulins and light chains were found in the ependymal cells and/or choroid stroma of each sle patient. the pattern or intensity of immunoglobulin deposition did not distinguish those patients with and without neuropsychiatric manifestations. no staining was seen in tissue from the controls. though the choroid plexus serves a filtering function, the finding of immunoglobulin deposits in the choroid plexus cannot be correlated specifically with any of the diverse manifestations of cns lupus. other clinicopathologic correlations must be sought. multinucleate cells (heterokaryons) are frequently found in rheumatoid synovium as well as in cultures of isolated, adherent cells from this tissue. using an experimental polyethylene glycol (peg) was used to fuse cells in monolayer cultures of rabbit synovial fibroblasts. fusion began within 30 minutes of peg treatment and continued for about 4 hours; approximately 40% of the cells developed 2 or more nuclei. in some experiments, up to 30% of fused cells contained 6 or more nuclei, indicating true giant cell formation. measurement of intracelluar (86 rubidium) to indicate the presence of a leaky cell membrane showed that immediately after peg treatment, intracellular (86 rb+) dropped to 30% of control levels. it began to approach normal by 5 minutes but did not recover fully for about 4 hours, at completion of fusion. during the first 24 hours after peg treatment, the treated cultures incorporated 1/5 as much (3h) thymidine as did control cultures, but incorporation of (3h) leucine into tca-precipitable radioactivity was unaffected. autoradiographic studies using (3h) thymidine revealed that peg depressed incorporation of the label into dna for at least 4 days. secretion of proteinases from peg-treated and control cultures in serum-free dulbecco's modified eagle's medium was compared. peg-treated cultures containing multinucleate cells secreted 3180 u (cumulative) latent collagenase into medium changed every 72 hours over 28 days, compared with 279 u produced by control cells during the same period (1 u collagenase degraded 1 kg collagen/hour at 37â°c. collagenase was activated from its latent precursor form by tpck trypsin-i0 wg/ml, 30 minutes, 25oc, followed by addition of excess soybean trypsin inhibitor.) neither peg-treated nor control cells released substantial or significantly different amounts of neutral or acid proteinases into medium during the same period. our data show that compared to controls, cultures of multinucleated cells have a decreased rate of cell replication and increased rate of collagenase production. multinucleate cells in rheumatoid synovium may amplify mechanisms for active collagenolysis. fourteen previously untreated polymyositis (pm) patients were treated with prednisone 60 mg/day until cpk values normalized and then with 40 mg/day for a total of 12 weeks. they were also randomly placed on either azathioprine (a) 2 mg/kg/day or placebo (p) in double-blinded manner. manual muscle testing, total prednisone dose required over 12 weeks, and muscle biopsy changes were used to detect differences in the two groups. the a group (7 patients) turned out to be weaker at onset (total manual muscle testing score -280) than the p group (7 patients with score of -170) but improved more (44 points to -236) than the p group (6 points to -164). however, the difference in improvement was not statistically significant. cpks in the two groups normalized a t about the same rate and each group therefore received comparable doses of prednisone over the 12 weeks. all followup muscle biopsies at the end of 12 weeks showed improvement in the majority of the eight microscopic features reviewed and in total numerical score. the trend was toward greater improvement in the a group but not significantly so. changes consistent with steroid myopathy were noted in 7 of 9 women but only 1 of 5 men (4 in a and 4 in p) and 5 of these 8 patients were weaker at 12 weeks. of special note is the observation that of 13 patients with normal cpk at i2 weeks, 8 still had significant inflammatory infiltrates. therefore, azathioprine plus prednisone is not dramatically better than prednisone alone in polymyositis over the initial 12 weeks of treatment. the development of steroid myopathy in over one half the patients is not influenced by a or p and at the prednisone dosages used complicates the evaluation of changes in muscle strength; nevertheless, the trend is toward greater improvement in the azathioprine group in muscle strength and also follow-up muscle biopsy scores. surprisingly, a normal cpk does not indicate resolution of inflammation in the muscle and cannot be used reliably to indicate full control of the disease. followup is continuing. with the appreciation of the relationship between immunogenetics and infection in the pathogenesis of rs, interest in this condition is increasing. in order to determine more precisely the natural history of rs, data are presented from two distinct california communities: university (pa) and community (sb). to date, 102 consecutive patients have been studied: 77 (pa) and 25 (sb). rs is defined as an asymmetric oligoarthropathy (predominantly lower extremity) accom-panied by one or more of the following symptoms: 1 ) urethritis, 2) diarrhea at onset, 3) inflammatory eye disease, 4 ) mucocutaneous manifestations consisting of balanitis, buccal ulceration, or keratodermia blennorrhagica. patients with ankylosing spondylitis, psoriatic arthropathy, or other rheumatic syndromes were excluded. nineteen (19%) of the 102 presented with diarrhea and 74 (73%) had at least a triad of the above features. there were more similarities than differences between sb and pa. for example, females represented 14% (sb) and 12% (pa). the mean duration of disease to date is 76.3 months (sb) and 82.8 months (pa), disease activity continuing (albeit, sometimes, episodically) in 82% of the sb practice and 90% of the pa patients. hla-b27 was present in 79% (sb) and 82% (pa). only 7% had a persistent monoarthropathy. twenty-four percent of patients had a positive family history for an inflammatory polyarthropathy. other similarities included keratodermia blennorrhagica in 24% (sb) and 25% (pa), tendinitis in 43% (sb) and 38% (pa) and heel disease in 50% (sb) and 40% (pa). sacroiliitis occurred in 23%, 50% of whom had asymmetric change. only 1 patient with sacroiliitis was hla-b27 negative. the esr (3-118, mean 42) appeared unrelated to disease activity. aortitis was present in only 3 (pa) patients. differences between pa and sb patients were minimal; balanitis and uveitis appearing more frequently in the former group. buccal ulceration was seen in 23% of the pa patients and 12% of the sb group. from this study we conclude: 1) rs is a major chronic rheumatic disease; at least two-thirds of patients have active disease at an 81 month followup. the prognosis for occupation requiring significant exertion should remain guarded. 2) there are minimal differences between an academic and community population of rs patients. 3) there were no discernible differences between disease severity in hla-b27 positive and negative patients. the activities of the major fragment of the third component of complement, c3b, are controlled by at least two proteins, c3b inactivator and b1h globulin. using in vitro model systems, we have previously demonstrated that 81 h physically binds to c3b and blocks its activity. in the present report immunofluorescent examination of renal biopsies has been used to demonstrate that similar c3b-blh interaction occurs in vivo. twelve biopsies from patients with systemic lupus erythematosus or acute poststreptococcal glomerulonephritis were examined. igg and complement components were found by immunofluorescence to be deposited in a granular fashion in the glomeruli of these specimens. by electron microscopy, mesangial, subendothelial, intramembranous and/or subepithelial electron dense deposits were found. a single biospy in which only c3 was found to be deposited, consistent with activation via the alternative pathway, was also studied, as was one patient with goodpasture's syndrome, whose biopsy contained linear deposits of proteins along the glomerular basement membrane. six specimens from patients with such nonimmune renal diseases as arteriolar nephrosclerosis and acute tubular necrosis were also examined. by use of direct immunofluorescence with anti-blh specifically shown to be free of any contaminating anti-c3, deposits of b1h were found in every instance (14 of 14 exams) in which c3 deposits were observed. this was true regardless of the underlying disease which resulted in the c3 deposits. the spatial distribution of p l h within the glomerulus was often congruent with the distribution of c3 in the same tissue, suggesting that these two proteins were intimately associated. n o deposition of b1h was observed in those with nonimmune renal disease. we conclude that binding of / 3 1h to fragments of c3, presumably c3b, occurs in vivo during immunologic activation of the complement system, just as we had previously demonstrated it to occur in vitro. hahn et al. (ann int med 76365, 1972) have shown a relationship between anti-hdz and anti-dna in 4 patients with hdz-sle, and yamauchi et al. (j clin invest 56:958, 1975 ) have shown immunologic cross reactivity between dna and hdz with rabbit antisera to hdz-albumin conjugates. to find if the development of antibodies to dna or dnp in patients taking hdz represents the production of anti-hdz which cross reacts with dna/dnp determinants, we followed prospectively 2 1 hypertensive patients taking this drug (average dose 100 mg/day) for one year. antibodies to hdz were measured by passive hemagglutination, to native dna by in the prospective group the major finding was development of increased levels of anti-dnp in 8 patients, 7 of whom also made anti-hdz. nine other patients made anti-hdz without anti-dnp. none made anti-ndna. one developed a mild hdz-sle syndrome with anti-hdz and anti-dnp. examination of sera of 4 other patients with hdz-sle syndrome by radioimmunoassay failed to show an immunologic cross reaction between hdz and dnp although such a reaction was found in guinea pig anti-hdz-bsa serum. the results show a relationship between production of antibody to hdz and to dnp in patients taking hdz but do not support the hypothesis that anti-dnp in such patients is antibody cross reacting with hdz. they are consistent with the findings of fritzler and tan (clin res 25:a483, 1977) that anti-dnp in drug-related sle reacts with the histone moiety. the relationship of the immune response to hdz to that to dnp remains unexplained. anti-idiotypic antibodies recognize antigenic determinants (idiotypes) uniquely associated with a given antibody molecule, or closely related molecules. the in vivo interaction between idiotypes and anti-idiotypic antibodies has been postulated to play a central role in the regulation of the immune system. the purpose of this study was to characterize the idiotypic antigens on three monoclonal igm anti-y-globulins and to determine if cross-reacting idiotypes were present in the sera of patients with rheumatoid arthritis (ra). anti-idiotypic antibodies against the purified igm ( k ) anti-y-globulins lay and si, and the igm (a) anti-y-globulin koh, were raised in rabbits. when analyzed by solid phase radioimmunoassay (ria), each antibody reacted only with the immunizing antigen, and not with pooled igg or other igm paraproteins lacking anti-y-globulin activity. studies with recombinant molecules reconstructed from lay or si light or heavy chains and light or heavy chains from heterologous proteins demonstrated that the idiotypic antigens were formed from a specific heavy-light chain interaction. the idiotypes were closely related to the antibody combining site, as judged by the inhibition of idiotype-anti-idiotype binding by antigen, i.e., igg, and of idiotype-antigen binding by anti-idiotype. idiotypes cross-reacting with the monoclonal igm ( k ) anti-yglobulin si, but not with the igm (a) anti-y-globulin koh, were found in increased concentration in the sera of patients with seropositive ra. the idiotype positive material, as analyzed by gel filtration, was found in the 19s but not the 7s fraction of serum. from these experiments we conclude: 1) that the idiotypes on igm anti-y-globulins are associated with the antibody binding site and are the product of a specific light-heavy chain combination; 2) igm rheumatoid factors in ra patients, although polyclonal, may have idiotypic antigens cross-reacting with those on monoclonal igm ( k ) anti-y-globulins; 3) igg rheumatoid factors probably have different idiotypic antigens than igm rheumatoid factors and may therefore have different active sites and antibody specificities; 4) anti-idiotypic antibodies may be useful for the selective suppression of anti-yglobulin production without a general depression of immune responsiveness. connective tissue activating peptides from lymphocytes (ctap-i) and platelets (ctap-111) are known to stimulate glycosaminoglycan synthesis, glycolysis, and mitogenesis in connective tissue cell cultures. direct evidence suggested that increased accumulation of cyclic amp was involved in the mechanism of action of these peptide agonists, and increased prostaglandin e synthesis was postulated on the basis of indirect evidence. in the present experiments, ctap-i and -111 were incubated with human and murine cells in culture, and prostaglandin e was measured by radioimmunoassay using antibody directed primarily to prostaglandin e,. both ctap-i and -111 markedly stimulated the elabo-ration of prostaglandin e, into culture medium, the earliest evidence of increased synthesis occurring at 4 hours with maximal concentration found at 24 hours. in a typical experiment, after 24 hours incubation, buffer, ctap-i and ctap-i11 treated cells produced, respectively, 39, 1150, and 580 pg of prostaglandin e,/1.5 x 1cp normal human synovial cells. substantial residual stimulation persisted at least through 48 hours. the lymphocyte factor (ctap-i) appeared to be more potent than ctap-i11 in stimulating prostaglandin synthesis. indomethacin these studies substantiate the postulated increase in synthesis of e series prostaglandins by human connective tissue cells on exposure of ctap-i and -111, and clarify the mechanism of action of these agonists on "activated" target cells. the importance of elevated extracellular concentrations of prostaglandins is uncertain, although they may potentiate the actions of ctap-i and -111. in order to examine the repair collagens produced by cells present in injured cartilage, the femoral articular surfaces of three groups of new zealand white rabbits were scarred in the following manner: superficial and deep lacerations, and drilled holes. eight weeks after surgery the rabbits were sacrificed and slices of injured articular cartilage harvested. the types of collagen produced at the site of these lesions were identified by labeling the recovered specimens with *h proline and characterized by sds gel electrophoresis, cmc chromatography, and cnbr peptide analysis. in all cases, tissue-specific type i1 ([ai( 1 1)18) cartilage collagen was synthesized. histologic examination revealed the chondrocytes bordering the cartilage injured by deep laceration and drill holes responded by increased cellular activity. grossly, the drilled holes were completely filled with tissue possessing staining and morphologic characteristics similar to that of hyaline cartilage. these data strongly suggest that in the lacerative type of injury, the surrounding tissue will produce enough matrix for repair only when the subchondral bone is violated. both repaired and normal cartilage produce tissue-specific type i1 collagen. we recently demonstrated that, from birth on, nzb splenic b cells spontaneously secrete 40 to 100 times as much pentameric igm as normal b cells (j immunol 119:1639 (j immunol 119: , 1977 . spleen cells from 6 to 10 week old mice have been examined in further study of this abnormality. nzb spleen suspensions contain 8 to 10 times as many spontaneous plaques to sheep erythrocytes and to tnp conjugated sheep erythrocytes as normal mice. there is a similar increase in the number of cells secreting igm as detected by a reverse plaque assay. after cold ethanol-acetic acid fixation, it can be shown that nzb spleen contains 4-696 cells with cytoplasmic igm compared to 0.2-0.4% in normals. nzb spleen cells were sorted by flow microfluorometry (fmf) into surface ig negative cells and 4 pools with increasing levels of surface igm or total ig. the surface ig negative fraction produced no igm but the positive pools each had the same level of igm production and cytoplasmic staining. when sorted into 4 pools on the basis of size, however, all the igm production was found in the largest fraction. nzb spleen suspensions contain more large cells than normal spleens by coulter volume measurements and fmf reveals more large surface ig positive cells in nzb than normal spleens. when surface ig was enzymatically removed before fixation, it was found that the nzb cells with cytoplasmic ig were 4 to s times brighter than those from normal mice. thus nzb spleen contains approximately 10 times as many cells which synthesize and secrete igm as normal mice. each such nzb cell appears to contain and presumably secrete about 5 times as much igm as do cytoplasmic ig positive cells in normal mouse spleen. because nzb mice spontaneously produce antibody to thymocytes, we utilized fmf at high gain to search for immunoglobulin on peripheral t cells. none could be demonstrated. after fixation, however, immunoglobulin was readily detected in nzb splenic t cells. whether this immunoglobulin is passively acquired and rapidly pinocytosed and thus not available for staining on the cell surface or endogenously synthesized by the t cell is under investigation. in an effort to define the cellular basis of abnormalities of polyclonal b cell activation previously noted in nzb mice, the surface immunoglobulin (sig) isotypes of spleen cells from young and old nzb mice were examined. after lactoperoxidase-catalyzed radioiodination, cells were lysed, the immunoglobulins bound to rabbit anti-mouse immunoglobulin and the immune complexes absorbed to s aureus. the complexes were solubilized and the cell surface immunoglobulins analyzed by sodium dodecyl sulfate (sds) polyacrylamide gel electrophoresis. cell surface immunoglobulin isotypes were also analyzed by staining spleen cells with fluorescein-conjugated rabbit anti-p, anti-6, and anti-ig sera and examining stained cells by fluorescence microscopy or by automated cytofluorometry on a becton-dickinson facs-111 cell sorter. spleen cells from both young (6 weeks old) and old (one year old) nzb mice were found to have markedly increased ratios of cell surface igm/igd compared to cells from balb/c control mice. the altered ratio of sig isotypes was not a consequence of increased proteolytic activity present in nzb cell suspensions since the addition of nzb cells to iodinated balb/c cells did not alter the balb/c ratio of sigm/sigd. it was not due to the presence of cytophilic antibody or autoanti-body since the sigm was found to have a sedimentation coefficient of 8s and was thus not of serum origin. when examined by fluorescence microscopy or by cytofluorometry, nzb spleen cells were found to have normal numbers of sigm+ and sigdf cells, indicating that the altered sigm/sigd ratio observed in the radioiodination experiments was due to abnormal densities of these isotypes on b cells rather than to the absence of an igd-bearing b cell subset. the increased sigm/sigd ratio may be a consequence of in vivo polyclonal b cell activation, since in vitro polyclonal activation of m o p e spleen cells has been shown to alter this ratio in a similar manner. since the increased ratio was noted as early as 6 weeks, these data provide support for the concept that polyclonal b cell activation precedes the onset of autoimmune disease in nzb mice. sera from patients with systemic lupus erythematosus (sle) will bind native dna (n-dna). poly dat, a synthetic n-dna, consists of regularly recurring base pairs. this results in a restricted antigenicity but ensures that the molecules do not contain single-stranded (ss) regions. many laboratory preparations of n-dna will be contaminated with either ss dna or structures with ss regions in predominantly duplex molecules. the aim of this study was to determine a relationship between antibodies to poly dat and native dna in individual sle sera. one hundred sera from 35 patients with sle were used in this study. antibodies to poly dat and n-dna were measured in duplicate in each serum using a millipore filter technique. poly dat was prepared from datp and *h labeled dttp in the presence of e coli dna polymerase. native ah labeled dna was extracted from hae 70 cells after incubation with 'h thymidine. optimal concentrations of each antigen were calculated for use in the assay. each preparation was structurally analyzed by both hydroxyapatite (hap) elution studies and ethidium bromide fluorescence (ebr) to determine the presence of ss dna or ss regions within a predominantly duplex molecule. the specificity of antibodies to poly dat and n-dna was assessed by passing sera over agarose columns bound to either circular pm2 dna or poly dat and measuring both n-dna and poly dat antibodies in serum and eluate. neither preparation contained ss dna assessed by hap elution. poly dat was entirely duplex as assessed by ebr fluorescence but our n-dna contained 15% ss regions within the predominantly duplex molecules. under optimal conditions each serum showed consistently less binding to poly dat than to n-dna. a significant correlation was seen between the binding to poly dat and n-dna (r = 0.91, p < 0.01). this correlation was consistent in all sera tested and no serum showed exclusive binding to only one preparation. binding to both antigens could be removed by passing a serum over an agarose column carrying either poly dat or n-dna. these results suggest that in clinical practice diagnosis and management of sle will be provided by measurement of antibodies to either poly dat or n-dna provided that the n-dna preparation is well characterized and does not contain ss dna or significant ss regions. despite its limited potential antigenic sites, poly dat provides a useful alternative to n-dna and does not possess ss regions to which other antibodies may bind with less diagnostic specificity. cultured adherent rheumatoid synovial cells (asc) produce collagenase and prostaglandins, particularly pge;. with age in culture and passage, collagenase and pge, production decrease; however, release of these substances can be stimulated up to > one hundredfold by a factor (apparent mol wt of -14,000) released by cultured human peripheral blood mononuclear cells (mcf). mcf stimulation of collagenase production by asc is enhanced by addition of low concentrations of indomethacin (indo), 1 nm; such concentrations of indo block pge, synthesis > 5wo even in the presence of mcf. at higher indo concentrations (1-10 fim) which inhibit pge, synthesis -1008, collagenase stimulation is usually inhibited by -60%. in some asc stimulated with mcf, this inhibition by indo (10 pm) is reversed by addition of small amounts of pge, (10 ng/ml). addition of these small amounts of pge, results in augmentation of collagenase stimulation to levels greater than those seen when cells are stimulated with mcf alone. addition of larger amounts of pge, (1 pg/ml) under these conditions tends to inhibit collagenase. asc respond to exogenous pge, with increased levels of camp. mcf also modulates this camp response: preincubation of asc with either indo or mcf plus indo augments camp response to exogenous p g b . it is possible that some of the effects of pge, on collagenase in asc are mediated through adenylate cyclase. pge, levels thus have profound effects on collagenase production by asc and modulate collagenase response to stimulation with mcf. therefore, pge, inhibition with pharmacologic agents in vivo could have beneficial or deleterious effects with regard to connective tissue destruction depending upon the type, sensitivity, and prior environment of the responding cell. ty cells, a subset of t lymphocytes, bear fc receptors for igg and have suppressor activity for antibody production after activation with igg immune complexes. levels of ty cells as well as total t lymphocytes were measured in 19 patients with systemic lupus erythematosus (sle), 11 with active and 8 with inactive disease, and in 47 normal subjects. mononuclear cells were separated on a ficoll-hypaque gradient, depleted of adherent and phagocytic cells, and pelleted with neuraminidase-treated sheep erythrocytes. rosette-forming cells were counted to determine the percentage of total t lymphocytes. rosettes were then mechanically dissociated and t cells separated from sheep erythrocytes through a ficoll-hypaque gradient at 37â°c. purified t cells were pelleted with ox erythrocytes sensitized with rabbit igg and rosette-forming cells recorded as ty cells. a total of 200 cells in each of three replicate assays was counted. active sle patients showed a significant decrease in total t lymphocyte percentages compared to normal subjects (p < 0.05), confirming previous reports. in addition, markedly depressed percentages of ty cells (p < 0.002) were found in patients with active disease. patients with inactive sle were not significantly different from normal subjects, both with regard to total t lymphocytes as well as ty cells. two acute sle patients who went into remission showed significant increases in both total t lymphocytes and ty cells with clinical and laboratory improvement. active sle patients show decreases both in total t lymphocytes and the ty subset of lymphocytes associated with suppressor activity. low levels of detectable ty cells in sle may reflect the presence of high levels of circulating immune complexes and/or the defect in suppressor activity reported in these patients. the 6-8 fold increase in red cell turnover in patients with sickle cell anemia results in uric acid overproduction. since uric acid overproduction in these patients begins within the first 5 years of life, we studied 91 patients with sickle cell anemia and normal gfr ranging in age from 6 to 48 to determine the natural history of serum uric acid and urate excretion in sickle cell disease. hyperuricemia (plasma urate 6.5 mg/dl) was observed in only 2 of 26 patients younger than age 13. however, urinary uric acid/creatinine ratios and 24 hour urinary uric acid levels were elevated in these patients, indicating uric acid over-production and hyperuricosuria. thirty-seven percent of adults (24/65) were hyperuricemic (mean plasma urate 8.6 f 0.5 mg/dl); 41 were normouricemic. of normouricemic adults studied while on a purine-free diet, 9 of 16 (56%) had 24 hour urine uric acid excretion greater than 600 mg. urate clearance in these 9 patients was increased (cur 14.8 f 0.8 ml/min), indicating that normouricemia was maintained by hyperuricosuria. urate clearance in the hyperuricemic subjects was decreased (cur 5.3 f 0.8 ml/min) as compared to both normal subjects (cur 8.2 f 0.6 ml/min) and normouricemic adults with sickle cell disease (cur 14.8 f 0.8 ml/min). urate clear-ance appears to be the major determinant of serum uric acid concentration even in sickle cell patients with urate overproduction. responses of urate clearance to probenecid and pyrazinamide were exaggerated in the normouricemic overexcretors [pza suppressible urate clearance (psur):ss, 12.6 f 0.8 ml/min; control 6.4 f 0.8 ml/min; probenecid (pb) response; ss, 48.8 f 9 ml/min; control, 40.4 f 8.0 mi/min] and were diminished in the hyperuricemic subjects with diminished urate clearance (psur, 4.9 f 1.2 ml/min; pb response, 15 f 2.2 ml/min). urate clearance was correlated pah clearance. these results suggest that changes in urate clearance were secondary to changes in tubular secretion in urate. the initial response to urate overproduction in ss disease is hyperuricosuria with increased urate clearance and maintenance of normal serum urate concentration. hyperuricemia is found in adults with diminished urate clearance and is associated with other evidence of impaired renal tubular function. nephropathy is a little known complication of polymyositis. thus, we describe the features of glomerulonephritis in 5 patients with polymyositis. the patients (4 men and 1 woman) aged 21-41 years each presented with symmetrical limb girdle weakness, widespread polyarthritis, myalgias, recurrent fever, and proteinuria. cpk, ldh, and sgot levels were elevated in all cases, and the diagnosis of polymyositis was confirmed by electromyography and muscle biopsy. although raynaud's phenomenon and sacroiliitis were each present in one case, no patient had the skin changes of dermatomyositis, and no evidence of other diffuse connective tissue disease or underlying neoplasm. ana was negative in all patients; cryoglobulins, le preparations, and asot were absent in 4 patients tested. c3 complement level was decreased in one case. rheumatoid factor was negative in 4 and latex positive ]:i60 in one case. blood urea nitrogen and serum creatinine were normal in each patient; however, the 24 hour urine protein excretion ranged from 1.7 to 4.4 gm. three patients had an abnormal urine sediment and one had myoglobinuria. renal biopsies (4 patients) showed a mild focal segmental mesangial increase. immunofluorescence was negative in one biopsy and mildly positive in a granular pattern for igg and igm in 2 others. treatment of all patients with high-dose corticosteroids led to rapid clearing of the proteinuria and slower improvement of the polymyositis. a 10-year review in our center of 70 polymyositis cases showed proteinuria exceeding 300 mg/24 hours in 9 patients and abnormal urinary sediment in 4. none of these latter patients had underlying malignancy. a focal glomerulonephritis associated with polymyositis may be more common than is generally appreciated. although the pathogenesis of the renal lesion in these cases is unknown, it may be related to myoglobin. two enzymes of the purine nucleotide degradation pathway, adenosine deaminase and purine nucleoside phosphorylase, are associated with specific immunodeficiency syndromes. a third catabolic enzyme, 5'-nucleotidase, was measured on the external surface of peripheral circulating lymphocytes from patients with immunoglobulin deficiencies. all 8 male patients with congenital agammaglobulinemia demonstrate reduced activity of lymphocyte ecto-5'-nucleotidase to 30 to 48% of the normal value. the mean activity is 5.7 f 1 .o nm/hr/lob cells for congenital agammaglobulinemia as compared to the mean normal value of 15.0 * 5.6. patients with common, variable hypogammaglobulinemia or selective iga deficiency have values within the normal range. two other lymphocyte plasma membrane ectoenzymes, atp'ase and nonspecific phosphatase, have similar values in lymphocytes from normal subjects or from subjects with congenital agammaglobulinemia. the activity of 5'-nucleotidase in lymphocyte lysates has similar values in normal and enzyme deficient subjects. ecto-5'-nucleotidase has similar activity in both t and non-t lymphocytes in all subjects and the deficiency occurs in both these categories of cells in the affected patients. ecto-5'-nucleotidase from normal and enzyme deficient subjects has a similar p h optimum'of 7.5, is similarly inhibited by adenosine-5'4, p-methylene diphosphonate, has hyperbolic kinetics and a similar michaelis constant of 25 pm for amp. these data suggest that the reduction of lymphocyte 5'-nucleotidase activity is an abnormality localized to the plasma membrane in this x-linked, b-cell immunodeficiency syndrome. in vivo clearance of exogenous single-stranded dna (ssdna) is extremely rapid and occurs primarily through the liver. with higher doses of ssdna, however, both liver uptake and blood clearance approach a maximum, enabling large molecular weight ssdna to persist in the circulation (emlen, mannik; j exp med, march, 1978) . in the present study, we examined the effects of prior saturation of the liver with immune complexes on the clearance kinetics of ssdna. heat denatured calf thymus '*'i ssdna was chromatographed on hydroxyapatite and a defined molecular weight was obtained by gel filtration over sepharose 4b. immune complexes were prepared at 5-fold antigen excess with human serum albumin and purified rabbit anti-human serum albumin. female c57bl/6j mice were injected at time 0 with immune complexes containing 5 mg antibodies or with buffer. at 3, 6, 12, 24, or 48 hours, animals were given 50 pg of '9 ssdna; serial blood samples were assayed for radioactivity and clearance velocities were calculated by linear regression analysis. clearance of ssdna was slowed in the mice pretreated with immune complexes. control mice cleared ssdna at a rate of 2.63 f 0.27 pg/ml/min, while animals pretreated with immune complexes cleared the ssdna at a rate of 1.70 f 0.04 at 3 hours (p < 0.01), 1.36 f 0.31 at 6 hours (p < 0.001) and 1.39 f 0.49 at 12 hours (p < 0.01). clearance returned to normal at 24 and 48 hours. the degree of suppression of ssdna clearance velocity at different times after administration of immune complexes correlated with the previously established values for the amount of immune complexes present in the liver at a given time. to examine the effects of pretreatment with immune complexes on the saturability of ssdna clearance mechanisms, 10, 50, or 100 pg of ssdna were given 6 hours after administration of immune complexes. with the 100 pg dose the clearance velocity approached a maximum, but this value was less than half of the maximum clearance velocity in normal mice. these observations indicate that immune complexes alter ssdna clearance by decreasing the number of sites in the liver to which dna can bind, or by decreasing access of dna to the liver. by altering clearance kinetics, immune complexes may contribute to the persistence of dna in the circulation of patients with sle. (supported by nih grant am11476.) rheumatoid arthritis (ra) frequently affects the wrist, leading to pain, loss of function, and destruction of normal anatomy. surgical treatment by synovectomy alone does not correct carpal deformities and ulnar deviation; fusion of the wrist, although effective in reducing pain, severely limits function. to prevent loss of upper extremity function while simultaneously correcting deformities and relieving pain, we combined synovectomy with distal ulnar excision, correction of carpal supination-subluxation, and repair of damaged extensor tendons. this surgical stabilization approach was performed 61 times in 50 patients with ra. all had definite or classic ra and had not responded to aggressive physiotherapy, including splints and local and systemic medications. all had synovitis with deformity and instability. the deformity most commonly seen was carpal supination-subluxation with relative prominence of the distal ulna. the primary reasons for operation were frank (23) or potential tendon rupture (3), persistent synovitis with pain at rest (21), and pain with significant deformity (14). the mean age was 48 years (range: 21-76). the wrist was exposed with preservation of the exten-sor retinaculum. following excision of the distal ulna, a synovectomy of the radiocarpal and intercarpal joints was carried out. any existing carpal dissociations were realigned, and the carpal supination-subluxation deformity was corrected by placing the wrist in pronation and reconstructing the triangular ligament using the volar capsule. extensor tendon repair and grafting were then carried out. length of followup was 30 months with a range of 1-8 years. results were classified as good if the patient was satisfied, had only occasional pain, a stable wrist, and no recurrence of synovitis. three patients were lost to followup and of the remaining 58, good results were obtained in 42. an additional 12 had objective improvement but experienced recurrence of synovitis and pain and an extensor lag of up to 1 5 o . less satisfactory improvement was found in 4 of the 58. the average loss of motion was 47'37, leaving patients with a combined palmar and dorsiflexion range of 42". although this stabilization procedure is not considered appropriate for the severely destroyed wrist, the overall 900/0 improvement (fair and good results) justifies its consideration in preference to either synovectomy or wrist fusion alone. the destructive inflammation observed in synovia of rheumatoid joints may be largely due to autoimmune processes. antibodies to types i, 11, 111 collagens have previously been detected in joint fluids of rheumatoid patients and cellassociated antibodies which react only with cartilage type i1 collagen can be locally detected in inflamed rheumatoid synovia. we are interested in finding evidence for cell-mediated immunity to collagen. immune lymphocytes challenged with antigen produce substances (lymphokines) that can grossly stimulate the secretion of the enzyme collagenase from macrophages. collagenase, which can degrade cartilage collagen producing cartilage destruction, is also secreted by macrophage-like cells in rheumatoid synovia and its secretion can be stimulated from synovial cells by a factor(s) produced by lymphocytes, unstimulated or stimulated with phytohemagglutinin. in the present study inflamed synovia were removed at surgery from 9 patients with classic rheumatoid arthritis. synovial fragments were maintained in a viable state in organ culture for up t o 2 weeks. culture media were changed every 2 days and the collagenase secreted into the culture medium was assayed. to these cultures were added type i or type 11 collagen in the form of diffusible immunologically active fragments prepared by cyanogen bromide cleavage of native collagen. in 3 of 9 rheumatoid patients, the addition of type i1 collagen fragments resulted in a significant increase in collagenase secretion compared with explants which had not been stimulated: artificial stimulation of lymphocytes in these explants with phytohemagglutinin also resulted in a similar increase in collagenase secretion. type i collagen peptides had no stimulatory effect. these results support the thesis that in some patients rheumatoid synovia contain immune lymphocytes which can respond t o type i1 collagen peptides produced by degradation of hyaline cartilage collagen. when challenged with antigen, these cells may produce factors that stimulate the secretion of collagenase from synovial cells leading to further cartilage destruction. although intraarticular corticosteroid injection has been considered a therapeutic adjunct in rheumatology for the last 25 years, scientific proof of efficacy is still lacking. the present study was undertaken, therefore, to determine the effect of intraarticular steroids on pain in the osteoarthritic knee by use of a controlled, double-blind protocol. thirty-four patients with symptomatic osteoarthritis of the knee were included in the study. patients previously injected with local steroids were excluded. half the patients were treated with 20 mg of triamcinolone hexacetonide injected into the knee, the other half with the same volume of placebo (vehicle without steroid). the two physician-experimenters performed the arthrocentesis and withdrew any synovial fluid, if possible. a nurse-assistant then injected either steroid or placebo through the same needle according to a predetermined, random schedule. thus, the physicians who conducted the study did not know the nature of the material each patient received. knee pain was quantified prior to, and at 1, 4, 6, and 8 weeks after injection. at 1 week the steroid group had significantly reduced knee pain compared with the pretreatment assessment. this reduction persisted through the 8 week evaluation period. the placebo group also exhibited a significant amount of pain relief at 1 week, but this was significantly less than that experienced by the steroid group. from 4 through 8 weeks there was no statistically significant difference between the 2 groups. whether or not synovial fluid was initially aspirated did not affect results. postinjection flares occurred as often with placebo as with steroids and did not affect the subsequent course. it is concluded that intraarticular steroids reduce the pain of osteoarthritis, but such a response cannot be distinguished from a placebo effect by 4 weeks postinjection. it has been reported previously that antibodies to histones can be demonstrated by immunofluorescence. when tissue sections are extracted with 0.1 n hci, histones are eluted from nuclei, and the extracted tissue contains only d n a devoid of nuclear proteins. histones can be reconstituted to nuclear d n a by incubation of acid-extracted tissues with purified calf thymus histones. sera containing antibodies to histones show positive antinuclear antibody (ana) staining on untreated tissue, become ana negative on acid-extracted tissue and regain ana positivity on histone-reconstituted tissue. sera from 23 patients with drug-induced lupus erythematosus (procainamide 19, isoniazid 2, nitrofurantoin 2) and 20 patients with idiopathic (not drug-induced) systemic lupus erythematosus (sle) were studied. all 23 drug-le patients had positive ana on control tissues but ana became completely negative on acid-extracted tissues. on histone-reconstituted tissues, 22/23 again became ana positive. in contrast, of 20 idiopathic sle sera which were positive for ana on control tissues, only 12 became negative and of these, 4 again became ana positive on histone-reconstituted tissues while the other 8 remained negative. three other sle sera showed partial reduction in ana titer but increased in titer on histone-reconstituted tissue. thus, 22/23 drug-le patients (96%) had antibodies to histones compared to 7/20 sle patients (35%). to determine the specificity of anti-histone antibodies, the h1, h2a-h2b and h3-h4 fractions of calf thymus histone were prepared by differential salt extraction and sephadex gel filtration and used in reconstitution experiments. it was shown that in 21/22 of the drug-induced sera antibodies were primarily against h2a-h28, while in the 5 idiopathic sle sera studied, antibodies to all classes of histone were found. a further difference was that idiopathic sle sera had antibodies to native dna and to the nonhistone proteins sm and nuclear rnp whereas drug-induced le sera did not. the heterogeneity of anas in idiopathic sle and the striking prevalence of anti-histone antibodies in drug-induced le, as demonstrated by immunofluorescence, have been valuable clinically in differentiating between these two syndromes. the etiology of pss is unknown and progress toward its understanding has been hampered by the lack of a model for the disease. it has recently been reported that skin changes similar to those of scleroderma develop in some long-term survivors of bone marrow transplantation (bmt). five such subjects, who lived an average of 19 months post-bmt, developed sclerodermatous cutaneous involvement and also underwent examination for visceral complications resembling pss. at present, 2 are alive and 3 have died. we compared their findings with the findings in 49 well-characterized patients with scleroderma to ascertain the similarities and differences between these two groups. taut hidebound skin was found in all patients in both populations and selected skin biopsies were also similar. restrictive lung disease, characterized by decreased vital capacities and diffusing capacities, was found in 88% of the pss patients and in 4/5 (8wo) of the bmt group. cardiac disease and raynaud's phenomenon, similar to that found in the pss group, were noted in one post-bmt patient, and pathologic evidence of pss-like esophageal involvement was found in another. in both the bmt and pss groups, the responses of lymphocytes to suboptimal concentrations of pha were d e pressed, while complement levels, screening tests for autoantibodies and circulating levels of iga and igm were essentially normal. the frequency of patients with abnormal ana and dna-bindings was higher in the pss group than in the bmt group. the incidence of abnormal levels of immune complexes, igg levels, anergy, and decreased percentages of b-and t-lymphocytes was higher in the bmt patients than in those with pss. even though only 19 months have elapsed since transplantation, these 5 post-bmt patients have begun to develop signs and symptoms of a pss-like disease. we suggest that bmt bears closer examination as a model for pss. this study was initiated to further investigate the inhibitory effect of igm rheumatoid factor (rf) in an antibodydependent cell-mediated cytotoxicity (adcc) assay. this assay employs human peripheral blood lymphocytes as effector cells and igg or igm sensitized, 51cr labeled ox erythrocytes as target cells. serum and synovial fluid were obtained from patients with seropositive classic rheumatoid arthritis (ra) and heat inactivated. rf was prepared by dialyzing the sera and synovial fluid against sodium acetate buffer (ph 4.0), fractionating on sephadex g-200 with the same buffer and pooling the fractions constituting the leading side of the 19s peak, dialyzing against pbs and further purifying by passage over an igg-coupled sepharose 4b column and eluting with acid. this igm-rf was tryspin digested (enzymexubstrate ratio = 1 : 50) at 60â°c for 20 minutes and the reaction stopped by adding soybean trypsin inhibitor. the fragments were fractionated on biogel a5m to separate undigested igm, fc,p and fab. the igm-rf, fc& and fab fragments were tested for inhibitory capacity in igm and igg induced adcc at both the effector and target cell levels. it was found that igm and igg induced adcc was inhibited by igm-rf and its tryspin digest fragments. inhibition at the effector cell level was achieved by rf fc& and fab whereas inhibition of the target cell level was achieved only by rf fab. these results indicate that r f may inhibit adcc at the effector or target cell level and thus modulate an immune response that is of potential importance in immune-complex disease. patients with seropositive rheumatoid arthritis (ra) of adult-onset were compared to control groups by utilizing two primary histocompatibility procedures, b-cell (ia) alloantigen typing and mixed lymphocyte culture (mlc) reactivity. by use of a two-stage microcytotoxicity assay with a panel of 30 alloantisera, the frequencies of various b-cell alloantigens in patients with ra were compared to those of control populations, including patients with multiple sclerosis. three b-cell alloantisera were identified that gave particularly high frequencies of reactivity with ra patients. delineation of the specificity of these 3 alloantisera on a panel of 36 b-cell lines derived from individuals homozygous for mlc alleles revealed that reactions were obtained only with those lines from individuals that were positive for hla-dw4, dw7, or dwlo. absorption of the alloantisera with b-cell lines from dw4, dw7, or dwlo positive individuals eliminated the seroreactivity with all b-cell lines having any of these three de-terminants, thus demonstrating that the alloantisera react with a b-cell antigen common to cells with either of the three mlc specificities. mlc testing of the ra patients with a panel of normal homozygous cells defined in the seventh international histocompatibility workshop revealed an increase in the frequencies of hla-dw4 and dwlo as compared to controls (26% versus 14.6% and 13% versus 6.9%, respectively), while the frequency of hla-dw7 was not significantly different (23% versus 18.6%). the individuals who were negative for the hla-d alleles dw4, dw7, and dwlo by conventional mlc testing yet positive for the shared b-cell alloantigenic specificity were of particular interest. the results indicate that a shared antigenic specificity exists among alleles of b-cell alloantigens that is, in turn, partially related to particular mlc alleles. thus susceptibility to ra may be a function of the inheritance of the molecules bearing this antigenic specificity. activation of mediator cells such as platelets and neutrophils plays an important role in the pathogenesis of gout. monosodium urate crystals (msu), the probable initiating agents of gouty inflammation, have been shown to stimulate suspensions of washed platelets or neutrophils. when msu crystals are coated with igg (as occurs in plasma), stimulation is markedly enhanced. these studies, using msu induced human platelet serotonin secretion as a model, examined the nature of cellular recognition mechanisms for the igg-coated msu crystal and the uncoated crystal. f(ab')2 fragments of specific anti fc antibody blocked and the lipopolysaccharide of s minnesota r595 enhanced human platelet secretion induced by igg-coated urate crystals. these agents had little effect on stimulation by uncoated crystals. this indicated that urate crystals stimulate platelets independently of fluid phase igg. urate crystals directly stimulated suspensions of washed rabbit platelets which lack fc receptors. in contrast to human cells, stimulation was blocked by igg. this again demonstrated igg-independent cell stimulation by urate crystals. calcium pyrophosphate dihydrate crystals could trigger human platelet secretion only when coated with igg. this suggests that when crystals were coated with igg, the surfacebound igg alone may be the stimulus to the cell. this was confirmed by the finding that polyvinyl pyridine-n-oxide, a hydrogen acceptor, blocked human platelet stimulation by uncoated but not igg-coated urate crystals. in contrast to igg, f(ab')2 fragments, igm, or iga did not enhance human platelet stimulation by urate crystals. this indicates that the effect of igg on urate crystal stimulation of platelets depends on the fc region of igg. these data demonstrate that urate crystals (and potentially other surfaces or particles) can stimulate a mediator cell by at least two mechanisms: by direct stimulation without the mediation of absorbed igg or when coated with igg, by triggering the cell via fc receptors. the correlation of serologic abnormalities, especially low serum complement and high levels of anti-dna antibody, with disease activity has been established in sle. the significance of abnormal serologic tests in the absence of active clinical disease is still unclear. this report describes a group of 14 patients seen in the course of a prospective study of sle in whom a discordance between clinical and serologic features was apparent. these patients had persistently positive le preps and antinuclear antibody tests, low serum complements, and high levels of dna binding. the mean lymphocyte response to con a mitogen was suppressed in these patients as compared to age and sex matched controls done on the same day. these patients did not differ significantly from other patients with sle, seen during the same period, in the frequency of skin manifestations, raynaud's phenomenon, and arthritis; they showed trends toward a lower frequency of photosensitivity, serositis, mucous membrane ulcers, and neu-ropsychiatric involvement and had a statistically significant lower frequency of alopecia and nephritis. they also resembled the larger group in the presence of most laboratory abnormalities with only hypergammaglobulinemia occurring less frequently. these patients have been followed untreated for a mean of 4% years without evidence of clinical exacerbations of disease. thus in individual patients with sle it would seem advisable to determine whether their clinical course and laboratory abnormalities are concordant. some patients will illustrate this pattern and thus therapeutic manipulation according to changes in laboratory variables may be indicated. some patients will display clinical-laboratory discordance and could then be treated for clinical exacerbations only, irrespective of laboratory changes. followup in our 14 patients would indicate that patients with such humoral and cellular immune abnormalities may safely be followed untreated for prolonged periods. independently and in two separate laboratories, cocultivation techniques have demonstrated the presence of viruslike particles (vlps) in rheumatoid synovial membrane cells (rsc). one technique consisted of cocultivation of long-term rsc cultures with human lung fibroblasts (wi 38); the other system utilized cocultivation of freshly dissaggregated rsc with fetal rabbit synovialcell cultures. controls in both laboratories consisted of similar cocultivations using cultures derived from nonrheumatoid membranes. all cocultivations were maintained for 2-3 months without subculture, during which time small foci of heaped-up cells were observed. by electron microscopy, all the rheumatoid cultures (15) but none of the control cultures (7) showed vlps. these appeared as spherical bodies with a spike-like external array, seen both in sonicated negatively stained (pta) preparations, and in thin sections of harvested rsc cocultivations. these vlps were found budding from the cell membranes, and also within cytoplasmic vacuoles. their morphology resembled that of budding rna viruses, although coronaviruses and myxoparamyxoviruses may also show these characteristics in certain situations. similar particles have not been previously demonstrated in rsc. serial passage of cocultivation extracts containing vlps onto other cell types resulted in repeated transient cytopathic effects. extracts of the rsc cocultivations were injected into suckling mouse brains, resulting in a reproducible illness not observed when control extracts were similarly injected. additional studies are under way to identify the agent and to explain its presence in the rsc cocultivation systems. the mechanism responsible for leukopenia in felty's syndrome (fs) is unknown. both increased peripheral destruction of leukocytes and decreased marrow production may play a role in this process. the present study was designed t o determine whether factors in the sera of patients with fs inhibit the maturation of granulocyte precursors from human bone marrow. sera were obtained from 19 patients with fs; 10 patients with rheumatoid arthritis, splenomegaly, and normal white cell counts; 9 patients with active ra, and 10 patients with osteoarthritis. nucleated cells were isolated from human marrow, and single cell suspensions were cultured in mccoy's medium and fetal calf serum. the serum to be tested, 0.1 ml, was incubated with 0.1 ml of the cell suspension (containing 10 x 10' cells/ml) and 0.1 ml rabbit serum, and the mixture was placed in 3% agar. at the end of 12 days, the number of cells giving rise to granulocyte-macrophage colonies (cfu-c) were counted. serum from a healthy adult was run simultaneously as a control. inhibition of maturation was considered to be present if the number of cfu-c in the agar incubated with the test serum was reduced by at least 25% as compared to healthy control serum. sera from 8 of 19 patients with fs caused a significant reduction in the number of cfu-c. seven of the 8 patients with serum inhibitors had undergone splenectomy; in 3 of these subjects the inhibitor was absent prior to splenectomy, but present after splenectomy. none of the sera from the 29 control subjects gave a positive response. these data indicate that the sera of certain patients with fs contain a factor that inhibits human granulocyte precursors in vitro. this factor may play a role in the induction of leukopenia in fs. the presence of a serum inhibitor of granulocyte maturation may also explain the failure of some patients with fs to respond to splenectomy. this study was designed to determine-whether lymphocytotoxic antibodies (lca) in patients with sle had specificity for the ii antigen system; the ii antigens are known to be present on the surface of all formed elements in blood. sera from 10 patients with sle were tested for lca using the standard microdroplet assay. following determination of the lca in whole sera, each serum was serially diluted twofold in saline and retested for lca. the dilution of the serum which gave a 50% decrease in cytotoxicity was used in the following absorption experiments. each diluted serum was absorbed threefold with either adult (rich in i antigen) or cord (rich in i antigen) red cells a t 4"c, and then retested for lca activity. eluates were prepared from 2 sera incubated with either adult or cord cells a t 4"c, and the eluates assayed for lca. the cytotoxicity of the whole sera was 79% or greater in all but one serum. seven of the diluted sera showed an obvious reduction in cytotoxicity following absorption with cord cells, but minimal or n o reduction after absorption with adult erythrocytes. of the remaining 3 sera, the lca were equally reduced by adult and cord cells in 1, and were unaffected by absorption in 2. in the 2 sera studied, eluates prepared from cord cells showed greater lca activity than eluates from adult cells. statistical analysis confirmed that the mean cytotoxicity values in the sera after absorption with cord cells were significantly different from those obtained after absorption with adult cells (p < 0.005). additional studies showed that there was a strong correlation between the level of lca and the titers of cold agglutinins against cord red cells. these data indicate that the lca in most sle sera react with i antigen. cold-reactive antibodies with i specificity have been associated with hemolytic anemia and thrombocytopenia in other disease, thus, the capability of lca to react with i antigen may explain the observation that lca were found in higher frequency and greater titer in sle patients who have hematologic abnormalities. clinical and experimental observations indicate that estrogens may play a part in rheumatoid arthritis (ra) and in the regulation of immune responses. in order to delineate this relationship, the effect of oophorectomy in an experimental immune synovitis resembling ra was studied. nineteen adult female rabbits were sensitized to homologous igg and then randomly divided into 4 groups. group i ( 6 ) underwent oorphorectomy. group i1 (7) had the same but were treated with 1 mg of estradiol intramuscularly every other week. group i11 (3) had a sham procedure; group iv (3) were unoperated controls. an immune synovitis was then induced in the right knee by the biweekly intraarticular injection of 1 mg of igg and the animal sacrificed after 8 weeks. skin testing to igg was done at 2 week intervals. serum cortisol levels were performed prior to sacrifice by use of a radioimmunoassay technique. gross and histologic assessment of synovial inflammation and cartilage changes was recorded using a "blind" grading previously reported from 0 for no changes to 3+ for severe involvement. synovial cathepsin d activity was analyzed by the anson technique. there were no changes in the initial positive skin tests to igg in any groups. cortisol levels in groups 11,111, and iv were 5.06, 3.53, 3.60 hg% without significant differences, while in group i, the 1.7 was significantly depressed. synovial inflamation in the oophorectomy rabbits was 1+, compared to the unoperated and sham groups of 2+. the reduced synovitis in group i was reflected by a significantly lower cathepsin d level of 1.2 p moles/hour/l mg of protein compared to 3.15 and 3.06 for the other groups. however, when oophorectomy rabbits were given estrogen replacement, there was an increased synovitis rated 3+ with a significantly elevated cathepsin d level of 5.3. cartilage changes were rated 2+ and appeared uniform throughout the groups. the results of this study indicate that in this model of immune synovitis, oophorectomy appeared to inhibit synovial inflammation while estrogen replacement seemed to enhance this response. whether this inhibition is by direct endocrine mechanisms or through the immune system remains to be studied. finally, since ra has a significant female predilection, these findings may be important in the pathophysiology of this disease. we investigated the role of suppressor cells in the depressed cellular immunity of patients with sarcoidosis. we have recently described a glass adherent prostaglandin producing suppressor cell that inhibits t-cell mitogenesis in phytohemagglutinin (pha) stimulated cultures of human peripheral blood mononuclear cells (pbmc) by secreting pge, (j exp med 146:1719 , 1977 . increased activity of this cell is responsible for the hyporesponsiveness to pha seen in pbmc from patients with hodgkin's disease (n engl j med 297:963, the mean response of 14 patients with active sarcoidosis to three concentrations of pha was significantly (p < 0.01) less than controls. passage of the cells over glass wool resulted in a 116% increase in the mean response to pha in the sarcoidosis patients and a 39% decrease in controls. the pha response of the active sarcoidosis patients went from 20% of control before passage over glass wool to 71% of control after. addition of indomethacin, a prostaglandin synthetase inhibitor, to pha cultures increased the response of the pbmc from patients with sarcoidosis 192 f 32% versus a 112 f 18% 1977). increase in controls (mean f sem, p < 0.01). the sarcoidosis patients had an increased percentage of. monocytes in the peripheral blood mononucleat cell preparations (31 f 5% monocytes for patients, 13 f 2% for controls, mean f sem, p < 0.01) and the percent monocytes in a pbmc preparation from patients with active sarcoidosis correlated with the percent increase in pha response after glass wool passage (r = 0.62, p < 0.05). thus, there appear to be at least three mechanisms operating in the depressed pha response of pbmc from patients with active sarcoidosis. first, there appears to be increased activity of the prostaglandin producing suppressor cell. second, the increased proportion of monocytes correlates with increased suppression, either via a diluting effect or perhaps through another active suppressive effect. blockade of these first two mechanisms by indomethacin and glass wool passage does not result in total restoration of the pha response of patients with active sarcoidosis, and therefore, a third, as yet unknown, factor must also contribute to the depressed pha response. the rheumatologist is aware of hand deformities in the adult. very little is written concerning the hand of the child with juvenile rheumatoid arthritis (jra). roentgenographic and clinical analysis was done, review of surgery and outline of treatment is given. five-hundred patients are under treatment. there are three basic types of onset in jra. systemic onset is similar to that described by still; polyarticular closely resembles the disease in the adult; pauciarticular involves four or less joints. jra has a much better prognosis than the adult type but tends to produce more stiffness. examination of 100 successive children showed 10% had radial deviation of the rnetacarpophalangeal (mcp) joint and 23% bad decreased flexion. seven percent had boutonniere deformity and 1% had a swan-neck contracture. there was decreased flexion of the pip in 27% and decreased extension in 10%. the wrist showed 55% decreased extension and only 22% decreased flexion. a statistical review of the records and x-rays of 200 children showed finger involvement in approximately 50%. wrist involvement in approximately 25%, and very few cases of boutonniere or swan-neck. ulnar shortening in 93 joints measured on roentgenogram showed a range of 1-10 mm with an average of 4.1 mm. ulnar wrist deviation was seen in 67 joints with an average of 13 degrees and radial deviation in 12 joints with an average of 11 degrees. metacarpophalangeal ulnar deviation was seen in 31 with a range of 2 to 25 degrees and an average of 8, and a mcp radial deviation also of 3 1 between 2 and 15 degrees with an average of 7.4 degrees. there was n o ara abstracts statistical correlation between ulnar shortening, ulnar deviation, and metacarpophalangeal radial deviation, as reported in previous literature. surgery between 1965 and 1975 was rare and included collateral ligament release in 2 patients, tenolysis on 5 occasions, carpal tunnel release, and reconstruction of thumb mcp. terminal vasospasm required finger amputation. synovectomy of 16 mcp and 10 pipjoints was done and studied between 1965 and 1970. n o recurrences were seen in late followup, but stiffness precluded continuing this procedure. the common problem of wrist flexion contracture is best managed by physical therapy and extension orthosis, and the loss of finger flexion by active exercises, occasional injection, and active splinting. we have previously demonstrated that the translational movement of glucose in pathologic synovial fluids is too rapid to be accounted for by bulk diffusion (fed proc 36:1069 , 1977 . such enhanced diffusivity is a property of a n isolated 2.5% matrix of ha as well, wherein both diffusivity and matrix structure are critically dependent on ca++ concentration (see semin arthritis rheum 7:141-152, 1977 for review). the current study examines translation movement of glucose in a i% matrix. human umbilical h a and agarose were prepared as 1% matrices in phosphate buffered saline at ph 7.0. the bulk diffusion coefficients (d) for glucose and sucrose within each matrix were determined in codiffusion experiments employing a capillary method. the d for both solutes in h a was indistinguishable from that in agarose. if the matrix was prepared in 50% horse serum, glucose diffusivity was enhanced twofold in h a while slightly diminished in agarose. a 50% dialysate of serum as a solvent enhanced glucose diffusivity in ha threefold; the dialyzed protein had little effect. the en-hanced glucose diffusivity in the presence of a 50% dialysate was inversely dependent on solute concentration. the diffusivity of sucrose in h a was little altered by these solvent changes. in order t o gain insights into matrix structure that might underlie the enhanced glucose diffusivity, darcy numbers were determined. permeability is dependent on h a concentration, has a ph optima of 7.0 in a 1% matrix, and is critically dependent on ca+ + concentration rising rapidly below 10 mm. matrix structure is highly dependent on the solvent environment particularly in the physiological range. constituents in the serum dialysate may induce a matrix configuration that supports the enhanced glucose diffusivity. the matrix configuration that exists in the presence of the dialysate is likely to be present in synovial fluid as well. this configuration can facilitate the translational movement of a nutrient such as glucose. facilitated movement may be homeostatic in inflammatory states such as rheumatoid arthritis where low synovial fluid glucose concentrations are well described. sera from 19 patients with mctd were examined for the presence of immune complexes (ic). the diagnosis was based on serologic and clinical criteria. all patients had antibodies to extractable nuclear antigen by passive hemagglutination. titers of 1 : 1,000,000 or greater were found in 17 patients and 1:655,ooo and 1 :40,960 in the remaining two. treatment of the antigen with rnase resulted in a 1,000-fold or greater decrease in titer in 18 patients and a 64-fold decrease in one. rheumatoid factors were present in 8 patients and antinuclear antibodies in 12. all patients had a mixed rheumatic syndrome. clinical features included: raynaud's phenomenon-16, swollen hands and/or sclerodactyly-12, myopathy-1 i , pleuritis--12, malar or diffuse rash-7, and arthritis-15. one patient had a stable iga membranous glomerulopathy. ic were measured in 46 sera by a raji cell radioassay (rc-ra), monoclonal rheumatoid factor radioimmunoassay (mrf-ria), and clq binding assay (clq-ba). ic were detected by at least one method in 45 (98%) sera. the single negative serum was from a patient with clinically inactive disease. ic were found by the rc-ra in 42 (91%) sera, by mrf-ria in 22 (48%), and by clq-ba in 20 (43%). mean values for each assay compared to normal controls were: 76.8 versus 1.7 pg agg.-igg equiv/ml for the rc-ra (p < 0.001); 12.0 versus 4.4 pg agg.-igg equiv/ml for the mrf-ria (p < 0.001); 19.2% versus 10.9% la51-clq bound for the clq-ba (p < 0.001). ic were detected by all three assays in 9 (20%) sera and by two methods in 2 1 (46%). thus, ic were found by two or more methods in 30 (65) sera. only three sera failed to react in more than one assay. a different pattern of reactivity was found in 89 rheumatoid arthritis sera where ic were detected at a higher frequency by mrf-ria (71%) and clq-ba (82%) while the frequency by rc-ra (28%) was lower. sufficient clinical data were available for 16 of the mctd patients to permit preliminary evaluation of the relationship of disease activity and ic. changes in levels of ic appeared to parallel clinical activity in 12 patients by rc-ra, in 9 by mrf-ria, and in 3 by clq-ba. these data indicating a high incidence of ic in mctd and a different pattern of reactivity from ra emphasize the wide spectrum of immune complex disease. hydroxyapatite (ha) has been found by others in synovial fluid and fluid leukocytes by electron microscopy and electron probe. we have developed a method to quantify ha in synovial fluid which avoids possible em artifacts. ("c) ehdp, (sa 10.6 pci/omole) was used in a final concentraion of 16 nm/ml in normal serum or phosphate buffered saline (pbs) containing standard ha or to joint fluid samples; serum or joint fluids were processed under oil. after rotation x 3h 4"c, radioactivity/o.l ml was determined before and after centrifugation (49000 x g), and expressed as percent loss of nuclide from the supernatant. standard ha binding in serum monsodium urate or calcium pyrophosphate dihydrate (cppd) crystals failed to bind in concentrations up to 0.5 mg/ml and 1 .o mg/ml respectively. the centrifuged pellets were washed in distilled water and dried to a spot which showed ha by x-ray diffraction. this method was sensitive to 25-50 pg ha standard. we confirm the finding of ha crystals in joint fluids, handled physiologically, in amounts equal to 1-15 pg ha standard/ml. such small amounts and the low wbc in these fluids fail to support a phlogistic role for ha crystals. patients with systemic lupus erythematosus (sle) have circulating immune complexes (ic) which are thought to be involved in disease pathogenesis. using a newly described method for in vivo evaluation of res igg fc membrane receptor function, we previously demonstrated a defect in untreated patients with active sle and related this res defect to the presence of circulating immune complexes. a serial study of the correlation of defective res fc receptor function, immune complexes, and disease activity was undertaken to further investigate the interaction of these factors. isologous yr-labeled erythrocytes were sensitized with human anti-rh(d) and their intravenous clearance determined. clearance rates (t-1/2) c' levels and ic levels by 1z51-clq precipitation were determined prior to therapy in 17 sle patients, 12 of whom had active disease. fifteen of 17 had markedly prolonged clearance rates (range = 80 to > 1 ,ooo minutes; average clearance t-1/2 in 14 normal volunteers = 40 minutes, range 35 to 50 minutes). the correlations of clinical activity with 1) clq binding and 2) prolonged clearance were statistically sig-nificant (p < 0.005 and p < 0.05, respectively; spearman rank coefficient). in addition, elevated clq binding and depressed c4 levels were significantly correlated (p < 0.05). at the end of from 1 to 19 months, 10 patients who initially had prolonged clearance were restudied. patients had been treated with either corticosteroids (7 of 10) or nonsteroidal, antiinflammatory drugs (3 of 10). clinically, 7 of 10 were improved; the remaining 3 continued to do well. although a clearance defect persisted in all 10 (t-1/2 from 88 to 600 minutes), in 7 of 10 t-1/2 was markedly improved, suggesting improved fc receptor function. in 7 patients, there was an apparent correlation between increased clearance and clinical improvement. there was also a significant correlation between clinical improvement and a decrease in clq binding (p < 0.05). these studies underscore the interrelationships between the presence of circulating immune complexes, defects in membrane fc receptor function which might lead to continued circulation of these complexes, and disease activity in sle. we have found immune complexes in patients with lyme arthritis, an apparently tick-transmitted illness that typically begins with the skin lesion, erythema chronicum migrans (ecm). many patients subsequently develop clinical attacks of synovitis histologically like that of rheumatoid arthritis; some also develop other systemic complications (aseptic meningitis, peripheral neuropathy, or carditis). we measured circulating immune complexes (cic) serially in 58 patients with ecm, by the l*si-clq binding assay. the table shows the first test result on each patient's serum in each of the designated categories. circulating immune complexes-an antigenic component of which may be related to the causative agent-were almost always present very early in the illness and in patients with systemic complications. in contrast, they were found less often during attacks of arthritis and still less often during remissions. quantitative levels tended to diminish with time; in individual patients they fluctuated in a dampened hemi-sinewave pattern in parallel with recurrent bouts of active disease. synovial fluid from all of 7 patients contained immune complexes, compared to only 3 of 7 concomitant sera. in the 3 positive sera the concentrations were much less than in the corresponding joint fluids. we suggest that the causative agent is introduced into the skin, where ecm is the initial clinical manifestation of a systemic, immune-mediated inflammatory disorder that often becomes localized to and propagated in synovium. thus, lyme arthritis can be considered a human model for an infectious etiology of rheumatoid arthritis. little attention has been given to analgesia, divorced from suppression of inflammation, in rheumatoid arthritis (ra), despite its importance to patients. this study was designed to determine the comparative efficacy of five often used analgetics for the pain of active ra synovitis. single doses of 650 mg aspirin (asa), 650 mg acetaminophen (ac), 65 mg codeine (co), 50 mg pentazocine (pe), 65 mg propoxyphene (pro), and placebo (pi) were given in a randomized double blind fashion, and on a prn basis, to each of 30 carefully selected hospitalized r a patients with painful synovitis of at least 4 joints. all other drugs and distracting activities were prohibited during the study period and at least 6 hours of observation required between doses of test agents. subjects recorded time of onset and duration of maximal relief, percent of relief achieved from each agent and all possible side effects, and, after taking all 6 agents, ranked their order of preference, with 1 as most effective. onset and duration of action of all agents were similar except ac and pi, which required longer to act and lasted a shorter time. side effects occurred significantly more frequently with pe, somewhat more often with co, but were otherwise insignificant. effectiveness was analyzed according to some of all ranks, mean percent relief and percent of subjects achieving 50% or greater relief for each agent. as expected, by parameters measured in this study and by cost to the patient, asa is a superior analgetic in ra. however, ac, by cost and effectiveness, is an acceptable alternative, as codeine would be except for its narcotic properties and cost. cost, side effects, and limited effectiveness restrict the usefulness of pe. pro seems no more effective than pi and of questionable usefulness in ra, except perhaps as a placebo. sum there is evidence that the inflammatory properties of crystalline material may be modified by their interaction with serum proteins. in this study, the activation of c3 by crystals is quantitated by two dimensional crossed immunoelectrophoresis (tdiep). in polypropylene tubes 0.5 ml aliquots of fresh human serum were mixed with 0.1 ml of buffer containing 2.5 mg of washed crystals. the tubes were incubated with agitation of 37oc for 1 hour and then centrifuged at 15,ooog for 4 minutes. aliquots of the supernatants were diluted appropriately in edta-containing buffer to inhibit further conversion of c3. by use of standard techniques, tdiep was performed using rabbit antisera to human c3. a method was devised whereby 6 samples could be compared simultaneously on a single plate. after staining, the combined area under the pic and bia peaks was measured using planimetry. the numerical results below are percentage points of total area under the p1a peak in excess of that seen in saline controls. the coefficient of variation of the ratio of the two peaks was 0.008 for a single specimen on the same plate. the areas could be corrected to indicate milligrams of c3 converted. sodium urate was highly active in the electrophoretic conversion of c3 with a dose and time related response. activation was prominent a t concentrations of crystals seen in gouty effusions. heating the crystals at 200' c for 2 hours reduced c3 conversion by nine-tenths, but also altered morphologic properties of the crystal preparations. conversion was inhibited by increasing concentrations of edta and eliminated by 0.005 m edta. the edta inhibition was not seen in the presence of excess calcium. with the conditions described above, potassium urate crystals were much less active (8%) than sodium urate (31%). eight different commercially available preparations of steroids gave varying but small amounts of c3 activation (-1 t o 5%). it is demonstrated that different crystals and altered forms of the same crystals vary in their interaction with serum and their ability to activate complement. it is not known what properties are responsible for these phenomena. the calcium requirement suggests that the interaction of sodium urate with complement occurs at or before c1. it is possible that immunoglobulin, which has a high affinity for sodium urate and is found in sodium urate crystals in vivo, is altered and initiates complement activation by the classic pathway. temporal artery pain, polymyalgia rheumatica, and blindness are well recognized manifestations of giant cell arteritis. however, the disease may often present in a less evident fashion. this is demonstrated by 22 (39%) of our series of patients. of 56 patients with biopsy-proved giant cell arteritis, the chief complaint was polymyalgia rheurnatica in 17; temporal pain in 15, and loss of vision in 2. in the remaining 22 patients, the predominant complaint that brought them to the physician did not suggest the diagnosis of giant cell arteritis, since headache and polymyalgia were either absent or so minimal as to escape notice. these complaints included fever of unknown origin in 6 patients; malaise, anorexia, weight loss, and abnormal liver function tests suggesting occult malignancy in 6; and unexplained anemia in 2. four patients presented with a neurologic syndrome; 2 had diplopia and 2 acute weakness of one arm. claudication was the chief complaint of 4 patients, involving the leg in one, the arm in one, and the jaw in 2. all patients responded well t o steroid therapy. the diagnosis of giant cell arteritis in patients such as this who do not present in typical fashion depends on detection of a very rapid erythrocyte sedimentation rate, biopsy of a temporal artery, and the awareness that the disease may manifest itself in a variety of ways. the administration of certain drugs induces clinical and laboratory features of systemic lupus erythematosus (sle) in man but few studies of this human disease model have been undertaken. hydralazine, a useful antihypertensive agent, is one of these drugs and has acquired a "bad press" because of such observations. a prospective study of 25 hypertensive patients with age, sex, and race matched controls was begun 3 years ago. all subjects had complete clinical examinations; anf, d n a binding; immunoglobulin and complement levels; skin tests to a battery of antigens and lymphoprolifera-tive responses t o mitogens and antigens obtained prior t o starting hydralazine and serially throughout the study. all subjects were a n f negative at the start of treatment. fifteen of the 25 have shown varying a n f response (in undiluted serum) but only 3 had a titer of 1:lo or above, one becoming positive at 12 months and 2 at 24 months. one of these subjects had clinical symptoms suggestive of sle and the drug was stopped. five subjects have shown a proliferative lymphocyte response to hydralazine and/or its metabolites (jci 56:958, 1975) including the 3 with positive anf. acetylation rates were obtained in 22 of the hypertensive patients. thirteen were slow and 9 fast acetylators. the 3 a n f positive subjects were all slow acetylators as were 4 of the 5 with proliferative lymphocyte responses to hydralazine and its metabolites. this study t o date suggests that hydralazine in its present form may be less likely t o cause serologic and clinical sle than previously reported and that screening by acetylation rates may define a susceptible population. this and other lupus related drugs continue t o be a rich source of data relevant to lupus in humans. (supported by an niamdd grant). normal neutrophils (pmns) develop intracytoplasmic inclusions after incubation with sle sera. the resulting inclusions stain for igg, igm, and c3 and are believed to be immune complexes removed from the sera in vitro. such inclusions have also been identified in the circulating pmns from sle patients. the present study examined the relationship between pmn inclusions and clinical and laboratory features of sle. sera were carefully collected at 37" from 35 sle patients. normal buffy coat cells were incubated in the sle sera for 90 minutes a t 37' and then centrifuged and washed. slides of the washed cells were prepared in the cytocentrifuge, stained with fitc goat anti-human igg, igm, iga, and c3, and examined under ultraviolet light. the composition of these complexes was also examined with a fluorescent probe for double-stranded polynucleotides with the aid of ethidium bromide (eb). eb was added t o sera known to contain high concentrations of anti-dna antibodies and then incubated with normal pmns as before. preparations included 1) no added eb, 2) eb only, 3) fitc anti-human igg (fianti-igg) or 4) f1-anti-igg and eb. inclusions containing both igg and igm correlated with clinical activity (p < 0.001), depressed serum complement (p = 0.026), cryoglobulinemia (p = 0.014), and anti-ndna antibodies (p < 0.001). igg inclusions alone were not significantly correlated with any of the parameters. c3 and igm appeared to be mutually exclusive, i.e. when one was present, the other was never present. eb staining inclusions (red fluorescence) suggested that polynucleotides were present. these findings suggest: 1) the pmn inclusions consist of immune complexes which contain double-stranded polynucleotides, 2) these complexes correlate with disease activity when igg and igm are both present, and 3) such complexes may contribute to a number of granulocyte disturbances seen in patients with sle. in previous studies on preservation of articular carti-(324,000 dpm/mg protein) permitted quantitation of the neulage performed in our laboratories, it was found that addition tral protease activity released into the media of the stored of a-tocopherol (200 pg/ml) to the culture media of stored cartilage explants. employing this assay we found that acartilage resulted in improved preservation of its biochemical tocopherol resulted in significant inhibition of this activity as and biomechanical properties when compared to cartilage shown in the table. stored in absence of the vitamin. although it has been shown that a-tocopherol stabilizes lysosomal membranes in other lage were due to stabilization of lysosomal membranes, we no. control + a-tocopherol control + a-tocopherol developed a highly sensitive assay for neutral protease activity employing a biosynthetically prepared protein substrate. this five further patients with sle have been treated with plasmapheresis. six to 8 liters of plasma per week were removed, using a haemonetics model 30 hood cell separator. from 12 to 32 liters were removed in all. levels of complement components were measured by immunodiffusion and by a hemolytic assay. dna antibodies were measured by passive hemagglutination of dna coated red-cells. immune complexes were measured in vitro by the raji-cell assay. two patients were critically ill. one had seizures, pericarditis, pleurisy, and severe edema, despite treatment with 100 mg prednisone daily for one month. the raji assay was 750 pg/ml (normal < 40 pg/ml). following the removal of 14 liters of plasma her condition improved dramatically, complement levels rose to normal, and raji assay fell to 150-200 pg/ml. the second acutely ill patient had lupus cerebritis, with a level of only 60 pg/ml on raji assay. after the removal of 18.6 liters of plasma there was no significant improvement in her clinical condition. the remaining three cases were less severely ill and had raji assays of 350, 257, and 94 pg/ml. they showed a reduction of circulating immune complexes after each plasmapheresis, and an overall, variable fall after 2 to 3 weeks of treatment, but no striking clinical benefit. those cases with a high raji-assay also showed increased binding of doublestranded dna. the raji assay appears to be of value in predicting the response to plasmapheresis: patients with a very high level may show a massive and sustained reduction, with a good clinical response. in those with lower levels, the response is less predictable. plasmapheresis appears to be a valuable accessory form of therapy in the severely ill patient with acute sle. to investigate the vascular lesion in scleroderma (sd), the effect on human endothelial cell (ec, umbilical cord) growth of sera from 21 patients with early sd was compared with 10 healthy control sera. ec growth was monitored by tritiated thymidine chtdr) uptake and by direct cell counts. shtdr uptake of ec was inhibited up to 97% by 11 of 21 sd sera compared to the remaining 10 sd and 10 control sera. mean 8htdr uptake was 2747 cpm for control sera (range: 1130-575l), 2448 cpm for the 10 noninhibitory sd sera (989-5162). and 182 cpm for the i i inhibitory sd sera (58-424). the sd patients with inhibitory serum activity tended toward shorter disease and symptom duration and were more likely to have peripheral edema and hypergammaglobulinemia than sd patients without inhibitory activity. furthermore, the inhibitory effect was no longer present in one patient after therapy with glucocorticoids which was associated with reduction of peripheral edema and improvement of myositis. the activity was further characterized in selected sera. sd serum completely inhibited the increase in ec 8htdr uptake seen with increasing normal serum concentration from 2 to 25%. 8htdr uptake correlated with reduction in ec number (30% reduction at 20% sd serum concentration versus 100% increase with control serum) and with cytotoxicity (52% cell death versus 6% in control serum). these effects were not observed when the target cells were mouse 3t3 or human fibroblasts, smooth muscle cells, or peripheral monocytes, suggesting specificity of the inhibitory effect of sd sera for ec. the inhibitory effect of sd serum was heat stable (56"c, 30 minutes), nondialyzable, present in both plasma and serum, and unaffected by mixing with control serum. when serum was fractionated on sephadex g-200 column, the inhibitory activity was found to elute in the position of serum albumin. sera from 7 patients with non-sd active rheumatic diseases (ra, sle, pan, dm) showed no inhibitory effect on human ec, fibroblasts or smooth muscle cells or mouse 3t3 cells. these preliminary observations describe an ec-specific cytotoxic serum factor(s) in patients with sd. the in vivo role of this factor(s) in the pathogenesis of the proliferative vascular lesions in scleroderma is unknown. activation? activation of the complement (c) system in ra has been amply evidenced by a) detection of c and immune complexes in ra synovial leukocyte inclusions; b) depressed ch50, c4, and c2 levels as well as split products of c3, c4, and fb in ra synovial fluids; c) split products of c3 and c4 in ra plasma samples; and d ) hypercatabolism of c3 in meta-bolic turnover studies. we measured the fractional catabolic rates (fcr) of radioactive labeled c4 and fb in ra patients and normal controls to assess the relative importance of classical and alternative pathways of complement activation in ra. our results demonstrated: 1) hypercatabolism of c4 in 10 of 15 ra subjects and inverse variation of c4 fcr with plasma c4 levels; 2) hypercatabolism of f c in 5 of 10 ra patients; 3) c4 fcrs disproportionately higher than fb fcrs in 3 of 5 ra subjects undergoing simultaneous studies; 4) correlation of c4 and fb catabolism and homologous igg catabolism; and 5) occurrence of hypercatabolism of c4 mostly in the extravascular space, a phenomenon previously observed by us also with the igg hypercatabolism of ra. patients with high fb fcrs had higher titers for rheumatoid factors. patients with high c4 fcrs had higher total joint counts and a greater number of manifestations of extraarticular disease. c4 and fb fcrs did not correlate with circulating immune complexes by the raji cell assay or with the sedimentation rate. the results suggest that complement activation in ra occurs primarily through the classical pathway and in the extravascular compartment, probably principally by immune complexes not detected by the raji cell assay, and with participation of the alternative pathway in some of the patients. studies from this laboratory have demonstrated that the pha and con-a responses of splenic lymphocytes from rats with adjuvant-induced disease are diminished. these diminished responses return to normal with spontaneous, corticosteroid (cs), or methotrexate (mtxtinduced remissions. the diminished pha and con-a responses are due to two types of suppressor cell: one which adheres to plastic ar glass, and another which adheres only to glass. the latter cell, which accounts for the bulk of the suppression, is more sensitive to the inhibitory effects of cs and mtx in vitro than are the mitogen-responsive lymphocytes. virazole (vzl), a synthetic nucleoside, is a noninterferon inducing antiviral agent which inhibits aid. four groups of highly inbred fisher rats were studied as follows: 1) untreated rats; 2) rats treated with 200 mg of vzl given 7 days prior to sacrifice; 3) rats treated with freund's adjuvant given 14 days prior to sacrifice; 4) rats treated with freund's adjuvant followed by vzl 7 days later. whole spleen cell suspensions were studied, as well as suspensions after removal of plastic-adherent cells by incubation in petri dishes or after removal of glass-adherent cells on columns of glass wool. each of these three types of suspension was also incubated with a range of concentrations of vzl and with either pha or con a. ah-rdr incorporation into nucleic acid was determined at 72 hours. spleen cells from untreated rats, rats given 200 mg of vzl 7 days prior to sacrifice, and adjuvant-treated rats given vzl had normal pha and con a responses. the spleen cells from rats given adjuvant alone had markedly diminished pha and con-a responses due to the two types of supprcssbr cells, both of which were more sensitive to vzl in vitro than were the mitogen responsive cells. this preferential sensitivity of suppressor cells to vzl was greater than that noted with cs or mtx. two possible interpretations are: 1) vzl has a more specific immunosuppressive effect on suppressor cells than cs or mtx; 2) suppressor cell function is due to a virus which is inhibited by vzl. twenty-four cerebrospinal fluid (csf) samples from 16 patients with systemic lupus erythematosus (sle) were evaluated for cyclic-gmp (c-gmp) concentration by radioimmunoassay. for comparison studies, the patients were divided into three groups based on their clinical status at the time of each lumbar puncture: those with 1) active neurologic and psychologic abnormalities (group i), 2) active neurologic abnormalities only (group ii), and 3) psychologic abnormalities only (group 111). groups i and i1 had mean csf c-gmp values of 3.2 nm f 0.64 (se) and 4.1 nm f 0.10 respectively, which were both significantly higher than the mean for group i11 (1.2 nm f 0.43) (p < 0.05) as well as for the mean of a previously studied group of patients with lumbosacral pain syndromes (0.68 nm f 0.14) (p < 0.001). other csf findings did not display this close correlation with activity of neurologic disease. the number of samples with abnormal csf leukocyte counts was significantly greater for group i1 (6/8) compared with that found for group 111 (0/11) (p < o.ol).however, no significant difference was found between group i and group 111, for this abnormality. in 4 sle patients significantly higher levels of csf c-gmp were found on serial sampling during times when neurologic abnormaltiies were active. neither prednisone nor psychoactive drug therapy had any demonstrable effect on csf c-gmp levels in these pa-tients. comparison of clinical markers of sle disease activity among the three groups revealed hematologic and serologic abnormalities not to be of significant value in distinguishing sle patients with active neurolgic involvement (groups i and 11) from those without (group 111). however, fever and, to a lesser degree, rash, proteinuria, and abnormal urinary sediment were present more frequently in groups i and i1 than in group 111. thus elevated csf c-gmp concentration may be a marker of active neurologic disease in sle. assay of csf c-g m p may therefore be helpful in the clinical assessment of sle patients with neurologic and/or psychologic abnormalities both with regard to their diagnosis and therapy. this study reports the first serial observations of c-gmp in a group of sle patients with correlation of manifestations of disease activity and extends our previous studies of cyclic nucleotides in sle. mp produces a chronic arthritis in mice with histopathologic features resembling rheumatoid arthritis. since the chronicity of arthritis appears to result in part from persistence of mp within the joints, elucidation of host mechanisms responsible for elimination of mp is of considerable importance. the role of complement in the pathogenesis of mp-induced arthritis was studied in mice congenitally deficient in the fifth component of complement (c5). in all experiments, at least 5.5-week-old male mice per strain free of detectable mycoplasmas were inoculated intravenously with 2 x 1p color change units of mp. subjective assessment of the arthritis was carried out by scoring ankles, wrists, qetacarpal, metatarsal, and digital joints on a scale of 0 to 3. in a preliminary study c5 deficient dba/2 mice and 4 strains of normal mice: t.o., c3h, cba, and c57bl/10 were used. in the normal strains the .arthritis peaked in severity within 15 days and declined thereafter. in contrast the arthritis in the dba/2 mice reached a plateau at 15 days and gradually progressed in severity for as long as 5 months postinoculation. a second study was carried out with 3 strains of c5 deficient mice, i.e. dba/2, akr and a strain and 2 normal strains: c3h and c57bl/10. the arthritis in the c5 deficient strains persisted at a high level 3 months after inoculation despite considerable variability in the severity of the acute arthritis among the strains. the arthritis in the normal strains rapidly declined as before. at 3 months postinoculation, mp was isolated from the joints, lungs, and spleens of c5 deficient mice more frequently and in larger numbers than from normal mice, consistent with the failure of cs deficient mice to eliminate mp. complement-fixing antibody to mp was detected in higher titers in c5 deficient strains. the results demonstrate the importance of c5 in the elimination of mp from the joints and organs of infected mice. moreover, the study may have clinical relevance since secondary complement deficiencies exist in a number of rheumatic diseases. it supports the concept that complement deficiency might contribute to the persistence of as yet undefined etiologic agents in human arthritidies. we have studied 34 american black patients with primary ankylosing spondylitis (as) unassociated with ulcerative colitis, crohn's disease, psoriasis, or reiter's syndrome. all the patients were unrelated and met the rome criteria for as. sixteen (47%) of them possessed hla-b27. we further studied the remaining 18 hla-b27-negative patients to see if there was an association with other b locus antigens belonging to the hla-b7 cross-reactive group (hla-b7, b27, bw22 and bw42). hla-b7 was present in 10 of these 18 patients (55.6%) compared to 14 of 59 (23.7%) b27-negative black controls (x' with yates correction = 5.11, p < 0.025, relative risk = 4). bw22 was found in one patient who also had b7. bw42 was not tested for. antisera used to detect b27, b7, and bw22 were free of noteworthy cross-reactivity. duration of disease, age at onset of as, skeletal manifestations, prevalence of acute anterior uveitis and frequency of a positive family history for as were compared between the 10 b7-positive (gp-i) and the 16 b27-positive (gp-11) patients. duration of disease at the time of study was not significantly different in the two groups. however, mean age at onset of as differed significantly: 33.6 years in gp-i and 22.2 years in gp-i1 (p < 0.005). skeletal manifestations of the disease did not differ significantly in these groups. uveitis occurred in 2 patients (20%) in gp-i and 8 patients (50%) in gp-i1 (p = 0.158). a family history of as could be obtained in none of gp-i patients and 6 (37.5%) of the gp-i1 patients (p = 0.034, using fisher's exact test). these data indicate that hla-b7 may be associated with the development of as in the b27-negative black popu-lation. the b7-positive black as patients are older at onset of their disease than the b27-positive patients and tend to lack obvious familial aggregation of the disease. the findings suggest that american black as patients lacking b27 but possessing b7 represent a subgroup of patients with this disease. prostaglandin synthesis inhibitors (pgsi) may affect renal function in humans, especially under conditions of renal injury such as lupus nephritis. to explore this preferential effect in systemic lupus erythematosus (sle), we measured basal excretion of urinary prostaglandin e (ipge) by radioimmunoassay in 10 female sle patients and 28 normal women on a constant 109 meq sodium diet. no patient or normal control received nonsteroidal antiinflammatory drugs during the study period but 7 of 10 patients were on maintenance low dose prednisone ( i 20 mg/day). six of 10 patients had biopsy-proved nephritis; 4 had insufficient urinary findings to prompt a diagnostic biopsy. all ipge values were determined in triplicate on at least three separate 24-hour collections except in one patient with two such collections. sle patients had a significantly higher mean ipge excretion than normals (45.8 f 5.3 versus 29.6 f 2.2 ng/hour; mean f sem; p < 0.005). sle patients with biopsy-proved nephritis had a higher mean excretion than those without biopsy (49.7 f 8.6 versus 39.9 f 3.2 ng/hour) although the difference was not statistically significant. urinary sediment and general disease activity did not correlate with urinary ipge. all 10 patients showed a decrease in creatinine clearance with pgsi administration but the percent change did not correlate with basal urinary ipge excretion. elevated urinary ipge excretion in sle may reflect renal inflammation due to immune complex nephritis and may explain the greater sensitivity of renal autoregulation to pgsi in this setting. the therapy of lupus glomerulonephritis has been evaluated in a randomized study of corticosteroid treated patients comparing bolus ivcy (5.5-26.7 mg/kg, every 3 months), combined oral cy + az (50 mg of each, daily), and prednisone alone (pr). five years after the start of the trial, 38 patients have been randomized: ivcy (12), cy + az (14), and pr (12). renal involvement at study entry has been characterized by urine sediment abnormalities (38), impaired function (crci < 100 ml/min) (33), nephrotic proteinuria (lo), glomerular proliferation on biopsy (31), and an estimated disease duration of less than one year (25). the assigned drugs have been continued without complications in 31 patients with a median course of 16 months. toxicity has necessitated discontinuation of the drugs in 4 courses; 3 cy + az (pulmonary infection, hemorrhagic cystitis, amenorrhea) and 1 ivcy (hepatitis). death has occurred in 3 patients; 2 ivcy (epiglottitis, sudden death), and 1 pr (pulmonary embolism). the changes of renal function of the 25 patients receiving a minimum of 6 months of continuous therapy have been assessed by evaluating the number of patients demonstrating a reduction of function (crci) by at least 10% (alo) or 25% (a25) from baseline determinations at study entry. this interim analysis of an ongoing trial is encouraging and suggests that ivcy and cy + az may be more effective than pr in the maintenance of renal function of patients with lupus glomerulonephritis. extended follow-up will be required to determine the duration of these effects and the development of drug related complications. the mechanism(s) underlying the destruction of bone observed in chronic inflammatory diseases such as rheumatoid arthritis or osteomyelitis remain(s) to be elucidated. soluble mediators derived from immunologically activated cells may play an important role in such phenomena. we have observed that alloantigen-stimulated human mononuclear leukocytes (mnl) release a factor(s) capable of resorbing fetal rat bones in vitro. we have investigated procedures for the large-scale generation and partial characterization of this bone resorbing factor(s). human mnl obtained by leukophoresis (ibm cell separator) of two nonrelated healthy individuals (yield = 1-2 x lolo cells/donor) were cultured together. (2.5 x 1v cells/ml, 40 hours, 37"c, mishell-dutton atmosphere). supernatant fluids were removed and assayed for bone resorbing activity by measurement of ca46 release from fetal rat bones in vitro. these supernatants effected significant bone resorption when compared to either control supernatants prepared from a single donor's cells or medium alone. indomethacin ( 5 x to 1 x 1wm) did not inhibit either the production or biological activity of this factor(s). bone resorbing activity eluted with molecules of 11-18,ooo daltons on sephadex g-75 and appeared in the breakthrough when applied to deae cellulose (0.005 m phosphate, 0.02m naci, ph 7.5). eluate containing bone resorbing activity after sephadex g-75 chromatography x 2 and deae cellulose chromatography was applied to amionic disc gels (4"c, ph 9.3, trisglycine). a single peak of bone resorbing activity was observed, although this activity could not be attributed to a stainable protein bond. the results indicate that alloantigen stimulated human mnl elaborate a low molecular weight bone resorbing factor(s) resembling the previously described osteoclast activating factor (oaf). methods presented for the large-scale generation and partial purification of this activity should facilitate clarification of the role of this factor in immune tissue destruction. inflammatory cells may play a role in modulating connective tissue growth and function in a number of rheumatic diseases. the interaction of lymphoid and connective tissue cells was studied in vitro by exposing human dermal fibroblasts (fb) to supernatants (sn) of peripheral blood mononuclear cells (pbmc). proliferation was assessed by bh-thymidine incorporation and direct cdl counts. protein synthesis was determined by ah-proline incorporation and collagen synthesis by protease-free collagenase releasable counts. sn of both unstimulated and pha-stimulated pbmc suppressed fb growth in a dose-dependent fashion. sn suppressive activity was non-dialyzable, heat-stable, not cytotoxic, and removed by absorption of sn with fb but not with pbmc. there was suppression of protein synthesis by pbmc-sn in direct proportion to the suppression of cell number (41 %), but disproportionately greater suppression of collagen synthesis (68%). culture sn of t-cell depleted pbmc were as active in suppressing fb growth as were sn of unfractionated mc (46-63% suppression, mean 54 f 7% for t-depleted versus 41-59%, mean 48 f 8% for unfractionated). supernatants of t-cell enriched populations had decreased activity (8-20%, mean 16 f 6% suppression). the growth suppression seen was due, at least in part, to stimulation of fb prostaglandin (pg) synthesis. immunoreactive pge, production by fb was increased 70-fold (1.3 versus 90.0 ng/106 cells) by pbmc-sn. when inhibitors of pg synthesis (indomethacin, sodium meclofenamate) were added to fb cultures just prior to addition of pbmc-sn, the growth suppressive effect was abrogated (30-100%). furthermore, exogenously added pge, (final concentration = 50 ng/ml) resulted in suppression of fb growth comparable to that seen with pbmc-sn. thus, lymphoid cells produce soluble factors which induce fb to modulate their own growth in vitro and which alter collagen synthesis. the effector of the growth autoregulation appears to be a pg. this system provides an in vitro model for the study of lymphoid and connective tissue cell interaction which may improve the understanding of proliferative and fibrotic human disease. although there is compelling evidence to support the view that altered endocytic-lysosomal functions of synovial tissue may play a role in the joint tissue injury seen in rheuma-toid arthritis (ra), there are no data which quantitate these functions in human synovial cells (sc). therefore, we investigated the rates of pinocytosis and intracellular digestion of a soluble protein, horseradish peroxidase (hrp), in monolayers (4-15 days in primary culture) of sc from ra and non-ra patients. pinocytosis was linear with increasing h r p concentrations (0.01-10 mg/ml), time (30-120 min), cell numbers (1-10 x 106) and cell protein (50-lo00 pg; 5 x i@ cells equivalent to loopg protein). specific activity (sa; ng h r p taken up/100 pg cell protein/2 hr) increased in direct proportion to cell concentration. sa of r a cultures (100-200 pg protein) after exposure to 1 mg/ml h r p was 270 f 182 (mean f sd; n = 5); an equivalent concentration of non-ra sc had a sa of 142 f 90 (n = 5). uptake in both r a and non-ra sc was inhibited most effectively by 10-3m potassium fluoride (80%) and 4â°c (98%). r a sc digested 50% of cell bound h r p (t%) in 6 hours, whereas t% for non-ra sc was 13 hours. uptake and digestion of immune complexes by ra sc were also studied. insoluble hrp-anti h r p complexes formed at equivalence were added to sc at 1.25 pg hrp/ml (total protein concentration 12.5 pg/ml). uptake of complexed h r p was at a rate (4.8% of this load/100 pg cell protein/hr.) approximately 600 times that for soluble h r p (at a load of 1 mg/ ml). only 16% of complexed h r p was digested by 24 hours. these studies indicate: 1) human sc have a relatively high rate of pinocytosis and r a sc demonstrate the greatest rates of uptake; 2) kinetics of pinocytosis in sc are primarily those of fluid phase uptake; 3) the rate of pinocytosis increases with increased cell density, a relation which may be important in ra synovial tissue; 4) r a sc can readily accumulate immune complexes, but their ability to digest them is much more limited than it is for soluble proteins. further quantitative studies of the mechanisms whereby sc process joint constituents, soluble antigens and immune complexes should lead to a more precise definition of the contribution of sc endocyticlysosomal functions to tissue injury in ra. this study was designed t o evaluate the effects of several nonsteroidal antiinflammatory drugs commonly used in arthritis therapy on gastric mucosa, and to evaluate the effects of 2 agents, tolectin and motrin, in subjects with demonstrated clinical intolerance to aspirin. gastroscopy was carried out before and after all treatment schedules, and gastric mucosa was graded by the endoscopist (fl) on a scale of 0-4+. photographs were taken and subsequently reviewed in a randomized double-blind manner by both the endoscopist (fl) and an impartial gastroenterologist (rn). remarkably consistent results were obtained: part i. forty volunteers were randomly divided into 8 groups with 5 subjects in each group and treated for 1 week with either motrin (2400 mg/day), motrin (1600 mg/day), indocin (1 50 mg/day), indocin (100 mg/day), naprosyn (750 mg/day), naprosyn (500 mg/day), aspirin (3.6 gm/day), and placebo. part 11. five normal volunteers who had developed 4 + hemorrhagic gastritis during a previous study after 1 week of aspirin (3.6 gm/day) were given motrin (2400 gm/day), tolectin (2000 mg/day), and placebo for 1 week in a randomized crossover manner with at least a 2-week washout between medications. results. part i. placebo subjects always showed the least pathology followed by motrin (1600 me), naprosyn (500 mg), motrin (2400 mg), indocin (i50 mg), indocin (100 mg), naprosyn (750 mg), and aspirin (3.6 gm). in 2 patients receiving naprosyn (500 mg) and indocin (100 mg) frank gastric ulcer was produced. differences between motrin 1600 or 2400 mg and aspirin were highly significant (p < 0.01) as were differences between aspirin and placebo (p < 0,001). differences between motrin (1600 mg) and indocin (100 mg), and between motrin (2400 mg) and naprosyn (750 mg) approached significance (0.05 < p < 0.10). part 11. of the 5 subjects taking tolectin, 2 had hemorrhagic gastritis of a degree comparable to that with aspirin, and 3 had similar but less extensive changes. one subject on motrin had extensive hemorrhagic gastritis as with aspirin, and 4 had minimal or no changes. in both parts i and 11, gastric mucosal biopsies, blood levels, and photographs confirmed endoscopic pathology. this study demonstrated that severe gastric mucosal hemorrhagic and ulcerative changes occur in subjects using nonsteroidal antiinflammatory drugs, and that significant differences exist between various drugs. seven patients with active gonococcal (gc) arthritis immunoelectrophoresis (cie). the diagnosis of gc arthritis were studied for the presence of gc antigen (ag) and anti-in all seven patients was made by typical clinical presentation, body (ab) in serum and synovial fluid by counter-positive local culture for ngonorrhoeae (ng) , and response to treatment. gram stain and culture of synovial fluid, as well as blood cultures, were negative in all patients. specimens were run against rabbit antisera to n g (difco) to detect gc a g and against a turbid solution of fresh isolates of n g to detect g c ab. glass slides were coated with 3 ml of 0.015 m agarose in 0.075 m barbital buffer p h 8.6. pairs of wells 3 mm apart and 3 mm in diameter were cut in the agar and filled with lop1 of samples-ag in the cathode well and ab in the anode well. slides were electrophoresed in barbital buffer for 45 minutes at room temperature at 4 mamps/slide, and examined for the presence of precipitin lines. results are shown in the table. g c a g was found in the serum of 1 patient and in the synovial fluid of 3 others. gc ab was detected in the serum of the remaining 3 patients and in simultaneous synovial fluid of 2 of these. g c ab was absent in convalescent serum in patients #6 and #7 at 2 months. only l of 30 patients with initial or recurrent active gc urethritis, cervicitis, or salpingitis had serum g c ab detected by cie; none had g c ag. these synovial fluid patient no. results suggest that the finding of gc a g or ab in synovial fluid o r serum by cie provides evidence for gc arthritis and may be positive when gram stain and culture are negative. cie therefore appears to be a useful test in the diagnosis of this condition. f m f frequently presents a diagnostic dilemma in childhood. its musculoskeletal manifestations may be confused with septic arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, or other rheumatic disease. although 80% of patients have symptom onset in childhood, diagnosis is usually delayed. we have followed 31 patients who presented before age 19 years. there were 16 male and 15 female children. the mean age of onset was 4.4 years (range 1-14); mean age at diagnosis was 9.3 years (range 2-18). there were 23 armenians, 3 sephardic jews, and 5 of other ethnic origins. all had recurrent fever and peritonitis, pleuritis, or arthritis, without evidence of other rheumatic or nonrheumatic disease. all were of mediterranean ancestry. presenting complaints included fever in loo%, abdominal pain in 90%, chest pain in 62%,joint pain in 45%, and skin rash in 7%. musculoskeletal manifestations ranged from arthralgias to arthritis. the knees were involved in 8 children, ankles in 6, shoulders in 2, sacroiliac joint in 2, and wrists in 1. both children with sacroiliac joint involvement had marked erosive changes and were initially thought to have ankylosing spondylitis. four patients had splenomegaly. n o patient had proteinuria o r other evidence of amyloidosis. all of 13 children tested were negative for hla-b27. fifteen children with frequent and severe attacks, mean age 11.6 years (range 3-18), were selected for colchicine therapy. patients were begun on the nearest whole tablet equivalent of 1 .o mg/meter squared. in nonresponders, the dosage was raised to a maximum of 1.8 mg/day. six patients were noncompliant, but the remaining 9 children have been followed for 4-52 months (mean 29) on continuous colchicine therapy. the mean number of attacks for the 3 month period prior t o initiation of therapy was 5.6 (range 3-12), but only 0.6 (range 0-2) for the 3 most recent months of colchicine therapy (p < 0.0025). the only side effect was transient diarrhea, which usually resolved without reduction in dosage. although the long term safety of colchicine in childhood has not been established, n o deleterious side effects have been observed. it appears that children tolerate colchicine well. colchicine therapy may allow children incapacitated by severe attacks of f m f t o assume a nearly normal life. a new amino acid, y-carboxyglutamic acid (gla), originally discovered in prothrombin where gla residues are sites for specific binding of ca++ ions, more recently has been identified in osteocalcin, a non-collagenous protein from bone matrix. based on amino acid composition and sequence of bovine bone gla-protein, osteocalcin is distinct from all other vitamin k dependent clotting proteins (factors 11, vii, ix, and x) synthesized in the liver. the posttranslational formation of gla, studied extensively in liver microsomes from warfarin treated rats, occurs in a vitamin k dependent enzymatic oxidative carboxylation of selected glutamic acid residues in prothrombin requiring bicarbonate ion and nadh or chemical reducing agent. to show that gla synthesis occurs in bone tissue and that the carboxylase enzyme has similar requirements in bone, microsomes were prepared from the long bones of 17 day old chick embryos. the eggs were injected at 12, 14, and 16 days with sodium warfarin, an inhibitor of gla synthesis, to allow for the accumulation of endogenous substrate available for specific labeling with nah14cob. the microsomes separated from bone homogenates by differential centrifugation incorporated 14c into gla only in the presence of vitamin k1. "c-gla was identified after alkaline hydrolysis of the microsomes and separation on an amino acid analyzer. further confirmation of "c-gla was obtained by acid hydrol-ysis of the putative gla component resulting in decarboxylation to glutamic acid and recovery of approximately 50% of the incorporated radioactivity in glutamic acid. the posttranslational enzymatic generation of specific calcium binding sites in bone suggests that the vitamin k dependent carboxylation reaction may have a possible role in regulating mineral deposition in calcified tissues. since the formation of a specific calcium binding amino acid in bone is vitamin k dependent, anomalies might be expected to occur in mineralized tissues under conditions of vitamin k deficiency or anticoagulant therapy. of significance is the finding of gla-containing proteins in ectopic calcifications (subcutaneous scleroderma and dermatomyositis plaques, atheromatous plaque) which suggests gla may be important in the formation and resorption of bone in a wide variety of disorders. a retrospective study was instituted on 10 patients in the ucla lupus nephritis clinic in an attempt to determine the ability of three serologic indicators-specifically immune complexes (ic), anti-dna antibodies (dna-ab), and c3-to predict the activity of sle renal disease as indicated by changes in 24 hour proteinuria, serum creatinine, and creatinine clearance. we chose not to assess urinary sediment because of difficulty in quantitating terms such as "rare," "occasional,'' and "full field" as used by different observers. patients' records and serum samples from the period 1975-1977 were employed. the mean number of matched clinical samples per patient was 20 with a range of 9-28. similarly, the mean number of matched serologic samples per patient was 16 with a range of 8-24. "normal" daily or short term variation for each clinical and serologic parameter was assessed from paired samples taken within 1-7 days of each other. these "normal" values were then used as a baseline, variations from which could be tested for abnormality by statistical techniques. by use of simple regression analysis it was noted that ic as determined by 4% polyethylene glycol precipitation correlated better than either dna or c3 with clinical parameters. intriguingly, ic correlated with neither dna-ab or c3. more importantly, when attempts were made to predict deterioration of clinical parameters 3-5 weeks after either a single abnormal serologic determination or after paired determinations of serologic parameters demonstrating significant deterioration, no test or combination of tests produced less than a 50% false positive rate. however, all tests were capable of predicting stability or improvement of clinical parameters with a true negative rate that averaged 92.3% for ic, 91.4% for c3, 90.8% for dna-ab. no combination of tests improved these rates. pyogenic infection originating within the intervertebral disc space, once thought to be uncommon, is becoming increasingly recognized. we report the clinical findings in 15 adult patients with non-tuberculous pyogenic infection of the intervertebral disc occurring in two teaching hospitals over an 18 month period. chills, rigors, and low grade fever were common prodromal complaints (7:ls). the acute onset of back and neck pain (14:ls) with point tenderness over the involved spinous process (8: 15) most accurately indicated the diagnosis. referred abdominal pain or sciatica were less commonly seen. acute paraparesis or quadaparesis occurred in 2 patients with epidural abscess. the sedimentation rate reflected disease activity in all patients. plain radiographs were positive in only 7 patients. tomography was helpful in another 3. ""'tc pyrophosphate bone scan was positive in 11:12 cases and responsible for diagnosis in 4 of 5 patients with normal radiographs. overall the bone scan was positive in 11 of 12 cases studied. responsible organisms were cultured usually from the involved disc space (7:9). staphlococcus aureus (4) and e. coli (3) were the most common pathogens, although other microorganisms were responsible. no pathogen was isolated in 6 cases. delay in diagnosis averaged 14 weeks. previous spinal roentgen abnormalities ( 9 14), disc surgery (3:14) , another site of active infection (3:14) , and alcoholism (2:14) were predisposing factors. intensive intravenous antibiotics followed by two months oral antibiotic therapy proved curative. ten of 15 patients could be followed one year after therapy and were asymptomatic except for 2 patients with pre-existing mecha-nial low back pain. none had evidence of active septic discitis. one patient died of pulmonary embolism post-discectomy. epidural abscess was the usual indication for surgery. the sedimentation rate returned to normal in all survivors. septic discitis is a common disease, but delay in diagnosis is usual despite a characteristic clinical syndrome. the sedimentation rate and bone scan are the most useful adjuncts to diagnosis. aspiration of the suspected disc space for responsible organisms is advocated. most patients are afforded excellent prognosis following judicious rest and prolonged antibiotic administration. musculoskeletal complaints of uncertain etiology have been noted in up to 30% of patients receiving a kidney transplant. we have studied prospectively 35 consecutive renal transplant recipients. joint pain, stiffness, and swelling were assessed 5-6 days in every week of the postoperative period. six patients experienced joint pain affecting chiefly knees but also shoulder, ankle, and temporomandibular joints. arthralgias were variable in severity and duration, and in time of onset after transplantation. in 2 patients pain coincided with reduction of steroid dosage and was abolished by modifying the treatment regimen. a third patient, who developed transient joint pain 17 days after appearance of a painless effusion, had hyperparathyroidism. joint effusions, confirmed by arthrocentesis, appeared in 18 knees of 11 patients. mean duration from transplantation to first joint aspiration was 17 days. effusions were generally unaccompanied by pain, except on extreme flexion, and persisted for 24 hours to 6 weeks. twenty-five synovial fluid samples were obtained from the 18 knees. the mean synovial fluid volume was 15 ml (range, 3-65 ml) and the mean cell count was 28/mma (range 0-150/mms). in every case viscosity and mucin test were normal and crystals were not found on polarization microscopy of the centrifuged fluids. joint x-rays showed only soft tissue swelling. electron microscopy of three fluids revealed no hydroxyapatite-like material. synovial biopsies in 2 patients were normal by light microscopy. no cases of aseptic necrosis have been identified. tests for serum ana, immune complexes, and rheumatoid factors were all negative. neither the arthralgias nor effusions bore any apparent relationship to the source of the donor kidney, renal function, fever, or use of anti-thymocyte globulin. no major transplant rejection episodes occurred in any of the patients with effusions or arthralgias, whereas no joint problems arose in any of 9 patients who rejected donor kidneys in the postoperative period. thus, benign effusions are common after renal transplantation. they appear unrelated to immunologic factors or crystal deposition and may be due to transudation associated with high-dose steroid treatment. to investigate the hypothesis that infection may precipitate certain arthritis syndromes, we performed a battery of infectious agent serologic titrations on 130 early-diagnosed arthritis patients and 60 normal controls. forty-five antigens were employed: 8 for common bacteria, 6 for mycoplasma, 9 for respiratory viruses, 5 for other viruses, and 17 for y enterocolitica. the arthritis patients included 25 with sle, 48 with ra, 14 with jra, and 43 with arthritis of undetermined diagnosis (aud). data were analyzed by diagnostic groups and b27 status. no significant difference was found among the arthritis and control groups in antibody titers to any of the common bacterial antigens or the mycoplasma species, 8 of the respi-ratory viruses, and 2 of the other viruses. in jra patients, adenovirus (group) and herpes simplex (types 1 and 2) virus titers were somewhat low, and in the ra and aud groups cytomegalovirus titers were low. in sle, mumps virus titers were slightly higher than in the other groups. however, in contrast to the occasional difference serologic titrations with previously mentioned antigens, highly significant differences in antibody titers to all 13 domestic y enterocolitica antigens (7 serotypes) were found among the groups. arthritis patients had consistently lower median levels than normal controls with the lowest titers found in sle, followed by jra, aud, and finally the ra group. the 4 finnish y enterocolitica antigens (serotypes 3 and 9) showed no significant difference among groups but yielded the lowest median titers of all yersinia antigens. evidence of yersinia reactive arthritis was not found in this patient sample, and median antibody titers of the 1 1 b27 positive and 76 b27 negative arthritis patients without either rheumatoid factor positivity or the diagnosis of sle were highly comparable. the results provide no serologic evidence of infectious agent association with early-diagnosed arthritis but reveal impressive hyporeactivity to domestic y enierocoliticu serotypes which suggests decreased natural igm agglutinating antibodies to gram negative organisms as previously reported in sle patients (baum j, ziff m: j clin invest 48:758, 1969) . among the various microvascular abnormalities present in the skin of patients with connective tissue diseases and demonstrable by in vivo microscopy, the most characteristic pattern of capillary abnormalities is found in patients with scleroderma (sd) (maricq hr, spencer-green g, leroy ec: skin capillary abnormalities as indicators of organ involvement in scleroderma (systemic sclerosis), raynaud's syndrome, and dermatomyositis. amer j med 6 1:862-870, 1976) . the prevalence of this sd pattern in a larger sample of patients with sd and related connective tissue diseases was studied in three separate arthritis centers and in 147 patients with the following diagnoses: sf (so), systemic lupus erythematosus (sle, a), mixed connective tissue disease (mctd, 26), and raynaud disease (rd, 11) . results from widefield microscopic observations and photomicrography show sd-type capillary abnormalities present in the following numbers of patients in each diagnostic category: sd, 41 (82%); sle, 1 (2%); mctd, 14 (54%); and rd, 1 (%). tortuous capillaries were noticed in 25 patients with sle (42%), 3 with sd (6%). 3 with mctd (12%), and 4 with r d (36%). only 2 patients (with mctd) had both a sd pattern and tortuous capillaries (8% of mctd or 1% of all patients). non-specific microvascular changes were present in 17 patients with sle (28%), 4 with sd (8%), 3 with mctd (12%), and 2 with rd (18%). many sle (17 patients, 28%) and r d (4 patients, 36%) patients showed no capillary abnormalities, as did 6 patients with mctd (23%) but only 2 with sd (4%). these results confirm the high frequency of sd-type capillary pattern in sd patients and separate them from patients with sle, who do not show a pathognomonic pattern by the present techniques. tortuous capillaries, frequent in sle, occur in other diseases and in normal subjects, although to a lesser degree. it is interesting to note that patients with mctd, an overlap syndrome having clinical features of both sd and sle, exhibited no specific pattern of capillary abnormalities. about half of the patients showed sd pattern, and the other half demonstrated tortuous capillaries, other non-specific changes, or no observable microvascular abnormalities. clinical criteria for classification of systemic sclerosis (ss) were derived from prospectively entered data on 799 early-diagnosed rheumatic patients contributed by 29 centers. cases included patients with definite or probable ss, or scleroderma in overlap. controls were patients with systemic lupus erythematosus (sle), polydermatomyositis, or raynaud's phenomenon alone. computer assisted and multivariate analysis techniques were used to construct criteria with minimal redundancy. careful definition of cutaneous involvement contributed the most powerful criteria. sclerodermatous involvement proximal to the metacarpal phalangeal or metatarsal phala-ngeal joints (ie, "proximal" scleroderma), whether in an acrosclerotic distribution (acrosclerosis), on the face or neck (scleroderma facies), or on the trunk or abdomen, was present in 91% of definite ss cases and in only 1% of combined controls. if the major criterion of proximal scleroderma was absent, ss cases could be distinguished by having at least 2 of 4 minor criteria, ie, sclerodactyly, digital pitting scars, pulmonary fibrosis, or large bowel sacculations. employing the major with minor criteria yielded 98% sensitivity in definite ss patients and 97% specificity in total controls, without using exclusions. when applied to an independent rheumatic patient the course of articular involvement in early rheumatoid arthritis (ra) is variable and has not been quantitatively characterized. in a prospective study of 50 younger adult, early ra patients (negative for hla-b27), detailed articular, systemic, laboratory, and therapeutic data were collected twice yearly for a mean followup of over 5 years. based upon the number of joints involved during followup, each patient was assigned to separate pain/tender (p/t) and swelling (sw) articular course patterns. monocyclic pattern was defined as one cycle of documented arthritis with articular remission observed for at least 1 year; intermittent as two or more arthritis cycles, each separated by joint remission of at least 6 months; continuing as continuous joint involvement but without persistent progression; and progressive as continued progressive involvement. the table shows numbers of cases in each pattern, mean numbers of joints involved at entry, and the mean annual change (a) in numbers of joints involved during followup, determined by regression analysis. monocyclic groups included significantly more males (p/t, p < 0.05; sw, p < 0.001), and seronegative cases (p/t, p < 0.05; sw, p < 0.01). only one woman experienced complete articular remission while progressive swelling occurred in only 2 cases. entry joint involvement correlated with subsequent p/t and sw articular patterns (p < 0.01 and p < 0.05, respectively). cross-sectional analysis of the numbers of joints involved at each protocol exam and longitudinal analysis of regression slopes of individual patient joint involvement both indicated trimodal distributions: monocyclic; chronic (ie, combined intermittent and continuing groups), and progressive patterns, which were independent of therapy. the data provide quantitative evidence for monocyclic, chronic, and progressive articular course patterns in early ra, with identification of significant sex and clinical correlates. in some postmortem cases of systemic lupus erythematosus (sle) associated with diffuse proliferative glomerulonephritis (dpgn), an antigen related to mammalian type c rna viral core (p30) protein is deposited in the glomerular lesions in an immune complex pattern (proc natl acad sci 73:233, 1976) . now an attempt is made to support this finding by examining the glomerular immune deposits in sle-dpgn for evidence of type c virus antibody. human immunoglobulins (igs) were eluted from the glomerular immune deposits in two fractions by sequential treatment with dnase to elute anti-dna antibodies, followed by acid buffer to elute remaining antibodies. the eluted igs were then assayed by a sensitive, specific, and quantitative enzymoimmunoassay, which compares favorably with radioimmunoassay, for the detection and measurement of anti-p30 antibody against purified p30 proteins of the four chief groups of mammalian type c viruses: murine, feline, endogenous feline rd-i 14/endogenous primate, and simian sarcoma virus type i (ssv-i)/infectious primate virus groups. human igs which showed specific anti-p30 antibody activity, particularly against p30 antigen of the rd-i 14 virus group and to lesser extent against p30 antigen of the murine and ssv-i virus groups, was eluted by acid buffer from the glomerular immune deposits in two cases of sle-dpgn that were known from previous study to contain deposits of viral p30-related antigen in the same tissue lesions. these findings support the hypothesis, stemming from studies of the new zealand mouse model of sle, that subinfectious antigenic expression of a type c virus is involved in the pathogenesis of dpgn associated with sle. (this work was supported by nci, dhew grant no. male b/w mice generally die after one year of age with lupus nephritis and/or lymphoid malignancy. these mice gradually develop a severe depression of cell-mediated immunity which may contribute to the subsequent fatal autoimmune and lymphoproliferative disease. we studied t lymphocyte function in 8 young (3-4 month) and 9 old (15-20 month) male b/w mice in separate paired experiments. the spleen cell response to phytohemagglutinin was 62,087 cpm (geometric mean) for young mice compared with 490 cpm for old mice (p < 0.oool). when spleen cells from young and old mice were mixed together, there was significant suppression of young cells by old splenocytes in all experiments (57-98% suppression). this suppression is mediated by a radio-resistant, nonadherent, mononuclear leukocyte, probably a small lymphocyte. although this suppressor cell is eluted in the "t cell fraction" of a nylon wool column, it cannot be identified as a t cell because it is resistant to anti4 and complement treatment . in a typical experiment, unfractionated old spleen cells incorporated 291 cpm compared t o 42,825 cpm for young spleen cells. a mixture of old and young cells incorporated 6,683 cpm, whereas the predicted incorporation for this 50/50 mixture was 21,558 cpm (69% suppression). old spleen cells from the t cell fraction of the nylon wool column were enriched for suppressor cells (95% suppression in the mixing experiment). in contrast, the b cell fraction was completely depleted of suppressor activity (actually 23% greater than predicted incorporation). anti4 and complement treatment of spleen cells from a n 18 month old b/w mouse demonstrated 80% suppression by the untreated cells and 98% suppression by the preparation depleted of &bearing cells. these results have been consistently reproduced. there is no evidence of a suppressor cell in old mice of four normal strains (dba/2, c57b1/6, balb/c, and nzw). we conclude that a mononuclear suppressor cell, probably a lymphocyte, contributes significantly to the severely depressed t lymphocyte function of aging b/w mice. this suppressor mechanism may participate in the pathogenesis of the lymphoproliferative and autoimmune disorder characteristic of the strain. none of the clinical and laboratory parameters presently used for the assessment of sle kidney disease is completely reliable. immunofluorescence of the urinary sediment (ifus) has been recently reported to accurately predict the rejection of renal transplants, and the purpose of this study was to evaluate its usefulness in renal sle. thirty consecutive patients who met the ara preliminary criteria for sle were included in the study. all had recent kidney biopsies and were treated with prednisone and immunosuppressors at usual doses with urinalysis, urinary light chains, c3, anti-dna binding, npn, and creatinine as parameters of activity. random urine specimens were collected weekly for 6 months and the urinary sediment was studied by direct immunofluorescence with antisera anti iga, igg, ig m, and fibrinogen degradation products. clinicai grading and fluorescence readings were done by independent observers. nine patients had normal kidney biopsies and ifus was always negative. nine patients had abnormal biopsies, were classified as inactive, and ifus was always negative. in 12 patients with abnormal biopsies and active renal disease, ifus was always positive, even before the appearence of proteinuria, hemoglobinuria, casts, and light chains. ifus was positive 1 to 3 months before c3 dropped in 4 patients and became negative 1 to 4 months before c3 returned to normal in 7 patients. in the presence of residual proteinuria, ifus became negative in 5 patients. there was no correlation be-tween the histological type and the immunofluorescence findings in the kidney biopsy with the type of immunoglobulin found in the urine. this procedure is easy to perform, not costly, and the results are available within 1 hour. our data suggest that it reflects the changes in renal status months before the other parameters and therefore it may be a valuable method for guiding a more flexible treatment of patients with sle kidney disease. fifteen percent or less of patients with juvenile rheumatoid arthritis (jra) have positive latex fixation tests (lft); whereas, approximately 45% have hidden rheumatoid factor (rf) (19s igm r f in the peripheral blood probably bound with 7 s igg and, therefore, not detectable by the standard lft). in this study, a complement-dependent hemolytic assay was used to determine the presence of hidden rf. sera of 59 children with seronegative jra, 2 with seropositive jra, 7 children with connective tissue diseases, 3 with leukemia, and 12 normal children were separated by gel filtration at ph 4.0 to obtain the igm-containing fraction. these igm fractions were subjected to the complement-dependent hemolytic assay in which sheep erythrocytes (srbc) are coated with reduced, alkylated, and acid-treated rabbit igg anti-srbc antibody and are hemolyzed by guinea pig complement in the presence of 19s igm rf. thirty-seven of 61 patients with jra, of which 23 of 33 polyarticular jra, 12 of 21 pauciarticular jra, and 2 of 7 systemic jra, had titers > 1 : 16. none of 12 normal controls and only 1 of 10 disease controls had titers > 1 : 16. the median titer of all jra patients was 1 :42 and healthy and disease controls, 1 : 7. estimates of the significance of the differences between the median titers of jra patients and of the controls were obtained by mann-whitney analysis. they were significant at p < 0.001. when data from patients with active disease were analyzed separately, the median titers for polyarticular jra were 1:97, pauciarticular 1:91, and systemic 1:23. patients with inactive disease did not have significantly different titers from controls. the active polyarticular and pauciarticular values were significant at p < 0.001 and p < 0.005. these results demonstrate: (1) 59% of seronegative jra patients have hidden rf by this procedure; (2) the hemolytic assay is more sensitive than the lft or sheep cell agglutination tests (scat) in determining the presence of hidden rf in jra; and (3) activity of disease correlated with higher titers in the hemolytic assay, and the assay was superior to the lft or scat as an indicator of disease activity. this study was devised to answer in relation to articular cartilage proteoglycans (pgs) whether degradation products or products of incomplete synthesis are detectable in the earliest pathological lesions of this rabbit model of osteoarthritis. an ultramicro transport method for obtaining s value polydisperse profiles for pg aggregates (pgagg) and subunits (pgs), and miniaturized extraction, dialysis and cscl density gradient ultracentrifugation techniques have allowed study of these pgs and various pg products within the s value range of 1-200 from histologically defined sites. thirty new zealand white rabbits (weighing 2-2.5 kg) were subjected to partial medial meniscectomy and killed 10-12 weeks postoperatively under nembutal anesthesia. micro-dissected medial femoral condyle samples included 1 ) ulceration, 2) 1 mm rim surrounding the ulcer often with fibrillation, and 3) cartilage from the medial and lateral femoral condyle which occasionally revealed fibrillation but usually appeared to be normal. amounts of cartilage equivalent to 3) above were obtained from the contralateral (left) knee for use as a primary control and from sham operated (right) knees for use as a second control. the cartilage from 4 rabbits was required to obtain a pool of 10-20 mg wet cartilage for each zone 1-3 above. these cartilages were diced and pgs extracted, dialysed, and partitioned. for pg extracted with 4.0 a4 gucl from left femoral articular cartilage of control knees, the profile shows a subunit peak with weight average sediment coefficient s; o of about 16 and an aggregate peak extending from 40 up to 120 s with an average of 62. similar profile data on the tibia1 cartilage provided an average sediment coefficient of 59. about 1/3 of pg was in aggregated form and the rest pgs. interestingly, the undissociated pgagg revealed an s value range of 40-180. pgs extracted without dissociation in 0.5 m gucl gave similar data. in regard to the profile of s values of pgs from small ulcers, the weight average sediment coefficent s: o of the peak was 16. peaks indicative of pg products with lesser values were found in 3 pooled samples with large ulcerations. a similar study run after isolation of pgs in a dissociative csci gradient confirmed this result. in conclusion, the major abnormality detected early was a disturbance in pg aggregation, and later pgs degradation. we recently observed the development of glomerulonephritis in 3 patients with sicca syndrome (ss) who did not fulfill the diagnostic criteria of systemic lupus erythematosus. these patients developed ss 5 to 17 years prior to the onset of glomerulonephritis. two were diagnosed as having membranoproliferative glomerulonephritis by light and electron microscopy, and 1 was found to have membranous glomerulonephritis. although immunofluorescent studies were not available, all clearly had microscopic evidence of immune complex (ic) deposition. moreover c'3 levels were decreased in all three a t the time of onset of renal disease, and all had high levels of circulating ic as determined by the lz6i clq precipitation test. these findings raised the possibility that ic plays a role in the development of certain aspects of tissue injury in ss. for these reasons 55 patients with ss were studied for the presence of circulating immune complexes with the lasi clq precipitation test. increased ' ' ' 1 clq binding activity (clqba) (10%) was found in 47 (86%) patients (mean clqba = 48%, range 2-98%). thirty-five patients (64%) had rheumatoid factor (rf) present in their serum. analysis by spearman rank correlation revealed a positive association between the clqba and the titer of r f as determined by the bentonite flocculation test (bft), (rho = 0.551, p < 0.0005). it was found that the highest positive correlation between clqba and bft titer existed for those patients with ss alone (rho = 0.687, p < o.oos), and then decreased for those patients with ss plus extraglandular disease (rho = 0.462, p < 0.05) and even more for patients with ss plus rheumatoid arthritis (rho = 0.429, 0.05 < p < 0.10). there was no apparent correlation between clqba and bft titer in patients with ss plus another connective tissue disease. pretreatment of 10 sera with 0.01 m 2mercaptoethanol to dissociate igm into monomers resulted in the eradication of rheumatoid factor as measured by bft but caused only slight to moderate reductions in clqba. thus circulating immune complexes exist in many patients with ss, are distinct from rheumatoid factor, and may contribute to certain aspects of tissue damage in this disease. we have recently described a high (69%), but not absolute, association of sicca syndrome (ss) with the hla-dw3 allelle, which is in linkage disequilibrium with hla-b8 (nejm 296:895, 1977) . in this study we determined the blymphocyte antigens of 24 patients (21 females and 3 males, ages 26 to 73) and 184 normal controls. in addition to ss, 3 of the patients have rheumatoid arthritis and 3 have systemic lupus erythematosus. immunoglobulin-bearing lymphocytes were separated from heparinized peripheral blood on goat anti-human igg (fab) coated plastic plates and tested with 60 antisera in complement dependent cytotoxicity tests. the antisera used were obtained from multiparous women and absorbed with pooled platelets to remove hla-a, -b, and -c antibodies. two antisera, 172 and ags, reacted with the b-lymphocytes from all the ss patients compared to 37 and 24% of the normal controls, respectively. four additional antisera (35,350,590, and 715) reacted more frequently (67,63,58, and 54%) with the patients' b-cells than with those of controls (17, 21,24, and 14%) . the remainder of the antisera tested had the same frequency of reactivity in this disease as in normals. t o determine if these antisera recognized the same or different antigens, their reactions with the lymphocytes from the normal controls were compared by the x ' test. antisera 172 correlated with both 35 and 350 but not with ags or 715. antisera ags correlated only with 7 15. similar statistical analysis was done for the patients. the antisera 172 and ags were excluded because of their 100% prevalance in ss. in the ss patient group antisera 35, 350, and 590 were associated with each other but not with 715. this association can be due t o either cross-reactivity or linkage disequilibrium. family studies are in progress to determine whether ss patients can be heterozygous for 172 and ags and, if so, whether they can be present in trans position. assuming that the b-lymphocyte antigens in humans are coded by loci of an ir region, our results suggest that two immune response genes may be involved in the pathogenesis of ss. in addition, the absolute association of 172 and ags antigens with these patients should provide a n additional aid in diagnosis of ss. glucocorticoid administration is associated with induction of osteopenia in man and in some laboratory animals. there is debate about dose and duration required, and whether bone loss is primarily trabecular o r cortical. accordingly, 18 adult female rabbits were divided into 3 groups: 6 received saline, 6 received 1.5 mg cortisol/kg body weight (wt), and 6 received 2.5 mg cortisol/kg wt. all received equal volumes subcutaneously once daily early in the morning. xylenol orange (50 mg/kg 13 days before kill) and tetracycline (1 5 mg/ kg 2 days before kill) were injected as bone markers. rabbits were given food and water ad libitum, and weights were measured every 5-6 days. bone mineral content (bmc) and width (w) were measured by photon absorptiometry before treatment and every 2 weeks. measurement site was the humerus 4 cm proximal to the bent elbow (all cortical bone). results are shown in the table. histopathology. the amount of bone was reduced in cortisol-treated rabbits. in the humerus this was localized to the endosteal surface. in the lumbar vertebrae the endosteal side of the ventral cortex was primarily affected with some thinning of trabeculae. calculation of rates of formation (f) and resorption (r) showed f was essentially nil in the cortisol groups. r was identical in the saline and low-dose cortisol rabbits, while r was accelerated in the 2.5 mg/kg treated rabbits. analysis of bmc and cross sections from the same site of the humerus for bone area showed a good correlation (r = 0.94). summary. rabbits treated with cortisol in the doses used developed generalized bone loss. this loss appears to involve more cortical than trabecular bone. bone marker studies show that the main effect is interference with f, and, with the larger dose, acceleration of r. in vivo measurement of bmc accurately reflects the progression of the bone loss. intermediate complexes are polymers of igg-rheumatoid factor which sediment between the 6.6s and 19s components of normal serum. they are best identified by analytical ultracentrifugation. their presence in serum can be inferred by a double "gullwing" precipitin line of the igg on immunoelectrophoresis due to accumulation of the molecular aggregates of igg around the well. in this study we have shown they can also be detected by evaluation of the difference between the electrophoretically determined y-globulin concentration and the igg concentration determined by radial immunodiffusion (rid). molecular aggregates of igg are quantified by serum protein electrophoresis, but they are underestimated by r i d because their effective or molar concentration is low relative to the monomeric igg standard. therefore, when molecular aggregates of igg are present, the difference between the electrophoretically determined y-globulin and the igg measured by r i d is abnormally high. sixty-one consecutive blood donor sera and 46 hypergammaglobulinemic sera from patients with diseases known not to be associated with the presence of intermediate complexes o r in which the presence of intermediate complexes was excluded by analytical ultracentrifugation made up the reference population. eleven sera with known intermediate complexes were examined. the mean and 95% tolerance intervals (covering 99% of the population with 95% confidence) of the reference population for the difference between the electrophoretically determined y-globulin concentration and the igg as measured by r i d was 0.31 f 0.73 g/dl. eight of i 1 patients' sera with known intermediate complexes fell outside the upper limit. all 3 sera which fell within the 95% intervals had concentrations of intermediate complexes less than 1.8 g/ dl. in addition, the degree of deviation from the reference mean showed a direct linear correlation with the level of intermediate complexes present. if the electrophoretically determined y-globulin concentration minus the igg concentra-tion by r i d is greater than 1 .o g/dl, intermediate complexes should be presumed to be present in concentrations greater than 1.8 g/dl. synthesis of antibodies to dna (anti-dna) in systemic lupus erythematosus (sle) is postulated t o result from a lack of control mechanisms which normally suppress autoantibody production. we investigated these mechanisms by measuring anti-dna synthesis by normal and sle peripheral blood lymphocyte (pbl) populations stimulated with pokeweed mitogen (pwm). pbl isolated from 5 patients with sle and 5 controls were further separated into tand b-cell enriched fractions by density sedimentation of spontaneous rosettes formed by aet treated sheep red blood cells and t lymphocytes. a portion of the t-cell fraction was irradiated with 3000 rads to inactivate were cultured with normal or sle-b cells (0.4 x iv) at ratios these studies show that sle-b cells from some patients are capable of synthesizing anti-dna which is best demonstrated in co-culture with suppressor inactivated, irradiated t cells. normal-t cells suppress the response, whereas suppression exerted by sle-t cells is variable from patient to patient. anti-dna the antibody produced by rheumatoid arthritis (ra) synovial lymphocytes may be directed at a uniquejoint antigen inciting the inflammation, and thus may be useful to identify the antigen. we have made permanent r a synovial lymphoblastoid cell lines capable of producing unlimited quantities of antibody. r a synovial membranes were obtained surgically, finely minced in medium (rpmi 1640, 20% fetal calf serum, glutamine, antibiotics), placed in 60mm linbro plates, infected with epstein-barr virus, incubated (37oc, air + 5% co,) and fed weekly. typical lymphoblastoid cell lines developed in 13 of 43 (32%) cultures after a mean 33 days, range 18-53. once established, the cell lines were put in large flasks and medium changed completely 3 times a week. eight of the 13 permanent lines were lost after a mean duration of 54 days (range 7-120) due to bacterial contamination or poor viability. the latter was due to suboptimal growth support by specific lots of rpmi or serum; subsequently these were pretested. the 5 remaining lymphoblastoid lines now have a mean duration of 159 days (range 120-210), during which their mean volume was 207 ml (range 98-332). maximum volume was 1.4 liters, but could be expanded indefinitely in 2 liters or larger flasks on shaker platforms at low speed. mean cell doubling time was 34 hours, range 24-48. igg concentration in harvested supernatant medium was measured sequentially by a radioimmunoassay described previously. mean igg production by each line was 55, 246,420,426, and 1405 pg igg/day, decreasing slowly with time. adjusted for cell concentration, mean production was 0.9, 3.9, 4.6, 5.8, and 9.7 pg igg/iv cells/day. total production to date was 2.418, 29.061, 36.684, 50.768, and 59 .0 milligrams. the culture producing the lowest amount may be producing a predominance of another immunoglobulin type. in 3 of 5 lines igg production tended to be significantly higher when cell concentration was lower (r =0.465 -0.673; p < 0.001). these ra lines produced more igg than previously reported for lymphoblastoid lines derived from other human tissues (1-3 bg/lp cells/day). they also produced 5-10 times more igg than our previous batch organ cultures of ra synovia. their increased igg production may be due to improved culture conditions and/or intrinsic immunologic hyperactivity of ra lymphocytes. these cultures may provide an excellent source of antibody specific for the antigen(s) inciting ra. recently we reported that partial disuse of a joint produced a defect in proteoglycan (pg) aggregation in human articular cartilage indistinguishable from that seen in osteoarthritis. the present study examines the rapidity with which defective pg organization develops after total immobilization of a limb, and the reversibility of the defect. the right hind limb of dogs was immobilized in a cast for 5 days to 8 weeks, at which time the animals were killed. during this period the dogs ambulated freely on 3 legs but bore no weight on the immobilized limb. in some cases the cast was removed after 6 weeks and the dogs then ambulated fully for up to 4 weeks prior to sacrifice. cartilage from the distal femur of the immobilized and the contralateral control knees was cultured for 24 hours in ham's f-12 nutrient mixture containing iwo fetal calf serum and 86s0,. pgs were extracted with 4 m guanidinium chloride (guhci) and purified by successive cesium chloride density gradient centrifugations in 0.4 m and 4.0 m guhci, ie, under conditions favoring forma-tion of pg aggregates and disaggregation of pgs, respectively. after only 5 days of immobilization a6s0, incorporation into pgs was suppressed by 5070, and this diminution in pg synthesis persisted through 8 weeks of immobilization. after 3 weeks of immobilization no evidence of pg aggregation could be found. at that time pgs from the second gradient were the same size as those from the first gradient (sepharose 2b k., = 0.63) and showed no shift in their sepharose 2b elution profile after incubation with hyaluronic acid (ha) in vitro, indicating that pg-ha interaction had not occurred. however, 1 week after removal of the cast, aggregates had again formed in the cartilage and were as large in hydrodynamic size as those in control cartilage. these results emphasize the importance of joint motion in maintenance of the normal organization of cartilage pgs. the pg aggregation defect which occurs with immobilization alters hydraulics of the cartilage, especially with impact loading, and may thus predispose to chondrocyte injury. in the course of examining host defenses against infection in patients with systemic lupus erythematosus (sle), we have found a previously undescribed serum inhibitor of complement (c5)-derived -chemotactic activity (ctxa). serum from 6 of 23 patients, when activated with zymosan, failed to attract polymorphonuclear leukocytes (pmn) comparably to normal zymosan-treated serum (zts) (measured by the "leading front" method of zigmond and hirsch). incubation of normal pmn with these sera did not affect their random motility or subsequent chemotactic response to normal zts. whereas levels of c3 and c4 (measured immunochemically) were modestly low in these sera, no gross abnormalities involving alternative complement pathway activation could be detected. when preincubated with normal zts (1: 1) at 37" for 30 minutes, these sera caused significant inhibition (30-1ooo/o) of ctxa. they also inhibited the ctxa of columnpurified c5-derived peptides (from normal zts), but had no effect on the ctxa of either the synthetic peptide, n-formylmet-leu-phe or the bacterial chemotactic factor from e coli. the inhibitor in these patients' serum was heat-stable (56â°c for 30 minutes) and acted specifically on c5-derived ctxa (not on pmn). mixing (without preincubation) of patient serum with normal zts failed to cause inhibition of ctxa. the inhibitor also acted reversibly; molecular sieve chromatography dissociated heat-stable inhibitory activity (a single peak with an apparent molecular weight of 50-60,000 daltons) from normal amounts of c5-derived ctxa in patients zts and in mixtures of normal zts incubated with patient serum. further characterization of the inhibitor has revealed it to be a basic protein (pi between 9 and 10) which can be inactivated completely by treatment with pronase. despite its effect on c5derived ctxa, the inhibitor did not influence two other c5derived biologic activities in zts: pmn lysosomal enzyme releasing activity and pmn aggregating activity. this heatstable inhibitor, uniquely specific for c5-derived ctxa in serum from some patients with active sle, may account, in part, for increased susceptibility to infections caused by pyogenic microorganisms. the evidence that type c viruses are involved in systemic lupus erythematosus (sle) is conflicting. we tried to detect type c expression in a total of 34 sle patients during various collaborative studies over the past four years. forty tissues (17 placenta or gestational products, 10 spleen, 6 kidney, 7 other) and/or cell cultures derived from them were tested a total of 110 times using 10 different methods. seventyfive percent of the tissues were tested by 2 or more methods and 33% by 4 or more. type c virus isolation was attempted using four distinct protocols: culture with sedimentation of sh-uridine-labeled virions, cocultivation with viral rna-dependent d n a polymerase assay, cocultivation with focus formation assay for helper virus rescue of the defective murine sarcoma virus genome (a albino), and triple cell fusion with viral polymerase assay. thirty-four tissues from 3 1 patients were tested a total of 55 times with negative results except for one type c isolate in a recent experiment. detection of both type c interspecies and primate species antigens was attempted using 3 different radioimmunoassays in 2 separate laboratories (g. j. todaro, h. p. charman), and indirect immunofluorescence (r. c. mellors). twenty-two tissues from 18 patients were tested a total of 37 times with negative results. electronmicroscopy of gestational products from 9 patients revealed type c-like particles in 4 placentas, but also in 2 of 3 normal controls (m. imamura). type c-related sequences were not found in cellular d n a from 9 patients using hybridization to a murine type c cdna probe (g. s. aulakh). various false-positive results were also encountered in most of the studies. the virus isolate-positive patient has not yet been tested by other methods, but the 4 patients with type c-like particles were each tested by 3 to 5 other methods with negative results. thus 5 of the total 110 tests were positive on 5 of the 40 tissues from 34 patients. these combined collaborative studies are the most comprehensive yet done in sle. if type c expression is enhanced in sle, it is not regularly demonstrable using current methods. a prospective clinical, serologic and histopathologic study was performed on 267 consecutive patients suspect for sjiigren's syndrome (ss) referred to an interdepartmental university clinic during the years 1972-1977. one hundred female and 25 male patients met at least two of the following three criteria: 1) lymphocyte focus sco.re greater than one (fs > 1) on labial salivary gland (lsg) biopsy, 2) keratoconjunctivitis sicca (kcs), 3) associated extraglandular connective tissue or lymphoproliferative disorder. of the 125 ss patients, lsg biopsy fs > 1 occurred in 96%, definite kcs in 55%, and associated extraglandular disease in 85%. rheumatoid arthritis was present in 27%, scleroderma in 7%, systemic lupus erythematosus in 6%, and polymyositis in 2%. lymphoproliferative disease or connective tissue abnormalities not fulfilling criteria for a coexisting connective tissue disease (ctd) were present in 43% of patients. patients with extraglandular disease and kcs almost always had a fs > 1 (33:35 patients), whereas patients with extraglandular disease and a fs > 1 did not necessarily also have kcs (only 38:77 patients). we conclude that lsg biopsy is more sensitive than kcs for detection of ss in patients with an underlying connective tissue or lymphoproliferative disorder, and may help establish a diagnosis in patients with clinically undiagnosed autoimmune or lymphoproliferative disease. moreover, lsg biopsy is far superior t o clinical symptoms or signs of salivary gland dysfunction which are not specific for ss. in fact, only 50% of patients with any symptoms suggestive of ss actually had the disease confirmed. local glandular disorders, anxietydepressive syndromes, and parasympatholytic drugs were common causes of oral and/or ocular complaints. our study suggests a new definition of ss as an autoimmune exocrinopathy based on the utility and diagnostic value of lsg biopsy. in view of the hla-b8 association and unique precipitating antibodies to nuclear antigens (ha, ss-a, ss-b) found in ss, autoimmune exocrinopathy might be considered a genetically and serologically distinct connective tissue disease related t o but separable from other connective tissue diseases. rheumatoid arthritis or a coexisting connective tissue disease occurs in only a minority of patients with autoimmune exocrinopathy and should not be a requirement for diagnosis. degradation of collagen at sites of tissue injury and inflammation is effected by the dual action of collagenase and nonspecific proteases present in neutrophils, macrophages, and other cells. monocytes eventually accumulate at such sites and as macrophages perform important phagocytic functions. mechanisms whereby monocytes are attracted to areas of in-flammatory reactions are incompletely understood, although several different chemotactic factors have been described. we have measured the chemotactic response of normal human peripheral blood monocytes to different types of human collagens and collagen degradation peptides by a modified boyden technique. chemotactic activity (ctx) expressed as monocytes per oil immersion field for various preparations tested were as follows: type i collagen (1.8 p m collagenase retained chemotactic activity. additional studies were undertaken with synthetic tri-and dipeptides containing amino acids common to the three different collagens. peptides containing proline or hydroxyproline (for example, gly-pro, gly-hyp, pro-hyp, gly-pro-hyp, and gly-pro-ala) were chemotactic for monocytes at concentrations ranging between lo-' and 10-bm. these data suggest that peptides generated as a result of degradation of collagen by collagenase and other proteases might function to chemotactically attract monocytes to sites of tissue damage and inflammation in vivo. although it is known that a small percentage of patients with sjbgren's syndrome (ss) may develop malignant lymphoma, the frequency of ss in lymphoma has not been established. therefore, 50 consecutive untreated patients with biopsy-proven non-hodgkin's lymphoma were screened to establish the prevalence of ss. patients were defined as having ss if they exhibited objective evidence of keratoconjunctivitis sicca and xerostomia. all were assessed by history, physical examination, schirmer test, rose bengal staining of the cornea and bulbar conjunctiva, and if possible a serial salivary scintiscan, serologic studies, and a lip biopsy. of the 50 patients seen, 13 were identified as having ss. of these, all 13 had a history of xerostomia and keratoconjunctivitis sicca, while 12 had a positive schirmer test, 5 had positive staining with rose bengal, and 3 had a markedly abnormal salivary gland scan. three had a positive anti-nuclear antibody (ana) and 1 anti-salivary duct antibody. of the 13 with ss, 6 had adequate lip biopsies. of these, 3 were normal, and 2 showed mild, non-specific lymphocytic infiltration, while 1 was highly iuggestive of ss with lymphocytic infiltration and salivary gland atrophy. five of the 13 had musculoskeletal complaints, 4 had classic or atypical raynaud's phenomenon, and 2 had a concurrent diffuse connective tissue disease (1 had scleroderma and 1 had probable rheumatoid arthritis). in summary, of 50 patients screened, 13 had ss as defined above. of these, 12 had positive schirmer tests, 5 had positive rose bengal staining, and 3 had abnormal salivary scans. three had a positive ana, 1 had anti-salivary duct antibody, and 3 had abnormal lip biopsies. five had musculoskeletal complaints, 4 had raynaud's phenomenon, and 2 had a concurrent diffuse connective tissue disease. thus, ss in patients with non-hodgkin's lymphoma appears to be more common than is generally appreciated. hydroxyapatite crystals have been recently suggested as a cause of crystal induced synovitis in humans. we have performed the following studies in an attempt to develop an experimental model to further study the relation of hydroxyapatite to inflammation. articular calcification was induced in six-week-old nzw rabbits with techniques similar to those described by selye in other tissues. eight rabbits were given a single dose of 500,000 u oral vitamin d, and the next day left knees were injected with i mg feci,. right knees were injected with saline. four rabbits received intraarticular feci, without vita-min d. cartilage and synovial membrane were studied by light and electron microscopy at 5, 15, and 45 days. synovianalysis, roentgenograms, and microradiography were also done. six other six-week-old rabbits were given vitamin d orally 500,000 u for 90 days without any intraarticular injection. six controls were followed without vitamin d and all were killed at 90 days for studies as above. mild synovial inflammation was seen in feci, injected joints without vitamin d. in rabbits given vitamin d there was tissue necrosis in the feci, injected joint with synovial calcification visible by light microscopy by the fifth day. calcifica-tions were all characteristic of hydroxyapatite by electron microscopy and could be seen in interstitium, occasionally on collagen fibers, and in vacuoles of synovial cells. small amounts of iron were seen in phagocytes without relation to the calcification. calcification increased over 45 days. cartilage was not calcified except for a small surface deposit at 45 days in one rabbit. synovial fluids had only very low leukocyte counts with predominance of mononuclear cells. joints not injected with iron were normal. the rabbits given vitamin d for 90 days all developed round, mid-zone articular cartilage calcifications similar to those spontaneously occurring in older rabbits. there was no synovial calcification, inflammation, or joint effusion. by electronmicroscopy all calcium deposits were in the interstitium and were hydroxyapatite-like needles. many chondrocytes showed degenerative changes. thus, different patterns of articular calcification can be produced in rabbits' knees with techniques described. acute crystal associated inflammation was not demonstrated. crystals formed and sequestered in synovial or cartilage tissue appear to be tolerated without inflammation. studies of systemic lupus erythematosus (sle) families and populations suggest that a gene(s) linked to the major histocompatibility complex (mhc) influences sle. we have examined the mhc in sle families, patients, and controls by determining the cytotoxicity of antisera which detect mhcdetermined antigens expressed selectively on b lymphocytes. b lymphocytes from 41 sle patients and 184 controls were tested against a panel of 47 pregnancy sera, and reaction frequencies of individual sera were compared. twenty-eight of the sle patients were also typed with a panel of hla-drelated sera from the 7th international histocompatibility workshop, hla-drw types assigned, and compared with 490 workshop controls. the hla-drw types and individual serum reactivities which were increased in the sle population are shown. sle families shows that one hla haplotype is usually shared among those individuals with sle and other autoimmune abnormalities in a given family. exceptions exist, however, indicating that the haplotype itself is neither necessary nor sufficient for the expression of autoimmunity. this study demonstiates that certain mhc-related bcell alloantigens, possibly products of immune response genes, are increased in sle. family study indicates that the requirements for sle development are not limited t o the mhc, however, and probably involve additional genetic and/or environmental factors. clinical observations of increased osteoarthritis (oa) with menopause and studies of oa in experimental animals suggest that androgens worsen and estrogens ameliorate oa. further studies have demonstrated that estrogens suppress and androgens increase 35s04 incorporation into nonarticular cartilage. using a rabbit partial meniscectomy model of experimentally induced oa, we compared nontreatment (18 rabbits), estradiol valerate 1.6 mg/kg intramuscularly every 2 weeks (20 rabbits), and testosterone cypionate 5 mg/kg intramuscularly every 2 weeks (20 rabbits) on the development of oa and on proteoglycan (pg) synthesis by cartilage. animals were killed 12 weeks post partial meniscectomy, and osteoarthritic lesions were noted. knee sections processed for histologic examination were stained with h and e and safranin-0 with fast green counterstain. cartilage metabolism in the 3 groups was examined by in vitro measurement of ""so, incorporation into articular cartilage after 2 hour incubation in dulbecco's modified eagle medium. medial and lateral components of femoral and tibia1 knee joint surfaces were studied separately. normal unoperated knees served as additional controls. frequency and severity of osteoarthritic lesions were the same in all 3 groups. osteoarthritic and normal articular cartilage from estradiol-treated animals revealed statistically significant reduction in s6s04 incorporation as compared to untreated animals. androgens had no significant effect on 36s04 incorporation. femoral s6s0, incorporation was uniformly greater than tibial 90, incorporation in all groups (p < 0.05). estradiol did not ameliorate nor testosterone worsen oa. both normal and osteoarthritic articular cartilage were susceptible to estradiol suppression of proteoglycan synthesis. the poor correlation between severity of oa and rate of pg synthesis may require reevaluation of the role of the latter in the oa disease process. variations in cartilage metabolism from different surfaces (femoral versus tibial) may relate to known differences in susceptibility of joints to development of oa. we have been analyzing antibody activities in sera from individuals working in various laboratories and in sera from a normal population. compared to the normal sample, the prevalence of elevated anti-dna activity was significantly greater in samples of sera from over 40 personnel in systemic lupus erythematosus (sle) laboratories (p < 0.001), from laboratories involved with nucleic acids (p < 0.001), and from routine hospital laboratory personnel (p < 0.01). evidence that this anti-dna activity was due to gammaglobulin was obtained by the presence of y -d n a bound to igg in a radioautograph. in addition, differences were found in the prevalence of dna reactivity between the normal sample and the samples from the three laboratory groups when anti-human gammaglobulin was used as the precipitating agent. also, we have isolated the material from a serum containing high dna reactivity by a dna-cellulose affinity technique, and the fraction with dna binding activity contained only igg. lymphocytotoxic activity was also increased in the sera from sle laboratory personnel compared to the other laboratories and to the normal sample (p < 0.0005), as previously reported (xiv international congress of rheumatology, abstract p. 131, 1977). immunoglobulin levels were analyzed by immunofluorescence. there was no significant difference in the igg and igm levels, but the mean iga level of the sle laboratory personnel was 2.27 f 0.96 mg/ml compared to 1.42 * 0.76 mg/ml in the normal sample ( p < 0.001). when sera from 17 of the laboratory personnel with the highest anti-dna activities were compared to 17 normal sera, additional abnormalities were found. not only was there a significant difference in the anti-dna activity (p < 0.001) and the iga levels ( p < 0.001), but also the mean igm level in the laboratory personnel (1.93 f 0.58 mg/ml) was significantly greater than in the normal sera (1.01 f 0.75 mg/ml)(p < 0.001). no difference in the mean igg levels was found. of 5 additional specific antibody activities quantitated by radioimmunoassay, one of these (anti-bovine gammaglobulin) was elevated more frequently in the laboratory personnel than in the normals (p < 0.02). the data suggest that laboratory personnel tend to have an increased immune reactivity, particularly those working with sle sera. this condition might be due to laboratory exposure to a stimulus, causing an immune response that includes autoantibody production. in most cases, amyloid arthropathy (aa) has been associated with multiple myeloma or demonstrable paraproteinemia. various modes of presentation and laboratory features have been described. we have analyzed clinical, laboratory, and radiographic features of 5 patients with aa seen over a 2-year period. none of these patients had identifiable diseases known to cause secondary amyloidosis. presenting symptoms were those of carpal tunnel syndrome (3: 5) and/or swollen hands with stiffness (4: 5 ) . periarticular tenderness of the hands and thickened palmar tendons were noted. pitting edema of the hands, at times massive, was noted in 4 of 5. knee and elbow effusions were present in 2 patients. sedimen-tation rates, rheumatoid factor, and antinuclear antibodies were normal or negative. serum and urine protein electrophoresis and immunoelectrophoresis failed to detect any paraprotein. joint fluids (4) were noninflammatory except for one aspirated during an acute attack of pseudogout. radiographs showed degenerative changes (3: 5) and chondrocalcinosis (4: 5 ) . synovium obtained by open biopsy (5: 5) revealed deposits of material which displayed metachromasia after crystal violet staining. in 3 cases, electron microscopy was performed and revealed typical fibrils in synovium. deposits were localized to perivascular and subsynovial locations. attention is called to the presentation of aa as edema of the hands with or without median nerve compression. edematous hands and seronegative arthritis should raise the possibility of aa. these patients are also unusual in that aa was not associated with dysproteinemia and absence of a paraprotein should not discourage invasive measures to document aa. the associated chondrocalcinosis has been previously recognized in only one case report. chondrocalcinosis in 4 of 5 patients may be a chance occurrence in an elderly population (age range 67-90) or may cause aa by chronic local inflammation. antimalarial therapy for connective tissue disease has been limited by its potential retinal toxicity. the present study was undertaken to assess visual problems in patients treated with long-term hydroxychloroquine given in standard dosage. all patients who received both treatment and ophthalmologic evaluation a t albany medical college were included and consisted of 99 patients treated for a t least 1 year. each patient was examined a t baseline and then every 6 months for visual acuity, central fields using a red test object, funduscopic abnormalities, accommodation, corneal deposits using a slit lamp, and keratoconjunctivitis by shirmer test. electro-oculograms (eog) were performed in 14 patients who had received high total doses or had abnormal central fields suggesting toxicity. almost all patients received hydroxychloroquine in the maximal daily dose of 400 mg. total dose ranged from 146 to 927 g (median 365) and duration of treatment from 13 to 68 months (median 33). diseases treated were rheumatoid arthritis in 58 patients, systemic lupus erythematosus in 31, juvenile rheumatoid arthritis in 4, mixed connective tissue disease in 2, and other in 4. ophthalmologic toxicity was minimal. no patients were precluded from taking hydroxychloroquine at baseline evaluation. no corneal deposits or accommodation defects were found. three patients had abnormalities in central fields: paracentral scotomata and/or minor field restrictions. all 3 had rheumatoid arthritis. toxicity occurred after total doses of 73, 206, and 316 g. visual changes were completely reversible in the first patient who was the only one in the series with funduscopic changes. she has now received a total dose of 535 g and has normal fundi, central fields, and eog. the second patient continues to have central field restriction but has had resolution of paracentral scotomata, a normal eog, and no visual complaints. the third had reversal of central field defects but had an abnormal eog when subsequently tested. we conclude that hydroxychloroquine in a dosage of 400 mg/day is safe from significant ophthalmologic toxicity if followed by appropriate testing, and we find n o evidence from an association of increased toxicity with higher total dose or with the diagnosis of systemic lupus erythematosus. the metal-dependent neutral proteoglycanase, extracted from 1,200 grams of human articular cartilage, occurred in four electrophoretic forms. these were purified 2,000-fold and produced single proteolytically active bands on disk electrophoresis. these forms were separated by preparative flat-bed isoelectric focusing and had approximate isoelectric points of 8.7, 8.3, 7.6, and 7.1. gel filtration and sds gel electrophoresis showed that they have an apparent molecular weight of 25-27,ooo and are composed of subunits of 13-14,000. gel filtration and dialysis indicated their tendency to disaggregate and reaggregate into monomers and dimers. their proteoglycanase activity passed through visking tubing and the passage continued on repeated dialysis with fresh buffer. this was prevented to a large extent by dialysis against zinc or cobalt ions. all the forms degraded the protein core of proteoglycan subunit optimally at ph 7.25 and were inhibited almost completely by addition of o-phenanthroline or passage through the chelating resin, chelex. however, they differed in their inhibition by edta, the most cationic forms being the least inhibited. also, the 3 most cationic forms had n o activity on casein, histone, and the link proteins, indicating a relative specificity for degrading proteoglycan. these enzyme forms which are active at the ph in cartilage matrix may have an important role in proteoglycan degradation in osteoarthritis. bxsb mice (developed by dr. e. d. murphy, jackson laboratory) spontaneously evolve a lupus-like disorder similar to the disease of nzb/nzw (b/w) mice. however, in contrast to b/w mice, the bxsb disease is more severe in males who die of immune complex nephritis at a mean age of 5 months. we have compared the formation of antibodies to dna in bxsb and b/w mice. the early appearance of autoimmunity in male bxsb mice is associated with a premature switch from igm to igg serum antibodies to dna. this is analogous t o the early switch from igm to igg antibodies in female b/w mice which is associated with accelerated disease and impending death. we have also measured the spontaneous synthesis of antibodies to dna by spleen cells cultured for 96 hours. culture supernatants contain immunoglobulin which binds dna specifically as determined by radioimmunoprecipitation. two month old male bxsb mice produce more antibody to d n a in culture than d o age-matched female mice of bxsb, b/w, balb/c, or c57 b1/6 strains. anti-dna production in culture increases with age in b/w mice. older female b/w mice subjected to prepubertal castration and treated with androgen produce significantly less anti-dna in culture as compared to sham or estrogen-treated controls (p < 0.017). the effect of hormone treatment on bxsb mice is currently under investigation. these results suggest that the male-dominant disease of bxsb mice is reflected in early synthesis of antibodies t o d n a which is apparent both in vivo and in vitro. female 43 the rheumatoid nodule : clinical significance although the subcutaneous nodule (scn) is the extraarticular hallmark of rheumatoid arthritis (ra), its clinical significance has not been established. it was our thesis that the presence of scn would serve to identify the subset(s) of rheumatoid patients with systemic and highly immunoreactive disease. all patients with definite (46) and classic (165) rheumatoid arthritis who had an initial unit admission during the interval january 1974 through june 1976 were selected for study. primary reasons for admission included active synovitis (57%), orthopedic surgery (17%), and a miscellany (26%) of therapeutic complications and intercurrent illness. of the 21 1 patients, 91 (43%) had scn on admission or by documented history. medical record analysis per protocol revealed patients with and without scn were comparable in terms of demography, duration, and activity of ra. similarly, systemic features did not differ significantly between two groups, as shown in the table. with regard to sero-reactivity, patients with scn and the anodular group were also alike in terms of the presence of rheumatoid factors (97% versus 85%), antinuclear factors (61% versus 42%), and hypocomplementemia (13% versus 10%). there was a tendency only for those with scn to have higher latex test titers. thus, contrary to expectations, subcutaneous nodules cannot be relied upon to screen out those patients with rheumatoid variants and systemic disease. twenty-four patients (18 women, 6 men, mean age 54 150 mg daily, versus placebo for 6 months, while continuing years) with definite rheumatoid arthritis were entered into a baseline non-steroidal antiinflammatory therapy and/or preddouble-blind study using the immunomodulator levamisole, nisone up to 10 mg/day. assessment criteria included articular index, grip strength, 50 feet walking time, duration of morning stiffness, subjective pain relief, sedimentation rate, and latex fixation titers. to date, 21 patients have completed the initial phase. eight o f 9 on levamisole improved while the remaining patient, who did not appear to respond, flared after drug discontinuation. two patients on levamisole were excluded because of skin rash. four of 10 on placebo appeared to improve but not to the degree seen with levamisole. the mean changes in articular index, number of swollen joints, and duration of morning stiffness were statistically significant a t 6 months for levamisole treated patients but not for placebo (p < 0.02). subjective pain scale responses were significantly better among levamisole versus placebo, but n o differences were observed between the groups in grip strength or 50 feet walking time. five levamisole treated patients converted to seronegativity after 6 months. sedimentation rates, however, remained stable in both groups: skin rashes developed in 5 patients on levamisole, and 2 were discontinued; in 2, rash was unrelated, and 1 continued on a lower dose. of interest, 3 of 10 on placebo had rashes. preliminary investigations of delayed skin hypersensitivity, lymphocyte responsiveness, phagocytic function, and cutaneous inflammatory responses failed to show any correlation with clinical responsiveness. levamisole is a potentially therapeutic agent in rheumatoid arthritis, but its mode of action is yet to be determined. since synovial fluid cells in patients with systemic lupus erythematosus (sle) have not previously been examined by electron microscope, we have studied 17 sle joint effusions with emphasis on the occurrence of le cells and tubuloreticular structures (trs) and on correlations with clinical and light microscopic findings. all patients fulfilled at least 4 ara preliminary criteria for sle and had antinuclear antibodies in their serum. three patients had drug-induced sle. synovial fluid volumes varied from 0.75-25 cc; leukocyte counts ranged from 875-58,000 but only 3 were over 15,000. polymorphonuclear leukocytes predominated in 3 effusions including the one with 58,000 cells. cultures were all negative. monocytes and large sudan positive macrophages were prominent. joint fluid le cells were found in 8 patients while 12 had extracellular hematoxylin bodies, and 4 had a variety of other smaller eosinophilic, hematoxyphilic, or cellular inclusions. le cells were identified by electron microscope in 10 patients. the major and often sole visible constituent of the inclusion was a clump of short filaments about 200 nm in diameter. these lay in vacuoles which also contained acid phosphatase. these filaments which appear to be products of nuclear chromatin also were seen extracellularly surrounding some cells and in smaller vacuoles. small acid phosphatase positive vacuoles also contained cell cytoplasmic debris, intact nucleii, erythrocyte fragments, and large amounts of finely granular protein-like material. the finely granular material was shown t o contain igg by immunoperoxidase electron microscope staining. t r s were found in 8 synovial fluids and were predominantly in mononuclear cells with dense bodies and rough endoplasmic reticulum. they were infrequent in small lymphocytes or transformed lymphocytes with polyribosomes. two l e cells had trs. buffy coat blood cells contained trs in the 2 cases studied. synovial fluid cell trs were seen in 2 of 130 inflammatory and noninflammatory synovial fluid controls but no control joint fluids had the filamentous le inclusions seen by electron microscope. n o correlation of any synovial fluid finding with therapy, effusion duration, or disease severity has been found. synovial fluids in sle contain trs, many phagocytic cells, and can have higher leukocyte counts and pmn percentages than often appreciated. le inclusions in joint fluid are composed predominantly of filaments derived from nuclear chromatin. the sera of 81 patients fulfilling the ara criteria for systemic lupus erythematosus (sle) were studied by immunodiffusion and the modified farr technique for the presence of antibodies to the following antigens: ss-a, ss-b, rana (rap antigen), rnp, sm, scl-1, and ds-dna. the incidence of antibodies to ss-b, rnp, sm, and ds-dna was similar to those previously reported. the incidence of anti-scl-l antibody was 0%, a finding which has not been reported pre-viously. the incidence of r a p (23%) was higher than the 7% previously reported. the incidence of anti-ss-a antibody was 26%. this antibody was found in high frequency in primary sjbgren's syndrome (70%) in the past but was not found in the small number of sle patients studied at that time. in this study where a large number of sle patients were studied and anti-ss-a antibody was present, only 48% had any evidence of keratoconjunctivitis sicca based on schirmer's tests. this indicates that not only is anti-ss-a an antibody "marker" for primary sjogren's syndrome, but it is relatively common in sle as well and furthermore, can exist in sle in the absence of keratoconjunctivitis sicca. serial studies were also performed on some patients. titers of anti-ss-a antibody were determined. these varied from neat to 2048 and correlated with disease activity. anti-ss-a often paralleled anti-ds-dna levels; however, anti-ss-a in some cases was an earlier predictor of clinical flares. thus, anti-ss-a antibody may be helpful not only in the management of sle patients, but with further investigation, aid in determining the pathogenesis of this disease. the phenanthridine dye ethidium bromide (eb) intercalates with native double-stranded d n a (dsdna) resulting in an enhancement of fluorescence when assayed fluorometrically. contamination of dsdna preparations by singlestranded dna (ssdna) does not affect the assay because there is no fluorescence enhancement when ssdna is added to eb. igg purified by deae column chromatography from the sera of 10 normal volunteers: 19 systemic lupus erythematosus (sle) sera with d n a binding activity as measured by the millipore filter (mpf) radioimmunoassay, and 5 sle sera without d n a binding activity were tested for their ability to compete with eb for binding to dsdna. in 18 of 19 of the sle with mpf-dna binding activity, there was greater than 2090 inhibition of the fluorescence normally shown when eb binds dsdna. in contrast, 0 of 10 control igg preparations and 0 of 5 of the sle igg from non-dna binders had any such effect. furthermore, the decrease in fluorescence due to the inhibition of eb binding to dsdna was linear with in-creasing amounts of igg, thus allowing direct quantitation of anti-dna activity. since the binding constant of eb is known to be >2 x 10-'m, this assay measures high avidity antibodies and is not affected by non-specific, low-avidity d n a binding molecules. the specificity of anti-dna antibodies in the eb assay was shown when absorption with excess nucleoside monophosphates did not alter the inhibition of eb binding in unabsorbed samples. however, the synthetic polynucleotides poly da, poly dt, poly dc, poly da:dt, and poly dg:dc were able t o absorb dna binding activity. therefore, the eb assay has demonstrated several features which make it an important adjunct in studying the specificity and binding characteristics of anti-dna antibodies. in addition, the assay is inexpensive, is easily and quickly performed, and it does not require the use of radiolabeled substrates making it practical for further development in investigative and clinical use. degradation of collagen in tendon, bone, and cartilage by collagenase is a significant factor contributing to the morbidity of the inflammatory arthritides. although considerable information is available as to the effect of drugs and mediators of inflammation on collagenase production and activity, the mechanisms during aging in the collagen fibril causing increased resistance to collagenase degradation are poorly understood. in this study, the effect of collagen cross-linking on collagenolysis was measured with an in vitro model system consisting of purified chick calvarium collagen fibrils and lysyl oxidase, the cross-linking enzyme. chick calvatia collagen fibrils were cross-linked by incubation with lysyl oxidase for varying time. controls consisted of collagen incubated without enzyme or with lysyl oxidase plus /3-aminopropionitrile, an irreversible inhibitor. the fibrils were subsequently measured for nascent cross-link content or incubated with purified rheumatoid synovial collagenase, and the rate of collagenolysis was measured. after synthesis of approximately 0.1 schiff base cross-links per collagen molecule, a ten-fold resistance to collagenase digestion was observed. inhibition of collagenase by edta prevented digestion. synthesis of collagen fibrils with higher cross-link content resulted in further resistance. these results demonstrate that the increased resistance of collagen to collagenolysis observed during fibril maturation in vivo is due to synthesis of native, unreduced schiff base cross-links. the cross-link content at which resistance to degradation develops is significantly less than that which affects fibril tensile strength in vivo. this suggests that resistance to collagenase is the earliest physiological effect of cross-linking in vivo. the rate of cross-link synthesis may be a significant factor regulating the rate of net collagen deposition in normal and pathologic states. vascular abnormalities have been shown to play an important pathogenic role in progressive systemic sclerosis (pss), especially in the group of patients who develop rapidly progressive kidney disease. as part of a study of the kidney in pss, we have also performed comprehensive investigations of coagulation parameters in order to evaluate the possibilities that: 1) coagulopathy occurs in these patients; and 2) coagulation abnormalities may be related to the development of renal disease in pss. seven patients with pss and normal renal function, as judged by normal blood pressure, creatinine clearance, and absence of proteinuria, were enrolled in the study. coagulation parameters studied included prothrombin and partial thromboplastin times, fibrin split products, fibrin monomer, sonoclot@ (which reflects fibrin monomer formation in recalcified whole blood), thromboelastograph (which reflects fibrin polymer formation), and platelet aggregometry. in addition, each patient underwent percutaneous renal biopsy. patients with pss showed platelet hyperreactivity t o collagen but not to other agents (91% aggregation versus control of 80.6% p < 0.01). the following abnormalities, indicative of a hypercoagulable state, were found: increased rate of clotting by thromboelastograph in 5 patients; increased rate of clotting by sonoclot in 4 patients; and premature onset of clotting by thromboelastograph in 3 patients. the patient with the most hypercoagulable profile has subsequently developed mild proteinuria. five patients had renal biopsy findings on light microscopy suggestive of pss kidney disease, including vascular intimal fibrosis, sclerosis, and fibrinoid necrosis. four of these 5 were hypercoagulable by one or more tests; both patients with normal biopsies had normal coagulation profiles. these data suggest that: 1) platelet-collagen interactions may be abnormal in pss; 2) certain patients with pss manifest laboratory evidence of hypercoagulability; and 3) hypercoagulability appears to correlate with renal biopsy abnormalities in pss patients with normal renal function. normal human mononuclear cells spontaneously lyse in vitro targets derived from cell lines of tumor origin. the natural killing of k-562, a tumor cell line derived from a patient with chronic myelogenous leukemia, varies in patients with systemic lupus erythematosus (sle) (27% f 21%) and controls (35% f 20%). t o evaluate the possible role of serum factors, normal peripheral blood mononuclear cells were incubated with 12 sle sera, 10 normal control sera, or media alone in the natural killing assay. 0.5 x l(p ficoll-hypaque purified mononuclear cells after pretreatment with sera or media were incubated for 4 hours with 1 x 10' s1chromium ("cr) labeled k-562 cells. cytotoxicity was measured by the release of into the supernatant. the mean 61cr release in the presence of normal human sera was 96% f 4% of that with media alone. in the presence of sle sera, mean 61cr release was 36% & 24% of that with media alone. the magnitude of natural killing suppression by sle sera did not correlate with complement levels, ana titer, or d n a binding. the natural killing suppression by an individual sle serum did not correlate with its inhibition of the antibody dependent cell mediated cytotoxicity of chicken red cells. the factors in sle sera responsible for suppression of natural killings are non-dialyzable, precipitable with 50% (nh,),so,, and removable by absorption with anti-ig antibody. the factors are excluded by a g-200 column. the major portion of the suppressive activity was found in fractions of igm or greater density on sucrose gradients. these findings are compatible with factors in sle sera, most likely immune complexes or antibodies, capable of inhibiting the natural killing of tumor cell lines. these factors may prevent cell mediated cytotoxic destruction of malignant cells in vivo and may explain the increased association of sle and malignancy noted by canoso et af. (arthritis rheum 17:383, 1974 ). full evaluation of any hyperuricemic patient requires these problems can be largely avoided by assessing uric acid assessment of uric acid excretion, but the standard 24-hour excretion per 100 ml of glomerular filtrate. this simple, physiurine method presents many problems. ill-timed and inologically sound parameter is obtainable from single, untimed complete collections may combine with bacterial con-urine specimens by multiplying urinary uric acid by plasma tamination and crystal precipitation to cause significant errors. creatinine and dividing by urinary creatinine (all in mg/100 ml). to minimize possible diurnal effects on uric acid excretion, all samples were taken in the morning. to evaluate the precision of the method, 15 normal men collected two 24-hour urines. spot urine specimens with concurrent serum samples were also obtained on two separate mornings. uric acid was measured spectrophotometrically, and creatinine was measured by standard autoanalyzer technique. the coefficient of variation (sd/x) was 3 i% for paired, quantitative 24-hour urine uric acid determinations, while spot, mid-morning assessments of uric acid excretion per 100 ml of glomerular filtrate had a more satisfactory coefficient of 21%. since most laboratories do not employ the enzymatic spectrophotometric method, urine uric acid determinations were also performed by a colorimetric, autoanalyzer technique. these values correlated well (r = 0.90) with the more specific spectrophotometric findings, but were an average of 7% higher. samples from 23 normal, adult men, the mean urinary excretion of uric acid in mid-morning was 0.405 f 0.098 mg/ 100 ml (sd), while 19 gouty men (studied between attacks and off hypouricemic drugs) had a mean of 0.679 f 0.423 mg/100 ml. included in the latter group were 5 significant overexcretors with values of 0.821, 0.905, 0.982, 1.45, and 2.01 mg of uric acid per 100 ml of glornerular filtrate. we believe that this assessment of mid-morning serum and urine samples effectively identifies overexcretors of uric acid. in addition, it is more convenient, more physiological, and more precise than the conventional 24-hour method. (supported by nih grant am13925.) in mixed connective tissue disease (mctd), a newly described rheumatic syndrome, no comprehensive histopathologic descriptions exist. we have followed 16 mctd children over a range of 1-1 1 years, (mean 6 years); 4 have died. three autopsies and 6 renal and 3 muscle biopsies were reviewed. among adults with mctd, tissues from 2 autopsies, and 18 kidney, 21 muscle, and 4 lung biopsies were reviewed. in children, proliferative vascular lesions, with intimal and medial thickening and luminal narrowing but without fibrinoid or inflammatory change, affected large vessels (aorta, coronary, renal), and small arterioles of many organs. inflammatory infiltration, predominantly plasmacytic, was marked in skeletal muscle 6:6, liver 3: 3, salivary glands 2: 2, intestine 3 : 3, and heart 3: 3. distinctive lesions of esophagus (atrophy of inner muscle layer), thymus (hyperplasia), kidney (membranous change), liver, lung, and salivary glands differed significantly from those expected in childhood systemic lupus erythematosus, polyarteritis, or scleroderma. many of these organs were not clinically involved during life. histologic estimates of numbers of t and b lymphocytes in spleen and lymph nodes, and degree of plasmacytosis (with hyperglobulinemia), differ from systemic lupus erythematosus and juvenile rheumatoid arthritis. in adults with mctd, muscle changes included: diffuse inflammatory infiltrate 15:21 (perior endomesial, and perior intravascular). by atp-ase, 5: i i had type i fiber predominance. on muscle immunofluorescence, 8: 10 had vascular, sarcolemmal basement membrane, or granular fiber staining, with igg or igm. in 20 kidneys there were included: mesangial proliferation 5, focal-local change 5 , membranous 3, membrano-proliferative i , proliferative vessels 1, normal 5 . lung tissue (6) revealed: vascular proliferation 2, vascular medial hypertrophy 2, and interstitial fibrosis 3. in mctd, children and adults have similar lesions: inflammatory lesions may predominate early, but can occur late. immunofluorescent data suggest an immune basis for injury; the late predominance of proliferative vascular lesions suggests that vascular sclerosis is a serious complication. the findings of many significant histologic lesions, without clinical signs or symptoms, suggest that features of this multi-system disease evolve slowly, and that the full spectrum of mctd is not yet known. with ra and 10 normal subjects. after isolation of the lymphocytes by isopycnic sedimentation, the cells were placed in tissue culture and half were infected with ebv. every 6 days for 1 month the supernatant fluid was removed from each culture and fresh medium was added. total igm and igm-rf secreted into the supernatant were measured by solid phase radioimmunoassay. independently the cells were examined for transformation and numerically graded. all infected cultures produced igm-rf, which correlated with a high grade of transformation. the amount of rheumatoid factor (rf) produced by ebv infected normal, but not rheumatoid, lympho the incubation of pmns with a chemotactic factor (cf), in the absence of a gradient, prevents the cells from responding with directional migration when, after washing, they are challenged with a gradient of the same or a different chemotactic factor (deactivation and cross-deactivation). structurally unrelated cfs, some shown to bind to distinct cell receptors, can cross-deactivate, which suggests that deactivation is not due to receptor blockade. we propose that deactivation is the result of generalized microtubule assembly induced by c f incubated with the cells in the absence of a gradient, thus rendering the cells incapable of responding to a c f gradient with distinctive localized assembly, a proposed requirement of normal chemotaxis. if this scheme is correct, the simultaneous preincubation of pmns with suitable concentrations of colchicine, a microtubule disrupting agent, and a c f should protect the cell against deactivation and colchicine-induced suppression of chemotaxis. human neutrophils were preincubated, 20' at 37oc, with colchicine to 10-'m); the chemotactic factors gly-his-gly (iopg) or crystalinduced chemotactic factor (ccf) 25pg alone; colchicine and either chemotactis factor; or hanks. cells were washed and tested for chemotactic response against the c f using a radioassay that utilizes y r labeled neutrophils. preincubation of cells with either chemotactic factor or colchicine alone resulted in a dose-dependent inhibition of chemotaxis. when cells were preincubated with both chemotactic factor (gly-his-gly iopg or ccf 20pg) and suitable concentrations of colchicine ( or io-o), a reversal of the inhibition of chemotaxis was noted. deactivation reappeared when the balanced ratio between colchicine and c f was altered. preincubation of c c f with colchicine had no direct effect on its chemotactic activity, and colchicine ( 10-6m) did not alter the specific binding of radiolabeled c c f to neutrophils. additionally, both c c f and gly-his-gly induced microtubule assembly by electron microscopy. functional evidence is presented with two distinct chemotactic factors, which suggests that the basis for deactivation is overpolymerization of microtubules that prevents the pmns from responding to a chemotactic gradient with directional migration. fever is a common occurrence and frequent reason for hospitalization of patients with systemic lupus erythematosus (sle). to assess the frequency, causes, and clinical and laboratory characteristics of febrile episodes in hospitalized patients with sle, the medical records of 568 admissions of 160 sle patients during the 5 year interval 1973 to 1977 were reviewed. eighty-three febrile episodes, defined as a n oral temperature 238oc and one blood culture drawn for evaluation of fever, occurred in 63 patients. the febrile episodes were classified: group a-clinically active sle only (50). group b-documented infection (l9), and group c-miscellaneous (14). clinically active sle accompanied 6 episodes in b and 2 in c. treatment a t the time fever developed included steroids in 64,84, and 100% and cytotoxic agents in 16,32, and 50% of episodes in a, b, and c, respectively. infectious causes of fever were bacterial septicemia (8), localized bacterial infections (7), herpes zoster (3), and miliary tuberculosis (1). miscellaneous causes were procedure-related fever (4). drug fever (3), addisonian crisis (i), myocardial infarction (i), and unidentified (5). the initial clinical impression was correct in 45 of 50 infectious episodes. two episodes of bacterial septicemia were unexpected; one occurred in a patient with concommitantly active sle. the clinical impression was correct in 47 of 51 episodes in a; infection (3), acute appendicitis (i), and drug fever (1 ) were suspected in the others. comparing a and b, patients' age, disease duration, and fever patterns were similar. shaking chills were more frequent with infection (p < 0.001) but were present in 28% of a. laboratory studies helpful in identifying patients with infection were leukocytosis > 12 x ip/mm (p < 0.001), neutrophilia >8 x ip/mm (p < 0.001) and normal dna binding (p < 0,001). four deaths occurred despite appropriate therapy: 1 in a with necrotizing lupus pneumonitis, and 3 in b with gram negative sepsis, 2 of whom had active sle. in summary, infection accounted for 23% of febrile episodes in hospitalized patients with sle. bacterial septicemia was found in 11% and was associated with a high mortality. the initial clinical impression was usually correct and the wbc count, absolute neutrophil count, and dna binding were helpful in distinguishing between infection and active sle. renal involvement in progressive systemic sclerosis (pss) is the most devastating form of the disease with one year mortality close to 100% unless hemodialysis or transplantation is successful. at present, there are no reliable predictive factors which allow us to separate those who will eventually develop renal disease from those who will not. because of this, we have studied 9 patients with pss who were normotensive and had normal renal function (mean serum creatinine 0.89 mg%, mean creatinine clearance 88 cc/min, mean protein excretion 55 mg/ 24 hours) and normal urinalysis. studies included percutaneous renal biopsies, evaluation of the renin-angiotensin system, and a cold pressor test to try to determine renal vascular reactivity. five of 9 renal biopsies demonstrated distinct vascular lesions on light microscopy with intimal fibrosis in 4, hyaline intimal sclerosis in 3, and arteriolar fibrinoid necrosis in 1. electron microscopy of the vessels showed changes similar to those on light microscopy. in addition, wrinkling of the glomerular basement membrane (gbm) with expansion of the mesangium by gbm-like material was seen in 8 of 9 biopsies. although nonspecific, these changes are suggestive of ischemia in the kidney and are compatible with the vascular changes of pss. immunofluorescence studies revealed c3 in vessels in all 9 biopsies. plasma renin activity (ppa) was performed in 8 of 9 patients. of the 5 patients with abnormal biopsies, 4 had elevated plasma renins (9.31 ng/ml/hr) at the time of biopsy and the fifth patient has subsequently developed significant elevation. the 3 patients with normal biopsies had normal plasma renins (2.38 ng/ml/hr). cold pressor testing with pra determination resulted in a mean maximal increase of 6.52 ng/ ml/hr in those with abnormal biopsies, 0.4 ng/ml/hr in those with normal biopsies, and 0.19 ng/ml/hr in 6 control subjects. these findings indicate that renal histologic vascular involvement may precede the onset of clinical renal disease in pss and correlates well with pra elevations and increased responsiveness of the renal vasculature. followup of these patients will determine the clinical significance of these findings. ninety-six patients with classic or definite rheumatoid arthritis were consecutively screened for the presence of early ocular manifestation of disease. each patient was followed for an average of 18.7 months and received an average of 4 ophthalmologic exams at an average of 6 month intervals. none of the patients demonstrated ocular lesions definitely attributable to rheumatoid arthritis. eighteen patients developed new ocular pathology consisting of chronic blepharitis, chronic iritis, corneal subepithelial defects, keratitic precipitates, keratitis, corneal abrasion, and abnormal tear film. twenty-one patients of this group and 15 normal adult controls were screened for keratoconjunctivitis sicca. slit lamp examination, rose bengal staining, shirmer's testing, and tear lysozyme concentration were measured in each patient. tear lysozyme concentration was indirectly determined spectrophotometrically by measur-ing lysis of the dried cell walls of micrococcus lysodeikticus. patients with rheumatoid arthritis demonstrated abnormal rose bengal staining (rheumatoid arthritis, 5 patients; control, none), significant decreased tear production (rheumatoid arthritis, 14.5 f 1.9 mm wetting shirmer strip; control, 24.0 f 2.6 mm wetting schirmer strip) p < 0.01, and markedly increased tear lysozyme concentration (rheumatoid arthritis, 41.0 f 4.4 pg/ml; controls 16.9 f 3.8 pg/ml) p < 0.01. thus, unlike patients with severe sjagrens syndrome who have low tear lysozyme concentrations, patients with rheumatoid arthritis demonstrate significant increases in tear lysozyme levels. this new finding, as yet unexplained, may represent the earliest lesion in the development of keratoconjunctivitis sicca in patients with rheumatoid arthritis. nzb/nzw mice develop autoimmune disease similar t o that of humans with lupus systemic erythematosus. treatment with cyclophosphamide (cp) protects against the disease but often is associated with a high incidence of tumors. tumors resulting from direct chemical carcinogenic activity increase with rising cumulative dose and time after exposure and are of diverse types. immunosuppression itself is reported to facilitate tumor growth or, perhaps, oncogene expression and has been primarily associated with sustained immunosuppression, predominantly with tumors of reticuloendothelial (re) origin. we compared tumor development in nzb/nzw mice given different cp regimens (see table) . reticuloendothelial tumors increased with daily dose but did not correlate with cumulative dose or duration of treatment. smaller numbers of other (non-re) types of neoplasms occurred in all groups. our results 1) suggest that re tumor development in autoimmune disease is more dependent on continuous, high doses of cytotoxic drug than on cumulative dose or duration of treatment and 2) are compatible with the possibility that re tumors result from immunosuppression or oncogene expression. no. the success of colchicine in the treatment of acute gouty arthritis is well established. however, the usefulness of this drug in the therapy of the pseudogout syndrome has been less enthusiastically regarded. in general, assessments of such therapy refer to lower success rates and somewhat inconsistent results. nevertheless the use of other antiinflammatory drugs to treat acute pseudogout is sometimes militated against by the presence of associated conditions commonly seen in the age group of these patients, including congestive heart failure, gastrointestinal disease, or neurological deficits. we have treated 7 consecutive patients with the acute arthritis of pseudogout with a standard regimen of colchicine by the intravenous (iv) route. there were 4 males and 3 females, ages 56 to 88, and all presented with acute mono-or oligoarticular arthritis. in all cases typical calcium pyrophosphate dihydrate (cppd) crystals were identified by red-compensated polarized light microscopy of the synovial fluid. none of the patients had elevated serum uric acid, except 1 which was on diuretics, and synovial urate crystals were absent. synovial fluid white counts varied from 2600 to 54000/ mma with 70-88% polymorphonuclear leukocytes; 4 of the 7 had radiologic evidence of chondrocalcinosis. all patients were treated within 54 hours of the onset of the acute arthritis. colchicine was usually given at a dose of 2.0 mgm iv over a period of 20 minutes followed by 0.5 mg iv every 6 hours for the next 24-48 hours. total iv colchicine dosage varied from 4 to 7.5 mgm. an excellent response occurred in 24-36 hours in 5 of 7 patients and in 37-48 hours in the other 2, with complete resolution of inflammation in all 7. we conclude that 1) iv colchicine may provide effective and consistent therapy for the acute arthritis of pseudogout; and 2) because of this, prompt response of an acute arthritis to iv colchicine remains an insufficient clinical criterion for the diagnosis of gout. the high pyrophosphate (ppi) concentrations observed in osteoarthritis (oa) and chondrocalcinosis (cc) synovial fluid (arthritis rheum 16:171, 1973) suggest that ppi may be a precursory ion involved in human articular mineral formation, whether in midzone sites in cc-cappi deposition-or in "tidemark" cartilage in oa. why predominantly hydroxyapatite and often some cappi (bjelle, personal communication) are seen in oa, and cappi is deposited in cc, might hypothetically depend in part on moderate and severe ppi hydrolase(s) deficiencies. such deficiencies are postulated to be associated with the calcifying subcellular apparatus in oa and cc articular cartilage, respectively. this postulated ppi deficiency would be relative to normal calcifying growth cartilage in which ppi in mineral is present in only trace amounts (wuthier et al., calc tiss res 10198, 1972). to examine this hypothesis, articular cartilage from 8 patients with oa (4 male, 4 female, age 49-74), 6 with cc (4 male, 2 female, age 60-79), and 4 normal controls was assessed for various phosphohydrolase activities. cartilage was sliced in a cryostat, homogenized, and extracted in triton x-100. the extract was centrifuged and the supernatant, once dialyzed, was passed through a de-52 column and enzyme fractions were eluted according to prevjously described techniques. although total protein and dna content were about equal from all cartilage samples, alkaline phosphatase activity was found in the following ratios: normal: cc: oa = i : 6: 60. in all samples, 2 peaks of alkaline phosphatase activity were eluted similar to the findings of arsenis et al. in calf growth cartilage (calc tiss res 20159, 1976). uniquely, with cc some of the total activity (14%) was found in the void volume of both peaks. characterization of alkaline phosphatase using various metals, inhibitors, and substrates was similar for all samples tested. however, analysis of the ratios of ppiase: alkaline phosphatase for growth cartilage (arsenis, above) versus oa and cc samples ranged from 6: 1 to 3: 1 respectively. in conclusion, the high ppiase to alkaline phosphatase ratio in growth cartilage compared to this ratio in cc and oa supports the view that reduced ppiase activity might play a role in 1) the probable elaboration of ppi into synovial fluid in oa and cc; 2) the in vitro elaboration from articular cartilage of ppi into eagle's medium in patients with oa and cc (j clin invest 56:1473, 1975); and 3) provision of ppi ion for variable cappi crystal deposition. despite the prominence of neuropsychiatric features in systemic lupus erythematosus (sle), no satisfactory method exists for the diagnosis and monitoring of central nervous system (cns) involvement. this study describes the application of a new technique for studying both cerebral metabolism and blood flow in sle patients by using inhaled molecular 15-oxygen and 15-oxygen labeled carbon dioxide. tracer amounts of 15-oxygen are inhaled, and after a period of equilibration, the brain is scanned with a gammacamera. the image produced represents cerebral metabolism. the procedure is repeated using carbon 15-dioxide, and the resultant image represents cerebral blood flow. twenty-eight scans were performed on 24 sle patients who had been classified as having clinically definite cns disease (13), clinically probable cns disease (7), or no clinical evidence of cns disease (8). the scans, which were reported blind, were classified as normal (showing a full pattern of both cerebral blood flow and metabolism), as showing a major abnormality, or as showing a minor abnormality. the results are shown in the table. scan abnormalities were seen in 25 of the 28 studies, usually afecting several cortical areas. in 6 patients in whom multiple recordings were made, improved 15-oxygen scan appearances correlated with clinical improvement. 15-oxygen brain scanning appears t o offer a highly sensitive non-invasive technique for the identification and study of cerebral involvement in sle. neutrophils stimulated at sites of inflammation release lysosomal enzymes. experimentally, a variety of agents are capable of stimulating neutrophils to release their lysosomal enzymes. neutrophil membranes contain fc receptors for igg, and thereby possess surface bound igg. in these studies we examined the hypothesis that specific antibodies could combine with surface bound igg, perturb the cell membrane, and provoke inflammatory responses of neutrophils. antisera were raised to immunoglobulins g , m, a, d, e, and isolated y chains, fc and f(ab'), of igg. these were rendered monospecific by solid phase immunoabsorbents. isolated normal human peripheral blood neutrophils were incubated with specific antisera and the release of lysosomal /3-glucuronidase, a-mannosidase, and lysozyme were measured. non-lethal release of lysosomal enzymes was observed with antisera to whole igg, iga and t o y and fc of igg. the igg fraction of anti-igg antiserum also stimulated non-lethal release, thereby suggesting that the response was not complement-mediated. specific igg and iga receptors were detected on neutrophils using immunofluorescent labeled antibodies. n o such receptors were detected for igm and igd. these results indicate that inflammatory responses of neutrophils can be triggered directly by anti-immunoglobulins, and suggest one mechanism for initiation of the inflammatory response in patients with diseases characterized by the presence of antibodies to igg and other immunoglobulins. incubation of polymorphonuclear leukocytes (pmn) with immune complexes trapped in micropore or collagen membranes results in the phenomenon of "frustrated phagocytosis," that is, enhanced release of lysosomal hydrolases. w e have previously shown immune complexes irreversibly trapped in joint collagenous tissues in antigen-induced rabbit experimental arthritis and in rheumatoid arthritis. the present experiments were designed t o investigate in vitro interactions between pmn and joint collagenous tissues obtained from rabbit joints with experimental arthritis or control tissues from saline or monosodium urate crystal-injected joints. experimental and control articular cartilage samples and menisci obtained from such animals were incubated for 1 hour with normal pmn isolated from rabbit peritoneal exudates or blood. after fixation, the tissues were examined by electron microscopy. fresh cartilage and menisci from arthritic joints showed only few damaged pmn near the articular surface. after incubation with pmn, large numbers of pmn attached to the articular surface were seen. in areas of superficial erosion, the pmn invaded the tissue several cell diameters below the lamina splendens. degranulated pmn were observed with immunoelectron microscopy in scattered areas to phagocytose amorphous material containing rabbit ig. following addition of pmn t o control tissues, only a few pmn became attached to the articular surface. in monosodium urate injected joints, after incubation with pmn, these cells were found attached to the surface in moderate numbers, but no invasion into the tissues was seen. these studies indicate that immune complexes trapped in joint collagenous tissues may induce the phenomenon of "frustrated phagocytosis" leading to enhanced release of lysosomal hydrolases. similar studies using rheumatoid joint tissues are in progress. (suppported by nih grant am16209.) half of the girls. intervals between onset of symptoms and diagnosis ranged between 1 month and 10 years (median 3 months). twenty-eight children were white, 5 black, 6 native american, 4 asian, and 2 of other races. most frequent presenting findings were fever and malaise, musculoskeletal complaints, skin rash, and renal disease. unusual presentations included cholecystitis, isolated nephrotic syndrome, and chorea. three patients presented with isolated thrombocytopenia. serious pulmonary manifestations occurred in 5 patients; 2 died within 2 months of disease onset. clinical evidence of nephritis occurred in 9 of 1 1 boys and 25 of 34 girls. thirty-six patients had 1 or more renal biopsies. three patients with normal urinalyses had histologic nephritis by biopsy. therapy consisted of prednisone (41 of 45 patients) and either azathioprine or chlorambucil (21 of 45 patients). since 1969 therapy has been geared to normalizing serum hemolytic complement values as well as controlling clinical manifestations of disease. chronic renal failure developed in 4 patients; only 1 had had adequate therapy early in disease. one renal failure patient received a renal transplant, and 2 are on hemodialysis. six girls and 3 boys have died (209'0). seven patients had active lupus at time of death with severe uncontrolled multisystem lupus (3), pulmonary lupus (2), central nervous system lupus (l), and renal failure (1). one patient with active sle also had a myocardial infarction (age 7) as a contributing cause of death. two patients died while in clinical remission, 1 of a myocardial infarction (age 20) and 1 of clostridial sepsis with hemolysis. infection played a major role in 4 deaths (fungal brain abscess, acute staphylococcal endocarditis, disseminated herpes infection, clostridial sepsis). thirty-three patients continue to have active sle requiring treatment; 2 are in remission and are not on medications; i patient was unavailable for followup in 1978. in patients with systemic lupus erythematosus (sle), abnormal unfractionated lymphocyte responses to phytohemagglutinin (pha) can be corrected by removal of adherent mononuclear (m) cells (arthritis rheum 20127, 1977) . the present study demonstrates that this abnormal response occurs when 1) autologous adherent mononuclear cells or 2) cell-free supernatant fluids from autologous and allogeneic adherent cells are added to t-cell cultures, and that 3) suppression does not occur when adherent cells are pre-treated with indomethacin. ficoll-hypaque (fh) separated peripheral blood mononuclear cells from normals and from patients with sle were further fractionated into t cells by passage over an antihuman f(ab), column (97% e-rosettes) and m cells by plating on glass (88% peroxidase-staining). cell number was constant in all experiments. supernatants from normal controls and from sle patients were obtained after 48 hour culture of fh, t, and m cells in rpmi containing ab serum. normal and sle t cells were tested for pha response in the presence and absence of autologous and allogeneic m cells or cell-free su-pernatants from fh, t, or m-cells. autologous but not allogeneic m cells suppress t cell responses. cell-free supernatants derived from m cells of 14 consecutive experiments with sle patients caused an average 44% suppression of normal t-cell response to pha (mean peak cpm relative to control). suppression was not due to media exhaustion. supernatants from normal m cells caused an average 35% suppression. supernatants from both normal and sle t cells did not suppress either autologous or allogeneic t-cell responses. when m cells of patients with sle were cultured in the presence of 1 pg indomethacin/ml, the obtained supernatant was not inhibitory. the data indicate that poor in vitro unfractionated lymphocyte response to pha in patients with sle is mediated by a soluble indomethacin-sensitive product derived from the adherent mononuclear cells, possibly prostaglandin. since supernatants from normal adherent cells were also inhibitory when m-cell concentration was held constant, it is likely that the difference between sle and normal lymphocyte responses is quantitative rather than qualitative. renal crisis in scleroderma, with malignant hypertension and rapidly progressive renal failure, is perhaps the most ominous of clinical syndromes in the rheumatic diseases. survival with medical management has not been reported, although a few patients have survived with renal dialysis or transplantation. we report 3 patients surviving scleroderma kidney with medical management alone for periods of 3,4, and 10 years. patient 1 with malignant hypertension in the fourth year of his classic systemic sclerosis was hospitalized with blood pressure of 220/ 140, generalized seizures, visual acuity decrease to virtual blindness, and striking grade 4 hypertensive retinopathy. creatinine rose to a high of 8.0. extraordinarily vigorous antihypertensive treatment with massive doses of 5 drugs reduced blood pressure to low normal ranges. renal function 4 years later is stable with a creatinine of 2.2 mg 8. patient 2 developed her malignant hypertension after 3 years of classic scleroderma, with blood pressure 200/130, grade 3 hypertensive retinopathy, generalized seizures, a creatinine of 2.9, and urine protein of 2 gms per 24 hours. plasma renin was 4400 ng/dl. aggressive antihypertensive treatment with 5 drugs reduced blood pressure to the low normal range, and over the following 3 years renal function has improved to a creatinine of 1.5. patient 3 developed malignant hypertension after several years of classic scleroderma. blood pressure was 250/150, creatinie rose to 2.8, hypertensive grade 4 eye changes were noted, and renal biopsy confirmed afferent arteriolar necrosis. hemorrhage following renal biopsy resulted in transient hypotension, with subsequent control of blood pressure in the low normal range with combination antihypertensive drug therapy. her renal function remains stable with a creatinine of 1.5 mg% after 10 years. skin lesions improved following hypertensive control in 2 of these 3 patients. the common denominator in these 3 successfully managed patients appeared to be aggressive and determined reduction of blood pressure to low normal ranges, with later reliance upon propranolol in 2 of the 3 patients. the percentage of patients who will respond to such management is unknown, but aggressive early treatment of hypertension appears indicated. the multi-system involvement and varied clinical presentation of systemic lupus erythematous (sle) make its treatment difficult and often controversial. to determine present clinical practices, 200 rheumatologists and nephrologists, selected randomly from the directory of medical specialists, were surveyed for their management practices for 8 hypothetical sle patients. responding physicians follow over 1900 patients with sle. patients were managed as follows: (1) joint and skin involvement only: ninety-four percent of respondents chose asa (12-16 tabs/day) with 9% also using hydroxycholoroquin. (2) early clinical nephritis: seventy-eight percent would initially treat with prednisone, usually at about 1 mg/ kg/day but with 25% choosing 0.5 mg/kg. (3) active nephritis with rising creatinine: ninety percent would initially employ prednisone, at doses approximating 1 mg/kg, and 21% would also use immunosuppressives. (4) serologicalfiare in an asymptomatic patient: forty-six percent would treat with prednisone and 51% would withhold treatment. (three percent demanded a renal biopsy). ( 5 ) central nervous system sle: uniformly treated with prednisone, at least 1 mg/kg, with 23% also employing immunosuppressants. (6) late stage arotemic in-active sle: fifty-nine percent elected to treat with prednisone. (7) prednisone taperingpractices: from a starting dose of 60 mg/day, physicians tapered to a mean of 33 mg/day after 3 months and 17 mg/day after 6 months. thirty-three percent used a divided dose initially, and 57% used alternate day schedules later. (8) flare while tapering: all physicians increased medication, with 87% increasing prednisone dose, 13% adding an immunosuppressant, and many doing both. rheumatologists and nephrologists were similar in treatment of most problems. however, nephrologists used immunosuppressive agents twice as frequently in early disease but treated late stage nephritis less vigorously than did rheumatologists. nephrologists also used fewer divided daily doses of prednisone and used alternate day schedule sooner and more frequently. treatment was highly case specific. substantive disagreement occurred when there was serological disease without clinical disease, and in treatment of end-stage nephritis. central nervous system episodes and early progressive nephritis were uniformly treated aggressively. immunosuppressive agents were employed only by a minority of respondents. antibody-dependent cell-mediated cytotoxicity (adcc) was studied in adult patients with rheumatoid arthritis (ra) and in healthy controls. three effector cell populations from the peripheral blood were studied. these included a mixed mononuclear population (8-12% latex positive), a monocyte-depleted fraction (less than 1% latex positive), and a monocyte enriched fraction (65-80% latex positive). the target cells were chicken erythrocytes coated with rabbit anti-chicken erythrocyte antibody (igg fraction); multiple effector: target ratios were studied. there was no significant difference in lymphocytotoxic antibody is found in the serum of patients with systemic lupus erythematosus (sle). the antibody is defined by its reaction with lymphocytes, but it also reacts with antigens on human brain tissue. the existence of an antibody in sle serum recognizing both brain and lymphocyte antigens prompted a search for a similar substance in the cerebrospinal fluid (csf) of sle patients, with and without central nervous system (cns) disease. csf was obtained from 17 patients with sle in the course of evaluations for fever, headache, or manifestations of cns dysfunction. an independent clinical analysis of the sle patients and their hospital records revealed that 10 of the 17 had cns dysfunction attributable to sle. criteria for cns disease were one or more of the following: seizures, transverse myelitis, chorea, ataxia, hemiparesis, psychosis, or hallucinations. multiple abnormalities were present in 5 of the 10 patients, with l patient having 3, and 4 patients having 2 of the above abnormalities. a population of patients without rheu-matic disease who were being evaluated for a variety of neurological abnormalities served a s controls. the csf was tested in a dye-exclusion microcytotoxicity assay using peripheral blood lymphocytes from 3 normal donors as targets. cytotoxicity was defined as greater than 15% killing of lymphocytes from 1 or more of the normal donors. nine of the 17 sle patients'had lymphocytotoxic activity in their csf compared to 1 of 31 controls. when lymphocytotoxicity of csf was compared with presence or absence of lupus cns disease, a positive correlation was found (x' = 3.82, p < 0.05). the mean % lymphocytotoxicity in the cnfs did not correlate with the serum titer of lymphocytotoxic antibody (r = 0.12). cytotoxic activity to human lymphocytes has been identified in the csf of patients with cns manifestations of sle. if the cytotoxicity is cross-reactive with neuronal antigens, as is the serum antibody, it may have a pathogenetic role in cns lupus. the role of bone marrow derived cells in determining genetic variation in susceptibility to experimentally induced amyloidosis in mice was investigated. mice of the amyloid susceptible cba strain were lethally irradiated and repopulated with marrow cells from the highly resistant a strain. lethally irradiated cba mice reconstituted with syngeneic marrow served as controls. to determine the effects of irradiation per se on amyloid production, normal cba mice, as well as a strain mice that were irradiated and reconstituted with a marrow, were also studied. following 4 weeks of daily subcutaneous casein injections, spleen sections stained with congo red were scored for the degree of amyloidosis by independent blind observers using the following 4 point scale: grade i-a rim of amyloid around 1 or more splenic follicles; grade 2-a rim of amyloid in more than 50% of splenic follicles; grade 3-a rim of amyloid in all splenic follicles; grade 4-diffuse splenic amyloid with bridging be-tween almost all follicles and some distortion of the splenic architecture. thirty percent of cba mice given a marrow failed to develop amyloid, with an average score of 1.1 for this entire group. in contrast, control mice given syngeneic marrow all developed significantly more amyloid, with an average score of 3.4. non-irradiated cba mice developed a n intermediate severity of lesions. irradiated a mice remained amyloid free. the progression of amyloidosis in cba mice was significantly retarded by grafting of marrow from the resistant a strain, even though irradiation seemed to accelerate amyloid production in cba mice. the resistance of the a strain to amyloidosis was not affected by lethal irradiation. these data demonstrate that bone marrow derived cells are an important factor in the pathogenesis of amyloidosis and that the genetic difference between susceptible and resistant mouse strains lies, at least partially, in their hematopoietic tissues. the biochemical features of 2 families with purine nucleoside phosphorylase (pnp) deficiency and t-cell immunodeficiency disease were compared. the erythrocyte enzyme from 2 brothers in family 1 had 0.45% normal pnp activity. plasma urate values in these patients are 2.6 mg/dl and plasma inosine levels are 41.2 and 45.2 p m . urine samples contain uric acid 454 and 527 mg/gm creatinine, inosine 5.1 and 4.7 millimoles/gm creatinine, and guanosine 1.4 and 1.8 milli-moles/gm creatinine. when compared t o normal, their enzymes showed a) a 10-fold increase in the apparent km for inosine, b ) a diminution of the isoelectric p h from normal values of 5.4 to 5.8 to 5.08 to 5.26, c) an inability of inosine to protect the mutant enzyme against thermal inactivation as compared to the protection of the normal enzyme, d ) a loss of the normal near optimum activity a t ph 7.4, and 3) a normal value for the stokes radius. the enzyme from a patient in family 2 has 0.07% of normal activity. this patient has a plasma urate of 1.8 mg/dl and a plasma inosine of 38 w m . urine samples contain uric acid 73 mg/gm creatinine, inosine 14.8 millimoles/gm creati-nine, and guanosine 4.8 millimoles/gm creatinine. the enzyme protein had a ) a 3 to 4-fold increase in the apparent km for inosine, and b ) only minor changes in enzyme activity over a ph range compared to normal. these observations suggest that a ) the degree of abnormality in uric acid and nucleoside concentrations in the plasma and urine reflect the severity of the enzymatic deficiency, b) structural alterations of the mutant pnp proteins result from structural gene mutations and genetic heterogeneity in the disease pnp deficiency. since pnp activity was found in every human tissue assayed, the systemic involvement of pnp deficiency can be accounted for. recently we reported the finding of an antibody, pm-i , directed toward one of the nuclear acidic protein antigens (napa), in sera of patients with polymyositis. this study was undertaken to investigate the relationship between pm-i antibody, polymyositis syndromes, and other rheumatic diseases. sera were tested against nuclear extracts by immunodiffusion and counterimmunoelectrophoresis for the presence of antibodies to pm-1 and other napa. clinical data were then obtained and analyzed. fifty-two patients were identified who had polymyositis syndromes as defined by significant muscle weakness, elevated serum muscle enzymes, myopathic electromyogram, and muscle biopsy typical of polymyositis. of these, 24 had polymyositis, 18 had polymyositis-scleroderma overlap, and 10 had derrnatomyositis. pm-i antibody was found in 50% of polymyositis, 73% of polymyositis-scleroderma, and 20% of dermatomyositis. polymyositis syndromes, a difference in the prevalence of the clinical characteristics was noted as shown in the table. blinded serum exchanges of 58 sera between 3 medical centers revealed the prevalence of the pm-i antibody to be 25% in childhood dermatomyositis (4 of 16), 17% in adult dermatomyositis (3 of 18). and 38% in polymyositis (3 of 8). patients with other rheumatic diseases were negative for pm-i antibody (0 of 16). we conclude that the pm-i antibody has a high specificity for polymyositis and that it may identify a subset of polymyositis patients who have other connective tissue disease manifestations. pm-i positive pm-i negative clinical characteristic n = 21 n = 25 the pm-1 antibody was found rarely ( < i % ) in lo00 patients other muscular disorders such as myasthenia gravis, muscular pulmonary disease dystrophy, and polymyalgia rheumatica. among patients with an association between viral infection and joint inflammation has repeatedly been reported. our recent studies showed that interferon inducer, double stranded polyinosinate-polycytidylate (poly l:c), as well as human interferon, stimulated both prostaglandin e (pge) and hyaluronic acid production by human cultured synovial fibroblasts. the present study was performed in order to evaluate further the role of interferon and poly i:c in joint inflammation. polynucleotides, mouse interferon, or phosphate buffered saline were injected intraarticularly in 10-week-old wistar derived male rats by methods previously described. animals were killed 24 hours later and the synovia were removed for histological examination and determination of pge. injection of poly i:c or mouse interferon induced an inflammatory response. poly a:u was only slightly active in this respect. by contrast, pbs or single stranded polyinosinate (poly (i)) or polycytidylate (poly (c)) did not induce any significant inflammatory response. rat knee joints injected with either poly i:c (50, 100, or 250 pg/joint) or interferon (500 or lo00 p/joint), appeared macroscopically swollen and edematous and contained an increased amount of viscous fluid. microscopically inflamed synovium contained variable amounts of inflammatory cells, most of them polymorphonu-clear. synovial pge levels were increased by poly i:c, poly a:u, and interferon but not by poly (i) or poly (c). mouse interferon also induced an inflammatory response when injected in mouse knee joint. we suggest that interferon may be a mediator in the initiation of inflammation by viruses. nineteen patients with musculoskeletal complaints after jejuno-ileal bypass for morbid obesity were reviewed for evidence of connective tissue disease and immunologic abnormalities. presence of arthritis or arthralgias and extraarticular connective tissue symptoms, elevated westergren sedimentation rate (esr), and absence of other significant forms of arthritis were criteria for diagnosis of postintestinal bypass arthritis. eleven of the 19 patients met these criteria. seven were females. six of the i 1 had joint swelling and 5 had only arthralgias. all had moderately elevated esr. extraarticular manifestations include erythema nodosum (3 patients), pleural effusion (2 patients), and carpal tunnel syndrome (1 patient). synovial fluid analysis in 5 showed mild to moderate inflammation. serum rheumatoid factor was present in 1 patient in low titer while 3 patients had positive ana in titers ranging from 1:128 to 1:1024. in 2 patients the ana reverted to negative with therapy, and in the third patient the ana became negative after the bypass was reversed. four had hypercomplementemia, 6 had elevated serum igg, and 3 had ele-vated serum iga. none of the 1 1 patients demonstrated cryoglobulins, but 1 had soluble immune complexes by complement inhibition technique. synovial biopsy in 1 case showed chronic inflammation and iga and c' deposition by immunofluorescence. hla typing of 6 of the 1 1 patients with post bypass arthritis showed no trend. in i patient the length of bypassed bowel was reduced, and in another the bypass was reversed. in both cases the arthritis remitted. two patients required low dose prednisone therapy for the arthritis; the others were treated effectively with nonsteroidal antiinflammatory agents. these 1 1 patients had an inflammatory arthropathy often accompanied by features suggestive of connective tissue disease, such as erythema nodosum, pleural effusions, and circulating ana. although none had cryoglobulins, which were previously reported by others, 1 had circulating immune complex. this suggests that immune mechanisms are involved in the pathogenesis of arthritis in some, if not all, of these patients. honig et a1 (arthritis and rheum 201065, 1977 recently reported that patients with active systemic lupus erythematosus (sle) usually do not have markedly increased serum concentrations (>200 pg/ml) of c-reactive protein (crp) and that such a finding suggests the presence of superimposed infection rather than active lupus. since a semi-quantitative capillary precipitin technique was employed in that study, in which only very high crp concentrations were regarded as positive, we investigated the significance of lesser increases in serum crp concentration in patients with sle, using a quantitative radial immunodiffusion method sensitive to 1.5 pg/ml. we retrospectively determined crp concentrations in 141 serum samples collected from a group of 17 patients with sle over a mean period of 19 months. thirtytwo episodes of significant increase in serum crp concentration were detected. careful review of clinical findings associated with each crp peak revealed that these episodes were associated with active lupus without infection in 20 instances (median crp concentration 21 pg/ml, mean 43.7, range 7.8-197.2) in 9 instances, elevations of serum crp concentration were attributed to proven or suspected superimposed infection or bone fracture (median 45 pg/ml, mean 52.6, range 16.3-118.2). three crp elevations of 7.3, 8.2 and 39.4 pg/ml occurred at a time when there was clinical evidence of neither lupus activation nor infection. there were 8 instances of onset or exacerbation of lupus activity at times when serum samples did not show elevated crp levels. in 2 of these, very active disease was present; crp elevations were noted in the next samples obtained, 13 and 33 days later. in a third patient with both infection and lupus activity, crp levels were found elevated 8 days later. these data indicate that moderate to marked increases in serum crp concentration may occur in the course of sle in association with activation of disease, as well as in association with infection or other cause of tissue necrosis. rarely no obvious clinical cause can be found. occurrence of serum crp elevation in patients with sle does not differentiate between lupus activity and infection. and immunologic study of the post-intestinal bypass arthritis-dermatitis syndrome medical versus surgical management of ischemic necrosis of bone in systemic lupus intestinal bypass surgery has become a successful means of effecting weight reduction in morbid obesity. as a complication of surgery, a fraction of patients develop an arthritis and tenosynovitis which may be associated with circulating cryoproteins. we have had the opportunity to study 6 patients with post-jejunoileostomy arthritis, all of whom had, as a striking feature of the illness, a widespread dermatitis.the patients were all female. the arthritis developed from 12 to 62 months after surgery and predilected both large and small joints of the upper and lower-extremities. associated with the arthritis were erythematous macules, ranging in size from 2 to 12 mm in diameter. these lesions developed into papules over 2 days, subsequently became pustular-vesicular, and predilected arms, legs, trunk, and face. lesions were found in different stages of evolution.cryoglobulins consisting of igg, igm, and c3, c4 were found in 3 of 4 patients. immune complexes (raji cell) were found in 3 of 3 patients, including 1 patient with absent cryoglobulins. synovial fluid analysis in 3 patients revealed california wbc ranging from 1200 to 5800 with from 20 to 75% pmn. immune complexes (raji cell) were found in 3 of 3 synovial fluids.excisional biopsies of the skin lesions were performed in 3 patients. there were no areas of fat necrosis. small capillaries and venules were infiltrated with pmn in all cases.immunofluorescent stains revealed deposits of igg and c3-c4 in vessel walls in 1 of 3 patients. fluorescein conjugated antisera against 5 bacterial pathogens revealed no staining in 1 patient. treatment with oral antibiotics was initiated in all patients but was not invariably successful in decreasing the dermatitis or arthritis. in 2 patients, intravenous antibiotics were also unsuccessful, but oral prednisone was promptly followed by decreased dermatitis-arthritis. in responsive patients, the serum cryoprotein titer decreased; the kinetics of this decrease and the reappearence of the cryoprotein after stopping antibiotic-prednisone treatment was studied.post-intestinal bypass surgery may cause a systemic immune complex disease primarily involving joints and skin. each met at least 4 sle classification criteria, had 2 clinical criteria of disease activity, and had elevated serum dna binding (30-93% by farr assay). three had low serum hemolytic complement levels (62-89 u). their drug regimen, including prednisone (20-45 mg/day), remained stable for 2 i days prior to, during, and for 28 days after frentizole therapy. rash (4 of 6), synovitis (4 of 6), mucosal ulcers (3 of 3), and pleurisy (1 of 1) improved during frentizole therapy. synovitis worsened in 1 case. the only toxicity was transient sgot and sgpt elevation in 2 cases. bone marrow and skin tests were not suppressed by the drug. frentizole had no effect on hemoglobin concentration, creatinine clearance, or urinary protein ex-response. other laboratory findings (mean f sem) are summarized in the table. these results suggest that frentizole is an active, relatively non-toxic immunoregulatory drug in sle patients. current long-term studies should define its role in the therapy of sle and other autoimmune diseases. clinical and laboratory findings of 45 children with systemic lupus erythematosus (sle) prospectively followed since 1962 have been summarized. mean disease duration was 5.3 years (range 2 months to 16 years, median 5 years). there were 34 girls and 1 1 boys, less of a female preponderance than that of most adult series. youngest age for onset of systemic complaints was 4 years. ten of 11 boys had disease onset prior to sexual maturation, whereas disease began after menarche in adcc activity between patient cells and control cells when either the mixed mononuclear population or monocyte-depleted population were studied as effectors. the monocyteenriched fraction from patients with ra, however, mediated a significantly increased degree of cytotoxicity (table) . enhanced cytotoxicity was more evident at low effector: target ratios and was independent of phagocytosis. adcc may be important in ra since it reflects both humoral and cell-mediated immune mechanisms. the enhanced effector function of the peripheral blood monocyte in this system may be an indication that mononuclear phagocytes are "activated" in patients with ra. the ara preliminary criteria for the classification of systemic lupus erythematosus (sle) were developed before the widespread use of tests for anti-dna antibodies and serum c3 levels. analysis of our data suggests that these tests should be included in the ara criteria and could replace 2 of the present 14 manifestations which are less specific and less sensitive than the serum c3 level and circulating anti-native dna antibody level (a-dna).three thousand three hundred thirty-four sera from 98 sle patients were studied. only patients observed by us and whose sera were tested both during periods of remission and disease activity were included in this study. an average of 34 serum samples per patient was studied. the average period of observation was 38.4 months. patients had at least 4 of the 14 manifestations of the ara criteria. c3 levels and a-dna were studied from 59 normal individuals, 118 patients with rheumatoid arthritis followed by us, and from patients with other diseases.two of the non-sle patients (1%) had a-dna level (expressed as % dna bound) of more than 41% (46%,52%). thus, a a-dna of >41% binding had a specificity for sle of 99%. eighty-two percent of the sle patients had a-dna of >41% binding as their highest value-a sensitivity of 82%. none of the non-sle patients had a c3 of <71 mg%-a specificity of 100%. fifty-five percent of the sle patients had a c3 level of <7 1 mg% as their lowest c3-a sensitivity of 54%.these data were analyzed using the method employed to select the 14 manifestations of the ara criteria. the average rate of correct classification (the arithmetic mean of the sensitivity and specificity) (arcc) for a-dna of >41% binding is 90.5 and for c3 of <71 mg% is 77. the arcc for the 22 items which comprise the 14 manifestations of the ara criteria varied from 52.0 to 94.9. of these, only the presence of le cells had a arcc greater than that for a-dna. if c3 and a-dna data had been available at the time the ara criteria were developed, they would have been included. the arcc for the mean of the 2 items combined for the cns manifestations and the arcc for the mean of the hematologic items were lower than those for either c3 or a-dna. thus, the c3 and the a-dna could replace these 2 manifestations. the data suggest that the ara criteria should be revised.the significance of ischemic necrosis of bone (inb) in sle and the cause of the bony lesion are not established. in this study, 36 patients with sle and inb have been compared to 124 patients with sle alone; and, in those with inb, the cause of the osseous lesion has been analyzed in relation to medical versus surgical management.those clinical features of sle significantly correlated with inb include raynaud's phenomenon (61% versus 13%, p < o.ool), myositis (25% versus 7%, p < o.ol), and vasculitis (50% versus 26%, p < 0.025). the course of 73 inb sites in the 36 patients initially grouped into medical (20 patients; 41 sites) and surgical (i6 patients; 32 sites) is shown in the table.the initial orthopedic procedure was uniformly core decompression. all patients were receiving corticosteroids. the failure of medical management alone is shown by the persistence of symptoms (39 or 95%), x-ray progression (35 or 85%), and need for reconstructive surgery (22 or 53%). in stages i1 and 111 of the surgical group, only 7 sites (25%) were symptomatic, 5 sites (17%) progressed on x-ray, and only 3 (10%) required further surgery. none in stage i advanced.differences in followup intervals would not appear to explain these statistically significant differences.thus, in the steroid-treated sle patient, especially with raynaud's, myositis, and/or vasculitis, the risk of inb is major and the need for early diagnosis essential if progression of the bony lesion is to be retarded by orthopedic intervention. acute abdominal syndromes have been increasingly recognized as major life-threatening events in patients with rheumatic disease. early diagnosis is critical to the institution of appropriate medical/surgical management and may be impeded by the masking effects of antiinflammatory, especially steroidal, therapy. it is our purpose here to report those features, clinical and laboratory, which appear to relate specifically to intraabdominal arteritis, our major cause of an acute abdomen in those with systemic lupus erythematosus (sle) and polyarteritis (pa).during the past 7 years, 15 of 140 patients admitted to the unit with sle and 4 of 8 patients with pa developed an acute abdomen. in 1 i (73%) of those with sle and all 4 with pa, the abdominal event was secondary to mesenteric arteritis. the remaining 4 patients with sle had polyserositis (2), pancreatitis ( i ) and, in the other patient, an undiagnosed recurrent syndrome which responded each time to increased corticosteroids alone. when patients with sle and an acute abdomen were compared to the 125 patients without abdominal syn-dromes, the significant discriminating features in the abdominal group were increased peripheral vasculitis (57% versus 25%, p < 0.025), thrombocytopenia (57% versus 19%, p < o.ooos), and presence of rheumatoid factor by the latex test system (92% versus 45%, p < 0.0005). of the 4 patients with pa, peripheral neuropathy was present in all, thrombocytopenia developed concomitant with the abdominal syndrome in all, and rheumatoid factor was uniformly present in the 3 patients tested (1 : 1280, 1 : 1280, i : 10,240). eight of those with sle and the 4 with pa died from the abdominal catastrophe. six of the 7 surviving sle patients represent those most recently seen.thus, in the patients with sle and pa, the presence of thrombocytopenia and circulating rheumatoid factor are poor prognostic signs and may pathogenetically predispose the patient to intraabdominal arteritis. outcome can only be favorably altered by early recognition and prompt institution of appropriate medical and/or surgical management. key: cord-355374-e8k72955 authors: clemens, e. bridie; van de sandt, carolien; wong, sook san; wakim, linda m.; valkenburg, sophie a. title: harnessing the power of t cells: the promising hope for a universal influenza vaccine date: 2018-03-26 journal: vaccines (basel) doi: 10.3390/vaccines6020018 sha: doc_id: 355374 cord_uid: e8k72955 next-generation vaccines that utilize t cells could potentially overcome the limitations of current influenza vaccines that rely on antibodies to provide narrow subtype-specific protection and are prone to antigenic mismatch with circulating strains. evidence from animal models shows that t cells can provide heterosubtypic protection and are crucial for immune control of influenza virus infections. this has provided hope for the design of a universal vaccine able to prime against diverse influenza virus strains and subtypes. however, multiple hurdles exist for the realisation of a universal t cell vaccine. overall primary concerns are: extrapolating human clinical studies, seeding durable effective t cell resident memory (trm), population human leucocyte antigen (hla) coverage, and the potential for t cell-mediated immune escape. further comprehensive human clinical data is needed during natural infection to validate the protective role t cells play during infection in the absence of antibodies. furthermore, fundamental questions still exist regarding the site, longevity and duration, quantity, and phenotype of t cells needed for optimal protection. standardised experimental methods, and eventually simplified commercial assays, to assess peripheral influenza-specific t cell responses are needed for larger-scale clinical studies of t cells as a correlate of protection against influenza infection. the design and implementation of a t cell-inducing vaccine will require a consensus on the level of protection acceptable in the community, which may not provide sterilizing immunity but could protect the individual from severe disease, reduce the length of infection, and potentially reduce transmission in the community. therefore, increasing the standard of care potentially offered by t cell vaccines should be considered in the context of pandemic preparedness and zoonotic infections, and in combination with improved antibody vaccine targeting methods. current pandemic vaccine preparedness measures and ongoing clinical trials under-utilise t cell-inducing vaccines, reflecting the myriad questions that remain about how, when, where, and which t cells are needed to fight influenza virus infection. this review aims to bring together basic fundamentals of t cell biology with human clinical data, which need to be considered for the implementation of a universal vaccine against influenza that harnesses the power of t cells. countless examples exist for influenza a viruses causing havoc on public health, from perpetual seasonal epidemics, worldwide pandemics, and zoonotic infections from animal reservoirs, yet our current vaccine methods do not arm us against the diversity of influenza viruses. influenza vaccines are the most widely used vaccines in the world, with over 500 million doses used annually [1] , due to seasonal epidemics and the recommendation of annual vaccination. however, the efficacy of the inactivated influenza vaccine (iiv) is moderate to poor, and is impacted by antigenic drift [2] , mismatch [3, 4] , pandemic emergence due to reassortment [5] , and egg adaptations during vaccine production [6] , which can all lead to reduced protection and increased incidence of infections. the efficacy of the live attenuated influenza vaccine (laiv)-mainly recommended for use in children-has also dropped in recent years [7] , possibly due to thermal stability issues [8] or antigen competition during priming [9] . overall, these factors have culminated in reduced public confidence in influenza vaccines [10] . current vaccine stockpiles for avian influenza viruses h5n1 and h7n9 have reduced immunogenicity compared to seasonal influenza viruses [11, 12] , requiring multiple doses, the use of adjuvant, and may not match future emergent versions of these viruses [13] . the 2009 h1n1 pandemic showed that we are only able to respond after the fact, as the monovalent pandemic vaccine became available after the peak of human infections, leaving the majority of the population to "ride out the storm" and public outcry at the spectre of the pandemic severity predictions. vaccine production methods have been significantly ramped up in the wake of the 2009 pandemic, but the timing of virus isolation, distribution, and large-scale production will encounter similar issues in future pandemics. overall, a substantial revitalisation of the current vaccination program is needed to combat influenza viruses, overcome vaccine production limitations, and pre-arm ourselves against diverse and divergent influenza a viruses. vaccination educates our adaptive immune system-specifically t and b cells-for a faster, stronger, and more specific response upon re-encounter with the matching antigen. however, current iivs and laivs are not efficient in inducing t cell immunity, potentially contributing to their limited efficacy and breadth of reactivity against diverse influenza viruses. importantly, current inactivated influenza vaccines tend to prevent the induction of cross-reactive cd8 + t-cells, which would otherwise be elicited by natural influenza virus infections and are our primary protection in case of a vaccine mismatch or pandemic outbreak [14] (figures 1 and 2) . t cells express a clonal t cell receptor (tcr), which recognizes linear fragments of viral peptides that are 9-15 amino acids long, presented by the major histocompatibility complex (mhc) molecules on the surface of infected cells or antigen presenting cells. access to the mhc presentation pathway utilises endogenously (direct presentation) or exogenously (cross-presentation) derived peptides generated by the cleavage of viral proteins by the immunoproteasome. this process has important implications for vaccine approaches that need to consider the design and delivery of antigen to these pathways for the activation of t cells. viral entry and translation are required for access to mhc i processing machinery; however, peptide presentation can precede virus replication. the protein composition and immunodominance hierarchies of the subsequent t cell responses predominantly reflect abundant virion proteins expressed during infection and the timing of that expression [15] . memory t cells can provide very effective heterosubtypic immunity, whereby t cells are able to recognise different and even unrelated influenza viruses due to peptide homology or the conservation of linear peptide sequences between different strains and subtypes of influenza [16] [17] [18] [19] [20] [21] . heterosubtypic t cell immunity is especially important when there is no existing antibody response, which can occur during seasonal antigenic drift, zoonotic infection, or a pandemic situation. the protective efficacy of cross-reactive cd8 + t cells has been demonstrated in extensive animal studies of mice [22] [23] [24] and ferrets [25] . for example, mice primed with h3n2 virus, boosted with h1n1, and finally challenged with a high dose of lethal h7n7 virus have no detectable virus replication [26] . thus, unlike the majority of b cells, t cells are capable of expanding immunological protection against diverse influenza viruses. although antibody-mediated immunity elicited by natural infection or current vaccine strategies is capable of providing sterilizing protection, this protection is generated primarily against the hemagglutinin (ha) and neuraminidase (na) epitopes on the virus surface. immune escape is more common for surface antigens via modifications of glycans and amino acid substitutions at key antigenic sites than internal proteins which are constrained by functional limitations of viral fitness [27, 28] . lee et al. identified 72 t cell peptide epitopes that are cross-recognised between h5n1 and seasonal h3n2 viruses by cd4 + and cd8 + t cells, and only one was derived from the ha surface protein [17] . the majority of t cell epitopes are derived from internal proteins, which have a conservation rate of >90% between different iav strains and subtypes, whilst ha and na surface proteins are only 40-70% conserved between different iav subtypes [29] . indeed, t cells that are cross-reactive against highly pathogenic h7n9 and h5n1 avian influenza viruses can be found in the peripheral blood of unexposed healthy volunteers [17, 19, 20] . furthermore, robust and expedient recruitment of such cross-reactive t cell memory correlates with improved infection outcomes, faster recovery, and survival from h7n9 infection [30] . while influenza a and b viruses share many features, there is little sequence identity between analogous proteins (range 7-37% amino acid identity), with the exception of the polymerase basic 1 (pb1) protein (58% amino acid identity) [31] . despite this, broadly neutralizing monoclonal antibodies against highly conserved ha and na epitopes of influenza a and b viruses have been reported (reviewed in [31, 32] ). in addition, a conserved cd4 + t cell epitope in the ha fusion peptide of influenza a and b viruses can elicit cross-reactive cd4 + t cells in humans, and a number of well-characterized cd4 + and cd8 + t cell epitopes in the pb1 protein show complete amino acid conservation or only slight variation amongst influenza a and b viruses [31, 32] . this hints at the possibility of truly universal pan-influenza a and b virus t cell immunity that could be harnessed by vaccination. the area of research on t cell cross-reactivity beyond influenza a viruses currently remains underexplored. t cells are represented by a family of phenotypically diverse subtypes in terms of function, location, and magnitude. cytotoxic cd8 + t cells recognize viral peptide in the context of mhc class i (mhci), which is expressed on the surface of all nucleated cells, allowing immune surveillance. cd8 + t cells mediate the direct lysis of virus-infected cells and produce anti-viral cytokines, effectively and directly reducing viral load and the length of infection. meanwhile, helper cd4 + t cells recognize viral peptides within mhc class ii (mhcii), which is expressed by professional antigen presenting cells, b cells, macrophages, dendritic cells, and other cd4 + t cells. helper cd4 + t cells are a critical cornerstone of establishing effective immune memory against influenza virus infection, and are necessary for the establishment of cd8 + t cell memory responses [33] and high avidity class switched antibodies (reviewed in [34] ). in addition, it has been proposed that mhcii is also expressed by type ii alveolar pneumocytes during infection [35] , enabling cd4 + t cells to target a minor population of infected cells in the lung tissue by direct cytotoxic mechanisms themselves. t cells express a clonally diverse and highly specific tcr on their cell surface. the tcr consists of an α and β chain heterodimer, with the fine specificity of antigen recognition provided by somatic recombination and non-germ-line encoded additions to the complementarity determining regions (cdrs) of each chain. the estimated diversity of the human t cell repertoire is 2 × 10 7 distinct tcrs [36] , whilst the tcr diversity of an individual epitope-specific t cell response can be oligoclonal or composed of up to 40 different distinct tcr sequences [37, 38] . unlike antibody responses, tcrs do not undergo affinity maturation and the thymus involutes after puberty, greatly diminishing the output of naïve t cells as we age. the capacity for heterologous immunity by t cell responses is imperative for protection against the huge array of possible pathogens encountered in a lifetime, which out-numbers the tcr repertoire; therefore, an overlap in tcr specificities is a necessity. heterologous t cell protection to different epitopes has been reported in mice between lcmv, vaccinia virus, and pichinde virus [39] ; and in humans between influenza and hepatitis c virus [40] ; papilloma viruses and coronaviruses [41] ; influenza and epstein-barr virus (ebv) [42, 43] ; dengue subtypes [44] ; and as discussed above, between different strains of influenza and variants of immunodominant influenza-derived epitopes [45] . t cells are derived from the bone marrow progenitor cells, mature and develop in the thymus, undergo positive and negative selection processes, then emigrate as non-self-reactive immature cells in the periphery for immune surveillance for cells expressing altered or non-self-antigens. immature naïve t cells circulate between secondary lymphoid organs surveying for cognate antigen, while mature memory subsets-depending on their phenotype and priming signals-can reside in the tissue parenchyma (t cell resident memory, trm), the lymph node (t cell central memory, tcm) and periphery (t cell effector memory, tem). t cell recognition of peptide-mhc in the context of further signalling (e.g., co-stimulation and inflammatory cytokines) leads to t cell activation, proliferation, and differentiation, amplifying the effective army of t cells against infection. the number of naïve epitope-specific cd8 + t cells has been estimated in the mouse model for immunodominant and subdominant epitopes at 39-72 cells/mouse and 247-315 cells/mouse, respectively [38] . the efficiency of recruitment from the naïve pool of t cells to respond during acute infection is dependent on numerous factors, such as: the peptide context, mhc allele restriction, antigen presenting cell, the timing of antigen expression during the virus life cycle, and the avidity of the tcr and peptide-mhc interaction ( [46] , and reviewed in [47] ). after tcr engagement, recruitment, and activation, naïve t cells then amplify up to 10,000-fold during the acute stages of infection. the rapid expansion and peak magnitude of virus-specific t cells coincides with drastic reduction in viral titres from days 5-9 of primary virus infection, whereas influenza-specific antibodies peak and plateau from days 14-20 post infection. following antigen clearance at about 14 days post influenza infection, the antigen-specific t cell pool contracts, whereby the most differentiated cells undergo activation-induced cell death by apoptosis, leaving behind a stabilised memory pool [48] . the kinetics of t cell responses in human influenza infection are more variable, with some studies reporting a rapid peak in virus-specific t cells at one-week post-infection with h1n1 that subsequently contracts within 3-4 weeks after infection [22, 49] , while others suggest a more protracted response dynamic, peaking 3 weeks after infection with only modest decline in cell numbers by day 78 and returning to baseline by day 700 [50] . to what extent these variations in dynamics reflect differences in virus strain, infection severity, or individual characteristics of the host response remains to be determined. the t cell memory pool remaining after infection is estimated to have a half-life of 8-15 years in humans, and remarkably can be detected after 70 years post small-pox vaccination [51] , but the lifespan of influenza-specific t cells has not been tracked past 10 years [52] due to repeated encounters by influenza virus infection making tracking difficult with time. multiple doses of laivs-and not iivs or subunit influenza vaccines-can induce long-lived, broad, protective immune responses in mice [53] [54] [55] . therefore, laiv appears to be more immunogenic in animal studies. however, human responses against laiv are disparate depending on age, and laivs only seem more effective than iiv in children, not adults, which coincides with boosted cellular immunity [56, 57] . this is possibly a result of children's more naïve immune states resulting in a robust primary cellular immune response [58] [59] [60] [61] [62] [63] [64] [65] [66] . laiv-induced humoral and cellular immunity could be maintained for at least one year [67] , but the longevity beyond 1 year has not been studied so far. the laiv vaccine currently has limited efficacy, with a reported vaccine efficacy of only 3% for laiv vs. 65% for iiv reported in 2017 [7] . however, laiv was a sincere attempt at a t cell-inducing vaccine by nasal delivery, but appears too mild, with reduced tissue tropism and inflammation [68] and has so far failed to increase cellular immunity or improve vaccine efficacy in adults. therefore, future universal vaccines must improve upon laiv by either the use of more immunogenic vaccine vectors (such as e1 deleted adenovirus [69] or mva [70] ), adjuvants (interleukin-15 (il-15) [29] , pam2cys [71] , or mf59 [72] ), or less attenuated influenza viruses (such as the use of codon bias mutants [73] , ns1 mutants [74] , or ha-signal peptide viruses [75] ). lessons should be learned from the limited efficacy and immunogenicity of laiv in human studies in the development of next-generation universal vaccines. t cells do not operate in isolation, but in synergy with multiple immune mechanisms to provide broad heterosubtypic protection ( figure 1 ). heterologous protection mediated by cross-reactive cd8 + t cells was shown to be dependent on an interaction with macrophages and fcr-dependent non-neutralizing antibodies [76] , similar to the inexplicable protection rates beyond t cell response magnitude observed in the laiv thailand/philippines clinical trial [77] . in addition, the timing of recruitment of memory cd8 + t cells, cd4 + t cells, nk cells, or neutralizing antibodies impacted h7n9 infection outcomes [30] . further cell types also contribute to heterologous protection, such as mucosal associated invariant t (mait) cells [78] and fcr + cells to recruit t cell responses [76] . thus, the next generation of t cell vaccines should combine multiple immune responses for heterologous protection. effective heterologous immunity against zoonotic influenza (h7n9) viruses requires synergy of multiple immune arms [30, 76, 78] . without the recruitment of two or more immune arms, protective immunity is diminished, as modelled on outcomes of infection from h7n9-infected patients. although multiple arms are likely to be activated at the same time, hospitalized patients clearly demonstrate that different arms had a more prominent role if one arm fails to respond. mait: mucosal associated invariant t. cd4 + t cells are phenotypically diverse, and depending on their surface receptors and cytokine expression, can be further characterised into numerous subsets: th1, th2, treg, t follicular helper (tfh), th17, th22, and th9 (reviewed in [79] ). the cd4 + t cell field is less developed than the cd8 + t cell field (reviewed in [80] ) for influenza research, due to the availability of mhci tetramers and transgenic mouse strains. however, continued efforts using adapted methods such as peptide scanning or whole virus for identification of influenza-specific cd4 + t cells have revealed their intrinsic necessity for heterologous protection against influenza virus infection [49] . characteristically, th1 type cd4 + t cells can secrete ifn-γ and are necessary for the formation of cd8 + t cell memory capable of recall and tfh cd4 + t cells for the induction of high affinity class switched b cell memory [81, 82] . induction of ha-stem-specific antibodies requires highly developed b-cell receptor sequences that have undergone affinity maturation, likely to be coordinated by cd4 + t follicular helper (tfh) cells [83] . immunization with current iivs activates tfh cells, correlating with an increase in the number of peripheral antibody-secreting and memory b cells [84] . an emerging body of work has demonstrated the critical role tfh cd4 + cells play in shaping the antibody profile, whereby conserved nucleoprotein (np)-specific cd4 + tfh cells compete with ha-specific tfh cells, resulting in reduced h7-ha antibody recall responses after priming with the h1-ha [85] [86] [87] . therefore, the order of priming for establishing immunodominance hierarchies for cd4 + tfh responses can impact the development of ha-stem antibodies in the germinal centre. the interplay of protein-specificity and order of immune memory or "imprinting" is important in the context of human immune memory, which is heterogeneous and individualistic depending on a subject's exposure background. imprinting of ha-subtype specific cd4 + t cell and b cell memory can have an epidemiological impact. ha-imprinting was recently described in an epidemiological model explaining the age association of h7n9 and h5n1 infections, and providing >75% protection from severe infection caused by these viruses. this was attributable to an individual's first ha-group encounter resulting in an age distribution of cases born after 1968 for h5n1 and before 1968 for h7n9 corresponding to the switch from a phylogenetic group 1 (including h1, h2, and h5 ha subtypes) to group 2 (includes h3 and h7 ha subtypes) ha during the 1968 hong kong h3n2 pandemic [88] . furthermore, in addition to helper functions, influenza-specific cd4 + t cells may be imperative to protection from heterologous influenza infection. in a transgenic mouse model, cd4 + t cells have also been found to be directly cytotoxic during influenza infection [89] , and human influenza-specific cd4 + t cells can express granzymes, perforin, ifn-γ, and exhibit killing function correlating with reduced severity of infection and viral loads [49] . furthermore, a t cell-inducing vaccine mouse model identified that depletion of memory cd4 + t cells but not the cd8 + t cell compartment removed heterologous protection from lethal influenza challenges [29] . new roles continue to emerge of the key role influenza-specific cd4 + t cells play during heterologous infection. overall, influenza-specific cd4 + t cells are a cornerstone of effective influenza responses, and their role is a dynamic interplay on the recall of heterologous t and b cell responses and infection outcomes ( figure 1 ). the long-term role of imprinting cd4 + t cell memory responses by universal vaccines will have an important implication in future vaccine design, and is not yet fully explored. epidemiological studies during past influenza pandemics, where there is an absence of neutralizing antibodies, have suggested that t cell-mediated immunity provided some heterosubtypic protection [90] [91] [92] . however, the complexity involved in conducting such studies (i.e., obtaining the right cohort that does not have pre-existing antibody response and sampling prior to infection) means that direct evidence for the role of t cells during influenza virus infections in humans has been limited. as such, a major challenge in the universal vaccine field is to determine the strength of a t cell-mediated "correlate of protection". unlike animal models, influenza virus infections in humans can result in a wide spectrum of symptoms that reflect either upper or lower respiratory tract involvement. the clinical disease and epidemiology is also distinct between seasonal and avian influenza cases. thus, in practise, the definition of "protection" in human studies has varied from study to study, and has generally meant a reduction in virus shedding or manifested symptoms. these symptoms have either been self-reported [93] [94] [95] , observed [49] , or clinically determined [96] [97] [98] . in seasonal influenza, the earliest direct evidence that cd8 + t cells can mediate protection by facilitating virus clearance after infection was shown in a human challenge study [99] . thirty years later, sridhar et al. [94] took advantage of an existing community-wide cohort to show this within the context of natural infections during the h1n1 2009 pandemic. they demonstrated that the magnitude of a subset of ifn-γ + il-2 − cd8 + t cells was most strongly correlated with reduced disease severity after infection. these cells were also shown to be late-effector memory t cells (cd45ra + ccr7 − ). this was in contrast with the findings from an earlier experimental h3n2 or h1n1 human challenge study, where cd4 + t cells were correlated with reduced viral load and disease symptoms instead of cd8 + t cell memory responses [49] . this discrepancy was attributed to differences in experimental designs (i.e., virus challenge using laboratory-grown viruses and hemagglutination inhibition (hai) negative subjects versus natural pandemic infection), and it serves to further highlight the challenges associated with studying t cell responses in humans. because t cells recognize highly conserved viral proteins, there has been great interest in investigating their protective role during human cases of avian influenza virus infections. while numerous studies have demonstrated the presence of cross-reactive t cells against avian influenza viruses in vitro [17] [18] [19] [20] 86] , actual evidence of protection against infection or disease in the field is still lacking since the sporadic and virulent nature of avian influenza cases precludes any systematic cohort studies. studies to date have been observational, and were based on a relatively small number of cases [30, 100] during the h7n9 outbreak in china in 2013. these studies suggested that the early induction and recruitment of memory cd8 + t cells was associated with improved prognosis [30, 100] . in contrast to cellular immunity data (which are scarce), there have been numerous studies investigating cytokine profiles in infected humans as prognostic markers and signatures of correlates of protection. pro-inflammatory cytokines such as interleukin (il)-6 and chemotactic regulators of innate immune cells such as il-8, mcp-3, and mcp-1 are commonly induced during the acute phase of disease [93, 101, 102] . however, cytokines that are important in inducing th1 and th2 responses have also been reported to be depressed in severely ill individuals [97, 98, 100] . of particular interest is il-12, a regulator of downstream th1-responses. concentrations of this cytokine were lower in severe seasonal and avian influenza cases, which incidentally also had lower levels of peripheral cd8 + t cells [97, 100] compared to mild influenza cases. however, the role of this cytokine needs to be further validated, since data from murine models suggests that high levels of il-12 cytokine can suppress the formation of lung cd8 + memory t cells in the airways [103] . the state of current knowledge of influenza t cell responses in humans is still trying to catch up with the lessons we have learned from animal models. in vitro and murine studies have demonstrated that t cells can mediate lung pathology (reviewed in [104] ), primarily through hypersecretion of soluble factors such as ifn-γ and tnf-α, causing direct lysis or "bystander damage" to the lung milieu [105] . although immune-mediated pathology has certainly been suggested as an important component of severe influenza infections in humans [98, 106] , a definitive causal role for t cells that is uncoupled from innate immunity or viral factors is still lacking. indeed, cytokines associated with cellular innate immunity appear to be better predictors of influenza disease severity [93, 101, 102] . most studies of t cell responses in humans have been conducted within the peripheral compartment due to the ease of sample access. however, peripheral and airway mucosal immunity are not always in concordance for respiratory infections [93, 102] . furthermore, viral replication can persist in the lower respiratory tract without detection in the upper respiratory tract, where nasal swabs are often collected [107, 108] for detection of viral rna. airway samples are difficult to collect-particularly those from the lower respiratory tract. nasal swabs contain mucosal inhibitors and have low cellularity, and are inappropriate for cellular immunity studies, while nasal aspirates are rare in human influenza studies. furthermore, sampling the lower respiratory tracts through bronchoalveolar lavage is often ethically precluded in healthy adults, and therefore such studies of local respiratory tissue t cell immunity are limited to severe infection cases that compares infection outcome rather than protection from infection or disease [93, 97] . in the few rare studies where such data are available, the number of influenza-specific cd4 + and cd8 + t cells, as well as the magnitude of cytokine response, are far greater in the lungs compared to the blood [98, 109, 110] . homologous virus challenge in ferrets that received the monovalent a/viet nam/1203/2004 (h5n1) vaccine showed that the cd8 + t cells in the airways, but not the blood, correlated with early viral clearance [111] . elegant studies in murine models have further identified a t cell population in the airways (tissue resident memory t cells, see section below) that are phenotypically and functionally distinct from those in the blood [112] and that have also been identified in humans [113, 114] . however, as it is likely that the peripheral compartment will continue to be a proxy of evaluating t cell responses in humans, the extent of how accurately the peripheral t cell responses reflect the airways, and crucially, how they translate to short-and long-term protection are important considerations for effective t cell-mediated vaccine development. because most adults have some baseline level of detectable influenza-specific t cells, some efforts have been made to quantitatively identify the t cell-mediated protective threshold as the "correlate of protection". in a study involving over 2000 children in thailand and the philippines, forrest et al. reported that after receiving two-doses of laiv, hai-seronegative children that had ≥100 spot-forming unit (sfu)/10 6 pbmc in an ifn-γ-elispot assay (using inactivated vaccine components as antigens) were protected against symptomatic disease during subsequent infection [77] . this study also made two notable observations: (i) the protective thresholds for the study populations in these two countries were different, and (ii) other mechanisms of protection not attributed to the measured response were noted with increasing vaccine dosage. thus, in attempting to provide a "quantitative" correlate of protection, this study also highlighted the population heterogeneity in the t cell compartment and the importance of other immune-mechanisms (i.e., those mediated by non-ifn-γ + cells, or non-hai-reactive antibodies) that were missed by the experimental approach. in a more recent study, hayward et al. [95] chose to focus on the immunodominant response against the influenza np-, rather than the ha-specific t cell response, and reported that ≥20 sfu/10 6 pbmc was associated with reduced viral shedding in community-acquired influenza. finally, sridhar et al. [94] used statistical modelling to predict that every 10-fold increase in the ifn-γ + il-2 − cd8 + t cell frequency (enumerated by elispot) is associated with a 7-fold reduction in the risk of developing febrile influenza. because of the different assays used across these three studies, it is difficult to interpret the importance of these quantifiable findings until they are further corroborated. it is worth noting that these studies evaluated symptomatic cases, and evidence for the role of t cells in asymptomatic cases-particularly within the community setting-is still lacking. clinical evidence thus far supports the role of existing t cells-particularly the cd8 + population-in reducing symptom severity. however, the heterogeneity in experimental design (i.e., study definition, endpoints used, and the use of different or unqualified assays to measure t cell responses, and elispot methods are not directly comparable between different studies) seems to suggest the phenomenon of "the blind men and the elephant", whereby each study is measuring the t cell response as an immune correlate in different ways, sometimes providing disparate conclusions. integrating the myriad clinical findings to identify a single quantifiable trait of t cells as the "correlate of protection" remains a major challenge. while some of these issues are surmountable with sophisticated study designs and further research, others-such as airway t cell responses-may remain challenging to address in humans. thus, in addition to understanding the basic biology, the field must also work on developing a robust system that includes standardized and qualified t cell assays for use in human studies. to date, vaccines intended to elicit t cell immunity against viral infection have generated disappointing levels of protection. this has provoked the recent reassessment of variables important for the successful generation of t cell-mediated immunity with the quantity and location of the memory t cell population now considered of critical importance. t cells require contact with their target cell to mediate their cytolytic function; in other words, they act locally. therefore, any effective t cell-based vaccine targeting the earliest stages of infection would require the deposition of significant numbers of memory t cells locally within the mucosal tissue. non-lymphoid tissues house a large proportion of the memory t cell pool [115] . previously, it was thought that these were simply circulating memory t cells trafficking through the tissue as part of their immunological surveillance. however, it is now accepted that the majority of the cells present within the tissue are in fact resident and represent a distinct memory t cell population [116] . these peripherally deposited memory t cells-termed tissue resident memory (trm)-play a critical role in local immune protection by directly killing pathogen-infected cells [117, 118] , releasing cytokines that render the surrounding/local environment non-permissive for pathogen replication [119] , and promoting the recruitment of other immune cells from the circulation [120] . both cd4 + and cd8 + t cell lineages can form trm, and can be distinguished from circulating memory t cells due to the expression of key surface markers. cd4 + trm upregulate cd69 and cd11a expression [121, 122] , while cd8 + trm also express cd69 as well as cd103, the α chain of the αeβ7 integrin [116] , although cd103-negative cd8 + trm cells have also been detected [123] ; thus, alternative markers for trm are needed. not all memory t cell subsets are equally protective against influenza virus infection, with only the trm pool being shown to be absolutely indispensable for providing optimal heterosubtypic immunity ( figure 2 ). following secondary encounter with influenza virus, both cd4 + [121, 122] and cd8 + [124] lung trm rapidly acquire effector function and respond swiftly, mediating enhanced viral clearance and survival to lethal influenza infection. why do trm provide superior protection against influenza virus infection? simply, they are in the right place at the right time! influenza virus gains entry into the body by inhalation and initiates its replication cycle within the respiratory tract. the early stages of infection, when virus titres are low in the infected host, provide the ideal window of opportunity for effective immune responses to limit disease progression. trm deposited along the respiratory tract are perfectly situated to combat the earliest stages of an influenza infection. human lung harbours a large number of memory t cells [114] , a significant proportion of which express the molecular signature and phenotypic profile of trm cells. the vast majority of influenza-specific memory cd4 + and cd8 + t cells present within human lung tissue adopt a trm phenotype [114, 122, [125] [126] [127] [128] . differentiation of these influenza-specific cells into trm is important for their long-term maintenance, as we find that the size of the influenza-specific cd8 + t cell population persisting within the lung directly correlates with the efficiency with which these cells differentiate into trm [128] . influenza virus-specific trm were shown to be highly proliferative and polyfunctional [123, 128, 129] . molecular profiling revealed that human lung trm constitutively express high transcript levels of numerous cytotoxic mediators and deployment-ready mrnas encoding effector molecules, which is reflective of these cells being poised for rapid responsiveness [123, 129] . influenza virus-specific cd8 + trm in human lung tissue also maintain diverse tcr profiles-a feature important for effective t cell function and protection against the generation of viral-escape mutants [128] . influenza-specific pulmonary trm are a core component of the natural heterosubtypic immunity developed following exposure to influenza virus ( figure 2) . thus, influenza vaccines designed to impart optimal heterosubtypic immunity should evoke this memory t cell subset. defining parameters that promote trm formation along the respiratory tract is a critical step towards the development of such a vaccine. so, what do we currently know about the factors that drive trm? exposure to interleukin-15 (il-15) [130] and transforming growth factor-β (tgfβ) [131, 132] , as well as down-regulation of krüppel-like factor 2 (klf2), s1p1r [133] , t-bet and eomes expression [130] , and up-regulation of hobit [134] have been proposed as universal trm developmental requirements. in some tissues (including the lung [125, 135, 136] and brain [137] ), trm development is also dependent on local cognate antigen recognition. can we rationally design vaccines to specifically evoke trm? in 2012, iwasaki and colleagues [138] published the first vaccination regime that specifically evoked trm at a site of potential viral exposure-they coined this vaccination regime "prime-pull". this approach relied on two steps: conventional parenteral vaccination to elicit systemic t cell responses (prime), followed by the topical application of inflammatory agents to lure (pull) the cells into the tissue where they could differentiate into trm [138] . while the "prime-pull" vaccination strategy has proven effective at lodging highly-protective trm pools in the skin [118] and reproductive tract [138] , this approach is not effective at depositing trm cells within the lung [131] . this is because trm differentiation in the lung-unlike skin and reproductive tract-requires local cognate antigen recognition [125, 135, 136] . as such, an extension of the "prime-pull" vaccination regime which incorporates the local delivery of cognate antigen was developed as a pulmonary trm vaccination strategy. several groups using a variety of modified "prime-pull" vaccination approaches, including (i) intranasal immunization with influenza peptide/protein alone [135, [139] [140] [141] or loaded onto dendritic cells [142] , (ii) intranasal delivery of non-replicative influenza virus [143] , or (iii) intranasal immunization with monoclonal antibodies linked to influenza antigens that target antigen to respiratory dendritic cells [131] , were all able to successfully generate lung cd8 + trm that were highly protective against influenza virus challenge. collectively, these vaccination studies demonstrate that the key to inducing pulmonary trm is the local (intranasal) administration of the vaccine antigen ( figure 2 ). such strategies might also be beneficial for the optimization of anti-influenza antibody responses through induction of mucosal secretory iga in the respiratory tract. vaccination strategies that deposit influenza virus-specific trm cells in the lung provide exquisite protection against heterosubtypic influenza challenge [131, 143] . unfortunately, this protection is transient. unlike populations in the skin and intestinal mucosa, trm cells in the lung undergo attrition [124] as a result of increased apoptosis [144] . mouse models confirm that within 7 months of their deposition, influenza virus-specific lung trm decay below a numerical threshold, leaving the lung susceptible to reinfection [124] . this observed decay of lung trm cells in mice is no reason to completely dismiss their potential to provide long-term immunity in humans, as just because mouse lung trm decay does not necessarily mean that human lung trm also undergoes this rapid rate of attrition. recent studies by ahmed and colleagues [145] , which utilize in vivo deuterium labelling to assess human memory cd8 + t cell turnover and longevity, show that the longevity of circulating memory t cells subsets in humans does not reflect the lifespan of these cells in mice. it will be important to determine the lifespan and turnover rate of human lung trm if these cells are to be incorporated into vaccines against respiratory pathogens, provide durable vaccine memory, and determine the vaccine schedule to maintain protection. nonetheless, while lung trm erode, their counterparts in the upper respiratory tract form a stable long-lived memory t cell pool [142] . influenza-specific nasal tissue trm are effective at limiting local replication of influenza virus and can block the transmission of influenza virus from the upper respiratory tract to the lung, and in doing so, prevent the development of severe pulmonary disease [142] . these cells have the potential to provide long-term immunity against influenza virus. to protect globally against influenza virus, we need to think locally! trm located along the respiratory tract are perfectly situated to mediate rapid protection following the inhalation of influenza virus, and are capable of providing potent protection against this inhaled pathogen. influenza vaccines designed to impart heterosubtypic immunity should evoke this memory t cell subset. how best to induce influenza virus-specific trm along the respiratory tract after vaccination will be one of the challenges to address in the coming years. the major antigenic targets of influenza-specific t cells are epitopes derived from highly conserved internal virus proteins, providing the basis for heterologous or "universal" immunity across multiple unrelated strains of influenza. studies assessing the relative contributions of different influenza virus proteins to human t cell responses have identified np, matrix protein 1 (m1), and pb1 as the major targets [16, 49, 95] , suggesting that a t cell vaccine may only need to focus on a few key viral proteins to achieve similar coverage to natural infection. the focus on priming t cell memory to conserved protein targets may also have implications for reducing interference with ha-imprinting in future vaccine design [88] . t cell responses to influenza virus differ between individuals, primarily due to the expression of diverse mhc alleles (known in humans as human leucocyte antigens, hlas) that determine the array of viral peptides presented to t cells for recognition. given the polymorphic nature of hla alleles [146] and the fact that each individual expresses six hla class i (hla-i; hla-a, -b, and -c) and six hla class ii (hla-ii; hla-dr, -dq, and -dp) alleles, there is potential for vast diversity in the epitopes presented for recognition during infection. despite this, t cell responses usually focus only on a few peptide + hla epitopes, with responses to the same epitopes typically observed across individuals expressing the same hla allele. responses to different epitopes often arrange into reproducible immunodominance hierarchies, wherein they can be defined as immunodominant (large) or subdominant (small) in magnitude. the relative size of a given epitope-specific t cell response reflects a complex interplay of virus and host factors (reviewed in [47] ), including the particular combination of hla alleles expressed [147] , and does not necessarily relate to the number of naïve epitope-specific precursors available. approaches combining in vitro infection of cell lines with mass spectrometry analysis have allowed direct identification of the array of peptides presented at the cell surface by individual hla alleles following influenza infection. to date, these results show the presentation of a reasonably small selection of virus peptides (nine for hla-a*02:01 and up to six for b*07:02), mainly derived from internal virus proteins, of which only a few (one and two peptides, respectively) consistently induce immune responses [148, 149] . understanding why some presented peptides are targeted while others are seemingly ignored remains a key question that may lead to strategies for optimizing the breadth of vaccine-induced t cell responses. hla-i allele expression is an important predictor of cross-reactive influenza-specific cd8 + t cell immunity, with a recent study identifying five alleles (a*02:01, a*03:01, b*57:01, b*18:01, and b*08:01) capable of eliciting robust cd8 + t cell responses against immunogenic np and m1 peptides that are conserved across all human influenza a virus, including the novel avian-derived h7n9 virus [18] . strong representation of these "protective" hla alleles in a population is therefore predictive of universal memory t cell responses with the potential to protect against multiple circulating and novel influenza a virus strains. as hla profiles are heritable and strongly influenced by ethnicity, the extent of this universal immunity shows an expected ethnic bias, with higher coverage anticipated in caucasian populations with enrichment of these key alleles (57%, based on hla allele expression) as compared to indigenous alaskans and indigenous australians (16%) [18, 150] . whilst certain hla alleles may confer a protective advantage through universal immunity to multiple influenza strains, the expression of other alleles may increase susceptibility to severe influenza disease. the h7n9 study mentioned above also identified four hla-i alleles (a*01:01, a*24:02, a*68:01, and b*15:01) that target unique h7n9 np and m1 peptide variants that are unlikely to elicit cross-protective immunity between seasonal and h7n9 infection [18] . thus, upon infection with h7n9, individuals with these hla alleles will need time to activate and amplify new primary cd8 + t cell responses to distinct h7n9 peptide variants rather than recalling t cell responses generated against seasonal influenza viruses, potentially resulting in longer time to recovery and greater risk of severe disease compared to individuals with pre-existing cross-protective cd8 + t cell memory. hertz et al. showed that hla-a*24 alleles also have low binding preference for peptides from conserved regions of the 2009 pandemic h1n1 (ph1n1) virus, and carriage of these alleles correlated with low influenza-specific t cell responses in ph1n1-infected patients [151] . moreover, at a population level, carriage of hla-a*24 alleles or the hla-a*68:01 allele was associated with increased mortality to ph1n1. these "risk" hla alleles may serve as useful markers to identify individuals or populations that are more likely to have low magnitude and less cross-reactive memory t cell responses, placing them at greater risk of severe influenza disease. interestingly, hla-a*24 and a*68:01 alleles are highly prevalent in certain indigenous populations [152, 153] , which could explain why these groups experience a higher burden of influenza disease and mortality to seasonal and pandemic influenza [151] . however, risk hla alleles may also have indirect effects on susceptibility to influenza through associations with other autoimmune or metabolic disorders. for example, hla-a*24 is associated with diabetes [154, 155] , a disease with increased incidence in indigenous populations [156] . nevertheless, the notion of protective and/or risk hla alleles has been demonstrated previously for human immunodeficiency virus (hiv) and hepatitis c virus (hcv) [157] [158] [159] [160] , and is clearly emerging as a factor in influenza infection linked in part to the capacity of certain hla alleles to present conserved (protective) or variable (risk) influenza peptides. ethnicity is a key determinant of risk for severe influenza disease with indigenous populations worldwide experiencing a disproportionate burden of morbidity and mortality caused by infection with influenza viruses. during the 2009 h1n1 pandemic, higher hospitalization and morbidity rates were observed for indigenous people in canada, the united states, new zealand, and australia [161] [162] [163] [164] [165] . likewise, indigenous populations experienced higher mortality rates during the 1918 pandemic (8.5% compared to 2.5%, worldwide [165] ). higher influenza virus infection rates in indigenous populations may be related to crowded living conditions, increased rates of chronic disease (lung, heart, and metabolic) and co-morbidities that exacerbate the severity of infection. nevertheless, recovery from influenza depends strongly on the ability to mobilize multiple arms of the immune system-in particular, an early cd8 + t cell response [30] . the severity of influenza disease and prolonged hospitalisation periods observed for indigenous people may therefore reflect a lack of pre-existing protective cd8 + t cell immunity that promotes rapid recovery. as mentioned, hla-a*24 alleles-a known risk factor for severe ph1n1 disease [151] -are enriched within several global indigenous populations [152, 153] , indicating a possible hla-related deficiency in t cell responsiveness to influenza that may contribute to the vulnerability of these communities. notably, the hla profile of indigenous australians is relatively restricted and quite distinct from non-indigenous australians, with predominant use of hla-a*34:01, 24:02, 02:01, 11:01, and hla-b*13:01, 56:01/02, 15:21, 40:01/02 [153] . as such, the dominant cd8 + t cell responses in indigenous australians are likely to focus on different peptide+hla epitopes compared to other ethnicities. since studies have mostly focused on identifying and characterising immunogenic peptides for common and widely expressed hla alleles such as hla-a*02:01, immunogenic influenza epitopes are yet to be defined for most (71%) of the hla-i alleles found in indigenous australians. however, analysis of hla-a*02:01-restricted m1 58-66 -specific cd8 + t cells found comparable magnitude, functional quality, and clonal characteristics in indigenous australians and non-indigenous australians, suggesting that a*02:01 + indigenous australians (representing 10-15% of this population) have robust cross-protective t cell immunity to any influenza a virus [45, 153] . however, accurately determining the extent and quality of cd8 + t cell immunity in indigenous australians and other indigenous populations worldwide will require the identification of prominent t cell targets for the relevant indigenous hlas. it will be of great interest to see how effectively these hlas elicit influenza-specific cd8 + t cell immunity. based on hla profile, targeting responses to a few prominent hlas in indigenous australians could achieve high levels of population coverage [153] , but may necessitate a tailored vaccine, as many of these hla alleles occur rarely in other ethnicities. clearly, much needs to be done to improve our understanding of influenza virus infection in indigenous populations before we can design better protocols to protect these populations, which are at greater risk of severe influenza disease and death. despite the challenges of hla diversity and possible confounding associations with metabolic and autoimmune disorders, understanding t cell responsiveness to influenza across a broad range of hla profiles will be an important part of designing and testing the efficacy of future vaccines. although response magnitude-typically measured in the periphery-is undoubtedly a critical aspect of effective anti-influenza t cell immunity [49, 94] , polyfunctionality in the t cell response is also linked with improved outcome to infection [166, 167] . hla genotype may be an important intrinsic factor shaping the magnitude and functional profile of t cell responses. hla-b alleles are significantly better than hla-a alleles at generating robust polyfunctional (ifn-γ and il-2) cd8 + t cell responses to hiv, cmv, ebv, and influenza [168] . while this reflects a general enhanced effect for hla-b alleles, there also appears to be hierarchy of association with polyfunctionality amongst hla-b alleles. the combination of hla alleles expressed can also impact the number and tcr repertoire of epitope-specific precursors, while altered peptide presentation, modulation of surface expression, and competition for overlapping peptides can shape the activation and proliferation of these precursors during infection in the context of different hla profiles [147, [169] [170] [171] [172] [173] [174] [175] . it is therefore very difficult to predict t cell response hierarchies to an epitope across the hla-diverse human population. however, focusing on the largest immunodominant t cell responses may not be all-important, as smaller subdominant t cell epitopes can also contribute to influenza virus clearance, and while they may be underutilized following natural infection, they could be harnessed by vaccination to achieve their full antiviral potential and provide a broad combined response that does better than natural infection [38] . increasing the breadth of the antiviral response by targeting subdominant epitopes may also reduce the potential for mutational escape in immunodominant epitopes [148, 176] . while an effective influenza vaccine should capitalize on the ability of protective hla alleles to present universally conserved viral components by self-selecting peptides for presentation and elicit broad spectrum t cell immunity, singling out specific peptides as vaccine targets could come at the cost of population-wide coverage across diverse hla profiles. approaches that incorporate whole protein antigens are more likely to provide coverage in the human setting of diverse hla types and avoid concentrated immune pressure that may drive the emergence of escape mutations in targeted epitopes. furthermore, strategies could be used to optimize peptide presentation by risk hla alleles through modifications of extra-epitopic processing sites by vaccine design. upstream extra-epitopic sequences have already been shown to alter cd8 + t cell responses to the immunodominant a2-m1 58-66 cd8 + t cell response, possibly via changes to the cleavage pattern of the m1 protein that influence the extent of m1 58-66 peptide presentation [177] . an ideal influenza t cell-based vaccine would therefore induce an overall robust response comprised of multiple cross-reactive epitope-specific t cell populations with high functional quality and engage multiple immune mechanisms for greater synergy of protection ( figure 2 ). it would encompass the importance of hla coverage even for minor ethnicities with rare hla types, and importantly, leverage our knowledge of key epitopes and cross-reactive t cell responses to do better than nature. influenza is an rna virus, and uses its own error-prone rna-dependent rna polymerase for replicating its genome. therefore, the virus is able to adapt rapidly, and is infamous for antigenic drift and resistance to anti-virals under selection pressure. virus escape from immune-mediated control undermines effective vaccination, and will also need to be considered for a t cell-inducing vaccine. while t cell-targeted proteins and peptides are more highly conserved than the antibody targets in the surface ha, variation and adaptation in t cell epitopes derived from influenza viruses have already been identified. cd8 + t cell escape has been observed in some t cell epitopes of naturally circulating influenza viruses [45, [177] [178] [179] , which raises the concern for vaccine-mediated t cell escape. as t cells recognize viral peptides within host mhc, viruses can mutate at mhc-anchor residues to reduce presentation [180] [181] [182] [183] [184] . alternatively, exposed residues can form contacts with the tcr for recognition, and variation at tcr contact sites can reduce recognition by existing t cell responses [176, 185] . however, due to tcr diversity, establishing a new t cell response to the new peptide variant is often subsequently possible [169, 176, 182, 183, 186, 187] . mutation of viral peptides at mhc anchor residues or tcr contacts can impact viral clearance [183] and the recall of established heterosubtypic memory t cell responses [176, 180] . however, this concept remains under-appreciated, and could undermine t cell-targeted vaccine-mediated control. our previous preliminary data from the isolation of viral rna from the lungs of infected mice shows that influenza escape can occur very early after the infection of an individual mouse and is common across individual mice, and thus is not a rare event [180] . mutations at anchor sites and tcr contacts for cd8 + t cell influenza epitopes were readily identified, and reverted in the absence of epitope-specific immune pressure [180] , which may suggest hla frequencies in the population will determine the rate of t cell-mediated immune escape. population sequence studies of drift in the np protein have identified positive selection pressure [28] , and furthermore, at the epitope level, anchor mutations were identified in the hla-b*27:05-and b*08:01-restricted np 383-391/380-388 epitope [179] . other evidence of immune escape in np and m1 cd8 + t cell epitopes showed that epitopes are functionally constrained, limiting the extent of variation possible, but t cells posed an evolutionary pressure on influenza virus [28, 188] . the influenza m1 58-66 epitope reproducibly selects a dominant public trav27/trbv19 + tcrαβ in hla-a*02:01 + donors that can cross-recognize naturally occurring m1 58-66 peptide variants, seemingly limiting the establishment of mutant strains within the circulating pool of human influenza a viruses and conferring universal immunity to influenza in a*02:01 + individuals [45] . conversely, in the immunodominant hla-b*07/b*35:01 restricted np 418-426 epitope, a different pattern of sequential viral variation has generated over 20 different peptide variants at tcr contact sites [189] , with t cell evasion occurring for selected peptides [169, 186] , resulting in the need for priming multiple t cell sets for the coverage of diverse np 418 variants. in this situation, successive waves of exposure to np 418-426 variants may drive diverse and unique tcrαβ repertoire recruitment with variable patterns of cross-reactivity against individual variants that favours, rather than controls, the emergence of additional mutants [45, 189] . therefore, pre-emptive priming of t cell subsets to common escape variants could circumvent immune escape [176] , and thus could be incorporated into future vaccine strategies that will need to consider contrasting patterns of epitope variation and immune selection. a tailored mosaic peptide vaccine might be the best way to circumvent influenza virus escape, but on a population level for vaccine rollout would be impractical considering hla selection for full-length proteins and immune competition during t cell priming, this area of research needs further exploration. the h5-derived np protein is already able to be interchanged in the leningrad laiv backbone, demonstrating the possibility of a mosaic approach to incorporating t cell peptide variants with current methods [190] . furthermore, a recent study demonstrated that hiv-infected children, as opposed to hiv-infected adults, were able to generate hiv variant-specific cd8 + t cell responses limiting the selection of escape-epitope variants early in infection [191] . a superior immune response against natural influenza virus infections has also been observed in children aged 4-14, but is not well understood [192] , identifying a key window for vaccination of the t cell compartment to do better than nature. together, these studies suggest that including common tcr escape variant epitopes in a t cell-inducing influenza vaccine might be most effective in young children and could prevent vaccine-mediated t cell escape. overall, more research is needed to establish whether combining t cell epitopes, tcr escape variants, and timing of vaccination will benefit t cell-inducing influenza vaccines. in addition, little is known about cd4 + epitope variation during influenza infection due to a paucity of defined epitopes. furthermore, data is also lacking on individual human viral quasi-species generated during infection, with only one example of sequencing of t cell peptides from a longitudinal infected paediatric patient-derived viral rna [180] . this area of research will need to be expanded to enable a vaccine design that thwarts viral escape. while the recent licensure of recombinant ha protein-based vaccines can overcome manufacturing delays and problems with egg-adaptations, they represent only a marginal improvement of an old method based on inducing ha-targeted antibodies. this approach will not improve the breadth of vaccine coverage, and thus new and novel approaches towards influenza vaccination should be considered. t cell immunity has the strongest potential as the immune correlate capable for true universal pan-influenza immunity (table 1) . current efforts on the clinical development of pandemic vaccines to utilise t cells do not match this potential, and should be further prioritised. vaccines are currently being developed to utilize t cell-based immune control with the potential for "universal" protection from influenza virus infection. a number of strategies are in clinical development [193] (figure 3) , including the use of vectors such as modified vaccinia ankara (mva) and simian adenovirus encoding the internal np and m1 proteins of influenza, adenovirus 5 vectored vaccines containing the ha alone, and recombinant peptide approaches for mosaic of conserved peptides or np with m2e proteins. viral vectors have different safety profiles to recombinant proteins and inactivated vaccines, but maybe the most promising approach for robust immune responses with the potential to do "better than nature". viral vectors can be replicating (such as vaccinia, adenovirus 5, and simian adenovirus) and not safe for use in the immunocompromised and elderly. non-replicating vectors (including mva, e1-deleted adenoviruses, influenza minus ha signal peptide) can also be used, and are safe in everyone. promising results have been reported for the mva+np/m1 vaccine, boosting robust t cell responses in adults and the elderly [70] and protecting from influenza challenge [194] . furthermore, t cell-inducing vaccine strategies have also been assessed in ferret transmission models, specifically the ha minus signal peptide vaccine, which shows reduced transmission time and peak viral loads from vaccinated ferrets to naïve ferrets [75] . t cell-inducing vaccines for targeting pandemic potential viruses are a significantly under-utilised resource, with only 13% of pandemic potential vaccines in clinical development capable of inducing robust t cell responses ( figure 3a ). the majority (83%) of avian h5n1 and h7n9 pandemic potential vaccine candidates are being produced as inactivated vaccines, with the addition of adjuvant to increase immunogenicity and potentiate t cell responses. adjuvants are used in 76% of non-t cell-targeted vaccines, while only 13% of t cell-targeting vaccines use adjuvant ( figure 3a ). the methods of delivery for t cell-inducing vaccines typically do not require exogenous adjuvant because viral vectors are self-adjuvanting, but their use is constrained in some populations, such as the elderly, young children, and pregnant women. [193] , including those that use adjuvant or are reactive against avian influenza viruses (h5, h7, or h9 subtypes). vaccines missing input or not defined were excluded. (b) proportion of total vaccines from (a). * denotes vaccines which are designed to stimulate t cell response. squalene-oil-in-water-based adjuvants (i.e., mf59 and as03) have been licensed for use with some inactivated influenza vaccines in europe and the us, and have been reported to improve cd4 + t cell responses after vaccination, presumably through the robust stimulation of innate immune responses, leading to enhanced antigen presentation [195] [196] [197] . the mf59 iiv [72] can induce igg isotype switching in a cd4 + t cell-independent manner, while still inducing a robust cd8 + t cell response. this relationship may be exploited in the context of cd4 + t cell imprinting. there is also a suggestion that these adjuvants can improve cd8 + t cell responses. the vaccination of ferrets with mf59 or as03-adjuvanted, inactivated h5n1 vaccines elicited a stronger cd8 + t cell response upon virus challenge compared to unadjuvanted vaccines [111] . however, these adjuvants' effects on the cd8 + t cell populations in humans is limited [198] . care must also be taken when applying inferences from animal data to the human setting, as the majority of animal studies use naïve animals for testing vaccine responses, whereas only the very youngest in the human population are naïve to influenza virus. consensus on an acceptable level of protection for t cell vaccines -need to consider the context of pandemic preparedness and threat of zoonotic infections. -whilst immunity induced may not be sterilizing, a vaccine that reduces symptoms, the length and severity of infection, prevents deaths and hospitalizations, and potentially decreases transmission in the community may bring sufficient health and economic benefits to be worthwhile. targeting heterologous t cell responses that induce broad protection against diverse influenza virus strains and subtypes. -greater knowledge of influenza epitopes recognised by cd4 + and cd8 + t cells. -need to define universally conserved virus components that elicit broad spectrum t cell immunity for incorporation in a t cell vaccine. providing population-wide coverage across diverse hla profiles and ethnicities. -approaches that incorporate whole protein antigens or complete virus rather than selected peptides. -employ strategies to optimize peptide presentation and boost responses to subdominant epitopes (e.g., modification of extra-epitopic processing sites). -consider tailored vaccine design for ethnicities with unique or 'risk' hla profiles that place them at high risk of severe influenza disease. circumventing virus escape of t cell immunity. -spread immune pressure over multiple epitopes through use of full proteins or whole virus. -pre-emptively prime t cell subsets to common escape variants at a young age. -combine with other immune mechanisms such as induction of stem antibodies to reduce immune pressure on t cell epitopes. establish local immunity at the site of infection seeding durable, effective trm memory populations in the lung and upper respiratory tract. -requires local (i.e., intranasal) administration of antigen. -further work needed to determine the lifespan and stability of human lung and airway trm, and their potential to provide durable vaccine memory. synergize multiple immune mechanisms combining long-lasting broadly-reactive t and b cell immunity. -t cell vaccines should be used in combination with improved antibody vaccine targeting methods and induce multiple responses (e.g., peripheral cd8 + t cells, lung-resident cd8 + trm, cd4 + t fh cells, cd4 + t h1 , memory b cells and cross-reactive stem-specific antibodies) -in a combinatorial vaccine, it will be important to consider the long-term effects of vaccine imprinting on t cell and b cell responses, particularly in the face of ever-evolving influenza viruses. in addition to broadly protective t cell vaccines, various vaccination strategies to elicit ha-stem-specific antibodies are currently under development (reviewed in [199] ). however, high concentrations of ha-stem-specific antibodies are required to induce sterilizing immunity [200] . failure to induce high enough ha-stem-specific antibody titres in some individuals combined with the continuous immune pressure on this region in the rest of the population could eventually lead to unforeseen ha-stem escape mutations. a universal influenza vaccine strategy will greatly benefit from an additional layer of long-lasting broadly-reactive immunity in the form of a t cell component to dampen the severity and limit the spread of an influenza virus that managed to escape the ha stem-specific antibody response. reciprocally, the induction of additional ha-stem-specific antibodies with vaccination could help prevent vaccine-mediated t cell escape, as most natural influenza viruses will be neutralized before they establish an infection, limiting their exposure to cd8 + t cell immune pressure. a true universal influenza vaccine would combine the best of both worlds, as a "one-two" punch against influenza viruses. this review has highlighted some under-appreciated aspects of influenza-specific t cell immunity for consideration in the design of a universal vaccine of the future (table 1 ). this includes some caveats, such as the potential short half-life of t cell resident memory, t cell immune escape, and the impact of antigen presentation timing for immunodominance hierarchies in the context of hla alleles and population-wide coverage. further research is needed in the t cell field to identify their protective capacity and the optimal vaccine design for safe delivery, resulting in the longest duration effective memory population. while statistical models have predicted that a t cell-inducing vaccine is likely to be more effective in antibody naïve t cell-primed subjects for h7n9 infection than seasonal h3n2 viruses [201] , wider efficacy trials are needed in combination with clinical data. another challenge faced by t cell-inducing vaccines is that they will need to improve the standard of care provided by the current inactivated vaccines. inactivated vaccines and vaccine vectors containing influenza viral proteins elicit different immune correlates of protection, and have so far not been compared head-to-head in a large-scale efficacy trial. unfortunately, this is not in the foreseeable future when the role of t cells mediating protection from infection are still being fundamentally discussed. the use of t cell-inducing vaccines will require the standardisation of assays to assess t cell memory. the hai assay is a relatively simple rudimentary measure of virus-specific antibodies which has been in use for over 70 years, and is now well standardised [202] . in contrast, the study of influenza-specific t cell memory responses from human subjects is undoubtedly more technically-demanding and resource heavy compared to serology approaches. there is currently some concerted effort towards standardizing and streamlining experimental approaches for t cell assays in vaccine studies [203] , although it will likely take some time before a standard t cell assay is identified. while t cells have a vast potential for breadth of recognition against influenza viruses, the field is in its relative infancy compared to antibody-based vaccines. a quantum leap will be needed in adapting vaccine production, safety and efficacy trials, and standardisation of assays for their assessment on a large efficacy trial scale for their realization. the authors declare no conflict of interest. the 2003 world health assembly resolution on seasonal influenza vaccination coverage and the 2009 influenza pandemic have had very little impact on improving influenza control and pandemic preparedness influenza activity-united states, 2014-15 season and composition of the 2015-16 influenza vaccine h3n2 mismatch of 2014-15 northern hemisphere influenza vaccines and head-to-head comparison between human and ferret antisera 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doi: 10.1016/j.berh.2020.101528 sha: doc_id: 349451 cord_uid: vak2p7ac there has been a progressive interest on the modifications of the human defense system following insults occurring in the interface between our body and the external environment, as they may provoke or worsen disease states. studies suggest the billions of germs that compose the gut microbiota influence one’s innate and adaptive immune responses at the intestinal level, but these microorganisms may also impact rheumatic diseases. the microbiota of the skin, respiratory and urinary tracts may also be relevant in rheumatology. evidence indicates that changes in the gut microbiome alter the pathogenesis of immune-mediated diseases such as rheumatoid arthritis and ankylosing spondylitis but also of other disorders like atherosclerosis and osteoarthritis. therapeutic strategies to modify the microbiota, including probiotics and fecal microbiota transplantation, have been received with skepticism, which, in turn, has drawn attention back to previously developed interventions such as antibiotics. helminths adapted to humans over the evolution process, but their role in disease modulation, particularly immune-mediated diseases, remains to be understood. the present review focuses on data concerning modifications of the immune system induced by interactions with microbes and pluricellular organisms, namely helminths, and their impact on rheumatic diseases. practical aspects, including specific microbiota-targeted therapies, are also discussed. the coevolution between germs and humans has been marked by mankind development-related modifications. migratory flows and industrialization have affected farming, food intake, housing, and clothing, causing a profound impact on the environment [1] . the most compelling evidence of genetics' relevance to the pathophysiology of rheumatic diseases is the association of the human histocompatibility antigen hla-b27 with inflammatory spondyloarthropathies (spa). hla-b27 prevalence varies across populations and some hla-b27 variants have been associated with a decreased prevalence of ankylosing spondylitis, highlighting the complexity of this association [2] . of interest, concordance rates for identical twins in rheumatoid arthritis (ra) vary from 15 to 30%, which is similar to the 30-40% concordance rate in type i diabetes, a disease considered to have a clear and strong genetic influence [3] . since most prevalent rheumatic diseases are not directly associated with specific genes, it is likely that various genes, together with environmental agents, influence the natural course of such disorders. the possibility that germs (microbes) present in the human body modulate the immune response is a classical concept. however, direct causality relying on koch's postulates has been difficult to prove, thereby limiting the association of a specific germ with a disease. fastidious germs and asymptomatic carrier states represent challenges when attempting to implicate a specific insulting agent as the cause of a disease. germs populating "open" body areas such as the skin, oral cavity, respiratory, and gastrointestinal systems, as well as "extracellular" and secretory components are collectively called the microbiome, a term that does not encompass multicellular agents that may also be relevant in this context like worms and fungi. modern biotechnology is based on complex dna sequencing rather than culture and staining procedures. although this has greatly improved the identification of germs, it has also added to the complexity of when attempting to understand parasite-host interactions [4, 5] . microbiome issues have attracted the interest of various researchers who have produced excellent articles and reviews on the subject of germs and human interactions. nonetheless, not much attention has been granted to helminths, which might potentially have a role in this scenario. a decrease in the prevalence of infections and infestations was previously associated with an increased prevalence of allergic and autoimmune diseases among populations living in western developed countries. moreover, water purification, massive vaccination and other sanitary measures provided health benefits to people living in industrialized countries [6] . still, these apparently healthful "cleaning" strategies could also reduce human microbiota diversity, which could in some way negatively impact one's health. likewise, improvement in health care practices can also render adverse events. recent outbreaks, such as the arboviruses epidemics in brazil in the last 10 years [7] and the ongoing coronavirus pandemic, lead to the immediate incorporation of individual practices aiming at the protection against contamination. attitudes like avoiding contact with other people and the use of handrails in public transportation, or wearing gloves and masks are deliberately taken in an attempt to prevent infection [8] . while this course of action is understandable during a pandemic, excessive handwashing or showers, avoidance of child contact with unprotected soil, grass, and animals, may compromise the stimulation of the human immune system, thereby jeopardizing the body's response to infections [6, 9] . although it is still debatable, reduced breastfeeding and increase in caesarean delivery as well as massive vaccination were also shown to have a negative impact on immune development, being associated with increased prevalence of allergic and autoimmune diseases [10] . following this line of thought, researchers have previously compared the immune system with the central nervous system based on the idea that establishing connections and plasticity are probably vital to the functioning of both systems. based on this assumption, avoiding contact with germs may result in more vulnerability [11] . one possible mechanism for the breastfeeding benefit is the presence of oligosaccharides in human milk, which could modulate the intestinal microbiota. it was recently shown that mice exposed to these oligosaccharides did not develop a type 1 experimental diabetes. that effect was linked to modifications of the intestinal microbiota, leading to an increase in short-chain fatty acid content in the gut. xiao et al. (2018) used their results to justify the benefits of exclusive breastfeeding in humans. we should consider that oligosaccharides isolated from human milk rather than mouse milk were offered to the animals. applying that premise to mammalian milk it might well be that cow's milk could be beneficial to humans, provided that both contain the same type and amount of oligosaccharides [12] . examples of a possible link between robust sanitary measures and increased incidence of immune-mediated diseases include the augmented prevalence of asthma in developed countries [13] , at the same time that autoimmune diseases appear less frequent or less severe in nigeria, where proper sanitation is still to be implemented [14] . inflammatory bowel diseases, e.g. crohn's disease, are considered perfect models of the association between changes in dietary habits and less exposure to germs with increased incidence of diseases considered to be immune-mediated. crohn's disease is currently more prevalent in wealthy populations living in the northern hemisphere, but numbers appear to be increasing in developing countries, possibly due to dietary modification and sanitary improvements [15] . other immune-mediated diseases including type 1 diabetes, systemic lupus erythematosus (sle), ra, and multiple sclerosis have also been reported to be less prevalent in developing countries [6] . "everything is autoimmune until proven otherwise" was once said, claiming for a ubiquitous role of autoimmunity in any disease state. thus, splitting diseases into immune or non-immune-mediated would merely serve didactic purposes [16] . innate immune mechanisms operate in close association with the adaptive immune responses, modulating the body's defense against tissue damage caused by a germ, trauma or cancer. this also applies to housekeeping processes such as digestion, breathing, and senescence, which generate by-products that may cause harm to the host. for instance, uric acid released in the event of cell death alerts the immune system, unleashing apoptosis to avoid systemic and potentially harmful inflammation [17] . atherosclerosis and cardiovascular damage have been associated to changes in the intestinal microbiota. of interest, studies have linked the processing of phosphatidylcholine in the intestine to increased atherogenesis by producing trimethylamine-n-oxide (tmao) [18] . accordingly, modification of the intestinal microbiota by dietary changes, repopulation of the gut flora or administration of probiotics could lead to a marked reduction in plasma and urine tmao levels, which was associated with a decreased risk of major cardiovascular events [19, 20] . although we may not consider atherosclerosis a bona fide immunemediated disease, the above-described results indicate that microbiota changes could alter disease course. limitations of these studies include assessment of tmao levels systemically as well as evaluation of the microbiota only at the intestinal level. in fact, antibiotics might have altered the microbiota in other sites such as the skin, oral cavity, and respiratory tract, which were not directly evaluated. although causality is hard to prove, the data reinforces the need to pursue the concept of an association between atherosclerosis and microbiota. it is beyond our scope to fully discuss all aspects of the microbiome. we will focus in bacteria and their products, and helminths as triggers or modulators of disease processes in rheumatic diseases. the microbiota has gained more and more relevance as a driver in the pathogenesis of various rheumatic diseases including ra, sle and, more recently, osteoarthritis (oa) [6, 21] . the gastrointestinal tract appears as a natural target, partially because it harbors a great and diverse number of bacteria. nutritional habits, antibiotic use, constipation as well as gastrointestinal infections are some factors that may alter the bowel's microbiome. considering the huge number of cells linked to immune mediation in the gut, e.g. cell population in peyer's patches, modifications of the gut flora definitely impact one's immune response [5] . animal studies showed that the modification of the intestinal bacterial flora influenced the severity of antigen-induced arthritis (aia) [22] and experimental allergic encephalomyelitis -i.e. the most commonly used animal model for human multiple sclerosis [23] . changes in the cytokine profile of those animals, with predominance of type 2 t helper (th) cell responses and increase in the number of t regulatory (treg) cells, were associated with improvement following restoration of a "normal" flora in germfree animals that received faecal transplantation [5] . issues regarding innate immune mechanisms are receiving more attention, given their importance to the physiology of the gastrointestinal tract. integrity of the mucosa with a protective layer of mucus, acid ph in the stomach, biliary fluid, maintenance of junctions between epithelial cells as well as presence of antimicrobial secreted peptides, e.g. defensins and cathelicidins, and immunoglobulin a constitutively present in the lumen represent first-line defenses against invaders. distinguishing "normal flora" from invading organisms can be made by pattern recognition receptors (prr) exposed in intestinal epithelial cells, which include toll-like receptors (tlr) and nucleotide-binding oligomerization domain (nod)-like receptors. sensing of pathogens or endogenous stimuli released from dying or damaged cells by prr leads to downstream activation of intracellular signal transduction inflammatory pathways [4, 5] . a concerted effort between both innate and adaptive immune responses is then constructed to face damage. it has been proposed that the cost to the immune system progressively increases in a stepwise process resulting from engagement or disruption of successive protective barriers [24] . simple as it may seem, normal peristalsis contributes to dispatch pathogenic material. during constipation, delayed bowel emptying favors proliferation of germs, and disturbance of vesical bladder clearance leads to recurrent urinary tract infections, particularly in women [25, 26] . in developing countries with poor sanitation, recurrent episodes of diarrhea compromise maintenance of a normal flora due to disruption of the gut epithelia, as well as to altered restoration of the normal flora secondary to speedy bowel movements [27] . on the other hand, seniors are more susceptible to constipation. this may further compromise gut flora and favor the development of diverticula, which are potential sites of inflammation [28] . tissue-resident lymphocytes, eosinophils, macrophages, and treg cells are present in the intestinal barrier acting to contain the inflammatory insult. breaches in the gastrointestinal barrier expose immunocompetent cells located in the lamina propria and increase systemic migration of germs or their constituents (antigens) allowing their encounter with professional dendritic antigen-presenting cells in mesenteric lymph nodes, amplifying an inflammatory response that should have been kept locally. in the appropriate unlucky host, bearing a susceptible genotype, the triggering of a complex immune response spreads systemically [4, 5, 24] . although unproven, one may speculate that the highly irrigated synovial tissue with fenestrated capillaries and easily accessible immune competent cells, such as type a synoviocytes, are a preferable harbor to those germs or their parts (antigens), where they can foster a sustained immune response. medications used to treat rheumatic diseases also modify our microbiome. sulphasalazine, which has antibiotic properties, is still an option to treat inflammatory bowel disease and chronic inflammatory arthropathies [29, 30] . minocycline, marketed primarily as an antibiotic, was until recently used as a disease-modifier drug in ra [31] . methotrexate, a cornerstone in the treatment of ra, as well as leflunomide may have part of their therapeutic benefit linked to changes in the host microbiome [32] . more recently, fexofenadine, a compound to treat chronic allergic diseases, was shown to inhibit tumor necrosis factor activity, revealing an immunomodulatory role to fexofenadine not hitherto described [33] . chloroquine also has antiviral activity and has just been shown to inhibit in vitro coronavirus replication in concentrations that could be achieved in clinical practice [34] . of interest, the recent worldwide arbovirus chikungunya outbreak has been associated with joint manifestations, which could derive from a direct viral activity or the triggering of existing subclinical inflammation in the joints [35] . in addition, other viruses like epstein-barr and parvovirus b19 have long been associated with the pathogenesis of immunemediated diseases. hence, the recent proposed antiviral chloroquine activity could well be an additional mechanism to explain its effective response to treat various rheumatic diseases, including the ability to ameliorate musculoskeletal manifestations following chikungunya infection [36] . vulnerability to infections particularly in the immunocompromised host occurs in "open systems" such as the skin, respiratory, urogenital, and gastrointestinal tracts. periodontitis has been shown to have a major role in the pathogenesis of ra. however, recent epidemiological data may question periodontitis relevance in ra pathogenesis. using modelling to extrapolate collected data, both the prevalence and burden of ra were reported to be higher in high-income populations where oral hygiene is presumably better [37] . edentulation is highly prevalent among low-income brazilians and chronic periodontitis is the major cause of tooth loss during adulthood [38] . yet, ra burden was shown to be milder in brazil and other low-income areas of the globe, as compared to wealthy regions. also, we have reported very low childhood health assessment questionnaire (chaq) scoring, meaning a favorable outcome, among low-income juvenile idiopathic arthritis (jia) patients living in one of the less developed areas in brazil. indeed, it is surprising that the chaq scores were lower than those collected among jia patients evaluated in the pediatric rheumatology international trials organization (printo) and childhood arthritis and rheumatology research alliance (carra) registries that gather data from high-income populations, with presumably better sanitation and oral hygiene. although it may just be coincidental, there is a high prevalence of helminthiasis in our low-income population leading us to speculate whether helminths or their products down-modulate inflammation in autoimmune diseases, thus decreasing severity [39] ( figure 1 ). in developing regions, despite low-income and low-literacy that negatively impact disease outcome, helminths' infestations may down-modulate inflammation, resulting in less damage. maintaining skin integrity is probably as relevant as avoiding gut damage for adequate immune homeostasis. ultraviolet radiation, trauma, and tick bites are common causes of lesions inflicted to the skin, paving a way to stimulate immune competent cells located in the dermis. stratification of skin layers and the presence of continuous capillaries and fat tissue keep inflammatory stimuli away from direct contact to "hidden" professional antigen presenting cells of the skin, such as the dendritic langerhans cells. in addition, innate immune mechanisms, including tight junctions between cells, pigmentation, hair follicles, sweat and sebaceous material prevent access to cells located in the dermis. as an analogy to the gut barriers, it seems that every effort is made in order to avoid access of germs, or their parts (antigens), to satellite lymph nodes where antigen presentation and systemic widespread inflammation ensues. it was suggested that a homeostatic response of intestinal cells to commensals is under tight regulation, including the production of antibodies to those germs. that tolerance could be broken during infections, triggering the proliferation of cd4 + t cells both to the invading pathogen as well as to commensals, developing memory cells to the new infecting agent. in a specific host that equilibrium could be lost leading to unresolving inflammation like that occurring in crohn´s disease [40] . similar mechanisms could be the reason for the occurrence of gastrointestinal manifestations in spa. a recent report has shown that skin-resident t lymphocytes constitutively expressing interleukin (il)-17a coexist with commensals, contributing to tissue integrity. harmful stimuli represented by trauma, infection or insect bite brake barriers, promoting cell plasticity so that the cytokine repertoire is now enriched with il-5 and il-13, leading to prolonged inflammation. therefore, skin barriers help maintain homeostasis by avoiding contact of stimuli to professional cells of the immune system [41] . once a certain stimulus, in a susceptible host, surpasses that initial defense, systemic inflammation is unleashed, provoking non-resolving autoinflammatory or autoimmune diseases, whether affecting mainly innate or adaptive immune responses, respectively. as mentioned above, worldwide epidemiological data show that the burden of ra varies across the globe, with numbers appearing to be higher in developed countries [37] . previous estimates of ra have suggested a 0.5-1.0% prevalence in wealthy regions, with lower prevalence in underdeveloped countries, varying from 0.1 to 0.5% [42] . however, we have to bear in mind that more accurate data were collected in developing regions. thus, despite some discussion, research suggests ra is less prevalent and/or less severe among low-income populations. paradoxically, delay in diagnosis, less access to specialists, failure to comply with treatment and unavailability of disease-modifying compounds presumably carry a negative impact to the treatment of chronic inflammatory arthropathies [43] . regarding sle, it was shown that low-income patients living in the northeast of brazil had similar long-term damage evaluated by the systemic lupus collaborating clinics' (slicc) criteria, compared to patients from developed countries [44] . helminthiasis are still highly prevalent in the northeast of brazil and, according to the recommendations of the world health organization, it is current practice to prescribe prophylactic anti-helminthics to decrease the burden of parasite infections and helminths' infestations (https://extranet.who.int/rhl/topics/preconception-pregnancy-childbirthand-postpartum-care/antenatal-care/who-recommendation-preventiveanthelminthic-treatment; downloaded in feb 15, 2020). helminths have for a long time been considered relevant modulators of the immune response [45] . there are reports on the benefit of the administration of thichuris suis eggs to treat patients with crohn's disease [46] . our group has shown that animals subjected to collagen-induced arthritis and treated with a crude ascaris suum extract have decreased inflammation with reduction of cytokine release into the inflamed joints [47] . we then identified the gene sequence of a peptide from the a. suum extract present in the celomic fluid of that worm, which shows antiinflammatory activity in mice subjected to aia. intramuscular administration of a plasmid with the correspondent gene sequence decreased inflammation in aia mice, apparently through a shift of the macrophage population into an m2 phenotype (unpublished data). it is worth mentioning that oral administration of a. suum extract, rather than viable worms as with t. suis experiments, was effective, providing a proof-of-concept to support the idea that helminthic products released into the bowel modulate the inflammatory response [47] . it has also been previously shown that an excretory-secretory product of acantocheilonema vitae has an immunomodulatory role, being able to decrease osteoclast-mediated bone resorption via modification of the gut microbiota [48] . helminths or their products might have beneficial and harmful effects to the host. difficulties in establishing a net result should not prevent efforts to dissect those mechanisms, both to understand the pathophysiology of immune-mediated diseases as well as to develop therapeutic alternatives (figure 2) [49, 50] . possible mechanism where secretory products from a. suum drive macrophage differentiation into an m2 phenotype, leading to the decreased release of inflammatory mediators. in 2006, inman wrote "perhaps a word of caution is needed for the critics and a word of encouragement for those hunting microbes in the joints", when referring to the possible role of infection in the development of rheumatic diseases, specifically spa [51] . he compared this concept to the relationship between helicobacter pylori infection and gastritis, which was at first met with skepticism, but is nowadays accepted as a fact [51, 52] . when talking about "microbes in the joints", one can discuss the topic from the perspective of infection and how it can contribute to the development of certain rheumatic diseases, but also in a broader sense, considering the human microbiome (more specifically, the gut microbiome) and how it can be related to these disorders, specifically inflammatory arthropathies [51, 53] . the understanding of the depth of this relationship could, in the future, yield new forms of treatment [54] . spa, as a group, are characterized by findings of asymmetric oligoarthritis, axial involvement, particularly of the sacroiliac joints, and inflammatory back pain, enthesitis, and characteristic extra-articular features, including acute anterior uveitis and psoriasis [51, 55] . ankylosing spondylitis (as), psoriatic arthritis (psa), reactive arthritis (rea), and inflammatory bowel disease (ibd)-related arthritis [55] all share an association with hla-b27 [51] . the hla-b2705 subtype is the one that's most commonly associated with spa [56] . in fact, the presence of this allele is the greatest risk factor for the development of spa [57] . parallel to the genetic predisposition, there has been mounting evidence for the role of infection in the pathogenesis of spa [58, 59] . the clearest example of this association is illustrated by rea [51] . for a long time, there has been evidence that chlamydia trachomatis (an obligate intracellular parasite of eukaryotic cells [60] ) plays a role in the development of inflammatory arthropathies [61] . the pathogenesis of chlamydia-related arthritis can be considered distinct from that associated with enteric bacteria since it involves metabolically active organisms residing long-term within monocytic cells in synovial tissues, after resolution of the primary genital infection and migration of the cells to the joint, a process that is known as persistence [56, [61] [62] [63] . there is also evidence that differences in the expression of heat shock protein-60 genes may contribute to organism persistence [64] . another interesting aspect to consider is that only a minority of patients with chlamydia genital infection develop rea. this could be explained by the findings of gérard (2010) that showed only ocular serovar group chlamydiae in synovial biopsies from arthritis patients, who were pcr-positive for c. trachomatis dna in that tissue and not genital serovar. this could mean that the composition of the initial inoculum influences the probability of arthritis development [65] . these data, if confirmed, could be relevant when thinking about a way to monitor which patients will go on to develop or have a greater risk of developing arthritis. nevertheless, it is important to note that these findings were based on the evaluation of only four patients [65] . similarly, c. pneumoniae has also been accepted as an etiologic agent for inflammatory arthritis [66, 67] . earlier, it was believed that antibiotic treatment of patients with chlamydia-induced arthritis was ineffective [59, 61, 63, 68] , but recent studies indicate antibiotic therapy might be beneficial [56, 69] . however, this efficacy is not complete as the proposed treatment (6 months of oral antibiotic therapy with either doxycycline or azithromycin, both combined with rifampin) improves joint symptoms in 63% of patients and leads to remission in 22% [51] , indicating other mechanisms might be involved in the development of arthritis which, if better understood, could lead to the development of novel therapeutic targets [54, 59] . besides chlamydia, enteric pathogens such as campylobacter jejuni, clostridium difficile, escherichia coli, salmonella, shigella, and yersinia, and respiratory pathogens like chlamydia pneumoniae or mycoplasma pneumoniae have also been shown to give rise to reactive joint processes. evidence for the benefit of antibiotic therapy directed at those bacteria is sparse, since it doesn't appear to alter the course of the arthritis [56] . the actual concept of rea makes history of infection a required criterion for its diagnosis, but, unlike in septic arthritis, inflammatory arthritis is not directly caused by infection of joint tissue (positive culture), instead it follows an extra-articular infection [51, 56] . as for the other spas, which comprise the majority of the diseases in this group, with rea accounting for less than 2% of the overall disease burden [70] , the role of infection (whether there is a role or what that role is) is not as well established. nonetheless, there has also been some evidence regarding this matter [51, 59, 71, 72] -various authors have discussed the role of chlamydia, not only in rea but also in undifferentiated spa [71] . rashid et al. (2016) reported a link between elevated levels of klebsiella antibodies and as in various geographical locations [73] , and the development of this disease appears to be related to the presence of k. pneumoniae aerogenes in the gastrointestinal tract [58] . the role of hla-b27 has been a focus of research when trying to understand spa. in rea, hla-b27 is present in 50 to 80% of patients [74] . there are several theories that attempt to define how exactly hla-b27 predisposes to spa and how it is implicated in its pathogenesis [56, 75] . this is of particular relevance in the context of the arthritogenic peptide hypothesis, according to which microbial peptides mimic certain self-peptides. specifically, hla-b27 may have structural features that are shared with bacteria, causing reactivity of hla-b27-specific, cd8 + bearing cytotoxic t lymphocytes, leading to autoimmunity and tissue damage and inflammation [58, 76] . zhang et al. (2018) stated that as seems to be related to k. pneumoniae infection in hla-b27 positive patients, based on consistent findings of raised anti-k. pneumoniae antibodies and the presence of molecular mimicry between the hla-b27 molecule and bacterial antigens [58] . because some patients who are hla-b27 negative also develop rea, other investigations have focused on the role of specific microbial factors in the pathogenesis of the disease. for example, in patients with rea and documented s. typhimurium infection it has been suggested that salmonella outer membrane protein is able to stimulate interleukin (il)-17/il-23 production in synovial immune cells, possibly contributing to arthropathy [77] , considering the well-established role of this pathway in spa [79, 81] . there has also been some focus on the role played by the microbes that inhabit the human body and do not represent a state of infection -in this case, the focus has been centered around the gastrointestinal microbiota (oral and gut microbiota) -and its possible implication in the pathogenesis of spa [80] [81] [82] . changes in the homeostasis and balance of the microbiota can lead to subtle but perhaps profound alterations in the balance with the host's immune system, leading to a state of inflammation that may contribute to disease. the correlation between hla-b27 and as has been known since the 1970's [83, 84] . this relationship is also true for the other spa, as stated above. studies indicate inflammatory bowel disease, or, at least, intestinal inflammation, is more prevalent in spa patients (as or others) and some genes associated with as are also associated with ibd [83, 85] , including genes related to gut physiology and immunology. based on these associations, it has been postulated that imbalances and defects in gut microbiota can play a role in the pathogenesis of spa [85, 86] , since there's a significant clinical, genetic, immunological, and microbiological overlap between ibd and spa [55] . not surprisingly, there has also been evidence that hla-b27 affects the gut microbiome and its metabolites, and perhaps the handling of bacteria during infection [82, 83] . the gut microbiota is acquired post-partum and its composition can be influenced by various factors during its development until around 3 years of age, when it seems to stabilize. during a person's lifetime, the gut microbiota can be affected by diet and nutrition and also antibiotic use -disturbances in its balance can be a factor for disease [53] . a study that analyzed biopsies from the terminal ileum of 9 as patients showed the microbiome in as patients was different from healthy subjects, with greater abundance of bacteroidaceae, lachnospiraceae, porphyromonadaceae, rikenellaceae, and ruminococcaceae and lower abundance of prevotellaceae and veillonellaceae [53] . data has shown that hla-b27 can determine an increase in the intestinal permeability, which results in continuous antigenic stimulation with activation of effector t cells [53, 82, 87] . hla-b27 can also induce an endoplasmic reticulum stress response and promote autophagy/unfolded protein responses (upr), resulting in the release of tumor necrosis factor (tnf), interleukin-17 (il-17), overexpression of il-23 by paneth cells, and interferon-γ and increase in th17 cells [83] . il-23 can regulate the maturation of autoreactive th17 cells and induce chronic inflammation by stimulating il-17, il-6, il-8, and tnf production in neutrophils and macrophages [88] . there has also been evidence that hla-b27 may interact directly with gram negative bacteria, either by provoking an immune response through antigen mimicry (as previously discussed) or by altering genes expressed by bacteria, thus affecting dendritic cell function and influencing the immune response [89] . despite the relationship between spa, hla-b27 and the microbiome, the best example of the influence of gut dysbiosis in the pathogeneses of rheumatic diseases is still ra and its relationship with citrullinated proteins [90] [91] [92] . proteins are ubiquitous in the human body, assuming different forms and specific functions. after syntheses in ribosomes, they are further altered and specialized through posttranslational modification (ptm), which includes various processes. one of such processes, involving the conversion of arginine amino acid into citrulline amino acid, is called citrullination and occurs physiologically during apoptosis. this "biological operation" is catalyzed by peptidylarginine deiminase enzymes (pads) and produces an amino acid that is not encoded by the genome, being solely produced by this process [93] . there has been extensive research and data that demonstrate the role of pads in the development of immune-mediated diseases [93] , and in the field of rheumatology, this is particularly relevant in respect to ra. in humans, there are five pad isoenzymes (1 to 6), expressed in various tissues and organs that contribute to many of the body's physiological functions. they are actively expressed in the intestinal epithelium, making it a source of citrullinated peptides [90] . changes in oral microbiota can also influence progression and outcomes in ra [53] and porphyromonas gingivalis (pg) has been ascribed a major role. it is present in the oral cavity and proliferates in patients affected by periodontitis. the pad expressed by pg can lead to citrullination and may explain the close association between ra and periodontitis, an inflammatory disease of the oral mucosa [92] . there are actually reports of a direct correlation between the serum level of antibodies and pg plus acpas in ra patients [94] . however, as we mentioned above, discrepancies concerning increased ra burden in regions with better oral hygiene may question the relevance of periodontitis in ra pathogenesis [37] . when considering the role of gastrointestinal microbiota and infection in rheumatic diseases, the best characterization was achieved in ra, through the understanding of the process of citrullination and its relationship with oral microbiome, mainly pg. in spa, altered intestinal permeability and hla-b27's role in the induction of that disruption may trigger antigenic stimulation with t cell activation [53] . it is also relevant to refer, in respect to citrullination, that it was thought that citrullinated proteins were specific to the synovium of ra patients, but recent reports revealed citrullinated proteins are also present in the synovium of non-ra inflammatory conditions [95] , which may account for its probable role in other inflammatory arthropathies. antibiotic use may be considered a mixed blessing in in the context of therapeutics. while it is undisputable that antibiotics are lifesaving in many cases, widespread and unnecessary use of such compounds, particularly those with a broad-spectrum profile, leads to the development of bacterial resistance as well as alterations to the microbiome. modifications of the immune response occur secondarily to the eradication of commensal microorganisms, which are important stimuli to intestinal cells. probiotics are commonly regarded as a useful and harmless strategy to reconstitute the gastrointestinal flora following antibiotic use. probiotics can be routinely prescribed by physicians and other health care professionals, and may also be found as over-the-counter products, on the premise of restoring homeostasis. however, there is a lack of adequate studies documenting those benefits, not to mention that probiotics are marketed without complying with the strict rules used for drugs' approval. by not being appropriately regulated, neither the safety nor the efficacy of probiotics have been specifically documented. it was recently shown that supplementation of probiotics to humans or mice that received broad-spectrum antibiotics led to delayed restoration of the previous normal flora. on the other hand, autologous fecal microbiota transplantation (fmt) shortened the time for reconstitution of the gut flora [96] . although suez et al. (2018) have correctly discussed limitations and extrapolations from their single study in healthy volunteers, the results justify a closer look into the advertised benefits of probiotic use, regardless of indication, highlighting the need for data to adequately document harms and benefits. antibiotics are also used as modulators of the immune response. sulphasalazine is indicated in spa and ra as a disease-modifying compound due to its immunomodulatory activity [29, 31] . until recently, tetracyclines (minocycline) were used as disease-modifying antirheumatic drugs because of their anti-inflammatory effects involving the inhibition of proinflammatory cytokines and matrix metalloproteinase activity [30] . gold salts had their efficacy at least partially linked to antimicrobial activity and were formerly in use for ra treatment. regarding rea, and despite the difficulties in pointing to a specific germ as the causative agent, prolonged antibiotic use has been associated with decreased symptoms and disease duration [58, 71] . other medications already in use may have unsuspected antibiotic activity as the recently described antiviral chloroquine response. however, the most common and effective treatment for chronic inflammatory arthropathies relies in down-modulating inflammatory response-related targets with immune suppressors, aiming for symptom relief and halting structural tissue damage. following the boom in microbiome research, other microbiota directed therapeutic interventions appeared. for instance, changing dietary habits as a means to increase the supply of short-chain fatty acids by ingesting fiber is also an appealing strategy to improve immune function, because it is perceived as a non-harmful alternative in addition to the low-calorie profile benefit. a high-fiber diet favors the production of short-chain fatty acids, particularly propionate and butyrate, which have antiinflammatory and immunomodulating activities, and were associated to bone-sparing effects in ra patients. mice subjected to short-chain fatty acids' administration had decreased bone resorption partially mediated by down-regulation of tnf receptor associated factor (traf)-6 and nuclear factor of activated t-cells, cytoplasmic 1 (nfatc1) genes [97] . as a therapeutic alternative to treat chronic inflammation, fmt attracts both interest and skepticism. the successful use of fmt in patients affected by clostridium difficile infections refractory to antibiotic treatment came as a proof-of-concept promising study [98] . despite its recognized benefits, the procedure has its risks. harmful contaminants can be supplied together with the "good guys" that would restore the intestinal flora after fmt. indeed, the u.s. food and drug administration (fda) issued a safety alert on june 13, 2019 about the serious adverse reactions due to the transmission of multi-drug resistant organisms following fmt (www.fda.gov/media/86440, downloaded on feb 02, 2020). the logistics behind the necessary standardization of appropriate fmt "components" include storage, "quality" control regarding donors, recipients, expiration date, to mention a few issues. given the difficulty in translating fmt into clinical practice and the inconsistent data on probiotic use, microbial active pharmaceutical ingredients have also been developed as treatment strategies for rheumatic diseases. biological therapies involving the delivery of live organisms are a major challenge and regulations are being developed, but it was not without great care that the industry introduced biologicals into the therapeutic armamentarium of immune-mediated diseases. a recent review addressed issues about the development of microbial therapeutics, which is still in its early stages. uncontrolled proliferation of a living organism, toxicity and hypersensitivity reactions as well as long-term stability are some of the challenges that need to be overcome. pharmacokinetics, pharmacodynamics and the supply chain are matters of concern, particularly if one considers worldwide distribution. gut barriers, including acidic ph, bowel movement, mucus layer, bile salts, to name a few, also affect the bioavailability of oral vaccines or stability of microbe-derived orally active compounds [99] . challenges represent opportunities for improvement. considering the interest of big pharma companies on live biotherapeutics, the marketing approval of a specific microbial therapy will likely happen in a near future. * identify causal relationships between microbiome perturbations and human rheumatic disease * define roles, for skin, respiratory, and urogenital tract microbiota in the pathogenesis of rheumatic diseases; * conduct high-quality clinical trials with pharmaceutical grade probiotics in rheumatic diseases; * identify mechanisms by which antibiotics modulate f rheumatic diseases; * for patients with rheumatic disease, determine "healthy" and "harmful" dietary components that are simple,affordable, and 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and citrullination an overview of the intrinsic role of citrullination in autoimmune disorders peptidylarginine deiminase from porphyromonas gingivalis citrullinates human fibrinogen and α-enolase: implications for autoimmunity in rheumatoid arthritis association of periodontitis with rheumatoid arthritis: a pilot study specific presence of intracellular citrullinated proteins in rheumatoid arthritis synovium: relevance to antifilaggrin autoantibodies post-antibiotic gut mucosal microbiome reconstitution is impaired by probiotics and improved by autologous fmt short-chain fatty acids regulate systemic bone mass and protect from pathological bone loss fecal microbiota transplantation--an old therapy comes of age microbial therapeutics: new opportunities for drug delivery 4 item group: ig000129 manuscript title: microbes, helminths and rheumatic diseases chapter andar rodolfo teófilo -60430-270 -fortaleza -ce -brazil phone e-mail address: arocha@ufc.br the authors declare no conflict of interest practice points * antibiotic use should be made within specific indications, thereby trying to limit changes to the microbiota; * attention should be given to innate immune mechanisms as modifiers of disease states; * proven dietary recommendations for the treatment of rheumatic diseases are not yet available * general recommendations, such as avoiding excess salt and fat, and consuming fresh vegetables, are current best practice * benefits of probiotic use, at least in rheumatic diseases, demand bona fide data key: cord-352150-ey9kc7zj authors: degauque, nicolas; brouard, sophie; soulillou, jean-paul title: cross-reactivity of tcr repertoire: current concepts, challenges, and implication for allotransplantation date: 2016-03-24 journal: front immunol doi: 10.3389/fimmu.2016.00089 sha: doc_id: 352150 cord_uid: ey9kc7zj being able to track donor reactive t cells during the course of organ transplantation is a key to improve the graft survival, to prevent graft dysfunction, and to adapt the immunosuppressive regimen. the attempts of transplant immunologists have been for long hampered by the large size of the alloreactive t cell repertoire. understanding how self-tcr can interact with allogeneic mhc is a key to critically appraise the different assays available to analyze the tcr vβ repertoire usage. in this report, we will review conceptually and experimentally the process of cross-reactivity. we will then highlight what can be learned from allotransplantation, a situation of artificial cross-reactivity. finally, the lowand high-resolution techniques to characterize the tcr vβ repertoire usage in transplantation will be critically discussed. being able to track donor reactive t cells during the course of organ transplantation is a key to improve the graft survival, to prevent graft dysfunction, and to adapt the immunosuppressive regimen. the attempts of transplant immunologists have been for long hampered by the large size of the alloreactive t cell repertoire. understanding how self-tcr can interact with allogeneic mhc is a key to critically appraise the different assays available to analyze the tcr vβ repertoire usage. in this report, we will review conceptually and experimentally the process of cross-reactivity. we will then highlight what can be learned from allotransplantation, a situation of artificial cross-reactivity. finally, the low-and high-resolution techniques to characterize the tcr vβ repertoire usage in transplantation will be critically discussed. keywords: tcr repertoire, transplantation, cross-reactivity, alloreactivity, tcr, mhc, t cell understanding the cross-reactivity shaping the t lymphocyte receptor repertoire through evolution, numerous processes have been selected to generate a diverse repertoire of tcrαβ able to protect mammalian from pathogenic insults (figure 1) . highly similar genes recombine to form functional genes and generate a highly diverse tcr repertoire. tcrβ chains are encoded by distinct variable (v; trbv), diversity (d; trbd), and joining (j; trbj) genes, whereas tcrα chains are encoded by distinct sets of v and j genes (trav and traj). junctional diversification further extends the combinatorial diversity by either trimming gene ends or adding nucleotides between the recombining genes (1) . in contrast to the ighv (v genes of immunoglobulin heavy chain) germline dataset compiled by the immunogenetics (imgt) group that greatly benefit from the advanced of deep-sequencing technologies, the human tcr germline has been only minimally changed since the complete sequencing of the tcr gene loci in 1996 (2, 3) . the 65 functional genes, orfs, and pseudogenes have been reported for the trbv, 54 for the trav and 2 for the trbd dataset. the analysis of the tcr cdr3 is still a very challenging process. the identification of the trbd genes figure 1 | understand the cross-reactivity of a highly diverse tcr repertoire. a highly diverse tcrαβ repertoire is generated by iterative processes selected through evolution. combinatory diversity results from the selection of variable (v; trav and trbv), diversity (d; trbd) and joining (j; traj and trbj) genes. junctional diversification further extends the combinatorial diversity by either trimming gene ends or adding nucleotides between the recombining genes. finally, the association of the tcrα and tcrβ chain constitutes the final steps of the numerous iteration processes that lead to the generation of a highly diverse tcr repertoire, which is able to efficiently protect individuals from pathogenic stimulations. tcrαβ adopts a stereotype docking geometry atop the mhc/peptide complex. this orientation leads to a spatial interaction between the germline-encoded cdr1 and cdr2 of the tcrα and β chains and the edges of the peptidegroove of mhc. the accumulation of reported crystallographic structures has challenged the stereotypic view of the angle of the tcr docking. however, the recognition of conserved motifs on the side of mhc molecules by cd4/ cd8 co-receptor constrained the tcr docking geometry. despite the high diversity of the tcr repertoire, a high degree of cross-reactivity has been reported that could be explained by the "natural" ability of tcr to interact with mhc molecules (mhc focus model) as well as the interaction of tcr to a limited number of amino acids of the peptide bound to the mhc peptide groove. march 2016 | volume 7 | article 89 2 frontiers in immunology | www.frontiersin.org cannot be performed due to the high degree of similarities of the trbd at their 5′ ends, the short length of the two genes, and the presence of g-rich n nucleotides at the 5′ ends that could be also added by the tdt enzyme. it is misleading to estimate the combinatory diversity by simply multiplying together the number of v, d, and j genes (4). rather than a random combination of the tcr genes, studies have shown that tcr genes are highly biased in their usage, and that only part of the theoretical diversity is selected (5, 6) . chromosomal recombination patterns can be explained by variations in enhancers and recombination signal sequences (rss) and organization of the trbj genes (a block of six and seven genes located respectively downstream from the trdb1 and trdb2 gene) that leads to a bias in d-j pairing. the diversity of the tcr repertoire is further broaden during the rearrangement process first by the addition of p nucleotides (palindromic nucleotides) thanks to recombination activating gene-1 and -2 (rag1 and rag2) (7) that form hairpin loops at the gene end and then by the addition of n nucleotides (with a biased toward g nucleotides) by the terminal deoxynucleotidyl transferase (tdt) (8) . insertions of nucleotides have a profound impact on the diversity of the complementary-determining regions 3 (cdr3) sequences and contribute to most (60%) of this diversity (9) . the coding ends of the genes can be also trimmed by exonucleases. however, given the limited number of amino acids, the removal of nucleotides by exonucleases is constrained to generate a productive codon and therefore limits the contribution of exonuclease trimming to the diversity of the tcr repertoire. finally, the association of the tcrα and tcrβ chain constitutes the final steps of the numerous iteration processes that lead to the generation of a highly diverse tcr repertoire, which is able to efficiently protect individuals from pathogenic stimulations. six cdr will engage the peptide/mhc complexes, endogenous and exogenous peptides being presented respectively by mhc class i and ii molecules. mhc class i grooves constrain the length of the presented peptides (8-14 amino acids length) while the open nature of peptide-binding cleft of mhc class ii molecules allow a broader range of peptides to be presented. the hla locus is the most polymorphic region of the human genome, with more than 13,000 variant alleles (10,297 hla class i alleles and 3,543 hla class ii alleles according to the imgt/hla). the high diversity of hla conferring an almost unique signature of hla for mankind is further extended by the combinatory diversity resulting from the association of six hla class i (two alleles of hla-a, -b, and cw) and six hla class ii molecules (two alleles of hla-dr, -dp, and -dq). the high mutation level of the hla loci is preferentially focused on the peptide-binding cleft that clustered most of the variability of the amino acid sequence. the focus of mutations underlines the function of the hla molecules, namely being able to display a very large array of peptides. garcia et al. were the first to report the crystallographic structure of a murine tcr 2c bound to peptide/mhc class i (h-2k b -dev8). the cytotoxic t cell clone 2c is one of the most well-characterized tcr and has been initially isolated from a balb/b mouse as an allospecific t cell that recognized l d on the mastocytoma p815. beside its primary antigen (peptide p2c), the 2c tcr can bind to different antigens, including the dev8 (10) and siyr (11) . they also showed that tcrαβ adopts a 45° diagonal orientation to the long axis of the peptide (12) . this orientation leads to a spatial interaction between the germlineencoded cdr1 and cdr2 of the tcrα and β chains and the edges of the peptide-groove of mhc (figure 1) . the highly diverse cdr3 region is facing the central portion of the bound peptide. the multiple crystallographic structures of tcr/peptide mhc complexes [more than 120 of crystallographic structures tcr repertoire in transplantation frontiers in immunology | www.frontiersin.org have been obtained (13) ] have revealed that the docking angle of the tcr is conserved with a stereotype position of a 75° diagonal orientation to the long axis of the peptide (14) . the conserved binding model has lead to the concept that tcr and mhc are hardwired to interact, resulting from a coevolution selection of conserved regions (codons) to lock in tcr onto mhc molecule. the stereotyped orientation of tcr atop mhc molecule is however more flexible than initially proposed, with the accumulation of crystal structures. the median docking angle of tcr is 63.2° (min-max 37-90°) with mhc class i and 76.4° (min-max 44-115°) with mhc class ii (13) (figure 1) . different theories have been postulated to explain the hardwire of tcr to mhc molecules (15) , including the key role of co-receptors of cd4 and cd8 that imposed steric requirements for concurrent associations of tcr, cd3, cd4/cd8, and mhc complexes allowing the appropriate signaling events to occur. indeed, the main role of co-receptor cd4 and cd8 is to recruit the src tyrosine kinase p65lck (lck) via its association with the cytoplasmic tail of cd4 or cd8. lck concentration promotes phosphorylation of immunoreceptor tyrosine activation motifs (itams) in the cytoplasmic tails of cd3 subunits and then initiates the cascade of signaling events leading to the full activation of the t lymphocyte. given the key role of co-receptor cd4 and cd8 in process, it was assumed that their ability to bind, respectively, the membrane-proximal α2 and β2 domains of the mhc class ii molecule and the protruding loop in the α3 domain of the mhc class i molecule will constrain the docking geometry of the tcr to the pmhc (figure 1) . a recent report by beringer et al. (16) had challenged the consensus idea of a highly stereotype docking of tcr atop mhc molecules (13) . crystallographic structures of two tcr binding to proinsulin peptide presented by hla-dr4 (hla-dr4 proinsulin ) have been obtained from two clones of induced regulatory cd4 t cells. the ternary complexes revealed a 180° polarity reversal compared to all other tcr-peptide-mhc complex structures. it remains to be address whether this singular observation could be generalizable, whether the reverse docking is a unique feature of regulatory cells and whether the potential signaling differences may influence the phenotype and the function of the t cells. cross-reactivity can be defined by the ability of a given tcr to interact with more than one pmhc complex with different presented peptides or mhc molecules. this new concept has been presented as early as 1977 by matzinger and bevan (17) . an alloreactive t cell clone was derived by owens et al. in 1984 with three h2-e reactivity (allo-e k specific, h2-e k , dba/b10 h2-e d , and self h2-e d ) (18) . since then, numerous reports have provided evidence of cross-reactivity. for instance, mouse 2c tcr can interact with syngeneic mhc h-2k b presenting dev8 (10) and siyr (11) and with allogeneic h-2k bm3 presenting dev8/k bm3 (10) and allogeneic h-2l d -p2ca (11) . the study by birnbaum et al. is an elegant attempt to quantify the cross-reactivity of a given tcr (19) . using five different cd4 tcr clones (three from mouse origin and two from human origin), high throughput screening of yeast libraries and deep sequencing, the authors demonstrate that a single tcr can interact with more than 100 different peptides. jerne et al. postulated in the mid-1950 that each cell exhibits a unique clonotype able to recognize only one antigen (20, 21) . don mason has been among the first to challenge the validity of this clonal selection theory (22) showing that the immune system will be highly incompetent to protect an individual from external insult if one and only tcr was able to recognize a single peptide presented in a given hla context. more than 10 15 t cells, which would weigh more than 500 kg, would be needed to provide efficient coverage of the potential foreign peptides. this clearly stated that the immune system could not efficiently protect individual if one tcr interacts with a single antigen. unlike the affinity maturation of b cell receptor, the protein sequence of tcr is fixed and naive t cells are required to recognize foreign antigens not encountered before. the number of potential antigens to be recognized is huge given the variability induced by the high diversity of peptide-binding groove of hla class i and ii molecules. from the 20 proteinogenic amino acids and given that peptides from 8-to 14-mer can be presented, an incredibly high number of peptides can be potentially generated (>10 15 peptides) (23) . the diversity can be further extending by the posttranslational modifications of amino acids. in a 2012 opinion paper, andrew sewell elegantly presents the necessity of the cross-reactivity (23), as the number of potential foreign peptide-mhc complexes that t cells might encounter dwarfs the number of tcrs available [the number of unique tcrαβ is estimated to be in the magnitude of 10 11 (24, 25) ]. the mechanisms described previously to generate a diverse tcrαβ have to be envisioned at the population level. given the relatively limited number of genes encoding for tcr chain α and β and the requirement of tcr to recognize the highly diverse hla molecules, the necessity of each t cell to recognize a large array of peptides is expected (22) . before presenting the experimental approach aiming to quantify the number of peptides recognized by a single tcr, we would like to present clear evidences of the cross-reactivity involving memory t cells without previous antigen encounter. it has been described few years ago that cd8 t cells with a memory phenotype can be found in mice (26) (27) (28) . cd8 t cells specific for ovalbumin and viral antigens (hsv, vaccinia) could be detected in mice despite their germ-free environment (28) . despite the absence of previous antigen encounter, these pre-existing memory cd8 t cells harbor traits of memory cells such as the ability to rapidly proliferate upon stimulation and to secrete rapidly pro-inflammatory cytokines. homeostatic proliferation, aging, and cross-recognition of alternate ligands have been postulated to drive the accumulation of these memory-like naive cd8 t cells (27, 29) . this observation has been extended to human settings in which cd4 t cells specific for hiv-1, cmv, and herpes simplex virus (hsv) epitopes were identified in healthy volunteers that had never been infected with these viruses (30) . again, these cells exhibit not only memory markers but also memory-associated features (rapid proliferation and cytokine secretion). the acquisition of memory characteristics could be a consequence of homeostatic proliferation (31) or a consequence of the cross-reactivity to other antigens in the environment. to support the latest hypothesis, su et al. have shown that hiv-1 specific t cells can recognize environmental peptides present in the gut and soil, bacteria and ocean algae, and plants. of interest, t cells specific for hiv-1 can even be purified from cord-blood (30) , demonstrating thereby the presence of t cells able to recognize self and non-self antigens in newborns. of interest, the phenotype of cross-reactive t cells was different between newborns and adults, with a naive and a memory phenotype, respectively. the concept of clonal deletion that occurred in the thymus is challenged by the aforementioned reports and compelling evidences suggest that from an evolutionary perspective, the necessity to protect an individual against pathogens is far more important than to limit the autoreactivity. a recent study from davis team further sustained this claim (32) . the frequency of cd8 t cells specific of a y chromosome specific antigen (equivalent to hy peptide) is only threefold lower in man as compared to women (32) . of interest, whereas cd8 t cells purified for their specificity regarding a pool of six self peptides do not proliferate after stimulation with the same set of self peptides, cd8 t cells specific of a pool of six non-self peptides exhibit a potent proliferative response (32) . the absence of response reported for self-specific cd8 t cells and not for foreign antigen-specific cd8 t cells has been linked to a different genetic programing as compared to the clones purified from woman, with a lower expression of il-2r, il-21r, and bcl-xl (32) . thus, evolution has favored the absence of hole over autoimmune disease (about 1% of incidence). it may seem awkward that the evolution has favor the escape of antiself specific t cells from thymic selection over a more stringent deletion of all anti-self t cells. a heavier burden of maintaining tolerance is needed to prevent the development of autoimmune diseases. however, the need to defend the immune system against pathogens, especially during childhood, is far greater than the need to prevent autoimmunity as for population's survival. by limiting the deletion of self-reactive t cells and thanks to the large cross-reactivity of t cells, the holes in the t cell repertoire that pathogens might take advantage of are constrained. the analysis of the immune system in monozygotic twins is enlightened in many aspects as such studies allow the dissociation between the inborn and the acquired contributions. the team of davis has recently showed that the heritability of t and b cells parameters declines very rapidly with age (33) . at the age of 40 years, the heritability explained less than 10% of the variation in t and b cell parameters. cmv infection is a protypical example of the influence of non-heritable factor on the whole immune system. indeed, 58% of all parameters measured in discordant twins were influenced by cmv infection (33) . the environment carves the immune system of each single individual, with each past immune response heavily imprinting the (present) immune system. since the initial observation that immunity against cowpox protects individual from smallpox (34) , numerous examples of cross-reactivity had been reported in mice and in human (35) . for instance, infections with bcg, influenza a virus (iav), lymphocytic choriomeningitis virus (lcmv), and murine cytomegalovirus (mcmv) all confer a level of protective immunity against vaccinia virus (36) (37) (38) . the benefit of cross-reactivity as for pan-virus protection is more difficult to assess for obvious reasons. nevertheless, the numerous example of a single tcr able to recognize different antigens [bcg and poxviruses (37) ; papillomavirus and coronavirus (39) ; influenza virus and epstein-barr virus (40) ]. the large cross-reactivity of t cells confers a more efficient protection cover using a limited number of t cells that need to screen an incredibly large array of peptides that can be presented by mhc molecules. beside the efficient use of limited t cell resource, cross-reactivity confers a spatiotemporal advantage to the immune system to scan any infected cells. cross-reactivity could also be envisioned as an evolution strategy to limit the immune recognition escape. recipient immune system can interact with foreign hla molecules under two very different circumstances: pregnancy and transplantation. thanks to evolution and adaptation of the maternal immune system to the presence of hla mismatch fetuses, allorecognition during pregnancy is not harmful and could even be beneficial as for mammalian sexual reproduction. immunological tolerance toward allogeneic fetus is obtained through a complex network of regulatory mechanisms including the lack of expression of classical mhc class i molecules by the placental trophoblast and the expression of non-classical mhc class i hla-e and hla-g. more surprisingly, hla mismatches have been proposed to be beneficial for pregnancy outcome. in the 1960s, billington reports that the placenta is larger in h-2 incompatible mouse as compared to compatible fetuses (41) . hla compatible fetuses (i.e., similar to maternal hla) have been shown to be more prone to be aborted (42) . in contrast, recipient immune system will potently eliminate an allogeneic graft in the absence of immunosuppressive therapy. despite the absence of thymic central selection (43) of potential graft-recipient t cells by allogeneic mhc motifs regarding their ability to recognize allogeneic potential hla, a large pool of t cells can be activated by donor hla molecules either through the direct pathway (i.e., donor hla presenting donor peptides) or the expected processing of foreign mhc molecules, coined as the "indirect pathway" (i.e., recipient hla presenting donor peptides) in transplantation immunologist jargon. the direct allorecognition pathway represents a unique example of functional and efficient cross reactivity. two main hypothesizes have been postulated to explain the basis of alloreactivity, emphasizing the role of either mhc molecule or peptide. the polymorphism between donor and mhc molecules could act as an "innate focus" that leads to the activation of unprimed recipient t cells or the allopeptide could be recognized as foreign antigen while allogeneic and self-mhc molecules exhibit a high degree of similarity (figure 1) . according to the mhc centric model, the peptide plays only a minor role in the process, and alloreactive tcrs recognize structural determinants on the mhc helices of syngeneic or allogeneic mhc. the bias of tcr to interact with mhc molecules supports this theory. crystal structures of allo-pmhc complexes such as 2c tcr with allogeneic h-2k bm3 presenting dev8/k bm3 (44) or bm3.3 tcr with allogeneic pbm1-h-2k b (45) have shown that alloreactive tcrs interact with allogeneic mhc in a similar fashion as with syngeneic mhc. to further support the role of mhc in alloreactivity, it has been reported that some hla mismatches between donor and recipient are associated with worse graft survival than others, leading to the notion of taboo mismatches based on shape rather than sequence differences (46) . for instance, despite a single amino acid in an hla class i antigen, mismatches between hla-b*4402 and hla-b*4403 is associated with transplant rejection (47) and acute graft-versushost disease (48) . the peptide repertoire bound to hla-b*4402 or hla-b*4403 have been shown to be very similar (49) . however, a recent report challenges this observation (50) . the single amino acid mismatch induced the presentation of more unique peptides by hla-b*4403 than hla-b*4402, consistent with the stronger t cell alloreactivity observed toward hla-b*4403 compared with hla-b*4402 (50) . this observation supports the notion of a peptide focus tcr allorecognition, in the same line as molecular mimicry. allorecognition could also involved cross-reactivity between mhc class i and mhc class ii or even xeno mhc (51, 52) . in 1986, schilman et al. reported that cd8 t cell clone could be activated by both mhc class i (h-2d b ) and mhc class ii (i-e k ) molecules (53). by-directional recognition of t cells between mhc class i and mhc class ii have been reported later (54) (55) (56) . these observations may have important implication in the attempt to minimize hla mismatches during the process of organ allocation. using a mixed lymphocyte reaction (57) , it has been shown that 1-10% of t cell in peripheral blood can be activated (58) . as mentioned before, the number of hla mismatches between donor and recipient is a primary driving force that mobilized a larger fraction of t cells than nominal antigens. whether alloreactive t cells are activated by the high number of new antigens presented by donor hla or by the large number of different allo-phla complexes (or both) is still under debate, and the two hypotheses are not mutually exclusives. the indirect pathway further enhances the reactivity of recipient t cells toward allogeneic graft. indeed, peptides presented by mhc molecules derived predominantly from mhc-related molecules (59) (60) (61) . the introduction of donor hla molecules will thus lead to the introduction of great pool of new peptides that can mobilized a large fraction of recipient t cells. it is now also well accepted that memory t cells generated prior transplantation constitute a major hurdle for long-term graft acceptance. chronic viruses such as ebv and cmv induce the generation of a large pool of memory t cells. for instance, 10% of both the cd4 and cd8 memory compartments in blood are reactive to hcmv (62) . the cross-reactivity between virus-specific t cells and allogeneic hla has been extensively documented (63) . ebv or cmv specific cd8 t cells exhibit frequently a crossreactivity toward allogeneic mhc class i complexes (64) (65) (66) (67) (68) . similar observations have been reported for cd4 t cells specific for ebv or cmv (69) (70) (71) . virus-specific t cells that cross-react with alloantigens have been shown in experimental models to proliferate in response to a transplanted allograft in vivo (72) . for instance, lcmv-specific cd8 t cells generated after infection of mice with armstrong strain of lcmv are able to vigorously proliferate in vivo after skin transplantation and ultimately to mediate skin graft rejection (72) . tracking anti-donor response by the investigation of tcr vβ repertoire: from low resolution technique to high throughput sequencing given the size of anti-donor t cell pool, great efforts have been paid to track the immune-response using the analysis of tcr vβ repertoire and to correlate specific usage of tcr vβ repertoire with graft status or graft outcome. before presenting the available reports, it is necessary to present the two major methods used to investigate tcr vβ usage; a low resolution (spectratype alone or tclandscape when combined with quantitative analysis) and, more recently, a high resolution (deep-sequencing of tcr vβ region) approach (figure 2) . the low-resolution technique is based on the analysis of the length of the cdr3 region whereas the high-resolution technique identifies the sequence of each tcr vβ and later quantifies the abundance of the different t cell clones. each tcr vβ family is composed of t cells with various lengths of their cdr3 region. the distribution of the cdr3 length can be assessed by spectratype (73, 74) . a broad spectrum of profiles can be identified ranging from a gaussian-like profile to a highly restricted profile, highlighting the absence of selection of t cell, or the expansion of t cell clones, respectively. different analytic tools have been used to characterize the cdr3 length distribution (75) (76) (77) (78) . the qualitative assessment of the tcr vβ repertoire can be complemented by the quantification of the different vβ families at the mrna level using qrt-pcr (79) (80) (81) or at the cellular level using flow-cytometry (82) . such techniques still offer several benefits over higher resolution techniques such as their cost, the short time frame to obtain results, and the generation of a reasonable amount of data can be also displayed as "visible" pattern as an "x-ray" of the global tcr alteration in a specific pathological context (83) (84) (85) (86) . a rapid survey of the usage of the tcr vβ repertoire can be efficiently performed, guiding further investigations focused on targeted tcr vβ families. at the other range of the resolution spectrum, deep-sequencing of tcr vβ obtains a full picture of the usage of t cell repertoire with deep or ultra-deep resolution. the availability of all tcr vβ sequences allows for the precise appraisal of the distribution of the different t cell clones especially across different biological compartments (76) . furthermore, with a complete tcr vβ sequencing, researchers can investigate the similarity of t cell figure 2 | characterization of the tcr vβ repertoire by low resolution and high resolution technique. immune challenge leads to the selection of t cells harboring specific tcrαβ among the highly diverse tcrαβ repertoire. antigen-specific t cells could be identified by low-resolution techniques (e.g., spectratype) or high-resolution techniques (e.g., ngs). low-resolution techniques are aiming to identify vβ families that exhibit monoclonal distribution of their cdr3 length distribution using vβ specific pcr and spectratyping. the clonality of the identified vβ families needs to be confirmed by the sequencing of the pcr product. vβ-specific t cell purification enables later to perform functional assay or to reconstruct the tcrαβ in order to identify the recognized antigen. deep-sequencing of tcr vβ region identify the sequence of each tcr vβ and intensive bio-informatic process is needed to quantify the abundance of the different t cell clones. given the burden of data generated, the next-generation sequencing is well-fitted to track t cell clones in time or across different anatomic sites. march 2016 | volume 7 | article 89 6 frontiers in immunology | www.frontiersin.org sequences between biological compartments or individuals and take advantage of public repository databases to assess the specificity of a sequence and potentially to reconstruct the tcr in order to search for the recognized peptides. however, the amount of data generated using this technique is extremely high and efficient bio-informatics tools specifically devoted to the analysis are needed to identify meaningful information in the ocean of data. the accessibility of deep-sequencing is likely to be broaden in the near future thanks to the advances in bio-informatics tools and the reduction of the cost. tcr repertoire in transplantation frontiers in immunology | www.frontiersin.org low-resolution techniques have been used to investigate the usage of tcr vβ repertoire in kidney transplant recipients with various clinical outcomes or at various time points posttransplantation (86) (87) (88) . using the combination of spectratyping and quantitative assessment of the tcr vβ transcript, we have been able to define direct or indirect allorecognition patterns in an experiment model of allograft in congenic rats (52, 79, 80) . using the same approach, we reported that patients with biopsy-proven chronic antibody-mediated (camr) rejection exhibits strong alterations of their tcr vβ repertoire correlating with the level of graft lesions classified with banff classification (87) . in contrast, operationally tolerant patients [i.e., patients off-immunosuppression for more than 12 months with a wellfunctioning graft (89) (90) (91) ] exhibit a polyclonal tcr vβ repertoire (87) . a large cohort of patients with stable graft function for more than 5 years post-transplantation had been prospectively recruited in our center with stringent clinical and demographic inclusion criteria in order to obtain a homogeneous population. nevertheless, we could highlight that the usage of tcr vβ repertoire is highly heterogeneous ranging from the absence of clonal selection (similar to operational tolerance) to an accumulation of selected t cells (as for camr rejection) (87) . the presence of altered tcr vβ repertoire has been previously reported in a rat model of camr (92) in which similar cd8 clones could be identified in the blood and in the graft (93) . in a large prospective study of kidney transplant recipients with a stable graft function for more than 5 years, we show that the altered tcr vβ repertoire was due to an accumulation of temra (t cell effector memory re-expressing cd45ra; cd45ra + ccr7 − ) cd8 t cells with an activated profile (cd27 − cd28 − ), a high expression of cytotoxic molecules, perforin (perf) and granzym b (gzm-b) , t-bet, and cd57 and the ability to secrete tnf-α and ifn-γ (88) . of interest, stable patients who have an increase in differentiated temra cd8 t cells have a twofold higher risk of long-term graft dysfunction (88) . of note, using a similar strategy, kim et al. recently reported that clonal cd8 t cell could be evidenced in human transplanted hand, with several tcr clonal selections persisting at least 100 days (among the 178 days of surveillance) (94) . collectively, these data highlight that a low-resolution technic provides key features as for the accumulation of selected t cell clones that can be used to monitor the kidney transplant recipients. a major drawback of spectratype-based method is its intrinsic low resolution as multiple t cell clones could share the same cdr3 length. it is necessary to sequence the tcr vβ chain with an altered cdr3 length distribution to assess the clonality of a given vβ family. however, we recently compared spectratype or next generation sequencing (ngs) techniques to characterize the tcr vβ repertoire in the blood, the cerebral spinal fluid (csf), and the central nervous system (cns) of patients with multiple sclerosis (76) . both methods were as efficient to highlight the similarity of tcr vβ repertoire between csf and cns (≈80% of tcr vβ clones identified in the cns were also found in the csf) and to identify ≈50% of the tcr vβ clones using blood cd8 sample (76) . as previously discussed, the size of donor-reactive t cell repertoire is large and constitutes a limitation to the use of deep-sequencing approach. it may thus be a naive approach to perform ngs on unfractioned t cells with the aim to identify t cell clones specific to a given situation, such as kidney transplantation or viral infection; a two-step approach is needed. the first step is to purify the t cell population of interest based on the expression of phenotypic (using tetramer for instance) or functional (e.g., cytokine secretion, proliferation) markers. the in-depth characterization of tcr vβ of t cell population of interest allows for the definition of a signature that can be later used as a tag when unpurified samples are analyzed. this approach has been used to track cmv-or bk-specific t cell clones (95) or alloreactive t cells (96, 97) . the first report hypothesizing such an approach in the transplant context has been published by the group of leventhal (96) . using healthy volunteers, this study aimed to assess breadth, clonal structure, and dynamics of the alloreactive t cell repertoire. after 7 days of mlr, the proliferating t cells were purified according to the dilution of cell division dye. by comparing the number of clones before culture and in the proliferated mlr responder, two types of alloreactive clones were identified, low-(i.e., unobserved in pre-culture sample and ≥10 t cells after mlr) and high-abundance pre-culture clones (i.e., present in pre-culture sample and ≥10× enriched after mlr). more than 11,000 low-abundant clones and more than 2,000 high-abundant clones were detected in the different experiments. these data provide new evidences of the large size of the alloreactive t cell pool. this approach was used recently to track donor-reactive t cells in kidney transplant recipients (97) . the fingerprint of donorreactive t cell repertoire was established before transplantation by deep-sequencing of proliferating cd4 and cd8 t cells after 6 days of mlr. the fingerprint of donor-reactive t cells was monitored later after transplantation without the need to perform mlr. the team of sykes provides evidences that tolerance induction protocol based on combined kidney and non-myeloablative bone marrow transplantation results in a reduction of donor-alloreactive t cell clones. however, this decrease was neither observed in the patient that failed to respond to the tolerant inducing protocol nor in patients with standard immunosuppressive regimens. pretransplant identification of donor-reactive t cell clones before transplantation could thus be a means to track the activation of the immune system by allogeneic graft. the studies of emerson (96) and morris (97) showed that the anti-donor fingerprint is stable over-time in healthy volunteers. given the design of the assay, only pre-existing clones could be tracked. it would be of great value to compare the anti-donor clone repertoire before and after transplantation, starting each time from a direct mlr assay to investigate if new anti-donor t cells arise after transplantation. indeed, infections that occurred frequently after transplantation could generate virus-specific t cells with an allogeneic crossreactivity potential (71) . moreover, not all proliferating cells after 6 days of mlr are per se donor-specific as proliferation of t cells could also be linked to bystander stimulation (98) . will transplant immunologists be able to track the rise and the expansion of donor-specific t cells and would this approach be widely available and useful to the clinical management are still open questions. high-through put techniques that have recently emerged are certainly an important step forward. nevertheless, the high cross-reactivity of t cells is a major hurdle to identify the trigger of the expansion of donor-reactive t cells, as donor antigen, viral peptides, and other environmental antigens can lead to the selection of donor-specific t cells. while promising, the study of tcr alteration has not overcome the double difficulties of offering an accessible technical presentation of the data and a validated correlation with clinical outcomes. therefore, longitudinal studies to test the reactivity of recipient t cells against donor antigens at different time points are needed. nd, sb, and j-p s wrote the review. this work was realized in the context of the labex igo program supported by the national research agency via the investment of the future program anr-11-labx-0016-01 and the labex transplantex [anr-11-labx-0070_transplantex], in the context of the ihu-cesti project which received french government financial support managed by the national research agency via the "investment into the future" program anr-10-ibhu-005 and in the context of a centaure foundation. the ihu-cesti project is also supported by nantes metropole and the pays de la loire region. this work was also supported by the fp7 visicort project that has received funding from the european union's seventh framework program for research, technological development and demonstration under grant agreement no 602470. we thank dr. yap and dr. haspot for their fruitful discussion. somatic generation of antibody diversity the complete 685-kilobase dna sequence of the human beta t cell receptor locus analysis of the 1.1-mb human alpha/delta t-cell receptor locus with bacterial artificial chromosome clones estimating the diversity, completeness, and cross-reactivity of the t cell repertoire different tcrbv genes generate biased patterns of v-d-j diversity in human t cells exhaustive t-cell repertoire sequencing of human peripheral blood samples reveals signatures of antigen selection and a directly measured repertoire size of at least 1 million clonotypes junctional sequences of t cell receptor gamma delta genes: implications for gamma delta t cell lineages and for a novel intermediate of v-(d)-j joining most alpha/beta t cell receptor diversity is due to terminal deoxynucleotidyl transferase statistical inference of the generation probability of t-cell receptors from sequence repertoires degenerate recognition of alloantigenic peptides on a positive-selecting class i molecule self-mhc-restricted peptides recognized by an alloreactive t lymphocyte clone an alpha beta t cell receptor structure at 2.5 a and its orientation in the tcr-mhc complex t cell antigen receptor recognition of antigen-presenting molecules reconciling views on t cell receptor germline bias for mhc a t cell receptor v beta segment that imparts reactivity to a class ii major histocompatibility complex product t cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex hypothesis: why do so many lymphocytes respond to major histocompatibility antigens? derivation from an alloreactive t-cell line of a clone which cross-reacts with a self h2-e-restricted minor alloantigen deconstructing the peptide-mhc specificity of t cell recognition the natural-selection theory of antibody formation the somatic generation of immune recognition a very high level of crossreactivity is an essential feature of the t-cell receptor why must t cells be cross-reactive? a direct estimate of the human t cell receptor diversity comprehensive assessment of t-cell receptor beta-chain diversity in alphabeta t cells nonrandom attrition of the naive cd8+ t-cell pool with aging governed by t-cell receptor:pmhc interactions derivation and maintenance of virtual memory cd8 t cells the antigen-specific cd8+ t cell repertoire in unimmunized mice includes memory phenotype cells bearing markers of homeostatic expansion diversity of the cd8+ t cell repertoire elicited against an immunodominant epitope does not depend on the context of infection virus-specific cd4+ memory-phenotype t cells are abundant in unexposed adults homeostasis of naive and memory t cells clonal deletion prunes but does not eliminate self-specific αβ cd8(+) t lymphocytes variation in the human immune system is largely driven by non-heritable influences the history of the smallpox vaccine heterologous immunity between viruses protective heterologous antiviral immunity and enhanced immunopathogenesis mediated by memory t cell populations cd4 t-cell-mediated heterologous immunity between mycobacteria and poxviruses specific history of heterologous virus infections determines anti-viral immunity and immunopathology in the lung human papillomavirus type 16 e7 peptide-directed cd8+ t cells from patients with cervical cancer are cross-reactive with the coronavirus ns2 protein broad cross-reactive tcr repertoires recognizing dissimilar epstein-barr and influenza a virus epitopes influence of immunological dissimilarity of mother and foetus on size of placenta in mice human leukocyte antigen matching and fetal loss: results of a 10 year prospective study t cell tolerance by clonal elimination in the thymus structural comparison of allogeneic and syngeneic t cell receptor-peptide-major histocompatibility complex complexes: a buried alloreactive mutation subtly alters peptide presentation substantially increasing v(beta) interactions crystal structure of a t cell receptor bound to an allogeneic mhc molecule association between specific hla combinations and probability of kidney allograft loss: the taboo concept bone marrow-allograft rejection by t lymphocytes recognizing a single amino acid difference in hla-b44 hla-b44-directed cytotoxic t cells associated with acute graft-versus-host disease following unrelated bone marrow transplantation characterization of natural peptide ligands for hla-b*4402 and -b*4403: implications for peptide involvement in allorecognition of a single amino acid change in the hla-b44 heavy chain a naturally selected dimorphism within the hla-b44 supertype alters class i structure, peptide repertoire, and t cell recognition human peripheral blood cd4 t cell-engrafted non-obese diabetic-scid il2rγ(null) h2-ab1 (tm1gru) tg (human leucocyte antigen d-related 4) mice: a mouse model of human allogeneic graft-versus-host disease characterization of an lyt-2+ alloreactive cytotoxic t cell clone specific for h-2db that cross-reacts with i-ek restricted mhc-peptide repertoire predisposes to autoimmunity how the t cell repertoire becomes peptide and mhc specific a single t cell receptor bound to major histocompatibility complex class i and class ii glycoproteins reveals switchable tcr conformers quantitative assay of antigenic disparity at hl-a-the major histocompatibility locus in man quantifying the frequency of alloreactive t cells in vivo: new answers to an old question predominant naturally processed peptides bound to hla-dr1 are derived from mhc-related molecules and are heterogeneous in size specificity and promiscuity among naturally processed peptides bound to hla-dr alleles sequence analysis of peptides bound to mhc class ii molecules broadly targeted human cytomegalovirus-specific cd4+ and cd8+ t cells dominate the memory compartments of exposed subjects advances in direct t-cell alloreactivity: function, avidity, biophysics and structure t cell receptor repertoire for a viral epitope in humans is diversified by tolerance to a background major histocompatibility complex antigen cross-reactivity of herpesvirus-specific cd8 t cell lines toward allogeneic class i mhc molecules an alloresponse in humans is dominated by cytotoxic t lymphocytes (ctl) cross-reactive with a single epstein-barr virus ctl epitope: implications for graft-versus-host disease cross-reactive memory t cells for epstein-barr virus augment the alloresponse to common human leukocyte antigens: degenerate recognition of major histocompatibility complex-bound peptide by t cells and its role in alloreactivity cross-recognition of hla dr4 alloantigen by virus-specific cd8+ t cells: a new paradigm for self-/nonself-recognition ebv-specific cd4+ t cell clones exhibit vigorous allogeneic responses cross-recognition of human alloantigen by cytomegalovirus glycoprotein-specific cd4+ cytotoxic t lymphocytes: implications for graft-versus-host disease allo-hla reactivity of virus-specific memory t cells is common allografts stimulate cross-reactive virus-specific memory cd8 t cells with private specificity circulating t cell repertoire complexity in normal individuals and bone marrow recipients analyzed by cdr3 size spectratyping. correlation with immune status molecular detection and in vivo analysis of the specific t cell response to a protein antigen statistical analysis of cdr3 length distributions for the assessment of t and b cell repertoire biases expanded cd8 t-cell sharing between periphery and cns in multiple sclerosis the blood of healthy individuals exhibits cd8 t cells with a highly altered tcr vb repertoire but with an unmodified phenotype t cell repertoire alterations of vascularized xenografts direct recognition of foreign mhc determinants by naive t cells mobilizes specific vbeta families without skewing of the complementarity-determining region 3 length distribution highly altered v beta repertoire of t cells infiltrating long-term rejected kidney allografts immune responses elicited in tertiary lymphoid tissues display distinctive features improved assessment of t-cell receptor (tcr) vb repertoire in clinical specimens: combination of tcr-cdr3 spectratyping with flow cytometry-based tcr vb frequency analysis blood t-cell vbeta transcriptome in melanoma patients serial blood t cell repertoire alterations in multiple sclerosis patients; correlation with clinical and mri parameters serial evolution of tcr beta chain transcript mobilization in hiv type-1-infected patients following vaccine immune stimulation and haart interruption operationally tolerant and minimally immunosuppressed kidney recipients display strongly altered blood t-cell clonal regulation analysis of the peripheral t-cell repertoire in kidney transplant patients expansion of highly differentiated cytotoxic terminally differentiated effector memory cd8+ t cells in a subset of clinically stable kidney transplant recipients: a potential marker for late graft dysfunction identification of a peripheral blood transcriptional biomarker panel associated with operational renal allograft tolerance the natural history of clinical operational tolerance after kidney transplantation through twenty-seven cases clinical operational tolerance after kidney transplantation indirect cd4+ th1 response, antidonor antibodies and diffuse c4d graft deposits in long-term recipients conditioned by donor antigens priming functional compartmentalization following induction of long-term graft survival with pregraft donor-specific transfusion clonal cd8+ t cell persistence and variable gene usage bias in a human transplanted hand tcr repertoire analysis by next generation sequencing allows complex differential diagnosis of t cell-related pathology defining the alloreactive t cell repertoire using high-throughput sequencing of mixed lymphocyte reaction culture tracking donor-reactive t cells: evidence for clonal deletion in tolerant kidney transplant patients high-resolution characterization of cytokine-producing alloreactivity in naive and allograft-primed mice the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-017184-1ewi3dka authors: nan title: primary immunodeficiencies date: 2008 journal: pediatric allergy, asthma and immunology doi: 10.1007/978-3-540-33395-1_22 sha: doc_id: 17184 cord_uid: 1ewi3dka primary immunodeficiencies (pids), once considered to be very rare, are now increasingly recognized because of growing knowledge in the immunological field and the availability of more sophisticated diagnostic techniques and therapeutic modalities [161]. however in a database of >120,000 inpatients of a general hospital for conditions suggestive of id 59 patients were tested, and an undiagnosed pid was found in 17 (29%) of the subjects tested [107]. the publication of the first case of agammaglobulinemia by bruton in 1952 [60] demonstrated that the pid diagnosis is first done in the laboratory. however, pids require specialized immunological centers for diagnosis and management [33]. a large body of epidemiological evidence supports the hypothesis of the existence of a close etiopathogenetic relation between pid and atopy [73]. in particular, an elevated frequency of asthma, food allergy (fa), atopic dermatitis and enteric pathologies can be found in various pids. in addition we will discuss another subject that is certainly of interest: the pseudo-immunodepressed child with recurrent respiratory infections (rris), an event that often requires medical intervention and that very often leads to the suspicion that it involves antibody deficiencies [149]. structural genes, and also perhaps on the lack of 2/4 c4 genes [506] . mbl deficiency is due to one of three point mutations in the gene for mbl, each of which reduces levels of the lectin by interfering with the protein oligomerization [351] . in children with this kind of deficiency, the level of mbl is 4.9 mg/l compared to the 143 mg/l in controls [487] . regardless of whether the children are homozygote (hz) or heterozygote (het) in relation to a given mutation, the defect appears to be more consistent in small babies aged 6-18 months [487] , who show an immaturity in providing immune response to capsular bacteria and in whom low levels of opsonin are incapable of compensating for this [506] . the risk of contracting infections is similar in hzs [175] and hets [486] , though it persists throughout life in hzs because of an abnormal allele, while it exhausts itself in the hets, where the frequency of abnormality is similar to that of the general population [175] . anomalies in immunoglobulins (ig) and in opsonization have been observed, respectively in 13%-20% of children suffering from frequent asthma and igg subclass deficiencies. children suffering from cystic fibrosis also present an elevated prevalence of immediate cutaneous reactions to aeroallergens, and although without primary defects of adoptive immunity, they are susceptible to severe rris; therefore it may be possible that they suffer from the mucosal antigenic exclusion [61] . unlike asthmatic children, in whom a relatively high concentration of ige for respiratory viruses was observed [172, 173, 457] , positive skin prick tests (spts) are more common for aspergillus fumigatus [61] . the hypothesis suggested by these observations is that atopy derives from an unbalanced immune response to foreign antigens, with a consequent lack of their early identification or the capacity to neutralize or eliminate them. this hypothesis is based on the evidence that id precedes the development of atopy: in the taylor et al studies, 22 newborn babies, the children and/or siblings of atopic patients, presented a significant reduction in serum concentrations of iga when aged 3 months: this was transient hypogammaglobulinemia (hgg) of infancy (thi). the association of very low iga levels with atopy has been proposed again in the classic prospective study on the association of viral respiratory infections (vri) and the onset of allergic manifestations, which proved serum iga levels at the lowest normal levels in the children studied [173] . this data has been confirmed within primary immunodeficiencies (pids), once considered to be very rare, are now increasingly recognized because of growing knowledge in the immunological field and the availability of more sophisticated diagnostic techniques and therapeutic modalities [161] . however in a database of >120,000 inpatients of a general hospital for conditions suggestive of id 59 patients were tested, and an undiagnosed pid was found in 17 (29%) of the subjects tested [107] . the publication of the first case of agammaglobulinemia by bruton in 1952 [60] demonstrated that the pid diagnosis is first done in the laboratory. however, pids require specialized immunological centers for diagnosis and management [33] . a large body of epidemiological evidence supports the hypothesis of the existence of a close etiopathogenetic relation between pid and atopy [73] . in particular, an elevated frequency of asthma, food allergy (fa), atopic dermatitis and enteric pathologies can be found in various pids. in addition we will discuss another subject that is certainly of interest: the pseudo-immunodepressed child with recurrent respiratory infections (rris), an event that often requires medical intervention and that very often leads to the suspicion that it involves antibody deficiencies [149] . in several pids (table 22 .1) [61, 65, 76, 101, 162, 351, 414, 480, 514, 543] , atopic symptoms are present: gastroenteric and rhinitis in selective iga deficiency (sigad), severe ad in wiskott-aldrich syndrome (was) and hyper-ige syndrome (higes), in which it spreads over the entire body, and to which other allergic manifestations can also be associated such as asthma, rhinitis and angioedema. various acquisitions indicate that pid is also an opsonization deficiency, observed in 5% of the normal population [469] . in this disease, microorganism phagocytosis by polymorphonuclear (pmn) leukocytes appears annulled, and the patient is subject to severe infections supported by capsular bacteria: the deficiency, described in association with severe and recurrent infantile infections [175, 485, 487] , depends on the lack of mannose-binding lectin (mbl) [487] , its primary immunodeficiencies a possible atopy dependence on iga underproduction rather than on ige hyperproduction ( fig. 4.1 ): in children with levels of iga at the minimum normal level, and followed from birth until the age of 18-23 months, a greater severity of atopic manifestations and an increased cumulative incidence of asthma, ad and otitis media with effusion (ome) were observed compared to controls. the close links between id and atopy are confirmed by symptoms similar to ad present in some forms of was (70%), higes (85%), xla (x-linked agammaglobulinemia) or autosomal recessive (ar), ataxia-telang1268 chapter 22 primary immunodeficiencies proposed that in these patients cd4-th2 levels are sufficient for modulating ige synthesis, but cd8 t-cell levels are inadequate for inhibiting ige synthesis, which results in increased ige synthesis. this hypothesis is supported by the observation that omenn syndrome, was and especially higes, with an immunological phenotype characterized by a quantitative and qualitative reduction of cd8 t cells, are accompanied by extremely high levels of serum ige [61, 162, 196] . lymphocytes in subjects with normal levels of ige are incapable of producing them, not even after stimulation with polyclonal activators such as pwm (pokeweed mitogen) or ebv (epstein-barr virus), while patients with high antibody levels spontaneously synthesize in culture sige (specific) levels between 200 and 2,000 pg/ml, also releasing factors capable of increasing ige secretion (ige-pf) [287] . supernatant derivatives from the t cells of patients with higes are in fact capable of inducing in vitro the pre-b cells to increase ige production; furthermore, when the t lymphocytes in these patients are isolated on the basis of receptors for the ige fc fragment, the remaining cells release ige-pf [287] . considering the suppressive activity of human lymphocytes with cd8 phenotype on sige, it has been observed that these lymphocytes are able to suppress sige synthesis in patients with high antibody levels; similarly cd8 + cells from a bone marrow transplant (bmt) can suppress ige production in the hla-compatible recipient [478] . the study of patients with id associated with hyper-ige has supplied useful information concerning ige system biology, although the immune defect essentially responsible for ige increased production and for severe atopic iectasia (ata), thymic alymphoplasia, scid (severe combined id) (48%) [44] and, occasionally, by digeorge syndrome (dgs), id with hyper-igm (higms) now cd154/cd40l deficiency, selective igm deficiency, biotin-dependent carboxylase deficiency, cgd (chronic granulomatous disease), primary neutropenia, and in netherton, nezelof, omenn and shwachman syndromes [434] . other forms, in addition to those discussed, are associated with gastrointestinal symptoms: diarrhea and malabsorption of xla and thi, diarrhea in was and dgs, food-related allergies (43%) in sigad and also an elevated frequency of asthma [36] .among secondary id, only aids is associated with ad (chap. 23). the association between a deficiency of t cells and high levels of ige, observed in patients with higes, nezelof syndrome, ata, was and other diseases, has been known for some time (table 22. 2) [62] . experimental studies on animals indicate that there may be an inverse correlation between serum ige levels and t-cell functions: this could be attributed to a t-lymphocyte deficiency in atopics, genetically determined, which makes them more vulnerable to the camp inhibiting activity, and consequently causing an imbalance between the two subclasses of t cells, which could lead to ige hyperproduction and atopy development; however, in no case is there evidence of a relationship between cd8 deficiency, ige levels and allergic symptoms. it has been 1269 serum ige concentrations 54 7 chronic granulomatous disease 10 6 m-17 y <1-3, 160 88 hyper -ige syndrome 11 3-31 y 3150-40,000 11,305 nezelof syndrome 3 8 m-3 y 5-7,000 55 non-x-linked agammaglobulinemia 15 6-35 y 1-10 3 other variable immunodeficiency normal infants and adults 106 2-55 y 2549 55 data from reference [62] . m months, y years, gm geometric mean. manifestations has not yet been identified. the most interesting syndromes from this point of view are the three syndromes analyzed above, characterized by common clinical indications such as early ad onset, increased susceptibility to all varieties of pathogens, as well as an exceptionally high ige serum level [162, 394] ( table 22. 2). several pids have autoimmune features, including chédiak-higashi syndrome, cgd, complement deficiencies c1q, c1r, c1s, c2, c4, griscelli syndrome, higms (cd154 deficiency), lad (leukocyte adhesion deficiency), hla class i deficiency, hla class ii deficiency, omenn syndrome, was, and xlp (17) , which will be dealt with subsequently. in 25 children with a mean age of 44 months, autoimmunity was chronic and severe requiring prolonged immunosuppression, however with no spontaneous remission of such manifestations [44] . definition pids ( fig. 22 .1) [413] consist of a heterogeneous spectrum of congenital, individual and combined anomalies of the immune system (humoral deficiencies, combined deficiency of b and t cells, the complement, phagocytes, neutrophils, etc.), as well as syndromes and diseases associated with id that are traditionally classified as pids. the updated classification (table 22 .1) has divided ª100 pids into six main groups, also including secondary id with infections (first among them all aids) that cause deficiency and immunosuppression [414] . the classifications of pids is based on characteristic clinical features and specific alterations in immune status. advances in molecular genetics now make it possible to complete the table according to the types of genetically altered molecules involved [63] . to complete this data, see table 22.1 and table 22 .3 [236, 246, 407, 453] showing the behavior of antibodies and circulating b and t cells. pids occur infrequently and are highly heterogeneous in nature, relatively few centers gain extensive experience in the diagnosis, so it is difficult to estimate the prevalence of these disorders from routinely collected health statistics [33] . studies in 13 countries on all continents have included 10,895 patients: tables 22.4 and data concerning incidence has increased considerably thanks to a greater availability of specific tests and more widespread knowledge in the medical profession related to these pids, including ata [94] . however, because 1271 primary immunodeficiencies [236, 453] ; other data from [246] (omenn syndrome) and [408] (jak3). ada adenosine deaminase, id immunodeficiencies, jak janus-family kinase, pnp purine nucleoside phosphorylase, ø decreased, øø markedly decreased, ≠ increased, -absent, + present, n normal. a progressive. b not functional. predominantly t-cell defects 3 the total may not correspond to the sum of the cases because it may include some pid with very low incidences. the figures should be divided into the years that were considered. a incidence × 10 6 live births; the thi figure includes probable cases. thi 14 60 11 selective igg subclass deficiency 10 39 autosomal hyper-igm syndrome 3 34 2 selective antibody deficiency with normal igs 4 20 10 cellular and antibody id syndromes associated with other major defects 20 ata 7 149 12 wasp syndrome 31 34 2 digeorge anomaly 1 18 6 hie 4 63 6 nijmegen anomaly 1 immunodeficiency associated with or secondary granulocyte dysfunctions 9 defects of phagocyte number and function cgd 14 85 3 cyclic neutropenia 1 11 1 kostmann's syndrome 4 14 schwachman syndrome 1 complement deficiency -undefined 1 total pid 166 1, 428 122 time period 15 years 20 years 11/1983-12/1999 latin america includes eight countries. xla x-linked agammaglobulinemia, cvid common variable immune deficiency, thi transient hypogammaglobulinemia of infancy, scid severe combined id, was wiskott-aldrich syndrome, ata ataxia-telangiectasia, cgd chronic granulomatous disease. to be reliable and can also be used for other recessive x-linked pids to identify cell lines with genetic defects, as is the case of was [537] . one must, however, properly consider the phenomenon of mutations, that can render useless the inactivation method, as has been proved in was, in xla and also in scid, in which the mutation is not in the maternal t cells but in the germlines [389] . the main clinical aspects of humoral and cellular pid are schematized in table 22 .6 [80] ; further in numerous pids there is a deficiency of chemotaxis (table 1 .65) as in cgd [416] . in antibody deficiencies, current treatment, while waiting for genetic treatment to become available, complicated in xla by several btk mutations, is based on the prophylactic administration of ivig, combined with quick antibiotic treatment during infectious episodes. the genes responsible for id linked to chromosome x have been recently mapped on the respective chromosome bands (fig. 22. 2): the bands on the short limb are designated "p" and those on the long limb "q" (table 22 .1). this was possible thanks to the refinement of dna recombinant technology (rdna), including dna probes (sequences of radio-marked dna) and restriction fragment length polymorphism (rflp). the closer the gene segregates to rflp, the lower the chance that they might be separated by recombination phenomena when meiosis occurs: the identification of deficient genes allows early diagnosis, even prenatal, and if necessary gene therapy or bmts [76] . furthermore, the observation that numerous pids are transmitted with an x-linked modality allows a relatively simple diagnosis of males with a positive family history (fh); if fh is negative (40%-50% of xla cases) or there are females presenting a clinical pattern of pid, or when sporadic cases are caused by a new mutation, carrier identification is based on the study of immunologically normal female carriers, with two populations of b precursors, using x-chromosome inactivation analysis. this test does not take into account the existence of possible gene mutations and the availability of already affected relatives, and it is also relatively simple and fast [537] . molecular studies follow the hypothesis that, at an early stage during embryogenesis, one of the two x chromosomes is randomly inactivated in the cells of all tissues of female embryos (persisting as barr's chromatin) [300] . therefore in normal conditions, one has a cell mosaic that actively expresses for 50% the paternal x chromosome and for the remaining 50% the maternal x chromosome (lionization) [300] (fig. 22 .3a) [355] . in female carriers of xla, the cell mosaic expresses 50% for an x chromosome with btk in an active form and the remaining 50% for an x with a mutated btk (bruton tyrosine kinase). this means that in the carrier mother it is inactivated in preference to the x chromosome carrier of the defective gene in b, which matures therefore in an unbalanced manner (not randomly), while in all other cells activation occurs randomly. it follows that in fixed carriers only the b lymphocytes that have the x carrier of a normal gene complete the differentiating route, while the precursors that express the x chromosome with a mutated btk do not mature into b cells, but remain blocked [100, 413] (fig. 22.3b) . in x-scid, the study of fixed carriers follows the corrected lyon hypothesis, because the cells with a normal active x develop into normal t lymphocytes; however, when t precursors with a mutant x reach the stage where the x is needed, they do not find it and consequently do not develop: thus female carriers have only one normal and active x, instead of the random mixture of cells with one of the two active x [389] . the inactivation test appears predominantly b-cell immunodeficiency inherited in an x-linked trait, only 50% of males have a fh positive for pid; female cases are also known, supporting an ar trait [100] . classically affected subjects present levels of igg at <100 mg/dl, with very low circulating iga, igm and b cells (table 22. 3), in which are found, in addition to bm, pre-b lymphocytes in an almost normal quantity [100] . xla is characterized by a blocking of b-cell differentiation that results in an arrest of the evolution of pre-b1a cells: low levels of cytoplasmic igm and high levels of surrogate light (l) chains (cd179b) into later-stage b cells [348] .the b-cell differentiation arrest in the majority of xla patients appears to be homogeneous, with approximately 80% of the pro b-cell compartment being negative for cytoplasmic igm expression [349] . the size and nature of the residual more mature b-cell population (leakiness) varied among patients, independent of the type of btk mutation. further, it appears that the pro b-cell compartment composition in bone marrow (bm) of some xla patients can be influenced by low levels of wild-type btk mrna [349] . on the contrary, t cells are normal both in function and in number, as is thymus architecture, including hassall's corpuscles and thymus-dependent areas of spleen and lymph nodes. b lymphocyte zones are typically depleted, with an absence of gcs, plasma cells, and cortical and medullar differentiation compared to normal (figs. 22.4 and 22.5) and an absence of adenoidal tissue (fig. 22.6 ). the intestinal lamina shows a similar deficiency [227] , even if both b and t cells use the same recombination (chap. 1). in the bm, increased pre-b lymphocytes without cd19 and cd21 can be observed. the pre-b cells are capable of transcribing and translating microgram intracytoplasmic (ic) h chains, but not the l chains [453] , thus pre-b only form microgram chains not associated with v h , while only 5% of normal cells produce incomplete chains [278] . experimental data currently indicate that the defect lies in the xla gene mutations that codify for btk [505, 513] . the xla gene is expressed by b cells during differentiation, but is not transcribed in the t cells, thereby explaining the b lymphocyte maturative block at the pre-b level [61] (fig. 2.6 ), immediately after their appearance in the bm [302] . xla, however, presents a genetic heterogeneity, explained by mutations in the 5 btk domain (ph, th, sh3, sh2, kinase), with a frequency proportional to the pertinent domain dimensions [514] . the mutation size was ascertained by finding 175 mutations in 236 patients ( fig. 22.7) . equally, genes codifying for marker proteins and receptors that are essential for b-cell maturation and development are also involved: in fact many of these proteins, including btk, hm chains and surface proteins are crucial for b-cell differentiation [395] . studying chil-dren of both sexes with xla, various mutations of hm germline have been identified, in addition to deletions affecting the d, j h and cm genes and other gene alterations capable of blocking h chain synthesis on b lymphocytes [548] . an equivalent molecular defect was observed in an infant girl with xla, with differentiation block preceding the ig gene rearrangements by early pre-b cells [316] . there are also the so-called leaky forms, with absent or few b cells and various antibody deficiencies [240] , which can be attributed to individual mutations of btk [267] , for example in the non-kinase domain, which permits the expression of normal btk levels [427] . however, btk mutations can be even more detrimental for b lymphocyte proliferation compared to the total kinase absence [370] . xla is clinically characterized from its onset in male babies, at 5-6 months of life (but also at the end of the 1st year), when the maternal igg passive protection ceases. it usually attracts attention due to delayed growth and mostly recurrent and severe bacterial infections dominate (sinusitis, otitis, bronchitis, [476] . although representing the phenotypic picture of humoral id, confirmed as a separate deficiency [459] , xla associated with ghd is mapped in the same region as the x chromosome of the isolated xla. the observation of hz deletion of one or more c h genes for the h chains of ig in 5%-10% of normal patients has led to the identification of various polygenic deletions concerning the genes of one or more isotypes and subclasses [282, 414] . some subjects are lacking in genes of all or some igg subclasses, associated with iga 1 and ige deficiency, with no clinical symptoms in 94% of cases [282, 414] . in italy these deletions have a frequency of 2.7% and the expected frequency in hzs is of 1:1,400 [385] . only some of the families that produce l chains and not the k chains are known. in one family the molecular bases of the deficiency were ascribable to two different punctiform mutations, one in each cκ allele that prevented the formation of -s-s bridges between the k and h chains. the k:l ratio in human ig is 2:1, and the relative alterations can be observed in numerous primary or secondary ids [414] . only one patient is known with an l chain deficiency, hgg and rri (upper and lower respiratory tract) [508] . [61, 79, 302] . rotavirus and echo viruses also cause severe meningoencephalitis in 5%-15% of patients [100] . phenotypic variability may occasionally be present, as in a family spanning three generations [332] . in 33 patients with a median age of 9.4 years the median age at the xla onset was 8 months and the median age of diagnosis was 4 years, with a median diagnosis delay of 33 months. the common infectious diseases were pneumonia, otitis, diarrhea, sinusitis, and arthritis. the most common chronic infections were seen in 75.8% of the patients: in the respiratory tract in 93.9%, in the gastrointestinal tract in 75.8%, in the central nervous system (cns) in 33.3%, and in the musculoskeletal system in 21.2% of patients [324] . bronchiectasis, malabsorption, arthritis, autoimmune and tumor-related diseases are the most common complications, as well as edema, contractures, etc. (fig. 22 .8). one must predict the onset of bronchiectasis and intervene quickly with specific physiotherapy, because forms that are initially localized later spread, causing respiratory failure in older children and adolescents. one-third of all cases start with mono-or rheumatoid arthritis (ra) caused by ureaplasma urealyticum with a sterile exudate, which usually regresses following treatment with ivig [355] .anti-polio vaccinations with live attenuated viruses should be forbidden, because they can cause very severe pneumonia [278] . about ten cases of xla associated with ghd are known. in addition to reduced growth, clinical symptoms are typical of xla, though it is not a variant, sporadic cases have been described, occasionally associated with sigad (10%-20%) and more often with ata (80%) and susceptibility to infections [227] or without rris [385] , differentiating patients with probable pid from those with low levels of igg 2 (table 22 .7) [387] , in whom it may represent delayed maturation [450] . selective deficiency of igg subclasses presents three different aspects: ∑ total lack of a subclass ∑ two sd levels below average ∑ inability to produce antibodies relative to the subclass in question, even when hematic concentrations are normal [465] the following selective deficiencies are present [283, 465] : ∑ isolated igg 1 : deficiency in only a few cases has been described, also because this subclass represents 60%-70% of all iggs (the others account for 25% [igg 2 ], 6% [igg 3 ] and 3% [igg 4 ]), its absence is very probably an indication of an evident hgg; these patients usually have a reduced level of total iggs and react normally to antigens with a polysaccharide capsule. ∑ other combined id (cid): for example igg 2 +igg 4 , igg 2 +igg 3 , igg 1 +igg 2 +igg 4 , igg 1 +igg 2 , igg 2 +igg 3 + igg 4 , some of which are associated with a deficiency of iga or its subclasses [153, 465] . the association of sigad and defects of igg subclasses is explainable in view of ig production ontogenesis by b cells: one starts with igm, moving on to igd, and then to igg ending up with iga passing by ige [296] ; therefore the deficiency could originate with an immunological defect involving the t lymphocyte regulating work or b cells secreting ig, with an effect on the final stages of their production. in rare cases, more or less extended deletions in chromosome 14 have been observed, in the region that codifies the h chains; in most cases the genome is instead intact, confirming a possible defect in b lymphocyte switching [367] . very rarely this deficiency depends on gene hz deletions [227] . this pid is probably the most common of all (tables 22.4, 22 .5), especially in nonselected populations of caucasian origin [367] . it is defined by the presence of a serum level of iga <5 mg/dl and the absence of siga in the total deficiency; in the partial sigad levels are <5 mg/dl but <2 sd compared to normal levels for age, with measurable siga [367] . in total deficiencies, igm and igg levels can be normal [414] (table 1. 15), but igg 2 and igg 4 levels are low [153] . in several cases, the partial deficiency is transient [383] , returning to normal levels of iga in 50% of cases by the age of 14 and in 80% by 18 ( fig. 22.9 ) [383] . the sigad is transmitted sporadically; however, cases of multifactorial and dominant ar, with a variable or incomplete expression [105, 536] transmitted within the same family have been reported. functional alterations reflect on the final maturing process of b lymphocytes, given that about 80% of b iga + lymphocytes show an igm+igd+iga+ membrane phenotype, a normal aspect only in newborn babies [103] . studies on chromosome 18 have not led to conclusive results, because deletions in children with sigad are associated with mental retardation, facial dysmorphisms, failure to thrive, etc. an association with hla haplotypes situated on chromosome 6 is instead more consistent, and common in patients with cvid. interesting indications for understanding the pathogenesis come from molecular genetic studies that have allowed the formulation of a hypothesis of multifactorial origin, given that the combinations of more widely involved hla haplotypes and extended haplotypes involve the class i-iii genes, to the extent that they are more often encountered in the general population [105] .among the class iii alleles the most studied is the gene that dictates the c4a, among class i and ii the most common are a1, a28, b8, b13, b40, cw6, dr1, dr3, dr7, dqw1, associated with haplotypes such as a1, b8, dr3; b13, dr7; a1, b14 or a28, b14. other haplotypes are extended, such as b8/sco1/dr3, bw65/sc2 [1, 2] /dr1, bw57/sc61/dr7 and b44f/fc31/dr7, the first of which is increased in patients with a combined iga, igg 2 and ige deficiency [170, 313, 536] . the association with dr3 gives sigad a risk factor of 13 (table 18. 3). one hla supertype is also found in deficiencies of 21-hydroxylase with a late onset, suggesting an important locus for iga differentiation close to class iii hla genes [99] . it has been thought that to induce sigad a non-hla gene or an environmental penetration factor might be necessary, due to the possible sigad discordant expression in hla-identical twins [536] . however, the analyzed sequence of involved alleles showed a significant sigad correlation with some alleles belonging to hla-dq locus, composed of a protective allele with aspartic acid and a susceptible one with valine or alanine in the b chain in position 57 [358] . we have observed that the presence of aspartic acid ensures protection in dm. furthermore, the immunological deregulation extends to the typical formation of auto-antibodies and characterized by the presence of igg anti-iga [61, 196] . in these subjects, there is a wide symptom range, mostly represented by rris, allergic and autoimmune diseases (aids), among which is diabetes. allergic diseases are twice as common in partial deficiencies, unlike aids [507] . from a clinical point of view, the frequency of chronic diarrhea and malabsorption, associated with celiac disorders and giardia lamblia infestation are not surprising, considering siga's prominent iga 1 , igg 2 , igg 4 and ige due to deletion of ig h chain constant region genes were associated with undue susceptibility to infection [384] (see "rri"). a few cases of selective igm deficiency are known, associated with rris and various other symptoms [414] . igm deficiency was detected in four children with rris. isolated igm defect was present in two children, and two more children had an associated igg 3 subclass deficiency [160] . cvid includes a heterogeneous group of unhealthy conditions that have in common hgg and rris; it has an incidence of between 1:50,000 and 1:200,000 [414] . the inheritance of two susceptibility genes within the hla on the short arm of chromosome 6: one located near the class ii region and the other near the junction between the class iii and class i regions is a serious risk for the development of cvid [441] . there are autosomal dominant or ar forms also linked to sex; sporadic cases are the most common [413] . the molecular bases are not totally clarified as yet: the pathogenetic mechanisms may depend on b lymphocyte (80% of patients) and t lymphocyte (20%) defects [227] . the b-cell intrinsic defect is attributable to an alteration of the differentiating line at different stages of maturation, resulting in a poor formation of antibodies, with hgg of variable degrees, while in patients with xla the circulating b lymphocytes are virtually absent. the iggs are <500 mg/dl (with a reduction in all the subclasses: a normal phenotype is observed in only 14% of patients) [367] . more often there is a hierarchical order in the shortage: igg 3 < igg 1 < igg 2 < igg 4 [528] . iga and igm antibodies are <5-50 mg/dl [102] , reflecting the potential cd154 underexpression, implying an activation deficiency [150] or a t-b cooperation defect [232] . a study of t lymphocyte subpopulations indicates various subgroups of patients: 60% have t cells with scarce il 2 , il 4 and il 5 levels, while 30% have a reduced cd4:cd8 ratio, with an increase in cd8 bearing the cd57 marker, which suppresses igg production, elaborates normal il 2 levels and increases ifn-g production [232] . it can also accompany a deficiency of interleukins (ils: il 10 , ifn-g), suggesting a defect in the signaling mechanisms based on the tcr/cd3 [191] . a t-lymphocyte deficiency is therefore difficult to evaluate [527] , also because this could be a vri effect [232] . cvid can also be observed following congenital rubella or ebv infections; it can also be induced by some drugs such as phenytoin [355] . in 2/9 cvid families, 5 subjects were identified with identical large mutations in the icos (inducible costimulator) gene, expressed on the surface role in the formation of a barrier against the penetration of polypeptidic macromolecules through the intestinal mucosa. sigad therefore facilitates the penetration of food antigens through the mucosa followed by the formation of specific antibodies. for example, 50% of patients present cics and precipitins to cm, 23% to bovine anti-serum and 13% to anti-serum of calf fetus [106] . symptoms affecting the respiratory tract are also caused by the absence of siga, as in 30/36 children aged 1-15 with increased susceptibility to rris [327] . patients balance the siga deficiency with the sigm, but in some cases the compensation is insufficient for exempting them from rris and asthma [227] . sigad should be diagnosed on the basis of both serum and secretory iga, because normal levels in adults are achieved at different times (table 1. 15) . some drugs such as phenytoin (an anticonvulsant) can determine sigad, sometimes persisting in time after the drug has been discontinued. the clinical symptoms in these cases are not different from those of patients with sigad [507] . there is no random treatment; these patients do not benefit from therapy with ivig, even when enriched in iga. there are no counter-indications for obligatory and optional vaccinations [507] . whole blood or plasma transfusions containing iga can sensitize patients or cause anaphylactic shock in those already sensitized [105] . life expectancy is excellent; however, the random discovery of sigad in asymptomatic children should not be underestimated. they should in fact undergo periodic clinical and laboratory controls so as to identify as early as possible any possible pertinent symptoms. at the same time, there is the need to ensure a good life quality with adequate prevention of rris in those patients whose respiratory tract is affected [383] . sadni translates into the inability to respond to certain antigens, especially if polysaccharide. while some individuals are normal, others contract sinopulmonary infections. the reduction of igg 2 levels is more of an associative relationship than a random one; igg 2 levels, on the other hand, do not predict antibody responses. subjects who do not respond to anti-hepatitis vaccination may fall into this category [414] . in one retrospective survey at a pediatric tertiary care center, sadni was the most frequent diagnosis, accounting for 23% of id diagnoses [236] . there are cases of patients in good health, without ige due to gene deletion [62] . in two siblings, deficiency of of activated t cells, which interacts with the icos ligand gene expressed on b cells. an additional 181 patients with sporadic cvid were examined, and no mutations were found. only 9 in 226 patients with cvid screened thus far (<4%) have been found to have icos mutations. one unexplained feature of cvid is that the onset of clinical symptoms does not occur until late childhood or adulthood [429] . pid is variable either in the clinical and immunological pattern, or in the onset period, more common during the school years or in adults, but also between the ages of 1 and 3 [102] . the acute bacterial recurrent and/or severe lower respiratory tract infections (lrti) are characteristic: sinusitis (60% of pediatric cases), otitis media (47%), bronchitis, pneumonia (87%) and/or digestive tract infections (diarrhea 57%) [213] . the prevalence of infections caused by mycetes has increased as well as cases of pneumonia caused by pneumocystis carinii, a signal for cell-mediated immunity (cmi) [152] . the gastroenteric tract is dominated by symptoms similar to those seen in celiac disease, with generalized malabsorption, steatorrhea, lactose intolerance, protein-losing enteropathy, inflammatory bowel disease (ibd), saccharidase deficiency and malabsorption of vitamin b 12 and folic acid, supported also in this case by intestinal infestation caused by giardia lamblia [527] . the tumor necrosis factor receptor family (tnfr) member taci (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) mediates isotype switching in b cells. in 4/19 unrelated subjects with cvid and 1/16 subjects with sigad there was a missense mutation in one allele of tnfrsf13b (encoding taci). none of these mutations were present in 50 healthy subjects. tnfrsf13b mutations cosegregated with the phenotype of cvid or sigad in family members of the 4 index subjects. b cells from subjects with taci mutations expressed taci but did not produce igg and iga in response to the taci ligand april (a proliferation-inducing ligand), probably reflecting impaired isotype switching [87] . other characteristics are hemopathies, hepatosplenomegaly, autoimmune hemolytic anemia (aiha) and x-linked lymphoproliferative disease (xlp), and cutaneous and internal organ granulomas (which differentiate it from xla), in particular ra, thrombocytopenia, and neutropenia [102] . offspring of cvid patients are at risk throughout their lives for cvid development and should be monitored with a high index of suspicion [441] . based on experimental evidence, it has been hypothesized that iga-and cvid-associated deficiencies may be the extreme opposites of one clinical spectrum: there is a block of b-cell differentiation, different only in the isotype involved. both defects often appear in different members of the same family groups and more or less the same alleles are present [313] . the most accredited hypothesis is that a number of extended haplotypes of the hla system are shared, to which gene duplications, deletions and polymorphisms codifying for some class ii and iii alleles correspond [22] . in fact, a number of common hla haplotypes, especially belonging to class iii, are observed in patients, and at least two haplotypes in 77% of cases [518] , such as hla-dqb1*0201, hla-dr3, c4b-sf, c4a, g11-15, bf-0.4, c2a, hsp-70-7.5, tnfa-5, hla-b8 and hla-a1, postulating therefore the existence of a common genetic basis [22] , with a susceptible gene (6p21.3) possibly the association marker [61] . for example in five members of a large family with one of the two pids, duplications of the c4 genes were associated with a selected group of hla class ii and iii genes [22] . the fact that four members without pid also had these haplotypes indicates that their presence alone is not sufficient for expressing pid, leaving room therefore for other factors [22] such as overlapping relations with celiac disease. the analysis of linked genes has confirmed a strong association with locus 4a, suggesting that an important role in both pid is played by the gene codifying c4a or an adjacent one [162] . see "x-linked hyper-igm (or hyper-igd) or cd154 deficiency (xhigms)" for further discussion. unlike transient hgg that occurs when the maternal iggs gradually disappear from circulation (table 1 .15), in the original study by taylor et al iga levels had fallen, becoming regular in newborn babies with thi after 1 year, corresponding to the nonatopic levels [493] . it consists therefore of a pathological delay in the normal antibody production maturing process. walker et al have calculated that the prevalence of thi is 23 × 10 6 in children, equal to the prevalence of symptomatic sigad (24 × 10 6 ) [525] . in all 15 children the igg and in 12/15 (80%) the iga were <5th percentile, 9/15 (60%) had igm levels <20th percentile resolved around the 22nd month; further confirmation consisted in the fact that the 12 children had symptoms either of ad or of fa or food intolerance [525] . from table 22 .4 the mean incidence is from 1 to 61 × 10 6 . during 7 years 30 children aged 6-46 months were diagnosed with thi and an incidence of 4.3/year [124] . in other studies the main defect was in the igg: in one it had normalized between 18 and 40 months [129] , in another trial 13/247 babies (5.3%) exhibited at the age of 10 months an absence of serum igg levels and of specific antibodies to viral agents, which in eight children were detected before the serum igg levels returned to normal, whereas in basis is an il 2 r deficiency [529] , more precisely of the g receptor mapped on chromosome xq13 [347] . the sole deficiency of il 2 r is not sufficient for producing an immunological phenotype as devastating as scid [529] . because the g chain of il 2 r (il 2 rg), a shared component of il 4 r, il 7 r, il 9 r, il 13 r, il 15 r, il 21 r and il 23 r [351] , gc mutations interfering with its link to the ils deprive the lymphoid progenitor cells of the crucial signals for normal lymphocyte intrathymic development [424] . mutations in any of the genes: il 2 rg, il 7 ra, jak3, artemis, rag1, rag2, cd38, ada, cd45 cause scid [68, 69, 211, 247, 272, 347, 350, 365, 391, 424, 443] . a total of 264 il 2 rγ gene mutations have been sequenced, of which 169 are unique [341] . each of these mutations has resulted in γc deficiency with varying degrees of id. the mutations are distributed throughout the eight exons of the gene, as well as in the regions necessary for proper transcription and translation. the penetrance of each of the above il 2 rg mutations is unknown. exons 5 and 7 have mutation hot spots. the types of mutations identified include missense, nonsense, insertions, deletions, splice mutations, and mutations that affect rna processing and translation [341]. among 93 mutations in 136 patients, the most numerous (67%) are punctiform mutations (fig. 22 .11) plus one missense related to amino acid residues [390] , with a lack of jak3 and gc interactions [424] . in an atypical form, the substitution with residual cysteine of the arginine at position 115 appears to be decisive for gc chain expression, probably a mutation reversion at the basis of the molecular defect, with a numeric and functional t-cell normalization [475] . the mutations, by inactivating the common g chain, render the t cells of boys with scid-x1 unresponsive to several ils. the result is a block in t-cell development and a severe deficiency of mature t cells. b cells, although pre-two children normal igg levels were detected even before the appearance of specific viral antibodies. igg levels usually normalize at between 15 and 36 months [78] , at the age of 2 years (fig. 22 .10) or before 36 months of age in 33/40 children; however, 7/40 still had low ig levels at 40-57 months of age [253] . at 27 was in 9/30 children ig levels were still <2 sd for age and in 5/9 various igg subclass deficiencies were detected [124] . a prospective study with an 8-year follow-up found that igg and iga deficiency is normalized by the age of 6, but in a minority of cases this may be a prodrome of sigad or another humoral deficiency [315] . a study with a 10-year follow-up of 35 children with igg deficiency as well as iga deficiency in 34% of the cases, observed multiform clinical symptoms. since thi can gradually normalize, some children have low antibody titers, and others low igg levels. however, both groups experienced significant infections [110] . in some cases, thi is asymptomatic; in others infections, especially of the respiratory tract, are present. the designation of thi may be a misnomer, and an alternative designation could be added to thi such as "with recovery" or "with development of other dysgammaglobulinemia" [315] . general characteristics of combined t-cell/b-cell immunodeficiency are summarized in table 22 .8 [453] . t -b + scid is a heterogeneous group with an incidence of between 1:50,000 and 1:75,000 livebirths [509] . xlinked fh is positive in 53% of cases [68] . the genetic sent in normal or even increased numbers than in other forms of scid, are dysfunctional [347] . b cells do not mature or produce antibodies due to a complete b-cell differentiation arrest at the pre-bcr checkpoint, showing the absence of complete vdj recombination [350] . other forms are also known with an attenuated phenotype and a partial t-cell function [162] . typical scid-x1 represents the most common form, with 45.5% of cases [68] (5.5% in tables 22.4, 22.5) . in the thymus, there is a severe hypocellularity, without lymphocytes and hassall's bodies where thymic epithelial cells predominate without grossly evident corticomedullary differentiation (fig. 22.12 ). severe lymphopenia is often associated with eosinophilia; nk cells are within the norm or rare. the majority of in-fants with scid-x1 lack both t and nk cells (t -b + nkphenotype) [68, 391] . cd3 t cells, if present, are of maternal origin, because the block, as also in scid ar, occurs at the level of cd4 -, cd8 -, cd44 -, cd3 + and cd1a + ; developing t cells and cd83 + thymic dc are reduced >50-fold when compared to age-and gendermatched control thymus [209] (fig. 2. 2, pre-t, tn), thus scid t -b + . the study of other subpopulations distinguishes the scid subtypes: t cells are reduced in all variants, the absolute cord blood (cb) number is 158-2,400 lymphocytes/mm 3 (tables 1.34, 1.35, so that any count below 4,000/mm 3 is lymphopenic). moreover, in ada deficiency (adenosine-deaminase) there is a maximum reduction in total lymphocytes, in scid-x1 and in jak3 defi-1284 chapter 22 primary immunodeficiencies alies) and against a common t and nk cell drop [68] . furthermore, in two cohorts [68, 474] the affected females had the same phenotype, indicating a possible complex molecular defect [68] . there is no response to delayed spts, and there is an absence of lymphocyte proliferative responses to mitogens and to a specific antigen such as tetanic toxoid [61, 189, 474] . the average age at diagnosis was 4.4 months in 31 children [21] , similar to the ages reported in >200 children with scid from all causes [68, 474] . the male:female ratio is 3:1 and diagnosis is often missed or occurs too late to save the lives of those infected infants who may manifest gvhd early on with morbilliform eruption in the first few days of life due to the transplacentally acquired maternal t cells, intractable diarrhea resulting in a severe malabsorption ( fig. 22 .13), severe interstitial pneumonia (fig. 22. 14), or giant cells caused by anti-measles vaccination or bcg (bacillus calmette guérin), with death caused by chickenpox or infections caused by pneumocystis carinii, herpes, adenovirus, cmv (cytomegalovirus), etc. [63] . the absence of tonsils is observed and also lymphoid tissue [508] and thymus [453] hypoplasia. these children must be transferred urgently to a specialized center and be placed in a sterile room to receive a bmt [474] . on rare occasions, il 2 rγ mutations have caused an atypical mild scid that presented beyond infancy [390] . up-regulation of bcl-2 by an il 2 r lacking il 2 rb tyrosine residues leads to increased cell survival after il deprivation; astonishingly, this survival signal does not occur when gc tyrosine residues are absent. thus, if ciency, the number of b cells is the highest and that of nk cells is the lowest (b + >t ->nk -); nk cells on the contrary reach their highest levels in the ar form [68] . in scid there can also be b alymphocytosis [474] . this divergent data is, however, characteristic of t -b + nkmolecular defects (gc, jak3 defects), b + t -nk -(ada deficiency), or t and b (possible recombination anomgc-dependent signals are revealed only in the absence of il 2 rb tyrosine, il 2 r engages at least two distinct signaling pathways to regulate apoptosis and ccl-2 expression [293] . in two clinical series, patients with mutations in il 2 rg represent 28%-45% of all scid cases [68, 474] . children with il 2 rg mutations have lymphopenia in 95% of cases, with total lymphocyte counts <2,000/mm 3 (normal levels, 4,000-13,500/mm 3 ), based on clinical case series [68, 474] . all patients have very low or absent t cells, and approximately 88% have low or absent nk cells [390] . ar mutated genes on autosomal chromosomes have been identified in ada deficiency, jak3 deficiency, and rag1 or rag 2 deficiency [65] . the existence of b-and t-lymphocyte lymphoid precursor differentiated defect is particular, in some cases a rag1 and rag2 mutation, the two genes that activate vdj recombination [443] was observed; however, this rag2 gene function has been questioned [411] since a rag defect is more present in t -b -scid and the omenn syndrome [491] . in babies suffering from scid, there is a marked reduction of t and b lymphocytes (table 22. 3) and all in vivo and in vitro responses are absent. onset and clinical and histological pattern is similar to that of x-scid. the jak3 gene mutation (tables 1.31-1.33) variant has a frequency of 5.9%-7.4% [68, 408] among babies affected by scid and in the absence of t (3±2%) and nk cells (1±1%) [407] . the molecular base is the mutation affecting the jak3, which prevents it from associating with the gc chain and from sending signals to the abovementioned ils [354] and to other marker proteins belonging to the jak-stat complex [301] . at the origin is a lack of t lymphocytes that transform into the scid phenotype [424] . these patients present b + t -nk -: the b (70±12%) with iga equal to 2±2% [407] , and those with x-scid present a defective differentiation, but are capable of producing elevated levels of ige in the absence of other isotypes [354] . this data indicates that gc and jak3 are essential for t-and nk-cell development [407] . the clinical characteristics are identical to those in x-scid, with the difference that the scid-jak3 phenotype is also observed in females (50%) [408] . furthermore, a jak3 deficiency could be an important cause of scid ar and should be considered in all patients with the b + t -nkphenotype, without an x-recessive heritage [68] . in a 6-month scid-x1 infant presenting with a history of recurrent infection and failure to thrive, a novel splice mutation, gc-dependent, was described, characterized by near-normal count of functionally deficient nk cells (b + t -nkcell phenotype). cell surface gc expression was undetectable on nk cells and in trace amounts in the minority of b cells. t cells were absent, igg and iga undetectable, and igm were within the normal range [183] . bmt is not a perfect therapy, because b-cell function developed in 3/9 children, and nk functions normalized in 2/9 children after bmt [408] . the family pedigree shows an inbred family with consanguinity across five generations. two brothers were diagnosed with scid. one, at the age of 4 months, presented with persistent oral thrush, oral ulcers, and failure to thrive. he had no palpable lymph nodes and no thymus shadow on a chest x-ray film. the second was diagnosed soon after birth and the third brother has always been healthy. three other male cousins died in infancy from severe infections consistent with scid; a 4 th cousin presented with oral candidiasis at the age of 2 weeks and failure to thrive. no thymic shadow was detected on chest x-ray film and peripheral blood lymphocytes showed persistent lymphopenia. he had no lymph nodes, failed to reject a skin allograft and did not show an increase in the blood igg and igm antibodies for dtp after three vaccinations. the three affected patients were hz for a caet transition at nucleotide 394 in exon 4, leading to a proline to serine substitution (p132s) in the extracellular domain of il 7 r. the cousins and their parents harbored both wild and mutant alleles. this partial deficiency is sufficient to block t-cell development and lead to a scid phenotype. the fh of severe pid with multiple affected male infants strongly suggested an x-linked inheritance. nevertheless, this family consanguinity is in favor of an ar inheritance [410] . defective il 7 r expression caused in three patients a t -b + nk + scid, indicating that the t-cell defect in scid-x1 resulted from inactivation of il 7 ra signaling. thus il 7 r-mediated signaling is required for t cells but not for nk ontogenes. mutations in the gene for the il 7 r chain on chromosome 5p13 were found in all three patients [391] . these infants resemble those with other types of scid with respect to their susceptibility to infection and the absence of functional t cells and b cells. however, they differ in that their circulating lymphocytes are primarily nk cells. rag1 and rag2 are required for the rearrangement of tcr and bcr genes [343] . half of the patients with t -b -scid had mutations in their rag1 or rag2 genes, thus highlighting the crucial role of these genes in normal v(d)j recombination machinery [443] . rag-thymocytes lack a functional pre-tcr and hence arrest at the cd44-/cd 25+ stage of differentation [491] : without rag1 and rag2, mature ig and tcr genes cannot be assembled, and lymphocyte development is arrested at very early stages [53] . abnormalities of the chest, scapula and iliac bones and short and stumpy limbs [219, 224] . x-ray abnormalities are documented in fig. 22 .17: the absent thymic shadow and a notable cupping and flaring of the ribs' ends (arrows) can be observed, while histological studies of the chondrocostal junctions document their total cellular disorganization (fig. 22.18 ). this deficiency is the object of a great deal of attention because it was the first to be treated using gene therapy [313] . the lack of ada is observed in 14.8% of patients with scid [68] . the ada enzyme catalyzes the conversion of adenosine and deoxyadenosine into inosine and deoxynosine; although ada is found in all cells (cd26 anchors ada to the lymphocyte cell surface (table 1. 2), the deficiency damages above all the immune system [219] . table 22 .9 [219] summarizes the biochemical foundations of this pid. more than 50 ada mutations are known, including >30 amino acid substitutions, deletions and punctiform mutations or anomalies of the gene itself, such as exon 1 deletion and exons 4, 5, 7 and 9-11 mutations with a total of 15, nine of these in patients with ada-scid and six in those with a partial deficiency [219] . an additional 29 mutant alleles have been found (28 missense and 1 single-codon deletion) [21] . adenosine and deoxyadenosine are also apparent suicide inactivators of the enzyme s-adenosylhomocysteine (sah) hydrolase, with consequent accumulation of sah, a powerful inhibitor of virtually all cellular methylation reactions [61] . the accumulation of metabolites, including camp, deoxy-atp and 2'-o-methyladenosine, has a toxic effect on the cells by blocking dna synthesis and dividing and resting t lymphocyte proliferation [61] (fig. 22.15 ). four different clinical phenotypes have been described for ada-deficient subjects (table 22 .10) [219] , which cover a broad spectrum of immunological aberrations, from the complete absence of b and t immunity, indicating scid (85%-90% of patients) (the thymus in fig. 22 .16) to forms with a delayed onset or partial deficiency (10%-15%) [219] . in children, the delay between onset of symptoms and diagnosis has been estimated to average 2 months [474] . if clinical symptoms indicate an early onset, in addition to typical scid symptoms, there are also x-ray pathognomonic skeletal abnormalities of the chondrodysplasia type, especially this very rare ar type of scid was observed for the first time in 1959 in identical twin male infants who exhibited a total lack of both lymphocytes and granulocytes in their peripheral blood and bone marrow. it has a frequency of 1% in cases of scid [68] . the children are symptomatic in 90% of cases within the first days after birth [46] and is usually fatal within the 3rd month of life without a bmt [224] . due to the common stem cell (sc) non-maturation [224] , it is characterized by total block in lymphoid and myeloid precursor differentiation, therefore not only by an extraordinary lymphopenia, but also by a marked cytopenia in all sections (table 22 .11) [474] , in the spleen, in the lymph nodes and in the gastroenteric tract, and a high frequency of severe successive infections [474] . the thymus is always much reduced in volume, no hassall's bodies are seen [224] . seven of the eight infants reported by who with this defect died between 3 and 119 days of age from overwhelming infections; the eighth underwent complete immunological reconstitution from a bmt [543] . an additional three of five children who required two hscts (hematopoietic sct stem-cell transplantation) and received intensive conditioning therapy before haploidentical hsct (matched for 3 of the 6 hla loci) are alive and well an activated phenotype and poor functional capacity [117] . studies involving hla typification and dna polymorphism show that t cells belong to the host, ruling out, therefore, the etiology of maternal cell engraftment [117] , unlike other types of scid [474] . the absence of circulating b is also characteristic [117] , equal to 3.8%-7.1% of normal levels [474] , reaching 0% [520] , high ige levels (526 ui/l), hypereosinophilia reaching 3,000¥10 9 cells/l (normal, 0-0.5 cells/l), and low ig levels at the beginning [246] then declining to the point of agammaglobulinemia [520] , comparable to that in reticular digenesis (table 22 .11) [474] . the marked b-cell depletion can also bear rag1 and rag2 gene missense mutations that decrease the efficiency of vdj recombination, which results in impaired but not absent rearrangement of both bcr and tcr. four missense mutations were detected in the rag-2 in 6/8 patients [491] . in 13/16 patients (81%) the mutations affected the rag1 gene, and in 3/16 (19%) the rag2 gene [515] . increased ige is linked to th2 primary infiltration, with spontaneous production of il 2 ifn-g, il 4 , il 5 and il 10 , which is down-regulated by ifn-g therapy [437] . clonal expansion of vb14 + cd3 + , cd4 -cd8secreting high il 5 levels and low il 4 and ifn-g levels [318] could indicate an analogy with the fas (cd95) defect. t lymphocytes show an activated phenotype and a spontaneous apoptosis associated with reduced expression of bcl-2 gene product, and a higher cell death of cd4 + cd45r0 + cells [59] . given that high cd30 levels in the lymph nodes, skin and serum of three children generated th2 lymphocytes [95] , a th2-mediated pathogenesis is possible: the cd30 are th2 markers (chap. 1). as in human scid, b and t cells are found in mice with scid, but with a final repertoire that is decidedly oligoclonal and lacks the heterogeneity characteristic of a normal immune system, so lymphopenic scid and omenn syndrome could be two aspects of the same disease with different clinical expressions, especially of time [224] . clinically, young babies soon after birth show a generalized exudative erythroderma and desquamation, often mistaken as ad, alopecia, widespread lymphadenopathy, hepatosplenomegaly, persistent and profuse diarrhea, failure to thrive with malnutrition ( fig. 22.19) , aiha, recurrent infections caused by common and opportunistic germs ( fig. 22 .20), and markedly elevated serum ige levels [136, 241, 474, 520] . this outline included four babies from the same family with the same symptoms until death occurred at 10-19 months, but who did not present hypereosinophilia and were diagnosed as dm [375] . differential diagnosis may be challenging since omenn syndrome and gvhd show dyskeratosis and basal vacuolation, but the first always shows acanthosis and usually parakeratosis. gvhd shows a flat epidermis and rarely parakeratosis. both can be distinguished after immunohistochemical staining for cd45 and cd68, which shows predominantly lymphocytes in the dermal infiltrate in omenn syndrome, and relatively more macrophages in gvhd [438] . with myeloid and t-and b-cell lymphoid reconstitution [46] ; another child is alive and well after 32 months [13] . b-scid, characterized by increased cell sensitivity to radiation secondary to mutations of the artemis gene, could carry a poorer prognosis because of defective repair of dna breaks [406] , occurring around the time of bmt, from the effects of chemotherapy, infections, and gvhd [206, 247, 332] . one group of patients with scid with an additional sensitivity to radiation was found to harbor large deletions or truncation mutations in the artemis gene mapped on chromosome 10p [65] , implying a role for artemis in dna double-strand break repair, which is mutated in human scid [350] . omenn syndrome is classified as a scid because newborn babies exhibit symptoms similar to a gvhd, due to 1a antigen expression and cd1a absence [241] , and because it can coexist in families with alymphocytosis [117] . this is an ar syndrome with an unknown pathogenesis, sharing characteristic clinical and immunological abnormalities with t + b -scid [515] . severe cutaneous lesions with hyperkeratosis, apoptotic malpighian necrosis and basal membrane destruction can be associated [241] . no lymphoid cells or hassall bodies are found in the thymus [241] . the immunological structure reveals histiocyte infiltration of the skin, bm and lymph nodes, with proliferation of t infiltrating the epidermis and the enteric mucosa, increased t cells with 1289 combined t-cell and b-cell deficiency igg (g/l) 7 2.28-6.16 igm (g/l) 0 neutrophils (cells/ml) 200 1,000-8,500 data from [474] . in a child with scid and circulating t cells within the norm, a gene transcription deficiency was ascertained [414] . a male infant of first cousin parentage presented at the age of 6 months with cmv pneumonia, persistent oral and esophageal candidiasis, adenovirus gastro-enteritis, and failure to thrive. he developed lymphadenopathy, hepatosplenomegaly, iron deficiency anemia with no evidence of hemolytic anemia, and chronic inflammation of his lungs and mandible. biopsies showed extensive lymphocytic infiltration of his lung, liver, gut, and bone. serum igg and igm were elevated, but iga was low. he had t-cell lymphocytopenia, with an abnormal cd4:cd8 ratio of 1:1. the t cells responded poorly to anti-cd3, phytohemagglutinin and other mitogens, and to il 2 . he was found to have a truncated mutation of the il 2 ra chain (cd25). he was given a successful allogeneic bmt after cytoreduction [452] . about 225 cases have been reported, [75, 290, 539] , 78% [75] or 100% [539] of which have the x-linked form of cd154 deficiency [75] . patients are generally male, but there can be a non-x-linked form [224] , in which 22% of patients are females [75] . an estimated minimal incidence was calculated of 1 in 1,030,000 live births. over half of 79 patients developed id symptoms and were diagnosed by 1 year of age, and over 90% by 4 years of age [539] .although carriers of xhigms are considered to be asymptomatic, an extreme lyonization of the normal x can lead to a mild expression of the xhigms which is similar to cvid [118] . it can be secondarily caused by environmental factors and also stem from congenital rubella [278] : this indicates its heterogeneity. mutations in the tnfrsf5 encoding cd154 in xhigm patients result in a lack of b-cell signaling by activated t cells [53] . however, 21 boys out of 56 failed to express cd154, and tnfrsf5 mutations were found in 20 of these boys, whereas no tnfrsf5 mutations were found in 16 boys with weak expression of cd154 [184] . as a result, xmigm b cells fail to undergo isotype switching and produce only igm due to a defect in the rna editing enzyme, activation-induced cytidine deaminase (aicda), an enzyme expressed only in b cells and required for the processes of class-switching and somatic hypermutation of ig genes [357] . the marked reduction of igg (<150 mg/dl), ige and iga is accompanied by a sharp increase in mature igm and circulating igd, but b cells do not express other ig [227] . interestingly, 25% of patients with confirmed xhigms who had tnfrsf5 mutations had low concentrations of igg, iga, and igm. most of the remaining patients with xhigms had the classic pattern of normal or raised igm with low concentrations of iga and igg [184] . the cd154 gene defect is usually expressed on the membrane by activated t lymphocytes, which therefore cannot bind b-cell cd40 [266, 346] . figure 22 .21 shows 75 cd154 localizations and mutation frequencies, in 39.5% of cases mistakenly. for example, a sense codon substitutes for a missense one, creates a premature stop signal: therefore specific pertinent mutations, such as g144e, tunistic infections. this disorder is related to mutations in the gene that encodes the nuclear factor kb (nf-kb), which is required for activation of the transcription factor nf-kb, or nemo (nf-kb essential modifier), also known as ikk (inhibitor of b kinase). the phenotype observed in x-higms-eda patients shows that the putative zinc-finger domain of nemo has a regulatory function and demonstrates the definite requirement of cd40-mediated nf-kb activation for b cell ig classswitching [233] . three other genes, expressed by b cells, have been associated with the higm phenotype giving place to higm 2-4. mutations of activation-induced cytidine deaminase (aicda) (higm2) and uracil glycosylase (ung) (higm4), both expressed by follicular b lymphocytes, lead to defective class switch recombination and somatic hypermutation. mutations of cd40, the cd154 receptor, cause a rare autosomal form with a clinical phenotype similar to cd154 deficiency (higm3). these rare pids may shed light on the complex events leading to the production of high-affinity, antigen-specific antibodies of different isotypes [146, 355] . early treatment with ivig associated with antibiotic prophylaxis have reduced the incidence of life-threatening infections and improved the growth of children with higms [290] . cycles of g-csf (granulocytecolony stimulating factor) in the presence of severe neutropenia are advised [290] . substitute therapies with soluble forms of recombinant or gene type cd154 [76] are being studied. a recent review of cd154-deficient patients showed that 75% develop liver disease and only 20% survive into the third decade of life [290] . bmt has a successful outcome in young children (65%); older patients with more ad-can interfere directly with the link site for cd40 ( fig. 22.22 ). consequently the signal which indicates that b cells should begin isotype switching, limited to the production of low-affinity igm, is missing [346] . without isotype switching, gc formation is minimal [508] (fig. 22 .23) and follicular dendritic cells (fdcs) are reduced in number, also having an abnormal phenotype [147] . as shown by figs. 1.31-1.33, the lack of cross-linking of cd40 by cd154 results in b-cell failure to up-regulate cd80 and cd86, important costimulatory molecules that interact with immunoregulatory molecules on t cells such as cd28 and ctla-4. two patients with normal levels of cd154 have also been described [359] . as in males with xla, infections start during the 12th month, those most often observed are otitis, pneumonia or sepsis cased by pyogenic bacteria, opportunistic infections, in particular caused by pneumocystis carinii [290] , and also ulcerative stomatitis, ra, neutropenia, aiha, lymphoproliferating complications and type b gastroenteric lymphomas with igm [224, 413] . the most prominent clinical infections were pneumonia (81% of patients), upper respiratory infections (urti) (49%-87%) including sinusitis (43%) and recurrent otitis (43%), lrti (82.1%) recurrent/protracted diarrhea (34%-55.3%), cns infections (12.5%-14%), sepsis (13%-14.3%), cellulitis (13%), hepatitis (9%-16.3%), and osteomyelitis (1%) [290, 539] . lymphoid tissues are normal or hyperplastic [75] . recently, a rare form of higms associated with hypohydrotic ectodermal dysplasia (eda) characterized by the absence or hypoplasia of hair, teeth, and sweat glands has been described. unlike patients with higms, these patients failed to have a history of oppor purine-nucleoside phosphorylase (pnp) deficiency, ar, for which 35 patients have been reported [53] , is characterized by the absence of an enzyme necessary for the catabolism of purines, which converts inosine, deoxynosine, guanosine and deoxyguanosine into hypoxanthine and guanine ( fig. 22 .15); the responsible gene has been mapped to chromosome 14q at position 13.1 [414] . this has also been observed in 33 patients with nezelof syndrome [304] . a variety of mutations have been found in the pnp gene in patients with pnp deficiency [432] . although ada and pnp are both purine salvage pathway enzymes, pnp deficiency does not lead to as severe an id as ada deficiency. patients have considerably reduced concentrations of serum and urinary uric acid. numbers of t cells fall progressively, more than that of b cells (table 22. 3), just like the proliferating responses to mitogens and antigens, especially because pnp deficiency causes an intracellular accumulation of deoxy-gtp (guanosine triphosphate) inhibiting ribonucleotide-reductase and t-and b-lymphocyte proliferation, so combined t and b defects are critical. pnpdeficient patients are as profoundly lymphopenic as those with ada deficiency, with absolute lymphocyte counts usually <500/mm 3 . ig levels and production of specific antibodies are all normal [19] . onset may be early, as for scid, but also delayed until the age of 3-5 years. the clinical pattern is dominated by recurrent bacterial, viral and fungal infections, with an abnormal susceptibility to opportunisic germs. two-thirds vanced liver disease may die because cryptosporidia infection that has progressed rapidly following pretransplantation cytotoxic conditioning therapy [252] . therefore, a patient with end-stage liver disease related to cd154 deficiency first received a liver graft, and as soon as liver-graft function was satisfactory, bmt was performed with a nonmyeloablative conditioning protocol of fludarabine and melphalan [208] . the screening for cd154 deficiency should include children with severe rri, and with dysgammaglobulinemia with a normal or increased igm level [197] . conventional allogeneic hsct from an hla-matched or a matched unrelated donor (mud) is curative and feasible, if performed before significant infections and organ damage occur [503] . an approach for high-risk patients including nonmyeloablative hsct was workable in a retrospective analysis of 38 european patients undergoing hsct for cd154 deficiency in eight european countries between 1993 and 2002. the donor sc source included 14 hla-identical siblings, 22 muds, and two phenotypically matched parental stem cells (scs) (12 tcd [t-cell depleted]). of these patients, 12 (32%) died from infection-related complications, with a positive result in 68.4% of patients [180] . carriers can be detected, and this is useful for making a prenatal diagnosis [407] . of patients suffer from neurological alterations, ranging from spastic symptoms and alterations, etc., to mental retardation and one-third from aids, the most common of which is aiha. the consequence of severe infections, generalized vaccination, severe chickenpox, lymphosarcoma and gvhd caused by blood transfusions in the first decade of life is death [304, 397] unless bmt is successful [25, 58, 67, 83, 98] . however, poor neurodevelopmental progression may result [25] or may not [98] . since the biochemical bases of pnp and ada deficiencies are similar, it is hoped that genetic treatment will also be effective in children with this pid [76] . this deficiency of hla molecule expression occurs in the more severe forms of pid if they are class ii: about 80 cases [140, 428] are known of this ar syndrome [140] , heterogeneous for the numerous complementation groups the patients are divided into [76] . hla class ii molecules are absent in all tissues [400] , to the extent that the cells of patients maintained in cultures for years preserve the negative phenotype [400] . there is a deficiency of class ii gene transactivator (ciita) codified by chromosome 15, the expression of which plays an important role in t-cell activation: its absence makes class ii gene expression impossible [473] . this function is shared with another protein mapped on chromosome 2, rfx5, with a binding site in the promoter region of genes codifying class ii chains [401] . two additional class ii-specific transcription factors are rfxap and rfxank [314] . these act on the class ii promoter region and are essential and also nonreplaceable, to the extent that alternative routes cannot compensate for their absence [311] . furthermore inactivation, or the deficiency of these factors, has a specific effect on the genes dictating hla class ii, the li chain and hla-dm, because there is no indication that other regulating systems may be involved [401] . the absence of hla class ii is associated with a cd4 lymphopenia. hla class i expression is normal in the patients tested and cd8 lymphocyte numbers are not reduced [428] . interestingly, in a twin study, despite the deficiency, there were antibody responses and class ii-dependent t cells; hence the authors envisage that this represented a hla class ii residual expression below the test sensitivity [540] . the clinical outline is dominated very early on, before the age of 6 months (range, 2 weeks to 12 months) [428] , by severe and recurrent gastroenteric and pulmonary infections, with a severe and prolonged course, associated with malabsorption and failure to thrive [77] . bacterial and viral infections, bronchopneumonia, hepatitis, cholangitis, viral meningoencephalitis and various autoimmune manifestations are common complications [140] . even though an hla class ii deficiency is clinical-ly less severe than scid, the result is uniformly fatal during the first or second decade of life [162] . the most evident immune defect consists in the complete lack of reactivity to exogenous antigens, which in vivo reflects an anergy to spts, as well as the complete lack of hla class ii expression and absence of cellular and antibody responses to antigen stimulation [140] , which are instead positive to mitogens (table 22 .8; fig. 22 .24) [508] . laboratory investigations show a normal b lymphocyte number, but children may be agammaglobulinemic [140] . the thymus and other lymphoid organs are remarkably hypoplastic, with a severe cd4 lymphocyte depletion, while cd8 and b-cell levels are normal. the syndrome involving a deficiency of hla antigens confirms an important hla biological role in the complex system of t-b cooperation [77, 140] . some studies suggest that there are more types of deficiency. when placed together in a culture, the b lymphocytes of these patients, previously transformed by ebv, the lymphocytes correct each other so as to allow hla class ii molecule expression. this has led to the identification of so-called complementary groups [251] . the specification that the gene is mapped on chromosome 19p13.3 can lead to an earlier prenatal diagnosis [166] . longterm survival seems to depend primarily on hla-identical and hla-haploidentical bmt performed in the first 2 years of life, before the acquisition of chronic virus carriage and sequelae of infections [257, 140] . a child recently received a transplant [428] with a novel protocol [208] : the cd4 count increased up to 300 cells/ml [428] . a direct correction of the genetic defect is based on the transduction of cells from patients with lentiviral vectors encoding ciita, rfxank, rfx5, or rfxap. the rfxank vector restored class ii expression in a t-cell line from one patient. the rfxap vector corrected primary cells from a second patient [314] . the study of the common association of hla class i molecule deficiency, already known as bare lymphocyte syndrome, has led to the identification of various patients with an isolated deficiency and of one patient with a deficiency associated with class ii, the most severe [85] . the deficiency is caused by tap-1-tap-2 mutation, accompanied by severe and chronic bacterial rris [115] . in two brothers with the ar hla class i defect, the onset of rris took place between the ages of 4 and 7; the poor expression of nk cells was so severe that it led to the development of bronchiectasis [125] . the immunological structure is characterized by few cd8: the deficient expression of hla class i molecules is diagnostic [115] (tables 22.1, 22.3). mutations with no symptoms referable to an id, therefore integrating a genetic heterogeneity. two other brothers and both parents were healthy [5] . while the ε chain deficiency produces modest clinical symptoms, the other two are severe also from an immunological point of view: in the γ chain deficiency resulting from the profound cd8 and cd45ra decrease caused by altered thymic activity that leaves the cd45ro unaffected [249] , and in those of the ζ chains due to the severe thymic atrophy [5] and thymocytes falling to 15% of normal levels, with limits at between 1% and 50% [249] . the cd3δ deficiency due to a heritable mutation of the cd3 gene that prevents the synthesis of the cd3 protein has been reported in 3 cases hz for the cd3 mutation. two cousins died at 2-3 months of age because of overwhelming infection. the thymus shadow is clearly visible on chest x-rays. the thymus becomes populated with developing thymocytes, with an arrest of differentiation at the cd4 -cd8stage of t-cell development. a girl (3rd patient) survives after a bmt [111] . this rare deficiency transmitted as an ar trait is caused by mutations of the zap-70 gene, a non-src family protein tyrosine kinase (ptc) important in t-cell signaling (tables 1.31-1.33). zap-70, known to be crucial for t cell activation, is a key player in tcr down-modulation and z degradation [136] . zap-70 has an essential role in the cd3g chain deficiency due to g or e gene mutations [19] determines a lack of cd8 and the absence of cd45ra [249] . in the first two cases described, one brother died at 31 months because of viral pneumonia after a clinical history indicating scid with severe aiha, while the other was asymptomatic at the age of 10 years, although with the same molecular defect [18] . the study of these brothers proved that, in spite of the absence of functioning g chains and 50% of the expressive levels of the cd3/tcr complex, the lymphocytes were normal. according to the authors, other chains may act in the place of missing ones; however, the correlated scarcity of cd8 may have negatively interfered with the mechanisms discriminating between self and non-self, while g chain deficiency could have modulated the onset of the deceased brother's severe autoimmune disease (aid) [18] . a cd3e deficiency was found in a 4-year-old child with mild rri symptoms and otitis media; the expression of the cd3/tcr complex was only 10%, but the stimulation with anti-cd3 induced a normal proliferating response. in fact, despite the ongoing mutation, a northern blot analysis showed production of a low amount of transcribed rna, corresponding to a small quantity of e normal chains, even though their dimensions were smaller than normal ones [471, 496] . the z chain deficiency found in the two brothers is similar to the deficient expression of cd3/tcr [5] . in the younger brother, the thymus, markedly reduced, showed no hassall bodies; the elder brother had similar chain [342] . in several babies (most of mennonite origin) with scid [20, 92, 139, 179] , the nonfunctional cd4 t cells (table 22. 3) were either normal or increased (cd3 + cd4 + , 75%). cd8 absence in the thymus and in circulation (cd3 + cd8 + , 0%-2%) [139, 179] suggests that the selective process is arrested during the transition from double-positive (dp) to mono-positive (mp) t cells [20, 140, 179] .arrested thymocytes had terminated rag gene expression and up-regulated tcr and bcl-2 expression, but failed to differentiate into mature cd4 or cd8 mp thymocytes, to be rescued from death by neglect or to sustain il 7 ra expression [294] . zap-70 deficiency results in an impairment of transendothelial migration that can be rescued by the transfection of zap-70 because cross-talk between the zap-70 signaling pathway and the chemokine receptor cxcr4 is required for t-cell migration [502] . although the thymic architecture is normal with presence of hassall bodies [179] and cd8 seem normal in the cortex, very few migrate to the medulla [20] . the near absence of cd8 + cells and an increased cd4:cd8 ratio dominate [27] . the few cd8 coexpress cd56 + , the nk-cell marker; b cells appear normal and functional, cd3 -cd19 + is at a level of 20%-40% [139, 179] , and serum ig values are normal [27] . the same phenotype was found in the brothers [92] ; other relatives were het [20, 139] . the absence of cd8 expression was shown to correlate with a missense mutation in both ig alleles of the cd8 a gene domain in a 25-year-old man and his sister, whereas high percentages of cd4 -cd8 -tcrab + t cells were found in the three siblings [114] . the proliferative responses in vitro to phorbol myristate acetate (pma) and ionomycin, pkc activators (protein kinase c), were normal, unlike pha (phytohemagglutinin), pwm, tetanic toxoid, anti-cd3, etc. [20, 139] . the positives operate below the tcr, while the negatives react directly with the cd3/tcr complex [92, 140, 179] , confirming the zap-70 deficiency [20] . the cd4 are present despite the deficiency because syk, the other member of the family, ensures a compensatory role in the infrathymic cd4 selection, although with a limited efficacy [179] . seven months after bmt, a child was clinically well and immunologically recovered [27] . studies in two siblings hz for a stop mutation in the tap-2 gene suggest that nk cells express still unknown inhibitory receptor(s) (the missing receptor, discussed in chap. 1) capable of down-regulating the nk cell cytotoxicity on binding to surface ligand(s) expressed by t cell blasts. functional analyses were consistent with the concept that this putative inhibitory receptor is expressed by virtually all tap-2/nk cells, whereas it is present only in rare nk cells from healthy persons. another prospect would be that tap-2/nk cells are actually missing this still unidentified triggering receptor involved in nk cell-mediated killing of pha blasts. since cells derived from patients displaying defective expression of either of the tap subunits are characterized by a strong reduction of mature hla class i molecules at the cell surface, a tap deficiency is connected with hla class i deficiency [517] . as discussed in chap. 1, nfat (nuclear factor of the activated t cells) is a transcription factor that forms a powerful transcriptional activating complex and, by linking with specific dna-regulating sites, plays a critical role in the synthesis of various t-cell ils which, due to the deficiency or excessive migratory mobility of nfat, although normal in number and in distribution, are incapable of activating and/or secreting the genes of il 2 , il 4 and ifn-g [86] . a 4-year-old girl with scid presented during infancy with severe recurrent infections and failure to thrive; her mrna was not produced for il 2-5 and ifn-g due to poor t-cell proliferation, although these were normal in number and in distribution, to initiate the transcription of the relative genes, regulated by nfat, with a binding site in the proximity in the 5¢ region. this severe clinical picture is accompanied by evident hgg [19, 86] . nk-cell deficiency is found in scid, cvid, reticular dysgenesis, chédiak-higashi syndrome, xlp, lad in tap-2 deficiency and in cfs (chronic fatigue syndrome), in particular cid such as scid, suggesting an association between nk-and t-cell deficiencies [478] . there is one known case of an adolescent with an isolated numerical and functional deficiency of nk cells and of precursors, recurrent neutropenia, severe and recurrent ebv, cmv, herpes simplex virus (hsv) infections and life-threatening chickenpox. another child, diagnosed at the age of 2.5 years with a cd8 deficiency, suffers from severe viral and bacterial infections although he has antibodies to various viruses [49] . the growing list of human genetic defects that impair nk-cell function has been recently joined by nemo-id [356] which occurs in a group of patients with antibody deficiency combined with exquisite susceptibility to infection with nontuberculous mycobacteria. infectious susceptibilities common to these disorders stress the important role for nk cells in host defense [59] . the natural history of three boys with nemo mutations outside of the 10th exon has been described. including these boys, there have been 22 families described as having nemo-id. the resulting estimated incidence of nemo-id is 1:250,000 live male births, making this disorder significantly less common [356] . cd45ra cells and a marked clinical improvement. these data indicate that the thymus is differentially required in the maintenance of the tcr repertoire complexity [380] . known also as idiopathic lymphocytopenia, primary cd4 t-cell deficiency is revealed by a profound and persistent reduction in circulating cd4 and with a cmi deficiency. it is documented in patients suffering from infections caused by opportunistic germs such as cryptococcus-induced meningitis and oral candidosis, also including ten children and a number of adolescents, for whom the following minimum levels of cd4 per age have been established: <1,000 cells/mm 3 from 0 to 23 months and <300/mm 3 from 2 to 12 years, or a total lymphocyte count of <20% on two separate occasions without being hiv-infected [463] . a family has been reported involving two brothers aged 13 and 18 with t counts between 150 and 200/mm 3 , recurrent respiratory, intestinal and cutaneous infections, and failure to thrive. the mother showed a low cd4:cd8 ratio [122] , while the entire family showed normal levels of ig and subclasses and hla molecules [128] . other symptoms included mental retardation, pansinusitis, bronchiectasis [168] , but no infections caused by opportunistic germs such as those reported by the who scientific group [414] . one case of primary cd7 deficiency is known of a child with scid without genetic transmission of the deficiency. t-cell proliferative responses to mitogens were defective and il 2 r expression was deficient on his t lymphocytes, and b cells did not differentiate into antibodysecreting cells when provided with the help of normal t cells [245] . the index patient for primary cd45 deficiency was the first child of consanguineous kurdish parents. she presented aged 2 months with a rash, pyrexia, hepatosplenomegaly, lymphadenopathy, pneumonitis, pancytopenia, and disseminated cmv infection. laboratory analysis showed absolute lymphopenia, low t cell numbers, with markedly low cd4 + and low cd8 + and normal b cell numbers. she responded well to anti-cmv treatment and at 8 months underwent a mud bmt. t-cell engraftment was demonstrated 3 weeks after bmt. despite continuous anti-cmv treatment, her undifferentiated scid human p56lck deficiency p56lck deficiency is an ar scid due to a defect of an src kinase critical for the generation of mature thymocytes in adult mice. p56lck is important in tcr signaling and phosphorylation of the itams of the cd3/tcr complex proteins. mutant mice lacking p56lck have pronounced thymic atrophy, a critical reduction in dp (cd4 + cd8 + ) thymocytes, no detectable mp thymocytes, and only a few peripheral t cells. both proliferation and development of a given defined cell subpopulation depend on meuse age. the absolute numbers and proliferation of dn and isp (immature single positive) thymocytes only proliferate during fetal and early postnatal life up to 14 days after birth, whereas the proliferation is significantly decreased beyond that age, thus lck may have differential roles in the proliferation and maintenance of dn, isp, and mp/dp thymocyte populations [151] . the first demonstration of a human scid patient with an abnormal expression of p56lck is an scid infant hospitalized at 2 months for dehydration, failure to thrive, and sepsis. the immune phenotype included hgg, selective cd4 lymphopenia, lack of cd28 expression on cd8 + t cells and poor t cell blastogenic responses to various mitogens and il 2 . p56lck protein expression was only minimal with an unusual mrna splicing pattern of the lck gene. the levels of p59fyn were normal and it is therefore possible that p59fyn played a role, albeit incomplete, in the development of his mature t cells. the child has since undergone an allogeneic bmt (at 32 months) from a matched unrelated donor (mud) [185] . unfortunately the boy died 2 months later due to cmv infection and gvhd (fd goldman, pers. comm., 8 nov. 2005 ). whn (winged-helix-nude) encodes for a transcription factor that is crucial for maturation of the thymus microenvironment [185] . nu/nu mice fail to develop a thymus and mature t cells due to a defect in the whn gene encoding a transcription factor necessary for terminal epithelial cell differentiation. a defective whn gene could lead to the disrupted early t cell development in the bm. t cell progenitors were associated with a lack of pta gene expression and a failure to give rise to mature t cells in adoptive euthymic hosts. wild-type hscs rapidly matured into functional t cell progenitors in the marrow of euthymic or thymectomized but not nu/nu hosts. therefore defects in bm prethymic t cell development can contribute to t cell deficiency in nu/nu mice [90] . in two sisters a severe scid caused by mutation of the whn gene was associated with complete alopecia. hla-identical bmt in one of the two girls resulted in a clear reconstitution of cd4 + and cd8 + cmv reactivated, and she died 55 days after bmt [74] . a 6-bp deletion in the gene encoding cd45 resulted in the loss of glutamic acid 339 and tyrosine 340 in the first fibronectin type iii module of the extracellular domain of cd45, identifying a region important for cd45 structural integrity and lack of surface cd45 expression. this was almost certainly responsible for the id in this girl [494] . a second child presented at 2 months of age with severe cid, showing similar t-cell defects. despite normal b-lymphocyte numbers, serum ig levels decreased with age [272] . introduction of a functional cd45 minigene was sufficient to overcome the main scid-associated defects and represents a potential route to a gene therapy for human cd45-deficient scid [516] . two male infants born to consanguineous parents had scid despite phenotypically normal blood lymphocytes. their t cells were unable to produce il 2 , ifn-g, il 4 and tnf-a [154] . another child with scid had defective transcription of il genes encoding il 2 -il 5 [86] . dna binding of activation protein 1 (ap-1), oct, creb, sp1, and nf-k b was normal, but the binding of nfat to its il 2 promoter response element [154] , or the ability of nuclear factors from the child's t lymphocytes to bind response elements present in the il 2 regulatory region [86] was barely detectable [86, 154] both before and after t-cell stimulation [154] . these results indicate that the nfat abnormality may underlie the multiple il deficiency in these boys. nezelof syndrome, also known as cellular id with ig, or combined with a predominant t-cell defect, or as a scid variant, clinically less severe compared to the previous ones, is characterized by a form of ad, concentrations of ige that may also be extremely elevated (table 22. 2), and normal or increased serum levels of other ig classes [62] . the cmi study emphasized the mature t-cell reduction or absence, various expressions of immature cells, with cutaneous anergy to spts and a reduced or absent in vitro lymphocyte response to mitogens. from infancy, patients present recurrent or chronic pulmonary infections, pondostatural retardation, oral and/or cutaneous candidosis, chronic diarrhea, recurrent cutaneous and urinary tract infections, gram-negative bacterial sepsis and a particularly severe form of chickenpox [394] . differential diagnosis must include pediatric aids, also marked by proportionably increased ig and a lack of antibody and t-cell function [79] . inherited through ar modalities, cd95 deficiency has been observed in 8 children, two of whom were brothers, with mutations of the fas gene, one hz and 7 het [166, 281] , as well as in 9 unrelated children [468] . these mutations most often arise as a result of mutations in the gene encoding the lymphocyte apoptosis receptor fas/apo-l/cd95. a novel mutation has been identified in the intracellular apoptosis signaling domain of fas in 11 members of a family, with several members monitored for up to 25 years [228] . thus, the deficiency is inherited in an autosomal dominant fashion but with a high degree of variability in clinical expression [228] , but also in an ar fashion [510] . the clinical picture is dominated by imposing hepatosplenomegaly with an early onset, even neonatal, accompanied by t-cell hyperproliferation, chronic and persistent lymphadenopathy, and failure to thrive [468] . an extensive lymphocyte infiltration of lymph nodes, spleen and liver is observed, with t cells reaching 35,000/ml [cd3 + , cd4 -cd8 -(dn) equal to 35-60 cells/ml compared to 0-3 in controls], as in omenn syndrome, also in the bloodstream, with possible oligoclonality of t cellularity. dn t cells expressed the a/b tcr [468] . immune dysregulation is associated with g and a hgg (hypergammaglobulinemia, auto-antibodies and aids, especially of the hematological type, such as aiha, and with a severe and recurrent thrombocytopenia [166, 281] . autoimmune features are discussed in chap. 18. an overlapping mechanism could belong to the etiopathogenesis of xlp and omenn syndrome. was has a prevalence of approximately 4 × 10 6 live births [402] . it is transmitted as a recessive hereditary trait linked to the chromosome x, localized in a pericentrometric position on the short limb of chromosome x (xp11.22-p11.3) [274] . it is therefore possible to identify the female carriers and to provide prenatal diagnosis [61] . the gene that codifies the was defective protein (wasp) has been isolated [458] and has 167 mutations distributed among all 12 exons of the entire gene, 110 of which are unique and 38 familiar, with two large deletions, one embracing exons 1-7 and one intron 8 [275, 444] (fig. 22.25 ). six novel mutations have been identified that involve nonsense mutations, or small deletions, all of which result in predicted truncation of wasp synthesis [57] . a new, recurrent mutation is v75m, due to a cpg island was found in a hz girl, who showed microthrombocytopenia and infections to the same degree as her hemizygous father and brother. the amount of was protein was about 10% in platelets and 15% in mononucleated white cells [388] . involved in ensuring the t lymphocyte functional polyvalence, also explaining why microvilli and platelet defects are absent [152] .wasp and several related proteins (the wasp family) are all involved in the organization of the actin cytoskeleton. to carry out vital functions, cells have to rearrange their actin cytoskeletons [467] . the characteristics peculiar to wasp as a meeting point for the marking pathways is illustrated in fig. 22 .26 [152] . the wasp function is absent in 135 cases of was, in ten with attenuated was and in 23 with xlt [372] . in normal subjects, it is found in the cyto-molecular biology has proven that wasp found only in blood cells binds the small gtpase cdc42h2 in the gtp but not in the gdp [23, 265, 490] . cdc42h2 plays a critical role in the assembly of actin filaments [490] and in t-cell polarization when they encounter a b-lymphocyte apc [481] . wasp activity is regulated by several proteins acting in concert to control wasp configuration. the wasp-interacting protein, when phosphorylated, releases wasp from its grip, allowing wasp to be activated by rho-family gtpases [433] . experimental data also indicate that cdc42, wasp and actin might be plasm but not in the nucleus of various cells such as platelets, t and b lymphocytes and monocytes [477] . the xlt gene is located on the same locus as the was and could therefore be a variant [444] ; the main immunological anomalies are summarized in table 22 .12 [287, 413] . children with was have significantly elevated levels of il 4 and ige (table 22. 2) and decreased levels of ifn-γ [217] . the pathogenetic mechanism unifying the symptom triad is not clear; the glycosylation defect has been proved, primarily concerning sialidation, therefore resulting in an instability on the membranes of platelets, neutrophils and lymphocytes expressing a glycoprotein sialopherin (cd43) [402] , localized on chromosome 16, which makes it an improbable candidate, even though cd54 is indeed the binding agent of cd43 and could therefore play a role in t-cell maturation, differentiation and activation, thereby acquiring marking capacities that are independent of tcr/cd3 [19] . however, the tcr-mediated signaling defect is characteristic of was [259] , in addition to the reduced expression of cd23 [458] , which can explain immune and hematological defects. in the lymph nodes, there is a shortage of lymphatic follicles and the thymus-dependent and -independent areas are depleted, moderately at the age of 4 years ( fig. 22.27 ) and to a greater extent at 8 years ( fig. 22.28 ). the predominant immunological outline is constituted by elevated iga and ige levels, low igm and all igg levels, as well as the absence of a response to polysaccharide antigens, which is why the children's serum lacks isohemagglutinin [278, 528] . in unweaned babies, the most striking finding is the cd4:cd8 ratio =5 [549] , compared to 2.65 in normal children aged 0.63-3.06 (tables 1.36-1.39). was usually starts at 13.7 months (range, 1-58) [132] with hemorrhagic manifestations, petechiae and prolonged bleeding from the umbilical scar or the circumcision site, observed in newborn babies [402] . the clinical triad is characterized by cutaneous lesions that are practically indistinguishable from rather severe ad (70%), congenital thrombocytopenia (100%), a marked susceptibility to rris (91%) [132] (figs. 22.29, 22.30) and gastroenteric symptoms such as hematemesis, melena and chronic diarrhea [132] . other complications may include neutropenia (25%), arthritis (29%), skin vasculitis (22%), cerebral vasculitis (7%), inflammatory bowel disease (9%), and renal disease (3%) [132] . a reduced thrombopoiesis (level <50,000/ml), with microthrombocytes and an accelerated turnover in boys must allow for a suspected diagnosis [413] . it has recently been proven that the classic presentation is more common in children aged 6.8 months than in those aged 7.2 months (60% compared to 25%), unlike platelet counts [549] . however, only 27% of 154 unselected children with persistent thrombocytopenia, positive fh, small platelets and defects associated with t and/or b lines had the classic triad and 20% only thrombocytopenia before diagnosis [484] . primary immunodeficiencies infections, appearing during the first months of life, are often marked by otitis media, pneumonia, meningitis and sepsis, caused by viruses (cmv and herpesvirus) and by bacteria (pneumococci or other capsular polysaccharide). these are followed by more common infections caused by opportunistic germs, pneumocystis carinii and mycetes such as candida albicans. differential diagnosis should also include a rare ar syndrome similar to was, also reported in female patients, characterized by ad, rris and thrombocytopenia with microthrombocytes [62] . when caring for these children one must monitor the platelet count, the immunological structure (ig, lymphocyte and subpopulation counts) and the potential onset of autoimmunity and tumors [224] .aiha may be found in 36% of children [132] . prophylactic treatment for infections is done with ivig; 500 mg/kg every 3 weeks) and sulfamethoxazole (25 mg/ kg/2 days) after diagnosis [132] . splenectomy may decrease the bleeding tendency [224] , but early relapse of thrombocytopenia after splenectomy is predictive of a poor prognosis [132] . on average death occurs around the age of 11 (8 in untreated children), but can occur between 0.5 and 4.5 [549] , with survival also >18. death is caused by massive hemorrhages (23%), tumors (26%) and severe infections (44%) [484] . the second largest group of patients with id given bmts since 1968 are those with was, with 78.8% of children aged <5 years [158] . fourteen out of 18 patients underwent phenoidentical (n=1) or haploidentical (n=13) hscts; the other four died before hsct could be undertaken [132] . boys who had received a mud hsct transplant <5 years had survival rates similar to those receiving hla-identical sibling transplants, but the success rate decreases dramatically at the age of 5-6 [158].wasassociated t-cell signaling defects can be improved upon retrovirally transduced hscts [258] . recently, correcting the t-cell defects has been proposed. the potential for correction of the t-cell defects has recently been demonstrated by transduction with an oncoretroviral vector encoding the wasp, which resulted in correction of the deficient proliferative response to tcr stimulation characteristic of was [483] . ata is a complex ar inherited syndrome, associated with neurological, immunological, endocrinological, hepatic and cutaneous abnormalities, characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, and increased susceptibility to rris [379] with an incidence estimated at 1:100,000-1,300,000 live births [61] . in italy the frequency on the general population, is of 1.3 × 10 6 , with an increase in hets from 1.7% to 3.43% [94] . it is characterized by a genetic heterogeneity, which is reflected in the division into four main groups of complementation, to which one must add the nijmegen and at-fresno variants, perhaps caused by other well-defined id syndromes the same gene, also localized on the long arm of chromosome 11q22.23 [176] . the 12-kb gene, called atm (at mutated) because of its mutations by defective splicing in all patients with ata, permits het identification [435] . a dna clone complementary to atm shows considerable affinity to factors responsible for signals involved in regulating the cell cycle and codifying a protein similar to phosphatidylinositol-3-kinase (pi 3k) [435] , involved in mitotic signal transduction, meiotic recombination, and cell cycle control. a result could be a recombination defect which interferes with b and t lymphocyte gene rearrangement, involving tcr and isotype switching, consequent to a damaged dna triplication and therefore accounting for ig deficiencies [176] . cells from these patients progress too rapidly from the g 1 phase, in which they receive ionizing radiations, to the s phase, then continuing irradiation, to the g 2 /m phase with further delay, evolving in apoptosis [279] . this hypothesis has received further credit after observing that the p53 gene expression does not increase in human cells exposed to radiations [250] . the p53 gene is part of the normal cell cycle and during the s phase provides time for the dna physiological repair after exposure to radiation that may also be cosmic [279] . the thymic tissue is either absent or degenerated with a fetal appearance (fig. 22.31) : some follicles, also with b cells, are visible at the age of 2 (fig. 22.32) , at 8 there is complete cellular depletion (fig. 22.33 ). immune deficiencies are humoral and cellular (cutaneous anergy and depressed proliferative responses) [70] . the karyogram shows that the lymphocytes have common rupture points at the chromosomal level with inversions and translocations involving precisely the tcr and ig genes [320] . most chromosomal translocations involve the genes encoding tcr on chromosome 7 and the ig h chains on chromosome 14: most breakpoints occur at the loci that encode ig and tcr for antigen (regions 7q35, 7p12, 14q32, 14q12) [264] , in areas typical for cod-ification of molecules of immunological importance (chap. 1). an important role is played by genes belonging to ig gene superfamily (igsf) (table 1.4) . possibly the progressive id of ata, like its apparently unlinked manifestations, is at least in part linked to the accumulation of clonal anomalies affecting the tcr and the igsf: this suggests the intervention of "illegitimate" recombinations damaging above all the t cells [162] . t-cell immunological deficiency is completed with lymphopenia, a decreased cd4:cd8 ratio due to the drop in cytotoxic cd8, and a rise of immature forms with tcrgd [80] . another consequence is the isotype deficiency: about 70% of patients present sigad; >50% are also affected by an igg 2 -igg 4 deficiency with igm becoming monoclonal, and 30% by serum igg deficiency [364, 379, 528] . fig. 22 .32,the degree of depletion of lymphocytes is extensive in both thymus-dependent and thymus-independent areas chromosome 11 into cells, the chromosomal aberrations induced by x-rays were suppressed [261] . the rare ar nijmegen breakage syndrome, so called because it was initially seen in two brothers of secondcousin parents living in that city, and at the moment observed in approximately 80 patients, has various characteristics of ata but without ataxia, telangiectasia, or high concentrations of afp. clinical characteristics are singular: short stature and microcephaly with prenatal onset, bird-like profile, prominent midface, a long nose, low-set ears, cutaneous depigmentation with caféau-lait spots, an almost normal intelligence, and also rris and bronchiectasis. humoral and cellular id includes reduction of antibodies and lymphoproliferative responses [70, 224] . during an 8-year period of observation, the id was found to be profound, highly variable, and with a tendency to progress over time in 40/50 children [193] . there is a high proclivity to expressing rearrangements of chromosomes 7 and 14 as in ata [70, 224] . dgs is usually sporadic, with known cases of positive fh [224] . it is caused by a defective development of the 3 rd and 4 th branchial pouches which takes place before the 12 th week of gestation, with consequent thymic hypoplasia or aplasia and parathyroid hypoplasia; the 5 th and 6 th pouches and branchial arches can also be affected [497] . the cause can be found in the neural crest cell incapacity to migrate and interact appropriately with endothermic cells of the brachial pouches and arches [295] . deletions (often microdeletions) at the pericentrometric region of chromosome 22q11-pter have been described in 80%-90% of cases [130] . a microdeletion 22q11.2 was recorded in 112 children aged 4-70 months, 54% of whom had developmental delays, mild hypotonia, as well as language and speech delays [181] . another 80 children had deficits in the areas of attention, story and visuospatial memory, arithmetic performance relative to other areas of achievement, psychosocial functioning [541] , and mental retardation in 73% of 44 children [11] , thus indicating the need for early intervention beginning in infancy [181] . overlapping alterations are present in the syndrome complex known as catch 22, which in turn includes the charge association. other cases of dgs can derive from microdeleted chromosome 10p (fetal-alcoholic syndrome, retinoic embryopathy, maternal diabetes) [414] . this variable phenotype is reliably referred to microdeletion 22q11.2; the greater it is the more complex is the associated phenotype [508] . another difference depends on the variable spectrum of t-cell abnormalities in individuals with dgs who might have normal t-cell numbers and function, low t-cell numbers but fairly normal t-cell proliferative function [32] or no t cells purkinje cells (pcs) and degenerated granular cells. that the number of basket cells, so called because they form with the axons bunches of fibrils distributed so as to form a nest in which the nucleus of each pc settles, is almost normal, proving that pcs are probably normal at birth and degenerate only later [176] . typical clinical manifestations are ataxia, telangiectasia of both auricular lobes and sclera, rris and an elevated incidence of neoplasia [489] . from a review of 331 patients [70] , the percentages of symptoms are as follows: progressive ataxia (100%), typically cerebellar, becomes evident when children start walking or a little later, affecting intentional movement and becoming complicated by dysarthria (100%) and involuntary choreic movements (92%), causing the majority to be unable to walk by about the age of 10-12 [276] . at a later stage it is possible to observe nystagmus (67%), strabismus, oculomotor apraxia (88%), reduction or absence of reflexes (77%), and dyslalia, increasingly amplified and, in some patients, also mental retardation [176] . telangiectasias develop between the ages of 1 and 6 on the bulbar conjunctiva (97%) (fig. 22.34) , on the flexor surfaces of the limbs and areas exposed to sun rays (17%). height and weight are <10th percentile (64%) and the appearance is progeric (63%). severe rris are common (70%), encouraged by antibody deficiencies. the pathogens involved can be bacterial or viral, often resulting in lung bronchiectasis, all starting after the onset of neurological manifestations [276, 489] . associated neoplasia (6%) is usually lymphoreticular, less common than adenocarcinoma, with an eightfold increased trend for all kinds of tumors [224] . in cultures the fibroblasts of these patients are three times as sensitive, compared to controls, to ionizing radiations and to radiomimetic chemical substances, but not to uv rays, unlike what is observed in the cells of subjects affected by xeroderma pigmentosum [70] . in addition, the persistence of elevated serum a-1-fetoprotein (afp) levels was observed in all patients. an interesting in vitro study has reported that by introducing a normal human 1303 other well-defined id syndromes fig. 22.34 . conjunctival telangiectasia in a girl with ata [305] . a second group is referred to as having partial dgs (dgsp) or transient forms (dgst), with mild symptoms. the designation "complete digeorge syndrome" (dgsc) is reserved for the third group of infants who have absence of thymic function in addition to other defects of the 3 rd and 4 th pharyngeal pouches, <1% of patients with dgs, although they can have high t-cell numbers that respond to mitogens [32, 305] . these patients have profound id, with its associated clinical findings [308] . dgst includes cases with a spontaneous quantitative and qualitative t lymphocyte recovery [162] . the thymus can also be ectopic: in dgsc the t zones are depleted, the cd4/cd8 markedly reduced both in number and in function with spt anergy, and b cells appear unaffected or increased [162] . in dgsp, the most common type, t-cell number and function are instead usually normal, as are the cd56/cd16 cells with a nk phenotype, or they may be moderately reduced [162] . the proliferative response to mitogens can be pathologically reduced [224] and the response to polysaccharide antigens may be absent [442] . from neonatal age, there are malformations of other structures that form during the first weeks of embryogenesis, presenting a suggestive but not pathognomonic picture (table 22 .13) [79] . diagnosis is usually suspected within the first 2 days after birth, due to the presence of hypocalcemic tetany caused by hypoparathyroidism and cardiac malformation. the facial dysmorphism is also characterized by a small mouth with thin lips described as fish-like [79, 192] (fig. 22.35) . the two rare cardiopathies indicated in table 22 .13 depend on neural crest nonintegration, as mentioned, which accounts for >50% of the alterations alone [295] . others can be observed affecting the right heart, such as fallot tetralogy, pulmonary athresia with an interventricular septum defect, and pulmonary infundibular stenosis [508] . babies surviving the neonatal period manifest from the very first months an increased susceptibility to infections, particularly those of the respiratory and digestive tract, viral and/or fungal, but also caused by pneumocystis carinii, which can be fatal in dgsc [162] . other findings include gastroesophageal reflux, speech delay, laryngomalacia, absent kidney, conductive or sensorineural deafness, 6th cranial nerve palsy, and hypothyroidism [535] . treatment with high doses of vitamin d and diets enriched with ca gluconate are needed immediately, also ensuring that calcemia remains at the lower limit of normal values so as to avoid snc and renal damage. subsequently the possible correction of cardiac malformations should be evaluated. id may be severe, but can regress spontaneously with reconstitution of cmi and t functions; compensating hyperplasia of the residual parathyroid tissue can make it possible to discontinue ca and vitamin d treatment [192] . dgs natural history is, however, complicated by mental retardation and the difficulties encountered in correcting cardiac malformations and in controlling hypoparathyroidism [224] . because of variability in the id severity, it is difficult to evaluate claimed benefits of bmt: in two cohorts of 8 [305] and 5 transplanted infants [306] , the survivors were 3 out of 13 (23.1%). recently, 5/6 and 7/12 infants underwent postnatal transplantation with cultured unrelated thymic tissue, with immunosuppression, with positive results [307] . del22q11.2 syndrome, characterized by a 3-mb deletion on chromosome 22q11.2 is the most frequent known chromosomal microdeletion syndrome, with an incidence of 1 in 4,000-5,000 livebirths. patients show 1304 chapter 22 primary immunodeficiencies clinical outlines not unlike gvhd [446] (fig. 22.36 ) [508] , suggesting a possible connection to a fas deficiency (cd95) or apoptosis syndrome. the sh2d1a gene was found altered in two families, thus indicating that xlp must be considered when more than one male patient with cvid is encountered in the same family, and sh2d1a must be analyzed in all male patients with cvid [330] . recently a critical revisitation of this experiment in nature has allowed the identification of links between id and allergy [62, 178, 196, 287] . the rare higes is associated with bacterial rris, chronic ad, coarse facial features and very elevated ige levels [60, 250] ( table 22. 2), up to 40,000 iu/ml [549] . linkage to a region on chromosome 4q has been demonstrated in several affected families; however, neither the fundamental host defect nor the defective gene has yet been identified [195] . fh is frequently positive for atopic disease, at times higes is combined with an unusual predisposition to staphylococcus aureus infections [62, 178, 196, 287] . in buckley's study, it was present in 36.4% of cases, of both sexes, indicating an autosomal dominant transmission with incomplete penetrance [62] . onset occurs in the pediatric age in 90% of cases [64] . clinical presentation is unusual: there are no complaints during the first months of life, toward the 3rd-4th month a severe form of chronic ad appears all over the body, which can be associated with other allergic manifestations, including asthma in 13.6% of cases [62] . skin biopsy specimens reveal spongiosis and perivascular dermatitis and/or folliculitis with a predominance of eosinophils [91] . there is an excessive predisposition to cutaneous and respiratory tract infections (deep and superficial abscesses, otitis, pneumonia, sepsis) ( fig. 22 .37), also encouraged by neutrophil chemotactic deficiency caused by defective cellular functions (table 1. 65), which, if present, is so pronounced that it becomes a characteristic, unlike ad where it is secondary [226] . the subcutaneous abscesses, described as cold, not covered by warm and reddened skin, are pathognomonic to higes but not essential to the diagnosis [144] . the abscess is filled with pus that always grows staphylococcus aureus; in some cases mucocutaneous candidosis and chronic herpetic keratitis are associated [178] . infections appear within the first 18 months [62] . the face shows coarse and dysmorphic features, midline facial defects such as a prominent nose and a high, arched palate, and disproportionate cheekbones and mandible; pondostatural growth notably retarded [64] , pneumatocele (fig. 22 .38) and osteoporosis caused by reduced bone density with a tendency to fracture [287] complete the picture. six consanguineous families have been reported with an ar form of higes, including 13 cardiac abnormalities, t-cell deficits, cleft palate facial anomalies, and hypocalcaemia. at least 30 genes have been mapped to the deleted region. recently, in 5/13 patients with del22q11.2 syndrome without 22q11 deletion mutations were found in t-box 1 that is a major genetic determinant of the del22q11.2 syndrome [545] . xlp is caused by a defect in the sh2d1a gene (table 22 .1), which binds to the cytoplasmic domains of cd150 slam (signaling lymphocyte activation molecule) and 2b4, and may regulate signals transmitted by these receptors in t and nk cells, respectively [345] . xlp has been reported in >270 males from >80 families [446, 460] , and in an other 27 males [381] , it is inherited with the x-linked model. it is set off in males aged 5-6 by an ebv infection that became manifest with a very polymorphous pattern, often with unusually severe or fatal infections mononucleosis caused by the immune system incapacity to respond to ebv, or evolving into a hgg with iga and igg deficiency and higms, or medullar aplasia and/or a burkitt type lymphoma [446] . the disease has been reported in 15 female subjects [381] . xlp polymorphism could be explained by the fact that the ebv receptor is expressed on differentiating b lymphocytes starting with the preceding isotypic conversion stage [500] . it has recently been verified that before ebv infection, males already suffer from dys-or pan-hgg, incapable of regulating the expression of ig and/or containing b or t lymphoproliferation. even after ebv infection, the immune system is unable to provide adequate th2 responses, and therefore releases cytotoxic alloreactive cd8 and th1-like t cell ils, causing extensive damage to the entire parenchyma, exemplified by fulminating hepatitis, cellular infiltrations and tissular necrosis. the lymphoid tissues with an altered structure are also affected by necrosis, with a high incidence of mostly nonlocalized lymphomas [381] . the thymus is also affected by thymocyte rarification, with 1305 other well-defined id syndromes fig. 22.36 . bone marrow biopsy specimen in a 3-year-old boy: numerous histiocytes in erythrophagocytosis affected children aged 15 months to 12.5 years, with ar-higes presenting with the classic immunological findings, including rri, eczema, elevated serum ige, hypereosinophilia, and severe recurrent fungal and viral infections [64] . notably, patients with ar-higes did not have skeletal or dental abnormalities and did not develop pneumatoceles, as seen in autosomal dominant-higes [404] . among the immunological characteristics (table 22. 14) [93, 287] , cutaneous anergy to several antigens such as candida and tetanic toxoid is characteristic, which is associated with the anomaly of proliferative responses by the t cells to antigens and mitogens, in contrast with the integrity of other functions tested in vitro [196] . t subpopulations appear to be normal [64] . however, the lymphocyte proliferation to anti-cd3/cd28 monoclonal antibodies can be impaired [226] . as noted, ifn-γ deficiency associated with a pathological th2 prevalence has a fundamental impact on ige hyper-production [178, 287] . in higes, some studies have confirmed ifn-g deficiency compared to controls [120, 368] , also due to an impaired response to il 12 [56] , while others have not [62, 512] ; however, compared to ad, normal levels of t producers of il 4 are characteristic [120] . considering the ifn-g/il 4 + correlation of ad, in higes no specific t-cell anomalies are noted, nor does the hyper-ige explain this pediatric abnormal susceptibility to infections: high ige levels are also seen in children with ad, who do not, however, have an unusual predisposition to abscess formation [226] . one typical characteristic is sige directed against microbial antigens: the anti-staphylococcal sige rise to 8.9% compared to normal levels of 0.2%-0.6%. another constant finding is the increase in 100% of cases of eosinophil concentrations, which make up 6%-12% of leukocytes [64] , reaching 30%-50% [178] . by expressing the cd40-cd154 duo, they stimulate the isotype b-cell switching to ige.we studied children affected by severe ad, chronic fa-induced diarrhea and asthma. the allergens responsible were cm and der p [73] . in case of higes caused by fa, atopic manifestations can clearly improve following an exclusion diet, reducing the frequency of infections and partially correcting the immune defect [420] , revealing how fa can induce several immunological anomalies. diagnosis is made on the basis of the data in table 22 .14; differential diagnosis with ad is schematized in table 22 .15 [287] . treatment with cromolyn is extremely effective, anti-staphylococcal antibiotic treatment [64, 226] and if necessary antifungal therapy provide good results [144] . griscelli disease, mapping to chromosome 15q21 [372] , is an ar syndrome caused by mutations in the myo5a (gs1), rab27a (gs2), or mlph (gs3) genes, all of which lead to a similar pigmentary dilution [50, 312] . the disease is also characterized by partial oculocutaneous albinism, predisposition to pyogenic infections and in most patients by abnormal regulation of the immune system, which results in a syndrome of macrophage hyperactivation, known as hemophagocytic lymophohistiocytosis [15] . mutations in the gtp-binding protein rab27a (gs2), which appears to be involved in an uncontrolled t lymphocyte and macrophage activation syndrome, leading to death in absence of bmt, occur in this syndrome [319] . a mutation was found in the myo5a gene (gs1) associated primarily with neurological impairment [319] . two identical twin boys aged 3 months were reported with persisting fever, mouth ulcers, hepatosplenomegaly, pancytopenia and failure to thrive [431] , as was an 8-month-old infant [397] . both infants had silvery-gray hair and pigment clumps on the hair shafts, and skin biopsy showed accumulation of melanocytes on melanosomes. their parents were first cousins and a sibling with similar manifestations had already died, as did the twins. a genetic study revealed a 5-bp deletion in the rab27a gene (510 del aagcc in exon 5) [431] . in a 4-year-old child with hemophagocytic syndrome, id, and secondary neurological disorders, typical melanosome accumulation was found in skin melanocytes and pigment clumps were observed in hair shafts. two heterozygous mutant alleles of the rab27a gene, a c-t transition (c352t) leading to q118stop and a g-c transversion on the exon 5 splicing donor site (g467+1c) were found [50] . the finding of gray strands of hair, gray eyebrows, and eyelids in childhood should alert pediatricians to considering griscelli syndrome since an early diagnosis is life-and health-saving [203] . the phagocyte system with the biochemical basis of cgd is analyzed within the framework of innate immunity. chronic granulomatous disease (cgd) has an overall prevalence of 1:500,000 to 1:10 6 , although this could be underrated (table 22 .4), considering that some subjects may have a very mild clinical phenotype that escapes diagnosis [501] . a us registry of birth rates found a prevalence of 1:200,000 to 1:250,000 live births for the period 1980-1989 [538] . the youngest patient was 27 days old [337] and in 12 children with cgd the mean age at the onset of infections was 5 months, with a median delay in diagnosis of 2.5 years [371] . otherwise the the clinical features of this rare ar disease include oculocutaneous albinism and susceptibility to especially s. aureus and b-hemolytic streptococcus [549] . approximately 85% of patients develop an accelerated phase of the disease, with deposition of lymphohistiocytes in the liver, spleen, lymph nodes and bm, resulting in hepatosplenomegaly, lymphadenopathy, bm infiltration hemophagocytosis, pancytopenia as well as fever, jaundice, prolonged bleeding, easy bruisability, neurological changes (nystagmus and neuropathy), mild mental retardation, and partial ocular and cutaneous albinism [285, 334, 526] . the cellular hallmarks of the disease include large lysosomal granules in leukocytes, giant melanosomes in melanocytes and affecting other cells of the body such as neural schwann cells, renal tubular cells, gastric mucosa, pneumocytes, hepatocytes, langerhans cells of the skin, and adrenal cells [229, 157] . the fundamental defect in this disorder was found to be caused by mutations in a gene mapped to chromosome 1q42-q43 [31] encoding a cytosolic protein on chromosome 1 named lysosomal-trafficking (lyst) regulator, encoding a 425-kd protein whose function remains unknown [285] . bmt is resolutive in these children [205] . normal protein expression (x91 + form), but with a total absence of oxidase due to incorrect binding [412] . ar-cgd is caused by a mutation in the genes encoding the remaining oxidases of 47 kd (p47 phox ) (phox, phagocytic oxidase) [538] (ncf-1), p22 phox of 22 kd (cyba), and p67 phox of 67 kd (ncf-2) [109, 501] . the ar-cgd forms (18.5%-22% of cases) [285, 458] are identified using the immunoblotting technique, depending on whether they affect the p22 phox , the p47 phox or the p67 phox [84, 97, 109, 501] , with greater prevalence in an american study [97] . patients with the x-cgd appear to have a more serious clinical phenotype than patients with the ar-cgd, based on the fact that they are diagnosed significantly earlier (mean, 3.01 years of age vs 7.81 years of age, respectively), have a significantly higher prevalence of infections and a higher mortality (21.2% vs 8.6%) [537] . mutations in any of the 6 structural molecules (table 22 .16) lead to cgd. mutation of rac2 (see lad), the predominant g protein in neutrophils, leads to defects in so production, as well as in chemotaxis [416] . activation of the nadph oxidase requires complex rearrangements between the protein median age at onset was 1.12 months, and the median age at diagnosis was 1.1 years [81] . the deficiency appears in two forms ( [335, 538] , with an h-chain deficiency, is divided into four x91 subtypes (table 22. 16 [84, 97, 109, 112, 185, 501] ), also identified on the basis of nbt results, depending on whether the x91 is absent (the most common form), reduced or present but inactive; subtype x91is divided into two variants: in one of them the nbt is slightly positive in 80%-100% of cells (6% of patients), in the other in 5%-10% (3% of patients) [84, 109, 412, 501] . more precisely, the four subtypes are caused by mutations in the four gp91 phox regions, many of which depend on cybb gene mutations, causing the x91 0 form, while 17 mutations depend on the nadphoxidase activity (x91form) and eight others lead to a 1309 phagocyte deficiency subunits, which are in part mediated by noncovalent binding between src-homology 3 domains (sh3 domains) and proline-rich motifs [447] . cgd is a hereditary disease (table 22 .16) characterized by severe recurrent pyogenic infections. this marked susceptibility is caused by the phagocytes' incapacity to kill in particular the catalase-positive bacteria, because of a genetic defect of the nadph-oxidase enzymatic system situated in the wall of the phagocytic vacuole. in cgd, phagocytosis occurs normally, but the nadph-oxidase is unable to markedly produce anion superoxide (o 2 •-), h 2 o 2 and other o 2 free radicals, thereby permitting the survival of microorganisms within the cells, where they are protected from the antibodies and from most antibiotics [501] . another consequence of the lack of o 2 radicals is the development with countless inflammatory episodes, which then result in typical granulomas [109] . the nitroblue tetrazolium (nbt) reduction test is based on the chemical characteristics: in fact, the phagocytes without o 2 •are unable to reduce the yellow nbt of products activated by pha aspecifically stimulated phagocyte o 2 , or specifically with corpuscle particles such as preopsonized yeasts (fig. 22.41) . the result was 0% in 14 children [81] . at a molecular level, the genes that codify the two subunits of flavocytochrome b588, gp91 phox and p47 phox have been cloned: respectively the cytochrome, h (b) and l (a) chains situated on the phagosome vacuole membrane, and also the cytosolic factors p40 phox , p22 phox and p67 phox , deriving from the nadph-oxidase activation, all proteins placed inside the cytoplasm and that belong to innate immunity. it has therefore been possible to identify molecular lesions at the cgd origin, with the exception of the p21 rac1 [16, 412, 533] . the clinical pattern is severe in the x91 0 form and variable in the other two x91 forms; onset occurs within the 1st year of life in 2/3 of cases, and in others within the 2nd year [159] , although it can appear also at the age of 16 [335] . purulent recurrent infections, with a granulomatous evolution, predominantly affect the epithelial surfaces normally colonized by bacteria, such as cutaneous, subcutaneous, mucous membranes, the respiratory tract and the intestine: cutaneous and mucosal infections, and lymphadenitis lead to suppuration and fistulation (fig. 22.42) , pneumonia or lung abscesses (fig. 22.43 ) are more frequently characterized by persistent fever and diarrhea [159] (table 22. 17) [109, 538] . pneumonia was the most prevalent infection in 369 patients (79%) (mostly by aspergillus), followed by suppurative adenitis (53%), subcutaneous abscess (42%) and liver abscess (27%); mostly by staphylococcus, osteomyelitis (25%) mostly by serratia, and sepsis (18%), and by salmonella [538] . in a long-term trial, pneumonitis was the most prevalent infection (91%) followed by lymphadenitis (83%), aphthous stomatitis (58%), liver abscesses (25%) and chronic lung disease the rates (%) consist of a first [109] and of a second number related to the european study [84] ; us data regarding ar cgd are in parentheses [97] . data from [84, 97, 109, 185, 501] . x x-linked, ar autosomal recessive, ad autosomal dominant inheritance, nd not done. a the superscript symbols indicate the level of immunoreactive proteins: 0 undetected,diminished, + normal protein levels. granulomas in the entire lung parenchyma, which are formed by mononucleates (fig. 22 .44a) with giant cells (fig. 22.44b) . the chronology of infection onset is summarized in table 22 .18 [335] : lymphadenitis is the earliest. osteomyelitis is usually a worrying complication: (58%) [371] . lymphadenitis, lung infections, enteral infections, and hepatic abscesses were the most frequent infections in a cohort of 48 children [335] . staphylococcal liver abscesses are almost pathognomonic of cgd [447, 538] . because the infections develop in areas drained by lymphatics, they tend to diffuse via the lymphohematogen route, thus causing arthritis and osteomyelitis and abscess formation, especially affecting the bones, which are the most severe manifestation, and hepatitis with common upsurge of hepatosplenomegaly. lung infections are almost the rule: those initially segmented and parallel tend to gradually spread over the entire lobe [109, 538] . histological examination shows widespread gastric outlet obstruction 10 15 urinary obstruction extensive bone destruction involves various segments, for example the vertebra, the metacarpus and the metatarsus, causing widespread damage, which is difficult to treat and is also irreversible [109, 501] . aspergillus, pulmonary, bone (fig. 22 .44c) or encephalic infections constitute a severe therapeutic problem and are threatening events, with a mortality rate of 26%, but with specific treatment the prognosis is good as far as recovery is concerned [335] . the treatment includes prophylaxis with trimethoprim-sulfamethoxazole (tmp/smx) (5 mg/day given in two divided doses), and ifn-g (50 mg/m 2 subcutaneously thrice weekly) in all patients with cgd, regardless of genotype [81, 416] . itraconazole therapy (5 and then 10 mg/kg/day) has an excellent tolerance in all cases and was effective in 29 of 32 children (90.6%) [336] . survival until the age of 21 and beyond is achieved by 20% of patients with cgd xl and 37% of those with cgd ar [339] . because the prognosis is uncertain, as observed, the only possibility for a definite resolution is with a bmt, from family donors who are x-cgd or x-cgd-identical [393] . bmt was successful in 27 children out of 31 (87.1%) (see table 22 .30), including a 4-year-old boy with x-cgd who underwent successful hla-identical peripheral blood sc transplantation during invasive pulmonary aspergillosis and osteomyelitis, which was unresponsive to antifungal treatment [48] . lad is due to mutations in the gene on chromosome 21 at position q22.3 encoding cd18 (table 22 .1). it is divided into five types: lad type i to lad type v [24, 71, 138, 367] . the three subunits of the cd11/cd18 complex are involved in pid (lad type i syndrome), ar, linked to the lack of a m b 2 equal to cd11b/cd18 (table 1.46) surface expression on all leukocyte populations caused by 20 different mutations in the cd18 encoding gene, often severe in infancy [145, 202] . children with a deficiency of these integrins have a defect above all in phagocyte action, suffer from severe infections from the neonatal period [202] due to absent b2-integrin activity, which impairs neutrophil ability to exit the circulation and travel to sites of infection. on the contrary, leukocyte movements are not prevented, indicating the normal involvement of cd54 and cd102 (table 1.4) . the clinical basis for defining this disease, described in over 200 cases [145] , dates back to a study at the soothill school in 1979 [215] . there are two forms of lad type i [71] : if the deficiency is full blown (no detectable cd18), the clinical symptoms (table 22. 19) [71, 138] are dominated by severe and recurrent infections with a negative prognosis in the first years of life unless corrected by an allogenic bmt, the only resolutive treatment [492] . if instead it is a partial deficiency with residual cd18 expression, the clinical outline is less severe and some patients, with appropriate treatment, can live to adult age [24] . lad type ii, ar (cd18 levels are 1%-10% of the normal levels), with a molecular base represented by an slex ligand (cd15s) is a defect common to cd62 e and p, which mediate neutrophil rolling. in the absence of a gdp-fucose transporter, the slex is not made. lad type 2 results from mutations in this transporter that takes fucose into the golgi apparatus for posttranslational fucosylation of newly synthesized proteins. this is the ligand for cd62e; without it, leukocytes cannot make initial attachment to vascular endothelium [7] . lad has been described in two children aged 3 and 5 with mental retardation, from different families, but both with parents who were blood relatives [145] . it has a lower mortality rate [138] . mice with a deficiency of both selectins show a lad-like syndrome, providing a useful model for studying these syndromes [171] . lad type iii shows defective tethering and adhesion and bleeding diathesis. this is a new syndrome where in vitro leukocytes showed normal rolling along endothelial cell cultures but defective tethering and tight adhesion. thus this is a defect in the capability of vascular integrins on circulating leukocytes to rearrange with their endothelial ligands at adhesive contacts and rapidly arrest on target vascular endothelium in response to endothelial-displayed chemoattractants. however, the expression levels of the major integrins on lymphocytes and neutrophils were largely conserved in the patient cells, ruling out a lad-i syndrome. patient leukocytes showed no lad-ii like fucosylation defect, since they expressed normal levels of the fucosylated marker cd15s, comprising the slex carbohydrate selectin ligand [7] . defects in both leukocyte and platelet functions that are biochemically and molecularly distinct from the adhesion disorders previously described suggest a mutation in an early myeloid pathway. the defect is associated with regulation of the gtpase activating protein rap1, as demonstrated by the intact rap1 expression and activation by phorbol esters, thus ruling out an lad defect in rap1 gtp loading [255] . lad type iv manifests defective cd62e expression or tethering. a girl developed pseudomonas omphalitis at 5 weeks of age, recurrent ear and urinary tract infections, and had clinical evidence of impaired pus formation reminiscent of a lad syndrome, but her neutrophils were functionally normal and expressed normal levels of cd18, cd62e, and slex. however, the patient showed an absence of cd62e from the endothelium, although e-selectin mrna was present. in contrast to patients with lad 1, she had mild chronic neutropenia but appropriate leukocyte increases in response to infections or gm-csf. a bm biopsy performed during a period of health showed normal cellularity for her age. her fh is remarkable only for a previous sibling who had died at 32 weeks of gestation of a staphylococcal infection of the fetus, amniotic fluid, and 1313 phagocyte deficiency placenta. she also has two half-sisters who are completely well. the fh is negative for recurrent infections in either parent or more distant relatives [121] . lad type v caused by rac2 deficiency. a 5-week-old boy born to unrelated parents had delayed uc separation, perirectal abscesses, poor wound healing, and absent pus at sites of infection in the setting of neutrophilia, suggesting a neutrophil defect. his neutrophils exhibited decreased chemotaxis, polarization, azurophilic granule secretion, as well as significantly reduced stimulated superoxide production but had normal expression and up-regulation of cd11b. rac2 constitutes more than 96% of the rac in neutrophils [9] . a 1-yearold boy who had multiple recurrent, life-threatening infections characterized by leukocytosis and notable for the absence of pus in the inflamed tissues was reported. the presence and density of cd11b, cd11c, and cd18 were normal. the expression of cd62p and cd62l were also normal. a bmt was curative. the boy shared a phenotype that closely mimicked that of a mouse mutant deficient in the rho gtpase, rac2 [534] . the disease was shown to be attributable to an ad mutation in the rho gtpase rac2 at an amino acid needed for proper interaction with other intracellular proteins. rac2 comprises >96% of the critically important g protein rac in neutrophils. each member of the family appears to control a distinct function of the actin cytoskeleton (chemotaxis and degranulation) and nadph oxidase (superoxide production) function [9] . a male child from the mother's first pregnancy was born at term from parents of arab ethnic origin who were first cousins. he had a severe genetic disorder associated with functional defects in multiple leukocyte integrins, reflected in recurrent infections, profound leukocytosis and a bleeding diathesis. platelet transfusions and antibiotic courses reduced the symptoms, which remained a significant clinical problem. at age 6 years, he died from disseminated fungal infection after a mismatched bmt. a younger brother presented with the same clinical and hematological phenotypes at birth and died at age 1 week from sepsis. g6pd converts g6p to 6-phosphogluconolactone, generating nadph and a h + ion from nadp + . nadph oxidase catalyzes the monovalent reduction of o 2 to o 2 •-, with the subsequent conversion to h 2 o 2 by superoxide dismutase [285] . in the form of a partial deficiency, known as the cause of hemolytic anemia or favism, the enzyme's residual activity (20%-25%) permits nor-mal bactericidal activity. the 400 g6pd variants have been classified by the level of residual enzyme activity and propensity for hemolysis and grouped into five classes: class i, severely deficient with chronic hemolytic anemia; class ii, severely deficient with occasional hemolytic anemia (<10% residual activity); class iii, moderately deficient (10%-60% residual activity); class iv, normal activity (60%-150%); class v, increased activity [310] . in a trial on 161 g6pd-deficient subjects originating from different parts of italy, a greater molecular heterogeneity than described by others was observed, especially in sardinia [310] . in a complete deficiency, sexually transmitted, whose gene is localized on the chromosome x at position p28 and characterized by several mutations and their variants, with a consequent deficiency of bactericidal activity, the neutrophils are unable to kill s. aureus, e. coli and serratia, and therefore there is an increased susceptibility to infections, rather like cgd [109] . the diagnostic work-up of children may reveal a child with recurrent infections who initially received the diagnosis of g6pd deficiency, subsequently shown to have the phenotype of x-linked cgd [3] . the disorder has a higher incidence in mediterranean countries and asia, in japan (10.6%) than in indonesia (4.3%), as ascertained with a novel screening kit [234] , and is low in newborns in tehran, iran (2.1%) [1] . within the framework of oxygen-dependent killing defects, hereditary myeloperoxidase deficiency (mpo) is the most common neutrophil biochemical defect and plays an important role in the host defense mechanism against microbial diseases. the neutrophil disorder characterized by the lack of mpo activity is speculated to be associated with a decreased level of immunity. mpo is unusually accompanied by a specific pathology. ar transmitted, it appears far more common than previously suspected (1:2,000 for the partial deficiency to 1:4,000 for the total deficiency). it is a disorder that is prevalently recorded in entirely healthy patients and therefore, in most cases, a random laboratory finding. in addition to three already-known mutations, the genetic characterization of an italian population showed the presence of six novel mutations: four missense mutations, a deletion of an adenine within exon 3 (c.325dela) and a mutation within the 3¢ splice site of intron 11 (c.2031-2a>c). the c.325dela deletion causes a shift in the reading frame with the occurrence of a premature stop codon within the pro-peptide. the activation of a cryptic 3¢ splice site located 109nt upstream of the authentic 3¢ splice site causes a shift in the reading frame that may lead to the generation of an abnormal mpo precursor lacking the enzymatic activity [303] . in a japanese patient with complete mpo deficiency, neutrophil function analysis revealed that mpo activity was alkaline phosphatase [414] . monocyte functional alterations in the second individual suggest that c/ebph plays a critical role in monocyte/macrophage development of humans and implicates abnormalities in monocytes/macrophages and neutrophils in the onset and development of the disorder [455] . severe congenital neutropenia (scn) and cyclic neutropenia are disorders of neutrophil production predisposing patients to recurrent bacterial infections. recently, mutations of the gene encoding neutrophil elastase 2 (ela2) have been indicated as the most common cause for scn as well as the cause for autosomal dominant cyclic neutropenia [225] . deficiency of ela2 leads to regularly fluctuating levels of neutrophils [112] . linkage analysis on 13 affected pedigrees have shown that cyclic neutropenia and sporadic cases of this disease are due to a mutation in the gene for ela2, located at 19p13.3 [112] . this enzyme is synthesized in neutrophil precursors early in the process of primary granule formation [225] . a mutation in the ela2 gene was detected in one of three apparently autosomal dominant kindreds with familial scn. no mutations were identified in the apparently ar families [12] . these results fit those showing that mutations were found in all five scn families [112] , but they suggest that not all cases of autosomal dominant scn caused by mutations in ela2 [12] . however, the high frequency of het mutations in the neutrophil elastase gene in sporadic scn confirms a previous report [112] . considering that four novel mutations and a low-frequency polymorphism were detected, nearly all cases of sporadic scn may result from de novo het mutations in ela2 [12] . in recurrent scn, an absolute neutrophil count of <200 cells/mm 3 (or <0.1¥10 9 /l) [12] oscillates with an approximate 21-day periodicity. circulating neutrophils vary between almost normal numbers and zero [225] . in about 30% of patients with cyclic neutropenia, however, the cycles range from 14 to 36 days [42] . in 26 children referred during a 22-year period pids were as follows: cyclic neutropenia (30.7%), shwachman-diamond syndrome (26.9%), kostmann syndrome (23%), and chédiak-higashi syndrome (19.2%). the mean absolute neutrophil count of children was 398.2±259.3 cells/mm (range, 74-1,152/mm) at the first visit. the children first experienced symptoms of infection suggesting neutropenia at a median age of 7.5 months (range 1 month to 10 years), also suffering from oral ulcer, otitis, pneumonia, diarrhea, cutaneous abscess, and oral candidiasis [398] . fever, stomatitis, and periodontitis and skin infections occur during periods when the neutrophil count is low. significantly diminished with slightly elevated superoxide production. mutational analysis of the patient revealed a glycine to serine substitution (g501s) in the exon 9 region [356] . because the granulocytes without mpo cannot kill candida, some subjects, presumably carriers of a more extensive mutation and in association with other diseases, present severe and recurrent candida infections. the mpo defect can be diagnosed via a cytochemical investigation or a quantitative count of enzyme levels [303] . neutrophil-specific granule deficiency is a rare autosomal dominant disorder characterized by recurrent pyogenic infections, defective neutrophil chemotaxis and bactericidal activity, and lack of neutrophil secondary granule proteins [284] . the markedly decreased level of mrna expression for the bactericidal/permeability-increasing (bpi) protein, the activation factor pu-1 and defensins in these patients suggests a role for ccaat/enhancer binding protein (c/ebpη) gene in earlier phases of the myeloid differentiation program [187] . c/ebpη is a member of the leucine zipper family of transcription factors, expressed primarily in myeloid cells [284] . recessive mutations in the c/ebpη gene were described in one patient; analyses of the c/ebpη locus indicated that the disorder could have resulted from hz recessive inheritance of the mutant allele from an ancestor shared by both parents [187] . loss of c/ebph function is the primary genetic defect in this disease [455] . in a second individual lacking functional c/ebph, analysis of peripheral blood leukocytes revealed aberrant expression of cd45, cd11b, cd14, cd15, and cd16 on the proband cells [455] . a male patient lacking neutrophilspecific granules died from complications of pneumonia at age 20 [284] . neutrophil-specific granules contain important microbicidal components (table 1.23) . among other deficiencies of oxygen-independent killing, this ar defect is characterized by severe recurrent bacterial deep-tissue skin infections without patients showing an increased susceptibility to a particular pathogen. they have defects in chemotaxis, disaggregation, and receptor up-regulation. deficiencies of the oxidoreduction and microorganism-killing mechanisms have also been described. the markedly decreased level of mrna expression for the bactericidal/ permeability-increasing (bpi) protein, the activation factor pu-1 and defensins in these patients suggests a role for c/ebph in earlier phases of the myeloid differentiation program [187] . the defect is identified through a blood test colored with a wright reactive in which polymorphonucleates do not present the specific granules that normally contain lactoferrin. from a morphological point of view, the nuclei appear bilobated and the nuclear membrane may show intro-and extroversions. it is also possible to identify the membrane's lack of cyclic neutropenia is an autosomal dominant disorder in which cyclic hematopoiesis causes intervals of neutropenia and susceptibility to opportunistic infection. in nine families whose children displayed typical blood patterns, pedigrees confirmed dominant inheritance without evidence of heterogeneity or decreased penetrance; three pedigrees suggested new mutations [369] . a wide spectrum of symptom severity, ranging from asymptomatic to life-threatening illness, was observed within the nine families. the phenotype changed with age. children displayed typical neutrophil cycles with symptoms of mucosal ulceration, lymphadenopathy, and infections [369] . patients are usually asymptomatic, but during the period of severe neutropenia, recurrent overwhelming infections, inflammation, and ulcers occur in about 10% of patients and can lead to significant chronic morbidity [298] . severe neutropenia was shown by 21 children, moderate by 4, and mild by 1: 16 of these children had leukopenia, 7 anemia, 2 thrombocytopenia, and 1 monocytosis. during follow-up, respiratory infections developed in 24, oral manifestations in 20 children. the most common infections, in descending order of frequency, were otitis media, abscesses, pneumonia, oral ulcers, acute diarrhea, cutaneous infections, oral candidiasis, and periodontits. sinusitis, cystitis, conjunctivitis, meningitis, and osteomyelitis were less frequently observed. hepatomegaly was also detected in 10 children and splenomegaly in one; 3 children died of recurrent infections. therefore, recurrent infections always deserve further evaluation for detecting such disorders [398] . abdominal pain must be assessed aggressively because of the high frequency of clostridium infections during the period of severe neutropenia [369] . during the course of scn, bm shows lack of maturation of granulocyte precursors beyond myelocytes, and there is myeloid hyperplasia during the remainder of the cycle. occasionally, there is a reduction in the severity of neutropenia and the accompanying infections over time [369] . a complete clearing of symptoms and a significant increase in quality of life is noteworthy in children [298] . however, while the disease is commonly described as benign, four children in three of the nine families died of clostridium or e. coli colitis, documenting the need for urgent evaluation of abdominal pain [369] . pediatric cyclic neutropenia is effectively treated with rhug-csf (recombinant human g-csf), usually at doses of 1-5 mg/kg/day (median dose, 2.5 mg/kg/day) [449] or twice weekly, or once a month. typically, children are noted in early infancy to have persistent scn with absolute neutrophil counts <0.2¥10 9 /l lasting for months or years [12] . in children aged 4 days to 19 months, the initial and lowest median absolute neutrophil counts were 0.29¥10 9 /l and 0.06¥10 9 /l, respectively [289] . usually, children suffer from long-term recurrent bacterial infections, and maturation arrest of myelopoiesis at the promyelocytemyelocyte stage of bm development [12] . the disease begins during the 1st year of life, and its infectious complications include cellulitis, perirectal abscess, peritonitis, stomatitis, and meningitis, commonly as a result of infections with s. aureus, e. coli and pseudomonas aeruginosa [42] . the numbers of circulating monocytes and eosinophils are often increased [42] . missing the most important cells in the defense against bacterial infections, the neutrophil granulocytes, children suffer from episodes of severe, often life-threatening bacterial infections [42] . they spend many days in hospital, requiring iv antibiotic treatment. recurrence of bacterial infections leads to irreversible tissue damage, for example in the lungs, requiring often disabling surgical interventions. a high incidence of significant bone mineral loss was seen in children with scn [545] . the presence of qualitative and quantitative abnormalities of primitive myeloid progenitor cells expressing g-csfr may play an important role in the impairment of granulopoiesis in these patients, thus nearly all patients have a response to pharmacological doses of rhug-csf: neutrophil counts rise, infection rates fall, and mortality is reduced [343] . since the introduction of rhug-csf, most children enjoy a normal life span and a greatly improved quality of life, although they still have problems with infections, especially chronic gingivitis and periodontitis [82] . it is more likely that the bone loss was caused by the pathophysiological features of the underlying disease, but it is possible that rhug-csf accelerates bone mineral loss [545] . prolonged administration of rhug-csf at a dose of 3 u/kg bw twice daily may be associated with increased bone resorption, mediated by osteoclast activation and leading to bone loss. in children, the resulting osteopenia can be successfully managed with antiresorptive bisphosphonate therapy with significant improvement in bone density [449] . a child maintained on long-term rhug-csf therapy developed acute myelogenous leukemia associated with a g-csfr mutation.after having undergone successful allogeneic bmt, both ela-2 mutation and g-csfr mutation became undetectable by pcr [237] . shwachman syndrome, a rare ar condition, characterized by pancreatic insufficiency, reduced mobility and neutrophil chemotaxis, cyclic neutropenia, thrombocytopenia, metaphyseal dysostosis, delayed growth and recurrent pyogenic infections, in two cases was associated with isolated gh deficiency [96, 268] . in addition to metaphyseal chondrodysplasia, neutropenia, and pan-poor to absent granuloma formation [242] . salmonella and certain viral infections [hsv, cmv, parainfluenza, and respiratory syncytial virus (rsv)] are also seen [126] . most patients bearing an ifn-gr1 deficiency present gross mutations that truncate the protein and prevent its expression, giving rise to severe mycobacterial infections and, frequently, a fatal outcome [6] . mortality in these children is high, and infections are severe and recurrent [242] , as in an 8-year-old girl before receiving a bmt [405] . a point mutation may be fatal: an individual, probably hz for the mutation, died from meningitis due to mycobacterium bovis [6] .a hz missense ifn-gr1 mutation was identified in two siblings who did not respond to low or intermediate concentrations, yet responded to high ifn-g concentrations, probably for a reduced affinity of ifn-gr1 for its ligand [243] . otherwise the mutation results in normal surface expression of ifn-gr1 that do not bind ifn-g [244] . a dominant deletion in the ifn-gr1 gene has been reported in a female patient hz for a 4-bp deletion in exon 5 of ifn-gr1 who developed postvaccinal disseminated bcg infection [417] . the ar form of partial ifn-gr1 deficiency was reported in 18 patients of 12 unrelated kindred with susceptibility to mycobacterial infection [403] . an 8-year-old girl with ifn-gr1 deficiency, also with recurrent mycobacterial infections and liver cirrhosis with portal hypertension, received red cell-depleted bmt from her hla-identical sister. the transplantation course was uneventful and 4 years later the child remains in excellent clinical condition and free of mycobacterial infections [405] . a complete ifn-gr2 deficiency was found in a child due to a hz dinucleotide deletion resulting in a premature stop codon in the protein extracellular domain. this gene defect emphasizes the critical role that ifn-g plays in host defense against mycobacteria [126] . a girl with bcg and salmonella enteritidis infection and a hz recessive deletion in the p40 subunit of il 12 leading to a complete il 12 p40 deficiency has been reported. a large hz deletion within the il 12 p40 subunit gene was found, precluding il 12 p70 (composed of p40 and p35 subunits) functional expression by activated dcs and phagocytes. the net result was a markedly impaired ifn-g production by lymphocytes. however, addition of recombinant exogenous il 12 p70 in the assay was able to restore normal ifn-g production in vitro [8] . the girl suffered from well-organized granulomas, possibly due to residual il 12 -independent ifn-g production [8] . another kindred [377] and two siblings and one unrelated patient [142] carried the same large deletion, also accompanied by disseminated infections. a 3-year-old female was repeatedly hospitalized since the age of 5 weeks for recurrent episodes of pneumococcal pneumonia with sepsis and other infections in the absence of creatic exocrine insufficiency, the findings in children are noted as variable extremity shortening, cup deformation of the ribs, metaphyseal widening and hypoplasia of the iliac bones, and increased echogenicity of the pancreas with no change in size [43] . recurrent infections begin during the 1st year of life and commonly involve the sinuses, lungs, bones, skin, and urinary tract [42] . neutropenia, either cyclic or intermittent, occurs in all patients, and 10%-25% of patients also have pancytopenia [464] . immune functions may be involved in this syndrome, including marked pan-hgg, especially of the iga, normal/increased cellular immunity, but depressed humoral and nk cell immunity [268] . in 13 patients diagnosed in infancy, a significant growth improvement and a decreasing frequency of infections were observed over time, in addition to improvement or normalization of exocrine pancreatic function [96] . a continuous spectrum from systemic bcg infection to local recurrent nontuberculous mycobacterial infection covered by the clinical features of affected children has recently helped to identify several genetic defects in the monocyte-macrophage-th1 t-cell pathway [509] . different types of mutations in four genes (ifn-gr1, ifn-gr2, il 12 p40, il 12 rb1) forming the ifn-γ/il 12 axis [123] have revealed both allelic and nonallelic heterogeneity and result in different disorders whose common pathogenic pathway is impaired ifn-g-mediated immunity [123, 403] . several children have been reported who presented a new kind of hereditary id with severe and/or recurrent infections caused by only one microorganism family, in opposition to other patients with classic pid. five new syndromes may encompass these children with a genetic predisposition to infectious diseases. if the ifn-g/il 12 axis is impaired, the host becomes highly susceptible to infection with organisms that replicate intracellularly (susceptibility to mycobacterial disease). stat-1 (signal transducer and activator of transcription-1) deficiency predisposes to viral disease, nemo and irak-4 (il 1 r-activating kinase-4) deficiencies predispose to infections caused by pyogenic bacteria [376] . this pid encompasses several defects: complete, partial, and ar ifn-gr1 deficiency, and complete, partial, and ad ifn-gr2 deficiency [122] . ifn-g and the cellular responses induced by it are essential for controlling mycobacterial infections. patients with ar mutations leading to complete loss of ifn-gr1 or ifn-gr2 expression have the most severe phenotypes, and they present early in life with disseminated severe infections, especially if they have received bcg vaccination, and have fever. she exhibited il 12 deficiency that was associated with an abnormality of the il 12 p40 gene. although present, ifn-g was reduced [211] . a genetic lack of il 12 rb1 surface expression predisposes to severe infections by pathogenic mycobacteria or salmonella and causes strongly decreased, but not completely abrogated ifn-g production [408] . the deficiency may be complete as well as partial [291] . several patients with these features have been reported [291] . three unrelated individuals with severe, idiopathic mycobacterial and salmonella infections were found to lack il 12 rβ1 chain expression. il 12 rβ1 sequence analysis revealed genetic mutations that resulted in premature stop codons in the extracellular domain [116] . a patient with severe infections as above and multiple adverse drug reactions had t cells unable to produce ifn-g or proliferate in response to il 12 , despite the expression of wild-type il 12 rβ1 and il 12 rβ2 [186] . defective il 12 r signaling leads to low t-cell and nk-cell ifn-g production [509] . il 12 rb1 and il 23 rb1 chains are associated in an ar deficiency with susceptibility to mycobacteria and salmonella infections [351] . the stat4 (2 forms, ad and ar [351] ) s721 mutant failed to restore ifn-g production in stat4-deficient il 12 rb2 transgenic cells [329] . stat1, -3, and -5 activation by il 12 was lost, an impairment specific for il 12 ; nor is activation of stat4 alone sufficient for il 12 -induced ifn-g production and proliferation [186] . two unrelated infants hz with respect to mutated stat1 suffered from mycobacterial disease, but unlike patients with ifn-gr deficiency both died of viral disease [133] the complement is an integral part of the humoral defense system against infections and also for promoting inflammatory process (figs. 1.63, 1.65 ). complement deficiency was found in 6/176 dutch patients (3.4%) over a 33-year period (0.1% × year) [156] . from the study of blood donors the prevalence may be of 0.03% in the general population [531] . congenital deficiencies have been described for most of the proteins it is composed of (tables 22.20 and 22 .1f [167, 169, 453, 531] , usually following the ar model. properdin deficiency is the only complement deficiency that is x-linked [531] . hets can be easily identified because their relevant component is present in the serum with a 50% concentration. the lack of one component at the hz level serologically involves the blockage of enzyme release below and the absence of hemolytic activity, while that of controlling proteins causes its uncontrolled activation, consuming the factor that is the object of control and, in various ways, also of successive components [137] . nonfunctional c1q variants have been observed, c1r and c1s deficiencies are often associated, probably because they are mapped on contiguous genes of chromosome 12 (c1q on 1) [167] . the b, c2 and c4 genes, situ-ated on the short limb of chromosome 6, constitute along with others the hla class iii (chap. 1). c6 and c7 are codified on chromosome 5p and have a similar structure; c8 shows a different structure, because the molecule consists in three a, b and g chains, united to form two subunits, a-g and b dictated by different genes [495] . alternative pathway deficiencies are extremely rare [328] . complement deficiencies are accompanied by an increased frequency of infectious pathologies [155] , although it is not rare to come across them in individuals who are apparently in good health, as in the case of c2 hereditary deficiency [422] . also frequent are teens and young adults with autoimmune manifestations (chap. 18). classic pathway deficiencies are often associated with sle-like diseases (systemic lupus erythematosus), id of the early components of complement (c1-c3) are associated with risks of infections caused by encapsulated bacteria such as streptococcus pneumoniae, haemophilus influenzae type b, as well as by meningococci [363] . the incidence of sle in patients with c1q, c4, or c2 deficiency is 90%, 75%, and 15%, respectively [378] . partial c4 deficiency is also associated with sle; 15% of patients with sle exhibit c4a deficiency [531] . several components are associated with development of membranoproliferative glomerulonephritis (table 22 .20 [167, 169, 453, 531] ). in alternative pathway id, the infections recognize pyogens as the most common etiological agents, while final common pathway id (c5-c9) or properdin (p) have been associated with recurrent or invasive infections by neisseria (n) gonorrhoeae or n. meningitidis, gramnegative bacteria, and asplenia, agammaglobulinemia [167, 169, 363, 531] . it is estimated that the frequency of meningitis in subjects with hz deficiency of the final c5-c9 pathway is 10%, 6,000-fold higher than in non-id individuals [363, 488] . some characteristics appear to associate the patients with complement deficiency and meningococcal disease: frequent recurrent episodes, an older age at the first onset, lower mortality compared to patients with a normal complement, and a prevalence of males [167] . over 50 patients with c1q, c1r and c1s deficiencies have been described, c1s deficiency only in two cases [239] . a selective and complete c1s deficiency in a 2-year-old girl with complex aids including sle-like syndrome, hashimoto's thyroiditis, and autoimmune hepatitis has been reported. exon-specific amplification of genomic dna by pcr followed by direct sequence analysis revealed a mz nonsense mutation in the c1s gene exon xii at codon 534. both parents were het for this mutation [127] . a deficiency in one of these proteins is sufficient to block the classic pathway activation; deficiency results as a consequence of non-synthesis, which in the unlike c2, hz c4 deficiency is very rare and is caused by the non-expression of all 4 alleles (2 of c4a and 2 of c4b, 2 maternal and 2 paternal alleles), which can occur due to punctiform mutations, gene deletions, or other gene alterations that prevent gene transcription [28] . the two 4a and 4b genes are polymorphous, just like c2, c3, c6, c4a and c4b and the b factor (bf); polymorphic variants of the other proteins are rare, with 12 different alleles for c4a and 23 for c4b identified at the moment. moreover, two loci c4a and c4b null alleles q0 (quantity 0), do not codify for any phenotype, although often present in the general population [167] . in a 4-year-old caucasian child who suffered from several bouts of pneumonia caused by respiratory viruses, eight episodes of acute otitis media, prolonged respiratory and urinary tract infections, molecular studies of the c4 gene region revealed hz deletion of hla class iii cyp21a-tnxa-rp2-c4b, generating total deficiency of case of c1q amounts to 60% of cases, while in the remaining 40% the molecules are malfunctioning but cross-reacting with the native molecule [328] . c1r and c1s deficiencies are usually combined, due to the contiguity of the two genes; typically in these patients c1r is absent and c1s levels are reduced (20%-40%) [422] . affected patients suffered from a sle-like syndrome and sporadically from an extended predisposition to infections [155] . associated symptoms in c1q deficiency are sle-like syndrome, rheumatic disease, and infection. several children suffered from meningitis, recurrent septicemia, recurrent otitis media, pneumonia, and stomatitis; two died from meningitis septicemia [241] . the incidence is based on the data from [167] ; the inheritance is always ar, with the exception of c1-inh deficiency (autosomal dominant) and factor p deficiency (autosomal recessive or x-linked). data from [167, 169, 453, 531] . gn glomerulonephritis, jra juvenile rheumatoid arthritis. c4b and the flanking 5¢ region up to c4a [230] . moreover, in 7/13 cases the c4a*q0 alleles were related to a c4a/cyp21p gene deletion within the hla-b8 c2c bfs c4aq0b1 dr3 haplotype. in 3/13 cases, the c4b*q0 allele was related to a c4b/cyp21p gene deletion within the hla-b18 c2c bff1 c4a3bq0 dr3 haplotype [212] . the c4 null allele incidence is so elevated that 60% of the population expresses all c4 genes, while 30% lacks 1-3 alleles [328] . the elevated number of q0 probably derives from the marked similitude of the two genes, which facilitates the unequal crossover, but this crossover in the hla can modulate the expression of three c4a alleles and one c4b or vice versa; the c4aq0 allele spreading amplifies the risk of contracting sle and juvenile ra (jra). hz c2 deficiency, the most common in the caucasian population, has an incidence that varies between 1:10,000 and 1:28,000, whereas the het carrier rate is 1.2% [456] . it is usually found in the a25, b18, dr2, bfs, c2q0, c4a, c4b2 haplotype context which, due to its considerable rarity, could assume a predictive value [238] . two different types of c2 deficiency are known: in type i the synthesis is deficient due to the protein nontranslation, in type ii there is a selective absence of secretion but not of synthesis, therefore c2 levels are 0.5%-2% of normal values [238] . hets have a nonfunctioning gene, the complement profile is characterized by serum c2 concentrations equal to 50% of normal values; about 50% are asymptomatic, while the other half exhibit frequent infections and quite a few suffer from sle and correlated syndromes [155] . het c2 deficiency was associated with a 28-bp deletion in the c2 gene (type i), mainly within the hla-a25 b18 c2q0 bfs c4a4b2 dr2 haplotype [212] . in a certain number of cases, a c2 deficiency is accompanied by a partial bf malfunctioning, genetically close to it. hzs can also have a deficient function of the alternative pathway [445] . possibly this deficiency, like other quite common ones, may not always be reported in the literature: the total number of cases therefore underestimates the real prevalence, as is also found in children with c7 deficiency [167] . c2 deficiency must be suspected in all patients presenting pneumococcal infections after the age of 2 years [239] . both c2 and c4 predispose to sle, but this is not the expression of a particular genetic association caused by the same gene localization, because c1q, r, s, deficiencies, which also cause sle, are situated as mentioned outside the hla system [422] . the molecular bases of this pid appear heterogeneous. the c3 gene exists in different allelic forms, some of which have reduced functionality. one must remember the c3 important role in immune responses, also as far as apc and b cells are concerned, as well as the defensive role played in innate immunity along with c4. since both pathways converge in the cleavage and activation of c3, there is no way that this defect can be corrected, and furthermore the opsonic power is greatly deficient, as is the c5 chemotaxis; therefore patients affected by hz deficiency mostly present clinical symptoms totally similar to a congenital hgg with severe recurrent infections and at times the symptoms of cic disease [239] . although id is severe, some patients apparently remain in good health and the syndrome may also in time become less severe, probably due to the higher number of immune experiences that allow a better effector function to antibody reactions mediated by the fc receptor [167] . c3 protein was defective in noninfected nigerian children with protein-energy malnutrition (pem), but rose significantly in the presence of bacterial infection, thus sharing the values found in healthy controls [137] . in its clinical expressions, c5 deficiency does not differ from the other deficiencies discussed here; the clinical consequences of absent c5a anaphylotoxin are unclear [328] . one-fourth of all patients are asymptomatic. in caucasians incidence is 1:60,000 [495] . deficiencies associated with c6-c7 are rare but reflect the close genetic proximity of their pertinent genes; in c6-c8 deficiencies, 63% of patients lacking one component experience at least one severe episode of neisseria infection and 5.5% one aid [239] . rare in europe, c7 deficiency is the second most common complement deficiency in the japanese (0.005%) [339] . in italian children it has a prevalence of 10% and has been identified also in healthy siblings [531] . in japan c7 deficiency is more associated with meningococcal meningitis than with neisseria infections [339] . in a highly inbred arab population, a c7 deficiency was associated with a mutation (g1135c) that is also prevalent among israeli jews of moroccan ancestry [34] . a total deficiency of this 150-kd protein, inherited as an ar trait, has been described in a young patient with a hemolytic-uremic syndrome and in one case in italy whose parents were first cousins [531] . among 21 relatives of the proband studied, encompassing 3 generations, ten had low factor h levels, including her two children, indicating a het factor h deficiency [157] . h deficiency results in uncontrolled breakdown of c3, and in depletion of bf, p and c5 [157] . c3 and c9 components are decreased in varying degrees, while c3 and c5 are found in plasma in traces and only as activated molecules [167] . inheritance of factor d deficiency is for the moment uncertain [488] (table 22 .20): a partial deficiency (6%-12% of normal concentrations) has been described in two mz twins, and a total deficiency in one male. this deficiency, serologically characterized by the non-functionality of the alternative pathway, is clinically accompanied by an increased susceptibility to neisseria infections [239] , leading us once more to emphasize the alternative pathway significance as a substantial means of defense for a broad spectrum of damaging actions caused by bacterial infections. properdin deficiency is the only one inherited as a characteristic linked to the chromosome x and only affects males [190] . at the moment, >50 cases have been described. the deficiency can be materialized by a total p absence, with levels reduced to 10%, with normal levels, however, showing an altered functional activity [239] . specific research has shown a remarkable reduction of c3a and b titers, which represent the c3-convertase proteins, with a consequent heightened consumption due to the alternative pathway spontaneous activation. males are affected by septic episodes caused by neisseria, sometime fulminating, with onset even occurring during the 1st year of life [488] . there is no evidence of increased susceptibility to cic diseases or infections caused by other organisms [155] , thus implying that a functioning alternative pathway is particularly important for a defense against infections [328] . correctly identifying children with complement abnormalities is important and worthwhile if any of the following factors are present: id (such as repeated or unusual infections with other organisms, fh, unusual course of the illness, etc.), repeated neisserial infections, infection with an unusual serogroup, fulminant disease in males (p deficiency), coexisting angioedema, autoimmune, or connective tissue disorders [220] . in the two forms of c8 deficiency (c8a + c8g and c8b mapped on chromosome 1), the subunit not involved is present in the serum, also with reduced levels, and accompanied by altered functionality of the one involved. c8 deficiency has different characteristics due to the diverse associations of the b and a-g chains; therefore caucasians with this deficiency lack the b chain (10%), while colored patients lack the a and g chains (90%) [495] . c9 deficiencies are poorly considered because they are often asymptomatic [339] ; however, in japan there is an incidence of 0.1% [339] and between 33% [155] and 25% of cases [339] present meningococcal meningitis. congenital c9 deficiencies are very common among the japanese (0.036%-0.1%) and represent 11.3% of all deficiencies (table 22 .20). there have been >150 cases of congenital c5-c9 deficiencies reported, distributed unevenly between the various ethnic groups: c5 and c6 deficiencies are prevalent in colored patients and the c7 deficiencies appear to be more common in caucasians [422] . an analysis of published studies shows that 14% of patients with sporadic meningococcal infections may have a c5-c9 deficiency [495] . clinical patterns are overlapping. the most common complement deficiency is c1-inh deficiency (c1 inhibitor), responsible for hereditary angioedema that causes symptoms in hets (chap. 8). at least 15 cases of factor i deficiency are known [328] , with autosomal co-dominant transmission because parents show normal complement levels at 50% of factor i [239] . as in an h deficiency, serologically one has the alternative pathway activation, due to c3b non-catabolization that continuously forms c3 conversion with severe c3 deficiency, the levels of which do not exceed 15% of normal values [155] . in hzs, in addition to subsequent pyogenic infections, as in c3 deficiency [422] , cutaneous rashes and urticaria caused by massive release of histamine and pro-inflammatory cellular products by anaphylotoxic fragments are reported [167] . the fact that a child during the initial period of life should experience a certain number of urtis or lrtis is within the norm: rris are mainly caused by immunological immaturity or inexperience, both transient. a typical symptom outline is difficult to define, and prevalence is also little known. in id children, a basic pathology is present instead, which encourages the recurrence of infections. although a distinction between infection and associated disease is important, childhood infections might have a key role in stimulating the maturation of the immune system, and the microbial burden in early life has been invoked as a protective factor against wheezing and asthma (chap. 4). preschool-age children have an especially high frequency of vris, with most having three to eight infections per year and 10%-15% have ≥12 vris per year. the rate in children aged 0-4 has been about 1 in 200 children compared with about 1 in 500 for children aged 5-10, and about 1 in 1,000 for those aged 11-17 [413] . rris may be defined as >six episodes of urti and/or >3 lrtis in the previous year, or based on age and ≥8 episodes per year if aged <3 or ≥6 episodes per year if aged ≥3. several risk factors can influence the onset and recurrence of infections [217] . it is clear that the younger the child is, the more he/she may fall ill: this is also related to serum ig levels (table 1 .15). the dogma of primary and secondary responses also may not apply to infections, at least those caused by rotavirus, which confers considerable protection only after various infectious events and in children aged 1 [511] . environmental factors in the absence of a basic pathology are important. it is also obvious that the more crowded the environment the child lives in, the more probable that infection becomes. in addition to the number of siblings, other factors such as socioeconomic status, age (preschool children), contact with outside persons, especially babysitter, early social contacts, exposure to passive smoke, indoor and outdoor pollution may be found to be related to the hygiene hypothesis (chap. 4). however, daycare attendance, which was considered to be an indicator of exposure to respiratory pathogens, and the presence of siblings, increased the risk of urti in preschool children aged 4-5 [273] , and in the 1st year of life for children with fha [88] . among children with fha, the protective effect of day care attendance in early life against the development of atopy only begins by 2 years, and against wheezing this may not be observed until after 4 years [89] . the particular ease of smoking parents and/or relatives who fall ill with influenza has been known for some time, to the same extent that the children who live with them are affected by rris (table 4 .24). the impact on rri incidence as it is related to children in kindergartens, has been reflected by a significantly increased morbidity observed in babies aged 3 months to 3 years who go to daycare, who show a number of more severe and longer lasting infections per year [523] , with an incidence of 6.5% in children who stay at home compared to 13.1% of those in kindergartens [524] and an increase of 49% of ome persistence (chap. 15). in children exposed to cigarette smoke, the risk increased 3-fold for lrti [221] or by 3.5-fold, equal to ≤3 episodes of respiratory infections each year [26] . studies on environmental pollution have identified the most damaging agents: conclusive data on fine particles in suspension and polluting derivatives is available, proving a significantly increased risk of infantile rris: table 4 .20 indicates that no 2 reduces immune defenses against rtis, provoking alterations of the epithelium and of the lymph node cells, with negative effects on mucociliary clearance and macrophages. the biological role played by no 2 in the domestic pollution derived from the home has been ascertained to be related to cooking and the smoke released by combustion [26] . using wood for heating leads to so 2 development, while radiators cause the air to dry, which in turn causes potentially infected particles to remain in suspension. pollutants are increasingly responsible for indoor pollution (chap. 4). although levels of micro-pollution are not easily ascertained, significant associations with acute rris and conditions such as polypnea and dyspnea have been reported, especially in <2 year-old children [507] . these children's capacity to evoke adequate responses is genetically controlled; however, it is commonly known that their parents or siblings have suffered similar illness as children. in subjects with physiological immaturity of the immune system, vris more easily cause infectious episodes, which are important factors to be considered only in the presence of recurrent or incompletely cleared conditions [165] . predisposing factors related to a basic pathology derive from perinatal factors, more common in premature babies, which can lead to respiratory tract alterations and consequently to bronchopulmonary dysplasia; anatomical anomalies; cystic fibrosis, which can become recurrent pneumonia; adenoiditis causing otitis and ome; congenital ciliary dyskinesia; humoral deficiency and pid characterized by recurrent sinopulmonary infections [101] (table 22 .21) [79] . viruses are the principal etiological agents, and over 159 a novel member of the coronavirus family has been characterized, which is associated with cases of sars (severe acute respiratory syndrome). phylogenetic analyses and sequence comparisons showed that sarscoronavirus is not closely related to any of the previously characterized coronaviruses [421] . as investigated by a 15-year study the overall prevalence is age-related, and different between children aged 0-4 ( fig. 22.46) [326] and those aged 5-19 ( fig. 22.47 ) [326] : rsv and rhinovirus have a different impact on the first group (58% vs 28%) and the influenza viruses on the second group (9%-48%). incidence in young children was 1.75-fold higher than in those aged 5-19 [326] . rsv causes bronchiolitis in breast-fed babies, with a higher frequency the younger the child is (tables 11. 24, 11.25) , and rhinitis in older siblings. even an infectious agent neglected for some time, such as ureaplasma urealyticum, causes a lung pathology in younger children while sparing those >3 years old [271] . the bacteria most commonly involved are: streptococcus pyogenes (pharynx and larynx); haemophilus in-1323 complement deficiency fluenzae (middle ear and larynx reaching the epiglottis); and streptococcus pneumoniae (middle ear). there are often secondary bacterial infections such as complications caused by vris, which certainly contribute to recurrent infections and/or the onset of chronicity [508] . in chap. 4 we reported several studies which concluded that early infections may protect from atopy development (hygiene hypothesis). we must distinguish pid outlines and pseudo-id in children with rris. summarizing the aforementioned, severe and recurrent lrti and sinusitis are the principal clinical manifestations in children affected by deficiencies prevalently involving humoral immunity (table 22 .1). these children fall ill during the first weeks of their lives and often contract infections caused by opportunistic agents, fungi, protozoa and viruses, and as months go by are also affected by malnutrition and failure to thrive. episodes affecting the airways, particularly common in cellular and combined id, tend to become longer or severe, especially if complicated by pneumonia [44] . chronic disorders such as sinusitis and bronchiectasis (sinobronchial syndrome) are not rare; interstitial pneumonia is in most cases caused by pneumocystis carinii and results in tachypnea and lung hyperinflation [409] . infections supported by the herpes simplex, ebv and cmv are also common: males with xlp exhibit a deficient response to ebv [446] . severe and recurrent sinopulmonary, was, higes and ata infections as well as severe rris in cgd, complement deficiencies and lad 1 to v must also be borne in mind. cases of recurrent pneumonia should be warning signals to rule in 4.8% pediatric cases of pids [392] . the second most important manifestation is chronic diarrhea: in some cases the infections are caused by rotavirus and enterovirus among which the echo: giardia lamblia, salmonella and campylobacter can also cause chronic enteric infection; malabsorption resistant to treatment can be ascribed to cryptosporidium [80] . rris are common in children. they reflect the immaturity of the immune system in its encounter with environmental antigens; this developmental delay during the first years of life fosters the development of rris. thus, rris are part of the growing-up process of any child [47] . the consequences of rris can be of a profound and sometimes protracted alteration of the different immune defense mechanisms, which place the child in an undefended position, similar to the condition observed in children with pids, compromising the phagocytes, lymphocytes, nk cells, antibody production, and ils at every occasion [419] . the responsible viruses for these infections have a development limited to surface mucous cells, spreading from cell to cell due to contiguity, while the viremic stage is absent or remains marginal. the incubation period is therefore brief, normally <3 days; consequently the immune response may not be capable of ensuring a protective function, or it only intervenes partially, clarifying the potentially unlimited number of infectious episodes [392] . from a pathogenetic point of view, the virus works by triggering the development of ige and allergic sensitization and/or damaging the immune structures [322, 519] . in the first case, it is known that experimental infection in mice with rsv is capable of significantly increasing the absorption of ovalbumin (ova) administered by aerosol, of igg, ige and anti-ova siga (fig. 4.26) and of increasing the synthesis of ige and specific igg to ragweed also administered by aerosol. the result is that the majority of infants become infected with rsv, although lrtis develop in only about 20% [382] . approximately 25%-50% of those subsequently experience recurrent acute asthma from vri [530] . the mechanism by which vris induce atopic sensitization in experimental models is identified with antigen penetration and sige synthesis [322] . studies show that viruses increase mucosal permeability, by modulating antigen uptake and altering antigen processing by the mucosa, which results in the ige-suppressor t-cell depression, while ifn-g-modulated histamine release further increases mucosal permeability [162] . it is probable that the immune deficiency is secondary to vris, because many viruses are capable of inducing transient [521] . the lack of nk cells, which are in the front line of defense against viral infections, and with which the altered production of il 2 and factors activating the phagocytes are associated, appears significant, but it is unclear whether the deficiency is primary or secondary to viral infections [419] . the basic question with potential therapeutic consequences therefore remains unanswered, and is probably destined to remain so until more sophisticated tests are available to clarify this issue, although the nk-cell reduction in these children corroborates the first hypothesis. recent data emphasizes the important defensive activity of cytotoxic cd8 t cells and by different ils, including il 10 , il 28 and il 29 active in antiviral defense: if the infected cells express on the surface the viral antigen processed in association with hla class i molecules, cytotoxic cd8 take care of killing the cell. normally the cd8 are activated by the virus itself or by soluble factors released by the infected cells and mediate the cellular lyses after recognizing hla class i antigens on the same cell. however, if the apc is a macrophage, it can be parasitized by the virus, with consequently reduced chemotaxis and microbicidal activity and in particular the capacity to cooperate with t cells [322] . considering that macrophages may have evolved specific mechanisms for directing t-cell development toward the th1, since cmi can solve some infections, it is clear that their dysfunctions appear in the insufficient nk cell activation and inadequate th1 development in response to infections. ifn-g, produced not as a direct consequence of infection, but probably by il 12 and/or il 15 , stimulates the nearby cells to block the nucleic acid transcription and therefore the viral replication, preventing the infection of those cells to which virus spreads due to contiguity. therefore the ifn-g species-specific antiviral activity takes place at the very first stages of the infection, preceding the antibodies [322] . the response to germs' capsular polysaccharides, particularly deficient until the age of 2, although encounters with these germs are abundant during this period of time, still remains to be evaluated [508] . however, selected children, although normal from an immunological point of view, may have a deficient antibody response even aged 4-8 [195] with a percentage of modifications of both humoral and cmi, therefore not only of antibody synthesis or phagocyte and neutrophil functions, but also of t lymphocytes and related ils [457] . it is also possible to hypothesize that persistent vris are capable of inducing th2 activation by antigens or super-antigens: th2 t cells with il 4 help induce virus-specific cd8 + to produce il 5 , which recruits eosinophils in the respiratory tract, thus reducing ifn-g secretion. thus there may be an increased interaction of ige mast cells in these subjects with immunoregulatory alterations, due to a marked lymphoproliferative response and the elaboration of other ils, which consequently amplifies ige production in the respiratory tract. the ige response is thought to lead to a greater production of bronchoconstrictor mediators by effector cells; viral infections themselves may induce these cells to release histamine. it is also known that humoral deficiency, especially of iga, very often opens the way to gram-positive germs causing viral and bacterial infections, thereby completing the circle [409] . interestingly, only 2/13 children have at least a significant production of antigen-specific salivary iga against klebsiella pneumoniae [418] . even if the alterations are transient and aspecific in children with rris, their occurrence and persistence for a number of months, for a year and even longer, leads to believe that the immunosuppressive mechanisms set off by the first episode occasion more severe, profound and lasting consequences for the immune functions than those occurring in their normal peers [36, 273] . having at least one physician-diagnosed lrti in the 1st year of life was significantly associated with recurrent wheezing (or, 2.0) and asthma (or, 2.5) [89] . at the base of this exclusive predisposition in children for contracting rris, there is a nk cell reduction [419] and immune deficiencies related to the global lymphocyte population, the cd4 and the cd4:cd8 ratio, unbalanced toward cd4 t cells, prevalent in children expressing coughing compared to those with bronchial hyperreactivity (bhr) [374] . other t-cell deficiencies in children with humoral anomalies include a considerable spontaneous production of il 2 and il 4 , both generated by th phenotypes, or alternatively by the th0, which express both ils [216] . it is similarly feasible that virus-specific cd8 deprived of cytolytic activity are converted into th2-like t cells when il 4 is present [216] . cmi in these children consists above all in the transient t-cell numeric and functional depression, coinciding with a deficiency of ils necessary for their activation, proliferation and differentiation. the virus toxic effect also acts directly on the t cells, inciting rough structural modifications including giant polynucleate cells, which jeopardize homing and recirculation capacities, to the point of immunosuppression, affecting the specific lymphocytes for that particular virus, thus favoring the attacking organism [552] . a condition of immunosuppression occurs also as a result of superantigen (sa) orchestration, which, stimulating a large num-nonreactive children of 4%-19% [143, 430] , rising to 13%-42% if with an iga and/or igg subclass deficiency [198, 216, 430] . while 50% of children have low antipneumococcus igg 2 , antibody responses are totally absent in 40% of dysimmunoglobulinemics with a virtual absence of iga and igg 2 [430] , confirming previous data [198] . iga and igg 2 -deficient children show a clinical pattern with elevated susceptibility to s. pneumoniae infections [430] . von waldeyer ring (nalt) is a significant constituent of immune response, and of resistance to nalt-dependent infections. among the consequences of chronic adenotonsillitis (table 15. 2) the statistically significant decrease in ig levels, including siga, must be evaluated eventually in relation to rri complications (chap. 15). igg subclass deficiencies can be present in children with chronic and/or severe asthma, associated or not with sigad, in children affected or not by asthma with severe rris or with chronic nonallergic respiratory clinical symptoms, in children with sigad, ata, was, cvid, scid and in healthy subjects [80] . the anti-virus antibodies generally belong to the igg 1 and igg 3 isotypes while the igg 3 protect from microbes with polysaccharide antigens, such as s. pneumoniae and group a and type b h. influenzae [153] . table 22 .7 shows normal values for igg subclasses in subjects aged 0-15. unlike total igg concentrations, igg 1 and igg 3 reach normal levels within the 1st year, igg 2 mature more slowly, ensuring an effective antibody response only after the 2nd year, and igg 4 develop even more slowly. various authors believe that the role played by igg subclasses is unique and vital in defending from infections: igg 2 deficiency is associated with an increased susceptibility to infections by bacteria expressing capsular polysaccharides, such as pneumococci, meningococci, h. influenzae, bordetella pertussis, etc., as well as other factors capable of setting off an inflammation [113, 465] . igg 4 deficiency instead seems associated with a marked predisposition for rris [333] . however, undetectable igg 4 subclass levels are a common finding in normal individuals and an accurate detection of very low levels of igg 4 is technically difficult to achieve [450, 465] . babies aged ≥1 month have levels of circulating lymphocytes secreting all four subclasses in a higher number than adults; therefore the capacity to produce antibodies exists well before a full humoral response is developed. subnormal igg subclass concentrations, especially of igg 2 , are also observed in healthy children who do not present an increased susceptibility to infections. low subclass levels do not necessarily indicate that the subject will experience immune disorders exclusively linked to these, nor do normal concentrations guarantee that the child will be spared complications. sub-jects have been observed both with normal igg 2 levels and with recurrent infections and with a subclass deficiency, which often do not form other types of antibodies [450] . igg subclass determination does not indicate what the humoral level restricted to that molecule is: this is a characteristic in children, unlike adults, even though research was carried out using the same methods in the two studies [465] . interesting hints come from studies involving children with rris: of children aged 2-10, with a confirmed diagnosis of susceptibility to infections, 67/567 (11.8%) had a igg subclass deficiency, almost all concerning igg 4 , with several associations [283] . in other children, the selective igg 4 deficiency was statistically significant compared to atopic controls without rri, associated not so much with a well-defined state of id as to a respiratory tract defense mechanism deficiency, in view of the fact that the relative prevalence of this subclass in secretions may indicate a role in the mucosal defense [333] . other children with typical symptoms of recurrent infections, lymphadenopathies, failure to thrive and hgg exhibited low igg 2 levels, confirming that normal levels of total igg do not exclude a subclass deficiency [454] . in a cohort of young babies, igg 4 deficiency was only present in 78/267 subjects (37%) [198] ; however, the absence of a control group makes the results incomparable. an igg subclass deficiency is therefore able to induce or worsen chronic respiratory symptoms in allergic and nonallergic children, especially if predisposed to developing these affections [113] , or in subjects with sigad [327] . with time these deficiencies, and eventually also those associated with iga, can normalize [29] . in conclusion, transient and persistent iga and/or igg 2 deficiencies have been reported in a small percentage of asymptomatic children [446] , but even if iga and igg subclasses are not always required as such for a normal immune response, their deficiency may predispose to rris [160] . as previously illustrated in chap. 11, the close links between atopy and rris are known, and this is confirmed by the observation that asthmatic children have a higher incidence of rris than their nonasthmatic siblings. it has been known that rris during the early periods of life can play a role in the development of bhr and atopy: in the classic study by frick et al [173] , in 11 out of 13 allergic children sensitization was propitiated by rris. with continued observation, the authors noted the presence of high ige levels, positive rast and histamine released by leukocytes after infections [172] . in a cohort of 73 asthmatic children aged 0.8-3.1, the 21 affected by 20th week of pregnancy is possible, so as to evaluate the immune phenotype in the fetal blood [491] . wccs (white-cell counts) in cb and differential counts can be used to detect the lymphopenia that is commonly present in infants with scid. however, subset analysis by flow cytometry is necessary to enumerate t, b and nk cells. subsequently, scid diagnosis will be suspected when overwhelming opportunistic infections occur [320] . depending on whether one suspects a humoral, cellular or innate immune deficiency, we begin with the algorithm in table 22 .23 [107] , positive if infants or children have ≥2 of these signs, then 478, 522] on laboratory tests can be consulted. however, children with variable levels of antibody id may end up with different diagnosis [269] . children with higms presented initially with a history of an increased susceptibility to infection including pneumocystis carinii pneumonia [539] in 43% of children [290] . in pids affecting phagocytes, because of the relatively narrow spectrum of disease-specific infections (such as aspergillosis in cgd), careful attention to the microbiology laboratory early in the course of evaluation of a patient suspected of having a pid is crucial to orient the work-up in the appropriate direction [416] . in a male newborn referred to hospital at 27 days of age for fever, hemodynamic failure and an inflammation syndrome caused by pulmonary infection, culture of tracheal, bronchoalveolar lavage samples and lung biopsy grew positive for a. fumigatus, enabling the diagnosis of cgd [337] . to investigate whether patients with undiagnosed id could be identified in diverse inpatient hospital populations, a scoring algorithm and computer screening method was updated [107] on the basis of icd-9 codes to survey the discharge diagnoses of all hospitalized patients over periods of time. thus 17 id patients were identified, eight of whom were children aged 2-10 (47%), two with neutropenia, two with igg deficiency, one with lad, one with dgs, etc. [108] . we also suggest including congenital phagocytic defects in the differential diagnosis of recurrent bacterial or fungal infections in a child [285] . a congenital complement deficiency should be suspected if levels of even one component are reduced [167] . early diagnosis is essential for choosing the necessary treatment [44] . the differential diagnosis of pid will emphasize the different characteristics schematized in table 22 .27, to which one must add objective rarity, while fh and child gender become important [10] . in subjects suffering from omenn syndrome, was, severe combined and cellular ids, the screening of clinical symptoms may be useful at birth and during the first few months after birth (table 22. 28) [61, 80, 399] . the localization of infections is multiple, the id child usually appears to be ill, and the peripheral lymph nodes and lymphatic rris had a higher incidence of fh positivity (p=0.015), increased ige (p=0.021), as well as a combined iga (p=0.038) and igg (p=0.018) deficiency [296] . ige hyperproduction could be the result, not only of the wellknown association between igg subclasses and ige and their coregulation of il 2 expression [296] , but also of a virus-caused unbalanced cd4:cd8 ratio [374] . these results link atopy to rris, confirming that the state of chronic inflammation and bhr induced by allergic sensitization is an ideal substratum for the adhesion and chronic evolution of bacterial and/or viral infections. there are no specific clinical outlines for rris. on the contrary, symptoms are extremely varied, with, as previously mentioned, infections caused by bacteria and viruses. urtis are common at age 4. during the last 12 months, 9.5% of the children experienced more than one bout of acute otitis media, 6.9% had more than one pharyngotonsillitis episode, 47.7% contracted >2 common colds, and 3.2% had rhinitis weekly or monthly [273] . there are children who, during the period of maximum exposure due to biological immaturity and immunological inexperience, suffer from one episode each month affecting different organ systems, as well as lymphadenopathies and failure to thrive. the capacity for inducing bhr in normal subjects and worsening the symptoms in those already ill are precisely caused by vri, also facilitating greater penetration of inhaled viral allergens [530] (table 11 .10); rris in turn predispose to sinusitis. lower ifn-g levels produced by 18 of 53 children at 6 months of age were even greater if the comparison was made between children with rris and those with no or maximally one rri during the follow-up period [382] . rhinovirus-induced infections (table 11 .11) take the appearance of common rhinitis, but stimulate mastocytes to release histamine, contributing to bhr development and the perspective of delayed reactions. a differentiating feature is the respiratory infections in the id child that may also result from opportunistic pathogens [80] (table 22 .21). respiratory infections should be under control. a screening of humoral immunity revealed low ig levels in 4.6%, low iga levels in 2.3%, and sigad in 1.3% of children [270] . during the last few years, increasingly sophisticated diagnostic techniques have permitted prenatal diagnosis in many cases (table 22. 22) [61, 166, 389, 406, 414, 491] : in forms supported by rag-1 and/or rag-2 mutations a diagnosis even at the 10th-12th or at the pharyngeal tissue are almost imperceptible [102, 196] . the seriously undernourished appearance should be noted, more often observed in children with scid [162] . rris can be observed in other cmi forms [162] : deficiencies of cd3 g and e chains [18, 249, 471] , zap-70 [92, 139] and hla class ii [77, 256] . finally, children with aids will seem to be in severe general condition and this disease is a paradigmatic example of how hiv can overturn the t lymphocyte immune defense with regards to opportunistic infections [79] . in some cases of pediatric aids there is hgg that is indistinguishable from pid and that belongs to the differential diagnosis of severe recurrent infections during the first few months after birth [79] . as far as tih is concerned, the confirmation of a normal presence of the b lymphocytes and low levels of intrinsically produced ig is resolutive, compared to agammaglobulinemia [61] . an articulate case history often identifies the familiarity of rris, usually with an absent basic pathology and the frequent predisposing environmental factors (table 22 .21), among which passive cigarette smoking stands out. maximum prevalence occurs during the first 2 years of life or during first contacts with school, the disease is limited in time, and there is usually a single location. in most cases the pseudo-immunodepressed child is clinically normal in all other respects [374] . in ten reported sars-infected children from hong kong, fever, cough, and runny nose were 1329 complement deficiency lateral pharyngeal x-ray to visualize adenoidal tissue cytokine production (il 2 , il 3 , il 4 , il 5 , ifn-g, il 12 ) chromosome fragility (ataxia-telangiectasia, bloom's syndrome, etc.) data from [101, 478, 522] . ada adenosine-deaminase, adcc antibody dependent cellmediated cytotoxicity, ctl cytotoxic t lymphocytes, ltt lymphocyte transformation test, pha phytohemagglutinin, pma phorbol myristate acetate, pnp purine nucleoside phosphorylase, ppd purified protein derivative. sars seems to have a less aggressive clinical course in younger children [223] . when in doubt, a broad spectrum of laboratory tests are available: cbc, proteinemia and protidogram, serum ig levels, or in secretions and igg subclasses (table 22 .7), immunoelectrophoresis (homogeneous components, k/l), dosage of isohemagglutinin, five other natural antibodies, the sweat test [10] , in strictly selected cases also a lymphocyte population and subpopulation count (tables 1.34-1.39), and x-ray of paranasal sinuses. analysis of the lymphocyte profile sometimes shows a number of deficiencies, statistically differentiated from those found in other children affected by an asthmatic pathology; however, none of the immunological deficiencies indicated (table 22 .29) are characteristic in pseudo-immunodepressed children. common diagnostic methods may not be capable of revealing a deficiency of igg subclasses or of selective igg 4 : the chance that there may be abnormal igg 2 or igg 4 levels is not excluded by the normality of igg serum concentrations [153] . furthermore, the distribu-tion of igg into four subclasses makes it difficult to identify these deficiencies simply by measuring total serum igg levels [113] ; only for the past few years have there been highly specific reagents for measuring individual subclass levels and methods such as radial immunodiffusion (rid) [198, 317] . the aaaai has recommended not relying on subclass levels [10] , especially igg 4 levels, which seem to be unmeasurable in 25% of the population [198] . rid, which has proven to be more sensitive than the elisa used by the cdc in atlanta, has shown that 25% of normal children have values below normal for at least one subclass [317] ; a similar deficiency was present in 58% of children with rris [198] . a recent study measuring the igg with both methods, has proved that the rid can show higher values of igg 1 and igg 2 in low serum levels of both ig [387] , data with an unquestionable negative effect in pediatrics. in conclusion, at the moment our knowledge suggests that we should also carefully interpret low levels of one or more subclasses, because on the one hand this might indicate a transient or paraphysiological condition, on [174] . igg levels in all patients also rose considerably compared to previous treatment: the average levels in different determinations was >700 mg [174] . finally, all children grew normally; the height achieved by each child is between the 3 rd and 50 th percentile, within the limits of theoretical values calculated on the height of their parents. substituting therapy with ivig has allowed patients to return to their normal activities, with a considerable improvement in quality of life. in all these years, we have never come across substantial unwelcome reactions or infectious complications [174] , as also found by other authors [177, 461] . ivig could also be effective for reducing the allergic symptoms discussed thus far: presuppositions are not lacking, such as the blocking of allergens and mastocyte fcr thanks to the modest quantities of igg 4 present in the preparations [196] . in addition, the increased understanding of the igg transplacental passage (chap. 2) can absolve the function of timing their transfusion in the case of mothers with antibody id, so that the fetal defenses can be complete and quantitatively adequate. in sigad common ivig preparations cannot be used, even if with a low content of iga, nor enriched, both because of the extremely short iga life-span, which would therefore suggest iga administration every 2-3 days, and because the infused iga do not reach the secretions [507] . should ivig be indicated for a deficiency associated with igg 2 , or should transfusions of blood derivatives become necessary, one must first investigate serum antibody anti-iga levels (igg and ige) and, should these be positive, avoid infusions or administer them in a hospital under strict medical supervision, or use washed red blood cells [507] . the same precaution must be taken for subjects with ata for whom ivig, if is appropriately administered, also ensure beneficial effects on quality of life, while there are no known therapies for contrasting neurological symptoms. in patients with humoral deficiency, alongside ivig, if appropriate, an antibiotic prophylaxis is suitable with monthly cycles, alternating amoxicillin, cephalosporin, co-trimoxazole, etc., bearing in mind family compliance. in cvid recurrent infections caused by giardia lamblia should be treated using furazolidone (8 mg/kg/day) or methronidazole (15 mg/kg/day) for 10 days, if necessary to be repeated. cvid treatment in specialized centers involves recombinant il 2 , il 10 and cimetidine [456] . some t-cell pids represent a severe clinical emergency, such as omenn syndrome, in which hypovolemic shock and reticular dysgenesis are immanent in the battle for survival. although precise figures are unavailable, thousands of patients worldwide with different forms of the other a modest deficiency can result in clear hgg [451] . subnormal igg 2 levels can indeed be associated with various manifestations of immune dysfunctions; it is therefore advisable in this case to proceed with specific investigations, measuring the response to polysaccharide antigens and studying the lymphocyte activity in vitro [451] . in children with normal serum levels, who are instead lacking in antibody responses to polysaccharide antigens [143, 430] , this is a conclusive investigation [470] (fig. 22.47) . a study of children aged >5, half atopic and half not, has confirmed this thesis, concluding that the answers were similar in both groups, therefore excluding a greater rri predisposition in the atopic children [344] . many patients with high ige levels do not present atopic manifestations: it is thought that an increased concentration is related to a reduced inhibiting activity of the thymus in ige synthesis [457] . recurrent sinopulmonary infections must make one also consider cystic fibrosis and immotile cilia syndrome [457] . children with malnutrition (chap. 21) suffer from numerous ids, prevalently concerning cmi; their vulnerability makes them succumb to severe bacterial me infections and urtis, often also risking death. obese subjects may also be affected by rris due to a possible adipose tissue hypovascularization or to a defect in the granulocyte microbicidal activity [457] . in the presence of antibody deficiency, antibiotic treatment is chosen as a preventive therapy in less severe cases, otherwise the preferable therapy consists in ivig (fig. 22.48 ). this treatment is restricted to a limited number of diseases, including some forms of id, secondary or cytopenic id, in which effectiveness has been proved in dbpc studies [135, 426, 439] , like other positive forms of intervention described, while it appears to be of no use in uncomplicated thi [426, 439] . two children aged 2.5 and 3 with higes and kawasaki disease were administered 400 mg/die of ivig for 5 days and one 2.5-year-old with higes received only one dose, with ige levels falling from 4,000-14,000 to 600-5,000 ui/ml on the 28th day. hence there was almost a normalization of ige production with symptom relapse after 6 months; similar results using a single dose were also obtained in two children with higes and severe ad [254] . the following data represent a number of clinical and immunological parameters in children suffering from humoral pid and ata, with igg levels <100 mg/dl. treatment with ivig, also at a higher dosage, was very well tolerated by patients: all children presented a clearverely affected by several factors [26] . either hla-identical marrow or t-cell-depleted (tcd) haploidentical parental marrow is the standard of care for scid ( fig. 22.51) . when histocompatible related donor bmt is unavailable, a bmt either with hla-identical unfractionated or tcd haploidentical parental marrow is the standard of care for scid [338] . all but one (95%) of 21 scid infants who received tcd identical or haploiden-genetically determined id have been given bmt in attempts to correct their underlying id [66] , including a recent series [14] . specific treatment for cellular pid consists in a bmt from a hla-compatible donor [10] . the ideal sc donor is normally a sibling who shares identical hla class i and class ii loci. without such a donor, these transplantations usually resulted in fatal gvhd. if death did not occur, event-free survival was se-1335 treatment tical bmt in the first 28 days of life are currently alive, with the period of survival ranging from 8 months to >19.2 years after transplantation. this compares favorably with a 74% survival rate of 96 infants receiving transplants at a median age of 190 days (range, 45-516 days) [338] .a girl with t -b -scid received a full matched bmt from her sister at age 2 weeks [491] . a worldwide survey conducted by buckley from 1994 through 1997, with subsequent additions of published cases from the literature, revealed that 239 of 302 (79%) patients with pid transplanted with hla-identical marrow during a period of 34 years were alive [65] . there are >375 patients worldwide who have survived scid as a result of successful transplantation of hla-identical or haploidentical bm [67] . most importantly, 34 of 35 infants (97%) undergoing transplantation in the first 3.5 months of life are currently alive [69, 338] , compared with a cut-off at 6 months (97% vs 86%, children younger vs those aged >6 months) receiving bmt (or 5.0 [14] ). we stress that neonates developed higher lymphocyte responses to phytohemagglutinin and higher numbers of cd3 + and cd45ra + t cells in the first 3 years of life than those receiving bmts late. t-cell antigens peaked earlier and with higher values in the neonatal bmts (181 days to 1 year) than in the late bmts (1-3 years) [338] . over the past 22 years 78% of all scid patients (110/142) receiving bmt at duke university medical center survive to varying ages up to ≥20 years after bmt. only 16 had an hla-identical donor. all others received rigorously tcd haploidentical bm from a parent, most often the mother. the soy lectin, srbc rosetting technique was used (r. buckley pers. comm. november 20th, 2004 and april 20th, 2005). an uncommon bmt to treat ar scid was undertaken in a 1-month-old girl. the donor was her hla-mismatched 6-year-old sister, who had previously received a bmt from her father [479] : presently, they are aged 5 and 13 and are affected by molloscum contagiosum infection [547] . bmt, both hla identical unfractionated and tcd hla aploidentical [63] .a recent trial found that because only 10%-15% of affected children have a familial hlaidentical donor (rid), the alternative therapeutic options are bmt from a mud or a haploidentical bmt or from hla-mismatched related donors (mmrds). only 40% of these children may find a matched donor; therefore, the remaining pid-affected children are candidates for a tcd haploidentical bmt [277] . mud hsct is successful in young children [132] , but the success rate decreases dramatically above the age of 5-6 years [158] (table 22 .30) [13, 15, 25, 27, 39, 46, 52, 55, 56, 58, 64, 67, 69, 80, 83, 85, 98, 111, 119, 131, 132, 163, 185, 188, 197, 205-208, 231, 233, 235, 237, 245, 252, 256, 257, 259, 260, 277, 280, 286, 288, 290, 305, 307, 334, 360, 366, 380, 405, 408, 428, 440, 448, 452, 456, 466, 474, 491, 496, 499, 503, 534] . bmt survived. compared with mmrd bmt, survival was significantly higher with rid or with mud (45/54 = 83.3%) [201] . when an hla-identical sibling as the donor is unavailable, a phenotypic hla-matched unrelated bmt is needed, also used in cd154 deficiency [290, 499] , with a clinical and immune outline normalization [246] and a variable effect on igg subclasses 1337 treatment [58] . c median (1.5-13 years after bmt). p, at the time of publication. [153] . possibly because of earlier diagnosis before untreatable opportunistic infections develop, the results have improved considerably during the last two decades [65] . bmts have been successful when applied within the first 7-24 days of life in 21 infants with scid, 20 (95%) of those still alive range from 8 months to 19 years, not justifying in utero transplants. a completely normal t-cell function was obtained within 82-118 days [338] and in an other 83 patients was still present after 10-17 years [373] . of the 58 children who received a mismatched parental bmt from 1980 to 1998, 43 (74%) remain alive with t-cell immune reconstitution, a median of 128.8 (range, 13-224. 3) months after bmt [362] . of 96 children out of 117 who received allogeneic bmt after the first 28 days of life, 71 (74%) are alive [69] . three out of five children who received a hsct are alive and well after 18-32 months [13] . breast feeding appeared correlated to an earlier reconstitution when the donor was the mother [338] . intra-amniotic gene transfer has been successfully carried out on a laboratory animal, registering in a dose-dependent manner the fetal gastroenteric and respiratory effects [222] . if confirmed in human beings, this method of treatment will certainly prove useful for prenatal correction of pid. bmt/hsct should be completed by conditioning regimens with busulfan and cyclophosphamide, less toxic than total lymphoid irradiation or a combination of nucleoside analogs and anti-lymphocyte antibody preparations [65] . for example, busulfan (16 mg/kg), melphalan (90 mg/m 2 ) and anti-thymocyte globulin (36 mg/kg) [83] . to enhance the engraftment rate in haploidentical bmt in pid, it was recently suggested to add donor peripheral scs after mobilization with g-csf (16 mg/kg for 5 days) and bm cells. with this procedure the cell load is increased, which allows intensification of the conditioning regimen for induction of faster engraftment [277] . in utero bmt suggested advantages include the sterile environment in utero, and immaturity of the fetal immune system enabling the prevention of clinical manifestations of the disease in the neonate, and the engraftment without the use of cytotoxic conditioning regimens: a child thus treated was well at age 11 months [164] . two series of six and four patients [386, 504] and two additional case reports [182, 532] of in utero transplants have been published, yet failure of b-cell engraftment and function may result in long-term dependence on ivig replacement [248] . better results than those published for in utero bmt for scid were implicit in 13 infants admitted and diagnosed at a median age of 3 days because of a fh of a previously affected infant. bmt was successful and all children are alive and well with follow-up to 11.5 years [248] or <30 days vs approximately 4 months [194] . however, it is suggested that in utero transplants may carry the risks associated with injecting the fetus and the inability to detect gvhd during gestation [65] . umbilical cb transplantation (ucbt) was done in two children affected by a zap-70 deficiency and an omenn-like syndrome. both are alive and well at 4.5 and 2.2 years after ucbt [148] . unrelated ucbt in eight children with severe t-cell id [260] and in three with was [259] resulted in consistent and stable t -, b -, and nk cell development [259, 260] . faster availability of ucbts is a meaningful advantage for patients requiring urgent transplantation: a median of 25 days more rapidly than did those receiving bone marrow [30] . in a large report [423] , 40 patients with scid, seven with was, and other unspecified pid received an unrelated ucbt. ucb was evaluated as a sc source for immune reconstitution in children with severe primary t-cell ids such as scid, reticular dysgenesis, thymic dysplasia, cid, dgs, and was when a matched sibling donor was unavailable, and has been used to date in more than 2,000 patients [194] . three infants who rejected a tcd-mismatched parental bmt without prior cytoreduction engrafted after infusion of ucbt [69] . a 4.5-year-old girl with hla class ii deficiency had a successful related ucbt for graft failure following tcd nonidentical bmt [52] . a girl with reticular dysgenesis failed to engraft following her first transplant, but fully engrafted after a second unrelated ucbt. five of six patients showed grade i gvhd, although one child experienced grade iv skin and gut gvhd. immunological ucbt resulted in consistent and stable t-cell, b-cell, and nk-cell development [260] . long-term event-free survival (≥27 months) with recovery of antigen-specific responses was reported following an unrelated ucbt in a child with omenn's syndrome [38] . gene therapy, which has revolutionized and could revolutionize even more pid treatment in the near future, is analyzed in table 22 .31 [76, 104] . the requisite for applying this form of genetic engineering treatment is that the responsible gene must be cloned; the most common techniques involve knock-out mice and inactivating a particle [104] , or employing retroviral vectors (table 22 .32) [263] , totally deprived of their genomic factors except for the normal copy of the gene to be inserted. this is indispensable for allowing the vector to reach the human nucleus, where it will integrate with the cellular genome [263] . in this case, pbls are collected through leukapheresis and cultivated in vitro with retroviral particles containing a normal gene rna copy: thus the healthy gene is introduced into the cell genome using a vector and the manipulated cells are reinfused, so as to restore normal immune functions. this system has been used to treat two children aged 2 months suffering from scid [508] and from ada deficiency by employing autologous pbls [51] , bm cells [54] , and cb cells [262] . three reports [4, 88, 204] of successful gene therapy in infants with x-linked scid [88, 204] and in t -b -scid [4] are a major step forward among repeated efforts to achieve better immune reconstitution in ada-scid with gene therapy than with bmt/sct [161] . immediately after the diagnosis had been made in two capacity of wild-type, replication-competent retroviruses to cause leukemia in immunologically immature neonatal mice [263] , and in humans (two children out of 11) [63, 88, 204] . retroviruses can cause insertional oncogenesis, a long-known potential complication of retroviral gene transfer attempts, because gene integration occurs at random in the genome, thus deregulating the expression of cellular oncogenes [263] . this complication has been thought to be unlikely with such vectors, because they are capable of inserting only once into the cell's chromosomes and cannot repeatedly reproduce and integrate. lentiviruses may be more effective than murine retroviruses for gene transfer into human hematopoietic scs and t lymphocytes [263] . in ada-scid, the safety and efficacy of hsc gene therapy combined with nonmyeloablative conditioning for the treatment of scid has allowed two children to live at home and clinically well, with normal growth and development [4] . on january 14, 2003, fda placed on "clinical hold" all active gene therapy trials using retroviral vectors to insert genes into blood scs after having learned that a second child treated in the french gene therapy trial developed a leukemia-like condition. gene therapy is on hold despite enormous promise for certain scid/ cid variants. survival. in scid, the european experience with unfractionated hla-identical and tcd or non-tcd haploidentical or mud bmts in patients with scid reported that between 1968 and 1999, a 3-year survival with sustained engraftment was significantly better after hla-identical than after mismatched transplantation (77% vs 54%).within the hla-identical group, survival after bmt from genotypically or phenotypically identical related or muds was 81%, 72%, and 63%, respectively [14] . in non-scid, 3-year survival after genotypically hla-matched, phenotypically hla-matched, mmrd, and mud bmt was 71%, 42%, 42%, and 59%, respectively [14] . in a retrospective analysis of bmts performed between 1977 and 1991 at 13 european centers in 149 children as young as 1 month with 11 different pids (excluding scid), the overall survival among 53 recipients of hla genetically identical bmt was 66%, 45.5% in 22 patients who received closely matched bmt, and 38% in 71 recipients of bmt with two or three mismatched hla antigens. a significant improvement in survival has been achieved in most pids (overall survival, 81.5% vs 51.7%, primarily because of a decrease in the frequency of infectious complications [163] . in the similar analysis performed in 193 children with scid at 18 european centers between 1982 and 1993, 116 out of 193 (60.1%) patients were alive with evidence of engraftment 6 months after bmt. however, 24 patients died >6 months post-bmt, mainly due to cgvhd and/ or viral infection. thus gvhd 6 months after bmt and b -scid vs b + scid were the main factors associated with a poor outcome [206] . the disease-free survival was significantly better for patients with b + scid (60.7%) than for those with b -scid (33.3%) [14] . in a children aged 8 and 11 months including a novel splice imitation in the common gc chain [183] , haploidentical cd34 + peripheral progenitor cells mobilized with gm-csf were isolated to a purity of more than 99%. these cells were infused with no prior chemoablation and no prophylaxis against gvhd. both children showed signs of t-cell reconstitution beginning 3 weeks after the cd34 + infusion and were weaned from continuous cures. they are in excellent health, without gvhd, 34 and 68 months after transplantation. one child does not need replacement ig. the other received a booster infusion of cd34 + scs from the original donor 1 year later to improve b-cell function and now receives ig every 3 months. both were followed for 10 months after gene transfer [88] . however, retroviral vectors have the 1339 treatment data from [4, 76, 104] . ciita class ii transactivator, cgd chronic granulomatous disease, xla x linked agammaglobulinemia; for other abbreviations see table 22 .1. a done with success, see text for details. b in utero transplant of maternal stem cells. trial on children with pid receiving bm from hla-nonidentical related donors or from hla-identical unrelated donors at 13 european centers between august 1990 and june 1993, 22 out of 28 children (76.6%) survived 22-58 months. bm was tcd by use of either erythrocyte rosetting or monoclonal antibodies to prevent gvhd [231] . additional survival rates were reported previously (table 22 .30). in a series of consecutive ud bmts 31/33 children with scid and non-scid pids who received a bmt with reduced-intensity conditioning (ric) regimen between 1998 and 2001 survived after a 3.3-year follow-up, as well as 10/19 children who received a bmt with myeloablative conditioning (mat) between 1994 and 1998 and survived after an 8.6-year follow-up. therefore a ric regimen results in improved survival and reduced bmt-related mortality compared with mat in hr children undergoing an ud bmt [396] . in 170 transplanted patients with was, the 5-year probability of survival differed according to donor type: 87% with hla-identical sibling donors, 52% with other related donors, and 71% with mud. significantly, boys who had received a mud transplant before 5 years of age had survival rates similar to those receiving hlaidentical sibling transplants [158] . however, the time required to develop immune function after haploidentical scts is quite different from that after unfractionated hla-identical bm. lymphocytes with mature t-cell phenotypes and functions fail to rise significantly until 3-4 months after bmt; normal t-cell function is reached between 4 and 7 months [338] . b-cell function develops much more slowly, averaging 2-2.5 years for normalization; many do not have b-cell function, despite normal t-cell function. [338] . ex vivo rigorous depletion of post-thymic t cells from donor marrow that cause gvhd is efficient and feasible, even in haploidentical settings [13] , presumably because of more effective infection-control measures and better transplantation strategy [514] . for non-scid, sct can provide a cure, and grafts from unrelated donors are almost as beneficial as those from genetically hla-identical relatives [45] . in most patients, deficient b-cell function persists after transplantation and requires lifelong ivig therapy [69, 207] , which is necessary to prevent bacterial and common viral infections [69, 514] . some patients also have persistent deficiencies of t-cell function after sct [206, 373] . in children with rris, depending on the nature of the infection, the pediatrician will prescribe the most appropriate symptomatic and/or antibiotic therapy. in the presence of persistent inflammation, or during the winter, when the risk of close acute recurrent episodes is higher, anti-inflammatory preparations will be prescribed via aerosol, chromones, ketotifen, b 2 -adrenergic and if necessary steroids for topical use, strictly depend-ing on the need. we suggest monitoring measures, such as keeping a clinical diary, in which each acute episode should be briefly noted, continuing registration until clinical symptoms have not regressed for at least 15 days and returning to keep notes in the diary each time there is a cough and/or nasal and/or bronchial inflammation, completing this with pef as well as some respiratory parameters right at the beginning and then every 6 months. it is obvious that if medical intervention is not resolutive a center specialized in infantile respiratory physiopathology should be contacted [47] . children with sars were treated with high-dose ribavirin, oral prednisolone, or iv methylprednisolone, with no shortterm adverse effects [223] . antibiotics must be used very carefully in these children because they can influence positively or negatively the innate, cellular or humoral immunity (chap. 18), interaction with ils and growth factors are not known, repeated use often causes phenomena involving allergy/ intolerance [470] , and most infection-prone children suffering from vris are given antibiotics unnecessarily. in italian children (54.6% of males) aged 6 months to 14 years (median, 4 years) with a history of rris, macrolide therapy of acute respiratory infections influenced the natural history of rris, probably because of their elective activity on atypical bacteria [508] . considering the emergence of antibiotic-resistant bacterial stock, as for example s. pneumoniae, immunotherapy has been proposed as a means of preventing rris by providing children with small doses of inactive bacterial antigens liable to trigger specific and protective immune responses (table 22 .33) [36] . for example, om-85 bv significantly reduces the urti rate, particularly in a dbpc study in 232 children aged 3-8 with a history of acute urtis [448] , is active in preventing rri episodes [36] with a meaningful reduction in the number of days of suffering acute urtis [448] . bacterial ribosomal and membrane proteoglycans of s. pneumoniae, which stimulate b cells with secretory responses, as well as memory cells, may be used for responding to future infections [36] . ribosomal immunotherapy appears to be not only well tolerated, but also ideally targeted to induce mucosal responses [37] . among the preparations reserved for specific use, a study of pidotimod in dbpc trials proved its effectiveness in a sample of 101 children with rris, also showing increased cd25, absent in placebo-treated children [72] . the use of immunostimulants should be limited to children with proven high susceptibility to acute urti, or overexposed children attending daycare facilities, or attending kindergarten or elementary school [508] . however, according to a meta-analysis, immunostimulants are an effective treatment for the prevention of acute urti in children [40] . furthermore the indiscriminate and purely empiric use of ivig must be discouraged in every child with rri, whereas in a prospective, dbpc study of ivig and co-trimoxazole, 106 of 130 children <8 years referred for recurrent bacterial rris became infection-free over a 4-month obser-if caused by pneumococci. other kinds of vaccines have provided disappointing results: fig. 22 .52 [340] indicates the immune bases of a specific immunization and the possibility of specific interventions. in children with pid, one should bear in mind all the aforementioned facts. until the past few years, there was a busy motion into the fundamental problems underlying a majority of these conditions. many have now been mapped to specific chromosomal locations, and an impressive number vation period [353] , in addition to having an extremely unfavorable cost-benefit ratio [426] immunization children with rris and deficient antibody responses to germs expressing a capsular polysaccharide can be successfully vaccinated, but avoiding the administration of live virus vaccines and integrating this if necessary with an igg replacement therapy [218] . furthermore, in view of the availability of conjugated vaccines, it will be possible to induce antipneumococcus-igg 2 , providing an effective treatment for children with rris, especially 1341 treatment of the fundamental biological errors have been identified. the pediatrician is entrusted with a more difficult job, that of identifying as early as possible the possible existence of pid, remembering the suggestions for case history in chap. 6, with the exception of clinical emergencies such as omenn syndrome and reticular dysgenesis. this specific research becomes a necessity thanks to the new diagnostic and therapeutic advances that have been conceived over the past few years: the earlier one acts, on the one hand with a prenatal diagnosis and on the other with a bmt or sct therapy, the greater the chance to 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genes required for t-cell development: il7r, cd45, il2rg, jak3, rag1, rag2,artemis, and ada and severe combined immunodeficiency neonatal bone marrow transplantation for severe combined immunodeficiency t lymphocyte receptor deficiencies a mammalian cell cycle checkpoint pathway utilizing p53 and gadd45 is defective in ataxia-telangiectasia three in vivo promoter phenotypes in mhc class ii deficient combined immunodeficiency bone marrow transplantation for cd40 ligand deficiency: a single centre experience transient hypogammaglobulinemia of infancy: clinical and immunologic features of 40 new cases high-dose intravenous g-globulin treatment for hyperimmunoglobulinemia e syndrome lad-iii, a leukocyte adhesion deficiency syndrome associated with defective rap1 activation and impaired stabilization of integrin bonds major histocompatibility complex class ii deficiency: clinical manifestations, immunologic features, and outcome bone marrow transplantation in major histocompatibility complex class ii deficiency: a single center study of 19 patients gene therapy for wiskott-aldrich syndrome: rescue of t-cell signaling and amelioration of colitis upon transplantation of retrovirally transduced hematopoietic stem cells in mice umbilical cord blood transplantation in wiskott aldrich syndrome kinetics of t-cell development of umbilical cord blood transplantation in severe t-cell immunodeficiency disorders suppression of x-ray-induced chromosome aberrations in ataxia-telangiectasia cells by introduction of a normal human chromosome 11 engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency gene therapy for t-cell immunodeficiencies the cytogenetics of ataxia-telangiectasia gene deletion in the human immunoglobulin heavy chain constant region locus: molecular and immunological analysis serum igg subclass concentration in patients with undue susceptibility to infections neutrophil-specific granule deficiency results from a novel mutation with loss of function of the transcription factor ccaat/enhancer binding protein epsilon immunodeficiency diseases caused by defects in phagocytes elective bone marrow transplantation in a child with x-linked hyper-igm syndrome presenting with acute respiratory distress syndrome clinical and immunologic aspects of the hyperimmunoglobulin e syndrome bone marrow transplantation for chronic granulomatous disease: long-term follow-up and review of the literature severe chronic neutropenia in chinese children in hong kong clinical spectrum of x-linked hyper-igm syndrome severe mycobacterium bovis bcg infections in a large series of novel il-12 receptor beta1 deficient patients and evidence for the existence of partial il-12 receptor beta1 deficiency primary immunodeficiency diseases in singapore -the last 11 years anti-apoptotic signaling by the interleukin-2 receptor reveals a function for cytoplasmic tyrosine residues within the common gamma (gamma c) receptor subunit restricting zap70 expression to cd4+cd8+ thymocytes reveals a t cell receptor-dependent proofreading mechanism controlling the completion of positive selection cardiovascular anomalies in digeorge syndrome and importance of neural crest as a possible pathogenetic factor reduced levels of igg subclasses and iga in young children with asthma incidence of primary immunodeficiencies in a population of south moravia, czechoslovakia cyclic neutropenia: an unusual disorder of granulopoiesis effectively treated with recombinant granulocyte colony-stimulating factor primary immunodeficiency syndrome in italy: a report of the national register in children and adults x-chromosome inactivation and developmental patterns in mammals mutations of jak-3 gene in patients with autosomal severe combined immunodeficiency agammaglobulinemia and insights into b-cell differentiation genetic characterization of myeloperoxidase deficiency in italy purine nucleoside phosphorylase deficiency complete digeorge syndrome: persistence of profound immunodeficiency transplantation of thymus tissue in complete digeorge syndrome thymus transplantation in complete digeorge syndrome: immunologic and safety evaluations in 12 patients complete digeorge syndrome: development of rash, lymphadenopathy, and oligoclonal t cells in 5 cases postnatal thymus transplantation with immunosuppression as treatment for digeorge syndrome molecular heterogeneity of glucose-6-phosphate dehydrogenase (g6pd) variants in italy a gene encoding a novel rfx-associated transactivator is mutated in the majority of mhc class ii deficiency patients mutations in mlph, encoding a member of the rab effector family, cause the melanosome transport defects observed in leaden mice progress in primary immunodeficiency direct genetic correction as a new method for diagnosis and molecular characterization of mhc class ii deficiency transient hypogammaglobulinemia of infancy: need to reconsider name and definition a human non-xla immunodeficiency disease characterized by blockage of b cell development at an early prob stage interpretation of igg subclass values: a comparison of two assays hematopoietic stem cell transplantation for severe combined immunodeficiency in the neonatal period leads to superior thymic output and improved survival inherited deficiency of ninth component of complement: an increased risk of meningococcal meningitis national human genome research institute (2003) xlinked scid mutation database essential role for zap-70 in both positive and negative selection of thymocytes abnormalities of primitive myeloid progenitor cells expressing granulocyte colony-stimulating factor receptor in patients with severe congenital neutropenia a comparison of the development of antibody responses to the polysaccharide antigen (candida albicans mannan) in atopic and healthy infants and children x-linked lymphoproliferative disease: genetics and biochemistry a 39-kda protein on activated helper t cells binds cd40 and transduces the signal for cognate activation of b cells interleukin-2 receptor g chain mutation results in x-linked severe combined immunodeficiency in humans genetic defect in human x-linked agammaglobulinemia impedes a maturational evolution of pro-b cells into a later stage of pre-b cells in the b-cell differentiation pathway composition of precursor b-cell compartment in bone marrow from patients with x-linked agammaglobulinemia compared with healthy children radiosensitive scid patients with artemis gene mutations show a complete b-cell differentiation arrest at the pre-b-cell receptor checkpoint in bone marrow primary immunodeficiency diseases: an update primary immunodeficiency in colombian children a prospective, double-blind, placebo-controlled trial of i.v. immunoglobulin and trimethoprim-sulfamethoxazole in children with recurrent respiratory tract infections signaling via il-2 and il-4 in jak3-deficient severe combined immunodeficiency lymphocytes: jak3-dependent and independent pathways expansion of cd3 + , cd4 -, cd8 -t cell population expression high levels of il-5 in omenn's syndrome mutations in rab27a cause griscelli syndrome associated with haemophagocytic syndrome meyn ms 1993) high spontaneous intrachromosomal recombination rates in ataxia-telangiectasia registro español de inmunodeficiencias primarias (redip) interactions of viruses with the immune system microbial antigens and superantigens. clinical and immunological significance xlinked agammaglobulinemia: a survey of 33 iranian patients differential requirement for p56lck in fetal and adult thyreopoiesis acute respiratory illness in the community: frequency of illness and the agents involved susceptibility to infections in children with selective iga-and iga-igg subclass deficiency complement deficiency and disease stat4 serine phosphorylation is critical for il-12-induced ifn-gamma production but not for cell proliferation alterations of the x-linked lymphoproliferative disease gene sh2d1a in common variable immunodeficiency syndrome phenotypic variability: clinical presentation between the 6th year and the 60th year in a family with x-linked agammaglobulinemia (letter) artemis, a novel dna double-strand break repair/v(d)j recombination protein, is mutated in human severe combined immune deficiency deficiency of igg4 in children: association of isolated igg4 deficiency with recurrent respiratory tract infection chédiak-higashi syndrome: four cases from northern finland incidence, severity, and prevention of infections in chronic granulomatous disease long-term itraconazole prophylaxis against aspergillus infections in thirty-two patients with chronic granulomatous disease chronic septic granulomatosis revealed by neonatal pulmonary aspergillosis antibody deficiencies novel missense mutation found in a japanese patient with myeloperoxidase deficiency the aid enzyme induce class switch recombination in fibroblasts different amino acids at position 57 of the hla-dq b chain associated with susceptibility and resistance to iga deficiency immunodeficiency with hyperimmunoglobulinemia m in two female patients is not associated with abnormalities of cd40 or cd40 ligand expression bone marrow transplantation for t-b-severe combined immunodeficiency disease in athabascan-speaking native americans the presentation and natural history of immunodeficiency caused by nuclear factor kb essential modulator mutation hematopoietic cell transplantation for immunodeficiency disorders indications for the immunological evaluation of patients with meningitis igg2 deficiency in ataxia-telangiectasia a critical role for il-21 in regulating immunoglobulin production bone marrow transplantation in 26 patients with wiskott-aldrich syndrome from a single center nuovi aspetti diagnostici e patogenetici dei difetti primitivi dell'immunità umorale selective deficiency of interferon-gamma production in the hyper-ige syndrome: relationship to in vitro ige synthesis genetics, phenotype, and natural history of autosomal dominant cyclic hematopoiesis atypical x-linked agammaglobulinemia (letter) long-term followup and prognosis of chronic granulomatous disease in yugoslavia: is there a role for early bone marrow transplantation? griscelli disease maps to chromosome 15q21 and is associated with mutations in the myosin-va gene thymic function after hemapoietic stem cell transplantation for the treatment of severe combined immunodeficiency il bambino che si ammala spesso: profilo immunologico-clinico xlinked immune dysregulation, neonatal insulin dependent diabetes, and intractable diarrhoea new hereditary immunodeficiencies and genetic predisposition to infective diseases in children inherited interleukin-12 deficiency: il12b genotype and clinical phenotype of 13 patients from six kindreds links between complement abnormalities and systemic lupus erythematosus ataxia telangiectasia syndrome: clinical picture and immunological abnormalities human equivalent of the mouse nude/scid phenotype: longterm evaluation of immunologic reconstitution after bone marrow transplantation immunodeficiency-related lymphoproliferative disorders: prospective data from the united kingdom children's cancer study group registry lowered yields of virus-induced interferon production in leukocyte cultures and risk of recurrent respiratory infections in children clinical heterogeneity and reversibility of selective immunoglobulin a deficiency in 80 children two siblings with deficiency of iga1, igg2, igg4 and ige due to deletion of immunoglobulin heavy chain constant region genes extensive deletion of immunoglobulin heavy chain constant region genes in the absence of recurrent infections: when is igg subclass deficiency clinically relevant? il trapianto prenatale e postnatale di cellule staminali emopoietiche in bambini affetti da immunodeficienza primitiva methodologic problems in establishing normal values for igg subclass concentrations in a pediatric population: comparison of radial immunodiffusion and elisa methods recurrent v75m mutation within the wiskott-aldrich syndrome protein: description of a homozygous female patient prenatal diagnosis and genetic analysis of x-linked immunodeficiency disorders the interleukin-2 receptor gamma chain maps to xq13.1 and is mutated in x-linked severe combined immunodeficiency, scidx1 defective il7r expression in t(-)b(+)nk(+) severe combined immunodeficiency a partial deficiency of interleukin-7r is sufficient to abrogate t-cell development and cause severe combined immunodeficiency the scid but not the rag-2 gene product is required for sm-se heavy chain class switching chronic granulomatous disease the primary immunodeficiencies primary immunodeficiency diseases: report of an iuis committee the common cold -principles of judicious use of antimicrobial agents phagocyte immunodeficiencies and their infections 561del4 defines a novel small deletion hotspot in the interferon-gamma receptor 1 chain naturally occurring immune response against bacteria commonly involved in upper respiratory tract infections: analysis of the antigen-specific salivary iga levels ridotta funzionalità natural killer in bambini con infezioni respiratorie ricorrenti a case of hyperimmunoglobulinemia e treated with cow's milk-and egg-free diet characterization of a novel coronavirus associated with severe acute respiratory syndrome the complement system outcomes among 562 recipients of placental-blood transplants from unrelated donors interaction of il-2rb and gc chains with jak1 and jak3: implications for xscid and scid primary immunodeficiencies in switzerland: first report of the national registry in adults and children intravenous immunoglobulin consensus statement brief report: a point mutation in the sh2 domain of bruton's tyrosine kinase in atypical x-linked agammaglobulinemia clinical course of patients with major histocompatibility complex class ii deficiency recurrent pneumonia as warning manifestation for suspecting primary immunodeficiencies in children disorders of the polymorphonuclear phagocytic system. in: stiehm er (ed) immunologic disorders in infants and children difetti dell'immunità cellulare e immunodeficienze combinate clonal selection and learning in the antibody system improved survival after unrelated donor bone marrow transplantation in children with primary immunodeficiency using a reduced-intensity conditioning regimen griscelli syndrome congenital neutropenia and primary immunodeficiency disorders: a survey of 26 iranian patients recurrent sinusitis and immunodeficiency congenital immunodeficiency with a regulatory defect in mhc class ii gene expression lacks a specific hla-dr promoter binding protein molecular defects in the bare lymphocyte syndrome and regulation of mhc class ii genes sialophorin (cd43) and the wiskott-aldrich syndrome impaired interferon gamma-mediated immunity and susceptibility to mycobacterial infection in childhood autosomal recessive hyperimmunoglobulin e syndrome: a distinct disease entity correction of complete interferon-gamma receptor 1 deficiency by bone marrow transplantation the repair of dna damages/modifications during the maturation of the immune system: lessons from human primary immunodeficiency disorders and animal models activation-induced cytidine deaminase (aid) deficiency causes the autosomal recessive form of the hyper-igm syndrome jak3) deficiency: clinical, immunologic, and molecular analyses of 10 patients and outcomes of stem cell transplantation host defense mechanisms in respiratory infection icos deficiency in patients with common variable immunodeficiency immunoglobulin isotype-specific antibody responses to pneumococcal polysaccharide vaccine in patients with recurrent bacterial respiratory tract infections a rare syndrome in the differential diagnosis of hepatosplenomegaly and pancytopenia: report of identical twins with griscelli disease direct evidence of autosomal recessive inheritance of arg24 to termination codon in purine nucleoside phosphorylase gene in a family with a severe combined immunodeficiency patient mechanism of recruitment of wasp to the immunological synapse and of its activation following tcr ligation cutaneous symptoms in primary immunodeficiencies a single ataxia-telangiectasia gene with a product similar to pi-3 kinase immunostimulation with om-85 in children with recurrent infections of the upper respiratory tract: a double-blind, placebo-controlled multicenter study t helper type 2-like cells and therapeutic effect of interferon-gamma in combined immunodeficiency with hypereosinophilia (omenn's syndrome) omenn's syndrome: differential diagnosis in infants with erythroderma and immunodeficiency indications for the use of intravenous gammaglobulin bone marrow transplantation (bmt) for the syndrome of pigmentary dilution and lymphohistiocytosis (griscelli's syndrome) the complex genetics of common variable immunodeficiency selective polysaccharide antibody deficiency in familial digeorge syndrome rag mutations in human b cell-negative scid waspbase: a database of was-and xlt-causing mutations defective activation of the alternative pathway of complement in patients with homozygous c2 deficiency: studies in two unrelated families x-linked lymphoproliferative disease: twenty-five years after the discovery genetic, biochemical, and clinical features of chronic granulomatous disease treatment of chronic granulomatous disease with myeloablative conditioning and an unmodified hemopoietic allograft: a survey of the european experience severe osteopenia in a young boy with kostmann's congenital neutropenia treated with granulocyte colony-stimulating factor: suggested therapeutic approach clinical and immunologic characteristic of healthy children with subnormal serum concentrations of igg2 subnormal serum concentrations of igg2 in children with frequent infections associated with varied patterns of immunologic dysfunction human immune disorder arising from mutation of the a chain of the interleukin-2 receptor laboratory assessment of immune deficiency disorders immunodeficiency presenting as hypergammaglobulinemia with igg 2 subclass deficiency phenotypic and functional alterations of peripheral blood monocytes in neutrophil-specific granule deficiency recent advances in the genetics of primary immunodeficiency syndromes recurrent and chronic upper respiratory infections and chronic otitis media defective expression of cd23 and autocrine growth-stimulation in epstein-barr virus-transformed b cells from patients with wiskott-aldrich syndrome confirmation of x-linked hypogammaglobulinemia with isolated growth hormone deficiency as a disease entity a new kindred with x-linked lymphoproliferative disease treatment of hypogammaglobulinaemia with intravenous immunoglobulin regulation of the polarization of t cells toward antigen-presenting cells by ras-related gtpase cdc42 primary immunodeficiency diseases in norway functional correction of t cells derived from patients with the wiskott-aldrich syndrome (was) by transduction with an oncoretroviral vector encoding the was protein a multi-institutional survey of the wiskott-aldrich syndrome molecular basis of opsonic defect in immunodeficient children associations of mutations in mannose binding protein gene with childhood infection in consecutive hospital series association of low levels of mannan-binding protein with a common defect of opsonisation complement deficiencies in infections with neisseria meningitidis genetic aspects of ataxia-telangiectasia wiskott-aldrich syndrome protein, a novel effector for the gtpase cdc42, is implicated in actin polymerization detection of rag mutations and prenatal diagnosis in families presenting with either t-b-severe combined immunodeficiency or omenn's syndrome two siblings with recurrent infections transient iga deficiency and pathogenesis of infantile atopy a deletion in the gene encoding the cd45 antigen in a patient with scid inherited deficiencies of the terminal complement components structural analysis of low tcr-cd3 complex expression in t cells of an immunodeficient patient embryologic and other developmental considerations of thirty-eight possible variants of the digeorge anomaly results of allogeneic bone marrow transplantation in patients with leukocyte adhesion deficiency brief report: correction of x-linked hyper-igm syndrome by allogeneic bone marrow transplantation genetically determined immunodeficiency diseases: a perspective unexplained opportunistic infections and cd4+ t-lymphocytopenia without hiv infection. an investigation of cases in the united states haematological abnormalities in shwachman-diamond syndrome igg subclasses t cell depleted haploidentical bone marrow transplantation for the treatment of children with severe combined immunodeficiency a family of wasps clinical, immunologic, and genetic features of an autoimmune lymphoproliferative syndrome associated with abnormal lymphocyte apoptosis defective opsonization. a common immunity deficiency immunology in the pediatrician's office independent mutations of the human cd3e gene resulting in a t cell receptor/cd3 complex immunodeficiency genetically determined immunodeficiency disease and malignancy report from immunodeficiency cancer registry complementation cloning of an mhc class ii transactivator mutated in hereditary mhc class ii deficiency (or bare lymphocyte syndrome) severe combined immunodeficiency: a retrospective single-center study of clinical presentation and outcome in 117 patients atypical x-linked severe combined immunodeficiency due to a possible spontaneous reversion of the genetic defect in t cells molecular genetic analysis of x-linked hypogammaglobulinemia and isolated growth hormone deficiency studies of the expression of the wiskott-aldrich syndrome protein new and old immunodeficiencies bone marrow transplantation in severe combined immunodeficiency from a sibling who had received a paternal bone marrow transplant immunodeficiency disorders: general considerations severe combined immunodeficiency in man with an absence of immunoglobulin gene rearrangement but normal t-cell receptor assembly chronic granulomatous disease signaling through zap-70 is required for cxcl12-mediated t-cell transendothelial migration allogeneic hematopoietic stem cell transplantation for seven children with x-linked hyper-igm syndrome: a single center experience stem cell transplants in utero for genetic diseases: treatment and a model for induction of immunological tolerance deficient expression of a b cell cytoplasmic tyrosine kinase in human x-linked agammaglobulinemia mannose-binding lectin: the pluripotent molecule of the innate immune system consensus conference: il bambino con deficit di iga: le caratteristiche cliniche, immunologiche e genetiche per una corretta gestione il bambino immunodepresso th1 t-cell and monocyte defects autoimmune lymphoproliferative syndrome (alps) in a child from consanguineous parents: a dominant or recessive disease? rotavirus infection in infants as protection against subsequent infections regulation of immunoglobulin (ig)e synthesis in the hyper ige syndrome the gene involved in x-linked agammaglobulinemia is a member of the src family of protein-kinases primary immunodeficiency mutation databases v(d)j recombination defects in lymphocytes due to rag mutations: severe immunodeficiency with a spectrum of clinical presentations a cd45 minigene restores regulated isoform expression and immune function in cd45-deficient mice: therapeutic implications for human cd45-null severe combined immunodeficiency analysis of natural killer cells in tap2-deficient patients: expression of functional triggering receptors and evidence for the existence of inhibitory receptor(s) that prevent lysis of normal autologous cells major histocompatibility complex class iii genes and susceptibility to immunoglobulin deficiency and common variable immunodeficiency release of leukotriene c4 in respiratory tract during acute viral infection affinity maturation and class switching evaluation of the child with suspected primary immunodeficiency frequency and severity of infections in day care upper respiratory tract infections in young children: duration and frequency of complications features of transient hypogammaglobulinaemia in infants screened for immunological abnormalities chediak-higashi syndrome: a clinical and molecular view of a rare lysosomal storage disorder clinical and cellular features of "common variable" hypogammaglobulinemia igg subclass levels in the serum of patients with primary immunodeficiency severe combined immunodeficiency due to a specific defect in the production of interleukin-2 clinical patterns and natural history of asthma clinical and laboratory evaluation of complement deficiency in-utero transplantation of parental cd34 haematopoietic progenitor cells in a patient with x-linked severe combined immunodeficiency phox , a third cytosolic component of the activation complex of the nanph-oxidase to contain sh3 domain dominant negative mutation of the hematopoietic-specific rho gtpase, rac2, is associated with a human phagocyte immunodeficiency a prospective cytogenetic study of 36 cases of digeorge syndrome family studies of iga deficiency carrier detection of the x-linked immunodeficiency diseases using x-chromosome inactivation analysis report on a national registry of 368 patients topical imiquimod for molluscum contagiosum in t-cell immunodeficiency mutations in the mu heavy-chain gene in patients with agammaglobulinemia immunological findings in infants with wiskott-aldrich syndrome chromoglycate treatment of patient with hyperimmunoglobulinaemia e syndrome (letter) primary immunodeficiency diseases in latin america: first report from eight countries participating in the lagid. latin american group for primary immunodeficiency diseases bcl10 activates the nf-kappab pathway through ubiquitination of nemo virally induced immunosuppression the xlinked hyper-igm syndrome: clinical and immunologic features of 79 patients brief report: twin boys with major histocompatibility complex class ii deficiency but inducible immune responses neuropsychological profile of children and adolescents with the 22q11.2 microdeletion case definition for surveillance of severe acute respiratory syndrome sars primary immunodeficiency diseases role of tbx1 in human del22q11.2 syndrome high incidence of significant bone loss in patients with severe congenital neutropenia (kostmann's syndrome) key: cord-023143-fcno330z authors: nan title: molecular aspects of viral immunity date: 2004-02-19 journal: j cell biochem doi: 10.1002/jcb.240591009 sha: doc_id: 23143 cord_uid: fcno330z nan mechanisms of t-cell mediated clearance of viruses from the central nervous system are poorly understood, but likely to differ from those employed in the periphery because the cns lacks lymphatic drainage and constitutive expression of mhc class i antigen, and the unique structure of the cns vasculature imposes constraints on access by leukocytes and soluble immune mediators. to study the mechanism by which viruses are cleared from neurons in the central nervous system, we have developed a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (oblv60). grew preferentially in the olfactory bulbs of balbk mice. using in situ hybridization, we found viral rna localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. virus was cleared rapidly from the olfactory bulb between 5 and 11 days. athymic nude mice failed to eliminate the virus demonstrating a requirement for t lymphocytes. immunosuppression of normal mice with cyclophosphamide also prevented clearance. both cd4+ and cd8+ t-cell subsets were important as depletion of either of these subsets delayed viral clearance. gliosis and infiltrates of cd4+ and cd8+ cells were detected by immunohistochemistry at 6 days. the role of cytokines in clearance was investigated using an rnase protection assay for il-la, il-lp, il-2, il-3, il-4, il-5, il-6, tnfa, tnfp and ifny. in immunocompetent mice there was upregulation of rna for il-la, il-lp, il-6, tnfa and ifny at the time of clearance. nude mice had comparable increases in these cytokine messages with the exception of ifny. induction of mhc-i molecules on cells in infected brains was demonstrated by immunohistochemistry in normal and nude mice, suggesting that ifny may not be necessary for induction of mhc-i on neural cells in vivo. luca g. guidotti, kazuki ando, tetsuya ishikawa, lisa tsui and francis v. chisari. the scripps research institute, la jolla, ca 92037 although cytotoxic t lymphocytes (ctl) are known to clear viral infections by killing infected cells, recent studies suggest that they can also suppress the replication of certain viruses by noncytolytic mechanisms. we have examined this area by monitoring the immunopathological and antiviral consequences of antigen recognition by hepatitis b virus (hbv) specific ctl in hbv transgenic mice that express the viral gene products in their hepatocytes. we have shown that intravenously injected ctl rapidly trigger their target hepatocytes to undergo apoptosis, but that the direct cytopathic effect of the ctl is minimal in comparison with the cytopathic effects of the antigen-nonspecific intrahepatic inflammatoly response that they activate. in addition to killing the hepatocyte, the same ctl also downregulate hbv gene expression and completely abolish hbv replication in the hepatocytes that they don't destroy. this noncytolytic antiviral ctl effect is mediated by at least two distinct processes in these animals. first, the ctl cause a quantitative reduction in the steady state content of all hbv mrna species in the hepatocyte, and this is followed by disappearance of all of the corresponding viral proteins in the liver and serum. the ctl initiate this process by secreting ifny and tnfa when they are activated by antigen recognition. since the regulatory effect of the ctl can he prevented completely by prior administration of the corresponding antibodies. nuclear run-on experiments reveal that viral mrna transcription is unaffected despite the profound reduction in hbv mrna content in the liver, suggesting that the ctl-derived cytokines accelerate viral mrna degradation in the hepatocyte. a second noncytolytic antiviral pathway is also activated by the ctl. we have recently shown that hbv nucleocapsid particles, and the replicative hbv dna intermediates that they contain, disappear from the transgenic mouse liver following either ctl administration or partial hepatectomy. the latter of which triggers hepatocellular regeneration without any change in hepatocellular hbv mrna content. these results suggest that preformed hbv nucleocapsid particles may be actively degraded during hepatocyte turnover, and they raise the possibility that similar events might also occur in nondividing hepatocytes that are activated by noncytolytic signals delivered by the ctl. we propose that, in addition to their pathogenetic effect, the comhined effects of the ctl response at die hbv mrna. nucleocapsid and rcplicative dna levels may represent a curative antiviral stimulus during hbv infection. since the virus must contain molecular elements that iespond to these ctl-induced antiviral signals. inactivating mutations at these loci could be very efficiently selected by immune pressure, because a single mutation could abrogate the antiviral effect of a wide spectrum of t cell responses, irrespectrve of epitope specificity. identification of these viral response elements and the intracellular pathways that interact with them may lead to the development of new strategies for antiviral drug design. human fibroblasts infected with hsv are resistant to lysis by cd8+ cytotoxic t lymphocytes (ctl), yet human b cell lines can be efficiently lysed by these ctl. the effect on human fibroblasts is rapid (within 2 hr of infection of cells), occurring before synthesis of mhc class i is altered by virus infection. a recombinant hsv, f-usbmhc, which expresses mouse mhc class i proteins does not render human fibroblasts sensitive to lysis by mouse ctl. mhc class i molecules are retained in the er of hsv-infected fibroblasts i n a misfolded, unstable form and stability of the mhc complex can be restored by addition of exogenous peptides. using a panel of hsv mutants and ad expression vectors we demonstrated that the hsv ie protein icp47 was both necessary and s f i c i e n t to cause retention of class i and icp47 expression in fibroblasts caused the cells to resist lysis by cd8+ t lymphocytes. icp47 is a soluble, cytosolic protein and we have found no evidence of membrane association. therefore, it appears that icp47 inhibits cytosolic stages of the antigen presentation pathway so that antigenic peptides do not reach the er. to date, polyclonal and monoclonal antibodies directed to icp47 have not specifically precipitated any of the previously described components of the antigen presentation pathway and we have not found icp47 associated with tap transporter proteins or proteosomes i n these experiments. the effects of icp47 are being assessed in proteosome and tap transporter assays. gst-icp47 fusion proteins tightly bind a 8.5 kda cytosolic cellular protein which is found in a number of adherent human cell lines but not lymphocytes. the protein has been purified and sequencing is in progress. in addition, radiolabelled icp47 binds to a single cellular protein of =55 kda on ligand blots. these proteins are good candidates as cellular targets of icp47 and as novel components of the antigen presentation pathway. preliminary experiments support the hypothesis that icp47 is very effective i n blocking cd8+ t lymphocyte responses in vivo, perhaps explaining the predominance of cd4+ vs. cd8+ anti-hsv ctl i n vivo. we expect that icp47 may be very useful, not only to elucidate antigen presentation pathways, but also to prevent immune recognition of gene transfer vectors and a s a immunosuppressive agent. susceftibility to polyoma virus-induced tumors is conferred by an endogenous mmtv superantigen. aron e. lukacherl, yupo ma2, john p. carroll2, sara r. abromson-leeman2, joseph c. laning2, martin e. dorf2, and thomas l. benjamin2. idepartment of pathology, emory university school of medicine, atlanta, ga 30322, and 2department of pathology, harvard medical school, boston, ma 02115. susceptibility to tumors induced by mouse polyoma virus varies among inbred mouse strains. we have previously shown that polyoma tumor susceptibility is controlled by products of mhc as well as non-mhc genes. in crosses between mhc-nonidentical strains differing in tumor susceptibility, resistance correlates with dominantkodominant inheritance of the resistant h-2 haplotype. we have observed the opposite pattern of inheritance of susceptibility in crosses between mhc-identical strains. in crosses between the highly susceptible c3wbida mouse and the highly resistant but mhc-identical (h-2k) c57bwcd.i mouse, polyoma tumor susceptibility is conferred by a single autosomal dominant gene, which we have designated pyvs. pyj does not encode cell receptors for the virus, affect viral dissemination or anti-viral antibody responses, or affect intracellular events essential for productive infection or cell transformation by the virus. whole-body irradiation renders cs7bwcd.i mice fully susceptible to polyoma-induced tumors, indicating an immunological basis for this strain's resistance. we hypothesized that p y j encodes an mtv superantigen (sag) that confers susceptibility to c3wbida mice by deleting precursors of polyoma-specific t cells. we found that tumor susceptibility in (c3wbida x c57bwcd.i) x c57bwcdj backcross mice cosegregated with mtv-7. inheritance of mtv-7 showed perfect concordance with absence of peripheral vp6+ t cells. genotyping of backcrossmice using markers of simple sequence repeat polymorphisms flanking mtv-7 showed no evidence of recombination between pyvs and mtv-7. strongly biased usage of vp6 by (a) polyoma-specific cd8+ ctl from virus-infected c57bwcdj mice and by @) cd8+ t cells infiltrating a polyoma tumor in a virus-immune c57bwcd.i host provide further evidence that t cells bearing this mtv-7 sag-reactive vp domain are critical anti-polyoma tumor effector cells. these results indicate identity between p y j and mtv-7 sag, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's t cell repertoire. infection of mice with lymphocytic choriomeningitis virus (lcmv) causes a transient to longlasting immunosuppression dependent upon virus-isolate dose of virus and age, h-2, non h-2, level of cd4+ t cells, of cd8+ t cells and kinetics of neutralizing antibodies of the host. the immunohistological analysis suggests that cd8+ t cell dependent disappearence of marginal zone macrophages of follicular dendritic cells and of virus infected cells in general correlates with immunosuppression. the details of mechanisms responsible for these findings are now being analysed. a role of this cd8+ t cell dependent immunosuppression in the establishment of a lcmv carrier state in immunocompetent mice is suggested by the following experiments: the otherwise slow and low neutralizing antibody response agamst lcmv is accelerated and enhanced by cd8+ t cell depletion at the time of infection, suggesting virus-specific immunopathology being responsible at least partially. the elisa antibody response is not significantly altered under the same conditions but is abrogated if lcmv-specific t cell receptor transgenic mice are infected with high doses of lcmv, indicating, that suppression of the specific antibody response depends upon the relative kinetics of ctl versus antibody responses. whether exhaustion of specific ctl responses is enhanced by similar mechanisms remains to be tested. the role of interleukins of the relative distribution of virus in the mouse and in the various aspects of immunosuppression are now being studied. immunosuppression, caused by cd8+ t cell-dependent immunopathology, may also be operational in hiv infection in humans. such a pathogenesis of hiv-triggered aids could explain several aspects of the disease process not readily fitting the (unproven) conventional idea that hiv is causing immunodeficiency via direct viral pathogenicity. the cellular immunity against two dna tumor viruses (i.e. human adenovirus type 5 (ad 5) and human papillomavirus type 16 (hpv16)) was studied with respect to possible immune escape mechanisms and to the development of ctl epitope based peptide vaccines. after identifying an immunorelevant ctl epitope in the ad 5e1a protein to which ctl clones were directed that could eradicate ad 5e1 induced tumors in nude mice, an amino acid replacement study of this epitope revealed a point mutation that totally eliminated the possibility to recognize the mutant peptide by the ctl clones directed against the wild-type peptide sequence. new viral constructs were made that contained this point mutation and used to transform mouse embryo cells. however, these mutant tumor cells were still immunogenic and ctl clones specific for these mutant tumor cells were shown to react with a peptide derived from the ad 5e1b protein. these ad 5eib specific ctl clones, however, were as effective as the ad 5e1a specific ctl clones in the eradication of ad 5e1 induced tumors in nude animals, indicating that a choice can be made of immunorelevant epitopes to which an immunization strategy could be developed. in addition, we discovered that by supertransfection of ad 5e1 induced tumor cells with the activated ras oncogen the possibility of ad 5e1b specific ctl to recognize the ad 5e1 induced tumors was eliminated whereas the ad 5e1a specific ctl could still kill these tumor cells. this might indicate a new mechanism of tumors to escape ctl. in an hpv16 induced mouse tumor model an immunosubdominant ctl epitope was identified in the e7 protein that could, upon immunization with that peptide,protect mice against a subsequent challenge with hpv16 induced tumor cells. by changing the anchor residues in that peptide an even more immunoprotective peptide could be generated. combined, these data indicated a successful use of a ctl epitope based peptide vaccine in the prevention of hpv16 induced tumors in mice. subsequently this led us to identify relevant ctl epitopes of hpv16, that is highly associated with cervical carcinoma in humans, for the major hla-a alleles (i.e. hla-a *0101, a *0201, a"0301, a*1101 and a *2401). together these alleles cover a majority of all humans. ctl epitopes were identified through peptide-mhc binding assays followed by in v i m peptide immunizations with high affinity binding peptides to induce primary ctl responses and immunogenicity studies in hla-a transgenic mice. thereafter, memory ctl responses were measured in cervical cancer patients against selected peptides. combined, these data led us to develop a ctl epitope based peptide vaccine that could be of use in hpv16 induced cervical cancer patients. a clinical trial for this disease is scheduled to start in the fall of 1994. class ii presentation of an endogenously synthesized glycoprotein. carol s. re is^'.'.^, shirley m. bartido', miriam stein', and stephanie diment3.4, biology department', and center in contrast to class i presentation which is well characterized to use peptide fragements of proteins sythesized in the cytoplasm, exogenously administered experimental antigens enter the class ii mhc pathway through endocytosis. we have been studying the recognition of the glycoprotein of vesicular stomatitis virus (vsv) which can enter either the exogenous or endogenous pathways for presentation to cd4 + t cells. investigations of the intracellular sites involved, the proteolytic processes involved in the epitope generation, will be discussed. the glycoprotein studied in detail is a truncated form of the wt type 1 glycoprotein, termed poison tail (gpt) . expressed with a vaccinia virus vector, the gpt remains endo h sensitive and never becomes endo d sensitive, indicating that it is restricted to the endoplasmic reticulum. gpt is degraded in the er, and w e believe the degradation products include the immunogenic epitopes recognized by a panel of lad and i-ed t cell clones and hybridomas. lmmunofluorescence studies have confirmed the er localization. flow cytometric evaluations s h o w that the gpt never appears on the cell surface, in contrast to the wt g. the peptides generated are not secreted; using an innocent bystander assay, gpt-infected cells are incapable of sensitizing 5'cr-labeled uninfected apc. this contrasts with the rapid ability of supernatants from wt g-vaccinia virus-infected cells to sensitize apc for t cell recognition. investigations of the characteristics of the enzymes contributing to the degradation of the gpt have shown that a reducing environment is essential, as diamide treatment of cells prevents degradation. lysosomotropic drugs (eg. nh,ci and leupeptin) d o not alter the half-life of the protein, but do prevent presentation of the peptides; this is inconsistent with an autophagic component to the proteolysis. ph optima are physiological, as ph8 environment inhibits the enzyme activity. inhibitors of enzyme classes are consistent with a trypsin-like, and not cystein-, cathepsin b-, or chymotrypsin-like class. supported by nih grant al 18083 to csr. (emcv) and mengovirus are related members of the cardiovirus genus of picomaviruses. their rna genomes encode a large polyprotein which is cleaved proteolytically in co-and post-translational reactions to yield all mature viral proteins necessary to establish an infection. although originally thought to be exclusively murine in host range, both viruses actually infect a wide range of mammals. emcv has caused devastating epizootics in captive primates (eg: macaques, chimps and baboons), domestic pigs and exotic zoo collections (elephants, lions and tigers). death, following ingestion of virus-contaminated material, is rapid, and caused by extensive meningoencephalitis and virus-induced damage to the cns. myocarditic lesions are common in older animals. when administered intracerebrally, the ld,, for emcv strain r is about 1 pfu. we are studying the pathogenesis of emcv and mengo with engineered cdna plasmids containing infectious viral sequences. many plasmids contain h'uncated versions of the unusual 5' noncoding homopolymeric poly(c) tract that is a hallmark of these cardioviruses. short poly(c) mengoviruses grow very well in tissue culture but are 106-10'z fold less pathogenic to mice than the wild-type strains. animals receiving sublethal doses of short-tract mengo strains develop high titers of neutralizing antibodies, exhibit potent ctl responses and acquire lifelong protective immunity against challenge with wild-type virus. the genetic stability of the short-tract strains, even upon serial brain passage, mark them as safe, efficacious live vaccines. currently, we believe the poly(c) phenomenon is due to interference by the wild-type virus sequences (long poly(c) tract) with normal cellular cytokine induction mechanisms (ie: ifa and ifp) during the initial stages of animal infection. the targeted cells are probably macrophages, and their singular ability to correctly respond or not respond to poly(c) tract length during the first few hours of infection determines whether an inoculated animal will live (protectively vaccinated) or die. the short-tract viruses probably induce if in the macrophages, and are consequently killed then rapidly cleared from the host in related experiments we've found that attenuated mengo strains can easily carry large heterologous insertions within their genomes, and express these sequences into protein them during replication in animals. the resulting immune response (b cell and ctl) to the chimeras is directed towards the foreign sequences (epitopes) as well as towards the mengo proteins. a chimeric hiv vaccine, a rabies vaccine and an lcmv vaccine have been developed and tested. the lcmv chimera seems especially effective, as a single pfu of this engineered mengo strain, administered orally to a mouse, is sufficient for complete immunogenic protection against intracerebral challenge with wild-type lcmv virus. rsv is the most common cause of serious viral lower respiratory tract disease in infants and children. we have recently renewed our efforts to generate a safe and effective live attenuated rsv vaccine for topical administration that will overcome the deficiencies of previously studied live and non-living rsv vaccines. this vaccine will be a bivalent vaccine consisting of subgroup a and b live attenuated virus components. since the peak incidence of severe disease caused by rsv is in the 2-month old infant, an rsv vaccine will need to be effective when given to 1-month old infants. based on the success of live poliovirus vaccines given early in infancy, it is anticipated that the intranasally administered live virus vaccine will infect and induce a protective local and systemic immune response even in infants with passively acquired maternal antibodies. the main approach that we have taken in this effort to develop the live rsv vaccine is to introduce one or more ts mutations by chemical mutagenesis into a cold-passaged virus (cprsv) that had been partially attenuated by the acquisition of host-range mutations selected by passage in cells of a heterologous host species. we have developed a large set of cprsv subgroup a rs mutants (termed cprs mutants) that contain the host-range mutations selected during cold passage and two or more ts mutations introduced by chemical mutagenesis. these mutants have been evaluated in virro for their level of temperature sensitivity and in vivo in rodents, chimpanzees, and humans. a large set of rsv subgroup b cpts mutants has been similarly produced and evaluated. the immunogenicity and protective efficacy of three candidate live attenuated rsv vaccine strains that represent a specaum of attenuation were evaluated for protective efficacy in chimpanzees. prior to infection some of these animals were given rsv immune globulin by the iv route to simulate the condition of the very young infant who possesses passively-acquired maternal rsv antibodies. the three candidate vaccine strains were immunogenic and induced significant resistance to rsv challenge in both groups of chimpanzees. interestingly, the chimpanzees infused with rsv antibodies prior to immunization were primed more effectively for an unusually high serum neutralizing antibody response to infection with challenge virus than chimpanzees which did not receive such antibodies. this high booster response occurred despite marked reshiction of replication of the challenge virus. the evaluation of two candidate vaccines in seronegative human infants will also be described. rs virus is immunologically interesting for at least t w o reasons: 1) upper respiratory reinfection occurs despite previous exposure and demonstrable immunological memory: 2) humans or rodents previously immunised against virus infection can show enhanced disease during reinfection. others have shown that passive transfer of antiviral antibody either protects against virus infection or has no effect, and there is no evidence of antibody enhancement of disease in vivo. by contrast, t cell immunity appears closely associated with disease augmentation. we have focused on examining the immunological mechanisms of disease enhancement in mice. initial studies showed that transfer of cd8+ cytotoxic t lymphocytes (ctl) causes rapid virus clearance from the lungs of rs virusinfected mice, but also increased disease severity with alveolar haemorrhage and polymorphonuclear (pmn) cell recruitment to the lung. this disease (reminiscent of shock lung) could sometimes be fatal, whereas normal mice recover well from similar doses of rs virus. next, we compared the effects of cd4' and cd8+ t cells, using polyclonal t cells separated immunomagnetically from mixed lines grown in vitro with viral antigen. cd4' t cells were more pathogenic than cd8+ t cells in a dose-for-dose comparison, but that the type of pathology varied depending on the type of cell injected. while testing recombinant vaccinia viruses expressing single rs viral proteins for their ability to protect mice against infection, we observed that animals sensitised t o the major surface glycoprotein g (attachment protein) developed lung eosinophilia after challenge with rs virus intranasally. t cell lines from the spleens of mice sensitised with various recombinant vaccinia viruses were established. those form mice primed with the m 2 (22k) protein were predominantly cd8' ctl, and that produced few cytokines. those from mice primed with fusion protein (f) generated mixed t cell lines with both t h l cd4+ t cells, and ctl. mice primed to g protein gave rise to predominantly cd4' t cells producing th2 cytokines. ln vivo transfer of these cell lines into na'ive rsv infected mice reproduces the patterns of disease seen in mice sensitised in vivo with the respective antigens. the mouse model of rs virus disease therefore has excellent potential for illustrating mechanisms of lung immunopathology. the eye is a complex organ whose function is to transmit light images through different cell and tissue layers and liquid media to a neurosensory retina. elements as could occur when invading pathogens arrive and an inflammatory response with its swelling, plasma protein extravasation, leukocyte infiltration and tissue damage results. inflammatory responses when possible and rely on immune defenses which do not involve tissue distortion and damage. restricting tissue damaging responses is not always effective and the process is best developed in response to agents delivered to locations such as the anterior chamber. where an inflammatory response is initiated which may result in ocular impairment. such herpetic stromal keratitis (hsk) is a common cause of blindness in man. animal model studies indicate that hsk is a multi-step process initiated by virus in an avascular structure. hsk fails to occur in the absence of t cells or replicating virus. disappears several days before a visible inflammatory response becomes evident. evidence will be presented that the secondary agonists which drive the inflammatory response may not be viral antigen(s) per s e . multiple cell types are involved in hsk, with the respective role of functional sets of lymphocytes changing according to the clinical phase of the disease. in addition, nonspecific inflammatory cells such as neutrophils and nk cells also influence the severity of lesions. basically the reaction begins with t cells that produce type one cytokines, particularly ifn-y, dominating the scene, but during remission type 2 cytokines, notably il-10, appear as mechanistically involved. from the use of knockout mice for various immunological parameters, evidence will be presented that numerous mechanisms of pathogenesis may be at play during hsk. damage to corneal tissues in all systems appear to involve tnfo. a second ocular damaging event in which immunopathology is at least partially involved is herpetic retinal necrosis. evidence that this disease may involve the immunopathological role of cd4 t cells and protective effects by cd8' t cells will be presented, as will be suggestions by which the pathological events are mediated at the molecular level. thus it is in the eye's functional interest to limit acute viral infections and live vaccines often confer long-term immunity the nature of t and b cell memory is different. b cell memory is manifested not only by the presence of memory b cells but also by continuous antibody production in contrast, the effector phase of the t cell response i s shortlived and long-term t cell memory is due to the presence of 'quiescent' antigen specific memory t cells that are present at higher frequencies and are able to respond faster upon re-exposure to virus due to increased levels of adhesion molecules in this talk i w i l l present our results on. (i) the bone marrow as a major site of long-term antibody production after acute viral infection, (ii) the role of c d 4 ' t cells and b cells (immune complexes) in maintaining cd8+ t cell memory, (iii) the role offas antigen in regulating t cell responses, and (iv) the efficacy ofvarious antigen delivery systems in inducing long-term t cell memory sendai virus is a natural respiratory viral pathogen of mice. intranasal infection of mice with the virus provokes a virus specific antibody-forming-cell reaction that exhibits a distinct kinetic pattern in the lymph nodes that drain the respiratory tract, in the spleen, and in the bone marrow. the bone marrow afc population is extremely long-sustained, and supports an active humoral response that essentially persists for the lifetime of the infected animal. thus the conventional categories of "primary" and "secondary" response may not apply to the humoral response of mice naturally exposed to respiratory viruses. paradoxically, the population of b cells that reacts most rapidly to sendai virus infection does not itself secrete antibody, but can he demonstrated by the recovery of hyhridomas that secrete "polyspecific" antibodies. the activation of this polyspecific b cell population is, like the humoral response, extremely persistant. viral infection thus sets in train multiple b cell "memory" processes. variation in the rules of development and turnover of different b cell populations constrains the mechanisms that may operate to generate these different forms of memory. establishment and maintenance of t cell memory to respiratory viruses, peter c. doherty, sam hou, christine ewing, david topham, anthony mcmickle, james houston, and ralph tripp, department of immunology, st. jude children's research hospital, memphis, tn 38105. the analysis of the development and memory phases of the cd4+ "helper" n h ) and cd8+ cytotoxic t lymphocyte (ctl) responses to the respiratory pathogens, influenza virus and sendai virus (parainfluenza type 1) have been characterized by a combination of limiting dilution analysis (lda) for determining th and ctl precursor @) frequency and facs separation of lymphocytes with different activation phenotypes. the interpretation at this stage, largely based on the analysis of the ctl response, is that the development phase of t cell memory and the primary response are synonymous. virus-specific ctlp are produced in considerable excess of the numbers required to provide the effector ctl that terminate the primary infection, with only a fairly small proportion localizing to the target organ (the lung) that supports virus growth. even when many of the proliferating ctlp are killed by administration of a small dose (20 mgkg) of the dna-targeted drug cyclophosphamide (cy), there is no indication of immune exhaustion. the cd4+ th response has, at this stage, not been analyzed through the course of the primary infection. use of the lda approach to determine thp frequencies is inherently more difficult, as the "read-out'' is lymphokine production and there is considerable "bystander" activation in these primary responses to respiratory viruses. memory thp and ctlp are characterized initially by the expression of an "activated" phenotype: cd44-high, l-selectin-low, cd49d (vla-4) high. after some months, an increasing proportion of the memory t cells revert to the l-selectin-high cd49d-low form typical of naive ctlp. the change, which is never absolute, seems to occur first with cd49d and the rate varies for different viruses. current experiments are addressing the possibility that intercurrent infection, particularly with the mouse y-herpesvirus 68 which causes persistent infection of lymphoid tissue, may be inducing a switch back to the activated pattern, as a consequence of "bystander" effects, or "low affmity" stimulation via the clonotypic tcr in responding lymphoid tissue. the question of such cross-reactivity and/or exposure to "high lymphokine" environments for the long-term maintenance of memory is also being addressed. to study the factors which regulate the generation and persistence of specific t cell memory we have used model systems utilizing t cell receptor transgenic mice as a source of enriched naive cells which can be either cultured in vitro to generate effector populations or restimulated in adoptive hosts. in either case one can visualize the development of an expanded effector population. we have documented that the proliferation and il-2 production of the naive t cells depends on their activation by apc expressing high levels of co-stimulatory molecules. we find that b7.1 and icam-i as costimulators strongly synergize and that increased t cell receptor triggering can both increase the magnitude of the response and decrease its dependence on costimulation. when cytokines il-4 vs il-i2/ifny are present at the initiation of the response of either cd8 or cd4 cells they dictate that the effectors generated will be polarized either towards il-4 and il-5 secretion or il-2 and ifny secretion, respectively. the fate of the effector population generated and followed in vitro, also is tightly regulated by ag, cytokines and probably by costimulation. cd4 effector cells not re-exposed to ag, produce no cytokines and they die within 3-4 days. effectors restimulated with ag make massive amounts of cytokines, regardless of the presence of cytokines, at low densities of ag and with little dependence on costimulation. when there is little il-2 produced and no cytokines added, effectors die rapidly by apoptosis. however the combination of il-2 and tgfp block apoptosis and support expansion of the effector population which is greatly enhanced by periodic ag stimulation. some conditions favor the reversion of effector-like cells to a more resting memory phenotype and these are being further explored. we have also examined the development and maintenance of memory after transfer of effector cells to adoptive hosts. long-lived polarized memory populations are generated from the polarized effectors and these persist for prolonged period in the absence of apparent ag stimulation. this supports the idea that factors other than antigenic stimulation, present in situ can support the expansion and maintenance of memory cells. the rabies glycoprotein (g) is the only external protein of the virion and is therefore responsible for any interaction that rabies makes with the host cell during the first steps of the virus cycle. the g protein is also the target of neutralizing antibodies. there are around 450 trimers of g at the virion surface which constitute the spikes visible by electron microscopy. upon exposure to slightly acidic ph, the glycoprotein undergoes a conformational change which results in ion er and less regular spikes. strikingly and quite differently from influenza hemagglutinin, this conformational change is reversible: if the p d is risen back to 7.0, the s ikes re ain their neutral configuration (1). probably as a consequence, the viral infectivity is totally preserved after an exposure of 2 hours at p 8 6.4 an cf 37t, which induces the conformational change, followed by an incubation at neutral ph. since the conformational change is reversible, there is a ph-dependant equilibrium between the native and the low-ph conformation: the higher the ph, the more spikes are in their native configuration. two main antigenic sites and several minor sites have been identified on the native rabies glycoprotein (2). specific amino acids belonging to each of the two major antigenic sites are important or essential for viral virulence. for instance a lysine in position 198, which is part of antigenic site 11, is important, although not essential, for the viral virulence. similarly, the arginine 333, which belongs to antigenic site 111, is essential for pathogenicity while dispensable for multiplication in cell culture (reviewed in 3). viral strains mutated at arginine 333 have lost the capability to penetrate certain categories of neurons, suggesting that this mutation affected the recognition of specific receptors or subsequent interactions necessaly for the penetration of the virus at nerve terminals. therefore the two main antigenic sites are regions of the glycoprotein which also interact specifically with neurons in the animals. we have found that neutralization requires the fixation of at least one or two igg for every three spikes, irrelevant of the anti enic site recognized by the antibody (4). most neutralizing antibodies recognize conformational epitopes which are accessible on the native configuration of the protein. some epitopes remain accessible also on the acidic configuration while others are not. in addition, a minority of antibodies recognize epitopes which are only accessible on the acidic conformation. this is not unlikely in view that each spike has a certain probability to undergo a conformational change, even at neutral ph. in consequence the surface of the virus probably fluctuates and g epitopes which are not accessible on the native glycoprotein could be transiently exposed. conformational flexibility at neutral ph and physiological temperatures has also been observed for poliovirus (5). structural flexibility of external proteins could have important implications in virus-host interactions. katpus, norlhwestern university medical school, chicago, il 6061 1 theiler's murine encephalomyelitis viruses (tmev) are endemic enteric pathogens of wild and colony-reared mice. lntracerebral inoculation of susceptible mouse strains leads to a chronic, progressive inflammatory demyelinating disease of the central nervous system (cns) characterized clinically by an abnormal gait, progressive spastic hind limb paralysis and urinary incontinence, and histologically by parenchymal and perivascular mononuclear cell infiltration and demyelination of cns white matter tracts. demyelination is related to persistent cns viral infection. due to the similarity in clinical and histological presentation, tmev-induced demyelination is considered to be a highly relevant model of multiple sclerosis (ms). our current interests are in determining the phenotype, fine specificity, lymphokine profile and tcr usage of cns-infiltrating cells involved in the effector stages of tmev-induced demyelination. based on a variety of experimental evidence, it is clear that demyelination induced in sjuj mice by infection with the bean strain of tmev is a thl-mediated event: (a) disease induction is suppressed in t cell-deprived mice and by in vivo treatment with anti-i-a and anti-cd4 antibodies; (b) disease susceptibility correlates temporally with the development of tmev-specific, mhc-class il-restricted dth responses and with a predominance of anti-viral lgg2a antibody; (c) activated (le., ll-2rc) t cells infiltrating the cns are exclusively of the cd4+ phenotype, and (d) proinflammatory cytokines (ifnq and tnf-p) are predominantly produced in the cns. we have mapped the predominant thl epitope on the virion to amino acids 74-86 of the vp2 capsid protein. a thl line specific for vp274-86 exacerbates the onset of demyelination in recipient mice infected with a suboptimal dose of tmev. tmev-infected sjuj mice fail to exhibit peripheral dth and t cell proliferative responses to the major myelin proteins, mbp and plp, and pre-tolerization with neuroantigens has no affect on the incidence or severity of tmev-induced demyelinating disease, whereas neuroantigen-specific tolerance prevents the induction of relapsing experimental autoimmune encephalomyelitis (eae). in contrast, tolerance induced with intact tmev virions specifically anergizes virus-specific thl responses and results in a dramatic reduction of the incidence and severity of clinical disease and cns dernyelination in sjuj mice subsequently infected with tmev. these results have important implications for a possible viral trigger in ms as they indicate that chronic demyelination in tmev-infected mice is initiated in the absence of demonstrable neuroantigen-specific autoimmune responses and are consistent with a model wherein early myelin damage is mediated via primarily by mononuclear phagocytes recruited to the cns and activated by pro-inflammatory cytokines produced by tmev-specific thl cells. the concept that prions m novel pathogens which are different fium both viroids and viruses has received increasing support from many avenues of investigation over the past decade. enriching fractions from syrian hamster (sha) brain for scrap= prion infectivity led to the discovery of the prion protein 0. prion diseases of animals include scrapie and mad cow disease; those of humans present as inherited, sporadic and w o r n neurodegenemive disorders. the inhecited human pion diseases m genetically linked to mutatim in the prp gene that result in non-conswative amino acid substitutions. transgenic v g ) mice expressing both sha and m o w @lo) prp genes were used to demonstrate that the "specie9 bank?' for -pie prions resides in the primary structure of pip. this concept was strengthened by the results of studies with mice expressing chimeric mdsha transgenes &om which "artificial" prions have been synthesized. similar chimeric mdhuman (hu) rp transgenes were constructed which differ from m o w by 9 amino acids between residues 96 and 167. au of the tg(mhu2m) mice developed neurologic drsease -200 days after inmulation with brain homogenates from three patients who died of creutzfeldt-jakob disease (cjd). inoculation of tg(mhu2m) mice with cjd prims produced mhu2mprpsc, inoculation with mo prions produced moprw. ihe patterns of meluzmprpc and mom% accumulation in the brains of tg(mhu2m) mice wen differenl about 10% of tg(huprp) mice expressing huf" and non-tg mice developed neurologic diseane >500 days after inoculation with cn, prions. the different susce@uies of tg(hw) and tg(mhu2m) mice to human prions indiate that additional species specific factors such as chaperone proteins are involved in prion replicaton. diagnosis, prevention and treament of human @on diseases should be faciliated by tg(mhu2m) mice. in other sindies, tg mice were compared expressing wt and mutant moprp. overexpression of the wtmoprp-a aansgene -8-fold was not deleterious to themiw but it did shorten scrapie incubation times from -145 d to -45 d after inoculation with murine m p i e pnons. in contrast, overexpression at the same level of l morp-a transgene mutated at codon 101 (corresponding to codon 102 in hurp) pmdnced spontaneous, fatal neurcdegeneration between 150 and 300 d of age in two lines of tg(mohp-pio1l) mice designated 2866 and 2247. genetic crosses of tg(moprp-p101l)2866 mice with gene targeted mice lacking both rp alleles ( p m -p ) produced anhats with a highly synchronous onset of illness between 150 and 160 days of age. the t g~o p r p -p l o l l ) 2 8 6~~ mice had numerous prp plaques and widespread spongiform degeneration in contrast 10 the tg2866 and 2247 mice that exhibited spongifonn degeneration but only a few prp amyloid plaques. another line of mice designated tg2862 overexpress the mutant transgene -32-fold and develop fatal neurodegeneration behveen 200 and 400 d of age. tg2862 mice exhibited the most severe spongiform degeneration and had numerous, large pip amyloid plaques. while mutant moprpccploll) clearly produces neurodegeneration, wtmoprpc profoundly modifies both the age of onset of illness md the mumpathology for a given level of transgene expression. our tidigs and those from other smdies suggest that mutant and wtprp interact, phaps through achaperone-like protein as noted above in sndies of tg(mhu2m) mice, to modify the pathogenesis of the dominantly inhe&ed prion diseases. anton, heidi t. link, and jonathan w. yewdell, laboratory of viral diseases, niaid, bethesda, md 20892-0440. cd8' lymphocytes (tcd8+) play an important role in host immunity to viruses and other intracellular parasites. virus-specific tcdi+ recognize mhc class i molecules in association with peptides of 8 to 10 residues derived from viral proteins. this presentation will focus on how and where antigenic peptides are generated by cells. to begin to characterize the nature of proteases involved in the generation of antigenic peptides from cytosolic proteins, we used a panel of recombinant vaccinia viruses expressing different forms of influenza virus nucleoprotein (np). we found that the efficiency of generation of two np peptides is related to the metabolic stability of the source gene product. there has been considerable speculation that such short lived proteins are degraded by proteasomes in a ubiquitin-targeted process. our observations, however, call into question the importance of ubiquitin targeted-proteolysis in generating antigenic peptides from exogenously provided or endogenously synthesized viral proteins. we also examined the extent to which antigenic peptides can be generated in the endoplasmic reticulum (er). we found that antigenic peptides could be produced from short precursors (17 residues) hut not from a number of full length proteins (influenza virus hemagglutinin, np, ovalbumin) that are targeted to the er by a nh2-terminal signal sequence. peptides were generated much more efficiently from the cooh-terminus of the 17 residue precursor than from the nh2-terminus. these findings indicate that the er has a much more limited capacity than the cytosol to generate antigenic peptides, but that er proteases (particularly aminopeptidases) could perform the final proteolytic steps in the generation of class i binding peptides from precursors imported from the cytosol by tap, the mhc encoded peptide transporter. potential advantages of synthetic peptide or engineered recombinant vaccines are that they can be limited to contain only the specific antigenic determinants for desired responses without other determinants that elicit unwanted responses, and that the sequences of the determinants themselves can be modified to enhance potency or breadth of crossreactivity. however, they can have the disadvantage that any single determinant may be presented by only a limited selection of major histocompatibility complex (mhc) molecules of the species. to overcome the problem of mhc polymorphism, we have identified determinants presented by multiple mhc molecules, and have also located multideterminant regions of the hiv-1 envelope protein that contain overlapping determinants each presented by different class i1 mhc molecules, so that the whole multideterminant region is presented by multiple mhc molecules of both mouse and human. we have made use of "cluster peptides" spanning these multideterminant regions of the hiv-1 envelope to provide help for neutralizing antibody (ab) and cd8+ cytotoxic t lymphocyte (ctl) responses to peptides attached to these helper regions. these synthetic peptide vaccine constructs containing the p18 peptide from the v3 loop of hiv-1 iiib or mn, elicited both neutralizing ab and ctl in multiple strains of mice. the cluster peptides inducing helper t cells were essential for elicitation of ab and ctl to the p18 segment of both iiib and mn strains of hiv-1 in mice of several mhc haplotypes. several adjuvants were compared for their ability to elicit both ctl and ab simultaneously, without one response inhibiting the other. a single formulation in incomplete freund's adjuvant (ifa) could elicit all 3 responses, neutralizing ab, ctl, and th1 helper cells. the ctl specific for the mn strain p18 peptide crossreacted with strains sc, sf2,2321, and cdc4. the peptides in f a also elicit high titers of antibodies in rabbits. boosting was found to enhance ctl responses as well as ab responses. these constructs are being prepared for a human immunotherapy trial. these vaccine constructs are potent and also avoid sites on gp160 that are known to elicit enhancing antibodies or autoimmune responses that might conmbute to disease pathogenesis. however, we can potentially improve on these by tinkering with the internal structure of the individual epitopes. we have found that replacing a negatively charged glutamic acid residue with an uncharged amino acid in one of the helper determinants makes it 10 to 100-fold more potent in binding to the class i1 mhc molecule and in eliciting murine helper t cells that still recognize the natural hiv-1 sequence. thus, such a modified peptide should be more potent as a vaccine, while retaining the ability to elicit t cells that will respond to hiv proteins that of course do not have the altered sequence. we are currently mapping the critical residues for presentation of one of these peptides by human hla-a2, with the intent of developing modified peptides that will be more potent as components of a human vaccine. thus, by leaming how these peptides bind to mhc molecules and tcell receptors, we can design internally modified determinants to construct more potent or more crossreactive second generation vaccines. we are testing these vaccine approaches in a mouse model in which mice can be protected against tumor cells expressing hiv proteins as would an hivinfected cell. dna vaccines, comprised of non-replicating plasmids encoding viral proteins, are capable of generating protective immunity in animal models of several viral diseases. in preclinical models of influenza infection, reduced viral shedding was observed in dna-vaccinated ferrets after challenge with the human clinical virus strain, a/georgia/93. cross-strain protection was conferred by dna encoding the major internal proteins (nucleoprotein, np, and matrix, m1) and the surface protein haemagglutinin (ha) from the antigenically-distinct previous virus strains, a/beijing/89 and a/hawaii/91. this protective efficacy was greater than that seen by immunization with the widely-used clinical vaccine composed of killed a/beijing/89 virus. thus, compared to a killed virus vaccine, protection seen with the dna vacane against a drifted virus strain was greater. we previously demonstrated that immunization of mice with np dna generated mhc class i-restricted cytotoxic t lymphocytes. mice likewise were protected from death and morbidity following cross-strain challenge'. ha dna vaccines generated neutralizing antibodies in mice, ferrets and primates, and provided protection in m i d and ferret models of influenza. in animal models of other viral diseases, immune responses and protection against viral challenge have been seen after immunization with dna encoding viral proteins. dna encoding hiv gp120 generated ctl and neutralizing antibodies in monkeys. antigen-specific proliferative responses and, in mice, secretion of high levels of yifn relative to levels of il-4, months after immunization were also observed . immunization of rabbits with dna encoding l1, the major viral capsid protein of cotton tail rabbit papilloma virus (crpv), resulted in neutralizing antibodies and protected against the development of warts after inoculation with crpv. mice immunized with dna encoding the glycoprotein gd from herpes simplex virus type 2 (hsv-2), developed neutralizing antibodies and were protected from death when subsequently challenged with hsv-2. dna vaccines were protective in animal models of various viral diseases. neutralizing antibodies, helper t cells (thl) and cytotoxic t cells were generated. cross-strain protection due to cellular immunity was demonstrated. ' science, 1993 2593745-1749 , 2dna cell biol, 1993 the profile of a neurovirulent virus is determined by its mechanism of entry into the cns (neuroinvasion), the type of cns cell in which it replicates (neurotropism) and its ability to cause pathologic effects in the brain (neurovimlence). whereas neuroectodermal cells, especially neurons, are the target cells of most neurovirulent viruses, the main target cell in the brain for siv and other lentiviruses is the macrophage. infection in, expression of viral antigens by and products of siv replication exported from these cells result in inflammation and degenerative changes in the brain and concomitant loss of neurons. siv strains that are mainly t-cell tropic cause transient activation of t-cells and during this period, infected t-cells cross the blood brain barrier and localize in the brain causing persistent hut minimally productive infection and minimal neuro pathologic effects. viral proteins but not virions are produced continuously. by virtue of the tropism of the virus for cd4 t cells, many infected animals eventually become immunosuppressed and develop aids, but not classical ueurological disease. viruses which are macrophage tropic invade the brain presumably also in t lymphocytes and the viruses infect macrophages in the brain. however, productive virus replication is minimized by antiviral cd8 t cells which suppress (kill?) all virus producing cells throughout the body, including the cns. productive virus replication in brain macrophages and accompanying inflammatory changes develop only when cd8 cells fail i.e. after profound immunosuppression sets in. the neurological disease that results from productive virus replication in macrophages in the brain therefore depends on presence of an appropriate macrophage-tropic viral phenotype invading the neuropil and development of immunosuppression in the host. the neurological disease could therefore be defined as one of the aids syndromes. the adenovirus (ad) early transcription region (e3) codes for more than 7 polypeptides, four of which have already been shown to alter the immune response to ad infection. the amount of the class i major histocompatibility complex (mhc) on the plasma membrane can be reduced by the binding of the ad e3 gpl9k protein to the mhc heavy chain, which prevents transport of the complex out of the endoplasmic reticulum. this process interferes with presentation of viral peptides to cytotoxic t lymphocytes. cytolysis by tumor necrosis factor-o (tnf) is inhibited by 4 distinct viral polypeptides, 3 of which (the ad e3 14.7k or the complex of the 10.4k and 14.5k proteins) are coded in the e3 region. the e3 polypeptides are translated from a family of viral mrnas, that are synthesized from a single viral promoter and processed by alternative splicing. we have studied the functions of the e3 polypeptides in several murine models. the goals of these experiments were to determine the effects of the ad e3 polypeptides in acute and persistent viral infections as well as in a transplantation model designed to measure whether these viral immunoregulatory proteins would abrogate allogeneic graft rejection. in a vaccinia virus (v.v.) pneumonia model, in which the isolated ad e3 14.7k or ad e3 gpl9k genes were inserted into the v.v. pathogen, the ad anti tnf polypeptide increased viral virulence but the ad anti mhc had no effects. in addition to manipulating the ad e3 genes in viral constructs, several transgenic mouse lines containing the ad e3 genes have been constructed for these experiments. the e3 genomic dna behind the rat insulin promoter (rip) has been used to generate transgenic animals. islets from rip-e3 transgenic animals (h-2b'd) have been transplanted allogeneically to h-2d recipients and remained viable, secreting insulin until the end of the experiment at 94 days; in contrast, control nontransgenic islets of the same genotype were rejected by 21-28 days. the e3 genes behind the native e3 promoter have been inserted into mouse embryos to generate transgenic animals, and the expression of the transgene monitored in multiple organs. the e3 promoter of the transgene is responsive to stimulation by the ad e1a following infection with an e3 minus ad 7001 and can also be upregulated by administration of bacterial lipopolysaccharide. the effects of this transgene on ad pathogenesis are currently being studied. thus, these viral immunoregulatory genes have been shown to alter viral pathogenicity during acute infection and to downregulate the host immune response sufficiently to permit islet cell transplantation. these results on manipulating the ad e3 genes for the control of the host immune response also have implications for designing adenovirus vectors for gene therapy. "emerging" infections can be defined as infectious diseases that either have newly appeared in the population, or that are rapidly increasing their incidence or expanding their geographic range. recent viral examples include aids, ebola, and hantavirus pulmonary syndrome (fnst identified in a 1993 outbreak in the southwestern u.s.). emerging viral infections show a number of common features. most "new" viruses derive from existing viruses that move into new areas or acquire new hosts ("viral traffic"). many are zoonotic (originating from animal sources) (even pandemic influenza appears usually to be a reassortant originating in wildfowl). ecological or environmental changes (either natural, or, often, man-made) may precipitate emergence of new diseases by placing people in contact with a previously unfamiliar zoonotic reservoir or by increasing the density of a mtud host or vector of a pathogen, increasing the chances of human exposure. upon introduction into a human population from a zoonotic reservoir, the newly introduced virus may cause localized outbreaks of disease. some may show rapid variation and evolution upon introduction, and some evidence suggests a role for immune selection in this process. a few viruses (such as hiv) may succeed in establishing themselves and disseminating in the human population, becoming truly "human" infections. human activities can also play an important role in establishment and dissemination. migrations from rural areas to cities, now an accelerating worldwide phenomenon, or other displacements, can introduce remote viruses to a larger population; the virus may then spread along highways and (globally) by air travel. the development of an effective system of surveillance and rapid response is essential, but resources for this are presently inadequate. vaccine development, production, and deployment problems also need to be addressed. immunopathology may be a key feature of many of these infections, a number of which manifest as hemorrhagic fevers. many of the life threatening complications are due to increased vascular permeability. the resemblances to septic shock suggest that cytokines (such as tnf) are likely to be important in the pathogenesis of these infections. the response of cells, such as the macrophage, that induce or synthesize key cytokines, may be an important element, and the ability to infect these cells may be one common denominator. why some viruses elicit this response, while other closely related viruses do not, cannot yet be predicted from molecular data. better understanding of these aspects of the immune response should lead to additional therapeutic strategies. (supported by nih grant roi rr03121.) genetic approaches have been used to detect and characterize numerous previously unidentified hantaviruses. puumalarrospect hilvsin nombre-like viruses or virus variants are present throughout north and south america, europe and russia. several of the american viruses identified are associated with the newly recognized hantavirus pulmonary syndrome (hps), a severe respiratory illness with high mortality. the genetic relationships of these and previously characterized hantaviruses have been studied by phylogenetic analysis of the nucleotide sequence differences located in pcr bgments amplified from the g2 encoding region of the virus m segments. the relationships observed are consistent with a long-term association of viruses with their primary rodent reservoirs and suggestive of coevolution of host and virus. a sin nombre virus isolate is now available and its genetic characterization has been completed. various virus antigens have been expressed and are being used to probe the interaction of the virus with the host immune system. hantaviruses cause significant morbidity and mortality throughout the world. more than 200,000 cases of hemorrhagic fever with renal syndrome (hfrs) are reported annually in asia, europe and scandinavia. the etiologic agents of hfrs are hantaan, seoul and puumala viruses, with hantaan virus causing the most severe form of the disease. in 1993, a new hantavirus was discovered in the united states (initially termed four comers virus), and was identified as the etiologic agent of hantavirus pulmonary syndrome (hi's). vaccines for hantaviruses are not readily available, although a number of inactivated viral preparations have been made and tested in asia. recurrent problems with inactivated hantaviral vaccines have been lot to lot variability, the need for repeated immunizations, and their inability to elicit long-lasting neutralizing antibody responses in immunized volunteers. because of such limitations on traditional vaccine development for these viruses, as well as the viruses' hazardous nature and slow, low-titer replication in cell culture, we used a recombinant dna approach to develop a vaccine for i-ifrs. our vaccine is a recombinant vaccinia virus expressing the m segment of hantaan virus under control of the vaccinia virus 7.5 k promoter and the s segment under control of the 11 k promoter. the m segment, which encodes the g1 and g2 envelope proteins, was included because of our findings that: (1) immunization with vaccinia or baculovirus-expressed g1 and g2 induced a neutralizing and protective immune response in hamsters; and, (2) neutralizing antibodies to g1 or g2 could passively protect hamsters from challenge with virulent virus. the s segment, which encodes the nucleocapsid protein (n), was included because of our finding that hamsters immunized with baculovirus-expressed n also were protected from subsequent infection. although the protective immune response to n is probably cell-mediated, the importance of such a response is presently not well defined. assessment of our vaccine in preclinical studies, indicated that immunized hamsters developed neutralizing antibodies and were protected from displaying viral antigen in their lungs after challenge. in a phase i, dose escalation, clinical study, the vaccine induced neutralizing antibodies in individuals immunized subcutaneously with approximately lo7 pfu of the recombinant virus. in addition to humoral responses, immunized volunteers developed a cell-mediated immune response as indicated in lymphocyte proliferation assays. larger clinical studies, including alternate routes or booster immunizations, are planned. based on these studies, we anticipate that the vaccine will be efficacious for preventing hfrs caused by hantaan and the antigenically closely related seoul virus. we are studying the cross-protective properties of this vaccine with more distantly related hantaviruses such as puumala virus. although we expect this vaccine to be safe as well as effective, we also are investigating the use of more attenuated pox-viruses as vaccine vectors. infection of mice with lymphocytic choriomenigitis virus (lcmv) results in a profound expansion in the number of spleen cd8 t cells and in the induction of virus-specific ctl activity. thereafter, the cd8 t cell number declines, and the ctl activity diminishes, though the frequency of lcmvspecific precursor ctl per cd8 cell, as assessed by limiting dilution assays (lda), is remarkably stable throughout long-term immunity. the decline in t cell and total spleen leukocyte number at the late stages of acute infection is associated with high levels of apoptosis, as detected by the in situ nucleotidyl transferase assay. apoptosis occurred in both the t cell and b cell populations, with the b cells dying in clusters. this apoptosis was also seen in tfansgenic mice ectopically expressing bcl-2 in the t and b cells and in c57bl/6 ipr/@r mice, which have a mutation in the fas gene. t cells from the infected animal underwent apoptosis in vitro when stimulated through the tcr with anti-cd3, thereby explaining some of the immunosuppression seen during acute viral infections. memory cells persisted for over a year and could be found in blast-size cell populations. challenge of lcmv-immune mice with either pichinde virus, vaccinia virus, or murine cytomegalovirus led to the reactivation of the lcmv-specific ctl response. lda analyses showed unexpectedly that these heterologous viruses crossreacted with subpopulations of lcmv-specific memory t cells. this memory t cell response to virus from an earlier infection was associated with enhanced immunopathology and enhanced clearance of virus during a heterologous virus challenge. over the course of the acute infection, ctl specific for the second virus were preferentially expanded over the crossreactive ctl, and after the acute infection, when the t cell response had subsided, ctl memory to the first infection had decreased. there is therefore a network of memory t cells which contribute to and are modulated by infections with putatively unrelated viruses, and apoptosis plays a homeostatic role in the course of these t cell responses. immune responses to live attenuated retroviral vaccines, r. paul johnson*?, cara wilsont, kelledy mansons, michael wyands, bruce walker?, ronald c. desrosiers* *new england regional primate research center, southborough, ma 01772 thfectious disease unit, massachusetts general hospital, boston, ma 021 14 â§tsi/mason, worcester, ma immunization of rhesus macaques with live attenuated retroviruses deleted in nef can induce protective immunity against challenge with pathogenic siv. development of protective immunity in these vaccinated animals occurs only after several months of infection, with maximal protection observed after one year. the specific immune responses responsible for mediating protection have not been defined, and little is known about the cellular immune responses in animals vaccinated with these live attenuated retroviruses. we have analyzed cellular and humoral immune responses in rhesus macaques and chimpanzees infected with live attenuated retroviruses. siv-specific neutralizing antibodies were present in vaccinated animals, but did not clearly correlate with protection against challenge. ctl specific for envelope and gag were identified in vaccinated macaques studied 12 or more months after vaccination. quantitation of siv-specific ctl activity in one of these animals using limiting dilution analysis revealed a relatively high precursor frequency of cytotoxic t lymphocytes, up to moo0 for gag and 1/8500 for envelope. cd8+ lymphocytes obtained from vaccinated macaques were also able to suppress siv replication in autologous cd4+ cells. suppression mediated by unstimulated cd8 autologous cells was maximal when cells were in direct contact with siv-infected lymphocytes, but cd8+ cells activated by an anti-cd3-specific monoclonal antibody were able to release. a potent soluble inhibitor of siv replication. in contrast to the relatively vigorous ctl response present in vaccinated macaques, we were not able to detect consistent ctl activity in chimpanzees infected with a hiv-1 molecular clone (nl43) or attenuated viruses at periods up to one year after infection, despite the use of a variety of stimulation techniques. proliferative responses to hiv p24 and gp160 were observed in chimpanzees infected with n u 3 and attenuated variants. although the relative contribution of these immune responses to protective immunity is not known, the relative vigor of the cellular immune responses observed in vaccinated macaques suggest they may play a role in mediating resistance to challenge. obiectives: to analyze the magnitude and specificity of the ctl response to hiv-1, and to determine the tcr usage by clonal ctl responses in infected persons, including persons with documented infection of up to 15 years with cd4 cells > 500/mm3. methods: hiv-l-specific ctl activity was evaluated in pbmc as well as in pbmc stimulated in vitro with hn-1 infected autologous cd4 cells, using target cells infected with recombinant vaccinia viruses expressing hn-1 proteins. ctl epitopes recognized by these individuals were determined using cloned effector cells. quantitative cultures were performed by endpoint dilution, and viral quantitation was determined by qc-pcr tcr analysis was performed by pcr, using both family-specific primers and anchored pcr, followed by sequencing. sequence analysis of ctl epitopes in autologous viruses was determined by pcr amplification and sequencing. clonal frequency was analyzed in pbmc by oligonucleotide probe to the cdr3 region of the tcr. studies performed in long-term non-progressing persons indicate the presence of a vigorous and broadly directed ctl response. detailed epitope mapping in a person infected for 15 years, who by qc-pcr had <i67 viral rna molecules per ml and who tested negative by bdna assay, revealed ctl clones directed at six different epitopes, with detectable recognition of three of these epitopes using freshly isolated pbmc as effector cells. the ctl clones were broadly reactive against many hown hiv-1 variants, and both the p17 and p24 epitopes were cross reactive with siv. sequence analysis of autologous virus within the three dominant ctl epitopes revealed the lack of immune escape variants in pbmc, plasma, and virus obtained from coculhue. supernatant. analysis of tcr usage by clones to defined epitopes in persons at different disease stages revealed heterogeneity in tcr usage and heterogeneity in recognition of virus variants withii these epitopes. analysis of clonal frequency using an oligonucleotide probe to the cdr3 region of the tcr in an individual with a vigorous response to gp41 suggested that approximately 6% of circulating t cells were a single hiv-l-specific clone. conclusions: our results indicate that cytotoxic t lymphocytes are part of the immune response in persistent non-progressive hiv-1 infection, and that prolonged infection can occur without the development of viral immune escape variation within defined ctl epitopes. in addition, vigorous circulating ctl responses can occur in the setting of an e x m e l y low viral load. heterogeneity in the ability of clones from different individuals to recognize sequence variation within ctl epitopes may be important in the pathogenesis of infection. ctl to conserved epitopes or ctl which are broadly reactive with multiple viral mains may be important in conmbuting to maintenance of the asymptomatic state. factor-i (irf-1) gene is essential for the transcriptional activation of inducible nitric oxide synthase (nos) in murine macrophages. it also plays an important role in the activation of interferon and il-6 and il-7 receptor genes. to investigate the role of irf-1 related systems of defense against viral myocarditis, the transgenic mice were innoculated with l o 5 pfu of coxsackie virus 83 (cvb3). homozygous irf-1 (+) and heterozygous irf-1 (+/-) mice were randomized into groups to determine heart and spleen viral titres, cardiac pathology and mortality rates at early and late stages of the disease. the mortality was dramatic in the irf-1 -imice. the hearts of the homozygous animals also had increased mononuclear cell infiltration, necrosis and viral replication. we condude that irf-1 regulated gene products such as inos and ifn are essential for the early defense against cvb3-induced myocarditis by attenuating viral replication and inflammatory responses. the flu season is a yeariy problem, especially in the elderly population. annual vaccination lacks broad protection and antibody titers induced in the elderly are frequently lower than those acheived by younger vaccinees. using the mouse model, we are investigating the differences in immune response between old and young mice to influenza vaccine and at the same time testing the ability of a potent adjuvant to boost the response in the the elderly. our studies show defects in the immune response of the elderly. antibody titers after immunization are lower in the elderly for both seronegative and seropositive mice. cmi studies show that helper t-cell response (proliferation) to in-vitro antigen stimulation is also decreased in the elderly. facs analysis of wbcs show the elderly mice have lower leukocyte and lymphocyte populations, lower cd4 and b-cell populations and a higher proportion of macrophages and cd8 cells than young mice. it was also noted that the young mice had very consistent wbc profiles while the elderly profiles had greater variability. serum cytokine studies show lower levels of il2, il4 and il5, but higher levels of il6 and ifng in the elderly as compared to the young mice. the addition of mf59 to the immunization increased the antibody titers of both naive and seropositive young and old mice. the helper t-cell response of the young mice was unaffected, while the response of the elderly group was increased. cytokine profiles show an increase in il2, il4 and il5 levels for both young and old, while il6 and ifng levels went up for young animals but remained constant for the elderly mice. coxsackievirus 8 3 (cvb3), a member of the picornavirus family, is a common cause of acute or chronic human myocarditis. however, whether cell damage results primarily from direct virusmediated effects or from immune-mediated destruction of the heart tissue during the infection remains unclear. we used a cardiovirulent variant of cvb3 which is able to infect several strains of mice. the infection results in a disease which strongly implicates immunopathogenetic mechanisms in myofiber necrosis. to characterize the role of the immune system in this disease we used various transgenic knockout (ko) mice with different immune defects. cvb3 is able to infect normal c57bl/6 mice and immunocompromised (cd4 ko and 62m ko) mice and causes a lethal disease after i.p. injection in a viral dose dependent manner. we found elevated ld5os in immunocompromised mice, which also die slightly later than normal mice. this virus replicates in the hearts of all mice used with maximal titers 3-5 days pi.. large pathological changes were detectable only in heart tissue of cd4 ko mice, and were maximal at 1-2 weeks after infection. these results suggest the possibility that cd4' t cells may limit tissue damage by an as yet undefined mechanism; the roles of cd4+ and cd8' t cells in this disease are under investigation. research supported by a grant of the daad, germany. mice lacking p56lck are resistant to coxsackievirus b3 induced heart disease. tamara martino, karen aitken, joseph penninger, jan0 nikhilanandhan, tak mak, fayez dawood, wen-hu wen, lily wee, martin petric, and peter liu, the toronto and princess margaret hospitals, university of toronto, canada. coxsackievirus 83 (cvb3) causes severe heart disease in adult mice, and is a useful model for the human cardiac conditions of myocarditis and dilated cardiomyopathy. in this study, we have demonstrated that transgenic mice lacking the t-cell signalling molecule p56kk are resistant to cvb3 induced heart disease. the virus replicates in the heart, and is cleared from the myocardium at a rate comparable to "normal" littermates, but there is only minimal mortality, or damage to the cardiac tissue. however, in ick-deficient mice depleted of natural killer (nk) cells prior to viral infection, there is increased cvb3 replication, and some infiltration by mononuclear cells in the myocardium. to further elucidate the role of the immune system in cvb3induced heart disease, cytokine expression in the hearts of cvb3 infected normal, ick-deficient, and nk-cell depleted mice is under investigation. we conclude that t-cell activation is critical to produce substantive myofiber necrosis after cvb3 infection, that nk-cells play an fundamental role in protecting the mice from severe virus infection, and that ick transgenic mice serve as an important model for understandina the dathogenic mechanisms involved. we now show that cd4+ t cells from 62m-/-mice exhibit antigen specific release of serine esterase and interferon-gamma. further, as quantitated by flow cytometry, the early decrease in cd4+ t cells seen in normal mice 3 days after lcmv infection, previously presumed to be due to the activity of cd8+ ctl, still occurs in 62m-/-mice. however, 621114mice show an earlier rise in percentage and absolute numbers of cd4+ t cells by 7 days after infection. cd4+ t cells from am-/mice rise to above baseline levels with kinetics paralleling the early rise in cd8+ t cells in normal mice after lcmv infection. cd4+ ciz activity is lower in 62m-/mice and have a later peak than cd8+ ctl from normal mice. these differences may explain the lower mortality and longer time course of immunopathologic meningitis in 621114-mice. mice can assume some of the cytotoxic functions of the cd8+ cell population from normal mice, including immune-mediated pathology. further, cd4+ t cells from 62m-/-mice have the capacity to release serine esterase, and show kinetics similar to cd8+ t cells from normal mice. mice genetically engineered to be deficient in 62-microglobulin in conclusion, these data suggest that cd4+ t cells from am-/australia. molecule is an essential component of the t cell help required for an antibody response. the interaction between cd40 and its ligand, in the presence of b cell acting cytokines, such as interleukin 4 (il-4), is sufficient to trigger b cells to proliferate and differentiate and to induce immunoglobulin class switching. a recombinant vaccinia virus encoding both factors, cd40l and il-4, did not, however, stimulate an enhanced antibody response in mice infected with the construct. instead, the antibody levels were lower than those of mice infected with a control virus. the reduced antibody response reflected the diminished growth of cd4ol-expressing viruses, to the extent that immunocompromised mice were able to resolve these infections. the kinetics of virus clearance indicate that the anti-viral mechanism of cwol is rapidly activated and has generalized tissue distribution. the cwolmediated clearance of virus is independent of the anti-viral cytokines, tnf and ifn-y. the anti-viral activity of c w l may represent a surprising and potent effector mechanism of t cells activated during a virus infection. mary's medical school, norfolk place, london w2 lpg, u. k. and *nationallnstirute formedica1 research, london, nw7 1aa. respiratory syncytial (rs) virus is the most important respiratory pathogen of infants, causing the majority of cases of bronchiolitis, it can be life threatening in very young infants and those with underlying cardiopulmonary disease or immunodeficiencies. t cell immunodeficiency can lead to prolonged infection in children and t cell transfers clear virus in the murine disease model. mice can be readily infected with rs virus and have proved a valuable model of its pulmonary pathology and the t cell response to it. il-3 has a broad spectrum of activities on proliferation and terminal differentiation of early blast cells and committed progenitors of many lineages. less is known about the effects of il-3 on t cell development and proliferation and its effect on the host response to infection by common pathogens. the il-3 gene was disrupted in 129/sv embryonic stem (es) cells by homologous recombination after transfection with omega-type constructs flanked with a herpes simplex thymidine kinase gene. these cells were used to produce a mouse strain deficient of il-3. mice were infected intranasally with a 2 strain rs virus; pathological changes in the lung were monitored by cytology of bronchial lavage, and virus persistence by nested rt-pcr of lavage fluid and lung homogenate. no differences were found in pathology or virus persistence between knockouts and normal mice. il-3 deficiency is therefore compatible with apparently normal responses to viral infection. recombinant adenoviruses are an atuactive vehicle for gene therapy to lung in the treatment of cystic fibrosis (cf). first generation viruses deleted of ela and elb transduce genes into airway epithelial cells in vivo, however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. in this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive nansfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. our studies indicate that mhc class i resmcted cd8+ cytotoxic t lymphocytes (ctls) are activated in response to viral antigens leading to destruction of virus infected cells and loss of transgene expression. mhc class ii associated presentation of viral antigens activates cd4+t helper (th) cells of the -1 subset and, to a lesser extent, of the tm subset. ldv establishes a life long viremic infection in mice which is not controlled by anti-ldv immune responses. anti-ldv antibodies are rapidly generated but they neutralize ldv infectivity only poorly. here we show that ldv-specific cytotoxic t cells (ctls)are generated within 7 days pi. c t l s were detected by h-2 specific lysis of ldv-specific target cells. these target cells were generated by transfection of 3t3-nm cells with a retrovirus vector carrying the gene (ow 7) for the ldv nucleocapsid (n) protein. spleen cells from ldv-infected mice were incubated in vitro with ldv-infected macrophages or transfected 3t3-nih cells and cultured for 5 days in the presence of il-2. the ctls had largely disappeared in mice by 30 days pi. the following experiments were conducted to further explore the mechanism of immune escape. in situ hybridization was used to localize ldvinfected cells in tissues of infected mice. these were found in persistently infected mice only in the liver. testis, and lymphoidal tissues. in addition large amounts of virion or virion debris accumulated in the germinal centers of the spleen and lymph nodes beginning about 3 days p.i. the accumulation of large amounts of ldv antigen in the germinal centers may be causally related to the suppression of the ctl response as well as to the polyclonal activation of b cells associated with ldv infection. to determine whether immune selection plays a role in ldv immune escape, ldv was reisolated from nude and immunocompetent balb/c mice at various times pi. the ows for the nand the two envelope glycoproteins were r t k r amplified and sequenced. the role of cytokines in initiating the immune response to venezuelan equine encephalitis (vee) was investigated. mice were infected in the left rear foot pad with either cloned virulent vee (v3000) or an isogenic single-site attenuated vee mutant (v3032) (single amino acid change at e2 glycoprotein position 209 from glu-lys) and at 1,6,24,48,72, and 96 hours post-infection, the popliteal lymph nodes and spleen were harvested and examined for tnf-a, il-12, ifn-y, il-2, il-4, il-6, and il-10 gene expression using a quantitative rt-pcr. in both strains elevations of tnf-a, il-12, ifn-y, il-10, and il-6 were detected in the lymph node, with no changes in either il-2 or il-4; however, whereas cytokine elevations were detected by 6 hours after injection of v3000, elevations were delayed until 24 hours following infection with v3032. cytokine mrna elevations in the spleen were less substantial, but elevated levels of ifn-y, il-10, and il-12 were also observed. these findings suggest that vee infection induces an early highly specific cytokine pattern associated with simultaneous elevations in both ifn-y and il-10 and that a single amino acid mutation can induce temporal changes in this pattern without alterring either the actual cytokines induced or the degree of their elevation. we are currently performing intervention experiments to investigate the effect of intravenous anti-il-12 andor anti-ifn-alp antibody administration on this early in vivo cytokine response to examine in particular whether these cytokines may be required for the observed elevations in either ifn-y or il-10. to confirm the importance of the immune system in clearing rotavirus infection we infected rag 2 knockout mice with murine rotaviruses. both knockout p u p s and adults developed chronic infections. to determine if a t cell independent antibody could be mediating the clearance of rotavirus infection in nude mice we measured igg, igm and iga in faeces and serum of rotavirus infected mice. a low and transient igm response was found in serum but not faeces of infected nude mice but not in age matched naive nude mice. the protein specificity of these antibodies is being determined. to determine if a primary infection in n u d e mice would induce protection (memory) against a secondary infection, we challenged six week old mice that had been infected as pups with the murine ew strain of rotavirus with the murine cambridge strain of rotavirus. the previously infected mice, as well as naive nude mice, both shed very low but equal levels of rotavirus in the stools. interestingly adult naive heterozygous littermates shed more virus than the nudes. the factors responsible for this relative resistance of adult nude mice to rotavirus infection as well as the mechanism for viral clearence in nude mice will be discussed. symp. 1990, 121:149-160) . the great majority of the poxvirus encoded proteins actively mediating the evasion of host defense are secretory proteins termed "virokines". viruses produce proteins which can either compete with or antagonizz molecules critically involved in generating immune responses. the vaccinia virus complement control protein (vcp) was the first virokine to be reported that has structural similarity to humadmouse complement control proteins, with the greatest similarity to the human complement 4b binding protein (bc4-bp) and the first protein to have a postulated role in the viral evasion of host defense (kotwal and moss, nature 1988, 355:176-179) . bioactive vcp, purified from serumfree medium has been shown to be more potent than the hc4bbp ( kotwal, am. biotech. lab., 1994) and has also been shown to bind to two important complement components, c3 and c4, and block the complement cascade at multiple sites in vitro (kotwal et al., science 1990, 2502327-830; mckenzie, kotwal et al., j. inf. dis. 1992 , 1661245-1250 . the importance of this protein was demonstrated in vivo using recombinant virus lacking an intact dna coding for vcp (isaacs, kotwal and moss, pnas 1992, 89:628-672) and further underscored by the smallpox virus genomic sequence analysis, indicating the presence of a highly conserved homolog of vcp ( massung et al., nature 1993,366:748-751) . in order to determine the precise effects of vcp at the site of infection, a mouse model has been developed. measurement of the specific swelling response and microscopic examination of the histological changes in balb/c and dbn2 mice has revealed that vcp is capable of regulating inflammatory response in vivo (jayaraman and kotwal, 1993 mol. immunol. 30:20) . in order to ensure that the possiblity of the unrelated mouse strains sharing identical h-2 haplotypes but differing at numerous other loci was not overlooked, we performed similar experiments in congenic strains. well characterized c5deficient mice injected with poxviruses in comparison to normal mice, produce a significant inflammatory response, accompanied with severe ulceration that persists for several days. the data suggests that the host complement response is a critical factor in egulating poxvirus pathogenesis. previous studies have suggested that immune response was one of the major factors that contributed to the host resistantlsusceptible mechanism to sendai virus infection (brownstein, 1981) . various immune regulatory molecules were investigated in inbred susceptible 129/j (129) mice and resistant c57bl/6j (b6) mice which share the same mhc haplotype. when they were given 200 eid, of sendai virus intranasally, 129 mice showed histologically diffuse interstitial pneumonia compared to the local moderate inflammation in 86 mice. virus was eliminated from the infected lung 3 days earlier in b6 mice than in 129 mice. the cytokine levels and cytokine-producing cells were investigated in the acute pneumonic lung. also the recall cytokine production in the draining mediastinal lymph node (mln) were studied. the maximal numbers of il-2, ifn-y, il-10, and il-4 producing cells were comparable for each strains of mice, while more il-6 producing cells were present in the lung of 129 mice. however, the numbers of these cytokine producing cells peaked in 129 mice 3 days later than in b6 mice. analysis of soluble cytokines in bronchoalveolar lavage fluid showed that at least two-fold higher levels of ifn-y, il-10, and il-6 were present in 129 mice than in 86 mice. recall cytokine production in the draining mln showed only the presence of il-2, ifn-y, il-10, and il-6, while no il-4 was detected. generally, higher levels of these cytokines were detected throughout the whole infection process in 129 mice. therefore, the susceptibility of 129 mice to sendai virus is not due to insufficient cytokine production in these two anatomical sites, yet heavier and prolonged virus burden in susceptible mice may drive the more rigorous and protracted induction of immune regulatory molecules. cytokines play a decisive role in determining the type of immune response elicited by an antigen, both in driving th cell differentiation along thl or th2 pathway and, ultimately, the effector and regulatory activity of these subsets. we have studied the role of cytokines in virus infections by constructing recombinant virus vectors encoding cytokine genes. during replication in mice, these vectors produce the encoded factor which is secreted from the infected cells with profound immunological consequences. cytokines studied todate fall into two categories; either altering viral pathogenesis or selectively stimulating specific immune responses. the expression of il-2, ifn-y, tnf-a or il-7 in recombinant vaccinia virus (rvv) dramatically alters pathogenicity, such that immunodeficient mice resolve the normally lethal infection. on the other hand, expression of il-4 by rvv suppresses the induction of cytotoxic t cells (ctl) inhibiting the clearance of virus which leads to an increase in viral pathogenicity. the expression of il-4 by rvv was shown to inhibit the host il-2 response required for the development of ctl's. no production was also significantly suppressed by il-4. rvvs expressing il-5 or il-6, although not affecting pathogenicity, preferentially stimulates iga reactivity to coexpressed antigens. encoding cytokines in recombinant viruses has clearly demonstrated the important role of cytokines as anti-viral effector molecules and in regulating appropriate immune responses. in mice infected with lymphocytic choriomeningitis virus (lcmv), interleukin-i2 (il-12) doses of >i00 ngld induce profound immunotoxicities characterized as almost complete inhibition of virus-induced cd8+ t cell expansion and ctl activation, and up to 2 log increases in viral replication [orange, wolf, and biron, j. immunol. 152:1253, 19941 . serum tumor necrosis factor (tnf) is also observed under these conditions. the studies reported here further characterize the expression and function of tnf in this context. northern blot and in sifu hybridization analyses demonstrated that il-12 induced tnf-cx expression and that lcmv infection synergized with il-12 for this induction. administration of antibodies neutralizing tnf reversed the il-12-induced immunotoxicities in lcmv-infected mice and restored anti-viral defenses. the tnf-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and cd8+ t cells isolated from lcmv-infected mice were more sensitive to tnfmediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. additional physiological changes were observed in il-12-treated uninfected mice and were dramatically elevated in il-12-treated virus-infected mice, including: 1) decreases in body weights; 2) elevation of circulating glucocorticoid levels: and 3) decreases in thymic mass. these changes were also reversed by anti-tnf. the results delineate a unique tnf-mediated immunotoxicity and have significant implications concerning detrimental consequences of in vivo tnf andlor il-12 for protective anti-viral responses. lactate dehydrogenase-elevating virus (ldv), a naturally occurring virus, causes a persistent infection in mice and presents an ideal model for the study of immune modulation during acute and persistent virus infections. within a few days following infection with ldv there is a pronounced polyclonal activation of b cells followed by the suppression of primary b cell responses to t-dependent ag. we investigated the effect of acute and persistent ldv infection on the development of a memory b cell response to the model protein antigen, horse cytochrome c (cyt), by employing a modification of the splenic fragment assay. about a 50% decrease in the frequency of responding agspecific memory b cells was observed in balb/c mice infected with ldv, whether the mice were immunized with cyt at the time of ldv infection or three weeks later. this may be due in part to a defect in t cell help, since in cultures of normal memory b cells and t cells derived from ldv acutelyinfected mice the frequency of responding b cells was also decreased two-fold. in situ hybridization using a cdna probe specific for ldv revealed two patterns of ldv rna within the spleen. twenty-four hr p.i. ldv rna was located within the marginal zone, surrounding each follicle. this pattern is consistent with permissive macrophages. during persistence viral rna could no longer be detected in the marginal zone, but was located within the follicles. the absence of ldv-permissive cells within the follicular region suggests that the source of ldv rna is not due to ongoing viral replication. one possibility is that circulating virus is trapped by a specific cell population within the follicle. the effect of virus trapping within the spleen provides a mechanism by which ldv and other viruses can modulate immune cell function during persistent infections. ifn-y can be produced by activated nk cells. this cytokine enhances immune responses by augmenting macrophage antigen presentation. viral infection induces ifn-dp and nk cell activation. changes in splenic architecture, cell trafficking, and cytokine expression were examined during viral infections of c57bu6 mice. at times coinciding with ifn-dp production and nk cell activation, there was a redistribution of nucleated cells from red pulp to white pulp regions in spleens isolated from mice infected with either lymphocytic choriomeningitis virus (lcmv) or murine cytomegalovirus (mcmv). cell transfer experiments with dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate-or pkh26-gl-labeled bone marrow cells isolated from normal mice demonstrated an infection-induced accumulation of non-t/non-b cell populations along recipient splenic marginal zones. flow cytometric analyses demonstrated that approximately 10% of the transferred bone marrow cells accumulated in spleens after 20 hrs and 30% of these expressed the nk cell marker, nkl.l+. in vivo antibody treatment procedures, to eliminate cell subsets in donor mice, demonstrated that the cells localizing at the marginal zone were derived from agmi+ and n k l . l + populations. a small subpopulation of marginal zone cells in infected mice were shown to be expressing high levels of ifn-7 mrna by in sifu hybridization. treatment with anti-agmi or anti-nk1 .i antibodies eliminated both endogenous nk cells and the ifn-y mrna positive cells. these data demonstrate that newly derived nk cells accumulate along marginal zones. the results also suggest that this trafficking pattern may act to enhance immune responses by facilitating delivery of cytokines to specialized antigen presenting cells. david segal, janet ruby, alistair ramsay and ian ramshaw. depamnent of cell biology, john curtin school of medical research, po box 334, canberra, act, 2601 australia cytokine expression has been shown to correlate with protective or ineffective immune responses in a number of disease models. recently there has been the suggestion that immunity to some retroviruses is associated with the production of ceaain patterns of cytokines. to explore this further we have have used rauscher murine leukemia virus (r-mulv) infection of c57bu6 (resistant) and balb/c (susceptible) mice to elucidate the role of cytokines immunity to retroviruses. initially the in viho proliferation of spleen and lymph node cells from infected mice was examined. in response to stimulation with immohilised anti-cd3 antibodies the proliferation of spleen hut not lymph node cells from infected mice. was found to he rapidly suppressed. suceptible balb/c mice exhibited a much greater suppression than resistant c57bu6 mice. the cause of this suppression is under investigation however, the immunosuppressive molecules nitric oxide and prostaglandins are not involved. in vitro cytokine production by spleen and lymph node cells from r-mulv infected mice was determined. in response to stimulation with immobilised anti-cd3 antibodies, spleen cells from infected balb/c mice produced diminishing amounts of ifn-7 and il-2. in contrast spleen cells from infected c57bu6 mice produced ifn-y and l-2 to levels that were only slightly less than uninfected controls. a-6 production by spleen cells from infected mice of both strains was at levels higher uninfected controls. anti-cd3 stimulated lymph node cells from infected mice produced elevated ifn-1 suggesting that suppressed cytokine production is spleen specific. expression of cytokine genes in vivo is currently being investigated using rt-pcr to detect cytokine mrna in the spleens of infected mice. we have previously shown that primary resting murine b lymphocytes are non-permissive for vesicular stomatitis virus (vsv), however, a productive infection can be induced when infected b cells are activated with anti-immunoglobulin (a-lg) plus il-4 or lipopolysaccharide (lps). we posit vsv in unactivated primary b cells provides a paradigm of persistently infected lymphocytes and activation dependent recall of an active infection. analysis of the behavior of virus in unstimulated b cells during long term culture and the requirements for subsequent induction of productive infection has been limited by the poor survival of primary cells in culture. we circumvented this limitation by using highly purified small b cells from mice transgenic for the bcl-2 proto-oncogene, expression markedly extends in vitro survival of unstimulated primary b cells. overexpression of bcl-2 does not alter b cell infection or induction of a productive infection by activators during acute infection. infection does not effect b cell survival in culture. unstimulated virus infected b cells produce primary viral mrnas but not viral proteins or infectious particles (pfu) during culture. persistently infected b cells stimulated with a-lg plus il-4 produced a fully productive vsv infection at all times analyzed, up to 3 weeks post infection. in contrast, vsv production in persistently infected b cells activated with lps markedly declined relative to acutely infected activated cells (50-1 00 fold by week 1 and 1,000 fold by week 2). cells were not completely refractory to lps activation as vsv protein was produced. the selective lps deficiency is unique to persistently infected cells as uninfected cultured b cells proliferate and differentiate to produce antibody upon lps activation. these data show that a persistent infection may selectively alter the host cell response to previously productive activators which may as a consequence interfere with immune regulation. rsv-g glycoprotein specific t cells preferentially secrete il-5 and predispose to pulmonary eosinophillia., anon srikiatkhachorn. and thomas j. braciale, the beirne b. carter center for immunology research and the departments of microbiology, pathology, and pediatrics, university of virginia health sciences center, charlottesville, va 22908 we studied the immune responses to two different glycoproteins of respiratory syncytial virus (rsv) in a murine model. balb/c mice were immunized with recombinant vaccinia virus expressing either rsv-fusion glycoprotein ( vac-f), attachment glycoprotein (vac-g) or 8-galactosidase (as a control). these mice were given rsv intranasally three weeks after priming and then sacrificed 5 or 14 days later. spleens and bronchial lymph nodes were harvested for in vitro culture and lungs were harvested for histologic studies . we found that bulk cultures obtained from both vac-f and vac-g immunized animals secreted both thl and th2 type cytokines when stimulated with rsv infected spleen cells . however, the levels of 11-5 and 1fn-y were higher in bulk cultures derived from vac-g primed animals while the levels of il-2 were higher in the bulk culture from vac-f primed animals. the il-4 and il-5 production was relatively short lived since spleen cells and bronchial lymph node cells obtaind form mice sacrificed 14 days after intranasal inoculation produced much lower levels of il-4 and 11-5 while the levels of il-2 and ifn-y production were comparable to bulk cultures obtained from mice at the peak of infection. there was little inflammatory response in the lungs obtained from mice immunized with the control vaccinia. in contrast , lungs from mice immunized with vac-f or vac-g showed significant infiltration of inflammatory cells. there was a striking infiltration of eosinophils in the lungs from mice primed with vac-g. these eosinophils could be detected aroud major bronchi and blood vessels, as well as, in some cases, in lung parenchyma. this study suggests that the immune responses to different viral glycoproteins may be distinct and may play important roles in viral pathogenesis. during infection of normal mice with lymphocytic choriomeningitis virus (lcmv), nk cell responses peak on day 3 and subside as cd8+ t cell responses are activated at day 7 post-infection. in contrast, 02m-/-mice, lacking cd8+ t cells, have dramatically elevated nk cell responses on day 7 postinfection. the 02m-/-response is evidenced by increased nk cell activity, as well as up to 5-fold increases in blast and total nki.i+cd3-cell numbers. nk cell responses in normal mice are cyclosporin a (csa)-resistant and interleukin (il)-2independent, whereas day 7 nk cell responses in 02m-/-mice are csa-sensitive and il-2-dependent. to investigate the role of additional cytokines in regulating cellular responses during acute viral infections, production and function of il-4 and transforming growth factor4 (tgf-0) were examined. induction of il-4 mrna, at late times post-infection of normal mice, was shown by in situ hybridization of t cell-enriched splenic leukocytes and polymerase chain reaction (pcr) amplification of cdna from rna. ellsas of media cor.aitioned with cells isolated on days 0, 3, 5, 7, 9, and 14 post-infection demonstrated delayed induction of il-4 protein as compared to ctl activation. tgf-0, evaluated in biological and elisa assays, was induced maximally at days 7 to 9 post-infection. the kinetics of tgf-0 production by cells from infected 82m-/mice was similar to that of normal mice. however, cells from 02m-/-mice produced il-4 at early but not at late times postinfection. together, these results suggest that either il-4 is a critical cytokine for shutting off nk cells during normal responses to viral infection, or that the 02m-l context modulates responsiveness of nk cell subsets to other late cytokines. studies are in progress to distinguish between these two possible mechanisms. the induction of fever in response to infection is an important host defense mechanism that enhances aspects of the immune response and restricts the replication of some microorganism. vaccinia virus, a member of the poxvirus family, is a complex cytoplasmic dna virus that encodes a variety of proteins that interfere with host immune functions, such as complement regulatory factors and soluble receptors for il-lp, tnf and ifny. here we show that expression of the vaccinia virus il-1p receptor (vil-lpr) in the w r strain prevents the febrile response and reduces the severity of infection in intranasally inoculated mice. fever was recorded on days 1-6 after infection of mice with a vil-lpr deletion mutant, but not in animals infected with wild type wr or a virus revertant. these studies were extended to other virus strains that were used as smallpox vaccines, and expression of the vil-lpr was consistently found to prevent the onset of fever. vaccinia virus induced a severe hypothermia after 6 days in infected mice that was independent on vil-lpr expression and correlated with virus replication in the brain, the organ that controls body temperature. these results represent the first example of a virus mechanism to inhibit the host febrile response and suggest a central role for soluble il-lp in the induction of fever in poxvirus infections. measles virus (mv) infection can depress cell-mediated immune responses for months following clinical disease. mv is known to infect the thymus during human illness and this may contribute to immune suppression. we have used the scid-hu m o w with co-implants of human fetal thymus and liver to determine the effect of virulent and avirulent strains of mv on the thymus. scid-hu mice were. infected by direct inoculation of the graft with 103 pfu of either a wild type strain of mv(chicago-1,chi-1) or an attenu-ated strain (moraten, mor) and sacrificed at intervals over 28 days. peak viral titers, as judged by plaque assay on vero cells, were reached by chi-1 on d4 (105.7 pfu/ third of implant), and moron d21 (103.2 pfu/ third of implant). hematoxylideosin stained sections of chi-1-infected thymuses showed marked distortion of the cortex and medulla by d4 with thymocyte poilolosis and decreased cellularity. by d14, these. implants were mostly devoid of normal thymocytes. mor-infected thymuses showed relatively preserved architecture and cellularity. suspensions of the cells from implants stained with mabs to cd3,cd4 and cd8 were analyzed by flow cytomehy. there were significant decreases in the cd4+cd8+ cell pop-ulation by d10 with complete loss of all such cells by d28 with chi-1, and only modest reductions with mor. immune fluorescence staining of sections with a mv mab to hemagluttinin(ha) and abs for either human cytokera-tins(ael/ae3) or cd15 co-localized mv predominantly to epithelial and monocytic cells. additionally, mv antigen was present diffusely by d4 in both cortex and medulla in chi-i infection whereas mor-infected implants had only patchy distribution by d21. only rare cells stained both with mv ha and cd2 or cd4. mv ha was not expressed over background on any cd4+ cells judged by facs. we conclude that mv replicates in the scid-hu thymic implant primarily in epithelial and monocytic cells, and that the attenuated virus reproduces more slowly and with less cellular disruption. little mv ha could be demonstrated in thymocytes, therefore the data suggest that significant infection of the thymic epithelial stroma disrupts the thymic microenvironment which normally supports and aids in selection of immature t cells. part of the long-term immune suppression seen in mv infection may be due to infection of the thymic epithelial stroma with subsequent loss of thymocytes. it is becoming increasingly evident that many poxviruses contain genes that enable the virus to evade the host's immune system. myxoma virus is a leporipoxvirus and is the causative agent of myxomatosis, a rapidly lethal disease in the european rabbit (oryctolagus cuniculus). one possible mechanism of immune evasion is virus-induced downregulation of cell-surface receptors important for an immune response. cell-surface levels of several receptors on a rabbit t cell lymphoma cell line (rl-5) were monitored by flow cytometry. following infection with myxoma virus, cellsurface levels of cd4 were found to drop dramatically. other cell surface antigens such as cd18, cd43, and cd45 were unaffected during infection with myxoma virus. further more, the downregulation of cd4 by myxoma virus could be inhibited by treating cells for an extended period of time with pma, suggesting that the downregulation was not simply a masking of the epitope via viral antigens. analysis of cd4 levels in the presence of cytosine arabinoside indicates that late gene expression is not necessary for the modulation. since the tyrosine specific protein kinase p56lck associates with the cytoplasmic domain of cd4 we have also examined the association of p56lck with cd4 as well as steady state levels of p56lck during viral infection. the modulation of surface cd4 has also been described in hiv infected t cells suggesting that the loss of cell-surface cd4 may be a common viral immune evasion tactic by lymphotrophic viruses. i n addition, stably-transfected cell l i n e s expressing e i t h e r u s 1 1 o r us2-6 gene products s i g n i f i c a n t l y reduced l e v e l s of mhc class i heavy chain. studies are i n progress t o f u r t h e r d e f i n e t h e mechanism by which t h e s e v i r a l gene products a l t e r immune recognition. cytotoxic t lymphocytes (ctl) may play a significant role in containing the spread of hiv in infected individuals. although hiv-infection is associated with immune suppression, a vigorous ctl response has been detected in infected adults. hiv can be transmitted from mother to child. one third of vertically infected children has a rapid evolution toward disease, with onset of aids before 18 months. the other two thirds remain asymptomatic for years. the bimodal course of disease evolution in hiv-infected children could be related to differences in the host immune control of viral replication. hiv-specific ctl response from fresh and in vitro activated pbmc of hiv-infected children was measured. the vast majority of infected chidren had detectable hiv-specific ctl, which where cds+cd8+. we previously showed that among children with a slow disease progression, fresh ctl were more frequent in the p2a(paucisymptomatic) group than in the pl(asymptomatic) and the p2b-f groups (symptomatic group). the cohort of children has now been followed during 4 years, and 46 children have been tested at least once. we found that ctl responses were less frequent in the children with a rapid disease progression than in the children with a slow disease progression at the same age. our data suggest that ctl response is an important factor in delaying disease evolution. we, as well as others. have proposed that sag function is critical to the ability of milk-borne m m n to infect mice. to determine whether this is the case, we created transgenic mice (hyb pro/cla) with a frameshift mutation int the sag gene. young hyb pro/cla mice (c 10 weeks of age) showed no deletion of their cognate vp14* t cells, unlike transgenic mice carrying a functional sag gene however, a slow, progressive loss was seen in the hyb prolcla mice as they aged, indicating that it was due to expression of wild type sag protein. thus, as the hyb pro/cla mice aged, there was production of virus that appeared to lose the cla mutation. the hyb pro/cla mice produced transgene rna in their lactating mammary gland and shed virus in their milk. their nontransgenic offspring of showed infection with transgene-encoded mmtv because they had the typical slow deletion of vp14+ t cells characteristic of c3h mmtv infection and because we detected transgene-derived m m n rna in their mammary glands. cloning and sequencing of the viral rna produced by the nontransgenic offspring of the hyb pro/cla mice showed that recombination between the mtv-1 endogenous viral rna and the transgene-encoded rna occurred, such that the frameshift introduced by the cia mutation was repaired. these results show that there is selection of infectious virus that contains a functional sag gene. thus, it appears that the only virus that is capable of being transmitted by the milk borne infection pathway is that which encodes a functional sag protein. hepatitis b virus (hbv) causes acute and chronic liver diseases and is closely associated with hepatocellular carcinoma. in order to understand the cellular immune response against hbv in chronic hbv infection, t cell proliferation, cytotoxicity and cytokine production were studied. we found that although the majority of asymptomatic hbsag carriers and patients of chronic hepatitis b (chb) had no proliferative response to hbsag, some individuals in both groups showed significant t cell proliferation against hbsag. in contrast, the proliferative t cell response to hbcag in asyrnpatomatic hbsag carriers was significantly stronger than that in patients of chb with acute exacerbation. in addition, the frequency of hbcag-reactive t cell precursors measured by limiting dilution assay was much higher in asymptomatic hbsag carriers than in patients of chb. therefore, t cell responses against hbsag and hbcag are regulated differently in chronic hbv infection. furthermore, we demonstrated hbsag-and hbcag-specific cytotoxic t lymphocyte (ctl) activity in asymptomatic hbsag carriers, using autologous hbsag-and hbcag-expressing lymphoblastoid cell lines (lcl) as target cells, respectively. the cloned ctl were able to produce ifn-y, tnf-a or gm-csf after stimulation. these findings demonstrate that t cell response to hbv is not completely suppressed in asymptomatic hbsag carriers. most of them have strong hbcag-specific response and some of them have hbsag-specific response. transcription and tax the human t-lymphotropic virus type i (htlv-i) promoter contains the structural features of a typical rna polymerase i1 (pol 11) template. the promoter contains a tata box 30 bp upstream of the transcription initiation site, binding sites for several pol i1 transcription factors, and long poly a+ rna is synthesized from the integrated htlv-i proviral dna in vivo. consistent with these characteristics, htlv-i transcription activity was reconstituted in v i m using tbp, tfiia, rtfiib, rtfiie, rtfiif, tfiih and pol 11. in hela whole cell extracts, however, the htlv-i ltr also contains an overlapping transcription unit (otu). htlv-i otu transcription is initiated at the same nucleotide site as the rna isolated from the htlv-i-infected cell line, mt-2, but was not inhibited by the presence of a-amanitin at concentrations which inhibited the adenovirus major late pol i1 promoter (6 pglml). htlv-i transcription was inhibited when higher concentrations of a-amanitin were used (60 pglml), in the range of a typical polymerase in (pol 111) promoter (va-i). purified tax, transactivates this promoter 5-to 10-fold in v i m . interestingly, basal and tax,-transactivated transcriptional activity of the htlv-i ltr could be reconstituted with the 0.5 m phosphocellulose fraction. these observations suggest that the htlv-i ltr contains overlapping tax,responsive promoters, a typical pol i1 promoter and a unique pol i11 promoter which requires a distinct set of transcription factors. tax, further in vifro transactivates a polymerase i1 template containing the 21 base pair repeats cloned upstream of the ovalbumin promoter and g-free cassette. tax,-transactivated transcription was concentration dependent and inhibited by low concentrations of a-amanitin. flaviviruses are arthropod-borne viruses whose route of infection is via the skin. they are mostly neurotropic and responsible for significant human morbidity and mortality. the classic cell-mediated immune response to a viral infection may be influenced by the ability of these viruses to modify expression of cell-surface molecules involved in the presentation of antigen to, and activation of, t cells. the skin langerhans cell is the prototypic nonlymphoid dendritic cell and as such is uniquely placed to participate in a response against epidermally-acquired viral infections. the migratory properties of these cells contribute to their role as initiators of t cell-mediated immune responses within the draining lymph node. we have previously shown infection of epidermal cells in vifro by the flavivirus west nile (wnv) results in an increase in mhc class i and i1 expression on the majority of epidermal cells and langerhans cells respectively. in this study a technique for infecting the epidermis with wnv in vivo was developed. tme-dependent increases in the surface expression of a number of antigens which are involved either directly or in a co-stimulatory capacity in initiating a cell-mediated immune response, were detected on both the majority of epidermal cells and the langerhans cell population using flow cytometry. these increases were detectable as early as 16 hours after infection. a significant decrease in the percentage of langerhans cells remaining in the epidermis was observed within 48 hours of infection. the phenotypic changes observed in vivo are analogous to those described following in vifro culture of langerhans cells. these results, together with the reduction in langerhans cell numbers, may represent the in situ maturation and concomitant migration of these. cells as a consequence of virus-induced cytokines within the skin microenvironment. which cause a wide variety of illnesses with high morbidity and mortality in humans throughout the world. their high genomic stability argues for a survival strategy related more to interaction with the vertebrate host immune response, than a dependence on viral genetic mutation. our previous work has shown that west nile virus (wnv) infection of many cell types directly induces functional increases in class i and 11 mhc expression. we report here that wnv infection of human embryonic fibroblasts (hef) results in the increased expression of cd54 by two distinct mechanisms. an early, direct cytokine-independent mechanism operates within 2 h of virus infection, while an indirect mechanism, regulated by type 1 interferon (ifn), operates within 24 h of virus infection. cd54 expression increased by 4-5 fold within 2h of wnv infection on hef, and by 6-7-fold within 24h. wnv-inactivated, conditioned supematants removed from infected hef cultures after 4 h incubation did not alter cd54 expression on unqimulated hef. whereas conditioned supernatants from 24 h-infected cultures increased cd54 expression by about 1.5-2-fold after incubation for 24 h, but not after 4 h, similar to cd54 induction by 200ulml of ifn-p. increased cd54 expression on hef by wnv was also cell-cycle dependent. cd54 increased only in quiescent, contact-inhibited infected hef in go phase. in contrast, induction of cd54 by types 1 and 2 ifn was not cell-cycle dependent. other viruses, including double-stranded dna viruses, vaccinia, and adenovirus 2 and 5, and the single, positive-stranded rna alphavirus, semiliki forest virus, did not induce cd54 expression on hef after 24 h. another alphavirus, ross river, was able to induce cd54 but only by the indirect mechanism of type 1 ifn-dependent release. poly i.c, also, increased cd54 expression to the same extent as ifn-p after 24 h, making it unlikely that the early increase was due to a nonspecific viral effect. the closely related flavivirus, kunjin, induced increased cd54 expression in a manner similar to wnv. the ability of flavivhses to induce increased cd54 expression directly within a few hours of infection may be an important virus-host survival strategy promoting cell-cell adhesion and hence possible further viral infectiodreplication. recognition of viral peptides presented on the cell surface in association with class i mhc molecules leads to lysis by cytotoxic t cells (ctl) and forms an important part of the immune response to hiv infection. hiv virus has a high mutation rate and variation in the region of the viral epitope may allow evasion of this immune response. variation could theoretically affect processing of the antigen, binding of the epitope to the hla molecule or recognition of the presented epitope on the cell surface. we have studied proviral sequence variation in gag and ctl responses in a number of hla b8 patients infected with hiv. amino acid substitutions, such as a lysine to arginine change at position 3 of the pi7 gag nonamer cckkkyklk, lead to loss of recognition of the peptide by ctl from the patient whose provirus contained this sequence. these variant peptides bind to hla 68 with comparable affinity to the index peptide suggesting that this loss of recognition is likely to be caused by changes in the interaction between the hla-peptide complex and the t cell receptor. other changes, such as lysine to arginine or glutamine at position 7, not only cause loss of recognition, but also lead to inhibition of lysis of targets bearing the index peptide. thus it appears that in addition to loss of recognition by cytotoxic t cells, naturally occurring epitope variants may act as "antagonists", as has been demonstrated in mhc class ii systems. antagonism may be an important mechanism allowing immune escape by the hiv virus. genes. subsequent complex formation between peptide, class i and p2microglobulin in the er results in stable cell surface expression of the trimeric mhc-1 molecule. in previous studies we showed that in hpv-16 positive cervical carcinomas there was a loss of mhc-1 protein expression, which correlated at the single cell level with loss of tap protein. in this study we investigated whether loss of tap and mhc-1 is mediated by an hpv-16 encoded protein. human keratinocytes were transfected withvarious hpv-16 constructs including pat16, the full length genome, pat16esx the full length genome with a premature stop codon in e5, puc.et16, the e6 and e7 oncogenes only, and pkve5, expressing e5 from mouse moloney ltr the different constructs were transfected into primary keratinocytes, cloned cells grown in medium supplemented with and without y-interferon ( y -a r ) for 48 hours. cells were harvested and total rna and protein harvested for northern and western blots respectively. western blots showed very low steady state levels of tap-1 and mhc-1 heavy chains in the cells with pat16 as well as those containing es alone, which was marginally increased by y-lfn. in contrast, primary keratinocytes, pat16esx and puc.et16 lines showed comparable tap-1 and mhc-1 protein levels, which increased a & y-ifn treatment. northem blots showed no differences in the amounts of tap-1 and mhc-1 mrna between the different cell lines. the data indicate that expression ofhf'v-16 e5 leads to post-transcriptional loss of mhc-1, presumably by interfering with tap. to map and characterize functional differences between e1a of ad5 and adl2, we previously constructed a series of hybrid ad5/12 e1a genes and used them with ad12 e1b to transform primary hooded lister rat kidney cells. at least two regions within the first exon of ad12 e1a were identified which influenced tumorigenicity. this study further examines the role of these regions in tumorigenicity by analyzing their affect on cell surface mhc class i expression and sensitivity to class i-restricted cd8+ as well as to non-class irestricted nks. the bcrfl open reading frame of epstein-barr virus exhibits remarkable sequence homology with the coding sequences of interleukin-10 from a variety of organisms. many of the numerous immunological properties ascribed to interleukin-10 are shared by the product of bcrfl and this has led to it being termed viral interleukin-10. in order to investigate the activity of viral interleukin-i0 (vil-10) and its interactions with the human interleukin-10 receptor we have expressed the protein in a bacterial and the eukaryotic cos-7 expression systems. the bacterially expressed vil-i0 was partially purified and used to set up two assays to measure i l l o activity: i)the increase in igm secretion from an ebv transformed b cell line -mt4.l and ii)the downregulation of class ii hla expression on the human monocytic cell line thp-1. a series of deletion mutants (both n-and c-terminal as well as an internal deletion to remove a putative heparin binding domain) were constructed to identify possible domains within the vil-10 protein that interact with the hil-10 receptor and confer its biological activity. a number of these mutants have been expressed in the cos-7 expression system and their structure and biological activity are currently being assessed. the identification of the domains within vil-10 that interact with the receptor or accessory proteins may aid in the understanding of the possible role of vil-i0 within the ebv life cycle and in the pathogenesis of the numerous diseases associated with the virus. generation. to further test the role of ctl in ad pathogenesis, viruses lacking the cll epitopes were tested when mutants that lack the immunodominate ctl epitope in eia where used, a second immun-ssive epitope in elb becorns the predominate target of clu. these findings arc important since human ad is currently being tested as a vector for gene therapy of cystic fibrosis. our data suggest that when consuucting ad vectors to be. used for gene therapy, one must retain either the 10.4k or 14.7k genes to decrease pathology and that meting the genes that encode the antigens that a n recognized by clu does not prevent the generation of ad specific clu. the interferons (ifns) a n ? a family of cytokines whose functions include the protection of cells against viral infection. type i ifns include the 15 ifna subtypes and ifnp that compete for binding to the same cell surface receptor, while type ii ifn (ifny) binds to a different receptor. the orthopoxviruses, of which vaccinia virus (vv) is the prototypic member, have developed a number of anti-ifn strategies. the vv e3l protein competitively binds dsrna and prevents the activation of ifninduced and dsrna-activated protein kinase (pkr), while the vv k3l protein shows sequence similarity to the eukaryotic initiation factor 2a (eif2a) that is phosphorylated and inactivated by pkr. the k3l protein competitively binds the kinase and blocks host eif2a phosphorylation and hence ifn-induced inhibition of host protein synthesis. onhopoxviruses also suppress cytokine action by expressing soluble cytokine receptors that bind and sequester the ligand; to date soluble receptors for interleukin-18, tumour necrosis factor and ifny have been described. supernatants from vv-infected cells were found to contain a soluble inhibitor of type i ifn that was conserved in most of the orthopoxviruses tested. the inhibitor was produced early in infection and did not inhibit ifny. the ifna/p inhibitor was mapped and the gene expressed from recombinant baculovirus. the inhibitor blocked the binding of 125i-ifna to u937 cells and binding of 125i-ifna to supernatants from baculovirus and vv-infected cells demonstrated that the inhibitor functioned as a soluble receptor for 1fnc1fp. direct binding of 1251-ifna to vv wr supernatants revealed that the soluble ifna/p receptor had a high affinity for type i ifn. deletion of the gene from the vv genome and ligand blotting of the soluble receptor demonstrated that ifn binding was encoded by a single protein. competitive binding curves using ifna from other species revealed that the poxvirus soluble ifndp receptor bound human and bovine ifn with high affinity but murine ifn with relatively low affmity. interestingly, the soluble ifncrip receptor is highly conserved in variola virus. given the importance of ifn in antiviral defense it is likely that the soluble ifndp receptor plays an important role in the virulence of the orthopoxviruses. endogenous processing of a viral glycoprotein for presentation t o cd4+ t cells has defined a previously under-investigated pathway in antigen processing and presentation. it may be important not only for pathogens, but also for self-proteins, and thus may be involved in self-tolerance. we have been characterizing the processing o f the er-restricted gpt glycoprotein of vesicular stomatitis virus (vsv) biochemically and enzymatically, by cellular localization using confocal immunofluorescence, cellular fractionation, and by t cell recognition assays. by flow cytometry, gpt is undetected on the plasma membrane; in contrast, the wild type protein (g) is readily found following infection of a20 cells with a vaccinia virus vector, leading t o endogenous synthesis. the gpt can be found exclusively in the er compartment using co-localization with markers for er (signal peptide binding protein, calnexin), and not in the golgi compartment (a-mannosidase 11, wheat germ agglutinin), endosome, lysosome, or surface plasma membrane. this is consistent with the characteristics o f the localization of the proteases which appear to be responsible for its degradation. work is in progress to localize the site of peptide binding to mhc heterodimers. supported by nih grant a118083 t o csr. presentation of an out-of-frame class i restricted epitope. t.n.j.bullock and l.c.eisenlohr, department of immunology, thomas jefferson university, philadelphia, pa 19107. antigen presentation by class i mhc molecules is thought to require the degradation of fully formed proteins in the cytosol. this degradative process supplies oligopeptide epitopes for transport into the endoplasmic reticulum (er) where they can interact with and stabilize class i molecules. stable class i molecules, associated with p2-microglobulin, can then proceed to the cell surface where they present the epitopes to t cell receptors. the generally accepted model for protein translation, the scanning hypothesis proposed by ko&, is thought to describe the traditional method of translation for the majority of proteins. we wished to test the hypothesis that any internal methionine that is in good translation initiation context can be a source of short peptides, which may then be processed into class i epitopes. nucleoprotein gene (np), the target of the ctl response of several inbred mouse strains. np contains three class i restricted epitopes at amino acids 50-57 (h2-kk), 147-155 (h2-kd) and 366-374 (h2-db). the frameshift was introduced 26 amino acids upstream of the h2-kd epitope. the mutated genes were then recombined with vaccinia virus and tested for presentation using ctl restricted to each of the epito s described above. we found that, whilst presentation of the h2-i@ epitope was unaffected by the frame shift, the epitope proximal to the frameshift (h2-kd) was no longer presented to appro riately restricted ctl. however, presentation of the distal h2-dg epitope was retained. therefore we have shown, using a viral protein and a viral expression system, that out-of-frame epitopes can be processed and presented to ctl. work is ongoing to c o n f m that internal methionines are capable of providing a platform for the initiation of translation for in-frame and out-of-frame epitopes. we have created a frameshift mutation in the influenza pr8 the fine specificity of t cell recognition of peptide analogues of the influenza nucleoprotein epitope np 383-391 srywairtr was studied using hla b27-restricted influenza-specific cytotoxic t cell (ctl) clones, of defined t cell receptor (tcr) usage, derived from unrelated individuals following natural infection. synthetic analogue peptides were synthesized containing single amino acid substitutions, and tested both for binding to hla b'2705 in vitro, and for presentation to ctl clones by hla 827positive targets. even conservative amino acid substitutions of the peptide residues p 4 , 7, and 8 profoundly influenced ctl recognition, without affecting binding to hla 8'2705. these amino acid side chains are thus probably directly contacted by the tcr. ctl clones which used the tcr v a l 4 gene segment (but not those using tcr va12) were also sensitive to p1 substitutions, suggesting that the tcr alpha chain of these clones lies over the n terminus of bound peptide, and that the "footprint" of certain tcrs can span all exposed residues of a peptide bound to mhc class 1. these results, taken together with previous structural and functional data, suggest that, for nonarner peptides bound to hla 827, p i , p4 and p8 are "flag" residues with tcr accessible side chains. the e3/19k protein of human adenovirus type 2 (ad2) is a resident transmembrane glycoprotein of the endoplasmic reticulum. its capacity to associate with class i histocompatibility (mhc) antigens abrogates cell surface expression and the antigen presentation function of mhc antigens. at present, it is unclear exactly which structure of the e3/19k protein mediates binding to mhc molecules. apart from a stretch of approximately 20 conserved amino acids in front of the transmembrane segment, e3/19k molecules from different adenovirus subgroups (b and c) share little homology. remarkably, the majority of cysteines is conserved. in this report, we examined the importance of cysteine residues (cys) for structure and function of the ad2 e3/19k protein. we show that e3/19k contains intramolecular disulfide bonds. by using sitedirected rnutagenesis, individual cysteines were substituted by serines and alanines, and mutant proteins were stably expressed in 293 cells. based on the differential binding of monoclonal antibody tw1.3 and cyanogen bromide cleavage experiments, a structural model of e3/19k is proposed, in which cys 11 and cys 28 as well as cys 22 and cys 83 are linked by disulfide bonds. both disulfide bonds (all four cysteines) are absolutely critical for the interaction with human mhc antigens. this was demonstrated by three criteria: loss of e3/19k coprecipitation, lack of transport inhibition and normal cell surface expression of mhc molecules in cells expressing mutant e3/19k molecules. mutation of the three other cysteines at position 101, 109 and 122 had no effect. this indicates that a conformational determinant based on two disulfide bonds is crucial for the function of the e3/19k molecule, namely, to bind and to inhibit transport of mhc antigens. previous studies have suggested that several abundant cmv proteins are major immunogenic targets in seropositive adults. we are interested in defining the major viral protein targets of a cd8' ctl response, in order to derive a vaccine strategy for individuals who are unable to mount immune responses which are lymphokinedependent because of immunosuppression. hla-typed and cmv-pgsitive normal volunteers who have hla-a alleles that represent -75% of the u.s. population are being tested to determine which of 5 abundant cmv proteins they recognize by a cd8' ctl response: p28, p65, p150, ie, and gb. t cell lines will be derived in order to unambiguously determine the hla restriction of the cd8' ctl response to each of these proteins. proteins which are recognized by the most hla diverse population will be further characterized in terms of mapping of class 1 epitopes through the use of t cell clones derived from the polyclonal cell lines by limiting dilution. the defined epitopes will form the basis of a vaccine strategy to augment the memory responses of seropositive volunteers against cmv. these epitopes will be used to boost the ctl precursor frequency of bone marrow transplant donors as a means to transfer cellular immunity to immunosuppressed hematologic transplant recipients. an alternative strategy is to immunize seropositive individuals with recombinant viral proteins as a means to boost immunologic memory. we are pursuing that strategy in a transgenic murine model of hla-a2.1 developed by dr. l. sherman (scripps institute, la jolla). we are vaccinating the transgenic mice with two well defined cmv proteins, p65 and gb together with either of two lipid-based adjuvants, commercially available d0tapm (bcehringer-mannheim) or mf5gth (chiron, emeryville, ca). our preliminary studies with hsv-2 gb demonstrate that both adjuvants are effective at eliciting murine class i restricted responses against the protein. current studies are evaluating the recognition properties of the adjuvant-cmv protein complexes by hwa2 as a restriction element in the transgenic model. the ctl response to sendai virus in c57by6 mice is directed almost exclusively to a single h-2kb-restricted epitope derived from the virus nucleoprotein, npj24-332 (sev-9). analysis of 18 independent t cell hybridomas generated from c57by6 mice following primary sendai virus infection has shown that a very diverse repertoire of tcr is selected in response to this epitope. crystallographic analysis of sev-9 bound to kb has shown that the side chaiis of peptide residues phpi, gl484, a d s , and alaps protrude towards the solvent and are potentially available for recognition by the tcr notably, residues gi484 and a d 5 protrude prominently from the peptide binding site due to their l o c a l i o n on a bulge in the center of sev-9. to determine the importance of each of these residues for t cell recognition, we analyzed hybridoma responses to sev-9 analogs substituted at each of these four positions. preliminary data showed there generally appeared to be dominant recognition of glyp4 and asnm. however, individual hybridomas exhibited distinct patterns of fine specificity for residues phep1 and alaps. thus, individual hybridomas were dependent on one, both, or neither of these residues for recognition of sev-9. these data are consistent with a critical role for the gi94 and a d 5 in governing tcr-sev-9eb recognition and suggest a structural basis for the diversity of the tcr repertoire selected by this @tope. previous results from this laboratoty demonstrated that the dominant influenza a epitope recognized by hla42.1 restricted ctl from hla-a2.1 uansgenic mice was the m1 peptide epitope that is immunodominant in human ctl responses. however, analysis of a large number of ctl lines revealed a subset of influenza a/pr/8/34-specific murine ctl that recognized an hla-a2.1 restricted epitope distinct from m1. using recombinant vaccinia viruses encoding werent influenza gene segments, the epitope recognized by these ctl was shown to be derived from the a/pr/8 nsl protein. because these ctl did not recognize targets infected with the a/alaska/6/77 saain of influenza, candidate peptide epitopes were synthesized based on sequences that included an hla-a2.1 specific binding motif and that differed between a/pw and nalaska all of these ctl recognized a nonamer and a decamer peptide which contained a common 8 amino acid sequence and two distinct sets of bmding mtif residues. however, the n0name.r peptide was able to sensitize ctl for half maximal lysis at 80-2500 fold lower doses than either the octamer or decamer. the homologous peptide derived from nalaska nsl contained conservative amino acid changes at positions 4 and 8 and was not recognized at any tested concentration, although it bound with higher &ity to hla-a2.1 than the peptide from a/pw8. the a/pr/8 nsl nonamer epitope was also recognized by human influenza a specific ctl derived from two individuals. these results substantiate the general utility of hla class i aansgenic mice for the identification of human cn epitopes for other pathogens. furthemore, the recombinant dhfr was functional in the induction of gb epitope-specific ctl response upon immunization of c57bv6 mice. these results indicate that an viral epitope expressed in a cellular protein can be. efficiently processed, presented and recognized by epitope-specific ctl, and suggest that the cellular proteins can be used to express ctl epitopes for induction of cd8+ immune responses. virus-specific cytotoxic t lymphocytes (ctl.) were generated a day later at this site. to determine which apc was capable of stimulating virusspecific ctl precursors in the mln, b, t and dendritic cells from the mln of influenza-infkcted mice were separated and examined for the presence of virus. the predominant cell type which contained infectious virus was the dendritic cell. b and t cells from the mln contained little, ifany, virus. the apc capacity ofthese populations was tested by their ability to stimulate vir~~-~pecific t cell hybridomas. only dendritic cells from the mln of influenza-infected mice were able to stimulate virusspecific t cell hybridomas, althwgh all apc populations from both naive and influenza-infected mice were effective stimulators after in y h pulsing with the appropriate intluenza peptide. potential apc populations were also separated from the lung. v i s was detected in bronchioalveolar macrophages and dendritic cells but not b or t cells. both macrophages and dendritic cells isolated from intlum-infected lungs could stimulate virus-specific t cell hybridomas. the ability of the mln and lung apc populations to stimulate naive cd8' t cells and generate virus-specific ctl is currently being examined. virus infected cells present only a very limited number of peptides intracellularly processed from a viral protein to ctl even when many peptides hearing the mhc class i-restricted binding motif are present in the protein. infection of h-2b mice w i t h lymphqtic choriomeningitis virus (lcmv) induces a cd8+ ctl response directed against three wellcharacterized epitopes presented by h-2db molecules: "396-404 (fqpq-ngqfi), gp33-43 (kavynfatcgi) and gp276-286 (sgven-pggycl). the h-2db motif is characterized by a sequence of 9 to 11 a.a. with two anchor residues: asn at position 5 and hydrophobic (met, ile, leu) at the c-terminus. the lcmv np and gp proteins contain thirly-one other peptides exhibiting the db motif. however, no ctl response against one (or more) of these peptides has been characterized. peptide binding to mhc is a critical step in antigen presentation. the aim of this study was therefore to analyze the binding properties of the potential db lcmv peptides. the 34 lcmv peptides and 11 known db-selective peptides were synthesized and their mhc binding affinities measured in two db-specific binding assays. most of the lcmv peptides (28/34) did not bind to db. the other 6 (including the 3 epitopes) and all the known db peptides showed good affinity. comparison of the sequences (good vs. non binders) allowed the identification of auxilliary anchors required for high binding affinity or of negative elements hampering mhc binding. in addition to the main anchors, the positive and negative factors at secondary residues play a crucial role in governing peptidemhc interactions. knowledge of such factors might he of importance for the prediction of mhcrestricted ctl epitopes. etienne joly, andrea gonzalez, carol clarkson, jonathan c. howard and geoffrey w. butcher. laboratory of immunogenetics, department of immunology, the babraham institute, cambs cb2 4at, uk. tap transporters from rats can be divided into two allelic groups, depending on their capacity to provide the rt1.aa molecule with an appropriate level of suitable peptidesl. recent results suggest that this might correlate with the rt1.aa molecule requiring arginine-ended peptides (powis et al., manuscript submitted), which the tapb allele of the transporter is unable to translocate across the er membrane efficiently2~3. rt1.a alleles are naturally linked with the tapa or the tapb allelic group4. we have set out to characterise various alleles for the rt1.a molecule, and find that, for the majority of tapaassociated rt1.a molecules, 3 acidic residues line the c/e pocket, dictating arginine as c-terminal anchor residue for the bound peptides. on the other hand, in tapb-associated rt1.a molecules, one acidic residue at the most is found in the c/e pocket, which certainly results in a different anchor residue for the bound peptides. the selective pressure of viral infections must have driven this coevolution which affects dramatically the array of peptides presented to cytotoxic t lymphocytes. cytotoxic t lymphocyte responses in hiv infection can be impaired due to variation in the epitope regions of viral proteins such as gag. we show here an analysis of variant epitope peptides in three gag epitopes presented by hla b8. seventeen variant peptides were examined for their binding to hla b8; all but one bind at concentrations comparable to known epitopes. all except two could be seen by ctl clones grown from hla b8 positive hiv-1 infected patients and were therefore immunogenic. however, in one haemophiliac patient studied in detail, there was a failure to respond to some of the peptides that represented virus present as provirus in his peripheral blood. in one case his ctl had previously responded to the peptide. thus there was a selective failure of the ctlresponse to variant epitopes. this impaired reaction to new variants and failure to maintain responses to some epitopes late in hiv infection could contribute to the loss of immune control of the infection. pira, anna ferraris, daniele saverino, peifang sun and annalisa kunkl; dept. immunology, san martino hosp. univ. of genoa, 16132 genoa, italy. th epitopes present on viral proteins can be recognized by specific th cells if appropriately expressed by antigen presenting cells (apc) as a result of uptake and processing. since viral epitopes are not simply present in the context of viral proteins, but also in the context of whole viral particles, it is important to determine the role of the molecular and/or structural context on antigen uptake-processing-presentation. therefore we have generated panels of cd4+ human t cell lines and clones specific for different hiv antigens (gp120, p66, p24), in order to test their ability to respond to the same epitopes present within synthetic peptides, recombinant proteins or inactivated virions (provided by g. lewis, dept. microbiology, univ. maryland, baltimore). we could identify t cell lines and clones that were able to discriminate the molecular and structural context of the epitops. certain t cells, in fact, responded to peptides and proteins, but not to viral particles, whereas other t cells were also able to proliferate when challanged in vitro with autologous apc and viral particles. the data suggest that in the human th cell repertoire specific for viral antigens t cells exist that can discriminate the molecularstructural context of th epitopes. it will be interesting to ascertain whether t cells specific for epitopes that can only be recognized when provided in the context of a soluble molecule, but not of a viral particle, have any relevance in viva protection, or are a simple by-product of the cellular immune response. eric g. pamer, merceditas s. villanueva, section of infectious diseases, yale university school of medicine, new haven, ct 06520 listeria monocytogenes is a gram positive bacterium that infects macrophages and secretes proteins into host cell cytosol. the murein hydrolase p60 is secreted by l. monocytogenes and is required for complete bacterial septation. in the infected macrophage secreted p60 is processed by the host cell into the nonamer peptide p60 217-225 and is presented to cytotoxic t lymphocytes by the h-2kd mhc class i molecule. we have used strains of l. monocytogenes that secrete different amounts of p60 to show that the rate of p60 217-225 production is proportional to the amount of antigen secreted into the host cell cytosol. p60 is degraded in the host cell cytosol with a half life of 90 minutes. the appearance of p60 217-225 is coupled to the degradation of newly synthesized p60. we have determined the rate of intracellular p60 secretion and by accounting for the rate of p60 degradation we estimate that approximately 35 p60 molecules are degraded to produce one p60 217-225 epitope. this ratio is maintained over a range of intracellular antigen concentrations. our findings provide an estimate of the efficiency of antigen processing and demonstrate the remarkable capacity of the mhc class i antigen processing pathway to accommodate new epitopes. we have isolated and characterized three cytotoxic t lymphocyte (ctl) clones from the peripheral blood of two acute seroconversion patients and one patient in the first trimester of pregnancy. these clones were cd8+ and class i hlarestricted by the b7 molecule. all three clones recognized lllb and rf but not mn strains of hiv-1. using vaccinia vectors expressing truncated versions of the hiv-1 envelope, the clones were found to recognize an epitope within amino acids 287-364, but not including 312-328 of gp120. further mapping of the epitope with synthetic 20-mer peptides overlapping by 10, or 25-mers overlapping by 8, was unsuccessful. the sequence of the region of gp120 recognized by these clones was compared to the predicted hla-87 peptide binding motif and a possible matching region was found. using shorter peptides corresponding to this potential epitope recognition site, the minimum epitope recognized by the clones was determined to be the 10 aa sequence rpnnntrksi spanning amino acids we have further pursued a strategy to define a minimal cytotoxic epitope for a vaccine against cmv infection using t cell clones derived from individuals who have the mhc 835 gene (kind gifts of drs. riddell and greenberg, fred hutchinson cancer research center and dr. robert siliciano, johns hopkins university medical center). we tested by chromium release assay (cra) the recognition of a series of 835 allelic variants of ebv-lcl. by 835 restricted and cmv or hiv-specific t cell clones. several conclusions quickly became apparent. the previously described 8'3501 peptide epitope from pp65 was not able to prime the autologous 835 ebv-lcl for killing by the pp65-specific ctl, whereas a recombinant vaccinia virus expressing whole pp65 could cause the same cell line to be recognized and killed in the same experiment. in addition, an hiv gp41-specific cd8' ctl which has a defined minimal cytotoxic epitope will only recognize and kill a subset of 835 ebv-lcl. the two t cell clones will not recognize each other's autologous ebv-lcl. the resolution of this interesting phenomena comes from sequence analysis of the hla class i b genes from both ebv-lcl. ebv-lcl which contain the b'3502 allele are recognized and killed by the pp65-specific t cell clone, and cell lines carrying 8'3501 alleles are recognized by the hiv gp41-t cell clone. we conclude that the reported cmv pp65 b"3501 restricted epitope is not correct, since the ctl in question will only recognize 6'3502 alleles in combination with the correct pp65 epitope. fragments with or without a signal sequence sensitize rma-s/kd to a similar limited extent. this data i s consistent with an inefficient movement of peptides from the cytoplasm into the er by a tap independent mechanism and does not reveal a processing competent compartment within the secretory pathway. peptide transport by the transporter associated with antigen processing (tap) was studied using a microsome system as previously reported by heemels et. al.. in this system, a radiolabeled synthetic peptide which can be n-link glycosylated is used as the indicator peptide for the transport studies. the transport efficiency of synthetic peptides corresponding to antigenic peptides restricted to the murine kd molecule was measured by inhibition of labeled peptide transported into the microsomes. the transport efficiency of three kd epitopes in the type a influenza virus "147-155, ha204-212 and ha210-219 was found to be similar. an 11 amino acid peptide corresponding to ha204-214 which contains the 204-212 epitope was transported at a similar efficiency as the 9 amino acid minimum epitope. however, when the peptide sequence is further extended by one amino acid to residue 215, this peptide is poorly transported. these results suggest that the flanking region of an epitope can dramatically influence the transport of the epitope. when the transport kinetics of tap was studied using the microsome system, the vmax for transporting the indicator peptide (a variant of np epitope that has the sequence tynrtrali) was found at 260.8 fmolelminute (+/-30.5). the km for this peptide was found to be 231.9nm(+/-31.8). bypassing a block in antigen processing for class i-restricted cytotoxic t cell recognition. amy j. yellen-shaw and laurence c. eisenlohr. thomas jeferson universitv. hiladelphia, pa., 19107. previous work from our laboratory showed that processing of an influenza nucleoprotein (np) epitope (amino acids 147-155) expressed endogenously from a recombinant vaccinia virus "minigene" is severely impaired when a flanking sequence (the dipeptide threonine-glycine) is appended to the cterminus of the construct (147-158/r-). the inhibition of processing is overcome by placing the unprocessed peptide in the context of the fulllength np molecule, demonstrating that regions of a protein outside the epitope itself critically affect the ability of the proteolytic machinery to fragment the protein appropriately. to determine the requirements for bypassing the block in antigen processing, we have constructed an array of "minigene"-expressing vaccinia recombinants in which the unprocessed epitope is extended by varying lengths toward either the c-terminus or the n-terminus of the np molecule. our results show that while an extension of the c-terminus by only one amino acid restores processability, a much longer extension of the n-terminus (75 < n < 100 amino acids) will also allow the substrate to be processed. it is therefore clear that a full-length, properly folded molecule is not required for liberation of the blocked epitope, and that probably more than one mechanism can contribute to enhancement of substrate proteolysis. we hypothesize that the c-terminal extension allows recruitment of an endopeptidase versus exopeptidase ("trimming") activity which is capable of cleaving the difficult bond. we considered the possibility that the n-terminal extension rescues processing by recruitment of the ubiquitin-dependent degradation system. to address this possibility we replaced all available ubiquitination sites (lysine residues) in one of the rescued constructs (50-158/r-) to see if the construct would still be processed and presented. the six available lysine residues were changed to arginine using pcr-based mutagenesis. the resulting construct (termed 6r) was recombined into vaccinia virus and tested for presentation to np-specific ctl. the 6r construct was presented at a level equivalent to that seen with the wild-type 50-158/rconstruct. this result provides clear evidence that entry into the ubiquitindependent degradation pathway is not responsible for rescue of presentation in this system and more importantly, that ubiquitination is not required for processing of all large substrates. chia-chi ku, li-jung chien ,and chwan-chuen king, institute of epidemiology, national taiwan university, taipei, taiwan, r.o.c. dengue virus (den) can cause dengue fever (df) and dengue hemorrhagic fever (dhf) i dengue shock syndrome (dss) and den-2 was the most common serotype found in dhf outbreaks globally. current hypotheses suggested that dhf may be associated either with antibodydependent enhancement (ade) or with viral virulence. den can replicate predominantly in monocytedmacrophages (mim), but whether peripheral blood lymhocytes (pbls) are the target cells of den still remain controversial. in order to compare whether various clinically derived den-2 will interact with mim and lymphocytes in different manners, we used two isolates --plo46 strain (obtained from a df patient during taiwan 1981 outbreaks) and 16681 strain (isolated from a dhf patient in thailand by cdc, usa) to infect primary mim and lymphocytes as well as several types of cell lines. primary lymphocyte culture was nonadherent cells obtained after 24 hr adherence of pbmcs, whereas the primary mim culture was collected by depletion of lymphocytes using anti-cd3icdi9 mab and complement prior to adherence procedure and the purity of mim culture was checked by cd14 surface marker staining. supernatants (sn) of virus were harvested at various time points post infection after with several or without treatments. our prelimanary data showed that dhf-associated den-2 strain had higher viral yield in certain age of mim and a promonocytic cell line (hl-cz) than taiwan df-associated den2 strain. in addition, this dhf-den2 strain was more likely to infect the promonocytic (hl-cz) than well differentiated monocytic (ctv-1) and lymphocytic (h9) cell lines and also had higher peak yields than den-i virus in hl-cz cells. interestingly, dhf-den2 strain replicated much more efficiently in primary lymphocytes no matter these cells were activated with pha or not, whereas taiwan df-den2 strain virus was hardly detectable in sn of both activated and non-activated lymphocyte cultures. therefore we conclude that (1) different strains of dengue virus could orchestrate quite differently with immune cells, (2) different stage of mim differentiation might be an important permissive determinants for dengue virus infection and replication, and (3) den virus strain virulence -a more important factor than lymphocyte activation status -seemed to determine whether this strain would infect human pbls. further studies should be focused on searching for detaied mechanisms of virus and immune cell interactions. (2) when viral yields were enhanced early than day5 post infection, it provided tremendous opportunity to attack the immune system and finally may lead to severe disease. hiv-1 using recombinant immunoglobulin molecules, marie-claire gauduin, graham p. allaway, paul j. maddon, carlos f. barbas, dennis r. burton, and richard a. university school of medicine, new york. ny 10016. primary isolates of hiv-1 have been shown to be less sensitive to neutralization by immune sera, monoclonal antibodies and cd4-based molecules than t cell line-adapted strains of hiv-i. we studied two immunoglobulin molecules for ability to neutralize primary isolates of hiv-i. lgg12 is an immunoglobulin molecule created from a combinatorial phage expression library and reacts with the cd4 binding site (cd4-bs) on gp120. cd4-lgg2 is a recombinant molecule in which the variable domains of both heavy and light chains of lgg2 were replaced with the first and second immunoglobulin-like domains of human cd4. both molecules have been previously shown to effectively neutralize hiv-i in vitro. ex vivo neutralizations were performed as follows: lgg12 and cd4-lgg2 were added at 25 pg/ml to wells containing serial dilutions of plasma from hiv-i-infected patients and phastimulated peripheral blood mononuclear cells from seronegative donors. p24 production was measured over 14 days of culture and an end-point titer of hiv-1 in the presence and absence of added antibody was determined. both igg12 and cd4-lgg2 were found to reduce the original hiv titer from seven plasma samples with high virus titer (>250 tcid50/ml) by up to 625-fold. this is in comparison to soluble cd4 which only reduced viral infectivity by 55-fold at the same concentration. in vitro binding and neutralization assays on isolates recovered from plasma confirm the potency and breadth of neutralization by these two molecules. these studies suggest that recombinant antibodies directed at the cd4-bs of hiv-1 gp120 are able to effectively neutralize primary isolates of hiv-1 and may be useful in dissecting the mechanisms of resistance to neutralization by other antibodies. dillner and p. heino, microbiology & tumor biology center, karolinska institute, stockholm, sweden hpv 16 the major cause of anogenital precancers in man. the search for neutralizing epitopes that could form the basis for a preventive vaccine has shown that the surface-exposed imunodominant epitopes of the capsid are strongly conformationdependent, which has precluded detailed epitope analysis. similarly, immunization with whole, denatured capsid proteins has only identified linear immunodominant epitopes positioned on the inside of the capsid. reasoning that linear surface-exposed epitopes should exist, but might be cryptic, a set of 66 overlapping synthetic peptides corresponding to the entire hpv16 capsid proteins was used to generate hyperimmune sera. several antisera against 3 different peptides were reactive with intact hpv16 capsids at titers up to 1:150.000. hiv-1 serum antibodies and mucosal iga. basil golding, john inman, paul beining, jody manischewitz, robert blackburn and hana golding. div. of hematology and viral products, cber, fda, and lab. of immunology, niaid, bethesda md 20892. previously, we showed that hiv-1 proteins conjugated to 8. abortus (ba) could generate anti-hiv-1 neutralizing antibodies in mice even after depletion of cd4* t cells. in this study a 14-mer peptide from the v3 loop of hiv-1 (mn) was synthesized 013) and coupled to ba and klh. balb/c mice were immunized twice i.p. with these conjugates at two week intervals. v3-klh induced mainly igg1, whereas v3-ba induced all igg isotypes but lgg2a predominated. fecal extracts from mice immunized with v3-ba were shown by elsa to contain iga antibodies. sera from these mice bound gp120, expressed on the surface of infected cells. sera from mice immunized with v3-ba inhibited syncytia formed between cd4' t cells and chronically infected [hiv-i (mn)] h9 cells. inhibition of syncytia, formed by other hiv-1 lab. strains correlated with the degree of their homology with the v3 region of hiv-i (mn). to mimic the efffect of hiv-1, mice were depleted of cd4' cells using anti-l3t4 at the time of primary or secondary immunization. following primary immunization, cd4+ t cell depletion abrogated v3-klh antibody responses, whereas responses to v3-ba were retained and sera from these mice were able to inhibit gp-120mediated syncytia. in secondary responses, cd4' t cell-depletion prevented boosting to v3-klh, but v3-ba increased anti43 and syncytia-inhibiting antibodies. these results suggest that: 1. 8. abortus, can provide carrier function for a peptide and induce both serum and mucosal antibody responses, and 2. that infection with hiv-1 with subsequent impairment of cd4' t cell function would not abrogate anti-hiv-1 antibody responses if 8. abortus is used as a carrier to stimulate memory responses. nucleotide sequence analysis of the vh genes revealed the usage of one particular vh germline element (vh61-1p) in all clones. this finding allowed the determination of somatically mutated positions in the vh regions. two vsv-ind neutralizing antibodies expressed vh and vl genes in complete germline configuration whereas the rest of the clones showed somatic mutations which obviously were antigen dependently selected for. however, binding affinities of mutated and unmutated antibodies were comparably high. in order to determine the influence of somatic point mutations on one single antibody we generated a monovalent single chain antibody (fv-ck) of a mutated clone and reversed it stepwise to germline configuration by means of site directed mutagenesis. surprisingly, already the germline configuration of fv-ck could neutralize vsv-ind, even though the binding affinty was lower than that of the mutated fv-ck. every single somatic point mutation tested improved the binding avidity although some mutations reduced affinity. thus, during the course of vsv-ind infection some antibodies are subjected to avidity maturation although this is not required for the generation of high affme, efficiently virus neutralizing antibodies, lisa hyland'", sam hou'.~, and peter c. doherty'. 'department of immunology, st. jude children's research hospital, memphis, tn 38 10 i, 2departments of immunology and microbiology, and 'pathology, university of otago,dunedin, new zealand. the b and t cell responses in c57bl/6j(b6) mice treated with the mab mel-14 to l-selectin have been analysed following i.n. infection with sendai virus. mel-14 treatment caused a 70-90% decrease in the lymphocyte recruitment to the mediastinal (h4ln) and cervical (cln) lymph nodes following infection with sendai virus. the cellularity of the spleen was unchanged. the clonal expansion of cd8+ ctl precursors in the mln was slightly delayed, but potent ctl effectors were present in the virusinfected lung by day 10 after infection and the overall magnitude of the response was not compromised. the prevalence of iga antibody forming cells (afcs) was greatly increased in both the mln and the cln of the mice given the mel-14 antibody. the igm response was prolonged and the igg response, particularly iggl, was delayed compared to controls. the altered pattern of the antibody response may reflect the limited availability in mel-14-treated mice of th cells secreting lymphokines which are involved in ig class switching, by blocking the entry of cd4+ th precursor cells into lymph nodes. facs sorting for l-selectin+, 8220+, and l-selectin-, b220+ cell populations from the mln and the cln of normal b6 mice 9 days post sendai virus infection, showed that the afcs were from the l-selectin-, b220+ cell population, a population which comprised 6-10% ofthe total cell population. we have distinguished targets of broadly neutralizing antibodies present in hiv-1 infected individuals by imunoselection in vitro and by the use of chimeric virus. one target of neutralizing antibodies, defined by an escape mutant with an ala to thr substitution at position 582 in gp41, is resistant to human monoclonal antibodies that map to a site closely congruent with that for cd4 binding. substitution of gly, ser, and val fail to confer resistance. a second, defined by an ala to val substitution at position 281, upstream from the v3 loop, does not involve the same site and does not involve v3. substitution of thr or ile also confers resistance. replacement of the v3 loop of hiv-l(mn) into a clone of hiv-l(iiib) allows the detection of two other broadly neutralizing targets. one recognizes the v3 peptide of mn but is affected by regions outside v3. the other appears to be conformational and outside v3, but its functional recognition is influenced by the v3 loop. all of these sites seem to depend on the overall conformation of the envelope protein rather than a single discrete linear epitope. antibodies against amino acids 579-613 of the hiv transmembrane (tm) glycoprotein have been shown to enhance hiv infection in vitro in the presence of complement. there has been no study demonstrating that enhancing antibodies to this region of hiv, despite increasing levels of infectious virus 10 to 100 fold in vitro, adversely affect disease pathogenesis. in two separate studies reported herein, it is shown that animals which have high levels of antibody against this region of siv, amino acids 603-622 of the envelope, fair poorly compared to animals with lower antibody levels against this region when subsequently challenged with siv. when actively immunized with a synthetic peptide from this region of siv, animals died earlier and failed to clear antigen at two weeks after infection compared to animals that received a control peptide (p<0.05). when animals were passively immunized with antibodies from a longterm survivor of siv infection, those animals that received higher levels of antibody against the tm peptide died within six months compared to longer intervals for those animals that had lower levels of antibody to this region. when taken together, these data suggest that antibody to the tm region of siv and hiv in general, and to this highly conserved peptide in particular, are detrimental to the host. therefore, immunization strategies that minimize the immune response against tm or treatment protocols that decrease antibody levels against tm may lead to prolonged survival following exposure to lentiviruses. we have developed a mouse model to examine the immune response to hpv 16 proteins when these proteins are presented to the immune system via the epithelial route. in this model animals are grafted with keratinocytes expressing hpv e6 a n d e7 genes using a transplantation procedure which permits epithelial reformation. animals so grafted when challenged intradermally with e7 either as protein or via a recombinant vaccinia virus exhibit a delayed type hypersensitivity response which is e7-specific and cd4+ t cell mediated. animals grafted with a sub optimal priming inoculum of cells develop immune non-responsiveness and have an abrogated dth response when challenged subsequently with a priming cell graft. in the present study w e have examined the antibody status in these animals. the e7 protein of hpv 16 was expressed in e. coli as a maltose binding fusion protein using the plasmid vector pmalc. after cleavage and affinity purification this protein was used in a n elisa assay to measure antibody levels in 4 groups of mice (1) those not challenged with e7 (2) mice not grafted but challenged with e7 protein in the ear (3) mice primed by grafting with 107 hpv e7 expressing cells and challenged with e7 protein (4) mice primed by grafting with 5 x 105 hpv 16 e7 cells on day 7, grafted again with lo7 hpv 16 e7 cells on day 14 and challenged with e7 protein in the ear. mice optimally grafted and challenged (group 3) exhibited high titres of igg antibodies, particularly elevated levels of iggza. mice sub-optimally grafted (group 4) exhibited igg antibody levels comparable to the control group (1). the possible mechanisms of this immune attenuation are discussed. the hepatitis c virus is a frequent cause of chronic liver disease. a proposed mechanism responsible for virus persistence is evasion of the host immune response through a high mutation rate of crucial regions of the viral genome. the portion of hcv genome coding for the amino-terminal part of the putative envelope protein (gp70) undergoes frequent mutation during the course of infection. we have cloned and sequenced the hypervariable region (hvri) of the virus isolated from an hcv asymptomatic patient at three time points during 18 months follow up. sequence analysis has allowed the identification of variants of this region and multiple antigenic peptides (map), corresponding to three hvrl variants, sequentially foundin the blood stream of the patient, have been synthesized. maps have been used as antigens for detection of specific antibodies in elisa. our results show that anti-hvri antibodies and their cognate viral sequence coexist in the blood stream but a viral sequence becomes undetectable when the specific antibodies reach maximum levels of reactivity. thus humoral immunity against the hvrl may play a role for virus clearence. the presence of anti-hvr1 antibodies was also investigated in 100 hepatitis c viremic individuals and 25 non-viremic patients. a high frequency of positive reaction (90%) against at least one of the three hvrl variants analysed in this study was detected in the viremic patients. finally, competition experiments show that antibodies crossreacting with more than one hvrl variant are produced by hcv infected individuals. this results suggest that complex cross-reactivity exist between hcv isolates for antibodies against the hvrl region as described for antibodies against the gp120 v3 loop of hiv. we propose as mechanism for viral escape in hcv chronic infections the one described as the "original antigenic sin", observed firstly in influenza, in togavirus, paramixovirus, enterovirus, and recently in hiv infection. using an adult mouse model to study active immunity against rotavirus infection, it was previously shown that oral immunization with some, but not all, animal rotavirus strains induced protection against subsequent infection following oral challenge witb the murine rotavirus strain edim (ward et al., 1992) . to determine i f a specific rotavirus protein could be associated with protection in this model, mice were immunized with a series of 18 reassortants between the fully protective edim strain and a partially protective heterologous rotavirus strain (rrv-g). reassortants that contained genes for edim proteins responsible for protection were anticipated to provide complete protection; however, no edim proteins were found to be both necessary and sd3cient for full protection. instead, protection was found to be highly correlated with viral shedding (p = ,005) and with serum rotavirus iga titers stimulated by the different reassortants (p < ,001). this indicated that protection was related to the intestinal replication properties of the different reassortants rather than to specific immunogenic properties of edim proteins. this conclusion was supported by the finding that the titers of serum rotavirus i& but not igg, stimulated in mice following oral immunization with a series of animal rotaviruses was directly related to protection against edim. if these findings can be extended to humans, they suggest that the efficiency of intestinal replication following oral inoculation with a live rotavirus vaccine candidate may be the primary determinant of successful immunization. h a l l medical center, 55 individuals with adequate serum samples were identified as either rapidly progressing (rp) or slowly progressing (sp) by clinical and surrogate marker criteria. anti-v3 profiles were determined using synthetic proteins derived from the amino acid sequences of the v3 region of 5 laboratory strains of hiv-1 in standard capture elisa format. serum obtained from each patient at multiple different time points was screened against these peptides. the majority of individuals in both groups demonstrated broad recognition, with reactivity to peptides corresponding to the v3 regions of mn, sf2, ny5 and han/sc. less than 50% of individual in each group recognized the v3 peptide derived from iiib, @=ns, between groups). as the rp progressed to aids there was significant nonspecific narrowing of response, while the sp remained broadly reactivity (p< .001). in v i m neutralizing activity of the homologous laboratory isolates was determined with cytotoxicity, cytopathic effect and p24 ag inhibition assays. although most patient serum was capable of inhibiting p24 ag production in homologous lab strains while aids-free, there was no relationship with the ability to inhibit homologous virus effects on target cells and anti-v3 profiles. model, we show that after resolution of the acute infection, when antiviral plasma cells in the spleen decline, a population of virus-specific plasma cells appear in the bone m w and constitute the major sou~ce of longterm antibody production. following infection of adult mice, wspecific antibody secreting cells (asc) peaked in the spleen at 8 days postinfection, but were at this time undetectable in the bone marmw. the infection was essentially cleared by 15 days and the asc numbers in the spleen rapidly declined while an increasing population of lcmv-specific asc appeared in the bone marrow. when compared to the peak response at 8 days post-infection, timepoints from 30 days to more than one year later demonstrated greater than a l@fold reduction in splenic asc. in contrast, jltvlv-specific plasma cells in the bone marrow remained at high numbers and correlated with the high levels of antiviral serum antibody. the prewnce of antiviral plasma cells in the bone marrow was not due to a persistent infection at this site, since virus was cleared from both the spleen and bone marrow with similar kinetics as determined by infectivity and pcr assays. the igg subclass profile of antibody m e t i n g cells derived from bone manuw and spleen correlated with the igg subclass distribution of lcmv-specific antibody in the serum. upon rechallenge with w , the spleen exhibited a substantial increase in virus-specific plasma cell numbers during the early phase of the secondary response, followed by an equally sharp decline. bone marrow asc populations and lcmv-specific antibody levels in the serum did not change during the early phase of the reinfection but both increased about 2-fold by 15 days post-challenge. after both primary and secondary viral infection, lcmv-specific plasma cells were maintained in the bone marrow showing that the bone marrow is a major site of long-term antibody production after acute viral infection. "memory" t cells, and associated with responsiveness to soluble and recall antigens. cd4+ lymphocytes staining bright, dim, or negative (equivalent to an isotype control) for cd29 were evaluated in 49 uninfected controls (group l), 84 hiv-1 positive patients with 220% cd4+ t cells (group 2), and 47 hiv-i-infected patients with ~2 0 % cd4+ t cells (group 3). most of these subjects also had 3-color staining for cd4\cd45ro\cd45ra. the appearance of positive cd29 and cd45ro on hivinfected and uninfected cells correlated well (r=.82 p<.ool). the percentage of cells staining cd4+\cd29+.(bright plus dim) was 43.3 (95%cl 37.3-49.4) in group 1, 28.9 (27.5-30.4 ) in group 2, and 10.2(8.6-11.9) in group 3. the respective values for these groups that were cd4+\cd2gbwm was 30.6 (26.9-34.3), 20.7 (1 9.3-22.2), and 7.4(6.3-8.6). values for cd4+\cd45ro+ were 33.7 (31.8-35.5), 21.8 (20.5-23.1), and 9.9 (8.5-11.3), respectively. in single factor discriminate function tests, the %cd4+\cd29+ cells best predicted subject group (87% correct), proving to be a better discriminator than %cd4+\cd29b'h' (77% ~orrect),cd4+\cd29~"" (5 l%), cd4+\cd45ro+ (75%) and cd4+\cd45ro+\cd45ra-(63%). overall, no advantage was seen to splitting the cd4+\cd29+ cells into bright and dim positive subsets in the subjects studied for the purpose of stratifying early vs. late hiv infection. likewise, splitting the cd4+\cd45ro+ compartment into cd45ra+ subsets did not improve the ability to distinguish between uninfected and early or late hiv-1 infected patients. the relationship between the virus-specific cytotoxic response in hiv infected patients and disease progression support the concept that a vaccine candidate should also induce a virus-specific ctl activity. immunization of uninfected adult volunteers by a hiv-gpl60 recombinant canarypox virus was carried out in a phase i trial.two injections of a recombinant canarypox expressing the hiv-l/mn gp160 were performed at month 0 and 1 and two boosts of recombinant gpl60mn/lai at month 3 and 6 in alum or incomplete freund adjuvant(1fa). hiv-envelope specific cytotoxic activities were detected from ctl lines derived from pbmc stimulated by specific stimulation with autologous hiv infected blasts. ctl lines were obtained from 18 out of 20 donors : seven out of eighteen (39%) were found to present envelope specific cytotoxic activity at months 2, 4, 7 or 12 post immunization ; this activity was characterized as a cd3+,cd8+, mhc class-i restricted cytotoxic activity, and for at least two volunteers, this activity was still present two years after the first canari-pox/env injection. because avian poxviruses are incapable of complete replication and undergo abortive replication in mammalian cells , this is a n example of the persistence of long term memory cd8+ cytotoxic t lymphocytes in the absence of the priming antigen, indicating that t-cell memory might be independent of continued antigenic exposure. the university of alabama at birmingham, al 35294. mhc class i restricted cd8' ctl activity plays an important role in the control of influenza virus infection as indicated in studies in mice and humans. cytokines such as il-2 and ifn-y regulate the generation of virus-specific ctl responses. we recently demonstrated a good correlation between the induction of influenza virus-specific ctl activity and the production of ifn-y by the cd8' t cells at the single cell level using an if?-specific elispot assay, secreted ifn-y by an elisa, and ifn-y specific mrna expression by rt-pcr. several recent studies have characterized cd4+ and cd8' t cells by their expression on the surface of distinct d45r isoforms. cd45ra is expressed on naive or virgin t cells, while cd45ro is expressed on memory t cells. in the present study, pbmc of healthy young adult subjects were stimulated with influenza a virus and then enriched for cd8+ t cells. the cd8' cells were stained for cd45ro' (pe) and cd45ra' (fitc) cells and sorted. ctl activity against virus-infected autologous target cells was determined in a 4 hour 'lcr release assay while ifn-y production and expression was assessed by elispot and quantitative rt-pcr, respectively. cds+/cd45ro+ (memory) cells exhibited significant mhc class i ctl while cds+/cd45ra+ cells exhibited no lytic activity. no activity was exhibited by freshly isolated or unstimulated cd8+/cd45ro+ t cells. similarly, cd8+/cd45rot t cells contained significantly higher numbers of ifn-y spot forming cells and higher quantity of ifn-y-specific mrna than cd8+/cd45rac cells. these data support our previous findings that ifn-y may serve as a useful surrogate marker for influenza virus-specific ctl activity in humans. in studying the kinetics of the cd8+ t cell response in lcmv infection we have observed a profound activation and proliferation of cd8+ t cells with a 10-40 fold increase in total number peaking at day 8-9 post infection. in c57bw6 mice, most of the viral antigen is cleared by day seven, and after day 9 the total cd8+ number per spleen drops about 10-fold. however, the relative specificity of the viral peptidespecific precursor ctl frequencies @ctwf) per cd8+ cell remains remarkably stable between day 7-8 of the acute infection and for many months thereafter. thus, the decline in the cd8' t cell number is not a function of the tcr specificities but is rather an across-the-board event. in contrast, we found that subsequent to the decline of the ctl response to a second heterologous virus infection such that the mouse was in a "resting, immune'' state, there often was a reduction in pctl/f to the first virus. for example, infections with w or mcmv substantially reduced the pctuf to lcmv or pv in all memory compartments, including spleen, lymph nodes, peritoneal exudate cells. reinfection with the original virus substantially elevated its pctuf and restored the pctuf that had been reduced by a heterologous viral infection. analyses of the progression of ctl responses during a heterologous virus challenge of a virus-immune mouse indicated a high frequency of crossreactive ctl appearing early during infection, but as the infection progressed there was a higher proportion of ctl specific only for the second virus. thus, we believe that when the across-the-board apoptosis of t cells occurs late in the infection, ctl specific for the first virus are diluted by those responding to the second virus. this may cause the reduction in memory to the first virus and may be one of the mechanisms contributing to the waning of secondary immune responses to certain viruses over time if there is no re-exposure to the original infectious agent. t-cells which arise after virus infection will aid our understanding of tcell memory and be useful in the design of vaccines which augment the memory response. to estimate the sendai virus specific precursor frequency in memory mice, cd4+ cells from c57bl6 female mice which had been infected with sendai virus intranasally (i.n.) more than two months earlier were subjected to limiting dilution analysis. responder cell populations were enriched for cd4+ cells either by magnetic bead depletion of non-cd4+ cells, or by facs after staining with anti-cd4 monoclonal antibody these enriched (>90% cd4+) responders were cultured with sendai virus-infected, irradiated, t-cell depleted splenic antigen presenting cells (apc). supernatants from these cultures were tested for activity on the cytokine-dependent ctll cell line. duplicate cultures of responders on uninfected apc were used to set the level of rejection (mean cpm + 3x std. dev.). using this type of analysis we were able to demonstrate a frequency of memory thp at 111600 cd4+ cells, compared to a frequency greater than 1/1ooooo in naive controls. the memory cd4+ cells were further characterized as cd45rb-low (1/472) , cd44-high (1/294), lselectin-low (1/364), and cd49d-high (vla-4-high) (v102). this is close agreement with other phenotyping studies on cd4+ memory cell specific for soluble antigens. t cells, ralph a. tripp, sam hou, anthony mcmickle, james houston and peter c. doherty, department of immunology, st. jude children's research hospital, memphis, tn 38105. the immune response of influenza a and sendai-virusspecific, memory cd8' cytotoxic t lymphocyte precursors (ctlp) have been analyzed in c57bu6 mice infected intranasally with unrelated or cross-reactive respiratory viruses. the numbers of influenza a-specific memory t cells increased in the regional lymph nodes (ln), spleen and bronchoalveolar lavage through the course of an irrelevant infection (influenza b). memory t cells showed evidence of enhanced steady-state activation. profiles of ctlp recruitment were analyzed in association with t cell proliferation and activation to determine whether signaling via the t cell receptor is necessary to induce "bystander" stimulation of the memory t cell pool. the extent of t cell proliferation was addressed by treating mice with low doses of cyclophosphamide (cy). "resting" sendai virus-specific memory t cells were unaffected by cy treatment, however upon challenge with influenza and treated 5 or 6 days later, the emergence of influenzaspecific ctlp was severely diminished. cell cycle analysis showed that cy eliminated the majority of cd8' t cells from the ln and spleen resulting in dna fragmentation of 12-18% ofthis lymphocyte subset. a decrease (though smaller) in the numbers of sendai virus-specific ctlp indicated that some of the cycling cells killed by cy were memory t cells, presumably activated in a "bystander" manner. the decrease in ctlp numbers for both influenza and sendai virus-specific ctlp was still apparent 9 days after cy treatment, long after the viral elimination. thus, immune responses to unrelated antigens may be a mechanism involved in maintaining the pool of memory t cells. experimentally vsv can result in an acute cns infection of mice. data from our in vitro experiments indicate that no has inhibitory effect on productive vsv infection. vsv infection at neuroblastoma nb41 a3 cells was significantly inhibited by loopm of a no donor s-nitro-n-acetylpencillamine (snap), while 1oopm of the control compound n-acetylpencillamine (nap) had no effect. when vsv infected nb41a3 cells were treated with 500pm of a constitutive no synthase (cnos) activator n-methyl-d-aspartate (nmda), a significant inhibition of vsv production was observed. inhibition by 500pm of nmda was reversed by 300pm of nos inhibitor n-methyl-l-arginine (l-nma). work is in progress to determine the effects of inducible nos (inos) in a glioma cell line c6 on vsv infection. levels of no and expressions of both cnos in neurons and inos in glial cells in the cns following vsv will be further investlgated. supported by nih grant a118083 to carol s. reiss. pediatrics, university of iowa, iowa city, ia. 52242 mouse hepatitis virus, strain jhm (mhv-jhm), is a neurotmpic coronavirus which causes acute encephalitis and acute and chronic demyelinating encephalomyelitis in susceptible rodents. 40.90% of suckling c57bu6 (kbdb) mice inoculated intranasally with mhv-jhm at 10 days and nursed by dams immunized against the virus develop a chronic demyelinating encephalomyelitis characterized clinically b hindlimb paralysis, at 3-8 weeks postinoculation. the chronic demyelinating encephalomyelitis nor the clinical symptoms. recently, it was shown that lymphocytes isolated from the central nervous system (cns) of c57bu6 mice both acutely and persistently infected with mhv-jhm display a cytotoxic t lymphocyte (ctl) response to the s protein of mhv-jhm. this response was further characterized by identifying the ctl epitopes that are recognized by a bulk population of ctls from the cns of mhv-jhm infected c57bv6 mice. three epitopes were identified using synthetic peptides and truncated forms of the s protein in primary ciz assays. the epitopes recognized were amino acids 510-518 (cslwngphl, db), 598-605 (rcqifani, kb), and 1143-1151 (nfcgngnhi, db). thus, the results indicate that cytotoxic t lymphocytes responsive to the s protein of mhv-jhm in c57bu6 mice recognize both kb and db-restricted cil epitopes. ctl lines and clones specific to these peptides and the entire s protein are being developed to test their biological significance in vivo with respect to the acute encephalitis and chronic demyelinating disease caused by mhv-jhm. a marked change in susceptibility to some neurotropic viruses during the first few postnatal weeks has long been recognised in rodents. infection of neonatal or suckling mice with the neurotropic alphavirus, semliii forest virus results in lethal encephalitis. infection of weaned animals is not lethal. earlier investigations focusing on changes in specific immunity have shown this not to be the explanation. infection of 3-4 week old mice with severe combined immunodeficiency does not result in acute rapidly fatal encephalitis. we have studied mortality, neuroanatomical distribution and spread of infection in mice of different ages and the effect of gold compounds on rendering infection of 3-4 week old mice lethal. neuroanatomical distribution of infection correlates with synaptogenesis. as this is completed in different systems within the first two weeks postnatal, systems no longer transmit virus and infection switches from disseminated to focal and restricted. complete productive replication and transmission of infection require smooth membrane synthesis which is present in neurones undergoing synaptogenesis, absent in mature neurones but inducible by administration of gold compounds. infection of neurones undergoing synaptogenesis is productive and virus is transmitted along neuralpathways, infection spreads rapidly around the brain, destroys cells and animlas die of a fulminant encephalitis. in mice infected after 14 days of age replication in mature neurones is restricted, nonproductive, cannot be transmitted, does not spread, is non-destructive and non-lethal. as a consequence, in the absence of immune responses virus can persist in isolated cns cells for life and can even be detected by reverse transcriptase pcr in immunocompetent mice months after infection. in the presence of an immune response, cd8+ t-cells recognise and destroy infected glial cells leading to dem yelination. a~k e r m a n n ,~ virology swine,' virology cattle,' and avian diseases3 research units, national animal disease center, usoa, agricultural research service, ames, ia 5001 0 a recombinant pseudorabies virus (prv) (lltbap) was constructed which contains a 3.0 kb deletion spanning the standard recombination junction of the unique long and internal repeat sequences replaced by e lacz expression cassette. this deletion interrupted the large latency transcript gene (llt) and truncated one copy of the diploid immediate early iel80 gene. replication and viral gene expression of lltbaz in madin-darby bovine kidney cells was similar to that of the parental virus and a virus rescued for the deleted sequences (lltbres). when inoculated intranasally in 4-week-old or 4-day-old pigs, lltba2 replicated efficiently at the site of inoculation yet caused markedly reduced fatality when compared to the parent or lltbres viruses. in particular, the lltba2-infected pigs did not exhibit neurological symptoms characteristic of prv infection. to further examine the pathogenesis of lltba2, 4-day-old pigs were infected intranasally with lltpa2 or lltbres and necropsied at various times postinfection. virus isolation from the nasal turbinate, tonsils, and trigeminal ganglia was comparable between the two viruses. although both viruses spread to the brain and induced an inflammatory response in cns tissues, virus isolation from brain tissues was reduced about 20-fold for lltpa2. abundant prv antigen was detected in the cerebrum and cerebellum of lltpresinfected pigs, but only a few antigen positive neurons were observed in the cerebrum of lltba2-infected pigs. while replication of lltbres in the brain progressed until death at 7 days post-infection, replication of lltpa2 in the brain ceased by 9 days post-infection and the pigs exhibited only mild clinical signs. since lltba2 is capable of spread to the cns, reduced neurovirulence of lltbaz is likely the result of its decreased ability to replicate in cns tissues. the cns is a target for hiv infection, and in individuals with aids this can lead to a devastatin dementia. only certain viral variants appear capable 07 invading the cns and infecting microglia and brain macrophages. in order to determine whether the virus entering the brain may be particularly pathogenic to the cns, we isolated microglia from the brains of siv-infected rhesus monkeys. transfer of these cells into naive animals indicated that productive siv infection could indeed be transferred. furthermore, cns infection occurred within a relatively short time span, and was associated with viral gene expression in the brain and pathology characteristic of hiv encephalitis. serial transfer of microglia into additional animals also resulted in successful transfer of infection, neuroinvasion, and neuropathology. behavioral analysis in a trained group of animals is ongoing. this result demonstrates that neuropathogenic virions partition into the cns during natural siv infection, likely driven by mutational events that occur during the course of infection. molecular characterization of the microglia-associated virus has revealed that a distinct pattern of sequence changes in the envelope gene occurs concomitantly with this in vivo selection. our approach will allow the dissection of functional neuropathogenic elements present in these viruses. in non-specific host defense mechanisms. ifn-y-induced nitric oxide (no) in murine macrophages was previously shown to inhibit the replication of poxviruses and herpes simplex virus type 1 (hsv-1) . we now demonstrate that murine macrophages activated as a consequence of vaccinia virus (vv) infection in viva express inducible nitric oxide synthase (ios). the vvelicited macrophages were resistant to infection with w and efficiently blocked the replication of w and hsv-1 in infected bystander cells of epithelial and fibroblast origin. this inhibition was arginine dependent, correlated with no production in cultures and was reversible by the nos inhibitor nqjmonomethyl-l-arginine. the mechanism of no mediated inhibition of virus replication was studied by treating vv-infected 293 cells with the noproducing compound, s-niuoso-n-afetyl-penicillamine. antibodies specific for temporally expressed viral proteins, a vv-specific dna probe and transmission electron microscopy were employed to show that no inhibited late gene protein synthesis, viral dna replication and virus particle formation, but not expression of the early proteins analyzed. further, we have also identified putative enzymatic targets of inactivation by no that results in inhibition vv replication. although antiviral ctl are important for virus elimination. they can only halt further virus spread, and cannot reduce the number of infectious particles already present. the beneficial effect of ctl-mediated lysis is apparent only if infected cells are lysed before assembly of progeny virus. if infectious virus was released from infected cells in solid tissues before the generation of neutralizing antibody or in sites where antibody did not readily penetrate, then recruitment of mononuclear phagocytes, which phagocytose and destroy infectious material and/or become non-productively infected, would definitely help control virus dissemination. in this context, inos induction in macrophages may be an important antiviral strategy. in addition, the inhibition of virus replication in infected contiguous cells by inos-expressing macrophages at infectious foci would prevent release of mature viral particles after lysis by nk cells and ctl. since viral early proteins are expressed in such infected cells, their recognition and subsequent lysis by ctls will not be hindered. cns persistence, tropism and genetic j. pedro s i s , anthony a. nash and john k. fazakerley, department of pathology, university of cambridge, cb2 iqp, uk theiler's murine enchephalomyelitis virus, a natural occuring enteric pathogen of mice, is a picomavirus belonging to the curdovirus genus. following intracerebral inoculation of 3-4 week old cba or balb/c mice, the bean strain causes a chronic persistent cns demyelinating infection in a proportion of the cba that survive acute infection. balb/c mice are resistant to chronic demyeliating disease. we have studied the tropism, persistence and genetic variability of bean, in cba and balb/c mice in the chronic phase of this disease. by in situ hybridisation and reverse transcription (rt) pcr and southern blot analysis, no viral rna could be detected in the cns of any balb/c mice later than day 60 post-infection. in contrast, in a large group of cba mice studied up until 393 days post-infwtion, viral rna could be detected by both techniques in 50% of mice until as late as 268 days post-infection. by employing a combination of, in situ hybridisation for viral genome followed by immunocytochemistry for cell phenotypic markers, bean rna was observed predominantly in oligodendrocytes and occasionally in astrocytes during persistent infection, in both brain and spinal cord. in the persistently infected mice, the striking total destruction of the pyramidal layer of the hippocampus, substantia nigra and anterior thalamic nuclei indicated that these were the mice that had had greatest dissemination of virus and highest virus titers during the preceeding acute phase of infection. direct pcr t h d cycle sequencing of uncloned rt-pcr products, revealed that during persistent infection, loops i and ii of the vpi capsid protein gene did not undergo any genetic variability. furthermore, no changes were detected in this region in sequenced pcr products amplified from the cns of mice with severe combined immunodeficiency in which no selective immunological pressure would have been operative. infection, thomas e. lane, michael j. buchmeier, dorota jakubowski, debbie d. watry, and howard s. fox, department of neuropharmacology, the scripps research institute, la jolla, ca 92037 our laboratory is interested in the effects of siv infection in the central nervous system of rhesus macaques. to enrich for neuroinvasive and neurovirulent viruses, microglia were isolated from infected monkeys and used t o infect new, uninfected monkeys. such microglia-mediated infection resulted in the production of neuropathological changes, including giant cells, macrophage infiltrates and microglial nodules in recipient animals within 4 months. microglial cells isolated from siv-infected monkeys produced virus in vitro as measured by reverse transcription (rt) and p27 production. treatment of microglia with recombinant human interferon alpha (rhulfn-a) resulted in a sharp decrease in viral activity (both rt and p27 production) suggesting that rhulfn-a is able t o modulate viral activity in infected microglia. we have analyzed slvenv sequences by pcr amplification directly from microglia dna preparations from monkeys. nucleotide sequence analysis results in an enrichment of unique sequences in the v1 region of the siv env gene. the majority (>95%) of nucleotide changes encoded amino acid changes, indicating that these envelope sequences evolved as a result of selection. moreover, sequential passage of sivassociated microglia resulted in an increase in potential n-linked glycosylation sites within the v1 region of the env gene when compared with the parental virus. these data suggest that sequential passage of microgliaassociated siv may select for neuroinvasive, neurovirulent variants. the adoptive transfer of ctl specific for an ld-restricted epitope within the nucleocapsid protein of the jhmv strain of mouse hepatitis virus both protect from acute infection and reduce virus replication in the mhc class 1 positive cells within the cns. the source of these ctl and the route of their delivery is critical in the outcome of this protection. for example, 10 fold less spleen cells activated in vitro with the pn peptide are required for protection via the direct i.c. route than the i.v. route. in addition, ctl clones are unable to protect via the i.v. route and are very efficient via the i.c. route. these data suggested the possibility that the cd4+ t cells within the polyclonal activated spleen cell population derived from in vitro culture on the pn peptide were facilitating access to the cns. to examine this question, polyclonal pn-specific t cells were either depleted of cd4+ t cells prior to transfer to infected recipients or untreated cells were transferred to recipients depleted of cd4+ t cells with monoclonal antibody gk1.5. both of these treatments eliminated the ability of the ctl to reduce virus replication within the cns, suggesting that cd4+ t cells in the peripheral compartment are required for the entry of ctl into the parenchyma of the cns during acute cns encephalomyelitis. division of retrovirology, walter reed army institute of research and henry m. jackson foundation, rockville, md 20850; department of retrovirology, armed forces research institute of medical sciences, bangkok, thailand background the hn-1 epidemic in thailand is largely due to two highly divergent subtypes of virus, b and e. dual infection with distinct hn-1 subtypes, which has not been reported previously, would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. merhoak: pcr typing and serologic typing were used to screen a panel of non-random convenience specimens from hiv-1 infected subjects in thailand. specimens that showed dual subtype reactivity in these assays were subjected to differential pmbe hybridization and nucleotide sequence analysis of multiple molecular clones to c o n f m the presence of dual infection. results. two individuals were shown to simultaneously harbor hiv-1 of env subtypes b and e (table) . additionally, both subtypes were identified in co-cultured pbmc from one individual. conclusions. these data provide the fmt evidence of dual hiv-i infection in humans and reinforce the need for polyvalent vaccines. infection by herpes simplex virus wsv) induces in man and in mice cytolytic t lymphocytes (ctl) which recognize the immediateearly protein icp27. because of its early expression during the hsv replication cycle, lcp27 represents a prime target for specific t cell responses susceptible of controlling virus replication. we have expressed in e. coli a remmbinant construct coding for a fusion protein consisting of a fragment of influenza virus non-structural protein4 (nsi) and the lcp27 sequence of hsv-2. the nsi-icp27 protein was purified by preparative eleclmplmresis and formulated in oil-in-water emulsions with monophosphoryl lipid a (mpl) and qszl adjuvants. balwc mice were immunized by two intrafootpad injections of formulations containing 5 pg of nsi-icp27. responder cells obtained from draining lymphnodes were re-stimulated in vitro with p815 cells lransfected with icp27 and then lesled for cytolytic activity on icp27-p815 and control p815. the induction of icp27 specific ctl by different formulations was observed and will be discussed. the induction of heterologous cytotoxic t lymphocytes (ctl) using cassettes of multiple conserved t cell epitopes derived from different proteins and/or virus strains is envisioned as a promising vaccine approach. to study the effects of antigen processing on peptide presentation from chimeric epitope precursors we are using a model system comprising two distinct viral epitopes which are immunodominant in the h-2d haplotype: a dd restricted epitope from the gp160 protein of hiv-1 and an ld restricted epitope from the murine hepatitis virus nucleocapsid protein (mhv n). the influence of proximity and flanking sequences of epitopes on antigen presentation was analyzed using vaccinia virus (vv) recombinants in which the epitopes were expressed as chimeras containing the individual epitopes in reverse order or separated by different spacer residues. whereas individually expressed epitopes were efficiently recognized by protein-specific ctl, recognition of peptides derived from tandem constructs varied significantly with closer epitope proximity and sequential order. following immunization with the recombinant viruses, the chimeras were all able to induce antiviral ctl specific for the native proteins. however, cn, frequency analysis indicated that the number of responder cells to the same epitope dramatically depends on its context within the chimera and correlates with antigen recognition in vitro. the profound effect of flanking regions on ctl induction suggests that the context of an epitope will require careful evaluation in the design of recombinant multivalent minigene vaccines to induce an optimal t cell mediated immune response. and robert e. johnston', departments of 'microbiology & immunology and %iochemistxy, univ. of north carolina, chapel hill, nc 27599 a hll-length cdna clone of venezuelan equine encephalitis virus w e ) has been altered to contain two strongly attenuating mutations and a second subgenomic rna promoter immediately downstream of the structural gene region. expression ofthe influenza ha protein from this second promoter in baby hamster kidney (bhk) cells was approximately 50?? of the level in influenza virus-infected cells, as measured by immunoprecipitation. fourweek-old cd-1 mice were inoculated subcutaneously with 2 x 10' p h of the ha vector, vector alone or diluent. expression of ha mrna was detected in the draining lymph node of ha vector-inoculated mice by in situ hybridization, consistent with the organ tropism of vee. mice were challenged three weeks after imnmization by intranasal administration of lo5 ed, of influenza h s . au 24 corn1 mice suffered severe disease and 50% died. only one of 12 ha vector-inoculated mice died, and another exhibited signs of disease for one day and recovered. the geometric mean elisa titer of anti-ha serum igg in the ha-vector inoculated mice was 246, while only three control mice had measurable serum reactivity, and that was at the lowest dilution tested, 150. in a parallel experiment, no influenza infectivity was detected in the lungs of 12 ha vector immunized mice at 4 days postchallenge. in contrast, 8/12 pbs-inoculated mice and 5/12 inoculated with vector alone were positive for influenza infectivity and had geometric mean titers of 3.04 and 1.93 x lo6 pwgm, respectively. this vector also has been used to express the h n w c a protein in a form recognized by patient sera and a specific antibody on western blots. these experiments demonstrate the feasibility of using vectors based on attenuated vee cdna clones for protective immunization against heterologous human and animal pathogens. dose/response curves have been used to compare different routes of immunization with plasmid dna encoding the h1 hemagglutinin glycoprotein of influenza virus. routes of inoculation included intramuscular, intradermal and gene gun delivery of dna. from 100 to 0.1 ug of dna was inoculated by intramuscular and intradermal routes. from 0.4 ug to 0.0004 ug of dna was inoculated by gene gun. each route was evaluated for single and boosted immunizations. antibody titers were followed over a 20 week period, following which animals were evaluated for protection against a lethal challenge. each of the routes raised both antibody and protective responses. gene gun-delivery of dna required 250 to 2,500 times less dna to raise responses than the intramuscular and intradermal inoculations. boosts did not have much of an effect on antibody titer or protection except at low dose inoculations (4 ng and lower for the gene gun). for each of the routes, antibody responses showed good persistence over the 20 weeks of the experiment. inoculation of mice with plasmid vectors carrying a microbial gene under the control of an appropriate promoter results in a full spectrum of immune responses to the vectorencoded antigen. using a murine rabies model a plasmid termed psgsrab.gp expressing the full-length rabies virus glycoprotein regulated by an sv40 promoter was shown to induce upon inmuscular inoculation a rabies virus specific t helper cell response. of the thl type, cytolytic t cells and virus neutralizing antibodies resulting in protection against a subsequent challenge with live rabies virus given either peripherally or directly into the cenaal nervous system. a response comparable in magnitude was also induce upon inoculation of a vector expressing a secreted form of the rabies virus glycoprotein. the immune response to the dna vaccine could be modulated by co-injection of the rabies virus glycoprotein-expressing vector with plasmids expressing mouse cytokines. inoculation of mice with the psg5rab.gp vector and a vector expressing granulocyte/macrophage colony stimulating factor (gm-csf) enhanced both the t helper and the b cell response to rabies virus thus improving vaccine efficacy. co-inoculation with vectors expressing interferon-g failed to improve the response. co-inoculation of the antigen-expressing vector with a plasmid encoding mouse i l 4 caused a reduction of both the t helper cell response and the b cell response to rabies vlns. hpvl6 e7 hpv associated cervical cancer cells express hpv16e7 protein and antibody to hpv16 e7 can be detected in the blood of cancer patients, yet the twnours are. not rejected. a mouse transgenic for the e7 protein of hpv16, and expressing e7 protein in the skin, has recently been described (1) and these mice develop spontaneous humoral immunity to e7 protein similar to patients with cervical cancer(2). to determine whether immunisation could induce immunity to e7 sufficient to allow tumour rejection, we firstly demonstrated that immunisation of h-zb mice with hpv16e7 protein with quil a as adjuvant could induce cytotoxic t cells able to kill hpv16 e7 expressing tumour cells in nrro. we then used similar immunisation with e7iquil a to induce e7 specific immunity in fvb (h-29) mice. h-2qskin @s expressing e7 were not rejected by e7 immunised h-zq mice, though immunisation induced antibody to e7, and similar grafts were rejected, as expected, across an doantigen mismatch in h-zb mice. we conclude either that hpvl6 e7 lack a tc epitope in the context of h-zq, or that expression ofe7 in the skin from the e7 transgenic mice is insufficient for recognition by primed effector cells, and further experiments will address this distinction. cervical carcinoma is strongly associated with infection by human papillomavirus (hpv) types 16 or 18, and continued expression of the e6 and e7 gene products. this provides an opportunity for an immunotherapeutic approach to the treatment of cervical carcinoma by activation of immune reponses directed against these virally encoded tumour specific antigens. we have constructed a recornbinant vaccinia virus expressing e6 and e7 from hpv16 and 18 with the aim of inducing e6 and e7 specific hla class i restricted cytotoxic t lymphocytes (ctl). the sequences have been inserted into the wyeth vaccine strain of vaccinia virus at a single locus in the form of two separate fused e6/e7 reading frames, each under the control of an early v a d n i a promoter, and each modified to inactivate the rb binding site. the virus has been characterised with respect to its ability to synthesise the expected hpv proteins, its genetic stability, and growth and virulence in a mouse model prior to use in human clinical trials. analysis of hpv16 â�¬7 specific ctl from c57bu6 mice immunised with this recombinant virus show the response to be equivalent to that generated by a control vaccinia recombinant expressing non-modified hpvi 6 e7 alone, with similar recognition of the defined immunodominant h-2db restricted epitope, e7 residues 49-57. ability of mice to resist influenza challenge, arthur friedman, douglas martinez, john j. donnelly and margaret a. liu, department of virus and cell biology research, merck research laboratories, west point, pa 19486 mice infected with the laboratory strains of a/pr/8/34 (hln1) or the mouse adapted a/hk/68 (h3n2) show complete protection against challenge with a different strain of influenza a. humans, however, undergo multiple influenza infections as previous infections appear to provide weak or short-lived protection against the continual antigenic change of strains. we have previously shown that immunization of naive mice with dna encoding the conserved internal antigen nucleoprotein (np) provides protection against both h1 and h3 strains of a/influenza. although such mice became infected they were resistant to weight loss and death this differed substantially from a/pr8 and a/hk recovered mice which were resistant to subsequent infection. to produce a more representative model of human infection, we infected the lungs of mice with currently circulating strains of human influenza. mice that had been given lung infections with a/beijing/92 were susceptible to subsequent infection with the a/hk/68 strain although they were resistant to weight loss and death. other strains such as a/beijing/89 or a/georgia/93 provided only marginal protection against weight loss and death against a/hk challenge. mice that were immunized with np dna had greater resistance to weight loss and death after a/hk/68 challenge than mice previously infected with a/bei/89 and a/ga/93, and were similar to mice that had been previously infected with a/bei/92. thus, infection with different virus strains provide various levels of cross strain protection and the level of protection provided by immunization with dna can exceed that induced by live influenza infection. the development of sendai virus-specific cytotoxic t lymphocyte (ctl) effectors and precursors (p) has been compared for mice that are homozygous (-/-) for a disruption of the h-21-ab class ii major histocompatibility complex (mhc) glycoprotein, and for normal (+i+) controls. the generation of cd8+ ctlp was not diminished in the (-/-) mice, although they failed to make virusspecific igg class antibodies. while the cellularity of the regional lymph nodes was decreased, the inflammatory process assayed by bronchoalveolar lavage (bal) of the infected lung was not modified and potent ctl effectors were present in bal populations recovered from both groups at day 10 after infection. there was little effect on virus clearance. as found previously with cd4-depleted h-2b mice, the absence of a concurrent class il-mhc-restricted response does not compromise the development of sendai virus -specific cd8+ t cell-mediated immunity. the importance of cytoxic t lymphocytes in defense against acute and chronic viral infections is gaining increasing recognition. our approach to investigating the structure-function relationship between immunogens and their in vivo ability to elicit cytotoxic t lymphocyte responses has been to formulate simple, well-defined structures that vary in their ability to introduce associated antigens directly into the cytoplasm of antigen presenting cells. we have introduced methods for the preparation of unique, lipid-matrix based immunogens, which are highly effective in mice and monkeys for stimulating strong cd8+ cytotoxic t cell responses, (ctl). antigens used have been proteins or peptides derived from influenza, parainfluenza, and hiv viruses, and whole formalin-fixed siv. ctl can be induced by parenteral as well as oral administration. comparing the physical and chemical nature of our formulations with those from other laboratories which have reported the use of subunit preparations to induce cd8+ ctl, leads us to propose that a minimal immunogenic formulation capable of eliciting cd8+, mhc class i restricted cytotoxic t lymphocytes includes: i) a peptide that represents a mhc class i epitope; ii) a component that enhances the aftinity of the immunogen for mhc class i positive antigen presenting cells ; iii) properties that can compromise the integrity of a lipid bilayer, facilitating delivery of the antigen directly into the cytoplasm for class i presentation. cd8+ responses to peptides, glycoproteins, and even whole fixed viruses, makes them attractive candidates for diseases where clearance of infected cells is important in protection and recovery. cani ne rabies is uncontrolled. rabies also is epizootifally active in several species in most areas of the wor!d. thm, vaccination of animals, both wild and domestic, as well as postexposure treatment of humans remains a global concern. unfortunately, in those countries in which,people most need postexposure prophylaxis, the best vaccines are expenswe and in limited supply, whereas available vaccines are of questionable immunogenic efficiency, are otten contaminated and may produce neurological complications. the goal of this study was to determine whether a rabies vaccine for global use is complete one round of replication have the potential to be used as vaccines. we have previously reported the abiliity of a ghdeleted herpes simplex virus type 1 (hsv-1) to protect mice and guinea-pigs from subsequent challenge with wild-type hsv. this virus, which we have called disc (disabled infectious single cycle) virus, can infect normal cells but the absence of gh in the progeny virus prevents further rounds of infection. as disc hsv clearly has potential as a vaccine, it is important to determine the durability of the immune response elicited by this virus. we have investigated the ability of disc hsv-1 to protect mice from a wild-type virus challenge six months post vaccination using the ear model of hsv infection. two immunisations on day 0 and day 21 resulted in a considerable reduction in virus titres in the challenged ears, and an almost complete absence of virus in the dorsal root ganglia. hsv-specific antibody titres as determined by neutralisation and ellsa were maintained for the six months period. it was possible to demonstrate an hsvspecific cytotoxic t-cell response in the disc hsv-1 vaccinated mice following challenge; this ctl activity was similar to that observed in mice vaccinated with wild-type virus and challenged after the same time period. animals vaccinated with inactivated virus or control mock-vaccinated mice showed a low level of ctl activity typical of a primary ctl response following challenge. these results indicate that an effective cell-mediated and humoral anti-hsv immune response can be maintained for at least six months following vaccination with disc hsv-1. viruses which lack an essential gene and thus can only the lmmunogenicity of two ctl @topes. influenza npl47-158 and plasmodium berghei cs protein 252-260 were studled in balblc mice. paptides were formulated as a) a iipopepudepeplkle conlugated to irlpalmltoyl-sgly~~l cysteine (pam3cyj) and dissolved in a 1% dmsolglycerol solution. b) micmparticles prepared with poly @.l ladide-coglywlide) using a solvent evaporation technique. the micropaltides were administered as a suspension in phosphate buffered saline or c) an emulsion prepared wilh egg lecithin and 10% soya oil in water. 1 wpg of peptide or controls (the welght equivalent of blanks) were administered to groups of 3 mice intra-peritoneally or subcutaneously at 1.10 and 20 days. 7 days following the last immunization splenocytes were cukured in vlro in the presence of appropriate pepwe or wntml m h rat con a supematant as a source of omwth fadors. ctl adivity was measured in a standard 4 hour chromium release assay and results expressed as % specific lysis. ctl could be elicited in vivo with all three formulations. at an ewedoctarget ratio of 1w:l the plasmodium berghei peptide encapsulated in micmpartides gave 47% iysls on peptide pulsed target calls. levels of lysis were similar for the peptide in emulslons. the iipopeplide p3ccs252-260 gave a level of lysis of 82% at an e:t ralioof1w1. these results demonstrate that peplides edminldered in a variety of formulations can induce a systemic ctl response in vivo. peplide vaccines using such formulations wuld be used to stimulate ctl responses as part of a prophyladic vaccines or as immunotherapeulics. attenuated the attenuated sabin strains of poliovirus have been used for many years to elicit protective immunity to poliovirus. oral vaccination with replicating polioviruses generates both mucosal and systemic immunity. therefore, use of recombinant polioviruses expressing heterologous antigens as vaccine delivery vectors should provide a system for generating protective immunity to those antigens. cdna copies of the poliovirus genome has been used to construct vectors containing a multiple cloning site for insertion of heterologous genes. a pilot enhanced-potency inactivated poliovirus vaccine (ejpv) with assumably improved immunogenicity containing win-treated type 3 poliovirus (shah sauketf) together with the regular type 1 and type 2 canponena was subjected to s*mdard safety and potency tesls in the labmatory and laken through wase i and i1 clinical aials. in balb/c mice, the lrypin-mcdit%d e-if' v cfryipv) was found to induce antibodies targeted ouaide the uypsin-sensitive bc-loop of capsid protein vp1. as previously shown for hypsin-mated type 3 poliovirus @vm alone. trypsin used to modify the type 3 component at the bulk phase was removed by the vaccine manufacturer (rivm) in the regular purification process. absence of uypsin in the final product was further confumed by immunizing mice and rabbits with 10-fold concentrated type 3 component of tryipv. assays for lrypin antibodies using eia and westem blot techniques were newve. in the clinical phase 1 aial six adult volunteers with existing immunity to poliovirus were given increasing doses of tryipv. already one tenth of the regular dose induced a booster effect in neuarlizing antibodies to both intact and mypsin-treated type 3 poliovirus. no unexpected sideeffects were recorded phase i1 trials comprised 50 adult volunteers with at least 5 years since the last dose of poliovirus vaccine and 50 children who were due to receive the third dose of the regular immunization schedule at about 2 years. in both groups. 25 individuals received tryipv and 25 were injected with the regular enhanced potency ipv (e-ipv). serum specimens drawn before injection and one month after were tested for neunalizing antibodies using standard microneuwlization assays (all mutypes) and the racina test (intact and uypsin-mated type 3 poliovirus). in all volunteers tryipv was at least as immunogenic t w the regular e-ipv according to all assays. no statistically significant differences in side effects were reported. a murine/influenza virus model has been used to evaluate the longevity of antibody and protective responses raised by gene gun delivery of a hemagglutinin-expressing dna. mice were immunized and boosted at one month with 0.4 ug of an h1 expressing plasmid dna (pcmvri1). antibody responses and protection against a lethal challenge were followed over the next year. antibody responses had good longevity exhibiting comparable titers at one year post boost as at 10 days post boost protection against the lethal challenge was complete at 10 days, 1 month and four months post boost, but only partial at one year. a transgenic mouse model for identifing htlv-1 t-cell epitopes: generation of hla-b*3501-restricted ctl directed against synthetic peptides and naturally processed viral antigens, christian schiinbach*, ai kariyone*, kiyoshi nokiharaa+, karl-heinz wiesmulle6 and masafumi takiguchil, departments of tumor biology* and immunology#, institute of medical science, university of tokyo, tokyo "tokyo university of agriculture and technology, tokyo +biotechnology instruments department, shimadzu corp., kyoto, japan $natural and medical science institute at the university of tubingen, reutlingen, germany the majority of human t-cell leukemia virus type-i (htlv-l), hla class i-resmcted t-cell epitopes have been identified by cloning htlv-1 patient-derived t cells. here we describe for the frst time a rapid method (reverse immunogenetics) for identifing t-cell epitopes, together with a transgenic mouse model as a guide for testing the cellular immune response to a mixture of the lipohexapeptide immunoadjuvant pamgcys-ser-(lys)4 and synthetic htlv-1 peptides which seem suitable for vaccine design. htlv-1 amino acid sequences were searched for eight to 14mer patterns carrying the anchor residues of the hla-b*3501 peptide motif at positions two and eight to fourteen. 65 candidate peptides were synthesized according to the matched sequence patterns. their hla-b*3501 affinity was quantitatively analyzed in an indirect immunofluorescence peptide binding assay using rma-s-b*3501 cells. the fourth group (controls) were inoculated with h3n2 (in) thereby providing heterotypic ctl immunity in the context of a natural infection without the confounding effects of humoral immunity against surface antigens. all four types of inoculations have been shown to protect normal (class i expressing) mice from a lethal challenge with influenza, presumably mediated by class i restricted cytotoxic t cells. the two groups inoculated via the intranasal route may gain additional protection by activating the mucosal immune system (iga). none of these types of inoculations has been evaluated in the context of class i1 restricted cytotoxic t cells, the only ctls found in class i deficient mice. for all four types of inoculations, mhc class i deficient mice lost significantly more weight than the class i expressing control groups (seven mice per group) indicating the importance of class i restricted t-cells in protection. within the class i expressing groups, there was no significant difference between the four types of inoculations; within the class i deficient groups the vac-np im immunized mice lost significantly more weight than the h3n2 group;the other two groups, vac-np in and genetically immunized groups had intermediary results. these data lend support for a protective role for mucosal immunity. results on both class i and class i1 ctl activity for the four types of inoculations will also be presented. we tested the pbmcs of patients participating in two vaccine therapy trials for their ability to recognize overlapping peptides of the gp120 la1 sequence. seventeen patients participating in a phase i gp160 protocol and 13 patients participating in a phase i gp120 protocol had their pbmcs isolated by ficoll separation of heparinized venous blood. the fresh pbmcs were plated, in triplicate, into 96 well plates containing peptides overlapping the la1 sequence of gp120, pulsed on day 7 with tritiated thymidine and harvested and counted on day 8. results: the percentage of patient's pbmcs from each trial with an lsi 2 5 to each peptide are depicted below. conclusions: the pbmcs of hiv-infected volunteers who have been multiply immunized with either gp160 or gp120 proliferate to multiple peptides within the gp120 molecule. reactivity from the end of c1 through early c2 (lai #i 12-21 1) is particularly prominent and contains previously undescribed th epitopes (asterisks). conspicuously missing is reactivity to the v3 loop peptide (la1 #300). although the percent reactivity to the entire gp120 molecule is similar between the immunization groups, there is differential recognition of some of the individual peptides, particularly peptides in early c3 (lai #319-348). the intracytoplasmic lifecycle of listeriu mmcytogenes (lm) enables it to be a convenient vaccine vehicle for the introduction of foreign proteins into the mhc class i pathway of antigen presentation. taking advantage of these properties, we have inserted the nucleoprotein (np) gene from lymphocytic choriorneningitis virus (lcmv) into the lm chromosome by site specific homologous recombination. infection of mice with recombinant lm expressing lcmv-np elicited a virus-specific ctl response. we were able to recover lcmv-np specific ctl precursers from recombinant lm vaccinated mice as shown by vigorous secondary ctl responses after in vitro stimulation. in contrast to mice immunized with wild type lm, mice vaccinated with np-recombinant lm were protected against challenge with immunosuppressive lcmv variants. protection was demonstrated by reduced viral titers or complete clearance of lcmv from serum and various organs including, spleen, liver, lung, kidney, and brain. the kinetics of the lcmv challenge indicate that mice vaccinated with recombinant lm were able to arrest viral growth early in the infection due to a strong ctl response and did not exhibit the immunopathology associated with infection of naive mice. since lm not only delivers antigens into the mhc class i pathway but also induces il-12 production, it has the potential to function simultaneously as a vehicle for expressing foreign antigens and as an adjuvant promoting cell mediated immunity. , latent membrane protein [lmp] 1 and 2a) in chromium release assays. we were fortunate in identifying one child from whom cryopresetved pbmc samples were available before. and during ebv seroconversion. ebv-specific ctl activity was demonstrated concurrent with initial detection of virus in the peripheral blood by ebv-dna pcr. in the absence of detectable serum antibody. ctl lines from all nine children recognized one or more ebv latent gene prcduct(s). all children demonstrated ctl responses against one or more ebna 3 proteins (3a, 3b, 3c). and ebna 3c was recognized most frequently. no ctl responses were detected against the ebv latent proteins ebna 1, 2, lp or lmp 1. the ebv-specific ctl lines expressed cd3/cd8 and mab blocking experiments demonstrated that the majority of target cell lysis was inhibited by antibody against mhc class-i but not antibody against mhc class-11. these results represent one of the first reports characterizing ebv-specific ctl responses in young children. the striking similarity between ebv-specific ctl responses described here in young children and those reported for adults suggests that the ebna 3 family of proteins and lmp 2a should be considered for inclusion in candidate ebv vaccines. evaluation of cellular immune responses to adenovirus vectors in the cotton rat. soonpin yei,' gary kikuchi,' ke tang' and bruce c. trapnell.' departments of virology' and immunology,2 genetic therapy, inc., gaithersburg, maryland 20878 replication deficient recombinant adenovirus (av) vectors are efficient gene delivery vehicles currently being developed for a variety of in vivo gene therapy strategies such a s for the fatal pulmonary component of cystic fibrosis. the cotton rat (sigmodon hispidus) is one of the most widely accepted animal models for studying these av vectors because wild type human adenovirus replicates in cotton rats and because of the histopathologic similarities of infected respiratory epithelial tissues from humans and cotton rats. despite this, methods for studying immunologic responses in the cotton rat have not been developed. importantly, recent studies in the cotton rat (gene tber. 1 :192-200; 1994) in our laboratory suggest that a dose-dependent specific immune response to av vectors can limit expression of the transgene. in this context, w e have established methods to evaluate cytotoxic lymphocyte (ctl) responses to av vectors in the cotton rat. to accomplish this, a ctl target cell line was established consisting of primary cotton rat lung fibroblasts (crlf). splenocytes from cotton rats exposed previously to an av vector were harvested, cultured in virro with irradiated, addb274nfected crlf. cultured (effector) splenocytes were then incubated with s'cr-labelled crlf (target) cells a t effectoctarget (e:t) ratios of 100, 50 and 10. in parallel, splenocytes from naive cotton rats served as negative controls. results demonstrated vector-specific ctl lysis of target cells significantly greater than controls: 80.3 f 1.3% vs 6.2* 0.5%. 49.6*1.6% v s 5.7*0.4%. and 22.8*3.5% vs4.8*0.5% (meanrts.e.m., n=3; p<o.ol, all comparisons) a t e:t ratios of 100, 50 and 10, respectively. thus, a specific ctl response does develop in cotton rats following in vivo av vector administration. this observation will be useful in guiding vector modifications in the rational development of improved vectors for clinical use. the identification of ctl epitopes is essential for subunit vaccine design against aids. a major portion of the anti-viral ctl responses after infection with htv in humans or siv in rhesus macaques is made up by anti-gag specificities. here, effector cell populations were established from hiv seropositive individuals by specific stimulation of pbl with pfa fixed autologous b-lcl infected with tw gag. expanded cultures were then tested for ctl activity against autologous b-lcl pulsed with overlapping 20-mer peptides derived from p24. two individuals (008 and 617) had ctl against p24.14 (a(mino) a(cids) 263-282, krwiilglnknrmysftsi), two others (038 and 157) recognized p24.17 (aa293-312, frdwdrfyktlraeqasqd). similarly we found two siv infected rhesus macaques reacting against the analogous aa sequences of siv; it. p26.14 (8653) and p26.17 (ivy). this led us to further fine map the human and rhesus ctl reactivities using sets of 9 aa overlapping 10-mers and to test for hiv-1isiv crossreactivity. both two p24.14 epitopes (possibly restricted by hla-a2) and the p26.14 epitope were non-overlapping, and all three epitopes were non-crossreactive. finemapping of the p24.17 epitopes is in progress. the sn p26.17 epitope was also non-crossreactive, despite only one aa difference (position 6, s+t) between the siv and hiv sequence. others have also mapped cl'l epitopes to the same regions of p24 in humans (johnson ji 147,1512 ,1991 , buseyne jv 67,694,1993 and to p26.17 in cynomolgus macaques (gotch jmprim 22.1 19,1993) . in conclusion, multiple non-cross reactive ctl epitopes are clustered within corresponding regions of hiv and siv gag. ctl hotspots may be important for inclusion into vaccines. forming virus) with cd-4 and cd-8 1-cells, b-cells and adherent cells, while an average of 5% of virus was associated with lps-stimulated 8-cells and adherent cells. in the second experiment the combined function of antigen presenting cells, 1-helper cells and 8-cells (humoral immune response) to a third party antigen (srbc) during the acute phase of enterovirus induced disease was increased at day 0, and decreased at days 2, 3 and 4 post-cvb3, infection relative to unlnlected animals. conclusions: these data indicate that cvb3, associates with and replicates in rnurine splenic immune cells, and that infection of susceptible mice with cvb3, modifies the immune response to a third party antigen during the acute stages of disease. an understanding of such cvb3,-i mmune cell interactions is criitical for gaining insight into the pathogenesis and persistence of enteroviral infection both within and outside of the immune system. understanding the pathogenic mechanisms of virus infections depends upon identification of viral gene products involved in host-virus interactions in vivo. many viral gene products which affect pathogenesis in vivo are nonessential for virus replication in vitro. therefore, we constructed insertion or deletion mutants in the hind i11 j and i regions of murine cytomegalovirus (mcmv) to assess the importance of these genes in in vivo replication of virus as well as interaction with immune regulatory and effector cells. three mutants replicated in nih 3t3 fibroblasts as efficiently as wild-type virus, thus confirming the nonessential nature of the genes. deletion of 2.8 kb in hind i11 j and of 7.7 kb in hind i11 j and i resulted in mutants with reduced replication in target organs in vivo. an insertion in hind i11 j had no significant effect on replication in mouse salivary gland, but curtailed growth of the virus in footpad fibroblasts and subsequent priming of mcmvspecific cpl precursors in the draining lymph node. in addition, the 7.7 kb deletion mutant was unique in its failure to replicate in a permissive, differentiated macrophage cell line. current studies aim to further define the role of this gene region in hcmv pathogenesis. that in vv5-4 progression o f v v life cycle is blocked at the the uncoating ii or replication stage. furthermore, host protein synthesis in v v 5 -4 cells is not shut-off after v v infection suggesting uncoatingll/replication is important for determining cell fate. since this mutant clone vv5-4 was isolated b y retroviral insertional mutagenesis. a cellular gene that was integrated by single retrovirus was isolate and codes for a 5kb transcript in northern blot analysis. a 2 kb cdna was isolated and codes f o r a novel polypeptide o f 800 amino acids that show no homology with known proteins. experiments are in progress to understand t h e role of this cellular gene in vv infection. in addition, a two-hybrid system will be employed to identify a n y viral gene product interacting with this cellular target u s i n g o u r laboratory's s t a n d a r d s t r a i n o f a/japan/305/57 in in uitro assays of viral infectivity of the mouse b-lymphoma a20-1.11. we have found that although infection sensitizes the a20 cells for recognition by both class i and class i1 restricted ctl.'s and leads t o high surface expression levels o f hemaglutinin (ha), the infected a20 cells grow through t h e infection, ha expression declines, and few of the infected cells die. in contrast, using a second s t r a b of a/japan/305/57 from a different passage history, we find that n o t only are the infected a20 cells sensitized for lysis by both class i and class ii restricted ctl's, exhibiting even higher levels o f ha surface expression, but in addition, most cells in the infected population die with characteristics of apoptosis. the two virus strabs appear to be highly related because in various assays we have found t h e m antigenically indistinguishable in both their t-and b-cell epitopes. using these two virus strains in a n in uiuo dose response assay, we have found that o u r laboratory's standard strain will kill all mice infected with a 10-2 the therapeutic and prophylactic antiviral efficacy of interleukin-12 (il-12) was studied using murine models of herpes simplex virus (hsv) and murine cytomegalovirus (mcmv) infection. therapeutic intraperitoneal administration of il-12 commenced 6 hours after mice were infected w i t h hsv, and was continued daily for a total of 5 days. il-12 therapy improved the survival rates of mice w i t h systemic hsv infection, compared to that of placebo treated infected mice. since neutrophilia and thrombocytopenia were common in dengue patients,we are investigating whether bm may be accompanied with abnormal hematopoiesis. using ficoll-hypaque separation, mononuclear cells prepared from the total bm cells. 1 .0x106we~ells/well of both adherent and nonadherent cell types were infected with two different den-2 virus strains : (1) thiland strain isolated from the dengue hemorrhagic fever(dhf1 patient and (2) taiwan strain isolated from the dengue fever(df) patient at moi=l and collected the suprnatants at various time points for assay. the preliminary data showed that : (a) adherent cells were infected with den2 and dhf virus strain replicated much more efficiently (2.3x104pfu/ml on day 3 p.i.1 than df strain; (b) nonadherent cells were 10 times less efficient to produce viral yields than adherent cells (dhf and2 df strains peaked at 6.3~10' pfu/ml and 1.5~10 pfu/ml on day 3 p.i., respectively); (c) addition of gm-csf to nonadherent cells increased virus infectivity and productivity. in conclusion, den viruses were able to infect bm cells and the more virulent virus strain isolated from dhf patients had better replication capability in human bone marrow cells. future studies on cytokines and immunologic evaluation are in progress. number of gene products whose function is to modify host responses. recent evidence suggests that a subclass of poxvirus-encoded host response modifiers interferes with host defense responses, and thus can be termed immune or inflammatory response modifiers (irms). several poxvirusencoded irms possess homology to mammalian proteins which are associated with host defenses, while other poxvirus irms have little sequence homology to immune-effector molecules or their receptors. the ultrecht strain of rabbitpox (rpv) virus induces a large reddish pock on the chorioallantoic membrane (cam) of the chicken embryo. rpv mutants lacking the spi-2/crmn38k gene trigger an inflammatory response by 36-48 hr after inoculation, similarly to that found for cowpox virus (brighton red strain) mutants. rpv mutant viruses with a deletion of the ps/hr gene, a gene which has recently been associated with virus host range and virion maturation, elicited an inflammatory response on the chicken embryo cam at 48-72 hr after virus inoculation. thus, another poxvirus-encoded gene product has been identified that has irm functional activity which would not have been predicted by sequence analyses. poxviruses have recently been found to possess a herbert w. virgin iv, center for immunology and division of infectious diseases, washington university school of medicine, st. louis, mo 63110 murine cytomegalovirus (mcmv) follows three stages of infection in the immunowmpetent host. following the initial acute infection, a persistent infection remains in which lytic virus resides in the acinar cells of the salivary glands. finally a third stage of infection exists which is characterized by a lack of detectable infectious virus. it is not known whether mcmv remains persistent at some low level during this thiid stage or establishes molecular latency. to test this, spleens, kidneys and salivary glands from mice 2-8 months post infection were evaluated by two sensitive assays. sensitivity was tested using virus co-cultured on murine embryo fibroblasts (mefs) for 14 days. by this method, 1 pfu mcmv was reproducibly detected in the presence of sonicated spleen, kidney and salivary glands diluted 1:lo. scid mice were used as a second detection assay for mcmv. using 46 scid mice and different doses of virus, the lds, of mcmv in scid mice was 2-3 pfu. mcmv infections were detected when 10-25 pfu were added to sonicated spleens, kidneys, and salivary glands prior to injection into scid mice. using both co-culture with mefs and injection of tissue into scid mice, we have tested a total of 34 spleens, 34 kidneys, and 37 salivary glands and have detected no persistent virus. while no preformed virus existed in these tissues, reactivation from explanted spleen, kidney and peritoneal exudate cells occurs in culture after 10-40 days. in addition, lytic mcmv was detected in scid mice 28 days after injection with 2(10)' kidney cells from latently infected mice. following identical transfers of latent spleen cells, mcmv was not detected in scid mice although explants from the same organs reactivated in vitro. using facs sorting, panning, magnetic bead separation, and reactivation assays, individual cell types have been analyzed for the presence of mcmv genome and the ability to reactivate virus. heat shock proteins (hsp) are expressed in all organisms in response to a variety of stresses, including virus infection. however, since viruses are not known to encode hsp genes, this represents expression of cellular hsps. we have found a dramatic induction of the 72kda hsp in the ovaries of w-infected mice, and using primary ovarian fibroblasts, demonstrated hsp72 mrna within virus-infected cells. the physiological role of hsps expressed during virus infection is unknown, although hsps act as molecular chaperones to facilitate protein folding and assembly in unstressed cells, and a number of bacterial hsps are essential for bacteriophage replication and morphogenesis. in order to determine whether hsp72 expression was beneficial to vv replication, we constructed a recombinant vv which encoded murine hsp72, but found that this virus grew to similar titres as control viruses, both in vitro and in vivo. in particular, kinetic studies of virus growth in an hsp72-negative cell line showed no difference from control viruses, and the presence of hsp72 did not influence the virulence of infection in immunocompromised nude mice. these results are interesting given that hsp70 proteins act as molecular chaperones and that hsp72 can be found in association with vv proteins. we suggest that this association may simply reflect the inherent affinity of hsp70 for non-native proteins, and the close physiological proximity of hsp and nascent viral proteins within the virus infected cell. our results indicate that the functional significance of these interactions are minimal with respect to the outcome of virus infection. furthermore, although the expression of hsw2 in vv-infected mice coincides with the peak anti-viral cellular immune response, hsp72 does not appear to be immunologically significant, as we cannot demonstrate any immunological responses to hsp72 during the course of vv infection. the effect of in vivo neutralisation of ifn-y on cytokine production during influenza infection was compared in cs7/bl6 and balb/c mice. similar cytokine profiles were obtained on in v i m restimulation of mln or spleen cells from virus-infected mice of each strain. at days 7 and 10 postinfection, mln or spleen cells from both strains of mice produced il-2, il-6, il-10 and ifn-y, but little or no il-4 or il-5. however, following in vivo treatment with anti-ifn-y antibody, production of 1l-4 and il-s was induced in mln and spleen cells of balb/c mice whereas those from cs7/bl6 mice showed little change in cytokine profile. an increase in il-6 and 1l-10 production was also observed on in virro restimulation of splenocytes from balb/c mice. however, significant amounts of 1l-2 and ifn-y were still secreted in these cultures. in v i m neutralisation of ifn-y in the restimulated cultures had little effect on the production of other cytokines, regardless of whether this cytokine had also been neutralised in vivo .despite the change in cytokine profile, anti-ifn-y treatment had no effect on the kinetics of viral clearance in balb/c mice. a similar situation was seen for c57/bl6 mice. congenic strains of mice are currently being used to determine whether the effect on cytokine profiles is associated with the mhc haplotype. wunner, and steven l. spitalnik, department of pathology and laboratory medicine, university of pennsylvania and the wistar institute, philadelphia, pa 19104 rabies virus glycoprotein (rgp) is the only glycoprotein on the viral surface, is the target of viral neutralizing antibodies, and is the viral protein that interacts with the host cell receptor. rgp of the era strain is a 505 amino acid type i membrane glycoprotein with 3 potential n-glycosylation sites at asn37, asn247, and asn319. due to rgps central role in the immunology and biology of rabies, it is important to determine its three-dimensional structure. towards this end we produced a recombinant form of rgp that may be more amenable to analysis by x-ray crystallography. to avoid the use of detergents, we constructed a soluble 434 amino acid form of rgp lacking a transmembrane domain (rgpt434). rgpt434 was secreted by transfected cells and was appropriately glycosylated, assembled, antigenic, and immunogenic. since n-glycosylation was required for rgpt434 secretion, we minimized the number of n-glycans by site-directed mutagenesis. interestingly, rgpt434 was secreted only when asn3l 9 was n-glycosylated. in contrast, full-length rgp was expressed at the cell surface a any of the three potential sites was n-glycosylated. soluble inhibitors of n-glycosylation were used to simplify the structures of the n-glycans on rgpt434. although inhibiting alpha-glucosidases with castanospermine blocked secretion, inhibiting any further processing with deoxymannojirimycin had no effect on secretion. finally, to simplify the purification process, a tail of 6 his residues was added to the cooh-terminus of rgpt434. this modification did not affect secretion, antigenicity, of assembly of the recombinant glycoprotein, but did allow purification using ni+2-agarose chromatography. in summary, we constructed a soluble form of rgp with a minimal number of homogeneous n-glycans and an "epitope" tag. this form of rgp is structurally similar to the extracellular domain of the full-length protein and may be amenable to analysis by x-ray crystallography. endogenous retroviral genes (ev) are well characterized in chickens. our laboratory has established chicken lines with single and multiple ev loci and, compared to ev negative birds, these lines exhibit a delayed and suppressed titer of neutralizing antibody in responses to avian leukosis virus (alv) challenge. to address the effects of ev genes on the cell mediated immune response we have developed an in-vitro assay to monitor the in-vivo induction of mhc restricted cytotoxic t cells (ctl) induced by alv. using this assay, we find the ctl response in birds expressing the ev-21 locus is reduced 85 to 95% compared to ev negative birds having nearly identical (eighth backcross) genetic backgrounds. other ev loci (1,6,10 and 11 ) also reduce the ctl response to alv in mhc matched chickens with different genetic backgrounds. ' national public health institute. helsinki. finland universities of tampere, qulu and 'helsinki, finland coxsackie b virus infections have been associated with clinical iddm in seveml previous studies but their initiating role in the slowly progressing beta-cell damage was for the fmt time directly implied in our recent prospective. nationwide dime study. siblings of the index iddm cases who progressed to clinical iddm experienced enterovim infections more frequently than nonprogressing siblings during prospective follow-up period. enterovim infections were initially demonstrated by detecting significant increases in serum class-specific cross-reactive enteroviral antibodies by using radioimmunoassay (ria) with the heavy-chain capture principle. causal association benveen these infections and the pathogenesis of iddm was suggested by temporal association of sercconvenions of viral antibodies with increases in islet cell antibody levels which are considered to be markers ok ficell damage. the possibility of the existmce of specific diabetogenic s & s of enternviruses was then considered. to identify the serotypzs responsible for the infections, successive sera from subjects exhibiting seroconvenion of autoantibodies coinciding with an increase in enteroviral antibodies were anlyzed by plaque n e u n a t i o n assay using a set of different enterovhes. the results indicate that several enternvirus serotypes may be associated with the progressive prediabetic p~ocess. not only different coxsackie b but also coxsackievirus a9 -infections were found to coincide with seroconvenions of islet-cell and insulin auwantibodies. it is known that an immunogenic region of gad 65. one of the major isletcell antigens, shows a high degree of homology with the 2c protein of cbv-4. studies on potential immunological cross-reactions between enteroviruses and two significant autoantigens, hsp60 and gad65, are in progress. this is carried out by using immunohistochemistry in infected cell lines and in frown sections of human pancreas with antibodies induced by synthetic peptides and natural and expressed viral proteins, the role of p53 alterations in human malignant pleural mesotheliomas (mpm) is unclear. immunostaining (ipox) of snap frozen mpm specimens revealed p53 expression in 31 of 52 (60%). p53 detection by ipox is usually associated with mutated p53. sscp for exons 5-9 revealed p53 alterations in 2 of 25 specimens, and loh at exon 4 was seen in only 2 of 12 evaluable specimens. thus, most of these specimens appeared to contain wild-type (wt) p53. we recently reported that sv40 induces mesotheliomas in rodents, and that sv40-like sequences are present and expressed in mpm. of note, sv40 large t antigen (tag) binds and inactivates wt p53 i n ! , abolishing its tumor suppressor function. we theorized that the immunohistochemical persistence of p53 may result from its physicdl binding with tag. tag was detected by ipox in 32 (62%) of these mpm specimens. expression of tag was significantly associated with coexpression of wt p53 (p2 = 0.008, fisher's exact text), and preliminary experiments suggest co-precipitation of p53 and tag. finally, we demonstrate that waf-1 is not detectable in mpm expressing wt p53 suggesting that p53 is inactive. we speculate that tag expression in mpm binds to and inactivates p53 contributing to the malignant phenotype. human papilloma virus (hpv) infection is involved in 90% of cervical carcinomas and possibly 95% of cervical intraepithelial neoplasia (cin). at present it is unclear whether patients infected with the transforming hpv types can mount efficient t cell responses. to address this issue we examined the ctl response of peripheral blood lymphocytes from patients attending a cervical neoplasia outpatient clinic. all were diagnosed hpv positive with various stages of c m and nine patients were selected for hla-a2 expression by facs analysis. pbmc were stimulated with caski, a cervical carcinoma cell line demonstrated to be hf' v type 16' and hla-a2'. culture media consisted of rpmi-10% human serum supplemented in three cases with 15 u/ml il-7, in another three cases with 3 u/ml il-2 and 15 u/ml il-7, and in the final three cases with 10 u/ml il-2 and 15 u/ml il-7. the ctl were screened for reactivity to caski and in addition the cervical carcinoma cell line c33a (hpv, hla-a2') transfected with either the hpv type 16 e6 or e7 genes. one patient's cells maintained with 10 uiml il-2 and 15 ulml il-7 showed hpv e7 protein specific cytotoxicity in a hla-a2 restricted fashion. these ctl lysed the caski stimulator cell line but not the nk sensitive target k-562. the ctl efficiently lysed a c33a-e7 clone but in contrast a vector only transfectant was not lysed. the lysis of c33a-e7 was blocked with a-cd3 and a-cd8 antibodies at 66% and 41% respectively. facs analysis determined that this ctl population was 92.8 % cd3'cds' and 7.3% cd3'cd4'. limited e6 recognition was also observed but has not been hlly characterized to our knowledge this report represents the first demonstration of hpv e7 responsive ctl isolated from the peripheral blood of hf' v infected patients. we have previously shown that proviral variants found in the peripheral blood mononuclear cells (pbmc) of hiv-1 infected individuals can interfere with recognition by autologous cytotoxic t-lymphocytes (ctl). abolition of recognition could be caused by interference with t h e processing of viral peptides, by a failure of the variant peptides to bind to mhc class-i molecules or by a failure of the mhc class-vpeptide complex to efficiently activate the tcr (t-cell receptor). these mechanisms could allow the virus to escape the immune surveillance by ctl. most studies so far have addressed the question of viral variation in t-cell epitopes by analysing proviral dna, extracted from the pbmc of hiv-1 infected individuals. however, the analysis of virion-associated rna offers several advantages. sequences found in viral rna are likely to be functional, a t least u p to the point of packaging into virus particles. furthermore, rna sequences represent virus which is currently actively produced. here, we present the sequence variation i n two hla-b8 restricted epitopes: p17-3 gag and amino acids 18-26 of the pol gene. both proviral dna from pbmc and viral rna sequences from plasma are discussed. the ability of viral variants to hind to mhc class-i molecules was assessed and the recognition of variant peptides by ctl was tested. variants were also tested for their ability to antagonize recognition of the index sequences. we show that viral variants with the ability to interfere with recognition by ctl are not only found in proviral dna but also in virion associated rna. objective: clinical course of hiv-1 infection may be influenced by an effective host immune response against hiv-1 and/or by viral properties. to investigate the cellular immune response to hn-1 we analyzed ctl activity against different hiv-1 proteins in long-term asymptomatic hiv-1 infection as well as during rapid progression to aids. methods: 10 longterm asymptomatic individuals with cd4 counts >500 celllpl after more than 8 years of infection were selected from the amsterdam cohort study on aids versus 10 subjects who progressed to aids < 5 years. ctl activity was measured on "cr labelled hla matched or autologous b-lcl, infected with rvv expressing hiv-1 ag. both bulk and limiting dilution ctl assays were performed longitudinally with pbmc after ag-specific stimulation. sequences of ctl epitopes were determined in homologous virus isolates resulrs: different kinetics of anti-gag ctl responses were observed in rapid progressors. in any case ctl responses disappeared during progression to aids. in long-term asymptomatic subjects persistent ctl responses were observed together with low viral load. conclusions: sustained, broad anti-hiv cellular immunity may correlate with maintenance of the asymptomatic state in long-term survival by controlling viral replication. enteroviruses are a large group of positive stranded rna viruses known to be responsible for a number of distinct disease entities. recombination is thought to be capable of generating new enterovirus strains that cause significant morbidity. for example, enterovirus 70 which was responsible for a pandemic of haemorrhagic conjunctivitis and poliomyelitis is thought to have originated by recombination between a coxsackie b like virus and another unidentified enterovirus. we are studying a group of echovirus 11 isolates from an outbreak of disease in southem india. sequence analysis within the 5' untranslated region reveals that these isolates fall into two groups that differ by -20% (equivalent diversity to that seen between between published sequences of poliovirus 1 and coxsackie a 9 virus). these two groups of viruses also differ in their cell tropism. isolates defined as group 1 by their 5'utr sequence grow equally well on ht29 cells (a human colon carcinoma cell line) and vero cells. isolates of group 2, with one exception, grow only on ht29 cells. analysis of the structural proteins of these isolates revealed differences in migration that correlated with their cellular tropism. thus, significant genotypic and biological diversity exists amongst these virus isolates. one virus isolate had the 5' untranslated region sequence of a group 1 virus but the protein profile and cellular tropism of a group 2 virus. the best explanation of these findings is that this anornolous isolate is a natural recombinant between the parented strains. both the ease with which viable recombinants are generated and the diversity present within this one enterovirus serotype increase the potential for the production of novel pathogenic enterovirus strains. dominant susceptibility to polyoma tumors in inbred wild mice, sharon r. nahill, yupo ma, john carroll and thomas l. benjamin, department of pathology, harvard medical school, boston, ma 021 15 polyoma virus (f' y) is a mouse dna tumor virus which, under appropriate conditions, causes tumors in a wide variety of cell types. generation of tumors is a function of both the viral and host genomes. lukacher et al. have recently described a dominant gene, pyv', carried by the c3-i mouse strain, which confers susceptibility to py-induced tumors mapping and immunological analyses indicate that py4 is the mouse mammary tumor virus 7 superantigen (mtv 7 sad gene, which deletes t cells required for py tumor immunosurveillance in h-2' mice. to determine the generality of endogenous superantigens as determinants of susceptibility and to reveal potentially novel mechanisms of susceptibility, we have looked for dominant susceptibility @ s ) gene(s) id newly established and genetically diverse inbred wild mouse strains, czech i1 and pedatteck (peru). both strains are susceptible to py as 100% of infected animals develop a full profile of tumors. crosses between cs7br, whose resistance is contributed by the major histocompatibility (mhc) locus, and susceptible peru or czech 11, yield f1 progeny which are fully susceptible, indicating a dominant inheritance pattern of susceptibility the incidence of tumor-bearing backcross animals [((peru x cs7br) x c57br) and ((czech i1 x cs7br) x c57br)i suggests that ds is due to at least one, but not more than two genes. amplification of genomic dna from the czech i1 and peru mice by pcr using primers specific for mtv 7 sag indicates that both strains are negative for proviral mtv 7 sag. furthermore, the mechanism ofds in these mice may be independent of all mtv sag as pcr using primers specific for the highly conserved region of mtv sag is unable to amplify mtv dna from peru or czech i1 genomic dna. these results indicate that, like the c3hibi, the pedatteck and czech i1 contain gene(s) which overide the resistance to py-induced tumors contributed by the mhc of the c57br parent and which may cause tumors via a novel, mtv sag-independent mechanism. we have initiated efforts to map the ds in peru and czech i1 mice using pcr and primer pairs flanking simple sequence length polymorphisms. fis-2 is a low leukemogenic, but relatively strong immunosuppressive variant of friend murine leukemia virus (f-mulv). this variant was originally isolated from t-helper cells of flc-infected adult nmrl mice. compared to f-mulv, fis-2 suppresses primary antibody response more efficiently in infected mice. some of the fts-2 infected adult nmri mice developed a disease resembling the acquired immunodeficiency syndrome induced by hiv. restriction mapping and nucleotide sequence analysis of fis-2 show a high degee of homology between this variant and the prototype f-mulv clone 57. in this study we have attempted to localize the genomic determinant of fis-2 which is responsible for induction of a strong suppression of primary antibody response. six chimeric viruses of fis-2 and f-mulv were constructed. the primary antibody response of the mice infected with these chimeric viruses were investigated. the results of these experiments will be presented. anti-fmdv antibodies, as measured in an elisa capture assay, were cross reactive. b) cellular: proliferative (cd4) t cell responses of peripheral blood mononuclear cells (pbmc) were low or undetectable during primary responses to vaccine or virus, and frequently low during secondary responses. for good t cell proliferation in vitro, multiple immunisation is required. this may reflect preferential stimulation of the th2 cd4 t cell subset. interestingly, when cd4 responses were observed, cd8 tcell responses were also detectable. 2 . recognition of individual viral proteins a) expression cloning: structural and non-structural protein pseudogenes were cloned from cdna by pcr. expressed in pgex-3xuc. and purified by sds-page. b) humoral: structural and non-structural proteins were recognised by infected animals. a good anamneetic antinon-structural response was only observed when the boosting serotype differed from the serotype stimulating the primary response. c) cellular: both structural and non-structural proteins were recognised and some were cross reactive. interestingly, vp1 was strain specific, and the polymerase (3d) was the most immunogenic and cross reactive. d) a construct comprising 3d and the immunodominant vp1 epitopes was prepared and tested. in common with other herpesviruses, the envelope glycoproteins of equine herpesvirus 1 (ehv-1; equine abortion virus) are major determinants of the infectious process and pathogenicity, and are inducers of humoral and cell-mediated immune responses. as such, they are candidates for components of subunit vaccines against ehv-1. to generate useful amounts of individual ehv-1 glycoproteins, we have constructed recombinant badoviruses capable of expressing glycoproteins c, d, h (gc, gd, gh ) in insect cells, and have evaluated the recombinant products as innnunogens in a murine model of ehv-1 infection. au three glycoproteins induced serum (elisa) antihodies to ehv-1, and ehv-1 gc and gd also induced neutralizing antibody responses. following intranad challenge with infectious ehv-1, protective immunity, as demonstrated by acelerated clearance of virus fiom respiratory tissues to below detectable levels, was evident in mice immunized with either recombinant gc or gd. in contrast, gh-immmkd mice did not develop detectable neutralizing antibody, and did not clear challenge virus more rapidly than controls. delayed type hypersensitivity and lymphoproliferation responses to ehv-1 antigen were observed for each of the ehv-1 glycoproteins, and in experiments with gdimmunized mice, a role for cell mediated immunity in protection was confirmed by adoptive transfer and t-cell depletion experiments. the data provide support for the potential of glycoproteins c and d as a subunit vaccine against ehv-1. molecular pathogenesis of ural infeetiom 52-331 enterovirus-immune cell interactions: implications in enterovirus-induced diseases we have also evaluated the effect of virus infection on the humoral immune response to cvb3, infection in adolescent c3h/hesnj mice. antigen presenting cell, 1-helper cell and 8-cell function were evaluated utllizlng a sheep red blood cell (srbc) plaque assay. mice were injected intrapentoneally (ip) with lo5 plaque forming units of cvb3, at day 0 and with lo7 srbc's at days 0, 2, 3 and 4 post-cvbb, infection. splenocytes were harvested 4 days post-srbc injection, mixed with target srbc's and guinea pig complement and incubated. plaques were then quantitated. results: cvb3, was associated with 12.9% to 17.4% of cd-8 positive t-cells and w a 11 % to 26% of adherent splenocytes. after mitogen (lps and con a) stimulation, b-cells and adherent cells were demonstrated to be permissive for viral replication. a 248% and 738% under non-stimulated conditions. an average of 1 % of virus is cell-associated (plaque north america. bruce anderson, teny yates, norah torrez-martinez, wanmin song, brian hjelle. university of new mexico, albuquerque, n.m.we recently identified a new species of hantavirus (hmv) associated with the harvest mouse reithrodontomys megalotis (hjelle b et al, j. viroj. 1994, in press ). an arizona woodrat (neotoma mexicana) was found to he infected with hmv, presumably through "spillover". hmv is most closely related to the four comers hantavirus (fcv) of deer mice (genus peromyscus). the nucleocapsid gene and protein of hmv differ from those of fcv by 24% and 15% of residues, and the 1896 nt s genome is shorter by 163 nt. we surveyed 174 reithrodontomys animals captured in the u.s. and mexico for hantavirus antibodies; 27 (15.6%) were positive. s segment cdnas were amplified and sequenced from seropositive animals captured in california (4), arizona (3), new mexico (l), and mexico (2). a monophyletic clade of hmv-like agents was identified at all sites, although an r. megalotis infected with an fcv-like virus was also identified in the state of zacatecas, mexico. nucleotide sequence distances among members of the hmv clade were up to 15.5%. but amino acid distances were less than 2%. hmv is enzootic in harvest mice throughout much of north america, and can also infect wood rats. htlv i-associated myelopathy/tropical spastic paraparesis (hamnsp) is a slowly pro ressive neurological disease characterized by perivascuaar mononuclear infiltrates in the cns. htlv i-specific cd8+ ctl are found in pbl and csf of infected patients with htlv i-associated neurological disease but not in htlv i seropositive individuals without neurological involvement. previous studies have shown that in hla-a2+ patients, htlv i-specific cd8+ ctl restricted by hla-a2 recognize a peptide derived from the htlv i tax protein (tax 11-19 llfgypvw). in the present study, we have analyzed the potential of these tax-specific ctl to recognize addtional peptides. our results demonstrate that a subpopulation of high affinity cd8' tax 11-19 specific ctl clones cross-react on a self peptide derived from the se uence of myelin-associated glycoprotein (mag 556-564 vl&sdfri) presented by hla-a2. these obsenatlons suggest that the demyelination process in hamltsp may be,due, in part, to virus-specific ctl recognition of a self myelin component that is independent of htlv i infection. development of pathology varies widely between different strains of mice after intracerebral inoculation with the so-called 'docile' isolate of lymphocytic choriomeningitis (lcm) virus. the c3hebfej and blo.br/sgsnj mouse strains have been of special interest because they display autoimmune hemolytic anaemia with varying degrees of apparent immunological involvement. in this study, we examined the role of cd4+ t helper cells in this autoimmune response by treating mice with the cw-specific gk1.5 monoclonal antibody. we also determined if polyclonal activation of b lymphocytes, induced either by lcm virus or by lactate dehydrogenase-elevating virus, another well known b cell activator, correlated with the development of anaemia in these mice. our results strengthened the central role of the immune system in the anaemia in c3h mice by showing that depletion of cd4+ cells largely, if not completely, abrogated this anti-erythrocyte autoimmune reaction. as reported by others, we found that the anaemia was more mild in b 1o.br mice than in c3h mice. however, we could not confirm the difference in the degree of b lymphocyte polyclonal activation between these mice. furthermore, lactate dehydrogenase-elevating virus had no apparent effect on erythrocytes, even though this virus also induced a sharp increase in plasma igg levels. one of the two class i mhc (h-pkd)-restricted immunogenic sites identified on the influenza strain aijapanl57 (h2n2) hemagglutinin (ha) encompasses two distinct partially overlapping epitopes, mapping to residues 204-212 and 210.219. when we investigated the magnitude of the ctl responses of balwc mice to the two overlapping epitopes, we found that while the nhrterminal nonamer epitope is immunodominant, eliciting vigorous ctl responses in njapanl57-immunized balb/c mice, the ctl responses to the cooh-terminal decamer epitope are weak and variable. the c-terminal epitope subdominance seems to be due to factors other than inefficient processing of the epitope in vivo because ctls generated by priming mice with recombinant sindbis viruses expressing only one of the ha 204-219 subsites displayed patterns of responsiveness similar to that of influenza virus primed ctls. limiting dilution ctl assays showed that the ctl precursor frequency (pctl) of the nterminal epitope is at least ten fold higher than the pctl of the cterminal epitope, implying that the low and variable pattern of cterminal specific responsiveness was due to the limited t cell precursors in the c-terminal specific ctl repertoire. this was further confirmed by the limited heterogeneity in the cross reactivity patterns displayed by the c-terminal specific ctl for an ig vh fragment and the ha 210-219 epitope of influenza strain a i m 5 7 in short term bulk cultures, and the facs analysis of tcr vg chain usage. taking these together with our previous observation that some jha 210-219 specific ctls can also crossrecognize an ig vh fragment. these studies had provided a strong evidence that ig gene products may influence t lymphocyte function and repertoire development. we have previously described the identification of homologous regions in the c-terminus of hiv-1 gp41 and in the n-terminus of hla class i1 beta chains. forty percent of patients infected with hiv-i virus were shown to have antibodies which bind to the homologous sequences, as well as to native hla class i1 molecules. affinity purified crossreactive antibodies (crab) were shown to have direct blocking effects on normal t cell responses to recall antigens, and could mediate adcc of hla class ii+ cell lines.in order to determine the contribution of such antibodies to disease progression, we obtained longitudinal plasma samples from patients in the macs study. in a first study, it was found that the presence of high titers crabs correlated with a more rapid disease progression (p = 0.027 by fisher two tail analysis)in a second, 7 year-longitudinal study of 12 progre.ssors and 12 stable patients we found: (1) the production of crab was seen in 70 -80% of rapid progresson, while the true stables produce only infrequent low-titers crab. (2) in rapid pmgressors, production of crab preceded by 2-3 years the marked drop in cd4 counts. (3) crab production did not correlate with the degree of hyperglobulinemia in these patients. (4) the presence of crab during the asymptomatic stage correlated with early loss of t-helper responses to recall antigens.we are currently establishing whether periodic measurements of crab in patients sera could be valuable in predicting a drop in cd4 counts and disease progression. the lymphokine ifn-y is i pleiotropic insnunomodulator and possesses intrinsic antiviral activity. we studied its significance in the development of antiviral immune responses using ifn-7 receptor deficient (ifn-yr-'.) mice. after inoculation with live attenuated pseudorabies virus (prv) the mutant mice showed no infectivity titers in various tissues and transient viral ag expression only in the spleen similar as in wild-type mice. however, the absence of the ifn-yr resulted in increased proliferative splenocyte responses. the prv-immune animals showed a normal ifn-1 and 11-2 production, without detectable 11-4, and with decreased 11-10 secretion in response to viral ag or con a. immunohistochemically, an increased ratio of ifny/i1-4 producing spleen cells was found. after immunization with either live attenuated or inactivated prv, ifn-yr"' mice produced significantly less antiviral antibody (ab), and more succumbed to challenge infection than the intact control animals. the reduction in ah titers in the mutant mice correlated with lower protection by their sera in transfer experiments. thes? findings are in line with the strong enhancing effect of exogenous ifn-y on rabies virusand prv-specific igg responses. our data demonstrate that a physiological ifn-y system is surprisingly not critical for the generation of antiviral th-i-type and the suppression of th-2-type cytokine responses. the lymphokine, however, is an important mediator in the generation of protective antiviral ab. key: cord-005487-vac061r8 authors: nan title: physicians abstracts: ebmt 2010 date: 2010-04-07 journal: bone marrow transplant doi: 10.1038/bmt.2010.40 sha: doc_id: 5487 cord_uid: vac061r8 nan b cells have been demonstrated to present antigen to t cells in vivo. cd40-activation dramatically improves antigen presentation by normal and malignant b cells and has therefore been studied as an approach to generate autologous "non-artifi cial" antigen presenting cells for active immunotherapy. furthermore, cd40-b cells have recently been shown to expand tumorantigen and viral specifi c ctl as well as regulatory t cells and are therefore of great interest for post-transplant immunotherapy. human b cells when activated via cd40-l/il-4 can be expanded from small amounts of peripheral blood in 12-14 days. cd40-activated b cells can prime naïve cd4 + and cd8 + t cells, expand memory t cells and express important surface homing molecules. nevertheless, it remains unclear whether cd40-activated b cells migrate to secondary lymphoid organs (slo) in vivo to attract and interact with t cells. to address this question we established a methodology to generate murine cd40-activated b cells. at day 14 of culture, these cells are >95% cd19 + and cd80/86/mhci/mhciihi. murine cd40-activated b cells present a 'homing phenotype'; migrate toward slo chemokines such as ccl19, ccl21 and cxcl13; and induce t-cell chemotaxis in vitro. upon cd40l activation, b cells up-regulate ccr7 while down-regulating cxcr5 expression which suggests direction of activated b cells toward the b-zone/t-zone boundary. we compared the homing of gfp + cd40-activated b cells to resting gfp + b cells and show for the fi rst time that cd40-activated b cells home to slo significantly more effi ciently than resting b cells. furthermore, cd40activated b cells localize in the b-cell areas, and a signifi cant fraction move to the b-t boundary close to the t-cell zone over time. to dissect t-cell-apc interactions on a single cell we analyzed three-dimensional migration in collagen matrix using time-lapse videomicroscopy. interestingly, antigen-loaded cd40-activated b cells differ from immature and mature dc by displaying a rapid migratory pattern undergoing highly dynamic, short-lived (7.5 min) and sequential interactions with cognate t cells. taken together, these data revealed that cd40-activated b cells can home to secondary lymphoid organs and interact dynamically with t cells thus underlining their potential as cellular adjuvant for cancer immunotherapy. long-term follow-up of upfront tandem autologous -ric (reduced intensity conditioning) allogeneic transplantation versus autologous transplantation (nmam2000) in multiple myeloma g. gahrton, b. björkstrand, s. iacobelli, u. hegenbart, a. gruber, h. greinix, l. volin, f. narni, p. musto, m. beksac, a. bosi, g. milone, p. corradini, h. goldschmidt, t. de witte, c. objectives: treatment of multiple myeloma with allogeneic hematopoietic stem cell transplantation is controversial. the nmam2000 study compares tandem autologous (asct)/ reduced intensity conditioning (ric) allogeneic transplantation (ricallo) to only asct or asct in tandem (asct2) in a prospective study based on genetic randomization. patients and methods: out of 358 myeloma patients accrued during 2001-2005 from 26 ebmt centres 109 with an hlaidentical sibling donor were allocated to tandem asct/ricallo and 249 without to asct only. previously untreated patients with at least stable disease after vad (vincristine, doxorubicine, dexamethasone)-like induction treatment were included at the time of conditioning for asct. single (n = 145) or tandem (n = 104) asct was optional in the asct arm. conditioning for asct was melphalan 200 mg/m 2 , and for ricallo fl udarabine 30 mg/m 2 x 3 plus tbi 2 gy. the two treatment groups were well matched for standard prognostic parameters and response status at asct. results: on an intention to treat basis the cumulative 24 and 60 months non-relapse-mortality (nrm) was 12 % and 16% with asct/ricallo and 3% and 4% with asct respectively (p = 0.0001 (gray test)). the cr rate within 60 months was 52% with asct/ricallo and 41% with asct (p = 0.0009: improved in time (fine/gray model)). at 60 months after asct relapse/progression rate was 49% and 78% (p = 0.0014: reduction in time fine/gray model)), pfs 35% and 18% (p = 0.0012 reduction of risk in time (cox)) and os 65% and 58% (p = 0.0048 reduction in time (cox)) (at 84 months 60% and 22%) for asct/ricallo and asct respectively. a comparison between those patients who received a second allo (n = 91) versus a second auto (n = 104) relapse/progression rate at 60 months from second transplant was 43% and 78% and pfs 39% and 19% in asct/ricallo and auto2 respectively. information about the chromosome 13 deletion (del(13q14)) was present in 214 patients. in 92 patients with the deletion os at 60 months was 69% and 55%, and pfs 31% and 11% for asct/ricallo and asct respectively. gvhd in ricallo was as expected (agvhd grade 0-1: 80%; grade 2-4: 20%; cgvhd limited: 36%; extensive: 27%). conclusion: the risk of myeloma relapse is lower with tandem asct/ricallo as compared to asct or tandem asct, both in an intention to treat analysis and in patients that received the correct treatment. nrm is higher, but considered acceptable in view of the improved pfs and os with tandem asct/ricallo. a fi ve biomarker panel predicts acute graft-versus-host disease s. paczesny (1) , d. bickley (1) , s. choi (1) , j. crawford (1) , t. braun (1) , s. pitteri (2) , j. hogan (2) , p. reddy (1) , s. hanash (2) , j. ferrara* (1) , j. levine* (1) (1)university of michigan (ann arbor, us); (2)fred hutchinson cancer research center (seattle, us) * have contributed equally to this work current evidence suggests that a composite panel of four biomarker proteins (il2ra, tnfr1, il8, hgf) is diagnostic and prognostic for acute graft-versus-host disease (gvhd). elafi n is a biomarker that has also been found to be diagnostic for gvhd of the skin, as well as prognostic for overall survival (os). we sought to evaluate whether these fi ve biomarkers are able to predict future occurrence of gvhd, non-relapse mortality (nrm) and os. we measured by sequential elisa, levels of these fi ve proteins in plasma collected prospectively from 485 allogeneic hct patients randomly divided into training (149 gvhd-, 175 gvhd + ) and validation (74 gvhd-, 87 gvhd + ) sets. we obtained samples 3-14 days before onset of gvhd (median day 29) and at equivalent time points in patients without gvhd. there were no signifi cant differences between sets in age, conditioning intensity, donor source, hla match or gvhd grade between training and validation sets. the median day of sample acquisition was day 20 and day 21, respectively. logistic regression determined that a linear combination of the fi ve proteins levels predict the occurrence of acute gvhd in the training set. the receiver operating characteristic curves of each of the fi ve individual biomarkers are shown in figure 1 with an area under the curve (auc) for the composite panel of 0.77 (95%ci: 0.72-0.82). when this model was applied to samples of the validation set, the corresponding auc was 0.76 (95%ci: 0.69-0.84). this 5-biomarker panel therefore discriminated between patients who later developed gvhd and those who did not. proportional odds logistic regression models determined that the panel gave prognostic information regarding the eventual maximum grade of gvhd (p<0.001 in training set, p = 0.05 in validation set). given this correlation, we next divided the patients into high and low risk groups based on their predicted probability of developing gvhd (high risk ≥ 0.7 and low risk < 0.7) and analyzed these groups for differences in nrm, relapse mortality and os. the differences in 1 year nrm and os between groups were signifi cant in both the training and validation sets (table 1 ). when adjusted for other known risk factors (age, conditioning intensity, donor source, and hla match), the difference in os remains signifi cant (p = 0.02) in both sets. in conclusion, a 5-biomarker panel can predict gvhd before any clinical manifestation and provides prognostic information including long term survival. s3 their interaction with dendritic cells. recent clinical data has indicated that the content of plasmacytoid dendritic cells (pdc) in an allograft is associated with the risk of relapse following allogeneic bone marrow transplantation (bmt). we have previously shown that the addition of donor pdc to a graft comprised of purifi ed hsc and t-cells led to enhanced th1 activation of donor t-cells with improved gvl activity without increasing gvhd in multiple murine bmt model systems (li and waller ji 2009 ). here we studied the mechanism that donor pdc augment gvl without increasing gvhd, and present a novel model for regulation of post-transplant immunity. methods: donor hematopoietic stem cells (hsc), t-cells and pdc in the allograft were rigorously purifi ed using immuno-magnetic selection and high-speed fl uorescent-activated cytometry (facs) in the h2b b6->h2k b10.br transplant model. donor cell subsets purifi ed from wild type (wt) or interferon gamma knock-out(ko),(ifn-gko), interferon gamma receptor ko (ifn-grko), and indolamine 2,3 dioxygenase (ido) ko (ido-ko) were combined to determine the role of the ifn-g and ido in the separation of gvhd from gvl in murine bmt. results: in the b6->b10.br bmt model, the addition of 50,000 donor pdc to a graft comprised of 3,000 hsc and 300,000 t-cells lead to th1 immune polarization and enhanced proliferation of donor t-cells with higher levels of donor t-cell chimerism and improved gvl activity without increasing gvhd. the absence of allo-reactivity was dependent upon donor t-cell synthesis of ifn-g and the presence of ifn-gr on donor pdc. co-culture of t-cells with syngenic pdc in one-way mlr lead to up-regulation of ido in donor pdc. increased gvhd after transplanting pdc from ido ko donors in combination with wt t-cells and hsc demonstrated the central role of ido in the initiation of counter-regulatory effects that limit gvhd. wt t-cells and pdc lead to the generation of donor-derived t-reg in bmt recipients that limited gvhd. pdc from ido-ko donors had markedly reduced numbers of donor t-reg. conclusions: donor pdc regulate the initial activation and gvl activity of donor t-cells and subsequently generate t-reg that limit gvhd. the dynamic regulation of the activation and alloreactivity of donor t-cells by donor pdc suggest novel clinical approaches to enhance gvl activity without gvhd. j. peccatori (1) , d. clerici (1) , a. forcina (1) , m. bernardi (1) , r. crocchiolo (1) , c. messina (1) , m. noviello (1) , s. mastaglio (1) , f. giglio (1) , s. malato (1) , m.t. lupo stanghelllini (1), s. marktel (1) , a. assanelli (1) , m. battaglia (1) , a. ferraro (1) , s. rossini (1) , m.e. bernardo (2) , a. bondanza (1) , m. g. roncarolo (1) , c. bonini (1) , f. locatelli (1) , f. ciceri (1) ( background and aim: rapamycin, in contrast to calcineurin inhibitors, allows t regulatory cell (t-regs) proliferation while inhibits effector t cell expansion. we investigated the safety of infusion of t-cell repleted unmanipulated pbsc from haploidentical donor with a combination of rapamycin, mycophenolate and atg as gvhd prophylaxis, to preserve early t-regs function (trramm study, eudract 2007-5477-54) . patients and methods: since 2007, fi fty-nine patients (pts) underwent sct for aml (39 pts), all (9), mds (3), cml-bc (4), nhl (2) or hd (2) . median age was 50 years (range 14-69). at transplantation 7 pts were in early phase, and 52 in advanced phase. median comorbidity index (ci) score was 1 (0-5). the conditioning regimen included treosulfan (14 g/m 2 for 3 days), fludarabine (30 mg/m 2 for 5 days) and an invivo t and b-cell depletion by atg-fresenius (10 mg/kg for 3 times) and rituximab (a single 500 mg dose). all pts received allogeneic unmanipulated pbsc from an hla-haploidentical related donor. gvhd prophylaxis consisted of rapamycin (target level 8-15 ng/ml, till day + 60) and mmf (15 mg/kg tid till day + 30). results: all patients engrafted and all but eight were in disease remission at fi rst marrow evaluation on day + 30. cumulative incidence of grade 2-4 and grade 3-4 agvhd were 29 and 13% respectively. only 12 patients developed cgvhd. cumulative incidence of trm and relapse incidence were 25% and 44% respectively. after a median follow-up of 8 months, projected os at 1 year is 43%. immunoreconstitution was fast and sustained with a median 221 circulating cd3 + cells/μl (range 43-1690) from day 30. the immune-reconstitution was polarized towards central memory (cd45ra-cd62l + cells 32.7% ± 4.8), il-2 producing cells (il-2 + cells 26.2% ± 5.3). we detected high levels of cd4 + cd25 + cd127-foxp3 + t regulatory cells (up to 30% of circulating cd4 + t lymphocytes) starting from day 30. these cells were able to suppress in vitro proliferation of autologous effector cells demonstrating to be regulatory t cells. conclusions: rapamycin-mycophenolate-atg are effective as gvhd prevention in t-cell replete unmanipulated haploidentical peripheral sct and are associated with an early t-cell immunoreconstitution characterized by the in-vivo expansion of earlydifferentiated t cells and t-regs. further studies are warranted to gain insight on the role of rapamycin as platform for exploitation of t-regs in allogeneic hsct from mismatched donors. direct interaction of human nk cells with aspergillus fumigatus induces a th1 immune response and provokes signifi cant fungal killing but not via their usual cytotoxic pathways m. bouzani, m. ok, o. kurzai, h. einsele, j. loeffl er wurzburg university (wurzburg, de) objectives: invasive aspergillosis (ia), caused mainly by aspergillus fumigatus (af), is a highly devastating disease for immunosuppressed subjects. host's defence is principally confi ned to innate effector cells like alveolar macrophages, neutrophils and dendritic cells. in our study, we questioned the possible interaction of af with another potent component of the innate immunity, the natural killer (nk) cells. methods: human nk cells were isolated after magnetic depletion of the peripheral blood of volunteers and they were used after 24h priming with 500 u/ml recombinant human interleukin 2, rhil2. interferon gamma (ifn-g) and tumor necrosis factoralpha (tnf-a) regulation were assessed after nk-af coculture. fungal damage was investigated through plate killing assays. to investigate the infl uence of rhil2 on nk cells, plate killing experiments were performed with resting and primed nk cells. transwell permeable membranes, nk cell granule depletion (treatment with strontium chloride), surface expression of degranulation markers cd107a/b and neutralization of nk death ligands (tnf-related apoptosis-inducing ligand [trail] and fasl) by blocking antibodies were used to evaluate the means of interaction. results: fungal germlings induced towards nk cells a th1 immune response with upregulation of ifn-g and tnf-a (p<0.05). nk cells displayed strong fungicidal effect against germlings (p<0.05), but they were inactive against conidia. priming with rhil2 (p<0.05) and direct effector-pathogen contact (p<0.001) were required for their interaction. the cytotoxic effect was not attributed neither to the release of perforingranzyme, nor to the engagement of nk cell death ligands. conclusion: human nk cells are stimulated in vitro by af, which triggers a th1 immune response and causes important fungal killing. this interaction requires priming of nk cells with rhil2 and conditions of direct contact between nk cells and fungus. interestingly, nk cells do not mediate their cytotoxic effect via perforin -granzyme pathway, neither through the engagement of death ligands, suggesting that another pathway is involved in nk cell -af interaction. our study attributes to nk cells anti-aspergillus properties, suggesting them as a future potential immunotherapeutic tool against ia. objective: to conduct a survey on indications, effi cacy and safety of growth hormone (gh) treatment in children and ado-lescents after haematopoietic stem cell transplantation (hsct) within the ebmt. methods: in this retrospective survey using a two step approach, we asked (1st questionnaire) for information on endocrine follow-up, the use of gh, reasons for not using gh, indications for gh treatment, number of patients treated with gh and diagnostic tests to diagnose gh defi ciency (ghd); and (2nd questionnaire, to follow) data on growth, diagnosis of ghd, interval between hsct and gh treatment, dose schedules, other endocrine defi ciencies. results: 1st questionnaire: 53 centres replied until 15.11.2009: endocrine follow-up (n = 50) was performed by the hsct-clinic and an endocrinologist in 34%, the hsct-clinic in 12%, an endocrinologist in 38%, a specialist in another clinic in 10% and the hsct-and another clinic in 6%. 63% (33/52) centres treated patients after hsct with gh, whereas 37% (19/52) did not. of the centres reporting, 3/10 centres from italy used gh, whereas 7/10 did not. in contrast, 6/7 centres from france used gh whereas 1/7 did not. german centres reported the use in 3/6 centres. all centres from finland (2), great britain (2), spain (2) and switzerland (2) reported the use of gh. indications for gh treatment were growth failure in 6% (2/32), growth failure or ghd in 50% (16/32) and ghd only in 44% (14/32). reasons for not using gh were: no indication in 74% (14/19), risk of side effects in 11% (2/19) and lack of fi nancial support in 5% (1/19) . the numbers of patients treated in centres was less than 5 patients in 28% (9/32), 5 to 10 patients in 31% (10/32), 10 to 20 patients in 22% (7/32) and more than 20 patients in 19% (6/32). diagnostic tests for ghd used were the insulin-test (n = 17), arginin-test (n = 14), spontaneous gh-secretion (n = 10), clonidin-test (n = 10) and ghrh-test (n = 9). conclusions: in the majority of centres the endocrine follow-up was performed by an endocrinologist. gh treatment was regularly used for the treatment of growth failure after hsct. about 50% of the centres used gh treatment for growth failure due to ghd only. in centres not using gh the main reason was that they saw no indication for gh treatment. only a minority did not use gh due to the risk of side effects. the insulin-test, arginintest and spontaneous gh-secretion were the major tests used for the diagnosis of ghd. a. rovo, m.t. van lint, m. aljurf, n. salooja, g. sucak, a. hunter, m within nonmalignant complication after hematopoietic stem cell transplantation (hsct) gonadal dysfunction with absence of sperm production leading to defi nitive infertility is a common long-term sequela. young age at hsct and longer interval time since hsct has been associated as a favourable factor for spermatogenesis recovery. the total body irradiation (tbi) used as part of the conditioning regimen plays a central role in posttransplantation infertility. we assessed in a retrospective multicenter study on a large cohort of male survivors, the probability of sperm recovery after hsct, and evaluated associated factors for fertility recovery. male patients in which at least one seminal fl uid analysis was performed after hsct being in complete remission and in whom the results were available were the target population. ninety centers reporting to the ebmt were asked to participate, 23 accepted and so far 15 centers contributed with 217 patients. the median age at hsct and at time of 1st sperm fl uid analysis (sfa) was 23 and 29 (5-64) years respectively. the median time interval between hsct and sfa analysis was 54 months . 206 (93%) received an allogeneic hsct, 169 (73%) had bone marrow as source of stem cells, 201 (94%) received a myeloablative conditioning and 149 (67%) received tbi (with a median doses of 12 gy (7.5-14.4) as part of the conditioning. during the follow-up 130 recipients (63%) had any grade of acute gvhd, 128 (64%) any type of chronic gvhd (cgvhd); 23 (17%) from them had ongoing cgvhd at sfa time. presence of at least one spermatozoa in sfa was considered as recovery of spermatogenesis. spermatozoa were detected in the semen in 58/217 (27%) patients; 19 (9%) of them showed normozoospermia. in the univariate analysis following associated factors were signifi cant (table 1) : younger age at hsct, longer time interval between hsct and sfa, conditioning without tbi, patients without ongoing cgvhd. in the multivariate analysis, absence of tbi (p<0.00001), longer time interval (p = 0.002), absence of ongoing cgvhd at sfa (p = 0.037) were signifi cant. in conclusion, this is the largest population of male survivors evaluating spermatogenesis after hsct. in this study we confi rm the established factors which infl uence recovery of spermatogenesis such as age (univariate analysis), time interval between hsct to sfa and tbi. here for the fi rst time evidence for graft versus testis effect can be demonstrated. introduction: development of leukemia or myelodysplasia derived from donor cells termed donor cell leukemia (dcl) is a rare but severe complication following allogeneic hematopoietic transplantation. the estimated incidence is low ranging from 124 cases of dcl per 100,000 transplants (mostly myeloablative conditioning, retrospective analysis) to 5,000 per 100,000 transplants (non-myeloablative, single institution experience). although about 60 cases have been reported in the literature, very little is known about pathogenesis and clinical management of donor cell leukemia. factors proposed to be involved in malignant transformation and development of dcl include immunoregulatory dysfunction, immunosuppression, host environment triggering malignant processes, replicative stress and epigenetic reprogramming as well as antigenic or viral stimulation. due to the increasing number of case reports throughout the last few years the late effects working party decided to analyze and evaluate the experiences with donor cell leukemia within the ebmt centers. methods: a fi rst, short questionnaire will be sent to all ebmt centers asking for observed, proven or suspected cases of dcl. centers reporting one or several cases will be asked to complete a second, more detailed questionnaire. the aim of the study is to identify and analyze a suffi cient number of cases to answer the following questions: how is the incidence of dcl evolving? is development of dcl associated with viral status before and viral reactivation/infection during as well as after transplantation? is there any infl uence of graft source and donor type (bone marrow vs. peripheral blood stem cells vs. cord blood and related vs. unrelated)? is the risk of malignant transformation of grafted cells increasing with donor age? is there any association with the form and extent of pretreatment (radiotherapy and chemotherapy) implicating a role of damaged microenvironment in the development of dcl? and fi nally, is dcl also observed as a very late complication of hematopoietic stem cell transplantation? conclusion: the answers to these questions will help to further characterize donor cell derived leukemia and provide new insights into leukemogenesis in general. a. crotta, a. tichelli, a. ruggeri, i. ionescu, a.l. herr, k. boudjedir, e. gluckman, v unrelated cord blood transplant (ucbt) is associated with a reduced incidence of chronic gvhd when compared to other sources of stem cells, however risk factors analysis for incidence is scarce in the literature. we retrospectively analyzed 1257 patients (pts), 755 children (age≤18) and 502 adults, receiving fi rst single (n = 1080) or double ucbt (n = 177) in ebmt centers, between 1995 and 2009 , for malignant and non-malignant diseases, who survived at least 100 days from transplantation with neutrophils recovery and without relapse or autologous reconstitution. median age was 12 years (0. , most common disease was acute leukemia (60%). 11% of units were hla-identical (antigen level for hla-a and b, allelic for drb1), while 41% and 48% had 1 or 2-3 mismatches, respectively. median tnc infused was 3.7x10 7 /kg. conditioning regimen was myeloablative (mac) in 75% of cases (tbi>6gy in 30%), ric in 25%. busulphan-based conditioning was used in 41% and atg was added in 75% of cases. csa + steroid was the most common gvhd prophylaxis (50%); in children prednisone was used in 70% of pts, mycophenolate mofetil was used in combination in 52% of adults. median follow-up was 28 months (3-157). incidence of cgvhd at 2y was 29±1% and 45±2% in children and adults respectively (p<0.001). due to statistical difference between children and adults, risk factors analyses were performed separately. gvhd was extensive in 42% of children and in 52% of adults. out of 412 pts who developed cgvhd, 219 had previously agvhd (126 out of 212 children and 93 out of 200 adults). in univariate analysis in children, factors associated with increased incidence of gvhd were: age (>5y), advanced status of disease at transplant (>cr2) and number of hla disparities; cgvhd was 25±2% and 36±4% (p = 0.03) in 6-5/6 cord blood unit and 4-3/6 respectively. in multivariate analysis only hla disparities (hr = 1.95, p<0.001) and status of disease at transplant (hr = 1.74, p = 0.006) were associated with increased incidence of gvhd. in adults, higher incidence of cgvhd was associated with male sex, advanced disease at ucbt and use of mac regimen. in multivariate analysis only the status at transplant was independently associated with cgvhd (hr = 1.46, p = 0.03). in conclusion, incidence of cgvhd was lower in children than in adults. status of disease at transplant was associated with increased incidence of gvhd in children and adults. number of hla disparities was associated with increased cgvhd only in children. in ms, progression free survival has been reported to range from 50 to 70% at 5 years after hsct. the most frequent conditioning regimen adopted in europe is the association of beam and atg, an intermediate-intensity scheme able to completely suppress mri activity for at least 2 years in most of patients. mortality has dropped in the last years from a relevant 7,3% to 1.2%, due to a better transplant management and patients selection. in rapidly progressive ssc 5-years mortality is estimated to be 40-50% [11] . the most commonly used conditioning regimen in europe is cyclophosphamide (200 mg/kg) and atg. in these patients, progression free survival after hsct has been showed to be above 50% at 5 years. furthermore, the use of high intensity conditioning regimens does not seem to offer special advantages over lower intensity schedules for this disease. the distribution of transplants per year shows a drop of activity in sle and especially in infl ammatory arthritis, following the introduction of new biological agents after 2000. the emergence of pharmacological resistance in long-term treatments may suggest a possible renewal of the activity also in this subset of patients. in chronic infl ammatory bowel diseases (ibd) an increasing activity has also been shown in the last 6 years. hsct is able to provide a high rate of long-term, immunosuppression-free remissions in ad patients resistant to conventional therapies. appropriate selection of patients is crucial for providing the best risk-benefi t equipoise. evidence of effectiveness and careful assessment of toxicity is expected from ongoing and future prospective clinical trials. data reported to the ebmt registry at december 2009 102 european activity in multiple sclerosis r. martin (1) , g. mancardi (2) intensive immunosuppression followed by autologous transplantation of hematopoietic stem cells (ahsct) has been investigated as a possible strategy for the treatment of severe autoimmune disorders, including multiple sclerosis (ms) . it has been demonstrated that ahsct leads to the elimination of self reactive t cells and complete or almost complete renewal of the immune repertoire. immune system renewal has been demonstrated at the level of the t cell receptor repertoire in cd4 + and cd8 + t cells and in subsets of nk-and t cell population. according to the database of the european blood and transplantation group (ebmt) registry, more than 400 ms cases have been treated with ahsct. the retrospective survey, carried out in 2006, of all the patients recorded in the registry, with a median follow up of more than 3 years, showed that improvement or stabilization of neurological conditions occurred in 63% of cases. transplant related mortality was very high (7,3%) in the 1995-2000 period, but subsequently dropped to 1.3% in the 2001-2007 years. the results appear to be particularly impressive in rapidly evolving severe ms cases unresponsive to conventional therapies. in these cases, defi ned also as malignant forms of ms, numerous european groups convincingly demonstrated the capacity of ahsct to suppress the progression of the disease, with an unexpected relevant improvement of the neurological conditions lasting for a long period of time. the establishment of ahsct as a rescue therapy in highly active ms cases unresponsive to other conventional therapies represents a very important step in ms clinical management, and the above mechanistic studies strongly support the rationale of this approach. despite these advances, the acceptance of ahsct as a rational treatment escalation remains low among neurologists, and this is probably one of the main reasons why the phase 2 study (astims), comparing ahsct vs. mitoxantrone with a mri primary endpoint, had to be terminated due to recruitment diffi culties. based on this experience, a new phase iib/iii study is currently being designed. this study will compare ahsct with best available and approved therapy in the group of highly active relapsing-remitting ms patients, who failed fi rst-line therapy and one escalation step. the main goals of this trial will be to establish effi cacy in a controlled trial and to address important mechanistic questions. the status of the current discussion toward the trial design as well as strategies to recruit interested centers with ms-specialized neurologists and experienced transplant physicians at one location and potential mechanisms of funding will be presented. systemic sclerosis is a potentially life-threatening chronic autoimmune disease characterized by skin thickening, vasculopathy, and visceral involvement, mainly of lungs, gut, heart and kidneys. the clinical manifestations are diverse and refl ect a spectrum of subsets, ranging from limited to diffuse disease. diffuse cutaneous systemic sclerosis (dcssc) generally runs a more aggressive disease course, requiring intensive immunosuppressive therapy. haematopoietic stem cell transplantation (hsct) has resulted in long-term improvements of skin thickening, stabilization of organ involvement, but at the expense of mainly cardiopulmonary toxicity which has resulted in death in 5-8% of cases according to a recent analysis by the ebmt working party autoimmune diseases. to assess whether the benefi ts outweigh the risks, a prospective randomized controlled trial is nearing completion comparing hsct with iv pulse cyclophosphamide in which 156 dcssc patients have been enrolled in 27 centres. the primary endpoint is event-free survival, defi ned as survival until death or development of major organ failure during 2 years follow-up. formal analyses of safety and effi cacy will be done in 2011. interim safety analyses have lead to changes in the transplant protocol and eligibility criteria to also target patients with very early disease. the rationale of the trial is that effective intervention in very early disease is needed to induce long-term remissions. all patients are followed-up for at least 7 years. in anticipation of the results from the astis trial, a new smallscale clinical trial will be done to comprehensively investigate the mechanism of action of hsct in ssc focusing on its effects on biomarkers of fi brosis, autoimmunity, infl ammation and vasculopathy. it is hoped that the results of the astis trial and those of the north american scot trial will provide solid data to decide on the design of a subsequent randomized trial. if astis and scot indeed prove that hsct is superior over conventional chemotherapy, then one option would be to modify the trans-plant regimen to reduce the risk of cardiopulmonary toxicity and/or enhance the intensity of the control arm. crohn's disease (cd) is characterized by chronic infl ammation in segments of the digestive tract leading to tissue damages. uncontrolled infl ammation is associated with excessive immune responses towards the microbiota. progress has been recently made in the management of cd, with increased use of immunosuppression and biologic therapies. early optimized use of these treatments in patients with poor prognosis may provide a satisfactory control of the disease in the majority of patients. however, a fraction of cd patients experiences severe disease refractory to medical management, which may result in progressive tissue damages, need for surgery and chronic disability. the fi rst evidence of effectiveness of hematopoietic stem cell transplantation (hsct) was reported with the observation that patients with cd, who underwent allogenic or autologous hsct for a haematological or solid malignancy, experienced long-lasting remission of their ibd. although few cases regarding autologous hsct in concomitant cd and malignant diseases have been reported, collected data have shown similar results to allogeneic transplantation in terms of remission. beyond case reports, autologous hsct as primary treatment for cd has been investigated in two single-centre phase ii studies. despite the low number of patients and their limited follow-up, autologous hsct was shown to have the capacity to induce complete clinical and endoscopic remission, with impressive results. the astic trial, designed to assess its effi cacy and tolerability, is ongoing. included patients undergo mobilization of stem cells, and are then randomized to transplantation after one month or after one year. autologous hsct may represent a therapeutic option in cd patients with refractory disease, and could change the natural history of the disease, inducing in some cases long term remission. haematopoietic cell transplantation for autoimmune diseases: ebmt/cibmtr collaboration m. pasquini (milwaukee, us), r. saccardi (florence, it) high dose immunosuppressive therapy with autologous haematopoietic cell rescue has the potential to halt the autoimmune process and result in a medication-free period. since 1996, more than 1000 patients in europe and 400 patients in north and south america who underwent transplantation for autoimmune disease (aid) have been registered in the ebmt and cibmtr databases. despite this activity, questions related to the timing of transplantation and what aid benefi ts more from transplantation remain unanswered. the development of a collaborative strategy has the potential to answer some of these questions and assist the development of prospective studies that can advance the fi eld. following this strategy the cibmtr and ebmt autoimmune disease committees joined forces in 2007 to expand the study portfolio and establish an infrastructure to facilitate future studies. the most common aid indication for transplantation is multiple sclerosis (ms). data collection from both registries was harmonized into a single disease-specifi c form developed by a group of investigators affi liated to both committees. collaborative project to focus on long term outcomes after transplant for ms was developed, especially to determine factors associated with prolonged disease-free intervals. two in person discussions on the topic leveraged the development of an international phase iii clinical trial with the objective to compare transplant with best medical treatment in a population with rapidly progressive ms. the next collaborative initiative is to focus on the second most common indication, systemic sclerosis (ssc). we plan to start harmonizing disease-specifi c forms and a collaborative study focused in this disease. lastly, as the majority of patients present to transplantation with advanced aid, studying patients with coexistent aid who present for allogeneic transplantation, represent an opportunity to understand the graft-versus-autoimmunity effect. thus, the cibmtr is planning to identify these patients at time of registration and subsequently enrolled them on a prospect cohort study to evaluate the impact of graft-versus-host disease on aid. outcomes database on transplant for aid are currently underutilized due to the heteroneity of diseases, scarcity of comprehensive level disease-specifi c information and loose integration with non transplant specialist. bringing the ebmt and cibmtr together in the last 3 years, helped engage investigators, including neurologists and rheumatologist to develop collaborative studies. an update on autologous stem cell transplantation in severe systemic lupus erythematosus and in early diabetes type 1 d. farge, r. cervera, d. jayne, a. voskuyl, j.m. van laar, a. doria, m. mosca, d. boumpas, r. saccardi, e. snarski, j.f objectives: to present the experimental and clinical rational for the ongoing ebmt or nih trials for ahsct in severe sle and in early diabetes type i. background: since 1996, autologous hematopoietic stem cell transplantation (ahsct) has been used successfully in severe systemic lupus erythematosus (sle) and early insulin type i dependent diabetes (t1d) to induce durable remission. in sle bilag a, the nih or eurolupus cyclophosphamide (cy) protocols and mycophenolate mofetil (mmf) for induction are associated with 20% failure, 50% relapse and 10-15 % death at 10 yrs. among 85 sle out of 900 ebmt pts with fi rst ahsct in 2009, 3 years pfs was 54 % (95% ci: 42%-66%) with 87% (95% ci: 79%-95%) overall survival (farge d haemetologica 2009 ). higher remission rates and also some relapses, were shown in the north american experience (burt r, jama, 2006) with the possibility of resetting the autoimmune response and inducing tolerance in sle after hsct and then in new onset type i diabetes (voltarelli and burt et al, jama, 2007) . in susceptible strains of mice, allogeneic hsct prevents insulitis and development of type 1 dm. in 2008, follow-up of the 23 ahsct for t1d with in brasil, showed that 14/21 remained insulin free (and normal hba1c) with longest insulin free follow-up beyond 4 yrs and no correlation with pre-hsct c-peptide, but ahsct must be performed within 3 months of diagnosis. eight patients with t1d (<6 weeks from diagnosis, c-peptide positive, anti gad -antibodies positive) treated in poland using 200mg/cy conditioning and ahsct plus antithymocyte globulin (atg genzyme) had no major complications after 6 (2-15) mths median follow-up and 8/8 pts became independent from exogenous insulin within 24 ( + 6-+ 60) days after ahsct. acarbose may have positive effects on the duration of remission of t1d after ahct. planned studies: these were the basis for 1) the multicenter, ebmt approved astil phase ii study, to analyse the effi cacy of ahsct followed by mmf as maintenance for severe sle 5 yrs since diagnosis with sustained or relapsed active bilag a sle after at least 6 months of best standard local therapy among 30 patients. after cy 4 g/m 2 mobilisation, unselected ahsct will use cy (200 mg/kg body wt in 4 daily doses) plus rabbit atg and maintenance by mmf (2 g/day). the primary effi cacy endpoint will be the proportion of patients alive who achieve clinical success at 12 months and maintenance of this status until 24 months after inclusion; 2) the nih approved phase i/ii study of ahsct in 15 newly-diagnosed t1d pts within 3 months of diagnosis, and a positive antibody to an islet cell autoantigen and fasting c-peptide > 0.2 nmol/l. to compare cy 4 g/ m 2 mobilization, unselected ahsct using cy (200 mg/kg body wt in 4 daily introduction: graft versus leukemia (gvl) effect of hematopoietic stem cell transplantation (hsct) is mostly based on donor t cell-mediated alloreactivity. this is particularly relevant in the context of hsct from family haploidentical and matched unrelated donors (mud), in which mismatched hla molecules are potent targets for donor t cells. still, upon in vivo selective pressure by donor t cells, leukemia can undergo genomic rearrangements which result in loss of the patient-specifi c hla, a mechanism which our group recently demonstrated to be frequently responsible for leukemia relapse after haploidentical hsct (vago et al., nejm, 2009 ). methods: 113 consecutive patients who underwent a partially hla-matched transplantation for myeloid malignancies from 2002 to present, were included in our analysis. for 76 patients the donor was family haploidentical, and for 37 was unrelated and mismatched for a median of 2/12 hla alleles. all patients received donor t cells as part of the hsct protocol. post-transplantation follow-up comprised monthly bone marrow examination, with short tandem repeat (str) chimerism analysis and hla typing performed in parallel. in cases of relapse with suspected loss of the mismatched hla, str chimerism and hla typing were performed also on purifi ed leukemic blasts. results: disease relapse occurred in 31 and 9 patients after haploidentical and mud transplantation, respectively. after haploidentical transplantation, 11/31 relapses (35.5%) were due to mutated leukemic blasts which had lost the patientspecifi c hla haplotype. interestingly, 1 of the 9 relapses after mud transplantation occurred through the same mechanism, in a patient who had received two subsequent transplants from hla-c-mismatched donors. upon detection of the mutated leukemic blasts, 9 of these 12 patients were enrolled to receive a subsequent hsct from a different donor, mismatched for the remaining hla haplotype. conclusions: our data consolidate the clinical relevance of this escape mechanism from the gvl effect mediated by alloreactive donor t cells. screening for these mutants should be encouraged in patients who relapse after partially hla-mismatched hsct, to guide therapeutic strategies, avoid predictably ineffi cacious donor t cell add-backs, and quickly enroll the patients to a salvage transplant. to improve this approach, novel diagnostic and therapeutic tools are warranted, to provide earlier molecular detection and specifi c targeting of these mutants. impact of allogeneic stem cell transplantation on prognosis of patients with high-risk aml: results of the aml shg 295 and 01/99 trials k. wagner, g. heil, m. zucknick, d. hoelzer, o.g. ottmann, h. martin, m. lübbert, j. finke, w. heit, w. fiedler, l. kanz, g. schlimok, h. kirchner, a. raghavachar, w. brugger, a. ganser, j objective: patients with high risk acute myeloid leukemia (aml) have a dismal prognosis after chemotherapy. allogeneic stem cell transplantation (allosct) in fi rst complete remission (cr) is widely recommended for these patients but its impact on outcome is uncertain. methods: in the prospective multicenter trials aml 295 and 01/99, we treated 825 adult patients with aml (except apl) up to 60 years. all patients received intensive double induction and early consolidation chemotherapy. high risk patients were defi ned by either bad response to induction i (persistent bone marrow blasts on day 15 after start of therapy) or high risk karyotype (all karyotypes other than normal or cbf-leukemias). all high risk patients with a matched related donor (mrd) were scheduled for an allosct as late consolidation. patients without a family donor in the 295 trial should receive an autologous stem cell transplantation, whereas in the 01/99 trial an allosct from matched unrelated donors (mud) was recommended. results: median follow up of the patients is 78 months. 393 of the patients fulfi lled the high risk criteria. median overall survival (os) of these patients is 17 months and the six-year os is 30%. in multivariate analysis, a complex or monosomal karyotype (hr 1.82; 95% ci 1.36-2.43) and age above the median of 47 years (hr 1.53; 95% ci 1.18-1.98) were identifi ed as adverse prognostic factors for os. 234 of the 393 high risk patients (59.5%) achieved a cr. median relapse-free survival (rfs) is 12.8 months and six-year rfs is 33%. leukocytes above the median (hr 1.65; 95% ci 1.18-2.29) and a complex/ monosomal karyotype (hr 1.61; 95% ci 1.06-2.44) were identifi ed as independent prognostic factors for rfs. of the 234 patients who achieved cr, 97 received an allosct from mrd (n = 65) or mud (n = 32) in fi rst cr. median os of the transplanted patients is not reached and six-year os is 57%. median rfs after allosct is 100 months and six-year rfs is 53%. outcome did not differ between mrd and mud allosct. the impact of allosct on prognosis was analyzed in a multivariate cox-model with allosct included as a time-dependent covariable. in this analysis, allosct in fi rst cr signifi cantly improved os (hr 0.48; 95% ci 0.34-0.69) and rfs (hr 0.48; 95% ci 0.33-0.69). conclusion: allosct from matched related or unrelated donors in fi rst cr improves overall and relapse-free survival of patients with high risk aml. cord blood transplantation for adults with acute lymphoblastic leukaemia -a survey of outcomes conducted by eurocord and the acute leukaemia working party of the ebmt d. purtill (1) cord blood transplantation (cbt) is considered an option for adults with acute lymphoblastic leukaemia (all) for whom stem cell transplantation is indicated. however, little information is available regarding outcomes of this procedure in this group of patients. we conducted a retrospective review of the eurocord database in order to describe outcomes of adults treated with single or double cbt for all at ebmt centres from 2000 until 2008. two-hundred and thirty six patients with a median age of 30 years (18-62 years) were included. most were transplanted in fi rst complete remission (cr1) (n = 101) or cr2 (n = 75). median white cell count at diagnosis was 13.9x10 9 /l (0.6-624) and 56% (n = 56) of patients in cr1 had t(9;22) or t (4;11). double cord blood transplantation was performed for 73 patients (31%). overall median total nucleated cell dose at freezing was 3.9x10 7 /kg (1.45-8.81 ) and it was 4.5 (2.1-8.8 ) and 3.4x10 7 /kg (1.5-7 .0) respectively for double and single transplants. most patients received cord units with one (n = 65) or two (n = 133) hla disparities. overall cumulative incidence (ci) of neutrophil recovery was 74±3% and acute graft-vs-host disease was 33±3%. transplant-related mortality (trm) was 41±3% and 35±3% for patients in cr1 and cr2 respectively, and 63±3% for the 60 remaining patients with more advanced disease (cr3, relapsed or refractory disease). ci of relapse were 20±6%, 27±8% and 33±11% respectively for cr1, cr2 and advanced disease. at the median follow-up of 2 years, leukaemia-free survival (lfs) was 30±3% for the whole population and 39±9% for cr1, 38±6% for cr2 and 4±3% for advanced disease. lfs for patients in cr1 with t(9;22) or t(4;11) was 39±7% and for double cbt it was 38±7%. on multivariate analysis, being in cr1 or cr2 at transplant was the only factor associated with improved lfs (hr 0.25, 95%ci 0.08-0.42, p<0.001). when this group was analysed separately, the use of a reduced intensity conditioning (ric) regimen was associated with improved lfs (57±8% vs. 32±5%, hr = 0.53, 95%ci 0.27-0.79, p = 0.01). in particular, the 27 patients who received the combination of cyclophosphamide, fl udarabine and tbi (2-6 gy) had an lfs of 64±10%. cord blood is an alternative source of stem cells for allogeneic transplantation for adults with high risk all. results with ric and double cbt are encouraging and should be further investigated in a larger series of patients. superior long-term survival with a high rate of allogeneic stem cell transplantation in aml (non-apl) patients below 60 years of age g. juliusson (1) introdution: allogeneic stem cell transplantation (allosct) prevents relapse in aml, but the indication in intermediate risk is unclear and depends on the transplant-related mortality. the swedish acute leukemia registry (blood 2009; 113:4179) includes 3878 patients (pts) with acute leukemia diagnosed 1997-2006 covering 98% of all cases in sweden, with 9 million inhabitants. using this population-based registry, we evaluated the sct rates in aml, and the long-term outcome. allo-sct rates in different swedish regions were also evaluated. transplantation data was individually validated in 2008, and survival was updated through the national population registry. patients and results: of 797 adult aml (non-apl) pts <60 yrs (median 51 yrs), 304 (38%) had an allosct (23% with secondary aml) in cr1 (n = 206; 26%), or at later stages (n = 92; 12%), with decreasing rates by age ( figure 1 ). donors were unrelated in 52%. median follow-up was 6.2 yrs, and 170 (56%) of allografted pts were alive. treatment-related mortality (trm, i.e., death in cr), and aml deaths according to age, donor, and status at allosct is shown in figure 2 . estimated 5-yr and 10-yr os was 41% and 35%, respectively. these results could be compared to recently published large aml studies from ecog, eortc-gimema, uk mrc and gamlcg, with allosct rates of 5-15% and 5-yr os of 18-38%. the health care region se, which had the highest allosct rate (61% of aml pts <60 yrs) had a 5-yr os of 52% for the total aml population, as compared to the other swedish regions with a mean allosct rate of 35% and a 5-yr os of 40% (log rank, p = 0.04). the difference in allosct rate was mainly due to more allografts among older pts: in the age cohort 50-59 yrs, 42% of cr1 pts underwent allosct yrs in region se as compared to 15% in the rest of sweden, associated with a more pronounced survival benefi t (p = 0.007). conclusions: this is a real world population-based study on allosct rates, treatment-related mortality and long-term survival in aml pts. our results indicate that allosct is performed in sweden with relatively low total non-relapse mortality, and with a moderate relapse rate. the association found between a high allosct rate in pts 50-59 yrs and a better survival is intriguing, is suggestive of a positive impact of a more frequent transplantation practice in this age cohort, and supports the need for prospective studies. role of the graft-versus-leukaemia effect after reducedintensity conditioning allogeneic stem cell transplantation as treatment for acute myeloid leukaemia: a survey from the acute leukaemia working party of the ebmt f. baron, m. labopin, m. mohty, n. basara, d. niederwieser, n. milpied, j.j. cornelissen, c. malm, l. vindelov, d. blaise, j.j.w.m. janssen, e. petersen, g. socie, v previous studies have observed a lower risk of relapse in patients (pts) who experienced chronic gvhd after ric allo-sct versus in those who did not. the objective of the current study was to further investigate the association between chronic gvhd and relapse in a large cohort of pts given ric allo-sct as treatment for aml. data from 1188 aml pts in fi rst or second cr transplanted between 2000 and 2008 following a ric regimen at ebmt affi liated centers were analyzed. patients were given pbsc from hla-identical sibling (mrd, n = 879), or from hla-matched unrelated donors (mud, n = 309). median pt age at transplantation was 55 yrs in pts given grafts from mrd, versus 57 yrs in those given grafts from mud. the impact of chronic gvhd on relapse risk, non-relapse mortality (nrm) and leukemia-free survival (lfs) was assessed by time-dependent multivariate cox models and in a landmark analysis. three-yr incidences of relapse, nrm and lfs were 35 ± 2%, 14 ± 2%, and 50 ± 2%, respectively, while 2-yr incidence of chronic gvhd was 49 ± 2%. in a landmark analysis at 18 months after allo-sct, 5-year relapse rates were 10 ± 2% versus 19 ± 3% for patients with or without chronic gvhd (p = 0.04), respectively. in multivariate cox models, cr2 versus cr1 (p = .003), pt age >55 yrs (p = .008), alemtuzumab use in the ric (p = .048), tbi-based ric (p = .006), high-risk cytogenetics (p = .001), and absence of chronic gvhd (p = .015) were each associated with higher risk of relapse. factors associated with high nrm were mud versus mrd (p = .003), grade ii-iv acute gvhd (p<.001), and chronic gvhd (p = .002). factors associated with lower lfs were cr2 versus cr1 (p = .003), pt age >55 yrs (p = .007), alemtuzumab use in the ric (p = .012), and high-risk cytogenetics (p = .003). in conclusion, in this cohort of aml patients transplanted in remission, chronic gvhd was associated with a lower risk of relapse while profound in-vivo t cell depletion with alemtuzumab was associated with higher relapse rate suggesting that gvl effects play a role in preventing aml relapse in patients given ric allo-sct. in spite of the presence of gvl effect, chronic chvd was associated with higher nrm and therefore there was no net impact on lfs. strategies to decrease nrm related to chronic gvhd should be further investigated in order to keep the benefi ts from gvl effect and improve lfs. finally, the impact of chronic gvhd duration and severity on lfs need to be studied. allogeneic stem cell transplantation in acute myeloid leukaemia with normal karyotype: a risk factor analysis in 247 patients, based on molecular markers and stage at transplantation t. pfeiffer (1) , m. schleuning (2) cytogenetically normal acute myeloid leukaemia (cn-aml) represents a heterogenous disease, that can be subdivided by molecular analysis. only limited data is available on sct in different molecular subgroups, particularly in advanced stage. therefore, we retrospectively analyzed data on 247 pts. with cn-aml, who uniformly had received the flamsa-ric regimen for sct in 14 european centres. pts. suffered from de novo aml (76%), saml/mds (21%), and taml (4%). median age was 52y (19-71). donors were matched or mm family, and matched or mm unrelated donors in 30%, 2%, 50% and 18%, respectively. sct was performed in untreated disease (6%), primary induction failure (pif,23%), 1st complete remission (cr1,14%), and >cr1 (57%). median follow-up of survivors was 19 mo. overall survival (os) and leukaemia-free-survival (lfs) at 2y from sct was 51% and 47%. the stage at sct was the most important factor (p = .001 for os, <.001 for lfs). results were promising after sct in cr1 (2y os/lfs 76%), and pif (2y os/lfs 69%), but were inferior after sct in untreated disease (2y os/lfs 34%), or >cr1 (2y os 42%, lfs 34%). information on molecular markers was available in 183 pts. (74%). as suggested (schlenk, nejm 2008) , analysis was based on 2 subgroups: 22 pts. with isolated npm1 mutation (npm1mut), and 161 pts. with other genotypes (flt3-itd, n = 66; or wildtype flt3/wildtype nmp1 [flt3wt/npm1wt], n = 95). pts. with npm1mut had a 4y os/lfs of 75/63% with identical outcome, when transplanted in pif, cr1, or >cr1. pts. with other genotypes showed an os/lfs of 51%/48% at 2y and of 40%/39% at 4y, without differences among pts. with flt-itd and flt3wt/npm1wt. however, in this subgroup, outcome was highly dependent on the stage at sct, with excellent results after sct in cr1 (2y os/lfs: 76%) or pif (2y os/lfs: 75%/74%), but inferior outcome after sct >cr1 (2y os/lfs 38%/33%; p = .004 for os, .001 for lfs). conclusion: allosct following flamsa-ric produces excellent survival in pts. with cn-aml, particularly when performed in cr1. encouraging results in pif support an early sct, regardless of molecular subgroup, when cr is not reached after double induction. in npm1mut, sct in advanced disease achieved identical results as in early stage, supporting the delay of sct until relapse has occurred. in contrast, pts. with flt3-itd or flt3wt/npm1wt achieved signifi cantly worse results when transplanted >cr1, arguing in favour of sct in cr1 for this molecular subgroup. comparison of outcomes after allogeneic sct for adult patients with aml in remission using either i.v. busulfan plus cyclophosphamide or tbi plus cy in the myeloablative conditioning regimen: an-alwp-ebmt survey a. nagler (1) , m. labopin (2) , a. shimoni (1) tbi/cy and oral bu/cy are the most common myeloablative regimens for adults with aml with comparable survival and relapse probabilities. intravenous (i.v.) bu in contrast to oral bu has more predictable pharmacokinetics and a more favorable toxicity profi le. outcomes with i.v. bu/cy, thus, may be superior to those achieved with tbi/cy. in order to address these issues, the alwp of the ebmt performed a survey comparing i.v. bu/cy to tbi/cy as conditioning regimen for adult pts. with aml undergoing allosct. overall, 1479 allosct were analyzed: bu/cy -332, tbi/cy -1147. median age was 40 (range, 18-60) and 39 (range, 18-65) years and median transplant year was 2007 (2000 -2007) and 2004 (2000 -2007) for the bu/cy and tbi/cy groups, respectively (p<0.0001). disease status at allosct was cr1 -81% vs. 80% and cr2-19% vs. 20% for the bu/cy and tbi/cy groups, respectively (ns). wbc count at diagnosis was 15x109/l in both groups. cytogenetic: good -6% and 8%, intermediate -26% and 42% and poor risk -6% and 6%, respectively (na -44%-62% of pts). 76% and 82% of both groups underwent allosct from sibling donors, while 24% and 22% from mud, respectively. follow up was 15(0.5-96) and 41 mo. for both groups, respectively. 75% and 47% of the bu/cy and tbi/cy groups received pbsc, while 25% and 53% received bm grafts, respectively (p<0.0001). engraftment was similar in the bu/cy vs. the tbi/cy groups, 97% and 97%, respectively. cumulative incidence of nrm at 2y was 16 + 2% and 18±1%, respectively (p = 0.46). acute gvhd both >ii-iv and iii-iv were signifi cantly lower in the bu/cy vs. the tbi/ cy groups: 21% and 32%and 8.5% and 19.5%, respectively (p<0.0001). while, vod was signifi cantly higher in the bu/cy vs. the tbi/cy groups, 7.8% vs. 1.7%, respectively (p<0.001). two year relapse incidence was comparable, 25 + 3% and 21 + 1% for bu/cy vs. tbi/cy, respectively (p = 0.73). lfs was comparable, as well, 59 + 3% and 61 + 2%, respectively (p = 0.73). in multivariate analysis poor prognostic factors for lfs were: poor cytogenetics (p≤10-4), disease status (cr2 vs. cr1) (p≤10-4), age >40y (p = 0.001), mud vs. sib. donor (p = 0.037) and pb vs. bm grafts (p = 0.042). in conclusion, in this retrospective ebmt survey, outcomes of allosct including engraftment, trm, rr and lfs was comparable, while acute gvhd probability was signifi cantly lower and vod probability signifi cantly higher in adult pts. with aml in cr using myeloablative i.v bu/cy vs. tbi/cy conditioning regiments, respectively. higher incidence of relapse with higher doses of cd34 + cells from leukapheresis products infused in adults with acute myelocytic leukaemia autografted during the fi rst remission n.c. gorin, m.m. labopin, d. blaise, f. witz, t. de witte, g. meloni, m. attal, d. carreras, v. rocha on behalf of the alwp purpose: the cell source for autologous stem cell transplantation has shifted from bone marrow (bm) to peripheral blood (pb). for patients with aml in cr1, we previously showed that the risk of relapse was greater with pb than bm and a poorer outcome was associated with a shorter interval from cr1 to pb transplantation (≤80 days) (jco 2009 in press). leukemic and normal progenitors bear the cd34 + antigen and can be mobilized together; we questioned whether there was a relation linking the doses of cd34 + cells infused to the outcome. methods: out of 1262 patients autografted with pb more than 80 days post cr1 and reported to the ebmt registry using medb form, the dose of cd34 + cells infused was available in 772. results: the cd34 + cell doses were categorized by percentiles to divide the whole group into fi ve categories containing the same number of patients. we identifi ed the fi fth percentile (>7.16 × 10 6 /kg) as the cut off point for relapse incidence (ri) and leukemia-free survival (lfs). patients receiving the highest dose had a higher ri (57 ± 4%, vs. 44 ± 2%; p = 0.008) and a lower lfs (34±4% vs. 49 ± 2%; p = 0.007). in a multivariate analysis adjusted for differences, ri was higher in patients receiving the highest cd34 + cell dose ((hazard ratio [hr], 1.48; 95% ci, 1.12-1.95; p = 0.005).and the lfs was worse (hr, 0.72; 95% ci, 0.55-0.93; p = 0.01). conclusion: for patients with aml in cr1 autografted with pb, risk of relapse is greater and lfs is lower in those receiving the highest doses of cd34 + cells. oral session 2: early side effects o122 dna transfer from donor to host cells as a mechanism for epithelial chimerism and genomic alterations after allogeneic haematopoietic cell transplantation m. waterhouse (1) , m. themeli (2) , h. bertz (1) , n. zoumbos (2) , j. finke (1) , a. spyridonidis (2) (1)albert ludwigs university of freiburg (freiburg, de) ; (2)university hospital patras (patras, gr) research in the fi eld of allogeneic hematopoietic cell transplantation (allo-hct) revealed hidden consequences of the co-existence of two genetically distinct populations in the transplant recipient. first, epithelial cells with donor-derived genotype emerge and second, epithelial tissues of the host acquire genomic alterations. we asked whether horizontal transfer of donor-dna to host epithelium is operating in (and linking) both phenomena. 176 buccal samples were obtained from 71 allotransplanted patients and analyzed with microsatellite markers for the presence of donor-dna and microsatellite instability (msi). the presence of graft-derived hematopoietic cells in the samples was excluded by immunocytochemistry. the results were associated with clinical data. genomic instability induction and dna transfer in epithelial cells by allogeneic lymphocytes was assessed in vitro. we found a high contribution of donor-dna (mean 26.6%) in buccal samples in 61 out of 68 evaluable patients. in addition, 32% of the samples were positive for msi (msi + ). the extent of donor-dna was signifi cantly correlated with the occurrence of genomic alterations (p<0.05). the age of the patient and a female-to-male transplantation were signifi cantly correlated with msi. there was a trend for increasing risk of msi for patients who experienced severe graft-versushost disease. during follow up secondary malignancy was diagnosed in 5 patients (14%) who exhibited msi but only in 1 (3%) with no msi. by applying a time-dependent statistical analysis we found that the probability of secondary tumor development was signifi cant higher in the msi + as compared to the msipatients (p = 0.024, hazard ratio 6.68, 95% . in an in vitro model, we demonstrated that apoptotic lymphocytes may not only induce genomic alterations but also transfer dna through a phagocytotic process to co-cultured epithelial cells. the ingested lymphocytic dna was also incorporated into the nucleus and integrated into the genome of the recipient epithelial cells, resulting in the creation of hybrid chromosomes. our results indicate that continuous charge of the transplant recipient with apoptotic donor-dna and its illegitimate integration into host epithelium may come in light as epithelial cells with donorderived genome or genomic instability events and may provide a new model for elucidating protean clinical manifestations after allo-hct, such as secondary malignancy. the haematopoietic cell transplantation-specifi c co-morbidity index and non-relapse mortality after reduced intensity conditioning: fi nal analysis from the alwp of ebmt in aml patients in fi rst complete remission m. mohty, m. labopin, n. basara, j.j. cornelissen, r. tabrizi, c. malm, j. perez-simon, a. nagler, n. kröger, b. rio, r. martino, m. eder, k. bilger, d. bunjes, g. socié, d. blaise, e. polge, v the current study was designed to test the performance of the adapted charlson comorbidities index (ci; so-called "sorror index") and its association with outcomes among a cohort of 345 patients (i) aged >50 y.; (ii) diagnosed with a single disease entity, aml in cr1, and (iii) who underwent a ric allo-sct reported to the ebmt registry between 1999 and 2006. in this series, the median year of allo-sct was 2004, and the median age was 58 y. (range, 50-76) . 32% of patients needed more than one induction course to achieve cr1. a fl udarabinebased ric regimen was used in 64% of patients, while 31% of patients received low-dose tbi as part of their ric, and 6% received other non-specifi ed ric regimens. 76% of the patients received allo-sct from an hla-matched sibling donor. based on score calculated with hazard ratios (hr) estimated on the population studied, 161 patients (47%) had a ci score of 0, 96 patients (29%) had a score of 1, and 49 (14%) had a ci score of 2. the remaining 39 patients (11%) had ci scores of 3 or more. in this cohort, 2 years overall and leukemia-free survival rates were 64±3% and 54±3% and the 2 years relapse and non-relapse mortality (nrm) cumulative incidences were 32±2%, and 15±2% respectively. the 2 years nrm incidences according to comorbidities score 0, 1, 2 and 3 + were 9±2%, 15±4%, 18±5% and 31±7%, respectively. in multivariate models (adjusted for recipient age, donor type, use of tbi or not, and cytogenetics risk group) comorbidities such as moderate active liver disease, obesity, prior history of renal dysfunction, and prior history of severe liver disease were associated with the highest hrs for 2 years nrm (varying from 2.11 to 2.76) and 2 years cumulative incidences of nrm varying from 22% to 44%, whereas previous solid tumor, diabetes, rheumatologic abnormalities, moderate pulmonary diseases, cardiac abnormalities (other than arrhythmia and valve disease) were associated with the lowest hrs (varying from 0.2 to 1.0) with 2 years cumulative incidences of nrm varying from 5 to 17%. results from this large study performed in a single disease entity and homogeneous allo-sct setting, suggest that the hematopoietic cell transplantation-specifi c ci is a simple, informative and useful tool for capturing pre-transplant comorbidities, and for predicting nrm after ric allo-sct for aml in cr1 in patients aged >50 y. such index may be used for clinical trials and patient counselling before ric allo-sct. fibrin glue directly applied on damaged bladder mucosa during cystoscopy is highly effective to treat severe, refractory, haemorrhagic cystitis after allogeneic transplant m.c. tirindelli (1) (1) background: hemorrhagic cystitis (hc) occurring after hematopoietic stem cell transplant (hsct) signifi cantly affects quality life of patients, prolongs hospitalization and in some cases can become a life-threatening complication. its management has not been established. fibrin glue (fg) is a hemostatic agent derived from human plasma with proven effi cacy in repairing damaged tissues. study design and methods: this study included patients who met the following criteria: grade ≥3 hc not responding to hyperhydration, bladder irrigation, antiviral treatments and transfusion support. fg was obtained using vivostat system, an automatic method for processing and applying fg. during conventional cystoscopy and maintaining bladder distension by a co2 insuffl ator, fg was accurately sprayed through a specifi c applicator on bleeding mucosa. fg polymerized on contact and set over several days. the response to the treatment was defi ned complete (cr) for disappearance of hematuria, partial (pr) for at least one grade regression of hc and no response (nr). results: from jan 06 to oct 09, 626 patients undergoing an autologous (n = 428) or allogeneic (n = 198) hsct were registered at the rtn. no autologous hsct recipients developed hc of severe grade, whereas 18 of 198 patients (9%) undergoing an allogeneic hsct met the criteria to enter the study. these 18 patients (6 m, 12 f) with a median age of 32.5 years (range, 18-53) had been submitted to a hsct from hla identical sib. (n = 4), unrelated cb (n = 4), mud (n = 2) or related haploidentical donor (n = 8) for different hematological malignancies. all patients, deeply immunosuppressed with positive bkv viruria >7x10 6 copies/ml, developed a very severe hc, refractory to all current treatments including antiviral therapy. at time of fg application, hc persisted for a median of 16 days (range, 7-65) and was grade 3 and 4 in 14 and 4 patients, respectively. the number of fg applications was 1 in 15 patients, 2 in 2 and 3 in 1 patient for a median of 11 ml (range, 6.3-16.2) of glue. the treatment was successful in 16 out of 18 patients (89%). all 14 patients with grade 3 hc responded and the response was complete in 12 (86%) and partial in 2 (14%), while of the 4 patients with grade 4 hc: 1 achieved cr, 1 pr and 2 nr. no patient died of hc. conclusions: fg therapy is a feasible, safe, easy repeatable, not invasive, small time consuming, lightly expensive and highly effective procedure in treating severe, refractory posttransplant hc. glutamine-enriched intravenous total parenteral nutrition doesn't improve mucositis and clinical outcome after stem cell transplantation for childhood malignancies. a prospective double-blind controlled study on behalf of aieop group c. uderzo (1) , e. marrocco (1) , p. rebora (1) , f. cichello (1) , m. bonetti (1) , s. cesaro (2) , s. varotto (2) , p. verlato (2) introduction: high dose chemo-radiotherapy followed by stem cell transplantation (sct) constitutes the principal cause of post-transplant severe mucositis and related complications. intravenous glutamine-enriched solution (ges) has been advocated as one of the support treatment capable to improve marked body protein wasting, oxidative stress which result in a mucosal damage, often "primum movens" of infectious diseases beginning from g.i.tract damage. patients and methods: 118 patients (79 males, median age at sct 8.1 years) with haematological malignancies have been treated from june 2005 to june 2008 with high dose chemotherapy and/or tbi followed by allogeneic sct (35 match related,65 match unrelated,18 mismatch.patients were randomly and double-blinded assigned to undergo either for total parenteral nutrition (tpn) or for ges tpn at the dose of 0,4 g/kg/day from day of sct until the end of tpn. the principal endpoint of the study was the mucositis assessment according to who criteria.the evaluation of the clinical,haematological and laboratory parameters served to perform the evaluation of secondary end-points.statistical analysis was set up to demonstrate a 20% difference in terms of mucositis incidence and severity with a power of 80% (alfa = 0.05; two-sided tests). results: both study groups were comparable for age, gender, diagnosis and type of sct. the ges patients (60) were clinically similar to the controls at the entry; however, they didn't experienced a reduction of rate and grade of mucositis, with an odd ratio of 0.98(0.26-3.63) adjusted for the type of sct. neither the type of analgesic treatment nor the duration of opiod treatment, were statistically different. days of both different tpn, length of hospital stay, trm at day + 180,acute or chronic gvhd, incidence of severe infectious diseases, immunological recovery, progression of malignancies were similar in both groups. nutritional status at the beginning and at the end of both types of tpn didn't show any difference at univariate analysis. no toxicity of ges was observed. conclusions: the current study is one of the few designed to demonstrate the utility of ges on mucositis in the setting of children undergoing sct for malignancies. ges failed to infl uence incidence and severity of mucositis after sct and didn't offer any advantage on clinical outcome, as claimed by other retrospective studies often performed in adult transplanted patients. clinical and genetic risk assessment for overall survival in haematopoietic stem cell transplantation a. dickinson (1) objectives: non-hla gene polymorphisms and clinical risk factors impact on outcome after hsct. age, stage of the disease, time from diagnosis to transplant, histocompatibility and donor and patient gender combination are key transplant risk factors for hsct. we have assessed the impact of non hla gene polymorphisms on the ebmt risk score for overall survival in a eurobank cohort of 915 hla identical sibling and matched unrelated donor (mud) transplants. methods: hsct patients with acute leukaemia (aml + all (al)) (n = 463), cml (n = 187) plasma cell neoplasia (n = 120) or lymphoma (n = 145) and their donors from 8 transplant centres were genotyped for non-hla polymorphisms within the tumour necrosis factor receptor ii (tnfrsf1b), estrogen receptor (esr1), vitamin d receptor (vdr), interleukin (il) 6 (il6), il-1 receptor antagonist (ilrn), interferon gamma (ifng) and il 4 (il4) genes. genetic factors were assessed using the log rank test (p value < 0.2). candidate factors were included in addition to the ebmt risk score in a stepwise cox regression procedure to select fi nal genes. predictive value of the model for overall survival was assessed through the concordance (c) index and prediction error curves. results: in the whole cohort the presence of allele c in the donor il-6 genotype (snp il6-17) was associated with lower survival time (especially in the cml sub group p = 0.025) and improved the prediction of the ebmt risk score. in the al subgroup the absence of patient il4 (any t) and presence of il1rn (any c) (high risk group) were associated with lower survival time (compared to the remaining patients (low risk group)), and taken together also improved the predictive value of the ebmt risk score. figures 1a and b show the lower probability of survival (and increased trm) in the high risk versus low risk group. conclusions: this study confi rms the importance of non-hla genotyping for risk assessment in allogeneic hsct. improvement of fi t of the ebmt risk score presents a powerful novel tool to assess this impact of gene polymorphisms in a complex heterogeneous hsct population. in a confi rmatory study the ebmt risk score was associated with survival for an al patient group. assessment of patient il4 (any t) and il1rn (any c) in this second cohort is in progress. background: cognitive impairments have been found among patients receiving chemotherapy and hsct. however, studies with representative samples are rare, but needed to clarify the short and long term impact of different conditioning regimens and hsct. in the present multicenter trial, we assessed the prevalence and course of cognitive dysfunctions in patients with hematological diseases undergoing allogeneic hsct. moreover, the role of neurotoxic intensity of conditioning regimens was investigated. methods: 102 patients (61% male) with mixed hematological cancers (41% acute myeloid leukemia) at an average of 48 years of age were assessed before (t0), 100 days after (t1) and one year following (t2) allogeneic hsct. 36% of the participants received intense neurotoxic conditioning regimens. a battery of neuropsychological tests using computer-and paperpencil-based methods was used covering the domains of attention, memory, executive and psycho-motor function. measures assessing self-perceived cognitive impairments and psychological distress were additionally applied. results: compared to published test norms at each assessment time point, signifi cant impairments in several neuropsychological test parameters across all cognitive domains were found. at baseline, patients had impaired cognitive functions in 50% of the test parameters. no signifi cant change in the prevalence of cognitive impairments was observed over time except for a mild increase in psycho-motor dysfunction at t2. patients who were classifi ed as having intense neurotoxic conditioning were more likely to show impairments in the domain attention at t1 (p<0.05, d = 0.53) and showed a different time course of performance in attentional tasks compared to patients with mild neurotoxic conditioning (p<0.01, eta² = 0.05). conclusions: a remarkable amount of patients was classifi ed as having cognitive impairments prior to hsct. possible explanations include the impact of the hematological cancer disease, invasive treatments applied prior to conditioning and hsct, and treatment related distress. a decline in cognitive functioning was primarily limited to psycho-motor functions. however, subgroups of patients and in particular those with intense neurotoxic conditioning regimens might be at greater risk for developing short term cognitive impairments particularly in the domain attention. acknowledgement of funding: this study was supported by a research grant from the german josé carreras leukemia foundation. clinical outcomes of second allogeneic haematopoietic stem cell transplantation for acute leukaemia and myelodysplastic syndrome relapsing after fi rst allogeneic transplantation t. yamashita, t. kikuchi, i. kamiya, r. hanajiri, y. nagata, s. wakabayashi, k. taoka, t. kobayashi, k. ohashi, h. sakamaki, h. akiyama tokyo metropolitan komagome hospital (tokyo, jp) background: allogeneic hematopoietic stem cell transplantation (hct) is an effective therapy in acute leukemia and myelodysplastic syndrome (mds). but even after hct, these diseases recur in some cases and prognosis of these patients is very poor. second allogeneic hct (hct2) may be one of the most effective therapies in some patients who relapsed after fi rst allogeneic hct (hct1). because of the heterogeneity of hct2, it is often diffi cult to fi nd useful prognostic factors and appropriate strategies. in our single-center study, we retrospectively analyze the clinical data of 465 patients with acute myeloid leukemia (aml), acute lymphoblastic leukemia (all) and mds who received hct1 in our hospital to investigate the factors that affect clinical outcomes of hct2. patients and methods: we included patients with aml, all and mds who received hct1 between 1986 and 2008 in our hospital. probability of overall survival (oas) and event-free survival (efs) were calculated using the kaplan-meier method. cumulative incidence rates were calculated using standard methods for hematopoietic recovery, acute and chronic gvhd, relapse and non-relapse mortality (nrm). multivariate analysis was performed with variables that can affect the clinical outcomes using cox proportional-hazards regression models. results: a total of 465 patients were eligible for this analysis. five years oas of hct1 is 50.8%. 5 years cumulative incidence of relapse (ri) 30.5% and 138 patients relapsed after hct1. relapsed patients group was signifi cantly lower incidence of chronic gvhd (p = 0.008). in this patients group, hct2 was performed for patients with aml (n = 19), all (n = 11), mds (n = 4). myeloablative conditioning was used in 28 cases. five years oas of hct2 was 15.2%, and oas and efs of myeloabative hct2 was 24.4% and 13.1%, respectively. five years ri was 57,2% and trm was 30.7%. multivariate analysis showed that 1) relapse to hct2 over 180 days, 2) age at hct2 under 33 years, 3) cr at hct2, 4) related donors are signifi cant favorable prognostic factors for oas of hct2. conclusion: our study shows that myeloablative hct2 brought sustained remission in a proportion of patients with aml, all and mds who relapsed after hct1. age and disease status at hct2 are important factors that infl uence the clinical outcomes. this study also suggests that the duration from relapse after hct1 and hct2 have impact on regimen-related toxicity of hct2. hepatic dysfunction is frequent and diverse before and after allo-ric and has a major impact on the transplant outcomes p. barba (1) introduction: hepatic dysfunction is one of the most frequent and less studied organ impairments in the setting of allogeneic stem cell transplantation (allo). to analyze the impact of pretransplant liver function on the outcome of allo-reduced intensity conditioning (allo-ric) and to determine the incidence, characteristics and risk factors of hepatic injury after allo-ric we conducted a retrospective study in two spanish centers. patients and methods: we analyzed 281 adult patients receiving an allo-ric. the median follow-up for survivors was 5 years (rg 0.2-10). ric consisted in fl udarabine 150 mg/m 2 plus melfalan 70-140 mg/m 2 or busulfan 8-10mg/kg. pretransplant indicators of liver injury analyzed were aspartate aminotransferase (ast), alanine aminotransaminase (alt), gammaglutamyl transpeptidase (ggt), alkaline phosphatase (ap), total bilirubin (bil), prothrombin time (pt) and the category of severe hepatic disease according to hct-comorbidity index (hct-ci_hep). posttransplant severe hepatic injury (shi) was defi ned as maximal total serum bilirubin (bil) level > 4 mg/dl or encephalopathy of hepatic origin (according to hogan, blood, 2004) . results: pretransplant liver laboratory tests abnormalities were found in 51 (18%) patients. we observed high levels of ast, alt, ggt, ap, bil, pt and hct-ci_hep in 5 (2%), 16 (6%), 26 (9%), 21 (7%), 14 (5%), 2 (1%) and 27 (9%) patients, respectively. among the pretrasplant indicators of liver injury analyzed, hct_ci hep showed to be the best predictor of transplant outcomes. in multivariate analysis (mva), hct-ci_hep high risk patients showed higher 100-days and 2-years nrm (hr 3.1 [95%ci 1.5-6.2] p = 0.002 and hr 1.9 [95%ci 1-3.7] p = 0.04, respectively), lower overall survival (os) (37% vs. 53%, p = 0.04), higher grade 2-4 agvhd p<0 .001]), higher grade 3-4 agvhd and a trend to higher cgvhd (1.6 [95%ci 0.9-2.8] p = 0.06). after allo-ric, a total of 182 patients (65%) developed grade iii-iv liver toxicities. a total of 74 (26%), 111 (40%), 160 (57%) and 51 (19%) patients developed grade iii-iv levels of ast, alt, ggt, and bil at a median time of 194 (rg 0-1629), 146 (rg 0-1932), 195 (rg 0-3126) and 74 days (rg 0-783), respectively. sixty-seven patients (24%) developed shi at 5 years with a cumulative incidence of 19% (95%ci 15-25). main causes of shi were gvhd (n = 36, 54%) and pharmacological toxicity (n = 7, 10%). in mva, risk factors for shi were unrelated donors (hr 3. a hallmark of acute graft-versus-host disease (agvhd) is the selective attack of certain tissues, namely the gastro-intestinal tract, liver and skin but not others such as the pancreas and kidneys. imaging techniques such as whole body in vivo bioluminescence imaging, dynamic multiphoton-laser-scanningmicroscopy and ultramicroscopy utilized by others and our own group help to elucidate the elusive mechanisms underlying alloreactive t cell traffi cking. here we dissected the molecular mechanism that direct alloreactive t cells via vascular endothelial cells to target tissues in a murine mhc major mismatch hematopoietic cell transplantation mouse model. as a strategy we employed endothelial ligand blocking antibodies during the agvhd effector phase after alloreactive t cells leave secondary lymphoid organs. immunohistochemical analysis of target and non-target tissues revealed organ dependent upregulation of infl ammation induced endothelial ligands. the identifi ed surface molecules subsequently served as therapeutic targets. we learned that during the agvhd effector phase multiple endothelial ligands need to be blocked simultaneously to avert agvhd target manifestation. blocking of single molecules such as madcam-1, vcam-1, e-selectin, p-selectin alone could not prevent alloreactive t cell traffi cking to the intestinal tract, liver and skin. when we treated transplanted mice with the combination of two or even three antibodies we observed a reduction of alloreactive t cells. however, only when we blocked four targets simultaneously, namely madcam-1, vcam-1, e-selectin, p-selectin in combination we could prevent gvdh target manifestation in the skin, in the liver and the small intestines. from these data we conclude that alloreactive t cell homing depends on the recruitment by infl ammation induced endothelial ligands of target tissues. after clonal expansion of alloreactive t cells simultaneous blocking of several endothelial ligands was required to effi ciently abrogate agvhd in target tissues. our data indicate that t cell homing remains highly attractive for therapeutic interventions. our results also point out that several pathways should be targeted simultaneously in order to prevent agvhd. distinct temporal and spatial roles for host conventional and langerhans cells in the development of graft-versushost injury f. fallah-arani, h. goold, b.r. flutter, j. sivakumaran, c.l bennett, r. chakraverty royal free and ucms (london, uk) background: following bone marrow transplantation, donor t cell reactivity can be confi ned to the lympho-haematopoietic system or, in the presence of infl ammation, can additionally extend to peripheral tissues leading to graft-versus-host disease (gvhd). in this study, we have explored the role of individual host dendritic cell (dc) subsets in regulating these processes using models involving inducible depletion following donor t cell transfer to allogeneic chimeras. methods: balb/c thy1.1 + t cells were transferred to established allogeneic chimeras generated following reconstitution of lethally irradiated b6 mice with a mix of balb/c and b6.cd11c-dtr bone marrow. cd11c.dtr mice express a primate diptheria toxin receptor (dtr) under the control of the cd11c promoter. in the resulting chimeras, host cd11c + conventional dc (cdc) were depleted at the time of donor t cell transfer by injections of diphtheria toxin (dt). in experiments to test the role of host langerhans cells (lc), balb/c thy1.1 + splenocytes were transferred to balb/c + b6 > b6.langerin. dtr, where co-treatment with dt leads to depletion of host, epidermal lc. in each of the above settings, infl ammation was induced in some chimeras by systemic or local application of a toll-like receptor (tlr) agonist at the time of t cell transfer. results: absence of host cdc at the time of delayed t cell transfer abrogated accumulation of donor cd8 cells in recipient spleens and reduced in vivo cytotoxicity against host target b cells. this was associated with a lack of conversion to full donor chimerism, demonstrating a requirement for host cdc in priming the lympho-haematopoietic graft-versus-host response in the steady state. we then explored the role of host cdc and lc in a model of cutaneous gvhd, where local gvhd is induced following application of a tlr agonist (imiquimod). maximal imprinting of e-selectin ligand expression upon donor cd8 cells within the draining lymph node required the presence of host cdc. however, even in their absence, donor t cells were able to access the epithelium and cause injury. selective depletion of host lc had no effect on imprinting or tissue infi ltration of donor t cells. however, the capacity of donor t cells to induce gvhd was severely impaired in this case. conclusions: these data show that host cdc are required for gvh reactivity in the steady state. in contrast, in a model of skin gvhd, host cdc are dispensable whereas lc have a unique post-priming role. failure to imprint donor cd8 memory and exhaustion may explain loss of gvt responses following donor leukocyte infusions b.r. flutter (1) , f. fallah-arani (1), s. sivakumaran (1) , c.l. bennett (1) , s. ghorashian (1) , g. freeman (2) , m. sykes (2) (1)university college london (london, uk); (2)harvard medical school (boston, us) background: donor t cell alloreactivity can be co-opted to deliver graft-versus-tumour (gvt) responses following donor leukocyte infusions (dli) given after bone marrow transplantation (bmt). however, the major reason for treatment failure following dli is relapse, suggesting a long-term failure of antitumour immunity. a distinctive element to the gvt response is that the antigens (ag) to which the response is directed are cleared from the haematopoietic system but continue to be expressed by non-haematopoietic cells. in this study, we have explored the infl uence of non-haematopoietic ag upon the antihost cd8 t cell response in a model of delayed dli. methods: b6 cd45.1 t cells were given after 8 weeks to allogeneic chimeras where ag was ubiquitous (b6 + bdf1 >bdf1) or restricted to the haematopoietic compartment (b6 + bdf1 >b6). in each setting, direct priming of the initial t cell response by bdf1 ag-presenting cells resulted in the eradication of bdf1 haematopoietic cells. however, in b6 + bdf1 >bdf1 chimeras, ag was still present in peripheral tissues, whereas in b6 + bdf1 >b6 chimeras, ag was cleared completely. the donor t cell response was tracked at timed intervals following transfer in both sets of chimeras. results: the continued presence of non-haematopoietic ag led to a failure to sustain donor cd8 cytotoxic and effector cytokine responses by 60d following transfer and this was associated with a failure to establish a central memory (t-cm) population. in contrast, where ag was absent in peripheral tissues, functional t-cm populations were preserved. the failure to generate donor t-cm in chimeras with ubiquitous ag expression occurred at two distinct levels. firstly, residual dli-derived cd8 t cells demonstrated a pd-1 hi/cd127 lo/ ifn-g neg 'exhausted' signature. consistent with this, cd8 functions were partially restored by in vivo antibody blockade of the pd-1 pathway. secondly, during the initial phase (<14d) of the response, we observed a lack of memory precursor formation when ag was ubiquitous. thus, when ag was ubiquitous, the capacity of activated, post-mitotic, cd8 t cells to establish recall immunity following transfer to ag-free hosts was much reduced. conclusions: these data demonstrate that alloantigen within non-haematopoietic tissues is not ignored, but rather blocks memory imprinting and drives eventual cd8 t cell exhaustion. while these effects may lessen the risk of gvhd, they might also impair the durability of the gvt response. background: adoptive transfer (at) of tcr-gene transduced t cells has become a promising therapeutic tool, however, limited in vivo survival and the development of an unresponsive state often termed 'exhaustion' has hampered reproducible clinical success. here we demonstrate that a) upregulation of pd-1 on donor-derived tcr-engineered t cells is associated with a loss of gvl effects after late at and b) pd-l1 blockade after hematopoietic stem cell transplantation restores robust gvl-effects. methods: we chose a mhc-mismatched allogeneic bmt model (b10.a mice (h-2a) into c57bl/6 (h-2b) mice) and a retrovirally encoded tcr (ot-1 anti-ovalbumin) that was introduced into b10.a-derived donor t cells for at after mhc-mismatched hct. after hct, leukemia-bearing (c1498-ova) mice (syngeneic hct-recipients served as reference) received at and were monitored for gvhd, gvl, and in vivo t cell persistence/ functionality. for pdl-1 blocking experiments pdl-1 antibodies (clone 10f.9g2) were used. results: 1) up to 1x10 9 tcr-transduced cd8 + t cells were generated in vitro within 7 days using a retroviral vector system linking the genes for the á-and â-chain via a 2a sequence. 40% of cd8 + t cells coexpressed the respective tcr chains vá2 and vâ5. 2) the introduced tcr mediated comparable specifi c cytotoxicity against c1498-ova in vitro being functional on autologous and allogeneic t cells. 3) after early at transduced t cells rescued up to 60% of mice in a dose dependent manner. (p = 0.001-0.01 versus mock-transduced controls). 4) after late adoptive transfer (day 56) autologous t cells peaked in numbers by day 3 after at and provided strong gvl-effects. in contrast, numbers of allogeneic tcr-modifi ed t cells declined rapidly within the peripheral blood and increasingly expressed pd-1. lower frequencies of allogeneic t cells in the periphery translated into nearly abolished gvl effi cacy upon leukemia challenge 3 days after at (p = 0.004). 5) pd-l1 blockade in vivo after allogeneic hct restored gvl-effi cacy of adoptively transferred t cells raising leukemia free survival from 0% to 60% (p = 0.001 compared to isotype controls) without increasing gvhd rates. conclusion: at with tcr-engineered t cells after allogeneic hct can decrease the risk of gvhd and provide potent gvl effects. however, increased expression of pd-1 on adoptively transferred t cells is associated with decreased gvl effi cacy and can effectively be reverted by pd-l1 blockade. allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative treatment for many hematologic malignancies or hematopoietic dysfunction syndromes in adult patients, but the application is still limited due to major complications, such as severe graft versus host disease (gvhd). diagnosis of agvhd is based on clinical features and biopsies. we have shown earlier that agvhd can be diagnosed using a proteomic pattern established by screening with capillary electrophoresis (ce) and mass spectrometry (ms). the agvhd-specifi c proteomic pattern has been evaluated prospectively and blinded on more than 960 samples collected from more than 141 patients undergoing allo-hsct at hannover medical school (mhh) and 7 additional clinics. since 2007 a new diagnostic proteomic pattern for agvhd was developed, using a higher resolution ms. thus, 192 patients from mhh and 133 patients from 4 collaborating clinics were subjected to double screening for development and prospective evaluation of the ms-17-agvhd-specifi c pattern. the majority of the patients included was transplanted for hematological malignancies (n = 313), 12 for hematopoietic failure syndromes (2 pnh, 9 saa) . conditioning regimens included reduced intensity conditioning regimens (ric) for about 60% of the patients of mhh, as well as standard conditioning regimens (mainly tbi + cy or busulfan + cy). gvhd-prophylaxis was cyclosporine a (csa) and mycophenolate (mmf) or csa metothrexate (mtx), as appropriate. in addition, about 80% percent of the patients received atg (antithymocyte globulin) prior to hsct. the new agvhd-specifi c pattern, ms_17, consisting of 17 differentially excreted proteins and peptides was developed and prospectively evaluated in parallel for the last 2 years. prospective and blinded evaluation of the patients included in this analysis for early recognition of patients at risk for agvhd development revealed the correct classifi cation of patients developing agvhd about 7 days (range: 2-21 days) prior to the development of clinical symptoms for agvhd with a sensitivity 76% and specifi city of about 85%. pre-emptive therapy ad ministered upon positivity of the protemic patterns reduced the incidence and severity of agvhd (p = 0.04). thus, a multicenter study has been initiated in germany to test the effi cacy and safety of pre-emptive therapy. hhv6 and acute gvhd c. pichereau, k. desseaux, a. janin, r. peffault de latour, m. robin, p. ribaud, s. chevret, g. socié hôpital saint louis (paris, fr) background: previous studies have suggested that hhv6 infection is correlated with acute gvhd following allogeneic hsct, but whether hhv6 triggers, is associated with, or is a differential diagnosis of gvhd is unknown. methods: 414 patients received an allogeneic hsct at the saint louis hospital (paris) between january 2004 and december 2007. whenever acute gvhd was suspected, hhv6 rq pcr and organ biopsy was performed. cumulative incidence of hhv6 reactivation was estimated using competing risks approaches. predictive factors of increased risk of reactivation or acute gvhd were assessed using cause-specifi c hazard cox models. effect of reactivation on further occurrence of acute gvhd was tested by introducing a time-dependent variable. results: 376 pts were tested for hhv6: 229 (61%) were male, median aged 40 years . 300 (80%) had malignancies and 76 suffered from non malignant disorders (aplastic anemia, sickle cell disease, thalassemia). 188 pts were grafted with related donors (50%). the conditioning regimen was myeloablative in 237 cases (63%). the source of hsct was bone marrow in 161 cases, peripheral blood in 157 cases and cord blood in 58 cases. 240 pts (64%) developed acute gvhd. hhv6 was detected in 100 pts, 66 of whom developed gvhd. hhv6 reactivation was signifi cantly associated with cord blood graft (59% versus 21%, p<0.0001) and unrelated transplant (39% versus 14%, p = 0.007). gvhd was signifi cantly associated with previous hhv6 reactivation (hazard ratio, 1.63; 95% confi dence interval, 1.01-2.62; p = 0.04). tissue biopsies were available for 64 of the 100 hhv6 infected pts (32 skin and 32 gut samples). there were no signifi cant difference between pts who had a biopsy and pts who hadn't, except for the incidence of acute gvhd (75% versus 50%, p = 0.01). 24 pts who had no or poor pathological evidence of gvh (grade 0 or 1), later on developed severe clinical acute gvhd (grade iii and iv), suggesting the role of hhv6 as a trigger of severe gvhd. beside this, 10 pts who didn't have clinical and histopathological gvhd (grade 0) showed a significant lymphoid infi ltrate on their biopsy. immunohistochemical analysis are ongoing, that could provide the evidence of "pure" hhv6-related skin rash after hsct. conclusion: this study suggests the role of hhv6 as a trigger of acute gvhd, particularly in its severe manifestations. beside this, it confi rms that hhv6 infection is a differential diagnosis of acute gvhd in a signifi cant proportion of patients. prochymal® improves response rates in patients with steroid-refractory acute graft-versus-host disease involving the liver and gut: results of a randomized, placebo-controlled, multicentre phase iii trial in gvhd p.j. martin (1) , j.p. uberti (2) background and methods: steroid-refractory acute gvhd (sr-gvhd) remains a signifi cant and life-threatening complication of allogeneic hematopoietic cell transplantation (hct). prochy-mal® (mesenchymal stem cells, msc, derived from unrelated volunteer adult donors) was evaluated in addition to standard of care, including institutionally selected second line treatment, in a randomized (2:1) trial in patients with sr-gvhd (protocol 280). patients received 8 infusions of 2 x 10 6 msc/kg over 4 weeks (or volume equivalent for placebo), with an additional 4 infusions administered weekly after day 28 in patients who had a partial response, defi ned as improvement in at least one organ without progression in others. the primary endpoint was durable complete response (dcr) for ≥ 28 days. additional prospectively defi ned outcomes included responses in patients by organ involvement. patients and results: 244 patients with sr-gvhd (skin involvement n = 144, gastrointestinal involvement n = 179, liver involvement n = 61) were enrolled and treated on a 2:1 basis: prochymal (n = 163), placebo (n = 81). there were no signifi cant differences in age, pre-transplant conditioning, graft source, hla-matching or second-line therapy between treatment arms. for the prochymal and placebo arms, respectively, the grades of gvhd at entry were b (22% vs. 26%), c (51% vs. 58%), and d (27% vs. 16%). the respective dcr rates were 35% vs. 30% (p = 0.3) in the intent-to-treat population and 40% vs. 28% (p = 0.08) in the per protocol population. results for secondary endpoints are shown in table 1. patients with gvhd affecting all 3 organs had overall complete or partial responses rate of 63% vs. 0% (n = 22, p<0.05, fisher's exact test) at day 28. patients treated with prochymal had less progression of liver gvhd at weeks 2 and 4 respectively (32% vs. 59%, p = 0.05; and 37% vs. 65 %, p = 0.05). the incidence of infections was not different between arms. incidence rates were 9% vs. 8% for recurrent malignancy, 1.8% vs. 2.5% for infusional toxicity, and 0.6% vs. 4.6% for ae-related discontinuation in the prochymal and placebo arms, respectively. conclusion: gvhd with liver or gut involvement is a life-threatening complication of hct. these results suggest that the addition of prochymal produced signifi cant improvement without additive toxicity in patients with sr-gvhd involving visceral organs. s18 o137 treatment of steroid-refractory acute gvhd with mesenchymal stem cells improves outcomes in paediatric patients. results of the paediatric subset in a phase iii randomized, placebo-controlled study p. szabolcs (1) , g. visani (2) background: successful treatment of steroid-refractory acute graft-versus-host disease (sr-gvhd) following allogeneic hematopoietic cell transplantation remains a signifi cant challenge. because of their immunomodulatory properties and safety profi le, adult mesenchymal stem cells (mscs) have been proposed as a treatment for sr-gvhd. intravenous allogeneic msc therapy (prochymal) for sr-gvhd was independently evaluated in the pediatric subset of a double-blind, placebocontrolled study. methods: pediatric patients (<18 years old) with grade b-d sr-gvhd were randomized to receive either prochymal or placebo in addition to standard of care, including institutionally selected second line agent. patients received 8 infusions of 2 × 10 6 cells/kg for 4 weeks (or volume equivalent for placebo), with 4 more infusions weekly in the case of a partial response (pr). the primary endpoint was durable complete response (cr 28 days); secondary endpoints included incidence of cr, pr and progression through 100 days, survival, and safety. results: twenty-eight children were randomized to prochymal (50% male, 79% caucasian) or placebo (71% male, 71% caucasian), with a median age of 7 yrs (range 1-15) and 10 years (range 1-18), respectively. the dominant transplant graft source was cord blood (71% prochymal, 57% placebo), with mostly unrelated donors (93% vs. 79%, respectively). the median duration of agvhd prior to enrollment was 20 days for prochymal and 8 days for placebo (p<0.05). at baseline, agvhd grades b:c:d were 3:8:3 for both arms. for prochymal, organ involvement was 64% skin, 43% gi, and 36% liver. for placebo patients, organ involvement was 57% skin, 79% gi, and 29% liver. durable cr was 64% for prochymal and 43% for placebo (p = 0.5). prochymal improved rates of cr and or (table) . the median time to cr was 25 days vs. 63 days. the safety data showed no infusional toxicity and no evidence of prochymal leading to ectopic tissue. there were no aes leading to discontinuation of therapy. conclusion: in a sr-gvhd population in which 79% of patients had grade c/d disease, the addition of prochymal to standard of care resulted into a faster and better cr. in view of their increased response rates and a well-tolerated safety profi le, mscs appear to be a safe and effective therapy in the treatment of pediatric patients with sr-gvhd. aim: response rate after induction and after asct. patients and methods: td consisted of thalidomide 200 mg/d and dexamethasone 40 mg on days 1-4 and 9-12 at 4-week intervals for 6 cycles. vtd was identical to td plus velcade 1.3 mg/m² on days 1,4,8,11 of each cycle. combination chemotherapy plus velcade consisted of 4 cycles of vbmcp/vbad followed by 2 cycles of velcade. results: as of december 31, 2008, 306 patients (median age: 57 yrs, m 156, f: 150; igg 183, iga 71, light chain 43, others 9) entered the study. 55 of 253 (22%) had high-risk cytogenetics (t(4;14), t(14;16) and/or 17p deletion). all 306 patients (td: 104, vtd: 102 and vbmcp/vbad/velcade: 100) were evaluable for response and toxicity to induction therapy. the cr plus vgpr rate was signifi cantly higher with vtd than with vbmcp/vbad/velcade (59 vs. 37%, p = 0.003) and td (59 vs. 28%, p<0.001). the progressive disease (pd) rate was higher with td than with vtd (22 vs. 8%, p = 0.005). in patiens with high-risk cytogenetics vtd was associated with a higher cr rate than td and vbmcp/vbad/velcade (42 vs. 0%, p = 0.003 and 23 vs. 0%, p = 0.04, respectively) as well as with a lower pd rate (0 vs. 37%, p = 0.003 and 0 vs. 23%, p = 0.04, respectively). the incidence of > grade 2 thrombotic events was higher with td than with vtd (8% vs. 1%, p = 0.01) and grade >2 peripheral neuropathy was higher with vtd than with td or vbmcp/ vbad/velcade (14 vs. 1% vs. 0%, p<0.001). 218 patients were evaluable for response after asct. the cr rate was higher with vtd (52%) than with vbmcp/vbad/velcade (49%) and td (37%) although the differences did not reach statistical signifi cance. the estimated os at 2 yrs. was 82% with no significant differences among the 3 arms. ttp and pfs were shorter with td (p = 0.05 and p = 0.012, respectively). summary: 1) induction with vtd produced a higher cr + vgpr rate, 2) in patients with high-risk cytogenetics, td resulted in a signifi cantly lower cr and higher pd rate than bortezomibcontaining regimens, 3) the pfs was shorter with td and 4) asct increased the cr rate in 23%, 21% and 26% in the td, vtd and vbmcp/vbad/velcade arms, respectively. interim analysis of a phase iii, prospective randomized trial of melphalan stem cell source was pbsc in 93% pts. 15% pts received myeloablative allogeneic sct as part of a planned tandem strategy, whereas 85% received a non myeloablative conditioning. overall response at allo sct was 84% and included complete remission (cr 19%), very good partial remission (vgpr 9%), partial remission (pr 56%). results: all pts except 3 had sustained donor engraftment. among pts, 36%, 13% and 20% achieved cr, vgpr and pr respectively at day 100. median follow-up from allo sct was 37 months (range 1-122). event-free survival (efs) was 58% (49-66) at 3 years. cumulative incidence of transplant related mortality (trm) at 100 days was 10% and was mostly related to pneumonia. a single line of treatment before tandem auto/allo sct was associated with improved 3-year-efs (p = 0.02). 28% pts experienced grade i to ii acute graft-versus-host-disease (gvhd) and 13% grade iii to iv. 11% pts had limited and 18% extensive chronic gvhd. whereas grade i to ii acute gvhd was associated to better 3-year-efs (p<0.0001), extensive chronic gvhd had no statistically signifi cant impact on efs. moreover the absence of deletion 13 was not associated with a better efs. conclusion: our results suggest that the number of treatment lines received before tandem auto/allo sct is an important issue, with an improved efs for pts treated by a single line before tandem. whereas better control of acute gvhd might further improve survival, impact of the deletion 13 on outcome seems limited. purpose: the outcome of mm patients treated with low dose tbi and allogeneic related sct following autologous sct has been shown to be superior to that from two autologous sct (gahrton et al.; bruno et al.) . since related donors are available for approximately 30% of patients only, the outcome of allogeneic unrelated sct in patients without a related donor was compared to that of patients with a related donor in an intention to treat analysis. patients and methods: mm patients (n = 44) with a median age of 51 (range 32-65) years were treated at the university of leipzig with autologous sct followed by allogeneic sct. autologous stem cells were mobilised with cyclophosphamide and transplanted after melphalan 200 mg/m 2 on day -3. allogeneic sct was performed at a median of 5 (range 2-11) months after autologous sct using fludarabine (30 mg/m 2 /d from days -3 to -1) and tbi (2 gy on day 0) followed by cyclosporine (6.25 mg/kg twice daily from day -1) and mycophenolate mofetil (15 mg/kg daily from day 0). patients received an unrelated (n = 22) or related (n = 22) stem cell graft. results: patients with unrelated donors (n = 22) showed hematological toxicity comparable to those of related donors. all patients had stable hematopoietic engraftment with t-cell chimerism of 100% 3-6 months after sct. with a median follow up of 41 (range 2-87) months overall survival (os) at 5 years was 51±12% and 52±13% (p = 0,19) in patients with unrelated and related donors, respectively. progression free survival (pfs) was 34±12% at 5 years for patients with an unrelated compared to 14±8% for patients with a related donor. this difference was due to a decreased relapse incidence (ri) in patients with unrelated donors (54±12%) compared to related donors (84±9%). non relapse mortality was 22±9% in the whole population with no difference between the two groups. of the 22 patients having received allogeneic unrelated sct, 7 (32%) are currently in cr, 2 (10%) in vgpr, 3 (14%) in pr and 1 (5%) has stable/progressive disease, with no difference between related and unrelated sct. conclusion: our data confi rm the feasibility of autologous sct followed by low-dose tbi conditioning regimen and unrelated sct. pfs seems to be higher and ri lower in unrelated compared to related sct. these feasibility data provide the basis for a phase iii study comparing auto-allo unrelated to auto-auto sct. background: poems syndrome is a rare paraneoplastic syndrome resulting from a clonal plasma cell proliferation producing a small monoclonal protein usually lambda type restricted. this syndrome is a multisystemic disease, its major clinical feature being a progressive peripheral neuropathy. single osteosclerotic lesions should be treated with radiation therapy only. in widespread disease, high-dose therapy followed by autologous hematopoietic stem cell rescue (asct) has shown to be an effective therapeutic option in short series, but with significant morbidity. patients and methods: between december 1999 and september 2009, 19 patients with poems syndrome (12 female/7 male) were treated with melphalan-200 (16 patients) or melphalan-140 (3 patient) followed by asct at 9 spanish institutions. median age was 54 years (range: 26-67). all of them presented a m protein lambda type (iga: 16; igg 3), peripheral polyneuropathy (18), osteosclerotic lesions (12), organomegaly (16), endocrinopathy (7), skin lesions (18), extravascular volume overload (14), papilledema (6), polycythemia (2) and thrombocytosis (12). four patients had pulmonary hypertension, two portal hypertension, and three castleman disease. four patients were previously untreated. the median number of prior therapies was 2 (range, 0-4). median time from diagnosis to asct was 8 months (range, 2-95). results: no transplant-related-mortality (trm) was observed. after a median follow-up of 45 months (range, 3-89), one patient has died of progression 90 months post-asct. five patients presented an engraftment syndrome and two a primary graft failure resolved, one with a back-up infusion on day + 26 and the other presented a delayed engraftment. sixteen patients were evaluable for response, 8 achieved a complete hematologic response (cr) (if-), 7 a near-cr (negative electrophoresis but if positive) and one died of progression 90 months post-asct. clinical improvement was observed at 4-6 months post-transplantation. all patients had a signifi cant organic improvement. the four patients with severe pulmonary hypertension and the two with portal hypertension improved of both pressures. conclusions: in this series, asct proved to be a highly effective therapy for patients with widespread poems syndrome. despite no trm was observed, these patients may have a delayed hematopoietic recovery and may develop an engraftment syndrome that must be diagnosed/treated promptly. a salvage treatment containing novel agents consolidated by allogeneic stem cell transplantation with reducedintensity conditioning improves outcome of multiple myeloma patients failing autologous transplantation f. patriarca (1), h. einsele (2) objectives: allogeneic stem cell transplantation (allo-sct) employing reduced intensity conditioning (ric) is a feasible procedure in selected patients with relapsed multiple myeloma (mm), but its effi cacy is still a matter of debate. we investigated the role of ric allo-sct in mm pts who relapsed after auto-sct and were then treated with a salvage therapy based on novel agents. our study was structured similarly to an intention to treat analysis and included only those pts undergoing a hlatyping immediately after the failure of auto-sct. the cohort of pts having a donor (donor group) was compared with the one not having a suitable donor (no donor group). patients and methods: one hundred thirty-six consecutive pts were retrospectively evaluated. fifty-seven found a donor and 50 (88%) underwent an allo-sct: 16 identical sibling (32%), 34 mud (68%). conditioning regimens were fl udarabine, melphalan±thiotepa in 21 patients (42%), fl udarabine + 2 gy tbi in 15 cases (30%), fl udarabine and cyclophosphamide in 8 patients (16%) and fl udarabine and treosulfan in the remaining 6 cases (12%). seven pts having a donor did not receive allo-sct for progressive disease or severe comorbidities. median age of donor group was signifi cantly younger than no donor group (53 versus 60 years, p = 0,000045). the 2 groups were balanced with regard of time between auto-sct and relapse, treatment of fi rst relapse, duration of salvage treatment, and quality of response after salvage treatment. results: the median follow-up for all patients was 15 months (1-97) after relapse. the median time to progression-freesurvival (pfs) and overall survival (os) for all patients were 16 and 25 months, respectively. two-year pfs was 41% in the donor-group and 18% in the no-donor group (p = 0.0003). twoyear os was 57% in the donor-group and 49% in the no-donorgroup (p = 0.08). in multivariate analysis the availability of a donor was statistically signifi cant for pfs (p = 0.003); moreover a signifi cant difference in outcome was observed comparing patients who achieved cr + vgpr versus pr versus sd/pd after salvage treatment (p = 0.05, p = 0.002, p = 0.0001 for pfs, p = 0.01, p = 0.001, p = 0.0001 for os). conclusions: this study comparing salvage treatment with novel agents consolidated by ric allosct versus salvage treatment alone after a failed auto-sct provides evidence for a signifi cant pfs benefi t and a trend for a prolonged os in mm patients having a donor. grouping patients based on d and non-d kir2ds4 allelic status revealed individuals homozygous for kir2sd4(d) had significantly shorter duration of fever, compared to those heterozygous or homozygous for kir2ds4 (non-d), (p = 0.003). this held true on multivariate analysis. no signifi cant association was seen between kir2ds4 genotype and neutrophil engraftment time or microbial isolates. of note, there was a signifi cant association between kir2dl3 positivity and gram-ve bacteremia (p = 0.017). these data support a role for kir genotype and infectious complications post-asct. kir genotyping may aid risk assessment of infectious complications post-asct and optimization of anti-infectious prophylaxis, surveillance and treatment in those deemed at risk of sepsis. these results suggest a functional role for the deleted kir2sd4 variant, previously believed non-functional in the innate immune response. in the era of novel therapeutic agents, high-dose chemotherapy and autologous stem cell transplantation (asct) remains an integral part of treatment for multiple myeloma (mm). therefore, the choice of new drug combinations for induction therapies must take into consideration the requirement to collect a suffi cient number of haematopoietic stem cells (hscs) for one or more courses of asct. lenalidomide and melphalan have been shown to impair hsc mobilisation, but induction therapies containing either thalidomide or cyclophosphamide do not have a relevant impact on the hsc collection yield. we considered the possibility that the combination of cyclophosphamide and thalidomide could have an additive impact on the hsc compartment and carried out a retrospective analysis of the outcome of peripheral blood hsc mobilisations performed in 111 mm patients. patients who had received induction therapy with ctd (oral cyclophosphamide, thalidomide, dexamethasone; n = 55) were compared with a control group of patients (n = 56) who had received vad (n = 30) or z-dex (idarubicin, dexamethasone; n = 26) during the same 4-year period. all mobilisations were performed with cyclophosphamide and g-csf. our standard collection target was 4 × 10 6 cd34 + cells/ kg, with a minimal target of 2 × 10 6 being accepted if patients failed the standard target. the total number of cd34 + cells harvested was signifi cantly lower in the ctd group (5.2 vs. 9.7 × 10 6 /kg, p = 0.002). the number of cd34 + cells harvested on the fi rst day of apheresis and per apheresis procedure were also lower in the ctd group (2.8 vs. 7.3 × 10 6 /kg, p = 0.002; 2.6 vs. 6.7 × 10 6 /kg, p = 0.002). more patients in the ctd group failed to achieve both the standard (36.4% vs. 16.1%, p = 0.021) and minimal (18.2% vs. 5.4%, p = 0.036) stem cell harvest target, despite a higher number of patients in the ctd group undergoing two or more apheresis procedures (52.8% vs. 32.1%, p = 0.012). the failure rate on the fi rst day of apheresis was also higher in the ctd group both for the standard (56.3% vs. 28.6%, p = 0.003) and the minimal target (36.7% vs. 16.1%, p = 0.041). age or number of induction therapy cycles did not have an impact on mobilisation failure in the entire cohort or the ctd group alone. these observations provide novel evidence that drugs with no previously demonstrated effect on hsc mobilisation can have a considerable negative impact when used in combination, which can result in a high rate of hsc collection failures. a. nagler (1) background: allogeneic transplantation of hematopoietic stem cells (allo-sct) from an hla-matched related (mrd) or unrelated donor (urd) is a curative option for patients (pts) with high-risk hematological disease (hrhd). in the absence of a mrd, pts have been offered investigational transplant strategies such as umbilical cord blood (ucb) or family haploidentical sct (haplo-sct). in our institution, all patients with hrhd are typed at entry; if a suitable mrd donor is missing a urd search is promptly activated. our policy is to offer an haplo-sct to adult pts lacking an mrd or urd in order to adequately treat hrhd in the ideal appropriate time according to clinical indications to allo-sct agreed in ongoing protocols for primary disease. methods: here we report the retrospective intention-to-treat (itt) analysis of alternative donor transplantation at our institution. data were obtained from institutional database. results: between jan-2004 and nov-2009, 361pts (100% of the following itt analysis; median age 48y) received indication to allo-sct according to ebmt recommendations. eightytwo pts (23%) received a transplant from a mrd; 170pts (47%) activated an urd search in the ibmdr registry; 78pts (22% of total pts, 46% of urd searching) received a urd transplant; 35pts (9.7%-20,5%) received an haplo-sct due to lacking of a suitable urd in the appropriate timing according to disease status, or absence of suitable criteria to engage an urd donor; 9 pts (2,5%-5,2%) received a ucb because lacking a suitable haplo donor. overall, 129pts received an haplo-sct (36%): 35 after ibmdr research, 94 up-front. nineteen pts died before receiving a transplant (5%), 44 (12%) are searching for a suitable donor. if we consider only pts with acute leukemia (213pts, median age 47y, range 15-72; over 50y 92pts) 47pts (22%) received a transplant from a mrd; 66pts (40%) activated an urd search; 37pts received a urd transplant (17%), 6pts (3%) a ucb, 97pts an haplo-sct (46%). eight pts died before receiving a transplant (4%), 18 (8%) are searching for a suitable donor. allo-sct was performed in complete remission in 104pts (62% alive), in persistence of disease in 83pts (20% alive). conclusion: in itt analysis, 83% of overall pts candidate received an allo-sct: 60% from an alternative donor, 23% from a mrd. the highly committed policy performed in the alternative-sct setting and the implementation of an alternative donor option are essential prerequisite to obtain these results. there was a marked variation in total numbers of transplants performed between countries ranging from 38 to 2629. the median age of hsct programs was 10 yrs (range 3-23). the total number of hsct performed per year has continued to increase and is yet to plateau. a greater proportion of transplants was allogeneic hsct (allo-hsct) compared to autologous hsct (asct) (77% vs. 23%). acute leukemia constituted the main indication for allo-hsct(37%). there was a relatively high rate of hsct for bone marrow failure (n = 1001, 17%) and hemoglobinopathies (n = 885,15%) when compared to data reported to ebmt and ibmtr. cml continued to be an indication for 8.7% of allo-hsct in 2007. the rate of unrelated donor hsct remained low, with only 2 non-umbilical cord unrelated donor transplants over the surveyed period. the use of peripheral blood stem cells varied between countries though increasingly constituted the main source of stem cells in allo-hsct. ric was used in 13.4% of allo-hsct in the the survey. asct rates continue to increase; while acute leukemia was the main indication for asct in the 1980s, overall the main indications for asct in the survey were lymphoma (45%) followed by myeloma (26%). conclusion: we present the fi rst survey of hsct activity in the em region over 23 years which refl ects the unique health conditions of this region, accounting for notable differences in transplant practices from europe and north america. annual hsct rates continue to rise. economic, logistic and other factors are likely to be responsible for disparities in activity within the region. this survey may be valuable in providing a basis for healthcare planning in the fi eld of hsct in the region. background: related haploidentical donors, as cord blood, can be alternative donor sources in stem cell transplantation (sct). severe graft-versus-host disease (gvhd), however, has interfered the progress of haploidentical stem cell transplantation (haplosct). to deal with this strong gvhd, t cell depletion has usually been used in european countries. on the other hand, based on the difference of ethnicity prone to less severe gvhd in japan, we have performed unmanipulated haplosct using myeloablative or reduced intensity preconditioning regimen for ten years. in this meeting, we will summarize our experience of ten years. , and patients over 45 years old or with comorbidities or repetitive sct (including second to fi fth sct) underwent reduced intensity preconditioning regimen consisting of flu/(ca)/bu/atg or flu/(ca)/mel/atg (haplomini, n = 146). high dose ara-c (ca) was optional to reduce tumor burden. atg (fresenius) dose was 2 mg/kg/day for four days. gvhd prophylaxis consisted of taclolimus (tac), methylprednisolone (mpsl) 2 mg/kg/day, short term mtx, and mycophenolate mofetil (mmf) 15 mg/kg/day in haplo-full, and tac, mpsl 1 mg/kg/day in haplo-mini, respectively. for elderly patients over 50 years old in haplo-mini, mmf was added. results: hematopoietic engraftment in haplosct was as rapid as that in hla-identical sct, except eight cases of graft rejection. acute gvhd (grade ii-iv) was observed in 30%. overall survival in fi ve years is 30% in haplo-full and 40% in haplo-mini, respectively. if limited to cr cases, overall survival reached over 60% in haplo-mini. there is no difference in survival rate among patients' diseases. discussion: unmanipulated haplosct is feasible and effective for refractory diseases. atg dose used in haplo-mini is rather low compared with that of european cases reported so far. although it should be too early to refer long term outcome, unmanipulated haplosct could be considered as an option to fi ght against refractory diseases. background: while chemotherapy + g-csf (g) is effective for autologous stem cell mobilization (scm) it exposes patients (pts) to risks and side effects. plerixafor (p) + g provides a safer alternative for mobilization of hematopoietic stem cells in pts with mm or nhl. to better understand the comparative effectiveness of standard and newer approaches for scm, we conducted a study aimed to: (1) determine the clinical outcomes and cost of scm with cyclophosphamide (cy) plus g (2) compare outcomes of cy + g to a clinical trial of p + g. methods: a retrospective study was conducted in all pts undergoing fi rst scm from 1/04 to 3/08 (n = 241) with cy (3 gm/m 2 ) and g (10 mcg/kg started 1 day after cy for 10 days with planned fi rst day of collection on day 11). apheresis was initiated when peripheral blood had >15 cd34 + cells/μl. positive clinical outcome (pco) was defi ned as >2 × 10 6 cd34 + cells/kg collected on planned day of collection and within 1 or 2 apheresis without a prior negative event that led to additional clinic/inpatient evaluation. the cost of drugs, apheresis, product processing, and clinical events were reported based on medicare part b physician, laboratory, and ancillary fee schedule. data on pts undergoing scm with p + g were obtained from published clinical trials in pts with myeloma (mm) or lymphoma (l) undergoing fi rst scm using g (10 mcg/kg without dose escalation) and a maximum of 4 days of p. results: among cy + g pts, 141 (61%) were males; 121 (50%) had mm, 115 (48%) had l (5-other diagnoses); and 61 (25%) pts. had prior radiation. pco was seen in 48 (20%) pts.; 23% of mm and 15.7% of l pts. median total cost of cy + g scm was $10,732 (range, 6988-30827). pco was associated with a lower cost than non-pco in the overall group, (mean, $10,371 vs. $12,870, p = 0.001), in mm pts. (mean, $10,511 vs. $12,152, p = 0.026), and in l pts (mean, $10,133 vs. $13,627, p = 0.006). assuming a similar distribution of pco in 100 pts. with mm and l, the projected per pt cost of scm would be $11,774 and $13,067 (mean, $12,421) with cy + g. projected costs of scm using p + g for 100 pts. with mm and l were $12,852 and $8986 (mean, $10,919). conclusion: scm with cy + g was associated with a lower rate of pco and higher cost than p + g, supporting front-line use of p + g for scm. future prospective studies should investigate whether scm with p + g translates into decreased resource utilization and improved quality of life for pts undergoing scm. .5x10 9 /l, platelet count (pt) >150 × 10 9 /l, and hemoglobin level (hb) >120 g/l], good partial response (gpr) [anc >1 × 10 9 /l, pt >50 × 10 9 /l, and a hb level >100 g/l] and poor partial response (ppr) [anc >0.5 × 10 9 /l, pt >20 × 10 9 /l, hb level >80 g/l and transfusion independency]. no response (nr) was defi ned as failing criteria for at least ppr. non-responding patients received a second-line therapy (hsct or androgens), a second course of ist with lg (15 mg/kg/day/ × 5 days) or tg (3.5 mg/kg/day/ × 5 days); or no treatment. subgroup analyses were conducted and differences in response were tested using the chi-square statistic test. immunoablation followed by immune reconstitution by transplantation of autologous peripheral blood stem cells is a promising approach to treat refractory or otherwise incurable s26 autoimmune diseases. it has recently been suggested that this treatment could lead to remission in some cases of the early diabetes type 1 if administered prior complete destruction of beta cells by autoimmune mechanisms. the objective of this study was to verify these fi ndings on an independent group of patients. methods: eight patients (age 19-32) with early diabetes type 1 (no more than 6 weeks from diagnosis, c-peptide positive, anti gad -antibodies positive) were qualifi ed for the treatment. the patients were subjected to 2-4 plasmaphereses to remove circulating immune complexes and then mobilized with cyclophosphamide and g-csf. leucaphereses were continued until >3,0 x 106 cd34 + cells/kg were collected. the conditioning consisted of cyclophosphamide (50 mg/kg/day on days -5, -4, -3, -2 prior to transplant) and antithymocyte globulin (atg genzyme -of 0.5 mg/kg/day given on day -5, 1.0 mg/kg/day given on days -5, -4, -3, -2 and -1). results: median follow up for the patients as of december 2009 is 10 months (range 6-19 months). no major complications were observed during the transplantation and in the posttransplantation period. all patients (8/8) became independent from exogenous insulin after the transplantation. median day of insulin withdrawal was + 24 (range + 6 to + 60). one patient resumed insulin 7 months post transplant. six out of 8 patients were given acarbose to reduce the potentially toxic infl uence of observed hyperglycemias. after the transplantation patients exhibited good control of glycemia -the average hba1c concentrations were 12.3% at the diagnosis, and 5.59%; 6.23%; 5.9% -3, 6, and 12 months after the transplantation respectively. this was correlated with increased levels of c-peptide after the transplantation. conclusion: this report independently confi rms that independence of exogenous insulin could be reached in diabetes type 1 patients following immunoablation and reconstitution of the immune system with autologous peripheral blood stem cells providing that the procedure is performed prior total destruction of beta cells in the pancreas by the autoimmune mechanisms. the use of acarbose may have potentially positive effect on the duration of remission of diabetes type 1 in the patients after the transplantation. new perspectives on the use of autologous haematopoietic stem cell transplantation in multiple sclerosis: three strategies of high-dose immunosuppressive therapy with autologous haematopoietic stem cell transplantation during the last decade high-dose immunosuppressive therapy with autologous hematopoietic stem cell transplantation (hdit + asct) has been used with increasing frequency as a therapeutic option for ms patients. we aimed to study clinical outcomes in multiple sclerosis (ms) patients after early, conventional, and salvage of hdit + asct. 155 ms patients (secondary progressive-54, primary progressive-28, progressive-relapsing-5, relapsing-remitting-68) were included in this study (mean age-33.0; male/female -63/92). beam or mini-beam conditioning regimens were used. 136 patients did not have any supportive treatment after hdit + asct; 29 patients with risk factors were administered mitoxantrone during a year after asct as a consolidation therapy. 68 patients underwent early transplantation (edss 1.5-3.0), 79 -conventional (edss 3.5-6.5), 8 -salvage transplantation (edss 7.0-8.5). median edss at base-line -4.0. the mean follow-up duration -22 months (range 6-125). neurological evaluation was performed at baseline, at discharge, at 3, 6, 9, 12 months, and every 6 months post transplant. transplantation procedure was well tolerated with no transplant-related deaths. the effi cacy analysis was performed in the group of patients who did not have consolidation treatment. out of 78 patients with the follow-up at least 9 months the following distribution of patients according to clinical response at 6 months post transplant was observed: 36 patients (46%) achieved an objective improvement of neurological symptoms; 41 patients (53%) had disease stabilization; 1 patients (1%) progression. in the patients who underwent early transplantation (n = 32) 15 (47%) patients stabilized and 17 (53%) improved. after conventional transplantation (n = 41) 22 (54%) patients stabilized, 18 (44%)-improved, and 1 (2%)-progressed. after salvage transplantation (n = 5) 4 patients were stable, and 1 improved. at long-term follow-up (median-26.5 months) out of 60 patients improvement was registered in 35 (59%) patients; stabilization -in 20 (33%) patients, and progression in 5 patients (1 patient after early; 4 -after conventional transplantation). no active, new or enlarging lesions were registered in patients without disease progression. thus, hdit + asct appears to be a safe and effective treatment for ms. further studies should be done to establish the best timing for transplantation and to evaluate consolidation therapy in patients receiving early, conventional, and salvage transplantation. in the last decade, the so-called nonmyeloablative or reduced intensity conditioning (ric) regimens for allogeneic stem cell transplantation (allo-sct) have emerged as an attractive modality to decrease allo-sct-related toxicities and nonrelapse mortality. indeed, ric allo-sct represents an attempt to harness the immune graft-versus-tumor effect while attempting to control or overcome toxicity. the work of different pioneering groups rapidly proved that this approach is feasible in several disease settings or patients' categories, and had the added benefi t of expanding the transplant option to patients who are ineligible for standard myeloablative allo-sct. currently, the use of ric allo-sct has challenged the need for high-dose conditioning regimens. unfortunately, and despite several thousands of patients receiving ric allo-sct, the true value of ric allo-sct in the management of hematological and non-hematological malignancies, especially aml is, as yet, diffi cult to delineate. currently, there are only very few, if any, prospective, randomized, or controlled trials that addressed the specifi c role of ric allo-sct versus other treatment strategies in aml. based on a large number of registry-based analyses, the ric subcommittee of the alwp has attempted over the last few years to better defi ne the role of ric allo-sct in aml patients through fi ne analysis of leukemia-free survival and overall survival balanced against treatment-related toxicity, complications, and death. a major advance was the identifi cation of the most important comorbidities that are likely to impact outcome after ric allo-sct for aml. another major achievement is the launch in 2010 of the multicenter/multinational ebmt randomized phase 3 study evaluating the role of ric allo-sct in elderly patients with aml. at present, the use of ric prior to allo-sct appears to be on the cutting-edge. the ric approach proved to be much more complex than originally thought. since the fi rst reports published in the late 90s', the ric allo-sct literature has exponentially expanded. the complexity of ric allo-sct practice is progressively deciphered and the optimism to regard ric allo-sct as a potential and promising treatment modality for many aml patients remains very high among investigators, warranting continuous and renewed clinical and therapeutic research in this area. transfusion of donor lymphocytes (dlt) for treatment of leukaemia relapse after allogeneic stem cell transplantation (sct) has been a milestone in the fi eld of immunotherapy against malignant disease. it can be regarded as the proof of principle for the graft-versus-leukemia effect. during recent years, the immunotherapy subcommittee of alwp has initiated a variety of retrospective studies to evaluate in detail the role of unmanipulated dlt in acute leukaemia. in contrast to cml and early stages of mds, dlt was less effective in the treatment of post sct relapse in highly proliferative diseases as acute leukaemia. nevertheless, when given as maintenance therapy after effective cytoreduction, dlt could induce long term remissions in selected patients in aml, both after standard and reduced intensity conditioning transplants. strategies that included dlt were more effective than treatment without the use of donor cells. in contrast, no such effects could be shown in all. these data have prompted the strategy to use donor cells already before the occurrence of overt haematological relapse, i.e in minimal residual disease, mixed or falling donor chimerism, or even prophylactically. this strategy is now widely used across european transplant centres and has shown promising results. future strategies include the combination of targeted therapies and donor cell based strategies, as well as the use of specifi c subsets of donor cells in order to augment the antileukemic effi cacy and control for side effects of dlt, such as gvhd. allogeneic stem cell transplantation (allosct) with reduced intensity conditioning (ric) is being increasingly used for acute myelogenous leukemia (aml) patients with high comorbidities not eligible for standard myeloablative conditioning. new compounds and formulations including intravenous busulfan, treosulfan and recently clofarabine have been introduced into the pre transplant conditioning regimens in an attempt to reduce transplant related toxicities while keeping or increasing the anti leukemic effect, overcoming the increased relapse rate usually associated with ric allosct. similarly, replacing bm by peripheral blood stem cell (pbsc) grafts may increase the gvl, albeit with the price of increasing toxicities, gvhd and trm. thus, the optimal conditioning regimen for adults with aml is yet unknown. in order to address these issues, the alwp of the ebmt recently performed several surveys analyzing the use of iv bu for allosct, comparing iv bu/cy to tbi/cy, comparing pbsc and bm, and is about to compare treosulfan to iv bu as conditioning regimens for adult patients with aml. in the iv bu/cy vs. tbi/cy survey that included 1479 patients, the fi ndings indicated that outcomes of allosct including engraftment, trm, rr and lfs were comparable while gvhd probability was signifi cantly lower and vod probability signifi cantly higher in adults patients with aml using iv bu/cy vs. tbi/cy conditioning, respectively. in a separate survey analyzing the use of iv bu allosct for aml the 1 year cumulative incidence of vod was found to be 8.4 + 2%. the factors associated with vod were mismatched unrelated donors and being not in remission. the pbsc vs. bm survey included 1537 patients. we observed signifi cantly higher incidence of gvhd and nrm and lower incidence of relapse rate with the use of pbsc, resulting in equivalent lfs. the treosulfan vs. iv bu survey is ongoing. in house comparison revealed similar lfs in aml patients in remission, while results were better using 4d iv bu combinations in patients with active disease. for mds the flu/treo conditioning regimen was the best. in conclusion, it is conceivable that introducing new compounds in the pre allosct conditioning regimens in combination with pre-and post-allosct targeted therapy will enable us to tailor the conditioning regimen to the specifi c disease category, reducing toxicity while improving the anti tumor activity. allogeneic hsct (allohsct) is generally considered the best consolidation option for younger patients diagnosed with an aml with high-risk cytogenetics. nonetheless, the cytogenetic high-risk category comprises different aml subtypes, and the specifi c results of allohsct for most of these cytogenetic entities are mostly unknown. moreover, several molecular lesions, such as mutations of flt3, mll or wt1 genes, identify subsets of patients with a poor-prognosis disease despite harboring a non-adverse-risk cytogenetic abnormality. therefore, the precise role of allohsct for the management of high-risk aml should be determined after a comprehensive analysis of the outcome of transplant in all these biological aml varieties with an adverse prognosis. in this context, the molecular markers sc is conducting a project to analyze the results of allohsct for diverse high-risk aml subsets, such as aml with t(6;9), aml with 3q abnormalities, and normal karyotype aml with internal tandem duplication of flt3 gene (flt3-itd three-year lfs was 28 9% and 18.9% for patients in cr1 and pif, respectively. finally, the prognostic impact of flt3-itd on the outcome of allohsct has been analyzed in a cohort of 120 patients (median age: 41, 18-60) with a normal karyotype aml. donor was an hla-identical sibling in 75% of cases, whereas conditioning regimen was a myeloablative regimen in all transplants. all patients received transplant in cr1. of note, 2-year lfs, ri, and nrm was 58.5%, 30.5%, and 13.3%, respectively, without a signifi cant difference between transplants from related and unrelated donors. in summary, the outcome of allohsct in patients with two high-risk aml subtypes such as aml with t(6;9) and flt3-itd aml showed a relatively favourable outcome, which compares favourably with reports from non-transplanted patients. on the contrary, the outcome of patients with aml and 3q26 rearrangement seems inferior, even in patients transplanted in an early phase of the disease. therefore, in order to elucidate the current role of allohsct for high-risk aml, transplant analyses should be focused on specifi c biological aml entities, since the results might differ largely among aml subtypes. finally, a careful collection of biological features at diagnosis is essential for addressing studies based on biological categories. hsct is a well-recognized option for the treatment of acute leukemia with the number of transplantations continuously growing over the last decades. however, the hsct rates vary strongly between countries. in particular, the activity in central and eastern (c&e) europe is markedly lower compared to the western part of the continent, suggesting the role of various socio-economic and geographic factors. using the human development index (hdi) as a surrogate marker we demonstrated that the socio-economic status (ses) of a country strongly correlates with the total number of hsct per population, as well as separately with sibling-hsct, unrelated-hsct and autologous-hsct in europe. we further speculated that in line with the transplant activity the results of hsct may also vary between countries or regions. direct comparison of hsct performed from sibling donors for aml in cr1 showed superimposable results obtained in c&e and western europe, however, the transplant characteristics differed with younger recipient age, longer interval from diagnosis to hsct and less frequent use of peripheral blood, tbi, t-depletion and reduced-intensity conditioning in c&e countries. subsequent analysis demonstrated that the outcome of sibling-hsct for acute leukemias in c&e europe improved over time. in particular the cumulative incidence of nrm decreased from 22 2% for patients treated between 1990-2002 to 15 3% for hsct performed between 2003-2006, despite increasing recipient age. in another analysis we demonsatrated the infl uence of the ses on outcome in europe, which, however, could not be explained by regional differences. best outcome (increased lfs and reduced ri) after myeloablative hsct from either related or unrelated hla-matched donor was observed in 8 countries with the highest hdi, while no differences could be demonstrated for the remaining 22 analyzed countries. interestingly, transplantations in countries with the highest hdi were characterized by increased recipient age and shorter interval from diagnosis to hsct. finally, we speculated that the center experience may infl uence results of hsct. using transplants with reduced-intensity conditioning (ric-hsct) as a model we analyzed results according to the team experience as defi ned by: 1) time from the fi rst allohsct and from the fi rst ric-hsct, 2) the total number of allohsct and the number of ric-hsct performed in a study period (2001) (2002) (2003) (2004) (2005) (2006) (2007) . we found that results in centers performing few ric-hsct (<15 in 7 years) were inferior compared to the remaining centers in terms of all, lfs, ri and nrm, which has been confi rmed in a multivariate model. altogether, in a series of retrospective analyses based on the ebmt alwp registry we demonstrated that both rates and outcomes of hsct for acute leukemia vary among countries and centers. both socio-economic factors and team experience infl uence results. we previously reported the ebmt experience with salvage high-dose chemotherapy (hdct) in pediatric patients with extragonadal germ-cell tumour (gct) (de giorgi u et al. -bjc 2005) . we analyzed a total of 23 children with extragonadal gct, median age 12 years (range 1-20), treated with salvage hdct with haematopoietic progenitor cell support. the gct primary location was intracranial site in nine cases, sacrococcyx in eight, retroperitoneum in four, and mediastinum in two. twenty-two patients had a nongerminomatous gct and one germinoma. nine patients received hdct in fi rst-and 14 in second-or third-relapse situation. no toxic deaths occurred. overall, 16 of 23 patients (70%) achieved a complete remission. with a median follow-up of 66 months (range 31-173 months), 10 (43%) have been continuously disease-free. of six patients who had a disease recurrence after hdct, one achieved a diseasefree status with surgical resection followed by chemotherapy and radiotherapy. in total, 11 patients (48%) were diseasefree at the time of analysis. eight of 14 patients (57%) with extracranial primary and three of nine patients (33%) with intracranial primary gct were disease-free at the time of analysis. hdct induced impressive durable remissions as salvage treatment in children with extragonadal extracranial gcts. after 7 years from this fi rst analysis (done in 2003 published in 2005), we decide to analyze the long-term results of this experience integrating these data with other from other cases from the ebmt registry previously not included, and with data from additional patients from italian registries (gitmo and aieop). high-dose chemotherapy and autologous stem cell transplantation in relapsed germ cell tumours: do we need a randomized study? p. pedrazzoli (1) nasopharyngeal carcinoma (npc) is an epstein-barr virus (ebv)-related malignancy expressing a restricted set of viral antigens. the outcome of patients with npc failing conventional radio-chemotherapy is poor, the median overall survival of patients receiving second line treatments being less than two years. hence, the need for alternative therapies capable of improving disease-free survival and associated with reduced toxicity. since 2001, we have implemented a t-cell therapy program for patients with npc failing conventional treatment. so far, we have treated 27 patients with disease resistant/relapsing after >2 lines of radio-chemotherapy in two sequential trials of cell therapy with autologous ebv-specifi c cytotoxic t-lymphocytes (ctl). in detail, ebv ctl (4 escalating doses to a maximum of 8 × 10 7 ctl/dose in the fi rst trial, or 2 doses of 3-4 × 10 8 ctl in the second trial) were administered in patients without or with preceding lymphoablative chemotherapy and recombinant human interleukin-2 (rhil-2). the proportion of patients achieving response (partial, pr, complete, cr) or stable disease (sd) lasting at least 3 months (recist criteria), as well as immunologic correlates of effective treatment, were evaluated. overall, the objective control of disease was 57%, with 6/27 patients showing complete (n = 1), partial (n = 4) and minimal (n = 1) responses, and 10/27 disease stabilization (median duration 7 months). no severe adverse events were observed after cell therapy; 4 patients showed an infl ammatory reaction at the tumor site. the use of preparative chemotherapy and increased ctl dose did not infl uence outcome. importantly, patients showing response to cell therapy showed the emergence of ebv lmp2 antigen-specifi c t-cells in their peripheral blood. ebv-specifi c ctl therapy is safe and associated with clinical benefi t in patients with advanced npc refractory to standard therapies. the use of ctl with higher specifi city for the ebv subdominant antigens expressed by the tumor, such as lmp2, at an earlier stage of disease, could further implement the strategy and ameliorate the outcome of patients with relapsing/refractory npc. this study was supported by a grant from the italian association for cancer research (airc). , and csa given orally (5 mg/kg/d). patients randomized to receive g-csf were given glycosylated g-csf from day 8 to 120 (150 microg/m 2 /d, sc). os at 6 years was 75%, it was 82% for patients with severe aa and 66% for patients with very severe aa (p = 0.001). survival decreased with increasing age from 100% (<20 years) and 92% (20-40 years) to 71% (40-60 years) and 56% (>60 years) (p < 0.001). there was no difference, overall and when stratifi ed according to age and aa severity in os (p = 0.64) and efs, defi ned by death, need for transplantation, relapse and non response as events (p = 0.36) between the study arms at 6 years ( table 1 ). the median neutrophil count was signifi cantly higher between day 30 and 240, in the g-csf group; this difference did not persist to day 360, when g-csf was stopped. there were fewer infections (36% no g-csf; 24% with g-csf; p = 0.006), and less days of hospitalization during the fi rst 90 days (p = 0.03) in the g-csf group. 44 patients died, mostly from infection (55%).there was no difference in death rates, cause of death and response rates between both groups. overall 73% of patients with and 62% without g-csf did respond to is (p = 0.49). 57 patients did not respond to fi rst-line therapy, 31 patients relapsed during the fi rst year of treatment (no difference between both groups). in conclusion, g-csf added to standard is increases neutrophil counts, and decreases rate of infections and days of hospitalization but has no impact on os, efs, remission, death and relapse rates. this has to be weighed against possibly higher risks of mds/aml, as suggested by previous studies. an hla-identical sibling (65%), 157 pts received a myeloablative regimen (78%) and 135 bone marrow (64%) with gvhd prophylaxis consisted in csa ± mtx in 154 pts (73%). during evolution, 14 pts did not engraft (7%). acute gvhd of grade >i developed in 52 patients (29%). chronic gvhd developed in 57 pts (26 extensive). after a median ±se follow-up time of 61±6 months, 64 pts died (35 infections, 18 gvhd). the 5-year os rate was 68 ± 3%. none of the common variables examined for association with os were statistically signifi cant, except for sct indications with an increasing risk from rsh, saa and thr ( figure 1 , p = 0.03). based on the 24 pairs obtained through the matching process, the 5-year os estimate after thr ( figure 2 ) was 45 ± 11% for pts with sct and 87±8% for pts without sct [hr 10.0 (95%ci 1.3 to 78.1); p = 0.01]. of note, the os among non-sct pts was signifi cantly worse (p = 0.01) in the non-matched than in the matched group, raising the question of pts over selection due to the matching process. concerning pts with saa complication, after exclusion of pts with thr and of non-sct pts with a follow-up time <6 months after saa, the 5-year os was 96±2% for pts without sct and 81±4% for pts with sct, but the 2 groups differ signifi cantly for age at saa and year of saa. conclusions: further matching processes are necessary to conclude on this large cohort of pnh pts in order to defi ne the exact place of sct in pnh, especially in the era of the eculizumab. introduction: currently peripheral blood (pb) is more commonly used as stem cell source than bone marrow (bm) in both autologous and allogeneic hematopoietic stem cell transplantation (hsct) (gratwohl, bmt, 2005) . however, pb is associated with an increased risk of chronic graft-versus-host disease (gvhd), which is a disadvantage in non-malignant diseases. a recent study of ebmt/cibmtr showed that incidence of chronic gvhd and overall mortality were higher after hsct with pb than after hsct with bm in young patients with severe aplastic anemia (saa) (schrezenmeier h, blood, 2007) . the ebmt transplant activity survey shows that the number of pbsct has increased rapidly since early 90's and exceeded the one of bmt in 1999 in family donor and in 2002 in unrelated donor (ud)-hscts. the number of pbsct has also increased in saa. we were therefore interested to review the current status of stem cell source selection in bone marrow failure ( in conclusion, pb has been increasingly used as a stem cell source in bmf despite of its higher risk of chronic gvhd. there were major differences in stem cell source distribution, regarding donor type, the global region as well as countries in regions. it may refl ect the differences in infrastructure in each center/country, donor and physicians preferences and policy of marrow donor program. clearly, recommendation for stem cell source is warranted in bmf. we conclude that 5q-syndrome can be cured by allogeneic transplantation, but additional abnormalities reduce the curability. the role of lenalidomide given before transplant will also be analysed. patients. an interim toxicity analysis was performed when the fi rst hundred patients had been included. the safety committee agreed to resume the trial. we will report the feasibility and the toxicity in both arms. an interim analysis is currently ongoing. other preliminary studies demonstrate that vtd is a highly active and relatively well tolerated regimen. the combination is used in the relapse setting, as well as fi rst line, consolidation and maintenance. in this protocol, the starting doses of velcade and thalidomide are relatively high and the duration of treatment is long. we will assess the superiority of vtd over td in the relapse setting as well as its toxicity. objectives: chronic lymphocytic leukemia (cll) is susceptible to well characterized graft-versus-leukemia effects. the leukemic clone is eradicated at the molecular level in >50% of patients. yet, up to 50% of patients have persisting disease or relapse after allogeneic hematopoietic cell transplantation (hct). the application of donor lymphocyte infusions (dli) appears to be attractive in this situation. the aim of this retrospective analysis is to study the long-term effects of dli in patients with cll. methods: data from patients with cll who received allogeneic hct between 1997 and 2008 and had received at least one dli were included. the outcome of dli was analysed in two situations. 1) "pre-emptive" dli prior to frank relapse or disease progression 2) "therapeutic" dli given after documented relapse. baseline and follow-up data were downloaded from the ebmt database. results: 217 patients fulfi lled the inclusion criteria. major characteristics were median age 52 years, a median of three prior chemotherapy regimens, 71% matched sibling donor hct, 74% reduced intensity conditioning, 85% received peripheral blood stem cells. atg or alemtuzumab was used in 37%, ex vivo t-cell depletion (tcd) in 12%, no tcd in 50% of the patients. the median follow-up after the fi rst dli was 20 months. in the cohort of 109 patients who received dli prior to relapse, 38 patients received more than one dli, 21 patients more than two dli, 11 patients more than three dli. grades ii to iv acute gvhd was reported in 9 out of 35 patients (26%) with informative data. pr or cr was documented in 10 out of 20 patients (50%). overall survival and progression-free survival 5 years after the fi rst dli was 42% (95% ci, 28% to 56%) and 22% (95% ci, 12% to 22%) respectively in this group of patients. 108 patients received dli as treatment of relapse. among these, 42 patients received more than one dli, 16 patients more than two dli, 5 patients more than three dli. the overall response rate was 38% (13 out of 34 informative patients). nine out of 50 patients (18%) experienced acute gvhd grades ii to iv. in this group the 5-year overall survival after the fi rst dli was 21% (95% ci, 9% to 33%). only 3 patients had a follow up of more than 5 years. conclusion: in patients with cll donor lymphocyte infusions appear to have only moderate long-lasting activity. further investigation to delineate factors associated with improved outcome is warranted. , 3 centres 8%, 5 centres 7.5%, 2 centres 6%, 5 centres 5%. 3 centres using 10% dmso washed cells prior to infusion although another 9 centres also washed cells in occasional patients mostly less than 10% of their patients. 43 centres added additional agents to the freezing mixture, mostly albumin (25), hydroxy ethyl starch (6), heparin (8) and tissue culture medium (5). the median amount of dmso given per centre per patient was 20 g, although the upper limit set by the centre was often considerably higher. 75% of centres did not use any delay between bags of stem cells and the median duration of infusion was 22 minutes. side effects were defi ned using nci criteria (version 3.0) and initially results were analysed by each centre. patients in centres who used washed cells or 5% dmso experienced less nausea and vomiting and 'severe other' effects, although hypotension and hypertension did not seem to be affected (p = 0.026, 0.014, 0.624 and 0.208 respectively). on a centre basis, the use of an upper limit to the amount of dmso which could be given (70 g or the amount given ≤20 g versus >20 g) did not result in any reduction of any of the groups of side effects. similarly when the amount of dmso given and the duration of dmso infusion were compared on a centre-basis, no signifi cant differences were found. further analysis will be undertaken using individual data where appropriate and the results presented. introduction: cmv infection and disease still remain serious and frequent complications after hsct. both morbidity and mortality have been reduced by prophylactic and pre emptive strategies based on biological tests and on more or less effective anti-viral drugs. however, in absence of comparative studies, there is no consensus for diagnosis, prophylaxis and treatment. then we conduct a survey to assess the current cmv management for patients less than 18 years in ebmt centres. materials and methods: in december 2009, a 40-item questionnaire about diagnostic tests, monitoring schedules, prophylactic and pre emptive strategies and treatment modalities was send to centres. defi nition of treatment failure, recourse to drug resistant cmv mutant research, practice of cell therapy and combined anti-cmv therapy were also tested. results: by the 31st december '09, we have received 35 responses from 14 countries. a second shipment will send in order to increase the number of responding centres. as expected, whatever the considered aspect i.e. monitoring, prophylactic or pre-emptive applied strategy, used drug in front or second and more line therapy etc. none centre report same procedure than another. all results will be presented and discussed during pdwp meeting in vienna. then, some proposals will be made regarding studies comparing results in different centres with "opposite" procedures. the fi nal goal of this work is to collect enough data to build consensual procedure. here we describe the results of the fi rst 87 patients. all cases were confi rmed by pcr in nasopharyngeal or bronchoalveolar lavage samples. the fi rst case was diagnosed on july-9. patients' characteristics are shown in table 1 . eight cases were considered nosocomial infections. in sct patients, the median time from transplant to infl uenza diagnosis was 588 days (7-6155). s-oiv characteristics are given in table 2 . the most frequent symptoms were: fever (86%), cough (84%), rhinorrhea (51%), odynophagia (26%), myalgia (22%), headache (11%) and dyspnea (11%). diarrhea was rare (2%). of those who presented with upper respiratory tract infection alone, 9 (14%) had progression to pneumonia. 30% had pneumonia, with no difference between sct patients (31%) and non-sct patients (28%). fifteen out 17 pneumonias in sct patients occurred in patients during the fi rst postransplant year or later but under immunosuppressive treatment. five patients were transferred to the intensive care unit (icu). four out of 5 of the most severe cases (admitted to icu or fatal course) had lymphopenia (<500/ mm 3 ) compared with 26% of those with less severe forms (p 0.010). compared with adults, children had more pneumonia (39% vs. 28%) although the difference was not statistically signifi cant (p 0.3). only one of the vaccinated patients against seasonal infl uenza were diagnosed with pneumonia compared with 41% who were not vaccinated (p .005). all patients except one were treated with oseltamivir for a median of 5 days (5-27). zanamavir was added (in combination or sequentially) in 4 cases. no major adverse events relating to anti-infl uenza treatment were reported. summary: the novel s-oiv is a serious disease in stc and oncohematological patients with a high incidence of pneumonia (26%) and signifi cant mortality (3%). diarrhea was not a frequent symptom. lymphopenia was linked to the most severe forms. vaccination for seasonal infl uenza might protect against the development of pneumonia. nevertheless, the severity of new pandemic infl uenza seems to be similar to seasonal infl uenza in this patient population. background: ebv-ptld (epstein-barr virus-related post-transplant lymphoproliferative disorder) is a rare but serious complication after hsct and number of patients at risk is increasing over time. available data do not refl ect general practice of diagnosis and treatment of this complication. objective: assessment of the current strategy of diagnosis and preemptive use of rituximab for ebv-ptld in ebmt transplant centers (tc). methods: in 2009 survey, data regarding ebv strategy from 74 participating tc were registered on specifi c forms and centrally analyzed. responses from 10 centers were excluded due to lack of specifi c strategy. results: regular monitoring for ebv after hsct is done by 47/64 (73%) tc, and in 7/64 (11%) tc is related to clinical situation. the monitoring is performed in all allohsct patients in 32/47 (68%) tc, while in remaining 22 tc in following subgroups: mud (17/22), t-depletion in vitro (8/22), t-depletion in vivo (21/22), family mismatched (14/22), cord blood transplants (16/22), and in single centers in patients with saa, ebv mismatch, or after autohsct for autoimmune disorders. quantitative ebv-dna by pcr is performed in 62/64 (97%) centers. the assay is performed in: whole blood -50/64 tc (78%), plasma -9/64 (14%), lymphocytes -4/64 (6%), serum -1 center. the test is repeated twice a week in 8/64 tc (12.5%), once a week in 39/64 (60.9%), once per 2 weeks in 8/64 (12.5%), once a month in 5/64 (7.8%) and adjusted to risk in 4/64 (6.2%) tc. the monitoring for ebv reactivation after hsct is performed for a period of: less than 3 months in 2/64 (3%), 3 months in 22/64 (34%), 6 months in 19/64 (30%), 12 months in 8/64 (12%) and adjusted to risk in 13/64 (20%). rituximab as a pre-emptive therapy for ebv-ptld is routinely administered in 51/64 (80%). the number of ebv-dna copies as an indicator for preemptive therapy with rituximab was given by 60 tc, but varied between the centers, and were based also on symptoms and signs (table) . conclusions: ebv-ptld strategy exists in most of the responding centers. differential approach regarding indications for preemptive therapy is seen between centers: rituximab is administered as preemptive therapy in 80% of participating transplant centers. objectives: the aim of this study was to analyze the clinical risk factors, donor and recipient cytokine gene polymorphisms associated with cytomegalovirus (cmv) infection within 100 days after allogeneic hematopoietic stem cell transplantation (allo-hsct). methods: we studied in a total of 203 pairs of recipients and their donors, who underwent allo-hsct at our center. we analyzed 12 single nucleotide polymorphisms (snps) in 6 pro-and anti-infl ammatory cytokines genes, tumor necrosis factor ( 92.6%of122 patients developed cmv positive antigenemia without disease, only 9 patients developed cmv disease (7 patients with pneumonia and 2 patients with enteritis). there was a higher incidence of early cmv infection in transplantation with unrelated donors (66.4% vs. 51.7%, p = 0.043) and in recipients who were cmv seropositive before transplantation(72.9% vs. 54.9%, p = 0.018). the recipient's tgf-beta1-509 and + 869 genotypes were signifi cantly associated with the incidence of early cmv infection in both the unrelated transplantation cohort and sibling transplantation cohort, but the infl uence of the donor's tgf-beta1-509 and + 869 genotypes was signifi cant only in the sibling transplantation cohort. multivariate analysis identifi ed two risk factors for early cmv infection: cmv seropositive recipients (rr: 1.712, 95%ci: 1.177-2.490, p = 0.005), recipients with tgf-beta1 + 869 c allele (rr: 2.225, 95%ci: 1.401-3.536, p = 0.001). donors with tgf-beta1-509t allele was found to be less signifi cant factor (p = 0.094). conclusion: although cmv disease has been reduced in the era of antiviral prophylaxis and preemptive therapy, our fi ndings suggest the incidence of cmv infection remains high and provide the fi rst report of relationship between genetic background of donor and recipient to the risk of cmv infection in a chinese population. hsct. toll-like receptors (tlr) are essential components of the innate immune system. c3h/hej mice that lack functional tlr4 did not develop cystitis after intravesical instillation of e. coli. individuals with the asp299gly polymorphism of the tlr4 gene showed a diminished infl ammatory responsiveness to endotoxin. because of the requirement of tlr4 in infl ammatory response we hypothesized that tlr4 asp299gly reduces the risk of bk virus-induced hc after hsct. 166 children (median age, 13 years) who underwent allogeneic bone marrow (n = 105) or peripheral blood stem cell transplantation (n = 61) in a single center and their respective donors were genotyped of tlr4 for rs4986790 (a896g, asp299gly) using taqman real-time polymerase chain reaction. the donor was hla-matched unrelated in 57% of transplants or hla-identical related in 33% of transplants. conditioning regimen was myeloablative in all cases. two forms of post-transplant immunosuppression predominated, cyclosporine a and methotrexate in 64% of transplants and cyclosporine a alone in 25% of transplants. the asp299gly polymorphism was present in 21 of the 166 patients (12.6%) and in 24 of the 166 donors (14.4%). interestingly, we found a signifi cantly reduced incidence of bk virus-induced hc in patients with the asp299gly polymorphism (0% vs. 22.8%, p = 0.009). in addition, we observed a significantly reduced incidence of bk virus-induced hc in patients who were transplanted from a donor with this specifi c polymorphism (4.2% vs. 22.5%; p = 0.05). in ten of the donor-patient pairs the polymorphism was present in both individuals and no hc was observed. the occurrence of the tlr4 asp299gly polymorphism, in either recipients or donors, had no signifi cant impact on acute and chromic gvhd, relapse rate, bacterial and fungal infectious complications, transplant related mortality, and overall survival. in conclusion, asp299gly polymorphism of the tlr4 gene in the recipient and/or the donor is associated with a signifi cant decrease of bk virus-induced hc after hsct in childhood. this study provides the fi rst evidence that the innate immune system through tlr4 signaling pathway seems to play an important role in the pathogenesis of bk virus-associated hc after hsct. design and methods: one hundred-seventeen patients, median age 52 (20-67) years, with various haematological malignancies transplanted between 1999 and 2007 entered the study. eighty-seven recipients negative for hbv surface antigen (hbsag), anti-hepatitis b core antigen antibodies (anti-hbc) and hbv-dna with hbsag negative donors were defi ned as at no risk of hbv reactivation whereas all the remaining 30 patients were defi ned as at risk. in accordance with the italian national guidelines for immunocompromised hosts, patients at risk transplanted after 2005 received lamivudine to prevent hbv reactivation from conditioning up to 12-18 months after discontinuation of all immunosuppressive drugs whereas before 2005 no prophylaxis was given. results: patients at no risk did not experience hbv reactivation/ hepatitis. among patients at risk, hbsag negative recipients from hbsag positive donors (3/3), hbsag positive recipients from negative donors (2/2) and 11/25 anti-hbc positive were treated with lamivudine. none developed hepatitis b after a median follow-up of 33 months (7-55). hepatitis developed in 3 anti-hbc positive untreated patients conditioned with a reduced intensity regimen up to 19 months after discontinuation of immunosuppression and in none of those on prophylaxis. conclusions: we observed: no risk of hepatitis b in recipients serologically negative for hbv, transplanted from hbsag neg donors; effi cacy of lamivudine in controlling hbv reactivation in both hbsag positive recipients and negative recipients from s38 hbsag positive donors; a signifi cant risk of hbv reactivation in hbsag negative/antihbc positive recipients and effi cacy of prophylactic lamivudine in this setting and the importance of prolonged prophylaxis even after the discontinuation of immunosuppressive drugs. republic -a single-centre experience p. hubacek (1,3) , d. boutolleau (2, 4) , c. deback (2, 4) , p. keslova (3) despite the improvement of infection monitoring and antiviral treatments, cytomegalovirus (cmv) infections remain an important cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant recipients (allohsct). aim was to investigate cmv resistance in paediatric and adult allohsct recipients and to clarify the cmv load in whole peripheral blood and target tissue in situation of cmv disease. between i/2002 and xii/2009, we tested 17504 whole blood samples from 427 adults (median 22/patient) and 211 children (median 32/patient) after allohsct. additional 1348 biological non blood samples were tested too.samples were tested for cmv and albumin gene quantity by rq-pcr and results were normalized to 100000 human genome equivalents. ganciclovir as fi rst-line therapy was initiated when cmv load exceeded 1000 normalized viral copies (nvcs) and switched to foscarnet or cidofovir in case of none response.clinical resistance was suspected after 2 weeks of unsuccessful well-conducted treatment.resistance was studied using ul97 and ul54 gene sequencing.in the patients deceased in consequence of cmv infection,dna quantity in the tissue was tested too. virostatic treatment was started in 142 adults (33%) and 63 (30%) children.clinical resistance was observed in 58 adults and 25 children.known genotypic cmv resistance was proved in 11 of them (5.4% of treated) detecting mutation c592g, a591v, a594v, l595s, l595f, g598s and del597-599 in ul97 and del981-982, n408k, v715m, p522s in ul54). natural polymorphisms and unknown phenotype changing were also detected in both genes. despite the virostatic treatment, symptomatic cmv disease was observed in 15 adults and 10 children. ten patients deceased in consequence of cmv infection,mainly combined with gvhd. typical pathological signs of cmv infection and antigen detection in the tissue were found in 1 patient only.despite this median of cmv quantity in mainly affected lung tissue was 9534 nvcs (range 1029-384456) while median in the whole blood was only 1065 nvcs (range 183-89072). quantifying of cmv dna is very useful in the treatment of cmv infection. unfortunately our observation suggests the limits in case of cmv disease. genotypic evidence of cmv resistance proved to be very useful in the improvement of therapeutic management of patients. further studies are required to ascertain the true nature of the novel mutations described within ul97 and ul54 same as compartmentation of cmv infection. supported by mz0fnm2005, mstm0021620813. cmv reactivation often occurs in patients after allogeneic hsct. recently, pdgfr-alpha was suggested as the cellular receptor for human cmv (soroceanu et al., nature, 2008) . pdgfr-alpha binding and activation of a downstream pi3kinase signaling pathway was essential for cmv propagation. many tkis, such as imatinib, effi ciently inhibit pdgfr-alpha at concentrations readily achievable in patients. the aim of this study was to retrospectively assess a possible link between cmv reactivation and tki therapy. . also, at 2 years, the relapse incidence was not signifi cantly associated with the type of ric regimen: 37±3%, 34±2% in the tbi-based ric and chemobased ric groups respectively (p = 0.17). finally, nrm was also comparable between both groups (21±3% and 20±2% in the tbi-based ric and chemo-based ric groups respectively; p = 0.60). despite its retrospective nature, results from this large study suggest that ric allo-sct is a valid option for aml patients not eligible for standard allo-sct. the overall outcomes (lfs, nrm and relapse) appear not to be signifi cantly different between aml patients in cr1 receiving a low dose tbi-based ric allo-sct versus those receiving a chemotherapy-based ric allo-sct using an hla-identical sibling donor. patients with all who relapse after allogeneic hct have a grim prognosis and the optimal salvage therapy remains unknown. aim of this retrospective analysis was to report the outcome of relapsed all after allogeneic hct, to identify factors which have a prognostic signifi cance and to evaluate whether any relapse treatment strategy was associated with better outcome. for this study the ebmt database was searched for: 1) adult patients with all aged >18 y at hct; 2) transplanted in remission; 3) with hematological relapse after an allogeneic hct; 4) hla identical or mud hct performed from 1995 to 2006; 5) suffi cient med-c data. 465 adult pts (median age 32 y, 18-66) met these eligibility criteria. diagnosis was 76% b-lineage all, 21% t-all, 33% ph + all and previous allograft (68% were hla identical and 32% mud) was performed in remission (cr1 65%, cr2 32%, cr3 3%). the median interval from transplant to relapse was 7 months (0.8-52). post-relapse interventions were: no further treatment in 13% pts, 43% pts received chemotherapy at a median of 5 d after relapse , 23% pts received dli at a median of 45 d post-relapse (4-344) and 20% pts underwent a second transplant at a median of 71d after relapse (7-286). 61% of ph + all pts in addition received a tki inhibitor. overall, 40% pts reached a subsequent cr. after a median follow up of 56 months (3-141), the estimated 1-, 2-and 3-year overall survival (os) was 32%, 19% and 10%. the table below shows the statistically signifi cant parameters associated with 2-year os. in the multivariate analysis, os was associated with disease status at hct (cr2 + vs. cr1 p = 0.005, hr: 0.57), time to relapse after hct (>vs< median 7 months, p<0.0001, hr: 2.27 ) and number of peripheral blood blasts at relapse (>vs<10%, p<0.0001, hr:0.49), but it was not signifi cantly infl uenced by cytogenetics (ph + vs. other), donor type (sibling vs. unrelated), stem cell source (bm vs. pb), type of conditioning (tbi vs. chemo) or type of treatment for relapse (dli or hct-based therapy vs. therapy without cell infusion). this study highlights the extremely poor outcome of all pts who relapse after allogeneic hct. better outcomes were found in patients transplanted in cr1, with late relapse after hct (>7 months) and lower tumor burden at relapse (<10% of pb blasts). these factors should be taken into consideration when discussing further therapeutic options for these patients. introduction: prognosis and survival in aml patients is associated primarily with cytogenetic abnormalities but also with molecular markers. the internal tandem duplication (itd) of the flt3 gene is one such marker associated with poor prognosis. we report here an analysis of the impact of allogeneic sct on clinical outcome of cytogenetically normal aml patients according to their flt3-itd status. patients and methods: flt3-itd mutation was analyzed by capillary electrophoresis after pcr amplifi cation of material frozen at the time of diagnosis from a total of 116/277 patients with normal karyotype entered between 1997 and 2008 into two osho studies (aml 96 and aml 2002). sct was performed after conditioning with cytoxan and 1200 cgy tbi followed by gvhd prophylaxis with cyclosporine and methotrexate. related and unrelated donor search was initiated as soon as possible and patients allocated to groups with or without a donor. a total of 46 patients were allocated to the donor group and 70 patients to the no donor group. results were analyzed as intention to treat. results: event free survival (efs) of all patients was 38% after 5 years. in patients (n = 46) allocated to receive hct from an allogeneic donor, efs was 44% compared to 33% in those allocated to the non-transplant treatment (n = 70). as previously described, detection of a flt3-itd mutation had a negative impact on efs at 5 years, which was 25% in flt3-itd positive and 46% in flt3-itd negative patients (p = 0.06). subsequently, efs was analyzed according to flt3 status and post-remission treatment. in the flt3-itd positive group, efs was inferior in patients without donor (19% at 5 years) compared to those with a donor (36% at 5 years). this difference was due to a decreased relapse incidence in the donor group (48%) compared to the no-donor group (80%). however, a difference in efs was also observed in flt3-itd negative patients in the donor (50% at 5 years) compared to the nodonor group (43% at 5 years). this difference was also associated with a reduced relapse incidence in the donor group (26%) versus the no-donor group (55%; p = 0.003). conclusion: in conclusion, allogeneic hct improves efs in aml patients with a normal karyotype by reducing relapse incidence irrespective of the flt3-itd mutation status. the median follow-up of living patients was 20 months (range, 1 to 53 months). at 3 years the overall survival (os) was 38% and progression-free survival (pfs) was 36%, while the incidence of non-relapse mortality (nrm) and relapse were 29% and 34%. we performed multivariate cox regression modelling for os and pfs while competitive event statistics were applied for nrm. in order to adjust for a potential selection bias the hct-specifi c comorbidity score, karnofsky performance status, delay between diagnosis and hct and age were forced into the multivariate model. we performed a complete case analysis based on 223 patients. the hazard ratios (hr) of a matched or a mismatched ud compared to a sibling donor were 1.1 (95% ci, 0.7 to 1.7) and 1.3 (95% ci, 0.6 to 2.0) for os and 1.3 (95% ci, 0.8 to 2.0) and 1.3 (95% ci, 0.8 to 2.1) for pfs. in interaction analyses we did not identify signifi cantly differing effects according to age and disease stage. of note, having a matched ud did not signifi cantly affect the risk of nrm (hr=0.8; 95% ci, 0.4 to 1.6). our results confi rm the current practice in high risk aml to deliver allogeneic hct from matched ud and sib. an updated analysis will be presented at the meeting. (14) ( this phase-iii randomized ebmt-intergroup trial studied the impact of a consolidating haematopoietic stem cell transplantation (autohsct) vs. wait and watch for patients with cll in binet stage a progressive, b or c, in cr, nodular pr or vgpr after fi rst or second line therapy. the primary objective was to show that autohsct increase the 5-year progression-free survival (pfs) by 30%. here we present a fi rst analysis based on 80% of expected follow-up forms. results: between november 2001 and july 2007, 223 patients were enrolled (sfgm-tc/fcllg n = 99, mrc n = 63, gcllsg n = 36, sakk n = 10, other ebmt centers n = 15). there were 74% males and 26% females. binet stages were progressive a 13%, b 67%, c 20%; 59% were in cr, and 41% in very good or nodular pr. of note, sfgm-tc/fcllg included only patients in cr. eighty three percent of the patients were enrolled in 1st, and 17% in 2nd line treatment. patients were randomized between group 1 (autohsct n = 112) and group 2 (observation n = 111) after an induction treatment which was left at the discretion of the investigators. median pfs was 26.6 months (18.3-35) in the observation group and 50.1 months (40-60.1) in the autohsct group; the 5-year pfs was 29% and 42%, respectively (p<0.001). accordingly, the 5-year relapse incidence was 54% vs. 70%; p<0.001. the cox modeling for randomization arm, binet stage, disease status, line of treatment, contributing group (country), and the interaction between randomization arm and contributing group confi rmed that autohsct signifi cantly improved pfs (hr 0.45 [0.30-0.66] p<0.001). the benefi cial effect of autohsct was stable over all contributing groups. at 5 years, the probability of os was 83% and 82% for autohsct and observation, respectively; p = 0.81. signifi cant differences in terms of non-relapse death were not observed. in conclusion, in patients with cll in fi rst or second remission, consolidating autohsct reduces the risk of progression (pfs) by more than 50%, but has no effect on overall survival. further analyses on variables affecting the outcome are underway and results from a quality of life study on both groups are awaited. improved long-term outcome after highly purifi ed peripheral blood cd34 + cell transplantation from here, we report about the long-term follow-up results of a prospective phase ii study in patients after transplantation of highly purifi ed peripheral blood cd34 + stem cells from hla-identical sibling donors for cml in 1. cp. a total of 58 pts with a median pretransplant ebmt risk score of 2 (range 1-4) have been included in this study. all patients received a myeloablative conditioning regimen with tbi, cyclophosphamid with/or without thiotepa and atg, but without any prophylactic immunosuppression post transplantation. one patient received an unmanipulated graft due to poor cd34 + donor cell mobilization, while two patients were successfully retransplanted with an unmanipulated graft from the primary donor after secondary graft failure. of the 58 pts, 56 were eligible for the application of dli, but 6 pts did not receive dli due to sustained molecular remission and complete chimerism. thirty-two pts (64%) received dli because of increasing bcr-abl transcript levels or hematologic relapse, and 18 pts (36%) as programmed t-cell add-back. the median starting dose was 0.33 (0.01 -10) x 10 6 cd3 + cells per kg with a median maximum dose 3.3 (0.17 -100) × 10 6 cd3 + cells per kg. dli alone induced a lasting reduction of median bcr-abl transcript levels (bcr-abl/gapdh ratio) of more than 3 log10 and the estimate of being in a complete molecular remission at 8 years is 76% ± 5%. 12 pts. (24%) did not respond to dli alone, but 8 of these pts. attained a cytogenetic and molecular response by imatinib (n = 9), dasatinib (n = 1) and/or interferon treatment. four patients were retransplanted with an alternative donor. the cumulative risk of grades ii-iv acute gvhd is 15% ± 5% for all study pts, and the risk of chronic gvhd is 25% ± 6%, respectively. after a median follow-up period of 88 months (range 6 -125) for all patients, the cumulative 10-year survival estimate is 91,3% ± 4%. including all therapies for molecular relapse after transplant, 50 of the 58 included patients (86%) were in molecular remission at the last time point of observation. causes of death (n = 5) were disease progression, secondary malignancy, liver failure, septicemia, and systemic capillary leak syndrome in one patient each. in conclusion, the concept of highly purifi ed pb cd34 + cell transplantation in conjunction with adoptive dli is associated with a particularly low risk of non-relapse mortality and allows induction of lasting molecular disease control in the majority of 1. cp cml. allogeneic stem cell transplantation (asct) after reducedintensity conditioning (ric) has become a reasonable treatment option for patients with advanced myelofi brosis. the role of characteristic molecular genetic abnormalities such as jak2v617f on outcome of asct is not yet elucidated. in 139 out of 162 myelofi brosis patients with known jak2v617f mutation status who received asct after ric the impact of jak2 genotype, jak2v617f allele burden and clearance of mutation after asct was evaluated. a non-signifi cant higher treatment-related mortality (31% vs. 19%, p = 0.1) and relapse incidence (30% vs. 21%, p = 0.2) was noted in patients harbouring jak2 wild-type, resulting in a decreased overall survival in a multivariate analysis (hr 2.23, p = 0.007). no signifi cant infl uence on outcome was noted for the mutated allele burden analyzed either as continous variable or after dividing in quartiles. achievement of jak2v617f negativity after asct was signifi cantly associated with a decreased incidence of relapse (hr 0.22, p = 0.04). in a landmark analysis, patients who cleared jak2 mutation level in peripheral blood 6 months after asct had a signifi cant lower risk of relapse (5% vs. 35%, p = 0.03). we conclude that jak2v617f mutated status but not allele frequency resulted in an improved survival and rapid clearance after allografting reduced the risk of relapse. early autologous stem cell transplantation in poor-risk chronic lymphocytic leukaemia. long-term results from the gcllsg cll3 trial focusing on incidence and type of secondary malignancies f. mcclanahan (1) (2), p. dreger (1) ( introduction: as previously reported, early autosct as conducted in the cll3 protocol is a feasible therapy option for younger patients (pts). purpose of the present analysis was to study the impact of fish karyotype and ighv mutational status on long-term pfs and os and the incidence and type of secondary malignancies occurring in this trial. trial design and patients: the design of the protocol has been extensively described previously. from 1996 to 2002, 216 pts were registered. as 47 cases had to be excluded due to screening failure (n = 21), withdrawn consent (n = 19) or other reasons (n = 7), 169 pts were eligible for the current analysis. male to female ratio was 5:1 and the median age at diagnosis was 51 yrs (range 27-60). results: sct was performed in 131 pts (78%) at a median time of 17 months (range 4-159) after initial diagnosis, whereas 38 pts did not proceed to sct (mobilization failure n = 14, disease progression n = 4, early death n = 3, pts preference n = 6, unknown reasons n = 11). at a median follow-up of 99 months (range 4-137), median os of all 169 pts was 10.5 yrs (10.5 yrs after sct, 6.1 yrs without sct, hr 0.26, 95% ci 0.13-0.54, p<.0001). median pfs was 6.3 yrs (6.8 yrs with sct and 4.8 yrs without, hr 0.39, 95% ci 0.23-0.67, p = 0.0007). diagnostic samples for assessment of ighv mutational status were available for 143 (85%). an unfavorable ighv rearrangement was present in 104 pts (73%), and was associated with signifi cantly worse pfs (p = .0001) and os (p = .017). fish was possible in 160 pts. whereas pfs (p <.0001) and os (p <.0001) were considerably reduced in pts with del 17p-, there were no signifi cant differences between the other subsets. altogether, 20 secondary malignancies were observed, with 6 cases of t-mds/ t-aml, translating into a 10-year incidence of 20% (95% ci 11-30%), with no signifi cant difference among pts treated with and without sct (p = 0.68). however, all cases of t-mds/ t-aml occurred after sct, yielding a 10-year incidence rate of 9% (1-18%). overall survival after secondary neoplasms was 22 months , with no difference between t-mds/aml and other malignancies. conclusions: unmutated ighv remains an adverse prognostic factor. while del 17p-is associated with a very poor outcome, autosct may eliminate the unfavorable impact of del 11q-seen with conventional therapy. secondary neoplasms are a serious problem after early autosct, but do not appear to occur more frequently than reported after sct for other diseases. reduced intensity conditioning (ric) has reduced non-relapse mortality (nrm) associated with allogeneic hsct in hodgkin lymphoma and relapse is now the commonest cause for treatment failure. however, there is little published evidence to guide management of relapse. we performed 81 ric allografts for hl (44 matched related, 37 unrelated donor) incorporating t cell depletion with alemtuzumab. 35 (43%) were primary or salvage-refractory, 47 (58%) had failed a prior autograft, and the median number of prior treatment lines was 5. patients were monitored by pet-ct and relapse defi ned by recurrence or progression of fdg-avid lesions in sites of prior disease. if fdg-avidity occurred only in new sites, relapse was confi rmed by biopsy if accessible (n = 7), otherwise an interval scan was performed at 6-8 weeks. following cyclosporine withdrawal and in the absence of gvhd, patients received dose-escalating dli for mixed chimerism (mc) or progression. the 3 year ci of nrm was 17% and of relapse was 45%. 44 patients received a total of 87 dlis (median 2, range 1-5). treatment for mc alone (n = 22) resulted in conversion to full donor in 15/20 evaluable patients, continuing improvement in 2, and 3 remained stable. only 1 of these relapsed (ci 7% at 3 years from initial dli), receiving further dlis following chemotherapy. relapse was treated in 23 patients with dli either alone (n = 13) or following debulking chemotherapy (n = 10). median maximal doses were 1 × 10 7 in unrelated and 1 × 10 8 cd3 + t cells/kg in related donor transplants. complete responses were seen in 12 and partial responses in 5 of 22 evaluable patients (response rate 77%), and did not differ signifi cantly according to donor source (10/13 mrd vs. 7/9 ud). the majority of responders developed gvhd (5 gd iii-iv acute, 4 extensive chronic). 7 cr are maintained at a median of 4.6 years from last dli (5 without prior chemotherapy), 3 died of gvhd-related complications, and 2 progressed (at 1.6 and 2.3 years). 3/5 with pr progressed. the projected 3 year os and pfs from relapse are 64% and 54% in this group. the os and current pfs for the entire cohort of 81 patients are 61% and 53% at 4 years. in conclusion, the data demonstrate favourable long term survival in this heavily pretreated hl cohort, a strikingly low relapse incidence in those receiving dli for mc, and the ability of dli guided by pet-ct to induce high response rates and durable salvage in the setting of relapse post t cell depleted transplantation. a recent update of 3 consecutive prospective trials with high-dose therapy and autograft, without or with rituximab, as primary treatment for advanced-stage follicular lymphoma shows a sizeable group of patients surviving in continuous complete remission c. tarella, m. ladetto, f. benedetti, u. vitolo, a. pulsoni, c. patti, v. callea, a. rambaldi, a. piccin, l. devizzi, m. musso, e. iannitto, p. spedini, a.m. liberati, f. ciceri, a. gallamini, f. rodeghiero, g. gini, a. de crescenzo, f. di raimondo, a. levis, t. chisesi, t. perrone, d. rota scalabrini, g. rossi, a.m. carella, g. parvis, i. majolino, r. passera, m. ruella, a. pileri, a.m ) or in the proportion of patients with systemic symptoms or with elevated serum lactate dehydrogenase. the median time from the diagnosis to asct was also comparable as well as the number of previous treatment lines and the use of rituximab before asct (49% vs. 53%). the elderly patients were less often in fi rst complete remission at the time of asct (42% vs. 53%, p = 0.003). the most common conditioning regimen used was beam (62% in the elderly vs. 58%) followed by cyclophosphamide plus total body irradiation (14% vs. 14%). non-relapse mortality (nrm) was comparable at 3 months (3.7% in the elderly vs. 2.1% in younger patients) and at 1 year (4.2% vs. 2.9%) but was higher in the elderly patients at 5 years (8.6% vs. 3.2%, p = 0.04). the most common causes of nrm were infections (53% vs. 44%). with a median follow-up of 21 months for the elderly patients and 25 months for the younger patients, the risk of relapse was 57% and 54% (n.s), respectively. progression-free survival was also comparable (38% vs. 40% at 5 years) without any plateau. overall survival was worse in elderly patients (60% vs. 69% at 5 years, p = 0.01). autologous stem cell transplantation is feasible in selected elderly patients with mcl. early nrm is low and comparable to younger patients. risk of relapse and progression-free survival are also comparable but overall survival is worse in the elderly patients. continuously increasing relapse risk indicates the need for improvements in pre-and posttransplant strategies in order to improve long-term outcome in this lymphoma type. phase ii study of radioimmunotherapy with yttrium(1) allogeneic hematopoietic cell transplantation (hct) using reduced intensity conditioning (ric) offers a potential curative therapy to patients with advanced indolent nhl. combined use of radioimmunotherapy (rit) with ric may increase anti-lymphoma activity of ric while avoiding additional toxicities. forty patients have been enrolled in a multicenter phase ii study of allogeneic hct using rit with yttrium-90-ibritumomab tiuxetan (y90-cd20, zevalin) with 0.4 mci (15 mbq)/kg on day -14 combined with ric using fl udarabine (30 mg/m² day -4 to -2) and 2 gy tbi (day 0) followed by allogeneic hct from matched related or unrelated donors. gvhd-prophylaxis consisted of cyclosporine and mycophenolate mofetil. diagnoses were follicular lymphoma (fl, n = 17), chronic lymphocytic leukemia (cll, n = 13), mantle cell lymphoma (mcl, n = 8), marginal zone lymphoma (n = 1) and immunocytoma (n = 1). median age was 56 (range, 35-68) years. pbsc grafts were either from matched related (n = 13) or matched unrelated donors (n = 27). all patients were "high risk" with refractory disease or relapse after preceding autologous hct. engraftment was rapid and sustained with no graft rejections. median time to >500 granulocytes/μl was 13 (range, 0-69) days, and to >20000 platelets/μl 4 (range, 0-69) days. in 13 patients platelets were never <20000/μl and in 6 patients granulocytes never <500/μl, illustrating the nonmyeloablative intensity of the conditioning regimen. trm in the fi rst 100 days was 10% (n = 4) and overall 40% (n = 16). no additional toxicity due to rit compared to our previous experience with the same ric as single modality was observed. incidence of grade ii-iv gvhd was 45% (ii = 3, iii = 12, iv = 3). to date, chronic gvhd occurred in 19 patients (48%, limited = 8, extensive = 11). deaths occurred due to infections = 8, gvhd = 7, relapse = 2 and cerebral bleeding = 1. 22/40 (55%) of all patients are alive with a median follow up of 672 (range, 251-1086) days, resulting in a kaplan-meier estimate 2 year survival of 51% for all, and 67% in fl, 49% in cll and 38% for mcl patients. risk factors for decreased overall survival and trm in multivariate cox regression analysis were non-fl histology (p = 0.032 and p = 0.024) and agvhd >grade 3 (p = 0.001). in conclusion, a combination of rit with ric is feasible with no additional toxicity due to rit and with stable engraftment in all patients. disease response and overall survival seems promising even in this elderly and heavily pretreated cohort of patients. allogeneic stem cell transplantation after autologous haematopoietic stem cell transplantation relapse in aggressive lymphoma patients: fi nal report of a retrospective gitmo study l. rigacci, a. bosi, b. puccini, p. corradini, l. castagna, n. cascavilla, g. milone, a. bacigalupo, r. scimé, g. specchia, a. rambaldi, p. leoni, f. ciceri, a. levis, s. guidi, b. bruno, r. oneto, r. fanin on behalf of gitmo autologous hematopoietic stem cell transplantation (ahsct) has been shown to be an effective therapy for patients (pts) with aggressive lymphoma. pts who relapse after an ahsct have a very poor prognosis. allogenic hematopoietic stem cell trasplantation (allohsct) has shown to be effective in the rescue of indolent lymphoma pts relapsed after conventional therapies. according to this data we have retrospectively analized all pts with diagnosis of aggressive lymphoma in the gitmo data-base who have performed an allohsct after an ahsct relapse. from 1995 to 2008, 181 pts were selected from the gitmo data-base. one of the principal objective in this fi nal report was the completeness of the data. data missing were: acute gvh was evaluable in 90% of pts, chronic gvh in 70% of pts, response after allogeneic transplantation in 66%, condition at the moment of allotransplant in 94%, therapy pre-allo-hsct in 77% of pts. the other characteristics were evaluable in all patients. one-hundred and one were male (56%), 160 presented a diagnosis of dlbcl. the stem cell donor was related in 115 pts, the stem cell source was peripheral blood in 151 pts and bone marrow in 30. the conditioning regimen was conventional in 55 pts and reduced intensity (ric) in 126 pts. the median time between ahsct and allo-hsct was 12 months. ninethy-seven pts (54%) obtained at least a partial remission before allo-hsct, 74 (41%) were treated with active disease and in 10 cases the data was not available. after allo 78 pts (43%) obtained a cr and 10 a pr with an orr of 49%, 69 pts (38%) showed a rapid progression of disease. ninethy-seven pts died, 46 due to disease and 51 to treatment related mortality (trm). acute graft versus host disease was recorded in 61 pts and a chronic one in 42 pts. with a median follow-up period of 16 months (4-28) from allo the os was 56% and after a median period of 12 months (6-17) from allo the pfs was 49%. in multivariate analysis the only factors which affected pfs were the status at allo and a cr after allo transplant. this conclusive retrospective analysis confi rms that the only one parameter affecting either os or pfs was the response status at the moment of all-hsct and does not confi rm that ric could permit to obtain better results in aggressive lymphoma. probably a myeloablative conditioning should be reconsidered in pts with aggressive disease because of the slow-acting graft versus lymphoma effect is overriden by the rapid growth of the tumor. patients (5%), ii in 100 (28%), iii in 80 (23%), and iv in 155 (44%). forty-nine percent of the patients had b symptoms, 7% bulky disease, and 54% extranodal involvement. karnofsky performance status < 80 was reported in 72 patients. treatment following relapse consisted on conventional chemotherapy and/ or radiotherapy in 294 patients (64%), second asct in 35 (8%), and allogeneic stem cell transplantation in 133 (29%). a significant higher proportion of patients, age < 50 years, received a second transplantation in comparison to older patients (39% vs. 16%, p =.006). after a median follow-up of 49 months (range 1-150), the os from relapse after asct was of 55% at 2 years and 32% at 5 years. in multivariate analysis, independent risk factors for os were early relapse (< 6 months), stage iv, bulky disease, poor performance status, and age > 50 at relapse. in conclusion, according to the ebmt database, most hl patients relapsing after asct were treated with chemo-radiotherapy approaches and some of them with a second transplantation. those patients in a good performance status presenting with a localized late relapse seem to be the ones showing the best prognosis. hematopoietic cell transplantation (hct) is the therapy of choice for a variety of malignancies. hct provides disease benefi t through both the high-dose conditioning regimen and an allogeneic graft versus tumor effect (gvt). however graft-versus-host disease (gvhd) still remains a major obstacle. it has been proposed that host conditioning may not only be crucial in the activation of alloreactive t cells but also determine acute gvhd organ manifestation. we wanted to investigate how the host-conditioning regimen affects the host target tissues in terms of infl ammatory cytokines and their role in donor t cell recruitment. utilizing a non invasive bioluminescence imaging (bli), and fl ow cytometry in a murine allogeneic hematopoietic cell transplantation (allo-hct) model, we compared lethally irradiated (8gy) vs. non-irradiated balb/c wild type, and balb/c rag-/-cgc-/-(dko) (h-2d) mice that received allogeneic luciferase + fvb/n t cells (h-2q). we observed that the proliferation (bli, cfse), acquisition of activation markers (cd25, cd44, cd69) and homing receptors (a4b7, aeb7, p-selectin ligand, e-selectin ligand) by alloreactive t cells occurred independently whether allogeneic recipients were conditioned or not conditioned before allo-hct. as t cell recruitment may have occurred as a result of alterations of the milieu infl ammatory cytokines in gvhd target tissues, we compared the cytokine profi le in conditioned vs. non-conditioned hosts. at days 3 and 6 after allo-hct, host tissues were analyzed for a th1/th2/th17a cytokines from the target tissues; liver, large bowel, small bowel, peripheral blood and a non target tissue: kidney. at day 3, high levels of inf-g, tnf and il-6 were detected in the balb/c wt conditioned compared to the non-conditioned host in all target tissues and most markedly in blood and the large bowel. more importantly both the balb/c rag-/-cgc-/-(dko) conditioned and non-conditioned host displayed very high levels of the infl ammatory cytokines, with balb/c wt and balb/c rag-/-cgc-/-(dko) conditioned hosts displaying higher levels. similar results with a reduced expression of the cytokines were observed at day 6 indicating that the cytokine storm peak was maybe at day 3. in summary host conditioning is not a requirement for alloreactive t cell activation rather induced infl ammatory cytokines such as tnf and inf-g are the determinant factors for effector t cell recruitment to gvhd target tissues. acute graft-versus-host disease (gvhd) after gender mismatched stem cell transplantation may be caused by female donor t cells recognizing hy minor histocompatibility antigens expressed by male recipients. hy-specifi c cd8 positive cytotoxic t lymphocytes (ctl) have been detected in peripheral blood samples collected at the onset of acute gvhd. however, it is still unclear whether these ctl reach acute gvhdaffected tissues. we validated the sensitivity and specifi city of fl uorescently labeled multimeric hla-a2 molecules containing hy peptide, i.e. hla-a2/hy dextramers, to stain cryosections prepared from skin biopsies obtained from healthy male hla-a2 positive volunteer donors. these skin explant tissues were cultured with female hy-specifi c ctl or control ha-1 specifi c ctl before cryopreservation. unlike conventional hla-a2/hy tetramers, hla-a2/hy dextramers stained hy-specifi c ctl, but not ha-1 specifi c ctl, when applied to already cryopreserved skin explant tissue. control staining of serial sections with hla-a2 dextramers containing infl uenza peptide showed no positive signal. next, the presence of hla-a2/hy dextramer positive cells was analyzed in cryopreserved skin biopsies derived from 7 male hla-a2 positive pediatric patients who developed acute gvhd of the skin after receiving a hematopoietic stem cell graft from a hla matched unrelated donor. while a few cd8 positive hla-a2/hy dextramer negative cells were detected in the skin of 4 boys who received male hematopoietic stem cells, significantly higher numbers of cd8 positive cells were detected in the skin of 3 boys who received a female graft; 50-75% of these cd8 positive cells were specifi c for hy. skin-infi ltrating hyspecifi c t cells were visualized as early as 2 weeks after sct when total peripheral blood lymphocyte counts were still low. our results underline the usefulness of this multimeric staining reagent for in situ characterization of donor-derived t cells that infi ltrate acute gvhd affected tissues. (1) granulocyte-colony stimulating factor (g-csf) is increasingly described as an immuno-modulatory agent for a diverse range of diseases and although the cytokine is usually associated with the regulation of immune responses, clear evidence exists that it can also exacerbate pathology in some settings. the clinical shift toward utilizing g-csf mobilized stem cell grafts has resulted in enhanced hematopoietic reconstitution, reduced relapse rates in advanced disease, similar levels of acute gvhd but a striking increase in chronic gvhd. the mechanisms by which stem cell transplantation (sct) promotes chronic gvhd are unclear. comparison of cytokine expression following tcr activation of splenocytes from naïve and g-csf treated b6 or balb/c donors (low and high responders respectively) showed little effect of g-csf on th1 (ifn-gamma and tnf) or th2 (il-4, il-5 and il-10) cytokine production. in contrast, il-17 production was dramatically enhanced in response to g-csf treatment in both strains but was highest in balb/c donors. the amplifi cation of il-17 production by g-csf occurred in both cd4 and cd8 conventional t cells and by using relevant knock-out mice or blocking reagents we demonstrated that this was independent of il-6, tgf-beta or il-23 signalling. however, the induction of il-17 by g-csf was completely lost in the absence of il-21 signalling. g-csf induced the production of il-21 in cd4 t cells, and this occurred independently of il-17 production itself. we next utilized multiple models of gvhd using g-csf mobilized b6 or balb/c wild-type or il-17 deficient donors, in both mhc matched and mismatched transplant settings. surprisingly, il-17 was critical for the induction of sclerodermatous chronic gvhd occurring late after transplant using either donor strain. importantly, il-17 controlled the dramatic sequestration of macrophages into skin that coincided with the fi brogenic response. this study provides a logical explanation for the propensity of allogeneic stem cell transplantation to invoke sclerodermatous gvhd and suggests a therapeutic strategy for intervention. non-hla gene polymorphisms contribute to the immune response leading to graft-versus-host disease (gvhd). we applied a systematic approach using microsattelite (ms) marker typing for a large number of immune response genes on pooled dna of japanese donors and recipients of haematopoietic stem cell transplants (hsct) to identify recipient and donor risk loci for gvhd. ms, due to their multiple alleles, are more informative than single nucleotide polymorphisms (snp). we selected 4,231 ms markers, tagging 3,093 target genes (representing the 'immunogenome') at close proximity (<100kb). we selected 922 unrelated hsct donor/recipient pairs from the japan marrow donor programme (jmdp) registry, based on clinical homogeneity (acute leukaemia, age 4-40 years, myeloablative conditioning, bone marrow source). 42.61% of pairs had a 10/10 hla match. the population was split into discovery and confi rmation cohorts with 460/462 pairs each. eight dna pools, four for each of the two independent screening steps were created using a highly accurate dna pooling method. while 4,321 ms were typed on the four pools of the 1st screening step, only markers positive here were typed on the 2nd screening pools. fisher's exact test for 2x2 (each ms allele) and 2xm chisquare tests were performed, comparing allele frequencies of recipients with gvhd grade 0-1 with gvhd grade 2-4 (donors accordingly). markers positive after both independent screening steps (p-value <0.05, same associated allele, consistent odd's ratio (or)) were genotyped for confi rmation on individual samples of all 922 pairs. the independent, 2-step pooled dna screening process has effectively reduced false-positive associations. in the fi nal analysis, 39 (recipient) and 58 (donor) ms loci remain associated with risk or protection from gvhd. (1), g. afram (2) we have evaluated the impact of nih score system in the outcome of 820 patients and studied additional prognostic factors among patients who develop cgvhd at 3 european centers. furthermore, we tried to determine the prognostic impact of the different organs involved in order to defi ne which ones must always be evaluated and which ones could be avoided in a routine examination. patients' characteristics: patients transplanted from 2000 to 2006 at 3 different institutions were analyzed; 77% received stem cells from a related donor and 23% from alternative donors. results: median follow up was 1087 days (range: 7 to 2944). 53% and 31% developed overall and extensive cgvhd, respectively. according to nih classifi cation 29%, 24% and 17% of the patients were categorized as mild, moderate and severe cgvhd, respectively. type of onset was de novo in 22%, quiescent in 27% and progressive in 12%. cumulative incidence of delayed acute gvhd was 10% while the respective value for overlapping syndrome was 17%. among patients with cgvhd, the extent of cgvhd infl uenced on survival (55% vs. 71% for extensive vs. limited cgvhd, p<0.001), as well as the development of overlapping syndrome (os 57% vs. 71% for patients who did or did not have overlapping syndrome, p = 0.01), of severe cgvhd (68%, 68% vs. 48% for patients with mild, moderate vs. severe cgvhd, p<0.001), and the type of onset (70% vs. 57% vs. 51% for de novo, quiescent and progressive cgvhd, p<0.001), while delayed acute gvhd did not infl uence on outcome. in multivariate analysis patients with severe cgvhd displayed a dismal outcome [hr = 0.31, 95%ci (0.17-0.58); p = 0,001]. the type of onset allowed to identify prognostic subgroups even among patients with severe cgvhd, so that the combination of both variables discriminate different patients subpopulations in terms of outcome [hr = 0.18, 95%ci (0.06-0.51); p = 0,001]. among targeted organs performance score at the time of cgvhd had the most signifi cant effect on survival [hr = 4.73, 95%ci (2.39-9.36), p<0.001]. also liver, gut and lung involvement adversely infl uenced on survival while eyes or mouth involvement had no infl uence on outcome. conclusion: nih scoring system plus cgvhd type of onset allows to defi ne patients subgroups in terms of survival. patients with overlapping syndrome have a poor outcome. among targeted organs, performance score was an independent prognostic factor while eyes and mouth involvement did not infl uence on outcome. chronic gvhd (cgvhd) remains the leading cause for late morbidity and mortality after allogeneic hematopoietic stem cell transplantation (hsct). the consensus conference summarized the current evidence on treatment of cgvhd and developed guidance for clinical practice. the consensus process included hsct centers within germany, austria, and switzerland participating in four meetings accompanied by two surveys on treatment of cgvhd send to all centers within the german and austrian working party on bone marrow and blood stem cell transplantation and the basel transplant center (switzerland). evidence was graded into: i (based on randomized trials), ii (based on case controlled or well designed phase ii trials), iii-1 (several case series), iii-2 (one case series), iii-3 (case reports) according to all available publications. recommendations were based on effi cacy and safety profi le: a (should always be applied), b (should generally be applied), c-1 (may be applied 1st-line), c-2 (may be applied 2nd-line), c3 (may be applied advanced line), c-4 (may only be applied in special circumstances). for 1st line treatment of cgvhd the following agents have been proposed: prednisone a i, calcineurin inhibitors (cni) c-1 ii, mmf c-1 iii. for 2nd-line treatment the following treatment options have been proposed: prednisone b iii-1, cni c-1 iii-1, mtor inhibitors c-1 iii-1, mmf c-1 iii-1, photopheresis b ii. a pulse of steroids c-2 iii-2 and rituximab c-2 iii-1 should be generally applied after 2nd-line treatment but may be used earlier in special circumstances. advanced line treatment options being proposed are: mtx c-2 iii-1, hydroxychloroquine c-2 iii-2, clofazimine c-2 iii-2, pentostatine c-2 ii, thoracoabdominal irradiation c-2 iii-2, and imatinib c-2 iii-1. additional agents in advanced line treatment are retinoids c-3 iii-2, azathioprine c-3 iii-1, and thalidomide c-3 ii. other treatment options have been rated as experimental and may only be applied in clinical trials or special circumstances: alemtuzumab c-4 iii-3, etanercept c-4 iii-3, alefacept c-4 iii-3. the low level of evidence of the proposed treatment options resulting in a low level of recommendation indicates the urgent need for further evaluation in clinical trials. moreover, the lack of indicators for response to certain agents requires a "trial and error" approach in choosing a treatment option and clinical trials for evaluation of biomarker for response are urgently needed. recently, we reported an elevation of immature cd19 + cd21-b lymphocytes in patients with active cgvhd. here, we investigated b lymphocyte subsets in cgvhd cohorts with abnormal serum immunoglobulin levels. patients and methods: seventy-fi ve patients were enrolled into our study a median of 42 months after hct. they consisted of 3 groups: 25 with cgvhd and high serum igg (>1600 mg/dl), 22 with cgvhd and low igg (<700 mg/dl) and 28 patients with resolved cgvhd and normal igg (control group). severe cgvhd was present in 64% and 45% with more than 2 organs involved in 44% and 36% in the high igg and low igg group. signifi cantly more patients in the high igg group than in the low one had autoantibodies (72% vs. 18%, p = 0.02). peripheral blood cells were analyzed by multiparameter fl ow cytometry after staining for cd19, cd21, cd27, cd10, cd38, cd95, and igd. results: while the high igg group had equal cd19 + b cell numbers, the low igg group had signifi cantly diminished ones compared to the control (400 vs. 160 vs. 400 × 10 6 /l p<0.0001). numbers of cd10high most immature b cells in the high igg, low igg and control group were 26, 7, and 7 × 10 6 /l. immature cd19 + cd21-b cell proportions were 9.2%, 16.5%, and 7.5% in the high, low igg and control group (p = 0.01). in the high igg group transitional b cells were signifi cantly increased (28 vs. 14 × 10 6 /l, p = 0.02) compared to the control. in the high igg group naive and class-switched memory b cells were equal to the control whereas in the low igg group both naïve (13 vs. 35 × 10 6 /l p = 0.02), class-switched (16 vs. 22 × 10 6 /l, p = 0.03) and non-class-switched b cells (4 vs. 10 × 10 6 /l, p<0.0001) were signifi cantly lower compared to the control. cd10-cd21low cd95high b cell proportions were the same in the high igg and control group, while they were signifi cantly elevated (23.4% vs. 9.6% p<0.0001) in the low igg group. conclusions: cgvhd patients with hypergammaglobulinemia have normal b cell numbers with elevated immature and transitional b cells but no signifi cant impairment of b cell maturation. in contrast, patients with hypogammaglobulinemia have both a signifi cant b cell defi ciency and a distortion of b cell homeostasis in the circulation. our data indicate different pathogenetic mechanisms of cgvhd leading to different clinical presentations. preliminary results of a phase ii trial of montelukast for the treatment of bronchiolitis obliterans syndrome after hsct k. williams (1) bronchiolitis obliterans syndrome (bos) after allogeneic hsct is a serious manifestation of cgvhd. current treatments yield poor and transient responses. although the pathogenesis of bos after hsct is unknown, a similar disease, bos after lung transplant is associated with elevated leukotriene levels. we present preliminary results from an irb-approved prospective, open label, phase ii trial to test the effi cacy of montelukast, a leukotriene inhibitor, for the treatment of bos after hsct. bos diagnostic criteria included: fev1<75%, fev1/vc < 0.7 or air trapping on ct and rv>120% or rv/tlc>120% in the absence of infection and presence of another cgvhd manifestation. subjects had stable or declining fev1 on stable or decreasing immunosuppression. eleven patients have enrolled. one withdrew prior to medication and 9/10 patients have reached the primary endpoint (6 months) and have continued on study medication (10 mg montelukast po nightly). study participants ranged from 15-64 years, 7/11 female, with baseline fev1 from 33 to 71% predicted, and 3/11 patients required supplemental oxygen. fev1 increased 6-10% predicted in 3 patients, remained stable in 4, and declined less than 15% in 2. comparison of patient pre-study fev1 decline to on-study fev1 values was generated using the slope of fev1 volume vs. days post-transplant. the difference in pre-and primary endpoint slope revealed: 7/9 improvement and 2/9 decline. three patients reduced immunosuppression on study with complete cessation of tacrolimus in 1, cessation of steroids in 1, and decreased tacrolimus in 1 (including 2 with stable fev1 and 1 with increase in fev1); 1 patient had a steroid increase less than 1mg/kg/day. two patients had worsening of other cgvhd manifestations, including skin fl are that resolved with local therapy (1) and gastrointestinal cgvhd fl are that improved with increased steroids and local therapy (1) . notably, after 6 months, 1 patient demonstrated fev1 increase of 14% predicted (from baseline) on tacrolimus taper after steroid cessation and 1 patient no longer required oxygen supplementation for exercise and sleep. montelukast was well-tolerated with only one grade ii attributable adverse event (insomnia) during the six-month collection period. using nih consensus criteria, improvements were also noted in 4/4 with buccal mucosa cgvhd and in 2/5 with evidence of liver disease. these fi ndings suggest that montelukast is a promising therapy for bos after allogeneic hsct. oral session 11: paediatric issues 1 o256 objective: allogeneic hematopoietic stem cell transplantation (hsct) remains the main treatment option for children with advanced primary or secondary myelodysplastic syndrome (mds). relapse rate following hsct for advanced mds ranges between 20%-50%. this analysis was performed to asses the outcome of patients treated with a second hsct (hsct2) for disease recurrence after hsct1. patients and transplant: within studies ewog-mds 98 and 2006, 73 patients with advanced mds relapsed after fi rst hsct (hsct1); a second allograft was performed in 32 of these patients. for the 32 patients with hsct2, diagnosis prior to hsct1 was primary advanced mds (n = 18), therapy-related mds (n = 9) and secondary mds after bone marrow failure disorder (n = 5). preparative regimen of hsct1 consisted of busulfan, cyclophosphamide and melphalan in 22 / 32 patients. median time to relapse was 13.8 months (0.9-77.3) after hsct1, time from relapse to hsct2 3.7 months (0.4 -14.8), and median age at hsct2 13.0 years (5.4 -25.3). twelve patients were transplanted from a matched sibling donor, 16 from a matched unrelated and 4 from an alternative family donor. in 18 cases hsct2 was performed using the same donor than in hsct1. stem cell source was peripheral blood (n = 22) or bone marrow (n = 10). conditioning regimen and gvhd prophylaxis varied widely according to the centers preferences, a tbi-based regimen was given to 12/32 patients. outcome: median follow up was 3.0 years (0.3-5.4) after hsct2. all but 2 patients engrafted. acute gvhd grade ii -iv was seen in 10 patients, chronic gvhd in 8 of 22 patients at risk. transplant related mortality occurred in 9 patients; 4 of these died from gvhd, 1 from sudden cardiac arrest 6.5 years post hsct2. a second relapse was noted in 12 patients, all of whom succumbed to their disease. there was no signifi cant difference in relapse rate according to the choice of donor (identical or different donor than in hsct1) or the occurrence of acute/ chronic gvhd. for the whole cohort of 32 patients, the probability of event free survival at 5 years was 0.35 (0.17-0.53). conclusion: we conclude that hsct2 after mds relapse is feasible and may rescue a considerable proportion of patients. reduced-intensity conditioning for children with refractory cytopenia: results of the ewog-mds study b. strahm (1) , p. bader (2) objective: refractory cytopenia (rc) is the most common subtype of childhood myelodysplastic syndrome (mds). hematopoietic stem cell transplantation (hsct) in rc following a myeloablative preparative regimen has resulted in an eventfree survival of 80% with a very low probability of relapse and a probability of therapy related mortality of 15%. this analysis was performed to asses the outcome of children transplanted for rc following a reduced intensity conditioning. patients and transplant procedure: fifty-six patients (32 male/24 female) were diagnosed with rc at a median age of 11.2 years (1.8-17.9). none of the patients had an abnormal karyotype. the median time to hsct was 0.71 years (0.12 -9.1). patients were grafted from a matched sibling donor (msd) (n = 15), an alternative family donor (n = 1) or an unrelated donor (ud) (n = 40). ud were matched 10/10 or 9/10 based on hla-a, -b, -c, -drb1 and -dqb1 molecular typing. stem cell source was bone marrow (n = 48) or peripheral blood (n = 8). all patients were prepared with thiotepa (3 × 5 mg/kg) and fl udarabine (4 × 40 mg/m 2 ). prophylaxis for graft-versus-host-disease (gvhd) was csa ± mtx, ± mmf for msd, and csa, mtx and anti-thymocyte globuline for patients transplanted from an ud. results: two patients each experienced primary or secondary graft failure. fifty-four patients reached neutrophil engraftment at a median time of 24 days (11-105). platelet engraftment was demonstrated in 48 patients at a median time of 28 days (1 -201) ; in 2 patients an additional stem cell boost was necessary. out of the four patients with graft failure three patients are alive after second hsct. the incidence of acute gvhd grade ii-iv and grade iii-iv was 23% and 5%, respectively. ten of 52 (19%) patients at risk developed chronic gvhd which was extensive in 5. one patient died of from veno-occlusive-disease with multi-organ-failure. viral infections were the most prevalent complication. fifty-four patients are alive with a median follow-up of 2.0 (0.1 -7.0) years resulting in a probability of overall survival of 0.95 and 1.0 for patients transplanted from mud and msd, respectively. conclusion: in summary the conditioning regimen with thiotepa and fl udarabine offered an excellent survival for patients rc. chronic gvhd and viral infections were the main complications. long term follow is needed to assess the expected reduction in long term sequelae. allogeneic stem cell transplantation (sct) is indicated in approximately 20-30% of children with acute lymphoblastic leukemia (all). unrelated umbilical cord blood (ucb) is an established stem cell source for sct. we retrospectively analyzed 532 children with all in complete remission (cr)1 (n = 186), cr2 (n = 238) and cr3 or advanced disease (n = 108) who received ucbt as fi rst transplant. patients were transplanted in ebmt centers from 2000-2008. median age was 6.8 years (y) (29 patients less than 1 y). the most common immunophenotype was b-cell precursor all and 17 patients had biphenotypic all. of 186 patients transplanted in cr1, 45% had poor risk cytogenetics (t4;11 or t9;22). grafts consisted of one (n = 504) or two (n = 28) units; 62% had 0-1 hla mismatches with recipients while 38% had 2-3 mismatches (antigen level for hla-a and b, allelic level for drb1). median tnc cell dose at freezing and infusion was 5.9 × 10 7 /kg and 4 × 10 7 /kg, respectively. conditioning regimen was myeloablative in 96% of cases and tbi >6gy was used in 52%. other regimens included busulphan with cyclophosphamide ± thiotepa or melphalan. atg was added in 88% of cases and gvhd prophylaxis was csa ± steroids in 75%. median follow-up was 18.5 months (3-109). cumulative incidence (ci) of neutrophil recovery, acute gvhd and trm were 82 ± 2%, 27 ± 3% and 21 ± 3% respectively. in multivariate analysis tnc infused >4 × 10 7 /kg (p = 0.001) and remission status at ucbt (p = 0.01) were associated with improved neutrophil recovery. ci of 2y relapse was 37±3% (31% for cr1, 34% for cr2, 50% for advanced). disease status at ucbt (hr = 0.36, p 0.001) and use of tbi>6gy (hr = 0.58, p = 0.01) were independently associated with lower ci of relapse. 2 y probability of leukemia-free-survival (lfs) was 38±2% (49% for cr1, 42% for cr2, 10% for advanced; p = 0.001). in multivariate analysis, disease status at ucbt was the only factor associated with improved lfs (hr = 0.32, p = 0.001). causes of death were infections or other transplantrelated events (n = 172) or disease progression (n = 95). in conclusion, in the absence of an hla identical donor, ucbt remains a valuable alternative option for children with high risk all. disease status at transplant and cell dose are the most important risk factors for outcomes. use of tbi in the conditioning regimen was associated with decreased incidence of relapse but was not associated with improved lfs. the role of minimal residual disease as a predictor of outcomes of ucbt is under investigation. treosulfan-based conditioning in children: retrospective analysis of the german and austrian experience r. beier (1) background: treosulfan (treo) is an alkylating agent with good myeloablative activity and a favourable toxicity profi le. it has been widely used in hematopoietic stem cell transplantations (hct) of high-risk adult patients. here we report on our retrospective analysis of children treated in germany and austria. patients and methods: all 25 pediatric transplant centers in germany and austria were asked to submit data on treo conditioning. nine centers reported a total of 92 transplantations, 3 autologous and 89 allogeneic. results: the median age at transplantation was 9,9 (range 0,1-20,4) years (n = 74). underlying diseases were malignancies (n = 44: 12 aml, 12 all, 6 mds, 5 ewing sarcoma, 2 neuroblastoma, 1 rhabdomyosarcoma, 1 b-all, 1 lgl, 1 biphenotypic leukemia, 1 nasopharynx carcinoma, 1 alcl, 1 nephroblastoma), immunodefi ciencies (n = 26: 8 scid, 4 cgd, 3 unclassifi ed, 3 hyper-igm, 2 interleukin-10 receptor defi ciency, 2 hlh, 1 was, 2 cvid, 1 mhc class ii defi ciency), hematologic diseases (n = 20: 8 thalassemia, 3 camt, 3 osteopetrosis, 2 sds, 1 sickle cell disease, 1 saa, 1 dkc, 1 scn), and metabolic disorders (n = 2: 1 mld, 1 mannosidosis). donors were matched sibling or matched related (n = 21), matched unrelated (n = 50), autologous (n = 3), mismatched unrelated (n = 2) or haploidentical family donors (n = 12). two patients received a combination of a cord and a haploidentical graft. treosulfan was given at a total dose of 30 g/m 2 (n = 8), 36 g/m 2 (n = 31), and 42 g/m 2 (n = 53). additional conditioning drugs included fl udarabine (n = 82), thiotepa (n = 43), melphalan (n = 22), cyclophophamide (n = 4), or tbi (n = 1, 4gy). atg (n = 37), alemtuzumab (n = 23), okt-3 (n = 11), and a combination of atg and okt3 (n = 3) were used together with csa ± mtx or mmf as gvhd-prophylaxis. eight patients experienced non relapse mortality, six of them had heavily pretreated malignancies. one engraftment failure (thalassemiahaplo) and three rejections (1 immunodefi ciency, 1 thalassemia, 1 scn) were reported. all other patients engrafted rapidly. toxicity and gvhd grades in these patients were mostly i and ii. event free survival after 3 years (data available for 65 patients) was 92% for non-malignant and 37% for malignant diseases. conclusion: treo based conditioning regimens in children were effective in terms of engraftment and survival. optimal treo dosing for children is still not well defi ned. pharmakokinetic studies in children are needed. objectives: hepatic veno-occlusive disease (vod) is a severe complication following hematopoietic stem cell transplantation (hsct), with a mortality rate of 20%-50%. the diagnosis of vod is based on clinical criteria. however, the late onset of clinical signs may delay the due treatment. studies in adults have addressed to plasminogen activator inhibitor-1 (pai-1) a possible role as marker of vod. our aim was to prospectively evaluate the role of fi brinolytic parameters to discriminate vod from other liver disorders occurring after hsct in a pediatric population. methods: we studied 161 children (males 96, females 65, mean age 7,91 ± 5,17 years) who underwent 195 hsct performed at the pediatric hematology oncology of padua university. the prevalence of vod was recorded. in 105 hsct the levels of alanine amino-transferase (alt), total bilirubin, pai-1 antigen (pai-1:ag) and activity (pai-1:act), t-pa antigen (t-pa:ag), d-dimer, pt, aptt, antithrombin, fi brinogen and platelet count were assayed before and weekly for 1 month after hsct. results: the prevalence of vod was 5.6% (11/195 hsct), and it was signifi cantly higher in patients with pre-existing risk factors for vod as compared with those without (9.7% vs. 1.9%; p<0.05). all but one patient who developed vod showed an early increase in pai-1:ag, pai-1:act, t-pa:ag and d-dimer levels, even before the clinical diagnosis of vod. the increase in fi brinolytic parameters, and in particular pai-1:ag, was statistically signifi cant in comparison with that observed both in patients without complications (pai-1:ag p<0,0001) and in those with non-vod liver disorders (pai-1:ag p<0.0001). similar fi ndings were also seen when vod patients were compared with subgroups of children with infections and/or different types of hepatic diseases complicating hsct. conclusions: our study demonstrates a role of fi brinolytic tests in the diagnosis of vod after hsct in the pediatric population. in particular, the early rise in pai-1 antigen levels may suggest an incoming vod, even in the absence of clinical signs. in addition, pai-1 antigen increase may help to discriminate vod from other hepatic complications after hsct. late onset non infectious pulmonary complications (lonipc) after allogeneic hematopoietic stem cell transplantation (hsct) such as chronic graft-versus-host disease (cgvhd) of the lung, bronchiolitis obliterans (bo), bronchiolitis obliterans organizing pneumonia (boop), sinu-bronchial syndrome and restrictive lung disease are well characterized. immunodefi ciency, with or without cgvhd, and recurrent respiratory tract infections may be a vicious circle which requires optimal supportive therapy. mucociliary dysfunction is described in chronic pediatric lung diseases like cystic fi brosis (cf) and primary ciliary diskinesia (pcd). treatment with dornase alpha as a mucolytic drug with anti-infl ammatory effect improves morbidity and mortality rate in children with various chronic lung diseases. we hypothesized that outcome of our patients with lonipc measured by mortality, lung function, inhalative therapy and systemic immunosuppressive treatment could be infl uenced by dornase alpha inhalations 2500 i.e. once daily. we analyzed 23 children with lonipc who have received an allogeneic hsct between 1994 to 2009 for malignant (n = 10) and non malignant (n = 13) diseases. we compared 12 children (study group) obtaining dornase alpha inhalations with 11 children without dornase alpha. characteristics of both groups are shown in table 1 . additional supportive care was similar in both groups. dornase alpha inhalations were well tolerated with no side effects and high compliance. children treated with dornase alpha had a lower mortality rate (17%) than in the control group (36%) and showed in the long-term follow-up an improved lung function, predominantly of the peripheral airways (mean expiratory fl ow (mef) after 25%, 50% and 75% of forced ventilation capacity), in the forced expiratory value after 1 second (fev1), in resistance (r eff) and in the intrathoracic gas volume (itgv) compared to the control group. treatment with dornase alpha reduced signifi cantly the inhalation frequency of salbutamol, fl uticason or tiotropium after 6, 12 and 24 months (4 ± 2 times per day vs. 2 ± 2, p < 0.05; vs. 2 ± 2, p < 0.05; vs. 1 ± 1, p < 0.05) compared to the control group. immunosuppressive medication was similar in both groups. our data showed that inhalation with dornase alpha in children with lonipc is a well tolerated treatment option which may improve mortality rate and lung function. additional inhalative therapy but not systemic immunosuppression could be reduced. in order to fi nd hallmark of protection we studied the functional composition and magnitude of cmv-specifi c t cell response in cmv-seropositive (cmvpos) and cmv-seronegative (cmvneg) healthy donors and compared it to children after hct. polychromatic fl ow cytometry was used for detection of cmvspecifi c immune responses. the ability of cd4 + and cd8 + tcells to produce cytokines: interferon-ã (ifng) and interleukin-2 (il-2) and to express activation marker cd40l and/or to mobilize the degranulation marker cd107a in response to cmv antigens was evaluated by intracellular cytokine staining method after in vitro stimulation. peripheral blood samples were collected from 61 patients who underwent allogeneic sct and 58 healthy donors. monitoring of viral load was performed weekly from day + 7 using quantitative rt-pcr. monitoring of cmvspecifi c t-cells begun on day + 28 and was performed weekly during the time of hospitalization and on day + 56, + 90, + 180 and + 365 after sct. we have compared functional signatures in patients controlling their pcr documented reactivations (controllers) and patients non-controlling reactivations (non-controllers). as a reference we have included healthy cmvpos and cmvneg donors. among cd8 + t-cells we found three functional signatures (ifng + , ifng + /il-2 + and ifng + /il-2 + /cd40l + ) that signifi cantly differed between controllers and non-controllers. whereas single ifng + cd8 + t-cells were the most frequent of cmv-specifi c t-cells, they were not restricted to cmv controlling individuals. we found signifi cant correlation between number of weeks with low viral load reactivations and frequency of ifng + cd8 + tcells (r = 0.4, p<0.0001). dual ifng + /il2 + cd8 + t-cells were present in majority of controllers, but they were virtually absent in non-controllers. triple ifng + /il-2 + /cd40l + positive cd8 + t-cells represented a rare subset of novel cd8 + helper phenotype. no cd4 + t-cell functional signature resolved controllers and non-controllers. we (1) ( aberrant dna methylation contributes to the malignant phenotype in cancer including myeloproliferative neoplasms and myeloid leukemia. we studied aberrant dna methylation in 14 candidate loci in blood and bone marrow samples of 87 children with juvenile myelomonocytic leukemia (jmml) and asked whether it is associated with clinical, hematologic or prognostic features of the disease. the pattern of hypermethylation allowed the categorization of jmml cases into several groups. high methylation was strongly associated with higher age and increased hemoglobin f level at diagnosis. importantly, hypermethylation at diagnosis characterized cases with signifi cantly increased probability of leukemia relapse after hsct: the 5year relapse incidence was 0.22 (0.11-0.45) in the no-methylation group but 0.69 (0.49 -0.96) in high-methylation cases (p<0.01). the predictive power of high methylation for outcome following hsct was also refl ected in a multivariate cox model which included age at diagnosis, sex, platelet count and mutational category (ras, ptpn11, cbl mutation or clinical diagnosis of nf1); here methylation was the only signifi cant prognostic factor (p = 0.013). future guidelines for intensity of graft-versushost prophylaxis in jmml will have to take the methylation status at diagnosis into account. ecfcs have recently been described as vascular progenitor cells with robust proliferative potential and vessel-forming capacity. vascular integrity depends on endothelial and pericyte functions. this study was performed to establish ideal conditions for the generation of stable vessels by ecfc/msccotransplantation. mscs were propagated as previously described. ecfcs were isolated with a novel recovery strategy and propagated under animal protein-free culture conditions with pooled human platelet lysate (phpl) replacing fetal bovine serum (fbs). ecfc long-term proliferation potential was monitored and phenotype was analyzed by fl ow-cytometry and immune-cytochemistry. genomic stability was assayed with chromosome g-banding and array-comparative genomic hybridization (array-cgh). additionally we compared telomere-length and telomerase-activity of ecfcs at different time points during culture with fl ow-fl uorescence in situ hybridization (flow-fish) and telomere repeat amplifi cation protocol-assay (trap). functionality was studied during vascular network assembly in vitro and in human vessel formation in immune-defi cient mice in vivo. a mean of four ecfc colonies/ml of peripheral blood could be recovered. the progeny of these cultures could be expanded to mean 1.5 ± 0.5 × 10 8 ecfcs within 11-25 days. consecutive analysis confi rmed ecfc purity, immune phenotype and sustained proliferation potential for >30 population doublings with preserved progenitor hierarchy. genomic stability was confi rmed by karyotyping and array-cgh. telomere-length analysis revealed longer telomeres in cord blood compared to peripheral blood-derived ecfcs and a constant shorting of chromosomal ends through passaging, which could be further confi rmed by a lack of telomerase activity. large-scale expanded ecfcs functioned to form complex vascular networks in vitro and assembled stable cd31 + /vimentin + /von willebrand factor + human vessels when transplanted together with msc in vivo under defi ned conditions. these human vessels were connected to the mouse circulation as indicated by a rich content of ter119 + murine erythrocytes. this demonstrates that functional and stable human vessels can be generated in vivo as result of ecfc/msc-cotransplantation. this procedure represents a promising tool to develop innovative experimental, diagnostic and therapeutic strategies. hematopoietic progenitor cells (hpcs) which circulate in the blood are increasingly recognized as co-regulators of the development of tumor solid tumors through their ability to support neovessel formation and the transduction of the hpcs with suicide genes offers a new modality targeted antitumor cellular therapy. further to clarify mechanisms of their tumor homing, we investigated the role of the small gtpases rac1 and 2 expressed in hematopoietic cells during tumor development in mice. mice with an inducible deletion of the rac1 gene in hematopoetic cells and/or constitutive deletion in rac2 were used. rac1 deletion was induced by polyic treatment in rac-1fl ox/fl ox mice harbouring a hematopoietic-specifi c inducible cre recombinase. mice had previously been inoculated with lewis lung carcinoma by s.c. injection. we found that in mice with hematopoietic specifi c deletion rac genes, llc tumors grew more slowly in the absence of rac genes 1 and 2 compared to wild type (wt) controls. at the same time, numbers of mononuclear cells and hpcs increased in the blood. mobilization was further increased in tumor bearing mice. in contrast, the content of cd45 + cells in tumors of mice with an induced defi ciency of rac1 and 2 in hpcs was greatly diminished as shown by immunohistochemistry. we then performed competitive homing experiments by co-injecting green and red fl uorescence-stained wt and rac1/2-/-hpcs in tumor bearing mice. this revealed a 60-70% decrease in numbers of rac1/2-/-hpcs homing to tumors compared with controls. next, we studied in vitro adhesion in parallel plate fl ow chambers to delineate specifi c adhesion defi ciencies of the rac deleted hpcs. we found decreased arrest and persitstent adhesion of rac1/2-/-hpcs on endothelial cells on sdf-1a precoated endothelial cells. this correlated with a decreased ability of these cells to migrate through gradients fo sdf-1a in transwells. moreover, analysis of fi rm adhesion on both, vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1, either alone or in the presence of sdf-1a revealed a critical role of rac1 and 2 genes in adhesion strengthening of hpcs. in conclusion, rac1 and 2 negatively regulate mobilization of hpcs in tumor bearing mice, but are critically involved in entry of hpc into tumor tissue in a process involving sdf-1a. modulation of rac activation in progenitor cells may thus be a tool to modify tumor growth and to target suicide gene delivery to tumor sites. the however, hoxa9, hoxb7, hoxc10 and hoxd8 were defi ned as good candidate markers to discriminate cb-msc and bm-msc from ussc. thus, our data suggest that the "biological fi ngerprint" based on the hox code can be used to distinguish functionally distinct stem cell populations from bone marrow and cord blood. this work was supported by a grant from the dfg ko2119/ 6-1. combined action of endothelial and mesenchymal niche cells to amplify haematopoietic progenitor expansion in a humanized system d. thaler (1) the hematopoietic stem/progenitor cell (hspc) niche is an anatomically confi ned space governing hspc proliferation, differentiation and self renewal. recent research identifi ed distinct compartments described as stromal and vascular niches. this study was initiated to directly compare mesenchymal stromal cell (msc) and endothelial colony-forming progenitor cell (ecfc) contribution to niche functions in a humanized co-culture system. mscs and ecfcs were propagated under animal protein-free conditions. autologous msc-ecfc pairs were established to avoid donor variation. purifi ed cd34 + hspcs were used as responders in a cytokine-driven (il-3, 6 ng/ml; flt-3-l, 50 ng/ml; scf, 20 ng/ml) niche cell-regulated co-culture system. different standard media were employed to compare serum-free conditions with humanized cultures supplemented with pooled human platelet lysate (phpl). primary expansion culture of hspcs with and without niche cell support was followed by colony forming cell (cfc) assays to determine maintenance of clonogenicity. liquid cultures supplemented with 10% phpl were more efficient than serum-free cultures resulting in a mean 16-84-fold increase in nucleated cell progeny within 11 days. mscs or ecfcs further amplifi ed hspc proliferation. interestingly, the supportive effect of mscs or ecfcs was constantly more pronounced in humanized phpl-supplemented compared to serum-free cultures, indicating an important role of the natural platelet-derived growth factors. the combination of mscs + ecfcs resulted in hspc proliferation with up to 567-fold nc increase. the hspc progeny was mainly comprised of differentiating myeloid cells. cfc assays revealed that phpl-supplemented liquid cultures were more effi cient than serum-free cultures resulting in a 1.2-4.5 fold cfc expansion. both msc and ecfc initiated a more than 6-fold increase of cfcs, whereas combined action of mscs + ecfcs resulted in a maximum of 23.5-fold cfc increase in phpl-supplemented cultures. the impact of human ecfc as compared to msc cotransplantation on human hspc engraftment and reconstitution in immune defi cient mice is currently under investigation. our data indicate that mscs and ecfcs are effi cient in supporting hspc proliferation and cfc amplifi cation in a humanized co-culture system. the combination of both niche cell types had no further additive effect. the humanized co-culture thus provides a novel model system for analyses of hspc-niche cell interactions. a. reinisch (1), n.a. hofmann (1) , a. ortner (1) , n. etchart (1), c. , c. dullin (2) , c. diwoky (2) osteosarcoma (os) and ewing's family tumors (eft) are the most frequent bone sarcomas in young adults. unresectable or metastatic presentations are currently characterized by an extremely severe prognosis, with a consequent great need for new therapeutic approaches. we conducted a preclinical study to investigate the potential effi cacy of cytokine-induced killer (cik) cells as adoptive immunotherapy for bone sarcomas. cik cells are a heterogeneous subset of ex-vivo expanded t lymphocytes presenting a mixed t-nk phenotype and endowed with a mhc-unrestricted antitumor activity. cik cells can be used both as autologous adoptive immunotherapy or even infused from a donor after allogeneic hct. we successfully generated cik cells from 6 patients with metastatic bone sarcomas (5 os; 1 eft). ciks were generated from pbmc, cultured for 3-4 weeks with the timed addition of ifn-a; ab anti-cd3 and il2. at the end of the culture we obtained a heterogeneous t cell population with a median of cd3 + cd56 + cells of 40% (15-45), 52% (51-55) were cd8 + and presented a high expression (>95%) of nkg2d, the receptor mediating most of ciks' antitumor activity. the median ex-vivo expansion, calculated on the cd3 + cd56 + fraction, was 50 fold (22-400). cytotoxicity experiments (n = 4) demonstrated that cik cells effi ciently killed 2 different os cell lines (86%, 85%, 81% and 68% of specifi c killing at a 40:1, 20:1, 10:1 and 5:1 effector/target ratio respectively). more striking, in selected experiments (n = 2), we could confi rm the potent killing activity of cik cells against the autologous os tumor (figure 1 ). the test was performed by staining target cells with cfse and co-culturing them with ciks at different ratios; after 5 hours data were analyzed by fl owcytometry. we confi rmed that both cell lines and autologous os cells presented a high expression of mic a/b, main ligands for the nkg2d receptor of cik cells. the tumor-specifi c cytotoxicity of cik cells was confi rmed by the absence of any signifi cant killing against non-tumoral mhc-mismatched targets. our data are the fi rst report of antitumor activity of cik cells against autologous os cells. our fi ndings are encouraging and support the designing of adoptive immunotherapy clinical trials with autologous cik cells for os patients. the observed safety across major hla-barriers suggests that also donor-derived cik cells might be considered in peculiar experimental clinical settings exploring allogeneic hct for solid tumors. m. nonn, j. knapstein, a. brunk, d. tomsitz, s.a. khan, e. distler, m. theobald, w. herr, u.f. hartwig johannes gutenberg university (mainz, de) donor lymphocyte graft engineering to separate graft-versushost (gvh) and graft-versus-leukemia (gvl) immunity is of central interest in allogeneic hematopoietic stem cell transplantation. therefore, we established a xenograft-transplantation model using nod/scid/il2r gamma chain null (nsg) mice to evaluate engraftment, residual gvh-reactivity and gvl-immunity of human leukemia reactive cd8 + t cell lines in vivo. previous studies demonstrated engraftment of resting or polyclonally stimulated human cd3 + t lymphocytes to high levels in spleen and bone marrow following adoptive transfer into nsg mice and induction of xenogeneic gvhd (x-gvhd) within 3-5 weeks depending on the applied t cell dose. the engrafted human t cells contained both cd4 and cd8 subsets in the same ratio as analyzed prior to transfer. further transfer experiments using isolated human cd4 + and cd8 + t cell subpopulations revealed long-term engraftment of cd4 + t cells and induction of xenoreactivity. cd4 + t cells isolated ex vivo from spleen from these mice demonstrated an activated and memory phenotype suggesting a xenoantigen driven response in vivo. in contrast, adoptively transferred cd8 + t cells did neither engraft nor induce x-gvhd in nsg recipients. similar results were obtained upon transfer of several leukemia-reactive cd8 + t cell lines generated by short-term mixed leukemia lymphocyte coculture of cd8 + donor lymphocytes and acute myeloid leukemia (aml) blasts. investigating different cd8 + t cell growth and differentiation promoting cytokines we found that administration of human interleukin (il)-2 or il-7 failed to improve cd8 + t cell survival and expansion in vivo, whereas co-transfer of 10% autologous, naïve cd4 + t helper cells or repetitive injections of human il-15 resulted in robust cd8 + t cell engraftment in nsg recipients. in addition to these data, fi rst results of studies obtained in a therapy model to investigate gvl-responses of hla-mismatched aml-reactive cd8 + cytotoxic t cells upon transfer into primary aml engrafted nsg mice in the presence of autologous t helper cells or il-15 will be reported. in summary, we have shown that cd8 + t cell engraftment and homeostatic proliferation in nsg mice is dependent on cd4 + t cell-mediated or, alternatively, il-15-driven signals. we conclude that our nsg transplantation model provided with these signals may be a valuable tool for evaluating gvh-and gvlreactivity of ex vivo modifi ed human donor t cell grafts in vivo. oral session 13: reduced-intensity conditioning o334 ric allo-hsct for haematological malignancies up to 70 years is feasible without extratoxicity: a retrospective study of 619 patients on behalf of the sfgm-tc p. chevallier, d. blaise, n. milpied, m. michallet, j.p. vernant, n. fegueux, m. renaud, b. rio, n. gratecos, j.y. cahn, g. socie, i. yakoub-agha, a. huynh, s. francois, j.o. bay, c. cordonnier, a. buzyn, n. contentin, e. deconinck, m the aim of this report was to assess the outcome of 619 patients aged ≥60 years who received a ric allo-sct, with a special emphasis on the comparison of the outcome of patients aged 60-65 years (y.) and patients aged >65 y. between 1998 and 2008, 619 patients aged ≥60 years with different haematological diseases were treated with a ric allo-sct, and reported to the sfgm-tc registry. patients twice allografted were excluded from this study. this series included 408 males (66%) and 211 females (34%). the median age for the whole cohort was 62 (range, 60-71) y. 369 patients (60%) were diagnosed with a myeloid malignancy. 468 patients (76%) had high risk disease features. 380 patients (61%) received a ric allo-sct from an hla-matched related donor. pbsc were used in 82% of the patients (n = 511). the conditioning regimen consisted of fludarabine and busulfan in 307 cases (50%), fludarabine and low dose tbi in 148 cases (24%). the remaining 164 patients (26%) received other socalled ric protocols. atg was used in half of cases. with a median follow-up of 23 (range, 1-125) months after allo-sct, engraftment was 96%, grade ii-iv and grade iii-iv acute gvhd occurred at a median of 31 days after allo-sct in 29% (n = 180) and 12% (n = 75) of patients, respectively. chronic gvhd was observed in 142 out of 528 evaluable patients (27%). nrm was 31% (n = 194). 2-year os was 48% (95%ci, 44-53%). in order to assess the applicability of ric allo-sct to the older age group, we next compared the outcome of patients aged from 60 to 65 y. (n = 509; median age 62 y.) and those aged >65 y. (n = 110; median 66 y.). except for age, in univariate analysis, these 2 groups were not statistically different in terms of demographic, disease or transplant characteristics. overall, the median time to anc>500/μl was 18 (range, 1-99) days in each group. the grade ii-iv acute gvhd (29% vs. 31%), the overall nrm (31% vs. 33%), the relapse (32% vs. 24%) and the deaths (54% vs. 54%) incidences, as well as the 2-year os (48.5% (95%ci, 44-53%) vs. 48% (95%ci, 38-57%)) were comparable between the younger population vs. the older one (p = ns). in a cox multivariate analysis accounting for all relevant factors, age >65 y. was not found to be a statistically signifi cant factor associated with worsened survival. despite its retrospective nature and the inherent selection biases, this data support the use of ric-allo-sct in patients up to 70 years. final results of uk multi-centre, phase ii study of campath-1h dose de-escalation prior to non-myeloablative hla-identical sibling stem cell transplantation g. orti, k. peggs, m. collin, r. clark, d. milligan, h. roddie, e. liakopoulou, c. crawley, a. clark, r. pettengel, n. chowdhry, m. roughton, j. snowden, e. morris, k. thomson, p. kottaridis, a. fielding, s. mackinnon, r. chakraverty on behalf of the uk collaborative group background: inclusion of 100mg campath-1h (cam) as part of a fludarabine/melphalan conditioning regimen is effective at preventing gvhd and reducing nrm following allogeneic sct. however, these benefi ts are offset by high rates of infection and potentially a loss of graft-versus-tumour effects. we reasoned that reductions in the dose of cam would permit improved immune reconstitution post-transplant. methods: we performed a multi-centre trial in which the total dose of cam was reduced step-wise in cohorts from 60 mg to 20 mg prior to hla-identical sibling transplantation (n = 101). eligibility criteria were patients with haematological malignancies aged 18-65, life expectancy >3 months and unsuitable for myeloablative conditioning. primary endpoints were nrm, incidence of gvhd or infection. the study received irb approval and all patients gave informed consent. the cam dose was reduced over 4 cohorts: 60 mg (group a, n = 26); 40 mg (group b, n = 24); 30 mg (group c, n = 27) and 20 mg (group d, n = 24). results: median follow up is 2.6 years. median age was 50 yrs (range 17-64). 3-yr os was 71% with no differences observed in between the groups. 3-yr nrm was 16% overall (23.1% in group a, 12.5% in b, 7.4% in c and 20.8% in d, with no significant differences between the groups). cumulative incidences of grade ii-iv acute gvhd was 3.9% in group a, 8.3% in b, 3.7% in c and 12.5% in d. 3-yr cumulative incidences of extensive chronic gvhd were 7.7%, 4.2%, 0% and 16.7% in groups a, b, c and d respectively. the incidence of infection and kinetics of cd4, cd8, total lymphocyte recovery did not differ between the groups. the lowest dose cohort (20 mg, group d) compared unfavourably to the other 3 cohorts combined (30-60mg, groups a-c), in terms of cumulative incidence of severe grade iii-iv acute or chronic, extensive gvhd (hr 4.2, 95% ci 1.1-15.6) and a trend for greater number of non-relapse deaths due to infection (hr 3.1, 95% ci 0.9-10). 2 patients died of grade iii-iv acute gvhd in group d with no deaths related to this complication in the other groups. conclusions: signifi cant dose de-escalation of cam prior to hla-identical sibling transplantation is feasible without increasing nrm, although reductions below 30mg are associated with a higher risk of severe acute and extensive chronic gvhd. in future studies, we will determine whether use of cam at a dose of 30mg for hla-identical sibling transplantation will translate into improvements in progression-free survival. comparison of bu-cy-based standard myeloablative conditioning vs. fl udarabine and busulfan (flu-bu)-based reduced-intensity conditioning m. mohty, m. labopin, g. socié, n. milpied, m. attal, d. blaise, o. ringden, p. brown, b. lorentz, m. brune, r.m. hamladji, r.f. duarte, a. nagler, v the aim of this analysis was to compare outcomes (lfs, nrm, and relapse incidence) between patients receiving the classical bu-cy myeloablative conditioning (mac) regimen vs. patients receiving a fl udarabine and busulfan (flu-bu) reduced-intensity conditioning (ric) regimen prior to allo-sct. since the use of ric in young patients is still limited, the analysis was restricted to patients aged >40 years, who were transplanted using an hla identical sibling donor for aml in cr1. in summary, in this specifi c setting of aml in cr1, lfs is not statistically different when using mac or ric allo-sct for patients older than 50 years. however, for younger patients (40-50 y. age group), the use of ric is associated with a higher relapse rate. prospective trials addressing the use of ric in patients younger than 50 years are needed. indeed, reducing toxicity without compromising disease control could be of signifi cant benefi t to many patients, but "more intensive" regimens, despite the hazard of increased toxicity, may be necessary in others. thus, the trade-off between dose intensity, toxicity, and disease control will remain to be assessed for each individual patient. long-term results of reduced-intensity conditioning with busulfan and fl udarabine ± atg prior to allogeneic hct: 10-year follow-up k. sockel, m. bornhäuser, n. kröger, d. beelen, a. fauser, r. schwerdtfeger, w. siegert, l. kraut, a. cook, t. zabelina, c. theuser, g. ehninger, j reduced intensity conditioning (ric) prior to allogeneic stem cell transplantation (hct) is being used for 10 years now. the long-term outcome of ric conditioning with respect to late relapse and late non-relapse mortality (nrm) is still subject to ongoing research. therefore, we sought to determine factors predictive for long-term nrm and overall survival (os). methods: we retrospectively analyzed data from patients who received ric based on busulfan and fl udarabine prior to allogeneic hct between march 1998 and july 2001 in six major german transplant centers. overall survival was analysed using multivariate cox regression models, while competitive event statistics were applied for nrm. results: we identifi ed 196 patients with a median age of 52 years (range, 12 to 67). the underlying hematologic diseases were: aml/mds (47%), cml (20%), cll (16%), lymphoma (10%) or other hematologic malignancies (7%). donors were matched siblings in 42%, other matched relatives in 5%, matched unrelated in 40% and unrelated with single mismatches in 12% of the patients. the incidence of acute gvhd grades ii-iv was 53%. the cumulative incidence of any episode of extensive chronic gvhd at 5 years after hct was 46%. 66 patients were alive with a median follow up of 9 years (range, 2 to 11). the 10-year os and relapse-free survival was 32% (95% ci, 21% to 35%) and 28% (95% ci, 21% to 35%). non-relapse mortality at 10 years was 30% (95% ci, 23% to 37% that, these 2 regimens produce similar 1 year os (primary endpoint). however, fba is associated with better early pfs and efs and socially acceptable cost-effectiveness ratio but worse early qol. fba is also associated with better long term disease control, whereas ftbi tends to produce lower trm and higher rejection rates. clinical data might help designing individual and optimal strategies for each candidate patient, while economical data may help hospitals to tailor their transplant program, depending on the patient population that they care for. allogeneic-related stem cell transplantation with reduced-intensity conditioning versus best standard of care in older patients with acute myeloid leukaemia in fi rst complete remission. interim analysis of a prospective multi center phase iii trial t.l. kiss (1) introduction: allogeneic transplants after reduced intensity conditioning (rict) are increasingly used in patients (pts) with acute myeloid leukemia (aml). there is however a lack of prospective data supporting this practice. we present an interim analysis focusing on safety of an ongoing phase iii multi center trial aiming at determining whether rict from an hla matched sibling donor (msd) leads to improved overall survival (os) compared to standard of care in elderly pts with aml in fi rst complete remission (cr1). patients and methods: elderly pts with de novo or secondary aml in cr1 with non favourable risk cytogenetics were enrolled if they had a potential sibling donor and assigned to the rict group or control group depending whether a msd was identifi ed. both groups were followed prospectively. in the rict group 85% of pts were conditioned with fl udarabine (150 mg/m 2 and busulphan 6.4 mg/kg iv or 8 mg/kg po). graft versus host disease (gvhd) prophylaxis consisted of ciclosporine with mycophenolate mofetil or methotrexate. the study is supported by the canadian blood and marrow transplant group. results: between 2003 and june 2009 101 pts (53 males, 48 females), median age 62 (53-74) yrs were enrolled and allocated to the rict (n = 61) or control (n = 40) group. median follow up for the rict and control group was 17 and 23 months, respectively. after a median of 2.3 months (1) (2) (3) (4) (5) (6) (7) (8) 49 pts in the rict group (80%) underwent allografting. 12 pts did not reach transplant due to non-relapse death (n = 3); relapse-related death (n = 6) or comorbidities (n = 3; infections, cardiac disease). six pts (12%) died after transplant without previous relapse, due to gvhd (n = 4), infection (n = 1) and vod (n = 1). relapse rate for patients assigned to the rict group was 34% (n = 21), for patients actually transplanted 31% (n = 15). in the control group there were 2 (5%) non-relapse deaths and 18 (45%) relapses. kaplan-meier estimates of 3 year os and pfs in the whole study group was 50% and 45% respectively, with no signifi cant difference between rict and control arms. conclusion: this early analysis indicates that rict can be performed with relatively low transplant related mortality and with a moderate relapse rate. the full potential of rict can only be assessed after inclusion of more patients, longer follow-up, and by reducing the rate of pts not reaching transplant due to early relapse. in this interim analysis, no conclusions can be drawn on the putative positive effect of rict in elderly patients with aml. t-cell depleted reduced-intensity transplantation followed by donor leukocyte infusions to promote graft-versuslymphoma activity results in excellent long-term survival in patients with multiply relapsed follicular lymphoma k. thomson (1) follicular lymphoma is an indolent disorder, which is treatable but considered incurable with chemotherapy alone. the curative potential of allogeneic transplantation using conventional myeloablative conditioning has been demonstrated, but this approach is precluded in the majority of patients with fl, because of excessive toxicity. reduced intensity conditioning regimens are therefore being explored. this study reports the outcome of 85 consecutive patients with fl transplanted using fl udarabine, melphalan and alemtuzumab. patients were heavily pretreated, having received a median of 4 lines of prior therapy, and 27% had failed previous autologous transplantation. median patient age was 45 years and 53% received stem cells from unrelated donors. with a median follow-up of 4 years, the non-relapse mortality was 15% at 4 years (8% for sibling, 21% for unrelated donor transplants), acute gvhd grade ii-iii occurred in 14%, and the incidence of extensive chronic gvhd was only 18%. relapse risk was 26%, and this was signifi cantly reduced where mixed chimerism had been converted to full donor chimerism by the use of dli (10% vs. 36%; p = 0.03). in addition, 10/13 (77%) given dli for relapse post-transplant remitted, with 9 of these responses sustained and one responding to further dli. in these patients, cr is currently ongoing at a median of 44 months (12-74) following last dli. current pfs at 4 years was 76% for the whole cohort: 90% for those with sibling donors and 63% for those with unrelated donors. the excellent long-term survival with associated low rates of gvhd and frequency and durability of dli responses makes this an extremely encouraging strategy for the treatment and potential cure of fl. we analyzed the incidence and the risk of secondary solid tumors for 17,997 adult recipients (5,598 related bone marrow s58 (bm) recipients, 3,747 related peripheral blood (pb) recipients, 6,530 unrelated bm recipients, 2,093 unrelated cord blood (cb) recipients objectives: allogeneic hematopoietic stem cell transplantation (hsct) can severely compromise patients' health related quality of life (hrqol), but there is lack of evidencebased data in this area. this is the first report to provide a longitudinal assessment of hrqol in transplanted thalassemia children and their families also using a parent proxy-report evaluation. hrqol scores in children and parents were prospectively evaluated as well as parent healthcare satisfaction. methods: fifty-eight thalassemia patients from middle eastern countries were evaluated at baseline. twenty-eight children (median age 9.5 years) were transplanted from an hlamatched donor (25 sibling, 2 unrelated and 1 haploidentical) in 2 italian hsct centers. the pedsql 4.0 generic core scales were administered to patients and their parents both before and after (3, 6, 18 months) transplantation. parent healthcare satisfaction was assessed using the pedsql healthcare satisfaction generic module. these questionnaires were also administered to 30 conventionally treated thalassemia patients and their parents, all coming from the same geographical area. change from baseline at 3, 6 and 18 months was assessed using the wilcox signed rank test. results: two-year overall survival, thalassemia-free survival, mortality and rejection were 89%, 79%, 11% and 14%, respectively. the cumulative incidence of acute and chronic gvhd was 35% and 18%, respectively. eighteen months after transplantation, total pedsqol scores were signifi cantly higher than baseline ratings, both in child ( + 8.3, p = 0.05) and parent reports ( + 13.4, p<0.01) (figure 1 ). while physical, emotional and social functioning scores generally tended to decrease after 3 months in comparison with baseline values, they all returned to higher levels at 18 months after transplantation. final pedsqol scores were also signifi cantly higher than those obtained for conventionally treated patients (p<0.05). conclusion: this is the fi rst longitudinal study showing a trend towards a better hrqol in thalassemia children at 18 months after transplantation compared to pretreatment levels and conventionally treated patients. also parent satisfaction with cure and doctor-family communication was high. these data could help to relieve some of the uncertainty surrounding the diffi cult choice of transplantation in a chronic disease like thalassemia. allogeneic stem cell transplantation (sct) is potentially curative therapy for patients (pts) with acute leukemia and mds. sct is associated with substantial mortality during the fi rst 2 years after sct due to relapse and non-relapse mortality (nrm) whereas after 2 years survival curves often reach a plateau. the pattern of late events was reported in myeloablative conditioning (mac) but is not well defi ned in the reduced intensity (ric) setting. to explore late outcomes we analyzed sct results in a cohort of 401 pts with aml/ mds and all. pts meeting standard eligibility criteria were given mac (bucy or cy/tbi)). pts considered at excessive risk for nrm were given reduced toxicity conditioning (rtc) such as fl udarabine (f) with high-dose busulfan (bu) or treosulfan or a ric regimen of f and reduced bu or melphalan. the 5-year overall survival (os) was 35% (95ci, 30-41) and was similar with the various regimens. we identifi ed 115 pts with aml/mds (n = 96) or all (n = 19) who were alive and leukemia-free 2 years after sct from related (n = 70) or unrelated donors (n = 45), using ric (n = 34), rtc (n = 37) or mac (n = 44). median age was 51 (17-72). at the 2-year time-point, 41 pts had a history of acute gvhd and 55 had active chronic gvhd which still required immune suppressive therapy (ist) in 48. the probability of remaining alive for the next 5 years was 80% (95ci, 70-90). it was 76%, 85%, and 83%, after ric, rtc and mac, respectively (p = ns). there were 16 deaths beyond 2 years; 9 due to relapse and 7 due to nrm. nrm included 3 deaths due to solid cancers. there were 4 additional secondary cancers in pts currently alive. two pts died of myocardial infarction and two of chronic gvhd. in all, the cumulative incidence of late nrm and relapse mortality was 9% (4-20) and 11% (6-22), respectively. advanced age (>50) was the most signifi cant predicting factor for shortened os; 64% and 96% in the older and younger groups, respectively (p = 0.005 results: recipients with mbl levels <1000 ng/ml were at increased risk to suffer from one or more major infections (p = 0.002) during entire follow-up. infectious susceptibility was increased after neutrophil recovery, particularly until 24 months (hazard ratio (hr) 3.4) with sustained effects afterwards (hr 2.9) (figure 1 ). in multivariate analysis mbl serum concentrations <1000 ng/ml were independently associated with major infections after neutrophil recovery (p = 0.009) when corrected for recipient age at transplantation, sex, diagnosis, history of total body irradiation, history of splenic irradiation or splenectomy, acute or chronic graftversus-host disease, and duration of immunosuppression. in subgroup analysis occurrence of severe herpes virus infections in particular was associated with signifi cantly lower mbl levels (p = 0.02). conclusion: our fi ndings indicate that low mbl levels may predict markedly increased susceptibility to severe infections with sustained effects even late after allogeneic hsct. determinations of mbl status should therefore be included into pretransplantation risk assessment. introduction: aim of this study was to evaluate frequency and risk factors of both secondary myelodysplastic syndromes/acute leukemias (smds/al) and solid tumors in lymphoma patients treated with the high-dose (hd) squential (hds) chemotherapy with peripheral blood progenitor cell (pbpc) autograft. patients and methods: data have been collected on 1,347 lymphoma patients treated at 11 centers associated to gitil ( figure 1 ). patients received either the original or modifi ed hds regimens, as shown in figure 2 . pbpc were collected after hdcyclophosphamide (cy), and, in a subgroup, after a 2nd round of mobilization with hd-ara-c. to reduce the incidence of acute and chronic graft versus host disease (gvhd) anti-t-cell globulins (atg) have been incorporated into the preparative regimen for allogeneic stem cell transplantation (sct) from alternate donors by many centers. different atg preparations are available and little is known about the optimal dosing. therefore we conducted this retrospective registry study utilizing specifi c questionnaires to participating centers. chronic myelogenous leukemia (cml) in chronic phase has been selected as underlying disease in order to have a rather homogenous patient population. a total of 1359 patients (pts) have been analyzed. 534 pts had received no atg, 288 atg-fresenius, 122 thymoglobuline® (genzyme), 261 other in vivo t-cell depletion, mainly campath, and 154 had received in-and ex-vivo t-cell depletion, utilizing thymoglobuline® for in-vivo depletion. a cumulative dose of less than 40 mg/kg atg-fresenius or less than 10 mg/kg thymoglobuline® has been defi ned as low-dose. the median follow-up for surviving pts is 62 months (range: with no statistically difference in the different pts groups. only the use of atg-fresenius and thymoglobuline® proved to be an independent positive prognostic factor for overall survival in multivariate analysis incorporating the ebmt risk score. this was due to decreased treatment related mortality. however, any of the analyzed t-cell depletion strategies increased the risk of relapse, which did not translate into overall survival, since relapse after allo sct is manageable in cml pts. when also analyzing the dosing of atg, the use of high dose atg-fresenius was associated with the best long-term overall survival of about 70%. when comparing high dose fresenius versus all others the use of high dose fresenius had the same impact on overall survival as the ebmt risk score, indicating that the use of high dose fresenius is an independent positive prognostic factor. similar effects were not seen with high-dose thymoglobuline®. interestingly, the positive effects of atg only became obvious after 4 months after transplant suggesting no protection against acute gvhd but protection against mortality from chronic gvhd. although unrelated allogeneic sct in chronic phase cml is nowadays a rather rare indication these data nevertheless prove benefi cial effects of in vivo t-cell depletion and also emphasize, that the different preparations are not interchangeable and that the dosing is of great importance. skewed t-cell receptor repertoires in very long-term survivors after allogeneic haematopoietic stem cell transplants a. rovó (1), c. arber (1), p. fisch (2), u. siegler (1) disturbed immune reconstitution and late auto-immune like phenomena have been observed after hsct and described as "late altered immunity" in very long-term survivors. in a previous cross-sectional study on 44 long-term survivors after allogeneic hsct (>10 years) and their respective donor, we demonstrated that recipients with cgvhd and a female donor had signifi cant telomere shortening of the hematopoietic cells, and subtle signs of altered late organ function as compared to their respective donor (baerlocher et al., blood, 2009 ). we were therefore interested to learn more about these differences and analyzed in the same cohort the t cell receptor beta (tcrb) repertoire diversity by spectratyping of peripheral blood t lymphocytes. we compared by visual analysis the gaussian like distribution of the peaks from 23 vb subfamilies. pattern of each vb subfamily was classifi ed as normal or skewed. normal refers to >19 vb subfamilies with a gaussian like distribution, abnormal to >5 vb subfamilies with a non gaussian pattern. the overall complexity of the tcrb repertoire (total number of peaks of all 23 vb subfamilies) from the recipients was compared with the respective donor. the median age at hsct and at time of the study was 22.5 (6-50) and 44 (27-62) years for the recipients; 23.5 (11-45) and 45 (31-61) for the donors. the median time interval between hsct and tcrb repertoire analysis was 20 years (12-25). during follow-up 4/10 analyzed recipients had cgvhd. the median number of skewed vb subfamilies was 8.5 (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) for recipients, and 2 (0-8) for donors (p = 0.008). the median overall complexity was 149 (101-170) for recipients and 159 (138-183) for donors (p = 0.041). 8/10 analyzed recipients showed an abnormal pattern of the tcrb repertoire. all but two donors had a normal repertoire (p = 0.001); one of them suffered of 2 neoplasms after donation one patient with an abnormal tcrb repertoire ( figure 1 , upn498) had still extensive, severe cgvhd and showed the shortest telomere on the leukocytes and t-lymphocytes: 3.7 and 3.6 kb respectively, versus median 6.5 (5.4-8.1) and 5.7kb (4.8-7.4) respectively. these data indicate that t cell repertoire diversity in long term survivors after allogeneic hsct is abnormal and differs from the repertoire of their respective donors. the cause of this skewed t cell repertoire remains open but gives a formal basis for the clinically observed late altered immunity. the clinical consequences thereof need to be evaluated in the future. a second allogeneic sct was given to 119 patients (median time from sct1 11 mo, from relapse 1.8 mo), using sic (43 %) or ric (57% background: center specifi c outcome data are being discussed as useful tools for patients, the insurance industry, and politicians to assess quality of specifi c centers and to guide decision making processes. hematopoietic stem cell transplantation (hsct) requires signifi cant infrastructure and is costly. hsct therefore qualifi es as a target for such an exercise. methods: we made use of the comprehensive database of the swiss blood stem cell transplant group (sbst) to evaluate center specifi c mortality rates. nine centers reported a total of 4717 hsct, 1427 allogeneic (30.3%), 3290 autologous (69.7%) in 3808 patients between 1997 and 2008 to treat acute or chronic leukemia (29%), lymphoid neoplasia (53%), non malignant disorders (3%) or solid tumors (15%). because of mandatory reporting by legal requirement these include data on all hsct performed in switzerland over this period. data were analyzed separately for recipients of allogeneic and autologous hsct for survival and transplant related mortality (trm) at day 100 and at 10 years. results: overall survival at 10 years was 49 + 4% after allogeneic and 40 + 3% after autologous transplantation. there were signifi cant differences among centers in unadjusted analyses of survival and trm. these differences became absent or marginal, when results were adjusted for disease, year of transplant and the ebmt risk score (incorporating patient age, disease stage, time interval between diagnosis and transplantation, and for allogeneic transplants donor type, and donor recipient gender combination) in multivariate analysis. conclusions: these data indicate comparable quality among centers in switzerland. they furthermore demonstrate that comparison of crude center specifi c outcome data without adjustment for patient risk factors may be highly misleading. mandatory data collection and outcome analysis including all cases within a comprehensive quality management system may serve as a model for other cost intensive therapies to ascertain quality. . contrary, the more relevant negative aspect is the increase of workload (38%). the major modifi cation produced by the qms is to recognize the critical situations (51%) and to report in proper forms the nca is useful also to resolve them (36%). also, 67% think that internal audits are helpful to check the activities. the last question aimed to understand the relevance of qms: 58% agree that qms is part of daily activity and only for 2% "there is too much to do". the collected data show that the quality culture is, today, part of the work background and this result is recognized by 70% of lab staff, 47% of nurses and 62% of physicians. in general, the perception of qms is good but there are still areas to be improved, particularly regarding the management and resolution of critical situations. it is clear that to study the perception of qms is a tool to verify his effi cacy and continuously improve it, to make clear the diffi culties and fi nd appropriate corrective actions. so, qt plans to repeat the survey at least once at year in order to build a complete evaluation's system for work environment' quality and staff satisfaction. background: the liver is the current site for pancreatic islet transplantation (tx) but has many drawbacks due to immunological and non-immunological factors as well as important technical limitations. we asked whether pancreatic islets could be engrafted in the bone marrow (bm), an easily accessible and widely distributed transplant site that may lack the limitations seen in the liver. methods: pancreatic islets were implanted into the bm of diabetic c57bl/6 mice. islet survival, function and morphology were evaluated in comparison with the liver site. moreover a phase i/ii open label pilot study to assess the feasibility and safety of bm as alternative site for islet tx was started in patients with type 1 diabetes mellitus. results: syngeneic islets engrafted effi ciently in the bm of c57bl/6 mice rendered diabetic by streptozocin (stz) treatment. for over a year post-tx these animals showed parameters of glucose metabolism that were similar to those of non-diabetic mice. islets in bm had a higher probability to reach euglycemia than islets in liver (2.4 fold increase, p = 0.02), showed a compact morphology with a conserved ratio between alpha and beta cells, and affected bone structure only very marginally. islets in bm did not compromise hematopoietic activity, even when it was strongly induced in response to a bm aplasia-inducing infection with lymphocytic choriomeningitis virus. in humans, 3 diabetic patients received an intra bm islet infusion at the level of iliac crest without any adverse effects. in all recipients islets engrafted as demonstrated by the presence insulin producing cell in addition, preferential pairing facilitated by introduction of an extra disulfi de bond in the constant regions of the tcr chains can increase cell surface expression of the transferred tcr and decrease formation of mixed tcr dimers. another strategy based on the fact that tcrs differ in their capacity to compete for cell surface expression, is to select recipient t cells with weak competitor phenotypes. both weak and strong competitor phenotype virus-specifi c t cells were used to assess improvement of ha-1-tcr cell surface expression using the different strategies. the most marked improvement in ha-1-tcr expression and functionality was observed after tcr transfer of a cysteine modifi ed and codon optimized ha-1-tcr resulting in 70% and 35% of ha-1 tetramer positive weak and strong competitor t cells, respectively. this resulted in effi cient recognition of primary leukemic cells that endogenously process and present ha-1, independent of whether the recipient t cells were strong or weak competitor t cells. furthermore, results demonstrate that next to increased ha-1-specifi c reactivity, neoreactivity after ha-1-tcr gene transfer was dramatically reduced by cysteine modifi cation of the ha1-tcr. based on these results, cysteine modifi ed and codon optimized ha-1-tcrs will be used for the planned phase i/ii clinical trial to treat patients with relapsed hematological malignancies. reactivation of latent cytomegalovirus (cmv) infection is a frequent complication in cmv-seropositive patients after allogeneic hematopoietic stem cell transplantation (hsct). the s66 cmv-related morbidity and mortality are particularly high, if the donor is cmv-seronegative and thus, no cmv-specifi c memory t cells are being transferred from donor to recipient during transplantation. grafting non-reactive t cells of cmvseronegative donors by virus-antigen specifi c t cell receptors (tcr) may be an effi cient means to transfer cmv-specifi c t cell function into allogeneic hsct recipients. in this study, we have reprogrammed t cells of cmv-seronegative donors with human tcr recognizing the immunodominant hla-a*0201-binding cmvpp65 peptide epitope 495-503. to overcome the limitations of retroviral tcr gene transduction that hamper clinical translation, we used in vitro transcribed rna encoding cmv-specifi c tcr for electroporation of non-reactive t cells. this procedure resulted in transient surface expression of the introduced tcr for at least 3 days as demonstrated on both cd4 + and cd8 + t cells by specifi c tcrvb chain and hla tetramer staining analyses. the cmvpp65 tcr rna-transfected t cells showed hla-a*0201-restricted ifn-g secretion and cytolysis against cmvpp65 495-503 peptide-pulsed target cells and against human fi broblasts upon cmv infection. we also observed that tcr rna transfection of cd4 + t cells turned them into potent cmvpp65/hla-a*0201-specifi c t helper cells. this was demonstrated by co-incubating them with immature dendritic cells (dc), which resulted in maturation of dc only in the presence of the cmvpp65 epitope. furthermore, we transfected pure naïve and memory cd8 + t cell subsets isolated from peripheral blood of cmv-seronegative donors. although 90% of naïve cd8 + t cells were cmvpp65/hla-a*0201 tetramer positive after electroporation, they mediated only marginal lysis toward cmv-infected fi broblasts. in contrast, memory cd8 + t cells showed strong cytotoxicity against cmv-infected fi broblasts for at least 3 days upon tcr rna transfection. in summary, our data demonstrate that non-reactive human t cells can be easily redirected with cmvpp65 tcr rna, thereby gaining cmv-specifi c t cell effector function for a considerable time period. we therefore believe that cmvpp65 tcr rna has the potential to be further developed as a therapeutic "off-the-shelf" reagent for cmv-seropositive patients who undergo allogeneic hsct from cmv-seronegative donors. functional cytotoxic t lymphocytes emerge in patients with ph + acute lymphoblastic leukemia under imatinib therapy and may be exploited for adoptive t-cell therapy c. quadrelli (1) an effective specifi c immune response against adult all has so far been reported only in the allogeneic stem cell transplant setting. we have recently demonstrated that bm-resident, ifng-producing, p190bcr-abl specifi c autologous t cells may occur in ph + all patients upon treatment with high dose im, and that the presence of these cells correlates with lower minimal residual disease (mrd) values and better clinical outcome. on the basis of these preliminary data, we proceeded to assess for the presence of cytotoxic t-cells among the bm resident, bcr-abl-specifi c t-cells, and characterized the cytotoxic t-cell populations identifi ed, with the aim of optimizing a protocol for the ex-vivo expansion of p190bcr-abl-specifi c cytotoxic t lymphocytes (ctls) to be employed in protocols of t-cell therapy to control minimal residual disease after chemotherapy or hsct. p190bcr-abl-specifi c ctls were identifi ed by co-colturing bm mononuclear cells (bmmc) of ph + lla patients with bcr-abl peptide-pulsed autologous dendritic cells; the peptides employed derived from the complete spanning of p190bcr-abl fusion region. the p190bcr-abl-stimulated t cell lines generated with this protocol were mainly cd8 + /cd3 + or cd4 + /cd3 + cells with effector memory phenotype, that exerted a specifi c lytic activity against bcr-abl fusion protein. in detail, we observed a p190bcr-abl specifi c lytic activity >100 lu10/106 in 7 of the 10 patients tested (median lysis against p190-derived peptidepulsed targets: 1355 lu10/106). in 3 of the patients, for whom autologous leukaemia blasts were available, we could demonstrate a strong leukemia-directed lytic activity (median lysis: 10000 lu10/106). the lysis directed towards autologous blasts was mainly mediated by cd8 + t-cells, and was hla class irestricted. lytic activity towards autologous, mock-pulsed pha blasts, or allogenic ph-blasts was low/absent. our fi ndings demonstrated that p190bcr-abl-specifi c t cell responses may emerge in patients with ph + all under im treatment, and may be stimulated and expanded ex-vivo. whether these p190bcr-abl-specifi c t cells may have a possible therapeutic role in ph + all patients, and may be expanded from healthy donors to be employed in protocols of adoptive cell therapy after hsct remains to be addressed in future studies. background: a positive selection of leukemia associated antigen (laa)-specifi c t cells would be highly desirable to amplify the gvl effect and to decrease the risk of gvhd. wilms tumor gene 1 (wt1) and the receptor for hyaluronic acid mediated motility (rhamm) are laas recognized by cd8 + t lymphocytes. streptamers constitute a novel multimer technology to select specifi c cd8 + t cells, available at good manufacturing production (gmp) level. material and methods: both tetramers and streptamers were used to detect the frequency of hla-a2 restricted cd8 + t cells in the naïve peripheral blood (pb) from both healthy donors (hds) and aml patients. rhamm-and wt1-specifi c cd8 + t cells were further characterized for the expression of cd27, cd28, cd45ra and ccr7. in the next step, laa-specifi c cells were positive selected by macs columns after labeling with streptamers and thereafter immunophenotyped. moreover, mixed lymphocyte peptide cultures (mlpcs) were performed to enrich wt1 specifi c t cells derived from the pb of hds. rhamm and wt1 specifi c cells were subjected to carboxylfl uorescein succimidyl ester (cfse) based proliferation and cytotoxicity assays. results: 21 of 40 hds showed naïve laa specifi c t cell frequencies of 0.5 to 1.6% of all cd8 + t cells. in aml patients in complete remission signifi cantly higher frequencies of laaspecifi c t cells than in patients at diagnosis could be detected, up to 6.0% of all cd8 + t cells. these cells revealed to be cd8 + cd27-cd28 + cd45ra + ccr7-effector t cells in fl ow cytometry. after positive selection by macs columns a purity of 20-87% could be achieved for laa-specifi c t cells. after a maximum of three rounds of mlpc, only a frequency of 2-5% could be achieved, thus demonstrating the power of the streptamer technology. both tetramer-and streptamer-based selections yielded similar amounts of laa-specifi c t cells. after positive selection, these t cells preserved an effector t cell phenotype and showed active proliferation in cfse staining. streptamerselected t cells showed a trend towards higher cd28 expression thus indicating a more activated t cell status. conclusion: in summary, the streptamer technology allows to select highly pure fractions of rhamm-and wt1-specifi c effector t cells with proliferative and cytotoxic properties. in analogy to dlis specifi c for viral antigens such as cmvpp65, production of leukemia specifi c dlis is feasible on a gmp level. further leukemia antigens are currently evaluated by our group. delicate balance between regulation and stimulation at the antigen presenting cell site determines the likelihood of successful priming and survival of antigen-specifi c t-cells from the naïve donor repertoire i. jedema, t.s. lam, m. van de meent, c. hoogstraten, j.h.f. falkenburg leiden university medical center (leiden, nl) although the in-vitro induction of tumor-and pathogen-specifi c t cells from the naïve donor repertoire has been shown to be feasible, the robustness of the procedure remains limited, hampering large scale clinical application. in this study, we investigated the role of individual parameters like the frequency of antigen-specifi c precursor t cells (tprec) and regulatory t cells (treg), the number of antigen presenting cells (apc) used as stimulator cells, and the number of targeted antigens (ag) on the ability to prime, enrich and expand ag-specifi c t cells from primary immune responses in-vitro. therefore, we developed an in-vitro model system allowing the monitoring of ag-specifi c activation and proliferation of individual naïve donor t cells in the fi rst 14 days of the immune response using pkh labeling, cd137 counterstaining and quantitative fl owcytometric analysis. in this model we exposed naïve tprec to allogeneic apc in different responder/stimulator (r/s) ratios in the presence of different numbers of innocent bystander cells. optimal t cell activation was seen at specifi c tprec/s ratios between 1/1 and 1/5, irrespective of the number of innocent bystander cells. lowering the number of stimulator cells per tprec resulted in incomplete activation and proliferation, but more importantly, exposure to an excess of stimulator cells resulted in induction of activation-induced cell death (aicd) of the antigen-specifi c tprec. next, we investigated the role of treg in the induction phase of the immune response. treg were like tprec attracted to the site of the apc and their activation further increased their inhibitory potential. especially when they were at a numeric advantage, treg were capable of impairing antigen-specifi c tprec priming. increasing the number of apc in the induction phase of the immune response could overcome this negative regulation since the number of treg per apc is diminished. however, at low tprec frequencies, in case of single peptide responses, this will lead to aicd of the antigen-specifi c tprec. this may be prevented by increasing the number of antigenspecifi c tprec by simultaneous targeting of multiple antigens. in conclusion, the in-vitro generation of antigen-specifi c primary immune responses can only be successfully and reproducibly performed by creating an optimal balance at the priming site of the immune response (e.g. the apc) between the number of negative regulators (treg) and responding cells (tprec). fistulas are an invalidating, often diffi cult to treat, complication of patients with crohn's disease (cd), a disabling, chronic, relapsing infl ammatory enteropathy caused by dysregulation of the immune tolerance towards intestinal bacteria in genetically susceptible individuals. mesenchymal stromal cells (mscs) represent a promising tool in approaches of regenerative medicine. we investigated the feasibility, safety and effi cacy of local injection of autologous bone marrow (bm)-derived mscs for refractory cd fi stulas. mscs were isolated and expanded ex vivo in the presence of platelet lysate from bm of 12 patients (7 males, median age 33 yrs, range 16-59). patients received intrafi stular injection of mscs scheduled every 4 weeks (median 4 infusions) and were monitored at time of each injection, and 1, 3, 6, 12 months after the last treatment. the cytokine profi le of mscs and their ability to infl uence apoptosis of mucosal t cells obtained from involved and uninvolved colonic areas were also analyzed. msc expansion was successful in all patients and no adverse event was recorded during and up to 12 months after treatment. intrafi stular injection of mscs was effective in inducing sustained closure of fi stulas, with the appearance of regenerative tissue along the tracks. in particular, 7 patients (70%) benefi ted from complete and sustained healing of fi stula tracks, while three had partial response. all patients showed a signifi cant reduction of both cd activity index (pre-and posttreatment median values: 294 sd 49 and 99 sd 32 at 6 months after the last infusion; p < 0.001) and perianal disease activity index (pre-and post-treatment median values: 13.0 sd 2.2 and 4.5 sd 2.4 at 6 months after the last infusion; p < 0.001) reaching disease remission usually after the second procedure. the immunephenotype of circulating t lymphocytes showed progressive increase of the number of cd4 + cd25bright foxp3 + cells, which became signifi cant (p<0.01) after the second procedure and remained stable up to 6 months after the last infusion. no modifi cation of serum cytokines was observed at any time point. mscs caused a sort of block of the rates of both apoptotic and living cells when incubated with t lymphocytes from diseased mucosa, whilst critically increased the apoptotic rate when incubated with t lymphocytes from apparently healthy mucosa. local injection of autologous bm-derived mscs appeared feasible, safe and successful in treating fi stulas associated with cd. we recently showed that nk cells require two signals to initiate lysis; the fi rst primes (s1) and a second triggers (s2). we found nk resistant cell lines which express s1 but lack s2 and that resting nk cells stimulated with these cells retain the primed state even after cryopreservation. these are termed tumour activated nk cells (tank). we are conducting a clinical trial in patients with aml in cr or pr with less than 25% blasts who have exhausted conventional treatment options. tank are generated from a haploidentical family donor by overnight incubation of nk cells with lysed ctv-1 cells. tank are then purifi ed from the lysate and released at a single dose of 10 6 nk/kg with a t cell contamination of <10 4 /kg and cryopreserved. pre-infusion conditioning -fludarabine (25mg/m 2 /day) for 3 days plus 2gy tbi on day 4. eleven patients have been enrolled to date, 6 of whom have been treated. patient characteristics: pt01 -57 yr female; previous autologous transplant, treated in cr3. pt02 -71 yr male; in pr after 5-azacytidine. pt03 -50 yr male; previous allogeneic transplant, treated in cr3. pt04 -71 yr male; treated during relapsing disease and circulating blasts. pt05 -73 yr female treated in cr1 following one course of induction chemotherapy. pt06 -67 yr male; treated in cr2. results: no infusional toxicity. all patients suffered a degree of bone marrow suppression needing in-patient supportive care. aplasia ranged from 3 weeks up to 45 days. pt01 remains in cr at month + 16; pt02 achieved and remained in cr until month + 11, relapsed and treated with second tank infusion; pt03 prolonged and severe pancytopaenia requiring cd34selected stem cell rescue, achieved and remained in cr until month + 11.5; currently undergoing re-induction chemotherapy. pt04 cleared peripheral blasts for 2 months; now with progressive disease. pt05 in cr, 3 months post infusion; pt06 is non-evaluable at present. extremely prolonged nk chimerism (up to + 6 months) has been seen in all patients in the absence most cord blood transplants (cbt) are performed using cord blood units mismatched with the recipient at one or two hla loci. it is not known whether the locus at which mismatches occur infl uences outcomes. we examined the effect of locusspecifi c mismatches on outcomes of 1305 hla-mismatched (one mismatch, n = 626, two mismatches, n = 679) single cbt performed for haematological malignancies at ebmt centres from 1994 to 2008. two digit typing for hla-a and hla-b and 4 digit typing for hla-drb1 was used. patients were transplanted for all (n = 563), aml (n = 411), mds (n = 166), chronic leukaemia (n = 87), lymphoma (n = 22) or myeloma (n = 10). among the 626 patients who received a cord with one hla mismatch, the mismatch occurred at hla-a (n = 191), hla-b (n = 247) or hla-drb1(n = 188). transplant-related mortality (trm) for this group at 2 years was 37±2% and was 35±4%, 35±3% and 42±4% for hla-a, -b and -drb1 mismatches respectively. on multivariate analysis the mismatched locus had no infl uence on trm when adjusted for age, year of transplant, stage of disease, cmv status and cell dose. there was a trend for less relapse for drb1 mismatches (21±3% vs. 29±3%, p = 0.051) but this did not lead to a difference in disease-free survival (dfs) between groups (38±4% vs. 39±3%, p = ns). neutrophil engraftment and acute graft-versus-host disease (gvhd) were unaffected by locus of hla mismatch. for the 679 patients who received a cord with two hla mismatches, mismatch combinations included a + b (n = 250), b + drb1 (n = 220), a + drb1 (n = 122) or two mismatches each at either a (n = 20), b (n = 34) or drb1 (n = 33). trm for this group was 42±2% at 2 years and was 41±3% (a + b), 41±4% (b + drb1), 39±5% (a + drb1), 55±12% (a + a), 42±9% (b + b) and 58±9% (drb1 + drb1) by mismatch group. the loci at which hla mismatches occurred did not infl uence trm on multivariate analysis, nor when grouped according to mhc class (class i only vs. class ii±i). however, double drb1 mismatch was associated with more grade ii-iv acute gvhd (50±8% vs. 32±2%, rr = 1.77, p = 0.03). in addition, on multivariate analysis a + b mismatches were associated with improved probability of dfs (34±3% vs. 30±2%, hr = 0.80, p = 0.03). a cell dose cut-point could not be defi ned in either population. in summary, for cbt with one hla mismatch, the locus at which mismatch occurs does not infl uence outcomes. however, for cbt with two hla mismatches, it seems that two mismatches at class i (hla-a and -b) are preferable to other mismatch combinations. matched unrelated donor is associated with lower relapse than matched related donor in reduced-intensity conditioning transplantation: implications for graft-versus-malignancy effect v. ho, h. kim, j. aldridge, g. kao, d. liney, j. koreth, p. armand, c. cutler, j. antin, r. soiffer, e. alyea dana-farber cancer institute (boston, us) as success of reduced intensity conditioning (ric) stem cell transplantation (sct) relies on graft-vs-malignancy (gvm) effect, the extent of minor hla antigen disparity in matched related donors (mrd) vs. matched unrelated donors (mud) could impact clinical outcomes. while convention dictates that mrd is preferred over mud, does this wisdom hold in ric sct? we conducted a retrospective review of 477 (279 mud, 198 mrd) ric sct performed at our institution from 9/01 through 12/08. all received uniform ric conditioning with busulfex (3.2-6.4 mg/kg) and fludarabine (120 mg/m 2 ). gvhd prophylaxis was mostly tacrolimus/mini-methotrexate ± sirolimus (98%). sc source was pbsc (97%), marrow(3%). all donors were 10/10 allele matched at hla a, b, c, drb1, dqb1. the mud and mrd cohorts were balanced in demographics, disease (mds/aml 44% vs. 45%), high disease risk (81% vs. 82%), prior sct (30% vs. 33%), and gvhd prophylaxis. median cd34 + dose was higher in mud (8.7 vs. 7.5 × 10 6 cd34 + /kg, p = 0.003). cumulative incidence of grade ii-iv acute gvhd and 2-yr chronic gvhd were similar, 21% vs. 16% (p = 0.21), and 50% vs. 47% (p = 0.17) in mud and mrd, respectively. there was no difference in transplant related mortality (trm), time to neutrophil or platelet engraftment, or % achieving over 90% donor chimerism at days + 30 and + 100. cumulative incidence of relapse was 52% for mud, 67% for mrd, p = 0.003. with a median follow-up of 25 and 28 months, 2-yr progression free survival (pfs) was 41% for mud and 29% for mrd, p = 0.004 ( figure 1 ). 2-yr overall survival (os) was 57% for mud, 49% for mrd (p = 0.35). improvement in pfs did not translate to os benefi t because many relapses were salvaged with second sct or other therapy. in competing risks regression, mrd was associated with increased risk for relapse compared to mud (hr 1.45, p = 0.004), but no difference for trm. in cox regression, mrd, prior sct, high disease risk, and mds/ aml diagnosis were associated with inferior pfs (table 1) . donor age, cd34 + dose, and year of sct were not associated with worse survival. in ric sct, mud is associated with signifi cant improvement in relapse and pfs compared to mrd, without increased gvhd or trm. this improvement is not related to younger donor age or higher cd34 + count, suggesting that hla minor antigen disparity in mud sct may mediate stronger gvm. while further investigation is warranted, these results could have great implications for the future selection of donors for ric sct. tolerance of g-csf-administration was different in the two groups: 12/171 (7,01%) sib donors stated, that they only poorly had tolerated g-csf administration, 4/106 (3,77%) mud donors said that their tolerance to g-csf had been poor. 3/171 (1,75%) sibs indicated, that the infl uence of pbsc donation to their health status had been negative, while 2/106 (1,88%) unrelated donors said that there had been a negative infl uence of pbsc-donation on their health status. 5/171 (2,92%) sibs described their current health status as "bad", while none of the 106 muds did so. the most serious adverse effects that occurred after donation were one case of hodgkin's disease in one of the related donors and one case of acute lymphatic leukaemia in an unrelated donor. furthermore, one case of squamous cell lung carcinoma occurred in a related donor and one case of mamma carcinoma in an unrelated donor. in addition to these malignancies the following health problems could be noted in the donors: subdural haematoma a few days after donation: 1 sib, 0 mud. sarcoidosis: 1 sib, 0 mud. cardiac disorder: 2 sibs; 0 mud. essential hypertension: 4 sibs, 4 muds. discus hernia: 0 sib, 4 muds. hypothyreosis: 0 sib, 1 mud. depression: 1 sib, 1 mud. tinnitus: 0 sib, 2 muds. the presented data show that serious health problems after donation may occur and therefore global collection of these data is necessary in order to calculate the actual risks of pbsc donation in advance. evaluation, consent and care of related donors and recipients at haematopoietic stem cell transplant centres in the united states: practice patterns and potential for confl ict of interest p. anderlini (1), t. pedersen (2), d. confer (2) background: a real or perceived confl ict-of-interest may arise in the situation where the same physician is responsible for the care of two individuals whose care is interdependent. in hematopoietic stem cell transplantation (hsct) from unrelated donors facilitated by the national marrow donor program, donors and recipients are under the care of separate physicians in order to provide unbiased care and eliminate the potential for a confl ictof-interest. however, the practice patterns of evaluation and care for related donors and recipients at centers performing hsct are unknown. material and methods: a practice pattern survey of transplant centers in the united states reporting to the cibmtr was conducted by the donor health and safety committee between december 2007 and july 2008. the survey was administered as an online survey tool sent to the center medical directors. the survey focused primarily on determining the type of provider involved in medical clearance, informed consent and medical management of the donor and the providers relationship to the recipient. results: of 222 centers surveyed, 98 evaluable responses were received (40%). the median number of related donor transplants per year at responding institutions was 15 (range: 1-400); the median total number of transplants per year was 70 (5-600). as shown in the table, transplant physicians in greater than 70% of centers were involved in overlapping care of the donor and the recipient during the donor evaluation, clearance and collection phases. these patterns were similar between transplant teams caring for adult or pediatric donors and recipients. conclusion: among responding centers, it appears that medical management of recipients and their related donors by the same provider is common, and may not be viewed as a potential confl ict-of-interest. whether this potential confl ict-of-interest affects donor care is unclear, and deserves further investigation. long-term follow-up and risk factors for outcomes after related hla-identical cord blood transplantation for patients with malignancies. an analysis on behalf of eurocord-ebmt a.l. herr (1) , n. kabbara (1), p. teira (1), c. bonfi rm (2) outcomes after related hla identical bone marrow or cord blood transplantation (cbt) in children have been reported to be comparable (nejm 2000) . in order to analyze risk factors for outcomes, we studied 149 patients with malignancies who had a fi rst single unit related hla identical unmanipulated cbt and were reported to eurocord-ebmt. cbt were performed since 1990, in 25 countries and 68 centers. median fu was 6.7 years (range 7 months to 17 years). nearly all patients were children, median age being 5 years. acute leukemia (al) was the main diagnosis (n = 109) followed by myelodysplasia (n = 17), chronic myeloid leukemia (n = 12), solid tumors (n = 6), lymphomas (n = 3) and hemophagocytic lymphohistiocytosis (n = 2). according to ibmtr criteria, 39 patients had low, 71 intermediate and 39 high risk diseases. all cb donors were siblings except 2: one 2nd degree relative and one child. cb units contained a median of 5.1 × 10 7 /kg total nucleated cells (tnc) at collection and 4.1 × 10 7 /kg tnc after thawing. the conditioning regimen was myeloablative for 144 patients, 50% including tbi. the most frequent gvhd prophylaxis regimen was cyclosporine alone. methotrexate (mtx) was used for 22 patients. the cumulative incidence (ci) of neutrophil recovery was 89% at d + 60 with 135 patients achieving a neutrophil count of 0.5 × 10 9 /l after a median time of 24 days. infused tnc ≥ 4.1 × 10 7 /kg (p = 0.002) and the lack of mtx in gvhd prophylaxis (p = 0.002) were independently associated with improved neutrophil recovery. ci of acute and chronic gvhd was 11% at d + 100 and 10% at 2 years, respectively. ci of non-relapse mortality was 9% and of relapse was 46% at 5 years. remission status (p = 0.04) and the use of tbi-containing regimens (p = 0.03) were independently associated with a lower relapse rate in al patients. for cbt performed in year <2000 vs. ≥2000, disease-free survival at 5 years was 34% vs. 55% (p = 0.009), and overall survival (os) at 5 years was 44% vs. 66% (p = 0.01). transplant year ≥2000 (p = 0.005), low or intermediate risk disease status at cbt (p = 0.008) and infused tnc ≥ 4.1 × 10 7 /kg (p = 0.04) were independently associated with improved os. five-year os in al patients was associated with remission status (p = 0.001): 77% for patients in cr1, 50% in cr2, 32% in cr3 and 21% in advanced phase of the disease. improving results over time and the low toxicity of related hla-identical cbt in malignancies support banking and use of family cb units with suffi cient cell dose. in the past, the outcome of adolescents with acute lymphoblastic leukaemia (all) in 2nd complete remission (cr) given hsct s72 prophylaxis was carried out with atg or okt3. primary engraftment occurred in 85%. after reconditioning, fi nal engraftment was achieved in 98%. gvhd grade 0-1 occurred in 86%. 12% had grade ii, 4% had grade iii. chronic gvhd occurred in 4 patients. no gvhd related mortality was observed. median follow up was 5.9 years. efs at 1 year was 56% (cr1), 51% (cr2) and 50% (≥cr3); efs at 5 years was 49% (cr1), 46% (cr2) and 27% (≥cr3). median survival of patients with active disease was 0.2 years. relapse rates at 1 year were 0.32 (cr1), 0.47 (cr2) and 0.46 (≥cr3); relapse rates at 5 years were 0.32 (cr1), 0.47 (cr2) and 0.64 (≥cr3). trm at 1 year was 14%, causes of death remained viral and fungal infections, no ebv lpd occurred. conclusions: positive selection of progenitor cells or depletion of t and b cells can minimize acute and chronic gvhd in both matched unrelated and mismatched related transplantations and may prevent gvhd related mortality. lethal infections occurred in 14% of the patients probably due to the delayed recovery of t cells. despite profound depletion of donor t cells, relapse rates were acceptable and remained on a stable level after the fi rst year in patients with complete remission. thus, absence of gvhd may not necessarily be associated with high relapse rates in childhood all. apart from t cells, other effector cells like nk cells are likely to exert gvl effects and may contribute to the favorable efs of our patients. fanconi anemia (fa) is a rare disorder characterized by progressive bone marrow failure, congenital abnormalities and a predisposition to cancer. bmt is the only treatment able to restore normal hematopoiesis in this disease. here, we update the brazilian experience using only cyclophosphamide 60 mg/ kg (cy60) as a preparative regimen for the treatment of pts with fa. objective: analyze the outcome of 70 pts with fa submitted to a related bmt using cy60 mg/kg. material and methods: period: 07/1999-07/2009; gender: 32f/38m age: 5-34 ys (m: 10,5). type of donors: matched siblings donors (msd): 60 pts; other related donors (ord): 10 pts. 68 pts were in aplastic phase while 2 had mds. all pts received a preparative regimen with cy60 mg/kg and gvhd prophylaxis with cyclosporine and methotrexate. statistical analysis was performed using spss. overall survival (os) was estimated using the kaplan-meier method. multivariate analysis was performed by cox method. results: 60 pts are alive between 135-3530 days (m: 1663) after bmt. os: 85,5% at 5 ys. pts transplanted below the age of 10 (37pts) had an os of 97,1% at 5 ys. there was no statistical difference between transplants performed in curitiba(56pts) and other bmt centers (86,1% × 83,2%). 2 pts died before d + 28 and were not evaluable for engraftment. only one pt had primary graft failure and he is alive and well 5 ys after the 3rd transplant. late rejection occurred in 6 pts (2 pts with mds, 2 pts with ord, 2 pts with msd) at a median of 234 days after bmt. 2/6 pts are alive and well after a 2nd bmt. cumulative incidence of rejection at 1 y was 10%. pts receiving > 25 transfusions had a lower survival when compared to those less transfused (91% × 62% p = 0,03). mucositis grade ii-iii occurred in the majority of pts;13/67 pts developed acute-gvhd grade ii-iv has been reported to be worse than that of children. in order to evaluate whether in recent years the outcome of adolescents has become more similar to that of children, we studied paediatric patients (age < 18 years at transplantation) reported to the aieop-hsct group registry who received allogeneic hsct for all in 2nd cr in a paediatric institution between 01/1990 and 12/2007. a total of 400 patients (64% males and 36% females) were anallyzed. 339 patients were children (<14 years old), whereas the remaining 61 patients where adolescents (14-18 years old). median age at hsct was 8.3 and 16.1 years, for children and adolescents, respectively. a mfd was employed in 50% of children and in 58% of adolescents; the remaining patients having received a mud hsct. conditioning regimen included tbi in 90% of patients, in most cases associated with thiotepa and cyclophosphamide. cyclosporine (csa) alone was used for gvhd prophylaxis in 88% of patients transplanted from a mfd, whereas the combination of csa, mtx and anti-thymocyte globulin (atg, 3.75 mg/kg from day -4 to day -2) was employed in 77% of patients given mud hsct. no difference between children and adolescents was observed for patient-and transplant-related variables, with the exception of the infused cell dose which was greater in children. as of october 2009, the probability of developing grade ii-iv, grade iii-iv acute gvhd or chronic gvhd was 54%, 19% and 32% for children, and 49%, 14% and 43% for adolescents (p = ns). eight-year probability of efs,trm and ri were 53%, 22% and 25% for children and 55%, 23% and 22% for adolescents, respectively (p = ns). univariate analysis showed that s3 + s4 bfm classes at relapse (p<0.0001), recipient male gender (p = 0.01), mud hsct (p = 0.02), grade 0 or iv acute gvhd (p<0.0001) were poor-risk factors for efs. only bfm class at relapse and acute gvhd (grade 0 or iv) maintained their prognostic signifi cance in multivariate analysis. adolescents and children <14 years of age, treated in paediatric institutions, had the same outcome. multivariate analysis confi rmed the gvl favourable effect in patients with grade i-iii acute gvhd.results obtained with mud hsct were comparable to those achieved with mfd hsct. adolescents should be referred for transplantation to treatment teams that have experience in the management of childhood all. long-term survival and relapse rate after transplantation of highly t and b cell-depleted stem cells from alternative donors in paediatric patients with acute lymphatic leukaemia p. lang (1) we present long term results in 68 children with all who received highly t and b cell depleted stem cells from matched unrelated (n = 17) or full haplotype mismatched related donors (n = 51) at our institution. our aim was to minimize gvhd and to avoid ebv lpd in both matched and mismatched transplantations. remission status was: cr1, n = 18; cr2, n = 23; ≥cr3, n = 12; non remission or second transplant, n = 15. graft manipulation was carried out with microbeads and the clinimacs tm device (indirect depletion of t and b cells with cd34 + positive selection (n = 50); or direct depletion with anti-cd3/anti-cd19 coated microbeads (n = 18)). t and b cell counts were reduced for 4-5 log with median numbers of residual t cells of 15 000/kg bw (cd34 + selection) and 49 000/kg bw (cd3/19 depletion). no pharmacological immune suppression was given after positive selection, whereas patients with cd3/19 depletion received mmf. the conditioning regimens were either tbi or bu based (n = 50) or a toxicity reduced protocol (flud, tt, mel) was used (n = 18). rejection more than 500 candidate donors were evaluated at our institution (median age 43 y, r 16-79 y; 185 donors ≥50 y; male 283). 460 donors were evaluated with marrow smear and cytogenetic test, 328/460 were eligible for donation (4 bone marrow harvest, 324 peripheral blood gcsf mobilization), 10 proceeded to stem cell donation without marrow evaluation. 393 out of 460 marrow were morphologically normal (85,43%), 58 (12,63% -median age 54 y, 31 donors >50 y) presented abnormalities, 9 (1,96%) were not evaluable. we registered 19 cases of plasma cells hyperplasia (10 not eligible to donation) 7 (1,52%, 5 donors > 50 y) presented abnormalities, 1 was not evaluable. we registered 5 cases of chromosome y deletion (4 not eligible to donation), 1 of pericentric inversion of chromosome 3 (p22;q29 additional follow-up will give us more important information on safety of hsc donation procedure. o373 impact of kir-ligand mismatches and kir genotypes on the outcome of patients receiving unrelated donor stem cell transplantation for lymphoid malignancies m. morelli (1), a. bermema (1) all pts received rabbit anti-thymocyte globulin for in-vivo t cell depletion. nineteen donor/recipient pairs were matched at molecular level for the hla-a, b, c, drb1, dqb1 loci and 30 were mismatched (27 at class i, 6 at class ii). the majority of pts had chemosensitive disease (n = 38, 78%). the median follow-up was 24 months (range 3-65). we studied kir genotypes using the kir-typing kit (miltenyi biotec) and hla-cw kir ligand using high-resolution molecular techniques. results: twenty-one pts were c1/c2 heterozygous, 16 were c1/c1 and 12 were c2/c2 homozygous. in the combination patient c1 absent/donor kir2dl2/kir2dl3 present (n = 12, 25%), we observed no trm and only 3 cases of acute gvhd (agvhd) as compared to other combinations (n = 37) in which 3 cases of trm and 11 cases of agvhd were observed. this combination was associated with a trend of better cumulative incidence of trm, os, and pfs os: 61% vs 67%) or in the recipient (n = 36, 74%) showed no signifi cant association with a better os or pfs. sibling donors (n = 8), haploidentical cd3/cd19 depleted grafts (n = 3), cd34 + selected graft with okt3 (1) hlh (5), leukocyte adhesion defi ciency (4), chronic granulomatous disease (3), severe immune dysregulation (3), other pid (15) mfd (13) hla matched 7-10/10). 16 of 45 were cords (7-10/10 hla matched). follow up is 2-59 months (median 16 months) 13 children died: 6/40 in the treosulfan/fl udarabine group, 7/30 in the treosulfan/cyclophosphamide group -hlh disease day-1, graft rejection and cmv, infection (4), gvhd, gvhd +infection, gvhd + cerebral infarcts, cerebral haemorrhage, pneumonitis, vod and mds/ aml. skin toxicity was common. vod occurred in 2 children in combination with cyclophosphamide and both had enterovirus in the gut. 2 patients rejected (mhc ii defi ciency successfully retransplanted autoimmunity after bmt in primary immunodefi ciency diseases: single-centre report of 184 children had aml (10 in cr1 at high risk, 10 in ≥cr2 and 2 in relapse), 5 had all (4 in cr1; 1 in relapse) and 1 had high grade nhl in relapse. conditioning was: 8gy single fraction tbi, thiotepa (4 mg/kg × 2), fl udarabine (40 mg/m² × 5), cyclophosphamide (35 mg/kg × 2) no gvhd developed in 24/26 patients, 2 developed ≥ grade ii gvhd. ten patients died (3 vod, 2 fungal pneumonia, 1 bacterial sepsis, 1 cns aspergillosis, 1 systemic toxoplasmosis, 1 adenoviral infection, 1 mof). cd4 and cd8 counts reached, respectively, 50/μl medianly on days 34 (range 19-63 days) and 24 (range 15-87)of cmv reactivation were signifi cantly fewer than after our "standard haplo" transplants. in kir ligand-mismatched transplants, speed of nk cell reconstitution/maturation and size of donor vs. recipient alloreactive nk cell repertoires were preserved. in conclusion, in the setting of haploidentical transplantation infusion of tregs makes administration of a high dose of t cells feasible for the fi rst time donor nkt cells and outcome following hla-identical peripheral blood stem cell transplantation d. nachbaur oral session 15: myelodysplasia / regulatory issues and quality management o350 monosomal karyotype, higher blast count and ex vivo t-cell depletion predict poor outcome after allogeneic stem cell transplantation for mds/saml patients with chromosome 7 abnormalities m. van gelder, j. schetelig, l. volin, j. maertens, g. socié, e. petersen, h. thomssen, a. biezen, r. brand, t. de witte, n background: high-risk mds/saml patients with a chromosome 7 abnormality have a poor prognosis with conventional therapies. these patients usually proceed to allogeneic sct (allo-sct). data on the effect of this approach are scarce. objective. description of outcome and identifi cation of risk factors of allosct in mds/saml patients with a chromosome 7 abnormality. methods: from the ebmt database 277 patients with mds/saml having any chromosome 7 abnormalities (median age 45 years) who underwent fi rst allosct between 1981 and november 2006 were identifi ed. stem cells from related donors were transplanted in 191 patients (177 hla-identical) while 85 received unrelated grafts. bone marrow was used as stem cell source in 148 patients. standard conditioning was applied in 222 patients. sixty-three patients fulfi lled the criterion for complex cytogenetic abnormalities (ca). a monosomal karyotype (mk), defi ned as at least one autosomal monosomy and at least one other chromosomal abnormality (breems da et al., jco 2008; 26:4791) , was present in 68 patients, of whom 24 did not have ca. results: median follow-up (fu) of patients alive at last fu was 5 years (range 0-18 years). estimate 5-year overall (os), and relapse-free (rfs) survival was 28 ± 6% and 26 ± 6% respectively. the relapse rate at 3, 12 and 60 months was 9 ± 3%, 29 ± 5% and 39 ± 6% resp. non-relapse mortality at 3, 12 and 60 months was 21 ± 5%, 36 ± 6% and 40 ± 6% resp. in multivariate models including age, mk, ca, % blasts at allosct, donor type, sex match, conditioning regimen, t-cell depletion and year of allosct three factors remained statistically signifi cant predictors for poor outcome: mk, ≥5% blasts at allosct and ex vivo tcd (adjusted hr for os resp. 1.65 [1.15 -2. 35 95%ci], 1.67 [1.14 -2. 46 95%ci] and 1.83 [1.21 -2.76 95% ci] resp.). when patients with ex vivo tcd were excluded, 5-year os in the remaining 97 mds/saml patients with any chromosome 7 abnormality and <5% blasts at allosct with or without mk was 8 ± 14% and 53 ± 12% resp. and for the 91 patients with ≥5% blasts 5-year os was 9 ± 12% and 29 ± 11% resp (see figure) . conclusion: this large study on outcome of allosct for patients with mds/saml and any chromosome 7 abnormality shows that long-term survival can be achieved in patients without the poor risk factors mk, ≥5% blasts at allosct and ex vivo tcd. for patients with mk new approaches that tackle the high and early relapse rate are warranted. bone marrow fi brosis on outcomes of patients with mds/saml undergoing allogeneic stem cell transplantation n. kröger, t. zabelina, a. van biezen, r. brand, t. bone marrow fi brosis has an important impact on the prognosis of patients with mds. we evaluate the impact of bone marrow fi brosis in 721 patients who underwent allogeneic hematopoietic stem cell transplantation (hsct) for mds/aml and were reported to the ebmt. no fi brosis was noted in 483 pts, mild or moderate fi brosis in 199 pts and severe fi brosis in 39 pts. diagnosis in the none, mild/moderate and severe fi brosis group were ra/rars (36%, 39% and 30%), raeb (43%,43% and 48%) and raeb-t (21%, 18% and 20%). stem cell source were from related (63%, 63% and 62%) or unrelated (36%, 36% and 38%) donors. leukocyte engraftment (>1.0 × 10 6 /l) was observed after a median of 16, 17 and 19,5 days for the days for the none, mild/moderate and severe fi brosis group, respectively (p = 0.002). there was a trend for more graft failure in severe fi brosis group (10%, p = 0.07). in a multivariate analysis bone marrow fi brosis did not infl uence non-relapse mortality, but severe fi brosis signifi cantly infl uenced risk of relapse (hr 5.7, p = 0.02), resulting in a reduced disease-free (hr 5.9, p = 0.01) and overall survival (hr 7.5, p = 0.006). other factors for reduced survival were advanced disease at transplant (hr 14.9, p = 0.005), non-cr before transplant (15.8, p = 0.001) and hla-mismatch (hr 5.3, p = 0.02). in summary, apart of the disease status, no complete remission at transplant, and hla mismatch transplantation, severe bone marrow fi brosis is an independent prognostic factor for disease-free and overall survival for mds patients undergoing allogeneic stem cell transplantation. management and outcome of myelodysplastic syndrome (mds) and secondary acute myeloid leukaemia relapsing after allogeneic stem cell transplantation -a retrospective analysis on 888 patients by the mds subcommittee of the ebmt chronic leukaemia working pa c. schmid, a. van biezen, r. brand, j. finke, l. volin, a. vitek, d. selleslag, d. heim, p. van dem borne, a. ganser, m. mohty, m. boogaerts, t. de witte, n the aim of this study was to obtain reliable data on management and outcome of relapse after allogeneic sct for mds and saml in adults. median age of 888 patients was 51 years. patients (pts.) had been transplanted for ra/rars (8%), raeb (19%), raeb-t (13%) and saml (60%), either previously untreated (20%), in cr (44%), relapse/progression (13%) or primarily refractory (24%). standard (sic) and reduced intensity (ric) transplants were performed in 59% and 41%, donors were id siblings (58%), mud or mm relatives (42%), and 5 syngeneic twins. median time from sct to relapse was 6 mo . median follow up from relapse of 188 survivors was 6.7 mo. overall survival (os) at 1, 2 and 4 years was 24%, 16% and 10%. in a cox regression model, the mds subtype at time of transplant (ra/rars vs. raeb [hr 1, 524, ci 1, 390, ci 0, 071 vs. saml hr = 2, 209, ci 1, 114, p<0.001) , and the interval from sct to relapse (1st vs. 2nd [hr = 0, 770, ci 0, 969] vs. 3rd [hr = 0, 609, ci 0, 768] vs. 4th quartile [hr = 0,404, ci: 0,318-0,513], p<0.001), were associated with survival. in 342 pts., data on the management of relapse were available. 223 pts had received dli. among them, median time from sct to relapse was 6.5 mo, median time from relapse to dli1 was 1 mo. 149, 42 and 32 patients received 1, 2, and 3 dli. median os from relapse was 7.6 mo. again, remission duration after sct (p<.001) and less advanced disease at time of sct (p = .038) were associated with better os. constitutional variability in genes involved in innate immunity (irf-3, hamp, ptx3) and in cell proliferation (atbf1 and nfat5) infl uences disease free survival after allogeneic stem cell transplantation b. martín-antonio, i. alvarez, f. márquez-malaver, p. trujillo, m. carmona, j. falantes, i. espigado, á. urbano-ispizua hospital universitario virgen del rocío (seville, es) genes involved in innate immunity and in regulation of cell proliferation may play an important role in infections and modulating the intensity of the infl ammatory response after allogeneic stem cell transplantation (allo-sct). thus, genetic variability in donor and recipient in these genes might be an important factor infl uencing the outcome of allo-sct. we have studied the potential infl uence of 15 single nucleotide polymorphisms (snps) in donor and recipient in genes of innate immunity (irf-3, hamp, ptx3, hbd2) and regulation of cell proliferation (atbf1, nfat5, akt2, nm1, cd151, tcirg1, sh3kbp1) on clinical outcomes after allo-sct, specifi cally on the incidence of acute gvhd, transplant related mortality (trm), relapse, and disease free survival (dfs). study population consisted of 106 donor-patient pairs undergoing hla identical sibling allo-sct in a single institution. patient median age was 38 years (range, 5-66). 42% of the patients were in advanced phase of disease. cumulative incidence for acute gvhd, trm, relapse and dfs was computed with the cmprsk package and with kaplan-meier. patient irf3 rs2304205 aa, donor atbf1 rs719327 aa and patient akt2 rs12460555 cc dominant genotypes, were associated with a higher incidence of relapse (p = 0.02, p = 0.04 and p = 0.002, respectively) and lower dfs (p = 0.02, p = 0.04 and p = 0.009, respectively). all of them retained signifi cance at multivariate analysis. variant rs719327 aa in atbf1 showed the same prognostic values when present in donor or in patient (relapse: 55% vs. 34% and dfs: 26% vs. 46%). when it was present both in donor and patient, the differences were more prominent (relapse: 69% vs. 33%, p = 0.003 and dfs: 15% vs. 45%, p = 0.01). donor hamp rs7251342 ag genotype and donor nfat5 rs6499244 aa dominant genotype were associated with a lower and a higher incidence of trm (p = 0.007 and p = 0.02, respectively) infl uencing in a higher and lower dfs (p = 0.04, p = 0.02, respectively). they retained its signifi cance at multivariate analysis. nfat5 is necessary for optimal t cell development and rs6499244 aa variant showed the highest mrna expression. interestingly, none of the 25 patients with a donor carrying ptx3 rs18040680 aa recessive genotype had trm but showed a higher tendency in the incidence of relapse (61%). in conclusion, genetic variability in innate immunity and in cell proliferation has a strong infl uence on the clinical outcome of allo-sct, which might be important when choosing allo-sct protocols. the factors to predict adverse events occurred within 30 days of post-peripheral blood stem cell donation at family donors y. kodera, s. kim, k. nagafuji, m. hino, k. miyamura, r. suzuki, a. tamakoshi, m . fukushima on behalf of the japan society for hematopoietic cell transplantationbackground: it has become obvious that certain adverse events might occurre at peripheral blood stem cell (pbsc) donors during or after the donation process. it is therefor crucial that certain factors which can predict the occurrence of such adverse events at normal donor are cralifi ed to prevent the adverse events. the jshct has continued the nation-wide consecutive follow up of pbsc family donors and in this project, the day 30 report of post donation was included. this time, we present the outcome of the day 30 report analysis. materials and methods: among 3,264 pbsc family donors who were consecutively pre-registered to jshct donor center between april 2000 and march 2005, 2,873 day 30 reports were obtained and subjected for analysis. the relationship between family donors' 1) gender, 2) age, 3) body-weight, 4) daily and 5) total dose of granulocyte-colony stimulating factor (g-csf), 6) current and 7) past health conditions and the occurrence of 1) thrombocytopenia, 2) prolongation of hospitalized period, 3) any adverse events (bone pain, fatigue, head ache, insomunia, anorexia, nausea, vomiting, splenomegary) as well as the mobilized cd34 + cell numbers were statistically examined. results: risk factors for thrombocytopenia were higher total dose of g-csf and older age. risk factors for prolongation of hospitalized period were higher total dose of g-csf, any present illness, older age and low body-weight. the risk factor for bone pain was higher body-weight. the risk factors for fatigue were female and lower body-weight. the risk factors for insomnia were older age and female and the risk factors for anorexia were female and low body-weight. predictive factors for lower cd34 + cell mobilization/donor's body-weight were older age and higher total g-csf administration, and predictive factors for higher cd34 + cell mobilization were male and younger age. discussion: it was revealed that certain adverse events which occurred within 30 days of post-donation and cd34 + cell numbers to be mobilized could be predicted by utilizing the basic information of donors and of pbsc mobilization/harvest process. to predict them, to notify them to donors and to prepare for possible events will contribute to keep pbsc donor's safety and trust. background and aim: allogeneic hematopoietic stem cell transplantation (hsct) has become an established therapy and the numbers of such procedures increase year by year. the risk for while 17/66 pts developed chronic-gvhd (extensive: 11 pts). in the multivariate analysis only acute-gvhd grade ii-iv and extensive chronic-gvhd were associated with a lower os. 3 pts developed oral squamous cell carcinoma (scc) between 3-5 ys after bmt (all had c-gvhd) and 1pt died. 10pts died between 12-2034 days after bmt. major causes of death were related to rejection or gvhd. trm at 100 days: 4% and at 1 y: 8%. conclusions: this regimen was well tolerated and had an excellent survival especially for pts below the age of 10. long term follow up is still needed to determine the incidence of cancer in this group of pts. survey of outcomes of unrelated cord blood transplant in patients with haemoglobinopathies: a retrospective study on behalf of cibmtr, nycb and eurocord a. ruggeri (1) allogeneic hematopoietic cell transplantation (hct) from hla-identical donors is curative in patients with hemoglobinopathies. in order to extend the availability of hct, unrelated cord blood transplantation (ucbt) has been investigated as an alternative. between 1996 and 2009, 51 patients receiving a single ucbt for hemoglobinopathy were reported to the eurocord and cibmtr registries. thirty-four had thalassemia major (thal) and 17 had sickle cell disease (scd). all thal patients were transfusion-dependent with a median time from diagnosis to ucbt of 26 months and 12 of 17 patients with scd had a history of stroke. median age at ucbt was 5 years and median follow-up was 2 years. cord units were matched at 6 of 6 (15%), 5 of 6 (34%) and 4 of 6 (51%) hla loci (antigen-level for class1, allele-level for class 2). median infused cell dose was 4.6 × 10 7 /kg (1.5-13). thirty-nine patients received a myeloablative conditioning regimen with busulfan (bu) and cyclophosphamide (n = 33) or bu with other agents (n = 6). reduced intensity conditioning regimens (ric) (n = 12) were fl udarabine-based with bu < 8 mg/kg or melphalan < 150 mg/m². cumulative incidence (ci) of neutrophil recovery at 60d was 72±6%, with 35 of 51 patients reaching neutrophil recovery at a median time of 22 days. higher cell dose (>4.6 × 10 7 /kg) (80% vs. 54%, p = 0.004) and ucbt performed after 2004 (75% vs. 61%, p = 0.001) were associated with improved neutrophil recovery. day-100 chimerism analysis was available for 47 patients: 19 patients had complete donor chimerism, 4 mixed chimerism and 24 autologous recovery. among 16 patients who did not achieve neutrophil recovery, 5 had available data on subsequent treatment. two had an autologous back-up and one received a second ucbt which engrafted. ci of grade ii-iv acute-graft versus-host disease (gvhd) was 23% and 9 patients had chronic gvhd. the 2-year probability of overall survival was 77%. thirty-eight patients (22 thal, 16 scd) are alive, 16 with full donor chimerism (8 thal, 8 scd). of the 13 deaths, 5 occurred prior to day-100, mainly due to infections. no deaths due to gvhd were reported. ten of 12 patients who received ric are alive, 4 with donor engraftment. despite the small number of subjects, these results show a particularly high risk of graft failure after ucbt for hemoglobinopathy. tnc dose plays a key role in engraftment. novel approaches modifying the conditioning regimen and/or increasing cell dose in prospective clinical trials are needed. graft rejection and second haematopoietic cell transplantation in patients with thalassaemia major s. santarone, e. di bartolomeo, p. bavaro, p. di carlo, p. olioso, g. papalinetti, p. di bartolomeo bmt center (pescara, it) graft rejection for patients with thalassemia major (tm) receiving haematopoietic cell transplantation (hct) includes early graft failure (gf) (no evidence of engraftment), late gf (lost of engraftment), and recurrence of tm. we examined our series of 126 consecutive patients aged 1 to 29 years (median 10 years) with tm who were transplanted either from hla-identical related (n = 123) or unrelated (n = 3) donor. conditioning regimen consisted of busulfan (bu) and cyclophosphamide (cy). graftversus-host disease (gvhd) prophylaxis included cyclosporine (csa) and methotrexate (mtx). all patients received bone marrow (bm) cells. the 10-years overall cumulative incidence of graft rejection was 9% + 0.07. the impact of pre-transplant patient risk factors (age, gender, number of transfusions, ferritin, splenectomy, ast, alt, hepatomegaly, hbv and hcv infection, liver fi brosis, cmv serology), donor characteristics (age, heterozygous state) and hct regimens (dose of bu, gvhd prophylaxis, bm dose) was studied in univariate analysis. no factor was univariately associated with signifi cantly increased probability of graft rejection. eleven events occurred: 4 early gf, 2 late gf, and 5 recurrences of tm. patients with either early or late gf underwent an urgent second hct, whereas 3 of 5 patients with recurrence of tm choose to be submitted to second hct. at all, 9 patients received a second hct from the same donor. their median age was 14 years (4-19). conditioning regimen for second hct was immunosuppressive for 5 patients (atg in addition to fl udarabine (flu) or cy) and myeloablative for 4 patients (bucy in 1 and thiotepa (th), treosulphan (treo) and flu in 3). gvhd prophylaxis consisted of csa alone for 5 patients and csa /mtx for 4 patients. six patients were given bm cells and 3 received peripheral blood stem cells. results: 2 patients died from hct related causes (heart failure at day 29 and acute gvhd at day 83, respectively). seven patients (78%) are living. one patient had no sign of engraftment and is now living with recurrence of tm following a third hct with double unrelated cord blood cells. tm recurred in an other patient at 3 months post-second hct. this patient is now living with transfusion therapy. five patients (56%), including the 3 patients transplanted with th-treo-flu regimen, are cured with a median follow up of 3 years (0,2-21). this study shows that the incidence of graft rejection following hct for patients with tm is low and second transplant is successful in an high proportion of patients. treosulfan is a bi-functional alkylating agent which causes less vod than busulphan and does not require anticonvulsant prophylaxis and drug monitoring. we report the use of treosulfan in 70 children undergoing hct for primary immunodeficiency (pid) at two supraregional transplant centres in the uk. children received 42 g/m² or 36 g/m² treosulfan in combination with either cyclophosphamide 200 mg/kg (30) or fl udarabine autoimmunity is a complication after haematopoietic stem cell transplantation in patients with primary immunodefi ciencies (pid) that sometime occurs when bmt is performed from alternative donors with extensive stem cell manipulation. the fact that only purifi ed stem cells (cd34 positive cells) are injected could lead to a slower and incomplete post-transplant immunological reconstitution. since the majority of children treated in our unit received haploidentical cd34 positively selected grafts, we retrospectively analysed the population of pid patients grafted in our institution between 1990 and 2008. of the 114 affected by severe combined immunodefi ciency (scid), 70 received an haploidentical transplant, while the others received a matched unrelated or a hla-identical transplant. 94 patients were affected by inborn errors; 15 received an haploidentical transplant, the others received a matched unrelated or a hla-identical transplant.we analysed the incidence of autoimmunity in these two groups of patients. out of 184 children, 17 experienced autoimmune symptoms after hsct. among this group, 11 had a diagnosis of scid, 2 of leaky scid, 2 of omenn's syndrome, one of chediak-higashi and one of wiskott-aldrich syndrome. autoimmune symptoms included autoimmune haemolytic anaemia (8), autoimmune hypotiroidism (6 patients), cutaneous autoimmunity (2), vasculitis (1) and chronic bronchoreactivity. all patients except for 2 received a myeloablative conditioning therapy. 10 of them received an haploidentical bmt and 7 a mud or a hla-identical bmt. no correlation was found analysing onset of autoimmunity and split chimerism in all groups of patients nor we could fi nd a correlation with immunsuppressive therapy. in conclusion we found a low incidence of autoimmunity despite the fact that the majority of our patients received a cd34 positive selected graft with an even distribution of the complication independently from the type of donor or stem cell manipulation procedure. introduction: there is still a discussion about the best bm harvesting method to obtain enough progenitor cells during the bone marrow (bm) harvesting procedure. the technique used in most bmt centers is the multiple aspiration of a small (2 ml) amount of bm from the iliac crest, sternum or tibia. this is proposed to minimize the dilution with peripheral blood. others are using few large amount aspirations. this procedure was not evaluated in children so far. to fi nd out possible differences in graft composition between this two methods and to evaluate the feasibility in children we initiated the following prospective study. patients and methods: 20 bm harvestings were done in 18 patients (median age 8,78, (2,48 -16,6 y), f 5, m 13). all patients were transplanted in our bm transplantation unit. there were 7 bm harvests for autologous bmt and 13 for allogeneic bmt. the autologous donors were median age 6,93 (2,5-17 y), f 1, m 5), the allogeneic donors were median age 19,75 (6,45-50 y), f 8, m 5). the amount of harvested bm was median 900 ml (440-2380 ml) and median 37 ml/kg bw of the recipient (20-55 ml/kg). the method a was defi ned as an aspiration of 2 ml bone marrow at most before the position of the needle was changed. the method b was defi ned as an aspiration of 100 ml at least before the location of the needle was changed. the bm was collected and analyzed among others for mnc, cd 34 positive progenitor cells, cfu, and t-cells. two operators carried out the bm harvest. one began with method a and changed in the second part to method b. the second operateur carried out the procedure in the opposite way. both operators collected the same amounts for each harvesting method so long that the amounts of bm harvested in one method was identically with the second. results: we found no statistically signifi cant difference between method a and b regarding the content of mnc, cd3 + t-cells, and cfu (mnc/ml 824572 a versus 725000 b wilcoxon test p = 0.7; mnc/kg 3.1 10e07 a versus 2.9 10e07, p = 0.3; cd3/ml 162500 a versus 300000, p = 0.3; cfu/10e05 mnc 1678 a versus 1315 b, p = 0.09), but a clinically not relevant but statistically signifi cant difference between cd34 positive cells (cd34/kg 2.62 a versus 2.09 b, p = 0.045). all transplanted patients showed a regular engraftment. conclusion: bm harvest by large amount few punctures method (b) is as suffi cient as the common used small amount frequent punctures method (a), and could be therefore used equally. haploidentical transplantation, with extensive t cell depletion to prevent gvhd, is associated with a high incidence of infectionrelated deaths. the key challenge is to improve immune recovery with allogeneic donor t cells without triggering gvhd. as t regulatory cells (tregs) controlled gvhd in preclinical studies, background: natural killer t (nkt) cells represent a small but signifi cant lymphocyte population which have been demonstrated to play a regulatory role in autoimmune disease as well as in gvhd/gvl effects. methods: the number of cd3 + cd56 + nkt cells in the graft was retrospectively correlated with clinical outcome of fi fty-eight patients receiving a fi rst allogeneic peripheral blood stem cell transplant (myeloablative conditioning, n = 31; reduced-intensity conditioning, n = 27) from their hla-identical sibling donors between dec 2004 and jul 2009. results: a median number of 0.15 (range, 0.01-0.57) × 10 8 /kg cd3 + cd56 + nkt cells was transplanted with the graft. nkt cells within the graft signifi cantly correlated with the number of cd3 + t cells, cd56 + nk cells, and mnc (p<0.05). overall survival for the entire cohort at two years was 55% (41%-69%, 95% ci) with no difference between pts. receiving a high (>0.15 × 10 8 /kg) or low (<0.15 × 10 8 /kg) nkt cells. the cumulative relapse incidence for the entire cohort was 40%, with a trend for a lower relapse incidence in pts. receiving a low vs. high nkt cell dose (32% vs. 47%). the nonrelapse mortality was 19% showing a trend for a lower nrm in patients receiving a high nkt cell dose (12% vs. 27%). the cumulative incidence of agvhd ii-iv was 38% for the entire cohort. the incidence of agvhd ii-iv was lower in pts. receiving a high nkt cell dose (30% vs. 45%). the cumulative incidence of cgvhd in pts. at risk was 48%, with no difference between pts. receiving low or high nkt cell numbers. by multivariate cox analysis including cd3, nk, nkt, treg, cd34 cell number, recipient age, and time interval between diagnosis and sct as numerical variables, and risk category by the underlying disease (standard risk vs. high risk), sex mismatch (male recipient/female donor vs. other combinations), and myeloablative vs. reducedintensity conditioning as categorical variables the most powerful predictive parameters for survival were standard vs. high-risk disease (p = 0.01), a high nk (p = 0.02) and a low nkt cell dose (p = 0.05). the most powerful predictive parameters for relapse were high risk disease (p = 0.01), a low nk (p = 0.02) and a high nkt cell dose (p = 0.08). discussion: these data indicate that in the setting of peripheral blood stem cell transplantation from hla-identical sibling donors a higher number of nkt cells in the graft might increase the risk of relapse by lowering the incidence of acute gvhd. objectives: adoptive transfer of nk cells with or without previous allogeneic progenitor cell transplantation may represent a promising therapeutic option in patients with hematological or oncological diseases. different nk cell isolation strategies have been pursued. here, two different methods were compared with respect to recovery, immunophenotype and cytotoxic capacity of purifi ed nk cells. methods: nk cells of healthy donors were isolated from a lrs chamber of a platelet apheresis. cells were enriched by (i) cd56 positive selection of t cell (cd3) depleted lymphocyte fractions (method i, mi) or (ii) depletion of non-nk cells (untouched protocol, method ii, mii) by magnet-activated cell sorting (macs). recovery of nk cells obtained by m i or ii were compared. nk cells derived by mi were activated by stimulation with interleukin (il)-2 (100u/ml) or il-15 (10ng/ml) overnight and analyzed for cytotoxic activity. for expansion of nk cells cd2/cd335 antibody coated macsibeads (miltenyi) were used as artifi cial stimulators in il-2 supplemented media (500u/ml) in a two weeks culture. finally, immunophenotype of effector cells and killing of target cells were assessed. results: median nk cell recovery was 35.9% (n = 4) and independent of strategy of enrichment (mi versus mii). overnight activation of nk cells led to a 2-fold enhanced killing of k562 cells. nk cell expansion led to a 100-fold increase in numbers of functional active nk cells. expanded nk cells showed characteristics of activated nk cells as indicated by the induced expression of nkp44 (cd336). surface expression of tac-tile (cd96), a marker for certain activated immune cells, was found with cd96 antibody th-111 elevated on bead-expanded nk cells in comparison to nk cells cultured only in il-2 conditioned media, which may enhance reactivity of expanded nk cells against the ligand of cd96, namely polio virus receptor expressing tumours. conclusion: t cell depletion and cd56 positive selection led to an acceptable recovery of enriched nk cells that could be successfully activated by il-2 or il-15. the described nk cell isolation and activation protocol can easily been transferred into a gmp laboratory. for clinical application, further expansion of isolated nk cells under gmp conditions may be preferable. nk cells enriched, expanded and activated by these means may be an adoptive cellular therapeutic option that in addition may be improved by combination with tumour antigen specifi c antibodies. s76 o386 a randomized trial comparing cd34 enriched grafts versus cd3/cd19 depleted grafts in partial t-cell depleted allogeneic stem cell transplantation n. schaap, d. eissens, f. preijers, a. van der meer, a. schattenberg, h. dolstra, w. van der velden, t. de witte, m. smits, n.m.a. blijlevens radboud university medical centre (nijmegen, nl) we initiated a randomized trial in aml, all and mds patients (pts) treated with partial t cell depleted allogeneic sct using immunomagnetic cd34 selection versus cd3/cd19 depletion. a benefi cial effect was hypothesized on long term engraftment and dfs using cd3/cd19 depletion. residual cell populations in the graft after cd3/cd19 depletion may play a protective role during early phase after sct and generate an increased gvl effect. from may 2006 until may 2009 pts with aml, all and mds were stratifi ed for diagnosis and randomized. median age of the pts was 46 and 45 yrs, respectively. stem cells were obtained after g-csf (10 ug/kg). t cell add back to a fi xed dose of 5 × 10e5 cd3 + t cells/kg bw was standard in all pts. pts without significant (> grade 2) acute and/or chronic gvhd were eligible for prophylactic dli. in this interim analysis we determined immune reconstitution of t cells, b cells and nk cells using fl ow-cytometry and functional essays and evaluated clinical outcome. reconstitution: nk cells are completely maintained in cd3/ cd19 depleted transplants. b cell depletion is equally effective. cd34 + cells are signifi cantly higher in the cd3/cd19 depleted grafts (median 4.3 versus 6.5 × 10e6/kg, p<0.05). all pts engrafted and the time to engraftment was not different. in the fi rst 3 months after sct, the immune reconstitution of lymphocytes, especially cd4 t cells, nk and nk-t cells was faster in the cd3/cd19 group. fourteen days after sct the cytotoxic nk and regulatory nk subsets were already present in the cd3/cd19 group whereas the cd34 group needed 2 months to full recovery. the cytolytic response against a leukemia target was stronger in the cd3/cd19 group (p = 0.02). nk receptor expression of nkg2a (inhibitory) was strongly decreased whereas expression of nkg2c (activating) was enhanced. clinical outcome; acute gvhd > grade 2 was 17% in both groups. extensive chronic gvhd was 13% (cd34) vs. 18% (cd3/cd19. one year trm, rr, lfs and survival using cd34 selection and cd3.cd19 depletion were 5% vs. 39% (p = 0.066), 36% vs. 39% (p = 0.385), 64% vs. 35% (p = 0.063), 74 vs. 41% (p = 0.016), respectively. conclusion: compared to cd34 selection, cd3/cd19 depletion resulted in a faster reconstitution of cd4 cells,nk-t and nk cells. however survival was signifi cantly better in pts transplanted with cd34 selected grafts. due to a loss of power in favor of our hypothesis (15% increase in overall survival) using statistical simulation models, this trial was stopped. (1) introduction: in haploidentical hematopoietic stem cell transplantation (hsct), the infusion of donor lymphocytes transduced to express the herpes simplex virus thymidine kinase (hsv-tk) suicide gene allows to control gvhd, to mediate gvl, and to rapidly provide an effective and polyclonal anti-infective t cell repertoire (ciceri and bonini et al., lancet oncology, 2009 ). even though their engraftment is necessary to achieve these effects, hsv-tk + cells represent the minority of lymphocytes circulating in treated patients. therefore, we investigated the putative role of hsv-tk + cells in promoting thymic activity and t cell development from graft progenitors. methods: thymic function was assessed in adult patient who underwent haploidentical hsct and infusion of suicide genemodifi ed donor t cells for hematologic malignancies, after validating the methods in healthy pediatric and adult controls. single joint t cell receptor excision circles (sjtrec) were quantifi ed by qpcr in peripheral blood mononuclear cells (pbmcs) and purifi ed t cells, and the proportion of cd31 + recent thymic emigrants (rtes) in cd4 + naïve t cells was measured with immunophenotype analysis in pbmcs. thymic output was correlated with thymic volume, assessed by computed tomography (ct) scans. t cell receptor repertoire was assessed by vbeta spectratyping. the relative contribution of hsv-tk + and hsv-tk-donor t cells to post-transplantation anti-host alloreactivity was studied by mixed lymphocyte cultures. results: at the moment of t cell immune reconstitution (defi ned as cd3 + cells > 100/μl peripheral blood), the cd4 + naïve t cell subset was almost entirely comprised by cd31 + rtes, and this percentage remained high, as compared to age, also in subsequent months. in informative patients, rte frequency before hsct and before hsv-tk + cell infusion was in line with agerelated healthy controls, suggesting a direct role of the infused cells in mediating the effect. accordingly, in treated patients ct scans documented an increase in thymic volume following hsv-tk + cell add-backs. finally, low absolute sjtrec counts could be detected, in line with the documentation of extensive peripheral proliferation and low rte absolute numbers. conclusions: these data show that after the infusion of suicide gene-modifi ed t cells a renewal of thymic activity takes place, which contributes to the recovery of the peripheral t cell repertoire. elevated pretransplant serum ferritin levels have been associated with an increased susceptibility for opportunistic infections and increased incidence of morbidity and mortality after allogeneic hct. we studied in 81 patients who underwent myeloablative allogeneic hct for acute myeloid leukemia pre-and posttransplant serum ferritin levels and correlated the serum ferritin levels with the tlr9 expression and the cellular immune reconstitution 3 and 12 months post transplant. further, we studied in vitro-experiments in kasumi 1 cells the tlr1, tlr2, tlr3, tlr5, tlr7, tlr9 and tlr10 expression after overwhelming iron exposure. the average pretransplant serum ferritin level was 1245 μg/ml (mean) in all aml-patients (mean 1100 μg/l for patients with aml in 1.cr and mean 1820μg/l for patients with aml > 1.cr). post transplant serum ferritin level increased up to 2080 μg/ml (mean) for all aml patients (mean 1290 mg/l for aml in 1.cr and mean 2350 μg/l for patients with aml > 1.cr). the application of 300 ng iron to acute leukaemia cell lines sd1, and kasumi-1 cells increased signifi cantly tlr1,tlr2, tlr3, tlr5, tlr7 and tlr9 expression in relation to the housekeeping gene abl measured by real-time rt-pcr. in kasumi-1 cells tlr1 increased up to 50,6% (p = 0.014) tlr2 35.5% (p = 0.046), tlr3 57,8% (p = 0.006), tlr5 62.9% (p = 0.005), tlr7 46.2% (p = 0.02), tlr9 44.2% (p = 0.026) and tlr10 54,7% (p = 0.07) compared to untreated kasumi 1 cells. further, patients with elevated post transplant ferritin level > 2000 μg/l had an increased tlr9 expression in mononuclear cells (tlr9/abl quotient 6485 versus 4543; p<0.05) 3 months post transplant. the number of cytotoxic t cells cd4 + cd8 + in patients with elevated serum ferritin level was signifi cantly elevated after transplant (mean 189 versus 95 cells/μl 12 months post transplant, p = 0.034), whereas no differences were found in the number of cd3 + cd4 + t helper cells, b19 + cells, nk cells. these results indicate that elevated ferritin levels might activate the innate immune system by increasing tlr expression. this might be of importance since we recently showed that increased tlr9 expression was associated with adverse impact on nonrelapse mortality in the transplant setting. further exaggerated tlr9 expression has been discussed to induce overwhelming immune responses as sirs or ards. more studies are definitely necessary to evaluate the role of elevated iron overload on the innate immune system. clinical scale generation of functional human natural killer cells from umbilical cord blood cd34-positive cells for immunotherapy j. spanholtz, m. tordoir, c. trilsbeek, j. paardekooper, t. de witte, n. schaap, f. preijers, h. dolstra umc st.radboud (nijmegen, nl) immunotherapy based on natural killer (nk) cell infusions is a potential adjuvant treatment for many cancers. we established an extremely effi cient cytokine-based culture system for ex vivo expansion of nk cells from hematopoietic stem and progenitor cells from umbilical cord blood (ucb). systematic refi nement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. the use of gbgm culture media in a clinical applicable protocol allows the ex vivo expansion and differentiation of cd34 + cells to more than 4-logs into cd56 + cd3-nk cells with very high purity. these ex vivo-generated cd56 + cell products contain nk cell subsets expressing cd94/nkg2a and kir as well express high levels of activating nkg2d and ncrs. functional analysis showed that cd56 + cell products containing alloreactive nk cells effi ciently kill myeloid leukemia and melanoma tumor cells as well as primary acute myeloid leukemia (aml) cells. we have currently translated the protocol to clinical scale production using a closed cell culture bioprocess ( figure 1 ). cd34 + selection from cryopreserved ucb samples (n = 9) using the clinimacs device was optimized and the selection resulted in a cd34 + enrichment with a mean number of 2,2 ± 1,6 × 10 6 cells and a purity of 71 ± 14 % cd34 + cells. validation experiments using these cells showed, that the generation of suffi cient numbers of nk cells without contaminating t-cells or bcells under a closed system process is feasible. the cell cultures using bags show a mean expansion of 1273 ± 506 fold (range 759-1770 fold, which generated 8,6 × 10 8 -1,9 × 10 9 nk cells from cord blood-derived cd34 + cells within maximal 6 weeks of culture. the mean purity of the nk cell product was 71 ± 9 % (range 63-80%) of the total cells in the bag cultures. in order to improve the purity of the product, we include bioreactors during the differentiation culture process. using this modifi cation we were able to synchronize the differentiation process in small (plate) and large scale (bag) experiments, which allows the generation of large scale nk scale products with a purity of more than 95% cd56 + cells devoid of t-and b cells (figure 2a and b) . these nk cell products will be used for immunotherapy in elderly aml patients. this study is a phase i dose escalation study in a series of 12 aml patients who have successfully achieved cr (<5% blasts in the bone marrow) after standard intensive chemotherapy. key: cord-014976-546zaoxn authors: nan title: publication only date: 2006-03-08 journal: bone marrow transplant doi: 10.1038/sj.bmt.1705327 sha: doc_id: 14976 cord_uid: 546zaoxn nan ischemic myocardial damage is an increasing cause of heart failure in the western world and has long been considered irreversible because adult cardiomyocytes are terminally differentiated and do not proliferate. stem cells are undifferentiated cells capable of self-renewal, proliferation, and differentiation into multiple lineages permitting tissue regeneration. a number of types of stem cells are now recognised, as well as partially differentiated progenitor cells that are capable of proliferation and differentiation to multiple lineages. reversal of heart failure would require myocardial revascularization, remodelling of the left ventricle and replacement of damaged myocyte. we investigated the safety of transplanting un-manipulated autologous bone marrow into infracted myocardium in two patients. these patients underwent coronary bypass using the pi-circuit technique and external reshaping of left ventricle in off-pump surgery. we evaluated the efficacy of this combined technique in the improvement of cardiac function. autologous bone marrow (300ml) was obtained by bilateral posterior iliac bone aspiration at the time of surgery. bone marrow mononuclear cells were isolated by means of a density ficoll-paque gradient. then the cells were exhaustively washed and resuspended in a normal saline solution containing 5% human serum albumin. cell count, viability and cultures were appropriately performed. following the operation the bone marrow mononuclear cells (30ml) were injected directly to the myocardium of the left ventricle. no significant complications were observed. the left ventricular ejection fraction at rest was improved significantly in both patients from 14 and 20% to 24 and 29% respectively, three months following the operation. furthermore, we observed significant reduction of the end diastolic volume of the left ventricle and improvement in the inferior-posterior wall motion, this area was not revascularized, in comparison to the previous one before the operation. these findings suggest that transplantation of unmanipulated autologous bone marrow into scar tissue of the human heart is safe and enhances cardiac function, when used in combination with myocardial revascularization and remodelling of the left ventricle. this benefit can be seen after 3 months of the bone marrow transplant and is maintained after 8 months of follow-up. a. symeonidis, m. tiniakou, a. spyridonidis, m. karakantza, e. triantafyllou, a. kouraklis-symeonidis, v. pesli, p. matsouka, n. zoumbos univers of patras med school (patras,gr) the haematopoietic sct unit in patras university was established in 1998, aiming to cover the requirements of the area of peloponese and south-western greece. since then 79 autologous and 23 allogeneic stem-cell transplants have been performed in 89 patients. auto-transplanted patients were 39 male and 30 female (median age 47, range 19-70 years), diagnosed as non-hodgkin's lymphoma (nhl=26), multiple myeloma (mm=26), hodgkin's lymphoma (hl=8), acute myelogenous leukemia (aml=7) chronic myelogenous leukemia (cml=1) and chronic lymphocytic leukemia (1) . nine mm patients received tandem auto-transplants, while 1 auto-and 3 allo-were second transplants. at transplantation 41 patients were in cr, 27 in pr and 11 had refractory disease. bone marrow (bm) grafts were used in 3 cases and peripheral stem-cell grafts (pbsc) in 76. patients were conditioned with melphalan (32), cbv (17), beam (14), busulfan-based regimens (12) and thio-tepa-based regimen (4). engraftment was achieved in 76/79 cases. three patients died early post-transplant. after a median follow-up of 31 months, 47 patients are alive (68%), 40 of them disease-free, and 22 have died, due either to transplant-related complications (5), or after an early (<1 year post-transplant) 9 (nhl=5, aml=3, mm=1), or a late relapse (> 2 years post-transplant) 8 (mm=6, hl=1, nhl=1). for all allo-transplants the donor was a matched sibling. patients were 15 male and 5 female (median age 38, range 18-56 years), diagnosed with aml (9), acute lymphoblastic leukemia (all=4), myelodysplastic syndrome (mds=3), aplastic anemia (2), cml (1) and myelofibrosis (mf=1). excluding patients with aa, disease status at transplantation was 1st cr for 11 patients, 2nd cr for 3, and active residual disease for four. stem-cell source was bm in 5 and pbsc in 18. a standard conditioning was used in 18 cases and a reduced intensity one in 5. engraftment was achieved in all cases. one patient died early, due to uncontrollable vod, and 7 (all=4, aml=3) due to disease progression, which was early (<8 months) in 5 cases, and late (at 17 and 21 months post-transplant) in 2. five patients developed acute gvhd grade i-ii, and one grade iv. after a median follow-up of 21 months, twelve patients are alive and disease free (aml=5, mds=3, aa=2, cml=1, mf=1) but 7 of them have manifested chronic gvhd, (extensive in 4). in one patient chronic gvhd emerged after dli, administered for smoldering relapse. activation of plts by collagen, measurement of hypotonic shock response (hsr) and extent of shape change (esc) were tested in addition. in os-pc and c-pc plt yield was 2.72±0.37 and 2.81±0.33x10 11 /unit (recovery 79.1% and 73.1%, p=0.003), residual erythrocytes were <0.6x10 9 /unit, residual leucocytes <0.16x10 6 /unit in all pc. on d1 values for po2 in os-pc and c-pc were 120.9±27.1 and 107.6±30.7mmhg, pco2 58.3±7.0 and 69.5±13.6mmhg (p<0.001), hco3 18.7±0.9 and 19.8±1.2mmol/l (p<0.001), respectively. on d7 values for po2 in os-pc and c-pc were 99.6±44.0 and 85.1±24.5mmhg, pco2 23.8±2.8 and 27.9±4.4mmhg (p<0.001), hco3 10.0±1.1 and 10.1±1.1mmol/l, respectively. results (mean±sd) of metabolic and activation markers and morphologic features on d1 and d7 are shown in the table. in both groups functional parameters revealed a sufficient capacity for aggregation during storage. compared to c-pc os-pc were significantly more efficient in recovery of plts, whereas cd62p and cd63 were significantly higher due to a higher extent of plt activation in the automatic system. a possible difference in clinical outcome of transfusion of os-pc compared to c-pc has to be investigated in further studies. objectives: recently the investigation of differentiation potential of bone marrow stromal cells becomes important because of their implementation in transplantation. but the structure of the stromal cells pool is still not fully known. the aim of the study -to investigate the differentiation ability of bone marrow clonogenic fibroblasts and to elaborate the standart assays for clonogenic proliferation and adipogenic and osteogenic differentiation (ad and od) of bone marrow fibroblasts in children. methods: the material -bone marrow aspirates from 11 healthy donors, 15 patients with acute lymphoblastic leukemia (all) and 15 patients with acute myeloblastic leukemia (aml). bone marrow stromal fibroblasts were cultured accoding a.fridenstain in our modification. od was induced by: b-glycerophosphat 7x10 -3 m; dexametasone 1x10 -8 m; ascorbic acid 2x10 -4 m. for ad were used: dexametasone 1x10 -7 m and insulin 1x10 -9 m. results: optimal conditions for maximum cloning efficiency were following: isolation bone marrow mononuclear cells; density by explantation -2x10 4 /ml cells; culture medium: 199 medium with 20% human serum of any blood group. osteogenic induction increased the proportion of fibroblasts colonies from normal bone marrow with alkaline phosphatase activity from 18,8% to 77,8%; the adipogenic inductors increased the proportion of colonies with lipid-rich vacuoles (detected by sudan) from 31,2% to 50,8%. the cloning efficiency of stromal precursors (the number of colonies per 1x10 5 explanted cells) in the patients with aml did not differ from normal donors (47,8 and 49,7) and was much lower in all patients (2,7). stromal cells from leukemic patients showed decreased (in comparison with normal donors) potency for od in the presence of inductors (from 28,6% to 58,4% for aml; from 17,5 to 45,9 for all patients). the ability of stromal cells to ad did not differ in patients with acute leukemias and normal donors. conclusion: standardization of stromal fibroblasts clonogenic cultivation assay is necessary for the evaluation of bone marrow stroma state. the using of osteogenic and adipogenic inductors demonstrate the possibility of in vitro differentiation of stromal progenitors and showed the differences between normal and leukemic stromal cells. initiation of leukapheresis for peripheral blood stem cell collection at a low level of circulating cd34+ cells l.k. tan, t.g. soh, b. tan, j. mah, t.c. liu, c.s. chen, p. law national university hospital (singapore, sgp) objectives: most patients or donors undergoing leukapheresis (lp) for autologous or allogeneic peripheral blood stem cell (pbsc) collection require multiple lp to achieve a sufficient cd34+ cell dose (e.g., ≥ 2.5 x 10 6 /kg). lp is initiated when peripheral blood (pb) cd34+ reached a certain level (e.g., ≥ 20 per microliter). the aim of this retrospective analysis is to summarize our institutional experience of initiating lp at a lower pb cd34+ cells level of 5 per microliter and to investigate the merits of this practice. methods: all patients or donors underwent lp (using cobe spectra or baxter amicus) processing 3 times the blood volume. a total of 192 procedures (130 autologous and 62 allogeneic) was performed in 87 patients or donors between jan 04 and oct 05. autologous patients were mobilized with chemotherapy and g-csf (10 mcg/kg) while allogeneic donors with g-csf (10 mcg/kg) alone. a "good" lp is defined as having ≥ 1 x 10 6 cd34+ cells/kg in the collection so that a minimum dose of 3 x 10 6 /kg can be achieved in 3 sessions. cd34+ cells were measured by sequential gating (staining with antibodies against cd45 and cd34 together with forward and side-scattering). results: each lp contained 6.44 x 10 8 wbc/kg (median, range: 1.3-17.5) and 2.48 x 10 6 cd34+ cells/kg (median, range: 0.14-24.9). cd34+ cells/kg in lp were correlated to pb cd34+ cell counts (r = 0.80). as shown in table 1 , initiating lp at higher levels of pb cd34+ cell (≥ 10 per microliter) increased the proportion of "good lp", whether "all" collections or only the first collections were considered. however, a substantial number of "good lp" (≥ 50%) would be missed if lp was initiated at 10 or 20 cd34+ cells per microliter in pb (table 2 ), but almost none at 5 cd34+ cells per microliter. conclusion: the result demonstrated that initiating lp at 5 pb cd34+ cells per microliter is helpful to some patients /donors. additional criteria may need to be adopted to determine whether lp should be discontinued if mobilization is adequate to minimize resource utilization. g-csf mobilised peripheral blood progenitor cells (pbpc) grafts are widely used in haematopoietic transplantation, but the effect of this cytokine on the cellular content of the graft is not fully understood. apart from the cd34+cells, t lymphocytes, nk cells and dendritic cells present in pbpc grafts play a crucial role in the haematopoietic and immune reconstitution following allogeneic transplantation. the aim of this study was to evaluate and compare cell populations present on g-csf mobilised pbpc collected on the 5th day of mobilisation and on peripheral blood (pb) prior to mobilisation of 15 healthy adult donors with a median age of 39 (range 19-62). the cell populations studied were t lymphocytes (cd4 and cd8), nk cells (bright and dim) and dendritic cells (myeloid and lymphoid). total nucleated cells (tnc) were determined by haematological counters, cell phenotypes were evaluated by flow cytometry using 4 colour staining, and each cell subset was determined using specific markers. t lymphocytes were divided in cd3+cd4+ or cd3+cd8+; nk cells into nkdim (cd3-cd56+) and nkbright (cd3cd56++); dendritic cell were considered lin-and hla-dr++ and then divided into lymphoid (ldc) cd123++ (high) and myeloid (mdc) cd11c+. in both pb and pbpc collections t lymphocytes were the main cell population present. with the mobilisation procedure there was a variable fold increase of cell populations (table1), namely 84 fold increase for tnc, while t lymphocyte, dc and nk populations showed 98,112 and 53 fold respectively. despite the differences in number, the t lymphocyte, dc and nk sub-populations kept the same ratio. there was a significant correlation between the pb tnc concentration and the fold increase of tnc, cd3+ cells and nk cells in the pbpc graft. there was no correlation with the increase in dc and any pb parameter evaluated. in summary, g-csf mobilisation while increasing the tnc number (up to 84 fold) does not affect nk, dc and t lymphocytes sub-populations ratio in the grafts from healthy adult donors. further studies are required to determine if the development, maturation and functional activity, namely cytotoxic and immune capacity of these cells are affected by the procedure. toxicity and efficacy of donor lymphocyte infusion after haematopoietic stem cell transplantation s. roncon, m. bini-antunes, f. campilho, i.l. barbosa, a. avila, s. ferreira, h. leal, c. pinho vaz, a. campos, r.b. ferreira, p. pimentel, a. carvalhais instituto portugues de oncologia -crop (porto, p) relapse remains one of the main complications after allogeneic haematopoietic stem cell transplantation (hsct). donor lymphocyte infusion (dli) is a therapeutic approach that is able to mediate antitumour effects and restore prolonged remissions. this antitumour effect has been well established in chronic myeloid leukaemia (cml) and less in other malignant diseases. we retrospectively analysed 40 patients (14f/26m) who relapsed or were at high risk of relapse after hsct between december 1994 and september 2005. patients median age was 32 years (6-55). they had the following diagnosis: cml 17, acute myeloid leukaemia (aml) 3, acute lymphoid leukaemia (all) 6, multiple myeloma (mm) 6, hodgkin disease (hd) 4, aplastic anemia (aa) 2, non-hodgkin lymphoma (nhl) 1, myelodisplastic syndrome (mds) 1. graft source was g-csf mobilized peripheral blood in 35 patients and bone marrow in 5. conditioning was myeloablative in 27 patients (in 19 of them graft was t cell depleted in vitro) and of reduced intensity (ric) in the remaining 13 patients. eight patients underwent a 2nd allogeneic transplantation and 4 of them later received dli. the indications for dli were relapse/disease progression in 35 patients and pre-emptive in 5. the median interval between transplantation and the first dli was 8 months . the number of dli was one for 21 patients, two for 17 and three for 2 patients. the median dose of cd3+ lymphocytes infused was 1x107/kg (0,05-10 x 107/kg). a complete response was observed in 33% of patients (4/9 acute leukaemia, 1/6 mm, 8/17 lmc) and a partial response in 2 (1 mm and 1 smd). there was no remission in 45% of patients (4/4 hd, 2/2 aa, 8/17 cml, 2/9 la, 2/6 mm). in two patients there was no available information. all the five patients relapsed after prophylactic dli. eight patients developed graft versus host disease (gvhd) "de novo" after dli. seven patients with cml did not achieve molecular remission after dli but did it when imatinib mesylate was associated. at the last follow up, 25 patients remained alive and 20 of them established full donor chimerism. in summary, complete remissions induced by dli were inferior to reported in literature. gvhd developed just in an acceptable number of cases and was controlled. further studies need to be performed to evaluate which patients really benefit from dli. report of stem cell transplantation in solid tumours in iran k. alimoghaddam, n. mahdavi, s. samiee, m. jahani, a. mousavi, a. ghavamzadeh horc (tehran, ir) introduction: the prognosis for many pediatric and young adult patients with solid tumors that have metastasized at the time of diagnosis or have relapsed after therapy remains very poor the lack of efficacy of chemotherapeutics, radiotherapy and cytokine-based immunotherapy for many patients with metastatic cancers has catalyzed, at least in part, enthusiasm for exploring allogeneic-based immunotherapy against solid tumors. while there is little doubt about the potential for its application in oncology, extensive use of nonmyeloablative hematopoietic cell transplantation (nmhct) in the treatment of solid tumors will likely remain limited until both the safety and efficacy of the approach are improved. materials and method: we collect the data of 25 patients who had undergone stem cell transplantation by reviewing their records from the date of their transplantation up to the date of last contact. analysis of the data has done via using spss software. results: of all the 25 patients 11(44.00%) were male, and 14(56.00%) were female. the main diagnoses before transplant are brought in the following table. (table1) the median age was 30 years old; ( 2-53 y/o) respectively. 22 (88.00%) patients received autologous and 3 ones (12%) received allogeneic stem cell transplantation. 22 ones (88.00%) had peripheral blood and 3 (12%) ones had bone marrow as graft type. the median duration of hospitalization for autologous transplanted patients was 20 days which was 28 days for allogeneic transplanted ones. transplant mortality rate in the first 100 days was 8%. the median follow-up duration was 396 days, with minimum and maximum of 16 and 4749 days respectively and during this period the overall survival (os) is 77.58% and the disease free survival rate (dfs) was 65.71%. palifermin for oral mucositis prophylaxis in autologous transplantation n. miranda (1) , p. santos (2), t. mendonça (1) , i. ferreira (1) , a. guimarães (1) , j.l. , m. abecasis (1) (1)instituto português de oncologia (lisbon, p); (2)hospital egas moniz (lisbon, p) objectives: oral mucositis is a near universal complication of intensive chemotherapy. the patient discomfort often require i.v. narcotics and is the main cause for total parenteral nutrition. some authors have associated the duration and the severity of mucositis with transplant outcomes. until very recently no effective treatment or prophylaxis was available. palifermin is the recombinant human keratinocyte growth factor and a previous study have shown its ability to decrease the length and severity of mucositis in conditioning regimens including total body irradiation (tbi). from august to november 2005 we have used palifermin for the prophylaxis of mucositis in adult patients (pts) under autologous bmt with conditioning regimens without tbi. methods: nine patients (4 females and 5 males) with a median age of 46.8 (range 27-56) have been included in an open label study included in an extended access program. the predominant diagnosis was non hodgkin's lymphoma (7). the conditioning regimen was beam in 7, vp16 + melphalan in 1 and carboplatinum + vp16 + thyotepa in 1. palifermin was given according to the manufacturer instructions 60 micrograms/kg body weight on 3 days before chemotherapy and on 3 days starting on day 0 after stem cell infusion. we analysed severity and length of mucositis, narcotic need, haematological reconstitution and side effects of palifermin. we have compared the results with a historical cohort matched for diagnosis, age, conditioning regimen and number of cd34+ cells transplanted. results: there was only one case of discontinuation of treatment due to toxicity (generalized skin oedema with severe hypotension). severe pruritus was present in 4 pts and generalized rash in all but one patient. a statistical significant increase in weight was observed in pts treated with palifermin on day +3 (median + 4 kgs versus -2 kgs without palifermin). the number of days under iv narcotics was lower on palifermin group (3.4 vs 4.8) and more patients did not require narcotics at all (4 out of nine versus 1 in 9). the mean of the number of days without any oral nutrition was lower on palifermin group (2.66 versus 5.22). all this differences did not reach statistical significance. the haematological recovery, antibiotic need, bacteriological isolates and length of stay on hospital were equivalent in both groups. conclusion: in our series palifermin decrease the severity and duration of mucositis and was associated with significant skin toxicity. clinical experiences with the new keratinocyte growth factor palifermin in 7 patients treated with high-dose chemotherapy followed by autologous stem cell transplantation m. fillitz, e. koller, e. schlögl, e. pittermann-höcker hanusch-hospital (vienna, a) oral mucositis is a common side effect of chemotherapeutic and/or radiotherapeutic treatment in malignant diseases resulting in pain, diarrhoea, malnutrition, gastrointestinal bleeding complications, local and systemic infection leading to prologation of hospitalization duration and increased medication and treatment costs. this side-effect of cancer treatment has to be expected in up to 50% of all treated cancer patients and the percentage is even higher in the hematopoietic stem cell transplantation setting. as the functional role of different cytokines such as tumor necrosis factor-alpha and different interleukines and probable protective factors like secretory iga in the pathological pathway leading to mucositis is not yet well understood, neither a widely accepted prophylaxis nor a therapy is available. we report our data of prophylactic application of the recombinant human keratinocyte growth factor palifermin in 7 autologous stem cell transplantation recipients(female 3; male 4). in 3 cases diagnosis was relapsed non-hodgkin´s lymphoma, 4 patients were suffering from multiple myeloma. the patients age ranged from 31 to 65 years, karnofsky status was 100% in all. palifermin was given as intravenous bolus injection on 3 consecutive days , third dose at least 30 hours before administration of high dose chemotherapy. second cycle consisted of another 3 doses that were given on 3 consecutive days starting with the day of reinfusion of the stem cell harvest product. the single injection doses ranged from 3.9 to 5.4mg according to the producer´s 60 mcg/kg/d recommendation. we experienced only one case of who-grade 3-mucositis which could not be distinguished from mucosal hyperproliferation due to the medication, all other patients showed no sign of oral mucositis. diarrhoea was maximum who-grade 1. treatment related side effects consisted of mucosal oedema (vagina, tongue, palate, lids), erythema of the face and upper body,dysphagia and disturbances of taste and sensibility. in one patient excessive proliferation of the oral mucosa lead to detachment from palate and tongue, in another patient mild temporary dyspnoea occurred. altogether those findings were well manageable and resolved without additional measure within 3 days after last injection. we observed no septic event. the hospitalization duration was slightly reduced to former comparable patients even though the first cycle of palifermin led to a 4 days prologation of the pre-transplant phase. prevention of oral mucositis in patients undergoing radiochemotherapy and allogeneic stem cell transplantation with recombinant human keratinocyte growth factor (palifermin): a single-centre experience w. rabitsch, p. kalhs, w. köstler, s. wöhrer, a. schulenburg, v. supper, m. mitterbauer, h. greinix bone marrow transplantation (vienna, a) objectives: oral mucositis is one of several common adverse effects of myeloablative therapy. it is particulary frequent in patients receiving high-dose chemotherapy with or without total-body irradiation for hematopoietic stem cell transplantation (hsct). palifermin is the first agent to be approved for the prevention of oral mucositis induced by myelotoxic therapy. so far, data on ist use in allogeneic hsct are rare. patients and methods: we analyzed the efficacy of palifermin on 4 consecutive patients (median age: 42, range 33-48 years) undergoing allogeneic peripheral blood stem cell transplantation (sibling donor; n=2; unrelated donor, n=2) at our institution. all patients received a conditioning therapy with cyclophosphamide and total body irradiation. palifermin was administered intravenously in a dosage of 60µg/kg/day 3 days before myeloablative therapy and 3 days after stem cell infusion. mucositis was assessed daily and graded according to the who-scale. results: one patient died on day +10 after transplantation due to gram-negative sepsis and multi-organ failure. the other 3 patients were eligible for assessment of mucositis. the 3 eligible patients experienced only who grade 2 mucositis. the drug was generally well tolerated without severe side effects. one patient developed erythema on the face, which resolved 24-hours after palifermin administration without the need of medication. in the other 2 patients no side effects were observed. only 1 patient experience acute graft-versushost disease (gvhd) of the skin grade ii. currently, 3 of 4 patients are alive, 1, 3 and 4 months after hsct. conclusion: palifermin represents the first drug approved by the us fda for reduction of incidence and duration of oral mucositis in a specific patient cohort. in our cohort of patients we observed no serious side effects and only low grade mucositis. the effect of palifermin on gut epithelia or gvhd incidence and severity has to be assessed in larger patient cohorts. pegfilgrastim in comparison with filgrastim after allogeneic stem cell transplantation c. lutz, g. massenkeil, i. tamm, t. terwey, s. neuburger, b. doerken, r. arnold university hospital charité (berlin, d) purpose: after autologous and allogeneic peripheral blood stem cell transplantation (pbsct) filgrastim (g-csf) is given daily to enhance neutrophil recovery and prevent risk of infection. peg-filgrastim (polyethylen-glykol-g-csf) has been approved in 2002. since the clearance of peg-filgrastim is mediated by neurophils, in neutropenia activity and serum concentration is prolonged and elevated. the aim of this study was to evaluate for the first time the efficacy of a single fixed dose (6 mg) in patients after allogeneic pbsct from matched unrelated donors. methods: on day +5 after allogeneic pbsct five patients (2 aml, 2 all, 1 cml; median age 29y) received 6mg peg-filgrastim s.c. the neutrophil and platelet recovery was compared to patients (n=21; 11 all, 6 aml, 2 mds, 1 cml, 1 mm; median age 41,5 y) treated with daily filgrastim (5µg/kg i.v. over 4h). definition of neutrophil recovery was ≥ 0,5/nl, platelet recovery ≥ 20/nl on three consecutive days and without transfusions respectively. g-csf serum levels were measured by elisa (r&d systems). results: peg-filgrastim was well tolerated. in patients treated with peg-filgrastim the neutrophil engraftment was 18 days versus 19 days in the filgrastim group. the platelet recovery in the peg-filgrastim group was 22 days and 20 days in the filgrastim group. these differences were not significant. g-csf levels were measured after the first administration of filgrastim or peg-filgrastim respectively. the results showed a neutrophil mediated clearance of peg-filgrastim with a peak serum level on day +2 that dropped in parallel to neutrophil recovery. the peak level of peg-filgrastim was about ten fold higher than the peak level of filgrastim (111 ng/ml vs 10 ng/ml). conclusions: our data suggest that a fixed dose of 6mg peg-filgrastim used after allogeneic pbsct is effective and gives comparable results to daily administration of filgrastim. larger prospective studies are needed. use of pegfilgrastim after high-dose melphalan and autologous peripheral blood stem cell transplant in multiple myeloma patients (1) , p. iacopino (1) (1)azienda ospedaliera bmm (reggio calabria, i); (2)policlinico universitario (messina, i) single dose of pegfilgrastim is equivalent to daily filgrastim after standard dose chemotherapy in decreasing the duration of neutropenia. daily filgrastim started within 1-4 days after autologous peripheral blood stem cell transplant (apbsct) leads to decrease in time to neutrophil engraftment. we undertook a study of pegfilgrastim after high-dose melphalan (hdm) and apbsct. in all, 13 patients with multiple myeloma (stage iii durie-salmon classification), eligible to undergo hdm and apbsct, were enrolled. patients were conditioned with hdm at a dose of 200 mg/m² intravenously on day -2.the day of apbsct was termed day 0. the median stem cell dose infused was 4 x 10 6 /kg (range 3-7). patients received a single dose of 6 mg pegfilgrastim subcutaneously 24 h after apbsct. there were no adverse events secondary to pegfilgrastim. neutrophil engraftment was defined as first of 3 consecutive days of an anc equal or greater than 0.5 x109/l after a previous nadir. platelet engraftment was defined as platelet count of equal or greater than 20 x 109/l. all patients engrafted neutrophils and platelets with a median of 10 days (range, 9-17) and 13 days (range, 10-26), respectively. the incidence of febrile neutropenia was 53.8% (7/13). the median duration of febrile neutropenia was 2 days (range, 0-7). the mean number of platelet and packed red blood cell transfusions were 3 + 6.2 u and 0.9 + 1.7 u. it's interesting to note that 9/13 patients didn't required packed red blood cell transfusion. one patient died for cerebral bleeding after engraftment, on day + 21; 2 patients experienced persistent reversible neurophilia. neutrophil and platelet engraftment were compared with a cohort of 38 patients (same diagnoses, method of stem cell collection, conditioning regimen and stem cell dose) treated at our institute, who received filgrastim at 5 mg/kg subcutaneously daily, starting at day + 3 or day + 5 and no statistical difference were shown (p= ns). in conclusion, pegfilgrastim given as a single fixed dose of 6 mg appears to be safe after hdm and apbsct. pegfilgrastim may be convenient to use in outpatient transplant units. the concept of treating chemosensitive leukemia, lymphoma and myeloma patients with high dose chemotherapy followed by reinfusion of peripheral stem cell has been implemented in the set-up of bone marrow transplantation, and often considered as transplantation procedure. with the use of growth factors to stimulate bone marrow and allow the harvest of peripheral stem cells with an apheresis machine haemoneticsr mcs3p and cryopreservation, the procedure of autologous peripheral stem cell transplant (apst) became feasible in hematology departments, and not necessarily in bone marrow transplant units. for this reason, we decided eight years ago to set up such a system in our institute. our team included three md hematologists, one nurse and one apheresis technician. we equipped our institute with the necessary separator machine and after a period of ten months trial of investigation of the harvest quality, we started to treat our patients in our institute, instead of referring them to a transplant unit. our institute is a part of a 600-bed university hospital and covers a population of 500 000 inhabitants. in this abstract we would like to present our results. using two haemonetics, approximately 300 harvests have been performed with achievement of an excellent yield of cd34 stem cells (median, 4.8x106/kg). the harvest was performed at the day care unit and then cryopreserved in liquid nitrogen. in most patients myeloablative chemotherapy was also given ambulatory. following the reinfusion of the harvest, patients were hospitalized in isolation rooms. the median time of recovery (neutrophils >0.5x109/l) was 11 days. at the end of the year 2005 (eight years activity), 120 auto-transplantations were performed, including 12 tandem. the diagnoses were: multiple myeloma in 49, non-hodgkin's lymphoma 33, hodgkin's disease 14, aml 4, all 2, amyloidosis 4 and cll 14 cases. treatment related mortality was 1.7%. we believe that such endeavor should be encouraged and advised for more hematology centers. hematologists in training and senior hematologists have the benefit of keeping their patients under close supervision with the challenge of further therapies, increasing the clinical level, the motivation and the interest in the field of hematooncology. h. dimitriou, e. linardakis, g. martimianaki, e. stiakaki, a. fillipidi, m. kalmanti university of crete (heraklion, gr) mesenchymal stem cells (mscs) are multipotent progenitor cells within the bone marrow (bm) capable of differentiating into various tissue specific cells. mscs form an integral part of the bm stroma, have immunomodulatory functions and play an important role in the support of hematopoiesis. their multipotentiality and ease of ex vivo expansion has raised great interest in the clinical use of mscs for tissue repair and gene therapy. in order to evaluate if malignant and non malignant hematological diseases quantitatively and qualitatively affect bm derived mscs, bone marrow from children with acute lymphoblastic leukemia (all diagnosis n=9, different phases of treatment n=29, end of therapy n=10), idiopathic thrombocytopenic purpura (n=16), autoimmune neutropenia (n=12) and control patients (solid tumors without bm involvement, n=30) was harvested and the mononuclear cell (mnc) fraction isolated. mscs were expanded in amem supplemented with 10% selected fcs, characterized and compared in terms of their phenotypic characteristics, clonogenicity and ability to differentiate into adipo-(a), osteo-(o) and chondrocytes(c). mncs at day 0 expressed high levels of cd34, cd45, cd29 and cd44, and very low levels of cd14, cd105 and cd90. expression of hematopoietic markers on cells at passage 1 (p1) and thereafter progressively diminished while expression of cd29, cd44, cd90 and cd105 increased approaching 100%. cell doubling time ranged from 3 to 4 days at all passages. high clonogenicity was observed in all samples at all passages as shown by the presence of cfu-f colonies (>50 cells) with the exception of all samples at diagnosis which showed impaired proliferation and clonogenicity that returned to normal since remission was achieved at the following phases of treatment till the end of therapy. at p2 or p3, mscs were differentiated towards the a, o, and c lineages by using specific induction media. differentiation was assessed by histochemistry and rt-pcr (lpl and ap2 for a, osteoprotegerin, osteocalcin and alp for o, aggrecan and col ii for c). p2 or p3 mscs from all groups exhibited bi-or trilineage differentiation. preliminary cloning experiments showed that msc population is composed of cells with differing proliferation potential and clonogenicity. these results indicate that blood diseases of childhood do not affect the characteristics of mscs which could have clinical applications particularly in hematopoietic reconstitution following transplantation. pegfilgrastim after autologous stem cell transplantation c.r. rinaldi, c. becchimanzi, a.m. risitano, n. marra, b. rotoli, g. de rosa university federico ii (naples, i) recombinant hemopoietic growth factors are not added routinely after autologous stem cell transplantation (asct) in our institution. in myeloma patients, a multicentric protocol to which we partecipate indicates granulocyte growth factor after re-infusion, at the dose of 5 ug/kg, from day +1 to hematological recovery. because of equivalence between a single peg-filgrastim (peg) dose and daily filgrastim doses in decreasing the duration of neutropenia after standard dose chemotherapy, we used peg after asct in patients affected by myeloma or lymphoma. from february 2004 to november 2005, we enrolled 53 patients, 30 suffering from myeloma (13 undergoing single-asct and 11 tandem-asct) and 23 suffering from lymphoma (13 non-hodgkin, 10 hodgkin), conditioned by mel200 and beam, respectively. all patients received a single dose of 6 mg pegfilgrastim subcutaneously 24 h after autologous stem cells infusion. all patients engrafted neutrophils and platelets with a median time of 10 and 13 days, respectively, (regardless the underlying disease and type of conditioning). the incidence of febrile neutropenia was 30 % (16/53) with a median duration of 12 hours. we observed no adverse events secondary to peg injection. no patient had clinically significant mucositis. we compared this cohort of myeloma patients with an historical group of autotransplanted myeloma patients treated by standard daily doses of filgrastim and of lymphoma patients transplanted without g-csf administration. time to neutrophil and platelet recovery was identical in both groups of myeloma patients, and appeared sensibly reduced in lymphoma patients treated with peg as compared to no-g-csf patients (days 21 and 19 in nhls, 15 and 11 in hls) . we conclude that a single dose of pegfilgrastim after asct is safe, well tolerated and accelerates neutrophil recovery, thus decreasing time of hospitalization it seems equivalent to daily dose of filgrastim. we also documented no differences between our cohort of patients and historical groups in order of febrile neutropenia and proved infections. since peg disappearance from the circulation is not due to renal or hepatic clearance, but only to uptake on granulocytic cells and their precursors, this drug can be given even early after stem cells infusion, and will be utilized as soon as the engraftment occurs. mesenchymal stem cells are able to stimulate alloreactive immune cells l. fang, c. lange, m. engel, a.r. zander, b. fehse university medical center eppendorf (hamburg, d) bone marrow-derived mesenchymal stem cells (msc) have been suggested to be "immune-privileged" while exerting a strong immune-modulatory function as "third-party" cells in an hla-independent manner. therefore msc are interesting candidates for cell and gene therapeutic applications. however, a better understanding of the mechanisms underlying their immune-modulatory potential would be very important for msc application in clinical settings. we investigated the interaction of msc with allogeneic immune cells by co-culturing them with un-matched pbmcs and using them as third party in mixed lymphocyte cultures. we present data demonstrating that the immune-privileged state of msc results from an interplay of stimulating and suppressing factors. the directly stimulating activity leads to both active lymphocyte proliferation and secretion of pro-inflammatory cytokines by allo-reactive lymphocytes. stimulation is, however, dominant only at low msc:effector cell (<0.1 under our experimental conditions), but outweighed with higher msc numbers by the suppressing activity. allogeneic mixed lymphocyte cultures as well as msc-mediated stimulatory effects are efficiently suppressed by the addition of mscconditioned medium underlining the important role soluble factors play in msc-mediated immune modulation. in conclusion, based on our data we suggest that the "immuneprivileged status" of msc reflects a sensitive balance of mhcmediated immune activation and the suppression of immunological reactivity largely conferred by soluble factors. circulating endothelial progenitor cells in children with non-malignant diseases following allogeneic bone marrow transplantation m. massa, v. rosti, r. campanelli, e. bonetti, v. meli, d. poddighe, t. mina, d. pagliara, d. lisini, f. locatelli irccs policlinico san matteo (pavia, i) bone marrow-derived endothelial progenitor cells (epcs) circulate in the peripheral blood (pb) of healthy subjects (hs). epcs seem to play an important role in maintaining vessel wall homeostasis, in the neo-angiogenetic processes and in the re-endothelization of the wall of injured vessels. the aim of the study is to assess the number and origin of circulating epcs in children with non-malignant diseases who received allogeneic bmt from an hla-identical sibling or a matched unrelated donor. we studied patients with thalassemia major (n=9), fanconi anemia (n=2), sickle cell disease (n=3), mucopolysaccharidosis (n=1), diskeratosis congenita (n=1), acquired aplastic anemia (n=3), or chediak higashi syndrome (n=1). we evaluated pb samples at 21, 45, 60, 90, and 120 days after transplant. the number of epcs was evaluated as cd34+vegfr-2+ or cd34+cd133+vegfr-2+ cells by cytofluorimetric analysis, and by in vitro culture. the analysis of pb samples from 10 age matched donors (hs) was included in the study. donor or recipient origin of epcs was assessed on at least 10 individually picked endothelial colonies by micro-satellite analysis. in patients tested 21 days after transplant the percentage of circulating cd34+vegfr-2+ cells (median 0.1%, 0-0.5) and the percentage of cd34+ co-expressing the cd133 and vegfr-2 antigens, representing a restricted subset of immature epcs (median 0.06%, 0-1.3), were comparable to those found in hs (median 0.0%, 0.0-6.7; median 0.6%, 0.0-15.1, respectively). the number of epc derived colonies was also comparable in patients tested at 21 days after transplantation (median 26/10 6 mononuclear cells, 0-91) and in hs (median 22.5/10 6 mononuclear cells, 11-43). neither the percentage of circulating cell subsets, nor the number of epc derived colonies, showed significant modifications during 120 day follow up (by anova test). microsatellite analysis was performed on the epc derived colonies of 3 patients, tested at time points ranging from 1 to 2 months. in 2 patients, all the analysed colonies were of donor origin; in the third patient all the analysed colonies were of patient origin (hematopoietic engraftment donor/recipient 95%/5%). circulating epcs are detectable in patients given allogeneic bmt from 21 days up to 4 months after transplantation. further studies are needed to definitively conclude their origin and to assess whether their recovery can be correlated to the clinical outcome of the transplanted patients. a. spiropoulos, e. goussetis, m. theodosaki, k. stefanaki, i. peristeri, v. kitra, e. petrakou, s. graphakos "aghia sophia" children's hospital (athens, gr) bone marrow and peripheral blood of adults contain a subset of progenitor cells, which are able to differentiate into mature endothelial cells, thus contributing to re-endothelialization and neo-vascularization. the number of these cells in healthy subjects is rather low; almost 0.002% of total mononuclear cells and a variety of factors may further influence their number. to investigate how allogeneic stem cell transplantation (sct) influence circulating endothelial progenitor cells (cepcs), we obtained peripheral blood samples from 13 transplant recipients at different time points (ranging from 8 months to 5 years after transplantation). peripheral blood mononuclear cells were separated by ficoll density-gradient centrifiguration and were seeded to fibronectin-coated well dishes containing endocult medium (stem cell technologies). in order to remove monocytes and mature endothelial cells, non-adherent cells, at day two of culture, were harvested and further cultured for an additional three days to allow formation of endothelial colonies. the phenotype of the cells that emerged in culture was characterized by immunohistochemistry, and their origin was determined using a polymerase chain reaction (pcr)-based assay for polymorphic short tandem repeats (strs). all samples gave rise to epcs colonies in 5 days. the mean number of epcs colonies/ 10 6 cells was 58 ± 25.3 (range; 46-96) and it didn't seem to correlate with the post-transplant time. the cultured cells expressed typical endothelial markers such as cd31 and vwf. for each patient and at all time points, str-pcr analysis showed that cultured cells came exclusively from the donor. these results demonstrate that cepcs are detectable after sct and that their number is independent of post-transplant time. early cd34+ cells recirculate after autologous peripheral blood stem cell transplant and peg-filgrastim administration for haematological malignancies s. de matteis, n. piccirillo, s. de vita, f. sorà, m. tarnani, l. laurenti, p. chiusolo, g. reddiconto, s. sica, g. leone università cattolica del sacro cuore (rome, i) cd34 protein is widely accepted as reliable marker for identifying hemopoietic stem or progenitor cells in bone marrow and in peripheral blood. cd34+ cells represent a heterogeneous cell population consisting of primitive uncommitted and pluripotent progenitors as well as committed stem cell. previous studies showed that these progenitors, mainly myeloid-committed subsets, are detectable in the early phase following infusion of autologous/allogeneic stem cell at day +9 (albo et al, haematologica 2004) . based on these findings, we investigated the kinetics of appearance of cd34+ cells after autologous peripheral blood stem cell transplant (apbsct) and administration of pegfilgrastim 6mg at day +1, and its correlation with haematological engraftment. we studied in 16 consecutive patients (pts), affected by haematological malignancy and treated with apbsct (table 1) , the percentage of cd34+ cell in peripheral blood every other day from day + 2 until patient discharge. these cells were detectable starting from day +10 after transplantation. the peak of the cd34+ cells was at day +12 (range 10-16), at this day wbc were 6015/mmc (range 1060-13940/mmc) and the number of cd34+/mmc were 7.85 (range 0.8-116). there was no correlation between total number of cd 34+/kg infused and day, absolute number and percentage of cd34+ peak. statistical analysis demonstrated a significant negative correlation (r-0.53, p=0.035) between age of pts and peak of cd34+, while a significant positive correlation (r0.58, p=0.019) between age of pts and day of cd34+ peak. when haematological reconstitution after apbsct we observed a significant positive correlation between day of cd34+ peak and time to absolute lymphocyte>0.5 x10 9 /l(alc) (r0.59, p=0.016), pmn>0.5x10 9 /l (r0.53, p=0.035), plt>100x10³/mmc (r0.71, p=0.002), length of hospitalization (r0.51, p=0.044). this data seems to link up the appearance of cd34+ cells with the bone marrow reserve: younger pts release higher number of cd34+ in peripheral blood after apbsct and these cells are detectable sooner than in the older pts. furthermore, time to alc>0.5 x10 9 /l and pmn>0.5x10 9 /l recovery, plt>100x10³/mmc and length of hospitalization are longer in pts that release later cd34+ cells, i.e. the older pts. early appearance of cd34+ cells after apbsct and peg-filgrastim might be considered as surrogate marker of bone marrow reserve. further analysis of cd34+ subset are ongoing to confirm these data and to clarify their significance. imbalance of osteoprotegerin/receptor activator of nuclear factor-kb ligand in bone marrow plasma and microenvironment after allogeneic stem cell transplantation c. selleri, l. tauchmanovà, p. ricci, a.m. risitano, m.c. martorelli, g. cerciello, i. imperatore, a. casale, t. musella, g. lombardi, a. colao, b. rotoli federico ii university (naples, i) persistent decrease in bone mineral density (bmd) is a known complication after allogeneic stem cell transplantation (allo-sct) due to the transplant procedure, gonadal failure, immunosuppression and deficit of osteoblast precursors in the marrow microenvironment. in addition, transplanted patients may develop chronic endocrine and immunological disorders, including chronic graft versus host disease, that can further affect bone turnover. osteoprotegerin (opg) plays a pivotal role in bone remodelling, by neutralizing the effect of rankl on differentiation and activation of osteoclasts. we investigated the relationships between densitometric values, circulating opg and interferon-gamma (ifn-gamma) levels; moreover, opg and rankl levels were measured in marrow plasma and in conditioned medium of long-term cultures (ltc) of marrow mesenchymal-derived osteogenic cells. thirty six allo-sct patients (17 females; age:35±9 yrs) were enrolled and compared to 36 controls matched for age, gender and body mass index. bmd was measured at lumbar spine and femoral neck by the dexa technique. lumbar and femoral bmd were lower in patients than in controls (0.9±0.16 vs 1.04±0.08, p<0.01). serum opg and ifn-gamma were significantly (p<0.05) higher in patients than controls. patients' serum ifn-gamma correlated with opg levels (r=0.54; p=0.04). by contrast, opg levels were lower in patients than in controls in marrow plasma (p<0.001) and in conditioned media after one (p=0.035) and 3 months (p=0.003) of ltc of marrow mesenchymal-derived osteogenic cells. rankl values were similar between patients and controls, being lower in conditioned medium than in marrow plasma (p<0.01, both groups). the rankl/opg ratio in ltc was significantly higher in patients than in controls, although this ratio progressively increased with the time of ltc in both groups (p<0.05). there was no correlation between serum, in situ opg levels and densitometric values. in conclusion, our findings suggest that after allo-sct: 1) the paradoxically high serum opg levels, which correlate with ifn-gamma, are likely due to deranged immune system; 2) low marrow and microenvironment opg levels confirm persistent quantitative and qualitative deficit of osteoblastic precursors; 3) the increased microenvironment rankl/opg ratio may negatively affect bone remodelling. mesenchymal stem cells (mscs) are endowed with multilineage potential and immunomodulatory ability, these properties rendering them attractive for tissue engineering and immunotherapy. however, it is still a matter of debate whether donor mscs have a sustained engraftment in the host bone marrow (bm) after allogeneic hematopoietic stem cell transplantation (hsct). in particular, studies on the fate of mscs transplanted with cord blood (cb) are lacking. the aim of this study was to analyse the donor/recipient origin of mscs in pediatric patients receiving an allogeneic hsct. thirty-six patients undergoing allogeneic hsct for either malignant (24 cases) or non-malignant disorders (12 cases) were enrolled in the study; 19 patients received cb transplantation (cbt, 13 from a related and 6 from an unrelated donor) and 17 patients bm transplantation (bmt, 7 from a related and 10 from unrelated donor). results were also compared with those obtained in 11 adults given hsct for either malignant (7 cases) or non-malignant (4 cases) disorders. mscs were grown from bm aspirates taken 2-17 months after hsct. msc samples at the third-fourth passage were phenotipically characterized and resulted to be positive for cd73, cd105, cd106, cd29, cd13 (>98%) and negative for cd34, cd45, cd14 (<2%). donor/recipient origin of mscs was assessed by amelogenin assay (in case of male recipient/female donor) and microsatellite analysis. mscs were grown from 29 pediatric patients; in 8 samples (4 after bmt and 4 after cbt) a confluent layer of cells did not grow, leading to an insufficient quantity of mscs for chimerism analysis. molecular analysis on mscs demonstrated a full recipient chimerism in 10/13 and in 10/15 of the assessable pediatric patients given bmt and cbt, respectively. a mixed msc chimerism with donor cells was observed in 3 patients transplanted with bm cells and in 5 children given cbt. chimerism analysis performed on peripheral blood mononuclear cells (pbmcs) of the same patients, showed a full donor chimerism in all children given bmt but one, while a mixed chimerism was detected in 6 out of 19 children given cbt. a full recipient msc chimerism was observed in all adult patients, who also displayed a full donor pbmc chimerism. these data suggest that bm soil of pediatric patients might be more favourable than that of adults for the engraftment of transplanted mscs and that mscs able to engraft in the host can also be transferred with cb. graft engineering r1080 cryopreservation of peripheral blood progenitors for autologous transplantation in haematological malignancies with different concentration of cryoprotectant -five-year single-centre experience a. pivkova, l. cevreska, n. siljanovski, z. stojanoski, o. karanfilski, s. genadieva stavrik, i. panovska, s. krstevska balkanov, s. trajkova, b. georgievski clinical center (skopje,mk) in this study we present our five year center experience with cryopreservation of pbsc and autologous transplantation in 42 patients with hematological malignancies treated in a period 2000-2005 at department of hematology, skopje. material and methods: diagnosis of patients were (9 aml, 11 nhl, 10 mm, 12 hd) and median age at transplant was 34 years (7-63). mobilization of pbsc was provided with etoposide (vp-16) + g-csf 10mcg/kg in aml patients, and high dose cyclophosphamide 4-5gr/msq+g-csf 10mcg/kg or alone g-csf 10 mcg/kg in patients with limphoproliferative diseases. collected pbsc were cryopreserved in solutions with 5% dmso in 20 patients and 10% dmso in 22 patients, computer programmed until -80c, and stored different period in liquide nitrogen on -196c. autologous transplant was preformed wid conditioning consisted of myeloablative highdose chemotherapy, bucy in aml patients, high dose mel in mm patients, beam or hd ice in nhl patients and beam in hd patients. cell viability was assessed by fluorescence microscopy using acridine orange dye exclusion. results: a total of 103 pbsc cryopreservation procedures were preformed in our group of patients with median 3 (2-5) apheresis procedures. median period from storage of cryopreserved pbsc grafts until thawing was 46 days (32-60). total number of infused cd34+cells was between 2,0-15x10 6 /kg and median number of mononuclear cells was 4,2x10 8 /kg (1, 2) . the amount of infused dmso solution ranged between 210-650ml (median 430ml) with dmso concentration ranging 23ml-50ml (median 35ml) in a group preserved with 10%dmso and 13-23ml (median 19ml) in 5% dmso cryopreserved grafts. the viability of the fresh harvests before storage vas median 97% (range 68, 5-99, 9%). the poorer viability was associated with harvest cell count. bellow 300x109/l the median viability was 98% and only 2/42 cases had <85% viable cells. harvests count above 300x109/l the median viability was 78% (67,8%-99%). in a group of patients that received pbsc grafts preserved with 10% dmso, also revealed signs of mild dmso infusion related toxicity (22%vs14%). hematopoietic recovery was similar in both groups, sor ne>0,5x10 9 /l on day +9 (8-10), plt >20x10 9 /l on day +12 (11-14). our results confirm that the infusion of cryopreserved autologous pbsc in hematological malignancies revealed successful engraftment in all patients and good cell viability. we did not registered "hard to mobilize" patients and graft failure. thermogenesis axp™ and bioarchive™ systems for automated cord blood banking l. dobrila (1), s. jiang (2), j. chapman (2), d. marr (2), k. kryston (2), p. rubinstein (1) (1)national cord blood program at new york blood center (new york, usa); (2)thermogenesis corp (rancho cordova, usa) background: good tissue practices (cgtp) in cord blood banking require product uniformity and reproducible mononuclear cell recovery and viability, suggesting that automation could be critical to facilitating cgtp-compliance for cord blood banks. processes that lend themselves to automation are cord blood volume reduction, controlled rate freezing, storage and retrieval that avoids unnecessary transient warming events. we have evaluated the autoxpresssystem (axp),that allows for automated volume reduction in a closed system. the autoxpress consists of a microprocessor-controlled device and a disposable closed blood bag set that provides for the separation of cb into a freezing bag, an erythrocyte bag and an excess plasma bag, the mononuclear cell product is concentrated into a uniform volume in the freezing bag, ready to be cryoprotected and fully compatible with the bioarchive system,a system that allows the controlled-rate freezing, liquid nitrogen storage and retrieval of 3,600 cbus. study design: the efficiency with which cord blood hematopoietic progenitor cells can be concentrated into the freezing bag of the autoxpress bag-set was determined using the cd34 cell marker and colony-forming unit (cfu) counts as principal indices. the product was cryoprotected with 10% dmso, frozen in the bioarchive system, stored for 2-4 weeks under the liquid n2 level and then retrieved and thawed using the standard clinical protocol. twenty-three consecutive cord blood units were evaluated for cell recovery by measuring the collection and product volumes, the hematocrit and the counts of total nucleated cells (tnc), mononuclear cells (mnc), cd34+ cells and colony-forming units (cfu),before and after axp processing. we also determined these indices after freezing, storage in the bioarchive system and thawing. results: results are presented as the mean s.d. (n=23) for all values. the axpprocess achieved mnc fraction volumes of 19.7 0.3 ml with a final average hematocrit of 29.8 2.6%. the post-processing recovery of cd34+ cells was 98.2 8.0% and those of cfu 94.6 7.0%, of mnc 97.9 4.9% and of tnc 84.8 9.2%. less than 1% of tnc were lost into the excess plasma bags. granulocytes, accounting for 15% of tnc and less than 0.5% of the cd34+ cells were lost into the red cell bags. post-thaw the recoveries of cfu and viable cd34+ cells were 96 4.8% and 94 2.1%, respectively. conclusions: the axpefficiently and reproducibly separates cord blood mononuclear and cd34+ cells into a consistent, uniform volume. these cells retained their viability post bioarchive freezing, storage and retrieval (>94%). thus, axpcoupled with the bioarchive system supports a very high quality standard for automated cord blood processing. the influence of cryopreservation and the duration of frozen storage k. nowak, s. giebel, m. krawczyk-kulis, j. wojnar, j. holowiecki silesian medical university (katowice, pl) among the factors which enable successful transplantation, the ability to store and subsequently recover sufficient viable hematopoietic stem cells to reestablish hematopoiesis is critical. the goal of this study was to evaluate the impact of freezing procedures and cryopreservation in liquid nitrogen into -176°c on cell viability, wbc, cd34+ cells recovery and clonogenic capacity. in the retrospective study 74 samples derived from patients with hematological malignancies (n=66) and healthy donors (n=8) were analysed. median duration of the product storage was 3.6 years (1 day -15.6 years). the viability (%), wbc (g/l), and cd34+ (%) all decreased significantly after cryopreservation (p<0.000001) with median relative changes of -15.3%, -13.2%, and -10.7%, respectively. after thawing the viability equaled 79.5% (36-96%), wbc 46.3g/l (1.9-190 .8g/l), and cd34+ cells 0.47% (0.01-7.34%). in multivariate analysis the following factors were associated with poor recovery: 1) viability: presence of malignant disease (p=0.000002), use of cyclophosphamide (ctx) for mobilisation (p=0.00008), 2) wbc: presence of malignant disease (p=0.003), older age (p=0.003), 3) cd34+ cells: presence of malignant disease (p=0.006), storage duration (p=0.000003). after thawing the median number of clones/10 4 cells was as follows: 1) cfu-gm on day 7: 11 (0 -70.2), 2) cfu-gm on day 14: 27.6 (14 -95.5), 3) bfu-e: 11.3 (4.7 -48), 4) cfu-gemm: 1.9 (0 -7.2). the clonogenity was negatively influenced by: 1) cfu-gm on day 7: presence of malignant disease (p=0.00004), chemotherapy-containing mobilisation regimen (p=0.01), ctx for mobilisation (p=0.00004), 2) cfu-gm on day 14: older age (p=0.048), presence of malignant disease (p=0.000002), chemotherapy-containing mobilisation regimen (p<0.000001), 3) bfu-e on day 14: presence of malignant disease (p=0.00002), chemotherapy-containing mobilisation regimen (p<0.000001), cfu-gemm on day 14: diagnosis other than cml (p=0.0009). we conclude that both diagnosis and mobilisation regimen have impact on recovery and clonogenity of cryopreserved hematopoietic stem cells. on the other hand, even long-term storage enables preservation of vial cells with high clonogenic potential, which may be used for transplantation. immune reconstitution after hla-haploidentical transplantation using unmodified marrow and cd6depleted blood stem cell: not different from hla-identical transplantation p. rujirojindakul (1) immune reconstitution (ir) is a key role of allogeneic hematopoietic stem cell transplantation (hsct) not only because persistent immune defects is related to posttransplant infectious morbidity, but also because it may influence the risk of relapse and the development of secondary malignancies after hsct. many factors have an impact on ir, especially the degree of genetic differences between donor and recipient. several studies have shown extreme slow ir in patients receiving t-cell depleted graft from human leukocyte antigen (hla)-haploidentical (hap) donor. differently, we have used unmodified marrow on day 0 and cd6-depleted mobilized blood cells (mbc) on day 6 for haploidentical transplantation. cd6-depleted mbc are devoid of cd4-positive cells, they contain cd34-positive cells, nkcells and a minority of cd8-positive cells. to compare the ir during the first year post transplant between hap and hlaidentical (id) recipients, we have carried out a prospective, longitudinal analysis of ir in 15 hsct patients in each group. we analysed reconstitution of naïve, memory t cells, b cells, natural killer cells ,dendritic cells and monitored thymic output by using tcr rearrangement excision circles (trecs). surprisingly, reconstitution did not differ between the groups. this study reveals that ir in hap transplantation using unmodified marrow and cd6-depleted mbc is not worse than id group. background: autologous graft versus host disease (gvhd) is a novel self-limited autoaggression syndrome, encountered in the autologous pbsct (peripheral blood stem cell transplantation) setting with spontaneous occurrence in patients receiving cd34+ enriched autografts. aim: to present the case report, concerning the occurrence of a rather rare form of gvhd in a patient with cd34+ enriched auto-pbsct for ewing sarcoma. case report: we present the case of a 14 years old female patient with ewing sarcoma of the scull and right hip who underwent autologous pbsct with a cd34+ enriched graft. the preparative regimen consisted of busulfan and melphalan. engraftment occurred on day + 11 for neutrophils and on day + 12 for platelets. at day +22 signs of acute gvhd involving only the skin occurred initially in the axillae, flexion sites of the elbows and popliteal region and shortly afterwards involving the face, neck, trunk and abdomen. beside maculopapular exanthema, bullae formation and large epidermal descuamation were observed, an image corresponding to stage iv of acute gvhd. the patient remained feverless, without pruritus or other signs or symptoms. at the time of onset the investigation of immune reconstitution presented marked lymphopenia (abslolute values per microliter):cd4+ = 44 ,cd8+ = 34 ,cd16+/cd56+ = 173).no other modified biological parameters could be found. skin biopsy revealed lymphoid, predominantly cd8+ infiltration. in 50 days the lesions progressively disappeared, with the maintenance of residual hyperpigmentation and nail keratosis. conclusion: autologous gvhd, confined to the skin without any clinical-biological evidence of internal organ disease is a possible self-limited complication of hct cd34+cell enrichment of the graft could be in our case a predisposing factor.the impact on the evolution will remain an issue to be assessed. why will an amotosalen-based protocol for extracorporeal photopheresis be valuable? j. e. hearst (1) , f. heshmati (2), s. talib (1) (1)university of california (berkeley, usa); (2)hopital cochin (paris, f) bone marrow transplantation has the potential of providing a complete cure of the disease symptoms of hematologic malignancies and, ultimately, with an appropriate safety profile, the symptoms of hemoglobinopathies as well. even in the case of related fully matched bone marrow transplantation, there is morbidity and mortality associated with graft-versus-host-disease (gvhd). successful application of mismatched (related-haploidentical and unrelated) bone marrow transplantation (bmt) in patients with leukemia or lymphoma requires that improved overall outcomes of bmt be obtained, coupled with the avoidance or successful treatment for the gvhd now experienced in existing mismatched bmt protocols. these two goals may require use of reducedintensity conditioning (ric) in support of the transplantation. extracorporeal photopheresis (ecp) has proven to be effective as a treatment modality for gvhd following transplantation and is gaining popularity as a treatment protocol. yet, there remain significant technical challenges in the application of ecp. as a solution to many of these problems, we propose the development of a more simple and less expensive process for the psoralen photochemical treatment of the transplant patient's autologous leukocytes. this process will define the therapeutic dose of photochemically treated cells required for clinical responses equivalent to those which have been observed to be most effective using presently approved ecp instrumentation and protocols. however, this new process uses amotosalen, a more photochemically efficient psoralen, and it uses lymphocytes collected by the conventional methods of blood banks. the greater photoefficiency of the psoralen compound results in far shorter exposures of the target leukocytes to uva light, leading to less non-specific damage of the cells, leaving them metabolically active, although unable to divide. it is our premise that these are the ideal properties that photochemically treated lymphocytes must have in order to s312 maximize the positive immunological responses associated with a successful photopheresis treatment. uva1 phototherapy as a treatment for sclerodermic chronic graft-versus-host disease refractory to immunosuppressive therapy g. marotta, m. pellegrino, s. sammassimo, m. tozzi, c. miracco, m. fimiani, f. lauria azienda ospedaliera universitaria senese (siena, i) chronic graft versus host disease (cgvhd) is the most common late complication affecting long-term survivors of allogeneic hsct. several organs are targets of cgvhd but the skin is the tissue mainly affected. two subtypes of involvement, cutaneous lichenoid and sclerodermoid, have been described, based on clinical and histopathological examinations. chronic gvhd is usually treated with immunosoppressive drugs which, however, are not effective in about 20-40 % of patients. in this setting of refractoty/resistent patients, extracorporeal photochemotherapy and ultraviolet a (uva) radiation often show a positive clinical outcome, but the results are limited by the low number of treated cases. in our institute, uva1 phototherapy has been used for treating 3 patients with advanced cutaneous cgvhd (generalized sclerodermoid skin involvement) resistant to conventional immunosuppressive therapy. patients enrolled gave fully informed consent and they underwent skin biopsies before and after uva1 phototherapy. moreover, the cutaneous elasticity has been evaluated by means of an elastometer. uva1 radiation (340-400 nm) was emitted by a gp-24h irradiation unit with a dose of 60 j/cm². irradiance was measured with a spectroradiometer and found to be 95mj/cm² at skin level. patients were treated 5 times per week for a total of 6 weeks (total 1800 j/cm²) and the cutaneous lesions were carefully inspected and palpated before starting every treatment. patients were conditioned with a reduced intensity regimen and received unmodified g-csf mobilized pbsc from matched related donors. gvhd prophylaxis consisted of csa and mtx. all patients had failed to respond to at least three lines of immunosuppressive therapy. at the end of sessions the clinical response was assessed subjectively and objectively, and it was graded as good (obvious softening), moderate (mild softening) and poor (no change). two patients had a good response and one a moderate response reporting remarkable softening of skin lesions after uva1 phototherapy without side effects. clinical responses were associated to an improvement of histopathological findings. the evaluation of skin elasticity showed a significant increase in resilience, hysteresis, and distensibility. our results demonstrate the efficacy of uva1 phototherapy. it appear, in the setting of cutaneous cgvhd, as a procedure well tolerated and effective particularly for patients who do not respond to standard immunosuppressive treatments. rituximab in chronic graft-versus-host disease of the lung i. von olshausen, a. spyridonidis, h.c. bremer, w. windisch, h. bertz, j. finke university hospital freiburg (freiburg, d) we report about a 24 year old male patient in whom severe obstructive chronic graft-versus-host-disease (cgvhd) of the lung improved with rituximab, an anti-cd20 antibody. the patient had undergone hla-matched unrelated allogeneic peripheral stem cell transplant for his acute myeloid leukaemia in august 2003. with discontinuation of cyclosporine after day +100 mild cgvhd of the skin and liver occurred, but improved spontaneously within a few weeks. in january 2005 the patient presented with severe dyspnoea associated with an afebrile upper respiratory tract infection. the work-up revealed severe obstruction on lung function tests, nodular infiltrates in computed tomografy scan (ct), acute bronchitis in bronchoscopy, but no infectious agent in bronchial lavage. initial and long term treatment with various antibiotics, antimycotics and several immunosuppressants (prednisone, rapamycin, mycophenolatmofetil, cyclosporine a, cyclophosphamide) was unsuccessful. after a single dose of rituximab (375mg/m²) dyspnoea improved for a few days, but symptoms reappeared. we started a regular weekly rituximab application in july 2005 resulting in marked clinical improvement, slight improvement on lung function (peak exspiratory flow, pef) and regression of the nodular infiltrates in ct scan despite tapering off other immunosuppression and intermittent respiratory infections. the patient was able to participate in household thresholds again. rituximab therapy was discontinued in november 2005 because of patient's admission with spontaneous pneumothorax. the patient is now being evaluated for lung transplantation. like in our case, monoclonal anti-cd20 antibody has recently shown efficacy in cgvhd and several autoimmune diseases, probably due to elimination of b cells which may act as antigen presenting cells for t cells and as a source of autoantibody production. conclusion: we propose regular weekly rituximab application as a treatment option in cgvhd of the lung. are peak level measurements of cyclosporin a useful in allogeneic stem cell transplantation? c. scheid, c. heinz, u. holtick, k. hübel, v. göde, t. zander, a. engert, m. weihrauch, m. hallek university of cologne (cologne, d) peak level plasma levels of cyclosporin a (csa) 2 or 4 hours after drug administration (c2 or c4) correlate with the degree of calcineurin inhibition and with the auc much better than trough levels (c0). in solid organ transplantation dosing according to c2 levels rather than c0 led to reduced rejection rates. in allogeneic stem cell transplantation csa doses are still adjusted according to c0 levels. to investigate whether c2 and c4 levels would be useful in csa dosing after stem cell transplantation these measurements were performed in consecutive patients in addition to c0 levels. if intravenous csa was given, peak levels were assessed after the end of a 4 hour infusion. so far 16 serial measurements were undertaken in 10 transplant patients (6 aml, 1 all, 1 saa, 1 myeloma, 1 cll). there was only a weak correlation between trough and peak levels of csa and between dose and plasma levels irrespective whether the drug was administered orally or intravenously. although all trough levels were within the target range of 250 -450 ng/ml peak levels were all below 1000 ng/ml. there was no evidence of so-called "late absorbers" as all c4 levels were lower than the c2 levels. two patients showed evidence of microangiopathic hemolytic anaemia, however peak and trough levels of csa were not different from other patients. all but two patients showed signs of acute gvhd. in view of the csa doses given and the trough levels, the peak levels were considerably lower than expected from results in liver or renal transplantation. this was even more surprising as most patients received itraconazole, a drug known to increase csa levels by interfering with csa metabolism in the liver. in summary c2 monitoring is feasible and demonstrates lower peak levels than expected from solid organ transplantation. these levels should lead to a lesser degree of calcineurin inhibition. whether this translates in increased rates of gvhd remains to be seen with a larger patient number. if this holds true csa dosing with higher c2 target levels might decrease acute gvhd after stem cell transplantation as previously shown for rejection rates after liver transplantation. clinical and histopathological features of oral chronic graft-versus-host disease following allogeneic reducedintensity conditioning haematopoietic stem cell transplantation: a single-centre blind study f. demarosi (1) introduction: oral involvement of chronic graft-versus-host disease (cgvhd) occurs in 80% of patients suffering from cgvhd and the oral cavity may be the primary or even the only site of cgvhd involvement. lichen planus-like lesions are the most distinctive oral changes of cgvhd. the histopathologic changes of oral cghvd include epithelial atrophy, apoptotic bodies, hydropic degeneration of the basal cells, and a mononuclear subepithelial cell infiltrate with lymphocyte invasion. aim: the aim of this blind study was to compare clinical and histological features of oral mucosa in patients who underwent allogeneic reduced intensity conditioning hematopoietic stem cell transplantation (ric hsct). patients and methods: this study enrolled 10 adult patients who consecutively underwent allogeneic ric hsct for hematological malignancies between october 2004 and october 2005. all patients were assessed for the presence of oral cgvhd by oral examination performed on the day +100 at the unit of oral pathology and medicine, university of milan. clinical lichenoid changes of the oral mucosa were regarded as positive for cgvhd. following informed consent, an incisional biopsy was taken from oral mucosa of all patients, either with or without oral cgvhd lesions. biopsies were examined by a pathologist who was unaware of clinical aspect of the mucosa (blind). results: biopsies were taken from 4 patients with clinical evidence of oral cgvhd and 6 patients with apparently normal oral mucosa. biopsies were performed at a time point ranging from 96 to 165 days (median 113.5 days) after transplantation. sites of biopsies were buccal mucosa and gingiva. histological cgvhd changes were detected in all the 4 patients having also clinical evidence of oral cgvhd, and in 4 out of 6 patients with apparently healthy mucosa. conclusions: while histological changes of oral mucosa without corresponding clinical changes are not sufficient to make a definite diagnosis of oral cgvhd, their detection might be of considerable help to predict the onset of the disease following ric hsct. a longer follow up of patients showing histological changes with no clinical counterpart, will possibly elucidate whether such changes are indeed predictive of the occurrence of clinically evident lesions. evaluation of safety and tolerability of extracorporeal photochemotherapy in paediatric patients affected with graft-versus-host disease c. del fante, c. perotti, gl viarengo, p. bergamaschi, d. romano, l. salvaneschi policlinico s. matteo (pavia, i) objectives: extracorporeal photochemoterapy (ecp) is a therapeutic option for treatment of acute and chronic graft versus host disease (agvhd and cgvhd) resistant to standard drug therapy. in the pediatric context, ecp procedure has some technical limitations when compared to adult subset. low body weight, venous accesses, hypersensitivity to hypocalcemia, fluid balance and transfusion demand may represent a limitation for ecp treatment in children. we report our experience in 15 very low body weight children (≤ 15 kg) affected with agvhd and cgvhd in terms of safety and tolerability of ecp. methods: ecp consists of 3 distinct steps: 1) collection of mononuclear cells by spectra cobe (version 6.0) cell separator device processing 2 blood volumes; 2) ex-vivo dilution with saline and addition of 8-mop to the bag, transfer of the buffy coat in a uv-a permeable bag and irradiation at 22°c; 3) reinfusion of the cells to the patient after 2 hours to avoid hypotermia. 15 patients, median age 4.2 years (range:2-5) median weight 11.5 kg (range:7-15) were treated. a central venous catheter was positioned in all patients. our treatment protocol consisted in 3 procedures per week in agvhd and 2 procedures per week in cgvhd, both followed by 2 procedures every 2 weeks for 2-3 weeks then by 2 procedures per month. 8 patients were affected with agvhd (grade ii-iv with skin, liver and gut involvement); 7 patients had extensive cgvhd (skin, mucosal, liver, and lung).the cell separator device was primed every time with filtered and irradiated rbc. calcium gluconate was continously administered by pump during the procedure. the acd/whole blood ratio was always setted at 1:20. all patients were monitored for blood pressure and heart rate during the entire procedure. results: the total number of ecp procedures performed was 375, with a median of 25 (range:8-65) procedures/patient. 5/15 (33.3%) patients experienced mild hypotension, 6/15 (40%) moderate hypotermia and 2/15 (13.3%) hypocalcemia (nausea and vomiting); no procedure was discontinued. the transfusion demand did not augment during all the course of treatment; no life-treathening infections were recorded. conclusion: we demonstrated that ecp is feasible and safe even in very low body weight patients on condition that ecp is performed adopting some simple precautions. our experience broadens the cohort of patients who can benefit of this therapeutic option. silesian centre for cellular transplantation, poland k. suchnicki, a. lange lower silesian center for cellular transplantation (wroclaw, pl) 89 haematological patients -malignancies (cml: n=29; aml:n=23; all:n=20) and saa (n=17); children (n=33) and adults (n=56) were transplanted with bm (n=72) and pbpc (n=17) from hla-identical sibling donors in years 1990 to 2000. all leukemia patients received myeloablative conditioning (bucy or bucyvp);saa patients were conditioned with cy or cyatg. chimerism was tested and proved to be complete in all but 5 cases (death before day +30). during 13 years of follow up:31 patients died of transplant related causes (regimen related toxicity:n=2, rrt/agvhd:n=2, infection: n=7, agvhd/multi organ failure/inf:n=8 , cgvhd/mof/inf:n=12) or relapse (n=8). agvhd was seen in 44,2% cases (grade1=19,7% of patients with hematological recovery, grade2=14%, grade>2= 10,5%). severe agvhd (grade>2) was seen only in leukemia patients, who received toxic conditioning, but not in saa conditioned only with cy+-atg. agvhd was more frequent and severe in pts with a high grade of toxicity (among patients with low rrt there were 63% cases of agvhd=0 and no cases of agvhd>2; among patients with high rrt there were 38% agvhd=0 and 22% agvhd>2). in addition to toxicity infections and inflammation seemed to aggravate agvhd as shown by the presence of an elevation of serum crp at the advent of severe agvhd. apparently the presence of toxicity and agvhd had an additive negative effect on survival. the incidence of cgvhd among all patients living>day+100 was 49% and increased with :i) diagnosis: saa(11,8% of saa cases),aml (47,4%),all(53,9%),cml(73,9%),ii)previous agvhd: all surviving patients with agvhd>1 developed cgvhd. cumulative survival is better in following :i)saa vs aml/all/cml (p=0,003),ii)all, cml early stage vs advanced (p=0,02),iii) conditioning bucy vs bucyvp (p=0,02), iii) age: children vs adults (p=0,26),iv) gender: female recipient from male donor better than all other pairs (p=0,67),v) rrt: toxicity grade 0-4 (sum of who degrees for toxicity of skin, mucous, liver, gastro-intestinal tract)vs grade 5-7 vs grade 8-14 (p=0,012), vi) crp: serum level of 0-25 vs level>25 (p=0,0031),vii) agvhd grade 0-1-2 vs grade 3-4(p=0,00069 ), viii) cgvhd grade lim better than no cgvhd better than extcgvhd (p=0,224) which reflects gvl surveillance of gvh cells (less relapses) without overt, life threatening reaction. this observation documented that the outcome of transplantation in sibling setting depends largely on toxicity, infections and agvhd which in turns aggravate themselves. the role of atg in reducing acute gvhd in related and unrelated donor transplants y. zalyalov, a. ganapiev, s. alexeev, a. smirnova, n. stancheva, n. ivanova, a. alyanskiy, b. afanasyev pavlov's state medical university (st. petersburg, rus) background: acute graft versus host disease (gvhd) remains the major complication of allogeneic haemopoietic stem cell transplantation (allohsct) with an incidence of 35-65% and mortality of up to 40-50%. atg is commonly used in the conditioning regimens for donor transplants to reduce the risk of rejection and gvhd. patients and methods: since 1998 up to date we have performed 96 allohsct (52 (54%) with atg and 45 (46%)without atg) for the following patients -aml (n=22), all (n=44), cml (n=13), non-hodgkin's lymphoma (n=3), aa (n=6), hodgkin's lymphoma (n=2), mds (n=2), kostmann's syndrome (n=1), hypereosinophilic syndrome (n=1). the average age was 18 years . dose of atg was 60 mg/kg.b.w. (45-120). myeloablative conditioning regimen consisted mainly of bu-cy (27 pts (52%), nonmyeloablative conditioning treatment -flu-cy (25 pts (48%). the gvhd prophylaxis was short term mtx and csa. results: rate of gvhd grade 0-ii in pts with atg was 85% (44 pts), gvhd grade iii-iv -15% (8 pts). rate of gvhd 0-ii in group pts without atg was 64%(29 pts) and gvhd iii-iv -36% (16 pts). tmr in group with atg was 56% (29 pts), without atg -67%(30 pts). os in pts with atg was 44%, without atg -33%. conclusions: the use of atg in conditioning regimen reduces the incidence of acute gvhd grade iii-iv and increases overall survival in patients after allohsct (p= .04). intracellular markers of eosinophils and mast cells in patients with lymphoproliferative and myeloproliferative disorders associated with high-grade eosinophilia l. komarova, y. zueva, n. mikhaylova, b. afanasyev, a. totolian pavlov state medical university (st. petersburg, rus) objectives: distinct eosinophilia rarely occurs in chronic graftversus-host disease (gvhd) after allogeneic hematopoetic stem cell transplantation (hsct), but is often seen in hematological malignancies, including lymphoproliferative diseases. a hypothesis exists that marked eosinophilia in chronic gvhd is mainly of non-clonal origin and, in general, is associated with favorable prognosis. the aim of this study was to investigate the serum levels of secretable eosinophil and mast cells proteins in the patients with chronic gvhd and other hematological malignancies. patients and methods: six post-transplant patients with chronic gvhd with eosinophilia were followed up after allo-hsct. thirty hematological malignancies in patients with marked eosinophilia (>0.4 x 10 9 /l) were also observed, including cases of lymphoproliferative (n=20) and myeloproliferative (n=10) diseases. eighteen patients with bronchial asthma (ba) comprised a reference group for polyclonal eosinophilia. high content of peripheral blood eosinophils served as the major criterion of patients' selection and was confirmed by flow cytometry. eosinophil cationic protein (ecp) and tryptase were measured in serum by fluoroimmunoenzyme assay (pharmacia, sweden). results: the percentages of circulating eosinophils in patients with lymphoproliferative (median: 11.7%; range, 2-78%) and myeloproliferative diseases (median 16.8%; range, 1-68%), and in cases of chronic gvhd after allo-hsct (median 16.3%; range, 3-70%) were significantly higher than in ba patients (median 10.2%; range, 5-23%); ð=0.01. the levels of total ecp were markedly increased in the patients with lymphoproliferative diseases (median: 18.8 ng/ml; range, 2-197 ng/ml) compared to ba (median: 10.5 ng/ml; range, 2.8-80 ng/ml); p=0.001. however, the serum levels of tryptase and ecp in total group of hematological patients (median: 6.5 ng/ml; range, 2 to 24.1 ng/ml) were not increased, as compared with asthma cases (median: 5.4 ng/ml; range, 1.9 to 24.4 ng/ml); p=0.05. likewise, in chronic gvhd with eosinophilia, the serum contents of both ecp and tryptase did not differ from those in ba. conclusion: the significant differences in ecp levels in blood serum in the patients with lymphoproliferative diseases and absence of differences in ecp and tryptase levels in blood serum of chronic gvhd and ba patients suggests intact functions of mature eosinophils in gvhd, thus reflecting nonclonal genesis of this feature in both states. natural killer cell activity in the early phase after allogeneic stem cell transplantation: impact of conditioning strategies l. fischer, o. penack, c. gentilini, e. thiel, l. uharek charité campus benjamin franklin (berlin, d) background: research into the role of natural killer (nk) cells in allogeneic stem cell transplantation (sct) has been greatly expanded recently. nk cells may contribute to the gvl reaction possibly without exerting a gvhd effect. using a novel flow cytometric assay, which detects the lytic granule membrane protein cd107a as a marker for nk cell degranulation, we investigated the effect of in vivo t cell depletion and the type of conditioning on nk cell function in the early phase after allogeneic sct. methods: peripheral blood mononuclear cells were collected at day +30 and +90 after allogeneic sct and incubated with the nk sensitive cell line hl60 (3 hours at 37°c, e:t ratio 1:1). pe-cy5 conjugated anti-cd107a antibody was added prior to incubation, monensin (0.05mm) was added after 1 hour of incubation. finally cells were further stained against cd56, cd3 and cd16 and the proportion of cd107a positive nk cells was measured by flow cytometry and the absolute number of degranulating nk cells was calculated. results were compared to values from 15 healthy controls. results: twenty two patients were investigated of whom 14 had been treated with a conventional dose conditioning regimen and 8 had received a reduced dose regimen. at day +30, the proportion of nk cells with cytotoxic activity (cd107a+/cd56+) was significantly reduced as compared to normal donors (2.6% vs. 5.6%, p<0.001). at day +90 the percentage of degranulating nk cells in five evaluated patients was within normal range (mean 5.7%). the predominant proportion of degranulating cells was in the cd56dim/cd16-subpopulation (mean 9.8%). at day +30 the percentage of cd107a+ cells averaged 1.9% after conventional conditioning compared to 4.0% after reduced intensity conditioning (p=0.21). the absolute number of degranulating nk cells was significantly reduced after conventional conditioning (4.1/µl vs. 19.8/µl, p=0.011). neither the percentage (2.5% vs. 2.9%, p=0.77) nor the absolute number (9.4/µl vs. 13.5/µl, p=0.59) of cd107a+ nk cells differed significantly in patients with and without atg induced t cell depletion at day +30. conclusion: according to our data cytotoxic activity of nk cells is reduced after allogeneic sct. the absolute number of nk cells with cytotoxic activity is significantly higher after reduced intensity conditioning which may impact on the outcome of this strategy. we saw no significant influence of antibody mediated in vivo t cell depletion by atg on nk cell activity during the first two months post sct. it is known that nk alloreactivity is involved in the control of neoplastic cells in the setting of aploidentical bone marrow transplantation (bmt) in patients with acute myeloid leukemia (aml). the role of nk alloreactivity in the setting of marrow unrelated transplant (mud) in lymphoid neoplasia is still controversial. in a series of 15 patients (9 acute lymphoid leukemia, 4 non-hodgkin's lymphoma l, 2 severe aplastic anaemia), we investigated whether nk alloreactivity is involved in the control of lymphoid neoplastic cells. in addition, using monoclonal antibodies (moabs), we evaluated the expression of killer immunoglobulin-like receptors (kirs), killer lectine-like receptors (klrs), and natural cytotoxicity receptors (ncrs) in patients under study. according to hla-cw alloreactivity, patients were separated into two groups: a group of 6 patients with potential nk alloreactivity (group a: 3 all, 1 nhl, 2 saa), a second group of 9 patients lacking nk alloreactivity (group b: 6 all, 3 nhl). the mean follow up was 24 months ± 6 months. 100% of group a patients were alive, whereas 66% of group b were still in remission, indicating a role for alloreactivity in lymphoid neoplasia. concerning expression of nk receptors, group a was characterized by the high expression of kirs, potentially involved in alloreactivity in 2 out of 6 patients. klr was represented by cd94/nkg2a in 5 out of 6 and in one case by cd94/nkg2c. this last case showed a recovery of cd94/nkg2a phenotype after 18 months. ncrs and nkg2d were usually expressed, with exception of nkp44. group b was characterized by an heterogeneous pattern of expression of kir, whereas cd94/nkg2a was expressed in 7 out of 9 cases and 2 cases expressed nkg2c. interestingly, one of these two cases relapsed during follow-up. ncrs were usually expressed, with the only exception of nkp44. our data indicate that nk alloreactivity might be an advantage for survival in patients affected by acute lymphoid neoplasia. no correlation could be demonstrated with expression of natural killer receptors (nkrs) and clinical behaviour, suggesting that analysis at clonal level is mandatory to get insights into the mechanism involved. this study was supported by a grant from fondazione città della speranza. a rare complication after allogeneic stem cell transplantation: acute axonal neuropathy a.d. moicean, d.n. colita, v.n. mirea, a.m. dumitrescu, t. puscariu, z. varady, a. tanase, i. constantinescu fundeni university institute (bucharest, ro) despite considerable progress in the management of allo-hsct, infection remains an important cause of morbidity and mortality after transplant. cmv is frequently involved in the infectious pathology after hsc transplantation. we present a 18-year old women with high-risk acute lymphoblastic leukemia who underwent matched sibling pbsc transplantation. she received standard conditioning regimen (endoxan 120 mg/kgc and tbi 12 gy). gvhd prophylaxis received was with csa and methotrexate. she developed grade ii gvhd (cutaneous and intestinal) on day +16 with resolution under corticotherapy. starting with day +50 she had weakness, osteoarticulary and muscular pain with functional impairment. neurological examen and the electrophysiological study showed an acute axonal neuropathy. she had more than 16 000 cmv copies in the blood pcr. she received 250 mg bid i.v ganciclovir for 21 days as induction therapy and than 300 mg/d for more 4 weeks with complete remission of the neurological symptoms a 15 year old female patient was diagnosed with cml in chronic phase and stem cell transplantation (sct) was planned from her one antigen mismatched sister. pre-transplant routine investigations revealed a high antibody titer against toxoplasma gondii (ift 1:5120, kbr 1:40, igm negative) indicating the history of a previous infection. cerebral magnetic resonance imaging (mri) ten days before transplantation showed no abnormalities. the patient was conditioned with busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg). because of the hla-a mismatch rabbit atg (fresenius, 60 mg/kg) was added. graftversus-host disease (gvhd) prophylaxis consisted of cyclosporine a and mtx. pbscs containing 7.2 *106 /kg cd34pos stem cells were transfused. engraftment occured rapidly on day +12. on day +11 the patient developed severe headache and hours later generalized seizures. computered tomography showed multiple patchy hypointense lesions in the subcortical white areas with corresponding nodular signal enhancements in t2 weighed mri analysis suggesting an infectious process. eeg revealed pronounced focal abnormalities in the left hemisphere extending from frontal to the temporal regions. liquor analysis revealed no causative infectious agent. assuming reactivation of toxoplasmosis we initiated a specific quatruple therapy consisting of pyrimethamin orally, sulfadiazin orally, clindamycin i.v. and tmp-smx i.v. clinically the patient improved rapidly. two weeks later cerebral mri showed regression of the temporoparietal white matter lesions with further improvement during the following weeks resulting finally in complete resolution. antibody titers against toxoplasmosis slowly regressed during the months following transplantation under continuation of oral specific therapy. the patient is now more than one year after stem cell transplantation alive and well without signs of toxoplasmosis and without neurological sequela. toxoplasmosis is a rare but dreaded complication usually developing between 2 and 6 months after stem cell transplantation with a high mortality rate. cml patients seem to be at increased risk for reactivation of toxoplasmosis after stem cell transplantation. the course of our patient, however, is unusual because of the very early manifestation after stem cell transplantation, the rapid course and prompt response to treatment. mucositis is a common side effect of chemotherapy and radiotherapy with no effective treatment. it occurs when cancer treatment destroys the rapidly dividing epithelial cells, particularly in the oral cavity, leaving the mucosal tissue open to ulceration and infection. the aim of this study was to assess the state of oral mucosa in a patient after allo-pbsct who has received palifermin, a recombinant human keratinocyte growth factor. materials and methods: a 19-year-old male was treated in the department of haematology of the medical university in warsaw due to the aml. conditional chemotherapy was applied, according to the bucy 4 + atg regimen and allogeneic haematopoietic cells transplantation from an unrelated donor. he was receiving palifermin (60 microg/kg/d) intravenously for 3 consecutive days immediately before the initiation of conditioning therapy (on days -13, -12, -11) and after allo-pbsct (on days +3, +4, +5). on day +3 the oral mucous membrane was pale and swollen, with linea alba visible on cheeks. superficial glossitis and viral pharyngitis were noted. beginning with day +5/+6 proliferative gingivitis was observed. on day +9 gingival contour was altered and the gingiva covered nearly completely tooth crowns of all teeth. the gingiva were whitened, as if covered by thick epithelium. slight gingival hyperplasia was still observed on day +24. during the forming of gingival hyperplasia the patient had a subjective "membrane growing" sensation with tingling and itching. he reported an oral cavity pain score of 0 in the 10-point pain scale. since day +4/+5 skin rash coexisted, spreading over hairy head skin, face, dorsum and chest. disseminated papulopustular (acne-like) lesions were observed. some of them were related to the hair follicles. skin changes were present till day +15. neutropenic fever was noted on day +6 (absolute leucocytosis 0.1 g/l). concomitant medications: orungal 2 x 200 mg p.o., heviran (aciclovir) 5 x 400 mg p.o., tazocin 4 x 4.5 g i.v. on days +2 and +3, maxipime 3 x 2.0 g i.v. on days +3 till +7, vancomycin 2 x 1 g on days +5 till +14, metronidazole 3 x 500 mg i.v. on days +4 till +14, neoral 2 x 100 mg i.v. since day +5 and 2 x 150 mg since day +13, zyrtec 1 tablet/d since day +6. conclusions: palifermin is an efficient pharmaceutical in mucositis prevention in patients after allogeneic pbsc transplantation. transient complication of hyperplastic gingivitis with a concomitant skin eczema of a papulopustular nature arose. progressive fatal respiratory failure due to pseudomembranous aspergillus tracheobronchitis following allogeneic stem cell transplantation p. sedlacek, p. hubacek, k. zdrahalova, p. pohunek, o. nyc, p. pavlicek, j. stary university hospital motol (prague, cz) invasive fungal infections are frequent and often fatal in patients following allogeneic hematopoietic stem cell transplant (hsct). long-term immunosuppressive therapy for graft versus host disease (gvhd) seems to be the most predisposing factor. we present a 16 year old girl with paroxysmal nocturnal hemoglobinuria (pnh). due to unfavourable course (severe attacks of hemolysis requiring hemodialysis, budd-chiari syndrome) she was indicated for hsct. she consequently failed to engraft two unmanipulated grafts from hla mismatched (b, cw alleles) unrelated donor. first time bone marrow was infused with cd34 7x10/6/kg, conditioning consisted of treosulfan, cyclophosphamide and atg. second time peripheral blood stem cells (pbsc) were used with cd34 8x10/6/kg after fludarabine, cyclophosphamide and atg. after 3 months lasting aplasia third allogeneic graft (pbsc -cd34 4,5x10/6/kg) was given with no conditioning surprisingly followed by rapid and full trilineage engraftment. she than developed severe acute and chronic extensive gvhd, treated with cyclosporine a, steroids, tacrolimus, mycophenolate, sirolimus and antihymocyte globuline. during post-transplant course she suffered from bkv hemorrhagic cystitis, repeated cmv reactivations and colitis, drug induced nephropathy and steroid diabetes. she had a long-term and lasting preemptive voriconazole prophylaxis. for acute hemodynamic instability due to severe gastrointestinal bleeding 7 months after hsct she was transferred to icu and electively intubated. at that time she was also heparinized for acute vein thrombosis. early on she started to desaturate, chest x-ray showed unilateral atelectasis. bronchoscopy revealed whitish membranes, plugs and casts causing extensive obstruction of both lungs. cultures grew aspergillus fumigatus. therapy including caspofungin, nebulized amphotericin b and repeated mechanical removal of obturating membranes failed to stop progression and patient died due to isolated respiratory failure 10 days upon arrival to icu. autopsy confirmed fungal involvement of trachea, larynx and bronchi. pseudomembranous mycotic tracheobronchitis may be a rapidly progressing complication in heavily immunocompromised patients. complex therapy including even combination of potent antifungals may fail to overt fatal course. even in patients on antifungals this complication must be actively looked for with early use of bronchoscopy and exact identification of pathogen. systemic candidiasis is a rare but life threatening complication in immunosuppressed patients undergoing allogeneic sct. combination of new antifungal agents might improve outcome in these patients. here, triple anti-mycotic therapy is described in an all patient in urgent need of allogeneic bone marrow transplantation. the patient with t-cell acute lymphoblastic leukemia of thymic differentiation achieved remission after treatment according to the gmall 07/03 protocol. two months after the consolidation therapy relapse resistant to treatment regimens containing fludarabin, cytarabin, etoposide and amsacrine occured. even after claeg (cladribine, etoposide, cytarabine and campath-1h) the patient still had 35% leukemic bone marrow blasts requiring high dose chemotherapy with allogeneic stem cell transplantation. one day after start of the conditioning regimen the patient showed mycotic skin manifestations and blood cultures became positive for candida cruzei despite fluconazol prophylaxis. because of the limited sensibility of fluconazol resistant candida species to liposomal amphotericin b and the high mortality rate in patients with systemic candidiasis voriconazol was added immediately to liposomal amphotericin. subsequently, the patients body temperature increased and caspofungin was added. since the mycotic skin manifestation responded to this triple anti-mycotic combination allogeneic peripheral blood stem cell transplantation from an unrelated donor could be performed. altough the fever resolved later the patient showed signs of a septic shock requiring intravenous administration of dopamine. with the unchanged triple antifungal therapy the patient became afebrile, skin manifestations showed complete resolution and cultures became negative. three months after the onset of systemic candidiasis the patient was fully active with no signs of fungal infection and in haematological and molecular remission. this case shows, that the entire intense conditioning chemotherapy could be administered and allogeneic stem cell transplantation is feasable in patients with systemic candidiasis when such combined antifungal treatment is given. i. vrelust (1), a. gadisseur (1), e. steel (1), w. schroyens (1), a. van de velde (2), z.n. berneman (1) (1 neutropenic patients, especially following aggressive chemotherapy, are at high risk for infectious complications. these are an important contributary factor to the treatment related morbidity and mortality (trm). because neutrophils represent the first line of host defense, granulocyte transfusion therapy is a tempting therapeutic approach. although such therapy has been employed sporadically for several decades, clinical benefit has been compromised by technical problems and low granulocyte yields resulting from inadequate donor stimulation. the discovery of granulocyte colony-stimulating factor (g-csf) as a means to elevate blood neutrophil counts in healthy donors has rekindled interest in granulocyte transfusion therapy. we describe a 55-year old female patient with an acute myeloid leukaemia (aml) who underwent remission induction chemotherapy. in absence of repopulation of the peripheral blood the bone marrow showed persistent aml. despite antifungal prophylaxis the patient developed an invasive pulmonary aspergillosis. voriconazole, g-csf and caspofungin were added, and granulocyte infusions were started because of the continuing neutropenia. the granulocytes were collected from donors with a compatible blood group through leucaferesis after stimulation with g-csf and dexamethasone (8mg), and then irradiated. the transfusions were given three times a week. conditioning for a reduced intensity (ric) allogeneic peripheral blood stem cell transplant (pbsct) with a hla-identical brother was started. in total 15 granulocyte infusions were given; of which 5 after transplantation, the last on day +10 after pbsct. they were discontinued when the anc count reached 1.0x109/l. all granulocyte infusions were tolerated very well. four months after transplantation she is in a complete hematologic remission without signs of graft-versus-host-disease (gvhd), and without signs of active pulmonary infection. the precise role of donor granulocyte infusions remains to be delineated, partly because of the lack of defined clinical trials. we conclude that granulocyte transfusion therapy may be useful for neutropenia-related fungal infections in patients with hematologic malignancies. the use of granulocyte infusions during a ric-pbsct procedure does not seem to lead to an increased risk of gvhd or hamper engrafment itself. a. kerguelen, m. canales, a. lopez, m. martin, t. cobo, d. hernandez, j. g-bustos, f. hernandez-navarro university hospital la paz (madrid, e) introduction: patients with haematological diseases previously diagnosed with invasive fungal infection (ifi) are considered to be at high risk of suffering reactivation of the infection during subsequent intensive chemotherapy. methods: in the last 2 years 3 patients with haematological diseases (2 aml and 1 acquired aplastic anaemia) and previous invasive aspergilosis have undergone allogeneic haematopoietic stem cell transplant in our centre. all patients received as primary antifungal therapy combination of liposomal amphotericin b (ambisome) and caspofungin displaying complete or good partial radiological resolution of the infection. itraconazole and voriconazole was continued as secondary prophylaxis. conditioning regimen consisted of busulfan and cyclophosphamide in patients with aml and atg and cyclophosphamide in the aplastic anaemia patient. cyclosporine in combination or not with methotrexate was the regimen administered to prevent ghvd. results: in a patient with aml no clinical or radiological signs of reactivation of fungal infection were observed through the transplant procedure. in the other patient with aml itraconazole was changed by liposomal amphotericin because of unresponsive fever. in the patient with aplastic anaemia combined therapy with amphotericin and caspofungin was initiated because of radiological worsening, but galactomannan antigen was negative in all analysis performed. no patient has died because of infectious complication during or after transplant. conclusion: availability of new antifungal agents does allow pre-transplant therapy of previous ifi, aiming to achieve a clinically undetectable state of infection, and an adequate antifungal treatment during transplant to diminish risk of reactivation of fungal infection in allografted patients. however, the optimal use of antifungal agents or their combinations remains to be determined and more studies are necessary to confirm our experience. the frequency of invasive fungal infections, in particular infections due to aspergillus and other moulds, has increased over the past two decades. whereas invasive aspergillosis mainly involves the respiratory tract, lung or sinus, the lower gastrointestinal tract is rarely affected, most often in the frame of secondary dissemination. primary invasive aspergillosis of the gut is a rare event associated with high mortality and has not been reported to date in patients after autologous stem cell transplantation (sct). we report on a 10-year-old boy who developed isolated intestinal aspergillosis soon after autologous stem cell transplantation for pnet of the central nervous system. the boy received broad-spectrum antibiotics because of neutropenic fever, and antimicrobial prophylaxis included trimethoprim-sulfamethoxazole, metronidazole, acyclovir, fluconazole and topical amphotericin b. antimycotic therapy was started because of persistent fever and abdominal pain, and rising serum levels of galactomannan and the isolation of aspergillus fumigatus from the stool suggested invasive aspergillosis. the boy underwent enterostomy on day +19, and diagnosis of intestinal aspergillosis was pathohistologically confirmed. no other site of invasive aspergillosis was evident. the patient was treated with antimycotic combination therapy consisting of liposomal amphotericin b, voriconazole and caspofungin. the clinical condition slowly improved over the next months. enterostomy was removed on day +120 and antimycotic treatment has been stopped soon after. currently, on day +140, the boy is at home without major gastrointestinal complaints. we conclude from this case that primary intestinal invasive aspergillosis can occur in patients undergoing autologous stem cell transplantation, and therefore, this diagnosis has to be considered in this setting in patients suffering from fever and abdominal pain. in case of positive galactomannan antigenemia, an extensive search for invasive aspergillosis should be performed and, at the same time, early antifungal therapy should be started. mycobacterial disease is a rare and difficult diagnosis in hematopoietic transplant recipients. we report the case of a twenty-year-old woman who underwent a second matched unrelated donor (murd) hsct for a null all in second complete remission. conditioning regimen consisted on cyclophosphamide 120 mg/msq; busulfan 16 mg/kg and thiotepa 750mg/msq. on day +44, while receiving acyclovir prophylaxis, and a previous cotrimoxazole prophylaxis, she developed an acute ascending paraparesis with anaesthesia and reflex loss and loss of sphincter control. mri imaging showed gadolinium contrast enhancement on cauda equina roots. csf analysis showed elevated cell count (116/mm³, normal range nr: 0-5/mm³), elevated csf protein concentration (261mg/dl: nr: 1-40mg/dl), and no hypoglucorraquia (54mg/dl with simultaneous plasma concentration of 84mg/dl). flow cytometry analysis showed 96% t lymphocytes, 0.09% non clonal b lymphocites, 3,5% monocytes and 0,4% neutrophils. bacterial, fungal and mycobacterium presence was ruled out and empirical antiviral treatment consisting on foscarnet (180 mg /kg /day), immunoglobulin (0.5g/kg/48hours) was started. metilprednisolone 1g per day for tree days flowed by 30mg per day for three consecutive weeks. no clinical response was observed and neurotrophic viral infection was excluded by pcr technique. a second lumbar puncture was performed 17 days later showing a lower number of lymphoid cells (10/mm³) but with an increase in protein concentration (321mg/dl) without hypoglucorraquia; moreover auramine positive rods were present. antituberculous treatment with rifampicin (600 mg/d), isoniazide (300 mg/d), ethambutol (2.5g/d) and pyrazinamide (2g/d) were started, with no clinical improve but no worsening. hepatic toxicity was developed and isoniazide an rifampicin were suspended after a month of complete treatment and ofloxacin (400mg/12h, ethambutol and pyrazinamide was started as a second-line therapy. this is to our knowledge the first reported case of transplantationrelated tuberculous arachnoiditis. pentastomiasis of the liver in a patient following unrelated stem cell transplantation with estimated hepatic acute graft-versus-host disease j. dahlke (1) , r. kobbe (2), a.a. oyekunle (1) , f. ayuk (1) , n. kröger (1) we report a case of human hepatic pentastomiasis (armillifer armillatus) in a 22-year old man with aml who immigrated to germany from togo in 2002 and died as a result of relapse, 37 days post hla-matched unrelated stem cell transplantation. at day +30 post transplantation, diarrhoea occurred and gastroscopy with excisional biopsy revealed an acute graft versus host disease of the stomach and upper small bowel. for treatment we started with a tapering schedule of methylprednisolone (5mg/kg body weight/day). three days later we observed a hyperbilirubinemia and slightly increased liver enzymes. the abdominal ultrasound showed an increased size of the liver and an increased portal perfusion. the findings were interpreted as a hepatic involvement of the acute graft versus host disease (agvhd) and a salvage treatment with mycophenolat mofetil was initiated. the patient died as a consequence of intracerebral bleeding in a cerebral infiltrate of the leukaemia. at autopsy, in addition to the cerebral findings, we found multiple pentastomides (documented by photographs) up to 3cm length (spec. armillifer armillatus) subcapsular, in the parenchymatous tissue and in the portal veins. no agvhdlike inflammatory infiltrate in the liver was observed. late effects and quality of life r1106 trilineage hypoplasia after initial neutrofil engraftment in patients with acute myeloid leukaemia undergoing autologous transplantation with bu/cy conditioning: frequency, outcomes and prognostic significance (case report) a. pivkova, l. cevreska, n. siljanovski, z. stojanoski, s. genadieva stavrik, i. panovska, s. krstevska balkanov, s. trajkova, b. georgievski clinical center (skopje, mk) myeloablative conditioning with autologous stem cell transplantation (asct) is a treatment option for aml patients with lack of sibling donor. hematopioetic engraftment may be prolonged depending on the type of myeloablative regimen, mobilization regimen, dose of mnc and patients age. in a 5 year period (2000) (2001) (2002) (2003) (2004) (2005) we realized a total of 9 autologous transplanations with cryopreserved peripheral blood cells (pbsc) and busulfan and cyclophosphamide conditioning. we present two cases (22%) of aml (standard risk) patients (two females 48 and 40 years at transplant) that underwent autologous transplantation at department of hematology, skopje. mobilisation of pbsc was preformed with vp-16 2000 mg/msq + g-csf 10µgr/kg in one patient and hd-arac 3gr/mscx3, idarubicine 10mg/msqx3+g-csf 10µgr/kg in the other patient. a minimum of 2,0x10 8 /kg mnc and 3,0x10 8 /kgmnc respectively were collected and preserved in 5% dmso solution. we registered engraftment for ne>0,5x10 9 /l on day +11 and +13 and for plt>20x10 9 /l on day +13 and +16. patient were followed up in outpatients and pancytopenia was registered one month after transplant with plt<20 x10 9 /l with mild haemorrhagic complications plt transfusions dependable, hb < 80g/dl and red cell transfusion dependence (on two week interval), wbc < 3,0 x10 9 /l with cytokine treatment (twice a week g-csf 375 µgr/kg). bone marrow biopsy revealed trilineage hypoplasia, no signs of organomegaly, no microbiological and viral findings. one patient has recovered completely 15 months after transplantation and the other is still in good physical condition but present pancytopenia 12 months posttransplant. we conclude that prolonged pancytopenia is due to bu/cy conditioning, the age could be a significant factor for starting myeloablative conditioning prior autologous transplant as well as minimum mnc dose could prolonge immune reconstitution. neurological long-term follow-up after allogeneic bone marrow transplantation p. sostak, c. padovan, m. holtmannspötter, g. ledderose, h. kolb, a. straube klinikum großhadern (munich, d) to improve our knowledge about the neurological outcome after allogeneic bone marrow transplantation (bmt), we have started a prospective study already 6 years ago. so far we could show that within the first year after transplantation a significant proportion of patients (65%) had developed neurological sequelae. besides well-defined neurological complications more than half of the study population suffered from new neurological abnormalities of unknown origin predominantely affecting the peripheral nervous system. in a small subgroup of patients already central nervous signs, cognitive deficits and white matter lesions could be detected and this was in relation to chronic graft-versus-host disease (gvhd)/immunosuppression. to determine whether central nervous system (cns) involvement during gvhd might manifest at a later time in more bone marrow recipients, the follow-up period now was extended and patients again received a neurological examination, underwent a neuropsychological test battery and standard mri sequences. long-term follow-up results will increase the insight onto the spectrum, incidence and etiology of neurological sequelae after allogeneic bmt. they will be discussed in relation to retrospective and experimental data, which suggest involvement of the cns during gvhd. keratoconjunctivitis sicca with recurrent calcium deposition in the cornea and severe visual loss due to gvhd p. keslova (1) we present severe keratoconjuctivitis sicca with recurrent calcium deposition in the cornea after keratoplasty in a patient with extensive chronic graft versus host disease (gvhd). a 17-years-old girl with myelodysplastic syndrome (raeb) underwent stem cell transplantation (sct) using peripheral blood stem cells of two alleles mismatched unrelated donor (b, cw). conditioning regimen consisted of busulfan, cyclophosphamide and melphalan, for gvhd prophylaxis combination of rabbit antithymocyte globulin (atg fresenius) and cyclosporine a (csa) with short term methotrexate were used. due to renal toxicity csa was early switched to tacrolimus and corticosteroids were started at day+22. week after she developed steroid resistant acute gvhd grade iv with skin and gut involvement. for that she was successfully treated with combination of steroids, mycophenolate mofetil and sirolimus (day+27 through day+71). during period of acute gvhd severe keratoconjuctivitis sicca with deep corneal defects has been developing and visual acuity had deteriorated. there were increasing pancorneal calcium deposits (calcareous degeneration). serum calcium and phosphate levels were normal at several times points. longterm immunosuppression improved symptoms of gvhd but healing of corneal defects was not reached despite local therapy of antibiotics and corticosteroids. at day+133 patient underwent removal of calcium deposits and both eyes were covered by amniotic membrane. at day+303 keratoplasty in the left eye was performed with transient improvement of visual acuity. two weeks after keratoplasty calcified plaques have recurred in the transplanted tissue and fast visual loss reappeared. we suggest that ectopic corneal calcifications are probably associated with persistent epithelial and stromal defects and keratoconjunctivitis sicca as a symptom of persistent active chronic gvhd. combined immunosuppressive therapy should therefore continue. we plan to repeat surgery at the later time under better control of gvhd. patient is now one year after sct with no signs of leukaemia relapse with full donor hematopoiesis, but prognosis of vision still remains at this moment uncertain. supported by vz fnm mzo 00064203 thyroid dysfunction is an important problem in patients receiving bone marrow transplantation. however there was no case of hashimoto encephalitis in a patient after stem cell transplant reported. we describe the case of 47 year old female patient who underwent allogeneic bone marrow transplantation and who developed hashimoto encephalitis. aml patient was subjected to allogeneic stem cell transplant with reduced intensity conditioning (flag-ida) in october 2002. early posttransplant period was complicated by reactivation of cmv infection and prolonged peripheral cytopenia. 24 months after transplant the patient chimerism analysis revealed graft rejection without aml relapse. 32 months after transplantation the patient developed fatigue, loss of appetite, vomiting but also psychiatric symptoms: hallucinations and paranoid ideations. ct scans does not revealed anything specific. typical psychiatric treatment was not efficient. later patient experienced episodes of epilepsy and developed cerebellar ataxia and progressive unilateral paresis with impaired consciousness. in accessory investigations -in csf elevated level of proteins and eeg abnormalities were noted, blood and csf cultures were negative, thyroid hormones level were slightly decreased, tsh and atpo antibodies titers were elevated. results of mri confirmed disseminated pathologic changes in white matter. hashimoto disease with encephalitis was diagnosed and the patient was treated with high dose methylprednisolone i.v. for 5 days with rapid improvement noticed within first 48 h of the treatment. the neurological state normalize within one week. maintenance treatment with decreasing dose of oral prednisone is carried out. occurrence of hashimoto encephalitis in described patients seems to be connected with chronic graft rejection process and impaired prolonged regeneration of immunity after transplant. the role of different infections mainly viral should also be investigated. bmt: care about donors a. tellier, c. bauchetet, z. marjanovic, j.-p. marie, b. rio hotel-dieu paris (paris, f) purpose: bmt improvement must take in consideration ethical issues regarding donors. based on donors refusal cases report, our analysis underlines the significance of identifying «unwilling donors». moreover, we are concerned about the process of obtaining informed consent of family membres to undergo hla histocompatibility tests in order to prevent psychological consequences of peripheral blood and bone marrow donation. background: studies about psychological issues of bone marrow donation show that donors may be worried about their health status (switzer et al., 1997 ; molassiotis et holroyd, 1999) . switzer et al. correlate donors difficulties to their hesitation during donation decision process and consider that screening donors motivations is useful to a psychoprophylaxis approach. other authors note that families donors are even more exposed to psychological problems (chang et al, 1998) . directly concerned by recipient reactions to bmt, family donors (wollcott et al,1986 ) may feel unconscious guilt in cases of unsuccessful or complicated bmt (futterman and wellisch, 1990) . deeper psychodynamics of bmt process indicates that donor can confuse biological (hla) and psychological identity and be anxious about being ill. (alby, 1988 (alby, , 1990 ascher, 1994 ascher, , 2004 . topall-rabanes et al. (2000) notice that about 20% of 202 studied family donors are classed as «reluctant» : for these subjects, donation decision is not considered as a real choice. they suffer from health problems and feelings of regret even longtime after donation. methods and results: we reviewed the records of donor refusal cases representing 2% of 400 allografts performed in our twenty years bmt practice. in-depth analysis of patients psychosocial situation, quality of social support, family members relationships, patient-family-staff communications modalities, in particular regarding to donation information process. we will present these cases and retrospective analysis to introduce ethical questions on hla histocompatibility testing. conclusion: regarding to our experience and to our data, it seems that information process of family membres about bone marrow donation may lead to an impossible choice. appointed by hla testing as donor, family member is moved to suffer of biological determination and to become «unwilling donor». protecting these persons is a medical responsability. more research is necessary on this subject. there are 14 patients with paranoiac reactions (persecution, sensitive, litigious, invention) were possible to allocate among the mental disorders at our patients. results: paranoiac reactions with ideas of persecution (n = 7) are the most often in our sample. in such cases patients suspect those around them, especially medical personnel, in preconceived attitude to them and, possibly, to conspire for damnification of patients. such ideas have a systematic character and are resistant for treatment. sensitive paranoiac reactions (n = 2) are joined with perception of physical handicap and characterized by sensation of slighting attitude and mockery those around patient and by confidence in dissemination of detractive rumours. litigious paranoiac reactions (n = 4) are characterized by lowsystematized and insufficiently well-founded requests and multiple joined with hematological malignancies such as requests to compensate the prejudice applied by disease or laying claims to medical personnel are at fault, in opinion of patient, in the prejudices joined with disease. invention paranoiac reactions (n = 1) in our sample are characterized by elaboration of self-treatment methods of his/her disease. treatment of examined states was significantly resistant and included antipsychotic and anxiolitic medications. conclusions: pr at the patients with hemato-oncological disorders after bone marrow transplantation are the separate problem demanding the special attention both the hematologist and the psychiatrist, especially relative to its treatment. b.v. afanasiev (1), n.v. stancheva (1), l.s. zubarovskaya (1), e.v. semenova (1), m.a. ovsyannikova (1), t.i. ionova (2), t.p. nikitina (2), a.a. novik (3) (1)st. petersburg state medical university (st. petersburg, background: quality of life (qol) is increasingly used as a treatment outcome along with traditional clinical outcomes in children with cancer undergoing bone marrow transplantation (bmt)/stem cell transplantation (sct). the aim of our study was to estimate qol parameters and symptoms in survivors of childhood blood cancer after bmt/sct. patients and methods: fifteen survivors were evaluated 1-13 years (median, 3 years) after allogeneic bmt/sct for acute leukemia (12), chronic leukemia (2) and myelodysplastic syndrome (1) . median age at transplantation -13 yrs (range 1.5-21.0), girls/boys-10/5. pedsql™ generic core scales and sf-36 questionnaires were used for qol assessment in the group younger than 18 yrs at the time of the survey and in the group 18 yrs and older, respectively. nj children cancer symptom inventory and md anderson symptom inventory were used for symptom assessment in the younger and older groups, respectively. for comparison 56 healthy controls (20for younger group; 36 -for older group) matched to survivors by age and gender were included in the study. results: no significant differences in qol parameters (physical, psychological and social functioning) between survivors and control group were revealed. only school functioning for children younger than 18 yrs was lower in the group of survivors (61 vs 77, p<0.05). seven survivors experienced moderate or severe symptoms (5 to 10 scores on 0-10 scale). four of them had pronounced psychological symptoms. other pronounced symptoms were chronic pain, fatigue, lack of appetite, shortness of breath, drowsiness and nausea. six survivors had at least two moderate or severe symptoms. qol parameters, namely, physical, psychological and social functioning in long-term survivors of childhood blood cancer post allogeneic bmt/sct is comparable to healthy people. however, nearly half of survivors experience different symptoms in long-term period after transplantation. this confirms the importance to monitor and control late related symptoms in order to preserve qol of long-term survivors of childhood blood cancer. differential diagnosis of t-cell lymphoproliferative disease after allogeneic haematopoietic progenitor cell transplant s. koschmieder, m. stelljes, a. schmidt, g. silling, t. spieker, g. köhler, c. renne, w.e. berdel, j. kienast university of muenster (muenster, d) two cases are presented of a rapidly developing cervical lymphadenopathy 19 days after allogeneic peripheral blood stem cell transplant for acute myeloid leukemia. patient #1, a 57 year-old female, developed painful bilateral cervical adenopathy (up to 2 cm) overnight, fever (39.2°c), and a fine maculopapulous rash. no further enlarged lymph nodes were found by computed tomography, and the right cervical mass was removed. histology showed infiltration of the lymph node by atypical lymphoid cells that expressed cd3 and cd4. molecular analysis revealed monoclonally rearranged tcr gamma chains. histology of the skin showed a leukocytoclastic vasculitis without any signs of acute gvhd. ebv pcr was consistently negative in peripheral blood and lymph node. the diagnosis of t-ptld was made, and the patient received a 10-day course of methylprednisolone. with this regimen, the patient´s condition rapidly improved, and the fever, lymphadenopathy, and rash resolved completely within a few days. patient #2, a 46 year-old female, developed painful acral bullous erythematous lesions on her arms, legs, and in her mouth on day 17 post transplant which progressed to generalized maculopapulous rash. histology showed acute graft versus host disease (gvhd) of the skin. on day +19, the patient presented with painful bilateral cervical adenopathy (up to 1.8 cm), and abdominal ultrasound and mri showed no other regions of lymphadenopathy. histology of one removed lymph node demonstrated a highly proliferating, diffuse infiltrate of cd3 positive t cells with 60% of t cells expressing cd4 and 40% expressing cd8, respectively, as well as scattered cd20 positive cells. molecular analysis detected a polyclonal pattern of tcr rearrangement and an oligoclonal immunoglobulin receptor rearrangement. ebv pcr was negative in peripheral blood and lymph node. the clinical scenario was interpreted as concomitant lymphadenopathy associated with acute gvhd, and the patient received methylprednisolone for 10 days and completely recovered. this report highlights the necessity to remove suspiciously enlarged lymph nodes, developing after allogeneic transplant, in order to distinguish between t-ptld and lymphadenopathy accompanying acute gvhd. the evaluation of t-cell receptor gamma gene expression for prognosis of relapse development after stem cells transplantation a. sipol, d. butlitskiy , u. vorobjeva , a. aljanskiy , m. zarayskiy , l. zubarovskaya, b. afanasyev st. petersburg state med pavlov university (st. petersburg, rus) background: the hematopoetic stem cells transplantation (hsct) in patients with hematological disorders is the most radical method of the therapy. the early diagnostics of relapse in post-hsct period could improve significantly the therapy effectiveness. the purpose of this study was to develop the minimal residual tumor cells detection method by studying the mediated mechanisms of organism's reaction to tumor clone. materials and methods: nineteen patients with different hematological malignances, who were undergone the hsct, have been included in the research. total mrna was extracted from peripheral blood leukocytes, sampling in the time of conditioning regime completion, just prior to stem cells transfusion. we performed the rt-pcr with primers specific to v-j-gamma junctions (tcr-gamma gene). specific signal was detected in 2% agarose gel. results: absence of tcr-gamma gene expression at day-0 were significantly more often in group of the patients who were staying in remission after hsct (p=0.013). in group of the patients with myeloablative pre-transplant conditioning the significantly differences in tcr-gamma gene expression depending on the fact of relapse has not been revealed. in group of the patients with reduce intensity conditioning regime the positive correlation between the presence of tcr-gamma gene expression at day-0 and relapse in post-hsct period was observed (p=0.047). conclusion: the definition of tcr-gamma gene expression before hematopoietic stem cells infusions is the method of a tumor process activity estimation during the therapy, especially in case of using the reduce intensity conditioning regime. we suggest this criterion as the early prognostic factor for the relapse developing in post-hsct. mrd-directed adoptive immunotherapy following allogeneic stem cell transplantation fails to permanently revert disease progression in childhood acute lymphoblastic leukaemia between viii/2000 and vii/2004 we performed allogeneic haematopoietic stem cell transplantation (hsct) in consecutively 36 children with acute lymphoblastic leukaemia (all). we analysed prae-and post-transplant minimal residual disease (mrd) levels using quantitative rq-pcr targeted to immunoglobulin and/or t-cell receptor rearrangements in 25 of them with available targets (with adequate sensitivity and specificity according to esg-mrd-all criteria). seven of patients with detectable mrd prior hsct (n=8) relapsed after transplant and one died in ccr before day +100 due to transplant related complications. in the group of prae-hsct mrd negative patients (n=17), only one relapse appeared. in a total of 4 patients, there was a time-frame for an attempt to avert the disease progression detected by rq-pcr. in one bcr/abl+ patient, imatinib mesylate dose was increased and 3 doses of dli (5.7x10 6 /kg cd3+ cell) were administered. despite this, patient relapsed +660 days after hsct. second bcr/abl+ patient developed molecular relapse despite chronic gvhd. therefore, immunosuppression was quickly discontinued and imatinib mesylate was administered. he achieved temporary molecular remission but 6 months later died of cns disease progression. third patient developed mrd positivity +60 days after hsct and therefore immunosuppression was quickly tapered. gvhd reactivation required steroid and csa treatment. after gvhd resolution immunosuppression was ceased and three escalating doses of dli (1x 10 6 , 1x 10 7 , 1x 10 8 cd3+/kg) were given. nevertheless, this did not avert haematological relapse. this patient after high-dose chemotherapy achieved second complete remission (cr) with mrd negativity prior to second hsct and now is 11 months after hsct in continuous cr and mrd-negative. the fourth patient (bcr/abl+) developed relapse despite three dlis and imatinib mesylate treatment given for positive mrd +90 after hsct. adoptive immunotherapy (rapid cessation of immunosuppression, infusion of dli in 4 to 6 weeks interval and/or use of imatinib mesylate in bcr/abl+ all) after the transplantation was not successful in our cohort in relapse prevention and might only postpone the manifestation of relapse and facilitate further efficacious chemotherapy and retransplantation. we are confident that all effort should be aimed to a better control of the pre-transplant mrd levels. grant support: fnm 9735, mz 6929-3, gauk 62/2004, mz 00064203 and msm0021620813. the dynamics of chimerism evolution determines the differential outcome of various transplant settings i. buño, p. balsalobre, g. iglesias, d. barroso, c. manzano, r. carrión, d. serrano, a. gómez-pineda, j.l. díez-martín hosp. g.u. gregorio marañon (madrid, e) background: several factors such as the intensity of the conditioning regimen, the t-cell content of the graft or the gvhd prophylaxis, influence the degree of chimerism after sct. objective: to evaluate the dynamics of chimerism after different sct settings (ablative, reduced intensity conditioning -ric-and t-cell depleted -tcd-) and its influence in the success of the procedure. patients and methods: 68 sct: 32 ablative (including 7 from mud), 19 ric and 17 tcd (including 8 from haploidentical donors and 2 from mud with 1 hla disparity). chimerism analysis was performed by fish or str-pcr (sensitivity 1%). samples: bone marrow (bm) and peripheral blood (pb) on days +30, +100, +180, +365 and once a year thereafter, as well as pb and leukocyte lineages (t lymphocytes cd3+, b lymphocytes cd19+ and myeloid cells cd15+ isolated (purity >95%) by immunomagnetic means, automacs, miltenyi biotec), every 2 weeks, starting on day +15 and until complete chimerism (cc) was achieved. results of chimerism follow-up were censored once the diagnosis of relapse or rejection was established. results: incidence of mixed chimerism (mc) on day +30 (ablative: bm 27%, pb 15%; ric: bm 40%, pb 41%, cd3 40%; tcd: bm 31%, pb 33% , cd3 54%), as well as its dynamics (mc on day +100: ablative bm 8%, pb 4%; ric bm 8%, pb 15%, cd3 22%; tcd bm 20%, pb 40% , cd3 37%) were different in the three sct settings. moreover, the percentage of recipient cells (%r) was significantly higher after ric and tcd than after ablative sct, as well as in t lymphocytes than in bm or pb (7/8 cases with simultaneous studies showed mc in t lymphocytes and cc in pb). all ric sct evolved to cc by day +180 while tcd sct showed persistent mc (2 patients with stable mc after one year). the incidence of rejection was greater after ric (2/19) and tcd (4/17) than after ablative sct (2/32). all these patients showed mc, mainly in t lymphocytes, which allowed early diagnosis and successful treatment with immunosuppression withdrawal and donor leukocyte infusion. patients with cc in pb/t lymphocytes on day +30 had a higher incidence of gvhd>i than those with mc. in the present series, however, a relationship between chimerism and relapse, disease free survival or overall survival, was not observed. conclusions: sct with greater incidence of mc (ric and tcd) favor immune tolerance between donor and recipient which reduces the risk/severity of gvhd at the expense of a higher incidence of graft rejection. reliable quantification of haematopoietic chimerism after allogeneic stem cell transplantation by real-time quantitative pcr analysis a. picardi (1) introduction: increasing mixed chimerism (mc) represents a poor prognostic factor after allogeneic stem cell transplantation (sct). moreover, to define the best timing of immune-suppression withdrawal and donor lymphocytes infusion, a strict monitoring of donor hemopoiesis is needed. methods: we evaluated 18 donor/recipient pairs using a quantitative real-time pcr (qrt-pcr) with the aims 1) to evaluate the informativeness of this chimerism assay and 2) to compare qrt-pcr analysis with standard methods such as fluorescence in situ hybridization (fish) for mismatched sex pairs or variable nucleotide tandem repeats (vntr) for matched sex pairs. qrt-pcr (lightcycler 2.0, roche) was performed on bone marrow and peripheral blood samples collected monthly, using eleven biallelic dna genetic system located on chromosomes 1, 6, 9, 11, 17, 18, 20, x and y. glyceraldeyde phosphate dehydrogenase (gapdh) gene was used as active reference. before quantification, donor and recipient dnas were genotyped using primers and probes specific for all genetic markers. patients had a median age of 43.5 years (range 26-70) and were affected by acute leukemia (n=12), or lymphproliferative disorders (n=6). standard regimen was used in 10 cases, reduced intensity conditioning in 4, while 4 patients underwent an unrelated sct. median follow-up of the 18 patients was 16.5 months (range 4.2-34.4). results: both qrt-pcr and fish detected donor/recipient differences in 100% of pairs, while vntr was not informative in 25% of sex matched pairs. mixed chimerism was observed in 8/18 patients (44.4%) using qrt-pcr and in 3 of the 16 patients (18.7%) evaluable with standard methods. overall, 5/18 patients (27.8%) relapsed; before relapse, mixed chimerism was observed in all patients by qrt-pcr and in 3/5 by fish/vntr. qrt-pcr detected mixed chimerism 45 days (range 0-315) earlier than standard methods. in 2 cases in which vntr was either not informative or not predictive for relapse, the interval between detection of mixed chimerism by qrt-pcr and relapse was 30 and 315 days, respectively. conclusions: chimerism determination using qrt-pcr is more informative than standard methods and may represent an useful tool for the follow up of allogeneic sct. reduced intensity transplant programme. a single-centre experience a. bonini, a. tieghi, f. merli, l. gugliotta asmn (reggio emilia, i) from 2001 we introduced a reduced-intensity conditioning regimen for allogeneic transplants(allo). this regimen is tailored for old patients (pts) or for those who have previously received an allo or autologous bmt. at now we have transplanted 15 pts; 2 of them received previously an autologous and 1 an allo bmt. they were 10 males and 5 females and the donors were 9 males and 6 females. the mean age of the pts was 50 yrs (range 22-68 yrs).the diagnoses were: 2 hd,7 nhl, 1 mds, 2 renal cancer, 1 sarcoma, 1 myeloma and 1 cml; the 3 solid tumors were all metastatic, 4 pts were in pr, 5 in ii cr, 1 in iii cr and the one with mds at the onset. the abo compatibility was:complete for 8, minor for 2 and major for 5. the cmv status was: donor/pt positive for 13 and positive donor/negative pt for 2. the conditioning regimen consisted of thiotepa, fludarabine and cyclophosphamide except for the pt with cml who received busulphan and fludarabine. the source of stem cells was peripheral blood in 14 and bone marrow in 1 and the stem cells were cryopreserved after the harvest. the gvhd prophylaxis consisted of csa and short course mtx. the 14 pts who received peripheral stem cells the mean cell dose was 4x106/kg cd34+ cells, for the one who received bone marrow was 2.9x108/kg mnc. the mean time to reach pmns >0.5x109/l and plts >20x109/l was respectively 15 days(range 13-20 days)and 24 days (range 15-55 days). the mean number of transfused rbc and plt units was respectively 4 (range 0-8)and 4 (range 1-7). the mean grade of mucositis (according to the who classification) was 2. the major complications during neutropenia were 5 fuo,5 gram + bacteremias and 2 cerebral aspergillosis. no cases of vod of the liver were observed. 8 pts had agvhd(1 grade 4 and 7 grade 2) and 1 limited cgvhd. two pts developed cmv reactivation after allo; the complication was cured with gancyclovir. nine over 15 pts died: 4 for disease (the 3 solid tumors but for the 2 with renal cancer a response even if transient was observed and 1 nhl), 1 for acute gvhd and 2 for cerebral aspergillosis. the chimerism was complete for all the evaluable pts at 30 days. none needed dli. six patients are in cr with a median follow up of 2 year (2 were in partial remission and 1 in ii chronic phase at transplant) and 1 relapsed. this pilot study demonstrated an acceptable regimen-related toxicity(according to the age and previous chemotherapy including transplants), the possibility to reach a very early full-donor chimerism and to cure high risk pts. introduction: autologous stem cell transplantation (autosct) is usually preferred to allosct, due to its widespread availability, lack of the immunological problems intrinsic to the development of graft-versus-host disease (gvhd), and the infrequent bone marrow involvement present in hodgkin's disease(hd), for patients undergoing high-dose chemotherapy/radiotherapy. allosct has been associated with a high transplant-related mortality (trm) in patients with hd due to a high incidence of gvhd and of fatal infectious events after transplantation. the poor outcome of these patients after allosct may reflect in part the advanced status of the disease at transplantation and the poor performance status of the patient population allografted. furthermore, the high trm present in the conventional allosct setting has never allowed a proper evaluation of a possible graft-versus-hodgkin's effect. in an effort to reduce the trm associated with allosct, low-intensity regimens have been developed; the curative potential of these protocols would rely on the graft-versus-leukemia effect of the allogeneic infusion more than in the conditioning regimen per se. case: in our hospital a total of 3 patients with relapsed hodgkin's disease underwent reduced-intensity conditioning (ric) allogeneic stem cell transplantation (allo-sct) from an hla-identical sibling. we explored reduced-intensity allografts using fludarabine-melphalan conditioning and early withdrawal of immunosuppression as an alternative to palliative chemotherapy. graft-versus-host disease (gvhd) prophylaxis was mini-methotrexate and ciclosporine with weaning at day 90. the age of patients was 26, 58 and 23 years. the time from initial diagnosis was 55, 120 and 27 months and from autograft was 31, 64 and 30 months. the 3 patients were in refractory relapse. time to neutrophil recovery (absolute neutrophil count ≥ 500/microl) was 17 days for 2 patients and 16 days for the other. time to platelet recovery ( platelet count ≥ 50 000/microl) was 14, 12 and 15 days.the 100-day mortality was 1. none of them developed acute gvhd. one patient developed mild chronic gvhd. as of november 2005 2 patients remain in complete remission. conclusion: although the number of hd patients allografted with reduced-intensity protocols is low and the follow-up still short, it seems that the reduced-intensity allogeneic stem cell transplantation is effective in relapsed and refractory hodgkin's disease where autografts have failed. remarkable reduction of acute gvhd and infectious complications after reduced-intensity conditioning and low-dose (30 mg) campath-1h p. reményi, á. bátai, b. kapás, a. sipos, s. lueff, i. bodó, m. réti, v. goda, t. masszi national medical centre (budapest,hun) during the four month-period from may, 2005 to september, 2005 we performed reduced intensity (ric) allogeneic transplantation for five patients with their hla indentical sibling donors. the median age of the 3 female and 2 male patients was 40.7 years (32.8-57.1). two patients had chronic lymphocytic leukemia (cll), one hodgkin's disease (hd), one follicular nhl grade i (fl) and one myelodysplastic syndrome (mds). the hd patient relapsed after previous autologous stem cell transplant. the fl patient was in complete remission, the others were in partial remission before transplant with low tumour burden. the conditioning regimen consisted of 1x30mg campath-1h and fludarabine 5x30 mg/m² for all patients, adding melphalan (140 mg/m²) for the lymphoid malignacies, and busulphan (8mg/kg) for the mds patient. for gvhd prophylaxis 3mg/kg cyclosporin a (continuously) +8mg/m² methotrexate was given on days 1, 3, 6. all patients engrafted. one patient developed grade i acute gvhd. two patients had febrile neutropenia, one developed central venous line infection. no one had cmv reactivation or disease. after a median of 173 day-follow up (100-180 days) all patients developed full donor chimera tested by vntr pcr. the two cll patients are in remission proven by flow cytometry (less than 1% cd19/cd5+ cells). the fl patient is in cr and received four courses of mabhtera (375mg/m²) as maintenance therapy. the mds patient is in cr according to bm histology, but still thrombopenic (60 g/l). the hodgkin's patient has active disease with minimal tumor burden with mixed chimerism at day 100, now waiting for dli. conclusion: low dose (30 mg) campath-1h+ csa/mtx gvhd prophylaxis is a well balanced regimen regarding the incidence and severity of acute gvhd, infectious complications and gvl effect after ric conditioning. these preliminary results -especially concerning the late infections complications -compare much better to those we observed in our previous series of ric transplants with higher doses (90 or 100 mg) of campath-1h, or that we read in the literature. with the publication of these preliminary results we would like to underline the message that less is better regarding campath-1h in reduced intensity conditioning. conditioning regimens consisted of fludarabine 30 mg/m²/day x 5 days plus melphalan 140 mg/m²/day x 1 day (n=25) or plus oral busulphan 4 mg/kg/day x 2 days (n=11) or plus cyclophosphamide 60 mg/kg/day x 2 days and globuline antithymocyte 2.5 mg/kg/day x 2 days (n=4). the patients were grafted with bone marrow (n=7), cord blood (n=4) or pbsc either unmanipulated (n=6) or cd34+ selection (n=20) or cd3/cd19 depletion (n=3). gvhd prophylaxis was performed with csa+ mtx (n=29), csa only (n=7) and csa + steroids (n=4). donors were either related (n=25) or unrelated (n=15). the median number of cd34+ cells infused was 5.75x10 6 /kg recipient bw (range 0.25-47.7). results: there were a rapid recovery of neutrophils (median 13 days; range 5-29) and platelets (median 15 days; range 5-56). the median length of hospital stay was 17 days ( range 9-77). with a median follow-up of 10 months (range 3-40) the incidence of agvhd and cgvhd were 16±6% and 24±8% respectively. the probability of trm was 10±5%. patients grafted with manipulated pbsc had the lowest trm (5±3%). the relapse incidence was 35±11%. high number of infused cd34+ cells (p=0.066) and cgvhd (p=0.01) was associated with a lesser ri. the event-free survival was 57±10%. nine patients died of relapse or progressive disease (n=5), agvhd (n=1), cgvhd (n=1) and other (n=2). conclusion: fludarabine-based ric provide a good alternative to myeloablative conditioning for allogeneic transplantation either malignant or non-malignant disease in children. induction: treatment related mortality is the trade off of allogeneic transplants. although the probability at two years has been reduced to 15-25% following non-myeloablative (reduced intensity conditioning) allogeneic transplant it remains a barrier to administer these transplants. we desired to identify mechanisms that compromise safety or would enhance safety. methods: the different outcome parameters in transplantation were defined as safety or efficacy parameter and per safety and efficacy parameter the literature was searched for investigational method, prophylactic, supportive or therapeutic measurement. furthermore the toxicity scales were reviewed and known side effects were listed and include in the search. results: outcome parameters defined as safety parameter included engraftment, disease recurrence; side effects of major concern, acute and chronic graft versus host disease, infections, death as well as life threatening side effects defined by the safety scales. more that first complete remission was considered and adverse safety parameter. 1) engraftment failure was noted after inadequate cytoreductive treatment such as 2 cgy tbi and cyclophosphamide or underdosing of fludarabine (<90 mg/m²); adequate dosing of cytoreductive and immune suppressive treatment appeared critical for rapid trilineage engraftment. 2) tbi appeared associated with higher risk of graft versus host disease and conditioning with drugs only. 3) in vivo t-cell depletion by f.e anti-thymocyte globulin was associated with high risk of cytomegali virus reactivation. 4) t-cell depletion of the graft is associated with increases risk of relapse 5) b-cell depletion of the graft seems to reduce the risk of chronic graft versus host disease 6) cmv positivity is not a prerequisite for cmv reactivation 7) a combination of immune suppressive agent reduces the risk of acute graft versus host disease discussion: based on these and less stringent criteria we defined a practice guideline and make recommendations for reduction of side effects and treatment related mortality. the method developed model will be presented. monitoring of mixed chimerism by single nucleotide polymorphism analysis in two children supported with granulocyte transfusions after allogeneic stem cell transplantation w. schwinger, p. sovinz, h. lackner, h. dornbusch, m. benesch, a. moser, g. lanzer, c. urban medical university graz (graz, a) objectives: neutropenia is a major risk factor for early transplant related mortality in children undergoing haematopoietic stem cell transplantation (hsct). in case of infection, refractory to antibiotic therapy the combination with allogeneic granulocyte transfusions is a logical approach to manage this problem. these patients are chimeras of at least three different cell populations (recipient -stem cell donorgranulocyte donor). two patients where followed closely by single nucleotide polymorphism (snp) analysis to reveal the duration and percentage of leukocytes derived from granulocyte donors. patients and methods: one patient suffering from evans syndrome and was transplanted with cord blood of a mmud after myeloablative conditioning with busulfan, thiotepa, etoposide and atg. the child received one neutrophil transfusion at day +9. the second patient was diagnosed with aml (fab m2) and was transplanted in persistent bm-aplasia after induction chemotherapy with bm of her hla-identical mother after reduced intensity conditioning (ric) with fludarabine, melphalan and atg. this child was transfused with eight granulocyte transfusions of three different allogeneic donors starting at day -3 until day +10 with two three days intervals. snp analysis was performed the day after each granulocyte transfusion and once weekly until stable allogeneic engraftment was achieved. results: both patients showed allogeneic engraftment of >99% donor chimerism at day 34 and 28 respectively. third party leukocyte dna could be detected from day +9 until day +55 in the first patient and from day -3 until day +21 in the second patient. in the second child up to five genetically different leucocyte dna populations where detectable and could be quantified. conclusions: granulocyte transfusions of third party donors after allogeneic stem cell transplantation are increasingly used. snp analysis allows precise monitoring of donor chimeras even in case of two or more donor cell populations after transplantation. this could be important especially in case of ric transplantations in which precise follow up of engraftment kinetics is very important. patients are alive and without disease. one year overall survival and disease free survival (dfs) is 60% (50-70%) and 50% (40-60%), respectively. transplant related mortality (trm) was the cause of death in 7 patients, 3 months and one year cumulative incidence (ci) of trm were 11 % (4-31%) and 22 % (10-49%), respectively. eight patients relapsed (median time: 184 days (75-593); one year ci 23% (12-47%). twelve patients developed acute graft versus host disease (agvhd); ci 39% (24-64%) fourteen of 24 evaluable patients developed chronic gvhd; ci 67% (50-90). the development of cgvhd was associated with better dfs: 74% (63-85%) vs. 25% (5-46%) p=0,04. conclusion: ric allo-sct is a curative option in patients with advanced age diagnosed of amd/mds with an acceptable trm. age should not be an exclusion criteria in patients with aml/mds for allo-sct. cgvhd is associated with better dfs. finaced in part by g03/008, 2004/xt0058. allogeneic stem cell transplant (allo hsct)is a curative approach for patients with hematologic malignancies but it is associated with high treatment related morbidity and mortality. because transplant related mortality increases with advanced age, advanced disease and unrelated donors, patients older than 50-55 years may be excluded from this procedure. reduced intensity conditioning and new preparative regimens are therefore explored to allow hsct to a wider patient population. the aim of this study was to evaluate efficacy and toxicity of the combination treosulfan (water soluble alkylating agent, busulphan derivative) and fludarabine as preparative regimen for allo-hsct in patients receiving match sibling or unrelated donors for advanced heavily pretreated hematologic malignancies. since july 2005 to november 2005 8 patients (3 all, 3 aml, 1 cml,1 mm ) entered this study. mean age was 41 years (range 22-6 ). conditioning consisted of treosulfan 12 gr/m² for 3 days, fludarabine 30 mg/m² for 5 days, cyclosporine plus short mtx and anti-thymocyte globulin (thymoglobulin) at a total dose of 6 mg /kg. all patients engrafted; mean time to neutrophil recovery >500 x109/l was 14 (range 11-18 ) days , to platelets >20000x109/l was 16 ( range 12-25) days. no conditioning regimen related deaths was observed. three (3) patients experienced gi toxicity (2 grade 1, 1 grade 4), 2 patients had grade 2 liver toxicity. no acute gvhd was observed. all patients are alive with a follow-up ranging from 20 to 120 days. despite the short follow-up, in this preliminary report we underline that treosulfan-fludarabine -atg conditioning is characterized by reduced toxicity; long term follow-up is necessary to evaluate os, dfs and relapse in these patients. background: prognosis of patients with aml relapsing within a year of allografting is poor. management options are limited and response to donor lymphocyte infusion(dli) is poor due to disease kinetics. second allografting using conventional conditioning is unpopular due to high transplant related mrotality(trm).there is paucity of data on second allografting with reduced intensity conditioning(ric) as a salvage for relapses post-allografting. herein, we report 2 such patients who were successfully salvaged and achieved a durable complete remission(cr) with ric and second allografting. case 1: a 16 year old male with normo-cytogenetics aml received an allograft from a hla identical sibling after myeloablative conditioning. he relapsed 5 months after transplant with loss of donor chimerism. he was salvaged with flag regime followed by serial dlis and achieved cr2 which lasted 3 months before relapsing again. he was reinduced with flag regime followed by pbsc infusion from the original donor. he received no post-transplant immunosuppression with the development of grade 1 graft versus host disease(gvhd). he has since achieved 100% donor chimerism and remains in cr3 for more than 12 months. case 2: a 59 year old male with myelofibrosis transforming into aml associated with del(20q) received his first allograft from a hla identical sibling after non-myeloablative conditioning. donor chimerism declined after 3 months and blast counts continued to rise despite serial dlis. he was reinduced with idarubicin and cytarabine, followed by a second allografting from the original donor with ric (fludarabine 25mg/m² d-7 to -4, melphalan 70mg/m² d-3 to-2). full donor chimerism ensued and he remains in cr for more than 12 months. immune suppression was tapered with development of grade ii gvhd. conclusion: remission durations after the second allografting exceeded those after the first. a ric regime with second allografting is a more effective modality than dli in the treatment of relapsed aml. a ric regime lessens the trm associated with second transplants, while allowing for engraftment of infused pbscs with execution of gvl effect. immune modulation by aggressive manipulation of posttransplant immune suppression also appears to be a key element in successful second allografting. this salvage approach offers a practical, well-tolerated and potentially curative treatment for patients who in most circumstances, would have been precluded from further active management. reduced intensity conditioning (ric) allogeneic stem cell transplantation (allo-sct) can reduce the frequency of transplant-related toxicities, at least in the early period after allo-sct. here, we analyzed the profile of platelets recovery and transfusion requirements in the first 3 months after ric in a single institution series of 90 consecutive patients receiving allo-sct from an hla-identical sibling. patients and graft characteristics were: age 49 y. (range, 18-63), diagnoses: 30 myeloid malignancies (33%), 34 lymphoid malignancies (38%), and 26 metastatic solid tumors (29%). 71 pts (79%) received a fludarabine, busulfan and atg-based ric, while 19 pts (21%) received a low dose irradiation-based ric. all patients received a pbsc graft. 47 pts (52%) developed grade 2-4 acute gvhd. platelets recovery (>20.000/l) was observed at a median of 11 days (range, 0-99). the kinetics profile of platelets recovery is shown in the figure below. in the whole study population, the nadir was observed around day +7 after allo-sct, and a plateau was reached by the end of the first month. in this series, patients needed a median of 1 unit (range, 0-53) of filtered and irradiated donor apheresis platelets. of note, 35 pts (39%) did not require any platelets transfusion during the follow-up period (median follow-up: 392 days). among the 55 patients (61%; 95%ci, 51-71%) who received at least one transfusion of platelets, 27 were not transfused beyond day +100 after allo-sct. when comparing these 55 patients, to the group of 35 patients who were never transfused, platelets count prior to ric allo-sct (median count 130.000/l vs. 161.000/l; p=0.07) and the occurrence of severe acute gvhd (p=0.0001; 100% of patients with grade 3-4 acute gvhd were transfused) were the parameters significantly associated with platelets transfusion needs. in this cohort, 69 pts could be assessed for platelets recovery at day +100: among them, 58 (84%) had a platelet count >50.000/l. at day +100 after allo-sct, a diagnosis of myeloid malignancy (aml, cml or mds) was associated with a delayed platelet recovery (p=0.03). overall, these observations show a significantly lower rate of platelets transfusions and a quicker kinetics of platelets recovery after ric allo-sct. in addition, the low level of myeloablation observed after ric, may offer a window of opportunity for testing of megakaryocytic growth factors, towards further improving the safety and outcome of ric or nonmyeloablative allo-sct. fungal infections have become the major cause of infectious morbidity and mortality in patients undergoing bone marrow transplantation (bmt). in many cases invasive aspergillosis infections create a major therapeutic dilemma and contraindication to marrow transplantation. we report on a 8 years old boy with secondary acute myeloid leukemia, who underwent unrelated donor peripheral blood stem cell transplantation (pbsct) with previously diagnosed pulmonary aspergillosis and successfully recovered from the infection. probable invasive pulmonary aspergillosis (ipa) was diagnosed in the patient acc. to eortc-criteria. a large diffuse wedge-shaped infiltration was observed in thorax ct scans two months before pbsct and throughout the early posttransplant period. liposomal amphotericin b (l amb) 5mg/kg/d i.v. and voriconazole p.o. 2 x 4-6 mg/kg/d were administered for the whole peritransplant period. after conditioning regimen incl. treosulfan 3x14g/m², melphalan 100mg/m², atg fresenius 4x5mg/kg the patient received pbsc (14.4 x 106 cells cd34+/kg recipient bw) from the unrelated donor, who was mismatched at 1 a*-allel. csa, mtx and atg were used as gvhd prophylaxis. a rapid and sustained allogeneic engraftment (neutrophils > 0.5 g/l on day +11, thrombocytes > 50 g/l on day +19) was observed. the posttransplant period was uneventful except for 2 weeks long lasting exhausting morning cough and subtle breathing difficulties requiring passive oxygen therapy. three weeks after pbsct, bronchoscopy with broncho-alveolar lavage revealed no pathogens. nevertheless it was decided to continue treatment with l amb 5mg/kg every second day. in control thorax ct the infiltration was considerably smaller and no indication was found to perform an open lung biopsy. the patient received further treatment with voriconazole p.o. for 5 months. regularly performed thorax cts revealed a continuous regression of pulmonary changes. the patient remains alive and well in cr 11 months from transplant without any pulmonary abnormalities, except for a slight thickening of the interlobar groove and is given itraconazole p.o. this report demonstrates that administration of full-dose antifungal therapy and shortening the neutropenia period due to peripheral blood stem cell transplantation allow the successful outcome, even in high-risk patients with previous aspergillosis. post-transplant pulmonary complications are not rare and their mortality is still very high. we present 11-old year girl with high-risk acute lymphoblasic leukemia after allogenic hematopoetic cell transplantation (hct) from matched related sibling donor. conditioning regimen consisted of fractioned total body irradiation (12gy) and high-dose etoposid (60mg/kg). as graft versus host disease (gvhd) prophylaxis cyclosporine a was used. graft contained 3,26x10 6 /kg cd34+ cells. on day +5 persistent fever occurred, antibacterial and antifungal treatment were administrated. on day +7 5ug/kg g-csf was added. we observed symptoms of respiratory failure. on day +10 wbc was 400/ul, cutaneous gvhd grade iii appeared and steroids were administrated. on day +12 she has all symptoms of pulmonary oedema, was intubated and mechanically ventilated. simultaneously rapid hematopoesis reconstitution was observed: leukocytes>1.0 g/l and granulocytes>0.5 g/l on day +11, and platelets>50 g/l on day +14. after few days of the gradual improvement, her status suddenly deteriorated. chest x-ray showed fluid in the alveolar space in both lungs. pentamidine was added to treatment. severe but stabile status (opportunity to controlled ventilation, fio2 above 80%) lasted about 3 months. she was treated with wide-spectrum antibiotics and antifungal drugs (liposomal amphotericin, voriconazole) although her blood cultures were still negative. in this time we also treated her with anti-tnf-alfa antibodies. because of probable invasive aspergillosis and candidiasis we introduced treatment with caspofungin. gradual improvement was observed: significant decrease of the requirement for oxygen, possibility to reduction of controlled pressures and respiratory rates and simultaneous improvement of radiologic changes. after few days of continuous positive airway pressure ventilation right pneumothorax appeared and continuous suction drainage had been used for three weeks. simultaneously she was detoxicated from thiopental and morfine using fenobarbital and methadone. on day + 128 mechanical ventilation was discontinued. requirement for passive oxygen therapy gradually decreased. nowadays chest tomography shows mild pulmonary fibrosis and bronchiectasia. the patients is alive, in good general status, under intensive physical and s327 pulmonary rehabilitation with stable full donor chimerism without immunosupression. nevertheless regular long-term pulmonary follow-up is still required. cns symptoms and their severity strongly correlate with outcome of patients with familial haemophagocytic lymphohistiocytosis (fhl). here we describe a case of a patient without primary cns involvement related to fhl who developed progressive cns damage of unknown aetiology after allogeneic stem cell transplantation (sct). the patient was diagnosed at the age of 2.5 years with fhl, mutation in perforin gene was not proved, no signs of reduced perforin expression were observed. he was treated according to protocol hlh94 and then transplanted in clinical and haematological remission of fhl from his haploidentical father (peripheral blood stem cell -cd34 positive selection; clinimacs) because no matched donor was available. myeloablative conditioning consisted of busulfan, cyclophosphamide and ratg. soon after engraftment he suffered from cmv reactivation and he consequently experienced acute graft rejection. following okt3 and steroids further t cell depleted graft sct was infused 26 days after 1st sct. early after second engraftment he developed severe acute encephalopathy and syndrome of inadequate adh secretion (hyponatremia, hypothermia, neurologic seizures,…), hhv6 variant b was at that time detected in cerebrospinal fluid (csf) and blood. this was early followed by marked ebv and b cell proliferation treated with rituximab. after engraftment, complete donor haematopoiesis was confirmed with exception of almost full autologous recovery of t lymhocytes (split chimaerism). at day+80 all t lymphocytes (3,8.10 9 /l) were activated (expressing hla dr) and were of host origin. eleven months after sct hhv7 was repeatedly detected in peripheral blood and later frequent pharmacologically almost uncontrolled epileptic seizures started initiating progressive mental retardation. throughout this whole period repeated mri scans and examinations of csf, blood and marrow failed to document involvement of cns due to uncontrolled fhl. one year after sct cytototoxic assays showed significantly decreased activity of t cells. despite the number of donor origin t cells started to predominate the host ones only 2 years after sct, there were no clear clinical signs of fhl. unfortunately the patient developed irreversible cns damage. we speculate that multiple herpetic infections were responsible for proliferation of activated autologous t lymphocytes that contributed to severe cns damage in this patient. supported by grants of mh cr no.7459, 00064203 and 0021620812 consecutive twenty seven patients with inborn errors: four inborn metabolism: three metachromatic leukodystrophy, and one mucopolysacaridosis type 1: hurler´s syndrome; also one osteopetrosis, two mayor thalassemia, two fanconi anemia, and eighteen inmunodeficiencies: twelve severe-combined immunodeficiency (scid), two wiskott-aldrich syndrome and four hemophagocytic lymphohistiocytosis, were transplanted in our centre during the last nine years. we report two cases of scid: scid type jak3 deficiency (t-, nk-, b+) and major histocompatibility complex (mhc) ii deficiency which were treated with double haploidentical parental donor positively selected hla-mistmached cd34+ progenitor cells were isolated from peripheral stem cells, after mobilization with granulocyte colony-stimulating factor (g-csf) at standard dose, by the isolex 300 (baxter)and clinimacs(miltenyi)systems selection devices respectively. the first case was a male, 3.5 months years old with jak3 deficiency (t-, nk-, b+) who was undergone to transplant without conditioning regimen; value of peripheral cd34+ cells infused was 30.53 x 106 /kg and the mean cd3+ cells number was 3.2x105/kg; and 3.68 log t-cell depletion. he had graft rejection at day +71 and therefore was undergone to second haploidentical from the same family donor: father, this time with no-myeloablative conditioning with fludarabine (f) + melphalan + anti-thymocyte globuline (atg) and prophylaxis for graft-versus-host disease (gvhd) with cyclosporine (cya), he died ten days after the second transplant by heart insufficiency and metabolic failure. the second patient was a female, 36 months years old with mhc class ii deficiency, she was undergone to haploidentical transplant with myeloablative conditioning based f+ busulfan and ciclophosphamide and atg. the value of peripheral cd34+ cells infused was 20.25 x106 /kg and the mean cd3+ cells number was 1.6x 105/kg, and 3.63 log t cell depletion; she had graft rejection at day +180 and mixed chimerism therefore was underwent to second haploidentical transplant from the same parental donor: mother, the conditioning regimen was based okt-3 and dexametasone, she died five days after the second transplant by lung bleeding. despite poor prognosis at diagnosis of these cases, and the engraftment failure regardless mega-doses of cd34+; this approach could be a feasible therapeutic option for patients lacking a suitable donor. introduction: mobilized peripheral blood stem cells (pbsc) represent the most important source for autologous stem cell transplantation, even in children with malignancy. the current practice is administration of g-csf alone or in combination with chemotherapy. recently, a polyethylene glycol (peg)conjugated form of g-csf (pegfilgrastim) has been licensed. preliminary data indicate it has the same effects of filgrastim in terms of elevation of absolute neutrophil count, mobilization of pbsc, and reduction of duration of chemotherapy-induced neutropenia, with the obvious advantage that these effects could be sustained for several days from a single injection without added toxicity. in a recent experience the efficacy of a single dose (6 microgr) of pegfilgrastim, in combination with salvage chemotherapy, was tested in an open-label phase ii study of 25 pretreated patients. the authors concluded that pegfilgrastim as an adjunct to chemotherapy is a predictable and highly effective mobilization regimen in pretreated lymphoma patients (isidorins 2005). very limited experience is available on the use of pegfilgastrim in children. in the only two available report it was used to shorten the duration of severe neutropenia after cytotoxic chemotherapy in five children with ewing sarcoma (te poele em 2005) and in seven pediatric cancer patients (wendelin g 2005). we are not aware of any report on the use of pegfilgrastim for mobilization in children. patients: we treated 3 children, 2 males, aged 9, 13 and 16 years, affected of rhabdomyosarcoma, ewing sarcoma, and b-nhl. all received chemotherapy with vp16 and edx and than a single subcutaneous dose (6 microgr) of pegfilgrastim. two patients had a good response, with the peak cd34+ count (95 and 49) on day +6 and +10, while the failed to mobilize and to collect a sufficient number of cd34+ cells even after conventional g-csf and bone marrow harvests. the only patient who so far has completed the treatment program underwent, according to the treatment protocol, autologous transplant repeated three times and engrafted (pmn > 500/microl) on day +11, +11, and +15. conclusion: mobilization with peg-filgastrim was safe and efficient in our patients; failure to mobilize was observed only in an heavily pre-treated patient with ewing sarcoma. stem cell transplantation (sct) is the treatment of choice for patients suffering from severe aplastic anaemia (saa) who do not respond to immunosuppressive treatment (ist). patients transplanted from an hla-identical sibling have an excellent prognosis compared to patients who were grafted from alternative donors. t-cell depletion by cd34 positive selection of peripheral stem cells reduces the risk of gvhd considerable. this, however is followed by an increased risk of graft rejection. most recently, it could be shown, that changing the graft processing from cd34 positive enrichment to depletion of cd3 and cd19 positive peripheral cells does facilitate and improve engraftment in children transplanted from hla-haploidentical parents. consequently, also patients with saa who are at highest risk for graft rejection might benefit from such a new graft processing technique. a 7-year-old girl who failed to respond to ist received an allograft from mud. the conditioning regimen consisted of fludarabin (5x40mg/m²), thiotepa (2x5 mg/kg), melphalan (2x70mg/m²) and okt 3 (17x 0.1 mg/kg). g-csf mobilized peripheral blood stem cells (pbsc) were purified using anti-cd3/cd19 microbeads (miltenyi). recovery of cd34+ progenitor, cd56+ nk cells and cd14+ monocytes was more than 70% each. residual t-and b-cells were < 0.008 and 0.02 %, respectively. in total, 12.05x10 6 cd34+ cells; 30.3x10 6 cd56+ cells; 33.6x10 3 cd3+ cells and 8.8x 10 4 cd20+ cells per kg were administered. without g-csf, engraftment occurred on day +12 for leucocytes and both neutrophils and platelets on day +13. acute toxicity was mild (grade i). post transplant immunosuppression was performed using mmf. gvhd grad i was observed, which responded to prednisolone. noteworthy, although t-cells were severely depleted, t-cell regeneration was fast with more than 100/µl cd3 and cd4 positive cells detectable already on day + 25. chimerism analysis showed complete donor chimerism. at day 60, good immune recovery with 304/µl cd3+, 67/µl cd4+, 239/µl cd8+ and 360/µl cd56+ cells was detected. in conclusion, cd3/cd19 depleted peripheral stem cells from a mud in combination with a reduced intensity conditioning regimen could be a promising option in the treatment of patients with aplastic anaemia not responding to immunosuppressive treatment and lacking a matched sibling donor. idiopathic myelofibrosis (imf) comprises myelofibrosis, extramedullary haematopoiesis, hepatosplenomegaly and pancytopenia. in adults imf represents a poor prognosis, progressive fibrosis and leukaemic transformation are frequent. allogeneic hematopoietic stem cell transplantation (hsct) is a treatment option but is connected with high risk of graft failure and toxicity. in children the disease is rare and variable, stable course or spontaneous remission has been reported. we describe two cases of imf in children. in a girl mild anaemia and thrombocytopenia were first documented at the age of 3 years. at 8 years pancytopenia was found, trephine bone marrow (bm) biopsy revealed normocellular haematopoiesis with myelofibrosis. she remained in a good clinical state with a stable blood count but further bm biopsies showed decreased cellularity with myelofibrosis. 2.5 years after the diagnosis her blood count dropped off, hepatosplenomegaly was noted and bm biopsy revealed marked myelofibrosis. 2 months later at the age of 11 years hsct was therefore performed (matched unrelated donor, flu+mel+ratg). anc engrafted on day 20, platelets on day 25, complete donor chimaerism achieved on day 21. corticosteroids started on day 160 for mild extensive gvhd. bm biopsy at day 180 remained hypocellular with myelofibrosis, however blood count was normal. 17-year-old male presented with pallor, quickly developed pancytopenia with blasts. bm biopsy showed myelofibrosis, increased blasts, trisomy +8. hsct from a hla identical sibling was performed 2 months later (flu+mel+ratg), anc engrafted on day 16, platelets on day 62. complete donor chimaerism was achieved on day 14, no gvhd. at day 180 bm biopsy did not display myelofibrosis, peripheral blood count was stable. clinical deterioration started 10 months post-hsct with fever, weight loss, hepatosplenomegaly, mixed chimaerism, thrombocytopenia, blasts and extramedullary infiltration of breast. trephine and breast lump biopsy confirmed relapse of imf in transformation to aml-m7 with trisomy +8 and trisomy +21. second hsct from the same donor was carried out 11 months after the first hsct (bu+cy+mel). gvhd grade ii treated with steroids manifested on day 11. anc engrafted on day 13, platelets not engrafted. despite artificial ventilation diffuse alveolar haemorrhage resulted in death on day 37. reduced intensity conditioning composed of fludarabine and melphalan is preparative regimen of choice for hsct in children with imf. there are no studies on the connection between graft versus host disease (gvhd) and angiogenesis. however, chen et al have shown in nat med (2004) that the vascular endothelial growth factor-c (vegf-c) -vascular endothelial growth factor receptor-3 (vegfr-3) -axis has an effect on alloimmunity, and that the blockade of vegfr-3 signaling is immunosuppressive. both vegf-c and angiopoietin 2 (ang2) are important in angiogenesis and lymphangiogenesis. in this study we measured the levels of these two factors in children after urd-sct (unrelated allogeneic stem cell transplantation). patients and methods: nine patients aged 4-16 yrs were included, six of whom developed significant acute gvhd (agvhd, gr ≥ 2) while three did not. the diagnoses of the agvhd patients were all (acute lymphoblastic leukemia) (n=3) and saa (severe aplastic anemia) (n=3). the non-gvhd patients also had all (n=2) and saa (n=1). gvhd prophylaxis consisted of cyclosporin and short methotrexate. the serum concentrations of vegf-c and ang2 were analyzed by elisa at 2-102 days posttransplant, 3-5 samples/patient. results: the vegf-c and ang2 concentrations (median, range) were 1400 (545-10960) pg/ml and 2160 (875-6685) pg/ml, respectively. the presence or absence of agvhd did not make any difference in the levels. neither did we find correlation between hemoglobin, white blood cell count, bilirubin, crp or sedimentation rate and these two angiogenic factors. the vegf-c levels were significantly lower than in our previous study on all patients at diagnosis. the individual maximal ang2 levels correlated with survival (≥ 7mo follow-up, p=0.014). both absolute lymphocyte count and platelet count correlated with vegf-c levels, probably because these cells are known to produce vegf-c. conclusion: our results did not support the hypothesis about correlation of the levels of angiogenic factors with gvhd. instead, there was a novel finding about low concentration of ang2 being predictive of a good outcome. our findings pose important questions on the emerging role of angiogenic factors in the evaluation of the pathogenesis of gvhd. background: hematopoetic stem cell transplantation (hct) does appear to be a therapeutic option for children and adolescents suffering from shwachman-diamond syndrome with severe cytopenias and/or myelodysplastic syndrome. the paucity of experience with children undergoing hct for sds has been the major obstacle for recommendations regarding time point, transplant regimen, and patient subgroup benefiting most from hct. most but three reported patients received a preparative regimen either consisting of bu/cy or bu/tbi. however, severe early toxicity with cardiomegaly, myocardial fibrosis, and cyclophosphamide associated cardiomyopathy have been described. therefore, we tested the feasibility of a cyclophosphamide free protocol using fludarabine, treosulfan, and melphalan as a conditioning regimen. methods: between 2004 and 2005 two children with sds were enrolled. age at transplantation was 17 and 7 years. both patients received conditioning with fludarabine (30 mg/m²/day x 6), treosulfan (12 g/m²/d x 4), melphalan (140 mg/m²/d x 1), and campath-1h. all children received a non manipulated fresh bone marrow graft. the first patient from a hla-identical sibling, the second from a 10/10 locus matched unrelated donor. mean cell doses transplanted were 3.7 x 10 8 nucleated cells/kg bw (2.9 x 10 8 /kg and 6.2 x 10 8 /kg). results: both patients achieved donor derived engraftment, no gvhd exceeding grade ii was observed, and both maintained donor chimerism at 100%. all patients developed grade iii mucositis. on patient experienced a cerebral seizure early after transplant most likely caused by csa toxicity. gvhd prophylaxis was switched to mmf and the patient fully recovered from this single event. apart from this, no rrt > grade ii was seen. all patients are alive after a follow up of 17 and 4 month. conclusion: a fludarabine based, cyclophosphamide free conditioning regimen seems to be a feasible approach for matched sibling and matched unrelated hct in children and adolescents with sds. larger numbers and a longer follow-up are needed to make these results more comparable to traditionally used preparative regimens. a prospective comparison of immune reconstitution after autologous haematopoietic stem cell transplantation in children v. wiegering, b. winkler, m. eyrich, p.-g. schlegel university of wuerzburg (wuerzburg, d) introduction: autologous haematopoietic stem cell transplantation (auto hsct) has become an established therapy for numerous advanced paediatric solid tumours. after haematopoietic stem cell transplantation all recipients experience a period of immunodeficiency. regeneration of adequate t-cell numbers and repertoire diversity are key elements in the recovery of immune competence. patients: immune reconstitution was studied in 28 children (34 transplants; median 5,5 years; range 13m-18y; 10 female, 18 male). blood samples were drawn before auto hsct, on days 14 (take), 30, 60, 100, 200 and 365 and >15months. methods: we analyzed lymphocyte subpopulations using flow cytometry. intracellular cytokines (ifngamma, il2, tnfalpha, il4, il5, and il10) were determined by facs after in vitro stimulation with pma, ionomycin and brefeldin for 24h. additionally, we measured il2, il15 and tgfbeta in unstimulated sera by elisa. additional we measured trec´s and spectatypes. results: as to lymphocyte subpopulations after auto hsct, nk-cells were the first to regenerate. in t-cells, an inverted cd4-cd8-ratio could be detected during the first year. in cd4+ t-cells the memory phenotype (cd45ro+) predominated. as to cytokine levels in unstimulated sera, we saw high levels of il4 shortly after transplantation, levels decreased to pretransplant values during one year. tgfbeta levels increased during the first year and decreased thereafter. ifngamma levels remained stable. in stimulated t-cells, ifngamma and tnfalpha increased in the first year and went down afterwards. interestingly, high ifngamma levels after transplantation correlated significantly with a better survival. non-irradiation containing conditioning regimen for children with fanconi's anaemia m. jarrar, m. sarhan, m. milhem, f. abdel-rahman, r. rihani, s. sharma, i. na'em king hussein cancer center (al-jubeiha, jor) fanconi anemia is an inherited disorder that leads to progressive bone marrow failure. the only curative treatment of the severe aplastic anemia that ultimately develops in these patients is allogeneic stem cell transplantation. patients with fanconi anemia have increased chromosomal fragility. as a result they are prone to both short and long term complications when conditioning regimens containing radiation are used. we report on 5 patients (3 males and 2 females) with fanconi anemia who were transplanted from matched siblings without using radiation as a part of conditioning. median age at time of transplant was 9 years (3-18 years). none of the patients had mds changes or leukemia prior to transplant. conditioning regimen consisted of fludarabine 120 mg/kg, cyclophosphamide 20 mg/kg and rabbit atg 20 mg/kg. peripheral blood was the source of stem cells in 3 patients, while bone marrow was the source in 2 patients. gvhd prophylaxis consisted only of cyclosporine. at a median follow up time of 368 days (48-568 days), 4 patients are alive with normal hematopoesis. one patient failed to engraft. he was transplanted again with a different conditioning; however he had late rejection and died of sepsis 259 days after second transplant. median cd 34+ cell/kg infused was 4.6 million (1.2-7.6 million). median time to neutrophil and platelet engraftment was 9days and 74 days, respectively. two patients had fever with atg; one patient had bacteremia during the neutropenic period. two patients developed fungal infections after engraftment. two patients had mild vod. three patients received vod prophylaxis consisting of urisidiol and spironolactone. cmv reactivation occurred in 4 patients and was treated with gancyclovir. grade 1-2 skin and liver acute gvhd occurred in 2 patients. limited chronic gvhd occurred in 2 patients. one patient developed extensive chronic gvhd. conditioning without radiation is well tolerated in fanconi anemia patients and results in prompt engraftment. infectious complications appear to be high. autologous peripheral blood stem cell transplantation in low weight paediatric patients: methods and clinical outcome a. galmes, m. canaro, m. guibelalde, m. morey, l. bibiloni, a. vila, a. gutierrez, j. besalduch son dureta hospital (palma de mallorca, e) peripheral blood stem cell (pbsc) collection may be difficult in low weight body pediatric patients, with technical and clinical problems related to vascular access, low total blood volume, citrate toxicity, high extracorporeal volume, and patient's tolerance. methods: we present our experience with 12 consecutive children weighing ≤ 20 kg, diagnosed with acute leukaemia (3) and solid tumors (9), which were collected and transplanted between september 1994 and february 2005 at our centre. patients mean body weight was 15 kg (range 7-20); median age was 4 years (range, 1-7 years) ( table 1 ).harvesting of pbsc was started after 5 days of cytokine alone (g-csf 10 mcg/kg/24hs. s.c.). procedures were performed using a baxter cs-3000 plus separator primed with a mixture of irradiated and white cell-depleted red cells resuspended in 5% albumin and diluted with saline to match the patient's haematocrit. heparin and acd-a was used for anticoagulation (heparin 5000 ui in 1000 ml acd-a) in patients weighing < 10 kg. the median number of leukapheresis was 2 (range 1-4), processing 2.5 volemia in each session. the platelet count decreased significantly after each procedure without requirement of platelet transfusions. special monitoring of toxicity was done. the children were no sedated and showed no serious side-effects. all pbsc were cryopreserved with dmso 5% and stored at -80ºc in mechanical freezer. results: the median time from cryopreservation to transplantation was 29.5 (17-130) days, and the median number of infused mononuclear cells and cd34+ cells were 5.05 (2.8-14.7) x10 8 /kg and 2.5 (0.5-4.3) x10 6 /kg, respectively. the median number of infused post-thawing cfu-gm was 23.3 (17.4-39.1) x10 4 /kg. all patients showed a safe and sustained engraftment. median time to reach 500 and 1000 neutrophil per microl was 12 (10-17) and 12 (10-16) days. median time to 20 and 50 platelets level per litter was 20 (10-50) and 33 (12-84) days (table 2) . long-term hematopoietic recovery at 6, 12 and 24 months was achieved in all cases (table 3) . conclusion: our experience shows that our pbsc collection and cryopreservation method is a safe and efficient procedure in children weighing less than 20 kg., with sustained haematopoietic reconstitution. an approach to retrospective validation and performance monitoring of pbsc collection and other white cell procedures using the cobe spectra cell separator: "cells collected" as a function of "cells processed" by 2 different methods k.w. douglas, j. sinclair, m. mcgarvey, a. mcphelim, s. taylor, m. drummond s.n.b.t.s. clinical apheresis unit (glasgow, uk) validation of cell separator machines for a specific apheresis procedure poses a number of challenges. in particular, for white cell collection procedures it is not feasible for individual centres to carry out a prospective validation process prior to introduction of the procedure, because there is no way of performing a "dummy run" of a white cell procedure without actually putting a donor on the machine. we therefore attempted retrospective validation of our cobe spectra cell separator machines for pbsc collection using the mononuclear cell (mnc) procedure by estimating efficiency of the mnc procedure for each of the individual machines, in terms of the total mononuclear cell dose achieved in the apheresis product as a function of the number of mononuclear cells processed by the machine. there should be a direct correlation between total mononuclear cells processed by the machine and the final mnc dose in the product. the difficulty is in estimating "total mononuclear cells processed by the machine", which will only ever be an approximation. we estimated "cells processed" using 2 different methods: method 1: the donor's peripheral mononuclear cell count (lymphocytes plus monocytes) multiplied by the run time; method 2: the number of total blood volumes processed multiplied by the donor's peripheral mononuclear count. retrospective audit was performed on 70 pbsc collections carried out on five spectra machines over an 18-month period. total mononuclear cell dose in the apheresis product was graphed as a function of "cells processed" as calculated by both methods above. both methods showed a clear, statistically significant linear correlation, but there was less scatter and a lower p value using method 1 (r=0.64, p<0.0001). it was noted that the gradient of the trend line is a measurement of the efficiency of pbsc collection. the data was therefore subdivided depending on which of the 5 spectra machines had been used for collection, and individual data analysis was performed for each machine. this showed that the five machines all performed the mnc procedure with very similar efficiency (gradients of trendlines 0.522 to 0.593). the same process can easily be applied to ongoing performance monitoring of the five machines, and our aim will be to do this annually from now on. m. miorin, e. brunetta, e. scquizzato, s. varotto, c. messina, s. cesaro, g. binotto, i. baesso, e. calore, m. facco, e. ave, m.v. gazzola, r. destro, g. semenzato, r. zambello, l. trentin university of padua (padua, i) allogeneic bone marrow transplantation (abmt) is one of the powerful therapies for several hematological malignancies. multiple mechanisms contribute to the graft success, such as the entity of primary disease, donor bone marrow availability, minimal residual disease (mrd) and complications including infections and acute graft-versus-host disease (gvhd). gvhd represents a major complication of abmt and is the main cause of morbidity and mortality. the present study takes into account the recovery of the t cell-compartment before and after abmt in 14 pediatric patients affected by different hematological malignancies. to evaluate the pattern of accumulation of t cells, we investigated the usage of t cell receptor (tcr-beta) chain variable regions (tcrbv) and the complementarity-determining region 3 (cdr3) up to 2 year follow-up after abmt. increased expression of some tcrvb families were observed in patients during the months after abmt. in the following months after abmt, we confirmed the presence of the same t cell clones and, sometimes, we showed the appearance of a new tcrbv family subset. we observed a random distribution of overexpressed tcrbv families and we did not show a preferential expression of a peculiar tcrbv. in order to clarify whether cells expressing a tcrbv region were clonally expanded, we performed cdr3 spectratyping and sequencing analysis. a predominant polyclonal pattern was observed in donors and patients before transplantation while at 1 and 3 months after bmt some clonal subsets were identified. in the majority of patients the presence of these subsets persists until 24 month after abmt. a skewed tcrbv repertoire and oligoclonal/monoclonal subsets we observed may explain the post-bmt immunodeficency detectable after transplantation and may reflect responses to pathogens or alloantigens in correlation with clinical gvdh. this study was supported by a grant from fondazione città della speranza. s. ganepola, k. rieger, c. loddenkemper, j. maul, a. muessig, e. berg, h. stein, e. thiel, r. duchmann, l. uharek university medicine berlin, charite (berlin, d) introduction: tregs are involved in the control of immune responses to foreign antigens and play an important role in the pathophysiology of gvh-reactions. they are characterized by expression of cd4+, cd25+ and the transcription factor foxp3. although tregs are of emerging interest in allogeneic cell therapy, their precise enumeration in heterogeneous cell products has been extremely difficult. using monoclonal antibodies (moabs) against foxp3 and immunenzymatic labelling at the single-cell level in paraffin-embedded tissues, we have investigated different techniques to identify tregs in cellular products and tissue biopsies. methods: 20µl of the anti-human pe conjugated foxp3 antibody (clone pch101, ebioscience) were used for intracellular labelling of 1x10 6 cells of cd19 depleted, macs sorted cd4+/cd25+ cell fractions of peripheral blood mononuclear cells. the abcam goat polyclonal foxp3 antibody was used for double immunoenzymatic labelling of foxp3/cd3 and foxp3/cd25 of pbmc cytospins, of macssorted cd4+cd25+ selected cell fractions and of paraffinembedded tissues. results: nuclear staining of the foxp3 and measurement by flow cytometry showed bright results in macs-sorted peripheral blood cells as well as in cytospins. gating on cd4+ cells of the cd19 depleted, cd25 enriched cell fraction, 67% of the cd4+ cells are positive for the foxp3 marker. the frequency of foxp3+ treg in the peripheral blood lymphocytes of healthy individuals ranged between 2-4 % of total lymphocytes, like previously described. in double labelling cytospin analyses of foxp3 and cd25, we confirmed co-expression of cd25 on all foxp3 positive cells. counting 10 high-power fields, 63% of the cd3+/cd25+ cells are foxp3+. in healthy individuals pbmc-cytospins we found 5% cd3+/cd25+/ foxp3+ cells. in double labelling analyses of foxp3 and cd25 in paraffin embedded tissues, we confirmed co-expression of cd25 on all foxp3 positive cells. only a few weakly cd25-stained cells were negative for foxp3 staining, indicating a preferential staining of the cd25high cell population. conclusion: direct staining of cellular products with moabs against nuclear foxp and subsequent flow cytometry can give similar results as nuclear foxp3-staining in cytospin preparations or the assessment of cd4+cd25+ cells by flow cytometry. these new techniques allow straightforward identification and quantification even of very low numbers of treg in peripheral blood subsets or other tissues. patient age and granulocytic contamination of apheretic harvests are important factors for adverse events after infusion of cryopreserved hsc g. milone, a. strano, s. mercurio, s. coppoletta, k. battiato, s. leotta, m. poidomani, r. giustolisi ospedale ferrarotto (catania, i) adverse events (ae) after cryopreserved cellular infusions are frequent and seldom can be life-threatening . dmso is considered important in their pathogenesis, however other factors could well play a role. we have prospectively collected data on ae presenting after 175 hsc infusions following high dose chemotherapy performed in our centre during a 4 y. period in patients affected with haematological neoplasm. stem cell source was pbsc in 153 cases while bone marrow in 22. in all cases an endotoxin-free dmso was used. one or more ae was registered in 51/175 infusions (28.6%). gastrointestinal complains were reported in 17% of all infusions, skin rashes in 5.7% of cases, shaking in 4%, cough in 5.1% , fever in 2.2%, shortness of breath in1.2%, dizziness in 1%, headaches in 0.5%. in univariate analysis patient age over 60 was significantly associated to a higher incidence of ae, in fact incidence of ae was 25 % below 50 years of age, 26% in 50-60 decade and 47% over 60 years (p=0.04). frequency of ae was higher after pbsc than after bm (32% versus 4.5%,chi-test: p=0.002). in univariate logistic regression factors found important for ae in pbsc group were total number of cells infused /kg ( p=0.01) and volume/kg of dmso infused (p=0.006). adverse events were more frequent also when total number of granulocytic cells/ present in pbsc harvest was >0.5x10 8 /kg in respect to infusions containing a total number of granulocytic cells below this threshold (14% versus 48%, chi test: p=0.0004). all aforementioned factors were evaluated in multivariate logistic regression and age of patient (p= 0.008) and granulocytic contamination over 0.5x10 8 /kg (p=0.001) were still significant while the total volume of infused dmso loose importance (p=0.1). no cardiovascular events were recorded during infusions, however we registered a statistically decrease of blood pressure and a statistically decrease of cardiac frequency. a significant correlation existed between reduction of cardiac frequency and volume/kg of dmso infused (r:0.220, p= 0.005). in conclusion while non cardiovascular ae are dependent from patient age and from granulocytic contamination of apheretic harvests, cardiovascular changes are influenced only by volume/kg of dmso infused. as far as non cardiovascular adverse events are concerned, particular attention should be paid in infusions of hsc in patients over the age of 60 years and when the grafts have a granulocytic contamination over to 0.5 x10 8 /kg. haematopoietic stem cell transplantation for haematological malignancies: a 5-year single-centre experience b. georgievski, l. cevreska, n. siljanovski, a. stojanovic, o. karanfilski, z. stojanoski, s. genadieva stavrik, i. panovska, s. krstevska balkanov, a. pivkova clinical center (skopje,mk) hematopoietic stem cell transplantation (hsct) is widely used therapeutic method in the treatment of patients with hematological malignancies. in this study we present our results in 5 years experience in transplantation for hematological malignancies. in a period 2000-2005 a total of 97 transplants have been realized, 30 allogeneic sibling and 67 autologous transplantations. in the group of patients treated with allogeneic sct, 60% of patients were in active disease prior transplantation, with diagnosis (19 aml; 6 cml; 1 aa; 1 nhl; 1 cll; 2 all); ratio males:females = 2:1, median age 34 years (20-54). bone marrow (bm) as source of hsc was used in 4 patients and 26 were preformed with peripheral blood stem cells (pbsc) with donor sex ratio 1:1 (15/15). conditioning was provided with bu/cy (13pts), bu/cy+mel (7pts), beam (2pts), hdice(1pts), nonmyeloablative flag/ida(4pts), flu/mel(1pt). the amount of infused fresh bone marrow was 1010ml(950-1100ml) with mnc 3.5x108/kg(2.5-4,5) and pbsc 4,32x108/kg (2,5-6.2). median number of transfused blood products was for er median 3 doses (0-6) and plt 19 doses (5-34). engraftment for ne>0.5x109/l and plt >20x109/l was recognized on day +12 (10-24). acute graft versus host disease (gvhd) gr iii/iv was noticed in 5 patients and chronic extensive gvhd in 3 patients. in the second group of 67 autologous recipients, 27 patients with diagnosis (19 aml; 4 nhl; 3 hd; 1 all) received fresh bm as source of hsc and the other group of 40 patients ( 9 aml; 2 all; 11 hd; 8 nhl; 10 mm) received pbsc previously mobilized with g-csf+chemotherapy. median age was 35 years (7-63), males:females=1:1. 40% of patients with limfoproliferative diseases were with refractory/relapsed disease and other patients were in complete remission before sct. conditioning regimens consisted of high-dose chemotherapy mainly bu-cy, beam, ice high-dose, melphalan. the amount of infused fresh bm was 1010ml(950-1250ml) with mnc 4,0x108/kg(2.9-5.8) and pbsc 3.85x108/kg (1.71-6.0). median number of transfused blood products was for er median 3 doses (0-6) and plt 35 doses (0-73). engraftment for ne>0.5x109/l and plt >20x109/l was recognized on +15(8-25). median follow up for both groups was 30 months (3-60), trm in allogeneic recipients was 4 patients (13,3%) with survival of patients transplanted in cr of 84,62%, and in the autologous group trm was 6 patients (8,9%) with survival of 62,69%. s. genadieva-stavrik, l. cevreska, a. pivkova, z. stojanoski, b. georgievski clinical center (skopje, mk) introduction: the administration of a combination of chemotherapy and cytokines g-csf is associated with a significantly increased efficacy of stem cell mobilization compared with either modality alone. method: the aim of this study was to evaluate the efficacy of g-csf preceded by chemotherapy (cyclophosphamide 4g/m sq for 1 dose) for hematopoetic progenitor cell mobilization for lymphoma and myeloma patients. we started g-csf as a fixed dose 480mu sq every day as soon as the leukocyte counts began to rise after chemotherapy induced myelosupression. leukapheresis was commenced at the time when leukocyte count rose up to 1000/ul, and repeated for 2-4 consecutive days until target number of cd34+ cell, at least 2x106/kg was collected. results: thirty-five patients (male to female, 19:16, age range 21-65, lymphoma 25, myeloma 10) underwent a total of 73 courses of leukapheresis for hematopoetic progenitor cell collection prior to autologous transplantation from april 2002 through may 2005. the target amount of marrow was harvest in all patients. all the patients achieved good engraftment after autologous transplantation. the mean days required for wbc count to be over 1,000/ul was 8-16 days. patient's age, sex, underlying malignancy, exposure to chemotherapy before mobilization did not show any statistically significant correlation. conclusion: we can conclude that chemotherapy followed by g-csf administration is an effective way for mobilization of hematopoetic progenitor cell and verified itself as a good mobilization method. cyclophosphamide + gcsf as mobilising schedule in multiple myeloma and malignant lymphoma patients: factors associated with mobilisation efficiency r. carrion, j. anguita, m. calderon, d. serrano, p. balsalobre, i. buño, a. gomez-pineda, jl. diez-martin hospital gregorio marañon (madrid, e) backgroung: cyclophosphamide (cy) at dose of 1.5 grs/m² and gcsf is commonly used to mobilize blood stem cells to support high dose therapy in patients (pts) with multiple myeloma (mm) or malignant lymphoma (ml). however, 20-25% of pts do not achieve the minimum stem cells dose needed for a rapid hematopoietic engraftment and risk factors for poor mobilization are not well known. methods: 93 pts diagnosed of mm or ml and candidates to autologous stem cells transplant (asct) between october 96 and march 05 were treated with cy 1.5 grs/m² (d0) followed by gcsf 10 mcg/kg/d from d+1. a first apheresis procedure was planned on d +7. all pts were treated as outpatients and all of them were naïve to mobilization procedures. we analyzed some clinical factors, in addition to cd34+ cells count, wbc and platelet count on the first day of apheresis. we defined failure as: preapheresis cd34 count less than 10/microl, or cd34 yield (1st day) lower than 1.5 x 10 6 /kg, or cd34 yield (1st round, three days) lower than 2.5 x 10 6 /kg. results: male/female 37/56; median age 55 (min 22, max 69); mm/ml 45/48 (11, hodgkin disease). according to diagnosis (mm vs ml) mm pts had: a higher proportion of women (53% vs 27%, p=0.01), older (median 58 vs 51, p=0.0001), with a lower number of previous courses of chemotherapy (4 vs 9, p=0.0001). asct did not performed in 14 pts: 7 due to poor mobilization (5 pts by disease progression, 1 by graft tumoral contamination, and 1 by a second neoplasm), 3 mm pts and 4 ml pts. twelve pts (12/79, 15%, 4 mm pts and 8 ml pts) had to undergo another mobilization program (most of them with gcsf alone) to achieve sufficient stem cells. as a whole, 19 pts (20%) would be considered as a failure to get an adequate stem cells collection. a median of 8 days was necessary to get a minimal preapheresis cd34 count. this interval was not different between mm and ml pts(p=0.59). significant correlations were seen between preapheresis cd34+ counts, cd34 yield (1st day) and total (1st round) (p=0.0001). some covariates were selected to explain failure of mobilization (bone marrow involvement in ml pts, number of chemotherapy courses, and preapheresis wbc and platelet). in contrast with other studies none factor associated with mobilization failure was found by logistic regression analysis. conclusions: 1) a combination of cy + gcsf is a safe, predictable and effective for the most of mm or ml pts (80%). s333 2) we have not identified any premobilization factors predicting poor mobilizer pts. k. ali moghaddam, s. samiee, n. mahdavi, l. nedaeifard, m. jahani, a. mousavi, m. iravani, b. bahar, a. ghavamzadeh horc (tehran, ir) introduction: umbilical cord blood (ucb) may be an alternative source for allogeneic transplantation for the treatment of hematological malignancies and genetic disorders in patients without suitable donors particularly in ethnic minorities. early experience with umbilical cord blood transplantation (cbt) demonstrated a lower incidence of graftversus-host disease (gvhd) even though the procedure was performed with hla-disparate grafts. the major drawbacks of cbt are slow hematopoietic recovery and a high incidence of graft failure, as a result of a lower number of progenitors infused. materials and method: we collect the data of 13 patients who had undergone cord blood transplantation by reviewing their records from the date of their transplantation up to the date of last contact. analysis of the data has done via using spss software. results: 13 patients received cbt which in our center during the past 6 years, consist of 8 talassemia, 2 severe combined immune deficiency (scid), 1 aml-m2, 1 mps-1 (hurler syndrome). of all the 13 patients 9 (69.23%) were male, and 4(30.77%) were female. the median age was 4 years old; (8 months -14 ys) respectively. donors were hlaidentical siblings in 9(69.23%) patients and unrelated in 4(30.76%) patients. the conditioning regimen for 76.92% of patients was busulphan, and cyclophosphamide. 8 patients had not gvhd, (3 of them died in first 100 days), 2 had only gi gvhd, 1 had gi and skin gvhd, and 2 had gi, skin and also liver gvhd. the median duration of hospitalization for transplanted patients was 50 days. transplant mortality rate in the first 100 days was 30%. the median follow-up duration was 189 days, with minimum and maximum of 7 and 1823 days respectively and during this period the overall survival (os) is 74.07% and the disease free survival rate (dfs) was 37.50% conclusion: cord blood is a considerable alternative source for hematopoietic stem cells for allogeneic transplantation for malignant or nonmalignant hematopoietic disorders. as our cases were few we can not conclude definitely about the advantages or disadvantages, but in our study cord blood recipient from hla-identical siblings had lower gvhd and mortality than unrelated donors. peripheral blood stem cell collection: from outsourcing to in-service process. an opportunity to optimise the procedure. results after one year at eio -milan d. laszlo, a. agazzi, a. alietti, l. orlando, m. cassatella, l. roveda, g. vasaturo, m. venturino, a. lettiero, c. massaro, c. rabascio, a. cocquio, g. martinelli european institute of oncology (milan, i) peripheral blood stem cell (pbsc) collection by leukapheresis (laf) represents the standard method to obtain blood stem progenitors. this procedure is usually referred to transfusion centers joined with transplant units. a possible pitfall of this kind of management could be the impossibility to perform "ad hoc" the procedure in any time. from 97 a transplant program with pbsc collection is ongoing at the eio; until june 04 the laf has been performed by the transfusionist team outsourcing and then governed by the medical-nurse staff of our division. aim of the study was to evaluate the outcome of this shift in management. methods: firstly we considered what kind of laf-related variable could be examined to optimise the service for the patients in terms of harvest quality and assistance and for the institute in terms of reduction of time and cost of the procedure. secondly we organized a special training with the aim to perform the laf in-service using our know-how and resources. successively a comparative analysis of the data after one year of our management has been performed and compared to the data collected during one year of the previous management. in particular all the following variables of the procedure have been analyzed by a specific data-base: a)quality of the stem cell product; b)comfort for the patient; c)time related to procedure; d)costs. results: from july 04 to july 05 we performed 94 consecutive laf. the staff was operative 24hours every day saturday and sunday included. the two populations of patients were matched for age, sex, diagnosis, stage of disease and previous treatment. the collection target were>4.0x10 6 /kg and >2.0x10 6 /kg cd34+ for donors and patients respectively. the table shows the collection results obtained. the best ratio optimal collections/patients obtained with our management (97%vs90%) could be attributed to the optimisation of the procedure timing and to the excellent cooperation between medical doctors and the nurses in the in-service experience. concerning the time for an optimal collection, no difference was observed into the two managements (only 7% of the patients needed three or more procedures). the activity of the internal staff has permitted to cut down significantly on expenses in charge of the institute for the transfusionist's performance. . conclusions: thanks to the constitution of an internal dedicated team, we were able to perform the pbsc collection with similar results and lower cost in comparison with the outsource management. (1) (2)leningrad regional clinical hospital (st. petersburg, rus) background: mobilized peripheral blood stem cells (pbsc) have become the main source for autologous transplantation in patients with haemathological malignancies or solid tumours. the aim of the study was to establish the influence of diagnosis, sex, age, number of previous courses of ct, mobilization regimen, bone marrow involvement on the outcome of pbsc mobilization. patients and methods: 145 patients with haemathological malignancies and solid tumors were included in the study (hodgkin's lymphoma, hl (n=24), non-hodgkin lymphomas, nhl (n=29), multiple myeloma, mm (n=32), acute leukaemias, al (n=15), solid tumors, st (n=21)). 79 (54%) were females, 66 (46%)males. 104 (72%) patients were >50 years of age, 41 (28%) patients were an age of less than 50 years.100 patients (82%) were mobilized with g-csf 10 mcg/kg for 5 days, a combination of ct and g-csf (5 mcg/kg) has been used in 21 patients (18%). 46 patients (49%) received more than six courses of ct and 47 (51%) less than six respectively. bone marrow (bm) involvement was diagnosed in 76 (56%) patients. apheresis was performed on 'spectra'v.5.1 (gambro). the following criteria for the mobilization outcome were used: non-mobilizable patients <1±106 cd34+/kg; poorly mobilizable patients >1±106xcd34+/kg; mobilizable patients >5±106xcd34+/kg (s.fu et al., 2000) . results: 14 patients (11%) were non-mobilizable; 61 patients (48%)-poorly mobilizable; 52 (41%)-mobilizable patients. according to the diagnosis we observe the following results: hl-9.0±3.4x106 cd34+/kg, nhl 6.8±2.2x106 cd34+/kg, mm-8.8±2.6x106 cd34+/kg, st-8.6±3.0x106 cd34+/kg, al-5.5±1.2x106 cd34+/kg. comparing all groups between each other we found no significant differences. there were also no influence of age and sex on stem cell mobilization (p= .94, .89 respectively). bm involvement does not seem to be an independent factor with significant adverse influence on pbsc mobilization (p= .78). in patients received less than six courses of ct stem cell yield was significantly higher (10.0±2.2x106 cd34+/kg against 5.5±1.7x106 cd34+/kg (p= .006)). better outcome was seen in patients mobilized with ct plus g-csf than g-csf alone (8.12±1.12x106 cd34+/kg against 6.9±0.9x106 cd34+/kg (p= .008)). conclusions: better stem cell yield was seen in patients received less than 6 courses of ct and in patients mobilized with g-csf combined with ct than g-csf alone. diagnosis, age, sex, bm involvement doesn't influence outcome of pbsc mobilization. inroduction: the phenomenon of nonspecific cell aggregation (cell clumping) can be observed in nucleated cell preparations obtained from bone marrow, peripheral blood and umbilical cord blood (ucb) following thawing. such preparations containing populations of both mature as well as immature hematopoietic progenitor cells are obtained by processing whole cord blood and freezing. different techniques may be applied for the thawing of such preparations. object: to evaluate the phenomenon of cell clumping, the influence on it was examined at increasing densities of nucleated cell suspensions which had been extracted from cord blood and then frozen. the two selected techniques of thawing were also evaluated and their influence on cell clumping. methods: the fraction of nucleated cells from the 80 ucb was obtained by sedimentation. samples containing the suspensions of these cells (5, 10, 20, 50 mln/ml)were cryopreserved. in vitro cultures of the colony forming cells (cfc) were performed before freezing and after thawing. results: the intensity of the cell clumping increased simultaneously with the increasing density of the cell suspension. it increased from approx. 17% in the 5 and 10 mln/ml groups to approx. 43% in the 50 mln/ml group, when thawed accordingly to "the classic" technique. if the solution containing dextran (rubinstein's technique) was applied post thaw to dilute the cell suspension, the clumping phnenomenon was markedly inhibited. it didn't exceed approx. 15% in any density group. independently to the intensity of the aggregation process, the number of the cfc among the whole pool that remained suspended after thawing (per 100 000 cells), maintained a quite stable level of approx. 70% of the pre-freezing value. conclusions: the intensity of the cell clumping phenomenon is directly influenced by the probability of the cell contact. neverthless, the substances like dextran may markedly inhibit this process. this phenomenon is not selective process and affects both early hematopoietic cells (cfc) as well as mature cells, independly of the initial density of the frozen suspension. it seems in order to protect thawed nucleated cell suspensions from clumping and preventing the associated looses, future studies will have to concentrate on the composition of adequate freezing mediums containing clumping inhibitors. centre of slovenia, ljubljana d. domanovic, z. dovc, a. nunar-perko, p. mali, m. cukjati blood transfusion centre of slovenia (ljubljana, svn) collection of peripheral blood stem cells (pbsc) by aphaeresis is safe and reliable procedure that is generally well tolerated. beside the adverse reactions associated with g-csf or gm-csf mobilization, some adverse effects related to the aphaeresis procedure can occur. in the period from january 2004 to the end of october 2005 we retrospectively analyzed 141 pbsc collections in 74 autologous and 14 allogeneic donors. pbsc collections were performed in the blood transfusion centre of slovenia, ljubljana, by standard volume aphaeresis procedures (two blood volumes processed) on the amicus blood cell separator (baxter). the targeted number of collected cd34+ cells was > 2 x106/kg of recipient's body weight (bw) and > 5 x 106 / kg bw in plasmocytoma donors. peripheral antecubital vein access was established in 117 (83 %), femoral catheter in 22 and subclavia catheter in 2 collections were placed without documented side effects. we have identified 20 (14.3 %) adverse effects and 5 (3.5 %) instrument troubleshooting. in 11 (7.9 %) collections, the adverse effects occurred during the establishment of venous access (repeated phlebotomy 7%, unsuccessful phlebotomy 1%). during the collection we documented 9 (6.4 %) adverse reactions. the citrate reaction in 5 collections (3.6 %), hematoma in 1 collection (0.7 %), fatigue in 2 collections (1.4 %) and heart arrhythmia in 1 collections (0.7 %). citrate reactions were mostly present as significant paresthesias and cured by oral calcium tablets and aphaeresis continued. there were no significant post donation decrease of platelet count and platelet transfusions were not necessary. the one troubleshooting was due to the unrecoverable stop (0.7 %) an four were during the disposable set instalation (2.9 %). the data confirmed the presence of mild but relatively common adverse effects in the collection of pbscs by apheresis especially in the autologous donors. the serious adverse reactions were not documented. predictive factors that affect the mobilisation of cd34+ cells in healthy donors treated with recombinant granulocyte colony-stimulating factor m. martino, i. callea, a. pontari, g. praticò, t. moscato, e. massara, g. console, e. quartarone, g. irrera, g. pucci, a. condemi, a. dattola, p. iacopino azienda ospedaliera "bianchi-melacrino-morelli (reggio calabria, i) no specific characteristics have been identified as predictors of peripheral blood stem cells (pbsc) mobilization in healthy donors. in this study, clinical characteristics and laboratory data for 122 healthy donors who underwent apheresis on day 5 of treatment with recombinant granulocyte colony-stimulating factor (g-csf) were retrospectively analyzed for correlations with cd34+ cell mobilization. the variables that were analyzed included age, sex, body weight, basal complete blood count and maximum white blood count (wbc) before apheresis, g-csf type and dosage. median age and body weight were 42.5 years (range 16-65) and 72.5 kg (range 47-121), respectively. by univariate analysis, male sex (p= 0.007), body weight (< 70 vs. >70 kg, p= 0.04) and donors age (<50 vs. > 50 years; p= 0.015) were correlated with the number of cd34+ cells mobilized. by multivariate analysis, donor's age and male sex were the only two variables that significantly predicted a high cd34+ cell level. in conclusion, our data suggest that male sex and younger age are the only factors that significantly affect cd34+ mobilization in healthy donors. hla-a19 is a "broad" specificity, which includes a few serologically defined antigens (spits): a29, a30, a31, a32, a33, a74. the hla-a19 occurs at a rather high frequency in most human populations, and the a19 splits are found at differing frequencies in different ethnic groups and populations. the distribution of a19 splits is very important for transplantation medicine, especially for bone marrow transplantation. incorrect assignment of a19 splits is one of a major problems in using serology for typing hla-a antigens. sometimes serology hardly discriminates a19 splits and the low resolution dna typing is required. the data of a19 split distribution in russians are few and ambiguous. the objective of our study was to determine the distribution of a19 splits in russian donors typed in research center for hematology (moscow) in 2005. methods: 121 donors were typed. serological typing was performed using commercial sera. in case of hla-a19 or homozygosity in hla-a locus revealed by serology the samples of donor blood were retyped using pcr-ssp ("protrans" or kit of research center for hematology, moscow). results: by serological typing hla-a19 was found in 24 donors (19.8%). the a19 splits were defined in 16. the hla-a*19 (by pcr-ssp) was detected in 23 donors (19%). the most frequent among hla-a19 splits was a*29 (n=7; 5.8%). a*30 and *31 were found with the equal frequency (n=5; 4.1%). a*32 was observed only in 2 cases (1.7%) and a*33in 4 cases (3.3%). in our donors we didn't reveal a*74. in one case serologically defined a19 was not confirmed by pcr-ssp. conclusion: these results confirm the studies that dna typing for hla class i improves the typing quality. serological typing is insufficient for hla-a19 split identification. objectives: the availability of unrelated donor hematopoietic stem cell transplantation(hsct) could be increased with the use of allele mismatch donors if the status of hla mismatching were better understood. with the use of different transplant and immunosuppression regimens according to the patient-risk grouping will allow donor selection criteria to be refined to meet the needs best of the individual patient. methods: we conducted a hla matched or mismatched unrelated donor hsct based on the hla high-resolution typing for 61 consecutive cases(37 male, 24 female) of adult acute myeloid leukemia(aml) patients. the median age was 32(range, 17-55) and the median follow-up duration was 25 months(range, . compared to the group of patients received hsct from hla perfect matched(8/8) unrelated donor(cohort 1), twentyfive(41% of total population as cohort 2) mismatched unrelated donor at the level of hla-cw antigen(n=9) or 1-2 alleles(n=14) at the hla-a, b, and dr loci were considered. two cases were mismatched at the hla-cw combined with 1 allele level. fortyeight patients were in 1st cr and 10 were in impending relapse status and most of them had a very poor cytogenetic profiles. the majority of patients(n=53) had intermediate or unfavourable cytogenetic features. the main conditioning regimen consisted in cyclophosphamide plus tbi(n=55) with our standard gvhd prophylaxis containing fk-506 plus short course methotrexate. some of cohort 2 patients(n=9) received additional mycophenolate mofetil from the day 21 posttransplant. the majority of patients received bone marrow as a stem cell source. the two groups had similar pre-transplant characteristics. results: all, except 1(cohort 2), transplanted patients were engrafted. the incidence of acute gvhd(29% vs 42%, cohort 1 vs cohort 2) was significantly different in the two cohort, but not in the chronic gvhd(23% vs 21%). five(20%) patients were relapsed in the cohort 2. the 2-year non-relapse trm was 0% vs 8%. the estimated probability of dfs and efs at 3-year was 77% vs 71% and 71% vs 58%, respectively. conclusion: these data suggest that allele and antigen mismatches can elicit more detrimental gvhd together with even a chance of graft failure that lead to increased trm. analysis of large hsct populations with a diversity of mismatches is necessary to define a tolerable mismatched unrelated donor so that we can enlarge the available allogeneic unrelated hsct donor pool. the majority of haematopoietic transplantation are currently performed with peripheral blood progenitor cells (pbpc). in donors, it is very important to optimize pbpc harvesting to obtain the target cd34+ cell dose required for transplant (>4.0x106/kg body weight of the recipient) with a reduced number of apheresis. in the present study we retrospectively analysed data from pbpc collections of healthy adult donors (n=156, 84m /72f) with a median age of 39 years (range 17 -73) performed between august 1995 and october 2005. donors were mobilized with daily administration of g-csf (10-16mg/kg) for 5 days, with generally mild to moderate side effects. total nucleated cells (tnc) and cd34+ cells present on pbpc grafts were evaluated with haematologic counters and flow cytometry respectively. on the collection day, the peripheral blood from most adult donors contained >20 cd34+ cells/µl, previously established as a marker of a good mobilisation. healthy donors had standard apheresis collection using the cobe spectra, with few adverse consequences, mainly related either to vascular access or to metabolic or hemodynamic changes. the majority of donors (n=58%) underwent one apheresis, whilst the remaining 38% and 4% of donors had 2 and 3 apheresis respectively. for adult donors the pbpc collected yielded a median 15.5 tnc x108/kg (2.9 -168.1) and 8.7x106 cd34+ cells/kg (1.0 -64.4) body weight of the recipient and only in 3 collections the target number of cd34+ cells was not reached. we found a correlation between age of the donor and the number of cd34+ cells collected per kg bw of the recipient, being in the older donors more difficult to achieve the required cd34+ cell dose. according to gender, male donors underwent one apheresis, had significantly higher pb cd34+ cell count prior to aphaeresis and cd34+ cell/kg bw recipient collected in comparison to the female donors who had a median of 2 apheresis and had less cells (pb cd34+cells: 79vs55 and cd34+ cell/kg bw recipient 10.7vs7.2 for male and female respectively). there was no correlation between the g-csf dose used in the mobilization and pb cd34+ cell count prior to apheresis or the total cd34+ cell collected. our results show that g-csf mobilisation and pbpc collection in adult healthy donors is feasible and safe for the harvesting of the graft for allogeneic haematopoietic transplantation and the main factors affecting collection are age and gender of the donor. thrombophilic screening in healthy donors treated with recombinant-human granulocyte-colony stimulating factor for mobilisation of peripheral blood stem cells m. martino, t. moscato, g. console, g. irrera, g. messina, e. massara, g. praticò, e. quartarone, c. mammì, f. luise, a. piromalli, p. iacopino azienda ospedaliera bmm (reggio calabria, i) the granulocyte-colony stimulating factor (g-csf) induces an activation state of endothelial cells and coagulation system increasing thrombotic risk. laboratory testing for the identification of heritable trombophilia in high-risk subject groups have become common practice. the objective of this study was to evaluate the effectiveness of thrombophilia screening in healthy donors prior to use g-csf to mobilize peripheral blood stem cells (pbsc). thrombophilia screening comprised of testing for factor v leiden (fvl) g1691a, prothrombin (ptm) g20210a, thermolabile variant (c677t) of the methylene tetrahydrofolate reductase (mthfr) gene, protein c (pc), protein s (ps), factor viii (fviii) and homocysteine (hcy) plasmatic levels, antithrombin iii (atiii) activity, and acquired activated protein c resistance (apcr). we investigated prospectively 72 healthy donors, 39 men and 33 women, with a median age of 42 years (range 18-65). five donors (6,9 %) were heterozygous carriers of fvl g1691a; two healthy donors had the heterozygous ptm g20210a gene mutation; c677t mutation in the mthfr gene was present in 34 (47,2%) donors in heterozygous and in 7 donors (9,7%) in homozygous.apcr was revealed in 8 donors of the study (11.1 %). the pc plasmatic level was decreased in 3 donors (4.2 %); the ps level was decreased in 7 donors (9.7 %). an elevated fviii dosage has been showed in 10 donors (13.9 %) and hyperhomocysteinemia in 9 donors (12.5 %). concentration of atiii was in the normal range for all study group donors. the fvl mutation was combined with the heterozygous ptm g20210a in two cases and with ps deficiency in one case; two healthy donors presented an associated deficiency of pc and ps. the basal screening of thrombofilia permitted to identify healthy donors with a higher risk of thrombotic events. a careful monitoring should be considered in these cases before administer g-csf. during g-csf therapy, we administered low-molecular-weight heparin in all donors and folic acid, vitamins b6 and b12 in healthy donors with c677t mutation in the mthfr gene and a hyperhomocysteinemia plasmatic level. our strategy of prophylaxis was correlated with the absence of short-and long-terms thrombotic and hemorrhagic side effects. no complications known to be related to the anticoagulant occurred in this cohort of healthy donors. differential levels of chimeric tolerance in a single patient. how much change in quantitative chimerism values is enough for clinical significance? d. kristt, j. stein, r. narinski, h. or, t. klein, d. kristt rabin mc (petach tikvah, il) chimerism monitoring based on strs is frequently used following sct. it is best implemented in terms of long-term tracking since engraftment is a dynamic process. when viewed this way, trends and fluctuations in the chimerism values can be observed. one fundamental question raised by such observations is what is the minimal change in chimeric status that has biological and/or clinical significance. as an indirect approach to this question, we present a case of thalassemia major which we were fortunate to follow over a 7 year multi-sample course with two scts each with its own stable % chimerism level. following the first sct the patient developed a stable chimeric tolerance of approximately 30% [27-35%]. however, he remained transfusion dependent, necessitating a second transplant two years ago. once again his chimerism stabilized but at a higher plateau of approximately 42% [39-44%]. he is currently healthy, and no longer requires transfusions. in conclusion, this interesting case afforded us the opportunity to compare two different, but stable levels of chimeric tolerance, differing by approximately 10-12%. since over the long-term, str-platform error is approximately 2-5%, the patient is likely to have sustained a stable elevation of approximately 10% chimerism that freed him from transfusion dependency. the case raises a number of important questions regarding the biological implications of chimerism values, such as: 1) is 10% a relative or absolute figure; 2) is there a minimum/threshold chimerism value for functionality of the graft in these metabolic diseases; 3) do all patients have the same % chimerism interval/differential and threshold? novel aspect of long-term chimeric tolerance in a patient with severe combined immune deficiency: in utero stem cell transplant suppressed by subsequent paternal iatrogenic transplant t. klein, i. yaniv, b. garti, d. kristt rabin mc (petach tikvah,il) severe combined immune deficiency (scid) is often treated with stem cell transplantation (sct). the successfully treated patient usually lives in a state of mixed graft-host chimeric tolerance. this relationship, however, may not be entirely static. we report here a case of a child in which a transfusion of maternal stem cells occurred in utero. this was documented by demonstrating a mixed chimerism in the child based on an evaluation of the child's blood using hla tissue typing and molecular dna methods. at age 3 mo the patient manifested scid, which necessitated an exogenous sct from the father. following the paternal sct, three dna sources were demonstrated, with four hla haplotypes. however, in the ensuing period progressive loss of the maternal component of the patient's tolerance state was observed. now, after 8 yr post-paternal sct, the patient is healthy, but there is no significant maternal dna signal demonstrated in the child, although he does have a stable chimeric level of 25-35%. in conclusion, the three-way mixed chimerism was not tolerated, and the in utero maternal stem cell component was ultimately suppressed by the subsequent paternal sct. this case suggests that even a tri-valent tolerogenic state may be regulated by the dynamic interactions between the host and graft, enabling one set of graft-host interactions to become dominant. successful treatment of graft rejection with immunosuppression withdrawal and/or donor leukocyte infusion after early diagnosis based on t-cell mixed chimerism i. buño, p. balsalobre, g. iglesias, d. barroso, c. manzano, d. serrano, r. carrión, a. gómez-pineda, j.l. díez-martín hosp. g.u. gregorio marañon (madrid, e) background: graft rejection is a serious and difficult to manage complication after sct. objective: to evaluate the usefulness of chimerism monitoring for the early diagnosis of graft rejection in different sct settings, as well as for the follow up after treatment with immunosuppression withdrawal (isw) and/or dli. patients and methods: 68 sct (32 ablative, 19 ric, 17 tcdincluding 8 haploidentical-). chimerism analysis was performed by fish for the sex chromosomes or str-pcr (sensitivity 1%). chimerism was analyzed in pb and leukocyte lineages (t lymphocytes cd3+, b lymphocytes cd19+ and myeloid cells cd15+ isolated (purity >95%) by immunomagnetic means, automacs, miltenyi biotec), every 2 weeks, starting on day +15 (except in ablative), and until complete chimerism (cc) was achieved. results: after initial engraftment in all patients, graft rejection was diagnosed in 8 (2 (6%) ablative, 2 (10%) ric, 4 (23%) tcd), either established (severe pancytopenia and bm aplasia) or incipient (progressive decrease in pb and bm cell counts) a median of 52.5 days (range 23-110) after sct. all patients showed mixed chimerism (mc) in bm and pb with higher percentages of recipient cells (%r) in pb. in 7/7 patients studied, t cells showed persistent mc with high %r (>50% in 6/7; <50% and >25% in 1/7). b cells showed mc in 5/6 patients studied, with lower %r (<15% in 3/5, >50% in 1/5). only 2 patients showed mc, transient in one of them, in myeloid cells. 2 patients were not treated due to concurrent multiorgan failure and subsequently died. reduction of is in 5 patients obtained 1 response (normal pb and bm counts, and cc). the other 4 patients underwent isw but no further response was obtained. one of them received a second sct while the other 3 were treated with dli, and all of them responded. the last patient (transplanted from a haploidentical family donor) who was not receiving is, responded to treatment with dli. time from therapeutic intervention to response was variable with a median of 2 months (range 1-7). 4 patients developed gvhd>i, which was the cause of death in one and was controlled in the other three. one patient died from sepsis in complete remission (cr) 9 months after the transplant. 4 patients are alive in cr a median of 50 months after sct (range 16-72). conclusions: the observation of mc, mainly in t lymphocytes, together with a decrease in pb and bm cell counts, allowed early diagnosis and successful treatment (6/6 patients) of graft rejection. immunosuppressive effects of nucleic acids -or how to learn from artefacts? t. yang, h.j. kolb, i. steinmann, m. svihla, r. buhmann gsf (munich, d) defibrotide, a single-stranded nucleic acid (ssna), was already shown to mediate immunosuppressive effects. in the current survey we investigated whether randomly chemically synthesized ssnas of different length and composition could provide similar effects. for this purpose, purified t-cells were stimulated in the presence of dntp´s or ssna with allogeneic, irradiated pbmc´s, pha or anti-cd3/cd28 dynabeads. cellular proliferation was assessed by incorporation of tritiumlabelled thymidine (³h) thymidine), respectively (³h)damp or by staining with cfda (carboxyfluorescein diacetate, succinimidyl ester). after 72h or 120h of incubation, the incorporation of (³h)thymidine, or (³h)damp as well as the cfda distribution was assessed. cell viability was measured by trypan blue exclusion. t-cell activation was measured after 72h by quantifying the number of cd3+ t-cells expressing the activation markers cd25 and cd69. cellular uptake of cy5labelled nas was detected by fluorescence microscopy. moreover, the interference of different nas or singular nucleotides with nucleoside analogues on t cell proliferation was tested by cfda-assays. na of different length, composition or concentrations (up to 5mm) did not cause cytotoxic effects to lymphocytes. but the incorporation of (³h)thymidine or (³h)damp was competed by na. these effects were found to be dependent on length, concentration and base-composition of the na. the proliferative capacity of the t-cells, as assessed by cfda-staining, seemed to be unaffected. moreover it could be shown, that nas interfere with nucleoside analogues and antagonized the antiproliferative and cytotoxic effects of these drugs. the standard approach to detect cellular proliferation by incorporation of tritium-labelled nucleotides or derivatives is not useful to assess changes in cellular metabolism or proliferation in context with nas. even more important, treatment approaches using nucleoside analogues like fludarabine, cytarabine e.g. in context with nas might be critical and diminish the efficacy of these drugs. e. elli, f. colnaghi, a. colombo, m. parma, v. rossi, e. terruzzi, l. verga, a. biondi, e. pogliani ospedale s. gerardo (monza, i) introduction: chimerism status (cs) analysis is useful for evaluation of donor (d) cells engraftment after allogeneic haematopoietic stem cell transplantation (hsct). adverse events are often described as associated to a loss of chimerism, but a strict correlation between cs and residual disease is still controversial. pcr-based assays analyzing polymorphic short tandem repeat (str) markers are actually the more employed methods in cs monitoring, even if a standardized specific panel is not still available. objective: we tested a semi-quantitative method, based on multiplex pcr amplification of 16 str markers using a commercial kit (powerplex 16 system promega), in order to evaluate its informativity in cs monitoring and the correlation between cs and some clinical variables. methods: the informativity of the assay was tested on peripheral blood samples from 36 allografted patients (pts) and their related sibling donors; perspective evaluation of cs was performed on 27 pts at 1, 2, 3, 4, 6, 12, 18 and 24 months after hsct. pts who showed no evidence of recipient (r) cells were considered to have a complete chimerism (cc), pts who presented each d and r cells were defined as mixed chimerism (mc). results: the multiplex assay gave at least one high informative marker (range: 1-4, median 2) in all the 36 pts. we analyzed 125 blood samples from 27 pts, 98 (78,4%) presented cc, 27 (21,6%) mc and none autologous reconstitution. some pts were defined as having an increasing mc (imc) when they shifted from cc to mc or when in a mc setting the r amount increased in two or more consecutive controls. we evaluated if there was a correlation between cs and some clinical variables: r/d gender, diagnosis, r/d sex mismatch, abo system incompatibility, conditioning regimen, cd34+ and cd3+ cell dose infused, disease status at hsct, stem cells source, acute and chronic graft versus host disease (gvhd), marrow relapse. chi-square analysis demonstrated a significant correlation between icm and a brief time marrow relapse, moreover a low incidence of chronic gvhd, male d and diagnosis of acute leukemia seem to be associated with increasing level of r dna. conclusion: pcr amplification of a panel of 16 str loci is an informative method to evaluate cs in pts after hsct. imc seems to be useful to predict marrow relapse; some clinical conditions such as male donor and acute leukemia diagnosis seem to limit a complete d engraftment; chronic gvhd is favorable for a stable cs. non-haematopoietic tissue repair r1162 gmp production of autologous cd133+ cells for intracoronary administration after acute myocardial infarction r. giordano (1) subjects affected by acute myocardial infarction (ami) with absent angiographic myocardial blush (mb) and lack of st segment elevation resolution after primary angioplasty, have short-and long-term poor clinical prognosis. we recently started a phase i/ii randomized controlled study based on the hypothesis that, in this target population, after primary angioplasty and stenting, intracoronary injection of cd133+ cells from bone marrow (bm, group a) or mobilized peripheral blood (mpb, group b) could enhance endothelial regeneration and improve heart function compared to controls treated with standard pharmacological therapy alone (group c). the study started in june 2004 and it is expected to enroll 15 patients (5 per each randomization group). up to november 2005, 12 patients have been included. in group a (n=4), bm was processed within 2 hours of collection. in group b (n=3), the administration of g-csf (5 µg/kg/day for 3-4 days), started from day 6-10 after ami and leukapheresis was performed following standard procedures. an automated cd133+ stem cell selection was performed with the clinimacs® plus instrument (miltenyi biotec) in our class b -iso 6 facility. the mean (±sd) number of cd133+ cells after immunoselection was 8.2x10 6 (±4.3) in bm samples and 147.7x10 6 (±182.3) in mpb samples respectively, with a purity of 62% (±8) in the group a and 91% (±8) in group b. the percentages of viable cells (propidium iodide) in the post-selection samples were 90 (±1) in group a and 99 (±1) in group b, respectively. the sterility tests for bacteria and fungi on the purified samples were negative. purified cd133+ cells were injected in the culprit vessel using a well-sized over-the-wire angioplasty balloon within three minutes of occlusion. no adverse events have been reported during and immediately after the cell administration. this results show that cd133+ selection is feasible and safe also in ami patients. the short and long term efficacy of this cell therapy approach in preserving the myocardial viability and function after ami is currently under investigation using pet-based techniques and echocardiography. defibrotide (df) is a polydisperse mixture of 90 % singlestranded polydeoxyribonucleotides with anti-thrombotic, profibrinolytic and anti-apoptotic functions. df is already successfully used in the treatment of hepatic veno-occlusive disease in allogeneic stem cell transplantation (sct). our observation that df can also protect endothelial cells (ec) from conditioning (fludarabine)-mediated apoptosis (1) prompted us to apply it prophylactically to patients (pts) at risk for endothelial complications. pending on the magnitude of risk, pts received 200-800mg every 6h in 2h-infusions, usually from day (d) -7 until d+21 post sct. circulating ec (cec) as a marker of conditioning-mediated endothelial toxicity (2) were detected by magnetic bead separation of cd146+ cells from edta blood of 33 sct pts (20 df, 13 non-df) and co-staining with ulex europaeus antigen lectin 1. cec maxima until d+100 post sct were compared between the two groups. df pts showed significantly lower maxima of cec than untreated pts (1142 [±1082] in the df treatment group vs. 2508 [±1987] cec/ml in non-df pts, respectively, p=0.008). similarly, when cec maxima were compared in the time period of df prophylaxis, again, df pts had less cell counts (499 [±615] vs. 1498 [±1709] cec/ml in control pts, respectively, p=0.01). in an overlapping cohort of 52 pts (21 non-df, 31 df) serum was assayed for its induction of apoptosis in a human microvascular endothelial cell line (hmec), a monitoring approach that had been found to correlate with episodes of gvhd and severe microangiopathy (3). apoptotis was determined by flow cytometric analysis of the cellular granularity of propidium-iodide-negative indicator hmec. similar to the cec measurements apoptosis inducing maxima until d+21 were compared between df and non-df pts and turned out to be significantly different (apoptosis by pts´ sera normalized to untreated control hmec: 4.4 [±2.0] in df pts vs. 5.9 [±4.2] in non-df pts, p=0.04). these preliminary analyses suggest the protective efficacy of df prophylaxis in the course of sct. the final proof of principle is to be validated in long-term clinical follow-ups. 1.g. eissner et al., blood 100, 334-340 (2002 ). 2.a. woywodt et al., blood 103, 3603-3605 (2004 ). 3.a. ganster et al., bone marrow transplant. 33, 355-357 (2004 . treosulfan, cyclophosphamide and anti-thymocyte globulin for allogeneic haematopoietic stem cell transplantation in severe aplastic anaemia s. giebel, j. wojnar, m. krawczyk-kulis, m. markiewicz, i. wylezol, t. kruzel, m. kopera, j. holowiecki silesian medical university (katowice, pl) graft rejection is the major cause of failure after allohsct in severe aplastic anemia (saa), when cyclophosphamide is used as a single agent for conditioning. to reduce the risk of this complication we decided to intensify the preparative regimen by adding reduced dose of treosulfan -an alkylating agent possesing both immuno-and myeloablative properties. between 2003 between -2004 years) were treated in a single institution with allohsct from either hlaidentical sibling (n=3) or an unrelated volunteer (n=3). conditioning regimen consisted of treosulfan 10 g/m²/d on days -7, -6, cyclophosphamide 40 mg/kg/d on d. -5, -4, -3, -2, and anti-thymocyte globulin (thymoglobulin, genzyme) 2 mg/kg/d on d. -3, -2, -1. each bone marrow and peripheral blood was used as a source of stem cells in 3 cases. all patients engrafted with the median time to neutrophil >0.5x10 9 /l and platelet >50x10 9 /l recovery of 16 (14-22) days and 21.5 (12-29) days, respectively. complete donor chimerism was achieved on day +30 in all cases. none of the patients developed grade iii-iv acute gvhd, one patient experienced grade ii acute gvhd. at one year the cumulative incidence of extensive chronic gvhd equaled 33%, overall cgvhd -50%. at the median follow-up of 12.5 (12-25) months all patients remain alive and disease-free with complete donor chimerism. at one year the karnofsky index equaled 100% in 5 patients, 70% -in one case. we conclude that treosulfan + cyclphosphamide + antithymocyte globulin is a well-tolerated preparative regimen and allows stable engraftment in saa patients. the use of treosulfan allows intensification of the conditioning without providing an additional non-hematological toxicity. clinical outcome in adults with severe aplastic anaemia. a retrospective single-centre analysis s. buchholz, e. dammann, c. koenecke, e. mischak-weissinger, m. stadler, m. eder, j. krauter, b. hertenstein, a. ganser hannover medical school (hannover, d) introduction: allogeneic bone marrow transplantation (bmt) is the treatment of choice in young patients suffering from severe aplastic anaemia (saa). due to improved, less toxic conditioning regimens and advances in prophylaxis against graft-versus-host-disease (gvhd) survival has improved steadily. nonetheless, long-term side effects, such as chronic gvhd, occur in up to 30% of patients requiring treatment and leading to an increased mortality. here we present the analysis of for patients with saa comparing. patients and methods: between 1987 and 2005, twenty one patients (13 male, 8 female) with saa and one patient (female) with paroxysmal nocturnal haemoglobinuria (pnh) were transplanted in our centre. the median age at transplantation was 24.5 years (range 17 -46). fifteen patients were transplanted with stem cells from their hlaidentical related donor, 5 patients from hla-identical matched unrelated donors and 2 patients were transplanted from a syngeneic donor. in 19 cases stem cell source was bone marrow (bm), 1 patient received bm and peripheral stem cells (pbsc) and 2 were transplanted with pbsc. conditioning regimen consisted of cyclophosphamid (cy) alone (n=14), or in combination with either total nodal irradiation (n=1), or with fludarabin (flu, n=3). one patient was treated with cy and total body irradiation (tbi) while1 patient received flu, cy and tbi. gvhd-prophylaxis was cyclosporin (csa) and methotrexate (mtx) in all but the 2 patients transplanted from syngeneic donors. all patients engrafted. acute gvhd developed in 9 patients (41%) and was readily controlled by immunosuppression. chronic gvhd occurred in 9 patients (36%; 6 limited, 2 extensive) within a median follow-up of 3 years (range 0. 1-15.5 years) . twenty one out of 22 patients are alive and free of haematological disease, one patient died because of toxoplasmosis before day +50. conclusion: the incidence and severity of acute and chronic gvhd is similar other studies. our data suggest that bmt is a favourable therapy for young patients with aplastic anaemia, showing good engraftment, controllable complications and a good clinical outcome. stem cell transplantation and immunosupressive treatment of severe aplastic anaemia: single-centre experience l. tukic, d. stamatovic, o. tarabar, v. glavicic, m. elez, l. simic, s. marjanovic, m. malesevic military medical academy (belgrade, cs) background: stem cell transplantation (sct) from an hlaidentical fully mached sibling donor (msd) is the best treatment option for severe aplastic anaemia (saa). patients without suitable msd should be treated with immunosuppressive therapy (ist). patients and methods: between 10/1995 and 11/2005 26 patients with newly diagnosed saa were treated either with sct from msd (11 patients) or with ist (15 patients). there were performed 12 allogeneic sct in 11 patients. all donors were hla-identical sibling (1 donor was identical twin). source of stem cells was bone marrow in 9 (one with second transplant) and peripheral stem cell in 3 scts. conditioning regimens were based on cyclophosphamide (cy) with atg in 9 and fludarabine with cy and atg in 3 scts. 13 patients received combined ist with antithymocyte or antilymphocyte globuline (atg/alg), cyclosporine a and steroids and 2 patients atg with steroids. the median interval from diagnosis to ish was 30 days (range 23 to 510). results: engraftment was documented in 10 patients with allogeneic sct (1 patients died without engraftment). one patient developed acute gvhd grade 3-4 and died after 48 days, and the other had pneumonitis interstitialis (cmv+) and died after 60 days. till november 2005 7 of 10 patients (70 %) are alive with sustained engraftment. median survival from sct is 44 months (range 6 to 104). concidering ist, 13 of 15 patients (86.7%) achieved response (4 had two or tree cycles of ist). one patient relapsed 1 year after ist. two patients from ist group died, major causes of death were infection and hemorrhage. overall survival in the ist group is 73.3% (11/15 patients) after a median follow up of 38.7 months (range 6 to 121). conclusion: our results confirm significant improvement in outcome of saa patients during last decades in due to modern induction front line therapy including allogeneic autoimmune diseases r1167 successful treatment of autoimmune thrombocytopenic purpura after bone marrow transplantation with anti-cd20 antibody: a case report b. giannini, c. bosi, m. stanzani, g. bandini, f. bonifazi seragnoli (bologna, i) we describe a case of persistent, severe autoimmune thrombocytopenia, refractory to prednisolone but responsive to chimeric monoclonal antibody anti-cd20 ( rituximab). a patient transplantated for hodgking disease (upn 576), developed autoimmune thrombocytopenia with severe bleeding, 150 days after unrelated bone marrow transplantation: at the same time there were no signs of graftversus-host-disease, cytomegalovirus infection, sepsis or microangiopathic process. high titer of antibody against platelet antigens was found. during treatment with prednisolone 1 mg/kg, for two days, the platelet count remain below 5000/µl. in spite of increasing the dose of cyclosporine and methilprednisolone (2 mg/kg/daily) with addition of intravenous immunoglobulin for five days, only a transitory partial response ( platelet 40000/µl) was observed. after four days from last dose of immunoglobulin, the platelet count fell again below 5000/µl; antiplatelet antibodies still highly positive. after two weeks of therapy with steroid, we started with anti-cd20 antibody with first dose of 375 mg/m² followed by dose of 100 mg/m² once weekly for 3 weeks. complete response was achieved after 6 weeks from therapy initation, with a complete normalitation of platelet count and with disappearance of antiplatelet antibody in peripheral blood. no apparent toxicity, or side effects that could be attributed to rituximab were observed. the patient is in complete response 5 mounths after therapy with anti-cd20. rituximab induced complete response in approximately 50% of the patient with immune thrombocytopenic purpura refractory to prednisone or splenectomy; it's also effective in patients with secondary itp. to our knowlege, only few cases of itp after bone marrow transplantation have been successful treated with rituximab. we suggest for an early use of anti-cd20 in similar cases. treatment of a malignant form of multiple sclerosis with immune ablation and autologous stem cell transplantation v. kimiskidis, i. sakellari, c. smias, k. kapinas, a. anagnostopoulos, a. kazis, a. fassas g.papanicolaou (thessaloniki, gr) malignant forms of multiple sclerosis (ms) are rare cases characterized by very aggressive demyelination and rapid progress to disability leading to death within 5 years from onset despite treatment, which fails to control the disease. we report a case of a young male patient of 19 years old with aggressive ms who was treated with a high-dose immunosuppressive regimen (hdis) using myeloablation followed by autologous blood stem cell transplantation (asct) that has induced a dramatic and long-lasting remission of the disease. the patient was diagnosed with ms in june 2000. at that time he had minor disability, his edss score being was 3.5, and active disease on mri showing 10 gadoliniumenhacing (gd+) lesions. he was put on steroids and interferon-beta which, however, had no effect, while disability was rapidly accumulating. by february 2001, i.e. within 8 months, the edss score rose to 6.5 and the patient was unable to walk unaided for more than 20 metres. on mri, gd+ lesions increased to 18, as did their volume. in march 2001 it was decided to treat him with hdis and, in order to mobilize blood stem cells, he received cyclophosphamide (cy) 4 g/m² plus gcsf 10 ug/kg. six days after cy infusion he had a disease flare with worsening of edss score to 7.5, and further increase in the number and volume of gd+ lesions. the patient was treated with steroid pulses for 3 days and showed some improvement which allowed the continuation of g-csf and subsequent stem cell collection. two months after cy infusion, he underwent asct with busulfan 16 mg/kg over 4 days plus antithymocyte globulin 7.5 mg/kg for conditioning. one month post asct the edss score dropped to 3.5 and no gd+ lesions were detected. the patient continued to improve over the following years. the last assessment at 52 months after asct showed nearly absent disability (edss score: 1.5) and, again, no gd+ lesions on mri. the spectacular responses of this case and also of the three similar cases reported in the literature support the role of asct in rapidly evolving, so-called malignant, forms of ms. an obvious amelioration in a case of scleromyxedema after successful double autologous peripheral blood stem cell transplantation followed by thalidomide and bortezomib consolidation s. ataergin, f. arpaci, a. ozet, m. demiriz, s. komurcu, b. ozturk, o. kuzhan gata (gulhane) objective: scleromyxedema is characterized by cutaneous deposition of mucin, dermal fibrosis and monoclonal gammapathy. the response to treatment has been variable after several treatment modalities including high-dose treatment (hdt) and autologous stem cell transplantation (asct). methods: we report a case of scleromyxedema who achieved amelioration after double hdt and asct, followed by thalidomide and bortezomib consolidation. a 38-year-old male patient was admitted with persistent pruritis. he was treated with anxiolytic and antidepressant drugs with diagnosis of neurodermatitis; however, the skin was thickened and papular lesions appeared. a skin biopsy revealed scleromyxedema. he was treated with topical corticosteroids and retinoic acid preparations. however, no amelioration were noted. topical cyclophosphamide, systemic corticosteroids were used for three months, then the patient has discontinued all the medications. low-dose interferon-a treatment (1.5 mu) was initiated for three times weekly; however, the treatment was stopped due to an acute and severe rhabdomyolisis after the third administration. on his admittance in our department, he had papular lesions of 0.3 mm without pain, erythema or desquamation all over his body. he had also some nodular formations of 1.3 x 1.5 cm, on his face and neck. the whole blood count and biochemical analysis were normal. erythrocyte sedimentation rate, crp were within normal limits and all viral markers were negative for hepatitis, ebv, cmv, toxoplasma, hiv as well as rheumatologic markers. serum immunoglobulin (ig) levels were within normal ranges except for ig g and ig light-chain lambda. the peripheral blood smear and bone marrow aspiration and biopsy revealed no pathology. he then underwent an asct after conditioning with melphalan (200 mg/m²) and a second transplant was done four months later using the same conditioning regimen (140 mg/m²). results: after the transplantation, the immunoglobulin levels have partially regressed. physical appearance has been ameliorated and the skin biopsy revealed a regression in mucin deposition in dermis. consolidation treatment was initiated with thalidomide followed by bortezomib. he is still on his follow-up without any progression for three years. conclusion: hdt and asct may be an alternative treatment in the amelioration of lesions related to scleromyxedema. (1) the depletion of autoreactive lymphocyte populations in the graft is mandatory for the success of autologous transplantation in patients with severe autoimmune disease. clinical grade cd34+ selection can be used to obtain lymphodepletion in autologous leukapheresis products. we report on the implementation of a gmp production process of autologous purified cd34+ cells from mobilized peripheral blood of patients affected by systemic sclerosis (ss) in our institution. for cd34+ cell mobilization, 4 patients (1 male, 3 females, aged 60 years ±16) with ss refractory to standard immunosuppressive therapy, received cyclophosphamide (cy) 4 g/mq and g-csf (filgastrim, 10 ug/kg/day s.c.) starting 5 days after the last cy administration until stem cell collection. leukapheresis was performed when wbc and cd34+ cell count were at least 2x10 9 /l and 20/ul respectively, using a fresenius hemocare as104 (c4y) instrument. the automated selection (clinimacs, miltenyi) was performed in 24 hours of leukapheresis collection. the selection procedure and the preparation of the final product for the cryopreservation were performed in our class b -iso 6 facility. the cells, resuspended in normal saline solution and human serum albumin, were cryopreserved in 10% final dmso. for quality control of the cryopreserved product, the cells from a satellite segment of the cryopreserved bag were thawed and their viability evaluated by trypan blue exclusion. the mean (±sd) wbc, cd34+ and cd3+ cell content of the pre-selection products were 22,075x10 6 (±12,926), 524x10e 6 (±357) and 1,522x10 6 (±1,331) respectively. after immunoselection we obtained a mean (±sd) cd34+ and cd3+ cell number of 312x10 6 (±169) and 0.7x10 6 (±0.8), with a purity of 87% (±12). the mean percentage of viable cells (propidium iodide) in the post-selection samples was 92 (±6). the sterilty tests for bacteria and fungi on the purified samples were negative. the viability of the post-thawed samples was 89% (±1). the conditioning regimen consisted of cy 50 mg/kg/day for 4 days i.v. and rabbit anti-thymocyte globulin 2.5 mg/kg/day for 3 days. neutrophil engraftment was reached at day +14 (±2), and all patients are alive, with stable or improved clinical conditions after 9 months (±4) after transplantation. these results demonstrate that gmp production of cd34+ cells for autologous transplantation is feasible, safe and could efficiently support a transplantation program for patients affected by ss. high-dose immune immunoablative therapy and autologous stem cell transplantation in severe resistant crohn's disease: profound response for 18 months followed by treatable relapse y. sorour, k. robinson, p. hurlstone, k. el-ghariani, a. lobo, j.a. snowden sheffield teaching hospitals (sheffield, uk) we report the case of a 25 year old female with severe resistant crohn's disease (cd) treated with high-dose immune immunoablative therapy (hdit) and autologous stem cell transplantation (sct). diagnosed with cd at age 11, initial disease control had been achieved with azathioprine and steroids, but, at age 14, colectomy and ileostomy were performed for a severe flare. from age 22, increased disease activity was unsuccessfully controlled with azathioprine, steroids, infliximab, methotrexate, combination rifabutin/metronidazole/clarithromycin, thalidomide and tacrolimus. from nov 2000-jan 2003, 5 surgical episodes had resulted in resection of 1.25m small bowel. dietary modifications had included an elemental diet, but from 2002 the patient was dependent on home total parenteral nutrition. severity of symptoms had resulted in recurrent and prolonged inability to work and to warrant care under a palliative medicine specialist. based on poor quality of life, risk of life threatening complications, and inability to control the disease effectively, the option of autologous transplant was pursued after examination of the case and proposed treatment protocol by the local research ethics committee, review by two independent gastroenterologists and one transplant haematologist, and obtaining informed written consent. treatment commenced in sept 2003 with mobilisation using cyclophosphamide (cy) 2g/m² and g-csf. in nov 2003 the patient was treated with cy 200mg/kg, rabbit atg 6mg/kg and methylprednisolone 1gx5 followed by 3.0 x10 6 /kg isolex enriched cd34+ cells. treatment was complicated by neutropenic sepsis, oropharangeal and stomal mucositis. engraftment time was within normal limits. discharge was on day+16. pre-and 12 months post-treatment data are summarised in the table. cd remained inactive until march 2005 with the development of increased stoma output and abdominal pain. relapse was confirmed by ileal biopsy. in contrast to pre-sct, disease control has been achieved with immunosuppressants and surgery, permitting ileal reanastomosis, pouch formation and reversal of ileostomy in sept 2005. the patient remains stable as of nov 2005. in conclusion, hdit and autologous sct may be an effective therapy for medium term control of severe, treatment resistant cd. it remains to be seen whether post-sct relapse is easier to control than cd activity before sct, as suggested by data in other autoimmune diseases. in addition to randomised trials, future studies could look at means of prolonging responses, such as maintenance treatments. hyperkalemia is one of the side effects of cyclosporin (csa). the mechanism of hyperkalemia is unclear. apparently many factors in pathogenesis of hyperkalemia may be involved. nephrotoxicity of csa that significantly impairs renal perfusion, glomerular filtration reduced k+ excretion and secondary hypoaldosteronism seemed to be the reasons for csa-associated hyperkalemia. there are publications about hyperkalemia in patients after renal transplantation but only few reports devote this phenomen in bone marrow transplantation patients. we report about 2 cases of hyperkalemia with bradyrhythmia during csa administration in patients with cml in 1 cp, male 27 and 46 years old undergoing bmt from hla -identical siblings. hyperkalemia (6,2 -6,5 mmol/l) and bradyrhythmia (46-50 /min) in 42-68 days after transplantation was observed. at that time a serious worsening in renal function (increase serum creatinine and serum urea, decrease filtration rate) and in serum csa level rise was found in both. after discontinuation of csa treatment all these symptoms disappeared. these observations suggest that csa may be a cause of hyperkalemia associated with bradyrhythmia in bone marrow transplantation patients. careful monitoring of csa level in blood and renal function may be important in prevention these complications. objectives: allogeneic haematopoietic stem cell transplantation (allo-hsct) is the only curative therapy for patients with chronic lymphocytic leukaemia (cll). however, transplant-related mortality remains relatively high and relapse is still a major problem. there are only few anecdotic reports of the use of thalidomide, an immunomodulating agent, in such patients. case report: 48-year old patient with resistant cll is presented. he was treated unsuccessfully with several cycles of different agent combinations, started with chlorambucilmethylprednisolone then fludarabine-cyclophosphamide and also with monoclonal antibody rituximab. as he has hla matched sister we proposed allo-hsct. on admission before transplantation patient was presented with massive lymphadenopathy, anemia (hb 76 g/l), wbc 24.8 x 109/l with absolute lymphocytosis (lymphocytes 98%) and thrombocytopenia 23 x 109/l. pre-transplant conditioning consisted of high-dose cyclophosphamide and total body irradiation. combination of monoclonal antibody alemtuzumab (campath-1h), cyclosporine a and short methotrexate were given for the prevention of acute graft versus host disease (gvhd). the patient received 2.4 x 106/kg stem cells. there were no serious complications in post transplant period. lympadenopathy completely disappeared. patient was discharged 4 weeks after transplantation, without gvhd and with normal complete blood counts (cbc). on bone marrow examination there was still residual leukaemic infiltration (70%). two months later acute gvhd developed, with skin involvement only. at that time lymph nodes massively enlarged again and high wbc with absolute lymphocytosis reappeared. cyclosporine immunosuppressive therapy was stopped and thalidomyde 100 mg/day was introduced combined with methylprednisolone 8 mg three times per week. during period of 4 months treatment he was readmitted once for lung aspergillosis which responded well to voriconazole. improvement appeared slowly, with regression of lymphadenoapthy. after 4 months he still has residual leukaemic marrow infiltration -about 30%, but normal cbc without absolute lymphocytosis. conclusion: our patient is new evidence that thalidomide may have significant antileukaemic effect in refractory cll. due to this and anti gvhd effect perhaps in the future it could be incorporated as a first line gvhd prophylaxis regimen in patients transplanted for cll. intensive combination therapy and autologous pbpct for blast crisis cml revisited d. heim (1) imatinib is the most active therapy for chronic myeloid leukaemia (cml) in all phases of the disease. overall survival of patients with blastic phase cml treated with imatinib monotherapy however is 6 months only. the reason for imatinib resistance in advanced phase cml is mostly due to bcr-abl independent mechanisms. therefore a combination therapy of imatinib with conventional high dose chemotherapy is often used for remission induction and allogeneic stem cell transplantation is performed if a suitable donor is available. autologous stem cell transplantation has drawn new attention in the treatment of cml since sufficient numbers of peripheral stem cells (pbpc) can be mobilized under concomitant treatment with imatinib and collection of bcr-abl negative autologous peripheral stem cell transplants has been reported. we tested in a pilot trial of 3 patients the feasibility of a treatment consisting of imatinib 600mg qd combined with 4 cycles of cytarabine 1000mg/m² x 7d in patients with cml in blast crisis (bc) who do not have a hla-matched stem cell donor. pbpc were mobilized after the 4th cycles of cytarabine with g-csf (filgrastim) 10ug per day. pbpc were cryopreserved and reinfused after a conditioning therapy with either cyclophosphamide 200mg/kg and busulfan 16mg/kg (bucy)(table 1: patient 2 + 3) or cyclophosphamide 120mg/kg plus 12 gy tbi (cy/tbi)(table 1: patient 1) the stem cell products of all 3 patients contained sufficient numbers of cd34+ cells after mobilization with g-csf after the 4th cycle of high dose cytarabine and imatinib given through. the autologous pbpc graft of one of the patients was bcr-abl negative, a second graft had detectable bcr-abl at low level in the q-rt-pcr (table1). no data on the presence of bcr-abl is available for the third graft. engraftments of the transplants were in the expected range. no excessive toxicity was recorded. treatment of cml in bc with imatinib combined with high dose cytarabine and autologous pbpct after high dose chemo-/radiotherapy is feasible and may result in sustained complete molecular remission. alemtuzumab and autologous sct in cll, experience in a small centre e. zappone, e. ortu la barbera, u. coppetelli, c. ciabatta, f. ciccone, s. nardelli, a. centra, g. potente, a. de blasio ospedale s.m. goretti (latina, i) alemtuzumab (al) as a single agent or in combination with chemotherapy is an effective treatment for chronic lymphocytic leukemia (cll) in refractory or relapsing patients (pts) and has been shown to induce complete molecular responses. autologous stem cell transplantation (sct) induce prolonged and durable remission in many hematological malignancies but its role in the treatment of cll is controversial. we included al in the treatment of refractory, relapsed or high risk cll pts prior to stem cell collection and high dose chemotherapy consolidation of the response obtained. we treated 7 pts with binet stage iii cll in partial remission (pr) or stable disease (sd) after 1 to 3 lines of chemotherapy, including fludarabine and 1 pt with advanced b-lymphocytic lymphoma in pr. chemotherapy debulking prior al was necessary in pts with high disease burden and large nodal involvement. disease status before al treatment was complete remission (cr) 2, pr 4, and sd 2. al was given subcutaneously, treatment duration was 4-12 weeks and total dose was 180-1000 mg. results after al were cr 5 and pr 3, all 8 pts had below normal lymphocytic counts, and lymphoid marrow infiltration below 20%; 5 out of 7 cll pts had cd5/cd19 expression in the marrow below 1%. after al 5 out of 8 pts were mobilized: 4 with cyclophosphamide 5-7 gr/mq and 1 with additional chemotherapy followed by aracytin; mobilization is planned in the last pt. stem cell collection was adequate: 4-40 cd34x10 6 /kg, in 1-4 apheresis procedures. two pts were not mobilized after being treated for cytomegalovirus (cmv) infection at the end of al treatment, they maintain cr without further treatment 11 and 18 months after al. four pts undergo sct: engraftment and clinical course were normal, 1 pt progressed before transplant. of the 4 transplanted pts 3 are in ccr at 24, 12 and 6 months post sct, 1 pt progressed 13 months post sct and was retreated with al with minor response. cmv reactivation occurred in 6 out these 8 pts and antiviral therapy was necessary in 4. al was effective in inducing significant clinical response in these high risk pts, stem cell collection was feasible and autologous sct could be performed without significant early or late complications. cmv reactivation occurs in the majority of al treated pts and must be carefully monitored. the results in this very small group of patients are encouraging but we can not draw any conclusion about the general application of this program in cll. at the time of imatinib era in cml treatment hsct became a disputable issue. in this study we compared the outcome of unrelated donor transplantation to that in sibling donor setting in cml patients receiving reduced toxicity conditioning. 19 patients received hsct from matched sibling donors, 30 patients from unrelated donors. reduced toxicity consisted of: : busulfan 8mg/kg, fludarabine 120mg/m² (allo sib) or 150mg/m² (mud) and atg. donors for unrelated transplant were matched for 10 specificities at following resolution levels : loci a, b, c at intermediate or high resolution and dr at high resolution level only. patients were stratified into 3 groups : i) allo-sib ( hla matched ; n=19) , ii) mud fully matched ( 10/10 match at intermediate or high resolution level for hla class i and high resolution for class ii ; n=11) , iii) mud mismatched ( at least 1 allele mismatched ; n=19 ). cumulative proportion survival was: i) 78% at the end of 4 y follow up period in sib hsct , ii) 65% in patients transplanted from mud donors fully matched in 10 specificities and iii) 35% in patients transplanted from donors mismatched in at least one allele. (figure) our results document, that optimal matching in five loci benefit the outcome of transplantation, which in unrelated donor transplantation can be similar to that obtained with sibling donors providing 10/10 matched donor at high resolution level. refractory graft-versus-host disease after stem cell transplantation in thalassaemia: pentostatin is safe and effective treatment g. leopardi, g. visani, c. giardini, g. sparaventi, f. d'adamo, g. nicolini, b. guiducci, s. barulli, m. lucesole, l. malerba, a. isidori san salvatore hospital (pesaro, i) toxicity, graft rejection with return of the thalassemia and graft-versus-host disease (gvhd) are the main causes of failure after stem cell transplantation (sct) in thalassemic patients, particularly in those at poor prognosis (i.e. class 2 and 3). steroid refractory gvhd is associated with a not negligible non-relapse mortality. few data are actually avalaible on the use of pentostatin for refractory gvhd in thalassemic transplanted patients. we analyzed four children, 2 females and 2 males, aged 8-13 (median 9.5 years), transplanted for beta-thalassemia major (1 class 1, 1 class 2, 2 class 3). two patients (class 1 and class 2) received unrelated sct (1 bone marrow, 1 pbsc). the two other children (both class 3) were transplanted from hla identical sibling after previous transplantation procedures, having rejected twice and once, respectively. three patients (2 unrelated and 1 hla identical sct) developed refractory acute gvhd grade iii-iv with multiple organ involvement at +29, +35 and +58 days, respectively. one patient had chronic extensive gvhd. in the patient receiving the third allogeneic hla identical sct both acute and chronic refractory gvhd occurred. patients affected by acute severe gvhd failed to respond to primary treatment with cyclosporine a and methylprednisolone at doses varying from 2 to 5 mg/kg/day and thus received salvage therapy with pentostatin 1.5 mg/sqm for 3 consecutive days. one presenting with acute multiorgan gvhd (skin, liver and gastrointestinal tract) had short term response. two patients responded: one developed a secundary, chronic extensive, gvhd. this child as well as the other with chronic severe extensive gvhd (involving skin, liver, eyes and oral mucosa in one case and skin and mouth in the other) starting at +256 and +526 days from transplant were treated with pentostatin (4 mg/sqm i.v. every 2 weeks) for 3 and 4 months. they are alive with significant improvement of skin and mouth symptoms and tapered concurrent immunosuppressive treatment. no toxicity or impairing chimerism due to pentostatin were observed. pentostatin thus appears as safe and effective treatment for acute and chronic severe gvhd after sct for thalassemia. supported in part by ail pesaro onlus pure red cell aplasia after mud stem cell transplant in thalassaemia major: successful treatment with rituximab m. lucesole, g. visani, c. giardini, b. guiducci, g. sparaventi, g. nicolini, f. d'adamo, g. leopardi, s. barulli, l. malerba, a. isidori hematology and transplant center (pesaro, i) pure red cell aplasia (prca) is a not infrequently observed complication of allogeneic sct performed across the abo complex and often refractory to standard strategies. the peculiar recovery of erythropoiesis after sct in thalassemia major could be a possible factor confounding for a correct diagnosis, as well as for the therapeutic choice. a young male patient was transplanted from abo incompatible (donor b rh+; recipient 0 rh+) hla-identical unrelated donor for beta thalassemia major. after standard myeloablative conditioning regimen (bu-cy-thio), he received cyclosporine a and mtx as gvhd prevention prophylaxis. the post-transplantation course was characterized by an incomplete haematological recovery. a poor graft function was observed at day +200. the bone marrow was hypoplastic with the apparent absence of erythroid precursors, and a possible rejection of the graft was suspected; nevertheless, fish analysis of chromosome y for the engraftment showed 94% of donor cells, with a normal count of the beta chains (100%); no evidence of haemolysis was recorded. all dna virus were negative. the diagnosis was prca. epo 10.000u/every other day was started by day +201; additionally we submitted the patient to plasmapheresis (3 procedures, last at day +210) without hematologic response (3-5 units rbc/weeks, 1-2 platelets for week). at day +215 a first dose of rituximab (rtx) (375 mg/sm) was administered; we observed a progressive increase in reticulocytes and platelet counts (respectively 106000/mm³ and 52000/mm³) 19 days after the infusion of rtx. insorgence of cystitis and pielonephritis (p.aeruginosa) delayed the administration of a following dose of rtx with a progressive, increasing transfusion dependency. after the resolution of the infective complications, a second dose of rtx was administered on day 272, and a third dose on day +299. two weeks after the last dose of rtx a response was observed: reticulocytes count increased, and the level of hgb slowly normalized .the patient is now in complete remission 18 months after transplantation, and with a complete hematologic take. rituximab could thus be a promising agent for treatment of not infrequent cases of prca, in transplanted thalassemia patients, refractory to standard therapies. a successful t-lymphocyte engraftment achieved by megadose cd34+selected peripheral blood stem cell s344 transplantation in a t-b-nk+ scid case without using conditioning regimen a. ikinciogullari (1), c. aytekin (1), f. dogu (1) , m. yuksek (1), a. yildiran (1) severe combined imunodeficiency (scid) is a genetic disorder characterized by profoundly defective or absent t and b cells functions. allogenic stem cell transplantation (sct) is to date the only curative therapeutic option for scid. here we report a t-b-nk+scid patient who was transplanted by megadose peripheral blood stem cells (pbsc) collected from his father without conditioning regimen. case: a 6 months old boy referred to us with the diagnosis of t-b-nk+scid. his physical examination showed disseminated hyperceratotic papular skin lesions. immunohistochemical investigation of the skin biopsy revealed hsv 2 infection. he was treated with acyclovir and foscarnet combination, but skin lesions didn't resolve. thus he received a megadose cd34+ selected (clinimacs, miltenyi biotec) pbsc (62x10 6 cd34+cells/kg) transplantation from his father without conditioning regimen. he received cyclosporine for gvhd. he engrafted at posttransplant day 34. detection of chimerism performed by str (short tandem repeat) pcr analysis and whole blood samples showed mixed chimerism with 100% donor t cells. acute gvhd grade i developd at day 56 rapidly resolved with systemic corticosteroid treatment. his chronic hyperceratotic papular herpetic lesions completely recovered at second month after sct. he is doing well with successful immunoreconstitution five months after sct. to our knowledge he is the first successfully engrafted scid case with t-b-nk+ phenotype following uncoditioned haploidentical pbsc transplantation. myeloid-related proteins 8 (mrp8) and 14 (mrp14), both s100 proteins, are the major calcium-binding proteins expressed in phagocytes during specific stages of differentiation. they form stable complexes and are present in circulating neutrophils and monocytes, representing the first cells invading inflammatory lesions. the protein complex is found in inflammatory fluids in distinct inflammatory conditions, including rheumatoid arthritis, allograft rejection, inflammatory bowel disease, and lung disease. prerequisite for its secretion is the contact of phagocytes with extra-cellular matrix proteins or inflamed endothelium, resulting in elevated intracellular calcium levels and activated protein kinase c. mrp8/mrp14 is thereby released specifically at inflammatory sites and leads to increased serum levels in correlation with the degree of inflammation, indicating an extra-cellular role of these molecules in inflammatory processes. we report a 4year-old girl with: a) severe anemia, b) neutropenia, c) inflammation and d) severe growth failure. bone marrow examination showed moderate dyserythropoiesis. we did not detect hemolysis, iron deficiency, hemoglobinopathies, immunological diseases or any autoantibody. serum levels of copper and ceruloplasmin were within normal range, although serum zn concentration was markedly increased (310 µg/dl). urinary zn excretion and erythrocyte zn concentrations were within normal range. family studies demonstrated normal zn and cu plasma levels. patient's plasma calprotectin concentration showed a 6000-fold increase (2900mg/l) compared to normal values. calprotectin concentration is known to be elevated in many inflammatory conditions but is generally below 10mg/l and thus far below the levels reported in this patient. we describe this case as an inborn error of zinc metabolism caused by dysregulation of calprotectin metabolism, which mainly presented with the features of chronic microcytic anemia and inflammation. we suggest that bone marrow transplantation could be the best clinical intervention for this new disease. sequential autologous peripheral blood stem cell transplantation with beam conditioning as salvage treatment for refractory high-grade lymphoma e. van hul, a. gadisseur, e. steel, w. schroyens, a. van de velde, z.n. berneman antwerp university hospital (edegem-antwerp, b) t(8;14) mature b-cell (burkitt's) lymphoma/leukaemia (bl) is classified as one entity in the world health organisation (who) classification. bl is a poor-risk, aggressive non-hodgkin lymphoma. despite significant improvements in the treatment of bl, outcomes of adults are generally inferior to those of children. further intensification of the chemotherapy regimens and the inclusion of up-front, high-dose therapy and autologous peripheral blood stem cell transplantation (asct) has significantly improved the duration of response and survival. strategies to improve survival in these poor-risk patients also include sequential asct, and asct followed by non-myeloablative allogeneic transplantation. we present a 54-year old male who was diagnosed with bl with a double translocation t(8,14) and t(14,18). he was in remission after cycle 1 of the hovon 37 study protocol (prednisone, vincristin, daunorubicin, asparaginase) but relapsed after the second cycle (ara-c, mitoxantrone). he was then treated according to the hoelzer protocol but after initial good response proved progressive after the second block, with increasing abdominal mass, acute renal failure and metabolic encephalopathy. salvage therapy was initiated with an autologous peripheral blood stem cell transplantation (pbsct) with beam (carmustine, cytarabine, etoposide, melphalan) conditioning resulting in a very good partial remission, which was then consolidated at day + 29 by a second autologous pbsct after beam conditioning. he was planned for an haploidentical allogeneic pbsct according to the perugia protocol on day +51 after the second pbsct but died unexpectedly of complications of an acute gastrointestinal bleeding before the conditioning (tbi, thiotepa, fludarabin, atg) could be started. we present what appears to be the first reported case of tandem asct with beam conditioning in an adult patient with burkitt's lymphoma/leukaemia . this intensive therapy proved feasible and relatively well tolerated, certainly in view of the bad condition of the patient at the start of the first conditioning. the use of palifermin (keratinocyte growth factor) in the future could further increase the tolerability of this regimen. beam has been proved to be a highly effective treatment in lymphoma but provokes a severe mucositis. nevertheless, tandem beam and autologous pbsct should be further developed as a salvage regimen in the treatment of patients with poor-risk aggressive lymphomas. an association between anbioimmunoblastic t-cell lymphoma and hcv infection: therapeutic difficulties g. mihailov, p. ganeva, n. vasileva national center of haematology and transplantation (sofia, bg) angioimmunoblastic t cell lymphoma (ail) is a rare lymphoproliferative disorder characterized by systemic lymphadenopathy, hepatosplenomegaly, fever, liability to s345 infections, skin eruption, polyclonal hypergammaglobulinaemia, hemolytic anaemia. clinicaly the disease runs a fatal course in the majority of patients even after multiagent chemotherapy, interpheron á, cyclosporine a, corticosteroids, danazol and recently purine analogues. fewer than 20% of patients survive 5 years after diagnosis. highdose chemotherapy (hdct) followed by autologous bone marrow transplantation represents a promising new treatment modality for patients with this type of lymphoma. we present a case of 48 year-old woman with association of ail and hcv+ infection with high replication of virus and interesting clinical course of her disease (cns infiltration and complication with bacterial meningitis). it is widely thought, but not yet explained that there might be a pathogenetic link between the infection of hepatitis c virus and onset of b non hodgkin's lymphoma (nhl). in our case we have association with t cell lymphoma. we discuss our treatment difficulties ( 4 courses imvp-16, 4 course for brain type lymphoma), following by fludarabine. there is an evidence that ail is susceptible to hdt and autologous stem cell transplantationshuold be considered to the patient. recent data suggest that high dose chemotherapy (hdct) and autologous stem cell transplantation (asct) may be of benefit in patients with aids-related lymphoma (arl). we report on a patient with refractory arl successfully salvaged by hdct and asct. in august 2004 a 43-year-old man presented with a rightlower quadrant abdominal mass. he was known to be hivpositive since 1984 (cdc category c3). highly active antiretroviral therapy (haart) was initiated in 1997. his latest cd4 cell counts was 232/µl and his hiv-load <50 copies/ml. abdominal ct-scanning confirmed a lesion of 10 x 7 cm in greatest dimension localized in the right iliac fossa. the spleen was enlarged at 13.8 x 8.4 cm. a biopsy revealed a stage iia diffuse large b-cell lymphoma. from august to december 2004 the patient received 6 courses chop resulting in a partial remission. because a pet-scan indicated residual active lymphoma 2 more courses r-choep (rituximab, cyclophosphamide, doxorubicin, vincristine, etoposide, prednisone) were applied and a complete remission (cr) was achieved. 2 months later he relapsed with an isolated tumor localized in the right iliac fossa. the lymphoma increased in size under 2 courses of bendamustine/rituximab. in july 2005 the patient received 2 courses of cisplatin/cytarabine/dexamethasone (dhap) supported by g-csf. a sufficient number of peripheral blood stem cells were harvested after the 1st cycle (9.16 x 106/kg cd34+ cells). the patient experienced stable disease and received another course ifosfamide/etoposide/epirubicin (iev). however, progressive disease caused an incomplete paresis of his right leg. in september 2005 hdct (beam) was administered followed by asct (9.16 x 106/kg cd34+ cells). haart was continued throughout the transplant period with the hiv viral load remaining negative. the patient developed neutropenic fever, a central venous catheter-related infection (koagulase-negative staphylococci), bacteremia (klebsiella pneumonia) and toxic enteritis who grade 3. broad spectrum antimicrobial therapy was given for 3 weeks. the granulocyte count reached 0.5 x 109/l on day +14 and platelets 30 x 109/l on day 21. a ct-scan performed 6 weeks after asct showed a partial remission. moreover, the paresis of his right leg has disappeared. the patient is doing well and currently undergoes consolidating radiotherapy to the initial tumor bulk. hdct and asct may be of benefit in selected cases of refractory arl. angioimmunoblastic t-cell lymphoma: treatment in two cases a. lópez de la guía, m. martin-salces, a. kerguelén, r. de paz, t. cobo, d. hernandez, j. g-bustos, m. canales, f. hernandez-navarro h.u. la paz (madrid, e) introduction: angioimmunoblastic t-cell-lymphoma (ail) is one of the mature t-cell neoplasm defined in the real-and who-classifications. although patients with angioimmunoblastic t-cell lymphoma (ail) have a poor prognosis with conventional treatment , there are no generally accepted treatment guidelines of proven effectiveness because of low frequency. in that way, it would be necessary to determinate new lineage-treatment to improve unfortunately evolution. methods: between 2002 and 2005 we reported the good development with high-dose chemotherapy (hdct) and autologous haematopoietic stem cell transplantation in two patients with refractory in our centre. results: the age at transplantation was 53 and 55 years-old respectively. treatment prior to bone marrow transplantation in one case was initially prednisone alone and cladribine, cyclofosfamide and prednisone for 3 cycles, and the other one was chemotherapy based of increased dose of schedule epoch; in this case was necessary secondary treatment with ifosfamide and etoposide ife for 2 cycles. cd34+ selected autologous peripheral blood stem cell transplantation was given like third line of treatment in the two cases. the regimen for the mobilization of peripheral blood stem cells (pbsc) included ifosfamide, etoposide and g-csf. in one case the median yield of pbsc was 7,55 x10 6 cd34+cells/kg and 1.2 x10 5 cd3+cells/kg and the other one was 4,15 x10 6 cd34+cells/kg and 0.8 x10 5 cd3+cells/kg. the conditioning treatment consisted of beam regimen. there was none treatment-related death. post-taspe complications were herpes zoster infection and biclonal gammapathy igg kappa and igg lambda in one case, and no evidence of acute complications in the second patient. the patients remain in complete remission after a following time of 29 and 5 months respectively and there is no evidence of relapsed. conclusions: our cases confirm previous experience that ail is susceptible to high-dose chemotherapy and cd34+ selected autologous peripheral blood stem cell transplantation, but more cases and longer observation time as well as better selection of patients with refractory ail would be necessary to determinate the standard treatment. (2)imperial college (london, uk) introduction: we describe the novel use of beam campath conditioning in an autologous setting. we used alemtuzumab ( campath 1h ) in combination with beam conditioning in two patients who underwent autologous peripheral blood stem cell transplantation for aggressive t cell lymphoma. case a: a 42 year old lady of carribean origin presented with htlv-1 driven atll. she was treated with 6 courses of chop + dacluzimab with good pr. she received dhap salvage therapy with stem cell collection followed by a beam campath 1h autograft. she developed cmv reactivation treated with valganciclovir. her day +90 re-staging showed cr with significant reduction in htlv-1 proviral load from 77 copies to 5.82 copies/100 pbmcs. case b: a 33 year old south american presented with massive splenomegaly & pancytopenia. he was diagnosed with gd hepatosplenic lymphoma and was started on chop 14 chemotherapy regimen with initial response followed by progression after 6 cycles. he received dhap chemotherapy followed by autologous peripheral blood stem cell transplant with beam campath 1h conditioning. post transplant, he developed cmv reactivation managed with valganciclovir. he received splenic irradiation 6 weeks post transplant and at 14 months post transplant, he remains in complete remission. role of alemtuzumab (campath 1h)-campath 1h targets all lymphocytes expressing cd52. this includes gd t cells. htlv-1 predominantly infects cd4+ t cells (90% to 99% of the proviral load). the expression of cd52 on htlv-1 infected cells has not been studied in humans but in nod/scid mice developing htlv-1 positive tumours, the 'leukemic' cells express cd 52 to a high level. conclusion: the use of beam campath 1h conditioning regimen in an autologous setting is a novel approach in selected patients with aggressive t cell lymphomas. it merits further investigation. unrelated bone marrow transplantation in a child with high-risk anaplastic large cell lymphoma: case report g. tezcan, v. hazar, v. uygun, a. kupesiz, a. yesilipek akdeniz university school of medicine (antalya, tr) the use of allogeneic stem cell transplantation in non-hodgkin lymphoma patients is not yet clearly defined, especially in children and adolescents. for patients who are in need of bmt, to have a chance for human leucocyte antigen (hla) identical sibling to serve as an allogeneic donor is only 30%.. for others, transplant from a matched unrelated donor (mud) is an alternative option but with higher risk for gvhd and graft rejection as well as infectious complications and organ toxicity. here, we describe a case of alcl treated with unrelated cord blood transplantation. 5 year old boy admitted with the complaint of mass in right medial thigh region was diagnosed as alcl and chemotherapy was started. at the beginning of second course, because of progression, chemotherapy was changed and daclizumab was added. by the beginning of third chemotherapy course, he was in remission and chemotherapy was continued while an unrelated donor was being searched since hla matched sibling donor was not available. at the end of forth course, one antigen mismatched cord blood was found and following marrow recovery, he was referred to our center for stem cell transplantation while he had been still in remission. the patient was conditioned with total body irradiation ( total 1200 cgy) given in six fractioned doses in association with etopside (40 mg/kg) and cyclophosphamide (90 mg/kg). he was infused with 1.79x107/kg mnc and 0.2x105/kg cd34 + cells. cyclosporine, metotrexate and atg were used for gvhd prophylaxis. engraftment was achieved for neutrophil and thrombocyte on posttransplant day 24 and 39, respectively. on posttransplant day 15, venooclusive disease developed and it was treated with defibrotide. he has not been experienced any sign of gvhd and he has been still in remission by the end of posttransplant three months. he was the first pediatric alcl case to our knowledge who was treated with unrelated cord blood transplantation. the increasing number of volunteer stem cell donors and although in very limited cases successful results published in the literature regarding the unrelated stem cell transplantation in this group of patients, make this therapeutic option acceptable in the treatment of high risk or treatment failure patients although more data and long term follow up in larger series are needed. high-dose chemotherapy and autologous stem cell transplantation for malignant lymphoma: influence of disease status at time of transplant s. genadieva-stavrik, l. cevreska, a. pivkova, z. stojanoski, o. karanfilski, b. georgievski clinical center (skopje, mk) high-dose chemotherapy with autologous stem cell transplantation (asct) could improve the survival of patients (pts) with malignant lymphoma, but the optimal time of asct is still not clear. this therapeutic method is considered as the optimal strategy in high risk patients in remission and relapsed malignant lymphoma patients. however the potential role in chemoresistent disease and primary refractory relapse is still controversial and optimal timing and indication is still to be assessed. methods: we analyzed 25 consecutive adult patients with malignant lymphoma who underwent asct in a period 2000-2005. m.hodgkin (hd) was diagnosed in 12 pts, non-hodgkin lymphoma (nhl) in 13 pts. the graft was non-purging, peripheral stem cell. the median follow-up was 30 months (from 1 to 60 months). in hd group the conditioning regimen was beam in all cases. status at asct was complete remission (cr) in 2 cases, chemo sensitive relapse in 5 cases and primer refractory disease in 5 cases. patients in complete remission are patients with adverse prognostic factors for hodgkin disease. in nhl group the conditioning regimen was beam in 11 cases, and other chemotherapeutic regiment in 2 cases. high grade histology was diagnosed in all patients, diffuse large bcell 9 pts, anaplastic large t-cell 3, lymphoblastic 1 pts. status at asct was complete remission (cr) in 2 cases, chemosensitive relapse in 5 cases, and refractory disease in 6 cases. according to the international prognostic index (ipi) which is validated scoring system predictive of survival in various types of de novo aggressive non-hodgkin lymphoma patients with cr has been evaluated as high risk patients. results: permanent complete remission was achieved in 4 out of 4 pts (75%) with cr at asct in 5 out of 10 pts (50%) with chemosensitive relapse. complete remission could not be achieved in refractory disease. conclusion: the retrospective analysis of our data showed that the asct in patients with malignant lymphoma is an effective salvage therapy. status of the disease at the time of asct is prognostic factors which strongly influenced the outcome of asct. the study suggests that the best results are achieved if asct is performed in complete remission compared with refractory/relapsed disease. patients with malignant lymphoma and initial high risk prognostic factors should be transplanted in first complete remission. autologous stem cell transplantation after radioimmuntherapy with ibritunomab-tiuxetan (zevalin) followed by high-dose cyclophosphamide is feasible in lymphoma patients with end-stage renal disease w. stein, f. gottschalk, o. knigge, u. aurich, m. wernicke, m. kiehl clinic frankfurt/oder (frankfurt/oder, d) we report on a 51 year old male patient suffering from follicular lymphoma grade ii, stage iiia, first diagnosed in 2004. initial therapy consists of 8 cycles r-chop (rituximab, cyclophosphamide, doxorubicine, vincristin, and prednisolone) with partial response. in april 05 patient became symptomatic with abdominal pain and a ct scan demonstrates enlarged lymph nodes paraaortocaval and a left hydronephrosis necessitating ureter splint. after verification of lymphoma relapse two cycles of dexabeam (dexamethasone, bcnu, etoposide, ara-c, melphalan) chemotherapy were initiated followed by stem cell harvest and as lymphoma shows progression we added one cycle of r-dhap (rituximab, dexamethasone, high dose ara-c, cisplatin) again without any s347 response. due to further lymphoma progression patient became haemodialysis dependent as creatinine level increase to 621 µmol/l and urea level to 29.8 mmol/l. furthermore, he suffers from oliguria, ascites and severe edema of the legs, scrotum, and abdomen. as no standard chemotherapy with a clear chance of response was feasible we decided to start a high dose chemotherapy followed by autologuous stem cell transplantation. conditioning regiment consist of radioimmunotherapy with ibritunomab-tiutexan (1,2 gbq) 22 days prior to high dose cyclophosphamide (60 mg/kgbw/day) on days -3 and -2. on day 0 1,99 x 106 cd 34+ cells per kg bw were given. platelet engraftment was observed on day +15 (20.000/µl) and day +31 (50.000/µl), respectively, as neutrophil exceed 500/µl on day + 10. patient did not show any problems associated with either radio-chemotherapy or neutropenia. as edema and ascites disappear during neutropenic phase kidney function resolve 3 weeks post transplant. mrt scan shows a very good partial response with residual lymph nodes paraaortocaval. from this case we conclude that in refractory follicular lymphoma radioimmunochemotherapy followed by high dose cyclophosphamide as conditioning regiment even in the case of renal failure necessitating haemodialysis is feasible and highly effective. twenty patients (male: female=13:7, ages ranging 15-62 with a median of 38) received high dose chemotherapy followed by peripheral blood stem cell transplant (pbcst) at inha university hospital. their diseases consisted of diffuse large b cell lymphoma (dlbcl, n=7), peripheral t cell lymphoma (ptcl, n=5), lymphoblastic lymphoma (lbl, n=2), anaplastic large cell lymphoma (n=2), burkitt lymphoma (n=1), nasal type extranodal nk/t cell lymphoma (n=1), mycosis fungoides (mf, n=1), and nodal marginal zone lymphoma (mzl, n=1). most of them were in stage via (n=9) or vib (n=5), and others were in stage ie (n=1), bulky iia/bulky iiae (n=4), or iiia (n=1). three patients had disease extended to cns at the very beginning of the treatment. chemotherapy including chop, r-chop, promace/cytabom, copblam-v, dhap, high dose methotrexate, or regimens for acute lymphoblastic leukemia (patients were exposed to a median of 9 courses of chemotherapy ranging 6-20) yielded cr1 (n=11), pr1 (n=1), cr2 (n=5), cr3 (n=2), and pd (n=3) right before pbsct. a total of 24 (3 allogeneic and 21 autologous) pbsct were performed for 20 patients. allogeneic pbsct was carried out in case of disseminated mf, recurrent ptcl after autologous pbsct, and recurrent ptcl in cr3 after tandem autologous pbsct. a patient with lbl received double autologous pbsct. the conditioning regimen was cbv (cyclophosphamide,bcnu, etoposide) for most autologous pbsct, cytbi (cyclophosphamide and total body irradiation) for mf, fludarabine based chemotherapy in other allogeneic settings. radiotherapy was given before or after pbsct in 6 patients (brain in 3, abdomen in 1, mediastinum in 1, and nasal cavity in 1). a fatal veno-occlusive disease developed in mf patient who died even after orthotopic liver transplant. fatal septicemia in 3 patients at immediate post-pbsct period hampered proper evaluation of the treatment efficacy. a median disease free survival duration was 6 months (range 0~75+), and overall survival duration 16.5 months (range 1~75+). all the patients died of disease who had metastatic disease in their brains. as of this writing, 9 of 20 (45%) are alive disease free at a medial of 26 months (range 4~75). it is of note that, among them, a ptcl patient who received triple pbsct is alive disease free at 54 months post-transplant, a patient with dlbcl in cr3 at 66 months, a patient with disseminated mzl in cr1 at 70 months, and a patient with ptcl in cr1 at 75 months. a. mousavi, s. samiee, m. iravani, b. bahar, m. jahani, k. ali moghaddam, a. khodanbandeh, i. bibordi, a. ghavamzadeh horc (tehran, ir) the non-hodgkin's lymphomas (nhls) are cancers of the cells that populate lymph nodes. it is classified according to its histology, its immunophenotype, cytogenetic and molecular biology. most nhls are cancers of b-lymphocytes. although some of patients with nhl are cured with chemotherapy with or without radiotherapy, the ones who relapse and those with primary refractory disease have poor outcomes with salvage regimen. over the past 17 years, several clinical trials using high dose chemotherapy or chemoradiotherapy with autologous stem cell transplantation or allogeneic stem cell transplantation in this setting have been reported. approximately 50% of patients appear to be cured using this approach. high dose therapy/autologous stem cell transplantation is standard therapy in two scenarios, in relapsed or refractory non-hodgkin's lymphoma and in patients with refractory aggressive lymphoma whose disease is shrinking with second-line chemotherapy. here we report 65 patients with nhl, who have undergone high-dose chemotherapy (hdct) followed by hematopoietic cell transplantation (hct). of all the 65 patients 47 (72.3%) were male, and 18(27.7%) were female. the median age was 27 years old; with minimum and maximum of 10 and 51 y/o respectively. the majority of patients who had immunophenotype study were diagnosed with b-cell lymphoma (62.9%). 54 patients (83.0%) received autologous stem cell transplantation and 53 ones (81.5%) had peripheral blood as graft type. before (hct) 52.3% of patients were in first complete remission. (including cr with salvage therapy) the conditioning regimen for the majority of patients (34.85%) was ccnu, etoposide, ara-c (cytarabin) and melphalan. the median duration of hospitalization for autologous transplanted patients was 24 days which was 37 days for allogeneic transplanted ones. transplant mortality rate in the first 100 days was 4%. the median follow-up duration was 253 days, with minimum and maximum of 2 and 3103 days respectively and during this period the overall survival (os) is 70.83% and the disease free survival rate (dfs) was 60.08%. zevalin therapy of a non-hodgkin relapsed lymphoma patient following an autologous peripheral stem cell transplantation l. rejto (1) the yttrium-90 (90y) -labelled ibritumomab tiuxetan (zevalin, idec-biogen, san diego ca) is an accepted therapy for relapsed, or therapy-refracter b-cell non-hodgin lymphomas, but it is not officialy recommended in patients who have failed an autologous stem-cell transplant. patients with recurrent lymphoma following an autologous transplant have limited treatment options. 11 cases so far only have been described in the literature whose relapse following autologous peripheral stem-cell transplantation (apsct) has been treated with zevalin. in the present paper the authors discuss the case of a 53 year-old male patient, who underwent lymph-node biopsy due to generalised lymphadenomegaly. the histology test proved positive for cd20 follicular lymphoma. following an 8 cycle chop (cyclophosphamide , doxorubicine, oncovin, prednison), a 4 cycle fnd (fludara, mitoxantron, dexamethasone), later a 3 cycle dhap (dexamethasone, high-dose ara-c, cisplatin) therapy, autologous peripheral stem-cell transplantation was performed. six months later due to a relapse a 4 cycle r-cepp (rituximab, cyclophosphamide, etoposide, prednison, procarbasine), then a 5 cycle hyper-cvad therapy was applied. the patient (had no compatible sibling) proved to be therapy-refractory, therefore zevalin treatment was started (after a preparatory rituximab therapy 1180 mbq zevalin was applied). the zevalin treatment did not caused any unusually serious side effect. during the 5 months since the start of the zevalin therapy the patient has been in a complete haematological remission. in nhl, in case of a relapse following apsct zevalin therapy has proved to be a good alternative. the use cd-20 monoclonal antibody in the treatment of bcell non-hodkin lymphoma with autologous stem cell transplantation o. tarabar, l. tukic, d. stamatovic, z. tatomirovic, b. balint, b. cikota, z. magic medical military academy (belgrade, cs) introduction: recent trials have shown that cd-20 monoclonal antibody, rituximab (r) may be effectively associated with autologous stem cell transplantation (asct) in the treatment b cell non-hodgkin's lymphoma (nhl). aim: to analyze the efficacy of the incorporation of r at different steps of autologous transplantation (r-asct) programs in patients (pts) with high-risk nhl. methods: between march 2000 to december 2004 r+asct was applied for the treatment of 7 pts with b cell nhl (2 low grade; 5 diffuse large cell lymphoma -dlcl). asct were performed after chop induction chemotherapy (ct) with r (r-chopx4) in 4 pts or without r in 3 pts. in the time of asct complete remission (cr) had 2 pts and others pts had partial remission (pr). regimen mobilization were g-csf with cy ± vp-16 in 3 pts, eshap ct in 1 pt, r+cy in 2 and megar-choep in 1 pt. a single infusion of r (375 mg/m² ) used as in vivo purging 3 days prior cy or 0 day of therapy megar-choep. the average number of colected mobilized cd34+ cells was 5,7x106/kg bm (range 4,1-8,2). all pts received cvb conditioning regimen. the posttransplant immunotherapy consisted of a single dose r every 3 months (m) started 2 m following asct in 6 pts. sequential monitoring of minimal residual disease (mrd) during and after treatment was performed by pcr. five of seven pts are available for mrd. results: at a median follow-up of 36 months (6-50), 6 pts are alive. after r+asct treatment, 3 out of 5 available pts became pcr negativ (low grade lymphoma-1; dlcl-2). four pts with dlcl are still in complete remission (cr) and two of them in molecular remission (mr) 45 and 46 months (m). the both pts with low grade lymphoma relapsed 23 and 24 m after transplantation, the one of them never attained pcr negativity and second pt reverted to pcr positivity after 15 m. this therapy is well tolerated, with no adverse effects on hematological recovery of incidence of infections. conclusion: this therapy was effective in the subset of pts with high risk dlcl. r+asct treatment is able to eliminate mrd, whereas pcr positivity is associated with a high risk of relapse. double syngeneic transplantation in plasma cell leukaemia c. barrenetxea, m. callis, s. iraheta, e. sanchez, v. pons vall d´hebron (barcelona, e) introduction: plasma cell leukemia (pcl) is a rare disorder, characterized by circulating clonal plasma cell. it accounts for less than 1% of all plasma cell dyscrasias and has a fatal prognosis. it can be primary or secondary, when there was a previously diagnosed plasma cell dyscrasia. the median survival is 7-12 moths in the first case and 2 moths in the second. case: we present a 54 years old man, diagnosed in november 2003, with multiple myeloma iga kappa, bence jones +, that presented weight loss, retinal hemorrhages, respiratory distress, hepatomegaly, splenomegaly, osteolytic lesions, the cariotype showed hyperploid, chromosome 13 monosomy, translocations t (1,12) and t (4,14). first, he was treated with tree cycles of a drug's combination with melphalan, carmustine, vincristine and dexametasone with no response, therefore, was changed to cyclophosphamide, adriamycin, vincristine methotrexate y citarabine. after two cycles, the patient got complete remission. the patient had a twin brother, and we decided, to do a double transplantion to consolidate the response. the first transplantation was condicionated with carmustine, etoposide, citarabine and melphalan (beam), and the second with cyclophosphamide and total body irradiation; the patient remained in complete remission. eight months after second transplant was admitted to the hospital with disorientation, bradipsiquia, headache, and sensoriomotor loss of lower extremity. laboratory examination showed differential count of leukocytes, haemoglobin and platelets were normal, ldh increase, absence of monoclonal gammophaty in blood and urine by inmunofixation, and the brain's computerized tomography showed multiple intraparenchymatous lesions, with peripheral edema, in both cerebral hemispheres, confirmed by magnetic nuclear resonance, all of that suggested a neoplastic disease. these lesions were biopsied with the result of multiple myeloma lambda. the patient died one day after biopsy because intracranial hypertension. conclusion: plasma cell disease has poor prognosis, and transplantation could be a good option for some patients, but in our case we only achieve to extend life a few months. delayed engraftment following autologous stem cell transplantation: risk factors apart from stem cell dose r. fineman, n. haddad, t. zuckerman, i. avivi, t. faibish, m. markovitz, l. dan, d. abu zemach, l. akria, j.m. rowe rambam medical center (haifa, il) background: the use of mobilized peripheral blood stem cells results in prompt engraftment of all three cell lines in both autologous and allogeneic transplantation compared to bone marrow. the stem cell (cd34+) dose is known to be a major factor for bone marrow recovery. although reinfusion of ≥ 5x10 6 cd34+ cells/kg is considered more than adequate in terms of rapid engraftment, there are still patients who engraft after day 21, despite sufficient cd34+ cell dose. we looked for other factors contributing to delayed engraftment apart from the stem cell dose. methods: retrospective data on lymphoma and multiple myeloma patients with delayed engraftment (absolute neutrophil count >500/ul after day 21) after autologous transplantation were analyzed. factors including stem cell dose, infectious complications and outcome were evaluated. median age at hsct: 56 (40-72), 8 pts > 60. diagnosis: mds=16, saml=13. twenty-seven pts received a median of 2 (1-4) chemo cycles before hsct; 2 pts received only red cells transfusions allo, 1 haplo, 3 mud). hsct source: pbsc 26 5%) pts; 1, 1, 6 and 0 after auto, allo, haplo and mud, respectively. causes of death: infection 8, disease progression 10, ards 1, other 1. all pts evaluable for disease status after hsct (n°=24) were in cr at first marrow examination median os from hsct is 258 (19-1466), median. at last update 10 pts (34.5%) are alive in cr after a median follow-up of 705 days haplo and mud, respectively. r1204 results of high-dose chemotherapy followed by autologous stem cell transplantation in children with advanced neuroblastoma in paediatric bone marrow transplant centres in poland from wojcik (1), k. kalwak (2), e. gorczynska (2), d. turkiewicz (2), a. dyla (2) pl) purpose: postransplant morbidity and clinical outcome in children with advanced neuroblastoma (nbl) who underwent high dose chemotheraphy followed by autologous stem cell transplantation were investigated. patients: the total 80 children with stage iii/iv neuroblastoma treated in five bone marrow transplantation units in poland from 1995 to 2005 were analysed. median age of childen was 4,95 years (range 1-14,5 years). 48 children were transplanted in 1 complete/partial remission (cr/pr), in 32 patients megachemotherapy was a part of the treatment of relapse (>1 cr/pr). aphaeresis was done in 64 patients. bone marrow was collected in 10 pts; bone marrow and stem cells were transplanted in 6 patients melfalan + etoposide + carbo in 10 pts.; melphalan in 3 pts.;thiotepa + ctx + carbo in 1 patient and thiotepa + topotecan + carbo in 1 patient. results: 47 (57,5%) children are still alive at median observation time 16 months. 34 (42,5%) children died dfs) is 0,6; expected 5 -year os and dfs is 0,42 and 0,4 respectively. os at 16 months in the group of children transplanted in 1 cr/pr was 0,74; dfs 0,61. os and dfs expected at 3 years were 0,54 and 0,52 respectively. in children transplanted > 1 cr/pr estimated os was 0,74, dfs 0,58 at 16 months and expected 3-year os and dfs were 0,25 and 0,42 respectively. conclusions: megachemotherapy followed by autohsct in patients with advanced neuroblastoma has not many adverse effects. estimated 5 -year os and dfs are higher in the group of children results: a total of 466 patients underwent an autologous stem cell transplantation for lymphoma and multiple myeloma at the rambam medical center between the years 1995 and 2005 (238 and 228 respectively). indications for transplantation included a chemosensitive relapse in lymphoma high risk patients and rarely also for refractory patients. in myeloma auto transplants were performed for patients achieving good response upon initial chemotherapy. patients with lymphoma received the beac/beam conditioning and patients with myeloma were given melphalan 200 mg/m². 12 patients showed a delayed engraftment, 9 of them were patients with multiple myeloma and only 3 with lymphoma. median time to engraftment was 26 days (range 21-41). in 11 patients the cd34+ cell dose was >10x10 6 /kg and in one patient it was 6x10 6 /kg. 2 patients died of severe infections, one with lymphoma on day 36 without engraftment, the other with myeloma although engrafted on day 21. the outcome of other patients was uncomplicated. there were no detectable differences in the clinical course or toxicity among those who engrafted eraly or the late engrafters. conclusion: the use of mobilized peripheral stem cells shortens time to engraftment. using high doses of stem cells is safe, but there remains a significant risk of delayed engraftment in 4% in myeloma compared with <1% in lymphoma patients. considering the homogenicity of the groups and the high stem cell dose reinfused, it is likely that bone marrow microenvironment, known to be impaired in multiple myeloma, has a role in fascilitating engraftment. a single-centre experience in autologous stem cell transplantation for patients with multiple myeloma h. kasparu, j. könig, o. krieger, m. girschikofsky, d. lutz elisabethinen hospital (linz, a) from oct. 1996 to aug. 2005 we performed 121 autologous stem cell transplantations (abct) in 71 multiple myeloma (mm)-patients (age 58 (median:37-67) years; female:31, male: 40). following conventional chemotherapy 33 patients (transplanted between 1996 -1999 or patients not eligible for a tandem transplantation concept due to late infections (n=2) or toxic side effects (carditoxicity (2), neurotoxicity (1), dermatitis (1), smds (1)) underwent a single course of abct and 38 patients multiple courses of transplantations (26 double and 12 tripple abct). no significant differences between both groups were seen according to age, sex distribution or stage of disease at time of abct. more patients who relapsed after conventional treatment (15 vs. 5 pts) were included in the single abct group. the conditioning chemotherapy consisted of mel 200 mg/m² for single and double abct and 100 mg/m² for tripple abct. all patients were transplanted with peripheral stem cells. the time to granulocyte recovery > 0,5 g/l lasted 8-13 days (median:11) and to platelet recovery > 50 g/l 8-55 days ( median:13) without a difference between the consecutive numbers of transplantation. all patients but one in each group responded to transplantation: cr 13 pts., pr 19 pts., trm 1pt. (single abct group) and cr 16 pts., pr 21 pts., failure 1 pt.( multiple abct group), resp. in the single abct group 22 pts. (67%) relapsed after 7-73 months (med: 17 mo) , in the multiple abct group 16 pts. (42%) relapsed so far after 6-44 months (med: 16 months). the median observation time is shorter in the multiple abct group (22 vs. 40 months). the median pfs and os in the single abct group is 20 and 69 months, resp. in the multiple abct group median pfs lasted 28 months, the median os is not reached yet. both transplantations were well tolerated even in older patients. there is a trend to longer pfs in patients after multiple abct, but due to different observation periods no final conclusions can be drawn concerning os. autologous haematopoietic stem cell transplantation for the treatment of multiple myeloma o. tarabar, l. tukic, d. stamatovic, m. elez, v. glavicic, l. simic, b. balint, s. marjanovic medical military academy (belgrade, cs) high dose therapy with autologous stem cell transplantation (asct) is the treatment of choice for patients (pts) with multiple myeloma (mm). we report here our centre experience in pts with mm who have undergone asct. between december 1998 and september 2005 27 pts (19 m/8 f) with a median age of 49 years (range 38-62) were transplanted. most pts (74%) were in stage iii (durie-salmon). before transplantation, 20 pts have received 1 line of chemotherapy (4-6 cycles vad). at the time of autograft 75% pts were responders (partial remission or very-good partial remission) and the others had minor response/refractory disease. all pts received peripheral blood stem cell support after conditioning with melphalan (5 pts) or melphalan associated to cyclophosphamide and busulfan (by/cy2+m). three months (m) after asct 16 pts received interferon (3 mu/s.c. t.i.w) and 4 pts thalidomide (100-200 mg daily) as maintenance therapy. after transplantation a complete (cr) or very-good partial response was achieved in 11/25 pts (44%); all other treated pts experienced a reduction of m-component >50%. with the median follow up of 21,5 m (range 2-72), 63% pts were alive. to date, in cr are still 8 pts with the median duration of remission from asct of 24 m. seven pts relapsed or progressed during 8 to 26 m after asct. two pts died from transplant related complication. asct is safe and effective procedure not only in chemotherapy sensitive pts with mm but also in resistant cases. our date confirm that asct is the current gold standard therapy for many pts with mm. dose intensity and efficacy of treatment in patients with multiple myeloma e. darskaya, s. bondarenko, a. smirnova, b. afanassyev pavlov state medical university (st. petersburg, rus) 63 mm patients were included in our study. induction therapy was vad-d-d or idad-d-d. 23 patients with mm were undegone intensification dexabeam. 14 patients received 1 cycle dexabeam. 6 was sensitive to the 1-st line therapy, 1dexabeam resulted in 4 (66 %) cr, 1 (17 %) ncr and 1 (17 %) pd. 8 patients was unsensitive and the resulte was 3 (37 %) cr, 3 (37 %) pr, 1 (13 %) sd, 1 (13%) na. 2 cycles dexabeam received 9 patients, 2 of them were in pr after the first line therapy, 4-in sd, 3-in progression of disease. 2 (29%) sensitive patients achieved ncr, 7 patients, resistant to the first line therapy achieved 2 (29 %) cr, 4 (57 %) pr, 1 (14 %) -sd. 28 patients of mm underwent 1 asct,18 patients (64,2 %) after conditioning regimen of melphalan 200 mg/m², 5 patients (18 %) -melphalan 180 mg/m², 4 patients (14,2 %) -140 mg/m². 6 patients with mm received tandem asct. 5-years dfs in sensitive to induction and intensification therapy patients was 20 %, dfs in group of the patients with progression or relapse before asct was no longer that 0.6 year. p = 0.03. the 5-years duration of a plateau phase of the patients, sensitive to the first line therapy and intensification, after 1-st asct was 25 %, the duration of a plateau phase of the patients with progression before asct, was no longer that 1.5 year. p = 0.06. the 6-years duration of a plateau phase of the patients, who received melphalan 200 mg/m² as conditioning regimen, was 23 %. in patients, received 180 mg/m² melphalan and less it was no longer that 5.8 years.s350 3 years dfs in group of the patients with tandem asct was 65 % in comparison with 15 % in group of the patients with single asct. p = 0.03. the conclusion: our preliminary results are that the increasing of dose intensity improves the efficacy of treatment of patients with multiple myeloma. successful myeloablative allogeneic haemopoietic stem cell transplantation in a patient with end-stage renal failure on haemodialysis a. alfred (1) introduction: end stage renal failure (esrf) has conventionally been considered a relative contraindication to hsct. although increasing numbers of patients undergoing autologous procedures with hd are being reported, documented experience in the allogeneic hsct setting remains extremely limited. to the best of our knowledge only three previous case reports have been published. case report: a 38 year old male, treated with regular hd for esrf from 2003, was diagnosed with myelodysplastic syndrome associated with monosomy 7 (ipss int-2) in 2004. after extensive counselling with the patient, careful consideration of donor issues, and collaboration between bmt and renal teams, a decision was made to proceed with allogeneic hsct with curative intent. in april 2005, the patient underwent myeloablative conditioning with total body irradiation 12 gy and cyclophosphamide 120 mg/kg followed by transfusion of g-csf mobilised allogeneic pbsc from his hla and abo-matched sister (cd34 cell dose = 6.8x10 6 /kg). recipient and donor cmv serology was negative. during conditioning, hd was performed on a daily basis to optimise biochemistry. the patient was closely monitored for cyclophosphamide cardiotoxicity with ecg and echocardiography. oral mesna was used to prevent haemorrhagic cystitis. hd was subsequently maintained at the regular thrice weekly schedule. graft-versus-host disease (gvhd) prophylaxis was ciclosporin and methylprednisolone. ciclosporin levels were little affected by hd and there were no significant problems maintaining the therapeutic range. otherwise careful consideration was given to drug dosing in relation to hd. regimen related toxicity was no more than expected in a patient with normal renal function. engraftment was prompt and discharge was on day +28. mild acute and chronic ghvd has been managed with additional corticosteroids. there have been two inpatient re-admissions for post-transplant complications. bone marrow examination at three months post hsct confirmed trilineage engraftment and 100% female karyotype with no evidence of monosomy 7. the patient is presently stable, in remission and on reducing immunosuppression at over 7 months post-hsct. conclusion: this case highlights the feasibility of myeloablative conditioning and allogeneic hsct in carefully selected patients with esrf on hd. ethical considerations should incorporate the additional impact on the donor of a potential future histocompatible living donor renal transplantation. liver complications in hematopoietic transplantation (hct) setting may be life threatening, and hepatitis b virus (hbv) infection increases the risk of hepatic complications in patients undergoing hct. we describe a 63 year old white women who was treated 5 years before with chemotherapy for non-hodgkin disease achieving complete remission. she acquired hbv infection because of a blood red cell transfusion. five years after complete remission she developed anemia, piastrinopenia and leucopenia. bone marrow biopsy histology excluded lymphoma relapse but revealed multilineage myelodisplasia with excess of blasts. cytogenetic examination detected 7qdeletion. according to ipss score myelodisplasia was classified at high risk. since an high viral b load (450560 copies/ml), despite normal alt and ast values, the patient underwent lamivudine prophylaxis. hct was considered and his brother resulted full hla match. he was succesfully mobilized with g-csf. before transplantation viral b load did not decrease so antihepatitis b prophylaxis was changed from lamivudine to adefovir dipivoxil that was also employed as the only antiviral prophylactic treatment along all the procedure. patient underwent reduced conditioning regimen: e.v. busulfan (7,2 mg/kg weight) plus fludarabine (150 mg/m²) and rabbit atg fresenius (25 mg/kg).graft versus host disease (gvhd) prophylaxis consisted in cyclosporin and metotrexate. 5,83 x 10 6 /kg cd34 and 7,6 x 10 8 /kg monocleated were infused. neutrophil engraftment was at +13, while platelet engraftment at +12. the patient developed steroid sensitive cutaneous grade ii acute gvhd. complete chimerism was achieved at + 21. at day 60 she developed abrupt alt and ast increase (>600 iu/l), without bilirubin increase and a concomitant viral b load more than 10 7 copies/ml. lamivudine was reintroduced and adefovir was continued. one week later transaminases began to decrease and at day 85 both alt and ast were under 100 iu/ml, while viral b load was 1 x 10 6 and the patient is well at day 100. a reduced conditioning regimen with fludarabin and e.v. busulfan together with rabbit atg revealed to be safe and efficacious in this 63 year old patient. moreover lamivudine and adefovir association was able to control hbv replication. adefovir alone successfully avoided every other viral infection (hsv, cmv etc.)along transplant procedure and it did not impair engraftment. haematopoietic stem cell transplantation in poor prognosis mds and saml: prolonged patients survival is achievable, but refining of infections management and reduction of treatment-related toxicities is required to improve patients outcome m. tassara, a. crotta, l. camba, f. lunghi, m. marcatti, j. peccatori, m. bregni, f. ciceri, m. bernardi san raffaele scientific institute (milan, i) introduction: allogeneic hematopoietic stem cells transplantation (hsct) is the therapy of choice for poor prognosis mds and saml patients (pts); a hla identical, related (allo) or unrelated (mud), or haploidentical familial (haplo) donor is available for most pts. autologous (auto) hsct is an alternative for pts without a donor. prolonged overall survival (os) and disease free survival (dfs) are reported in a minority of mds/saml pts after hsct. major drawback of allo, haplo and mud hsct is the high incidence conclusions: prolonged os and dfs are achievable with hsct in poor prognosis mds and saml pts, also in the elderly. prevention of pts contamination before hsct, mainly from aspergillus, and reduction of early trm, mainly in the haplo subset of pts, could improve pts survival. trials for primary/secondary anti-fungal prophylaxis are ongoing and reduced-toxicity conditioning regimens are under investigation at our institute. auto in cr is an alternative if hsct from a donor is not feasibile; to reduce the relapse rate after auto an experimental maintenance treatment should be proposed. high-dose chemotherapy and autologous peripheral blood stem cell transplantation followed by a successful extremity sparing surgery in a case of osteosarcoma arising in osteogenesis imperfecta s. ataergin, f. arpaci, k. erler, b. demiralp, a. kaya, a. ozet, m. basbozkurt gata (gulhane) faculty of medicine (ankara,tr) objective: osteosarcoma (os) arising in osteogenesis imperfecta (oi) is reported as only nine proven cases in the english literature; among these one case had a limb sparing surgery, but none underwent a high-dose chemotherapy (hdc) and autologous peripheral blood stem cell transplantation (apbsct). methods: we recently demonstrated the effectiveness of hdc and apbsct in localized osteosarcomas. this case is the first one of os occurring in oi who underwent a hdc and apbsct. results: a-20-year male patient had been followed-up since his birth with type i oi and had recurrent traumatic bone fractures. he was admitted to orthopedics clinic with complaints of bone pain, edema and swallowing in right proximal tibia. an incisional bone biopsy revealed conventional osteosarcoma. the stage was iib and two cycles of neoadjuvant chemotherapy consisting with cisplatin 30 mg/m², adriamycin 20 mg/m², ifosfamide 2.5 g/m² and mesna 2.5g/m² were administered in three consecutive days, every three weeks, according to our institutional treatment protocol for osteosarcoma. fifteen days later, stem cells were mobilized using g-csf (10 µg/kg/day in two doses) and collected by cobe spectra cell separator (cobe bct inc., lakewood, co). the amount of cd34+ cells was 6.69x106/kg. hdc (ifosfamide 12 g/m², carboplatin 1.2 g/m², etoposide, 1.2 g/m² and mesna on a dose of 140-160% of the total ifosfamide dose were given in dividing doses on six consecutive days) and apbsct were performed thereafter. extremity sparing surgery (wide en-bloc resection and reconstruction prosthesis) was applied after the neutrophil and platelet engraftment. postoperative tumor necrosis rate was 99% (by histopathology) and the same induction chemotherapy regimen was given for three additional cycles as consolidation chemotherapy. conclusion: the patient is still disease-free with a good functional score and no fracture has occurred in 11 months after the last follow-up. improved survival in neuroblastoma by autologous peripheral blood stem cell transplantation: a single-centre experience h. kook, j.y. kim, h.j. baek, d.k. han, h.r. yi, h.j. kim, t.j. hwang chonnam university hwasun hospital (hwasun, kor) objectives: neuroblastoma is the most common extracranial solid tumor of childhood, and its outcome in advanced diseases has been very poor. in this study, the author evaluated the treatment outcome and prognostic factors in advanced neuroblastoma. methods: the study group comprised of 48 patients who were diagnosed and treated with neuroblastoma at chonnam national university hospital from january, 1996 to may, 2005. data were obtained from the retrospective review of the medical records. patients were classified according to the evans group. the conventional treatment including surgery, radiotherapy, pre-and post-operative chemotherapy was given to stage i, ii, and iii patients. for stage iv, relapsed patients, high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (pbsct) was administered. the chemotherapy consisted of cisplatin, doxorubicin, etoposide, and cyclophosphamide (ccg 3881, 3891). conditioning for pbsct was modified vamp-tbi(cisplatin, doxorubicin, etoposide, melphalan, and total body irradiation). all patients who completed cytotoxic therapy were then either received no further therapy or treated with 13-cis-retinoic acid for six months. results: among 48 patients, 27 were males and 21 females. the median age at diagnosis was 36.5 months (range, 1-167 months). the primary sites were the adrenal glands in 25 patients, followed by retroperitoneum in 10, and thoracic cavity in 10. most of the patients were in advanced stages: stage iii in 11; stage iv in 26. autologous pbsct was done in 14 cases. the 5-year event-free-survival (efs) rate was 41% in all study patients with 100% for stage i, 67% for stage ii/iii, 35% for stage iv, 50% of iv-s. in cases with stage iv neuroblastoma, the efs rate at 4 years after diagnosis was better among the patients who underwent autologous pbsct than among the patients who received chemotherapy (51 % vs. 20 %; p = .05). also, efs was better in patients who received 13-cis-retinoic acids after pbsct than those who did not (100% vs. 14%; p < .005). conclusion: treatment with high dose chemotherapy and autologous pbsct improved efs among children with advanced neuroblastoma. in addition, treatment with 13-cisretinoic acid was beneficial for patients who underwent transplantation. prospective randomized study is warranted to further improve survival for subset of advanced patients who might fail to current management strategies. key: cord-005478-5iu38pr6 authors: nan title: the 45th annual meeting of the european society for blood and marrow transplantation: physicians – oral session date: 2019-07-03 journal: bone marrow transplant doi: 10.1038/s41409-019-0562-9 sha: doc_id: 5478 cord_uid: 5iu38pr6 nan methods: study aim was to evaluate the schedule of ist given in combination with pt-cy as gvhd-prophylaxis post-haplo for acute leukemia (al) and reported to the alwp/ebmt registry. patients were divided into 3 groups: received cyclosporine a-mycofenolate-mofetil(csa-+mmf) initiated at day+1 (group-1, n=124) or csa +mmf both started at day+5 (group-2, n=170) and tacrolimus + mmf from day+5 (group-3, n=215). transplants were performed from 2006-2017 and median follow up is 21 months (range 11-36). pt-cy was given on day+3 and day+5 in group-1 and on day+3 and day+4 in group-2 and 3. results: acute myeloid leukemia (aml) was the most common indication for haplo (76%) and approximately 45% of patients were transplanted in cr1. there were some differences among groups: patients in group-1 were younger (median age 46 years, p< 0.02) were transplanted in more recent year (2015, p< 0.001), received more frequently a regimen based on tbf (thiotepa, fludarabine and busulfan) (83%, p< 0.001) and bone marrow (bm) as source of stem cells (77%, p< 0.001), with no atg (100%, p< 0.001). probability of os at 2 years was 59%, 48% and 44%, for the 3 groups, respectively, p=0.15. probability of lfs and grfs at 2 years were 52% and 46%, 43% and 36%, 39% and 33%, for the 3 groups, respectively, (lfs p=0.05, grfs p=0.01. overall the cumulative incidence (ci) of grade ii-iv acute gvhd was 18%, 39% and 25%, for the 3 groups, respectively, p< 0.001, and the ci of chronic gvhd was 23%, 21% and 25%; p=0.28. the ci of relapse at 2 years was 26%, 37% and 35% (p=0.01) and background: patients with acute myeloid leukaemia (aml) often achieve remission but subsequently die of relapse driven by chemotherapy resistant leukemic stem cells (lscs). here we hypothesized that lscs must also escape immunosurveillance to initiate and maintain cancer and investigate the interplay with nkg2d, a danger detector expressed by cytotoxic lymphocytes such as natural killer (nk) cells that recognizes stress-induced ligands (nkg2dl) of the mic and ulbp protein families on aml cells. methods: 175 de novo aml were stained with antibodies against mica, micb and ulb2/5/6 or an nkg2d-fc chimeric protein recognizing pan-nkg2dl. nkg2dl pos and nkg2dl neg aml cells sorted from the same patient were analysed in colony forming assays, leukemogenesis assays in nsg mice, by rnaseq, gene expression arrays, qrt-pcr and targeted next generation sequencing. aml cells co-cultured or not with nk cells (control or anti-nkg2d pre-treated) were co-stained for additional stem/immunological markers. parp1 expression was analysed by qrt-pcr and immunoblot, and binding to nkg2dl promoters by chromatin immunoprecipitation. parp1 inhibition (parpi) in aml cells was performed in vitro or in vivo using sirnas or inhibitors (ag-14361, veliparib) . results: heterogeneous nkg2dl expression was detected among leukemic cells of the same patient (fig. 1a) . interestingly, when compared to nkg2dl pos subpopulations, nkg2dl neg aml cells isolated from the same patient showed immature morphology, enhanced in vitro clonogenicity (39±47 colonies vs. 1±4, p< 0.001, n=32 aml patients) and selective abilities to initiate leukemia (nkg2dl neg , 64/70, 91%; nkg2dl pos , 0/78, 0%; p< 0.00001, fig. 1b , n=19 aml patients) and survive chemotherapy in nsg mice devoid of functional nk cells. in mice, nkg2dl neg aml cells generated both nkg2dl pos and nkg2dl neg progeny of which again only latter was able to induce leukemia in re-transplant assays. similar leukemia-specific mutations were detected in nkg2dl neg compared to nkg2dl pos aml cells from the same aml but published lsc (fig. 1c ), hsc and 17genes stemness score signatures were specifically enriched in nkg2dl neg subfractions. nkg2dl neg cells enriched for lscs defined by alternative markers (cd34 + , cd38 -, gpr56 + ) but could identify cells with functional and molecular lsc activity also in cd34 non-expressing aml (n=11 analyzed patients). nkg2dl expression was repressed by parp1 recruitment at nkg2dl promoters. parp1 inhibition (parpi) induced nkg2dl surface expression in lscs and co-treatment with parpi and nk cells (but not with either alone) suppressed leukemogenesis in patient derived xenograft (pdx) models (fig. 2c ) cotransplanted with nk cells. low nkg2dl surface or high parp1 mrna expression associated with poor outcome in aml patients. furthermore, nkg2dl neg and cd34 + lscs showed reduced expression of other immune stimulatory molecules (e.g. cd112, cd155, cd80, cd86) and different expression of immune or inflammatory response gene signatures (gsea). conclusions: these data indicate that lscs escape nk cell recognition by selectively suppressing the surface expression of nkg2dl and other immunostimulatory molecules. absence of nkg2dl can identify lscs across genetic aml subtypes (including cd34 negative amls). this lsc specific mechanism of immune evasion could be overcome by treatment with parp1 inhibitors, which in conjunction with functional nk cells holds promise to eradicate lscs and promote immune-mediated cure of aml. disclosure: c.l.: sanofi, novartis, otsuka (consultancy); roche (research funding) background: in contrast to imatinib, data on the use of 2 nd and 3 rd generation tyrosin kinase inhibitors (tki) in the treatment of minimal residual disease (mrd), molecular and hematological relapse (mr/hr) after allogeneic stem cell transplantation (sct) in philadelphia chromosome positive (ph+) acute lymphoblastic leukemia (all) are scarce. methods: we performed a retrospective, ebmt registry based analysis, including patients with documented use of 2 nd or 3 rd generation tki given for persisting mrd, mr or hr after allosct in 2006 -2016 choice of tki, efficacy, and toxicity of tki and patient outcome were analysed. results: 140 patients (female 58, male 82) were identified, out of which 136 had also received a tki (78% imatinib) before allosct. median age at transplant was 48 years (18-66), 81% were transplanted in first complete remission (cr1), 51% of the patients were in molecular cr. conditioning was myeloablative in 71% and reduced in 29%, donors were matched siblings (48%), unrelated (41%), haploidentical (6%) and cord blood (5%). after allosct, 111 patients developed hr, 23 mr and 6 had persisting mrd. for treatment, patients received dasatinib (n=104), nilotinib (n=18) and ponatinib (n=18). median interval between diagnosis of persisting mrd or mr/hr and first application of a tki was 10 days, median duration of tki treatment was 154 days (range 4 -2193). fifty-eight patients were treated with tki only (dasatinib, n=50, nilotinib, n=3, ponatinib, n=5) , while 82 received additional treatment such as dli, chemotherapy, or second allosct. main toxicities of dasatinib were effusion, edema, or other pulmonary complaints (10 -15% of patients) and infections (13%). no particular side effects were reported for nilotinib and ponatinib (no vascular events). dose reduction of tki was required in 32%. response rates were 71% (entire cohort) and 72% (patients receiving tki only). for the entire cohort, 2-and 5-year overall survival (os) from first application of tki was 49% and 33%. two-year os was comparable in patients treated for persisting mrd/mr and for hr (48% and 50%). among patients treated with tki only, 2/5-year os was 38%/33%. rate of cgvhd was 11% for the whole population and 14% for the tki alone cohort. conclusions: the use of 2 nd and 3 rd generation tki, given alone or in combination with other therapies for treating persisting mrd, mr or hr after allosct in ph+ all was not associated with increased toxicities. dasatinib was the most frequently used drug. outcome compared favorably with published results on relapse after allosct in ph negative all, suggesting that treatment with tki could improve survival after post-transplant relapse, even when given as single therapy. type of relapse did not influence response rates and outcome. disclosure: nothing to declare multi-state modelling of the interplay between remission-induction chemotherapy and consolidation with allosct in newly diagnosed aml patients reduced rate of relapse in the clofarabine arm, discovering a significant difference between the treatment arms in the hazard of relapse only after allosct (hr 0.65, 95% ci 0.46-0.94, p-value = 0.02), and not before allosct (hr 0.81, . in addition, we found increased nrm in the clofarabine arm before allosct (hr 1.95, 95% ci 1.15-3.31, p-value = 0.01), and not after allosct (hr 0.80, 95% ci 0.50-1.28, p-value = 0.34). these effects are statistically significantly different (interaction test hr 0.41, 95% ci 0.20-0.83, p-value = 0.01). at two years after registration, 20.4% (95% ci 16.6-24.1) of the patients in the control arm, and 19.9% (16. 1-23.6 ) in the clofarabine arm were alive relapse-free in cr without allosct, while 20.9% (16.7-25.1) and 26. 8% (22.3-31.4 ) were alive relapse-free after allosct. conclusions: presented results suggest that the rate of relapse after allosct is lower among patients whose induction therapy includes clofarabine. these results could possibly be explained by higher rate of mrd negativity achieved in the clofarabine arm before proceeding to allosct. we also observed a higher nrm rate in cr before allosct in the clofarabine arm, indicating that the favorable effect of clofarabine on relapse may be compromised by toxicity. clinical trial registry: hovon 102 study is registered at netherlands trial registry #ntr2187 disclosure: nothing to declare haploidentical transplant with post-transplant cyclophosphamide for t-cell acute lymphoblastic leukemia: outcome strongly correlates with disease status; a report from the ebmt acute leukemia working party background: partial tandem duplication of mll (mll-ptd) is an infrequent mutation in aml which produces a number gain of 5' acetyltransferase domains of kmt2a protein as a result of a repeated exon 3-9/10 gene sequence. mll-ptd leads to a disturbed histone acetylation and upregulation of determined hox genes. mll-ptd aml defines a specific aml entity, distinguishable from cases of aml with mll rearrangement, with a characteristic pattern of co-mutations, including a high association with flt3-itd (q-y sun et al., leukemia 2017) . prognosis of mll-ptd-aml is remarkably poor, with initial chemoresistance and high relapse rate; as a consequence, allogeneic hematopoietic cell transplantation (allohct) in early phase is recommended to overcome its high-risk prognostic impact (jp patel et al., nejm 2012; v grossmann et al, blood 2012) . nonetheless, studies focusing on transplant outcome have not been previously addressed. methods: for this purpose, we analysed the outcome of mll-ptd aml adult patients reported to ebmt who had received an allohct from matched related or unrelated donors in cr1 during the period 2000-2016. molecular screening of mll-ptd was performed locally, but the presence of this mutation was verified specifically by a focused questionnaire among participating centres. results: overall, we identified 58 patients fulfilling inclusion criteria (median age, 52.8 years; 29/58 female patients). most patients harboured an intermediate risk cytogenetics (50/56; 89% of available) and 14 (14/54, 26% of available) patients presented with concomitant flt3-itd. donor was a matched sibling in 27 transplants (47%) and an unrelated donor in the remaining cases. conditioning was myeloablative (mac) in 35 procedures (61%) and reduced intensity in 22 (40%). in vivo t-cell depletion was used in 36 (64%) of transplants. at two years, cumulative incidence of relapse (cir) was 33% (95% ci: 21-45), and non-relapse mortality (nrm) 16% (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) ; 100-day acute gvhd of grade iii-iv was 7% (2-16) and 2-yr chronic gvhd was 38% (extensive, 13%, 6-23). two-year overall survival (os) was 56% (45-71), with leukemia-free survival (lfs) and graft-and relapse-free survival (grfs) of 51% (40-65) and 39% (27-58) , respectively (figure) . multivariate analysis did not identify any prognostic factor for lfs; notably, presence of flt3-itd, conditioning regimen intensity or donor type did not influence outcome. conclusions: these results support the potential clinical benefit of allohct performed in cr1 in patients with mll-ptd aml, with a decreased relapse risk compared to previously reported series, suggesting the existence of a tangible graft-versus-leukemia effect (gvl) in this setting. disclosure: nothing to declare o017 safety and efficacy of reduced intensity conditioning regimen combined with anti-thymocyte globulin and post-transplantation cyclophosphamide as graft versus host disease prophylaxis for acute myeloid leukemia background: we aimed to evaluate the safety and efficacy of the use of reduced intensity conditioning regimen (ric) combined with anti-thymocyte globulin (atg) and posttransplant cyclophosphamide (ptcy) for graft versus host disease (gvhd) prophylaxis in patients diagnosed with acute myeloid leukemia (aml). methods: one hundred four adult patients were included. all patients received the same ric regimen including fludarabine (30mg/m2/day day -5 to -2), busulfan (3.2kg/ m2/day day -3 and -2), and total body irradiation (200 cgy) (day -1) combined with rabbit-atg (4.5 mg/kg: day -3 to -1), ptcy (50mg/kg/day: day +3,+4), and cyclosporine. unmanipulated peripheral blood stem cells were infused. last follow-up was november 2018. median follow-up was 19 months (range 5-35). results: findings are summarized in figure 1 . one year overall survival (os) progression-free survival (pfs) and non-relapse mortality (nrm) was 67.7% (95% ci 58.6-76.8), 60.8% (95% ci 51. 1-70.5 ) and 16.4% (95% ci 6. 9-25.8) respectively. main causes of death were relapse (18%) and infection (13%). three patients had residual disease prior-transplant and they had a significant worst os (p=0.000) and rfs (p=0.002). patients who had karnofsky performance status (kps) ≤80% had a significantly worse os (p=0.004) and pfs (p=0.014). the achievement of ≥95% chimerism of background: indications for hematopoietic stem cell transplantation (hsct) for adults with all evolve over time and vary among countries. methods: the goal of this study was to assess general trends in the number of various types of hscts performed between years 2001 and 2015 in europe. data reported to the ebmt registry were used for this analysis. in addition, we evaluated hsct rates with respect to the incidence of all in selected european countries. results: altogether, 15346 first allogeneic (n=13460) or autologous (n=1886) hscts were performed in the period 2001 -2015 . comparing years 2013 -2015 and 2001 -2003 , the number of allohscts performed in first cr increased by 136%, most prominently for transplantations from unrelated (272%) and mismatched related donors (339%). the number of hscts from matched sibling donors increased by 42%, while the number of autohscts decreased by 70%. the increase of the use of allohsct, irrespective of the disease stage, was stronger for ph-pos (166%) than ph-neg all (38%). among patients aged >55 years, the number of allohsct increased by 559% while among younger adults (18-55 years), by 59%. between 2001 and 2003 , peripheral blood was used as source of stem cells in 61% cases of allohsct, compared to 84% between 2013-2015. the use of bone marrow decreased from 38% to 16%, respectively. the proportion of allohsct with reduced-intensity conditioning (ric) increased from 6% to 27%. among myeloablative transplantations, regimens based on total body irradiation were the preferable option (app. 80% over the whole study period). in contrast, among ric regimens, the use of chemotherapy predominated (84% between 2013-2015) . in most of analyzed individual countries, the estimated rates of allohsct (no. hsct per 100 newly diagnosed all) for patients in cr1 increased over time. however, the values for a period 2013-2015 varied strongly, being highest in finland (57.9), followed by the netherlands (35.4) and sweden (35.4) while lowest in russia (2.6) . conclusions: results of our analysis indicate continued trend to increased use of allohsct for adults with all, which may be attributed to increasing availability of unrelated donors. however, it may also be speculated that the introduction of tyrosine kinase inhibitors allowed higher proportion of patients with ph-pos all proceeding to transplantation. finally, the implementation of ric regimens contributed to wider use of allohsct among older adults. limitations of the analysis include any assumptions made regarding all incidence for the specified time period and possible variation in reporting to the ebmt registry from different countries over time. year type of hsct 2001 hsct -2003 hsct 2004 hsct -2006 hsct 2007 hsct -2009 hsct 2010 hsct -2012 hsct 2014 hsct -2015 disclosure: sg has received honoraria for amgen. as and sw are employees of amgen and own shares in amgen. mm has received honoraria for speaking for amgen and pfizer. outcome of allogeneic-hsct in adult patients with phpositive-all in the era of tki: a retrospective analysis of università degli studi e ospedale maggiore policlinico di milano, milano, italy, 33 genova, italy, 34 azienda socio sanitaria territoriale papa giovanni xxiii, bergamo, italy background: we conducted a retrospective, nationwide analysis to describe the clinical outcome of adult patients with philadelphia chromosome-positive acute lymphoblastic leukemia (ph+all) undergoing allogeneic hematopoietic stem cell transplantation (hsct) after being treated with a tki based therapy. methods: a total of 441 patients were included in the study. the median age at hsct was 44 (range: 18-70). all 441 patients (100%) received tki before hsct (performed between 2005-2016) . of these patients, 404 (92%) were in cytologic complete remission (cr) while 37 (8%) had an active disease at the time of hsct. molecularly measurable residual disease (mrd) was negative in 147 patients (36%) at the time of hsct. the donor was unrelated in 46% of cases. the prevalent source of stem cells was peripheral blood (70%). the conditioning regimen was myeloablative in 82% of cases (tbi-based in 50%) and included atg in 51% of cases. results: with a median follow-up after hsct of 39.4 months (range: , the probability of overall survival (os) at 1, 2 and 5 years was 69.6%, 61.1%, and 50.3%, respectively, with a median os of 62 months. progression free survival (pfs) at 1, 2 and 5 years was 60.2%, 52.1% and 43.7%, respectively. os and pfs were significantly better in patients with cr and mrd-negativity at the time of transplant compared to patients with cr but mrd-positive (50% os not reached vs. 36 months, p=0,015; 50% pfs not reached vs 26 months, p=0.003). the cumulative incidence of relapse (cir) at 5 years was significantly lower in patients with cr and .4%, p=0.001). the non relapse mortality (nrm) after 1, 2, and 5 years was 19.1% (95%ci: 15.5-22.9), 20.7% (95%ci: 17-24.7), and 24.1% (95%ci: 20-28.5), respectively. the subgroup of patients with mrdnegative both at hsct and at 3rd month after hsct had a better outcome (5 year os 70%). conversely, the 37 patients who underwent hsct with active ph+all had a median os and pfs of 7 and 5 months, respectively. background: haploidentical (haplo) donors have expanded patient transplant access. however, outcome of patients with acute lymphoblastic leukemia (all) undergoing allogeneic stem cell transplant (asct) with haplo donors in argentina has not been reported. we aimed to analyze the outcome of asct in patients with all, particularly results with haplo donors. methods: we collected data from patients with an all diagnosis who underwent asct in first complete remission (cr1) and subsequent remissions (cr2+) in 15 centers in argentina, affiliated to gatmo, between 2008 and patients that underwent asct with matched donors (sibling and unrelated) and haplo donors (with post-transplant cyclophosphamide) were included. both donor categories were compared in terms of overall survival (os), nonrelapsed mortality (nrm) and cumulative incidence of relapse (cir). graft versus host disease (gvhd) was also evaluated. multivariate analysis was performed by cox regression for os and fine-gray for ci of competing events. a further propensity score (ps) adjustment was performed by donor group. results: in a 10-year period, 236 patients were included (mean age 31y; range 16-64; male 63.1%); 188 (80%) during last 5 years. all phenotype was b (79%) and t (21%). at diagnosis, 47/236 (20%) had cns involvement and 75/236 (32%) were philadelphia chromosome positive. asct was performed in cr1 (n=126; 53%) and in cr2+ (n=110; 47%) after a median time from all diagnosis to asct of 9 and 26 months, respectively. comorbidity index (hct-ci) was 0-1 in 199/236 (90%). donors were matched (n=175; 74%; 146 related and 29 unrelated) and haplo (n=61; 26%). conditioning regimen was myeloablative in 215/236 (91%; 170 patients with total body irradiation), and this conditioning was more frequent in matched (95%) than haplo (79%) (p=0.001) donors. two-years os was 54 % (95%ci 46-60) for the entire population; 55% (95ci 47-63) for matched donors and 49% (95% ci 34-62) for haplo donors (p=0.350). in the multivariate analysis, pretransplant status (cr1 vs cr2+; hr 2.06, p< 0.001), cns status at diagnosis (yes vs no; hr 1.70; p=0.019) and unrelated donors (yes vs no; hr 1.77; p=0.036) were independently associated with os; donor category had not impact in the os. by adjusting the ps term (roc area 0.787), no difference was found by donor category. twoyears nrm was 24% (95%ci 18-31) for matched and 22% (95%ci 12-33) for haplo (p=0.999) donors; older donors (p=0.049) and unrelated donors (p=0.001) were associated with higher nrm. two-years cir was 25% (95%ci 19-33) for matched and 38% (95%ci 24-51) for haplo (p=0.137) donors; only male donors were associated with higher cir (p=0.031). ci of grade 3-4 acute gvhd was 20% vs 17% (p=0.784) and chronic gvhd was 35% vs 27% (p=0.057) for matched and haplo donors respectively. in both groups, matched and haplo donors, the half of deaths were due to relapse. background: relapse is the most important cause for treatment failure in pediatric b-precursor acute lymphoblastic leukemia (bcp-all) occurring in 10-20% of patients. mechanisms of ineffective graft-versus-leukemia (gvl) effects or t-cell responses against all remain to be investigated. methods: we analyzed parameters of immunosurveillance in bone marrow (bm) samples of 100 pediatric patients to identify potential mechanisms of t-cell suppression. expression of co-stimulatory/ co-inhibitory molecules was analyzed to identify implications for gvl. expression was correlated with clinical outcome (8 years mean followup) . t-cell immunoglobulin and mucin-domain containing-3 (tim-3) overexpression and crispr/cas9-mediated knockout (ko) in primary t cells were performed to analyze its role for anti-leukemic t-cell functionality. to induce an interaction of t cells with leukemic blasts, anti-cd19/-cd3 bispecific t-cell engager (bite) was added and t-cell activation/ proliferation were analyzed. fold change (fc) was created by comparing levels of t-cell activation/ proliferation before vs. after co-culture. transcriptome analysis of primary bm samples identified expression levels of known tim-3 inducers. results: flow cytometric analyses of 100 bcp-all samples showed increased tim-3 expression on cd4 + bm t cells at initial diagnosis in patients with relapse in the course of disease. multivariate analysis confirmed 7-fold increased relapse risk in tim-3 high (n=37) vs. tim-3 low expressing (n=37) patients. pd-1 expression on bm t cells alone had no impact on relapse-free survival (rfs), whereas patients with high percentage of tim-3/pd-1 double positive cd4 + bm t cells showed significantly decreased rfs (87.8% vs 41.7%). co-culture experiments revealed that tim-3 is induced in primary t cells by contact with leukemic cells (mean tim-3 expression 51.1% vs. 29 .7% on t cells with vs. without addition of leukemic cells, n=3). transcriptome analysis was performed to identify expression levels of known tim-3 ligands/ inducers in bm samples with high vs. low tim-3 expression. no significant differences in expression levels of high-mobility-group-protein b1 (hmgb1), carcinoembryonic antigen-related cell adhesion molecule 1 (cea-cam1) or galectin 9 were observed. known tim-3 inducers il-12, il-15, il-27, il-7 or transforming growth factor beta 1 (tgf-β1) were not differentially expressed indicating that another mechanism must be responsible for tim-3 overexpression. tim-3 overexpression and crispr/ cas9-mediated tim-3 ko were performed to analyze functional relevance of tim-3 expression in an in vitro leukemia model. t cells of healthy donors were co-cultured with leukemic cells and anti-cd19/-cd3 bite to induce anti-leukemic t-cell response. tim-3 ko t cells showed significantly increased activation compared to wildtype t cells (fc of cd69 expression 5.0 vs. 3.2, n=3) . in contrast, proliferation of tim-3 overexpressing t cells was significantly impaired (fc 1.6 vs. 2.3, n=3) , whereas tim-3 ko t cells showed higher proliferation levels compared to controls (fc 6.5 vs. 2.4, n=3) . conclusions: tim-3 expression on cd4 + bm t cells is a strong predictor for pediatric bcp-all relapse and is induced by t-cell interaction with leukemic cells. tim-3 expression decreases anti-leukemic t-cell activation and proliferation and thus constitutes a new mechanism of immune escape and potentially insufficient gvl effects in pediatric bcp-all. targeting the tim-3 axis can be of interest to improve future immunotherapy of advanced bcp-all. disclosure: nothing to declare. abstract already published. multiparameter flow cytometric minimal residual disease before myeloablative allogeneic hematopoietic cell transplantation in acute myeloid leukemia influences patients survival in first and second complete remission background: the growing evidence from the literature strongly suggest that multiparameter flow cytometric (mfc) minimal residual disease (mrd) assessment in aml can be used to risk-stratify patients at the time of allogeneic hematopoietic stem cell transplantation (allohct). we sought to determine the significance of mfc-mrd status in patients with aml in first or second complete remission (cr) treated with myeloablative conditioning (mac) allohct at six centers of the polish adult leukemia group (palg). methods: mrd was assessed by 6-color (8-color since 2017) mfc performed on bone marrow aspirates obtained as routine assessment before allohct. all consecutive patients undergoing mac allohct were included in the analysis if they underwent pre-hct mfc-mrd analysis from may 2013 until january 2018. the abnormal population was quantified as a percentage of the total cd45 + white cell events. residual disease at a ≥ 0.1% level was considered mrd-positive (mrd+) . results: we identified 83 adult patients (median age 41 years, range 19-64) with aml undergoing allohct from either hla-identical sibling (n=30) or unrelated donor (n=53), in cr1 (n=72) or cr2 (n=11), who were conditioned with i.v. busulfan given in myeloablative dose (9,6-12,8 mg/kg) in combination with flu (n=46) or cy (n=37). gvhd prophylaxis consisted of calcineurin inhibitor combined with mtx plus atg in allohct from unrelated donors. positive mrd status before allohct was detected in 30/72 (42%) pts in cr1 and 6/11 (55%) pts in cr2. the mrd(-) and mrd(+) groups did not differ in terms of gender, age, eln cytogenetic and molecular genetic risk, first and second cr, conditioning regimen, hsc source, and type of donor. the 3-year overall survival (os) for mrd(-) and mrd(+) patients were 77% and 49% (log-rank p= 0.023). the respective 3-year leukemia-free survival (lfs) were 61% and 37% (log-rank p=0.012). in univariate and multivariate cox proportional hazard model the only significant adverse prognostic factors for lfs were mrd(+) (hr 2.45, p=0 .025) and high eln genetic risk (hr 3.70, p=0.0009) . the same factors significantly influenced os [hr 2.48, p=0.036 and hr 3.47, p=0 .004 for mrd(+) and high eln risk, respectively] . conclusions: our findings confirm that pre-transplant residual disease at a ≥ 0.1% level assessed by mfc is independent risk factor for both lfs and os in patients undergoing allohct. in addition, the results of our study show that mac allohsct outcomes in patients with aml in first and second cr are significantly influenced by both mfc-mrd status and eln cytogenetic and molecular genetic risk. [[o023 image] 1. leukemia-free survival according to pre-transplant minimal residual disease level] disclosure: nothing to declare o024 second allogeneic stem cell transplantation in acute lymphoblastic leukemia patients in second complete remission or relapse: a study on behalf of the alwp / ebmt background: second allogeneic transplantation (hsct2) is a therapeutic option for patients (pts) relapsing (rel) after first hsct (hsct1) however most of the available data is in acute myelogenous leukemia (aml) and there is very limited data on hsct2 in patients (pts) with all. therefore, the alwp of the ebmt performed a large registry analysis to study the outcome of hsct2 in pts with all. methods: we studied 245 pts receiving hsct2 as a salvage treatment between the years (y) 2000-2017 for rel following hsct1 in cr1. median follow-up of surviving pts was 58 months (iqr: 24-98). results: 142 pts (58%) were males and median age at hsct2 was 34.6 years (range: 18-74). median time from hsct1 to hsct2 was 463 (63-5482) days (d) and from rel to hsct2 it was 114 (15-348) d. at the time of hsct2 157(64 %) pts were in cr2 while 88 (36%) had advanced disease.101 (41%) pts received sibling donor (msd) and 144 (59%) unrelated donor (ud) hsct2 (10/10-63; 9/10-10, missing hla-71). in 34% of pts with available data the hsct2 was performed from the same donor. the majority of pts with available mrd data transplanted in cr2 (64/84 pts) were mrd negative pre-hsct2. karnofsky performance status was >90 in 60% of pts. 93% were transplanted with pb graft. 204 (83%) pts received chemotherapy based conditioning (reduced intensity 63%, myeloablative 37%) while it was tbi based in 41(17%) pts. 109 (52 %) pts received in vivo t-cell depleted (tcd) grafts. 94% of the pts engrafted. acute graft-versus-host disease (agvhd) ≥ ii and ≥ iii-iv occurred in 33% and 17% of the pts. incidence of 2y total and extensive chronic gvhd was 38% and 19%, respectively. main causes of death were leukemia recurrence in 54%, gvhd in 18% and infections in 19%. at 2 and 5 y, the cumulative incidence of nrm, ri, lfs, os and grfs were 24% & 26 %: 56% & 62%, 20% & 12 %, 30% & 14 % and 12% & 7%, respectively. in mva no factor predict nrm. in multivariate analysis, ri was independently associated with time from hsct1 to rel, agvhd ≥ii after hsct1 and in vivo tcd and lfs the prognostic factors were time from hsct1 to rel and kps≥90 at hsct2. factors associated with os were age (per 10 years), time from hsct1 to rel, ric at hsct1, kps>90 at hsct2 and ud vs msd. longer time internal from rel to hsct2 and in vivo tcd was associated with inferior cgvhd. lastly for grfs the prognostic factors were time from hsct1 to rel, agvhd ≥ii after hsct1, ric at hsct2 and kps≥90 at hsct2. conclusions: results of hsct2 in all pts with rel or cr2 are poor with only 14% os and 7% grfs at 5 y with very high ri of 62%.the prognostic factors are similar to those previously reported for hsct2 in aml. the future goals are to prevent and treat relapsed all by mrd driven novel monoclonal antibodies and car-t cell therapy. disclosure: nothing to declare. aplastic anaemia outcomes of allogeneic stem cell transplantation (hsct) for older patients (> 50 years) with severe aplastic anaemia using alemtuzumab-based ('fcc') regimen: king's college hospital experience background: treatment of older patients with severe aplastic anaemia (saa) is problematic with poor long-term survival after treatment with antithymocyte globulin (atg) and/or ciclosporin (csa). use of fludarabine, low dose cyclophosphamide (cy) and atg ('fcatg') conditioning suggests better outcomes among older patients transplanted from matched sibling donors compared to high dose cy/ atg conditioning, but gvhd remains a serious concern. we have transplanted saa patients aged > 50 years, predominantly from unrelated donors, using alemtuzumabbased ('fcc') regimen. methods: from our fcc saa database of 65 patients, 27 aged > 50 years were transplanted between 2007-18. median age was 61 years (range 51-71); 12 aged 50-59 and 15 aged ≥ 60 years. donor was matched sibling (msd) in 6 (22%), 10/10 matched unrelated (mud) in 18 (66%), 9/ 10 unrelated (mmud) in 3 (12%). conditioning was fludarabine 30mg/m 2 x 4, cy 300mg/m 2 x 4, alemtuzumab 0.2mg/kg daily from day -7 to -3. post graft immune suppression was csa alone. 2gy tbi was added to fcc for mmud hsct. all patients received peripheral blood (pbsc) as stem cell source. 10/27 (36%) patients were hla alloimmunised. pb telomere length (tl) by multiplex qpcr measured in 17 patients, was < 1 st centile in 3 (17%), < 10 th centile in 2 (11%) and normal in 12 (70%) patients. first line hsct was performed in 2/6 (38%) msd and 3/21 (12%) among unrelated donors. hct comorbidity index (htc-ci) score was 0-1 in 10 (37%); 2 in 7 (25%) and >2 in 10 (37%). results: three patients had invasive fungal infection at time of hct and died day +14 to 21, and one patient died at 11 months from multiorgan failure with recurrent parainfluenza virus and cmv. median cd3 chimerism was 71% (1-100), 77% (23-100) and 60% (22-98) at day +100, iyr and 3yr post hsct. one late graft failure at 6 months was associated with low csa blood levels, and was followed by successful 2 nd transplanted with no gvhd. 5-year os was 86%, compared to 96% among 27 patients aged < 50years (p=0.14). os was 89% and 82% for patients aged 50-59 and ≥ 60 year, respectively, p=0.8. os for msd, mud and 9/10 mmud was 100%, 80% and 100%, respectively. htc-ic score of >2 was associated with worse os of 72% compared to 94% with score < 2, p=0.13. outcomes were comparable irrespective of telomere length (84% vs 80% for normal vs short telomere, p=0.76). cumulative rates of acute and chronic gvhd were 5% and 12%, respectively. all cases of acute gvhd were confined to skin and grade i/ii only, and no cases of severe chronic gvhd. 17 (62%) patients needed dose reduction of csa with addition of mycophenolate due to renal dysfunction. rates of cmv and ebv reactivation were 25% and 27% respectively, with no cmv or ebv disease. conclusions: fcc conditioning regimen enabled high survival and low risk of gvhd among older patients with hct-ci score < 2 and who did not have established invasive fungal disease at time of hsct. clinical trial registry: not applicable disclosure: no conflicts of interest to declare haploidentical transplantation with post-transplant cyclophosphamide (haplo-ptcy) for 71 patients with acquired or inherited bone marrow failure syndromes (bmf): the experience from curitiba, brazil background: availability of unrelated donors as well as time to find a donor and the costs related to graft acquisition are important limitations in countries with ethnical minorities and fewer resources. methods: we describe the experience of 78 transplants in 71 patients(pts) with acquired or inherited bmf submitted to an haplo-ptcy transplantation between 04.2008 and 08.2018. the median age was 9ys, 70% were male and 94% were cmv positive. haplo-ptcy was the 1 st transplant for 62 pts, second or third for 9pts. diagnosis: fanconi anemia (fa,n=48), acquired severe aplastic anemia (asaa,n=10), telomere diseases (n=6); other inherited bmf (n=7). all pts had failed prior therapies and 96% had previous blood transfusions. the majority received a ric regimen with low dose tbi (n=69, 97%). donors were father(n=28), mother(n=31), other relatives(n=12). bone marrow was the stem cell source in 70pts. all pts received gvhd prophylaxis that included ptcy followed by cyclosporine and mycophenolate mofetil. fa pts received a modified preparatory regimen and ptcy at a total dose of 50mg/kg (n=32) or 60mg/kg (n=16) while other bmf received 100mg/kg results: fa pts: 14 pts did not receive atg in the preparatory regimen and all engrafted, despite the presence of donor specific antibodies(dsa) in 2 pts. three pts had aml and 2 are in remission 3 and 6ys after transplant. 7pts died due to gvhd (n=5); toxoplasmosis/cmv pneumonia (n=1) or relapse (n=1). 7/14 pts are alive with a median follow-up(fu) of 6.7ys. in the subgroup of fa pts receiving r-atg(n=34), 3pts presented primary or secondary graft failure(gf), none had dsa and all died despite a 2 nd haplo-ptcy with different donors. 6pts had advanced disease and 4 are in remission at the last fu. 26/34pts are alive at a median of 4 ys after transplant. eight pts died due to gvhd (n=4); rsv pneumonitis (n=1) and gf (n=3). all 10 pts transplanted with asaa are alive and fully engrafted at a median of 5.6ys after transplant and none developed gvhd, nine out of 13pts transplanted for other inherited bmf are alive at a median of 3ys after transplant. gf occurred in 4pts, all received a 2 nd haplo-ptcy from different donors and 2 are alive and engrafted. 4pts died due to gvhd (n=1), gf (n=2) and tma (n=1). altogether cmv reactivation occurred in 51pts (72%), at a median of 31 days (range:15-90) and hemorrhagic cystitis in 31pts (44%) at a median of 44 days (range:8-65). after transplant. conclusions: haplo-ptcy for pts with acquired or inherited bmf should be offered for those who need an immediate transplant but lack a matched donor. 70% of pts are alive at a median fu of 3ys but gvhd is a major complication for pts with inherited bmf, especially fa. new approaches to gvhd prophylaxis and treatment are needed in order to improve quality of survival for these pts. disclosure: nothing to declare relationship between plasma rabbit anti-thymocyte globulin level and response to immunosuppressive therapy in patients with severe aplastic anemia: results of a multicenter, prospective, randomized study background: patients with acquired aplastic anemia (aa) who do not have hla-matched donors receive immunosuppressive therapy (ist) with anti-thymocyte globulin (atg). previous studies have suggested several variables that predict response to ist. however, no studies have investigated the plasma atg level as a variable. in this study, we assessed the relationship between plasma rabbit atg (r-atg) level and response to ist in patients with severe aa. methods: patients with severe aa who required initial ist were enrolled from may 2012 to october 2017. the ist regimen included r-atg (thymoglobulin®, sanofi, cambridge, 2.5 or 3.5 mg/kg/day for 5 days) and cyclosporine a (6 mg/kg/day for minimum 6 months). plasma r-atg level was measured using a rabbit igg elisa kit on days 14 and 28. response rate was defined as complete and partial responses at 6 months. receiver operator characteristic curves were generated to discriminate between response and no response to ist. results: a total of 81 patients (aged 1.7-67.9 years) were randomized; 43 and 38 patients received 2.5 and 3.5 mg/kg of r-atg, respectively. in the 2.5 mg group, the response rate was 63%, which was comparable with that in the 3.5 mg group (58%) (p = 0.820). plasma r-atg level greatly varied in both groups. median r-atg level on days 14 and 28 after ist was 15.2 (0.0-97.7) and 1.8 (0.0-74.9 μg/ml), respectively, which was not significantly different between two dosages of atg groups (day 14, p = 0.498; day 28, p = 0.404). according to the r-atg level, response rates were significantly higher in the group with higher r-atg level than in that with lower atg level (day 14, 88% vs. 52%, respectively; p = 0.006 and day 28, 79% vs. 46%; p = 0.005) (figure) . cut-off levels at days 14 and 28 were 21.6 and 4.8 μg/ml, respectively. the vast majority (90%) of patients with levels higher than cut-off levels on day 14 responded to ist. in multivariate analysis, higher atg levels at day 28 were independent favorable predictors of response to ist at 6 months (or = 0.29; 95% ci: 0.09-0.93; p = 0.037). there were no significant differences in the kinetics of lymphocyte subsets among patients treated with different dosages of atg. however, higher atg level was associated with lower cd4+ t and regulatory t cell numbers for the entire 6-month period. conclusions: the present data indicate interindividual variability in plasma r-atg level. higher atg level resulted in improved response to ist and correlated with prolonged immune reconstitution. individualized dosing of atg via a pharmacokinetic model may improve the response rate to ist and reduce the number of patients who require allogeneic stem cell transplantation following ist. clinical background: radiation and dna alkylating agents used in hematopoietic cell transplantation (hct) can cause organ damage, malignancy and death. these risks are heightened in patients with genetic bone marrow failure (bmf) syndromes driven by defects in cellular proliferation or dna repair, including dyskeratosis congenita (dc), which arises from impaired telomere maintenance. we hypothesized that proliferative defects in hematopoietic cells of patients with bmf and very short telomeres might permit myeloid engraftment following hct without the need for radiation or dna alkylating agents. we conducted a multi-center prospective trial (nct01659606) evaluating engraftment after hct without these agents. methods: we enrolled bmf patients with genetic validation of dc or lymphocyte telomere length < 1 st percentile by flow-fish. we performed hct using bone marrow allografts from related or unrelated donors matched at 7 or 8 of 8 hla alleles after a preparative regimen consisting of only fludarabine and alemtuzumab. graft versus host disease (gvhd) prophylaxis consisted of cyclosporine a and mycophenolate mofetil. the primary endpoint of the trial was donor myeloid engraftment, defined as an absolute neutrophil count ≥500 cells/μl by day +42 and donor myeloid chimerism >50% by day +100. [[o029 image] 1. engraftment and survival after radiation-and alkylator-free hct for bmf with very short telomeres] results: twenty patients (age 1.7-31.5 years old at hct) received treatment between august 2012 -october 2018 at 7 institutions. eighteen of the 20 patients received unrelated donor grafts (15 matched, 3 single-allele mismatched). primary myeloid engraftment was achieved in 19 of 20 patients (95%) at a median 22 days post-hct (range 1-35 days). the single patient with primary graft failure had dc-related liver disease and hypersplenism; in this case, splenectomy at day +47 promptly revealed donor myeloid engraftment. of the other 19 patients, 16 had sustained myeloid engraftment, with a median post-hct follow-up of 21 months (range 1-74 months). three patients had secondary graft failure. two of these had early graft rejection and underwent successful repeat hct using higher intensity regimens. the third patient maintained high donor chimerism after primary engraftment but developed severe neutropenia in the setting of multiple viral reactivations, and died of a fungal infection 90 days post-hct. there was one other death, due to dc-related gastrointestinal complications 19 months post-hct. none of the 16 patients who engrafted durably under the protocol regimen had acute gvhd. four had chronic gvhd (3 limited, 1 extensive), treated successfully with limited courses of topical or oral steroids. conclusions: we conclude that this radiation-and alkylator-free hct conditioning regimen is an effective strategy for bmf in patients with dc or very short lymphocyte telomeres. eliminating dna damaging agents may reduce hct complications including gvhd and enable transplant in patients with high-risk comorbidities. clinical background: the standard treatment of acquired aplastic anemia (aa) is either intensive immunosuppressive therapy (ist) or allogeneic hematopoietic cell transplantation (hct). as supportive measures, red blood cells and platelet transfusions are the mainstay of therapy and patients are often multitransfused, which in turn can lead to anti-human leukocyte (hla) alloimmunization. in acquired aa the rate of hla-alloimmunization has previously shown a higher frequency in patients with aa compared to hematological malignancies. however, these results date back before the general introduction of leukoreduction of blood products and photochemical pathogen reduction of platelet components, and are based on cell-based assays. in recent years, leukoreduction and pathogen reduction of blood products became standard in switzerland and the solid-phase assay (luminex® technology) is now widely available to test for hla-antibodies, allowing a more extensive and detailed characterization of hla-antibodies. with these techniques, less is known on the exact incidence of hla-antibodies and their magnitude, associated cofactors and its impact on treatment outcomes in acquired aa. methods: we retrospectively investigated 54 aa patients treated with ist (n=44) and/or hct (n=25) at the university hospital of basel and the university children's hospital of basel (switzerland) regarding hla antibodies since the introduction of testing with the luminex® at our center in 2008. at least one hla antibody measurement before and/or after therapy was available per patient. all patients received leukoreduced blood products and as of 2011 platelets treated with intercept® (uv+ amotosalen). results: overall, hla-antibodies were detected in 40 (74%) patients with a higher rate of hla alloimmunziation by severity of aa (p< 0.01). the median number of hlaantibodies in each patient before therapy (i.e. ist or hct) was 3 (iqr 0-25). in patients undergoing hct hlaantibodies were more frequent before treatment start as compared to patients with ist treatment (median 13 (iqr 0-45) versus 2.5 (iqr 0-16.5), p< 0.05). differences between treatments remained after adjusting for all covariates (p< 0.01). there was no statistically significant difference regarding the hla antibody mean fluorescence intensity (mfi) between the two treatment forms (overall mean mfi of 1580 +/-2075). the highest mean hlaantibody mfi before therapy was 4747 (+/-7221) with a maximum of 24020. females showed a significantly higher number of hla-antibodies (p< 0.01) and also higher mean mfi (p< 0.05). furthermore, the number of pregnancies was associated with higher numbers of hla-antibodies (p< 0.01), however the number of transfusions did not have significant impact on hla-antibody number and mfi. regarding outcome, there was no significant association between the number of hla-antibodies and engraftment as well as bleeding events. conclusions: hla-alloimmunization is still frequent in patients with acquired aa but today number of pregnancies and gender seem to be more important for development of hla-alloimmunization than number of transfusions. interestingly, patients treated by hct show a higher rate of hla-alloimmunization before treatment start in comparison to ist, thereby emphazing the importance of blood management and donor selection in hct in acquired aa as hla-antibodies can cause platelet refractoriness and can represent donor-specific antibodies in the setting of mismatched hct (e.g. haploidentical). disclosure: nothing to declare. health-related quality of life in systemic sclerosis before and after autologous hematopoietic stem cell transplant -a systematic review background: autologous hematopoietic stem cell transplantation (ahsct) for severe rapidly progressive systemic sclerosis (ssc) allows significant regression in skin and lung fibrosis and improvements in overall and event free survival up to 7 years after transplant. we undertook this study to synthesize the evidence on changes in healthrelated quality of life (hrqol) associated with ahsct for ssc. methods: autologous hematopoietic stem cell transplantation (ahsct) for severe rapidly progressive systemic sclerosis (ssc) allows significant regression in skin and lung fibrosis and improvements in overall and event free survival up to 7 years after transplant. we undertook this study to synthesize the evidence on changes in healthrelated quality of life (hrqol) associated with ahsct for ssc. results: the search returned 656 articles. eight were selected: 3 uncontrolled phase i or ii trials, 2 cohort studies and 3 rct (assist, astis, scot). hrqol data from 289 ssc patients treated with ahsct and 125 with intravenous cyclophosphamide (cyc) as a comparator were extracted. hrqol was assessed using the health assessment questionnaire-disability index (haq-di) (n=275 patients), the short-form health survey (sf-36) (n=249 patients) and the euroqol 5 dimensions (eq-5d) (n=138 patients). hrqol was analyzed as a secondary outcome in all studies. quality of the data was assessed as high. haq-di improved significantly more with ahsct compared to cyc (-0.58 vs -0.19, p=0.02 at 2 years in astis; 53% vs 16% improved at 4.8 years in scot). scores also improved pre-post ahsct in the uncontrolled studies (ranging from -0.6 to -1.7 points at one year (all p< 0.05), and up to -1.5 points at 7.5 years (p< 0.001)). sf-36 physical component summary scores were significantly better in subjects treated with ahsct compared to cyc (between group differences ranging from 26 points at one year in assist and 6.1 points at 2 years in astis (p=0.01); 56% vs 15% improved at 4.8 years in scot (p=0.02)). similar differences pre-post treatment scores were also reported in an uncontrolled study (increase of 20 points, p< 0.0001). in assist, there was a trend for the sf-36 mental component summary score to improve in the ahsct arm (46 vs 58, p=0.07) and worsen in the cyc arm (56 vs 42, p=0.04) at one year. there were no significant differences between the ahsct and cyc arms in astis and scot with 2.0 and 4.8 years of follow-up, respectively. post-treatment scores improved significantly compared to pre-treatment in an uncontrolled study (from 51 to 64 points, p=0.005). astis showed a significant difference in the index-based utility score of the eq-5d (0.29, p< 0.001) and a non-significant difference in the general health visual analogue scale (6.7, p=0.36) at two years, in favour of ahsct compared to cyc. conclusions: although there is heterogeneity in the reported data, ahsct in ssc was consistently associated with marked and sustained improvement in hrqol. this analysis provides additional compelling data for the role of ahsct in ssc when assessing patient's point of view. clinical trial registry: we hypothesised that ahsct induces a rebooting of thymic function, resulting in the re-development of a functional, tolerant immune system. we aimed to examine cellsurface and dna markers of recent thymic emigrants (rte's) longitudinally in a cohort of ms patients post-ahsct in order to identify markers that correlate with a durable treatment response. methods: peripheral blood mononuclear cells (pbmncs) were collected from patients enrolled in the phase ii trial at st vincent's hospital sydney for ahsct in ms (moore et al, jnnp in press). a multicolour flow cytometry panel to optimally identify rte's was performed on 10 patient samples at 0, 6, 12, 24 and 36 month timepoints, allowing us to track changes in thymic output longitudinally following ahsct. dna markers of thymic function -sj:b trec ratio was performed in the same cohort of patients, on the same bio-banked sample to enhance the validity of observed changes. statistical analysis was performed with graphpad prism. results: a sustained, significant increase in rte's and sj:b trec was detected between pre-transplant and 24 month post-transplant specimens (p = 0.024). in patients where similar analysis was able to be performed at 36 months a trend to increased markers of thymic output was observed. a correlation between rte's and sj:b trec was observed (r=0.70, p = 0.003). contrary to other publications in the field, trec as a marker of thymic output did not appear to be lower when patients were analysed by age (< 30 yrs vs. >30 yrs). greater thymic output as determined by sj:b trec was observed at all timepoints in patients who had evidence of sustained disease remission as opposed to patients who experienced disease relapse. conclusions: we have seen evidence that sustained thymic reactivation is a component of immune reconstitution following ahsct for ms. previous studies have only demonstrated thymic activity at 12 months but this study confirms that thymic activity remains prominent at 24 and even 36 months. this thymic regeneration may contribute to a durable clinical response in patients with ms post hsct. clinical background: allogeneic hsct offers the potential replacement of an aberrant immune system. this retrospective study assessed long-term outcomes of this strategy in patients treated for severe autoimmune diseases (ads), reported to the ebmt registry. methods: among the total 126 patients who received allogeneic hsct between 1997-2014, we received detailed questionnaires on long-term outcomes from 64 patients. the diagnosis of ad was hematological (n=21) and nonhematological (n=43), among pediatric (=45) and adult (=19) populations. the median age of patients at hsct was 11.14 years (pediatric: median 8.07 years, range 1.22-17.77); adult : median 31.30 years, range 21.43-51.57). all patients were refractory to previous immunosuppressive therapies (median of 4 lines of treatments, range 1-13), and eight of them received a previous autologous transplant. the graft source was pbscs in 38, bm in 24, and cord blood in 2 patients. donors were as follows; 41% mrd, 42% mud, 9% mmrd, 5% syngeneic and 3% cord blood. conditioning was mac in 35 and ric in 29 patients. serotherapy with atg was given in 16 patients, while 30 patients received alemtuzumab. post-transplant gvhd prophylaxis was cyclosporine-based for the majority of patients (n=54). results: median follow-up was 67 months (range 7.9-189 months). toxicity profile was similar to allogeneic hsct in other contexts. the incidence of grades ii-iv acute gvhd was 16.4% (95% ci: 8.4 -26.8 ) at 100-days; severe acute gvhd was reported in 6.5% of them. cumulative incidence of chronic gvhd was 32.5% (95% ci: 20. 8 -44.8) at 5-year; extensive manifestations were reported for 58% of chronic gvhd. overall graft rejection rate was 4.9%. seven secondary ad and one case of new malignancy (lymphoma) occurred. viral reactivations were reported in a total of 33 patients, including cmv (n=14), ebv (n= 10), adenovirus (n=5), bk virus (n=3), hsv (n=2), hhv6 (n=3) and vzv (n=2). seven cases of invasive fungal infection were reported (one aspergillosis and three candidiasis). ten bacterial infections (only 4 patients developed infection from gram-negative bacteria) and four pneumonia were observed. at the last follow-up complete clinical response was obtained in 69.4% of patients, while partial remission was reported in 6.5%. relapse incidence (ri) was 21.9% (95% ci: 11. 8 -33.9 ) at the last follow-up. post-hsct autoimmune disease specific treatment was required for 12 patients. in subgroup analysis among different diseases, the os rates were similar between immune cytopenia and other ads. at 5 years, os was 76% (95% ci: 64. 8 -87.3) , nrm was 13.3% (95% ci: 6.1 -23.2) and pfs was 64.8% (95% ci: 52.1 -77.6), with no differences between immune cytopenia (73.8%) and other ads (64.3%). by multivariate analysis, only one prognostic factor remained significantly associated with long-term outcomes: a more recent year of transplant (better os, p=0.007; lower chronic gvhd, p=0.047). conclusions: this large retrospective survey of the ebmt registry confirms the potential of allogeneic hsct to produce long-term disease remission in a large proportion of refractory ads, with acceptable toxicities and nrm. results: twenty-seven patients were evaluated before and at 6 months after transplant, 22 of which were additionally evaluated at 12 months. at 6 and 12 months after ahsct, patients presented significant improvement of mrss (p< 0.01), mip (p< 0.01), mep (p< 0.01), 6mwt distance (p=0.02), and physical (p< 0.01) and mental (p=0.02) components of the sf-36, when compared to pretransplant evaluations. no changes were observed in fvc after treatment. despite a transient decline in dlco at 6 months (p< 0.01) after transplant, dlco levels at 12 months were not different from baseline (pre-transplant). significant correlations were observed between the 6mwt distance and physical component score of quality of life (r=0.62, p< 0.01). no significant correlation was observed between pulmonary function and the other evaluated variables (mrss, respiratory muscle strength, physical capacity and quality of life). conclusions: ahsct significantly improves the functional status of ssc patients in the first year of follow-up. although the pulmonary function remained stable after ahsct, there was significant increase in the physical capacity and quality of life of patients. these results can be interpreted as positive outcomes of ahsct for ssc. disclosure: nothing to declare. model of multidisciplinary and multicenter approach for hsct for children with multiple sclerosis: long background: hsct for children with multiple sclerosis (ms) proved effectiveness and safety. it is required to improve the results by analysis of long-term follow-up and late effects. several challenges identified in multidisciplinary collaboration for successful treatment as well as a problem of switching these patients to the adult healthcare. we aimed to create a model of organization of help for children with severe refractory multiple sclerosis based on multidisciplinary and multicenter approach. methods: fifteen children with ms received autologous hsct (ahsct) from january 2010 to may 2018. gender: females -11, males -4. mean length of ms prior ahsct was 22.7±8.4 months and mean age of ms debut was 12.7 ±2.1 years old. all patients had severe refractory ms treated with corticosteroids, interferons, plasmapheresis and mitoxantron with negative results. these patients presented signs of neuroinflammation. mean baseline edss before the start of mobilization was 4.8±1.4. procedures included mobilization with the help of cyclophosphamide and filgrastim and conditioning: cyclophosphamide 200 mg/kg and hatg 160 mg/kg. all patients received at least 2 x 10˄6/kg cd34 + hematopoietic stem cells (mean 4.3 x 10˄6±2.6 x 10 ˄6). we analysed the incidence and nature of late effects in patients with at least one year of follow-up (based on the standard protocol for late effects). fertility preservation proposed for patients. ahsct as well as pre-and posttransplant care was done by multidisciplinary team involved both transplant and neurological team. technology of transfer patients to adult center for post-transplant observation was identified. results: all patients survived. mean time to engraftment was 11 ±1.6 days. eleven patients experienced culture negative fever, one patient -cystitis, and one patient had cmv reactivation within 100 days of the transplant. no patient experienced an edss increase post-hsct above baseline, and all patients improved. mean improvement of edss was 2.8±1.2 during the first 60 days after ahsct (fast recovery). in-time transplanted patients improved better. improvement confirmed by immunological data (increasing of immune regulation index and t-regs in comparison with the baseline). median follow-up period was 48 months (8-93 months) . four patients (26.7%) experienced exacerbations (both neurological and mri) in median of 2 years (1-3 years) after ahsct. no onsets of secondary autoimmune disease and malignancies was seen. cardiocascular late effects were seen in 6 patients and endocrine -in 3 patients (all females). all these late effects were successfully treated. patients after the age of 18 years old were transferred to partnering adult center. this center uses the same protocol for hsct (in adults) and posttransplant observation. detailed scheme of transfer developed. conclusions: ahsct for pediatric patients with severe refractory ms appears to be safe and effective method and in-time hsct can significantly improve the outcomes. most of the patients did not experienced exacerbations. late effects found in patients were successfully treated. we provide a best care for these patients in both childhood and adulthood by transferring them to adult center. thus, a unique multicenter and multidisciplinary model of care for children with severe refractory ms was found. disclosure background: neuromyelitis optica spectrum disorder (nmosd) is an inflammatory central nervous system disorder characterized, despite immunotherapy treatments, by life-long, severe, and disabling attacks of optic neuritis and myelitis. the aim is to determine if autologous nonmyeloablative hematopoietic stem cell transplantation could be an alternative treatment option. methods: following stem cell mobilization with cyclophosphamide (2 g/m 2 ) and filgrastim, patients were treated with cyclophosphamide (200 mg/kg) divided as 50 mg/kg intravenously (iv) on day -5 to day -2, ratg (thymoglobulin) given iv at 0.5 mg/kg on day -5, 1 mg/kg on day -4, and 1.5 mg/kg on days -3, -2, and -1 (total dose 6 mg/kg), and rituximab 500 mg iv on days -6 and +1. unselected peripheral blood stem cells were infused on day 0. aqp4-igg antibody status was determined by clia validated elisa or flow cytometry assays. cell killing activity was measured using a flow cytometry based complement assay. results: twelve (eleven aqp4-igg positive) patients were treated with a median follow-up of 54 months. ten patients are more than five years post-transplant. at five years, 80% of patients were relapse-free off all immunosuppression (p< 0.001). at one and five years after hsct, edss improved from a baseline mean of 4.3 to 2.8 (p<0.001) and 3.25 (p<0.001), respectively. nrs improved after hsct from a baseline mean of 69.5 to 83.8 at one year (p<0.001) and 85.9 at five years (p<0.001). the sf-36 quality of life total score improved from mean 34.2 to 55.1 (p=0.03) and 62.1 (p=0.001). aqp4-igg serostatus converted to negative in nine patients and complement activating and cell killing ability of patient serum was switched off. two patients remained aqp4-igg seropositive (with persistent cell killing ability) and relapsed within two years of hsct (p< 0.01). conclusions: prolonged drug-free remission with aqp4-igg seroconversion to negative following nonmyeloablative autologous hsct warrants further investigation in larger randomized controlled trial. clinical trial registry: identifier: nct00787722 clinicaltrials.gov disclosure: nothing to declare o038 abstract already published. autoimmune haemolytic anaemia after haematopoietic stem cell transplantation in children: a french multicenter study background: autoimmune cytopenias (aic) are a rare but serious complication of haematopoietic stem cells transplantation (hsct). the auto immune haemolytic anaemia (aiha) is the most frequent of these complications in paediatrics and is difficult to treat. so far, there has been no nationwide post transplantation aiha study. methods: this observational, retrospective and multicentric study focused on french paediatric cases of aiha after hsct between january 2007 and january 2018. data was collected from national reference databases and direct interview of physicians. the inclusion criteria were patients between 0 to 18 years old who developed an aiha or an evans syndrome after hsct. data concerning patient, primary diagnosis, hsct procedure, pre-transplant viral status, blood group compatibility, characteristics of cytopenia, delay transplant-cytopenia, graft vs host reaction. laboratory characteristics and therapies were analyzed as well as the response to first, second or third line treatments. results: 2856 paediatric hsct were performed in 16 french paediatric centers between 2007 and 2018. among them, 36 children developed an aic: 30 aiha, 1 pancytopenia and 5 evans syndromes. the median age at hsct was 4,9 years old (0, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] 8) . average delay between transplantation and aic was 192 days (20-600). the median follow-up after transplant was 35,8 months (5-94) . 23 patients were transplanted for non-malignant disease (12 benign hemopathies, 11 immunodeficiencies) and 13 for a malignant one. stem cell source was bone marrow for 50% of patients, peripheral blood stem cells for 29% and cord blood for 12%. the donor was unrelated for 65% of hsct, matched related for 15% and 12% of procedures were haploidentical. 76% of patients received als as part of their conditioning regimen, it was myeloablative in 65 % (tbi based for 6%) and reduced intensity in 20%. direct anti-globulin test (dat) was positive to igg +/-c3d for 52% of patients, 16 patients had a blood group incompatibility (8 minor and 8 major). 64% of patients had an acute gvh and chronic gvh on time of aiha was find in 12% of patients. for 70% of patients the first line treatment was a combination of steroids (2mg/kg/day), intravenous immunoglobulin and rituximab (375mg/m 2 per week). for 41% of patients a second or third-line treatment was required (imurel, cellcept, cyclophosphamide, sirolimus, campath, bortezomib). 8 patients died (4 relapse, 2 infections and 1 severe anaemia, 1 multiple organ failure). conclusions: this is the first study describing precisely the aiha post hsct in pediatrics in france along with the various treatments used. it's a rare but severe complication with multiple risk factors associated with a high mortality rate and no standardized therapy at this time. further studies would allow us to understand the disease better in order to prevent its occurrence and treat it more efficiently. disclosure: nothing to declare background: the prognosis of relapsed/refractory acute lymphoid leukemia (all), non hodgkin lymphoma (nhl) or chronic lymphocytic leukemia (cll) is very poor, particularly in patients relapsing after autologous (autohct) or allogeneic hematopoietic cell transplantation (allohct). in the last decade, several chimeric antigen receptor anti-cd19 (car19) constructs have been developed. two of them (tisa-cel and axi-cel) are already approved by the fda and ema for all and nhl. however, the availability and affordability of these commercial carts remains a challenge in europe. methods: the first academic pilot clinical trial (clinicaltrials.gov nct03144583) using our fully academic (a3b1:cd8:4-1bb:cd3z) car19 was approved by the spanish agency of medicines on may/2017. eligibility criteria included r/r all (adult and pediatric), nhl and cll who had failed standard available therapy. the primary objective of the study was safety, and secondary objectives were response rate and its duration (progressionfree survival). here we report an updated analysis of this trial. results: as of december/2018 we have included 35 patients in the study. of these, we performed lymphoapheresis to 34 and we processed the cells of 33, although for the moment we have only infused to 25 of them. the diagnoses of these 25 patients were all in 20 (13 adults), nhl in 4 and cll in 1. median age was 23 years and 40% were women. of the 20 patients with all, 17 had relapsed after allohct, while 3/4 patients with nhl had relapsed after autohct. after conditioning with fludarabine (90 mg/ m 2 ) and cyclophosphamide (900 mg/m 2 ), we infused 0.4-5 x10 6 ari-0001 cells /kg, first as a single infusion (first 19 patients, cohort 1), and then in 2-3 fractions (last 6 patients, cohort 2). we have observed 5 (20%) cases of severe cytokine release syndrome (crs) and the non-relapse mortality (nrm) was 12% (3/25) . these deaths were due to crs (2) and pseudomembranous colitis (1), and all happened in cohort 1. we had no cases of severe neurotoxicity, and grade ii neurotoxicity was only seen in 2 patients. of the 14 patients with active all at inclusion, 11 had enough follow-up for efficacy analysis, and all of them achieved a cr (9 of them with negative mrd). with a median follow-up of 8.7 months (range, 1.3-14.8) , relapses were observed on days +223 (cd19+), +105 and +284 (cd19-), leading to a progression-free survival of 53% at 12 months. in patients with nhl/cll we have documented cr in 2, one of them (nhl) relapsing at day +259. one nhl patient has not been evaluated yet and the other 2 patients did not respond and have died due to their lymphoma. the cll patient, refractory to 5 lines of treatment, achieved a cr, which is maintained at day +377. conclusions: treatment with ari-0001 cells is effective with a response rate of 78.5% in all (pfs of 53% at 1y) and 50% in nhl/cll. we also observed cases of severe crs in 20% and a nrm of 12% of patients, though dose fractioning seems to improve safety profile. still, longer median follow-up and further patient inclusions are needed. clinical background: bcma car-t cells have demonstrated substantial preclinical and clinical activity for relapsed/ refractory multiple myeloma (rrmm) patients. in different clinical trials, the overall response rate (orr) varied from 50% to 100%. complete remission (cr) rate varied from 7% to 60%. previous studies indicated higher car-t cell expansion in vivo achieved better remission. here we developed a bcma car-t cell product manufactured via lentiviral vector-mediated transduction of activated t cells to express a second-generation car with 4-1bb costimulatory domain. methods: our trial (chictr1800017404) was initiated to evaluate the safety and efficacy of autologous bcma car-t treatment for rrmm. the enrolled rrmm patients either had received at least 3 prior treatment regimens, including a proteasome inhibitor, an immunomodulatory agent, anti-cd38 monoclonal antibody or were double or triple-refractory, and have over 30% bcma expression. patients were subjected to a lymphodepleting regimen with flu daily for 3 days (30mg/m 2 , d4 to d2) and cy (500mg/ m 2 , d-3 to d-2) prior to the car-t cell infusion (d0) at a dose range of 1-5 x10 6 car+ cells/kg. results: as of the data cut-off date (nov 28th, 2018), 17 patients, median age 61.8 (49 to 75) years old, with a median of 4.9 (0.4 to 10.8) years since mm diagnosis, were infused with bcma car-t cells. patients had a median of 14 prior regimens (range 4 to 24) ( figure 1a ). all the 17 patients were eligible for initial evaluation of early clinical response with a median observation period of 12 (0.9 to 40) weeks. orr was 100%. all the patients achieved mrd negative in bone marrow by flow cytometry within 1-2 weeks after car-t infusion. partial response (1 pr, 5.9%), very good pr (6 vgpr, 35.3%), and complete response (10 cr, 58.9%) within 12 weeks post car-t infusion were achieved. durable responses from 4 weeks towards the data cut-off date were found in 15/17 patients (88.2%). the disease progressed in 1 patient from pr by week 5 ( figure 1b ). one patient died of severe infection by day 9. all patients had detectable car-t expansion by flow cytometry from day 3 post car-t cell infusion. expansion peaks were found between day 6 to day 17. the peak car-t cell expansion in cd3+ lymphocytes of peripheral blood (pb) varied from 35% to 95% with a median percentage of 83.5% ( figure 1c ). the peak absolute number of car-t cells in pb varied from 530 to 23000 per μl with a median number of 10300 per μl. cytokine release syndrome (crs) was reported in all the 17 patients, including 2 with grade 1, 4 with grade 2 and 11 with grade 3. car-t -related aes were 9 pancytopenia (52.9%), 14 fever (82.3%), 8 nausea (47.1%), 1 heart failure (5.8%). no patient died of crs complication. conclusions: our data showed bcma car-t treatment is safe with prominent efficacy. we also observed high expansion level and long term persistence of bcma car-t cells contribute to potent anti-myeloma activity. these initial data provide strong evidence to support the further development of this anti-myeloma cellular immunotherapy. clinical trial registry: chictr1800017404 disclosure: nothing to declare chimeric antigen receptor (car)-mediated bcl11b suppression in lymphoid progenitor cells propagates natural killer-like cell development background: the transcription factor b cell cll/lymphoma 11b (bcl11b) is indispensable for t lineage development of lymphoid progenitors. pre-fabricated t cell products, allowing for a wider choice of effectively targetable antigens, being applicable to a wider range of patients, and minimizing the risk of long-term sequalae from on target/off tumor effects would be highly desirable. here we hypothesized that antigen receptor engineering of hematopoietic stem cells would fundamentally impact lymphoid progenitor cell fate and as a consequence biological properties thereby allowing to generate an ubiquitously available lymphoid cell product for targeted immunotherapy across mhc barriers. methods: murine hematopoietic stem cells were transduced with a broad panel of 1 st and 2 nd generation murine cars containing different costimulatory domains and numbers of active immune receptor-based activation motifs (itams) against cd19 in a lentiviral backbone and consecutively differentiated into lymphoid progenitors using the op9-dl1 feeder layer system. resulting products were comparatively assessed in vitro and in vivo (facs, functional assays, microarray gene transcript analysis, western blotting, timely controlled transgene expression, leukemia challenge experiments, in vivo lymphoid depletion experiments) upon co-transplantation into a mhc-mismatched myeloablative transplantation model for relapsed leukemia. results: car expression on early ex vivo generated lymphoid progenitors suppresses bcl11b ( figure 1b ) and leads to decreased t cell-associated gene expression. concomitantly, suppressed bcl11b permits lymphoid progenitors to acquire nk cell-like properties ( figure 1a ) and upon adoptive transfer into hematopoietic stem cell transplant recipients they differentiate into carinduced killer cells (carik) that mediate potent antigendirected antileukemic activity across mhc barriers ( figure 1c ,d). a cd28, but not 4-1bb, costimulatory domain and active itams are critical for a functional carik phenotype. conclusions: these results give important insights into differentiation of lymphoid progenitors driven by synthetic car transgene-expression and inform the potential use of ex vivo generated carik as an "off-the-shelf" product for targeted immunotherapy. disclosure: m.v.d.b has ip licensing with seres therapeutics and juno therapeutics. m.v.d.b. has also received honorariums from flagship ventures, novartis, evelo, seres, jazz pharmaceuticals, therakos, amgen, merck & co, inc., acute leukemia forum (alf), and dkms medical council (board) and research support and has stock options with seres therapeutics. a.g. has received research support from aprea therapeutics and infinity therapeutics. all remaining authors have no conflict of interest. [[o043 image] 1. figure 1 : car-induced killer (carik) cells provide strong anti-leukemia effects in vivo.] (a) c57bl/6 (b6) recipients received 3 x 10 6 b6 tcd-bm only or additionally with 8 x 10 6 syngeneic im1928z1lymphoid progenitors. numbers of nk1.1 + carik cells and frequencies of cd3 + tcrβ + progeny within the tom+ gate in spleens on day 28 (im1928z1, n= 5; itom, n= 4). statistics was performed by using students t-test (twotailed). data represent means ± s.e.m. (b) western blot analysis for bcl11b in lysates from itom lymphoid progenitors, im1928z1-lymphoid progenitors or b6 wt thymocytes. (c) recipients co-transplanted with either syngeneic (syn) or mhc class i and ii mismatched (allo) im1928z1-expressing lymphoid progenitors (n= 10 mice, respectively) received 1.2 x 10 6 c1498-mcd19 leukemic cells on day 21 after transplantation and monitored for survival. (d) car-lymphoid progenitor-co-transplanted mice were treated with weekly i.p. injections of either an anti-nk1.1 antibody (clone: pk136; 200μg/dose) or with pbs for control (n= 10 per group). all mice received 1.2 x 10 6 c1498-mcd19 cells on day 21 after transplantation and were monitored for survival. survival curves were compared using mantel-cox (log-rank) test. significant differences are indicated by *p < 0.05, ***p < 0.001, ****p < 0.0001. characterization of an hla-dpb1 specific t-cell receptor for adoptive immunotherapy sebastian klobuch 1 , elisabeth neidlinger 1 , carina mirbeth 1 , wolfgang herr 1,2 , simone thomas 1, 2 background: hla-dpb1 mismatches occur in up to 80% of allogeneic hematopoietic stem cell transplantations from hla 10/10 matched donors and were shown to be associated with a decreased risk of leukemia relapse. therefore, targeting hla-dpb1 mismatched antigens by donor t cells seems to be an attractive strategy to enhance graft-versusleukemia effect. we recently established a reliable method to generate and isolate hla-dpb1 mismatch reactive t cells receptors (tcr). tcr-modified t cells showed leukemia eradication of primary human aml blasts in a xenogeneic nod/scid/il2rgc -/-(nsg) mouse model. however, human fibroblasts used as surrogate cells for graft-versus-host (gvh) reactivity were also recognized by hla-dpb1-specific t cells upon ifn-γ pretreatment, which up-regulates hla-class ii expression on these cells. in this project, we aim at the isolation of tcrs recognizing mismatched hla-dpb1 only on hematopoietic cells, which might lower their risk for gvhd. methods: naive-enriched cd4 t cells were stimulated with autologous dendritic cells expressing allo-hla-dpb1 alleles upon rna transfection. to drive the outgrowth of cd4 t-cell populations expressing 'cd4-independent' tcr which allow the redirection of both cd4 and cd8 t cells, we blocked the cd4/hla interactions by the addition of cd4 binding antibody. allo-hla-dpb1 reactive cultures were analyzed for their recognition of primary aml blasts in ifn-γ elispot as well as 51 chromium-release assays. highly reactive cd4 t cell populations were further analyzed for their ifn-γ secretion against nonhematopoietic cells. tcrs from most promising cd4 t cell clones with an hla-dpb1 specific recognition of hematopoietic but not non-hematopoietic cells were isolated and further analyzed. results: two cd4-independent t cell clones with reactivity to the hla-dpb1*03:01 mismatch allele specifically lysed hla-dpb1*03:01 + primary aml blasts. most importantly, one of these t cell clones did not show ifn-γ secretion upon co-culture with ifn-γ pretreated primary fibroblasts. therefore, we isolated this tcr and transferred it into cd4 and cd8 t cells from healthy donors. tcr dp03 re-directed cd4 and cd8 t cells specifically recognized and lysed primary aml blasts from several hla-dpb1*03:01 + patients in vitro. again, cells of non-hematopoietic origin (fibroblasts) were not recognized even after ifn-γ pretreatment and hla-dp upregulation. to optimize tcr expression and activity, we exchanged the constant domains of the tcr by their murine counterparts. this modification not only led to a higher ifn-γ production and lysis of aml blasts, but also induced recognition of ifn-γ pretreated fibroblasts. however, tumor cell lines overexpressing hla-dpb1*03:01 were also recognized by cd4 t cells engineered with the wild-type or murinized tcr dp03 , suggesting that recognition of hematopoietic and non-hematopoietic cells is rather triggered by the avidity between the t cell and the target cell than by the tcr target epitope. conclusions: in conclusion, our data suggest that allo-hla-dpb1 specific tcrs are powerful therapeutic off-theshelf reagents in allogeneic t-cell therapy of leukemia. the isolation of allo-hla-dpb1 specific tcr without crossreactivity to non-hematopoietic cells might be one strategy to avoid hla-dpb1 specific gvh reactivity upon inflammatory situations (e.g. viral infections). however, our data also indicate that finding of the most suitable tcr candidate is challenging. disclosure: nothing to declare. abstract already published. infusion of memory t cell (cd45ra-depleted) dli improves cmv-specific immune response early after abt cell-depleted hsct: first results of a prospective randomized trial background: abt cell depletion effectively prevents severe gvhd in mismatched hsct, but in a proportion of cases delayed immune recovery leads to increased infection risk and nrm. we've shown in a pilot study that infusion of low-dose memory t cells (cd45-ra depleted) is safe after engraftment among recipients of ab t cell-depleted grafts (pmid:29269793). we initiated a prospective trial to directly test the efficiency of this approach. we report here an interim result of a prospective, randomized, single-centre trial (nct02942173). methods: a total of 100 paediatric patients with malignant disorders (all, n=56; aml, n=30; nhl, n=8; acute mixed lineage leukemia, n=5 and mpd, n=1) were enrolled between october 2016 and september 2018. patients were randomly assigned to receive cd45ra-depleted dlis (experimental arm), n=54, or not (control arm), n=46. median age at hsct was 8.9 years, m:f ratio -42:58. the conditioning consisted of either treosulfan (n= 50) or tbi (n= 50) in combination with fludarabine and thiotepa. gvhd prophylaxis included tocilizumab at 8 mg/kg at day 0, abatacept at 10 mg/kg at day 0, +7, +14 and +28, and bortezomib at 1,3 mg/m 2 at days -5, -2, +2, +5. neither antithymocyte globulin nor calcineurin inhibitors were used. donors were hla-haploidentical (n=94) or matched (n=6). all donors and 76% of the recipients were cmv seropositive. pmbc grafts were split and tcrαβ/cd19 depletion and cd45ra depletion were performed with clinimacs prodigy. the median dose of cd34+ cells was 10x10 6 /kg, αβt cells -28x10 3 /kg. in the experimental arm memory dlis were infused on day 0 at 25x10 3 /kg and on days +30, +60, +90, +120 at 50x10 3 /kg. in the control arm 8 patients received dli after engraftment to prevent relapse (n=6) or treat infections (n=2). primary endpoints were the cumulative incidence (ci) of cmv viremia (>500 copies/ml) by day +100 and the ci of acute gvhd grade ≥ ii. results: median follow-up for survivors was 1 year (0,2-2). engraftment of wbc and platelets was achieved in 99 pts, one patient died at day +8. wbc and platelets engrafted at a median of 11 days and 14 days, respectively. the incidence of cmv viremia was 45% (36-56) overall, 41% (30-56) in the experimental arm vs. 50% (38-67) in the control arm, p=ns. the ci of agvhd ≥ grade ii was 10% (6-18) overall, 10% (4-23) in the experimental arm vs. 9% (4-24) in the control arm, p=ns. two patients died, one per treatment arm, resulting in 2% (0-14) ci of trm at 1 year among the whole cohort. causes of death were preengraftment bloodstream infection and disseminated adenovirus infection. patients randomized to experimental arm acquired anti-cmv reactivity significantly earlier, according to ifn-g elispot assay on day +30 after hsct (p=0,0001). conclusions: co-infusion of donor-derived memory dli with the αβ t cell-depleted graft is safe and improves recovery of virus-specific immune responses. replacement of atg with targeted blockade of cd28/cd80 costimulation and il-6 receptor does not compromise engraftment and gvhd control, and is associated with low rate of non-relapse mortality. [[o046 image] 1. disclosure: nothing to declare o047 conditioning prior to cd19-specific car (19-28z) t cells predicts response and survival in pediatric relapse/refractory (r/r) b-all background: cd19-specific car t cells have demonstrated clinical benefit in patients with r/r b-cell acute lymphoblastic leukemia (b-all). several factors have been associated with response including conditioning chemotherapy, cd4/8 ratio, and post infusion car t cell expansion. methods: we studied the feasibility of a multi-center trial of a msk-developed cd19-specific car (19-28z) for the treatment of r/r b-all, the toxicity following infusion, and performed predictor analysis for optimal response. pediatric and young adult patients with r/r b-all were eligible for infusion. patients received a cyclophosphamidebased (cy) conditioning of high dose (hd; 3g/m2) or low dose (ld; 1.5g/m2) chemotherapy. outcomes of interest were complete response (cr), overall survival (os). variables considered were conditioning regimen (hd-cy vs ld-cy), pre-treatment disease burden (mrd vs morphologic), complete remission (cr) status, absolute lymphocyte count (alc) change, and in vivo car t cell expansion. results: 25 patients were included; 17 patients received hd-cy and 8 received ld-cy prior to car t cell infusion. among evaluable patients (n=24), cr or cr with incomplete count recovery was demonstrated in 94% and 38% for hd-cy vs ld-cy cohorts respectively (p=0·01). os was superior in the hd-cy cohort as compared to the ld-cy cohort (median os = not reached; nr) vs. 4·7 months (p=0·004; figure 1 ). lymphodepletion (delta alc: prior to and following cy) was significantly higher in the hd-cy cohort as compared to the ld-cy cohort (p< 0·001; figure 2 ). the in vivo car t cell expansion (peak car t cell vector copy number/ml) in peripheral blood was higher in the hd-cy cohort as compared to the ld-cy cohort (p=0·01; figure 2 ). to less extent, disease burden prior to treatment with conditioning chemotherapy and car t cells impacted response. disease response was 93% (13/14) in low disease burden group (mrd-cohort) compared to 50% (5/10) in the high disease burden group (morphologic cohort) (p=0·05). os was also superior in the low disease burden group (median os = nr) compared to high disease burden group (median os = 4.3 months; p=0·01). combined response for hd-cy/mrd was 100% (12/12), hd-cy/morphologic 75% (3/4), ld-cy/mrd 50% (1/2), and ld-cy/morphologic 33% (2/6). grade iii/ iv toxicity occurred in 32% (8/25) of patients including severe cytokine release syndrome (scrs) in 16% of patients and severe car-associated neurotoxicity in 28% of patients. conclusions: in this preliminary analysis we demonstrate that dose intensity of conditioning chemotherapy positively correlated with response and overall survival for patients treated with car t cells and confirms, to a lesser extent, pre-treatment disease burden impacts both response and overall survival. clinical trial registry: nct01860937 disclosure: the authors acknowledge william lawrence and blanche hughes foundation provided funding for the conduct of this study with juno therapeutics providing funding for an extension cohort of patients. k.j.c. has received research support from juno therapeutics; has consulted, participated in advisory boards, or participated in educational seminars for juno therapeutics, and novartis. r. j.b. m.s. i.r. are co-founder, stock holders, and consultants for juno therapeutics. c.s.s. has received research support from juno therapeutics; has consulted or participated in advisory boards for juno therapeutics, kite and novartis. early and late hematologic toxicities in children and adults treated with cd19-car t cells elad jacoby 1,2 , shalev fried 1,2 , abraham avigdor 1,2 , bella bielorai 1,2 , amilia meir 1 , michal besser 1,2 , jacob schachter 1,2 , avichai shimoni 1,2 , arnon nagler ,1,2 , amos toren 1, 2 background: autologous t cells transduced with cd19directed chimeric antigen receptors show notable remission rate in advanced patients, leading to approval by the fda and ema for treatment of relapsed and refractory acute lymphoblastic leukemia (all) and non-hodgkin lymphoma (nhl). the most common adverse events reported are cytokine-release syndrome (crs) and neurotoxicity. here we study and profile of hematologic toxicity of patients following locally produced car t cells. methods: we studied the first 38 patients treated on a phase 1b/2 clinical trial using cd19 car-t cells for b-cell malignancies, focusing on hematologic toxicities (neutropenia, thrombocytopenia and anemia), from the initial lymphodepleting regimen till progression or an additional treatment was administered. cytokine levels were studied using milliplex map, human cytokine/chemokine panel ii. results: between july 2016 and march 2018, 38 patients were enrolled on the trial, and 35 patients who received car-t cells and survived more than 21 days were included in this analysis. neutropenia, thrombocytopenia and anemia occurred frequently (94%, 80% and 51%, respectively) after car t cell infusion, and were associated with a prolonged or biphasic nature: in 93% of patients hematologic toxicity occurred or were ongoing after 21 days from cell infusion, and in 52% (neutropenia) and 44% (thrombocytopenia), two trough levels were noted, the second trough occurring after day +21. later events of cytopenia, following more than 42 days from car infusion and in absence of further therapy, occurred in 62% (neutropenia), 44% (thrombocytopenia) and 17% (anemia requiring prbc transfusion) of patients. we identified a strong correlation between the late hematologic toxicities (thrombocytopenia and neutropenia, p=0.018, thrombocytopenia and anemia, p< 0.0001, anemia and neutropenia p=0.05). on univariate analysis, factors affecting late cytopenia were prior hsct (p=0.0015, 0.0083 and 0.02 for anemia, thrombocytopenia and neutropenia respectively) and higher crs grade p=0.003, 0.018 and 0.04 for late anemia, thrombocytopenia and anemia respectively). diagnosis (all vs nhl) or age were not correlated to the incidence of early or late post-car cytopenia. to further study potential causes of late events in patients who were in remission, and in absence of signs of hemophagocytosis, patients' serum was analyzed for chemokine panel at different time points. as expected, serum thrombopoietin levels were correlated with the platelet count. we observed that only in late events (more than 21 days from infusion) sdf-1 serum levels correlated to neutrophil count (r 2 =0.3, p=0.04). conclusions: cytopenia are common events following cd19 car t cell therapy, and may have a prolonged and even biphasic course. patients at risk include those following a recent hsct or with high grade crs. late neutropenia events which occurred later than expected recovery from conditioning chemotherapy and following resolution of crs, were correlated with serum sdf1 levels, similar to prior observations with late onset neutropenia related to rituximab (dunleavy, blood 2005 background: acute myeloblastic leukemia (aml) represents 40% of all leukemias of western countries, being the second malignant hemopathy in pediatric population. in the last decades, the survival rate has maintained around 60%, being relapse the main problem. it has been highlighted the role of immune system for the control of aml and new therapeutic strategies have been developed. in this setting, the use of alloreactive natural killer (nk) cells could play a key role not only in the context of hematopoietic stem cell transplantation (hsct) but also as adoptive immunotherapy in patients with minimal residual disease. in this project we proposed including the cellular therapy with haploidentical activated and expanded nk cells as adjuvant therapy in pediatric patients affected by aml in complete remission and without indication of hsct. methods: it is a multicentre, open, prospective and no randomized phase ii clinical trial, to evaluate the efficacy and safety of allogenic nk cells infusion from haploidentical donor after a lympho-ablative chemotherapy in pediatric patients affected by low and intermediate risk aml in first complete cytological remission. patients were treated according to spanish protocol (sehop 2002) including 2 inductions plus 2 consolidation cycles. after chemotherapy patients received the infusion of activated and expanded nk cells (nkae) after a lympho-ablative treatment based on cyclophosphamide 60mg/kg and fludarabine 125mg/kg. nk cells were obtained from 250 ml of peripheral blood from haploidentical donors selected based on alloreactivity of kir inhibitors and kir activators receptors. nk cells were activated and expanded for 3 weeks trough co-culture with the cellular line k562-mbil15-41bbl suitable for clinical use in humans (good manufacturing practices, gmp). results: at this time 14 products in 7 patients have been infused, with the following characteristics: nk cells (cd45 + cd56 + cd3 -) 86.64 ± 11.36%; t lymphocytes (cd45 + cd56 -cd3 + ) 2.94 ± 3.55%. one product was rejected for quality criteria. a mean of 51.14 ± 53.99 x 10 6 nk cells/kg and 0.69 ± 0.35 x 10 6 t cells/kg were infused. median age of the patients were 7 years (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) . treatment and infusions were well tolerated. the main adverse event was neutropenic fever in one patient. after a median follow up of 530 days (371-813) all the 7 patients are alive. only one patient showed relapse at day +307 after nk cells infusion and was submitted to an hsct from a matched unrelated donor. conclusions: the preliminary results of this trial suggest that nk cells infusion as consolidative strategy in pediatric patients with aml, is feasible and secure. the therapeutic effect should be confirmed in a larger cohort of patients. clinical trial registry: clinicaltrials.gov (nct02763475) and eudract (2015-001901-15) . disclosure: authors have no conflict of interest. the trial was funded by "fundación mutua madrileña" grant o050 tcrαβ/cd19 depleted and il-15 stimulated donor cell infusions can exert anti-tumor effects after allosct without inducing gvhd in vitro and in vivo background: natural killer (nk) cells and γδ t-cells have been shown to play a significant role in gvl effects after allogeneic stem cell transplantation (sct). both cell types are not restricted by mhc molecules which makes them unlikely to elicit graft versus host disease (gvhd) even in mismatched sct and therefore are suited also for cell-based posttransplant immunotherapy. incubation with cytokines strongly enhances their spontaneous and antibody dependent cellular cytotoxicity (adcc). methods: thus, we investigated the efficacy of a combination of il-15 stimulated nk-and γδ t-cells from mismatched donors in vitro and based on urgent medical need in ultra high risk patients. results: in vitro data: spontaneous cytotoxic activity and adcc with tcrαβ/cd19 depleted pbmcs from healthy donors against ls (neuroblastoma) and nalm-6 (leukemia) target cells after overnight stimulation with il-15 was significantly increased compared to non-stimulated cells (e: t ratio 20:1; ls cells: 43% and 61% vs 19%; nalm-6 cells: 44% and 68% vs 26%). the results for cd107a assays are in line with the cytotoxicity approaches. nk and γδ t-cells showed a significantly stronger stimulation after il-15 incubation +/-adcc, as measured by secreting cytokines (cd107a, infγ, tnfα). moreover, 7 patients at extremely high risk for relapse received nk-/ γδ t-cell infusions after previous transplantation from haploidentical (n=5) or matched donors (n= 2); diagnoses: t-all, relapse after 1 st sct n=1; b-lineage-all, cr after 2 nd sct, n=3; aml, relapse after 1 st sct, n=1; relapsed neuroblastoma, pr after 2 nd sct, n=1. for that purpose magnetic microbeads and the clin-imacs ® device were used for tcrαβ/cd19 depletion of leukapheresis products from the original donors. afterwards, remaining cells (nk, γδ t and myeloic cells) were stimulated with il-15 (10 ng/ml) overnight. efficacy of the stimulation procedure was measured in 3 leukapheresis products: after incubation with target cells (ls) both, nkcells as well as γδ t-cells showed a significant increase in cd107a secretion compared to non-stimulated effectors (n=4) from healthy donors (nk-cells: 75% vs 12%; γδ tcells: 44% vs 2%). a median number of 12.4x10 6 nk-cells and 1.66x10 6 γδ t-cells and 1.08x10 3 residual tcrαβ cells/ kg were infused. four patients had received therapeutic mabs (anti-gd2, anti-cd19 or anti-cd38) within 48 hours after cell infusion to induce adcc. side effects were treatable cytokine release syndrome in 3 patients; no gvhd occurred. infused cells could be tracked by cd69 expression for up to 26 days in peripheral blood. outcome: 2 patients with t-all and neuroblastoma responded and achieved another cr, 2 patients maintained their previous cr for up to now 55/222 days; 3 patients progressed/relapsed after 26/69/89 days. additional therapies were applied in 4/7 patients. 4 patients are alive (median follow up: 248 days after infusion), 2/4 are disease free. conclusions: infusion of a combination of nk/ γδ tcells can increase anti-tumor effects and adcc with appropriate therapeutic mabs without inducing gvhd even after mismatched sct. further investigations are needed to evaluate the role of this cellular therapy. disclosure: nothing to declare o051 abstract already published. identifying chimeric antigen receptor (car) centers and car activity in europe, a survey on behalf of the cellular therapy & immunobiology working-party (ctiwp) of ebmt background: the use of chimeric antigen receptor (car) t cells has shown outstanding efficacy in patients with relapsed/refractory b-cell lymphoid malignancies, as reported by many groups in united states (us) and china. car-t cell based activity in europe is still at early stages. few european academic and pharma-sponsored clinical trials are currently opened to inclusions, and access to the two ema-approved autologous car-t products remains limited for the majority of european centers, since they must undergo a tedious and non-harmonized qualification process imposed by the manufacturer as part of the drug label. improved awareness of car-t centers and ongoing car-t activities in europe are essential to promote european networking activities, improve competitiveness of eu medical centers, and develop clinical trials, education, accreditation, and registration/long-term follow-up, thus fulfilling the needs of stakeholders. methods: a survey questionnaire was prepared by the committee members of the ebmt cellular-therapy-&-immunobiology-working-party (ctiwp). questions were formulated to identify car-t centers and car-t based activity in europe, and to know the characteristics of centers and the organization for implementing a clinical car-t program. using monkey survey, the survey was sent to 566 ebmt-centers. results: 123 centers replied to the survey. 340 patients (all 45%, nhl 32%, mm 10%, cll 5%, aml 1.6%, solid tumors 7%) have been treated so far in 31 ebmt centers from 12 countries. most of these centers are jacie-accredited (90% for clinical activities; 93% for cell collection activities). 54 additional centers reported plans to start car-t cell administration within the following 6-months. most patients were treated in the setting of academic clinical trials (58.6%l) or pharma sponsored trials (36%l). few patients (7%) received marketed car-t cells. pcr (36%) and flow-cytometry (60%) were used to monitoring the persistence of car-t cells after infusion. cytokine levels were measured in 78% of the cases, and ngs was performed in 45% of the centers. half of the centers reported to use "point of care" manufacturing. patient management was mainly carried out by bone marrow transplant (bmt) physicians (85%). however, 77% of the centers reported to have also implemented a "car-t team", as a multidisciplinary team managed by a coordinator, with weekly clinical meetings. in more than 90% of the centers, there were specific rules for transferring patients to the intensive-care-unit. in contrast, only 40% of the centers had a nurse coordinator for management assistance of these patients. 77% of the centers plan to expand the capacity of their own department for car-t program. conclusions: this survey is the first attempt to gather information about car-t cell activity in europe. this first results confirms active and growing involvement and high quality standards of european centers having car-t cell program. the expanding use of these complex atmp approaches could be facilitated through harmonization of the clinical practice, shared analysis of good quality data, and by a centralized european clinical trial office. the cellular therapy car-t form is readily available to capture in more detail safety and efficacy of this intervention and will allow sharing in a transparent process registry data with stakeholders. methods: this is an open-label, phase 1 study (chictr1800017404). eligible patients had rrmm, confirmed as bcma-positive by flow cytometry or pathological examination. autologous t cells transduced with a bcmatargeted chimeric antigen receptor (car-bcma t cells) are used(1×10^7/kg car+t cells), following pre-infusion treatment--intravenous administration and lymphodepletion conditioning (fludarabine and cyclophosphamide). the primary outcome measure is incidence of adverse events (aes) especially clinical events, including crs, neurotoxicity and coagulopathy, et al. additional outcome measures are duration and expansion of bcma car t cells. results: 19 patients (median age of 61, range 41 to 71) with rrmm have received treatment. the median %pc in bm at enrollment was 30.23% (range 2% to 72%). 4/19 had high tumor burden, defined as ≥ 50% bone marrow plasma cells. 3/19 had prior autologous stem cell transplant. as of data cut-off (dec 1,2018), crs grade 1-2 occurred in 8 patients, grade 3-4 occurred in 11. surprisingly, not a few patients developed coagulation disfunction, manifesting elevated d-dimer, prolonged aptt, pt, tt, and decreased fibrinogen, with 0 case of organ bleeding. laboratory values correlating with crs reaching grade 3-4 (n=11) compared to those with milder crs (n=8) included peak ferritin (mean: 31095.23 vs 1665.53 ug/l, p< 0.005), peak il-6 (mean: 8001.07 vs 266.92 pg/ml, p< 0.005), peak il-10 (mean: 324.89 vs 43.81 pg/ml, p< 0.01), and peak ifn-γ(mean: 2936.46 vs 58.21 pg/ml, p< 0.05). no significant difference was seen in peak crp, il-2 and tnf-α. patients with crs 3-4 had a higher △aptt (mean: 38.86 vs 12.09 s, p< 0.01), △d-dimer (mean: 44509.18 vs 13347.75 ug/l, p< 0.05) and △fibrinogen (mean: -1.68 vs -0.34 g/l, p< 0.05) compared to those with milder crs. there was no significant difference in △pt and △tt.the changes of d-dimer and aptt were in line with the indictors of crs, such as crp, il-6 and il-10 in some individual cases. the significant correlation between aptt and crp (p< 0.05) appeared on 17 patients (89.5%), while the significant correlation between il-6 and aptt as well as between il-6 and d-dimer only appeared on 7 patients (36.84%). conclusions: early safety and efficacy results of bcma car t therapy in rrmm are encouraging. aes of coagulation were observed. changes of aptt, d-dimer and fibrinogen of patients with high crs level were more significant than those with milder crs. on the individual level, aptt correlated with crp in the course of therapy. given extensive cross-talk between inflammation and coagulation, coagulation-related indictors are more convenient for monitoring crs. also, our study suggests the importance in diagnosis and early management for coagulopathy to avoid crs-related mortality. clinical trial registry: chictr1800017404 http:// www.chictr.org.cn/abouten.aspx disclosure: nothing to declare o054 abstract already published. point-of-care production of cd19 car-t cells in an automated closed-system bioreactor: report of first clinical experience background: cd19 car-t cell products are approved as therapy for advanced b-cell malignancies. the predominant manufacturing model is a centralized industrial-type production process. an alternative approach to car-t cell production and delivery to the patient is via a point-of care manufacturing process. methods: between february 2018 to november 2018 a total of 15 pts with relapsed/refractory b-cell malignancies (4 female, 11 male[gr1], median age 12 y) were screened, 10 pts were enrolled for single center, phase i-ii trial of safety and clinical efficacy of automatically produced cell therapy product, 5 were eligible for compassionate use of therapy. seven patients had relapsed b-all after hsct, 6 pts had refractory relapse after chemotherapy, 1 pt had refractory induction failure, 1 pt had refractory primary mediastinal bcell lymphoma (pmbcl). eight patients received previous blinatumomab infusion. eight patients had high disease burden >5% blast cells in bm, median 89% (14-100), 6 pts had minimal residual disease (mrd) in bm. the clinimacs prodigy t cell transduction (tct) process was used to produce cd19 car-t cells from fresh patientderived leukapheresis product. automatic production included cd4/cd8 selection, stimulation with macs gmp t cell transact, transduction with lentiviral (second generation cd19.4-1bb zeta) vector (lentigen, miltenyi biotec company) and expansion over 10 days in the presence of texmacs gmp medium and macs gmp il-7/il-15 combination. final product was administered without cryopreservation after fludarabine/cyclophosphamide preconditioning. all patients received prophylactic tocilizumab at 8 mg/kg. results: all production cycles were successful. median transduction efficiency was 59% (32-75). median expansion of t cells was 36 fold (18-49). cd4/cd8 ratio in the final product was 0,6. the cell products were administered at 1*10 5 /kg of car-t cells in 5 pts, 5*10 5 /kg in 5 pts, 1*10 6 /kg in 5 pts. cytokine release syndrome occurred in 8 (53%) pts: grade i in 5 pts, grade ii in 2 pts, grade iv in 1 pts. car-t cell related encephalopathy occurred in 4 (26%) pts, including one fatal brain edema. grade i neurotoxicity had 2 pts, grade ii -1 pt, grade v -1 pt. four patients were admitted to the intensive care unit (icu). three patients died until day 28 (2 due to sepsis, 1 due to fatal brain edema and sepsis, on autopsy in the brain vessels of this patient were found klebsiella pneumoniae emboli) twelve patients were evaluable for response at day 28. three pts had persistent leukemia, without evidence of car-t expansion. mrd-negative responses were achieved in 8 cases among 11 evaluable cases with bone marrow involvement. patient with pmbcl had a decrease in metabolic activity on pet/ct scan. cd19(-) relapse after initial response was registered in 1 case at 109 day. conclusions: production of car-t cells with the clinimacs prodigy tct process is a robust option that provides a point-of-care manufacturing approach to enable rapid and flexible delivery of car-t cells to patients in need. robustness and consistency of this approach provides a solid basis for multi-center academic trials in the field of adoptive cell therapy. disclosure: nothing to declare background: in the last decade, several prognostic scoring models such as the international prognostic scoring system (ipss), the dynamic ipss (dipss) or dipss-plus have been developed for diagnosed primary myelofibrosis (pmf) and are currently used for decision finding regarding allogeneic stem cell transplantation. furthermore, the prognostic relevance of mutation profile resulted in a mutation-enhanced system (mipss70) in transplant-age pmf patients (70 years or younger). for secondary myelofibrosis, the mysec-pm was developed. while all scoring systems were developed in nontransplant populations and may be useful in decision finding regarding transplantation, uncertainty remains regarding usefulness of these systems to predict posttransplant outcome and thus counseling patients whether to proceed to transplant. methods: bone marrow or peripheral blood samples were obtained before transplantation and mutations were detected using next-generation sequencing. the following myelofibrosis-associated genes were sequenced: jak2, calr, mpl, asxl1, idh1/2, cbl, dnmt3a, tet2, sf3b1, srsf2, u2af1, ezh2, tp53, nras, kras, runx1, and flt3. cytogenetic analysis and reporting were performed according to the international system for human cytogenetic nomenclature criteria using standardized techniques. we examined 361 myelofibrosis patients, of whom 260 had pmf and 101 smf at time of transplantation. a training cohort of 205 patients was used to create a clinical-molecular transplant scoring system (mtss) predicting survival from cox models, internally validated by use of bootstrap and cross-validation. model discrimination was measured by the concordance index (c). the final mtss was externally validated in a cohort of 156 patients and was furthermore applied to posttransplant non-relapse mortality as a secondary objective. results: multivariable analysis on 5-year survival identified age ≥ 57 years, karnofsky performance status < 90%, platelet count < 150 x 10 9 /l and leukocyte count > 25 x 10 9 /l at time of transplantation, hla-mismatched unrelated donor, asxl1 mutated and calr-/mpl-unmutated genotype being independent prognostic factors for outcome. the uncorrected concordance index for the final survival model was 0.723 (0.713-0.733), and bias-corrected indices were similar. a weighted score of 2 was assigned to transplantation from an hla-mismatched unrelated donor and an calr-/mplunmutated genotype, whereas a score of 1 was assigned to older age, leukocytosis, thrombocytopenia, asxl1 mutation, and poor performance status. the mtss consisted of four distinct risk groups showing 5-year survival in the validation cohort of 83% (71-95%) for low (score 0-2), 64% (53-75%) for intermediate (score [3] [4] , 37% (17-57%) for high (score 5), and 22% (4-39%) for very high risk (score > 5), respectively (p < 0.001). increasing score was predictive of non-relapse mortality (p < 0.001) and remained applicable to pmf ( conclusions: we show here that this internally and externally validated mtss accurately discriminated different risk for death and may improve counseling patients with respect to transplant compared with currently existing systems, as well as facilitate design of clinical trials in the transplant setting. disclosure: nothing to declare. response to up-front azacitidine in juvenile myelomonocytic leukemia (jmml): interim analysis of the prospective european multicenter study aza-jmml-001 background: jmml is a chemotherapy-resistant neoplasia of early childhood. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative therapy, saving approximately 50% of these children. relapse is the major cause of treatment failure, with chemotherapy prior to hsct being notably unsuccessful. novel therapies controlling the disorder prior to hsct are urgently needed. methods: we conducted a phase 2, multicenter, open-label study to evaluate pharmacodynamics, safety, and antileukemic activity of azacitidine monotherapy prior to hsct in patients with newly diagnosed jmml. azacitidine was administered at 75 mg/m 2 /day intravenously on days 1-7 of a 28-day cycle for 3 to 6 cycles. the primary endpoint was the number of patients with clinical complete remission or clinical partial remission (cpr) at cycle 3 day 28 (c3d28); secondary endpoints included overall survival following hsct. results: eighteen jmml patients (13 ptpn11-, 3 nras-, 1 kras-, 1 nf1-mutated) aged 0.2-7.0 years were enrolled. median (range) wbc, platelet count and spleen size were: 19.7 (4.3-59.0) × 10 9 /l, 28 (7-85) × 10 9 /l, and 4 (2-14) cm below the costal margin, respectively. dna methylation class (lipka et al. nat comm 2017; n=17) was high, intermediate, or low in 10, 5, and 2 patients, respectively. sixteen patients completed 3 cycles of therapy and 5 of them completed 6 cycles. two patients discontinued treatment before completing 3 cycles due to disease progression. six patients (33%) experienced ≥ 1 grade 3 or 4 manageable adverse event, consistent with the known azacitidine safety profile. eleven patients (61%) achieved cpr at c3d28 and 7 had progressive disease either at c3d28 or prior. importantly, 8 of the 15 patients who needed platelet transfusions before or shortly after treatment initiation did not require transfusions at the time of hsct. seven of these 8 platelet responders had normalized their platelet count (≥ 130 × 10 9 /l). palpable spleen size decreased in 11 responders by a median of 3.5 cm after 3 cycles and ranged from 0-2 cm below the costal margin after 6 cycles. sixteen patients received allo-hsct from a family or compatible unrelated donor following a busulfan-(n=15) or treosulfan-based (n=1) preparative regimen after a median of 57 days (36-112) from last azacitidine dose. thirteen transplanted patients were leukemia-free at median follow-up of 15.7 months (0.1-31.7) after hsct. two children (both high methylation class) given hsct relapsed after the allograft. sixteen of the 18 patients were alive at a median follow-up of 19.8 months (2.6-37.3). one patient who discontinued treatment before cycle 3 died from disease progression, and 1 non-responder died from graft failure. conclusions: this study shows azacitidine monotherapy was well tolerated in children with newly diagnosed jmml. although the long-term advantage of azacitidine therapy remains to be fully assessed, both the decrease in spleen size and significant platelet responses observed demonstrate that the drug was effective in jmml and provided clinical benefit to jmml patients in this study. this clinical trial has shown that azacitidine therapy prior to hsct may be considered for patients with jmml. clinical trial registry: nct02447666 disclosure: charlotte niemeyer is a member of a board of directors or advisory committee and provides consultancy for celgene. claudia rössig is a member of a board of directors or advisory committee at amgen, eusapharm, roche, celgene, novartis, pfizer, bms; honoraria from amgen, roche, pfizer. andré baruchel provides consultancy (includes expert testimony) with celgene, novartis, servier, jazz pharma; research funding and honoraria from shire; honoraria from novartis, jazz pharma. susana rives has received honoraria (for talks in industria sponsored satellite symposia) from novartis, jazz pharma, baxalta, shire, servier; speaker's bureau at novartis, jazz pharma, baxalta, shire, servier, amgen, erytech pharma; membership on an entity´s board of directors or advisory committees at novartis; received travel and accommodation expenses for medical congresses from novartis, jazz pharma, baxalta, shire, servier, amgen, erytech pharma. marco zecca has received honoraria from chimerix and jazz pharma. marry m. van den heuvel-eibrink received honoraria from celgene (consultation fee). bouchra benettaib, noha biserna, jennifer poon, mathew simcock, meera patturajan are employees of and hold stock or other equity ownership in celgene. christian flotho, daniel lipka, jan starý, karsten nysom, gérard michel, thomas kilngebiel, franco locatelli, giuseppe basso, concetta micalizzi, irith baumann, markus schmugge liner have nothing to disclose. ruxolitinib before allogeneic hematopoietic stem cell transplantation (hsct) in patients with myelofibrosis: a preliminary descriptive report of the jak allo phase ii study marie robin 1,2 , raphael porcher 3 , corentin orvain 4 , background: allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment for patients with myelofibrosis; os is from 30 to 60% depending on age, comorbidities, disease status and type of donors. jak1/2 inhibitors have been reported to decrease constitutional symptoms and spleen size in one half of patients with a possible advantage of os as compared to best current treatment. retrospective studies show that ruxolitinib has been used in patients before hsct with apparent good tolerance. methods: in 2012, we initiated a phase ii prospective french collaborative (filo and sfgm-tc) trial testing the role of ruxolitinib given before allogeneic hematopoietic transplantation in patients with myelofibrosis. patients could be included if they were intermediate or high risk according to lille score or ipss. patients had to start ruxolitinib after inclusion and were transplanted in case a donor has been identified within 4 months. primary aim was progression-free survival at one year after hsct. results: 76 patients could be included. 53 (70%) had a primary myelofibrosis and 23 (30%) had a myelofibrosis secondary to essential thrombocythemia (n=10) or polycythemia vera (n=13). at time of inclusion, 43 (57%) patients had constitutional symptoms, 72 (95%) had palpable splenomegaly, 48 (63%) patients had hemoglobin < 10 gr/dl, 23 (30%) patients had peripheral blast cell count > or = at 1% and 16 (21%) had thrombocytopenia < 100 g/l. median follow-up was 31 months. at 4 months, one patient has died, 11 patients had no donor, 18 had an hla matched sibling donor, 31 had an hla matched unrelated donor and 15 had a 9/10 hla mismatched unrelated donor. 59 (78%) patients with a donor at 4 months could be transplanted of whom 18 underwent a splenectomy before transplantation. conditioning regimen was fludarabine 90mg/m2 in combination with melphaln 140mg/m2. gvhd prophylaxis was cyclosporine and mycophenolate mofetil with atg in unrelated donor. partial response under ruxolitinib was 26% while the whole complete response incidence was 46% at one year. os and pfs at 2 years were 68% (95% ci: 59-80) and 53% (95% ci: 43-66). os was significantly better in patients with hla matched sibling donor as compared to patients without a donor or with an unrelated donor (p=0.008, figure 1 ). cumulative incidence of acute gvhd on day 100 was 66% of whom 44% had grade 3-4 acute gvhd. cardiogenic shock generally associated with severe sepsis or "sepsis like" syndrome occurred in 7 patients: 2 patients were not transplanted because of this complication while in the 5 other patients cs occurred within the 15 days after transplantation. three renal failures secondary to tumor lysis syndrome were declared just after conditioning regimen initiation. figure 1 . overall survival in jak allo patients. conclusions: os was very good in patients with hla matched related donor and significantly better than in patients with an unrelated donor. more analyses are currently ongoing to determinate the potential role of cytokine release in peri-transplantation time, as well as specific myelofibrosis and quality of life questionnaires assessing the impact of ruxolitinib in patients. background: while cytogenetics may influence outcome in primary myelofibrosis (pmf) from diagnosis which lead to several prognostic systems incorporating different cytogenetic risk classifications, its definitive role specific after allogeneic stem cell transplantation is still unclear. here, we aim to evaluate the role of currently existing cytogenetic risk classifications included in the dynamic international prognostic scoring system (dipss)-plus and in the mutation-enhanced system (mipss70-plus version 2.0). methods: in this multicenter retrospective study, we examined 149 pmf patients undergoing allogeneic stem cell transplantation. current cytogenetic risk stratifications used in dipss-plus and mipss70-plus version 2.0 were evaluated. according to dipss-plus, an unfavorable karyotype includes +8, -7/7q, i(17)q, -5/5q,12p-, inv (3), and 11q23 rearrangements while mipss70-plus version 2.0 consisted of very high risk (vhr: single/multiple abnormalities of -7, i(17q), inv(3)/ 3q21, 12p-/12p11.2, 11q-/11q23, +21, or other autosomal trisomy, not including +8/+9), favorable (normal karyotype or sole abnormalities of 13q-, +9, 20q-, chromosome 1 translocation/duplication or sex chromosome abnormality including -y), and unfavorable (all other abnormalities). results: the median follow-up period of all 149 pmf patients was 56 months and the median time from diagnosis to transplant was 26 months. after five years, 51 (34%) patients had died. the median age at transplant was 57 years and 63% of patients were male. most allografts were applied using peripheral blood (97%) and were received from an hla-matched unrelated donor (42%), followed by mismatched unrelated (34%), matched related (23%), and mismatched related or haploidentical (1%). reduced intensity conditioning was applied to 75% of patients. splenectomy before transplant was undergone in 15% and ruxolitinib was received by 27%. frequencies according to driver mutation genotype were: calr (19%), jak2 (59%), mpl (5%), and triple negative (17%). asxl1 mutation was present in 38% while 64% had more than two mutations overall. 94 (63%) patients had normal karyotype. most frequent abnormalities were trisomy 8 in 11 (7%), trisomy 9 in 5 (3%), deletion in 20q in 17 (11%), deletion in 13q in 11 (7%), chromosome 7 in 5 (3%), chromosome 5 in 5 (3%), and chromosome 1 translocation/duplication in 2 (1%). more than two abnormalities were present in 9 (6%) patients. high cytogenetic risk category according to dipss-plus was present in 24 (16%) while 12 (8%) had vhr and 25 (17%) had unfavorable risk according to mipss70-plus version 2.0. regarding outcome, an unfavorable karyotype according to dipss-plus showed 5year os of 62% (52-72%) vs 63% (57-70%), with causespecific hazard being 1.16 (p=0.69). 5-year relapse and nonrelapse mortality rates were not significantly different showing 9% and 33% for unfavorable karyotype vs 17% and 28% (p=0.31 and 0.52). with respect to the three-tiered classification of mipss70-plus version 2.0, 5-year os of vhr was 74% (62-86%) and unfavorable was 52% (42-62%) vs favorable risk of 64% (58-70%), with cause-specific hazard of 1.09 (p=0.74). relapse and non-relapse mortality were 9% and 17% for vhr, 4% and 48% for unfavorable, and 19% and 26% for favorable risk (p=0.19 and 0.48). conclusions: current cytogenetic risk stratifications do not predict outcome in pmf after allogeneic stem cell transplantation. [[o060 image] 1. background: the aim of this study was to assess the outcome of patients with myelofibrosis allografted before and after 2010, in two transplant centers (genova san martino and rome gemelli). methods: we have studied 147 patients, divided in two groups: 2001-2010 (n=59) and between 2011 and 2018 (n=88). the age was significantly older in the most recent group (53 vs 58 years, p< 001), and there was a greater use of alternative donors (38% vs 80%, p< 0.0001), and more patients with dipss-r high score (40% vs 47%, p=0.1). the transplant score (based on transfusions >20 and splee size >22 cm) was intermediate-high risk in 82% and 58% (p< 0.01) of patients respectively. the conditioning regimen was a combination of thiotepa, busulfan fludarabine (tbf) in 1% and 87% of patients grafted before and after 2010 (p< 0.00001). the transplant risk score (ts) was based on spleen size (>22 cm) and pre-graft transfusion (>20) as previously described (bone marrow transplant. 2010 mar; 45(3) :458-63) results: outcome. the cumulative incidence (ci) of acute grade ii-iv (25%) and of chronic gvhd (18%) was comparable in the two time periods. the 5 year ci of non relapse mortality (nrm) was reduced overall from 37% to 20% (p=0.01) and the 5 year ci of relapse from 47% to 11% (p=0.00006). as a consequence the overall 5 year actuarial survival has improved from 35% to 64% (p=0.003). predictive factors. the following factors predicted survival in multivariate cox analysis: high risk transplant score (hr 2.5, p=0.01), high risk dipss-r (hr 2.0, p=0.01), the use of tbf (hr 0,50, p=0.02), alt donor (ht 1.8m p=0.04), donor age >29 years (hr 1.8, p=0 .06) and abo match (hr 0.64, p=0.08). dipss and transplant score. when combining dipss-r (high) and transplant score (int2-high) we could identify 3 groups : score 0 (dipss not high and ts not high) (n=40) score 1 (either high dipss or high ts) (n=51), score 2 (both dipss a ts high risk) (n=55) . nrm was 9%, 27% 40% in these 3 group (p=0.0004), relapse was 17%, 29%, 25% (p=0.1) and 5 year survival was 78%, 55%, 20% (p< 0.00001). in the current transplant era (>2010) the 5 year disease free survival of these 3 groups is 78%, 70%, 35% (p< 0.00001). abo matching further increases dfs for score 0 patients. nrm mortality for these 3 groups is currently 6%, 16%, 36% (p=0.01). conclusions: the 5 year survival of allografts in patients with myelofibrosis has improved overall from 35% before 2010, to 64% beyond 2010, despite the current use of 80% alt donors. predictive factors are the transplant score (transfusions and spleen size), dipss-r and the use of 2 alkylating agents (tbf), the latter being the major change in the 2 transplant eras. patients received ruxolitinib before the transplantation, 7 were transfusion-dependent. all the patients underwent pbsc infusion, except one who received bmsc. in 7 cases the source was a sibling donor, in 9 a mud, in 2 a hla-mismatched ud (1 mismatch). conditioning regimen was mostly based on the combination of singlealkylating agent and fludarabine (12 bu/flu; 3 mel/flu/ bcnu; 1 mel/flu; 1 treo/flu) with atg infusion. six patients received a fully myeloablative schema, 12 reduced-intensity conditioning. only one patient received conditioning with tbi. results: before the transplant, a panel of molecular analysis based on next-generation-sequencing revealed, in addition to the mpl mutation, alterations in asxl1 (22%), srsf2 (11%), sf3b1 (5.5%), ezh2 (11%), idh1 (5.5%), idh2 (5.5%), tet2 (33.5%), tp53 (5.5%). after the transplant, the incidence of acute gvhd was 72%; only 3 patients (16.5%) experienced acute gvhd grade 3-4. chronic gvhd was registered in 50% of cases (9 patients: 8 mild, 1 moderate, 0 severe). relapse occurred in only 1 case. nrm incidence was 16.5% of cases, occurring in the first year after transplant. with a median follow up of 55 months, 5-year overall survival was 83.5%, and 5-years pfs reported the same value, beeing the only relapse at >10 years after transplantation. the relapse occurred in the only patient who harbored mutations in both asxl1 and ezh2 genes. conclusions: these retrospective data suggest that the particular group of mpl-mutated myelofibrosis may have a good outcome after stem cells transplantation. in particular, our data revealed very low rate of disease relapse (5.5%) in comparison with the available historical controls regarding myelofibrosis in toto. [ background: a significant proportion of cml patients undergoing allogeneic stem cell transplantation (allo-hsct) restart tki following transplant to prevent relapse. there is however no data to support if tki can be discontinued following allo-hsct and whether such patients can safely discontinue tki remains controversial. practices varies among transplant centres depending on countrywide practices, centre experience, duration of molecular remission, patients 'wish and analysis of retrospective data on the outcome of patients who discontinue tki after transplant may provide further insight to help elaborating future guidelines. the present study objective is to investigate the outcome of patients with cml who discontinue tki therapy after restarting tki following allo-hsct. this retrospective study may be helpful to support future guidelines. methods: through the ebmt database of patients who received an allo-hsct between march 2004 and september 2013, we identified 81 cml patients who restarted tki treatment post allo-hsct and stopped it after at least 3 months of tki therapy. results: out of 81 patients who discontinued tki, 21 were in first chronic phase (cp1) at the time of transplant, 31 in second or third chronic phase (cp1 and cp2) and 28 in accelerated or blastic phase (ap/bp) or primary refractory disease. one patient had missing data at the time to transplant. allo-hsct conditioning was of reduced intensity (ric) in 27 patients and myeloablative (mac) in 54 patients, including tbi in 29 patients. tki therapy was (re)started in all patients after a median time from transplant of 4.4 months (range, 0.4 to 57.6 months) for a duration of 9.7 (range, 3.1-62.3 months). after a median time from tki discontinuation of 65.8 months (range 51.8-71.1), 5-years progression free survival (pfs) and overall survival (os) were 51% (95%ci 39 to 62%) and 62% (95%ci 51 to 73%) respectively. patients in cp1 at the time of transplant had a significantly higher 5-years os compared to those in cp2/ cp3 or ap/bp, 100% vs 37% (95%ci 17 to 58%) and 59% (95%ci 39 to 80%) respectively (p< 0.001, figure 1 ). causes of death (cod) in the cp2/3 and ap/bp groups were relapse (12/31 and 5/28 respectively) and nrm (4/31 and 4/28 respectively, 1 missing cod in each group). conclusions: post-transplant tki discontinuation appears safe in patients who receive an allo-hsct while still in first chronic phase. however, such approach in patients who transform to advanced phases before allo-hsct remains a matter of concerns given the high incidence of post allo-hsct relapse. further analysis to identify reason for restating tki post-transplant and for subsequent discontinuation will be performed after further data is collected through the ongoing data quality initiative (dqi) in cml. [ background: allogeneic stem-cell transplantation (allo-sct) is a well-established treatment modality for high-risk hematopoietic malignancies. however, the optimal intensity of myeloablation with a reduced-toxicity conditioning regimen to decrease relapse rate after allo-sct without increasing trm has not been well established. thiotepa is an alkylating compound with antineoplastic activity and immunosuppressive properties, as well as the ability to penetrate the blood brain barrier. thiotepa has become an integral part of the thiotepa, busulfan iv (busilvex), and fludarabine (tbf) conditioning regimen, which is being used with increasing frequency, particularly for haploidentical and cord-blood transplants. however, few studies have focused on analyzing the effect of thiotepa dose in the tbf conditioning. methods: the aim of this study was to evaluate the optimal dose of thiotepa, as part of the tbf conditioning for allo-sct in adults with aml transplanted in complete remission (cr), by comparing the transplantation outcomes of patients who received 5 mg/kg thiotepa and 2 days of busilvex (6.4 mg/kg) (t1b2f) versus those who received 10 mg/kg thiotepa with 2 days of busilvex (t2b2f) or 3 days of busilvex (9.6 mg/kg) (t2b3f) using a large dataset from the ebmt registry. results: we identified 639 aml patients allotransplanted between january 2009 and june 2018 from matched related or unrelated donors or t replete haplo-identical donors. 127 patients (20%) received (t1b2f); 113 patients (18%) received (t2b2f); the remaining 399 patients (62%) received (t2b3f). all the patients were in cr at transplant. median follow-up was 20 months (iqr: 9-37). outcomes are summarized in the table 1. acute gvhd grade ii-iv was 15%, 17% and 19% (p=0.14) respectively. at 2 years the non-relapse mortality (nrm) was 22%, 25% and 21% respectively (p=0.62); the relapse incidence (ri) was 18%, 19% and 17% (p=0.37) respectively; the leukemia free survival (lfs) was 60% vs 56% vs 63% (p=0.21) respectively and the overall survival (os) was 67% vs 62% vs 67% (p=0.56) in the 3 groups, respectively. the 2year grfs was 50%, 43%, and 55% respectively (p=0.02). in multivariate analysis, acute gvhd was higher for patients receiving t2b2f (p=0.01; hr 2.25) or t2b3f (p=0.02; hr 2.05) as well as for patients receiving transplant from haploidentical donor or peripheral blood stem cells, whereas nrm was higher for older patients (p=0.001; hr 1.56), patients receiving t2b3f (p=0.008; hr 2.28) or haploidentical transplant (p=0.009; hr 2.2). importantly, os was lower for older patients (p=0.001; hr 1.4) or for patients receiving t2b3f (p=0.004; hr 2.09). the multivariate analysis was adjusted to all the different factors between the 3 groups. conclusions: t2b2f is associated with higher incidence of acute gvhd compared to t1b1f whereas t2b3f associated with higher nrm, a higher incidence of acute gvhd and a lower os compared to t1b1f. with the limitation of the retrospective nature of registry data, these results suggest that a lower dose-intensity of thiotepa and busilvex in the tbf regimen in general may yield better results in aml patients transplanted in complete remission. background: thethiotepa-busulfan-fludarabine (tbf) based conditioning regimen is widely used in t-cell repleted haploidentical transplantation (haplo) with post-transplant cyclophosphamide. however, the use of anti-thymocyte globulin (atg) has not been well established. it decreases the incidence of graft versus host disease however some claim that it's at the cost of increased relapse. we conducted this multi centric study to compare the outcomes of patients who underwent haplo with tbf conditioning regimen with atg to those without. methods: this is a multicentric retrospective study. data was collected from 4 centers, the american university of beirut medical center, hospital saint antoine paris, institute paoli calmette marseille, and humanitas research hospital milan. we included all consecutive adult patients who underwent haplo with tbf conditioning. the conditioning consisted of thiotepa 5 mg/kg per day infused on days -7 and/or -6, fludarabine 30 mg/m2 infused on day -5 to day -2; and busulfan 130 mg/m2 infused on day -5 to day -3. graft versus host disease (gvhd) prophylaxis consisted of post transplantation cyclophosphamide 50 mg/ kg per day on day +3 and day +5, cyclosporine on day +6 and readjusted according to level, and mycophenolate mofetil 500 mg every 6 hours beginning on days +6 to +28 or +35 depending on the center. patients who received atg received a dose of 2.5 mg/kg per day. results: we included a total of 268 patients, 69 of whom (26%) received atg (group 2) as part of the conditioning chemotherapy. patients who received atg had a younger median age compared to the second group without atg (group 1) (53 and 58 years respectively; p value 0.004). (63% vs 61%) of each group had acute leukemia, and (71% vs 70%) were in complete remission at the time of transplant, while 47 patients (24%) in the group 1 had progressive disease at transplant. 151 patients (56.5%) had an intermediate disease risk index (dri). in the atg group, 59 patients (30%) compared to 50 (73%) in the other group received 5mg/kg thiotepa, while 140 (70) and 19 (27%) received 10mg/kg respectively. peripheral blood stem cells were the most common graft source in both groups (83% and 88% respectively). at a median follow-up of 15.4 months, patients receiving atg had a statistically significant decreased risk of acute graft versus host disease (agvhd) (rr 0.47; p value 0.031), and non-relapse mortality (nrm) at 24 months (rr 0.5; p value 0.027). atg also resulted in higher progression and overall survival at 24 months, which was not statistically significant (66.2% and 59.8%; p value 0.168, with 76.6% and 67.8%; p value 0.056 respectively). conclusions: atg as part of the pre-transplantation conditioning leads to significant reduction in agvhd and nrm at 24 months without significant effects on pfs or os. disclosure background: high-risk leukemia is associated with poor prognosis and inferior outcome. in elderly or comorbid patients allogeneic sct with myeloablativ conditioning regimen as the most effective treatment option is not available. sequential regimen combining cytoreductive therapy with ric has shown high antileukemic activity for high-risk patients with acceptable toxicity profile. this study is based on the observation that antileukemic effects have been described previously for the nucleoside analogue clofarabine. methods: the trial was designed as an investigatorinitiated prospective, multicenter, open-label, two-arm, parallel-group phase ii study comparing clarac to flamsa regimen. flamsa regimen consists of fludarabine (30 mg/ m2, days -13 to -10), amsacrine (100 mg/m2, days -13 to -10) and cytarabine (2000 mg/m2, days -13 to -10). clarac regimen consists of clofarabine (30 mg/m2, days -16 to -12) and cytarabine (1000 mg/m2, days -16 to -12). both cytoreductive therapies were combined with bu/cy (busulfan, 4 x 0.8mg/kg, days -6 to -5 and cyclophosphamide 60 mg/kg, days -4 to -3). as gvhd-prophylaxis atg, csa and mmf were used. patients with high risk aml or advanced mds (ipss ≥ int2) with contraindication for conventional conditioning or refractory to induction therapy were eligible. primary objective was to demonstrate that event-free survival is improved by clarac instead of the flamsa. secondary objectives were overall and relapse-free survival, mortality rate, safety profiles and cardiac toxicity. results: between 2011 and 2017, 62 patients were recruited, 2 patients did not meet the in-/exclusion criteria. a total of 60 were randomized with 30 patients each in the clarac and flamsa group. mean time to event was 656.6 ± 84.6 days for flamsa and 565.6 ± 49.2 days for clarac, respectively (p=0.177, figure 1 ). in total 38 of the adverse events were serious with fatal outcome of 3 patients in the clarac and 4 patients in the flamsa group. cardiac toxicity was observed in 26 patients in the clarac treatment arm, whereas 27 patients were affected in the flamsa treatment arm (p=0.730). overall survival for clarac was numerically, but not statistically inferior to flamsa (p=0.134). a part of 16/30 (53.3%) patients died until the end of the study in the clarac treatment arm, whereas only 12/30 (40.0%) died in the flamsa treatment arm (p=0.134). conclusions: this study did compare two different conditioning regimens for allogeneic stem cell recipients with high risk aml/mds. 62 patients have been included and 60 were randomized. the treatment arms were wellbalanced at study baseline for relevant covariates. superiority of the clarac treatment regimen over the flamsa regimen could not be demonstrated. consistently hazard ratios for event free survival, overall survival and relapsefree survival were in favor of the control group with flamsa treatment. no differences were found regarding cardiac toxicity, rate of engraftment, or chimerism. regarding general safety parameters, no relevant differences between the two treatment strategies were found. clinical trial registry: background: currently, there is no direct evidence to recommend specific conditioning intensities in myelofibrosis undergoing allogeneic stem cell transplantation. moreover, recent risk stratifications for diagnosed myelofibrosis (mf) included specific mutation profiles as prognostic factors. using clinical-molecular data from four different centers from germany and france, we sought to determine whether molecular genetics have an impact on outcome after reduced intensity (ric) and myeloablative conditioning (mac) stem cell transplantation in mf. methods: previously published methods were used to sequence myelofibrosis-associated genes (i.a. calr, jak2, mpl, asxl1, srsf2, ezh2, idh1/2, dnmt3a, tet2, tp53). risk stratifications according to dynamic international prognostic scoring system (dipss), mutation-enhanced system (mipss70), genetic inspired prognostic system (gipss), prognostic model for secondary myelofibrosis (mysec-pm), cytogenetics as well as other clinical and transplant-specific variables were included in analyses. cox model with hazard ratios (hr) was used for survival (os) and cumulative incidence for relapse and non-relapse mortality (nrm). risk ratios (rr) were obtained for subgroup analysis. results: the study included 361 mf patients (260 primary and 101 secondary mf) of whom 230 received ric and 131 mac. the median follow-up was 61 months in ric and 73 months in mac and the median age was 57 and 54 years. patients receiving ric were at higher risk according to dipss, mipss70, and mysec-pm, whereas frequencies of driver mutation genotype (calr, jak2, mpl, triple negative) as well as asxl1 mutation were similar. hla-mismatched unrelated donors were more frequent in ric. most common conditioning regimens for ric were bu/flu (83%) and flamsa (12%), and flu/mel (37%), treo/flu and tbi/flu (21%, respectively) for mac. no significant difference was found for ric versus mac with respect to os (62% vs 57%; p=0.35), relapse (16% vs 13%; p=0.60) or nrm (28% vs 30%; p=0.68). early relapse within five months was increased after ric (7% vs 3%). including molecular variables, ric showed higher but not significantly different os rates in patients with < 3 mutations, in triple negative driver mutation genotype, asxl1 mutation ( figure 1 ). when evaluating patients with an asxl1 mutation, those 88 patients who had only one asxl1 or one additional mutation seemed to benefit from ric showing 5-year os of 65% vs 20% for mac (p=0.001), whereas no difference was identified when more than two additional mutations were present (57% vs 65%; p=0.27) . furthermore, in patients with an asxl1 mutation and one additional driver mutation (calr, mpl, jak2), 5year os was significantly different showing 63% in ric vs 18% in mac (p=0.005). regarding current risk stratifications, ric showed significantly improved os only in high risk dipss and mysec-pm, whereas no difference was found regarding the remaining systems such as dipss-plus, mipss70 and gipss ( figure 1 ). conclusions: the evaluation of different conditioning intensities in mf did not favor ric or mac regarding currently existing risk stratifications. with respect to molecular genetics, a proportion of patients specifically harboring up to two mutations including asxl1 may benefit from ric with respect to os. [ background: sequential conditioning regimens (sr) have shown substantial activity in relapsed/refractory acute myeloid leukemia (r/raml). the original prototype sr is the flamsa-cytbi regimen. various modifications of this protocol have been developed in recent years (see table 1 ). in the current study, we compared the outcomes of patients suffering from r/raml that had received their first allogeneic stem cell transplantation (allosct) after conditioning with a sr. methods: adult patients with r/raml who had received their first allosct following sr conditioning between 2000 and 2017 and were reported to the ebmt registry were analyzed. patients were grouped according to the type of sr used as shown in table1. the flamsa-cytbi group served as comparator for all others. the primary endpoint was leukemia-free survival (lfs). secondary endpoints were overall survival (os), relapse incidence (ri), nonrelapse mortality (nrm), refined graft-versus-host-diseasefree, relapse-free survival (grfs), acute (a)gvhd and chronic (c)gvhd. multivariate analysis was done using cox regression. results: patients' characteristics are detailed in table 1 . there were more patients with transformed nhl in the beam (18%vs7%). the median time to neutrophil count > 0.5 g/l and platelet counts > 50 g/l were 10 (4 -24) and 10 (1-124) days for beeam and 12 (1-34) and 12 (9-50) days for beam, respectively. twenty-nine (22%) patients in the beeam and 35 (15%) patients in the beam groups relapsed after a median time to relapse of 6 and 9 months, respectively. after a median follow-up from transplant of 33 months, 54 (15%) patients died, 24 (18%) in the beeam and 30 (13%) in the beam groups, respectively. the main causes of death was lymphoma in 31 patients, (beeam:13, beam:18), infections in 16, (beeam:7, beam:9), secondary cancers in 2 patients (beeam). there were no significant differences between beeam and beam regimens for os: hr=1.02 [(0.55-1.86 conclusions: in this matched pair analysis, beeam and beam resulted in equivalent nrm, lfs and os suggesting that both conditioning regimens may reasonably be employed in patients with dlbcl. the higher relapse rate following beeam requires further evaluation. a prospective randomized study will be required to confirm the equivalence of the two regimens. [[o071 table] 1. background: inflammatory bowel diseases (ibd) are thought to increase the risk and severity of graft-versus-host disease (gvhd) and non-relapse mortality (nrm) after allogeneic hematopoietic stem cell transplantation (allo-hsct). thus, ibd have been included in pre-transplant comorbidity risk scores, although formal analysis of allo-hsct outcomes in patients with ibd are lacking. methods: with this background, we designed an ebmt registry retrospective case-controlled analysis to assess outcomes of allo-hsct in patients with ibd. the aim was to compare the incidence of gvhd, nrm and overall survival (os) after allo-hsct in the 2 groups of patients. each patient with ibd was matched with 3 controls according to following factors: patient sex, patient age, disease, intensity of conditioning, donor type and hla disparity, cell source and year of transplant. group comparisons were done using logrank test or gray test for competing risks outcomes. results: between 2011 and 2015, 175 patients with ibd who underwent allo-hsct for a hematologic malignancy were reported to ebmt. the cohort comprised 94 males and 81 females, with a median age of 53 years (range, 18 to 69 years) at the time of transplantation. the most frequent malignancies in the ibd group were acute leukemia (106 patients; 61%) and myelodysplastic/myeloproliferative neoplasm (42 patients; 24%). the donor was an identical sibling for 66 patients (38%), and a matched unrelated donor for 56 patients (32%). 93 patients (53%) received a myeloablative conditioning regimen while 82 patients (47%) received a reduced-intensity conditioning regimen. with a median follow-up of 37 months (range, 32-45) for the patients with ibd and 36 months (range, 33-39) for controls, the cumulative incidence of grade ii-iv acute gvhd was 27% for the patients with ibd and 28% for controls (hazard ratio (hr) for ibd versus controls, 0.95; 95% ci, 0.66 to 1.36; p=0.77). the cumulative incidence of extensive chronic gvhd at 36 months was 25% in patients with ibd and 17% in controls (hr, 1.54; 95% ci, 1.03 to 2.28; p=0.03). nrm at 36 months was 27% for the patients with ibd and 25% for controls (hr, 1.07; 95% ci, 0.76-1.52; p=0.68). the relapse incidence at 36 months was 32% in patients with ibd and 33% in patients without ibd (hr, 1.19; 95% ci, 0.94-1.49; p=0.82). os at 36 months was 47% for the patients with ibd and 49% for matched controls (hr, 1.04; 95% ci, 0.81-1.33; p=0.79). finally, gvhd-free relapse-free survival (grfs) at 36 months was 29% for the patients with ibd and 34% for matched controls (hr, 1.19; 95% ci, 0.94-1.49; p=0.14). conclusions: we report the largest matched-controlled study of allo-hsct in patients with ibd conducted so far. contrary to our expectations, we found no significant differences in acute gvhd, nrm or os between the ibd group and controls. however, ibd patients had significantly more extensive chronic gvhd than the control group. our results suggest that allo-hsct should not be contraindicated if ibd alone is considered a comorbidity. however, ibd patients have a higher risk for developing severe forms of chronic gvhd, which could considerably impair their long-term quality of life. clinical background: the easix (endothelial activation and stress index) score is associated with non-relapse mortality (nrm) and overall survival (os) after reduced intensity (ric) allohct (luft et al, lancet haematol 2017). we aimed to validate the prognostic ability of easix in a cohort of both unmodified and cd34-selected allohct. methods: between april 2008 and december 2016, 509 adult patients (pts) underwent unmodified ric or non-myeloablative (nma) allohct (n=149) with uniform gvhd prophylaxis of sirolimus/tacrolimus and low/ dose mtx or myeloablative conditioning (mac) allohct using ex vivo cd34-selection (n=360) with the clinimacs cd34 reagent system (miltenyi biotech) as gvhd prophylaxis. the easix formula (ldh*creatinine/thrombocytes) was calculated at multiple timepoints (pre-allohct, day 30, day 100 and onset of acute gvhd). a log transformation using base 2 (log2) was applied to all easix variables to reduce skew. a one unit increase in log2 easix is associated with a doubling (one-fold increase) of easix on the original scale. relapse and death or relapse, were considered competing risks for nrm and acute gvhd, respectively. results: median age was 55.6 years (19.6-78.7) and most pts were male (59%). most pts had myeloid malignancies (72%) and received mac (70%). with a median follow up of 4.8 years (0.7-10) among survivors, 1 and 3-year os were 79.9% (95% ci, 76.2-83.2) and 63.2% (95% ci, 5.7-67.3), respectively. the 1 and 3-year nrm were 13.2% (95% ci, 10.4-16.3) and 22.2% (95% ci, 18.6-25.9), respectively. the 1-year cumulative incidence of grade 1-4, 2-4 and 3-4 acute gvhd was 44.4% (95% ci, 40.0-48.7), 33.0% (95% ci, 29.0-37.1) and 10.4% (95% ci, 8.0-13.3), respectively. causes of death in 211 pts at last follow up included relapse (34%), gvhd (25%) and infection (20% higher easix score at d30, d100 and at onset of acute gvhd was significantly associated with increased risk of death and nrm (table 1 and figure 1). conclusions: higher easix scores at day 30, day 100, and at onset of acute gvhd are associated with higher nrm and inferior os with a more prominent association in calcineurin inhibitor-based unmodified allohct compared to cd34-selected allohct. endothelial damage is an important contributor to poorer outcomes after allohct and easix formula provides an easy complimentary tool to predict outcomes in these patients. background: subsequent malignant neoplasms (smns) are one of the most important complications of hematopoietic stem cell transplantation (hsct) and result in considerable morbidity and mortality. the reported rate of smns after hsct in adults ranges between 1-11% at 10-years. there is limited data on smns after pediatric, adolescent and young adult (aya) hsct, where the potential years of life gained is greater than among older adults. the objective of this study was to assess the incidence and types of smns in a cohort of survivors of childhood and aya hscts that were performed for malignant indications. methods: all hsct patients (age 0-30 years at time of transplant) who survived at least 2-years after hsct in the province of ontario, canada between 1994 and 2017 were identified from 3 transplant centers. clinical data were linked to provincial administrative databases and the ontario cancer registry that identifies cancer cases based on pathology reports and electronic patient records. results: four-hundred and forty-six 2-year allogeneic hcst survivors were included in this study. of them, 36 (8%) developed 45 smns at a median follow up of 13.1 years (range: 0.25-22.8 years). the 10-year cumulative incidence of smn was 4% (2.1-6.3%) and the 20-year cumulative incidence of smn was 18% (12.4-24.6%). several patients developed more than 1 smn. the most common smns were: papillary carcinoma of the thyroid (n=11); secondary leukemia/lymphoma (n=10); squamous cell carcinoma of the skin/oral mucosa (n=9); and adenocarcinoma of colon/lung (n=5). ten other types of smns were found including sarcoma, melanoma, nerve sheath tumor and breast cancer. nine survivors died at a median of 7.7 months after smn diagnosis. the 10-year cumulative incidence of smn for 190 acute lymphoblastic leukemia survivors who received total-body irradiation was 5% (2.4%-10.6%). conclusions: our findings corroborate the observation that children and aya who undergo allogeneic hsct are at a significant risk for developing smn. careful observation in the survivorship period is required for potential early detection. clinical 1979 and 1998 and who survived at least 20 years post-hct while continuing follow-up at our centre. we documented performance status, comorbidities, number of medications and occurrence of secondary malignancies at 20 years, as well as survival following the 20-year time-point. results: the median age of the cohort at 20 years post-hct was 56 years (range 37-77), 157 (91%) of patients underwent transplant using a related donor. eighty patients (46%) underwent hct for cml, 44 (25%) for aml, 14 (8%) for all, 12 (7%) for aplastic anemia, 23 (13%) for other indications. bone marrow grafts were used in 163 (94%) patients. myeloablative conditioning was used in 163 (94%) patients. individual comorbidities were categorized into five major groups: endocrine 79 (46%), cardiac 59 (34%), secondary cancer 46 (27%), psychosocial 39 (23%), and other organ dysfunctions 94 (54%). at the 20 year mark, median karnofsky performance status was 100 (range 30-100). no comorbidities were seen in 24 (14%) patients. the most frequent individual comorbidities were dyslipidemia (n=54, 31%), hypertension (n=54, 31%), osteoporosis (n=31, 18%) and hypothyroidism (n=28, 16%). at the time of the 20-year post-hct follow-up, the median number of medications patients were taking was 2 (range 0-25). follow-up data after the 20-year mark was available for 146 (84%) patients. median follow-up of survivors after the 20-year mark was 59 months (range 4-219 months). fiveyear overall survival of the 146 patients was 92% (95%ci 85-96%) and at 10 years was 89% (95% ci 79-94%). when grouped by age at the 20-year mark, there was no significant difference in 5-year os survival between ages 35-49 (n=48, 5-year os 91%), 50-60 (n=58, 5-year os 95%) and 61-75 (n=40, 5-year os 87%) (p=0.90). when grouped by the number of concurrent comorbidities, there was a significant difference in os between the groups with 0-1 (n=54), 2-3 (n=53) and ≥4 comorbidities (n=39) (10-year os 98%, 87% and 67% respectively, p=0.0001, figure 1 ). when grouped by the number of medications patients were on at the 20-year mark, there was a borderline significant difference between the groups on 0-1 (n=61), 2-4 (n=46) and ≥5 (n=39) different medications (10-year os 93%, 93% and 76% respectively, p=0.05). conclusions: long-term allogeneic hct recipients may develop a number of long-term comorbidities that negatively influence survival even past the 20-year mark. these findings warrant the continuous long-term medical followup of allogeneic transplant patients, regardless of age or time that has lapsed post-hct. background: no standard procedure is in use to predict sinusoidal obstruction syndrome/venoocclusive disease (sos/ vod). recently the sos/vod cibmtr clinical risk score (age, karnofsky, sirolimus, hepatitis b/c, conditioning regimen, disease type) has been established using the cibmtr database (biol blood marrow transplant. 2018; 24:2072) . the endothelial activation and stress index (easix), based on the simple formula 'ldh(u/l) x creatinine(mg/dl) / thrombocytes (10 9 /l)', has been proven to predict mortality after gvhd (lancet haematol 2017; 4:e414) . the aim of the current study was to test prediction of sos/vod by easix compared with the cibmtr score in two independent european cohorts. methods: sos/vod was defined according to the 2016 ebmt criteria. the capacity of easix and of the cibmtr score for predicting sos/vod was tested retrospectively in 556 consecutive adult patients undergoing allosct at a single institution between 2001 and 2013 (training cohort). the primary endpoint was prediction of sos/vod by the cibmtr score or by easix-d0 (easix measured at the day of allosct). results were validated in an independent cohort of 446 adult allosct recipients from another single institution transplanted between 2013 and 2015. incidence of sos/vod was assessed by uni-and multivariable cox regression analyses using age, easix and the cibmtr score as confounders. results: sos/vod was diagnosed in 35 patients (6.3%, median onset day +8) in the training cohort and in 45 patients (10.1%, median onset d +9) in the validation cohort, respectively. in the training cohort, increasing easix-d0 was significantly associated with sos/vod incidence on multivariate analysis (hr per log2 increase 1.27, 95%ci 1. 03-1.57, p=0.023) . similarly, easix-d0 predicted the incidence of sos/vod in the validation cohort (hr per log2 increase 1.35, 95% ci 1.14-1.59, p< 0.001). also, the cibmtr score showed an association with sos/vod incidence which was however significant only in the training cohort (hr per log2 increase 1.55, 95% ci 1.09-1.62, p=0.016) but not in the validation cohort, (hr per log2 increase 1.43, 95% ci 0.98-2.08, p=0.060). these results are visualized by comparing easix-d0 and cibmtr-vod scores in patients grouped according to later vod development within the observation period ( figure 1 , kruskal-wallis tests; 1a-b easix-d0 and 1c-d cibmtr risk score). the association of easixd0 with vod incidence was independent from cibmtr score. conclusions: easix-d0 is very easy to test and predicted sos/vod in two separate cohorts of allosct recipients independent of the cibmtr-vod score. patients with high easix-d0 scores might be candidates for clinical evaluation of intensified strategies to prevent sos/vod after allosct. [[o076 image] 1. figure 1 ] disclosure: the authors thank jazz pharmaceuticals for providing financial support that was used for data collection. the company had no active part in this project, did not have access to data and was not involved in analyses or writing/editing of this abstract. early hyperbilirubinaemia without sos/vod -a link between endothelial dysfunction and early mortality after allogeneic transplantation thomas luft 1 , david schult 1 , joshua majer-lauterbach 1 , sihe jiang 2 , aleksandar radujkovic 1 , peter dreger 1 , olaf penack 2 1 university hospital heidelberg, heidelberg, germany, 2 charité universitätsmedizin berlin, berlin, germany background: endothelial dysfunction is a risk factor for early mortality after allogeneic stem cell transplantation (allosct) and is linked to transplant-associated thrombotic microangiopathy (tam). similar to tam, diagnosis of sinusoidal obstruction syndrome / venoocclusive disease (sos/vod) is based on consensus criteria, and several scores have been proposed that include earlier or later stages of the diseases. early hyperbilirubinaemia occurs frequently after allosct. the pathophysiology is often elusive, and only in a subset of allosct recipients with hyperbilirubinaemia sos/vod is identified as the underlying mechanism. the aim of the current study was to explore clinical impact and pathophysiological context of early hyperbilirubinaemia without sos/vod. methods: in two independent cohorts of 864 and 446 patients, serum bilirubin levels before allosct and on days 0, 3, 5, 7, 14, 21 and 28 were retrospectively retrieved. sos/vod was defined according to the 2016 ebmt criteria. patients with at least one bilirubin value of >3mg/dl between days 0-28 were grouped as "early hyperbilirubinaemia". we assessed overall survival (os), non-relapse mortality (nrm) and time to relapse (ttr) with and without early hyperbilirubinaemia depending on coincident sos/vod, tam, and refractory acute gvhd, and we investigated the impact of statin-based endothelial cell prophylaxis (pravastatin plus ursodeoxycholic acid). serum bilirubin levels were correlated with the endothelial activation and stress index (easix, 'ldh(u/l) x creatinine(mg/dl) / thrombocytes(10 9 /l)'), measured before allosct (easix-pre) or on the day of transplantation (easix-d0). similarly, bilirubin levels were correlated with the sos/vod cibmtr clinical risk score, and with serum markers of liver damage (alanine transaminase, alt, gamma-glutamyltransferase, ggt) and endothelial cell distress markers (angiopoietin-2). results: early hyperbilirubinaemia was diagnosed in 156 (18%) and 141 (32%) patients of the training and validation cohort, and vod diagnostic criteria were met by 23% and 30% of patients with early hyperbilirubinaemia, respectively. in all patients, early hyperbilirubinaemia was associated with increased nrm and os (nrm, training: hr 2.03, 95%ci 1.47-2.80, p< 0.001; nrm validation: hr 2.12, 95%ci 1.48-3.06, p< 0.001). increased nrm in recipients with hyperbilirubinaemia without sos/vod was independent from tma or refractory acute gvhd ( figure 1 ). easix-pre and easix-d0 correlated with day 0-28 bilirubin in both cohorts. easix-pre, easix-d0 and the cibmtr-vod score predicted risk of early hyperbilirubinaemia. however, only easix predicted risk of nrm in patients without sos/vod. endothelial protection with statins and ursodeoxycholic acid was associated with reduced incidence of and reduced nrm after early hyperbilirubinaemia. furthermore, pre-transplant angiopoietin-2 correlated with early hyperbilirubinaemia, whereas alt and ggt did not. conclusions: early hyperbilirubinaemia represents a novel risk factor for nrm independent of tma and refractory acute gvhd, even in patients not meeting the diagnostic criteria for vod. the endothelial relationship of this condition is underlined by the observation that angiopoietin-2, easix-pre and easix-d0 but not markers of liver damage associate with the incidence of early hyperbilirubinaemia. therapeutic strategies aiming at normalization of endothelial dysfunction after allosct are attractive. as a first example, our data demonstrate reduced incidence of early hyperbilirubinaemia and reduced nrm thereafter in allosct recipients prophylactically treated with statins and ursodeoxycholic acid in the peri-transplant period. [[o077 image] 1. figure 1 background: accumulating evidence has suggested complement activation in transplant-associated thrombotic microangiopathy (ta-tma), mainly in the pediatric setting. to further understand its pathogenesis in adults, we hypothesized that both complement and neutrophils are activated in ta-tma as previously observed in distinct thrombotic disorders. methods: we enrolled adult ta-tma (international working group/iwg criteria), acute and/or chronic graftversus-host-disease (gvhd) and control hematopoietic cell transplantation (hct) recipients in a 1:1:1 ratio (january 2015-june 2018). complement activation was detected in patient sera and plasma with the modified ham test (mham), soluble c5b-9 and activated c3 fragments; while neutrophil activation with neutrophil extracellular traps (nets) using extracellular dna and myeloperoxidase/ mpo-dna. results: we studied 10 ta-tma, 10 gvhd and 10 control patients. ta-tma patients suffered from severe acute and/or extensive chronic gvhd. ta-tma presented at median +109 (9-930) day post-transplant. full donor chimerism was evident in all patients. no significant difference in transplant characteristics was observed among groups, except for the significantly lower gvhd rate in the control group. c5b-9 and mham levels were significantly increased in ta-tma compared to gvhd (p=0.004 and p=0.006, figure a ) and control patients (p=0.001 and p=0.005, figure b ), while no significant difference was observed in activated plasma c3 levels in the plasma. in the multivariate analysis, c5b-9 levels were an independent predictor of ta-tma diagnosis (p=0.026). we next sought to determine the cutoff value of c5b-9 that distinguishes ta-tma from other hct recipients. in the receiver-operating characteristic curve, we found a significantly high area under the curve (0.823, p=003). values higher than 321.5 ng/ml conferred a specificity of 70% with a very high sensitivity of 91.7% for ta-tma diagnosis. regarding nets, both extracellular and mpo-dna were significantly increased in patients with ta-tma compared to gvhd (p< 0.001 and p=0.042, figure c ) and control patients (p< 0.001 and p=0.029, figure d) . interestingly, increased complement activation markers (c5b-9 and mham) were strongly associated with mpo-dna (r 2 =0.461, p=0.001 and r 2 =0.403, p=0.018, respectively) and extracellular dna (r 2 =0.555, p=0.001 and r 2 =0.502, p=0.002, respectively). lastly, we studied changes of complement and neutrophil activation in 3 patients that received complement inhibition by eculizumab. despite delayed initiation in the first two patients (28 and 18 days post ta-tma diagnosis respectively), we observed laboratory response including evidence of reduced hemolysis, schistocytosis and transfusion needs. both extracellular dna and c5b-9 levels were significantly reduced post 2 doses of eculizumab (p< 0.001). however, all patients succumbed to complications of endstage renal disease and infections after a median of 3 (2) (3) (4) (5) (6) (7) (8) doses of eculizumab. conclusions: our findings demonstrate for the first time a crosstalk between complement and neutrophils in adult ta-tma. in addition, we were able to set a cut-off c5b-9 value for distinguishing complement activation in unselected patients diagnosed with the iwg criteria. although complement inhibition by eculizumab seems to hinder this pathophysiological process, further studies are needed to clarify changes and identify optimal therapeutic targets in this complex setting. [[o078 image] 1. background: hepatic vod/sos with multi-organ dysfunction (mod; typically, renal or pulmonary) may be associated with >80% mortality. defibrotide is approved for treating severe hepatic vod/sos post-hematopoietic stem cell transplantation (hsct) in patients aged >1 month in the european union, and for hepatic vod/sos with renal or pulmonary dysfunction post-hsct in the united states and canada. per prescribing guidelines, defibrotide 25 mg/kg/day (4 divided doses) is recommended for ≥21 days. this pooled analysis examined time to complete response (cr) of vod/ sos and mod symptoms relative to day of defibrotide initiation in patients who received defibrotide 25 mg/kg/day. methods: time to cr and safety data were pooled from 2 studies that included patients with vod/sos and mod post-hsct who were treated with defibrotide: a phase 3 trial (n=102) and a phase 2, randomized dose-finding trial (n=74 receiving 25 mg/kg/day). duration of therapy in patients who discontinued due to cr in an expanded-access program (t-ind) in vod/sos patients with and without mod post-hsct (n=1000) was analyzed separately due to differences in the patient population and data monitoring protocol (eg, cr data by day were not collected). vod/sos diagnosis was defined by baltimore criteria/biopsy for the phase 2 and 3 studies; in the t-ind, modified seattle criteria also was permitted. minimum recommended treatment duration was ≥14 days (phase 2) or ≥21 days (phase 3, t-ind). results: the pooled phase 2 and 3 trials had 60 patients with cr (n=34 and n=26, respectively) and 116 patients without cr (n=40 and n=76, respectively). of 60 patients with cr ( figure) in the phase 2 and 3 trials, the median time to cr was 24.5 days (range: 7-123); of these, 53.3% achieved cr after 4 or more weeks of treatment. in the t-ind, 390 patients discontinued treatment due to cr (median time to discontinuation, 22 days after initiation of defibrotide; range, 2-64), with 57 patients (14.6%) discontinuing on or after week 4 of treatment. in the phase 2 and 3 studies (n=176), 58 patients (33%) had treatment-related adverse events (traes); most common were gastrointestinal (gi) hemorrhage (4.0%), epistaxis (4.5%), hypotension (5.7%), and pulmonary alveolar hemorrhage (5.7%). in the t-ind (n=1000), 210 patients (21.0%) had ≥1 trae; most common were pulmonary hemorrhage (4.6%), gi hemorrhage (3.0%), epistaxis (2.3%), and hypotension (2.0%). conclusions: among patients with cr in the phase 2 and 3 studies, a significant number of patients achieve cr (40%) after 4 weeks of persistent treatment, highlighting the importance of continued therapy as per label indication. support: jazz pharmaceuticals figure. time to cr among 60 patients achieving cr in defibrotide phase 2 and 3 studies] trial registry: clinicaltrials.gov: nct00003966, nct00358501, and nct00628498 disclosure: paul g. richardson has served on advisory committees and as a consultant, and has received research funding from jazz pharmaceuticals. angela r. smith and leslie lehmann have nothing to disclose. nancy a. kernan received grants from gentium during the conduct of the study, and her research was supported by the national cancer institute of the national institutes of health under award number p30 ca008748; the content is solely the responsibility of the author and does not necessarily represent the official views of the national institutes of health. she has a research grant from jazz pharmaceuticals. robert ryan and william tappe are employees of jazz pharmaceuticals and hold stock and/or stock options in jazz pharmaceuticals plc. stephan a. grupp has served on a steering committee and as a consultant to jazz pharmaceuticals. sperm counts and prevalence of testosterone substitution at long-term follow-up after myeloablative allogeneic hsct in childhood background: little is known about the long-term effects of pediatric hsct on the male reproductive axis. we investigated sperm counts and prevalence of testosterone substitution twenty years after pediatric hsct and aimed at identifying risk factors for azoospermia and testosterone substitution. methods: this cross-sectional follow-up study included two national cohorts of adult males (≥ 18 years) treated with myeloablative allogeneic pediatric hsct before age 17 between 1980-2010 in denmark and finland. the study included medical history; physical examination including testicular volume and screening for chronic graft-versushost-disease (cgvhd); sex hormones and a semen sample. cumulative (pre-hsct and hsct) gonadal irradiation including tbi and cumulative cyclophosphamide equivalent doses (ced) were estimated from patient files. results: 98/181 (54%) of eligible patients (age 18-47 years) participated with a median (range) follow up time of 18 years. 76% had malignant diagnoses and 74% were treated with tbi-conditioning. 91 delivered a semen sample. results of semen and sex hormone analyses are listed in table 1. 30/98 (31%) had detectable sperm counts (0.010-524.8 million), of these 15 were treated with chemotherapy only and 15 with tbi (2 with 2 gy tbi and 13 with 10-12 gy tbi including 5 with gonadal shielding). in patients with detectable sperm in the ejaculate, increase in sperm counts was associated with time from hsct (β=6.6 million per year 95%ci ((-0.18)-13.32), p=0.056, time range 9-32 years) indicating late spermatogenic recovery. testicular irradiation was a strong risk factor for azoospermia (or=6.2 95%ci (2.0-20.7), p< 0.001) and testosterone substitution (or=5.0 95%ci (1.3-28.3), p=0.012) and no patients with cumulative testicular irradiation doses >12 gy had detectable sperms, figure 1 . pre-pubertal stage at hsct was a risk factor for later testosterone substitution (or=10.4 95%ci (1.5-453), p=0.0060). risk of testosterone substitution was associated with time from transplantation (or for +1 year 1.1 95%ci (1.0-1.2), p=0.025). cumulative ced adjusted for testicular irradiation was not a risk factor for azoospermia or testosterone substitution, nor was cgvhd. [[o080 image] 1. figure 1] conclusions: late spermatogenic recovery is possible 10-30 years following myeloablative hsct but depends on cumulative testicular irradiation dose; azoospermia was present in all patients treated with >12 gy. pre-pubertal stage at hsct increases the risk for later testosterone substitution supporting the hypothesis that pre-pubertal leydig cells are more sensitive to irradiation than more mature ones. additionally, the risk of testosterone substitution increased with time from transplantation indicating a potential early androgen insufficiency in male hsct recipients. thus, close follow-up and focus on cumulative irradiation doses are needed. disclosure background: hematopoietic stem cell transplantation (hsct) has become a standard component of therapy for several malignant indications. as hsct may improve survival for some cancers, the risk for late complications is of increasing concern. the frequency of hospitalizations can serve as a proxy measure of severe morbidity. however, knowledge regarding late hospitalizations is limited. the objectives of the study were to describe health care utilization as measured by hospitalizations beyond 2 years following transplant in survivors of pediatric/adolescent and young adult (aya) hsct for a malignant indication. methods: we linked data from 3 ontario hsct centers and provincial health care utilization data housed at institute for clinical evaluative sciences (ices) to describe all hospitalizations and their indications. we also described intensive-care unit admissions. the study population included ontario residents with cancer age 0-30 years who underwent hsct between 1992 and 2015, who survived more than 2 years from transplant (index date). hospitalizations were described from the index date until dec 2017 or death. results: the cohort consisted of 446 survivors who were followed for a median of 11.4 years from index date (interquartile range (iqr) 2.6-18.5). indications for transplant included: acute lymphoblastic leukemia (n=190, 42.6%); myeloid malignancy (n=231, 51.8%); and lymphoma (n=25, 5.6%). of these, 150 (33.6%) received a related-donor bone-marrow hsct. at the time of hsct, ages were: 0-9 (n=132, 29.6%); 10-18 (n=137, 30.7%); and 19-29 (n=177, 39.7%) years. there were 269 patients (60%) with at least 1 hospitalization beyond 2 years from hsct. there were a total of 1879 hospitalizations, resulting in a hospitalization rate of 0.43 per follow-up year. average length of hospital stay was 6.1 days. a total of 97 intensivecare unit admissions were documented among 57 (12.8%) patients. the most common indications for hospitalization were: graft-versus-host disease (gvhd) (n=486, 25.9%), relapse (n=303, 16.1%), infection (n=186, 9.8%), orthopedic procedures/fractures (n=90, 5.4%), benign neoplasm (n=96, 5.3%) and subsequent malignant neoplasm (n=83, 4.7%). among those who did not relapse, 190/320 (59%) were hospitalized. at 10 years following hsct, the proportion of patients hospitalized was 10.5%. an underlying diagnosis of acute myeloid leukemia (aml) (p=0.001) and chronic gvhd (p=0.014) were associated with increased rate of hospitalization. in the follow-up period, 73 (16.4%) patients died. conclusions: we identified a high rate of late hospitalization in pediatric/aya survivors who underwent hsct for a malignant indication, even among those without relapse. a diagnosis of aml and chronic gvhd were associated with increased risk for hospitalization. careful observation in the survivorship period is required for potential prevention of hospitalization. clinical background: engraftment syndrome (es) is a clinical complication characterized by inflammatory signs and symptoms occurring during neutrophil recovery after stem cell transplantation (sct). its incidence varies depending on the clinical criteria used. the objective of this study was to analyze the incidence, clinical characteristics, risk factors and clinical outcomes of es after haploidentical-sct with post-transplant cyclophosphamide (haplosct) in a single center. methods: 105 consecutive haplo-sct performed between 2010-2016 in our center were retrospectively reviewed. gvhd prophylaxis was performed with cyclophosphamide 50mg/kg/day days +4 and +5, mmf and csa from day +5. g-csf was started in all cases from day +5 until engraftment. 11 cases were excluded from the analysis (9 due to death before engraftment and 2 due to primary graft failure). maiolino and spitzer´s diagnostic criteria were used to define es. results: characteristics of the 94 transplant included are shown in table 1 . the es incidence was 27.6%, with median time to diagnosis of 17 days (iqr, (15) (16) (17) (18) (19) . median time to neutrophil engraftment in the es cohort was 17 days (iqr, (14) (15) (16) (17) (18) (19) (20) . fever (100%) and skin rash (77%) were the most frequent clinical findings. there were other 5 cases of fever and skin rash during the peri-engraftment period with a final diagnosis of gvhd considering clinical outcome. 18 patients (69%) received high doses of corticosteroids, with favorable response in 78% of cases. of note, 28% cases also needed intensive supportive care. there were no deaths secondary to es. univariable analysis showed a higher risk of es with use of brother/sister as cell donor (61% cases of es; p=0,003). no other risk factors were identified. no association was noted with acute or chronic gvhd. there was no significant difference in nrm, overall survival and progression-free survival between es and non-es patients. conclusions: in our experience, es is a frequent complication of haplosct with post-transplant cyclophosphamide. to our knowledge, this is the largest study including only haploidentical sct. most cases of es had a self-limited course or good response to corticosteroids, and there were no associated mortality. however es can progress to multi-organ dysfunction with need of intensive supportive care. in our analysis, incidence of es was higher with the use of sibling haplo-donors, and further studies are needed to confirm these results. we have not found relationship between es and gvhd. specific biomarkers may contribute to an early identification of this entity in order to install therapeutic measures. disclosure: nothing to declare features and outcome of early cardiac toxicity associated with post-transplant cyclophosphamide in allogeneic stem cell transplantation background: data on risk factors and incidence of early cardiac events (ece) after post-transplant cyclophosphamide (pt-cy) are scarce. thus, we compared clinical outcomes between patients who received pt-cy and patients who did not in a cohort study including all consecutive patients allografted in our center. methods: we analyzed all ece occurring within 3 months after hsct in 331 patients. transthoracic echocardiography and ekg were performed before hsct, at day +90 and in case of ece. prior to transplant, 276 patients (83.4%) had at least one cardiovascular risk factor, 72 (21.8%) cardiovascular disease history and 31 (9.4%) left ventricular systolic dysfunction (lvsd) (defined by left ventricular ejection fraction < 53%). median age was 55 years (range, 15-76) and 60.4% of patients were males. patients were transplanted for aml (n=153, 46%), all (n=49, 15%), lymphoma (n=44, 13%), multiple myeloma (n=8, 2.4%), mds (n=35, 11%) and mpn (n=42, 13%). disease risk index was high or very-high in 93 patients (28%). conditioning regimen were mac (n=131, 40%), ric (n=85, 26%) or sequential (flamsa-like) (n=115, 35%). donors were matched related (n=89, 27%), unrelated (n=124, 37%) or haploidentical (n=118, 36%). stem cell source was peripheral blood (n=310, 94%) or bone marrow (n=21, 6%). gvhd prophylaxis included cyclosporine in all patients associated with mycophenolate mofetil (n=267, 81%), short courses of methotrexate (n=23, 7%) and/or antithymocyte globulin (n=312, 94%). in the pt-cy group, 136 patients received pt-cy at 50 mg/kg/day for at least 1 day and 100 patients for 2 days, including 13 patients with unrelated donor and 6 patients with matched related donor, either because of hla-mismatch or renal insufficiency, or inclusion in a clinical trial. results: in univariate analysis, cumulative incidence of ece was 21.3% in the pt-cy group and 8.2% in the no pt-cy group (p< 0.001). the main complication was lvsd (15% of patients in the pt-cy group and 3% in the no pt-cy group, p< 0.001). other ece included acute pulmonary edema (n=13, 4%), arrhythmia (n=12, 4%), pericarditis (n=7, 2%) and coronary artery syndrome (n=3, 1%) in the whole patient group. ece resolved in 38 patients (78%). cardiovascular risk factors and the cumulative doses of anthracycline were not significantly associated with the incidence of ece. in multivariate analysis, the main factors associated with ece were the use of pt-cy [hr=2.8, 95% ci 1. after a median follow-up of 36.5 months (iqr, 32-40), the 2-year cumulative incidences of nrm, relapse, os and dfs were 28% vs. 22%; 23% vs. 19%; 56% vs. 63%; 49% vs. 59% in the pt-cy and no pt-cy groups, respectively (p values non significant). at last-follow-up, 136 patients have died. the main causes of death were disease relapse (n=51), gvhd/infection (n=48) and ece (n=8). conclusions: incidence of ece is significantly higher in the pt-cy group. in elderly patients or with a history of pretransplant cardiac event, an alternative to pt-cy should be considered to prevent ece. disclosure: nothing to declare severe iron overload, measured by liver mri at the preallo-hsct, significantly impaired the long-term outcome of the procedure methods: once approved by the clinical trials and ethics committee, a liver mri was systematically offered to patients who were admitted to undergo an allo-hsct in our center. among the 131 pts consecutively transplanted between june 2015 and july 2018, 100 pts signed the informed consent and underwent a pre-hsct mri to assess the hepatic iron load. a signal intensity ratio (sir) method was employed for the measurement. results: 64 pts were male, and 36 were female. median age was 54 years (range: 12-69). the baseline diseases were: 37 aml, 24 nhl, 16 all, 8 mds, 8 cmpd, 6 mm, and 1 bmf. 67 underwent alternative donor transplants (56 unrelated, and 11 haplo-identical), and 33 hla-id family donor transplants. stem cell source was pb in 96, and bm in 4 cases. conditioning regimen was intensive in 51 pts, and ric in 49; no non-myeloablative allo-hsct were performed. based on hepatic iron overload at pre-hsct mri, the patients were classified into the following groups: 23 pts (23%) showed severe io (lic > 80 micromol/g or 4.5 mg/g), 25 pts (25%) moderate io (lic 36-80 micromol/g or 2.1-4.4 mg/g) and 52 pts (52%) no significant io (lic < 36 micromol/g or 2.1 mg/g). as shown in table 1, majority of patients with severe iron overload had been heavily transfused, and had a high pre-hcst ferritin level. surprisingly, pre-hsct chelation had been employed only in 3 pts (13%) of this group. overall mortalities at days +100, +180 and +365 in the global series, and in severe versus non-severe io group are reflexed in table 2. conclusions: 1) our data shows that pre-allo-hsct iron-overload correlates with previous prbc transfusion load; 2) it also makes evident that io is an important risk factors for post-transplant mortality. 3) our real-life study reflects that only a minority of heavily transfused pts had received chelation therapy previously to the allo-hsct. 4) considering the relevance of pre-allo-hsct iron overload, we strongly suggest to referring physicians to employ chelation therapy for patient candidates to transplant during the treatment of the underline disease. background: neurologic complications (ncs) are associated with relevant morbidity and mortality after allo-hsct. the aim of this study was to analyze the incidence, characteristics, risk factors and outcomes of patients developing ncs after allo-hsct. methods: we evaluated 971 consecutive adult patients (>17 years) who underwent allo-hsct at our center between january 2000 and december 2016. we collected data on neurological symptoms, diagnostic methods, time of onset and cause. nc was defined as any neurological event that occurred after starting the conditioning regimen and before relapse. nc due to central nervous system (cns) infections o neoplastic infiltration were excluded. we considered both cns and peripheral nervous system (pns) complications. results: the current series comprised 467 allo-hsct from matched sibling donor (msd), 381 from umbilical cord blood (ucb), 49 matched unrelated donor (mud) and 74 haploidentical donor (haplo). median age was 41 years and most patients had acute leukemia (63%). median follow-up of surviving patients was 71 months (range, 11-213). there were differences in median follow-up according to the donor source, being longer in ucb and msd, 87 and 67 months respectively, and shorter in mud and haplo, 23 and 24 months respectively (p< 0.0001). overall, ncs were documented in 149 cases, 64 after msd transplants, 74 after ucb, 9 after mud, and 5 after haplo. cns complications (68%) were more common than pns events. the most frequent nc was encephalopathy (31%) followed by myopathy (13%) 1-year and 5-year cumulative incidence of ncs was 11% and 13%, respectively. 5-year cumulative incidence was 11% after msd, 17% after ucb, 16% after mud, and 6% after haplo transplants (p=0.014). conclusions: ncs are common and diverse after allo-hsct. ncs were more frequent in recipients allografted from alternative donors, recipients older than 40 years, and in those developing gvhd. cns complications, but not pns, are associated with poor os. disclosure: nothing to declare. abstract already published. using ciclosporin's area under the curve (auc) to predict risk of acute kidney injury in non-myeloablative haematopoietic stem cell transplantation vaidie julien 1 , jean-baptiste woillard 1 , stéphane girault 1 , pierre marquet 1 , franck saint-marcoux 1 , arnaud jaccard 1 , pascal turlure 1 background: non myeloablative allogeneic stem cell transplantation (hsct) by limiting toxicity, can be proposed to elderly patients. however, acute renal injuries related to anti-calcineurin, which are frequent in this population, can negatively impact the outcome. currently, the exposure indexes to follow and the target to use are not consensually established. however, using the area under the curve (auc) for therapeutic drug monitoring (tdm) is theoretically the best method to describe the patient's exposure. the primary objective of this study was to determine an auc target for ciclosporin associated to the occurrence of acute kidney injury in hsct patients. methods: we retrospectively studied all consecutive patients who received a non-myeloablative allogeneic stem cell transplantation at limoges university hospital from june 2009 to december 2015. patients received fludarabine 30 mg/m2/day between d-6 and d-2 before allograft and busulfan 3.2 mg/kg/day at d-4 and d-3. gvh prophylaxis consisted in rabbit anti-lymphocyte serum at the dose of 2.5 mg/kg at d-2 and d-1, and ciclosporin at the beginning dose of 3mg/kg per os twice a day. mycophenolate mofetil was added for patients with hla-matched or mismatched unrelated donors. tdm of ciclosporin was done based on trough concentration (c0) twice a week with concomitant renal evaluation using creatininemia. dose adjustments were done in according to the sfgm-tc recommendations and renal tolerance. ciclosporin's auc was evaluated at day 1, day 14 and day 28 after allograft using bayesian estimation from a limited sampling strategy and a population pharmacokinetics model previously published. the association between ciclosporin auc and acute kidney injury was investigated using a joint model. a roc curve was then constructed to investigate an auc threshold associated with the best sensibility/specificity ratio for acute kidney injury (aki . interestingly, a very low correlation was observed between ciclosporin c0 and auc (r² =0.55 for the overall period). additionally, an higher intra-individual variability was observed with c0 than auc (coefficient of variation= 36% and 26% respectively). conclusions: we report in this study that a ciclosporin auc=5.15 mg*h/l could be used as a high threshold for aki. new evaluations of auc in prospective studies are needed to better define the relevance of this marker in clinical practice. disclosure background: human cord blood (cb) provides an attractive source of hematopoietic stem cells for allogeneic transplantation of patients with a variety of diseases. a sufficient hematopoietic stem cell (hsc) dose, currently measured as cd34+ cells/kg recipient, is essential for successful engraftment. nevertheless, the frequency of true hsc within the cd34+ population and the dynamics of their clonal offspring remain poorly understood and may differ between donors. methods: here, we use cellular barcoding and multiplexed high-throughput sequencing to determine the frequency of repopulating cells among cd34+ cells from 20 individual human cb donors, and to quantify their contribution to each of the blood lineages over time in murine nod/scid/il2ry -/-(nsg) xenografts. results: in total, we detected a median of 14.3 (range 5-168) clones in blood and 15.0 (range 1.7-85) clones in bone marrow, corresponding to hspc frequencies of 1:100 to 1:1000. the number of retrieved clones correlated to barcoded cd34+ cell dose (spearman r=0.85, p=0.001), yet could vary up to four-fold between mice transplanted with the same cell dose. clonal patterns in blood early after transplant differed markedly from those at later time points, and became increasingly deterministic over time. the majority of clones displayed multilineage output, yet clones with bias towards lymphoid or myeloid lineages were also present. similar to recent data on murine hsc clones, human cb clones were distributed asymmetrically across different bone marrow sites. conclusions: in conclusion, the frequency of nsgrepopulating cells among cord blood cd34 + cells is low, and highly variable between individual cb donors. heterogeneity in hsc frequency, proliferation and lineage fate decisions may contribute to (non-)engraftment upon hsct. future research will be aimed at identifying the underlying mechanisms guiding hsc behavior upon transplantation and expanding our findings to human hsct recipients. [[o089 image] 1. background: chronic granulomatous disease (cgd) is a rare genetic immune disorder that leaves patients susceptible to life-threatening infections, chronic inflammation and often long hospital stays. x-linked cgd (x-cgd) is caused by mutations in cybb encoding the gp91phox subunit of the phagocyte nadph-oxidase, which regulates cell ph and ionic content for efficient microbial killing. allogeneic hematopoietic stem cell transplant (hsct) has been a potentially curative approach for x-cgd patients, but is often complicated by lack of hla-matched donors and risks of graft versus host disease, graft rejection, and procedure-related fatality. previous attempts at autologous ex vivo gene therapy for x-cgd using gammaretroviral vectors have met with limited efficacy due to transient engraftment of gene corrected cells, gene silencing, and mutagenic activation leading to myelodysplasia. here we report on 9 patients with a history of severe x-cgd-related complications, who received autologous ex vivo gene therapy (gt) using a novel self-inactivating lentiviral vector (g1xcgd lv) designed to limit the risk of mutagenesis through preferential expression of the missing g91phox subunit in mature myeloid cells. methods: similar trials of gt with g1xcgd lv were initiated in the uk (n=3, plus 1 compassionate use patient) and in the usa (n=5, 3 sites). all patients had histories of severe, persistent infections, and inflammatory disease. g-csf and plerixafor-mobilized cd34+ selected hematopoietic stem and progenitor cells were transduced ex vivo with g1xcgd lv. subjects received near-myeloablative conditioning with single agent busulfan, targeted to net area-under-the-curve of 70,000 ng/ml*hr. freshly prepared or cryopreserved quality-tested gene-modified cells, manufactured on-site, were administered intravenously. primary endpoints were efficacy, as determined by percent of oxidase positive granulocytes by dihydrorhodamine [dhr] flow cytometry, and safety at 12 months. results: we report results for 7 patients (2-27 years) with 1-2.5 years of follow-up; 2 additional patients were treated but died within 3 months of gt from complications deemed related to pre-existing disease-related co-morbidities (severe pulmonary disease, anti-platelet antibodies). gt was welltolerated, only one serious adverse reaction (a systemic inflammatory process at engraftment of functional neutrophils) was reported as possibly related to gt. patients experienced typical conditioning-related transient neutropenia, thrombocytopenia and mucositis. there has been no molecular evidence for clonal dysregulation or gene silencing through cpg dinucleotide methylation. followup demonstrated sustained stable persistence of 12-46% oxidase (+) neutrophils for >12 months in 6/7 surviving patients (one, who remains clinically well, had a decline to < 5% after 3 months) [ figure] . these patients have maintained restoration of biochemical function and immunity (defined as ≥10% of oxidase (+) by dhr) as of december 2018. patients have been well, without new x-cgd-related infections, and 4 are successfully weaned off prophylactic antibiotics. conclusions: these results are the first demonstration of effective autologous lentiviral gt at 12 months in severely affected x-cgd patients without evidence of genotoxicity. corrected neutrophil function has been observed in 6 patients for >12 months and has been associated with significant clinical improvement, freedom from infections, and resolution of chronic inflammation. results are supportive of extended clinical trials evaluating the safety and efficacy of g1xcgd lv-based gene therapy. updated results from the ongoing northstar-2 (hgb-207) trial of lentiglobin gene therapy in patients with transfusion-dependent β-thalassemia and non-β 0 /β 0 genotypes franco locatelli 1 , alexis thompson 2,3 , janet kwiatkowski 4,5 , suradej hongeng 6 , john porter 7 , martin sauer 8 , adrian thrasher 9 , isabelle thuret 10 , heidi elliot 11 , ge tao background: allogeneic hematopoietic stem cell (hsc) transplantation is potentially curative for patients with transfusion dependent β-thalassemia (tdt); however, it is associated with risks of morbidity and mortality and is limited by donor availability. gene therapy has the potential to be an effective treatment option for patients with tdt, but without some of these limitations. lentiglobin gene therapy contains autologous cd34+ hscs transduced ex vivo with the bb305 lentiviral vector encoding β-globin with the t87q amino-acid substitution. lentiglobin is being studied in patients with tdt and non-β 0 /β 0 genotypes in the ongoing, phase 3 northstar-2 study (hgb-207; nct02906202). methods: northstar-2 is enrolling patients with tdt who had a history of ≥100 ml/kg/year of red blood cells (rbcs) or ≥8 rbc transfusions/year. to generate drug product (dp), autologous cd34+ cells were collected by apheresis after g-csf and plerixafor mobilization and transduced with the bb305 lentiviral vector. patients received myeloablative conditioning with single-agent busulfan before dp infusion. primary endpoint was the proportion of patients achieving transfusion independence (ti, weighted average hemoglobin [hb] ≥ 9 g/dl without rbc transfusions for ≥ 12 months). patients are followed for 2 years and offered participation in a long-term followup study. statistics are presented as median (min-max). results: as of 14 september 2018, 16 patients (age 19 years; 9 patients ≥ 18 years) have been treated (follow-up 9.3 [0.7-20.4] months). patients received a cell dose of 7.7x10 6 (5.0-19.4) cd34+ cells/kg with a dp vector copy number of 3.1 (2.1-5.6) vector copies/diploid genome and 82% (53-90%) of cells were transduced. baseline liver iron content (lic) was 6.4 (1.0-41.0) mg fe/g dw. outcomes by age and baseline lic will be reported. times to neutrophil and platelet engraftment were 19 (13-32) and 44.5 (20-84) days, respectively; 1 patient (1.0month follow-up) and 4 patients (1.0-2.1 months follow-up) had not achieved neutrophil and platelet engraftment, respectively, at time of analysis. of 11 patients with ≥ 3 months of follow-up, 10 (6 ≥ 18 years old) stopped chronic rbc transfusions and two have achieved ti. at last study visit, total hb in these 10 patients was 11.1-13.3 g/dl consisting of 7.7-10.6 g/dl gene therapy-derived hb, hba t87q . one treated patient had no rbc transfusions for 11 months, then re-initiated transfusions due to low hb. non-hematologic grade ≥ 3 adverse events post-infusion in ≥ 3 patients included stomatitis, febrile neutropenia, epistaxis, pyrexia, and veno-occlusive liver disease (vod). one grade 3 event of serious prolonged thrombocytopenia after platelet engraftment was considered possibly related to lentiglobin. the three grade 4 serious vod events were attributed to myeloablative conditioning ( table 1 ). the events resulted in extended hospitalization and resolved following defibrotide treatment. there were no deaths or graft failure and no evidence of vector-mediated replication of competent lentivirus or clonal dominance. conclusions: in northstar-2, 10/11 patients with tdt and non-β 0 /β 0 genotypes treated to date produced sufficient gene therapy-derived hb, hba t87q , to stop chronic transfusions following lentiglobin gene therapy. total hb in patients off rbc transfusions remains stable at > 11 g/dl. the safety profile of lentiglobin remains generally consistent with myeloablative busulfan conditioning. background: metachromatic leukodystrophy (mld) is an ultra-rare and devastating demyelinating lysosomal storage disease caused by mutations in the arylsulfatase a (arsa) gene, currently with no approved treatment. we report an interim analysis of the safety and efficacy results of 20 early-onset mld subjects treated with experimental autologous, ex-vivo, lentiviral-mediated hematopoietic stem cell gene therapy (hsc-gt) followed for up to 8 years posttreatment, as part of an ongoing, open-label, study. the study has completed the enrollment period; follow up visits are currently on-going. methods: gt consists of a formulation of autologous cd34 + cells transduced ex vivo with a self-inactivating lentiviral vector encoding for the human arsa gene and administered intravenously after busulfan conditioning. twenty early-onset mld subjects (pre-symptomatic or early symptomatic) were enrolled and treated (9 late infantile [li] and 11 early juvenile [ej]). co-primary efficacy endpoints were improvement in gross motor function measure (gmfm) score (10%) and a significant increase in arsa activity in peripheral blood mononuclear cells (pbmcs), evaluated 24 months after treatment. safety endpoints include engraftment failure and long-term safety and tolerability of lentiviral-transduction. results: 18/20 subjects are alive after a clinical follow-up of 3-8 years. two ej subjects, treated after onset of symptoms, died due to rapid disease progression 8-and 15months post-treatment. there was no treatment-related mortality, no evidence of abnormal clonal expansion, and no adverse events related to the medicinal product. durable and stable engraftment of gene-corrected cells were observed beginning 1-month post-treatment, with persistent vector copy number in cd34 + bone marrow cells and pbmcs throughout the follow-up for all 18 subjects. reconstitution of arsa activity in the hematopoietic system was observed in both populations (li and ej), stabilizing at normal to supranormal levels within three months. arsa activity in csf showed a similar pattern; normal levels were observed 9-12 months post-treatment, demonstrating effective enzymatic production in the central nervous system (cns). the majority of li and ej subjects treated before the onset of overt symptoms showed normal motor development, stabilization of motor dysfunction or a significant delay in disease progression, as measured by gmfm total score and gross motor function classification (gmfc)-mld. cognitive function (measurements included performance and verbal iq scores) was maintained within normal range for most subjects, independent of their symptomatic status at the time of treatment. improvement or stabilization of central demyelination and peripheral nervous system (pns) abnormalities were observed in most subjects treated. conclusions: this interim analysis demonstrates that hsc-gt continues to be a safe and well-tolerated treatment for all mld subjects treated with a clinical follow-up ≤8 years. all subjects achieved high levels of multi-linage engraftment, polyclonal hematological reconstitution and central and peripheral arsa activity reconstitution within or above normal levels. patients treated prior to symptom onset achieved a sustained clinical benefit in motor and cognitive function as well as on instrumental biomarkers of pns and cns demyelination, suggesting that autologous, ex-vivo hsc-gt is a highly promising therapeutic approach for li and ej mld pre-symptomatic subjects. further research is needed to support the benefit:risk profile in ej patients. clinical trial registry: nct01560182 https://www.clinicaltrials.gov/ct2/show/nct01560182? term=nct01560182&rank=1 disclosure: the san raffaele telethon institute for gene therapy (sr-tiget) is a joint venture between telethon and ospedale san raffaele (osr). ada-scid gene therapy (strimvelis) was licensed to glaxosmithkline (gsk) in 2010 and received european marketing authorization in 2016. alessandro aiuti is the pi of the ada-scid long-term follow up clinical trial sponsored by gsk. strimvelis was licensed to orchard therapeutics (otl) in april 2018. lentiglobin gene therapy in patients with sickle cell disease: updated interim results from hgb-206 background: β-globin gene transfer may reduce or eliminate complications in patients with sickle cell disease (scd). lentiglobin gene therapy (gt) comprises drug product (dp) made from autologous hematopoietic stem cells (hscs) transduced with the bb305 lentiviral vector (lvv) encoding β-globin with an anti-sickling t87q substitution (hba t87q ). the safety and efficacy of lentiglobin gt in adults with scd is being evaluated in a phase 1 study, hgb-206 (nct02140554). patients were initially treated with dp made from bone marrow harvested (bmh) hscs (group a, fully enrolled), then from dp made from bmh hscs but using a refined manufacturing process (group b, fully enrolled), and subsequently from plerixafor mobilized hscs (group c, currently enrolling). methods: adults with severe scd (history of recurrent vaso-occlusive crisis, acute chest syndrome, stroke, or tricuspid regurgitant jet velocity of > 2.5 m/s) were enrolled. autologous cd34+ cells, collected by bmh or apheresis following mobilization with 240 μg/kg plerixafor, were transduced with bb305 lvv. after myeloablative busulfan conditioning (area under the curve goal of 5000 [range 4400 -5400] μm*min daily), patients were infused with the transduced cells and monitored for safety and efficacy. summary statistics are median (min-max). results: as of may 15, 2018, 22 patients had hscs collected, 18 patients had dp manufactured and 15 patients were treated. eleven patients (9 in group a, 2 in group b) underwent bmh and 12 patients (1 in group b [who also had bmh], 11 in group c) underwent mobilization/ apheresis. median of 4.3 (0.1-10.8) x 10 6 and 10.4 (3.8 -21.6) x 10 6 cd34+ cells/kg were collected per bmh (n=26) and per mobilization cycle (n=17), respectively. eighteen grade 3 adverse events (aes) in 6 patients were attributed to bmh and 5 grade 3 aes in 3 patients to mobilization/apheresis. dp and treatment characteristics are shown in table 1 . dp characteristics were improved in group b and group c vs group a. the safety profile post-dp infusion was consistent with myeloablative conditioning and underlying scd; most common non-hematologic grade ≥ 3 aes were stomatitis, febrile neutropenia, and vasoocclusive pain. no grade ≥ 3 dp-related aes, graft failure, veno-occlusive liver disease, replication competent lentivirus detection or clonal dominance were reported. at last visit (table 1) , hba t87q levels were higher in group b (3.2-7.2 g/dl) vs group a (0.5-1.2 g/dl). in 4 group c patients at the 3-month visit, hba t87q (4.1 [3.2-6 .0] g/dl) levels were equal to or exceeded hbs levels (3.3 [2.8-3.8 ] g/dl). in 1 group c patient at the 6-month visit, hba t87q was 8.8 g/dl and total hb was 14.2 g/dl. conclusions: these data support the safety and feasibility of plerixafor-mediated hsc collection in patients with scd. hgb-206 protocol changes have improved lentiglobin dp characteristics yielding higher hba t87q levels. additional data will determine the clinical effect of increased hba t87q /hbs ratios. background: the prognosis of most patients with chemotherapy-refractory or multiply-relapsed cd30+ (a cell membrane protein) non-hodgkin's lymphoma (nhl) or hodgkin lymphoma (hl) still remain poor. targeting cd30 with monoclonal antibodies in hl and anaplastic large cell lymphoma (alcl) was shown to induce remarkable clinical activity; however, occurrence of adverse events (mainly neuropathy) may result into treatment discontinuation in many patients. immunotherapeutic approaches targeting cd30 by chimeric antigen receptor (car) has been demonstrated to be of value in two independent clinical trials (pmid: 27582488) (pmid:28805662), although clinical benefit was sub-optimal. methods: we designed two 3 rd generation, clinical-grade retroviral vectors carrying the cassette anti-cd30 singlechain variable fragment linked via cd8 hingetransmembrane domain, to the signaling domains of two costimulatory domains, namely either cd28/4-1bb or cd28/ox40 and cd3-ζ. the inducible caspase-9 (icasp9) safety switch was also included in the constructs with the goal of promptly controlling undue toxicity. as a selectable marker, we added, in frame with the car molecule, a peptide derived from cd34 antigen. the in vitro anti-tumor efficacy was evaluated by using karpas299, l428 or hdml-2, in both short-term cytotoxic assay (represented by cr 51 release assays) and long-term co-cultures (7 days). cytokine profile upon antigen stimulation was characterized, as well as tcell exhaustion and memory marker profile. to assess the expansion, persistence, and antitumor effect of car.cd30 t cells in vivo, we used a nsg mouse model engrafted i.v. with human lymphoma cell lines (karpas299 and l428) genetically modified with ff-luciferase, this allowing the monitoring of tumor growth by ivis imaging system. persistence of car.cd30 t cells was evaluated every 15 days, together with a deep characterization of memory profile and policlonality of persisting t cells. results: independently from the costimulatory domains cd28/ox40 or cd28/4-1bb, the generated retroviral vectors showed similar transduction efficiency of t cells (86.50±5.08% and 79.30±5.33%, respectively). nevertheless, car.cd30 incorporating cd28.ox40 costimulatory domains was associated with more stable expression of the car over time, during extensive in vitro culture (84.72 ±5.30% vs 63.98±11.51% car+ t cells at 30 days after transduction; p=0.002). this finding was also associated with the evidence that car.cd30-cd28.ox40 t cells showed a superior anti-lymphoma in vitro activity as compared to car.cd30-cd28.41bb t cells, when challenged at very high tumor/effector ratio (8:1). moreover, antigen-specific stimulation was associated to high levels of th1 cytokine production, with car.cd30-cd28.ox40 t cells secreting a significantly higher amount of ifnγ (8306.03±3745.85 pg/ml), il2 (13492.68±5837.77 pg/ml) and tnfα (17661.00±11113.27 pg/ml) as compared to car.cd30-cd28.41bb t cells (6617.81±3025.67 pg/ ml, p= 0.040; 7616.67±4464.06 pg/ml, p=0.008; 5824.63 ±1823.73 pg/ml, p=0.02; respectively). in nsg mouse lymphoma models, we proved that car.cd30-cd28. ox40 t cells had an extensive superior anti-tumor control than car.cd30-cd28.41bb t cells, leading to a significant reduction of bioluminescence at day 45 (3.32x10 6 vs 2.29x10 10 , p=0.04) and an increased overall survival of the treated mice (60% vs 10%, at 180 days, p=0.0014). conclusions: overall, these data indicate that, in the context of car.cd30 t cells, the costimulatory machinery of cd28.ox40 is crucial for improving both persistence and ultimately the antitumor efficacy of the approach. disclosure: nothing to declare background: acute graft-vs-host disease (agvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). less than 50% of patients (pts) achieve sustained responses with first-line corticosteroid (cs) treatment. retrospective studies demonstrated clinical benefit with the janus kinase (jak)1/jak2 inhibitor ruxolitinib (rux) in pts with steroid-refractory (sr) agvhd. here we present 6-month follow-up data from patients enrolled in reach1 (nct02953678), a phase 2 trial evaluating rux plus cs in sr agvhd. methods: reach1 was a single-arm, open-label, multicenter study. eligible pts were aged ≥12 years and developed grade ii-iv sr agvhd following allo-hsct from any donor source for hematologic malignancies. sr agvhd was defined as gvhd that progressed after 3 days or had not improved after 7 days of primary treatment with methylprednisolone ≥2 mg/kg/d (or equivalent), development of gvhd in another organ after receiving ≥1 mg/kg/d methylprednisolone for skin or skin plus upper gastrointestinal gvhd, or inability to tolerate cs taper. pts received rux 5 mg twice daily (bid), with optional increase to 10 mg bid in the absence of cytopenias. the primary endpoint was day 28 overall response rate (orr), and the key secondary endpoint was 6-month duration of response (dor). orr was defined as the proportion of patients demonstrating a complete response (cr), very good partial response, or partial response. results: the study enrolled 71 pts. median age was 58 years, and 49.3% were male. treatment was ongoing in 11 pts (15.5%) at data cutoff (2 jul 2018) . at day 28, orr was 54.9% (cr, 26.8%). responses were observed irrespective of agvhd grade and sr criteria. best orr at any time during treatment was 73.2% (cr, 56.3%). median (range) time to response was 7 (6-49) days. median dor with minimum 6 months follow-up was 345 days for both day 28 responders ( figure 1 ) and for pts who had a best overall response at any time during treatment. four pts (5.6%) had malignancy relapse. overall, nonrelapse mortality at 6 months was 44.4%; results varied by day 28 response (day 28 responders, 21.2%; other responders, 64.1%; nonresponders, 78.9%). median overall survival had not been reached for day 28 responders. the most frequently reported hematologic treatment-emergent adverse events (teaes) were anemia (64.8%), thrombocytopenia (62.0%), and neutropenia (47.9%). cytomegalovirus infection (12.7%), sepsis (12.7%), and bacteremia (9.9%) were the most frequently reported infections. fatal rux-related teaes were sepsis and pulmonary hemorrhage (1 pt each) and were attributed to both rux and cs. conclusions: in this first prospective trial of rux in sr agvhd, orr was 54.9% by day 28 and 73.2% at any time during treatment. responses were rapid and durable. the ae profile was consistent with expectations for rux and pts with sr agvhd. rux represents a promising therapeutic strategy. background: chronic graft-versus-host disease (cgvhd) remains a major complication after allogeneic hematopoietic cell transplantation (allo-hsct). over the last decade, clinical success in patients with cgvhd has been hampered by the lack of insight into the complex pathobiological mechanisms of the disease and the paucity of specific therapeutic targets. although, it is now evident that the clinical manifestations of cgvhd are the result of a highly complex immune pathology involving both donor b cells and t cells as well as other cells. current work on immune cells involved in cgvhd pathobiology is limited by the number of parameters that conventional flow cytometry (fcm) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. mass cytometry time-offlight (cytof) substitutes rare earth elements for fluorophores to label antibodies, which allows simultaneous measurements of more than 40 parameters in single cells without correction for spectral overlap, and empowers us to understand cgvhd biology at the single-cell level. methods: we used mass cytometry with extensive antibody panels to perform in-depth immune profiling of peripheral blood samples from 34 patients following allo-hsct, in which 11 patients were without cgvhd, 7 patients experienced moderate cgvhd and 16 patients experienced severe cgvhd. the involved organs in patients with cgvhd are skin, liver and lung. results: we simultaneously stained cells with 42 antibody panels created for this study. the t cell panel was designed to identify different populations of naive, memory, effector, regulatory, and exhausted t cells. the panels also included markers for the identification of b cells, natural killer cells, nkt cells, dc cells, plasma cells, granulocytes, and myeloid cells. in 4 million measured cells, we identified 40 immune cell phenotypes, in which there were 22 t cell phenotypes, 6 b cell phenotypes, 6 monocyte phenotypes and 6 granulocyte phenotypes. to generate a comprehensive view of the immune ecosystem of cgvhd, we generated two-dimensional maps of the data using the dimensionality reduction algorithm t-sne. this analysis showed a strong overlap between cgvhd of moderate and severe grades, but seperation from patients without cgvhd. seven immune compositions were identified to be cgvhd-associated. five distinct immune cells were correlated with specific cgvhd-involved organs (skin or lung), thereby presenting an in-depth human atlas of the immune cells in this disease. conclusions: this study revealed potential biomarkers and targets for immunotherapy of cgvhd and validated cytof as a valuable tool that can be used for immune profiling of cgvhd. disclosure: nothing to declare o100 t cell costimulation blockade with abatacept for acute graft-versus-host disease prevention in matched and mismatched unrelated donor transplantation: results of the first phase 2 trial background: we performed a phase 2 trial in adults and children to test abatacept for agvhd prevention ('aba2'), based on our promising preclinical and pilot patient data. methods: aba2 had 2 cohorts: a) hla mismatched ('7/ 8', n= 43), a single-arm study with pre-specified cibmtr matched analysis (vs cni+mtx or cni+mtx+atg). b) hla-matched ('8/8', n= 142) , randomized double-blind, comparing cni+mtx+placebo vs cni+mtx+aba ('aba'). abatacept dosing was 10mg/kg on d -1, +5, +14, +28. aba2 was designed as a screening phase 2 trial, with relaxed type 1 error (0.2) and standard type 2 error (0.2). power analysis assumed reduction of gr 3-4 agvhd from 30%-->10% in 7/8s and 20%-->10% in 8/8s. median follow up = 708 days (7/8s) and 369 days (8/8s) . results: reduced grade 3-4 agvhd: aba was associated with decreased d180 gr 3-4 agvhd. in 7/8s, gr 3-4 agvhd =2.5% (aba) vs 31% (cni+mtx) and 22% (+atg), (1-sided p =0.001, 0.005). in 8/8s, gr 3-4 agvhd =6.85 % in aba vs 14.6% in placebo, (1-sided p =0.068). reduced grade 2-4 agvhd in 8/8s: aba was associated with decreased d180 gr 2-4 agvhd. in 7/8s, gr 2-4 agvhd =42% (aba) vs 54% (cni+mtx) and 45% (+atg, 1-sided p =0.098, 0.25). in 8/8s, gr 2-4 agvhd =44.5% in aba vs 62.3% in placebo (1-sided p =0.004). chronic gvhd: for 7/8s, 1 yr cgvhd =38.8% (aba) vs 43.5% (cni+mtx) and 35.5% (+atg, p =0.4, 0.99). in 8/8s, data collection is ongoing. safety indicators: there was no difference in neutrophil or platelet engraftment, cmv and ebv reactivation between aba and controls. cumulative incidence of relapse in 7/8s at 2 yr 9.4% (aba) vs 20.6% (cni+mtx) and 23.4% (+atg) (p=0.115, 0.085). in 8/8s, at 1 yr, it was 13.8% (aba) vs 20.5% (placebo, p =0.7). statistically significant survival advantage in 7/8s: for 7/8s, 1 yr non-relapse mortality (nrm) = 10.5% (aba) vs 32.7% (cni+mtx) and 26% (+atg, p =0.024, 0.365). for 8/8s, nrm = 7.1% vs 14.6% at 1 yr (p =0.5). severe agvhd free survival at 6 months for 7/ 8s =97% (aba) vs 55% (cni+mtx) and 59% (+atg, p =0.001, 0.006). for 8/8s = 89.0% (aba) vs 76.8% (placebo, p = 0.049). for 7/8s, relapse-free survival (rfs) = 73.7% (aba) vs 38.7% in cni+mtx and 48.7% in +atg (p =0.001, 0.027). for 8/8s, rfs = 79.1% for aba vs 64.9% (placebo, p =0.38). for 7/8s, overall survival (os) =71% (aba) vs 47.5% (cni+mtx) and 58% (+atg, p =0.01, 0.145). for 8/8s, os =83.2% (aba) vs 76.6 (placebo, p =0.32). conclusions: our results suggest that short-course aba can safely prevent agvhd without compromising relapse. despite the modestly sized study, the comparative event size for 7/8s was high enough that the protective effect of aba against gr3-4 agvhd was highly significant. for 8/ 8s, there was a statistically significant improvement for gr 2-4 gvhd and a trend toward an advantage in all parameters. for both cohorts, severe agvhd free survival was statistically-significantly improved. these results are the first to demonstrate efficacy of in vivo t cell costimulation blockade in preventing agvhd. background: in the nih cgvhd diagnostic classification, patients with gvhd after 3 months are classified as either late agvhd or cgvhd. to date, this is only a clinical classification, with no biological differences identified. recently, the pbmtc 1202/ applied biomarker in late effects of children and adolescent (able) study completed accrual of 302 pediatric allogeneic hematopoietic cell transplantation (hct) patients. we used day 100 biomarkers to identify biological differences between cgvhd and late agvhd. methods: the pbmtc1202/able study with 26 centers in canada, us, and europe prospectively collected peripheral blood samples at 3, 6, 12 month post hct and at the onset of cgvhd in 302 children. a comprehensive analysis for previously identified cgvhd immune cell markers by flow and cytokines by elisa on plasma and streck tubes shipped overnight and centrally evaluated at bc children's hospital. clinical data was collected centrally with a thorough central clinical adjudication by the pbmtc study committee. of those enrolled, 228 were evaluable at day 100 and classified as a) late agvhd (n = 58), b) cgvhd (n = 44), and c) controls that did not develop cgvhd (n = 132). univariate analysis was performed comparing late agvhd, cgvhd, and no gvhd controls. significant differences were defined as a biomarker with both a roc auc ≥0.60 and p value ≤0.05 compared to controls. results: the profile of cgvhd included a cluster of abnormalities in memory and transitional b cells, conventional naïve and follicular helper t cells, and a loss of both recent thymic emigrant regulatory t cells and cd56 bright nk regulatory cells. four inflammatory cytokines, st2, aminopeptidase n, cxcl9 and mmp3 (see table 1 ) were increased. patients clinically identified as late agvhd had a more restricted biomarker pattern of limited b cell abnormalities and st2. conclusions: late acute gvhd is limited to restricted b cell and elevation of st2. cgvhd is characterized by the identical b cell abnormalities but with the additional loss of regulatory function in cd56bright nkregs and rte treg cells. with the loss of regulatory function in cgvhd, there is an increase in cd21 lo b cells, follicular t helper cells, and additional cytokines. these prognostic markers findings may suggest therapeutic targets that differ for late agvhd compared to cgvhd. background: we are reporting the outcome of 69 patients with steroid refractory acute graft versus host disease (sr-gvhd), treated with an anti-cd26 monoclonal antibody (begelomab r ). methods: twenty-eight patients were enrolled in two pilot studies eudract no. 2007-005809-21 and no. 2012-001353-19 , whereas 41 patient were treated on a multicenter follow up compassionate use of the antibody. the median age of the patients was respectively 42 and 44 years. at the time of anti-cd26 treatment, gvhd was overall recorded as grade ii in 8 patients, grade iii in 33 and grade iv in 28 patients. in the pilot sudies patients had failed 1 line of treatment, wheas in the follow up compassionate use, patients had failed one line (n=18), two lines (n=11), three lines (n=11) or 4 lines of treatment (n=1). results: there were no adverse events attributable to the antibody. day 28 response was recorded in 75% and 63% in the pilot studies and follow up patients. response in grade ii gvhd was evaluable only in the pilot studies (57%); response in grade iii gvhd was recorded in 80% and 83% patients in the two groups; response in grade iv gvhd was recorded in 66% and 56% of patients in the two groups. overall there were 60% responses for skin and liver stage 3-4, and 70% responses for gut stage 3-4 gvhd. the cumulative incidence of non relapse mortality (nrm) at 6 months was 28% and 38%. for day 28 responders, this figure was 19% and 22%, for non responders it was 57% and 66% in the two groups. the overall survival at 1 year was 50% for the pilot studies and 33% for the follow up patients. conclusions: in conclusion, begelomab induces a high remission rate on day+28 in patients with sr-gvhd, including a significant proportion of patients wih severe gut and liver gvhd. clinical background: cgvhd is characterized by an imbalance between effector and regulatory arms of the immune system that results in overproduction of inflammatory cytokines including il-17 and il-21. moreover, a persistent reduction in the number of regulatory t (treg) cells limits the ability of the immune system to recalibrate this pro-inflammatory environment. kd025 is an orally available rho-associated coiled-coil kinase 2 (rock2) selective inhibitor. in vitro data suggest that kd025 modulates immune homeostasis by shifting the th17/treg balance towards a treg phenotype. methods: kd025-208 is an open-label phase 2a study in patients with steroid-dependent cgvhd after no more than 3 prior lines of treatment. three cohorts (c1: 200mg qd (n=17), c2: 200mg bid (n=16), and c3: 400mg qd (n=21)) were enrolled. the primary endpoint is overall response rate (orr), defined per the 2014 nih consensus criteria. results: as of 13-september-2018, the median duration of treatment was 37, 33 and 27 weeks for c1, c2 and c3, respectively. the median age was 52 years (range 20-75) and median time from cgvhd diagnosis to kd025 treatment was 19 months. 67% of patients had received ≥2 prior lines of therapy and 48% had ≥4 organs involved at baseline. 20 patients remain on treatment with kd025, with median duration of treatment of 89 weeks (n=6), 68 weeks (n=3) and 34 weeks (n=11) for each cohort, respectively. the orr was 65% in c1, 63% in c2 and 52% in c3. responses were rapid, with 75% of responders achieving a response at the first assessment (8 weeks). among responders, 82%, 50% and 36% have sustained responses for ≥20 weeks in each cohort, respectively. responses were observed across all affected organ systems, including crs in upper gi, lower gi, esophagus, mouth, skin, joints/fascia, eyes, and liver. two patients with lung cgvhd achieved pr. 69% of patients achieved reductions in corticosteroid dose and 7 patients discontinued corticosteroid treatment while receiving kd025. 72% of responders achieved a clinically meaningful improvement (≥7-point reduction) in the lee symptom scale (lss) score. kd025 has been well tolerated. commonly reported aes (≥20% patients) were urti, ast/alt elevations, fatigue, nausea and diarrhea. grade ≥3 aes occurring in >3 patients were ggt elevations (n=6) and hyperglycemia (n=4). no saes were considered related to study drug. two patients discontinued treatment due to aes considered possibly related to kd025 (headache, diarrhea). three fatal events occurred (relapse of leukemia; lung infection; cardiac arrest); none were considered related to kd025. no increase in incidence of infection was observed. consistent with the postulated kd025 mechanism of action, th17 cells decreased and treg cells increased in patients receiving kd025 background: antithymocyte globulin (atg) treatment significantly decreases later development of chronic graftversus-host disease (cgvhd). one phase 3 trial evaluating atg was the canadian bmt group (cbmtg) 0801 study that found that atg treatment resulted in significantly less cgvhd and dependence on immunosuppressive treatment at 1 year. the exact mechanism by which atg decreases cgvhd is not known. we hypothesized that using known prognostic day 100 cgvhd biomarkers in the wellcontrolled cbmtg 0801 trial represented an optimal approach to understand atg's biological impact on cgvhd in humans. methods: a separately developed cbmtg cgvhd biomarker study opened while cbmtg 0801 was ongoing and accrued 40 patients (n = 25; atg treated and n = 15 controls) of the 203 cbmtg 0801 patients. samples were collected at 3, 6, and 12 months and at the onset of cgvhd and evaluated at bc children's' hospital research institute, vancouver, bc. patients were evaluated for day 100 immune cellular markers previously associated with later cgvhd including: a) naive helper t (th) cells, b) recent thymic emigrant (rte) th cells; c) cd21 low b cells; d) cd56 bright nkreg cells; and e) treg cells. frequencies in the atg treated and control group were evaluated to detect significant difference using non-parametric t-test mann-whitney test. results: patients of this subpopulation (aged 16-70 years) were shown to be representative of the larger cbmtg 0801 study population. the atg treated group had a significant decrease in total t cells, rte th cells, and naïve th cells at day 100 compared to the control population (p < 0.0001 each population -see table 1 ). atg treatment had no impact on tregcells, cd19+ b cells, and cd21lowb cells but there was a significant increase in cd56bright nkreg cells (p < 0.0001). we evaluated the ratio of naïve th effector cells to the regulatory nkregcell population and saw a >100 fold difference the atg treated group (naïve th cell:nkregcell ratio = 0.06) compared to untreated patients (ratio of 9.2; p < 0.0001). in this small population we found that the naïve th cell:nkregcell ratio was also high prognostic for later development of cgvhd (1.37 vs. 0.13; p < 0.0001). conclusions: these results suggest that atg's major mechanism of action is related to its ability to simultaneously inhibit naïve th cells and enhance cd56 bright nkreg cells after transplantation. while these results require confirmation, they support strategies that target the ratio of nk reg cell and naive cd4+ t cells to modulate cgvhd. clinical trial registry: nct01217723 disclosure: none of the authors have any conflicts of interest to declare o105 immune reconstitution -based score at diagnosis of cgvhd predicts gvhd severity and overall-survival: a novel prognostication tool for gvhd treatment tailoring background: allogeneic stem cell transplantation (hsct) survivors are at a relevant risk of developing chronic gvhd (cgvhd), which importantly affects quality of life and increases morbidity and mortality. early identification of patients at risk of development of severe cgvhd related morbidity would be a relevant tool to tailor preventive strategies. we have previously demonstrated the role of immune reconstitution (ir) as predictive biomarker of occurrence of cgvhd. the aim of this study was to evaluate the prognostication power of ir at cgvhd onset through a new ir-based score. methods: we analyzed clinical data from 383 adult patients consecutively undergoing first allogeneic hsct transplant between january 2011 and december 2016 at our institution. a written consent was given for the use of medical records for research. patients were divided into a test cohort (307 pts) and a validation cohort (76 pts). median follow-up for surviving patients was 4 years.we built a cox multivariate models for os in patients with cgvhd of any severity. variables included in the models were: patient age (according to median value), r-dri score, type of donor (matched related donor vs matched unrelated vs haploidentical), main gvhd prophylaxis platform (atg-based vs ptcy-based vs neither of the two), ir values (cd4, cd19, nk, iga, igm according to median values) at cgvhd diagnosis, history of prior agvhd, karnofsky ps, plt < 100.000/μl, alc< 1000/μl, eos < 500/μl.once we identified the variables independently predicting os by multivariate analysis, we derived a formula for a prognostic risk index by using the β coefficients found in the model. each patient was then assigned a score and we defined three groups of os risk (low, intermediate and high) by dividing the score into three classes using the first and third quartiles. finally, to evaluate predictive performance of the ir-score we calculated the receiver operating characteristics (roc) curve via the area under the curve (auc), to summarize the ir-score ability to correctly classify events and non-events. results: 115 patients (87 test-cohort, 28 validationcohort) were evaluated for cgvhd and outcome. our multivariate model defined the variables independently predicting os at cgvhd onset: cd4+ count >233/ mcl, nk count < 115/mcl, igm < 0.45 g/l, karnofsky ps < 80%. final score was calculated as follows: 2,4 (if cd4 >233/ mcl) + 2,1 (if nk < 115/mcl) + 2,1 (if igm < 0,45) + 4,3 (if karnofsky < 80). low risk patients were defined as having a score ≤2.4, intermediate >2,4 and ≤4.5, high risk >4.5. the 3y-os for low risk patients was 96%, for intermediate 76% and for high risk 27% in the test-cohort and 100%, 66% and 35% in the validation-cohort ( figure 1a-b) . the roc curve analysis supports the validity of the ir-score in our cohort of patients -auc 85.5%, with 95% confidence intervals higher than 50%. furthermore ir-score was able to stratify across nih-severity classification (figure 1c). conclusions: immune-reconstitution score at diagnosis of cgvhd predicts gvhd severity and overall-survival. irscore could be adopted to identify patients at high risk and modulate cgvhd treatments accordingly. disclosure: chiara bonini has research contract with intellia therapeutics. the other authors declare that they have no conflicts of interest. haploidentical transplantation with sirolimus-based gvhd prophylaxis and unmanipulated pbsc graft: background: haploidentical transplantation has emerged as a viable option for patients lacking a fully matched donor. we firstly explored the association of sirolimus and atg, later followed by sirolimus with pt-cy as gvhd strategy. herein, we describe long-term outcomes of haploidentical hsct using sirolimus-based gvhd prophylaxis. methods all patients received sirolimus and mmf as gvhd backbone prophylaxis plus atg in 203 patients, and pt-cy in 151. conditioning regimen was based on treosulfanfludarabine; recipients of pt-cy transplants were more likely to receive a regimen intensified by a 2 nd alkylating agent (melphalan or thiotepa). median follow up was longer in atg group (70 vs 26 monhs, p< 0.01). there were no differences in dri. results: the majority of patients reached the neutrophil (89% in atg group vs 86% in pt-cy group) and platelet (76% vs 77%) engraftment within 30 days after hsct. immune-reconstitution was broad and fast, reaching more than 100/ml cd3+ t cells within a median of 35 vs 33 days. the two groups were similar in terms of survival and main transplant outcomes. in the atg group, the cumulative incidence of grades ii-iv and iii-iv acute gvhd at 100 days was 26% and 20%. corresponding rates after pt-cy were 35% and 20%. the cumulative incidence of overall and severe chronic gvhd was 31% and 10% at 3 years in atg group and 42% and 16% after pt-cy .the cumulative incidences of relapse and nrm in atg group were respectively 41% and 31% at 3 years. corresponding rates after pt-cy were 35% and 27%. in atg group, 3-year os was 36%, while grfs was 24%. the corresponding probabilities after pt-cy were 44% and 24%. the only difference reported was a better pfs in favour of pt-cy (38% vs 29%, p=0.04 conclusions: extended follow-up in 354 patients confirms sirolimus-based gvhd prophylaxis as feasible and safe in haploidentical hsct based on unmanipulated pbsc graft. both atg and ptcy association to sirolimus provide an effective prevention of gvhd and translate into a similar long-term overall survival. a significant advantage of sir-pt-cy on relapse rate warrants further investigation. background: steroid-refractory graft-versus-host disease (sr-gvhd) is still responsible for high mortality in patients undergoing allogeneic stem cell transplantation; a number of agents is currently available in case of steroid-refractoriness, yet there is so far no consensus about a standard second-line treatment and overall survival (os) remains poor.α1-antitripsyn (αat) is a circulating 52-kda serine protease inhibitor found to enhance the production of anti-inflammatory cytokines and to favor the expansion of regulatory t-cells; it has therefore been tested in situations of altered tolerance and disproportionate inflammation, including gvhd. two studies showed that treatment of sr acute gvhd with αat is feasible and effective. methods: we retrospectively analyzed a series of patients who received exogenous αat for sr acute gastrointestinal gvhd or overlap gvhd with acute gut features. sr-gvhd was defined and graded according to standard criteria. αat was administered intravenously at a loading dose of 90 mg/kg at day 1 followed by 30 mg/kg /day every other day for a total of 8 doses. response to treatment was defined according to published criteria; os was estimated with the kaplan-meier method.a panel of 46 cytokines and immune cell subsets were measured before treatment and once weekly during treatment by a luminex assay and by flow cytometry, respectively. results: sixteen patients were treated for gut gvhd between september 2016 and march 2018. median age was 50 years (range 18-56). αat was administered at a median time of 104 days from transplantation (range 38-215) and of 65 days from gvhd onset (range 12-198) . acute gvhd was scored as grade ii in 28% of patients, grade iii in 66%, grade iv in 6%. sixty-seven percent of patients had already received one or more lines of treatment other than steroids, including ruxolitinib, etanercept, atg; orr was 44% with a cr rate of 27%; median time to best response was 21 days (range 6-26), with a continued orr at day 56 of 39%. the overall rate of gastrointestinal responses was 61%. median follow-up of living patients was 440 days (range 84-602); median os was 138 days and 1-year os was 48% (95% ci 26% -74%).the most common infectious event was cmv reactivation (29%); 2 grade 3-4 infectious complications were recorded. there was no quantitative deficiency of blood aat levels before treatment (1.72 g/l ± 0.46); blood αat rose significantly during and after treatment. baseline αat level didn´t differ between responding and nonresponding patients. a cytokine profile was evaluable in 12 patients; no statistically significant increase or decrease in cytokine plasma concentration after αat infusion was observed. surprisingly, a decrease in circulating t regs after exposure was found (p=0.002), regardless of patients´responding status. conclusions: treatment with αat was safe and effective in a cohort of sr-agvhd high risk, pre-treated patients and should be considered as a possible alternative. changes in the cytokine milieu and t-cell subsets shown in murine models were not observed in a real-life setting. table 1 . peripheral blood was used as graft source in 87% of the patients in the atg group and in 78% in the pt-cy group. gvhd prophylaxis consisted in atg 2mg/m 2 days -4 to -2, mtx days +1, +3, +6 and +11, and csa from day -1 in the atg group. the pt-cy group received cyclophosphamide 50 mg/kg/d on days +3 and +4, followed by either csa or tacrolimus and mycophenolate mofetil (mmf) from day +5 in 30 patients (42%), cyclophosphamide on days +3 and +5 combined with csa or tacrolimus from day 0 in 26 patients (36%), or cyclophosphamide on days +3 and +5 combined with tacrolimus and sirolimus from day +5 in 16 patients (22%). cumulative incidence at 100 days of grade ii-iv (67% vs 46%, p=0.02) and iii-iv (41% vs 3%, p=0.002) acute gvhd, were significantly higher in the mtx-csa group ( figure 1a ). there were no differences in the 2-year cumulative incidence of chronic moderate to severe gvhd between the atg and the pt-cy group (30% vs 24%, p=0.6). after a median follow-up of 90 months for the atg group and 26 months for the pt-cy group, 2-year overall survival (os) was higher in the pt-cy group (45% vs 60%) although not statistically significant (p=0.09) ( figure 1b) . we found no differences between both cohorts in 2-year event-free survival (efs) (46% and 50%, p=0.55) and the composite endpoint of gvhd-free and relapse-free survival (gfrs) (38% vs 40%, p=0.253). the 2-year cumulative incidence of relapse was significantly higher in the pt-cy group (26% vs 6.6%, p=0.04) and non-relapse mortality (nrm) at 2-years was higher in the atg group (40% vs 22%) but not statistically significant (p=0.06). conclusions: in our experience, in spite of the limited number of patients, gvhd prophylaxis using pt-cy combined with additional immunosuppression after mud hsct, using mostly peripheral blood as graft source, reduced the cumulative incidence of agvhd compared to standard prophylaxis with mtx-csa. prospective studies with longer follow-up are needed to confirm these observations. disclosure: nothing to declare o109 abstract already published. methods: 44 consecutive patients who underwent allo-sct for hematological malignancies between january 2016 and august 2018 were included in this prospective singlecentre protocol. all patients had at least one baseline risk factor predicting development of severe gvhd (e.g. hla mismatch, fem-to-male sex mismatch). pt-cy in combination with a second immunosuppressive drug was used as gvhd prophylaxis. results: patients characteristics are summarized in table 1 . median age was 56 (range, 19-67) years, with 6 male patients (14%) receiving a graft from a female donor. more frequent allo-sct indications were acute leukemia and mds (54.5%) followed by nhl (18%). eleven patients (25%) were transplanted in advanced status. donor was a sibling, matched unrelated or mismatched unrelated in 34%, 25% and 41% respectively. seven patients (16%) received a myeloablative conditioning regimen including tbi (8 or 13.5 gy) while the remaining (84%) received a ric/rtc regimen based on fludarabine, busulfan or melphalan +/-thiotepa (3mg/kg). median follow-up for survivors was 365 days (range:64-959). median time to neutrophil and platelet engrafment were +23 (12-36) and +22 (10-50) days, respectively (g-csf not routinely used). early toxicity was low, without cases of thrombotic microangiopathy, only 2 cases of drug-related renal failure (4.5%) and 1 case of possible vod. before the introduction of mini-thiotepa in the ric protocols (flubu/ flumel) there was 1 case of primary graft failure (gf) and 5 cases of late graft failure (6/28; 21%); 4 cases were successfully regrafted with the mini-thiotepa ric. there have been no further cases of gf after its introduction (n=8 evaluable cases + 4 second salvage allorics). additional potential risk factors for gf were cd34+ ≤3x10e6/kg (p=0.07) and a high lymphocyte count at stem cell infusion (p=0.06). the ci of grade 2-4 acute gvhd at day + 120 was 28% (95%ci: 14-48) with only 2 cases of refractoriness to steroids. of the 37 evaluable patients, only 1 developed moderate chronic gvhd leading to a 1yr-ci of 3%. nrm was 10.5% at 1yr and the ci of relapse was 31% (95% ci: 21-40). all relapses occurred in patients with intermediate/ high rdri. 1yr-os was 76% and the estimated 1-year dfs was 63.6%. conclusions: these outcomes confirmed the feasibility of both ric and mac allo-sct using pt-cy as soc with a single immunosuppressive drug in patients at high risk of gvhd. an important observation was the high rate of gf with "classical" alloric platforms (flubu/flumel), which appears to be lower after introducing the mini-thiotepa. although these data need confirmation in larger cohorts, the current results suggests that pt-cy may pave the way to improving the quality of life of transplant survivors by markedly reducing severe gvhd. protection of the endothelium during steroid-refractory gvhd background: clinical data demonstrated that endothelium related factors predict mortality after the diagnosis of agvhd, suggesting that the endothelium may be involved in the pathobiology of steroid-refractory agvhd (sr-agvhd) (j clin oncol. 2018 mar 10;36 (8) methods: intestinal biopsies from patients after allosct. murine agvhd models balb/c→b6, b6→bdf and lp/ j→b6 with and without steroid treatment. immunostaining, electron microscopy, light sheet fluorescence microscopy (lsfm), facs. in vivo and in vitro assays for endothelial dysfunction. treatment with phosphodiesterase type 5 inhibitor (pde5) in sr-agvhd models. results: we found a significant higher percentage of apoptotic vessels in duodenal and colonic mucosa biopsies of patients with grade iii-iv agvhd compared to no gvhd ( figure 1a ). in murine experimental agvhd, we detected severe microstructural endothelial damage and reduced endothelial pericyte coverage accompanied by reduced expression of endothelial tight junction proteins leading to increased endothelial leakage in agvhd target organs. during intestinal agvhd, colonic vasculature structurally changed, reflected by increased vessel branching and vessel diameter ( figure 1b) . we analysed human biopsies and murine tissues from sr-gvhd vs. naive (untreated) agvhd and found significantly lower lymphocyte infiltration in sr-gvhd, demonstrating low inflammatory activity ( figure 1c ). our findings suggest that endothelium-related and t cell independent mechanisms play a previously unrecognized role during sr-agvhd, providing the rationale for t-cell independent treatment strategies. as a first example for such an approach, we tested the endothelium-effective pde5 inhibitor sildenafil and found reduced apoptosis as well as improved metabolic activity of endothelial cells in vitro. in accordance, sildenafil treatment resulted in improved survival and reduced target organ damage during experimental sr-agvhd ( figure 1d ). conclusions: we show profound endothelial involvement after allo-hsct and demonstrate that endothelialprotection with sildenafil ameliorates sr-agvhd, providing a novel non-immunosuppressive treatment approach. these results can serve as rationale for translational development of endothelium-based therapies for sr-agvhd. disclosure: the authors declare no confilct of interest relevant to this study o113 abstract already published. the therapeutic effect of immune-modifying microparticles in an acute graft-versus-host disease model john galvin 1 , sara beddow 2 , stephen miller 2 1 university of illinois chicago, chicago, il, united states, 2 northwestern university, chicago, il, united states background: inflammatory monocytes are recruited to target organs during acute graft versus host disease (agvhd). as seen in other autoimmune disorders, inflammatory monocytes play an important role in antigen presentation and cytokine production. these actions allow for a sustained activation and proliferation signal to t-cells. previous studies have shown that imp treatment in mouse models of colitis, encephalitis, myocardial infarction and peritonitis markedly reduced monocyte accumulation in the affected end-organs --promoting tissue repair; reducing disease symptoms and increasing survival. therefore, our objective was to test clinical outcomes after imp treatment in a mouse model of agvhd. methods: murine agvhd model: balb/c mice were given 800 cgy total body irradiation, irradiated balb/c mice were transplanted with 5×10 6 c57bl/6 bone marrow cells and 1×10 6 c57bl/6 spleen cells via tail vein.imp treatment: imps were made with plga (phosphorex inc, hopkinton ma) was administered to the recipient mice (1.4 mg/kg body weight) by iv daily starting from day 5 to day 10 after bone marrow transplantation (bmt). pbs at the same volume was used as vehicle control. in vivo bioluminescence imaging: mice were given an intraperitoneal injection of luciferin (150 mg/kg body weight) and then anesthetized and imaged using the ivis imaging system (xenogen). imaging data were analyzed and quantified with living image software (xenogen). results: imp treated mice had significantly less severe acute gvhd symptoms (average score of 2.48) than the untreated bm+sp group (average score 3.96) starting at the time of imp treatment (days 5-10) and remained with significantly reduced symptoms for the 30 day course (figure 1 ). imp treatment also rescued bm+sp mice from agvhd associated mortality with a 30-day overall survival of 62% compared to 4% in the untreated bm+sp group (figure 2 ). intestinal tissue from the imp treated mice compared to the bm+sp mice demonstrated less evidence of agvhd (an average score of 1.25 and 2.75, respectively). hepatic tissue from the imp treated mice compared to the bm+sp mice demonstrated less evidence of agvhd (an average score of 1.5 and 2.42, respectively). imp treatment also significantly reduced inf-γ levels in the intestinal tissues of treated mice compared to untreated bm +sp mice. in the mice infused with lymphoma cells (a20-luc), imp treatment reduced agvhd symptoms and death while preserving the gvl effect. conclusions: our results demonstrate that imps significantly reduce symptoms and mortality in a murine model of agvhd while preserving gvl. the reduction in inflammatory monocytes with imps leads to a reduction in inflammatory cytokines, hepatic lymphocyte infiltration and intestinal mucosal denudation. these findings highlight the potential of imp therapy as a specific and potentially safe treatment in acute gvhd. [[o114 image] 1. figure 1 background: despite significant improvements in the supportive sickle cell disease (scd) causes substantial morbidity and mortality. allogeneic hematopoietic stem cell transplantation (hsct) is currently the only curative option but is only offered if a matched sibling donor (msd) is available. with a msd availability of < 20% t cell depleted hsct from a haploidentical donor (t-haplo-hsct) is a potential alternative. methods: 29 patients (pts) with advanced stage scd (asscd) were transplanted with a cd3 + /cd19 + or αβ/cd19 + depleted t-haplo-hsct (20 pts, median age 13 years, range 3-31 years) or with bone marrow (bm) from a msd, 9 pts, median age 14, range 9-25 years). indication for hsct was asscd with multiple scd related complications. all pts underwent exchange transfusion before hsct. in all pts the conditioning regimen consisted of treosulfan, thiotepa, fludarabine and atg. immunosuppression was carried out with cyclosporine a or tacrolimus and mycophenolate mofetil. the control group received a msd bm allograft. results: in the t-haplo-sct group the pts received a cd3 + or αβ + t-and cd19 + b-cell depleted peripheral stem cell allograft with 13,1 x 10 6 cd34+ cells/kg body weight (range 8,1 to 77,9) . all pts with a median follow up of 22 months (range 4-57 months) in the msd group and 18 from 20 pts with a median follow up of 17 months (range 5-58 months) in the t-haplo-sct group are alive. engraftment was achieved in all pts with stable chimerism over 90%, except for 4 pts with a stable mc in the t-haplo sct group and 1 patient in the msd group, but complete engraftment of red cell precursor in the bm. all pts are off immunosuppression with a stable almost complete chimerism. the conditioning regimen was well tolerated with no case of high-grade transplant related morbidity. the post-transplantation complications were comparable in both groups. one patient developed after severe rotavirus gastroenteritis a severe cmv pneumonitis and succumbed to an uncontrolled cmv pneumonitis. one patient in the t-haplo sct group suffered from a late graft failure and developed a macrophage activation syndrome. he died in a septic event.none developed a glucksberg grade iii-iv agvhd and in the t-haplo sct group 4 pts (20%) and in the msd group 2 pts (22%) developed a steroid sensitive mild to moderate cgvhd with symptoms of fasciitis, oral as well as mild cutaneous gvhd. in both groups no severe or steroid refractory cgvhd was observed. conclusions: our results demonstrate increasing evidence for the safety and efficacy of cd3 + /cd19 + or αβ + /cd19 + depleted haploidentical hsct in asscd. the treosulfan based conditioning regimen was an excellent alternative to busulfan with a low incidence of transplant related morbidities and therefore most suitable for pts with scd. these results open the option of a curative therapy for almost all scd pts without a msd. disclosure: nothing to declare. abstract already published. hematopoietic stem cells o117 abstract already published. comparison of outcomes post allogeneic hematopoietic cell transplantation using fresh versus cryopreserved peripheral blood stem cell grafts background: cryopreservation is routine practice with autografts, however in the allogeneic hct setting the effects of cryopreservation have not been thoroughly investigated. we sought to compare allogeneic hct outcomes using fresh versus cryopreserved grafts in a large single centre cohort. methods: between 2003 and 2017, we retrospectively reviewed 951 consecutive adult patients who underwent allogenic peripheral blood hct at our centre. outcomes assessed included platelet (≥20x10e9/l) and neutrophil (≥0.5x10e9/l) engraftment, occurrence of acute graft-versushost disease (gvhd) in the first 100 days, overall survival (os), cumulative incidence of relapse (cir) and non-relapse mortality. results: median follow up of survivors was 47 months (range 4-177 months). fresh grafts were received by 525 patients, 426 received cryopreserved grafts, median age at hct was 53 and 54 years respectively. transplant indication was myeloid malignancy in 711 (75%), lymphoid in 229 (24%) patients. myeloablative regimens were used in 506 (53%) patients. the majority of fresh grafts were from unrelated donors (82%) while most cryopreserved grafts were from matched related donors (90%). in vivo t-cell depletion was performed in 65% of fresh and 18% of cryopreserved transplants. median time to neutrophil engraftment for fresh versus cryopreserved grafts was 15 and 15 (10-48) days respectively, while median time to platelet recovery was 16 (12-186) and 17 days, respectively. for fresh versus cryopreserved grafts, grade ii-iv acute gvhd was seen in 51% and 54%, respectively (p=0.42) while grade iii-iv acute gvhd was seen in 28% of patients in both groups (p=0.96). on univariate analysis, os for the entire cohort at 2 years was 50% (95%ci 47-54%) and at 5 years was 40% (95%ci 36-43%). two and 5 year os was 47% (95%ci 43-52%) and 39% (95%ci 34-44%) respectively for fresh grafts and 54% (95%ci 49-59%) and 41% (95%ci 36-46%) respectively for cryopreserved grafts (p=0.21). cumulative incidence of relapse (cir) of the entire cohort at 2 years was 18% (95%ci 16-21%) and at 5 years was 21% (95%ci 19-24%). two and 5 year cir was 16% (95%ci 13-20%) and 19% (95%ci 15-22%) respectively for fresh grafts and 21% (95%ci 17-25%) and 25% (95%ci 21-29%) respectively for cryopreserved grafts (p=0.02, figure 1 ). [[o118 image] 1 . figure 1] multivariable analysis for os verified no significant difference between fresh versus cryopreserved grafts (p=0.25). for cir however, cryopreservation was the only independent predictor of relapse (hr 1.43 for cryopreserved, 95%ci 1.07-1.91, p=0.02), while for nrm cryopreservation was not an independent predictor of increased risk (p=0.06). when the multivariable analysis was repeated for related donor transplants only (n=474, 385 cryopreserved grafts, 89 fresh), this confirmed the independent increased relapse risk for cryopreserved grafts (hr 2.30, p=0.01) . conclusions: we confirmed on univariate and multivariable analysis that there is no significant difference in os between allogeneic transplants performed with fresh versus cryopreserved peripheral blood stem cell grafts, however there is a significant increase in relapse risk associated with cryopreservation. disclosure: nothing to declare. background: high dose post-transplantation cyclophosphamide(ptcy) used in haploidentical transplantation (haplo-sct) has demonstrated to be highly effective in acute and chronic gvhd prophylaxis; however it is associated with high relapse rates. anti-t-lymphocyte globulin (atg-fresenius®) is also effective as gvhd prophylaxis but its benefit in overall survival (os) and relapse free survival (rfs) is unclear. the aim of this study was to compare the effectiveness of two gvhd prophylaxis regimen employed in high risk transplantation: ptcy-haplo-sct and low doses of atg-f used in peripheral blood(pb) and mismatched transplantation. the primary endpoint was to evaluate the incidence and severity of agvhd and cgvhd. as secondary endpoints we analysed the os, rfs and grfs (considering as events severe agvhd, systemic therapy-requiring cgvhd, relapse or death). we also evaluated mortality related to transplantation(trm) and post-transplant complications. methods: we retrospectively analysed 111 allo-sct performed in our institution between 2012 and 2017. we analysed two cohorts: 49 haplo-sct with ptcy (50 mg/ kg, days +3,+4) followed by tacrolimus and mycophenolate (mmf); and 62 pb and/or mismatched transplants with low dose atg-f (7 mg/kg days -3,-2,-1) associated to calcineurin inhibitors starting on day -1, with short course mtx (days +1,+3,+6) or mmf. in both cohorts, mmf was stopped on day +28 and calcineurin inhibitors were tappered on day +50. comparing both groups, we found differences in diagnosis (lymphoproliferative disorders 16 vs 4,p=0.003), high dri-score (18 vs 11, p=0.04), previous transplantation (12 vs 5,p=0.02), reduced-intensity conditioning regimen (36 vs 20,p< 0.001) and bone marrow as stem cells source (38 vs 9, p< 0.001). results: median time to neutrophil engraftment was similar in both groups: 17 vs 16 days. conversely, median time to platelet recovery was longer in ptcy cohort (33vs18 days, p=0.016). there were no differences in agvhd incidence (ptcy 30.6% vs atg 36.4%) or severe agvhd (ptcy 4.1% vs atg 9%, p= 0.852). the global cgvhd incidence was pcty 56.1% vs atg 66%. mild, moderate and severe cgvhd incidence was 31.7%, 19.5% and 4.8%, for ptcy vs 28%, 26% y 12% for atg(p=0.475).with a median follow-up of 27 months (28 months for ptcy and 22 months for atg) the os at 12 and 24 was: pcty 67.8 % and 61%, and atg 68.8% and 59.9%,p=0.971). rfs (12 and 24 months) was: 61% and 57.9% for ptcy, and,69% and 54% for atg(p=0.839). grfs at 12 and 24 months was 46.5% and 42.9%, for ptcy patients and 40.7% and 33% for atg patients (p=0.433). ptcy cohort seemed to develop more relevant non-infectious complications, but there were no differences among infectious complications. image 1. trm was similar in both cohorts: pcty 18.4% vs atg 22.5% (p=0.250). we neither found differences in early toxic mortality (< 100 days): pcty 16.3% vs atg 14.5% for atg (p=0,421). conclusions: regardless the different transplant scenarios in which they were used, ptcy and low dose atg seem to be equally effective in the prophylaxis of severe forms of acute and chronic gvhd, offering similar grfs. moreover,they show similar early toxicity and rate of infectious events. disclosure: we have nothing to disclose. conclusions: emapalumab treatment promoted disease control, blunting the exacerbated immune response typical of the disease, with a favorable safety and tolerability profile. the results of the study also suggest that emapalumab may contribute to optimize post-transplant outcome of patients given hsct. based on these data, emapalumab received marketing authorization in the us from the food and drug administration for the treatment of patients with primary hlh with refractory, recurrent or progressive disease or intolerance with conventional therapy. background: allogeneic hematopoietic stem cell transplantation (hsct) is a potentially curative treatment for some inherited disorders, including selected primary immunodeficiencies (pids). in the absence of a wellmatched donor, hsct from a haploidentical family donor (hifd) may be considered. various approaches are being developed to mitigate the risks of graft failure and graftversus-host disease (gvhd) and to speed-up immune reconstitution. among those, high-dose, post-transplant cyclophosphamide (ptcy) is increasingly used in adult recipients. however, data on ptcy in children and those with inherited disorders in particular are scarce. methods: we reviewed the outcome of 27 children transplanted with hifd and ptcy for pid (n= 22) or osteopetrosis (n= 5) in a single center. median (range) age was 1.5 years (0.2 to 17)). patients in our series had major risk factors for poor post-hsct outcome such as active viral infections at the time of transplantation (n=9), ebvrelated lymphoproliferation in partial remission (n=3), previous kidney transplant (n=1). hsct with ptcy was a primary (n=21) or a rescue procedure after graft failure (n=6). conditioning regimen was myeloablative in most primary hscts and non-myeloablative in all rescue procedures. results: after a median follow-up of 17.3 months, 24 of the 27 patients engrafted. twenty-one patients are alive and have been cured of the underlying disease. the two-year overall survival rate was 77.7%. the cumulative incidences of acute gvhd grade ≥ ii, chronic gvhd and autoimmune disease were 49%, 26.6%, and 26.5%, respectively. there were only two cases of grade iii acute gvhd, all cases of cgvhd were limited and allowed to stop systemic immune-supression, autoimmunity consisted in 2 autoimmune hemolytic anemia in remission at last follow up, 1 vitiligo and 2 thyroiditis. the cumulative incidence of blood viral replication and life-threatening viral events were 58% and 15.5%, respectively. there was evidence of early t cell immune reconstitution including early anti-viral responses. conclusions: in the absence of an hla-identical donor, hifd hsct with ptcy is a viable option for patients with life-threatening inherited disorders. disclosure: nothing to declare background: griscelli syndrome (gs) is a very rare autosomal recessive disease, characterized by skin hypopigmentation and silvery-gray hair. gs type 2 (gs2) patients suffer immunodeficiency and potentially fatal episodes of macrophage activation known as accelerated phases during early childhood. the only curative treatment modality for these patients is allogeneic hematopoietic stem cell transplantation (hsct). we report the outcome of hsct in 35 children with gs2. to date, this is the largest cohort of gs patients who have undergone transplantation at a single center. methods: we retrospectively reviewed 35 consecutive patients with gs2 who underwent hsct at our institution between january 1993-december 2017. median age at diagnosis and at transplant was 0.3 (0-12) and 0.9 (0.3-15.1) years respectively. prior to hsct, 28 (80%) and 16 (46%) patients had life-threatening accelerated phases (hlh) and cns involvement respectively. all such patients were treated with chemotherapy and achieved remission at the time of hsct. the source of grafts was matched related marrows in 19 (54.3%) pts and partially mismatched unrelated cord in 14 (40%) pts. two patients received haploidentical and matched unrelated marrows respectively. conditioning regimens were myloablative doses of busulfan/cyclophosphamide and busulfan/fludaribine in 27 (77%) and 8(23%) patients respectively. all patients received gvhd prophylaxis. growth factors were administered in 33 (94.3%) and median cd34 dose used was 5.46 (2.14-11.94) 10^6 and 5.45 (1.8-13 .5) 10^5 per kg of body weight for marrow and cord respectively. results: post infusion rate of engraftment was 88.6% (31 cases) with median time to neutrophil and platelets recovery were 15 (8-30) and 29 (14-61) days respectively. cumulative incidence of acute gvhd was 28.6% (10), with an overall grade of i, ii and iv as 20%, 60% and 20% respectively. the post-transplant course was complicated by cmv infection, ebv viremia and veno-occlussive disease in 14(40%), 7(20%) and 7(20%) patients respectively. chimerism studies at the last contact were available for 24 patients. full donor cell chimerism (100%) was seen in 16 (67%) of the transplanted patients. post-transplant two patients experienced disease reactivation at a median time 45.5 (28-63) days. with a mortality rate of 37.1% (13) and a median follow-up time of 87.7 months, five-year cumulative probability of overall survival (os) for our cohort of patients was 0.625 ± 0.083. transplant related mortality counted as death within day 100 was 28.6% (10). prominent causes of death were septic shock followed by ards. cumulative probability of five years overall survival was significantly better in those who did not have hlh prior to sct (1.0±0.0 vs. 0.529±0.096, p-value: 0.037). of the 16 patients with neurological involvement before hsct, 8 survived with residual sequelae in 3 patients. os at five years was 0.500±0.125 and 0.729±0.104(p-value: 0.328) in pts with and without cns involvement at presentation respectively conclusions: hsct in patients with gs is potentially curative with long-term, disease-free survival. early hsct before the development of the accelerated phase showed better result. [ background: primary immunodeficiencies (pids) are heterogeneous inborn disorders resulting in impaired cellular and/or humoral responses. pid present a wide range of clinical manifestations, ranging from infections to autoimmune, inflammatory and/or malignant complications. allogeneic stem cell transplantation (allosct) is curative for pediatric pid with an excellent safety. in adults on the other hand, this therapeutic approach remains controversial. methods: this is a retrospective, monocentric study of 26 consecutive adult patients with pid who underwent an allosct between 2011 and 2018. the objective was to assess the feasibility, effectiveness and safety of this procedure. results: twenty-two (85%) of 26 patients presented an inherited t or b cell deficiency and 4 (15%) a phagocyte impairment. twenty (77%) patients had a genetic diagnosis. besides infectious complications, 8 (31%) patients had an history of lymphoma and 13 (50%) an history of autoimmune/ inflammatory complication. the median age at transplant was 24 years (range 17-37). twelve (46%) patients received a myeloablative conditioning (mac), 12 (46%) a reduced intensity conditioning (ric) and 2 (8%) had no conditioning (second transplant for severe combined deficiency). mac included fludarabine (flu)/buslfan (bu, dosage: 12.8 mg/kg) based regimen (n=11) and bu (12.8 mg/kg)/cyclophosphamide (cy) based regimen (n=1). ric included based flu/bu (range dosage: 6.4-9.6 mg/kg) regimen (n=9), flu/cy based regimen (n=1), thiotepa/flu/bu regimen (n=1) and low dose tbi based regimen (n=1). among the 24 patients who received a conditioning, 4 (17%) received alemtuzumab and 11 (46%) received antithymocyte globulin as part of the conditioning. the stem cell source was bone marrow for 8 (31%) and peripheral blood stem cells for 18 (69%). the donor was matched related for 10 (38%), matched unrelated for 12 (46%), mismatch unrelated for 2 (8%), and haplo-identical for 2 (8%). all assessable patients had a successful engraftment. eight (31%) presented a grade ii-iv acute gvhd (one grade iii, 0 grade iv), and 5 (23%) a chronic gvhd (2 limited and 3 extensive). with a median follow-up of 29 months (3-90 months) post-transplant, the 3-years overall survival (os) was 80.8%. the transplant related mortality was 19.2% with 5 deaths. among them, both patients transplanted with an haplo-identical donor died. all deaths occurred in the firstyear post-transplant. thus, the use of an haploidentical donor was associated with an adverse outcome. conversely, neither the conditioning nor the stem cell source was associated with a worse outcome. except one patient with an history of aggressive b cell lymphoma who relapsed few months after allosct, no patient with an history of lymphoma relapsed. after a salvage treatment, the relapsing patient remained in complete remission 5 years later. at last follow-up, all surviving patients had a stable, mixed donorrecipient or full donor chimerism without any sign of active infection. conclusions: allosct in the pid setting is an effective therapeutic with an acceptable toxicity and should be considered in case of severe infectious, inflammatory and/or malignant complications in young adult patients with pid when an appropriate donor is available. disclosure methods: we analyzed the results of allogenic hsct with tcrαβ/cd19 graft depletion in 148 patients with various pid (excluding classic scid) who received hsct from may 2012 to september 2018 in our center. the median age at hsct was 3,5 years (range 0,43-17,63). 112 patients received hsct from matched unrelated, 31haploidentical donors, 5 -siblings. the conditioning regimens included: fludarabin (flu) 150mg/m2 with 1 alkylator (treosulfan (treo) 36-42g/m2) in 33 patients, with 2 alkylators (treo 36-42g/m2 with melphalan 140mg/m2 or thyotepa 10mg/kg) in 74. twenty-five patients received 2 alkylators with addition of g-csf 30mg/kg and plerixafor 24mg/kg. seventeen patients with nijmegen breakage syndrome (nbs) received reduced intensity conditioning with busulfan 4mg/kg or treo 30mg/m2, cyclophosphamide 40mg/kg and flu 150mg/m2. in all but 2 patients serotherapy was used: 10 patients -horse atg -90mg/kg, 133 -rabbit atg (thymoglobulin) 5mg/kg, 3 -1mg/kg anti cd52 monoclonal antibodies. in 55 patients two or more immunosuppressive drugs were used after transplantation (combination of calcineurin inhibitor (cni) with short course of methotrexate, or mycophenolate mofetil, or abatacept), 66 patients received one immunosupressive agent (cni). from november 2017 no posttransplant immunosuppression was used (27 patients). primary end points were: incidence of acute gvhd, graft failure (gf) and transplant related mortality (trm). because of the supposed influence of conditioning regimen on gf and trm incidence, to the analysis of gf and trm were included only 74 patients with similar conditioning regimen with 2 alkylators (excluding patients with nbs, with 1 alkylator and addition of g-csf and plerixafor in conditioning regimen). median follow up after hsct was 1,95 years (range 0,04-6,3 years). results: overall survival (os) in 148 patients was 0,85 (95% ci 0,79-0,91). cumulative incidence (ci) of acute gvhd in patients with two and more immunosupressants was 0,164 (95% ci 0,09 -0,3), with one -0,17 (95% ci 0,1 -0,29), with no immunosupression -0,11 (95% ci 0,04 -0,32), p=0,843. ci of graft failure was: in patients with two and more immunosuppressive agents (n=25) -0,24 (95% ci 0,12 -0,48), with one (n=32) -0,17 (95% ci 0,1 -0,29), without immunosuppression (n=17) -0,06 (95% ci 0,01 -0,44), p=0,172. ci of trm in patients with two and more immunosuppressants was 0,22 (95% ci 0,1 -0,47), with one -0,1 (95% ci 0,02 -0,28), without immunosuppression-0,26 (95% ci 0,09 -0,73), p=0,42. conclusions: we conclude that in our group of pid patients presence or absence of immunosuppression after hsct with tcrαβ/cd19 graft depletion and its extent made no significant difference on the incidence of acute gvhd, graft failure and trm. considering these results hsct with tcrαβ/cd19 graft depletion without posttransplant immunosupression could be recommended for patients with pid. disclosure background: lps-responsive beige-like anchor protein (lrba) deficiency is a severe primary immunodeficiency with a variable clinical phenotype, including features overlapping with common variable immunodeficiency, autoimmune lymphoproliferative syndrome, and immune dysregulation polyendocrinopathy enteropathy x-linked syndrome and an association with lymphoma. recent findings strongly support hsct in patients with severe presentation of lrba deficiency. however, there are no up-to-date follow-up and survival data of patients not undergoing transplantation beyond previous publications of large cohort studies. this study was conducted to increase the knowledge on transplant experience and to elucidate the clinical course of both transplanted and untransplanted patients with lrba deficiency under various targeted treatment modalities. methods: we performed an international inborn errors working party-and european society for immunodeficiencies-wide survey in 2018 to collect information about the hsct experience and the clinical course of both transplanted and untransplanted patients with lrba deficiency. we included an assessment of the disease activity and treatment responses with a specially developed immune deficiency and dysregulation activity (idda) score, which weighs the sum of organ involvement (scored by 0-4) by days of hospitalization and performance scores, taking into account the type of therapy and response per treatment phase, which we herewith introduce. data were obtained in accordance with the declaration of helsinki and an institutional review board review by means of retrospective chart review by local physicians. results: we analyzed the data of 68 lrba deficient patients from 23 centers, including 35 unpublished patients. 20 patients of our cohort underwent hsct between 2005 and 2018. overall survival of patients undergoing transplantation (median fu 30 months) was 70% (14/20 patients); all deaths were due to transplant-related mortality and occurred within 3 months of hsct. 83.3% (40/48) of untransplanted patients (2-68 years) are currently alive. out of the surviving transplanted patients (n=14) 6 are in complete remission, 4 in good partial remission (no treatment) and 4 in partial remission (receiving therapy), implying that 10 out of 14 transplanted patients (71.4%) are currently without treatment. only 5 out of 40 living untransplanted patients (12.5%) do currently not receive therapy. most commonly used drugs in lrba deficiency were steroids (27.7%), sirolimus (18.8%) and abatacept (18.8%). disease activity, measured by idda score, was significantly lower in patients treated with abatacept (p< 0.0001). no immunosuppression-associated malignancy was detected in our cohort. analyzation of the idda score of all living patients revealed significantly lower disease activity in patients transplanted and alive, than in those who did not undergo hsct (p= 0.01). conclusions: in conclusion our findings do, concordant with previous results, support hsct, especially in patients with a severe phenotype of lrba deficiency. however, the question of hsct indication and optimal time point cannot yet be generally resolved. in terms of conventional treatment, abatacept is clearly favorable in untransplanted patients. nevertheless, it is not available in many countries and the long-term dependency on this treatment with its associated potential risks remains. finally, our newly introduced idda score for assessment of disease activity may also prove useful for other syndromes with immune dysregulation. disclosure: nothing to declare background: cytomegalovirus (cmv), is a major complication of allogeneic hematopoietic stem cell transplant (hct) recipients. to overcome morbidity associated with cmv infection, antivirals are given, and while they successfully suppress viremia, they do so in the absence of immune reconstitution. a different approach is to use a vaccine to bolster immunity to cmv early post-hsct and sustain it at least until the patient is immune reconstituted. we selected a highly attenuated, non-proliferating viral vector referred to as modified vaccinia ankara (mva) to insert 3 immunodominant cmv antigens pp65, ie1-exon4, and ie2-exon5. this novel vaccine (triplex) demonstrated excellent tolerability and immunogenicity in a clinical study of 24 healthy adults (la rosa, et al. blood 2017) . these findings prompted us to conduct a test of vaccine protective efficacy in at-risk hct recipients in a multi-center, randomized and placebo-controlled phase 2 clinical trial (nct02506933). methods: eligible patients were cmv-seropositive and undergoing hct for hematologic malignancies from matched related/unrelated donors without the use of exvivo or in-vivo t cell depletion. the randomization (1:1) was stratified by donor serostatus and hct center. patients were enrolled pre-hct, and on d28 were requalified for eligibility based on lack of prior cmv reactivation, ≥grade 3 gvhd, high-dose steroids, or other significant transplantrelated morbidity. after randomization patients received triplex or placebo injections on d28 and d56 post-hct and followed for one year. the primary endpoint is defined as any cmv reactivation [≥500 cmv genome copies (gc)/ ml], low-level reactivation prompting antiviral therapy, or cmv disease prior to d100 post-hct. based on the objective of reducing reactivation from 30% to 10% or from 40% to 15% on the vaccine arm, a sample size of 102 was determined to provide at least 90% power at a one-sided 0.10 level of significance by log-rank test. results: the patient clinical/hct characteristics were balanced between the vaccine arm (n=51) and placebo arm (n=51). the vaccine was well tolerated with no significant difference in grade 3-4 aes or saes in both arms. triplex displayed potent immunogenicity, as many vaccine recipients with either cmv-positive or -negative donors showed strong reconstitution of both cd4 and cd8 cmv-specific immunity that initiated soon after the first injection and was elevated for at least 100d post-hct. most notably there was a reduction of primary endpoint-defining reactivation through d100 in the vaccine arm (5 events, 9.8%) compared to the placebo arm (10 events, 19.6%, p=0.08). greater detail regarding protocol-specified secondary endpoints including time to viremia, its duration, frequency and duration of antiviral drug treatment, late cmv viremia, time to engraftment and incidence of acute and chronic gvhd, relapse and non-relapse mortality will be presented. conclusions: in summary, triplex was well tolerated in seropositive hct recipients, and was significantly better than placebo in preventing viremia cases accompanied by improved cmv-specific t cell reconstitution. background: due to the risk of invasive pneumococcal disease (ipd) after allogeneic transplant, the ecil7 guidelines recommend 3 doses of pneumococcal conjugate vaccine (pcv) starting 3 months after transplant, followed by either one polysaccharide 23-valent vaccine (psv23) dose or a 4th pcv in case of chronic gvhd. however, no data strongly support recommendations from the 2 nd year. the aim of this study was to assess the anti-pneumococcal antibody (ab) concentrations, years after different schedules of vaccination. methods: patients who received allogeneic hct more than 18 months ago and were seen in consultation between jan-nov 2018 were screened for igg ab concentrations for the 7 pneumococcal serotypes shared by pcv7, pcv13, and psv23 (4, 6b, 9v, 14, 18c, 19f, 23f) , using modified enzyme linked immunosorbent assay (wernette et al.2003) . patients were considered protected if their ab concentrations were > 0.35μg/ml for the 7 serotypes tested. from 2001, all patients received 3 pcvs during the 1 st year of transplant, then either one dose of psv23 or (from 2009) a 4 th pcv. for patients transplanted for more than 12 months when pcv became available, we locally recommended > 2 pcv and one psv23. results: sixty-one patients were assessed at a median of 8.3 years (range: 1.7-39.1) after transplant. the mean age was 53 years (range: 21-79), m/f sex ratio 0,85. most patients had acute leukemia (32, 52.5%) or lymphoproliferative diseases (13, 21.5%). the donor was hla-identical in 42 (69%) cases. conditioning regimen was myeloablative in 37 (61%), reduced-intensity or non-ma in 12 (19.5%) each. none patient had experienced ipd since transplant nor had received immunoglobulins for at least 6 months or rituximab for at least 23 months at time of assessment. fifty-four patients (88.5%) received 3 consecutive pcv: 46 (75.5%) during the first 12 months and 8 (13 %) thereafter. five (8%) patients received 2 pcv and only one patient (1.5%) received a unique dose of pcv. the 1 st pcv was administered at a median of 3.5 (range: 3-201) months after hct. fifty-seven (93.5%) patients received one psv23 at a median of 13 months (range: 0.9-385) and 33 (54%) of them received a 2nd psv23 thereafter. finally, 39/ 61 (64%) patients received the recommended program. the overall protection rate was 29/61 (47.5%) at a median time of 8.3 years after hct. no differences were observed between patients who received the recommended program (18/39, 46%) and the others (11/22, 50%). however, this latter group was often vaccinated > 10 years after hct and therefore not protected for years, but may have developed a better response to only one or 2 pcv. in all groups, the ab concentrations were heterogeneous from one serotype to an other. recipient age, donor age, underlying disease, conditioning, donor type, previous gvhd, lymphocyte count or gammaglobulin serum levels were not associated with seroprotection. conclusions: although the early antipneumococcal vaccination program is now well established, half of the patients vaccinated according to the guidelines were not protected anymore years later. prospective studies are needed to establish an optimal long-term programme. disclosure: nothing to declare , letermovir vs. placebo) and was well tolerated overall. we evaluated risk factors associated with development of cs-cmvi to further inform the clinical use of letermovir prophylaxis and future trial designs. methods: the 495 participants without detectable cmv dna at randomization (primary efficacy population) were analyzed. cs-cmvi was defined as cmv viremia requiring antiviral preemptive therapy (pet) or cmv diseasepatients without cs-cmvi who died or withdrew from the trial on or before week 24 were censored at the time of those events for this analysis. potential risk factors for cs-cmvi by week 24 post hct were examined using univariate and multivariate cox proportional hazards models. candidate covariates were included in the multivariate cox model if they were associated with cs-cmvi in the univariate analysis or had been previously identified in the literature as significant risk factors. to avoid collinearity, the trial high-risk for reactivation of cmv stratification covariate was replaced with an updated covariate that only included patients who underwent hct with a mismatched donor, cord blood, ex-vivo t-cell depletion, or received alemtuzumab. the haploidentical hct and matched unrelated donor type were considered separately from the trial high-risk cmv categories. graft-versus-host disease (gvhd) and systemic glucocorticoid exposure were modeled as time-dependent covariates. race was dichotomized into asian and non-asian. the effect of study treatment was not included in the model due to nonproportional hazards. instead, letermovir treatment was used as a stratification variable. hazard ratios and 95% confidence intervals (ci) were calculated. results: there were 128 cs-cmvi events (25.9%) among 495 patients by week 24 post-hct for an incidence rate of 0.19/100 patient-days (95% ci, 0. 16-0.22 conclusions: donor cmv seronegativity, haploidentical hct, gvhd, glucocorticoid use, atg use, and asian race conferred significant risk of cs-cmvi through week 24 post-hct in a phase 3 study of letermovir prophylaxis. these results identify hct patient groups that would benefit the most from letermovir prophylaxis. methods: 769 adult patients (age 18-60) with de novo aml in complete remission (cr) perusing t-cell replete haplo-hsct with atg+g-csf protocol were consecutively enrolled at peking university people's hospital between 2010 and 2016. all patients were evaluated for donor-patient cmv serostatus before hsct. cmv dna emia were positive for >1000 copies/ml cmv by pcr tests on peripheral blood (pb). refractory cmv infection was defined as cmv dnaemia lasting for >2 weeks in spite of treatment. pb samples of 8 patients with refractory cmv infection (4 relapse and 4 without aml relapse) were sequencing for cmv derived small rna. cmv-mir-us4-1/ul-148d were then chosen as target mirnas and tested by stem-loop taqman qpcr in consecutive patients (n=157). cumulative incidence of relapse (cir) and treatment related mortality (trm) were calculated using competing risks, cox model was tested with patient age, sex, wbc count at diagnosis, cr status, courses to achieving cr, cytogenetic risk group, minimal residual diseases before hsct, cmv infection, cmv-mir-us4-1/ ul-148d expression. us4-1/ul-148d were further explored for the association with t cells and natural killer (nk) cells reconstitution and target mrna validation. results: in the total cohort of 769 patients, the 3-year overall survival (os) and leukemia-free survival (lfs) were 75.3 % and 78.4%, cir and trm were 15.9% and 8.8%. cmv infection occurred in 71.5% recipients at a median time of 4 weeks after haplo-hsct, and the duration was 2 weeks. 49.7% recipients experienced refractory cmv infection. cmv infection (yes vs. no, refractory vs no) did not affect cir in univariate analysis, while in multivariate analysis, cytogenetic risk group was identified as the only independent prognostic factor affecting cir (hr 1.72, 95% ci, p=0.032 ). in the cohort testing for cmv microrna, 35.0% patients were identified expression of us4-1, 40.1% patients for ul-148d, while 29.3% patients had co-expression. the relapse incidence was significantly lower in patients with vs. without co-expression of mir-us4-1/ul-148d. co-expression of mir-us4-1/ul-148d was the only independent risk factor for reducing cir (hr 0.414, 95% ci, 0.165-1.036; p=0.019). us4-1/ul-148d as also found to promote reconstitution of nkg2c+ nks. luciferase assay identify plzf as target of us4-1 while klrc1 targeted by mir-ul-148d, which suggest potential role of shifting nkg2c/nkg2a balance to nkg2c domination. conclusions: the present study is the first prospective trial to evaluate cmv infection on relapse for aml patients following t-cell replete haplo-hsct with atg+ g-csf protocol. our results suggested cmv-mir-us4-1/ul-148d co-expression rather than cmv infection reduces the relapse incidence, the stronger gvl effect might be associated with strengthening nkg2c+ nk cells reconstitution and alloreactivity via plzf/klrc1 pathway. [[o129 image] 1. mir-us4-1/ul-148d reduces relapse in aml after haplo-hsct] clinical trial registry: chictr-och-10000940 disclosure: nothing to declare o130 a randomised, placebo-controlled phase 3 study to evaluate the efficacy and safety of asp0113, a first-inclass, dna-based vaccine in cmv-seropositive allogeneic haematopoietic cell transplant recipients adjudicated cmv-specific avt or adjudicated cmv eod and mortality through 1 year post transplant. results: overall, 514 patients were randomized, of whom 501 received ≥1 dose of randomized treatment (asp0113 n=246; placebo n=255). there was no statistically significant difference in the proportion of patients who achieved the primary endpoint between the asp0113 (n=87 [35.4%]) and placebo (n=77 [30.2%]) groups, respectively (odds ratio 1.27; 95% confidence interval p=0.205 ). there were no statistically significant differences between groups for any secondary endpoints (table) . the incidence of teaes was similar between groups, except for a greater incidence of drugrelated teaes in the asp0113 group (n=194 [78.9%] ) compared with placebo (n=74 [29. 0%]) attributed to injection site-related teaes. mean t-cell response to pp65 increased over time in both groups and was significantly greater with placebo compared with asp0113 (p=0.027). there was no statistically significant difference between groups for mean gb-specific antibody response. conclusions: asp0113 did not demonstrate efficacy in the reduction of overall mortality and cmv eod through 1 year post transplant. asp0113 demonstrated a similar safety profile to placebo, with the exception of injection siterelated teaes that were more frequent in the asp0113 group. participants in this study will be followed up to 5.5 years post transplant for long-term safety assessments. disclosure: jm: personal fees and non-financial support from astellas, basilea, cidara, f2g, gilead, merck and hsct) and 893 autologous hsct (auto-hsct). during the observation period 40 probable and proven rare ifd (eortc/msg 2008 criteria) cases were diagnosed in children and adults with hematological malignances and non-malignant hematological diseases after allo-hsct (n=30), auto-hsct (n=2), and chemotherapy (n=8). the median age was 23 (2-59) y.o., males -60%(n=24). the median follow up time for rare ifd cases was 3 months; for survivors -30 months. results: incidence of rare ifd in hsct recipients was 1,5%, it was higher after allo-hsct (1,6%) than auto-hsct (0,2%) (p< 0,01). in eight patients, this complication developed after ct and four of them proceed to allo-hsct. the most frequent underlying diseases were acute lymphoblastic leukemia (33%) and acute myeloid leukemia (30%). the median time of onset of rare ifd after allo-hsct was 104 (21-1057) days, auto-hsct -138 (60-216), after start of . etiology of rare ifd was identified by culture in 65% cases: rhizopus spp. -27%, paecilomyces spp. -7%, fuzarium spp. -5%, malassezia furfur -5%, trichosporon asahii -3%, scedosporium apiosperium -2%, scopulariopsis gracilis -2%, rhizomucor pusillus -2%, lichtheimia corymbifera -2%, mix rare ifd with rhizopus spp. + paecilomyces spp. -5%, paecilomyces spp. + fuzarium spp. -5%. 35% cases (mucormycosis) were diagnosed by microscopy. in 45% cases rare ifd developed after or in combination with invasive aspergillosis, and 2 patients had both preexisting invasive aspergillosis and co-infection with mucormycosis. the main site of infection were lungs (82%), the main clinical symptom -febrile fever (95%). antifungal therapy was used in all patients: lipid amphotericin b -30%, lipid amphotericin b + caspofungin -22,5%, voriconazole -17,5%, posaconazole -12,5%, lipid amphotericin b + posaconazole -10%, and echinocandins -7,5%. surgery was used in 10% patients. overall survival at 12 weeks from the diagnosis of rare ifd was 50%. the 12-weeks overall survival was better in patients after ct and auto-hsct (80%) than allo-hsct (42%), p=0,048. conclusions: the incidence of rare ifd in hsct recipients was 1,5% and depends on type of transplantation. rare ifd is a late complication after chemotherapy and hsct and usually develops after or in combination with invasive aspergillosis. higher incidence and worst prognosis rare ifd is observed in allo-hsct recipients. disclosure: nothing to declare o133 incidence and outcome of kaposi sarcoma after hsct: a retrospective analysis on behalf of idwp background: kaposi's sarcoma (ks) is an angioprolipherative disease which occurs in immunosuppressed patients, often associated with infection by human herpes . in solid organ transplantation (sot) ks is relatively common, the risk being 60-500 folds higher than that of general population and representing around 23% of secondary cancers. also hematopoietic stem cell transplantation (hsct) is a risk factor for the development of ks but until now only few case reports were published. we assessed retrospectively the incidence, the clinical characteristics, and the outcome of ks in the ebmt database. methods: the cases of ks were identified by ebmt registry (promise) and by inviting all 569 ebmt centers to notify ks cases. the clinical features, type of therapy, survival rate and causes of death were retrieved from of promise or, if lacking, by specific case report form sent to participating centers. the center response rate was 74/ 569 (13%). results: fourteen centers reported 17 patients with ks, all ks were diagnosed from 2004 to 2017, but one case that was diagnosed in 1987. the analysis was limited to 13/17 patients because ks was diagnosed before hsct in 4 patients: they were 13 patients, 3 females and 10 males who developed ks after allo hsct (10) and auto hsct (3); moreover, ks occurred after a second hsct in 2 patients and after a third hsct in 1 patient. the search of hhv8 in tumour tissue was done in 9 cases and resulted positive in 8/ 9. the underlying disease were: 46% leukemia, 23% lymphoma, 15% myeloma, 8% myelodysplastic syndrome (mds), 8% bone marrow failure. the source of stem cells was bone marrow in 4 patients (31%) and peripheral blood in 9 patients (69%). the median age at ks diagnosis was 49.7 years (range 6. 3-61.4) . considering the number of hsct performed in the participating center from 2004 to 2017, the incidence rate was 0.17% in allogeneic transplantations (9/5345), 0.05% in autologous transplantations (3/5857) and 0.11% (12/11202) in the whole group. the interval of time between hsct and the development of ks was 7 months (range -0. 3-61.2) . the organ involvement was: 62% skin (8 patients), 31% lymph nodes (4), 8% gingival (1) . apart from withdrawal of immunosuppression, 2 patients received chemotherapy, 3 patients received radiotherapy, and 1 patient received radio and chemotherapy; moreover 5 patients received antiviral treatment with ganciclovir or foscarnet. eight patients (62%) are alive whereas 5 patients (38%) died at a median time of 8.1 months, range 0.5-12.3. the causes of death were infection in 2 cases, secondary malignancy/ptld in 2 and relapse/progression in 1 whereas no case of death directly associated to ks. conclusions: ks is a rare complication of the immunosuppressive status related to hsct that generally occurs within the first year after hsct. the low prevalence and the rarity of this complication do not justify the adoption of screening program for hhv-8. on the other hand, the role of the virus in febrile status in immunosuppressed patients and the risk factors for the development of ks are not well known. disclosure background: autologous stem cell transplantation (auto-sct) is considered the standard treatment for patients with relapsed or refractory (r/r) hodgkin lymphoma (hl). for those with high-risk disease, an alternative consolidation strategy with allogeneic sct (allo-sct) could be a potential option to improve the outcome. however, allo-sct with a reduced-intensity conditioning (ric) needs around 3 months for the graft-versus-lymphoma effect (gvl) to develop, thus in patients with an aggressive hl the disease might progress before this happens. in this setting, a tandem auto-ric-sct approach has the potential of combining cytoreduction to keep the lymphoma under control and the potential benefit of a gvl effect. to better understand the safety and efficacy of a tandem auto-ric-sct approach we conducted a retrospective analysis of patients treated with this strategy between january 2004 and december 2015 and reported to the ebmt registry. methods: patients were included if they had received an auto-sct followed by a planned ric-sct in < 6 months with no disease relapse between the procedures. the primary endpoint was progression-free survival (pfs) after the tandem procedure. secondary endpoints were overall survival (os), cumulative incidence of non-relapse mortality (nrm), incidence of relapse (ir) and graft versus host disease (gvhd). results: one-hundred and thirty patients [58% male, median age at auto-sct, 30 years (range: 18-65)] fulfilled the inclusion criteria. the median time between diagnosis and auto-sct was 16 months (range: 2-174) and the median number of lines prior to auto-sct 2 (2) (3) (4) . disease status at auto-sct was complete response in 32%, partial response in 27% and the remaining 41% were transplanted with active disease. the median time from auto to allo-sct was 3 months (1-6). forty percent underwent an identical sibling allo-sct, 39% unrelated and 21% haplo. tbi was used in 35% of the patients as a part of ric. gvhd prophylaxis was cyclosporine-methotrexate in 36% of the patients, cyclosporine-micofenolate mofetil in 16% and post-transplant cyclophosphamide in the remaining 17%. 91% of the patients engrafted after ric-sct. after a median follow-up of 44 months (6-130), 33% of the patients background: allogeneic stem cell transplantation (allo-sct) is a valid option in patients with refractory/relapsed lymphoma but gvhd remains the major cause of mortality and morbidity. calcineurin-inhibitors (cni) combined with methotrexate or atg is the conventional strategy for preventing gvhd, resulting in an incidence of cgvhd of 41-60%. post-transplant cyclophosphamide (pt-cy) reduces the risk of severe cgvhd and improves survival in acute leukemia patients receiving allo-sct from matched sibling (msd) or unrelated donors (ud). the aim of this retrospective registry-based study was to compare pt-cy-based gvhd prophylaxis to standard prophylaxis in the setting of msd or ud for patients with lymphoma. methods: three thousand eight hundred sixty-four lymphoma patients undergoing an hla -id sct(hla identical stem cell transplantation) registered in promise were included in the study (table 1) . outcomes between pt-cy vs no-pt-cy were compared a) with a multivariate cause-specific cox model adjusted on ric/mac, donor type, source of stem cell, age of the patient, donor gender, patient gender, diagnosis and, disease status at sct; and b) by matching one pt-cy patient (118) with two no-pt-cy patients (217) using the same covariates. results: in univariate analysis, comparing pt-cy and no-pt-cy, the 100-day ci of grade 2-4 agvhd was 26% and 27% (p=0.7), the 1-year ci of non-relapse mortality (nrm) was 12% and 16% (p=0.6) and the 3-year relapse incidence (ri) was 38% and 33% (p=0.2), respectively. the 1-year ci of cgvhd was 32% vs 42% (p=0.1), 3-year pfs for pt-cy and no-pt-cy was 43% and 46% (p=0.5) and 54% and 57% (p=0.9) , respectively. in multivariate analysis, prophylaxis with pt-cy was not associated with a reduced risk of agvhd, overall or extensive cgvhd, nrm, ri, nor with an improved pfs or os. likewise, in the matched-pair analysis pt-cy did not impact on any of these outcomes. conclusions: this study demonstrates that in lymphoma patients who underwent an hla-id sct, gvhd prohphylaxis strategies employing pt-cy-based achieve equivalent transplant outcomes to those seen with cni-based strategies. myeloablative conditioning may contribute to disease control after stem cell transplantation in blastic plasmacytoid dentric cell neoplasia background: blastic plasmacytoid dentric cell neoplasia (bpdcn) is a rare and clinically aggressive hematopoietic malignancy, which preferentially involves the skin, the bone marrow, and, occasionally, the lymph nodes. it mainly affects elderly patients and has a poor prognosis with conventional chemotherapy. the treatment for this condition is heterogeneous, some patients treated with lymphoma-type regimens and others receiving intensive acute leukemiatype chemotherapy. although preliminary case series suggest that hematopoietic stem cell transplantation (sct) could provide sustained disease control in patients with bpdcn, the role of sct and potential graft-versus-tumor effects (gvt) in this condition is yet undefined. methods: between 2009 and 2017 26 patients were included in an ebmt prospective non-interventional study (nis) on the value of sct in bpdcn. these were compared with 133 patients with bpdcn registered with the ebmt during the same time period outside the nis. no differences with regard to overall survival (os), line of treatment or year of transplant was observed between patients included in the nis and the remainder from the ebmt database, and they were therefore analyzed together. results: one hundred and forty-two patients were treated with an allogeneic sct (allosct) and 17 patients with autologous or syngeneic sct (autosyn). the median follow-up was 17 months with no differences between the allosct and autosyn groups. disease status at sct was complete remission (cr) in 88%, and 90% patients received the sct as part of first-line treatment. two-year os after autosyn was 70% (95% ci 46-99%), whereas it was 65% (95% ci 53-83%) following allosct. of those patients who received an allosct, 71% were transplanted from an unrelated donor (ud), and 29% from an identical sibling donor (sib). reduced intensity conditioning (ric) was used in 47% and myeloablative conditioning (mac) in 53% of the patients. in allotransplanted patients, multivariate competitive risk analysis of nrm and relapse incidence considering age, conditioning intensity, line of treatment, remission status prior to allosct and donor type revealed a statistically significant reduction of relapse incidence (p=0.02, hr 0.2 ci 0.1-0.7) without increased nrm for patients who received mac compared to ric. multivariate cox regression analysis for os considering the same co-variates confirmed that mac was associated with a significantly reduced mortality risk (p=0.05, ci 0.4, hr 0.1-0.99) along with being in cr at allosct (p< 0.001, hr 9.1, . conclusions: this study confirms on a large data set that sct is an effective and potentially curative treatment for patients with bpdcn. the superiority of mac and the efficacy of autosyn suggest that apart from gvt, highdose therapy might be an important contributor to long-term disease control in this condition. clinical background: allogeneic hematopoetic cell transplantation (allo-hct) is a curative therapy for patients with relapsed/ refractory and high-risk non-hodgkin lymphoma (nhl). however, no large studies have evaluated the trends in the utilization of allo-hct in elderly nhl patients (≥65 years). using the cibmtr registry we report here a time trends analysis of allo-hct use in elderly nhl subjects methods: we identified 727 nhl patients (≥65 years of age) undergoing a first allohct during 2000-2015 in the united states (u.s.). study cohort was divided into the following time-periods for analysis; 2000-2005 vs. 2006-2010 vs. 2011-2015 . primary outcome was overall survival (os). secondary outcomes included progression-free survival (pfs), relapse/progression (r/p) and non-relapse mortality (nrm). results: baseline patient characteristics are shown in table1. during the three study time-periods median patient age (67-68-year), use of reduced-intensity conditioning regimens and proportion of patients with chemosensitive disease remained stable. in the most recent era (2011-2015) a higher proportion of patients had t-cell nhl, history of prior autografts, good performance status (kps 90-100) and high hct-ci, while fewer subjects received a hlamatched sibling hct. the cumulative incidence of day100 grade 2-4 acute graft-versus-host disease (gvhd) for 2000-2005 vs. 2006-2010 vs. 2011-2015 cohorts was 25% vs. 35% vs. 31% respectively (p=0.47). the cumulative incidence of chronic gvhd at 1 year was 21% vs. 34% vs. 31%, in similar order (p=0.07). the 4-year probabilities of nrm and r/p of 2000-2005 vs. 2006-2010 vs. 2011-2015 time-periods were 34% vs. 29% vs. 30% (p=0.69) and 48% vs. 40% vs. 40% (p=0.39), respectively (figure) . the 4-year probabilities of pfs and os (2000 -2005 vs. 2006 -2010 vs. 2011 -2015 showed significantly improved outcomes in the most recent time-periods as following: 17% vs. 31% vs. 30% (p=0.02) and 21% vs. 42% vs. 44% (p< 0.001), respectively (figure) . on multivariate analysis, compared to the 2000-2005 cohort, the 2011-2015 cohort showed a 28% reduction in the risk of mortality (rr=0.72, 95%ci=0.52-1.008, p=0.056). the most common cause of death was relapse of primary disease in all time-periods. conclusions: utilization of allo-hct has steadily increased in elderly nhl patients in the u.s. since 2000. in the recent years despite decline in the use of hlamatched sibling donors and transplanting elderly patients with higher hct-ci and more heavily pretreated disease, survival outcomes have improved. age alone should not be a determinant for allo-hct eligibility in nhl. methods: four medical experts who had managed patients with dlbcl using different car t-cell therapy protocols and products independently reviewed the extracted adverse event data from the case report forms and re-graded crs using the more commonly used lee scale (lee, blood, 2014) and the nt grading for encephalopathy (modified car t related encephalopathy syndrome (mcres) (neelapu, nature reviews in clinical oncology, 2018) while blinded to the original trial grading and other experts' grading. re-grading assessments and disagreements concerning the assigned lee and nci ctc grades were later discussed and reconciled among reviewers during a live meeting. as per the investigational charter, in cases that could not be reconciled, the most conservative final assessment of any reviewer determined the final grading for any individual case. results: crs: 64 of 111 (58%) patients were originally recorded as crs by penn scale, and 63 were regraded as crs by lee criteria. 18 of 64 subjects received anticytokine therapy overall. with this blinded reassessment, more patients were categorized as grade 1 (lee vs penn: 26 vs 17), fewer patients as grades 2 and 3 (18 vs 23 and 10 vs 15, respectively) and the same number of patients as grade 4 (9 vs 9). nt: results were compared with data on nt determined by nci ctc criteria of tisagenlecleucel, in which nt was broadly defined as the occurrence of any nervous system or psychiatric ae (eg, anxiety, dizziness, headache, peripheral neuropathy, and sleep disorder). 68 of 111 subjects were identified as having nt by ctc criteria: 34 (30.6%) patients were identified as having grade 1/2 nt, 11 (9.9%) patients as having grade 3 nt,& 5 (4.5%) patients as having grade 4 nt. evalution by mcres grading system revealed low rates of encephalopathy/delirium: 5 (4.5%) patients had grade 1/2 nt, 6 (5.4%) patients had grade 3 nt, and 8 (7.2%) patients had grade 4 nt. no grade 5 events were seen and the presence of crs was associated with higher likelihood of concomitant nt. conclusions: these results demonstrate difficulties in identifying cross protocol toxicity assessments. harmonized grading scales being developed for future studies will facilitate comparison of the safety profiles of different car t-cell and other immune effector cell products. further analyses are ongoing of these data with the new asbmt consensus crs guidelines. recent studies show that mouse bone marrow tregs localize in the hematopoietic stem cell (hsc) niche, where they contribute to hscs maintenance and promote donor engraftment and b cell lymphopoiesis. we are investigating if human tregs promote b cell reconstitution and immunity in preclinical models and in haplo-hct patients. methods: b cell reconstitution was analysed monthly by facs in bone marrow (bm) and peripheral blood (pb) samples from 51 patients who underwent either treg/tcon haplo-hct (33 patients), or t-cell depleted haplo-hct (8 patients) or haplo-hct with high dose post-transplant cy (ptcy, 10 patients). diagnosis was acute leukemia in 39 patients, lymphoma in 9 and multiple myeloma in 3. pb total immunoglobulin (ig) and anti-cytomegalovirus (cmv) igm were also monitored together with cmv viremia. for the mouse model, donor derived human treg and cd34 + hematopoietic stem cells (hscs) were coinfused in sublethally irradiated (2 gy) immune-deficient nsg mice and donor engraftment and b cell reconstitution were analysed in mouse pb twice a month by facs. results: b cell reconstitution was faster after treg/tcon haplo-hct when compared to other haplo-hct protocols. b cell counts were higher in pb of patients that received treg/ tcon haplo-hct (p = .02) and were comparable to those of healthy subjects by 4 months after transplant (131±121 cells/ mm 3 , fig.1a ). we could detect early frequencies of cd34 + cd38 + cd10 + cd127 + common lymphoid progenitors, cd45 + cd10 + cd38 + cd19 -pre/pro-b, cd45 + cd10 + cd38 + cd19 + pre-b, and pro-b cells in the bm of these patients, that resulted in an increased production of cd38 + cd19 + cd5 -igm + immature b cells, cd38 + cd19 + cd5 + igm + transitional b cells and cd19 + cd20 + mature b cells. we used a mouse model of xenotransplantation to understand whether donor b cell reconstitution in treg/ tcon haplo-hct is boosted by a treg-mediated effect on donor human hscs. we found that infusion of human tregs facilitated donor hsc engraftment. hsc-derived mature b cells were rapidly abundant and easily detectable 60 days after hsc infusion in pb of treg-treated animals. to evaluate donor b cell function we analysed ig production in response to cmv reactivation in transplanted patients. post-transplant hypogammaglobulinemia was rapidly corrected in treg/tcon haplo-hct patients. total igm were higher compared to other haplo-hct protocols and reached normal levels by 3 months after transplant (152 ±194 mg/dl, fig.1b ). cmv reactivation rate was similar among haplo-hct protocols (~73%), but it occurred later after treg/tcon immunotherapy (45+/-17 days vs 33 +/-11 days). new production of anti-cmv specific igm was documented in 60% of cmv seropositive patients 76 +/-33 days after treg/tcon haplo-hct, while anti-cmv specific igm were undetectable after other haplo-hct protocols within the first 6 months after transplant. [ background: allogeneic cell transplantation (hsct) success prediction is partly based on minimal residual disease (mrd) and hematopoietic chimerism testing. we developed a droplet digital pcr platform (dpcr) for the simultaneous detection of mrd and hematopoietic chimerism. methods: a panel of 12 deletion/insertion polymorphic markers and frequent molecular targets used for mrd testing: npm1, runx1-runx1t1 (t 8;21), dnmt3a, mll-ptd, cbfß-myh11 (inv 16), kras, mll-af10, idh 1/2, ckit, bcr-abl (t9; 22), evi1 and wt1 expression were included in a single dpcr platform for mrd and hematopoietic chimerism analysis. a total of 225 patients were evaluated with a mean follow-up of 653 days (range: 30-5895 days). results: hematopoietic chimerism analysis revealed mixed chimerism (mc) in 93 patients (41% of all patients). mc was detected more frequently in patients with reduced intensity conditioning (68%) when compared with full conditioning (47%). the mean percentage of host derived dna in peripheral blood was 13% (range: 0.1-90%). three different patterns of mc were observed: increasing, decreasing or stable mc. in those patients with stable or decreasing mc (n=46), as well as patients with complete donor chimerism (n=132) the molecular targets used for mrd monitoring were not detectable. in 47 out of 93 patients, increasing mc was detected. this group of patient showed in addition either a positive mrd marker, increased wt1 expression or both. in 37 patients with increasing mc, a positive mrd marker and increased wt1 expression hematologic relapse of the underlying disease was observed. in patients with increasing mc and a positive mrd marker, we analysed whether mc or the molecular target for mrd was first detected. in 40% of the cases mc was detected before the molecular marker used for mrd assessment, while in 13% of the patients mrd positivity was detected before mc. in the remaining patients (47% of the patients) mc and mrd positivity were detected simultaneously. the mean time between either mc or mrd detection in peripheral blood and relapse was 116 days (range: 15-385 days). patients with increasing mc and mrd positivity, whether or not they responded to treatment, showed a similar kinetic pattern for the chimerism and mrd markers. in those patients that responded to molecular relapse treatment (n=10) the mean time to achieve complete donor chimerism or mrd pcr negativity was 305 days and 294 days respectively. conclusions: the combination of mrd and chimerism markers in a dcpr platform represents a sensitive and accurate diagnostic tool for the comprehensive assessment of the molecular remission status after hsct. in addition, by using the developed dpcr platform costs and turnaround times can be reduced. disclosure background: minimal residual disease (mrd) monitoring can help to indicate impending relapse of acute leukemia after allogeneic hematopoietic stem cell transplantation (allo-hsct). because impending relapse can be altered with early detection of low-volume disease and timely therapies, preemptive intervention is a reasonable option for patients with mrd which can spare those in remission from further therapy. chemotherapy plus donor leukocyte infusion (chemo-dli) is the most important preemptive intervention, but it may lead to several complications, such as severe graft-versus-host disease (gvhd) and pancytopenia. hypomethylating agents (hmas) represent another potential preemptive intervention, but it only delayed the time to hematologic relapse and the long-term efficacy may be unsatisfactory. thus far, few studies had identified the longterm efficacy of preemptive intervention with drugs in patients with mrd after allo-hsct.two prospective studies (nct02027064 and nct02185261) reported that preemptive interferon-α (ifn-α) treatment can help clear minimal residual disease (mrd) and prevent relapse after allogeneic hematopoietic stem cell transplantation (allo-hsct). in this extension study, we aimed to identify the long-term clinical outcomes of preemptive ifn-α treatment in acute leukemia patients who were mrd positive after allo-hsct (n=118). methods: mrd was monitored by multiparameter flow cytometry (mfc) and taqman-based reverse transcriptionreal time polymerase chain reaction (pcr). a patient was considered to be mrd-positive when a single bone marrow sample tested positive by pcr or mfc. recombinant human ifn-α-2b injections were administered subcutaneously for 6 cycles (twice or thrice weekly in every 4-week cycle). results: the 4-year cumulative incidence of total chronic graft-versus-host disease (cgvhd) and severe cgvhd after ifn-α treatment was 58.5% (95% ci, 49.5-67.5%) and 10.2% (95% ci, 4.7-15.7%), respectively. the 4-year cumulative incidence of relapse and non-relapse mortality (nrm) after ifn-α treatment was 16.1% (95% ci, 9.4-22.8%) and 5.6% (95% ci, 1.1-10.1%), respectively. the 4year probabilities of disease-free survival and overall survival (os) after ifn-α treatment were 78.3% (95% ci, 70.6-86.0%) and 84.0% (95% ci, 77.1-90.9%). in multivariate analysis, severe acute gvhd was associated with a higher risk of nrm and poorer os, and mild to moderate cgvhd was associated with a lower risk of relapse and better survival. conclusions: these data confirmed that preemptive ifnα treatment showed long-term efficacy in patients who were mrd-positive after allo-hsct. clinical trial registry: the study was registered at http://clinicaltrials.gov as #nct02185261 and #nct02027064. disclosure: the authors declare no competing financial interests. a phase ii clinical trial of leuprolide for enhancement of immune reconstitution after ex vivo cd34+ cell-selected allogeneic hct with tbi-based conditioning gnrh agonist use, which has been associated with thymic cellular degeneration in mouse (velardi, jem 2014). as direct gnrh antagonism might circumvent this effect, a follow-up phase ii trial evaluating peri-transplant degarelix for enhancement of immune reconstitution is in progress. background: in allogeneic hematopoietic stem cell transplantation (allo-hsct) recipients, cytomegalovirus (cmv) reactivation and disease are frequent causes of morbidity and mortality, that may be evaded by cmv-specific t cell reconstitution. methods: we designed a prospective, single-center observational study to assess if the kinetic and quality of cmv specific t-cell reconstitution impact the incidence and severity of cmv reactivations. we report data on the first 54 consecutive patients affected by hematological malignancies receiving allo-hsct followed by cyclophosphamide and rapamycin between december 2017 and august 2018. patients received allo-hsct from family (sib-lings=9; hla haploidentical=21), unrelated hla matched (n= 23) donors or cord blood (n=1). the cmv serostatus of host (h) and donor (d) pairs was: h + /d + (n=36, 67%), h + /d -(n=17, 31%) and h -/d + (n=1, 2%); h -/dwere excluded. cmv dnaemia was assessed weekly in whole blood (wb). absolute numbers of polyclonal and cmv-specific t cells were quantified by flow cytometry using trou-count™ tubes (bd) and dextramer® cmv-kit (immu-dex), respectively, in the graft and fresh wb at days -7, +30, +45, +60, +90, +120, +150 and +180. dextramers permit the identification of cmv-specific lymphocytes restricted for several hla class i molecules: a*01:01/ *02:01/*03:01/*24:02 and b*07:02/*08:01/*35:01. these alleles allowed the longitudinal evaluation of 48 (89%) patients. results: at a median follow-up of 150 days post-hsct, 28 (58%) patients experienced a cmv-related clinically relevant event (cre, median +50 days), including 7 patients (15%) with cmv disease. for each time-point, we compared the absolute number of cmv-specific lymphocytes in patients experiencing or not a subsequent cre. at +45 days, we observed lower cmv-specific cd8 + t cells in patients prone to reactivate cmv than in not reactivating patients (median cmv-specific cd8 + cells/ ml= 0.125 vs 1.69, p=0.026). furthermore, patients with any dextramer positivity at +45 days displayed a lower incidence of cre compared with subjects who were negative (cre probability: 0.55 vs 0.83, p=0.038). patient stratification based on different thresholds of dextramerpositive cells confirms the inverse association between cre and cmv-specific immunity (cre incidence in patients with: 0 cells/ml=0.83, < 1 cells/ml= 0.66, ≥ 1 cells/ml= 0.36; p=0.046). we observed a higher cre incidence in cmv h + /dpairs than in h + /d + (0.89 vs 0.44, p=0.03). taking advantage of the hla mismatched-hsct setting, we then dissected cmv-specific t-cell response according to hla restriction elements (h/d=shared n=40, drestricted n=12, h-restricted n=19). in h + /d + pairs, we observed a fast and similar kinetic of reconstitution of cmv-specific lymphocytes restricted by h/d and d hlas. conversely, in h + /dpairs, we detected only cmv-specific cd8 + lymphocytes restricted for h/d haplotypes. hostrestricted cells remained undetectable for the first 150 days after hsct. conclusions: when the donor is cmv seropositive, a rapid and effective reconstitution of cmv-specific d-and h/d-restricted memory t cells occurs. if the donor is cmv seronegative, only h/d-restricted lymphocytes are observed early after allo-hsct in h + /dpairs. these findings indicate that cmv reactivation can prime h/d-restricted t cells presumably educated in the donor thymus; conversely, d-and h-restricted donor-derived lymphocytes have not yet undergone neither cross-priming nor thymic education, which might be required for full protection from cmv. disclosure: c.b. received research support from molmed s.p.a. and intellia therapeutics. l.b. is employed by immudex aps. non of the other authors has any relevant conflict of interest to disclose. o148 5-azactidine is safe and effective therapy for prevention of disease relapse in high-risk patients with acute myeloid leukemia and myelodysplastic syndrome following allogeneic stem-cell transplantation ivetta danylesko 1 , noga shem-tov 1 , adriana del-giglio 1 , ronit yerushalmi 1 , arnon nagler 1 , avichai shimoni 1 background: allogeneic stem-cell transplantation (sct) is curative approach in patients with aml or mds. however, disease recurrence is the major cause of treatment failure. 5azacitidine has been used in standard treatment of mds and also in aml patients not eligible for standard chemotherapy. there is limited data on the safety and efficacy of azacitidine after sct. we explored the use of low-dose azacitidine for prevention and treatment of relapse of aml/ mds after sct. methods: patients in cr after sct who were considered to be at high-risk for relapse were given azacitidine at 32mg/m 2 for 5 days every 28 days, planned for 2 years. patients with overt relapse after sct were given 32-75 mg/ m 2 for 5 days until progression. results: ninety-four patients, median age 64 years (24-77) were given azacitidine after sct from hla-matched sibling (n=29), matched-unrelated (n=64) or haploidentical (n=1) donors. diagnosis was aml (n=66) or mds (n=28). the conditioning regimen was myeloablative (n=56) or reduced-intensity (n=38). 22 patients were given prophylactic azacitidine; 15 were in cr after sct but at high-risk for relapse due to active disease (n=7) or positive minimal-residual disease (mrd) (n=3) prior to sct, or poor-risk cytogenetics (n=5). seven patients were given azacitidine as secondary prevention after achieving cr with chemotherapy for post-transplant relapse. 19 patients were given azacitidine pre-emptively for mixed-chimerism, positive mrd after sct or early relapse (< 10% marrow blasts). patients in the combined prophylactic/ preemptive group (n=41) started azacitidine in a median of 2.4 months (1.2-14.9) after sct and received a median of 8 courses . 4 patients were also given donor-lymphocyte infusions (dli) concomitantly with azacitidine. 23 are still on therapy, 12 died or progressed, 2 stopped after long remission, 3 stopped due to patient request and only 1 discontinued due to toxicity. 17 of 22 patients given prophylactic azacitidine remained in cr. 16 of 19 patients given azacitidine preemptively achieved cr and 10 remained in cr. with median follow-up of 14 months , 30 of the 41 patients in the prophylactic/ pre-emptive group are alive and 11 died. the estimated 3-year os and pfs are 58% (95%ci, 29-86) and 50% (95%ci, 27-74), respectively. the expected pfs in this group of high-risk patients is less than 20%. 53 patients were given azacitidine for overt relapse after sct. patients were given a median of 3 courses (2-36) and 16 were also given dli. 10 patients achieved cr, 10 stable disease and 33 progressive disease. 5 patients are still on therapy, 44 died or progressed and 4 had to discontinue due to hematological toxicity. with median follow-up of 15 months , 12 are alive and 5 are progression-free. and 7% (95%ci, 0-13), respectively. conclusions: azacitidine is safe and effective therapy when used prophylactically in high-risk aml/mds patients to prevent relapse or preemptively to treat mrd or early relapse after sct. azacitidine maintenance may improve outcome in this high-risk patient group and should be further explored. results of azacitidine treatment in overt post-transplant relapse are limited. disclosure: nothing to declare novel mass cytometry analysis identifies reciprocal changes in nkreg and cd4 em as the dominant early immune reconstitution signature associated with subsequent acute gvhd after ric-ahst background: treatment failure after allogeneic haematopoietic stem-cell transplantation (ahst) using reducedintensity conditioning (ric) results from either too much alloreactivity and harmful acute graft-versus-host disease (agvhd) or not enough (reducing graft-versus-tumour effects). studies have identified individual reconstituting immune cell subsets associated with development of clinical alloreactivity but the functionally dominant parameters remain unknown. we therefore used mass cytometry (ms) technology to determine multiple parameters simultaneously to identify dominant cellular immune reconstitution signatures associated with development of clinical alloreactivity after ahst. methods: phenotypic markers identifying >30 t, b and nk cell subsets known to influence alloreactivity were combined in a single ms panel. peripheral blood from 52 patients with haematological cancer was analysed at d+30 after t-replete hla-matched ric-ahst using uniform conditioning. test samples were spiked with cd45barcoded healthy control cells. three complementary high-dimensional analytic tools were used to identify immune signatures in cd45 + lineage + cells across the whole cohort and identify differences between patients grouped by subsequent occurrence of agvhd. results: significant batch effects were effectively reduced with a novel r-based algorithm normalising data to control cells. unsupervised clustering analysis using phenograph and flowsom algorithms identified 24 and 40 phenotypically distinct clusters respectively. diversity analysis demonstrated lower cluster diversity (p=0.06) in the 20 patients who subsequently developed agvhd consistent with perturbation of phenotypic clusters in these patients. comparison of individual cluster abundance identified 2 cluster groups significantly different between patients who subsequently developed agvhd and those who did not. a cluster containing cells with a cd56 bright cd16 neg cd27 +/regulatory/tolerant nk cell (nkreg) phenotype was significantly reduced in patients who subsequently developed agvhd using both phenograph and flowsom algorithms (p< 0.001). a differentiating cell population with this phenotype was also identified in forward analysis using the citrus algorithm. notably, this reduction in nkreg was accompanied by a significant increase in abundance of a cluster of ccr5 + cd45ra -ccr7 -cd4 effector memory t-cells (tem) in patients who subsequently developed agvhd. there was a significant negative correlation (p=0.01) between nkreg and tem. in contrast there was no inverse correlation between cd4 regulatory t-cells and cd4em. these changes were independent of clinically significant cmv reactivation. finally, we determined the impact of time to agvhd on this novel immune signature. reciprocal changes in nkreg and cd4 tem abundance were more significant in patients who developed agvhd before d60, consistent with a dynamically evolving immune signature. conclusions: we show proof-of-concept that a novel pipeline can be applied to ms data to measure multiple immune reconstitution parameters after ahst. importantly, this pipeline identified concomitant loss of nkreg and increase of cd4 tem in patients who subsequently developed agvhd. this is consistent with loss of nk cell-mediated control of alloreactive cd4 tem cells as the dominant immune process preceding the development of agvhd after ric-ahst. our data provide mechanistic insight into evolution of alloreactivity and support the development of strategies to maintain or expand nkreg numbers early post-transplant to reduce harmful agvhd. [[o149 image] 1. dominant immune reconstitution signature early after ric-ahst associated with subsequent acute gvhd] multiple myeloma o150 abstract already published. impact of high-risk cytogenetics in newly diagnosed multiple myeloma undergoing upfront stem cell transplantation: a study from the ebmt chronic malignancies working party background: current consensus identifies t(4;14), t(14;16), t(14;20), gain and/or deletion in chromosome 1, and del(17/ 17p) as high-risk cytogenetics in newly diagnosed multiple myeloma (ndmm). however, evidence on outcome of specific abnormalities after transplantation as first-line treatment is limited. we analyzed high-risk ndmm patients reported to the european society for blood and marrow transplantation (ebmt) registry undergoing upfront stem cell transplantation. methods: upfront transplantation was defined as first autologous transplant within 12 months from mm diagnosis. survival and cumulative incidence were calculated from date of first transplant (95% confidence interval). end points were progression-free survival (pfs), overall survival (os), relapse and non-relapse mortality (nrm). cox model with hazard ratios (hr) was used for multivariable os analyses and cumulative incidence method for relapse incidence and nrm. results: within the ebmt registry, 623 high-risk ndmm patients according to cytogenetics underwent single autologous (n=446), tandem autologous (n=105), autologous-allogeneic (n=72) stem cell transplantation between 2000 and 2015. the median follow-up of all patients was 58 months (95% ci, 52-63 months), the median age was 59 years (range, 25-76 years) and the median time between diagnosis and transplantation was 5.6 months (range, 2.2-11.7 months). frequencies according to cytogenetic were: del(17) (n=333, 54%), t (4;14) (n=344, 55%), gain or deletion in chromosome 1 (n=85, 14%), t(14;16) (n=19, 3%). two or more cytogenetic abnormalities were documented in 143 patients (23%). 56% of patients were male and most patients had igg (53%) or iga (26%) paraproteins. a karnofsky performance status < 90% had 29% of patients while frequencies according to international staging system (iss) i/ii/iii were 21%/57%/22%. complete remission (cr) at time of transplantation was achieved by 18%. in univariable analysis, presence vs absence of del (17) conclusions: in ndmm patients with at least one highrisk cytogenetic abnormality undergoing upfront transplantation, outcome was similar between del(17) and t(4;14) while the presence of two or more high-risk cytogenetic abnormalities showed significantly worse os compared with only one high-risk abnormality. disclosure: nothing to declare. the role of renal impairment at diagnosis in multiple myeloma undergoing autologous transplantation. a retrospective analysis of the cmwp background: renal impairment (ri) is frequent in newly diagnosed myeloma patients and is considered to be a risk factor for worse overall survival. with active myeloma therapy renal function often improves or even normalises. however, it is unclear whether renal impairment at diagnosis is a persisting biological risk factor or rather a potentially reversible organ complication. methods: from the ebmt calm study database all myeloma patients having received a first autologous transplant between 2008 and 2012 with information on renal function both at diagnosis and at transplant were extracted. renal function was classified according to the calculated glomerular filtration rate (gfr) rate as normal ("normal", gfr > 50 ml/min), moderately impaired ("moderate", gfr 30-50 ml/min) or severely impaired ("severe", gfr < 30 ml/min). categorial variables were tested by chi-square test. os was determined from transplantation and calculated by kaplan meier with logrank testing. results: 1905 patients fulfilled the selection criteria and were included. at diagnosis, 1447 patients had normal, 184 moderate and 274 severe ri. median age at diagnosis was 58, 60 and 59 years in the ri subgroups. genetic information was available in only a subgroup of patients. t(4;14) was present in 14%, 22% and 13% respectively and del17 was found in 7%, 8% and 4%. bortezomib-based induction therapy was given in 53%, 58% and 64% of cases (p=0.018). os differed significantly between the ri groups with a median of 84, 72 and 62 months, respectively (p< 0.001, fig 1) . in contrast renal function at transplant had no impact on os with a median of 78 months (no ri at transplant), 79 months (moderate ri at transplant) and not reached (severe ri at transplant). most of the 274 patients with severe ri at diagnosis had improved their renal function by the time of transplantation. however, this did not positively impact on os: patients with no ri at transplant had a median os of 37 months (n=132), while it was 65 months for moderate ri (n=71) and not reached in patients transplanted with persisting severe ri (n=71,p< 0.001). conclusions: from this large analysis including almost 2000 myeloma patients renal impairment at diagnosis has been found to be a risk factor for os, while renal function at transplant did not impact on post transplant survival. these findings support the safety and efficacy of autologous transplantation in patients with severe ri at transplant. on the other hand improving renal function between diagnosis and transplant does not seem to improve prognosis.. our analysis supports the notion that ri at diagnosis appears to be a surrogate parameter for a more aggressive disease course. disclosure: nothing to declare fig.1 oerall survival according to ri at diagnosis] analysis of outcomes in patients with myeloma who had a second allohct either for disease relapse or graft failure: an ebmt cmwp study background: the options for patients with myeloma (mm) who relapse or develop graft failure after an allosct are limited. a second allohct is occasionally feasible though is high-risk. we performed a retrospective analysis to assess outcomes in this cohort. methods: data on patients with mm who underwent a second allohct at ebmt centres between 1994 and 2017 were obtained from the ebmt registry. results: a total of 273 patients (165 m,108 f) with mm (51% igg, 22% iga, 23% lc) underwent a second allosct. the median (range) age at the first allohct was 48.5 (20.1-67.9) years and 78.1% were >/=pr. when comparing the indications for the 2 nd allohct -relapsed mm (74%) or graft failure (26%) -patients with graft failure were significantly more likely to have received a mismatched (related/unrelated) or unrelated donor for the 1 st allosct (58% vs. 31%) (p=0.001), were more commonly female (57% vs. 38%) (p=0.018) and were more likely to have had a ric as opposed to a mac allosct (79% vs. 59%) (p=0.015). the median (range) interval between the 1 st and 2 nd allohcts was 3.5 (7.7-66.6) months in cases of graft failure and 40.5 (68.6-170.4) months for those who had relapsed. at a median (95% ci) follow-up of 107.2 (84.9 -141.7) months following the second allohct, overall survival (os) was 41% (35-47%) at two years and 26% (20-32%) at five years. there was no difference in os at five years based on the indication for allohct: 25% (17-32%) for relapse and 32% (19-45%) for graft failure (p=0.595) (figure 1 ). neutrophil engraftment following the 2 nd allohct was achieved by day +28 in 94% (90-98%) and 80% (69-92%) of the relapse and graft failure patients, respectively. the cumulative incidence of agvhd (ii-iv) and cgvhd (at five years) following the 2 nd allohct was 27% (21-33%) and 39% (32-47%), respectively.relapse-free survival was 12% (7-17%) at 5 years, 7% (2-13%) in those transplanted for disease relapse and 22% (10-35%) in those transplanted for graft failure (p=0.061). the cumulative incidence of relapse and nrm at five years was 64% (57-71%) and 24% (18-30%), respectively. the five-year os following the second allosct was 14% (1-28%) for those relapsing between 12 and 24 months after the first allohct and 31% (21-40%) for those relapsing later than 24 months (p=0.07).on univariate analysis, os at five years was superior in patients who had had hla identical sibling donors as opposed to other donor sources: 32% (24-40%) vs. 17% (9-25%) (p< 0.001). on multi-variate analysis, donor source (hla identical sibling vs. other) remained a predictive factor for os (p=0.014). conclusions: in this high-risk mm cohort, one quarter of patients remained alive five years after the 2 nd allohct with similar outcomes seen following disease relapse and graft failure. however, the relapse-free survival rate was low in those transplanted for relapsed mm. later relapses after the first allohct appear to fare better and the best outcomes are seen using matched sibling donors. a 2 nd allohct therefore remains an option to be considered for selected mm patients. disclosure: nothing to declare minimal residual disease (mrd) ratio before and after autologous stem cells transplantation (asct) in multiple myeloma (mm) riccardo boncompagni 1 , michela staderini 1 , chiara nozzoli 1 , elisabetta antonioli 1 , barbara accogli 1 , riccardo saccardi 1 1 careggi university hospital, florence, italy, background: in the last ten years, multiparametric flow cytometry (mfc) has been standardized and routinely applied for the detection of mrd as a prognostic factor in mm patients across different lines of therapy. we assessed the mrd carried out before and after asct in a series of consecutive mm patients in order to investigate whether the ratio of the two determinations might increase the prognostic potential. methods: we collected bone marrow samples for mrd assessment at the end of induction therapy and 3 months after asct from 61 mm patients treated between 2013 and 2017 achieving at least a very good partial remission (vgpr) with a bortezomib-based induction therapy, according to the most recent international myeloma working group (imwg) criteria (kumar s et al, lancet oncol 2016) . mfc-determined mrd was evaluated according to euroflow recommendations (kalina t et al, leukemia 2012) . all patients were examined with 18fluorodeoxyglucose positron emission tomography/computed tomography (fdg-pet/ct) scan before and after the asct. results: post-induction therapy mrd was found predictive of post-asct mrd status. indeed, patients transplanted in a mrd positive status had a significantly increased risk to maintain a mrd positivity status after transplantation (odds ratio -or -15,053, p = 0,002). detection of post-asct mrd had a negative impact on median pfs (28 months vs not reached respectively, p = 0,001). in cox-regression analysis, a complete remission status (cr) with an undetectable mrd after the asct resulted to be the major protective factor from relapse (hazard ratio -hr -0,012, p = 0,005), while patients with a detectable mrd before and after the asct had the worse pfs (22 months, hr 2,958; p = 0,029). risk analysis showed 3 different pfs risk groups: "high" for the patients with mrd detectable before and after the asct, "intermediate" for patients with mrd positivity before the asct who achieve a negativity after, and "low" in the case of mrd undetectable before and after. in our study, response evaluated by fdg-pet/ct showed no correlation with pfs. conclusions: multiparametric flow cytometry is a relatively recent method to assay mm mrd, and its role in mm therapeutic path is still under investigation. according to our data, a detectable mrd after the asct is a major relapse risk. interestingly we found that it can be early predicted by the post-induction mrd status and its negativization after asct has a modest impact on this. therefore, we support the concept of treatment escalation when a cr is not reached after the induction treatment, in order to undergo to the asct in the best possible response. however, double mrd determination before and after vtd, with ≥60% of patients achieving a best response of ≥vgpr. the orr in patients receiving 'other' induction therapies was 71% and the ≥vgpr rate was 29%. finally, following auto-sct, the orrs for patients receiving vtd induction were around 80%, 60%, and 33% in lines 2-3, 4, and 5+, respectively. conclusions: this analysis provides prospective, realworld data on therapy of patients with mm receiving auto-sct. vtd is the most widely used induction regimen prior to auto-sct. moreover, the response rates are in line with reported rates in phase iii clinical trials. while other induction regimens are being developed, vtd is likely to remain a standard of care, because access to novel agents will continue to vary greatly from country to country due to factors such as affordability, local guidelines/restrictions, and regulatory decisions. background: car-t cell therapy against the cd19 antigen is a breakthrough treatment for patients with relapsed/ refractory (r/r) b-cell non-hodgkin lymphoma (nhl). despite impressive outcomes, non-response and relapse with cd19 negative disease remain challenges. through dual b-cell antigen targeting of cd20 and cd19, with a first-in-human bispecific lentiviral car-t cell (lv20.19car), we attempt to improve response rates while limiting relapses due to cd19 antigen loss. production was optimized with point of care automated manufacturing using the clinimacs prodigy, a compact gmp compliant tabletop device in an iso7 clean room. methods: patients were treated on our phase 1 dose escalation + expansion trial (nct03019055) to demonstrate feasibility of point of care manufacturing and safety of a bispecific 41bb/cd3z lv20.19car t cell for adults with r/r b-cell nhl. safety was assessed by incidence of dose limiting toxicities (dlts) within 28 days postinfusion. dose was escalated in incremental 3+3 fashion with a starting dose of 2.5 x 10 5 cells/kg and a target cell dose of 2.5 x 10 6 cells/kg. lymphodepletion was with fludarabine 30 mg/m 2 x 3 days and cyclophosphamide 500 mg/m 2 x 1 day. patients received either fresh uncryopreserved car-t cell infusions (n= 7) or cells thawed (n= 3) after cryopreservation. results: 10 patients have completed treatment: 9 patients in dose escalation and 1 patient in dose expansion. median age was 55 years (46-67) and histology included dlbcl in 4 patients, mcl in 4 patients, and cll in 2 patients. in dose escalation, 3 patients were treated at 2.5 x 10 5 cells/kg, 3 patients at 7.5 x 10 5 cells/kg, and 3 patients at 2.5 x 10 6 cells/kg with no dlts to report. no patient experienced grade 3-4 cytokine release syndrome (crs) or grade 3-4 neurotoxicity (ntx) allowing start of a dose expansion cohort at the 2.5 x 10 6 cells/kg level. in total, 6 patients had grade 1-2 crs and 3 patients had grade 1-2 ntx. mean time to crs was day +9 post-infusion and no patient required icu level care. 4 patients required 1-2 doses of tocilizumab. the day 28 overall response rate for all patients was 80% with 5/10 achieving a complete response (cr) and 3/10 achieving a partial response (pr). all patients in cr remain in remission, the longest 15 months from treatment. car-t persistence is demonstrated in figure 1 . two patients had progressive disease (pd) at day 28 and 2 patients with pr, eventually progressed. all progressing patients underwent repeat biopsy, and all retained either cd19 or cd20 positivity. target dose lv20.19 car t cells were produced in all patients indicating 100% feasibility of our manufacturing process. conclusions: phase 1 results from the first-in-human bispecific lv20.19 car t clinical trial demonstrate that near patient manufacturing and infusion of 2.5 x 10 6 cells/ kg is safe for further investigation with no dlts among treated patients. point of care production logistics aided the administration of fresh car-t cells in the majority. with limited toxicity and 60% sustained response in this relapsed refractory population, this approach to car-t production and dual b-cell targeting merits further investigation. o159 car-t cell therapy bridging to allogeneic hematopoietic cell transplantation for patients with refractory and relapsed acute lymphoblastic leukemia jia chen 1,2 , yi fan 1,2 , yang xu 1,2 , suning chen 1,2 , huiying qiu 1,2 , xiaowen tang 1,2 , yue han 1,2 , chengcheng fu 1,2 , depei wu 1, 2 background: refractory and relapsed (r/r) acute lymphoblastic leukemia (all) always leads to a dismal outcome. allogeneic hematopoietic cell transplantation (hct) is the only potentially curative modality for r/r all, but the long-term survival post-hct remains unsatisfying. car-t cell therapy targeting to cd19 produces promising response for r/r all patients, but the recurrence is the major concern. we investigated the effectiveness of a tandem protocol using car-t cell therapy followed by allogeneic hct. methods: we conducted a prospective study to enroll the patients with r/r all. major inclusion and exclusion criteria are: 1) definitely diagnosed as all; 2) primary refractory (failed to achieve cr after induction) patients or relapsed patients with no response for salvage therapy; 3) with an available donor for allogeneic hct; 4) without severe organ dysfunction or uncontrolled infection. patients enrolled received car-t cell therapy targeting to cd19 with a total dose of 5~10×10 6 /kg of recipient weight, and the preparative regimen before car-t cell infusion consisted of fludarabin and cyclophosphamide. after the evaluation at 30 days post car-t cell infusion, patients started the allogeneic hct procedure using a myeloablative conditioning (modified bu/cy). the control group consisted of patients with r/r all and underwent allogeneic hct without car-t therapy in the same time frame. results: totally 24 patients were enrolled in this study from december, 2016 through april, 2018, including 17 primary refractory patients and 7 relapsed patients (details in table 1 ). twenty patients (83.3%) achieved cr after car-t cell therapy, and 6 patients (25%) developed grade 2 or higher crs. no irreversible toxicities emerged and all the patients moved to the hct procedure. when comparing with the control group (figure 1) , the 1-year cumulative incidence of relapse was 14.8 ± 8.0% for the trial group versus 46.4 ± 12.4% for the control group (p = 0.090), and 1-year overall survival was 71.4 ± 17.1% versus 54.0 ± 11.4% (p = 0.067). conclusions: we concluded that car-t cell therapy bridging to allogeneic hct is a promising approach for r/r all patients, which leads to both high response and low risk of relapse. besides, car-t cell therapy is a safe modality as salvage treatment for r/r all patients, which had little negative impact for following hct. methods: to test the hypothesis that haploidentical hct would be a valid option for high-risk pediatric aml patients lacking a matched donor, we designed a diseasespecific, multi-centre study. we retrospectively analyzed 179 consecutive patients under 18 years with high-risk aml underwent matched sibling donor (msd) (n=23) or haploidentical donor (hid) hct (n=156) between july, 2013 and dec, 2017. a 1:3 ratio matched pair analysis was implemented with the following matching factors: cytogenetic risk, disease status (cr1/cr2/>cr2), age and sex of patients, sex of donor, and graft type. results: all patients achieved myeloid recovery with a median time of 15d and 13d for msd cohort and hid group (p=0.002). the cumulative incidence of grade ii-iv acute graft-versus-host-disease (gvhd) in msd cohort (13%) was significantly lower than in hid group (35%, p=0.048); the incidence of chronic gvhd was comparable between the two groups. the cumulative incidence of relapse in msd cohort (39%) was significantly higher than in hid group (16%, p=0.037); the incidence of nrm was 0 and 10% (p=0.12), respectively. the 3-year overall survival (65% versus 75%, p=0.68) and leukemia free survival (61% versus73%, p=0.29) were comparable in msd-hct compared with hid-hct. in a multivariate analysis, hid-hct remained a significant factor for reduced relapse rate (hr 0.259(0.092-0.731), p= 0.011) in comparison with msd-hct. in subgroup analysis for patients with known cytogenetics and transplanted in the first complete remission (n=58), the cumulative incidence of relapse in msd cohort (50%, n=14) was significantly higher than in hid group (9%, n=44, p=0.001); and leukemia free survival (50% versus 81%, p=0.021) were significantly lower in msd-hct compared with hid-hct. in a multivariate analysis among these subgroup of patients, hid-hct remained a significant factor for increased lfs (hr 0.304(0.102-0.905), p= 0.032) in comparison with msd-hct. conclusions: in conclusion, unmanipulated haploidentical-hct achieves outcomes comparable to those of isd-hct for high-risk pediatric aml patients and even exerts greater gvl effect in some circumstances. such transplantation was proved to be a valid alternative treatment for high-risk pediatric aml patients lacking a matched donor. larger prospective studies are needed to confirm these findings. disclosure: nothing to declare. background: allogeneic hematopoietic stem cell transplantation (hsct) is the only curative option for patients with beta thalassemia major. although limited study in the literature has evaluated the impact of age on success of transplantation, more data are needed. in this study, we aimed to evaluate the effect of age of the patients on transplant outcome in cases who underwent hsct with the diagnosis of thalassemia major. methods: all cases who underwent stem cell transplantation with thalassemia major were included. all thalassemia major patients with a median age of 7 years (range 7 month,17.7 years) underwent allogeneic hsct using myeloablative conditioning regimen. cyclosporine and methotrexate were used as gvhd prophylaxis. in total, 169 patients underwent hsct at age younger than 7 and 159 patients underwent at age older than 7 years and all patients were assigned to two different groups according to transplantation age. the distribution of donor type and stem cell sources by age groups is shown in table 1 . no statistical difference was found between the two age groups in terms of donor type and stem cell source. patients in two different age groups were compared with cox regression analysis in terms of overall survival, thalassemia free survival and thalassemia-gvhd free survival. results: a total of 299 patients; 162 patients under 7 years of age, 137 patients aged 7 and over, were engrafted and remained transfusion independent with full or mixed chimerism. four patients, two from each age group, did not engrafted and had primary rejection. four patients under seven years of age developed secondary rejection, whereas in the group of patients older than 7 years of age, 17 patients experienced secondary rejection (p< 0.05) . a total of 42 patients developed acute gvhd (12.8 %)and their rates were similar in both age groups (10.6% vs 15%). chronic gvhd development rates in two group was also similar (9% vs 7%). the median follow-up time was 34 months (range 0.5-93 months). the 3 -year overall survival rates (os), thalasemia-free survival rates (tfs), thalassemia-gvhd free survival rates (dfs) were shown in the table 2. both thalassemia-free survival and thalassemia-gvhd free survival were higher in patients who underwent transplantation under seven years of age . there was no difference in overall survival. conclusions: the results of our study show that the rates of rejection are high, thalassemia free and thalassemia / gvhd free survival are low in patients who underwent stem cell transplantation over seven years of age. in the light of successful transplantation results from unrelated donors, the delay in age of transplantation in thalassemia patients should also be avoided. disclosure: nothing to declare o167 hla-haploidentical transplantation with regulatory and conventional t-cell adoptive immunotherapy in pediatric patients with high-risk acute leukemia background: post-transplant relapse is still a major cause of treatment failure in high-risk acute leukemia (al) patients. in order to separate the gvl effect from gvhd, we investigated the role of a thymic-derived cd4 + cd25 + foxp3 + regulatory t cells (tregs). the perugia center reported results from 69 adult high-risk al patients who received an hla haploidentical t-cell-depleted hematopoietic transplant and adoptive immunotherapy with donor tregs and conventional t cells (tcons) (and no posttransplant pharmacologic immunosuppressive gvhd prophylaxis) (di ianni et al., blood 2011 , martelli et al. blood 2014 . adoptive immunotherapy with tregs and tcons prevented post-transplant leukemia relapse and largely protected patients from gvhd. in this report we present a pediatric cohort of high risk leukemia patients who received a haploidentical treg/tcon-based hematopoietic transplant. methods: twelve pediatric patients, median age of nine years (range, 5-19) with high-risk acute leukemia underwent hla-haploidentical stem cell transplantation with regulatory and conventional t-cell adoptive immunotherapy between september 2016 and december 2017. eleven had all (three ph+), one secondary aml. seven patients were transplanted in cr1 (3 ph+ all, 1 all in cr after second-line induction, 1 all with extramedullary leukemia, 1 all with t(19;11), 1 secondary aml after medulloblastoma), two patients in cr2, three in cr3. median time from diagnosis to transplantation was 18.5 months (range, 5-48), median time from relapse to especially for aml pts. our analysis suggests that early αβ t cell recovery is associated with a relatively low nonrelapse mortality and relapse rate. disclosure: nothing to declare background: enhancing stem cell performance can improve the results of hematopoietic stem cell transplant (hsct) for diseases in which engraftment is unpredictable, and in patients receiving pre-hsct conditioning regimens of progressively decreasing intensity. in a series of preclinical studies, we explored the use of tat-myc, a chimeric recombinant protein, to improve the performance of hematopoietic stem cell grafts. methods: tat-myc recombinant protein encompasses 9 amino acids from the n-terminal nuclear localization domain of hiv-tat, coupled to the entire coding sequence of c-myc, with an appended histidine tag to aid in protein purification. the construct was expressed in bacteria and purified to pharmacological grade purity under glp conditions. results: brief (1 hour) culture of fibroblasts or hematopoietic cells in medium containing 10 microgram/ ml tat-myc results in rapid nuclear localization of the recombinant protein, whence it disappears within 48 hours as measured by western blot. flow cytometric assays have been validated to measure the uptake of tat-myc recombinant protein into nucleated marrow cells. marrow homing of tat-myc recombinant protein-treated murine marrow increased 5-fold as compared to that of control cells. incubation of activated murine t cells with tat-myc recombinant protein conferred resistance to granzyme b cytotoxicity, but did not protect cells from effects of cyclophosphamide. tat-myc recombinant protein-treated marrow could be serially transplanted in mice for three generations. murine bone marrow harvested from 5fluorouracil treated mice and briefly incubated with tat-myc recombinant protein outcompeted control marrow when transplanted in sub-lethally irradiated immunedeficient mice even at ratios of 1:9 treated:control cells. t-and b-cell reconstitution following transplantation in immune deficient mice was superior following tat-myc vs control incubation of murine marrow. engraftment of human umbilical cord blood (ucb) cells in sub-lethally irradiated immune deficient mice was markedly improved following tat-myc incubation as compared to that of control ucb cells. the transforming potential tat-myc protein was extensively explored. tat-myc culture hematopoietic cells did not display aneuploidy in cytogenetic or spectral karyotypic analyses. intramuscular injection of 10 micrograms of tat-myc protein in p53 +/mice for 12 consecutive weeks did not result in tumor formation. to exaggerate potentially transformative effects of tat-myc protein, murine marrow was co-incubated with both tat-myc and tat-bcl2 recombinant proteins prior to transplantation into irradiated immune deficient mice. mice were followed for 24 weeks and none developed malignancies. serially transplanted marrow incubated with both tat-myc and bcl-2 proteins did not result in malignancies in recipient mice. of >500 mice that have been exposed to tat-myc recombinant protein in our experiments, none has developed a tumor. conclusions: in preclinical studies, brief incubation with tat-myc recombinant protein enhances homing, engraftment and immune reconstitution of murine and human cells in recipient mice following transplantation. brief exposure to the recombinant protein (as opposed to transduction of the myc gene) does not cause malignant transformation of cells. we are currently developing clinical trials using tat-myc protein to enhance engraftment following hsct. disclosure: greg bird, brian turner, thomas payne, yosef refaeli are employees and or shareholders in taiga biotechnologies. jerry stein has received laboratory support from taiga biotechnologies graft γδ t-cell receptor sequencing identifies public clonotypes associated to hsct efficacy in aml patients and unravels cmv impact on repertoire distribution lucas cm arruda 1 , ahmed gaballa 1 , michael uhlin 1,2,3 relapse patient group had an increased proportion of long cdr3 sequences (54-57 nucleotides) compared to relapse patients [0.41%vs0.27% (p=0.02) and 0.11%vs0.04% (p=0.04)]. grafts from cmv-positive donors presented significantly reduced diversity (inverse simpson's di: 30.70vs81.21, p=0.02), decreased proportion of cdr3 sequences having 24, 27, 42 and 51 nucleotides [0.27%vs0.61% (p=0.01), 0.57%vs1.12% (p=0.02), 5.11%vs7.54% (p=0.007) and 0.61%vs2.1% (p=0.04)], and an increase of sequences with 30-39 nucleotides (5.32%vs4.13%, p=0.03). hyperexpanded clones took up 2.5 times more space in the cmv positive grafts (49.33% vs19.38%, p=0.007), who presented a skewed non-gaussian distribution. for all samples, the segments trgv9 and trgjp were the most frequent. nonrelapsing group received grafts with lower usage of trgv4, trgv5 and trgjp2 segments and higher usage of trgjp1 compared to relapse patients [4.13%vs7.46% (p=0.04), 1.12%vs4.77% (p=0.02), 3.02%vs5.52% (p=0.02) and 6.62%vs3.23% (p=0. 02) ]. cmv-positive donor grafts presented a lower trgv2 and trgjp expression (6.29%vs8.99%, p=0.04, and 30.99% vs60.63%, p=0.01) as well as a higher trgjp1 gene usage (11.04%vs3.90%, p=0.04). the v9-jp combination was the most frequent pairing in all samples. non-relapse patients received grafts with lower usage of the pairing v4-j2, v5-j2, v8-jp2 [3.12%vs6.57% (p=0.04), 1.06% vs4.38% (p=0.02) and 1.33%vs1.91% (p=0.03)] and higher usage of v2-jp1 pairing (1.28%vs0.36%, p=0.03) than relapse patients. the tcr usage of the sequence pairs v2-j2, v2-jp2, v4-jp2, v9-jp, and v9-jp2 was lower in cmv-positive grafts [4.62%vs6.94% (p=0.04), 0.59%vs1.33% (p=0.04), 0.31%vs0.09% (p=0.02), 30.75%vs60.30% (p=0.02) and 0.07%vs0.29% (p=0.01)]. we identified 12 public clones shared exclusively between the grafts received by non-relapsing patients in addition to four private over-represented sequences exclusively present in grafts given to nonrelapse patients, taking from 2.00% to 6.23% of the trg repertoire and longer than 45 nucleotides. we also identified five private over-represented and one public cdr3 sequence associated to cmv infection. additionally, cmv-positive grafts presented the highest percentage or repertoire taken by private over-represented clones, ranging from 13.72% to 41.61%. conclusions: our findings show that the trg composition is not associated to agvhd incidence, cmv infection reshapes the trg repertoire and several public sequences are associated to clinical remission. disclosure: the authors have nothing to disclose. background: pediatric patients with high-risk alveolar rhabdomyosarcoma (arms) above the age of 10 years cannot be cured by conventional therapies. immune cells targeting erbb2 with a chimeric antigen receptor (car) were recently considered for these patients. cytokineinduced killer (cik) cells already capable of natural killer (nk)-like anti-tumor capacity additionally redirected with an erbb2 car may provide overall disease control in these high-risk tumors. methods: erbb2-car modified cik cells were generated from conventional cik cells (wt-cik) by lentiviral gene transduction on day 4 of culture. the codon-optimized car sequence consists of an igg heavy-chain signal peptide, an erbb2-specific antibody fragment scfv (frp5) and a modified cd8α hinge region, as well as cd28 transmembrane and intracellular domains and a cd3ζ intracellular domain. 1x10 5 luciferase gene-transduced rh30 (arms) cells were engrafted in immunodeficient nod/scid/γc -(nsg) mice. mice were randomly selected into 5 different treatment groups (dbps on day +1, 2.5x10 6 wt-cik or erbb2 car-cik cells on days +1 and +36, 2.5x10 6 wt-cik or erbb2 car-cik cells on days +22 and +57). mice were monitored by bioluminescence imaging (bli) until day +100. tumor engraftment and immune cell homing at tumor sites were analyzed by facs, chimerism and immunohistochemistry analyses. results: human rms xenografts were established in all mice treated with dbps only. control-mice showed a median survival of 62 days. human rms was identified in all analyzed organs, with the highest tumor burden seen in livers of dbps-treated mice. mice injected with wt or erbb2-car cik cells on days +1 and +36 showed a significant improved (p < 0.014 and p < 0.01) disease-free survival, respectively. furthermore, no signs of tumor engraftment were shown by bli in erbb2 car-cik cell treated mice while some of the mice treated with wt-cik cells developed positive tumor signals between weeks 7 and 10. in 4 out of 6 (64%) wt-and in all (8 of 8, 100%) car-cik cells treated mice no residual tumor cells were identified by pcr-based analysis. in contrast, tumor cells were detectable in all mice with delayed anti-tumor treatment applied on day +22 and +57. however, tumor growth was lower in these groups. correspondingly, bli showed delayed tumor engraftment in mice with wt-and even more with car-cik cell treatment given on day 22. treatment on day 22 resulted in a significantly improved survival of erbb2-car cik cell treated mice (p < 0.01), while survival was not improved after wt-cik cell infusion (p > 0.07). within all treatment groups, immune cells were detected by chimerism and facs analyses. facs analyses showed a significant increase of nk-like t cells (p < 0.01 and < 0.05, wtand erbb2-car cik cells). additionally, a higher, but not significant, amount of effector memory and stem cell memory t cells were detected. conclusions: these pre-clinical in vivo results indicate that erbb2-car redirection of cik cells improves both homing and nk-like cytotoxicity of cik cells in the presence of erbb2-positive tumors, implying that this therapy may represent a step forward in the treatment of patients with resistant, relapsed and advanced rms. disclosure: michael merker, juliane wagner, vida meyer, thomas klingebiel, winfried s. wels and eva rettinger have nothing to declare. peter bader declares the following potential conflicts of interest: novartis (consultancy: included expert testimony, speaker bureau, honoraria), medac (research funding, patents and royalties), riemser (research funding), neovii (research funding), amgen (honoraria) . genesis -a phase iii randomized double-blind, placebocontrolled trial, evaluating safety and efficacy of bl-8040 and g-csf in mobilization of hcs's for autologous transplantation-multiple myeloma hemda chen 1 , zachary d. crees 2 , keith stockerl-goldstein k 2 , abi vainstein 1 , ella sorani 1 , osnat bohana-kashtan 1 , john f dipersio 2 1 biolinerx, tel aviv, israel, 2 washington university in st. louis, st. louis, wa, united states background: cxcr4 mediates retention of hematopoietic stem cells (hscs) in the bone marrow (bm) niche. bl-8040, a novel, high affinity cxcr4 antagonist is a potent mobilizer of hscs to the peripheral blood with numerous potential clinical applications, including mobilization of cd34+ cells for autologous hsc transplantation (auto-hsct) in multiple myeloma (mm). this study aims to evaluate the efficacy of single dose bl-8040 plus g-csf in mobilization of ≥6.0x10 6 cd34+ cells/kg in up to 2 apheresis sessions for auto-hsct in mm. methods: a phase iii study composed of an open-label, single-arm lead-in part1 followed by a randomized, doubleblinded, placebo-controlled part2. eligible mm patients age 18-78 will receive g-csf (10 μg/kg; sc) daily for up to 8 days and one dose of bl-8040 (1.25 mg/kg; sc) or placebo on day 4 followed by up to 2 apheresis sessions; and if needed a second dose of bl-8040 or placebo on day 6 followed by up to 2 apheresis sessions. progressive disease at time of ldc did not respond although car-t cells could be seen morphologically under the microscope. this might be explained by multidrug related phenomenon protecting refractory leukemia from car-t cell attack. conclusions: commercial available car-t cell product tisagenlecleucel (kymriah®) showed high efficacy in r/r-all patients to re-induce cr. clinical trial registry: commercial available car-t cell product tisagenlecleucel (kymriah®) showed high efficacy in r/r-all patients to re-induce cr. disclosure: pb: novartis (consultancy: included expert testimony, speaker bureau, honoraria); medac (research funding, patents and royalties); riemser (research funding); neovii (research funding); amgen (honoraria) . aj: novartis and bluebird: (consultancy) . all other author declare no coi. the main causes of death were sct-related in 53% and disease in 35%. 36-months pfs, os, ir and nrm were 53% (44-63), 72% (64-80), 34% (25-43) and 13% (8-20), respectively. cumulative incidence of grade 3-4 acute gvhd at 100 days after ric-sct was 10% (5-16) and chronic gvhd at 36 months 48% pfs and ir were influenced by patient sex (p=0.025 and 0.04) and disease status at allo-sct 039) and stem cell source (p=0.04); acute grade 3-4 gvhd by donor type (p=0.037) and chronic gvhd by allo-sct conditioning (p=0.016) and donor sex (p=0.01). conclusions: this is the largest series analysing the efficacy and safety of a tandem auto-ric-sct approach in r/r hl. the low nrm and ir with promising pfs and os suggest that this might be an effective post-transplant cyclophosphamide-based gvhd prophylaxis compared to standard prophylaxis in patients with lymphoma receiving hla identical transplantation: a retrospective study from the lwp of ebmt luca castagna 18 ebmt lymphoma worky party clinical trial registry: nct02445248 disclosure: richard t. maziarz: honoraria, membership of advisory committee and research funding employment: oregon health & science university (ohsu); the potential conflict of interest re: consultant services to and payment from novartis has been reviewed and managed by dava oncology; honoraria and research funding: genentech; membership on an entity's board of directors or advisory committees membership on an entity's board of directors or advisory committees and research funding: novartis honoraria and membership on an entity's board of directors or advisory committees: nordic nanovector. vadim v. romanoff: employment: novartis employment: novartis. james signorovitch: employment: analysis group, which received research funding from novartis. solveig g. erickson: employment: novartis. david g. maloney: research funding other: scientific advisor: kite pharma, novartis disclosure: nothing to declare o155 multiple myeloma treatment in real-world clinical practice: a focus on induction regimens prior to autologous stem cell transplantation from the prospective, multinational, non-interventional emmos study cic, ibmcc (usal-csic) 13 state budget healthcare institution of moscow. city outpatient clinic 68 of healthcare use of unmanipulated hla-haploidentical donor transplants (haplo-hsct) is constantly increasing in the last years. few cases of haplo-hsct using posttransplant-cyclophosphamide (pt-cy) for pediatric patients were reported by single center and registry studies, with an incidence of grade ii-iv agvhd ranging from 30% to 40% and cgvhd approaching up to 40%, although with low incidence of extensive disease. methods: we investigated the outcomes of children (< =18y) undergoing haplo-hsct using pt-cy as gvhd prophylaxis disease status at haplo-hsct was cr1 for 31%, cr2 43% and advanced for 26%. poor-risk cytogenetics was reported in 36% of aml, and 11% had ph+ all all patients received pt-cy, in association with tacrolimus/mmf in 33% or csa/mmf in 28% or engraftment rate was 89% with 22 patients experiencing graft failure. cumulative incidence (ci) of day-100 acute gvhd grade ii-iv and grade iii-iv were 33% and 14% respectively, and ci of 2-y chronic gvhd was 20% (9% extensive disease). 2-y ci of nrm was 19% and relapse 41%. disease recurrence and infections were the most common causes of death. 2y-os and lfs were 49% and 40%. 2y-os was 53% and 47% (p=0.91) for aml and all; it was 80%, 47% and 22% (p< 0.001) for patients transplanted in cr1, cr2 and advanced disease. for 2y-lfs, no significant difference was found according to the type of conditioning regimen (39% macchemotherapy-based, 43% mac-tbi based and 38% for ric, p=0.97). the use of pbsc was associated with higher ci of grade os: cr2 vs cr1 conclusions: pt-cy is effective in preventing severe gvhd in children with leukemia receiving an unmanipulated haploidentical-donor transplant. disease status remain the most important factor for outcomes. the use of pbsc as stem cell source increases the risk of grade ii-iv agvhd. the effect of long-term complications, and morbidity related to gvhd results: all patients achieved primary, sustained fulldonor engraftment (median neutrophils engraftment 14 days, range 9-19; median platelets 15 days, 9-19). five patients (42%) developed ≥ grade 2 agvhd (2/5 had concomitant hcv hepatitis and developed liver agvhd), none developed cgvhd. the immune recovery was good in all patients despite immune suppressive therapy in patients with agvhd causes of nrm were: 1 agvhd, 1 invasive aspergillosis, 1 thrombotic microangiopathy. nine of the 12 patients are alive at a median follow-up of 19 months (2-22 months), cgvhd/ leukemia-free survival is 75%. conclusions: these preliminary data in 12 very high risk pediatric patients showed that hla-haploidentical transplantation with regulatory and conventional t-cell adoptive immunotherapy russian federation background: relapse, gvhd and associated non-relapse mortality are the main obstacles to successful hsct in children with leukemia. αβ t cell depletion was developed to prevent gvhd and improve immune reconstitution in recipients of mismatched grafts either melphalan (n=41) or thiophosphamide (n=31) or etoposide (n=16) were added, fludarabine was used in all pts. two types of gvhd prophylaxis were used: type 1 (n=18): hatg 50 mg/kg and post-hsct tacro/mtx, type 2 (n=71): thymoglobulin(ratg) 5mg/kg the median dose of cd34+ cells was 8 x10 6 /kg, aβ t cells -16 x10 3 /kg. results: five patients (5,6 %) died before engraftment due to septic event. primary engraftment was achieved in all evaluable pts (100%) with full donor chimerism. among the whole cohort the ci of gvhd grades ii -iv and iii -iv was 26-74), p=0,002. ratg was also effective in prevention of cgvhd: ci at 2 year after hsct was 15% vs 33%, p=0,08. 3-year ptrm was in the group with available immune reconstitution data (n=68) αβ t cell recovery at day +30 was associated with a trend to decreased incidence of relapse, ci of relapse was 29% (95% ci:15 -54) in those with αβ-t cell count < median vs 15 % (95% ci: 6-38) in those with αβ-cell count >median italian bone marrow donor registry hematopoietic stem cell transplantation (hsct) from hla-a, -b, -c and -drb1-matched unrelated donors (8/8 ud) is performed across hla-dpb1 barrier in more than 85% of cases. clinically tolerable (permissive) mismatches (mm) at hla-dpb1 locus have been classified by different immunogenetic models. here we compare the prognostic value of these models ii) a similar model subdividing alleles in 4 tce groups (tce4, crocchiolo iii) differences in "delta functional distance" scores of 12 polymorphic aminoacids in hla-dpb1 peptide-binding groove (crivello while the first three models were applicable to all 382 hla-dpb1 mm patients, the latter was restricted to 229 of them. the tce4 model appeared the most restrictive one, with only 36% of mm considered to be permissive. median follow-up was 3.2 y. results: hla-dpb1 permissive (p) mm pairs defined by tce4 model (n=135) had superior 3-y overall survival (os) and gvhd-free & relapse-free survival (grfs) compared to non-permissive (np) mm (n=247) (60±8% vs 49±7% cgvhd (hr 1.6, p .03) and extensive cgvhd (hr 3.6, p< .01). the predicted hla-dpb1 mismatched allele expression in the recipient was associated with 100-d ci of grade≥2 agvhd: 32±10% in high expression (n=76) versus 16±6% in low expression (n=153), p< .01. this was confirmed in multivariate analysis for grade≥2 agvhd (hr 2.2, p< .01), however, without higher hazards for trm and overall mortality. the overlap among the four models and their adjusted hr for os is shown in figure 1. conclusions: functional hla-dpb1 matching is of prognostic value in 8/8 ud-hsct outcomes. in our cohort, tce4 appears superior to other models in predicting survival and stratifying risks of trm and cgvhd however, it remains unclear how outcomes of patients with all treated with a haploidentical donor (haplo) compare with hla matched unrelated donor (mud) transplants. methods: we, therefore retrospectively compared outcomes of 506 patients with all who underwent haplohct with ptcy, reported from the participating centers (hit-rc and ebmt) from 01/2005 to 6/2018, with a matched cohort of 1012 patients (1:2) who underwent mud-hct and were reported to ebmt. patients were matched for sex, age at transplant (≤40 or >40), disease type (b-all vs. t-all), disease stage (cr1 vs. cr2 vs. other), disease risk (high vs. others), philadelphia chromosome status (positive vs negative), and conditioning regimen (mac vs. ric/nma) in multivariable analysis, os, pfs, nrm, and relapse rate were not statistically different between patients receiving hct from haplo or mud, regardless of the intensity of the conditioning regimen; (table 1). conclusions: in conclusion, in this large retrospective analysis, outcomes of patients with all undergoing transplant from a haploidentical donor with post median follow-up was 14,6 months and was similar in both cohorts (p=0.78). 2-year overall survival (os) was 41.5% for the all patients and did not differ between transplants from a ird or a crd (36% vs 47%, p=0.33; table i). 2-year progression-free survival (pfs) and gvhd/relapse-free survival (grfs) was 39% and 37%, respectively. 1-year non-relapse mortality (nrm) was 22% and was similar between ird and crd (p=0.62, table i). 6-months cumulative incidence of grade 2-4 acute gvhd, grade 3-4 acute gvhd and 1-year moderate-severe chronic gvhd was 23%, 8% and 19%, respectively. again, there was no difference between crd and ird transplants in terms of grade 2-4 acute gvhd and moderate-severe chronic gvhd (table i) conclusions: our results confirm previous findings that a crd haploidentical transplant is a viable option for haplo-sct when a first-degree donor is available. main long-term outcome are not different in this matched pair analysis. extending our analysis to a larger cohort of patients receiving a crd is warranted to confirm our preliminary results. references: 1 disclosure: the authors have no conflict of interest to disclose stem cell mobilization, collection and engineering o173 a recombinant chimeric protein, safely enhances graft performance following hematopoietic stem cell transplantation median follow-up time for survivors was 27 months. results: overall survival (os) and relapse-free survival (rfs) rates at 2 years after transplantation were 49% 37%, respectively. cumulative incidences of non-relapse mortality (nrm) and relapse at 2 years were 21% and 43%, respectively. in multivariable analysis, performance status (ps) over 1 (ps>1 vs ps < =1, hr 2.49, p=0.005) and lymphoma progression at transplantation (cr/cr-u/pr vs others, hr 1.95, p=0.043) showed significant negative impacts on os. cbt was strongly associated with better os compared to unrelated bmt/pbsct (hr 0.34, p=0.003) and comparable to related bmt/pbsct. lymphoma control status at transplantation was significantly associated with relapse (relapse/induction failure vs cr/cr-u/pr, hr 2.61, p=0.017). poor ps over 1 at transplantation (ps>1 vs ps < =1, hr 3.35, p=0.023) and reduced-intensity conditioning (ric) regimen (ric vs myeloablative, hr 3.54, p=0.025) were associated with higher risk of nrm. conclusions: allo-hsct could improve overall survival of patient with mature t-or nk-cell lymphomas, if performed at appropriate timing with good disease control of partial remission (pr) or better. cord blood unit could be a favorable alternative donor source when related donors are not available. on the other hands, ric regimen did not decrease the risk of nrm in allo-hsct for mature t-and nk-cell lymphoma patients in our setting. out study also suggested that major problem of allo-hsct is still a high frequency of relapse after transplantation. better disease control is mandatory to improve the outcomes of allo-hsct for mature t-or nk fabio ciceri 1 , luca vago 1 , katharina fleischhauer 5 unite´de recherche mixte en sante´(umr_s) 938, inserm netherlands, 11 institute of hematology and blood transfusion ptcy is largely adopted as gvhdprophylaxis backbone in haploidentical transplantation. the encouraging results prompted investigations to assess ptcy also in unrelated donor (ud) setting. high-resolution hla-matching contributes to ud-hsct success; however, due to the selective in-vivo deletion of alloreactive t-cells, ptcy could modulate hla-matching impact on ud-hsct. methods: we compared the outcomes of acute leukemia patients receiving 10/10 (n=431) and 9/10 (n=234) hlaallele matched ud-hsct with ptcy gvhd-prophylaxis table 1 illustrates patients' characteristics. the power to detect a 2-years 10% difference grfs between the 2 groups was 83% (alpha 2-sided=5%). results: outcome endpoints at 2 years were not different between ±7%, p=0.5; lfs: 56±5% and 55±7%, p=0.7; os: 63±5% and 60±8%, p=0.9, respectively). the 100-day ci of grade≥2 and grade≥3 agvhd were comparable for 10/10 and 9/10 ud (31±5% and 28±6%, p=0.4 and 10±3% and 9 likewise, the 2-y ci of cgvhd and extensive cgvhd were similar between 10 the 2-y ci of trm was 19±4% after 10/10 and 17±5% after 9/10 ud-hsct (p=0.4). relapse incidence at 2-y was 25±5% for 10/10 and no interaction was found between donor type and additional atg use. variables associated with grfs were active disease (hr 2.1 compared to 1 st cr, p< 10 -5 ) and karnofsky ps≥90% (hr 0.6, p< 10 -5 ). conclusions: in the present series of acute leukemia patients transplanted with ptcy, we report comparable survival with 9/10 and 10/10 hla-matched ud-hsct, across all disease stages, suggesting that this platform could alleviate the detrimental effect of single hla-allele mismatching. these results warrant prospective comparative trials of ptcy versus standard use of atg-based gvhd disease status: cr1 disclosure: nothing to declare o178 myeloablative conditioning for first allogeneic hsct in pediatric all: ftbi or chemotherapy? -an update of the retrospective multicenter ebmt-pdwp study rose-marie hamladji 14 , cristina diaz de heredia 15 , elena skorobogatova 16 czech republic, 4 hôpital robert debré and paris-diderot university methods: this update was done to extend the time of follow-up (fu, date of analysis: 01/oct/2018). to compare outcomes of ftbi vs cht-conditioning, we performed a retrospective ebmt-registry study. children aged 2-18 years (y) after mac for first allo-hsct of bm/ pbsc from msd/ud in cr1/cr2 between 2000-2012 were included. propensity-score-weighting was used to control pretreatment imbalances of observed variables. this statistical method ensured that analyzed groups differed only in the parameter under investigation (here: conditioning) busulfan/cyclophosphamide/ etoposide (bu/cy/eto) was the most frequently applied cht-regimen in cr1 (66 (31%)) and bu/cy in cr2 (68 (32%)). the remaining conditionings bu/ cy (68) or other-chemo (143) with median-fu of 6.2, 5.2 and 5.8 y. in weighted univariate analysis, 5-y-os was 31.1% after other-chemo, 43.5% after bu/cy and 58.8% after ftbi. in weighted cox-model, pts having received other-chemo had a higher risk to experience an event compared to ftbi (hr=2.00, p=< .0001). 5-y-lfs was 25 coxmodel, pts having received bu/cy and other-chemo had a higher risk to experience an event compared to ftbi (hr=2.06, p=.006; hr=2.13, p< .0001). 5-y-nrm (range: 18.3% (bucy) to 21.1% (other-chemo)) did not show significant differences in weighted cox-model. conclusions: this recent study-update ensured a substantial fu. we confirmed the clear superiority of ftbiconditioning compared to cht with regard of lfs and ri for all-pts undergoing allo-hsct in cr2. for pts in cr1 we could not find significant differences between ftbi and cht-conditioning. these retrospective findings are currently re with data monitoring committee (dmc) review after each cohort. part2 will include 177 patients randomized 2:1. results: part1 enrolled 11 patients, median age following these promising results, dmc recommended early continuation to part2 of the phase iii trial. conclusions: the genesis lead-in results demonstrate bl-8040 is a potent mobilizer of hscs, with potential to improve mobilization rates while minimizing mobilizationrelated healthcare costs. clinical trial registry: nct03246529 disclosure: hemda chen -medical director, biolinerx abi vainstin -vp of medical affairs, biolinerx ella sorani-vp of r&d, biolinerx osnat bohana-kashtan-project manager markus y. mapara 1 , john f. tisdale 2 , julie kanter 3 , janet l. kwiatkowski 4,5 , lakshmanan krishnamurti 6 , manfred schmidt 7 , alexandra l. miller 8 , francis j. pierciey jr. 8 background: phlh is a rare, genetic, hyper-inflammatory syndrome driven by high production of interferon (ifn)-γ. emapalumab (ni-0501) is a monoclonal antibody that neutralizes ifn-γ and is developed as a treatment for hlh.methods: this open-label phase 2/3 study (nct01818492, data cut-off is july 20 2017) includes 34 patients ≤18 years old with phlh based on genetic confirmation, family history, or presence of ≥5/8 hlh-2004 diagnostic criteria. patients were treatment-naïve (7) or had failed previous conventional hlh therapies (27) . emapalumab (1 mg/kg intravenously every 3-4 days, increased up to 10 mg/kg based on clinical and laboratory response parameters) was administered with dexamethasone (5-10 mg/m 2 /day with tapering permitted). treatment duration was 8 weeks with possible shortening to a minimum of 4 weeks or extension up to allogeneic hematopoietic stem cell transplantation (hsct). the primary endpoint, overall response rate (orr), was objectively assessed based on normalization or ≥50% improvement in pre-defined criteria: fever, splenomegaly, cytopenias, hyperferritinemia, fibrinogen and/or d-dimer levels, central nervous system (cns) abnormalities, with no sustained worsening of scd25 levels. an exact binomial test at one-sided 0.025 significance level was used to evaluate the null hypothesis that orr be at most 40%. following completion of the study, patients entered an extension phase (nct02069899).results: patients (median age 1.0 yr, range 0.1-13 yr) entered the study with broad spectrum of phlh clinical abnormalities, >30% with cns involvement. mutations in phlh-associated genes were present in 79% of patients. orr was significantly higher than the pre-specified null hypothesis, thus meeting the primary efficacy endpoint (table) . response rates based on investigator's clinical judgement were 70.6% and 70.4% in the two groups.emapalumab was safe and well tolerated. mild to moderate infusion-related reactions occurred in 27% of patients. infection caused by pathogens potentially favored by ifn-γ neutralization occurred in 1 patient (disseminated histoplasmosis, resolved with treatment). no off-target effects were observed. most patients proceeded to hsct (50% of patients received myeloablative conditioning and 50% reduced intensity conditioning) with favorable outcome (engraftment in 86% and 89% of patients, respectively) and 90% of patients receiving hsct were alive at 1 year post transplant.background: asp0113 is a first-in-class, dna-based vaccine designed for the prevention of cytomegalovirus (cmv) infection; it contains two plasmids encoding glycoprotein b (gb) and phosphoprotein 65 (pp65). this study aimed to investigate the efficacy, safety and immunogenicity of asp0113 compared with placebo in allogeneic haematopoietic cell transplant (allo-hct) recipients.methods: this was a randomised, double-blind, placebocontrolled phase 3 study. cmv-seropositive allo-hct recipients received five intramuscular 1 ml doses of 5 mg/ ml asp0113 or placebo (phosphate buffered saline) in a 1:1 ratio on days −14 to −3, 14 to 40, 60±5, 90±10 and 180 ±10, relative to the day of transplant (day 0). plasma cmv viral load was determined through 1 year and analysed at the central laboratory. treatment-emergent adverse events (teaes) were recorded through 30 days after the last randomized treatment injection. immunogenicity was measured by t-cell responses to pp65 and gb-specific antibody levels through 1 year after the first randomized treatment injection. the primary endpoint was the proportion of patients with a composite of all-cause mortality and adjudicated cmv end-organ disease (eod) through 1 year post transplant. secondary endpoints were cmv viraemia rate, adjudicated cmv-specific antiviral therapy (avt) rate, a composite of protocol-defined cmv viraemia and adjudicated cmv-specific avt use, first occurrence of background: haploidentical stem cell transplantation (haplo-sct) with post-transplant cyclophosphamide (pt-cy) represents a potential curative strategy for patients with hodgkin lymphoma (hl) when a matched related or unrelated donor is not available 1 . while bone marrow (bm) was originally the preferred stem cell source, more recently peripheral blood stem cell (pbsc) is more often used. some retrospective studies suggest that the risk to develop acute and chronic graft-versus-host-disease (gvhd) is higher with pbsc than bm, while pbsc may reduce the risk of relapse 2 . here we analyzed the effect of stem cell source in 91 hl patients receiving haplo-sct with pt-cy, with the aim to evaluate if the final outcome is modified by the use of pbsc or bm.methods: from april 2009 to january 2017, 91 consecutive patients with poor prognosis hl received a haplo-sct with pt-cy either from a pbsc (n=38) or bm (n=53). the two cohorts were similar for most characteristics, but the pbsc group had more patients with an unfavorable hematopoietic stem cell transplant comorbidity index (hct-ci) score ≥3 (p=0.002) and had received a non myeloablative conditioning (nmac; p=0.001).results: cumulative incidence of neutrophil>500/ul at day +30 and of platelet >20000/ul at day +60 were 96% (95% ci: 89-98) and 96% (95% ci: 88-99), respectively, with no significant differences between the pbsc and bm cohorts. with a median follow-up of 22.8 months, there was no difference between pbsc and bm graft in terms of cumulative incidence of grade 2-4 acute gvhd (29% vs 21%, p=0.3), grade 3-4 acute gvhd (3% vs 4%, p=0.7) and moderate-severe chronic gvhd (9% vs 7%, p=0.7). this was also confirmed by multivariate analysis. in the whole population, the 2-year overall survival (os), 2-year progression-free survival (pfs) and 1-year gvhd/relapse free survival (grfs) rates were 67%, 58% and 58%, respectively. we observed a trend for improved os (74% vs 62%, p= 0.07) and pfs (62% vs 56%, p= 0.1) for recipients of pbsc relative to bm cells, but pre-transplant disease status was the only significant variable by univariate analysis (table i) . by multivariate analysis, pre-transplant active disease status, transplant from a bm and hct-ci ≥ 3 remained the only independent predictors of adverse outcome in terms of os; pfs and grfs (table i) . nonrelapse mortality was not affected by graft source both by univariate and multivariate analysis, while pre-transplant disease status was the only variable affecting the chance of disease relapse.conclusions: overall these data suggest that pbsc is associated with better outcome, in terms of os, pfs and grfs, relative to bm cells as graft source for patients undergoing haplo-sct with pt-cy. in addition, the risk of acute and chronic gvhd is not increased after pbsc relative to bm graft.references disclosure: the authors have no conflict of interests to disclose o139 consensus crs and neurotoxicity regrading of "juliet": phase ii prospective study of tisagenlecleucel therapy in patients with relapsed/ refractory large b cell lymphoma background: t-cell depletion using ex vivo cd34+ cell selection reduces gvhd risk after allogeneic hct, but delayed immune reconstitution, particularly t-cell reconstitution, has limited improvement in survival. sex steroids negatively impact lymphopoiesis, likely by thymic atrophy, and our preclinical models have shown that androgen blockade with the gnrh agonist leuprolide enhances thymopoiesis (goldberg, ji 2009; velardi, jem 2014) . we hypothesized that peri-hct leuprolide could improve immune reconstitution among recipients of cd34selected hct.methods: this was a phase ii clinical trial of leuprolide in cd34-selected myeloablative allogeneic pbsct for hematologic malignancies in patients aged 18-60 (nct01746849). all participants received conditioning with tbi 1375cgy, thiotepa 10mg/kg, and cyclophosphamide 120mg/kg; antirejection prophylaxis with rabbit atg 5-7.5mg/kg; and palifermin 60mg/kg/d on days -13 to -11 and 0 to +2. patients received a 3-month depot of leuprolide 11.25mg 2-6 weeks before conditioning and a second depot 3 months later. primary endpoint was an absolute cd4+ count >=200 by 6 months post-hct. patients who died, relapsed, or otherwise did not have flow data available at day +180 were excluded from primary endpoint analysis but included in outcome analyses. we excluded flow data after secondary cell infusions (dli, ctls, second hct, cd34+ cell boost) but followed recipients of these interventions for survival analysis. descriptive statistics summarized absolute levels of lymphocyte subset counts at select time intervals. a kruskal-wallis rank sum test compared counts among patients who received leuprolide/palifermin, historical controls who received palifermin alone, and historical controls who received neither. kaplan-meier functions estimated os/ rfs. cumulative incidence functions estimated gvhd/ nrm.results: thirty-two patients received at least one dose of leuprolide. median age was 39 years (range 21-57). twenty-six(82%) had acute leukemia, 3(9%) mds/mpn, and 3(9%) cml. all but one had an 8/8-matched donor. at median follow-up of 24 months among survivors (range 7-61), estimated 2y os was 74%(95%ci 60-91%) and rfs 62%(95%ci 46-83%). ci of trm at 2y was 10%(95%ci 2-24%). ci of grade iii-iv acute gvhd was 3%(95%ci 0.2-14%). of 27 patients with evaluable flow data, 30% achieved a cd4+ count >=200 by 180+/-30 days, not significantly different from historical controls. median lymphocyte counts at 180+/-30 days did not differ significantly among groups (table; figure) .conclusions: this phase ii study did not demonstrate significant quantitative improvement in immune reconstitution after leuprolide with palifermin in recipients of tbibased cd34-selected hct. tcr sequencing to identify possible improvement in t-cell diversity in this cohort is forthcoming. one potential explanation for these results lies in the initial surge in sex steroid levels immediately after background: the prospective, multinational, noninterventional emmos study aimed to document, and describe real-world treatment regimens and disease progression in patients with mm at different stages of the disease.methods: adult patients initiating any new mm therapy between 2010 and 2012 were eligible. a multi-staged patient/site recruitment model was applied to minimize selection bias, and enrolment was stratified by country, region, and practice type. patients' medical/disease features, treatment history and remission status were recorded at baseline, and prospective data on treatment, efficacy and safety were collected electronically every 3 months until 2 years after last enrolment. responses were investigatorassessed. overall findings from emmos were previously reported. here, we are presenting additional analyses focusing on the induction regimens used in the subgroup of patients who proceeded to auto-sct frontline.results: a total of 2358 patients (775 with stem-cell transplant [sct] and 1583 without) were enrolled. patient demographics/baseline characteristics were as expected. of 380 recipients of sct after enrolment, 299 (79%) underwent auto-sct frontline. 90% of the auto-sct patients were aged ≤65 years. among these 299 frontline auto-sct patients, the majority had a single transplant (87%). the most frequent induction regimen was bortezomibthalidomide-dexamethasone (vtd; n=95; 32%), bortezomib-dexamethasone (vd; n=56; 19%), bortezomib-cyclophosphamide-dexamethasone (vcd; n=49; 16%), doxorubicin-bortezomib-dexamethasone (pad; n=26; 9%), and cyclophosphamidedexamethasone-thalidomide (ctd; n=26; 9%). only 1% of patients received a bortezomib-lenalidomidedexamethasone (vrd) induction regimen, while lenalidomide was shown to be the most frequently used agent in lines 2 and 3 at time of relapse. in the vtd subgroup, most patients received 100 mg thalidomide dose during induction. the majority of administration schedule was based on 21 days cycles, while a few other schedules were seen corresponding to 35 days cycles or to delay due to adverse events or other specific reasons. the most prevalent number of vtd cycles was 3 and 4 (42% and 38%, respectively). lower or higher number of cycles was only marginal (6% and 14%, respectively). out of the 95 patients with vtd induction, 39% achieved cr as best response, 28% ncr or vgpr and 16% pr. after auto-sct, the best overall response rates (orr) at any time during frontline therapy were >85% for those patients whose treatment included 24 university medicine greifswald, greifswald, germany background: we explored the effect of dinutuximab beta (ch14.18/cho) on outcome within the hr-nbl1/siopen trial population by comparing an era prior immunotherapy availability.methods: the analysis cohort consists of the immunotherapy population (ip) (2009-2013) and a matched control population (cp) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) . all patients had rapid cojec induction, up to two tvd courses and high-dose chemotherapy (bumel or cem) followed by autologous stem cell reinfusion (hdc/ascr) within 9 months since diagnosis; local control included surgery and local radiotherapy (21gy) followed by maintenance with six cycles of isotretinoin. ip patients had additional five cycles of dinutuximab beta short-term infusions with or without subcutaneous interleukin-2. cp patients had to be part of the hdc randomization. the median time between ascr and initiation of immunotherapy was 109 days in the ip. only patients without progressive disease at this landmark time point were included in the cp. median follow-up was 5.8 years (iqr: 4.2 to 8.2 years) for 844 eligible patients.results: the 5y-efs of the ip (378 patients) was 57% ±3% compared to 42%±2% for the cp (466 patients; p< 0.001). both populations were balanced for sex, age, stage 4, mycn amplification and response prior hdc. multivariate analysis showed an independent higher risk for the cp (p=0.0002, hr 1.573), for cem (p=0.0029; hr 1.431), a response < cr prior to maintenance therapy (p=0.0043, hr 1.494) and for >1 metastatic compartment at diagnosis (p< 0.001, hr 2.665). after adjustment for risk factors, the benefit of immunotherapy was confirmed in bumel-(p=0.0066; hr 1.439) and in cem-treated patients (p=0.0107; hr 2.334).conclusions: results suggest a patient benefit from dinutuximab beta based immunotherapy with or without il2 within the hr-nbl1 trial.clinical trial registry: the trial was registered with clinicaltrials.gov (number nct01704716) and eudract (number 2006-001489-17) . https://www.siopen-r-net.org disclosure: the academic data supported apeiron to obtain the dinutuximab beta product licensure in may 2017 in the european union (ema). siopen and ccri had an agreement in place with apeiron regarding the provision of academic data. ruth ladenstein and holger lode acted as consultants for apeiron on behalf of siopen for the ch14.18/cho development.the other authors declared no conflicts of interest. 19 out of 24 (79%) received serotherapy with alemtuzumab (n=12) or atg (n=6) before viral reactivation. where possible immunosuppression was withdrawn in combination to bcv therapy. viral load was detected in blood by pcr as copies/ml. data on response to bcv was divided into complete response (cr) with undetectable virus in blood and resolution of symptoms, partial response (pr) with at least 1 log drop in viral load after bcv, no response (nr) with no change in the viral load and stationary disease and progressive disease (pd) with evidence of at least a log rise in viral load by pcr or organ disease progression.results: the median viral load at the start of bcv was 5.4 million copies/ml (range: 4142-85 million copies/ml). bcv was used as a first line treatment in 8 cases and as second line in 16 cases after failure of first line therapy (n=8), toxicity (n=5) or both(n=3).13/24 (54%) patients had evidence of viral induced organ disease at time of bcv administration; 11 adv disease (encephalitis, pneumonitis, hepatitis and colitis), 1 ganciclovir resistant cmv retinitis and 1 bk haemorrhagic cystitis. 19/24 (79%) patients achieved either a cr (n=15) or pr (n=4) and 8/13(61%) patients with organ disease achieved a cr.two patients with adv disease and pr received donor derived cytotoxic t lymphocytes to achieve cr. at a median follow-up of 20 months (range: 7-53.8), patients who were in cr or pr did not show any evidence of viral reactivation after bcv discontinuation despite no evidence of immune reconstitution (ir). four patients had evidence of disease progression with significant rise in viral load while on bcv therapy and all 4 died. the patient with ganciclovir resistant cmv attained cr of cmv retinitis.among 22 patients with adv viraemia ± disease; 18 (81%) achieved either cr (n=14) or pr (n=4). nine cases had concomitant bk± cystitis at the time of bcv therapy and all had nr. toxicity was observed in 4/24 cases; renal impairment (n=1), transaminitis (n=1) and diarrhoea (n=2). median cd3, cd4 and cd8 were persistently low both pre-bcv and at the end of treatment; 100 cells/ul vs 290 cells/ul, 40 cells/ul vs 70 cells/ul and 20 cells/ul vs 120 cells/ul; p=0.2, p=0.34, p=0 .5 respectively (figure 1 ). at last follow-up, 15/24 (63%) were alive. of these, viral infection related mortality was 4/24 (17%).conclusions: bcv is an effective and well tolerated treatment in immune compromised patients with adv infection with a response rate of 80%.[[o163 image] 1. background: although the impact of donor graft composition on clinical outcome after hematopoietic stem cell transplantation (hsct) has been studied, little is known about the role of intra-graft γδ t-cell receptor (tcr) repertoire on clinical outcome following hsct. using high-throughput sequencing we sought to analyze the tcr γ-chain (trg) repertoire of γδ t-cells within donor stem cell grafts and address its potential impact on clinical response in the corresponding patients.methods: we analyzed twenty peripheral blood stem cell grafts from matched unrelated donors and classified as cmv-positive/negative. the respective acute myeloid leukemia recipients were followed for disease relapse and acute graft-vs-host disease (agvhd) development post-hsct. γδ t-cells were isolated using magnetic beads and the gdna extracted for next-generation sequencing (immunoseq, adaptive biotechnologies). trg characteristics were assessed using vdjtools, vdjviz, tcr and immunoseq analyzer platforms.results: deep sequencing showed similar median total/ unique reads in all grafts as well as similarly low unique cdr3 trg ratios. grafts presented multiple clonal overrepresentations, with no differences on tcr richness between patient groups. grafts received by the non-background: prognosis of mature t-and nk-cell lymphomas remains poor despite development of novel therapeutic agents. accordingly, these lymphomas are still good candidates of allogenic hematopoietic stem cell transplantation (allo-hsct) to achieve long-lasting remission of the diseases. however, the analysis of transplantations for these lymphomas is scarce, mainly due to the rarity of these lymphomas. hence, we analysed the data of these transplantations operated in 15 different japanese institutions as a multi-institutional joint research and examined factors that affected the outcomes in aims of figuring out better transplant strategies against these lymphomas.methods: a total of 116 patients who received allo-hscts for mature t-and nk-cell lymphomas (ptcl-nos, n=38; nk/t cell lymphoma nasal type, n=22; aitl, n=20; alcl, n=12; eatl, n=8; other lymphomas, n=16) from 1998 to 2016 in 15 institutions were examined. median age at transplantation was 50 (range, 18-69). forty patients received transplantation from related bone marrow transplantation (bmt) / peripheral blood stem cell background: children, adolescents and young adult patients with all with second relapse, relapse after allogeneic sct or patients with primary refractory disease have a poor prognosis with conventional treatment concepts. in this patient group several studies using second generation cd19 chimeric antigen receptor t-cells (car-t cells) demonstrated high efficacy with two year survival rates of up to 65%. recently, two different car-t cell products were approved by the fda and in 2018 also by the ema in europe: tisagenlecleucel (kymriah®) for the treatment of patients with b-precursor all who are i) refractory, ii) in second relapse or iii) who relapsed after allogeneic sct (relapsed/refractory all; r/r all) as well as for diffuse large cell lymphoma (dlbcl) and axicabtagen ciloleucel (yescarta®) for the treatment of b-cell lymphoma. here we report our first results using commercially available car-t-cell product tisagenlecleucel (kymriah®) in patients with all which were treated by the university hospital for children and adolescents frankfurt am main (n=9), the department of medicine iii, university hospital lmu munich (n=1), and the von hauner kinderspital, lmu munich, germany (n=1).methods: between october and december 2018 eleven patients received apheresis for car-t cell generation. nine patients suffered from r/r c-all, and two from r/r bprecursor all. eight patients had relapsed after allogeneic hsct, one patient each suffered from first r/relapse, second r/relapse or from primary r/all. in 9/11 (82%) patients car-t cell production was successful after one and in 2 patients (18%) after a second apheresis.median patients' age was 16.7 years (1.1-25.4 ). between apheresis and start of lymphodepleting chemotherapy (ldc), 10/11 patients received low dose chemotherapy according to the frapostall protocol (willasch et al. 2016 ) and one patient was treated with inotuzumab. production slots were immediately available, resulting in turn-around-time from apheresis to product delivery of 3-4 weeks. disease status at start of ldc was cr w/o mrd (n=4), cr mrd pos. (n=2), cri (n=1), persistence of blasts (n=2), and disease progression (80-100% blasts, n=2). ldc consisting of flu-cyc was given to 9/10 patients, one patient did not receive ldc.results: car-t cells could be transfused to 10/11 patients at a median dose of 1.5 mio/kgbw (0.145 mio 8.5). in one patient, in whom a second viral transduction procedure was necessary; neither ldc nor car-t cell transfusion could be given because of diseases progression and deterioration of the patient's general condition. cytokine release syndrome (crs) grade i was observed in one patient; 8/10 patients did not develop crs. cytokine related encephalopathy syndrome (cres) grade ii was observed in 1/10 patients. at day +28 8/10 patients (80%) achieved mrd negative cr. the two patients with key: cord-023055-ntbvmssh authors: nan title: immunogenicity date: 2004-02-19 journal: j cell biochem doi: 10.1002/jcb.240410506 sha: doc_id: 23055 cord_uid: ntbvmssh nan ia moyecules with respect to their roles as peptide receptors and target structures for tcr interaction. particular attention has been paid to distinguishing between local and distant effects of amino acid substitutions on ia function and to determining which residues interact with peptide antigen and which (if any) with the tcr. this ex erimental approach has led to the identification of several regions of the pofvorphic amino-terminal domains of the a and p chains as playing critical roles in chain-chain association and quaternary ia conformation. the a1 and p l putative helical regions have been found to have distinct degrees of structural lability, with the a1 helix showing much greater susceptibility to conformational change due to allelic variation in other re ions of the molecule. allelically polymor hic residues in the a1 and p 1 domainstave been shown to play important roles in &e activity of the assembly/folding control regions, and hence, analysis of local binding roles of specific residues in ia molecules must take this additional effect of substitutions at these positions into account. by controlling for large scale conformational effects, individual residues in the p chain have been assigned to desetopic ( eptide interaction) and histotopic (tcr interaction) roles. in the cytochrome c molel, a putative peptide bindin "pocket" involvin residues from both the postulated p l a helix and also the p-stran% floor has been defined, residues controlling both the extent of binding and the orientation of the bound peptide have been identified, and at least one residue with tcr interaction potential without obvious peptide binding properties has been localized. combining these data with those of other investigators leads us to propose a general model of class i1 mhc structure-function relationships. we have shown previously that memory b cells transferred into k-allotype distinct congenic rats in the absence of any priming antigen are deleted from the adoptive host within a matter of weeks (half-life of 1-2 weeks). in contrast co-injection of antigen with the cells facilitates their survival and the maintenance of a donor response for periods in excess of one year. in the experiments reported here we ask if the persistence of t cell memory is also dependent on antigen. 10. carrier (klh) primed t cells were transferred in the presence or absence of antigen into irradiated, k-allotype distinct adoptive host. a t various times after transfer these rats were injected with 2x10' hapten-carrier (dnp-klh) primed b cells together with 50 ig of soluble dnp-klh. this limiting number of b cells makes a secondary type response only if carrier-specific memory t cells survive in the adoptive host. we found that already at 6 weeks following transfer without antigen, no memory t cell help was available for these b cells. in contrast t cells transferred together with 10 ~g klh provided help for secondary type donor responses at 6 and 12 weeks after transfer. we conclude that longterm memory at both the t and b cell levels does not reside with small, very long-lived, resting cells but. with active clones that are maintained by small amounts of antigen that may persist for long periods. once antigen is lost from lymphoid tissues both t and b cell memory wanes within a relatively short time. t cells recognize antigen in the form of short peptides associated to class i or class i1 mhc molecules. each mhc molecule has the ability to bind a large number of peptides and peptides with unrelated sequences can compete for binding to the same mhc molecule, as well as in vitro. in vivo competition strictly correlates with the capacity of the competitor peptide to bind to the mhc molecule presenting the antigenic peptide and its extent dependes on the molar ratio between antigen and competitor. competition among different peptides derived by processing of hen egg-white lysozyme (hel) appears to exert a major influence on the immunodominance of antigenic determinants recognized by t cells. thus, the h l peptides 1-18 and 25-43 are both generated by hel processing and are both able to bind to the i-e molecule but only 1-18 becomes immunodominant because it has th ability to compete in vivq with other hel peptides, such as 25-43, for the available sites on the i-e molecule. however, two immunodominant t cell epitopes, such as those in hel peptides 51-66 and 112-129, both interacting with i-ak molecules, do not compete with each other when injected together at equimolar concentrations. such a coexistance is anticipated between peptides that bind with relatively high affinity to the presenting molecule and thus have both the chance to occupy a number of binding sites sufficient for t cell activation. r v s e iii xen ic tr lantation. in v i m lnvesti$ation uslng mocloml a n t m i e s r e 4 e -t e x e y skin gracs-val on m i c e w a s significantl pr:lrd l g anti-antihdy trea-t but n o t a b anti-antibody: w i d e r the saue animals but a n t i c d 4 antibody did prolong minor a n t w -d i allqrdts. in v i m studies r m that primary proliferation a n f z . 2 prcdwtion & -f i cells in response to mmkey stimulators was weak conpared to allogeneic reqonses. secondary responses t o xenogeneic stinulation were strong after in v i m priming but required the presence of responder nc's. assays for c y g t s t cell effectors in m i c e which had rejected monkey skin revealed few such cells. zhese results est that widely d i a t e xencgeneic processing and presentation, since xenogeneic antigens require that such presentation be in association with the.= antigens of regponder apc's, the xenogeneic r a f t s have a functional similarity t o aff2 leted allografts. shoved t h a t f e t a l r e n a l and f e t a l and p o s t n a t a l testis a l l o g r a f t s survived longer than corresponding a d u l t t i s s u e i n non-immunosuppressed outbred r a t hosts. the c u r r e n t study a s k s v h e t h e r t h e d i f f e r e n c e i n s u r v i v a l betveen r e n a l and t e s t i c u l a r g r a f t s and between g r a f t s of d i f f e r e n t ages is r e l a t e d t o d i f f e r e n t i a l t i s s u e expression of class i and class i1 mrna t r a n s c r i p t s or s u r f a c e antigens. and i f t h e s e p a t t e r n s change w i t h t r a n s p l a n t a t i o n . congeneic mice w e found t h a t prolonged s u r v i v a l of c57bl/6 f e t a l r e n a l (n=42; p<0.008) and f e t a l (n=14; ~( 0 . 0 5 ) and p o s t n a t a l (n= 8; ~( 0 . 0 5 ) t e s t i s mouse a l l o g r a f t s t r a n s p l a n t e d beneath t h e r e n a l c a p s u l e of a d u l t r e c i p i e n t bio.a mice and t h i s s u r v i v a l c o r r e l a t e s i n v e r s e l y w i t h t h e expression of class i and class i1 mrna (northern a n a l y s i s ) and p r o t e i n s (immunohistochemistryy) and t h a t both p r o t e i n and mrna increased throughout ontogeny f o r both t h e testis and kidney. after t r a n s p l a n t a t i o n t h e r e vas a marked i n d u c t i o n of mhc mrna t r a n s c r i p t s f o r both testis (n=207) and kidney (n=320). implanted f e t a l kidney t i s s u e t h a t survives. however. f a i l e d t o express d e t e c t a b l e mhc p r o t e i n , i n d i c a t i n g t h a t some p o s t -t r a n s c r i p t i o n a l modification i n t h i s t i s s u e occurs. t o a f f o r d it p r o t e c t i o n from r e j e c t i o n . implanted testis shoved i n d u c t i o n of both mrna and p r o t e i n v e l l above i t s much lower baseline. i n d i c a t i n g t h a t i t s r e g u l a t i o n , i n c o n t r a s t t o t h e kidney may be t r a n s c r i p t i o n a l . thus t h e f e t u s may lower t h e mhc burden as a s t r a t e g y t o escape r e j e c t i o n e i t h e r by p o s t t r a n s c r i p t i o n a l modification of p r o t e i n expression a s i n t h e kidney or by t r a n s c r i p t i o n a l modification of mrna as i n t h e testis. culture of thymus tissue in 2-deoxyguanosine (2dgua) is thought to reduce tissue immunogenicity by selectively depleting highly immunogenic, thymic immigrants of bone marrow origin. in the mouse 2dgua treated thymus tissue survival is markedly enhanced compared to untreated tissue when transplanted under the kidney capsule of allogeneic recipients. these experiments were repeated in the rat. as expected, strain da neonatal thymus tissue was rejected when transplanted under the kidney capsule of normal allogeneic strain pvg rats. surprisingly. acute rejection occurred even when the tissue was cultured for 14 days in 4 mm pdgua (3x the effective dose in mice). by in vitro criteria this dose was very effective in destroying thymocytes. to test whether residual marrow derived cells that escaped pdgua treatment were responsible for inducing rejection we "parked" the 2dgua treated da tissue in t cell depleted pvg rats. our working hypothesis was that the few remaining donor derived cells of marrow origin would be overgrown by host type cells. when pdgua-treated da thymus tissue was transplanted into t cell depleted pvg recipients graft rejection did not occur. however da pdgua treated thymus tissue, parked for as long as 200 days in t cell depleted pvg rats, was acutely rejected when retransplanted into normal pvg recipients. we interpret these results to suggest that rat thymic epithelium devoid of marrow derived cells is innately immunogenic. c 104 corinne amiel, violaine gugrin, thierry may, philippe canton, gilbert c faure, laboratoire d'immunologie and maladies infectieuses, chu de nancy, facult6 de mgdecine, 54500 vandoeuvre les nancy, france. lfal is a dimeric membrane molecule composed of a specific alpha chain (cdlla) and a beta chain (cd18) common to three members of the lfa family. lfal is physiologically expressed on all white blood cells, while other molecules of the lfa family (with cdllb and cdllc alpha chains) are restricted to cells of myeloid lineage. a defective expression of lfal has been described in some congenital immune deficiency and in aids. we investigated the lfal defect on peripheral blood lymphocytes from 100 hiv+ patients. three different monoclonal antibodies were used, respectively directed to chain-specific epitopes of cdlla (spvl7, sanbio) and cd18 (iot18, immunotech) and to a conformational epitope involving both chains (iot16, immunotech). cell suspensions were stained in indirect immunofluorescence and a flow cytometer (epics profile, coultronics) was used to assess the percentages of stained cell, the fluorescence intensity and the shape of fluorescent peaks. our data suggest that lfal expression is impaired in hiv+ patients both through the quantitative expression of each chain and through conformational alterations. the adhesion molecule lfa-1 is known to be important in antigen presentation. we have previously shown that both monocyte and t cell lfa-1 play a role in the interaction between these two cells (eji d; 943, 1987) . antibody to icam-1 (known to act as a ligand for lfa-1) also inhibits antigen presentation, although icam-1 is not thought to be expressed on resting t cells (eji 18: 35, 1988) . we have looked at the expression of icam-1 on t cells after incubation with 12 cytokines and found that only il-2 consistently effects an increase in both the percentage of icam-1 positive cells and in the level of expression. in addition we have found that a proportion of resting t cells express very low levels of icam-1. double labelling experiments have shown that these cells are part of the memory t cell population as defined by antibodies to uchli, lfa-3 and lfa-1, and furthermore that icam-1 negative cells are unable to respond to to antigens such as ppd and flu but are able to respond to pha. this suggests that icam-1 represents an additional marker on the memory t cell population which more precisely defines the subset able to respond to recall antigens icam-1 expression on t cells, anne-marie buckle and nancy hogg, macrophage lab. icrf, lincolns inn fields, london, wc2a 3px, u.k. immunization, francis r. carbone and michael j. bevan, department of immunology, research institute of scripps clinic, la jolla, ca 92037. ctl recognize peptide forms of processed, foreign antigens in association with class i molecules of the mhc and are usually directed against endogenously synthesized "cellular antigens" such as those expressed by virusinfected cells. in vifro studies have shown that small exogenous peptides can directly associate with class i molecules on the cell surface and mimic the target complex derived by intracellular processing and presentation. we have recently generated ova-specific, h-2kb-restricted ctl by immunizing c57bl/6 mice with a syngeneic tumor line transfected with the ova cdna. the ctl recognize the ova transfectant eg7-ova and the synthetic peptide ova but fail to recognize the native protein. we reasoned that given the potential for direct peptide/class ?'&$%ation observed in vifro, ova2s,-2ra may induce ctl after in vivo priming. however, we found that this is not the case. ova,,,-,,, and peptides of increasing lengths up to which are all able to form the target complex in vitro, are inefficient at priming ~% -%~~ specific ?h%sponses following intravenous injection. this is also true for both native and denatured ova. in contrast to these results, the synthetic peptide ova22g:z76 corresponding to a peptide in a partial tryptic digestion of ova can efficiently prime c57bl/6 mice in vrvo following intravenous injection. this peptide elicits ctl which appear identical to those derived from animals immunized with syngeneic cells producing ova endogenously. it is now well established that human t lymphocytes can be activated via the t cell specific cd2 antigen. in order to determine if a factor@) other than the single cd2 polypeptide is involved in cd2 mediated signal transduction, we have stamy transfected murine l cells with the human cd2 cdna. we report that such transfectants expressed hah levels of cd2 at the cell surface. formed sheep erythrocyte rosettes and expressed the three cd2 epitqm previously defined on human t lymphocytes, including the "activation associated' t i 13 epitope. the latter observation unequivocally demonstrating that expression of the ti13 epitope, in contrast to a previous report, is entirely independent of t cell specific factors. combinations of cd2 mabs that are potent stimulators of human t cells. however, failed to elicit either an increase in the concentration of intracellular free calcium or augment [3h]-thymidine inmrporatbn in the transfectants. these results provide both formal identification of the cd2 cdna and dearly demonstrate that the single cd2 polypeptide expressed in an heterokgous cell system devoid of t cell specific factors, cannot alone transduce intracellular signals in response to stimulatory combinations of cd2 mabs. the results are therefore consistent with the notion. that the functional cd2 antigen expressed in human t lymphocytes, requlres the association of another, as yet, undefined factor@). this conclusion was based on several lines of evid nce incl ding the observation that mabs specific for the class i a3 domain of either h-zl8 or l 2 d b interfered with t e generation of cd8-dependent (low substitution at position 227 in the a3 domain are not lysed by cd8-dependent primary ctl but are lysed by secondary cd8-independent (high affinity) ctl generated in the presence of antibody to the a3 domain. populations of ctl. we have isolated and characterized a c d w , cd4-da-specific cpl line. this line is cd8-independent and is capable of lysing the addition, we are currently generating clones from primary $-specific ctl cultures to obtain cd8-dependent (low affinity) ctl. directed rnutagenesis are being tested with the cd8-dependent and cd8-independent clones to define additional residues important for cd8 recognition. the comparison of ctl clones with different cd8 dependencies will allow us to more precisely define the role of cd8 in t cell recognition. percolle from the buffy coat of one unit ot blood. these cells (=400x10 ) are introduced into a curame 5000 elutriation centrifuge (rotor speed of 3000 rpm; loading flow of 10 ml/min). nine fractions can be obtained. the first three containing >90% lymphocytes; fraction 4 (3000 rpm-18 ml/min) and fraction 5 (2900 rpm-18 ml/min! contain both lymphocytes and monocytes and the next three fractions contain >90% monocytes; the finat fraction (rotor off) contains monocytes + granulocytes. cells from each fraction (5x10 /well) are incubatee for five days with tetanus toxoid (1.5lf/well) and an enriched population of t cells (5x10 /well). quadriplicate samples are then pulsed for 16 hours with 'h methyl thymidine. maximum apc activity is found in fractions 4 and 5 representing 4 to 7% of the mononuclear cells. apc activity for these two fractions can be further purified by selective absorption of the cells onto gelatin coated surfaces that have been preincubated with plasma. the non adherent lymphocytes are rgmoved after two hours. after overnight incubation spontaneously released cells (1-5x10 ) can be harvested which have a higher apc activity than cells rotated by elutriation alone. these methods are now highly reproducible in our laboratory, so we can now begin to characterize and study these cells. the male s p e c i f i c h-y a n t i g e n h a s been shown t o behave as a minor histocompatibility a n t i g e n in man and mouse. i n t r a n s p l a n t a t i o n , male t i s s u e may t r i g g e r t h e c l o n a l expansion of h-y reactive hhc r e s t r i c t e d effector cells of female o r i g i n . although male epidermal cells (ec) can induce an anti-h-y t cell response in female mice, so far in v i t r o techniques have f a i l e d t o i d e n t i f y t h e cell-defined h-y a n t i g e n on murine ec (1). here w e developed a 51cr release assay t o use human c u l t u r e d k e r a t i n o c y t e s (k) as t a r g e t cells for hla-a2 specific and ma-a2 r e s t r i c t e d h-y s p e c i f i c t cell clones. hla-a2+ but n o t h l a -a t k were l y s e d by anti-ma-a2 ctls i n a dose dependent manner. low but d e t e c t a b l e l e v e l s of anti-h-y k i l l i n g were found a g a i n s t ma-a2+ male k b u t n o t a g a i n s t h l a -a t male or hla-a2+ female k. both l e v e l s of a l l o r e a c t i v e and h-y s p e c i f i c l y s i s were d r a m a t i c a l l y enhanced after exposure of k t o ifn gamma. these r e s u l t s s t r o n g l y suggest t h a t h m a n male s k i n cells are d i r e c t l y s u s c e p t i b l e for h-y d i r e c t e d t c e l l k i l l i n g through t h e expression of f u n c t i o n a l h-y/hla complexes on t h e i r cell s u r f a c e . i n view of t h e s e f i n d i n g s , t o g e t h e r w i t h our r e c e n t s t u d i e s on t h e expression of h-y ctl determinants on h m a n hematopoietic p r o g e n i t o r c e l l s (21, t h e r o l e of h-y a s a t a r g e t s t r u c t u r e f o r c e l l mediated immunity i n o l i n i c a l t r a n s p l a n t a t i o n should be s e r i o u s l y taken i n t o account. 1. steinmuller d. and burlingham w.j. t r a n s p l a n t a t i o n 19@4,37,1,22. 2. voogt p.j., goulmy e., fibbe w.e., e t a l . j . clin. invest. sept.1988. c 116 diphteria toxoid (dt) presentation by hla dr7 transfected murine fibroblasts bismuth, laboratory of c e l l u l a r and t i s s u l a r immunology, chu p i t i e s a l p b t r i b r e , p a r i s , france and veterans medical c e n t e r , iowa c i t y , usa. l t r a n s f e c t a n t s e x p r e s s i n g s i n g l e type of human mhc c l a s s i1 molecules produced by dna conjugate formation has been studied with cloned t cell lines and a b cell hybridoma and with t cells and b cells from normal mice. resting t cells and b cells do not form appreciable numbers of conjugates but conjugates are formed between t cells stimulated with alloantigen for four days and b cells activated by 24 hour culture with lps. irrelevant lymphocytes do not affect the rate of specific conjugate formation in suspensions of cells agitated by gentle rocking but impair conjugate formation when cells are allowed to settle in round bottom tubes. in further experiments, it was shown that the conditions for the induction of lymphokine secretion by the t cell were not indentical to the conditions for conjugate formation.the significance of these and other observations for the interaction of t cells and b cells in vivo will be discussed. of the primary mixed leukocyte reaction (mlr) and that this reaction occurs in multicellular dendritic cell-cd4+ t cell clusters [cellular immunology 111, 183-195(1988) dendritic cells are able to contact, cluster, and retain allogeneic t cells and induce these alloreactive cells to proliferate and divide. tions labeled with a vital flvorescent dye, we show that only dendritic cells efficiently form stable clusters. labeled monocytes and b cells do not form clusters with t cells. when labeled monocytes and unlabeled dendritic cells are used to stimulate t cells, unlabeled clusters form. labeled monocytes do not move into the clusters until the third day of the mlr. significant levels of il-2 and a-ifn appear in the culture supernatant by the first or second day. blast transformation by the second day of the mlr as demonstrated by giemsa staining of cluster cytopreps. also been studied by immunoperoxidase staining. it is known that human peripheral blood dendritic cells are potent stimulators using purified dendritic cell populathe distribution of certain adhesion molecules within clusters has c1m microbiology and immunology, emory university, atlanta, ga 30322. immunization of sjl/j mice with myelin basic protein (mbp) induces the t cell-mediated autoimmune central nervous system disease, experimental allergic encephalomyelitis. response against a dominant epitope (residues 89-101) leads to disease. lymph node t cells from mbp-immune mice react against several epitopes in addition to 89-101 indicating that the i-as molecule is able to form immunogenic complexes with several mbp peptides. the question asked in these studies was whether subdominant epitopes from the same molecule would compete with the dominant epitope for binding sites on the i-as molecule. to address this question two t cell clones, one specific for 89-101 (sp4.2) and a second specific for a second epitope present in peptide 89-170 (sp4.7) were tested for responsiveness when cultured with the dominant epitope alone or with mixtures of peptides containing dominant and subdominant epitopes. reactivity of sp4.2 against peptide 89-101 was inhibited by peptides 1-37 and 43-88. reactivity of sp4.7 against peptide 89-170 was not inhibited by peptide 89-101 although peptides 1-37 and 43-88 were inhibitory. controls indicated that inhibitory reactivity was not due to toxicity at high concentrations of peptides. these findings imply that subdominant epitopes are able to compete with dominant epitopes of mbp for binding sites on i-as molecules. linda r. gooding, frances c . rawle, david i . kusher, w i l l i a m s . m. wold+ and barbara knowles*. department of microbiology and immunology, emory university school of medicine, atlanta, ga 30322, 'institute f o r molecular virology, s t . louis university school of medicine, s t . louis, mo 63110 and *the wistar i n s t i t u t e , philadelphia, pa 19104. i n several v i r u s systems e a r l y non-structural proteins localized predominantly i n the nucleus of infected cells are major t a r g e t antigens f o r cytotoxic t lymphocytes (ctl). whether early synthesis o r nuclear l o c a l i z a t i o n are important factors i n immunodominance is not known. w e have recently developed a system f o r studying the ctl response t o human group c adenoviruses i n mice. by us ng both transfected t a r g e t s and virus deletion mutants w e have shown t h a t , response t o wild type ad5. there are two e1a t r a n s c r i p t s , 12s and 1 3 s . which both encode major e a r l y nuclear antigens d i f f e r i n g by a 46 amino a c i d insertion: both antigens are recognized equally w e l l by ctl. the e3 encoded 19k glycoprotein (gpl9k) of ad5 binds t o mhc c l a s s i antigens i n the endoplasmic reticulum preventing t h e i r translocation t o the c e l l surface and strongly inhibiting l y s i s by ad5 specific ctl. however, the presence of gpl9k i n the priming v i r u s does not a f f e c t the s p e c i f i c i t y of the ctl generated f o r e l a , so the immunodominance of t h i s protein cannot be due t o the fact t h a t i t is the only major protein synthesized before gpl9k i n the course of infection. using virus deletion mutants we are investigating whether ctl s p e c i f i c f o r other ad5 antigens can be induced i n the absence of ela, and whether e1a is also the dominant antigen recognized i n mice of other mhc haplotypes. respond to antigens present on non-replicative virions. in contrast, we have obtained balc/c i-erestricted t hybridomas specific for the neuraminidase (na) glycoprotein of a/pr8 influenza which recognize infectious, but not non-replicative virus, closely resembling recognition requirements observed for most class i mhc-restricted responses to influenza. recognition correlated with the rte nova synthesis of viral na within antigen-presenting cells, but did not depend strictly upon the amount of na present in cultures, since high na concentrations could be achieved by addition of non-replicative virus without being stimulatory for na-specific t cells. recognition of a neo-antigen was ruled out, since, in high concentration, na isolated from purified virions, even if reduced and alkylated, was recognized by the t hybridoma clone. isolated na was recognized when added to pre-fixed apc, suggesting that this form of antigen was able to bypass the usual processing pathway of exogenous proteins. this suggests that endogenously-synthesized antigen may use different pathways to achieve class 11-associated presentation. t lymphocyte activation is a complex event which is influenced by a variety of distinct cell surface molecules. in order to determine the role of individual molecules in the activation process, we have developed an efficient methodology for generating cell variants in which expression of molecules is selectively inhibited by expression of anti-sense rna from an epstein-barr virus episomal replicon. in a previous study, we reported that marked inhibition of cd8 cell surface expression could be achieved in a human t cell clone using this approach. we have now extended this strategy to another t cell surface molecule, cd2, as a first ste towards ascertaining its role in t cell activation. to this end, we s nthesized a &-mer oli onucleotide corresponding to a sequence in. the 5: end of the c d i n g re ion of human cd'i and inserted it in an anti-sense orientation into this replicon. this a-c%2/rep3 construct was electroporated into jurkat cells. analysis of stable a-cd2irep3 transfectants by immunofluorescence staining and flow cytometry demonstrated complete and selective inhibition of cd2 expression. in contrast to the nontransfected arent, this cd2-variant demonstrated a partial loss in its ability to form conjugates a n 8 to secrete interleukin 2 when stimulated with anti-cd2 monoclonal antibodies. however, stimulation of the cd2-variant with a23187 and pma did result in interleukin 2 secretion. several observations suggest that cd8 functions not only as an adhesion molecule recognising mhc class i on the adjacent cells but also potentiate the transducting capacity of the tcr/cdg complex. comparison of the mouse ly 2 protein sequence with the homolog rat ox8 and human t8 sequences revealed most highly conserved regions in the membrane and cytoplasmic part of the molecule. the conservation of the transmembrane and cytoplasmic sequences in different species may be significant for the function of the cd8 molecule. in order to initiate the functional dissection of the cd8-molecule we constructed mutations in different parts of the molecule. by transfecting the a and b chain genes donated by a cd8 dependent cytotoxic t cell clone(kb5 c20) into the mhc class i1 restricted agd cd4 t cell hybridoma do-11.10 we were able to reconstitute the ability to respond to k only if the transfer was done with the ly 2 molecules (gabert et al., 1987. cell, 50. 545-554) . in this system surface expression of mutated and non mutated ly-2 molecules were checked by facs-analysis and the molecuar size of the proteins were analysed by immunoprecipitation with the anti-ly-2 monoclonal antibody 19/lj8. finally functional effects of the mutations were investigated in response towards the k alloantigen. we have simulated graft versus host and host versus graft reactivity in vitro by studying primary anti-minor h responses in a limiting dilution culture system. the ability of bmm and peripheral blood mononuclear cells (pbm) to stimulate and respond in this system were compared by estimating the number of proliferating cells. in gvh-direction the combination of donor-bmm (d-beim) and host-pbm (h-pbm) was 2 to 15 times more effective in stimulating proliferation than any other combination; the same applied to the combination h-pbm/d-bmm in hvg-direction.-using these combinations the median frequency of proliferating cells in gvh-direction was 1/5300 (range 1/59c-1/18100) in 10 pairs, in hvg-direction (7 pairs) 1/2330 (range 1/115-1/6100). 85% of the proliferating cells had the phenotype of mature t-cells.-using the same combination of responder/stimulator cells we have also estimated the number of cytotoxic cells specific for the hla-identical target cell. in gvh-direction the median estimate (n=8) was 1/10300 (range 1/66o-1/45700), in hvg-direction (n=5) 1/2250 (range 1/400-1/6500). by split well analysis similar or higher frequencies of cytotoxic cells with specificity for nk-targets were detected (gvhr: 1/5750, hvgr: 1/3620). it was however possible to identify a significant number of minor h-specific clones by segregation analysis; their specificity could be confirmed after clonal expansion. the clones had the phenotype of typical cytotoxic t-cells.-the relevance of the two cytotoxic subpopulations described above to clinical events such as gvhd, graft rejection and relapse needs to be clarified.molecular cloning of murine icah-1, k.j. horley, b. baker, and f. takei, terry pathology, university of british columbia, vancouver, b.c., canada. we have previously reported a novel cell surface antigen expressed on activated and proliferating murine lymphocytes. the antigen, termed hala-2, is absent or present at low densities on thymocytes, lymph node cells, and fibroblast cell lines, indicating it is not a universal proliferation antigen. some cells of the spleen and bone marrow express mala-2 at a high density possibly representing in vivo proliferation in these tissues. apparent molecular weight of 95-100 kd under both reducing and nonreducing conditions, and is susceptible to endo f digestion. the monoclonal antibody yn1/1.7 that reacts with this antigen, profoundly inhibits mlr. a xgtlo cdna library was constructed from ns-1 cells that express a high level of mala-2, and screened with synthetic oligonucleotides resulting in the isolation of a full length cdna clone (-3.2 kb). the cdna sequence has high homology with the human icau-1 sequence, indicating that hala-2 may be the murine homologue of this characterized protein. hines, trudeau i n s t i t u t e , inc., p.o. box 59, saranac lake, ny 12983 a tumor c e l l l i n e , et-5, has been derived from an apparent fibrosarcoma t h a t arose i n a c57bl/6 male mouse. antigens. mice t h a t have r e j e c t e d et-5 become imnune t o these minor h antigens, judged by accelerated s k i n g r a f t r e j e c t i o n , and t h i s imnunity can be t r a n s f e r r e d t o imnunod e f i c i e n t mice w i t h lymphoid c e l l s . however, spleen c e l l s from mice t h a t have r e j e c t e d according to the widely accepted view, cd2 (t11, sheep erythrocyte receptor) is the first t cell-specific antigen to appear on differentiating thymocytes during ontogeny. it follows that cd2 should be expressed on all immature and mature t cells. using two-color cytofluorometry i have here identified subsets of cd2-cd3+ t cells both in fetal human thymus or spleen and in adult peripheral blood. cd2-cd3+ t cells constitute 1-25% of fetal thymocytes and 0.1-0.8% of peripheral blood t cells. il-2-dependent longterm clones of cdi-cdj+ cells do not react with a panel of monoclonal antibodies (mab) directed against the t1ll, tlll or t113 epitopes of cd2 and do not transcribe cd2 mrna. fetal tissue-derived clones react with the tigammaa mab and thus express a functional tcr gamma chain, while cd2-cd3+ clones from peripheral blood are bha031+ and express a full-length 1.3 kb tcr c s .ria. the clones established here are currently being characterized with respect to functional capacities. i conclude that expression of cd2 is not an absolute prerequisite for the expression of the cd3/tcr molecular complex on human t cells. if they are added after 24 hours. these interactions are bidirectional. since both cdlla and cd18. and t h e i r ligand i-cam 1. are expressed on the presenting c e l l s as well as the t cells. however, a l l such e a r l y adhesion related events are not bidirectional since anti-cdz and anti-lfa-3. which are expressed d i f f e r e n t i a l l y on t c e l l s and presenting c e l l s respectively are also effective as inhibitors. antibodies, a n t i cd4 and a n t i cd25 antibodies do not i n h i b i t clustering but do i n h i b i t p r o l i f e r a t i o n , and t h i s i s seen irrespective o f when the antibodies are added i n t o the assay. our findings suggest t h a t there are two mechanisms involved i n dendritic c e l l -t c e l l interaction, f i r s t l y an inrnediate cell-cell adhesion step and l a t e r a secondary signal transduction process possibly mediated v i a cytokines. the q u a l i t a t i v e d i fferences between dendritic c e l l and b c e l l induced i m n o g e n l c i t y may thus l i e i n e i t h e r o f these two steps. king, department o f eathology, the bland-sutton i n s t i t u t e . university college and middlesex school i n contrast, a n t i class i1 m c lmmunogenicity c130 cultvred tissue is capable of stimulating an rggwwse when l " s p l m l e d ~e n e i c w y , robert j. ketchum and orion d. hegre, dept. of cell biology and neumanatoay, university of uinnesota, minneapolis hn 55455. neonatal rat islets derived by culture-isolation have teen shown to k free of mlc class 11+ cells, and are immunologically silent when transplanted to either syngeneic or allogeneic hosts. allogeneic transplantation of cultured neonatal non-islet pancreatic tissue, which is known to contain class 11+ cells, results in rapid allograft rejection. unexpectedly, m i c transplantation of cultured non-islet ductal tissue also resulted in lononuclear lm,me cell (hnc) infiltration of the graft in 86% of grafts examined. highly purified syngeneic islets and ductal elements grafted syngeneically at remote sites display an i u n e response in the ductal element graft, while the islet graft is free of any imnme cell infiltrate. this syngeneic imune response does not result from the use of xenogeneic serum in the medium, since cultures carried out using syngeneic rat serum supplemented medium yielded identical results. uncultured neonatal pancreatic tissue grafted syngeneically does not result in iqk: infiltrate, thi6 i.rmne response to a syngeneic stimulus correlates with the presence of class 11+ (antigen presenting) cells. in grafts free of class 11+ cells (culture-isolated islet grafts) no i.rmne responrrc to syngeneic stimulus was observed, while a response was present when syngeneic ductal elements, known to include class 11+ cells, were grafted. this indicates a need for cells capable of antigen presentation to stimulate this syngeneic rerrponne, and suggests that either a modified self antigen or a nomally sequestered antigen is being presented. this syngeneic imune response demonstrates many of the same characteristics of, and may be analagous to, the in vitro syngeneic, or autologous, mixed lymphocyte reactions. indicating this response is not to developnental antigen. c 132 the presence of "self" mhc class i1 (ma-dr) antigens determines whether blood transfusions ihmunise or suppress. el lagaaij, a termijtelen. e goulmy, & jj van rood, leiden university hospital, the netherlands. blood transfusions can immunise the recipient, as well as induce prolonged allograft survival. it is not known what makes that some transfusions inrrmnise the recipient whereas others induce immune suppression. we investigated if certain mhc compatibilities or differences between recipient and transfusion donor and organ donor are required to induce the beneficial "transfusion effect" in man. we studied graft survival and blood transfusion induced changes in cellular and humoral immunity in 4 different patient groups. the patients received a single blood transfusion of a randomly choosen donor. we found in all 4 groups that to induce a beneficial "transfusion effect" compatibility for at least 1 hla-dr antigen between recipient and transfusion donor is required. if the transfusion and recipient are mismatched for both ma-dr antigens, the recipient is immunised, resulting in an increased antibody production (p=o.ool), an increased cytoxicity (cml) (p=o.oos), an increased mixed lymfocyte reaction (mlr) (p=o.oos) and a decreased graft survival (p-0.003). after a beneficial (ma-dr sharing) transfusion. the in vitro test remain unchanged or decrease. graft survival increases with the number of shared antigens between transfusion donor and organ donor (p=o.o2), suggesting that a donor specific suppression is induced. recent experiments have revealed a direct interaction between the cd4 molecule and hla-dr antigen. to address the nature of this interaction we have used a xenogeneic system in which a human cd4 cdna was expressed in the murine cd4-and cd8-negative hybridoma 3dt52.5.8. the tcr of 3d3752.5.8 recognizes the murine class i molecule od. a class i 1 expressing dd-positive cell line was obtained by cotransfection of the human class i1 cdnas together with the murine od gene int.0 the murine fibroblast line dap3. coculture of 3dt52.5.8 and dap3 expressing dp-dd resulted in a 20 fold increase in il-2 production and in rosette formation only when both cd4 and dp were present on the responding t hybridoma and the presenting cell, respectively. we are using this system to map regions of the cd4 molecule that interact with the class i1 mhc ag. the cd4 molecule has also been shown to be the receptor for the human immunodeficiency virus (hiv) via the gp120 molecule. since gp120 and class i1 both interact kith cd4, we have used our functional assay to verify if gp120 exerts an inhibitory function on cd4 class i 1 interaction. recombinant gp120 inhibits the functional interaction and rosette formation in a concentration dependent fashion with maximal inhibition at about 10 pg/ml.. this inhibition is specific since it can be reversed by recombinant soluble cd4. the fact that recombinant. gp120 can inhibit the functional interaction between cd4 and its physiological ligand (class i1 ags) suggests that the use of gp120 on a vaccine against hiv infection could alter the immune response of such individuals. this work was supported by src, mrc and nci. t lymphocytes discern self from non-self molecules through the interaction of their antigen-specific receptors and proteins encoded by the mhc. although the nature of this association is not well-defined, a model has been proposed whereby the v-segments of the t cell receptor interact with residues along the 2 alpha helices of the class i antigen (davis et al.; 334:395, 1988) . we have recently shown that ctl generated against the class i molecule qiod crossreact on several unrelated murine class i antigens containing the shared qiod residues at amino acid positions 152, 155, and 156 (mann et al.; j x 168:307, 1988) . these residues contributed by the a-2 domain occur in the alpha helical portion of the class i molecule and amino acids 152 and 155 could interact directly with the t cell receptor. to further characterize the role of these amino acids, we are in the process of determining whether insertion of these 3 residues by site-directed mutagenesis into a human class i molecule will allow for the antigen's recognition by anti-910 ctl. here the t cell repertoire becomes restricted, so that foreign antigen can be recognized only when associated with the mhc products of the host, and mature t cells are tolerized to self antigens, a process which also seems to be mhc-restricted. thus, t cells should be non-reactive to self antigens when they are associated with mhc products present on the tolerance-inducing thymic cells, whereas they may still react to the same self antigens when associated with different mhc products. to examine mhcrestricted tolerance in vivo, a model system must have: a) self antigen in the context of one mhc haplotype. and b) tolerance to both that and a second mhc haplotype. chimeras were prepared by aggregation of preimplantation embryos of two strains of mice, c57bl/6 (b6) and balb/c. the thymus of such chimeras should be composed of two distinct and completely intermixed populations of cells, one from each parental strain (isozyme analysis indicates no detectable fusion of cells). thus, t cells maturing in the chimeric thymus should be exposed to and tolerized to minor histocompatibility antigens (mhas) of one parental strain only in association with the mhc of that strain. for example, mice might be expected to express b6 mhas only with h-2b (the 8 6 mhc). however, our chimeras were fully tolerant to f1 skin grafts, which have "hybrid" combinations of mhas and mhc (e.g., b6 mhas with h-2d). these results are most consistent with either, a) "wholesale" antigen processing and presentation of all mhas by the tolerizing thymic cells, and/or, b) functional sharing of mhc products between the parental thymic cell populations. many of the events critical to the maturation of t lymphocytes occur in the thymus. in the case of t-cell responses against viruses such response defects are associated with a marked increase in disease susceptibility as illustrated by class i mhc controlled susceptibility to lethal pneumonia induction by sendai virus. certain class i or class i1 mhc determined tc response defects (four out of six tested by us) can be restored by imunization in vivo and/or restimulation in vitro with dc. dc are the most effective apc. their superior apc capacity is due to 1) a very high absolute number of class i and class i1 mhc molecules, and 2 ) a low degree of sialylation of mhc and other surface molecules, reducing negative charge and facilitating access of the t-cell receptor to the mhc groove presenting the antigenic peptide and/or improved clustering with t cells. the more effective antigen presentation by dc allows a more prominent role for a cd4+ q cell independent pathway of cd8+ tc activation. it is postulated that the more effective direct triggering of cd8+ tc precursors lowers the threshold for il-2 production by cd8+ cells, reducing the requirement for il-2 production by the cd4+ cells. failure of dc to overcome certain mhc-linked specific tc response defects probably reflects complete failure of any foreign peptide derived from the processed antigen to interact efficiently with the mhc or a true tc repertoire defect. donald pious, department of pediatrics, university of washington, seattle, w 98195 mhc class i1 molecules bind inmunogenic peptides derivsd fro13 soluble antigen and the complex is recognized by specific t cells. ue have isolated eight independent mutant b -l u clones which are altered in their ability to present antigen. in standard proliferation assays using four different soluble protein antigens, the mutants are unable to stimulate the majority of t cell clones restricted to hlfl dr or dp. cllthough unable to present *hole hepatitis b surface antigen (hbdg), they effcctively present a hbdg peptide to a dprestricted t cell clone. the fact that both dr and dp restricted antigen presentation is abnormal in these mutants made it likely that the class i 1 structurual genes are unaltered. this hypothesis is supported by the finding that dno sequences from the dr genes of one mutant are norml. however, two observations indicate that the uture class i1 di-expmsccd by the mutants are structurally altered. binding to the mutants with tuo polymorphic anti-dr antibodies and one anti-dp antibody is reduced, although the level of cell surface class i1 expression is normal. second, the class i1 diners from the mutants dissociate into no-rs under in vitro conditions (sds-pwe) which preserve dimers in the progenitor line. together, these functional and structural data suggest that the mutants are defective in a molecule that either associates with or post-translationally modifies class i1 molecules and is required for the physiologic formation of an twc/antigen complex. they do not function as restriction elements presenting foreign antigens to t-cells. to investigate the nature of this functional defect we have constructed 3 different recombinant class i genes using dna segments from the b10 q9 (qa-2) or h-2db genes. structural protein encoded by the recombinant genes is derived entirely from the q9 gene whereas the cis-acting transcriptional regulatory elements or the dna segment encoding the membrane anchoring domain is derived from the h-2db gene. into fertilised cba/ca embryos by microinjection and transgenic lines were established. to date we have established 13 transgenic lines. we have shown that the q9 (qa-2) antigen encoded by the recombinant genes behaves as a major transplantation antigen in skin grafts and provokes strong secondary cytotoxic t-cell responses in grafted animals irrespective of the tissue distribution or mode of membrane anchorage of the q9 antigen. at present, we are investigating whether the q9 antigens encoded by the recombinant genes are able to present influenza virus or mouse minor histocompatibility antigens to t-cells during immune responses and hence whether they can function as restriction elements. is a distinctive system because it permits an analysis of the activation requirements for antigen specific, resting t cells. been isolated following culture with anti-ig-sepharose and compared to dendritic cells as stimulators of cd4' t cells in the mix. i1 mhc products and independently stimulated the 1 ' mlr and the production of several t derived lymphokines, including il-2 and 11-4. however, the relative potencies of dendritic cells and anti-ig blasts as 1'mir stimulators varied in a strain dependent fashion. times more active in stimulating hls-mismatched. mhc-matched t cells, relative to syngeneic t cells. anti-ig blasts when stimulating acroas an mhc barrier and were likewise more effective in binding iqic-disparate t cells to form the clusters in which the mix was generated. dendritic cell-t cell clustering was resistant to anti-lfa-1 mab, while b blast-t cell clustering was totally blocked. thus, anti-ig b lymphoblasts and dendritic cells, two cell types which differ markedly in phenotype, also differ in efficiency and mechanism for initiating responses in allogeneic t cells. only anti-ig blasts could stimulate across an mls barrier, being at least 100 in contrast, dendritic cells were 10-30 times more potent than the as the lymphocyte f u n c t i o n antigen 3 (lfa-3) and the i n t r a c e l l u l a r adhesion molecule (icam). we i n t e r p r e t t h i s as an increase i n the membrane expression o f these s t r u c t u r e s f o l l o w i n g incubation. the increase i s blocked by the t r a n s l a t i o n i n h i b i t o r , cycloheximide, implying t h a t p r o t e i n synthesis i s involved. helper t cell responses to soluble globular proteins require processing of the protein by ia-expressing antigen presenting cells (apc). antigen is internalized into acidic vesicles, proteolyzed, and peptides containing t ceu antigenic determinants are transported to the apc surface where they are recognized by the antigen-specific t cell in conjunction with ia. most ia-"pressing cells are competent apc, however, only b cells have antigen-specilic receptors on their surface auowing bound antigen to be processed and presented at 1/lw the antigen concentration required by nonspecific apc little is known about b cell antigen processing function during differentiation, or if ig-mediated apc function is altered at different maturational stages, thus allowing regulation of b cell-helper t cell interactions. neonatal acquisition of apc function was examined in mice ages day 1 to day 15. splenic cells from d l to d10 mice process and present pigeon qochrome $, pg at 0-20% of adult levels. by d15 neonatal spleen cells acquire the ability to process and present soluble pg at 3 5 4 of adult levels. the ability to internalize antigen through ig rwptors was determined using an antigen-antibody conjugate, p$-&(ab')z. neonatal spleen cells acquire the ability to process antigen through ig simultaneously with the ability to process soluble antigen. lack of prowsing by neonatal spleen cells prior to d10 is not attributable to insufficient levels of surface ia. since d3 neonate spleen cells are able to activate t cell hybrids to 50% of adult levels when provided with p$ 81-104, containing the t cell determinant. dlod15 neonate presentation of p@1-104 is indistinguishable from adult levels. b cell maturation into memory b cells was identified by the loss of the jlld differentiation marker. splenic jlld'o b cells increase from 540% following immunization and return to nonimmune levels after 4 weeks. during antigen-induced b cell maturation, jllb b cells are indistinguishable from splenic b cells in the ability to present antigen introduced into the processing pathway either pinocytotically or via surface ig. p p antibody conjugates specific for mouse f(ab')z i n , igd, or igg are presented equally well by both splenic and jll@ b cells. thus, acquisition of b cell processing function appears to be developmentally regulated and may play an important role in b cell tolerance meshanisms. once b cells have acquired the ability to process antigen this function is maintained and is not regulated during maturation into memory b cells. we are currently investigating the role of ig isotype during neonatal acquisition of antigen processing. (supponed by nih grants ai-18939, ai-12001, and ai-2317) as the preliminary studies suggested that carrier sc (apc) for ts vs. tcs activation might be distinct, studies were done to directly address this possibility by assessing tha ability of s3 coupled to various cell populations to activate ts and tcs. the results indicated that ts activation required that s3 be coupled to plastic adherent cells which bear both i-a and i-j determinants. these cells are nonadberent to anti-ig and nonfunctional in cyclophosphamide (cy) treated mice. i n contrast activation of tcs required coupling of s3 to plastic non-adherent and anti-ig adherent cells. these cells are functional in cy treated mice and bear the b cell markers jlld and i-a but not 1-3. thus s3-specific ts are activated by i-a+ i-j+ adherent cells (presumably macrophages) whereas tcs are activated when antigen is presented by b cells. (nih grant ca25054.) interleukin-2 activated killer (iak) lymphocytes (also known as lak cells) which destroy a broader spectrum of tumors invitro than nk cells have been used sucessfully in an adoptive immuno-therapy protocol for the treatment of patients with a variety of advanced cancers. the cell suface molecule(s) on tumor cells that a r e involved in specific binding to iak cells and in programming iak cells for cytolysis (iak acceptor molecules) have not been characterized. inorder to identify such acceptor molecules a crude membrane digest of the lung carcinoma cell line a549 was biotinylated and adsorbed to iak cells or to unstimulated human peripheral blood lymphocytes (upbl) (each from the same person). proteins from the washed solubilized cells were separated by page, western blotted and probed with streptavidin-alkaline phosphatase. several experiments demonstrated that different tumor membrane proteins bound to iak cells compared to upbl. the unstimulated cells bound one tumor membrance protein (about 40kd) not found on the iak-adsorded blot. the iak cells bound three tumor proteins (approximately 30,46 & 50kd) not found on the upbl-adsorded blot. three other proteins (about 35,44 & 55kd) were found to adhere equally well t o iak cells and upbl. utilizing a streptavidin affinity column, solubilized tumor membrane proteins that bound to iak cells could be separated from solubilized iak membrane proteins. the isolated tumor membrane proteins that adsorded to iak cells inhibited iak mediated lysis of a549 tumor cells by >85%. these studies suggest that specific cellular adsorption techniques may be useful in isolating and characterizing tumor membrane proteins involved in interactions unique to cytolytic lymphocyte-tumor cell target binding and lysis. activation of human t lymphocytes occurs via the t cell receptor-cd3 complex but can also be induced through the non-antigen-specific cd2 molecule. selected combinations of mabs or the soluble cd2 ligand, namely lfa-3 and a unique anti-cdz mab (cd2.1) induce human t cell activatlon. cd4 is an accessory molecule implicated in t h e activation of human t lymphocytes. this molecule may exert this function by increasing intercellular avidity through binding to mhc class i1 molecules and/or by transmitting intracellular signals. we have investigated the action of mabs directed against different epitopes on the cd4 molecules in the activation of human t cells via the cdz pathway. we show that anti-cd4 mabs inhibit cd2 induced t cell proliferation in an epitope-depe dent fashion. this inhibition does not appear to be linked to the lower cd2 mediated [ c a ' + ] response induced by anti-cd4 mabs, since [ca"] response is equally affected by anti-cd4 mabs whether or not they inhibi ed t cell proliferation. in conclusion, the partial inhibition of the cd2 induced [cb'] response of t cells by various anti-cd4 mabs suggest that : 1) this inhibition does not totally account for the inhibitory effect of anti-cd4 mabs, 2) and the proliferation induced by anti-cdz mabs may not be completely ascribed to the [ca2+] response of t cells. rosenberg. and alfred singer, experimental immunology branch, nci, nih, bethesda, md 20892 . we have devised a model to study the in vivo generation of suppressor cells by using mice congenic at qa-1, a class i-like molecule encoded to the right of h-2d. disparate tail skin grafts (tsg) unless a second graft with additional helper determinants was also present. without any source of additional help, failed to reject their qa-1 graft, and were unable to reject them even upon the subsequent addition of exogenous help. thus, exposure to qa-1 disparate grafts, in the absence of additional help, either led to qa-1 specific tolerance or suppression. mice failing to reject qa-1 allografts revealed the presence of qa-1 specific suppressor cells that inhibited the in vivo activation of antigen specific effector cells capable of rejecting qa-1 bearing allografts. experiments using t cell subpopulations should allow for further characterization of these qa-1 specific suppressor cells. we found that b6 mice did not reject qa-1 however, animals engrafted with a qa-1 graft alone, in the thymus, the t-cell receptor genes are rearranged, the t cells learn to recognize their own major histoconpatibilitp complex "hc). and they learn to respond to foreing hec. these events seem to be linked to the interaction between t-cell precursors and the stromal cells of the thyms. thus increasing evidence points to an essential role for the thymus epithelial cells (te cells) in development of at least hec class i1 recognition by the t cells. to be able to study the importance of te cells in t cell maturation. we have developed a method for growing murine t cells in serum-free pediun with well defined constituents. the m d i u a allows far growth of te cells without concomitant growth of bone marrow derived cells as macrophages and fibroblasts. data obtained by en and lmmunocytochenistry showing the epithelial nature of the cultured cells, as well as autoradiographic data on the growth pattern, and characterization of tb cell supernatants will be provided in addition to results obtained from co-culture of te cells and t-cell precursors (cm-cds-thymytes). lmmunogenicity c 152 branch, national cancer institute, bethesda, md 20892 the effector limb mediating skin allograft rejection is highly antigen specific, rejecting cells that express allogeneic mhc antigens while sparing those which fail to express allogeneic mhc determinants. disparate skin grafts are completely rejected in spite of the fact that only a small percentage of the cells within the graft express ia antigens. thus, it is possible that mhc class i1 disparate grafts are rejected by a mechanism that does not assess the expression of mhc determinants on each cell. we assessed the specificity of the rejection of ia disparate grafts by using allophenic skin grafts in an adoptive transfer system and concluded that skin grift rejection across an mhc class i1 disparity required recognition of allo-ia determinants expressed by every cell in the graft. therefore, we reasoned that mhc class i1 antigens must be induced on these ia negative populations. indeed, injection of mice with gamma interferon dramatically induced ia antigens on previously negative keratinocytes. we next tested whether the induction of allogeneic ia determinants on keratinocytes was necessary for graft rejection by engrafting parental strain mice with skin from f >parent bone marrow chimeras. such grafts failed to be rejected, in spite of the speciic rejection of the allogeneic langerhans cells, indicating that the failure of keratinocytes to express allogeneic class i1 determinants leads to graft preservation. conclusion, mhc class i1 disparate skin allografts are rejected in a highly antigen specific fashion, secondary to the induction of mhc class i1 antigens on skin cells that fail to constitutively express them. to address whether the extensive polymorphism characteristic of class i molecules influences cd8 binding, we have screened a panel of transfectants expressing individual class i mhc alleles. of 18 alleles tested, only aw68.1 did not bind. all other molecules dld bind, including a2.1 and aw69, which differ by 13 and 7 amino acids respectively from aw68.1. position 245 in the alpha 3 domain was identified by sitedirected mutagenesls as the critical residue differing between a2.1 and aw68.1 which determines binding. a mutant aw68.1 molecule containin alanine at position 245 bound cd8, while a mutant of a2.1 with valine at 245 did not. alanine is found at position 245 of all human and murine class i molecules sequenced to date except aw68.1 and aw68.2, which have valine at that position. bulk cultures of a2-allospecific ctl were also sensitive to this substitution, and preferenhally recognized both molecules with alanine at 245. this study shows that aw68.1 differs from other class i molecules in its capacity to bind cd8, and raises the possibility that aw68.1 may not function as a restriction element as effectively as other class i alleles. t cell hybridomas derived from h-zd lnc recognked s and pres2 antigens in a i-ad restricted way, while t cell hybridmas from h-2k lnc manifested a specificity for either pres2 in association w i t h i-ak or for s in association with i-!& the activation of lhese hybridomas by antigen and antigen presenting cells (af'c), as measured by il-2 secretion, was found to be sensitive to prostaglandines and could be completely inhibited by anti-lfa-1 moncclonal antihxlies. different aft populations were tested for their capacity to present spresz particles to these t cell hybridomas. various macmphage like populations such as resident, con a induced, thiiglycolate induced peritoneal exudate cells as well as splenic adherent cells were found to present efficiently the spres2 antigen. in conhast b cells and la+ b cell lines (ta3, m12.4) could not function as accessory cells in the spresz specific stimulation of these t cell hybridomas. the inability of these cells to present this antigen was not due t o inhibitory effects since these cells did not inhibit the presentation capacity of other potent apc's. funhcnnore addition of apc's of a different haplotype could not complement for the defective presentatiw of spresz by b cells and b ceu lines indicating that mhc independent accessory factors are not implicated in this process. hence it is clear that maaophage-like apc's and b cells d i f f a in their capacity to process and present spdz antigens. since spresz is a very stable particle composed of lipids and proteins it is conceivable that such antigen quires a smng degradation and swh plocessing might occur in cenain -phage-like apc's but not in b cells. recombinant human insulin biosynthetically labeled with k n d " s at several amino acids was used as an antigen and was exposed for varyillg lengths of time to ta3 mouse b cell apc. subcellular fractionation and hplc chromatography permitted several of the processed peptides distributed throughout the insulin molecule to be monitored. many insulin peptides localized to both the extracellular (8 peptides) and intracellular (4 peptides) compartments of ta3 cells were detected. membrane-associated peptides is in progress. many of the peptides processed by ta3 apc in situ co-elute with those obtained upon digestion i n vitro by the insulin-specific insulin degrading enzylg5 (ide). f o r the processing of 51-labelled human insulin suggest that insulin may be processed in b cell apc into immunogenic peptides by an enzyme(s) present on the plasma-membrane, intracellularly and extracellularly. (supported by mrc and cda) . we investigated the pathway of antigen processing 5 itu in cell apc. hurine ctl clones specific for hia-a2 were generatef with the human cell line jy. four of five ctl clones were found to lyse a2k transfected murine cells more effectively than a2 transfectants. anti-cd8 specific mab inhibited t 3 lysis by these four clones, and this inhibition was more pronouncgd for a2k one clone, which lysed a2 and a2k transfectants equivalently, was shown to be insensitive to anti-cd8 antibody inhibition. these findings indicate that a2-specific t r i n e ctl clones possess greater avidity for murine target cells expressing the a2k hybrid molecule relative to those expressing the a2 molecule. this implies that a cd8 interaction with the same molecule seen by the t cell receptor is important for target cell recognition. hla class i1 antigens are highly polymorphic cell surface proteins involved in initiation and regulation of the immune response. allelic sequence variation primarily affects the structure of the first external domains of the a and j3 component chains. here we provide evidence for other types of allelic polymorphism for these genes. the sequences of two cdna clones corresponding to the hla-dqp mrnas from an hla homozygous cell line exhibit both alternative splicing and readthrough of polyadenylation. furthermore, the alternative splicing event is associated with only a subset of hla-dqb alleles, while the polyadenylation site readthrough is found in a larger subset. this suggests that polymorphic & acting elements within the hla-dqp gene control both processing steps. proteins, presumably encoded by the alternatively spliced mrnas lacking transmembrane exons, are immunoprecipitated with a monomorphic monoclonal antibody directed against hla-dq. these proteins are found in supernatants of cultured cell lines for which secretion is predicted, but not in those of cell lines which do not contain the alternatively spliced mrnas. such secretion class of i1 allelic products could profoundly affect interactions between effector and target cells in an immune response. departments of biology and chemistry and the cancer center, q-063, university of california at san diego, la jolla, ca 92093. we are studying the murine icam-1 gene and the effects of icam-1 on antigen recognition. using the human icam-i cdna, we have isolated cdna and genomic clones encoding the murine homologue. the murine icam-1 gene is a single-copy gene that consists of multiple exom spanning 25kb of dna and encodes a 2.5kb mrna that is expressed at high levels in a wide variety of different cell types. sequence analysis indicates that murine icam-1 is 50% and 6096 homologous to human icam-1 on the protein and dna levels, respectively, and is a member of the immunoglobulin gene superfamily, consisting of several v-like domains linked tanddy. we are also studying the effects of icam-1 on antigen recognition. the t-cell clone 14d m s f 1988). this response is blocked with the anti lfa-1 antibody fd441.8 when antigen is presented by blo.a(2r) spleen cells but not when antigen is presented by dcek, a fibroblast transfected with i-ek. we are currently aansfecting the murine icam-1 cdna into dcek in order to determine if we can enhance the 14d response and to determine if the enhancement is lfa-1 dependent. wth an alp a beta tcr that recognizes moth the t cell differentiation anti en, cd4, is expressed by mhc class i1 restricted t lymphocytes. mhc class i1 products. the association between cd4 expression and restriction by mhc class i1 rfioducts has led to the hypothesis that cd4 may interact with monomorphic determinants of a large body of experimental evidence suggests that cd4 interaction with mhc class i1 molecules leads to an increase in the binding avidity of t cell-stimulator cell interactions. a direct test for a functional cd4-mhc class i1 interaction in t cell activation requires a separate evaluation of cd4-ia interactions from tcr-a /ia recognition. however, a separate evaluation proves difficult since the t cell receptor and cb4 may interact with the same mhc class i1 molecule. in this report, we use a t cell activation protocol, where tcr-ag/ia recognition is replaced by tcr complex-antlcd3 antibodies interactions. using this activation protocol, we pave analyzed the effects of monoclonal anti-mhc class i1 antibodies on the activation o$a cd4 hybridoma in the absence of its tcr restr$tinj mhc class i1 molecule (ie ) but in the presence of unrelated mhc class i1 molecules (ie ,ia ). the data obtained clearly indicate a functional role for cd4-mhc class i1 interactions in t cell triggering. we have targeted hen egg lysozyme (hel) to murine b cells using heterocrosslinked antibodies which specifically bind to surface igd or different mhc molecules. occurred more quickly with targeting to igd than to mhc structures as assessed by fixation and pronase stripping experiments. hel was internalized quickly into acidic compartments when targeted to igd but was detected much later when targeted to mhc molecules, as assessed by shifts of fluorescent signal of internalized fitc-hel. however, the data indicate that not all endocytosed hel entered low ph (<5.5) compartments. degraded hel was released from b cells following endocytosis of 125-i-hel. this release was detected earlier with targeting to igd than to mhc structures. interestingly, the total amount of internal 125-i-hel decreased with time after endocytosis via igd, but the internal 125-i-hel was almost entirely whole undegraded hel at all times following endocytosis. these data and those of chloroquine and leupeptin inhibition studies indicate differences in the fate of antigen entering b cells via igd or mhc structures, and support the notion of a neutral ph storage compartment for antigen endocytosed via surface igd on normal splenic b cells. internalization and presentation of hel to hybridoma t cells laboratories, department of surgery, university o f iowa, iowa city, ia 52242 target cell lysis by cd8' ctl is a highly specific phenomenon in vitro, as we have confirmed repeatedly in reverse labelling tests by showing that admixed "third party" target cells are not lysed in the presence of specific ctl-mediated cytolysis. however, when mixtures of ctl and their specific targets are inoculated into the skin of hosts syngeneic to the ctl, host cells at the site of inoculation are destroyed, often to an extent that results in grossly observable, full -thickness necrotic lesions. we have evoked these "innocent bystander" reactions in mice with ctl directed against single and multiple non-h-2 antigens and tnphapten and influenza a virus-specific antigens. thus, the ability to trigger bystander tissue destruction appears to be a general characteristic of ctl-target cell interaction in vivo. our current evidence suggests that host inflamatory cells recruited and activated by factors stimulated by ctl-target cell recognition actually mediate the tissue destruction. these ctl-initiated bystander reactions may be the basis of the non-specific tissue destruction that contributes to allograft rejection and that is observed in many serious virus infections and in intense dth reactions and contact dermatitis. rong h a lin. baael i n s t i t u t e f o r i u m l o g y , baael, snitzerland. w e have investigated the b a d e for i n u n i t y or tolerance t o a m a e aezw proteinthe f i f t h caponent of c o l p l a c n t (a). i n c5 deficient nice t h i s protein is absent from s e r m and as a cnrwquence they are not tolerized t o cs. c5 deficient dca generate uy bearing t c e l l s which recognize c5 i n the c m t e x t of clasa 11. i n contrast, c5 s u f f i c i e n t mice i n which c5 protein is continuously probced d m t mtmt t c e l l reaponsea againmt u. we have tested i f t h i s self protein i s proceseed and presented with clase i1 i n n o m l mice and can be recognized by c5 specific t c e l l s i n the absence of exogenewsly added antigen. a l l clasa i1 bearing c e l l s fm c5 s u f f i c i e n t l i c e activated c5 specific t c e l l clonea without additional antigen. presentation was mt a cansaquence o f c5 secretion by macrophages i n culture but was a h t o be derived prom endqlenewaly generated cs/cl.ss i1 caplexea. thus t h i s self protein is e f f i c i e n t l y preaented ,inin and available f o r tolerance in&xtion. although c5 deficient dca cannot secrete c5 they s t i l l synthesize a precursor mlecule, pro-c5, i n accumulating evidence from a number of models suggests that unique subsets of antigenpresenting cells a r e responsible for the induction of specific t cell-mediated responses. w e have previously described an age-dependent maturational defect in the ability of the sjl strain of mice to activate dth-inducer t cells to a wide variety of antigenic stimuli. none of the other 14 strains tested exhibited a similar defect and all other accessory cell dependent responses were unaffected in the dth unresponsive sjl. w e have also shown that the adoptive transfer a macrophage from older dth responsive sjl or other dth responsive las strains can overcome this defect in dth responsiveness. w e have recently found that a subpopulation with the mac-i+, mac-3' and mac-2-surface phenotype a r e able to transfer responsiveness. facs analysis indicate that the mac-3 phenotype is expressed on less than 20% of macrophages. titrations of the mac-3' cells isolated by facs indicate that adoptive transfer of o ly 100 mac-3' cells can overcome the defect in dth responsiveness. by contrast, transfer of 10 mac 3-or mac 2' cells were unable to overcome the defect. our data suggest that the induction of cd4' antigen specific cells dth-inducer t cells is mediated by a phenotypically unique small subset of macrophage accessory cells. in our studies, we have examined the effect of administering fab' fragments of anti-l3t4 moab (fabl-gk1.5) on the inhibition of humoral immunity. treatment of klh-primed mice with 0.5 mg fabl-gk1.5 depleted l3t4' cells from lymph node tissue while leaving other lymphocyte subpopulations intact. after injection of klh in complete freund's adjuvant, these t,depleted mice were unable to produce anti-klh antibodies. long-lasting unresponsiveness against klh (12 weeks) was observed despite the apparent regeneration of the t, population of the lymph node. the results obtained using either fab' or intact gk1.5 antibody were comparable and suggest that a transient depletion of t, does not account entirely for the long-term humoral unresponsiveness. the aim of this study was to gain a more detailed insight into the molecular aspects of antigen processing during the imune response. as a first approach, endosomal vesicles were isolated from bovine alveolar macrophages and their proteolytic activity with respect to a model protein antigen, sperm whale myoglobin (mb), was characterized. during the first stage of digestion of mb by the endosomes, a limited number of fragments were preferentially released from the antigen. we have isolated and identified these fragments. the digestion of myoglobin is completely prevented by pepstatin, a specific inhibitor of aspartic proteinases, and only marginally by other proteinase inhibitors. when mb fragments preferentially released upon digestion with purified bovine cathepsin d, an aspartic proteinase abundant in macrophages, were identified, almost all coincided with the fragments released by the endosomes. to define in more detail the selectivity of cathepsin d under the mild conditions applied, other protein antigens were similarly treated with the enzyme and the peptides released were identified. the location of the preferential cleavage siteswhen related to known t-cell epitopessuggests a dominant role for cathepsin d in the processing of protein antigens to yield fragments for presentation to t-cells. possibly, the observed selectivity of the enzyme may account for the structural similarities among t-cell epitopes, noted by others. actively acquired tolerance in mice to the antigens of the mhc (h-2) is induced by exposure of the animals to allogeneic lymphocytes within 24 hours of birth. actively acquired tolerance to the mhc in humans (hla) cannot be studied in the same way. however, we have evidence for the existence of actively acquired tolerance in humans in a study of 26 highly sensitized patients waiting for a renal allograft. they had developed complement dependent antibodies to the hla antigens of almost all unrelated caucasoid donors. the sera of these highly sensitized patients were tested against a panel of lymphocytes that were mismatched for only one hla class i antigen. we found for these 73 patients hla class i antigens that, although different from those present in the recipient, did not lead to a positive crossmatch. we called such antigens "permissible mismatches" and show that they often included those hla antigens of the patient's mother that the patient had not inherited (noninherited maternal antigens; nima). in 15 of the 26 patients, the permissible class i mismatches included the nimaa. the noninherited paternal antigens (nipas) were analyzed as a control; only two of the 25 nipas tested were acceptable mismatches, which emphasized the preferential nonresponsiveness to nima. recent experiments indicate that what holds true for antibody formation also holds true for t cell activation. of hla class i and hia class i1 allospecific cd8-positive ctl clones. monoclonal antibodies (mcab) directed against the cd8 structure were only found to inhibit antigen-specific cytotoxicity of a series of class i allospecific cd8-positive ctl clones and not of a class i1 allospecific cd8-positive ctl clone. however cytotoxicity induced by cd3 mcab (used at suboptimal concentrations) or cd2 mcabs in both types of ctl clone was blocked by cd8 mcabs. the absence of cd8 mcab blocking of antigen-specific cytotoxicity of the class-11-specific cd8positive ctl clone may be explained by assuming that it results from a triggering signal which is to strong to be overcome by the down-regulatory signal of the cd8 antigen. these combined findings clearly suggest a functional involvement of cd8 not only in tcr/cd3 activation, but also in tcr/cd3 controlled alternative activation routes, such as the cd2 activation pathway. moreover it shows that even an hla class i1 allospecific cd8-positive ctl clone expresses a functional active cd8 antigen. the absence of hia class i expression on the target cells (daudi cells) used in the experiments described indicate that the cd8 antigens not act solely in an adhesion-like fashion, but exhibit also a more general regulatory function in t-cell activation. this regulatory role of cd8 may be explained by assuming the induction of a threshold for activation, which is triggered after binding of cd8 mcab or binding to its natural ligand, hla class i. in our view, cde-mediated regulation of t-cell activation could therefore prevent non-specific triggering of cytotoxicity by interactions of insufficient affinity. *this study was supported by a grant from the dutch kidney foundation. the cd4 t-cell surface antigen 1s felt to have the dual functlon of stabilizlng the interaction of the t-lymphocyte with the antigen presenting cell (apc) a s well as transduclng an independent signal that can potentiate the actlvatlon related alteratlons generated through the t-cell receptor. we have found that upon antibody-mediated cross-linklng of the cd4 molecules of cloned murlne t-lymphocytes there is a time and temperature dependent decrease in the abundance of the lymphocyte-speciflc tyroslne klnase p66lok. this co-modulation is speclflc for cd4 and p66lck slnce cross-linking of other t-cell surface antlgens (cd3. t200, thyl.2) does not result in detectable alteratlons in the abundance of the lck protein and slnce cd4 cross-llnklng does not induce any alteratlon in the abundance of p60*.. another srcrelated tyrosine klnase highly expressed in t-cells. such data suggest that cd4 and the internal membrane lck protein are in close proximity within the cell. further analysls has revealed that slgnlflcant amounts of lck can be immunopreclpitated by antl-cd4 antibodies. in addltlon. cd4 can be speciflcally preclpltated by anti-lck antibodies. our data imply that cd4 and p661ok are physically associated in cd4+ t-lymphocytes. the flndlngs that cd4 is msoclated to the lck proteln in either murlne or human t-cells and that cd8 is also complexed to p66lck ln cd8* t-cells suggest that the lck tyroslne klnase is involved in the functlon of the cd4 and cd8 accessory molecules. these apc do not appear to present processed klsa determinants. in light of these findings, of apparent interest is the issue of which cells types are responsible for hlsa-specific t cell tolerance induction. studies in mice treated from birth with anti-p antibodies suggest an important but perhaps not exclusive role for b cells in this process. we are currently pursuing the identity of other cell types which may be involved. in addition, lmmunogenlclty c173 university of texas southwestern medical center at dallas, dallas, texas75235 qlo is a soluble class i-like major histocompatibility antigen produced specifically by the liver. previously, it has been shown that mice possessing soluble qlo can generate anti-q10 cytotoxic t lymphocytes (ctl), suggesting that this soluble molecule does not function as a tolerogen. we have recently constructed c3h transgenic animals which express an exon shuffled q10 (al, p2)/ld (03, tm) molecule. this qio/ld molecule is expressed specifically in the liver on hepatocytes but not on nonparenchymal liver cells, spleen, thymus, kidney or brain. the expression of qio/ld in the transgenic hepatocytes is equivalent to la expression on balb/c hepatocytes, suggesting the animals are expressing physiologic levels of the transgene. the presence of membrane bound qio/ld in c3h animals has not caused anti-910 ctl precursors to be deleted, however, because primary in vitro ctl assays show these transgenic animals can specifically lyse qio/ld targets. histopathologic examination of the livers of these animals does not show extensive lymphocytic infiltration or inflammation. in addition, serum levels of alanine aminotransferase. aspartate aminotransferase, and alkaline phosphatase are also normal, confirming that these animals do not show overt signs of liver rejection. results are ampatable with a t least two different pathways of antigen hardling, a pathway for degradaticm of antigen, and a "pmcessiq" pathway for antigen presenbtim. my+ l monocytcq enes appear t o i n t e r f e r e with t h e processing pathway, either by i n h i b i t i n g production of antigenic material t h a t can associate with ia o r by i n h i b i t i n g putative intracellular event@) imr0lvb-q the binding of ia to processed antigen and tmnsprt of annplexes to the cell surface the immunogenicity and antigenicity of synthetic peptides (sp) derived from the sequences of a streptococcal antigen were investigated in macaque monkeys. immunization with the free peptides of 17 and 21 residues failed to elicit serum antibodies or t cell responses. however, both serum antibodies and lymphocyte responses were elicited by immunization with the sp linked to tetanus toxoid (t) as a carrier. indeed, spl7-lt and sp21-tt elicited serum antibodies and proliferative responses of lymphocytes, not only to the sp but also to the native strtptococcal antigen. recall of sp17-tt or sp21-1t immunized monkeys w i t h suboptimal doses of the native srreptococcal antigen resulted in a significant increase in antibodies, both to the sp and native antigen, confirming that the two sp share antigenic epitopes with the native antigen. the b and t cell epitopes were then determined and the b cell epitopes resides in residue 8-13, whereas the t cell epitope overlaps and consists of residue 7-15. the t cell epitope has an amino-terminal leucine and carboxy-terminal glycine and alanine added to residue 8-13 of the b cell epitope. in spite of the b and t cell epitopes being expressed in sp17 (residues 1-15), the monomer failed to induce serum antibodies without a carrier. however, immunization with dimers of peptide-linked or disulphide-linked residues 1-15, without a canier, elicited both serum antibodies and proliferative responses of lymphocytes. the results suggest that the monomeric sp17 is not immunogenic, whereas the dimeric peptide elicits both antibodies and t cell responses. the minimal t cell-b cell structure required for immunogenicity is now being determined. lmmunogenlclty section a departments of immunology and rheumatology, mayo clinic, rochester, pin 55905. susceptibility to collagen induced arthritis (cia) in mice maps to the i-a loci in h-29 mice. however, swr (h-29) mice are cia resistant, suggesting a role of non-mhc genes. we have recently shown gene complementation between h-29 from swr and tcr v genes from several non-susceptible strains. cia has been induced in b10, c3h.a and a fackcrosses with swr with similar high incidences; 63, 71 and 67% respectively. c57l shares a similar background with b10 and is h-2b, but has the same v tcr mutation as swr. backcrosses showed a very low incidence (17%) of cia, and tte arthritis observed was of a much milder and transient nature. the invariant chain associated with hla class i1 molecules is a 31-33 kd glycoprotein implicated in antigen processing and assembly and intracellular transport of class i1 molecules. class i1 molecules and invariant chain are expressed primarily by b lymphocytes and antigen-presenting cells such as macrophages and can be induced by interferon-y in a variety of cell types. to define sequences involved in the human invariant chain gene regulation, 790 bp 5 ' to the initiation of transcription were subcloned upstream of the cat gene. transfection into invariant chain-producing cell lines and non-producing cell lines demonstrated that this 5 ' region displayed tissue specificity and responsiveness to interferon-y. deletion mutants were constructed to ascertain the functional properties of specific regions of the invariant chain upstream regulatory regions. these deletion mutants have led to the identification of 3 putative regulatory regions: 394 to 239, 2 3 to 216, and 216 to 165 bp 5 ' to the cap site of the invariant chain gene. deletion of any one of these 3 regions results in decreased cat activity. protein-dna interactions of these sequences have been characterized by mobility gel shift assay and dnase i footprinting. two regions have been identified that exhibit cell type dependent binding of nuclear proteins. two color flow cytometry was used to characterize the surface phenotypes of human bronchoalveolar lymphocytes (n=32). the cd4/cd8 ratio was highly variable (0.3-6.6, mean-2.1). a high proportion of the t cells expressed hla-dr (9-38%, mean=2l%) indicative of t cell activation. however, detectable levels of the i+-2 receptor were expressed on <3% of the cells. cd45r was absent from cd4 cells in most preparations (0-10% mean=3%) suggesting that the cells are inducers of ig synthesis. uchl1, a marker of memory cells was present on 68-100 % of lung t cells. uchll+ cd45r-lung lymphocytes responded poorly to pha and cona but did respond to il-2 in the presence of accessory cells. together these data suggest that lung lymphocytes are recently activated memory cells. il-2 induced lung t cell lines were also characterized for antigen expression and w ( activity. high lak activity was obtained in preparations containing a high proportion of cd8 cells. these cultures appeared to be suicidal. in contrast, lines with a high proportion of cd4+ had low or absent lak activity but proliferated in the presence of il-2 for at least 3 months expressing a cd2 cd45r-phenotype. this abstract is a proposed presentation and does not necessarily reflect epa policy. the polymorphic second exons of the hla-dp, and dpd genes have been specifically amplified in vitro by the polymerase chain reaction (pcr) method, using the thermostable dna polymerase of aauaticus. sequence analysis of mi3 clones containing the amplified dp sequences from a panel of thirty-four df typed cell lines revealed only the two previously characterized alleles for dp, . fourteen allelic variants were defined for dpw eight of these are associated with the t-celldefined dpwl-6 types; two subtypes were found for both dpw2 and dpw4. six additional dp alleles which were previously typed in the t cell assay as blanks were also idenlfied. based on this sequence information, non-isotopic sequence specific oligonucleotide probes have been developed and used to type a margarita betz, dominic dordai, brian e. lacy, and barbara s. fox. department of medicine, university of maryland school of medicine, baltimore md 21201. murine type 2 helper t cells (th2) secrete interleukin 4 (il 4) in response to antigen. despite the likely importance of these cells, little is known about their priming and expansion in vivo. we have demonstrated il 4 production in response to a cytochrome p peptide following t cell expansion in vitro. this antigen has not previously been shown to induce th2 cells. bio.a mice were primed with a peptide fragment of pigeon cytochrome c in cfa. lymph node cells were restimulated in vitro with antigen for 5-7 days, ficolled and rested for 3 days without antigen. cells were then tested by limiting dilution for the presence of antigen-specific il 4 producing cells. il 4 was detected using the il 4 sensitive cell line ct4s (provided by dr. w. e. paul, nih). the specificity of the response was confirmed by blocking with the anti-ll 4 antibody 11 b1 1. following in vitro restimulation of the primed lymphocytes with antigen, il 4 production was detectable from as few as1 03 cells per well. il 4 secretion was antigen dependent and required both in vivo priming and restimulation in order to be detected. it is not clear why primed lymph node cells, placed in limiting dilution culture directly after removal from the animal, failed to secrete detectable amounts of il 4 in response to antigen. suppression is an unlikely mechanism as fresh primed lymph node cells were unable to inhibit il 4 production by restimulated cells. we are now investigating the factors that may regulate the development of il 4 producing t cells. mark r. boothby, ellen gravallese, hsiou-chi liou. and laurie h. glimcher, department o f cancer biology, harvard school o f public health, boston, ma 02115. regulated pattern, and normally expression is limited to certain cell types such as 6 cells and macrophages. cells is accompanied by the loss o f class i 1 mhc expression. these genes also respond to external stimuli such as the cytokine il-4, which increases b cell ia. a region o f the aa mhc gene activated expression of a cat reporter gene in a b lymphoma cell line but not in a myeloma cell line. a nuclear protein that bound to two sites within this region was found. this binding activity was present in spleeiis that lack t cells and in b cell lines, but it was absent from all three myeloma cell lines tested. il-4 treatment of normal and athymic mouse spleen cells greatly increased the binding of this nuclear protein to its a a target sites, concomitant with increased aa transcription; "thus, 6 cells contain a sequence-specific binding activity regulated both by il-4 and by differentiation. the differentiation o f b cells to plasma lmmunogenicity c210 peter van den elsen3. ldepartment of immunology, the netherlands cancer institute, amsterdam, zdepartment of immunology, erasmus university, rotterdam, 3department of immunohaematology, academic hospital, leiden, the netherlands. human tcr y6 occurs in disulphide-linked (type 1) or non-disulphide-linked (type 2) forms, dependent on the use of the cyl or cy2 gene segment. the cyz gene segment can contain a duplication or triplication of exon 2 , which gives rise to different protein forms (types 2bc or zabc). it is not known whether functional differences exist between these receptor types. protein chemical analysis of type 1 and type 2bc receptors on functional human t cell clones derived from peripheral blood (pb) has indicated that not only the y chains, but also the 6 chains have a molecular mass and charge which set apart type 1 and q p e 2bc receptors. two sets of fifteen clones were randomly generated from pb of two normal donors after selection with the anti-tcr y6-1 mab, which recognizes all receptor types. dna rearrangement and mrna expression analysis of y and 6 genes allowed us to map the specificity of the anti-tcr y6 mabs 6tcs-1 and tiya to the v6l and vy9 gene segments respectively. subsequently it could be concluded from the analysis of random clones that the majority of type 1 receptors use vy9, while this preference seems absent in type 2 receptors. the great majority of type 1 receptors do not use v6l. while the majority of type 2 receptors do. this was confirmed by fluorescence analysis of pbl of a large panel of normal donors. we conclude that vy and v6 gene segments in functional tcr y6 in pb are used in non random combination and that their expression is correlated with rearrangement of the y gene to cyl or cy2. we have previously shown that polymorphic residues in the nh2-tenninal half of the p1 domain (amino acids 1-48; hypervariable regions 1 and 2 [phvl and 21) determine with which allelic or isotypic a chain a particular b chain can achieve efficient cell surface beterodimer expression. this result might be understood in terms of the current model for ia smcture which predicts that w v l would lie adjacent to region. therefore, to examine the role of ahvl residues in conmlling hetcroduncr expression. a mutant a d cdna was created in which the codon for amino acid 11 was mutated to code for the auk residue at this position. in addition, recombinant a d and a& cdnas, in which the segments encoding the three a hypervariable regions were exchanged between the two alleles, were used to study the connibutions of other a chain polymorphisms to this process. interestingly, the polymorphic residues in ahv2 are predicted to lie in a region of the a a chain a-helix which is adjacent to the phv4 region of the p chain a-helix. allelic substitutions in this latter region of ad have been shown to similarly affect surface ia h e t e r o d i i expression. taken together, these results suggest that there are at least two spatially separate areas in which the a and p chains interact and that these interactions are affected by polymorphic rtsidues in both areas, conmbuting to the efficiency of heteroditner expression and, most likely, ia quartcmary conformation. the aim of this project is to identify contact residues of the t cell receptor (tcr) with antigen and/or mhc class i1 molecules. as a model system, a vp17-containing tcr has been chosen since the majority of vb17' t cell hybrids react with ie molecules of the k,s,d, and b haplotype. t cell hybrids have been made which have a dual reactivity: they are vp17+ and recognize ie molecules but also show reactivity towards a known antigen, namely chicken ovalbumin (ova). one such hybrid has been mutagenized with ethyl methane sulfonate (ems). mutants were selected on the basis of their survival after stimulation by either antigen or ie. it was expected that mutations in all different kind of genes involved in t cell recognition and t cell activation would be found. mutants obtained fall into two major groups: 1) loss variants of tcr a or fl chains,t3 or l3t4: 2) mutants with point mutations in one of these genes. we are currently analyzing the mutants biochemically and functionally in order to identify the particular gene affected. point mutations in the a and p genes of tcr mutants will be localized using the polymerase chain reaction in combination with dideoxy sequencing. activation of t lymphocytes requires the intracellular fragmentation of foreign antigens and their presentation by class i or class i1 major histocompatibility complex glycoproteins. the direct binding of peptides to class i1 molecules has been shown in a number of experimental systems and its specificity compared to that of t cell activation. in contrast, direct binding of peptides to class i molecules has been difficult to detect; although peptide sensitization experiments and the crystallographic structure of hla-a2 persuasively argue for its occurence and importance. in this study, we demonstrate specific binding to hla-a2 of an influenza matrix peptide (flu-m1 residues 56-68) that has previously been shown to act as a target for certain hla-a2 restricted influenza-specific cytotoxic t lymphocytes. we estimate that less than 0.3% of the purified hla-a2 molecules were able to bind the added peptide. we and others have shown that allorecognition by cytolytic t lymphocytes (ctl) is analogous to t cell recognition of foreign antigens in that both can occur via presentation of antigenic peptides by products of the major histocompatibility complex. we have used peptides corresponding to the alphal helix of selected hla molecules to analyze t cell recognition of this polymorphic region. the alpha alpha helix of hla-b44 and -b13 are identical, and show a high degree oh homology with those of hla-bw58, -b47, and -b27. peripheral blood lymphocytes from 5 normal donors were stimulated in vitro with targets expressing hla-b44 to derive allospecific ctl lines and clones. in some individuals, the allospecific response was almost totally directed against the alphal helix. the ability of peptides corresponding to the alphal helix of these hla molecules to inhibit and induce lysis as well as to modify other assays of t cell activation will be discussed. diseases and *national institute of child health and human development, d bethesda, md 20892 . classical transplantation antigens are constitutively expressed on cells of all tissues exce t brain. transaiption is regulated by the interaction of nuclear factors with 5' flanking regions tiat include the class i re latory element (cre). previously, the cre has been divided mto 3 re om on the basis of nuclear t%or blndin . several studies have implicated the nuclear protein (ri) wfich binds to the inverted repeat geggattcccca) of re 'on i as necessary for gene transai tion although region i is identicarin all se uencedouse $d and l enes, it is not conservefin qa r y genes. a comparison of the c& from h-2ld with that of 610, a qa region gene expressed o y in the liver and fetal olk sac, shows that there are two changes within the inverted r eat se uence (tgaggactcc$a). these differences disru t the dyad symmetry. another nugotide diierence between h-2l and qlo falls within re 'on ifof the cre. however, qlo can bind to the nuclear factor (rii) that binds to region ii of the fit2ld cre, whereas qlo region i can not bind to the nuclear factor (ri) that binds to the region i inverted re at. to test whether the differences m region i contribute to the restricted tissue e ression of q l r w e have used site-directed in vitro mutagenesis to make the inverted repeat of]cglo region i like that of the classical class i genes. a change at either base enhances transcription as measured in a transient transfection system. either change also allows binding of the nuclear factor that binds to the classical r alterations in the cre regon i contribute to the limited tissue expression of310. the presence of disrupted cre region i in other region genes likely contributes to their tissue restricted expression. on i sequence. thus, molecular analysis of t cell receptor structure/function in sperm whale myoglobin specific t cell clones. jayne s.danska, alexandra m. livingstone, toshi isihara and c. garrison fathman, stanford university medical school, stanford, ca. we have undertaken structural characterization of the t cell receptors (tcr) utilized by a well defined panel of murine dba/2 t cell clones that recognize epitopes within the 110-120 peptide of sperm whale myoglobin (sp wmb) presented by i-ad or i e . only 2 of 14 independent clones show alloreactivity for 10 whc haplotypes. using the polymerase chain reaction (pcr) and dna sequencing of the tcr a and fc chains from matched sets of clones bearing either whc restriction or epitope specificity in common, we are addressing structural relationship between tcr and mhc/antigen for this model system. among 6 i-ed restricted t cell clones reactive with spwmb 110-120, all have highly homologous tcr 6 chains associated with a minimum of three different tcr a chains, some of which are derived from novel v gene families. to further characterized the specificity of these clones we are generating substituted peptides to identify residues within the epitope important for interaction with tcr or restricting mhc molecule. functional verification of the relationship between given tcr primary sequences, and recognition capability will be addressed by transfer of the a and/or 8 chafns cdnas created by pcr amplification into t-cell hybridomas expressing endogenous tcr genes of known sequence and specificity. with mhc fine specificity. the differential impact of substitutions with the n-terminal and c-terminal portions of the apl domain is consistent with models of auap structure in which the n-terminus interacts with peptide while the c-terminus interacts with both peptide and the tcr. or aauapu-resmcted t cell clones. the antigens tested were l-tymsine-p-lmmunogenicity c 218 expression of the q4p gene, patricia m. day, katherine e. lapan and jeffrey a. frelinger, department of microbiology and lmmunolog university of north carolina at chapel hill, chapel hill, nc 27599. generally, the transcription of class i genes tom the q a a region is limited to tissues of hematopoietic origin. previous work in our lab demonstrated widespread transcription of the q4 gene in the 81 0.p mouse, with high levels of mrna found in liver, lung, lymph node, spleen, testes and thymus. less rna was present in muscle and brain tissues. however,,it is not known whch individual cell types within these tissues are responsible for the transcription of the q4 gene. we raised polyclonal antisera against a synthetic peptide, derived from the predicted amino acid sequence of the q4p transmembrane region. we selected this region since it is the most locus specific. this antisera immunoprecipitates a class lsized protein. a monoclonal antibody, directed against the same peptide, has also been produced. sv40-transformed h-2p fibroblasts show an abundance of q4 message. suprisingly, indirect immunoluorescent staining with the monoclonal antibody reveals a cytoplasmic localization of the protein with a perinuclear concentration. different patterns have been observed in examination of the h-2b em onal carcinoma cell lines 402ax and pcc4. qgspecific antibodies allow us to identify the cell ty es which express the24 gene product. the application of in situ hybridization techniques will correlate the cellular site ofmrna synthesis and protein detected by antibodies. understanding the paltern of expressim of the q4 gene is the first step in determining the so far elusive function of these mhc genes. and il2r mrna a f t e r mitogenic stimulation. antibodies against cd45r, but not against c045 common determinants, synergise with suboptimal doses o f mitogen t o induce il2 and il2r mrna expression, suggesting t h a t cd45r molecules are operative i n transmembrane s i g n a l l i n g i n immature thymocytes. there i s also an i n d i c a t i o n from northerns using cd45 probes t h a t cd45p180 mrna i s not induced i n activated cd348-thymocytes as i t i s i n mature t c e l l s . these r e s u l t s support the idea t h a t cd45r+ molecules are essential f o r generating signals required f o r c e l l survival w i t h i n the productive intrathymic lineage. we examined a panel of thl and th2 t cell clones for the ability to induce antibody synthesis in a mishell-dutton culture system under cognate b-t cell conditions: our findings indicate that both thl and th2 t cells are heterogeneous, i.e., some but not all thl and some but not all th2 clones have the capacity to induce antibody under these conditions. we examined the effect of 7-irradiation or rnitomycin-c pretreatment of our thl and th2 clones on their ability induce antibody synthesis. asano et al. (j. immunol 138:667) have reported that th2 clones are exceedingly sensitive to 7-irradiation, with doses as low as 500 rads abrogating the ability of th2 clones to induce antibody synthesis. we found that while the helper activity of th2 clones was very radiation sensitive, helper activity in thl clones was very radiation resistant. thl clones given 2 0 0 0 rads of irradiation were as effective as unirradiated clones in inducing anti-tnp plaque forming cells (pfc). moreover, when used at higher t cell/b cell ratios in culture, irradiated thl clones were more effective than unirradiated clones in inducing antibody synthesis. the effect of 7irradiation on th2 clones was not simply due to inhibition of proliferation, since mitomycin-c pretreatment of the clones had little effect on helper activity. conservation, alexander l. dent, pamela j. fink and ste hen m. hedrick, department of biology, university of california, san diego, 8a 92093. the p chain gene of the murine t cell receptor has been shown previously to have an alternative1 spliced form of message. this message contains a novel exon, termed c& which is inserted between the vdj and constant region exons. we have studi6d expression of the cpo exon at the mrna level by rnase protection. we have found that about 1% or less of of p messages in normal t cell clones contain the cgo exon, whereas p m e s a es. in the thymus contain the exon at 10-20 fold higher levels. to address t i e importance of the c 0 exon in the immune ,system, we have undertaken a phylogenetic approach. \y cloning and sequencin the rat analogue of cpo, we have found that while the rat exon is very simifar to the mouse exon, both donor and acceptor rna splice signals are defective in the rat cpo gene. this implies that rat cpo cannot be spliced into rat p m e s a 8s. furthermore, we have sequenced the analo ous region to mouse cpo in 8 e human p chain locus, and have found no stretct of sequence remotel homolo ous to cpo. because cpo is not conserved evolutionarily, we beieve that ! he cpo gene element does not sewe an important function to the immune system of most vertebrates. 1988) . we found that one arrdno acid substitution at a vdjp juntional region position found to be highly conserved in pigcon cytochrome c-specific tcr's results in a change in antigen fine specificity, while an* change abolishes all detectable responses characteristic of the d6 tcr. we will present the results of mutagenesismansfection analyses of two other pigeon cytochrome c-specific tcr's. the murine ctl response to human class i molecules is 1-2 orders of magnitude lower than the response to murine alloantigens, due to structural differences between human and murine homologs. we investigated whether this discrepancy could be overcome by exposure of developing t cells to human class i molecules in transgenic c57bl/6 mice expressing hla-a2.1. ! c222 zhe bdixujiar basis of . lbve digiustn ard ed p d l u b 2 r , divof s ch mice expressed hla-a2.i in spleen, bone marrow and thymus at levels similar to those of endogenous h-2 molecules. however, the frequency of ctl specific for other human alloantigens remained similar to that of normal mice. the frequency of hla-a2.1 restricted influenza specific ctl was 1-2 orders of magnitude less than the frequency of h-2 restricted ctl. these results indicate that the poor response of murine ctl to human class i antigens is not determined by selection in the thymus, but by species-specific constraints on the interaction of mhc antigens with t-cell recognition structures. while the mice are tolerant to hla-a2.i expressed on murine cells, they still respond to hla-a2.1 expressed on human cells. the epitopes defined by such clones are present on hla-a2.1 positive human cells derived from several different tissues. such epitopej are not dependent upon the species of p2m associated with the class i molecule, nor upon the structure of the attached carbohydrate. the results suggest that one or more highly conserved normal human proteins contribute to the formation of such epitopes, and provide an explanation for the failure of ctl raised against class i molecules on human cells to recognize the same molecules expressed on murine transfectants. this suggests that normal endogenously expressed molecules may also be important in the formation of epitopes on class i antigens recognized by allospecific ctl. we previously demonstrated t h a t several subclones derived from a c03+, cd4-/cd8-t-cel i l i n e have undergone secondary rearrangements a t t h e t-cell receptor (tcr) a locus w h i l e maint a i n i n g i t s o r i g i n a l tcrb and igh d-j rearrangements (marolleau e t . al., i n press). these secondary rearrangements r e s u l t i n t h e j o i n i n g of germline va and j a gene segments which replace the p r e -e x i s t i n g va-jacmplexes of t h e parental t-cell line. i n an e f f o r t t o examine t h e molecular mechanism responsible f o r these va-ja gene replacements, t h e s t r u c t u r e s o f tcra cdnas prepared from both t h e parental and subcloned t-cell l i n e s were determined. i n addition, northern b l o t and southern b l o t analyses were performed on both t h e parental and subcloned t-cell l i n e s using a panel o f va and j a probes. our r e s u l t s i n d i c a t e t h a t : 1) secondary rearrangements r e s u l t i n both productive and non-productive va-ja j o i n s , 2) the mechanism whereby secondary rearrangements occur i s a d e l e t i o n event t h a t involves germline va genes 5 ' t o t h e p r e -e x i s t i n g va-ja complex j o i n i n g t o j a the class ii major histocompatibility complex (mhc) antigens are a family of integral membrane proteins whose expression is tissue-specific and developmentally regulated. a pair of consensus sequences, x and y, separated by an interspace element, is found upstream to all class ii genes. deletion of each of these sequences eliminates expression of class ii genes in vitro or in transgenic mice (1-3). furthermore, the absence of a specific binding protein for the hla dr a x box in patients with severe combined immunodeficiency disease whose cells lack class ii suggests a critical role for these proteins in class ii gene transcription (4). report the cloning of a agtll cdna encoding a dna binding protein (human x-box binding protein, hxbp-1) which, like the proteins in whole nuclear extract, recognizes both the x box and interspace elements of the human dra and murine aa genes. the hxbp-1 cdna hybridizes to two rna species, 2.2 kb and 1.8 kb in human, that are expressed in both class ii positive and class ii negative cells. hxbp-1 does not cross-hybridize to two murine aa x box binding cdnas recently isolated in our laboratory which also recognize the dra and a ax boxes. these observations provide evidence for the existence of multiple x box binding proteins which recognize a common or overlapping motif. chromosome mapping studies demonstrate that hxbp-1 arises from a multi-gene family two of whose members map to human chromosomes 5 and 22. taken together, these data suggest a high degree of complexity in the transcriptional control of the class ii gene family. france . in an attempt to analyze positive or negative in vivo regulation of clonal expansion of cytolytic t lymphocytes (ctl), we immunized blo.br mice with the kb specific ctl clone kbs-cu). and we tested whether t cells obtained from such mice would influence the in vitro development of the ctl clone kbs-c?o. a clone-specific helper effect has k e n observed, which is mediated by cd4+ splenic cells from immunized mice. control immunizations of b1o.br mice with ti negative variants of suggest that this growth regulation involves the recognition of the ti of kbsc20. the precise nature of the antigen recognized on the ctl clone, the possible involvement of ti determinants with or without mhc products u e now under investigation. we have shown that thy-]+ dendritic cells present in the epidermis of mice (dec) express cd3 associated v73 and v61 gene products. we have produced a monoclonal antibody directed against v73 and found that a wave of cells appearing at the earliest stages of fetal thymic development express v73. phenotypic and functional analysis of v73+ cells in the early fetal thymus indicates that they have characteristics in common with the v73+ dec. both populations express high levels of ly-1 and are ly-6c+. neither express cd4 or cd8. interestingly, the v73+ fetal cells express elevated levels of il-2 receptor, indicating that they may have been activated. functional analysis demonstrated that, unlike other fetal thymocytes, the v73+ cells can be stimulated to produce lymphokines and lyse a panel of target cells which are also lysed by the adult thy-l+ dec. these results raise the intriguing possibility that the first receptor-bearing component of the t cell system to appear in ontogeny might give rise to the thy-]+ dec. (3ritical to an understanding of the function of cells bearing the gamma-delta t cell receptor will be an understanding of when and where such cells function. in order to investigate this, we have used a variety of techniques (in situ hybridisation, cdna cloning, and pcr) to examine m a s of tcr gamma delta gene expression. one conspicuous site of expression is the intestinal epithelium, which is by contrast almost devoid of tcr alpha beta expression. interestingly, the v gene segment usage in this location is quite specific and is different to the specifcity that we have found in the spleen and in the thymus, and that others have found in the skin. this specificity suggests in turn that expression of the resmcting elements recognised by gamma delta may also be spatially nxticted. extensive analysis of junctional diversity can pmvide infomation on the diversity of antigen nxogniscd by gamma delta. the basis for selective expnssion of v gene segments may in part lie in different requkments of the v-gannna gene pmoters. to examine this, the wnscriptional capabilities of the various gamma gene promoters in t cells murine tcr gamma genes: distinct spatial restriction of v-gene segment usage, adrian thyday*, susan kyes*, simon carding#, charles a. janevay, being compared by linkage to the chloramphenicol acetyl transferase gene. biochemistry, university of wisconsin-madison, madison, 11 53706. murine strain a sublines a/j and a/wysnj have a genetic polymorphism that regulates serum immunoglobulin responses to several protein antigens. strain a/j secondary igg1 responses to bovinv gamma globulin, oralbumin, hemocyanin and galactosidase l-ere 50-, l o -, i -and 4-fold greater, respectively, than a/wysnj responses. subline a/hej is a low responding strain like .l/wysnj. analysis of h-2 class 1 and class i 1 molecules provided no evidence for a breeding error to account for the genetic polymorphism. instead, an important immune response gene outside h-2 may hare been heterozygous when the sublines diverged, and the polymorphism resulted from sezregation and differential allele fixation. a mutation subsequent to subline divergence is also a possible source of the polymorphism, but is less likely. the high responder phenotype inheritance pattern in (a/wysnj x a/j)fl, f2, and backcross mice was consistent with segregation of a single, recessive gene. we named this locus l a for the strain a sublines that define it; strain a/j represents the k a h allele and strains a/yysnj and a/hej represent the allele. hayes, keith d. hanson, faye nashold, and david j . miller, department of the secondary iggza responses were also affected. several different proteolytic digests of denatured seb have been tested for their ability to stimulate t cell hybrids to produce il-2. a tryptic digest that retains activity has been fractionated by hplc and the stimulatory component is being analyzed. examination of the amino acid sequence of seb and the proteolytic cleavage sites has led us to synthesize several peptides for analysis. these peptides, and their analogues, will be tested for their function in vivo and in vitro. to understand the interactions involved in the famation of peptide-mhc complexes, an assay has been developed to detect dr specific binding of peptide analogues of t e l l determinants to cell surfaces. ebv msformcd b cell lines (bcls) w m incubated with biotinylated peptide followed by fltc conjugated streptavidin, and then anaiysed by flow cytomctg. a panel of bcls homoygous for diffmnt dr types bound analogues of peptide 307-319 from influenza virus haemagglutinin (previously shown to be a helper t cell determinant restricted through dr1) to varying degrees, w h m no binding was observed to the dr-bcl rj225. binding could be specifically inhibited by the natural unbiotinylated t cell determinant or other drl restricted determinants. competition by a range of peptides revealed quantitative diffmnces in their ability to bind drl. the assay is currently being used to generate a detailed model of the complex formed betweenha307-319anddrl. and trp for leul 6. and hla-a2.3 differs from hla-a2.i by the substitutions of thr for ala 4 9 glu fo??a1152 and trp for zeu156. residues 9 and 95 in the 8-sheet of the molecule, and residues 149, 152, and 156 in the a-helix are thought to interact with bound peptide or the tcr. to evaluate the role of these residues on ctl-defined epitopes, two genes were constructed that encoded novel molecules which differ from hla-a2.1 only at residues 9, 43, and 95, or at residue 156. the effect of a-helix substitutions on serologic and ctl-defined epitopes that varied between hla-a2.i and hla-a2.3 were evaluated by constructing genes that encoded the individual differences at residues 149, 152, and 156, as well as additional non-naturally occuring substitutions at these same positions. hla-a2.1 specific ctl were found that were: (1) insensitive to substitutions at either residues 9, 43, and 95, or residue 156, but were lost when all four positions were changed; (2) dependent upon the residues 9, 43, 95, but not residue 156; (3) dependent upon residue 156, but not residues 9. 43, and 95; and (4) dependent upon residues 9, 43, 95, and residue 156. further epitope mapping with the a-helix mutants demonstrated that a substitution at residue 152 often destroys an epitope not affected by substitution at residue 156. even conservative substitutions at position 152 were more disruptive than nonconservative changes at residue 156. residue 149, while important in defining an mab epitope, had no effect on any ctl epitopes. these results indicate that spatially separate residues in the a-helix and 8-sheet of the molecule can contribute to the epitope recognized by a given ctl. furthermore, considerable complexity must exist in the spectrum of t cell receptors utilized to recognize hla-az, as 28 ctl clones exhibited 21 distinct fine specificity patterns. to follow the evolution of these class i types, to discern the chief selective pressures on its members and thus indicate the probable functional properties of the antigens. a cosmid library was screened for class i genes. 91 clones were mapped and could be grouped into 17 clusters of contiguous dna spanning 1,264 kb. by hybridisation studies, 61 class i genes/ gene fragments could be distinguished. transfection analysis revealed that 10 genes could be expressed as cell surface antigens: two genes, in a block of duplicated dna encoded serologically defined rt1.c products, the other 8 genes gave rise to novel class i antigens detected by the xeno-antibody 0x18. using region specific probes, we could detect clear rat homologues of the mouse qa and h-2 genes, however there were only two rat genes with limited homology to the mouse tla genes. the analysis showed extensive remodelling of the class i region in the evolutionary gap between rat and mouse. while the immunological role of t cells bearing the ap t cell receptor (tcr) has been well characterized, much less is known about the function of t cells bearing the $3 tcr. we investigated the role of tcr $cells in the immune response to complete freund's adjuvant (cfa). after immunizing mice with cfa, we observed a greater than 26-fold increase in the number of tcr $3 cells present in lymph nodes draining the sites of immunization, compared to a 3-4-fold increase in the number of tcr ap cells. there were at least three different species of 3tcr's expressed on these cells in the draining lymph nodes, including two protein products derived from the rearrangements of cyl and cp, and one product derived from cy4. 37% of tcr ys cells from immunized lymph nodes expressed the il-2 receptor in vivo. and these cells constituted roughly 50% of the proliferative response of total lymph node t cells to 11-2. tse, et al. (j.lmmun..vol.l25, p.491.1980) have demonstrated that at least three cell types are involved in the t cell proliferative response to antigen, including an antigen specific-t cell, an antigen-presenting cell, and a t cell that is found in unprimed lymph nodes or spleen, which has been termed the recruitable cell. we have utilized their approach of analyzing the slope of log cell number-log response curves to examine whether tcr @ cells can function as "recruitable" cells. we found that tcr ys cells as well as tcr ap cells can function as recruitable cells in this system. these data suggest that tcr $3 cells can participate in the immune response without being specific for the antigen. analysis of the membrane associated phosphoprotein profiles of b cells harvested from cultures of resting cells exposed to il-4 for 18-24 hrs reveals the presence of phosphoprotein with an mr in the range 75-80,000. destroy the autoradiographic signal from this phosphoprotein suggesting that it is phosphorylated upon tyrosine residues. appearance of this molecule, and lps also apparently fails to result in the presence of a 75kd structure in the phosphoprotein profiles. anti-il-4 antibody. 11811, in the cultures prevents the appearance of the 75kd phosphoprotein. the genes for t n f -a and tnf-p are tandemly arranged on mouse chromosome 17, with only 1.1 kb separating the 3' end of the tnf-p mrna from the 5' end of the tnf-a mrna. yet, the two genes are independently regulated. in vitro transcription and nuclear run-on experiments indicate that the two genes are transcribed from independent promoters. in macrophages, which express tnf-a but not tnf-p, only the tnf-a promoter is active. in t lymphocytes, which can synthesize both proteins, both promoters are active. activation of either cell type results in a moderate (up to 10-fold) increase in the level of transcription, while mrna levels increase more than 1wfoid under the same conditions. interestingly, the tnf-p gene is aanscribed 10-fold less than the tnf-a gene in t lymphocytes, although the corresponding mrna is more abundant. these results indicate that the accumulation of both tnf-a and tnf-p mrna after cell activation and their relative steady state levels are controlled mostly at a post-transcriptional step. acanomycin d chase experiments reveal that tnf-a mrna stability in macrophages is not significantly altered after activation by lf's, and therefore that stabilization done cannot account for the observed accumulation of tnf-a mrna. in order to examine more closely which elements are required for the regulation of tnf-a and tnf-p mrna abundance, we constructed hybrid genes combining putative control regions of tnf-a and tnf-p with known constitutive control elements. results obtained from the transfection of these hybrid genes into various cell types indicate that elements located both 5' and 3' of the coding sequence are required for the proper regulation of tnf-a and tnf-p mrna abundance. celiac disease is characterized by small intestinal mucosal injury and malabsorption. disease is activated when a genetically susceptible host ingests wheat gliadin or similar proteins (i.e., prolamins) in rye and barley. d region specif icities -dr3 and -dqw2. class i1 d-region haplotype associated with celiac disease is extended and also includes genes in the hla-dp subregion. chain gene with those encoding dr3 and dqw2 may indicate that the hla haplotype associated with celiac disease exhibits an unusual degree of linkage disequilibrium or, alternatively, that disease susceptibility involves the gene products of more than one hla locus. to characterize possible hla structural variants unique to celiac disease, the polymorphic second exons of the expressed dr, dq and dp genes were amplified from genomic dna of celiac disease patients, and their nucleotide sequences determined. our studies indicate the presence of a unique constellation of d region genes associated with the celiac haplotype, and exclude the presence of a disease specific dr, eq or dp structural gene variant in this disease. disease susceptibility is strongly associated with the hla class i1 we recently determined that the hla this same population of t cells contains a high frequency 0 1 % ) of cells which will respond to a given allogeneic mhc protein, or to differences at two other genetic loci termed mls, in conjunction with mhc. we have transfered the a and b chain genes from a pigeon cytochrome c/el specific, alloreactive. and mis' specific murine t cell clone into an unrelated host t cell. we demonstrate that the genes encoding a single a b receptor chain pair can transfer the recogntion of self mhc molecules c m p l e x e d with fragments of antigen, allogeneic mhc molecules. and an m1sc (hls-2) encoded determinant. in this case the transfer of antigen specificity and alloreactivity requires a specific a8 receptor chain combination, whereas mlsc reactivity can be transfered with the 6 chain alone into a recipient expressing a randomly selected a chain. site directed mutagenesis of the ja region has also been performed in an attempt to identify sites involved in the alloreactivity of this t cell clone. in addition. we demonstrate that a single amino acid change in the v-j junction of the a b receptor can alter mhc restriction a s well a s antigen fine specificity. department of genetics, washington university school of medicine, st. louis, i(0 63110. the s49 tumor sublines are variants isolated from a sing1 parent balb/c tumor which demonstrate locus-specific shut-off of their kd, dd and l8 genes. four phenotypically different sublines were characterized at the dna and rna level. southern blot analysis indicated that no major chromosomal deletions have occurred, and treatment of the sublines with 5-azacytidine had no effect on class i expression. between loci are unlikely. none of the repressed class i antigens could be induced with interferon even though the expressed antigens were fully inducible. northern blot analysis revealed message only for the expressed antigens, showing that the repression mechanism is acting at the transcriptional level. rnase protection analysis confirmed this result and demonstrated that the transcriptional repression is exquisitely specific for the kd, dd and ld genes as other "class i-like'' messages are present in. all the cell lines. expressing class i antigens from both fusion partners, but the negative class i antigens originating from the s49 partner were not expressed. lymphokine gene expression was examined in a panel of 116 short-term murine t lymphocyte clones derived by single-cell micromanipulation from allogeneic mixed leukocyte cultures. about 30% of clonable t cells, including both cd4+cd8-and cd4-cdw cells, could be expanded for assay at an average of 22 days after cloning. following stimulation with concanavalin a or anti-cd3 antibody, all clones secreted detectable granulocyte-macrophage colony stimulating factor (gi(-csf), interleukin-2 (il-2) and il-3, but cd4+ clones on average secreted higher 'levels of each lymphokine than cd8+ clones. clones (85%-96%) expressed detectable gm-csf, interferon-y and il-3 mrna and 11% expressed il-4 mrna. when the frequencies of co-expression of any pair of lymphokine mrnas vere determined, all were found to correspond to the values predicted for random assortment of the individual frequencies. for example, among 13 il-4-positive clones, 11 also transcribed interferon-y, giving the frequency of double-positive clones expected for random association (9.6% 10.8%). expression of the four lymphokine genes therefore segregated independently among the clones and did not allow the division of t cells into subsets vith distinct patterns of lymphokine synthesis. greater than 20-fold in the adult liver cell line, 2 to 3 fold in the macrophage cell line and just slightly in l-cells. we have subcloned the region 5' to the li gene which contains sequences that may be important to regulating expression of the li gene. this region includes a 15-mer (cctagaaacaagtga) which occurs 5' to many ifn?i regulated genes. current research has been directed towards identifying and comparing proteins from nuclear extracts prepared from control and ifn-7 treated cells which bind to this region (-260 to -1 1). this data indicates the li molecule may be expressed in cells not known to be directly involved in the immune response. although there has been considerable interest in the recently identified gamma, delta t cell receptor, relatively little is known as to its function. during our studies of the human immune response to autologous b cell lymphomas, we generated cytotoxic t lymphocytes (ctl) specific for tumor idiotype. these ctl lysed only autologous tumor cells and none of a large panel of other autologous and allogeneic cells. inhibitable by anti-idiotypic and anti-immunoglobulin antibodies but not by a panel of classical anti-mhc antibodies. phenotypic analyses showed that these ctl were cd3+, cd4-, cd8-, and express the delta, and presumably gamma! t cell receptor. such ctl can be used to gain new insights into the function of the gamma, delta t cell receptor and t cell recognition of immunoglobulin, and may prove clinically useful in adoptive immunotherapy. tumor lysis was for ebv-induced antigens. furthermore, lcl variant .221, which does not express any hla -a, -b. or -c determinants. is killed by cultures primed to lcl-.180. antibody blocking experiments suggested that this killing was mediated by t cells, and was not restricted by known class i antigens. depletion of leu 19 positive cells from the effector population did not eliminate cytotoxicity on lcl-.221. cold-target blocking studies further suggested that the class 11-nonexpressing lcl-.180 and the class i-nonexpressing lcl , 2 2 1 share residual deterninant(s) other than hla class i or class i1 that can restrict cytotoxic t cell responses to ebv-induced antigens. national jewish center for immunology and respiratory medicine, denver, co 80206 it is uncertain to what extent lymphokines can be differentially produced by activated primary t cell populations. to determine if il4 and ifnr were differentially regulated in uncloned human t cells from adults (ad) and neonates (nt), these mrnas vere measured by in situ hybridization after maximal stimulation by ionoaycin and pma. il4 mrna was detected in 1.2% of total (tl), 3.5% of cd4', 10% of cd4' cd45r-, and 0.1% of cdb' ad t cells, but in none of the tl, cd4+, or cd8' nt t cell populations (virtually all nt t cells were cd45r'). in contrast, ipnr mrna was found in 39.42% of tl, 34.36% of c d 4 ' , 51% of cd4' cd45r-, and 58% of cd8+ ad t cells, but only 2.3% of tl, 2% of c d 4 ' , and 4% of cd8' nt t cells. these results agreed with other estimates of il4 and ifnr production based on ria of cell culture supernatants, rna blotting, and gene transcription assays. in contrast to il4 and ipnr, il2 was expressed in similar amounts by ad and nt t cell fractions, as well as the ad cd4' cd45r' and cd45r' subsets. thus, the capacity for increased il4 and ifnr production by ad t cells appears attributable, in large part, to the postnatal acquisition of the cd45r' subset (putative memory t cell population). aowever, additional mechanisms exist which act transcriptionally to limit il4 production by both neonatal and adult t cells. such selective expression may be important for restricting the potentially pleiotropic effects of certain lymphokines t o appropriate responder cells. we observed significant inhibition (>70% at 800 ng/ml) of the presentation of wova and of ova 323-339 by the anti-ap 57-78 peptide mab's. exhibited significant inhibition. 61 peptide mab's. after incubation with antigen +/-mab, indicate that the inhibition occurs at the level of antigen presentation. dg11, was also observed for the anti-p chain peptide mab's and to a lesser extent by the anti-a chain peptide mab's. peptide sequences are capable of interfering with antigen presentation, in vitro. supported by nih grant, ai-14764. the ovalbumin (ova) i-ad restricted t cell hybridoma, do1l.10 was used to the anti-i-ad mab, mkd6, also much less inhibition was observed with the anti-% 43-experiments with glutaraldehyde fixation of the b1d.p cells before or inhibition of i-ad allorecognition by the t cell hybridoma. these results indicate that mab's generated against class i1 rijllinghoff, institute for clinical microbiology, university of erlangen-nurnberg, 8520 erlangen, f.r.g. and the *institute for clinical immunology and rheumatology, university of erlangen-niirnberg, 8520 erlangen. f.r.g. recently we have shown that cloned l . major-specific l1/1 t-helper cells of type 2 (th2cells), when stimulated with antigen, are able to induce polyclonal b-cell proliferation (1). we here present evidence demonstrating that this process is dependent on a direct cellcell interaction between t-and b-cells. which in the effector phase, i.e. during stimulation of the b-cells by activated t-cells, can be mediated by a mechanism other than cognate interaction. this conclusion is derived from experiments, in which highly purified resting b-cells were polyclonally stimulated by l1/1 t-cells triggered by an anti-t3 monoclonal antibody, in the absence of antigen. the triggering process was independent of the presence of the fc part of the antibody and occurred in cultures devoid of macrophages. thus, the well established cognate recognition does not appear to be the only way of b-cell induction by t-helper cells of type 2. studies show that a proportion of the peripheral blood cd3' t lymphocytes do not express cd4 or cd8 and are called double negative t cells. they normally have a 1 6 tcr. however, another population of double negative t cells exists that expresses the a@ heterodimer. w e have purified and expanded such a population isolated from the peripheral blood of a healthy individual and studied i t s phenotypical and functional characteristics. the c e l l s are cd3' cd4-cd8-, positive for wt31 and negative for the nk markers. they express a and p mrna b u t lack ymrna. from surface iodinated cells were precipitated w i t h monoclonal pf1 two closely running bands (46 & 48 kd) . functional studies demonstrate that they proliferate to anticd3 and pha, t h i s response was blocked by cyclosporin a. there was no nk lysis b u t anticd3 induced l y s i s of target cells. the cells responded t o il-2 and il-4 as previously shown for other t c e l l s , b u t also t o il-3, a lymphokine thought t o affect mainly stem cells and not previously shown t o stiaulate growth of mature cells. long term growth of these c e l l s was also maintained by these cytokines . roberto biassoni , silvano ferrini , rafck p. sekaly , and eric 0. long , laboratory of immunogenetics, national institute 2f allergy and infectious diseases, nih, beth-md 20892, and istituto nazionale per la ricerca sul cancro , 16132 genova, italy. cd3-cells grown in vim in the presence of il-2 acquire the ability to l~s e a wide variety of tumor cells in an mhc-unrestricted manner. we have previously shown that cd3-16 clones expressed the cd3 epsilon gene but no functional transcript from cd3 gamma, cd3 delta, tcr alpha, tcr beta and tcr gamma genes. this result suggested that these cd3-16' cells represented an early stage in t cell differentiation. to test for expression of the tcr delta gene in these cells, rna from a panel of cd3-16' clones and from three highly enriched populations was hybridized with several dna fragments of the delta locus. abundant transcripts were detected with a c delta probe and a j delta 1 probe in 6 out of 8 clones and in all three populations. at least four different transcripts were present with sizes similar to those found in cd3' tcr gamma-delta' cells. however, the tcr delta transcripts in cd3-16' cells are most likely derived from unrearranged genes because no rearrangement could be detected in dna from an enriched population using a j delta 1 probe, and because these aanscripts hybridized to a dna fragment corresponding to the unrearranged genomic sequence 5'-upstream of j delta 1. expression of unrearranged tcr delta genes in cd3-cells provides further evidence that these cells belong to the t cell lineage. functional capabilities and by differential release of either il2 or il4 upon activation. we have produced a new monoclonal antibody to cd45 which has allowed us to separate normal murine cd4+ cells into two populations based on the density of expression of cd45 epitope. the separated populations seem to be analogous of subsets found in cloned t cell lines. cd4+ t cells with high density of cell surface cd45 after polyclonal activation produce il2 and mrna encoding ifw and il2. it does not produce il4 or il4 mrna. cd45 low density population on the other hand transcribes mrna for il4 and secretes il4 protein. data will be presented to demonstrate that the two subsets of normal cd4+ cells also differ in their proliferative response to mitogenic stimuli and to exogenously added growth factors. the substitution of v to l at 95 was the only change that could be discriminated by 2 of 9 allospecific ctl lines. suggesting that those 2 ctl lines recognize a2.1 plus a peptide whose presentation andlor binding is affected by the v to l substitution in the floor of the peptide binding site. in contrast, the l to w substitution at 156 (but not the other 2 substitutions) abolished the ability of the a2 molecule to present the viral peptide to 24 out of 25 peptide-specific a2.1-restricted ctl lines, suggesting that this substitution alters the presentation of the influenza matrix peptide but does not inhibit the ability of the peptide to bind to the a2 molecule. although y8tcr.s have a great potential for diversity, it remains to be determined whether this potential is realized in terms of expressed y6tcrs. preliminary studies in several laboratories have indicated that y6tcrs expressed in earlg t h r c y t e s and adult epithelial tissues are more restricted in diversity com ared to adult tc expressin thymocytes. we have derived a panel of cloned dendritic epigrmal t cells (jetc, lines and5ybridomas that express at least three types of y6 receptors -c 8, cy26 and c n . immunoprecipitation, northern and southern blot analyses, and sequence anazses of l gt 10 cloned cdna or olymerase chain reaction ( k r ) amplified cdna segments have been used to anal ze in detail &e extent of diversit in the expressed y and 6 chains and whether restricted airin o?y and 6 chains occurs. our resu& indicate that for this panel of cloned cell lines 7 and ! irkg is nonrandom and that variability in certain types of receptors appears to be restricted. %owever, we have observed significant 6 chain diversity in these cells that is obtained by the use of multiple v-regions, and n-region and junctional diversity. we are investigating whether the observed y and 6 chain pairing, and pattern of 6 chain diversity are present in other $tcr bearing cells or whether they are only characteristic of detc. activation of ctl precursors from murine unprimed spleen cells with ril-2 or ril-4 results in distinct lytic spectra, depending on which lymphokine is present. we have used allo-stimulation in limiting dilution analysis with subsequent testing on an allo-specific target (a20) and an mhcdeficient, non-specific target (rle). in the presence of ril-4 exclusively allo-specific ctl are generated, while ril-2 supports a proximately equal numbers of precursors that k~ll a20 and rie targets. dose response analysis of ril-2-supported killing activity indicates that the lytic spectrum is independent of the amount of ril-2 used, and therefore this il-2 effect is intrinsic in its activity on unprimed spleen cells. mixing experiments indicate that ril-4 can partially override the effect of il-2 on the generation of non-specific killer cells. split well analysis and cold target inhibition experiments are in rogress to ascertain the actual proportion of specific killer cells which can be generated with ril-2. be. are also testing the ability of cofactors, such as il-1 and il-6, to optimize the response of il-4 generated ctl. we conclude that il-4, not il-2, must be used when ctl are generated from unprimed spleen cells in mice. t r a n s c r i p t s i n y/6 tcr populations. i n t e r e s t i n g l y , these same v genes, as well as a further+crosshybridizing v gene previously designated va7.2, are expressed by peripheral a& tcr c e l l s as 1.6kb tcra transcripts. these data suggest t h a t b2a2-dn th represent a developmentally unique subset i n which both v6 and vg segments are non-randomly expressed. furthermore they i n d i c a t e t h a t there i s considerable overlap between the v a and v6 gene repertoires . indianapolis, in 46285 in order to detect the small amounts of lymphokines generated in vivo following antigen stimulation, we developed a co-culture system which allows for detection of il-2/4, il-3/csf and tnf from ln cells stimulated in vivo with picryl chloride (pcl). utilizing thb system in combination with facs analysis and receptor binding studies, we examined the production of these lymphokines in primary and secondary immune responses. during a primary immune response, the production of il-2 was not readily detectable on dl, peaked on d3 and was gone by d5. at no time were we able to demonstrate the presence of il-4. alternatively, the presence of il-3/csf and tnf was w i l y detected on dl, but olso peaked on d3. in comparison to primary responses, secondary immunization lead to at least two alteraticns. (i) peak production of all lymphoki es shifted towards dl. (2) although most lymphokines did not demonstrate increasea in the amount produced/lo cells, the amount of lymphokine generated/ln was vastly increased due to an increased number of cells. utilizing single and dual color facs analysis we also examined the ln cells for alterations in t cell subpopulations. during the course of the primary response: (i) the percentage of thy i+ and l3t4+ cells decreased until d3 and then began to recover, (2) the percentage of thy-i+, t4-,t8-cells peaked at the time of greatest lymphokine production (i.e.-d3) and (3) the il-2 receptor was expressed solely on thy-i+ cells, was detected on both t4+ and t8+ subsets and peaked on d3. most of these alterations also occurred during the secondary response, but their timecoune was shifted so that maximal effects occurred earlier (e.g., dl). finally. the maximal binding of radiolabeled il-2 by the ln cells following both primary and secondary sensitization correlated with the expression of the il-zr as detected by facs analysis. in addition, binding of radiolabeled il-4 demonstrated similar patterns except for the detection of significant binding on dl. these results demonstrate that (i) an ordered timecourse of lymphokine production occurs in vivo following exposure to antigen and (2) the secondary immune response to pcl is characterrd by an accelerated tempo of lymphokine production, rather than an increased level of lymphokine production/lo cells. activation as direct g-protein activation by a1f4 pi-hydrolysis using phorbol diester stimulation of pkc restores the inhibi$$ble phenotype and the ability to upregulate c-fos. even more interesting, sig-linked ca responses by vs2.12-c1.2 are equivalent to those observed in the wildtype wehi-231. resul$g suggest that contrary to current thought, sig-generated signals may not be coupled to ca fluxes via inositol phospholipid hydrolysis. thus, vs2.12-c1.2 is a new and powerful tool with which to analyze signalling through sig at the molecular level. unlike the wildtype, crosslinking of sigm on vs2.12-c1.2 did the signaling defect in vs2.12-cl.2-appears to be proximal to phospholipase c triggers pi-hydrolysis and bypassing these latter lmmunogenicity c 302 analysis of t cell receptor 7 chains from adult cd4-,cd8-thymocytes mark w. moore, i. nicholas crispe and michael j. bevan, department of immunology, research institute of scripps clinic, la jolla, ca 92037. the role of tcr 7 genes in t cell development has not been determined. to extend out understanding of the repertoire of tcr 7 expression, we prepared a cdna library from cd4-,cd8-adult balb/c thymocytes and cloned and sequenced 15 tcrq genes from this cdna library. we found that 2 clones were transcripts of the unrearranged c7, gene and that 3 clones terminated in the j7, region. nine of the remaining clones were v71,2-j7 c7 genes and five of these were in frame. only one clone corresponded to c71 and was v7 -jy c7jofned'in frame. sds-page analysis of the 7-chain proteins from the surface of both balb/c anzcdbl)6 adult cd4-,cd8-thymocytes did not detect the 32,000 mw vy c7 protein, but did detect the 35,000 mw v7c7 protein. these results suggest that despite the abundanc$%f pull-length, functionally joined, v7 c7 transchpts in the thymocyte subset, the protein product is not expressed on the cell surface as the prehfcted 32,000 mw 7 protein. finally, our analysis of the v-j jointing of the 7 genes reveals both flexibility at the v-j junction and extensive n-region nucleotide addition that lead to diversity of the predicted protein sequence. il5 in response to the same stimuli. e identification of these two subsets of cd4' helper cells is mostly based on studies performed with long-term cultured t cell lines and it is not clear whether these two subsets exist in vivo and represent distinct lineages of t cells. in particular, the frequency, tissue distribution and ontogeny of cells capable of secreting il4 in vivo is not known. these studies have been hampered by the fact that freshly isolated t cells from unprimed animals failed to secrete detectable amounts of ila and il5 when stimulated in vitro by lectins or alloantigens, whereas iu is readily detectable in these same cultures. data presented here indicate that freshly isolated t cells from unprimed animals can be induced to produce il4 in a receptor-de endent, antigen-independent manner upon stimulation by anti-cd3 antibodies. our results also stow that only cd4' and not cdst cells can be induced to secrete il4 and that cross-linking of the receptor is required for o timal activity. we believe that this approach will be useful in identifying in vivo cells recomittefto the th2 pathway and study their ontogeny, activation requirements and tissue distriiution. hlb, brussels, belgium. we have studied the murine tcr repertoire against the c-terminus of cytochrome c in association with certain alleles of the mhc class i1 molecule, eakepk (iek) and eakepb (ieb). for mice possessing these alleles, the majority of responsive t cells utilize one member of the variable val1 gene family in conjunction with a limited set of vp genes. as an extension of these studies, we have examined ie specific, alloreactive hybridomas derived from ie non-expressing (eab) cytochrome c non-responder mice to determine their usage of va and vp genes. tion assay showed that fourteen utilized the same val1 gene segment used by the majority of cytochrome c specific, ie restricted t cells and eight utilized a closely related val1 gene that also is associated with this antigen response. element most commonly used by cytochrome c-specific t cells was not found among the alloreactive hybridomas tested, @ genes less frequently used in the cytochrome response were expressed by seven of the 22 alloreactive hybridomas whose va segments were defined by rnase protection. determining recognition of ie molecules both in mhc-restricted, antigen specific immune responses and in alloreactive responses. the t-helper cells of seven mouse strains, representing 5 class i1 haplotypes (ias, ia4, iab, iakiek, iadied) were responsive to immunization and restimulation with parent peptide. the ied determinant was shown to be a presenting element by monoclonal antibody blocking and by use of l-cell-transfectants as af'cs to purified t cells and to t cell hybridomas. a series of overlapping synthetic peptides identified two minimal t-cell sites within the parent peptide: mice expressing ia and ie responded to a fragment at the n-terminus of the parent peptide (site 1) while mice expressing only ia responded to a distinct but overlapping fragment at the c-terminus (site 2 ) . these minimal sites identified in vitro could be used to immunize mice in vivo in an mhc-restricted manner. the human 6 tcr locus is strategically located within the atcr complex between the cluster of va/v6 region and the ja segments. which can be spliced to ca in pre t cells, separates 6 from the ja segments. pulse field gel mapping as we11 as molecular cloning link diversity (ds), j g , c,5 and tea within 35 kb. considerable 6 tcr diversity is generated despite the predominant use of one v 6 and j 6 segment. d61 and d62 are 9 and 13 bp long, are frequently recombine as d,1/d62? and reveal exonucleolytic trimning with extensive "n" segment addition. specialized 5' and 3 ' 6 deleting elements, 6 rec and p j a , separate the 6 locus from the a locus. cells with 6 rec/$b ja recombinations comprise most 6 deletion events although 6 rec recombines with 2 other major acceptor sites in fetal and post-neonatal thymic dna. the 5' 6 deleting element ( 6 rec) is evolutionarily conserved in the mouse and functional comparisons are underway. delete the 6 locus may prove to be the pivotal event establishing separate y6 and ae lineages. to study the mechanism of t-cell tolerance, transgenic mice were generated that expressed the mlsa reactive t-cell receptor (tcr) o-chain vb8 1 on -90 % of peripheral t-cells. in transgenic mice bearing mlsd, the numbers of high tcr expressing thymocytes and of thy 1.2+ peripheral t-cells were reduced. the cd4/cd8 ratio of peripheral t-cells was decreased fourfold compared to negative littermates. both mlsa and mlsb tcr &transgenic mice were able to mount a t-cell dependent antibody response against viral antigens whereas the capacity to generate alloreactive and virusspecific cytotoxic t-cells was impaired in tcr &transgenic mlsa, but not in transgenic mlsb mice. rna analysis and immunof luorescence with tcr vb-specific mab further revealed, that the expression of endogenous tcr 0-genes in these mice was suppressed. tolerogen-reactive lymphocytes, as measured in the mlr, in spite of their long-term acceptance of a skin graft bearing the tolerated antigens. lymphokine production by mlr+ tolerant lymphocytes is different from that of syngeneic normal lymphocytes. normal lymphocytes produce only il-2 in primary response to tolerogen, while tolerant lymphocytes produce il-2 and il-4. using limiting dilution analysis, we have to estimated the frequencies of pil-2 and pil-4 (precursor) cells in these cultures. after primary k vitro stimulation, normal responders have a low but measurable frequency of pil-4 cells, while tolerant responders have a much higher pil-4 frequency. however, following subsequent & restimulations, the pil-4 frequency of normal responders rises and begins to approach that of the tolerant responders, such that the two populations are indistinguishable based on pil-4 frequencies following the third round of in vitro stimulation. these data suggest that the high frequency of il-4 producers (presumably t,, cells) among the tolerant lymphocytes resembles unexpectedly a "primed" state, rather than "unprimed"as in nontolerant responders (where th1, dominate the early response). the existence of "primed" t cells in phenotypically tolerant animals raises the possibility that precocious activation of tr1 (by neonatal exposure to tolerogen?) suppresses the later emergence of t,,, which would be expected to contain the cells responsible for graft rejection. a large number of cd4+ t-cell clones, obtained from peripheral blood t lymphocytes by direct limiting dilution, allowed us to address the question whether functional heterogeneity exists within the human cd4+ t-cell subset. six out of 12 cd4+ clones were able to lyse daudi or p815 cells in the presence of anti-cd3 antibodies. the remaining 6 cd4+ tcell clones tested did not acquire this cytotoxic capacity during a culture period of 20 weeks. in the absence of anti-cd3 mab, no lytic activity against daudi. p815 and k562 target cells was observed under normal culture conditions. these two types of cd4+ t cells showed high reactivity with anti-cdw29 (4b4) mab and no reactivity with anti-cd45r (284) mab. the cd4+ clones without anti-cd3 mediated cytotoxic activities (th2) consistently showed a higher expression level of cd28 antigens. th1 cd4+ clones did produce il-2, ifngamma and tnf-alpha.beta. whereas the th2 t-cell clones produced minimal amounts of il-2. ifn-gamma and tnp-alpha. beta in response to anti-cd3 mab and pma. not all cd4+ clones did release il-4, but there was no correlation with cytotoxic activity. moreover, as compared to the th1 cd4+ clones, tr2 cd4+ clones proliferated moderately in response to anti-cd3 mab. however, anti-cd3 mab induced proliferation of only the th2 cd4+ t-cell clones was enhanced by anti-cd28 mab. both cd4+ subsets provided help for polyclonal b-cell activation with anti-cd3 mab. our data suggest that the human cd4+ subset, in analogy to the murine system, comprises two functionally distinct t-cell subpopulations. in the mouse, when la antigens are isolated immunochemically, the predominant species isolated are the isotypic matched pairs, aaap and eaep. however, when la ap dimer expression is studied using an l cell transfection model, it is found that the isotype-mismatched dimer apdea is readily expressed at the cell surface. these results suggest that differences in assembly andl or transport of different la pairs may be most readily visualized in a competitive environment where multiple distinct la chains are available. to investigate this possibility, the relative efficiency of inter-and intra-isotypic dimer formation and expression was evaluated using a sequential l cell transfection system. l cells already expressing an ap dimer on the cell surface (apdea or apdaad) were supertransfected with a third la gene (aad or ea, respectively). synthesis of this second a or p protein led to competition for the unique partner chain. individual clones were scored for cell surface expression of the distinct dimers (e.g., apdea vs epdea or apdaad vs apdea) using facs analysis with chain specific monoclonal antibodies. in addition, each species of mrna was quantitated by northern blot hybridization using bcus specific probes. our results indicate that in the h-2d haplotype. isotype-matched dimers are expressed with 3-4x the efficiency of isotype-mismatched dimers. this result suggests that, regardless of the cell type studied, if each of the four murine la genes is expressed at equivalent levels, intraisotypic dimers will be expressed to the virtual exclusion of the interisotypic dimers. however, if chain synthesis asymmetry occurs, the isotype mismatched pairs may be expressed at immunologically relevant levels. differential we have identified a series of discrete stages among the cd3-double negatives which seem to form a sequence, with tcr gene rearrangement and rna expression gradually progressing, but with potential for expansion and repopulation of irradiated thymuses diminishing along the series. on this pathway cd3 must be expressed late or after the acquisition of cd4 and cd8. cell cycle analysis shows the highest rates of cell division to be among the rsa+ il-2r-pgp-1-population which probably precedes the transition to cd4+cd8+ and tcr expression. thus it seems unlikely that tcr/antigen interactions play a role in cellular events occurring among the double negative cells which lead on to mainstream t-cell development. egr-l is a murine early growth factor inducible gene which encodes a protein with zinc fingers. its expression was investigated in murine b-lymphocytes stimulated through their antigen receptor (sig) with anti-recptor antibodies (anti-ig) . rapid (by 15 minutes) upregulatlon of egr-l mrna expression was observed at doses of anti-ig sufficient to drive the majority of go cells into cell cycle. agonists and inhibitors of protein kinase c (pkc) showed that expression was coupled to the pkc component of receptor immunoglobulin transmembrane signalling. interestingly, signalling through sig on the murine b lymphoma wehi-231 did not upregulate egr-l expression even though similar signalling pathways are associated with this receptor in these cells. southern analysis showed that egr-l is not deleted or translocated in this cell line. importantly, cell growth and proliferation of wehi-231 is inhibited by anti-ig stimulation suggesting a relationship for egr-l expression and differential processing of receptor ig signals. this notion is further supported by the finding that murine b lymphomas whose proliferation is not inhibited by anti-ig showed receptor immunoglobulin coupled egr-l expression. reeulation of exdression of a class 1 wc transeene. dinah s . the expression of the transgene product. the patterns of expression of the transgene parallels that observed in situ, indicating that regulatory elements necessary for normal patterns of expression are contained within the injected 9 kb dna segment, and that trans acting factors involved in its regulation function between species. included among these elements are those specifying preferential expression in b cells relative to t cells. in vivo treatment of transgenic mice with a/@-interferon results in increased expression of the transgene in a number of tissues. the response parallels that observed for the endogenous h-zkb, but differs markedly from qa-2. analysis of the chromatin structure of the transgene reveals a single constitutive dnase i hypersensitive site present in both spleen and thymus, which is not altered by interferon. both a novel negative and positive regulatory elements have been identified in the 5'flanking region of the transgene. the negative regulatory element reduced the activity of both the homologous class i promoter and a heterologous viral promoter. in vivo competition experiments indicated that the functions of the positive and negative elements are mediated by distinct cellular trans-acting factors. the negative regulatory element requires the presence of a positive regulatory element to function. this interaction between elements represents a novel mechanism for regulating gene expression. mcdevitt. department of microbiology and immunology, stanford university school of medicine, stanford, ca 94305. published data show that encephalitogenic h-zu murine t cell clones with specificity for the n-terminal eleven amino acid peptide of myelin basic protein display a restricted fine specificity when tested on substituted analogs of the native peptide. for example, substitution of alanine at certain positions in the peptide totally abolishes the response of each clone (acha-orbea et al.. 1988. cell 54:263) . recent experiments also have shown that the ability of some peptide analogs to bind to h-zu i-a gene products does not always correlate with their ability to stimulate the t cell clones (see accompanying abstract by david c. wraith and hugh 0. mcdevitt). this suggests that h-2u mice may lack a t cell repertoire capable of recognizing these peptides complexed to h-2u i-a gene products. to test this possibility, h-2u mice were immunized with a panel of peptide analogs, as well as the native peptide. the in vitro t cell proliferative response to each of the peptides then was measured. the results show that in vivo immunogenicity of the peptide analogs also does not strictly correlate with their capacity to stimulate the t cell clones. in this way, the polyclonal t cell repertotre of h-zu mice for the myelin basic protein peptide analogs was examined, and could be compared with the i-a binding characteristics of the peptides. terms of antigen and mhc recognition. this response involves a limited repertoire of t cells which crossreact on species variants of the antigen. in addition, t cells specific for the antigen in association with syngeneic mhc can recognize antigen on similar allogeneic mhc molecules. the groupin of clones by functional phenotypes defined by these crossreactivities allowed us to corrcfate tcr gene usage with either antigen or mhc recognition. some of the pigeon cytochrome c-specific clones within one functional phenotype use receptors that differ by as few as two amino acid residues. other clones e y s s very different tcrs but exhibit similarities in antigen/mhc reco nition. the efect of these tcr differences on recognition was assessed using a pane? of anti en analogs with single amino acid substitutions presented on different mhc molecufes. each clone exhibited a unique pattern of res onse to the antigen analog panel, even clones with very similar receptors. also, eace residue in the antigenic region of the peptide was critical for interaction with at least one t cell receptor. therefore, the antigen must either be a linear molecule with each residue available to interact with the tcr or be able to assume several conformations to interact with mhc and the tcr. lmmunogenicity c323 thy-1+ cd3+ ly-5(b220)+ cd4-cd8-tcrx-6' helper cells. anne i. we have found that these cells can be preferentially stimulated to proliferate when cocultured with the b lymphoma, ch12. one to 2% of nylon wool non-adherent, ia-, jlld-, and cd8-lymph node cells from normal unimmunized mice have the phenotype thy-l', cd3', cd4-. and cd8-. these cells proliferate when co-cultured with a syngeneic surface ig' lymphoma, ch12, even in the absence of any added antigen, mitogen, or fetal calf serum. prior to stimulation we find that approximately 30% of thy 1.2' cd3' cd4-cd8-express the marker ly-5(b220), however after culture with ch12 the majority of cells with this phenotype express the marker ly-5(b220). after ch12 dependent proliferation the ly-5(b220)' t cells are able to provide help for secretion of ig by fresh ch12 b cells. surface labelling and precipitation of t cell receptor molecules reveals that most of the thy-1' cd3' ly-5(b220)* cd4-cd8-cells express tcr(r-6). furthermore, cd3 precipitation shows that as many as four different 7-6 heterodimers are utilized within the entire responding population. this suggests that a heterogeneous population of double negative tcri-6 cells are involved in the response to ch12. college of kedicine at east tennessee state university, johnson city, tn 37614 interferon-producing (t 1) and interleukin 4 (il4) producing (t 2) clones were assayed for their ability to diregtly induce cytostatic activity in macro:hages generated from splenic myeloid precursors (m -c). in the presence, but not in the absence, of antigen, t 1 clones activated the m -c to inhibit the growth of p815 tumor cells in vitro. th2 cjlones were not able to activate such effector activity in the i4 -c. effectively present antigen to the t 2 clones as evidenced by the proliferation of t 2 cells cultured with antigen in the pfesence, but not in the absence, of m -c. thereyore, although both t 1 and t 2 were activated by cognate interaction with antigen presenting (ba) or nippostrongylus brasiliensis (nb). spleen cells from these mice were cloned at limiting dilution with alloantigen stimulation, and every two weeks, lk production in response to con a was measured. clones derived from, and stimulated with, cells from unimmunized mice initially tended to secrete low lk levels, with few clearly defined th1 or th2 clones. by 56 days after cloning, some clones had acquired th1 or th2 patterns. cfa, ba and nb-imnunized mice gave rise to clones that were mostly th1 or th2 even at early times. cfa and ba immunizations induced almost exclusively th1 clones, whereas nb induced more th2 clones. these results are consistent with a model in which resting, previously unstimulated t cells produce low amounts of lks, and progress through stage(s) where they secrete both th1 and th2 lks before finally differentiating into th1 and th2 cells. the results with cfa, ba and nb-primed mice suggest that this process occurs in vivo as well as in vitro. strains as carriers of melioidosis antigens to the immune system, deja tanphaichitra, mahidol university, p.o. box 4-217, bangkok 10400, thailand the attenuated gale mutant, salmonella typhi strain, tyzla, served as the recipient in a conjugal dna transfer experiment. conjugal dna transfer was obtained by the mating procedure on an appropriate blood agar medium. were examined serologically. one selected strain was found to have the serological characteristics of the recipient s . typhi, tyfla strain and also expressed the pseudomonas the donor strain was a pseudomonas pseudomallei mu107. the resulting antigen clones were repurified by restreaking on the medium and pseudomallei antigen. the s. typhi transconjugant strain is due to the presence of the pseudomonas pseudomallei plasmid. a group of subjects when received four doses of this bivalent vaccine strain in this study it appears that pseudomonas pseudomallei synthesis in developed antibodies against pseudomonas pseudomallei up to 70%. pseudomallei, an intracellular pathogen, produces a characteristic antigen probably to be plasmid coded, we considered that the gale salmonella typhi tyzla oral vaccine strain, highly effective against typhoid fever, might be modified so as to be protective also against melioidosis due to pseudomonas pseudomallei. terminal deoxynucleotidyl transferase (tdt) is a lymphoid-specific nuclear enzyme present in early lymphocytes. to investigate the regulation of tdt gene expression, pre-b and pre-t cells were treated with phorbol 12-myristate 13-acetate (pma) o r three analogs, and tdt steady-state mrna levels were determined by northern blot analysis. treatment of early lymphocytes with pma results in a rapid and reversible decline in steady-state tdt mrna levels within six hours. this rapid decline can be blocked by pretreatment of the cells with a protein kinase c inhibitor, implicating protein kinase c activation in the decline of tdt mrna. nuclear run-off studies demonstrate that tdt transcription is rapidly down-regulated within 45 minutes after pma treatment, indicating that this regulation occurs mainly at the level of transcription. furthermore, cycloheximide blocks the decline in tdt in rna showing that new protein synthesis is required for transcriptional inactivation. the nucleoprotein gene from the influenza virus a/nt/60/68 was stably cloned into the attenuated aroa-strain of salmonella typhimurium sl3261. nucleoprotein purified from pnp -3261 was tested for the ability to generate virus-specific immunity. immunization with recombinant derived nucleoprotein induged immunity to all type a influenza tested but not against type b viruses. cd4 helper t cells were primed but no evidence was found for priming of class i restricted ctc. mice immunized with recombinant nucleoprotein were protected against a subsequent challenge of influenza virus. the information obtained from the study of the immunity and protection generated by the purified recombinant protein was then used to design experiments to investigate the possibility of using the attenuated salmonella vector to deliver the nucleoprotein molecule to the immune system by the parenteral or enteral routes. we characterized the extrachrom-1 circular i n i s in 19-day-fetal and 4-week-old m u r i n e thpmcytes and 8-week-old m u r i n e splenocytes. f popllation of circular chias was clone3 into the kgtll phase vector. we screened ca. 10 tna cl-by plaque hybridizations with all far kirds of tcr gene probes derived from jal , val 0, db1, db2, jyl , j61 and 562 loci. cut of 10,000 cna cl-from fetal and 4-week-ld thymocytes, 30 hybridized with tcr aprobes and 5 hybridized with tcr &probes. positive cl-with tcr yand 6probes were 3 to 7 in fetal thymocyte4erived library, but few in 4-week-old thymocyte. of 7 fetal tcr 6 clcnes analyzed, 6 cl-had dd or vd reciprocal joints and 1 clcne had vd ar dd d i n g joint. relative frequencies of circular dna clones for four different tcr genes are consistent with the order of the expression of the genes the t cell developnent. of 10,000 tna clsignalling could be studied. llzmambxane signalling was maasured by 9 ability to t?z3nslocate fkc frcrm the cytcplasa to the nw2leus after surface i-a was banrl by a or p dxdn specific monoclcnal antibody. i(pmwing either 6 or 12 amino rids fmn the a chain cvtoplasnic (cy) damin did not affect the ability of tkse i-a r m l d e s to trarslocate pkc to the nucleus. normal splenic b c e l l s were rendered non-responsive t o subsequent challenge w i t h lps, as measured by a decreased a b i l i t y t o generate antibody forming c e l l s (afc), by incubation overnight (18-24 hours) w i t h 10 ug/ml a n t i -i g . both i n t a c t and f(ab)', a n t i -i g , as well as monoclonal anti-igm (bet2 and b-7-6) , were able t o induce 6 c e l l non-responsiveness t o subsequent lps challenge, suggesting t h a t sig/fcr i n t e r a c t i o n s are not necessary i n the induction o f lps non-responsiveness. i n contrast, induction o f nonresponsiveness t o subsequent challenge w i t h fitc-prucella abortus required i n t a c t a n t i -i g . the a b i l i t y o f mitogenic a n t i -i g (rab f(ab)', o r 6-7-6 northern blot analysis and bioassay data were used to analyze 9 separate lymphokines as well as the il-2 receptor (murine tac). northern blot comparison of fresh and primedt4 enriched rna revealed that primed t cells produced 10-fold more lymphokine than the fresh t cells. the only lymphokine that showed equal amounts of mrna for both fresh and primed t cells was il-2. a time course of fresh and primed t4+ cell lymphokine production was also analyzed. the primed cells produced a short burst of lymphokine mrna that peaked between 7.5 and 13 hr after con a stimulation and declined after 18 hr. the fresh t cells produced a longer burst of lymphokine mrna that peaked 18-44 hr after stimulation. the il-2 receptor @-2r) mrna time course from activated primed cells showed different kinetics than lymphokine mrna. this suggested that molecular regulation of the il-2r might be different than lymphokine regulation. to further examine molecular regulation in the primed t cells polysome profiles were evaluated for lymphokines, l 2 r , and other cellular genes. the recently developed method of gene amplification by the polymerase chain reaction (pcr) has proven to be particularly suited for the analysis of t cell receptor (tcr) genes. we adopted existing methods for the preparation of cytoplasmic rna from as little as 1000 cells and used this material as template for first strand c-dna synthesis. pcr amplification of this c-dna, using v-and c-specific oligonucleotide primers yielded enough material to produce single-stranded dna in a second pcr which could then be sequenced without cloning. in case of unknown v-usage, the pcr was employed for screening for v-beta elements by sequential reactions with different v-beta specific primers. we have used this method to reinvestigate the h-2b restricted cytotoxic t cell response to tnp in c57b116 mice. beta chain sequences of 26 ctl clones obtained by direct cloning of immune spleen cells were compared to sequences of 11 clones obtained by cloning of individual short-term in vitro ctl lines. it was found that a) in vitro bulk-stimulations reduced the heterogeneity of the beta-chain responses to tnp, b) similarities between different tcr-beta-chains concentrated on the usage of certain jb-elements ( jb2.6, 2.5, 2.1 ) rather than v-region or nid-region sequences, and c) the majority of jb2.6 containing beta-chains was associated with alpha-chains expressing v-segments of the val 0 family. these expression of genes which encode the t cell antigen receptor is cenval to the generation of the t cell repemire. our labomtory has been investigating genes for both the alpha and beta chains of this receptor in inbred strains of runus norvqicus (the laboratory rat), a species in which several autoimmune disease models have been developed. and which is used extensively in transplantation studies. using genomic southern blots and mouse probes specific for five different v a subfamilies, we have estimated the size of the v a repertoire in ten inbred strains of rat. results show a significant increase in the size of one subfamily and suggest increases in two others in all ten strains. the rat v a l subfamily has about twice as many members as the mouse, while the va2 and vu5 subfamilies, depending upon the enzyme used, show a similiar duplication. the va6 and va9 subfamilies have a comparable number of members in both species. these data are most easily explained by a single duplication event in the rat invoking at least one and perhaps three subfamilies, but not encompassing the entire v a locus. this implies that the val subfamily (perhaps together with va2 and va5 subfamilies) is regionally clustered and not interspersed with either the va6 or va9 subfamily. based on restriction fragment length polymorphisms, we find evidence for six distinct v a haplotypes in the ren strains tested. we have also cloned eight unique germline v a l gene segments. one of these has been sequenced. and has a coding region 87% identical to the most closely related mouse v a l sequence. this degree of relatedness is similiar to ra#nouse vg homologues. which share 8548% nucleotide sequence similarity. we are using these clones to generate angle copy probes from flankiig regions to further map the v a l locus. current approaches to mhc-peptide binding studies require either large quantities of highly purified mhc protein and/or the use of sophisticated detection apparatus. i n order to simplify detection of peptide-mhc interactions we have investigated the use of photosensitive-crosslinkers. two reagents have been successfully tested. a benzophenone derivative of peptide 1-16 from rat myelin basic protein (rmbp) was only effective after the introduction of a glycine spacer residue between peptide and crosslinker. an azido-nitro-benzoyl derivative of peptide 7.4, a heteroclitic analog of rmbp 1-11 (1). had a high affinity and bound specifically to the peptide binding site. the 7.4 photoaffinity probe has been used to test the binding properties of other analogues of rmbp 1-11 and is currently being used to define (a) the kinetics, (b) ph and (c) temperature dependence of the binding event. this particular photoaffinity conjugate retains both the mhc binding and biological properties of the original peptide and is helping us to define the roles of "determinant" versus "t cell repertoire" selection in the mhc linked autoimmune response to mbp the antigen-specific t cell repertoire is diverse in its ability to recognize a wide universe of foreign antigens. this t cell repertoire is composed of a set of clones each of which is specific for a given foreign antigen. therefore the precursor frequency of t cells specific for any give foreign antigen is extremely low. however, two prominent exceptions to this general rule exist, and these are the t cells present at high precursor frequency which are specific for foreign hhc products or for the products of the minor lymphocyte stimulatory (mls) genes in the mouse. the present studies were undertaken in order to examine factors involved in t cell repertoire formation by assessing the relationship between t cell repertoire for conventional foreign antigens and for mls products. studies indicate a striking degree of overlap between the set of t cells specific for pigeon cytochrome c and the set of t cells specific for mlsc gene products. demonstrate that the basis for this overlap lies in the predominant expression of one tcr vp gene, vbs, by those t cells which recognize mlsc. involvement of specific tcr afl dimers in recognition of mlsc and further suggest that t cell reactivity to these gene products may play an important role in establishing the t cell repertoire for foreign antigens. conclude that, rather than destruction of some essential apc structure, ecdi fixation prevents the apc from actively responding during the encounter with the t cell. this results in a failure to express new structures (probably located on the apc plasma membrane) that appear to be essential for stimulating t cell proliferation. these structures are distinct from ia or il1. the induction of these structures during t-apc interaction occurs in six hours, requires protein synthesis, and can be elicited by il1, il4 or lps, but not ifn-gamma. in the absence of these induced structures, the apc stimulates a partial t cell response, il4 release, but the t cells fail to proliferate. these induced structures on the apc may be either adhesion molecules that stabilize the t-apc interaction, or they may provide additional stimuli to the t cell. were not c m n t o the three s t r a i n s o f mice (balb/c, regions 1, 3, 5, 6 and 7; c3h/he, regions 2, 3, 5, 6, 6', 7 and 7'; and c57bl/6, regions 2, 4, 5, 6 and 7). immunisation with type i1 collagen (cii) leads to development of arthritis in mice with certain mhc haplotypes and is associated with an immune response against cii. we have been studying the t-cell response in the arthritis susceptible strain dbm1 (h-2q) . analysing the proliferative response in cultures of lymph node cells from immunised mice a s well as t-cell lines and clones established from such cultures it was found t h a t li the t-cell response after immunisation with heterologous cii was preferentially directed against foreign determinants on the cii molecule with little o r no crossreactivity against autologous cii. 2 ' both the primary response and the reactivity of established lines and clones were directed against the cbll fragment of the cii molecule, using c b l l fragments prepared from chick, bovine or rat cii. 3/ pepsin present in cii preparations after using pepsin digestion for solubilisation of the collagen is strongly immunogenic even in very small amounts and it was therefore necessary to use cii prepared from lathyritic cartilage without pepsin digestion for immunisation. in contrast to the pattern in lymph node cultures from immunised mice we found that when culturing spleen cells from unimmunised mice there was a t-cell response against collagen that was preferentially directed against autologus cii. since we earlier have found that autologus cii may induce an immune response and also arthritis in dbn1 mice we conclude that there exist t-cells capable of reacting with autologus collagen and inducing an immune response as well as arthritis but that these cells are under regulation so that they not readily can be activated into proliferation but may be induced to perform certain effector functions. tested. the characterization of these two cd6 rdsas will be presented. analysis of hla polymorphism using sequence specific oligonucleotide probe hybridization to amplified dna, lee ann baxter-lowe, jay b . hunter, and jack gorski, the blood center of southeastern wisconsin, milwaukee, wisconsin 5 3 2 3 3 . hla polymorphism plays a key role in antigen:mhc interaction. the polymorphism of the first domain encoding exon of the hla-dr p chain has been studied by in vitro dna amplification and use of sequence specific oligonucleotide probe hybridization (ssoph) to detect polymorphic sequences. a 230 bp segment of genomic dna was amplified and hybridized with synthetic oligonucleotide probes (12-19 bases) under conditions that detect single base pair mismatches. identification of these mismatches can be used to predict micropolymorphism in the protein products, including single amino acid changes. haplotype specific patterns of oligonucleotide probe hybridization were defined for a panel of homozygous typing cells. analysis of family data demonstrated the expected inheritance patterns. most known serological specificities are encoded by multiple allelic forms of dr p chains and ssoph can identify these differences. this was exemplified by detection of unique ssoph profiles for subtypes of dr4, drw6 and drw52 alleles. this procedure was also used for analysis of hla-dr polymorphism in large numbers of heterozygous individuals, including an hla-deficient scid patient. the ssoph data were correlated with serological specificities and will be useful for delineation of hla restriction in alloand autoimmunity. different cell membrane receptors have been shown to be involved in human t lymphocyte activation induced by either monoclonal antibodies or mitogenic lectins. these t cell surface molecules can be divided into two categories : a) the t cell antigen receptor (tcr) associated with the non-polymorphic cd3 antigen b) t cell differentiation molecules not linked to cd3/ti such as cd2 (t11) and tp44 (9.3). monoclonal antibodies directed against these t cell surface structures triggered different t lymphocytes functions : mitogenesis, il-2 receptor expression, il-2 secretion. our knowledge about early events involved in t cell membrane activation is not complete, especially involving the transduction mechanism mediated by gtp-binding proteins ; nevertheless, numerous authors have demonstrated that cd3/ti complex triggering induces the activation of phospholipase c, leading to the phosphoinositide cascade associated with an increase of free cytoplasmic calcium ions. in the present report, we show that different activating cell molecules (con a , pha and pma) can trigger oxygen free radical liberation when incubated with the human jurkat tumor t cell line. since membrane oxidative metabolism has been shown to be related to the stimulation of the phospholipase a2, and to be the final consequence of a membrane nadphoxidase : this could represent a previously undescribed pathway of t lymphocyte activation. high affinity monoclonal antibodies (mab), specific for staphylococcal nuclease (nase), were produced and characterized. competitive inhibition assays were conducted resulting in a series of complementation groups that define eight overlapping epitopes. it is estimated that these epitopes account for 70% or more of the accessible surface of nase. mutagenesis of the coding sequences for nase was carried out to produce a series of variant molecules (each differing from wild-type. nase and from each other by a single amino acid) that will enable mapping of nase epitopes, determination of residues involved in antibody binding, and the contribution of various physical and chemical factors to affinity and fine specificity. screening some of these mutants with the panel of mab enabled us to map several nonoverlapping epitopes and further subdivided some of the mab complementation groups. oligonucleotide-directed mismatch mutagenesis has been done on codons encoding the original amino acid residue and other surface residues in its immediate vicinity. determination of enzyme activity and structural analysis by cd spectropolarimetry of several of the mutant proteins suggests that any structural changes that may occur are local and not global. supported by grants ai20745, l32ca09109 and s07rr05431 from the national institutes of health. activation of t cell proliferation is believed to occur via the hydrolysis of inositol phos holipids, which, through the second messengers inositol-1,4,5-tris hosphate and diacylglycerof(dag), promotes the elevation of intracellular calcium levels anjactivation of protein kinase c (pkc), respectively. the role of pkc in t cell activation was investigated by comparing the effects of stimulation by 12-0-tetradecanoyl phorbol acetate (tpa), and the dag, oleoylacetyl glycerol (oag), on a >99% pure population of t cells cultured in rpmi 1640 medium containing 10% autologous serum. treatment with either tpa or oag caused down-regulation of the t cell rece tor, a consequence of its hosphorylation, but only tpa, in syner leuiin 2 receptor (il2-r), expression and, sgsequently, proliferation. immunohistochemical staining with antisera specific for the pkc subspecies a, pi, pii and 7 shows that restin t cells express a, pi and pii pkc subspecies which are diffusely distributed throughout the celt. after 20 minutes treatment with either oag or tpa all three subspecies are redistributed to a focal area within the cell. the redistribution is transient in oag stimulated cells, where the pkc distribution is similar to that in untreated cells after 1 hour of treatment. in tpa stimulated cells, however, the pkc redistribution is prolonged, becoming more marked until mitosis occurs after 48-72 hours of treatment. these results suggest that transient intracellular redistribution ofpkc causes phosphorylation and down-regulation of the t cell receptor, but that prolonged redistribution is required or t cell proliferation. sm is a nucleoprotein complex associated with small rna molecules in eukaryotic cells. the spontaneous generation of anti-sm antibodies is specific for patients with systemic lupus erythematosus (sle) and develops in 2 5 % of mrl mice. the response has been shown to be t-cell dependent in mrl/lpr mice. t-cells specific for sm are found only in mrl (h-2k) mice and mice bearing h-2s and h-2f haplotypes (which do not develop anti-sm antibodies). we are currently working to define the variable regions of the t-cell receptor genes used in the sm response. a series of t cell hybridomas from mrl mice has been generated and are being screened for sm positivity. a technique has been designed to amplify specific alpha and beta chain tcr genes using the polymerase chain reaction allowing for a more rapid sequence analysis. it is also our intention to locate the sm specific epitopes of the t-cell hybridomas. d. bloom, p.l. cohen, and s.h. clarke, department medical institute and experimental immunology branch, nci. nih, bethesda. md 20892. to determine whether prior activation history affects t cell receptor mediated activation of t cell clones, the murine type i helper clone ae7 was maintained in tissue culture by stimulation every ten days with either (1) antigen (cytochrome c), irradiated h-zk spleen cells. and il-2 or (2) il-2 alone. ae7 cells grown with antigen and antigen presenting cells (ae7-ag) proliferated and produced t cell growth factor activity (tcgf) in its culture supernatants following stimulation with immobilized anti-t3 antibody. the tcgf activity was shown by bioassay using indicator cell lines and specific blocking antibodies to be almost entirely due to gm-csf with little or no il-2 activity detectable. detectable il-2 mrna levels. (ae7-ilz) displayed substantially greater anti-t3 induced proliferation than did ae7-ag cells. in contrast to ae7-ag cells, ae7-il2 cells produced large quantities of il-2 in response to anti-t3 stimulation. furthermore. one cycle of stimulation of clone ae7-ag with il-2 in the absence of antigen and irradiated spleen cells was sufficient to cause this clone to produce substantial amounts of il-2 upon subsequent anti-t3 stimulation. these data suggest that t cell receptor mediated stimulation of t cell clones by specific antigen and antigen presenting cells inhibits subsequent anti-t3 induced il-2 production. t cell proliferative responses and sera antibody levels of myasthenic patients to several synthetic peptides representing different epitopes of the human achr were examined. we detected significant differences in the humoral and cellular responses of mg patients compared to healthy controls to peptides of the human achr alpha-subunit with sequences p195-212, p257-269 and ~310-327. proliferative responses of lymphocytes from myasthenic patients to p195-212 and to p257-260 correlated significantly with hla-dr5 and with hla-dr3, respectively. in order t o investigate further the immune responsiveness to selected sequences of the human achr, t cell lines and clones specific for peptides p195-212 and p259-271 were established from lymph nodes of c3h.sw mice. the recognition specificities of these lines were tested by examining crossreactivity to a series of shortened and/or extended peptides of the above sequences. deletions of amino acids in positions 211 and 212 (211=p, 212=l) resulted in a decrease of the peptides' stimulatory activity on the ~195-212 specific t cell line, whereas deletion up to position 200 on the n-terminal end had no effect on the triggering potential of the peptides. similar results were obtained when deleting residues 270 and 271 (270=v, 271=p) in stimulation assays of the p250-271 specific t cell line. help in determining important t cell epitopes on the human achr. the role of guanine nucleotide binding regulatory proteins (g proteins) in the regulation of phosphorylation of the y subunit of the cd3 antigen has been examined. cd3 y chain phosphorylation in isolated t cell microsomes or permeablised t cells was stimulated by the g protein activator, guanosine 5'-0 thiotriphosphate (gtpys), but other nucleotides such as camp or gdpbs were ineffective. dependent. these data are consistent with the involvement of a g protein in the signalling mechanisms that regulate the phosphorylation of the cd3 y chain. the regulatory effects of calcium and gtpys were compared in normal peripheral blood derived t cells and jurkat cells. there were differences regarding g protein regulation of cd3 y chain phosphorylation in normal t cell and jurkat cells and current models explaining these differences will be described. expression of the gamma-delta t cell receptor has been thought to first occur in a population of thymocytes shortly after their precursors populate the thymus between 11 and 13 days of gestation. in the course of our studies investigating the ontogeny of t cell receptor expression in the mouse embryo we have identified an extrathymic site of gammadelta expression in a population of cells present at distinct times of gestation. evidence will be presented demonstrating two periods of activity of the murine gamma locus in the developing embryo. are colonizing the thymus from the liver and the gene segment useage detected is different to that first expressed in cells of the developing thymus. around the time of birth) involves the functional rearrangement and expression of a gamma gene segment corresponding to the initial functional rearrangements detected earlier in gestation in the thymus, which can occur independently of thymic influence. demonstrate a new site of gamma-delta receptor expression in the liver of newborn mice that can occur in the absence of any thymic influence. the primary (in vivo) response of cs7bl/6 animals to the class i antigen qa-1 is a helper (th) dependent event as indicated by the requirement for copriming with a distinct antigen capable of activating helper cells. in contrast, the secondary (in vitro) response to qa-1 demonstrates no need for costimulation with the helper antigen. in attempts to more closely examine the helper requirements for activation of primed ctlp, we have observed that depletion of l3t4 cells from spleens of qa-1 primed mice abrogates the in vitro generation of anti-qa-1 effectors. the response is restored by the addition of concanavalin a induced supernatant (cas) or by the addition of syngeneic but not qa-1 allogeneic l3t4 cells. indeed, even in the presence of cas, l3t4 cells expressing the qa-1 alloantigen specifically suppress the activation of anti-qa-1 ctl in a manner reminiscent of that seen with lyt-2 veto cells. although the mechanism whereby l3t4 cells exert suppression is unclear, we have determined that ctlp are susceptible to veto only within approximately the first 48 hours of culture, after which they resist suppression. results from further studies of the nature of suppression and the l3t4 veto cell will be presented. group i1 proteins induce ige ab responses in -90% of mite allergic patients. murine mab and human igg and ige ab. unrelated. crossreactive epitopes on gpi and gpii allergens from different mite species. in contrast, igg ab in balb/c mice immunized with loug specific" epitopes and <1% was a n t i -u i (a gpi homologue, with -80% amino acid sequence homology to i). four non-over lapping epitopes were defined by mab, with one species specific immunodominant site on each gpi allergen. cross-reactive gpi epitope and this mab could inhibit human ige ab binding by -40%. specificity of the murine anti-gpi response was not h-2 restricted, but could be altered by immunizing balb/c mice with lower ag doses (lug) in alum or 8 . dertussis. using these regimes, up to 52% of the murine igg ab responses was gpi cross-reactive. responses to gpii allergens appear to be strain dependant. unresponsive to gpii. however, balb/b, a/j, cba, c3h 6 c57b16 all produce gpii cross reactive igg ab. anitgenic sites on gpi allergens are conformational, whereas those on gpii may be sequential. known to affect ige expression in mice may also affect the epitope specificity of igg ab. we have compared the b cell epitopes on these allergens using panels of however, ag binding ria on 73 sera showed that human igg and ige ab recognize the gpi and gpii allergens are antigenically i in cfa was directed against "species murine ab balb/c are completely thermal denaturation and reduction and alkylation expts suggest that the results with the gpi allergens suggest that immunization regimes which are c425 this report demonstrates for the the exclusive recovery of 72-84-specific t cell clones c 426 further molecular analysis should identify and characterize achr reactive autoimmune clones and/or suppressor cells. cohplex, mogens h. claesson, p e t e r bkams a n d s t e e n d i s s i n g , l a b . e x p . h e m a t . immunol., d e p t . med. anatomy a, a n d d e p t . g e n e r a l p h y s the ly-6 alloantigens represent a family of phosphatidylinositol anchored proteins that function as accessory molecules in the process of t lymphocyte activation. the expression of these alloantigens is often induced on t and b lymphocytes after activation by mitogens or antigens. previous studies have shown that the induction of ly-6 alloantigens in t cells is at least in part due to the action of ifn-a/b or ifn-7. in the present study, we have demonstrated that ifn-7 also induced ly-6 molecules on b lymphocytes and bone marrow cells. furthermore, we now show that tnf also participates in the induction of at least one of the ly-6 proteins, ly-6a/e. tnf was found to synergize with ifn-7 to induce ly-6a/e expression in thymocytes, t lymphocytes, and bone marrow cells, but not b cells. for t lymphocytes, the synergistic induction of ly-6a/e by tnf was restricted to cells from the ly-6.1 haplotype whereas ifn-7 was sufficient to fully induce ly-6a/e expression in cells from the ly-6.2 haplotype. this result is consistent with the notion that there is more complex regulation of the ly-6afe molecules in t cells obtained from the ly-6.1 haplotype. for t cells from balb/c (ly-6.1) mice, ly-6a/e, but not ly-cc, molecules were induced by ifn-7 and tnf. furthermore, when compared to ly-6a/e, the regulation of mhc class 1 molecules in these t cells by tnf was minimal. the induction of ly-6afe molecules on balbfc t cells resulted in an enhanced capacity to activate these cells through the ly-6 t cell activation pathway. one transformed t cell line, 5.1.2. was also identified whose ly-6a /e molecules were synergistically induced by ifn-7 and tnf. optimal expression of ly-6a/e molecules on 5.1.2 cells required continuous culture of this cell line with these two cytokines and resulted in the detection of optimal levels of cytoplasmic ly-6afe mrna by northern blot analysis. this latter result suggests that ifn-7 and tnf regulate ly-6a /e at the level of transcription and/or mrna stabilization. ut southwestern medical center, dallas, texas 75235. an igm antlcd3 mab (38.1) was found to modulate cell surface cd3 on highly purified human t cells within 5 hours in the absence of a secondary antibody or accessory cells. inhibition could be overcome with accessory cells or il2. the inhibitory effects of 38.1 could be mimicked by briefly pulsing cells with the calcium ionophore, ionomycin. 38.1 or ionomycin pulsed cells were inhibited in their subsequent capacity to resp nd to pha even when exposures were carried out in the presence of egta to prevent increases in [cap*]. from extracellular sources. inhibition was not the result of an inability to respond to pha 'by increasing [ca2+] .. moreover the newly expressed cd3 molecules were capable of generating increases in [ca2*].' after reacting the cells with anti-cd3 + a cross-linking secondary antibody. these studies dem'onstrate that a state of nonresponsiveness in resting t cells can be induced by modylating cd3 with an anti-cd3 mab in the absence of co-stimulatory signals. a brief increase in [ca ' 1. resulting from the mobilization of intracellular calcium stores appears to be sufficient to induce'this state of t cell nonresponsiveness. cd3. laurie s. davis, mary c. wacholtz, and peter e. lipsky, dept. of internal medicine, lmmunogenicity c a central lab.blood transf.service, lab. of exp. and clin.immunology of the univ. of amsterdam, amsterdam. the netherlands monoclonal antibodies (mab) directed against the human cd3 molecular complex induce a strong proliferation of t cells, when immobilized on microtiter wells. this activation system, that was shown to be independent of accessory cells, accessory-cell derived factors or lfa-1 mediated intercellular adhesion (1). allows one to study the requirements for t-cell proliferation and differentiation in a well defined manner. il-2 and ifn-gamma but no il-4 could be detected i n culture supernatants of coated anti-cd3 stimulated t cells. the addition of ril-1 or ril-2 had only a moderate effect on t-cell proliferation, vhereas helper activity for ig production was strongly enhanced in the presence of these factors. in this system differentiation of precursors to cytotoxic t lymphocytes (ctl), as measured in anti-cd3mediated cytotoxicity, could be demonstrated within 2 days after initiation of the activation. allospecificity of the induced effector ctl was demonstrated using a panel of hla class-i p815-transfectants. in this system the regulatory role of the cd28 molecule in tcell activation and differentiation was studied. addition of anti-cdz8 mab to t cells stimulated with coated anti-cd3 mab enhanced il-2 production, proliferation as well as ig production. interestingly, pctl differentiation was also enhanced by anti-cd28 mab. this system seems valuable for the analysis of requirements for differentiation of human t cells subsets. 1. van noesel et al., nature 333, 850-852, 1988 analysis of the requirements for human t-cell differentiation, rolien de jong. vivienne rebel, g i j s van seventer. miranda brouwer, frank miedema, rene' van lier, the newly described t cell receptor (tcr) 6 locus is located inside the tcr a locus between va and ja . despite this unique situation, a highly efficient regulatory mechanism results in the complete independence of these two loci. we have recently described, in humans, a site specific recombination which joins a 5' deleting element (srec) to the send of the ja's (yja) resulting in the deletion of the tcr-6 locus in t lymphocytes expressing the a/b tcr. rearrangements of the tcr as well as immunoglobulin genes are mediated by a unique recombination machinery and therefore, the specificity of these rearrangements is thought to be the result of a differential accessibility of the dna involved in the recombination process. as a consequence (and/or cause) of the opening of a segment of dna, the region involved is fxst transcribed as a sterile transcript prior to the rearrangement. in that regard, we have found that the 2 kb of dna u p s a u~n of yja are actively transcribed ('t early a" transcript, tea) early during fetal development. the presence of the tea transcript presumably reflects the opening of the tea sequence prior to the tcr-6 deletional rearrangement. in order to better understand the mechanisms involved in the dna accessibility model, we started to look for dna-binding proteins which might play a putative role in the opening or blocking of the tea sequence. by the technique of "gel shift assay" we found such a negative regulatory protein in the nuclear extract from a non-lymphoid cell. the binding activity appeared to be specific as it was competed out by an excess of unlabeled autologous dna and not by an excess of irrelevant dna. further studies are now in progress to determine first wether the presence of this binding activity can be correlated with a "closed" configuration of the tea region and second to determine the precise location of the dna binding region. cohen, laboratory of chemical biology, niddk, national institutes of health, bethesda, md 20892. mabs retard lymphoproliferation as well as autoimmunity. interesting, so does the adminisuation of a mab to l3t4 , thus suggesting that the t helper subset, which is not part of the unusual expanding population, is required for initiating the pathology in these animals. as a means of characterizing the expanding population of abnormal cells as well as the phenotypically mature (l3t4+) cells that may be associated with them, i have generated a series of t cell hybridomas from the enlarged lymph nodes and spleen of m p r mice. in parallel, i have derived a series of control (non-lprflpr) hybridomas from mrulpr x balb/c f1 animals (which show no sign of pathology), and a series from mrl, mice (which have a delayed onset of autoimmunity without lymphadenopathy). very few hybridomas (20-30) were obtained in the non-lpr derived fusions. when i con a stimulated the lymphocytes from non-lpr mice prior to fusion however, many more hybridomas were obtained(l20-220). this is in contrast to the fusion efficiency obtained from lprnpr mice which did not require in viuo lymphocyte stimulation to obtain a comparable number of hybrids. this result suggests that the m u p r lymphocytes are activated in situ. in addition, while less than 15% of the lymphoid mass is comprised of t helper (l3t4+) cells , over 50% of the hybrids are l3t4+. the fact that a dispropomonate number of t helper cells are rescued by fusion suggests that the cells activated in situ may be autoreactive t helper cells. currently i am characterizing these t helper cells for their lymphokine production, t cell receptor gene usage and auto-specificity and will compare them to the hybridomas obtained from non-diseased animals. goodnow, s. gilfillan, h-j. garchon, j. erikson and m. davis. stanford university, stanford, ca 94305. we have made transgenic mice bearing gene constructs encoding the t cell receptor a and p chains from a cytochrome c-reactive t cell hybridoma. despite a lack of tissue-specificity in mrna expression, cell surface expression of uansgene-encoded protein was limited to t cells, presumably because both chains require cd3 proteins in order to assemble on the cell surface. in mice carrying only the a chain consuuct, the transgene was expressed in the thymus as early as day 15 of fetal life, 1-2 days before endogenous a chain mrna. the first detectable cell surface expression of a transgene was on 25% of day 15 fetal thymocytes. this vast increase in up-bearing cells in fetal thymus was due to pairing of transgenic a chains with endogenous p chains, of which a substantial number are. normally rearranged by day 15 of fetal liie. the balance between ap-expressing t cell supopulations was grossly disturbed in these mice, the most marked abnormality being an increase in the number of l3tnyt-2-cells both in thymus and in peripheral lymphoid organs. it therefore appears that premature expression of surface ap t cell receptor may disturb t cell differentiation pathways by allowing t cells to leave the thymus without expressing l3t4 or lyt-2. mice carrying the p construct showed no increase in surface expression of t cell receptor in fetal life, since endogenous a chain rearrangement was limiting. in mice carrying either the a or p chain mansgenes, the number and surface phenotype of t cells expressing y& t cell receptors was unaffected in early fetal liie. suggesting that the a p and y5 t cell lineages diverge before thc rearrangement and expression of the appropriate subset of t cell receptor genes. department of microbiology and immunology, institute and the university of pennsylvania, philadelphia, pa. 19104.the thymic stroma plays a major role in initiating the colonization, organization and differentiation of precursor stem cells into functionally mature t cells. a variety of cell types including thymic nurse cells, cortical and medullary epithelial cells, nonepithelial dendritic cells, and macrophages, combine to form the thymic stroma. the differential role of such cells in thymic development is unclear. we have isolated a number of morphologically distinct stromal cell lines from the thymuses of sv40 transgenic mice. several of the cell lines are of epithelial origin, while others have features consistent with non-epithelial "dendritic" cells. we have focused on one of these cell lines, bearing the phenotype of a cortical epithelial cell, for its ability to support the growth and differentiation of stem cells from the fetal liver and fetal thymus, and cloned pre-t cells obtained from adult mice. the cortical epithelial cell line produces factors that induce the dramatic proliferation of fetal liver and thymic stem cells . in addition, fetal liver cells cocultured with this cell line are induced to rearrange and express their t cell receptor (tcr) genes. a cloned pre-t cell line is also induced to rearrange its tcr oenes in resoonse to sianals mediated bv this cell line. gugrin, marie c. b6n6, corinne amiel, nadia coniglio, jacques leclsre, laboratoire d'immunologie and clinique endocrinologique, chu de nancy and facult6 de mgdecine, 54500 vandoeuvre les nancy, france. the lfal molecule, an adhesin of the lfa family involved in cell-cell interactions, is physiologically expressed on all white blood cells. it is absent in some congenital immune deficiencies ((id), and is expressed on a decreased number of peripheral blood lymphocytes (pbl) in aids. we investigated its presence on pbl from 60 patients with auto-immune disorders of the thyroid. a monoclonal antibody (iot16, immunotech) directed to a conformational epitope involving both chains of lfal was used in indirect immmunofluorescence on pbl from blood drawn at a similar time in all patients. a calibrated flow cytometer (epics profile, coultronics) was used to measure the percentage and numbers of positive cells, as well as the mean fluorescence (mf) and shape of the fluorescent peak. data were correlated with clinical information,therapeutic, and other pbl features such as the cd4icdb ratio. the percentage of lfa1+ cells was significantly decreased in patients with graves' disease, hypothyroidism and hyperthyroidism. the mf was lower and the shape of the fluorescent peak seldom displayed the bimodal characteristic noted in controls. these data suggest the participation of the altered expression of lfal in the pathogenesis or evolution of auto-immune diseases. pt=ciilat.pd that excess hla r l a s s tt expression, ciinmonly found i n a i t i v e human nutoinimiine tlisrases maintdin:? the *rctivatinn of dutnreactivr t c e l l s which in turn prnducr mediators which maintain r.la:;s i t expression. this hypothesis has been tested i n many ways i n t h y r o i d i t i s . crj t i c a l l y autoreactive t cell:? are â�¬nilrid i n thyroid autoinnline tissues which a r e rrstimnlatrd hy thyroid f o l l j c u l a r r r l l s . more rrcently we have been exploring the s p e c i f i c i t y of the autoantigen reactive t c e l l s in hashinoto's t h y r o i d i t i s where thyroy-lobulin s p e c i f i c clones have been found, i n contrast t o graves' disease, where thyrocyte recognizing clones do not react wi.th tliyroglnhulin. tn rheumatoid a r t h r i t i s , collagen type i1 clones have been found, persistently in the activated (il-2r') t c r l l pmil over several years i n t.he same p a t i e n t . to verify t h a t antigen present,ation is involved i n rhrumatoid art.hritis ( r a ) a disease i n which, unlike thyroi.dj t i s the nature of the major antigen presenting c e l l (apc) is unknown, thc e f f e c t of ,~nticl.ass i1 dntibodies a t 1-oncentration which block *ari;j vation of t r-el is mi the synthesis n â�¬ rla-dr mrna wa:j waluat.rd. the inhibitory effect supports d i.rifira1 role nf an d s yet iiriknown apc i n maintaining the i:hronj.ci t y o f r a conversely, all the clones were unable to respond to a substitution at 91 (tyr to asp). nase mutant proteins were constructed with the same single amino acid substitution and t cell responses to peptides and mutants were compared. preliminary evidence suggests that the mutant proteins like the peptides, substituted at residue 88 and 91, will not induce t cell clone responsiveness. these data suggest that the overall structure of the protein will not compensate for the lose of a particular amino acid which is necessary for t cell recognition. medicine, baltimore, md 21201. to explore the variables important in t cell priming, an adjuvantfree immunization regimen was developed. bio.a mice were primed subcutaneously with syngeneic spleen cells that had been pulsed with high concentrations (100pm) of the peptide 81-104, a cnbr cleavage fragment of pigeon cytochrome e. the t cell response was assessed using a sensitive limiting dilution assay that measures lymphokine production with the ctl-l cell line. the precursor frequency of antigen-specific cells in the draining lymph nodes of mice primed with antigen-pulsed spleen (aps) was 1 in 4000, indistinguishable from the frequency of 1 in 3400 found in mice primed in the footpads with 10 nmol of 81-104 in complete freund's adjuvant (cfa) (data are given as geometric means, n=5, s.e.m = x/t 1.7 and 1.3, respectively). despite the apparent similarity in the t cell compartment of mice primed using these different regimens, antibody induction was strikingly different. mice primed with 81-104 in cfa developed serum igm and igg responses against the peptide, with antibody detectable in an ellsa assay at a 1 :3000 dilution. mice primed with 81 -1 owaps, however, produced no detectable anti-peptide antibodies. maximal t cell clonal expansion therefore appears to be possible in the absence of antigenspecific b cells. these data argue against the hypothesis that antigen-specific b cells play an obligate role in t cell proliferation in vivo. the reasons for the lack of antibody induction are currently under investigation. cell receptor (tcr) complex of jurkat cells. the coprecipitation of these peptides with tcr requires treatment with monoclonal antibodies (mabs) directed against tcr (c305 or r140) prior to cell lysis and immunoprecipitation. treatment of jurkat cells with mabs directed against cd2 (9-1 or 9.6) or hla (w632) does not not induce the association of these peptides with tcr. the signal-transduction mutant cell lines, j.cam1 and j.cam2, have previously been described (1,z). these cell lines, derived from jurkat, fail to activate the inositol-phopholipid second-messenger pathway in response to anti-tcr mabs. treatment with mab c305 induces the association of the 35 and 39 kd peptides with tcr in j.cam2 cells but not in j.cam1. j.cam1 modulates tcr normally in response to anti-tcr mab treatment (1). hence, these observations suggest that the two peptides are involved in the signal-transduction pathway of the t cell receptor complex rather than receptor internalization. sle is an autoimmune disorder associated with several different hla class i1 antigens. we studied a large sle patient population by sequencing of the pcr amplified first domain of the dqb and dqa chains and by sequence specific oligonucleotide probes to further define these associations. shared dqb sequences at amino acid positions 26=1eu, 30=tyr, and 57fasp may predispose some individuals with hla dr1,2,4, or 6 to develop sle. a novel dqb sequence found in two drw6 dqwl sle patients shares these amino acids in the dqb hypervariable regions. the association of the drz dqwl.azh gene was greatly increased in the sle patients with lupus renal disease. the hla association may be directly due to structural aspects of the hla genes. when parent -+ f1 chimeras are prepared with supralethal irradiation (1300 rad + 900 rad), the donor-derived cd4+ cells differentiating in the chimeras show partial tolerance to host-type h-2 determinants, despite the apparent absence of host-type apc. donor-derived cd4+ cells give only low proliferative responses to host-type apc in primary mixedlymphocyte reactions (mlr); furthermore, in i-e-+ i-e+ combinations, the donor cd4+ cells show molecules. this finding implies that tolerance is induced intrathymically, presumably through contact with a non-marrow-derived component of the thymus, e.g. epithelial cells. in support of this possibility, thymectomized & + ( a x b)f1 chimeras given strain j? marrow cells and a strain thymus graft (irradiated) show no detectable tolerance to host-type strain b determinants: the strain & cd4+ cells differentiating in these chimeras give strong mlr to strain b and do not show deletion of v 11+ cells. = 70% deletion of cd4+ cells expressing i-e-reactive v 11 t cell receptor b measured by these two parameters applies not only to lymph node (ln) cd4+ also to cd4+ cells recovered from the thymus. interphotoreceptor retinoid-binding protein) is a glycoprotein of 1264 residues (bovine) which localizes in the retina and pineal gland and induces inflammatory changes in these organs (eau and eap, respectively) in immunized animals. the experimental disease is considered a model for certain uveitic condiiions in man. we have recently shown that irbpderived synthetic peptides can also induce eau/eap in lewis rats. the present study compared two such peptides, "r4" (residues 1158-1 180) and "r14" (1 169-1 191). peptide r14 was found to be immunodominant, shown by its being recognized by lymph node cells (lnc) or line cells sensitized against whole irbp. in contrast, peptide r4 was not recognized by the whole irbp-specific lymphocytes and is considered nondominant. in addition, lnc sensitized to r14, but not to r4, responded to intact irbp. r14 was superior to r4 in producing eau/eap and cellular immunity (minimal doses: 0.06 vs 67 pg/rat). on the other hand, the two peptides were comparable in their capacity to stimulate presensitized lymphocytes. moreover, lnc sensitized against r4 were similar to those sensitized against r14 in their capacity to adoptively transfer eau/eap to naive recipients. this study thus provides a unique system in which both immunodominant and nondominant peptides produce autoimmune disease and can be compared for their immunological features. the age-related diminution in immune responsiveness has been shown to result from increased regulatory mechanisms and not from a paucity of immunobgical recruitment (aging: immunology and infectious disease 1,47, 1988). we present evidence based on "libraries" of monoclonal antibodies (mabs) omained from young and aged donors that there occurs with aging an increase in autoimmunity which is possibly the result of the accumulation of liielong "original antigenic sins". the resultant increased connectivity of the immune system is represented by mabs obtained from aged donors which are multiply anti-self cross-reactive. furthermore increased connectivii is supported by the evidence that anti-2.4,6-trinitrophenyl mabs are ad8 positive, 2d3 positive as determined by i n h b i i n studies using mabs anti-idiitypic reagents. analysis of the vh and vk region genes utilized by these mabs indicate a nonrandom gene usage. life bng stochastic immunological events lead to a pattern of cross-reactiities and non-random usage of vh genes. these immnological events lead to the emergence of the patterns which are partially elucidated by the data presented. these patterns mimick those seen early in ontogeny, but indicate a possible convergence to an ever-increasing connectance of the idiitypic repertoire expression. in other words, life-long immunological experiences contribute to a down-regulation resulting in both paucity of drimaw immune reswnses and an increase in autoimrmnity which are both the earmarks of immunity in aging. (supported by usphs grants ag-04042 to eag and al 18316 to cab) lmmunogenicity c449 stimulation of these cell lines in suspension with saturating levels of mab okt3 produces total and fractional inositol phosphate accumulation linearly related to receptor number, (r > 0.9 ). this technique also allows an approximation of the minimal number of reccptors which must be engaged for second messenger generation in this system, which we estimate as 6.5~102 receptors per cell. or terminate t cell activation. since this molecule plays an important role in human t cell development we sought to identify the murine homologue of cd28 in order to determine its expression on murine t cell and its role in activation. we have used a human cd28 cdna clone to isolate a full length cdna encoding the murine equivalent of cd28 from an el4 t cell lymphoma library. this clone shows similar domain organization and a high degree of homology to the human cd28 molecule. the murine cdna clone has been used to examine mrna expression of cd28 in normal and activated murine t cells, and in various t cell tumors. peptides generated from the translated sequence will be used to produce antisera to correlate the surface expression of cd28 with mrna expression, and to biochemically and functionally characterize this molecule. pat happ and ed palmer, basic sciences division, dept. of pediatrics, national jewish center for immunology and respiratory medicine, denver, co 80206. we have attempted to determine the frequency of rearrangement and expression of the individual a and g chain v gene segments that make up an unselected, untolerized t cell repertoire. in order to do this we generated over 500 t cell hybridomas from freshly-isolated thymocytes of newborn c57blllo mice and subjected rna from these hybrids to northern dot blot analysis using 11 va, 16 vp. cy and c6 probes. comparison of the expressed repertoire of vp gene segments in this newborn thymocyle population with similar data previously generated from an adult peripheral t cell population reveals two vg genes, vp12 and vpl5, whose expression is decreased in the periphery, possibly due to the effects of tolerance. two additional vp gene segments were expressed more frequently in the peripheral population than in the newborn thymus, vp5 (4.5 times higher in the periphery) and val0 (2 times higher). it is possible that these represent ewo instances of positive selection of t cells which is determined primarily by the receptor's vg gene segment. va gene segments were expressed in only 15% of newborn thymocyte hybridomas (compared to 58% expressing vp) and determination of va rearrangement frequencies was complicated by the unexpectedly large number (67%) of hybrids expressing cs mrna. further examination revealed that several va gene probes were actually detecting rearrangements to cs. the most notable of these was va7, which accounted for approximately 34% of the expressed va repertoire but was rearranged exclusively to cs. barbara bergman, brenda bradley, kevin lafferty and mary portas. barbara davis center for childhood diabetes, u. colo. health sci. ctr., denver, co 80262. we have produced a panel of islet-specific t cell clones by culturing lymphoid cells obtained from non-obese diabetic (nod) mice in the presence of nod islet cell antigen and antigen-presenting cells (apc). these clones were selected to the panel on the basis of (a) their antigen-specific reactivity to islet cells and apc in an in vitro proliferation assay and (b) their ability to mediate islet graft rejection in vivo in a tissue-specific manner. we have further characterized these lines for cell surface phenotype, 11-2 production, and proliferative response to non-nod islet antigen. all of the clones tested to date are of the cd4 phenotype and make il-2 in response to islet antigen and nod apc. nearly all of the clones we have tested also make good proliferative responses to islet cell antigen obtained from mouse strains other than the nod or to a mouse beta cell tumor line. preliminary results indicate that at least one of these clones can lead to islet cell damage in a disease transfer experiment in which the cloned t cells are injected into a non-diabetic nod f1 recipient. we are currently carrying out tests to further characterize lymphokine production by these cloned cell lines, to analyze differences in antigen recognition and mhc restriction requirements among the clones, and to determine their effectiveness in mediating the disease process in nondiabetic animals. in an attempt to identify the epitopes on class i mo ecules recognized by alloreactive cytotoxic t lymphocytes (ctl) we have examined k -specific ctl for tpir recognition of synthetic peptides with sequences derived from the native k molecule. co secutive overlapping peptides molecule were tested for their capacity to inh&bit k -specific ctl clones in their recognition of cells expressing the native k molecule. in these studies inhib9ion by peptide was found to be an extremely rare event, although one peptide (k 111-122) did inhibit recognition by a particular ctl clone (clone 13). in a separate set of experiment8 it was observed that clone 13 could recopize kblll-122 when presented by h-2 class i molecules. as clone 13 was of h-2 origin, this finding led to conclude that inhibition may be due to class i-restricted recognition of the k pegtide on the surface of the ctl clone, peptide and native kb for the t cell receptor. we present evidence in favor of this conclusion. the pepscan method is used for the systematic identification of sequential b cell epitopes i n protein molecules (geysen, meloen, barteling. pnas 81: 3998; 1984) . it was designed for the synthesis and subsequent testing for antibody binding of large numbers of overlapping peptides directly on their solid supports. the mhc dependent presentation required for t cell recognition seemed prohibitive for the use of pepscan to identify t cell epitopes. however we now have shown that by a novel modification the peptides can be recovered from their solid supports and used in t cell assays. holmdahl, department of medical and physiological chemistry, box 575 uppsala university. s-75123 uppsala, sweden. both autoreactive t cells and autoantibodies play an important role in the pathogenesis of type i 1 collagen (cii) induced arthritis in mice. we have earlier reported that only strains with h-2q , h-2w3, h-2w17 and h2-r were responders to autologous mouse cii and only these strains developed arthritis after immunization with autologous or heterologous cii. however, heterologous cii induced a more acute and severe disease and a more pronounced autoantibody response. this findings indicate that 1) the ability to mount an immune response against autologous cii is a prerequisite for the susceptibility to collagen arthritis and 2) that a crossreactive autoantibody response after immunization with heterologous cii may further enhance development of arthritis. we have now studied activation of autoreactive b cells after primary immunization of den1 mice with rat cii. in hybridoma collections, obtained 9-11 days after immunization, 30.80% of the hybridomas produced igg reactive with autologous cii, 10-15% produced multispecific igm and a significant number produced igg rheumatoid factors. the anti-cii antibodies recognized at least 5 different epitopes on the cii molecule and originated from many different vh and v kappa gene families. furthermore, none out of 6 investigated anti-cii hybridoma expressed cd5 rna message. we therefore suggest that the primary anti-cii autoantibody response involves activation of memory b-cells. these memory b cells have most likely been earlier activated by cii autoreactive t cells. in these aspects the origin of the anti-cii autoantibody response is principally different from the origin of "natural" autoantibodies. t cell receptors (tcr) recognize antigen in association with self mhc molecules, usually following processing to smaller peptides. the t cell repertoire to an antigen, therefore, reflects not only the ability of a given mhc molecule to interact with antigen, but also the affects of initial repertoire selection by self mc. we have t#tn analyzing the tcr repertoire specific for beef insulin (61) in balb/c mice (h-2 ), which are high responders to the antigy. these studies revealed that vp.3 is dominantly used in the tcr's specific for bi/a and our preliminary data suggests that the vgb.3 chain may be involved in mhc restriction. we have now obtained several t cell hybridmas specific for bi from (balb/cxa/j) f1, animals. a/j mice (h-2a) are low responders to bi while the f 1 m ce a e high responders. most of the balb/cxa/j hybridmas were restricted to the hin the balb/c hybridmas. interestingly, the analysis of v gene usage demonstrated that vg8.3 was not used in the balb/cxa/j hydridonas. the relevance of these results to the development of the tcr repertoire in different mouse strains will be discussed. this work was supported by the mrc of canada. ctl specific for the q k molecule were generated from normal splenocytes by & vitro culture with a2k bearing stimulators. these ctl have been shown to lyse transfected targets expressing hla-a2 regardless of their murine haplotype, and they specifically kill a2 bearing human target cells. furthermore. the effector function of these ctl can be inhibited with an hla-a2 specific monoclonal antibody. thus, the transgene product functions correctly as a tolerogen and is recognized directly as a class i antigen. although transgenic mice have been shown to be tolerant to a2k expressed by murine cells, transgenic ctl specific for hla-a2 on the surface of human cells have been generated. these the major virulence factor of m.pneumoniae was shown t o be a 168 kda protein which is located in the tip structure membranes of these cells. beside the adhesin function this protein is also involved in first massive humoral and cellular responses of the human host during the acute phase of upper respiratory tract infections and interstitiel pneumonia. intranasal inoculation of guinea pigs with the isolated 168 kda protein led to lympho-histiocyte infiltrations around bronchi and small vessels of the lungs which are characteristic infiltrations after an infection with live m.pneumoniae cells. furthermore one peptide ( 1 7 amino acids long) which was synthesized according to the amino acid sequence of the adhesin, showed a proliferative activity to in vitro cultivated t-cells of bronchial washings, whereas synthetic peptides with th e sequences of the direct neighbourhood showed no in vitro activity. most interestingly this t-cell proliferative activity is located on a surface loop of this protein which is also responsible for the adhesin f uction. christopher a. smith, gwyn t. williams, rosetta kingston and john j.t. owen. department of anatomy, university of birmingham, medical school, vincent drive, birmingham 815 2tj, uk. rearrangement of t-cell receptor a and b chain gene segments during t-cell development results in a diverse array of receptor specificities. to avoid auto-immune responses, cells that have generated self reactive receptors are thought to be eliminated or inactivated, to produce self tolerance. recent studies have provided compelling evidence that clonal deletion of immature receptor bearing cells within the thymus makes an important contribution to this process, although the mechanisms involved are not uderstood. of immature mouse thymocytes with anti-cd3 antibodies added to thymus organ cultures, induces dna degradation and cell death through the endogenous pathway of apoptosis. is in marked contrast to the activation of mature t-cells by the same anti cd3 preparation and is specific to the extent that apoptosis is not induced by either anti-cd-4 or anti-thy-1. to organ cultures suggesting a role for changes in intra-cellular c a w levels in the signalling pathway leading to the induction of apoptosis in immature cells binding. thus activation of the process of apoptosis in immature cells binding self antigens may be the mechanism responsible for the selective deletion of cells that could generate an auto-reactive response if allowed to mature. we have now obtained evidence that engaging the cd3/t-cell receptor complex this in addition, calcium ionophore (ionomycin) also causes apoptosis when added anderson cancer center. houston, tx 77030 immunization of patients with bcg and irradiated tumor cells induces specific delayed-type hypersensitivity (dth) to tumor cells and not to normal colon cells. since igg antibodies may require t-cell help, we wished to characterize the igg-defined tumor-associated autoantigens (taaa) of human crc so as to define a subset of the t-cell repertoire for crc. western blots of detergent extracts of 73 primary and metastatic human colorectal carcinomas and paired normal tissues were probed with autologous igg. nine taaa were recognized by 20% or more of the sera: 74, 72, 58, 52, 45, 41, 38, 29 . and 26 kda. these taaa may be normal colon differentiation antigens, since they were present in extracts of normal colon. autoantibodies are more frequently present to the 41 kda antigen in patients with metastases (79%) than in primary tumors (47%. p<o.o5) while there is more reactivity to the 38 kda antigen in primary tumors (38%) than in metastases (7%, pso.05). only 1 of 8 patients sensitized with bcg and irradiated autologous tumor cells, developed antibody to new taaa; whereas 2 of 12 control patients developed antibody to new taaa without sensitization. the taaa in the western blots that stimulate autologous lymphocyte proliferation are greater than 200 kda. finally, immunization with a human crc failed to induce dth in mice to a syngeneic murine colon carcinoma that expresses at least 1 of the igg-defined human taaa. thus, taaa that presumably require t-cell help in patients do not appear to be part of the t-cell repertoire for dth to human crc. in several rodent species and strains, the emergence of specificity to a discrete immunodominant epitope of guinea pig basic protein (gpbp) results from selection of cell lines with whole gpbp. these epitope specificities have been found to be strain-specific and define the specificity of the t helper cells which transfer experimental autoimmune encephalomyelitis (eae) in adoptive transfer experiments. line from aci-strain rats which was encephalitogenic and specific for a new t cell epitope of gpbp. the cell line responded in vitro to the peptide cokkesponding to the region 39-54 of gpbp but not to peptides corresponding to other regions of gpbp. this cell line is restricted by i-a but not i-e and transfers a dth response to gpbp as well as eae into naive recipients. these results provide support for the hypothesis that the in vitro immunodominant epitope specificity defines the specificity of gpbp-specific encephalitogenic t cells. the definition of a new encephalitogenic epitope in the aci strain also suggests that the mechanism of eae in rats depends on strain-specific extrisic properties of antigen recognition such as the mhc class i1 genotype or the composition of the t cell antigen-receptor repertoire. we have produced a gpbp-specific t lymphocyte medical center, stanford, ca 94305. a wide variety of t lymphocyte surface antigens have been isolated and shown to play important roles in the t cell response. we outline here a strategy to isolate and characterize surface proteins unique to activated t cells. a rabbit anti-serum was generated by immunizing with allo-activated human t cells and subsequently absorbing with the t cell tumor hpb-all, a tumor that expresses all functionally relevant t cell surface antigens previously described. this absorbed antiserum was able to recognize an uncharacterized group of surface proteins on t cells that was absent from hpb-all. furthermore, the absorbed anti-serum was able to inhibit i n virro cytotoxic and mlr responses; an effect that was abolished by carrying out an additional absorption with the t cells used for the initial immunization. monoclonal antibodies to activation antigens were generated by immunizing with complexes made by adding the absorbed anti-serum to solubilized ctl. the absorbed anti-serum was also used to screen a lcgtll cdna library. one cdna clone so isolated is expressed on ctl as evidenced by northern analysis. dna sequence data indicates no similarity to other sequences in available databanks. childrens hospital of orange county, orange, ca 92668, and university of california, irvine, ca 92717. we examined the ability of cytokines to stimulate the proliferation of pbm from 41 new-onset iddm, 11 long-term iddm, 10 non-diabetic pediatric control, 5 type i1 diabetic, and 3 non-diabetic adult control subjects. initial results demonstrated the ability of human pbm culture supernatants containing peak natural il-1 activity to stimulate a significantly higher proliferative response from pbm of new-onset iddm than from pbm of long-term iddm, type i1 diabetics, or non-diabetic controls. more recent results have shown that human recombinant il-la, -lb, -3 , -4 and -6 , interferon gamma, lymphotoxin, and tumor necrosis factor, as well as con p.-stimulated rat spleen supernatants, also stimulated a significantly higher proliferative respcnse from pbm of new-onset iddm than from pbm of controls. however, human recombinant il-2 stimulated equivalent levels of proliferation from pbm of both new-onset iddm and controls. these data indicate that, in new-onset iddm, there 2.e present pbm which are in an early stage of activation not revealed by increased responsiveness to recombinant il-2. in the case of t cells, this may represent a stage prior to an il-2-dependent stage of activation. this would be consistent with the active induction of newly responsive cells during a stage of iddm when islet cells are still present. this response is not the result of the metabolic aspects of iddm but a reflection of the immunologically activated state of t cells, b cells andlor monocytelmacrophages in iddm. houghten, c. david pendleton, james l. comette, charles delisi and jay a. benofsky, metabolism branch, national cancer institute, national institutes of health, bethesda, md 20892. analysis of known helper t cell m) cpitopes suggests that th epitopes tend to be amphipathic alpha helices, that is, helices with hydrophobic residues on one side and hydrophilic residues on the other side. based on this hypothesis, a computer program "amphi" was generated which may aid in prediction of th epitopes. within residues 1 to 55 of spermwhale myoglobin (swmb), a th epitope is known to exist but has not yet been found. as an approach to further test the hypothesis, this undetermined th epitope was sought both by an unbiased test of ten evenly overlapping 15-residue peptides that span the region, and by computer analysis. of the ten peptides, only one was able to stimulate two clones that are specific for the 1-55 region. this peptide gave remarkably high stimulation index. analysis of this region by the computer program also predicted the site covered by this peptide (residues 2640) to be the most likely site for th epitope.. therefore, unbiased in v i m testing and the computer prediction correlated well. the peptide was able to prime t cells of b1o.br mice in vivo for in v i m response to the native swmb as well as to the peptide fragment of residues 1-55. immunization of mice with swmb showed that, of the ten overlapping peptides, the major site of response is to the identified peptide. finally, a peptide of residues 24-42 was made to increase the amphipathic score. this extended peptide induced greater proliferation of the clones. thus, this study has identified a new dominant epitope of swmb and confirmed the utility of the amphipathicity hypothesis in a prospective test. amino acids a t a selected site; 2 ) t-cell antigenicity, that is analysis of prirrary structures with 9-and 11-amino acid templates obtained from database of knawn y-epitopes; gly, hhydropkbic, pplar or charged amino acid (2); 4) prediction of the secondary structure according to gamier (3) to exclude sites having high turn or coil forming ptential. ccanparison of t h i s set with published program (1,4) shows that it has high predictive f r . so we used it fo search for probable t-epitopes i n hepatitis a virus capsid proand for mediators of delayed hypersensitivity (tdh ~ assayed by ear swelling induced by co-injection of responder cells with irradiated tumor cells). lymph node cells recovered 14 days post-inoculation contained tdh cells and ptc (freq 1/1,200) , but tc effector cells. by contrast, graft-infdtrating cells not only contained tdh and ptc (l/l,oao), but direct tc effector cells (21 lytic units). all three sets of t cells were antigen-spedfic. the finding of significant frequencies of ptc within the lymph nodes and the graft site, but tc q& at the latter, suggests that differentiation of tc from unprimed ptc may be a multistep process, the first of which occuis in the draining lymph nodes. the data further suggest that the terminal step@) in this process take place after the donally expanded ptc have disseminated to the graft site. we suspect that the final step requires help provided uniquely by tdh cells within the graft. the fmal step may not occur (as might have been expected) in the lymph node because priming of ptc and tdh take place at noncontiguous anatomic sites. interference & seed, pnas a, 8573, 1987) . the n-terminal amino acid sequence of normal t cell cd28 was found to match the hpb-all sequence except in residue 10 and 11 (...ts...) . in principle this might reflect somatic mutation in hpb-all or cd28-gene polymorphism: erroneous residue assignment in the gas phase sequencing cannot be excluded entirely. to investigate the significance of this sequence variation a genomic cd28 dna clone of a normal human t cell clone was analysed around residues 10. 11 and found to be identical with the hpb-all cdna sequence. sumtic mutation in hpb-all cd28 therefore can be ruled out. the molecular characterization of t cell receptor (tcr) structure in a panel of mouse t cell clones specific for sperm whale myoglobin (spw mb) has shown that 6 1-ed-restricted clones specific for the spw mb region 110-120 use highly homologous v bchains. four of these clones are indistinguishable in their response pattern to a panel of overlapping synthetic peptides spanning the spw mb sequence 110-120, yet use 3 different v a gene segments. we are currently trying to find fine specificity differences among these 4 clones, in order to correlate them with tcr expression. t cell hybridomas have been made from these clones, and are being screened on panels of substituted peptides. we hope that conservative substitutions at positions important for interaction with the tcr will reveal fine specificity differences among these clones. sloan-kettering institute, new york, ny 10021 we previously showed that clonal deletion, non-suppressor mediated immunological tolerance neonatally induced to mls allo-determinants was specific in that the frequencies of t cells responding to a variety of other stimuli were unaffected while the frequency of t cells responding to mls were drastically reduced. here we show that mls tolerance is detectable in the thymus prior to a detectable positive response in the periphery (spleen and lymph node). tolerance induced by spleen cells from mlsa congenic mice in balb/c mice is as "potent" as that induced by whole background incompatible dba/2 spleen cells, indicating that incompatibility at the ml s locus ( the ly-6 alloantigens have been shown to participate in the process of t cell activation based on the ability of anti-ly-6 monoclonal antibodies to induce il-2 production and proliferation of t lymphocytes. in the present investigation, we have demonstrated that peripheral t lymphocytes from a strain mice exhibited abnormally low proliferative responses after stimulation through ly-6a/e and ly-6c molecules when compared to responses of t cells from numerous other mouse strains. the abnormal activation of the ly-6 pathway of a strain t cells was not due to ineffective fc receptor cross-linking of the anti-ly-6 monoclonal antibodies, to inappropriate cellular expression of the ly-6a/e alloantigen in a strain t cells, or to an active suppressive phenomenon. identification of this defect provided an opportunity to compare t cell activation by the ly-6 and tcr pathways. interestingly, t lymphocytes from a strain mice proliferated normally when the cells were activated by monoclonal antibodies to thy-i or the cd3/tcr complex suggesting that this defective activation was selective for the ly-6 t cell activation pathway. cell separation studies of t cells and accessory cells demonstrated that this defect was quantitative rather than qualitative and that it was complex, residing at both the t cell and accessory cell levels. these results suggest that activation of t lymphocytes via the ly-6 molecule may involve unique signalling pathways and that activation via cd3/tcr can proceed normally in the absence of a fully functional ly-6 pathway. the h-u)d transfected l cells were recognized by the cil when they were infected with either influenza a virus or a vaccinia recombinant virus encoding the pb2. thirty-five synthetic peptides derived from pb2, which had either an amphipathic suucture or contained a rothbard' epitope, were tested for their ability to render uninfected targets susceptible to lysis by influenza specific effectors. three epitopes were identified, each of which contained a rothbard epitope. department of microbiology and inununolag, albany medical college, albany, ny 12208 our laboratozy has developed a new, powerful technique for hvestigating the inmplne ~n s = to peptide epitopes, involving the covalentcoupling of peptide to phosphatidylethanol-, follmed by amplexing with additional lipids and phospholipids to form a peptide-phoqholipid complex. these cmplexes can be used to inununize mice in the absence of protein carriers or adjuvants, thus facilitating the study of the hmne response to a mall chemically defined antigen. use of this technology has allmed us to identify two t helper cell epitopes in conserved regions of kiv gp 160 not previously identified by computer algorithims, defined by amino acids 485-518 and 585-615. inununization with these peptides in peptide-phoqholipid mmplexes results in the prduction i g and igg antibodies, which truss react with cloned fragments of the whole protein. us& this tecfinolosy we have begun to characterize the irmnune reqonse to individual peptide antigens. ?he response of h2-k mice to amino acids 494-518 of gp 160 of hiv, has been analyzed. lhe optimal dose of a peptide containing both b and t cell epitopes was found to be 15-30 ug, depending on the mute of administtation. im mhization required less antigen for optinarm antibody response than did ip. anchorage in the phospholipid cmplex is a strict requirement for an antibody response. additional variables, such as phospholipid m i t i o n and method of cross-linking have been studied and will be discussed. we believe that the use of this peptide-phospholipid ccnnplex technology will be significant both for studying the i n m u m response to single epitops and for vaccine developrent. virology, national bacteriologica; laboratory, 105 2 1 stockholm, departments of neurosurgery, virology and immunology, karolinska institute, 104 01 stockholm, johnson & johnson biomedical research, la jolla, california, pentadecapeptides, sequentially overlapping by 10 a.a., were syntesized based on the htlv-i11 sequences of gag and env proteins and used as antigens in igg-subclass elisas andt-cell stimulation assays. sera and cells were obtained from 30 asymptomatic, hiv-infected homosexuals. reactivity in all subclasses could be found to parts of the gag-protein. extensive areas of iggl and 3 reactivity were indentified mainly in the p17 and n-terminal half of p24 with an additional region in the n-terminal end of p15. the highest iggl and 3 reactivities were detected in a.a. 8-33, rich in glycine, arginine and lysine and thereby hydrophilic with a high segmental flexibility; iggz and 4 epitopes were found in the hydrophilic cooh-terminal of p1j; the second highest iggl and 3 reactivities were found in the hydro-?hilic n-terminal of p15. for gp120, the iggl epitopes identified were in the putativeloopregion (a.a. 296-331) and in the hydrophilic cooh-terminal end of gp120 (a.a.489-503). the immunodominant region of gp41 (a.a. 582-617) showed some igg2, 3 and 4 responses inaddition to igg1. t-cell proliferative responses were detected against many peptides usually in areas not binding igg. still, simultaneously t-and b-cell reactive peptides could be detected, and the showed a distinctly preference for igg1. the variation between different patients was considerable regarding both t-cell reactivity to peptides and the subclasses of reactive igg. a mapping of subclass restricted epitopes allows an elucidation of anti-viral imunological mechanisms. kristin komschlies, laboratory of experimental immunology, brmp, dct, and bcdp, program resources, inc., national cancer institute-frederick cancer research facility, frederick, md 21701 and jackson laboratories, bar harbor, me 04609. "double positive" (dp) thymocytes are precursors for cd4+ and cd8 mature subsets have been based on indirect evidence with in vivo anti-cd8 or anti-cd4 treatments. we have approached this question directly by isolating subsets of dp thymocytes from fetal or adult thymus and have shown that only a subset of dp cells ( 4 0 % of adult thymocytes) can generate donorderived thymocytes after i.t. injection into irradiated hosts. low-density dp cells are injected i.t., up to 20-40% donor-derived cells can be seen at 6 to 18 days after transfer. cd4' cells can constitute up to 30% of these donor-derived cells at day 10. in contrast, an equivalent i.t. transfer of dull ly-l/cd5 (dly-1). early precursor thymocytes would not generate cd4' cells until days 12-13 but would produce some cd8' cells by 18 days. thus we have detected differentiation of cd4+ cells and not cd8' cells from adult dp cells. depleted host and recovered donor-derived thymocytes 8 to 10 days after i.t. transfer of dly-1 cells, when most of the donor-derived cells are dp. when retransferred, these dp cells displayed a more rapid progression of thymic localization patterns than dly-1 cells, but were unable to produce enough donor thymocytes to analyze subsets. thus cd4' cells appear to differentiate via a very minor (low density) subset of dp thymocytes. may not be generated from these intermediate dp precursors but directly from dly-1 cells. the intrathymic differentiation process by which bone marrow-derived progenitor cells develop into mature, immunocompetent t lynyhocytes remains poorly understood. the majority of all intrathymic lymphocytes are cd4 8 "double positive" cells that have no well-documented function in thymic differentiation, but have been proposed to be either a critical intermediate between cd4-8precursor cells and mature t cells, or, alternatively, a "dead-end" cell type. furthermore, although signals mediated through the tcr complex are critical for t cell tolerance induction and repertoire selection, the functions of the cd4 and co8 differentiation antigens expressed by the vast majority of thymocytes are unknown. in the present study we addressed the possibility that cd4 functions as a signalling molecule during t cell development. we engaged cell surface cd4 on murine thymocytes by in vivo injection of anti-cd4 mab, and monitored the effects of that engagement on distinct thymocyte subsets. c d 4 ' 8 + cells respond to cd4 cross-linking by dramatically increasing their cell surface expression of cd3 and tcr, and decreasing their cell surface expression of cd4. in contrast, mature cd4'8-cells respond to cd4 engagement by decreasing their cell surface expression of both cd3 and cd4. these results provide clear evidence that cd4 engagement regulates cd3/tcr expression on developing thymocytes, and, most interestingly, results in increased tcr expression by cd4+8+ double positive thymocytes. thus, cd4 engagement is a membrane event to which c d 4 ' 8 ' thymocytes respond significantly, indicating that cd4 may be a signalling molecule in t cell development and may promote t cell differentiation by inducing increased cd3/tcr cell surface expression on cd4+8+ thymocytes in vivo. f.g. terpstra. p.th.a. schellekens and r.a.w. van lier, central lab.blood transf.service, lab.exp.clin. immunol. of the univ.of amsterdam, the netherlands t cells can be activated through several membrane molecules that may use distinct intracellular signalling pathways. t-cell activatiep threygh both the cd3/tcr complex and cd2 is accompanied by riaea in free intracellular ca (ca ) and pkc activation. in contrast, evidence from our laboratory suggests that triggering h a cdz8 occurs through a pkc-independent route. in this study we used mab to various t-cell membrane antigens to localize the activation defect in t cells from asymptomatic hiv-infected men that we previously showed to have an intrinsic defect in their proliferative response to soluble anti-cd3 nab. compared to hiv-homosexual controls, the monocyte-dependent response of t cells from hiv+ men to soluble anti-cd3 and a mitogenic combination of anti-cdz mab was decreased as was the monocyte-independent resgpnse to immobilized low-dose anti-cd3. in t cells from both groups a normal rise in (ca ) . was induced by anti-cd3 and anti-cdz mab. however, t cells from hiv-infected men respondad poorly to direct pkc activation by pma in the presence of healthy donor monocytes, whereas control t cells responded vigorously. in contrast, induction of proliferation by anti-cd28 mab was comparable in t cells from hiv+ and hiv-men. our results indicate in hiv+ men a selective qualitative t-cell defect in activation routes that are dependent on pkc activation, whereas pkc-dependent alternative activation appears to be unaffected. these findings will be discussed with repect to hiv immunopathology and normal t-cell physiology. we have studied the role of the protein kinase c (pkc) in the activation of a prototype th-2 cell, d10.g4.1. blocking of this enzyme with the drug h-7 [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazinet leads to an increase in cellular proliferation of d10 cells activated with specific antigen or mitogen. n addition, an increase in the amount of il-4 and il-5 mrna is seen under these conditions compared to the level observed in mitogen activated cells in the absence of h-7. this superinduction of il-4 and il-5 lymphokine mrna is also detected in mitogen activated d10 cells that are depleted of pkc protein by pretreatment of the cells with phorbol esters. in contrast, mitogen activation of s lenic t cells measured by proliferation is blocked in the presence of h-7. these data indicate that jkc has a regulatory role in th-2 cells by exerting a negative feedback on the events that occur after cell activation avoiding an uncontrolled response. this effect is opposite to that seen in a bulk t cell population that represents mainly th-1 type cells. the mechanism of signal transmission is, therefore, distinct in the two types of cd4 + t cells. aberrant expression of class i1 mhc proteins on non-immune somatic tissue has been implicated in several autoimmune diseases, but it was unknown whether this expression was involved in the initiation of disease or resulted from induction by the immune reaction. we ha e engineered several transgenic mouse lines expressing tissues using eith r the pancreatic elastase promoter or the class i kb gene promoter. the i-as on the pancreas is fully capable of presenting peptide antigens to i-ad restricted hybridomas. h-zb mic expressing i-ad on pancreatic acinar cells but not in thymus are not tolerant to i-as in mixed lymphocyte reactions in vitro, but do not spontaneously produce an immune respon e to the pancreas in vivo. peripheral antigen presentation, i-a restricted immune response, i-a expression by t-cells, and widespread somatic i-ad expression. expression, whereas another has only medullary expression. the effect that these different patterns have on mhc restriction is being examined. ctr., stanford, ca 94305. a transient increase in the intracellular cat+ ion concentration has been shown to occur rapidly following activation of t lymphocytes. this response i s a more immediate and direct indication of t cell activation than either production of interleukins or proliferation. we examined ca++ flux analysis using indo-l and the facs to measure ag-speclfic t cell activation and also to sort for an ag-specific t lymphocyte subpopulation. human t cell clones were exposed to elther allostlmulatory 6 lymphoblastoid cells (which cause proliferation of the t cells) or nonstimulatory 6 lymphoblastoid cells. in both cd4+ and cd8+ clones, we observed an ag-specific cat+ flux response similar t o an lonomycin positive control. we then attempted to enrich for antigen specific t cells from a mixed population. two phenotypically different human t cell clones were mixed in equal amounts. after coculture of the cell mixture w i t h 6 lymphoblastoid cells shown to stimulate only one of the t cell clones, the cell mixture was sorted on the facs based on specific cat+ flux. a ten fold enrichment was observed for the ag-specific clone. this approach allows rapld observation of t cell activation and provides a means of sorting antlgen reactive t cells from a heterogeneous cell population. previously attemps to characterize the function of beta-2-microglobulin (beta-2-m) have used anti-beta-2-m antibodies, to block the functional activity of the protein. we now demonstrate that human beta-2-m and the proteolytic derivates beta-2-m lys58 and beta-2-m deslys58 added in nanomolar concentrations to allogeneic, pha and con-a stimulated mouse lymphocyte cultures modulate the response induced. thus beta-2-m and especially beta-2m deslys58 when added day 0 and 1 augment the generation of specific cytotoxic t lymphocytes in mlc, problably due to stimulation of endogeneous lymphokine production in the culture. both the con-a and pha induced response were modulated. in the presence of beta-2-m and its proteolytic derivates the proliferation was optimal at 48 hours, as opposed to the 24 hours optimum in the abscence of beta-2-m. these results strongly suggest an active role of beta-2-m or proteolytic derivates hereof in the t cell activation and generation of specific cytotoxic t lymphocytes. a (1.2 fold) . the increase peaked at 2 min and returned almost to baseline by 10 mi?. igm b cells showed random x inactivation, while more mature igg and iga b cells had only the normal x active. we conclude that the xla gene product is required for b cell development, while the xscid gene product is required for both t and b cell maturation. gene mapping has placed xscid in xq13-21.3, linked to phosphoglycerate kinase (pgk), lod 3.1, 8=0. mouse xid, which affects only b cells, is also linked to mouse pgk, suggesting the possibility that xscid and xid belong to related gene families. c 530 institute at denver, national jewish center, denver, co 80206. mapping data for the bxd and akxd recombinant inbred strains shows that the mhc and the products of at least two other genes control the expression of vp3. one of these is linked to ly-7, and the second for the akxds is linked t mtv-13 on chromos me 4. a backcross of b1o.br x (c3h x b1o.br)fl (mls-2 x ( m l~-2~ x mls-2 ) ) has been used to confirm the linkage of one of these controlling genes to ly 7. using the vp3-specific monoclonal antibody, exceptions have been found to the general rule, that strains which have eliminated t cells expressing a particular vp specific for a defined self antigen in the thymus, also express this self antigen on their spleen cells, and so these cells are capable of stimulating t cell hybridomas bearing the particular vp in quest ion. the t cell receptor (tcr) of the alloreactive t cell clone 2c recognizes the h-2ld specificity. the cloned a (va 3.1) and p (vg 8.2) tcr cdnas have been inserted into plj retroviral vectors containing ltr and regions of the momulv and neomycin resistance genes. three different constructs have been used to transfect v2 cells. g418-resistant clones showing the highest titer of viral particle psoduction have been selected, and injected subcutan ously lacking the h-2l vt cells producing retroviruses carrying the a gene have been examined for the expression of the new specificity on the infected t cells. northern blot analysis using the va and the neo-probes showed, in the lymphoid tissues, a transcript with an expected size of 3.6 kbs hybridizing with both probes. in the spleen of treated balbfc mice, but not in dm2 mice, the expression of the exogenous va 3 resulted in proliferation of infected t cells, as assessed by an auto-mlr proliferative assays. into newborn balb/c mice (h-2 l +) o r into the congenic strain dm2 loose most of their lytic activity as well as the ability to form conjugates with specific tc. preliminary experiments show that spdp-anti-cd3-derivatization of specific tc overcomes the trypsin effect, so that both conjugation and lysis are observed between trypsin-treated pel and sdpd-anti-cd3-derivatized specific tc. many investigators, the ti/cd3 complex on pel surface is central to specific lysis. the observed inhibition of trypsin may be due to its action upon it. since trypsin-treated pel could be triggered toward conjugation with and lysis of tc by anti-cd3, the cd3 at least must have remained intact and is the important molecule for both the critical conjugation step and lysis. trypsin treatment possibly disturbs the interaction of tc with ti/cd3 of pel so as to prevent proper engagement of the cd3. concanavalin a (con a) treatment of tc appears not able to bring back the lytic activity or the ability to form conjugates of trypsin-treated cells, indicating a dichotomy in the mechanism of killing by derivatization with anti-cd3 versus con a mediation. comparing pel and their blasts ipeb). unlike pel, peb do not show ldcc or lytic activity against spdp-anti-cd3-derivatized nonspecific tc. ldcc by con a has been proposed to function via ti/cd3 interaction. unlike pel, peb also do not show fluorescence. it may be that the ti/cd3 complex on peb differs from the one on pel. at the same time, total lack of cd3 is contraindicated for peb kill specifically. the region important in ti interaction for ldcc and that recognized by anti-cd3 may be within the aberrant area of cd3, so neither the con a nor the anti-cd3 trigger could be transmitted on peb. the importance of the cd3 is indicated further by immunolabeling both cells with anti-cd3 revealed that c533 combination of cd4+ and cd8+ islet specific t cell clones are pathogenic in the nod mouse, eva-pia reich and charles a. janeway. jr., department of pathology, section of imunobiology, yale university school of medicine, 310 cedar st., new haven, ct 06510. although diabetes in the nod muse is thought to be initiated by t lymphocytes, thus far no t cell clone has been derived from nod islets. we therefore isolated islets from acutely diabetic nod mice and islet derived lymphocytes were propagated in il-2 and stimulated every 2 weeks with fresh mitomycin-c treated nod islets to maintain islet specificity (i.e. a proliferative response to nod islets). t cell clones were generated by limiting dilution followed by recloning by single-cell manipulation. facs analysis showed that all clones were positive for thy-l+ and cd3+, confirming that receptor bearing t cells had been cloned. cd4+cd8-and cd4-cd8+ clones were isolated that proliferated in the presence of nod islets. respond to nod but not balblc or b1o.br islets, while the two c d 8 ' clones respond to nod and balb/c islets but not blo.br. islet-specific, i-anod restricted, while the c d e clones are islet-specific and dd restricted. injection of individual cloned lines into young, irradiated nod mice leads to no overt or microscopic disease. a syndrome of dehydration, rapid deterioration and death about 10-14 days after transfer. mice sacrificed at this time show lymphocytic infiltration around the islets. this suggests that the cd4+ clones are however, mixture of c d 4 ' and cd8+ cloned lines causes these cloned lines may be useful in the search for islet cell autoantigens. dusseldorf 1 (frg). autoantibodies to nucleolar components are a common serological feature of patients suffering from scleroderma, a collagen vascular autoimmune disease. while animal models which spontaneously develop abundant anti-nucleolar antibodies have not yet been described, high titers of such antibodies may be induced by treating susceptible strains of mice with mercuric chloride. we have identified the nucleolar autoantigen against which the hgci2-induced igg autoantibodies from mice of strain b1o.s are directed. it is a protein of apparent molecular weight of 36 kda and pi value of about 8.6 which is associated with the nucleolar snrna u3, and by these criteria, is identical with a polypeptide called fibrillarin. it is striking that scleroderma patients spontaneously form autoantibodies against the same u3 rnp protein. the hgc12-induced murine and the scleroderma-specific human anti-u3 rnp autoantibodies were indistinguishable in their reactivities towards fibrillarin. they further resemble each other in so far as both recognize epitopes on the 36-kda protein which have been highly conserved throughout evolution. our results provide a basis to investigate at the molecular level whether similar immunoregulatory dysfunctions may lead to the preferential a n t i 4 3 rnp autoantibody formation in the animal model and in scleroderma patients. forty bp-specific cd4+ t-cell clones were isolated from the peripheral blood o f a patient with multiple sclerosis. studies with a panel of xenogeneic bps of known amino acid sequence and with laree fragments of the bp molecule demonstrated the existence of at least 10 epitopes recognized by human t-cells. at least 7 of these epitopes were potentially clustered in the middle or at the c terminus of the molecule. in studies with synthetic peptides corresponding to residues 86-105 and 152-170 of human bp (hbp), 9 clones proliferated in response to 86-105 and 19 recognized 152-170. ctl assays were carried out with targets consisting of an autologous b-cell line coated with hbp. nineteen of the 40 clones demonstrated bp-specific ctl activity; these were the same 19 that had recognized peptide 152-170 i n proliferation assays. these clones also killed targets coated with peptide 152-170 but not targets coated with 86-105. clones that proliferated in response to peptide 86-105 or to epitopes located outside of these two regions failed to kill hbpcoated tarpets. to a lack of binding of 86-105 to the target cell, as the same b-cell line successfully presented peptide 86-105, in proliferation assays, to clones that had previously recognized this peptide when presented by e-cells. activity against bp is more restricted than is the specificity demonstrated for proliferative activity. the lack of killinp of targets coated with 86-105 could not be ascribed prystowsky, department of pathology and laboratory medicine, university of pennsylvania, philadelphia, pa as an approach to studying regulatory factors important during t cell proliferation, the expression of genes induced during il2-stimulated t cell growth was examined. to identify genes induced by il2, a cdna library was made from il2-stimulated cloned t cells in late g1 of the cell cycle and was screened by differential hybridization, selecting those clones which had higher steady-state mrna levels after il2 stimulation. of 40,000 clones originally screened, 90 positive clones were identified, which represented 21 different genes. when partially sequenced and compared with sequences in the nih and embl databases, 6 clones were identified as cdnas encoding glycolytic enzymes, 4 were cdnas of cytoskeletal genes, 3 were cdnas coding for proteins important in protein synthesis, and 8 clones were not identified. the maximal steady state mrna levels generally occurred during late gl/early s phase, and the induction of most m a s was not inhibited by cycloheximide at a dose that inhibited cell proliferation. nuclear run-on transcription demonstrated an increase in the rate of transcription for gapdh. in addition, there is post-transcriptional regulation of expression, as some mrnas were stabilized upon il2 stimulation. having cloned these mrnas, we can now begin to study the regulation of expression of selected genes. supported by nih grants. cyciosporin a (csa). a potent immunosuppressive drug, caused organ-spedfic autoimmune disease, such as gastritis with anti-parietal cell autoantibodies or oophoritis with anti-oocyte autoanliies, in balwc mice when the drug was administered daily for one week to newborns. administration to adult mice did not. csa abrogated the production of l3t4' t cells and lyl-2' t cells in the thymus. consequently, these t cells were substantially depleted from the peripheral lymphoid organs, especially when the drug was administered from the day of birth. autoimmune disease was prevented when csa-treated newborn mice were inoculated with splenic t cells from normal syngeneic mice. on the other hand, removal of the thymus immediately after neonatal csa treatment produced autoimmune disease with a higher incidence and in a wider spectrum of organs, i.e.. thyroiditis, sialoadenitis of the salivary gland, gastritis. insulitis of the endocrine pancreas, adrenalitis, oophoritis, or orchitis. each autoimmune disease was accompanied by the development of autoantibodies specific for the corresponding organ antigens. it is likely that csa caused organ-specifc autoimmune disease, when administered to newborns, by depleting certain regulatory t cells (suppressor t cells) controlling self-reactive clones. (this work was supported by a grant from the lucille p. markey charitable trust.) artur scherf, philippe dubois, marika plat and luis perelra da sllva, instltut pasteur, 25-28, rue du dr. roux. 75015 paris, 'u93 inserm, hopltal st. louis. 2, place du dr. alfred fournier. 75010 paris, france. we isolated from a genomlc expression library of the malarla parasite p. falciparum a clone coding for an antigen associated with the membrane of infected erythrocytes. intensive sequence analysis of this gene, called 11-1, showed that the major part of this molecule consists of at least 200 repeats of nine amino acids. the consensus sequence of these degenerated repeats is e-e-v-v-e-e-v-v-p and secondary structure analysis predicts t h a t they have a high tendency t o form amphipathic alpha helices. the 8-and t-cell response t o the two most frequent types of repeat were analysed in five different h-2 congenlc mice strains using two synthetic peptides (pqa: y-p-(e-e-v-v-e-e-v-v-p)n-k: pqb: the 8-cell response t o both peptides is restricted to the h-2d and h-2k xaplotype. pca and pqb specific antibodies do not dlscriminate between the two peptides (measured by elisa). the mhc restriction was confirmed by anaiyslng the tcell response to both peptides. t-cell lines speclfic for each of the peptides pqa/pqb were derived from h-2d responding mice. although the peptides differed in four positions of the hydrophoblc part of the helices (valine replaced by isoleucine and leucine) no signiflcant dlfferences were observed in the antigen presentation between peptide pqa and pqb. in contrast, both peptides were unable to induce a proliferation of the heterologous t-cell ilne. suggesting t h a t the variable hydrophobic amino acids of the repeats are involved in tcell recognition. we are currently investigating if other variant repeats of the 11-1 antigen are restricted t o a different mhc haplotype and that the totality of repeats may be subjected to no restriction. glven the type of amino acid replacements within the two repeats tested, the t-cell receptor appears t o discriminate between very similar amino acids. auphan, claude boyer, annick guimezanes, claire langlet, cenae dimmunologie inserm-cnrs de marseille-luminy, case 906, 13288 marseille, cedex 9, france. the yinterferon gene is activated upon cell surface triggering of the aansmembrane t cell receptor (tcr)/cd3 complex or via phosphatidylinositol anchored thy-1 molecules cell clones. both these activation events are inhibited by antibodies directed at the lyt-2jlyt-3 molecules and are lost on tcr a-chain deletion variants (1, 2). biochemical analyses failed to reveal any association between the tcwcd3 complex and either lyt-wyt-3 or thy-1 molecules. however, such studies consistently revealed a non-covalent, cis-type association between lyt-ulyt-3 and h-2 class i products as well as between thy-1 and h-2 class i products. the structural features of the molecules required for such interactions are being determined using hybrid engineered molecules and possible implications of such interactions for t cell activation are being evaluated. (1) schmitt-verhulst, a.-m. et al. nature 325, 628-631 (1987) . (2) guimezanes, a. et al. cellular immunol. 113,435-446 (1988) . i t i s increasingly apparent that the association of both chains is critical to reconstitute t,he 4g-mhc binding site. combinational mechanisms for generating diversity play a major role in increasing the total size of the repertoire. however, evidence for preferential associations have been described between other heterodiaeric proteins of the immunoglobulin gene superfamilg. i t is therefore important to determine if association between different sets of vas and ybs occur at random or if, alternatively, preferential associations exist. we have devised a system which should enable us to determine, by dna mediated gene transfer of cloned cdnas encoding different vas o r vbs, if such preferential associations do exist. we hare also used a panel of monoclonal antibodies specific for a particular va or vb to derive t cell clones expressing these vas or vbs. in a polyclonally activated t cell population expressing a specific va or vb chain, we will present data indicating that the association between these chains is preferential or random. !nteractions between t cells and mhc molecules are thought to select a t-cell repertoire skewed towards recognition of antigens in the context of self mhc molecules. in addition, t cells which react strongly to self mhc molecules are eliminated by a process called self-tolerance. we have recently described generation o f transgenic mice expressing the a6 t-cell receptor (tcr) from the cytotoxic t lymphocyte 2c (sha et al., 1988 nature 335:271) . the clone 2c was derived from a ealf3.e (h-zb) anti-eale/c (h-2d) mlc and is specific for the ld class i mhc antigen. in transgenic h-2b mice, a large fraction of t cells in the periphery expressed the 2c tcr. these t cells were predominantly cd4-cd8+ and were able to specifically lyse target cells bearing ld. in the periphery of transgenic mice expressing ld, functional t cells bearing the 2c tcr were deleted. this elimination of autoreactive t cells appears to take place at or before the c04+cd8+ stage in thymocyte development. in addition, we report that in h-2s mice, a non-autoreactive target haplotype, large numbers of cd8' t cells bearing the 2c tcr were not found providing strong evidence for the positive selection o f the 2c tcr specificity by h-26 molecules. the requirements for cd4 expression during t cell differentiation in fetal thymus organ culture woc) were investigated by analysis of the effects of addition of anti-cd4 mab. control etoc cult& for 5 days contained predominantly cd4+cd8+ cells as well as cd4 or cd8 single positive cells expressing high levels of cd3. t cells from ftw cultured for 5 days in the presence of anti-cd4 m a b expressed markedly diminished cd4 expression. when anti-cd4 m a b was removed 20 hours prior to phenotypic analysis, it was found that cd4 was recxpressed on cd4+ cd8+ t cells. after 12 days of culture, control roc contained high frequencies (23-3046) of cd4+cd8and cd4-cd8+ thymocytes expressing high levels of cd3. in contrast, anti-cd4 mated cultures, while containing cd4-cd8+, cd3+ t cells, were well depleted of cells which reacted with anti-cd4 antimy. when anti-cd4 mab was removed 20 hrs prior to analysis, cd4 was reexpressed on cd4+cd8+ cells, and low frequencies (6%) of cd4+ cd8-t cells were detected, but these cd4 single-positive cells did not express high levels of cd3 or detectable ap heterodimrs. while normal frequencies of cd4-cd8+,cd3+ cells expressed vg8 determinants, the a n t i -w mated cultures were relatively enriched in cd4-cd8+cd3and cd4-cd8-cd3-t cells compaml to control 12 day eoc. these data demonstrate that while cd8+cd3+ t cells can differentiate in the presence of anti-cd4 mab, differentiation of cd4+cd3+ cells is inhibited and cell surface cd4 is down-modulated. cd4+cd8+ cells can develop in the presence of anti-cd4 m a b and do express t cell receptors. these results suggest that cd4 expression, while not required for development of cd3+cd4+cd8+ t cells, is necessary for differentiation of mature cd4+cd8-cd3+ t cells, and that the interaction of 0 4 with its ligand is critical for thymus selection of class-ii specific t cells. uppenkamp, and alfred singer, experimantal immunology branch, nih. bethesda, md 20892. in order to investigate the role of the t cell receptor (tcr) in thymocyte maturation, we have studied thymocytes from the immunodeficient c.b-l7/scid mouse, which due to a genetic defect, is unable to express tcr genes. despite this mutation, scid thymocytes were functional as they produced lymphokines and proliferated in response to stimuli (pma, ionomycin, il-2, il-4). phenotypic analysis revealed that, like normal immature thymocytes, sci+dd'&mocytes were large, thy 1.2+, cd4-, cd8-, tcr-and were enriched in cd5 , il-zr+, pgp-lf, and hsa+ cells. however other tcrpopulations present in normal mice, (i .e. cd4-8+tcr-and c d 4 ' 8 ' t c r -cells) were absent from scid mice. in order to determine if tcr expression was required for cd4 and cd8 expression, we analyzed thymi from the occasional scid mouse which possessed small numbers of tcr-bearing thymocytes. interestingly, expression of cd4 or cd8 was detected only in those scid thymi that possessed tcr' thymocytes, although cd4 and cdb expression was not confined to those tcr-bearing cells. further, the introduction of tcr' cells from thy 1 congenic normal mice into tcr-scid mice consistently promoted the expression of cd4 and cdb (but not tcr) by scid thymocytes. moreover, scid thymocytes from these chimeras possessed cd4+8+ cells, however no mature, single positive thymocytes of scid origin were detected. together, these results suggest that tcr-bearing cells within the thymic milieu are required for the expression of cd4 and cd8 and for the differentiation into cd4+8+ cells. however, final maturation into mature single positive thymocytes requires that the thymocyte itself express tcr. tcr in thymocyte differentiation: evidence from the immunodeficient the cd4 t-cell surface receptor has been implicated in the stabilization of lntercellular interactlons. in addition, it is felt to mediate the transduction of an independent lntracellular signal. we have recently shown (veillette et al.. in press) that the cd4 molecule expressed in t-lymphocytes is complexed to the internal membrane tyrosine kinase p66'ck. suggestlng that alterations in the properties of thls enzyme mediate the cd4 related signal. to gain further insight into the structure and function of the cd4-lck complex, we have generated nih3t3 flbroblasts that co-express the cd4 and lck proteins. nih3t3 flbroblasts expressing a murine lck cdna were transfected by calclum-phosphate preclpitatlon with a construct containing a murine cd4 cdna and the neomycin resistance gene. stably transfected clones were selected for resistance to the amlnoglycoslde c418. we have now demonstrated that the cd4 and ~6 6 '~~ expressed in these non-lymphoid cells can form a stable non-covalent complex. the structure and function of the cd4-lck complex expressed in murine fibroblasts is currently being evaluated. h-2b t c e l l s from h-2b+hh-2bxd responder animals fail to generate one (type a) of these two major clonotypes. environment i s permissive for the t cell specificities of both poly-18 clonotypes (type a and b) and also rovides the determinants for t h e i r selection. the absence of type a clones in the h-2b+h-2gxd chimeras can therefore only be attributed to the failure of h-2b t c e l l s to generate the appropriate t cell receptors. these results suggest a genetic hole in the t cell recognition repertoire of h-2b mice for the lysine-containing epitope of the poly-18 antigen. we have used oligonucleotide-primed gene amplification to study the association between hla class i1 alleles and autoimmune disease. pemphigus vulgaris (pv) is a severe autoimmune skin disease highly associated with dr4 and drw6. we have shown that the nucleotide sequence of the first (and most polymorphic) domain of a new dq allele is found in 1 3 / 1 3 israeli drw6 pv patients but only 1/13 or& healthy israeli controls. this allele differs at only position 57 from two other dq alleles not associated with pv. further, we show that a shared setuence in the third hypervariable region of the first domain of two common subtypes o f dr4 and drw6 cannot be the sole determinantdrgf susceptibility to pv. none of 7 dr4 /drw6+ pv patients had this epitope. we also report a new dr first domain sequences from a drw6 pv patient. the frequencly of t h ! $ allele in patients and controls is being assessed. similar studies to identify disease associated class i1 sequences are underway in patients with multiple sclerosis and rheumatoid arthritis. the frequency of these autoimmune responses is determined genetlcally. and is essentially 100% in the projeny derived in crosses d transgenic mice to some inbred strains of mlce [e.g. those of h-2d haplotype). and yet only 1096 in others (e. g. h-2b) . recently. we initiated an analysis of the i m m~~l~g i d nonresponsiveness in two lineages of insulin/t antigen transgenic mice with early onset of expression of the transgene (rip-1 tag #2 and rir tag #2). while the presence of the transgenic protein durlng the prenatai/neonatal period is the primary requirement for the nonresponsive phenotype, it further appears that the levels of t antigen expression durlng embrydnic development quantjtatively influence the subsequent nonresponstvmess in adult me. an additional modification is exerted by a genetic component whlch seems to map to the mouse major histocompatibility complex [h-2). interesiingly, it appears that the h-2d haplotype. whlch is associated with the most complete impairment of immune responses when t antigen is expressed prenatally. is also assodated with the highest frequency of autoimmune responses when the developmental expression of the transgene is delayed. this correlation is considerably strengthened by the observation that outcrosses to c57b1/6j m-zb) produce mlce with either a weaker tolerance in the case of the early expressers. or a less hquent and weaker autoimmune response in lines with a delayed onset of expression of the transgene. thus. the two distinct biolcgical consequences of the presentation of this antigen (tolerance or autolmmunity) are &ly to result from differences of the developmental maturity of the immune system, and both effects appear to be similarly mhc restricted. lmmunogenicity c 549 mapping the pzb geneti2 contribution t o lupus-like autoimmune disease. i-division o f basic science, national jewish center for immunology and respiratory medicine, 1400 jackson street, denver co 80206; 2-the jackson laboratory, bar harbor, me. nzb mice carry a geneb) that contributes to the lupus-like autoimmune disease seen in (nzw x nzb) fi hybrids. however, the precise locus of this gene has yet t o be determined. to map i t s location, we are breeding a panel of nzbxsm/j recombinant inbred (ri) with nzw mice. by determining which of the ri strains produce fi animals that develop autoimmunity, the location of this nzb gene can be deduced based on the strain distribution pattern of the known genetic markers among the nzbxsmlj ri strains. cells were then fractionated on a percoll gradient to obtain small dense 'resting" b celb and large 'activated' b cells. the small dense b celb demonstrated increased inhibition at every dose of ic examined, when compared to large b cells. for instance, pulsing with 10 ug of ic resulted in a 96% reduction in anti-fl, but not anti-trinitrophenyl, plaque forming cell (pfc) response for small dense b cells. in contrast, the pfc response of large "activated' b cells was reduced only 66%. in addition, when subtolemgenic doses of ic were used (10 ng) prostaglandin â�¬2 (5x10-8y) could function as a potent secondary negative signal rendering these b cells susceptible to negative signalling. we concluded that small dense b cells are more sensitive to ic induced unresponsiveness and that prostaglandin â�¬2 may further sensitize b cells along this pathway. .5 were developed in the mouse against antigens present on humans, hamster and rat nk and t lymphocytes. pre-incubation of lymphocytes with moab did not block the ability of the nk cells to bind to targets but did block their ability to lyse nk sensitive targets. the pre-incubation media contained lytic factors that lysed nk sensitive targets in a 20 h chromium release assay. interestingly pre-incubation of thymus cells or resting peripheral t lymphocytes with the moab induced the t lymphocytes to release lytic factors and acquire nk activity against nk-sensitive targets. lytic factors and acquired lytic activity were unable to lyse lak targets. signalling events did not involve cross linking or fc receptor participation since factors were immunoprecipitation and western blot analysis using the moabs defined a molecule of 27 kd on the surface of thymus or lung mononuclear cells. isolation and amino acid sequence analysis is being done to assist in the characterization of the g1. we have been studying in vitro activation of cd4-, cd8-thymocytes. in the present studies, highly purified double negative thymocytes were cultured with il 1 with or without il 2. following a 72 hour culture with il 1 + il 2 (in the absence of mitogens) significant proliferation was detected, although all cells remained cd4-, cd8-. the frequency of cells staining for cd3 cell surface protein increased from 10% to 60% and tcr vbeta8 from 4% to 40%. facs sorted cd3-cells from the cd4-, cd8population showed similar increases in cell surface tcr suggesting differentiation. northern blot analyses showed an increase in c -m rna at 24 hours, no change in cd3 delta or epsilon expression at 24, 48, and 72 hours, and a marked increase in the ratio of 1 . 3 to 1.0 tcr beta transcript between 24 and 48 hours. these data suggest that il 1 + il 2 can induce cd3 cell surface protein expression on progenitor thymocytes. furthermore, this induction is not associated with increased cd3 transcription, but rather is associated with an increased proportion of functional tcr beta transcripts. the p28 antigen of schistosoma mansoni has been shown to be able to induce protective immunity against schistosomiasis in rodents and primates. synthetic peptides were used to localize the major b and t-cell epitopes of this protein. the primary structure of the p28 molecule was analyzed using various predictive algorithms : surface localization, hydrophilicity, flexibility, secondary structure, amphipathicity of predicted a-helical regions, rothbard pattern. according to these criteria, several peptides were synthesized and tested in immunological assays in vitro and in vivo. correlations between these results and the physico-chemical predictions will be shown. simian virus 40 (sv40) t antigen is recognized by cytotoxic t lymphocytes (ctl) in association with class i major histocompatibility antigens.we have identified four distinct antigenic sites on sv40 t antigen defined by h-2db restricted ctl clones. we have therefore attempted to map these antigenic sites precisely by using overlapping synthetic peptides corresponding to sv40 t antigen amino acids. the synthetic peptides of 15-20 amino acid length were incubated for five hours with 5lcr labelled-non-sv40 transformed h-2b cells in the presence of ctl clones specific for each of the four antigenic sites; and the released radioactivity was used as a measure of specific recognition of synthetic peptides by the ctl clones. the results demonstrated that the antigenic site i resides between amino acids 205-219; sites ii and 111 between amino acids 220-233. site 111 can be separated from site ii as it was found to reside between amino acids 225-239. the antigenic site v was localized between amino acids 489-503. these results show that sv40 t antigen is the target protein for ctl and the regions of t antigen recognized by ctl are tightly clustered in the amino terminal half. attempts to identify amino acids that selectively interact with the class i antigen and the t cell receptor are in progress. peripheral blood lymphocytes of cancer patients demonstrate an early humoral and specific response to application of tumoral extracts. the response is measured as a change in the spectrum of fluorescence polarization of fluorescein molecules. this is probably a measure of sol-gel transformations induced in the cytoplasm upon cellular activation of resting lymphocytes. this measurement is carried by epifluorescent microscopy on single lymphocytes, sitted on a regular holes matrix that is scanned under computer control. it is possible to follow changes within each cell in a population of upto ten thousand cells. since the cells are sitted safely in their holes, it is possible to rinse them with various reagents and follow the kinetics of individual responses by repeated scanning. further studying of the stimulative process might be useful in identifying the responding lymphocytes and the tumoral reagents. this test of the immune system is intended for early and specific detection of cancer and for follow-up of immunotherapeutic manipulations as well. c 559 katharine s. ullman, j.p. shaw, elizabeth emmel, and gerald crabtree. stanford university, stanford, ca 94305. following antigenic stimulation, t cells undergo an orderly series of changes that result in cell division and immunologic function. in hopes of working backward in the cascade which links the membrane events to the genes essential for these differentiated functions, we have begun an analysis of the nuclear proteins responsible for gene activation in stimulated t cells. in previous studies, using a panel of internal deletion mutations of the 1l-2 enhancer, we found that when the region from -13 to -82 was mutated, the level of induction dropped 20-fold. this region is protected in a dnaase footprinting assay by a protein, nf-ilz-a. although the protection is seen with nuclear extracts from both stimulated and resting jurkat cells, in vivo this is a site of dnaase hypersensitivity found only in activated jurkats and peripheral blood t cells. a tetramer of the protected site directs transcription from an unrelated promoter in jurkat cells only when the cells are stimulated by lectin or antibodies to the antigen receptor. furthermore this activation can be blocked by 1 ng/ml of cyclosporin a. as a means of detecting more subtle changes in protein binding following stimulation. the methylation-interference patterns of stimulated and non-stimulated jurkat cell nuclear extracts were compared and found to be identical. we propose that though the dna-binding protein, nf-il2-a, is present constitutively, it becomes functional only following specific signalling through the antigen receptor. further characterization of this protein will help to clarify its particular role in the activation of il-2 expression following t-cell stimulation. the negative e f f e c t of f l a n k i n g sequences on t c e l l lmmunogenicity c561 central lab. blood transf.service, lab. exp. and clin. immunology of the univ. of amsterdam, the netherlands the hydrolysis of phosphatidylinositol-diphosphate and the formation of inositol-triphosphate (ip3) and diacylglycerol (dg) re among the first events that can be monitored after t-cell triggering. ip3 mobilizes c a 2 ' from intracellular stores, whereas dg is the physiological activator of protein kinase c (pkc). although it is suggested that activation of pkc is essential in the induction of mitogenic t-cell responses, no direct evidence for this hypothesis has been presented yet. in this study we used two novel pkc inhibitors, 1.e. 1-alkyl 2 methyl-glycerol (amg) and staurosporine to assess the role of pkc activation in human t-cell stimulation. we observed that the inhibl$ors did not interfere with the anti-cd3 or anti-cdz monoclonal antibody (mab) induced ca rises in peripheral blood t cells, whereas cytoplasmatic alkalinization initiated by the same stimuli or the phorbol ester pma was strongly reduced. amg as well as staurosporine gave a dose dependent inhibition of both interleukin 2 (il-2) production in jurkat cells and t-cell proliferation. in contrast, the induction of il-2 receptor expression was highly resistent to the action of both compounds. it was shown that anti-cd28 mab were able to partly overcome the action of both inhibitors. these studies support the important role of pkc-activation in human t-cell triggering. moreover, since anti-cd28 mab were able to counteract the action of the pkc-inhibitors, it is suggested that mitogenic stimuli generated via different t-cell membrane molecules may use distinct intracellular signal transduction pathways. a f t e r incubation w i t h ndlal-26 o r al-17, t a r g e t s o f @ phenotype were lysed by a n t i -a k i l l e r c e l l s . the a n t i t h e s i s was a l s o observed: a phenotype t a r g e t s were lysed by a n t i -8 t c e l l s a f t e r incubation w i t h ndlgl-17. ndla4 we have developed a monoclonal antibody that recognizes an apparently t cell-specific antigen, ltac, expressed only in the late stages of t cell activation. when peripheral blood lymphocytes are activated with allogeneic stimulators or pha and examined by facs, ltac is expressed only after day 6 or 7. ltac is expressed by all t cell clones tested, both cd4' and cd8'. a wide range of cell types were tested and found negative for ltac expression, including b cells, fibroblasts, endothelium, and neural cells; thus ltac expression seems to be restricted to the t cell lineage. immunoprecipitation of cell-surface labelled proteins and sds-page revealed a 160 kd band under reducing conditions and three bands of 240, 180, and 160 kd under non-reducing conditions. ltac is distinct from all other reported t cell activation antigens on the basis of its pattern of expression and molecular weight. we have purified ltac protein using wheat germ agglutinin-agarose chromatography followed by monoclonal antibody-agarose chromatography, and have used it to produce an antiserum suitable for screening bacterial cdna expression libraries. the regulation and function of molecules expressed in the late stages of t cell activation is an important frontier in understanding the immune system, and reagents such as monoclonal antibodies may have important diagnostic and therapeutic uses. balb.h.p7 mice were capable of responding to all 24 antigens tested but the magnitude of the response to some of these antigens (1 1 of 24 antigens) was reduced 2-5 fold when compared with balb/c mice. responses to the remaining antigens were either comparable (11 of 24 antigens) or occasionally even enhanced (2 of 24 antigens) compared to balwc mice. these results suggest that jp2 and dg2 elements are required to maintain the strength of the t cell repertoire and that, in the wild, mice carrying this deletion a u l d be at a significant selective disadvantage. the identification of 11-1 was carried out in radiolabeled murine macrophages by immunoprecipitation and sds-page, using antibodies to were predominately of mr 31.000 and 37.000, respectively. a s assessed by estimating radioactivity in each band, 11-larwas about 4 fold more abundant than 11-16. most of the cell associated 11-1 activity, as measured by the d10.g4.1 proliferation assay, could be inhibited by anti 11-iswhereas no inhibitory effect was observed with anti 11-1b. after removal of 11-ltc from cell lysates by immunoadsorbtion the residual activity accounted for only about 5 % of the total 11-1 activity, and was completely neutralized by anti 11-1b. however, in culture supernatants anti i1-1r reduced the 11-1 activity by approx. 50 % whereas a combination of both, anti 11-leand anti 11-10 completely neutralized the activity to background levels. interestingly, only one radiolabeld polypeptide of m, 37.000 was precipated from the extracellular medium by the anti 11-16 antibody. these data therefore suggest, that externalization is required in order to generate bioactive 11-18 molecules, but apparently a proteolytic processing step of the precursor is not involved. moreover, in view of previous reports, demonstrating that cellular interactions during antigen presentation can be mediated via a membrane form of 11-1, o u r experiments substantiate the hypothesis that membrane-associated, biologically active 11-1 is predominantely i1-1q. david l. brandon and joseph w. corse. usda agricultural research service, western regional research center, albany, ca 94710 in order to prepare antibodies which would recognize 0-glucosides of cytokinins (purine phytohormones), we investigated an alternative to carbodiimide reagents. carbodiimides induce the formation of amide bonds, and thus can be used to couple haptens containing carboxyl or amino groups to proteins. the use of water-soluble carbodiimides is limited by the sparing solubility of some haptens in water and by the formation of the intermediate 0-acylisourea, which can be less soluble than the hapten and which can itself act as an unwanted hapten. to overcome these problems, we have used 2-morpholinoethyl isocyanide and a suitable catalyst to effect amide bond formation. these reagents are the basis of recently developed chemical procedures for peptide synthesis (aigner et al.. 1980) . the reaction conditions permit coupling compounds which are soluble or insoluble in water to proteins in a simple, homogeneous system. we have demonstrated that this procedure is widely applicable for preparing enzyme-labeled and immunogenic conjugates of cytokinins and other biologically relevant compounds with an available carboxyl group. we hypothesize that conjugates made with these reagents will not induce the allergic responses and unwanted crossreactivities associated with the use of carbodiimides. supported, in part, by bard grant us-711-83. mlman r i g 2 , not uwuse rig2, uxluces a furctional1y-btut-e subset of mdlse thymqtes to. secrete -4, shichin turns iniuces cells w i t h i n this subpapllation to becane cytotcouc. nolmal mxls~ thymocvtes beaw ctl in to con a * il-2, and that the cplresponses induced by-agentsm blocked canpletely by the addition of the anti--4 nyib 1lb11. in addition, normal muse thymxytes produced il-4 in response to con a f i t 2 . the fijmtionally-ture lobster agglutinin (mgl)-positive t h y r e p subset responded to con a f i g 2 in a similar mmer. ylhen the functionally-nunature subset of mgl-negative thymzyk subsets was incubated with con a f muse r i g 2 , they did not becane ctl and &d not prcduce il-4. haever, when wl-negative thymcytes were cultured w i t h con a f human r 1~2 , cells w i t h i n this subpsxllatim developea gccd ctl activity and also secreted 'ihese data qmnstrate that cpl generatim and i g 4 pmauction occur mnccanitantly i n dl thymqte m a t i o r r s studied. momever, stirmli which f a i l to induoe cn generation also f a i l tostinulate erd0qencu.s iir4 praduction. immunity to the typhus group of rickettsiae is largely dependent on the effector function of several classes of t lymphocytes including those which produce gamma interferon. since the surface protein antigen (spa) derived from typhus group rickettsiae has been shown to be an effective immunogen in animal models, human t cell clones specific for the spa of rickettsia tvoh were isolated and tested for their antigenic specificity as well as for their ability to produce gamma interferon. eighteen cm-positive clones specific for the spa of r. t v p b exhibited considerable diversity in their response to the spas derived from two strains of p. orowa zekii and from r. c a n a b . the vast majority of clones also recognized the spa's from & or0 w w but not from r. c w . two heteroclitic clones demonstrated significantly higher proliferative responses to the spa's derived from one or both of the of r. orowazek i i strains than to the spa of r., and one clone demonstrated a significantly higher response to the spa of r. t v o k as compared to the other spas. all 18 clones produced gamma interferon in response to spa stimulation. one of the clones demonstrated significant proliferative responses to a fragment of the spa produced in e. coli infected with a recombinant lamda gt 11 phage containing a 1 kb fragment of the spa gene from r.. we conclude that the spa's from typhus group rickettsiae can elicit both a diverse t cell response in humans as well as the efficient stimulation of gamma interferon-mediated immunity. a monoclonal anti-mouse rbc antibody (g-8) has been prepared which appears to represent a pathogenic autoantibody related to those that arise spontaneously in aging nzb mice and which cause autoimmune hemolytic disease (aihd). this autoantibody recognizes native erythrocytes from mice but not from other species, and northern blot analysis revealed strong hybridization with the j558 probe but not with the 5 other vh gene family probes tested. thus, g-8 is clearly distinct from autoantibodies that recognize bromelain-treated mrbcs and which belong to a unique vh gene family. when g-8-producing hybridoma cells were grown as tumors in balb/c mice, the mice developed aihd characterized by a decrease in the number of erythrocytes (hematwrit) and the development of coombs-positivity. an anti-idiotypic antibody (e8) was prepared against g-8 and was found to recognize an idiotypic determinant present on most autoantibody forming cells derived from old (coombs-positive) nzb mice. previously, we demonstrated that treatment of spleen cells from young nzb mice with g-8 + c or with anti-ly2 antibody + c resulted in the elimination of suppressor cells and the generation of afc specific for mrbc. treatment with a control nzb igm mab (g-4) + c or with a rabbit anti-mrbc antiserum + c had no effect on the afc response to mrbc. similar deletion of suppressor cells was obtained following treatment of spleen cells from balb/c mice with g-8 + c. greater than 80% of the resulting afc expressed the g-8 idiotype. the results indicate that the response to self-erythrocytes is comprised of autoantibodies that express a recurrent idiotype. in an attempt to determine the optimal molecular parameters for the synthesis of anti-pathogen vaccines, we have studied the murine antibody responses to two types of model vaccines. the vaccines were: (1) purified polysaccharide type 3 derived from pneumococcal bacteria, and (2) synthetic conjugates made from a peptide region of the circumsporozoite coat protein of the murine malaria pathogen, p. berghei, coupled with dextran of high molecular weight. in both cases the molecular size was systematically vaned, and an optimal size was found to be important in determining the level of immune responsiveness. in the case of the malaria peptide-dextran conjugate, epitope multiplicity (epitope density) was also found to be an extremely important parameter, immunogenicity increasing with increasing epitope density. the james p. allison. cancer research laboratory, university of california, berkeley, ca. 94720. we have produced an antiserum to a synthetic peptide corresponding to a portion of the murine cd2 gene product. this antiserum reacted specifically with cells expressing cd2. flow cytometric analysis of normal lymphoid tissues revealed that thymocytes were heterogenous in the level of cd2 expression while splenic t cells expressed cd2 at uniformly high levels. cd2 expression with that of other t cell differentiation antigens (il-2r, j l l d , cd5 and cd3) in the murine young thymus and during fetal ontogeny was analyzed using three-color flow microfluorometry. our results indicate that cd2 expression is correlated with the stage of thymocytes maturation and demonstrate that il-2r expression in the thymus is not induced via stimulation of the cd2 pathway. klinische argeitsgruppen fur rheumatologie/immunologie,schwabachanlage 10, 8520 erlangen. accessory cell depleted resting (la) t cells are not able to produce autocrine growth factors when stimulated via the t cell receptor complex (tcr), although il-2 receptor expression takes place. the addition of accessory cells restores the proliferative capacity. we have tested a series of recombinant lymphokines and growth factors for their ability to substitute this accessory cell function for human cdb and cd4 t cells. triggering via the tcr was achieved by a combinatorial stimulation with anti-cd3 and either anti-cd4 o r anti-cd8 that gives rise to il-2 receptor expression only on the corresponding t cell subpopulation. ll-3, tnfa, if+, il-1, il-4, ll-6 and gm-csf were either negative or induced only a minor proliferative response. however, the combination of il-1 with il-6 was nearly as efficient as il-2 in cd4 t cells. cd8 t cells were much less stimulated by il-1+ il-6. effect, which is in contrast to some reports on accessory-cell depleted mouse t cells whose function require costimulation with il-1. this observation might be important for the understanding of t cell proliferation in chronic inflammatory tissues in which il-1 as well as il-6 is produced in large amounts. structural and serological studies of these secreted effector molecules have suggested that they are disulfide-linked dimers bearing t cell receptor u and 6 chain d terminants. a t cell hybridoma, mts 79.1, constitutively antibody staining indicated mts 79.1 expresses a t cell receptor containing a vg8 element. northern and southern analyses indicated mts 79.1 used v68 and va4 genes to encode the t cell receptor. the secreted mts 79.1 suppressor molecule was bound by monoclonal anti-v68 antibodies and by antibodies specific for constant region determinants of the u chain. to assess the role of a and 6 chain genes in encoding the secreted suppressor molecule, we have generated v68-and vu4-negative variants from the mts 79.1 parent. experiments performed to date indicate that the loss of the v68 gene results in the inability to produce the suppressor molecule. the results are consistent with the idea that the t cell receptor u and 6 chain genes are used to encode these hapten-specif ic/mhc-restricted secreted suppressor molecules. producing a dnp-specific/h-zk 5 -restricted suppressor molecule was generated and studied. sensitivity to clonal deletion and selection for mhc restriction a m to be a feature of a particular stage of thymqte &el*, specifically l a t e mrtical cw+8+ cells. in mcent studies we have slwm that early events i n signal trarrwluction i n respnse to antigen r e c p x crosslinkiq differ in the bmdture cw+8+ p p l a t i o n and the mature cw'8-or a>rl-8 p p l a t i o n . m t s indicate that antigen receptolg on hath inmature and mature -itive t cells tmnsdwe signals via calcium mobilization, h-er the maqnitu3e of fnflux of e&acellular q'+ wfiich follckfi birding of antireceptor a n t q d i f f e r s tetmen these pqulaticns. specifically, imnature cells shcw a m& reduced q influx espcnse carpared to mature cells. we dmw here that dligation of ~~n p has different amepexe w i t h regard to q'+ nnbilization in mature and inmnture cells, no sucfi difference is seen f o l l a d r q ligaticm of the receptnr's transducer, a. ihe zpsults suggest that the signall* cascade leading to the influx of extracellular is intact hen c d~ is ligated, but is inccnplete w i e i i m p , the physiological liw, is ligated. in addition, ligation of cw or cd8 on bmdture t cells i n a~~ influx of extracellular a ' + canparable to that sem i n mature t cells. a clonal population has been isolated frcm inmnture thymocytes whir31 has the characteristic signal tramdmtion pxqerties of the tulk of inmnture thymdcytes. 'ihese f i r d h p suggest that "signal" transfer frcm xpnp to 03 may be inefficient in cd4+8+ cells. struchual analysis of the xpnp/cd3 canplex in hnature and mtum t cells is in progress. flood and alan friedman, dept. of pathology, yale university school of medicine, new haven, ct, 06510. protective immunity to the ultraviolet (w) light-induced sarcoma 1591-re is directed toward a single tumor-specific transplantation antigen expressed by the 1591-re tumor cells. termed the a antigen. a progressive variant line of 1591-re, termed 1591-pro4, lacks only the expression of this a antigen. immunization of mice with 1591-re tumor cells haptenated with trinitrophenyl (tnp-1591-re) leads to the subsequent rejection of tnp-haptenated progressive tumors and to increased delayed-type hypersensitivity and ctl responses to tnp in normal, immunocompetent syngeneic mice. however, little or no humoral immunity to tnp is seen in animals injected with tnp-1591-re tumor cells. pro4 did not exhibit tnp-specific tumor protective or cell-mediated immunity, but rather exhibited tolerance to subsequent immunizations with more immunogenic forms of tnp. biochemical and molecular genetic studies have revealed the a antigen to exist on the cell surface as a complex of 3 class i mhc-like molecules. transfection studies with dna encoding each of these molecules into 1591-pro4 reveals that the expression of one, and only one, of these three molecules mediates this increased immunity. these experiments suggest that an mhc-like antigen expressed on the 1591-re sarcoma acts as a natural adjuvant to increase cellmediated but not humoral immunity to linked antigens. the mechanism of this increased immunity is discussed. efforts to immunize cattle against economically important gastrointestinal nematodes showed that inrmunity is manifested by: 1) a response that reduces the fecundity of established and subsequently acquired worms. and/or 2 ) a reduction in the number of worms developing upon challenge infection. however. the ability of individuals to mount such immunity is highly variable. extending these studies to naturally infected populations indicate that there is a great difference in the number of eggs excreted by individual young calves on pasture. to delineate whether these differences were the result of host genetics and to begin to elucidate the mechanisms of resistance to parasite infection, a genetically defined cattle herd was assessed for parasite levels by determining fecal eggs per gram. three years of sampling of the calves during their first grazing season indicates that: 1) certain individuals in the herd will consistently excrete high or l o w numbers of parasite eggs, 2 ) the high or low phenotype is significantly controlled by the genetic make-up of the calf, and 3 ) the high or low phenotype is highly heritable (heritability -40). susceptibility is currently under investigation. calves have been determined and mhc class i1 typing is currently in progress. information is being used to assess the role of the bovine mhc in controlling immunoresponsiveness to parasite antigens. we have been studying the differential effects of il 1 + il 2 versus il 4 on the growth and differentiation of cd 4-, cd 8-thymocytes. culture of highly purified cd 4-, cd 8-thymocytes with il 1 (4 u/ml) + il 2 (100 u/ml) resulted in marked proliferation and increased cell size without change from the cd4-, cd0-phenotype. culture with il 1 or il 2 alone did not cause proliferation. a substantial contribution to the proliferation was secondary il 4 release: addition of anti-il 4 (11b11) blocking antibody inhibited proliferation induced by culture with il 1 + il 2 . il 4 mrna was demonstrated by northern blot analyses after 4 8 and 7 2 hours of culture with il 1 + il 2 whereas none was detected at culture initiation and very little was present at 24 hours. effects of il 1 + il 2 on il 4 transcription rate will also be reported. despite a marked inhibition of proliferation with anti-il 4 , there was no affect on expansion of cd 3+ cells following culture with il 1 + il 2 (increase from 10% to 60% in 7 2 hours). thus, in this system, il 4 enhances proliferation of progenitor thymocytes but does not contribute to induction of t cell receptor. carplex. lhese cwplexes can be used to inmmize mice in the absence of protein carriers or adjuvants, thus facilitating the study of the inmum to a sml1 ckmically defined antigen. use of this tecfinology has allawed us to identify two t helper cell epitopes in cclllserved regions of hn gp 160 not previcusly identified by computer algorithims, defined by amino acids 485-518 and 585-615. inummization with these peptides in pptide-@nqhlipid cwplexes results in the prpauction i* and i* antibodies, which cioss react with cloned fragmnts of the w l e protein. us& this &logy we have begun to characterize the innume response to individual peptide antigens. ?he reqonse of h2-k mice to amino acids 494-518 of 9p 160 of hiv, has been analyzed. lhe optimal dose of a peptide containing both b and t cell epitopes was found to be 15-30 ug, depending on the route of administration. im d z a t i o n requir& less antigen for opthum antibody resporrsf, than did ip. ment for an ant-response. additional variables, as phosfholipid carpasition and method of cross-linking have been studied anl will be . webelievethattheuseof this peptide-phospholipid canplex tehmlogy will be significant both for studying the innwe response to single epitcpes and for vaccine develolment. based on assays in which t cell proliferation was induced via oxidative mitogenesis and exposure to mhc alloantigens, it has been reported that langerhans cells (lc) isolated from normal mouse skin aquire maximum capacity to activate t cells only after 72 hours of culture. we have studied lc from balb/c mouse skins for their capacity to present ovalbumin (ova) and ia doantigens to unprimed t cells and to antigen-specific t cell hybridomas. the data reveal that both fresh and cultured lc presented ova and alloantigens with equal efficiency to previously primed responders (and with 10 fold greater efficiency than spleen cells or the b cell lymphoma a20.1-11). by contrast g& cultured lc displayed the capacity to present antigen to ynorimed t cells. we propose that the antigen presenting potential of freshly prepared and cultured langerhans cells, respectively, reflect the in vivo functional properties of intraepidermal lc and of lc that have picked-up antigen in the epidermis and migrated via dermis to the regional lymph node. if so, these data suggest that resident epidermal lc are fully prepared to present cutaneous antigens to memory/effector t cells (efferent limb), whereas resident lc must leave the influence of the epidermis in order to develop the capacity to meet the more stringent conditions required for antigen presentation to porimed t cells (afferent limb). we have investigated the structural restrictions placed on residues contained within a minimal t cell determinant, using the balb/c class i1 restricted t cell response to the site 1 determinant of the influenza hemagglutinin molecule as a model system. to delineate which of the residues comprising the site 1 determinant are involved in interaction with the t cell receptor, we have determinaed the response of a large panel of site 1 specific t cell hybridomas to a collection of peptide analogs differing by single conservative or non-conservative substitutions at 9 positions. the fine specificity patterns of the t cell panel is extremely diverse; t cells varied in both the location and number of residues within the antigenic peptide that effected recognition. our results implicate at least 6 out of 9 residues within the antigenic pepetide as being involved in interaction with the t cell receptor. this result suggest that peptides comprising the site 1 determinant do not form alpha helical structures when in association with mhc molecules. rubella-specific isotype and igg subclass responses were evaluated using elisa techniques in 44 rubella ha1 seronegative adult females undergoing rubella immunization (ra 27/3 strain). responses were evaluated prior to immunization and at 1,2,3,4,5,6,12 and 24 wks post-immunization. pre-immunization sera showed detectable levels of rubella-specific antibody in the igg class (17/44); iga class (32144) and in one or more of igg subclasses (22/44). post-immunization, i 1 subjects failed to develop igm class responses by the ha1 (sdg) technique while 43/44 developed igm antibody by elisa techniques. iga responses were detected at low levels in all vaccinees beginning at 3-4 wks and declining by 24 wks post-immunization. antibody in iggl and igg3 subclasses by 2-3 wks post-vaccine with sustained iggl levels but significant decline in igg3 levels noted between 6 and 24 wks post-immunization. no seroconversion was noted in the igg2 subclass although 9 individuals had detectable pre-immunization iggz rubella antibody present. igg4 levels were detected in all vaccinees post-vaccine with a delayed and progressive rise over the study period. subsequent correlation was then performed between rubella-specific antibody responses and the presence or absence of adverse joint reactions occurring in association with rubella vaccine administration. all individuals produced detectable c624 t lymphocyte responses to varicella zoster virus. anthony hayward, abbas vafai, roger giller & eileen villanueba. departments of pediatrics and microbiology, university of colorado school of medicine, denver co 8 0 2 6 2 61 university of iowa school of medicine, iowa city i0 the proliferative response of blood lymphocytes from varicella zoster virus (v2v)-immune donors to live vzv, extracted vzv antigens or purified glycoproteins is predominantly by cd4+, hla-d restricted t cells but little is known of the specificies of the responder cells. we restimulated t cells cloned by limiting dilution from vzv-stimulated cultures with purified vzv glycoproteins gpi, gpii and gpiii and found that t cell clones with specificity for each of these mediated both help for antibody responses and hla-dr restricted vzv-specific cytotoxicity. 5 polypeptides of 10 to 14 amino acids length corresponding to predicted amphipathic sequences in the primary structures of g p i, gp i1 and gp iv were synthesised. proliferative responses were observed to 3 of these peptips (one from each glycoprotein) with responder cell frequencies in the 1:lo blood t cells range. the gp i peptide additionally defined an epitope recognised by serum antibody. an immunomodulatory approach to treating hsv-1 corneal disease, hendricks rl, departments of ophthalmology, and microbiology/immunology, university of illinois school of medicine, chicago, il 60612 herpes simplex virus type i (hsv-1) corneal infections are a leading cause of blindness worldwide. we and others have demonstrated that the cellular immune response to hsv-1 contributes to the elimination of virus from the cornea, but in doing so causes the tissue destruction that is responsible for the blinding complications of the disease. we have demonstrated that specifically suppressing the cytotoxic t lymphocyte (ctl) response to hsv-1 renders mice resistant to corneal disease following topical corneal hsv-1 infection. in agreement with this observation was our recent finding that in vivo depletion of wt4' (t helper/inducer, and most dth effector cells) neither reduced susceptibility to corneal disease, nor increased susceptibility to disseminated disease. the corneal lesions in wt4 depleted mice contained numerous lyt-2 (t suppressor/cytotoxic) cells, and no l3t4 cells. the wt4 depleted mice exhibited normal hsv-specific ctl precursor frequencies. experiments designed to determine the effect of in vivo lyt-2 depletion on susceptibility to corneal disease are in progress. our goal is to identify cellular immune responses to hsv-1 that maximize protection, while minimizing immunopathology in the cornea, and identify hsv-1 epitopes that preferentially activate those responses. supported we found that affinity purified antibodies to bsa, klh and diptheria toxoid all contain a substantial amount of specific anti-idiotypic activity. against bsa react with mouse anti-bsa antibodies, which suggests that we are dealing with internal image antibodies. 9 mrl-lpr/lpr mice develop spontaneous autoimmunity. we found that these mice make anti-anti-(self h-2) antibodies prior to making appreciable amounts of pathological autoantibodies such as anti-dna, anti-rnp.sm, and rheumatoid factor. the anti-anti-self antibodies are detected using an inhibition of antibody mediated cytotoxicity assay, that also detects anti-anti-(self h-2) in ordinary allogeneic anti-sera. the antibodies are not rheumatoid factors, although the animals do make rheumatoid factors later in the development of the disease. anti-self activity is fully developed at 2 months, when the other autoantibodies are typically barely detectable. important role in the etiology of the disease. the anti-we conclude that anti-anti-self antibodies could play an c 627 feedback regulation of 11-1 synthesis in monccytes by t cell products: dual effect of 11-4. mikko hurme, tessa palkama and marja sihvola, department of bacteriology and immunology, university of helsinki, sf-00290, helsinki, finland. il-i production of human monocytehnacrophages is regulated by several cytokines some of which are themselves able to activate the il-1 production (e.g. tnf and il-2) while others (e.g. ifn-y) modulate the production activated by other signals. we have now examined the effect of 11-4 on the 11-1 synthesis. 11-4 alone did not induce any 11-1 bioactivity or il-la or 11-1 0 mrna expression in freshly isolated peripheral blood adherent cells. in contrast, il-4 effectively suppressed the lps induced 11-1 production. this suppression took place without any decrease in the steady-state levels of il-la and il-i 0 mrna, suggesting that this downregulative effects is posttranscriptional. monocytehnacrophages are known to rapidly loose their ability to produce il-i when cultivated in vitro. if ifn-yis present in the culture fluid, the cells remain capable of producing 11-1. as ifn-yand have been reported to have similar "priming" effects on macrophages (e.g. increasing the tumoricidal capacity and mhc class ii antigen expression) we cultivated monocytes for 24 h in the presence of either ifn-y or 11-4, and after washing the cells they were stimulated with lps. il-1 activity could be detected both in the ifnyand il-4 preincubated cultures (but not in the cultures preincubated with medium alone). these data suggest that il-4 can also display a similar upregulatory function in il-i production as ifn-y. gahreston, tx 77550 development of immunity to members of the spotted fever group of rickettsiae is a t-cell dependent response. we have used t-cell hybridomas and cloned t-cell lines from immune animals and convalescent humans to identify the rickettsial antigens that induce antigen-responsive t-cells. in these studies we found that the 155 kda antigen of rickettsia tickettsii. the causative agent of rocky mountain spotted fever, is one of the immunodominant tcell antigens. t-cells from immune animals and humans were responded in culture to a recombinant 155 kda antigen. both sources of t-cells were of the t-helper type (l3t4* and 04' respectively) and produced 1l-2 and interferon. it was found that soluble antigenic material of b. rlckettsii obtained by extraction with hypotonic buffer maximally stimulated the t-cell lines. this material was enriched far the high molecular weight polypeptides of 1s5 kda and 120 kda. also. ethirwwi ' will induce a long-lived immunity against infection with r. rickettsii. infected guinea pigs develop a minimally cross-reactive antibody response to b. nckettsii. in contrast. a strong cross-reactive t-cell proliferative response is produced. studies are in progress to determine the nature of the common protective antigen of r. humans infected with the parasitic nematode ascaris lumbricoides vary considerably in antibody responsiveness to a 14 kda component of the parasite. this molecule is secreted by the parasite, and is also abundant internally. this heterogeneous reactivity has been modelled in laboratory rodents, and the antibody response to it is h-2-and rt1-restricted in mice and rats, respectively. using inbred and congenic animals, only mice of h-2' and rats of rtiu were, so far, found to be responders, and this restriction only operated in the context of infection. the specificity of the ige response in these animals was assayed by passive cutaneous anaphylaxis, and in an ige-specific elisa assay. the data show that the above mhc restriction also applied to the specificity of the reaginic antibody response, although animals of all mhc haplotypes responded to other ascaris allergens. amino acid analysis of the 14 kda equates it to a previously identified "allergen a" of the parasite, and we now have its sequence available. these findings have implications for the genetic control of allergic responses in general, and, in particular, to the hypersensitivity responses which are such a feature of infections with parasitic nematodes. there are also implications for the generation of hypersensitivity responses by recombinant vaccines involving certain parasite antigens. the cns. immunohistochemial analysis of both frozen sections prepared from the brains of animals immunized in this manner and of highly enriched glial cell subpopulation cultures for viral gp70 expression indicated that oligodndrocytes and possibly a subset of astrocytes were the targets of this infection. further, microscopic analysis of frozen sections failed to reveal any overt signs of gross pathologic changes associated with the viral infection. we have been able to demonstrate the presence of virus specific antibody in the serum of these mice as well as virus specific cytolytic t cells in the peripheral lymphoid organs. ments are currently underway to determine whether the lack of pathology associated with wb91 infection in light of the previously shown virus specific immune responses in these mice is due to a failure of antigen presentation within the cns or some other form of immunoregulatory phenomena. the t lymphocyte proliferative response to pigeon cytochrome 5 in bio.a mice is restricted to the egk:e,k ia molecule and specific for the c-terminal determinant comprised of residues 93-104. blo.a(3r) and blo.a(sr) mice are nonresponders to pigeon cytochrome 2 nonetheless, the t cell repertoire of blo.a(3r) or (5r) contains some t cell clones capable of recognizing and proliferating to pigeon cytochrome c w h e n presented by bio.a antigen-presenting cells (aft). therefore, one would expect to stimulate such clones in allogeneic bone marrow chimeras of the type bio.a +blo.a(3r) or (5r) b1o.a apcs and a blo.a(3r) or (5r) t cell reperto4!6~.~espectively. ,en(isea9c22rzave were primed with pigeon cytochrome cytochrome 5, they showed a good antigen specific proliferative response in vitro. surprisingly, however, if pigeon cytochrome 5 was used for priming, no response was detected, even at priming doses as high as 400 nmol per mouse. 81-104 could only be achieved by treating the allochimeras with an anti-cdb monoclonal antibody in vivo during the priming step. clones specific for purified protein derivative (ppd) in the same chimera. thus the regulation which involves cd8 positive cells is antigen specific. transfer of pigeon cytochrome 81-104 primed lymph node cells from the chimera into naive bio.a mice prevented priming of the recipient for a t cell proliferative response to pigeon 81-104, but not priming to the moth synthetic fragment. chimeras of an antigen-specific suppression mechanism involving cd8 positive cells. faculty of medicine, kyoto university, kyoto 606, and department of oncology, nagasaki university school of medicine, nagasaki 852, japan. sera from b6 mice immunized with a syngeneic ctl specific for fbl-3 tumor of b6 origin blocked the cytotoxic activity of only the immunizing ctl clone. therefore, a monoclonal antibody (mab) n9-127 was produced by fusion of the b6 spleen cells immune to a syngeneic fbl-3-specific ctl clone (no. 8). the specificity of the mab n9-127 was confirmed by immunoprecipitation, blocking of cytolytic activity, stimulation of proliferation, and induction of tcr-mediated nonspecific cytolysis of the ctl clone no. 8. in some b6 mice, 3-13% of the anti-fbl-3 mltc cells were positive for this n9-127-defined idiotype, and formed a well demarcated population upon examination by flow cytomehy. even in mice in which no such population was observed some ctl clones established by limiting dilution culture were also positive for this idiotype (10 out of 89 clones from 3 mice). the cytotoxic activities of these ctl clones were blocked by n9-127, which in turn induced the nonspecific cytolysis in redirected assay. however, no positive cells were detected in non-cultured normal or fbg3-immune spleen and lymph node cells. this indicates the presence of cross-reactive (dominant) idiotype in the b6 anti-fbl-3 cytotoxic t cell responses and may provide a potent tool for analyzing the idiotype-mediated regulation of the anti-tumor immune responses. slade andsylvie gillard, max-planck-institut fur immunbiologie, d-7800freiburg, federal republic of germany t cells play a n essential role in t h e protective immune response to malaria and a r e associated with s o m e of t h e pathological consequences of t h e disease. however, t h e n a t u r e of their responses and t h e antigens t o which they respond a r e not well defined. w e have developed a limiting dilution assay system in which specific t cell responses to malaria antigens c a n be monitored a t t h e clonal level. i t is possible to determine t h e nature of t h e responding t cell by t h e growth f a c t o r s they s e c r e t e and by their ability to a c t as helper cells for t h e antibody response to malaria antigens. our d a t a suggest t h a t t h e t cell response changes during t h e primary infection and in hyperimmupe animals. o n e to t w o weeks a f t e r initiation of a blood s t a g e infection t h e major cd4+ t cell which proliferates in response t o parasite antigens s e c r e t e s il-2 and ifn-y but is not a n efficient helper cell for antibody responses. in c o n t r a s t l a t e r in infection and in immune animals t h e r e is an e f f e c t i v e helper cell response and many of t h e s e cells a r e distinct from those secreting ifn-y and il-2. we a r e currently investigating whether these cells retain these phenotypes when grown in long-term in vitro culture and whether defined antigens of t h e erythrocytic parasite elicit different t cell responses. we have localized linear neutralization epitopes on the coronaviruses ibv, mhv, fipv and tgev. the results can be summarized as follows: 1. linear epitopes of the spike proteins (1162-1452 residues) could be mapped to a resolution of a single residue by expression of gene fragments in the prokaryotic pex plasmids and/or pepscan peptide synthesis. 2. the length the epitopes varied from 4 to at least 20 amino acid residues. we present evidence that the larger epitopes, although conformation-independent according to operational criteria, are nevertheless discontinuous. 3 . in ibv, we localized several overlapping but different epitopes within an immunodominant region of 30 residues. this region is recognized by all polyclonal antisera tested. we propose that its immunodominancy is a consequence of its structure and function and does not depend on antigen presentation or idiotypic networks. an immune response against the mouse testis-specific antigen ldh-c4 reduces fertility by 70 percent in female baboons. an immune reaction to human ldh-c4 would be expected to be more effective in primates. since the human testis enzyme is not readily available in large quantities, recombinant dna technologies were uscd to create a source of human ldh-c4. antibodies to mouse ldh-c4 were used to screen a xgtll human testis cdna expression library. a full length human ldh-c clone was identified, sequenced, and the ldh-c cdna was engineered for expression in e.coli. the 5' and 3' untranslated sequences were removed by restriction enzyme digestion, and synthetic linkers were added adjacent to the start and stop codons of translation. the modified cdna was subcloned into the prokaryotic expression vector pkk223-3 and introduced into w3110 l a c iq cells. cells were grown to mid-log phase, and induced with iptg for positive regulation of the strong hybrid tac promoter. induced cells overexpressed the 35 kd subunit which spontaneously formed the enzymatically active 140 kd tetramer. human ldh-c4 was purified 196-fold from liter cultures of cells by two step affinity chromatography to a specific activity of 90 i.u./mg. the 20 n-terminal amino acids sequenced were identical to those predicted from the nucleic acid sequence. antibodies to synthetic peptide epitopes of human ldh-c4 cross-reacted with the enzyme produced in e.coli. two mg human ldh-c4 were expressed per liter of bacterial cells. the purified protein is now available for innunogenicity and fertility studies. it is now generally accepted that the principal effector mechanism in the host's defence against leishrnaniasis is gamma-interferon (ifn-y) which activates infected-macrophage to eliminate intracellular parasites. mice by prior sublethal whole body irradiation or treatment with anti-igm or anti-cd4 antibody. protection can also be induced by repeated intravenous or intraperitoneal immunisation with killed parasites or purified antigens. ly immunised mice produce little or no il-3 or il-4 but substantially elevated levels of ifn-y when stimulated with leishmania1 antigens in vitro. lymphoid cells from balb/c mice with progressive disease can inhibit the maf (macrophage activating factor) and leishmanicidal activities of the culture supernatant of lymphoid cells from mice recovered from l. major infection. maf appears to be ifn-y, whereas the maf inhibiting factors are il-3 and il-4. system can be reproduced with recombinant ifn-y, il-3 and il-4 and the maf inhibiting activity of the suppressive supernatant can be reversed by specific anti-il-3 and anti-il-4 antibodies. the disease by influencing the ability of macrophage to kill the intracellular parasite. the development of efficacious vaccines against malaria requires an understanding of the mechanisms involved in protective immunity. f'revious studies with plasmodium berghei demonstrated that sporozoite immunity is dependent upon antibody responses specific for the repeat region of the circumsporozoite (cs) protein and cell mediated mechanisms involving cd8+ t cells. in this study we analyzed the splenic t cell repertoire directed against epitopes on the cs protein of p. berghei and determined whether sporozoite-immune cd4+ and cd8+ t cells respond to shared or distinct epitopes. sporozoite-immune spleen cells, cd4+ and cd8+ enriched t cell populations of balb/c (h-m), c3h @i-%), and c57bv6 (h-2b) mouse strains were cultured in the presence of irradiated sporozoites or synthetic peptides representing 70% of the complete cs protein. surprisingly, none of the cultures proliferated to any of the peptides tested, although proliferative responses to sporozoites were observed in unfractionated spleens and cd4+ t cell populations. cd8+ t cells did not respond to any of the antigens tested, even in the presence of exogenously added 11-2. titration of cd8+ cells into proliferating cd4+ cell cultures did not suppress the anti-sporozoite response. the lack of anti-peptide reactivity contrasts with uniform responses to sporozoites and may be the result of the context in which cs antigens are presented to t cells. functional analysis of accessory splenic b cells and macrophages revealed that while the anti-sporozoite proliferative responses were not affected by the removal of macrophages, sporozoite-primed b cells were essential for the responses. these data suggest that the cs protein on sporozoites is not processed extensively by macrophages to yield many potential t cell epitopes, but instead is presented by immuncdominant b cells that resmct responses to a limited number of t cell clones. of the primary infection and is also required for optimum protection against reinfection. current studies have demonstrated that relatively few of the viral antigens tested to date ( 7 viral envelope glycoproteins or 2 nonsmctural nuclear proteins) are recognized by hsv-1 immune ctl populations generated in several different strains of mice (h2 haplotypes h2b, h2d. or h2k). this failure of hsv specific ciz to recognize the cloned gene products in in v i m assays was demonstrable at the clonal level and could not be attributed to a peculiarity of the recombinant vaccinia conshucts used because studies with adenovims vectors or tranfected l cell constucts yielded the same results. surprisingly, despite their inability to be recognized by hsv specific ciz in vim, when used to immunize mice several of the vaccinia virus constructs would induce memory ciz populations capable of lysing hsv-1 infected autologous cells. for example, hsv-1 glycoprotein c (gc) was recognized by h2b restricted but not h2k restricted hsv specific ctl. however, immunization of either haplotype of mice with a vaccinia gc recombinant induced ctl populations which upon in v i m restimulation with hsv-1 would lyse histocompatible cells infected with hsv-1. this demonstrates that despite the presence of suitable epitopes (intrinsic factors) the context of the immunogen (extrinsic factors) will also influence it's ability to induce ctl. the results of further studies into the nature of these extrinsic factors will be presented and discussed with relevance to future sub-unit vaccine design. w d ~upponrd by public ~~l t h service g~mu, ai 14981 md ai 24471 fran the ti-^ ~n,litu= md lnrcniour d ,~~~~~~. infection of mice with hsv-1 induces a brisk ciz response which is necessary for the subsequent resolution we have investigated the structural basis for antigen mimicry by anti-idiotypic internal image antibodies. two mouse monoclonal antibodies (mabs) that bear internal images of a well-defined protein epitope, i.e., the rabbit immunoglobulin (ig) a1 allotype, were produced and the variable region sequences were determined by rna primer extension sequencing. the results showed that the mab light chains did not contain any allotype-related residues; however, both heavy chain v regions contained a unique sequence homologous to the nominal antigen but in opposite orientation. this reversed sequence was expressed within cdr2 of both mabs. synthetic peptides corresponding to the putative antigenic regions of rabbit ig and the mab internal images, respectively, were tested for the ability to mimic the al-like determinant. although the homologous residues were presented in opposite orientations, both peptides completely inhibited at similar concentrations the binding of rabbit ig to anti-a1 antibody. a paired thr and clu was necessary for expression of the a1 epitope as revealed by conservative substitutions in the peptide sequence. computer-generated, energy-minimized models of rabbit ig and the mabs revealed that the critical a1 residue side chain placements could be almost superimposable in either context. thus, it appears that an antigenic epitope can be determined solely by md 20892. proliferation of murine type i cd4+ t cell clones quires simultaneous occupancy of the t cell antigen receptor and delivery of an accessory cell-duived costitnulamy signal in contrast, isolated t cell receptor occupancy induces the cell into a state of reduced proliferative responsiveness to antigen. based on the observation that pkc-activating phorbl esters can at times substitute for the p s e n c e of accessory cells in t cell proliferative response. to mitogens or anti-cd3 mnodonal antibodies, we investigated the requkment for accessory cells in the antigen-and con a-induced hydrolysis of p m and activation of pkc. the presence of normal accessory cells was found to be unnecessary for the development of pkc-dependent phosphorylations and the addition of normal accessory cells had no effect on the activity of pkc. cell il-2 synthesis and proliferation presents a paradox. we have studied the effects of ueatment with a calcium ionophore and p h h l ester on t cells and find that increased [ca2+]i and pkc activation are in fact insufficient biochemid second messengers in the induction of proliferation. while pliferation was induced at high t cell density in response. to these stimuli, incubation of t cells at decrrased cell density drmonsuated markedly reduced proliferation, and single t cells failed to divide. this suggested that cellular interactions were. q u i r e d in the response. additions of either il2 or normal accessory cells allowed p l i f d o n at low density, consistent with a requirement for an accessory cell-derived costimulatoq signal in the induction of i l 2 synthesis, even in the plifcrative response to ionomycin and pma. this result underscons the importance of an accessory cell-duived costimulatory signal, acting independently of t cell receptor-mediated increases in [caz+] i and pkc activation, in the induction of t cell proliferation. we describe experiments designed to determine the molecular requirements for recognition by fluorescein-specific ctlps and ctls derived both from n a i v e and from immunized mice. we the production of prostaglandin e, a major immunesuppressor secreted by the macrophages was inhibited by the addition of 0.1 m indomethacin to the cultures of monocytes harvested from patients suffering from pulmonary tuberculosis and those from equal number of normal controls. the il-i activity was estimated i n the supernatants of these cultures by their ability to proliferate mice thymocytes. it was found that the supernatants from cultures with indomethacin showed a greater il-i activity than the ones without it(44% p 0.001). this indicates the possibility of pge offering a negative feedback control over il-i production. the defective cell mediated immunity i n patients with pulmonary tuberculosis may be explained through the inhibition on il-i production by pge whose enhanced production is reported i n our earlier studies. the results and our hypothesis on the autoregulation of il-i production w i l l be presented and discussed. in variant viruses which differ from the parental virus (gv) at specific epitopes recognized by monoclonal antibodies directed against the env gene product, gp70. biological clones isolated from gv express the gv phenotype suggesting that the loss of specific epitopes is the result of selective de novo processes in the immunocompetent host. additionally, inoculation of adult mice with a biological clone expressing the gv phenotype also results in similar variant viruses. however, inoculation of gv into neonatal or nonlethally irradiated mice results in a population of viruses expressing only the gv phenotype suggesting that the emergence of antigenic variants may be influenced by neutralizing antibodies and/or cellular host res sds-page analysis of immunoprecipitates of sg~s~~,elled lysates of fibroblasts infected with clones expressing gv or variant pehnotypes shows a size difference of the gp70 precursor. additionally, the recognition of a neutralizing epitope (e-55) associated with gp70 by mab55 is dependent on the appropriate native conformation of the epitope which appears to require glycosylation for expression. experiments are in progress to further examine the immunogenetic basis for the generation of these variants and to determine the molecular changes in the virus genome responsible for changes in epitope expression. investigated the capacity of murine splenict cells depleted of acceso cells ( ac ) t o proliferate in response t o stimulation by con a, @ cd3 ab and activated t cells. the zepletion procedures consisted of carbonyl iron treatment, 2x "panning " on anti-ig coated flasks, 2x anti-la cytotoxic treatments and percoll gradient purification of small resting t la-cells. the appropiate concentration of con a (10 ng/ml ) and plastic-bound (pb) @cd3 lg or its f (ab)' 2 fra ments induce proliferation, 112 r expression and 112 (but not 114 secretion in t la-cellscultured for 48% at 5x105 cells/well . responsiveness of tlacells t o con a and dcd3 in low density cultures (5x104 cells/well) is restored by the addition of irradiated th2 cloned cells but not thl ,splenic cellsor r l l l + rll2 + r114. likewise, responsiveness t o non activating doses of con a (lnglml) or soluble @cd3 is restored by the addition of irradiatedth2 costimulatory cells . these experiments demonstrate that the ability of t cells t o proliferate in the absence of ac is critically dependent on t-t interactions. t cell subsets prepared by either negative (l3t4-and ly2'cells) or positive selection proliferate in response t o pb @cd3 lg . although the proliferative responses of both l3t4-and ly2-cells are maximal at 48h, the l3t4-cells require l o x more pb @cd3 lg for maximal stimulation and their res onses decline much faster than those of ly2cells. in addition, l3t4-cells are not stimulated by pb &cd3 f(ab)'2 fra ments and their responses t o @cd3 i are inhibitable by anti fcr as well as anti-lfa abs . re onsesof%oth l3t4-and ly2-cells are !nhibita%le by @i12 and @i12 r abs but not by @l3t4, @ly2 or b l l 4 . these experimentsdocument interesting differences in the triggering requirements of l3t4-and ly2 'cells. supported by nih grants po1 ca 19266, t 326m-07183 and gm 07281. the 19k glycoprotein encoded in the e3 region of ad2 and ad5 (gpl9k) binds to class i mhc antigens in the endoplasmic reticulum and prevents their translocation to the cell surface. this has been proposed as a mechanism by which virus infected cells can avoid recognition by the host cytotoxic t lymphocyte (ctl) response. we have shown that gpl9k can inhibit target cell lysis by adenovirus specific ctl, but the effectiveness of this inhibition varies greatly between different mouse strains. this is due in part to differences in the affinity of gpl9k for different mhc class i molecules, but this cannot account for a l l the variation observed. i t has been shown that cd4+8+ immature thymocytes fail to secrete il-2 or express il-2 receptors in response to activation signals. furthermore. they cannot induce il-2 gene transcription. several tumor lines have now been characterized which have a cd4+8+ phenotype and fail to secrete i l -2 or express il-2 receptors in response to stimulation with ionomycin plus pma. these cells also do not express il-2 mrna after stimulation, as determined by northern blotting and rnase protection. to determine the molecular mechanism for this lack of transcriptional activity, nuclear extracts were analysed for the presence of the d n a binding factor nfat-i. this nuclear factor i s present only in ac we have undertaken an mhc analysis using the polymerase chain-reaction (pcr) and dot blot analysis of the amplified lyme arthritis patients dna with allele specific oligonucleotide (aso) probes. genomic dna for the first domain of the dq beta chain and of the dr/pi from 20 patients with lyme arthritis has been amplified and we are analyzing the distribution of dridrr and w a l l e l e s in this population to test the hypothesis that the mhc class i1 genes might be involved in presentation of selected spirochete epitopes whose recognition by t lymphocytes leads to lyme arthritis. most inbred strains of mice do not respond to porc insulin (pins). experiments were conducted to elucidate the mechanism of the non-responsiveness in h-zk mice: 1) purification of cd-4' t c e l l s from pins-immune b1o.mbr mice revealed pins-specific t helper (th) cells, 2) these pins specific th cells could be activated by i-ak and i-ek expressing l929-fibroblasts. therefore, both i-ak and i-ek molecules can present pins in an immunogenic manner and activate pins-specific th cells. by means of different cell-fractionation procedures, it was found that antigen-specific t suppressor (ts) cells regulated the pins immune response. these ts cells were of the fcr-, cd-4-, cd-8'. thy-1' phenotype, and they were present in normal mice. we believe that these experiments indicate that antigen-specific ts cells exist and are important regulators of immune and autoimmune responses. the possibility of functional inactivation of cd4+ clones by ts-cells was investigated. m leprae-responsive cd4+ clones were preincubated with ts cd8+ clones, apc and antigen for 1 ; hours. after which the cd8+ cells were removed from culture. the cd4+ clones were then restimulated with e. leprae and apc. cd4+ clones incubated with cd8+ cells and antigen were unresponsive to restimulation by antigen, although they were not killed and could respond well to il-2. addition of il-2 in the preor post-incubation culture neither prevented the induction of unresponsiveness nor reversed it. earlier models of tolerance have suggested that receptor occupancy in the absence of second signals induces tolerance in b-and t-cells. we would suggest that in the presence of ts-cells. a second signal may be negated leading to th-cell unresponsiveness. university of texas southwestern medical school, dallas, tx 75235 graft versus host disease (gvhd) poses a serious threat to the survival of patients with bone marrow transplants. the state of immunosuppression established in gvhd results in a variety of immunological abnormalities at the humoral and/or cellular level. we have developed a murine model of chronic gvhd across a minor histocompatibility (mh) barrier. in this model, immunosuppression develops. spleen cells from mice undergoing this type of gvhd are unable to respond to the polyclonal activators lipopolysaccharide and concanavalin a . however, the response against the b cell leukemia bcll remains intact. the protective immune response against bcll is directed towards the mh antigen h-40 and is mediated by cytotoxic t lymphocytes. thus, the specific t cell response against a mh antigen can occur in the presence of chronic gvhd despite the absence of a polyclonal b and t cell response lymphocytic choriomeningitis virus (lcmv), a member of the arenavirus family, has a biseqmented rna genome which encodes at least three polypeptides. the smaller rna segment encodes two virus structural proteins, the slycoprotein (gp) and the nucleoprotein (np). upon infection of mice with lcmv a cytotoxic t cell immune response directed against these proteins is measurable in vitro and in vivo. it can be demonstrated that depending on the haplotype of the mice, one or the other protein may play a major part in the immune response. in order to define the immunogenic epitope(s) of the nucleoprotein which are recognized specifically by the t cell receptors of cytotoxic t cells, stepwise 3 ' truncated qene fraaments encodina the nucleoprotein were cloned and expressed in vaccinia virus. with these recombinant vaccinia viruses, protection experiments in mice aqainst lcmv infection were performed in parallel with in vitro studies, namely specific recoonition of target cells expressing truncated fragments of the nucleoprotein by lcmv primed spleen cells. cdna clones encoding the mouse and human t cell il-1 receptors have been isolated and expressed in mammalian cells. the recombinant receptor binds il-1 indistinguishably from the natural il-1 receptor, and is functional in signal transduction. deletion of the cytoplasmic portion of the receptor abolishes its signal transduction abilities. sequence and secondary structure analysis suggest that the cytoplasmic segment of the il-1 receptor binds a nucleotide. experiments designed to test this hypothesis and to examine the mode of signal transduction will be presented. also to be discussed are the mechanism of triggering of the receptor by il-i. and the nature of il-1 receptors expressed in other cell types such as b cells. it became evident that these subsets reflect different stages of helper t cell maturation before and after activation. therefore, these t cell subsets have been designated as naive t cells (cd45r/2h4+, cdw29[4b4]-) and memory t cells (cd45r/2h4-, cdw29[4b4]+). we analysed the expression of these antigens in dermal lymphohistiocytic infiltrates from different benign skin diseases and cutaneous t cell lymphomas (chronic contact dermatitis (n=14), parapsoriasis en plaques (n=5), lymphomatoid papulosis (n=4), mycosis fungoides (n=15), sezary's syndrome (n-2), pleomorphic t cell lymphoma (n=6) and high grade t cell lymphomas (~4)). in almost all cutaneous t cell infiltrates memory t cells were preferentially found whereas in the peripheral blood both subsets are equally distributed. this implicates, that t cells infiltrating the skin already have had contact with their respective antigen. where the switch from naive to memory t cells takes place can not be answerded by our findings, as we have investigated rather longstanding skin diseases. however, these memory t cells, which can be activated more easily, make diseased skin more e f f e c t i v e in the nmdlification of an immune resnonse. organs and after several days of stimulation with antigen or mitogen and lymphokines. we find that fresh th synthesize and secerte -iu,ifng,il3 and gmcsf but very little il4 or il.5 within 24-48 hours. this pattern resembles the pattern of lymphokines secreted by thl cell lines. the th responsible for this secretion are cd4 positive t cells which are long-lived since they disappear very slowly following adult thymectomy. they are also sensitive to the in vivo administration of ats(antithyrn0cyte serum) and they express high levels of pgp-1. the kinetics of lymphokine secretion and the phenotype of the cells suppon the hypothesis that lymphokine secretion from fresh lymphoid cells comes from a population of memory cells. in contrast we fmd that we can also stimulate a separate, ats-resistant population to become lymphokine-secreting cells after four days of in v i m priming.these primed cultures rapidly synthesize an secrete large amounts of i u and il5 in addition to ifng , il3 and gmcsf( a phenotype which could be combination of both thl and th2 helpers), when they are restimulated with ag or mitogen. the cells whic are responsible are cd4 positive and have a shorter liespan since they decline considerably after adult thymectomy. we suggest that the lymphokine secreting cells detected after priming come from a population(s) of helper t cell precufiofi which have differentiated to become effectors. this generation 0: effectors requires lymphokines, especially e-4 andlor ilz and apcs. thus the development of helper th appears to follow a similar pathway as that of cells of the b cell lineage developing into ab-secreting cells and cd8 positive t cells which develop into cytotoxic effectors from precursors. we have studied the secretion of lymphokines by helper t cells freshly obtained from lymphoid interleukin-2 production by t cells has been shown to be required for both humoral as well as cell-mediated imune responses. thus, il-2 production was measured in syphilitic rabbits as a function of their imune response. maximal il-2 production induced by con a at 10-14 days post-infection was only l/2 that observed for uninfected rabbits and this correlated well with a decrease in t cell proliferation ( < 35% that of normal rabbits) upon stimulation with con a . this decrease in il-2 production in infected rabbits was restored upon removal of most of the adherent cells. furthermore, the il-2 production by 10-14 day infected spleens was restored above normal levels upon addition of indomethacin. this decrease in il-2 levels was not due to an increase in the ability of infected spleen cells to adsorb il-2. finally, studies assessing il-2 production at various times postinfection indicated that at 4 days post-infection il-2 levels were higher than normal, however as early as 10-14 days after infection il-2 levels decreased below normal levels and continued to be depressed as late as 30 days post-infection. these results may explain why all organisms are not eliminated during primary infection w i t h ' . pallidum and why secondary and tertiary phases of the disease may develop. has been shown that especially the antigenic presentation of the fusion protein is important for eliciting a functional immune response. to study the immunolo ic properties of the f protein, we expressed the f gene in e.coli as a $galactosidase-f-fusion protein after insertion in a pex vector. we constructed deletion mutants with fragments generated with restriction enzymes and with the polymerase chain reaction method. using a panel of monoclonal antibodies a rough epitope mapping has been performed. two arears were found on the protein, with one area two monoclonal antibodies react and with an other area four monoclonal antibodies react. both area's were found in f1, the c-terminal part of the protein. the pepscan method was used to fine map the epitopes of the monoclonal antibodies reacting with the second area on the primary sequence. in at least one viral system, cd8+ effector cells can be induced in animals lacking cd4+ t cells. since cd8+ effector cells are important in immunity to malaria sporozoites, we wished to know if they,too, could be generated without help from cd4+ cells. we depleted balb/c mice of their cd4+ t cells by injection of an anti-cd4 monoclonal antibody, and then tried to immunize them with irradiated plasmodium yoelii sporozoites. when challenged with infectious sporozoites, these mice were not protected against malaria infection. although they did not make antibodies to sporozoites, passive transfer of hyperimmune serum into these animals still did not protect them against a sporozoite infections. cd8+ t cells from these animals functioned normally in in vitro assays against tnp labelled targets. it appears that, unlike viral systems, the generation of cd8+ effectors in malaria requires cd4+ helper cells. thus both cd4 and cd8 epitopes should be included in any synthetic vaccine against malaria sporozoites. univ. pennsylvania, philadelphia, pa 19104 migrate from fetal liver or bone marrow. rearrange t cell receptor (tcr) genes, express tcr. undergo thymic selection and finally emerge as mature single positive t lymphocytes. most studies of thymic t cell development have been performed by using polyclonal populations of t lymphocytes, which have made the interpretation of the results complicated. cells (0 clone) from nude mice by culturing nylon wool non-adherent cd4-cd8-spleen and lymph node cells in the presence of wehi3 supernatant and con a supernatant. clone was thy-1-cd3-cd4-cd8-il2r(il2 receptor)-and they have been maintained more than 16 months without changing phenotype. when the c9 clone was stimulated with il4. ili/il2. illnl6. gm-csf, the cells were induced lo express thy-i, tcr and il2r proteins. however, culture of the cells with gm-csffll2 did not induce the expression of these molecules. southern blotting of the dna isolakd from gm-csffll2 culture suggested that they have undergone partial db 1 -jp 1 rearrangement. the cultured cells were then recloned twice by limiting dilution. the cloned cells were again shown to induce expression of cd3 complex by the stimulation of il4 enriched medium. therefore. we have established a system in which to induce differentiation of cloned pre t cell line into tcr+ cells in vitro. the human lymphocyte differentiation antigen cd8 is encoded by a single gene which gives rise to a 32 kda glycoprotein expressed on the cell surface as a dimer, and in higher molecular weight forms. we demonstrate that the mrna is alternatively spliced such that an exon encoding a transmembrane domain is deleted. that is secreted and exists primarily as a monomer. messenger rna corresponding to both forms is present in peripheral blood lymphocytes,(pbl), con a activated pbl and three cd8+ t cell lines with the membrane form being the major species. ratio of mrna for membrane cd8 (mcd8) and secreted cd8 (scd8) exist. in addition, the splicing pattern we observe differs from the pattern found for the mouse cd8 gene. this mrna is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted giving rise to a cell surface molecule which differs in its cytoplasmic tail from the protein encoded by the longer mrna. neither protein i s secreted. this is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. this represents one mechanism of generating diversity during speciation. cd4' t cells in the rat can be divided into two non-overlapping subsets by theii reactivity with the monoclonal antibody mrc ox-22 which binds some of the high molecular weight forms of the cd45 antigen. recent work, to be described has shown that the two subsets represent different stages of t cell maturation, with distinct t cell functions. the lymphokine repertoire of the memory t cell pool will be discussed with reference to the antigenic environment fmm which the cells are obtained. pancreatic islet allografts, gill, ronald g. and lafterty, kevin j., barbara davis center for childhood diabetes i univ. colo. health sciences center, 4200 e. 9th ave, box b-140, denver, co. 80262 we studied the cellular interactions requkd for the rejeaion of cultured mhc class i-dispiuate islet allografts. this model was suitable for studying t-t collaboration in that islet allograft immunity is cd4 dependent but rejection of the cultured islet graft is mediated by the cd8 cell. recipient c57by6 (b6) mice were grafted with mhc class idisparate b6.c-h-2bml (bml) islets beneath the mal capsule. islet grafts wen preaated for 7 days in 95% oxygen culture to reduce immunogenicity. thirty days after grafting, recipient mice wen immunized with 1oe6 live spleen cells from the strains indicated below. rejection of the established graft was not trig-by challenge with donortype bml spleen cells, indicating that the mhc class i stimulus was insufficient to initiate all@ immunity. further, immunization with a mixture of 1 m bml and 1oe6 mhc class ii-disparate b6.c-h-2bm12 (bml2) spleen cells failed to trigger host immunity. however, challenge with loe6 (bml x bml2)fl spleen cells t r i g g d acute rejection of the established bml islet grafts. the requirement for l i i presmtatidrecognition of class i and class 11 all* antigens to trigger allograft immunity indicates that the antigen-presenting (apc) plays an essential role for t-t collaboration in vivo. uature hla-dr complexes are purported to spontaneously internalize from and recycle to the plasma membrane of b but not t lymphocytes. using a neuraminidase protection assay, we have radiolabeled surface class i1 antigens on intact cells and cultured these cells under conditions which permit or prevent endocytosis; subsequently, surface glycoproteins on viable cells were desialylated and class i1 molecules were analyzed by iamunoprecipitation and two-dimensional gel electrophoresis. a panel of buman b lymphoblastoid cell lines and activated tonsillar b cell blasts failed to exhibit any internalization of class i1 complexes; control transferrin receptor molecules were endocytosed as ascertained by insensitivity to neuraminidase digestion. class ii+ pba blasts and sezary cells of the t lineage were also deficient in detectable hla-dr internalization. results did not vary regardless of the time allowed for efficient endocytosis (1251 ok 3~saethionine). or the addition of anti-class i1 monoclonal antibody during the chase period for endocytosis. therefore. within the limits of sensitivity of this assay, class 11 complexes do not appear to be internalized, either spontaneously or uben crosslinked by antibody. recycling represent a dynamic pathvay for regulating surface expression of class i1 antigens or a means of associating with and presenting foreign antigenic peptides. supported in part by usphs grants #5 t32 cao9058-14 and #5 eo1 a123081-03. in previous studies of antigen-specific t cell responses in two distinct models of autoimmune tubulointerstitial nephritis (tin), viiia villosa lectin binding (vv+) t cells have been shown to be necessary for effector t cell expression and mediation of tin. in anti-tubular basement membrane disease, antigen-specifi vv+ t cells direct the phenotypic selection of cd8+ nephritogenic t cells in susceptible mouse strains (j. immunol. 141, nov. 1,1988) . this function is mediated by an antigen-binding, i-js+ soluble protein factor. current studies investigate the role of the t cell glycoprotein which binds vv lectin in mediating w+ t cell function. using the previously described effector t cell induction assay, we found that n-acetyl-d-galactosamine (galnac) (at 25 mm but not 2.5 mm) inhibits vv+ t cell function and cd8+ effector t cell selection. when cd8+ effector t cell differentiation occurs in the presence of soluble factors derived from antigen-primed vv+ t cells, galnac is not inhibitory. these studies suggested that soluble gal nac may competitively bind to a soluble protein which stimulates vv+ t cells, in part by binding to the w lectin receptor, to synthesize andlor secrete their biologically active soluble factor. as an additional test of this hypothesis, we prepared detergent solubilized membranes from vv+ t cells and purified vv lectin binding proteins by affinity chromatography. like galnac. these membrane derived lectin binding proteins also inhibit vv+ t cell function and cd8+ effector t cell selection. inhibition by soluble 'lectin receptors' is dose dependent and is demonstrable with lectin binding glycoproteins derived from 15-20 x lo6 cells, in an assay utilizing 10 x106 vv+ cells. we are now further characterizing the lectin receptor and its endogenous ligand. elementary bodies and outer membranes of chlamydia trachomatis produce a high-titered igg response in rabbits and mice as measured by elisa and microscopic immunofluorescence assays. western blot analysis of total elementary body protein identifies a 40kd major outer membrane protein (momp) as the predominant antigen. to identify the cbmical structure of the epitope, purified momp was subjected to chemical and enzymatic fragmentation and the resulting peptides were purified by hplc and assayed for immunoreactivity. an immunoreactive 6kd cyanogen bromide peptide was amino-terminal sequenced and a series of overlapping synthetic peptides were synthesized and assayed for immunoreactivity. sequential single amino acid deletions at both the nhz and cooh termini allowed us to identify the precise epitope as a 12 amino acid peptide spanning residues 291-302 of momp. two amino acid substitutions at positions 293 (phe-gly) and 300 (pro-gly) completely eliminated antibody binding. the 12-amino acid synthetic peptide is a potent immunogen producing high-titered antibody responses that are specific for the momp molecule. analysis of an independently derived mutant harboring the same defect has shorn that this trans-acting gene is not required for transport of class i1 molecules. class i heavy chain is synthesized in this cell line and associates with b2m. transport of the class i appears to be blocked in the er or cis4olgi as the majority of the class i glycoproteins are not processed to the endo b resistant form. the ability of the cell to significantly increase expression of surface class i when the incubation temperature is lowered from 37oc to 22oc suggests that this gene may function to stabilize a particular conformation of the protein. consistent with this is the increased sensitivity of class i molecules in the mutant as compared to the parent to degradation when cell lysates are incubated at elevated temperatures. the inability to immunoprecipitate class i antigens in the mutant is possibly due to the action of endogenous proteases present in these lysates. two complementing approaches are being employed to isolate this gene and further analyze its role in class i biosynthesis. the first involves inactivation of the trans-acting gene by insertion of a retroviral vector and subsequent pcr amplification of regions flanking the vector. in another approach a cdna library will be introduced into the mutant cell line and the cdna will be reisolated from cells reexpressing surface class i. houston. tx. we have induced a panel of highly immunogenic (imm+) vanants of the murine fibrosarcoma mca-f using i-methyl-3niml-niuasoguanidine (mnng). 5-aza-2'-deoxycytidine (5-azacdr). and uv radiation. these tumors grew m immunosuppressed mice. but were complelely rejecled by normal syngeneic hosts. mice thac had rejected large numbers of imm+ also developed a smng, tumor. specific immunity to the parental mor. lmmunizalion with low numbers of lmm+ engendered only variant-specific immunity. the frequency of imm+ variant g e n e d o n was similar for the three induction different protocols (64% to 92%), suggesling lhat generauon of imm+ was more closely relalcd to the cell line used than to the inducing agent however, the swngth of the imm+ phenaypc was related lo ihe agent used, since mnng induced clones had the m g e s t immunogeniciues and uv-b ihe weakest the smng neoantigens expressed by mnng induced imm+ were varunt-sppifc. while uv and s -d d r induced clones displayed significant cross-reactivilies not attributable to the parental t u n a antigen. increased or inappropriate expression of class-l mhc antigens did not correlate with the imm+ phenotype. we investigated the phenotypes of the spleen cells medrating tumor rejccuon using the local adopllve lransfer a s a y (lata). variant-specific immunity w mnng, 5-azacdr and uv induced imm+ were all m d a t c d by thy1.2+. l3t4+. lyc2.1-t cells. afrw immunization with high numbers of imm+ w engender both anti-lmm+ and anu-parental immunity. both cd4+ and cdw effectors rejecled the imm+ in lata, while only ihe c w + t cells could wnsfer resistance w the parent immunity 10 the parenlal tumor anugen engendered by the imm+ suggeslcd associative recognition of the parental and neoantigem wgelher on ihe cell surface. this hypochfsis was supported by failure of lmm+ u) pmlect againu an antigenically disunct tumor (mca-d) admixed with it, either at the lime of immunization or at challenge. fusion of ihe h m * vanant with mca-d yielded a unique, hybrid parental umor antigen that was associatively recognized with rhc original imm+ neoanugen, demomirating the importance of antigen cocxpression. grant rr-5511-23. w e have recently demonstrated and reported that substitution of anionic side chain carboxylic groups with aminoethylamide groups on protein antigens exhibits a pattern of enhanced immunogenicity both in vivo and in vitro. this enhanced immunogenicity was also observed in low responder strains of mice and we investigated the mechanism by which it is achieved. we examined antigen processing and presentation of native (nbsa) and modified bsa (mbsa) to t helper c e h isolated from c57/bl low responder mice. a greatly reduced amount of mbsa than nbsa was required to activate both nbsa and mbsa primed th. proliferation of nbsa and mbsa primed t cells increased in proportion to the amount of time of exposure of the antigen presenting cells (apc) to nbsa, peaking at 8h. conversely, apc required less than 30 min exposure to mbsa to achieve optimal activation, indicating rapid uptake of mbsa. paraformaldehyde fixed apc recognized mbsa without a lag phase processing, indicating that this event also occurred quite rapidly. apc processed nbsa w a s presented to primed t cells more effectively than the soluble antigen m shown by the increased rate of t cell proliferation. in contrast, mbsa was equally well presented to th cells by apc m in soluble unprocessed form. our data demonstrate that the reduced response in low responders is greatly enhanced by a modified antigen which is rapidly taken up and processed by apc. b cells which bear surface innunoglobulin (sig) receptors specific for a particular antigen are abile t o present fragments of that antigen very efficiently t o t cells. this i s due. in part, t o the high affinity of the receptor, which facilitates antigen binding at low concentrations. using tnp-abc and specific antigen, we have demonstrated that the tnp-abc process antigen very effectively. w e have compared specific antigen with i t s polyclonal analog, anti-ig, and demonstrated differences in the kinetics of degradation of anti-ig and tnp-antigens by tnp-aex. both antigen and anti-ig bound by tnp-abc are degraded into small fragments which are released into the supernatant. however, the following differences have been found: 1) the rate of release of small fragments of tnp-antigen parallels the rate at which these cells become able to directly conjugate with t cells (a lneasure of antigen presentation), reaching a plateau between 4 and 6 hours. in contrast, the degradation of anti-ig and release of fragnents continues for 12 hours. 2) analysis of initial kinetics demonstrated that release of fragments of tnp-antigen begins 15 minutes after binding; there i s no significant release of anti-ig fragrnents u n t i l about 30 minutes. 3) in contrast to anti-ig where there i s significant accumulation of degradation intermediates within the cells, there i s very l i t t l e intracellular accumulation of intennediate-size fragments of tnp-antigen. thus, we propose that the processing of antigen bound via specific sig may involve a specific intracellular pathway and that intracellular routing may be determined either by the degree of cross-linking of sig induced by antigen vs anti-ig or the mode of interaction of the various ligands with sig. alt*, departments of biochemistry (*) and medicine (+), college of physicians and surgeons of columbia univerity, new york, new york 10032. we have recently analysed the structure of the 7 / 6 t cell receptor (tcr) expressed by the normal human thymocyte clone cii. cii expresses a c ' 2 constant region that is a polymorphic form lacking a copy of an izternal exon; the sequence of this constant region accounts for the size of the 7 chain and noncovalent linkage of 7 and 6 chains in the cii tcr. in order to elucidate its role, this 7/6 tcr will be reconstituted in immortalized t-cell lines. in addition, the productively rearranged human 7 / 6 receptor will be transgenically introduced into mice in order to assess the effect of the complete receptor on the development of t cells. the humoral immune response to human immunodeficiency virus has been shown to contain antibodies which act to mediate the uptake of virus through fc receptor mediated mechanisms. it is therefore possible that vaccination with the entire envelope polypeptide may present immunologic determinants that enhance infection. one means by which to generate an immune response to hiv that shall possess neutralizing activity in the absence of infection enhancing activity is to generate anti-idiotypic abs that bear the internal image of neutralizing human antibodies directed against hiv. we affinity purified human antibodies from hiv+ patients on a viral lysate column. we have produced 15 monoclonal anti-idiotypic antibodies directed against these abi's. two of these monoclonals were shown to he ag inhibitable by their ability to inhibit the binding of p o l y e l o n a l human antisera to hiv viral lysate on ortho hiv ab t,est, wells. one monoclonal, 0 b 0 , when coupled t.o klh and used to immunize mice, produced an abs that. bound to viral lysate in an elisa assay. an affinity column containing rbs was used to purify an abi that was shown to bind to p24 and p17 hy western blot analysis. these data suggest that 8br may be a potential vaccine candidate. we have recently described a transgenic mouse model which co-expresses the tcr u and fl chains from the 2c cell line (recognized by the 1b2 anti-clonotype). t celh bearing the transgenic clonotype are positively selected by elements of the h-zb mhc for expression on cd8' cells. thus in the periphery of h-zb animals 40-80% of the t cells are 1bz1/cd8*. the same peripheral expression is observed when the transgenes are expressed in f1 animals bearing a "neutral" mhc haplotype (eg. h-zb'*). however, when the transgene hi expressed in f1 animals which also express the h-2ld gene product, negative selection occurs by clonal deletion. however, this deletion is functional rather than structural as the 1b2 clonotype is present on 10-40% of peripheral t cells. these cells are unusual in that they express neither of the characteristic peripheral molecules cd4 or cd8. the absence of cd8 expression on the 1b2* cells appears to allow these potentially self-reactive clones to exist without evidence of autoimmunity. the original clone as well as 1b2+/cd8+ cells from h-2b animals are strongly inhibited by anti-cd8 reagents. in an effort to understand the process of negative selection and self-tolerance we have examined the capacity of these cells to be activated directly by the anti-clonotype rather than antigen (h-2ld). the results demonstrate that the clonotype is fully functional on these double negative cells, indicating a normal maturation in the thymus. further examination of their surface phenotype also supports the conclusion that these are fully mature cells which are phenotypically distinct from double negative cells which exist in the thymus of h-2b animals. of imunohematology. azl, leiden, the netherlands;2praxis biologics, rochester, new york, usa and 'university of southampton, uk. immunity to disease caused by neisseria menineitidis is associated with the presence of bactericidal and opsonic antibodies to the capsular polysaccharide (cps), lipopolysaccharide and to outer membrane proteins (omps). the cps of group a and c meningococci are proven efficacious vaccines, although the immunogenicity in infants is poor and the immunity is of short duration. the combination with t-helper epitopes will certainly improve the immunogenic properties of these t-independent (ti,) antigens. the group b cps is poorly immunogenic in humans probably because of tolerance due to structural similarity to host glycopeptides and/or glycolipids. we have focused our research onto the class 1 omps which show limited heterogeneity amongst meningococci. murine monoclonal antibodies to these proteins are highly bactericidal in vitro and will be used to map b-cell epitopes. t-epitopes have been identified by theoretical prediction of immunodominant sites by analysis of the amino acid sequence of the omp followed by their solid phase synthesis and subsequent testing for polyclonal activation of t-lymphocytes obtained from hl4-typed volunteers immunized with the omp. in addition human t-cell clones are generated with omps and maintained with antigen, ebv-transformed b-cells, fresh feeders and ril2. the clones are tested for antigen specificity, io vitro helper function, mhc restriction element, expression of surface markers and recognition of common meningococcal t-cell epitopes. c 642 demonstration o f p-azobenzene-arsonate-l-tyrosine (aba-tyr) speclfic t cells in low responder h-26 mice by il-1 supported t cell proliferatlon previous studies have shown that h-zb mice immunized with aba-tyr fail to produce aba specific delayed-type hypersensitivity and show little or no t cell proliferation in vitro to aba-tyr. these observations suggest that h-zb mice are deficient in th1 cells that respond to aba-tyr. by contrast, immunization of h-2b mice with tnp conjugates of aba-tyr revealed good cognate help, suggesting that these mice do possess aba-tyr specific th2 cells and that such cells are not revealed in conventional lymphoproliferative assays. because such assays are widely used to evaluate ir gene control and to map t cell epitopes, the databases generated from such studies may seriously under represent the total number of responder phenotypes and t cell epitopes. because of this concern, we established culture conditions that wlii support aba-tyr specific t cell proliferation in h-2b mice. in these studies, c57bu6.l mice were immunized s.c. with aba-tyr and 7 to 14 days later the draining lymph nodes were cultured with varying doses of aba-tyr or with varying doses of aba-tyr and varying doses of recombinant il-1 alpha (rll-la), a known costimulator of th2 cells. culture with aba-tyr alone produced no proliferation. by contrast, culture with aba-tyr and rll-la revealed t cell proliferation that titrated with the dose of aba-tyr and the dose of rll-la specific for conalbumin presented on ryngeneic antigen presenting cells and dependent on il-1 for its proliferation, was used a s an indicator cell for the ability of neonatal murine spleen cells to present antigen and produce il-1 and il-2.the antigen presenting capacity of neonatal spleen cells is low. during antigen presentation there is an augmentation of il-1 and il-2 production by the antigen presenting spleen cell population. however, neonatal spleen cells do not respond as well a s adult cells. the low levels o f il-1 can not be attributed t o a low potential for producing il-1 since neonatal cells produce high levels of il-1 after induction by a crude il-1 inducer factor (il-1-if).the this impairment leads t o a decreased stimulus of the 1-helper cell to produce inducer factors which leads t o low levels of il-1 and il-2 production by the neonatal cells during antigen presentation. no suppressor mechanisms responsible for the l o w interleukin production were detected. human or murine class i genomic dna was transfected into a b-lymphohlastoid x t-lymphoblastoid hybrid cell line. this fusion hybrid has lost both t cell derived copies of chromosome six and contains deletions spanning the class i1 region on both copies of chromosome six derived from the b-cell parent. previous data have described a transacting factor within this region that is responsible for class i antigen expression. hla-bw58 and b7 glycoproteins, although synthesized, were not transported to the plasma membrane in the hybrid. were surface expressed. these data suggest a fundamental difference between human and mouse histocompatibility antigens in their requirements for intracellular transport. the role of glycans in this transport dicotomy is currently under investigation. in addition, hybrid human-murine genes are being used to identify regions of the class i molecules involved in this transport phenomenon. we probed t h e means by which t h e a n t l g e n p r e s e n t l n g c e l l (apc) handles t h e a n t i g e n produce p e p t i d e s t h a t bind t o mhc-molecules. we propose t h e e x i s t a n c e o f a new type o f i n t e r n a l image i n which immunoglobulin v-region peptides. formed by processing, imitate peptides from conventional a n t i g e n s . w e r e f e r t o such denatured i n t e r n a l images as r e s i d u e internal images, since t h e y a r e a s s o c i a t e d w i t h t h e r e s i d u e of p e p t i d e s remaining a f t e r processing. in some cases, r e s i d u e internal images may be actual sequence images, i . e . , the v-region sequence m y be i d e n t i c a l t o the conventional a n t i g e n sequence.to be class i h-2ka-restricted cytolytic t lymphocytes (ctl) are directed against two immunodominant sites on the a/jap/305/57 influenza hemagglutinin (ha) that can be mimicked by synthetic oligopeptides spanning residues 202-221 in the ha1 and 523-545 in the hydrophobic, transmembrane region. analysis of the fine specificity of hal-specific ctl clones demonstrated that these ctl clones can be subdivided into at least two group based on their patterns of recognition of closely related influenza h2n2 field strains and a monoclonal antibody derived variant of a/guiyang/4/57. using a series of nested synthetic peptides spanning the 202-221 region, the minimal amino acid residues necessary for recognition by the two groups of ctl clones were defined and found to consist of two separate but overlapping sites. sequence comparison of the ha of the a/jap/305/57, the influenza field isolates and the monoclonal antibody derived variant has identified two amino acids, asn at position 207 and gly at position 215, that are critical for t cell recognition. thus, animo acid substitutions induced either by antigenic drift or by monoclonal antibody selection can affect class i ctl recognition. pretreatment with a n t m e s reactive with class 11 mhc anti-has previously been reported to be successful in exov~~kj a n t i g e n -w i r q dendritic cells (m) fran rodent tissue grafts. we have extended these exper-to inta? whole organ grafts. ilia pnmxses also demcnstrated a prolonged survival (17 f 2 days) ccapared to controls (11 t 1 days) (p < 0.01) tihen v l a n t e d into streprozatocin treat& da recipients. antigenic variation in the haemagglutinin (ha) of influenza a viruses frequently introduces new oligosacchekide attachment sites ( aan-x-serlthreo) and carbohydrate addition prevents antibody recognition by steric hindrance. acid substitution in mutant viruses of the h3n2 subtype (ha1 63 asp+asn), that introduces an n-glycosylation site (hn6gcys6&thr65), abrogates antibody and cd4+ t recognition. infected with x31 virus recognise a synthetic peptide corresponding to antigenic site e, ha1 56-76, and are sensitive to a single substitution (ha1 63 asp-basn) in mutant viruses. virus infected target cells, thereby confirming that carbohydrate addition prevents cd4' t cell recognition. here ve show that an amino cell i-ad restricted, ha specific t cell clones f r m balblc mice-reviously recognition of mutant viruses is restored however by tunicamycin-treatment of key: cord-005480-yg7salqt authors: nan title: oral sessions and working party date: 2008-03-26 journal: bone marrow transplant doi: 10.1038/bmt.2008.30 sha: doc_id: 5480 cord_uid: yg7salqt nan basic science award: o100 mt1-mmp and reck inversely regulate haematopoietic progenitor cell egress a. avigdor* (1) , y. vagima (2) , p. goichberg (2) , o. kollet (2) , s. shivtiel (2) , m. tesio (2) , a. kalinkovich (2) , i. petit (2) , o. perl (1) , e. rosenthal (1) , i. resnick (3), i. hardan (1) , a. nagler (1) , t. lapidot (2) (1)the chaim sheba medical center (tel-hashomer, il) ; (2) the weizmann institute of science (rehovot, il); (3)hadassah medical center (jerusalem, il) hematopoietic progenitor cell release to the circulation is the outcome of signals provided by cytokines, chemokines, adhesion molecules, and proteases. yet, the mechanisms of progenitor cell egress during g-csf mobilization are not fully understood. membrane type-1 metalloproteinase (mt1-mmp) and its endogenous inhibitor, reck, are established key regulators of tumor cell motility. we detected higher mt1-mmp and lower reck expression on circulating human cd34+ progenitors and maturing leukocytes as compared to immature bone-marrow (bm) cells. mt1-mmp expression was more prominent on cd34+ cells obtained from pb of g-csftreated healthy donors whereas reck was barely detected. g-csf mobilization in nod/scid mice, previously engrafted with human cells, increased mt1-mmp and decreased reck expression on human progenitors and maturing leukocytes, in a pi3k/akt1-dependent manner, resulting in elevated mt1-mmp activity. blocking mt1-mmp function impaired g-csf mobilization, while reck neutralization promoted egress of human cd34+ progenitors. targeting mt1-mmp expression by sirna or blocking its function reduced the in-vitro sdf-1 induced migration of human progenitors via matrigel and impaired the bm homing capacity of transplanted human progenitors in nod/scid mice. in accordance, neutralization of reck function facilitated the migration of human bm cd34+ cells in vitro. furthermore, following g-csf mobilization, we also observed a reduction of cd44 on human cd34+ progenitors in the bm of chimeric mice. this was accompanied by accumulation of cd44 cleaved products of molecular weights, expected for mt1-mmp activity, in the bm supernatants. blocking mt1-mmp function in chimeric mice resulted in less cleavage of cd44 upon g-csf mobilization, whereas in the absence of a mobilizing signal, increasing mt1-mmp activity by reck ab injection facilitated cd44 proteolysis on the bm cells. finally, mt1-mmp expression correlated with the number of cd34+ cells, collected on the first apheresis day in consecutive healthy donors and patients mobilized with g-csf. in conclusion, our results indicate that g-csf inversely regulates mt1-mmp and reck expression on cd34+ progenitors, resulting in net increase in mt1-mmp activity. mt1-mmp proteolysis of cd44 diminishes progenitor adhesion to bm components, leading to cell egress. these previously undefined cell autonomous changes in the course of g-csf treatment might serve as target for new approaches to improve mobilization. morbidity and mortality associated with treatment-related organ toxicity is a major factor limiting success of allogeneic hematopoietic stem cell transplantation (hsct). conservative therapeutic strategies have been ineffective in part, resulting in high rates of progression or complete organ failure and death. over the last decades, solid organ transplantation (sot) has been increasingly used for the treatment of terminal organ failure in hsct recipients. to date, information regarding the use of sot as treatment attempts in patients after hsct is limited. as well the risk factors accounting for the necessity of sot after hsct as well as the incidence and outcome of this therapy are not well defined. a questionnaire survey was carried out within ebmt centres. 107 centres participated in this survey, covering allogeneic hsct between 1984 and 2005 . 31 cases of sot were identified. in more detail, 13 liver-, 8 kidney-, 9 lung-, and one heart transplantations were performed in 25 different centres. indications for liver transplantation were infections leading to cirrhosis (n=4), sinusoidal obstruction syndrome (n=3), and gvhd of the liver (n=2). rejection of the transplanted liver or terminal organ failure occurred in one patient respectively. other complications after liver transplantation were infections (n=2), bleedings (n=2) and kidney failure (n=2). most kidney transplantations were performed because of chronic kidney failure due to drug toxicity (n=6). transplant rejection and/ or kidney failure did not occur. interestingly, two of the kidney donors were also stem cell donors for the transplant recipient. lung transplantation was performed in all cases because of bronchiolitis obliterans and/ or gvhd which led to respiratory failure. rejection occurred in 4 patients and in one patient terminal transplant failure occurred. other major complications in the lung transplant recipients were kidney failure (n=3) and infections (n=2). heart transplantation was performed in one patient because of pre-terminal heart insufficiency due to drug treatment. in summary, very few sot for terminal organ failures were performed. the overall survival of patients receiving an organ graft after hsct was 72.5% at 5 years with a median follow up time of 23 months. complications after sot, like infections and organ rejection were frequent, but manageable. we conclude that sot offers a viable therapeutic option for patients who develop terminal organ failure after hsct. influence of immunisation timing on the response to conjugate-pneumococcal vaccine after allogeneic stem cell transplant: final results of the ebmt idwp01 trial c. cordonnier* (1) , m. labopin (2) , v. chesnel (2) , p. ribaud background: pneumococcal infections are causes of death after sct. the efficacy of the polysaccharide 23-valent vaccine (ppv23) is limited before 6 mo after sct, or if graftversus-host disease (gvhd) . previous studies with the wyeth heptavalent conjugate prevnar® vaccine (pcv7), using different schedules after allogeneic sct, showed a response around 65-85 % of the patients. however, the optimal timing of vaccination is yet not defined, and pneumococcal infection may occur early in the first months after sct. our objective was to show that the response to early (e) (3 mo) immunization is not inferior to a late (l) (9 mo) immunization. methods: patients ≥ 5 year old and at 3 months after allogeneic myeloablative sct were randomized to receive 3 doses of pcv7 at 1 month interval, followed by a ppv23 6 months later, from 3 (e) or from 9 (l) months after transplant. the primary endpoint was the % of responders (≥ .15 µg/ml of each of the 7 pcv7 serotypes) 1 month after the 3rd dose of pcv7 (s3). ab levels were blindly measured by elisa. all patients were followed until 24 months after transplant, or until death, whichever occurred first. results: 158 patients were randomized: 75 in the early (e), and 83 in the late (l) group. most patients were adults with acute leukemia, transplanted from an hla-identical donor. 114 patients were evaluable for the primary endpoint (e: 57, l: 57). the response rate was respectively 79% vs 82% at s3, and not inferior in the early, when compared to the late group (90% ci; 8.6) . however, at 24 months, significantly less e patients were still protected when compared to l patients (26/44; 59% s 35/42; 83%, p=.013). in the lack of difference in the response between groups, the 2 groups were pooled to analyse the impact of transplant characteristics on the percentage of responders at s3. donor age (> 36y) and chronic gvhd were the only factors impairing the response in the multivariate analysis. conclusion: the ab response 1 month after 3 doses of pcv7 after allogeneic sct is about 80% and non inferior when started at 3 than at 9 months. we therefore recommend starting immunization at 3 months to offer an earlier protection. however, the e vaccination offers a significantly lower protection at 2 years, suggesting the need for a boost during the second year after e immunization. the authors are grateful to the safety committee: d. engelhard, p. reusser, and p. reinert depletion of the autoreactive immunological memory followed by autologous haemopoietic stem cell transplantation in patients with refractory sle induces long-term remissions through de novo generation of a juvenile and tolerant immune system t. alexander (1) , a. thiel (2) , g. massenkeil (1) , a. sattler (1) , s. kohler (2) , h. mei (2) , h. radtke (1) , g.r. burmester (1) , a. radbruch (2) , r. arnold (1) , f. hiepe* (1) (1)charité -universitätsmedizin berlin (berlin, de) ; (2) german arthritis research center (berlin, de) clinical trials have indicated that immunoablation followed by autologous haemopoietic stem cell transplantation (asct) has the potential to induce clinical remission in patients with refractory systemic lupus erythematosus (sle). to elucidate the mechanisms mediating the beneficial long-term clinical responses, we investigated the immune reconstitution in sle patients receiving asct as part of a monocentric phase i/ii clinical trial. seven patients with sle were evaluated during a long-term follow-up (median follow-up period 60 months) who were immunoablated with cyclophosphamide and rabbit antithymocyte globulin, followed by transplantation of purified autologous cd34+ haemopoietic stem cells. previous failure of conventional immunosuppression, including cyclophosphamide, had been an inclusion criterion. humoral immunity was evaluated, peripheral t and b lymphocytes were immunophenotyped and frequencies of t lymphocytes specific for distinct antigens of interest were assessed after short-term stimulation ex vivo. in all patients clinical and serological remission was observed, accompanied by disappearance of anti-dsdna autoantibodies and protective antibodies from serum. one patient developed a relapse 18 months after asct. in the responding patients, cd31+ cd45ra+ cd4+ t cells, i.e. recent thymic emigrants, recurred with a doubling in absolute counts compared to agematched healthy controls until 4-yr post-transplant (p=0.014). absolute numbers of cd4+ foxp3+ regulatory t cells (tregs) normalised after asct and tcr repertoires cd4+ t cells displayed a broad clonal diversity as compared to the pretransplant status. early after asct, often high frequencies of virus-specific effector t cells were detected. autoreactive t cells specific for nucleosomes or smd1 were not detectable. a normal b cell compartment developed within 12 months after therapy, as compared to the pre-existing b cell deficiencies, which had included naive (igd+) b cell lymphopenia (p=0.031), relative predominance of memory (igd-) b cells (p=0.016) and expansion of cd27high cd20plasma blasts. our data demonstrate that the long-term therapy-free clinical remissions observed in sle patients after complete immunoablation and asct are accompanied by a loss of immunological memory and a fundamental reset of the immune system. depletion of the autoreactive memory and reactivation of thymic education probably are the basis for regeneration of self-tolerance and clinical remission. m. themeli* (1), l. petrikkos (2) , m. waterhouse (2) , h. bertz (2) , n. zoumbos (1) , j. finke (2) , a. spyridonidis (1) (1)university of patras medical school (rion-patras, gr); (2)freiburg university medical center (freiburg, de) we previously demonstrated frequent genomic alterations measured by microsatellite instability (msi) in non-neoplastic epithelial tissues of pts who underwent allogeneic hematopoietic cell transplantation (hct) but not in pts after autologous hct (blood 2006; 107:3389) . confirmation in larger independent patient cohort and in an in vitro system was needed. 176 buccal samples from 71 unselected pts obtained 30-3722 days (median 322) after allogeneic hct were analysed for msi. control subjects (16 healthy and 15 pts after auto-hct, 47 samples) were negative for msi. msi was observed in 37 (52%) allo-transplanted pts. msi+ pts were significantly older than msi-patients (median age 60y vs 48y, p<0.05). by using logistic regression analysis we found that the relative risk for msi was 2-fold higher in pts who experienced extensive chronic gvhd as compared to pts with no gvhd. although the median follow up in msi+ pts was significantly lower than in msi-(336 vs 669 days, p<0.05), secondary malignancy (5 skin-and 1 adeno-ca but none in the oral cavity) was diagnosed in 5 (14%) of the msi+ pts) and only in 1 (3%) msi-pt (p<0.05). other clinical features were not significantly different between msi+ and msi-pts. in an vitro mutation analysis model we tested the hypothesis that an alloantigenic stimulus is substantially involved in the mutation process. briefly, keratinocyte (hacat) cells were transfected with a plasmid vector carrying a neomycin selectable marker, a hygromycin resistance (hygr) sequence and a (ca)13 repeat. in this system, dna slippage mutations become detectable after hygromycin treatment as hygr+ colonies. the mutant fraction was expressed as the number of hygr+ colonies corrected for relative cell survival. untreated cells served as controls. treatment of stably transfected hacat cells with tnfa (25-100ng/ml, 24h), tgfb (5-20ng/ml, 24 h) and supernatant from a mixed lymphocyte culture (mlc, 24h) didn't cause any detectable induction of genomic instability (gi). treatment with h202 (20-40µì, 1-24 hours) resulted in a time and dose dependent gi induction (max 3.5 fold). cocultivation of hacat cells with stimulated lymphocytes from mlc resulted in a 3.9 fold induction of the mutant fraction. in conclusion, our in vivo and in vitro data indicate that "alloantigenic reactions may induce genomic instability in the allotransplanted pts which might predispose them to secondary neoplasia. the ebmt risk score predicts outcome after allogeneic hsct in all haematological disease categories and is independent of stem cell source or conditioning intensity a. gratwohl*, m. stern, j. apperley, t. de witte, j. passweg, v. rocha, a. sureda, r. brand, d. niederwieser information on factors associated with outcome after allogeneic hsct is a prerequisite for risk adapted strategies. five key factors form the basis of the ebmt risk score: stage of the disease (early 0, intermediate 1, advanced 2), age of the patient (< 20 y 0, 20-40 y 1, > 40 y 2), time interval from diagnosis to transplant (< 1y 0, > 1 y 1), histocompatibility (hla-id sibling 0, others 1) and donor recipient gender combination (other 0, female donor for male recipient 1). they were identified and validated in several independent series of cml patients, but not yet in other diseases. we examined 53140 patients, 34 y of age (median, 0-77y range), 58.7% male with an allogeneic hsct for aml (15126; 28.6%), all (10756; 20.2%), cml 12321; 23.2%), mds (4112; 7.7%), mps (1184; 2.2%), lymphoma (4165; 7.8%), myeloma (1329; 2.5%) or saa (4057; 7.6%) between 1980 and 2005. donor was a hla id sibling in 78.2%, other donor in 21.8%. stem cell source was bone marrow in 64.6%, peripheral blood in 34.9% and cord blood in 0.5%.conditioning was standard in 86.5%, reduced in 13.5%. each risk factor was tested individually by multivariate analysis and confirmed as cumulative dose response risk in all subcategories with two exceptions: stage was not applicable in saa, time interval was not applicable in patients in 1st cr. cumulative incidence of transplant related mortality (trm) at 5 years increased with the risk score from 15.4% (score 0, 3500 pts) to 22.1% (score 1, 10428 pts), 27.6% (score 2, 13677 pts), 32.4% (score 3, 11729 pts), 37.9% (score 4, 8159 pts), 42.6% (score 5, 4843 pts) and 49.7% (score 6/7, 1250 pts). after stratification by risk score, underlying disease had only a minor impact on the rate of trm. inside the risk score categories, trm improved significantly during the period of observation (rr 1980 (rr -1989 rr 1990 rr -1999 rr since 2000 0.50 ). absolute trm rates declined less markedly (1980-89 36%; 1990-1999 31%; since 2000 27%) due to a shift towards higher risk patients in more recent years. the ebmt risk score separated risk categories in all diseases, for all donor types, for all stem cell sources and for patients with reduced or standard conditioning. these data show that risk categories for outcome after allogeneic hsct can be defined. they can be integrated into risk assessment algorithms and form the basis for individualised risk adapted strategies when transplant and non transplant strategies are available as treatment options. working party solid tumours 114 reduced-intensity allogeneic transplantation for breast cancer d. blaise* (1) , a. gonçalves (1) , s. fürst (1) , j.o. bay (2) , c. faucher (1) , m. michallet (3) , j.m. boiron (4), j.y. cahn (5) , n. gratecos (6) , m. mohty (1) , c. chabannon (1) , g. gravis (1) , b. esterni (1) , j.m. extra (1) , p. viens (1) (1)institut paoli-calmettes (marseille, fr) ; (2) centre jean perrin (clermont ferrand, fr) ; (3)chu edouard herriot (lyon, fr) ; (4)chu haut lévèque (bordeaux, fr); (5)chu jean minjoz (besançon, fr); (6)chu cimiez (nice, fr) we initially treated 18 pts with allo sct for advanced metastatic breast cancer. all pts (age: 45 (27-57)) underwent asct after the same reduced intensity conditioning (ric) (fludarabine (150mg/m2), busulfan (8mg/kg) and thymoglobulin (2,5mg/kg) or tli (1 cgy)) from a hlaidentical sibling (bm: 22%; pbsc: 78%) followed by csa. prior to asct a median of 3 lines of treatment (1-7) were administered over a period of 1452 days . nine pts underwent autologous sct at a median time of 1130 days (88-3012) prior to asct. all pts were measurable and had a median of 2 metastatic sites (1-4) (liver:72%, bone:50%, lung:22% and brain:11%): according to recist criteria, 13 (72%) and 5 (28%) pts had progressive (pd) and stable disease (sd) respectively. none of the 18 pts died from trm. two of them achieved partial remission (pr) at 60 and 150 days respectively (objective response (or): 18% (0-36). all pts but 1 eventually progressed and died from disease (2 year overall survival (os): 22%(9-45)). results are dramatically different in regards to disease status at time of transplant. while outcome is uniformly poor for pts with pd, patients with sd achieved a 40% (0-80) or rate for a 50% (19-80) os at 2 years with 3 (60%) patients surviving more than 2 years (640+, 834-and 1246-), which is significantly different from pts with pd. we established that ric-asct can be safely performed in brc pts, whereas highly pd pts do not benefit from this approach. we run a second trial in less advanced disease to confirm encouraging results (present accrual: 15 pts). all 33 patients will be presented. however it seems that curability in brc will be achieved only in pts in the initial disease phase: target population would need a careful selection on individual prognosis factors indicating their poor short term poor outcome: this represents the ultimate goal for future investigations. supported in part by a grant from the french ministry of health (phrc 2000; phrc 2003 ) and a special grant (pole areca) from the association pour la recherché contre le cancer (arc) we have utilized autografting to achieve maximum tumor reduction before proceeding to non-myeloablative allografting. this strategy could provide the benefit of a conventional allograft, but with reductions in the typical acute toxicities and associated mortality of myeloablative conditionings. between september 1997 and april 2004, we enrolled 17 patients with metastatic breast carcinoma. median age was 41 years. at the time of autografting, the patients had received a median of 3 (range, 2-5) previous chemotherapy lines; 14 patients had received hormone therapy, and seven patients had undergone radiotherapy on bone lesions. the primary endpoint of this study was the decrease of non-relapse mortality (nrm) from the current 20-35% noted after myeloablative allografting. patients received autografting at a median of 53 months (range, 14-152) from the diagnosis of breast cancer. no patient died after transplant. one patient who had been in complete remission and two who had been in partial remission before autografting remained in complete or partial remission. no non-relapse mortality was noted in the first 100 days after non-myeloablative allografting. thirteen patients achieved full chimerism. five patients (29%) developed grade ii-iii acute gvhd, while six patients developed chronic gvhd (five patients with extensive disease) and needed intensive immunosuppressive therapy. we have recently reported a subsequent patient transplanted from her hla-identical sister. disappearance of liver, adrenal, mediastinal, pleural, and diffuse nodes and bone metastases, observed simultaneously with clinical chronic gvhc 5 months after non-myeloablative allografting, suggested a profound graft-versus-tumor effect. renal cell carcinoma (rcc) has recently been identified as being a target for gvt effect. since 1999 there has been a number of publications describing gvt effects in patients with rcc undergoing mostly reduced intensity transplantation. at the nhlbi, patients have been conditioned with cyclophosphamide (60mg/kg x 2) and fludarabine (25mg/m² x 5) then transplanted with a g-csf mobilized blood stem cell allograft from their hla identical or single antigen mismatched related donor. twenty-nine of 74 patients have had disease regression consistent with a gvt effect (39.2 % cumulative incidence of a complete response + partial response). a better understanding of the immune cells and their target antigens that mediate tumor regression could potentially lead to the development develop more effective hct approaches for solid tumors. recently, t-cells with in vitro tumor cytotoxicity patterns consistent with recognition of minor histocompatibility antigens and tumor restricted antigens have been identified in some responding patients. the identification of tumor restricted antigens targeted by donor immune cells could lead to the development of transplant approaches that enhance gvt effects while avoiding gvhd through tumor vaccination or the adoptive infusion of in vitro expanded donor t cells with tumor antigen specificity. we detected rcc-reactive cd8+ t-cells by elispot analysis in the blood of several responding patients with metastatic rcc following hct that were absent before transplantation. we successfully generated donor cd8+ t-cell clones from lymphocytes obtained from these patients that have direct cytotoxicity against the patient's rcc cells. in one responding patient, cytotoxic t-lymphocytes and t-cell clones with rcc-specific tumor cytotoxicity were isolated from the blood after transplantation. utilizing cdna expression cloning, we identified an hla-a11-restricted 10-mer peptide (named ct-rcc-1) to be the target antigen of these rcc-specific tcells (takahashi y, harashima n. et al-j clin invest 2008 in press) ct-rcc-1-specific t-cells were detected by tetramer analysis in the patient's blood after tumor regression but not before hct. tetramer analysis of 8 hla-a11+ rcc transplant recipients showed ct-rcc reactive t-cells expanded significantly in all 3 responders in contrast to the 5 non-responders, where only 1/5 showed an increase in ct-rcc-1 reactive t-cells. the genes encoding the ct-rcc antigen were found to be derived from a human endogenous retrovirus (herv)-e previously unknown to be expressed in any human tissues; this herv-e was found to be expressed in the majority of rcc tumor lines and fresh rcc tissue but not in normal kidney cells or other normal tissues. this is the first solid tumor antigen identified using allogeneic t-cells from a patient undergoing hct. these data suggest this herv-e is transcriptionally active in rcc, encoding an immunogenic antigen that is over-expressed in rcc which could be a potential target for cellular immunity. update of the results of high-dose chemotherapy as primary or salvage therapy in germ cell tumours g. rosti* (1) , u. de giorgi (1) , p. pedrazzoli (2) , m. bregni (3) ( cisplatin-containing regimens cure nearly 80% of patients with advanced germ cell tumors. hdct has been extensively used in the last 20 years with somehow controversial results. the ebmt it-94 study on relapsing good-risk patients has not shown any difference in overall survival comparing four courses of standard second line therapy versus one late intensification single shot approach, even if patients achieving cr did significantly better if randomized in the high-dose arm. a phase iii us trial in patients with poor prognosis, treated upfront, even if not showing an advantage for hdct, has shown a significant difference for those with unsatisfactory marker decline. an input of the possible role of hdct in relapsing/refractory patients came from the retrospective data of the indiana university of tandem hdct with carboplatin and etoposide in a large series of consecutive men with metastatic testicular cancer that had progressed after receiving cisplatin-containing combination chemotherapy. this study shows 70% and 50% four-year disease-free survival in patients who received hdct as second-line or third-line or later therapy, respectively. as it is a retrospective review, one may argue that the results are biased by patient selection. this does not seem to be the case, however, as even patients with very poor prognosis achieved long-term disease-free survival -50% of survivors were classified high-risk by the igcccg classification and 45% had platinum-refractory disease. it is important to note that all patients in this series received peripheral-blood progenitors as sources of hematopoietic stem cells. this strategy allowed a rapid engraftment, thereby permitting the administration of two courses of high-dose in addition, peripheral-blood progenitors were enriched for cd34+ hematopoietic cells, a procedure which may have a role in eliminating possible cancer cells from the graft. we believe that, on the basis of the robust data provided by einhorn and colleagues, a well-designed randomized trial of hdct versus conventional-dose chemotherapy should be performed in patients with poorprognostic clinical features who relapse after initial chemotherapy. at present, there should be no debate on the use of tandem-hdct in patients with cisplatin-refractory germ-cell tumors and those who have failed second-line therapy. gitmo and igg (italian group for germ cell cancer) are planning a network of centres in italy to refer such patients for the tandem hdct. autologous stem cell transplantation international multiple sclerosis trial (astims, eudract number 2007-000064-24, supported by ebmt; www.astims.org) is now a multicenter, prospective randomized phase ii study. the primary endpoint is the number of new t2 lesions on mri. the investigational treatment comprises mobilization with cy and g-csf and conditioning with beam followed by asct and atg compared to 6 monthly i.v. pulses of mitoxantrone at 20 mg followed by 1gr of methylprednisolone. at the moment 18 patients have been enrolled (january 2008): 4 in barcelona, 3 in genova, 3 in modena, 3 in florence, 2 in chieti, 2 in reggio calabria and 1 in bergamo. the astims trial is therefore still going on, with the aim to arrive within 2 years from now at the new target of 30 enrolled cases. in the meantime, more than 400 are the ms treated in the world with ahsct and a few phase i/ii are running with the aim to identify the clinical characteristics of the patients who can really take advantage from the procedure or to evaluate the efficacy of low intensity conditioning regimens. the study of all the 58 cases treated in italy in the last 10 years with the same regimen (beam and atg), same inclusion criteria and followed by the same neurohematological teams involved in the prospective study supported by gitmo, showed that patients with a relapsing remitting clinical course respond significantly better than secondary progressive cases to ahsct, indicating the population of patients who have to be selected in the future for the design of prospective studies with a clinical endpoint. the astis trial j.m. van laar* (1) , d. farge (2) , a. tyndall (3) , o. astis investigators (4) (1) newcastle university (newcastle, uk); (2) hopital st louis (paris, fr); (3)basel university hospital (basel, ch); (4)jcuh (middlesbrough, uk) background: high dose immunosuppressive therapy (hdit) and hematopoietic stem cell transplantation (hsct) is a novel treatment for patients with severe systemic sclerosis (ssc) . previous studies showed durable responses in two thirds of patients up to 7 yrs after hsct (1) . this treatment modality is now further investigated through the astis-trial (autologous stem cell transplantation international scleroderma trial), a prospective, controlled, randomized trial to compare safety and efficacy of hdit + hsct versus monthly i.v. cyclophosphamide in ssc patients at risk of major organ failure or early mortality. objectives: to evaluate whether hdit + hsct is superior over conventional treatment in terms of safety and efficacy in ssc patients, and to assess potential predictive factors of response. methods: ssc patients with early active diffuse disease with or without major organ involvement are eligible. ssc patients randomized to the transplant arm undergo mobilization with cyclophosphamide 2x2 g/m², conditioning with cyclophosphamide 200 mg/kg, rbatg 7.5 mg/kg, followed by reinfusion of cd34+ selected autologous hsct. those randomized to the control arm are treated with 12x monthly i.v. bolus cyclophosphamide 750 mg/m². the primary endpoint is event-free survival, defined as survival until death or development of major organ failure during 2 years follow-up. progression-free survival is the main secondary endpoint. results: one hundred eleven ssc patients have been enrolled in 25 centers per january 2008: 43 male, 68 female, mean age 43 yrs, mean modified rodnan skin score 26, mean disease duration 1,8 yr, mean vc 81%, mean dlco 59%. sixty-one patients were randomized to the transplant arm, 50 to the control arm. no unexpected toxicities have yet been observed in either arm with a median follow-up of 36 months (range . grade 3,4 toxicities occurred in 15/43 transplant patients and in 13/48 controls (p=0.42). atg-related toxicity led to its discontinuation in 12/35 transplant patients. two fatalities in the transplant arm were categorised as probably treatment-related. conclusion: the ongoing astis trial has enrolled 111 patients sofar. treatment-related mortality and number of patients with serious adverse events of stem cell transplantation are lower than previously reported in registry analyses. references: 1. van laar jm, farge d, tyndall a, on behalf of the ebmt/eular scleroderma study group. the astis-trial, hope on the horizon. ann rheum dis 2005;64:1515. standard nih or eurolupus cyclophosphamide (cy) protocols and mycophenolate mofetil (mmf) as induction therapy in severe bilag a sle is still associated with 20 % failure, 50% relapse and 10% to 15 % death at 10 years in the absence of a single standard treatment worldwide for refractory sle, phase i-ii studies analysed the use of: a) rituximab (anti cd20 mab) in more than 1 000 patients showing complete to partial early response around 100% with relapse in 50 to 60% of the cases; b) autologous hematopoietic stem cell transplantation (hsct) since 1997 under the auspices of the joined ebmt-eular working party, reporting durable remission with reduced or no immunosuppressive drug requirement in 66%, one-third of whom later relapsed to some degree with a 74 ± 7% (n= 62/79) overall survival at 5 years for sle among the 863 hsct procedures registered: in 2007 in the ebmt data base. the north american, mostly single centre experience showed higher rates of remission with also some relapses. maintenance immunosuppression after induction of remission may decrease the return of disease activity. this was the basis of the ebmt approved astil trial: a prospective randomized open, multicenter, phase ii b study to compare the efficacy of autologous hsct with rituximab as remission induction, followed by mmf (2 g /day) as maintenance in both arms for severe sle patients with disease duration ≤ 5 years since the diagnosis and sustained or relapsed active bilag a sle. this analysed describes the outcome of pediatric patients receiving hematopoietic stem cell transplantation (hsct) to treat severe refractory autoimmune cytopenias. the registry of the ebmt contains data on 16 patients receiving 19 transplants. patients had autoimmune haemolytic anemia (7), evans's syndrome (7) , immune thrombocytopenia (3), pure red cell aplasia (1) and autoimmune lymphroliferative synrome (1) . 15 patients were males with a median age at diagnosis of 4 years (range 0.3-16 years) and a median age at transplant of 7.8 years (2-17 years) . the median disease duration prior the transplant was 41 months (range 2-115 months) and all patients failed multiple prior treatments. transplant were autologous for 7 and allogeneic for 12 patients, 6 of these transplanted from an hla identical donor, 2 from a family mismatched donor and 4 from a matched unrelated donor. one patient received 2 autologous transplant while another patient received an allogeneic transplant because a relapse after the first autologous transplant . the stem cell source was mobilized pbsc in 1 transplant, bone marrow in 6 and cord blood in 2 patients. the graft was t depleted in 4 of 7 recipients of autotransplant and 3 of 12 allotransplant recipient. the conditioning regimen used were heterogeneous. 3 patients died of treatment related mortality, 2 in the allo and 1 in the autologous group for a trm of 14 % eights patients had a complete and continous response after the transplantation although 1 of these died for secondary malignancy. 3 patients relapsed after the procedure (1 in the allo and 2 in the autologous group) and one of these died for disease progression. 3 patients were not evaluable for response. the present analysis has some limitations because treatment protocols, mobilization and conditioning regimen were heterogenous and doesn't allows a detailed analysis of these factor moreover these preliminary data suggest that autologous and allogeneic hsct may induce response in half of patients with severe autoimmune cytopenia of long duration unresponsive to several therapeutic options. 124b long-term follow-up of autologous stem cell transplantation for juvenile idiopathic arthritis n.m. wulffraat wilhelmina children's hospital (utrecht, nl) the majority of children with juvenile idiopathic arthritis can nowadays be treated adequately. however despite the use of combinations of antirheumatic drugs, corticosteroids and the newer so called biologicals (blocking the tnf, interleukin 1 or interleukin 6 pathways) a proportion of children with arthritis remain resistant also to these therapies and suffer from a very severe, debilitating and potentially fatal disease. for such children autologous stem cell transplantation (asct) is successfully performed since 1997. here we describe the long term outcome of the initial cohort of children with resistant juvenile idiopathic arthritis, treated with asct. the initial cohort of children was treated with a conditioning regimen containing cyclophosphamide, anti thymocyte globulins and low dose total body irradiation. overall favourable responses were seen, with a drug free remission rate of 50-55 %. in the more recent years late relapses were noted with lower percentages for drug free long term outcome. special emphasis is given on 2 cases showing very late relapses, occurring after 7 and 9 years. the observed relapses are often less severe compared to the situation before sct and can be treated successfully with conventional drugs in the majority of cases. more recently, asct was performed in 4 jia children with a fludarabin containing regimen in stead of low dose tbi. with a 4 to 5year follow up, these 4 patients are all in drug free full remission. allogeneic transplant with an hla matched family donor was reported in 2 jia cases. follow up of 1 and 3 year is sofar show clinical disease remission and tapering of medition. in conclusion, given the favourable long term outcome, sct remains a valuable treatment option for children with drug resistant jia. s7 126 multipotent mesenchymal stromal cells in the treatment of autoimmune diseases a. tyndall* (1) , f. dazzi (2) multipotent mesenchymal stromal cells ( msc) isolated from the bone marrow and other sites are currently being studied to determine their potential role in the pathogenesis and/ or management of autoimmune diseases. in vitro studies have shown that they exhibit a dose dependent antiproliferative effect on t and b lymphocytes, dendritic cells, natural killer cells and various b cell tumour lines, an effect which is both cell contact and soluble factor dependent. these soluble factors include tgf beta, il-10, indoleamine 2,3 dioxygenase, il-1ra, and hla-g among others. anti proliferative and immunomodulatory mechanisms are probably multiple and most likely due to the induction of arrest of the cell cycle in g0/g1. a plethora of phenotypic definitions and experimental conditions accounts for some of the variation in in vitro phenomena being reported previous assumptions that msc are immunoprivileged have been challenged by recent animal data in non immunosuppressed hosts. animal models of autoimmune disease and tissue injury (ischemic kidney, chemically induced lung fibrosis and liver toxicity) have mostly shown a positive clinical response, with some early warning signs in a melanoma model concerning tumour surveillance. a limited number of patients suffering from acute graft versus host disease have been treated with msc as well as sporadic case reports and small uncontrolled series in multiple sclerosis and crohns disease. prospective phase i trials are starting in multiple sclerosis and crohns disease and being considered in inflammatory rheumatic diseases. an international interdisciplinary data base has been developed to exploit the collective experience. sirolimus is associated with veno-occlusive disease of the liver after myeloablative transplantation c. cutler*, k. stevenson, h. kim, p. richardson, v. ho, c. revta, r. ebert, d. warren, j. koreth, p. armand, e. alyea, r. soiffer, j. antin dana-farber cancer institute (boston, us) veno-occlusive disease of the liver (vod) is an uncommon but important cause of mortality after allogeneic transplantation. to determine if use of the immunosuppressive mtor inhibitor, sirolimus, is a risk factor for vod, we performed a retrospective review of vod incidence and risk factors at our institution since 2000, when we began using sirolimus. methods: review of electronic medical records of all transplant patients undergoing tbi-based myeloablative transplantation with adult stem cell donors was performed. results: 510 patients transplanted between 1/2000 and 5/2007 were identified and stratified by sirolimus use (260 exposed, 250 unexposed). sirolimus patients received sirolimus/tacrolimus ± methotrexate; all others received tacrolimus/methotrexate as gvhd prophylaxis. there were no differences in the age, gender, donor-recipient gender match or diagnoses between cohorts. sirolimus patients were more likely to have unrelated or mismatched donors, were more likely to have received pbsc (p<0.001 for both) and were less likely to have gr. ii-iv acute gvhd (26 vs. 38%, p=0.004) in comparison with non-sirolimus patients. the incidence of vod in the sirolimus group was 15% and was 6.4% in the unexposed (rr 2.3, p=0.003), but vod occurred later among sirolimus patients (22 vs. 15 days, p=0.12) . among mrd recipients, the rr was 2.6 (12.2 vs. 4.6%, p=0.06). when adjusted for age, gender match, stem cell source, hla match (mrd vs. urd/mismatch), and transplant risk (standard vs. high), sirolimus use remained a significant risk for vod (adjusted or 2.54, p=0.006). cause-specific mortality related to vod was similar in sirolimus and non-sirolimus patients. despite the increase in vod, treatment-related mortality was similar in all sirolimus and non-sirolimus patients and among mrd sirolimus and non-sirolimus patients at 1 year (19 vs. 21%, and 12 vs. 13%, both p=ns). in addition, there was a trend towards increased overall survival (os) for all sirolimus patients (3 yr os 54 vs. 49%, p=ns) and for mrd sirolimus patients (3 yr os 67 vs. 53%, p=0.08). in a cox regression model, age > 50 (p=0.001), donor match (p= 0.01) and vod (p<0.001) but not sirolimus use (p=0.14) were significantly associated with overall survival. conclusions: sirolimus is associated with vod after tbibased myeloablative transplantation. despite this association, transplant outcomes appear equivalent or better than standard tacrolimus/methotrexate based immunosuppression. physical health can be compromised in very long-term survivors after hsct compared to their respective donors but not mental health: a paired analysis t. daikeler, a. rovo*, m. stern, j. halter, j.d. studt, a. buser, d. heim, j. rischewski, m. medinger, a. tyndall, a. gratwohl, a. tichelli university hospital (basel, ch) with the improvement of prognosis, health status and functional well being of long term survivors after hsct become an important issue. we performed a cross-sectional prospective study on 44 long-term survivors and their respective sibling donors at a median follow up of 17.5 years (range 11-26) after hsct. the median age of the recipients and donors at time of the study was 44.3 (24-63) and 43.4 years (22-61) respectively. both recipients and donors were seen on the same day for evaluation. the short form-36® (sf-36) health survey, which provides a generic health status measurement through 36 items assessing 8 concepts of health was used. three of the items measure physical health (pf, rp, bp), two measure both physical and mental health (gh, vt), and three measure mental health (sf, re, mh). in addition there are two summary scores for physical (pcs) and mental (msc) health. for statistical analysis norm-based scoring (nbs) was applied, where 50 is the mean score, and 10 the standard deviation of a defined general population. paired analysis between donors and recipients were performed for detecting differences. all scored items of recipients as well as of the donors were within the range of one standard deviation of the norm-based population. all scores concerning physical well being except one, (rp), were statistically lower in the recipients than in their donors. in contrast, there was no difference in scores concerning mental well being. this is confirmed by the summary measurements of physical health (pcs) with 52,8 in the recipients and 57,1 in the donors , (p=0.001) and mental health (mcs) with 50,8 versus 52,9 (p=0.831) . physical health (pcs) was lowest in patients with severe chronic gvhd compared to their donors (47,2 versus 57,2) (p=0.05), age older than 25 years at hsct 50,7 versus 56,2 (p=0.024), older than 42 years at the last control 52,2 versus 55,63 (p=0.05) and for female patients 51,8 versus 57,7 (p=0.024) . none of the factors had a statistical impact on mental heath status (mcs, p>0.05). in summary, quality of life of long term survivors after hsct measured with the sf-36 questionnaire is still within the normal variation of the general population. however, when compared to their respective donors, the physical health status is significantly compromised in the recipients. severe gvhd, older age and female gender are associated with an inferior physical health status. a retrospective analysis of sexual function, fertility and endocrine status in male long-term survivors of allografts for haematological malignancy i.h. gabriel*, r. szydlo, m. klammer, r. patterson, n. swan, n. salooja, e. olavarria, e. kanfer, d. marin, a. rahemtulla, j.f. apperely imperial college healthcare nhs trust, hammersmith hospital (london, uk) steady improvements in the outcome of allogeneic stem cell transplantation (allo-sct) have resulted in significant numbers of long-term survivors and an increasing focus on factors impacting quality of life (qol). post transplant infertility and sexual dysfunction are two such factors. using a questionnaire we audited 150 male survivors of allografting for haematological malignancies at our centre. 81 men (54%) responded. the median age at sct was 37.5 yrs. the median time from transplant was 6.9 yrs and 97% were >3 years from sct (63% >5 years). 76% had returned to full pre-sct sexual activity, however, a number of problems in sexual function were reported. 49% complained of new persistent erectile dysfunction (ed) (normal prevalence 2-20%). ed affected men of all ages (40%, 47% and 51% at < 30 years, 30-40 years and >40 years respectively). ed was seen in 52% of recipients of tbi vs 22% of those who had not received tbi. 30% experienced penile glans dryness, previously unreported which appeared to be closely associated with chronic gvhd(p 0.019). in addition urethral constriction, phimosis, genitourinary infection and peyronies's disease all complicated sct. 48% of men reported reduced libido with 37% of ed patients reporting normal libido. 13% and 17% of men suffered premature or painful ejaculation respectively. 43% of male survivors described a negative impact of infertility on themselves or their partners and 9% had utilised assisted fertility/donor insemination. other problems included dyspareunia, inability to use condoms, sicca syndrome, and poor body image. factors forming barriers to new, or worsening existing, relationships were most prominent in younger patients. compared to values pre-transplant fsh was raised in 100% by 6 months post-transplant and remained high at 5 yrs (p=0.003), indicating long-term damage to sertoli cells. lh was significantly elevated at 12 months in 15% compared to baseline (p=0.005) and normalised by 5 yrs suggesting leydig cell recovery. testosterone levels were normal in 98% of men at 12 months. lack of physician continuity, language, presence of visiting fellows/students and the impression that physicians are only concerned with managing malignancy were barriers discussing these issues at routine follow-up appointments. a high prevalence of sexual dysfunction exists post-sct. increased awareness of these complications and their effects on patients and their families would permit prompt and appropriate management. secondary malignancies in recipients of allografts for chronic myeloid leukaemia in chronic phase using hlamatched sibling or volunteer donors i.h. gabriel*, s. avery, r. szydlo, n. salooja, e. olavarria, e. kanfer, m. klammer, a. rahemtulla, j. goldman, j.f. apperley imperial college healthcare nhs trust, hammersmith hospital (london, uk) during the last decade imatinib has replaced allogeneic sct as first line treatment for chronic myeloid leukaemia (cml) and second generation tyrosine kinase inhibitors now compete with allografting as second line therapy. however over the same period there have been steady improvements in the outcome of allografting and therapeutic choices are increasingly complex. improved information regarding the long-term side effects of both chemotherapy and allografting might help inform decision-making. we now update the incidence of secondary malignancy in 481 consecutive patients of median age 33.9 years, who underwent allografts for cml in first chronic phase (291 from sibling and 184 from unrelated volunteer donors). the median follow-up is 13.9 yrs. all patients received cyclophosphamide with either total body irradiation (tbi) (n=474) or busulphan (n=7). gvhd prophylaxis was cyclosporin, methotrexate, ± t-cell depletion. the probability of developing a secondary malignancy post allo-sct was determined by the method of cumulative incidence (ci). the overall incidence in this group was 5.95% with 27 patients developing 29 new tumours. 31% were diagnosed within 5 years, 62% within 15yrs, 72% within 20 yrs. men and women were equally susceptible to secondary malignancies. the overall incidence of developing a new malignancy in our cohort is 5.95% which is higher than in an age-matched population. involved sites included: skin (5 including 2 melanoma, 2 squamous cell and 1 basal cell carcinoma), breast (4), high grade b-lymphoma, (4), tongue (3), colo-rectal (2), cervix (2), osteosarcoma (2), testis (1), bladder (1), mucoepidermoid (1), oesophagus (1), lung (1), penile shaft (1), and mds (1). 2 patients had 2 malignancies. pre-bmt treatment consisted of hydroxyurea alone in 10, busulphan in 2 and interferon in 1 and some combination of these in 9 others. the ci of secondary neoplasia increased with time reaching 1.7%, 3.3%, 6.3% and 14.2% at 5, 10, 15,and 20 yrs respectively. 13 patients have died, 10 directly attributable to the secondary tumour. as all patients had the same genetic abnormality and received only minimal prior cytotoxic therapy, this increase in neoplasia is likely to be due to sct. these data highlight the need for close follow up and screening. prospective evaluation of oxygenation index and noninvasive-ventilation in patients suffering from acute lung injury after allogeneic transplantation m. wermke*, s. schiemanck, g. höffken, g. ehninger, m. bornhäuser, t. illmer university hospital (dresden, de) objectives: respiratory dysfunction is a major cause of allogeneic transplant-related-mortality (trm). little is known about how to recognize and treat acute lung injury (ali) in this setting. we present the first prospective randomized clinical trial evaluating the use of non-invasive-ventilation (niv) for treatment of ali in allogeneic transplantation. methods: all patients (n= 530) undergoing allogeneic transplantation at a single center were investigated from 2001 to 2005. oxygenation-index (pao2/fio2) was monitored twice daily from the beginning of the conditioning regimen. patients meeting criteria for ali were randomized to receive either oxygen or intermittent niv with positive end-expiratory pressure. patients not responding to assigned therapy were allowed either to switch from oxygen to niv or proceeded with treatment on intensive care unit (icu). results: of 90 eligible individuals, 44 were randomized to receive oxygen and 42 to niv, 4 patients withdrew consent. oxygen-, niv-and control-(440 patients without ali) group were well balanced regarding known risk factors for trm. only tbi was significantly more frequent in patients with ali. patients with ali showed significantly shortened short-and long-term overall survival (os) when compared to controls (100-day-os 65 vs. 85%; median-os 7 vs. 22 months). oxygenation index was not only a major adverse prognostic factor; it also suggested ali long before clinical parameters raised suspicion. of 42 patients randomized to niv only 10 (24%) did not respond and had to be transferred to icu. in contrast 18 of 44 patients (41%) assigned to oxygen did not improve. of these, 17 switched to niv but only 12 patients had to be transferred to icu. thereafter, there was no significant difference between both groups in need for intubation (oxygen: 11 patients, niv: 7 patients). niv did not lead to improved survival in our study (100-day-os niv: 61%, oxygen: 68%; median-os niv: 6 months, oxygen: 7 months). survival of intubated patients was poor, as only 1 of 18 intubated patients survived for more than 100 days. conclusion: oxygenation-index is an easily measurable early indicator of ali and poor survival in allogeneic transplantation. niv seems to reduce the need for icu and consecutive intubation in patients not responding to oxygen. we were not able to show significant survival benefits for niv-patients, presumably because patients not responding to oxygen were allowed to switch to niv. what to do when the first allogeneic stem cell transplantation fails? m. kedmi, i.b. resnick, b. gesundeheidt , l. drey, s. samuel, r. or, m.y. shapira* hadassah -hebrew university medical center (jerusalem, il) the failure of allogeneic stem cell transplant (allo-sct) is usually cumbersome, we have retrospectively evaluated our experience in a 2nd allo-sct. patients: out of 1533 allo-transplantees, 145 patients (93 males, 52 females, median age 20.7y(8m-68.4y)) underwent 2 or more allo-sct. the indications for the 1st transplant were acute leukemia (93), chronic leukemia (17), lymphoma (3), other malignancies (3) and non malignant (29). the 1st to 2nd interval was 18d to 13.25y (median 98d). the most frequent indications for the 2nd sct were basic disease (45), rejection (23) and engraftment failure (15). the 2nd sct conditioning was radiation based (59)or chemotherapy based in the others and was myeloablative or reduced intensity (ric) in 64 and 81 respectively. in 89 of the scts the original donor was used. 2nd donor matching was full in 92 transplants (family-84, unrelated-8) or mismatched in 53 transplants (51 and 2, family and unrelated donor respectively). graft source for the 2nd sct was bm (65) and pbsc (80). 38 of the grafts were t cell depleted. gvhd prophylaxis was given in 31 2nd procedures. the median survival from 2nd sct was 70d. despite the low rate of gvhd prophylaxis used only 51 and 16 of the patients developed agvhd and cgvhd respectively. 29/145 patients (20%) transplanted survived a year after the 2nd sct (figure 1). trm was 66% including sepsis, liver and renal failure, neurologic toxicity, rejection and gvhd. factors indicating higher chance for survival were non malignant disease, longer time between procedures (more then 1y), hla matching and the use of ric (figure 2). age at transplantation, the indication for transplantation (relapse vs. others), the development of acute gvhd, radiation based vs. chemotherapy based conditioning, gvhd prophylaxis (either pharmacological or t cell depletion) or graft source were not shown to indicate better or worse prognosis. with a median follow up of 4.5y, 25 patients (17.2%) are still alive, out of which 18 are disease free. conclusion: although highly toxic, a 2nd allo-sct may be beneficial to some patients and lead to long term survival. it seems that the patients whom will gain the most out of 2nd allo-sct are those who had a longer period between scts, have a matched donor or have non malignant diseases although 14 patients with malignant diseases (some with refractory disease) survived at least a year from the 2nd procedure while in cr. it is also noteworthy that the use of ric improved outcome. s10 o136 treatment of donor graft failure with autologous or allogeneic stem cell boost or a second allogeneic transplantation based on chimerism testing a. shimoni*, n. shem-tov, a. rand, e. ribakovsky, r. yerushalmi, i. hardan, a. nagler chaim sheba medical center (tel-hashomer, il) donor graft failure (gf) is a life-threatening complication of allogeneic stem cell transplantation (sct). we performed this analysis to determine the rate and outcome of gf in the era of modern sct. we retrospectively reviewed data of 491 scts from hla-matched siblings (n=284), matched-unrelated (mud, n=180) or alternative donors (mismatched-related, haplo-identical and cord-blood, n=27) performed in a single institution since 1/2001. gf was diagnosed in 25 patients (pts), cumulative incidence (ci) 5.2% (95%ci 3.5-7.6). gf was determined when anc had not reached 0.5 x 10*9/l by day 21 (primary gf, n=21) or when anc decreased irreversibly after engraftment (secondary gf, n=4). ci of gf was 2.5%, 6.8% and 23.4% after sct from siblings, mud or alternative donors, respectively (p<0.001) but was similar following myeloablative or reduced-intensity conditioning (5.7% and 4.6%, respectively). pts with a predominant donor population in chimerism testing were given donor cell boost with no additional conditioning (n=10). pts with a predominant host population were given autologous back-up cells (n=8) or a second sct from a different donor (sibling-1, haplo-3, mud-1) with nonmyeloablative conditioning. 18 pts survived > 1 week after second graft infusion and are evaluable for engraftment. 16 pts engrafted within a median of 10 days (range, 5-15). the probability and pace of engraftment was similar in the different approaches. 11 pts (44%) were able to be discharged home and 14 died; 2 early after diagnosis of gf with no intervention, 5 within one week of second graft infusion and prior to engraftment, 2 with no engraftment and 5 early after engraftment from infection (n=3), organ failure (n=1) and gvhd (n=1). with a median follow-up of 19 months (range, 3-68), 6 are alive and 5 additional pts died (relapse-3, gvhd-1, infection-1). the projected 2-year survival for all pts was 23% (95%ci 5-41). interestingly, 4 pts given autologous cells had donor cell recovery, 1 had spontaneous autologous reconstitution within 3 weeks, 2 died within 2 months (gvhd-1, infection-1) with persistent donor cells and 1 remained complete donor until she relapsed 2 years later. in conclusion, treatment of gf with a chimerism directed method can salvage a subset of pts with gf. reserving autologous and/or donor backup cells or an alternative donor is advisable in pts at high-risk of gf. the observation of allogeneic recovery after autologous boost is intriguing and of unknown mechanism. at home autologous stem cell transplantation for haematological malignancies: the role of preparative regimens f. fernández-avilés*, m. rovira, c. martínez, a. gaya, c. gallego, a. hernando, s. segura, l. garcía, j. güell, m. valverde, e. carreras, e. montserrat hospital clínic (barcelona, es) background: the aim of this study was to investigate the impact of the most commonly used regimens on the engraftment, toxicity and readmission rate after autologous stem cell transplantation (asct) in patients managed at home. patients and methods: at home asct (since day +1) was offered to all patients with a good performance status, a travelling time to the hospital of less than 60 minutes, and a caregiver available 24 h per day. in all patients the preparative regimen was administered at the hospital. patients with lymphoma were treated with intensified beac or beam (mg/m 2 ) (bcnu 300, etoposide 1600, cytarabine 800, and cyclophosphamide (cy) 6000 or melphalan 140), patients with myeloma received melphalan 200 mg/m 2 and patients with leukaemia total body irradiation (tbi) 12 gy and cy 120 mg/kg. all patients received the same supportive care, including prophylactic i.v. ceftriaxone once daily. indications for re-admission to the hospital were: patient's or caregiver's desire; uncontrolled nausea, vomiting or diarrhea; mucositis requiring total parenteral nutrition or i.v. morphics; persistent fever; hemodynamic instability, pneumonia, or cardiac and/or respiratory distress. results: seventy-five patients were included in this study. forty-five received beac (n=10) or beam (n=35) (n=45, group a), 19 melphalan (group b) and 11 tbi-cy (group c). recovery (days) of granulocyte count above 0.5x109/l (group a: 11 (9-22), b: 12 (10-26) and c: 12 (10-22)) was significantly faster for patients from group a (a vs. b, p=0.02; a vs. c, p=0.05). fever occurred in 87%, 47% and 73% of patients in the groups a, b and c, respectively (a vs. b, p=0.003). the median (range) days with fever were 2 (1-11), 1 (1-4) and 1 (1) (2) (3) (4) (5) for a, b and c groups, respectively (a vs. b, p=0.01; a vs. c, p=0.05 and who mucositis grade upper to 2 was observed in 40%, 5% and 27% (a vs. b, p=0.006). in group a, 9 (20%) patients needed re-admission by pneumonia (n=4) and persistent fever (n=5), as compared to only 1 (3.3%) because of persistent fever in the rest of patients (p=0.04). conclusions: despite a faster granulocyte recovery, patients managed at home after beac-beam presented a higher incidence and duration of febrile neutropenia, severe mucositis, and high rate of hospital re-admission, especially when compared with patients receiving melphalan. based on this information we have established a more frequent and strict follow-up of patients having received beac-beam and managed at home. outcome of allogeneic stem cell transplantation in first remission for philadelphia chromosome-positive acute lymphoblastic leukaemia following three schedules of imatinib-based chemotherapy b. wassmann, n. goekbuget, h. pfeifer, d.w. beelen, j. dengler, n. kröger, m. stelljes, k. kolbe, w. bethge, m. bornhäuser, h.-j. kolb, m. lübbert, m. stadler, h. serve, d. hoelzer, o.g background: allogeneic sct in cr1 is considered the best curative treatment option in philadelphia chromosome-positive acute lymphoblastic leukaemia (ph+all) pts., but relapse and transplant related mortality (trm) remain significant limitations. although imatinib (im) is considered standard element of front-line therapy, the impact of different treatment schedules is not known. objective: to evaluate overall survival (os), trm and relapse risk (rr) in ph+all pts. following allogeneic sct in cr1 (n=108) after three different im-based chemotherapy regimens. patients: in a prospective, multicenter gmall study, three successive pt. cohorts received im according to one of three schedules: in cohort a (n=43) im was administered alternating with chemotherapy beginning immediately after induction phase ii (ipii)(med. age 44(23-62)yrs.). in cohort b (n=72)im was started after completion of induction phase i (ipi) and given concomitantly with chemotherapy throughout ipii, 1st consolidation (cons.i) and up to yrs.). pts. in cohort c (n=41) received front-line im starting after a 5 day prephase and continued parallel with ip i+ii, cons.i up to yrs.). results: the proportion of pts. transplanted in cr1 was 33(77%), 49(68%) and 26 (64%) in cohorts a, b and c, respectively. median follow-up since allogeneic sct was 11 (0.8-68) , 24 (0.3-51)and 9 (1-22) mo.. median os was 11 mo., 37 mo. and not reached. estimated os at 22 and 48 mo. was 39%, 57% and 71% and 33% and 47%, respectively. trm (35/108; 32%) was mainly due to infections (n=12; 34%) or gvhd (n=11; 31%). 29/35 (82%) trm deaths occurred in pts. aged >35 yrs.. trm at 3 and 12 mo. in pts. aged ≤ 35 yrs.(n=32) was 7% and 17% compared with 21% and 36% in pts. aged >35 yrs.(n=76)(p=0.05). rr was significantly lower in pts. with low or undetectable pretransplant bcr-abl levels (33% at 66 mo. vs. 100% at 18 mo., p=0.0002). conclusion: all tested schedules enable allogeneic sct in a high proportion of pts.. there was a trend towards superior os in cohort c with no significant difference in trm in the 3 cohorts. age and pretransplant mrd levels had the greatest impact on treatment outcome: trm was significantly higher in pts. >35 yrs. both low and negative pretransplant bcr-abl levels were predictive of a low probability of relapse, whereas high levels were associated with a 60% relapse incidence within the 1st year. the impact of posttransplant im on relapse risk remains to be determined. impact of flt3-itd on the outcome of hla-identical stem cell transplant for adult aml with normal cytogenetics: substantial probability of leukaemia-free survival despite increased relapse risk. a retrospective analysis of the ebmt-alwp s. brunet *, m. labopin, a. gratwohl, a. buzyn, j. harousseau, j. jouet, g. socié, a. rambaldi, m. mothy, g. cook, j. sierra, v the prognosis of patients with acute myeloid leukemia (aml) and flt3 internal tandem duplication (flt3-itd) is poor. it is unclear whether this mutation has impact on the outcome of allogeneic stem cell transplantation (allo-sct) for aml in patients with normal cytogenetics (nc). different cooperative groups have obtained controversial results. for this reason, registry data including large numbers of patients are of interest. we analysed the predictive value of flt3-itd on relapse incidence (ri), non-relapse mortality (nrm) and leukemia-free survival (lfs) in patients with aml in 1st cr with nc who underwent a myeloablative allo-sct from hla-identical siblings and were reported to the ebmt. the series included 131 patients, 89 negative itd/flt3 and 42 (32%) flt3/itd positive reported between 2001 and 2007 with a median follow up time of 16 and 10 months, respectively. no significant differences were observed between the two groups regarding gender, median age (38 vs 43 yrs), fab classification, number of induction courses to achieve cr, interval diagnosis to transplant, conditioning regimen, in vitro t-cell depletion, female donor to male recipient and cmv serostatus. in contrast, patients with flt3-itd+ had higher leukocyte counts (wbc) at diagnosis (16 vs 67.9 x10 9 /l, p=0.001) and more frequently peripheral blood was the source of stem cells (43% vs 67%, respectively). univariate (table1) and multivariate analyses demonstrated the adverse impact of flt3-itd for lfs (hr 0.29, p=0.01) and relapse incidence (hr=3.17, p=0.008). it is remarkable, however, 59% of flt3/itd positive patients transplanted for aml in first cr remain alive and disease-free at 2 years. other independent prognostic factors for lfs and relapse were: more than one induction course to achieve first cr. for relapse, higher wbc (hr=2.9, p=0.05) was also a significant prognostic factor. in summary, this analysis shows an adverse impact of flt3-itd on transplantation outcome. despite this finding 59% of flt3/itd positive aml patients in first cr and normal karyotipe remain disease-free at 2 years, a proportion that seems higher than with other treatment options. it is becoming clear that a higher lymphocyte count one month after allogeneic stem cell transplantation (sct) is associated with better transplant outcome in patients transplanted from an hla-identical sibling. however, a predictive role of the day 30 post-transplant absolute lymphocyte count (lc30) in unrelated transplants is not defined. we studied the relationship between lymphocyte counts and other engraftment parameters on outcome in 102 patients with myeloid leukemia (54 acute myeloid leukemia, 38 chronic myeloid leukemia, and 10 myelodysplastic syndrome) receiving myeloablative sct from an hla-a, -b and -dr matched unrelated donor at karolinska university hospital, stockholm from 1996-2006. median recipient age was 37 years (range 0.5-58), 22 patients (22%) were under 18 years. conditioning consisted of cyclophosphamide with busulphan (n=61) or total body irradiation (n=41). bone marrow (bm) was given to 44 patients and mobilized unmanipulated peripheral blood stem cells (pbsc) to 58. median stem cell dose was 6.8 x 10 6 /kg (0. 2-56.4 ). sixty-three patients (62%) received g-csf post-graft. immunosuppression used post graft was cyclosporine with four doses of methotrexate in 97 patients and other treatments in 5. overall survival at 5 years was 61% and relapse-free survival was 56%. the incidence of agvhd grades ii-iv was 62% in patients with an lc30 of <0.2x10 9 /l, 33% if the lc30 was 0.2-1.0 x10 9 /l and 25% in patients with an lc30 >1.0 x10 9 /l (p=0.008). transplant related mortality (trm) was 34% in patients with an lc30 <0.2x10 9 versus 19% (lc30 of 0.2-1.0 x10 9 ) and 0% (lc30 >1.0 x10 9 )(p<0.001). survival was significantly higher in 17 patients with an lc30 >1.0x10 9 /l, compared to 67 patients with an lc30 0.2-1.0 x10 9 /l, and 18 patients with <0.2x10 9 /l (91% vs. 60%, vs. 36% p=0.02 and 0.001 respectively). when analyzed as a continuous variable in multivariate analysis, a higher lc30 was associated with a lower incidence of acute gvhd grades ii-iv, improved survival, less relapse and higher relapse-free survival (see figure) . these results indicate that the lc30 is a robust prognostic factor for transplant outcome in matched unrelated as well as matched related sct for myeloid malignancies receiving either bm or pbsc with or without irradiation conditioning. further research to identify the transplant conditions leading to prompt lymphocyte recovery might lead to global improvements in sct outcome in unrelated sct. s12 o141 gitmo survey on the outcome of 2333 acute myeloid leukemia patients receiving autologous stem cell transplantation in the old and recent era: final results of the multivariate analysis a. olivieri*, b. bruno, g. meloni, m. falda, a. rambaldi, w. arcese, e. alessandrino, r. scime', r. lemoli, m. cimminiello, a. bacigalupo, a. bosi on behalf of gruppo italiano trapianto midollo osseo (gitmo) gitmo registry data files from 2333 acute myeloid leukemia (aml) adults, autotransplanted from 1985 to 2004, have been evaluated to assess the outcome according to age, conditioning and stem cell source; 2032 patients received one autologous stem cell transplantation (asct); 94 two or more asct; 207 allogeneic transplant after failure of asct. patients were categorized in 2 cohorts basing on the transplant era (before or after 1998); the two cohorts were significantly different as regard: age distribution (33% pts older >55 yrs transplanted after 1998 vs 10% pts >55 yrs before 1998); less pts <30 yrs received asct after 1998 (13% versus 28%); preparative regimens: tbi or bu-cy were less common in the recent era; status of disease: 92% received asct in 1rst or 2nd cr before 1998, compared to 90% after 1998; stem cell source: more pts (75%) received pbsc after 1998 versus 16% before 1998. with a minimum 8 yrs followup, os did not differ in the two cohorts: 37% in the 1111 patients autotransplanted after 1998 vs 36% in the 1220 autotransplanted before 1998. as a preliminary analysis suggested a possible role of pbsc in reducing the overall nrm after 1998 (from 14% to 11% after 1998), we made a multivariate analysis to evaluate the main variables (age, stem cell source, transplant era, disease status at transplant and type of conditioning) influencing the outcome, in terms of os, nrm, dfs and relapse incidence. age>55 yrs significantly worsened nrm (1,76 hr), os (1, 76) and dfs (1,5 hr) regardless the transplant era; asct performed in advanced disease (>2nd cr) was associated with a 2,9 times increased risk of relapse and nrm. stem cell source did not significantly influence os and nrm, but bm source, instead of pbsc, was associated with significantly reduced risk (0,79 hr; p=0.012) of relapse. finally tbi regimens were associated with increased nrm compared to bu-cy (1,6 hr). some conclusions can be drawn from this survey: 1-the advanced age (>55 yrs) remains an adverse factor both for nrm, os and for lfs; 2-these data definitely show that pbsc are associated with increased risk of relapse after asct, without major impact on os; 3-regimens including tbi are not recommended being associated with an increased nrm without a significant reduction of relapse. the main efforts in the future should be aimed to reduce the relapse incidence probably by designing new conditioning regimens (and by targeting the minimal residual disease post-transplant) and by a cautious use of pbsc in patients receiving a short consolidation. up-front allogeneic stem cell transplantation as part of induction therapy in newly-diagnosed high-risk acute myeloid leukaemia -an update of a prospective phase ii trial u. platzbecker*, m. füssel, m. schaich, t. illmer, b. mohr, j. schetelig, a. kiani, c. theuser, c. thiede, g. ehninger, m. bornhäuser university hospital (dresden, de) poor-risk cytogenetic aberrations, bad response to the first cycle of induction chemotherapy (ic) or the presence of an flt-3 receptor mutation define high-risk aml (hr-aml) and result in an increased risk of failure of long-term disease control. as a matter of fact, only a minority of this patient group proceed to hsct due to treatment failure or death from infectious complications during ic. therefore, there is substantial need to improve the outcome of these patients with hr-aml. we report results of an ongoing prospective trial evaluating an "early" hsct applied during induction-chemotherapy induced aplasia in forty (n=40) newly-diagnosed hr-aml patients with a median age of 50 years (17-68). a median of 12 days (range 6-34) after the first (n=18) or second (n=22) cycle of ic patients received a reduced-intensity regimen that was based on fludarabine combined with either busulfan (n=4) or melphalan (n=36) followed by allogeneic g-csf mobilized peripheral blood stem cells (pbsc) from related (n=12) or unrelated (n=28) donors. twenty-six patients were not in complete remission before conditioning therapy was started with a median marrow blast count of 20 % (range 6-85). patients with unrelated grafts received antithymocyte globulin and gvhd prophylaxis was performed with cyclosporine a only in all patients. all patients engrafted and went into remission. acute gvhd grade ii-iv occurred in 35 % and extensive chronic gvhd in 30% of patients. with a median follow-up of 17 months (range 1-91) the probability of overall and disease-free survival at 24 months is 68%. early allogeneic hsct as part of primary induction therapy seems to be an effective strategy in high-risk aml patients. comparison of up-front versus minimal residual disease triggered imatinib after stem cell transplantation for philadelphia chromosome-positive acute lymphoblastic leukaemia: interim results of a randomised phase iii study b. wassmann*, h. pfeifer, w. bethge, m. bornhäuser, j. dengler, m. stadler, d. beelen, n. basara, r. schwerdtfeger, k. schäfer-eckart, l. uharek, h. serve, d. hoelzer, o.g. ottmann on behalf of the gmall study group background: detection of minimal residual disease (mrd) following allogeneic sct for philadelphia chromosomepositive acute lymphoblastic leukemia (ph+all) is highly predictive of evolving relapse. we have previously shown that only about 50% of pts. with ph+all who convert to mrd positivity after allogeneic sct achieve renewed pcr negativity in response to interventional imatinib (im), started a median of 4 mo. after sct. failure to rapidly achieve a complete molecular response was almost invariably associated with hematologic relapse. we hypothesized that earlier initiation of im in the setting of a lower leukemic cell burden would increase response rates and improve dfs. objective: to determine whether the earliest possible initiation of im after sct is superior to delayed im triggered by reappearance of bcr-abl transcripts with respect to feasibility, tolerability and duration of molecular and hematologic remission. patients: six wks. after allogeneic sct pts. were randomly assigned to receive im either up-front (cohort 1)(n=17) or subsequent to detection of bcr-abl positivity as determined by real time quantitative and/or nested rtpcr (cohort 2)(n=17). target dose of im was 600 mg, but a lower dose of 400 mg was permitted if deemed necessary because of tolerability. im was scheduled for a total duration of one year of pcr negativity. results: to date, 34 patients have been enrolled. of the 17 pts. randomized to each cohort, 15 each underwent sct in first complete remission (cr1), 2 each in cr2. im was started in 13/17 pts. in the up-front im and 8/17 in the mrd-triggered cohort, with most pts. receiving 400 mg im (10/13 pts. and 5/8 pts., respectively). med. time from sct to start of im was 45 (cohort a) and 82 days (cohort b). after a med. follow-up of 260 (29-985) and 281 (31-1016) days in cohorts a and b, respectively, none of the 30 pts. transplanted in cr1 and 1 of 4 with sct in cr2 relapsed. three pts. (cohort a) died in cr, only 1 one of whom had actually received im. im was discontinued prematurely in 5/13 pts. in the im up-front arm and 3/8 in the mrd-triggered im cohort, due mostly to gastrointestinal toxicity and gvhd. conclusions: with rigorous monitoring of mrd, both schedules of post-sct im (up-front versus mrd-triggered) are associated with a remarkably low relapse rate, although follow-up is still short. tolerability of im is poorer than generally experienced in non-transplanted patients. the routine use of im after sct is a promising strategy to improve outcome of pts. with ph+all. we compared reduced intensity conditioning (ric, n=488) with myeloablative conditioning (mac, n=1596) in patients with aml undergoing hematopoietic stem cell transplantation (hsct), using hla-a, -b and -drb1 identical unrelated donors, transplanted between 1999 to 2005 and reported to the ebmt. in the ric group, median age was higher, transplant was performed more recently, time from diagnosis to transplant was longer, t-cell depletion was less commonly used, and more patients received pbsc vs. bone marrow (p<0.0001). in patients below 50 years of age in cr1, transplant-related mortality (trm), relapse and leukemia-free survival (lfs) were similar in the two groups. in patients in cr2-3 below 50 years of age, 2-year probability of relapse was 31% in the mac group, compared to 51% in the ric patients (p=0.006). in these patients, trm was 36% vs. 26%, and lfs was 44% vs. 36% at two years in the two groups, respectively (ns). in patients with aml advanced disease below 50 years of age, trm, relapse and lfs did not differ significantly between ric and mac patients. at two years, lfs was 22% with mac vs. 12% using ric (p=0.19). in patients above 50 years of age in cr1 and advanced disease, relapse was not statistically different in the two groups. in cr2-3, mac patients above 50 years had a probability of relapse of 18% vs. 47% for ric patients (p=0.009). there were no statistically significant differences in trm or lfs, using mac or ric in patients above 50 years. to conclude, using ric as an alternative to mac in patients with aml undergoing transplants with matched unrelated donors resulted in: no significant difference in trm in patients above and below 50 years of age, significantly increased risk of relapse in patients treated with ric in cr2-3. lfs was similar using the two conditioning regimens. higher incidence of relapse with peripheral blood as source of stem cells in adult patients with acute myelocytic leukaemia autografted in first remission n.c. gorin (1) in the past 30 years the modalities of autologous stem cell transplantation (asct) in first remission (cr1) for patients with acute myelocytic leukaemia (aml) have evolved: the age limit for asct has been gradually extended up to 70 years . total body irradiation (tbi) pretransplant has declined in favour of chemotherapy (ct). leukapheresis products (pb) have replaced bone marrow (bm) as source of stem cells. we were interested in evaluating the potential impact of these modifications on the outcome post asct. a total of 7648 asct (2947 bm, 4701 pb) were reported from january 1985 to december 2006. 79% of all pb transplants were done after 1994. therefore, we compared bm versus pb in patients patients transplanted after 1994: 1226 patients received bm and 4605 pb. the median follow up in the two groups were 46 and 16 (1-161) months respectively. patients receiving pb were older (48y vs 42,p<0.0001), had more aml of the m5,6,7 categories (p<0.0001) and received less tbi (15% vs 30%, p<0.0001). by multivariate analyses, age was a significant factor with lower trm, higher relapse incidence (ri) and lower lfs above 45 years. failure to reach cr within 40 days was associated with a higher (ri) and a lower lfs. the use of pb as compared to bm significantly resulted in a higher ri (51 ± 1% vs 43±2%, p=0.003), and a lower lfs (44±1% vs 50±2%,p=0.04) with only a trend for a slight reduction in trm (9±1% vs 12±1%, p=0.1 ). tbi was not a significant factor for outcome. we finally focused on good risk patients (cr within the first 40 days): the population consisted of 844 patients grafted with pb and 326 with bm. patients receiving pb were older (p<0.0001), and received less tbi ( p<0.0001). again in this good risk population the ri was significantly higher with pb (48 ± 2% vs 35±3%, p<0.01), with a trend for a worse lfs (47±2% vs 57±3%,p=0.1). we conclude that the shift to pb as a source of stem cells in the past 15 years has resulted in increasing the relapse incidence possibly through mobilisation of leukemic cells and /or insufficient purging of the autograft. s14 experimental stem cell transplantation/ stem cell research o146 co-transplantation of placental derived mesenchymal stromal cells produces superior engraftment of umbilical cord blood compared to double unit umbilical cord blood transplantation s. hiwase, p. dyson, s. young, b. to, i. lewis* imvs (adelaide, au) double-unit umbilical cord blood transplantation (ucbt) has been shown to overcome some of the limitations of ucbt, particularly in adult recipients. whether this simply reflects a cell dose effect has not been established. co-transplantation of mesenchymal stromal cells (mscs) has also been suggested as a means of enhancing engraftment and may be appropriate in patients where two suitably matched cords cannot be identified. in this study we have directly compared engraftment rates of double-unit ucbt with msc cotransplantation. mscs were obtained from placental tissue by enzymatic digestion and isolated by plastic adherence. placental mscs demonstrated fibroblastic morphology, immunophenotype and differentiation potential similar to bone marrow derived mscs. in a nod/scid mouse model 4 groups of mice were compared: group 1 received 5 x 10 4 cd34+ cells from a single cord unit (u1); group 2 received 5 x 10 4 cd34+ cells from u1 + 4 x 10 4 mscs; group 3 received 2.5 x 10 4 cd34+ cells from u1 + 2.5 x 10 4 cd34+ cells from u2; group 4 received 2.5 x 10 4 cd34+ cells from u1 + 2.5 x 10 4 cd34+ cells from u2 + 4 x 10 4 mscs. in 4 independent experiments mean engraftment rates were: group 1 28%, group 2 47%, group 3 24%, and group 4 44%. hence msc co-transplantation produced superior engraftment when compared with either single unit (p=0.05) or double unit transplantation (p=0.04). combining results demonstrated the superiority of msc co-transplantation with mice receiving mscs showing mean engraftment of 45% compared to mice who did not receive mscs having engraftment of 25% (p=0.005). transplantation of double ucbt did not improve engraftment when compared with a single ucbt of equivalent dose. additionally, the quality of engraftment was enhanced with msc co-transplantation producing superior engraftment of cd34+ cells. in conclusion, at equivalent cell dose single and double ucbt lead to similar engraftment, suggesting the enhanced engraftment seen with double ucbt reflects a cell dose effect. msc co-transplantation enhances engraftment of both single and double unit cords and may be a potential strategy to be explored in the clinic. infusion of allogeneic mesenchymal stromal cells can delay but not prevent gvhd after murine transplantation m. kambouris *, b. turner, l. sinfield, h. cullup, d. hart, k. atkinson, a. rice mater medical research institute (south brisbane, au) multipotent, mesenchymal stromal cells (msc) are emerging as a means of immunosuppression for patients with steroid refractory graft-versus-host disease (gvhd). despite clinical use, pre-clinical data is still lacking. we established an in vivo model using msc to control gvhd to determine their mode of immunosuppression. we showed in a mixed lymphocyte reaction that msc are highly immunosuppressive and significantly reduce t cell proliferation. we then examined the effects of donor-derived intraperitoneally (ip) or intravenously (iv) injected msc on gvhd in a myeloablative conditioned, full mismatched model of haematopoietic stem cell transplantation (hsct) [ubi-gfp/bl6(h-2b)->balb/c (h-2d)]. 4x10 5 donor-derived msc/mouse were injected 4hrs pre or 24hrs post hsct then mice were monitored daily for gvhd. only mice given msc ip 24hrs post hsct showed a signficant delay in death from gvhd, where median survival was increased by 10 days (day 7 vs day 17, p<0.001, (fig 1) . we then investigated if msc delivered ip pre or post hsct altered cytokine-driven trafficking of cellular effectors known to play a role in gvhd via timed sacrifice. control mice (hsct and no msc or msc and no hsct) were also sacrificed daily for 6 days following hsct. we found that msc given post-hsct enter an environment of significantly increased activated dendritic cells (dc) in the spleen ( fig 2) and at day 3 and day 6 post hsct, mice given msc pre hsct had increased levels of activated splenic dc compared to mice treated with msc 24hrs later. at day 3, we also saw more ifn-gamma in spleen washings in mice treated pre hsct compared to controls. we then determined the role of msc in gvhd control in a minor mismatch model [ubi-gfp/bl6 (h-2b)->balb.b (h-2b)]. msc were given on day 1, 7 or 14; or to mice with established gvhd (>25% weight loss with other surrogate gvhd markers more than 14 days post hsct). mice given msc therapy after gvhd onset showed no increase in survival compared to controls. however, msc administered prophylactically at day 1 (4x10 5 /mouse) or day 7 (1x10 6 /mouse) but not at day 14, showed significantly increased survival compared to controls suggesting that the timing of msc administration is important for their impact on gvhd. in summary, ip injection of msc influences gvhd and survival and may have in vivo influence on activated dc. elucidation of the mechanism by which msc control gvhd may ultimately lead to wider application of their use to assist hsct. s15 o148 immune reconstitution after cord blood transplantation by direct intrabone injection of cells a.m. raiola*, a. ibatici, v. pinto, a. kunkl, f. guialn, f. gualandi, d. occhini, a. dominietto, c. di grazia, s. bregante, t. lamparelli, m. mikulska , g. piaggio, m. podestà, f. frassoni, m. van lint, a. bacigalupo s. martino's hospital (genoa, it) introduction: cord blood transplantation (cbt) has been increasingly used to treat hematological malignancies. since march 2006 we have been investigating a pilot study of cbt in adults by injecting cb cells directly into the bone to overcome the cell dose barrier. pre-clinical transplant models have shown that intrabone (ibm) injection reduces the incidence of gvhd in mhc disparate donor/recipient pairs, suggesting that the way of transplant may affect the immune reconstitution. we have analysed immune reconstitution (t/b/nk cells) at different time-point after ibm-cbt in adults recipients. materials & methods: thirty-three consecutive patients with advanced haematological malignancies a single-unit graft cbt. median age was 35 years (18-62) and follow-up was 8 months (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) for 27 patients survivors longer than 30 days after cbt. median cell dose was 2.6 tnc/kg (range, 1.5-4). donor/recipient hla matching was highly disparate: 5/6 in 27%, 4/6 in 66%, 3/6 in 7%. all but 5 pts received a myeloablative conditioning regimen (tbi 1200cgy in 26/28 pts). gvhd prophylaxis consisted of csa, mmf and pretransplant atg. immunophenotype on peripheral-blood lymphocyte subsets was assessed on day +30, +60 +100, 6 months and 1 yr after cbt. results: early death occurred in 7 pts. all 26 were evaluable and engrafted 100% donor. overall survival was 50% and relapse-related death was 15% (5 pts). median time to neutrophil and platelet recovery was 23 and 40 days, respectively. grade ii acute gvhd was seen in 7 cases (27%) and grade iii in 1 case (4%). moderate/extensive chronic gvhd developed in 6/26 (23%). on day +30, median cd4+ and cd8+ count were as low as 11 and 7 cells/mmc, respectively and remained below normal values up to 6 months. at 1 year, cd4+ and cd8+ count normalized (397 and 250 cells/mmc). nk values reached values as high as 381 and 465 cells/mmc on day +30 and +60, then remained stable up to 1 yr. b cells were extremely low up to day+60, peaked to 445 on day +100 and slighty expanded over time. conclusion: data from our series showed that immune reconstitution is as delayed as in patient who received cb grafts intravenously, with prolonged t-cell lymphopenia and bcells spike since day+100 (komanduri, blood 2007). we also confirmed early nk expansion which could be due to the high donor/recipient pair hla disparity associated with the low cd4+ count. if this may have an impact on the relatively low relapse incidence observed in advanced patients warrants further studies. the effect of mesenchymal stem cells in a rat model of experimental arthritis is dose-and time-dependent e. yannaki (1) , a. papadopoulou* (1), m. yiangou (2) , e. athanasiou (1) , i. batsis (1) , a. athanasiadou (1) , a. xagorari (1) , a. anagnostopoulos (1) , a. fassas (1) (1)george papanicolaou hospital (thessaloniki, gr); (2)aristotle university (thessaloniki, gr) mesenchymal stem cells (mscs) have recently generated a great deal of excitement, due to their potential of differentiation into a broad spectrum of tissues and immune regulation by non-mhc-restriction. the latter capacity has resulted in an effective control of gvhd in several studies; however, the role of mscs in autoimmune diseases has not been extensively investigated in animal models. we aimed to explore the effect of rat mscs on cultured fibrobiast-like synoviocytes (fls) and t-cells from the spleen after adjuvant arthritis induction (aa) as well as their in vivo immunomodulatory potential in a rat model of aa resembling human rheumatoid arthritis. culture of aa-fls in the presence of supernatant from syngeneic msc culture when compared to fls in the absence of supernatant, reduced the aa-fls proliferation (p=0.00004) as well as the proliferation of cona-stimulated aa t-cells (p=0.04). coculture of activated t-cells with syngeneic mscs produced stronger inhibition (p=0.004) in a dose-dependent manner. the inhibitory effect of allogeneic mscs in activated aa t-cells was even stronger and similarly dose-dependent, either by secreted agents (p=0.017) or by cell to cell contact (p=0.00017). low doses of mscs (0.5-5x10 5 cell/recipient) administered iv, intrasplenic or intrabone marrow, at single or multiple infusions, didn't produce significant differences in disease scores of msctreated as compared to control rats. on the other hand, repeated, higher dose (6x10 6 cell/rat), iv infusions of syngeneic or allogeneic y+mscs to female recipients, before the onset of aa (d4 and d9 post aa), resulted in significantly lower arthritic scores with maintenance of a rather normal joint architecture with mild focal synovial hyperplasia/pannus formation and reduced abnormal chondropiasia or bone erosion as compared to control rats. in contrast, symptom worsening occurred when the same cell dose was injected after arthritis onset (d13 and d20 post aa). at the time of sacrifice (d30 post aa), no y+mscs were detected in the spleen or in cultured fls from synovial membrane, by pcr or fish, suggesting that the observed benefit was not due to mscs homing to the target tissues or that migration of mscs may have happened earlier. our data suggest mscs as a potential new therapeutic approach for autoimmune arthritis. however, the benefit of infused mscs seems to be dose-and time-dependent and further studies are required before the results can be clinically translated. objectives: mesenchymal stem cells (msc) suppress alloantigen-induced proliferation, interferon-gamma (ifngamma) production and cytolytic killing in vitro and infusion of third-party msc appears a promising therapy for acute gvhd. however, little is known about the specificity of immunosuppression by msc and in particular the effect on cell-mediated immunity to infectious pathogens. we have studied the effect of msc on virus-specific t-cell responses. results: peripheral blood mononuclear cells (pbmc) from 6 normal donors were stimulated for 5 days with autologous lymphoblastoid cell lines (lcl) (ebv), pp65 peptides (cmv), an adenoviral vector ad5f35 (ad) or allogeneic pbmc (allo), in the presence or absence of third-party msc (msc/pbmc ratio 1:10). msc significantly suppressed proliferation in response to allo (mean 61% suppression, p=0.003), but had less effect on the response to ebv (mean 42% suppression, p=0.016) and no suppression of the response to cmv or ad. msc had no effect on expansion of ebv and cmv pentamerspecific t cells after stimulation with their cognate antigen. elispot assays demonstrated that msc significantly inhibited ifn-gamma production in response to allo (mean sfc/10 5 cells 1125 ±274 without msc, 263±49 with msc) and to a smaller extent to ebv (3181±548 vs 2147±387), but not to cmv (2535±374 vs 2532±311). established ebvspecific cytotoxic t-cells (ebv-ctl) from 5 donors were stimulated with autologous lcl with or without msc for 5 days. the percentage of ebv-pentamer specific ctl was not affected by the presence of msc (mean 1.59% vs 1.52%). msc did not inhibit ifn-gamma production by ebv-ctl (6039±1644 vs 5885±1608). furthermore, cytolytic killing of lcl by ebv-ctl was not suppressed by msc (mean specific lysis 47±3.6% vs 52±4.7% at e:t ratio 30:1). finally, we studied anti-cmv immunity in 2 patiens who received msc for acute gvhd of the gut evolving into chronic gvhd, with a good transient response. pbmc from these patients showed persistence of pp65-pentamer positive t-cells and retained ifn-gamma response to cmv post msc infusion (sfc 69/10 5 pbmc pre-msc, 297 at 1 month and 231 at 3 months). conclusion: msc have little effect on t-cell responses to cmv, ebv and ad, which contrasts to their strong immunosuppressive effects on alloreactive t cells. these data have major implications for immunotherapy of gvhd with msc and suggest that the effector functions of virus-specific t-cells may be retained after msc infusion. intracerebral transplantation of human mesenchymal stem cells after hypoxic-ischaemic brain damage in rat h. huang* (1), j. fan (1), x. lai (1) , q. yu (2) , w. ding (1) , y. wang (2) (1)zhejiang university school of medicine (hangzhou, cn); (2)zhejiang chinese medical university (hangzhou, cn) mesenchymals stem cells (mscs) have dramatic potential of proliferation and multiple differentiation, and recently, neural transdifferentiation of mscs provoke extensive interest. our study was designed to explore the migration, differentiation and the therapeutic benefit of hmscs after hypoxic-ischemic brain damage in rats. hmscs were prelabeled with bromodeoxyuridine (brdu) for 72h before transplantation. animal models of hypoxic-ischemic brain damage (hibd) were built in one month old wistar rats, and three days after hypoxia-ischemia, hibd rats in hmscs-treated group (n=18) received intracerebral transplantation of 5±10 5 hmscs, while hibd rats received pbs of the same volume in control group (n=18). in sham-operated group (n=6) and hibd group (n=6) which just had hypoxic-ischemic brain damage but did not receive any treatment. all of the groups did not receive any immunosuppression agents. behavior tests (alternative electro-stimulus y-maze, by single blind method) indicated that pace memory capacity of rats in hmscs-treated group is significantly better at 4 weeks post-transplantation. he straining also showed that the damage caused by hypoxicischemic had lager pathological changes with bulks of neural cells necrosis and neuropil cavitations formation, even disappearance of normal architecture in cortex and hippocampus, however, it would be significantly improved in pathology in hmscs-treated group. hmscs stayed around the local area of transplantation in three days after transplantation, then migrated mainly along the ventricular system. four weeks later, hmscs were observed to migrate to the parenchyma and distribute throughout the cerebra. three days after transplantation, some hmscs expressed gfap in the local area of transplantation, and a few of cells expressed neurofilament (nf) near blood vessel. four weeks after transplantation, brdu and gfap co-express cells increased which mainly distributed in the local area of transplantation, the cortex, hippocampus and ependymal layer. nestin positive cells did not be detected at any time point. conclusion: after being transplanted intracerebrally, hmscs could migrate to the lesion area, express nf and gfap which indicated their differentiation into mature neurons and neurogliocytes, and promote tissue repair and functional recovery of hibd. furthermore, these results suggested that hmscs could be a promising treatment for hypoxic-ischemic brain damage. identification and enrichment of human bone marrow derived mesenchymal stem cells by monoclonal antibody, zuc3 x. lai, h. huang*, l. huang, j. cao, f. zeng, j. zheng zhejiang uninversity school of medicine (hangzhou, cn) human mesenchymal stem cells (mscs) could be well isolated and expanded from bone marrow and have been widely studied, however, there is no specifically definitive marker of mscs till now. in our previous study, a novel murine monoclonal antibody (mcab) zuc3 was produced by hybridoma technology, which was specifically reactive with human mscs, while showed negative cross-reactivity when screened against a variety of human tissues. flow cytometric analysis showed that zuc3 antigen expression by cultured mscs and mononuclear cells derived from bone marrow were 91.31±2.92% and 0.96±0.28% respectively, and western blotting demonstrated the molecular mass of antigen was about 33kd. zuc3 antigen positive and negative cells were separated from bone marrow mononuclear cells by immunomagnetic activated cell sorting, and plated respectively in human mscs medium consisting of 10% fbs, lg-dmem. the purity of the recovered fractions for zuc3 by macs was 76.82±6.32% by flow cytometry. the positive cells have adhered to culture flask in vitro, and the quantity of adhered cells that had fibroblast-like morphology increased and proliferated during primary expansion period, while the negative cells were observed as round shape cells without any proliferation. it was demonstrated that zuc3 antigen positive cells continued growth with spindle-shape, expansion in long-term culture. phenotype of zuc3 antigen positive cells was analyzed by flow cytometry, the culture-expanded positive cells were uniformly positive for cd29, cd44, cd105, cd106, and lack typical hematopoietic antigens such as cd14, cd34, cd45, hla-dr, which demonstrated that zuc3 positive cells sorted from bone marrow mononuclear cells were mscs. with proper medium, the zucs antigen positive cells could be successfully induced to differentiate into adipocytes, osteoblasts, and neuro-like cells which were positive of neuron markers such as nestin, nse and nf-m. conclusion: zuc3 mcab was a specific surface marker against human mscs for cell sorting. the zuc3 antigen positive cells separated from bone marrow mononuclear cells had potential capacity of high proliferation and multiple differentiation. everolimus enhances the immunomodulatory properties of cd271 positive selected human mesenchymal stem cells r. racila*, w. melchinger, j. finke, r. marks medical hospital university of freiburg (freiburg, de) immunomodulatory properties of human mesenchymal stem cells (msc) suggest their use as a potent cellular therapeutic agent for acute graft versus host disease in allogeneic hematopoietic stem cell transplantation (hct). everolimus is a mtor inhibitor which is currently being tested for gvhd prophylaxis, but its effect on mscs is not yet understood. since mtor inhibition by everolimus might interfere with protein biosynthesis, we tested whether mscs maintain their immunomodulatory properties in the presence of everolimus. in order to obtain a homogenous msc population, positive selection of cd271+ cells from bone marrow aspirates from volunteers using anti-cd271 antibody coated magnetic beads were used. the one-step procedure resulted in an enrichment of cd271highcd73+cd45lowcd34-msc with copurification of cd271lowcd73-cd45high hematopoietic cells. expansion culture resulted in a pure cd271lowcd45lowcd73highcd105highcd34low msc population. we assessed the immunomodulatory properties of cd271lowcd45lowcd73highcd105highcd34low mscs when cocultured with autologues and allogeneic t cells stimulated with anti-cd2/cd3 antibodies or third party peripheral mononuclear cells. the presence of msc in allogeneic t cell cultures did not result in a proliferative t cell response. moreover, stimulation of allogeneic msc/t cell cultures with anti-cd2/cd3 antibodies resulted in suppression of t cell proliferation as determined by thymidine incorporation. a 65-70% reduction of maximal t cell proliferation was seen when a ratio of 1:10 (msc to t cell) was used. in order to examine the effect of mtor inhibition onto the immunomodulatory properties of msc, we incubated the msc for 4 hours with 10 nm everolimus. after several washes the everolimus treated and irradiated msc were used for coculture (ratio 1:10) with stimulated allogeneic t cells. interestingly, everolimus treated mscs showed enhanced suppressive properties and a 85-90% reduction of maximal t cell proliferation. we conclude that everolimus enhances the immunomodulatory properties of cd271 positive selected human mscs and the clinical use of this mtor inhibitor might result in significant immunsupressive activity in allogeneic hct. fibrosis regression after autologous haematopoietic stem cell transplantation in systemic sclerosis l. vija, f. verrecchia, o. verola, a. de raignac, d. sibon, l. michel , d. farge st louis hospital (paris, fr) background: significant regression of clinical skin sclerosis, as assessed by repeated measure of the rodnan modified skin sclore (mrss), has been shown after autologous stem cell transplantation (abmt) in diffuse scleroderma (ssc) patients. objectives: to analyse wether 1) the clinical and histological extent of skin fibrosis can regress after hsct and 2) extra cellular matrix organization and pro-fibrotic signals elicited by tgf-b in ssc fibroblasts derived from skin biopsies vary according to the modified rodnan skin score (mrss) before and after hsct . methods: 38 ssc patients underwent 75 skin biopsies with simultaneous measure of mrss before or after treatment by immunosuppressive drugs with or without autologous hsct. the histological presence and distribution of sclerosis was assessed as : 0 = no fibrosis, + = light fibrosis, ++ = moderate fibrosis, +++ = extensive fibrosis within the papillary (pd), the superficial reticular dermis (srd), the median reticular dermis (mrd) and the deep reticular dermis (drd). human fibroblasts were established by explanting skin punch biopsy (ssc patients or healthy controls) in dulbecco's modified eagle's medium and cells were used for experiments between passages 3 and 8. immunoblotting analyses with anti-phospho smad3 (mayo clinic, rochester, mn), anti-smad3 (clinisciences, ca), and anti-bactin antibodies (santa cruz biotech, ca) and rt pcr for col1a1, col1a2 and pai-1 have been performed. results: double blind optic microscopy analysis of the biopsies seriate sections on standard matrix stains allowed to define 3 histological subgroups : 9 with grade 1 weak fibrosis, 24 with grade 2 moderate fibrosis and 42 with grade 3 severe fibrosis with significant correlation (p< 0.0001) between the grades of fibrosis and the mrss. in skin fibroblast cultures, smad3 phosphorylation levels, representative of tgf-b receptor activity, increased in parallel with the mrss. when compared to pretransplant values, a significant regression of the degree of fibrosis was observed both in the papillary and in the reticular dermis after hsct (n=7), correlated with a decreased mrss. conclusion: in ssc patients, we confirm that the histological extent of skin fibrosis correlates closely with the mrss and further demonstrate that both parameters regress after treatment by hsct. the extent of tgf-b signaling activation in ssc skin fibroblasts appears to parallel the severity of disease. we report here an update of the phase 2 multicenter, prospective study of high-dose immunosuppressive therapy and autologous haematopoietic cell transplantation in 21 nonprimary progressive ms patients showing high disease activity on the basis of both brain magnetic resonance imaging (mri) and sustained clinical deterioration despite conventional therapies. the treatment consisted of stem cell mobilization with cyclophosphamide (4 g/m²) and filgrastim, and conditioning with bcnu (1,3-bis(2-chloroethyl)-1-nitrosourea), cytosine arabinoside, etoposide, and melphalan (beam) followed by antithymocyte globulin (atg, 10 mg/kg) and infusion of unmanipulated peripheral blood stem cells (pbscs). the median follow-up is now 66 months (range 21-105). confirmed progression-free survival was evaluated according to edss disability score as the probability of being alive without clinical progression as compared to baseline. progression-free survival is 58,16% at 8,5 years after hsct (figure 1). seven patients achieved a sustained improvement in their edss score, while 6 patients remained stable and 8 worsened of at least 0,5 points in edss score. disease progression was not associated to clinical relapse. only one patient showed a clinical relapse at +52 months, spontaneously remitted without deterioration of the edss score.since transplantation patients were treated only with symptomatic therapy. hsct was capable to induce sustained progression and treatment-free survival in a large cohort of rapidly deteriorating ms patient with a modest transplantrelated toxicity. experimental animal data and single case reports have documented that allogeneic haematopoetic stem cell transplantation (hsct) potentially alters the course of severe autoimmune disease (aid). experience with allogeneic hsct for these indications is still limited. we therefore undertook a retrospective analysis of the ebmt database to identify patients, which received allogeneic hsct for aid. we identified 31 patients (pts), who underwent 34 allogeneic hsct for aid between 1984 and 2007. follow up was requested by a questionnaire, sent to the referring physicians, asking for diagnosis, transplant procedure, complications, disease status and last follow up. the completed questionnaire was available at the time of abstract submission for 15 pts. median age at transplantation for all pts was 20.07 (0.36-59.3) years; (16 male/ 14 female/ 1 not known). sixteen hsct were performed for non-haematological aid and 18 for haematological aid. three pts had two hsct for aid. eight pts had a previous autologous hsct. mean follow up time was 37.1 months (iqr 6. 2-85.2) . responses were complete for 12 pts (35%), partial for 7 (20%), no change for 6 (17%) and unknown for 9 pts (26%). complete or partial response rate was independent of underlying disease; (haematological vs. non-haematological aid) (p=0.5), gender (p=1.0) or age (p=0.69). cyclophosphamide based conditioning was associated with a better response rate (p=0,066), while atg had no influence on the response. overall mortality was 35%. three patients (9%) died of progression of aid, eight patients (25%) died of treatment related reasons. the median followup time of non-survivors (11 pts) was 5.2 months (range 2. 1-28.4 ) and of survivors (20 pts) 67.8 months (range 28. 2-86.1) . kaplan maier curves displayed similar survival rates for haematological and non-haematological aid. best survival was observed for twin donors (3 pts all survived) and matched donors (related or unrelated). tbi conditioning showed a trend to a higher mortality (p=0.09). in conclusion allogeneic hsct has the potential to induce complete remission in refractory aid. the high mortality rate limits its general use. for selected patients allogeneic hsct based on cyclophosphamide conditioning from a matched donor could be an option and should therefore be prospectively evaluated. 37 patients with intractable form of ms were included in the phase ii clinical trial involving the high dose chemotherapy with autologous peripheral blood progenitor cell (pbpc) support between 1998 and 2006. 33 patients underwent high dose conditioning beam. t cell depletion in vitro was performed on 20 grafts depending on number of harvested progenitors and available resources. 13 patients with not purged graft received atg 4mg/kg i.v. d+1, d+2 after pbpct. in 3 patients pbpc mobilization failed (cyclophosphamide 4g/m 2 + g-csf). one patient refused transplantation after improvement in disability following mobilization. median follow-up is 60 months (12 -108). median edss (expanded disability status scale) of grafted patients at the time of inclusion was 6.5 (5.0-8.5), median edss of grafted patients at last follow up was 7,0 (6.0-10.0). two patients died 31 and 58 months resp. after transplantation because of progression of ms and the cause not related to transplantation, respectively. two patients were lost for follow up. there was no treatment related mortality. at last follow up, the significant improvement (by 1.0 point and more on edss) remains in 1 patient, stabilisation of the disease occured in 23 patients (70%), 9 patients gained disability significantly (by 1.0 point and more on edss). in 3 patients occured transient significant neurological improvement lasting 12-30 months. the development of disability between the group grafted with in vitro purged graft and the group with atg i.v. was not significant. cumulative survival without significant deterioration was 80% in the group with purged grafts and 64,3% in the group without purging in vitro (p=0,178). among 4 mobilized but not transplanted patients 1 improved by 1.5 point on edss, 3 patients worsened by 1.0 point. three serious adverse events related to transplantation were observed: respiratory failure after the onset of mucositis in the patient with severe pontomesncephalic impairment, sepsis with respiratory failure following bilateral pulmonary hemorrhage, both patients needed temporary artificial ventilation and recovered. bleeding related to the inhibitor of fviii occured in 1 patient, remmission has been achieved after the immunosuppressive therapy. as majority of patients with otherwise intractable ms at least stabilized in their disability, we consider the results to be promising and requiring confirmation in a randomized trial involving also less handicapped patients. long-term results of a gitmo retrospective study on haematopoietic stem cell transplantation for paroxysmal nocturnal haemoglobinuria s. santarone*, e. di bartolomeo, a. bacigalupo, e. tagliaferri, a. iori, a. risitano, s. tamiazzo, f. papineschi, a. rambaldi, a. spagnoli, e. angelucci, p. di bartolomeo on behalf of the gitmo allogeneic haematopoietic stem cell transplantation (hsct) may cure paroxysmal nocturnal hemoglobinuria (pnh). in this study we report the results of allogeneic hsct in 26 patients (16 males and 10 females) affected by pnh who were transplanted between july 1988 and may 2007. the median age at time of hsct was 32 years (22-60). the median time from diagnosis to hsct was 33 months (3-208). all patients had received various treatments before hsct including steroids, immunosuppressive drugs and growth factors. twenty-one patients were transfusion-dependent. the median number of packed red blood cells and platelet concentrates received before hsct was 30 (4-500) and 33 (6-86) respectively. the median peripheral hematological counts at transplant were: polymorphonucleates (pmn) 1780 (20-10400) x10 9 /l, hemoglobin 8,7 g/dl (4.6-11), platelets (plt) 79 (6-355) x10 9 /l. thirteen patients developed thromboembolic episodes before hsct. twenty-four patients were transplanted from hla identical sibling and 2 from matched unrelated donors. the donor's median age was 33 years (20-59). the conditioning regimen was myeloablative for 15 patients (busulfan and cyclophosphamide), whereas 11 patients received a reduced intensity conditioning including fludarabine, cyclophosphamide, melphalan and total body irradiation. as graft-versus-host disease (gvhd) prophylaxis, 11 patients received cyclosporine (csa) alone and 13 were given csa and short course methotrexate. two patients received t-cell depleted marrow cells. twenty patients were given bone marrow cells (median nucleated cells 4.0 (2,2-7,5) x10 8 /kg) and 6 received peripheral blood stem cells (median cd34+ cells 3.5 (1.7-8.4) x10 6 /kg). twenty-five patients engrafted with a median time of 17 (10-38) days to reach >0.5 x10 9 /l pmn and 26 days to reach >50 x10 9 /l plt. the probability of developing grade ii-iv acute gvhd and extensive chronic gvhd was 42% and 16% respectively. the transplant related mortality at 6 months was 34%. causes of death were infection in 4 patients, acute gvhd in 1, chronic gvhd in 2, multi-organ failure in 1 and ebv-related disease in 1. as of december 2007, 16 patients are alive with complete hematological recovery and no evidence of pnh at a median follow-up of 109 months (8-212). the 10-year kaplan-meier probability of disease-free survival for the 24 patients transplanted from related donor is 70%. no patient developed thromboembolic disease following hsct. this study confirms that hsct is a curative treatment for the majority of patients with pnh. subcutaneous alemtuzumab is safe and effective for treatment of global or single-lineage immune-mediated marrow failure: a pilot study from the working party aplastic anaemia (wpsaa) a. risitano*, l. marando, c. selleri, b. serio, e. seneca, a. camera, l. catalano, g. scalia, l. del vecchio, a. iori, s. maury, a. bacigalupo, g. sociè, a. tichelli, j. marsh, h. schrezenmeier, j. passweg, b . rotoli on behalf of the wpsaa acquired marrow failure syndromes may globally affect all hematopoietic lineages, as in aplastic anemia (aa), or may selectively involve single lineages, as in pure red cell aplasia (prca) or in agranulocytosis (agr). standard treatment for these conditions includes immunosuppression (is), because of their common cellular immune-mediated pathophysiology. here we report a phase ii/iii pilot study with alemtuzumab (mabcampath®) (ale) followed by low-dose cyclosporine a (csa) as alternative is regimen in 24 patients suffering from marrow failure (13 aa, 7 prca and 4 agr). all patients received subcutaneous ale as escalating dose in consecutive days (3-10-30-30-[30] mg, total dose 103 for aa and 73 for prca and agr), premedicated by betamethasone, clorpheniramine and paracetamol. six patients received one or more additional courses as a result of relapse, so a total of 32 courses were administered. all patients started oral low dose csa (1 mg/kg) on day 7. an intensive anti-infectious prophylaxis has been exploited, which included oral valgancyclovir and cotrimoxazol as anti-cmv and anti-p. carinii agents, respectively. all patients completed the treatment with no relevant injection-related side effect (with exception of fever in some cases), nor significant clinical or laboratory abnormality. a complete lympho-ablation was observed in all patients within 2-3 days, which persisted for several weeks; transient worsening of neutropenia and/or thrombocytopenia were observed in some patients. at a median follow-up of 9 months, infectious events were irrelevant: in cumulative 184 patient-months, 2 hsv and 1 flu have been recorded, all resolving quickly. no cmv reactivation was demonstrated. immune reconstitution was delayed up to several months, especially affecting the cd4+ compartment. patients with adequate follow up (more than 3 months) were assessed for treatment efficacy: 8 aa patients showed 2 cr, 4 pr, 2 nr. in the 5 prcas, there were 4 cr and 1 nr; 3 out of 3 agrs obtained cr. among responding patients, 2/3 saas, 3/4 prcas and 1/3 agr experienced relapses, which were successfully treated by additional course of ale. in conclusion, subcutaneous ale is a feasible and safe is regimen for patients suffering from immune-mediated marrow failure syndromes. preliminary results suggest excellent response rate, and support efficacy even in case of relapse; such favorable risk-to-benefit ratio predicts for this regimen a leading position in the future is strategies. stable mixed chimerism has been described as being beneficial in patients with aplastic anemia after stem cell transplantation, as these patients were protected from severe graft versus host disease and from rejection, the major drivers of mortality in these patients. patients with marrow failure syndrome do not need full donor chimerism as they do not benefit from a graft-versus-malignancy effect. here we describe a protocol resulting in high rate of stable mixed s20 chimerism in patients conditioned with cyclophosphamide + atg and transplanted with peripheral stem cells depleted by campath with add-back of t-lymphocytes post-transplant at different dose levels, according to donor-recipient relationship. we included 10 patients in this protocol, median age was 35,5 (15-58) years, 5 were male, donors were identical siblings in 7, mismatched related in 1 matched unrelated in 1 and mismatched unrelated in 1, respectively. stem cell dose was 11.5 (5.4-40.2 ) cd34 x 10 6 /kg. time to neutrophil engraftment was 15 (8-17) days and to platelet engraftment 14 (8-24) days. median follow-up of surviving patients is 627 days (13-2533) acute grade ii gvhd was observed in 1 patient (with the mismatched unrelated donor), this patient died subsequently, while all other patients remain alive. there is mild chronic gvhd in 1, none of the patients lost the graft. chimerism analysis showed full donor chimerism in 2, transient mixed chimerism (defined as mixed chimerism developing into full donor chimerism) in 1, and stable mixed chimerism in 6, limited to mononuclear cells in 1 and observed in the granulocyte and mononuclear compartment in 5. mixed chimerism remained stable for up to 4 years. a protocol of conditioning with cyclophosphamide and atg and gvhd prophylaxis with t-cell depletion using campath and t-cell addback may induce stable mixed chimerism in a large proportion of patients with aplastic anemia with low gvhd and graft failure risks. i. resnick* (1), a. maschan (2), m. shapira (1), p. trakhman (2), e. skorobogatova (2), d. balashov (2), m. aker (1), r. or (1) (1 introduction: ten years ago we introduced a fludarabine (flu) based conditioning for hematopoietic stem cell (hsc) and umbilical cord blood transplantation [1, 2] for fanconi anemia (fa). this approach dramatically improved results of fa treatment. following initial case reports and small series [3, 4] , two large studies were published: by an italian group and of european experience [5, 6] . aim: here we present an update of our two centers' experience of hsc transplantation for fa. methods: thirty patients (age 3.2 to 31, median 10.3 years) underwent allogeneic hsc for treatment of fa. used conditioning protocols were flu based in 19: flu 150 or 180 mg/m 2 + cyclophosphamide (cy) 20 mg/kg or busulfan (bu) 4 mg/kg or both + antithymocyte globulin (atg) atgam 90 mg/kg or fresenius 40mg/kg. an alternative conditioning (11 patients) was cy-tai/tli or bu8cy40. follow up till december 1, 2007 was 3 to 112 months. results: in the group of patients who got flu based protocols disease free survival (dfs) is 84% vs 27% in pre-fludarabine protocols; p=0.006; or=14.2 (2.5-82.1). difference of dfs in patients transplanted from matched family donor (92%, n=12) vs matched unrelated donor (71%, n=7) was insignificant. causes of death were acute graft-vs-host disease (gvhd) grade ≥2 (n=6) in combination with sepsis (n=3), multiorgan failure (n=4), liver veno-occlusive disease (n=3), bleeding (n=2), chronic extensive gvhd (n=2), secondary malignancy (n=1). there was no clear advantage in using a combination of cy and bu compared with single agent protocols (dfs 3 out of 4 patients). three patients suffered a rejection and underwent a 2nd transplant (2 are alive and well). all surviving patients are disease free and in an excellent clinical condition. conclusion: combination of fludarabine with atg and low dose cy and/or bu is safe, demonstrates low rejection rates and is well tolerated by fa patients. lower doses of flu (150 mg/kg) can be safely used without a pronounced risk of rejection. references: 1.kapelushnik j et al. bone marrow transplant. 1997; 20:1109 . 2.aker m et al. j pediatr hematol oncol. 1999 21:237. 3.bitan m et al. biol blood marrow transplant. 2006; 12:712. 4.maschan aa et al. bone marrow transplant. 2004; 34:305. 5.gluckman e et al. biol blood marrow transplant. 2007; 13:1073 . 6.locatelli f et al. haematologica 2007 92 (10) results: cb recipients were more likely to have advanced leukemia at conditioning (relapse or induction failure, cb vs. bm = 47% vs. 31% in aml patients, and cb vs. bm = 28% vs. 21% in all patients, p<0.0001, p=0.087, respectively). the proportion of all with philadelphia chromosome abnormality was higher (38% to 25 %, p=0.001) among cb recipients. human leucocyte antigen (hla) was serologically mismatched in 93% of patients with aml and 93% of patients with all among cb recipients, while hla a, b, and dr were all matched genotipically for bm recipients. in controlled comparisons using multivariate analyses, among patients with aml, higher rate of treatment-related mortality (trm) (hazard ratio [hr]=1.51, 95% confidence interval [ci], 1.11-2.05, p=0.008) was observed in recipients of cb, which contributed to decreased overall survival (hr=1.45, 95%ci, 1.14-1.84, p=0.003). relapse rate did not differ between the two groups of aml patients (hr=1.27, 95%ci, 0.91-1.79, p=0.16). in patients with all, the relapse rate was higher with marginal significance among cb recipients (hr=1.45, 95%ci, 0.98-2.14, p=0.064). there was no significant difference between these groups for trm (hr=1.36, 95%ci, 0.94-1.95, p=0.10) or overall mortality (hr=1.25, 95%ci, 0.94-1.67, p=0.12) for patients with all. there was no increase in the incidence of acute graft versus host disease in cb recipients among patients with either aml or all despite hla disparity. conclusions: matched or mismatched cb is a favorable alternative stem cell source for patients without a suitable donor. we found different outcomes between patients with aml and all, which indicates the importance of disease-specific analyses in alternative donor studies. decreasing trm is required to improve the outcome for cb recipients, particularly for patients with aml. . the original minneapolis conditioning regimen was used in 106 (96%) pts and modified in 6 (4 or 6 gy tbi: 4 pts; atg : 2). characteristics of the grafts: a single unit was infused in 77 pts (69%), two in 35 (31%). hla compatibility was 6/6 in 6 pts, 5/6 in 36, 4/6 in 60, ≤ 3/6 in 6; 43 pts were abo matched. infused nucleated cells (nc) was 3.1x107/kg (1-9): 2.9 x 107/kg in single units and 3.7 x 107/kg in double units. csa and mmf were used for gvhd prophylaxis in all pts. results: neutrophils recovery was 85±4% at a median of 19 days (0-48) ; 14% pts experienced autologous recovery; 14% had mixed and 72% full donor chimerism at d+100. univariate analysis indicated the low weight, previous transplantation, double units and hla compatibility as significant factors for neutrophil recovery; however multivariate analysis did not find any significant factor. acute gvhd was observed in 34±5% of pts: 21, 12 and 5 pts had grade ii, iii or iv agvhd respectively and chronic gvhd in 16%. non relapse mortality was 12±3% at 6 months; relapse: 22±5%; overall survival: 72±5%. causes of death were relapse in 17 pts, gvhd in 2 pts, venocclusive disease and multiorgan failure in 5, infections in 4 and other toxicity in 3. dfs at 6 and 24 months were 68±5% and 65±5%, respectively. multivariate analysis identified 3 independent risk factors: hla disparity(0+1 vs 2+3), cell dose (<3.1x 107/kg) and age (>44y). this assessment of ucbt after nma confirms the good results of the minneapolis group (brunstein et al. blood 2007) . few events were observed between 6 and 24 mo and dfs remains high and ucbt is a good option in absence of other source of stem cells. background: cord blood transplants (cbt) are associated with delayed or failed engraftment in a significant proportion of patients. we hypothesized that direct intrabone (i.b.) transplant of cord blood cells could improve hematologic recovery. methods: unrelated cb cells were selected for 37 consecutive patients (25 patients. 4/6, 11 patients 5/6 hla and one pt. 3/6 antigen matched). median transplant cell dose was 2.5 x10 7 /kg (range 1.4 -4.2). cb cells were concentrated in 4 syringes of 5 ml each and infused in the supero-posterior iliac crest (spic) (11 patients bilaterally; 26 patients monolaterllay) under rapid general anesthesia (10 min. with propofol). patients' median age was 40 years (18-63); 30 had acute leukaemia (21 with refractory or relapsed disease and 6 high risk first remission leukemia); 2 chronic myeloid leukemia in advanced phase; 2 refractory hodgkin's disease; 2 lnh in advanced phase and 1 aplastic anemia/mds. most patients (n=28) were prepared with conventional tbicyclophosphamide. results: the infusion of cells i.b. in spic was uneventful. six patients are not evaluable because they died of multiorgan failure within 14 days from transplant. the patient with saa/mds did not engrafted and was re-transplanted. all the other patients surviving more than 14 days engrafted (100%). median for pmn engraftment (>0.5x10 9 /l) was day 25 (14-44), whereas for platelets (>20x10 9 /l) it was day 40 (range 22-64). four patients died of infection; one patients died of ptld on day +140. four patients relapsed and died. twenty-two patients are alive in remission at a median follow up of 9 months (range 2-21). from day +30 full donor chimerism was documented in cd3, bone marrow cells and progenitor cells from both the injected and non-injected spic; from day +30, cfc progenitors reached the values of normal individuals in bilateral sites documenting the colonization of the hematopoietc system and possibly an improvement of seeding efficiency. only 4 patients experienced acute gvhd grade ii and 2 grade i; 4 patients have moderate chronic gvhd and one patient extensive cgvhd. conclusion: direct intra-bone transplant of cb cells overcomes the problem of graft failure even when low numbers of hla mismatched cb cells are transplanted. low incidence/severity of acute gvhd was observed. nearly all patients for whom a cb unit was searched were able to undergo cbt. this approach may change the policy of hemopoietic cell transplants. background: the impact of donor-recipient abo matching on outcomes after allogeneic stem cell transplantation (sct) has been a matter of controversy. objective: to evaluate whether abo matching has a significant influence on overall survival(os) in patients(pts) receiving sct for hematologic malignancies. methods: we conducted an individual patient data-based meta-analysis using a pooled dataset extracted from 7 centers(ctrs) not included in previous pooled analyses. we analyzed pts who had received bone marrow or peripheral blood transplantation for hematological malignancies. the primary endpoint was os, comparing pts receiving an abo matched (ma) graft with those receiving a major (mj), minor (mi), or bidirectional abo mismatched (bi) graft using a multivariate (mv) cox model. in addition, os and mortality within 100 days were analyzed with adjustment for confounders. results: a total of 1208 sct, including 697 ma, 202 mj, 228 mi, and 81 bi cases, were analyzed. they included 709 related sct (rsct) and 184 unrelated sct (ursct) from western ctrs; 214 rsct and 101 ursct from asian ctrs. the median age of recipients was 39 years (range,1-69). the probabilities of os [95% confidence interval(ci)] at 5 years among ma, mj, mi, and bi groups were 48% (44-52), 48% (40-56), 45% (38-51), and 37% (26-49) with a median followup of 37 months (range,3-268). overall, adverse impact on os was observed for bi group [hazard ratios (hr) adjusted by age and sex:mj 1.01 (0.80-1.27), mi 1.21 (0.99-1.50), and bi 1.38 (1.01-1.87)]. among recipients of rsct, we observed no significant difference in os between the ma group and any other group. in contrast, mi and bi groups among ursct recipients experienced worse os. sources of heterogeneity were pts who received bone marrow; who had acute leukemia; who underwent transplant at asian ctrs. in mv analysis of os and early mortality adjusted for confounders, mi and bi groups showed inferior os among the subset of ursct, and an increased risk of early mortality was observed only among mj group regardless of donor type [hr (95%ci): mj 1.50 (1.09-2.05), mi 1.23( 0.90-1.70), and bi 1.21( 0.75-1.93)]. conclusion: our meta-analysis demonstrates an overall adverse association between bi transplants and survival, largely accounted for by adverse impact of minor or bidirectional abo mismatching on os in ursct. abo mismatching appeared to have little or no impact on os in rsct. larger studies including ursct of various ethnic backgrounds should be performed. intra-bone marrow injection of umbilical cord blood: no impact on rate of haematopoietic recovery c. brunstein, j. barker, d. weisdorf, t. defor, j. wagner university of minnesota (minneapolis, us) neutrophil engraftment after umbilical cord blood (ucb) transplantation is slower than that observed after adult peripheral blood or marrow transplantation.in order to augment engraftment, we evaluated the safety and potential efficacy of ucb intra-bone marrow injection(ibmi). it was hypothesized that direct ibmi to the marrow microenvironment would reduce hematopoietic stem cells (hsc) loss and improve homing. based on our prior experience with two partially hla matched ucb units, the median time to neutrophil engraftment was 23 days (r:15-41). trial success required: 1) absence of severe adverse events within 48 hours of ibmi, and 2) a 5-day reduction in days to neutrophil recovery (i.e., median anc>500/mcl by day 18). based on these measures of success, 29 patients were planned. all received cyclophosphamide 120mg/kg, fludarabine 75mg/m²/day and total body irradiation 1320 cgy with mycophenolate mofetil and cyclosporine a immunoprophylaxis. all pts received 2 ucb units randomly assigned to either iv infusion or ibmi. the ibmi unit was volume reduced to ~20ml with ~10ml infused into each posterior superior iliac crest under local anesthesia at the pts bedside. ten pts were evaluable (2 were disqualified due to inability to volume reduce the ibmi unit and ibmi unit was not randomized). median recipient age was 35 years (20-44) and median weight 73.6 kg (56-92). seven had acute leukemia and 3 had non-hodgkin's lymphoma. median infused cell doses for the iv and ibmi units were 2.0x10 7 tnc/kg (1.2-3.0) and 1.8x10 7 tnc/kg (1.4-3.4), respectively. pts received two 6/6 (n=1), two 5/6 (n=2) or two 4/6 (n=7) hla matched units. no adverse events were reported with the ibmi procedure. nine of 10 engrafted. median time to neutrophil and platelet recovery (>50,000/mcl) was 21 (17-49) and 69(30-272)days, respectively. complete chimerism was observed with one unit engrafting long term (ibmi unit in 4 and iv unit in 5). seven of 8 evaluable pts had acute graft-versus-host disease (grade ii in 5 and grade iii in 2). with a median follow up of 10 months, the probability of survival is 47%(95%ci,14-80%) at 1 year. based on this interim analysis, the study was prematurely discontinued based on the fact that the predetermined required rate of neutrophil recovery (median 18days) was unlikely with high degree of certainty. while technically easy and safe, neither neutrophil nor platelet recovery after ibmi were not improved to warrant additional patient accrual. j. fernandes*, v. rocha, d. setubal, m. bierings, m.a. champagne, r. pasquini, g. socié, primary graft failure (pgf) is a fatal complication after allogeneic hematopoietic stem cell transplantation (hsct). unrelated donor cord blood transplantation (ucbt) is frequently reported as having a delayed neutrophil recovery and the incidence of pgf varies from 10-25%. second transplants for pgf have been associated with a high treatment related mortality (trm) and a poor outcome, with an overall survival (os) varying from 0 to 30%. we retrospectively analyzed 35 patients who received a second ucbt following pgf after a first ucbt for hematological diseases, between 1995 and 2007. were defined as pgf patients that did not achieved a neutrophil count >500x10 6 /l until 60 days after ucbt or that received a second transplant for pgf in this period. patients having relapsed in the first 100 days after ucbt were excluded. median age was 11.3 years (range, 1-51). diagnoses were: acute leukemia (n=17), myelodysplasia (n=4) and bone marrow failure syndromes (n=14 -2=aplastic anemia and 12=fanconi anemia). patients received either one (n=31) or two (n=4) unrelated cord blood units matched 3-6/6 (hla a and b low resolution and drb1 allelic typing) as donors for the first transplant. median time between first and second transplants was 55 days (22-116) and median follow-up after the second transplant was 18 months. concerning the second transplants, 3 patients received mismatched unrelated bone marrow grafts, 7 haploidentical related grafts, 15 single ucb and 10 double ucb grafts. t-cell depletion was used in 8 cases. conditioning regimen was of reduced intensity in the majority of cases, the most frequent being fludarabine-based regimens. low-dose tbi was administered to 10 patients and serotherapy (alg/atg/other moab) was used in 16 patients. median time to neutrophil recovery was 19 days (10-47) and 19 of 35 patients engrafted (4 of 15 receiving a single cb unit, 7 of 10 receiving double cb units and 7 of 10 other alternative donors). chimerism analysis for these patients at engraftment showed: 16=complete donor and 3=mixed chimerism. twelve patients (34%) developed acute graft-versus-host disease (gvdh) grades ii-iv and chronic gvhd was observed in 5 of the 18 evaluable patients (28%). at 1 year, trm was 39%±1% and os was 41%±8%. in conclusion, these results suggest that second transplants in this set of extremely high risk patients are effective and should be considered early following primary graft failure after ucbt. umbilical cord blood (ucb) is increasingly used as a source of hematopoietic stem cells (hsc) for transplantation in patients (pts) with leukemia. initial reports suggested that ucb transplantation was associated with a 20-30% leukemia freesurvival (lfs). therefore, we evaluated the outcomes in 184 consecutive patients transplanted with ucb after a myeloablative therapy between 1995 and 2007 for the treatment of acute myeloid leukemia (aml,n=81), acute lymphoblastic leukemia (all,n=84), chronic myeloid leukemia (cml,n=13), and myelodysplastic syndrome (mds,n=6). conditioning consisted of either cyclophosphamide (cy) 120mg/kg, fludarabine (flu) 75mg/m²/day and total body irradiation (tbi) 1320 cgy with mycophenolate mofetil (mmf) and cyclosporine a (csa) immunoprophylaxis or the same cy/tbi with equine anti-thymocyte globulin (atg) 90mg/kg and methylprednisolone (mp) immunoprophylaxis. the median age was 16yrs (r: 0.5-52), median weight 57.3kg (r: 8-148). most were male (57%), cytomegalovirus seropositive (51%). grafts were composed of two units in 45% and hla mismatched at one (41%) or two (51%) antigens. the median infused cell doses were 3.4 x 10 7 nucleated cells/kg (r: 0.9-14), 3.9 x 10 5 cd34 cells/kg (r: 0.4-35), 1.2 x 10 7 cd3 cells/kg (r: 0.1-3.2). pts were classified as standard risk (acute leukemia in cr1-2 or cml in first chronic phase, n=138) or high risk (n=46). median follow-up for survivors is 2.8 years (r: 0.7-9.2). engraftment occurred in 164 patients (90% (95%ci, 86-94%). incidence of grades ii-iv and iii-iv acute and chronic gvhd was 43%(95%ci,36-50),14% (95%ci, (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) and 15% (95%ci, 10-20%), respectively. incidence of treatment-related mortality at 2 years and relapse at 4 years was 27% (95%ci, 21-33) and 26% (95%ci, 19-33), respectively. lfs was 43%(95%ci,35-51) and overall survival was 45%(95%ci,37-53) at 4 years. in multivariate analysis, disease risk group was the only risk factor for poor lfs (rr 2.0;95%ci,1.3-3.1;p<.01) and not year of transplant, pt age and weight, nc and cd34 cell dose, treatment regimen, gvhd, cmv serostatus and hla disparity. clearly, lfs after ucb transplantation, despite hla mismatch, represents a valuable alternative to bone marrow and peripheral blood especially for pts requiring urgent transplant or lacking an hla matched unrelated adult volunteer donor. double umbilical cord blood transplantation using reduced-intensity conditioning: a single-centre experience double umbilical cord blood transplantation (ducbt) reduces the time to engraftment and as a result may reduce associated transplant-related mortality. in this report, we describe our experience with ducbt after reduced intensity conditioning. methods: 51 subjects were treated on 2 sequential protocols between 2003-2007. all patients received fludarabine (30 mg/m²/d x 6), rabbit atg (1.5 mg/kg/d x 4) and melphalan (100 mg/m² x 1) as conditioning. gvhd prophylaxis began on day-3 and was administered through day 100 and then tapered. cyclosporine/mycophenolate mofetil (cy/mmf) was given to the first 21 patients, and sirolimus/tacrolimus (siro/tac) was given to the remaining 30 patients. all patients received 4/6 or better allele level hla-matched cord units and a supportive care regimen of antiviral, antibacterial and antifungal prophylaxis. g-csf was administered from day+5 through engraftment. results: the median age was 49 yrs (19-67) and 30 patients (59%) were male. malignant diagnoses were similar although there was a higher proportion of advanced lymphoid malignancy in the siro/tac group. subjects received a combined total of 4.4 x 10 6 tnc/kg and 1.9 x 10 5 cd34 cells/kg, without cohort differences. engraftment kinetics did not differ based on gvhd prophylaxis, with neutrophil engraftment (anc of 500) at a median of 21 days (13-70), and platelet engraftment (20 000/microl) at a median of 42 days (21-185). 5 patients experienced graft loss between days 35 and 102 (3 cy/mmf, 2 siro/tac). grade ii-iv acute gvhd occurred in 20% (33% cy/mmf, 10% siro/tac, p = 0.05). chronic gvhd occurred in 24%, without cohort differences. 100-day trm was 12%, and the risk of relapse at 1 year was 19% (cy/mmf 5%, siro/tac 32%, p = 0.03). atypical and viral infections were common, and ebv-ptld occurred in 5 patients. with a median follow-up of 20 months, relapse-free and overall survival at 1 year are 59 and 74%, without cohort differences. causes of death included infection(6), relapse(5), ebv-ptld(4), and other(4). conclusions: dubct following reduced intensity conditioning results in early engraftment and low 100-day treatment related mortality. the use of siro/tac gvhd prophylaxis is associated with less gvhd than cy/mmf prophylaxis. this approach may lead to a higher relapse incidence, although this may be explained by differences in subject baseline characteristics. dubct should be considered for all adults in whom a suitable adult donor does not exist. (1) rna encoding the wilms tumor protein (wt1) is overexpressed in the vast majority of patients with acute myeloid leukemia (aml) and peptide vaccination studies have demonstrated that wt1 is a target for active specific immunotherapy. preclinical data from our laboratory and that of hans stauss have shown that wt1 mrna-electroporated dendritic cells (dc) stimulate wt1-specific t cells in vitro (van driessche a et al. leukemia 2005; 19:1863 -1871 . therefore, we started a phase i/ii dose escalation trial in which patients with aml received intradermal injections with wt1 rnaloaded dc. feasibility, safety, immunogenicity and antileukemic activity of the vaccine were investigated. high risk aml patients (all but one of them in complete remission) underwent a leukapheresis and monocytes were isolated using cd14 immuno-magnetic beads by clinimacs. dc were generated in 6-day cultures in clinical-grade medium supplemented with serum, gm-csf and il-4 and matured with pge2 and tnf-alpha. keyhole limpet hemocyanin (klh) was added during maturation as a cd4+ t-helper antigen. mature dc were harvested, electroporated with wt1 mrna and used as vaccines. eight patients received four biweekly dc vaccines. a delayed-type hypersensitivity (dth) test was performed 2 weeks following the last vaccination. patients were monitored for minimal residual disease (mrd) by analyzing wt1 rna expression in peripheral blood by qrt-pcr. before and after the vaccination cycle, peripheral blood was collected for immunomonitoring purposes. there was successful dc generation and vaccine production in all patients selected. no serious adverse events or toxicity was seen. a decrease in wt1 rna expression was observed during the course of the vaccination in 4/6 patients who had an increased wt1 mrna level in peripheral blood at the start of dc vaccination. a vaccine-specific immune response was demonstrated in 8/8 patients by dth both to klh as well as to wt1. preliminary data from immunomonitoring in pre-and post-vaccination t cell samples from 4 patients show a mixed t helper (th)1/th2 response towards the klh and the wt1 protein following vaccination. we conclude that vaccination of aml patients with wt1 rnaloaded dc is feasible and safe. furthermore, the vaccine elicits anti-vaccine t-cell responses in vivo and a decrease in wt1 rna expression levels was observed during mrd monitoring in some vaccinated patients, strongly suggestive of antileukemic activity of the dc administered. reduced-intensity allogeneic stem cell transplantation using related haploidentical donors for relapsed or refractory lymphomas: evidence of anti-tumour activity in poor prognosis disease p. corradini*, c. carniti, a. raganato, a. vendramin, l. farina, v. montefusco, r. milani, m. milanesi, p. longoni, l. gandola, c. lombardo, a. dodero istituto nazionale dei tumori (milan, it) haploidentical stem cell transplantation (haplo-sct) has disclosed new possibilities for the treatment of hematologic malignancies in patients (pts) who do not have a hla compatible sibling or a fully matched unrelated donor. the role of haplo-sct in relapsed lymphomas is, at present, unknown. we devised a new strategy potentially useful also for pts without nk alloreactive donors (nk alloreactivity in lymphomas is untested). we report the results of a phase i-ii trial evaluating early add-backs of cd8-depleted donor lymphocytes (dlis) (from 1x10 4 up to 5x10 4 cells/kg from day+45 to day +105 at monthly intervals) after a reduced intensity conditioning (ric) regimen in 22 pts with relapsed lymphomas. ex-vivo and in-vivo t-cell depletion were carried out by cd34+ cell selection and alemtuzumab, respectively. histologies were non-hodgkin's lymphomas (nhl, n=13) [chronic lymphocytic leukemia (cll), n=5, aggressive nhl (hg-nhl), n=7 and follicular cell lymphoma (fcl), n=1] and hodgkin's disease (hd, n=9). the median age was 33 years (range, 15-65). pts received a median number of 4 lines and 86% failed autograft. at median follow-up of 29 months (range, 3-60 months), 10 pts (44%) were alive (n=7 in durable cr) and 12 died of disease (n=8) or non-relapse mortality (n=4). two-year os and pfs of the whole group were 44% (95% ci, 22%-64% ) and 33% (95% ci, 12%-56%), respectively. the main factors influencing 2-year os were age (≤ 45 years: os of 51%) and chemosensitivity of disease at time of sct (75% versus 25% for chemosensitive versus chemorefractory, p<0.02). before dlis, only 2 of 22 pts (9%) developed acute gvhd. cd8-depletion, perfomed using an immunomagnetic method, reduced the content of cd8+ tcells by a median value of 3.3 log (1.7-4). a total of 36 cd8depleted dlis were administered to 16 pts. three pts (18%) developed acute gvhd grade ii-iv following dlis. eight pts receiving cd8-depleted dlis never experienced a relapse. we observed a statistically significant expansion of cd4+ and cd19+, but not of cd8+ and nk cells, at day +120 following cd8-depleted dlis. eight pts had measurable trec/µg at 1 year after sct. spectratyping of t cells demonstrated a polyclonal vß repertoire: pre-transplant 97%, post-transplant 70%. escalated doses of cd8-depleted dlis increase the level of cd4+ in the first 4 months after haplo-sct and are feasible. this is the first report of long-term remissions in lymphomas after ric haplo-sct. k. perruccio*, f. topini, a. tosti, a. carotti, t. aloisi, f. aversa, m. f. after haploidentical stem cell transplantation, immune recovery is slow due to decaying thymic function and extensive t-cell depletion of the graft which is needed to prevent graft-versus-host disease (gvhd). consequently, infectious related mortality is about 30-40%. to address this problem, we investigated the efficacy of adoptive immunotherapy after photodynamic purging of alloreactive t cells (theralux tm , kiadis pharma, amsterdam, the netherlands) in preventing gvhd and improving immune s25 reconstitution. optimized protocol conditions provided 3,260 ± 450 (mean ± sd)-fold allodepletion, full retention of tregulatory cells, and preservation of pathogen-specific t-cell responses (against aspergillus, candida, cytomegalovirus (cmv), adenovirus (adv), herpes simplex virus (hsv), varicella zoster virus (vzv), toxoplasma antigens). here we present the preliminary results of a clinical trial. escalating doses of photodynamically allodeleted donor t cells, i.e., 1.25 x 10 5 /kg, 2.5 x 10 5 /kg, 5 x 10 5 /kg and 1 x 10 6 /kg, were infused into groups of haploidentical transplant recipients. only 1 patient developed grade iii agvhd at the 1 x 10 6 /kg cell dose and responded to immune suppressive treatment. immune assessment analyses revealed that infusion of cell doses equal or greater than 5 x 10 5 /kg are associated with significant reconstitution of t-cell counts and appearance of pathogen-specific t-cell responses. three weeks after infusion, cd4+ and cd8+ t cells were 124 ± 54/cmm and 327 ± 42/cmm (versus 11 ± 4/cmm and 8 ± 4/cmm respectively, in patients receiving t-cell doses below 5 x 10 5 /kg, p = 0.0007). aspergillus, candida, cmv, adv, hsv, vzv, toxoplasmaspecific cd4+ and cd8+ t-cell responses had recovered to frequencies within the normal ranges while they were absent in patients who received t cell doses under 5 x 10 5 /kg (p = 0.0002). in conclusion, this study demonstrates the feasibility, safety and preliminary indications of efficacy of adoptive immunotherapy after photodynamic purging of alloreactive t cells in recipients of haploidentical stem cell transplantation. a larger study will evaluate the impact of these t-cell infusions on transplant related mortality and disease free survival. leukemic stem cells (lsc) are a self-renewing subpopulation of malignant cells which is thought to play a central role in the pathogenesis of acute leukemia. lsc are likely contributing to both disease initiation and relapse and therefore represent an important therapeutic target. resistance of lcs to standard chemotherapy agents is an important consideration for the development of new therapies. in this study, we asked whether lsc of acute myeloid leukemia (aml) can be recognized and eliminated by the anti-tumor function of natural killer (nk) cells. using cd45dimcd34+cd38-as a phenotypic characteristic of the lsc population in aml, we purified these cells from peripheral blood of patients with de novo aml and examined their sensitivity to alloreactive nk cells. for this purpose, the recently described nk cell lines with single kir specificities and mismatched with respect to hla-class i allotype of target leukemic cells (diermayr et al, blood 2007 online) were employed. using the hematopoietic colony forming unit (cfu) assay in methylcellulose, we demonstrated that purified lsc gave rise to hematopoietic colonies which were maintained upon a serial passage. when lsc were preincubated with alloreactive nk cells the growth of aml cfu was significantly reduced. next, we demonstrated that cd45dimcd34+cd38-aml cells do not express the cell surface ligands, mic and ulbp, for the major nk cell activating receptor nkg2d. incubation of purified cd45dimcd34+cd38-cells with histone deacetylase (hdac) inhibitor valproic acid (va) for 2 days, resulted in an up to 2fold upregulation of mic or ulbp ligands in 8/12 tested aml samples. the va treatment had no impact on aml cfu formation in the absence of nk cells, but abrogated the clonogenic growth when applied together with alloreactive nk cells. importantly, the effect of va was limited to lsc since normal hematopoietic stem cells were unaffected, both with respect to expression of nkg2d ligands as well as the colonyforming efficiency. taken together, our results indicate that aml lsc are susceptible to nk cell-mediated cytotoxicity and that expression of nkg2d ligands makes lsc more accessible to nk cells. we propose that adoptive transfer of alloreactive nk cells in combination with pharmacological use of hdac inhibitors merit evaluation as novel approaches to prevent relapses of aml with nk cell immunotherapy. these approaches are now evaluated in an in vivo model of human aml transplanted into nod/scid mice. survival in patients receiving a transplant from an hlamismatched donor (at 10/10 alleles) can be significantly improved by selecting a donor mismatched for hla-dpb1 b. shaw* (1, 2) , n. mayor (2) recipient/donor hla matching is an important determinant of outcome in transplantation using volunteer unrelated donor (vud). there is evidence that matching for hla-a, -b, -c, -drb1, -dqb1 results in a beneficial outcome. the impact of matching for hla-dpb1 is more controversial and few studies have considered the additive effect of --dpb1. we investigated the outcome, dependent on the degree of hla matching at 12 alleles, in 488 recipients of vud transplants for leukaemia. the patients had aml (188), all (157) and cml (143). the majority of patients had t cell depletion as gvhd prophylaxis. 318 patient/donor pairs were matched for 10/10 alleles. of these, 89 were matched in addition for dpb1. 118 pairs had a single allele mismatch (9/10). of these, 30 were matched and 88 were mismatched for dpb1. 52 pairs had multiple mismatches (≥ 2), in 14 dpb1 was matched. we found a significant difference in overall survival when comparing these groups (fig 1, p=0 .006). survival was not significantly different in the 10/10 matched transplants dependant on dpb1 matching (p=0.13). however, there was a significant difference in survival dependant on dpb1 matching within the hla mismatched group (n=170) (4 years: 39% dpb1 mismatched compared 21% dpb1 matched, p=0.008). in particular, in pairs with a single hla mismatch at -a, -b, -c, -drb1, -dqb1, the presence of a dpb1 mismatch improved overall survival (4 years: 43%) compared to dpb1 match (25%, p=0.05). in multivariate analysis including disease, stage, patient/donor age, patient cmv status and gender, the significant survival benefit of dpb1 matching persisted (or 0.502; 95% ci 0.32, 0.78; p=0.002). this effect appeared to be mediated both by a decrease in relapse in the dpb1 mismatched pairs (4 yrs: 47% compared to 87% in dpb1 matched pairs, p=0.004) and a decrease in trm (4 yrs: 37% compared to 64% in dpb1 matched pairs, p=0.006). no differences in acute or chronic gvhd were seen. we conclude that in patients who receive hla-mismatched grafts (at hla-a, -b, -c, -drb1, -dqb1), a donor who is dpb1 mismatched in addition results in a superior outcome compared to one who is dpb1 matched. as physicians frequently have a choice between mismatched donors, we believe that selection by dpb1 within this group will be possible and beneficial. additionally, dpb1 is the only hla allele for which matching status impacts on disease relapse. we speculate that this may suggest a difference in the functional mechanisms of dpb1. the cxcl16/cxcr6 chemokine pathway specifically targets alloreactive t-cells to the bone marrow of mice with leukaemia r. van der voort, v. verweij, f. maas, j. vos, m. philippens, t. de witte, h. dolstra* umc st radboud (nijmegen, nl) allogeneic stem cell transplantation is an effective treatment for patients with leukemia. this therapeutic effectiveness is largely attributed to the graft-versus-tumor (gvt) response during which alloreactive donor t cells eradicate minor histocompatibility antigen (miha)-expressing tumor cells in the bone marrow (bm). unfortunately, subsets of alloreactive t cells recognize miha in healthy tissues, such as the gut, skin and liver, resulting in graft-versus-host disease (gvhd). thus, there is a strong need to separate gvt responses from gvhd. an appealing strategy to achieve this goal would be to increase the migration of alloreactive t cells to the tumorcontaining bm and/or block the infiltration of gvhd-prone organs. although the homing receptors for migration into the gut and skin are extensively studied, those involved in t cell trafficking to the bm are largely unknown. here, we characterized the pathway that alloreactive t cells exploit to infiltrate leukemia-containing bm. t cells were isolated from bm and spleen (control organ) from mice with or without acute myeloid leukemia (aml) that had previously received an allogeneic bm-transplant (bmt). in addition, we used naïve mice as controls. the expression of activation markers and a panel of 14 putative homing receptors was determined by flow cytometry, and the level of several chemokines by real-time pcr and elisa. migratory behavior was analyzed by transwell migration assays and flow chamber assays. where bm of naïve and bmt mice contained few t cells, amlcontaining bm was heavily infiltrated with cd4+ and cd8+ t cells with an effector/effector memory phenotype. interestingly, bm of mice with leukemia predominantly recruited cd4+ and cd8+ t cells expressing the chemokine receptor cxcr6. in contrast, the number of cxcr6+ t cells in bm from naïve or bmt mice without aml, and in all spleens was generally low. furthermore, the ligand for cxcr6, cxcl16, was expressed by activated macrophages and dendritic cells and its expression level increased in bm of mice with aml as compared to controls. finally, we show that cxcr6+ t cells are strongly attracted by soluble cxcl16 and show increased binding to the endothelial adhesion molecule vcam-1 upon stimulation with cxcl16. in conclusion, our data suggest that the cxcl16/cxcr6 axis is a novel pathway that specifically regulates the recruitment of alloreactive t cells into leukemia-containing bm. the pd-1/pd-l1 co-inhibitory pathway is involved in functional impairment of alloreactive cd8+ t-cell responses against leukaemia w. norde (1) , f. maas (1) , h. fredrix (1) , a. schattenberg (1), m. kester (2) , f. falkenburg (2) , r. van der voort (1) donor lymphocyte infusion (dli) following allogeneic stem cell transplantation (sct) is a potent treatment for hematological malignancies. the effectiveness is attributed to the graftversus-tumor (gvt) response, during which donor cd8+ t cells become activated by minor histocompatibility antigens (miha). consequently, these alloreactive cd8+ t cells clonally expand, acquire effector functions and kill mihapositive malignant cells. after the response, most cd8+ t cells die through apoptosis, while a small population remains to form a pool of long-lived memory cells. unfortunately, tumor relapses still occur despite the presence of miha-specific memory t cells in the patient for many years, suggesting that these memory t cells become impaired over time contributing to tumor evasion. recently, a crucial role for the co-inhibitory pd-1/pd-l pathway in inhibiting the function of virus-specific cd8+ t cells was demonstrated in chronic viral infections. here, we investigated the role of the pd-1/pd-l pathway on the functionality of miha-specific cd8+ t cells. initially, we analyzed expression of activation and co-signaling molecules on miha-specific ctl following restimulation with irradiated miha-positive ebv-lcl plus il-2. we observed that pd-1 expression was upregulated on miha-specific ctl within 24 hours after stimulation. in addition, we analyzed miha-specific cd8+ t cells from leukemia patients after dli. we observed increased levels of pd-1 on miha tetramer-positive cd8+ t cells, compared to tetramer-negative cd8+ t cell subsets in the same patient. knowing that sustained expression of pd-1 is associated with diminished proliferative capacity of virusspecific cd8+ t cells, we determined whether blocking the ligation of pd-1 could increase the capacity of miha-specific cd8+ t cells to proliferate in vitro. interestingly, we revealed that the addition of anti-pd-l1 antibody augments the proliferation of miha-specific cd8+ memory t cells. notably, addition of peptide alone or in combination with an irrelevant antibody did not induce proliferation of these cells. finally, we observed that primary leukemia cells and various hematological cancer cell lines express the pd-1-ligand pd-l1 following stimulation with ifng. these data suggest that pd-1/pd-l1 interactions in the tumor environment may impair miha-specific cd8+ t cell function, and intervening with this co-inhibitory pathway may result in improved gvt immunity after allogeneic sct. the graft versus-leukemia-effect in b-cell chronic lymphocytic leukemia (b-cll) appears to be less efficient compared to myeloid leukemias. since the aberrant coexpression of cd5 in association with cd19 is a hallmark of b-cll, we asked if both molecules might serve as a combinatorial t-cell target. b-cll cells as third party in mixed-lymphocyte reactions (mlr) induced t-cell anergy. we used recombinant proteins comprising a single chain (sc) fvcd5 antibody (ab) fused to human interleukin (il)-2 via the hinge region of human immunoglobulin (ig)g1 in conjunction with a bispecific s27 cd3x19 ab (bsab; clone okt3xhd37) to saturate cd19 and cd5 binding sites on b-cll cells. consistent with the absent immunogenic potential, b-cll cells pretreated with scfvcd5 ab, native il-2 and cd3x19 bsab were not recognized by allogeneic resting t cells. in contrast, b-cll coated with the dimeric scfvcd5-il-2 and cd3x19 bsab induced profound stimulator cell dependent allogeneic and autologous t-cell proliferation. in addition, t cells from fresh peripheral blood mononuclear cell samples from b-cll patients (n=4) pretreated with dimeric scfvcd5-il-2 and cultured in the presence of cd3x19 bsab expanded by 2-3 log within 7 days. notably, addition of soluble exogenous il-2 to both types of culture remained inferior compared to the cd5-targeted il-2 delivery. t-cell proliferation and expansion also resulted in cell-mediated cytotoxicity. in conclusion, a dual targeting approach using aberrantly expressed tumor cell surface molecules for membrane delivery of dimeric il-2 molecules using a fusion cytokine in conjunction with a bispecific antibody as shown for b-cll represents a strategy to reverse tumor-induced t-cell anergy. hurler's syndrome (hs), the most severe form of mucopolysaccharidosis type-i causes progressive deterioration of the central nervous system and death in childhood. allogeneic-stem cell transplantation (sct) before the age of two years halts disease progression and prolongs life. graft-failure and mixed-chimerism (40-50%) limit the success of sct for hs. unrelated-cord blood transplants (ucbt) are suggested to be a good alternative option for bone marrow, however, little is known about risk factors for outcomes after ucbt for this disease. we have analyzed 93 hs children that received an ucbt from 1995 to 2007 and were reported to eurocord or transplanted at duke university. median age at ucbt was 1,3 (0,2-4) yrs, and median follow up was 24 (3-140) mths. the donor was hlaidentical (hla-a and b by low resolution and hla-drb1 by high-resolution) in 13 cases (16%) and incompatible in 67 cases (84%: most with 1 (54%) and 2(26%) hla disparities). the median nucleated cell dose/kg and cd34+/kg at infusion were respectively 7.2 (2-22)x10 7 and 2,3 (0,5-17)x10 5 . with the exception of 5 patients, all received a busulfan/cyclophosphamide (+fludarabine: 6) regimen. all patients received atg or campath (4). results: median days to neutrophil and platelet recovery were 22 (10-46) and 35 (13-82) days, respectively. mixedchimerism was found in only 4%. all patients had normal enzyme levels after engraftment. in multivariate analyses for neutrophil recovery, a cd34±dose of >2.3x10 5 /kg (hr=2.0; p= 0.015) was associated with increased probability of recovery. acute-gvhd (grade ii-iv) was observed in 27%, while chronic-gvhd was seen in 10% at 2 years. two years overall survival (os) and disease/event free survival were 78% and 70%, respectively. for 2 years os, time from diagnosis to ucbt more than 6 months was associated with increased risk of death (6% for those children transplanted earlier and 30% for those transplanted later: p=0,04). in conclusion, outcomes following ucbt for hurler syndrome are encouraging. ucb appears to be a good alternative allogeneic stem cell source to transplant children with hs. earlier transplantation and higher cell dose are associated with better outcomes after ucbt for hs patients. introduction: transplant for mpsih has been associated with high rates of morbidity, mortality and graft failure. some of the difficulty of the transplant reflects pre-transplant co-morbidities including significant cardiac dysfunction, respiratory including upper airway compromise and visceral organ enlargement. in recent pharmacological enzyme replacement therapy (ert) has been available that will ameliorate many of these comorbidities. manchester protocol. we give 12 weekly doses of ert prior to conditioning and continue until donor cell engraftment. patients and methods. we have performed 71 transplants in 54 patients in manchester. 19 of these patients have received ert with transplant. in 15 of these 20 there has been full intensity conditioning with pk guided oral (hd) busulfan (40mgs/m²/dose, n=7) or iv busulfan with pk monitoring but not adjustment. results: of these 15 patients 7 received oral busulfan and the most recent 8 have received iv busulfan. of these 15 patients 14 are alive with full donor cell engraftment. one patient experienced primary graft failure following a cord transplant and was successfully transplanted with a second cord and despite achieving full donor chimerism died of adenovirus 18 months after the graft. donor source for these 15 patients was mud cord in 9, other mud in 2 (pbsc donations) and matched family donors in 4 (pbsc in 2). efs survival for this group is therefore 93% with a median follow up of 21 months. our engrafted survival rates for previous patients treated on previous protocols are 68%, including second grafts to maintain donor chimerism. 4 previous patients receiving ert and reduced intesity grafts experienced 3 rejections. administered busulfan was significantly higher in those receiving hd busulfan -they received a mean of 26mgs/kg compared with the previous dose of 20mgs/kg -(p<0.001, students t-test). discussion: manchester is the largest centre for mps transplants in europe and this is the largest collection of patients treated with both ert and sct. there were concerns that pre-transplant ert would increase rates of graft rejection. these data refute those concerns. we believe that the improving results reflect reduced patient co-morbidity after ert, improved delivery of full intensity chemotherapy with busulfan and better donor matching including use of unrelated cord blood as a donor source. s28 o180 haematopoeitic stem cell transplantation for chronic granulomatous disease -a single-centre experience e. soncini* (1), m. slatter (1) , l. jones (1), s. hughes (1), t. flood (1) , d. barge (2) , g. spickett (2) chronic granulomatous disease (cgd) is a primary immunodeficiency affecting phagocytes; mutations in genes coding for subunits of nicotinamide adenine dinucleotide phosphate cause failure of oxygen metabolite generation, resulting in impaired microbial killing, susceptibility to bacterial & fungal infections and lung & gut inflammation. antibacterial & antifungal prophylaxis with cotrimoxazole & itraconazole improve short and medium term survival but do not cure cgd; patient quality of life is burdened by frequent hospitalizations, recurrent diarrhoea, inflammatory lung damage, failure to thrive and a 50% risk of death by the third decade of life. haematopoietic stem cell transplantation (hsct) can cure the disease. we report our single centre experience. between 1998 and 2007, 20 patients underwent hsct for cgd in newcastle. a retrospective analysis of medical records to november 2007 reviewed age at hsct, donor type, hla matching, conditioning, immuno-reconstitution, significant post-hsct complications, growth, lung function and outcome. 15 received bu/cy, 3 flu/melph, 1 flu/bu, 1 bu/mel conditioning. 10 had sibling, 10 unrelated donors. 17 had 10/10 hla-match, 2 9/10 hla-match (a, c mismatch), 1 8/10 hla-match (dr & dq, hvg direction only). 15 received marrow, 3 pbsc and 2 cord blood transplants. 18 of 20 (90%) patients were alive with functioning neutrophils with follow up 0.25-9.25 years. 3/4 with significant inflammatory lung disease had an improvement in lung function. all 9 patients with colitis had resolution of symptoms, with impressive catch-up growth. 1 patient had cytopenias secondary to post hsct-cmv infection, there were no other significant infections, only 2 had significant gvhd, 1 liver that resolved with treatment and 1 gut gvhd that resolved with prolonged steroid treatment, with resulting osteoporosis and avascular hip necrosis. 2 patients died, both of complications relating to active fungal infection. 13/18 survivors are off prophylactic medication with excellent life quality. hsct is curative for patients with cgdcomplications are less frequent and outcome better in those without pre-existing infection or inflammation. hsct should be considered early in the treatment of cgd, particularly if a suitable donor is available. long-term outcome and quality of life after haematopoetic stem cell transplantation in osteopetrosis: a single-centre experience d. moshous (1) background: malignant infantile osteopetrosis (miop) is caused by defective osteoclast function. impaired bone resorption is leading to increased bone density resulting in progressive marrow failure and extramedullary haematopoiesis. the abnormal bone remodelling generates also compression of cranial nerves, especially the optical nerve, causing various degrees of visual impairment up to complete blindness, starting early in life. some patients present also neurological deterioration which is not clearly explained by the pathophysiology of miop. the genetic basis of op is heterogeneous, until now four genes with autosomal recessive inheritance have been identified: tcirg1, clcn7, ostm1 and rank-l, but there seem to be also autosomal dominant forms. in the absence of treatment, children with miop die early in life often because of bleeding or infection. the only curative treatment available consists in haematopoetic stem cell transplantation (hsct). patients and methods: we report on 40 hsct which were performed in 35 patients (17 females and 18 males) in our unit between 1984 and 2007. stem cell sources were bone marrow, peripheral blood stem cells and one unrelated cord blood unit. the donors were haploidentical related donors (26/40), hla-identical siblings (9/40), related matched donors (2/40), mud (1/40) and unrelated cord blood (1/40). the majority of patients received hsct before the age of three months (16 days to 26 months), with a mean age at first transplant of 5,5 months. two patients have been transplanted at an older age, at 3 years 4 months and 8 years 5 months respectively. conditioning consisted mostly in busulfan and cyclophosphamid based regimens, in 5 cases fludarabine was added, atg was used in 12 cases. the injected cell number varied from 0,2 -19,4 x 109 nucleated cells/kg, corresponding to 2,3 -73,2 x 106 cd 34+ cells. outcome: for two patients the follow up is still too short to conclude. out of 33 evaluable patients 16 survived (overall survival: 49%). the causes of death were interstitial pneumonia in 4 patients, veno-occlusive disease in 2 patients, multiorgane failure in 2 patients, rejection in 4 patients, transplanted related toxicity in 3 patients, bleeding in 1 and infection in 1 patient. survival was better in the setting of hlamatched hsct 8/12 (66,7%) versus hla-mismatched hsct 8/21 (38%. three patients received 2, one patient 3 hsct. detailed data especially on long-term neurological development will be provided. (2) ). there was no graft rejection; mixed donor chimerism occurred in 36%. the incidence of an acute gvhd ≥ ii was 19 %, that of a chronic gvhd ii 11 % with no difference between mrd and mud. cgvhd was associated with more than expected mri changes 2 years after hsct (p < 0.05). moreover, patients with unexpected disease progression often displayed poor immunologic reconstitution and late viral problems. 14 of 18 pts. (= 78 %) in group ii/iii remained neurologically stable with good quality of life; 12 of these continued to visit a regular school. all pts. in group i deteriorated after hsct; however, 4 out of 9 pts. stabilized and full dementia could be prevented. conlusion: the above results confirm the role of a favorable disease stage for hsct outcome, as already shown (c. peters, blood, 2004) . in addition, the data demonstrate the importance of transplant-associated factors (e.g. cgvhd). in our view, the superior neurologic outcome after mrd-bmt allows a less restrictive indication for hsct in more advanced ccald patients. j.-s. kühl* (1), g. strauß (1), b. weschke (1), w. köhler first successful bone marrow transplantation for x-linked chronic granulomatous disease using a sibling donor selected after preimplantation female sexing and hlamatching j. reichenbach* (1), h. van de velde (2), m. de rycke (2), c. staessen (2) , p. platteau (2), p. baetens (2), t. güngör (1), (1), i. libaers (2) (1 objective: allogeneic hematopoietic stem cell transplantation (hsct) from an hla-identical donor is currently the only proven curative treatment for chronic granulomatous disease (cgd). hsct with alternative donors is associated with higher morbidity and mortality. the objective was therefore to perform in vitro fertilization (ivf) and preimplantation hlamatching combined with female sexing for hsct in x-linked cgd. methods: ivf followed by preimplantation genetic diagnosis (pgd) was used to identify a female hla-genoidentical embryo in a family who needed a suitable donor for their boy affected with cgd. two pgd cycles were performed. results: in the second pgd cycle two hla-genoidentical female embryos were transferred and a pregnancy was obtained. the mother had a normal delivery of a healthy girl at term. conventional hsct had to be performed, due to insufficient cell numbers in the cord blood source, at age 12 months of the donor and age 5 8/12 years of the recipient and resulted in complete stable donor chimerism and immunological reconstitution up to 17 months post hsct. conclusion: hsct after ivf and combined female sexing and hla matching offers a new therapeutic option for patients with x-linked primary immunodeficiency such as cgd needing hsct but lacking an hla-genoidentical donor. a multicentric comparative analysis of outcomes of hla identical related cord blood and bone marrow transplantation in patients with beta-thalassemia or sickle cell disease n. kabbara* (1), f. locatelli (2) , v. rocha (1) most patients with beta thalassemia major (tm) or sickle cell disease (scd) can be cured by hematopoietic stem cell transplantation (hsct) from either cord blood (cb) or bone marrow (bm). one advantage of cb is the absence of risk associated with donation. in order to compare outcomes after hsct with cb or bm, we studied 388 patients with tm or scd who received hla identical sibling cb (n=72) or bm (n=316) allografts between 1994 and 2005. in order to avoid center and period effect, only centers that performed both types of hsct during the same period were included. we compared the incidence of hematopoietic recovery, acute and chronic graft-versus-host disease (gvhd), disease-free survival (dfs) and overall survival (os) after cb and bm transplantation. compared to bm, cb recipients were significantly younger (median of 6.2 y versus 7.2 y), smaller (19 kg vs 24 kg), and were transplanted more recently (in 2001 vs 1998) . the bm group consisted of 127 (40%) scd and 189 (60%) tm patients, and the cb group, 26 (36%) scd and 46 (64%) tm patients. the indications for transplantation in scd were not statistically different between cb and bm groups. more tm patients belonging to pesaro ii-iii risk classes received bm (65%) compared to cb (36%) (p=0.004). there were also differences in the conditioning regimen (more frequent use of atg/alg in the bm group and of fludarabine and thiotepa in the cb group) and gvhd prophylaxis (more methotrexate-containing therapy in the bm group compared to the cb group). in addition, the nucleated cell content was 10 times higher in bm compared to cb. the table below shows the non-adjusted univariate analysis for outcomes for all patients according to the stem cell source used. in tm patients, the 5 year-dfs rates were 87% and 83% for bm and cb recipients, respectively, and in scd patients, 92% and 85%, respectively. in a multivariate analysis adjusted for age and type of hemoglobinopathy, dfs was not statistically different between cb and bm recipients (rr=1.4, p=0.34). in conclusion, patients with tm or scd had excellent outcomes after hsct whether they received stem cells of cb or of bm from an hla identical sibling. these results strongly suggest that cb transplantation from hla identical siblings should be pursued when possible to avoid the discomfort and risks of a bone marrow harvest. s30 o185 communication biases during informed consent for unrelated bone marrow transplantation in adult thalassaemia patients g. caocci* (1), s. pisu (2) (1), g. la nasa (2) (1)r. binaghi hospital -asl8 (cagliari, it); (2)cagliari university (cagliari, it); (3)gimema data center (rome, it); (4)oxford university (oxford, uk) although there are numerous guidelines to evidence-based medicine, few explain how to build the information into patient oriented decision-making. several factors may hamper physician-patient communication and compromise the informed consent process. in an attempt to eliminate some of the barriers to understanding, we investigated the main factors of communication between physicians and adult thalassemia patients transplanted from an unrelated donor twenty-five transplanted thalassemia patients and 12 physicians were given a questionnaire to investigate the communicated, perceived and recalled risk for mortality and graft-versus-host disease (gvhd), the acceptable risk percentage, besides the motivation and external influences underlying the choice of undergoing hsct. the risks of dying or gvhd perceived by the patients were significantly lower than those communicated by the physicians (p=.002, p<.001, respectively). also the perception of severe gvhd as a life-threatening condition was much lower (p=.004). younger patients perceived a significantly higher risk of dying than older patients (p=.003). females perceived a significantly higher risk of developing gvhd or dying than males (p<.05 in both cases). the median percentage communicated for the risk of dying was significantly higher than the percentage remembered by the patients (30% vs 20%; p=.003). the median percentage considered to be acceptable for the risk of dying was significantly higher in the patients compared to the physicians (30% vs 20%; p=.008). hence, the balance between the risks and the benefits of the transplantation procedure had not been fully understood by the patients. therefore, it is important that physicians make every effort to overcome communication bias, heuristics and distorted processes of remembering or understanding that could possibly influence the informed consent and decisionmaking process. in adults with aml (n=24578), 43% received an hla identical family hsct, 35% an autograft, 14% an hla matched unrelated hsct, 8% an hla mismatched hsct(3% haplo, 4% hla unrelated and 1% cord blood). in adults with all (n=10932), 44% received an hla identical family hsct, 23% an autograft, 21% an hla matched unrelated hsct, 12% an hla mismatched hsct (4% haplo, 6% hla unrelated and 2% cord blood). for adults in first cr with all or aml given a hla identical sibling donor, 5-years overall survival (os) was 57%; it was 49 % for those given an autograft and 49% for those patients given a hla matched unrelated transplant. for adults with aml given an unrelated cord blood or a haplo-transplant in first cr, overall survival at 2 years were 62% and 48% respectively. many studies have been performed and published on behalf of the alwp with the collaboration of many ebmt centres. thanks to the ebmt centres, physicians and data managers, we have been able to collect specific information for those studies. therefore, in 2007, 5 studies and one editorial have been published in the most important journals in the field of medicine, hematology and transplantation, 13 retrospectives and 2 prospective studies are on going and 3 of 5 abstracts were oral presentation at the last ash meeting. moreover, the establishment of 5 subcommittees within the alwp, in the different fields of transplantation has brought enthusiasm among participants and increased the number of participants to up to 50 in the alwp meetings. we would like to thank w arcese, b labar and o ottman for their input as heads of alternative donor, developing centres and phi+all subcommittees, respectively. they have accomplished two years as subcommittee's heads. we would like to welcome a nagler, s giebel and j esteve as new heads of alternative donor, developing centres and molecular markers subcommittees, respectively. i take also this opportunity to thank m mohty and c schmid for their activity in the alwp. we hope that the enthusiasm inside the alwp can motivate young physicians and more ebmt centres to join us with their ideas and input to perform important studies for the ebmt community and importantly to continue improving the field of transplantation for patients in need. the main objective of this new subcommittee is the analysis of the impact of main molecular markers on the outcome of stem-cell transplantation. several molecular lesions described in recent years are contributing to a biological characterization of acute leukemia and provide relevant prognostic information. in this regard, mutations of flt-3, nucleophosmin (npm1) or cebpa genes, frequently found in the large subgroup of aml with normal karyotype, arise as the most relevant prognostic factors in this subset of patients. nonetheless, the precise role of different transplant modalities for each of these aml categories remains to be defined. thus, a recent analysis performed within the alwp confirmed a higher risk of relapse associated with flt-3 itd after myeloablative allotransplant transplant from an hla-identical sibling, although the procedure seemed to benefit a significant proportion of flt-3 itd aml patients transplanted in first cr, according to the survival plateau observed. nonetheless, the optimal transplant policy in these molecularly-defined high-risk patients is not firmly established. the sc is also aimed to investigate the results of transplant in patients with rare subtypes, such as aml with translocation t(6;9)/dek-can or aml with 3q26/evi1 rearrangement. given the low frequency of these entities, only those studies performed in large cooperative groups have the potential to elucidate the role of different transplant strategies for these specific subtypes. another objective of the sc is the design of biologically oriented studies analysis on the outcome of transplant in adult all, which might help to clarify the controversial role of transplant in early phases of the disease. thus, specific studies for wellrecognized prognostic categories of adult all such as mllrearranged, hypodiploid varieties or results of transplant among different t-all subtypes can be sponsored by the alwp. finally, the emergence of molecularly targeted therapy such as tyrosine kinase inhibitors in philadelphia positive all or conjugated anticd33 monoclonal antibody in aml are changing the natural history of the disease and, therefore, might modify indications and results of transplant in patients treated with these agents. reduced-intensity conditioning m. mohty* institut paoli-calmettes (nantes, fr) in the last decade, the so-called non-myeloablative or reduced-intensity conditioning (ric) regimens for allogeneic stem cell transplantation (allo-sct) have emerged as an attractive modality to decrease allo-sct-related toxicities. indeed, ric allo-sct represents an attempt to harness the well-documented immune graft-versus-tumor (gvt) effect while attempting to overcome toxicity. the work of different pioneering groups rapidly proved that this approach is feasible in several disease settings or patients' categories, and had the added benefit of expanding the transplant option to patients who are ineligible for myeloablative allo-sct. unfortunately, and despite several thousands of patients receiving ric allo-sct reported to international registries, the true value of ric allo-sct in the management of haematological malignancies is, as yet, difficult to delineate. several reasons can help understanding these difficulties. a consistent definition of "non-myeloablative" or 'ric' regimens is still lacking. the different ric regimens comprise a continuum that overlaps with standard myeloablative regimens. where in theory, reduction of the "inflammatory" component of the conditioning as in ric regimens may lead to the reduction of the incidence of gvhd, gvhd remains a matter of concern after ric allo-sct, raising questions about the need for continuous immunosuppression and its corollary of long-term infectious complications. also, the notion or definition of "ineligibility" for conventional standard allo-sct is not clearly defined. the goal of the ric subcommittee within the acute leukemia working party of ebmt is to address through retrospective and prospective studies, the specific role of ric allo-sct in net health outcomes that should include, in a specific disease setting, an analysis of disease-free survival and overall survival balanced against treatment-related toxicity, quality of life, complications and death. transfusion of donor lymphocytes (dlt) for treatment of leukaemia relapse after allogeneic stem cell transplantation (sct) has been a milestone in the field of immunotherapy against malignant disease. it can be regarded as the proof of principle for the graft-versus-leukemia effect. however, in contrast to the impressive results obtained in cml, results in acute leukemias have been inferior, although there is a small subgroup of patients that obviously responded. the immunotherapy subcommittee of alwp has set the goal to study the role of cellular immunotherapy more in detail with respect to different diseases as well as distinct variants of allogeneic sct. hence, a large retrospective analysis of dlt in relapsed aml has been performed, and similar investigations have been started in all. further, the role of dlt after haploidentical and ric transplants is in the focus of ongoing studies. finally, the conditions and the results of prophylactic or preemptive dlt, given after sct to chimeric patients in cr or in a minimal residual disease status, are currently investigated. use of hsct for patients with acute leukemia is increasing in europe. therefore many ebmt centers have been established in all countries, mainly in the new eu countries. although the number of transplants in eastern countries is growing and the results are improving, the scientific activity appears still insufficient. enhancement of the scientific collaboration in eastern europe is a principle goal of the developing centres subcommittee. to reach this goal it is planned to create a panel of young investigators representing major centres from the region. this panel is expected to discuss current clinical practice as well as to share laboratory experience. on this background it will be able to propose and coordinate multicentre studies including: 1) prospective clinical trials, 2) retrospective analyses, 3) biological studies on hsct for acute leukaemia. other goals of this subcommittee is to evaluate outcomes of hsct performed in eastern countries as well as to develop studies and methods for analyzing the role of center effect on the hsct outcomes. with these objectives two studies have been performed. in the first one, we have analyzed 640 patients (aml, 459; all, 181) treated with hla matched related donor in first complete remission in 10 countries, from1990 to 2006. with this study we have observed that results of mrd-hsct for acute leukemia in eastern europe improved over time, as a consequence of decreased nrm. another study performed was the update of the lancet paper which has demonstrated a center effect in major ebmt centers. we have updated this study and we were able to show a persistent centre effect but improvement of lfs of hla identical hsct for adults with aml in first cr over the period 1987-2005. other study proposals have been discussed such as the use of economic and social markers in order to evaluate transplant outcomes in the different eu countries in collaboration with yearly survey of a gratwohl. currently, we can find an alternative donor for almost all patients without an hla identical sibling donor, such as hla matched unrelated, haploidentical and cord blood unrelated donor. the objective of this subcommittee is to study the feasibility of those different strategies and their outcomes and compare their results. with the active collaboration with eurocord, we are able to perform studies of unrelated cord blood transplantation. a recent study in collaboration with eurocord, that will be presented during this meeting, has shown the impact of kir ligand mismatching on decreasing relapse and improving lfs in ucbt recipients with acute leukemia. also comparative studies of outcomes after ucbt and haplo have been performed in children and adults. one of the main objectives of this subcommittee is to improve the hla data of the database and therefore be able to study the impact of specific hla alleles on outcomes of hsct recipients, such as the impact of permissive hla-dpb1 alleles. with the collaboration with the immunobiology wp and donor registries, we hope to improve the data of hla high resolution typing of unrelated donors. other projects are i) to study the influence of gene polymorphisms with outcomes of alternative hsct ii) to compare various conditioning regimens in this setting; iii) to target therapy with the novel drugs preand post-alternative transplants and iv) to compare immune reconstitution after mud vs. haplo vs. cbt. update of the registration study v. rocha*, e. polge, m. arat, v. koza, h. wandt, t. ruutu, a. ferrant, g. milone, p. defabritis, w. arcese, l. verdonck, a. bosi, b. allione, a.l. herr, f. pinto, c. arrais, s. nabhan, g. socie, g. dini, e. gluckman, m the registration study is a joint prospective, multi-center, non randomized trial of ebmt al and pds wps and eurocord. primary aim is to evaluate the contribution of different strategies of treatment (matched sibling donor;msd and an alternative hsct) for adults with al. secondary aim are to compare, in a first step, policy of each centre, feasibility of each transplant modality, time to transplant. in a second step, to compare in a intent to treat analysis lfs, rr and trm according to different transplants strategies. the date of the registration of the patients is considered as the date of hla typing test. for each patient, a specific questionnaire has to be completed specifying the initial strategies and each change in those strategies : at registration, after 3-6 months after the resgitration and , each 3 months during the first year after hsct, and twice a year for the following 2 years. the intention to treat will be defined, for each patient by the choice of the treatment planned and reported by the each center and the real modality performed according to the last option, and donor availability. between december 2003 and december 2006, 727 adults, enrolled by 34 ebmt centrers, were registered for the study, 498 had aml, 216 all and 13 biphenotipic al. hla typing was performed for 448 patients at diagnosis, 179 after first cr, 43 in primary refractory la, and 53 in more advanced phase (18 in cr2 or more and 35 in relapse). a msd was found for 283 patients. the choice registered for the remaining patients was: 215 alternative donor (search for ud, cb, or haplo); autologous hsct (88 pts). chemotherapy alone (141 pts).the registration of patients has been closed on december 2006.the follow up of patients will proceed for 3 years. the final analysis will be performed on december 2009. a randomised phase iii study comparing conventional chemotherapy to low dose total body irradiation-based conditioning and haematopoietic cell transplantation from unrelated donors as consolidation therapy for elderly (>60y) patients with aml in first complete remission d. niederwieser* on behalf of osho /fhcrc/hovon sakk/austrian/french and alwp-ebmt study objectives: efficacy of allogeneic related and unrelated hematopoietic cell transplantation (hct) after reduced intensity conditioning as a consolidation treatment for patients with aml in complete remission or refractory anemia with excess of blasts (raeb) in comparison with a non-transplant approach. patients with a matched sibling or with an unrelated donor, who have entered cr1, will be eligible for randomization in a 2 (transplantation):1(non-transplantation) fashion. patients without a donor will receive post-remission therapy without transplantation. blaise, f. frassoni, m. kuenz, b. rio, n. russel, p. rebulla, g. sanz, j. garcia, j. cornelissen, c. navarette, d. niederwieser, a. nagler, g. socié, a. sureda, v umbilical cord blood (ucb) cells from unrelated donors have several advantages as compared to other sources of stem cells for allogeneic use, such as prompt availability, decreased risk of graft-versus-host disease and easy collection with little risk to the mother or to the newborn. ucb has been shown to contain sufficient progenitor cells to provide durable engraftment. however, because of low infused cell doses, single ucb transplantation (ucbt) in larger children and in adults may be associated with delayed engraftment and increased risk of graft failure. transplantation of double cord blood units (ducbt) represents a strategy to overcome this limitation. the results of both pediatric and adult ducbt studies suggest that this approach is safe, and is associated with higher engraftment rates and improved transplant outcome, in both the nonmyeloablative and myeloablative settings, as compared to single ucbt. some preliminary data also indicates a reduction in relapse with double ucb transplantation. in this view, we will discuss a proposal of a randomized trial comparing single versus double ucbt for patients with hematological malignancies. a writing committee has been established to discuss all the aspects of this protocol that will be presented during the alwp session. overall the leukaemia free survivals (lfs) at five years for patients autografted in first (cr1) and second (cr2) remission have been respectively 49 and 36% in adults and 66 and 49% in children, with some improvement in outcome after 1994. recent prospective trials from single institutions or national groups have confirmed a lfs of 50% at 5 years in adult patients autografted in cr1. several randomized studies in the pre "high dose ara-c" era had shown superior outcomes of asct over conventional chemotherapy. the only randomized study using hdara-c in the chemotherapy arm (us intergroup study,p cassileth nejm 1998, m slovak blood 2000) indeed has shown better results for asct in good risk patients by cytogenetics, better results for hdara-c in intermediate risk patients and better results for genoidentical allogeneic transplants in the high risk group. numerous retrospective studies using the ebmt registry have shown equivalence for lfs and overall survival (os) when comparing asct to allogeneic transplantation with unrelated donors and/or allogeneic transplants with reduced intensity conditioning (ric), with a higher relapse incidence (ri) for asct but a reduced transplant related mortality (trm). despite this situation, the annual reports of ebmt activities indicate a clear drop in asct for aml associated to a steady increase of allogeneic transplant activity, using ric and all cell sources including cord blood. the practice of asct itself has changed over the years: less than 15% of the patients still receive marrow as a source of stem cells and mobilization of stem cells in peripheral blood has become the s33 standard. total body irradiation (tbi) which has been shown to have the highest anti leukemic effect is used in only 15% of the patients for conditioning. in vivo purging consisting of chemotherapy consolidation courses given before mobilisation is not standardized, and higher cd34+ cell yields have been shown to contain leukemic cells and to be associated with a higher relapse incidence (n feller, leukemia 2003) . a recent retrospective survey of the awlp-ebmt (presented at this meeting) indeed indicates that the number of consolidation courses given before autografting with peripheral blood influences the outcome. in patients autografted with marrow, peripheral blood early (interval cr1-transplant<80 days) and peripheral blood later (>80 days) , the lfs at 3 years have been respectively 52,46 and 36%. the source of stem cells and the interval from cr1 to transplant have been significant in multivariate analyses. another recent retrospective survey from alwp-ebmt (nc gorin, jco, 2008 in press) has compared asct to allogeneic stem cell transplantation in patients with core binding factor mutations (inv 16 and t(8;21)) and shown equivalence in outcome for patients transplanted in cr1 with lfs around 70% at 3 years. overall the data accumulate to confirm previous and already old observations that asct remains an interesting alternative therapeutical approach for the treatment of : -older patients -patients considered for ric and/or unrelated donors transplants, with bm,pb or cord blood -chemosensitive aml (so called good risk patients) including thoses reaching cr1 rapidly (rapid remitters) and those carrying a cbf mutation. since pb has replaced almost totally pb as source of stem cells, for practicality reasons, all effort should be made at increasing in vivo purging aggressively, with, as much as feasible, minimal residual disease monitoring by molecular biology. there is still a need for randomized studies in good and intermediate risk patients, while high risk patients should be allografted whenever possible. in the largest prospective mutlicentre study initiated by the ebmt and comparing filgrastim-mobilized peripheral blood progenitor cell (pbpc) to bone marrow, patients transplanted with pbpc developed more often chronic gvhd. however outcomes, which where overall and disease free survival, relapse and transplant related mortality were similar in both cohorts. at the time of first publication we were aware that long term observation was needed to ultimately determine the role of both source of stem cells. indeed, outcome differences due to difference in the composition of the graft might become apparent only years after transplantation. we therefore planed a follow-up study in order to asses the long term outcomes, general health status, social integration as well as the occurrence of malignant and non-malignant late effects in both treatment groups. the patient accrual of the initial study took place between february 1995 and september 1999, including 350 patients transplanted for leukaemia from their hla-identical sibling. in september 2007 we sent out a questionnaire to all centres which had participated to the study. the questions included information on survival, cause of death, relapse, secondary malignancies, chronic gvhd and its treatment, the occurrence of non-malignant late effects such as hypothyroidism, bronchiolitis obliterans, sicca syndrome, blood counts at last follow-up as well as question on general health status and social integration. so far, 20 (48%) from the 42 centres responded, including 105 of 212 patients (49%) alive at 3-year follow-up. ninety-eight of the 105 patients reported patients are alive, and 7 have died after the 3rd year of follow-up (gvhd n=4, relapse n=2, other n=1). chronic gvhd is still present in 23 patients (22%), secondary malignancies occurred in 4 cases (5%), non-malignant complications in 24 (22%). the median karnofski score as a measure of general health was > 90% (range 70-100). eightyeight (83%) patients have returned to work or to school. during the late effect working party meeting 2008 in florence, italy, we will provide preliminary data on the longterm outcome, and compare both patients groups, patients transplanted with pbpc to those who had received bm. this is retrospective multicenter analysis on the incidence and risk factors of cardiovascular events after allogeneic hematopoietic stem cell transplantation (hct), in 548 long term survivors treated in 10 ebmt transplant centers. these patients received hct between 1990 and 1995 and surviving ≥1 year after transplantation. the median age of the patients at last follow-up or at time of a vascular event was 35 years (range 3-72 years), and the median follow-up time 9 years (1-16 years). twenty (3.6%) out of 548 patients developed an arterial event in at least one arterial territory. the cumulative incidence of first arterial event 15 years after hct was 6% (95% ci, 3%-10%). the cumulative incidence for patients with a high global cardiovascular risk score, including arterial hypertension, diabetes, dyslipidemia, physical inactivity and smoking was 17%, as compared to 3% in those with a low risk score. in univariate analysis, older age at last follow-up, all cardiovascular risk factors taken individually, and acute gvhd were associated with a higher risk of a cardiovascular accident. in multivariate analysis age older than 30 years at last follow-up, and patients with a high global cardiovascular risk score had a 6.4-fold and 9.8-fold increase, respectively of the relative risk for an arterial event after hct. thus, long term survivors after allogeneic hct are likely to develop cardiovascular risk factors, and present an increased risk for premature cardiovascular accidents after allogeneic hct. update on the phase ii/iii study of the incidence and outcome of vod with the prophylactic use of defibrotide in paediatric stem cell transplantation (vod-df study) s. corbacioglu* university children's hospital (ulm, de) along with graft versus host disease (gvhd) and cytomegalovirus (cmv) infection, veno-occlusive disease (vod) is one of the most frequently encountered serious complications after stem cell transplantation. the currently reported overall incidence of vod ranges from 5% to more than 60% in children who have undergone stem cell transplantation. defibrotide (df) is a polydisperse oligonucleotide derived from porcine intestinal mucosa with antithrombotic and protective properties on the microvasculature but minimal hemorrhagic risk. in large, multicenter, international phase i/ii trials targeting patients with severe vod df has emerged as a promising therapy for vod. the vod-df study is a prospective international multicentre phase ii/iii study with the aim to assess the beneficial effect of defibrotide on the incidence, morbidity and mortality of vod in children at high risk to develop vod after myeloablative stem cell transplantation (nih trial number: nct00272948, eudract number:2004-000592-33) . the study, co-sponsored by gentium and the ebmt, is open for recruitment since january 2006 with currently 27 participating centres in 10 countries. the prospective recruitment period is 3 years with a sample size of 270 patients. the current recruitment status as of january 2008 is 220 patients with an average recruitment rate of 10 patients per month. in november 2007 the first data safety monitoring board (dsmb) meeting evaluated safety data and mortality rates of the first 120 study patients who completed 30 days of follow-up. as conclusion of this meeting the types of adverse events described were considered typical for this patient population with no unexpected toxicities. no significant difference was observed in the number of adverse events between the prophylaxis arm compared to the control arm. therefore it was unanimously concluded that there were no safety considerations of concern arising from the defibrotide-treated patients on the prophylactic arm at the present time. for the determination of the finale sample size an interim analysis will be performed at 240 patients. the recruitment of this sample size should be completed by early this year. in the meantime the following centres either completed or will complete the initiation process in order to join the study: ankara, antalya, istanbul, and prague. the registration study is a joint prospective, multi-center, non-randomized trial of ebmt al and pds wps in cooperation with the eurocord. primary aim is to evaluate the contribution of different strategies of treatment (msd and alternative hsct) for children with al. secondary aim are to compare, in a first step, policy of each centre, feasibility of each transplant modality, time to transplant. in a second step lfs, according to different transplants strategies, rr, trm will be compared. the date of the registration of the patients is considered as the date of hla typing test. for each step, a specific questionnaire has to be completed: at registration, after 3-6 months, at hsct; each 3 months during the first year after hsct, and twice a year for the following 2 years, followup forms will be completed. the intention to treat will be defined, for each patient by the choice of the treatment reported to be planned by the centre in each questionnaire, the real modality performed according to the last option, and donor availability. between december 2003 and november 2007, 271 children, enrolled by 24 ebmt centres, were registered for the study. children had all (at diagnosis or in cr1, 94 patients; at first relapse or in cr 2, 73 patients; 12 in more advanced disease), or aml (at diagnosis or in cr1, 71 patients; with refractory disease, at first relapse or in more advanced phase, 17 patients). a msd was found for 83 children, the choice registered for the remaining 183 children was: chemotherapy alone (45 pts it is estimated that every year in europe about 500-600 children become donors of hematopoietic progenitor cells for siblings undergoing allogeneic stem cell transplantation. the aim of the study is the analysis of donor safety and occurrence of early side effects related to bone marrow (bm) or peripheral blood stem cells (pbsc) collection in pediatric siblings qualified to be a donor. the study is based on questionnaires send to transplant centers. following endpoints are analyzed: complications during anesthesia, complications related to bm collection, complications of catheter placement, complications related to apheresis, number of nights spent in hospital and psychological issues in donor immediately after stem cell collection and during one year follow-up. since january 2007, 112 donors (median age 9 years, range 11 months -18 years) were registered. the source of stem cells was pbsc and bone marrow, in 43 and 69 patients, respectively. preliminary data indicate that the rate of complications is very low, thus the procedure is safe for donors. due to low patients accrual, retrospective donors registration over a period of 1 year before the start of the study is being proposed. the 6th meeting of the ebmt pediatric diseases working party will be held june 2-4, 2008 in poznan (poland) and for the first time will be accompanied by the meeting of ebmt pediatric nurses. the second announcement with detailed information concerning preliminary scientific programme, call for abstracts, registration, hotel accommodation, travel, important dates etc. is available on the meeting web page www.bokiz.pl/ebmt-pds-wp-2008, which is also accessible using the link from the ebmt pds wp web page. physicians sessions (monday and tuesday, june 2-3), nurses sessions (tuesday, june 3), and joint session (wednesday, june 4) will create an excellent opportunity to summarize recent progress in the field of hsct from pediatric point of view, to initiate new ideas, and to improve an understanding and collaboration between nurses and physicians involved in pediatric hsct. apart from that the meeting will be a unique platform for initiation new trials, interaction, and cooperation. more than 30 outstanding speakers will present various aspects of topics of the meetings. however, organizers expect also a lot of interesting original oral as well as poster presentations, and an exciting and stimulating discussion. therefore, all participants are invited and encouraged to submit their own experimental and clinical results for presentation during the meetings. the deadline for abstract submission is monday, march 17, 2008. early registration is possible until monday, march 31, 2008. twenty grants from the ebmt covering registration fees and local accommodations will be awarded by the scientific committee on a competitive basis to physicians or nurses (information concerning application for grants is available on the meeting web page). pharmaceutical companies, manufactures of technical equipment and software as well as publishers are invited to display their products at the industrial exhibition which will part of the meeting. detailed information about poznan, including information how to get to poznan, is available on the web page www.city.poznan.pl. it is a real honor and a great pleasure to invite everybody interested and involved in pediatric hsct to poznan to attend the 6th meeting of the ebmt pediatric diseases working party and 1st meeting of the ebmt pediatric nurses, poznan (poland), june 2-4, 2008. 235 why is important to built a paediatric nurse group inside pds wp? s. calza* (1), v. van de crommert (2) (1)children's hospital (genoa, it); (2) transplantation group initiated a prospective, randomized, parallel-group, open-label phase iii, multicenter study, comparing vtd (arm a) with td (arm b) for mm patients progressing or relapsing after autologous transplantation. inclusion criteria were: patients in first relapse after at least one autologous transplantation, including those who may have received velcade or thalidomide before transplant. exclusion criteria: subjects with severe neuropathy or non secretory mm. 366 patients will participate (183 in each arm). primary study end point was time to progression. secondary end points included toxicity, response rate, eventfree survival and overall survival. treatment was scheduled as follows: velcade 1.3 mg/m² will be given as an i.v bolus on days 1, 4, 8 and 11 followed by a 10-day rest period (days 12 to 21) for 8 cycles (6 months) and then on days 1, 8, 15, 22 followed by a 20-day rest period (days 23 to 42) for 4 cycles (6 months). in both arms, thalidomide will be given at 200 mg/day per os for one year and dexamethasone 40 mg/day per os four days every three weeks for one year. thrombosis prophylaxis was strongly recommended as well as valacyclovir prophylaxis (in arm a) against reactivation of varicelle zoster virus. patients reaching remission could proceed to a new stem cell harvest. however, transplantation, either autologous or allogeneic, could only be performed after achieving the one year treatment. response was assessed by ebmt criteria, with additional category of ncr. adverse events are graded by the nci-ctcae, version 3.0. as of january 15, 2008, 118 patients entered the study. 79 in france (ifm 2005-04 study), 10 in italy, 9 in germany, 8 in switzerland (a sakk study), 5 in belgium, 3 in austria and the czech republic, 1 in the uk. 118 are assessable: 74 males, 44 females; median age: 60 yrs (range35-72), number of autologous transplant: one: 38, two: 80. of these patients, 60 were randomly assigned to receive vtd and 58 to receive td. treatment was discontinued in 11 patients. an interim toxicity analysis was planned to be performed when the first hundred patients had been included. this interim analysis is ongoing (january 2008). other preliminary studies demonstrate that vtd is a highly active and relatively well tolerated regimen. the combination is used in the relapse setting, as well as first line, consolidation and maintenance. in this protocol, the starting doses of velcade and thalidomide are relatively high and the duration of treatment is long. we will assess the superiority of vtd over td in the relapse setting. protocol eu-dract number: 2005-001628-35. tyrosine kinase inhibitors (tkis) are the accepted first line treatment of choice for the majority of patients with chronic myeloid leukaemia (cml) although allogeneic stem cell transplantation (sct) remains the only potentially curative procedure. although second generation tkis have shown promising results, in young patients resistant or intolerant to imatinib and with a low ebmt score allogeneic sct is still considered the best second line therapy. however, despite its curative potential, a significant proportion of patients relapse following allogeneic sct. the gold standard for the treatment of relapse following allogeneic sct is the infusion of lymphocytes from the transplant donor (dli). however, dli may result in significant complications including graft versus host disease and bone marrow aplasia and using escalating doses protocols, responses may be protracted. there is consequently a need for an alternative treatment for patients that relapse following sct, that is both efficacious, faster acting, easier to administer and safer. dasatinib has been shown to be effective in treating patients that are resistant or intolerant to imatinib and as a result constitutes a good candidate treatment option for imatinib intolerant or resistant patients that relapse following sct. the cml sub-committee has launched a prospective phase ii to investigate the efficacy of dasatinib in this setting. this study will concentrate on patients 18 years of age with ph+ cml whose disease has relapsed after transplantation from an hla-identical sibling or an hla-matched unrelated donor and have not responded to withdrawal of immunosuppressive treatment where this is possible. enrolled subjects will be commenced on dasatinib 100mg qid and receive treatment for 12 months. continuation of treatment beyond 12 months will be at the investigator's discretion. the primary end-point will be complete molecular remission at 12 months secondary end-points will include complete cytogenetic remission rates, overall survival and proportion of patients requiring dli. donor lymphocyte infusions will be administered to all patients in whom dasatinib has been discontinued indefinitely or there is evidence of disease progression during dasatinib therapy or there is evidence of disease relapse after initial response tom dasatinib. the study will open in the summer of 2008 and will aim to recruit 50 patients in 3 years. interested ebmt centres are invited to participate. (11) allogeneic hematopoietic stem cell transplantation (hsct) as first line therapy for patients with chronic myeloid leukaemia (cml) has been replaced by imatinib. the role as second line therapy in patients who failed imatinib treatment is a matter of debate. second generation tyrosine kinase inhibitors (tki) have already proven their efficacy in this setting. early transplant related mor¬tality of allogeneic hsct is considered to be too high. however, transplant outcome of young cml patients with a low risk for transplant related mortality has not been analyzed recently. method: in order to better counsel patients with a hlaidentical sibling confronted with this situation we performed a retrospective analysis of transplants reported to the ebmt be¬tween 2002 and 2005. we selected for patients who had a low risk (ebmt-) score for transplant related mortality and who were transplanted from an hla identical sibling. we analysed the outcome of those only who were transplanted in 1st chronic phase and who received best current treatment, defined as standard conditioning, no t-cell depletion and bone marrow as stem cell source. results: 214 patients (8% of all 2737 patients transplanted for cml in this time period) with a median follow up of 12 months (0-60 months) who fulfilled these criteria were identified. they were 46% males and 54% females with a medium age of 31 years (range 6 to 59 years). 21% (46 patients) were less than 20 years old and 20% were above the age of 40. the time interval from diagnosis to transplantation was less than 1 year in 86% of patients. about one third each had an ebmt risk score 0, 1 or 2. data were obtained from 81 teams in 33 countries. the probability of survival at 5 years in a competing risk model was 88% (95% c.i. 83-93) with a cumulative incidence of death without relapse of 10% at 14 months and no additional death from transplant related mortality thereafter until 60 months of follow-up ( figure 1 ). 5-years post hsct, 27% of patients were estimated to be alive after relapse (and hence the relapse free survival was 61%). conclusions: these results show the current transplant outcome which is achievable by selecting only patients with a low risk for transplant related mortality. in this context, the data shown is valid even without information on pre-and/or posttransplant therapy. allogeneic hsct is a valuable option as second line therapy after imatinib failure for cml patients with a low transplantation risk. t prolymphocytic leukemia (t-pll) is a rare, aggressive neoplasia of t lymphoid lineage which is characterized by poor survival of less than one year. incidental reports suggest that both autologous and allogeneic hematopoietic stem cell transplantation (hsct) might be effective in this disease. a comprehensive retrospective analysis of 44 patients registered in the ebmt database will be a subject of independent presentation at this meeting. however, given the limitations of conventionally collected registry data (dubious follow-up information and extreme heterogeneity), and realizing the impossibility of performing an international formal prospective trial under the current regulatory framework, we developed a new concept of two complementary projects: the first is called "ebmt prospective observational audit on allogeneic and autologous transplantation in t-pll" and means that transplant centers will be encouraged to register their patients with t-pll very timely with the ebmt, followed by mandatory submission of ebmt medb and follow-up forms. the second is the "ebmt/eln frame of orientation for allogeneic and autologous transplantation in t-pll". its purpose is mainly to avoid transplants in situations where they are very unlikely to be successful and to avoid excess heterogeneity of eventual transplants performed, thereby facilitating scientific analysis. this expert opinion-based framework covers criteria for the diagnosis of t-pll, transplant eligibility, pre-transplant remission induction strategies, remission requirements, timing of hsct, donor compatibility criteria, conditioning, gvhd prophylaxis, and mrd monitoring. with these two complementary components it should be possible to largely improve the usual quality of registry-based data and to generate scientifically sound knowledge on hsct in an orphan disease such as t-pll. primary plasma cell leukaemia (pcl) is a rare variant of plasma cell dyscrasia, associated with poor prognosis, median survival 8-12 months, significantly shorter than for multiple myeloma. treatment with alkylating agent therapy is ineffective though polychemotherapy may offer modest improvement in survival. autologous transplantation is now widely used in the treatment of pcl and this report summarises the european blood and marrow transplant (ebmt) experience. a retrospective study was carried out of 20844 patients with common type myeloma (58% igg, 21% iga and 19% light chain) and 272 patients with pcl undergoing first autologous transplant between 1980 and 2006. all patients were reported using med-a (limited data set) or med-b (extensive data set) forms and included in the study regardless of availability of complete data. comparisons used the chi-squared test for categorical data and mann-whitney test for continuous data. overall survival and progression-free survival were calculated using the kaplan-meier method and comparisons made using the log-rank test. relapse/progression and death without relapse or progression were computed by the proper non-parametric estimator for outcomes with competing risks and compared by the gray test. there was no significant difference in age, gender, calcium or albumin at presentation. haemoglobin was significantly lower (9g/dl v 11g/dl, p=0.000), creatinine significantly higher (122 micro mol/l v 92 micro mol/l, p=0.000) and b2 microglobulin significantly higher in the pcl group. there was no difference in graft type or use of total body irradiation but the pcl group was transplanted closer to diagnosis (6.0 v 7.7 months, p=0.000). while no significant difference in engraftment, pcl patients were more likely than myeloma patients to enter cr post-transplant. overall survival for pcl patients was greatly inferior to myeloma patients -25.7 (ci 19.5-31.9, p=0.000) v 62.3 months (ci 60.4-64.3), attributable to response of short duration and increased relapse-related mortality. this is the largest reported study of pcl patients and suggests improved outcome with use of autologous transplantation. it is however dispiriting to note that outcome is greatly inferior to that in myeloma despite likely pre-selection for fitness of the pcl group. there is urgent need for collaborative study of alternative approaches including highly effective induction with novel agents and optimal stem cell transplant strategy. 356 myeloma patients from 22 centres that had undergone hla typing were included in the trial. study inclusion was at the time of conditioning for first autologous transplant at the achievement of a response status of at least stable disease after vad-like induction treatment of previously untreated patients. patients with an hla-identical sibling were allocated to the auto+allo (aual)-arm (n=108) and patients without a matched sibling donor to the auto (au)-arm (n=248); single or tandem autografting was optional. conditioning for asct was melphalan 200 mg/m 2 , and for allo ric was fludarabine 30 mg/m 2 x 3 plus tbi 2 gy. the accrual period was from february 2001 to february 2005. the two treatment groups were well matched for the standard prognostic parameters such as beta-2-microglobulin, karyotype, gender, mm subtype, stage, albumin, creatinin, calcium and response status at transplantation. median age at transplantation was significantly higher in the au-arm (57 vs 53 years). the cr rate was 43% in the aual-arm and 40% in the au-arm (p=0.49). cumulative 24 months non-relapse-mortality was 11% in the aual-and 4% in the au-arm (p=0.05). at 3 years after transplantation, there was no significant difference between the treatment arms with respect to os (aual 67%, au 70%), rfs (aual 46%, au 46%) or relapse rate (aual 43%, au 48%).. however, looking at the os curve for all patients in the aual-arm, a survival plateau on the 60%-level seems to be emerging from 3 years and onwards. we conclude that no significant differences in outcome was observed in this early analysis, but longer follow-up is warranted before any definite conclusions can be drawn. updated results will be presented. allogeneic stem cell transplantation (sct) can cure patients with mds or aml. the major disadvantage of allogeneic stem cell transplantation is the high treatment related mortality. recently the introduction of dose-reduced conditiong followed by allogeneic stem cell transplantation has lowered the treatment related mortality in comparison to standard conditioning, but a prospective comparison between both approaches is lacking. the subcommittee mds of the clwp launched a multicenter, prospective phase iii-study comparing dose-reduced versus standard conditioning followed by allogeneic stem cell transplantation from related or unrelated donors in patients with mds or secondary aml. the primary endpoint is treatment related mortality at one year. the hypothesis is that a dose-reduced conditioning will reduce the non-relapse mortality from 40 % to 20 % at one year after allogeneic stem cell transplantation. a total of 160 patients is needed to achieve this goal. patients should have mds or saml (less than 20% blasts) and should be eligible for standard and dose-reduced conditioning and aged 18 -60 years if donor is a hla-matched unrelated donor (hla-a, hla-b, hla-drb1 and hla-dqb1) (one mismatch allowed)and aged 18 -65 years if donor is a hla-matched related donor ((hla-a, hla-b, hla-drb1 and hla-dqb1) (one anti¬gen-mismatch allowed). the patient will be randomised between a dose reduced conditioning (arm b) and a standard conditioning (arm a). the standard conditioning (arm a) consisted of busulfan (16 mg/kg bw orally or 12.8 mg/kg intravenously) and cyclophosphamide (120 mg/kg) and reduced conditioning consisted busulfan (8 mg/kg bw orally or 6.4 mg/kg intravenously) in combination with fludarabine (150 mg/m²). so far the protocol is activated in germany, the netherlands, russia and italy. detailed protocol as well as recruitment will be presented. a randomised trial of rabbit anti-thymocyte globulin, given on day+7 after alternative donor transplants a. bacigalupo*, f. ciceri, p. di bartolomeo, t. lamparelli, g. milone on behalf of the gitmo background: transplant mortality (trm) can be predicted by using laboratory values, on day+7 after an allogeneic hemopoietic stem cell transplant (hsct) (bone marrow transpl.2003; 32: 205) : increased mortality is mainly, but not exclusively, due to increased acute graft versus host disease (gvhd). in a pilot study, a low dose of rabbit anti-thymocyte globulin (atg) given on day+7 reduced gvhd and trm. aim of the study: the aim of this study was to test in a propsective randomized trial, whether intervention on day+7 in patients with a high risk score would reduced the risk of trm and gvhd. patients: eligible were 170 patients undergoing hsct from family hla mismatched (n=25), or unrelated donors (n=145). all patients received atg 3.75 mg/kg x2 pre-transplant, were stratified for intermediate and high risk day+7 score, and were randomized to receive an additional dose of ratg (1,25 mg/kg day +7 and day+9) (n=84) or no additional treatment (n=86) (control arm). the two groups were balanced for age, disease phase, day+7 score, and donor/recipient sex mismatch. results: the predictive value of day+7 score on trm was confirmed in the control arm (18% vs 42% for intermediate and high risk patients, p=0.03), whereas in the day+7 atg arm , there was no difference (29% vs 29%, p=1.0). trm was overall reduced from 35% in the control arm to 29% in the atg day+7 arm (p=0.37) : the difference was more pronounced in patients with early disease and high risk on day 7 (35% vs 15% p=0.08), and in hsct with female donors in male recipients (65% vs 20%, p=0.02). acute gvhd grade iii-iv was reduced overall from 16% to 6% (p=0.04), and chronic gvhd was reduced 32% to 14% (p=0.02). conclusions: we confirm that patients with different risk of trm can be identified on day+7 after hsct: in patients at greater risk of trm, the administration of atg on day+7 reduces gvhd and trm. additional intensified supportive care (including anti-infectious treatment ) may further reduce trm in patients with a high risk score on day+7. our group reported a strong association of polymorphisms (snps) within the antibacterial defense receptor nod2/card15 with severe gvhd following allogeneic sct. however, functional studies explaining these effects were so far missing. therefore, we analysed gastrointestinal biopsies from 11 controls, 6 patients (pts) prior to sct and 56 pts following sct. biopsies post sct were obtained at the time of first symptoms indicating gastrointestinal gvhd. slides were evaluated microscopically for occurrence of apoptotic cells, loss of crypts and infiltration with lymphocytes, eosinophils and neutrophils. in addition, immunohistochemical analyses for cd4, cd8, cd68, mib1 and cd25 expression were performed. all pts and donors were typed for presence or absence of nod2/card15 snps. semiquantitative histological results were compared with clinical parameters such as stage of gvhd, use of corticosteroids and nod2/card15 status. comparison of controls with pts post-transplant showed a significant increase of apoptotic cells / crypt loss associated with enhanced lymphocyte and neutrophil infiltration. whereas the number of cd8 cells in the lamina propria significantly increased after sct, cd4 cell numbers were strongly diminished. within post-transplant biopsies, loss of crypts (score 0.5 in gvhd 0-1, 1.2 in gvhd 2 and 1.3 in gvhd 3/4 ), changes in neutrophil infiltrates (score 0.3 in gvhd 0-1, 0.8 in gvhd 2 and 0.1 in gvhd 3/4 ) and reduction of cd4 infiltrates (score 1.20 in gvhd 0-1, 0.85 in gvhd 2 and 0.39 in gvhd 3/4) were clearly correlated with stages of gastrointestinal gvhd, whereas cd8 cells showed an increase and cd25 positive cells were unchanged in pts with severe gvhd. presence of nod2/card15 snps themselves resulted in a significant reduction of neutrophils (p 0.04) and cd4 cells (p 0.006) but had no impact on further parameters. this effect could not be explained indirectly by more severe gvhd in pts with nod2/card15 snps and was confirmed in multivariate analysis. our data indicate for the first time functional changes in gastrointestinal biopsies from pts after allogeneic sct in relation to the nod2/card15 genotype. the observed reduction of neutrophils and cd4 cells may result from a reduced expression of chemokines attracting these cells to inflammatory sites, as il-8 production is strongly regulated by nod2/card15 dependent activation of nf-kappab. presence of cd4 cells and neutrophils may be required to prevent dysregulated inflammation. introduction: the nih staging and response criteria offer for the first time criteria for standardized diagnosis and staging of severity as well as evaluation of physical functioning and quality of life (qol) of chronic graft-versus-host disease (cgvhd). we present the interim analysis of a prospective germany multicenter validation study on the nih staging criteria in cgvhd. methods: 102 patients (median age 45 years, range 18-67) after allogeneic hematopoetic stem cell transplantation (hsct) for hematologic malignancies were evaluated according to the nih criteria based cgvhd activity assessment, the lee cgvhd-symptom-scale, fact-bmt, human activity profile (hap), sf 36, berliner social support scale (bsss), 24 item adjective measure (24-am), hospital anxiety and depression scale (hads) and the nccn-distress-thermometer. enrolment occurred between day 100 and 1 year after hsct or in the presence of active cgvhd without time limit. follow-up surveys were conducted at 1, 2, 3, 5, 8, 12 and 18 months after baseline survey. at all time points disease status, co-morbidities and medication were documented. results: sixty five patients had cgvhd (mild n=18, moderate n=26, severe n=21) while 37 patients did not have cgvhd. the comparison of the severity grading (mild-moderatesevere) of the physician and severity grading of the patient revealed a high correlation (p<0.01, r=.66), while the comparison of 10 point scale of patient and physician revealed differences between patient and physician in the range of 3-5 points (physician), where patients graded more severity compared to physicians. the cgvhd nih consensus grading correlated inversely with fact physical well being (r=.41, p <0.001). the hap maximum activity score correlated inversely with severity of cgvhd (p<0.05, r=.35). the cgvhd symptom scale summary score correlated with physician severity grading (r=.66, p<.0001), the fact-g score (r=.66, p<0.0001), mental health (r=.56), energy and vitality (r=.5) and hap maximum score (r=.37, p < 0.001). beside fact physical and functional well being the hap maximum score was independent of other aspects of qol. discussion: the results demonstrate, that severity of cgvhd as assessed by the nih consensus grading correlates with impairment of physical well being as well as daily activities and qol. since the cgvhd symptom scale covers severity of cgvhd and aspects of qol it should be applied together with the hap in clinical routine. naturally occurring regulatory t cells (tregs) have been reported to play an important role in modulating graft-versushost disease (gvhd), a major complication after allogeneic haematopoietic stem cell transplantation. despite striking findings in animal models supporting the therapeutic use of tregs in gvhd, the data from human studies is limited and their mechanism of action remains elusive. in this study, treg modulation of cd8+ lymphocyte induced in situ graft-versushost reactions (gvhr) was evaluated using a unique in vitro human gvhd model. tregs were defined as cd4+cd25hifoxp3+ and isolated from buffy coat of healthy blood donors using robosep following rosettesep enrichment of cd4+ cells (stemcell technology). isolated tregs were expanded in vitro with anti-cd3cd28 mab coated dynabeads (invitrogen) prior to use. the alloreactive immune reactions were set up by co-culturing "donor" cd8+ lymphocytes with hla unmatched allogeneic "recipient" monocyte derived dcs (mo-dc) in the absence or presence of "donor"-derived tregs (1:4 ratio for treg: cd8+ lymphocytes) for 7-8 days. following magnetic depletion of mo-dc and tregs, allo-antigen stimulated "donor" cd8+ lymphocytes were co-cultured for 3 days with "recipient" skin tissue. the severity of histopathological changes in skin tissue was scored as grades i-iv according to the lerner gvhd grading system. in 4 out of 4 experiments the presence of tregs significantly reduced the severity of skin gvhr from grade iii to grade i. the levels of ifng, tnfa and il-5 cytokines in the supernatants from the primary co-culture of allogeneic mo-dc and cd8+ lymphocytes were significantly reduced in the presence of tregs (ifng: 2179pg/ml vs 40.41pg/ml, p<0.0001; tnfa: 128.2 vs 19.55, p=0.0001 and il-5: 1084.7 vs 11.52, p=0.0006). following allogeneic mo-dc stimulation there was a 5.5 and 7.6 fold reduction in the percentage of cd8+ lymphocytes expressing the activation marker cd69+ (13.2 vs 2.4, p=0.026) and intracellular ifng (18.2 vs 2.5, p=0.029) in the presence of tregs. cd8+ lymphocyte proliferation measured by 3h-thymidine incorporation and cfse dilution was found to be markedly suppressed (67%-96% inhibition) in the presence of tregs in alloreactions. further investigations are underway to explore the mechanisms and characterise the modulation of gvhr by tregs in an allo-antigen specific setting. donor-recipient hla class i ligands and kir-haplotype a are associated with severe acute graft-versus-host disease in unrelated haematopoietic stem cell transplantation for beta-thalassaemia r. littera (1) killer immunoglobulin-like receptors (kirs) regulate the activity of human natural killer cells, mainly through recognition of hla class i molecules. two broad haplotypes of kir genes have been defined. the a haplotype is characterised by a single activating kir gene (2ds4), whereas the b haplotype is characterised by two or more activating kir genes (2ds1, 2ds2, 2ds3, 2ds5 and 3ds1). many studies have investigated the impact of kirs and their ligands on hematopoietic stem cell transplantation (hsct) in patients affected by acute myeloid or acute lymphoblastic leukemia. however, the results of these studies remain controversial. allogeneic hsct in talassemia patients offers an ideal study model since this cohort of patients is not biased by the variability of conditioning regimens and the different clinical and immunologic characteristics of patients transplanted for oncohematologic disorders. we studied 66 thalassemia patients transplanted from an unrelated donor. the conditioning regimen was the same in all patients. donor and recipient pairs were typed for the hla-a, b, cw, drb1, drb3, drb4, drb5, dqa1, dqb1 and dpb1 loci using high resolution molecular typing techniques. kir genes were typed using kir-gene-specific primers. out of 66 transplanted patients, 52 are alive and well (disease-free survival 78.7%), 8 rejected and 6 died. twentytwo patients (22/66 -33.3%) developed acute graft-versushost disease (agvhd). in 6 of these patients agvhd was grade iii-iv. patients who were heterozygous for hla-cw groups 1 (hla-cwasn80) and 2 (hla-cwlys80) had a higher risk of developing acute gvhd than c1/c1 or c2/c2 homozygotes (14/29 vs 8/37; rr=3.3; 95% ci: 1.63-9.76; p=.03). conversely, 7/8 patients who rejected were c1/c1 or c2/c2 homozygotes (rr=6.5; 95% ci = 0.75 -56.54; p=.06 when compared with heterozygotes). these findings confirm the results of a previous study performed on a cohort of 45 thalassemia patients. in the present study, 5 of the 6 patients (83.3%) with severe grade iii-iv agvhd had been transplanted from donors who were homozygous for kir haplotype a (rr=23.4; 95% ci: 1.19-457.96; p=.008). in conclusion, it would seem that c1/c2 heterozygosity associated with donor homozygosity for the a haplotype is likely to favour donor alloreactivity and thereby increase the risk of severe gvhd. analyses of these genetic markers may help modulate conditioning regimens and the intensity of gvhd prophylaxis in patients undergoing unrelated hsct. interleukin-13 (il-13) is an immunoregulatory cytokine secreted predominantly by activated t-helper 2 (th2) cells. it suppresses the cytotoxic action of macrophages, inhibits the production of pro-inflammatory cytokines and is a central mediator of allergic inflammation. a single nucleotide polymorphism (snp) exists within exon 4 of the il13 gene at position +2044. the a allele of this snp and high il-13 levels have been linked with several inflammatory conditions and il-13 mixed lymphocyte culture (mlc) levels have been associated with graft-versus-host disease (gvhd) following haematopoietic stem cell transplantation (hsct). consequently the roles of the il13 +2044 snp and il-13 mlc levels in hsct were examined in this investigation. polymorphism studies were carried out on a cohort of 923 hsct recipients and donors from 7 transplant centres across europe. il13 genotyping was performed using pcr and rflp analysis. il-13 levels were measured in a cohort of 91 mlc supernatants using a cytometric bead array and correlated with gvh reaction (gvhr) grades from an in vitro model of gvhd. in all statistical analyses p values <0.05 were regarded as being significant. multivariant analysis of the whole hsct cohort demonstrated that the il13 +2044 a allele was significantly associated with the development of both acute (grades iii-iv) and chronic gvhd (p=0.028 and p=0.026 respectively). these associations remained significant when the cohort was stratified for transplant type and conditioning regimen. significant associations were also observed in a subset of patients diagnosed with cml; in hla-matched siblings possession of the il13 +2044 a allele was linked with a decreased susceptibility to chronic gvhd, whereas in mud transplants possession of the a allele was a risk for chronic gvhd. depending on the subset analysis, several clinical factors were also significantly associated with gvhd. analysis of the mlc data demonstrated that il-13 levels increased with gvhr grade, with significantly higher levels being observed in mlc supernatants with gvhr grades iii-iv (p=0.015). to our knowledge this is the first investigation examining the roles of both il-13 mlc levels and the +2044 snp in hsct. the findings are encouraging, indicating that il-13 may be involved in the immunopathology of gvhd. consequently, il-13 levels, as well as snp analysis could provide key pretransplant information on gvhd prognosis and be potential novel targets for post-hsct gvhd therapy. allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative treatment for hematologic malignancies, but the application is slimited due to major complications, such as severe graft versus host disease (gvhd). diagnosis of acute gvhd is based on clinical features and biopsies, a proteomic pattern specific for agvhd has been published and evaluated blindly on 902 samples collected from 168 patients undergoing allo-hsct at mhh between 2005 and 2007. the majority of the patients included were transplanted for hematological malignancies (n=158), 10 for hematopoietic failure syndromes (saa, n=6; pnh n=1; omf, n=3). forty-five patients were treated with dose-reduced conditioning regimens; gvhd-prophylaxis consisted of cyclosporin (csa) plus methotrexate (mtx) or mycophenolic acid (mmf), and antibodies respectively. most patients were transplanted from matched unrelated donors (mud, n=100),63 received stem cells from matched related (mrd, sib, 1 syngeneic), 3 from haplo-identical related, and 2 from mismatched related donors. based on the positivity of the agvhd pattern we initiated a pilot trial of pre-emptive therapy, treating patients upon pattern positivity with 1mg prednisone/kg bw and compared the outcome of the treated group to those patients who did not receive pre-emptive therapy. in 2005 90 patients were transplanted and screened for agvhd at mhh, but not pre-emptively treated. forty one patients developed agvhd (45%), 22 (24%) had agvhd >ii and were treated with standard protocols. eight patients developed agvhd grade iii or iv (8/22=36%) and 6 of these died. between april 2006 and june 2007 78 patients were transplanted at mhh. in 30 patients the agvhd proteomic pattern showed a clear correlation for gvhd >ii. twenty six (33%) of 78 transplanted patients had agvhd >ii, 12 received pre-emptive therapy, while 14 were treated upon clinical signs of agvhd according to standard treatment protocols.two patients in the preemptive treatment group (2/26=7.9%; 2 of 12 pre-emptively treated:16 %) had agvhd iii or iv and died (2/26=7.9%; 2/12: 16%). of the 14 standard therapy patients 6 developed agvhd grade iii or iv and to date 3 of these have died (6/26 =23% or 6/14 of the standard therapy group=42%). thus, taken together our results indicate that pre-emptive treatment may decrease the severity of agvhd and probably leads to a better overall survival. donor-derived t cells emigrating from the graft after solid organ transplantation have been shown to promote immunological tolerance thereby improving long-term graft survival. however, donor t cells are also able to induce lifethreatening allo-reactive graft-versus-host disease (gvhd) in the transplant recipient. the exact mechanism by which donor t cells influences this delicate balance between tolerogenicity and allo-reactivity has not been elucidated. we observed two liver transplant patients with severe gvhd who developed a donor t-cell chimerism in peripheral blood and bone marrow up to 100%, which is far above those of previously described cases. this enabled us to isolate donorderived t cells in sufficient numbers to allow for a detailed analysis of phenotypic and functional features ex vivo. we found that the donor t cells died by apoptosis over time without any evidence of rejection by host t cells. interestingly, the host-versus-donor reactivity appeared to be selectively impaired, as anti-viral t cells were still detectable in the host repertoire. these results indicate that graft t cells are able to specifically eliminate donor-reactive t cells from the host repertoire thereby preventing donor cells from subsequent rejection. since substantial donor t-cell chimerism persisted in both patients beyond resolution of gvhd, we investigated potential mechanisms of immunotolerance. we observed that the recovery from gvhd was not accompanied by an expansion of immunosuppressive cd4/cd25/foxp3-positive t cells in peripheral blood. however, we obtained formal evidence that host-reactive donor t cells were controlled by an alternative negative regulatory pathway, executed by the immunoinhibitory receptor programmed death-1 (pd-1) and its ligand pd-l1. we did not only find an exceptionally high level of pd-1 expression on host-reactive donor t cells ex vivo, but also discovered that blocking pd-l1 on host cells significantly enhanced anti-host reactivity by donor cd8 t cells in vitro. we thus suggest the interference with the pd1/pd-l1 pathway as a novel therapeutic strategy to control host-reactive donor t cells in solid organ transplant-associated gvhd. our observations might be also of relevance for other clinical scenarios of misdirected allo-reactivity, such as graft rejection as well as severe gvhd after allogeneic hematopoietic stemcell transplantation. (2) purpose: to determine risk factors of outcomes after umbilical cord blood transplantation (ucbt) for patients with advanced lymphoid malignancies. patients and methods: we evaluated 104 adult patients (median age, 41 years) who underwent unrelated donor ucbt for lymphoid malignancies. ucb grafts were 2 antigen hla mismatched in 61%, and were composed of one (n=78) or two (n=26) units, with a median cell dose of 2.5x10 7 nucleated cells/kg and 1.05x10 5 cd34 cells/kg. diagnoses were non-hodgkin lymphoma (nhl, n=62), hodgkin lymphoma (hl, n=29), and chronic lymphocytic leukemia (cll, n=13), with 85% having advanced disease and 60% having failed a prior autologous transplant. sixty-four percent of patients received a reduced-intensity conditioning regimen and 44% low-dose total body irradiation (tbi). median follow-up was 14 months. results: cumulative incidence (ci) of neutrophil engraftment was 85% by day 60, with greater engraftment in recipients of higher cd34+/kg cell dose (93% vs. 78%, p=0.0001). ci of non-relapse-related mortality (nrm) was 28% at 1 year, with a lower risk in patients treated with low-dose tbi (13% vs. 47%, p=0.007). ci of relapse or progression was 31% at 1 year, with a lower risk in recipients of double unit ucbt (9% vs. 38%, p=0.02), and those with chemosensitive disease (19% vs. 40%, p=0 .01) and indolent nhl (19% vs. 35%, p=0.04) . the probability of progression-free survival (pfs) was 41% at 1 year, with higher survival in those with indolent nhl (61% vs. 34%, p=0.04), chemosensitive disease (55% vs. 31%, p=0.004) and who received low-dose tbi (58% vs. 27%, p=0.002). conclusion: ucbt is a viable treatment for adults with advanced lymphoid malignancies. diagnosis of indolent lymphoma, chemosensitive diseases, and use of low-dose tbi and were factors associated with significantly better outcome. positron emission tomography scan performed before reduced-intensity conditioning allogeneic stem cell transplantation has a prognostic value in patients with relapsed and chemosensitive hodgkin's lymphoma or aggressive non-hodgkin lymphoma a. dodero* (1), r. crocchiolo (2) (1) ( positron emission tomography (pet) scan using 18fluorodeoxyglucose [18f-fdg] has a recognised prognostic value in patients (pts) with hodgkin lymphoma (hl) or aggressive non-hodgkin lymphoma (hg-nhl) receiving chemotherapy or autologous stem cell transplantation (sct). thus, we retrospectively assessed the prognostic role of pet scan before reduced-intensity conditioning allogeneic sct. between 2000 and 2007, 82 consecutive pts with hg-nhl or hl, responding to salvage therapy, were evaluated with a pet scan before allografting. presence (pet-pos) or absence (pet-neg) of abnormal 18f-fdg uptake was correlated to progression-free survival (pfs) and overall survival (os).interpretation of pet scan was obtained with visual assessment alone by a nuclear medicine physician (evaluation of maximal suv in pet-pos cases is ongoing). median age of pts was 36 years (range, 17 -68 years). histologic subtypes included: 38 hg-nhl [b phenotype (n=25), t phenotype (n=12), other (n=1)] and 44 hl. fortyseven pts (57%) were allografted from a hla-identical sibling donor, 16 from a haploidentical donor and 19 from an unrelated donor. sixty-eight pts (83%) failed autograft, the median number of prior regimens was 3 (range, 1-6). pet scans were performed at a median of 30 days prior to allograft: 41 out of 82 pts were pet-pos [hg-nhl (n=18), hl (n=23)] whereas 41 were pet-neg [hg-nhl (n=20), hl (n=21)]. pts with pet-pos or pet-neg scans were well balanced in terms of diagnosis, previous treatments, and type of donor. at a median follow-up of 30 months (range, 6 -86 months), 54 pts are alive and 28 died [toxicity n=12, disease n=16]. overall, the estimated 3-year pfs in pts with pet-neg or pet-pos scans were 68% (95% ci, 49% -81%) versus 30% (95% ci, 15% -47%), respectively (p<0.003). for hg-nhl pts, the estimated 3-year pfs was 70% for pet-neg as compared to 41% for pet-pos (p<0.02) whereas for hl pts, the estimated 3-year pfs was 68% as compared to 17%, respectively (p=0.05). a statistically significant higher cumulative risk of relapse was observed in pts with pet-pos scan before allograft as compared to the pet neg scan (53% versus 21%, p< 0.022). the estimated 3-year os in pts with neg or pos pet scans were 77% (95% ci; 60% -87%) versus 41% (95% ci; 24%-57%), respectively (p< 0.002). our study shows a better pfs and os for pts being pet neg before allografting. pet scan should be incorporated in pretransplant work-up to validate our findings prospectively. l. rigacci*, a. bosi, b. puccini, p. corradini, l. castagna, n. cascavilla, g. milone, a. bacigalupo, r. scimè, g. specchia, a. rambaldi, p. leoni, f. ciceri, a. levis, s. guidi, b. bruno, r diffuse large b cell lymphoma (dlbcl) is the most common lymphoid malignancy in adults. autologous hematopoietic stem cell transplantation (ahsct) has been shown to be an effective therapy for patients with dlbcl who relapsed after complete remission (cr). patients who relapse after an ahsct have a very poor prognosis and usually can not be cured with standard or high dose chemotherapy. allogeneic hematopoietic stem cell transplantation (allohsct) has shown to be effective in the rescue of lymphoma patients relapsed after conventional or high dose therapies. according to this data we have analysed all patients with diagnosis of dlbcl who have performed an allohsct from 2000 to 2005 after an ahsct relapse. seventy-five patients were selected from the data-base, 46 were male (61%), 71 presented a diagnosis of dlbcl and 4 were anaplastic large cell lymphoma. the stem cell donor was related in 61 patients (81%) and unrelated in 14 patients (19%). the stem cell source was peripheral blood in 61 cases and bone marrow in 14 cases. the conditioning regimen was conventional in 24 patients and reduced intensity in 51 patients. the median time between ahsct and allohsct was 13 months (range 1-68 months). twenty-five patients (33%) performed allohsct after the obtainment of at least a partial remission or a controlled disease, 36 (48%) were treated with active disease and in 14 cases the data was not available. after allohsct 75% of patients obtained a response (cr or pr) and did not have evidence of disease, 25% of patients did not respond and progressed. the treatment related mortality (trm) was 32%, 14 out 24 (58%) patients died in conventional regimen and 10 out 51 (20%) in reduced intensity arm. acute graft versus host disease (agvhd) was observed in 28 patients (grade iii-iv in 8 patients), and chronic (cgvhd) in 65%. after a median follow-up of 58 months from the diagnosis (range 11-196 months) and a median follow-up of 9 months after allohsct (range 0-82 months) the overall survival was 45%. the overall survival was significantly higher in patients treated with reduced intensity conditioning in comparison with patients treated with conventional conditioning (p:0.001). this retrospective study confirms that allo-transplant it is feasible and it could be really effective in a poor prognosis group of patients. moreover the use of reduced intensity conditioning improves these results. (2) median time from diagnosis to mud-sct was 25 months (range, 3 -205). 64% of the patients had failed previous autologous transplant (asct), and 25% were transplanted with chemorefractory disease. peripheral blood was the source of hematopoietic stem cells in 70% and reduced intensity conditioning regimens (ric) were used in 52% of the cases. after a median follow up for living patients of 35 months, the estimated 3-year non-relapse mortality (nrm), relapse rate (rr), progression free survival (pfs) and overall survival (os) for the whole series were 32%, 38%, 30% and 38.5%, respectively. grade ii-iv acute graft-versus-hostdisease developed in 32% of patients. patients selected for ric protocols were older (median age of 44 years vs 38 years, p = 0.02) and more heavily pre-treated; 75% had failed autograft compared with 53% in the conventional conditioning(cc) group (p = 0.01). despite these unfavorable factors, nrm for patients receiving ric was significantly lower than observed in patients treated with a conventional regimen:23% vs 41% at 3 years (p = 0.02). however, this advantage was offset by an increased rr in patients undergoing ric-mud (3-yr rr: 46% vs 30%, p = 0.2), resulting in a very similar pfs and os for both types of conditioning regimens. the prognostic factor with highest impact on pfs was refractory disease at transplantation (rr = 1.8; 95%ci 1.1 -3.1, p = 0.02). patients transplanted with chemosensitive disease had a 3 yr pfs of 35% irrespective to the conditioning regimen applied, whereas patients transplanted with chemorefractory disease had a 3 year pfs of 16% only. in conclusion, mud sct provides a true chance for cure for select patients with dlbc lymphoma who failed conventional therapies, particularity if transplanted with cemosensitive disease. ric, knowm to be associated with a reduced nrm rate, should be especially considered in patients with chemosensitive patients, whereas cc might be the preferred option for patients with more aggressive disease. prospective evaluation of 18f-fluorodeoxyglucose (fdg) positron emission tomography as a predictor of residual disease and subsequent relapse in patients with diffuse large cell ymphoma and hodgkin's lymphoma undergoing hdc and asct s. akhtar*, a. al-sugair, y. al kadhi, a. al-zahrani, m. abdelsalam, s. bazarbashi, d. ajarim, i. maghfoor king faisal specialist hosp. & res. centre (riyadh, sa) background: there is emerging data indicating poor outcome in diffuse large cell ymphoma (dlcl) and hodgkins lymphoma (hl) patients with positive 18f-fluorodeoxyglucose (fdg) positron emission tomography (pet) before high dose chemotherapy (hdc) and autologous stem cell transplant (asct). we initiated this prospective trial to evaluate the impact of pet as a predictor of post hdc residual disease and relapse in patients with dlcl and hl undergoing hdc asct. patients and methods: from july 2005 to june 2007, 115 patients with relapsed or refractory dlcl and hl were enrolled as a potential candidate for hdc asct. 43 patients did not have hdc asct due to progression (10), refusal (4), noncompliance (5) or other reasons (24). all eligible patients received eshap as salvage chemotherapy, responding dlcl or responding / stable hl patients received same chemotherapy as mobilization regimen for stem cell collection followed by beam as hdc. prior to the initiation of eshap, each patient had ct scan + other radiological studies if needed and pet ( ct-1 and pet-1), after 2-3 cycles of salvage chemotherapy / prior to hdc asct (ct-2 and pet-2) and approximately 100 days post hdc asct or earlier if clinically indicated (ct-3 and pet-3). 72 patients had hdc asct and 49 of them had both pet-2 and pet-3 available at the time of this analysis. results: patients characteristics are male: 28, female: 21, dlcl: 9 and hl: 37. relapsed 21 and refractory 28. median age at asct is 32 years (16 to 62). median follow-up from asct is 12 months. 27 patients were pet-2 negative prior to hdc asct. at the time of this evaluation, of these 27 patients, 4 (15 %) had an event, event free survival (efs) 85%. 22 patients were pet-2 positive prior to hdc asct, 10 (46 %) had an event, efs 54%. efs for pet-2 negative vs pet-2 positive has p value of 0.027. using kaplan-meier method, positive pet-2 has 53% probability of an event vs 16% for a negative pet scan (p=0.005). efs for ct-2 negative vs ct-2 positive patients is 83% vs 68%, p = 0.466. kaplan-meier for ct-2 positive showed 36 % probability of event vs 18% for ct-2 negative, p=0.198. conclusions: prior to hdc asct, positive pet scan indicates high risk of residual disease or progression. many of these patients are likely to suffer from treatment failure. these patients are potential candidate for more aggressive and experimental therapies. for many patient with pre-transplant negative pet, post transplant pet scan can be omitted. radioimmunotherapy with haemopoietic stem cell transplantation for treatment of malignant non-hodgkin lymphoma: multicentre study on thirty patients a. mele (1) (7), g. console (7), n. cascavilla (2) , p. scalzulli (2) /kilograms (range 2.55-21.6). all patients engrafted. the median number of red blood cell and platelet transfusion were 4 (1-7) and 6 (1-8), respectively. the median time to platelet and neutrophil counts higher than 20x10 9 /l and 0.5x10 9 /l were 14 (range, 9-35 days) and 10 days (range, 8-14) , respectively. mucosites occurred in all patients (grade iii and iv in 13 and 4 cases). febrile neutropenia occurred in 80% of cases. six pneumonitis and 8 blood stream infections were documented. one patient developed an atrial fibrillation. twenty-one of 30 patients were valuable for 90-day response. the 90-day overall response was 86% with 72% of cr. four early deaths before day-90 occurred: 1 case for septic shock (day +6), 1 for viral encephalites (day +60) and 2 for progression disease (day + 30, 65). the kaplan-meyer estimated treatment related mortality (trm) is 9%. seven cases (1 cr, 4 pr and 2 not response) progressed at a median follow-up of 95 days post hst (range, 60-300). twenty-four of 30 patients are alive at a median follow-up of 175 days post hst (range, 6-590). six patients died (20%): 3 for progression, 1 in cr for ards (day + 230) and 2 for trm. sixteen (53%), 5 (17%) and 2 of 30 patients are alive in cr, pr and progression, respectively. one case is not valuable for response (day+15). we analyzed the characteristics of 14 alive patients in cr: 9 had aggressive lymphoma and 13 were at least in pr before hst (p=ns). the kaplan-meyer estimated 2y-efs is 73%. conclusion. the use of rit plus transplant induces 70% of or (53% cr) with sustained engraftment, an acceptable extra-haematological toxicity and a rapid immunological recovery in patients who failed to achieve cr after first line chemotherapy. the power of this program needs to be assessed in a larger series of patients. hdt/asct has been planned either in front-line pts with high prognostic score/bulky mass/stage iii-iv or at relapse. first line therapy were mainly abvd/mine for hd and acvbp/chop ± rituximab for nhl. fdg-pet was performed after 1-5 chemotherapy cure. mine and dhap were the most frequent salvage chemotherapy used for refractory front-line therapy pts. the conditioning regimen was mainly bicnu-etoposide-aracytine-melphalan. 46 pts (53%) were pre-hdt/asct pet negative and 41 positive (47%). 8/41 pre-hdt/asct positive pts (19.5%) converted to negative by additional cross chemotherapy. after hdt/asct, 22/33 others pre-hdt/asct positive pts (67%) converted to post-hdt/asct pet negative. one negative pre-hdt/asct pet converted to positive. residual disease of positive pts was mainly treated by local radiation. after a median follow-up of 3.2 years (range 0.4-8.4) after pre-hdt/asct pet, 29 pts relapsed, 21 dead (with 18 of 29 relapses), 3 remained resistant disease. survival was measured from pre-hdt/asct pet to death (overall survival os) or relapse/death (event-free survival efs) with censoring time at the time of last follow-up. median os and efs for the two groups were not reached. estimated 3 years os was 80% and 73%, respectively for pre-hdt/asct pet negative and positive pts. estimated 3 years efs was 74% and 61% respectively for pre-hdt/asct pet negative and positive groups. a positive fdg-pet after induction chemotherapy is highly predictive of poor survival in hd and nhl pts but an additional risk-adapted treatment strategies before hdt/asct by salvage cross chemotherapy and after hdt/asct by targeted radiation may improve pre-hdt/asct pet positive pts outcome. donor lymphocyte transfusions (dlt) after allogeneic stem cell transplantation have been shown to be very effective in treatment of recurrent mylogenous leukemia but displayed limited use in chronic lymphocytic leukemia (cll) and highly malignant non-hodgkin lymphoma (nhl). here we studied whether bi20 (fbta05), a novel trifunctional bispecific antibody targeting cd20 on lymphoma cells and cd3 on t cells could induce graft-versus-leukemia / lymphoma responses in combination with dlt or mobilized peripheral blood stem cells (pbsct) after allogeneic transplantation in these diseases. six patients (3 cases each with p53 mutated cll, and highgrade nhl) refractory to standard therapy were treated with escalating doses of bi20 (range 10 -2000 µg) followed by dlt or sct. in 4 out of 6 patients, a prompt, but transient clinical and hematological response was observed. side effects (fever, chills and bone pain) were tolerable and appeared at lower dose levels in cll (>40 µg) than in high grade nhl (>200 µg). the cytokine profile was characterized by transient increases of il-6, il-8 and il-10. neither human anti-mouse antibodies (hamas) nor graft-versus-host disease (gvhd) developed allowing repeated treatment courses. in summary, the trifunctional antibody bi20 induced prompt antitumor responses in extensively pretreated, p53 mutated, alemtuzumab and rituximab refractory patients indicating its therapeutic potential. d. stachel*, k. kirby, l. corey, m. boeckh fred hutchinson cancer research center (seattle, us) background: although cmv viral load is a predictor of cmv disease in hct recipients, regulatory agencies presently do not accept it as primary endpoint for studies that evaluate new therapeutics. the aim of this study was to examine whether cmv viral load predicts transplant-related mortality (trm) or overall survival (os) in the era of preemptive therapy. methods: 2896 consecutive patients following a first hct at fhcrc between 1995 and 2005 were analyzed; 1481 of them were cmv seropositive. patients underwent weekly testing for cmv viremia by culture and quantitative pp65 antigenemia (ag) during the first 100 days after hct. preemptive antiviral therapy was given for any pp 65 ag. using univariate and multivariable cox models, we analyzed the association of initial, mean, peak cmv load, and cmv area under the curve (auc) for their association with os and trm at 1 year after hct. viral load parameters were analyzed in quartiles. pp65 ag results are expressed as cells/200,000 cells. results: trm occurred in 25.2% of all patients and 27.8% of seropositive recipients. in a model that included cmv seropositive recipients, the adjusted hazard ratios (hr) for the upper quartile of initial viral load (ag > 5), peak viral load (ag >10) and auc (ag>100) were 1.6 (95% ci 1.2-2.1), 2.2 (95% ci 1.6-3.0), and 1.7 (95% ci 1.3-2.3), respectively. the statistical models were adjusted for recipient and donor age, hla match, donor relationship, year of hct, acute/chronic gvhd and postengraftment neutropenia (to account for the toxicity associated with preemptive therapy); additional factors evaluated (but not significant in univariate analysis) were race, donor cmv serostatus, cell source, type and intensity of conditioning, t cell depletion, risk of the underlying disease, and gvhd prophylaxis. similar models that included all patients and those that used os as endpoint also showed significant associations for first and peak viral load and the auc. results are presently being validated in a separate cohort that underwent pcr surveillance. conclusion: initial and peak viral load as well as the viral auc are independently associated with trm and os in allogeneic hct recipients receiving preemptive antiviral therapy. the peak viral load showed the strongest association with trm and os. these data further support the use of parameters of viral dynamics as primary endpoints for studies that evaluate immune augmentation or drug prevention strategies. objectives: to evaluate the effect of immunoglobulin (ivig) and cytomegalovirus-hyperimmune immunoglobulin (cmv-ivig) prophylaxis in patients undergoing hematopoietic stem cell transplantation (hsct). methods: systematic review and meta-analysis of randomized controlled trials comparing systemic ivig or cmv-ivig with placebo or no intervention for prophylaxis in patients undergoing hsct. the cochrane library, medline, conference proceedings and references were searched until 2007. the primary outcome was all-cause-mortality at end of follow-up. two reviewers independently appraised the quality of the trials and extracted data. relative risks (rr) with 95% confidence intervals were estimated and pooled. results: twenty trials fulfilled inclusion criteria, 12 assessed ivig and 8 assessed cmv-ivig. for ivig vs. control there was no difference in all-cause mortality, rr 0.99; 0.88-1.12, 8 trials, fig 1. results were similar when only patients following allogeneic hsct were assessed (3 trials), when mortality was assessed at day 100-200 (5 trials) or after 2 years (3 trials), and when high-dose ivig was used (3 trials). trials' methodological quality did not impact results. overall, there was a reduction in the number episodes of interstitial pneumonitis (ip), rr 0.64; 0.45-0.89, 7 trials, although the reduction in cmv infections was not statistically significant, rr 0.84; 0.66-1.07, 6 trials. there was no difference in clinically or microbiologically documented infections, rr 1.00; 0.90-1.10 and rr 1.00; 0.88-1.15, respectively, 7 trials both). there was no difference with regard to acute graft vs. host disease (gvhd), rr 0.93; 0.83-1.04, 7 trials. veno-occlusive disease (vod) was significantly more frequent with ivig, rr 2.73; 1.11-6.71, 4 trials, fig 2, an open-label randomised study of oral valganciclovir versus intravenous ganciclovir for pre-emptive therapy of cytomegalovirus infection after allogeneic stem cell transplantation l. volin* (1), l. barkholt (2) cytomegalovirus (cmv) remains a leading cause of infectious complications after allogeneic stem cell transplantation (sct) and without preventive measures patients with cmv infection have a significant risk to develope cmv disease. a preemptive treatment approach with iv-ganciclovir or ivfoscarnet is used to restrict the use of potentially toxic drugs for patients at risk for cmv disease. recently, valgan-ciclovir, an oral prodrug of ganciclovir, has become available. the nordic bmt group started a study with the aim of demonstrating that treatment outcome of the first cmv dnaemia after sct with oral valganciclovir is not inferior to that obtained with iv-ganciclovir. the primary study endpoint was achievement of quantitative cmv pcr (qpcr) negativity on day 28 of treatment or earlier. qpcr was carried out weekly for the first three months after sct, and if positive, patients were randomized to receive either oral valganciclovir 900 mg twice a day or ganciclovir iv 5 mg/kg every 12 h for 14 days (adjusted according to renal function). qpcr was studied twice a week. if qpcr was negative on day 13-14 of treatment, the treatment was discontinued, and if negativity was not achieved but the copy number was decreasing, maintenance treatment once daily was administered for another 14 days. thereafter the treatment was carried out according to local policy. of the 96 patients receiving an allogeneic sct, 74 patients were treated for a hematological malignancy and 22 for a solid tumor. the patients in the poand iv-groups did not differ by recipient/donor cmv serostatus, age (median 55 vs 51 years), diagnosis, donor (sibling/ unrelated), or the time of pcr positivity (day +40 and +38 after sct, respectively). the median cmv dna copy number/ml at diagnosis was 2525 (200-160000) in the pogroup, and 1425 (200-41900) in the iv-group. the maximum copy number/ml was 5031 (300-160000), and 2750 (200-41900), respectively. the incidence of agvhd ≥ grade 2 was 30% in the po-group and 19% in the iv-group. the primary endpoint of the study was achieved in 30/49 (61%) of the popatients and 25/47 (53%) of the iv-patients. during the study three patients with a solid tumor de-veloped cmv disease, two in the po-group and one in the iv-group. oral valganciclovir therapy seems not to be inferior to iv-ganciclovir therapy in the preemptive treatment of the first cmv dnaemia after allogeneic sct, and the practical advantages of oral treatment are obvious. genetic variations in the nod2/card15 gene are associated with bacteraemia and sepsis after allogeneic stem cell transplantation m. grube, d. veith, g. rogler, j. brenmoehl, h. bremm, j. hahn, r. andreesen, e. holler university hospital regensburg (regensburg, de) purpose: single nucleotide polymorphisms (snps) of the nod/card15 gene resulting in a diminished nuclear factor-kappab (nf-kappab) response to bacterial cell wall products have been recently associated with an increased incidence of crohn`s disease as well as transplant related mortality and gvhd following allogeneic transplantation. in addition, a recent study from our group revealed nod2/card15 variants as independent predictors of death from septic shock in nonhematological patients. therefore, we now analysed the direct interaction of nod2/card15 variants with febrile bacteremia and sepsis in patients receiving allogeneic sct. experimental design: we retrospectively analyzed 139 donor/recipient pairs for single nucleotide polymorphisms (snps) of the nod/card15 gene (snp8, 12 and 13). bacteremia was determined by bacterial growth in the blood culture. septic syndrome was determined by classical criteria. results: 94/139 (68%) patients had unmutated snp`s (wild type group), in 45/139 (32%) patients either the recipient or the donor had any mutated snp (r or d any mutation). 68/139 (49%) patients showed febrile bacteremia, whereas 17/68 (25%) developed lethal sepsis syndrome. the cumulative incidence of bacteremia was 48% in the wilde type group and 59% in patients with any mutation (r or d any mutation). the cumulative incidence of lethal sepsis syndrome was 5% in the wilde type group and 39% in the patients with any mutation (r or d any mutation) (p< 0,001). in a more detailed analysis of individual donor and recipient snps, presence of snp8 in the recipient and any snp in the donor (either snp8, 12 or 13) were signifikant risk factors whereas the highest cumulative incidence of sepsis was found if the donor had a snp13 mutation (p < 0,001). since bacteremia/sepsis syndrome even occurred in patients without gvhd or prior to onset of gvhd our data argue against an induction of bacteremia/sepsis syndrome secondary to gvhd. in line with this view, a multivariate analysis including gvhd showed snp`s of the nod/card15 gene as independent risk factors for lethal sepsis syndrome (p<0.01). conclusion: our results suggest that defective signalling of nod2/card15 either in epithelia (recipient) or monocytes/macrophages (donor) may be directly involved in a diminished antibacterial defense. translocation of bacteria may be an important step in subsequent sct related complications. infection control interventions for cancer patients: efficacy evaluation through systematic review and metaanalysis m. paul*, a. schlesinger, a. gafter-gvili, l. leibovici rabin medical center; beilinson campus (petah-tikva, il) background: currently used infection control measures for hematological cancer patients are frequently applied with an unclear evidence basis resulting in a heterogeneous application of these interventions. methods: systematic review and meta-analysis. included were all prospective comparative studies assessing the effects of non-pharmacological interventions applied for infection prevention and control among cancer patients following chemotherapy. interventions were classified according to the transmission modality targeted by the intervention; airborne, contact or endogenous flora. the primary outcome assessed for all interventions was 100-day all-cause mortality. relative risks (rr) with 95% confidence intervals are reported. results: we identified 23 studies assessing protective environment (pe) including high particulate air filtration (hepa) ± laminar airflow (13 randomized trials, 12 hematological cancer, 9 for hsct), 10 studies comparing outpatient vs. inpatient management for hsct (all nonrandomized) and 5 miscellaneous studies assessing diet, footwear, gowns or specific room design (3 randomized trials, 2 for hsct). pe resulted in a remarkable reduction in allcause mortality at day 100: rr 0.79, 0.73-0.87 overall and rr 0.78, 0.66-0.92 in adequately randomized trials alone. the rr was 0.60, 0.50-0.72 for 30-day mortality and 0.86, 0.81-0.91 for the longest follow-up, up to 5 years. similar survival benefit was observed for allogeneic hsct patients and other patients. significant reductions were observed for all infection-related outcomes, except mold infections (table) . when isolating the effects of the different transmission modalities it became apparent that endogenous suppression using antibacterial (usually combined with antifungal) prophylaxis was the major contributor to the beneficial effect of pe ( figure) . outpatient hsct (3 allogeneic and 7 autologous) was non-inferior to inpatient treatment, rr 0.81, 0.46-1.45 for mortality; rr 0.93, 0.67-1.29 for infections; and rr 0.35, 0.15-0.80 for bacteremia. conclusions: protective isolation offers an overall significant benefit to the patient, since the assessment of all-cause mortality encompasses both infection, cancer and treatmentrelated outcomes. the additional value of hepa and strict contact isolation over antibiotic prophylaxis is unclear and probably depends on the local prevalence of mold infections. outpatient hsct appears safe and should be explored in randomized trials. m. dettenkofer*, r. babikir, h. bertz, a.f. widmer, w.v. kern, e. meyer, p for surveillance of nosocomial bloodstream infections (bsi) and pneumonia during neutropenia in adult patients undergoing bone marrow transplantation (bmt) or peripheral blood-stem cell transplantation (pbsct), an ongoing multicenter surveillance project was initiated by the german national reference centre for surveillance of nosocomial infections in 2000 (onko-kiss). methods: nosocomial infections are identified using cdc definitions for laboratory-confirmed bsi and modified criteria for pneumonia in neutropenic patients [for detailed information see : cid 2005; 40: 926-31, or in german language: http://www.nrz-hygiene.de/surveillance/onko.htm]. results: over the 60-month period from july 2002 up to june 2007 26 centres participated. altogether 4,909 patients with 72,449 neutropenic days were investigated. of these, 2,873 (59%) had undergone allogeneic and 2,036 (41%) autologous bmt or pbsct. the mean length of neutropenia was 14.8 days (9.2 d after autologous and 18.7 d after allogeneic transplantation). in total, 827 bloodstream infections and 403 cases of pneumonia were identified. site-specific incidence densities are shown in the table. there was a trend to lower incidence densities over the five years reported. following allogeneic transplantation, 17.7 bsi/100 patients and 10.4 cases of pneumonia/100 patients occurred whereas following autologous transplantation 15.7 cases of bsi/100 patients and 5.1 cases of pneumonia/100 patients were observed. the main pathogens associated with bsi were coagulase-negative staphylococci (51%). conclusions: the ongoing onko-kiss project adds to the improvement of quality of care in hct-patients by providing sound reference data on the occurrence of bsi and pneumonia during neutropenia. since 2006, surveillance is extended to neutropenic patients with acute leukemia to allow participation for centres not performing hct. t-cell-mediated immunity is an essential host factor in the control of hcmv latency in patients undergoing an allohematopoietic-stem-cell transplantation. our aims were to identify patterns of hcmv-specific immune responses associated with multiple or prolonged reactivations. we analyzed 116 recipients during the course of infection/reactivation and latency. the cd8+t-cell responses were weekly determined using hla-class i tetramers together with extended phenotypic analyses. our results showed that recipients from unrelated donors were more susceptible to multiple reactivations and that the donor hcmv serologic status influenced the occurrence of prolonged reactivations. we found that the lack of hcmv-specific t-cells during the first episode of reactivation was associated with multiple further reactivations. the sequential phenotypic follow up showed that patients with uncontrolled reactivations were unable to develop hcmv-specific t-cells of the late differentiation phenotype cd45ra+cd27-cd28-. our data indicate that the longitudinal evaluation of cd27 and cd45ra expression within the tetramer positive subset could help to identify patients developing a protective immune response. the evaluation of hcmv-specific immune responses during the first episode of reactivation, together with extended phenotypes could improve immune monitoring, especially in recipients from unrelated donors and other situations at risk of uncontrolled viral reactivation. invasive aspergillosis in allogeneic stem cell transplant recipients from alternative donor: incidence, risk factors and outcome a.m. raiola*, m. mikulska , b. bruno, m.t. van lint, f. gualandi, d. occhini, a. dominietto, c. di grazia, s. bregante, a. ibatici, t. lamparelli, e. furfaro, f. frassoni, c. viscoli, a. bacigalupo s. martino's hospital (genoa, it) introduction: invasive aspergillosis (ia) remains an important complication with high morbidity and mortality in patients undergoing haematopoietic stem cells transplant (hsct) according the donor type. materials and methods: we determined, with retrospective analysis, the incidence, risk factors for ia and outcome in 306 patients who received hsct from unrelated donor (mud), family mismatched donor, or cord blood between january 1999 and december 2006 in our unit. the diagnosis of ia was documented as proven or probable according to the 2002 european organisation for research and treatment of cancer eortc/niaid international consensus. we have also considered proven ia the presence of fungal invasion on autopsy. results: a total of 306 patients were included in the study, with a median follow-up of 297 days after hsct, (range 1-2709 days) (types of hsct: matched unrelated 60%, mismatched related 23%, mismatched unrelated 11%, cord blood 6%). there were 8 cases of proven and 37 probable ia, with prevalence of 14,7%. the median time to onset of was day + 53 (range: 4-449), with 30 (66%) cases diagnosed in neutopenia (before take o secondary neutropenia). there were 7 cases diagnosed on autopsy as a proven disease while antemortem they were classified as probable (2) or possible (4); no clinical suspicion was present in 1 patient. the diagnosis of probable ia was made by galactomannan platelia test in 29 patients and by sputum culture in 4. in 3 patients both criteria were present. in 29 cases ia was present within lungs only, whereas 14 patients developed disseminated ia. multivariate analysis identified the following risk factors for ia: late take of neutrophil cells (p=0.001) and steroid therapy (p=0.004). among 45 patients with ia, 34 died and 30 deaths were related to the mould infection. mortality was 76% (p<0.001). multivariate analysis, among patients with ia, for overall survival identified the following risk factors: atg use in conditioning regimen (p=0.046) steroid therapy, relapse, iga and cholinesterase at diagnosis of ia (p=0.009, 0.03, 0.041 and <0.001, respectively). conclusions: the prevalence if ia remains high among alternative hsct recipients, with few risk factors for ia. clinical and radiological presentation is highly aspecific and invasive procedures are rarely feasible. about concern survival of ia immuno deficiency at diagnosis seems to be important. henze (2) minimal residual disease (mrd) quantified prior to allogeneic sct has been shown to predict outcome in children with relapsed all in retrospective meta analysis. based on these results intense discussions were started as to whether transplant procedures should be adapted according to the mrd levels pre transplant. within the all-rez bfm group we have started a prospective trial evaluating the impact of pre-transplant mrd load in a well defined group of children who received their transplant in second or subsequent remission. between march 1999 and july 2005, 91 children with relapsed all treated according to the protocols all-rez bfm 96 or 2002 and receiving allogeneic sct in 2nd (n=77) or 3rd cr (n=14) have been enrolled. mrd quantification was performed within 40 days prior to sct by real time pcr using t-cell receptor and immunoglobulin gene rearrangements as clone-specific targets with at least 1 marker with a sensitivity of 10-4. probability of event free survival (pefs) and cumulative incidence of relapse (cir) in 45 patients with mrd ≥10-4 was 0.27 (±0.07) and 0.57(±0.08) compared with 0.60 (±0.08) and 0.13 (±0.06) in 46 patients with mrd <10-4 (p [efs, log-rank]=0.004; p [cir, gray] <0.001). clinical and therapeutical parameters were equally distributed between both subgroups. the difference in pefs and cir was more prominent in intermediate risk patients s2 (n=35) compared to high risk patients s3/s4/cr3 (n=56). thus, s2 patients with mrd ≥10-4 (n=14) showed pefs of 0.20 (±0.12) and cir of 0.73 (±0.15), where as patients who entered transplantation with mrd <10-4 (n = 21) had a pefs of 0.68 (±0.12) and a cir of 0.09 (±0.09); (p[efs] =0.020; p[cir] <0.001). high risk patients s3/4/cr3 who entered transplantation with a mrd load of <10-4 (n=25) showed a pefs and cri of 0.53 (±0.11) and 0.18 (±0.08), respectively. in contrast, pefs and cri were 0.30 (±0.09) and 0.50 se (±0.09) in patients who entered transplant with higher mrd load of > mrd-4. multivariate cox regression analysis revealed mrd as the only independent parameter predictive for efs (p=0.006). mrd prior to allogeneic sct proves to be the most important risk factor for outcome post transplantation. new strategies with modified sct procedures including conditioning regimen, graft manipulation, and gvhd prophylaxis and or post transplant intervention strategies are warranted and will be performed to improve prognosis in patients with a high risk of relapse. acute lymphoblastic leukemia (all) remains the most common indication for unrelated donor bone marrow transplantation in children. there have been major advances in conditioning regimes; supportive care and availability of donors, all of which have made transplant a viable treatment option for patients with relapsed or high-risk disease. we retrospectively analyzed data from 371 consecutive allogeneic transplants for all performed at the royal bristol hospital for children from october 1987 until september 2007. 371 transplants were performed in 357 patients with all. disease status at the time of transplant was complete remission(cr)-1, n=72; cr-2, n=227, cr-3, n=59 , children not in complete remission n=13.(see table 1 ) all patients were pre-treated on the current mrc-ukall protocols for the time period. eighty-six patients received stem cells from identical siblings or relatives. matched unrelated donors were used in 171 children, mismatched unrelated donors in 93, haplo-identical family donors were used in 21 children. included in these totals are 7 umbilical cord blood donations. cyclophosphamide and total body irradiation was used as conditioning therapy with the exception of children under the age of two and children receiving second transplants. ciclosporin a was used for graft verses host disease (gvhd) prophylaxis. short course methotrexate was given to mismatched and sibling donors. in the unrelated group t-cell depletion was effected using campath monoclonal antibodies. the majority of patients died due to disease relapse. the incidence of chronic graft verses host disease was low with correspondingly high karnofsky scores. this study offers a unique opportunity to analyze data from a large number of patients consistently pretreated according to united kingdom national leukaemia protocols and with a uniform approach to transplant conditioning, gvhd prophylaxis and supportive care over a 20 year period. background: intravenous bu has been used in the two dutch pediatric stem cell transplantation centers in various myeloablative regimens with different targets for the area under the curve (auc) and different dosing regimens of bu (once or four times daily). we retrospectively analyzed the association between bu exposure expressed as auc and clinical outcome. methods: all children, transplanted between 2001-2006, receiving intravenous bu as part of a myeloablative regimen, were included. patients were separated into quintiles based on the total auc of four days of treatment. the association with the primary endpoints death, graft-failure or relapse, efs and the secondary endpoints (acute graft-versus-host disease grade 2-4 (agvhd), veno-occlusive disease (vod) and mucositis grade 3-4), were tested using uni-and multivariate cox regression analysis. the lowest auc group was used as index group. results: 102 patients were included (46 malignant indications; 56 non-malignant indications). median age at transplantation was 3.1 years (range 0.2 to 21 years). the overall efs and survival were 68% and 72% respectively. in multivariate analyses a total auc between 72.5-80 mg*h/l was associated with highest efs (85%, p=0.029) and survival (90%, p=0.021). a lower auc bu was significantly associated with higher incidence of relapse or graft failure, whereas in the highest auc-percentile a high incidence of treatment related mortality (trm) was seen. covariates were hla-disparity and age. hla-mismatched donors and older children showed a significantly lower efs, the latter mainly due to a higher trm. agvhd occurred in 16%, vod in 22% and mucositis in 12% of patients. these side effects significantly correlated with a high auc bu in combination with the addition of melphalan to the conditioning regimen. the once daily dosing versus fourtimes daily did not influence any of the results. in conclusion a total auc of bu between 72.5-80 mg*h/l is associated with highest efs in malignant and non-malignant disease. a lower auc correlated with more relapse/graft failure especially in hla-mismatched donors. a higher auc was associated with more trm negatively influencing efs especially in older children. the occurrence of severe agvhd, vod and severe mucositis was mainly related to combination of bu and melphalan. dosing of busulfan in children by tdm to a target of 72.5-80 mg*h/l might improve efs. mechanical ventilation in a recent cohort of children after allogeneic haematopoietic stem cell transplantation: risk factors and outcome s. van gestel*, c. bollen, m. bierings, j. boelens, n. wulffraat, h. van vught university medical center utrecht (utrecht, nl) introduction: in previous studies, median intensive care unit (icu) mortality in children after hematopoietic stem cell transplantation (hsct) was 74% (range 25% -91%). it has been suggested that icu mortality decreased over time, but we could not confirm this in a recent meta-regression analysis (van gestel et al, submitted) . conclusions on mortality trends were limited, since recent outcome data from children requiring mechanical ventilation after hsct were scarce. objective: to assess risk factors for and outcome of mechanical ventilation in a recent cohort of children after allogeneic hsct. design: retrospective chart review. setting: a pediatric icu and hsct centre in a university hospital in the netherlands. patients and methods: all children who received an allogeneic hsct between january 1999 and april 2007 were included. patients who required endotracheal mechanical ventilation for more than 24 hours were identified. potential risk factors for the requirement of mechanical ventilation and for icu mortality were recorded. uni-and multivariable analyses were done to identify risk factors. results: 175 hscts were performed in 150 patients. thirtyfive patients (23% of hsct recipients) received mechanical ventilation on 38 occasions. none of the potential risk factors was significantly associated with icu admission. there was a trend towards an increased probability of icu admission over the years. this suggests that transplantations were carried out in sicker patients over time. sixteen admissions resulted in death on the icu, giving a case fatality rate of 42%. icu mortality was mainly associated with (persistence of) multiple organ failure on the first days of admission. none of the pre-admission transplantation characteristics significantly influenced icu mortality. based on their pediatric risk of mortality (prism) scores, our patients had a higher acuity of illness on icu admission than patients in previous studies. six-month survival in patients discharged from the icu was 82%. conclusion: we found a significantly lower icu mortality in a recent cohort of children after allogeneic hsct compared to previous studies, even though our patients were sicker. this can most probably be explained by improvements in transplantation medicine and icu treatment strategies. results from our study are promising, but need to be confirmed in other, preferably multi-centre, studies. early and comprehensive vaccination coverage in paediatric recipients of related and unrelated stem cell transplantation following coadministration of the pneumoccoccal conjugate vaccine prevenar™ and the hexavalent combination vaccine infanrix hexa™ r. meisel (1) children undergoing allogeneic hematopoietic stem cell transplantion (ahsct) lose protective immunity to vaccinepreventable disease and thus are at significant risk for lifethreatening infections. however, important issues on reimmunization after pediatric ahsct remain controversial due to the lack of prospective clinical trials. in the prospective multicenter vaccination trial ikast (nct 00169728) a total of 77 pediatric allohsct recipients (median age 8.3 (1.4-17.0) years) were therefore immunized with a primary series of three monthly doses of the hexavalent tetanus, diphtheria, pertussis, poliomyelitis, haemophilus influenzae type b and hepatitis b combination vaccine infanrix hexa (6vcv; glaxosmithkline pharma) along with the heptavalent pneumococcal conjugate vaccine prevenar (pcv7; wyeth pharma). vaccination was started at 6 months after transplantation, irrespective of immunosuppressive therapy or gvhd, with a subsequent booster dose at 18 months. immunogenicity was analysed by assessment of antibody concentrations and adverse events were prospectively collected. prior to immunization only 12.8% and 8.2% of patients (pts) exhibited protective antibodies towards pcv7 and 6vcv vaccine antigens, respectively. as a result of highly significant increases in mean antibody concentrations (p<0.001) protection to all pcv7 and 6vcv antigens was achieved in 85.1% and 89.8% of pts after primary immunization within the 1st year after allohsct, and this was independent of patient age, conditioning regimen, stem cell source, and most importantly, donor type (related vs. unrelated) and in vivo t cell depletion (all p-values>0.05). nine months later, prior to booster vaccination, 63.8% and 83.7% of pts retained protective antibody concentrations. however, mean antibody concentrations had dropped by a factor of 1.9-4.8 (p<0.001) except for pneumococcal serotype 14, thus underlining the need for subsequent vaccination. following booster immunization, antibody concentrations increased 2.4-19.1 fold (p<0.001) indicating robust memory responses, and 91.5% and 100% of pts achieved protective antibody levels against all pcv7 and 6vcv antigens. vaccination was well tolerated with no vaccine-related saes. our data show that early immunization of pediatric ahsct recipients according to our simple revaccination schedule is safe and provides early and comprehensive vaccination coverage during the first 2 years following ahsct from both related and unrelated donors. treosulfan-containing regimens achieve high rates of engraftment associated with low transplant morbidity and mortality in children with non-malignant disease and significant co-morbidities b.f. greystoke* (1), s. bonanomi (2) (1), p. naik (1), k. rao (2) , n. goulden (1) , p. amrolia (2) , r.f. wynn (1) , p.a. veys (2) (1)pendlebury children's hospital (manchester, uk); (2)great ormond street hospital (london, uk) treosulfan is an alkylating agent with both immunosuppressive and myeloablative properties, which has recently been introduced as a conditioning agent in allogeneic and autologous haemopoietic stem cell transplantation (hsct). early studies have been performed principally in adult patients with malignant disease in whom the drug appears to be well tolerated with a low incidence of regimen-related toxicity. we report the use of treosulfan in 32 consecutive children undergoing sct for non-malignant disease: immunodeficiency (n=18), metabolic disease (n=9), osteopetrosis (n=4) and other (n=1). patients received a total treosulfan dose of 36 or 42gms/m²/patient given in 3 divided doses on successive days. a range of other conditioning agents and serotherapy was administered to patients who underwent family donor sct (n=11), or unrelated donor sct (n=21). bone marrow (n=17), peripheral blood (n=9) and cord blood (n=5) were used as a stem cell source and in one patient a combination of marrow and cord blood from the same sibling donor was used. one patient (3%) died of early transplant-related complications. transplant morbidity was limited and there was no vod other than in this patient. mucositis was mild and many patients did not require parenteral nutrition. nappy rash with ulceration was noted in many patients. twenty-eight patients (87.5%) established donor cell engraftment which was full in 24 (85.7%). in 25 patients (78%) there was stable donor engraftment, including sufficient lineage specific chimerism, to correct the underlying condition. four patients required additional transplant procedures to maintain adequate donor-derived haemopoiesis, and two patients are under consideration for further hsct. twentyseven patients survive with a median follow up of 417 days. there were 4 late deaths due to progression of the underlying disease, gvhd or infection. treosulfan based conditioning regimens achieve excellent engraftment with reduced regimen-related toxicity and in children with non-malignant disease at high risk for both regimen-related toxicity and graft failure. preliminary results indicate that in a haploidentical setting cd3/cd19-depleted grafts may be advantageous regarding engraftment and immunreconstitution. since effector cell with potential antileukemic activity are cotransfused, such grafts may be suited in particular for patients with insufficient remission. nk cells have been shown both in vitro and in vivo to mediate positive effects regarding engraftment, gvhd, immunreconstitution and relapse and are an important part of the cd3/19 depleted grafts. we analyzed nk activity in 30 cd3/19 depleted grafts, which was low compared to fresh isolated pmncs. several cytokines and cytokine combinations for activation of the grafts were tested and the strongest enhancement in nk activity could be obtained with il-15. we therefore developed a protocol for the overnight incubation of the grafts according to gmp. stimulated grafts were transplanted seven times either at day 0 or as an immunotherapeutical approach after transplantation and were well tolerated. median cell counts for infused cells/kg bodyweight were 46x10 6 cd56+/3-, 13x10 3 cd3+, 9x10 3 cd19+, 88x10 6 cd14+ and 3,1x10 6 cd34+ cells. no occurrence of agvhd was seen in these patients after infusion of the cells. enhanced nk activity after stimulation could be demonstrated in vitro against k562 or primary leukemic blasts. nk cells showed increased proliferation capacity in brdu assay and [3h]-thymidine incorporation assays, especially after additional stimulation with il-2 while t cells showed only a moderately enhanced proliferation against third party stimulators or with okt3. therefore activation of haploidentical t cell depleted grafts with il-15 is a promising tool to enhance the activity of infused nk cells and should be further investigated in clinical trials. background: development of an effective strategy for patients with epmd. methods: 328 patients (pts) were registered. median age is 16.2 years (yrs) (0.4-49). primary site was extremity in 98 pts and axial/other in 217 pts (45% in the pelvis). metastatic spread was bone marrow (bm) only in 33 pts, bone only in 137 pts and bone & bm in 105 pts, other (mainly lymph nodes) metastatic sites in 33 pts. tumour volume was above 200 ml in 200 pts. six vide induction cycles were completed by 88%. local treatment included surgery when possible and/or radiotherapy (rx) as indicated. recommended hdt was busulphan (bu) 600mg/m² and melphalan (mel) 140 mg/m² with pscr. median follow up is 3 years (range, 0-7.5). results: partial remission or better was achieved after cycle 6 in 76%. the overall survival at 3 years is 32%±3. in patients with bm/bone metastasis, significantly favourable univariate factors in the unselected cohort at diagnosis (dx) were age < 14 yrs (event free survival at 3 yrs (efs) 39%, p<0.001), metastatic site (efs: bm only 47%, bone only 22%, bm+bone 15%, p=0.003), single bone lesions only (efs 37%, p=0.004), primary tumour site (efs: extremities 33%, chest/spine/hn 27%, abdomen pelvic 22%, p=0.035) and tumour volume of <200ml (efs 44%, p<0.001). ). multivariate analysis identified four major risk factors at dx: primary tumour volume >200ml p<0.001 (rr 2) and > 5 bone metastases p=0.055 (rr 1.7), age above 14 p=0.012 (rr 1.6), bm metastasis p=0.042 (rr=1.4). for pts receiving bumel it is noteworthy that 46pts of <14a and epmd achieved an efs of 46% in comparison to older counterparts >14a (efs 23%, p=0.007). conclusion: further strategy refinement and validation of hdt appears necessary within investigational , ideally randomised studies. allogeneic haematopoietic cell transplantation for chronic lymphocytic leukaemia with 17p deletion: a retrospective ebmt analysis j. schetelig*, a. van biezen, r. brand, d. caballero, r. martino, m. itala, j. garcía-marco, l. volin, n. schmitz, r. schwerdtfeger, a. ganser, f. onida, b. mohr, s. stilgenbauer, m. bornhäuser, t. de witte, p. dreger on behalf of the chronic leukemia working party, ebmt purpose: patients with advanced chronic lymphocytic leukaemia (cll) and 17p deletion have a very poor prognosis even after intensive chemotherapy. while allogeneic hematopoietic cell transplantation (hct) has the potential to cure patients with advanced cll it is not known whether this holds true for patients with 17p deletion. patients and methods: patients with 17p-cll who had received hct were identified by an ebmt-based survey. baseline data were downloaded from the ebmt database. additional information on the course of the disease, the cytogenetic diagnosis and last follow up was collected by a questionnaire. data were analysed as of february 2007. results: 56 patients were identified. twelve patients with autologous hct, haplo-identical donors or the detection of 17p-after hct were excluded from further analysis. 44 patients had received an allogeneic hct between march 1995 and july 2006 from a matched sibling donor (n=24) or an alternativ donor (n=20). the median age at hct was 54 years. the diagnosis of deletion 17p-was made by fish in 82% and by conventional banding in 18% of patients. the median interval between first diagnosis and detection of 17pwas 2.4 years and the median interval between detection of 17p-and hct was 0.5 years. patients had received a median of 3 chemotherapy regimens, including fludarabine in 98% of patients. at hct, 53% of patients were in remission. reduced intensity conditioning was applied in 89% of patients. 93% of the patients received peripheral blood stem cells. gvhd prophylaxis was performed heterogeneously. one patient experienced primary graft failure. acute gvhd grades ii to iv occurred in 44% of patients and extensive chronic gvhd in 46% of patients. after a median follow-up of 23 months (range, 2 to 90 months) of 24 patients who are alive, 18 were in complete remission, 4 in partial remission and 2 patients had progressive disease at last follow up. 4-year overall survival and progression-free survival was 47% (95% ci, 29% to 65%) and 38% (95% ci, 20% to 56%). the cumulative incidence of relapse at 4 years was 35% (95% ci, 12% to 57%). no additional relapse occurred in six patients with a follow-up between 4 and 7.5 years. conclusion: allogeneic hct has the potential to induce longterm disease-free survival in selected patients with advanced 17p-cll. given the otherwise very dismal outcome of this disease, prospective studies on allogeneic hct earlier in the course of 17p-cll seem warranted. separating patients with myelofibrosis with myeloid metaplasia into risk groups for allogeneic haematopoietic cell transplantation -results of a german multicentre analysis f. collenbusch, r. schwerdtfeger, m. schleuning, c. schmid, j. finke, m. stadler, m. bornhaeuser, d. messerer-schmid, h objective: we aimed to derive risk factors for allogeneic hematopoietic cell transplantation (allohct) in patients with s53 myelofibrosis with myeloid metaplasia (mmm) from retrospective analysis. patients: between 1999 and 2006 97 patients (pts) from 15 german centers, median (md) age 51 (19-66), were grafted from 41 related and 56 unrelated donors. 74 pts had primary, 23 secondary mmm. at allohct 32 pts were dupriez score 0, 37 score 1 and 28 score 2, 49 needed red cell transfusions. chromosomal and molecular analysis was available from 75 and 51 pts resp.: 51 pts had favourable, 24 unfavourable cytogenetics, 31 pts were jak2 mutated. conditioning was of standard, intermediate and reduced intensity in 27, 57 and 13 cases. 28 pts received bm, 69 pbsc. 5 boosts and 16 donor lymphocyte transfusions were given for 16 relapses, 2 incomplete chimerisms and one graft failure. results: at a md follow up of 985 (100 -2630) days probability of overall and relapse free survival (os and rfs) was 62 and 39%. neither time from diagnosis to transplant, age, bsymptoms, marrow fibrosis, splenomegaly, jak2, hemoglobin <10 g/dl, platelets <100/nl, ldh, comorbidity (cci or hct-ci), conditioning, type of donor nor graft source were predictive for os. log rank test showed a trend towards lower survival in cases with dupriez score ≥1 (p=0.06) and transfusion dependence (p=0.1). solely circulating blasts >1% (p=0.016), monocytes >1/nl (p=0.008) and cytogenetics (p=0.009) were predictive for survival after allohct. cox regression analysis within pretransplant variables revealed cytogenetics (p=0.016) and monocytes (p=0.036) as independent factor for os. for rfs only cytogenetics retained independence (p=0.003). combining cytogenetics, monocytes, blasts and transfusion dependence to a risk score from 0 to 4 allowed to discriminate between good (0-1), intermediate (2) (3) and high risk (4) pts for os (83, 45, 0%, p=0.0008) and rfs (68, 34, 0%, p=0.0013). following allohct pts with limited cgvhd had a projected os and rfs of 84% and 81% as opposed to 43% rfs for no and 20% rfs for extensive cgvhd, resp. (p=0.018). 7 pts treated with cellular immunotherapy achieved a partial and 7 a complete response. conclusions: pts with mmm can be scored as low, intermediate and high risk for allohct according to pretransplant disease characteristics. evidence for a potent graft-versus-mmm-effect suggests exploitation of adjuvant cellular immunotherapy to improve current results. syngeneic bmt for cml in chronic phase: update of the seattle results a. fefer*, j. radich, t. gooley, l. holmberg, m.e. flowers, s. pavletic, r. storb, f. appelbaum fred hutchinson cancer research center (seattle, us) to determine the efficacy of syngeneic bmt as treatment for chronic phase cml (cml-cp), we reviewed the results of all patients (pts) we transplanted between 1976 and 2000, before the gleevec era. of 34 pts, 5 died within 4 months (mos)due largely to pulmonary problems. of the 29 pts who went into a complete cytogenetic remission (cr), 15 relapsed at a median time of 2 years after bmt, but 14 remained in cr for up to 31 (median,19) years. all 9 pts tested were negative by pcr when last tested at 2-23 (median,13) years. thus, enduring cytogenetic and molecular cr's were achieved with syngeneic bmt, in the absence of an allogeneic graft vs leukemia (gvl) effect. at 20 years, the estimated probability of relapse, overall survival and relapse-free survival was 46%, 42% and 28%,respectively. to assess the influence, if any, of the conditioning regimen on the relapse rate, we subdivided the 34 pts into 3 groups transplanted in 3 time periods with 3 different conditioning regimens, as follows: group i (n=14), 1976-81, dimethylbusulfan (dmb) 5mg/kg iv, cyclophosphamide (cy) 60mg/kg/day x2 and total body irradiation (tbi) 10gy at a single exposure; group ii (n=10), 1982-88, cy 60mg/kg/d x2 & tbi 2gy/day x6; group iii (n=10), 1989-2000, busulfan (bu) 2mg/kg/day x4 po, cy 30mg/kg/day x2 and tbi 2gy/day x6. the median time from diagnosis to bmt for the 3 groups was 11, 3 & 5 months, respectively. relapses occurred in 5 pts in group i at a median of 4 years, 8 pts in group ii at a median of 1 eayr, and 2 pts in group iii at 1 and 4 years. thus, the relapse rate with the cy/tbi regimen was significantly higher than that for the regimens containing dmb or bu (hazard rate (hr) of relapse for group ii vs i was 4.17, p=0.01, and for group ii vs iii, hr 4.80, p=0.05). however, cy/tbi was less toxic, with no early bmt-related deaths in contrast to 2/14 and 3/10 deaths in groups i and iii. finally, we compared the risk of relapse in groups i and iii combined i.e.twins conditioned with dmb/bu-containing regimens, with that of 366 cml-cp pts who underwent bmt from allogeneic matched siblings after cy/tbi at our center. the risk of relapse is quite similar (hr=1.06, p=0.87). these results suggest that either the dmb or bu can eradicate a number of cml cells similar to that destroyed by an allogeneic gvl effect and/or that the dmb or bu can somehow induce a gvl effect exerted by syngeneic cells. we have previously shown that levels of the polycomb group (pcg) gene bmi-1 rna were significantly higher in advanced phase than in chronic phase (cp) chronic myeloid leukaemia (cml). in addition, in patients treated with hu and ifn-a, low bmi-1 expression was associated with an improved overall survival (os) (blood 2007). here, we investigated whether bmi-1 and other previously established prognostic genes (cd7, pr-3 and ela-2) are implicated in the prognosis of cml in the context of allogeneic stem cell transplantation (allo-sct). we studied 84 cp-cml patients who received allo-sct from hla-identical related donors. cd7, pr-3, ela-2 and bmi-1 expression was assessed by q-rt/pcr in the recipients pbmcs collected before allo-sct. the median expression level for each gene was used to segregate the patients into 2 groups (low: gene expression <median, and high: >median). the median fu post-allo-sct was 9.9 (range, 1.7-23.9) years. the median ebmt-gratwohl score was 3. none of the 4 tested genes showed any significant association with engraftment or with graft rejection. cd7, pr-3 and ela-2 expression was not associated with os. however, in contrast to our previous findings in the non-allo-sct setting, patients displaying a high bmi-1 expression level prior to allo-sct had significantly better os than those with low expression (p=0.005). when bmi-1 was included in a multivariate survival model and adjusted for the other prognostic variables, a high expression was found to be an independent marker associated with better survival (rr=2.72, 95%ci;1.1-6.9;p=0.034). given the impact of bmi-1 expression level on os, without a significant association with relapse, and since neither bmi-1, nor the other genes showed any significant association with leukemia-free survival, we assessed their impact on trm. there was a striking and significant association between acute gvhd and bmi-1 expression, not only in overall incidence (low bmi-1: grade 0-1 (n=21), grade 2 (n=10), grade 3-4 (n=9); high bmi-1: grade 0-1 (n=32), grade 2 (n=9), grade 3-4 (n=1); p=0.005), but also in cumulative incidence at day 100 (48% vs. 24%, p=0.016). in multivariate analysis, a low bmi-1 expression level was associated with an increased risk of grade 2-4 acute gvhd (rr=2.85, 95%ci; 1.3-6.4; p=0.011). these results suggest that bmi-1 can serve as a biomarker for predicting outcome in cp-cml patients receiving allo-sct. such measurement allows for tailored therapeutic intervention, including informed recommendation for allo-sct in patients failing tyrosinekinase inhibitors. haematopoietic stem cell transplantation in tprolymphocytic leukaemia: a retrospective ebmt analysis w. wiktor-jedrzejczak*, r. brand, a. van biesen, e. carreras, v. leblond, g. cook, m. ethell, a. nagler, t. ruutu, m.l. brune, j. schetelig, t.m. de witte, p. dreger on behalf of the clwp ebmt t prolymphocytic leukemia (t-pll) is a rare, aggressive neoplasia of t lymphoid lineage which is characterized by poor survival of less than one year. incidental reports suggest that both autologous and allogeneic hematopoietic stem cell transplantation (hsct) might be effective in this disease. however, no larger series on the efficacy of hsct in t-pll has been reported to date. therefore the purpose of the present study was to analyze the outcome of transplants for t-pll registered at the ebmt database. results: eleven patients who had undergone autologous transplantation (auto-sct) and 33 patients who had undergone allogeneic transplantation (allo-sct) were identified from the database and verified as t-pll. in the auto-sct group, there were 9 males and 2 females, with a median age of 59 (37-64) years. 5 patients were transplanted within the 1st year of diagnosis, and the remainder in later phases. 2 patients received tbi in their conditioning, while 8 received chemotherapy only. engraftment and recovery was prompt. there was one case of non-relapse death (nrm). 5 patients relapsed within the first 2 post-transplant years, translating into a median event-free survival (efs) of 14 months and a median overall survival (os) of 22 months. a single patient had prolonged disease control until relapse after 53 months.in the allo-sct group (20 males/13 females), median age was 51 (24-71) years; 18 patients (55%) had been transplanted within the 1st year of diagnosis. status at allo-sct was complete or partial remission in 18 patients (55%), and more advanced disease or unknown in the remainder. 14 patients (42%) received reduced intensity conditioning. donors were hla-identical siblings in 16 patients (48%), matched unrelated donors in 16 patients, and a mismatched unrelated donor in one patient. engraftment was documented in 29 of 30 patients (97%) with information available. 2-year nrm was 25%. 10 of 11 relapses observed occurred during the first post-transplant year, mainly in patients transplanted in advanced disease, translating into biphasic survival curves with initial steep decline (median efs 7 months, median os 11 months) but still 32% efs at 24 months suggesting sustained disease control in a subset of patients. conclusions: these data indicate that allo-sct may provide effective disease control in selected patients with t-pll. prospective investigation of allo-st in eligible patients with this otherwise fatal disorder seems to be warranted. high-dose chemotherapy with autologous stem-cell support versus standard-dose chemotherapy: metaanalysis of individual patient data from 15 randomised adjuvant breast cancer trials t. demirer*, n.n. ueno, m. bregni, d with autologous haematopoietic stem cell transplantation (hsct) for the treatment of primary poor risk breast cancer (bc) patients (pts) has lost favour over recent years in the oncology community. this is mainly due to the worrisome high mortality and morbidity of the procedure, and the lack of a clear survival benefit in early randomized studies. recently reported trials have demonstrated that hdc with hsct could still have a role in selected subgroup of pts, namely pts with ≥ 10 positive axillary lymph nodes (ln) and in pts with her-2 negative tumours. aim of this study is to re-evaluate toxicity and efficacy of hdc with hsct in a large cohort of pts receiving hdc in italy between january 1, 1990 and december 31, 2005. 1294 bc pts receiving hdc for poor risk bc were identified in the gitmo registry. in 1183 patients with >3 ln, a thorough data set including biological characteristics, toxicity and follow up was available. median age was 46 years (24-66), 62% of pts were pre menopausal at treatment, 71% had an endocrine responsive tumours and 43% had a her-2+ tumour. median number of positive axillary ln was 15 (4-63), with 23% of pts having ≥ 20 ln+. 73% of pts received alkylating agents-based hdc as a single procedure while 27% received epirubicin or mitoxantrone-containing hdc, usually within a multi-transplant program. transplant related mortality (trm) at 100 days was 0.7%, while late cardiac and secondary tumour related mortality were around 1% overall. with a median follow up of 74 months, median disease free survival (dfs) and overall survival (os) in the entire population were 6.5 and 7.5 year, respectively. exploratory subgroup analysis demonstrated that os was significantly better in endocrine responsive tumours (p=0.0000), while menopausal or her-2 status did not affect survival. in 85 poor prognosis pts with er, pgr and her-2 negative tumours (median ln+ = 18), median os was 110 months. median os was significantly better (p=0.0000) in patients receiving multiple transplant procedures, this effect being particularly evident in the ≥ 10 ln+ population. in conclusion, our retrospective analysis suggests that hdc with hsct has lower trm than expected and high efficacy in well defined subgroups of patients. multiple transplants seem more active than single hdc procedures. this analysis could be useful in selecting well defined patient populations in which to re-address the role of hdc as adjuvant treatment. long-term follow-up of metastatic renal cancer patients undergoing reduced-intensity allografting m. bregni*, m. bernardi, p. servida, a. pescarollo, r. crocchiolo, e. treppiedi, f. ciceri, j. peccatori scientific institute san raffaele (milan, it) objective: stem cell transplantation from a hla-compatible sibling donor has been advocated as adoptive immunotherapy for cytokine-resistant, metastatic renal cancer (rcc). however, the recent introduction of several targeted therapy compounds has reduced the interest in this therapeutic strategy. we have reanalyzed our transplant series with the aim to detect long-term benefit form allografting. methods: from february 1999 to may 2005, 25 patients with cytokine-refractory rcc received a reduced-intensity allograft from an hla-id sibling donor. median age was 53 years; most (24) had clear-cell histology. median number of previous treatments was 1 (0-3). median days from diagnosis to allograft were 822. all patients received a thiotepa, fludarabine, and cyclophosphamide conditioning regimen, and a cyclosporine-based gvhd prophylaxis. six patients received dli at escalating doses for progressing or nonresponding disease. results: one-year-os was 48% (95% ci: 28-68), and 3y-os was 20% (95% ci: 4-36). at a median observation time of 65 months, 5 patients are alive, one in cr, one in vgpr, and three with disease. we have analyzed the correlation of the following variables with survival: pre-transplant disease status, age at transplant, time from diagnosis to transplant, total infused cells, infused cd34+ cells, infused cd3+ cells, bm chimerism at +30, post-transplant disease status at +30 and +90, best response, day of best response, csa withdrawal day, infusion of dli, occurrence of gvhd. at univariate analysis, numbers of cd34+/kg infused cells, best response, and disease status at +90 significantly correlated with survival. at multivariate analysis, only disease status at +90 retained statistical significance (p=0.002), while cd34+ cells/kg retained marginal significance (p=0.059). conclusion: twenty percent of cytokine-refractory rcc patients are alive at a median 65 (40-72) months after allografting. all these patients have received >5x10 6 cd34+ cells/kg, and had stable or responding disease at +90 after transplant. three patients are receiving sorafenib, and two are in cr/pr without further therapies. reduced-intensity allografting is able to induce long-term disease remission in a fraction of relapsed rcc patients. it is unknown if relapse or pd after targeted therapy will be susceptible to allograft-mediated gvt effect. the place of allografting in the treatment of metastatic rcc, alone or in combination with targeted therapies, needs reappraisal. haploidentical family donors represent the ideal solution to offer to every patient with high risk leukemia the potential cure of marrow stem cell transplantation. extensive application of haploidentical transplantation (haplo-sct) is limited by high rate of late transplant related mortality (trm) and relapse associated with the delayed immune reconstitution secondary to the procedures for severe graft-vs-host-disease (gvhd) prevention. in a haplo-sct phase i-ii multicenter, open, no-randomized, trial sponsored by molmed spa, we infused donor lymphocytes genetically engineered to express the suicide gene herpes simplex thymidine kinase (tk-dli) to induce early immune reconstitution, while selectively controlling gvhd. between september 2002 and september 2007, 51 patients (pts) -median age 48-with high-risk hematologic malignancies were enrolled, 29 out of 51 pts were in complete remission (cr). after myeloablative conditioning regimen, 48 pts received a median 13x10 6 /kg cd34+ and 1.0x10 4 /kg cd3+ (median time to engraftment: 2 weeks). no immune reconstitution were observed in absence of tk-dli. twenty-seven pts received tk-dli: 22 pts obtained prompt immune reconstitution with cd3+>100/mcl at day+75 (median) from haplo-sct and day+23 from tk-dli. eleven pts developed gvhd (10 acute gvhd grade i-iv and 1 chronic gvhd) that was always abrogated by the suicide gene induction. the 3-year trm was 26%, with last infectious event at day 166 post transplant, for pts treated with tk-cells, and 69% for pts who didn't receive tk-cells (p<0.0001). immune reconstitution obtained with tk-cells infusion correlated with: 1. rapid development of a wide t-cell repertoire, 2. detection of high frequencies of t-cells specific for opportunistic pathogens, 3. abatement of the incidence of infectious adverse events (ae) and serious ae. the 3 years lfs was 45% for pts who achieved immune reconstitution and 9% for pts who failed immune reconstitution (p:<0.0001). this strategy is feasible and effective in providing immune reconstitution in haplo t-cell-depleted setting. in uni-and multi-variate analysis both status at transplant and immune reconstitution are significant risk factor. infusion of tk-cells could significantly extend the application of haplo-sct. a randomized phase iii study comparing tk-dli versus any t cell repletion strategy after haplo-hsct in high risk acute leukemia is now starting. ie-1 and pp65 specific peptide pools for the generation and expansion of cmv-specific donor t-cells after haploidentical stem cell transplantation: feasibility and first clinical experiences s. ganepola* (1), p. reinke (2) , c. gentilini (1) , m. hammer (2) , m. schmidt-hieber (1), c. tietze-bürger (1), k. freyberg (1), d. volk (2) , e. thiel (1) , l. uharek (1) (1)hematology/oncology/transfusionmedicine (berlin, de); (2)charite berlin campus mitte (berlin, de) for immunocompromised patients, rapid reconstitution of cytotoxic t cell function is mandatory for the control of human cytomegalovirus (cmv) infection and disease. the cmvassociated proteins pp65 and ie-1 are important components of a relevant cmv-directed immune response. here we report the first clinical experiences concerning feasibility and safety with a novel method for the generation of cmv-specific t-cells by stimulation with a peptide-pool containing pp65 and ie-1. material and methods: after apherisis of 1-1.2x10 9 pbmc from 4 healthy donors (2 igg positive, 2 igg negative) and 2 patients with proven cmv disease, cells were further processed under gmp-conditions. cells were stimulated with peptide pools representing the pp65 (ul83) and ie-1 (ul123) proteins and ifng producing cells were selected with the cytokine-capture-assay (miltenyibiotec) and further expanded in cell-culture conditions on a 24-well-plate. after performance of intern and extern quality controls, cells were freshly retransfused and/or kryoconserved in defined portions for further redonations. results: in 5/6 rounds we could expand cmv-specific t-cells. expansion-rates ranged from 1.2 fold to 71.1 fold (median 9.4) of the initial cell count. in a median time of 15 days (range 14-23 days), cell counts expanded from 1.2 fold up to 71.1 fold (median 9.4). facs-analysis at the end of the culture revealed that in median 80% of the cells were cd8+ (range 2.5-92.3%) and, respectively, 20% were cd4+ (range 7.2-89.6%). in cytotoxicity assays, a lysis of 50-83% (median 53%) of lcl-line cells could be achieved. so far, two patients with therapy refractory cmv disease received in vivo expanded polyvalent t-cells against multiple (ie-1/pp65) cmv epitopes. no serious adverse events occurred during the first days after administration and cmv antigenemia was decreasing in one patient. however, since both patients finally died from a septical multi-organ failure, we were not able to assess the long lasting impact of the manoeuvre on the control of cmv-disease. conclusion: selection and subsequent expansion of cmvspecific t-cells is possible with this method which allows an up to 70-fold expansion of cd8+ cells. the use of pp65 and ie-1 specific peptide pools for the prevention or treatment of cmv infection is feasible and could be of superior effectiveness as compared to approaches directed against a single cmv epitope. lmp2-specific tcr gene therapy for hodgkin's lymphoma d.p. hart, s. thomas, s. xue, h.j. stauss, e.c. morris* university college london (london, uk) ebv-positive hodgkin lymphoma typically demonstrates latency ii antigen expression, characterised by loss of most ebv antigens except for the latent membrane protein (lmp) 1 and 2 and the ebna-1 protein. reed sternberg cells expressing lmp2 can be a target for antigen-specific immunotherapy, but lmp2-specific autologous ctl for adoptive immunotherapy are difficult to generate due to poor immunogenicity. t cell receptor (tcr) gene transfer using retroviral vectors containing the tcr alpha and beta chain genes can reproducibly redirect the antigen specificity of a t cell population. the aim of this study was to generate a retroviral tcr construct suitable for the rapid and efficient production of lmp2-specific ctl. retrovirally introduced tcrs compete with endogenous tcrs for cd3 molecules required for assembly of the tcr complex. this competition may limit surface expression of the introduced tcr resulting in a transduced t cell with poor functional avidity. in an attempt to generate a 'highly competitive' lmp2-tcr the following modifications were made to the retroviral vector construct: i) nucleotide sequences were codon-optimised for efficient translation in human cells; ii) the constant region of each tcr chain was altered to contain murine sequences to enhance cd3 binding; and iii) the tcr alpha and beta chain genes were linked by a self-cleaving 2a sequence. the unmodified hla-a2-restricted lmp2-specific tcr was poorly expressed in primary human t cells (up to 2.5% of viable cd3+ t cells, as detected by facs analysis using monoclonal anti-vbeta13 antibodies), suggesting that it competed inefficiently with endogenous tcr chains for cell surface expression. however, retroviral transfer of the modified lmp2-tcr into human t cells improved lmp2 tcr expression to 55-65% cd3+ t cells. the transduced cells bound hla-a2/lmp2 pentamer, showed peptide-specific ifngamma and il2 production and killed target cells displaying the lmp2 peptide. importantly, expression of the introduced lmp2-tcr suppressed expression of almost the entire repertoire of endogenous tcr combinations, including 'mis-paired' tcrs. 'mis-paired' tcrs contain an introduced alpha chain paired with an endogenous beta chain and vice versa. the antigen specificity of such mispaired tcrs is unknown and could be auto or allo-reactive. modified tcr sequences, producing 'dominant' tcrs may improve the efficacy and reduce the potential risks of tcr gene therapy a novel form of adoptive immunotherapy. allogeneic stem cell transplantation in patient with major histocompatibility complex class ii immunodeficiency: a single-centre experience h. al-mousa*, z. al-shammari, a. al-ghonaium, h. al-dhekri, s. al-muhsen, r. arnaout, a. al-seraihy, a. al-jefri, a. al-ahmari, m. ayas, h. el-solh king faisal specialist hospital (riyadh, sa) background: major histocompatibility complex class ii (mhc ii) deficiency is a rare combined immunodeficiency disease. allogeneic bone marrow transplantation (bmt) is considered the only available curative treatment. survival rate post bmt is lower than other forms of primary immunodeficiencies. these differences were observed for both bmt performed with matched and non-matched donors. patients and methods: between june 1994 and august 2007, thirty two children with mhc ii deficiency underwent thirty seven bmt procedures at king faisal specialist hospital and research centre, riyadh, saudia arabia. five patients required second bmt trial. median age at bmt was 27 months (range, 1-129 months). the source of stem cells was unmanipulated marrows from hla-genoidentical siblings in 22 pts, hla-phenotypically identical related donors in 9 pts and umbilical cord for 1 patient. conditioning was with one of 4 regimens, regimen a (19 pts): busulfan (bu), cyclophosphamide (cy) and etopside (vp-16), regimen b (3 pts): bu/cy and anti-thymocyte globulins (atg), regimen c (2 pts): bu/cy/vp-16 and atg and regimen d (13 pts): fludarabine, melphalan and atg. median cd34 dose was 8.3 x 10 6 /kg (range, 1.5-20.7 x 10 6 /kg). graft-versus-host disease (gvhd) prophylaxis consisted of cyclosporine (csa) and methorexate (mtx) in 21 pts, csa alone in 15 pts and csa and steroid in 1 patient. results: 24 pts had adequate immune reconstitution and sustained engraftment (assessed by short tandom repeats) ranged from 24-100% for lymphoid line and 5-100% for myeloid line. seven pts (4 pts were post second transplant) died secondary to sepsis, multiorgan failure and primary disease. acute gvhd was seen in 20 pts and 4 pts developed chronic gvhd. the overall disease free survival rate was 75% with a median follow up of 5.3 years (range 0.5-10.2 years). low survival rate (20%) was seen in patients who underwent second bmt. conclusion: bone marrow transplantation (bmt) can cure the disease, provided it is performed before complications leading to severe organ failure develop. second bmt is associated with high rate of mortality. further studies and long term follow up are required to determine the appropriate conditioning regimen. to analyze the effects of cd34-trail+ cells on tumor vasculature, tumorbearing mice were perfused with sulfo-biotin and tumor endotelial cells (tec) were then revealed by horseradish peroxidase (hrp)-conjugated streptavidin. as compared with cd34-mock-or soluble (s)trail-treated mice, a 24-hour treatment with cd34-trail+ cells significantly (p ≤.001) reduced microvessel density (1850 ± 1139 vs 2227 ± 915 vs 757 ± 562 vessel per 1 x 10 5 tumor cells, respectively) and increased the thickness of the vessell wall (3.7 ± 1 µm vs 3.4 ± 1 µm vs 6 ± 1 µm, respectively), suggesting that cd34-trail+ cells induce an early vascular disruption leading to a progressive disintegration of the vascular bed. confocal microscopic imaging of tumor sections double-stained with anti-cd31 and anti-trail-r2 showed that this receptor was expressed by 8 -12% of large tumor vessels. interestingly, upon treatment with cd34-trail+ cells, but not strail, tunel staining revealed an extensive apoptosis of tec. forty-eight hours following injection of cd34-trail+ cells, a 21-fold increase of apoptotic index was detected, which was associated with extensive necrotic areas (20% to 25% of tissue section). these data show that: (i) tumor homing of cd34-trail+ cells induces extensive vascular damage, hemorrhagic necrosis and tumor destruction; (ii) the antitumor effect of cd34-trail+ cells is mediated by both indirect vascular-disrupting mechanisms and direct tumor cell killing. targeting of a therapeutic suicide gene to human alloreactive memory t-cells with stem-cell features requires il-7 and il-15 a. bondanza* (1), l. hambach (2) in a phase ii clinical trial investigating the prophylactic infusion of suicide gene-modified donor t cells after haploidentical hemopoietic cell transplantation (haplo-hct), we observed a rapid and effective immune reconstitution. after activation with anti-cd3 antibodies, t cells were modified with a retroviral vector (rv) encoding for the herpes simplex thymidine kinase (tk). tk+ cells displayed an effector memory (em) phenotype (cd45ra-cd62l-, cd28±cd27+, il-2±ifn-g+). when needed, graft-versus-host disease (gvhd) was controlled upon administration of the prodrug ganciclovir (gcv). the graft-versus-leukemia (gvl) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. genetic modification with rv is limited to memory t cells. em tk+ cells have a reduced alloreactivity. central memory (cm) t cells (cd45ra-cd62l+, cd28+cd27+, il-2+ifn-g±) share many characteristics with stem cells, namely the ability to selfrenew and to differentiate into effector cells. recently, it has been proposed that alloreactivity may be confined to memory t cells with stem-cell features. since alloreactivity is the common ground of both graft-versus-host disease (gvhd) and the gvl effect, crucial to the success of the strategy is the suicide gene-modification of cells with such properties. we found that addition of cd28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as il-7 and il-15, generates central memory (cm) tk+ cells. cm tk+ cells were highly alloreactive, both in vitro and in vivo in a humanized animal model of gvhd based on the grafting of human skin onto nod/scid mice. gcv administration abrogated gvhd. stimulation of cm, but not of em tk+ cells with autologous dendritic cells pulsed with hla2-restricted peptides from the minor histocompatibility alloantigen (mhag) ha-1 or h-y efficiently induced mhag-specific t cells that lysed natural ligand expressing hla-a2+ targets. a fraction of mhag-specific tk+ cells expressed il-7ra. only il-7ra+ mhag-specific tk+ cells could self-renew and differentiate into effector cells. when infused in nod/scid mice harboring human mhag+hla-a2+ leukemia, tk+ mhag-specific t cells significantly delayed disease progression. altogether, these data suggest that targeting of a suicide gene to human alloreactive memory t cells with stem-cell features requires il-7 and il-15 and warrant their use in the clinic for a safe and powerful gvl effect. loss of foxp3 expression after in vitro expansion of human cd4+cd25+cd127-regulatory t-cells p. hoffmann* (1), t.j. boeld (1) , r. eder (1) , j. huehn (2) , s. floess (2) in animal models the adoptive transfer of donor-type cd4+cd25+ regulatory t cells (treg) protects from graftversus-host disease (gvhd) after allogeneic stem cell transplantation (sct), as shown by us and several other groups. exploring this strategy in human sct, we currently perform a first phase i clinical trial using freshly isolated treg. for future trials requiring large cell numbers for repetitive treatments, we described in vitro culture conditions that permit a more than 3-log polyclonal expansion of treg (blood 104:895; 2004) . a highly enriched starting population proved to be crucial for the generation of pure treg cell products, a criterion fulfilled by the naïve, cd45ra+ subpopulation of cd4+cd25high t cells (ra+ treg) (blood 108:4260; 2006 ). an alternative isolation strategy for treg relies on the exclusion of cd127+ cells, as activated cd4+cd25+ conventional t cells express high levels of cd127 while cd4+cd25+ treg show no or only weak expression. for a direct comparison of the two isolation protocols, we sorted cd4+cd25+cd127low/neg t cells (cd127-treg) and ra+ treg from the same leukapheresis products and analyzed the cells after 2 and 3 weeks of expansion. whereas both populations were > 94 % foxp3+ upon isolation, only ra+ treg maintained foxp3 expression throughout the expansion period (93 % [range: 78 to 97 %; n=11] after 2 and 87 % [range: 71 to 97 %; n=9] after 3 weeks). in contrast, cd127-treg cultures contained only 82 % (range: 56 to 96 %; n=11) foxp3+ cells after 2 weeks and highly variable and significantly lower numbers of foxp3+ cells than ra+ treg cultures after 3 weeks (57 % [range: 18 to 93 %; n=9; p=0.006]). further analysis identified cd45ra-foxp3+ memory-type cells within the cd127-treg starting population as a major source of foxp3-as well as il-2-and ifngamma-producing cells emerging during in vitro culture. in addition, we analyzed the dna methylation status of a defined region within the foxp3 locus termed tsdr (t reg-specific demethylation region), which has been shown to be demethylated exclusively in natural t reg cells (baron et al., eji 37:2378; 2007) . upon isolation, ra+ and cd127-treg showed complete demethylation of the tsdr, but only ra+ treg maintained this demethylated status throughout the entire culture period. based on these findings, we suggest that expansion of naive cd45ra+cd4+cd25high t cells represents the most promising strategy for adoptive treg cell therapies. several eortc aml trials were based on a 2x2 factorial design. in elderly patients, aml-13 trial was set-up to assess the value of g-csf during/after induction, and of infusional vs non-infusional mini-ice as consolidation. in the eortc-gimema aml10 study (patients ≤ 60 years old), comparing 3 anthracyclines in induction and consolidation, the value of allosct vs autosct has been assessed based on intent-totreat analysis. the "donor vs no donor" comparison was performed, overall, and according to cytogenetic subgroups and age. in the eortc-gimema aml12 (age ≤ 60 yrs) study the accrual for the 1st question (hd-ara-c vs sd-ara-c in induction) had to be expanded, due to insufficient number of patients randomized for the 2nd one (il-2 vs no il-2 after autosct). new statistical considerations were set-up for the 1st question as the trial expanded to 2000 patients. 302 statistical planning of clinical trials: an emphasis on sample size determination a. latouche* ebmt (paris, fr) an essential step when planning a trial is the calculation of the sample size or the number of patients to recruit to detect a relevant effect with sufficient power. patients enrolled in a clinical trial may experience exclusive failure causes, which defines a competing risk setting. the main decision regards the relevant quantity to assess the treatment effect: either cause-specific hazard or cumulative incidence function. the analysis should then be performed according to the method used for sample size computation, if one does not want an under-powered trial. from practical point of view, the practitioner has different options to plan a study accounting for competing risks. in this work, we thus compare these approaches. in medical applications multi-state models arise in attempting to understand complex disease or treatment processes. in these models individuals can move among a finite number of states defined by specific conditions of health, often including death. they are natural extensions of the traditional survival models in which transitions between multiple states are considered. multi-state models give more insight into the disease-recovery process because they take into account the occurrence intermediate events. in this talk i discuss some of the advantages (and disadvantages) of multi-state models and their application to bone-marrow transplantation. long-term follow-up of hcv infected patients; updated results of the ebmt prospective study p. ljungman*, a. locasciulli, a. békássy, l. brinch, i. espigado, a. ferrant, i. franklin, v. gomez-garcia de soria, j. o'riordan, m. rovira, p. shaw, h many long-term survivors after sct are infected with hcv. it has been reported that a high proportion of these patients develop late occurring liver cirrhosis. treatment options include interferon with or without the addition of ribavirin but the knowledge about these options' effectiveness is limited. therefore in 1992, the idwp of the ebmt initiated a prospective study of hcv infected sct patients having survived at least 6 months after sct. the intention is to every five years ask for follow-up on living patients. 247 patients were included in the study cohort between 1992 and 1997. this is the 3rd report of this cohort. due to limited follow-up data, 49 patients have been excluded. thus, the report is based on 198 patients with a median follow-up of 16 years (0.52 -32.8). the kaplan-meier estimate of survival is 73.9%. 43 patients have died of whom 7 have been reported to have died from liver associated complications including one after liver transplantation. two additional patients are alive after liver transplantation. 79 patients have been treated with interferon with or without the addition of ribavirin or with ribavirin alone. 37/79 patients became hcv pcr negative of whom 3 have relapsed with positive hcv pcr. the side effect profile was similar to other patient populations treated for hcv. conclusion: in this prospectively followed cohort, the prognosis of chronic hcv infection was quite favourable. treatment is feasible and associated with acceptable efficacy and toxicity. current status of the apbmt registry y. kodera*, a. yoshimi, r. suzuki, y. atsuta, l.l. chan, a. li, p.-l. tan, w.y.k. hwang, t.v. binh, t.j. chiou, a. ghavamzadeh, l. dao-pei, p.the data was submitted through the national registries in my, jp, and tw. in other countries/regions, the data was collected either by apbmt data center or by regional coordinators. the total number of hsct has been steadily increasing in most of the countries/regions and most dramatically in cn and in ir. the annual number of hsct of 2005 reported was 4,598 (data of 7 countries/region), which was doubled in these 10 years; of those, 65% were allogeneic and 35% were autologous. the number of allogeneic sct has been steadily increasing year after year; meanwhile the number of autologous sct has been recently stable. the distribution of related and unrelated donors (ud) differed widely among the countries (ud= 0% [vn] to 62% [jp] in 2005) . ud-sct is increasing consistently in cn, jp and hk, but not in other countries/regions. interestingly, cord blood appeared to be a common stem cell source in asia, accounting for 35% of ud-hsct (11% in cn to 100% in ir/vn, data of 2005). unrelated donor peripheral blood stem cell transplantation is not common in asian countries/regions except for china. in summary, this simple survey provided us basic information on current situations and trends of hsct in asia, which may be helpful to advance the patient registry. recently apbmt developed an electric data collecting system called trump and the group also agreed to share the common basic survey items with ebmt and cibmtr (med-a/ted) and join the global transplant activity survey under the umbrella of world-wide group for blood and marrow transplantation (wbmt). these international collaborations may facilitate future advances of the hsct. background: ebv associated ptld is a serious complication after sct.several risk factors may increase the risk of ptld. analysis of ebv dna viral load has been suggested to be useful for monitoring and used as the basis for preemptive therapy with rituximab with the aim to reduce the risk for ptld. methods: patients who underwent sct from 030101 until 070515 were included in this analysis. ebv serological mismatch between donor and recipient, primary ebv infection, cord blood grafts, and diagnosis of lymphoma were regarded as risk factors and used to split the patients into a high risk and a standard risk group. before 050701 (early group), patients were tested on clinical suspicion of ebv associated symptomatic infection while after 050701 (recent group) high risk patients were monitored by quantitative pcr while standard risk patients were to be sampled on clinical suspicion of symptomatic ebv infection only. 274 patients were analyzed; 150 patients in the early and 124 in the recent group. in the early group 53 were classified as high and 97 as standard risk. in the recent group, 41 were defined as high and 83 as standard risk. the ebv viral load was measured in serum by a taqman pcr technique. after 050701, rituximab was to be given in high risk patients at an ebv dna level of >10.000 copies/ml. in the early group and in standard risk patients of the recent group, rituximab was given on suspicion of ebv symptomatic infection. results: a total of 971 blood samples were analyzed for ebv dna. more samples (median 4 in the early and 6 in the recent group) were taken in the high risk compared to the standard risk group (median 2 and 3 samples, respectively). in the early group, 25/53 (47%) high risk patients compared to 31/97(32%) standard risk patients had ebv detected at least once. the corresponding numbers in the recent group were 14/41(34%) in the high risk compared to 11/83 (13%) in the standard risk group. in the early group, 13/150 (8.6%) received rituximab. while 11/124 (8.9%) in the recent cohort was given rituximab. high risk patients received more often rituximab than standard risk patients in both the early and recent cohorts (data not shown). in the early group 6 (4%) developed ptld of whom 4 died (2.6%) while in the recent group, 4/124 (3.2%) developed ptld of whom 2 died (1.6%). conclusions: a strategy of targeted monitoring to high risk patients can be safely utilized with a low risk for development and fatal outcome of ptld. a retrospective ebmt survey on the use of cidofovir for bk-related haemorrhagic cystitis after allogeneic haematopoietic stem cell transplant s. cesaro* (1), y. koc (2) bk virus has been recently associated to post-engraftment hc. cidofovir (cdv) is often used for bk-related hemorrhagic cystitis (bk-hc) treatment although data on safety and efficacy are scarce for this indication. we collected retrospectively the experience of cdv for bk-hc among the ebmt centres. 61 episodes of bk-hc in 60 patients from 16 centres were recorded. all patients but 5 had been transplanted for a malignant disease. median age at haematopoietic stem cell transplant (hsct) was 21 yrs (range 2-64) and 23/60 patients were children or adolescent (age < 18 yrs). the source of stem cell was pb in 52%, bm in 29%, and cb in 19% whilst the type of donor was sibling in 30%, unrelated donor in 57% and other related donor in 13%. myeloablative regimen + tbi was used in 74% of patients. bk-hc occurred after a median of 41 days from hsct (range 3-577) but only 8 episodes were diagnosed after day + 100. hc was scored as follows: 7 grade i, 16 grade ii, 28 grade iii, 10 grade iv, and lasted in median 33 days (range 5-749). concurrent morbidity was represented by fever in 30%, hypertension in 26%, vod in 7% and ttp in 3% of episodes. most of patients received hyperhydration plus rc and plt transfusion during hc whilst only 51 of episodes were treated with bladder irrigation through vesical catheter. cdv was started a median of 6.5 days after hc (range -22 before to 135 days after hc) and was administered for a median of 3 doses (range 1-15). the most frequent schedule was 3-5 mg/kg/weekly or fortnightly with probenecid (used in 70% of all treatments). 6 patients received intravesical cdv via catheter. cdv toxicity was reported in 20% of episodes and was limited to kidney: 3 grade i, 4 grade ii, 2 grade iii, 3 grade iv. complete resolution or clinical improvement of hc was reported in 35 (57%) and 8 (13%) of episodes, respectively. although the datum is not available for all episodes cdv treatment was associated to negativization of bk viremia in 69% (27/39) and of bk viruria in 37.5% (9/24) of episodes. we conclude the cdv therapy is a potential useful option for the treatment of bk-hc with limited moderate or severe kidney toxicity. prospective data are needed to define the best timing and schedule of treatment and the predictive factors for clinical and virological response. single-centre prognosis analysis and validation of the seattle, french and strasbourg prognosis indexes of invasive aspergillosis in adult patients with haematological malignancies or after haematopoietic stem cell transplantation r. parody*, r. martino, f. sanchez, j.l. piñana, j. sierra sant pau hospital (barcelona, es) in this retrospective study we analyzed the outcomes of 117 adults hematological patients who had a history of proven (n:23), probable (n:61) and possible (n:33) invasive aspergillosis (ia) between january 1995 and june 2007, at santa creu i sant pau's hospital of barcelona. forty-one patients (35%) were recipients of an allogeneic hematopoietic stem cell transplantation (allohsct). the main goal of the study was to analyze the prognosis factors predicting the outcome of 4-month aspergillosis free survival (afs) and overall survival (os), in order to compare the results with previously published prognosis indexes. analysis was performed in all patients and separately in allohsct and non-allohsct patient cohort. a total of sixty seven patients (57%, 30 recipients of allohsct)) died in the first 4 months after ia diagnosis [median days: 20 (range1-117)]. invasive aspergillosis was identified as the main cause of death in 51 patients (43%, 23 recipients of allohsct) [median days: 16 (range ]. according with autopsy findings, 27 cases (11 possible and 16 probable) could be recategorized as proven cases. diagnosis of ia at or before 2000 had a negative impact in both 4-month afs and 4-month os. in all patients and allo-hsct patients, 4 variables (excluded the year of diagnosis) decreased 4-month afs in multivariate analysis: (i) disseminated ia and monocytopenia <0.1 x 109/l, respectively (ii) impairment of one organ, (iii) impairment of 2 or more organs and (iv) high-doses steroids and an alternative donor, respectively. a risk model for progression was generated for each group, according with the presence of 0-1 factors,( 82 and 100% afs), 2-3 factors, (27 and 45% afs) or 4 factors, (0% afs)[p<0.001]. in base of similar results previously published our prognosis index model could be validated with both the french and seattle models for allo-hsct recipients and with the strasbourg model for hematological patients. one-year microbiological survey with molecular typing method of pseudomonas aeruginosa in a bmt unit b. bartolozzi* (1), r. fanci (1) pseudomonas aeruginosa is one of the most common nosocomial pathogens in intensive care and in oncohematological units and it still represents an important cause of morbidity and mortality. molecular epidemiological survey is a very important tool to understand the nosocomial pattern of each hospital and to identify outbreaks. from may 2006 to june 2007 we performed a "real time" aflp (amplified fragment-length polymorphism) monitoring of all p.aeruginosa strains isolated in patients within the bmt unit of florence. the patients admitted in the unit during this period were 99 (70 autologous transplants, 15 unrelated allogeneic transplants and 14 identical sibling allogeneic transplants. p.aeruginosa was responsible for ten bloodstream infections (bsis) and of two microbiologically documented infections without bacteremia. the aflp analysis showed the persistence within the unit of two clones of p.aeruginosa. one was responsible of two outbreak episodes in patients allocated in the same single room. the first episode occurred in may-october 2006 involving 4 patients; a team consisting in the bmt head physician, the bmt head nurse, the infection control officer and the infection control physician was actively involved in a strict bacteriological surveillance that allowed to isolate the same strain from a irgasan soap sample. for this reason, hyperclorination of the water network, sink tap changing, water filters installation and weekly soap monitoring were performed. nevertheless the same clone was isolated after several months in a water sample, showing the inefficacy of the control measures taken. in may-june 2007 the same p.aeruginosa clone was isolated in three infected patients and in a shower tap sample. hydraulic works inside the room were performed and until now no other case of p.aeruginosa was observed. the other persistent clone was responsible of contamination of 4 sample of irgasan soap taken from the ward cloakroom (collected from november 2006 until april 2007) and of a bsi in a patient (march 2007) . our results showed that p.aeruginosa is largely and consistently found in environment which may represent an important source of infection, difficult to eradicate. moreover, punctual microbiological surveillance and molecular typing methods are essential to early detect nosocomial outbreaks, to identify p.aeruginosa reservoir and to guide in decision making in order to understand and possibly stop the epidemic chain. donor lymphocyte infusion (dli) following t cell depleted allogeneic stem cell transplantation (sct) is an effective treatment for relapsed hematological malignancies. dli often results in a profound graft versus leukemia (gvl) effect with relatively limited graft-versus-host disease (gvhd). both gvl reactivity and gvhd are likely to be mediated by donor derived t cells recognizing polymorphic minor histocompatibity antigens (mhag) on patient cells. it has been hypothesized that t cells recognizing mhags expressed on non-hematopoietic cells are responsible for gvhd, whereas t cells recognizing hematopoiesis restricted mhags may be selectively involved in gvl reactivity. to analyze the diversity of alloreactive t cells involved in gvl and gvhd we analyzed the immune response in a patient responding to dli following hla-matched sct for aml. six weeks after dli, gvhd limited to skin and mouth developed coinciding with a rapid and sustained conversion to 100% donor chimerism. we clonally isolated and characterized activated hla-dr expressing t cells at the onset of gvhd (10% of the circulating t cells). in total 133 cd8+ t cells clones and 241 cd4+ t cell clones were tested for alloreactivity as defined by cytotoxicity or ifng production upon recognition of patient derived ebv-lcl and not donor derived ebv-lcl. 49% of the cd8+ t cell clones and 20% of the cd4+ t cell clones were identified as alloreactive t cell clones. next, recognition of patient skin-fibroblasts as a target for gvhd was determined both in the absence or presence of ifng to mimic an inflammatory environment. none of the t cell clones recognized unmanipulated fibroblasts. only after co-culture with ifng, 28% of the alloreactive cd8+ t cell clones but none of the cd4+ t cell clones reacted with patient fibroblast. by blocking studies, panel studies and t cell receptor-vb analysis the diversity of this immune response was determined. 32 and 25 different reactivities were found for the cd8+ t cells and cd4+ t cells, respectively. in conclusion, isolation of activated t cells identified a very polyclonal immune response directed against multiple mhags in all hlaclass i and hla-class ii alleles. despite the polyclonality of this immune response, reactivity of the t cell clones was relatively hematopoiesis specific, resulting in 100% donor chimerism, persistent complete remission and only moderate gvhd limited to the skin. since t cell recognition of fibroblasts was observed only upon co-culture with ifng, this may reflect a secondary reactivity induced by the ongoing gvl effect. background and aims: chronic graft-versus-host disease (cgvhd) is a recognized cause of genital complications in the vulva and vagina in female patients (pts) after allogeneic stem cell transplantation. however, clinical features and consequences of genital cgvhd are poorly described in the literature. in a retrospective study, we assess prevalence, symptomatology and effect on sexual life of genital cgvhd. in a prospective study, we try to prevent progression and sequelae of genital cgvhd. here we report early results from these ongoing studies. patients and methods: all women allografted 1996-2005 in the western region of sweden are asked to participate in a study comprising (i) structured anamnesis and validated questionnaires for the identification of depression and sexual dysfunction; (ii) gynecological examination; (iii) biopsies for pathological examination. prospective pts are seen by the gynecologists up to 3 years post-transplant. clinical findings of genital cgvhd is a dry, thin, painful mucosa, inflammation, lichenoid bands, vulvar synechiae and vaginal stenosis. systemic and local estrogens do not resolve those signs. histology findings were those of chronic inflammation, lichenoid reaction and fibrosis. results: fifty-eight consecutive pts with 1-10 yrs of follow-up were approached and so far 30 women have been examined. median age was 46 (26-71) yrs) and follow-up post-transplant was 5.5 (1-10) yrs. fifteen pts (50%) had a clinically diagnostic cgvhd, and using pathology examination (n=13) the diagnosis was confirmed (n=6) or suspected (n=4). another 12 of the 30 pts had signs suggestive of cgvhd and histology was confirmatory in one and suspect in 6 cases. two of 30 pts had vulvar synechiae and 10 (33%) had vaginal adhesions. in the prospective study, 19 pts have been included and the follow-up is now 12 (3-24) months. after 12 months of follow-up (n=14) clinically cgvhd had been diagnosed or suspected in 8 cases. local corticosteroid or takrolimus cream was commenced in 4/19 cases. female sexual distress scale intervention performed as sceduled revealed sexual dysfunction at at least once in 23 (46% of all) and beck depression inventory indicated depression 18 (37%). conclusions: genital signs and symptoms, including vaginal stenosis, are common features of cgvhd and are associated with sexual dysfunction and depression. gynecological surveillance and early intervention may reduce the risk of severe sequelae after genital cgvhd. langerhans cell chimerism early after t-cell depleted allogeneic haematopoietic stem cell transplantation k. schneiker*, t. schmitt, a. konur, j. hemmerling, k. bender, e. von stebut, a. hadian, k. kolbe, c. huber, w. herr, r.g. meyer university clinic mainz (mainz, de) skin is the most frequently affected organ in acute graft versus host disease (gvhd). data from murine studies suggest that the interaction of residing host epidermal langerhans cells (lc) and donor t cells is crucial for the initiation of acute gvhd. in an ongoing clinical protocol applying alemtuzumab-based t cell depleted (tcd) allogeneic stem cell transplantation (sct) we observed acute skin gvhd occurring very early after transplantation despite of low t cell counts in the peripheral blood (meyer, blood 2007; 109:374) . we therefore intended to analyze the lc chimerism in these patients. up to now, lc-chimerism analysis in humans has been performed by the detection of the sex-chromosomes restricting it to sex-mismatched donor / recipient pairs. in our patient-population, this would limit the analysis to less than 1/3 of the patients. consequently, we aimed at a method to isolate lc from small skin samples with high purity for a sensitive str-based chimerism analysis of general applicability. epidermal skin layers were prepared from 6 mm punch biopsies by digestion with dispase i. they were further split and used for both immunofluorescent staining and digestion with trypsin to generate a single cell suspension and cd1a/mhc-class ii-positive lc were subsequently sorted by flow cytometry. the density of lc on day + 20 after hsct was much lower compared to before transplantation or to that of healthy individuals. but still, lc could be purified in all 10 analyzed patients. the isolated lc numbers ranged from 10 to >1000. we confirmed that the purity was exceeding 93.5% in 5 patients in a facs-reanalysis, thereby reproducing the findings with skin of healthy individuals. applying two alternative str-based protocols, we obtained reliable results for lc chimerism in 8 of 10 patients and could detect signals with as few as 35 isolated cells. in 2 patients, the majority of isolated lc was of donor origin whereas the other 6 patients had predominantly host lc. after day +50 post hsct, 2 further patients showed only a few remaining lc of host origin. in summary, we have established a sensitive method that enables the chimerism analysis on highly purified lc independent of sex-mismatched donor / recipient pairs. our results on a few patients' samples cannot yet be related to clinical events. but the method allows the investigation of lc´s chimerism and potentially of other tissue-resident antigen presenting cells to study their impact on gvhd in humans. we and others have data on the activation of coagulation and fibrinolysis during and after allogeneic stem cell transplantation (sct), which may have pathogenic implications in the subsequent recovery. we characterised the outcome including graft versus host disease (gvhd) in association with adaptive mechanisms of anti/coagulation after allogeneic sct in 30 patients with a hematological malignancy. they were given myeloablative conditioning with cyclophosphamide and total body irradiation. 19 patients received the transplant from a sibling and 11 from an unrelated donor. gvhd prophylaxis consisted of cyclosporine were "unresponsive, non-progressive". the prognostic features, including cytogenetics, were similar in both groups. 70% of the patients responded to the first hdt (cr/ncr 8%, pr 48%, mr 13%). 37 patients were given a second transplant (26 "auto", 11 "allo"). 41% who received a second "auto" up-graded their response (cr 9%, pr 14%, mr 18%) while 42% who underwent "allo-ric" increased their response (cr 28%, pr 14%). median survival of the whole series was 3 years. patients progressing while on therapy had a shorter survival than the "no-change" group (median 2 yrs vs not reached, p=0.00002). finally, the 50 "non-responsive, nonprogressors" patients had similar survival than the 716 with chemosensitive disease intensified with hdt. conclusions: 1) hdt in patients with primary refractory mm results in a low cr rate, 2) patients progressing while on initial therapy have a short survival despite the intensive approach and 3) patients with "non-responding, non-progressive" disease have similar survival than chemosensitive patients. whether this good outcome is due to the impact of hdt or to the natural history of a more indolent disease remains to be further investigated. background: primary amyloidosis (al) responds poorly to conventional therapy and has poor prognosis. autologous stem cell transplantation (asct) results in a significant response rate although the procedure is hampered by a high transplant-related morbidity and mortality. aim. to analyze the outcome of a series of 34 patients with al who underwent asct at a single institution during a 10years period. patients and methods: thirty four patients (16 m, 18 f; median age 54 years, range: 33-66) who received an asct between november 1997 and september 2007 were included. fourteen patients had received previous therapy and 20 were newly diagnosed. in 71% of the patients the light chain was of lambda type. the median number of involved organs was 2 (range, 1-4) including kidney (26 patients), heart (19), liver (11) , peripheral nerve (9), autonomic system (5) and gastrointestinal tract (5). thirty-eight percent of the patients had more than 2 organs involved. all patients were mobilized with g-csf alone and the intensive regimen consisted of mel-200 in 23 patients and mel-140 in 11 patients. the median interval between diagnosis and asct was 9 months. results: the overall transplant-related mortality (trm) was 27%. there was a trend towards a higher trm in patients transplanted from november 97 -april 02 (n=14, trm: 42%) as compared to those transplanted from june 02 -sept 07 (n= 20, trm 15%) (p= 0.07). in the 26 patients who underwent asct before nov. 2006 (minimum follow-up of 1 year) the response and survival on an intent-to-treat basis were as follow: cr (28%), pr (12%), no response (26%), early death (34%).organ response was observed in 11 patients (42%). the median response duration and the overall survival were 50 and 59 months, respectively. favorable prognostic features for survival were: cardiac septum <14 mm (p= 0.03), normal beta 2m (p=0.04) and normal nt-probnp (p= 0.006). updated results on the overall series of 34 patients will be presented at the meeting. conclusion: 1) asct in al results in significant cr rate with prolonged response duration an overall survival and 2) although trm is high, there is a trend towards a lower trm over the years this most likely reflecting both a better patient´s selection and general management. outpatient-based peripheral blood stem cell transplantation for patients with multiple myeloma a. ghavamzadeh, m. khani*, a. karimi, k. alimoghadam, a. manokian, r. maheri, m. asadi, f. afshar, a. shamshiry hematology, oncology and bmt resarch center (tehran, ir) intrduction: the aim of this study was to explore the feasibility and safety and cost-benefit of performing asct on an outpatient basis. material and methods:total of 86 patients affected by mm and in complete remission (cr) or partial remission (pr) were selected to receive asct on an out-patient or in-patient basis . in the in-patient group 31, 12 patients received 200mg/m² and 140mg/m² melphalan as conditioning regiment respectively . in out-patient group 12 patients received 140mg/m2 and 30 patients recived 200mg/m 2 melphalan. in out-patient group all the patients were programmed to go home the day after asct and to be rehospitalized in the case of febrile neutropenia or other sever toxicities. we used caregiver, general physician, staff nurse as an out-patient and visit team and also unequipped routine house of the patients during neutropenia. results :median ages were 50±7.5 years, median hospital stay were 28, 6.5 days in in-patient and out-patient respectively. there were not significant difference between these groups in aphresis days ,granulocyte colony stimulating factor(gcsf) requirement for mobilization and mononoclear cell (mnc) or cluster of differentiation(cd)34 + cell parameters (p<0.1), but statistically significant reduction in total cost and hospitalization (p<0.017).there were also significant reduction (p<0.001) in parentral antibiotic,blood product requirement and need for total parentral nutrition. conclusion :many different authores have explored the feasibility of autografting patient on an outpatient basis. the ease of administration of hdm as well as the lack of excessive extramedullary toxicity, including nausea and vomiting renders patients with mm more suitable for outpatient management, in the present study, we describe an outpatient program based on management of the patient in his/her house during aplastic phase . our results clearly indicate that such a proceder is feasible and safe in a patient population with a median age of 55 years. with an accessible caregiver, the most frequent cause of readmission in other study were febrile neutropenia and sever mucositis need tpn. in particular, it is worth nothing that transplant cost, requirement for blood product and hospital stay decreased significantly in this method of transplantation. treatment of multiple myeloma with sequential autologous and low dose total body irradiation based allogeneic unrelated sct c. pfrepper*, t. lange, r. krahl, m. cross, h.-k. al ali, w. pönisch, n. basara, d. niederwieser university hospital of leipzig (leipzig, de) purpose: the limited availability of related donors poses an important limitation in the treatment of multiple myeloma (mm). the use of unrelated donors would alleviate this problem. furthermore, outcome after unrelated transplants following reduced intensity conditioning (ric) has been shown to be superior in other diseases including cll and aml, due to increased graft-versus malignancy reaction. in this study, the feasibility of autologous followed by allogeneic unrelated ric sct was tested and the results compared to autologous followed by related allogeneic ric sct and allogeneic ric sct after relapsing following autologous sct. patients and methods: we report the outcome of 39 mm patients with a median age of 55 (range 33-64) years treated with high dose chemotherapy (melphalan 200mg/m² on day -3) followed by autologous sct. subsequent ric-sct was performed using fludarabine (30mg/m²/d from days -3 to -1) and tbi (2 gy on day 0) followed by cyclosporine (6.25 mg/kg twice daily from day -1) and mycophenolate mofetil (15 mg/kg daily from day 0). eleven patients received an unrelated graft (group 1) and 18 a related graft (group 2) within median 5 (range 2-8) months from the autologous hct. ten further patients (group 3) underwent either related or unrelated sct 13 (range 2-42) months after insufficient response following autologous transplantation and additional chemotherapy. results: durable engraftment was obtained in 97% of all patients. overall survival (os) at 2 years was 62±15%, 82±10% and 67±16% (p=0.089) in groups 1, 2 and 3, respectively at a median follow up of 23 (range 2-66) months. nine patients (23%) were in cr, 15 patients (38%) in pr, 3 patients (8%) had progressive disease after allogeneic sct. however; progression free survival (pfs) was 55±15%, 20±10% and 11±10% in the three groups, respectively (p=0.014). non relapse mortality (trm) was not different between the three groups, but relapse incidence was lowest in patients after unrelated transplantation. conclusion: we conclude that autologous sct followed by low dose tbi based sct from matched unrelated donors provides rapid and sustained engraftment for mm patients comparable to that of related donors. our limited data suggest a tendency to higher pfs following unrelated compared to related sct. these results provide the basis for a phase iii study designed to compare auto-allo unrelated to auto-auto sct. background: a significant proportion of patients with multiple myeloma have a long-lasting response after autologous stemcell transplantation (asct). however, others relapse relatively quickly. the aim of this study was to determine if the presence of monoclonal plasma cells (mpc), detected by flow cytometry, in the apheresis product, and in pre-and posttransplant bone marrow samples predicts a shorter time to progression (ttp). patients and methods: we included patients diagnosed with multiple myeloma and treated with high dose therapy followed by asct between november 1, 1998 and february 1, 2007. in all casas, flow cytometric analysis was performed in the apheresis products and in bone marrow samples, both prior and after asct. the variables evaluated as possible prognostic factors were: age, sex, type of monoclonal component, durie-salmon stage, iss stage, presence of mpc (cd138+/cd38+/ cd19-/cd45-or cd45dim) in apheresis products, and in bone marrow samples (30 days before, and 100 days after asct), and accomplishment of a complete response prior or after asct. results: 55 patients were included: 21 male, 34 female. median age at asct was 58 years (range, 30-69 years). twenty-nine patients had progressed. on univariate analysis, age .14); p<0.023], iss stage [or 7.61 (2.14-27.14); p=0.08], mpc in apheresis products (p=0.005), mpc in pretransplantation bone marrow (p=0.007), mpc in postransplantation bone marrow (p=0.06), and the achievement of complete response prior and after asct (p=0.01) had a negative impact on time to progression. on multivariate analysis, the presence of mpc in apheresis [or 3.1 (1.24-2.65), p=0.01] and the presence of mpc in pretransplantation bone marrow (or 14.2, p=0 .012) were identified as independent predictors of a shorter ttp. conclusions: the presence of mpc in apheresis products and in pretransplantation bone marrow were identified as independent predictors of shorter ttp. both parameters can identify a group of patients with multiple myeloma which could benefit from most aggresive conditioning regimens or additional maintenance therapies. long-term follow-up of patients with systemic al amyloidosis treated with high-dose melphalan and autologous stem cell transplantation after induction and mobilisation chemotherapy s. o. schonland*, t. bochtler, m. hansberg, a. mangatter, j.b. perz, a.d. ho, h. goldschmidt, u. hegenbart university of heidelberg (heidelberg, de) introduction: longterm survival in al amyloidosis (al) patients (pts) has been shown recently after high-dose melphalan (hdm) (sanchorawala, blood 2007) in 21% of the patients. however, no advantage of hdm compared to conventional chemotherapy has been observed in a randomized multicenter trial (jaccard, nejm 2007) . this was probably due to high transplantation related mortality (trm) and a substantially longer time to the start of chemotherapy in the hdm group. whether treatment intensification using induction and mobilization chemotherapy prior hdm can improve results has not yet been definitively shown. methods: we have updated 23 pts with al (perz, bjh 2004) who received vincristine/adriamycine/dexamethasone as induction and additional mobilization chemotherapy (mostly cyclophosphamide based) prior to hdm in our center from 1998 until 2004. inclusion criteria were age < 70 years, nyha stage < iii and a who performance status < 3. median age was 57 years; median number of involved organs was 3. fifteen and 13 pts had symptomatic renal or cardiac involvement, respectively (on dialysis at hdm, n=4). results: the median overall survival (os) has not been reached at a median follow up of 66 months post hdm (figure). trm was 9%. a hematological response (hr) to induction and mobilization chemotherapy occurred in 47% (14% of pts achieved a complete remission (cr)). hr and cr rate increased to 84% and 67% after hdm, respectively. median time to cr from hdm was 4 months. two patients had already an organ response (or) after induction and mobilization chemotherapy. or was observed in 68% and the median time to or was 11 months post hdm (range 0-40 months). pts with cr have an estimated os of 90% after 6 years; for pts not reaching cr the median os is 48 months (p=0.002). hematological relapse or progression occurred in about half of the patients; however 6 pts (26%) are still in first cr with a sustained or and a median follow-up of almost 7 years. conclusion: we observed a very high cr and or rate using this intensive treatment approach. patients with cr after hdm have an excellent survival. therefore, the role of induction therapy in al, e.g. with bortezomib or lenalidomide, should be further investigated in randomized trials in experienced centers. the main goal of treatment in al remains achievement and maintenance of a cr of the underlying plasma cell disorder. the results of reduced-intensity conditioning allogeneic stem cell transplantation (ric allo-sct) for multiple myeloma (mm) are still under considerable debate. while ebmt data did not support the universal use of ric for mm allografts, the italian randomized multicenter study suggested that in newly diagnosed myeloma, survival in recipients of a hematopoietic stem-cell autograft followed by ric allo-sct from an hlaidentical sibling is superior to that in recipients of tandem stem-cell autografts. the aim of this multicenter retrospective national study was to identify prognostic factors for outcome of high-risk patients with mm after allo-sct prepared by ric. data from 219 patients (median age 52 years, range 27-66), who received grafts from a sibling (n=197) or unrelated donor (n=22) were analyzed. at time of transplant, only 37 patients (17%) received ric allo-sct in cr or vgpr, while 134 patients (61%) were transplanted in pr. 48 patients were transplanted either in stable disease (n=15) or were in refractory/progressive disease (n=33). all patients have received at least one autologous transplant prior to ric allo-sct. the graft source was pbscs in the majority of patients (n=183). 21% of the patients received the seattle fludarabine and low dose tbi ric regimen, while 53% of patients received fludarabine, busulfan and atg. 32 patients (15%) died of transplant-related complications. the incidences of grade 2-4 acute gvhd and extensive chronic gvhd were 37% and 20% respectively. at 3 years, overall and progression free survivals (os, pfs) were 41% (95%ci, 34-49) and 19% (95%ci, 14-27) respectively. disease status (cr, pr, sd vs. progressive) was significantly associated with overall survival (p=0.0002; fig. below) . in multivariate analysis, disease status at time of ric allo-sct, was the strongest parameter associated with an improved os and pfs (p=0.005 and p=0.004 respectively). despite its obvious caveats, the relatively low trm observed in this series, suggest that there is still space to investigate ric allo-sct for mm. however, ric allo-sct appears to result in a durable response only if it is applied early in the disease history, especially when patients are still chemosensitive. since the latter results are also expected to be further improved with the systematic and early use of maintenance therapies (bortezomib and/or lenalidomide) after ric allo-sct, randomized or quasirandomized prospective studies are still warranted. the role of reduced-intensity conditioning (ric) allogeneic stem cell transplantation (allo-sct) for adult patients with acute lymphoblastic leukaemia (all) is still under debate. all encompasses a group of chemosensitive diseases, raising concerns that significant reduction of the intensity of the preparative regimen may have a negative impact on leukemic control. in this multicenter retrospective study, the outcome of 601 adult (age at transplant >45 y.) patients with all who underwent transplantation in complete remission (cr) with an hla-identical sibling donor, were analyzed according to 2 types of conditioning: ric in 97 patients, and standard myeloablative conditioning (mac) (or high-dose) in 504 patients. both groups were comparable in terms of gender, cr status (cr1 and cr2), interval from diagnosis to allo-sct, and r/d cmv serostatus. patients in the ric group were older (median 56 y. vs. 50y in the mac group;p<0.0001). most of the patients in the mac group received high dose tbi (80%), while the majority of the ric regimens included either low-dose tbi or were atg+chemotherapy-based regimens. the majority of patients (88%) from the ric group received pbscs. in the mac group, the stem cell source consisted of bone marrow in 42% of patients. with a median follow-up of 13 months (range, , the incidences of grade ii-iv and grade iii-iv acute gvhd were: 35%, 14%, and 28%, 10% in the mac and ric groups respectively (p=ns). the cumulative incidence of nonrelapse mortality at 2 years (nrm) was 32% (mac) vs. 22% (ric) (p=0.04). the cumulative incidence of relapse at 2 years was 30% (mac) vs. 42% (ric) (p=0.0007). however, the latter differences did not translate into any significant difference in term of leukemia-free survival (lfs) at 2 years: 38% (mac) vs. 37% (ric) (p=0.42). in multivariate analysis for lfs, the status at transplant was the only factor associated with an improved lfs (p<0.0001, rr=0.55, 95%ci, 0.42-0.72). the results of this study suggest that ric regimens may reduce nrm rate after allo-sct for adult all when compared to standard mac regimens, but with a higher risk of disease relapse and no impact on lfs. the latter represent promising findings, since patients who received ric are likely to have serious comorbidities, which led the transplantation center to choose ric, and surely most of these patients would not have received a standard allo-sct in most institutions. therefore, ric allo-sct for adult all (>45 y.) may represent a valid therapeutic option when a conventional standard conditioning is not possible, warranting further prospective investigations. reduced-intensity conditioning allogeneic stem cell transplantation for patients with acute myeloid leukaemia: long-term results of a "donor" versus "no donor" comparison m. mohty *, h. de lavallade, j. el-cheikh, p. ladaique, c. faucher, s. fürst, n. vey, d. coso, a.m. stoppa, j.a. gastaut, c. chabannon, d. blaise institut paoli-calmettes (marseille, fr) the issue of possible higher relapse rates after reducedintensity conditioning (ric) allogeneic stem cell transplantation (allo-sct) is still under considerable debate. this report describes the updated long term results (initial publication, leukemia 2005) of 95 consecutive acute myeloid leukaemia (aml) patients, diagnosed between 1999 and 2003 in a single centre. using a genetic randomization through a "donor" versus "no donor" comparison, our aim was to assess the true benefit of ric-allo-sct for adult aml. in this series, 35 patients (37%; "donor" group) had an "identified" hlaidentical sibling donor, while the remaining 60 patients had no hla-matched related donor ("no donor" group). as per institutional policy, hla-mud were not considered during the study period. no significant differences in patients or aml features were found between the two groups. in the "donor" group, 25 patients (71%; median age, 51 (range, 26-60)) could actually proceed to the ric-allo-sct. the current median follow-up is 60 months. in an "intention-to-treat" analysis, the km estimate of leukemia-free survival (lfs) was significantly higher in the "donor" group as compared to the "no donor" group (p=0.003; 60% versus 23% at 7 years). when restricting the analysis to patients who could actually receive the ric-allo-sct (median follow-up, 40 months from time of allo-sct), the difference in lfs was also significant between this group of 25 patients ("transplant" group) and the remaining 70 patients ("no transplant" group; p=0.0002; 72% versus 24% at 7 years). no major toxicities were encountered during ric administration (fludarabine, busulfan and atg), and only 3 patients died from toxicity, for a cumulative incidence of trm of 12% (95%ci, 3-32%) at last follow-up. this relatively low trm translated towards a significantly higher overall survival (os) in the "transplant" group as compared to the "no transplant" group (p=0.0003). in the "intention-to-treat" analysis, os was still significantly higher in the "donor" group as compared to the "no donor" group (p=0.003; fig. below) . after controlling for all relevant factors, in the multivariate analysis, only actual performance of ric-allo-sct (p=0.0005; rr=4.1; 95%ci, 1.8-9.1), was significantly predictive of an improved lfs. based on these long term results, we conclude that if a matched related donor is identified, ric-allo-sct should be proposed since it represents a valid and potentially curative option for aml patients not eligible for standard myeloablative allo-sct. graft failure after reduced-intensity conditioning b. hertenstein*, e. dammann, r. brand, a. van biezen, d. niederwieser, t. de witte, t reduced intensity conditioning (ric) regimens use doses of chemotherapy and/or radiotherapy which per se do not eradicate the malignant cells but which should be nevertheless sufficient to allow sustained engraftment. the incidence of graft failure might therefore be higher in ric and may be dependent on the regimen used. to evaluate the incidence of graft failure and the potential risk factors in patients transplanted after ric, data files of such transplantations were selected from the clwp data base. in all records chemotherapeutic drugs and dosages as well as tbi dosages were individually checked and only records meeting the ebmt definition of ric were included. the analysis was further restricted to patients surviving > 28 days. 1720 ric transplants were identified. a chemotherapeutic ric regimen was used in 1066 patients (flu/bu 571, flu/mel 296, flu/cy 60), tbi only in 97 and a combination of chemotherapy and tbi in 557 patients. stable engraftment occurred in 1640 patients (95.3%), no engraftment in 45 patients (2.6%) and a graft loss in 35 patients (2.0%). survival for patients with graft failure was 76% at 100 days and 38% at one year. 29 patients received a second transplant and survival at one year was 60% for these patients. graft failure was lowest in patients with myeloma (1.6%) and highest in patients with mds/mps (8.7%). with the use of bone marrow as graft source graft failure was much higher than with peripheral blood stem cells (11.8% vs. 3.7%) . graft failure occurred in 3.4% of transplants from hla-identical siblings, in 6.6% from matched unrelated donors and in 12.3% from mismatched unrelated donors. there was no difference between the different conditioning regimens, neither between chemotherapy alone, tbi or combined regimens (3.4% vs. 2.3% vs. 4.4%) nor between the major chemotherapy regimens themselves (flu/bu 1.9%, flu/mel 2.6%, flu/cy 4.3% for hla-id siblings). abo match and donor-recipient sex mismatch had no impact on graft failure. in multivariate analysis underlying disease and graft source were significantly correlated with graft failure. in summary the incidence of graft failure after ric is low. mds/mps as underlying disease and the use of bone marrow as graft but not the type of the conditioning regimen were risk factors for graft failure. (2), h. esperou (2) , m. attal (2) , n. milpied (2) , b. lioure (2) , p. bordigoni (2) , i. yackoub-agha (2) , j. bourhis (2) , b. rio (2) , e. deconninck (2) , m. renaud (2) , n. raus (1) , d. blaise (2) (1)hôpital edouard herriot (lyon, fr); (2)société française de greffe de moelle et de thérapie cellulaire (saint-denis, fr) this report updates a retrospective study from sfgm-tc registry concerning 1108 patients who underwent allogeneic hematopoeitic stem cell transplantation (hsct) after reduced intensity conditioning (ric) from hla identical siblings (84%) and unrelated donors (16%) for hematological malignancies. at time of conditioning, 442 patients were in cr, 337 in pr, 107 in stable disease (sd) and 222 in progressive disease (pd). as conditioning, 255 patients received fludarabine and tbi (2 grays), 465 patients fludarabine, busulfan and atg and 388 patients other regimens. after transplant, 336 patients (30%) developed an acute gvhd grade ii (grade ii: 178, iii: 80 and iv: 78). a chronic gvhd was present in 388 patients (35%) (185 limited and 203 extensive). with a median followup of 30 months, the 3 and 5-year probability of overall survival (os) were 43.5% (40-47) and 32%(29-35) respectively and the 3 and 5-year probability of event-free survival (efs) were 35%(31-39) and 28% (24.5-31) respectively. the trm at 1 year, 2 years and 3 years was 15% (13-17), 18% (15.5-21) and 20% (17-23). a mixture model, gfcure with splus statistical package determined the percentages of long-term survivors and its adequacy was verified graphically. the probability to be a long-survivor was 24% (17.5-32.5) (fig.1 ) and to be a long event-free survivor was 23% (19-28) (fig. 2) . the multivariate analysis has tested recipient and donor age, disease status pre-transplant, number of transplants before rict, hsc source, sex matching, hla matching, cmv status and abo compatibility. the only factor which had a significant impact on long-term survival after rict was the disease status just prior conditioning: pr versus cr: hr: 3.63 [1.14-9.18] p<0.001 and pd versus cr: hr: 4.35 [2.22-8.51] p<0.0001. in conclusion, these updated data demonstrate that allogeneic hsct after ric was able to possibly cure 23% of patients with haematological malignancies and the most important factor to take into account remains to be in cr pre-transplant. the haematopoietic cell transplantation-specific comorbidity index predicts survival and non-relapse mortality in lymphoma and myeloma patients receiving a ric allograft l. farina* (1), b. bruno (2) the allogeneic hematopoietic cell transplantation-specific comorbidity index (hct-ci) has been recently developed to identify patients at high risk of morbidity and mortality after an allogeneic stem cell transplant (allosct). reduced-intensity conditioning (ric) regimens have decreased non-relapse mortality (nrm) in heavily pre-treated patients. we performed a retrospective study to assess whether comorbidities, according to hct-ci, may influence the outcome of lymphoma and multiple myeloma patients undergoing a ric allosct. between 2000 and 2007, 197 patients received a ric allosct from a hla identical sibling (n=123) or unrelated (n=74) donor in three italian transplant units. median age at transplant was 52 years (range, 17-69) and 41% of the patients were 55 or older. diseases included non hodgkin's lymphoma (n=103), multiple myeloma (n=68) and hodgkin's lymphoma (n=26). median number of previous treatments was 3 (range, 0-8). disease risk according to kahl et al. (blood 2007) was low, intermediate and high in 29%, 42% and 29% of patients respectively. patients with hct-ci of 0, 1-2 and ≥ 3 were 64 (32%), 61 (31%) and 72 (37%), respectively. variables included in multivariate analysis were age (< 55 or ≥ 55), disease risk (low, intermediate and high), number of previous lines of therapy (≤ 2 and >2), hct-ci (0, 1-2 and ≥ 3), karnofsky perfomance status (ps, >80% and ≤ 80%). oneyear os and pfs were 87%, 60%, 60% and 91%, 75%, 70% in patients with hct-ci of 0, 1-2 and ≥ 3 respectively. cumulative incidence of nrm was 6%, 22%, 25% at 1 year and 6%, 24% and 27% at 2 years, whereas relapse mortality was 5%, 20%, 17% at 1 year and 7%, 27% and 23% at 2 years. by multivariate analysis only karnofsky ps (p=0.00051) and hct-ci (p=0.0038) were correlated with os, whereas pfs was influenced by disease risk category (p=0.013), number of previous therapies (p=0.038), and both karnofsky ps (p=0.031) and hct-ci (p=0.0009). interestingly hct-ci was the only significant factor that could predict nrm (p=0.034) while karnofsky ps failed to show a significant correlation (p=0.1). of note, age (≥ 55) was not statistically significant either in os, or pfs or nrm. although the data need to be confirmed in prospective trials, these results showed that hct-ci may be a useful tool to predict os, nrm and also pfs after ric allosct in lymphoma and myeloma patients. in the clinical setting hct-ci and not age should be used for patient risk assessment. venous thromboembolism (te) occurs as a consequence of genetic and environmental factors. important genetic risk factors are deficiencies of natural anticoagulants, antithrombin-iii (at-iii), protein c (pc) and ps, and genetic mutations of factor v leiden (fvl) and prothrombin (pth a20210). thrombotic complications after hct are usually catheter-related thrombosis (crt), pulmonary te (pte) and deep vein thrombosis (dvt). our aim in this study was to evaluate the effects of the deficiencies of atiii, pc and ps, and the gene mutations of fvl and pth on the incidence of the development of te complications and liver sinusoidal obstruction syndrome (sos) at the early or late period post-hct. in our center, pre-transplant work-up includes routine thrombophilia tests. between apr 1999-jan 2007 260 patients (m/f: 145/115, median age: 34 years) admitted to our transplantation center were retrospectively analyzed for the relation of the presence of thrombophilia and the frequencies of the occurrence of a te complications and liver sos. all but 6 patients (n=254) had an hla identical sibling donor. the ratios of the detection of the plasma activation level below 50 % for pc, ps and at-iii were 5.8 % (12/206), 15.7 % (32/204) and 25.4 % (66/260), respectively. gene mutations were studied in 198 patients prior to transplant and the frequencies of gene mutations were detected in 11.6 % patients (n=23) either fvl (n=14) or pth (n=9) gene. none of the patients had both mutations, fvl and pth. at the peri-or posttransplantation period we observed venous te in 24 patients (17 crt, 2 pe and 5 dvt), liver sos in 23 patients and myocard infarction at the early period in only one patient. in 4 out of 12 patients with low pc activity crt occurred, and liver sos was observed in only one patient. in 6 out of 24 patients with genetic mutation developed a te complication, 5 crt and 1 pte. crt occurred in 4 of 14 patients with fvl mutation, while 1 crt and 1 pte among 9 patients with pth mutation were observed. liver sos was seen in only one patient with pth heterozygote positive. we found the presence of low pc level or genetic mutation increased the frequency of the development of te (or: 6.9 and 3.7, respectively), but no effect on the liver sos. in conclusion, the use of thromboprophylaxis at peri-transplant period in patients with genetic mutations is still a controversial topic; which should be elucidated with controlled studies. iron overload (io) is an adverse prognostic factor in patients who undergo allogeneic haematopoietic stem cell transplantation (hsct) for thalassemia and appears to play a similar role in patients with other hematological disorders. estimation of io is primarily based on serum ferritin, however many confounding factors particularly in hsct recipients may result in frequent ferritin overestimation. aim of the study was to quantify io by uperconducting quantum interference device (squid) after hsct and evaluate the impact on infections and gvhd; additionally the feasibility of iron-depletion has been investigated in this pilot study we evaluated io in 89 consecutive adult patients who received hsct from a matched sibling (n=66) or a matched unrelated donor (n=23). primary diagnosis included aml/mds in 54% of cases. assessment of io after hsct included serum ferritin and in those with hyperferritinemia (> 1000 ng/ml), liver iron concentration (lic) was evaluated by squid magnetic susceptometry. io assessment was performed in patients in remission at a median time of 698 days after hsct (range 48-5239 days). median serum ferritin was 821 ng/ml (range 18-11110).thirtynine of 89 (44%) patients had serum ferritin level >1000 ng/ml; lic (microgfe/g liver wet-weight) evaluated by squid was available for 35/39 patients with elevated ferritin values. overall, 26 patients (29%) had moderate (lic levels 1000-2000 microg/gww) to severe (lic levels>2000 microg/gww) io; median lic values were 1419 microg/gww (range 1030-3253). nine patients (10%) had normal lic values (lic<400) despite high ferritin. patients with lic>1000 received a median of 41 packed red blood cells before study evaluation (range 7-79). the rates of bacteremias, invasive fungal disease and chronic gvhd were higher among patients with lic>1000 as compared to patients with lic<1000 (24%, 11% and 41% vs 16%, 5% and 25% respectively; p=ns). eighteen of the 26 patients with lic levels > 1000 were treated by regular phlebotomy. for 15 of these the phlebotomy program is still ongoing, while 2 patients completed the program with ferritin normalization; one patient was switched to deferasirox due to poor tolerance. in 5 cases phlebotomy was considered contraindicated; 3 patients who relapsed did not receive any treatment. our preliminary data show that one third of hsct recipients may present moderate/severe io as assessed by squid, and iron-depletion resulted feasible in 73% of these subjects. a trend for higher rates of infectious complications and chronic gvhd was observed in patients with io. despite recent advances, mortality rates after allogeneic hematopoietic stem cell transplantation (hsct) remains high and cannot be accurately predicted. several reports from the seattle group suggested the use of comorbidity indexes (ci) to provide valid and reliable scoring of pre-transplant comorbidities that predicted non-relapse mortality (nrm) and overall survival [blood 2004 (cci) , 2005 (hct-ci) & ann int med 2006 (pam)). however, whether such indexes could be used by other groups, and if one index better predicts survival than another, is yet unknown. hct-ci and pam required grading according to pulmonary function tests (pft), which were lacking for a majority of our patients. we thus designed a modified hct-ci and a corrected pam, without pft. survival curves were estimated using kaplan-meier method. cumulative incidences (ci) of non-relapse mortality were analyzed, with relapse treated as competing event. the likelihood ratio statistics in proportional hazards models was computed for each index, with its associated p-value, as a measure of association between the comorbidity indexes collapsed into risk groups and the outcomes. discriminative performance of comorbidity indexes was then evaluated by the c-index. standard errors of c-indexes were computed from that of somers' dxy rank correlation coefficient. p-values for comparison of c-indexes were obtained using a nonparametric bootstrap procedure. we thus retrospectively studied 286 patients (169 male, 117 female) who received their first allogeneic hsct at saint louis hospital between april 2004 and december 2006. the median age was 31 years (4-64), 149 patients (52%) were transplanted from a matched related donor and 248 patients received an hsct for intermediate or high-risk diseases (86%). using cci, 25% of our patients had indexes of 1 or more; median reduced hct-ci was 1; and median corrected pam score was 24. the discriminative properties of the 3 ci were rather low in our patient population with c-index of 0.515, 0.499 and 0.582 for the cci, reduced hct-ci and corrected pam indexes, respectively. comparison of patients-and transplant-characteristics between our and seattle group's cohorts however revealed significant differences with more children, more cord blood hsct and hsct for fanconi anemia in saint louis. finally, direct comparison of scoring items and multivariate analysis revealed that age, unmatched related donor and hepatic disease were associated with nrm in our cohort. primary graft failure occurs in 10-20% of unrelated cord blood transplantation (ucbt) patients. its occurrence is associated with increased risk of death due to relapse (when associated with autologous recovery) or complications of pancytopenia. salvage transplant is possible in patients with absence of neutrophil recovery (anr): clinical tools are required to promptly identify these patients at risk. we conducted this retrospective study to identify risk factors for anr. from 1996 to 2007, 98 consecutive children underwent a first ucbt in our center. anr was defined as absolute neutrophil count ≤ 100/microl in patients alive without disease on day +42. patients for whom a salvage transplant conditioning regimen was initiated before day +42 were excluded from this analysis (n=4, 2 of them are late survivors). ninety one (91) children were eligible for analysis. median time for neutrophil recovery (≥ 500/microl) was 27 days (range, 0 to 51). anr occurred in 7/91 (7.7%) and was confirmed by bone marrow biopsy. of these 7 patients, 2 underwent a 2nd transplant, one of which succeeded. five (86%) died between day +48 and +72 of transplant complications that occurred at a median of 38 days (range 29-70) of their first transplant. in 4 of them, complications were related to persistent neutropenia and/or lymphopenia. our current strategy of waiting until day +42 for a 2nd transplant decision carries a significant risk of mortality in patients with anr. when analyzed for demographics and graft characteristics, the group with anr did not differ significantly from the group with neutrophil recovery. the specificity and sensitivity for anr prediction of white blood cell (wbc) < 200 /microl at different time points is as shown in table 1. wbc < 200 /microl on day +28 and day +35 carries a risk of day +42 anr of 58% and 100%, respectively. the negative predictive value of wbc < 200 /microl on day +35 is 100%. therefore, in order to reduce the risk of mortality associated with anr, we now proceed to 2nd transplant evaluation for children with wbc < 200 /microl by day +28, and initiate the therapy on day +35 if wbc counts remains < 200 /microl. purpose: the aim of this retrospective study was to determine whether pre-transplant iron overload assessed by serum ferritin level predict the non-relapse mortality (nrm) and overall survival (os) rate after haematopoietic cell transplantation (hsct). patients and methods: 283 patients with hematological disorders underwent myeloablative or reduced-intensity conditioning followed by the first hsct from related or unrelated donors at keio bmt program from 1995 to 2005. 35 patients were excluded from this analysis because ferritin and crp levels were not available or measured at the time of transplant. demographic data and information on comorbidities was obtained from the keio bmt database. comorbidity was scored according to the hct-specific comorbidity index (hct-ci) proposed by the seattle group without any modifications. results: median value of ferritin was 448 ng/ml(range 26-3010 ng/ml). the percentage of patients with ferritin 193 ng/ml did not vary significantly among diseases. in all but 7 patients crp were within normal range at the time of measuring ferritin. there was a strong relationship between pretransplant ferritin and os. the 5-year os for patients was 76.2% in the first quartile (ferritin 0-193 ng/ml) (q1), 58.6% in q2 (194-448 ng/ml), 50.7% in q3 (449-1060 ng/ml), and 43.6% in q4 (more than 1061ng/ml) (p=0.001). 5-year os was also significantly higher for those with ferritin level more than 1000 ng/ml compared with those with ferritin equal or less than 1000 ng/ml (61.8% vs 43.8% p=0.004). the 5-year nrm was also significantly associated with pretransplant serum ferritin level. the 5-year nrm was 12.2% for patients with ferritin more than 1000 ng/ml, and 28.8% for those with ferritin equal or less than 1000 ng/ml (p=0.004). the mean score of hct-ci was 1.3 for those in q1, 1.6 in q2, 1.6 in q3, and 2.5 in q4. the mean score of hct-ci was significantly higher in patients with hyperferritinmia (ferritin more than 1000 ng/ml) than those without (2.5 vs 1.5 p=0.001). conclusion: these results suggested that pretransplant level of ferritin could be a marker of predicting nrm, os after hct and thus useful for patient counseling before hct. whether the addition of ferritin level into hct-ci scoring system give a better prediction of posttransplant nrm and os needs further investigation. increased incidence of renal impairment after radioimmunotherapy as part of the conditioning regimen before allogeneic stem cell transplantation t. zenz* (1), r. schlenk (1) intensifying the conditioning regimen with radioimmunotherapy (rit) is feasable for high risk leukemias and mds patients. while additional radiation exposure by rit is greatest in the bone marrow, significant exposure of the kidney occurs and an increased frequency of bmt nephropathy has been observed in patients receiving rit using the rhenium188 labelled anti-cd66 antibody. in order to obtain a precise picture of the incidence of renal impairment we compared two large cohorts receiving allogeneic stem cell transplantation (sct) with or without the use of rit from our center. between 1998 and 2004, 261 patients with a median follow-up of 58 months received hla-identical or mismatched allogeneic sct. of these 123 received an intensified conditioning regimen with a rhenium188 (n=87) or yttrium90 labelled (n=36) anti-cd66 antibody. the cohorts were similar with respect to sex, stem cell source and use of nephrotoxic medication. there was an increased proportion of patients with aml, all, and haploidentical donors in the group receiving rit (p<0.01), while the conventional group had more patients with cml, tbi in the conditioning regimen, cyclosporine use and incidence of gvhd (p<0.01). the clinical characteristics were documented for 5 years post sct. we defined relevant renal insufficiency as a creatinine level above 200 µmol/l and the failure to reach creatinine values below 150 to exclude intermittent drug toxicities. the incidence of renal failure was 13% in patients with conventional conditioning regimens and 24% in patients receiving rit (p=0.02). the competing risk analysis showed a significant increase in the cumulative incidence of nephropathy (p=0.01). even though the dosimetry showed decreased renal doses in patients with y90 this did not lead to a decreased incidence of renal impairment. other factors as tbi, cyclosporine use, donor source or gvhd did not influence the development of renal failure. in a multivariate model including rit, csa, donor source, gvhd and the combination of csa and rit, the combination of rit and csa was the only significant risk factor (p=0.01). in conclusion, rit lead to an increased frequency of continuous renal impairment. the combination of cyclosporine with rit appeared detrimental while tbi (with renal shielding) did not lead to an increased incidence of renal failure. strategies to decrease the incidence of renal impairment may include nephroprotection with ace inhibitors or alternative immunosuppression. prospective evaluation of oral mucositis in allogeneic stem cell transplant recipients receiving conventional myeloablative conditioning or reduced-toxicity conditioning with treosulfan and fludarabine h. uotinen*, l. volin, e. juvonen, a. nihtinen, t. ruutu helsinki university central hospital (helsinki, fi) oral mucositis (om) is a frequent and often severe complication of allogeneic stem cell transplantation (sct). the aim of this study was to evaluate the incidence and duration of severe (who grade iii-iv) and ulcerative (grade ii-iv) om in patients receiving different conditioning regimens. between sept 2005 and nov 2007, 131 patients were prospectively observed daily from the start of conditioning until hospital discharge. conventional (convent.) myeloablative (ma) conditioning (cy/tbi n=82, bucy n=10) was given to 92 patients (34 aml, 21 all, 7 mds, 7 cml, 7 mm, 6 mf, 10 others) and treosulfan (treo)-based conditioning to 39 (13 mds, 12 aml, 9 mm, 5 others) patients. treo-based conditioning consisted of treo 3x14 g/m² (n=28) or 3x12 g/m² (n=11) and fludararabine (fld) 150 mg/m². treo-fld conditioning is regarded as ma at least at the treo dose level of 3x14g/m² but with reduced non-hematological toxicity. treobased conditioning was given to patients not eligible for convent. full intensity conditioning because of age, comorbidity or heavy preceding cytotoxic treatment. there were no significant differences in the om results with the two treo dose levels used. the median age of the patients was 48 (18-62) years in the convent. ma group and 57 (19-65) years in the treo group. in the convent. ma group the donor was sibling in 40 cases and unrelated in 52 cases. in the treo group the donor was sibling in 17 cases and unrelated in 22 cases. the incidence of severe om was 40 % in the convent. ma group and 3 % in the treo group (p< 0.005). severe om episodes had a mean duration of 4 (1-26) days. the incidence of ulcerative om was 72 % in the convent. ma group and 33 % in the treo group (p<0.005). the ulcerative om episodes had a median duration of 6 (1-34) and 3 (range 1-8) days (p=0.003), respectively. om reached maximum who grade on day 13 in the convent. ma group and on day 16 in the treo group after the start of conditioning. the median duration of hospitalisation from the sct was 24 (17-52) days in patients with ulcerative om vs. 21 (range 17-43) days in patients without (p=0.110). in conclusion, conditioning with treosulfan and fludarabine caused significantly less severe and ulcerative mucositis than conventional ma regimens, and the duration of ulcerative mucositis was shorter. this supports the role of treo-fld conditioning for allogeneic transplantation especially in the treatment of patients with increased risk of toxic complications. patients receiving cbt had significantly slow neutrophil and platelet recovery in multivariate analysis. the incidences of acute and chronic gvhd were not significantly different. unrelated bmt showed better trm (25% versus 38% at 1 year, p<0.01), relapse (15% versus 26% at 3 years, p<0.01) and dfs (57% versus 29% at 3 years, p<0.01) results compared with cbt. next, we analyzed 498 bmt (n=370) and cbt (n=128) recipients who have taken total body irradiation (tbi; >8gy) containing myeloablative regimen and calcineurin inhibitors (cyclosporine or tacrolimus) plus methotraxate (mtx) for gvhd prophylaxis without history of prior transplants. in this subpopulation, multivariate analysis revealed no significant difference between bmt and cbt in dfs (57% and 51% at 3 years; hazard ratio (hr):1.34; 95% confidence interval (ci): 0.89-2.01; p=0.16). no statistically difference was also seen in trm (25% at 1 year after bmt and 23% at 1 year after cbt; hr: 0.90; 95%ci: 0.58-1.39; p=0.64). however, the cumulative incidence of relapse was significantly lower in bmt than in cbt (16% and 21% at 3 years; hr: 1.90; 95%ci: 1.12-3.22; p=0.017). the current japanese registration data in mds showed overall results of unrelated bmt were better than those of unrelated cbt by competing risk regression models. these data also suggest that unrelated cbt could be safely and effectively used as same as unrelated bmt when adequate transplant procedures are selected. comparative studies have shown that cb transplant is characterized by a lower risk of gvhd. cb units have been reported to contain tregs, but minimal data are available on these cells. aim of this study was to compare the suppressive functions of tregs expanded from cb units with those expanded from the peripheral blood (pb) of patients who have undergone an allogeneic sct. tregs were purified from mononuclear cells obtained from cb units or pb using the cd4+cd25+ regulatory t-cell isolation kit (miltenyi biotec) and expanded for 6 days in 96-well u-bottom plates coated with anti-cd3 and anti-cd28 moabs plus il-2. to assess the suppressive functions, expanded tregs from cb units or pb were seeded with naïve autologous effector t cells stimulated with allogeneic dendritic cells (dc) pulsed with apoptotic leukemic blasts, then incubated with [3h]-thymidine and counted in a beta-counter. suppressor activity was measured as [3h]-thymidine incorporation in the presence or absence of tregs. the proportion and the immunophenotypic analysis of tregs present in the cb units (n = 9) -in terms of expression of surface cd4, cd25, cd62l, cytoplasmic ctla-4 and foxp3was comparable to those obtained from the pb of allografted patients (n = 9). in addition, tregs from cb units and from the pb of these patients showed an equivalent expansion capacity [mean fold increase (range), cb units 8.6 (1.8-24); pb patients 9.1 (1.5-16.5) ]. on the contrary, preliminary data show that tregs expanded from cb units (n = 4) exert a higher suppressive function on the proliferative reaction of t cells stimulated by allogeneic dc compared to tregs expanded from the pb of allografted patients (n = 2) [mean fold reduction (range), cb units 9.01 (2.66-15.08); pb patients 3.83 (1.49-6.17) ]. moreover, immunofluorescence analysis demonstrated that tregs expanded from cb units (n=2) are highly positive for cytoplasmic il-10 (mean 82%, range 65-99) and negative for ifn-gamma. these results indicate that tregs contained in cb units exert a potent suppressive function in mixed lymphocyte reaction culture assays and offer further insights into the understanding of the biology of cb transplant. double negative tregs are reduced in allo-transplanted patients developing graft-versus-host disease b. serio* (1), z. mciver (1), a. risitano (2) , c. selleri (2) , j. maciejeski (1) (1)cleveland clinic (cleveland, us); (2) federico ii university of naples (naples, it) during development of a healthy immune system, central tolerance is induced in the thymus by the negative selection of t lymphocytes that have a high affinity for self antigens. after bmt, central tolerance may be impaired by thymic involution and conditioning regimens resulting in dysregulated alloreactivity. various subpopulations of regulatory t cells including the tregs or suppressor t cells with cd4+cd25+foxp3+ phenotype (foxp3 tregs), nk t-cells and the cd3+cd4-cd8-cd56-a/atcr+ t regs (double negative [dn] tregs) had led to the mechanisms of peripheral tolerance. we investigated behavior of dn following human allogeneic hsct with regard to the occurrence of graft versus host disease (gvhd) and restoration of t cell receptor (tcr) repertoire. a cohort of 40 patients was investigated; 16 underwent matched unrelated hsct and 24 received matched sibling grafts. frequency of dn and tcr repertoire of cd4 and cd8 cells was measured serially and at the time of diagnosis of gvhd by flow cytometry. our patient population demonstrated skewing of tcr repertoire and we identified a very strong and linear relationship between total number of vâ family expansions and the grade of gvhd (grade 1-4, n=28, p=.005); this relationship held true when considering separately both the cd4 and cd8 compartments (p=.009 and p=.023, respectively). the median frequency of dn tregs was calculated to be 0.54% of the total cd3+ t-cell population (range 0.04 -3.11). by using the total number of vâ family expansions as a gauge of alloreactivity, we observed a reduced number of dn tregs in those individuals that developed 4 or more vâ family expansions (n=25, mean 1.16 vs 0.56 cell/ìl, p=.03). in addition we also noted a significant difference in both the mean percentage and mean absolute values of dn tregs for those individuals that developed gvhd (grade >2) when compared to those that did not (n=29, 0.40 vs 1.32%, p=.004, and 0.8 vs 2.6 cells/ml, p=.024 respectively). the size of dn treg compartment inversely correlated to the grade of gvhd (grade 1-4) as both percentage and absolute value (n=29, p<.001, and p=.019 respectively). in conclusion we found that vb family expansion are associated with degree of alloreactvity and gvhd. in addition we show that dn tregs are reduced after bmt suggesting their important role in peripheral tolerance and alloreactivity. this work contributes to better define the regulatory role of dn and to develop future therapeutic applications. patients with myelofibrosis. a study of the mds subcommittee of the chronic leukaemia working party of the de witte on behalf of the mds subcommittee of the chronic leukemia working party of the european group for blood and marrow transplantation hla disparities were 4/6 match (n=49), 5/6 (n=10), and 6/6 (n=1). , respectively. twenty-one patients received cyclosporine, and 39 patients had tacrolimus alone as gvhd prophylaxis from day c1. neutrophil engraftment was achieved in 86% (median day; 20). cumulative incidence of pir, grade 2-4 and grade 3-4 acute gvhd were 60, 45 and 31%, respectively. estimated 2-years survival was 47% (95% ci: 34-68%) sierra hospital de la santa creu i sant pau (barcelona, es) background: neurological complications (nc) of allogeneic hematopoietic stem cell transplantation (allo-hsct), involving central (cns) and/or peripheral nervous system (pns) are common and remain life-threatening in most cases. their incidence and characteristics after allo-hsct have not been well defined we excluded patients with nc due to cns relapse of their malignancies as well as encephalopaty in the context of pre-mortem multi-organ failure cns complications included seizures in 7 cases, 5 non-focal encephalopaties, 5 meningoencephalitis and 5 strokes or hemorrhages. pns complications consisted of 4 cases of neuralgia/neuritis and 4 cases of demyelinating axonal neuropathies (1 guillain-barré syndrome) piñana is supported by grants isciii (cm06/00139) o404 haematopoietic stem cell transplantation for advanced primary mds in children: results of the ewog mds study group b. strahm* (1) ); (5)wilhelmina children´s hospital and methotrexate, and additional methylprednisolone in case of a sibling donor. the median clinical follow-up time for the patients was 37 months (3-46 months). several anti/coagulation activities associated with endothelial cell activation were serially assessed up to 3 months: prothrombin fragments 1+2 (f1+2), thrombin time (tt), fviii:c, activated protein c-protein c inhibitor (apc-pci) complex and protein c act (pc). during conditioning (on day-2) as an early sign of thrombin generation, f1+2 and apc-pci complex increased 2-3 fold. after engraftment, fviii:c increased steadily to reach its high maxi-mum on day +24 (273 % ±104 %, median±sd, p<0.001). interestingly, pc rose (189 % ± 63 %) in parallel with fviii:c with a 5-fold individual variability. after the engraftment fviii:c and pc were highly interrelated. gvhd developed in 8 patients and was predicted by early low pc activity (<90%) during conditioning (p=0.007) (or=16.7). gvhd was also associated with elevated level of f1+2 (> 0.7 nmol/l) (p=0.014) and was predicted by short tt (<15 s) (p=0.004) (or= 33.1) after the transplantation (on day +10). no patient whose f 1+2 was < 0.7 nmol/l (n=11) developed gvhd. elevated level of f 1+2 (>1.5 nmol/l) after the transplantation associated with non-relapse mortality. 3 patients with the highest thrombin generation after sct all died (7-23 months). in con-clusion, early up-regulation of thrombin generation and down-regulation of pc associated with the appearance of gvhd. after allogeneic sct procedure there is an intimate relation between endothelium regulated coagulation and development of gvhd, suggestive of a new therapeutic target. final results from a phetema study l. rosiñol* (1) , j.j. lahuerta (2) , a. sureda (3), j. de la rubia (4), j. , m. hernández-garcía (6), b. hernández-ruíz (7), j.a. , j.l. bello (9), d. carrera (10), m.j. peñarrubia (11) , e. abella (12) , a. león (13), c. poderós (14) , j.c. j. besalduch (16) , r. , i. p. ribas (19), j. san miguel (8) , j. bladé (1) (1)hospital clinic (barcelona, es) ; (2) background: two randomized trials showed that tandem transplant result in a significantly longer efs and os in patients failing to achieve complete remission (cr) or near-cr with a single transplant. however, other studies failed to show survival benefit from a second transplant but there was no survival plateau. promising results have been reported using dose-reduced intensity conditioning (allo-ric), especially after debulky with an autologous transplant.aim: to investigate the efficacy in terms of response upgrading and survival from a second transplant intensification in patients with chemosensitive disease who failed to achieve cr or near-cr with a first transplant. patients and methods: patients diagnosed with mm from oct 1999 to dec 2004 younger than 70 years received 6 courses of vbmcp/vbad and responding patients were intensified with busulphan/melphalan or mel-200 followed by stem cell support. patients not achieving cr or near-cr were planned to undergo a second transplant (second auto with cvbcyclophosphamide, etoposide and bcnu -or mel-200 intensification or an allo-ric with fludarabine/mel-140 conditioning, if sibling donor available. results: eighty-five patients received a second autologous transplant while 26 underwent an allo-ric. the cr rate was significantly higher with allo-ric (35% vs. 10%, p= 0.02). there was a trend towards a higher trm with the allogeneic procedure (5% vs. 15%, p=0.09). after a median follow-up of 51 months for alive patients, there were no significant differences in efs (48 mos. vs. nt reached) and os (80 mos vs. not reached for autologous tandem vs. auto/allo-ric). however, there is a plateau in the "allo-ric" group beyond 3 years of the second procedure not observed in the autologous arm. a final update will be presented. conclusions:1) an allo-ric transplant after an autologous procedure results in a significantly higher cr rate than a second autologous transplant, and 2) although we found no significant differences in survival between the two transplant modalities, there is a plateau in the allogeneic group. (2) background: it has been assumed that patients with primary refractory myeloma benefit from early high-dose therapy/stem cell support (hdt. however, in the reported series patients with "unresponsive-progressive disease" vs those "nonresponding-non progressing" were not analyzed separately aim: response and survival after early hdt in the two populations of truly primary refractory multiple myeloma (i.e., patients with progressive disease versus those with "no change" or "stable disease" while receiving the initial therapy). patients and methods: from oct 1999 to dec 2004, 829 patients with mm received 6 cycles of vbmcp/vbad and at least one transplant. 81 of the 829 patients were refractory to vbmcp/vbad. these resistant patients were scheduled to receive a tandem transplant, the first with bu-12/mel-140 or mel-200 and the second "autologous" with cvb (ciclophosphamide(etoposide/bcnu) or mel-200 or "allo-ric" (if donor available) with fluda/mel-140. response and progression were defined by the ebmt criteria. results: 31 of the 81 primary refractory patients had progressive disease under the initial chemotherapy while 50 allogeneic stem cell transplantation (sct) is the only curative treatment approach in patients with myelofibrosis. the major limitation is the high treatment related mortality, which exclude mainly elderly patient from this treatment procedure. to determine the toxicity and efficacy of a dose-reduced conditioning regimen, consisting of busulfan (10 mg/kg), fludarabine (180 mg/m²) and anti-thymocyte globulin (atg fresenius: 3x 10 mg/kg for related and 3 x 20 mg/kg for unrelated sct) followed by allogeneic sct, we performed a prospective multicenter trial in elderly patients with myelofibrosis in 18 centers in three countries. from 2002 to 2006, 104 patients with a median age of 55 years (r.,32-68) were included and 102 were evaluable for outcome. risk profile was low risk with constitutional symptoms (18%), intermediate risk (n= 58%) and high risk (n=19%). all but 3 patients received peripheral blood stem cells as graft source either from related (n=31) or unrelated donor (n=69). all but one (1%) patient showed leukocyte and platelet engraftment after a median of 18 and 21 days, respectively. the median duration of leukocyte aplasia was 9 days (r., 3-21). acute graft-versus host disease (gvhd) grade ii o iv occurred in 19% and severe agvhd iii/iv in 7%, while chronic gvhd was seen in 32% of the patients. non-relapse mortality at 1year was 17% (95% ci: 11-27%) and significantly lower for patients younger than 50 years of age (4% vs 24%, p<0.001) and for patients with low risk vs intermediate/high risk disease (9% vs 26%, p= 0.07), while a higher nrm was seen for patients transplanted from hla mismatched donors (66 vs 17%, p= 0.006). the cumulative incidence of relapse at 3 year was 25% (95%ci: 15-43%) and influenced by time from diagnosis to sct of less or more than 24 months (18 vs 40%, p=0.05). patients with splenectomy had higher incidence of relapse (60 vs 18%, p=0.003). the estimated 3 year overall and event-free survival was 73 and 58%, respectively. the overall survival was influenced by age less than 50 years (92% vs 59%, p=0.009 and low vs intermediate/high risk (94% vs 60%, p=0.03 and hla mismatch (39 vs 70%, p= 0.02), while no impact on survival was seen for cytogenetic abnormalities, jak2 mutation status and donor (related vs unrelated) these results of a prospective multicenter study show excellent outcome of a busulafan/fludarabine based reduced conditioning regimen followed by allogeneic stem cell transplantation in patients with myelofibrosis. graft rejection after stem cell transplantation following reduced-intensity conditioning is influenced by the underlying disease, the donor type, disease stage and the cd3 content of the graft g.-n. franke*, a. mikolajewska, s. leiblein, h.-k. al-ali, e. hennig, w. pönisch, d. niederwieser, t. lange university of leipzig (leipzig, de) objectives: stem cell transplantation (sct) after reduced intensity conditioning (ric) is routinely used as a curative approach for older and medical impaired patients with haematological malignancies. in contrast to sct with conventional conditioning, graft rejection (gr) remains an important issue. we analyzed patients with sct after 2 gy total body irradiation (tbi) with or without fludarabine (flu) conditioning to identify risk factors for gr. patients and methods: 330 patients with a median age of 58 (range 17 -74) years, underwent allogeneic sct (bm=11, pbsc=319) from a related (n=115) or unrelated (n=215) donor for aml (n=121), cml (n=34), nhl/mm (n=102), mds/mps (n=38) or other diseases (n=35). conditioning regimen consisted of 2 gy tbi at day 0 and flu 30 mg/m2 from -4 to day -2 (n=305) followed by cyclosporin a and mycophenolate mofetil. results: 28 (8.5%) patients developed primary (n=23) or secondary graft failure (n=5) defined as a t-cell chimerism of <10% donor cells. in univariate analysis and also in multivariate analysis, diagnosis of cml (p=0.011), unrelated donor (p=0.029), early disease stage prior to sct (p=0.048) and low cd3 cells in the graft (p=0.002) were identified as independent predictors for gr. the relative risk (rr) to experience gr was 4.8, 4.2, 3.3 and 1.8, respectively. even in a subgroup analysis with pbsc recipients only, cml, unrelated donor and disease stage were associated with higher risk of gr. furthermore, a lower cd3 count increases the risk of gr by a rr 1.5 (p=0.06) in the multivariate model (median 3.2x108/kg bw). an increase in cd3 cells was not associated with increased incidence of acute gvhd grad ii-iv until day 100 (p=0.935). in contrast, the cd34-content of the graft had no impact on gr either as categorical (>median vs. <median) or as continuous variable (p=0.454). conclusion: in contrast to given risk factors for graft rejection, the t-cell content of the graft can be individually requested in every patient. since higher cd3 cells in the graft reduces the risk of gr without subsequent acute gvhd in our cohort of patients, we recommend the administration of cd3 cells >3.2x108/kg bw especially in patients with cml, unrelated donor and early disease stage in ric-sct. unrelated cord blood transplantation for adult patients with acute myeloid leukaemia/myelodysplastic syndrome using a reduced-intensity conditioning regimen consisting of fludarabine, melphalan and total body irradiation k. masuoka*, k. ishiwata, m. tsuji, s. takagi, h. yamamoto, d. katoh, y. matsuhashi, s. seo, n. matsuno, n. uchida, a. wake, s. miyakoshi, s. taniguchi toranomon hospital (tokyo, jp) objectives: to evaluate the efficacy of unrelated cord blood transplantation (ucbt) in a reduced intensity (ri) conditioning regimen consisting of fludarabine (125mg/m²), melphalan (80mg/m²) and total body irradiation (400 cgy), we analyzed retrospectively the results of 60 adult patients with acute myeloid leukemia (aml)/ myelodysplastic syndrome (mds) in our hospital. patients and methods: we reviewed medical records of 60 patients with aml/mds who had received single cord blood unit between november 2003 and july 2007 at toranomon acute leukaemia 2 / myelodysplasia o397 myeloablative allogeneic stem cell transplantation with unrelated donors for high-risk acute myeloid leukaemia: no increase in relapse with alemtuzumab-depleted grafts p. kottaridis* (1), k. thomson (2) the role of t cell depletion in myeloablative allogeneic stem cell transplantation for acute myeloid leukemia (aml) remains uncertain. dose intensification may compensate for any potential loss of graft-versus-leukemia effect, and the use of t cell replete grafts is associated with significant morbidity and mortality, particularly when using unrelated donors. this study describes the results in 44 patients with high-risk aml transplanted with an alemtuzumab-containing myeloablative regimen, using unrelated donors. median age at transplant was 34 years and 20 patients (45%) had donors mismatched at 1-3 hla loci. all patients were high-risk for relapse, as defined by induction failure, adverse cytogenetics, >cr1, or secondary disease (4 preceding myelodysplastic syndrome [mds] , 3 therapy-related). two patients had untreated relapse at the time of transplant, and 9 of the remaining 42 (21%) had refractory disease. the conditioning regimen was cyclophosphamide 60mg/kg for 2 days, fludarabine 30mg/m² for 3 days and total body irradiation (tbi) 14.4gy in 8 fractions over 4 days. stem cell source was peripheral blood in 37 and bone marrow in 7, and graft-versus-host disease (gvhd) prophylaxis was with 20mg alemtuzumab added to the stem cells prior to infusion and cyclosporin 3mg/kg. median follow-up for surviving patients was 36 months. estimated event-free survival (efs) was 60% at 1yr and 55% at 4 years. acute gvhd grade ii occurred in 11% (no grade iii-iv) and extensive chronic gvhd in 23%. non-relapse mortality (nrm) was 19% at 4 years with no events beyond 10 months post-transplant, and estimated relapse risk was 27% at 4 years with no events beyond 15 months. for efs and relapse risk, the only significant variable was chemosensitivity pre-transplant, with inferior efs (65% at 4 years for chemosensitive patients vs 27% for chemorefractory, p=0.026) and worse relapse risk (15% at 4 years for chemosensitive patients vs 55% for chemorefractory, p=0.010) in those not in complete remission at transplant. there was no impact on nrm or relapse risk depending on the presence of hla mismatch, or of significant gvhd (>grade i acute, or any chronic gvhd). use of this regimen therefore permits the successful transplantation of patients with high-risk disease and hla-matched/mismatched unrelated donors, with minimal acute gvhd, relatively low nrm and no evidence of an excessive relapse rate, particularly in those in complete remission at transplant. haematopoietic stem cell transplants (hsct) using unrelated donors (ud) has become an important and viable option in the treatment of acute leukaemia (al). we have previously shown an increased risk of relapse with hla-dpb1 matching and independently, with nod2/card15 genotype. in light of these data, we have analysed a larger ud-hsct cohort in order to establish the impact on outcome when both variables are considered. hla and nod2/card15 typing was performed on 304 al ud-hsct pairs. transplants were between 1996 and 2005. diagnoses were all (47%) and aml (53%). 67% of the cohort were 10/10 hla matched and 16% were also hla-dpb1 matched. myeloablative conditioning regimens were used in 74% of transplants. 82% of conditioning protocols included t-cell depletion. bone marrow was used in 72% of transplants, the remaining 28% using peripheral blood stem cells. two forms of post-transplant immunosuppression predominated, cyclosporine a and methotrexate (47%) and cyclosporine a alone (38%). based on our previous data, the cohort was grouped according to their relapse risk, group 1 (dpb1 matched; nod2/card15 snp, n=24), group 2 (dpb1 matched; nod2/card15 wild-type (wt) or dpb1 mismatched; nod2/card15 snp, n=112) and group 3 (dpb1 mismatched; nod2/card15 wt, n=168). disease relapse differed significantly between the three groups (1 year: group 1 68%, group 2 48%, group 3 30%, p=0.006). this finding persisted in multivariate analysis where being in either group 2 or 3 was protective towards relapse as compared to group 1 (rr 0.321; p=0.001 and rr 0.478; p=0.031 respectively). in group 1 (high risk), this resulted in decreased overall survival (os) (33% vs 54% in group 3, rr 0.617; p=0.080). the best os was seen in group 3 (low risk) where in addition to low relapse, there was increased acute and chronic graft-versus-host disease (gvhd) (p=0.0019 and p=0.002 respectively). in this cohort, limited cgvhd was s74 associated with reduced relapse (p=0.01) and better os (p<0.0001). in accordance with our theory, being hla-dpb1 matched and nod2/card15 snp predicts for the worst outcome with significantly increased relapse and reduced os. the ideal pairing is hla-dpb1 mismatched and nod2/card15 wt. these data suggest that prospectively typing al patients for both variables will allow the prediction of transplant outcome and will allow the effects of being independently hla-dpb1 matched or nod2/card15 snp to be offset by intelligently selecting a suitable, less precarious donor. 8 (t8) is the most common chromosomal abnormality in aml. the prognostic impact of t8 as a sole aberration in aml remains unclear, conferring either an intermediate or a poor prognosis. indeed, patients with t8 occurring with other cytogenetics abnormalities seem to have the prognosis conferred by the accompanying aberration. the aim of this study was to describe the results of allogeneic transplantation in a large series of aml patients exhibiting isolated or associated t8 and to compare outcome with intermediate risk aml patients exhibiting normal caryotype and receiving allo-hsct. 182 aml patients were identified (males n=100; females n=82) with isolated (n=136) or associated (n=46, favourable group n=8, intermediate n=30; high-risk n=7; unknown n=1) t8, allografted with an hla identical sibling (n=113) or an unrelated donor (n=69) between 1990 and 2007 and reported to the ebmt. median age was 37 years (range: 17-68). median interval between allo-hsct and diagnosis was 165 days (range: 71-946). the proportion of patients in cr1, cr2/cr3 or active disease was 59%, 13% and 28%. myeloablative and reduced intensity conditionings were performed in 148 and 34 patients respectively. gvhd prophylaxis consisted of csa/methotrexate or csa/mmf in 49%. engraftment was observed in 171 patients (95%). grade ii and iii/iv acute gvhd occurred in 21% and 11%. chronic gvhd developed in 45%. with a median follow-up of 48 months (range: 3-180), 5-year lfs, relapse rate and nrm were 45%, 30% and 25%. status at transplant (cr vs others) (p<0.0001, hr 0.3, 95% ci: 0.2-0.46), female sexe (p=0.03, hr 1.61, 95% ci: 1.05-2.48) and hla sibling donor (p=0.02, hr 1.64, 95% ci: 1.08-2.49) were significant predictors for better lfs. 5-year lfs was similar between aml patients with isolated or associated t8 (41% vs 55%, p=0.11). whenconsidering only patients allografted in cr1, 5-year lfs was similar between isolated t8 aml patients (55%, n= 82, median follow-up 51 months), associated t8 aml patients (55%, n=26, median follow-up 51 months) and aml patients with normal caryotype (58%, n=1782, median follow-up 36 months). we conclude that allogeneic transplantation in first linetherapy is a valid therapeutic option in patients exhibiting isolated or associated t8. isolated or associated t8 do not confer bad prognosis and aml patients exhibiting such aberrations have to be considered as intermediate risk patients as they likely have the same outcome of aml patients with normal karyotype allografted in cr1. regimen intensity in acute myelogenous leukaemia: addition of 400cgy total body irradiation to a myeloablative fludarabine/busulphan/thymoglobulin allogeneic transplant regimen reduces relapse without increasing transplant-related mortality j. russell* (1), w. irish (2) , l. savoie (1) some attempts to intensify myeloablative stem cell transplant (sct) conditioning protocols for acute myelogenous leukaemia (aml) have reduced relapse only at the expense of increased transplant-related mortality (trm). a regimen of intravenous busulphan, fludarabine and thymoglobulin has been well tolerated but followed by a substantial relapse rate. we report the results of a study to enhance the antileukaemic effect of this protocol by adding a low dose of total body irradiation (tbi). 179 patients were treated between 1999 and 2006 with fludarabine 50mg/m 2 daily x 5 and intravenous busulphan 3.2 mg/kg daily x 4. 88 had additional total body irradiation (tbi) 200cgy x 2 on day -1 or 0. graft-versus-host disease (gvhd) prophylaxis was cyclosporine a, methotrexate and thymoglobulin (genzyme) 4.5 mg/kg total dose. median age was 46 (range 18-66) years in tbi recipients, 42 (16-65) for the non-tbi group. there was no difference in the proportions of sct with good risk (gr, cr1 or cr2) recipients (65% vs 56%), alternative (unrelated or mismatched related) donors (49% vs 40%) cmv +ve donor +/or recipient (60% vs 68%), female donors to male recipients (18% vs 19%) and high risk (hr) recipient cytogenetics (20% vs 14%) between the tbi and no tbi groups respectively. more tbi recipients received blood cells (91% vs 74%, p = 0.001) and consequently higher cd34+ cell doses (median 5.9x10 6 /kg, range 0.75-17.69 vs median 4.310 6 /kg, range 0.64-23.87, p <0.0001). follow-up of survivors was 12-83 months (median 31) for tbi recipients and 13-100 months (median 79) for those not given tbi. there was no difference in incidence of acute gvhd grade ii-iv at 23% vs 16±4%, acute gvhd grade iii-iv at 9% vs 9% and chronic gvhd at 55% vs 64% with and without tbi respectively. outcomes at 3 years are shown in the table: after adjusting for all the above risk factors the cox proportional hazard ratio for relapse was 0.32 (95% ci 0.17-0.6) in favour of tbi (p = 0.004). there was no effect of gvhd on relapse. the impact of tbi on relapse without affecting trm resulted in a significantly decreased risk of mortality with a hazard ratio of 0.56 (0.33-0.93, p = 0.03). the hazard ratio for disease-free survival was 0.46 (0.28-0.77, p = 0.003). this regimen allows some intensification by adding a low dose of tbi without an effect on trm but a reduction in relapse, confirming the importance of regimen intensity in transplantation for aml. s75 o401 long-term survival in patients suffering from aml with a complex aberrant karyotype after early allogeneic stem cell transplantation using the flamsa-ric regimen: results from a prospective phase ii trial c. schmid* (1), m. schleuning (2) introduction: patients suffering from acute myeloid leukemia (aml) with a complex aberrant karyotype (i.e. ≥ 3 cytogenetic aberrations) usually show poor response to chemotherapy and have a grim prognosis. allogeneic stem cell transplanttaion (allosct) is the treatment of choice, although the unfavorable prognostic value of a complex karyotype was preserved in most studies. in contrast, in the flamsa-ric pilot trial, a post-hoc subgroup analysis revealed similar results for patients with a complex karyotype as compared to more favorable cytogenetic subgroups (schmid, schleuning et al, jco 2005) . therefore, a prospective phase ii trial for patients with complex karyotype aml was initiated within the german aml cooperative group, to evaluate the role of allosct, performed as early as possible after diagnosis. the flamsa-ric preparative regimen contained the sequence of intensive cytoreductive chemotherapy (flamsa), followed three days later by reduced intensity conditioning (ric; 4gy tbi; cyclophosphamide, atg). patients and methods: 20 patients from 4 centers (median age: 50,1, range 20-63 years), with a median of 7 (range: 3-15) cytogenetic aberrations were included. median time from diagnosis to transplantation was 91 days. eight patiens had received one, 12 had received two courses of conventional chemotherapy. stage at start of flamsa-ric was cr1 in 7, first cytogenetic relapse in 1, and primary induction failure in 12 patients, including 8 with persistent disease after high-dose arac. donors were hla-identical siblings in 9, matched unrelated donors in 8, and 1-ag-mismatched unrelated donors in 3 cases. results: nineteen patients engraftet (median day: +18), one died in aplasia. 17 patients achieved molecular cr, 1 regenerated with blasts, and one had cytogenetically persistent disease. agvhd developed in 13 patients, but reached > grade ii only in 3. five patients developed cgvhd. relapse occurred in 2 patients. after a median follow up among survivors of 23 (range: 3-33) months, 8 patients have died from leukemia (n=3) or treatment-related causes (n=5). overall survival at one and two years from transplantation is 74% and 58%, the corresponding leukemia free survival is 69% and 58%. conclusion: to our knowledge, this is the first trial specifically addressing patients with a complex aberrant karyotype. although the numbers are small, the results suggest a promising activity of early allosct in this otherwise very unfavorable subgroup of patients with aml. the iron (fe) chelator, desferrioxamine (dfo), has been shown to have both antiproliferative and apoptotic effects in tumor cells. fe depletion results in g1/s cycle arrest and cell apoptosis and dfo acts as hypoxia-mimetic agent by accumulating the hypoxia-inducible factor-1 alpha protein which regulates the cellular response to hypoxia by cessation of growth. in this retrospective study we evaluated the effect of dfo administration and iron overload (iro) in relapse incidence in 127 consecutive patients (pts), allografted for malignant diseases (myeloid: 76, lymphoid: 51). the disease phase was early in 45, intermediate in 38 and advanced in 44 pts. thirty received non ablative and 97 ablative conditioning. peripheral blood (116) or bone marrow (11) grafts were donated from 101 siblings, 23 matched unrelated and 3 haploidentical donors. graft vs host disease (gvhd) prophylaxis was consisted of cyclosporine or prograf plus methotrexate. non-responders or relapsed pts within 2 months post allotransplant, were excluded. according to our center policy, pts with established engraftment and iro, as evidenced by ferritin levels>2000ng/ml, elevated liver enzymes or/and liver mri indicating hemosiderosis, receive dfo. among 95/127pts with iro, 31 were treated with dfo (50mg/kg x 5days/week, iv or sc for 2 months at least). the 5year relapse rate (rr) was significantly lower in pts treated with dfo than in non-treated pts (22% vs. 53%, p=0,003) and this benefit was restricted to myeloid malignancies only. in a multivariate analysis we examined dfo, chronic and acute gvhd, disease phase, graft source, type of donor, origin of malignancy and preparative regimen as risk factors for relapse. dfo administration retained its significance (p=0,02) while the absence of cgvhd and the advanced disease phase were significant factors for relapse (p<0,03). in order to explore whether iro affects the relapse incidence we separately examined the fe-overloaded pts who received no chelation (64) vs the non-fe-overloaded pts (32). the 5-year rr for untreated pts was 60% vs 33% for those with ferritin values <2000ng/ml (p=0,04). iro remained as significant risk factor for relapse in the multivariate analysis (p=0,03). this is the first clinical study to investigate the role of dfo in relapse incidence post allotransplant suggesting a possible role for dfo therapy post-transplant. prospective studies are needed to clarify if dfo may have a role in relapse prevention. equivalent disease-free survival results after cbt and bmt from unrelated donor using tbi containing myeloablative regimen and calsinulin inhibitors plus mtx methods in patients with mds in japan: multivariate analysis by competing risk regression models s. takahashi*, t. yamaguchi, m. monna-ooiwa, s. taniguchi, h. akiyama, t. morii, y. nagari, y. takaue, s. okamoto, k. miyamura, h. sao, t. nagamura, s. kato, t. kawase, y. the result of single institutional analysis in japan has shown cord blood transplantation (cbt) is promising in adults with hematologic malignancies including myelodysplastic syndrome (mds) and is comparable with bone marrow transplantation (bmt) from unrelated or related donors. we evaluated safety and efficacy of both bmt and cbt from unrelated donors using the data of mds patients within the japan marrow donor program and the japan cord blood bank network registry database and related those to biological and procedural factors. clinical data of 965 patients with mds including transformed acute myelogenous leukemia who received unrelated bmt (n=532) or unrelated cbt (n=433) between 1993 and 2006 were collected. the median periods of follow-up for survivors were 21 months for bmt and 12 months for cbt. we analyzed the hematopoietic recovery, incidences of graft-versus-host disease (gvhd), risks of transplant-related mortality (trm), relapse and disease-free survival (dfs) using competing risk regression models.advanced primary myelodysplastic syndrome (mds) represents a subgroup of childhood mds characterized by bone marrow (bm) dysplasia and an increased blast count in peripheral blood (pb) and/or bm. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment. here we report the results of 105 patients (pts) (71 males/34 females) enrolled in the prospective ewog mds study. according to the highest who type prior to hsct pts were classified as raeb (59), raeb-t (30) and mdr-aml (16). median age at diagnosis was 10.6 yrs (1.0-18.2) and the median time from diagnosis of advanced mds to hsct was 4.1 mo (1.0-31.2) . cytogenetics revealed monosomy 7 in 37 pts, a complex karyotype in 11 pts, other abnormalities in 22 pts, no abnormalities in 31 pts and was unknown in 4 pts. intensive chemotherapy was given to 31 pts prior to hsct. 43 children were transplanted from an hla matched family donor (mfd), while the remaining 62 were given an allograft from a matched or 1 ag mismatched unrelated donor (ud). the preparative regimen included busulfan, cyclophosphamid and melphalan in all cases. stem cell source was unmanipulated bm, pb and cord blood in 73, 30 and 2 pts, respectively. cyclosporine-a alone was used as graft versus host disease (gvhd) prophylaxis in most children transplanted from a mfd, while the majority of pts transplanted from an ud were given cs-a, mtx and anti-thymocyte globulin. with a median follow up of 3.1 yrs (0.1-10.2) 62 out of 105 pts are alive and disease free, 19 experienced relapse, and 24 died of transplant related complications. the 5-year probability of event-free survival (efs) is 0.56 [0.46-0.66] while the 5-year cumulative incidence of transplant-related mortality (trm) and disease recurrence are 0.23 [0.16-0.33] and 0.21 [0.14-0.32], respectively. the cumulative incidence of grade ii-iv gvhd is 0.47 [0.37-0.57]. there is no significant difference in efs, trm and disease recurrence according to donor, mds subtype, karyotype or the treatment applied prior to hsct. age at hsct >12 yrs, an interval from diagnosis of advanced mds to hsct ≥ 4 mo and the presence of gvhd contribute to a significant increase in trm. these results indicate that a large proportion of children with advanced mds can be rescued by allogeneic hsct. trm remains a main cause of treatment failure especially for children older than 12 yrs. for these patients new strategies to reduce trm will have to be defined. introduction: patients with a history of an allogeneic haematopoietic stem cell transplant(hsct)have an increasing risk to develop a secondary cancer.the association of the hla system to cancer is well known.sibling donors share the same hla alleles, and the question is,if they have an increased tumour incidence compared to the general population.this is of concern because the possibility of a tumour induction as a result of mobilisation with g-csf has been discussed. methods: all hla-identical sibling pairs with an hsct from 1974 to 2001 for a malignant disease and living in switzerland were evaluated. general data of the donors were taken from the patient's charts of the transplant unit.donors were contacted per call or mail and asked about their current health status.in case of a malignant cancer,information on date of diagnosis,localisation,treatment was obtained.data were compared with the age-,and sex adapted cancer incidence rate of the swiss association of cancer registries(sacr).the same information was retrieved for the patients. results: 318 pairs were identified,in 291(92%)the donors (142 men(m),149 women(w))could be contacted. median observation time was 13.8 years(y)(range 5-32y).146 donors were <50y,89 between 50-59y,29 between 60-64y,20 between 65-69 y and 7 >70y old, hence 85% were <60y old.seventeen (6%)donors,12 bone marrow and 5 peripheral blood donors,had developed a total of 18 cancer of 9 different localisations (mamma,prostate,skin,bone marrow, colon,bronchus,stomach,bladder,orl).according to the incidence rate of the sacr,3.3 tumours in m and 6.8 in w would have been expected,3 (m) (rr0.91) and 4 tumours (w) (rr 0.84) were found in donors between 0-49y.in the age category 50-69 y,4.5 tumours in m and 4.8 in w were expected and 5(rr1.11)respective 6(rr 1.23)observed.in the subgroup of men between 60-64y 1.07 tumour were expected and 4 s77 observed (p<0.02).no donor >70y developed a tumour.4 of the 291 donors had died,3 from the tumour,1 of cardiac disease.in 12 patients a secondary tumour was diagnosed post hsct. conclusion: we observed a definite number of donors with a malignancy after hsc donation. absolute numbers were similar to the numbers of tumours in their transplanted siblings, observed versus expected rates were similar to an age and sex matched population except in male donors between 60-64 y by now. data from this single centre cohort remain yet inconclusive but underline the need for international collaborative donor follow up. the number of transplants is consistently increasing for aml, all, and lymphoid malignancy in all countries/regions except for vietnam. meanwhile, the number of hsct for nonmalignant diseases such as aplastic anemia and hemoglobinopathy has been stable in all countries except for iran, in which country it is recently increasing. the proportion of hemoglobinopathy in all hsct differed widely among countries; 0% in japan and china, 2% in singapore, 3% in taiwan, 5% in hong kong, 10% in malaysia, 13% in vietnam, 22% in iran, (all hsct until 2006, only 2006 in taiwan) . the trends changed quite differently among countries and regions over time for other diseases, most strikingly in cml. in japan, the number of hsct for cml had consistently increased until 2000 (n=307 in 2000) and then rapidly decreased due to introduction of imatinib (n=98 in 2005). in contrast, it is dramatically increasing in china (n=8 in 2000 china (n=8 in to n=98 in 2005 and stable in other countries. the number of hsct for mds and multiple myeloma is consistently increasing only in japan (and iran for myeloma), but not in other countries. hsct for solid tumors had been commonly performed only in japan, but the number has decreased since 1997 (n=337 in 1997, n=163 in 2005) . in summary, hsct is developing in most of the asian countries/regions. however, disease indications and trends differed widely among them probably due to different economics situations, health service systems, and availability of donors and agents such as imatinib for cml. short-and long-term side effects in the healthy donors of allogeneic haemopoietic peripheral cells mobilised with lenograstim: a single-centre experience m. martino*, g. console, e. massara, e. spiniello, i. callea, f. gatto, g. messina, t. moscato, r. fedele, g. irrera, p. iacopino u.o. ematologia con trapianto (reggio calabria, it) healthy allogeneic donors, who were mobilized with lenograstim and underwent hemopoietic peripheral cells (hpc-a) collection at our institution, were enrolled in a shortand long-term surveillance protocol for a 10 year period. to date, 171 donors have been assessed with a median followup of 55 months (2 -145) ; for 71 subjects, the follow-up is > 60 months and for 20 subjects is > 96 months. healthy donors received lenograstim at a median dose of 9.9 µg/kg (range 7-15). bone pain was reported as the most common adverse event (70.2 % of donors). common associated symptoms included fatigue (16.4 %), fevers (5 % ), headache (26.3 %), nausea (12.9%), insomnia (16.4 %). spleen size increased in 4.1 % of donors (> 2 cm exceeding the marginal cost at physical examination ). all donors experienced side effect resolution within 2-4 days of lenograstim discontinuation. leukocyte mean peak values were 48 x 109/l and the nadir of platelet counts reached mean values of 93 x 109/l and; however, such a decrease was not complicated by bleeding manifestations. the hemoglobin concentration decreased slightly but not significantly. leukocyte and platelet counts returned to normal values in about one week. no vascular disorders and cardiac disease occurred. long-term observation included adverse events in donors after 30 days from hpc-a mobilization and any neoplastic or not disease developed any time post donation. 4 donors showed persistent, slight leucocytopenia until the second month, with recovery in the fourth month of follow-up. 18 donors showed an ast and alt result 2.5 times the upper limit of normal until the second months of follow-up. transit ischemic attack occurred in 1 donor, (39 months post after donation). 1 autoimmune event has been reported 28 months post-g-csf (anckylosing spondylitis); 1 donor with a history of chronic obstructive pulmonary disease developed a secondary polyglobulia (50 months post-g-csf); 1 donor developed a gastric tumor, 19 months post-donation. no hematological malignancy was observed. in conclusion, our main findings are that the primary toxicity of g-csf administration is bone pain and that no cardiovascular events was related to the donation. with a median follow-up near to 5 years, no hematological disease was observed in our cohort of donors. effect of the search of an unrelated stem cell donor in patients with high-risk leukaemia: prospective, singlecentre study on intention to treat analysis a.p. iori* (1), v. valle (1) allogeneic stem cell transplant (hsct) plays a major role in the treatment of acute leukemia (al) patients with high risk (hr) features at diagnosis or in ≥ 2nd complete remission (cr). between 1995 and 2007, 164 patients -median age 12 years (1 -59) -with hr al followed at our center and lacking a family hla compatible donor were addressed to a search of hsc donor through the bmww registry and cord blood banks. the aim of this prospective study was to assess the effect of the search on the outcome of patients with hr al on an intention to treat analysis. forty-three patients started the s78 search in i cr, 84 in ii cr, 4 in >ii cr, 33 in relapse. fifty-five % of the 131 patients who entered into the study in cr showed a disease relapse at a median of 4 (1-20) and 2 (1) (2) (3) (4) (5) (6) (7) (8) months from the start of the search for patients in i-ii cr and > ii cr, respectively. sixty-nine patients (42%) underwent an hsct, 32% in a more advanced phase compared to the start of the search. nineteen of the 33 patients (57%) who started the search in relapse obtained a cr and underwent an hsct. globally, 45% of patients failed to undergo a transplant because of lost eligibility due to disease progression. for the entire population, the 12 year survival probability and dfs were 19%. disease progression was the major cause of death. when the variables affecting outcome parameters were analyzed in univariate analysis, the occurrence of relapse during the search period and the transplant procedure affected os (p=0.001) and dfs (p<0.0001). a more advanced phase of the disease at the start of the search and no transplantation were the factors negatively affected the relapse (p=0.01, p< 0.0001). for patients who underwent an hsct, the factors which negatively affected os and dfs were a relapse during the search and a more advanced disease phase at transplant. both these factors plus the disease phase at the start of the search affected the relapse for transplanted patients. in conclusion, by decreasing the length of the search (4 months for patients in i-ii cr and 2 months for patients with a more advanced phase of the disease) the risk of relapse can be reduced, thus increasing the possibility of carrying out a transplant and the transplant success. moreover, starting the search in relapse we may obtain the cr while waiting for the hsct. therefore, during the search of an hsc donor the "timing" of the transplant must be considered the major strategic factor for patients with hr al. the goal of this prospective study was to evaluate the impact of donor's and recipient's genetic polymorphisms regarding components of innate immunity on outcome of allohsct. 102 consecutive patients with hematological malignancies, aged 32(18-58)y, treated with allohsct from either sibling (n=34) or matched unrelated (n=68) donors were included. the conditioning was myeloablative. gvhd prophylaxis consisted of csa, mtx ± atg. donors and recipients were tested for single nucleotide polymorphisms(snp)8,12,13 of the nod2/card15 gene, tlr4(299), tlr4(399), tlr5(stop codon c1174t) and il23r(11209026), as well as kir genotype. in addition, immune reconstitution was studied. os rate at 2y was significantly lower in allohsct with at least one activating kir mismatch compared to transplants with full compatibility (62%vs.86%, p=.01). in particular, the presence activating killer immunoglobulin-like receptors (kir) in the donor with its absence in the recipient (d+r-) was associated with decreased rates of os (60%vs.78%, p=.01) and dfs (58%vs.82%, p=.005), as well as increased nrm (27%vs.7%, p=.01). kir2ds1 and kir3ds1 d+r-mismatches resulted in increased risk of grade ii-iv acute gvhd, while kir2ds3 and kir2ds2 d+r-mismatches were associated with increased risk of chronic gvhd. d+r-activating kir mismatches correlated with increased cd8+/cd4+ t cell ratio up to day +100. in all cases of incompatibility regarding kir2ds1, kir2ds2 and kir3ds1, t cells with expression of respective receptors could be detected up to 360 days after allohsct. the presence of snp8 of the nod2/card15 gene in the recipient was associated with decreased probability of os (20%vs.70%, p=.005) and dfs (20%vs.70%, p=.01) as well as increased nrm (60%vs.17%, p=.01) and grade iii-iv acute gvhd (67%vs.8%, p=0.02). in a multivariate analysis adjusted for other potential risk factors, increasing number of d+r-activating kir mismatches as a continues variable appeared to independently influence os, dfs, nrm, grade ii-iv acute gvhd, and chronic gvhd. recipient snp8 of nod2/card15 was predictive for os, dfs, nrm, grade iii-iv acute gvhd, and chronic gvhd. snps of tlr and il23r genes had no impact on outcome. conclusions: both activating kir d+r-mismatches and recipient snp8 of nod2/card15 appear to enhance alloreactivity and independently influence survival after allohsct. evaluation of these polymorphisms may contribute to better donor selection and optimization of the allohct procedure. the interpretation of the role of hla-dpb1 in unrelated haematopoietic stem cell transplantation is subject to discussion. we have investigated the role of hla-dpb1 allele matching on haematopoietic stem cell transplantation (hsct) outcomes in 161 recipients who were hla -a, b, c, drb1, dqb1 matched with their unrelated donors at the allelic level (10/10). additionally, we analysed the association of polymorphic amino acids mismatches of dpb1 molecule with hsct endpoints, and the permissiveness concept of zino and colleagues (zino et al., 2004) . dpb1 allele mismatches were significantly associated with an increased incidence of acute graft-versus-host disease (agvhd), transplant-related mortality (trm) and worse overall survival (os). we observed that the mismatch at amino acid position 69 increased the risk for both agvhd and trm. risk factors for agvhd also included mismatches at positions 8, 9, 35, 76 and 84 . this is, to our knowledge, the first report of an in vivo effect of single amino acid mismatches on hsct outcomes. in our study, grouping of allelic mismatches into permissive and nonpermissive categories and their association with transplantation endpoints proved relevant for trm but not for other clinical endpoints. g.f. torelli*, r. maggio, n. peragine, m.s. de propris, b. lucarelli, m.g. mascolo, m. screnci, s. salvatori, l. malandruccolo, a.p. iori, a. guarini, r. foà sapienza university (rome, it) studies in mouse models of stem cell transplant (sct) have shown that the infusion of culture-expanded regulatory t cells (tregs) can be effective in preventing and suppressing gvhd, while still allowing a gvl effect. cord blood (cb) stem cells are now broadly used in the unrelated sct setting and key: cord-313474-1gux1gsi authors: nan title: physicians abstracts date: 2015-03-20 journal: bone marrow transplant doi: 10.1038/bmt.2015.27 sha: doc_id: 313474 cord_uid: 1gux1gsi nan introduction: the incidence of cgvhd after allogeneic sct is higher when peripheral blood stem cells are used as stem cell source. there is a strong need for preventing cgvhd after asct without increasing the risk of relapse. materials (or patients) and methods: we performed a multicenter, multinational, open-label, randomized study comparing anti-lymphocyte globulin (atg-fresenius s ) 10 mg/kg on day -3, -2 and -1 with no atg in patients with aml (n ¼ 110) or all (n ¼ 45) in 1 st complete remission (cr; n ¼ 139) or 2 nd cr (n ¼ 16) who received peripheral blood stem cells from their hla-identical sibling after standard tbi (12 gy)/ccclophosphamide (120 mg/kg) or busulfan (16 mg/ kg)/cy (120 mg/kg) based myeloablative conditioning regimen. standard gvhd prophylaxis consisted of cyclosporine a and a short course of mtx (10 mg/m 2 on day þ 1, þ 3, þ 6 and þ 11). the primary study aim was to compare the cumulative incidence of cgvhd at 2 years after asct. results: out of 161 randomized patients from 27 centers and 4 nations 6 were withdrawn before conditioning and asct due to leukemia progression, or cancellation of the donor. 155 patients were analyzed for safety and efficacy; 83 were randomized to atg and 72 to non-atg. the treatment groups were comparable regarding recipient and donor age and sex, cmv serostatus, disease (aml vs all), 1 st or 2 nd cr. the median time to leukocyte (41.0x10e9/l) and platelet (4 20x 10e9/l) engraftment was significantly delayed in the atg group (18 vs. 15 days, po 0.001 and 20 vs 13 days, po0.001). the incidence of acute gvhd grade i-iv was 25% for the atg arm and 35% for the non-atg arm (p ¼ 0.32 ) and for severe grade iii/iv acute gvhd 2% and 8%, respectively (p ¼ 0.2). the cumulative incidence of cgvhd at 2 years was 32% (95% ci 22-47%) in the atg and 69% (95% ci 51-74%) in the non-atg arm (po0.0001). regarding limited and extensive cgvhd the ci at 2 years was 26% vs 53% (p ¼ 0.002) and 19% vs 53% (po0.001), respectively. there was no difference in infectious complications, cmv and ebv reactivation between both arms. the cumulative incidence of therapy related mortality at 2 years was 14% (95% ci 8-24%) for the atg arm and 12% (95% ci 6-22%) for the non-atg arm (p ¼ 0.60), resulting in 2 year relapse-free and overall survival of 59% (95%ci 48-69%) and 74% (95% ci 63-82%) for the atg group and of 65% (95% ci 51-75%) and 78% (95% ci 66-86%) for the non-atg group (p ¼ 0.46 and p ¼ 0.21, respectively). chronic gvhd free survival at 2 years was 50% for the atg and 23% for the non atg arm (po 0.001). a composite endpoint cgvhd and relapse-free survival at 2 years was in favor for the atg treated patients (30% vs. 17%, p ¼ 0.005). conclusion: this is the first randomized cgvhd prevention study providing evidence that atg-fresenius s (3x 10 mg/kg) is highly effective in preventing limited and extensive cgvhd in hla-identical sibling pbsc transplantation when used within a myeloablative preparative conditioning regimen. the use of atg-fresenius did not result in an obvious increase of infectious complications and relapse, resulting in similar overall survival rates, but improved cgvhd/relapse free survival. introduction: the establishment of large transplant registries and introduction of novel statistical techniques have paved the way for large scale data analysis. nevertheless, contemporary tools for risk prediction of transplant related mortality (trm) following allogeneic (allo) hematopoietic stem cell transplantation (hct) are of limited clinical use, owing to a sub-optimal predictive accuracy. apart from inherent procedural uncertainty, methodological factors impeding prediction might be attributed to the statistical methodology used, number and quality of features collected, or simply the population size. using an in-silico approach (i.e. iterative computerized simulations), based on machine learning (ml) algorithms, we aimed to define prediction limiting factors of day 100 trm and rank variable contribution. ml is a field of artificial intelligence dealing with the construction and study of systems that can learn from data, rather than follow explicitly programmed instructions. commonly applied in complex data scenarios, such as financial settings, it may be suitable for outcome prediction of hct. materials (or patients) and methods: study population was a cohort of 28,236 adult acute leukemia allo-hct recipients from the ebmt-alwp. twenty four variables were analyzed, including recipient, donor and transplant characteristics. study design involved two phases. the first, focused on development and comparison of several ml based prediction models of day 100 trm. in the second phase, a repetitive computerized simulation was applied. factors necessary for optimal prediction were explored: algorithm type, size of data set, number of included variables, and performance in specific subpopulations. models were assessed and compared on the basis of the area under the receiver operating characteristic curve (auc). results: six ml based prediction models for day 100 trm were developed on the entire dataset. optimal aucs ranged from 0.65-0.68 . depending on the algorithm used for prediction model development, the in-silico experimental system yielded the following results: predictive performance plateaued on a population size ranging from n ¼ 5647-8471; a range of 6-12 ranked variables, selected by a separate feature selection algorithm, were necessary for optimal prediction; disease status and donor type were consistently top ranking variables. predictive performance of models developed for specific subpopulations, ranged from 0.59 to 0.67 for patients in second complete remission and patients receiving reduced intensity conditioning respectively. conclusion: we present a novel experimental system for assessment of prediction boundaries in hct. the present approach has clinical implications. we show that predictive performance of day 100 trm is unlikely to improve with the data routinely gathered on hct recipients, as different algorithms reach approximately the same performance. in addition, an exhaustive search for variable importance, reveal that few variables "carry the weight" with regard to predictive influence. predictive performance converged when sampling more than 5000 patients, reflecting the importance of large registry studies. overall, it seems we have reached a point of predictive saturation. improving predictive performance will likely require additional types of input like genetic, biologic and procedural factors. disclosure of interest: none declared. introduction: allogeneic stem cell transplantation (sct) can provide long-term disease control in selected patients with relapsed refractory nhl. restricted availability of a matched sibling donor limits its use especially for patients with rapidly progressing disease in whom unrelated donor search cannot be awaited. because data of alternative donor transplants in nhl is sparse, we aimed to compare outcome of haploidentical and cord blood transplants with conventional relatedand matched unrelated donor transplats for nhl. materials (or patients) and methods: information of patients with mantle cell lymphoma (mcl), dlbcl, t-cell lymphoma (tcl) and follicular lymphoma (fl) who received an sct from a sibling donor (sib), 10/10 matched unrelated donor (mud), haplo-identical donor (haplo) or cord blood (cord) between 2007 and 2012 was downloaded from the ebmt database. results: 2798 patients with nhl were identified in the ebmt database meeting the inclusion criteria. 2065 received a transplant from a sib, 447 from a mud, 167 from cord (18 mcl, 36 dlbcl, 43 fl, 70 tcl) and 119 from a haplo donor (16 mcl, 30 dlbcl, 22 fl, 51 tcl) . active disease (po0.01) and karnofsky index (ki) below 80% was also more common in the haplo group (p ¼ 0.02). other variables were balanced. median follow-up after sct for all patients was 27 months (ci 25 to 29). relapse incidence after conventional transplants (sib, mud) and alternative donor transplants (haplo, cord) was not significantly different within the whole group (haplo: hr 1.2 95% ci 0.9-1.8 p ¼ 0.23; cord: hr 1.1 95% ci 0.7-1.4, p ¼ 0.74 ) and across all studied disease entities. in contrast, nrm incidence was not significantly different between sib and mud, but significantly higher with alternative donor transplants (haplo: hr 1.8 95% ci po0.001 ; cord: hr 1.9 95% ci po0.001) . with the exception of fl where mud in addition to haplo and cord transplants had a significantly higher nrm incidence than sib transplants. because patients who received a haplo transplant had more commonly active disease at transplant and worse ki, we performed multivariate modeling of relapse-and nrm incidences adjusting for the above mentioned covariates. no different relapse incidences between donor groups was observed. nrm incidence in contrast, was significantly higher in mud (reference sib, hr , p ¼ 0.033) and cord (reference sib, hr , p ¼ 0.001) but not in haplo transplants. most interestingly, acute gvhd incidence was significantly increased in mud compared to sib (p ¼ 0.003) transplants but not in haplo (p ¼ 0.08) or cord (p ¼ 0.34) transplants. multivariate adjustment for diagnosis (mcl, dlbcl, fl, tcl), remission prior to sct, ki (kio90% vs. 490%) and conditioning intensity (ric vs. mac) did not reveal worse os for haplo but a worse os for cord (hr po0.0003 introduction: retrospective studies in mds/saml suggest that reducing the intensity of the conditioning regimen prior to allogeneic stem cell transplantation reduces the risk of nonrelapse mortality but is associated with a higher risk of relapse, but prospective randomized studies for mds are lacking so far. materials (or patients) and methods: within the chronic malignancies working party (cmwp) of ebmt, we performed a prospective randomized trial comparing a busulfan based (busulfan 8 mg/kg orally or equivalent dosis intravenously (iv) plus fludarabin 180 mg/m 2 ) reduced intensity conditioning regimen (ric) and a standard myeloablative busulfan (busulfan 16 mg/kg orally or equivalent dosis iv plus cyclophosphamide 120 mg/kg) based regimen (mac) in patients with mds or saml (o20% blasts). between may 2004 and december 2012, 129 patients were included from 18 centers and 7 nations and 127 could be analysed. major inclusion criteria were: mds (according to fab: ra, rars, raeb, raeb-t), cmml, and saml, blasts less than 20%, matched related or unrelated donor (hla 8/8, 1 mismatch was allowed), age 18 -60 years (for unrelated) and 18 -65 years (for hla-identical sibling). included patients were stratified according related vs unrelated donor and blast countoor4than 5%. the primary endpoint of the study was 1 year non-relapse mortality.the median age of the patients was 51.4 years (r.19-64y) . donors were hla-identical sibling (n ¼ 33), matched unrelated (n ¼ 74) and mismatched related or unrelated (n ¼ 20). the patients were well distributed in both arms regarding age, gender, ipss risk profile, number of blasts at transplantation, donor source and mismatch donor. results: graft failure occurred in 1 after mac and 2 after ric. median time to leukocyte (4 1.0 x 10e9/l) occurred after 14days after mac and 15days after ric and and platelet (420x10e9/l) engraftment occurred after 14 days after mac and 15 days after ric, respectively. acute gvhd ii-iv was noted in 28% after ric and 31% after mac. chronic gvhd was seen in 64% after ric and 63% after mac. the cumulative incidence (ci) of non-relapse mortality (nrm) after 1 year was 17% (95% including 12 pediatric/adolescent cases o18 years), 74 with myeloablative conditioning (67%; 34 tbi and 40 non-tbibased), 64 from hla-identical siblings (58%), and 9 using ex vivo t-cell depletion. in addition to the above matching criteria, cases and controls were also balanced for other factors such as donor gender and gender mismatch, cmv serostatus, in vivo t-cell depletion and karnofsky's performance status. compared to hiv-neg, hiv-pts had lower rates of neutrophil engraftment (92.6% vs 97.5%, p ¼ 0.03), higher incidence of grade iii-iv acute gvhd (21% vs 13%, p ¼ 0.05), higher nrm at day 100 (17% vs 11%, p ¼ 0.04) and 2 years (33% vs 23%, p ¼ 0.04), and similar incidence of relapse (29% vs 25%, p ¼ 0.96). overall, hiv-pts had poorer pfs (39% vs 52%; p ¼ 0.03; hr 1.42 [1.04-1.94] ) and os (47% vs 59%; p ¼ 0.004; hr 1.60 [1.16-2.22 ]) at 2 years than hiv-neg cases.outcomes within hiv-pts were comparable for myeloablative vs reduced intensity conditioning and among peripheral blood, bone marrow and cord blood stem cell sources (data not shown). finally, while hiv-pts' outcomes were comparable for allo-hct from hla-identical siblings and alternative donors (os: 48% vs 40%; p ¼ 0.38) , the use of such alternative donors in hiv-pts was less common than in hiv-neg hct recipients (42% vs 50%, p ¼ 0.02). conclusion: this study showed that the outcome of allo-hct is poorer in hiv-pts than in the general population, primarily driven by higher nrm, and in keeping with their inferior overall life expectancy despite haart. even so, allo-hct is feasible in hiv-pts with hematologic indications, with a 47% os at 2 years. in view of the current reduced use of alternative donors despite comparable results to matched sibling donors, hiv-pts requiring an allo-hct should be granted access to donor search and consideration for transplantation at the same level as hiv-neg counterparts. these data are key to inform allo-hct strategies in hiv-pts, including its investigational use to eradicate hiv infection. disclosure of interest: none declared. introduction: haplo-hsct is a therapeutic option for patients with high risk hematologic neoplasms with the advantages of quick availability, easy programation and logistics, and a committed donor. it has shown promissing results in patients diagnosed with relapsed or refractory hodgkin lymphoma (hl) at least comparable to allogeneic transplant from siblings or unrelated donors (burroughs lm et al. biol blood marrow transplant 2008; 14:1279 -1287 . materials (or patients) and methods: we retrospectively evaluate the results of haplo-hsct with iv busulfan (bux) based ric regimens (fludarabine 30 mg/m2 x5 days (-6 to -2), cyclophosphamide14,5 mg/kg x2 days (-6 to -5) , bux 3,2 mg/ kg x 1 (bux1)or 2 days (bux2) on days -3 to -2) and gvhd prophylaxis based on ptcy (50 mg/kg on days þ 3 and þ 4) and a calcineurin inhibitor plus mycophenolate from day þ 5 performed in geth centers to patients diagnosed with relapsed or refractory hl. results: from march-2009, 43 haplo-hsct have been performed in patients diagnosed with relapsed or refractory hl in 11 geth centers. median age was 31 years (17-53), 67% were males and all were in advanced phases of their disease, after a median of 4 prior treatment lines (2) (3) (4) (5) (6) (7) (8) . autologous hsct was previously employed in 79%, and allogeneic hsct in 7%. five patients (11.5%) have received more than 2 prior transplants. disease status at haplo-hsct evaluated by pet was complete remission in 14 (32%) and persistent disease in 29 (68%). bone marrow was employed in 11 (26%) and peripheral blood in 32 (74%), without t-cell depletion in all cases. the haploidentical donor was patients mother (20), father (3), siblings (19) or daughter (1) . the ric regimens employed were bux1 in 14 (32.5%) and bux2 in 29 patients (67.5%). median neutrophils engraftment was day þ 18 (13-44) and platelets 420 k was day þ 26 (13-150). graft failure with autologous reconstitution happened only in 1 patient (2.5%). the day þ 100 cumulative incidence (ci) of non-relapse mortality (nrm) was 7% (3/43) and 16% (7/43) at 1 year posttransplant. the day þ 100 ci of grade ii-iv acute gvhd was 43%, and grade iii-iv was 14.5%. chronic gvhd ci was 26.5% at 1 year, being extensive in 6%. after a median follow-up for survivors of 13 months , the event-free survival (efs) was 59.5% and overall survival (os) was 84%. the 1-year ci of relapse or progression was 25%. factors related with better 1year efs were cr prior to haplo-hsct (93% vs 45%; p ¼ 0.017) and receiving less than 4 treatment lines prior to haplo-hsct (100% vs 51.5%; p ¼ 0.018). no significant differences were seen when comparing bux1 against bux2 in terms of nrm, efs or os. conclusion: haplo-hsct with ptcy and bux based ric conditioning in relapsed or refractory hl patients, renders long-lasting remissions with acceptable toxicity and gvhd, obtaining better results in those transplanted in cr and with less than 4 treatment lines prior to haplo-hsct. disclosure of interest: none declared. introduction: b-lineage acute lymphoblastic leukemia (all) is the most common childhood cancer. although this disease can be successfully treated in 80% of patients, prognosis for primary refractory or relapsed disease is very poor. even after allogeneic stem cell transplantation (sct), relapse rates are considerable and correlate significantly with persistent minimal residual disease (mrd) prior to and after sct. mrd constellations represent favorable effector-target ratios and thus might be optimally suited for immunotherapeutic intervention with therapeutic antibodies. materials (or patients) and methods: we developed an fcoptimized cd19 antibody (4g7sdie) and produced it in pharmaceutical quality. 4g7sdie mediates markedly enhanced antibody-dependent cellular cytotoxicity (adcc) through its improved capability to recruit fcgriiia bearing effector cells including nk cells and gd t cells. 4g7sdie was applied on compassionate use to mrd-positive pediatric patients with relapsed or refractory all (cr1 n ¼ 3, zcr2, n ¼ 11). results: side effects such as headache and fever were negligible. in all patients complete cd20 þ b-cell depletion was observed during therapy. after discontinuation of 4g7sdie therapy b cell counts recovered rapidly to normal levels. in 9/14 patients mrd was reduced byz1 log or fell below mrd-detection threshold of 10 -4 over the course of treatment. 2/9 responders were receiving additional treatment. 6 patients relapsed, 1 patient died of cns chemotherapy associated toxicity and 1 patient died of late posttransplant sepsis. 6 patients have been in sustained remission for 264-1115 days (median follow-up 720 days). in initial cytotoxicity screenings, performed in an allogeneic setting, significantly increased lysis of all blasts by pbmc after adding 4g7sdie was observed. nk cells and gd t cells were identified as main effector cell populations. cd19 expression on patient blasts was confirmed by quantitative flow cytometry (mean 1.71x10 4 molecules/ cell, ± 0.54x10 4 ). cytotoxicity assays using patient pbmc on autologous blasts confirmed sustained functionality of patient effector cells over the course of 4g7sdie treatment. cytotoxicity assays were performed using pbmc from transplanted patients obtained at different time points of 4g7sdie treatment. lysis of autologous all blasts was increased when 4g7sdie or autologous patient serum taken after antibody infusion was added. after infusion of 20 mg/m 2 -40 mg/m 2 4g7sdie serum half-life was 20 h -43 h. serum levels of 4g7sdie remained above saturating concentrations ofz700 ng/ml (ec 50 ¼ 65 ng/ml) until the following application in the bi-weekly treatment cycle. notably, in 2/2 analyzed patients under 4g7sdie therapy, a down-modulation of cd19 surface expression on the leukemic blasts was observed. in vitro antigenic shift assays on patient blasts showed considerable but very heterogeneous shift of cd19 surface expression. furthermore, a positive correlation between cd19 surface expression levels and 4g7sdie mediated lysis was observed. these observations hint at in vivo tumor escape mechanisms and moreover indicate selective pressure exerted by immunotherapy with 4g7sdie, underlining its therapeutic potential, but also delineating possible limitations. conclusion: promising anti-leukemic effects of the 4g7sdie antibody have been observed in vitro and in vivo. we are currently preparing a clinical phase i/ii trial. disclosure of interest: none declared. introduction: allogeneic hct with myeloablative conditioning is considered a standard of care for adults with high risk acute lymphoblastic leukemia (all). however, with improving results of conventional-dose chemotherapy and the introduction of novel agents on one hand, and the improvement in transplantation techniques on the other, the indications for allohct require re-evaluation, taking into account patient-and procedure-related factors associated with the risk of nonrelapse mortality (nrm). the aim of this study was to analyze the results of myeloablative allohct treatment for patients with all according to recipient age and donor type. materials (or patients) and methods: 4859 patients treated with allohct in first complete remission during the period 1993-2012 were included. the outcomes were analyzed for the periods 1993-2002, 2003-2007 and 2008-2012 , in various age groups, separately for hla matched sibling (msd, n ¼ 2681) and unrelated donors (urd, n ¼ 2178). results: for msd-allohct recipients treated during the period 2008-2012, the following two-year probabilities of os were obtained: 76% for the 18-25 years old (y.o.) group, 69% for both the 26-35 y.o. and 36-45 y.o. groups and 60% for the 46-55 y.o. group. the incidence rates of nrm were 12%, 11%, 15% and 24%, respectively for those same age groups. in comparison with the 1993-2007 period, significant improvements were observed for all age groups except for the 26-35 y.o. patients. the improved survival rates were a consequence of reduced nrm and a tendency towards a reduced risk of relapse. among urd-allohct recipients, the os was 66% (18-25 y.o.) , 70% (26-35 y.o.) , 61% (36-45 y.o.) , and 62% (46-55 y.o.) , while the respective incidence rates of nrm were 21%, 20%, 21% and 19%, the improvement of os over time was documented for 36-45 y.o. (p ¼ 0.005) and 46-55 y.o. (0.0007 ) patients due to the reduced incidence of nrm with no significant effect on relapse. in a multivariate analysis adjusted for disease-, patient-, donorand procedure-related factors, transplantations performed for the period 2008-2012, when compared to 1993-2007 , were associated with significantly reduced risks of the overall mortality (hr ¼ 0.72, p ¼ 0.0003), treatment failure (either relapse or nrm, hr ¼ 0.77, p ¼ 0.002), and nrm (hr ¼ 0.73, p ¼ 0.01) and showed a trend towards reduced risk of relapse (hr ¼ 0.81, p ¼ 0.06). the overall mortality was reduced for transplants with tbi-based compared to chemotherapy-based conditioning (hr ¼ 0.71, p ¼ 0.005) as a result of reduced risk of relapse (hr ¼ 0.55, p ¼ 0.00004). type of donor (msd vs. mud) had no significant effect on survival (hr ¼ 0.9, p ¼ 0.24). conclusion: results of allohct for adults with all improved significantly over time in all age groups, mainly due to the reduction of nrm. importantly, results obtained with matched unrelated transplants were comparable to sibling transplants. total body irradiation should still be considered as the preferable type of myeloablative conditioning for all. disclosure of interest: none declared. cmv seronegativity is associated with a 20% decrease in 1-year survival in patients undergoing reduced intensity sibling-donor transplantation for treatment of myeloid malignancy d. j. lewis 1,* , c. holmes 1 , k. peggs 2 , a. peniket 3 , m. nikolousis 4 , s. nagra 5 , g. pratt 1,4 , c. craddock 1,5 , r. malladi 1,5 , p. moss 1, 5 1 school of cancer sciences, university of birmingham, birmingham, 2 university college hospital, london, 3 oxford university hospital, oxford, 4 birmingham heartlands hospital, 5 centre for clinical haematology, university hospital birmingham, birmingham, united kingdom introduction: reduced intensity (ric) allogeneic stem cell transplantation is a highly effective treatment for acute myeloid leukaemia and the immunological 'graft versus leukaemia' effect is believed to be a major mechanism of disease control. cytomegalovirus reactivation has been suggested to reduce the rate of disease relapse in acute myeloid leukaemia (aml). we investigated the influence of cmv serostatus on the clinical outcome of patients who underwent t cell depleted ric allografts for myeloid malignancy, and went on to examine reconstitution of lymphoid subsets. materials (or patients) and methods: we studied patients who underwent ric allografts for aml (n ¼ 272) and mds (n ¼ 82) from four uk centres, with fludarabine and melphalan conditioning þ /-alemtuzumab. overall survival was calculated. relapse rate and non-relapse mortality were calculated with competing risk analyses. results: the median overall survival was 2.17 years. the relapse rate of the entire cohort was 23% at 1 year with a nonrelapse mortality of 22%. the overall survival for the 'cmv at risk' group was 64.7% at 1 year compared to 57.9% for cmv (-/-). this difference was most marked in patients transplanted from sibling donors (n ¼ 140); the overall survival at 1 year was 79% in cmv-at-risk patients (n ¼ 99) compared to only 59% in the cmv seronegative group (n ¼ 41) (p ¼ 0.027). this 20% increment in survival was due to a 37% reduction in the rate of disease relapse in patients that were cmv-at-risk (1 year relapse rate of 21% versus 33%). there was a non-significant trend towards improved overall survival in those that experienced cmv reactivation amongst the cmv-seropositive patients (1 year os 83% versus 61%, p ¼ 0.19) , mainly due to a reduction in the rate of relapse (1 year relapse rate 17% versus 28%). because of this large difference in relapse risk, we went on to examine the effects of cmv serostatus and alemtuzumab use on reconstitution of lymphocyte subsets at 3 months post transplant. alemtuzumab led to 5-fold decrease in the t cell count at 3 months compared to transplants in which t cell depletion was not used. however, within this alemtuzumab group, positive cmv serostatus in the donor or recipient was associated with a relative 7 fold and 35 fold increase in the cd3 þ and cd8 þ t cell count compared to cmv seronegative pairs. indeed, cd8 þ t cells were virtually undetectable at this time point in cmv seronegative transplant/donor pairs. conclusion: cmv seropositivity is markedly beneficial for patients who undergo a sibling donor reduced intensity allograft for myeloid malignancy and in whom alemtuzumab is used for conditioning. this effect is most likely to be due to the profound influence of chronic viral replication on boosting t cell immune reconstitution in the early post transplant period. cmv seronegative patients with aml should be considered at risk of impaired survival for certain subgroups of stem cell transplant. disclosure of interest: none declared. introduction: allogeneic stem cell transplantation is the only curative option for patients with high risk acute myeloid leukemia (aml) and for those experiencing relapse. either matched sibling donor (msd) or unrelated donor (ud) is indicated. materials (or patients) and methods: with the aim to compare the outcomes of both strategies we have retrospectively analysed 1554 adults with aml receiving either msd (n ¼ 961) or ud (n ¼ 593) in ebmt centers from 2000-2012. for ud, 481 were 10/10 hla matched and 112 were 9/10. median follow up was 28 (range 3-157) months. there were statistical differences between the 2 groups. compared to msd recipients, ud transplants were older (49 vs 52 years, p ¼ 0.001), were performed more recently (2009 vs 2006, p ¼ 0.001) , had longer interval between diagnosis to transplant (10 vs 9 months, p ¼ 0.001), had more often secondary aml (13% vs 19%, p ¼ 0.002) and were transplanted with higher proportion of cmv negative donors (38% vs 37%, p ¼ 0.001). peripheral blood stem cells (pbsc) was used as graft source in 90% of patients in both groups, p ¼ 0.14. conditioning regimen was more frequently myeloablative for patients transplanted with a msd (61% vs 46%, p ¼ 0.001). msd received more often busulfan and cyclophosphamide as mac (16%) or a tbi based regimen (18%). for ud, bu-fludarabine was the most frequent conditioning used (19%). results: cumulative incidence (ci) of neutrophil engraftment was similar (93% vs 92% for msd vs ud, respectively, p ¼ 0.07). grade ii-iv acute gvhd was 26% vs 30% (p ¼ 0.11 ) and ci of chronic gvhd was 25% vs 24% (p ¼ 0.9) for msd and ud, respectively. for msd and ud respectively, ci of relapse (ri) was 57% vs 49%, p ¼ 0.001; ci of non-relapse mortality (nrm) was 23% vs 24%, p ¼ 0.24. probability of leukemia-free survival (lfs) at 2 years was 21% vs 26%, p ¼ 0.001, and overall survival (os) was 26% vs 33% p ¼ 0.004, respectively. chronic gvhd as time-dependent variable was associated with lower ri (hr 0.78 , p ¼ 0.05), higher nrm (hr 1.71 , p ¼ 0.001), and higher os (hr 0.69, p ¼ 0.001). according to hla-match for msd, 10/10 ud and 9/10 ud, ci of relapse (ri) was 57% vs 50% vs 45%%, p ¼ 0.001; ci nrm was 23% vs 23% vs 29%%, p ¼ 0.26 and probability of lfs at 2 years was 21% vs 27% vs 25%, p ¼ 0.003, respectively. in a multivariate analysis adjusted for differences between the 2 groups, ud was associated with lower ri (hr 0.76, p ¼ 0.001) and higher lfs (hr 0.83, p ¼ 0.001) compared to msd. when analyzing according to hla-match, there was no differences for patients transplanted with an ud 9/10 or a 10/10 for ri (hr 0.77, p ¼ 0.10) , and lfs (hr 0.92, p ¼ 0.53) . the other factors independently associated with better outcomes were the interval between diagnosis and transplant (ri hr 0.62, po0.001) , and lfs (hr 0.67, po0.001) . conclusion: unrelated donor transplant was associated with better lfs due to lower ri compared to msd for high-risk patients with aml transplanted in first relapse. there were not differences for the ud 9/10 match probably due the graftversus-leukemia effect in the setting of patients transplanted with active disease. disclosure of interest: none declared. introduction: allogeneic reduced intensity transplantations (rict) in elderly patients (pts) with aml in cr1 has become a commonly used treatment modality. however, no prospective studies to date have been reported to support use of this strategy. the aim of this prospective, multicenter study is to compare outcomes of patients receiving rict with patients being treated with conventional chemotherapy. in 2012 an amendment allowed also the use of matched unrelated donors. the study is currently ongoing in 8 countries with a total of 250 pts included. only pts included as per the original protocol are accounted for in this interim analysis which focuses on safety. materials (or patients) and methods: the study was designed by the transatlantic leukemia group (tralg) consisting of centers associated with the swedish and canadian bmt groups, and centers from germany, norway, finland and new zealand. patients should have intermediate or high risk aml in cr1 and not being eligible for a myeloablative transplant due to age or comorbidities, and have at least one potential sibling donor. date of inclusion was defined as the date of hla-typing of the first sibling, and pts were allocated to the donor (d) or no-donor group (nod) based on hla match. overall survival at 3 yrs is the primary endpoint; secondary endpoints include rfs, gvhd, non-relapse (nrm) and transplant related (trm) mortality. conditioning consisted in the vast majority of patients of fludarabine 150 mg/m 2 iv and busulphane 8 mg/ kg po or 6.4 mg/kg iv. gvhd prophylaxis was cya/methotrexate or cya/mmf. the study started to accrue patients in 2004. results were analyzed on an intent to treat basis. results: from 2004 pts were included in sweden (n ¼ 62), canada (n ¼ 56), germany (5) , norway (13), new zealand (5) and finland (5) . 12 patients were excluded from analysis; 3 favorable risk, 9 protocol violations and 134 pts; 60 females, 74 males, median age 62 (51-74) yrs, 21 saml, were allocated to the d group (n ¼ 75), or the nod group (n ¼ 59). in the d group, 16 pts (21%) did not reach transplant, 15 of these died (12 after relapse, 3 other causes). cytogenetic risk groups were categorized as per eln criteria. the distribution of total disease risk categories was 25% adverse, 66% intermediate, 9% unknown. median follow-up of surviving pts at dec 6 th 2014 was 4.6 (2.6 -10.1 ) yrs after inclusion. ninety-one pts relapsed (75%). kaplan-meier estimates of os and rfs in the whole group at 3 yrs were 45% and 37% respectively. for adverse and intermediate risk pts os and rfs at 3 yrs were 36% and 21%, and 51% and 44%, respectively. in the transplanted pts acute gvhd grade 0-1 occurred in 73% of pts, grade 2-4 in 20%. extensive gvhd occurred in 42%, limited in 15%. 89 pts (66%) have died, 17 without and 72 after aml relapse. in the nod group 5 (8%) pts died from non-relapse causes. in the d group, 3 pts died before transplant, while the trm was 9/59 (15%) with causes of death: 6 gvhd, 3 other. conclusion: selected patients with aml in cr1 tolerate rict or standard management with low mortality. disease control remains a major issue. this multicenter prospective protocol will continue to accrue patients until relevant conclusions can be drawn comparing a rict to standard treatment. the current rapid inclusion of pts and participation of new sites in australia, greece and estonia will help to complete the study. disclosure of interest: none declared. introduction: the wilms' tumor gene 1 (wt1) is overexpressed in 480% of acute myeloid leukemias (aml) and myelodysplastic syndromes (mds) and proved to be a good marker for minimal residual disease (mrd) monitoring. although allogeneic haematopoietic stem cell transplantation (allo-hsct) is the most effective treatment for aml/mds, disease recurrence remains a major problem. after allo-hsct, quantitative wt1 monitoring can represent a useful tool to detect mrd and tailor immunotherapeutic strategies. materials (or patients) and methods: we included in this retrospective analysis 111 pts with aml/mds (99 and 12 respectively) overexpressing wt1 and allotransplanted in our institution from 12/2007 to 01/2014. twenty two pts were transplanted from a matched related donor, 26 from a matched unrelated donor, 57 from a mismatched related donor and 6 from cord blood units. 101/111 pts received a reduced toxicity conditioning treosulfan-based. at the time of transplant, 73/111 pts (66%) were in cr, while 38/111 (34%) had active disease. median follow up (fu) of our cohort was s10 599 days (range, 55-2538 days). wt1 mrd monitoring was performed by rq-pcr and considered positive for values4250 copy numbers of wt1 per 10e4 abl [1] . after allo-hsct, detection of positive wt1 was followed by immunomodulatory therapeutic interventions according to the time from transplant, the presence of active graft-versus-host disease (gvhd) and the general clinical conditions: tapering and/or discontinuation of immunosuppressive drugs (is), donor lymphocytes infusions (dli), administration of hypomethylating agents. median time to disease relapse was calculated from the time of detection of wt1 value above the threshold. results: at day 30 post allo-hsct, 109 out of 111 pts (98%) were in cr. forty-five out of 109 (41%) cr pts had wt1 levels persistently negative during follow up (fu) and 23/45 remained in cr at the last fu. sixty-four out of 109 pts (59%) had at least one increase of bm wt1 levels above the threshold during observation and were evaluated for preemptive treatment. in 45/64 (70%) is was tapered until suspension and/or dli þ /-azacytidine was administered. median time from first wt1 positive value to treatment was 30 days (range, 15-45 days). in 16 out of these 45 pts (35%) wt1 level normalized, whereas 29 pts (65%) progressed to overt disease. all the 16 that normalized wt1 remained in cr. no grade iii or iv acute gvhd was observed, while severe chronic gvhd occurred in 2/45 pts (4%). median time to disease relapse for the 45 treated pts was 116 days (range, 20-951 days). in 11/64 pts (17%) with a post allo-hsct wt1 positivity, the presence of an active form of acute or chronic gvhd prevented from applying further immunotherapy strategies. among these pts, 5 remained in cr, 6 relapsed. median time to disease recurrence for these 11 pts with gvhd was 156 days (range, 32-204 days). finally in 8/64 pts (13%) concurrent clinical issues (e.g. active infection) did not allow any attempt to prevent relapse and 4/8 relapsed. median time to disease relapse for untreated pts was 35 days (range, 15-82 days). conclusion: in our series, pre-emptive immune-modulatory maneuvers targeting wt1 levels proved to be feasible and safe and of potential clinical benefit to postpone overt relapse in high risk aml/mds pts post allo-hsct. introduction: allogeneic stem cell transplantation (sct) with both myeloablative (mac) and reduced intensity conditioning (ric) is effective therapy in aml. several studies have shown that leukemia-free survival (lfs) is similar after sct with mac and ric. however, there is paucity of data on the long term outcome (beyond 10 years) following ric due to the relative recent introduction of this approach. the alwp of ebmt published in leukemia 2005 the largest study until that time, comparing the outcome of aml patients (pts) given ric (n ¼ 315) and mac (n ¼ 407). the median follow-up was 13 months. in multivariate analysis, non-relapse mortality (nrm) was significantly lower and relapse rate was significantly higher after ric resulting in similar lfs. materials (or patients) and methods: in order to better predict long-term outcome and late events we have now updated sct outcomes in a larger cohort of pts, age z50 years (n ¼ 1423) after sct from matched sibling donors, in the years 1997-2005 with a median follow up of 8.3 years (0.1-17) . results: 722 pts were given ric and 701 mac regimens. the median age at sct was 57 (50-75) and 54 (50-72) years, respectively (po0.001). 21% of ric recipients had advanced disease at sct compared to 25% of mac recipients. the percentage of pts in cr1 and cr2/ later cr was 62% and 17% compared to 63% and 12%, respectively (p ¼ 0.02). ric recipients were more likely to receive pbsc (92% vs 73%, po0.001 ) and in vivo t-cell depletion (33% vs 12%, po0.001). 16% and 20% had poor-risk cytogenetics, respectively (p ¼ 0.19 1.4, po0.001 ) and poor cytogenetics (hr 1.7, po0.005) . the conditioning regimen did not predict 10-year lfs. nrm rates were higher after mac than ric throughout the late post sct course, while relapse rates were only mildly decreased at the late phases. in pts surviving leukemia-free 2 years after sct the subsequent nrm was 15% and 9%, respectively (p ¼ 0.03). subsequent relapse rates were 14% and 9% (p ¼ 0.12) and lfs was 71% and 73%, respectively (p ¼ 0.76). in pts surviving leukemia-free 5 years after sct, subsequent nrm was 9% and 4%, respectively (p ¼ 0.06). subsequent relapse rates were 5% and 6% (p ¼ 0.53), and lfs was 86% and 90%, respectively (p ¼ 0.27). conclusion: lfs remains similar after ric and mac even 10 years after sct. the trend for excess nrm with mac remains throughout the late course, however excess relapse after ric is more obvious in the early phases. pts remaining leukemia-free 5 years after sct can expect excellent subsequent outcome with both regimens. long-term follow-up studies (beyond 10 years) are of significant importance when assessing sct outcomes. disclosure of interest: none declared. myeloablative busulfan based strategies in transplantation particularly of children with non-malignant diseases. a reduction in short term mucosal and hepatic (sos) toxicity and the absence of long term pulmonary toxicity have been demonstrated. it is unclear, however, whether this reduction of toxicity is accompanied by an equal or inferior myeloablative capacity compared to busulfan based myeloablative regimens. materials (or patients) and methods: we performed a retrospective analysis of consecutive patients with nonmalignant diseases transplanted in 4 german pediatric transplant centers (hamburg, hannover, munich and ulm) with a treo based regimen (treo and fludarabin (flu) ± thiotepa (tt)±serotherapy) in the period between january 1st 2000 and june 30th 2013. results: we identified 153 patients with inborn errors including primary immunodeficiencies, hemoglobinopathies, hemophagocytic lymphohistiocytosis, mucopolysaccharidosis and osteopetrosis. the median age at transplantation was 4.8 years (0.1-22) . all pts received treo/flu. tt was added in 102 of these pts, serotherapy in 139 pts. the os after 2 years was 89%, the efs 81%. the incidence of agvhd 1ii-1iv was 19% and 5.4% for cgvhd 11-13. primary engraftment of donor cells was present in 97% of patients. however, mixed chimerism at any time point was found in 59% and disease recurrence in 11%. an additional cellular therapy (act: stem cell boost, dli, or 2nd transplant) was applied in 28% of patients. act was more often needed after transplantations from mmfds and muds than from msd/mfds (p ¼ 0.08). there was a significant difference in patients who received alemtuzumab as serotherapy early (d-13 to d-4) versus late (d-3 or later) during conditioning, with 32% act in the early and 52% act in the latter group (p ¼ 0.02). cell source (bm or pbsc) and addition of tt did not affect the cumulative incidence (ci) of act. conclusion: there is an excellent overall survival and efs with treo based conditioning in the transplantation of patients with non-malignant diseases. despite a good primary engraftment, a high rate of mixed chimerism, disease recurrence and need for additional cellular therapy was observed. interestingly, early administration of serotherapy was correlated with a higher probability of stable donor engraftment and has to be considered as a relevant factor for transplant outcome. a randomized controlled prospective study comparing conditioning regimens with treo against busulfan in nonmalignant diseases is needed. disclosure of interest: none declared. long-term outcomes of reduced-intensity conditioning allogeneic stem cell transplantation for adult high-risk acute lymphoblastic leukemia in first complete remission s. lee 1,* , k. introduction: reduced-intensity conditioning (ric) allogeneic stem cell transplantation (sct) has emerged as an option designed to lower nonrelapse mortality (nrm) for older patients and those with comorbid conditions. however, the role of ric-sct in adult acute lymphoblastic leukemia (all) remains unclear because the interpretation of transplantation outcome is mainly limited by the small sample size, short follow-up duration, various regimens for conditioning and graft-versus-host disease (gvhd) prophylaxis, and the heterogeneity of the criteria used to select patients for ric-sct. previously, we conducted a phase 2 trial of ric-sct in adults with high-risk all and showed the potential role of this strategy, especially in patients in first complete remission (cr1). here, we report on the updated results of ric-sct by analyzing 93 consecutive adult high-risk all transplants in cr1. materials (or patients) and methods: during the period between 2000 and 2012, 93 consecutive patients in cr1 (median age, 51 years [range, 15-65 years]) were given an identical ric regimen consisting of fludarabine (150 mg/m 2 in total) and melphalan (140 mg/m 2 in total). the indications for ric-sct were advanced age (z50 years; n ¼ 53; 57.0%) and comorbid conditions (n ¼ 40; 43.0%). graft sources were peripheral blood stem cells (n ¼ 91; 53 8/8-matched sibling donor, 15 8/8-matched unrelated donor, 23 7/8-matched unrelated donor) and bone marrow (n ¼ 2; 1 8/8-matched unrelated donor, 1 7/8-matched unrelated donor). the median time-to-transplantation was 161 days (range, 106-291 days). gvhd prophylaxis was attempted by administering calcineurin inhibitors (cyclosporine for sibling donor transplants, tacrolimus for unrelated donor transplants) plus methotrexate. antithymocyte globulin (2.5 mg/kg in total) was administered to the patients who received allele-mismatched unrelated donor grafts. if residual leukemia was detected in the absence of gvhd at 3 months after transplantation, calcineurin inhibitors were rapidly discontinued. results: fifty-two patients developed grade ii to iv acute gvhd (42 grade ii, 6 grade iii, 4 grade iv). the cumulative incidence of acute gvhd at 100 days was 55.9%. of the 87 patients who survived for at least 100 days with sustained engraftment after transplantation, 61 developed chronic gvhd (30 limited, 31 extensive), resulting in a 5-year cumulative incidence of 65. 6% . after a median follow-up of 60 months (range, 24-174 months), the cumulative incidence of relapse (cir) and nrm at 5 years were 28.1% and 22.9%, respectively, and the 5-year disease-free survival (dfs) and overall survival (os) rates were 54.4% and 60.4%, respectively. within the cohort of ph-negative all transplants (n ¼ 43), the 5-year cir, nrm, dfs, and os rates were 26.4%, 15.5%, 61.7%, and 67.4%, respectively. in a subgroup of ph-positive all transplants (n ¼ 50), the 5-year cir, nrm, dfs, and os rates were 29.7%, 28.9%, 48.6%, and 55.0% respectively, and for these patients, minimal residual disease kinetics (early-stable molecular response vs late molecular response vs poor molecular response) and the presence of chronic gvhd were closely related to cir and dfs. conclusion: our data suggest that ric can be considered as a reasonable choice for providing a long-term disease control for adult high-risk all patients in cr1. disclosure of interest: none declared. substitution of tbi by intravenous busulfan for elderly aml/mds patients within the flamsa-ric protocol is feasible and yields comparable results m. schleuning 1,* , d. judith 1 , i. burlakova 1 , h. baurmann 1 , r. schwerdtfeger 1 , g. stuhler 1 1 centre for hematopoietic cell transplantation, dkd helios klinik, wiesbaden, germany introduction: the flamsa-ric protocol is a highly effective conditioning protocol for high risk myeloid leukaemia patients (pts). however, many of these pts are beyond the age of 60. in this population the use of total body irradiation (tbi) might be toxic, even with the reduced dose of 400 cgy, as described in the original flamsa-ric protocol. in an attempt to further reduce toxicity for elderly (460y) or comorbid pts we substituted tbi by intravenous busulfan (ivbu; 8 x 0.8 mg/kg bw) within the flamsa-ric protocol. materials (or patients) and methods: retrospective study to analyze the results of ivbu in comparison to those achieved in pts receiving the classical flamsa-ric protocol with tbi during the same time period. results: from november 2006 to october 2012 173 pts with high-risk aml or mds received an allogeneic stem cell transplant after flamsa-ric conditioning. eighty-three pts (median age 47y) received tbi and ninety pts (median age s12 64y) received ivbu. in the tbi group 76 pts suffered from aml and 7 pts from mds and in the ivbu group diagnoses were aml in 74 and mds in 16 pts. unfavourable cytogenetics or molecular genetics were found in 64% of tbi and 50% of ivbu pts, respectively. in the tbi group 43% were transplanted with active disease as compared to 71% in the ivbu group. in both groups stem cell grafts from unrelated donors were used in approximately 75% of pts. all pts engrafted. after a median follow-up of 5.5 y for surviving pts the probability of leukaemia-free survival at 5 y after transplant is 40% in the tbi group and 35% in the ivbu group (p ¼ 0.133). twentyseven relapses occurred in the tbi and 21 in the ivbu group. non-relapse mortality (nrm) for the tbi cohort was 10% and for the ivbu group 24% at day þ 100 (p ¼ 0.052) and 33% and 41% during the entire observation period (p ¼ 0.004). the difference in nrm is probably owing to the older age of the pts in the ivbu group and was due to more infectious complications. no significant difference in survival was observed in mud or sibling transplants in either group. conclusion: in conclusion, substitution of tbi by ivbu is feasible with no enhanced relapse rates observed and should be further evaluated in prospective clinical trials also for younger pts. introduction: to ascertain the therapeutic potential of non-tbi-based conditioning for cd34 þ hpc-selected, t celldepleted allografts, we conducted a trial comparing our standard regimen, arm (a) 1375 cgy hftbi þ thiotepa,5 mg/ kg/day x 2 days þ cyclophosphamide 60 mg/kg/day x 2 days vs. arm (b) busulfex 0.8 mg/kg/6 h x12 (dose adjusted) þ melphalan 70 mg/kg/day x 2 þ fludarabine 25 mg/m 2 /day x5 and arm (c) clofarabine 20 mg/m 2 /day x 5 þ melphalan 70 mg/m 2 /day x 2 þ thiotepa 5 mg/kg/day x2, as preparation for t-cell depleted cd34 þ pbsc transplants from gcsfmobilized leukocytes fractionated with the clinimacs cd34 þ reagent system. materials (or patients) and methods: primary endpoints were engraftment, gvhd, transplant-related mortality (trm) and 2 yr os and dfs (confer table) . stratification of pts to arms a (standard), b or c was based on the patient's disease, disease stage and clinical factors such as age, prior therapy or comorbidities enhancing risks of tbi. arm b was the non-tbi arm predominantly used for myeloid and arm c for lymphoid malignancies. prior to transplant, recipients of hla-matched or non-identical transplants received rabbit thymoglobulin at 2.5 mg/kg/day x2 or 3 days respectively, to prevent graft failure. no gvhd drug prophylaxis was given post transplant. results: a total of 215 consecutive patients, accrued between 5/13/2010 and 11/20/2014, were analyzed (84 in arm a, 103 in arm b, 28 in arm c). these pts have been followed for a median of 19.7 months. donors were related or unrelated and hla-matched for 73% of the patients and 1-2 hla alleles disparate for 27%. median age for the entire group was 47.8 years, with older pts predominating in the non-tbi groups (medians arm a, 31.2 yrs; arm b, 58.8 yrs; arm c, 24 yrs). the cd34 þ pbsc transplant provided a mean dose of 9.7 x 10 6 cd34 þ progenitors/kg (range 1.4 -89.7 ) and 4.5x10 3 cd3 þ t-cells/kg (range 0.6-25.3 ). all pts engrafted; but 2 pts (1.9%) in arm b experienced late graft failure, one of whom was reconstituted after a secondary graft. overall the incidence of grade ii-iv acute gvhd was 6%, 6% and 7% for arm a, b and c respectively. trm at 1 year was 9% in arm a, and 15% in arm b and 16% in arm c. two year os and dfs for each arm are: arm a -62.2% and 56%; arm b -68% and 59% and 48% and 48% for arm c. for the 115 pts who received standard risk transplants (i.e., pts with high risk forms of aml, all or nhl in 1 o cr, aml in 2 o cr, mds ra/rcmd, cml in 1 o cp or mm in cr1), 2 year os and dfs are: arm a -60% and 59%; arm b -74% and 65%; arm c -50% and 50%, with relapse rates at 2 yrs of arm a -23%, arm b -10.4%, and arm c -11.5%. cumulative incidence of relapse (cir) stratified by risk group reveals that the probability for relapse is significantly higher for the pts with high risk disease (p o0.0005), whereas the cumulative incidence of non-relapse mortality (nrm) is comparable (p ¼ 0.36)( figure 1 ). the median time to relapse has not been reached in either group. the estimates of relapse at 1 year were 23.2% (95% ci, 16.5-30.0% ) for high risk pts and 11.6% (95% ci, 6.7 -16.6%) for the other group. the estimated 2-year cir was 31.4% (95% ci, 23.5-39.4%) for the group at high risk and 14.6% (95% ci, as measured from allosct prior to dli bm progression and focal progression patterns were highly similar with cumulative incidences of bm progression of 23%±13% and 23%±13%, and of focal progression of 19% ± 12% and 21% ± 12%, after 12 and 24 months post-transplant, respectively. in contrast, as measured from dli bm progression and focal progression patterns showed strong dissimilarity: at 12 and 24 months after dli cumulative incidences of bm progression were 14%±13% and 17%±14% respectively, whereas focal progression was 48% ± 19% and 62% ± 18%, respectively, illustrating a potent immunological response in bm with only limited effect of dli on focal lesions. conclusion: disease progression patterns of multiple myeloma after tcd-ric allosct diverged from initially similar bm and focal progression patterns in the absence of alloimmune responses towards disease control in bm with focal progression after dli. this finding illustrates failure of donor lymphocytes to target extra-medullary/focal disease in multiple myeloma. disclosure of interest: none declared. engineered t cells modified to express a cs-1-specific chimeric antigen receptor (car) confer anti-myeloma activity in vitro and in pre-clinical in vivo models introduction: adoptive immunotherapy with t cells that were modified by gene-transfer to express tumor-targeting chimeric antigen receptors (cars) has therapeutic potential in advanced b-cell malignancies. we are pursuing the glycoprotein cs1 (slamf7/cd319) as candidate target for car t cells in multiple myeloma (mm), due to its restricted high level expression on malignant plasma cells in a significant proportion of mm patients. here, we evaluated the anti-mm function of t cells that we modified with cs1-specific cars in vitro and pre-clinical in vivo models. materials (or patients) and methods: we constructed two cs1-specific cars with antigen-binding domains derived from the huluc63 and luc90 mabs that target distinct cs1 epitopes, each comprising a signaling module of cd3zeta and a cd28 co-stimulatory domain, and encoded both constructs in lentiviral vectors for gene-transfer. results: cd8 þ and cd4 þ t-cell lines expressing the huluc63 and luc90 cs1-cars could be readily generated from healthy donors and mm patients (n ¼ 5/3), and propagated and expanded in vitro with similar kinetics as t cells expressing a cd19-specific car that we included as a reference. in functional experiments, cd8 þ t cells expressing either of the cs1-cars conferred specific high level lysis of mm lines (mm1.s, nci-h929 and opm-2), primary mm, k562 that had been stably transfected with cs1, but not native cs1-negative k562. we also detected high level production of ifng and il-2 (cd44cd8), and productive proliferation after stimulation with cs1 þ target cells, with significantly superior anti-mm reactivity mediated by the huluc63 compared to the luc90 cs1-car construct. we confirmed the anti-mm efficacy of cs1-car modified t cells using a xenograft model in immunodeficient mice (nsg/mm1.s). mice were inoculated with fireflyluciferase labeled mm1.s myeloma by tail vein injection and 14 days later, when mice presented with disseminated disease, administered a single dose (i.v.) of a cell product consisting of equal proportions of cd8 þ and cd4 þ t cells modified with either the optimal cs1-car, the cd19-specific car or mock transduced. we observed rapid and durable complete rejection of established mm from bone marrow and resolution of extramedullar mm manifestations in all of the mice treated with cs1-car t cells (n ¼ 4), whereas mice treated with cd19-car t cells, or control t cells had to sacrificed due to progressive disease (n ¼ 4/4). of interest, in this in vivo model, we observed similarly effective anti-mm responses mediated by cs1-car t cells that had been derived from healthy donors and mm patients. conclusion: our data suggest the potential of t cells expressing cs1-specific cars to confer anti-mm activity in clinical settings. the experience with anti-cs1 mab huluc63, that as single agent has only minute anti-mm activity, indicates targeting this molecule will be safe and not be associated with toxicity to normal tissues. we observed stronger anti-mm reactivity with cs1-cars targeting a proximal (huluc63) rather than a distal (luc90) epitope on cs1 protein, in line with our previous observation that the targeted epitope on a given antigen affects tumor recognition of car t cells. experiments to analyze the function of our cs1-cars against panels of primary mm in our nsg model are ongoing to inform our efforts of clinically translating car t-cell therapy in this entity. 4;14) , t(14;16), del17p by fish and/or del13q by karyotyping]. all pts had to achieve at least a partial response from preceding salvage chemotherapy (n ¼ 32) or second salvage auto hsct (n ¼ 12). pts underwent allo tcd hsct with busulfan (0.8 mg/kg x 10 doses), melphalan (70 mg/m 2 x 2 days), fludarabine (25 mg/m 2 x 5 days) and rabbit atg (2.5 mg/ kg x 2 days). tcd was performed by positive cd34 selection (isolex) followed by rosetting with sheep erythrocytes for the initial 13pts (2008-09) and by cd34 þ enrichment by the miltenyi device in 31pts thereafter, achievingo10 4 cd3 þ /kg for all grafts. none of these pts received immuno suppressive therapy post tcd hsct. pts with 10/10 hla matched donors were also eligible to receive low doses of dli (5x10e 5 -1x10e 6 cd3 þ /kg) no earlier than 5mos post allo hsct. results: 44 pts with a median follow up of 24.8 mos (range: 11.1-81.2 mos) of survivors are reported, median age 56 years (range 32-69). all pts engrafted promptly (median d þ 10, range þ 9 -þ 12).trm (grade ii-iv) at 12mos is 18% (95% ci: 8% -31%). acute gvhd was 2% (95% ci: 0% -11%) and chronic gvhd was not observed in any pt. the overall survival (os) and progression-free survival (pfs) with their 95% confidence intervals (ci) are shown in table 1 . factors associated with worse outcome were disease status and number of previous treatments prior to tcd hsct. (1) . in this study, we have analyzed the molecular consequences of del(8)(p21), an abnormality we and others have previously shown to have an adverse impact on survival of mm patients (2) (3) (4) . materials (or patients) and methods: in a cohort of 140 patients that were diagnosed with mm between 2001 and 2012, we have investigated the clinical impact of del(8)(p21) on time to progression (ttp) and overall survival (os). moreover, response rate of 84 patients to 1 st line bortezomib treatment was investigated. we have also analyzed the expression profiles of genes located near the 8p21 region in patients with and without del(8)(p21). additionally, we have analyzed the in vitro response of primary mm cells with and without the deletion to bortezomib-mediated killing and sensitization to trail/apo2l-triggered apoptosis in an attempt to understand why mm patients carrying 8p21 deletion respond poorly to bortezomib treatment. results: we found that mm patients carrying del(8)(p21) deletion had significantly shorter ttp compared to patients without the deletion (p ¼ 0.011) and most importantly these patients had significantly shorter os compared to patients without the deletion (p ¼ 0.001). in a cohort of 84 patients, we observed that patients with del(8)(p21) (n ¼ 24) responded poorly to bortezomib, 50% showing no response while 90% of patients without the deletion (n ¼ 60) responded to bortezomib treatment. in vitro analysis revealed that mm cells from patients with del(8)(p21) show higher resistance to bortezomib treatment possibly due to upregulated expression of genes such as ptk2b, ccdc25, rhobtb2, nfkb, myc and bcl2 while showing downregulated levels of tp53 and scara3 when compared to mm cells without the deletion. furthermore, we have observed that mm cells with del(8)(p21) express higher levels of the decoy death receptor, trail-r4 and fail to upregulate the pro-apoptotic death receptors trail-r1 and trail-r2 that are located in the 8p21 region. as a result, mm cells with del(8)(p21) were largely resistant to bortezomib and trail/apo2l-mediated apoptosis. conclusion: substantiating the clinical outcome of the patients, our data provides a potential explanation regarding the poor response of mm patients with del(8)(p21) to bortezomib treatment. furthermore, our clinical evaluation suggests that including immunomodulatory agents such as lenalidomide in the treatment regimen may help to overcome this negative effect, providing an alternative thought in planning treatments of patients with del(8)(p21). introduction: autologous stem cell transplantation is the standard treatment in patients with multiple myeloma (mm). however, there is discrepancy over the optimal mobilization regimen. therefore a randomized study was conducted to compare cellular composition of the collected grafts as well as early hematopoietic and immune recovery in mm patients receiving g-csf with or without low-dose cyclophosphamide for mobilization of blood grafts after induction with lenalidomide, bortezomib and dexamethasone. materials (or patients) and methods: thirty patients with mm were included into this prospective multicenter study. there were 16 males and 14 females with a median age of 62 years (range 43-70). fourteen patients were mobilized with cyclophosphamide plus g-csf (arm a) whereas sixteen patients were mobilized with g-csf alone (arm b). melphalan 200 mg/m 2 was used as high-dose therapy and patients having graft cd34 þ cell countso3 x 10 6 /kg (measured before freezing) were scheduled to receive g-csf after the graft infusion. cryopreserved graft samples were analyzed with a flow cytometry for t and b cells (cd3/cd8/cd45/cd19) as well as for nk cells (cd3/cd16 þ cd56/cd45). also cd34 þ cell subclasses were analyzed (cd34/cd38/cd133/cd45). complete blood counts were evaluated on day þ 15 and one month post-transplant and a flow cytometry for blood lymphocyte subsets (t, b, nk) was performed one month after the graft infusion. results: the blood grafts in arm a contained significantly higher amounts of cd34 þ cells and the grafts of the arm b contained significantly higher proportion of primitive cd34 þ cd133 þ cd38cells and t, nk and b lymphocytes ( table 1) . the median amount of infused cd34 þ cells was comparable between the arms (3.9 â 10 6 /kg in group a vs. 3.1 â 10 6 /kg in group b, p ¼ 0.056). the number of platelets was slightly lower in the group b at d þ 15 (p ¼ 0.094) otherwise the course of early hematological and immune recovery was comparable between the groups. the use of g-csf alone instead of a combination with cyclophosphamide seems to enrich the blood grafts with significantly higher number of t and b lymphocytes and a higher proportion of more primitive stem cells. the hematological and immune recovery was comparable between the arms. the possible effects of graft composition in long-term patient outcomes will be further evaluated in the ongoing goa study (graft and outcome in autologous stem cell transplantation). disclosure of interest: none declared. introduction: pet is a useful tool that allows deeper assessment of response beyond that measured by m protein levels. it has been reported to predict outcome following both asct. to be able to integrate pet-ct negativity to internationally accepted response criteria the cut-off level needs to be validated by independent investigators. this prospective study was initiated to elucidate the prognostic role of pet-ct in the asct setting utilizing the cut-off found in our patients in ankara university (3.35 ) comparing with those initially reported by barlogie et al (3.6 ) and zamagni et al (4.2) materials (or patients) and methods: 85 consecutive patients diagnosed and transplanted in ankara university with pre-and post-asct fdg-pet-ct imaging were included. patients were: median age 56.6 þ /-8.8 (m/f: 45/40), iss i/ii/iii: 37/33/15, renal impairment (8,2%), bone involvement (94,1%), del13q (40.9%), t (4;14) and/or p53(26.5%), ldh high(10,5%), induction with bortezomib (72,9%). pre-asct clinical response 3 vgpr: 52.9%, post-asct clinical response 3 vgpr:77.5%. overall survival (os): median: 33 months (4.2-141 months). pasw statistics for windows program was used for statistical analysis. results: as reported previously roc analysis revealed 3.35 as a significant cut-off level (p ¼ 0.005; os). pet-cr was defined fdg uptake less than 4.2 or 3.35 depending on the analysis. post-asct pet (44.2) was predictive for pfs (p ¼ 0.05) but not os (p ¼ 0.096) . however pet (43.35 ) was predictive for os (p ¼ 0.037) but not pfs. depending on the cut-off more (suv 3 4.2: 43/64) or less (suv 3 3.35 : 29/64) patients met the criteria for pet-negativity (or remission) following asct. expert pet assessment resulted with pet-cr 39/85 similar to the suv 3.35 frequency. as shown in figure patients to converted to cr after asct (positive/negative group) displayed a better pfs than those who had reached cr prior to asct. this analysis was significant if cut-off was 3.35 but not 4.2. expert assessment was also able to differentiate patients with better prognostic features. 0.0-0.49 ). 23 leukaphereses were analized for mrd: the median plasma cells value was 0,03% (0,00-0,7%); in 6 pts pcs wereo0,01% (cut off for mrd negativity). conclusion: mobilization with cy-bor-dx þ g-csf and borbased asct is safe and effective in elderly mm patients. this schedule allows the collection of an adequate dose of cd34 þ cells, with a very low rate of mobilization failure (2%), also in elderly mm pts. a low rate of clonal pcs contamination in the harvest was also observed. this approach allows to perform asct in most elderly pts, achieving high response rate and promising outcome with a short term treatment:20 weeks compared to the non-asct programs (54 weeks in the vmp program). disclosure of interest: none declared. mica expression levels were investigated in n ¼ 180 gut biopsies with sybr green s qrt-pcr. histological grades of the gastrointestinal gvhd (gi gvhd) were determined by the pathology department at the university clinic, regensburg and severity was grouped by assigning an apoptotic score (0 ¼ absence of apoptosis, 3 ¼ maximum apoptosis). a protein biochip array (evidence investigator s , randox) was utilised for measuring mica serum levels and evaluated in n ¼ 129 samples from allo-hsct patients collected at pretransplantation, day-7, day þ 14, day þ 28 and 3 months post transplantation. results: our analysis showed that the methionine allele in rs1051792 was associated with an increased risk of relapse (p ¼ 0.029). the same allele was also found to be associated with a reduced overall survival (p ¼ 0.041) which was more severe for non-t cell-depleted allo-hsct (p ¼ 0.001). vice, versa, the presence of the valine allele was associated with the development of agvhd (p ¼ 0.044). in the gut, mica expression was investigated in patients treated with low doses of steroids (r 20 mg/kg), as high dose steroid treatment strongly suppressed mica expression. higher levels of mica were associated with an apoptotic score ¼ 0 (no apoptosis) (p ¼ 0.044) and the absence of active gi gvhd (p ¼ 0.046). increased soluble mica levels at 3 months post-transplantation were significantly associated with agvhd (p ¼ 0.0123). conclusion: mica molecules have been shown to play prominent roles in immune processes and therefore are also potential agvhd biomarkers. in this study, we showed that the methionine (mica-129met) allele was associated with the incidence of relapse while the valine (mica-129val) allele was associated with an increased risk agvhd. a low overall survival for patients who did not have had the t cell depletion treatment was also associated with the presence of the methionine (mica-129met) allele. in the gut of patients treated with low doses of steroid, mica gene expression levels were higher with the absence of gvhd. this may indicate that the isoforms are able to meditate nk-cell and t cell inactivation, and down-regulate nkg2d with high levels of soluble mica contributing to the development of agvhd. eleven patients needed gvhd treatment: 4 pts received ganciclovir iv (gcv 5 mg/kg/12 h/14 days), 7 pts valganciclovir per os (vgcv 900 mg/12 h/14 days). both gcv and vgcv were effective in control clinical manifestations of gvhd in a median of 13 days (range 7-52) and resulted in a significant reduction in numbers of circulating tk-cells, without reduction of cd3 þ tk-negative lymphocytes resulting in no effect on long-term immune reconstitution. in five patients additional concomitant treatment with low-dose steroid (prednisone o0.5 mg/kg per day for a median of 2 weeks) was given. a pt who presented severe gut and liver gvhd and one pt who received at transplantation an high dose of unmanipulated lymphocytes (5.4x10 5 /kg) -were succesfully treated with a combined therapy of prednisone and cyclosporine or rapamicine in association with gcv. one patient developed a severe classic de novo c-gvhd, with sclerodermatous lichenoid skin and mouth features plus moderate dry-eye symptoms that was successfully treated with vgcv and a transient course of mycophenolate mofetil (2 g per day) over a 2 months period. no cases of quiescent or progressive c-gvhd was observed after a median follow-up of 679 days (range 139/4035). conclusion: in our long-lasting clinical application of haploidentical tk-cells, an effective induction of immune reconstitution and a complete control of gvhd, provided a long-term immunosoppressive therapy free survival in absence of gvhd related deaths or longterm complications. introduction: despite major improvements in allogeneic hematopoietic cell transplantation (allo-hct) over the last decades, severe corticosteroid-refractory acute and chronic graft-versus-host-disease (gvhd) still remains a life-threatening complication characterized by high mortality rates (40-70%). since preclinical and early clinical evidence indicated anti-inflammatory effects of ruxolitinib, we collected the outcome data from multiple stem cell transplant centers using the jak1/2 inhibitor ruxolitinib as salvage treatment in patients suffering from corticosteroid-refractory gvhd. materials (or patients) and methods: a total of 13 stem cell transplant centers in germany, france, switzerland and united states reported outcome data from 52 patients who received ruxolitinib for corticosteroid-refractory gvhd (skin, mucosa, intestine, liver, lung, musculoskeletal) between 01/ 2012 and 12/2014. patients were classified as having acute (n ¼ 32) or chronic (n ¼ 20) gvhd. the median number of previous gvhd-therapies was 4 for acute gvhd (range: 1-7) and 3 for chronic gvhd (range: 2-10). results: the overall response rate was 84.3% (27/32) in acute gvhd comprising 10 crs (31.2%) and 17 prs (53.1%). in chronic gvhd the overall response rate was 80% (16/20). clinical improvement was rapid with a median time to response of 1.5 (1-4) weeks and 1 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) week after initiation of ruxolitinib treatment in acute and chronic gvhd, respectively. all responders were in persistent remission at last follow-up. the median follow-up was 18 (2-58) and 13 (2-70.5) weeks for acute and chronic gvhd patients, respectively. non-responders (acute gvhd: 5/32, chronic gvhd: 4/20) received other salvage therapies. cytopenias (anemia, leukopenia or thrombocytopenia) and cmv reactivation were observed during the time of ruxolitinib treatment in both acute (18/32, 56.2% and respectively 11/32, 34.3% ) and chronic (4/20, 20% and respectively 3/20, 15%) gvhd patients, sometimes however cytopenias already preceded ruxolitinib treatment. ruxolitinib treatment was stopped or reduced in 2 patients because of cytopenia. cmv was controlled by antiviral therapy in all patients. 3 ruxolitinib responders died, one because of leukemia relapse 4 weeks after ruxolitinib was stopped, and two from gvhd progression. in one of the patients who died, ruxolitinib was stopped due to impossibility of oral drug application. conclusion: overall, these data collected in multiple centers using different strategies for gvhd prophylaxis and treatment suggest that ruxolitinib is a very promising agent in the treatment of corticosteroid-refractory acute or chronic gvhd and may be successfully used to treat a major subset of patients beyond 2nd line of gvhd treatment. a prospective randomized multicentre clinical trial testing therapeutic jak1/ 2 inhibition as salvage treatment in gvhd is planned to verify the efficacy of ruxolitinib and to identify potential biomarkers that may be predictive for response. disclosure of interest: none declared. transplant-associated renal microangiopathy is associated with a high risk of refractory acute gvhd and characterized by a specific biomarker signature introduction: there is increasing evidence that endothelial damage is involved in the pathogenesis of steroid-refractory graft-versus-host disease (refgvhd). recently, serum soluble st2 (suppressor of tumorigenicity, il-33 receptor), an independent risk factor of cardiovascular death, has been identified as a risk factor of refractory gvhd. however, the pathomechanism of endothelial cell dysfunction which is associated with mortality from gvhd is yet poorly characterized. renal transplant-associated microangiopathy (tma) is another endothelial complication of allosct, and its association with severe gvhd and with biomarkers of endothelial damage (st2, scd141 (soluble thrombomodulin)), and endothelial function (vegf) is investigated in this study. materials (or patients) and methods: evidence for renal tma was studied in a cohort of 508 patients who underwent allosct between 2002 and 2013 at our institution and who have provided informed consent for this observational study. criteria to diagnose renal tma included an otherwise unexplained 50% rise in creatinine and lactate dehydrogenase (ldh) levels (or a pre-existing ldh above 400 u/l), a 50% drop in platelet counts (or a pre-existing platelet count below 50/nl) and at least 4% schistocytes. cytokines were measured in sera taken prior to allosct and on the indicated days thereafter and stored at minus 80c. statistical analyses were performed using spss19 and included a cumulative incidence analysis of causespecific hazards and the non-parametrical median test of independent probes. results: both renal tma and refgvhd were rare complications after allosct but were significantly associated with each other (tma only 18/508 (3.5%), refgvhd only 26/508 (5.1%), both 20/508 (3.9%), chi 2 0.000). median time intervals from allosct to renal tma and refgvhd were 1.93 (0.23-60.16 ) months and 1.23 (0.16-12.4 ) months respectively. in the overlap group, gvhd onset usually occurred before renal tma (0.69 months, -55.4 to 11.2) nrm rates were significantly increased in all three cohorts but approached 100% in patients with both complications. serum stm levels as well as soluble st2 levels increased between transplantation and day 100/day 200 after allosct in all cohorts, refgvhd, tma and both. in contrast, vegf levels (day 100) were significantly lower specifically in patients with tma with or without refgvhd, but not in patients with refgvhd without tma. conclusion: this study identifies renal tma as an endothelial cell dysfunction associated with extremely high mortality rates in the context of gvhd. the absence or presence of renal tma defines two separate subsets of refgvhd with a different prognosis. biomarkers of endothelial damage or vulnerability, such as stm, st2, and vegf can help to dissect tma and refgvhd, and might be useful to identify and guide management of patients with endothelial dysfunction who are at high risk of fatal complications after allosct. disclosure of interest: none declared. introduction: snps of the key cytokines and chemokines involved in the pathogenesis of agvhd have become an object of major interest recently. here we present snps rs3774937 cc/ct/tt and rs3774959 aa/ag/gg of the nf-kb1 gene in association with agvhd. materials (or patients) and methods: in our single-center study we analyzed 70 patients allografted for the following hematological malignancies: aml (37%), all (14%), cll (13%), mds (13%), cml (6%), nhl (6%), imf (3%), cmml (4%), and other hematological disorders (4%) between 2009-2014. the median age of the study group was 51 (21-62) years. patients were allografted after myeloablative (11%), non-myeloablative (14%) and reduced (intensity/toxicity) conditionings (74%). gvhd prophylaxis was done by solo cyclosporine-a (80%), cyclosporine-a with mycophenolate mofetil (19%) and cyclosporine-a with short-methotrexate (1%). ''in vivo'' t-depletion with thymoglobuline was used in 64% of recipients. patients were allografted from hla identical donors (related 46%) with median age 37 (18-63) years. the female donor/male recipient combination represented 14% of all pairs. the grafts contained median 4.6 (2.8-8.0 ) x106 cd34 þ cells/kg and median 6.8 (1.9-27.1) x108 mnc /kg. snps analysis was done from genomic dna isolated from edta-treated peripheral blood. genotyping was performed with sequenom massarray platform using allele-specific maldi-tof mass spectrometry assay (sequenom, san diego, ca, usa). primers were designed using the sequenom snp assay design software version 3.0 for iplex reactions. univariate analysis was performed to find significant difference in agvhd among the patient groups with different nf-kb1 profiles. the asymptotic pearson's chi-square test was used in cross tabulation with significance level set to 0.05. results : out of 70 patients 46 patients (66%) are still alive, 24 patients (34%) died (12 died of transplant-related complications). the median post-transplant follow-up was 1.6 (0.1-4.5) years. agvhd developed in 21 patients (30%), grade iii-iv in 7 of them (10%). both nfkb1 snps were completely correlated in the sense that knowing the genotype of one fully determined the genotype of the second one (5 patients did not follow this correlation and were excluded from the analysis). consequently, we defined a common predictor nf-kb1 which codes the information carried by both nf-kb1 snps in the following way: all patients carrying the rs3774937 cc genotype were also positive for rs3774959 aa allele and were marked as nf-kb1 ¼ i, all patients carrying the rs3774937 ct genotype were also positive for rs3774959 ag allele and were marked as nf-kb1 ¼ ii and finally all patients carrying the rs3774937 tt genotype were also positive for rs3774959 gg allele and were marked as nf-kb1 ¼ iii. the nf-kb1 profile was found to be significantly associated (p ¼ 0.002) with agvhd in the following way: patients in the nf-kb1 ¼ i group are more probable to suffer from agvhd than in the nf-kb1 ¼ iii group. conclusion: this is the first report showing the association of nf-kb1 gene snps with agvhd. the transcription factor nf-kb has been implicated in the regulation of cellular stress and inflammatory signals. according to our pilot data patients with inherited genetic abnormalities of the nf-kb1 gene may be prone to agvhd. patients.the two groups -control/treatment -were well balanced in terms of diagnosis (p ¼ 0.9), and disease phase (p ¼ 0.4) : the most frequent diagnosis was aml (n ¼ 75), followed by all (n ¼ 38) and mds (n ¼ 10). the median age for control/treatment was 46 years (1-69) vs 38 yers (0, , (p ¼ 0.06); the proportion of patients over 50 years was 51% in the control and 49% in treatment group (p ¼ 0.8). the donor type in the control/treatment arms was as follows: hla identical siblings n ¼ 36/n ¼ 34, unrelated cord blood (cb) n ¼ 6/n ¼ 2, unrelated donor (ud) n ¼ 36/n ¼ 42, and haploidentical family donors (haplo) 6/7 (p ¼ 0.4). skin biopsies.a skin biopsy before randomization, was not mandatory: it was performed in 38 patients. gvhd was diagnosed as proven, probable and possible respectively in s23 21%, 32% and 29%of the patients.these different reports were equally distributed in treatment and controls (p ¼ 0.7). results: the cumulative incidence of acute gvhd grade ii (primary end point), was 50% in controls and 35% in treatment patients (p ¼ 0.02). this difference was maintained in different subgroups. the ci of transplant related mortality (trm) was 18% (control) vs 27% (treatment) (p ¼ 0.1), despite a non significant younger median age in treatment patients. excess mortality in the treatment arm was due to an excess incidence of infections. actuarial 1 year survival was 80% (control) vs 78% (treatment) (p ¼ 0.1). cases of death in the control/treatment groups were as follows: gvhd 10% -13%; infection, 4%4 8%; interstitial pneumonia, 2% -0%; toxicity, 1% -2%; leukemia relapse 19% -18% (p ¼ 0.3). there was no significant difference in trm among different centers (p ¼ 0.5) progression of gvhd and skin biopsies.the proportion of patients progressing to gvhd grade ii þ was simlar in proven, probable, possible gvhd (62%, 50%, 45% ) (p ¼ 0.7). however the proportion of gvhd related deaths was 37% for proven gvhd, and 8% for probable/possible gvhd. introduction: graft versus host disease (gvhd) is a common and severe complication after allogeneic stem cell transplantation. during pregnancy, the placenta and the fetal membranes function as an immunological barrier, protecting the fetus from the mother's immune system. we have isolated stromal cells from the decidual layer of term placentas. decidual stromal cells (dscs) are of maternal origin and strongly inhibit the alloreactivity of t-cells in vitro. the effect is mainly contact dependent, and decreases the production of several key cytokines involved in the cytokine storm promoting the continuation of gvhd. materials (or patients) and methods: to investigate the effect of dscs on acute gvhd we enrolled 34 patients diagnosed with acute gvhd and clinically non-responsive to standard therapy. the protocol was modified after 17 patients, the dscs were then thawed and infused in infusion solution with albumin instead of ab-plasma and given repeatedly and earlier upon diagnosis. this led to the formation of two treatment groups (group 1 n ¼ 17 and group 2 n ¼ 17), which were compared to matched historical controls (n ¼ 54). we also performed a retrospectively corrected analysis of steroid refractivity. results: group 1 received a median of 1 infusion on day 11 after standard treatment compared to group 2, who received a median of 2 infusions (p ¼ o0,05) on day 7 (ns). no adverse events related to the treatment were observed. at 4 weeks after treatment, 60% of the patients in group 1 hade responded to the treatment. in contrast, all patients in group 2 responded (p ¼ o0,05). all patients in the treatment groups received fungal prophylaxis. the overall cumulative survival (os) at one year was 28% for the controls as compared to 47% for group 1 and 80% for group 2 (p ¼ o0.001). when the groups were corrected for steroid-refractivity, the os was 3% for the controls, 38% for group 1 and 70% for group 2(p ¼ o0.001). in the steroid-refractory control group (n ¼ 32), the risk of dying from gvhd at one year was 81%, whereas no patients in the steroid-refractory group 2 died from gvhd(n ¼ 13, p ¼ o0.001). in the last 19 months, no patients have died from acute gvhd at our center. in conclusion, dscs might be an effective treatment of acute gvhd. the infusion should be prepared in albumin, given as early as possible and in repeated doses. to confirm these striking findings, we will extend the followup time and enroll more patients in our study. disclosure of interest: none declared. oral session: stem cell source and donor type o041 unmanipulated haploidentical stem cell transplantation after reduced intensity or ablative conditioning regimen for the treatment of acute leukemia-a report from the acute leukemia working party of the ebmt m. introduction: haploidentical hematopoietic stem cell transplantation(haplo-hsct)is feasible option for patients with acute leukemia(al)at high risk of relapse who do not have hla-matched related or unrelated donors. haplo-hsct was associated with severe acute graft-versus-host disease (agvhd) in unmanipulated transplants and a high incidence of graft rejection in t-cell depleted transplants because of the high frequency of t cells that recognized major class i or ii hla disparities between donor and recipient. two approaches were developed to overcome these problems:megadose of t-cell depleted hematopoietic progenitor cells without any posttransplant immunosuppression and t-cell replete grafts with innovative pharmacological prophylaxis of agvhd.posttransplant cyclophosphamide(ptcy)is regarded as a gvhdspecific immunosuppressant in adults but its feasibility is unknown in children. purpose: to evaluate the feasibility and outcome of haplo-hsct in children and adolescents with acute leukemia in active disease depending on conditioning regimens and methods of harvesting haplo-graft. primary end points: overall survival (os), transplant-related mortality (trm). secondary end points: engraftment rate, agvhd, cgvhd, relapse rate. materials (or patients) and methods: 56 patients(range from 1-21y.o. median 9 y.o.)with al(all-32pts, aml-24pts)in progressive disease (pd)(cytoreduction chemotherapy (ctx) prior conditioning regimen-15pts, without-41pts) were analysed.mac þ atg regimen based on giac protocol received 20pts, mac þ ptcy 50 mg/kg on d þ 3, þ 4-5pts, ric þ atg regimen based on flu-18pts, ric þ ptcy 50 mg/kg on d þ 3, þ 4-13pts. all pts received prophylaxis of agvhd based on csa-30pts, tac-8pts, tac þ sir-18pts. g-csf-primed t-cell replete bm was used as a graft source in 33pts (median cd34 þ cells 4,7x10 6 /kg), g-csf mobilized peripheral blood (cd34 þ selected by clinimacs, miltenyi biotec) and g-csfprimed bm-23pts (median cd34 þ cells 11,3x10 6 /kg). [o042] results: 3-year os was 33,3%. ptso9y.o. had significantly higher os vs pts49y.o. 46,7% and 18,5% respectively (p ¼ 0,01). 3-year os in pts receiving ctx prior conditioning regimen was 50% vs 26,8% in pts with leukemic burden(p ¼ 0,02). significantly difference were observed in 3year os in pts transplanted g-csf-primed t-cell replete bm 45,5% vs 13% in pts after g-csf mobilized peripheral blood and g-csf-primed bm (p ¼ 0,03). pts receiving ric þ ptcy had 3-year os 61,5%, ric-11,1%, mac-35%, mac þ ptcy-20% (p ¼ 0,06). trm after haplo-hsct in pts with al in pd was 38%. engraftment was sustained in 82,1% pts. full donor chimerism was achieved in 73,2% pts on d þ 30. median anc engraftment (40,5x10 9 /l) d þ 19,plt recovery(420x10 9 /l) d þ 17. cumulative incidence of grade 2-4 agvhd was 27,3%,cgvhd-23,9%. cumulative incidence of relapse was 39,3%. conclusion: haplo-hsct g-csf-primed unmanipulated bm is an effective method of achieving remission with good sustained engraftment rate in children and adolescents with resistant disease. ric regimen followed by t-cell replete haplo-hsct with ptcy on d þ 3, þ 4 was associated with good os, low incidences of gvhd and trm. before any definitive conclusions can drawn, a randomized study is required. disclosure of interest: none declared. the detection of donor specific anti-hla antibodies in recipients of unmanipulated haploidentical blood and marrow transplantation is predictive of poor graft function introduction: our previous study suggest that choosing young, male, non-inherited maternal antigen-mismatched donors is reasonable following unmanipulated haploidentical blood and marrow transplantation (hbmt). recently, a correlation between the presence of donor-specific anti-hla antibodies (dsa) and graft failure has been demonstrated in haploidentical transplant settings. in our protocol, approximately 99% patients can achieve sustained, full donor chimerism. however, poor graft function (pgf) remains one of complications after unmanipulated hbmt. therefore, we determined the effect of dsa on primary pgf in order to provide further evidence for donor selection. materials (or patients) and methods: three hundreds and fourty-five patients with hematological diseases receiving hbmt were enrolled in this prospective study. the median age of the patients was 26 years (range, 2-58 years). these patients were randomly selected as training group (n ¼ 173) and validation group (n ¼ 172). peripheral blood serum were collected pre-conditioning regimens. dsa were determined using the luminex-based assay. results: in all 345 patients, the percentages of dsa positive cases were 11.3% (39/345). the incidence of dsa in female patients was higher than that of male cases (16% vs. 8%, p ¼ 0.020). in the training set, a cutoff value of dsa (mfi ¼ 2000) were developed. multivariate analysis showed that the presence of dsa (patients with mfiz2000 vs. cases with mfiz2000) was associated with primary pgf. in the validation set, the association of dsa with primary pgf following transplantation was also confirmed. the association of pgf with inferior overall survival (os) was demonstrated both in the training group and in the validation group. in all 345 patients, the median time to platelet recovery in dsa positive (mfiz2000) patients was slower than that of dsa negative ones (25 days vs. 18 days, p ¼ 0.004). the incidence of primary pgf and primary graft failure was 5.5% and 0.9%, respectively. dsa positive patients experenced higher incidence of primary pgf (31% vs. 3 introduction: transplacental trafficking of maternal and fetal cells during pregnancy establishes long-term, reciprocal microchimerism in both mother and child because of exposure of the two immune systems to the non-self alloantigens (maloney et al., j clin invest. 1999 ). studies show the immune system in the mother is capable of being sensitized by paternal histocompatibility antigens. for example, antibodies directed against paternal hla-antigens (van rood et al., nature. 1958 ) and t lymphocytes directed against paternal major (van kampen et al., hum immunol. 2001 ) and minor histocompatibility antigens (verdijk et al., blood. 2004) are frequently detected in multiparous women. we previously demonstrated mother/child immune interactions positively influenced the outcome of mother to child hla haploidentical t cell-depleted hematopoietic transplantation. in a series of adult and pediatric patients we demonstrated mother donors conferred protection against leukemia relapse and improved transplant related mortality (trm), which was largely due to infection, and improved survival (stern et al., blood 2008). materials (or patients) and methods: the kaplan-meier method evaluated leukemia-free survival. cumulative incidence estimates were used for relapse and trm, as they are competing risks. multivariate analysis assessed the impact of diverse variables on transplantation outcomes. results: we analyzed the outcomes of 238 adult acute leukemia patients after t cell-depleted haploidentical transplantation. when compared with transplantation from all other family members, transplantation from mother donors was associated with significantly lower trm (largely infectious) (27% vs 50% from all other donors, p ¼ 0.01). multivariate analyses demonstrated transplantation from mother donors was an independent factor predicting improved survival (hazard ratio 0.41, 95% confidence interval 0.12 to 0.95, p ¼ 0.03). in an attempt to elucidate the mechanism, we analyzed donor t cell repertoires that were specific for cmv antigens presented by recipient antigen-presenting cells (by elispot and by limiting dilution cloning). unlike all other donor/recipient pairs, mothers possessed cmv-specific cd8 cell clones that killed child's and father's cmv-pulsed dendritic cells (dcs). such clones were nonalloreactive as they did not kill the child's or father's non-cmv-s26 pulsed dcs. mothers also possessed cd4 t cell clones that produced ifn-gamma in response to child's and father's cmvloaded dcs. such clones were non-alloreactive as they did not respond to child's or father's non-cmv-pulsed apcs. thus, mothers possessed a t cell repertoire that recognized cmv antigens also when presented by the unshared, father's, hla haplotype. in fact, they showed twice as many t cells that recognized cmv antigens presented by the child's apcs than all other donor/recipient pairs (po0.05). conclusion: therefore, pregnancy resulted in the generation of an additional t cell repertoire that specifically recognized pathogen antigens presented by the unshared paternal hla haplotype antigens on the child's apcs. apparently, upon mother to child t cell-depleted hematopoietic transplantation, such repertoire expands over time and helps reduce infectious mortality. further studies are needed to elucidate the mechanisms underlying mother t cell selection/education by paternal hla haplotype antigens on the child's apcs. disclosure of interest: none declared. uni-directional and bi-directional non-permissive hla-dpb1 t cell epitope group mismatches have similar risk associations in 10/10 matched unrelated donor hct , was analyzed after separating uni-directional from bi-directional non-permissive mismatches. non-permissive mismatches were defined as unidirectional hvg when the donor but not the patient carried an hla-dpb1 allele from a tce group not present in the patient, and vice versa as uni-directional gvh. bi-directional nonpermissive mismatches were present when none of the hla-dpb1 alleles in patient and donor were from the same tce group. the associations with clinical endpoints of overall survival (os), transplant related mortality (trm), relapse, acute gvhd (agvhd) and chronic gvhd (cgvhd) were studied using multivariate proportional hazards methods. results: the number of transplants with permissive or nonpermissive uni-directional hvg, uni-directional gvh and bidirectional mismatches was 1537, 527, 529 and 143, respectively. in the trm analysis, non-permissive uni-directional hvg (hr 1.32, p ¼ 0.001) and gvh mismatches (hr 1.28, p ¼ 0.005) had significantly higher relative risks (rr) of trm compared to the permissive group. the bi-directional group had similar rr (hr 1.35 , p ¼ 0.05). in pairwise comparisons, there were no statistical differences between the uni-and the bi-directional non-permissive groups for any of the outcomes tested. introduction: there are several alternative sources of donor stem cells available for patients (pts) who need an allo-sct and especially for those who lack a hla-matched donor. outcomes of mismatched-unrelated-donor (mm-urd) transplant have recently improved, and a comparison between matched and mm-urd sources in a uniform cohort of pts has not been performed after ric regimens. materials (or patients) and methods: pts, aged z50 year, who underwent fully matched or mm-urd ric pbsct or bmt from 2000-2012 were included in the study. all donors were hla-matched (10/10) or mismatched at one or two-loci (9/10 or 8/10). the kaplan-meier-estimator, the cumulative incidence function and cox proportional hazards regression models were used where appropriate. results: in total 3197 pts receiving matched or mismatched ric-urd allo-sct were included in the study (aml 2947, all 250). 2370 10/10 hla-matched pts were compared with recipients receiving 9/10 (n ¼ 712) or 8/10 (n ¼ 115) after ric mm-urd allo-sct. median age of 10/10, 9/10 and 8/10 hlamatched recipients were 60 years. higher female donor to male recipients were in 8/10 cohorts (20%) compared to 10/10 (12%) and 9/10 (14%) group (p ¼ 0.02). more pts with cr2 and advanced-disease were among 9/10 and 8/10 cohort compared to 10/10 matched donor recipients (cr2 24, 24 and 20%; advanced disease 26, 25, 24% respectively; p ¼ 0.04). also higher percentage of pts with secondary leukemia were in 8/10 cohorts compared 10/10 and 9/10 matched donor (35, 27, 25%; p ¼ 0.07). percentages of engraftment (97%, 95%, 97%, p ¼ 0.16) were no different between the 3 groups. acute gvhd grade ii-iv was 27%, 33%, 33%, and grade iii-iv 10, 13, and 10%, respectively for 10/10, 9/10 and 8/10 matched donors, respectively (p ¼ 0.003 and 0.03). in univariate analysis, 2-year survival rate was significantly higher for pts receiving 10/10 donor ric-urd allo-sct in cr1 compared to 9/10 or 8/10 mm-urd (os: 55%, 46%, 46%, p ¼ 0.01; lfs: 51%, 43%, 45%, p ¼ 0.03, respectively). however, among the cr2 and advanced disease groups there were no differences in outcome between fully matched or mm-urd (9/ 10 or 8/10) donor (cr2 þ : os 49%, 43%, 48%, p ¼ 0.30; lfs 42%, 37%, 45%, p ¼ 0.36; advanced-disease: os 38%, 31%, 31%, p ¼ 0.39; lfs 32%, 26%, 28%, p ¼ 0.50, respectively). there was no difference in ri between the 3 groups and nrm was higher after fully matched donor compared to mm-urd (9/10 or 8/10) only in pts with cr1 diseases (p ¼ 0.02). multivariate analysis showed higher nrm after 9/10 (hr 1.33, p ¼ 0.001) compared to fully matched donor and no difference in nrm between 9/10 vs. 8/10 mm-urd. there was no difference in ri between 10/10 vs. 9/10 or 9/10 vs. 8/10 mm-urd donor. os and lfs were superior after fully matched donor vs. 9/10 mm-urd (os: hr 1.24, p ¼ 0.001; lfs: hr 1.19 , p ¼ 0.002). however, there was no difference in adjusted os and lfs between 9/10 vs. 8/10 mm-urd ric allo-sct. chronic gvhd rate was not different between matched or mm-urd allo-sct groups. conclusion: despite the limitations of a retrospective registrybased study, our analysis shows no significant outcome difference between 9/10 and 8/10 mm-urd allo-sct after ric regimen in patient aged z50 year. in the absence of prospective data, we conclude that mm-urd ric allo-sct is a therapeutic option for acute leukemia pts not having fully matched donor. disclosure of interest: none declared. introduction: the use of minors as hsc donors is medically and legally accepted and is increasing. however, there is a lack of understanding of the physical and psychosocial effects of pediatric hsc donation. the goal of this investigation was to longitudinally investigate hrqol in this group. materials (or patients) and methods: participants were related pediatric donors (n ¼ 105) who donated at domestic u.s. centers between 4/10 and 5/13. data were collected from donors and their parents via structured telephone interviews at pre-donation, and 4 weeks and 1 year post-donation. a healthy age/gender matched pediatric sample was generated from existing data for normative comparisons. interviews gathered socio-demographics, psychosocial characteristics, and multidimensional hrqol using the well-validated pediatric quality of life inventory (pedsql) which produces a total score and physical, emotional, social, school and psychosocial subscores. t-tests were used to compare hrqol from donor self-report, parental proxy-report, and the normative sample across the three assessment points. mixed logistic models were used to examine the effects of pre-donation variables on post-donation hrqol. results: donors were 5-17 yrs (median ¼ 11 yrs) and all but one were sibling donors. most parental respondents were mothers (74%; median age 40 yrs), 87% were married, and 36% had at least a bachelor's degree. donor vs proxy. across all hrqol domains except emotional functioning and at all three assessment time points, donor self-reported hrqol was significantly lower than that reported by parental proxies. donor vs norm. at pre-donation, as compared to the normative sample, donors reported significant hrqol deficits across multiple subdomains and in total hrqol (t ¼ -2.76,po.01). at 4 weeks post-donation, donors reported deficits in physical functioning (t ¼ 5.99, po.001 ) and total hrqol (t ¼ -2.82,po.01). at 1 year post-donation, donors reported deficits in physical (t ¼ -2.51,po.05) and school functioning (t ¼ -2.12,po.05). donors at hrqol risk. across the three assessment time points, 21%, 19%, and 17% of donors respectively had self-reported pedsql total scores below the standard cutoff indicating significant clinical risk of poor hrqol -scores below the cutoff are similar to those of chronically ill children. sixteen percent, 13%, and 5% were below the cutoff at only one, two, or all three assessments respectively. the youngest donors (5-7 yrs) were at significantly greater risk of being below the cutoff than were their older counterparts with 37%, 39% and 82% of this group below cutoff at each of the three assessments respectively. in multivariable analyses, the pre-donation factor most strongly and consistently associated with below cutoff pedsql scores at 4 weeks and 1 year post-donation was pre-donation donor self-reported pedsql score (likelihood ratio: 9.17,po.01; 15.54,po.001). conclusion: these findings suggest that there may be significant hrqol deficits among pediatric hsc donors and that in this particular context, parents are not able to accurately report those deficits. these findings also indicate that research to identify predictors of poor hrqol and the development of interventions to screen and address hrqol deficits are urgently needed. disclosure of interest: none declared. introduction: in allo hct patients (pts) with disseminated adv disease, mortality is reported to be up to 80%. antiviral treatment (tx) usually consists of iv cidofovir (cdv), which has a significant risk of nephrotoxicity. bcv is an orally-available, lipid-conjugate of cdv with no evidence of nephrotoxicity in clinical trials. the pilot portion of the phase 3 advise (cmx001-304) study was initiated in march 2014 to enroll b100 allo hct and other immunocompromised adv pts with, or at risk of progression to, disseminated adv disease, to guide the final study design. as of 10nov2014, 73 subjects have been enrolled and entered into the database, including 60 allo hct pts (48 with disseminated adv disease), 7 solid organ transplant pts and 6 ''other'' pts. preliminary safety and virologic results for the 48 allo hct pts with disseminated disease are described. materials (or patients) and methods: all subjects receive open-label bcv 100 mg (z50 kg) or 2 mg/kg (o50 kg) twiceweekly for 12 wks, extendable up to 24 wks for pts at high-risk of relapse, and are followed for 24 wks post-tx. adv dna viral load (vl) in plasma is measured using a quantitative pcr test (limit of detection [lod] 2 log 10 c/ml). results: baseline (bl) characteristics for the 48 subjects are: median (range) age 12 (0.7, 69) y, 65% o18 y; 69% male; median (range) plasma adv vl 4.6 (o lod to 7.6 ) log 10 c/ml (n ¼ 45); 42% adv positive by qualitative pcr in respiratory secretions, 60% in urine, 65% in stool; 29% with cmv in plasma, 6% ebv in plasma and 42% bkv in urine; 42% received prior iv cdv. as of 25nov2014, 5 subjects had completed tx and 21 had discontinued tx prematurely. the most common reasons for tx discontinuation were death (n ¼ 11) and adverse event ([ae] n ¼ 4). median (range) tx duration was 38 (1, 141) days (n ¼ 45). virologic response in cdv-naïve and exposed subjects with detectable plasma adv vl at bl are summarized in the table. in subjects with positive adv pcr at bl, 65% (13/ 20) cleared adv in respiratory secretions, 55% (16/29) in urine and 48% (15/31) in stool. through 08dec2014, 40% (19/48) of allo hct subjects with disseminated adv disease had died, with a median 71-day observation period for living subjects. no death was attributed to bcv. aes leading to permanent tx discontinuation attributed to bcv were vomiting and abdominal pain in 1 subject, and acute gvhd in 1 subject. median (range) change in adv vl from bl (log 10 c/ml) median (range) time to minimum on-tx (days) proportion ?3 log 10 reduction in adv vl or to undetectable at nadir minimum on-tx last on-tx cdv-naive (n=23) -2.0 (-5.1, 0.5) 1.8 (-5.1 , +2.1) 15 (3, 106) 57% (13/23) cdv-exposed (n=18) 1.5 (-5.4 , +0.6) 1.2 (-5.4 , +0.6) 15 (4, 77) 72% (13/18) s29 conclusion: the observed mortality rate was 40% for allo hct pts with disseminated adv disease in advise, which is lower than literature rates reported for this pt population (50-80%; ison 2006, sandkovsky 2014). bcv showed potent virologic activity in cdv-naïve and exposed pts with no new safety concerns. these preliminary data support expansion of the pilot portion to a definitive phase 3 study. introduction: the guidelines for immunization of hematopoietic stem cell transplant (hsct) recipients recommend 3 doses of anti-pneumococcal conjugate vaccine (pcv) from 3-6 months after transplant, followed by a dose of polysaccharide 23-valent (ppv23) vaccine at 12 months in case of no chronic graft-versus-host disease (gvhd), or an additional pcv dose in case of gvhd. however, due to lack of long-term data, there is no recommendation for boosts after 12 months. our goal was to assess the retainment of anti-pneumococcal antibodies in allogeneic hsct recipients vaccinated 10 years ago. materials (or patients) and methods: in 2009, the idwp published the results of the idwp01 trial that compared the immune response assessed one month after 3 doses of pcv7, started either at 3, or at 9 months after myeloablative hsct 1 . additionally, all patients received 1 dose of ppv23 at 12 or 18 months after transplant. all surviving patients had been assessed for anti-pneumococcal antibodies against the vaccine-serotypes 24 months after hsct. this study was the basis of the current guidelines for anti-pneumococcal immunization after allogeneic hsct. the present study included 30 surviving patients from the idpw01, who were assessed for antibody levels against the 7 pcv7-antigens and against 2 of the ppv23antigens (pn1 and pn5), between 8.3 and 11 years after transplant, i.e. 6 to 9 years after the last assessment in the initial study. the mean age was 39 y (18-55), and 18/30 had acute leukemia. only 7 had chronic gvhd (limited: 6, extensive: 1) and 2 had suffered a leukemia relapse. eleven (37%) had received an additional dose of ppv23 at a mean time of 6.5 years (2-11 years) after transplant, according to local procedure. the rates of persistent responses to all 7 antigens of pcv7 were 65.5% for an ab cut-off of 0.15 mg/ml, and 40% for a cut-off of 0.50 mg/ml. compared to the response rate at 24 months after transplant, these rates were not significantly decreased but showed important serotype-specific variability. similar findings were observed for pn1 and pn5 antibody levels. neither the recipient or donor age, donor type, source of stem cells, gvhd, nor the administration of an additional dose of ppv23 (given to 11/30 patients) influenced the maintenance of the response. the timing of the initial vaccination was the only parameter influencing the long-term response; patients who were vaccinated lately after hsct (from 9 months) had a significantly better maintenance of the response than patients vaccinated early (from 3 months) after transplant. the 3 patients who were not responders at 24 months and who received an additional dose of ppv23 at 39, 40 and 72 months after transplant, respectively, did not respond. conclusion: in long-term hsct survivors without severe chronic gvhd vaccinated against s pneumoniae according to the current guidelines, the specific immunity is not fully maintained a decade later. patients, who received an additional ppv23 dose after 24 months post-transplant, do not seem to benefit from this boost. boosts with pcv should be explored. so far, the optimal schedule of anti-pneumococcal vaccination in hsct recipients after 12 months remains to be established. references : early cytomegalovirus reactivation -a potential factor for early robust t cell reconstitution and possibly a prognostic factor for agvhd after hsct p. r many previous studies have shown that agvhd puts patients at risk of cmv reactivation, most likely due to more intensive immunosuppression. however, recent studies and case reports also show that that cmv-r could be a risk factor for agvhd. here, we studied the effect of cmv-r on t cell reconstitution in patients with and without agvhd. materials (or patients) and methods: 106 cmv r þ /d þ patients transplanted 2005-2013 in our institution were included in this study. all the patient samples were monitored for the cmv viral load (cmvpp65 expressing cells/400,000 leukocytes) and t cell reconstitution (cd3, cd4, cd8 and all available hla specific cmv tetramers for each patient) within the first 100 days after hsct. patients were subdivided into five groups: group 1: no agvhd but cmv-r (no-agvhd-cmv-r), group 2: agvhd after cmv-r (agvhd-after-cmv-r), group 3: agvhd before cmv-r (agvhd-before-cmv-r), group 4: agvhd but no cmv-r (agvhd-no-cmv-r) and group 5: no agvhd-and no cmv-r (no-agvhd-no-cmv-r). results: the characteristics for onset of cmv reactivation and agvhd in the different subgroups are provided in table 1 . cd3, cd4, cd8 and cmv specific t cells were analyzed on day 50±10days after hsct. in order to investigate the potential influence of cmv-r in the absence of agvhd on t cell reconstitution, we compared the t cell numbers in the groups cmv-r þ /-subsequent agvhd (i.e. group 1 þ 2) with group 5 (no-agvhd-no-cmv-r). we found significantly more cd3 (p ¼ 0.021) and cd8 t cells (p ¼ 0.0057) in groups 1 þ 2 compared to group 5. there were no differences in t cells between the groups with cmv-r þ /subsequent agvhd (i.e. groups 1 þ 2) compared to the groups with agvhd þ /-subsequent cmv-r (i.e. groups 3 þ 4). moreover, there were no differences in t cell numbers between the groups with agvhd þ /-subsequent cmv-r (i.e. groups 3 þ 4) compared to group 5. to study the impact of cmv-r on t cell reconstitution in patients with subsequent agvhd we compared the t cell numbers in group 2 (agvhd-after-cmv-r) with group 1 (no-agvhd-after-cmv-r). there were significantly more cd4 t cells (p ¼ 0.0041) and a trend for more cd3, cd8 and cmv-ctls in group 2 (agvhd-after-cmv-r) compared to group 1 (no-agvhd-after-cmv-r). subsequently, we compared the potential influences of cmv-r and of agvhd on t cell reconstitution. we found significantly more cd3 (p ¼ 0.0138) and cd8 t cells (p ¼ 0.0125) in group 2 (agvhd-after-cmv-r) compared to group 4 (agvhd-no-cmv-r). moreover, we studied the overall potential influence of cmv-r in the presence of agvhd on t cell reconstitution. we found a trend for more t cells (cd3, cd4, cd8 and cmv-ctls) in group 2 (agvhd-after-cmv-r) compared to group 3 (agvhdbefore-cmv-r). in conclusion, patients with agvhd after cmv-r had considerably more cd4 t-cells on day 50 compared to patients with cmv-r but no agvhd and significantly more cd3 and cd8 t cells compared to patients with agvhd but no-cmv-r. these results suggest that early cmv-r enhances overall t cell reconstitution which could be a potential risk factor for developing agvhd after hsct. single and double cbt were used in 289 and 159 cases, respectively. tbi was part of the conditioning regimen in 263 cases (59%). in vivo t-cell depletion by atg was used in 61% of patients. at least one pcr with a viral load 43 log/ml of blood was sufficient to define hhv-6 reactivation after the graft. the impact of hhv-6 reactivation on cbt outcomes has been studied as a time-dependent variable. in multivariate analysis, hhv-6 was independently associated with graft failure (hr: 1.44 conclusion: our study confirms that hhv-6 reactivation is a risk factor for graft failure in cbt recipients after myeloablative conditioning regimen. this result has to be confirmed prospectively and in the setting of reduced-intensity conditioning cbt. this paves the way also to test prospectively the indication of ganciclovir or foscarnet use as anti-hhv-6 prophylaxis in cbt recipients. disclosure of interest: none declared. introduction: candida is the second more frequent cause of invasive fungal infection in haematological immunocompromised hosts, especially in the patients who undergo an haematopoietic stem cell transplantation (hsct). the aim of this study was to analyse retrospectively the outcome of patients with candida infections acquired in the first 100 days after allogeneic hsct. materials (or patients) materials (or patients) and methods: in prospective study 173 allohsct recipients were included from dec 2012 to jul 2013. the median age was 34 y.o., males -54%. most of pts had high-risk acute leukemia (70%). allohsct from mud were performed in 57%, mrd -24%, haplo -11%, mmud -8%, predominantly with ric (80%). eortc/msg 2008 criteria for diagnosis and response to therapy were used. since 2011 active diagnostic strategy, including bronchoscopy with bal, in pts with ct-scan lung lesions before allohsct has been introduced to the routine practice. ''active ia'' is the ia diagnosed just before hsct. results: incidence of ia before allohsct was 22,5% (n ¼ 39/ 173). according to eortc/msg 2008 criteria 92% of pts had probable ia and 8% proven ia. the main sites of infection were lungs -95%, central nervous system -3%, and colon -3%, other localizations were observed mostly in a combination with lung involvement: sinuses -5%, spleen -3%, and liver -3%. antifungal therapy before allohsct was administrated in 69% pts (voriconazole -95%, other -5%) with the median duration of therapy -2 months. complete response to antifungal therapy was registered in 10 (26%) pts, partial response or stabilization in 17 (43%), and ''active ia'' in 12 (31%) pts. after allohsct all pts received antifungal therapy with voriconazole (first line -31%, continuation of treatment -43%, and secondary prophylaxis -26%). median length of treatment was 166 days (37-394). cumulative incidence of relapse or progression of ia after allohsct was 12,4% (n ¼ 6). relapse of underlying disease was the main risk factor for the relapse or progression of ia after allohsct (6% vs 33%, p ¼ 0,007). progression of ia after allohsct was treated with voriconazole 600 mg per day (n ¼ 1) and combination vori þ caspo (n ¼ 3). relapse of the ia after allohsct was treated with voriconazole 400 mg per day (n ¼ 2). no toxicity of the antifungal treatment was registered. complete response was achieved in 4 pts, and stabilization -2. 12-weeks overall survival (os) after the start of antifungal therapy was 67%. two pts died with the progression of the underlying disease. 100days os after allohsct was 70%, 1-year os after allohsct was 57%. there was no significant difference in os in pts with or without ia before allohsct. conclusion: incidence of the ia before allohsct was 22,5%. cumulative incidence of relapse or progression of ia after allohsct in pts with proven and probable ia before allohsct was 12,4%. relapse of the underlying disease was the main risk factor for relapse or progression of ia after allohsct. secondary prophylaxis with voriconazole should be used in pts with ia before allohsct. relapse or progression of ia after allohsct didn't impair os. ia is not a contraindication for allohsct. disclosure of interest: none declared. pt. 12 patients had acute kidney injury which was managed conservatively without the need for renal replacement therapy. 3/6 survivors have normal lft's, 2 patients have a residual mild increase in transaminases due to cgvhd, whereas 1 patient has a moderate increase in lft's due to cgvhd. conclusion: busulphan based conditioning were the most important risk factor for vod. myelofibrosis had a strong trend towards causing vod (p-0.06). early intervention with defibrotide along with supportive management was able to completely resolve vod in most of the cases and the 100 day mortality was only 3/13 (23%). only 1 death was directly attributed to vod and 1 death each due to sepsis and biopsy proven drug induced liver failure. disclosure of interest: none declared. validation of two new prognostic scores to predict nonrelapse mortality in patients undergoing reduced-intensity conditioning allogeneic hematopoietic cell transplantation p. barba introduction: in 2014, 2 new pretransplant predictive models of non-relapse mortality (nrm) for patients undergoing allogeneic hematopoietic cell transplantation (all-hct) have been created based on modifications of the hct comorbidity index (hct-ci) and the ebmt score. the first model (hct-ci/ age) consisted of the addition of an extrapoint for patients440 years to the hct-ci (sorror et al. jco.2014 ). the other model developed by the alwp of the ebmt combined 16 categories of the hct-ci and the ebmt score into an integrated score (versluis et al. leukemia. 2014 ). none of these models have been validated in independent cohorts. materials (or patients) and methods: we analyzed the predictive capacity of these new models and compared it with the hct-ci and the ebmt score in a population of reducedintensity conditioning allo-hct (allo-ric) consecutively transplanted patients in 2 spanish centers during an 11 year period (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) . the scores of all models were calculated by a single investigator as originally defined. risk-groups stratification was also performed as originally defined, except for the ebmt score in which patients were divided into low-(score 0-3), intermediate-(score 4-5) and high-risk (scores 6) according to percentiles 33, 66 and 100. for the hct-ci/age patients scoring 0 points (n ¼ 3) were grouped with those scoring 1-2 points. the predictive capacity of all models was calculated by means of the harrell's c-statistic and were compared by calculating a z-score and p values from the estimated standard error. results: a total of 232 patients were included. median age at hct was 55 years (range 18-71). most patients received allo-hct from hla identical sibling donors (n ¼ 170, 73%) mainly for acute myeloid leukemia and myelodisplastic syndromes (n ¼ 73, 32%). median follow-up for survivors was 5.5 years (range 0.3-11) . the median hct-ci/age and the alwp model scores were 4 (range 0-14) and 5 (range 1-15), respectively. the median hct-ci, ebmt scores were 3 (range 0-13) and 5 (range 1-7), respectively. risk group distribution of patients according to each model is summarized in introduction: systemic inflammatory response syndrome (sirs) is defined as an inflammatory state induced by infections or toxic damages. sirs is diagnosed when two or more of the following criteria are met: body temperatureo36 1 c or438 1c, heart rate490 beats/minute, tachypnea420 breaths/minute or paco2o32 mmhg, leukocyteso4000 cells/ mm 3 or412000 cells/mm 3 or presence of410% immature neutrophils. the goal of this study was to assess the incidence of sirs early after an allogeneic stem cell transplantation (allosct) (from day 0 to hematopoietic recovery) and evaluate whether sirs may influence the occurrence of acute s36 graft-versus-host disease (agvhd) and non-relapse mortality (nrm introduction: pure red cell aplasia (prca) after allogeneic hematopoietic stem cell transplantation (hsct) is a relatively rare complication after major abo incompatible allogeneic transplant. although reduced intensity transplant was a potential risk factor for prca, the impact of stem cell source has not been fully evaluated. we conducted a retrospective risk factor analysis for developing prca in 163 major abo incompatible transplant including 106 cord blood transplantation (cbt) and 74 reduced-intensity conditioning. materials (or patients) and methods: we reviewed the medical records of 668 adult patients who underwent allogeneic hsct for the first time at the toranomon hospital from 2006 to 2013. prca after hsct was defined as anemia with low reticulocyte counts (o1%) in peripheral blood for more than 60 days after transplantation in association with neutrophil engraftment and a lack of erythroid precursors in bone marrow. results: one-hundred and sixty-three patients with major or bi-directional abo incompatibility who achieved neutrophil engraftment and survived more than 60 days after hsct were included in this study. seventy four patients received reducedintensity conditioning, 106 patients underwent cbt, 39 did bone marrow transplantation (bmt) and 18 did peripheral blood stem cell transplantation (pbsct). reticulocyte engrafted (reticulocyte 4 ¼ 1%) in 160 patients with a median time of 29 days after hsct during this study, which was significantly longer after cbt compared to bmt/pbsct (31 days vs. 26 days, p ¼ 0.008). in 9 patients, reticulocyte count remained o1% beyond 60 days post-transplant, 5 of whom were diagnosed as prca with a cumulative incidence of 3.1%. prca was not observed in cbt patients, and the cumulative incidence of prca was significantly lower after cbt compared to bmt/pbsct (0% vs. 8 is the only curative treatment in fa. immune reconstitution after hsct is increasingly recognized as a critical determinant of morbidity and mortality in hsct. the aim of the study was to better understand the kinetics of immune reconstitution in children with fa who underwent allogeneic hsct after a fludarabine based reduced intensity conditioning regimen. materials (or patients) and methods: in this study, lymphocyte subgroups of children who underwent hsct were evaluated before hsct and 1, 3, 6, 12, and 24 months after hsct. children with fa (n:21) comprised the study group and children with non-malignant diseases (n:36) comprised the control group. in addition to classical lymphocyte subgroups; activated t lymphocyte subgroups including cd8/57( þ ), cd8/56( þ ), cd3/hla-dr( þ ), cd4/25( þ ), cd4/ 28( þ ) t lymphocytes were evaluated in study and control groups. results: when absolute levels of lymphocyte subgroups were evaluated in children with fa, cd3( þ ) lymphocyte count returned to pre-hsct levels at 12 months. cd4( þ ) t lymphocyte count reached to pre-hsct levels at 24 months. cd8( þ ) t lymphocyte count returned to pre-hsct levels at 3 months. cd19( þ ) b lymphocyte count turned to pre-hsct levels at 3 months. cd4/8 ratio returned to pre-hsct levels at 12 months. cd16/56( þ )cd3( þ ) nk-t and cd16/56( þ )cd3(-) nk lymphocytes returned to pre-hsct levels within 1 month after hsct. among hsct related complications; acute gvhd developed in 2/21 (9.5%) children in study group and in 10/36 (27.7%) children in control group. on the other hand, chronic gvhd developed in 1/21 (4.7%) children in study group and in 5/36 (13.8%) children in control group. when specific subgroups reflecting lymphocyte activation were evaluated in study and control groups; activated cd8/ 56( þ ) nk lymphocyte and cd8/57( þ ) t lymphocyte count returned to pre-hsct levels at 3 months in both groups. while activated cd3/hla-dr( þ ) t lymphocyte count returned to pre-hsct levels at 1 months in both groups; activated cd3/ hla-dr( þ ) t lymphocyte count was higher at 1, 6, and 12 months in control group. activated cd4/25( þ ) t lymphocytes returned to pre-hsct levels at 12 months in study group and those returned to pre-hsct levels after 24 months in control groups. cd4/25( þ ) activated t lymphocyte count was higher at 24 months in study group (pr0.05). cd4/28( þ ) activated t lymphocytes reached pre-hsct levels at 12 months in control group and at 24 months in study group. besides, cd4/28( þ ) activated t lymphocyte count was higher at 6 and 12 months in control group (pr0.05). in this study, we show that the kinetics of recovery of the lymphocytes subgroups in children with fa after hsct follows those patterns also described for children with other diseases: early recovery of nk cells (1 month), followed by effector cytotoxic t cells (3 months) and b cells (3 months) , and finally, cd4( þ ) t-helper cells (24 months). high levels of cd3/dr( þ ) activated t lymphocyte count at 1, 6, and 12 months and high levels of cd4/28( þ ) t lymphocyte count at 6 and 12 months in control group are attributable to the high frequencies of acute and chronic gvhd in control group than those of study group. disclosure of interest: none declared. introduction: although hla haploidentical hsct has been largely employed in children with life-threatening nonmalignant disorders, the survival of patients given this type of allograft has been reported to be inferior to that of patients transplanted from a compatible unrelated volunteer (uv). we implemented a novel method of ex vivo t-and b-cell depletion based on the selective elimination of ab þ t cells and b cells. we herein report an update of 31 children with non-malignant disorders who were given this type of allograft. materials (or patients) and methods: twenty-two patients were males and 9 females, median age at hsct being 3.5 years (range 0.3-13.2) . nine patients had severe combined immunedeficiency (scid), 8 fanconi anemia (fa), 4 severe aplastic anemia (saa), 2 thalassemia major, 2 hemophagocytic lymphohistiocytosis (hlh) and 1 each immunedeficiency with polyendocrinopathy enteropaty x-linked (ipex), kostmann syndrome, hyper ige syndrome, osteopetrosis, swachmann-diamond syndrome and congenital amegakaryocytic thrombocytopenia (camt). all patients were transplanted from 1 of the 2 parents (21 from the mother and 10 from the father), the median number of cd34 þ and ab þ t cells infused being 22.57 x 10 6 /kg and 4 x 10 4 /kg. the original conditioning regimen consisted of treosulphan and fludarabine (flu) þ thiotepa in 13 (9 scid, 1 ipex, 1 camt, 1 kostmann syndrome and 1 swachmann-diamond syndrome), flu and cyclophosphamide þ single dose tbi in 12 (8 fa and 4 saa) and busulphan, flu and thiotepa in 6 (2 thalassemia, 2 hlh, 1 osteopetrosis and 1 hyper ige syndrome). no patient received immunosuppression after hsct. all patients received fresenius rabbit atg (4 mg/kg/day) on days -5 through -3 before allografting and rituximab (200 mg/m 2 ) to prevent ebv-related ptld on day -1. results: all patients but 6 engrafted, the median time to reach neutrophil and platelet recovery being 13 days (range 9-23) and 9 days (range 7-40), respectively. the 6 patients (2 with saa and 1 each with thalassemia, fa, hlh and osteopetrosis) who had primary graft failure were successfully re-transplanted (2 from the same parent, 3 from the other relative and 1 from an 1-hla locus disparate uv figure 1a ). in the latter group, agvhd started median 21 days after hsct and was zgrade iii and steroid refractory in 3/6 cases. a higher agvhd rate was also observed in male patients who received a graft from a female donor (f-4m mismatch) compared to all other sex-matches (5/9 vs. 2/16, p ¼ 0.02). in multivariate cox regression analysis, both omission of mtx and f-4m mismatch were associated with agvhd occurrence. in line with this, the protective effect of mtx was most pronounced in the subcohort of patients with a f-4m mismatch: grade ii-iv agvhd occurred in 5/5 f-4m mismatched non-mtx recipients whereas 0/4 f-4m mismatched mtx recipients developed agvhd (p ¼ 0.005). all relapse occurred in 9/25 patients (36%). we did not observe differences in relapse rate between non-mtx recipients and mtx recipients (4/11 vs. 5/14, p ¼ 0.97, figure 1b) . although non-mtx recipients were more often transplanted for all in 2 nd remission, pre-transplantation minimal residual disease (mrd) status did not differ between the groups. in line with earlier reports, mrd positivity was the most important risk factor for all recurrence. in mrd positive patients, the omission of mtx and agvhd occurrence did not prevent relapse after hsct. conclusion: mtx prophylaxis reduced the occurrence of severe agvhd, without compromising relapse free survival in pediatric all patients after t cell replete hla-identical bone marrow transplantation. prevention of agvhd reduces morbidity and the need for high-dose immunosuppressive agents, allowing for alternative immunotherapy-based therapeutic interventions in individuals at high risk for disease recurrence. introduction: hematopoietic stem cell transplantation (hsct) has contributed to improved outcome in childhood acute leukemia (al). however, post-hsct relapse is associated with a dismal prognosis and its optimal treatment remains unclear. we aimed to compare patients' related factors and treatment strategy, in case of relapse or progression post-allogeneic hsct in children with al in a recent ten-year period. materials (or patients) and methods: a total of 334 children who received a first allogeneic hsct for all or aml from january 2000 to december 2009 experienced a relapse or progression thereafter. they were treated in the 33 centers of the sfgm-tc, among them 279 cases were analysable. primary endpoint was overall survival (os) after diagnosis of relapse or progression post first hsct whatever the treatment post relapse was. . lymphocyte (sub)populations were analysed frequently post transplantation by flow cytometry. cd3 þ t-cell recovery was defined as appearance ofz100 cells/ml, b-cell and nk cells recovery asz50 cells/ml. the median active atg serum concentration at time of t-cell or b-cell reappearance was 0.03 au/ml, the maximum level was 1 au/ml. in 25% of the patients, nk cells re-appeared when the median active atg level was higher (0.16 au/ml) up to a maximum level of 7 au/ml (see figure) . for alemtuzumab the median concentration at t-cell recovery was 0.008 mg/ml, max. 0.2 mg/ml, but in 80% of the patients nk cells reappeared at a higher concentration up to 2.2 mg/ml. for both drugs, t-cell recovery was significantly correlated with the serum level of the drug (po0.001 for atg, and po0.01 for c1h), whereas a significant correlation was absent for nk cells. conclusion: atg and alemtuzumab are both able to deplete t-, b-, and nk cells. for both drugs, the exposure is highly variable even in patients with an equal weight receiving the same dose. here, we report that t-cell recovery is closely related with serotherapy exposure. nk-cells are the first cells that re-appear post hsct . all of the patients received the same ric regimen based on the use of fludarabine in combination with melphalan and antithymocyte globulin (atg). prophylaxis against gvhd was achieved via cyclosporine and methylprednisolone. results: all patients were engrafted. the median times to neutrophil and platelet engraftments were 11 days (range: 8-33), and 22 days (range: 10-67), respectively. patients underwent hsct from hla matched sibling donors (n ¼ 9), full matched other related donors (n ¼ 6), unrelated matched donor (n ¼ 1) and unrelated mismatched donor (n ¼ 3). the source of graft were peripheral blood (n ¼ 11), bone marrow (n ¼ 6) and cord blood (n ¼ 2 materials (or patients) and methods: twenty-nine patients with miop were treated by hsct at the university childreń s hospitals in paris (n ¼ 9, since 2008) and ulm (n ¼ 20, since 1998). miop was caused by mutations in tcirg1 (n ¼ 20), clcn7 (n ¼ 5), snx10 (n ¼ 1), rank (n ¼ 2), and fermt3 (n ¼ 1); age at transplant was between 2 and 72 months (median 6 months); donors were haploidentical family donors (n ¼ 16), mud (n ¼ 7), phenoidentical relatives (n ¼ 4), and msd (n ¼ 2). thiotepa and serotherapy was added to the busulfan and fludarabine based regimen in all patients with donors other than msd. results: all but 6 patients showed a primary and sustained engraftment; 4 of 6 patient, who rejected their haploidentical graft, could be rescued by a second graft from the second parent. severe vod, which had to be treated by ascites puncture, was seen in 2 patients only. only one case of gvhd4 12 and no case of chronic gvhd was observed. cause of death in 4 patients were liver toxicity in conjunction with cmv and fungal infection after prolonged aplasia (mud transplant at age of 1 month) and complications in conjunction with engraftment failure ( introduction: bmt is the only proven curative treatment available for haemoglobinopathies. however, the number of patients who can benefit is seriously restricted by the lack of hla-matched related donors not suffering from the condition and the limited number of unrelated donors available for the ethnic groups in which these conditions are prevalent. in order to expand the donor pool, haploidentical transplantation with a post-infusion of stem cells cyclophosphamide approach has been developed for young adults, but whilst well tolerated it has resulted in relatively high rates of rejection and the need for a prolonged period of immunosuppression 1 . materials (or patients) and methods: 12 consecutive parental haploidentical transplants (11 for sickle cell disease and one for b halassaemia major) were performed at st. mary's hospital, london, from june 2013 to november 2014. the median age was 9.5 years of age (range 3 to 14). all patients lacked a suitable hla-matched related donor and an unrelated search had not identified a 10/10 or 9/10 donor. endogenous haemopoieis was suppressed with hypertransfusions, hydroxycarbamide 30 mg/kg and azathioprine 3 mg/kg for at least two months pre-transplantation. the conditioning included fludarabine 150 mg/m 2 , thiotepa 10 mg/kg was added, cyclophosphamide 29 mg/kg, tbi 2 gy and atg (thymoglobulin) 4.5 mg/kg. gvhd prophylaxis was provided with cyclophosphamide 50 mg/kg on days þ 3 and þ 4, mmf and sirolimus. the minimum follow-up was 40 days post-transplantation and half of the patients are 4150 days post-transplantation and have completed all treatment. the source of stem cells was g-csf primed bone marrow in all cases, aiming 48 x 10 8 tnc/kg. results: all patients engrafted, though one patient subsequently suffered secondary graft failure following macrophage activation syndrome and died. the median neutrophil engraftment was 17 days (range 16 to 19). all 11 surviving patients are cured from the manifestations of the original disease. none of the patients suffered vod, though infectious complications occurred at a higher rate than seen for related transplants for the same conditions at our institution. five patients had no evidence of acute or chronic gvhd. five patients developed stage 1 acute gvhd (median presentation day þ 36, range 21 to 66) responding to topical steroids; one patient suffered skin gvhd stage 3 on day þ 35 and one patient gut gvhd stage 4 on day þ 24, both treated with msc. all patients responded to first line treatment with no recurrence of disease. all patients but one achieved 490% donor fraction both in whole blood and t cells. one patient requires immunosuppression beyond day þ 180 with stable 59% donor fraction in whole blood and 10% in t cells. introduction: bone marrow transplantation (bmt) offers a definitive cure for thalassemia in over 90% of low-risk children with a matched related donor. many centers currently incorporate thiotepa in busulfan-or treosulfan-based bmt regimens for thalassemia. this combination, however, may permanently impair fertilty in most patients. in the era of increasingly effective supportive care in which many thalassemia patients may have children, this is concerning. very longterm follow up studies have shown how the standard bu-cy regimen may be associated with birth rates comparable to the control population (la nasa et al. blood 2013). this study retrospectively compares bmt outcomes in two groups of low risk patients (defined as livero2cm and ageo12y) with severe thalassemia (st) (defined as a thalassemia syndrome with spontaneous hemoglobino7 g/dl), receiving oral busulfan (14 mg/kg), cyclophosphamide (200 mg/kg) and either thiotepa (10 mg/kg) (tt-bu-cy) or rabbit atg (fresenius 16 mg/kg or thymoglobulin 4 mg/kg total doses from day -12 to -10) (atg-bu-cy) as preparative regimen. standard cyclosporine and short-term methotrexate plus low dose methylprednisolone were used for gvhd prophylaxis. materials (or patients) and methods: this is a retrospective multicentre comparative study of the safety and efficacy of substituting tt with atg in low-risk st patients undergoing matched-related bmt. between january 2009 and july 2013, a group of 35 patients were transplanted after conditioning with tt-bu-cy, while between august 2013 and july 2014, 30 patients were conditioned with atg-bu-cy. results: the actuarial overall survival in the tt-bu-cy and atg-bu-cy groups is 91.3% and 93.4%; thalassemia-free survival is 88.4% and 93.4% at a median follow up of 23.5 and 3.1 months respectively, with no statistically significant difference by logrank test. conclusion: substituting thiotepa with atg in the standard bu-cy context seems safe and effective. higher fertility rates are expected for patients on the atg-bu-cy regime. disclosure of interest: none declared. materials (or patients) and methods: data has been collected retrospectively from 234 infants with scid who received transplants at 8 different centers over a 20-year period . the differences between groups were compared by using chi-square or fisher's exact test, where appropriate. a p value of less than 0.05 was considered statistically significant. results: 145 boys (62%) and 89 (%38) girls with scid whose ages ranged between 0.25-176 months (median 5 months) at the time of diagnosis were transplanted. parental consanguinity was identified in 171 (76%) of 224 infants. 72% of the patients had received bcg vaccination before diagnosis. b þ and b-phenotypes were detected in 41.8% (n ¼ 97) and 51.3% (n ¼ 119) respectively, while ada deficiency was recognized in 4.7%, rd (reticular dysgenesis) in 2.2% of the cases. rag1, jak3, rag2 and artemis defects were the leading genotypes among the patients with molecular diagnosis (42.3%; n ¼ 101). out of 234, 114 patients (48.7%) had either a matched sibling or a family donor, while 81 (34.6%) and 39 (16.7%) children received haploidentical (mmfd) and mud transplants respectively. the hsct source was bone marrow in 113 (48.3%), peripheral blood in 89 (38%) and cord blood in 32 (13.7%) of the patients. among a total of 24 (10.25%) retransplants, 18 received a second transplant while 6 children received a boost only. 153 children survived, 80 died and 1 was lost to followup. the overall survival rate was 65.7% over a 20 years period. it increased from 54% (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) to 69% (p ¼ 0.052) during the latter 10 years (2005-2014) and even to 72,9% during the last 4 years (2010-2014). the survival rates with relation to donor types were as follows: msd ¼ 85.7% (n ¼ 77), mrd ¼ 70.3% (n ¼ 37), haploidentical 47.5% (n ¼ 80) and mud 59% (n ¼ 39). age at diagnosis significantly (r5 months or 45months) influenced the survival rate of the patients (p ¼ 0.002). immunophenotype did not seem to have an effect on survival rate and immunoglobulin (ig) requirement following hsct did not differ between b þ vs b-phenotypes (p4 0.05). conclusion: this is the first multicenter study with the largest data obtained from scid patients transplanted in turkey. the median age at diagnosis was 5 months, b-phenotype and rag were the most common among other defects. age at diagnosis (45 months), and donor type (haploidentical) (po0.01) were two major factors significantly related to poor outcome. expanded donor availability, advances in intensive care facilities, diversity of transplantation centers and specialized teams are among major factors contributing to the longterm outcomes of hsct. however, newborn screening is of paramount importance in ensuring early diagnosis and timely transplantation thus improving the survival of scid patients in turkey. disclosure of interest: none declared. oral session: stem cell mobilization & regenerative medicine haematology, bmt unit, hospital clínico universitario virgen de la arrixaca, imib, university of murcia, 2 surgery, hospital clínico universitario virgen de la arrixaca, 3 haematology, bmt unit, hospital clínico universitario virgen de la arrixaca, imib, university of murcia, murcia, spain introduction: amniotic membrane (am) is a non-tumorigenic tissue attributed with various biological properties (low immunogenicity, anti-inflammatory, anti-fibrotic and antimicrobial effects) related to its ability to synthesize and release cytokines and growth factors. am, that is usually discarded after birth, is in our experience an easily obtained tissue which processing, storage and management can be included in the daily routine of the cryobiology laboratory. ma can be used as a ''biologic bandage'' for healing management of chronic wounds in diabetic and non-diabetic patients. in our hospital there is an ongoing clinical trial to study the use of am to improve epithelialization (nct01824381). here, we describe the results obtained prospectively after the compassionate use of cryopreserved human amniotic membrane allografts in 6 patients with chronic diabetic foot ulcers. materials (or patients) and methods: am was obtained from healthy mothers who had programmed an elective caesarean operation for obstetric reasons after they signed the informed consent. donors were screened by reviewing their medical records and by performing laboratory test to discard transmissible disease agents. am processing was done under sterile conditions in the gmp facility; the process involves: washing the am to eliminate blood traces, cut am into several fragments, sew each fragment on an impregnated dressing sheet and introduce them on cryopreservation bags adding the cryoprotectant solution based on human albumin, tc-199 medium and 9% dmso. the am fragments were storaged at -1961c and delivered once we the negative viral serology of the donor was confirmed 3 months later. cryopreserved am was applied to six consecutive patients with diabetic foot ulcers under a compassionate use program of the diabetic foot unit from may to november 2014. wound size reduction and rates of complete healing were evaluated. results: patients were aged between 45 and 65 years. they were affected by grade ii diabetic foot ulcers in the wagner ulcer classification scale that had failed previous treatments for periods between 2 months and 4 years. am was applied weekly or every ten days until complete healing or partial reepithelialization of the ulcers. a median of 5 (3-8) cryopreserved am fragments were applied for an average treatment period of 45 days. the mean size of the ulcer was reduced by 76%.l wreduced in size by 76%. at last follow-up, 4 out 6 patients have total epithelialization of the ulcer. no adverse events related to its application were observed. conclusion: our results show that the application of cryopreserved amniotic membrane is a feasible and safe treatment in complex diabetic foot ulcers. more rapid healing may decrease clinical operational costs and prevent long-term medical complications. furthermore, the treatment achieves re-epithelialization of long evolution wounds that were not reached with conventional therapies. disclosure of interest: none declared. bone as a regulator of human hematopoietic stem cell (hsc) trafficking: study of biochemical markers of bone remodeling and angiogenic cytokines during hsc mobilization, in patients with lymphoma and myeloma p. tsirkinidis 1,* , e. terpos 2 , g. boutsikas 1 , a. papatheo-dorou 3 introduction: bone is not considered just a structural, supportive tissue of bone marrow, but an hsc-niche regulator. data regarding the role of bone turnover in hsc mobilization in humans are scarce. the aim of the present study was to study bone remodeling and vessel equilibrium during hsc mobilization in lymphoma and multiple myeloma patients. materials (or patients) and methods: forty-six patients (32 lymphoma and 14 multiple myeloma) were studied. serum samples were collected at two time points: before mobilization (pre-mobilization sample) and on the day of hsc collection, which coincided with the peak circulating cd34 counts (collection sample). in 19/46 patients, 3 additional serum samples were collected, between mobilization and collection. the following molecules were measured by elisa in patients' sera: 1) bone resorption markers: carboxyterminal telopeptide of collagen type 1 (ctx), aminoterminal telopeptide of collagen type 1 (ntx), tartrate resistant acid phosphatase isoform 5b (tracp-5b), 2) bone formation markers: bone alkaline phosphatase (balp), osteocalcin (osc), osteopontin (opn), 3) the osteoblastogenesis inhibitor dickkopf-1 (dkk-1) 4) the osteoclastic regulators: receptor activator nf-kb ligand (rankl), osteoprotegerin (opg), 5) angiogenic cytokines: angiopoietin-1 (angp1), angiopoietin-2 (angp2), angiogenin (ang). values were compared with non-parametric methods. patients who had either a collection of cd34 þ cells o2.0x10 6 /kg, or a circulating cd34 count peak o20/ml were considered poor mobilizers. results: the comparison of the aforementioned molecules between the pre-mobilization and collection samples revealed: balp (p ¼ 0.000) and opn (p ¼ 0.049) increased significantly, while osc, a marker of bone turnover, and dkk-1 decreased significantly (p ¼ 0.000 and p ¼ 0.041, respectively). these findings reveal a significant increase of bone formation during mobilization. rankl (p ¼ 0.000) and opg (p ¼ 0.000) increased significantly, leading to an increase of rankl/opg ratio (p ¼ 0.000), consistent with osteoclastic activation. however, there was no evidence of increased osteoclastic activity, as ctx decreased significantly (p ¼ 0.026), while both ntx and tracp-5b did not change. angp-1 showed a dramatic reduction (p ¼ 0.000), while angp-2 increased (p ¼ 0.000), resulting in a significant decrease of the angp-1/angp-2 ratio, a finding indicating vessel destabilization during mobilization. these results were further supported by the intermediate measurements, which showed a straightforward alteration of bone metabolism early in hsc mobilization. poor mobilizers had significantly higher ctx levels both at premobilization (p ¼ 0.004) and collection samples (p ¼ 0.001), higher ntx levels at collection (p ¼ 0.02), lower angp-1 premobilization (p ¼ 0.004) and higher osc at collection (p ¼ 0.000) compared to good mobilizers. thus, ctx, ntx s48 and angp-1 pre-mobilization levels may serve as reliable predictors of poor mobilization. conclusion: our study showed for the first time in humans, that bone plays a dynamic role during hsc mobilization: bone formation and vessel destabilization are the two major events and osteoblasts seem to be the orchestrating cells during this process. osteoclasts are stimulated, but not fully active. moreover, some of these markers may identify poor mobilizers. disclosure of interest: none declared. introduction: allogeneic bone marrow transplantation is a curative treatment for leukemia and genetic disorders. although some patients are cured of their underlying illness, they are at risk of developing potentially fatal graft-versus-host disease (gvhd). a recent phase 3 randomized trial conducted by the canadian bmt group (n ¼ 230) comparing the impact of g-csf mobilized peripheral blood (pb) to bone marrow (bm). a representative group of 85 donor samples were evaluated and identified that high concentrations of cd56 bright nk cells in the donor product is strongly associated with a lack of development of both acute and chronic gvhd (odds ratio 0.11 ; p ¼ 0.0002) in g-csf stimulated bm. we found that the cd56 bright nk population was cd335 (nkp46) positive with comparable expression of cd337 in both sub-populations consistent with regulatory nk cells (nk reg ). we hypothesized that alternate strategies utilizing the mobilizing agent, plerixafor, may enrich for cd56 bright nkp46 cells in pb thus reducing the risk of gvhd and circumventing the need of harvesting donor cells from the bm. materials (or patients) and methods: we performed a pilot study to examine the impact of plerixafor, that blocks the sdf-1 cxcr4 interaction, in mobilizing cd56 bright nk reg cells in the pb and bm, to determine the whether we can optimize a donor source with the highest potential for post transplant tolerance, resulting in a lack of both agvhd and cgvhd. we recruited 9 healthy adult human volunteers. five subjects received one dose of plerixafor at 240 micrograms/kg/day and pb and bm samples were harvested prior to, at 4, and 24 hours after plerixafor administration. the subsequent 4 participants each received 4 daily doses of granulocyte colony stimulating factor (g-csf) at 5 micrograms/kg/day for 4 days, followed by a dose of plerixafor on day 5. pb and bm samples were collected prior to g-csf, after g-csf, and at 4 and 24 hours after plerixafor administration. we used multi-parametric flow cytometry to identify cd56 bright nk cells. the phenotype of cd56 bright nk cells was cd56 bright cd335 þ (nk46 þ ) cd3 -cd16perforingranzyme b -. results: we observed a significant rise in pb nucleated cells at both 4 and 24 hours after plerixafor administration with a peak increase at 4 hours. plerixafor alone induced a significantly higher proportion of cd56 bright nk reg cells in pb when compared to g-csf for 4 days (2.5 vs. 0.94 fold, p ¼ 0.041) and g-csf for 4 days followed by plerixafor treatment (2.5 vs. 0.43 fold, p ¼ 0.011). moreover, cells collected 4 hours after plerixafor alone had a significant increase in the proportion of cd56 bright nk reg cells (2.5 vs. 0.89 fold, p ¼ 0.031) in pb compared to bm. this study suggests that plerixafor alone is able to mobilize a high number of cd56 bright nk reg cells in pb harvested after 4 hours. we were not able to increase that population in a similar manner by using g-csf alone or g-csf plus plerixafor. these findings suggest that allogeneic donor mobilization of peripheral blood may give a product with a low rate of gvhd potentially superior to g-csf stimulated marrow and warrants further testing in a randomized clinical trial. disclosure of interest: none declared. introduction: only small studies have compared performance of the novel spectra optia apheresis system (terumo bct) with the widely used com.tec (fresenius healthcare) and the late cobe spectra (terumo bct) device in allogeneic stem cell collections. our collaborative working group compared performance data of all 3 devices from two collection centers to analyze device-as well as center-specific performance parameters. materials (or patients) and methods: we analyzed 5288 firstday apheresis collections in g-csf-stimulated healthy donors that were performed in cologne (cgn) using spectra optia mnc (n ¼ 1818), cobe spectra mnc (n ¼ 877), or com.tec (n ¼ 1657) and in dü sseldorf (dus) using spectra optia mnc (n ¼ 63) or cobe spectra mnc (873). peripheral blood and product samples were analyzed in center-specific laboratories. results: in both centers, irrespective of the apheresis device, similar yields of cd34 þ cells per kg donor bodyweight were reached. in cgn, collection rates (collected cd34 þ cells per kg donor bodyweight per cd34 þ cell count in peripheral blood before apheresis) were similar between the three apheresis systems. the continuously collecting cobe spectra yielded more cd34 þ cells over time (cr per h: 0,034 ± 0,009) than the discontinuously collecting apheresis systems 030±0, 009, po0, 001) and spectra optia mnc (0,025 ± 0,008, po0,001). the com.tec collected cd34 þ cells with highest efficiency (ce2: 63 ± 15% vs 61 ± 16%, po0,001 and 55 ± 15%, po0,001 for spectra optia mnc and cobe spectra, respectively). in comparison to cobe spectra (178±49 min), procedure times were longer when using com.tec (199±50 min, po0, 001) or spectra optia mnc (237±58 min, po0,001) systems. the product purity, measured as percentage of mnc was highest in products collected with the spectra optia mnc (79 ± 15% vs 76 ± 10%, po0,001 vs 75 ± 14%, po0,001 for cobe spectra and com.tec, respectively). the relative differences between cobe spectra and spectra optia collection performance parameters were similar in the dus apheresis center: shorter procedures with higher cr per h for cobe spectra, higher mnc purity in spectra optia mnc products. absolute results differed between the two centers. conclusion: the highest collection efficiency (ce2) was seen when the com.tec device was used. however, this was accompanied by low product purity. compared to any other apheresis device that has been analyzed in this study the novel spectra optia allows collection of higher mnc purity apheresis products from allogeneic donors. however, this was associated with a significant prolongation of procedure timethe major disadvantage of this device. the cobe spectra collects more cells over time by allowing higher mean inlet flow rates despite inferior collection efficiency. therefore, a continuous collection procedure on the spectra optia device that allows high inlet flow rates would be an ideal collection setting in terms of collection efficiency and collection rate per unit of time. disclosure of interest: none declared. introduction: our group has previously established the safety and effectivity in terms of cd34 þ cell recovery and viability of the automated washing of cryopreserved hematopoietic progenitors (hp) with the sepax â device (biosafe) using normal saline supplemented with 2.5% albumin (nsa), as well as the absence of infusion-related events and of a negative impact on engraftment dynamics (sánchez-salinas et al. 2012 ). in our present study we compare this solution with a ready-to-use free of human derived components solution: 6% hydroxyethyl starch 130/0.4 in 0.9% sodium chloride injection (voluven â , fresenius kabi). materials (or patients) and methods:: 411 peripheral blood hp units apheresis cryopreserved using autologous plasma plus 9% dmso corresponding to 158 autologous peripheral blood hp transplants were studied. after rapid thawing in a water bath at 371c, an automatic wash with the sepax (2 washes cycle) was performed using either nsa (229 units) or voluven (182 units). nucleated cell levels determined by an hematology analyzer, flow cytometry cd34 þ cell counts, trypan blue cell viability and granulocyte macrophage (gm-cfu) and erythroid (e-bfu) colony forming cell cultures were performed on aliquots collected prior to and after the washing technique. statistical analysis was performed using descriptive statistics and a simple-measures anova. results: the mean total nucleated cell (tnc) and cd34 þ cell recovery was 75,12% ±14,66, and 113,18% ±57,76 respectively for nsa and 79,08% ± 14,75 and 110,02% ± 44,02 for voluven. the mean gm-cfu and e-bfu cell recovery was 163,80% ± 152,64 and 144,62% ± 176,61 respectively for nsa and 187,59% ± 232,48 and 141,82% ± 148,75 for voluven. the mean viability recovery was 102.02 ±17,79 for nsa and 101,59 ±15,04 for voluven without differences between both solutions (p 0,795). there were no significant differences between both solutions in none of this parameters in spite of the tnc significant loss (p o0,001), there were no significant differences between the pre and post-washing cd34 þ cell numbers (p ¼ 0,146), gm-cfu (p 0,051), e-bfu (p 0,952) or viability (p 0,537). in contrast with the 40% of untoward reactions recorded in our historical data of 226 dmso containing cell infusions, we observed just three adverse effect with the washed cells with voluven (1,6%) and none with nsa. one patient experienced a epileptic fit related to the infusion speed, another two suffered grade 1 nausea and transient hipotension respectively. median time to neutrophil engraftment (4500 cells/ml) and platelet engraftment (420.000 cells/ml) for nsa were 11,29 ± 3,32 and 13,19 ± 6,8 days respectively and 12,02 ± 3,01 and 14,77 ± 14,87 for voluven. when comparing both solutions, there were no significant differences in neutrophil engraftment (p 0,169) or in platelet engraftment (p 0,376). conclusion: both nsa and voluven are equally effective for washing cryopreserved hp, ensuring a good cd34 þ cell recovery and preserving their viability and engraftment potential. both solutions avoid the dmso infusion related adverse events. as so, voluven constitutes an excellent alternative free of human-derived products and ready-to-use solution to our previous nsa standard washing solution. references: sánchez-salinas a. et al. transfusion. 2012 nov; 52(11) :2382-2386 disclosure of interest: none declared. introduction: ruxolitinib is a jak inhibitor that was recently approved for treatment of primary and secondary mf and shows impressive symptom control by suppression of inflammation. ruxolitinib is also a promising drug for treatment of acute and chronic gvhd. the immune-modulatory effects of ruxolitinib are at least in part due to an inhibitory effect on dendritic cell biology (heine et al., blood 2014) . dendritic cells (dcs) are important antigen-presenting cells. upon antigen contact they migrate into the draining lymph nodes to prime t cells. the aim of this study was to define the impact of the jak inhibitor ruxolitinib on dc migration. materials (or patients) and methods: cd14 þ cells isolated from human buffy coats were differentiated for seven days in the presence of gm-csf and il-4 to modcs and finally matured with lps. murine bone marrow-derived dcs (bmdcs) were generated by flushing bone marrow from femur and tibia of mice, plating the cell in medium containing gm-csf and maturing the cells with lps. migration of dcs was analyzed in transwell assays or dynamically by time-lapse microscopy within three dimensional collagen gels towards ccl-19 gradients. signaling events were analyzed by western blot to evaluate changes in phosphorylation levels. results: 2d migration of ruxolitinib-exposed dc is dosedependently reduced in vitro. by analyzing the migratory phenotype of human modcs within three dimensional collagen gels, ruxolitinib-exposed dcs are still able to sense chemokine gradients and form lamellipodia at the leading edge of the cell, whereas the retraction of the uropod is inhibited. additional in vivo studies could show that the jak inhibitor potently reduces homing of bmdcs into draining lymph nodes in mice. sirna knockdown experiments revealed that this inhibitory effect is jak1-and jak2-independent. on a molecular level we could show a reduced phosphorylation of rho-associated protein kinase (rock) in ruxolitinib-treated modc upon ccl-19 stimulation. finally, the migration phenotype of modc exposed to ruxolitinib could be mimicked by the rock inhibitor y-27632. conclusion: rhoa family members are key proteins controlling important steps of cell migration, such as protrusion formation at the front of the cell and consequently cell polarization. rock is a downstream effector of rhoa and leads to stabilization of the actin cytoskeleton via cofilin and actomyosin ii contraction by the myosin light chain. the observed reduction of rock phosphorylation may reveal an important mechanism of ruxolitinib-induced inhibition of dc migration. our current efforts focus on the validation of rock as offtarget jak1/2-independent target kinase of ruxolitinib as potential mediator of the observed effects, which may at least in part also explain the potential therapeutic impact of ruxolitinib for therapy of gvhd. introduction: alemtuzumab, a monoclonal cd52-antibody, is used for t-cell depletion (tcd) in the context of allogeneic hematopoietic stem cell transplantation (hsct) to prevent graft versus host disease (gvhd). when we applied this approach in a clinical trial, nearly half of our patients developed acute gvhd (agvhd) overall grade i-ii1. since regulatory t cells (treg) play a major role in controlling gvhd, we hypothesized that they might be functionally impaired in our patients after alemtuzumab based treatment and further investigated on these cells. materials (or patients) and methods: we analyzed peripheral blood samples of 20 patients with agvhd, 10 patients with chronic gvhd, and 12 patients who never developed gvhd after alemtuzumab-mediated tcd. treg were identified as cd3 þ cd4 þ cd25 þ cd127-or cd3 þ cd4 þ cd25 þ foxp3 þ and subsets described by expression of cd52. since cd52 is linked to the membrane by a glycosylphosphatidylinositol (gpi)-anchor, we used flaer to stain for gpi-anchors themselves. to further investigate treg activation, we stained additional markers: cd39, cd44, gitr, cxcr3, ccr5, ctla-4, garp and granzyme. to observe how treg reconstitute after hsct, we analysed samples from patients with agvhd at different time points after transplantation and correlated our findings with the clinical course of gvhd. treg function was evaluated in cfse-suppression-assays: patient derived ex vivo treg were facs-sorted according to their cd52 expression and incubated with proliferating cfsestained cd4-effector t cells from healthy donors. reduced cd4-proliferation was used as indirect marker for the suppressive function of the cd52 positive and negative treg. results: patients with agvhd showed significantly elevated percentages of cd52 negative treg: mean 55.3% (range 34.4-79.7% ) in comparison to only 10.1% (range 1-21.3%) in patients with chronic our without gvhd. by flaer-staining we confirmed that loss of cd52 correlates with the absence of gpi-anchors on the cell surface, these cells do also lack other gpi-anchored proteins (e.g. cd56, cd59). patients who overcame agvhd over time reconstituted gpianchor positive treg, whereas gpi-anchor negative treg remained the dominant population in patients with ongoing agvhd. the fraction of activated garp positive treg was mainly detected among the gpi-anchor positive treg population. all other markers showed a heterogeneous expression profile with a tendency towards lower expression on gpi-anchor negative treg. suppression-assays showed a higher suppressive capacity of gpi-anchor positive ex vivo treg in contrast to gpi-anchor negative treg of the same patient. conclusion: cd52 negative / gpi-anchor negative treg reconstituted in patients after alemtuzumab mediated tcd and mainly persisted in patients with acute gvhd. these treg were less likely to express the activation marker garp. functional assays demonstrated that gpi-negative treg were functionally impaired -in patients developing acute gvhd, these impaired treg represented the major treg-population. our preliminary data suggest that gpi-anchor negative treg, like other gpi-anchor negative t-cell subpopulations (previously shown), are functionally altered. we hypothesize that this might promote acute gvhd. these gpi-anchor negative treg could be useful to diagnose and guide immunosuppressive therapy in patients with acute gvhd. disclosure of interest: none declared. introduction: we previously showed that enhanced human invariant nkt (hinkt) lymphocytes recovery after allogeneic stem cell transplantation was associated with a reduced risk of acute graft versus host disease (agvhd). using a humanized mouse model of gvhd, we aimed to determine whether hinkt cell subsets could be involved in the regulation of allogeneic immune response and to elucidate their mechanisms of action. materials (or patients) and methods: human inkt were obtained by in vitro expansion of total peripheral blood mononuclear cells (pbmc) from healthy donors followed by electronic sorting of cd4 þ and cd4 -inkt subsets among the cd3 þ cd1d-tetramer positive total inkt population. xenogeneic gvhd was assessed in 2 gy irradiated nod/scid/ gamma-c -/-(nsg) mice previously injected with hpbmc alone or hpbmc enriched with cd4 -(pbmc þ inkt4 -) or cd4 þ (pbmc þ inkt4 þ ) hinkt cells. the effects of hinkt lymphocytes on the survival and phenotype of human monocyte derived dendritic cells (hmo-dc) were assessed by flow cytometry (on the expression of annexine v, propidium iodide, and that of cd86, cd80, cd40, respectively). the effects of cd4or cd4 þ hinkt cell subsets on the expression of the t cell activation marker cd25 and on the cytokine expression profile of cd4 þ t lymphocytes was assessed in vivo in the nsg model and in vitro, during the allogeneic mixed lymphocyte reaction (mlr). results: in vivo, nsg mice transplanted with pbmc þ inkt4cells showed a significantly prolonged overall survival in comparison to nsg mice transplanted with pbmc alone (p ¼ 0.001) or with pbmc þ inkt4 þ inkt cells (p ¼ 0.01). improved survival observed with hpbmc þ inkt4cells was associated with lower clinical and histological gvhd scores. in vitro, at 2 inkt/ 1 dc (2 n) ratio, hcd4 -inkt cells significantly increased the apoptosis of mature hmo-dc compared to the cd4 þ inkt subset (p ¼ 0.001). hmo-dc apoptosis in the presence of cd4 -inkt cells was contact dependent (21% in contact vs. 3% in transwell, po0.0001), occurred as early as 4 hours after cell-contact and was associated to the degranulation of cd4 -inkt cells as shown by cd107 expression. while h inkt cd4lymphocytes could inhibit the maturation of hmo-dc in vitro, their cd4 þ counterpart strongly induced high levels of expression of cd80, cd86 and cd40 on mo-dc in a contact dependent way. when allogeneic mlr was performed, in the presence of cd4 -inkt lymphocytes, proliferating alloreactive t cd4 þ lymphocytes showed a lower median fluorescence intensity of cd25 (p ¼ 0.02), and intracellular inf-g (p ¼ 0.005) il-17 (p ¼ 0.002) and il-21 (p ¼ 0.021) in comparison to mlr without addition of inkt cells. compared to nsg mice injected with pbmc alone, mice injected with pbmc þ inkt4cells showed lower cd25 expression on circulating t cd8 lymphocytes (p ¼ 0.001), and reduced intracellular expression of il-17 on circulating t cd4 lymphocytes (p ¼ 0.01). conclusion: these results are in line with our clinical observations and suggest that h cd4 -inkt cell subset could directly modulate the allogeneic immune response by downregulating antigenic presentation and subsequently t cell activation and th1/th17 cytokine production. the nsg preclinical mouse model suggest that developing a cellular therapy based on inkt cd4lymphocytes might be interesting for the prevention of human agvhd. disclosure of interest: none declared. in vivo expansion of host type regulatory t cells via a selective tnfr2 agonist protects from acute gvhd medicine ii, wuerzburg university hospital, 2 pathology, wuerzburg university, wuerzburg, germany introduction: cd4 þ foxp3 þ regulatory t cells (tregs) suppress graft-versus-host disease (gvhd) after allogeneic hematopoietic stem cell transplantation (allo-hct). by controlling the magnitude of the alloresponse tregs still allow for efficient anti-leukemia (gvl) effects of transplanted conventional t cells in preclinical mouse models. current clinical study protocols for donor tregs in the treatment or prophylaxis of gvhd rely on their ex vivo expansion and infusion in high numbers. here we present a fundamentally novel strategy for inhibiting gvhd based on the in vivo expansion of recipient tregs prior to allo-hct, exploiting the crucial role of tumor necrosis factor receptor 2 (tnfr2) in treg biology. materials (or patients) and methods: to expand tregs in vivo we developed a highly selective tnfr2 agonist (that would not bind to tnfr1) and treated allo-hct recipients two weeks before allo-hct. expansion of radioresistant host type tregs, alloresponses of conventional t cells, and tumor progression were monitored with bioluminescence imaging, fluorescence microscopy, and flow cytometry in different mhc major mismatch mouse models (c57bl/6, h-2 b 4balb/c, h-2 d and fvb/n, h-2 q 4c57bl/6, h-2 b ) of gvhd and gvl. results: in vitro, this new tnfr2-agonist expanded natural tregs from wild type but not from tnfr2-deficient mice. accordingly, a human variant of this tnfr2-specific agonist expanded human tregs in vitro. in vivo treatment of healthy mice with the murine tnfr2-agonist significantly increased treg numbers in secondary lymphoid organs and peripheral tissues, particularly in the gastrointestinal tract, a prime target of acute gvhd. consequently, pre-treatment of recipient mice with this novel tnfr2-agonist expanded host-type radiation resistant tregs prior to allo-hct in two models across mhc barriers. tnfr2agonist pre-treatment resulted in significantly prolonged survival and reduced gvhd severity when compared to tnfr2-deficient recipients or untreated allo-hct recipients. this was accompanied by reduced donor t cell proliferation and infiltration into gvhd target organs. in vivo depletion of host derived tregs completely abrogated the protective effect of tnfr2-agonist pre-treatment. while in vivo tnfr2-agonist pre-treatment protected allo-hct recipients from gvhd, antitumor effects of transplanted t cells remained unaffected in two different murine b cell lymphoma models. conclusion: our novel strategy demonstrates that the expansion of host tregs by selective in vivo tnfr2-activation significantly improves the outcome after allo-hct and results in prolonged tumor-free survival. disclosure of interest: none declared. introduction: we reported that mir-155 expression is upregulated in donor t cells during agvhd and mice receiving mir-155 knock-out (ko) donor splenocytes do not exhibit lethal gvhd and have improved survival as compared to mice receiving wild type (wt) splenocytes. while we showed that mir-155 does not affect the alloreactive or homeostatic proliferative potential of t cells, a significant decrease in the expression of the homing receptors ccr5, cxcr4, and s1p1 were found on mir-155-ko t cells, suggesting that the loss of mir-155 could impair the migration of these cells to the peripheral target organs resulting in less agvhd. here, we further investigate the impact of mir-155 expression in t cell migration and elucidate the t cell population responsible for agvhd modulation. materials (or patients) and methods: lethally irradiated balb/c or b6d2f1 recipients were infused with t cell depleted bone marrow (bm) cells (5x10 6 ) and gfp expressing mir-155 ko or gfp-b6 wt t cells (1x10 6 ). transplants with cd4 þ (2x10 6 ) only t cell subsets were performed to identify the lymphocyte population that contributes to the mir-155 mediated effects on agvhd. results: on days 7, 14 and 21 post transplant, recipient mice were sacrificed, and tissues harvested in order to study the kinetics of mir-155 ko t cell migration following allogeneic hsct. there was a dramatic decrease in t cell infiltration of peripheral organs (peyer's patches, liver, lung and skin) in recipients of mir-155-ko t cells as compared to wt t cells as evidenced by confocal microscopy of gfp labeled donor cells. we reasoned that these effects could be due to the modulation of ccr5 and other chemokine receptors by mir-155. we found that a recently described long noncoding rna (lincr-ccr2-5 0 as) located in the vicinity of several chemokine receptor encoding genes including ccr1, ccr2 and ccr5 and that is involved in chemokine regulation and t cell migration has 3 conserved potential mir-155 binding sites. we then set out to determine if mir-155 regulates the expression of this lncrna in relationship with ccr5 expression. there was a significant decrease in ccr5 mrna expression in mir-155-ko versus wt donor t cells obtained from recipient mice at the time of agvhd, but no significant difference in the levels of lincr-ccr2-5 0 as. a luciferase reporter assay, however indicate that there was an interaction between mir-155 and lincr-ccr2-5 0 as. our result does not exclude the possibility that mir-155 might influence the activity rather than lincr-ccr2-5 0 as levels, a hypothesis that is currently being tested. to identify the lymphocyte population involved in mir-155 mediated modulation of agvhd we performed a b6 into f1 transplant using cd4 þ t cells alone as the source of donor t cells. median survival of recipients of t cell depleted bm þ wt cd4 þ t cells (n ¼ 12) was 48 days as compared to 100% survival of all recipient mice of mir-155 ko cd4 þ t cells (n ¼ 12) on day 100, (p ¼ 0.02). recipients of mir-155 ko cd4 þ t cells exhibited also significant lower agvhd clinical (po0.01) and pathological scores (po0.01) than wt recipients. conclusion: our data suggest that mir-155 may exert its modulating effects in agvhd by affecting t cell migration. our results also point to the cd4 þ t cell subset seems to play an important role in the mir-155 regulation of agvhd. experiments are currently underway to determine the role of mir-155 in regulatory t cells and cd8 þ t cell subsets in the modulation of agvhd. disclosure of interest: none declared. oral session: solid tumors introduction: hdct has a recognized indication in the salvage setting of advanced gct and is steadily utilized worldwide. while the prognostic impact of response to prior lines of ct (i.e. definition of chemoresistance) is ascertained, that of response to induction/mobilization ct preceding single or multiple hdct cycles is unknown. we present the results of a retrospective study of the ebmtstwp. materials (or patients) in the multivariable model for pfs, tumor primary (overall p value ¼ 0.039), igcccg category (overall p value ¼ 0.033), and progression to induction (hr: 2.02, 95%ci: 1.31-3.12 , p ¼ 0.001) were significantly prognostic. the latter was also significantly prognostic for os (hr: 2.36, 95%ci, po0.001) together with the transplant setting (overall p value ¼ 0.014). results for response to induction ct were confirmed in the models that included missing data. the c-index of the model was 0.62 for pfs and 0.63 for os. conclusion: progression to induction ct prior to hdct was independently and significantly associated with shorter pfs and os, while response or progression to prior ct lines was not. this information could have important implications to refine patient eligibility to transplantation and enhance the prognostic risk grouping. these data need external validation. disclosure of interest: none declared. kir-ligand incompatibility in the graft-versus-host direction improves progression-free survival in patients with primary high risk neuroblastoma after umbilical cord blood transplantation with nonmyeloablative conditioning y. takahashi introduction: donor nk cells expressing inhibitory killer cell immunoglobulin-like receptors (kirs), which do not recognize their cognate ligands ('kir ligands') on recipient targets, are free from hla inhibition, resulting in a decreased incidence of relapse and improved the outcome after hla haploidentical stem cell transplantation (hsct) (ruggeri, blood 2007) or umbilical cord blood transplantation (ucbt) (willemze, leukemia 2009) in leukemia patients. venstrom reported that significant association between kir/hla genotypes predictive of missing kir ligands have a better outcome without allogeneic hsct following anti gd2 monoclonal antibody therapy in patients with high risk neuroblastoma (venstrom, clin cancer res 2009). these observations led us to start the clinical trial of allogeneic ucbt from kir ligand incompatible donor for patients with high risk neuroblastoma in 2008. materials (or patients) and methods: eligibility criteria of this study were newly diagnosed stage iv neuroblastoma patients with one of the following 1) chemo-resistant disease defined as mibg positive score after 4 courses of induction chemotherapy, 2) older age defined as 10 years old and older at diagnosis or 3) mycn amplification defined as greater than 10 copies per tumor cell. kir ligand mismatched ucbt donor was prospectively selected from the japan cord blood bank network according to the genotyping of hla-c or b as previously reported (willemze, r. leukemia 2009). we scheduled ucbt with reduced intensity conditioning regimen about three months after conventional high dose chemotherapy followed by autologous peripheral blood stem cell transplantation. reduced intensity conditioning regimen for cbt was flu, l-pam 140 mg/m2 and tbi 2 gy. tacrolimus and methotrexate were used for gvhd prophylaxis. single inhibitory kir expressed nk cells with no corresponding recipient's hla were monitored by flowcytometry before and after ucbt to access the expansion of alloreactive nk cells in vivo. we retrospectively analyzed the outcome of 82 patients with high risk neuroblastoma treated in nagoya university hospital between april 1982 and june 2014. results: fifty four patients were treated before dec. 2007 . after this study started in jan. 2008 , 15 patients underwent kir ligand incompatible cbt who match eligibility criteria (7 chemo-resistant, 6 mycn amplification and 2 older age) and 13 patients received standard treatment. all patients achieved engraftment in ucbt group and no patients developed grade iii or more acute gvhd and chronic gvhd. only two patients died in this group because of bu related lung toxicity. surprisingly, no patient in this group relapsed with the median follow-up period of 51 (6-71) months. on the other hand, cumulative incidence of relapse in others was 51.0%. 3year progression free survival in cbt group was significantly better than in others (83.6% vs 40.7%, p ¼ 0.048). single inhibitory kir expressed nk cells significantly expanded after cbt (p ¼ 0.0009). finally, multivariate analysis revealed only kir ligand incompatible cbt and stage iii were significant better covariate factors for relapse. conclusion: this is the first report that kir ligand incompatible allogeneic ucbt significantly reduced the incidence of relapse in high risk neuroblastoma. disclosure of interest: none declared. better prognosis for brca-mutated breast cancers treated with high-dose chemotherapy and autologous hematopoietic progenitor cell transplantation. a singleinstitution retrospective study l. boudin 1, 2, 3, * , a. goncalves 1, 2, 4 , j. m. extra 1, 2 , r. sabatier 1, 2 , h. sobol 2, 5 , f. eisinger 2, 5 , j. moretta 2, 5 , c. tarpin 1, 2 , j. camerlo 1, 2 , b. calmels 2, 6 , c. lemarie 2, 6 , a. granata 2, 6 , 4, 7 , f. bertucci 1, 2, 4 , a. madroszyk 1, 2 , p. viens 1, 2, 4 , c. chabannon 2, 4, introduction: hereditary brca genetic mutation is responsible for approximately 2-3% of breast cancers (bc).the objective of this study was to compare the outcome of brca-mutated (brca mut ) versus brca wild type or unknown (brca wt/uk ) bc following treatment with high dose chemotherapy (hdc) and autologous hematopoietic progenitor cell transplantation (act). materials (or patients) and methods: all female patients treated for breast cancer (bc) with hdc and act at institut paoli-calmettes between 2003 and 2012 were included. patients were divided into 2 groups depending on the indication of hdc with act: metastatic breast cancer (mbc) or high risk breast cancer (hrbc) including inflammatory breast cancer (ibc) and/or massive lymph node involvement (lni). all patients were examined for the presence of known brca 1 or 2 mutations. information regarding patient, tumor characteristics and treatment was also collected. the main objectives were the analysis of overall survival (os) and progression-free survival (pfs) according to the brca mutation status in the two groups. survival curves were generated using kaplan-meier method and compared using log-rank test. results: a total of 377 patients were included, 235 mbc and 142 hrbc (73 lni and 69 ibc). among mbc and hrbc, 10 (6 brca1, 4 brca2) and 5 (3 brca1, 2 brca2) patients were brca mut , respectively. in mbc, median age was respectively 36 (range 29-53) and 42 years (range 24-61) for brca mut and brca wt/uk ; 70% of brca mut patients had triple negative (tn) bc subtype (her2-negative and hormonal receptor-negative) versus only 21% in brca wt/uk individuals. in brca mut and brca wt/uk median number of metastatic sites was 2 and 1, with 90% and 69% visceral metastases, respectively. in hrbc, median age for brca mut and brca wt/uk was 36 (range 28-58) and 49 (range 20-62), respectively; 60% of brca mut had tn bc subtypes versus 24,6% of brca wt/uk . ibc represented 49% of hrbc in brca wt/uk and 40% in brca mut . in mbc, 90% (all except one) of brca mut remained alive at 5 years; the median overall survival (os) was not reached, compared with a median os of 3.62 years for brca wt (ci95% ¼ 3.07-4.68), (p ¼ 0.048 log-rang test); median progression free survivals (pfs) were 26,4 months (cl95% ¼ in brca mut patients versus 15,6 months (cl 95% ¼ 11. 64-18.48) , and in brca wt/uk patients (p ¼ 0.073 log-rank test). in hrbc, 8 years probabilities of os were respectively 1 (all patients alive) vs for brca mut and brca wt/uk individuals, while 8 years probabilities of disease free survival (dfs) were ) and , respectively (p ¼ 0.374 log-rank test). conclusion: the prognostic impact of brca mutation in bc remains controversial. in this series of mbc and hrbc, we have reported an outstanding survival outcome in a small subset of patients with documented brca mutation. in spite of a higher proportion of tn and more aggressive features, brca mut bc had a better outcome than their brca wt/uk counterpart, the difference reaching statistical significance in mbc population. hypersensitivity to dna-damaging agent, possibly due to defect in homologous recombination associated with brca mutation, could explain these results. despite the low numbers of cases in our series, hdc with act, may be considered as an option for brca mut , women with advanced bc. disclosure of interest: none declared. introduction: paclitaxel-based regimens are now commonly employed for second or third-line salvage therapy for gct. this might have an impact on the results with subsequent salvage hdct in these patients (pts). the ebmt-stwp is sponsoring a retrospective study on the outcomes of hdct administered in the last 10 years. hence, we aimed to study outcomes with hdct after relapse to paclitaxel-ct to identify the level of chemoresistance in these pts. materials (or patients) and methods: data have been collected from ebmt registry, including 24 european centers. eligibility included adult male patients (pts) with gct, and treatment with second or further-line hdct between the years 2002 and 2012. both paclitaxel used in prior ct lines of therapy and in induction-mobilization regimens pre-hdct were considered. multivariable cox regression analyses (mva) were undertaken to evaluate the association of prespecified factors (site of tumor primary, igcccg category, response to induction ct, response to prior lines [chemosensitive vs chemorefractory], line of hdct, year of hdct) including prior-paclitaxel therapy with progression-free (pfs) and overall survival (os). results: since 10/2013, 442 pts have been registered, 324 of them were suitable for present analysis. 165 pts (51%) received hdct in second-line, 102 (31%) in third and 57 (18%) beyond the third-line. hdct regimens were as follows: 177 (55%) hd-cbdca-vp16 (ce), 41 (12%) adding ifosfamide (cei), 106 (33%) other mixed regimens. 76 (23%) were taxane-containing. 120 pts received a single hdct course, 99 pts double and 104 multiple courses (1 missing). median follow up was 36 months (iqr: 19-70). prior paclitaxel was significantly associated with shorter os in the univariable model (p ¼ 0.032). however, on mva prior paclitaxel-therapy was not significantly prognostic for both pfs and os, as shown in the table. a separate model evaluated the interaction between prior paclitaxel-therapy and taxane-containing hdct: no significant interaction was found (p ¼ 0.221 1.18-5.76 .017 conclusion: the administration of paclitaxel-based regimens before hdct did not affect pfs/os. results were confirmed when excluding pts who were administered taxane-containing hdct. line of hdct was not significantly prognostic too. there is no evidence to disallow patients who have been treated with taxanes in second or third line to receive hdct as futher salvage therapy. these data need external validation. disclosure of interest: none declared. favorable outcome of a cohort of metastatic breast cancer patients treated with high dose chemotherapy and autologous transplantation at a single institution does not change the prognostic significance of histopathological subtypes l. boudin introduction: studies of high dose chemotherapy (hdc) in breast cancer (bc) often lack of biomarker information, notably the human epidermal growth factor receptor 2 (her2) status. the objective of this study was to evaluate the outcome of patients affected with different subtypes of bc following treatment with hdc and autologous hematopoietic progenitor cell transplantation (act). materials (or patients) and methods: all female patients treated for metastatic breast cancer (mbc) with hdc and act at institut paoli-calmettes between 2003 and 2012 were included. patient, tumor and treatment characteristics were collected. patients were categorized in three subtypes based on hormonal receptor (hr) and her2 status of the primary tumor: luminal (l), (hr þ /her2-), her2 (her2 þ , any hr), and triple negative (tn) (her2-and hr-). main study endpoints were overall survival (os) and progression-free survival (pfs) categorized by bc subtypes. treatment related mortality (trm) was also evaluated. survival curves were generated using kaplan-meier method and compared using log-rank test. results: a total of 231 mbc patients are included; median age was 47 (range 24-61); metachronous and synchronous mbc were 64% and 36%, respectively. 96% patients received hdc with act as part of their first-line treatment following diagnosis of metastases. median number of metastatic sites was 1 (range 1-6), including 69% of visceral metastases. l, her2 and tn subtypes were 61, 18 and 21% of patients, respectively. all but one her 2 patients (98%) received trastuzumab during the metastatic phase of the disease before (88%) and/or after (91%) oral session: acute leukemia 2 introduction: the outcome of patients 455 years old with all is poor with no clear recommendations regarding the indications for hct. these patients are usually considered ineligible for myeloablative allohct and offered transplantation with reduced intensity conditioning (ric). autologous hct is an alternative. however, the role of both treatment options has not been established so far. the aim of this study was to retrospectively compare results of ric-allohct and autohct in all 455 years old and to identify factors affecting outcome. materials (or patients) and methods: 267 patients treated with ric-allohct from either hla-identical sibling (n ¼ 154) or matched unrelated donor (n ¼ 113) and 179 treated with autohct in first complete remission between 2000 and 2011 have been included in this analysis. median age in both groups was 60 (55-74)y and 60 (55-76)y, while median interval from diagnosis to hct was 5.9 months and 6.6 months, respectively. the proportion of ph( þ ) all among those with reported cytogenetics was 71% and 66%, respectively. results: with a median follow-up of 33 months, the probability of os at two years was 44% for ric-allohct and 57% for autohct (p ¼ 0.02), while lfs rates were 34% and 41%, respectively (p ¼ 0.06). relapse incidence at two years was comparable for ric-allohct and autohct (42% vs. 48%, p ¼ 0.39) while non-relapse mortality was significantly reduced for autohct (23% vs. 11%, respectively, p ¼ 0.002). the advantage in favor of autohct was significant for ph(-) all (os: 61% vs. 38%, p ¼ 0.02; lfs: 54% vs. 21%, p ¼ 0.005) while no significant differences could be observed for ph( þ ) all (os: 55% vs. 47%, p ¼ 0.6; lfs: 42% vs. 35%, p ¼ 0.4). in a multivariate analysis adjusted for recipient age and gender as well as interval from diagnosis to transplantation the use of autohsct was independently associated with reduced risk of mortality (hr ¼ 0.69, p ¼ 0.01), treatment failure (hr ¼ 0.76, p ¼ 0.03) and non-relapse mortality (hr ¼ 0.39; p ¼ 0.0004) with no effect on relapse incidence (hr ¼ 0.98, p ¼ 0.88). in the ric-allohsct subgroup lfs was negatively affected by female donor/male recipient combination (hr ¼ 1.64, p ¼ 0.01). lfs rates for both sibling and mud transplants were comparable (32% vs. 35%, p ¼ 0.18). the use of peripheral blood cells compared to bone marrow was associated with reduced risk of relapse (hr ¼ 0.5, p ¼ 0.03). in the autohsct setting there was a tendency to higher risk of treatment failure with increasing recipient age (hr ¼ 1.05, p ¼ 0.06). other variables including type of conditioning (tbi-based vs. chemotherapy-based) did not affect survival in any of the study cohorts. conclusion: considering poor overall prognosis of all patients 455 years old, results of both ric-allohct and autohct appear enhancing and both types of transplantation may be considered valuable treatment options. potential advantage of autohct as suggested by results of our analysis should be further explored including data on disease-related prognostic factors and the status of minimal residual disease. disclosure of interest: none declared. introduction: the karyotype of the leukemic cells at diagnosis is one of the strongest prognostic factors in acute myeloid leukemia (aml). but the major cytogenetic risk categorizations were based on the large clinical studies of conventional chemotherapy for aml. in this retrospective study, we analyzed the influence of the cytogenetics of aml at diagnosis on the outcomes of allogeneic hematopoietic cell transplantation (hct). materials (or patients) and methods: from the database of jshct, we extracted the data of adult patients with aml who receive first hct between 2006 and 2010. a total of 4,241 recipients were included. additional survey for the recipients who had been reported to have cytogenetic abnormalities at diagnosis (n ¼ 2,384) confirmed the data of karyotype of 1,360 cases. results: cytogenetics at diagnosis were categorized into (a) normal karyotype (41.5%), (b) inv(16) or t(16;16) (2.9%), (c) t(8;21) (9.1%), (d) 11q23 abnormality (4.9%), (e) complex karyotype (4 ¼ 3 abnormalities) (12.3%), (f) monosomal karyotype (mk) (6.3%), and others. overall survival (os) and cumulative incidence of relapse at 2 years of all cases were 48.2% and 37.5%, respectively. these recipients were classified into 4 groups: favorable (fav) included (b) (n ¼ 121); intermediate (int), (a), (c), t(9;11) and others (n ¼ 3,169); unfavorable-1 (unf-1), (d) except t(9;11), and (e) except (f) (n ¼ 404); unfavorable-2 (unf-2), (f) (n ¼ 265). adjusted os at 2 years were 65.2% in fav, 53.2% in int, 37.8% in unf-1 and 24.2% in unf-2 ( figure) . risk of overall mortality (rom) and risk of relapse (rr) were compared among these four groups, adjusted by age, sex, disease status at transplantation and stem cell source. in fav group, rom (hr 0.71, , p ¼ 0.0038) and rr (0.44, 0.27-0.71 , p ¼ 0.0007) were significantly lower than in int group. on the other hand, compared with int group, recipients in unf-1 and unf-2 group had significantly (po0.001) higher rom (unf-1 1.48, 1.29-1.70 ; unf-2 2.22 1.93-2.57 ) and rr (unf-1 1.65, 1.41-1.92 ; unf-2 2.35, 1.94-2.73 ). these results showed that the karyotype of leukemic cells at diagnosis was also one of the powerful prognostic factors in hct for aml. but the impact of each cytogenetic presentation on the outcomes of transplantation may have some differences from that of chemotherapy. to improve this risk stratification system, molecular abnormalities of aml at diagnosis are also considered for the influence on outcomes of hct. disclosure of interest: none declared. late intensification with autologous hsct after nonmyeloablative beam conditioning has shown to be effective approach in adults with t-cell acute lymphoblastic leukemia treated by non-intensive protocol: results of the rall study group e. parovichnikova 1,* , l. group is conducting the all-2009 trial where the main principle is non-intensive but non-interruption treatment and autologous hsct after beam followed by maintenance in t-cell all pts without hla-identical donors (ebmt 2014, op-009). the autologous hsct was planned for all patients as late high dose consolidation (on the 20-22 weeks of the protocol). in patients with early and late t-cell immunophenotype having hla-identical siblings allogeneic bmt was an option. from nov, 2008 , till nov, 2014 , 30 centers enrolled 264 all patients. in 6phenotype was unknown (2,2%). b-cell precursor phenotype was diagnosed in 62,6% (n ¼ 166), t-cell precursor phenotypein 34,8% (n ¼ 91); biphenotypic al -in 0,4% (n ¼ 1). t-cell all patients were young -28 y (16-56); male gender prevailed -33 f/58 m. t-all subtypes distribution in our study was as follows: 44 (47,8%) patients had early t-all (t-i/ii), 36 (40,1%)thymic (t-iii), 11 (11,1%) -mature (t-iv). for the whole group medians were: hb-112 g/l (42-180), l -22,3*10/ 9 l (0,5-313), plt -90*10/ 9 (5-943), b/m blasts -74,9% (0-99), ldh -995 iu (131-12 000). no born marrow involvement was detected in 4 pts, b/m blasts 5-25% -in 10 (so t-lbl -14,5%). cytogenetics was available in 63,2% of pts (n ¼ 55/87), 45,5% of them (n ¼ 25/55) had normal (nk), 25 pts -abnormal karyotype, 5 pts (9%) had no mitosis. mediastinum involvement was registered in 48/87 (54,5%) and cns disease -in 11/87 (12,5%). results: induction and follow-up data were available in 87 pts. cr was achieved in 78/87 (89,8%): after prephase ¼ 12, after 1 st ind.phase ¼ 45, after 2 nd ind.phase ¼ 21. induction death occurred in n ¼ 5 (5,7%). primary resistance and even progression during induction was registered in n ¼ 4 (4,5%). 28 pts proceeded to autologous hsct, almost all (n ¼ 26) were successfully harvested at a median time of 20 weeks. 2 pts were poor mobilizers and bone marrow exfusion was carried out. no data on mrd level at time of harvesting is available so far. auto-hsct was applied at a median 6 months from cr. allogeneic hsct from sibling donors was performed in 6 cr pts (4 with ti/ii and 2 with t-iv). no deaths in allo-hsct group have occurred. the land mark analysis at 6 months for chemotherapy group (n ¼ 27) and at day of hsct for transplanted groups was done. disease free survival constituted for chemotherapy -55% at 5 years, and 100% -in both transplanted groups. none of the transplanted patients relapsed so far. the overall and diseasefree survival in the whole cohort of t-cell all patients was 58% and 68%. in a multivariate analysis only autologous hsct influenced dfs. conclusion: our study demonstrates that t-all may be treated by non-intensive, but non-interruption approach. even without high dose consolidation 5-years os and dfs (land mark analysis) constituted 55%. autologous hsct with beam conditioning after mild induction/consolidation followed by prolonged maintenance seems to add benefit to the overall optimistic results decreasing the relapse rate from 34% to 0% within 5 years. disclosure of interest: none declared. with a median follow up from time of hla typing of 14 months (range, 0.3-97) for all patients and 30 months (range, 3-97) for survivors, the 1-and 8-year probability of survival for all 225 eligible aml patients was 68±3% and 40±4%, respectively. at the same time points, for the 172 transplanted patients the probability of survival from time of graft was 60 ± 4% and 43 ± 4%, respectively. the probability of survival was particularly dismal for the 37 patients transplanted in advanced disease phase (4±4% at 4 yrs), whereas for the 135 patients transplanted in early (cr1 þ cr2) phase the 8-yrs probability of survival was 54±5% and, by excluding the low number of cb recipients, it was 55 ± 7% for 56 hla id sib, 58 ± 8% for 40 hrd and 68 ± 9% for 28 mud recipients (p ¼ ns). conclusion: rtn policy allows a donor identification in 94% of all evaluable aml patients and provides an allogeneic transplant to 86% of them with no substantial differences in s58 terms of long-term survival between initially eligible and definitively transplanted patients or by comparing the different donor stem cell sources. transplant results should be given following information on the specific transplant policy and only the itt analysis allows to know the real impact of each transplant program. disclosure of interest: none declared. materials (or patients) and methods: oral pan was administered in two sequential schedules, either thrice weekly (tiw) every week (a) or every other week (b). arm a, in which pan was started at a dose of 10 mg tiw and escalated to 30 mg tiw using a 3 þ 3 design was followed by arm b, in which pan doses increasing from 20 mg tiw to 40 mg tiw were investigated. pan was initiated between day þ 60 and þ 150 after hsct and given for up to 1 year. eligibility criteria included: ancz1,000/ml, plateletsz75,000/ml, adequate organ function and no severe gvhd. dlt was defined as prolonged g4 hematologic toxicity or any non-hematologic toxicityzg3 unrelated to disease progression within 28 days of the first pan dose. results: 24 pts (21 aml, 3 mds), median age 52 yrs (range, 21-71), are evaluable for mtd. cytogenetics were classified as low (n ¼ 2), intermediate-1/2 (n ¼ 11) or adverse risk (n ¼ 11) according to eln criteria. pan was started a median of 98 days (range, 60-147) after hsct from a mrd (n ¼ 8), mud (n ¼ 10) or mismatched donor (n ¼ 6) which was performed in active disease (n ¼ 17, median bone marrow blasts 23%, range, 8-77), cr1 (n ¼ 5) or cr2 (n ¼ 2). the rp2d was 20 mg tiw in arm a and 30 mg tiw every other week in arm b based on 5 dlts: fatigue g3 at 20 mg, colitis and nausea/emesis g3 each at 30 mg (arm a), diarrhea and headache g3 at 40 mg each (arm b). pan-related or unrelated g3/4 aes were reported in 20 of 24 pts (83%) and included hematologic toxicity (50%), laboratory alterations (42%), infections (29%), constitutional (29%) and gastrointestinal symptoms (25%) and neurologic, pulmonary, pancreatic/hepatobiliary or vasculary toxicity (4-8% each). aes were rapidly reversible after interruption and/or dose reduction (n ¼ 6) and no study-related deaths occurred. fifteen pts (63%) have completed one year of pan, 9 pts discontinued treatment prematurely after 7-217 days due to aes (n ¼ 8) or relapse (n ¼ 1). prophylactic or preemptive dlis (1-6) were administered to 15 pts (63%). eleven pts (45%) developed mild (n ¼ 6), moderate (n ¼ 3) or severe (n ¼ 2) chronic gvhd. patients died of aml (n ¼ 3) or severe chronic gvhd (n ¼ 1 introduction: our previous research has provided the evidence that an autoreactive immune system can be ?reset? into a healthy, tolerant state by immunoablative treatment to eradicate pathogenic effector cells, followed by transplantation of hematopoietic progenitor cells (hsct). here, we present the clinical and serologic responses and long-term immune reconstitution in 20 patients with severe ads for up to 15 years after receiving immunoablation and asct. materials (or patients) and methods: since 1998, 20 patients with refractory autoimmune diseases (sle, n=10; ssc, n=4, ms, n=2; polychondritis, n=1; panniculitis, n=1, granulomatosis with polyangiitis, n=1, and chronic inflammatory demyelinating polyneuropathy, n=1) received a cd34 + -selected autologous stem cell transplantation after immunoablation with atg (neovii, formerly fresenius) and cyclophosphamide as part of a prospective monocentric phase i/ii clinical trial. autoantibody titers were evaluated with elisa, peripheral tand b lymphocyte subsets immunophenotyped using multicolor flow cytometry. results: with a median follow-up of 12 years after immune reset (range, 24 months to 16 years), 15 of 20 patients (75%) achieved a progression-free survival (pfs), defined as survival without major organ failure. 50% of these patients showed durable clinical remissions for up to 15 years despite discontinuation of immunosuppressive treatment, while 25% of patients had a stabilization of their underlying disease under maintenance therapy. disease relapse occurred in three sle patients (at 18, 36, and 80 months, respectively), two of whom died later from uncontrolled disease and infection, respectively. 3 of 20 patients died from infection (n=2) or cardiac failure (n=1). anti-dsdna antibodies completely normalized in all sle patients and ana significantly declined from a median titer of 1:5120 at baseline to 1:160 six months after transplantation in patients with connective tissue diseases. recovery of recent thymic emigrants (rtes) was completed between 12 and 24 months after immune reset, reaching on average 3.6 to 5.1 times the baseline levels. recurring foxp3 + tregs had significantly higher expression levels for cd31 and cd45ra compared to age-matched healthy controls, indicating their recent thymic origin. numeric recovery of the naÿve b cell compartment was completed within 1 year after immune reset in responding patients. conclusion: these data confirm our assumption that the reprogramming of an autoreactive immune system into a juvenile and self-tolerant immune system is feasible and associated with long-term remissions. our findings propose that chronic autoimmunity is not an end point depending on continuous treatment with specific anti-inflammatory agents, but may be cured by combining specific targeting of autoreactive memory and effector cells with a reactivation of thymic activity. a future challenge is to make this therapeutic approach attractive for a larger number of patients by reducing the rate of severe infections. this may be achieved by either using a more selective graft purging, e.g., depletion of t cell receptor alpha/beta and cd19 + cells from apheresis products with the clinimacs device, or an adoptive transfer of microbe-or virus-specific memory t and/or b cells. disclosure of interest: none declared. in the present report we summarize the long term effects of transplantation. materials (or patients) and methods: the mobilization protocol included cyclophosphamide and g-csf. the conditioning consisted of cyclophosphamide (50 mg/kg/day on days -5,-4,-3, -2 prior to transplantation) and antithymocyte globulin (thymoglobulin -of 0.5 mg/kg/day given on day -5, and 1.0 mg/kg/day given on days -4,-3, -2 and -1) except for three patients, who received atg fresenius at adjusted doses. results: one of the transplanted patients died during neutropenia due to the sepsis and its complications. the mean time of observation of remaining 23 patients as of december 2014 was 57 months (range 33 -80 months). the independence of exogenous insulin after the transplantation was achieved in 20 of them. three patients were lost to follow up. the median time without exogenous insulin for 20 patients in follow up was 30 months (range 0-80 months). four out of 20 (20%) remain in remission of diabetes (exogenous insulin free) with median follow up of 54 months (range 34-80 months). notably, three patients in the series were treated with atg fresenius (due to transient problems with thymoglobulin availability) -the median follow-up of these patients is 65 months with 58 months of remission of diabetes. the treatment led to significant reduction of hba1c and good glycemic control in patients. no attempt was made to repeat procedure in relapsing patients. the hsct leads to remission of new onset t1dm in majority of patients but the response in most of them is limited to average of 30 months. patients receiving atg fresenius tended to have longer remission but small number prevented drawing conclusions. there is a need to develop procedure to either prolong response or to effectively treat the relapse of diabetes. disclosure of interest: none declared. hematopoietic stem cell transplantation for refractory crohn's disease: feasibility and toxicity j. introduction: autologous hematopoietic stem cell transplantation (hsct) is a salvage treatment for severe refractory crohń s disease (cd) patients. we evaluated feasibility and toxicity of autologous hsct for refractory crohn's disease (cd). materials (or patients) and methods: in this prospective study, refractory cd patients with an aggressive course despite medical treatment, impaired quality of life and no surgical options were included. hematopoietic stem cells were mobilized with cyclophosphamide and g-csf and collected by leukapheresis from pheripheral blood. in a second step, conditioning regimen with cyclophosphamide and rabbit antithymocite globulin (ratg) was used and previously collected stem cells were infused. toxicity and complications during the procedure and within first-year after transplantation were evaluated, along with the impact on safety after the introduction of supportive measures over the study. results: twenty-six patients were enrolled. during mobilization, 16 patients presented febrile neutropenia, including 1 bacteremia and 2 septic shocks. neutropenia median time after mobilization was 5 days. five patients withdrew from the study after mobilization and 21 patients entered into the conditioning phase. hematopoietic recovery median time for neutrophils (40.5x10 9 /l) was 11 days and for platelets (420x10 9 /l) was 4 days. ninety-five percent of patients suffered febrile neutropenia and 3 patients presented worsening of perianal cd activity during conditioning. among non-infectious complications, 6 patients presented antithymocyte globulin reaction, 12 patients developed mucositis and 2 patients had hemorrhagic complications. changes in supportive measures over the study, particularly antibiotic prophylaxis regimes during mobilization and conditioning, markedly diminished the incidence of severe complications. during the first 12-month follow-up, viral infections were the most common observed complications, and one patient died due to systemic cytomegalovirus infection. conclusion: autologous hsct for refractory cd patients is feasible but extraordinary supportive measures need to be implemented. we consider that this procedure should only be performed in highly-experienced centers. disclosure of interest: none declared. autologous haematopoietic stem cell transplantation in aggressive multiple sclerosis: a uk cohort from two centres j. clay 1,* , p. 0.5 (range 0.5-4.5) , whilst the remaining 4 had a deterioration of 0.5-1.0 materials (or patients) and methods: 25 cb units (cbu) were frozen and thawed across the participating banks, according to local sops; pre-freezing and post-thaw samples were assessed for clonogenic potential (cfu) and cd34 þ recovery and viability through standard ishage protocol (si) (brocklebank am 2001). a modified ishage gating strategy (mi) was developed by reduction of the debris gate and by extension of lympho-monocytes gate in order to detect cd34 þ events with abnormal physical parameters (cd34 þ gate). facs sorting was performed to collect cd45 þ /cd34 þ events included inside (p8) and outside(p9) the si cd34 gate. sorted cells were then characterized by fc, confocal microscopy analysis and cfu assay in order to confirm that cd34 þ cells detected out of the si cd34 gate are not an artifact. confocal microscopy analysis was performed by labeling with a cd34 ab recognizing a different epitope and marked with a nuclear counterstain. results: si and mi strategies showed no significant discrepancies when determining cd34 þ absolute number and viability in pre-freezing cbus. the recovery of viable cd34 þ cells assessed by si or mi in the thawed products did not show any differences statistically significant, whilst the recovery of total cd34 þ cells was significantly lower in si (58,3 ± 18%) than in mi (80,1±19.7%)-analyzed samples (mean±sd, po0,05 with pair t test). this difference resulted in a lower cd34 þ viability with mi than si (79,5% ± 15.9 vs 96,7% ± 3%, respectively, po0.02). fc post-sorting analysis showed that the sorted p8 (si cd34 gate) population was cd45 dim cd34 þ cd133 þ cd5 -7-aad-, whereas p9 (mi extended cd34 gate) showed the same markers expression but a 7-aad þ staining, therefore determining such events as cd34 þ dead cells. this data was confirmed by confocal microscopy analysis. finally, p8 events showed a 67% clonogenic efficiency, whereas p9 events were not able to generate any colony. conclusion: a cd34 þ count after cbu thawing lower than the fresh sample is often reported, usually with a high rate of viability. we showed that the standard methodology for cd34 þ counting leads to the exclusion of a number of dead cd34 þ cells, thus resulting in a viability overestimation. we propose here a modification of the ishage counting standard for thawed samples, which provides a more reliable assessment of cd34 þ viability. the content of pdc in bm grafts had a significant beneficial effect on post-transplant survival, an association not seen in gpb grafts 1 . the 20% improved survival seen in recipients of more donor bm pdc was due to decreased deaths from graft rejection and acute and chronic gvhd. donor bm pdcs in murine bmt favorably modulated t-cell activation, decreasing gvhd through gamma-interferon-dependent upregulation of ido, while preserving gvl effects 2 . the present study tested whether the biological activity of pdc from different graft sources are explained by the differential expression of homing receptor molecules or immuneregulatory pathway genes. materials (or patients) and methods: facs isolated pdc from allogeneic stem cell grafts obtained from 10 bm, 8 gpb, 4 umbilical cord blood (ucb) and 2 following mobilization with plerixafor (plxpb) samples. expression of chemokine receptor, integrin and selectin were measured by flow cytometry. gene expression on flow cytometry-sorted pdc was analyzed with illumina chips. differentially expressed genes using a 2-sided t-test comparing groups of replicate samples with po0.05 were selected for exploratory analyses of regulatory pathways. mhc mismatched mouse h2b-h2k transplants compared outcomes with pdc from ccr7 ko vs wt donor. indirect presentation of allogeneic peptides by pdc and classical dc was studied using h2b-h2 b/d transplants with h2kd peptide recognition by tea transgenic donor t-cells. results: pdc from bm and plxpb grafts had higher cd62l expression but lower ccr7 and cxcr3 expression than pdc from gpb and ucb grafts. high cxcr4 and low ccr9 expression were seen in pdc from all graft sources. while differences in ccr7 expression between pdc from bm vs gpb grafts suggested homing differences might explain observed differences in trm associated with the pdc content of the allograft, donor pdc from ccr7 ko mice had equal ability to modulate gvhd as pdc from wt mice. pdc in grafts from both human and mice were phenotypically immature and were relatively ineffective in activating t-cells through indirect presentation of alloantigen peptide, indicating that immune regulatory effects might be more important than antigen presentation. supporting this hypothesis, gene expression patterns of human pdc from bm showed lower expression levels of genes related to adaptive immunity, and higher [o108] expression of genes associated with innate immunity, than pdc from gpb, ucb, or plxpb grafts. conclusion: functional differences in immuneregulatory genes explain some of the observed differences in transplant outcome when stratifying recipients based upon between the content and source of donor pdc. bm pdc appear to be polarized towards activation of innate immunity while pdc from other graft sources are more polarized towards antigen presentation. novel approaches of stem cell mobilization, such as plerixafor, may increase the content of immature pdc enriched for expression of that might be effective in recapitulating the beneficial effects of bm pdc with respect to preventing early posttransplant transplant related mortality. references : results: with a median follow-up of 43, 43 and 49 months in the 10/10 urd, 9/10 urd and ucb, hematopoietic recovery was slower in ucb compared to urd recipient: the cumulative incidence of neutrophils recovery at day 28 and platelet recovery 450 â 10 9 /l at day 180 were respectively 73% and 56% in ucb against 96% and 85% in 10/10 urd and 95% and 75% in 9/10 urd (po0.0001). while there was no significant difference in the day 100 cumulative incidence of grade 2-4 agvhd: 31% in 10/10 urd, 39% in 9/10 urd and 32% in ucb, the 2 years cumulative incidence of cgvhd was significantly lower in ucb recipients: 16% against 37% in 10/10 urd (po0.0001) and 29% in 9/10 urd (p ¼ 0.004). in multivariate analysis, there was no statistically significant difference in nrm between ucb recipients and 9/10 recipients (hr 1.58, p ¼ 0.13) or 10/10 recipients (hr 1.35, p ¼ 0.25) . in multivariate analysis, the cumulative incidence of relapse/progression was significantly lower in 10/10 urd recipients compared to ucb recipients (hr 0.60, p ¼ 0.02) and there was also a trend towards a decreased incidence of relapse in the 9/10 urd recipients (hr 0.62, p ¼ 0.07). compared to ucb transplants there was no significant difference in pfs after 9/10 urd (hr 1.17, p ¼ 0.29 ) and 10/10 urd (hr 1.10, p ¼ 0.49 (table 1) . results: there were no significant differences in pre-transplant characteristics between both groups ( table 1) . the 30day cumulative incidence of neutrophil engraftment was similar in both groups (93% vs 100%), with a median time of 16 and 17 days, respectively (p ¼ 0.28). similarly, the 60-day cumulative incidence of platelet engraftment was 70% vs 76%, in a median time of 39 and 35 days, respectively (p ¼ 0.11). four cases among the haplo-cord group showed primary cb graft failure (3 of them showing third party donor cells engraftment) who were rescued by a second cb transplant in two cases and haplo-sct in two cases. there were no graft failures in the haplo-sct group. grade ii-iv acute gvhd was more frequent in the haplo-sct group (20% vs 41%, p ¼ 0.06) and chronic gvhd showed a higher tendency in the haplo group (18% vs 35%, p ¼ 0.24). with a median follow-up of 51 months (10-122) in the haplo-cord group and 19 months in the haplo-sct group, os was 52% and 78% (p ¼ 0.15), respectively. efs was 41% vs 72% (p ¼ 0.06), relapse rate was 22% vs 20% (p ¼ 0.71) , and trm was 22% vs 8% (p ¼ 0.16), respectively. conclusion: in our experience, albeit differences in follow-up and limited number of patients, myeloablative single cord supported by hla-mismatched cells and haploidentical transplantation with post-transplant cyclophosphamide both offer valid alternatives for patients with acute leukemia. engraftment rates are similar in both groups as well as relapse rate. early toxic mortality is higher in the haplo-cord group while gvhd rates seem to be higher in the haplo-sct patients. further analysis including larger series and longer follow-up are needed to confirm these observations. disclosure of interest: none declared. introduction: studies on conditioning regimens prior to autologous stem cell transplant (asct) in lymphomas generally present a major pitfall, the miscellaneous mix of histologies composing the series, resulting in a reduced statistical power when focusing on a specific subset. we previously demonstrated (visani et al, blood 2011) the safety of a new conditioning regimen with bendamustine, etoposide, cytarabine, and melphalan (beeam) prior to asct in resistant/relapsed lymphoma patients (eudractnumber2008-002736-15). the regimen showed long-lasting significant antilymphoma activity, with a 3-year pfs of 75%. materials (or patients) and methods: we thus designed a single histology, phase ii study to evaluate the efficacy of the beeam conditioning in resistant/relapsed diffuse large b-cell non-hodgkin lymphoma (dlbcl) patients. the study was registered at at european union drug regulating authorities clinical trials (eudract) n. 2011-001246-14. the primary endpoint of the study is to evaluate the 1-year complete remission rate. fixing the lowest acceptable rate as 55% and the successful rate as 70%, with a significance level a ¼ 0.05 and a power 1-b ¼ 0.90, the sample size was estimated in 88 patients. results: until now, 53 patients (median age 54 years, range 19-69) were enrolled. at the time of writing, we have data available for 44 patients with resistant/relapsed diffuse large b cell non-hodgkin lymphoma were consecutively enrolled in the study. briefly, 33/44 patients had advanced stage disease (iii-iv), 14 were primary refractory and 30 had relapsed after a median number of 2 lines of therapy (range: 2-3). eleven patients had 1 or more relevant comorbidities (range: [1] [2] [3] [4] [5] . 22 patients were in ii or subsequent cr after salvage therapy, whereas 19 were in pr and 3 had progressive disease. a median number of 5.90x10 6 cd34 þ /kg cells (range 2,8-9,42) collected from peripheral blood was reinfused to patients. all patients engrafted, with a median time to anc40.5x10 9 /l of 10 days. median times to achieve a platelet count 420x10 9 /l and 450x10 9 /l were 12 and 16 days respectively. ten out of 44 patients presented a fever of unknown origin (23%), whereas 21 patients (47%) presented a clinically documented infection. all patients received g-csf after transplant for a median time of 8 days (range: 8-13). one patient died due to an incomplete hematological recovery after transplant, producing an overall transplant related mortality of 2.7%. thirtyeight out of 44 patients are evaluable up to now for response to treatment. 31/38 (81.5%) obtained a cr, 3/38 a pr, whereas 4/38 did not respond to therapy. after a median follow-up of 12 months after transplant (range 2-30), 4/38 patients were refractory, 7/38 relapsed, and 27/38 (71%) are still alive, in continuous cr. conclusion: in conclusion, this data provide evidence that the beeam regimen is safe and has promising efficacy in resistantrelapsed aggressive diffuse large b cell lymphoma. pts with hl at risk of relapse post-asct (clinicaltrials. gov #nct01100502). the primary results showed that bv significantly improved progression-free survival (pfs) per independent review vs. placebo. efficacy analyses by investigator review were performed in subgroups as these data may provide useful information for making treatment decisions. materials (or patients) and methods: the aethera trial is a phase 3, randomized, double-blind, placebo-controlled study. after asct, pts received bv 1.8 mg/kg q3wk or placebo for up to 16 cycles. pts were enrolled in 1 of 3 high-risk categories: refractory to frontline (fl) therapy, relapse o12 mos after fl therapy, and relapse z12 mos after fl therapy with extranodal disease. the primary endpoint was pfs per independent review. pfs by investigator was also evaluated. results: 329 pts were randomized at 78 sites in the us and europe. median age was 32 yrs (range, 18-76) and 53% were male. asct conditioning regimens were beam: 61%, cbv: 11%, or other regimens: 28%. 6% of pts received radiation as part of their transplant regimen. all pts had completed the treatment phase as of aug 2013. subgroup analyses by demographics, stratification factors, pt characteristics, and number of risk factors showed that pfs by investigator was improved across all groups; the hazard ratio comparing bv to placebo was o1 for all analyses (table 1) . in an ad hoc analysis, pts with increasing numbers of risk factors for progression had progressively greater pfs benefit with bv vs. placebo. the most common adverse events (aes) in the bv arm (vs. placebo) were peripheral sensory neuropathy (56% vs. 16%) and neutropenia (35% vs. 12%). a higher incidence of herpes simplex and zoster infections were observed with bv (11%) vs. placebo (3%); otherwise, opportunistic infections were rare and balanced between the arms. the incidence of pulmonary toxicity (by meddra smq) was low, but slightly higher with bv vs. placebo (5% vs. 3%). two pts died r40 days of last dose of bv; cause of death was acute respiratory distress syndrome. response to salvage therapy pre-asct cr 123 0.78 (0.42, 1.46 ) pr 113 0.44 (0.26, 0.76 ) sd 93 0.40 (0.23, 0.71) hl status after frontline therapy refractory 196 0.55 (0.37, 0.83 ) relapse o12 months 107 0.45 (0.27, 0.93 ) relapsez12 months with extranodal disease 26 0.30 (0.08, 1.16) 42 treatments pre-asct 149 0.32 (0.19, 0.54 ) b symptoms after failure of frontline therapy 87 0.29 (0.15, 0.55) extranodal disease at time of pre-asct relapse 107 0.37 (0.20, 0.68) risk factors a z1 329 0.50 (0.36, 0.70 ) z2 280 0.40 (0.28, 0.57 introduction: although high-dose chemotherapy (hdc) is the gold standard for the treatment of many relapsed or refractory lymphomas, the outcome is still unsatisfactory in some subsets of patients with adverse prognostic features. we treated 111 high-risk patients with a tandem strategy associating debulking with hdc followed by autologous stem cell transplantation (asct) and subsequent allogeneic sct (tandem auto-allo) materials (or patients) and methods: adult patients consecutively treated at two centers were included. criteria for receiving tandem auto-allo were: hl and nhl refractory to first-line therapy, less than cr after first salvage treatment, relapse after prior asct, multiple relapses, histology of transformed follicular, mantle-, t-and nk-cell lymphoma and documented infections during hospital stay in 30 (27%), without significant differences between beam and hd-mel groups. among the 65 patients with active disease before asct, the overall response rate was 86% (n ¼ 56 responders) and those in cr were 34 (52%). no difference was observed in terms of response in the beam and hd-mel groups (p ¼ 0.28). allogeneic sct donor was either hla-identical (n ¼ 86), mud 9/10 (n ¼ 2), haploidentical (n ¼ 20) or cord blood (n ¼ 3). 3-y os of entire cohort was 68% (95% ci: 59-77), 3-y pfs was 61% (52-70), rates of acute gvhd grade 2-4 and chronic gvhd were 28% and 38% respectively. trm rate was 18% (n ¼ 20). no difference between beam and hd-mel group was observed for os (73% and 64% respectively, p ¼ 0.40) or trm (19% and 13%, p ¼ 0.44). of note, os of patients in cr before and after asct and os of those obtaining cr after asct was superimposable ( figure 1) figure) . a multivariate analysis revealed that late allo-hsct was a unfavorable prognostic factor for os with a statistical significance (hazard ratio, 1.46 ; 95% ci, 1.01-2.11; p ¼ 0.04 (3) or complete response after autosct after a third line of chemotherapy (9 pts). donors of allosct were hla identical siblings in 38% of patients, matched unrelated in 35% and haploidentical donors in 27% of patients. median age at transplant was 33 (range, 17-60). at allosct, 43% of patients were in cr, 30% were in pr, 27% in sd or pd. 83% patients performed allosct having relapsed o12 months from autosct or with primary refractory disease, 17% having relapsed 412 months after autosct. results: median follow-up of surviving patients was 4.8 years (range, 0.5-10.5) . overall survival (os) was 76% at 1 year, 59% at 3 years and 55% at 5 years of follow-up. in multivariate analysis, only disease status before allosct significantly impacted the os (p ¼ 0.003, hr 1.6, ci95% 1.2-2.2) , whereas donor and timing or relapse/refractoriness did not change os (p ¼ 0.149, hr ¼ 1.3, and p ¼ 0.501, hr ¼ 1.3, respectively better control of the disease: this has occurred together with changes in donor type (from unrelated to family haploidentical), in gvhd prophylaxis (from mtx cya atg to post transplant cyclophosphamide), and in the conditioning regimen, combining thiotepa and intravenous busulfan; in addition dipss was somewhat lower in more recent patients, and also spleens were smaller. we believe these data suggest that alternative donor grafts are currently a therapeutic option for patients with mf. disclosure of interest: none declared. long follow-up data show that survival rate after allogeneic stem cell transplantation in patients with cll is higher for younger patients because of significantly lower nrm during the whole follow-up period: a retrospective study of the cmwp m. van gelder 1,* on behalf of cmwp . among pediatric pts, 28% were aged 0-23 months, 52% were aged 2-11y, and 20% were aged 12-16y. day þ 100 survival data by age group are available for 526 pts post-hsct and 62 pts post-ct (table) , with rates of svod post hsct of 55% in pediatric pts and 50% in adults. hpse has been shown to be involved in inflammation and may therefore play an important role in the pathogenesis of vod of the liver. the purpose of this study was to identify an association between hpse gene single nucleotide polymorphisms (snps) and vod of the liver after allogeneic hsct in childhood. materials (or patients) and methods: 160 children (median age, 14 years) who underwent allogeneic bone marrow (n ¼ 91) or peripheral blood stem cell transplantation (n ¼ 69) in a single center and their respective donors were genotyped of hpse for rs4693608 and rs4364254 using taqman real-time polymerase chain reaction. the donor was hla-matched unrelated in 63% of transplants and hlaidentical related in 25% of transplants. conditioning regimen was myeloablative in all cases and based on busulfan in 46% of transplants or total body irradiation in 33% of transplants. two forms of post-transplant immunosuppression predominated, cyclosporine a and methotrexate in 50% of transplants and cyclosporine a alone in 30% of transplants. results: 160 donor/patient pairs were analyzed. cell samples from the patient were available in 155 cases and from the donor in 153 cases. genotype ag of hpse rs4693608 snp was found in 82 patients (53%), aa in 49 patients (32%), and 24 patients were homozygous for gg (15%). analysis of hpse rs4364254 snp revealed a similar distribution for tc (n ¼ 69, 44%) and tt (n ¼ 68, 44%) and a frequency of 18 patients (12%) for cc. vod of the liver was observed in 12/160 patients (8%). if vod of the liver was diagnosed all of our patients were treated with defibrotide as early as possible. altogether, 4/12 patients with vod of the liver (33%) died of vod, whereas 8/ 12 patients with vod of the liver (67%) survived vod. early medical intervention is most probably the reason for this high cure rate. patients with hpse genotypes gg or ag of rs4693608 (g4a) had a significantly reduced incidence of vod of the liver on day 100 after hsct compared to patients with genotype aa (5% vs. 14%, p ¼ 0.038). in addition, incidence of vod in patients with genotype cc or ct of rs4364254 (c4t) was significantly decreased in comparison to patients with genotype tt (2% vs. 15%, p ¼ 0.004). interestingly, no patient with genotype cc developed vod. because both snps co-occur in vivo, we generated subsets: aa-tt, gg-cc and a group with remaining snp combinations. we found significant differences between all three patient groups (p ¼ 0.035). patients with aa-tt showed the highest incidence of vod of the liver (17%), while vod was not observed in patients with gg-cc (0%) and residual combinations were numerically in-between (5% allogeneic, n ¼ 42) were considered at risk of hbv reactivation for the following criteria of positivity: 1) donor and/or recipient anti-hbc and/or 2) anti-hbe and/or 3) anti hbs þ /-. lamivudine prophylaxis was given to 51 out of 58 patients (88%). overall, 4 patients, 1 autologous and 3 allogeneic recipients, developed hbv reactivation at a median time of 40 months (range, 28-53) after hsct. of these 4 patients, 3 were at risk of reactivation and 2 of them were not receiving prophylaxis. one was hbv-negative at the time of pretransplant screening. the cumulative incidence of hbv reactivation was 4,9% for patients at risk and 1%.for the entire hsct population studied. one patient reactivated hbv infection during lamivudine prophylaxis. hbv isolate genotype was studied in all reactivated patients. two isolates showed hbsag escape mutations (123 n, 124y, 126i, 145 k, 145 r, 144e, r122k, t140s) and in 1 lamivudine drug resistance mutations (181 s and l801l), 3 of them resulted hbv genotype d and one resulted genotype f. conclusion: the low rate of reactivation in our cohort is probably due to the accuracy of pre-transplant donor/ recipients screening and to the extensive prophylaxis program. however, a case of hbv reactivation occurred in a patient negative at serological screening (sero-negative hbv occult infection). the detection of immune-escape hbsag mutations, associated with lack of recognition by neutralizing immune activity and by diagnostic assay in most hbvreactivated patients, supports the role of these mutations in the process of immune-suppression driven hbv reactivation. disclosure of interest: none declared. introduction: polyomavirus (pv) hemorrhagic cystitis (hc) is a severe complication after haploidentical hematopoietic stem cell transplantation (haplo hsct). in the setting of solid organ transplantation, the use of tacrolimus (fk) has been associated with higher risk of pv-hc compared with cyclosporine a (csa). the aim of our study was to investigate risk factors of pv-hc in haplo-hsct recipients, with particular focus on immunosuppressive agent used as graft-versus-host disease (gvhd) prophylaxis. materials (or patients) and methods: we retrospectively analyzed the incidence, risk factors and outcome of pv-hc in 149 consecutive adult patients (pts) undergoing haplo hsct due to hematological malignancies between 2009 and 2014 at our two institutions. all hscts were t-cell replete and included post-transplant high-dose cyclophosphamide as part of gvhd prophylaxis. pv-hc was defined as pv detection in urine by pcr testing in association with clinical symptoms of hc without other concurrent genitourinary conditions. variables tested as potential risk factors to develop a pv-hc were: conditioning regimen, age, diagnosis, stem cell source, levofloxacin prophylaxis, acute gvhd, interval between diagnosis and hsct, pre-hsct therapy lines, previous subdiaphragmatic radiotherapy, n. of cd34 þ cells infused, neutrophil and platelet engraftment, fk vs csa-based immunosuppressive regimen. results: main pts' characteristics are shown in table 1 . after a median follow-up time of 13 months from transplantation, ten (7%) pts developed a pv-hc. pv-hc occurred in 6/52 (12%) pts receiving fk and 4/97 (4%) pts receiving csa (p ¼ 0.10). etiology was bk virus in 9 (90%) cases and jc virus in one (10%) case. median time of pv-hc diagnosis was 30 (7-68) days post-hsct. in the multivariate analysis, myeloablative (mya) or reduced-intensity conditioning (ric) regimens (hr ¼ 4.25, 95% ci: 1.18-15.33 ; p ¼ 0.03) and fk-based immunosuppression (hr ¼ 3.78, 95% ci: 1.05-13.64 ; p ¼ 0.04) were the only two independent factors associated with pv-hc. regarding treatment, two pts received cidofovir therapy, one pt benefited from immunosuppression tapering, whereas only supportive therapy was required for the remaining 7 pts. notably, all pts achieved complete remission of symptoms. 3.9 (0.8-14.0) relapse who would benefit from further treatment intensification early after transplant. introduction: the prognosis for patients with hematologic malignancies (hm) who relapse after allohct is dismal, and immune checkpoint modulation with ctla4 blockade is a novel strategy to enhance the graft versus tumor (gvt) effect. materials (or patients) and methods: this is a phase i/ib study of the ctla4 blocking antibody ipilimumab to treat patients with any hm histology who relapse after allohct. the primary objectives are to determine the mtd and evaluate safety. secondary objectives include a preliminary evaluation of efficacy and changes in immune cell phenotype. ipilimumab was given at 3 mg/kg or 10 mg/kg iv q3 weeks for 4 cycles of induction, followed by maintenance dosing q12 weeks up to 1 year. disease-specific response criteria were assessed at the mid-point (7 weeks), end of induction (13 weeks), and throughout maintenance. immunophenotyping was performed by 8-color flow cytometry and analyzed by facsdiva. results: twenty-three patients are enrolled to date. in the phase i portion, 6 patients were treated at 3 mg/kg and 7 patients were treated at 10 mg/kg. an mtd was not reached, and 10 patients subsequently enrolled in the phase ib expansion cohort at 10 mg/kg. the median number of prior therapies excluding transplant was 3 (range 2-11), and 14 patients had received prior therapy for their post transplant relapse. histologies included chl (n ¼ 7), aml (n ¼ 7), nhl (n ¼ 4), and mds (n ¼ 2), and 1 patient each had mm, mpn, and all. the median age at enrollment was 55 (range 22-75). immune-related adverse events (iraes) were observed in 4 patients, and included itp (n ¼ 1), diarrhea (n ¼ 2), pneumonitis (n ¼ 3), and colitis (n ¼ 1), all of which were generally reversible with steroids with most patients resuming ipilimumab dosing. three dlts have been observed, including 2 cases of chronic gvhd (both liver, mild) and one patient death due to presumed sepsis in the context of severe iraes, including grade 3 colitis and grade 4 pneumonitis. nine patients remain on treatment, and 11 patients discontinued due to progressive disease, 2 patients due to cgvhd, and 1 patient with trm. in an interim efficacy analysis, 7/19 (36.8%) patients evaluable for response had anti-tumor activity with clinical benefit. four patients achieved formal responses by disease-specific criteria, including a chl patient with pr with dramatic reduction in nodal and extranodal disease with a complete marrow response at 7 weeks (baseline 90% involvement), a mm patient with a pr with near resolution of a lung plasmacytoma, and two patients with aml who achieved cr by 7 weeks. the median follow-up time among survivors is 5.4 months, and 6 month overall survival is currently 57%. immunophenotyping studies revealed that the ratio of regulatory t cells to conventional t cells decreased between 24% and 41% after ipilimumab treatment. conclusion: multiple doses of ipilimumab given to patients with relapsed hm after allohct were generally well-tolerated, with cgvhd and iraes observed in a small number of patients. anti-tumor activity was observed, both in lymphoid malignanices and for the first time in myeloid malignancies, including 2 patients with aml who achieved cr. the ratio of regulatory to conventional t cells decreased with ctla4 blockade. the phase ib expansion cohort at 10 mg/kg continues to accrue and will provide additional efficacy, safety, and correlative data. disclosure of interest: none declared. adverse risk cytogenetic (p ¼ 0.04). in patients who relapsed between 6 and 12 months post-transplant more than one course of chemotherapy to achieve cr1 (p ¼ 0.02), adverse risk cytogenetics (p ¼ 0.05) and flt3 itd positivity (p ¼ 0.00002) all predicted for relapse. finally only cmv positivity predicted for relapse risk for relapse more than 12 months post-transplant (p ¼ 0.05). conclusion: this study demonstrates for the first time that a complex interaction of disease specific and transplant factors determine the kinetics of relapse post-transplant. in addition to identifying that heterogenous factors related to transplant characteristics and disease biology determine the timing of disease relapse these data confirm the importance of early intervention post-transplant in patients allografted for aml and and will assist in the design of novel therapeutic strategies. disclosure of interest: none declared. introduction: bone marrow (bm) is the recommended stem cell source in hematopoietic stem cell transplantation (hsct) for bone marrow failure (bmf). recent large studies in aplastic anemia showed that the use of peripheral blood stem cells (pbsc) increased the risk for chronic graft-versus-host disease without reducing graft failures and lead to an inferior survival after hsct. however, a substantial proportion of hsct is still performed with pbsc. ease of collection and a more rapid engraftment might favor pbsc in the short term and more background information to clarify the situation is warranted. materials (or patients) and methods: the current global practice of hsct for bmf was studied for potential macroeconomic factors associated with the selection of stem cell source. introduction: the outcome of alternative donor hematopoietic stem cell transplantation (hsct) for the patients with severe aplastic anemia (saa) who lack an hla-matched familial donor has improved recently. this study was aimed to compare the treatment outcome of immunosuppressive therapy (ist) with alternative donor hsct in children with saa. materials (or patients) and methods: medical records of saa patients who received ist and/or alternative donor hsct from umbilical cord blood (ucb), haploidentical peripheral blood stem cells (pbsc), unrelated bone marrow (ubm), or unrelated pbsc (upbsc) between june 1996 and december 2012 were reviewed, and data were analyzed retrospectively. the kaplan-meier method was used to calculate the estimated survival rates which were compared with log-rank test. results: of 59 patients with saa, 42 patients received ist and/ or alternative donor hsct. nineteen patients received ist as an initial treatment modality, of whom 13 patients failed to respond at 6 months from the initiation of treatment. thirtyfour patients received alternative donor hsct either following ist (n ¼ 11) or as frontline therapy (n ¼ 23; 11 ubmt, 11 upbsct, 8 ucbt, 4 haploidentical pbsct). the failure-free survival rate (ffs) of ist group was 31.6%, while that of frontline hsct group was 91.3% (po0.001). patients who received hsct following ist showed an inferior event-free survival rate as compared with those who received frontline hsct (50.9% vs 91.3%; p ¼ 0.015). conclusion: the outcome of alternative donor hsct in our cohort was higher than usually expected, especially in those who received hsct without prior ist. these results suggest that frontline alternative donor sct might be a better treatment option than ist for children with saa who lack an hla-matched familial donor. disclosure of interest: none declared. increased risk of development of malignancies in adult patients treated with antithymocyte globulin as first line treatment for aplastic anaemia during a follow-up period of thirty years. j. v. d introduction: adult aplastic anemia (aa) is considered to be an immune-mediated disease with bone marrow aplasia and pancytopenia. patients can be treated with either hematopoietic stem cell transplantation (hsct) or immunosuppressive therapy (ist) with antithymocyte globulin (atg) and achieve long-term survival. treatment related long term toxicity and outcomes should guide the decisions about first line therapy. a long-term toxicity of both treatments is the development of malignancies. follow-up studies until 15 years after ist in aa patients showed cancer development in up to 25% of patients. analysis of cohorts with a longer duration of follow up are lacking and the incidence of malignancies in aa patients was never compared with a control population and the contribution of second line hsct to this risk is not clear. we assessed the long-term cumulative incidence of malignancies in a cohort of adult patients with aa who were treated with atg as first line treatment at leiden university hospital between 1980 and 2008 with hsct and death as competing risks and compared this incidence to cancer development in the general dutch population. materials (or patients) and methods: 93 patients treated with first line atg between 1980 and 2008 were entered in this single-centre retrospective cohort study. primary endpoint was the cumulative incidence of malignancies with death and second line treatment with hsct as competing risks. secondly, the cumulative incidence in our cohort was compared to that of the sex-and age matched general dutch population derived from the dutch cancer registration, using standardized incidence ratio (sir). furthermore, several patient-dependent (age), disease-dependent (severity of disease) and treatmentdependent (addition of ciclosporin to atg) variables were assessed as possible prognostic factors for cancer development, using cumulative incidence curves, gray's test and the cox proportional hazard model for the cause-specific hazard. spss statistics 20.0 and r were used for all data analyses. ethical approval for data collection and data analysis was obtained. results: median length of follow-up of surviving patients without hsct was 17.4 years (range 0.2-32.2 years) , with a cumulative malignancy rate of 29% after 30 years. the incidence of malignancies was increased compared with the general dutch population (sir: 3.35 (95% ci: 1.98-5.08 )). the cumulative incidence of developing mds/aml was 6% after 30 years (occurring at 5-17 years after start of treatment). in the multivariate analysis, age at time of ist was significantly associated with a higher malignancy rate (hr: 1.52 per 10 years; p ¼ 0.01). hr for addition of ciclosporin was1.87 (p ¼ 0.25) and hr for nsaa vs (v)saa hr was 1.65 (p ¼ 0.32)). the risk of the development of malignancies in aa patients treated with atg is increased compared to the risk in the age and sex matched dutch population for both solid and hematologic malignancies. we are not able to discern whether the increased risk is due to the treatment with ist or due to the biology of the disease. long follow up studies in age matched aa patients receiving hsct are needed to compare this long term risk between both treatments. these results underscore the recommendation in international guidelines to follow all patients with aa after initial ist lifelong. disclosure of interest: none declared. 1 hematopoietic stem cell transplantation, 2 molecular biology, dmitriy rogachev center for pediatric hematology, oncology and immunology, moscow, russian federation introduction: results of matched unrelated and, to a lesser extent, haploidentical transplantation in severe aplastic anemia have improved over the last decade. the major factors behind this improvement are high-resolution hla-typing, intensified immune suppression with fludarabine and alemtuzumab and modern supportive care. we implemented a new graft manipulation method, tcr-alpha/beta depletion, to further improve gvhd control and overall results of transplantation in a group of saa patients, refractory to two courses of atg/csa immune suppression. materials (or patients) and methods: twenty six patients with saa, median age 11,4 (2,5 -22) years, 16 male/ 10 female, received mud (20) or haploidentical (6) transplantation with tcr alpha/beta and cd19 depletion between 01. 09.2012 and 01.09.2014 . patients received a median of 2 atg/csa courses. core preparative regimen included cyclophosphamide 25 mg/ kg x4, fludarabine 30 mg/kg x 5, atg (horse) 25 mg/kg x 4 and total lymphoid irradiation 6 gy total dose. one patient received alemtuzumab instead of atg due to anaphylaxis. in all cases g-csf-mobilized pbsc were used as graft source. tcr-alpha/beta and cd19 depletion were performed with clinimacs plus according to manufacturer's recommendations. final graft contained 10x106 of cd34 þ /kg and 17x103 of tcra/b þ /kg. post-transplant immune suppression included short methotrexate and tacrolimus till day þ 45-60. results: primary engraftment was registered in all patients, median 15 days for neutrophils and 14 for platelets. secondary graft failure/rejection developed in 2 patients, both transplanted from mud. both patients were salvaged with a second mud transplantation. acute gvhd was registered in 4 patients, grade 2 skin in 2 and grade 3 skin þ gut in 2 patients. cumulative incidence of agvhd grade 2-3 was 15%(95%ci:6-38). chronic gvhd was registered in 1 patient, cumulative incidence 4%(95% ci:0,7-30). two patients (1 from each cohort) died of cmv pneumonia at 4 and 8 months respectively. in both cases cmv sero status was d-/r þ . at 2year follow-up cumulative incidence or trm is 12%(95%ci:0,3-46), km estimte of overall survival is 88%(95%ci:72-100), 92%(95%ci:76-100) in the mud group, 67%(95%ci:13-100) in the haplo group. conclusion: tcr alpha/beta depletion is a highly effective method of gvhd prophylaxis in saa patients. use of this platform makes alternative donor transplantation in saa a safe therapeutic option. further improvement will depend on new strategies to control viral infections. disclosure of interest: none declared. tp:129 (34) vs 173 (36) cm/s, respectively; (po0.001), and were decreased more significantly following hsct than on tp: mean(sd) d: -42 (31) vs þ 6 (34), respectively. the percentage of patients with normal velocities was significantly higher post-hsct (29/32) than in the tp arm (16/32) (p ¼ 0.001) conclusion: this prospective national trial comparing tp vs. hsct in sca-patients with a history of abnormal velocities shows for the first time that hsct significantly results in a greater decrease in velocities than tp, and has very little toxicity. these results suggest hsct is the treatment of choice for sca-children with a history of abnormal-tcd and genoidentical donor. disclosure of interest: none declared. introduction: busulfan (bu) is the backbone of the conditioning regimen for patients with sickle cell anemia (sca) undergoing bmt. patients with sca might predispose to transplant-related neurological and pulmonary toxicities due to pre-existing disease-related cerebrovascular and lung injury. bu therapy appears to be an important contributing factor in this context. to date, no studies have evaluated the pharmacokinetic (pk) parameters of intravenous bu (ivbu) for subsequent doses in patients with sca. materials (or patients) and methods: we studied ivbu pk parameters and clinical outcomes of 36 children with sca undergoing bmt from hla matched siblings. the median age of patients was 10.4 (range, 1.7-17.1 ) years. conditioning regimen consisted of bu/cy200/atg (n ¼ 12) or flu/bu/cy200 (n ¼ 24). six patients (17%) had stroke, 11 (31%) exhibited gliosis due to previous brain injury, 13 (36%) had repeated acute chest syndrome, and 8 (22%) patients were on chronic transfusions. ivbu was administered every 6 h for 4 days with pk-guided dose adjustment to target a conservative area under the concentration versus time curve (auc) range of 900-1350 mmol*min. the role of glutathione s-transferase (gst) polymorphisms has also been investigated. results: all patients had sustained engraftment. a repeated measures anova showed that the first-dose bu clearance (4.24 ml/min/kg) was significantly higher than after dose 5 (3.70 ml/min/kg; po0.0005), dose 9 (3.58 ml/min/kg; po0.0005), and dose 13 (3.42 ml/min/kg; po0.0005). such differences in clearance have never been described in patients with sca. after the first-dose, 69% of patients achieved the target range.none of the patients developed hepatic vod. no patient developed grade z3 toxicity. there was no association between gst polymorphism and the pk of bu. we adapted a new dose-adjustment strategy targeting exposures to the lower end (900 mmol*min) of the auc range after the first dose of bu to avoid unnecessary dose increases on subsequent days due to differences in clearance. this strategy enabled most patients (90%) to maintain the auc within therapeutic range following dose adjustments. a 3-year probability of os and sickle-cell free survival were 91% (95% ci, 75-97%). there was no correlation between any bu pk parameters and survival, toxicity, acute gvhd, chronic gvhd, and transplant-related mortality. conclusion: this study found that the pk behavior of ivbu in children with sca is characterized by significantly higher bu clearance after the first-dose compared with subsequent daily doses. this finding allowed us to adapt a new dose adjustment strategy after the first dose of bu, which effectively prevented subsequent dose readjustments. conservative auc range and targeting exposures to the lower end of the range after the first dose was associated with negligible toxicity, and high engraftment and sickle cell-free survival rates. disclosure of interest: none declared. however, the probability to find a suitable door is only 25-50%. since most of these patients (pts) could not find the potential donor, we would like to investigate the haploidentical donor hsct (hapo-sct) in thalassemia. materials (or patients) and methods: between jan 2013 and december 2014, a total of 116 severe thalassemia patients (pts) underwent hsct in our center. sixty five pts underwent mrd-hsct, 33 pts underwent mud-hsct and 18 pts underwent haplo-hsct. for haplo-hsct, 10 subjects were male and 8 were female. the median age was 16 yrs (range; 2-22). thirteen of 18 received stem cells from mother and 5 from father. ten of 18 were high risk class 3. these high risk class 3 pts received hydroxyurea 20 mg/kg/d for at least 3 months prior to hsct. all pts received 2 courses of pre-transplant immunosuppression (ptis) consisting of fludarabine (flu) 40 mg/m 2 /d together with dexamethasone (dex) 25 mg/m 2 /d for 5 days. after 2 courses of ptis, all pts received a reducedtoxicity conditioning (rtc) regimen consisting of thymoglobulin 1.5 mg/kg/d (d-11 to d-9) , flu 35 mg/m 2 /d i.v. (-7 to -2) each dose immediately followed by busulfan (bu) 130 mg/ m 2 once daily i.v. (d-7 to d -4) gvhd prophylaxis consisted of cyclophosphamide (cy) 50 mg/kg/d (d þ 3 to d þ 4). tacrolimus or sirolimus was given for 6 months to 1 yr started together with mycophenolate mofetil on d þ 5, the latter was quickly tapered after 2 months. t-cell repleted peripheral blood stem cells (pbsc) were given to all pts, targeting a cd34 þ dose of 7-16 x 10 6 cells/kg. results: sixteen of 18 were engrafted with full donor chimerism (100%) while 2 pts suffered graft failure. however, these 2 pts received second transplant on day þ 30 with minimal conditioning regimen and additional pbsc after which they achieved full donor chimerism. the median time to neutrophil engraftment was 18 days (range; 14 -22) . six pts had acute gvhd gr i, 3 grade ii and 1 gr iv. only one had extensive chronic gvhd. at this time, 17 of 18 pts survive thalassemia-free and have sustained full donor chimerism (100%). event free survival (efs) and overall survival (os) rates are 95%. the median follow up time is 12 months (range 4-20). the efs rates among mrd, mud and haplo-hsct in our center are 88%, 82% and 95% respectively (p ¼ 0.46). conclusion: haploidentical hsct for high risk thalassemia pts with our novel approach is safe and should be considered as modality to secure thalassemia-free survival with a low risk for graft rejection and treatment-related mortality. in view of our results, we suggest that all thalassemia pts even those with high risk class 3 features should be offered the chance for cure with hsct. disclosure of interest: none declared. introduction: allogeneic hematopoietic stem cell transplantation (hsct) is a curative option for many patients with hematological malignancies. graft versus host disease (gvhd) mediated by donor alloreactive t cells remains a major obstacle and limits its wider application. it is now well established that t regulatory cells (tregs) are critical for the maintenance of self-tolerance, and a deficiency or dysfunction in these cells is thought to be a key factor in the development and progression of gvhd. numerous murine studies have shown the benefit of adoptive transfer of tregs in the setting of hsct. notably, the success of this approach is improved if the transferred tregs are specific for antigens expressed by the host. a robust method to generate homogeneous populations of alloantigen specific tregs in humans would be a major step towards realizing the goal of using these cells to suppress gvhd. traditional approaches to generate antigen specific t cells include repetitive cycles of re-stimulation with antigen in vitro, sorting of tetramer positive cells, or over-expression of a t cell receptor for a specific antigen. these methods produce limited cell numbers and require modification for each individual patient depending on their own tissue type and that of the transplanted patient. we hypothesized that a more efficient approach to generating antigen specific tregs would be to genetically engineer them to express alloantigen-specific chimeric antigen receptors (cars). materials (or patients) and methods: we generated an hla-a2 specific car by cloning the immunoglobulin heavy and light chain variable regions from an anti-hla-a2 antibodysecreting hybridoma. these regions were fused into a single chain antibody, which was then linked to intracellular signalling domains from human cd28 and cd3zeta. surface expression and antigen-specificity of the a2-car was confirmed by flow cytometry to detect expression of a myc epitope tag on the extracellular portion of the car and binding to an hla-a2 tetramer, respectively. cd4 þ cd25 hi cd45ra þ naïve tregs (ntregs) and cd4 þ cd25 lo cd45ra þ naïve tconv (ntconv) cells were sorted from the blood of hla-a2donors, stimulated with artificia apcs and transduced with lentivirus encoding the a2-car or a control car specific for her2. results: transduced tregs maintained their expected phenotype, including high levels of foxp3, ctla-4, cd25 and helios, and low levels of cd127 and il-2 compared to tconv cells. to test antigen specificity, the transduced t cell lines were stimulated with k562 cells expressing hla-a2 or her2: only k562 cells expressing hla-a2 stimulated proliferation of a2-car expressing tregs and tconv cells. car-stimulated tregs also suppressed the in vitro proliferation of car-stimulated tconv cells. to demonstrate the in-vivo suppressive capacity of a2-car tregs, nsg mice were reconstituted with hla-a2 þ pbmcs in the absence or presence of different ratios of control-transduced or a2-car transduced tregs. preliminary results suggest that a2-car tregs have a superior ability to delay the onset of gvhd compared to polyclonal tregs. conclusion: human tregs can be efficiently transduced to express functional, alloantigen-specific cars that confer antigen-specific specific suppression of effector t cells both in-vitro and in-vivo. these data supports the feasibility of this strategy to re-direct tregs for transplant-related therapies. disclosure of interest: none declared. car spacers including ngfr domains allow efficient t-cell tracking and mediate superior antitumor effects m. casucci 1,* , l. falcone 1 , b. camisa 2 , f. ciceri 3 , c. bonini 2 , a. bondanza 1 1 innovative immunotherapies unit, 2 experimental hematology unit, 3 clinical hematology and bone marrow transplantation unit, san raffaele hospital scientific institute, milano, italy introduction: chimeric antigen receptors (cars) frequently include an igg1-ch2ch3 spacer conferring optimal flexibility for antigen engagement and allowing the selection and tracking of car-expressing t cells. a serious drawback of ch2ch3-spaced cars is however their interaction with fcg receptors (fcgrs). indeed, this antigen-independent binding may lead to the unintended elimination of cells expressing these receptors (mainly phagocytes), foster the development of non-specific immune reactions and drastically decrease the efficacy of the strategy due to the premature clearance of transduced t cells in vivo. materials (or patients) and methods: we designed and constructed novel car backbones by substituting the igg1-ch2ch3 spacer with regions from the extracellular portion of the low-affinity nerve-growth-factor receptor (lngfr), differing for the length and potential binding to ngf. in particular, we used our recently developed cd44v6-specific car as a model for comparing the antitumor activity of the different lngfr-based designs both in vitro and in vivo. results: after transduction, all constructs could be identified on the t-cell surface using anti-lngfr antibodies, indicating that they were correctly processed, mounted on the cell membrane and still recognized by anti-ngfr antibodies. as a consequence, all the lngfr-based spacers allowed selecting car-t cells with immune-magnetic beads coupled to anti-ngfr antibodies, without interfering with their expansion and functional differentiation after activation with cd3/cd28 beads plus il-7 and il-15. most importantly, lngfr-spaced car-t cells maintained potent cytotoxic, proliferative and cytokine-release activity in response to cd44v6-expressing leukemia and myeloma cells, while lacking antigen-independent recognition through the fcgrs. noticeably, even at supraphysiological ngf concentrations, the lngfr-spaced cd44v6-car.28z car t cells were not induced to proliferate, indicating the absence of signaling via soluble ngf. strikingly, lngfrspaced car-t cells better expanded and persisted in vivo compared to ch2ch3-spaced car-t cells and mediated superior antitumor effects in a well-established tumor disease model. interestingly, we demonstrated that the premature disappearance of ch2ch3-spaced car-t cells was due to engulfment by mouse phagocytes, a phenomenon not occurring with lngfr-spaced t cells. in conclusion, we have demonstrated that the incorporation of the lngfr marker gene directly in the car sequence allows for a single molecule to work as a therapeutic and as a selection/tracking gene and shows an increased efficacy/safety profile compared to the igg1-ch2ch3 spacer. disclosure of interest: none declared. university children's hospital, würzburg, 10 clinic of pediatric oncology, heinrich-heine-university düsseldorf, 11 university children's hospital, essen, 12 university children's hospital, münster, 13 department of pediatrics, jena university hospital, germany introduction: viral infections represent an important cause of morbidity and mortality in immunosuppressed patients post hematopoietic stem cell transplantation (hsct). as viral infections often remain refractory to pharmacologic treatment, alternative treatment strategies such as immunotherapy are required. adenovirus (adv) is the predominant diseasecausing pathogen in pediatric hsct. materials (or patients) and methods: in a clinical trial we analyzed safety and efficacy of ex vivo adoptive t-cell transfer (act) with hexon-specific t cells, predominantly of t helper -1 phenotype. thirty patients suffering from chemo-refractory adv disease or viremia after hsct were treated with advspecific t cells generated by ifn-g capture technique. results: adv-specific t cells were successfully isolated in 100% of cases using the adenoviral hexon antigen and were directly infused into the patients without further ex vivo expansion steps. adv-specific t-cell grafts were composed of a mixture of naïve, central memory, effector memory and effector t-cell populations with a predominance of late effector stages, indicating proliferation and effector potential of the transferred t cells. in all thirty patients, act was feasible without acute toxicities or significant onset of graft-versus-host disease. act led to antiviral immunity in vivo up to 6 months with viral control, resulting in complete clearance of viremia in 86% of patients with antigen-specific t-cell responses. efficacy of adoptive t-cell transfer was independent of the initial t-cell dose. after a follow up of 6 months post act, overall survival was markedly increased in responders (mean: 122 days, 15 survivors) as compared to non-responders who all died shortly after act (mean: 24 days, no survivors). adv-related mortality was 100% in non-responders compared to 9.5% in responders (z1 log reduction of dna copies/ml post act), indicating a strong correlation between virus-specific immunity and virus control. conclusion: in conclusion, ex vivo act of adv-specific t helper -1 cells was well tolerated and led to successful and sustained restoration of t-cell immunity, correlated with virological response and protection from virus-related mortality. this cellular immunotherapy is a short-term available and broadly applicable treatment. disclosure of interest: none declared. [o164] putkonen 6 , t. kuittinen 1 , j. pelkonen 2,7 , p. mäntymaa 7 , k. remes 6 , v. varmavuo 1 , e. jantunen 1 o094 o096 comparison of ric-allohct and autohct for 455 years old patients with acute lymphoblastic leukemia: an analysis from acute leukemia working party of the ebmt s united kingdom, 7 erasmus mc-daniel den hoed cancer centre double-blind, placebo-controlled phase 3 study of brentuximab vedotin in patients at risk of progression following autologous stem cell transplant for united states, 4 szent istvan & szent laszlo corporate hospital hematology & stem cell dept united states, 6 department of bone marrow transplantation & oncohematology, maria sklodowska-curie institute of oncology united states, 10 istituto nazionale dei tumori, milano, 11 irccs azienda ospedaliera universitaria united states introduction: the aethera trial was initiated to evaluate whether brentuximab vedotin (bv) can prevent progression in conclusion: bv improved post-asct pfs across all subgroups of pts. pts with more risk factors had the most benefit from bv. aes were consistent with the known safety profile of bv references: 1. lion t. adenovirus infections in immunocompetent and immunocompromised patients european guidelines for diagnosis and treatment of adenovirus infection in leukemia and stem cell transplantation: summary of ecil-4 adoptive transfer and selective reconstitution of streptamer-selected cytomegalovirus-specific cd8 þ t cells leads to virus clearance in patients after allogeneic peripheral blood stem cell transplantation lowest numbers of primary cd8 þ t cells can reconstitute protective immunity upon adoptive immunotherapy donor ebv status has introduction: epstein-barr virus (ebv) has been a major cause of post-transplant lymphoproliferative disorder after allogeneic stem cell transplantation (hsct). the impact of the donor (d) and recipient (r) serologic status on survival, relapse-free survival, relapse incidence, non-relapse mortality and incidence of graft-versus-host disease was unknown so far. objective: we analyzed the influence of the donor's and recipient's ebv status on allo-hsct transplant outcomes. materials (or patients) and methods: 11,364 allo-hscts performed due to acute leukemia ebv-seropositive donors also had no influence on relapse-free survival 57), and non-relapse mortality hsct recipients receiving grafts from ebv-seropositive donors had higher risk of acute gvhd after adjusting for confounders (donor type, conditioning, stem cell cource, patient age, gender match, t-cell depletion, year of transplant), d þ serostatus had an impact on development of acute gvhd p ¼ 0.09, and for cgvhd all patients engrafted, with a median time to neutrophil and platelet recovery of 18 days (13-45) and 16 days (9-100), respectively. post-hsct recovery of lymphocyte subsets was broad and fast: median time to cd34100/ml was 28 days, to cd44200/ml 41 days and to cd1940/ml 41 days. circulating t cells comprised naïve and memory subsets, with a recovery of cd31 recent thymic emigrants (rtes) from day 30. all patients had a significantly higher proportion of rtes at day 30 and 180 compared to their pre-hsct levels, suggesting an improvement in their thymic function after hsct. with a median follow-up for living patients of 15 months (5-24), the 1-year cumulative ci of nrm and relapse were 17% and 35%. three of the 11 acute leukemia relapses were hla-loss variants. notably, one was observed for the first time in all. ci of agvhd grade ii-iv and iii-iv at 6 months were 17% and 7%, while 1-year ci of cgvhd was 20% conclusion: myeloablative haplohsct with pbsc, pt-cy and sirolimus is a valid option for patients with aggressive/ advanced disease. the acceptable rates of gvhd and nrm as well as the favorable immune reconstitution profile open the way for combining it with novel immunomodulatory/ cellular therapies to improve dfs in patients at high risk for o142 factors determining the kinetics of disease relapse after allogeneic stem cell transplantation (allo-sct) for acute myeloid leukaemia (aml): a survey from the acute leukaemia working party of the ebmt c o143 a novel quantitative pcr approach targeting insertion/ deletion polymorphisms (indel-pcr) for chimerism quantification: finally high sensitivity and quantification capacity together a post-hematopoietic stem cell transplantation (sct) chimerism monitoring is important to assess engraftment, anticipate relapse and provide information on the development of graft versus host disease, facilitating therapeutic intervention. the aim of this study was to test the technical efficacy and clinical utility of a novel quantitative pcr approach targeting insertion/deletion polymorphisms of note, analysis of artificial mixtures provides evidence of significantly (z2 logs) higher sensitivity by indel-pcr (0.01%) than by str-pcr (1%, table 1). moreover, indel-pcr shows unprecedented quantification capacity (table 1). out of the 113 samples analyzed, 29 were positive and 6 negative by both methods, while 78 were positive only by indel-pcr (95% with o1% recipient). hematological relapse occurred in 5 patients, molecular relapse/persistence in 2 patients. all of them presented a positive indel-pcr (with increasing %r in 4/ 5) and a negative str-pcr result in the sample before relapse (table 2). the 12 patients in complete remission, although presented positive indel-pcr, showed stable or decreasing %r chimerism dynamics in 10/12 (data not shown). conclusion: this novel indel-pcr is a simple and accurate technique that, in comparison with the current gold standard str-pcr, shows very good concordance and provides higher rates of informative loci per patient, as well as unprecedented combined sensitivity and quantification capacity introduction: the immune recovery after cd34 þ cell selection is slow and patients tend to remain susceptible to opportunistic infections for several months after hsct. to hasten and improve post-transplant immune reconstitution broad repertoire various strategies under this approach, a rapid immunological reconstitution and very promising outcome have been reported in pediatric patients. with the aims of confirming these results even in adults, we have recently launched this programme and here we report our preliminar clinical data in 22 leukemia patients we have so far treated over the past 26 months. materials (or patients) and methods: twenty-two patients, median age 44 years (range 19-67), with aml (n ¼ 16), all (n ¼ 5), mds (n ¼ 1) entered the study. eight patients were in cr1, 3 in cr2, and 11 in advanced-stage disease at transplant. conditioning consisted of atg 1,5 mg/kg from day -13 to day -10, treosulfan 12gr/sqm from -9 to -7, fludarabine 30 mg/sqm from -6 to -2 and thiotepa 5 mg/kg on days -5 and -4. no patient received any post transplantation pharmacologic prophylaxis for gvhd. ten mg/kg g-csf was used to mobilize pbpcs from one-haplotype mismatched donors (3 mothers median cd4 þ cell/ml counts at 30, 60 and 90 days since the transplant were 36, 80 and 110, respectively. cmv antigenemia reactivation occurred in 6 cases (in 2, cmv serology was unfavourable). no patients has so far developed cmv disease. invasive fungal disease was prevented in all cases using l-amb-based prophylaxis over the neutropenic phase. overall,10 patients have so far died (7 relapse,3 non-hematologic causes). 12 survive (10 diseasefree,2 in early relapse) at a median follow-up of 13 months (range 2-24) (fig.1). conclusion: the infusion of ab/cd19-depleted grafts was safe and effective also in adult setting, resulting into rapid engraftment and fast immunological reconstitution haploidentical stem cell transplantation in very poor risk cytogenetics acute myeloid leukemia: results in 40 consecutive patients cytogenetic prognostic risk was defined according to the revised medical research counsil (mrc) classification, from diagnostic bone marrow samples with standard methods and in accordance with international system of human cytogenetics guidelines. os and disease free survival (dfs) were calculated using the kaplan-meier methods. results: median age of the patients at time of transplant was 50 years (range 28 to 67).cytogenetics:chromosome 7 abnormalities 16 pts, monosomal karyotype 3 pts and complex karyotyping 21 pts non relapse mortality (nrm) was 17% at 1 year after transplant.estimated lfs from day þ 30 after transplant was 34.5% and 28% at 3 years. conclusion: haploidentical stem cell transplant (haplo-sct) is a valid treatment option for the patients with very poor risk aml. references: 1 haploidentical, unmaipulated, primed bone marrow for pts with high risk hematologic malignancies haploidentical transplantation using t cell replete pbsc and myeloablative conditioning in pts with high risk hematologic malignancies who lack conventional donors kodera 7 on behalf of the worldwide network of blood and marrow transplantation o149 graft depletion of tcralpha/beta and cd19 in matched unrelated and haploidentical transplantation for severe aplastic anemia: high survival with low incidence of graftversus-host disease and transplant-related mortality m at enrollment all patients were on tp and paired analysis showed that mean (sd) maximum velocities had significantly decreased (po0.001) under tp:169 (46) cm/s vs 219 (26) cm/s at tp initiation. following hla-typing, 35 without genoidentical donor were included in the transfusion arm and 32 with genoidentical donor were transplanted in 6 hsct-centers. during the 12 months follow-up, no stroke was observed but one patient in the tp arm experienced a hyperammonemic reversible coma, without mri/mra alteration. in the hsct arm, all patients successfully engrafted, one grade ii and two grade iii acute gvhd, and no chronic gvhd were observed. complications were seizures (n ¼ 2), cmv (n ¼ 9) or ebv replications (n ¼ 5), hemorrhagic cystitis (n ¼ 4), aspergillosis (n ¼ 1), prolonged but reversible thrombopenia (n ¼ 2), transitory hemolytic anemia (n ¼ 1) granda ospedale maggiore policlinico, 5 hematology and bone marrow transplantation unit metabolic syndrome (ms) is defined as a clustering of five factors including (1) hyperglycaemia (2) hypertriglyceridaemia; (3) low hdl cholesterol; (4) hypertension; (5) obesity (high waist circumference or body mass index (bmi)). it is associated with raised risk of cardiovascular disease (cvd) and is increasingly recognised in patients after hct and revised guidelines for long-term hct survivors recommend screening for ms. previous studies have been small and the definition of ms variable, although harmonised criteria are now agreed materials (or patients) and methods: this was an ebmt approved cross-sectional, non-interventional study of consecutive hct patients aged 18 þ years and a minimum of 2 year post-transplant attending routine follow-up hct and/or late effects clinics in 9 centres. centres completed med c forms incorporating routine recording of the ms parameters (given above) as well as performance status (ecog); evidence of cardiovascular events using the harmonised definition of ms (at least 3/5 factors), the prevalence of ms was 47.2%. there was no difference in time since hct but there was a significant difference in prevalence by age at diagnosis, hct, follow-up (all po0.001 with increasing age). as expected, statistical differences (po0.001) between patients with and without ms were observed for bmi routine screening and early intervention may reduce the risk of cardiovascular events in hct survivors, and should ideally be tested in a randomised controlled trial setting. meanwhile, screening and management of reversible features of the metabolic syndrome should be robustly integrated within routine hct long-term follow-up care. references: disclosure of interest: none declared. introduction: the introduction of less toxic conditioning regimens for hematopoietic cell transplantation (hct) has led to an increase in eligible patients, although their benefit on patient's perceived wellbeing remains unclear. we aimed to prospectively study patients' quality of life (qol) and emotional wellbeing (ew) in consecutive hct recipients depression and anxiety (hads), and sleep quality (psqi) at pre-hct, at hospital discharge (hd) and at 3 months post-hct. results: out of 223 transplanted patients, 191 (86%) consented to participate. those who refused (n ¼ 32, 14%) more frequently had active disease at hct (31% vs. 21% for pr/cr, p ¼ 0.032) and/or had a prior hct among included patients (57% men; median age 53 83 received an auto-hct (n ¼ 79 at hd; n ¼ 52 at þ 3 m) 55 received an allo ric-hct (n ¼ 41 at hd; n ¼ 33 at þ 3 m) at baseline, clinically significant depressive symptoms were reported by 5% of the patients, with a slight increase (7%) at hd and at þ 3 m (p ¼ 0.058). again, there was a strong interaction between depressive symptoms and hct groups in the early post-hct phase (p ¼ 0.002); depression decreased in auto-hct after hd and, on the contrary, it increased in the same time-point in mac-hct (p ¼ 0.041). borderline differences were seen between auto-and ric-hct (p ¼ 0.062) but not between ric-and mac-hct (p ¼ 0.827). clinically significant anxiety was observed at baseline in 14% of the patients and significantly decreased at the time of hd (6%) and afterwards (5%) conclusion: mac-hct recipients reported the greatest impairment in the parameters studied; other variables such as gender, age and baseline ew/qol should be considered for specific psychological/clinical follow-up and eventual intervention unit of molecular and functional immunogenetics, 4 unit of hematology and bone marrow transplantation conflict with: scientific consultant of molmed s.p.a. o161 erbb2-chimeric antigen receptor (car) modified cytokineinduced killer (cik) cell intervention for refractory solid tumors after allogeneic stem cell transplantation m already at day 10 of culture, up to 90% of transduced cells showed surface expression of the erbb2-car (n ¼ 4). there were no significant phenotypic differences (cd3 þ /cd56 þ /cd4 þ /cd8 þ /tcr-a/b and tcr-g/d) between unmodified, empty control vector and erbb2-car transduced cik cells. erbb2-car cik cells efficiently lysed erbb2-overexpressing breast carcinoma cells (mda-mb 453) in a 3 hour short-term cytotoxicity (europium release) assay in vitro. compared with unmodified and empty-vector transduced cik cells e:t; 46.0% vs. 18.1% specific lysis, n ¼ 4; po0.019). long-term cytotoxicity analysis (16 h, brightfield imaging cytometry) demonstrated comparable results even at low effector to target ratios of 1:1 (36.3% vs. 11.8% specific lysis, n ¼ 3). comparable results for shortand long time cytotoxicity could be obtained for all other tested rhabdomyosaroma cell lines in vitro. conclusion: erbb2-car engineered cik cells are highly specific and efficient against erbb2-antigen expressing tumor cell lines in vitro. our experiments may help to develop an approach for improved treatment of patients with high-risk adoptive t-cell immunotherapy with hexon-specific thelper-1 cells as a treatment for refractory adenovirus infection after allo-sct -safety and efficacy results from a anna children's hospital university children's hospital, frankfurt, 5 dr. von haunersches kinderspital introduction: allogeneic hematopoietic cell transplantation (hct) offers the chance of cure for patients with nontransformed follicular lymphoma (fl) but is associated with the risk of non-relapse mortality (nrm). the aim of this study was to identify subgroups of fl patients who benefit from hct. materials (or patients) and methods: the minimum essential a data of 146 consecutive patients who received hct for fl between 1998-2008 were extracted from the database of the german registry ''drst''. diagnosis of fl was verified by contact with reference pathologists. results: the median patient age at time of transplantation was 48 years (range 29-71). prior to allogeneic hct 90/146 patients (62%) had undergone autologous hct. at time of hct, 110 patients (77%) had sensitive disease while 33 patients (23%) had chemorefractory disease (rd). engraftment was achieved in 99% of evaluable patients. day 100 nrm was 16%. the median follow-up of surviving patients was 9.1 years (range 3.6-15.7) . estimated 1, 2, 5, 10-year overall survival (os) was 67%, 60%,53%, and 48%, respectively. the corresponding estimates for efs were 63%, 53%, 47%, and 40%, respectively. 40% of the 33 patients with rd at time of transplantation survived long-term. of the n ¼ 116 patients with documented cr after hct only n ¼ 17 (15%) relapsed. only two late relapses (beyond year 3) were diagnosed among the 77 patients with a follow-up45 years. patients with chronic gvhd (irrespective of stage) had a lower risk of relapse, if transplanted in cr, and a higher chance to achieve cr, if transplanted in pr or with pd (no chronic gvhd: 19/48, chronic gvhd: 11/75, p ¼ 0.0019). therefore a reduced intensity conditioning approach might be considered in future prospective trials. hsct is most successful prior to leukemic transformation. given implications for treatment decisions and donor selection gata2 mutation screening should be performed on all patients with molecularly undefined mds and bmf disorders and potential related donors. disclosure of interest: none declared. pre-transplant weight loss predicts non-relapse mortality and relapse rates in patients with myelodysplastic syndrome after allogeneic stem cell transplantation a. radujkovic 1, * introduction: we have recently provided evidence that weight loss and minor metabolic changes prior to allosct were able to predict relapse and death of acute myeloid leukemia patients using data from two independent patient cohorts. this retrospective study investigated the influence of pre-transplant weight loss on the outcome of mds patients after allosct in three independent cohorts. materials (or patients) and methods: a total of 111 patients (59% male) with a median age of 52 years were included into the analysis. patients have been diagnosed with mds according to who criteria and received an allosct between 2000 and 2012 in three different german referral centers (heidelberg, dresden and berlin). weight data were raised from medical records by three independent researchers in three independent institutions. weight loss (expressed in percent) was calculated on the basis of recorded weight data at the time of allosct and the maximum weight in the time period of 3-6 months prior to allosct. the mds who subtype was ra(rs)/rcmd in 31 patients (28%), raeb1 in 30 patients (27%) and raeb2 in 49 patients (45%). according to ipss 34%, 45% and 21% of the patients were in the risk groups intermediate-1, intermediate-2 and high, respectively. the majority of the patients (n ¼ 72, 65%) was previously untreated. nineteen patients (17%) and 14 patients (13%) received hypomethylating agents and induction type chemotherapy prior to allosct, respectively. thirty-one patients (28%) received transplants from related donors, 59 patients (53%) from matched unrelated donors and 21 (19%) from mismatched unrelated donors. ninety-three patients (84%) received reduced intensity conditioning and 18 patients (16%) received standard myeloablative conditioning. survival times were measured from date of allosct. overall survival (os), relapse-free survival (rfs), relapse incidence and non-relapse mortality (nrm) were calculated from date of allosct to the appropriate endpoint. cox regression analysis was applied for os, rfs, relapse and nrm. relapse and nrm were considered as competing risks. results: estimated median follow-up at the time of analysis of surviving patients was 36 months. a total of 34 (31%) patients experienced weight loss 42% with 17 (15%) patients losing more than 5% weight in the period of 3-6 months prior to allosct. patient, disease and transplant characteristics did not differ between patients with weight loss (42%, n ¼ 34) and those without (n ¼ 77). in multivariate analysis, weight loss and donor type were independently associated with shorter os and rfs (po0.001 and po0.05, respectively). nrm was predicted by donor type (p ¼ 0.006), ipss (p ¼ 0.015) and pre-transplant weight loss (p ¼ 0.014) in multivariate analysis. furthermore, weight loss was also an independent predictor of relapse (cause-specific hr 11.52 95%ci po0.001) . in a mixed effect model with weight loss as outcome only ipss prior to allosct had significant impact on weight loss (p ¼ 0.046 introduction: hla-c-encoded kir ligands (c1/c2) have been identified as important factors for the outcome of unrelated allogeneic hsct: in a previous retrospective study cml recipients bearing at least one c2 ligand showed worse survival when compared to c1c1 recipients (hr 5.9 , po0.01), especially when peripheral blood progenitor cells (pbpc) were used or in advanced disease stages. these initial findings were confirmed in a second cohort in advanced aml/cml, (but not in mds or all/nhl) receiving pbpc. notably, hla-c allele matching contributed differentially to the transplantation outcome: it was beneficial in c1 patients, but was detrimental in c2 recipients (increased trm, hr 3.5, po0.012 ; increased relapse, hr 2.7, p ¼ 0.06). we hypothesized that c1 patients have a high frequency of immuno-competent nk cells (icnk) enabling eradication of residual disease due to the genetically hard-wired sequential acquisition of kir receptors during early reconstitution phase post hsct, with c1-specific nk cells emerging first. alloreactive t cells -resulting from hla-c mismatch-might thus not have an additional beneficial effect in c1 patients and might even be detrimental (e.g. increased gvhd), but may serve an important function in relapse control in c2 patients. the lack of disease control in hla-c-matched c2 patients would thus be explained by a combination of insufficient numbers of icnk cells in the early phase and the lack of alloreactive t cells later post hsct. consequently, this group exhibited poorest clinical outcome of all four groups defined by recipients kir ligands and hla-c allele matching in the investigated cohorts. materials (or patients) and methods: the aim of the present retrospective study was to determine the influence of hla-c allele matching on the background of hla-c encoded kir ligand status in a large patient cohort (n ¼ 7327, provided by cibmtr). statistical analysis was performed by cibmtr. patients received unrelated allografts between 1988 and 2009 , with the majority of patients after 2000 (70%). 30% of the recipients were younger than 30y, 67% younger than 40y. 70% of patients had early, 5% intermediate, and 25% advanced disease (aml 40%, all 21%, cml 22%, mds 17%). 56% received bone marrow, 44% pbpc. 80% received a myeloablative conditioning. endpoints were os, agvhd ii-iv and iii-iv, extensive chronic gvhd, relapse, dfs, and nrm. due to multiple comparisons and multiple endpoints, po0.01 was considered as significant. all models were adjusted for significant clinical covariates. stratification was used in cases of non-proportional hazards. patient-donor pairs were classified according the recipient hla-c kir ligand expression (c1c1, or c2 ( ¼ c1c2 or c2c2)) and the degree of the hla-c allele match: results: introduction: hepatic veno-occlusive disease (vod), also called sinusoidal obstruction syndrome, is a potentially fatal complication of hematopoietic stem cell transplantation (hsct). severe vod (svod), clinically characterized by multiorgan failure (mof), has been associated with a 480% mortality rate; it may develop in a substantial number of highrisk patients (pts). defibrotide (df), a sodium salt of complex single-stranded oligodeoxyribonucleotides, is thought to protect injured endothelium and to restore thrombo-fibrinolytic balance. in a phase 3 trial in svod, df improved complete response rate and survival at day þ 100 post hsct vs historical controls, with a favorable safety profile. in the european union, df is approved for treatment of severe hepatic vod in hsct therapy in adults and children. in the us, df is available through an expanded access, protocol-directed treatment ind (t-ind) collecting data on safety/efficacy in children and adults with svod and non-severe vod post hsct or post chemotherapy (ct). the original t-ind protocol required vod diagnosed by baltimore criteria (total bilirubin z2.0 mg/dl with z2 of hepatomegaly, ascites, or 5% weight gain) plus mof (renal and/or pulmonary) following hsct; the study was amended to include non-severe vod (ie, materials (or patients) and methods: in a single center retrospective study 382 patients who underwent allogeneic hematopoietic stem cell transplantation (hsct) for various diseases (51% acute leukemia) were genotyped for cyp 1b1 (c432g) expression and their influence on outcome was analyzed. genotyping of cyp 1b1 (c432g) was performed by real-time pcr.results: 169 patients (44%) were genotyped as homozygous wild-type (wt) gene c/c, 157 (41%) as heterozygous genotype c/g and 56 (15%) as homozygous gene mutated g/g. calculated genotype frequencies did not differ from that reported earlier by other studies for caucasians. patients' demographic and treatment characteristics showed no difference between the three groups except that cyp 1b1 cc was more common in females 52% than in males 38% (po0.02).five-year estimate for overall survival (os) was 58 þ 4% for the cc group and 48 þ 3% for the c/g-and g/g groups (po0.036). surprisingly, this difference was primarily evident in males (po0.009), where the group with cyp gene mutations did significantly worse (3-year estimate for os: 65 þ 5% vs. 47 þ 4%), whereas it was virtually absent in females (p ¼ 0.99). trm and rr were higher for the group with mutated genes in regard to the group with wt gene (although not significant). 2006 . in phylogenetic analysis of the e3 gene, a two-step pcr amplification of almost the entire adenovirus e3 gene was performed using primers designed from the known sequence of the hadv a31 reference strain (am749299.1). results: all 9 patients had been admitted to the ward, but the two last patients (patient 8 and 9) had no timely connection to other known hadv a31 cases ( figure) . in addition, four of the patients (1, 5, 6 and 7) made visits to the out-patient clinic on the same day as one or several other hadv positive patients.sequencing the hexon gene resulted in 100% homology between the patient samples but also to the reference strains of hadv a31 (accession number ab330112. 1 (cmv) is an important cause of morbidity after allogeneic hematopoietic stem cell transplantation (hsct). the latent virus reactivates in immune-compromised patients, due to both post-hsct immunosuppressive therapy and impaired t-cell reconstitution/function. here we report the impact of mismatches in hla-molecules between donor and recipient on cmv-reactivation and cmv-specific immune reconstitution. materials (or patients) and methods: this retrospective study included 752 patients, who received a transplant from a matched related donor (mrd n ¼ 234), matched unrelated donor (mud n ¼ 384), mismatched unrelated donor (mmud n ¼ 115) or mismatched related donor (mmrd n ¼ 19). hlatyping (10/10) of patients and donors was conducted via highresolution multiplexed pcr. blood samples were routinely monitored for cmv pp65 antigen expressing cells per 400,000 leukocytes. cmv-cytotoxic lymphocytes (cmv-ctl) reconstitution was analyzed in the blood from 246 patients at days þ 50, þ 100, þ 200, þ 300 after hsct, using 6 hla-cmv tetramers. results: the fisher's exact test was used to analyze the data. the outcome was that hla mismatch (class i or ii) showed significant influence on recurrent (multiple) cmv reactivations (mcmv-r) (po0.001). we analyzed the relative risk (rr) in the subgroups with different levels of hla-matching for 1cmv-r and mcmv-r. the group transplanted from mrd served as reference (ref. ) . shortly, we found significantly higher risk for mcmv-r in the mmud group (rr 2.6, 95% ci 1.63-4.15 , p ¼ 0.0001). in the mrd 24 (10%) patients had mcmv-r, while in the mud 59 (15%), in the mmrd 3 (16%) and in the mmud group 31 (27%) had mcmv-r. furthermore, we investigated the mean numbers of cmv-ctl/ ml of blood in the groups with different levels of hla-match. we divided cmv-ctl levels into 3 ranges: o1, 1-10 and 410 cmv-ctl/ml. in the mmud group we observed a trend for an increased risk for the lack of cmv-ctl (25 (48%) patients; rr 1.5 , 95% ci 0.96 to 2.38, p ¼ 0.07) compared to 21 (32%) patients from the mrd group. significantly less patients (17; 33%) had more than 10 cmv-ctl from the mmud group (rr 0.6, 95% ci 0.39 to 0.95 thus the focus of the present study was the selection of hadvstreptamer þ t-cells and ebv-streptamer þ t-cells.materials (or patients) and methods: cells from leukapheresis healthy donors were prepared in large (1 à 6 â 10 9 ) and small (25 x 106) cell batches. whereas the larger batch was directly labelled with streptamers to select hadv and/or ebvspecific t-cells (large-scale), the smaller batch was used to generate in vitro virus-specific t-cell lines before streptamerlabelling for streptamer selection (small-scale). isolation of hadv-and/or ebv-specific t-cells was performed using the clinimacs device.results: the purity of hadv-streptamer þ t-cells among cd3 þ cells, obtained from large-scale selection was only 7.6%, but reached up to 56% when hadv-and ebvstreptamers were applied simultaneously. a further increase in purity of hadv-specific t-cells reaching up to 98% was achieved by small-scale selection. all final products fulfilled the microbiological and chemical release criteria. ifn-g-response indicating functional activity was seen in 6/9 hadv and 2/3 ebv large-scale selections and in 2/3 hadv small-scale selections.conclusion: the use of hadv-streptamers for clinical applications is feasible particularly when combined with other streptamers or when performed after a previous in vitro expansion period. in this cohort of 149 t-cell replete haplo hscts using post-transplant high-dose cyclophosphamide, we found that a higher intensity of conditioning (mya or ric vs non-mya) as well as the use of more immunosuppressive calcineurin inhibitor (fk vs csa) were both significantly associated with a higher incidence of pv-hc. results: in cd34 þ lin -cd10cells, 1609 probes were deregulated between patients without agvhd and patients with agvhd (1560 of this probe were up-regulated and 49 were down-regulated, po0.05, fold change41.5) . in cd34 þ lin -cd10 þ cd24progenitors, 987 probes were deregulated between patients without agvhd and patients with agvhd (941 of this probe were up-regulated and 46 were downregulated, po0.05, fold change41.5). 273 probes were deregulated in both cd34 þ lin -cd10 þ cd24and cd34 þ lin -cd10populations. genes from ribosome protein biogenesis, translation machinery (eef1d, eef1g, eif3k) and cell cycle (ccnd1, cdk6) were over-expressed in cd34 þ lin -cd10 þ cd24and in cd34 þ lin -cd10populations from patients without agvhd compared with those from patients affected by agvhd and from healthy donors. expressions of genes from the oxidative phosphorylation metabolic pathway (ndufs2, sdha, atp5a1) and genes involved in stress resistance (btg2, mgst3, hpx) were specifically increased in cd34 þ lin -cd10 þ cd24lymphoid progenitors and not in cd34 þ lin -cd10non-lymphoid progenitors from patients without agvhd compared with patients suffering from agvhd and from healthy donors. we show for the first time that circulating lymphoid t-cell progenitors undergo profound changes in metabolism favoring energy production and response to stress after allo-hsct in humans. these mechanisms are abolished in case of agvhd, indicating a persistent cell-intrinsic defect in addition to the impact of agvhd on the bone marrow environment. disclosure of interest: none declared. introduction: post transplant interventions such as donor lymphocyte infusion (dli) or administration of pharmacological agents, represent important novel strategies with the potential to reduce the risk of disease relapse after allo-sct in acute myeloid leukaemia (aml). such approaches are critically dependent on timely intervention post-transplant but despite this the factors determining the kinetics of disease relapse in patients allografted for aml have not been defined. materials (or patients) and methods: 1052 adults who received an allo-sct for aml in first complete remission (cr1) between 2000 and 2012 were studied. 544 patients were transplanted using a sibling donor and 508 from an adult matched-unrelated donor. 538 patients received a myeloablative conditioning (mac) regimen and 514 a reduced intensity (ric) regimen. a series of landmark analyses were performed at 3, 6 and 12 months in order to identify prognostic factors of relapse for patients alive and well at the beginning of each time interval. the probabilities of relapse were calculated by using the cumulative incidence estimator to accommodate for death as a competing risk. factors predicting relapse were studied using cox regression model including time dependent variables. a backward stepwise procedure was used for variable selection. results: with a median follow-up of 26 months, 244 patients relapsed. the 3 year cumulative incidence of relapse was 26% [95%ci: 23-28]. overall 84% of patients destined to relapse did so within the first year post-transplant. the overall factors predicting disease relapse for the whole population were more than one course of chemotherapy to achieve cr1, flt3 itd positivity, adverse risk cytogenetics, shorter interval from cr1 to transplant. the occurrence of acute gvhd grade ii or greater (p ¼ 0.05) and chronic gvhd (p ¼ 0.03) were both associated with a lower risk of relapse. using landmark analyses the factors determining relapse at different stages post transplant were observed to differ. in the first 3 months post-transplant the significant factors determining relapse risk were: patient age (p ¼ 0.03), prolonged interval from diagnosis to cr1 (p ¼ 0.05), flt3 itd positivity (p ¼ 0.002), adverse risk cytogenetics (p ¼ 0.02) and use of in vivo t cell depletion (p ¼ 0.003). the only factors observed to determine relapse risk between 3 and 6 months post-transplant were introduction: the number of haematopoietic stem cell transplants being performed worldwide is increasing as is interest in side effects. despite the increase in published data on late effects in the last decade, data on very long term survivors (425 years) is lacking. in this study we describe the outcome of all the patients transplanted at our centre more than 25 years ago. materials (or patients) and methods: between june 1979-january 1990, 216 patients had received allogeneic sct for haematological malignancies at the hammersmith hospital. most patients (180/216) were transplanted for cml with cy/ tbi conditioning and the majority were in chronic phase at sct (n ¼ 140). at the time of analysis in december 2014, 151/216 (70%) patients had died. of 65 presumed survivors, 14 patients had moved abroad and an additional 13 patients were considered lost to follow up as they had had no contact with our centre within 5 years of the study. of the remaining 41 patients, detailed follow up information was available for 34. results: the majority of deaths (94/151) occurred within 2 years of sct. a further 27 patients died between 2-10 years after sct the most frequent cause of death being relapse (17/ 27) and infection (5/27). between 10-20 years after sct there were 18 deaths; the most frequent causes were relapse (n ¼ 4), second malignancy (n ¼ 4) and gvhd (n ¼ 4). between 20-35 years there have been 9 deaths and the most frequent causes were second malignancy (n ¼ 3) and respiratory (n ¼ 2). the latest recorded relapse was at 14.8 years. for 34 survivors for whom we had detailed follow up information the median follow up time was 29y 10 months (range 25y 8 months -35 years 7 months). the median age at follow up was 61y (range 45-80). 11/34 patients had had a diagnosis of cancer at the following sites: skin (bcc or melanoma) n ¼ 5, oral or tongue n ¼ 3, oesophagus n ¼ 1, breast n ¼ 1 and a further patient had had testicular and bladder cancer. 16/34 patients had dyslipidaemia and 14/34 were being treated for hypertension. 5/34 were diabetic and 12/34 were hypothyroid. of 15 male patients, 3 had low testosterone levels requiring treatment. 8/34 had vascular complications including three with ischaemic heart disease one of whom also had pvd, two with venous thrombosis, one with a tia and two patients with renovascular disease. dxa data was available for 25/34 of these patients and bmd was recorded as low (osteopenia or osteoporosis) in 12/25. conclusion: we conclude that late deaths more than 20 y after sct are more likely to be due to second malignancy than relapse. appropriate screening identifies a large number of abnormalities in the surviving patients most of which are amenable to treatment. disclosure of interest: none declared. the aim was to evaluate the survival and late toxicities, defined as any disease condition other than lymphoma occurring after at least 6 months after asct. the median patient age at asct was 52 years (range, 20-70). all patients relapsed after at least one chemotherapy line (previous treatments range 1-8), 26% of them received radiotherapy. at asct, 76% of patients were in cr, 15% in pr, 1% stable, 8% in progression. full dose beam was given to 87% of patients while 13% received dose reductions for comorbidities. results: the median follow-up was 5.4 years (range 0.5-12.2) . the 5-year os and pfs were 81 and 69% (median not reached for both). the non-lymphoma-associated mortality was 5%, 8% and 9% at 3, 5 and 7 years of follow-up. the os was impacted in multivariate analysis by disease status before asct (p ¼ 0.001, hr 2.2, ci95% 1.4-3.6 ), and radiotherapy (p ¼ 0.017, hr 6.5, ci95% 1.4-30.7) . pfs was impacted by female gender (p ¼ 0.029, hr 0.4, ), pre-transplant disease status (p ¼ 0.001, hr 1.9, ), and radiotherapy (trend, p ¼ 0.064, hr 2.7, . none of the factors analyzed impacted late non-lymphoma-associated mortality, except for a trend given by age (p ¼ 0.075). late toxicities after beam asct occurred in 61% of patients, and included infection (32% -most frequently pulmonary), hypogammaglobulinemia (30%), pulmonary complications (21% -mostly reduction of pulmonary function tests scores), metabolic syndrome (17%), cardiovascular complications (12%), second tumors (10%), hypothyroidism (8%), diabetes (5%), chronic kidney failure (4%), hepatitis b reverse seroconversion (2%) and ocular complications (1%). the cumulative incidence of second tumors was 1, 6, and 10% at 3, 5, and 7 years of follow-up, and reached a plateau of 16% at 10 years of follow-up. 17 patients had a second cancer, of whom 12 had a solid tumor (skin [4] , colorectal, prostate, lung [2 each], breast and oropharingeal [1 each]), 5 a hematologic tumor (secondary mds or aml [4] or nhl [1] ). age (p ¼ 0.013, hr 1.5 per year, ci95% 1.0-2.1) , and male gender (p ¼ 0.043, hr 0.05 favorable for female sex, ci95% 0.0-0.9) , increased risk of a second tumor. of 13 patients who died without lymphoma, 6 died of second tumors, 2 died of cardiovascular complications, 2 of late infections, and 3 for other causes. in multivariate logistic regression, the incidence of second tumors was associated with age (p ¼ 0.04), and there was a trend for patients receiving radiotherapy for late cardiovascular complications (p ¼ 0.07). conclusion: beam conditioning is associated with a 61% crude incidence of late effects, mostly infections, hypogammaglobulinemia, and pulmonary complications. the most important preventive measures for late mortality could be the screening for cancer, especially for older patients, screening for heart disease particularly for patients receiving radiotherapy, and prompt and aggressive treatment of late infections. disclosure of interest: none declared. introduction: the advent of highly active antiretroviral therapy (haart) in 1996 had led to a suppression of hiv viral load, to an improved immune function resulting in a significant reduction of opportunistic infections and hiv related morbidity and mortality. consequently, more intensive treatments, including autologous stem cell transplantation (asct), have been extended also to the hiv-positive population. however, in the literature data are scarce concerning the long-term events (incidence of lymphoma relapse, of second cancers and aidsdefining conditions) in hiv-positive patients (pts) affected by relapsed lymphoma who underwent asct. materials (or patients) and methods: we treated 36 hivpositive pts affected by relapsed/refractory lymphomas with asct consecutively in our cancer center. ten pts died during or early after asct due to progressive disease (4 pts), chemotherapy toxicity (1 pt) and infection (5 pts). we analyzed the post-transplantation long-term data of 26 hiv-positive lymphoma pts, reaching a complete response after asct. eighteen pts were male (69%) and 8 pts were female. our cohort of pts included 17 non-hodgkin's lymphomas (nhl) and 9 hodgkin's lymphomas (hl), respectively. twenty-two pts (85%) received one, and 4 pts received two second-line chemotherapy regimen before asct, respectively. the majority of the pts were submitted to a single (beam conditioning regimen) and only two pts to a tandem asct procedure (high dose melphalan followed by beam conditioning regimen). all pts received haart concomitantly to cancer treatment. results: two pts experienced a lymphoma relapse, after 4.27 and 3.08 years from asct, respectively. three pts presented with a secondary malignancy (1 pt an anal squamous cell cancer, 1 pt a squamous cell carcinoma of the larynx and 1 pt a cin2, respectively), with a median time of 3.01 years from asct. eight pts had opportunistic infections (oi): 2 pts developed a pneumocystis carinii pneumonia, 1 pt a cytomegalovirus pneumonia, 1 pt a mycobacterium avium complex pneumonia, 1 pt a herpes simplex chronic ulcer, 3 pts cutaneous relapsing herpes zoster, respectively. the median time of oi appearance was 0.25 years (iqr: 0.11-2.33 ). two pts died: one of lymphoma relapse, the other of car accident. with a median of 6-years follow up (iqr: 4.55-9.87 ) the os and pfs of the entire sample of pts were 91% and 36% at 10 years, respectively. our results may be summarized as follows: 1) 24 out of 26 pts are still alive and in long-term complete remission after asct. these data confirm the long-term efficacy of asct in hiv-positive pts affected by relapse/ refractory lymphoma. 2) the appearance of oi is earlier than that of second malignancies after asct. 3) the secondary malignancies developed by our pts are non-aids-defining cancers, in agreement with the increased incidence reported by the literature in the haart-era and at least two cases are linked to a viral pathogenesis (hpv for both anal cancer and cervical cancer precursor lesion). 4) both oi and second malignancies in our pts series were successfully managed and cured and the only long-term death occurred due to lymphoma relapse. disclosure of interest: none declared. introduction: allogeneic hematopoietic cell transplantation (hct) is an effective therapeutic option for high risk hematological malignancies; 80% of those who survive the first 2 years are expected to become long-term survivors. the prevalence of chronic health conditions approaches 75% among hct survivors and that for severe or life-threatening conditions exceeds 20%. 1 materials (or patients) and methods: a standardized followup of hct survivors is applied at our center, according to jacie standards. here we report the analysis of data collected between nov 2013 and nov 2014 in 249 adult patients (ptsmedian age at follow-up 54y -r19-81) who underwent an hct between 1992 and 2013 at our institution. data on 7 items were prospectively collected in an institutional database. a written consent was given by pts allowing the use of medical records for research in accordance with the declaration of helsinki. results: overall 40% of pts received an haplo, 30% a mud and 30% a match related hct; 13 pts deceased in the last year (7 because of disease relapse, 4 of late major infectious complication, 2 of second cancer). at a median follow-up of 4y (r1-22; cumulative follow-up 1277y) we observed: -chronic graft-versus-host-disease (c-gvhd): at a median follow-up of 3y (r1-21) 61 (25%) pts are presenting c-gvhd features. according to nih 2004 consensus criteria 15 cases were classified as mild, 25 moderate, 21 severe. median number of involved organs 3 (r1-4), 32 pts were experiencing skin lesions, 38 eyes impairment, 23 mouth alterations.-late infectious manifestation: 54 (22%) pts present late infections, 4 pts deceased. pneumonia was reported in 22 pts, varicella zoster virus reactivation in 12, encephalitis in 5 (3 virus related, 2 toxoplasma related), hepatitis in 4, ebv reactivation in 2.-second cancers: second malignancies were diagnosed in 32 (13%) pts, 5 pts are actually under work-up for diagnosis. nonmelanoma skin cancer was the most frequent diagnosis (17 cases); 3 pts were diagnosed with cervix cancer, 2 with prostate cancer, 2 with lung cancer and 2 with bladder cancer. single cases of thyroid, parathyroid, colon, gastric, kidney, larynx, endometrial and breast malignancies were also reported. all pts were treated according to standard policy for general population, 30/32 are alive.-thyroid dysfunction: 38 (15%) pts presented overt hypothyroidism.-cardiovascular diseases: arterial diseases were reported in 17 pts, atrial fibrillation in 5 and cardiomyopathy in 2 pts -overall 10%.-metabolic syndrome (ms): 84 (34%) pts were presenting features of ms (3/5 features among hypertension, dyslipidemia -raised triglycerides and lowered high-density lipoprotein cholesterol-, raised fasting glucose and central obesity). -secondary hemosiderosis: iron overload was documented (with mri and blood parameters) and treated in 38 pts (15%). according to donor source no difference were observed (chisquare test -p ns) except for higher incidence of moderate/ severe gvhd incidence in match related hct (p 0.0097) as compared to alternative hct. conclusion: hct survivors are at a defined relevant risk of developing long-term complications that have a direct impact on quality of life, morbidity and mortality. 1 introduction: long-term survivors of allogeneic hsct now form an expanding and unique patient population with often complex physical and psychological late effects (le) and associated unmet needs. despite international guidelines 1 , optimal delivery models of le services are unclear from clinical, organisational and economic viewpoints. materials (or patients) and methods: in order to scope current models of care for le service delivery within the uk, we undertook a survey of the 27 nhs adult allogeneic hsct centres during 2014. centres were invited to participate in an online survey composed of 30 questions examining service organisation, multi-disciplinary team (mdt) provision, access to other specialist services and patient engagement. results: a 100% response rate was achieved from programme directors or delegated specialist staff. around half of centres also treated patients r18 years and all centres had achieved or were working towards jacie accreditation. in 480% of centres, the le service was led, coordinated and delivered by consultant medical staff, with the remainder being nurse-led. most centres (490%) provided follow-up in a dedicated allograft or le clinic for the first year, but thereafter attrition resulted in b50% of patients being followed after 5 years, and b30% after 10 years. most centres had easy access to medical specialities necessary for le management, but specialist interest in long-term hsct complications was uncommon. only 18% of centres had access to a le mdt, often limited to patients o25 years. despite specific jacie competency s91 standards, a third of centres had held no le educational event in the previous three years. most centres (70%) had an sop for long-term monitoring and le management, with the focus predominantly on physical le with only 28% including a formal psychological screening assessment. only 39% of centres had audited the performance of the sop. screening for endocrinopathies, iron overload and cardiovascular complications was near universal, but access to mammography and cervical smear testing was more limited. revaccination rates were high, but only 23% of centres routinely tested antibody responses. despite recommendations, most (59%) centres never used standard templates 2 to communicate le risk to gps or referring consultants. only 41% of centres had a patient support group accessible to hsct survivors with equivalent numbers having undertaken patient satisfaction surveys related to le service provision. the most commonly perceived barriers to implementation of le services were funding of psychological and other clinical staff and extra investigation costs.conclusion: this survey has demonstrated variation and limitations in the provision of long-term follow up of allogeneic hsct survivors within the uk nhs. although patients are seen in specialist clinics and have access to other specialities, there are limitations in sustaining long-term screening, mdt working, education, audit and patient engagement, as well as perceived barriers to resourcing staff and investigations. further work is warranted to optimise effective, sustainable and affordable models of care for delivery of le services in this expanding specialised patient population. references: 1. majhail et al. biol 2 hematology department, hospital sant pau, 3 hematology department, hospital vall d'hebrón, 4 epidemiology department, hospital de sant pau, 5 hematology department, hospital del mar, barcelona, spain in vivo and to unravel the requirements for their long-term persistence directly in humans. materials (or patients) and methods: we studied the immune system of 10 patients who underwent haploidentical hsct and infusion of donor lymphocytes transduced to express tk suicide gene (median dose: 1.9x10 7 cells/kg) for high-risk hematologic malignancies. in case gvhd, proliferating tk-cells were promptly eliminated upon ganciclor (gcv) administration with complete resolution of the adverse reaction without immunosuppressive treatments. results: at a median follow-up of 7 years after hsct (range 2-12.3), a complete recovery of nk cells, b lymphocytes and ab or gd t cells was observed. the cd8 þ and cd4 þ t cell compartment of tk patients were characterized by level of naïve and memory cell comparable to age and sex matched healthy controls. tk-cells were detected in all patients, at low levels (median ¼ 4 cells/ul), even in patients treated with gcv. ex vivo selection of pure tk-cells confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory t cells to persist for years. importantly, gcv sensitivity was preserved in long-term persisting tk-cells, independently from their differentiation phenotype. longitudinal follow up revealed that tk-cells circulated in patients at stable levels and displayed a conserved phenotype comprising effector memory (t em ), central memory (t cm ) and stem memory (t scm ) t cells. the low level of ki-67 positivity suggested the maintenance of a pool of gene-modified memory cells through homeostatic proliferation. polyclonality was demonstrated by sequencing among tk-cells of thousands of diverse tcrs with a broad usage of v and j alpha and beta genes. the number of tk-cells persisting at the longest follow-up did not correlate with the amount of infused cells, but instead with the peak of tk-cells measured within the first months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory t cells. accordingly, we documented the persistence of cmv and fluspecific tk-cells only after post-transplant cmv reactivation or after flu infection. we observed that the number of infused t scm cells positively correlated with early tk-cell expansion and with their long-term persistence, suggesting that t scm might play a privileged role in the generation of a long-lasting immunological memory. conclusion: after infusion, gene-modified memory t cells persist for up to 12 years within a physiological immune system. antigen exposure and a t scm phenotype were associated with long-term persistence of infused tk-cells. further studies on tk-cell tcr repertoire and vector integrations are currently being performed to elucidate the in vivo dynamics of infused memory t cells. use of zoledronic acid after tcrab/cd19-depleted haploidentical transplantation to enhance gd t cells anti-leukemia effect p. merli introduction: hsct is a potentially curative option for a number of malignant disorders; however, up to 30% of patients lack a suitable hla-matched either related or unrelated donor. in order to optimize haploidentical transplantation, we recently developed a new method of graft manipulation (i.e. tcrab/cd19 negative selection), which retains in the final product large numbers of effector cells, namely nk and tcrgd lymphocytes. relapse remains the main cause of treatment failure. based on preclinical data showing bisphosphonates-mediated improvement of tcrgd cells-blast killing through accumulation of phosphoantigens, we started a prospective trial based on post-transplant infusion of zoledronic acid, with the aim of enhancing tcrgd cells anti-tumor effect. materials (or patients) and methods: enrolled in the study were 35 pediatric patients (median age at transplantation 10.3 years, range 1-18) affected by either all and aml (26 and 9 patients, respectively) at very-high risk for relapse/trm due to disease status (cytogenetic/molecular characteristics, lack of remission or previously failed hsct). all of them underwent a tcrab/cd19-depleted hsct from an hla-haploidentical donor (one of the two parents). according to the model of kir/kir ligand mismatch, 13 patients were transplanted from an nk-alloreactive donor. the median number of infused gd þ t cells was 7.9 x 10 6 /kg (range 0.9-42.7) . zoledronic acid was administered monthly at the dose of 0.05 mg/kg per dose (maximum dose 4 mg), starting from day þ 30 after transplantation. patients underwent zoledronic acid infusions, together with oral calcitriol and calcium supplementation, in the outpatient unit. results: a total of 102 infusions were administered with a mean of 2.9 infusions per patient (range [1] [2] [3] [4] [5] ; only one episode of symptomatic hypocalcemia (at first administration) occurred and was rapidly corrected with parenteral calcium supplementation. none of the patients experienced de novo onset or worsening of previously developed acute gvhd, this finding supporting the observation that gd t-lymphocytes do not cause gvhd. in the study period, six patients relapsed and 2 died due to infectious complications. with a median follow up of 9 months (range 4-22) the 2-year kaplan-meyer estimate of os and lfs were 88.1% (se 6.6) and 62.2 (se 10.1), respectively ( figure 1a) . the cumulative incidence of relapse and trm were 31.3% and 6.6%, respectively (figure 1b) . repeated infusions of zoledronic acid (i.e. more than 3) seem to offer an advantage in terms of dfs (87.5% vs 48.6%, p ¼ 0.13), although the difference was not statistically significant (figure 1c and d) .conclusion: these data suggest that the infusion of zoledronic acid after tcrab/cd19-depleted haplo hsct is safe. repeated infusions of zoledronic acid after haploidentical hsct seems to be more effective in preventing leukemia recurrence. more patients and a longer follow-up are needed to establish the efficacy of this approach. disclosure of interest: none declared. inducible t-cell receptor expression in precursor t-cells for leukemia control introduction: the co-transplantation of hematopoietic stem cells (hs) with those that have been engineered to express tumor-reactive t cell receptors (tcrs) and differentiated into precursor t cells (prets) may optimize tumor reduction. since expression of potentially self-(tumor-) reactive tcrs will lead to negative selection upon thymic maturation, we investigated whether prets forced to express a leukemia-reactive tcr under the control of a tetracycline-inducible promoter would allow timely controlled tcr expression thereby avoiding thymic negative selection. materials (or patients) and methods: using lentiviral vectors, murine lsk cells were engineered to express a tetracyclineinducible tcr directed against a surrogate leukemia antigen. tcr-transduced lsk cells were co-cultured on t cell development-supporting op9-dl1 cells to produce prets. lethallyirradiated b6/ncrl recipients received syngeneic t celldepleted bone marrow and 8 â 10 6 syngeneic or allogeneic (b10.a) tcr-engineered prets. an otherwise lethal leukemia cell (c1498) challenge was given 28 days later. results: after in vivo maturation and gene induction up to 70% leukemia free survival was achieved in recipients of syngeneic tcr-transduced prets (po0.001) as shown in figure 1 a. importantly, transfer of allogeneic gene-manipulated prets increased the survival of recipients (po0.05) without inducing graft versus host disease (gvhd). nontransduced prets provided significantly lower leukemia protection being not significantly superior to the pbs controls. the progenies of engineered prets gave rise to effector and central memory cells providing protection even after repeated leukemia challenge. in vitro transduction and consecutive expansion of mature t cells required at least 40 â 10 6 cells/ recipient to mediate similar anti-leukemia efficacy, risking the development of severe gvhd if of mismatched origin, and providing no long-term protection. importantly, while transgene induction starting immediately after transplant forced cd8 þ t cell development and was required to obtain a mature t cell subset of targeted specificity, late induction favored cd4 differentiation and failed to produce a leukemiareactive population due to missing thymic positive selection. conclusion: co-transplanting tcr gene-engineered prets is of high clinical relevance since small numbers of even mismatched hs can be transduced at a reasonable cost, expanded in vitro, stored if needed, and provide potent and long lasting leukemia protection. disclosure of interest: none declared. key: cord-023364-ut56gczm authors: nan title: education day monday: plenary session 1 monday: parallel sessions date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00651.x sha: doc_id: 23364 cord_uid: ut56gczm nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-023354-f2ciho6o authors: nan title: tuesday plenary session 3 tuesday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00654.x sha: doc_id: 23354 cord_uid: f2ciho6o nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-023346-8sqbqjm1 authors: nan title: monday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00652.x sha: doc_id: 23346 cord_uid: 8sqbqjm1 nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-009567-osstpum6 authors: nan title: abstracts oral date: 2008-04-23 journal: am j transplant doi: 10.1111/j.1600-6143.2008.02254.x sha: doc_id: 9567 cord_uid: osstpum6 nan abstracts evl (target 3-8ng/ml), aza or mmf with standard (sd) or reduced (rd) csa. data at 6 months post-tx is presented here. results. the proportion of patients with cmv events, including serious adverse events and laboratory evidence of cmv infection, has remained low and broadly similar following introduction of cc administration of evl and use of concomitant rd-csa. cmv syndrome and cmv organ involvement occurred in <3% of recipients receiving evl in each of the studies. the highest rates of all cmv parameters occurred with mmf + sd-csa or aza + sd-csa. conclusion. the low incidence of cmv infection and cmv-related events observed in heart tx patients receiving fd-evl and sd-csa as de novo immunosuppression has been maintained in newer regimens of cc-evl with rd-csa. cmv syndrome and cmv organ involvement are rare with evl-based immunosuppression after cardiac tx. the low rate of cmv infections in evl-treated patients may contribute to improved long-term outcome and a lower incidence of cav; confirmatory data are awaited. fibronectin-α 4 β 1 interactions enhance p38 mapk phosphorylation and metalloproteinase-9 expression in cold liver ischemia/reperfusion injury. sergio duarte, 1 xiu-da shen, 1 takashi hamada, 1 constantino fondevila, 1 ronald busuttil, 1 ana j. coito. 1 1 the dumont ucla transplant center. expression of endothelial fibronectin (fn) is an early event in liver ischemia/reperfusion (i/r) injury. we have recently shown that cs1 peptide facilitated blockade of fn-α4β1 leukocyte interactions regulates metalloproteinase-9 (mmp-9) expression in steatotic orthotopic liver transplants (olt). this study tests the function of the cs1 peptide therapy upon mmp-9, and further dissects putative mechanisms, in an alternate model of cold ischemia/reperfusion (i/r) injury. methods and results: cs1 peptides were administrated through the portal vein of sprage-dawley (sd) rat livers before and after 24 h cold storage (500 µg/rat). sd recipients of olts received an additional dose of cs1 peptides 1h post-olt. cs1 therapy significantly increased the 14d olt survival rate (100% vs. 50%, n=8/gr, p<0.005). cs1 peptides reduced sgot levels (u/l) at 6h (1413±420 vs. 2866±864 p<0.008) and 24h (1350±142 vs. 4000±1358, p<0.006) post-olt. cs1 treated olts showed good preservation of lobular architecture, contrasting with severe necrosis and sinusoidal congestion in controls. moreover, cs1 treated livers were characterized by a profound decrease in t (31±3 vs. 64±8, p<0.0002), nk (19±2 vs. 41±3, p<0 .0003) and ed1 (21±7 vs. 57±16, p<0.008) cells as early as 6h after i/r. neutrophils, as indicated by mpo activity (0.48±0.02 vs. 3.18±0.94, p<0.02) were depressed by cs1 therapy. this correlated with decreased mrna expression of tnf-α (0.032±0.04 vs. 0.83±0.26, p<0.005) and cycloxygenase-2 (0.8±0.1 vs. 2.2±0. 6, p<0.05) . leukocyte transmigration is dependent upon adhesive and focal matrix degradation mechanisms. mmp-9, which is inducible and expressed by infiltrating leukocytes, was profoundly depressed in 6h cs-1 peptide treated olts, at both mrna (0.1±0.1 vs. 0.5±0.2, p<0.03) and protein (0.15±0.07 vs. 0.7±0.14, p<0.03) levels. moreover, mmp-9 activity evaluated by zymography was reduced by ∼3-fold in the cs-1 group. interestingly, phosphorylation of mitogen-activated protein (map) kinase p38 (0.05±0.01 vs 0.14±0.06, p<0.03) was selectively downregulated in the 6h cs-1 treated olts. in conclusion, this work supports a broad regulatory role for fn-α4β1 interactions on mmp-9 expression by leukocytes, likely mediated through activation of p38 mapkinase, and matrix pathological breakdown associated with leukocyte infiltration. this data provides the rationale for the development of novel therapeutic approaches in cold liver i/r injury. ischemia reperfusion injury is the most common cause of acute kidney injury in both native and allograft kidneys. the pathogenic mechanisms of renal ischemia reperfusion injury include changes at the level of the microvasculature as well as the tubules. within the microvasculature, leukocyte-endothelial interactions likely play a role and contribute to the well-established microvasculature dysfunction (e.g., endothelial permeability) and alterations in endothelial-leukocyte interaction occur during ischemic acute kidney injury. our previous studies have demonstrated that t cells modulate renal ischemia reperfusion injury in a murine model, we hypothesized that t cells could mediate changes in renal vascular permeability during ischemia reperfusion injury. we performed a 30 min bilateral renal ischemia followed by reperfusion in c57bl6 wild type mice and in t cell deficient (nu/nu) mice with or without t-cell adoptive transfer from their wild type littermates and evaluated rvp by evans blue dye extravasations (ebde). the time course studies of rvp showed marked increases in renal ebde within the early 3-6 hrs after ischemia. cd3 positive pan t-cells but neither cd4 nor cd8 t cells were found to infiltrate into post ischemic kidney within 6 hrs, and comparison was made with other leukocytes using immunohistochemistry technique. gene microarray analysis demonstrated that the gene for tnf-α (tnfaip1), a potent mediator of microvascular permeability as well as a t cell product, was increased in the kidney early after ischemia. tnf-α, ifn-γ and il-4 protein by an intracellular cytokine staining technique was found increased early in peripheral circulating t cells and later in renal t cells after renal ischemia. the rise in rvp was significantly attenuated in t cell deficient mice nu/nu at 6 hrs compared to the wild type littermates and this attenuated rvp in t cell deficient mice was restored after adoptive transfer of splenic t cells from wild type littermates into these nude mice. these data demonstrate that t cells traffic early into post ischemic kidney, produce tnf-α and other cytokines that can increase rvp, and directly participate in the increased rvp during acute ischemia reperfusion injury. t cell-endothelial interactions are a likely mechanisms underlying the pathophysiologic role of t cells in renal ischemia reperfusion injury . background: ischemia/reperfusion injury (iri) is a major cause of organ dysfunction after intestinal injury and transplantation. the interaction between the innate and adaptive immune systems has become a major area of focus in this field. our preliminary work showed that mice undergoing intestinal iri experienced worse survival and tissue injury in conjunction with increased infiltration of pmn and cd3+ cells as compared to sham mice. the purpose of this study was to investigate the role of the t cell in intestinal iri through the use of genetically deficient mice. methods: under anesthesia, male c57bl6 wild-type mice (wt) and cd4 knockout mice (cd4ko) underwent 100 min of warm intestinal iri by clamping of the sma. separate survival and analysis groups were performed. intestinal tissue was harvested at 4h and 24h. tissue was analyzed by histology, cd3 immunostaining, myeloperoxidase activity (mpo), and semi-quantitative pcr for several cytokines/chemokines. results: wt had significantly worse survival compared to cd4ko (30% vs. 100%, p=0.002), and worse histopathological injury mostly involving mucosal sloughing. wt had higher mpo activity than cd4ko (1.9±0.88 vs. 0.70±0.73 at 4h, p=0.059, and 1.5±0.05 vs. 0.22±0.065 at 24h, p=0.004) and increased cd3+ cell infiltration. there was also increased mrna production of cytokines/chemokines in wt vs. cd4ko at both timepoints; specifically, the data with respect to b-actin at 24h was: il-2 (0. conclusion: this study demonstrates for the first time in the intestine that cd4+ t cells are important mediators of iri. in the absence of cd4+ t cells, better outcomes were associated with less pmn infiltration implying a link between the innate and adaptive immune systems. an alteration in chemotactic signaling potentially effected by cd4+ t cells may play a role in this process. these results confirm the important function of cd4+ t cells in intestinal iri and justify further investigation into their complex role in this process. messenger rna assessment in urine sediments from renal transplant patients is rapidly evolving as a non-invasive diagnostic tool. t cells, macrophages, and often b cells are present in rejecting kidney grafts. we questioned whether urinary mrna levels of markers representing these cell types ((regulatory) t cells: cd3ε, foxp3, cd25, tgf-ß; macrophages: cd68, s100a9; b cells: cd20) are associated with rejection. materials in our institute 520 urine samples a year from over 100 renal transplant patients are being collected for mrna quantitation purposes. urine sediments are pelleted and stored in rna-preserving solution. we initially investigated the effect of incubation time of urine on mrna integrity. next, in a case-control study 17 urine samples taken at time of biopsy-proven rejection were compared to 17 samples taken during stable graft function. median time of sampling (55 days versus 43 days posttansplant) did not significantly differ between groups. rna (0.90 ± 1.18 µg) was extracted using rneasy spin columns. message of the markers mentioned above was quantified with q-pcr and normalized. results incubation of urine for 7h or longer at room temperature after acquisition resulted in a 75% decrease in 18s rrna signals (p<0.05). however, storage of urine for up to 24h at 4ºc did not result in decreased mrna expression. in the case-control study, all markers tested showed the highest expression levels in urine rejection samples. when corrected for multiple comparisons, only tgf-ß (26-fold, p=0.007) and foxp3 (21-fold, p=0.002) expression was significantly increased in rejection samples compared to non-rejection samples. tgf-ß levels highly correlated with foxp3 levels (r = 0.90, p<0.00001), suggesting a mechanistic relationship in vivo between the two molecules. conclusion rna integrity in urine samples stored at 4ºc is maintained for at least 24h. detection of increased tgf-ß and foxp3 message in urine from patients with a kidney transplant represents a means for indicating occurrence of graft rejection. this implies that mrna urinalysis renders a suitable molecular tool for non-invasive patient monitoring in clinical practice. the data furthermore suggest that rejection is associated with tgf-ß-mediated immune mechanisms in which regulatory t cells play a role. the significance of b cell and plasma cell infiltration in renal allografts remains controversial. we previously established that transcript sets associated with ifng effects or t cells reflect the inflammatory burden in renal allografts. in the present study, we identified b cell associated transcripts (bats) and immunoglobulin transcripts (igts), reflecting b cell and plasma cell infiltration, respectively. using microarrays, we analyzed bat and igt expression in relationship to histologic lesions, diagnosis, and renal function in 177 renal allograft biopsies. immunostaining confirmed that bat and igt expression was associated with b cells and plasma cells in the graft. expression of bats and igts was increased in biopsies with rejection (1.2 ± 0.2, 1.6 ± 0.9) compared to non-rejection (1.1 ± 0.2, 1.1 ± 0.5), but was not different between t cell (1.3 ± 0.3, 1.5 ± 1.1) and antibody mediated rejection (1.2 ± 0.2, 1.5 ± 0.9) and also occurred in some non-rejecting biopsies (recurrent gn). bat and igt scores correlated strongly with time post transplant (fig1), which was best modeled as a dichotomous relationship: biopsies ≤ 5 months did not express bats or igts above the level of control kidneys. other inflammatory markers were not time dependent. in biopsies ≥ 5 months, bat and igt expression correlated with interstitial inflammation, tubular atrophy, and interstitial fibrosis. in a multiple regression analysis, only time post transplant and interstitial inflammation were independently related to bat and igt scores. when correcting for time post transplant, bat and igt scores did not correlate with renal function at the time of biopsy or future function 6 months post biopsy. bats and particularly igts are a time dependent feature of injured and inflamed renal allografts. corrected for the effect of time, bats and igts do not correlate with outcomes, indicating that b cell and plasma cell infiltrates have no specific role for the mechanism of injury independent of the inflammatory burden. their accumulation in late allografts may indicate emergence of specialized lymphoid compartments in tissues with long standing low level inflammation. pearson correlation coefficient between pbts expression and renal function pbts egfr at %change in egfr from biopsy 6 months after biopsy baseline to biopsy biopsy to 6 months after qcats -0.17* -0.16* -0.19* -0.05 grits -0.23** -0.25** -0.20** -0.10 irit_d1 -0.02 -0.01 -0.09 0.13 irit_d3 -0.51** -0.29** -0.36** 0.16* irit_d5 -0.28** -0.22** -0.19* -0.02 kt2 0.27** 0.13 0.22** -0.02 * p<0.05, ** p<0.01 thus the pbts have prognostic value. surprisingly, the transcripts in the biopsy with greatest prognostic value for future gfr or recovery of gfr were not related to rejection (cytotoxic t cell or ifng associated) but to the degree of injury response. we propose that the common pathway linking various injuries (immune and non immune) to gfr is through the degree of injury response these events induce in the epithelium, particularly those in the irit_d3 set. early rejection from anamnestic response in offspring-to-mother or husband-to-wife kidney transplant. kwan tae park, 1 song chul kim, 1 duck jong han. 1 1 surgery, asan medical center, seoul, korea. accelerated rejection can be developed in the immediate post-transplant period resulting from anamnestic response due to the exposure to fetal hla antigen during the previous pregnancy in case of offspring-to-mother or husband-to-wife. however, accelerated rejections in these groups have been rarely reported. 81 cases of offspring-to-mother (offspring group) and 53 cases of husband-to-wife (spouse group), who has been sensitized to spouse via their children, underwent kidney transplants from january of 1997 to august of 2007 at our institution and retrospectively reviewed. control group was female kidney recipients transplanted at the same period from living related donors other than offspring and from living unrelated donors other than husband. acute rejection (ar) rate within 3 months after transplant were 19.7% (16/81) in offspring group and 18.8% (10/53) in spouse group. the ar rates were not different between the two groups, however they were significantly higher than control group in both groups, namely 10.2% (24/235) for offspring control and 13.5% (10/74) for spouse control. the mean onset of ar was 7.5 day (0-29) and it was significantly later than that of control groups (16.5 day, p<0.05) and 54% of ar were accelerated rejections within 7 days after transplant. proportion of acute humoral rejection was significantly higher in both groups than control group (37.5% vs 8.3% in offspring group, 60% vs 10% in spouse group). most of the ar was successfully reversed by steroid pulse and/ or plasmapheresis, ivig, rituximab. any risk factors for the ar such as number of pregnancy, preoperative cdc cross matching, pra, immunosuppressant and antibody induction couldn't be identified. serum creatinine level in ar patients were higher than ar free patients by postoperative 1 month, but last follow up creatinine level didn't show any statistical difference (1.43 mg/dl in ar vs 1.14mg/dl in ar free). 5 year graft/patient survival rate were not different between ar and ar free groups and study and control groups. risk of higher rejection rate accompanied with higher accelerated and humoral rejection was identified in female kidney recipients from offspring or spouse donor in comparison with control group. considering the anamnestic response of this cohort of female recipients, more prudent preoperative screening and careful immediate postoperative immune monitoring are required for the avoidance of rejection and impaired graft survival. kaplan-meier survival curves showed that ∆upc > 0.2 were associated with decreased patient and allograft survival. these data show for the first time that regardless of the histopathology, ∆upc > 0.2 one month after rejection is associated with poor patient/allograft outcomes. from histology to microarrays the histopathological hallmark of t cell mediated rejection (tcmr), is interstitial infiltration. assessing infiltration by histology is arbitrary, limited in reproducibility, and has never been assessed against independent standards. we recently reported that a set of cytotoxic t cell-associated transcripts (qcats) could quantitatively assess the t cell burden in tissue. objective of the present study was to re-examine the current diagnostic criteria in relationship to the qcats. in 129 renal allograft biopsies, we assessed how histology predicts the qcat burden. an independent diagnostic threshold for qcats was established in control samples. applying this qcat threshold revealed current diagnostic criteria for histology to be flawed; the threshold for interstitial infiltration is too high (100% specificity, 37% sensitivity), the types of infiltration to be considered (i.e. i-banff) are wrongly defined, and tubulitis is not increasing diagnostic accuracy. changing the criteria by lowering the histological threshold for cortical infiltration from 25% to 10%, taking into account all interstitial cellular infiltration (= i-total) and ignoring tubulitis increased sensitivity to 91% with a decreased specificity of 50%. but, this includes biopsies having interstitial infiltration without qcats, indicating that histology might not discriminate between 'active' and 'inactive' infiltration. both the refined histological and the qcat threshold had prognostic value in terms of future renal allograft function. we propose a refined histological scoring system that better predicts the active t cell burden and outcome than the current banff criteria for renal allograft rejection. there has been an increasing and well justified interest regarding the long term renal consequences of kidney donation. the collective evidence suggests, however, that kidney donors enjoy a normal life span and their lifetime risk of esrd may not be different from non-kidney donors. assessing kidney function utilizing serum creatinine is not without limitations. therefore, to better assess kidney function, 5 yeas ago, we began a large effort to measure gfr using the plasma disappearance of iohexol and first void urinary albumin/creatinine ratio (acr) in randomly selected kidney donors who donated at our institution. results: we have performed over 3600 donor uninephrectomies since the inception of our program in 1963. of these, 242 donors underwent iohexol gfr at our general clinical research center. microalbuminuria is defined as acr between 30-300mg/g and macroalbuminuria as acr>300mg/g. the mean age was 53.0±9.6 years, 11.9±8.8 years have elapsed since donation, hemoglobin was 13.7±1.23g/l, systolic blood pressure (sbp) was 121.7±14.7mmhg and diastolic blood pressure (dbp) was 73.2±9.1mmhg. 82.6% of donors had a gfr greater than 60ml/min/1.73m 2 and 17.4% had a gfr between 30-60ml/min/1.73m 2 . the detailed renal profile of these donors is shown in the table below. multivariate analysis that adjusted for age, gender, ethnicity, time from donation, sbp, dbp and body mass index (bmi) identified age at donation, female gender and bmi as independent predictors for gfr<60ml/min/1.73m 2 and time from donation and systolic blood pressure as independent predictors for micro and macroalbuminuria. conclusion: this is the largest effort describing measured gfr in previous kidney donors. it is reassuring to find out that the majorities have a preserved gfr and only a minority has albuminuria. the risk factors for reduced gfr and albuminuria are analogous to what has been described in the general population. cystatin c levels and long-term donor health markers cystatin-c <1 (n=65) psychosocial and physical health of lkds following donation is of utmost importance. unfortunately, this issue has been neglected to a large extent. we sought to examine the current practices regarding psychosocial follow-up of lkds after surgery across transplant (tx) centers. we conducted a 68-question online survey regarding this practice and issues related to lkd. the survey was e-mailed via listservs of 2 professional tx societies. several questions allowed for more than one response. characteristics of the 69 tx centers that participated in the survey are found in table 1 . only 38.3% actively follow donors regarding mental health, substance abuse, or quality of life issues after donation. the professionals most often involved are social workers (72%) and nurse coordinators (69%). the majority of follow-up is conducted via phone (83.3%); though appointments (69.4%) and questionnaires (11.1%) are also utilized. 65% initiate follow-up with donors 1-4 weeks post-operatively, whereas only 27% of programs maintain contact at both 3-6 months and 12 months. 83.3% offer post-operative psychosocial support; 62% only under certain circumstances. this support is provided by social workers (90%), psychologists (30%), and/or psychiatrists (28%). support is available indefinitely in 68% of programs. 30.5% assume the costs of post-operative medical and psychosocial care indefinitely; 50.8% for a specified period of time. 40.7% bill the recipient's insurance and 15.2% bill the donor's insurance. 83.1% of centers accept donors without health insurance and only 5.1% purchase insurance on behalf of donors to cover post-operative health care needs. the results of this survey clearly demonstrate that post-operative psychosocial follow-up of lkds is uncommon and that current practices are widely variable. without routine follow-up of donors, tx centers are less likely to capture post-operative psychosocial issues that may result from organ donation. standardized post-operative follow-up of lkds should become a mandatory part of their care, which will require increased support from health care policy makers. the role of hepatitis c and race in patient and graft survival in combined kidney and liver transplantation. dilip moonka, 1 ravi k. parasuraman, 2 kim a. brown, 1 alissa kapke, 3 dean y. kim. 4 1 gastroenterology, henry ford health systems, detroit, mi; 2 nephrology, henry ford health systems, detroit, mi; 3 biostatistics, henry ford health systems, detroit, mi; 4 transplant institute, henry ford health systems, detroit, mi. the influence of hepatitis c (hcv) and race are not well understood in combined kidney-liver transplant (klt). hcv has a negative impact on patient and graft survival in liver recipients whereas african-american (aa) race is a negative prognostic factor in kidney recipients. aim: to determine the influence of hcv and aa race on patient and graft survival in klt. methods: the unos public use database was used to identify 2296 patients undergoing klt who had known hcv ab status. 13% were aa and 68% were non-hispanic white (nhw). 39.5% were hcv ab positive. groups were assessed for patient and graft survival. results: there is a significant gradient in patient survival and both kidney and liver survival from nhw patients without hcv to aa patients with hcv (table) . the patient survival at five years drops from 72% to 54% along this gradient. there is also a 14% drop in liver and an 18% drop in kidney graft survival. on pairwise testing, the difference in patient survival at 5 yr between all patients without hcv and those hcv ab positive drops from 72% to 60% (p=0.0003). the difference in 5 yr survival between all nhw and aa patients (regardless of hcv status) drops from 68% to 57% (p=0.221). on multivariate analysis, hcv and the combination of hcv and aa race remains associated with diminished survival but race alone does not. conclusions: patients with hcv who undergo a klt transplant are at increased risk for poor overall survival and poor survival of kidney and liver grafts. this effect appears even more pronounced in aa patients. this knowledge is critical to individual centers in assessing risk and benefit from klt in these groups and these groups represent an opportunity for improved interventions. kidney: pediatrics background: pediatric renal transplant recipients have excellent short-term outcomes but long-term success is compromised by complications of chronic immunosuppressive medications and chronic allograft nephropathy. studies show that calcineurin inhibitors and steroids can be individually avoided in pediatric renal transplantation. building on that experience we designed this study to optimize short and long-term renal allograft function with minimal chronic immunosuppression using a steroid-free, calcineurininhibitor withdrawal protocol in low risk pediatric renal transplant recipients. methods: unsensitized pediatric recipients of a first living donor kidney transplant received 2 doses of campath-1h ® (0.3 mg/kg), 1 day pre-and post-transplant. subjects received tacrolimus and mmf immediately post-transplant until week 8-12 when they underwent protocol renal biopsy and were changed to sirolimus and mmf if rejection free. the planned 35 subjects have been enrolled; this report describes the clinical outcomes of the 23 with 1 year of follow up. results: the mean subject age is 12.9 yrs; 59.3% are female and 70.4% caucasian. at transplant, 16/23 were cmv seronegative and 16/23 were ebv seronegative. protocol therapy was discontinued in eight subjects due to: rejection (3), mouth ulcers (2), leukopenia (1) , unrelated (2). clinical acute rejection (ar) occurred in 4 subjects (17%) and 2 had subclinical ar; ar was cellular rejection in 5 subjects, and humoral in 1 at 4 days post-transplant who had an undetected positive crossmatch to class ii hla. there were two graft losses, one due to recurrent fsgs and one due to medication non-adherence. there were no cases of ptld and no deaths. leukopenia occurred in 11 subjects (47.8%). there were 9 infections of which 7 were urinary tract infections (34.7%) and 2 were pneumonia (8.7%). conclusions: minimization of immunosuppression using a steroid-free, calcineurinwithdrawal protocol in low risk pediatric renal transplant recipients appears to be well tolerated with acceptable rates of clinical ar and no serious infections 12 months after transplantation. male; 57% white). substantial increases in bmiz were observed within the first 6 mo.; no changes were seen from 6 to 48 mo. baseline bmiz category (<-2.0, -2.0 to +2.0, >+2.0) influenced the pattern of change. subjects with low bmiz (<-2.0) at baseline experienced the greatest increases in bmiz, but overweight was rare; increases tended to result in a bmiz in the healthy range. those with high bmiz (>+2.0) at baseline demonstrated no significant change in bmiz post-tx. younger age at tx (highest risk for those 2 to 5 y. at tx) more remote date of tx, and baseline bmi between the 25 th and 75 th percentiles were significant independent risk factors for unhealthy weight gain both at 12 mo. and persisting at 48 mo. post-tx. weight gains occur early after tx and tend to persist. counseling focused on prevention of weight gain should be a routine part of post-tx care, with the most intense efforts concentrated on the highest risk patients: young, healthy weight children. avoidance of early weight gains may have an important impact on bmi over the long term. referral. lindsey a. pote, 1 jennifer trofe, 1 erin h. wade, 1 jorge baluarte, 3 alden doyle, 2 simin goral, 2 karen warburton, 2 robert grossman, 2 jo ann palmer, 3 roy d. bloom. 2 1 pharmacy, hosp of the univ of penn; 2 nephrology, univ of penn; 3 transplant surgery, children's hospital of philadelphia. intro: few data exist on outcomes in pediatric kidney recipients who transition to an adult transplant program. purpose: to examine outcomes in pediatric kidney recipients who transition to an adult transplant program and to identify characteristics associated with non-adherence related graft loss. methods: retrospective, single center analysis of 41 pediatric kidney recipients who transitioned to an adult program. results: for the cohort overall, transition to the adult program occurred a mean of 82 ± 8.7 months following transplantation. mean serum creatinine at transition was 2.6±3.2 mg/dl and mean patient age 21.2 ± 0.3 years. 61% of patients received living donor kidneys. within 31±3.5 months of transition, 13 (31.5%) of patients experienced graft loss. causes of graft loss included admitted non-adherence (n=6), recurrent disease (n=3), chronic progressive graft dysfunction (n=3) and bk nephropathy (n=1). graft loss occurred a mean of 16±7 months post transition for non-adherent patients and 20.5± 3 months for adherent patients (p=ns). the characteristics of the cohort is shown in the table according to whether or not patients had documented non-adherence related graft loss. conclusions: 1) graft loss commonly occurs within 3 years following transition and is attributable to both patient non-adherence and late referral by the pediatric transplant program, 2) non-adherence related graft loss was more common in males, 3) factors unassociated with non-adherence include ethnicity, prior transplantation, age at transplant, & duration of dialysis, 4) the development of collaborative pediatric-toadult transition clinics may enhance adherence and lead to improved graft outcomes in this population. this 2-year, prospective randomized pilot study compares the effect of conversion to sirolimus (srl) vs. continued mycophenolate meofetil (mmf) in patients with chronic allograft nephropathy (can), on histological progression. we present 2-year data on safety and renal function. participants >1 year post-transplant with can (banff ≥ci1, ct1) on tacrolimus, mmf and prednisone were randomized to continue mmf or convert to srl (target 8-12 ng/ ml). tac dose was minimized (target 3-5 g/l), and renal biopsy performed at baseline, 1, 2 years. 3-month interval monitoring included adverse event (ae) reporting, egfr (schwartz) and immunosuppressant levels. 16/20 (mmf=10, srl=10) have completed 2-year follow-up. baseline gender, ethnicity, previous acute rejection episodes, can grade, proteinuria (upcr) were similar. srl had lower baseline egfr (79±17 vs. 109±49 ml/min/1.73m 2 , p=0.22). 285 aes were reported (mmf=117, srl=168), 43 serious ae (sae: mmf=25, srl=28). common saes were similar: dehydration & elevated creatinine (30), gastroenteritis (10) and rejection (ar). 2 episodes (1 patient) of ar occurred in mmf group and 5 (2 patients) in srl group, all attributable to non-adherence. there were no sustained change in electrolytes, hg and total wbc counts in the 1 st 2 year, except neutrophil counts were lower in the srl group (24 mo: mean 4.7 vs. 2.8, p<0.05). platelets were significantly lower at 3 months (srl) but not thereafter (24 month: mean 271±71 vs. 196±61 x10e9). cholesterol and tg increased early (p<0.05), but only cholesterol persisted after 2 years (mean 5.2 vs. 3.7 mmol/l, p<0.05). in srl group only, upcr increased over 2 years (∆upcr 94±87 mg/mmol, p<0.05). this was not associated with hypoalbuminemia at 2 years. egfr was lower in srl vs. mmf after 18 months (p<0.01), but the rate of change in egfr from baseline was similar between groups (-28±23 vs. -30±39 ml/min/1.73m 2 , p=ns). serious adverse events did not differ significantly between the srl and mmf groups. sustained srl treatment was associated with mild neutropenia, hypercholesterolemia and proteinuria after 2 years. egfr was reduced at baseline compared with mmf, and both groups had similar rates of decline in gfr over time. allograft recipients. maarten naesens, 1 oscar salvatierra, 1 li li, 1 minnie sarwal. 1 1 department of pediatrics, stanford university school of medicine, stanford, ca. background: in contrast to adult kidney recipients, little is known about the long-term evolution of tacrolimus pharmacokinetics in pediatric kidney transplant recipients. methods: one-hundred five pediatric recipients of a kidney allograft, all treated with a corticosteroid-free immunosuppressive protocol, were included. the evolution of tacrolimus doses and exposure was recorded at 3, 6, 9, 12, 18 and 24 months after transplantation, as well as all pre-dose trough levels (c 0 ; n=9376) obtained in the first 2 years after transplantation. results: dose-corrected tacrolimus exposure (c 0 /dose/kg) increased in the first 2 years after kidney transplantation in pediatric recipients (table 1, figure 1 ). this decrease in dose requirement by time was only significant in children older than 5 years at the time of transplantation ( figure 1 ). in addition, the younger patients had significantly higher dose requirements compared to older recipients, which translated in marked underexposure in 72-79% of patients <12 years of age in the first days after transplantation. conclusion: pediatric kidney transplant recipients exhibit maturation of tacrolimus pharmacokinetics with time after transplantation. this can not be explained by differences in corticosteroid use, as all patients were treated with a corticosteroid-free protocol. the higher dose requirements for younger recipients and the absence of tacrolimus maturation in the youngest recipients suggest that age-dependent changes in tacrolimus intestinal first-pass effect, metabolism or distribution play a role. whether age-specific tacrolimus dosing algorithms will improve outcome needs further study. determinants of dose-corrected pre-dose trough levels (c0/dose/kg) aih may present as acute hepatitis in 25% of patients (pts) and result in alf. early administration of corticosteroids may obviate the need for liver transplantation (lt), but features which identify aih in pts with alf have not been determined. aim: to identify clinical and histological features which distinguish alf due to aih. methods: 197/1033 pts in the alf study group registry had no evidence of viral, metabolic, vascular, and drug/toxic liver injury. all had alf defined by acute disease, encephalopathy, and coagulopathy. based upon admission clinical features, 52/197 had probable aih, and 145 were considered "indeterminate." liver biopsies (lbx) available from 56 pts were reviewed by a blinded expert hepatopathologist. clinicopathologic correlations were analyzed retrospectively. results: all lbx available from pts with suspected aih had classical histologic features of aih. moreover, 23/46 (50%) lbx from pts with indeterminate alf also had aih features: extensive necrosis (100%), fibrosis (84%), cirrhosis (22%), interface hepatitis (75%), and plasma cell-rich inflammation (69%). of all 33 lbx with aih features, centrilobular lesions associated with acute, severe aih (am j surg path 2004;28:471) were frequent: plasma cellrich central venulitis (97%), exclusive centrilobular necroinflammation (19%), and pericentral dilatation/congestion (46%). clinical characteristics and outcomes of the 75 pts with aih based upon laboratory and histologic findings differed significantly from pts whose alf remained indeterminate: aih pts were older (44 v 38 y), predominantly female (75 v 56%), and had longer jaundice-encephalopathy interval (20 v 12 d) , lower alt (660 v 1949 u/l), higher globulins (3.7 v 2.7 g/dl), lower creatinine (1.7 v 2.3 mg/ dl), and a higher prevalence of ana (68 v 24%) and asma (51 v 29%) (all p<.01). although spontaneous survival did not differ, more aih pts underwent lt (63 v 34%), and more survived 1 month after enrollment (75 v 59%; p<.0001). conclusions: using histologic criteria to classify pts with indeterminate alf, aih accounted for at least 7% of the alf study group registry, and represented 50% of indeterminate alf. lbx should be performed in all patients with alf of obscure etiology, in particular to identify centrilobular necroinflammatory lesions, which appear to be specific indicators of acute and severe aih. (u-01 58369 from niddk). (2002) (2003) (2004) (2005) (2006) . mikel gastaca, 1 miguel montejo, 1 lluis castells, 2 antonio rafecas, 3 antonio rimola, 4 ramon barcena, 5 federico pulido, 6 magdalena salcedo, 7 martin prieto, 8 manuel de la mata, 9 jose r. fernandez, 1 jose m. miro, 4 the spanish lt in hiv-infected patients working group. 1 hospital de cruces, bilbao, spain; 2 hospital vall d´hebron, barcelona, spain; barcelona, spain; univ. of barcelona, barcelona, spain; 5 hospital ramon y cajal, madrid, spain; 6 hospital 12 de octubre, madrid, spain; 7 hospital gregorio marañon, madrid, spain; 8 hospital la fe, valencia, spain; 9 hospital univ. reina sofia, cordoba, spain. background and aim: we report on the preliminary results of the prospective multicenter spanish study in hiv-1 infected patients who underwent olt. methods: the prospective multicenter spanish study fipse-olt-hiv 05-gesida 45-05 was initiated in january 2002. inclusion criteria follows the rules previously described in the spanish consensus document. hiv-infected patients transplanted between january 2002 and december 2006 were included in this study. results: 89 olt were consecutively performed in 85 hiv-1 infected patients in the period of the study. median (iqr) follow-up is 16 (8-29) months. median (iqr) age was 42 years (39-46), 74% of the recipients were male and former drug abuse was the most common hiv-1 risk factor (78%). 83% of the patients were transplanted due to hcv-related cirrhosis and 6% due to hbv cirrhosis. median (iqr) meld score was 14 (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) . pre-olt median (iqr) cd4 cell count was 288 (165-486) cells/mm3 and 64%patients had undetectable plasma hiv viral load. immunosuppression was based on tacrolimus in 70% of the patients. haart was re-started in a median (iqr) time of 10 (5-21) days after olt and was based on efavirenz in 48% of the cases. acute rejection occurred in 38 patients (43%). twenty patients died (22.5%) mainly due to hcv recurrence (8%). antiviral therapy with peg-interferon plus ribavirin was initiated in 18 patients obtaining a sustained viral response in 6 of them (33%). patient survival (95% confidence intervals) at 1, 2, 3 and 4 years was 90% (81-95%), 74% (60-84%), 67% (52-79%) and 67% (52-79%), respectively. conclusion: for selected hiv-1 infected patients under haart therapy, olt is a safe and effective procedure at mid-term. as in the hiv-negative population, hcv recurrence is the mayor cause of concern and response to antiviral therapy is still disappointing. hepatitis c virus and objectives: describe the 12-year experience of a university hospital treating chronic hepatitis c relapse, histologically diagnosed after liver transplant. material & methods: from september 1991 through july 2006, all the patients subject to liver transplant with histological diagnostics of chronic hepatitis relapse were submitted to antiviral treatment. treatment was suspended if there was a severe adverse reaction to the drugs used, non-adherence, severe rejection or no response after at least 12 months. hcv positive serology patients, alt increase (1,5x nl) and hepatic biopsy with metavir rating showing structural 1 and/or periseptal or parenchymatous portal inflammatory activity /= 2 were included. from 1995 to 2003, all patients were treated with conventional interferon and ribavirine, regardless of the genotype. from october 2003 on, patients with genotype 1 were treated with ribavirine and pegylated interferon and genotype 3 were treated with conventional interferon and ribavirine. results: 47 patients were treated during this time period, thirty-seven male (78.7%). their average age was 48. 51.1% were genotype 1, 25.5% were genotype 3. average time between transplant and beginning of treatment was 30 months. average treatment was 16 (4-36) months. 74.5% received conventional interferon, 6.4%, pegylated alpha 2a and 19.1%, pegylated alpha 2b. 48.9% had one or more cellular rejection episodes before vhc relapse diagnostics and were treated with corticosteroids. sustained virological response (svr) occurred in 48.8%. three patients had a virological response at the end of treatment (evr) and have not completed six months post treatment. out of 21 patients with svr, 20.9% were genotype 3, 9.3% genotype 1 and 18.6% were not genotyped. considering genotypes, svr was detected in 16.7% with genotype 1, in 75% with genotype 3. srv was detected when we examined hepatic biopsy, in 85.7% of the f1/f2 patients (18 out of 21), 9.5% of the f3 patients (2), 4.8% of the f4 patients (1) . survival period of 5 years for svr patients was 94% (20 out of 21) and 68.2% for patient without svr (p = 0.03 -kaplan-meier). five-year survival was 79.2% for patients with genotype 1 and 100% for patients with genotype 3. conclusions: our results show it is possible to get good five-year survival rates for treated patients. however, handling adverse reactions and long term treatment still pose difficulties in pursuing better svr rates. the impact of alcoholic liver disease (ald) and background: although liver transplant survival benefit has been shown to be associated with meld score, disease-specific analyses have not been reported. we evaluated, using srtr data, the effect of ald and/or hcv infection on transplant waitlist and post-transplant mortality, and survival benefit of deceased donor liver transplants. methods: 38,889 patients age ≥18, who were listed between sept 2001 and dec 2006, and followed until dec 31, 2006, were classified into 4 cells according to hcv or ald status: hcv+, ald-: 12,322; hcv+, ald+: 4,220; hcv-, ald+: 6,581; hcv-, ald-: 15,776). cox regression was used to estimate waiting list mortality and post-transplant mortality separately. survival benefit, which encompasses both pre-and post-transplant events, was assessed using sequential stratification; an extension of cox regression which matches transplant recipients by meld and organ procurement organization to patients on active on the waitlist at the time of transplant. results: hcv significantly (p=0.0001) increased waitlist mortality, with a covariate-adjusted hazard ratio (hr) for hcv+ vs. hcv-of hr=1.19 (p=0.0001). the impact of hcv+ was greater among ald+ candidates (hr=1.36; p<0.0001), but was also significant among ald-candidates (hr=1.11; p=0.02). the contrast between ald+ and ald-waitlist mortality was only significant among hcv+ candidates (hr=1.14; p=0.006). post-transplant mortality was significantly higher among hcv+ vs. hcv-recipients (hr=1.26; p=.0009); there was no difference between ald+ vs. ald-recipients. survival benefit of liver transplantation was significantly lower among hcv+ compared to hcv-recipients with meld 9-29, but significantly higher for hcv+ recipients with meld scores of ≥30. a diagnosis of ald did not influence the survival benefit of transplantation at any meld score. conclusion: despite higher waitlist mortality, hcv+ recipients had significantly lower liver transplant survival benefit than hcv-recipients, within categories defined by meld score. in contrast, transplant survival benefit was not influenced by ald. transplanted for hbv-related cirrhosis. giuseppe tisone, 1 daniele di paolo, 1 ilaria lenci, 1 laura tariciotti, 1 andrea monaco, 1 manuele berlanda, 1 linda de luca, 1 giuseppe iaria, 1 alessandro anselmo, 1 irene bellini, 1 mario angelico. 1 1 hepatic surgery and transplantation, university of rome tor vergata, rome, italy. background and aim: post-transplant active immunization with hbsag vaccine is a potential prophylaxis strategy against hbv-recurrence after liver tranplantation due to hbv-related disease. previous studies showed conflicting results using standard vaccines, whereas the use of the new adjuvant 3-deacylated monophosphoryl-lipid-a (mpl) significantly increased patient's immunization rate. we investigated the efficacy of a long-term (12 months) accelerated (monthly doses) vaccination schedule using the mpl-adjuvanted vaccine administered with and without concomitant hbig. methods: 18 patients (m/f:13/5) transplanted for hbv-related cirrhosis 73±38 months earlier were recruited. all were hbsag and hbv dna negative in serum and cccdna negative in liver tissue; 5 (27.7%) were co-infected with hcv and 5 (27.7%) with hdv. study protocol consisted of 12 consecutive monthly intramuscular vaccine doses (hbsag 20mg plus mpl 50mg) given together with lamivudine (100 mg/daily). each of the initial 6 doses (first cycle) was administered within 7 days after 2000 iu hbig i.v. infusion, while the last 6 doses were given after complete hbig withdrawal (second cycle). hbsab titre was determined before each vaccine dose and during the follow-up. all patients were maintaiened on low-level immunosuppression. preliminary results: all patients completed first vaccination cycle; 14 (77.7%) patients received 12 adjuvanted vaccine doses (first and second cycles) and were monitored during 3 months follow-up after vaccination end. no side effects occurred, nor evidence of hbv recurrence. at the end of first cycle all patients achieved an anti-hbs titre >100 iu/l (mean 343±209 iu/l) and 3 (16.6%) a titre greater than 500 iu/l. at the end of follow-up 8/14 (57.1%) and 4/14 (28.5%) had an anti-hbs titre greater than 100 (mean 411±599 ui/l) and 500 iu/l, respectively. conclusions: nine months after hbig withdrawal more than half of the patients reached and maintained a protective anti-hbs titre (>100 iu/l). this intensive schedule using the mpl-adjuvanted vaccine, given in combination with hbig and lamivudine, seems to be more effective than previous hbv vaccination protocols, although a longer follow-up is needed to assess its final effectiveness. heterologous immunity via alloimmune responses to hepatitis c virus replication after liver transplantation. hideki ohdan, 1 masahiro ohira, 1 yuka tanaka, 1 kohei ishiyama, 1 nobuhiko hiraga, 2 michio imamura, 2 kazuaki chayama, 2 toshimasa asahara. 1 1 surgery, hiroshima university, hiroshima, japan; 2 internal medicine, hiroshima university, hiroshima, japan. the immunosuppressive environment in liver transplantation (lt) recipients infected with hcv is believed to be associated with the progression of hcv reinfection. in the present study, we simultaneously monitored hcv levels and alloimmune status in hcv-infected lt recipients. for evaluating the immune status of 33 hcv-infected lt recipients, we employed a mixed lymphocyte reaction (mlr) assay using a cfselabeling technique. the kinetics of the stimulation index of anti-donor reactive cd4 + t cells clearly mirrored that of the hcv rna titer, and a significant reverse correlation was observed between the 2 parameters (r 2 = 0.67). we did not observe a similar relationship between the stimulation index of an anti-third party cd4 + t cells and the hcv rna titer (r 2 = 0.03). one possible explanation for this phenomenon might be that cytokines outputted by t cells responding to allostimulation display the anti-hcv activity either directly or indirectly through the activation of bystander immunocytes. to investigate such possibilities, we performed a transwell culture assay comprising a mlr culture in the upper chamber and genomic hcv replicon cell culture in the lower chamber to mimic the anatomical features of the interaction between hcvinfected hepatocytes and alloreactive t cells that infiltrate the portal area in the liver. when one-way mlr was performed using one-halpoidentical combination in the upper chamber, hcv replication was significantly suppressed in the lower chamber containing hcv replicon cells. hcv replication was further suppressed when mlr was carried out with a complete allogeneic combination. this inhibiting effect was dependent on their ifn-γ secreting activity. in addition, a similar result was obtained when ifn-γ-secreting nk/nkt cells stimulated with il-2 were cultured in the upper chamber. in conclusion, there was a close relationship between the anti-donor and the anti-hcv immune status in the lt recipients infected with hcv. cytokines such as il-2 and ifn-γ may be produced in response to allostimulation, and even these cytokines do not cause graft rejection, display anti-hcv activity. the elucidation of such a possible heterologous immunity via alloimmune responses to hcv replication might lead to the establishment of a novel method to prevent the progression of hcv reinfection in hcv-infected lt recipients. hepatitis c e2 region diversity after liver transplantation: a report from the hepatitis c iii trial. juan f. gallegos-orozco, 1 hugo e. vargas, 1 georges netto, 2 gary l. davis, 3 , and 7.3% are morbidly obese . the goal of this study was to quantify the effect of bmi on access to transplantation (att), likelihood of receiving a transplantation (lrt), turndown rates (tr), and survival benefit (sb). methods: att was defined as either registering for the deceased donor waiting list or receiving a live donor transplant, and was analyzed based on the usrds registry using logistic regression. lrt was based on time spent in active status on the waiting list before receiving a transplant, and was analyzed based on the unos waiting list dataset using cox proportional hazards time-to-event analysis. tr was defined as the relative likelihood of being turned down for an organ offer by a provider other than the patient, and was analyzed based on the unos organ turndown dataset using negative binomial regression. sb was defined as survival after kidney transplantation versus survival on the deceased donor waiting list, and was analyzed based on the unos waiting list dataset using a time-dependent cox survival benefit model. all models were adjusted for known factors influencing those outcomes. lgbp lsg lsg mean follow-up (months) 15.4 (range 3-24) 9 (range 3-18) 12.5 (12, 13) patients > 9 months since operation (n) 6/7 4/6 2/2 mean % ewl at > 9 months 61 (range 41-75) 33 (range 24-40) 62 (50, 73) transplant candidate at > 3 months 7/7 5/6 2/2 underwent transplant 0/7 1/6 1/2 complications developed in two patients (both with cirrhosis) and there was no mortality. mean follow-up was 12.3 months, and mean ewl at 9 months or later was 61% (esrd), 33% (cirrhosis) and 61.5% (esld). obesity associated comorbidities were improved or resolved in all patients. serum albumin and other nutritional parameters 9 months or later after surgery were similar to preoperative levels in all 3 groups. at most recent follow-up, 14 out of 15 patients (82%) have reached our institution's body mass index (bmi) limit for transplantation and are awaiting transplant; one patient with esld has undergone a successful lung transplant and one patient with cirrhosis has undergone a successful liver transplant. factors that contribute to inequitable access to the transplantation network (unos/ optn) include socioeconomic status, geographic location and delayed referral. the goal of the meld allocation system was to assign priority to the sickest candidate and assure equitable access to liver transplantation (lt). the meld has been validated as a reliable marker for liver disease severity. at the time of referral to the transplant center or listing a high meld (hm) is an indirect measure of delayed referral. therefore, the aim of this study is to identify factors associated with a hm at the time of listing for lt. method: using the unos database, we identified all adult candidates listed for lt from 2002 to 2006. patients who received meld upgrade (i.e. hcc, fhf) and those listed for multi-organ transplant were excluded. the data collected included demographics, insurance payor, diagnosis, and meld score. meld score at time of listing was categorized as low (< 20) and high (>20) and insurance type as private, medicaid and government (excluding medicaid) . results: during the study period 15,323 candidates were added to the lt waiting list. of these, there were 10,302 (67.2%) males with a median age of 53 (range 18 -83) . caucasians were 11,511 (75.1%), hispanic (11.6%), african americans (8.9%) and the rest (4.4%). the underlying liver disease was hepatitis c (31.9%), alcohol (15.1%), alcohol/hepatitis c (7.2%), pbc/ psc (9.9%), cryptogenic (7.5%) and others (28.4%). age, gender and liver disease etiology were not associated with a hm at listing. a hm was associated with ethnicity: aa (49.5%), hispanic (44.2%), caucasian (36.6%)[p<0.001] and insurance type: medicaid (46.3%), government (38.8%) and private (36.5%)[p<0.001].there was no strong interaction between ethnicity/race and insurance in combination as predictors of hm i.e. both were independent. conclusion: (1) aa and lt candidates with medicaid insurance are more likely to have a hm score at initial listing (2) hispanics had the lowest rate of private insurance compared to caucasians with the highest rate. the above results suggest that type of insurance and ethnicity are independently associated with a hm (i.e. sicker patients) at listing. since a hm score is associated with increased mortality, implementation of strategies that result in timely and equitable access to the transplantation network regardless of the insurance type or ethnicity of the candidate(s) should be a priority. marked increase in the use of inactive status on the kidney transplant waiting list. kim nguyen, 1 valarie ashby, 1, 2 further wait time accrued only after active status was restored. on 11/5/03, the optn implemented a change in policy that provides for the accrual of waiting time during the entire interval that wl candidates are designated as s7. methods: we investigated the impact of this policy change on the patterns of s7 designation, using the srtr/optn database. initial and subsequent status on the wl were determined for 130,986 candidates placed on the ki tx wl from 11/5/00-11/4/06. the probability of becoming active (including receiving a living donor tx) on the wl was calculated for those who were s7 at listing before and after the policy change. results: table 1 shows trends in the use of s7 before and after the policy change. of the 1,396 candidates listed as s7 before the policy change, 76% became active within 6 months and 83% within 1 year of wl. for the 11,248 candidates listed as s7 after the policy change, the corresponding figures are 48% and 60% respectively. figure 1 demonstrates that, since the implementation of the new policy, the % of patients initially wl in s7 at most us tx centers has increased. conclusion: since the implementation of this policy, the number and % of pts wl as s7 at most ki tx centers, and the duration of s7 for those initially wl as s7 has increased dramatically. the implications of this practice on access to ki tx and survival warrants further investigation. can abstracts (11%) patients were retransplanted, and 13 (9%) patients had panel reactive antibody >30. the mean cumulative thymoglobulin dose administered was 5.7 mg/kg, and the primary maintenance immunosuppression started prior to discharge was a sirolimuscontaining regimen (59%). at the end of the follow-up period, 98 (65%) patients had functioning grafts and 52 (35%) patients experienced graft loss. of the 52 patients with graft loss, 15 (10%) patients experienced graft loss secondary to death. no pertinent differences were identified between groups. the graft survivals for african american recipients with african american or caucasian donors were 95% vs. 91% at 1 year and 83% vs. 72% at year 2. conclusions: our study results suggest that donor race does not adversely affect graft outcomes in african american cadaveric kidney recipients with modern immunosuppression. donor racial differences may not play a primary role in the inferior graft outcomes of african american cadaveric kidney recipients. cardiac evaluation before kidney transplantation: are we screening too often or not enough? krista l. lentine, 1 m. a. schnitzler, 1 j. j. snyder, 2 d. c. brennan, 3 p. hauptman, 1 p. r. salvalaggio, 1 b. kasiske. 4 evaluation for ischemic heart disease (ihd) is a common but non-standardized practice before kidney transplant. we retrospectively studied pre-transplant cardiac evaluation (ce) practices among a national sample of renal allograft recipients. methods: we examined usrds data for medicare beneficiaries transplanted in 1991-2004 with part a and b benefits from dialysis initiation through transplant. clinical traits defining "high" expected ihd risk were defined as diabetes, prior ihd, or >2 other coronary disease risk factors. pre-transplant ce were identified by billing claims for noninvasive stress tests and angiography. we quantified individuals with claims for coronary revascularization procedures between ce and transplant, and abstracted post-transplant acute myocardial infarction (ami) events from claims and death records. results: among 27,786 eligible patients, 46.3% (65.4% of high-risk and 20.4% of lower-risk) underwent ce before transplant. overall, 9.5% of patients who received ce also received pre-transplant revascularization, including only 0.3% of lower-risk patients studied by ce ( table a) . the adjusted odds of transplant without ce (higher or for no ce, table b ) increased sharply with younger age and shorter dialysis duration. increased likelihood of transplant without ce also correlated with black race, female sex, and certain geographic regions. post-transplant ami rates in patients transplanted without ce allow assessment of whether ce was appropriately deferred in those who indeed face low ihd-risk after transplant. among patients transplanted without ce, the 3-yr incidence of post-transplant ami was 3% and 10% in lower and high-clinical risk groups, respectively, but varied by clinical traits within these groups. in lower-risk patients transplanted without ce, blacks patients faced increased ami-risk compared to whites (adjusted hr 1.52, 95% ci 1.16-2.00). conclusions: observed ce practices demonstrate a low yield of pre-transplant revascularization but also raise concern for socio-demographic barriers to evaluation access. women who become pregnant in the first two years after kidney transplantation have a higher risk of graft loss. nadia zalunardo, 1 olwyn johnston, 1 caren l. rose, 1 john s. gill. 1 1 division of nephrology, university of british columbia, vancouver, bc, canada. existing information regarding the risks of pregnancy is derived from voluntary data sources. using medicare claims files from the usrds (1990 usrds ( -2003 , we examined pregnancies (defined by presence of an inpatient icd-9 billing code for pregnancy or pregnancy-related complication) in the first 3 years after kidney transplantation (ktx) among women aged 15-45 years whose primary insurance payor was medicare (n=16,195) . patients were followed until graft failure, death or december 31, 2003. there were 530 pregnancies identified in 483 women during the first 3 post-transplant years. women who became pregnant during the 1st or 2nd post-transplant year had a shorter time to graft loss than women who became pregnant during the 3rd year (figure). to minimize the survivor bias among women who became pregnant during the 3 rd posttransplant year, we determined the association between the timing of pregnancy and graft survival among the subset of women (n=455) who had graft survival of at least two years using a cox multivariate regression adjusted for age, race, cause of esrd, donor source, calendar year of transplant, dialysis vintage, maintenance immunosuppression, and gfr at 6 months after ktx. pregnancy in the 1 st year (hr 2.01, 95% ci 1.35 -2.99), and second year (hr 1.79, 95% ci 1.23 to 2.62) was associated with an increased risk of graft loss compared to pregnancy during the third post-transplant year. we concluded that women who are able to wait should be counseled to become pregnant after the second post transplant year. the aging donor and recipient populations have led to new challenges in simultaneous kidney-pancreas transplantation (skpt). the purpose of this study was to retrospectively review our single center experience in skpt with respect to extended (ex) donor (d) and recipient (r) criteria. methods: over a 65 month period, we performed 83 skpts with enteric drainage (79 portal venous drainage). ex ds were defined as age <10 (n=4), >45 (n=12, mean age 50.2 yrs), or donation after cardiac death (dcd, n=4). all dcd donors were managed with extracorporeal support. ex rs were defined as age >50 (n=20, mean age 55.8 yrs) or those with a pretransplant serum c-peptide level >2.0 ng/ml (n=7, mean 5.7 ng/ml). all rs received depleting antibody induction (57 ratg, 26 alemtuzumab) with tacrolimus, mmf, and tapered steroids (39 steroid-free). results: a total of 20 ds (24%) and 23 rs (28%) met the above ex criteria. median waiting time was 10 months, mean pancreas preservation was 17 hours, and median length of stay was 10 days. with a mean follow-up (f/u) of 28 months, patient (pt) (95% ex d vs 94% non-ex d), kidney (90% ex d vs 89% non-ex d) and pancreas graft survival (85% ex d vs 81% non-ex d) rates were similar between d groups (all p=ns). the incidences of delayed kidney graft function (5% in each group) and early pancreas graft loss due to thrombosis (5% ex d vs 8% non-ex d) were also comparable between d groups. with regard to r groups, pt (87% vs 97%, p=0.13) and kidney (87% vs 92%, p=ns) graft survival (gs) rates were slightly lower in the ex r group compared to the non-ex r group, respectively. however, death-censored kidney gs rates (100% ex r vs 95% non-ex r) were comparable between groups. uncensored pancreas gs rates (83% ex r vs 82% non-ex r) were similar. the incidences of acute rejection, surgical complications, infection, and other morbidity were comparable regardless of d or r group. at 1 year (or latest) follow-up, renal and pancreas functional parameters were similar between d groups. however, the ex r group demonstrated slightly compromised renal and pancreas allograft function and a greater need for oral hypoglycemic agents. conclusion: intermediate-term outcomes in skpt from selected ex ds or rs have comparable outcomes, although ex r criteria may represent a risk factor for pt survival and functional outcomes. conclusion: spk recipients with functioning pancreas grafts have significantly improved kidney and patient survival compared to ld ka and dd ka. however, early pancreas graft failure results in kidney and patient survival rates similar to ka recipients. spk provides optimal outcomes for patients with dm1, but long-term risk associated with early pancreas loss may be a consideration when selecting spk vs ka transplantation. the impact of long-term metabolic control on renal allograft and patient survival in type 1 diabetes. christian morath, 1 martin zeier, 1 bernd dohler, 2 jan schmidt, 3 peter p. nawroth, 4 gerhard opelz. 2 1 nephrology, university of heidelberg; 2 transplantation immunology, university of heidelberg; 3 transplantation surgery, university of heidelberg; 4 endocrinology, university of heidelberg. it is a matter of debate whether pancreas allografts independently contribute to renal transplant and patient survival in type 1 diabetics who received a simultaneous pancreas kidney transplant (spk). using the data of the collaborative transplant study (cts), we studied type 1 diabetic recipients of deceased donor kidneys (ddk), living donor kidneys (ldk), or spk performed during two time periods: 1984 to 1990 and 1991 to 2000 . we analyzed graft and patient survival rates for a maximum of 18 years. ddk recipients showed inferior graft and patient survival compared to ldk and spk recipients in both time periods. ldk recipients had a superior graft survival rate initially, but the survival rate of kidneys in spk recipients reached the level of ldk toward the end of the follow up period. the results of patient survival paralleled those of kidney graft survival: an early advantage of ldk as compared to spk faded away during follow up. multivariate analysis, in which pretransplant cardiovascular risk assessment was appropriately considered, showed that patient survival of spk recipients was superior to that of ldk recipients beyond the tenth year after transplantation (hazard ratio hr = 0.55, p = 0.005). this was reflected by a lower cumulative cardiovascular death rate in recipients of spk (37.0 %) compared to recipients of ddk (45.8 %) or ldk (49.3 %) . the early survival benefit of ldk compared to spk is lost during long-term follow up, probably related to improved glycemic control in spk recipients. proposal for a grading rejection schema in pancreas allograft biopsies from a multidisciplinary panel of pathologists, surgeons and nephrologists. 1.normal 2.indeterminate septal inflammation that appears active but the overall features do not fulfill the criteria for mild cell mediated acute rejection. active septal inflammation involving septal structures and/or focal acinar inflammation. -grade ii / moderate acute cell mediated rejection multifocal (but not confluent or diffuse) acinar inflammation with spotty acinar cell injury and drop-out and/or minimal intimal arteritis -grade iii / severe acute cell mediated rejection diffuse, (widespread, extensive) acinar inflammation with focal or diffuse multicellular /confluent acinar cell necrosis.and/or moderate or severe intimal arteritis and/or transmural inflammation -necrotizing arteritis chronic active cell-mediated rejection. chronic allograft arteriopathy (arterial intimal fibrosis with mononuclear cell infiltration in fibrosis, formation of neo-intima) 4.antibody mediated rejection =c4d positivity + confirmed donor specific antibodies + graft dysfunction -hyperacute rejection -accelerated antibody mediated rejection severe, fulminant form of antibody mediated rejection with morphological similarities to hyperacute rejection but occurring later (within hours or days of transplantation). -acute antibody mediated rejection specify percentage of biopsy surface with interacinar capillaries positive for c4d. 5.chronic allograft rejection/graft sclerosis stage i (mild graft sclerosis) <30% of the core surface stage ii (moderate graft sclerosis) 30-60% of the core surface. stage iii (severe graft sclerosis) >60% of the core surface 6. other histological diagnosis e.g. cmv pancreatitis, ptld, etc. a simple, reproducible, clinically relevant and internationally accepted schema for grading rejection should improve the level of diagnostic accuracy and positively affect patient care. rank test). similar results were obtained when death was included as a cause of graft loss (data not shown). conclusion: par is associated with worse long term kidney graft survival than kar. it is likely that patients that experience par within the first year have also experienced undiagnosed sub-clinical kar. this adds associative data to the argument that the likelihood of concordance between par and kar is high and therefore, the need for increased monitoring of kidney function after par is warranted. the most efficacious immunosuppressive (immuno) regimen for spkt is debated, and information on the best regimen to prevent amr is particularly scarce. we performed a retrospective comparative cohort study that included 136 spkt patients (pts) transplanted in 2002-2005, who received maintenance immuno with tac, mmf and steroids. two groups were compared: pts who received induction with alemtuzumab (alem) (n=97) and pts who were induced with basiliximab (basilmab) (n=39). donor and recipient characteristics were similar. kt acute cellular rejection (acr) was more frequent with basilmab (2-yr 12.8% vs 3.1%, p=0.04), but the incidence of biopsy-proven kt amr was similar (2-yr 18% with basilmab vs 13.8% with alem, p=ns). no differences in prevalence were detected considering early amr (<90th day) or late amr (>90th day) separately. multivariate analyses showed that the only significant risk factor for amr was female gender (adjusted hr 2.97, p=0.016). the biopsy-proven kt rejection was associated with clinical pt rejection in 13 out of the 21 amr cases (62%), without differences between the groups. post-rejection kt graft survival was similar in both groups (2-yr basilmab/alem 94.7/91.2%), but deathcensored kt survival was lower with alem (100/91.2%, p=0.05). the predominant cause of kt loss in pts induced with basilmab was death-with-function (n=3), while in pts induced with alem acute or chronic amr was the most common cause of kt attrition (n=5). pancreas survival was similar between both groups. more pt losses due to ar occurred in alem-treated group than in the basilmab group (4 vs 1) . nearly all early episodes resolved with treatment and were not associated with worse graft survival. conversely, late amr episodes carried a worse prognosis, with decreased 2-yr kt (61.5% vs 96.2%, p<0.0001) and pt graft survival (47.7 vs 92.3%, p<0.0001). late amr was associated with graft loss in multivariate cox models (kidney loss hr 3.34, p=0.05 and pancreas hr=3.22, p=0.049) . amr is common in spkt recipients. acr is better prevented with alem than with basilmab, but no relevant difference is found between alem and basilmab in amr. late (as opposed to early) amr episodes are associated with significant reduction in spkt survival rates despite treatment. preventive and/or different treatment strategies are required to address late amr in spkt. intraoperative fluorescence imaging (ifi) in pancreas transplantation (pt) to determine vascular patency and allograft perfusion. edmund q. sanchez, 1 srinath chinnakotla, 1 marlon f. levy, 1 robert m. goldstein, 1 goran b. klintmalm. 1 1 baylor regional transplant institute, dallas/ft. worth, tx. thrombotic complications of pt are well known. we report ifi using the spy device to assess immediate vascular patency and allograft perfusion. our controlled experience is presented here. methods: pts were imaged intraoperatively using the spy device under our irb approved study. indocyanine green solution (2.5 mg/ml) was injected into central venous catheters after completion of the vascular anastomoses of whole pancreaticoduodenal allografts. the pancreas transplants were performed in the retroperitoneal portal and enteric drained technique described by boggi, et al. imaging of the allograft vasculature, perfusion of the pancreas, and perfusion of the duodenal segments was performed and recorded intraoperatively. all video sequences were archived for later review. results: ifi on 7 pancreas transplants (6 simultaneous pancreas-kidney and 1 pancreas after kidney) was performed and video sequences were recorded. all pancreas allografts demonstrated intraoperative vascular patency and complete pancreatic and duodenal perfusion. there were no side effects seen. all pancreas transplants had immediate graft function. one patient was re-explored on postop day 0 due to persistent acidosis and hypotension. repeat ifi demonstrated vascular patency and perfusion, despite an ischemic external physical appearance. there were no vascular complications that required reoperation, nor were there any graft thromboses. characteristic slow venous outflow was seen in each case. conclusion: ifi with the spy device is a simple and effective method to determine immediate patency and perfusion of the whole pancreaticoduodenal allograft. its use is beneficial in re-exploration to rule out infarcted pancreas allografts. further development in quantification of vascular flow rates and allograft perfusion indices by this device is abstracts in progress. once these are established, this information will be studied in a controlled fashion, and will be compared with clinical outcomes. we have demonstrated safety and developed a protocol for using the spy device in ifi of pancreas transplants. tolerance/immune deviation i tolerance without immunosuppression: exploiting epigenetic regulation as a new approach to achieving donor-specific allograft tolerance. liqing wang, 1 ran tao, 1 joel a. friedlander, 1 wayne w. hancock. 1 1 pathology & immunology, children's hospital of philadelphia & upenn, philadelphia, pa. given toxicities of all current immunosuppressive agents, plus complications of malignancy, infection and chronic rejection, maintaining the search for new approaches to tolerance induction is essential. while weaning of immunosuppression is fraught with risks and costimulation blockade has not yielded the expected boon, use of a broad histone deacetylase inhibitor (hdaci), such as trichostatin a (tsa) or suberoylanilide hydroxamic acid (saha), promotes foxp3 acetylation of foxp3 and binding to target genes, leading to enhanced treg suppressive function, and 2 wks of therapy with hdaci and low-dose rapamycin can induce allograft tolerance. we now show that in addition to acetylation, modulation of a second major epigenetic mechanism, dna methylation, has salutary effects, such that combined use of an hdaci and a dna methyltransferase inhibitor (dnmti, e.g. 5-aza-2 deoxycytidine) has potent effects. microarray analysis of the effects of hdaci on tregs showed down-regulation of multiple genes associated with dna methylation, including dnmt, methyl-cpgbinding domain and associated proteins, leading us to test the effects of dnmti use on treg function. dnmti administration decreased foxp3 gene methylation, enhanced treg gene expression, and increased treg suppression in vitro, and led to a dose-dependent prolongation of cardiac allograft survival (balb/c->c57bl/6) in vivo (p<0.01). the combination of hdaci and lower doses of dnmti, administered for just 2 wks, led to permanent engraftment (>100 d, p<0.001), and the permanent acceptance of second donor allografts but acute rejection of third-party (cba) cardiac allografts indicated induction of donor-specific tolerance post-epigenetic therapy. analysis of long-surviving cardiac allografts showed a minor infiltrate consisting primarily of foxp3+ tregs and an absence of chronic rejection (transplant arteriosclerosis, myocardial fibrosis), whereas combined epigenetic therapy led to only minor prolongation of allograft survival in treg-depleted recipients. in summary, brief use of 2 clinically approved agents (an hdaci plus an dnmti), can synergistically enhance treg function and induce tregdependent donor-specific allograft tolerance without use of any immunosuppression. we conclude that insights gained through epigenetic targeting may provide completely new approaches to the taming and regulation of otherwise powerful immune responses post-transplantation. a novel epigenetic approach to generate mouse and human cd4 + cd25 + foxp3 + regulatory t cells. girdhari lal, 1 nan zhang, 1 william van der touw, 1 yaozhong ding, 1 jonathan s. bromberg. 1 1 gene and cell medicine, mount sinai school of medicine, new york city, ny. background: constitutive foxp3 expression is required for stable and suppressive cd4 + cd25 + regulatory t cells (treg) . we previously showed that demethylation of an upstream cpg island of the foxp3 promoter is characteristic of stable and suppressive treg. using dna methyltransferase inhibitors, we present a novel method to generate antigen-specific treg from mouse and human cd4 + cd25 -t cells. methods: naïve cd4 + cd25 -t cells and natural treg (ntreg) were purified from wild type or foxp3-gfp transgenic mice. human naïve cd4 + cd25 -t cells were purified from pbmc. t cells were cultured with irradiated antigen presenting cells in the presence of il-2, anti-cd3ε mab, tgfβ or the dna methyltransferase inhibitor 5-aza-2'-deoxycytidine (zdcyd), which demethylates the upstream promoter. foxp3 expression was determined by flow cytometry and qrt-pcr. results: naïve t cells cultured under stimulatory conditions with zdcyd express increased foxp3 mrna (30 fold) and intracellular foxp3 protein (35% of cells vs 2% without zdcyd). foxp3 expression is synergistically enhanced with tgfβ (76 ± 4.6% vs 38.8 ± 5.3% zdcyd alone or 44.6 ± 1.4% tgfβ alone), similar to ntreg (92.4 ± 2.5%). tgfβ in combination with other dna methyltransferase inhibitors (procainamide, hydralazine, rg108) also induces foxp3. zdcyd plus tgfβ induced treg stably express foxp3 and similar surface markers and cytokine mrna as ntreg. zdcyd plus tgfβ induced treg suppress proliferation of cd4 + cd25 -t cells, prevent cd4 + cd25 -cd45rb hi induced colitis in scid mice, and enhance islet allograft survival. zdcyd plus tgfβ induce foxp3 expression in antigen-specific wild type or t cell receptor transgenic cd4 + t cells stimulated with alloantigen or peptides, and in naïve cd8 + cd25 -t cells. in vivo administration of zdcyd preferentially preserves thymic cd4 + cd25 + foxp3 + treg generation. zdcyd alone or zdcyd plus tgfb induce strong foxp3 expression in human cd4 + cd25 -t cells compared to tgfβ alone. zdcyd plus tgfβ induced human treg suppress the proliferation of naïve cd4 + cd25 -t cells, whereas tgfβ-induced treg do not. conclusion: dna methyltransferase inhibitors induce stable and suppressive foxp3 + treg from peripheral cd4 + cd25 -t cells. these finding have important implications for understanding t cell development and differentiation, and provide a clinically applicable technique for manipulating epigenetic regulation for the generation of treg for tolerance. macrophages driven to a novel state of activation can promote tolerance. katharina kronenberg, 1 seiichiro inoue, 1 james a. hutchinson, 2 beate g. brem-exner, 2 gudrun e. koehl, 1 hans j. schlitt, 1 fred fandrich, 2 edward k. geissler. 1 1 surgery, university of regensburg, regensburg, germany; 2 surgery, university hospital schleswig holstein, kiel, germany. background: the use of immunomodulatory cells in organ transplantation (tx) is a promising tolerance-induction strategy. here we describe a novel macrophage population capable of enriching t regulatory cells (treg) and promoting tolerance. methods: balb/c bone marrow, spleen and blood mononuclear cells were cultured with m-csf for 5 days, with a 24 hr pulse of ifn-γ on day 4; the resultant adherent cells are referred to as ifnγ-induced monocyte-derived cells (ifnγ-mdc) . residual lymphocytes from these cultures were recultured with the adherent ifnγ-mdc for an additional 3 days. ifnγ-mdc were phenotyped by facs and immunomodulation of lymphocytes was determined by cell counting and facs analysis for tregs. mouse heterotopic heart tx was used for in vivo testing. results: ifnγ-mdc highly express cd11b/c, f4/80 and pd-l1. compared to classically-activated (m1) macrophages, ifnγ-mdc express less cd40/cd86, but higher cd11c. ifnγ-mdc profoundly delete activated, but not resting, lymphocytes (>60%), with cell contact (via transwell system) and caspases (via inhibitors) being essential. most interestingly, ifnγ-mdc highly enrich lymphocytes for cd4 + cd25 high foxp3 + treg (41±2%; n=27). the same, or higher, proportions of treg developed when ifnγ-mdc were produced from macs-purified cd11b + and cd4 + cells (1:1) . ifnγ-mdc culture supernatant showed increased il-10 (elisa) vs. monocyte control cultures (183±42 pg/ml vs. <30 pg/ml, respectively; n=6), suggesting il-10 as one potential mediator. ifnγ receptor signaling and cd40 interactions are required for treg enrichment, as confirmed using ifnγ receptor and cd40 knock-out mice (c57bl/6 mice). ifnγ-mdc derived from cd11b + cells and lymphocytes of ova-transgenic otii mice showed enrichment of treg by 10-fold with high-affinity ova peptide (323-339) vs. treg levels using a low-affinity ova peptide (ova 323-334), suggesting ag-specific treg cells can be enriched with ifnγ-mdc. finally, a single post-heart tx (d+1) i.v. injection of 5x10 6 donor (c3h)-derived ifnγ-mdc prolonged allograft survival in balb/c recipients from 8.5±0.8d in controls (n=5) to 21.4±8.3d (n=11; p=0.001). conclusions: ifnγ-mdc are a novel macrophage population capable of deleting activated t cells and highly enriching remaining lymphocytes for tregs. therefore, ifnγ-mdc could be useful in a clinical setting for promoting transplant tolerance. plasmacytoid dc therapeutic potential is independent of ido induction due to elevated dap12 expression. tina l. sumpter, 1 bridget l. colvin, 1 zhiliang wang, 1 andrew l. mellor, 3 angus w. thomson. 1,2 1 starzl transplantation institute, dept. of surgery, university of pittsburgh, pittsburgh, pa; 2 immunology, university of pittsburgh, pittsburgh, pa; 3 immunotherapy center, medical college of georgia, augusta, ga. optimizing the mechanisms of pdc tolerogenicity may facilitate their use in the development of cell-based tolerance induction strategies. pdc may undermine t cell function via inducible expression of ido, a tryptophan catabolizer that enhances t cell apoptosis. ido is negatively regulated by dap12. in some systems, loss of dap12 correlates with immunostimulatory activation of pdc, while in others, its absence correlates with enhancement of pdc tolerogenicity through induction of ido. in these studies, we characterized the ability of wt and ido-deficient pdc to attenuate murine heart allograft rejection in order to define the contribution of ido and its regulation by dap12 relative to the immunoregulatory potential of pdc. methods: pdc and control myeloid (m) dc were generated from bm cultures of wt or ido ko c57bl/6 (b6; h2 b ) mice. wt mdc, pdc or ido ko pdc were pulsed with alloag (balb/c; h2 d ), stimulated with cpg, and then injected i.v. into syngeneic (b6) recipients 7d before heart transplant. donor ag-specific activation of t cells was analyzed by mlr and elisa. dap12 expression was evaluated by rt-pcr and abrogated with sirna. results: administration of ido ko pdc 7d before transplant prolonged graft survival beyond that seen with control mdc, but not to the extent seen with wt pdc, suggesting involvement of ido. pdc cultures from ido ko mice exhibited a more mature phenotype than their wt counterparts with reduced expression of co-regulatory molecules (e.g., icosl). although ido ko pdc induced enhanced t cell alloactivation compared to wt pdc, use of the ido inhibitor 1-mt indicated that wt pdc do not produce functional ido. further analyses showed wt pdc exhibited high levels of dap12 mrna expression. silencing dap12 had no effect on the ability of wt pdc to activate allogeneic t cell proliferation, but significantly enhanced ifnγ secretion. addition of 1-mt to mlr with dap12-silenced pdc increased t cell proliferative responses to alloag, verifying ido induction in dap12-silenced pdc. conclusions: these data underscore the prophylactic potential of pdc for cell-based therapy that may be independent of ido, due, in part, to elevated dap12 expression. additionally, our data support a role for dap12 in both immunostimulation and immunoregulation by pdc. rapamycin preferentially blocks the expansion of potentially tolerogenic plasmacytoid dendritic cells in vivo. heth r. turnquist, 1 angus w. thomson. 1, 2 1 starzl transpl inst and dept of surgery; 2 dept of immunol, univ of pittsburgh sch of med, pittsburgh, pa. rapamycin (rapa), a 'tolerance-sparing' immunosuppressant with anti-proliferative properties, inhibits myeloid (m) dendritic cell (dc) differentiation/maturation in vitro. rapa decreases cd11c + dcs in the mouse spleen and suppresses the expansion of total cd11c + dc following administration of the dc growth factor, fms-like tyrosine kinase 3 ligand (flt3l). however, the influence of rapa on plasmacytoid (p) dc,the principal type-1 ifn producers in the body, known to regulate innate and adaptive immune responses, and reported to promote experimental transplant tolerance, has not been evaluated. methods: dc were propagated from bone marrow for 10 days (d) in flt3l, in the absence or presence of a clinically-relevant dose of rapa (10 ng/ml). in addition, dcs were mobilized in c57bl/10 mice by i.p. flt3l (10 mg/d for 10 d; d1-10). untreated and flt3l-treated groups also received rapa (0.5 mg/kg/d i.p.; d3-10). on d11, dc were isolated by density gradient centrifugation and/or cd11c + positive selection. mdc (cd11c + cd11b + ) and pdc (cd11c lo cd11b -b220 + ) were identified by flow cytometry. results: rapa suppressed pdc and mdc generation in vitro; rapa-dc-treated cultures had only 22% of the pdc and 40% of the mdc found in control conditions. in normal mice, both mdc (54% of control) and pdc (24.5%) numbers were reduced significantly by rapa administration. rapa, when given concurrently with flt3l, blunted the typical profound expansion of mdc. specifically, flt3l and rapa-treated mice displayed a 46±8x increase over control (steady state) numbers compared to a 145±39x increase in mice treated with flt3l alone. flt3l-induced expansion of pdc was to a much greater extent impacted by rapa, as absolute numbers of pdc increased only 6.5±2.2x over control numbers compared to 51±3x increase in absolute splenic pdc in flt3l-treated mice. conclusion: these data identify rapa as a selective suppressor of pdc generation, as described for corticosteroids. this has significant implications, given the use of rapa following organ transplantation, and the suggested importance of secondary lymphoid tissue pdc for promotion of transplant tolerance mediated by treg. due attention to these disparate effects of rapa on regulating cell populations will be necessary to optimize therapeutic regimens for safe promotion of tolerance. the liver is tolerated better than other transplanted organs. this may reflect the inherent tolerogenicity of liver dendritic cells (dc) and interactions with foxp3 + regulatory t cells (treg). recent studies have shown that cd103 expression on dc correlates with induction of foxp3 + treg, and can be enhanced in the presence of transforming growth factor-β (tgf-β) and retinoic acid (ra), both of which are produced in the liver. this study evaluates cd103 expression on liver plasmacytoid (p) and myeloid (m) dc and the potential of liver dc subsets to induce tregs. methods: pdc and mdc were magnetically isolated from livers and spleens of c57bl/10 mice and co-cultured with cfse-labeled allogeneic (balb/c) cd4 + cd25 -t cells. after 4 or 5d of co-culture, t cells were assessed for cfse dilution and intracellular foxp3 expression. rhtgf-β and either all trans or cis-ra were added to co-cultures. cd103 expression was evaluated by flow cytometry. results: cd103 expression was elevated on liver pdc and mdc compared to spleen pdc or mdc, with higher expression on liver mdc. pdc from the liver and the spleen were poor inducers of foxp3 in cd4 + cd25 -t cells compared to mdc. foxp3 induction was enhanced with tgf-β when splenic pdc were used as stimulators. however, when liver pdc were used as stimulators, tgf-β had no effect on foxp3 induction. ra (either all trans or cis) enhanced induction of foxp3 + cells in cd4 + cd25 -t cells in the presence of tgf-β when splenic pdc but not liver pdc were used as stimulators. tgf-β and tgf-β with ra also enhanced foxp3 induction in cd4 + cd25 -t cells when either spleen or liver mdc were used as stimulators, though splenic mdc were superior inducers of foxp3. conclusions: many studies have focused on naturally-occurring foxp3 + tregs in systemic tolerance. our data show that liver mdc and pdc are poor inducers of foxp3 in t cells, even in the presence of tgf-β and ra. these data suggest that the inherent tolerogenicity of liver dc subsets may be independent of treg induction or that liver dc interact with treg through alternative mechanisms. background: t cell-mediated immune rejection occurs in organ transplantation. in addition to mhc-tcr signaling, t cell activation requires costimulation from antigen presenting cells. b7 molecules (cd80/cd86) and cd40 are critical costimulatory molecules in t cell activation. insufficient or lack of costimulation results in inactivation or tolerance. we hypothesized that blocking the costimulation pathway using small interfering rna (sirna) expression vector can prolong allogeneic heart graft survival. method: vectors that express hairpin sirnas specifically targeting cd40 and cd80 were prepared. recipients (balb/c mice) were treated with cd40 and/or cd80 sirna vectors, 3 and 7 days prior to heart transplantation. control groups were injected with a blank vector and sham treatment (pbs). after sirna treatment, a fully mhcmismatched (balb/c to c57/bl6) heart transplantation was performed. result: allogeneic heart graft survival (>100days) was approximately 70% in the mice treated simultaneously with cd40 and cd80 sirna vectors. in contrast, allogenic hearts transplanted into recipients treated with blank vector and pbs stopped beating within 10 days. hearts transplanted into cd40 or cd80 sirna vector-treated recipients had an increased graft survival time compared to negative control groups, but did not survive longer than 40 days. real time pcr and flow cytometric analysis showed an upregulation of foxp3 expression in spleen lymphocytes and a concurrent downregulation of cd40 and cd80 expression in splenic dendritic cells of sirnatreated mice. an mlr, using splenic dendritic cells (dcs) isolated from tolerant recipients, showed a significantly lower t cell proliferation capacity in cd40-and cd80-sirna vector-treated mice, compared to control groups. tolerant dcs from cd40-and cd80-treated recipients promoted cd4+cd25+foxp3+ regulatory t cell differentiation. finally, tissue histopathology demonstrated an overall reduction in lymphocyte interstitium infiltration, vascular obstruction, and edema in mice treated with cd40 and cd80 sirna vectors. conclusion: this study demonstrates that the simultaneous silencing of cd40 and cd80 genes has synergistic effects in preventing allograft rejection, and may therefore have therapeutic potential in clinical transplantation. costimulation blockade (cob) of the cd28/b7 pathway has long been suggested as a means of attaining indefinite, well-tolerated, antigen-specific prophylaxis from allograft rejection. although effective in rodent models, cd28/b7 blockade is not alone sufficient to prevent rejection in more robust primate models. the relative presence of allocrossreactive memory t cells is one mechanism of cob resistant rejection. lfa3-ig is an approved agent for the treatment of psoriasis, shown clinically to deplete tem cells in psoriatic lesions. we hypothesize that lfa3-ig specifically targets cob resistance cells, including heterologous alloreactive t cell memory, while avoiding interference of peripheral mechanisms that foster cob-mediated allograft acceptance. rhesus monkeys underwent mhc mismatched renal allotransplantation. ctla4-ig was given 20 mg/ kg iv on days -1, 0,3,7,14,21,28 and 10 mg/kg iv on days 35,42,49,56. lfa3-ig, (0.3 mg/kg iv) was given on day -1,0,3,7 then weekly (8 wks). sirolimus was given orally (1mg/kg) days 0-90 and donor specific transfusion (7 ml/kg iv) on day -1. animals were followed by polychromatic flow-cytometry to quantify t cell subsets. lfa3-ig synergized with ctla4-ig successfully preventing renal allograft rejection in this model and leading to indefinite survival in 2/5 animals (table) . this enhanced survival was associated with a significant reduction in tem cells in animals treated with lfa3-ig compared to animals without lfa3-ig treatment (figure). lfa3-ig withdrawal led to tem re-population and rejection. this therapy regimen, all clinically available agents, promotes cob-mediated graft acceptance without the use of calcineurin inhibitors, steroids, or gross t cell depletion. the aim of this study was to investigate the immunological pathways that regulate t cell dependant allograft responses in the hope of finding novel immunomodulatory compounds. islet (and skin) allografts were transplanted into full mhc-mismatched wild type (wt) and baff-transgenic (baff-tg) recipient mice. allograft function/ rejection was monitored by blood glucose levels and/or confirmed by nephrectomy. histology, splenocyte populations & t cell functions were analyzed. the b cell activation factor from the tnf family (baff) is critical for b cell survival. t cells express baff receptors suggesting that baff may also play a role in t cell responses. contrary to expectations, we found that baff-tg mice accepted a full mhc-mismatched islet allograft for >100 days. in addition, baff-tg mice also showed delayed skin graft rejection. this was due to a t cell intrinsic change in allo-responsiveness as shown by a failure of purified baff-tg t cells to reject an allograft in adoptive transfer experiments. however, baff-tg t cells were not anergic per se as they proliferated normally to mitogens in vitro and to antigenic challenge in vivo. intriguingly, baff-tg mice harbored an increased frequency of t regulatory cells in the periphery as compared to wt mice. elimination of cd25 + t cells restored normal allograft rejection to baff-tg mice, demonstrating that the increased number of treg cells were responsible for their altered allo-immunity. in a second approach, baff-tg t cells depleted of cd4 + cd25 + t cells were adoptively transferred to transplanted rag recipients, and in this case the baff-tg cd4 + cd25 -t cells rejected their allograft. proliferation assays showed that excessive baff was not promoting treg expansion by driving proliferation in the periphery. adoptive transfer of gfp expressing syngeneic splenocytes did not exhibit enhanced survival in the presence of excessive baff. however, analysis of treg cells in the thymus of baff-tg mice showed an increased intrathymic frequency. together these results demonstrate that baff-tg mice harbor an expanded number of treg cells that prevent th1-type immune responses including allograft rejection. furthermore, we demonstrate that manipulating baff levels may provide a means to generate immunosuppression free dominant allograft tolerance. we have previously reported the outcome of pig-to-baboon thymokidney transplants from galt-ko miniature swine using an immunosuppressive regimen designed to facilitate the induction of tolerance. although the results were superior to results using hdaf thymokidneys, there was a high rate of early post-operative complications. we investigated whether the elimination of steroids and whole body irritation (wbi) from the treatment protocol would decrease the complication rate in the perioperative period. methods: 7 baboons received thymokidney transplants from galt-ko miniature swine. the immunosuppressive regimen was based on the previously published protocol, but eliminated steroids and substituted rituximab for wbi. it consisted of thymectomy day -21, splenectomy day 0, atg, rituximab, mmf and anti-cd154 mab. renal function was assessed by serum creatinine levels. evidence for baboon thymopoiesis in the pig thymus was assessed by facs and immunohistochemistry. results: one animal died due to a drug reaction at pod 18 with normal renal function and no evidence of infection. the remaining 6 baboons showed no signs of rejection although igm deposition was observed, likely due to preformed non-gal nab. there were no deaths due to rejection. although early infectious complications were not seen, late complications leading to mortality were still observed, including systemic cmv infection (pod 28), pleural effusions (pod 40, 57) , ards with pulmonary hemorrhage (pod 49, 81) and acute myocardial infarction (pod 83). two animals showed evidence for early thymopoiesis in the pig thymus by facs and immunohistochemistry. cd4+/cd8+ thymocytes were seen in the thymic grafts, indicating t cell development was supported by the pig thymus. the average survival of the steroid-free group was 51 days including the animal with the drug reaction and 56 days excluding it, compared to 34.6 days in the regimen that included steroids/wbi. conclusions: a steroid-free and wbi free regimen designed for the induction of tolerance across the pig-to-baboon xenogeneic barrier had fewer early post-operative complications than previous regimens. greater than 50-day average survival of recipients of life-supporting xenogeneic thymokidneys was observed without rejection. this regimen also appears to permit baboon thymopoiesis in the pig thymus. purpose: the effects of human decay accelerating factor (hdaf) addition to the galactosyl transferase knock-out (galt-ko) background in transgenic pig kidney xenotransplantation in baboons has not been previously studied. methods: baboon recipients of galt-ko (n=4) or galt-ko/hdaf (n=2) pig kidneys received flolan, heparin and/or aspirin. steroids (s), cobra venom factor (v), atg (a), mmf (m), and/or a low-dose cd154 blockade (c) regimen were given. results: pre-transplant (tx) anti-non-gal antibody (ab) titers were inconsistently associated with early galt-ko kidney xenograft failure. high d-dimer levels 4 hours post-tx were closely associated with early galt-ko graft loss but not when assessing levels of c3a, btg, or increased cd62p expression on circulating platelets. regimens lacking mmf and/or cd154 blockade elicited by day 7-14 large anti-donor non-gal ab responses . post-operative creatinine levels for the galt-ko/hdaf recipients were lower along with superior graft survival compared to galt-ko; d-dimer, anti-donor non-gal ab, c3a, btg, and f1+2 measurements are in progress for the galt-ko/hdaf recipients. galt-ko/hdaf v,a,m,s,c >10* tbp 239 1.9; 1.5 * graft survival at time of abstract submission as graft still functioning at time of abstract submission; tbp --to be processed ! --anuric; non-life supporting, but appeared viable, pink, and well-perfused until day 7 conclusion: early failure of vascularized galt-ko organs is closely associated with activation of coagulation pathways; whether this is triggered primarily by anti-non-gal ab or by other mechanisms is not addressed by our study. preliminary results using galt-ko/hdaf pig kidneys show promise in attenuating or possibly preventing these and/or other such mechanisms. the established efficacy of cd154-based costimulation blockade with mmf to prevent induced anti-non-gal antibody elaboration is confirmed by our preliminary findings. recovery of cardiac function after pig-to-primate orthotopic heart transplant. christopher g. a. mcgregor, 1 william r. davies, 1 keiji oi, 1 henry d. tazelaar, 1 randall c. walker, 1 krishnaswamy chandrasekaran, 1 guerard w. byrne. 1 1 mayo clinic, rochester, mn. xenotransplantation has been proposed as a method to alleviate the shortage of donor organs. we report survival of up to 8 weeks of three orthotopic transplants after pig-tobaboon cardiac xenotransplantation. pig-to-baboon orthotopic transplantation was performed using an adult baboon recipient and cd46 transgenic pig. the recipients were treated with an α-gal polymer to block the effects of anti-gal antibody and with atg induction and a tacrolimus and sirolimus based maintenance immunosuppression. heart function was continuously monitored by intramyocardial electrocardiography and every 3 -4 days by serial echocardiography. standard hematological, serum chemistry and cardiac troponin levels were monitored every 2 -3 days. the animals survived 34, 40, and 57 days in a healthy condition. mortality was a result of pneumonitis, respiratory failure and bowel infarction respectively. one recipient (survival 40 days) showed impaired perioperative left ventricular function with an ejection fraction of 25% on echo. over the next two weeks, the ejection fraction in this animal returned to normal (60%) and troponin levels normalized. this study shows the longest survival of orthotopic cardiac xenografts to date with recovery of impaired heart function after early ischemia reperfusion injury. this significant improvement in ventricular function suggests that the normal reparative processes appear to function across the xenotransplantation barrier, supporting the potential clinical potential of cardiac xenotransplantation. purpose: to determine whether the galactosyl transferase gene knock-out (galt-ko) protects pig lungs from hyperacute lung rejection (halr) by human blood. the effects of adding human decay accelerating factor (hdaf) to the galt-ko background will also be examined for the first time. methods: heparinized fresh human blood was used to perfuse galt-ko pig lungs (n=10). lung function was assessed by flow, pulmonary vascular resistance (pvr), oxygen transfer, and tracheal edema using pre-defined survival endpoints. historical wild-type (wt) pig lungs (n=15) provide context for analysis. additionally, two pig lungs expressing galt-ko/hdaf were recently examined, one of which received treatment with xigris. results: median lung survival in galt-ko lungs was 114 minutes (range 15min to >240min) vs. 10 minutes in wt lungs (range 4min to 36min, p= 0.01); 2/2 galt-ko/hdaf pig lungs survived >240 min. pvr at 5 minutes for galt-ko lungs was 62±35mmhg-min/l vs.wt lungs (308±178 mmhg-min/l at 5 minutes) vs. 23mmhgmin/l for each galt-ko/hdaf lung. complement activation (∆c3a) at 15 minutes was significantly lower with galt-ko (∆c3a 133±243 ng/ml) than wt lungs (vs. 2080±1332 ng/ml, p=0.039). galt-ko was also associated with reduced platelet activation: only 4±4% of circulating platelets express cd62p at 15 min. vs. 16±13% in the wt group (p=0.05); ∆βtg at 15 min [223±151 iu/ml (galt-ko) vs. 1196±1186 iu/ ml (wt)], and less thrombin formation (∆ f1+2) at 15 minutes (galt-ko: 5±7.9 nm vs. wt: 22±39 nm). ∆c3a, % of circulating platelets expressing cd62p, ∆βtg, and ∆ f1+2 measurements are in progress for galt-ko/hdaf. platelet sequestration was delayed as mean % of the initial platelets remaining in the galt-ko lung perfusate was 52±19% at 15min vs. 27±9% in the wt lung group (p=0.02). neutrophil sequestration was also diminished in galt-ko (58±15% residual) vs. wt lung (5±3% residual, p=0.004). conclusion: galt-ko pig lungs are significantly protected from several facets of halr: complement and coagulation cascade activation, neutrophil sequestration, and platelet activation are all partially attenuated by this modification alone. preliminary results suggest that galt-ko/hdaf lungs offer even further protection. disseminated intravascular coagulation is associated with tissue factor expression on recipient platelets and monocytes. chih che lin, 1, 3, 4 mohamed ezzelarab, 1 corin torres, 1 david ayares, 2 anthony dorling, 3 david k. c. cooper. 1 1 thomas e. starzl transplantation institute, university of pittsburgh, pittsburgh, pa; 2 revivicor inc., blacksburg, va; 3 department of immunology, imperial college london, hammersmith hospital, london, united kingdom; 4 department of surgery, chang gung memorial hospital, kaohsiung, taiwan. purpose acute humoral xenograft rejection (ahxr), frequently associated with disseminated intravascular coagulation (dic), remains a challenge in pig-to-primate xenotransplantation (tx). a previous in vitro study showed that recipient platelets and monocytes were induced to express tissue factor (tf) after incubation with porcine endothelial cells, and we speculated this may contribute to thrombosis. the present study investigated whether circulating (extragraft) monocytes and platelets express tf and whether this relates to the development of dic after pig tx. methods baboons (n=7) received an aortic patch or kidney from either a wild-type (wt) or α1,3galactosyltransferase gene-knockout (gtko) pig, with or without immunosuppressive therapy. baboon monocytes and platelets were isolated from blood before and after tx. surface tf phenotype was determined by flow cytometry. functional tf activity was determined by clotting time assays after mixing monocytes and platelets with recalcified factor vii (fvii)-deficient plasma with or without added fvii. before tx, monocytes and platelets did not express tf or display tf-dependent procoagulant activity. after gtko aortic patch tx, the baboons were euthanized by day 35 without developing dic. however, by day 28, circulating platelets (but not monocytes) expressed tf and promoted tf-dependent clotting in recalcified plasma. in the absence of immunosuppression, wt kidneys survived <1 day before the onset of dic, at which time tf activity was detected on both platelets and monocytes. after gtko kidney tx in immunosuppressed baboons, platelets expressed tf as early as day 3. in contrast, monocytes began to express tf only at the onset of dic. this study links expression of tf on recipient monocytes and platelets with the development of dic. expression on platelets appeared to predict the subsequent development of dic, whereas that on monocytes was associated with the onset of dic. whilst our observations do not establish a causal relationship, they provide the basis for further study. international human xenotransplantation inventory. antonino sgroi, 1 leo buhler, 1 megan sykes, 2 luc noel. 3 1 surgical research unit, department of surgery, geneva university hospital, geneva, switzerland; 2 transplantation biology research center, massachusetts general hospital, boston, ma; 3 world health organization, geneva, switzerland. background: xenotransplantation carries inherent risks of infectious disease transmission to the recipient and even to society at large, and should only be carried out with tight regulation and oversight. a collaboration between the international xenotransplantation association, the university hospital geneva and the world health organization has established an international inventory (www.humanxenotransplant. org) aiming to collect basic data on all types of xenotransplantation practices on humans that are currently ongoing or have been recently performed. methods: we collected information using publications in scientific journals, presentations at international congresses, internet-based information, and declarations of ixa members. an electronic questionnaire is available on the website www. humanxenotransplant.org, which can be filled out and sent to the office in geneva. results: we identified a total of 16 recent or current human applications of xenotransplantation, eight were currently ongoing and one will start soon. the source animal was: pig (n=7), sheep (n=3), calf (n=1), rabbit (n=2), blue shark (n=1), hamster (n=1), and unknown (n=1). all trials transplanted xenogeneic cells, i.e. islets of langerhans (n= 6), hepatocytes (n=1), kidney cells (n=1), chromaffin cells (n=1), embryonic stem cells (n=2), fetal (n=4) and adult cells (n=1) of various organs. the treatments were performed in 10 different countries, 7 in europe, 2 in russia, 2 in asia, 2 mexico, 1 in usa and 1 in africa. six countries had no national regulation on xenotransplantation. conclusion: several clinical applications of cell xenotransplantation are ongoing around the world, often without any clear governmental regulation. this information should be used to inform national health authorities, health care staff and the public, with the objective of encouraging good practices, with internationally harmonized guidelines and regulation of xenotransplantation. cells from α1,3-galactosyltransferase gene-knockout pigs additionally transgenic for human membrane cofactor protein demonstrate resistance to human complement-mediated cytotoxicity. hidetaka hara, 1 cassandra long, 1 mohamed ezzelarab, 1 peter yeh, 1 carol phelps, 2 david ayares, 2 david k. c. cooper. 1 1 surgery, thomas e. starzl transplantation institute, university of pittsburgh, pittsburgh, pa; 2 revivicor, inc., blacksburg, va. purpose: (1) to compare the antibody binding and complement-mediated cytotoxicity (cdc) of human sera to pig peripheral blood mononuclear cells (pbmc) and porcine aortic endothelial cells (paec) from (i) wild-type (wt), (ii) α1,3-galactosyltransferase gene-knockout (gtko), (iii) human membrane cofactor protein transgenic (mcp) and (iv) gtko/mcp pigs. (2) to investigate the effect on binding and cdc of human sera following activation of paec. methods: pooled human serum was tested by flow cytometry for binding of igm and igg (5% serum concentration) and cdc (25% serum concentration) to pbmc and paec from wt, gtko, mcp, and gtko/mcp pigs. paec from all 4 pig types were activated by ifn-γ, and again tested for antibody binding and cdc. results: there was higher binding of igm and igg to wt and mcp than to gtko and gtko/mcp pbmc and paec, but there were no differences in binding (i) between wt and mcp cells or (ii) between gtko and gtko/mcp cells. cdc of wt pbmc and paec was significantly greater than of gtko, mcp, and gtko/mcp pbmc (wt 73%; gtko 37%; mcp 30%; gtko/mcp 4%) and paec (wt 51%; gtko 18%; mcp 1%; gtko/mcp 0%) (both p<0.05). importantly, there was no lysis of gtko/ mcp paec. after activation of paec, although cdc was increased against wt and gtko paec, mcp and gtko/mcp paec demonstrated significant resistance to lysis (wt 73%; gtko 41%; mcp 5%; gtko/mcp 0%). conclusions: (1) cdc of all 3 types of genetically-engineered pbmc and paec was significantly lower than of wt pbmc and paec, with lysis of gtko/mcp paec being significantly lower than to gtko or mcp cells. (2) paec from both mcp and gtko/mcp showed resistance to lysis even after activation of the cells. (3) organs from gtko/mcp pigs should provide considerable protection against cdc. role for cd47-sirpα signaling in human t cell proliferation in response to stimulation with porcine antigen presenting cells. hiroyuki tahara, 1 hideki ohdan, 1 kentaro ide, 1 toshimasa asahara. 1 1 department of surgery, hiroshima university, hiroshima, japan. we have previously proven that genetic induction of human cd47 on porcine cells provides inhibitory signaling to signal regulatory protein(sirp) α on human macrophages; this provides a novel approach to preventing macrophage-mediated xenograft rejection. a recent report indicated that the similar cd47-sirp system negatively regulates the functions of both t cells and antigen presenting cells(apcs) in humans. we hypothesize that the interspecies incompatibility of cd47 may also act as an additional barrier of t cell-mediated xenograft rejection. we have analyzed the frequency and proliferative activity of human t cells responding to either porcine or allogenic apcs by using in vitro mixed lymphocyte reaction (mlr) assay with a cfse-labeling technique. irradiated stimulator porcine or human peripheral blood mononuclear cells(pbmcs) were cultured with cfse-labeled responder human pbmcs. by fcm analysis, the number of division precursors was extrapolated from the number of daughter cells of each division and from the mitotic events, and precursor frequencies in cd4 + and cd8 + t cell subsets. using these values, stimulation index(si) was calculated. the frequencies of alloreactive and xenoreactive cd4 + t cell precursors were almost identical, 4.5±0.8% and 4.4±0.9%, respectively (n=10-12, for each type of precursor). however, the si of xenoreactive cd4 + t cells was significantly higher than that of alloreactive cd4 + t cells, indicating a stronger reaction by a single xenoreactive cd4 + t cell. in the presence of human cd47-fc(containing the extracellular domain of human cd47 fused to the fc portion of human ig, 10µg/ml), the si of xenoreactive cd4 + t cells was significantly reduced to a level similar to that of alloreactive cd4 + t cells (n=12, p<0.05), although the frequencies of their precursors were absolutely uninfluenced. the frequencies of alloreactive and xenoreactive cd8 + t cell precursors were also identical, i.e. 3.5±0.3%, 3.9±0.9%, respectively (n=10-12). the si of both alloreactive and xenoreactive cd8 + t cells did not differ: however, that of xenoreactive cd8 + t cells was significantly suppressed in the presence of human cd47-fc (n=12, p<0.05). these results suggest that the t cell responses to porcine cells are stronger than those to allogeneic cells because of the interspecies incompatibility of cd47. moreover, genetic manipulation of porcine apcs to induce human cd47 expression might attenuate human t cell-mediated xenograft rejection. reactivation of human cytomegalovirus (hcmv) is a potential risk following the clinical application of pig-to-human xenotransplantation. since little is known about undesirable side-effects arising from hcmv infection of porcine organs, we investigated the capabilities of various hcmv strains to infect porcine endothelial cells (pec) and putative immunological consequences thereof. pec from different anatomical origins were incubated with the hcmv laboratory strains ad169 and tb40/e or a clinical isolate at a multiplicity of infection (moi) ranging from 0.1 to 100. viral replication kinetics, evolution of cytopathology, and lytic end points were analyzed. consequences of pec infection on human nk cell activation were evaluated using xenostimulation assay assessing nk cell ifng secretion and cytotoxicity. infection was evident and a maximum percentage of infected cells was reached at a moi of 10. hcmv replicated in all tested pec types, with a fraction of infected cells ranging from 1% to 50%. ad169 infection of 2a2 (microvascular ec) resulted in cytopathic effect (cpe) development by 3 dpi and in lysis of 70% of the cells at 7 dpi. contrary, no cpe was observed in ped (aortic ec) up to 15 dpi. infection of 2a2 with tb40/e was non-lytic and resulted in accumulation of both extra-and intracellular virus. virus titers reached a maximum at 5 dpi, peaking at levels 20-fold higher than residual input virus. we then determined whether infected pec supported a complete replication cycle and produced viral progeny. after 5 dpi pec supernatants and lysates revealed the production of significant amounts of virus. preliminary results showed that coculture of human nk cells and infected pec resulted in increased nk killing and ifng production. altogether, these findings provide evidence that pec are permissive and support the complete productive replication cycle of hcmv. however, cpe are cell-type and strain dependent. moreover, pec infection leads to modification of the xenogeneic cytotoxicity mediated by human nk cells. our findings allow a better estimation of the potential role of hcmv cross-species infection following xenotransplantation and may be crucial to guide future clinical trials. chronic rejection/injury: innate and adaptive immunity i testing for donor-specificity of antibodies post-transplantation increases the predictability of chronic rejection. mikki ozawa, 1 arundhati panigrahi, 2 paul i. terasaki, 3 narindar mehra. 2 1 one lambda, inc., canoga park, ca; new delhi, india; 3 terasaki foundation laboratory, los angeles, ca. purpose: post-transplant hla antibodies have been shown to be linked to chronic graft failure, yet some recipients do well in the presence of antibodies. we investigated whether testing for donor-specificity of antibodies improves the predictive value of antibodies in regards to chronic rejection. methods: in a prospective study of 400 renal transplant recipients, post-transplant sera were tested for hla and mica antibodies, and positive sera were tested by single antigen beads to determine whether the antibodies were donor-specific. one year after testing, biopsy and clinical data on the graft status were collected. patients who did not have follow-up or died with function were excluded from analysis. results: of the 350 patients with follow-up and abdr typing info, 102 (29%) were found to have antibodies. sixteen of those had donor-specific a, b, or dr antibodies (dsa), while 26 had non-donor specific ab's (ndsa). sixty patients had only dp, dq, cw, or mica antibodies, and were grouped into "donor-specificity unknown", as these typing were not available at the time of this report. table 1 summarizes the antibody groups and transplant outcome one year later. interestingly, the mean serum creatinine values at the time of antibody testing were very similar among the groups, regardless of the presence of antibody or the type (ranged 1.34 to 1.73mg/dl). one year later, however, a striking 63% of dsa patients had either returned to hemodialysis, were regrafted, had died, or had biopsy-proven chronic rejection, compared to only 23% in ndsa patients and 13% in those who were negative (dsa vs. neg, p=0.00009). mica antibodies also showed significant association with graft failure or can (p=0.03), and donor mica typing is underway. conclusion: in this prospective study of 350 renal patients, dsa detected posttransplantation were highly correlated with chronic rejection. inclusion of specificity analysis in post-transplant antibody monitoring would significantly improve the predictability of chronic rejection. donor cd4 t cells within solid organ transplants contribute to graft rejection. thet su win, 1 sylvia rehakova, 1 margaret negus, 1 kourosh saeb-parsy, 1 martin goddard, 2 eleanor bolton, 1 andrew bradley, 1 gavin pettigrew. 1 1 university of cambridge; 2 papworth hospital, united kingdom. this study examines the contribution of donor cd4 t cells within heart allografts to the development of graft vasculopathy, by providing help to recipient b cells for generating effector autoantibody responses. b6 mice were transplanted with mhc class ii-disparate bm12 hearts. the allo-and auto-antibody responses were quantified, and their contribution to allograft vasculopathy assessed by incorporating b cell deficient mice as recipients. the role of donor cd4 t cells in providing help for autoantibody was examined by removing cd4 t cells from donor hearts before transplantation by three approaches: treating with anti-cd4 mab, administering 1300gy lethal irradiation, and using bm12 donors that are genetically deficient in t cells. the contribution of donor cd4 t cells to autoanitbody development was further assessed by challenge with bm12 cd4 t cells two weeks prior to transplantation. bm12 heart grafts developed progressive vasculopathy and were rejected slowly (mst=95 days, n=17). the contribution of antibody-mediated effector mechanisms was confirmed by histopathological evidence (fibrinoid necrosis and vascular proliferation), by complement c4d staining, and by a reduction in the severity of vasculopathy and long-term graft survival in b cell deficient recipients. surprisingly, no alloantibody was detected, but recipients instead developed autoantibody. the autoantibody response was completely dependent upon the provision of help from donor cd4 t cells; it was abrogated by transplanting cd4 t cell deficient hearts. to further confirm an effector role for autoantibody in vasculopathy development, b6 mice were primed for humoral autoimmunity, but not for alloimmunity, by injecting with highly purified bm12 cd4 t cells prior to transplantation. heart grafts were rejected more rapidly (mst=29 days, n=4, p<0.0001), and demonstrated severe vasculopathy. finally, bm-chimeric recipients that lacked mhc class ii expression only on b cells did not develop autoantibody, suggesting that cognate interaction between the donor cd4 t cells and mhc class ii of recipient b cells is crucial for post-transplant humoral autoimmunity. our results demonstrate the novel finding that help for autoantibody production is provided by graft-versus-host cognate recognition of recipient b cell mhc class ii by donor cd4 t cells. this autoantibody contributes to the development of vasculopathy independently of alloantibody. allograft rejection. gang chen, 1 huiling wu, 1 jainlin yin, 1 josette m. eris, 1 steven j. chadban. 1 1 collaborative transplantation research group/renal medicine, university of sydney/rpah, sydney, nsw, australia. toll like receptors (tlrs) are innate immune receptors that play an important role in innate immunity but also provide a link between innate and adaptive immunity. myd88 is a key tlr signal adaptor. a recent clinical study reported that activation of innate immunity in heart-transplantation recipients through tlr4 contributes to the development of chronic rejection after cardiac transplantation. in this study, we aimed to determine whether tlr4-myd88 signaling is required for the development of chronic allograft damage using tlr4 -/and myd88 -/mice in a murine model of chronic cardiac allograft rejection. methods: cardiac transplants were performed: b6.c-h-2 bm12 (b6.h-2 bm12 ) hearts to wt, myd88 -/and tlr4 -/mice (all c57bl/6-h-2 b -single mhc class ii mismatch) as allografts (n = 6-8/group) and c57bl/6 to c57bl/6 isografts (n = 5). blood and tissue were harvested at day 49 after transplantation. cell infiltration, fibrosis and vasculopathy were assessed histologically (grade 0 -5). cd4 + foxp3 + cells in the blood and spleen were measured by flow cytometry analysis. results: all hearts remained pulsatile until sacrifice. histology of tlr4 -/and myd88 -/recipients showed protection from leukocyte infiltration (1.2±1.1&1.0±1.1vs 3.2±1.4, p < 0.01), fibrosis (0.75±1.0& 0.6±1.2 vs 2.6±2.0, p < 0.05) and vasculopathy (0.28±0.6 & 0.04±0.1 vs 1.9±2.2) at day 49 post transplantation compared to wt recipients. by facs, the ratio of cd4 + foxp3 + regulatory t (treg) cells to total cd4 + cells at day 49 post transplantation in spleen was significantly increased in tlr4 -/and myd88 -/recipients versus wt allograft and isograft mice (18.1±0.2 & 19±0.9% vs 12.9 ±1.1 & 11.8±0.2 %, p <0.01). conclusion: absence of tlr4 and myd88 signaling reduces chronic allograft damage in a murine model of chronic cardiac rejection. one mechanism of protection may be enhancement of regulatory t cell function. promotion of treg generation via the tlr4/ myd88 pathway may be one important consequence of cross-talk between innate and adaptive immunity. pathway: implication in transplant arteriosclerosis. thibaut quillard, 1 stéphanie coupel, 1 flora coulon, 1 juliette fitau, 1 maria-cristina cuturi, 1 elise chiffoleau, 1 béatrice charreau. 1 1 inserm u643, itert, nantes, france. endothelial cell (ec) activation, injury and apoptosis are the key events associated with transplant arteriosclerosis (ta) and chronic allograft rejection (cr). notch is a major signalling pathway controlling vascular function and injury. nonetheless, the involvement of notch, at endothelial level, in both ta initiation and progression remains unknown. using a fully mhc mismatched rat cardiac allograft model of cr, we found that ta at day100 correlates with a strong decrease in both expression and activity of the notch pathway in transplant as compared to tolerant and syngeneic controls. the present study investigates the contribution of notch activity to ec survival. in this purpose, a recombinant adenovirus, encoding the notch2 intracellular domain (nicd) and the reporter gfp cdnas was constructed and used to maintain notch activity in cultured primary human arterial ec. our results demonstrate that nicd protects ec from cell death induced by tnfα in the presence of chx or by anoïkis as measured by annexinv and dna content staining. nicd mediates cytoprotection through the regulation of key-apoptosis molecules. using an apoptosis-dedicated qpcr array, we found that 14 out of a panel of 88 apoptosis-related genes were significantly regulated. nicd upregulated bcl2, bcl2a1and bcl-xl (2.5-, 8.2-and 2.8-fold compared to noninfected cells, respectively). in addition, pro-apoptotic genes bim, drp1, bok and cd40 were markedly downregulated in transduced cells (6.7-, 11.4-, 3.9-and 14 .5-fold decrease, respectively). western blotting analysis confirmed the induction of protective molecules (bcl2 and bcl-xl) and the inhibition of pro-apoptotic proteins (bad, bak and bax). consistent with these data, nicd conducted phosphorylation of akt as well as a reduction of pten expression, suggesting that the cytoprotective activity of nicd may be mediated by recruitment of the pi3k survival pathway. to conclude, our findings indicate that ta correlates with a decreased notch signaling in transplant and that activation of the notch pathway in vascular ec prevents apoptosis by promoting protective gene expression and survival pathway. these data suggest that controlling notch activity may prevent ec dysfunction associated with ta and cr. donor age intensifies the early immune response. christian denecke, 1 xupeng ge, 1 irene kim, 1 daman bedi, 1 anne weiland, 1 anke jurisch, 1 steven mcguire, 1 johann pratschke, 2 stefan g. tullius. 1 1 div. of transplant surg, bwh, transplant surg res lab, boston; 2 dept. of surgery, charite berlin, germany. increasing numbers of organs from elderly donors are currently utilized for transplantation. advanced donor age may not only be associated with physiological impairments but also with a modified immune response of the recipient. we hypothesized a more potent early immune response following the transplantation of elderly donor organs and we analyzed the immune response in a mouse heart transplant model. young b6 mice received heart allografts from 3, 12 and 18mths old bm12 donors. the recipients immune response and intragraft changes were analyzed. elderly, non-manipulated hearts contained overall significantly elevated frequencies of cd4 + and cd8 + t-cells and dcs (cd11c + ) (18mths vs. 3mths: cd4 + : 2.77% vs. 1.35%, cd8 + : 3.90% vs 1.71%, cd11c + : 46.1% vs.11.8%, p<0.05). following engraftment of 18mths old heart grafts numbers of activated dc's (cd11c + i-a b+ and cd11c + cd40 + ) had significantly increased in recipient spleens (day 14:p<0.05). in parallel, frequencies of effector/memory phenotype t-cells (cd4 + cd44 high cd62l low and cd8 + cd44 high cd62l low ) were significantly elevated with increasing age (3 vs. 12 vs, 18mths: cd8 + cd44 high cd62l low :6.7% vs. 8.4% vs. 12.2%, respectively, p< 0.01). in addition, tregs (cd4 + cd25 + foxp3 + ) were also elevated (3 vs. 12 vs, 18mths: 2.9%vs.9.6%vs.11%,p<0.05) t-cell alloreactivity, as measured by ifnγ-production, increased with donor age (3mths vs. 12mths vs.18mths: 24.4±1.5 vs 74.4±21.9 vs. 73.2±7.8 ifnγ-producing spots/5x10 6 cells, p<0.05). mixed lymphocyte reaction (mlr) at day 14 revealed a gradual increase in splenocyte proliferation with advancing donor age (p=0.0002) indicating an enhanced immunogenicity of older organs. immunohistochemical staining confirmed augmented cd4 + and cd8 + t-cell infiltrates and an intense ki67 positivity of gics in 18mths old heart grafts, emphasizing an intensified immune response towards organs from old donors. in summary, old native heart transplants contain an overall higher number of passenger leukocytes contributing to an increased immunogenicity of these organs. after transplantation, a more potent dc and t-cell activation and intragraft t-cell infiltration was observed in elderly organs. nox and chronic kidney allograft interstitial fibrosis. shannon reese, 1 madhu adulla, 1 surmeet bedi, 1 jose torrealba, 1 deb hullett, 1 arjang djamali. 1 1 nephrology, uw madison, madison, wi. to determine the role of oxidative stress (os) in kidney allograft fibrosis, we are developing strategies that would decrease the generation of reactive oxygen species (ros) instead of using ros scavengers, the standard approach to antioxidant therapy so far. we therefore started to examine the expression of nadph oxidase (nox) subunits (nox2, nox4, p22 and p47phox) and their distribution in human and rat kidney allografts with chronic interstitial fibrosis (iftanos). using double-staining immunofluorescent studies in human allografts (n=16) we showed that nox2 was present in injured tubules costained with αsma ( figure 1.1) similarly, interstitial macrophages (cd68 + -nox2 + ) and myofibroblasts (αsma + -nox2 + ) but not cd3 + t cells or s100a4 + fibroblasts expressed high nox2 levels, suggesting that nox is involved in the pathogenesis of allograft fibrosis via epithelial-tomesenchymal transition (emt), macrophage and myofibroblasts activation. we then examined the coexpression of nox2, nox4 and p22phox in normal and transplant kidneys with iftanos and showed that these molecules were upregulated in the latter group and that nox2 and nox4 were associated with p22phox expression. these results were confirmed in the fisher to lewis rat kidney transplant model (figure 1 .2). immunoblot analyses at 3 weeks and 6 months showed that nox4 and nox2 levels were increased in the allogeneic (n=7) compared to syngeneic transplants (n=3). greater nox levels were composite tissue allotransplantation (cta) is a recently introduced option for limb replacement and reconstruction of tissue defects. as other allografts, a cta grafts can undergo immune-mediated rejection, and standardized criteria are required for characterizing and reporting severity and types of rejection. this manuscript documents the conclusions of a symposium on cta rejection held at the ninth banff conference on allograft pathology in la coruna, spain on june 26, 2007, and proposes a working classification scheme, the banff cta-07, for the categorization of cta rejection. this classification was derived from public international consensus discussions regarding all published scoring systems for cta rejection. given the current limited clinical experience in cta, a formal histological classification was established for acute skin rejection with the understanding that other types of rejection involving other tissues will be developed with periodic review of this emerging field. it was agreed that the defining features to diagnose acute skin rejection would include inflammatory cell infiltration with epidermal and/or adnexal structure involvement, epithelial apoptosis, dyskeratosis, and necrosis, and that severity of rejection will be graded under five categories. this classification refines proposed schemas, represents international consensus on this topic, and establishes a working collective classification system for cta reporting. the role of skin biopsies in diagnosing clinical rejection in hand composite tissue allotransplantation (cta). christina l. kaufman, 1, 4 ruben n. gonzales, 1 kadiyala v. ravindra, 3, 4 brenda w. blair, 1 joesph f. buell, 3, 4 warren c. breidenbach. 1, 2, 4 1 christine m. kleinert institute, louisville, ky; 2 kleinert kutz and associates, louisville, ky; 3 jewish hospital transplant center, louisville, ky; 4 surgery, university of louisville, louisville, ky. aim: traditionally allograft biopsy has dictated treatment of allograft rejection. we hypothesize that histological grade of the biopsy may over diagnose rejection in cta hand cta is unique in that the organ is external and early signs of rejection can be viewed directly. skin is the first tissue in the cta graft to show rejection. methods: for this study, rejection by defined by requirement of treatment. these episodes were compared with histological grade, and hand appearance. we reviewed 127 skin biopsies taken during 9, 7 and 1 years of follow up. results: three observations surfaced. first, a rejection grading scale for cta is still evolving. a review of the literature showed at least four different criteria are in use. the criteria used to grade biopsies at our center changed over the 9 year follow up. bias towards calling higher grades of rejection, and more aggressive treatment occurred in the first two patients. a re-read of random biopsies taken from the first two patients showed a down-grade of rejection to grade ii or i in five cases. secondly, concomitant cmv infection in the last patient prevented the aggressive treatment of rejection. despite high grade histology, the swelling, rash and redness responded to topical immunosuppression. systemic treatment was not necessary. analysis indicated that swelling and/or rash, and the percentage of skin involvement seemed to be more informative markers of existing (or resolving) alloreactivity than was histology. the biopsies taken from the graft shown below showed a grade iii histology. thirdly, changes in hand function were not associated with changes in biopsy grade. conclusion: the histologic grade of skin biopsies from hand allografts appears to over estimate rejection. alterations in immunosuppression shoud be based on appearance of the allograft, and clinical course as much as on the histologic grade of the biopsy. expression of molecular mechanisms of lymphozyte trafficking correlates closely with skin rejection in human hand transplantation. theresa hautz, 1 bettina zelger, 2 gerald brandacher, 1 hans g. mueller, 3 andrew w. p. lee, 4 raimund margreiter, 1 stefan schneeberger. 1 1 dept. of general and transplant surgery, innsbruck medical university, austria; 2 dept. of pathology, innsbruck medical university, austria; 3 dept. of dermatology, innsbruck medical university, austria; 4 div. of plastic surgery, university of pittsburgh. introduction: to understand in greater depth the molecular mechanisms involved in skin rejection in hand transplantation, we investigated 8 key molecular markers of lymphocyte trafficking, cellular rejection and antibody mediated rejection in human hand transplantation. methods: a total of 130 skin biopsies taken from three bilateral hand transplants were assessed by h&e histology (grades as per previously a published classification 0-4b) as well as immunohistochemistry using antibodies for following markers: lymphocyte function-associated antigen (lfa)-1 = cd11a, intercellular adhesion molecule (icam)-1 = cd54, selectin e = cd62e, selectin p = cd62p, ve-cadherin = cd144, human leukocyte antigen (hla) ii (dp, dq, dr), psoriasin = s100a7 and c4d. levels of expression were assessed (0, +, ++, +++) and read in the light correlated with the rejection as well as time after transplantation. results: rrejection ranged between grade 0 and 4a with an average score of 0.9. in healthy skin, none of the markers investigated was consistently up-regulated. upon rejection, cd54, cd62e and cd62p staining in endothelial cells was significantly increased. expression of cd62e and cd62p correlated well with severity of rejection. the majority of infiltrating lymphocytes stained positive for cd11a. interestingly, also kerationcytes were highly positive for cd11a at the onset of rejection. cd144 was detected on endothelial cells, but its occurrence did not correlate with rejection. psoriasin expression was observed in keratinocytes in a basal and focal pattern and correlated well with rejection. for c4d, no consistent staining pattern was observed indicating that antibody mediated rejection did not play a role in these patients. conclusion: molecular markers involved in lymphocyte trafficking are up-regulated upon skin rejection after hand transplantation and represent promising target for prophylaxis and treatment of rejection in composite tissue allotransplantation. investigation of the immunomodulatory phenotype of infiltrating lymphozytes in skin rejection of human hand allografts. theresa hautz, 1 gerald brandacher, 1 bettina zelger, 2 hans g. mueller, 3 andrew w. p. lee, 4 raimund margreiter, 1 stefan schneeberger. 1 1 dept. of general and transplant surgery, innsbruck medical university, innsbruck, austria; 2 dept. of pathology, innsbruck medical university, austria; 3 dept. of dermatology, innsbruck medical university, austria; 4 div. of plastic surgery, university of pittsburgh. introduction: skin rejection has complicated the postoperative course in human hand transplantation. to better define the characteristics of the lymphozytic infiltrate human hand transplant biopsies have been investigated for expression of foxp3 and indoleamine 2,3-dioxygenase (ido), a key regulatory enzyme to induce t-lymphocyte unresponsiveness. methods: a total of 104 skin biopsies taken from three bilateral hand transplant recipients during the first 6 years after transplantation were assessed by h&e histology (graded as per previously published classification 0-4b) as well as immunohistochemistry for ido and foxp3. levels of expression were assessed (0, +, ++, +++) and interpreted in the light of clinical courses, time after transplantation, severity of rejection as well as markers for lymphozyte migration. results: overall, rejection ranged between grade 0 and 4a with an average score of 0.94. ido was found constitutively expressed in the endothelium independent of rejection. ido expression in the cellular infiltrate was significantly increased upon and correlated well with severity of rejection (rejection grade 1, 0.91+/-0.85: rejection grade 3, 2.30+/-0.82 p<0.001). foxp3 positive t-cells were mainly found in severe rejection (rejection grade 1, 0.14+/-0.35: rejection grade 3, 0.75+/-0.75 p=0.019). ido expression correlated well with foxp3 expression, although the overall staining intensity for foxp3 was lower. a strong tendency towards higher expression of ido as well as foxp3 towards later time-points after transplantation was observed (year 1 -foxp3 0.07+/-0.26, ido 0.75+ /-0.92 [n=68] , year 3 -foxp3 0.45+/-0.74, , year 5 -fox 0.25+/-0.45, ). expression of ido correlated closely with expression of e-selectin, p-selectin, icam1 and lfa-1. conclusion: characteristics of the cellular infiltrate indicate a strong tendency towards self limitation of the alloimmune response towards the skin with both time after transplantation as well as severity of rejection in human hand transplantation. further studies are warranted to clarify the clinical relevance of these findings. prospective analysis of the immunologic profile of a hand transplant recipient in the first year. kadiyala v. ravindra, 1 warren c. breidenbach, 2 joseph buell, 1 suzanne t. ildstad. 3 1 department of surgery, university of louisville, louisville, ky; 2 kleinert, kutz, and associates, louisville, ky; 3 institute for cellular therapeutics, university of louisville, louisville, ky. introduction: a major obstacle to the wider application of hand transplantation is the long term complications associated with immunosuppression. minimization of immunosuppression is an important goal in all transplant recipients. currently there are no accurate tools to evaluate the immunological responsiveness which might help tailor the level of immunosuppression for an individual patient. the response of recipient lymphocytes to pha, candida, and alloantigen may represent laboratory tool towards this end. it has been reported that these 3 responses are hierarchical with response to alloantigen being the first to be lost, followed by candida and finally pha. a 54 year old male received a proximal forearm transplant in november 2006. immunosuppression included induction with a single 30 mg dose of alemtuzumab and maintenance with tacrolimus and mycophenolate mofetil. the patient developed an episode of cytomegalovirus infection followed by acute rejection after reduction of his immunosuppression during the 3 rd post-operative month. these were successfully treated with ganciclovir and topical tacrolimus & steroids respectively. blood samples were drawn at selected time points, and subjected to phenotyping of lymphocyte subsets and immune monitoring for circulating peripheral blood regulatory t cells (t reg ) and proliferative responses to phytohemagglutinin (pha), candida, and alloantigen. results: alemtuzumab induction resulted in profound lymphopenia. at week 2 and 1 month, the response to pha was intact (stimulation index 110 and 24 respectively), but response to alloantigen and candida suppressed (si <3). a similar immunologic profile persisted up through 6 months. at 1 year, the pha and candida responses are robust (si 69 and 38 respectively), but alloresponses have not returned. current immunosuppression consists of tacrolimus (6-9 ng/ml) and mycophenolate mofetil (500 mg b.i.d.). there is no gross evidence of acute or chronic rejection. conclusions: induction with alemtuzumab alters the recovery of immune response: recovery to candida was delayed beyond 6 months and to alloantigens beyond a year. in light of this, further reduction of immunosuppression may be contemplated in future hand transplants without the risk of rejection. composite tissue allotransplantation has achieved significant clinical advances despite an adequate preclinical model to study technical and immunosuppressive strategies. we have developed a non-human primate model of facial composite tissue allografts (cta). unilateral lower hemi-facial cta (bone, muscle, skin) transplants were performed between mismatched cynomolgus monkeys. immunosuppression consisted of 28 days of continuous iv tacrolimus monotherapy followed by tapered daily im doses. six animals received prophylactic gancyclovir. all animals had serial transplant biopsies. ten transplants have been performed, with one loss secondary to line infection on day 27 without evidence of rejection. two ctas had evidence of chronic rejection (day 56, 99); with development of alloantibody after 30 days. five ctas had prolonged survival (day 60, 86, 108, 150, 177) , but developed ptlds resulting in experimental endpoints. all animals had clinically normal grafts, but 3 animals showed histological evidence of mild rejection not treated with any additional immunosuppressive therapy. ptld tumors were analyzed using short tandem repeats (str) to define donor or recipient origin. str analysis demonstrated donor origin of 4 ptld tumors and recipient origin in 1 animal. none of the ptld animals had clinical evidence of rejection of skin, bone, or muscle. ebv was not detected in the serum of 2 tested animals, and ganciclovir therapy had no effect on the development of tumors. tacrolimus levels of ptld animals were higher than animals with rejected grafts (45 vs. 34 ng/ml; p=0.02). two additional animals have healthy grafts (day 24+, 52+) without evidence of rejection or ptld, and have been converted to rapamycin after day 28. we have developed a preclinical model for facial cta transplantation that achieves prolonged graft survivals with tacrolimus monotherapy. the high incidence of ptld tumors of donor origin represents an outcome similar to bone marrow transplantation in contrast to its rarity in solid organ transplantation. our findings are a cautionary note regarding ctas that include vascularized bone elements. immunosuppressive protocol modifications have been made in an effort to decrease the incidence of these donor-derived ptlds. heterotopic heart transplantation: the united states experience. jama jahanyar, 1 tarek a. sibai, 1 matthias loebe, 1 michael m. koerner, 1 guillermo torre-amione, 1 george p. noon. 1 1 dept. of surgery, baylor college of medicine, houston, tx. heterotopic heart transplantation (hht) is utilized in patients (pts) who do not qualify for standard orthotopic heart transplantation (oht). specific indications include refractory pulmonary hypertension and a donor-recipient size mismatch. the objective of this study was to analyze the unos database and compare outcomes of hht to oht. the unos database with more than 58000 pts undergoing thoracic organ transplantation in the u.s. between 1987 and 2007 was reviewed (based on optn data as of may 1, 2007) . primary endpoint of this study was overall survival and subgroup survival [pts with transpulmonary gradient (tpg)>15, ischemic (icm) and dilated cardiomyopathy (dcm)]. secondary endpoint was assessment of pretransplant criteria. exclusion criteria were retransplantation and missing transplant dates. of 41379 who underwent oht and 178 who underwent hht, 32361 and 111 respectively, were enrolled in this study. [5] [6] [7] [8] [9] [10] survival after oht is superior to hht. this survival benefit however, disappears in pts with a tpg>15. overall the survival after hht is superior to the reported survival in pts who undergo lvad implantation as destination treatment (rematch-trial/1year survival of 52%). thus in selected pts, especially those with elevated tpgs, hht should be considered a viable option with overall good results. ventricular assist device as a bridge to heart transplantation in children: can we afford it? william t. mahle, glen ianucci, robert n. vincent, kirk r. kanter. sibley heart center cardiology, emory university school of medicine, atlanta, ga. ventricular assist devices (vads) allow children with severe heart failure to be bridged to successful heart transplantation (ht). vads are being used with increasing frequency in the pediatric population and newer devices allow even young infants to be supported. vad implantation and maintenance, however, is quite expensive and the cost-effectiveness of vad use in adults has been questioned. to date, an economic analysis of vad support in children has not been undertaken. methods: we used pediatric health information system (phis), an administrative database of the child health corporation of america (a consortium of children's hospitals in north america), to determine the costs related to vad use in children. data on subjects <18 yrs of age from 2002-2007 were reviewed. hospital charges were converted to costs based on hospital-specific cost-to-charge ratios. projected survival for subjects who were successfully bridged to ht was derived from published data. costutility was expressed as cost per quality-adjusted life years (qalys) saved, expressed in 2006 us dollars. all future costs and benefits were discounted at 3%. results: the median age at implantation from the phis database was 11.2 years, range 2 days to 17 yrs. the mean hospital cost per patient was $522,489. estimated survival to heart transplantation was 77% and estimated successful explantation without transplantation was 4%. the calculated cost-utility for vad as a bridge to transplantation was $123,232 /qaly saved. if one assumes that the children who survived to vad explantation would otherwise have a high risk of hospital death (65%) without vad support, then the calculated cost-utility would be $117,235/ qaly saved. even if abstracts survival to transplantation exceeds 90%, vad implantation does not achieve a favorable cost-utility ratio. vad support in pediatric heart only becomes cost-effective if recovery and explantation can be achieved in over 22% of subjects. conclusions: vad supports serves as an effective bridge to heart transplantation in children. however, the cost-utility of this strategy is above the generally accepted threshold for cost-effectiveness ($100,000/qaly). in a setting of limited healthcare resources vad as a bridge to transplantation may not be justified. purpose: heart transplantation (tx) in patients with hla sensitization presents challenges in organ allocation. virtual crossmatch (vxm), in which recipient hla antibodies, identified by labscreen pra beads, are compared to the prospective donor hla-type, could increase the use of allografts from distant donors (dd). accuracy of vxm and outcomes of this approach in heart tx are not known. methods and materials: to increase time-efficiency at the time of organ allocation, crossmatch testing is frequently initiated on pre-set trays which contain sera of multiple prospective sensitized recipients. we used results from these studies to determine expected accuracy of vxm. we assessed outcomes of allocation algorithm implemented in 2001 in sensitized patients. conventional prospective crossmatch was done when allografts were procured from local donors (ld), while vxm was utilized when allografts were offered from dd. there were 257 direct t-cell ahg crossmatch tests done with sera of 12 potential allograft recipients who had preformed hla-antibodies of known class i hla specificities. as shown in table 1, the positive and negative predictive values (ppv, npv) of vxm were 92% and 79%. table 2 shows outcomes in 30 sensitized patients who were eligible for vxm approach. 14 received allografts from ld with negative prospective crossmatch while 16 (53%) received allografts from dd with negative vxm. three dd patients had a positive retrospective crossmatch -npv of vxm was 81% in this cohort. vxm has high negative and positive predictive values, accurately predicting results of standard direct crossmatch in most patients. vxm allows use of allografts from dd and is likely to improve organ allocation in the disadvantaged group of sensitized patients. single center experience with new heart allocation system implemented by united network of organ sharing. biljana pavlovic-surjancev, 1 nilamkumar patel, 1 linda dusek, 1 james sinacore, 1 jennifer johnson, 1 cassie bessert, 1 alain heroux. 1 1 heart failure/heart transplant program, loyola university medical center, maywood, il. purpose: in july 2006, united network of organ sharing (unos) implemented a new allocation system for adult heart transplant (tx) candidates with the following sequence: local status 1a→local status 1b→zone a status 1a→zone a status 1b→local status 2. the purpose of this study is to evaluate the impact of new system on heart tx patients at a single center in region 7. methods: patients transplanted during 1 year (y) prior to new system (7-11-05 to 7-11-06, group 1, n=19) and 1 y following new system (7-12-06 to 7-12-07,group 2, n=26) were compared for unos status at the time of transplant, waiting time, ischemia time, length of the hospital stay (los) before and after transplant, donor age, procurement-team travel distance and cost. results: new system significantly decreased median waiting time, but increased median ischemia time without affecting short-term survival: 1 patient died in each group. number of transplants increased 37% in group 2 mostly due to increased number of status 1a patients supported by iabp without change in number of status 1b and 2 patients. median pre-transplant los increased 16-fold in group 2 (p=0.072), whereas mean pre-transplant los increased by 5 days. in group 2, thirteen patients had donor heart procured in zone a and thirteen patients had donor heart procured locally, whereas in group 1, all donor hearts were procured locally. median procurement-team travel distance increased 15-fold and median travel cost 10-fold in group 2 (p<0.05). clinical outcomes associated with simultaneous heart-kidney transplantation. tariq shah, 1, 3, 4, 5 suphamai bunnapradist, 2 jagbir gill, 2 steven k. takemoto. 1 the figure below indicates recipients of shk transplants (open symbols) had lower rates of rejection when compared to heart (square) or kidney (diamond) recipients. survival rates were initially higher for kidney recipients with rates for shk similar to heart recipients. the hazard ratio (hr) for graft loss was higher for shk recipients compared to kidney, but lower when compared to heart recipients. the lower hazard for shk in heart allografts might be attributed to risk associated with pretransplant dialysis (hr=4.09, 3.81-4.39, p<0.001) . approximately 10% (2,122) of cardiac transplant recipients initiated dialysis prior to transplantation. the differing rates of shk rejection observed in the heart and kidney analytic files might be attributed to susceptibility or treatment of heart and kidney allografts, rejection monitoring or reporting. conclusion: retrospective examination of data provided to the optn indicates simultaneous heart-kidney transplantation seems to be effective for cardiac transplant candidates who require dialysis. abstract# 123 objective: krp203, a novel structural analog of fty720, has been documented to display 5-fold greater selectivity of agonism for sphingosine-1-phosphate (s1p) type 1 (s1p 1 ) receptor compared with s1p 3 receptor. clinical trial has shown that fty720 produced dose-limiting toxicity-bradycardia, due to its effect on s1p 3 receptor. we have tested the effect of krp203 on the survival of islet allografts either alone or combined with local delivery of cd4 + cd25 + foxp3 + t regulatory (treg) cells. previous studies using knockout mice have documented a key role for the integrin cd103 in promoting allograft rejection. these data are consistent with a critical role for cd103 expressing cells in this process. however, a direct test of this hypothesis has proven problematic due to the lack of mabs that efficiently deplete cd103 + cells in wild type hosts. to circumvent this problem, we conjugated the non-depleting anti-cd103 mab, m290, to the toxin, saporin (sap), to produce an immunotoxin (m290-sap) that selectively depletes cd103-expressing cells in vivo. treatment of naive mice with m290-sap selectively depleted cd103 + cd8 + cells and dramatically reduced the overall frequency of cd8 + lymphocytes in diverse compartment including intestinal intraepithelial lymphcyte,spleen and mesenteric lymph node. m290-sap also depleted cd103 + dendritic cells (cd11c + ) and t regulatory cells (tregs, cd4 + cd25 + ) in the above compartments. in the thymus, m290-sap depleted cd103-expressing cells in both cd4 -cd8and cd4 -cd8 + subpopulations, both of which express cd103, leading to a dramatic reduction in the number of thymocytes(77.08 ± 5.74 ×10 6 vs. 2.71 ± 0.82 ×10 6 , p<0.0001, in control vs. treated respectively). we next assessed the effect of m290-sap in a fully allogeneic islet transplantation model (balb/c→c57bl/6). m290-sap produced long-term (lt) graft survival (>100d, n=9,vs. untreated median survival time 13d, n=8 ). unconjugated m290 or isotype control (igg-sap) did not significantly prolong islet allograft survival. graft histology showed little, if any, lymphocyte infiltration surrouding islet transplants in lt mice. in contrast, intense lymphocyte infiltration with disruption of islet morphology was observed in untreated mice. pretreatment of donor islets with m290-sap did not significantly prolong allograft survival indicating that the immunosuppressive effect of m290-sap resides at the level of host. interestingly, we found a 3-4 fold increase in both percentage and number of foxp3 + cd4 + cd25 + cells in the spleen and draining lymph node from lt mice, though it remains to be determined whether such cells account for graft acceptance in m290-sap treated recipients. in summary, these data document that depletion of cd103 expressing cells promotes long-term islet allograft survival. these findings point to a novel strategy for therapeutic intervention in islet allograft rejection. pancreatic objective: to engineer pancreatic islets in a rapid and efficient manner with a novel form of fasl protein chimeric with core streptavidin and test the efficacy of engineered islets for long-term survival in allogeneic hosts. methods: balb/c pancreatic islets were engineered first by cell surface modification with biotin followed by the display of a chimeric form of fasl protein that consists of extracellular domain of fasl fused c-terminus with core streptavidin (sa-fasl). sa-fasl-engineered islets were transplanted into streptozotocin diabetic c57bl/6 mice under transient cover of rapamycin. unmodified islets or those engineered with streptavidin protein (sa) served as controls. results: all the islets showed effective engineering with sa-fasl, which persisted on the surface of islets for weeks in vitro as assessed by confocal microscopy. all the islets (n=23) engineered with sa-fasl survived over the observation period of 100-400 days without detectable signs of rejection. in marked contrast, all the unmodified (n=9) and sa-engineered (n=14) islets underwent acute rejection within 40 days. the observed tolerance was localized to the engineered islets as unmodified second set of islets transplanted under contralateral kidney of long-term (>90 days) graft recipients were rejected in a normal tempo (mst=17 ± 9 days) without any effect on the survival of primary islets. conclusions: engineering pancreatic islets with exogenous immunomodulatory molecules, such as sa-fasl, in a rapid (∼ 2 hrs) and efficient (100% of targeted islets) manner represents a novel means of immunomodulation with considerable therapeutic potential for the treatment of type 1 diabetes. supported in parts by nih (r21 dk61333, r01 ai47864, r21 ai057903, r21 hl080108), jdrf (1-2001-328) the ability of embryonic stem (es) cells to form cells and tissues from all three germ layers can be exploited for the generation of cells that can be used to treat diseases. in particular, successful generation of hematopoietic cells from es cells could provide safer and less immunogenic cells than bone marrow cells, that require severe host preconditioning, when transplanted across mhc barriers. in the past, it has been difficult to derive hematopoietic cells from es cells. it has now become clear that this was due to the lack of self-renewal properties by these newly developed progenitor cells. here, we exploited the self-renewal properties of ectopically expressed hoxb4, a homeobox transcription factor, to generate hematopoietic progenitor cells (hpcs) that successfully induce high level mixed chimerism and long-term engraftment in recipient mice. hoxb4-transduced 129svj es cells (h2 b ) were allowed to form embryoid bodies. these were dismantled after 5 days and the cells treated with a cocktail of hematopoietic cytokines. by day 26, es cells had formed hpcs. these newly generated hpcs were cd45+, cd34+, cd117+ but poorly expressed mhc class i molecules and no class ii. the hpcs fully restored splenic architecture in rag2 -/-γ c -/immunodeficient mice, abstracts comparable to bone marrow. additionally, hpc-derived newly generated t cells were able to mount a peptide-specific response to lymphocytic choriomeningitis virus (lcmv) and specifically secreted il-2 and ifn-γ upon cd3 stimulation. further, hpc-derived antigen presenting cells (apcs) in chimeric mice efficiently presented viral antigen to wild type (wt) t cells. in syngeneic recipient mice, hpcs engrafted and formed more robust t and b cell populations. the majority of the hpc-derived cells were however, gr-1 + , suggesting a bias towards myeloid cells by the hoxb4. interestingly, these cells successfully engrafted in allogenic mrl (h2 k ) and balb/c (h2 d ) recipients without the need for immunosuyprresion. this ability to form mixed chimerism across mhc barriers is a consequence of their lack of mhc, cd80 and cd86 expression. our results demonstrate for the first time that leukocytes derived from es cells ectopically expressing hoxb4 are immunologically functional and escape immunological rejection when transplanted across mhc barriers allowing the induction of mixed chimerism. normalization the de is derived from the anterior segment of the primitive streak which corresponds to the early and mid-gastrula organizer during early embryo development, from which many of the major visceral organs, including the liver, pancreas, lung, thyroid and intestines are derived. es cells were cultured in a serum-free medium containing activin a and bfgf for 6-8 days, the differentiated cells developed into an epithelial monolayer yielding more than 70% of cxcr4 expressing cells. cxcr4 has been reported as a cell surface marker of the definitive endoderm. molecularly, the differentiated es cells express typical definitive endodermal genes in particular, foxa2, sox-17, gsc, and hnf4α. the cxcr4 + definitive endodermal cells were further purified using immunomagnetic bead separation to more than 99% purity in order to eliminate teratoma-forming cells. to study their engraftment and regenerative capacity, these newly differentiated cells were intravenously infused into a mouse with carbon tetrachloride-induced liver injury. harvested livers from these animals showed large de-derived cells positive for albumin suggesting that they were de novo generated hepatocytes. a second cell type was ck-19 expressing, suggesting that the engrafted cells also differentiated into cholangiocytes. therefore, we further transplanted these de cells into a factor viii null mouse and asked whether these cells could correct factor viii activity. plasma factor viii activity (coamatic assay) fully normalized to that of wild type mice and has remained stable over 60 days. these data suggest that es cell-derived de progenitor cells can restore factor viii activity in hemophilia a mouse model, presumably through protein production in de novo generated hepatocytes. more importantly, none of these animals developed teratomas. thus, es-cell derived cells can potentially be coaxed to form cellular transplants with curative capabilities. objective: we have established a novel approach, protex, to rapidly and efficiently engineer primary cells, tissues, or organs to display on their surface exogenous proteins of interest for immunomodulation. this approach involves generation of chimeric proteins with core streptavidin, biotinylation of cells, and the transient display of chimeric proteins on the cell surface. in this study, we displayed a chimeric form of fasl (sa-fasl) on the surface of bone marrow cells and tested the efficacy of these cells to establish mixed chimerism in allogeneic hosts under nonmyeloablative conditions. methods: balb/c bone marrow cells were engineered with sa-fasl and 30 million of these cells were transplanted into c57bl/6 mice subjected to various doses of total body irradiation 2 days earlier. a short course of rapamycin was used to enhance the tolerogenic effect of sa-fasl. bone marrow cell recipients were typed for multilineage chimerism at various times post-transplantation and tested for donor-specific tolerance using skin grafts. results: all the animals (n=8) treated with 300 cgy total body irradiation and transplanted with sa-fasl-engineered donor cells showed significant levels of chimerism (10-60%) on day 14 post-transplantation that showed a steady increase overtime and reached to 40-90% on day 60 post-transplantation. in marked contrast, none of the control animals (n=11) receiving bone marrow cells and a short course of rapamycin showed detectable chimerism. chimerism was multilineage and associated with donor specific tolerance since chimeric animals accepted donor, but rejected third party skin grafts. conclusions: engineering bone marrow cells in a rapid (∼2 hrs) and efficient (100% targeted cells) manner with exogenous proteins having immunoregulatory functions provides a new and effective means of immunomodulation to establish mixed allogeneic chimerism under nonmyeloablative conditions with significant potential in clinical bone marrow transplantation. funded in parts by nih (r21 dk61333, r01 ai47864, r21 ai057903, r21 hl080108), jdrf (1-2001-328) , ada , and aha fellowship 0725348b. chronic allograft dysfunction is still a major clinical problem in organ transplantation. morphologically it is characterized by changes suggestive of an alloantibody mediated mechanism such as glomerulopathy and vasculopathy or by non-specific changes such as interstitial fibrosis and tubular atrophy. alloantigen dependent as well as -independent factors contribute to the pathogenesis of these changes. in this study we analysed allospecific t cells from the peripheral blood of kidney transplant patients under different immunosuppressive protocols with or without chronic allograft dysfunction. 107 renal allograft recipients of our renal transplant clinic were screened at least six months after transplantation. all patients were on a calcineurin-based immunosuppressive protocol consisting of cyclosporine (csa)/mycophenolate mofetil (mmf)/steroid, tacrolimus (tac)/mmf/steroid, or csa/steroid. patients had to be mismatched for one or more of the five candidate hla-dr antigens for which synthetic peptides were available (dr1, dr2, dr3, dr4, and dr7). patients with biopsy proven chronic allograft nephropathy (can)with an elevated serum creatinine level of ≥1.6 mg/dl were compared with patients with stable allograft function (serum creatinine <1.6 mg/dl). t cell lines were generated from peripheral blood lymphocytes of renal transplant recipients against donor-derived hla-dr peptides presented by self apc. t cell lines generated from patients with can produced significantly more ifn-γ, while those generated from stable patients produced il-10 associated with a low proliferation index in response to the donor-derived mismatched hla-dr allopeptide in vitro. moreover, significantly more cd4+cd25+foxp3+ t cells were found in stable patients on tac and mmf as compared to patients on csa and mmf. interestingly, a higher gene expression of cd4, cd25, ctla-4, foxp3, and il-10 was observed in those patients. taken together a th1 (ifn-γ) alloimmune response is deleterious and promotes chronic graft damage, while a th2 (il-10) response seems to be associated with a lower incidence of chronic allograft nephropathy. an immunosuppression based on tac and mmf seems to favour cd4+cd25+fosp3+ t cells and to allow long-term engraftment with stable renal function. cyclosporin induces epithelial to mesenchymal transition in renal grafts. the expression of epithelial to mesenchymal transition (emt) markers is a reliable predictor of the progression towards interstitial fibrosis and tubular atrophy of the renal grafts. in vitro experiments suggest that calcineurin inhibitors (cni) can induce emt of tubular epithelial cells. although no evidence was ever provided in vivo, this suggests that emt could be involved in the pathogenesis of renal fibrosis induced by cni. we have previously reported the results of a prospective randomized trial comparing the elimination at month 3 of either cyclosporine (csa, n=54) or mycophenolate (mmf, n=54) from a triple drug regimen in 108 de novo renal transplant patients. all of them had 2 systematic graft biopsies at months 3 and 12 post engraftment. in the leftover material, we retrospectively detected in tubular cells and by immuno-histochemistry the expression of two validated markers of emt: the de novo expression of vimentin (vim) and the cytoplasmic translocation of b-catenin (cat). we were able to measure the emt score at both months 3 and 12 in a total of 68 patients (34 in each group). in the csa group, the vim and cat scores had progressed between 3 and 12 months from 1. calcineurin inhibitors (cni) are efficacious but nephrotoxic immunosuppressives. arteriolar hyalinosis is one of the characteristic histological correlates of this toxicity. yet, time course of this lesion, its reversibility, dose-dependency and the discrimination between drug-related effects, diabetic and hypertensive vasculopathy remain unclear. aim of this study was to evaluate the prevalence and time course of cni-related vascular changes after renal transplantation (tx) in protocol (pbx) as well as in indication biopsies (ibx) and to correlate this to the cni blood levels, blood pressure and diabetes. from 491 patients, a total number of 1239 pbx, taken at 6 weeks (n=380), 3 (n=420) and 6 months (n=439) after tx and 360 ibx were classified according to banff-criteria. assessement of cni toxicity included: isometric vacuolisation of tubules (isovac), intimal arteriolar hyalinosis (iah) and nodular arteriolar hyalinosis (nah) and vacuolisation of small vessel smooth muscle cells (vsm). 96% of patients received either cyclosporine or tacrolimus. in pbx, isovac was present in 15%, 17% and 15% of patients at 6 weeks, 3 and 6 months post-tx, respectively; iah in 11%, 9% and 10%; nah in 5%, 5% and 7%; vsm in 19%, 19% and 15%. in late ibx (>2 years post-tx) the prevalence of the analyzed parameters apart from isovac (9%) was markedly higher: 42% for iah, 22% for nah and 38% for vsm. in pbx, vsm, iah and nah were associated with each other but not significantly dependenct on blood pressure, rejection episodes or diabetes. through levels of cnis were not different between patients with and without vascular hyalinosis. in patients with vsm and iah in late ibx one third had these lesions already present in earlier biopsies. conclusion: the prevalence of presumed morphological signs for vascular cni-toxicity in pbx is low and constant and apparently, not associated with cni blood levels, hypertension or diabetes. in contrast, in ibx later than two years after transplantation, prevalence of vascular cni-toxicity signs is much higher. this emphasises that cni reduction protocols should be regarded within the first six months after transplantation, when vascular changes are still marginal. further elucidation of precursor lesions in pbx would help to find out patients at risk for cni-induced vascular changes. donor introduction: achieving donor specific tolerance has been the goal of the transplant community. success has been reported with donor stem cell transfusion in animal studies and prevention of chronic rejection in cardiac allograft recipients in the clinic. this report details the results of the initial phase of a study in humans. methods: a prospective phase i/ii fda approved pilot protocol was initiated to evaluate the effects of donor graft facilitating cell (fc)/stem cell infusion in kidney transplant recipients. conditioning was performed with 200 cgy of total body irradiation. bone marrow processed to remove gvhd-producing cells but retain cd8 + /tcr -fc and stem cells was infused 24 hours post-operatively. the dosage of stem cells was limited by the t cell dosage. the starting dose was 1 x 10 5 t cells. this was increased in steps of 2 x 10 5 per patient. as the study spans a 7-year period, the immunosuppression changed: 3 patients received cyclosporine (cya), mmf and prednisone; 3 were induced with basiliximab and maintained on cya(2)/fk(1), cellcept and prednisone; and 3 received alemtuzumab induction and maintenance with fk and mmf. all patients underwent tolerance testing and immunoprofiling studies. results: of the 9 patients, 6 received live donor kidney transplants. one graft was lost from arterial thrombosis on day 2. delayed graft function was seen in 3 patients. good long-term graft function was seen in the other 8 patients. acute rejection was noted only in 1 and infectious complications (cmv-1, histoplasma-1) in 2 patients. two patients died with functioning grafts -one at 4 years from lung cancer and another from complications from diabetes at 6 years. six patients are alive with functioning grafts at mean follow-up of 4.3 years with a mean creatinine of 1.3 mg/dl. no gvhd was detected in any of the patients. macrochimerism was not detected in any of the patients at any point. notably, in spite of the fact that durable engraftment was not yet achieved, none of the patients were sensitized as a result nor did they experience immunologic sequelae. conclusions: in our patients there were no untoward sequelae related to either the conditioning regimen or marrow infusion. the incidence of acute rejection even on longterm follow up has been low. we currently propose to reduce the immunosuppression further in these patients. a pilot study was performed to evaluate whether immune cell depletion with alemtuzumab would permit post-transplant weaning of maintenance immunosuppression in well-matched renal transplant recipients. patients received alemtuzumab 30 mg intravenously on the day of the transplant and the subsequent 2 days while sirolimus and tacrolimus were started on day 1. tacrolimus was discontinued at day 60 in all patients. extensive immune monitoring was performed at 1 year. at current follow-up (22 to 34 months), all patients are alive with a functioning graft (median mdrd gfr=46 ml/ min). one patient experienced clinical and biopsy-proven rejection at 9 months. all other patients remain on sirolimus monotherapy. four patients have been weaned to 1 mg of sirolimus daily as their sole immunosuppressive agent, with resulting blood levels of 3-4 ng/ml. these 4 patients have no evidence of donor-specific alloantibody, are unresponsive or hyporesponsive to donor cells by the cytokine kinetics test, and have a regulator phenotype to soluble donor antigens by trans-vivo dth. flow cytometry of peripheral blood demonstrated increased foxp3 expression in the cd3+cd4+ population (p=0.002). naive b cells (cd19/cd27neg) cells increased in 9 of 10 patients (p=0.02) and memory b cells increased in all 10 recipients (p=0.0005) when comparing pretransplant to 1 year timepoints. other than the patient with rejection, 12-month protocol biopsies did not show any evidence of rejection, although 2 of 9 showed focal c4d positivity and 1 diffuse positivity. these 3 patients also had evidence of alloantibody by luminex xmap testing. in conclusion, the cytokine kinetics test, alloantibody testing, and trans-vivo dth assay abstracts results correlated with clinical evolution of patients who successfully weaned both tacrolimus and sirolimus without rejection or alloantibody. the flow cytometry findings described occurred regardless of clinical evolution and may represent alterations of the immune system inherent in the treatment protocol independent of individual patient responses to the graft. however, the functional assays of cytokine kinetics assay and trans-vivo dth may be of potential use to correlate with the clinical immune status of the kidney transplant recipient. we have previously reported the short-term results of alemtuzumab (campath-1h) pre-conditioning with tacrolimus monotherapy and subsequent spaced weaning in living donor kidney transplantation (ldkt). we report here our 5 year experience. methods: we performed 411 consecutive unselected ldkt (donor kidneys were removed laparoscopically) from 12/11/2002 to 11/26/2007 using 30 mg (0.5mg/kg) alemtuzumab and tacrolimus monotherapy. at 6 months post-transplant and every 2 to 6 months interval, we used clinical data (including elisa antibody titers, cylex t-cell activation assay, and identification of donor specific antibodies) to wean tacrolimus when possible (bid-->qd-->qod-->tiw-->biw-->qwk). the recipients included 5 hiv+, 35 pediatric recipients, and 60 re-transplants. the mean follow up was 843.1+478.7 days. results: actuarial recipient survivals at 1-, 2-, 3-years were 98.4%, 95.6%, and 92.7%, respectively. graft survivals at 1-, 2-, 3-years were 97.6%, 90.4%, and 85.4%, respectively. the mean creatinine (mg/dl) at 1-, 2-, 3-years were 1.46+0.62, 1.56+1.08, and 1.54+0.89, respectively. the mean gfr (ml/min/1.73m 2 ) at 1-, 2-, 3-years were 73.0+28.5, 71.8+28.8, and 68.9+28.6, respectively. the cumulative incidence of acute cellular rejection (acr) at 6-, 12-, 18-, 24-, 30-, 36-, 42-, and >42 conclusions: in the current era low risk patients infrequently have ar, and have excellent short and long term graft survival without the use of depleting antibodies. given the increased costs of these drugs, the indications for using depleting antibodies in low risk ktrs of scd kidneys should be further clarified. kidney transplantation prolongs survival in hepatitis c virus-positive (hcv+) patients with end-stage renal disease (esrd). however, the effects of induction therapy and chronic immunosuppression are unknown on the course of hcv infection and potential for cirrhosis in renal transplantation (rtx) recipients. we have retrospectively assessed parameters of liver function, child-pugh (cp) and meld scores in hcv+ esrd patients who received induction therapy with t-cell depletion (group 1: thymoglobulin, n=30) or an il-2 inhibitor (group 2: basiliximab, n=46). pre-rtx liver biopsies were similar in group 1 and 2. patients were followed for a mean of 826 days (range 47 to 2679 days) following rtx and received tacrolimus, mycophenolate mofetil, and sometimes steroids post-transplant. overall graft survival was 84% in group 1 and 85% in group 2 (p > 0.05). data were analyzed pre-rtx, at 30 and 365 days and at time of last follow-up. serum ast, alt, platelets, inr, albumin and bilirubin did not change following rtx in either group. cp scores in group 1 were not significantly changed after rtx (5.5 ± 0.9 to 5.7 ± 0.9 at last follow-up, p=0.53). group 1 patients on steroid-free protocols (n=8) demonstrated declining cp scores from 6.0 ± 1.0 to 5.3 ± 0.5 at last follow-up that were not statistically significant (p=0.31). group 1 patients on steroids showed opposite trends of cp: 5.3 ± 0.6 pre-rtx vs 5.8 ± 1.1 at last follow-up that similarly did not reach statistical significance (p=0.18). group 2 cp scores declined from 5.8 ± 0.8 to 5.4 ± 0.9 at last follow-up (p=0.04). there was no difference between cp or meld scores at any point between the groups. as expected, meld scores improved significantly following rtx and remained low up until the final visit (p=0.001); this was attributed to the drop in serum creatinine post-rtx. neither the use of thymoglobulin or basiliximab resulted in acute hepatitis resurgence or the development of cirrhosis post-transplant. we have not identified any association between choice of induction agent or maintenance immunosuppression regimens, including steroid withdrawal, with impaired hepatic function or progression to liver cirrhosis in hcv+ rtx patients. t cell depletion was well-tolerated by hcv+ rtx patients and resulted in good graft outcomes. interestingly, cp scores declined after renal transplantation in the basiliximab induction group. kidney: complications i prevalence background: a few years ago we observed an expansion of blood gd t cells following cytomegalovirus (cmv) infection in kidney transplant recipient (ktr). we recently demonstrated that these cells share a strong reactivity against cmv infected cells and tumor epithelial cells in vitro. an implication of gd t cells in the immune surveillance against cancer has been demonstrated in mouse and strongly suggested in human. we tested here the hypothesis of a protective role of cmv-induced gd t cells against neoplasia in ktr through: 1/ a longitudinal case / control (ktr with cancer / ktr without cancer) study where gd t cell percentages were determined before and after cancer diagnostic (n=63), 2/ a retrospective follow-up of 131 ktr for 8.23 years looking for risk factors for malignancy. results:the median of gd t cell percentage in patients with malignancies was significantly lower when compared to control patients 18, 12 and 6 months before the diagnostic of the cancer (p<0.005). using a conditional logistic model, we determined that patients with a gd t cell percentage above 4 % were protected from cancer (p<0.008). a significant association between increase of the vd2 neg gd t cell subset and lower cancer occurrence was only retrieved in the ktr who experienced pre-or post-graft cmv infection. finally, using univariate and multivariable analysis, absence of pre-or post-graft cmv infection in ktr was associated with a risk of cancer 5.69 times more elevated (p=0.006). this study reveals an unexpected protective role of cmv against cancer in ktr most probably via the expansion of gd t cells cross-reactive against cmvinfected and tumor cells. background: viral infection (vi) is a morbidity factor in transplant recipients (tx pts). induction therapy (ind-rx) is a known risk factor for vi. although cam is thought to be a more potent ind-rx than zen, we have previously shown similar cmv infection rates in each. we have also shown that cmv-tc analyzed by cytokine flow cytometery (cfc) are consistently detectable in cmv sero(+), but not sero(-) individuals. cmv-tc(-) was associated with persistence of cmv infection in tx pts. here, we report on the effect of ind-rx on cmv-tc in kidney tx pts. methods: 58 pre-tx samples from 39 cmv-sero(+) pts and 62 post-tx samples from 44 pts were submitted for cmv tc-cfc. whole blood was incubated with a pooled overlapping peptide mixture consisting of 138 peptides from cmv pp65 and brefeldin a at 37 degrees for 6 hours and room temperature overnight. ifnγ+cd8+ cells were enumerated by cfc and results were expressed as ifnγ+cd8+ cell%. results >0.2% were considered as (+ infection associated graft loss during the entire study period is shown in figure1. infections contributing to renal allograft loss increased significantly from 1990 to 2006. this may be due to increase use of both induction agents and potent maintenance regimens. this is an important cause for poor long-term graft outcome despite decreasing rejection rates and a balance has to be maintained between prevention of rejection and avoidence of infection. serum creatinine (scr) at procurement was 101±51 µmol/l. the incidence of donor hypertension, diabetes, and death from cerebrovascular origin was 31%, 15%, and 55% respectively. multivariate analysis showed that the only clinical parameters associated with a low egfr were donor scr and donor hypertension. nyberg or pessione scores were not significantly associated with a low egfr. regarding d0 biopsies, univariate analysis showed that % of sclerotic glomeruli (sg, p=0.02), arteriolar hyalinosis (p=0.03), mean remuzzi score (p=0.03) and mean cadi score (p=0.04) were all significantly associated with a low egfr. a logistic regression showed that an integrated score including: i) donor scr (±150 µmol/l), ii) hypertension, and iii) sg (±10%) had the highest performance in predicting a low egfr at 1 yr compared to clinical or histological parameters alone. using this composite score, the adjusted or for the prediction of a low 1-yr egfr ranged from 1 if none of the 3 factors were present, to 7.1 (if sg >10% was associated with one of the 2 clinical factors), and to 27.5 (if the 3 factors were present, p=0.0003). conclusion: this study highlights that d0 biopsies are useful to predict graft outcome particularly in md population, and may perform better than clinical scores alone. in this population, a simple and routinely applicable integrated scoring strongly predicts a poor graft outcome, which may allow an optimized allocation of marginal donors. prospective kidney transplantation from small pediatric donors is increasingly being utilized as a means to optimize the organ supply, however the single most common specified reason for the discard of pediatric kidneys is vascular damage, such as shortening of the suprarenal aorta or injury to the renal artery orifices, which often precludes en bloc transplantation (ebk). at our center, damaged kidneys were salvaged by transplantation as singles (sk background: in an effort to maximize the number of recipients transplanted per donor, transplant centers in our donor service area (dsa) voted to preferentially allocate local and imported en-bloc pediatric donor renal allografts to centers willing to transplant two individuals with single allografts. after 1 year of implementing this policy into action, we report on our initial experience. methods: from july 2006 to june 2007 we reviewed our experience with 12 adult single allograft recipients of pediatric donors less than 60 months of age. there were no exclusions based on age or size with exclusion criteria consisting of donor age < 2 months and single allograft size < 5 cm. all but 1 recipient received rabbit anti-thymocyte globulin induction, tacrolimus, mycophenolate mofetil, and rapid steroid withdrawal. results from this cohort were compared to 86 consecutive recipients of adult single allografts from standard criteria donors with the same immunosuppression protocol used as historical controls. results: 12 pediatric single allografts with median donor age of 24 months (range 8-58) were transplanted into 12 adults with median age 46 years (range 24-67). showed that non-white race was associated with increased risk of death (p<0.01). this effect of race was attenuated when ltx center was taken into account [b] . finally, with the addition of meld in the model, the effect of race on waitlist mortality all but disappeared [c] . conclusions: on the surface, minority patients may appear to have higher mortality on ltx waitlist compared to caucasian counterparts. however, this association is predominantly a result of minority patients having a higher meld score, although ltx center-specific mortality may contribute. these data suggest that waitlist outcome may be improved by optimizing referral of minority patients. background: in cirrhotic patients awaiting liver transplantation (lt), low serum sodium (na) predicts short term pre-lt mortality, independently of meld. incorporation of na into meld has been recommended to improve prognostic accuracy (meld-na; gastroenterology 130:1652 gastroenterology 130: , 2006 ). however, short term interventions such as water restriction that improve na may have little effect on prognosis. hypothesis: the lowest level of serum sodium in the preceding 30 (na30), 90 (na90) or 180 days (na180) may be better than the current serum sodium (nac) for predicting pre-lt cirrhotic mortality. methods: we reviewed electronic records of 764 cirrhotic veterans referred for consideration of lt, 2/28/02-6/30/07. date of most recent na at referral was chosen as time zero for determining nac, na30, na90, and na180 and for assessing subsequent survival. findings: within 90 days, 94 patients died pre-lt (12%) and 27 underwent lt (3%). na at all time points was associated strongly (p<.001) with prelt death (censored at lt). areas under receiver operating characteristic curves (aurocs) for na30, na90 and na180 as predictors of 90d prelt mortality (mean±se) were .808±.026, .807±.026 and .783±.025, respectively, compared to .714±.032 for nac (all p<.05 vs. nac). on multivariable logistic regression analysis, meld and na90 were independent predictors of 90d prelt mortality, with best discrimination given by the following model: meld-na90 = meld + (135-na90)*1.04 with value of na90 capped at 135. aurocs for meld-na90, meld-na, and meld were .875±.027, .865±.025 and .838±.027, respectively. findings were similar when patients with hcc at referral (n=155) were excluded (aurocs .884±.025, .876±.026 and .840±.031, respectively), and when 90 day survival endpoint was changed from "death censored at lt" to "death or lt" (auroc's .890±.021, .882±.022, and .855±.026, respectively). conclusion: short term improvement in na may mask true prelt mortality risk. the lowest na in the preceding 90 days is a better prognostic indicator than current sodium. substitution of meld-na90 for meld would permit more accurate "sickest first" organ allocation, while at the same time allowing prelt correction of na without loss of priority. prospective validation of meld-na90, in comparison to meld-na and meld, is warranted. hyponatremia does not affect survival following liver transplantation. byung cheol yun, 1 w. ray kim, 1 y. s. lim, 1 joanne t. benson, 1 walter k. kremers, 1 terry m. therneau. 1 1 gastroenterology and hepatology, mayo clinic college of medicine, rochester, mn. background: hyponatremia is a common yet important complication of cirrhosis. serum sodium (na) has been found to be an important predictor of survival in patients with cirrhosis. models incorporating na have been proposed for liver allocation. concerns have been raised, however, that liver transplantation (ltx) in hyponatremic patients will adversely affect the outcome. in this work, we assessed the effect of pre-ltx na on the short term survival following ltx. methods: patient-level data on all waitlist registrants in the us for 2005 and 2006 were obtained from the organ procurement and transplantation network. demographic, clinical and laboratory data at the time of ltx and outcomes following ltx were extracted. the relationship between na pre-ltx and survival post-ltx was analyzed using multivariable regression analyses. results: there were 7411 primary transplants that met the inclusion criteria between 2005 and 2006. the median na in meq/l at the time of ltx was 136 (interquartile range, iqr: 132-139). there were 1255 patients who had a na ≤130 meq/l. the mean meld score was 22.5 (sd 9.2). median follow up was 279 (iqr: 155-371) days. the overall 30-and 90-day survival post-olt was 96% and 93%, respectively. in a multivariable logistic regression model, meld was associated with 1.04-fold increase in 90-day mortality (95 confidence interval: 1.03-1.05), while na did not have impact on survival (hr=0.99, 95% ci: 0.98-1.02). the figure represents the risk of 90-day mortality according to na after adjustment for meld, which clearly shows absence of mortality increase over a wide range of na. conclusions: hyponatremia at the time of ltx has no detrimental impact on short term patient survival following ltx. although these data do not address morbidity (e.g., central pontine myelinolysis), there is no evidence that incorporation of na in organ allocation will lead to diminished survival. objective. this study examined the relationship between meld at liver transplantation (lt) and post-lt quality of life (qol). methods. adult lt recipients (n = 247) at two centers completed the sf-36 and transplant symptom frequency questionnaire (tsfq) 1-year post-lt. high sf-36 scores indicate better qol; high tsfq scores indicate more symptomatology. clinical (lab) meld at lt, demographic characteristics, presence of ascites, encephalopathy, and variceal bleeding pre-lt, current employment status, presence of co-morbid medical conditions, and bmi were collected from medical records. results. primary lt indication was viral hepatitis (57%), cholestatic liver disease (17%), or hepatocellular disease (27%), and 63% had ascites, 51% encephalopathy, and 29% gastroesophageal bleeding. mean meld at lt was 20±9. there was almost no correlation between meld and sf-36 physical (r = 0.11) and mental (r = 0.001) functioning. statistically significant yet weak correlations were found between meld and physical functioning (r = -0.15) and role functioning -physical (r = -0.15). meld was not significantly correlated with any other sf-36 scales (r's -0.11 to -0.01). meld was not significantly correlated with any tsfq domains: affective distress (r = 0.08), neurocognitive symptoms (r = 0.05), gastrointestinal distress (r = -0.02), physical appearance changes (r = 0.09), appetite and weight changes (r = 0.09), and miscellaneous symptoms (r = 0.08). older age (ß = -0.28), female sex (ß = -0.26), viral hepatitis (ß = 0.67) or cholestatic disease (ß = 0.45), higher bmi (ß = -0.62), and >1 medical co-morbidity (ß = 0.57) were significant predictors of lower qol as measured by the sf-36 (adj r 2 = 0.12, f = 4.4, p < 0.001). older age (ß = 0.27), female sex (ß = 0.29), higher bmi (ß = 0.71), history of variceal bleeding (ß = 0.19), and >1 medical co-morbidity (ß = 0.17) were predictive of more symptoms on tsfq (adj r 2 = 0.10, f = 4.1, p < 0.001). meld was not predictive of qol. conclusions. higher disease severity, as measured by meld, at lt does not portend a worse qol outcome for patients 1-yr after transplantation. other pre-lt indicators of decompensation also do not predict post-lt qol. post-lt qol is affected more by other variables, including age, sex, bmi, and medical co-morbidities. introduction: racial disparities in access to cadaveric renal allografts have been well described for renal transplantation. however, little is known about differences in orthotopic liver transplantation (olt) rates for patients of minority racial groups following listing. the purpose of the current study was to determine if there is difference in rate of transplantation among racial groups and to examine the potential reasons for the disparity. methods: the united network for organ sharing (unos) database was obtained. data was extracted for adult olts greater than 18 years of age performed from 2/2002-12/2005. transplants for which recipient race or model for end-stage liver disease (meld) score at listing were unknown and patients active on the list were excluded. rates of transplantation as well as differences in reasons for de-listing (transplantation, death/deterioration, and improvement) were examined. in an effort to examine only patients with chronic liver disease, further analysis was performed excluding patients with acute fulminant liver failure and retransplants. results: the database contained complete meld and race information on 17,916 olts. seventy-four percent of patients were caucasian, 12% hispanic, 9% african-american, and 4% were asian. as seen in table 1 , laboratory meld score at removal differed between racial groups. examining pair-wise comparisons of the three minority groups to caucasians, only hispanics differed in reason for delisting (table 1) . subgroup analysis excluding acute hepatic failure patients and retransplants showed similar results with hispanic patients being more likely to die/deteriorate as compared to other racial groups (31% deaths vs. 24% deaths for caucasians), and being less likely to receive a transplant (69% of hispanics vs. 75% of caucasians, p<0.001). conclusion: hispanic patients, although listed with higher meld scores, are transplanted less often than caucasian patients and are more likely to die/deteriorate while awaiting olt. reasons for this discrepancy are unclear and merit further attention. background: since the implementation of the model for end-stage liver disease (meld) for liver allocation, an increasing number of candidates with renal insufficiency have undergone orthotopic liver transplantation (olt). since candidates with renal insufficiency have higher post-transplant morbidity and mortality, meld-based allocation may be shifting some waiting list mortality to the post-transplant period in these candidates. the objective of this study was to evaluate the survival benefit among candidates with renal insufficiency who underwent olt. methods: scientific registry of transplant recipients data for adult candidates age ≥18 initially listed for olt between 9/1/01 and 12/31/2006 (n=38,899) were analyzed. the effect of serum creatinine on the survival benefit (contrast between waiting list and post-transplant mortality) was assessed by sequential stratification, an extension of cox regression. each recipient was matched with candidates active on the waiting list in the same organ procurement organization with the same meld score. results: for meld scores 12-40, the survival benefit of olt significantly decreased as serum creatinine increased. among candidates transplanted at meld 12-14, the 23% with serum creatinine >1.1 mg/dl (23%) experienced no significant survival benefit ( figure) . candidates transplanted at meld ≥15 experienced significant olt benefit irrespective of serum creatinine level. conclusions: comparing two patients with meld ≥12, the patient with higher creatinine experiences significantly less survival benefit from liver transplantation. almost onequarter of patients transplanted at meld 12-14 experienced no survival benefit from olt based on 5 years of follow-up. therefore, more careful assessment of candidates is required in order to maximize the survival benefit gained by the wait-listed end-stage liver disease population as a whole. liver: living donors and parial grafts i assessment introduction: consideration of the risks and benefits of a procedure are critical in medical decision making. however, relatively little is known about risk tolerance amongst donors and transplant professionals in live-donor liver transplantation (ldlt). we conducted confidential semi-structured interviews in a convenience sample of donors, non-donors (individuals who had been assessed for donation but did not donate) and transplant team members. in addition to examining issues surrounding decision making for ldlt donation, we sought to assess the tolerance of participants, above which they would no longer contemplate donation, for a number of potential outcomes following ldlt. the outcomes that participants were asked to consider included their tolerance for risk of donor death, risk of serious donor complication, as well as risk of recipient death following transplantation. the interviews were conducted sequentially, data was coded quantitatively, and the study terminated once saturation was reached. (pre, weeks 1, 4, 12, 26 and 52 post-donation) . ambivalence detected by staff or described by donor was recorded. donor and recipient characteristics were examined and compared between ambivalent and non-ambivalent groups. results: staff identified and self identifed ambivalent donors were not equivalent. staff assessments indicated 20 ambivalent donors (16 male, 4 female). 18 donors self-identified as ambivalent (11 male, 7 female); 7 donors were on both lists (5 male, 2 female). the combinations of brother to brothers and sons to fathers were the most common pairs among ambivalent donors and more common than in total donor cohort. recipient diagnosis of alcohol or hepatitis c related liver disease was more common in ambivalent donors. ambivalent donors were more likely to be college educated and to express significant religious affiliations than the total rhl donor group. all but 1 ambivalent donor indicated that they would donate again on the 1 year qol survey. conclusions: ambivalence about rhl donation is present in approximately 20% of candidates who complete donation. staff-identified and self-identified groups showed only 20% overlap; however, both groups showed similar characteristics. brother-tobrother and son-to-father pairings and recipients with perceived self-induced liver failure were more common in both groups compared to total donor cohort. ambivalent donors had more education and stronger religious or spiritual identification than the entire cohort. only 1 donor indicated persistent doubt about donation. these results suggest that expressed or perceived donor candidate ambivalence may represent a process of careful consideration and should not be used sole basis for donor disqualification. the impact of donor age on recipient outcome for adult right-lobe living donor liver transplantation (rldlt) is unclear. aim: to analyze the effect of donor age on recipient outcome following rdldt. methods: since 2000 we have performed 226 rldlt (mean donor age 37 years, range 18-60 years), including 20 donors age 55 years or older. we analyzed the effects of donor age, as a continuous or categorical (< 54 vs > 55years) variable, on recipient outcome. recipient outcome measures included biochemical markers of hepatocytes injury (ast, alt) and graft function (inr, bilirubin), postoperative infections, bleeding, biliary complications, acute cellular rejection, as well as patient and graft survival. analyses were carried out stratified for higher recipient meld scores (< vs. >25), recipient age (< vs. > 60 years), and hepatitis c virus (hcv) infection (presence vs. absence). results: 5-year patient and graft survival after rldlt was 80% and 78%, respectively. donor age as a continuous variable was associated with increased ast (p=0.04) and alt (p= 0.022) release after transplantation, while no effect was observed on inr or bilirubin. rldlt using donors above 55 years of age resulted in an increased incidence of biliary strictures (20% vs. 6%, p= 0.028), postoperative cholangitis (25% vs. 9%, p= 0.023). no effect of donor age was found for the following recipient outcome measures: the number of bile ducts supplying the graft, type of biliary reconstruction required; rejection, hemorrhage, pulmonary or urinary tract infections, renal failure, or length of hospital stay. 5-year patient survival was identical for patients receiving grafts from donors below or above 55 years of age (81% vs 77%, p= 0.86). similarly, 5-year graft survival was comparable for young and old grafts (78% vs 77%, p= 0.71). recipient age (< vs > 60 years), recipient meld score (< vs >25), or hepatits c status of the recipient did not impact on the effect of age on patient or graft survival. conclusion: in this single center series of 226 rldlt, the use of selected older donors did not impair graft and patient survival, but was associated with an increased rate of biliary strictures. background: biliary stricture rate after living donor liver transplant (ldlt) in adults remains relatively high in comparison to the stricture rate after adult cadaveric liver transplant or ldlt in pediatric patients. the etiology or risk factors for biliary stricture development at present time are uncertain. purpose: to determine the risk factors for biliary stricture after right lobe (rl) ldlt. methods: from 5/99 to 12/07, 109 ldlt procedures were performed in 109 adult recipients. eleven patients were excluded from analysis due to <90 days follow up or need for retransplant. the following data was prospectively collected: 1. demographics, 2. acuity of illness, 3. number of bile ducts, 4. type of biliary reconstruction, 5. graft to recipient weight ratio, 6. hemodynamic parameters, 7. outcomes. these parameters were compared in patients with and without strictures. results: mean follow-up for 98 patients is 1642 days (range: 120-3052). 8 of 109 patients died during the follow-up range and 3 required whole liver re-transplants. 33 patients (34%) developed a biliary strictures during the follow-up period. comparison of risk factors in patients with and without strictures revealed the following results: mean meld >1 bile duct grwr* < 1. neither meld score, number of bile ducts or type of biliary reconstruction appear to be contributing factors to the development of bile duct stricture following rl ldlt. the biliary stricture rate was related to the volume of transplanted liver and post transplant graft recovery. therefore, the development of biliary strictures in some patients may represent yet another feature of small-for-size syndrome. background: ox40 and cd154 can be expressed by both foxp3+ tregs and activated t effector cells. however, the question as to how ox40 and cd154 function, individually or collectively, in regulating such functionally different t cell subsets in transplant models remains poorly understood. in some models, blocking cd154 costimulation is remarkably effective in prolonging graft survival, but targeting cd154 alone rarely creates tolerance. but the role of ox40 in regulating the cd154 blockade induced tolerance is completely unknown. in the present study we critically examined the role of ox40 in the activation of cd154 deficient t effector cells as well as in the regulatory function of foxp3+ tregs. we also examined the effect of ox40 on the induction of new foxp3+ tregs/th17 cells from activated cd154 deficient t effector cells. the impact of ox40 in the induction of allograft tolerance was examined using an islet transplant model. we found that cd154 deficient foxp3+ tregs constitutively expressed ox40 on the cell surface, but the cd154 deficient t effector cells did not. however, when the t effector cells were sorted and stimulated in vitro, ox40 expression could be abstracts readily induced on the t effector cells. to further examine how ox40 regulates such functionally different t cell subsets, we found that ox40 delivers potent costimulatory signals to t effector cells, which prevent the induction of new foxp3+ tregs from activated t effector cells but promote their differentiation to th1 cells but not th17 cells. surprisingly, ox40 costimulation to cd154 deficient foxp3+ tregs completely inhibited their regulatory functions. in an islet transplant model, we showed that cd154 deficient mice can reject the dba/2 islet allografts, but blocking ox40 costimulation readily induced donor specific tolerance (mst>150 days), and this tolerant status was critically dependent on the induction of foxp3+ tregs. in contrast, treatment of cd154 deficient recipients with a agonist anti-ox40 mab precipitate rapid islet allograft rejection, suggesting that ox40 costimulation is critically important in the induction of transplant tolerance. conclusions: our data suggest that ox40 is a costimulatory molecule to t effector cells but a powerful negative regulator for foxp3+ tregs. thus, a key role for ox40 in the induction of transplant tolerance is the control of t cell mediated regulation. background: foxp3 is a winged-helix family transcription factor that is the master regulator for the development and function of regulatory t cells (treg). we investigated the molecular mechanisms important for regulation of foxp3 expression, and defined the structure of the active foxp3 promoter in cd4 + t cell lineages. methods: purified cd4 + cd25 -foxp3 -gfp -t cells (naïve) and cd4 + cd25 + foxp3 + gfp + treg were cultured with antigen presenting cells in the presence of il-2, anti-cd3ε mab, tgfβ or the dna methyltransferase inhibitor 5-aza-2'-deoxycytidine (zdcyd). foxp3 promoter structure and activity were monitored with methylation-specific pcr, disulfite-sequencing, chromatin immunoprecipitation (chip) assays, electrophoretic mobility shift assay (emsa) and luciferase promoter assay. the foxp3 promoter has an upstream cpg island ∼5kb from the transcriptional start site. disulfite-sequencing and methylation-specific pcr analysis showed that this region is heavily methylated in naïve cd4 + t cells and tgfβ induced peripheral treg, but demethylated in thymic derived natural treg (ntreg). chip analysis showed that the methylated cpg island is bound specifically by the dna methyltransferases 1 and 3b. zdcyd causes demethylation of the cpg island, and in combination with tgfβ, synergistically induces foxp3 expression. chip assays for acetylated histone 3 and sp1, both markers of gene activation, showed that the cpg island is acetylated and bound by sp1 in ntreg and zdcyd plus tgfβ induced treg, but not in activated cd4 + t cells or tgfβ induced treg. emsa likewise shows the cpg island binds sp1. in contrast to the upstream promoter, the structure of the first intronic promoter differs markedly between ntreg and tgfβ induced treg, but is not affected by zdcyd. the upstream cpg island also possesses enhancer activity that is repressed by dna methyltransferases. zdcyd plus tgfβ induced treg have stable foxp3 expression and enhanced suppressive functions in vitro and in vivo. conclusion: these results demonstrate that ntreg and tgfβ induced treg are genetically distinguished from each other by the epigenetic structure of a unique upstream cpg island of the foxp3 promoter. the function of this region is regulated by dna methylation and histone acetylation. zdcyd demethylates the promoter, leading to enhanced and stable expression of foxp3 and suppressor activity, similar to ntreg. this has important implications for biology, and generating treg for tolerance. chemokine background: trafficking of lymphocytes through lymphatics to secondary lymphoid organs is crucial for immune responses. we previously showed that regulatory t cell (treg) function required trafficking from the inflammatory graft site to the local draining lymph node (dln). since the mechanisms that regulate migration through afferent lymphatics are poorly understood, we explored the role of chemokine receptors on treg for afferent lymphatic migration in an islet transplantation model. methods: islets were transplanted from balb/c mice into foxp3 gfp c57bl/6 mice. treg from wild type, ccr2 -/-, ccr4 -/-, ccr5 -/-, or ccr7 -/-c57bl/6 mice were isolated, labeled with red dye pkh26, and transferred intravenously, or locally into the islet allograft. treg migration to islet grafts and dln was determined by flow cytometry and immunohistochemistry. endogenous foxp3 gfp+ treg and transferred pkh26 labeled treg were sorted from the islet grafts and the dln, and chemokine receptor and sphingosine 1-phosphate receptor (s1p1) expression were determined by rt-pcr. islet allograft survival was determined by measurement of blood glucose. results: freshly isolated treg expressed s1p1 and the chemokine receptors ccr2, ccr4, ccr5, and ccr7. endogenous treg, and both intravenously and locally transferred treg, that were recovered from islet allografts and dln expressed similar levels of ccr2 and ccr5. ccr4 was expressed preferentially on islet migrating treg, while s1p1 and ccr7 were expressed preferentially in dln migrating treg. locally transferred treg migrated to the dln, but ccr7 -/-treg were not able to migrate to the dln. ccr2 -/and ccr5 -/-treg were impaired in their ability to migrate to the dln. this suggested that these three chemokine receptors all regulated treg entry into afferent lymphatics and migration from the graft to the dln. in contrast, ccr4 -/-treg migrated normally from the islet to the dln. importantly, ccr2 -/-, ccr5 -/and ccr7 -/-, but not ccr4 -/-treg, were impaired in their ability to prolong islet allograft survival when transferred locally in the islet allograft. conclusion: treg migrate from the inflammatory site of the allograft to draining secondary lymphoid tissue through afferent lymphatics. this process depends on ccr2, ccr5, and ccr7; and is crucial for full treg function in vivo. these results demonstrate a novel role for sequential migration from the graft to the dln in treg function and suppression. epigenetic regulation of gene expression provides a major, and especially beyond oncology, largely unexplored means to regulate host immune cell functions. our ongoing analysis of histone deacetylase (hdac) expression by foxp3+ naturally occurring murine regulatory t (treg) cells showed tcr-activated tregs had 5-6 fold more hdac6 mrna than corresponding resting treg or non-treg cells. in various cell types, hdac6 deacetylates alpha-tubulin, cortactin, and hsp90, abrogates formation of the aggresome, and blocks the unfolded protein response, though nothing is known regarding these pathways in tregs. we found that an hdac6-specific inhibitor, tubacin (but not the control compound, niltubacin), increased treg suppressive function in vitro (p<0.01), in association with increased expression of ctla, il-10, gitr, pd-1 and other treg-associated genes (p<0.05), and increased treg foxp3 protein (though not mrna) expression. tubacin enhanced the conversion of cd4+cd25-cells into cd4+ foxp3+ treg in vitro, and globally decreased cytokine production, with the exception of il-10 and il-17 mrna. comparable and dose-dependent effects were seen using the hsp90 inhibitor, geldanamycin, suggesting that the effects of hdac6 inhibition were mediated, at least in part, by blocking the chaperone effect of hsp90. use of tubacin in vivo significantly decreased the severity of colitis in two murine inflammatory bowel disease models (p<0.05), dextran sodium sulfate-induced colitis and the cd4+cd62lhigh adoptive transfer model of colitis, as assessed by standard clinical and histologic criteria. in addition, 14 days combined use of tubacin and a subtherapeutic dosage of rapamycin led to significantly prolonged cardiac allograft survival (balb/c->c57bl/6) compared to use of either agent alone (p<0.05). our data show that use of the first known small molecule inhibitor of one specific hdac has important therapeutic effects, including enhancing the production and suppressive function of tregs. while ongoing studies are directed towards unraveling the interactions of hdac6-dependent pathways and treg functions, the current data indicate the importance of understanding the functions of hdacs to the development of entirely new ways to regulate host immune responses. dendritic cells supply paracrine il-2 for treg cell functional activity. regulatory cd4+cd25+ t cells (tregs) are important for the maintenance of immune tolerance, and immunotherapy with tregs is being explored for organ and cell transplantation. treg development, expansion and function depend on il-2. because tregs do not make il-2, they must obtain il-2 from another cell. although cd4+ teffectors are a logical candidate, the identity of the paracrine source of il-2 for tregs is not substantiated. we explored whether dendritic cells (dcs) could serve as the paracrine source of il-2 for treg and rd6, a cd4+cd25+ regulatory hybridoma. using four dimensional live cell imaging we demonstrate that treg and rd6 cells establish tight contact with dcs, and cd25 is localized at these contacts. using the il-2 elispot and real-time rt-pcr we found that splenic dcs and the jawsii dc cell line constitutively make il-2. lps and cpg increases dc production of il-2. co-culture with jawsii dc cell line significantly upregulates cd25 expression on alloreactive do11.10 tregs and rd6 cells, but not on do11.10 cd4+ teffector cells. tregs and rd6 cells are functionally suppressive after activation by wild type but not il-2 knock-out allogeneic dcs, and anti-cd25 inhibits the function of treg and rd6 cells in a dose response fashion. in contrast, wild type and il-2 knock-out dcs are equally able to activate alloreactive cd4+cd25-cells. supplemental il-2 at high (1000 u/ml) but not low doses (100 u/ml) restores the function of alloreactive tregs and rd6 that were activated by il-2 ko dcs. these data indicate that treg cells acquire il-2 from dendritic cells for their gain of function and validate dendritic cells as a paracrine source of il-2 for treg. introduction: previously, it has been demonstrated that foxp3, a gene required for the development and function of regulatory t cells, was highly expressed in the graft during cardiac rejection, suggesting infiltration of regulatory t cells in the transplanted organ during an allogeneic response. in this study, we investigated whether graftinfiltrating t cells expanded from rejecting human cardiac allografts exhibit immune regulatory activities. methods: graft-infiltrating lymphocytes (gils) cultured from endomyocardial biopsies (emb; n=13) with histological signs of acute cellular rejection were expanded in the presence of donor-antigens in il-2/il-15-enriched medium for 2-3 weeks. flow cytometry was used to analyze the expression of cd3, cd4, cd8 and foxp3. to analyze the immune regulatory function, we performed mlrs with peripheral blood mononuclear cells (pbmc) of the patients and irradiated donor or third party spleen cells in the absence and presence of gils (ratio 5:1). results: of the cd3 + gils, 9% (median; range: 1-21%) stained positive for foxp3. this foxp3 expression was detected in both cd4 + and cd8 + t-cell population (median: 8% and 9%, respectively). functional analysis demonstrated that gils suppressed the antidonor proliferation of responder t cells (range % inhibition: 55-81%). interestingly, this suppression was predominantly achieved by cd8 + gils: depletion of cd8 + cells from the gils population diminished the inhibitory effect, whereas addition of solely cd8 + gils to the mlr abundantly suppressed the anti-donor response (range % inhibition: 62-77%). in contrast, gils did not inhibit the proliferation of t cells stimulated with third-party antigens. the figure below depicts a representative example. graft-infiltrating lymphocytes expanded from rejecting cardiac allograft exhibit donor-specific immune suppressive activities. these results suggest that during acute cellular rejection, graft-infiltrating lymphocytes not only consist of graft-destructing effector t cells, but may also comprise immune regulatory cells of the cd8 + phenotype. the context: it has been previously suggested that a liver allograft is immunoprotective and able to decrease the rate of rejection of a donor-specific allograft of another organ. it has been recently proposed that allografts other than the liver may also be immunoprotective. objective: the aim of this analysis was to examine one year rejection rate and the incidence of rejection free survival of all combined transplants in the collective us experience to gain insight to any possible protective effect of one organ for another. methods: the united network of organ sharing (unos) provided de-identified patientlevel data. analysis included all recipients transplanted between january 1, 1994 and october 6, 2005 who were 18 years or older (except intestinal transplants). rejection at one year was defined as treatment for one or more episodes of rejection. results: analysis included a total of 83,424 patients who received either one, or combined, simultaneous or sequential, organ transplants in all possible combinations. results are summarized in figure 1 (one-year organ allograft rejection rate). the collected data demonstrate that the rejection rate of donor-specific organ allografts which accompanied primary liver, kidney, and heart transplants was significantly lower in combined transplants as compared to that of the primary allograft transplanted alone. this was not true, however, for intestinal and pancreatic allografts where protection for the accompanying organ was not observed. we further demonstrate that transplantation of two organs of the same type (double kidneys or double lungs), i.e. increase in antigen load, also leads to decreased rates of rejection of the allografted organs. conclusions: in combined simultaneous transplants, the heart, liver, and kidney allografts appear themselves to be protected, and to protect the other organ from rejection. increased antigenic load of identical antigens in case of double lung and double kidney transplants appears to also offer immunologic protection against rejection, perhaps by different mechanisms. background: a2all (9-center adult-to-adult living donor liver transplantation cohort study) has identified risk factors for mortality after aaldlt, including center experience. the aim of this study was to determine if a2all findings are reflected in the national experience. methods: aaldlt at a2all (n=681) and non-a2all centers (n=1598) from 1/1/98 to 8/31/07 in the scientific registry of transplant recipients database were analyzed. cox regression models adjusted for recipient and donor characteristics were fitted to test associations with mortality risk after aaldlt, including center type (a2all vs. non-a2all) and case number (for each aaldlt at each center). results: aaldlt were performed at 9 a2all and 63 non-a2all centers. there was no significant difference in overall mortality risk between a2all and non-a2all centers. significant predictors of death (both groups combined) included donor age (hazard ratio (hr)=1.14 per 10 years, p=0.003), recipient age (hr=1.25 per 10 years, p<0.001), diagnosis of hcv (hr=1.22, p=0.04) or hcc (hr=1.99, p<0.001), and earlier center experience (aaldlt case number ≤15, hr=1.58, p<0.001). there was no significant effect of transplant year after adjusting for experience. cold ischemia time >4.5 hours was associated with higher mortality (hr=1.83, p=0.004); this effect was similar in a2all and non-a2all centers. there were no significant interactions between center type and any predictor except center experience ( figure) . compared to later experience, earlier center experience was associated with significantly higher mortality risk in both a2all (hr=2.24, p<0.0001) and non-a2all centers (hr=1.38, p<0.004). survival during early experience was significantly worse at a2all vs. non-a2all centers (hr=1.42, p=0.024), but survival in later experience was similar. conclusions: after the first 15 cases, aaldlt survival was similar at a2all and non-a2all centers, and similar significant mortality risk factors were identified, including center experience. these analyses support the generalization of findings from a2all centers to others performing aaldlt. abstract# 173 rejection with hemodynamic compromise (hc) and chronic allograft vasculopathy (cav) impact survival in pediatric heart transplantation (phtx). we showed that high pro-inflammatory / lower regulatory cytokine gene polymorphism (gp) profile increased the risk for acute rejection. in this analysis, we assessed the effect of genetic factors on hc and cav. methods: 406 phtx with clinical and gp data for cytokines (tnf-α a-308g; inf-γ t+874a; il-10 g-1082a, c-819t, c-592a ; il-4 c-590t; il-5 t-746c; il-6 g-174c), growth factors (tgfβ-1 t+869c, c+915g; vegf a-2578c, c-460t, g+405c), effector molecules (fas a-670g; fasl c-843t) and pharmacogenomics (abcb1 c3435t, g2677t/a) were analyzed regarding hc and cav. results: adjusting for recipient black race and age with cox regression models, we identified the following risk factors: il-10 high was associated with lower rates of hc. low th1 (inf-γ, tnf-α) with high th2 (il-4, il-5) cytokine gp profiles were protective for hc in combination with il-10 high. carriers of fas high experienced higher rates for hc and cav and high fas-fasl combination doubled the relative risk for cav. abcb1 3435cc/2677gg genotypes were also associated with lower rates of hc (table 1) . conclusion: in this large multi-center study gps with higher regulatory profiles and increased drug transport were associated with a lower incidence of hc. a genetic proapoptotic profile might contribute to the pathogenesis of cav. sponsorship: this work was supported by 5p50 hl 074 732-03 from the national heart lung and blood institute, national institutes of health. it has recently been reported that cd1d-restricted nkt cells that express invariant tcr (inkt cells) play an important role in the production of autoantibodies through the interaction with b-1 cells. this observation prompted us to investigate the possible role of inkt cells in the production of antibodies (abs) against transplant-related antigens, such as abo blood group carbohydrates and histocompatibility complex allopeptides, in a mouse model. we have previously demonstrated that b cells with receptors for blood group a carbohydrates were found exclusively in a cd11b + cd5 + b-1 subpopulation of mice, resembling humans with blood group o or b. immunization with human blood group a red blood cells (a-rbcs) elicited the extensive production of anti-a igm and igg. furthermore, the number of b-1 cells with receptors for a carbohydrates increased in the peritoneal cavity. in cd1d -/and vα14 -/-balb/c mice, which lack inkt cells, such elicited production of anti-a igm was not observed, even after immunization with human a-rbcs. however, class ii -/-balb/c mice, which lack cd4 + t cells but maintain normal levels of inkt cells, exhibited levels of anti-a igm production comparable to those in wild-type (wt) balb/c mice. moreover, anti-a igg production was absent in cd1d -/-balb/c mice even after the immunization, indicating that although inkt cells crucially contribute to anti-a igm production and igg class switching, helper t cells do not. notably, the proportion of b-1 cells in the livers of cd1d -/-balb/c mice was significantly reduced (2.66 ± 0.43%, n = 4) when compared to that in wt mice (4.76 ± 1.93%, n = 4). we next immunized cd1d -/and wt balb/c mice twice with 2 ×10 6 allogeneic b6 mouse thymocytes, and thereafter detected the anti-b6 (allopeptides) abs by flow cytometry. in the cd1d -/mice, anti-b6 igm production was comparable to that of wt mice, and igg class switching also occurred normally. these findings indicated that inkt cells play a pivotal role in the production of abs specific for blood group carbohydrate determinants that are believed to be t cell independent, but are not required in the production of abs for allopeptides that are believed to be t cell dependent. the depletion of inkt cells or the suppression of their function might constitute a novel approach for preventing antibody-mediated rejection in abo-incompatible transplantation, or in xenotransplantation, which involves similar carbohydrate antigens. background static cold storage (cs) is the most widely used organ preservation method for deceased donor kidney grafts. retrospective analyses have indicated that preservation by hypothermic machine perfusion (mp) may lead to improved outcome after renal transplantation. however, there is a lack of sufficiently powered prospective studies to test the presumed superiority of mp. in an international prospective randomized controlled trial we enrolled kidney pairs of 336 consecutive deceased donors and randomly assigned one organ to mp and the contralateral kidney to cs preservation. follow-up was directed at all 672 recipients of these grafts. the primary endpoint was delayed graft function (dgf). mp significantly reduced the risk of dgf (or 0.63; p=0.02) and more than halved the incidence of primary non-function after transplantation, when compared to cs (2.1 vs. 4.8%; p=0.04). furthermore, mp significantly reduced the risk of graft failure in the first 6 months post-transplant (hr 0.46; p=0.05). in recipients who developed dgf, 6-month graft survival was better if their transplanted kidney was machine perfused (87 vs. 76%; p=0.05). hypothermic machine perfusion reduces the risk of delayed graft function, primary non-function, and graft failure in deceased donor kidney transplantation when compared to static cold storage. furthermore, mp alleviates the deleterious effect of dgf on graft survival. we investigated the trafficking of cells after skin and heart transplantation in a dynamic fashion through the use of in vivo microscopy. antigen presenting cells were followed using mhc-cl-ii-gfp and cd11c-gfp transgenic mice. vascularized and non vascularized skin grafts as well as heart transplants were used in syngeneic as well as allogeneic settings. after syngeneic non-vascularized skin transplantation, we observed an early and massive cellular infiltration of host cells into the graft as early as 3 hours post-transplant with a gradual accumulation in the dermis. the accumulation of host-derived cells was accelerated after graft vascularization at day 4/5 post transplantation. this graft infiltration by recipient cells was more pronounced with vascularized skin grafts, and to a higher degree in heart transplants. recipient cells similarly infiltrated allogeneic grafts early on and in larger numbers than for syngeneic grafts by day 4/5 post-transplantation. when visualizing mhc-cl-ii-gfp recipient cells in a syngeneic skin transplant, recipient dcs invaded the graft early on and, by 3 weeks post transplant gradually replaced graft dcs in the dermis (dermal dcs) and the epidermis (langerhans cells). donor dcs could still be seen in the graft up to 8 days post transplant. however, virtually all donor langerhans cells were eventually replaced by recipient ones in a concentric fashion suggesting that the new langerhans cells originate from the recipient skin adjacent to the graft and not from centrally-derived precursor cells. the vascular endothelium of a syngeneic transplant was partially replaced by recipient vascular endothelial cells in a centripetal fashion with more recipient-derived vascular endothelium present at the periphery of the graft and more donor-derived endothelium remaining in the center of the graft. therefore, the graft can be seen as a "chimera" of cells from donor and recipient origin. the presence of recipient endothelial vascular cells and dcs within the graft may be important for maintaining the indirect response thought to be responsible for chronic rejection. objective: maturation resistance and tolerogenicity can be conferred on dendritic cells (dc), -crucial regulators of t cells, by exposure to rapamycin (rapa), a tolerance-sparing immunosuppressant. the mechanisms underlying this acquired unresponsiveness, typified by diminished responses to toll-like receptor (tlr) or cd40 ligation, have not been identified. thus, our objective was to elucidate a molecular basis for rapa-induced dc maturation resistance. methods: rapa administration was used to condition splenic dc in vivo and bone-marrow derived dc in vitro. dc maturation was monitored by assessment of co-stimulatory molecule expression, cytokine production, and t cell allostimulatory capacity. to identify negative regulators of maturation, microarray analysis and quantitative rt-pcr was completed, and findings confirmed via western and flow cytometric analyses. results: in vitro or in vivo exposure of myeloid dc to rapa elicited de novo production of il-1β by otherwise immature dc (cd86 lo ). interestingly, dc il-1β production, acting in an autocrine/paracrine fashion, promoted dc overexpression of the il-1 receptor(r) family member, st2l, and enhanced its surface expression. st2l is the receptor for il-33, an il-1 family member, and has also been implicated as a negative regulator of tlr signaling. consistent with this regulatory function, il-1β-induced st2l expression suppressed the responsiveness of rapa-conditioned dc to tlr or cd40 ligation. conclusion: rapa causes de novo production of il-1β by immature dc, upregulating st2l, and establishing a barrier to dc maturation following exposure to tlr or cd40 ligation. as such this work identifies a novel mechanism by which a clinically-important immunosuppressant impedes the capacity of dc to mature and consequently stimulate effector/adaptive t cell responses. these findings are particularly relevant to the potential use of rapa-conditioned dc as "negative" cellular vaccines to block alloag-specific responses, as exposure to endogenous and exogenous inflammatory stimuli can induce dc maturation and negate the tolerogenic properties of immature dc. exosomes are nanovesicles (50-100nm) released to the extracellular milieu by different cell types. exosomes secreted by dendritic cells (dcs) and other apcs express mhc ag, adhesion molecules and costimulatory molecules oriented on the membrane surface with their binding domains facing outwards. thus, exosomes released by graft-infiltrating leukocytes (gils) could function as "ag-presenting vesicles" or as vehicles to transfer alloag between recipient's apcs during elicitation of t-cell allo-immunity. aims: to test if (i) gils activate anti-donor t-cells in secondary lymphoid organs by releasing exosomes with alloag into systemic circulation; or (ii) gils that traffic to the spleen as passenger leukocytes use exosomes as a local mechanism to transfer alloag to recipient's dcs. methods: exosomes were isolated from supernatants of bm-derived [c57bl/6(b6), ia b ] dcs pulsed with the balb/c iea 52-68 allopeptide and purified by ultra-filtration and ultra-centrifugation on a 30%sucrose/d 2 o gradient. we used pkh67 + exosomes and cd45.1 congenic b6 mice for traffic studies, heart (heterotopic) and skin transplantation models (balb/c→b6, thy1.2 + ), and cfse-labeled 1h3.1 tcrtg cd4 t-cells (thy1.1 + ) specific for ia b (b6) loaded with iea 52-68 (balb/c). dcs were genetically engineered to release exosomes expressing green fluorescent protein (gfp). we have previously shown that blood-borne exosomes carrying balb/c alloag are reprocessed by different subsets of splenic dcs for presentation to indirect pathway 1h3.1 cd4 t-cells. here, we demonstrated that although gils of cardiac and skin allografts release exosomes ex vivo, they did not secrete enough concentrations of exosomes with alloag into circulation to stimulate donor-reactive t-cells in lymphoid organs. instead, our findings indicate that migrating dcs (generated in vitro or isolated from gils), once homed in the spleen, they transfer exosomes expressing gfp and carrying allopeptides to spleen-resident dcs of the recipient, identified by the congenic marker cd45.1. conclusion: exchange of exosomes between dcs in lymphoid organs might be a mechanism by which passenger leukocytes transfer alloag to recipient's apcs in secondary lymphoid organs. t cell activation is critical in initiating adaptive immunity, and pkcθ, a novel member of the pkc family, mediates non-redundant functions in the t cell receptor; however, its role in the mediation of allograft rejection remains unclear. this study is aimed at investigating whether alloimmune response can be alleviated by a deficiency of the pkcθ molecule, and whether transgenic expression of anti-apoptotic bclmethods. wild-type (wt) cardiac allografts were transplanted into pkcθ -/mice, with or without sub-therapeutic anti-cd154 mab. purified pkcθ -/or pkcθ -/-/ bcl-x l t cells were adoptively transferred into rag2 -/mice engrafted with cardiac allografts. lymphocyte proliferation assays were performed (cfse). nf-kb activation was assessed by bioluminescence imaging (bli) using luciferase transgenic mice under the control of a nf-kb promoter. results. the cardiac allografts were rejected in a delayed fashion in pkcθ -/mice with increased nf-kb activation; however, sub-therapeutic anti-cd154 mab (that normally delays rejection of cardiac allograft) induced long-term survival of cardiac allografts. the cardiac allografts were permanently accepted in rag2 -/mice with adoptive transfer of pkcθ -/-t cells, and the rejection can be elicited by transfer of pkcθ -/-/ bcl-x l t cells. in a lymphocyte proliferation assay, pkcθ -/-t cells displayed greatly reduced proliferation. in response to cd3 and cd28 stimulation, pkcθ -/-t cells underwent accelerated apoptosis and reduced th1, th17, and treg subsets compared to the wt t cells. bcl-x l restored the survival of the pkcθ -/-t cells. conclusions. the results suggest that pkcθ mediates the alloimmune response. bcl-x l transgene prevents pkcθ -/-t cell apoptosis and re-elicits allograft rejection. tolerogenic dendritic cells (dc) are immature, maturation-resistant(mr) or alternatively-activated dc that express mhc molecules and low levels or absent costimulatory signals. although mrdc administration has successfully prolonged allograft survival in murine models, the mechanism of action in vivo remains unknown. aim: to test in vivo if the down-regulation of the anti-donor response induced by donor-derived tolerogenic dc is due to: (i) direct interaction of the tolerogenic dc with donor-reactive t cells or (ii) by reprocessing of the tolerogenic dc into alloantigen (alloag) by recipient apc for interaction with indirect pathway t cells. methods: dc were generated in vitro by culturing balb/c bone marrow cells for 6-8 days in medium with gm-csf + il-4 supplemented with 10nm 1α,25-(0h) 2 vitamin d 3 (vd 3 ). we used a model of heterotopic vascularized allogeneic heart transplantation [balb/c into c57bl/6 (b6)] and cd4 t cells from 1h3.1 tcrtg mice that recognize b6 ia b loaded with the balb/c allopeptide ieα 52-68 (indirect pathway) . results: we demonstrated that vd 3 renders dc maturation resistant (vd 3 -mrdc) as vd 3 -mrdc fail to up-regulate co-stimulatory molecule expression, release il-12p70, or stimulate allo-responsive t cells after challenge with potent dc-maturation stimuli. adoptive transfer (i.v.) of balb/c vd 3 -mrdc (day -7) significantly prolonged survival of balb/c heart grafts in b6 mice in the absence of immunosuppressive therapy. interestingly, we found that in vivo, balb/c vd 3 -mrdc induced proliferation of indirect pathway 1h3.1 cd4 t cells in the spleens of b6 recipient mice, indicating that reprocessing of the balb/c dc by host (b6) apc does occur. proliferation of 1h3.1 cd4 t cells in response to balb/c vd 3 -mrdc resulted in defective activation (cd62l high , cd69 low ) of 1h3.1 t cells, leading to their peripheral deletion and outgrowth of cd4 + foxp3 + treg cells. reprocessing of balb/c vd 3 -mrdc was performed by recipient splenic cd11c high cd8α neg dc, and donor alloag continued to be presented through the indirect pathway for 3 days after donor dc administration. conclusion: these results suggest that dc-based therapies downregulate t cell allo-immunity and prolong allograft survival, at least in part, through reprocessing of the tolerogenic dc into alloag by recipient apc. early introduction: alloreactive memory t cells are present in all transplant recipients due to prior direct sensitization or heterologous immunity. these cells are known to circumvent tolerance induction and/or prevent indefinite graft survival in several models, but mechanistic details of their function are unknown. the goal of this study was to test the hypothesis that cd8 memory t cells initiate alloreocognition and express effector functions within hours of reperfusion. methods: syngeneic or a/j (h-2 a ) hearts were transplanted into wt c57bl/6 (h-2 b ), cd4-/-, cd8-/-, or rag1-/-recipients. rna and protein were prepared from total graft homogenates and analyzed by qrt-pcr and elisa. rag1-/-mice received 1x10 6 wt or ifng-/-2c cells and were used as recipients 10 weeks after reconstitution. donor-specific cd8 memory cells were purified from wt spleens 8 weeks after a/j skin grafting, and donor-specific effector cd4 cells were purified from spleens of cd90.1 mice 8 days after a/j heart transplantation. flow cytometry was used to quantify graft infiltrates. results: allografts contained elevated levels of ifng and cxcl9 mrna at 24, 48 and 72 hrs post-transplant vs. isografts. detectable cxcl9 protein was produced in allografts from wt and cd4-/-recipients but not in isografts or allografts from cd8-/-or rag1-/recipients. treatment with ctla4-ig and mr1 failed to reduce cxcl9 production. reconstitution of rag1-/-mice with ifng sufficient or deficient 2c tcr transgenic cd8 cells indicated that early allospecific cxcl9 production absolutely requires ifng made by recipient cd8 cells. although donor-specific ifng production was undetectable in splenocytes until day 4-5 post transplant, graft-infiltrating cd44 hi cd62l lo cd8 t cells were present as early as 24 hrs post-transplant. in adoptive transfer studies, effector-memory cd8 t cells reconstituted early allospecific cxcl9 production in cd8-/-mice. lastly, primed cd4 t cells adoptively transferred at day 2 post-transplant readily infiltrated allografts in control but not cd8 depleted recipients. conclusions: cd8 memory t cells infiltrate allografts rapidly post-transplant, produce ifng, and propogate an inflammatory environment which optimizes recruitment of primed effector t cells. successful neutralization of this early allorecognition pathway should provide valuable adjunctive therapy to improve graft function and survival. background: allogeneic t cell stimulation requires not only antigen-specific signals but also costimulatory signals, most importantly between cd80/86 on the antigen presenting cell (apc) and cd28 and ctla4 on the t cell. engagement of the t cell receptor without costimulation can lead to anergy and the induction of regulatory t cells (tregs). t cell activation is also controlled by expression of the tryptophan-catabolising enzyme indoleamine 2,3-dioxygenase (ido). depletion of this essential amino acid, and/or the production of tryptophan metabolites inhibits t cell proliferation. methods: a genetic approach to confer tolerogenic properties on murine dendritic cells (dcs) has been explored using lentiviral vectors, based on the equine infectious anaemia virus. firstly, an intracellular method that prevents costimulation has been developed: a fusion protein consisting of ctla4 and kdel [an endoplasmic reticulum (er) retention signal] is expressed in dcs. the ctla4-kdel binds to cd80/86 in the er and prevents expression of these proteins on the dc surface. a second approach uses an elevated expression of the ido enzyme by transduced dcs. results: ctla4-kdel-or ido-transduced dcs were unable to induce allogeneic t cell proliferation. however, using two-stage dc:t cell co-culture assays, it was shown that ctla4-kdel-, but not ido-transduced dcs, can induce donor-specific t cell anergy in vitro and in vivo. tolerance to both the direct and indirect pathways was shown using ctla4-kdel-transduced dcs. linked suppression was mediated by the generation of donor-specific tregs. ido-transduced dcs did not generate tregs. furthermore, it was shown separately that dcs expressing ido whilst lacking cd80/86 expression for potential ligation by ctla4 (although ctla4-cd80/86 ligation upregulates ido, it downregulates t cell activation) failed to generate or even sustain foxp3+ treg populations. the ability of the transduced dcs to induce tolerance to allografts was assessed in a complete mismatch and cbk→cba (indirect pathway) corneal graft model. these results support a clinical strategy to induce treg-mediated, donorspecific transplantation tolerance using ctla4-kdel-, rather than ido-expressing dcs. indirect cd4 t cells that recognise processed alloantigen on recipient apc can provide help to alloreactive cytotoxic cd8 t cells that recognise intact mhc i alloantigen on donor apc, but exactly how such 'un-linked' help is provided is not clear. the respective abilities of direct and indirect pathway cd4 t cells to provide help for cytotoxic cd8 alloimmunity were examined in a mouse model of heart graft rejection in which the recipients contain only monoclonal helper cd4 t cells, specific for self-restricted h-y antigen (female b6 mar/rag1 -/mice). mice were additionally reconstituted with 10 6 b6 cd8 t cells, and then challenged with female balb/c (no cd4 t cell help), or male balb/c (indirect pathway help), or male b6xbalb/c f1 hearts (direct pathway help) . un-reconstituted mar/rag1 -/mice lack effector b and cd8 t lymphocytes, and consequently all heart grafts survived indefinitely. in contrast, reconstituted mar/ rag1 -/mice rejected male f1 grafts rapidly (mst 8d), whereas female balb/c grafts survived indefinitely, confirming a cd4-dependent effector role for the transferred cd8 t cells. cd4 t cell help through the indirect pathway, although sufficient to elicit graft rejection, was less efficient than direct pathway help, because male balb/c grafts were rejected more slowly than the f1 grafts (mst 12d, p<0.001). we next considered whether indirect pathway cd4 t cells provide help through recognition of mhc ii complexes on the surface of alloreactive cd8 t cells, in analogous fashion to the cognate interaction between b and t lymphocytes. in support, reconstitution of mar/rag1 -/recipients with instead, mhc ii-deficient cd8 t cells, resulted in slower rejection of male balb/c hearts (mst 21d, p<0.01), whereas male f1 grafts, that still permit provision of linked help, were rejected at the same tempo. most tellingly, mar/rag1 -/mice that received simultaneously a female balb/c heart and male b6 apc (to activate mar cd4 t cells) rejected their grafts rapidly when reconstituted with male cd8 t cells (mst 9d). in contrast, grafts survived indefinitely when female cd8 t cells were transferred. flow cytometric analysis of mitogenstimulated cd8 t cells revealed surface mhc ii expression. indirect allorecognition can provide help for generating cytotoxic alloimmunity, but not as effectively as through the direct pathway. indirect pathway help is potentiated by linkage through recognition of cd8 mhc ii. purpose: tolerogenic properties of dendritic cells (dc) are supported and preserved by conditioning with the immunosuppressant rapamycin (rapa). the ability of rapaconditioned, recipient-derived dc pulsed with alloantigen (alloag) to suppress both direct and indirect alloag-specific t cells in the absence of immunosuppression has been demonstrated in a murine allograft model. dc can acquire intact mhc from cells or cell lysates. however, the ability of alloag-pulsed rapa-dc to immunomodulate directly-reactive alloag-specific t cells has not been formally demonstrated. methods: dc were generated from c57bl/6 (b6; h2 b ) bone marrow cells in gm-csf and il-4. rapa was added to indicated cultures (rapa-dc) beginning on day (d) 2. on d7, cd11c + bead-purified rapa-dc or non-treated control dc (ctr-dc) were incubated with balb/c (h2 d ) splenocyte lysates ("alloag pulsing"). following incubation, the dc were harvested and the level of donor and recipient mhc molecules on cd11c + cells determined by flow cytometry and immunofluorescent imaging. surface levels of cd86, b7-h1 (programmed death ligand-1; pd-l1), and fas-l were compared. pulsed-dc were also incubated with cd8 + t cells from rag -/-2c mice for 3d. 2c cd8 + cells express t cell receptors specific for h2-l d , a mhc class i molecule of balb/c. following incubation, 2c cell proliferation and apoptosis were both assessed. results: ctr-and rapa-dc presented detectable levels of directly-transferred mhc class i and ii on their surface after incubation with allogeneic balb/c cell lysate. donor mhc presented by "pulsed" recipient dc stimulated directly-reactive, alloag-specific 2c t cell proliferation. however, only rapa-dc induced apoptosis in the overwhelming majority of these cells responding via the direct pathway. induction of apoptosis correlated with an increased level of surface fas-l on rapa-dc and their comparatively low level of cd86 relative to pd-l1. conclusions: rapa-conditioned dc can present intact mhc molecules acquired from lysates of allogeneic splenocytes and concurrently induce apoptosis of directlyreactive alloag-specific cd8 + t cells. as such, we provide mechanistic insight into a mechanisms by which alloag-pulsed, recipient-derived rapa-dc may facilitate allograft tolerance. background: t regs actively regulate alloimmune responses and promote transplant tolerance. atg, a widely used induction therapy in organ transplantation, depletes peripheral t cells but may preferentially spare t regs . sirolimus is thought to expand natural t regs . b7 t cell costimulatory blockade inhibits effector t cell (t eff ) expansion and may promote regulation. we investigated the effect of combining mouse atg (matg), ctla4ig and sirolimus on stringent skin allograft survival, and studied the mechanisms by determining t reg /t eff balance in vivo using a unique model (abm-tcrtg-foxp3/gfp reporter mouse conclusion:this is the first report to establish that t cell depletion with matg combined with ctla4ig and sirolimus synergize to prolong stringent fully allogeneic skin allograft survival by promoting regulation and tipping the t reg /t eff balance by both preserving t regs and facilitating generation of new t regs by a conversion mechanism. these results provide the rationale for translating such a novel therapeutic combination to promote regulation and tolerance in primates and human organ transplantation. expansion of cynomolgus cd4+cd25+foxp3+ regulatory t cells using low dose anti-thymocyte globulin. to test low dose atg in vivo, 2 mg/kg (10% of depleting dose) was administered thrice (day 0,1 and 2) to a naïve monkey and to a monkey that was treated concurrently with sirolimus (trough 15-20 ng/ml) after heart transplantation. in the naive monkey, lowdose atg led to expansion of cd4+cd25+foxp3+ tregs in peripheral blood (baseline 0.57%, day 7=4.09%, day 12=2.7%) and in lymph nodes (baseline 2.47%, day 7=9.35%, day 12=7.08%) without causing t cell depletion. similarly, in the transplanted monkey peripheral blood tregs expanded from 1.4% at baseline to 4.64% on day 6. low dose atg is not only able to expand tregs ex vivo by proliferation of natural cd4+cd25+ cells, but can equally induce tregs in vivo without lymphodepletion. these findings provide the rationale for development of tolerance inducing strategies based on enhancing regulatory mechanisms in human transplant recipients. immunological background⁄aim: previously, we have shown that combination of human anti-cd40 mab, 4d11 and tactolimus exerts additive immunosuppressive effect and markedly prolongs renal allograft survival in cynomolgus monkeys. in this study, we further evaluated the immunological aspects among these transplant recipients. method: kidney transplantations were performed across mhc mismatched cynomolgus monkeys. transplant recipient was given either no-treatment, tacrolimus (1 mg⁄kg⁄day, po), 4d11 (5 mg⁄kg, iv) or tacrolimus+4d11 (n=3⁄group). peripheral lymphocyte population, mlr and serum anti-donor antibody levels and graft histology were assessed. results: mean graft survival for no-treatment, tacrolimus, 4d11 and tacrolimus+4d11 treatment groups was 6.0±1.0, 27.7±3.2, 120.3±90.6 and 213.7±28.0 days, respectively. peripheral cd20 + cells partially declined in both 4d11 alone and 4d11+ tacrolimus given animals at the early post-operation period, although the numbers recovered thereafter. cd4 + and cd8 + cells were unaffected. cd4 + effector memory population was reduced by addition of tacrolimus to 4d11 (fig. 1a ). mlr against donor and 3rd party antigens were suppressed in both 4d11 and tacrolimus+4d11 groups (fig. 1b) . addition of tacrolimus further reduced graft cd4 + , cd8 + and cd20 + cellular infiltration (fig. 1c ). anti-donor antibodies were detected in sera during the treatment course of 4d11; however, they did not develop under the tacrolimus+4d11 treatment. graft c4d deposition correlated with serum anti-donor antibody levels. the 4d11 inhibits both cellular and humoral responses against donor antigens. addition of tacrolimus strengthens these immunosuppressive effects of 4d11, leading to further prolongation of graft survival. objectives: allogeneic islet transplantation offers the potential for cure from diabetes. application of this therapy, however, is limited by immunologic mechanisms requiring medical therapy to prevent rejection of the islets. costimulatory blockade of the cd28/ cd80/cd86 and the cd40/cd154 pathways has shown promise in ameliorating the immune response to allow engraftment and function of islets. we have evaluated a new drug regimen consisting of induction therapy with 3a8, a murine anti-cd40 antibody, and basiliximab and maintenance treatment with ctla4ig and sirolimus in diabetic rhesus macaques which received allogeneic islets. methods: allogeneic rhesus macaque islets (14,100 ie/kg ± 1,802) were transplanted intraportally into diabetic rhesus macaques (n=4) under the following immunosuppressive regimen: short term administration of anti-il-2 receptor (basiliximab) and anti-cd40 (3a8), with maintenance immunosuppression using sirolimus for 134 days and abatacept (ctla4ig) for long term therapy. weekly peripheral blood flow cytometric and cmv viral load monitoring was performed. results: recipients treated with this immunosuppressive regimen had immediate return to normoglycemia following islet transplant. the graft survival in the first three animals was 141, 283 and 297 days. the fourth animal continues to exhibit good glycemic control at his current post-operative day 30. each of these animals had monthly intravenous glucose tolerance tests with monitoring of blood glucoses and c-peptides with further evidence of glycemic response and c-peptide generation. flow cytometry confirms cd40 blockade during the administration of 3a8 and return of cd40 after cessation of therapy. the treatment was well tolerated with minimal evidence of cmv reactivation and no evidence of thrombocytopenia or thromboembolism. conclusions: these preliminary results indicate that cd28/cd40 costimulatory-based immunosuppressive regimens can protect allogeneic islets from rejection. furthermore, 3a8 appears to adequately block cd40 to facilitate this engraftment and function as demonstrated by flow cytometry. iwami, 1,2 qi zhang, 1 osamu aramaki, 1 nozomu shirasugi, 1 katsuya nonomura, 2 masanori niimi. 1 1 surgery, teikyo university, tokyo, japan; 2 renal and genitourinary surgery, hokkaido university, sapporo, japan. many studies have shown immunosuppressive effects of dietary intake of fish oil containing eicosapentaenoic acid (epa) in various models such as autoimmune diseases and transplantation. however, its mechanisms remain uncertain. furthermore, there have been no studies examining the effect of purified epa. here we determined the ability of purified epa to inhibit alloimmune response in mouse cardiac transplantation model. methods: cba recipients (h-2 k ) were given single injection of purified epa intraperitoneally on the same day as transplantation of a heart from c57bl/10 donors (h-2 b ). mixed leukocyte reaction (mlr) assay and enzyme linked immunosorbent assay (elisa) were also performed to evaluate the effect of purified epa on cell proliferation and cytokine production. to determine the presence of regulatory cells, adoptive transfer study was conducted. results: untreated cba recipients rejected c57bl/10 cardiac allografts with median survival time (mst), 8 days. in contrast, cba recipients treated with purified epa (1.0g/kg) had significant prolongation of allograft survival (mst, >100 days). cba recipients treated with 0.1g/kg purified epa eventually rejected allografts (mst, 13 days). in mlr assay, treatment with 1.0 g/kg purified epa suppressed alloproliferation of splenocytes in the recipients. the treatment also inhibited production of il-2, il-12 and ifng by the splenocytes in the recipients. when splenocytes were harvested from the recipients treated with 1.0g/kg purified epa 50 days after cardiac allografting and were adoptively transferred into naïve secondary recipients, the adoptive transfer induced significant prolongation of cardiac allograft in nave secondary recipients (mst >30 days, compared to that in the recipients with adoptive transfer of naïve splenocytes, mst, 10 days). conclusions: purified epa induced significantly prolonged survival of fully mismatched cardiac allografts, and generated regulatory cells. background: chronic allograft nephropathy (can), the most common cause of late kidney allograft failure, is not effectively prevented by the current regimens. activation of extracellular signal-regulated kinases 1/2 (erk1/2) mediating intracellular signal transduction from various growth factor stimuli is required for tgf-β production, which plays a key role in the development of can. hence, the therapeutic potential of disruption of erk1/2 signaling to prevent can was examined in an experimental model. methods: kidney donors from c57bl/6j mice (h-2 b ) were transplanted to bilaterally nephrectomized balb/c recipient mice (h-2 d ). the recipients were treated with ci1040 (mek-erk1/2 inhibitor) or vehicle after 14 days post-transplantation for 28 days. can was evaluated with the banff 97 working classification. results: all six allografts receiving ci1040 treatment were survived, while two out of seven grafts were lost in vehicle-treated group. at the end of experiment, the function of grafts in ci1040 treated recipients had been maintained, indicated by lower levels of serum creatinine and bun (30±6 µm and 22±8 mm, n=6) as compared to those (94±39 µm and 56± 25 mm, n=5) in vehicle group (creatinine, p=0.0015; bun, p=0.0054). pathological evaluation indicated that ci1040 reduced can, reflected by a lower can score in ci1040-treated group (3.93, n=4 ) as compared to that (9.96, n=5) in vehicle controls (p=0.0234). further examinations showed that ci1040 treatment resulted in inhibition of phosphorylation of erk1/2 and reduction of tgf-β levels in grafts. in vitro ci1040 potently suppressed not only growth factors-stimulated erk1/2 activation and tgf-β biosynthesis in renal tubular epithelial cells, but also attenuated alloantigenstimulated t cell proliferation. conclusion: our data suggest that interference of erk1/2 signaling with pharmacological agent (i.e. ci1040) has therapeutic potential to prevent can in kidney transplantation. objective: this is the first study to investigate the role of a novel jak3 and sykinhibitor, r348, in the prevention of obliterative airway disease (oad), the major obstacle after lung transplantation. methods: trachea from brown-norway (bn) donors were heterotopically transplanted in the greater omentum of lewis (lew) rats. recipients were treated for 28 days with r348 (10, 20, 40, or 80 mg/kg), rapamycin (0.75 or 3 mg/kg), or left untreated. allografts were recovered and processed for histological evaluation determining degree of luminal obliteration, percentage of respiratory epithelial coverage, and mononuclear cell infiltration. donor reactive (igg) antibodies from the recipient's serum were determined using flow cytometry. results: r348 at 20, 40, and 80 mg/kg significantly inhibited luminal obliteration in a dose dependent manner (69±20%, 20±13%, 15±7%; p=0.003 vs. no medication). rapamycin in both concentrations significantly inhibited luminal obliteration (37±15%, 11±6%; p<0.001 vs. no medication) similarly to r348 at 40 and 80 mg/kg. r348 at 40 and 80 mg/kg significantly preserved respiratory epithelium compared to r348 at 10 and 20 mg/kg (49±35%, 76±27% vs. 0±0, 3±7%; p=0.004) and was superior to rapamycin in epithelial preservation (49±35%, 76±27% vs. 27±17%, 36±15%; p=0.01). all r348 and rapamycin-treated recipients expressed decreased numbers of peritracheal mononuclear cells in a dose dependent manner (p<0.0001). r348 20, 40 , and 80 mg/ kg treated recipients had significantly reduced igg levels versus untreated recipients (381±136, 371±61, 230±56 vs. 853±207; p<0.03). all r348 treated recipient thymus and spleen weights were significantly lower compared to the untreated group (p=0.001). bun, cr, and cholesterol levels were unaffected in r348 treated recipients. conclusion: r348 potentially exhibits its inhibitory effect by preserving the respiratory epithelium, rather than by rapamycin's mechanism of reduced smooth muscle cell (smc) proliferation. r348 occupies a beneficial pharmacokinetic profile, lacks nephrotoxic and atherogenic properties, and provides a favorable alternative to rapamycin in the treatment of chronic rejection in lung transplant recipients. genz-29155 is a novel, oral immune-modulatory agent identified in a high-throughput screen designed to find inhibitors of tnfα-induced apoptosis. the molecular target of the compound remains under investigation but is likely downstream of the tnfα cell surface receptor. in vitro studies have shown genz-29155 to be an effective inhibitor of the tnfα-triggered caspase cascade but not anti-cd3 or fas-mediated apoptosis, and thus may act by inducing allograft resistance to immune attack rather than suppressing the alloimmune response per se. it has been shown to synergize with sirolimus in murine heterotopic cardiac allotransplant models. in order to test this promising new agent in a more clinically relevant model of solid organ transplantation, we studied genz-29155 in a mismatched nhp (rhesus macaque) renal transplant model. genz-29155 (n=3) was administered (3mg/kg, iv, days 0-14) with sirolimus (1mg/kg, po, days 0-30). five control animals received only sirolimus and vehicle (1mg/kg, po, days 0-30). all animals were followed serially by polychromatic flow cytometry to determine the relative and absolute number of cd4+ and cd8+ t cell subsets. time to allograft rejection, the primary end point, was determined by a significant rise in serum creatinine and bun, as identified with biweekly monitoring. after diagnosis of rejection, allografts were removed for histological and transcriptional studies, along with splenocytes for immune function assays. in this pilot study, prolongation of rejection-free survival was significantly improved with genz-29155 and sirolimus combined vs. sirolimus alone (42.67 days vs. 17.20 days, respectively, p = 0.05). given these initial results, we have initiated a larger study (n=12) to optimize the dose and duration of genz-29155. five animals, transplanted within the past month remain alive and well in this study. further investigation of this agent will allow us to better understand the benefit of inhibiting tnfα-mediated apoptotic effects in both cellular alloimmune response and allograft injury in solid organ transplantation. targeting purpose: allospecific t memory cell responses are present in transplant recipients from exposure to cross-reacting antigens. we have previously reported that lfa-1 inhibition suppresses primary cd8-dependent rejection responses which are not controlled by any conventional immunosuppressive strategy. these studies were conducted to analyze the efficacy of this anti-lfa-1 ab for control of cd8-dependent responses in sensitized hosts. methods: fvb/n (h-2 q ) donor hepatocytes were transplanted into c57bl/6 (h-2 b ) or cd4 ko (h-2 b ) recipients. memory responses were analyzed by retransplantation with a second fvb/n allogeneic hepatocyte transplant. cohorts of mice were treated with anti-lfa-1 mab and observed for hepatocyte survival or magnitude of cd8 + t cell mediated allospecific cytolytic activity. results: the untreated secondary cd4 ko and c57bl/6 recipients rejected hepatocyte allografts with enhanced kinetics in comparison to the primary graft (mst= day 10 vs day 14, and mst= day 7 vs. day 10, respectively; p < 0.05). anti-lfa-1 mab treated cd4 ko recipients demonstrated delayed rejection (mst= day 35 vs day 10; p=0.001) compared to secondary rejection in untreated cd4 ko hosts. anti-lfa-1 mab treatment did not delay rejection in sensitized c57bl/6 recipients (mst= day 7) but did significantly reduce the in vivo allospecific cytotoxic effector function in c57bl/6 secondary recipients (20.6±4.1%; p=0.001) as compared to untreated controls (97±1.3%). the residual cytotoxicity observed in anti-lfa-1 mab treated c57bl/6 recipients is comparable to the in vivo cytotoxicity of cd8-depleted c57bl/6 secondary recipients (12.1±8.9%) and is likely mediated by alloantibody. in fact, the level of allospecific cytotoxicity in anti-lfa-1 mab treated sensitized c57bl/6 recipients correlated with the amount of alloantibody present in recipient serum. conclusion: in conclusion, treatment with anti-lfa-1 mab delayed (cd4-independent) cd8-dependent rejection in sensitized recipients but did not delay rejection in sensitized cd4-sufficient c57bl/6 recipients. despite the efficacy of treatment with anti-lfa-1 mab to significantly reduce the in vivo allospecific cytotoxic effector function in sensitized c57bl/6 mice this strategy did not delay rejection. this is likely due to alloantibody mediated rejection in sensitized c57bl/6 (but not cd4 ko recipients) which is not suppressed by treatment with anti-lfa-1 mab. abstract# 194 cytomegalovirus (cmv) represents a major cause of infectious complications after transplantation. recently, chronic infections with lcmv, hiv or hcv were shown to be associated with functionally anergic t-cells characterized by high expression of the programmed death (pd)-1 molecule. this study was carried out to characterize functional exhaustion of cmv-specific cd4 t-cells as determinant of impaired cmv-control and to elucidate whether the pd-1 pathway may be operative in active cmv-infection after renal transplantation. cmv specific cd4 t cells from 13 controls, 31 hemodialysis patients, and 51 renal transplant patients were quantified using flow cytometry and analysed for their expression of pd-1 and cytokines ifnγ and il2. cmv specific proliferation was analysed by cfda-se dilution. in viremic transplant-recipients, a significantly higher proportion of cmv-specific cd4 t-cells were pd-1 positive (median 40.9%) as compared to non-viremic transplant patients (8.8%), dialysis-patients (8.8%) or controls (3.1%, p<0.0001). in line with functional impairment, pd-1 positive t-cells produced significantly less ifnγ per single cell as compared to pd-1 negative t-cells (mean fluorescence intensity 129.4±52.6 versus 256.6±96.1, p<0.0001). moreover, unlike controls or non-viremic patients, the majority of cmv-specific t-cells from viremic patients showed a long-term loss of il-2 production. interestingly, functional anergy of pd-1 positive cmv-specific cd4 t-cells was reversible in that antibody-mediated blockade of pd-1 signaling with its ligands pd-l1/-l2 led to a 10fold increase in cmvspecific proliferation. in conclusion, expression of pd-1 defines a reversible defect of cmv-specific cd4 t-cells, and blocking pd-1 signaling may provide a potential target for enhancing the function of exhausted t-cells in chronic cmv-infection. differential background: some patients with cmv disease may be simultaneously infected with multiple viral strains. it is unknown if different strains clear differently with the commencement of antiviral therapy. we assessed response to antiviral therapy in patients with simultaneous co-infection with multiple strains of cmv. methods: pcr-based strain typing of cmv was performed using the glycoprotein b gene of cmv (gb1-4) in a cohort of organ transplant recipients with cmv disease. from this, 89 patients were identified that had simultaneous infection with ≥ 2 cmv strains. quantitative assessment of each of the strain types was performed at regular intervals after starting antiviral therapy. results: the different types of multi-strain infections were gb1+gb2 (11/89, 12%), gb1+gb3 (22/89,25%), gb1+ gb4 (7/89, 8%), gb2+gb3 (10/89, 11%), gb2+gb4 (6/89, 7%) and gb3+gb4 (8/89, 9%). 25/89 (28%) were simultaneously infected with 3 or 4 different genotypes. within individual patients, there was trend for gb1 cmv load (4.27log genomes) to be lower than the other genotypes (p=0.05-0.07) at the onset of disease. decay kinetics for all genotypes showed a bisphasic response with a 1 st phase decline of ∼0.7 days and a 2 nd phase of ∼3days. 1 st phase delines were fastest for gb1 (p=0.0001 vs gb2 and 4) while gb4 decline was slower than gb3 during the 1 st phase. 2 nd phase declines were similar between gb1 and 3 (3days and 2.9 days) but were slower for gb2 and 4 (3.45 days; p=0.01). there was a significant correlation between 1 st phase decline and log decline from baseline by day 21 (r=0.37; p=0.002). relative fitness calculations revealed complex fitness dynamics between genotypes although gb3 was always less fit than gb1, 2 and 4, and gb4 and 2 were always less fit than gb1. conclusion: in patients with cmv disease who have simultaneous coinfection with multiple strains, the 1 st and 2 nd phase declines in gb1 are significantly slower than either gb2 or 4 and have a lower log decline from baseline by day 21. these data indicate that either a significant fitness difference exists between cmv strains or that antiviral control of replication may be linked to cmv gb genotype and should aid our understanding of treatment success and failure. one introduction: parvovirus b19 (pvb19) is a single-stranded dna virus that was first reported to affect transplant (tx) recipients around 20 years ago. in the kidney tx setting pvb19 has been reported to cause anemia and proteinuria. reported incidence in a general kidney transplant cohort has been reported to be between 0%-12% and as high as 38% in an anemic kidney tx population. here we report our incidence of pvb19 infection over a 12 year span. patients and methods: all records of kidney tx recipients from 1996 until 2007 were reviewed for the presence of pvb19 infection. there were 834 kidney tx performed during this period. diagnosis of pvb19 infection was made either by detection of pvb19 via pcr in a blood/tissue sample or by detection of virus on renal tissue by immunostain. in patients found to have pvb19 infection; presence of anemia, proteinuria, concurrent infection and acute rejection rates were examined. response to treatment with ivig was also evaluated. results: incidence of infection was 2.4% as 20 patients were found to have evidence of infection. average time from tx to diagnosis of infection was 21.6 months (range 3 days-86 months). average creatinine at diagnosis was 2.32 mg/dl. anemia was present in 85% of patients with an average hematocrit of 30.8%. proteinuria was present in 45% of patients with evidence of pvb19 infection. co-infection was noted in 5 patients (4 cmv, 1ebv) and acute rejection was noted in 30% of individuals within 3 months of diagnosis. collapsing glomerulopathy (cg) was present in 5 patients and they all had subsequent graft loss at an average of 6 months after diagnosis. 4 of the 5 patients with cg had proteinuria along with anemia and were caucasian. 90% (18/20) of all patients with evidence of pvb19 infection received ivig and cleared their infection. one of the remaining 2 pts without ivig spontaneously cleared their virus. conclusion: although the incidence of pvb19 infection in our kidney tx cohort was very low, its presence portends an unfavorable outcome. the presence of cg associated with pvb19 is an especially devastating lesion with very poor outcomes. response to treatment with ivig and reduction of immunosuppression is variable. based on our data it seems reasonable to screen all tx patients with unexplained anemia and concurrent proteinuria as early detection of pvb19 may be crucial. background: prior to transplant, screening for latent tuberculosis (ltbi) by tuberculin skin test (tst) is recommended. the accuracy of tst in end stage renal disease however may be limited. the quantiferon®-tb gold assay (qft) detects interferon-δ produced by peripheral blood t-cells in response to tb specific antigens and may be more accurate for diagnosis of ltbi. methods: this prospective single center study compared the tst to qft for the diagnosis of ltbi in a cohort of adult patients listed or undergoing workup for renal transplantation. all patients had both tst and qft performed. additional data collected included demographics, tb risk history and chest x-ray results. based on demographic and radiographic findings, patients were classified as high or low risk for ltbi. a positive tst was defined as ≥5 mm and positive qft as ≥0.35 iu/ml. results: a total of 45 patients were enrolled. complete data was available for 38 subjects (5 did not return to have tst read). the mean age was 50.4 +/-12.2 years with 21 (55.3%) males and 17 (44.7%) females. the most common etiologies of renal diseases were diabetes (52.6%) and glomerulonephritis (26.3%). most subjects (35 of 38) were on renal replacement therapy (hemodialysis in 73.7% and peritoneal dialysis in 18.4%). twenty (52.6%) subjects had received bcg and 10 (26.3%) were born in or lived in a country in with tb prevalence rate > 20/100000 population. fifteen (39.5%) subjects were considered to be at high-risk for ltbi. overall 5 (13.2%) had a positive tst and 3 (7.9%) had a positive qft. the qft was indeterminate in 1 subject due to a low mitogen response. agreement between the tests was 92% (k=0.72, p<0.0001). in low-risk subjects (n=23) the tst was negative in all and the qft was negative in 22 and indeterminate in 1. in clinically high-risk subjects, 5 (33%) had a positive tst and 3 (20%) had a positive qft. the 2 subjects with discordant results, both from tb endemic countries, had both completed treatment for ltbi 4 years prior and remained tst positive, but were qft negative. in renal transplant candidates, the tst and qft are comparable for the diagnosis of ltbi. the qft has the advantage of being completed in a single visit and in our cohort indeterminate results were uncommon. optimal utilization of htlv i/ii positive organs -a nationwide survey. objective: we recently presented data from the unos database that demonstrated no significant difference in graft or patient survival between htlv i /ii (+) and (-) liver recipients. several organ procurement organizations (opo) including our own, do not offer htlv i /ii positive organs while many others find it difficult to place them. despite this, the number of htlv (+) organs is increasing with 31 utilized in 2006 alone. this prompted us to evaluate the practical difficulties in placing these organs so as to improve utilization of these "high risk" life saving organs. medthod: a telephone/email survey of all the 65 opos in usa was done over a 2 month period from october to november 2007. results: of the 65 opos, 42 responded. all screen patients for htlv i/ii with elisa. 38 centers confirm with repeat elisa, 27 confirm with western blot and 6 centers do not pursue further. 36 of the 42 centers offer the htlv i/ii positive organs. there were a total of 231 positive donors in the past 5 years of which organs from 109 donors (47.2%) were placed. 27 centers offer all the organs while 15 offer one or more organs selectively based on accepting centers. none have been able to place the pancreas. only liver and kidney were commonly accepted. several centers noted a high false positive rate. based on the unos regional analysis data, 43% of the organs are utilized in ny state alone. many opos did not know which particular centers accept these organs and consequently spend a lot of time and effort in order to place them. a majority wanted to have a list of transplant centers that accept these organs. conclusions: htlv i/ii organs are being underutilized. moreover, our prior analysis of unos data shows that these life saving organs are shared more nationally than loco-regionally which is associated with a poorer outcome. increased knowledge of successful htlv (+) donation and the centers that are willing to utilize these organs in the appropriate setting will help expand the donor pool and decrease mortality on the waiting list. increasing traditional two-drug chronic immunosuppression (is) used in organ transplantation (tx) is associated with development of ebv-driven complications because of impairment of anti-viral cd8 + t cell surveillance. since the long-term impact of alemtuzumab preconditioning combined with tacrolimus monotherapy on ebv immunity after tx has not been studied, here we aim to analyze the frequency and function of peripheral blood ebv-specific cd8 + t cells. thirteen ebv + stable kidney transplant (ktx) recipients and 12 ebv + healthy controls were recruited to this cross-sectional study. all patients received alemtuzumab preconditioning, followed by tacrolimus monotherapy. blood samples were collected at least 1 year post-tx to allow immune reconstitution. the ebv-specific cd8 + t cell phenotype and function were screened by flow cytometry and ifng elispot assay. hla-a201 restricted ebv-lytic (bmlf1) and latent (lmp2a) peptides were used to generate tetramer (tmr) probes, and for functional screening in elispot. circulating cd8 + t cells from ktx patients had recovered by 1 year, and were comparable to those of healthy controls (28.6%±12.9 vs 23% ±6.6, p=0.2). moreover, the memory distribution and the frequency of ebv-specific cd8 + t cells detected in patients and controls (bmlf1-specific: 0.56%±0.96 vs 0.88%±1.08, p=0.46, and lmp2specific: 0.05%±0.06 vs 0.24%±0.40 p=0.09) were similar. in contrast, the frequency of functional type-1 (ifn-g producing) ebv-specific cd8 + t cells was significantly lower in ktx patients than in healthy controls (bmlf1: 47±24 spots/10 5 cd8 t cells vs 164±134 p=0.06, and lmp2: 9±8 vs 64±54 p=0.03). accordingly, on average, only 8-12% of circulating ebv-specific cd8 + t cells from ktx patients produced ifn-g, while 20-30% of effector cells were functional in healthy controls. addition of il-2 (20iu/ml) during the elispot assay reversed the hypo-responsiveness of type-1 (ifn-g) ebv-specific cd8 + t in patients (range 3-8 fold increase), suggesting that these effector cells were anergic. these results support the notion that alemtuzumab-induced lymphocyte depletion followed by tacrolimus monotherapy renders ebv-specific cd8 + t cells anergic in vivo, a state that can be readily reversed by cytokines such as il-2, which are commonly released during immune activation. background: the epidemiology of the transmission of cytomegalovirus (cmv) from organ donors to recipients is not completely understood. we studied donor to recipient transmission patterns by analyzing viral genomic variants through the use of cmv glycoprotein b (gb) genotyping by real-time pcr. polymorphisms in gb ul55 allow discrimination of 4 distinct genomic variants (gb 1-4). methods: organ transplant recipient pairs or triplets were included in the study if: a) they had cmv infection, b) they received an organ from a cmv seropositive donor, and c) there was at least one other recipient from the same donor that also developed cmv infection. genotyping (gb 1-4) was performed by quantitative real-time pcr on stored blood samples. clinical charts were reviewed to evaluate the clinical characteristics and outcome of cmv infection. results: of the 78 cmv seropositive donors screened, 21 were multiple organ donors for which 2 or more of their recipients developed cmv infection. the total number of recipients from these 21 donors was 86 (median of 4 recipients per donor). of these recipients, 47 (55%) had cmv infection (16 recipient pairs and 5 recipient triplets). the prevalence of genotypes was gb1 (n=22; 47%), gb2 (n=11; 23%), gb3 (n=4; 9%), gb4 (n=0). mixed infection with two concurrent genotypes was present in 10 patients (21%). overall concordance between cmv gb genotype in recipient pairs was 62.5% (10/16). if both recipients were cmv seronegative (d+/r-) the gb concordance in recipients was 67% (2/3 pairs). gb concordance was 70% (7/10 pairs) if one of the recipients was seronegative and the other seropositive. concordance was 33% (1/3 pair) if both recipients were seropositive. concordance between genotypes was seen in 2/5 (40%) recipients triplets. in seropositive recipients with cmv viremia, the origin of the cmv strain was thought to be donor derived in 11/16 (69%) and of reactivation of the recipients own virus in 5/16 (31%) of the cases. no difference in clinical outcome or organ tropism was seen between genotypes. based on an analysis of strain concordance within recipients from common donors, transmission patterns of cmv can be assessed. in d+/r+ transplant patients, donor strain superinfection accounts for the approximately two-thirds of cmv infection. backgroud: although map kinases have been implicated in the pathophysiology of liver iri, their functional significance in the mechanism of tlr4 mediated pro-inflammatory immune regulation, remains to be elucidated. methods: map kinase activation in a murine model of liver warm iri (90 min. ischemia, 6 h reperfusion) was determined by western blots. chemical inhibitors of erk (u0126, 10 µm in vitro or 160 mg/kg in vivo), jnk (sp600125, 50 µm or 30 mg/kg), and p38 (sb203580, 10 µm, 100 mg/ kg) map kinases were utilized in vitro in primary bm-derived macrophage cultures stimulated with lps (10 ng/ml); or in vivo in liver pro-inflammatory immune responses induced by lps (1 µg/mouse, i.p.) or iri. results: erk and jnk, but not p38, map kinase activation were readily detected in liver iri. in primary macrophage cultures, lps induced pro-and anti-inflammatory genes, including tnf-α, il-1β, il-6, il-10, inos and cxcl10. erk inhibitor mainly suppressed il-1β and il-10 (80% and 90% resp), whereas jnk inhibitor suppressed the majority of genes. in lps-induced liver inflammation, erk inhibitor suppressed il-6, il-1β, inos and il-10 by >50%, but failed to affect tnf-α/cxcl10. jnk inhibitor, on the other hand, preferentially inhibited pro-inflammatory genes, but marginaly affected il-10 (<30%). and produced comparable suppression of pro-/anti-inflammatory genes (figure1). interestingly, tnf-α was the least responsive gene subjected to map kinase regulation. conclusion: erk and jnk map kinase activation: 1/ are required for tlr4 activationinduced pro-inflammatory gene induction; 2/ play critical role in the development of ir-mediated liver immune response/tissue injury. background: the jak/stat signaling is one of the major pathways for cytokine signal transduction. the signal transducer and activator of transcription 1 (stat1) is mainly activated by ifn-α/ß/ifn-γ. the activation of stat1 by ifn-γ has been implicated in hepatic inflammation. we have shown that activation of toll-like receptor (tlr) 4 complex initiates pro-inflammatory response leading to liver ischemia/reperfusion abstracts injury (iri). indeed, tlr4 signaling in vitro activates stat1, which in turn triggers production of type-1 ifn-dependent cxcl10 (ip-10). this study was designed to analyze the cross-talk between stat1 and the map kinase (erk) downstream of jak/ stat signaling pathways. methods & results: we used a mouse liver model of partial warm ischemia (90 min), followed by reperfusion (6 h) . first, we employed stat1 ko (n=17) and control wt (n=16) mice. the hepatocellular damage, as measured by salt levels (iu/l), was significantly decreased in 10/17 stat1 ko mice (p<0.005); the remaining 7/17 of stat1 ko showed salt levels comparable with wt. hence, we distinguished two groups of stat1 "protected" vs. "nonprotected" ko recipients. histology revealed minimal sinusoidal congestion without edema/vacuolization or necrosis in stat1 ko "protected" group. the induction of mrna coding for tnfα/ il-6 was higher in stat1 ko "nonprotected" livers. the expression of cxcl10, the product of stat1 activation downstream of tlr4 in type i ifn pathway, was profoundly and selectively depressed in livers from stat1 ko "protected" mice, as compared to iri susceptible livers. similarly, western blot-assisted phospho-erk expression was up regulated selectively in the stat1 ko "protected" group. in the second series, c57bl/6 mice were treated 1 h prior to liver ischemic insult with jak-2 inhibitor (tyrphostin ag490; 40 mg/kg, i.p.; n=5), or vehicle (n=5). the hepatocellular damage, as measured by salt levels (iu/l), and histology was significantly decreased in ag490 group, as compared with controls (mean = 12770 vs. 28770; p<0.05). the disruption of jak/stat signaling by inhibiting jak2 uniformly ameliorates the inflammatory immune response in liver iri. however, the blockage of stat1 alone is insufficient to reproducibly exert cytoprotection. as jak2 is upstream of stat1 as well as upstream of map kinase (erk), this study highlights the role of both signaling pathways in hepatic iri. purpose: tlr4 is required for maximal ischemic injury of the heart, liver, lung, and kidney. to better understand the mechanisms of tlr4 action, we investigated a murine model of ischemic kidney disease and examined endothelial tlr4 expression. methods: 1. animal ischemia reperfusion injury(iri): the right kidney of wildtype(wt) c57bl/10, or tlr4-deficient(ko) c57bl/10scn mice was removed. the left pedicle was clamped for 23 min, followed by 4 hr reperfusion. sham animals were controls. 2. bone-marrow chimera: four groups of bm chimeras were created: wt→wt; ko→wt; wt→ko; ko→ko (8 recipients/grp). 8 wks later, chimerism was confirmed by tail and blood genotyping, and mice subjected to renal iri. 3. tlr4 mrna detected by dig-labeled antisense; tlr4 on endothelium by anti-tlr4 and anti-cd31. 4. total genome mrna expression on ischemic vs. sham wt mice, ischemic vs. sham ko mice(4 mice/grp) determined using affymetrix mouse genome 430.2 genechips followed by genesifter analysis. quantitative real-time rt-pcr confirmed candidate genes. 5. ms1 endothelial cells were treated with h 2 o 2 (500um) for 30 min, cultured for 4 hr and then expression of tlr4 and endothelial genes determined. results: tlr4-deficient mice had less renal injury as assessed by pathology and function. radiation chimeras showed that radioresistant parenchymal cells and radiosensitive leukocytes were both required for maximal injury. immunohistology and in situ hybridization identified endothelia in the outer medulla as a major tlr4 expressing cell type at 4 hr post-reperfusion. genechip analysis revealed a panel of cytokine, chemokines, proinflammatory and cell-cycle related genes that were differentially expressed on iri versus sham kidneys. real-time rt-pcr confirmed that the following pro-inflammatory endothelial genes increased after ischemia in wildtype but not tlr4 ko mice: tlr4, pentraxin related gene(ptx3), and endothelial cell-specific molecule 1(esm1). real-time rt-pcr showed that in vitro h 2 o 2 treatment of endothelial cells induced expression of tlr4(6.6-fold), ptx3(4.6-fold), and esm1(69-fold). we found that endothelia in the outer medulla increase their expression of tlr4 after ischemia, and that the endothelial genes ptx3 and esm1 increase only in wt ischemic kidneys. we also found that h 2 o 2 , which mimics reactive oxygen species generated during iri, directly increases these same endothelial genes in vitro. nkg2d is an activating receptor expressed on nk cells and cd8 + t cells. ligands for nkg2d including rae-1, mult-1 and h60 in mouse, may be upregulated by tissues in response to stress. a study in mouse macrophages described upregulation of rae-1 in response to stimulation through tlr4 by lps. we have shown that tlr4 mediates kidney ischemia reperfusion injury (iri) and also observed rae-1 upregulation in iri kidneys. we now determined whether: 1) kidney iri could induce the expression of nkg2d ligands: 2) expression of nkg2d ligands is tlr4 dependent; 3). bone marrow (bm) derived cells or parenchymal kidney cells express nkg2d ligands. methods. kidney-ischemia was induced in tlr4 -/-, myd88 -/and wt mice for 22 min. blood and tissue were harvested at days 1, 3 and 5. primary cultures of mouse tubular cells (tecs) were also subjected to ischaemia (mineral oil overlay for 1 hr) or tlr4 activation (lps stimulation). bm chimeric mice were generated by transplanting bm into irradiated recipient mice before iri was induced. results. rae-1 mrna level in iri kidney was increased from day 1 to day 5, peaking at day 3 compared to sham operated kidney (14-48 fold increase, p < 0.001) measured by real time pcr. rae-1 protein was detected in renal tubular cells from ischemic kidney but not from shamoperated control by flow cytometry. mult-1 mrna expression was also increased from day 1 to day 5 (15-51 fold increase, p <0.005). both rae-1 and mult-1 mrna levels were reduced in tlr4 -/and myd88 -/-iri kidneys (2-8 fold reduction) versus wt controls (p < 0.01). tlr4 -/and myd88 -/primary cultured tecs submitted to iri in vitro also showed less rae1 and mult-1 mrna expression than wt controls (p < 0.01). lps stimulated rae-1 and mult-1 expression in wt but not in tlr4 -/-tecs. tlr4 -/mice bearing wt hemopoietic cells had significantly lower kidney rae-1 and mult-1 mrna expression after iri versus wt mice with tlr4 -/-bm (p < 0.05). conclusion. kidney iri causes rae-1 and mult-1 expression and kidney parenchymal cells are the dominant source. tecs can be stimulated to express rae-1 and mult-1 by ischemia or lps via the tlr4 pathway in vitro and deficiencies in this pathway provide protection against iri and rae-1 and mult-1 expression in vitro and in vivo. thus, kidney iri causes upregulation of nkg2d ligands by parenchymal kidney cells via tlr4. background: intestinal ischemia/reperfusion injury (iri) is a major clinical problem. although toll-like receptor 4 (tlr4) has been implicated as a potential link between the innate and adaptive immunity, little is known on its role in intestinal iri. our preliminary research in intestinal iri has shown that, compared to sham controls, wt mice had decreased survival, worse tissue injury/apoptosis, increased pmn infiltration, increased cd3+ cell infiltration, and increased production of tlr4, chemokines, and adhesion molecules. here we used tlr4ko mice to further investigate the role of tlr4 in intestinal iri and its effects on cytokine/chemokine programs and apoptotic signaling. methods: c57bl10 wt and tlr4ko mice underwent 100 min of total jejunoileal warm iri by clamping of the sma. separate survival and analysis groups were performed. intestinal tissue was harvested at 4h and 24h. tissue analysis included histopathology, cd3 immunostaining, myeloperoxidase (mpo) activity, rt-pcr for chemokines/ cytokines, and western blots for apoptotic and ho-1 protein expression. results: tlr4ko had superior survival compared to wt (100% vs. 33%, p<0.02). on histopathology tlr4ko had near normal-appearing villous architecture, while in contrast, wt showed mucosal erosions and villous congestion/hemorrhage. tlr4ko had reduced cd3+ cell infiltration as compared to wt (3.9±0.7 vs. 6.2±1.4 per hpf at 4h, p<0.001; and 4.8±1.1 vs. 7.0±2.3 per hpf at 24h, p<0.02). early mpo activity was also reduced in tlr4ko (0.11±0.06 vs. 0.88±0.5 u/g at 4h, p<0.005). rt-pcr analysis demonstrated decreased production of mrna for ip-10, mcp-1, rantes, and ifn-γ and increased production of il-13 in tlr4ko. there was decreased protein expression of caspase-3 and increased expression of bcl-2 and ho-1 in tlr4ko mice. conclusion: the genetic absence of tlr4 exerts protection against intestinal iri, demonstrating for the first time that tlr4 is required for intestinal iri. the absence of tlr4 signaling reduces iri through reduced neutrophil and t cell chemotaxis, and up-regulation of protective molecules. these results support data that tlr4 is a mechanistic link between the innate and adaptive immunity, implicating tlr4 as a potential therapeutic target for the prevention of intestinal iri. it is well known that liver steatosis increases hepatic vulnerability to ischemia/ reperfusion (i/r) injury as part of the transplantation process. endotoxin (lps) is thought to be a major contributing factor to the pathogensis of i/r. during portal occlusion, lps is translocated across the mesenteric tissue barrier into the portal circulation, and is delivered as a large bolus to the liver at the point of reperfusion. at this time, lps is mainly recognized by toll-like receptor 4 (tlr4). this leads to downstream signaling and the production of proinflammatory products that ultimately lead to cellular inflammation, necrosis, and apoptosis. it is well known that steatotic livers are highly sensitive to endotoxin as compared to their lean counterparts post-i/r, and we have previously seen that monoclonal antibody blockade of endotoxin dramatically improves animal survival after i/r. therefore, we propose the novel hypothesis that tlr4 signaling is a major contributor to cellular damage after steatotic hepatic i/r. to test this hypothesis, we subjected male 4-week-old c57bl/10j (control) or c57bl/10scn (tlr4 deficient, tlr4ko) mice to a high-fat diet (hfd) for four weeks. then, we subjected the animals to 35 minutes of total hepatic ischemia and 1 or 24 hours of reperfusion. there was a dramatic improvement in animal survival in the hfd tlr4ko animals versus control hfd animals at 24 hours (74% vs. 31% in control hfd animals, p<0.05). there was significantly more liver necrosis (as measured by a grading scale from 0-3) in the control hfd animals as compared to the tlr4ko hfd animals (1.4±0.3 in control vs. 0.4±0.2 in tlr4ko, p<0.05). in addition, we see significant increases in the message level of the proinflammatory cytokines il-6, il-12, and ifn-γ at one hour in the control hfd animals that is abrogated dramatically in the tlr4ko hfd animals. we do not see these dramatic changes in the control animals fed a normal diet. despite the significant increases in inflammation in the control hfd animals versus the tlr4ko hfd and normal diet control animals, we do not see changes in the tlr4 message level or endotoxin boluses, implying an increased sensitivity in the absence of an increased number of receptors. tlr4 is a critical molecule in the pathogenesis of steatotic liver ischemia/reperfusion, and represents a potential therapeutic target for expansion of the donor pool. the background: neutrophils are considered crucial effector cells in the pathophysiology of organ ischemia and reperfusion injury (iri). particularly, neutrophil elastase (ne) accounts for a substantial portion of the neutrophil function. this study was designed to explore the role of, and mechanism by which ne exerts its function in a mouse model of liver warm iri. methods: partial warm ischemia was produced in the left and middle hepatic lobes of c57bl/6 mice for 90 min, followed by 6-24h of reperfusion. mice were treated with ne inhibitor (nei; 2mg/kg p.o.; gw311616a; n=6) or control (n=6) at 60 min prior to the ischemia insult. after 6h or 24h of reperfusion, sast/salt levels and intrahepatic neutrophil accumulation (myeloperoxidase [mpo] activity) were assessed. the pro-inflammatory cytokine (tnf-α, il-6), chemokine (cxcl-1, cxcl-2, ip-10) and toll-like receptor (tlr) 4 gene expression profiles were screened by rt-pcr. liver samples were collected for histological grading, and detection of neutrophil infiltration by the naphtol as-d chloroacetate esterase stains. results: nei treatment significantly reduced sast/salt levels, as compared with controls (17695±3413 vs 10990±2411; p<0.05 / 33650±3793 vs 13510±6809; p<0.01 at 6 h, and 11778±3113 / 14483±3972 vs 3565±957 / 4810±1063; p<0.01 at 24h). the expression of pro-inflammatory cytokines, and chemokines was significantly reduced in the nei treatment group (tnf-α in 6h; p<0.05, il-6 in 6h and 24h; p<0.01 and p<0.05, cxcl-1 in 24h; p<0.01, cxcl-2 in 6h and 24h; p<0.01 and p<0.05, ip-10 in 24h; p<0.01). the mpo activity (u/g) was also significantly reduced following nei treatment (14.1±7.7 vs 4.0±1.2; p<0.05). tlr4 expression was selectively diminished in nei pretreated livers (6h; p<0.01). histological examination of liver sections has revealed that unlike in controls, nei treatment markedly reduced edema, diminished centrilobular ballooning/sinusoidal congestion, ameliorated hepatocellular necrosis, and decreased local neutrophil infiltration. conclusion: the inhibition of ne ameliorated hepatocellular damage, reduced local inflammatory responses, and neutrophil activity/infiltration in a stringent mouse liver model of warm iri. interestingly, it also downregulated the innate tlr4 signaling. this study documents the previously unrecognized ne -tlr4 cross talk, and implies neutrophil elastase in the signal transduction pathway instrumental for liver iri. lymphocyte lymphocytes are involved in the early pathogenesis of ischemia-reperfusion injury (iri) in kidney; however, their role during healing is unknown. this has direct clinical consequence since lymphocyte-targeting agents are currently administered to prevent rejection during recovery from iri in renal transplants. c57bl/6 mice underwent unilateral clamping of renal pedicle for 45 min, followed by reperfusion, and were sacrificed at day 10. mice were treated with saline (c), methylprednisolone (pred) or mycophenolate mofetil (mmf) i.p. daily from day 2 until sacrifice (n=12/group). lymphocytes were isolated from the kidneys, counted and stained with monoclonal antibodies. kidney damage (% damaged tubules) and proliferation (ki67 assay) were assessed. flow cytometry analysis demonstrated increased numbers of tcrβ + cd4 + and tcrβ + cd8 + t and tcrβ -nk1.1 + nk, but not cd19 + b cells at day 10 in the ischemic (ir) kidneys compared to contralateral. regulatory t cells, tcrβ + cd4 + cd25 + foxp3 + , and t cell subsets tcrβ + cd8 + cd122 + and tcrβ + nk1.1 + also increased. moderate tubular damage in cortex, severe injury in outer medulla and increased proliferation in both compartments characterized the repair phase. pred improved histological damage in ir kidneys, while mmf worsened it. proliferative index correlated with histology in outer medulla. pred reduced the total counts and activation of tcrβ + cd4 + and tcrβ + cd8 + t cells in ir kidneys, and increased the percentage of tcrβ + cd8 + cd122 + among total tcrβ + cd8 + t cells. mmf reduced all lymphocyte subsets, decreased the percentage of tcrβ + cd4 + cd25 + foxp3 + among total tcrβ + cd4 + t cells, and lowered il-10 tissue levels. il-6 and platelet derived growth factor-bb protein levels were also decreased in ir kidneys from mmf-treated mice. in conclusion, specific trafficking and phenotypic changes of kidney-infiltrating lymphocytes occur during recovery from renal iri, and lymphocyte-targeting agents, pred and mmf, alter tubular cell structure, proliferation, and inflammatory response in the repair phase. background: ho-1 plays an important cytoprotective role in a variety of organ injury models. we have shown that ho-1 exhibits potent cytoprotective effects against liver i/r injury. this study explores the function and mechanism of ho-1 in liver i/r injury by using sirna that suppress ho-1 expression both in vitro and in vivo. methods: using a partial liver warm ischemia model, c57bl/6 wide-type (wt) mice (n=6/gr) were injected with ho-1 sirna/nonspecific control sirna (2 mg/kg, i.v. at day -1) or ad-ho-1/ad-β-gal (2.5x10 9 pfu, i.v. at day -2). sham control wt underwent the same procedures, but without vascular occlusion. mice were sacrificed at 6 h of reperfusion; liver tissue and blood samples were collected for future analysis. in in vitro studies, ypen-1 endothelium cells were transfected with ho-1 sirna (100 nm) or ad-ho-1/ ad-β-gal. results: ho-1 sirna treated mice showed significantly increased sgot levels (iu/l), as compared with nonspecific control sirna or ad-ho-1 (7593±2859 vs. 3104±1777 and 211.5±40, respectively; p<0.05). these correlated with histologic suzuki's grading of liver i/r injury, with ho-1 sirna showing significant edema, sinusoidal congestion/cytoplasmic vacuolization, and severe hepatocellular necrosis; nonspecific control sirna showed moderate edema, sinusoidal congestion/cytoplasmic vacuolization. in contrast, ad-ho-1 revealed only minimal sinusoidal congestion without edema or necrosis. ho-1 sirna significantly increased local neutrophil accumulation and caspase-3 activity, and increased the frequency of apoptotic cells (31.8±5.5 vs. 18.5±5.4 and 3.5±2.2, respectively; p<0.005), as compared with nonspecific control sirna or ad-ho-1. both ypen-1 endothelium cells and wt mice treated with ho-1 sirna revealed markedly increased caspase-3 activity and reduced ho-1 expression. in contrast, ad-ho-1 significantly decreased caspase-3 activity and increased ho-1 and anti-apoptotic bcl-2/bcl-xl expression. conclusion: this study provides evidence that ho-1 exerts cytoprotection against i/r injury by regulating liver apoptosis and inhibiting caspase-3 activation pathway. organ specific sirna is not only a powerful tool to study local gene function, but it may also provide novel therapeutic application in transplant recipients. islet cell transplantation has recently emerged as one the most promising therapeutic approaches for diabetic patients to improve glycometabolic control. one major problem of the procedure is the requirement of an immunosuppression protocol capable of counteract both auto and allo-immune response. recent data suggest that anti-thymoglobulin (atg) can halt efficiently the mounting of an alloresponse and the recurrence of autoimmunity in nod mice by expanding antigen specific t-regulatory cells. we retrospectively reviewed our casuistry type 1 diabetic kidney-transplanted patients who underwent islet transplantation using an immunosuppressive protocol based on atg or daclizumab (as induction treatment) plus cyclosporine and mmf as maintenance therapy. 45 patients underwent islet after kidney transplantation in our center. thirty-four patients received a time course of atg as induction (125 mg per day for 7-10 days), (number of islet infused=499,596±28,780), and 9 patients received daclizumab at induction (1 mg/kg every 2 weeks for 10 weeks); (number of islets=486,108±70,644). no major adverse events were recorded in our center; no malignancies or infections outbreaks were evident. patients in the atg induction group showed a better islet survival rate compared to daclizumab (p=0.01), according to c-peptide>1ng/ml. a sustained and prolonged c-peptide secretion was evident in the atg group; while in the daclizumab group only 1 patient was functioning at 1 year. interestingly, in the atg, which reached a longer follow-up, we cannot observe a loss of beta cell mass according to our metabolic test, suggesting the preservation of islet mass. in conclusion, atg can provide a good protection towards both allo and auto immune response. the next step will be to use atg in a calcineurin free protocol, allowing a better expansion of t-regs. introduction: despite consistent achievement of insulin-independence, recent data indicate that the long term success of islets transplants using the edmonton protocol is <10% at 5 years. the cause of the nearly universal late islet allograft failure remains unknown but hypotheses include: allo or autoimmune injury, marginal mass exhaustion, hepatic site related dysfunction, and immunosuppression toxicity. we examined the series of islet transplants performed at our institution and noted marked differences in the outcome and complications in ia and iak groups. our results may provide insight as to the cause of chronic islet loss. methods: thirty-one islet infusions were administered to ia (n=9) and iak (n=8) type-1 diabetics between 9/2001-11/2007. ia and iak had similar demographics and transplanted islet mass (13,838 vs 13,411 ieq/kg). ia received edmonton like immunosuppression with zenapax induction and cni/srl, whereas iak patients received zenapax and: cni/mmf/pred (6), cni/mmf (1), cni/srl (1)). results: insulin-independence was achieved in all but 2 patients who completed therapy (2 others withdrew). compared with ia, iak exhibited better glycemic control (6-month mean hba1c 6.5 vs 6.1, stimulated c-peptide 2.3 vs 6.0), and improved islet survival; all ia eventually failed and only 1/8 were insulin free for >2-years, whereas only 3/7 iak grafts have failed with 4 exhibiting continued robust function at >38,>38,>46, and >58 months with 3/4 fully insulin independent. in addition, all ia patients demonstrated immune sensitization post graft failure versus 0/8 iak. all 9 ia developed mouth ulcers versus only 1/8 iak. conclusions: ia and iak exhibit striking differences in outcome and complications. the absence of mouth ulcers and lack of sensitization in iak may relate to steroid use and continued immunosuppression for the kidney graft, respectively. the superior outcome of the iak cohort may be a result of differences between the two groups including the use of maintenance steroids, prior exposure to thymo or the chronically immunosuppressed state of the iak recipient. perhaps the most interesting correlation with outcome is the absence of the anti-proliferative agent sirolimus in the iak group. our results provide clues to the cause of chronic islet transplant failure and may lead to novel approaches to avoid it. islet background: although islet transplantation has become an option for treatment of type 1 diabetes, all currently used immunosuppressive protocols have significant renal and islet toxicity. we describe a novel immunosuppressive protocol using sirolimus and the anti-lfa 1 antibody efalizumab that permits prolonged islet allograft survival without the need for steroids or calcineurin inhibitors (ci). methods: between february and august 2007, 4 consecutive type 1 diabetic patients with hypoglycemic unawareness and normal renal function received allogeneic pancreatic islet transplants. induction immunosuppression consisted of 2 doses of thymoglobulin given on pre-transplant days -2 and -1, efalizumab (5mg/kg sq/week starting on d -1), and sirolimus. maintenance immunosuppression consisted of sirolimus and efalizumab. results: all patients achieved insulin independence after single islet infusions (mean ieq/kg=8,905). three of 4 remain insulin independent 4 or more months after transplant (table 1) . patient 1 resumed low dose insulin (approximately 15% of original dose) 6 weeks after transplant and is awaiting a second islet infusion. her blood glucose control is markedly improved and she has not experienced any hypoglycemic episodes. all patients show persistent c-peptide secretion and have stable renal function. side effects due to efalizumab were limited to transient irritation at the injection site. conclusions: thymoglobulin induction followed by sirolimus and efalizumab maintenance is well tolerated and allows prolonged islet allograft survival. this protocol is the first ci/steroid free islet regimen resulting in insulin independence with a single donor islet infusion. by eliminating ci, this protocol minimizes renal and islet toxicity and may thus improve long-term islet survival and function. long . clinical and metabolic profiles were assessed every 3 months for18 months. results: si-exn group was on this drug for median time of 171 days pre-si. both groups were similar except for duration from post-completion to graft dysfunction (648±34 vs 221±58 in si-exn and si-c group respectively, p<0.05) and duration of graft dysfunction before si (664±83 vs 326±93, p=0.03). si-c and si-exn groups received mean of 8713±2123 and 5613±485 ieq/kg, respectively (ns). only 3/5 of si-c patients achieved insulin independence for 303, 403 and >1670 days after si. all subjects in si-exn group achieved insulin independence for more than 443, 621, 641, 654 days. at 18 months insulin independence was 20% in si-c and 100% in si-exn group. comparing pre and post-si, si-exn group had significantly lower a1c at 3-18 months and lower auc glucagon at 6 and 15 months (p<0.05). auc c-peptide in si-exn group was significantly higher than si-c at 3 and 9 months. intravenous glucose tolerance test showed significantly increased acute insulin responses to glucose at 3-18 months in si-exn and at 3 and 6 months in si-c group. si-exn group had more acute c-peptide response to glucose than si-c at 3 and 6 month (p<0.05). acute exenatide administration during intravenous glucose tolerance test at 15 month in si-exn revealed significantly increased acute insulin and c-peptide response to glucose which indicates improved first phase insulin release. conclusion: supplemental islet infusions under exenatide lead to insulin independence, restore first phase insulin secretion and result in long term insulin independence. exenatide this prospective phase 1/2 trial aimed to demonstrate safety and reproducibility of allogeneic islet transplantation (tx) in type 1 diabetic (t1dm) patients and implement a strategy to achieve and maintain insulin-independence with minimal islets. ten c-peptide negative t1dm subjects with hypoglycemic unawareness received 1-3 intraportal allogeneic islet tx. four subjects (group 1) received the edmonton immunosuppression regimen (daclizumab, sirolimus, tacrolimus). the next 6 subjects (group 2) received etanercept, exenatide and the edmonton regimen. we followed all subjects for 15 months after the first tx. the primary efficacy end point was insulin independence. secondary endpoints were hba1c, fructosamine, ogtt, mixed meal test, glucagon stimulation test, ivgtt and hypoglycemia. to study the effect of exenatide, we compared frequently sampled ivgtt, c-peptide, proinsulin, amylin and glucagon with and without exenatide. two self-limiting bleeds occurred in 18 infusions. all subjects became insulin independent. group 1 received a mean total number of islets (ein) of 1,460,080±418,330 in 2 (n=2) or 3 (n=2) tx, whereas group 2 became insulin independent after 1 tx (537,495±190,968 ein, p=0.028). all group 1 subjects remained insulin free through the 15-month follow-up. two group 2 subjects resumed insulin: one after immunosuppression reduction during an infectious complication, the other with severe gastroparesis and exenatide intolerance. hba1c reached normal range in both groups (6.5±0.6 at baseline to 5.6±0.5 after 2-3 tx in group 1 vs. 7.8±1.1 to 5.8±0.3 after 1 tx in group 2). baseline hba1c was significantly higher in group 2 than group 1 (p=0.046). pre-and post-tx hypo scores were 841.8±1217.9 and 0 in group 1 vs 812.8±941.7 and 33.5±50.0 in group 2. glucagon levels decreased significantly in all subjects. in group 2, the decrease in glucagon levels after challenge tests with and without exenatide was 18.7 fold more significant after exenatide in all subjects ( background istx has been investigated as treatment for type 1 dm. however, the hope this approach would result in long-term freedom from exogenous insulin has failed in practice. techniques for isolating islets have advanced and with availability of new is agents, strategies can now be developed specifically for istx that will provide greater immunologic protection w/o diabetogenic side effects. rejection/ vascularization still remain major limitations for success of istx. we hypothesize that bm is an accessible, immunologic privileged space with natural well-developed vasculature and may be a suitable site for istx. method wistar rats were used as donors/ recipients. dm was induced by iv-streptozocin. rats who had morning glc levels > 300 mg/dl on two separate occasions were used as recipients. ptx was performed as normal rodent standard. islets were isolated from pancreas by distending pancreatic duct with liberase. islets were separated on a discontinuous histopaque density gradient, further purified, counted, divided into aliquots transplanted into different sites (liver, bm). bw, glc and c-pep were measured in all groups before/after tx. background: in february 2007, the islet community was notified of a possible bovine product contamination in the collagenase enzyme (liberase hi) used for human islet isolations. to eliminate the potential hazard of bovine spongiform encephalopathy, we successfully adapted our human islet processing procedure to utilize a different gmp collagenase and neutral protease (nordmark/serva). here we describe what we consider the most important factors for achieving reproducible and clinically useable islet isolations. methods/results: a standard isolation protocol involving controlled enzymatic digestion followed by density gradient purification was used. seventeen donor pancreata were processed and ten were ultimately used for clinical transplantation. eight of these successful isolations were performed using the nordmark/serva enzymes (table 1) . the following factors were identified as being important for ensuring successful islet yields: 1) donor age and size: male donors 17-45 years old who were tall (>180cm) and heavy (>100kg) conclusions: incorporation of several important modifications into our existing islet isolation protocol has allowed us to routinely obtain high quality islet isolations using an alternative, bovine product-free gmp enzyme. (n=187)], and in 121 pts immuno was cni-free. tac-treated pts were younger, more sensitized and more frequently re-kt. dgf was more frequent in pts receiving cni-free immuno. as a group, these pts were older, more frequently received a kt from a donor after cardiac death and less frequently from a living donor. acute cellular rejection (acr) was diagnosed in 41 pts (6.8%), 13 occurred before day 90th (e-acr), and 28 after this date (l-acr). c4d-positive amr was diagnosed in 128 pts (21.2%), of which 58 were early (e-amr) and 70 were late episodes (l-amr rates of acute rejection (ar) and background: an increasing number of hs patients are being transplanted using desensitization protocols. these patients are considered high risk for ar, particularly antibody mediated rejection (amr). here we examined our ar rates and treatment outcomes for our hs kidney transplant (kt) recipients using our most current desensitization strategies. methods: between june 2004 (when rituximab was introduced to our desensitization and treatment protocols) to july 2007, 130 hs kt patients were transplanted using combinations of high-dose ivig, rituximab, and plasmapheresis (pp) for desensitization. we examined the overall ar, c4d-cell-mediated (cmr), and c4d+amr rates. amr episodes were treated with steroids, high-dose ivig, and rituximab. refractory and rapidly progressive amr was treated with pp. treatment outcomes and differences in ar rates between deceased (dd) and living donors (ld) was examined. recent reports suggest that treatment with the monoclonal anti-cd20 antibody, rituximab (rtx) may improve renal graft survival in antibody mediated rejection (amr); however optimal dosing for this indication is unknown. we examined the efficacy of a single low dose (500mg) of rtx for treatment of refractory amr in order to limit significant infective complications associated with conventional dosing regimens (375mg/m 2 ). rtx was used in seven consecutive patients who had refractory amr as judged by ongoing biopsy evidence of amr after 4 weeks of standard therapy consisting of pulse methylprednisolone and plasma exchange with low dose ivig (100mg/kg) (pe/ivig). all patients received tacrolimus and mycophenolate mofetil. amr was defined as 1) characteristic histology on biopsy (banff criteria), 2) graft dysfunction and 3) presence of a donor specific anti-hla antibody (dsab). b-cell counts (cd19) and serum creatinine (scr) were monitored. pe/ivig was ceased in all cases after rtx dosing. the average follow-up since rtx dosing is 16.8 months (range 5.3-28.9 mths). all patients still have functioning grafts (100% 12 month graft and patient survival), with current mean scr levels (156±17µmol/l) significantly lower than mean peak rejection levels (514±355µmol/l) p=0.05. cd19 counts fell to zero and remained <5x10 6 /l for >6 months in all patients. dsabs remain detectable by luminex flow beads in all patients despite stabilization of scr. two of 7 patients developed an infective complication post rtx dosing. one patient developed bacterial pneumonia requiring hospital admission whilst a second patient developed cmv viraemia and later bk nephropathy which has not led to significant graft dysfunction. hence, infectious complications are far less than those reported for multiple standard dose regimens in similar patient groups. large clinical trials are required to confirm the efficacy of rtx in treating amr as well as optimal dosing. standard dosing is based on oncology treatment regimens which are likely to be excessive for amr in patients already markedly immunosuppressed. our data suggests single low-dose rtx for refractory amr results in excellent patient and graft survival and leads to low rates of serious infective complications in the short-term. renal the demographic and clinical data were similar between +cxm and negative cxm groups. the rejection rate was significantly higher and the length of stay was significantly longer in +cxm group as shown in the table. the ra survival rates were 8%, 7% and 6% lower at 1, 3 and 5 years post transplant respectively among + cxm recipients. however, this did not reach significance mostly due to small sample size (p=0.11). the inferior ra outcome is seen during the initial few years after transplantation that disappears after 9 years. in clkt, pre-transplant +cxm can result in higher ra rejection rate and longer length of stay in hospital. la may not always confer immunological protection to ra in + cxm clkt and its true burden may be under recognized since cxm is not routinely performed in all clkt. study of antibody specificities or la volume to determine the effect of +cxm on ra outcome is essential. skpt in patients with positive cdc b-cell and/or flow cytometry crossmatch is associated with high amr rate despite low dose ivig and r-atg/alemtuzumab induction. pancreas graft survival is inferior in patients with positive crossmatch while kidney graft and patient survival are similar to that of negative crossmatch recipients. majority of amr can be reversed with treatment but further long-term follow-up is needed to determine the impact on graft survival. ten the first european phase iii trial with tacrolimus in the early 90´s clearly showed advantages for tacrolimus in terms of acute rejection (ar) vs the original cyclosporin formulation. however, no advantages were seen in survival rates. the present investigator-initiated, observational follow-up study collected data on patients included in the original cohort of 448 patients who participated in this large study and who were randomised to a triple immunosuppressive regimen consisting of tacrolimus, azathioprine and steroids (tac/aza/ster, n= 303) or cyclosporine, aza and steroids (csa/aza/ster, n=145). efficacy and safety parameters assessed at follow-up included: acute rejection; patient and graft survival; renal function, vital signs, basic lab results and immunosuppressive regimen for the patients 10 years after completion of the original study. results: currently, data are available from 75% of the patients. all assessments were conducted following the intent-to-treat principles. more patients in the tac than in the csa group remained on the randomised treatment. while kaplan-meier (k-m) estimates for patient survival at year 11 show comparable (72% tac vs 72%csa) results, k-m estimates for graft survival demonstrate an advantage for the tacrolimus cohort (51% tac vs 46% csa). graft half-life estimates (gjertson and terasaki method) yielded overall results of 14.2 years (tac) and 11.4 years (cs). in both treatment groups, ar was associated with inferior long-term results. for patients with ar, half-life estimates were 11.1 and 7.8 years for tac and csa, respectively, while in patients without ar half-lives were 19.2 (tac) and 15.8 (cs) years. mean serum creatinine after 10 years was significantly lower in the tac cohort vs the csa group (1.65mg/dl tac vs 2.01mg/ dl csa, p<0.05). mean glomerular filtration rates (mdrd4 estimate) were 48.7 ml/ min in tac and 41.9 ml/min in cs. after ten years of follow-up, the mean (c-0) levels were 7.6 mg/ml (tac) and 120 mg/ml (cs). conclusion: analysis of this long term follow-up of a large study confirms the benefits of a tacrolimus-based therapy. it also confirms the concept that freedom from early acute rejection is associated with superior long term results in renal fuction. therefore, long term renal allograft function is remarkably good in tac patients. cyclophosphamide a novel therapy for ivig/plex-resistant antibody-mediated rejection. (c)). moreover subgroup analyses within the different groups were made to identify potential differences. groups were analyzed for survival, graft rejection and graftvasculopathy (cad). kaplan-meier analysis was used and log-rank test was performed to detect differences. results: a total of 81 transplants (8.9%) were blood group compatible. the majority (n=32) were 0a transplants (40%) followed by 0b (n=19; 23%), aab (n=17; 21%), bab (n=7; 9%) and 0ab (n=6; 7%). overall survival comparison showed no significant difference in long-term survival (10-year) recent data has shown the utility of urinary biomarkers to predict delayed graft function early following renal transplantation, but their ability to predict longer-term allograft function is less clear. we evaluated whether urinary biomarkers measured in the early post-transplant period would predict allograft function at 6 and 12 months after renal transplantation. urinary biomarkers including n-acetyl-β-d-glucosaminidase (nag), kidney injury molecule-1 (kim-1) and matrix metalloproteinase-9 (mmp-9) were measured in 20 patients at hours 0, 6, 12, 24 and days 2 and 3 following renal transplantation. glomerular filtration rate (gfr) at 6 and 12 months were calculated using the mdrd equation. all 20 patients had functional allografts at 6 months and 2 patients lost allografts after 6 months. levels of individual biomarkers at different post-transplant time points were correlated with 6 and 12 month gfr using spearman's correlation (rho) and are shown in the urinary nag levels at post-transplant hours 0, 6, 24 and days 2 and 3 showed significant negative correlation with 6-month gfr. nag levels at hour 24 and days 2 and 3 maintained the significant negative correlation with 12-month gfr. urinary kim-1 levels at the 24 hour point showed significant negative correlation with 6-month gfr and kim-1 levels at hour 24 and day 2 displayed significant negative correlation with 12-month gfr. urinary mmp-9 levels at no time points had significant correlation with either 6-month or 12-month gfr. urinary biomarkers particularly nag shows promise as a tool in the early prediction of longer-term allograft function following renal transplantation. in kidney disease chemokines are involved in the recruitment of leukocytes causing kidney damage and progression to endstage renal failure. similar mechanisms are proposed for chronic allograft failure in renal transplant recipients. the chemokine attracted leukocytes produce proinflammatory and profibrotic cytokines contributing to fibroblast proliferation, matrix production and tubular atrophy. the role of chemokines in the acute post-ischemic tubular necrosis early after renal transplantation and their impact on long term allograft outcome has not been investigated yet. methods 30 patients (23m, 7f, mean age 49.9yrs) with dgf (with a median number of 6.4 dialysis sessions) developed biopsy proven acute tubular necrosis during the first three weeks after transplantation. the biopsies were studied by immunostaining for ki67, ccr1, ccr2, rantes, mcp-1, cd20 and cd68, respectively. the patients were followed for a mean time of 4.2yrs. none of them developed a rejection episode in the follow up time. after follow up the mean serum creatinine was 2.4mg/dl (0.8-6. the goal of a tx is for the recip to achieve long-term survival (surv.), with continued graft function, equivalent to the gen. population. we studied subsequent outcome in 2202, 10-yr kidney tx survivors (tx 1963-1997) ; 62% living donor (ld); 87% 1 st tx; 41% female; 28% type 1 diabetes; mean age at tx (±se), 33±15 yrs. actuarial 25-yr surv. is shown in table 1 ; ld patient, graft, and death-censored graft surv. is signif. ↑vs. deceased donor (p<.0001). mean creatinine (± se) at 10 yrs was 1.7±.8; 15 yrs, 1.7±.9; 20 yrs, 1.5±1; 25 yrs, 1.6±1. the 2 major causes of late graft loss (gl) were death with function (dwf) and chronic rejection (can). by multivariate analysis, risk factors for gl after 10 yrs were age ≥50 yrs at tx; type 1 diabetes, retx, hla mm ≥3; ≥1 acute rejection episode, and pretx cardiac, peripheral vascular, or liver disease (p<.05). the most common cause of dwf was cardiovascular disease (cvd); however, >20% deaths were due to malignancy. the use of kidneys from donors with a positive serology for hcv into hcv(+) recipients still remains controversial. in 1990, our units adopted this policy, subsequently modified in 1993, so these kidneys were limited to recipients with a positive rna hcv before transplantation. the aim of the present analysis was to review the long-term safety of our policy. since january 1990 to february 2007, 474 hcv(+) patients with a negative hbsag and not treated with interferon received a kidney transplant in our units. 313 patients were transplanted from an hcv(-) donor(group 1) and 161 from an hcv(+) donor(group 2). median follow-up time was 65(ir 24-117) months. remarkably, group 2 showed a significantly higher donor age (46.6±13.6 versus 41.1±18.8 years;p<0.0001) and recipient age (50.4±13.1 versus 44.9±14.6 years;p<0.0001), as well as a worse hla compatibiliy than group 1. immunesuppressive therapy did not significantly differ between the two groups. group 2 notably, only 5 patients died because of a liver disease (3 in group 1 versus 2 in group 2) and only 2 patients developed a hepatocarcinoma (both in group 1). in summary, no significant differences were observed in hcv(+) recipients according to hcv serology of the donor, in terms of death censored graf survival, patient survival and evolution of liver disease. therefore, our experience clearly demonstrates that the use of kidneys from hcv(+) donors into hcv(+) recipients is a safe strategy in the long-term and a wise way of using these kidneys, that otherwise would be lost at a moment of shortage. introduction: deceased donor kidneys are allocated to adult candidates in the u.s. primarily by waiting time and hla similarity. we investigated two alternative allocation systems, lyft-scd and lyft-dy, that prioritize offers of kidneys to adults based in part on lyft. methods: based on characteristics of each candidate and donor, lyft was calculated as the difference between expected median years of life for the candidate with that kidney donor transplant (tx) versus without a kidney. years on dialysis were weighted at 0.8 of years with a functioning graft. the relative risk of graft failure for each donor was calculated from donor factors using a cox model and the donor profile index (dpi), the percentile score of that risk among kidneys used in 2003 (100%=greatest risk of graft failure). using actual candidates and deceased donors and acceptance patterns from 2003, we modeled allocation with the kidney-pancreas simulated allocation model (kpsam). kpsam sequentially offers kidneys to candidates according to user-specified allocation rules, and models the probability for kidney placement and post-tx survival. the lyft-scd model allocated scd kidneys by lyft and ecd kidneys by dialysis years (dy). the lyft-dy model allocated organs according to the formula [.8lyft(1-dpi)]+[dy(.8dpi+.2)]+[4pra/100] to adult kidney tx candidates. results: the table compares a year's worth of kidney-alone recipient characteristics, lifespan after tx and additional lifespan (v. dialysis) due to tx among all kidney (including spk) recipients, and the donor/recipient age correlation (r) under current national kidney allocation, allocation using lyft for scd, and allocation using lyft-dy within each donation service area. conclusions: allocation systems incorporating lyft have the potential to increase the number of years of life attainable from tx and are being considered for the allocation of kidneys within the u.s. introduction : organ shortage for transplantation has led to the use of marginal kidneys, which has been associated with poorer but still acceptable results. in our center, the transplantation of two very marginal kidneys into a single recipient was used as a strategy to increase the number of organs available. the present study reports renal function and survival of dual-kidney transplants (dkt) performed at our institution. methods : from october 1999 to june 2007, 392 transplants were performed at our center, from which 63 were dkt. kidney selection for a dkt was based on refusal of the kidneys for single kidney transplantation (skt), donor's age ≥ 60 years and ≥15% of glomerulosclerosis on pre-implantation biopsy. the calculated creatinine clearance (crcl, ml/min), graft and recipient survival from dkt were compared to those of skt from ecd (based on unos criteria, n=66) and ideal donors (id, aged 10 to 39 y, n= 63) over a seven-year period. results : dkt donors were significantly older than the two other groups (dkt: 69 year old, ecd: 62, id: 24). dual transplants were offered to older recipients by design (60, 50, 44). delayed graft function was more prevalent for dkt and ecd than with id (27%, 29%, 11%). twelve, 36 and 84-month crcl were similar for dkt and ecd but lower than id (twelve months: 58, 59, 81; 36 months: 54, 60, 82; 84 months: 62, 51, 86, respectively). patient survival, actuarial graft survival and actuarial graft survival censored for death were similar between the 3 groups. conclusion : to our knowledge, this study reports short and long-term outcome of the largest cohort of dkt performed at a single institution. it shows that dual-kidney transplantation is a more complex intervention with more risks of surgical and post-op complications (data not shown). however, with adequate surgical expertise, dkt patients can expect long-term results comparable or even better than ecd. in our center, the use of dkt has increased the number of transplantations by 19% for the period of study. it allowed older patients to receive a graft within shorter delays. moreover, it permitted a more judicious allocation of a resource that is still limited. an results: very few differences resulting from donornet were seen. for regionally exported kidneys, cold ischemia time (cit) was 22.9h before donornet (bd) and 23.3h after donornet (ad); 6.4% of regionally exported kidneys had cit>36h bd, as compared with 9.0% ad. for nationally exported kidneys, cit was 27.3h bd and 26.8h ad; 17.9% of nationally exported kidneys had cit>36h bd, as compared with 16.1% ad. the same hucs of exported kidneys bd were also hucs ad. of the top 10 centers utilizing exported kidneys bd, only one was not within the top 10 centers utilizing exported kidneys ad. when we looked at cit for kidneys exported to hucs, again there was little difference before or after donornet, with a trend to possibly longer cit for hucs. for kidneys regionally exported to hucs, cold ischemia time (cit) was 23.1h before donornet (bd) and 23.4h after donornet (ad); 7% of regionally exported kidneys had cit>36h bd, as compared with 10.4% ad. for kidneys nationally exported to hucs, cit was 27.2h bd and 27.4h ad; 17.5% of nationally exported kidneys had cit>36h bd, as compared with 16.8% ad. conclusion: the first 6 months of national implementation of donornet were not successful in decreasing cit for regionally or nationally exported kidneys. furthermore, the centers to which a high proportion of these kidneys were exported did not change as a result of donornet, and for these centers allocation efficiency is no better and might even be worse than before. machine cold machine preservation has been shown to improve outcome following transplantation of deceased heart-beating donor kidneys. to determine whether cold machine preservation also improves outcome of kidneys donated after cardiac death (dcd) we undertook a multicentre randomized controlled trial in the uk comparing machine perfusion with simple cold storage. one kidney from each dcd donor was randomized to machine perfusion (organ recovery systems lifeport device with kps-1 solution), the other to perfusion with uw (viaspan) solution followed by simple cold storage. the primary outcome measure was the need for dialysis in the first 7 days (delayed graft function, dgf); secondary outcome measures included gfr at 1 week (mdrd technique); 3 and 12 month graft and patient survival. at the time of writing, one week outcome data are available for all patients. a sequential design was used with the data inspected after 60 transplants and then every 20. all recipients received basiliximab, mycophenolate sodium, tacrolimus and prednisolone. the significance of the initial results has been tested using paired t tests and mcnemar's exact tests. recruitment was stopped after 46 donors when the unadjusted sequential analysis concluded that there was no difference in the incidence of dgf. 91 of the 92 kidneys were transplanted; one was not used for anatomical reasons. one kidney suffered primary non function; it had been machine preserved. the results shown below represent intention to treat. introduction: split liver transplantation has been adopted as an alternative to expand the organ donor pool. while the adult/child split liver (a/csl) in which an extended right graft (erg) is transplanted into an adult and a left lateral segment (lls), transplanted into a child, has gained a wide acceptance within the transplantation community, adult/ adult split liver (a/asl) in which the liver is split into a full right (fr) and full left (fl) is still performed very rarely. we analyzed the experience at our center with split liver grafts over a period of 10 years. material and methods: between october 1997 and october 2007 we performed a total of 676 liver transplants in 625 recipients (370/328 children and 306/297 adults). among the 593 (310 children and 283 adults) recipients of a primary isolated liver transplant, 281 (247 children and 34 adult) were transplanted with a a/c or a/a sl. the recipients of a whole size graft (249 adults and 63 children) were used as a control. results: adults: 34 patients received a split liver graft (26 erg graft and 8 fl/fr grafts) and 249 a whole liver graft. overall the incidence of biliary complications using a split liver graft was 29% and with a whole liver graft 15% (p= 0,0506).for the recipients of a split liver graft 1 and 5 years patient and graft survival was 91%/88% and 88%/85%. recipients of a whole liver graft had a 1 and 5 years patient / graft survival of 85%/84% and 80%/78% respectively. children: 247 children received a split graft (230 lls, 12 erg and 5 fl) and 63 a whole size graft. biliary complications occurred in 42% of the recipients of a split liver graft and 5% of the recipients of a whole size graft (p < 0,0001). among the recipients of a split graft 1 and 5 years patient / graft survival was 91%/84% and 87%/80% respectively. the recipient of a whole size graft had a 1 and 5 years patient / graft survival of 84%/78% and 83%/74%. conclusion: incidence of biliary complications is significantly higher using a split graft. at a high volume center, use of split liver grafts reached comparable and even better results of a whole liver graft in terms of patient and graft survival. thus, the use of split grafts should be strongly encouraged to expand the donor pool. a groups of primary ldlt and ddlt recipients were identified by matching diagnosis, meld score, and recipient age. cost data, acquired from the hospital financial database and inflation adjusted and clinical outcomes were compared between the 2 groups. background: liver transplant recipients with (gw/rw) < 0.8% are thought to have a higher incidence of post-operative complications, including small for size syndrome (sfss), and overall inferior outcomes. we analyzed a cohort of partial liver graft recipients and compared those with gw/rw < 0.8% to those with gw/rw > 0.8%. results: between 1999 and 2007, 107 adult patients underwent partial graft liver transplant. seventy-six grafts were from live donors (ldlt) and the remaining 31 were from deceased donor split-liver transplants (slt). of these, 22 patients had gw/ rw < 0.8% (12 ldlt, 10 slt), and 85 had gw/rw > 0.8% (64 ldlt, 21 slt). median follow-up was 46.1 months and did not differ between the two groups. baseline demographics including donor and recipient age, and meld at the time of transplant also did not differ between groups (table) . three-month and 1-year graft survival for the two groups was comparable. three recipients with gw/rw < 0.8% developed sfss (13.6%); all three had inflow modification with splenic artery ligation (sal), and 1 of the 3 grafts was lost at one-year. eight recipients with gw/rw > 0.8% developed sfss (9.4%) (p = ns); six underwent sal and none had graft loss at one-year. there was a trend toward increased hepatic artery thrombosis with the smaller grafts that did not reach statistical significance (p = 0.10). the incidence of other surgical complications was similar between the two groups (table) . the diagnosis of rejection in post-transplant kidney disease depends largely on the assessment of biopsy pathology, which suffers from arbitrary scoring, subjectivity, and sampling variance. with microarray gene expression data from 186 biopsies for cause, we used predictive analysis of microarrays (pam) to build a rejection classifier. because of the imperfect gold standard, the classifier cannot and should not have extremely high predictive accuracy. the goal was to see if microarrays could detect a robust rejection signature despite this uncertainty, and to identify the top genes distinguishing rejecting from non-rejecting biopsies. by examining discrepancies between pathology diagnoses and pam predictions, and using clinical follow-up information, we combine the strengths of each method to produce a more accurate diagnostic system. biopsies with rejection plus clinical episodes (fig. 1 ) have higher positive predictive value than those lacking episodes. the majority of episodes occur in the top part of fig.1 , indicating that a valid rejection signature is being detected. of the exceptions, most had been treated with steroids before the biopsy, suppressing their gene expression patterns. samples called borderline rejection separated into two distinct high/low probability groups using pam, with all the cases classified as clinical rejection episodes occurring in the high probability group. of the 39 genes chosen by the classifier, 36 had previously been annotated in our studies of mouse kidney graft rejection. gzma, gzmb, and prf1 ranked 26th, 32nd, and 38th respectively. the top five genes were: cxcl11, gbp1, cxcl9, indo, and cxcl10, all previously annotated in rejecting mouse kidneys as interferon-γ induced genes. in summary, gene expression-based classifiers, combined with histopathology, promise more accurate diagnostic assessment of the state of kidney transplants at the time of biopsy. moreover our data driven classifier independently identifies the genes previously annotated in experimental studies. influence of donor background: renal-cell associated tlr4 activation was found to be important in mediating ischemia reperfusion injury to murine kidneys. we hypothesized that genetic variations within the tlr4 gene of the kidney donor affects the rate of delayed graft function (dgf). methods: we genotyped the functional tlr4 polymorphisms (snps) d299g (rs4986790) and t399i (rs4986791) in 268 kidney donors from 2 centers and correlated them with the occurrence of dgf. dgf was defined as the need of dialysis within 7 days post-transplant or less than 25% drop in creatinine within the first 24 hours after transplant. in a sample of 67 patients from one center, we analysed intragraft hmgb-1 (high mobility group box protein-1) gene expression in pre-and post-implantation biopsies. statistical analyses were performed using the spss statistical package. results: both tlr4 snps were in hardy-weinberg equilibrium, and were combined for further analysis. recipients of a tlr4 mutated kidney showed a significant lower rate of dgf (p=0.005), which was persistent after correction for known donor-derived risk factors (donor age, ecd vs. dcd-kidney, cold ischemia time, p=0.007, hr 4.42, cl 1.51-12.94). additionally, we confirmed the presence of a possible tlr4 ligand, hmgb-1, in pre-and post-implantation biopsies. we also confirmed the presence of a functional tlr4 receptor in human proximal tubular cells which expressed mcp-1, il1-β, il-6 and tnf-α after specific tlr4 agonist treatment. conclusion: human renal tubular epithelial cells express tlr4 and a functional tlr4 snp in donor kidney is associated with lower rate of delayed graft function. background: methylation of promoter cpg islands has been associated with gene silencing and demonstrated to lead to chromosomal instability. we postulate that differences in methylation patterns observed in tissues ranging from normal to cirrhosis to hcc may lead to the discovery of direct precipitating events that contribute to tumorigenesis in hcv-hcc patients. methods: dna from 20 hcv-hcc tumors and corresponding non-tumor hcv cirrhotic tissues as well as 20 independent hcv cirrhotic tissues and 20 normal liver tissues were bisulfite treated and hybridized to the illumina goldengate methylation beadarray cancer panel i for interrogating cpg sites in the promoter regions of 808 distinct genes. for each cpg site, a summary statistic representing "percent methylated" was estimated. for each cpg site, a jonckheere-terpstra test was applied to identify whether there was a significant monotonic trend in percent methylated across the independent normal, cirrhotic, and hcc tissues. in addition, the paired hcc and non-tumor cirrhotic tissues were analyzed using a paired t-test. the q-value method for estimating gene-wise false discovery rates (fdr) was used to adjust for multiple comparisons; cpg sites with an fdr<0.05 were considered significant. to identify if serological responses to allogenic non-hla renal compartmentspecific antigens can be detected after renal transplantation (txp). methods: 36 paired pre-and post-transplant (mean time 25 months) serum samples from 18 pediatric kidney allograft recipients were used for identification of de novo non-hla antibody formation assessed using invitrogen protoarray (v3), measuring signal post-pre. probes from the protoarray and cdna microarray platforms were re-annotated to current ncbi entrez gene identifiers using ailun. 34 cdna arrays (gse:3931) from 7 normal kidney regions (inner and outer cortex, inner and outer medulla, papillary tips, renal pelvis and glomeruli; see table) were analyzed for compartment-specific genes by sam, and the highest-ranked genes in each compartment (ks two-sample test p < 0.001) and were ranked against each individual patient's numerical antibody response across the 5057 proteins on the protoarray. results: in 83% of patients (15/18), kidney-specific serological responses against the renal pelvis were the first to be detected, followed by responses against renal cortex. control gene expression data sets from other solid organs (gse: 1133; lung, heart, pancreas) did not demonstrate immune-response enrichment. the presence of these antibodies was not associated with donor specific hla class i or ii antibody levels. conclusion: we demonstrated de novo formation of circulating non-hla non-abo antibodies after txp, irrespective of hla antibodies, are detected against kidneyspecific protein targets. as these antibodies are not seen against other solid organs, the response is likely allogeneic. the renal pelvis and renal cortex have the highest immunogenic potential. we conclude that urinary cell mrna profiles that include levels of mrna encoding tubular proteins and mrna encoding proteins implicated in emt offers a noninvasive means ascertaining renal allograft status with respect to presence or absence of can. functional functional annotation of these genes identified cell cycle pathway and carboxylic acid metabolism pathway as potentially relevant for both datasets. altogether, these data suggest that as compared with recipients requiring on-going immunosuppression, operationally tolerant liver recipients exhibit traits of immunological quiescence and activation of natural killer related pathways. liver and kidney tolerant states appear to be fundamentally different biological processes, although some common pathways could be relevant to both settings. genetic introduction: both normal and fibrotic renal allografts develop a large number of persistent changes in pro-inflammatory gene transcripts early after transplantation (1) . the aim of the current study was to determine if this "transplant effect" is attenuated by immunosuppression, fibrosis or hla-match. methods: we studied 41 histologically normal implantation biopsies (t0) and 45 12m protocol biopsies (t12) from living donor kidney transplant recipients who had no identifiable post-transplant complication (no acute rejection, polyoma virus, etc). patients were stratified into 4 distinct clinicopathologic groups. the first three groups had normal histology at both t0 and t12 and included: 1) hla identical-tac treated (n=10); 2) non-hla identical-tac (n=19); 3) non-hla identical srl (tac-free, n=8). a fourth group was histologically normal at t0 but developed mild fibrosis by t12 (n=7). mrna expression from these biopsies was measured using the u133plus2.0 microarray and custom taqman low density arrays (tlda). data for each group was tested using a generalized linear model and changes common to each dataset identified (p<0.05). results: in addition to transcript changes specific for each clinicopathologic condition, a large number of transcripts (n=1152) were altered in all groups. 68% (n=778) had higher expression in all comparisons at t12 versus t0, including fibronectin and collagen iv. 32% (n=374) of transcripts were considered down-regulated, including interleukin 6 and 1β. gene ontology and signaling pathway analyses showed many of the transcripts to be involved in pro-inflammatory and immune response signaling pathways (ex. tlr, il-10/-2, etc). custom tldas were used to study several genes considered not detected by microarray. this included tgf-β1, cd-3e/-28/-74 and vegf, all of which were significantly up-regulated in multiple comparisons. conclusions: persistent changes in pro-inflammatory transcripts occur in all renal transplants. this "transplant effect" cannot be explained by calcineurin-inhibitors (occurs in tac-free immunosuppression), increased alloreactivity (similar in hla identical/ non-identical) or fibrosis. a likely cause is persistent changes from ir injury. the role of the "transplant effect" in long-term allograft survival is unclear, but it likely interacts with other forms of graft injury. background: a critical 26 gene-set for acute renal rejection (ar) diagnosis in peripheral blood has been previously identified by our group using cross-platform hybridization of 91 unique peripherla blood samples with matched biopsy diagnosis, with ar (n=39) and without ar (sta; n=52), with cross-hybridization across 3 human microarray platforms: 30k cdna lymphochip, 44k agilent and 54k affymetrix hu133plus. in this study we proposed to verify and validate this gene-set for ar diagnosis and prediction by peripheral blood pcr-based analysis. method: 57/91 peripheral blood samples from the microarray gene-set were selected for 384-well format multiplex abi-taqman qpcrv validation of ar diagnosis across the 26 gene-set. an independant set of 66 new, time and immunosuppression matched, peripheral blood samples (30sta, 36ar) were used as a test-set for blinded prediction of ar diagnosis using qpcr across the most informative 8 genes. 42 sequential samples from 14 ar patients (at ar, 1-3 months prior to, and after ar) were also examined by qpcr for these 8 genes for blinded ar prediction, prior to biopsy proven ar. prediction models were generated using logistic regression analysis and p<0.05 considered significant. result: in the validation set of 57 samples, 8/26 genes were highly significant for confirming peripheral blood-ar diagnosis (p<0.03). in the test group of 66 samples, ar was predicted with a very high level of sensitivity and specificity (ppv and npv> 90%). in the group of 14 ar patients with sequential samples pre-and post-ar, expression for these 8 genes were significantly elevated in the pre-ar samples (vs. sta, p<0.01) but were not different between pre-ar and ar values. conclusion: a highly specific and sensitive biomarker panel of 8 genes has been derived and tested by cross-platform microarray analysis. thsi gene-set has now been validated and further verified by multiplex pcr, for ar diagnosis and prediction, even 3 months prior to the rejection event. utility of this gene-set should be further validated in real-time by serial longitudinal peripheral blood testing, for more senstive and specific minimally invasive monitoring for graft rejection. tolerance/immune deviation ii background: our previous studies showed that regulatory t cells (treg) execute suppressive function in both the inflammatory site of the allograft and the draining lymph node (dln) to suppress allograft rejection. we explored how treg influenced the migration and function of antigen specific effector t cells and dendritic cells (dc) at these two sites in an islet transplant model. methods: treg were sorted from wild type or ccr7 -/-c57bl/6 mice, labeled with pkh26, and transferred directly to islet allografts (balb/c into c57bl/6). effector cd4 t cells (te) from alloantigen specific t cell receptor transgenic mice were labeled with csfe and transferred intravenously. treg and te migration to islet grafts and dln were analyzed with flow cytometry and immunohistochemistry. chemokine expression was determined by rt-pcr. cx3cr1 gfp mice, in which dc are internally labeled with gfp, served as islet donors, and donor dc migration was tracked with fluorescence microscopy. results: during priming and rejection, islet allografts expressed a panel of inflammatory chemokines shortly after transplantation that recruited te to the islets. te accumulated and proliferated in both the islet allograft and the dln. concomitantly, donor derived dc migrated from the islet to the dln as early as 1 day after transplantation. local transfer of treg into the islet allograft inhibited islet chemokine expression, inhibited donor dc migration in an il-10 and tgfb dependent fashion, and reduced te migration to and proliferation in the graft. the transferred treg also migrated from the islet allograft to the dln, and directly inhibited te accumulation and proliferation in the dln, and migration of te to the islet. ccr7 -/-treg, which could not migrate to the dln but were retained within the graft, and were far less effective than wild type treg in inhibiting te migration and proliferation into both the islets and dln. conclusions: treg migrate to the islet and suppress parenchymal cell chemokine expression, dc migration, and te responses in the islet allograft. treg also migrate to the dln to suppress te proliferation and migration. treg trafficking from the allograft to the dln is crucial for suppressive function in order to target the separate effector mechanisms. these results demonstrate novel and important functions for migration in treg induced suppression and graft survival. tregs cd4+foxp3+ regulatory t cells (tregs) play an important role in transplant tolerance, yet basic aspects of treg biology including the mechanisms involved in their induction remain unclear. α-cd45rb is a potent tolerogenic agent that induces a 2x increase in number of tregs in wt mice, even in the absence of allo-antigen. here we use foxp3red fluorescent protein reporter knock-in mice to study treg homeostasis and identify the mechanisms by which α-cd45rb induces tregs. cfse-stained highly sort-abstracts purified foxp3+ or foxp3-cells were adoptively transferred into fully replete naive wt congenic mice. whereas only 5-10% of transferred foxp3-cells undergo homeostatic proliferation (hp) over 10d, 50-70% of foxp3+ cells proliferate -a surprisingly high rate of hp. moreover, treatment with α-cd45rb markedly enhanced hp by transferred foxp3+ cells, resulting in a 10x increase in cell number. α-cd45rb induced de novo foxp3 expression in 13-25% of transferred foxp3-cells (many of which subsequently underwent hp). thus, α-cd45rb induces both conversion of foxp3-to foxp3+ cells and promotes hp in foxp3+ cells in the absence of exogenous antigen. we then addressed the signals controlling basal hp of tregs and both hp and conversion mediated by α-cd45rb. cfse-stained congenic cd4+ cells adoptively transferred into naive wt mice were untreated, or received csa, α-cd45rb, or both and foxp3+ and foxp3-cd4 cells were assessed on d10. calcineurin inhibition greatly reduced basal hp of transferred foxp3+ cells compared to untreated mice. moreover, csa completely abrogated increased treg hp induced by α-cd45rb, although conversion still occurred. next we assessed the role of tgfβ. we found that blocking tgfβ signaling with α-tgfβ shortened allograft survival, but had no effect on basal treg hp, or on treg hp or conversion by α-cd45rb. finally, although exogenous antigen is not required, basal and α-cd45rb-induced hp may still require recognition of self-ag bytregs. indeed, preliminary results reveal that when cfse-labeled cd4+ cells are transferred into mhcii ko mice, basal hp by tregs is reduced and not restored by α-cd45rb. thus, α-cd45rb alters normal controls regulating hp by treg. moreover, both basal and α-cd45rb-mediated hp requires intact calcineurin activity and tcr signaling, but not tgfβ. understanding the signals controlling hp in treg may reveal new therapeutic targets for tolerance induction. we report the first demonstration of a tolerance-inducing strategy based on posttransplant administration of allo-ag-specific treg (aastreg) and rapamycin (rapa), in a fully allogeneic, unmanipulated mouse heart transplant model. enrichment of naturally-occurring aastreg represented the first step to counterbalance the high frequency of alloreactive t cells. selection was achieved by co-incubation of freshly-isolated cd4 + cd25 + t cells with donor bone marrow-derived dendritic cells (dc). the source of stimulatory factors necessary to sustain treg proliferation was the supernatant of cd4 + cd25 -t cells co-cultured with allogeneic dc (mlrsup). use of mature (cd86 high ) dc favored extensive expansion of foxp3cells capable of il-17 production (t h 17). co-culture of treg with immature dc in the presence of mlrsup rendered a t cell population with regulatory phenotype: foxp3 + , gitr + , ctla-4 + , ccr7 + , cd62l -. these cells inhibited in vitro effector t cell proliferation at 1:20 and 1:40 treg:t cell ratios. the suppressive activity was ag-specific; no significant inhibition was evident when effector t cells were stimulated by third party dc. aastreg were then tested for their ability to induce transplant tolerance in a mouse heterotopic heart allograft model (balb/c to c57bl/10; unmanipulated). rapa was used: i) to inhibit effector t cell proliferation, while sparing treg activity and thus enhancing the in vivo treg:effector t cell ratio; ii) to promote resolution of the inflammatory state and preserve the susceptibility of conventional t cells to treg suppression. graft recipients received rapa (1mg/kg/d; d0-9) and 2x10 6 aastreg i.v. on d7. in comparison to the untreated group (median survival time, mst=11d; n=14), rapa alone extended mst to 30 d (n=7), with no long-term graft survival. under cover of rapa, aastreg exerted a profound tolerogenic effect: >80% recipients exhibited longterm graft survival (mst>150d; n=6). this effect was stronger than polyclonal treg administration: 40% long-term survivors (mst>50d; n=5). moreover, the tolerogenic effect was ag-specific, as aastreg selected against third party dc (c3h/hej) did not prolong graft survival in comparison to the rapa-only control group. these results indicate the feasibility and therapeutic potential of ag-specific tolerogenic cell therapy based on post-transplant administration of selected aastreg. direct intravenous immunoglobulins (ivig) is an effective treatment for t-cell mediated graft rejection and autoimmune diseases. ivig treatment is associated with rapid clinical improvements without the side effects of global immunosuppression. this prompted us to ask whether ivig might enhance directly the suppressive function cd4+cd25+foxp3+ regulatory t cells in vitro and in vivo. in vitro, mouse cba/ca (h2 k ) total cd4 + or cd4 + cd25responder cells were stimulated with c57bl/10 (h2 b ) splenocytes, and human cd4 + or cd4 + cd25cells were stimulated with allogeneic antigen presenting cells. in vivo, 1*10 5 total cd4 + or cd4 + cd25 -t cells of cba/ca mice were adoptively transferred into cba/rag1 -/mice, and one day later transplanted with a tail skingraft of c57bl/10 mice. ivig was administered i.v. on day 1,3,7,10 and 14. human serum albumin (hsa) was used as a control. binding of ivig and activation status of foxp3 + cd4 + t cells were determined. in vivo, only when total cd4+ t cells, but not cd25-t cells, were adoptively transferred, ivig protected against t-cell mediated rejection of the fully mismatched skingraft (mst: ivig >100 vs hsa 16 days, p<0.01). ivig binds to 16±2% of foxp3 + t cells, while binding to foxp3 -t cells was minimal. this binding was partially fcγ receptor mediated. furthermore, ivig treatment resulted in activation of cd25 + t cells as detected by increased expression of phosphorylated zap70/syk, which could be abrogated by specific tyrosine kinase inhibition. in vitro, ivig inhibited the alloproliferative response of murine cd4 + t cells by 75±25%, but after depletion of cd25 + t cells, this inhibition decreased to 17±12% (n=6, p<0.01). similar results were found in human mlr, where depletion of cd25+ t cells resulted in twofold reduction in ivig mediated suppression. significantly, incubation of human cd4 + cd25 + with ivig enhanced their ability to suppress allogeneic t-cell proliferation (ivig 63±8% vs hsa 37±10% of inhibition, n=5, p<0.05) . our results identify a novel pathway through which ivig treatment induces direct functional activation of both mouse and human regulatory t cells. immediate binding and activation of regulatory t cells is one of the mechanisms in the immunomodulatory repertoire of ivig, which allows rapid inhibition of allogeneic responses, and therefore can be a valuable tool after organ transplantation. generation foxp3 is a dna-binding protein that is necessary for regulatory t cell (treg) function, though recent data indicate that detection of foxp3 mrna or protein alone does not necessarily indicate whether the associated foxp3+ cell is functional or not. to that end, our analysis of foxp3 sequence showed the presence of an rxxr motif, conserved between mice and humans, that is located 12 amino acids (aa) from the carboxy terminus of foxp3 and is a potential recognition sequence for cleavage by proprotein convertase enzymes. we found several proprotein convertases are expressed by naturally occurring tregs, with further upregulation upon tcr activation. we generated an antibody against the 12 aa carboxyl peptide of foxp3, and by western blotting detected a peptide of about 1.3-kda, consistent with a 12-aa long carboxy-terminal foxp3 cleavage product. both cleaved and uncleaved forms of foxp3 were resolved by sds-page, and the cleaved form was found only in the dna-bound fraction. the requirement of proteolytic cleavage, at the intact tetrabasic rxxr motif (414rkkr417), for full treg function was shown by abolishing the motif through mutagenesis, followed by retroviral expression of mutants, and analysis of proteolytic cleavage by western blotting. assays of treg function by cells expressing either long-or short-foxp3 mutants showed short-foxp3 (missing the carboxy-terminal 12-aa) suppressed teff cell proliferation significantly more effectively than wt foxp3 or an engineered and cleavage-resistant mutant, termed long-foxp3 (p<0.01). moreover, adoptive transfer of cells expressing short-foxp3 prevented experimental colitis more effectively than wt-foxp3 (p<0.01), and both were more effective than cells expressing long-foxp3. animals that received cells expressing short-foxp3 gained weight and were protected from disease. thus, function of foxp3 is regulated at a post-translational level by proteolytic cleavage and the short form of foxp3 represents the active form. our data shows proteolytic cleavage of foxp3 is key to the generation of functional tregs, with enzymes(s) of the proprotein convertase family likely playing an important role in this process and providing an extra and hitherto unrecognized important level of regulation for foxp3. blocking since tgf-β is secreted in a latent form and active tgf-β is rapidly cleared from circulation. in order to make long-lasting active form of tgf-β, the mutant tgf-β was fused to a human igg fc component. the secreted human mutant tgf-β/fc (hmtgf β /fc) fusion protein stained with both anti-human tgf-β and anti-human igg antibodies, confirming the cytokine and isotype specificity of tgf-β moiety and fc domain. moreover, in vitro bioassay, hmtgf-β/fc inhibited il-4 dependent ht-2 cell proliferation in a dose dependent manner and had a circulating half-life of 36 hours in mice. in vitro, anti-cd3 and anti-cd28 triggered cd4+ or cd8+ t cell proliferation, measured by 3h-thymidine uptake, could be synergistically inhibited by tgf-β and rapamycin. at the same time, the two reagents when added together could synergistically promote the induction of cd4 + foxp3 + t cells from peripheral naïve cd4 + foxp3 -t cells. in a pancreas islet transplantation model in which the fully mhc mismatched dba/2 islets were transplanted into c57 bl/6 mice rendered diabetic by streptozotocin, mean survival time of islet was 19 days in control recipients (n=6). administration of muttgf-β/fc resulted in a delayed islet allograft rejection (mst; 38 days, n=5). treatment with rapamycin prolonged islet grafts survival in 63% of recipients. in contrast, combined treatment with tgf-β/fc and rapamycin by four doses produced indefinite islet allograft survival in 83% cases. we have produced the long-lasting active hmtgf-β/fc fusion protein. the tgf-β/fc is synergistic with rapamycin to convert naïve cd4+foxp3-t cells into cd4+foxp3+ tregs and promote long-term islet allograft engraftment. the the recognition of microbial motifs by the innate immune system leading to the stimulation of adaptive immune responses may explain the relationship between infections and susceptibility to graft rejection. a number of infectious agents have been reported to prevent the induction of allograft tolerance, however, none of these can reverse established tolerance, a situation that is highly relevant to clinical transpalntation. we here report that established allograft tolerance (induced with anti-cd154 + donorspecific transfusion) can be reversed in a cardiac transplantation mouse model following infection with listeria monocytogenes. this reversal of tolerance was dependent on the presence of both cd4+ and cd8+ cells, as well as of the tlr/il-1/il-8 adaptor molecule, myd88. we hypothesized that the reversal of tolerance requires that alloreactive conventional t cells (tconv) escape dominant regulation by pre-existing tregs. these alloreactive tconv can then proliferate resulting in an increased ratio of tconv:tregs favoring the reversal of established tolerance. indeed, we observed that tolerant grafts harbored low numbers of infiltrating cd4 + and cd8 + t cells (1-1.4 x 10 4 /heart), with 10-43% of the infiltrating cd4 + cells co-expressing foxp3 (n=10). following the reversal of tolerance and allograft rejection, a 7-10 fold increase in cd4 + foxp3and cd8 + foxp3 -tconv was observed in the graft, but no increase in the foxp3 + subset. in contrast, we observed no significant changes in the splenic t cell subsets, raising the possibility that the escape from tregs occurs locally within the graft. infection of tolerant il-6-or ifnar1-deficient recipients with listeria monocytogenes failed to reverse tolerance, consistent with a hypothesis that the initial escape of tconv from regulation may depend on il-6, while their proliferation may be type i ifn-dependent. in summary, the conditions for the reversal of tolerance is more stringent than the prevention of tolerance, and requires myd88-signalling and the presence of both cd4 + cells and cd8 + cells, as well as il-6 and type i ifn signaling. these studies point to the potential impact of bacterial infections on established tolerance, and to novel strategies to monitor and facilitate the maintenance of tolerance in the clinic. th17 our laboratory has identified kα1 tubulin, as an epithelial autoantigen to which immune response occurs following human ltx. further, we have shown a strong correlation between the presence of antibodies (abs) to kα1 tubulin and development of chronic rejection ie bronchiolitis obliterans syndrome (bos). goal of our study is to test the hypothesis that epithelial damage due to allo immunity results in remodeling exposes otherwise cryptic self antigens including kα 1 tubulin and collagen type v (coll v) leading to cellular immune reactivity and ab production against these auto-antigens which may play a role in the pathogenesis of bos. 10 patients who developed anti-hla ab and 10 patients who had no detectable anti-donor hla abs post-ltx by luminex assay were analyzed for development of abs to kα1 tubulin and coll v using elisa method developed in our laboratory. the elisa for kα1 tubulin used recombinant protein (1µg/ml) purified on affinity resin. the elisa for collagen v uses commercially available human collagen v (1µg/ml). elispot assay for ifnγ and il-10 were performed for 5 bos-and 5 bos+ patient pbls (at the time of bos diagnosis), after 3 stimulations with kα1 tubulin protein. the levels of anti-tubulin abs and anti-coll v abs were increased significantly in bos+ ltx recipients with anti-hla compared to those with no anti-hla, table 1 (p<0.05). . surprisingly, both cd4 and cd8 subsets induced long-term survival of secondary test grafts (>100 days). however, only the cd4 + t-reg prevented tvs (ni=19+5, 21+6% of vessels), as compared to cd8 + t-reg (ni=40+9 in 50+15% of vessels). the cytokine profile indicated a dominant il-10 response. allo-antibody analysis showed up-regulation il-4/il-12 dependent igg1 and igg2c. conclusion: allochimeric protein-therapy and csa treatment generate t-reg that prolong graft survival, but only cd4 + t-reg generated from allochimeric protein-therapy prevent tvs. this cd4 + t-reg may be responsible for long-term graft maintenance through its unique ability to control anti-inflammatory and alloantibody responses. immunodominant h60 background: minor histocompatibility ags (mihas) are self peptides, derived from proteolytic processing of normal cellular proteins. miha incompatibility can induce t cell response to dominant mihas and facilitates expansion of ctls and graft rejection. however, the contribution of ctls recognizing these mihas in solid organ transplantation has not been fully evaluated. methods: balb.b (h-2 b ) donor hearts were transplanted heterotopically to c57bl/6 (h-2 b ) recipients. in vivo alloreactive cd8 t cells were monitored with peptide/mhc multimers. α-lfa-1 mab was given to recipients to prevent rejection. various numbers of h60-specific cd8 t cells or cd8 t cells lacking h60 specificity were adoptively transferred into balb.b graft bearing b6.scid recipients. results: 50% of transplantation recipients developed acute rejection and showed markedly increased numbers of h60-specific cd8 t cells at day 8-12 in spleen. abundant cd8 infiltration was found and selective infiltration of h60-specific cd8 t cells in the graft was confirmed with flow cytometry and in situ tetramer staining. α-lfa-1 mab treatment prevented acute rejection (mst>100) and allogeneic t cell expansion. it also profoundly attenuated neointimal hyperplasia ( graft-bearing b6.scid mice. we found that the degree of acute rejection positively correlated with the number of h60-specific cd8 t cells transferred. however, transferred h60-specific cd8 t cells did not cause chronic rejection. interestingly, greater numbers of cd8 t cells with h4 immunodominance were found in spleen, blood and graft at 50 days after adoptive transfer of cd8 lacking h60 specificity compared to h60-specific cd8 t cell transfer. conclusion: h60 responses dominate other miha immune responses during acute rejection after balb.b to b6 cardiac allograft. we confirmed a role of immunodominant h60 specific cd8 t cells as pathological effector t cells in acute rejection but not in chronic rejection. maintaining a h60 response may be beneficial in allotolerance to suppress miha responses that could induce chronic rejection in long-term grafts. chronic lung allograft rejection, bronchiolitis obliterans syndrome (bos), affects up to 60% of transplant survivors 5 years post-ltx. human neutrophil peptides (hnp 1-3/ α defensins), have been identified in the lavage fluids from patients with chronic rejection by proteomics. goals of our study were to determine the role of anti-hla abs and defensins in bos and to define the interactions between α-1antitrypsin (aat), and defensins in regulating inflammation leading to epithelial cell proliferation and bos pathogenesis. bal and serum samples (post-ltx and pre bos) from 21 bos+, 15 bos-patients and 12 normals were analyzed by elisa for α defensins (hnp1-3), human β defensin2 (hbd2) and aat. small airway epithelial cells (saec) were treated with hnp1 or 2, with or without equimolar aat or with anti-hla abs and analyzed for levels of hbd2 production (elisa), cytokine and chemokine (luminex), and cell surface adhesion molecules (icam, vcam) by facs. bal and serum from bos+ patients had high levels of hnp1-3 and hbd2 compared to bos-recipients or normal sera (p<0.05) ( table 1 ). there was also a significant decrease in aat levels in bos+ compared to bos-or normal serum (p=0.01) ( table 1) . saec produced human β defensins following anti-hla ab stimulation or by hnp treatment (table 2) . there was increase in adhesion molecules (icam, vcam, 2 folds) cytokines {il-6, il-1r α (2 folds), il-1 β, il-13(20 folds)}, il-10(2 folds decrease), chemokines (il-8, mcp1, 2-4 folds increase) and growth factors (egf and vegf, 2.5 folds increase) in response to treatment with hnp1 or hnp2 compared to untreated saec, that was inhibited by aat. increased defensins and decreased aat levels were seen in lavage and serum of ltx recipients with bos. anti-hla abs further stimulate defensin production by saec. we conclude that chronic stimulation of epithelial cells both by defensins and anti-hla abs can lead to increased growth factor production contributing to the pathogenesis of bos. under tubular atrophy/interstitial fibrosis (ta/if) remains a major cause of late kidney allograft loss and epithelial mesenchymal transformation (emt) is now appreciated as a key feature in this process. we hypothesize that macrophages play a direct role in the development of ta/if by creating a profibrotic microenvironment and stimulating emt within the allograft. in human kidney allografts with ta/if (n=128), macrophage infiltration detected by immunostaining was significantly upregulated (mean score 2.8±0.5) compared to biopsies without corresponding pathological changes from recipients with stable function (n=51; 2.0±0.1; p<0.0001) and the extent of this staining also correlated to the magnitude of graft dysfunction (p<0.0001). rt-pcr in ta/if grafts also showed marked upregulation for emt markers αsma (3.0±1.1-fold; p<0.05), s100a4 (8.7±2.7-fold; p<0.05), and vimentin (4.2±2.3-fold; p<0.01), compared to stable function allografts. to explore the macrophage-emt relationship, we co-cultured freshly isolated human monocytes over a primary culture of human proximal tubular epithelial cells (ptecs) using culture inserts allowing media exchange but forbidding contact between the two cell populations. by rt-pcr, co-cultured epithelial cells showed significant downregulation of bmp7 (0.45±0.09-fold; p<0.001), a negative regulator of emt, and epithelial marker e-cadherin (0.001±0.001-fold; p<0.01), with marked upregulation of emt genes s100a4 (2.43±0.40-fold; p<0.001) and αsma (5.31±2.23-fold; p<0.05) compared to ptecs cultured in media alone. investigating the mechanism of monocyte driven emt, we evaluated the effects of am80, a synthetic retinoid that also inhibits il-6 and vegf signaling. am80 markedly reversed the transcriptional profile with reduction of emt markers s100a4 (0.34±0.10-fold; p<0.01), αsma (0.29±0.06-fold; p<0.05) and vimentin (0.45±0.13-fold; p<0.01), and a simultaneous increase in expression of bmp7 (151.7±53.9-fold; p<0.01), thus reversing the pro-emt milieu. these results indicate that human monocytes induce transcription of emt related genes in ptecs, even in the absence of physical contact. moreover, this phenomenon appears to be mediated in part by il-6, vegf, and other pathways mediated by the retinoic acid receptor. thus, in addition to their typical immune functions, macrophages support a milieu within allografts that promotes ta/ if in humans. further investigation into this novel pro-ta/if pathway could ameliorate chronic injury and improve graft survival. old donor kidneys are more likely to develop long-term graft failure, especially if they are exposed to stresses (e.g. rejection). this limited ability of old tissue to withstand stress may be due to its reduced capacity of replication and regeneration. telomere shortening determines lifespan and regenerative capacity. we showed that telomere shortening occurs in old human kidneys. late-generation terc ko mice with critically short telomeres have a reduced lifespan, show an accelerated aging phenotype and are an ideal model to resemble the situation in old human donors. this study addressed the question whether iri causes greater damage in kidneys from late-generation terc ko mice. we studied terc wildtype (wt), early-(g1) and late-generation (g4) terc ko mice 1 day (wt:n=7; g1:n=8; g4:n=8), 3 (wt:n=8; g1:n=8; g4:n=5), 7 (wt:n=6; g1:n=8; g4:n=8) and 30 days (wt:n=8; g1:n=9; g4:n=6) after iri. acute tubular necrosis was found mainly on days 1 and 3 and was significantly higher in terc ko g4 kidneys. tubular atrophy (ta) and intersitial fibrosis (if), reflecting chronic damage, were first detected at day 7 and increased in all mice with a significantly higher extent of ta/if in terc ko g4 kidneys at day 30 ( fig.) . the cell cycle inhibitor p21, a downstream mediator of senescence induced by telomere shortening, was significantly upregulated in all groups after iri. p21 levels were highest in terc ko g4 kidneys at day 30 ( fig.) . proliferative capacity, as measured by ki-67 immunostainings at day 30, was significantly lower in tubular, glomerular and interstitial cells of kidneys from terc ko g4 mice. we show that late-generation terc ko mice with critically short telomeres have a greater susceptibility towards acute injury and develop more chronic renal damage. this is likely due to the reduced capacity of these kidney to proliferate and thereby to regenerate. our data strongly suggest a pathogenetic role of telomere shortening, an important senescence mechanism, for the development of long-term graft failure. results: belatacept treatment had no effect on the number of peripheral blood treg cells and t cell suppression assays. the percentage of foxp3 cells was significantly elevated in rejecting kidney allografts in belatacept-treated patients compared to cnitreated patients. conclusions: following chronic belatacept therapy, the number and function of peripheral blood treg cells are maintained in kidney transplant patients. belatacept enhances the treg population in the allograft in patients with acute rejection. therefore, our data suggest that co-stimulation blockade with belatacept does not affect treg homeostasis. the increased number of treg cells in rejecting allografts in belatacepttreated patients may provide a novel mechanism whereby belatacept can mitigate the severity of acute rejection and improve graft survival. the steady-state auc of n-desmethyl-aeb071 was minor in comparison to aeb071 and similar between the patient groups: 200 ± 90 ng.h/ml in transplantation vs 393 ± 220 ng.h/ml in psoriasis (p=0.08). demographic covariates: in the first week posttransplant with patients receiving fixed-dose aeb071, intersubject variability for c0 was 78% and for auc was 49%. aucs were similar in men vs women (8350 ± 4163 vs 10784 ± 5369, p=0.26). age, which ranged from 18-64 years, did not influence auc based on regression analysis (r 2 = 0.005, p=0.65). there was a borderline-significant negative correlation between weight (range, 51-110 kg) and auc (p = 0.07); however, its clinical relevance was low in that it could explain <9% of the variability in auc (r 2 = 0.086). there was a significant positive correlation between aeb071 c0 and auc (r 2 = 0.724, p<0.001). conclusions: (1) in the first week posttransplant, patients achieved aeb071 blood levels anticipated for this regimen. (2) there was notable intersubject pharmacokinetic variability at this time but it was not attributable to standard demographic factors such as sex, age, or weight. (3) a good correlation was noted between c0 and auc suggesting that c0 might serve as a marker for total drug exposure. a we studied which is protocol would be best after rapid discontinuation of p. between 9/01 and 4/06, 440 1st and 2nd kidney tx recips were randomized: csa-mmf (n=151) vs. high tac-low srl (n=149) vs. low tac-hi srl (n=140) (for tac and srl levels, high = 8-12, low = 3-7). all received thymoglobulin (tmg) 5 doses, and p for 5 days; tmg was continued in dgf. min f/u = 1 yr; mean = 42±19 mos. there was no diff between groups in recip age, gender, ethnicity, prim dis, donor source, % retx, pra; or in donor age, gender, ethnicity. there was no signif diff. between groups (intention to treat) in actuarial patient, graft, death-censored (dc) graft, acute rejection (ar), or biopsy-proven chronic rejection rates (cr), serum cr level or calculated (mdrd) gfr or in studied side effects. cr(sd) 1.6(.7);1.7(.9);1.8(1.3); 1.9(2);1.6(.7) 1.5(.4);1.7(1);2(2.5); 1.7(1.1);1.6(.6) 1.5(.5);1.5(.7); 1.6(.6);1.6(.6);1.7(.7) mdrd gfr(sd) 63(24),58 (21),60(26), 61(26),62 (20) 64 (20),65(24),61(27), 56(23),63 (28) 65 (20) the most common cause of graft loss was death with function (csa 40%, high tac 35%, low tac 57%). the majority of recips in each group remained p-free (csa 72%, high tac 86%, low tac 74%) but a large % were not on the medications which they were randomized to (csa 16%, high tac 51%, low tac 42%). we found a signif ↑ of new onset diabetes (nodm) (p=.03) in the tac-srl groups: csa 2%, high tac 11%, low tac 5%. there was a trend towards more use of lipd lowering medications in the tac/srl groups. in summary, all 3 is protocols were effective; with min f/u 1 yr for each group, patient and graft survival and ar rates are similar. we found an increased nodm and possibly hyperlipidemia in the tac/srl groups. purpose: we present preliminary 2-year results from a worldwide trial comparing 2 srl regimens with tac+mmf. methods: renal transplant recipients (n=451) were randomly assigned to the following treatment regimens: group 1: srl (8-15 ng/ml, then 12-20 ng/ml after week 13) + tac (6-15 ng/ml) with elimination at 13 weeks (n=155); group 2: srl (10-15 ng/ ml through week 26, 8-15 ng/ml thereafter) + mmf (up to 2 gm/day) (n=155); or group 3: tac (8-15 ng/ml through week 26, 5-15 ng/ml thereafter) + mmf (up to 2 gm/day) (n=141). all patients received corticosteroids and daclizumab. in june 2006, group 2 was terminated (after all patients were accrued) because of increased acute rejection (ar) rates. results: demographic characteristics were similar between groups except for more females in group 3. patient and graft survival were also similar among groups (see table) . biopsy-confirmed ar (bcar) was significantly greater in group 2 compared with groups 1 and 3, p<0.001, and most occurred in the first 6 months posttransplant with preponderance during the first 3 months. subtherapeutic srl trough concentrations were reported in a large number of rejectors in group 2. most of the rejections in group 1 occurred within the first 3 months before tac was eliminated. all ars were mild to moderate; grade ii ars were proportionally greater in group 3. mean nankivell gfr was numerically higher in group 2. preliminary results at 2 years show excellent patient and graft survival and similar renal function among treatment groups, despite higher ar rates in group 2. early adequate exposure to sirolimus is mandatory to achieve desired (low bcar rates) results. the goal of rapid discontinuation of prednisone (rdp) after kidney transplantation is to minimize prednisone-related side effects without increasing acute rejection (ar) rates or decreasing long-term graft survival. to date, studies have shown that rdp is associated with decreased prednisone (p)-related sided effects, and randomized trials of rdp (vs. long-term p) have shown little or no ↑ in ar rates. however, concern remains that long-term graft survival will be worse with rdp protocols. we studied t½ (the time it takes for ½ of the grafts surviving at 1 year to subsequently fail) for 1st tx recipients treated with rpd (1999-2007) (n = 652) (antibody, cni, antimetabolite [mmf or srl] , and rdp) vs. 1st tx historical controls (1995) (1996) (1997) (1998) (1999) (2000) (n = 388) treated with a protocol of antibody, cni, antimetabolite [mmf] , and long-term p. table 1 shows characteristiscs of the 2 groups. pra; rdp were more likely to get a living unrelated donor transplant. t ½ is shown separately for living (ld) and deceased (dd) donor transplants in table 2 . there was no significant difference in t½ between groups. we conclude that, compared to historical controls, rapid discontinuation of prednisone can be done without any detrimental impact on long-term outcome. a prospective, randomized study to confirm this observation is necessary. successful 1a) . we enrolled 152 patients (table 1) with an average follow-up of 19.7 + 11.7 months. subjects 19 -65 years old included primary and re-transplants, with primary endpoints of renal function and can. follow-up averaged 16.6 + 9.5 months in 47 patients withdrawn from ci (26 from group 3, 21 from group 4). we found no increased rejection after ci discontinuation and that both ratg induction dosing regimen and ci discontinuation significantly impacted renal function and the development of can. we successfully discontinued both calcineurin inhibitors and steroids with either single or divided-dose ratg induction, and single-dose ratg induction independently associated with improved renal function and reduced can. study demographics single-dose ratg (6 mg/kg x 1) divided-dose ratg (1.5 mg/kg x 4) group 1 (n = 38) group 3 (n = 38) group 2 (n = 37) group 4 (n = 39) age 45.7 ± 11.9 45. in subset analysis, we also find an enhanced negative impact of srl when combined with steroids (str) in patients without dgf. conclusion: this data supports previous findings that srl may be associated with a sustained survival disadvantage apparent early post transplant, and that this effect appears exacerbated when combined with dgf or str. several potential explanations for this effect may include srl-associated prolonged early graft dysfunction, hyperlipidemia or proteinuria. however, patients initiating srl after discharge may not evidence this survival disadvantage, and this question was not addressed here.these findings may be of particular importance to centers employing combined srl and str therapy, as well as to define the best mode of support during recovery from dgf. prospective randomized studies would be necessary to evaluate these. table 1 shows cai in both groups from 1 to 5 years. in slr group the doses of cin were significantly lower from one through 5 years (p=0.05). cai due to interstitial fibrosis/ tubular atrophy was significantly lower in slr group at 5 years. our data shows that 5 year patient and graft survival, graft function, bpar and scar were comparable between mmf and slr groups despite lower prevelance of interstitial fibrosis/tubular atrophy in slr group. registry analyses suggest that tac/mpa immunosuppression is associated with superior kidney graft survival vs. tac/srl. large single-center experience may assist in clarifying these findings, by examining outcomes related to specific utilization practice. we retrospectively examined the outcomes of 529 consecutive first renal transplants (55% deceased donor, 45% living donor) at a single center, treated with tac/srl or tac/mpa. graft and patient survival, acute rejection rates, and 1yr egfr were analyzed by era of transplant (2000-2002 vs. 2003-2006) . changes in tac/srl utilization between eras included elimination of the srl loading dose and a reduction in tac target trough concentrations. summary. compared to tac, srl+cya was associated with a 55% decreased risk of skin ca that was statistically significant. although srl+cya was associated with a 32% decreased risk of de novo solid ca, it did not reach statistical significance. this reduced risk may not reflect the unique aspects of srl itself, but may be a reflection of practice pattern, patient selection or center effect. the backgrounds: a number of studies have observed increase of malignancies following renal transplantation. however, the incidence and the site of malignancies were quite different by the follow-up times, the era and the region. methods: we reviewed the records of 694 renal transplant recipients in our institute between 1970 and 2007 and recorded the incidence and types of de novo malignancies. they were divided into two groups by immunosuppressive era; azathioprine (aza) era (1970.4-1982.3: n=172) and calcineurin inhibitor (cni) era (1982.4-: n=522) . results: a total of 57 (27 in aza era and 30 in cni era) kidney recipients out of 694 developed 60 malignancies. the tumors included 10 gi-tract cancers, 9 liver cancers, 12 skin cancers, 4 tongue cancers, 6 breast cancers, 6 renal cell carcinomas, 2 thyroid cancers, 5 leukemia, 3 lymphoma, one lung cancer, one uterus cancer and one kaposi's sarcoma (ks). the average interval between transplantation and development of malignancy was 134±86 (8-340) months. mortality was high in liver cancer (89%) and leukemia (100%). cumulative incidence of malignancies of all 694 recipients in 5, 10, 20, 30 years were 2.3%, 4.5%, 10.7% and 14.3%, respectively. graft-loss censored cumulative incidence, which was calculated to see the incidence among graft survivors under continuing immunosuppression, of all recipients in 5, 10, 20, 30 years were 2.8%,6.3%, 16.3% and 26.2%. that of 5 ,10 and 20 years in cni era was 3.0%, 6.8% and 13.9%, while that in aza era was 1.8%, 4.9% and 19.5%, showing early higher incidence in cni era outstripped by aza era by 12 years. site of malignancy in cni era occurring within 3 years, which was never observed in aza era, was focused on liver, leukemia (including atl), ks and ptld. discussions:our results demonstrated that recent potent immunosuppressive regimen shortened the interval between transplantation and viral-related malignancies. however, long-term incidence of whole malignancies has been decreasing by minimizing chronic immunosuppression in our institute. the impact of transplant center practice on the association between pulsatile perfusion and delayed graft function. jagbir gill, 1 david gjertson, 1 suphamai bunnapradist, 1 michael cecka. 1 1 ucla, la, ca. the use of pulsatile perfusion (pp) is increasing in the us, but practice varies widely among transplant (tx) centers. we describe the variability of pp use and its impact on post transplant outcomes. methods: we identified all cadaveric kidney tx from 2000-2005 using optn/unos data. the cohort was stratified by tx center pp use as follows: low pp centers (0-10% pp use), med pp centers (10-30% pp use), and high pp centers (>30% pp use). donor characteristics and the incidence of dgf were compared between and within each strata. results: pp was used by 97 % of centers, however most centers used pp <10% of the time (70.7%). compared to low and med pp use centers, kidneys pumped in high pp centers were from donors that were younger, had a lower mean terminal serum creatinine, and had a lower incidence of cva and hypertension. the overall incidence of dgf was lowest in the high pp centers (13.3%), compared to the med (24.7%) and low pp (24.8%) centers. the rates of dgf within each strata (high, med, and low pp centers) for tx performed using pp versus cold storage (cs) are outlined in the table below . within each strata, the rate of dgf did not differ between tx performed with and without pp. in ecd transplants, pp was associated with lower rates of dgf across all center groups. however, the impact of pp on scd and dcd transplants was less significant and varied across centers by pp use. hla sensitized patients (20-100%) in our dsa are now transplanted at a rate of 40%, which is significantly higher (p = 0.02) than the 23% rate when ua weren't entered into unet. furthermore, of 9 kidneys imported into our dsa and allocated to the dsawide renal candidate list (since ua entry started), 63% (5/9) were transplanted into sensitized candidates (85% to 27%), each of whom had a negative flow or ahg t cell igg crossmatch. conclusion. the higher transplantation rate for hla sensitized patients in our dsa shows that virtual a, b, & c crossmatching yields a dsa-wide ranked list of sensitized candidates likely to have a negative final class i (t cell) crossmatch and be transplanted. the data also lend support to the notion that sharing kidneys across dsa boundaries for hla sensitized candidates, based on a negative virtual hla class i crossmatch, has merit. predicting introduction: predicting graft outcome after renal transplantation based on donor histological features has remained elusive and is subject to institutional variability. we propose a pre-transplant donor path scoring system that reliably predicts graft outcome regardless of recipient ® characteristics. methods: we retrospective analyzed 286 imported cadaveric renal transplants which were initially rejected by other centers due to donor parameters between 1/00-6/05. all kidneys were re-biopsied at our center prior to implantation. morphometric analysis performed consisted of measuring glomerular-size arterioles, interlobular, and arcuate/ interlobar arteries and wall to lumen ratio (wlr) was calculated; calculating % glomerulosclerosis (gs); the presence of arteriolar hyalinosis (ah), scar, periglomerular fibrosis (pfg), and acute tubular necrosis in the biopsies. the patients were followed for a mean of 33 months. multivariate cox analysis was done to evaluate the predictive value of these path variables to graft outcome.results: ah, gs>15%, wlr>0.5, pgf, and scar were found to independently predict graft outcome. the unos board of directors has approved the change from using panel reactive antibodies (pra) to calculated pra (cpra). the cpra is a formulated pra based upon the frequency of the specificities of hla antigens found in the donor pool and is expected to standardize the degree of patient sensitization. donor organs expressing unacceptable antigens will not be offered to a recipient with donor (hla) antigen specific antibodies (dsa). highly sensitive, single antigen bead and solid phase assays (flow pra and luminex) are used to identify these hla antibodies (abs). each transplant center can determine the criteria used for identifying an unacceptable antigen, for example, based upon dsa titer or the fluorescence intensity (fi) coming from the donor-specific single antigen bead. it is unclear whether abs identified by these techniques are clinically relevant for organ allocation. we retrospectively evaluated flow-pra, flow cytometry crossmatching (fcxm), hla ab specificities and titers of 300 pre-transplant (tx) sera from transplant recipients of deceased renal allograft donors transplanted following a negative cytotoxic-anti-human globulin crossmatch. the two year graft survival of 91% for the recipients (44/54, 81%) with low-titer (≤ 1:16) donor specific hla ab and a negative (-) fcxm was significantly better when compared to the 60% two year graft survival of the 19% (10/54) of recipients presenting with (+) dsa but a (+) fcxm (p< 0.001). recipients (55/110, 50%) with non-donor abstracts specific hla abs (high or low titer) and a (-) fcxm also experienced a better two year graft survival of 89% compared to the 74% for the other 50% of recipients with (+) fcxms (p < 0.001). these data suggest that in the presence of donor-specific or non-donor-specific hla abs you can not predict the crossmatch outcome without actually performing the crossmatch which will then influence donor organ allocation and graft survival outcome. in the face of low-titer dsa and a (-) fcxm recipients experienced excellent graft outcome when compared to recipients with (+) fcxms. therefore, donor organ allocation based on cpra (ab specificity and unacceptable ags) utilizing highly sensitive single antigen bead and solid phase luminex assays may disadvantage recipients (no donor crossmatch) who could otherwise be successfully transplanted. aim: to assess vegfr2 expression in hcc and the adjacent benign cirrhotic parenchyma and its correlation with tumor differentiation, vascular invasion, and tumor morphologic parameters. background: hcc is a highly vascular tumor in which angiogenesis is mediated in part by vegf. vegf is highly expressed in hcc and mediates its effects through multiple receptors including vegfr2. the tyrosine kinase inhibitor sorafenib inactivates the vegfr2 receptor and exhibits anti-tumor effects in hcc. clinical significance of vegfr2 expression with respect to tumor parameters has not been evaluated. patients and methods: immunohistochemical staining for vegfr2 was performed in hcc and corresponding adjacent cirrhotic liver from 79 patients undergoing liver transplant. 57 patients had hcc within mc. stains were scored by estimating the % of positive surface area in veins, arteries, and sinusoidal lining cells. data are presented as median [p25, p75]; wilcoxon signed rank and wilcoxon rank sum tests were used. results: vegfr2 levels in hcc were significantly correlated to levels in adjacent non-tumorous liver. higher levels of vegfr2 in non-tumorous liver were associated with higher levels in hcc from the same patient. vegfr2 levels were significantly higher in hcc compared to adjacent areas (p<0.05). vegfr2 levels in hcc were not significantly different between patients who fell within mc and those beyond mc. however, vegfr2 levels were significantly higher in the adjacent arteries of nontumorous liver (15.8 [0, 60] vs. 0 [0, 35]; p=0.03) in those patients with hcc beyond mc. subjects with moderate or poor differentiation had significantly higher levels of vegfr2 in sinusoids and veins of hcc and in the sinusoids of adjacent non-tumorous liver. there was no correlation with vascular invasion. conclusions: elevated vegfr2 in hcc correlates with elevated vegfr2 in adjacent cirrhosis, suggesting that high expressing hcc arise in a high vegfr2 expression, pro-angiogenic environment. moreover, higher vegfr2 expression in background cirrhosis correlates with advanced hcc (beyond mc), suggesting that anti-angiogenic agents may prevent tumor formation or progression in cirrhotic patients. this novel concept warrants further study. . we further attempted to find a subgroup of patients combining tumor size, number of nodules and various levels of afp which together would correlate strongly with pdiff tumors. however, the distribution was erratic and even in extreme outliers (>4 nodules, > 8 cm in maximum size, with or without high afp), where transplantation is currently not indicated according to any current expanded tumor inclusion criteria, the incidence of pdiff did not exceed 42%. conclusions: pdiff is most highly associated with tumors that exceed the milan criteria and the expanded criteria currently used for liver transplantation. thus, tumor biopsy would not appear to be of benefit except perhaps in those patients with a high afp level. however, if tumor criteria are further extended in the future, tumor biopsy may be justified in those with higher tumor burdens. < 2 cm) ), 2,511 (85.7%) at ls=t2 (1 tumor < 5cm or 3 tumors ≤ 2 cm), and 111 (3.8%) with ls>2. results: overall survival at 36 months was 76.5%, with significant differences seen for listing stage (ls 1= 82.2%, ls 2=75.9% ls>2=73.5%, p=0.0395) and by histologic stage as determined by pathology (p<.0001). survival for patients with histologic stage 4b hcc was only 29% at 36 months, versus 83% for stage 1. patients with tumors greater than 3cm both by listing stage and by pathology fared poorly compared to those with smaller tumors (p=0. 0.002). patients listed who received ablation treatment (at) preoperatively and who had their tumors "down-staged" had better results (p= 0.0061). we observed no difference in at types (tace vs rfa). those with micro or macrovascular invasion had lower survival rates, at 66.8% and 30.5%, respectively (p <.0001). afp >500 continues to be a significant predictor of lower post-transplant survival, with a survival rate of 57.9% at 36-months (p<.0001). conclusions: we conclude that listing and histologic tumor size, presence of micro/ macrovascular invasion, and high afp are associated with poorer lt results. at is emerging as potentially effective treatment for improving lt outcomes for hcc recipients. introduction: so far, milan criteria are used to select patients with hepatocellular carcinoma (hcc) for liver transplantation (lt). herein we compare prognostic markers in patients with pretreatment by transarterial chemoembolization (tace) to a second cohort transplanted without tace pretreatment. patients and methods: between september 1997 and october 2007, 134 patients with hcc underwent lt at our institution. eighty-two patients were pretreated by repeatedly performed tace whereas in 52 patients none or other forms of pretreatment had been used (non-tace group). tace was performed using lipiodol and mitomycin. every 6 weeks tace was repeated until transplantation. tumor response was assessed by ct scans (6-week intervals). results: sixty-seven percent of the patients transplanted after tace pretreatment exceeded the milan criteria compared to 37 % in non-tace patients. the proportion of recurrence-free patients was comparable in the tace and non-tace group (77.32 % and 78.45 %, respectively). in the univariate analysis grading, angioinvasion and progress-free tace were significant predictors for recurrence after lt in patients with tace pretreatment. progress-free tace was the only significant predictor of recurrence in the multivariate analysis. in the non-tace group t classification, number of nodules, grading, angioinvasion, milan criteria and underlying disease (hcv versus all other diseases) were significant. after tace pretreatment freedom from recurrence was 92.3 % in patients with stable disease or regress but only 38.4 % in patients with progress during tace. conclusions: tace pretreatment is capable of selecting a biological entity of tumors which differs significantly from untreated tumors. this statement is deduced from the remarkable differences of predictors for tumor recurrence in patients with and without tace pretreatment. moreover, tace patients can be separated into two groups: those who experienced tumor progress during repeatedly performed tace and those who did not. stable disease during the continued tace before transplantation resulted in remarkably low tumor recurrence. liver with a median follow-up of 30 months, there was no statistical difference in the 5 year overall survival in the m (76%) and m+ (69%) groups (p=0.40). the 5-year disease free survival was significantly higher in the m (80%) vs. m+ (61%) groups (p=0.02). when stratifying for ucsf criteria tumors, the 5-year disease free survival was milan (80%) vs. ucsf (72%) vs. beyond ucsf (54%) groups (p=0.01). univariate analysis demonstrated the following factors to be associated with disease recurrence: intermediate waiting time 2-4 months (p=0.01), preoperative tace (p=0.002) or resection (p=0.04), more than 3 tumors (p=0.03), and max tumor size over 5cm (p=0.03). multivariate analysis controlling for age, gender, and waiting time demonstrated that preoperative resection hr2.78 (95%ci 1.1-7.2), more than 3 tumors hr2.3 (95%ci 1.1-4.7) and max tumor size greater than 5cm hr2.68 (95%ci 1.2-5.9) were independently associated with disease recurrence. conclusions: the overall survival after liver transplantation is excellent in the m and m+ groups, far exceeding survival rates that can be obtained via any other modality. the current unos hcc algorithm should be reconsidered, to allow extra listing points for selected patients with hcc that exceed both the milan and ucsf criteria. postoperative use of intense insulin therapy in liver transplant recipients. lama m. hsaiky, 1 iman e. bajjoka, 1 dhaval patel, 1 marwan s. abouljoud. 1 1 transplant institute, henry ford hospital, detroit, mi. hyperglycemia and insulin resistance are common post liver transplant, even in patients with no history of diabetes. in the general surgical intensive care unit (sicu) population, the use of intensive control of blood glucose has recently been shown to reduce both morbidity and mortality. thus far, limited data exist in the liver transplant population. purpose: assess the impact of intensive insulin therapy to maintain blood glucose at or below 110 mg/dl immediately post liver transplantation in the surgical intensive care unit (sicu). methods: a retrospective evaluation of liver transplant recipients who received two different insulin protocols in the sicu was performed. prior to january 2003, patients were assigned to receive sliding scale insulin therapy (ssit) to maintain blood glucose (bg) less than 180 mg/dl. the intensive insulin therapy (iit) was implemented in august 2005 with a goal bg level between 80-110mg/dl. the following data was analyzed: bg ranges, need for mechanical ventilation, blood transfusions, infection rate in the sicu, rejection episodes and patient survival. results: a total of 97 liver transplant patients were evaluated; of which 47 patients were in the ssit group and the other 50 in the iit group. demographic characteristics were comparable between the two groups. in the iit, 24% of the bg readings were maintained at < 110 mg/dl versus 5% in the ssit. the incidence of hypoglycemia (bg <60mg/dl) was less than 1% in both groups. the need for mechanical ventilation was 3.0 days vs. 2.3 days and the overall number of blood transfusion was on an average of 5.0 vs. 2 units (p<0.05) in the ssit vs. iit group, respectively. iit also reduced overall sicu infection rate by 15% (p=0.01). the rate of acute cellular rejection at 3 months post transplant was less in the iit, 14% vs. 25% in the ssit group (p=0.02). moreover, mortality during hospital stay was reduced from 5% in the ssit to 2% in the iit group. conclusions: the use of intense insulin therapy immediately post liver transplantation has resulted in reducing infection rate and rejection episodes and a trend for reduced morbidity and mortality among post surgical liver transplant recipients without the adverse effects of hypoglycemia. medical epidemiology of patients surviving ten years after liver transplantation. kerri a. simo, 1 stephanie e. sereika, 1 david a. gerber. 1 1 abdominal transplantation division, department of general surgery, university of north carolina, chapel hill, nc. background: as the population of long term survivors of liver transplantation (olt) grows, their medical epidemiology has become increasingly important. the goals of this study were to define a collective profile of liver transplant recipients ≥ 10 years post olt and to compare their co-morbidities with those of the general population. in 2005, the national health survey reported that 22% of the us population had hypertension, 14.4% had diabetes/impaired fasting glucose, and 16.8% had chronic kidney disease. methods: a retrospective review of a prospectively collected database of 125 adult patients who underwent olt at a single transplant center from september 30, 1991 to october 1, 1997 was performed. inclusion criteria consisted of survival ≥10 years post olt with >1 year follow up. results: seventy-one patients met inclusion criteria. ninety percent of patients had ≥10 years follow up. the mean age at transplant was 43 (range 18-67). the mean calculated meld score was 22 (range 9-62, median=22). indications for olt were hcv(32%), alcohol(28%), cryptogenic(18%), autoimmune(10%), hbv(7%), psc(7%), pbc(7%), and other(10%). seven patients required retransplant during the first 10 years. an additional 38 patients underwent other operations: 10 arterial or biliary revisions, 13 hernia repairs and 19 non-transplant related (7 abdominal). during analysis, the following medical co-morbidities were found: 51 patients(72%) had hypertension (41 new onset), 20(28%) diabetes (10 new onset), 33(46%) renal insufficiency and 8 renal failure (28 new onset), 10(14%) cardiovascular disease (all new onset). nine patients (13%) were diagnosed with de novo cancer. medications for chronic health problems included 18 patients on diabetic medications, 46 on antihypertensives and 18 on lipid lowering agents. initial immunosuppression consisted of 100% on steroids, 61% on cyclosporine, 40% on tacrolimus, 24% on mycophenolate mofetil (mmf) and 23% on azathioprine. immunosuppression at 10 years consisted of 42% on cyclosporine, 41% on tacrolimus, 18% on steroids, 13% on mmf, 8% on sirolimus, 1% on mycophenolate sodium, 1% on azathioprine. no patients were on triple therapy, 29 were on dual therapy, and 42 were on monotherapy. summary: patients alive 10 years post olt have a significantly higher incidence of hypertension, diabetes, and renal disease than the general population. this study supports conscientious medical follow up to ensure continued meaningful survival. liver transplantation in the morbidly obese (bmi>40). c. quintini, 1 l. kauzman, 1 k. hashimoto, 1 p. ding, 1 t. doago uso', 1 n. sopko, 1 j. rosenblum, 1 f. aucejo, 1 c. winans, 1 d. kelly, 1 b. eghtesad, 1 d. vogt, 1 j. j. fung, 1 c. miller. 1 1 general surgery -liver transplant service, cleveland clinic oh. morbid obesity (mo) is a problem seen with increasing frequency among candidates for olt. mo is considered a contraindication for olt in some centers without clear evidence to support such a practice. our aim is to describe outcomes for olt in patients with a bmi>40kg/m 2 in a single center. methods. between 1/2004 and 4/2007, 364 olts were performed in 347 patients. 20/347 patients with a bmi>40 were compared to all other olt patients with a bmi< 40. we analyzed patient and graft survival, operative time, blood transfusion requirements, and post operative events (icu and overall length of stay los, surgical complications and infections). we also analyzed the post transplant weight records of our study group at 1-3 and 6 months. results. results are summarized in table 1. outcomes of olt in the morbidly obese are no worse than those of other patients undergoing olt. bmi is often artificially elevated in end-stage cirrhotic due to severe fluid retention. these patients exhibit rapid weight loss following transplantation that is likely due to extra-cellular fluid loss from the improved homeostatic milieu provided by the new liver and possible improvement in renal function. the presence of obstructive cad was not associated with increased peri-operative morbidity or mortality. these patients experienced similar patient and graft survival irrespective of the degree of cad. significant unmodified cad should not represent an absolute contraindication to liver transplantation. olt was also examined. we found that using a trj of 3.0 m/s as a cut-off created two age-matched groups with significantly different survival curves at one year (p = 0.006) with a relative risk of mortality of 3.98 for trj ≥3.0 m/s. this study underscores the importance of screening tte for the presence of pph in the evaluation of patients for olt, suggesting that clinically significant pph may be present at lower trj than typically prompt right heart catheterization. some of the limitations of the study are the variability of the time from pre-olt echo to transplant, and the assumption of rap = 5 without assessment of ivc size. we are using these data as a framework for further prospective trials to better assess the clinical applications of the findings. hyperlipidemia has been shown to predict faster chronic kidney disease (ckd) progression over the long term in lung allograft recipients. it is unknown whether disordered lipid metabolism may also aggravate the early loss of renal function often seen in this patient population in the immediate post-operative period. we studied 230 lung allograft recipients transplanted between january 1997 and december 2003. pertinent demographic and clinical variables were recorded at baseline and one month post-transplant, including creatinine levels and fasting lipid panels. logistic regression models were created to investigate an independent association between lipid levels and change in renal function by one month post-transplant. mean +/-sd baseline creatinine was 0.8 +/-0.2 mg/dl and low density lipoprotein (ldl) was 110 +/-35 mg/dl, the latter remaining unchanged at one month. in contrast, by one month post-transplant the mean creatinine level of survivors increased significantly to 1.2 +/-0.6 mg/dl (p < 0.001), with a overall mean increase of 52% above baseline. the highest quartile of patients that fared the worst experienced a rise in creatinine > 72% above baseline. on univariate analysis, there was a strong trend toward those with one month ldl values in the highest quartile (i.e., >140 mg/dl) having an increased odds of experiencing a rise in creatinine by one month > 72% above baseline (or 1.8, p=0.09). after controlling for age, gender, pre-transplant creatinine, bmi, and the presence of diabetes prior to transplant an ldl value in the highest quartile by one month post-transplant was the only variable independently predictive of a rise in creatinine > 72% above baseline at one month (or 2.5, p=0.05). in summary, hyperlipidemia occurring early post-lung transplant predicts faster loss of renal function soon after surgery. though speculative, given the known beneficial effects of lipid lowering agents such as statins on the rate of ckd progression, perhaps timely initiation of these medications after surgery may also attenuate the early decline in renal function often observed in this patient population. the risk for acute cellular rejection (acr) following lung transplantation (ltx) is still a problem despite heavy multidrug immunosuppression. induction therapy with potent t-cell depleting agents have facilitated the implementation of minimal post-transplant immunosuppression.the impact of this protocol on the activation of proinflammatory cytokines and effector molecules that affect the cellular rejection process is not well determined. in this study, we evaluated the relationship between upregulation of t-cell and macrophage-dependent inflammatory cytokines detected by molecular methods and the clinical status of ltx patients. we studied 28 ltx patients who received anti-lymphocytic induction therapy(thymoglobulin n=8 or campath-1h n=20) followed by maintenance immunosuppression with tacrolimus and low-dose steroids. we analyzed 137 bronchoalveolar lavage (bal) mrna samples (3-8 per ltx patient) by real-time pcr for gzmb, ifn-γ, il-15, mcp-1, rantes, tnf-α, and gapdh (control). the abi prism 7700 sds and 7500 fast real-time pcr systems were used and data were analyzed by the dct and 2-ddct methods. we determined the relationship of categorical outcomes (rejection-no rejection) with continuous variables (number of cycles) by anova. early acr that occurred within the first 60 days post-ltx was associated with an increase (>3-6 fold) of macrophage-specific mrna (mcp-1, rantes, tnf-α). 7/8 thymo-treated ltx patients experienced early acr while only 5/20 campath-1h-treated patients had early acr (p<0.005). in contrast, late acr that occurred greater than 60 days post-ltx was associated with a significant upregulation (4-64 fold) of t-cell dependent mrna (gzmb and ifn-γ) in addition to increases of rantes and mcp-1 mrna. overall, ltx patients who experienced early or late acr (n=20) exhibited higher mrna levels for the above mediators compared to stable patients (n=8). our data indicated that in early post-t-cell depletion, the acr phenotype was characterized mainly by macrophage activation. in contrast, greater than 3 months post-ltx with the recovery of cd8+ t-cells, the acr phenotype was associated with cytotoxic t-cell activation. furthermore, sensitive molecular methods may detect the activation of pro-inflammatory mediators within the allograft prior to the diagnosis of rejection by transbronchial biopsies and may impact optimal patient management. antibody-mediated rejection (amr) is defined by the presence of donor-specific alloantibodies, markers of complement activation and the clinical phenotype of organ dysfunction. compared to renal and cardiac allografts, very few amr cases are documented in lung transplantation (ltx). methods. we report here on seventeen ltx patients exhibiting: 1) donor-specific hla antibodies (dsa); 2) linear, continuous subendothelial c4d deposition in lung allograft; 3) lung allograft dysfunction. the presence and specificity of dsa were determined by elisa and/or luminex. c4d deposition was assessed by immunohistochemistry in transbronchial biopsy paraffin blocks. allograft dysfunction was considered when either biopsy-proven acute cellular rejection (acr, ≥ ishlt a2) or bronchiolitis obliterans (bos) were diagnosed. the average detection of dsa occurred in the first year post-ltx, on 283±218 postoperative day (pod), range 14 to 734 days. thirteen (76%) of amr patients were females. an anamnestic humoral response was encountered in 4 cases (one pregnancyrelated and three re-transplant patients), while de novo dsa were detected in 14 cases (82%). the percent-reactive antibody (pra) was lower in de novo cases (24%) when compared to memory response (71%, p<0.05). anti-class i dsa were found in 7 cases, anti-class ii in 5 cases, while 5 patients exhibited both class i and class ii dsa. specific vascular c4d deposition was detected in 16 (94%) patients. lung allograft dysfunction was considered in 14 (82%) cases, while three patients with dsa and specific c4d deposition fulfilled the criteria of sub-clinical amr. in patients where plasma-exchange/ ivig were applied, antibody titers dropped from 1:32 to 1:4 or 1:2 in two cases; in a third case, antibody titer remained high post-pheresis (1:512) and the graft failed. conclusions: both anti-class i and anti-class ii, low pra or high pra, pre-formed or de novo dsa can be detrimental for lung allograft. the presence of donor-specific alloantibodies, vascular c4d deposition and allograft dysfunction shows that amr criteria can also be met in ltx. rationale: recently, inhaled cyclosporine has been shown to reduce mortality and bos. previously, we demonstrated that apically-dosed cyclosporine poorly transmigrated differentiated human airway epithelial cells in vitro (using a transwell system). only ∼5% of the apically deposited cyclosporine passed through the epithelial layer whereas most of the drug accumulated within the epithelium. thus inhaled cyclosporine may not be capable of inactivating airway allogeneic t cells since it may not reach the airway wall in high concentration. in this study, we hypothesized that inhaled cyclosporine alters airway epithelial signaling cascades that may ultimately result in reduced inflammatory cell recruitment to the lung. methods: human tracheobronchial epithelial (htbe) cells from healthy donors were grown in ali media to confluence at an air-liquid interface in millicells. differentiated htbes were treated on their apical surface with cyclosporine concentrations of 1000 and 10,000 ng/ml or vehicle for 24hr to mimic the effects of inhaled cycolsporine. the basilar and apical compartments (n=8-12 for each) were then assayed for cytokine secretion (il8, il6, il1, il-12p40, tnf, eotaxin, mcp-1, gm-csf, rantes and egf) by luminex assays in pg/ml. results: 10,000ng /ml of cyclosporine markedly blunted the basilar secretion of il-6 (250 ±142 vs 71± 24, p= 0.002), rantes (22±8 vs 9±3, p=0.003), gm-csf (21±16 vs 3±3, p=0.008) and mcp-1 (181±112 vs 28±19, p=0.002). il8 and eotaxin were unaffected; tnfα, il1β and il12p40 were undetectable. at 1000ng/ml cyclosporine, cytokine secretion was decreased to a lesser extent. a similar pattern of diminished cytokine secretion was seen in the apical compartment of this system. last, apical, but not basal, egf secretion was augmented by cyclosporine (2231±817 vs 1114±614 pg/ml, p=0.02). conclusion: cyclosporine decreased the secretion of critical cytokines and chemokines from human airway epithelial cells. these mediators are known to enhance mononuclear and t cell recruitment in a large variety of animal models of disease as well as in clinical studies. inhaled cyclosporine may work by reducing cell recruitment. this hypothesis will require in vivo testing. funded by: cf foundation. background: cytomegalovirus (cmv), human herpes virus -6 and -7 (hhv-6 and -7) are β-herpesviruses that commonly reactivate and have been proposed to trigger acute rejection and chronic allograft injury. the role of these viruses in the development of bos after lung transplantation remains unclear. we assessed the contribution of β-herpesvirus infection in the allograft by a prospective molecular assessment of serial broncho-alveolar lavage (bal) samples in lung transplant recipients. methods: quantitative real-time pcr of bal samples were performed for cmv, hhv-6 and hhv-7 in a prospective cohort of lung transplant recipients. a time-dependent cox regression analysis was used to correlate the risk of bos and acute rejection in patients with and without β-herpesviruses infection. results: 93 patients were included in the study over a period of 3 years. a total of 581 samples from bal were obtained (median 6 per patient). 61/93 patients (66%) had at least one positive result for one of the β-herpesviruses: 48 patients (52%) for cmv, 19 patients (20%) for hhv-6, and 19 patients (20%) for hhv-7. median time to detection was 152 days (range 8-839) for cmv, 309 days (range 28-928) for hhv-6, and 286 days (range 17-928) for hhv-7. median peak viral load was 3,419 copies/ml (range 102-41,600,000) for cmv, 258 copies/ml (range 106-131,075) for hhv-6, and 665 copies/ml (range 103-66,100) for hhv-7. acute rejection (≥ grade 2) occurred in 46% and bos (≥ stage 2) in 19%. in the time dependent cox regression model, the relative risk of bos or acute rejection was not increased in patients with cmv, hhv-6, or hhv-7 reactivation. for example, the hazard ratio of cmv and bos was 1.04 (95% ci 0.62-1.73, p=0.9) and for cmv and acute rejection was 0.81 (95% ci 0.55-1.20, p=0.3). in many of the patients, β-herpesvirus reactivation occurred after the acute rejection episode likely reflecting augmented immunosuppression. abstract# 317 mannose binding lectin in this large cohort of lung transplant recipients, local reactivation of cmv, hhv-6 and hhv-7 in the allograft was very common. however, despite high viral loads in many patients, infection was not significantly associated with the development of acute rejection or bos. introduction: the role of chemokine receptors in regulating donor-specific responses to allografts is poorly understood. cd4 + cd25 + t cells regulate alloreactive cd4 t cell responses and acute rejection of single class ii mhc-disparate cardiac allografts in c57bl/6 mice. ccr5 is expressed by a small proportion of cd4 + cd25 + t cells but the requirement for these cells in regulating alloreactive t cell responses remains poorly understood. the goal of this study was to investigate the role of ccr5 + t regulatory cells in acute rejection of single class ii mhc-disparate cardiac allografts. methods: wild-type c57bl/6 (h-2 b ) and b6.ccr5 -/received heterotopically transplanted b6.h-2 bm12 mice heart grafts. the presence of cd4 + foxp3 + t cells in the recipient spleen and in heart allografts was determined by flow cytometry. foxp3 mrna expression in the heart grafts was analyzed by qrt-pcr. donor-specific cd4 + t cells producing ifn-g or il-4 in allograft recipient spleens were enumerated by elispot assay. cell sorted naïve wild-type cd4 + cd25 + t cells and cd4 + cd25 -t cells were adoptively transferred to wild-type and ccr5 -/mice before the cardiac transplant. results: in wild-type recipients >80% b6.h-2 bm12 cardiac grafts survived more than 100 days whereas ccr5 -/recipients rejected the allografts within 24 days (18 days mean survival) with intense cd4 + t cell infiltration in the graft. donor-reactive ifn-g and il-4 producing cd4 + t cell numbers were increased 2-fold in the spleens of ccr5 -/vs. wild-type recipients at day 7 post-transplant and in contrast to wild-type recipients these numbers were sustained for at least 7 more days. allograft infiltrating cd4 + cd25 + foxp3 + cells and intra-graft foxp3 mrna expression were clearly present in allografts from wild-type recipients and were virtually absent in allografts from ccr5 -/recipients. transfer of purified wild-type cd4 + cd25 + t cells to ccr5-deficient mice resulted in the long-term survival of 60% of b6.h-2 bm12 cardiac allografts. conclusion: ccr5 + regulatory t cells control the magnitude and function of the alloreactive t cell immune response to single class ii mhc-disparate cardiac allografts. profile that were distinct from those of cd4+cd25+ tregs, naïve cd4+cd25-t-cells, and activated cd4+ t-cells. furthermore, the cd4+ converted dn t-cells were highly potent in suppressing antigen specific alloimmune responses in vitro. in this study, we further characterized and test the functional potential of the converted dn t-cells in vivo. we showed that the converted dn t-cells retained a stable phenotype after re-stimulation in vitro and in vivo. il-2 was capable of breaking the anergic status and reserving the suppressive function of dn t-cells. in an immunocompetent mhc completely mismatched islet transplant model, the transfer of 13 x 10 6 dn t-cells (converted from cd4+cd25-t-cells of naïve c57bl/6 mice by co-culture with mature dba/2 dc plus ril-15 in mlr for 6 days) resulted in a statistically significant prolongation of alloantigen specific dba/2 strain, but not third party c3h strain, islet allograft survival in c57bl/6 recipients in comparison with that of untreated control group. as il-2 was capable of breaking the anergic status and reserving the suppressive function of dn t-cells, we added il-2/fc, a long-lasting form of il-2, and low dose rapamycin with dn t-cells in a mhc mismatched skin allograft model. the single transfer of 5 x 10 6 dn t-cells plus 28 days il-2/fc and low dose rapamycin treatment significantly prolong dba/2 skin allograft survival in c57bl/6 recipients in comparison with untreated group (mst 71 days vs. 11 days, p=0.0049) and il-2/fc plus rapamycin treated group mst (71 days vs. 34 days, p=0.0091). the results of using ex vivo cd4+ t-cells converted dn t-cells in skin and islet transplantation models support the concept and the feasibility of potentially utilizing this novel cell-based therapeutic approach clinically for the prevention of allograft rejection. jessamyn bagley, 1 jonathan g. godwin, 1 joren madsen, 2 john iacomini. 1 introduction: it has been suggested that natural killer (nk) cells are critical mediators that connect the innate and adaptive immune response. cytokine production by nk cells contributes to the polarization of immune responses to t helper 1, and nk cells express co-stimulatory molecules that may affect t cell proliferation. recent work has shown that nk cells are involved in the chronic rejection of parental cardiac grafts by f1 recipients. we hypothesized that given the role of nk cells in t cell activation and proliferation, nk cells may play a role in the development and function of t regulatory cells (treg) which control alloreactive responses. methods: nk, cd4+ t cells and cd4+cd25+ treg were purified from the spleens of c57bl/6j mice using macs bead separation followed by fluorescence activated cell sorting. naïve cd4+cd25-t cells from c57bl/6j mice (h-2b) were placed in culture with tgf-beta in the presence or absence of nk cells, and the development of treg was monitored by assessing foxp3 expression by intracellular cytokine staining and flow cytometry. in addition, the effect of nk cells on the function of treg was measured by elispot assay. results: following 4 days of culture with tgf-β and anti-cd3 antibody, cd4+cd25-t cells acquire foxp3 expression. the addition of activated nk cells to cultures with cd4+cd25-t cells and tgf-β prevented the acquisition of foxp3 expression by cd4 cells. tregs induced by stimulation of cd4+cd25-t cells with anti-cd3 in the presence of tgf-β are capable of suppressing the production of il-2, ifn-γ and il-4 by cd4 t cells in response to fully allogeneic balb/c stimulators. however, in the presence of activated nk cells, induced tregs fail to function, and production of il-2, ifn-γ and il-4 by cd4 t cells is restored. these experiments were repeated with natural cd4+cd25+ tregs isolated directly from mice without induction, and found that the ability of natural treg to suppress a cd4 t cell response to alloantigen was impaired in the presence of activated nk cells. conclusions: the presence of activated nk cells in culture can prevent the development of induced treg. furthermore, the presence of activated nk cells interferes with the function of mature treg in culture and allows a productive cytokine response by cd4 effector cells in response to alloantigen. results: both the syngeneic b6 cells and allogeneic dba/2 cells survived nicely in the rag-/-il-2rg-/-mice. however, the allogeneic dba/2 cells, but not the syngeneic b6 cells, were readily killed by the nk cells in the rag-/-mice. however, both rag-/-and rag-/-il-2rg-/-mice accepted the dba/2 skin allograft long term without any sign of rejection (>100 days). thus, nk cells by themselves, though cytolytic to dba/2 cells, fail to reject the dba/2 skin allografts. to test the hypothesis that the activation status of nk cells may dictate their alloreactive potential, we treated the rag-/-mice with il-5/il-15ra complex to maximally stimulate the nk cells in vivo. we found that il-15 is remarkably potent in stimulating nk cells in vivo; and nk cells stimulated by il-15 express an activated phenotype and are surprisingly potent in mediating acute skin allograft rejection in the absence of any adaptive immune cells, as il-15 treated rag-/-, but not the rag-/-il-2rg-/-mice, readily rejected the dba/2 skin allografts (mst=18 days). nk cell-mediated graft rejection doesn't show features of memory responses. and suggests that the fate of the allografts may depend on the activation status of nk cells and the availability of nk stimulating cytokines. background: t-bet is a transcription factor that promotes th1 development. both t-bet and the cytokines ifng and il-4 have been implicated as negative regulators of th17. in contrast, il-6 promotes th17 development. il-17 production is associated with granulocytic pathologies in several disease states. hence, this study assessed the relationship between t-bet, ifng, il-4 and il-6 in th17 induction and granulocytic infiltration of cardiac allografts. (ifng-/-) mice were transplanted with balb/c cardiac allografts. recipients were left untreated, depleted of cd4+ or cd8+ cells, treated with anti-cd40l mab, and/or treated with neutralizing anti-il-4 or anti-il-6 mab. graft histology was assessed and primed donor-reactive th responses were quantified by elispot. intragraft expression of il-17 and the th17 transcription factor rorgt were quantified by real-time rt-pcr. results: wt and t-bet-/-mice rejected their allografts at a similar tempo but with distinct pathologies: allografts in t-bet-/-recipients were heavily infiltrated with granulocytes while graft infiltrating cells in wt recipients were primarily mononuclear. while th1 and th17 responses were readily detectable in t-bet-/-recipients, th1 dominated the response in wt mice and th17 were not detectable. depletion of cd4+ cells prolonged graft survival in wt, but not in t-bet-/-recipients suggesting that cd8+ cells mediated rejection independent of cd4+ help in t-bet-/-mice. cd8+ cells were the source of il-17 in t-bet-/-recipients. anti-cd40l therapy promoted long-term allograft survival in wt recipients, but not t-bet-/-mice unless cd8+ cells were depleted. additionally, anti-cd40l therapy inhibited th1 responses in both wt and t-bet-/-recipients, but not the cd8+ th17 response in t-bet-/-mice. eliminating ifng and il-4 failed to induce il-17 production, while neutralizing il-6 reduced the th17 response in t-bet-/-mice. conclusions: while cd4+ th17 have been described in detail, cd8+ th17 have received less attention. in t-bet-/-allograft recipients, cd8+ th17 emerge independent of cd4+ help. cd8+ th17 are resistant to anti-cd40l therapy and are associated with granulocyte infiltration of the graft. these data implicate t-bet, as opposed to ifng and il-4, as a negative regulator of the th17 response while il-6 is required for cd8+ th17 induction. nan zhang, 1 bernd schroppel, 1 girdari lal, 1 jordi c. ochando, 1 jonathan s. bromberg. 1 1 background: cd4 + cd25 + foxp3 + regulatory t cells (treg) are important in suppressing immunity to prolong allograft survival. treg migration and its effects on suppressive activity are poorly understood. we determined treg migration patterns and effector function in an islet allograft model. ccr2 -/-, ccr4 -/-, ccr5 -/-, ccr7 -/-, l-selectin (cd62l) -/-, and fucosyltransferase (fuct) iv-vii -/-c57bl/6 mice were used to generate treg. treg were transferred intravenously, or locally to islet allografts, following islet transplantation (balb/c into c57bl/6). transferred treg were labeled with red dye pkh26 and migration to islet grafts, draining lns (dln), peripheral lns and spleen were tracked with fluorescence microscopy and flow cytometry. islet allograft survival was determined by measurement of blood glucose. results: treg expressed p-selectin ligand, cd62l, and a panel of chemokine receptors similar to other t cell subsets. intravenously transferred wild type treg migrated to both islet grafts and dln, and prolonged allograft survival. cd62l -/or ccr7 -/-treg, which migrated to islets but not dlns, prolonged allograft survival as potently as wild type treg. fuct iv-vii -/-treg, which lack e-selectin and p-selectin ligands, migrated to dln, but not islets, and did not prolong graft survival. similarly, ccr2 -/-, ccr4 -/-, and ccr5 -/-treg migrated to dln, but not islets, and did not prolong graft survival. when locally transferred to the islet graft, treg also migrated from the allograft to the dln, and prolonged graft survival even longer than after intravenous transfer. locally transferred ccr2 -/-, ccr5 -/-, or ccr7 -/-treg were not able to migrate from the islet to the dln, and were impaired in their ability to prolong islet survival. conclusion: treg migration to allografts is essential for their suppressive function; migration to lymphoid tissues alone is not sufficient to prolong graft survival. treg migration from the islet allografts to the dlns, via afferent lymphatics, is also required for optimal suppressive function and graft survival. the sequential migration from the site of inflammation and then to dlns is necessary for treg to execute fully their suppressive program. these results demonstrate a novel and important aspect of migration in treg suppression and tolerance. abstracts failure is unknown. methods:40 patients with iothalamate gfr <40ml/min (n=34) or on dialysis (n=6) at the time of liver transplant evaluation had undergone a percutanous ct guided renal biopsy. prior to the biopsy an inr ≤1.5 and platelet count ≥50,000/ ml were achieved in the majority of cases. all patients were monitored overnight for complications. candidates were listed for slk if pathology showed ≥ 40% glomerulosclerosis (gs) or ≥30% interstitial fibrosis (if). results:13 patients were eligible for slk and 27 for lta.creatinine was higher in slk candidates but not clinically different. background -it is well known that delayed graft function (dgf) is costly for those who received a renal transplant. however, the true cost of dgf is unknown. methods -we estimated the cost of dgf for adult cadaveric renal recipients in the usrds 1995-2004 who had medicare as their primary payer. those included were restricted to single organ recipients as well as those who had no previous transplants. cost was defined as the accumulated average cost per day for everyone with a functioning graft on that day. we examined the total cost of dgf, cost associated with dialysis, and non-dialysis cost for all patients combined and separately by donor type; standard criteria donors (scd), expanded criteria donors (ecd), and non-heart beating donors (dcd) as well as time to graft failure or no graft failure. background: based on adverse outcomes during the first year post-transplant, the optn membership and professional standards committee (mpsc) peer-review process flags transplant programs for further review using one of two methods. programs performing at least 10 transplants during at 2.5 year cohort are flagged based on the comparison of observed to expected event counts (death or graft failure) and the corresponding p-value (<0.05). programs performing 9 or fewer transplants are flagged if they have any adverse events. this leads to different flagging rates for programs of different sizes. the p-value has low sensitivity to identify poorly performing programs with a "moderate" number of transplants (10 to 20) whereas flagging every event results in a high false positive rate for "small" centers with <10 transplants. during the july 2007 review, 27% of "small" centers and 7% of centers with 10+ transplants were flagged for review (all organs). the scientific registry of transplant recipients (srtr) has developed alternative approaches for flagging centers with more consistent flagging rates. methods: the new method would allow for different choices of sensitivity (rr) and specificity (p-value) for different purposes and would flag program if either {p-value < .05 (a different value could be chosen)} or {observed / expected > rr}. the resulting false negative rate is less than 50% for centers with the given rr. this approach avoids an arbitrary definition of small versus large programs, and has sensitivity (or power) > 50% to flag programs with the selected rr, regardless of size. results: the following discussion: this approach gives a more balanced distribution of flagging across programs of different sizes and has sensitivity >50% for transplant programs of all sizes. with the choice of rr=2.5, the overall number of centers flagged would be nearly unchanged from current methods. center performance ratings are of increasing importance to the transplant community with the introduction of the cms final rule. ongoing debate exists regarding how much center ratings are directly a reflection of quality of care or whether ratings can be substantially influenced by exogenous factors. the study examined data from the national srtr database from 2000-2007. centers' semi-annual graft and patient survival were calculated along with a comparison with expected outcomes adjusted for covariates used by the srtr. centers meeting three criteria for poor performance were categorized within each cohort. patient characteristics at each center were compared with performance evaluations. overall, half of transplant centers met criteria for low performance in at least one of the semi-annual intervals. several center factors investigated were not significantly associated with the likelihood to meet low performance criteria including the proportion of older, obese and privately insured patients. in contrast, centers with higher levels of ecd transplants (most ecds=70% vs least ecds=40%), african american recipients (most aas=75% vs least aas=35%) and patients with low albumin level (lowest albumin =60% vs highest albumin =32%) were more likely to meet low performance criteria. approximately half the centers that initially met criteria for low performance no longer met criteria when excluding ecd transplants and african american recipients from the performance assessment. conclusions given the extreme implications of performance ratings for transplant centers including possible loss of funding to centers with low performance, it is critical that we recognize potential weaknesses and biases of performance ratings. our results are important towards understanding factors related to performance ratings and raising questions as to whether risk adjustment techniques are adequate for fair transplant center performance evaluations. rather than only risk adjustment, stratified evaluations may be a partial solution to remove disincentives to performing higher risk transplants. it is also important to recognize factors not associated with low performance such that centers do not unnecessarily limit access to groups based on perceived deleterious impact on ratings. model. olaf boenisch, 1 takaya muramatsu, 1 francesca d'addio, 1 robert padera, 1 hideo yagita, 2 nader najafian. 1 1 transplantation research center, brigham and woman's hospital and children's hospital, boston, ma; 2 immunology, juntendo university of medicine, tokyo, japan. t cell immunoglobulin and mucin domain (tim)-3, a molecule expressed on terminally differentiated th1 cells, is an important regulator of th1 autoimmunity and in induction of transplantation tolerance. its functions in alloimmune responses during acute and chronic rejection are unknown. tim 3-23, a novel blocking antibody of tim-3, was administered to recipients in various donor-recipient strain combinations on days 0 (500ug), 2, 4, 6, 8 and 10 (250ug) after transplantation. the frequency of ifng-, il-4-, and granzyme b-producing splenocytes was measured by elispot. these data establish the regulatory functions of tim-3 in alloimmune responses in solid organ transplantation models. the inhibitory actions may be secondary to modulation of effector or regulatory t cells and appear to dominate in conditions of low levels of t cell activation, due to a restricted degree of allogeneic mismatch or absence of cd28 costimulation. the i-r injury in transplanted kidney is a major cause of dgf, an event associated with an increased risk of acute rejection. adaptative immunity was suggested to play a role in the pathogenesis of renal i-r injury, although the influence of the th1/th2 bias in this scenario is still debated. thus, the aim of the present study was to evaluate the features of t cell response during i-r injury at the peripheral and tissue level in renal graft recipients with dgf. the mrna levels of specific th1 (t-bet) and th2 (gata-3) transcription factors were evaluated in circulating lymphomonocyte of kidney transplant recipients with early graft function (egf) (n=10) and dgf (n=10), before (t0) and 24 hours after transplantation (t24) by real time pcr. infiltrating lymphocytes were characterized in graft biopsies of patients with dgf (n=40) and in a control group of patients with tubular damage by acute cni toxicity (n=10) by immunohistochemistry. in addition, we evaluated the th1/th2 bias at the renal level in a pig model of i-r injury. t-bet/gata-3 mrna ratio was similar in the 2 groups of patients at t0. at t24 the dgf group presented a significantly higher increase of t-bet/gata-3 ratio compared with the egf group (798±346 vs 288±147% of t0, p<0.001). moving to the tissue level, dgf patients presented a number of interstitial cd4 + (8.0±5.1 vs 2.6±2.1, p=0.04) and cd8 + (10.8±6.7 vs 4±2, p=0.02) t cells significantly higher compared to the control group, while no significant differences were observed in cd20 + cells number between the two groups. also at the tissue level the ratio between t-bet + and gata-3 + cells was significantly higher in the dgf compared with the control group (3.5±1.8 vs 1.1±0.9, respectively, p=0.005). to confirm that these changes were due to i-r, we investigated the presence of t-bet + and gata-3 + cells in a pig model of i-r injury. interestingly, the ratio was significantly increased after 24 hours of reperfusion (basal 2.5± 0.9 vs 24 hours 8.1±3.6; p=0.02). in conclusion, our results suggest that kidney transplant recipients with dgf present a bias toward a th1-driven immune response both at the peripheral and at the tissue level. this event, due to the i-r process, as suggested by the animal model, may represent a link between dgf and acute graft rejection. as expected a strong negative association between duration of dialysis and patient survival was seen in caucasian recipients. in contrast the relationship between patient survival and duration of dialysis was u shaped in minorities with the worst patient survivals seen among preemptive transplants and those patients with over 60 months of dialysis. the difference in outcomes between caucasians and minorities could be related to the biologic differences in the effect of dialysis on subsequent transplantation or to a differential selection bias introduced by duration of dialysis in the two populations. anti-carbohydrate natural antibodies. lorenzo benatuil, 1 jonathan g. godwin, 1 shamik gosh, 1 john iacomini. 1 1 transplantation research center, boston, ma. background. we constructed immunoglobulin gene knock-in mice lacking expression of the αgal epitope that carry rearranged vh and vl genes encoding antibodies specific for the carbohydrate antigen galα1-3galβ1-4glcnac-r (αgal). here, we describe two novel populations of b cells in the spleen of these m86vhvlgt 0 mice and their role in the production of αgal specific antibodies. methods. m86vhvlgt 0 mice were sacrificed and single cell suspensions from spleen, bone marrow, peripheral lymph nodes and peritoneal cavity were prepared and stained with different antibodies and with fluorescently labeled αgal-bsa for flow cytometric analysis. using multiparameter cell sorting, we purified marginal (mz) and follicular (fo) zone b cells, in addition to two novel splenic b cell populations. cells were adoptively transferred into b cell deficient µmt/gt 0 double knock-out mice. serum samples were collected, and production of αgal specific igm antibodies was assessed by elisa. to investigate these b cell tolerance mechanisms, we employed mice with naturally occurring antibodies (abs) against human blood group a carbohydrates in their sera and possessing b cells with receptors for blood group a determinants. b cells with receptors for a carbohydrates in mice belong to the cd5 + b-1 subset, with phenotypic properties similar to those of human b cells. when these cells were temporarily eliminated by injecting synthetic a carbohydrates, subsequent treatment with cyclosporin a or tacrolimus, which blocks b-1 cell differentiation, completely inhibited the reappearance of b cells with receptors for a carbohydrates in mice. it is probable that calcineurin inhibitors used for preventing t cell-mediated rejection simultaneously suppress b-1 cell differentiation. however, despite a very limited dose of calcineurin inhibitors, the b cell tolerance toward blood group a antigens was persistently maintained in the blood group a-to-o liver transplant recipient. b cell tolerance after abo-incompatible transplantation might be a consequence of presentation of blood group carbohydrate antigens by cells in the engrafted liver. immune fluorescence staining of the human liver reveals that blood group antigens are predominantly expressed on the liver sinusoidal endothelial cells (lsecs). we have previously proven that lsecs, which constitutively express fas-l and pdl-1 have the capacity to tolerize alloreactive t cells. taken together, we hypothesize that blood group antigen-reactive b cells are also tolerized through the interaction with the lsecs. in order to address this possibility, we used α-1,3galactosyltransferase-deficient mice. when the α-gal-expressing lsecs isolated from wild-type mice were adoptively transferred via the portal vein into the splenectomized congeneic α-gal-deficient mice, these mice lost the ability to produce anti-α-gal abs (n = 4). this finding suggests that the lsecs expressing blood group carbohydrates play a pivotal role in the tolerization of newly developed b cells specific for the corresponding carbohydrate antigens after abo-incompatible liver transplantation. success in kidney transplantation has resulted from control of t-cell-mediated acute rejection. however, little has been done to improve the fate of patients who possess pretransplant donor-specific antibody (dsa), and no proven therapies exist to specifically prevent dsa formation post-transplant. while methods to detect and characterize dsas are clearly useful, the b-cell subsets that produce dsas or more importantly sustain their production are poorly characterized. this study set out to investigate the fundamental mechanisms determining the formation of donor-specific memory b cells and plasma cells in novel mouse models of dsa formation. we have developed two complementary and novel systems to track the phenotypic and functional properties of polyclonal donor-specific b-cells. the first system involves allosensitization between normal c57bl/6 and balb/c mice. we used advanced flow cytometric methods to track donor-specific b cells with h-2k b or h-2k d tetramers. tetramers are comprised of four identical h-2 molecules bound to a fluorescently labeled steptavidin molecule that is able to bind the donor-specific b-cell antigen receptor (surface immunoglobulin). in our second system, we use donor mice that constitutively express full-length membranebound chicken ovalbumin (mova) protein in all tissues. analogously, ova specific-b cells can be analyzed flow cytometrically with the use fluorescently-labeled ovalbumin. both systems allow us to identify donor-specific memory b cells (7aad -cd4 -cd8 -ova/ tetramer + igd -b220 + cd138 -) and plasma cells (7aad -cd4 -cd8 -ova/tetramer + igd -fb220 lo cd138 + ) during skin graft rejection (day 14 post transplant). we were also able to track the development of a stable memory cell population in the bone marrow and spleen for > 90 days cells.for the ova system quantitative elisa and elisot measurements for serum dsa and dsa-secreting cells, respectively, correlated with the development of donor-specific memory b cells. using these assays we will be evaluate polyclonal donor-specific b memory subsets under multiple conditions of immunity and tolerance and for the first time, characterize their functional properties and migration patterns. these experiments will provide new information on the basic biology of the memory b-cell response to allografts in mice and facilitate the development of these methods in sensitized patients that may lead to critical therapeutic opportunities for dsa production. in the tnf-related b-lymphocyte survival factor, blys/baff, is critical for primary follicular (fo) and marginal zone (mz) b-cell survival. in vivo neutralization of blys/ baff, using a newly developed monoclonal antibody, depletes the fo and mz b-cell compartments. here, we hypothesized that targeting b-lymphocytes, via the blys/baff pathway, could promote humoral tolerance to islet allografts. cohorts of stz-diabetic c57bl/6 mice were transplanted with isolated islets from balb/c donors. treatment with anti-blys/baff alone (100mcg/mse x2 doses+15mcg/wk/mse) did not protect islet allografts from acute rejection (mst=14d; n=4) . on the other hand, a 14-day course of rapamycin (1mg/kg) prevented acute allograft rejection (mst=35d; n=9). when rapamycin was combined with the anti-blys regimen, islet allograft survival was markedly prolonged (mst>150d; n=4). importantly, islet allograft survival was coincident with the absence of detectable serum alloantibodies and blunted donor specific t-cell responses. following treatment with anti-blys, b-lymphocyte compartment reconstitution was detectable at 30 days following treatment and was characterized by stringent selection at the transitional→fo b-cell tolerance checkpoint. overall, these data indicate that the blys/baff pathway may be a logical target of immunomotherapy for the achievement of humoral transplantation tolerance. reza tavana background: highly sensitized patients' ability to be transplanted is severely compromised because of high level of antibodies against various hla antigens. interleukin-21 is a type i cytokine that signals through a receptor composed of the il-21r and the common cytokine receptor -chain ( c ). it is produced by t-cells and has been shown to be the contributing factor in terminal differentiation of memory b-cell to anti-body producing b-cell and plasma cells. objective: we evaluated the effect of il-21 co-stimulation on antibody production capacity of b-cells and also measured the expression of il-21r on b lymphocytes of highly sensitized patients compared to non-sensitized patients on the kidney transplant list. methods: patients with a pra level >20% (sensitized) were compared to non-sensitized patients from the transplant waiting list. after consent was obtained, peripheral blood was taken from patients before initiating hemodialysis. leukocytes were labelled with anti-cd19, anti-il-21 antibodies and paraffin fixed after rbc lysis. il-21r expression was measured by flow-cytometry over facscan machine on the same day. the expression of the il-21r is significantly higher on b-lymphocytes of highly sensitized patients (hsp) compared with non sensitized patients (nsp) (fig 1.p<0.05 ). in-vitro co-stimulation of isolated peripheral b-cell with il-21 and anti-cd40 results in higher igg1 production compared with anti-cd-40 or il-21 stimulation alone (fig 2) . conclusions: il-21 is an important cytokine in b-lymphocyte stimulation and increases igg1 production. il-21 receptor on b-lymphocytes is up-regulated in sensitized kidney transplant recipients. il-21 to il-21r interaction between t and b-lymphocytes may be an important pathway in antibody production in highly sensitized renal transplant recipients. we created mixed bone marrow chimeras in irradiated µmt (b cell-deficient) recipients by transplantation of syngeneic bone marrow from µmt, wildtype (wt) and mhcko (lack mhc i & ii expression on all cells) mice. µmt+mhcko chimeras lack expression of mhc i & ii on b cells but not other professional apcs such as dcs hence antigen presentation specifically by b cells is disrupted. allograft rejection and development of alloreactive memory t cells in µmt+mhcko chimeras was compared to µmt+wt chimeras that had intact antigen presentation by both b cells and apcs. skin allograft rejection was comparable between µmt+mhcko and µmt+ wt chimeras (mst = 17 and 16 days, respectively, p = 0.6, n = 5/grp). however, heart allograft rejection was significantly delayed in the µmt+mhcko chimeras compared to µmt+ wt chimeras (mst = 23 and 15 days, respectively, p=0.03, n = 5/ grp). development of alloreactive memory t cells was assessed in chimeras at 8 weeks after skin allograft rejection by quantitation of antigen-specific ifnγ producing t cells. alloreactive cd4 and cd8 memory t cells were significantly fewer in µmt+ mhcko chimeras (7-fold fewer cd4, p = 0.02, and 5-fold fewer cd8, p = 0.003, n = 5/grp) than in µmt+ wt chimeras. these results show that the disruption of antigen presentation by b cells significantly delays heart but not skin allograft rejection. development of alloreactive cd4 and cd8 memory t cells is significantly impaired in the absence of antigen presentation by b cells. conclusions: antigen presentation by b cells accelerates heart allograft rejection and leads to development of alloreactive memory t cells. these findings emphasize antibodyindependent functions of b cells in promoting alloimmune responses and highlight the need for b-cell targeted therapies to improve long-term allograft survival. purpose: to test whether depletion of cd20+ b cells at the time of engraftment alters the prevalence of anti-donor alloantibody (ab)or severity of cav in the context of therapeutic immunosuppression with (csa) or high dose cd154 inhibition. methods: forty-five mlr-mismatched heterotopic cardiac cynomolgus allograft recipients were treated with high dose anti-cd154 monotherapy (αcd154; n=14, 5 with atg induction) or αcd154 with additional anti-cd20 therapy (rituximab 20mg/ kg q wk for 4 weeks: αcd154 + αcd20; n=18, 11 with atg). thirteen other animals received therapeutic csa (target trough >500ng/ml), five of which received additional αcd20. graft survival was censored at 90 days. ab was deemed (+) if present (by flow cytometry) around time of explant. results: 12 animals died with beating grafts, mainly with atg-associated lung pathology or infectious etiologies and are excluded from this analysis. 3 animals that had sustained αcd154 levels <100µg/ml after protocol day 30 were considered "subtherapeutic" for this reagent and also excluded from further consideration. graft survival with αcd154 + αcd20 (median >90d) and proportion of grafts surviving to 90 days (6/6) was significantly increased relative to αcd154 alone (median 43d, p=0.007; 4/14 >90d). graft survival with csa + αcd20 (median >90d) and proportion of grafts surviving to 90 days (4/4) was increased relative to csa alone (median 66d, 3/6 >90d). with therapeutic αcd154 (trough level >100 µg/ml until graft explant), 12/13 (92%) developed ab vs. 1/6 with αcd154 + αcd20 (17%) (p=0.006). average cav score for the αcd154group was 2.44 ±0.44 vs. 0.7 ± 0.8 in the αcd154 + αcd20 group (p=0.006 using unpaired t test). preliminary cav scoring suggests that added αcd20 inhibited cav (scores ranging 0.0-0.1) relative to csa alone (median 2.1; range 1.5-2.4); ab analysis is in progress for these groups. conclusions: using αcd20 is associated with significant attenuation of cav when used with either "therapeutic" αcd154 or csa. our findings demonstrate for the first time that αcd20 reduces the severity of cav in conjunction with both conventional immunosuppression and costimulation pathway blockade. mechanisms include inhibition of ab production, and perhaps others that remain to be defined. subsaturating concentrations of anti-hla antibody ( sensitized recipients having donor specific anti-hla abs have been successfully transplanted following a conditioning regimen employing plasmapheresis with or without pooled human immunoglobulin. in vitro studies have shown that exposure of ecs to subsaturating concentrations (ssc) of anti-hla ab (priming) followed by subsequent exposure to high concentrations (hc) of anti-hla induces the expression of protective genes, bcl2 and ho-1 and confers protection to complement mediated lysis of ecs. however, the molecular events following priming with exposure to ssc of anti-hla ab still remains undefined. to determine the priming events and to define the kinetics of protective gene expression, we exposed human aortic ecs to ssc of anti-hla ab for 72 hrs and re-exposed them to saturating concentrations of anti-hla abs. ecs were collected at 24, 48, and 72 hrs after exposure to ssc as well as 24 hrs after exposure to hc. expression profile of signaling intermediates (mapk, wnt, nf-kb, hedgehog, pi3kinase, stress pathway, tnf family) in the ecs were analyzed by gene array. analysis of the bcl2 and ho-1 expression showed no significant increase in expression following exposure to ssc of w6/32 (priming) or control ab alone at any of the time points ( background/aims: microcirculation disturbance, endothelial injury and cytokine overproduction are implicated in the pathophysiology of hepatic ischemia reperfusion injury (iri). thrombomodulin (tm) is a membrane-bound endothelial thrombin receptor that accelerates thrombin-catalyzed protein c activation, inhibits thrombin-induced fibrin formation, and also regulates inflammation. in the present study, we investigated the effects of recombinant human soluble tm (rhstm) on hepatic iri in the rat. methods: wister hannover rat was used for preparing ischemia/reperfusion model. hepatic iri was induced by subjecting 70 % area of the rat liver to 90 minutes of ischemia followed by 6 h of reperfusion. the rats were randomly assigned to a group receiving an intravenous injection of rhstm (3 mg/kg body weight) and to a group treated with saline 60 minutes prior to the beginning of reperfusion. sinusoidal endothelial cells (secs) and kuppfer cells (kcs) were isolated using centrifugal elutriation. the plasma levels and the concentration in cultured cell supernatant of il-6 and tnfα were measured by specific enzyme immunoassays. results: the plasma alt, ast and hyaluronic acid levels were significantly decreased, and the histological damage of the liver was attenuated in rhstm-treated rats as compared to control rats. using laser doppler flow-meter we found that rhstm treatment improved hepatic microcirculation. the intrasinusoidal fibrin deposition, injury of secs and liver dysfunction during hepatic iri were weaker in rhstm-treated rats than in control rats. tm activity in secs was significantly recovered, and plasma il-6 and tnfα were significant decreased in rhstm-treated rats as compared to control rats. further, il-6 and tnfα production in isolated kcs was also significant decreased in rhstm-treated rats as compared to control rat . conclusion: the present results suggest that rhstm is useful for the prevention of secs damage and kcs activation induced by iri. our present study also suggests that disturbance of hepatic microcirculation is induced in part by intrasinusoidal microthrombus formation and by locally released inflammatory cytokines from kcs. protective effects of preservation solution including activated protein c in small-for-size liver transplantation in rats. background: small-for-size liver graft is a serious obstacle of partial orthotopic liver transplantation (olt). however, various therapeutic strategies including surgical innovations, pharmacological agents and gene therapies to protect small-for-size liver graft have not yet been developed. aims: activated protein c (apc) is known to have cell protective properties via its anti-inflammatory and anti-apoptotic activities. this study aimed to examine the cytoprotective effects of preservation solution containing apc on olt using smallfor-size rat liver graft (20% partial liver). methods: liver grafts were assigned to two groups: in the control group, the grafts were flushed and stored in histidine-tryptophan-ketoglutarare (htk) solution alone for 6 h; in the apc group, in htk solution containing apc for 6 h. results: the apc group significantly increased 7-day graft survival from 60% to 100%, decreased levels of transaminase, and improved histological features of hepatic iri compared to the control group. myeloperoxidase activity demonstrated that the apc group markedly suppressed the infiltrations of neutrophil. hepatic expressions of tumor necrosis factor-α and il-6 of the apc group were remarkably decreased. the apc group significantly reduced serum hyaluronic acid levels, indicating attenuated sinusoidal endothelial cell injury. moreover, the apc group markedly increased hepatic levels of nitric oxide caused by upregulated endothelial nitric oxide synthesis (nos) together with downregulated inducible nos, and decreased hepatic levels of endothelin-1. finally, hepatocellular apoptosis of the apc group was remarkably suppressed by downregulated hepatic caspase-8 and caspase-3 activities. conclusions: preservation solution containing apc inhibited pro-inflammatory cytokine synthesis, which leads to hepatocellular apoptosis and liver injury. one of the cytoprotective effects of the apc treatment was to upregulate hepatic enos, followed by increased expression of hepatic no, and to decrease expression of hepatic et-1, resulting in the prevention of microcirculatory disturbance. preservation solution containing apc is a potential novel and safe product for small-for-size liver transplantation to improve liver graft function and animal survival. deletion of cd39 on natural killer cells attenuates hepatic ischemia/ reperfusion injury. living donor liver transplantation (ldlt) has emerged as a solution to ease organ shortage in orthotopic liver transplantation. however, ldlt is often complicated by small-for-size liver graft that is highly susceptible to injury and shows decreased liver regeneration. suppression of liver regeneration in small-for-size grafts correlates with impaired priming as a result of limited nf-κb activation and decreased production of the priming cytokines tnf and il-6. we have shown that the hepatoprotective protein a20 promotes liver regeneration, partly through blockade of the cyclin dependent kinase inhibitor p21. however the impact of a20 expression in livers on il-6 production and/ or signaling were still unknown. in this study we demonstrate that secretion of il-6 (elisa) following treatment with lps or tnf was moderately lower in hepatocytes transduced with a recombinant a20 adenovirus (rad) as compared to non-transduced or rad.β-galactosidase transduced cells. this indicates that il-6 production in hepatocytes is not solely nf-κb dependent (not totally blocked by the nf-κb inhibitor a20). despite similar or lower il-6 levels, il-6 signaling as evaluated by phosphorylation of stat3 (western blot; wb) was enhanced in a20 expressing hepatocytes. this was confirmed in experiments showing that a20 increases stat-3 phosphorylation in response to exogenous human il-6. accordingly, hepatocyte proliferation was significantly higher in rad.a20 transduced hepatocytes as opposed to controls. the pro-proliferative function of a20 mapped to the 7zn domain. since the balance of il-6 signaling in hepatocytes is finely regulated through a negative feed-back loop provided by the il-6/stat-3 dependent induction of suppressor of cytokine signal-3 (socs3), we investigated whether a20 affects il-6 signaling by modulating socs3 expression. our results indicate that a20 indeed decreased il-6 mediated upregulation of socs3 (wb). this later result was confirmed in vivo. improved regeneration and survival in a20 treated livers correlated with a substantial decrease in socs3 levels before and 24 hours following extended (78%) liver resection in mice. these results suggest that a20 enhances priming of hepatocytes by il-6 likely through down-regulation of socs3. this added to the effect of a20 on p21 would further enhance its pro-proliferative function in hepatocytes to benefit survival and function of small-for-size liver grafts. background: we have previously demonstrated that silencing inflammatory, apoptosis and complement genes can prevent ischemia-reperfusion (i/r) injury occurring in heart transplantation. however, the method for efficiently delivering sirna into donor organ has not been established. this study was designed to develop a new method to induce gene silencing by coronal artery infusing with sirna solution for prevention i/r injury in heart transplantation. methods: multiple sirnas that specifically target tnfa, caspase 8, and c5a receptor (c5ar) genes were generated and selected. sirna protection of donor organs was evaluated in a rat heart transplantation model. heart grafts from lawis rats were infused with sirna solution via coronal artery and preserved in hkt solution at 4° c for 18 hrs, and subsequently transplanted into syngeneic lawis rats. cardiac functions were assessed by heart beating rate. gene expression at mrna level was determined by qpcr. the i/r injury was assessed by immunohistochemistry. results: after donor heart perfusion with sirna solution, sirna was found to enter the myocardial cells, indicated by the fluorescence emitted from the dye labeled sirna. the levels of tnfa, caspase 8 and c5ar genes were significantly up-regulated in the grafts after ex vivo preservation for 18 hrs. these up-regulated tnfa, caspase 8, and c5ar genes were significantly knocked down by sirna infusion. using a sirna infused organ as a donor, the graft survival was significantly prolonged in heart transplantation. while sirna solution-treated heart grafts retained strong heartbeat up to the end point of observation (>10 days), the control grafts lost function within 3 days. in addition, an improved cardiac function was observed in the graft preserved in sirna solution. the protection of graft by sirna solution is associated with prevention of i/r injury. sirna solution-treated organs exhibited almost normal histological structures as well as less neutrophil and lymphocyte infiltration, compared with control solution-treated organs. conclusions: this study developed a novel ex vivo sirna delivery system using coronal artery infusion, which can effectively silencing genes in donor hearts and prevent cardiac i/r injury. background: ischemia/reperfusion (i/r) insult is a prime factor leading to liver dysfunction. apoptosis plays key role in the early graft loss following orthotopic liver transplantation (olt). bcl-xl has been showed to exert an anti-apoptotic function both in vitro and in vivo. this study was designed to evaluate potential cytoprotective effects and mechanisms for bcl-xl in liver i/r injury by ad-bcl-xl gene transfer. methods: a mouse model of partial 90 min warm hepatic ischemia followed by 6 h of reperfusion was used. balb/c wide-type (wt) mice (n=6/gr) were injected with ad-bcl-xl or adβ-gal reporter gene (2.5x10 9 pfu, i.p. at day -2). sham control wt mice underwent the same procedure, but without vascular occlusion. mice were sacrificed after 6 h of reperfusion; liver tissue and blood samples were collected for future analysis. results: ad-bcl-xl treated mice showed significantly lower sgot levels (iu/l), as compared with ad-β-gal or wt controls (509±361 vs. 2819±706, and 2212±841, respectively; p<0.005). these correlated with histologic suzukis grading of hepatic i/r injury, with wt/ad-β-gal controls showing significant edema, sinusoidal congestion/cytoplasmic vacuolization, and severe hepatocellular necrosis. in contrast, wt mice treated with ad-bcl-xl revealed minimal sinusoidal congestion without edema/vacuolization or necrosis. ad-bcl-xl gene transfer significantly reduced local neutrophil accumulation and apoptosis (3.8±2.9 of tunel+ cells in ad-bcl-xl vs. 21.5±5.3 and 23.5±6.5 of tunel+ cells in wt or ad-β-gal treated mice; p<0.01). unlike in controls, intragraft expression of mrna coding for tnf-α, e-selectin/icam-1, and ip-10/mcp-1 remained depressed in the ad-bcl-xl group. ad-bcl-xl gene transfer markedly depressed the activation of nf-κb, caspase-3, and increased ho-1, a20, and bcl-2/bcl-xl expression, as compared with wt/ad-β-gal controls. conclusion: this study demonstrates that inhibition of nf-κb activation contributes to the cytoprtective effects after bcl-xl gene transfer in hepatic i/r injury. the induction of anti-oxidant ho-1 and anti-apoptotic a20, bcl-2 by bcl-xl gene transfer exerts synergistic cytoprotective effect against antigen-independent hepatic inflammatory injury induced by i/r. hepatocyte background: primary graft non-function (pnf) affects survival and function of renal allografts. pnf, secondary to the ischemic and inflammatory injury in the peri-transplant period, leads to acute tubular necrosis and predisposes to acute rejection. defining new preconditioning regimens to reduce pnf are desirable. a20 is part of a negative antiinflammatory loop aimed at inhibiting nf-κb in renal proximal tubular epithelial cells (rptec). hepatocyte growth factor (hgf) is a pleiotropic growth factor upregulated in acute kidney injury and acute rejection likely to modulate inflammation and promote repair. in the present study we evaluated the effect of hgf on rptec and hypothesized that some of its protective functions may relate to the upregulation of a20. methods and results: treatment of rptec with hgf (50ng/ml) led to a 2.4±0.8 (n=4; p=0.04) fold increase in a20 mrna (real time-pcr), which translated into a significant 3.0±1.5 fold increase (n=5; p=0.04) in a20 protein by 6h, as shown by western blot (wb). two lines of evidence suggested that upregulation of a20 by hgf was nf-κbindependent. hgf did not degrade iκbα in rptec (wb) nor upregulated the nf-κb dependent molecule icam-1, as shown by flow cytometry analysis (facs). further, a20 was still upregulated in rptec expressing the nf-κb inhibitor iκbα, both at the mrna and protein levels. upregulation of a20 by hgf protected rptec from a subsequent inflammatory insult, here mimicked by the addition of tnf. pretreatment of rptec with hgf for 6 hours blunted tnf-induced (10u and 25u/ml) upregulation of icam-1, as analyzed by facs. conclusion: to our knowledge this is the first demonstration that a20 could be upregulated in rptec, in a non-nf-κb dependent manner. further studies are carried out to elucidate the transcription factors involved in hgf-induced upregulation of a20. from a clinical standpoint, these results highlight the unique ability of hgf to protect rptec from inflammation by inducing the anti-inflammatory protein a20, remarkably without triggering other pro-inflammatory signals. we propose that hgf-based therapies could serve in preconditioning regimens to prevent ischemia/reperfusion injury and reduce pnf and acute rejection in renal transplantation. background. liver ischemia reperfusion injury (iri) is one of the main causes of graft dysfunction and rejection in liver transplantation. it has been documented that iri is associated with inflammatory and complement pathway activation. this study was designed to investigate the efficacy of small interfering rna expression vector (shrna) targeting tnf-α and complement 3 (c3) genes in the protection of mouse liver iri. methods. shrna expression vectors were constructed for tnf-α and c3 genes. mice received shrna by hydrodynamic injection prior to iri, which consisted of interrupting blood supply to the left lateral and median lobes of the liver for 45 minutes followed by reperfusion. iri was evaluated using liver histopathology, as well as levels of serum alanine transferase (alt) and aspartate transaminase (ast). neutrophil accumulation was determined by a myeloperoxidase (mpo) assay. lipid peroxidation was assessed by malondialdehyde (mda) levels. realtime pcr was used to test gene silencing efficacy in vitro and in vivo. result. we demonstrated that iri is associated with an increase in tnf-α and c3 mrna levels in liver tissue 6 hours after reperfusion. shrna-treatment effectively down-regulated tnf-α and c3 expression in iri livers. in comparison with vehicle control, the serum levels of alt (9843.31 ±2610.66u/l vs 1960.00 ±1361.98 u/l) and ast (7188.25 ±3295.99u/l vs 1405.13 ± 774.65u/l), were significantly reduced in mice treated with tnf-α and c3 shrna. additionally, the neutrophil accumulation and lipid peroxidase-mediated tissue injury, detected by mpo and mda respectively, were improved after shrna treatment. tissue histopathology showed an overall reduction of injury area in shrna-treated mice. conclusion. this is the first demonstration that liver iri can be prevented through gene silencing of inflammatory genes and complement genes, showing potential for shrna-based clinical therapy. kidney -acute rejection: antibody-mediated rejection the (2) in the first three days after transplantation, a temporary decrease in dsa was observed in all amr cases, and all of them quickly rebounded thereafter; (3) c4d can be detected very early (can be seen on day 1, 89% in day 4 protocol biopsies, frequently in the absence graft dysfunction, and 100% in index biopsies); (4) the pathologic changes observed in sequential biopsies were c4d deposition followed by acute tubular injury, then interstitial inflammation and peritubular capillary margination seen in index biopsies; (5) pure amr occurred early (100% at day 3), usually evolving into mixed amr with accompanying cellular rejection (87% in index biopsies); (6) most recipients (87%) had initial graft function before developing amr; (7) background: antibody-mediated rejection (amr) has been recognized as a major problem in abo-incompatible (abo-i) renal transplantation (rtx). however, little is known about the long-term impact of amr in the abo-i renal transplant setting, especially after the introduction of a tacrolimus (fk)-based immunosuppressive regimen. the aim of this study was to assess the long-term impact of amr on the clinical and pathological outcomes in abo-i rtx. methods: fifty-eight patients who underwent abo-i rtx at our institution between march 1999 and december 2004 under an fk-based immunosuppressive regimen were enrolled in this study. protocol biopsies were performed regardless of renal function at one month and one year after rtx. fifty-six of the 58 patients received the biopsy at one month and 43 of 56 patients underwent biopsy at one year posttransplant. amr was diagnosed by morphological features based on the banff '05 update and other characteristic findings for amr previously reported, such as mesangiolysis, interstitial hemorrhage, and cortical infarction. we evaluated graft survival, incidence of chronic rejection characterized by transplant glomerulopathy (tgp) at one year posttransplant, and renal function using serum creatinine at three years posttransplant according to the incidence of amr at one month posttransplant. the overall graft survival rate at 3, 5, and 8 years after rtx was 93%, 87%, and 63%, respectively. the incidence of amr at one month was 34% (19/56). the graft survival rate of the patients with amr was significantly lower than that of the patients without amr (p<0.01, 3 years: 79% vs 100%, 8 years: 38% vs 94%). the incidence of tgp in the patients with amr was significantly higher than that of the patients without amr (p<0.001, 57% vs 7%). the serum creatinine concentration at three years after rtx was significantly higher in the patients with amr than in those without amr (p<0.001, 1.95 mg/dl vs 1.32 mg/dl). in this study, we revealed that amr in abo-i rtx is associated with not only graft loss but also the progression of chronic renal impairment, functionally as well as pathologically, even after the introduction of an fk-based immunosuppressive regimen. further studies are needed to establish a more effective immunosuppressive regimen, such as rituximab induction therapy; against amr in abo-i rtx. conclusions: de novo dsa in ar is an independent predictor of graft loss and its degree of influence is comparable to other established risk factors (aa race, dgf, increased baseline creatinine). additional studies are warranted to: 1) confirm the predictive ability of dsa and 2) determine whether reduction/eliminattion of dsa will allow improvements in graft survival. was performed in all the recipients before and six months after lrkt. graft biopsies were performed as well within and after six months of the transplantation (tx). all the data of 87 recipients were collected prospectively during the period of follow-up. humoral rejection rate, donor specificity, and time of appearance of the de novo abs were retrospectively studied. results among the 87 lrkt recipients, 47 (54%) showed negative/negative results, 15 (17%) showed positive/positive results, 12 (14%) showed positive/negative results, and 13 (15%) showed negative/positive results (de novo abs) in the pre-/post-transplant flow-pra analysis. among the 13 cases with de novo abs, 5 (38%) had donorspecific abs (dsa) and the remaining 8 (62%) had non-donor specific abs (ndsa) as determined by lab single antigen analysis. four of the five recipients (80%) with dsa showed evidence of both vascular and humoral rejection in the graft biopsies performed within 6 months of the transplantation, while one of the eight recipients (13%) with ndsa showed evidence of cellular rejection during the same period. a 5-year graft survival rate of the recipients with de novo abs was 69%, compared with 96%, 88% and 93% in other groups without de novo abs (p=0.009). conclusions lrkt recipients with developing de novo abs has much higher incidence of humoral rejection and worse prognosis, especially those with donor-specific de novo abs. cautious monitoring for the appearance of anti-hla antibodies should be adopted after transplantation, even in patients without anti-hla ab prior to the transplantation. despite significantly higher response than the 4 males w/o amr (p<0.03), the other 5 females did not experience amr. conclusions: 1) cfc is a novel assay to measure allo/ do cd3-cell responses, assess the degree of sensitization, and predict amr in hs, 2) allo/do cd3-cell numbers are elevated in many hs, but not nc, 3) hs w/ high(+) cfc are at increased risk for amr and may need additional pre-tx desensitization, 4) allo/do reactivity are higher in hs females, which may explain their higher rate of amr, 5) cfc cut off levels for amr prediction may be higher in females than males, 6) monitoring hs using the cfc pre-and post-desensitization may help determine the efficacy of desensitization and risk for amr. were treated with plasmapheresis, ivig and rituximab, and pts with l-amr received ivig and rituximab. the 2-year gs post-amr in pts with e-amr and focal c4d staining was 33% vs. 50% in pts with diffuse staining; while cases of l-amr with focal c4d deposition had a gs of 55% vs. 60% in cases with l-amr and diffuse staining. the number of cases with focal staining was low, and the numerically evident differences were not statistically significant (log-rank p=ns). notably, when losses due to death with a functional graft were censored, post-amr gs was significantly lower in pts with e-amr and focal staining than in their counterparts with diffuse c4d deposition (41% vs. 67%, log-rank p=0.04). in this retrospective single center study, focally positive c4d amr carries a worse prognosis than previously thought, and causes a significant reduction of gs. whether any degree of c4d staining in the context of kt dysfunction should be treated as amr remains a pending question. association time of biopsy was 29.1±43.0 mo after kt. however, 172 cases were biopsied in the 1st year posttransplant. the extent of c4d staining was graded as <10% (0), 10-50% (1), and ≥50% (2) of ptc, and the intensity was graded as none (0), light (1), and strong (2) staining. these findings demonstrate the significant discordance between detection of dsa and c4d, which is a relatively specific histological evidence of ab-mediated injury. this factor should be taken into account when clinical decisions for treatment of patients with either c4d or dsa positivity are made. the observed discrepancy could be partially due to the technique used for staining of the biopsy specimens, inability to detect anti-hla dsa with the available technology, or non-hla dsa. long term follow up data are needed to evaluate the impact of these markers on graft outcomes. introduction epithelial to mesenchymal transition (emt) is a potential mechanism of tissue fibrogenesis. in a previous study, we had reported that the early expression of emt markers was associated with the progression of renal grafts towards interstitial fibrosis and tubular atrophy (if/ta). here, we report the long-term follow-up of this cohort, paying a special attention to the evolution of graft function. patients and methods 83 patients engrafted with a kidney from a cadaveric (n=63) or a living (n=20) donor, and in whom sequential protocol biopsies had been performed at 3 and 12 months, were included. the phenotype of epithelial cells was studied at three months according to the expression of vimentin (an intermediate filament normally expressed by fibroblast-like cells) and to the cellular localization of β-catenin. grafts in which these two markers were abnormally expressed by more than 10% of tubular cells were considered as emt+ grafts. serum creatinine and creatinine clearance (estimated abstracts by gault and cockcroft index) were collected from 12 to 24 months post-transplant and compared according to the emt status of the graft. results multivariate analysis demonstrated that the early expression of emt markers was an independent risk factor of the progression of graft fibrosis between 3 and 12 months. more importantly, these early phenotypic changes were associated with a progressive and sustained deterioration of the graft function : emt+ patients had a statistically higher serum creatinine from twelve months after transplantation, and a significantly lower creatinine clearance from 18 months after transplantation (emt+ 49.4±4.5 ml/min vs emt-61.1±2.3 ml/min, p=0.01). the difference was persistent at 24 months. conclusion the expression of emt markers by tubular epithelial cells at an early time point post-transplant (three months) is highly suggestive of an ongoing fibrogenic process, and has repercussions on the long-term graft function. therefore, these epithelial phenotypic changes are relevant and promising biomarkers for an early detection of if/ta. we recently reported that 73% of transplant glomerulopathy (tg) has evidence of alloantibody-mediated injury in biopsies for cause. we found that 1/3 of tg is c4d+ab+ and 1/3 is c4d-ab+ suggesting that c4d staining is not sensitive enough to detect all biopsies with antibody-mediated injury. we aimed to develop a new laboratory test to detect biopsies with antibody-mediated rejection (abmr) which are missed by c4d. using affymetrix microarrays, we analyzed gene expression in 173 renal allograft biopsies for cause. we previously reported that both abmr and t cell-mediated rejection (tcmr) biopsies show increased expression of transcript sets associated with cytotoxic-t cells (cats) and gamma-interferon effects (grits) compared to biopsies without rejection (p<0.05). however, abmr biopsies were discriminated by a selective increased expression of 25 "endothelial cell-associated transcripts" (endats). these genes included established endothelial markers such as vwf, pecam1, sele, cd34, and cadherin 5, which are involved in endothelial-cell activation. hierarchical clustering of 82 biopsies with ab+ using endats identified a group of c4d-ab+ biopsies (n=20) clustered with c4d+ ab+ biopsies (n=18). thus 25% of biopsies with antibody (20 of 82) had increased endat-scores despite being negative for c4d. these c4d-ab+ biopsies with high endats, had higher scores for cats and grits, increased incidence/severity of tg, tubular atrophy/interstitial fibrosis, and worse future graft function (p<0.05), but similar incidence of tcmr or borderline lesions, in comparison to c4d-ab+ biopsies with no increase in endats. the c4d-ab+ cases with endothelial activation show extensive inflammation in the allograft, as measured by the gene sets, which is similar to c4d+ abmr. there are a significant number of cases with alloantibody and no c4d that show increased expression of endothelial genes. thus the transcriptomics detects deteriorating c4d-allografts with ongoing alloantibody mediated injury. we conclude that increased expression of endothelial genes provides a new feature of abmr, and can be used as a new diagnostic test to detect and treat c4d-abmr. probabilistic ( 001) . the bayesian network model was analyzed and, interestingly, cd86 was critically related to tg, suggesting a b-cell mediated process. tg was also predicted by upregulation of ccl2, ccl3, ccl5, cxcl9, il-8, il-10, and icam gene expression. ten percent of the samples were excluded randomly from the initial model, and subsequently used for cross validation. in the validation analysis, the model effectively predicted tg (auc of 0.92, 90% ppv) and sf (auc of 0.866, 83% ppv). this study provides a compelling and clinically relevant example of the combination of quantitative gene expression with probabilistic bayesian modeling to predict renal allograft pathology. potentially important molecular pathways associated with transplant glomerulopathy were also identified. the application of this integrated approach has broad implications in the field of transplant diagnostics and interpretation of large data sets. 1, 6, 12, 24, 36, 48 and 60 months respectively. the daily dose and blood levels of tac were significantly lower in tac/slr group compared to tac/mmf group. renal function is shown. despite lower prevelance of cai in tac/slr group long-term graft function and patient and graft survival are comparable between tac/mmf and tac/slr groups. objective: the aim of this study was to determine if ethinicity impacts graft outcomes in kidney transplant patients converted to sirolimus (srl) and either maintained on calcineurin inhibitors (ci) or mycophenolate (mmf) with steroids. methods: this was a retrospective analysis of all kidney transplants converted to srl and transplanted from 7/91 to 4/07. patients were divided into 4 groups: group 1: aas converted to srl + continued on ci; group 2: non-aas converted to srl + continued on ci; group 3: aas converted to srl + continued on mmf; group 4: non-aas converted to srl + continued on mmf. pediatrics and multiorgan transplants were excluded. results: a total of 257 patients were included (61% aa). demographics, baseline immunosuppression, and reason for srl conversion were similar between groups. table 1 displays characteristics and outcomes. patients converted to srl+ci regimens had higher rates of acute rejection before srl conversion (p<0.02), but equal rates after conversion. development of proteinuria was similar across groups. figure 1 displays the graft survival rates for each group. aa patients converted to srl+mmf tended to have poorer outcomes compared to aa patients converted to srl+ci. non-aa patients converted to srl+mmf tended to have better graft outcomes compared to non-aa patients coverted to srl+ci, although this did not reach statistical significance(p=0.186). conclusion: aas converted to srl may benefit from continued ci, while non-aas converted to srl appear to have better outcomes with mmf. further prospective studies are warranted to confirm these findings. aa srl+ci (n=113) there are no large registry studies evaluating the correlation of allograft failure for recipients of kidneys from the same deceased donor. we examined outcomes in such recipient pairs using data from the united states renal data system. methods: we studied the correlation of graft failure events within 19,461 pairs of same-donor recipients transplanted during 1995 through 2003. analyses were limited to patients with functioning grafts 3 months post-transplant (tx) and adjusted for known donor, recipient, and tx management factors. we estimated odds ratios to measure the increased risk for 1, 2, and 3-year graft failure and death-censored graft failure when the contralateral kidney had such an event. we also evaluated the effect of recipient pairs transplanted at the same center vs different centers. results: there is a strong correlation in outcomes for 2 recipients with the same donor (table) . the correlation was stronger within pairs transplanted at the same center than for those transplanted at different centers. differences in the correlation of graft failures within pairs transplanted at the same versus separate centers diminished over 2 and were absent by 3 years post-tx. results for death-censored allograft failure were similar. conclusion: unmeasured donor factors contribute significantly to the correlated graft failure outcomes in paired recipients of deceased donor kidneys and need further study. kidney tx outcomes in the first year may be affected by differences in management between transplant centers, more so than in subsequent years post-tx. 1 odds ratio of death-censored graft failure and graft failure in recipients of a donor pair, given that the outcome occurred in the recipient of the contralateral kidney, with both recipients being transplanted at the same (s) center or at different (d) centers. all odds ratios significant with p<0.001. the deterioration of kidney allograft function (dekaf) study is a nih-funded multicenter observational study of late allograft (ktx) loss. the study examines two cohorts: a "long-term cohort" (ltc) of prevalent ktx with scr < 2.0 mg/dl with deterioration of function (25% increase in scr or proteinuria) and a "prospective cohort" (pc) of incident ktx developing a persistent >25% increase in scr. we examined the pathologic features of the first renal biopsy (bx) obtained for new onset deterioration in each cohort. all bx were read and scored centrally using a modified banff schema. on average, bx were obtained at 6 (pc) and 75 (ltc) months post-tx. mean scr was similar, but more patients (pts) in ltc had proteinuria. moderate to severe interstitial fibrosis and tubular atrophy (ta) were more prevalent in ltc than pc (33 vs 13% ci score ≥2; 33 vs 9% for ta). the rate of vascular sclerosis >25% was similar (10.87% pc vs 15.46% ltc); however, hyaline arteriolar sclerosis >25% was more common in the ltc (30 vs 4.35%). interstitial inflammation (i) and tubulitis (t) scores were similar in both cohorts. however, more pts in ltc had peritubular capillary infiltrates (>5 cells) and evidence of tx glomerulopathy. while rates of interstitial inflammation and tubulitis sufficient to warrant diagnosis of acute rejection are similar in the prospective and long-term cohorts, long term cohort pts had more proteinuria, interstitial fibrosis, tubular atrophy, hyaline arteriolar sclerosis, and transplant glomerulopathy. analyses of histologic findings and renal outcome are ongoing. the term chronic allograft nephropathy (can) has been abolished by the last banff meeting report (am j transplant, 2007) and 2 categories have been introduced for chronic changes: chronic active t cell-mediated rejection and chronic active humoral rejection (cahr). aim of the study was to review all cases of can diagnosed in the last 4 years and to identify immunohistochemical markers of chronic rejection. a cohort of 79 cad pts with biopsy-proven can was analyzed. each case was reviewed and assigned into 3 groups according to banff 2005 criteria: chronic rejection (cr), chronic calcineurin toxicity (cnit) or chronic lesions not otherwise specified (nos). cd4+, cd8+, cd20+, cd68+ cells and c4d deposits were assessed by immunohistochemistry. twenty-eight pts were classified as cnit,34 as cr,19 of which were cahr, and 17 as nos. serum creatinine and 24h proteinuria at renal biopsy, extent of interstitial fibrosis and glomerulosclerosis were not significantly different among 3 groups (table1).the number of cd4 + cells was higher at ti level in cr compared to cnit (table1;*p=.05). cd8 + cells were higher at ti and g level in cr compared to cnit (table1;*p=.05). ti and g cd20 + cells were not different among the 3 groups (table1). the number of g cd68 + cells was increased in cr compared to cni and nos (table1;*p=.05). no significant difference in cd20, cd4, cd8, cd68 expression was found at ti and g level between c4d + and c4dcases of cr. cd68, cd8, cd4 but not cd20 expression at ti level correlated with ti fibrosis (r 2 =.105, .077, .156, respectively, p<.05) at the univariate analysis. only ti cd4 + cells independently correlated with fibrosis at multiple regression analysis. in conclusion, our data suggest that: morbid obesity limits access to kidney transplantation and predicts adverse transplant outcomes. there are limited data on the safety and efficacy of gastric bypass (gb) as a weight reduction therapy among transplant candidates and recipients. methods: we examined usrds registry data to identify medicare-insured kidney transplant candidates and recipients with billing claims for gb procedures. gb were categorized according to occurrence before listing, on the waitlist, or after transplant. we studied the clinical characteristics of gb-treated patients, and subsequent outcomes including progression from listing to transplant and 30-day mortality. usrds surveys bmi data at dialysis start, waitlist entry, transplant, and transplant anniversaries.we computed changes between most recently reported body mass index (bmi) values preceding and following gb, when available. results: we identified 72 transplant candidates treated with gb before listing, 29 who underwent gb on the waitlist, and 87 gb cases after transplant. patients treated with gb were most commonly female, white race, and without diabetic or hypertensive renal failure (table) . 30-day mortality after gb, calculable for listed and transplanted patients, was 3.4% and 3.4%, respectively. 1 transplant recipient experienced graft failure within 30 days of gb. of 29 patients treated with gb on the waitlist, 20 proceeded to transplant. post-gb weight loss was detected for 60% with gb pre-listing, 70% with gb on the waitlist, and 90.7% with gb after transplant. among patients listed for transplant in the same era and bmi >35 at first dialysis who were not treated with gb, 22% had lost weight between dialysis start and listing. conclusions: gb has been performed in small numbers of kidney transplant candidates and recipients, and is followed by weight-loss in the majority of cases. peri-operative mortality is comparable to reports in patients without kidney disease. gb warrants prospective study as a strategy for reducing complications of obesity in esrd. introduction: the present study investigated the incidence of posttransplant diabetes mellitus (ptdm) and calculated the risk of developing ptdm under a tacrolimus and mycophenolate mofetil (mmf)-based immunosuppression based on clinical characteristics, tacrolimus-pharmacokinetics, and genetic polymorphisms related to tacrolimus-pharmacokinetics, cytokines and diabetes mellitus. methods: seventy-one non-diabetic adult kidney recipients (male 37, female 34) were studied. patients with continuous high plasma glucose levels, over 6.5mg/dl of hemoglobin a1c, or requiring insulin and/or oral anti-diabetic agents for more than 3 months after transplantation at 1-year after transplantation were diagnosed as having ptdm. fifteen genomic polymorphisms were assessed. results: one year after transplantation, 21 recipients (29.6%) developed ptdm. positive risk factors were age (p=0.028) and body mass index (p=0.048). there were no significant differences in acute rejection rate, total steroid doses, tacrolimuspharmacokinetics or its related to genetic polymorphisms between the two groups. the frequencies of ptdm were significantly higher in patients with adiponectin t45g tt genotype than in those with the g allele (p=0.034), and in patients with glucocorticoid receptor (nr3c1) bcl i cc genotype than in those with the g allele (p=0.004). conclusions: the incidence of ptdm at 1-yr after transplantation was 29.3% in our cohort. elder or obese patients were risky for the development of ptdm. the presence of the adiponectin t45g tt or nr3c1 bcl i cc genotype may also be risk factors for ptdm, suggesting that insulin and glucocorticoid sensitivity-related genes are associated with the development of ptdm. analysis of these genotypes is a possible method of predicting a patient's risk for developing ptdm and would be a valuable asset in selecting appropriate immunosuppressive regimens for individuals. pharmacokinetics persistent hyperparathyroidism (hpt) with hypercalcemia is common after renal transplantation. studies have shown that treatment with cinacalcet corrects hypercalcemia and lowers pth levels in these patients. so far cinacalcet's steadystate pharmacokinetics and their correlation with pharmacodynamics (pk/pd) have only been studied in hemodialysis patients, but not in renal transplant recipients with persistent hpt. to gain further insight into cinacalcet's effects on calcium-phosphate homeostasis, we determined its steady-state pharmacokinetics and pharmacodynamic effects in these patients. in a prospective, single center, open label study we examined the effect of a 2-week treatment with 30 mg and subsequent 2-week treatment with 60 mg cinacalcet daily on calcium-phosphate homeostasis over 24 hours and determined the steady-state pharmacokinetics of cinacalcet in stable renal allograft recipients. the urinary calcium excretion was determined in timed urine samples. median auc 0-24 was 784.8 ng*h/ml and c max was 68.5 ng/ml for 60 mg cinacalcet which is higher, and oral clearance (cl/f) was 76.9 l/h which is lower in renal transplant recipients compared to previously published data of hemodialysis patients (50 mg cinacalcet auc 0-24 179, c max 17.2, cl/f 279). we also observed a non-proportional increase of auc 0-24 after doubling of the cinacalcet dose. the once daily administration of cinacalcet dose-dependently reduced ipth and serum calcium. cinacalcet and parathyroid hormone (pth) concentrations showed an inverse correlation and were fitted to a simple emax model (e max =80 % reduction vs. baseline, ec 50 =13 ng/ml). the 8-hour fractional urinary excretion of calcium was increased after 60 mg cinacalcet (baseline 0.85±0.17 %, 30 mg 1.53±0.35 %, 60 mg 1.92±0.37 %). renal function remained stable. cinacalcet's higher and non-proportional increase of auc 0-24 in transplant recipients compared to hemodialysis patients evokes the possibility of a pharmacokinetic interaction with concomitant cyclosporine treatment. cinacalcet effectively corrected the biochemical abnormalities of persistent hpt. the transient calciuria could potentially favor nephrocalcinosis and reduce bone mineral density, suggesting that higher doses of cinacalcet need to be used with caution in renal transplant recipients with severe persistent hyperparathyroidism. screening for proteinuria in the kidney transplant clinic. bryce a. kiberd, 1 romuald panek. 1 1 dalhousie university, halifax, ns, canada. proteinuria is a predictor of progression in kidney disease. it is not clear whether measuring albuminuria will have greater clinical utility over measurement by dipstick or total proteinuria in kidney transplant recipients. there has also been a trend away from using 24 hour collections to using spot urine albumin/creatinine (ac) and protein/creatinine (pc) ratios. we compare the prevalence of proteinuria estimated by dipstick, ac and pc in prevalent patients (>6 months post transplant) in the kidney transplant clinic. significant albuminuria defined as ≥30 mg/g was present in 52% (211/403). albuminuria was seen in 29% (70/245) with negative and 67% (34/51) with trace dipstick proteinuria. significant predictors of albuminuria in a mulitvariate logistic analysis were egfr (or 0.977 per ml/min/1.73m 2 , 95% ci 0.965-0.989 p=0.001), diastolic bp (or 1.028 per mmhg, 95% ci 1.008-1.048 p=0.007), and mmf use (or 0.50, 95% ci 0.32-0.77 p=0.002). macroalbuminuria (ac>299 mg/g) was seen in 17.4% (70/403) and significant predictors in a mulivariate logistic analysis were lower egfr and higher systolic bp. sirolimus use was associated with more macroalbuminuria and mmf use with less macroalbuminuria. in a subset of patients followed for >2 years prior gfr loss was considerably greater (p=0.021) in patients with albuminuria (-0.88 ml/min/1.73m 2 /year) compared to those without (-0.15 ml/min/1.73m 2 /year). however other measures of proteinuria were also significantly (p for trend) associated with prior gfr loss (ml/min/1.73m 2 /year) as shown in the <0.13 (n=185) 0.13-0.49 (n=109) >0.50 (n=80) ∆ egfr/year -0.33 -0.26 -1.40 0.02 * a pc cut point of 0.129 g/g had a sensitivity and specificity for albuminuria > 30 mg/g of 87% and 88% respectively (c=0.92, 95% ci 0.89-0.95), and a pc cut point of 0.49 g/g had a sensitivity and specificity for macroalbuminuria >300 mg/g of 100% and 94% respectively (c=0.98, 95% ci 0.97-0.99). ac may be more sensitive and therefore have more clinical utility than other measures of proteinuria for progression. however prospective follow up of renal function change and cv outcomes is required. serum creatinine is a crude marker of gfr in renal transplant recipients and changes in gfr are frequently not accompanied by commensurate changes in serum creatinine concentration. serum cystatin c and estimates of gfr (egfr) based on cystatin c have been shown to be more accurate than serum creatinine and creatinine-based egfr in renal transplant recipients. the purpose of this study was to determine whether the filler, lebricon and rule cystatin c-based egfr equations were better able to detect changes in true gfr than the mdrd and cockcroft gault creatinine-based egfr equations. we performed two measures of 99m tc-dtpa gfr, serum creatinine and serum cystatin c on each of 183 stable renal transplant recipients at least 6 months apart. we calculated and compared the percent annual change in the measured gfr and the estimated gfr using the various gfr estimation equations. we also determined the sensitivity, specificity, positive predictive value and negative predictive value of each prediction equation for the detection of decline in measured gfr. results are presented below: the cystatin c and creatinine-based egfr equations all demonstrated poor sensitivity and diagnostic performance to detect a decline in gfr. novel equations derived and validated in the transplant population are needed to accurately assess kidney function over time. background: hypercalcemia, hypophosphatemia and renal phosphate wasting are common after kidney transplantation and are related to persistent hyperparathyroidism and hyperphosphatoninism. animal data suggest that these alterations in mineral metabolism may contribute to nephrocalcinosis and progressive graft dysfunction. supporting clinical data are limited. aim: to test the hypothesis that nephrocalcinosis is highly prevalent in the early posttransplant period and is related to a disturbed mineral metabolism. methods: biomarkers of mineral metabolism (including albumin-corrected serum calcium [ca c ], serum phosphorus [p], biointact pth, calcidiol, calcitriol and alkaline phosphatase) and renal calcium and phosphorus excretion parameters were prospectively assessed in 201 renal transplant recipients (62% male, mean age 55 ± 14 yrs) at the time of their 3-month protocol biopsy. these protocol biopsies were screened for the presence of microcalcifications. intratubular, interstitial and/or cytoplasmatic microcalcifications were observed in 30.4% of biopsies. calcifications were more prevalent in recipients of a living related donor as compared to cadaveric donor. high serum ca c levels, high serum pth levels, a high urinary ca×p product and high fractional excretion of p and low serum p levels were significantly associated with renal microcalcifications (see figure below). microcalcifications were not related to the fractional excretion of ca, use of diuretics, immunosuppressive regimen, serum alkaline phosphatase level and history of delayed graft function. the extent of microcalcifications correlated significantly with the severity of mineral metabolism disturbances. conclusion: our data demonstrate that nephrocalcinosis is highly prevalent in the early posttransplant period and suggest that a disordered mineral metabolism is implicated in its pathogenesis. polymorphism in abcb1, the gene encoding for p-glycoprotein, predicts recovery of graft function early after kidney transplantation. the pharmacokinetics of cyclosporine (csa) is characterized by wide inter-individual variability. this might be particularly relevant in the early post-transplant period, due to the detrimental effects of the drug on the kidney. p-glycoprotein (p-gp), the product of the abcb1 gene, plays a key role in the distribution of csa at cellular level. single nucleotide polymorphisms (snps) of abcb1 might potentially influence the response of patients to csa. in particular, the snp in position 3435 of the exon 26, despite its silent nature, has been recently associated with altered specificity for its ligand, such as csa (kimchi-sarfaty et al, science 2007, 315:525) . whether p-gp pharmacogenetics would help to guide csa treatment early post-transplant remains ill defined. we sought to evaluate the effects of the snps in the exon 26 on the rate of recovery of graft function, as estimated gfr early postoperatively, in 150 kidney transplant patients given csa as part of their immunosuppressive regimen. the frequency of dgf (as need for post-operative dialysis) among the different abcb1 genotypes was also estimated. of the 150 kidney transplant recipients, 35% had the abcb1 wild type (c/c) genotype in exon 26, 40% were heterozygous (c/t) and 25% were homozygous (t/t) for the polymorphic variant in position 3435. gfr values were significantly lower in patients carrying one of the two mutant alleles than in the wild type ( figure) . the frequency of dgf was 15%, 28% and 30% in patients with the cc, ct and tt genotypes, respectively. these findings demonstrate that in patients carrying the ct or tt mutant alleles in exon 26 of the abcb1 gene and given csa, the recovery of graft function is less prompt, and the risk to develop dgf higher than in wild-type cc genotype. pre-transplant screening for abcb1 polymorphism would help to identify patients who may safely receive csa early post kidney transplantation. cigarette cigarette smoking has shown to reduce graft and patient survival in renal transplant patients. however, whether it could directly produce allograft disfunction has not been investigated. the aim of this study was to assess the smoking influence on renal graft function. we studied a cohort of 827 adult renal transplant patients, transplanted from jan/96 to dec/05 and followed until dec/06. smoking habits were recordered at the time of transplant (never, former, current smoker). during summer of 2007, a telephonical survey allowed us to obtain complete information about smoking habits in 642 patients (aged 47.6 ±13, 65% male): status (never, former, current), years of habit, years of quit, and number of cigarette smoked per day. number of "pack-years" was calculated. renal function was measure by inverse serum creatinine at 3 rd month and then annually. time to decline a thirty percent in inverse serum creatinine was registered. patients were divided in two groups: those who always smoked during all transplant period (smokers, n=94) and those who did not (n=546 renal insufficiency occurs frequently after extrarenal transplantation as a result of acute tubular necrosis at transplantation, high blood pressure, and cni toxicity. we performed 56 renal biopsies in 54 patients after heart (4), lung (20) , liver (22), bone marrow (9), and cornea (1) transplantation since 2000. the time from transplantation to biopsy was 49±53 months in general and was longest after liver (60±61 months) and shortest after bone marrow transplantation (37±43 months). the histologic changes were: tubular atrophy/interstitial fibrosis of ≥20% in 52% of biopsies (heart biopsies 75%, lung 70%, liver 46%, bone marrow 22%); acute tubular changes in 54% (heart 25%, lung 65%, liver 46%, bone marrow 56%); arteriolar hyalinosis in 41% (heart 75%, lung 65%, liver 23%, bone marrow 22%); arterionephrosclerosis in 41% (heart 50%, lung 50%, liver 36%, bone marrow 22%); glomerular sclerosis of ≥20% in 21% (heart 50%, lung 25%, liver 18%, bone marrow 22%); glomerulonephritis in 13% (heart 25%, liver 18%, bone marrow 22%; that means iga-nephropathy after heart and liver, immune complex nephritis twice after liver, mpgn after liver, and membranous glomerulonephritis and minimal changes after bone marrow transplantation); thrombotic microangiopathy in 7% (lung 10%, liver 5%, bone marrow 11%); finally one case of polyoma nephritis after lung transplantation. among 1702 heart and lung transplantations, 13 patients needed kidney transplantation (0.8%) after 96±40 months; and among 2221 liver transplantations, 11 needed kidney transplantation (0.5%) after 112±65 months (sign. later, p=001). conclusion: as expected, most histologic changes were those of cni toxicity and hypertension. surprising is the high number of glomerulonephritis under immunosuppression. thrombotic microangiopathy without the typical clinical signs were interpreted as cni-related toxicity and seems to occur more often after extrarenal than after renal transplantation. patients with heart and lung transplantation reach end-stage renal failure more often and earlier than patients with liver transplantation. the context: living kidney transplantation, a superior therapy to deceased donor kidney transplantation, is underutilized. states have enacted legislation and the federal government has launched initiatives to compensate living organ donors, but the effect of policy on improving living kidney donation rates in the united states is unknown. objective: to determine whether public policies are associated with changes in living kidney donation rates in the continental u.s. design, setting, and study subjects: series of cross-sectional analyses using records of state legislatures in 48 continental states and living kidney donation rates from the united network for organ sharing. main outcome measures: living kidney donation rate during each year from 1988-2006 and change in donation rates before and after legislation enactment in each state and launch of federal initiatives. results: from january 1990 through december 2005, 28 states enacted legislation for living donors (24 mandating paid leave, 8 tax deductions, 3 unpaid leave, 2 encouraging paid leave). few states (n=5) enacted legislation prior to 1999. there was a steady increase in the mean living kidney donation rate in the continental u.s during the study period (mean (standard deviation) annual increase in donations 1.5 (0.04) donations per 1,000,000 population). in analyses accounting for length of time state legislation had been enacted, the types of legislation enacted, and the incidence and prevalence of esrd in each state, there was a slightly (but not statistically significantly) greater average annual increase in donations after compared to before state legislation enactment (annual increase in donations per 1,000,000 population [95% confidence interval ( introduction: accurate and precise renal function assessment is essential in the evaluation of prospective kidney donors. while direct measurement of gfr is the "gold standard", it is not widely available. moreover, creatinine (scr)-based estimation equations are suboptimal to assess kidney function in this setting. ct scans are increasingly being used to study renovascular anatomy in donors and has replaced angiographic exams in many institutions. 3d imaging reconstruction allows for kidney volumes (kv) measurements which have been shown to highly correlate with measured gfr in this population. thus, the purpose of this study was to develop a model to estimate measured gfr that not only incorporates scr and demographic data but also kv as measured by 3d ct scans. methods: 244 individuals who underwent donor evaluation were identified. an automated segmentation algorithm was used to measure renal parenchymal volume from preoperative abdominal cts. patient demographics and scr values were obtained from the medical records. gfr (normalized for bsa) was measured by i 125 iothalamate renal clearances (igfr). an analysis of covariance model was created to correlate measured igfr with kv, patient age, sex, race, weight, height and scr. pearson's correlation coefficient was calculated for each variable. results: kv (p<0.001), age (p<0.001), scr (p<0.001) and weight (p<0.001) significantly correlated with igfr. sex (0.60), race (0.90) and height (0.76) were not statistically significant. the new fitted regression model is: kv-egfr (ml/min/1.73 m 2 ) = 70.77 -(0.444*age) + (0.366*weight) + (0.200*volume) -(37.317*scr). we then compared the performance of the kv-egfr model to the re-expressed mdrd equation using calibrated scr assay. the r 2 was 0.61 vs 0.50, respectively; signed median % difference was +1.8% vs -12.7%, respectively (% difference b/w estimated gfr and igfr); and accuracy within 30% (% of estimated gfr values that fall within 30% of igfr) of 96.3% vs 89.8%, respectively. finally, the kv-egfr model was closer to igfr (in absolute values) than the mdrd eq. in 177/244 (70.5%) cases vs 72/244 (29.5%) cases, respectively. conclusions: kidney volumes highly correlate with igfr and the proposed gfr estimation model outperforms the mdrd equation in potential living kidney donors. the kv-egfr model could be used to estimate donor gfr in lieu of i 125 iothalamate gfr which is less clinically available. for donor selection, current reports identify unsuspected renal pathology by time 0-biopsy. aim: to explore whether the findings at time 0-renal biopsy (bx) correlates with pre-donation clinical data including renal function. methods: kt databases from 2 institutions were reviewed. time 0-renal bx are routinely performed from the upper pole during back-table and evaluated by 2 nephropathologist for interstitial fibrosis (if), tubular atrophy (ta), arteriolar hyalinosis (ah), mesangial increase (mi), and glomerulosclerosis (gs). pre-donation data gathered from the donors were demography, body weight, bmi, systolic/diastolic bp, scr, proteinuria, and egfr by levey equation clinical data is summarized in the table. and gs showed no correlation. multivariate analysis failed to sustain the significant associations found on bivariate analysis, most likely due to a low event/parameter relation. conclusions: a significant correlation was observed between time 0-bx findings and clinical pre-donation parameters. whether these histological findings at the time of kidney donation represent a higher burden/risk for the remaining kidney ought to be evaluated during follow-up. in an era where living donation is increasing, we should advise a closer surveillance of these donors in order to modify risk factors that participate in kidney damage progression. predictors of poor early graft function following laparoscopic donor nephrectomy (ldn). matthew cooper, 1 abdolreza haririan, 1 stephen jacobs, 1 michael phelan, 1 benjamin philosophe, 1 stephen bartlett, 1 joseph nogueira. 1 1 dept of surgery, urology, and medicine, university of maryland, baltimore, md. ldn has become the standard of care in many transplant centers. poor early graft function remains an important complication. we conducted a retrospective study to evaluate the risk factors for slow or delayed graft function following ldn methods: donor and recipient records from the first 1000 ldn were reviewed (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) . results: slow graft function (sgf) was defined as cr>3.0mg/dl at pod5 and dgf as the need for dialysis within the first week following transplantation. donor variables examined included age, sex, race, bmi, egfr, and number of renal arteries. recipient variables included age, sex, race, bmi, prior tx, pre-tx dm, history of smoking and drug usage. additional variables evaluated included degree of relationship (lurt), hla mismatch, antilymphocyte induction, de novo cni usage, r v. l nephrectomy, wit, total or time, and performance of simultaneous deceased donor pancreas tx (splk). univariate analysis was performed with significance defined as p<0.05. significant variables were then included in the multivariate analysis. background: a kidney exchange program is the logistic solution for patients with positive cross match (x + ) or abo incompatible donors. a major problem in all kidney transplantation programs is the sensitized recipient. we analyzed the success rate for immunized recipients in the dutch kidney exchange program. methods: from january 2004 till december 2007 242 donor-recipient pairs were registered. there were 117 couples in the x + group while 125 pairs were abo incompatible. in the x + group the median pra was 46% (2-100%). to create new combinations a match program was run 16 times (every 3 months) with a median of 46 (16-66) participating couples. allocation criteria included bloodtype (first identical, then compatible), hla match probability within the actual exchange donor pool to ensure that highly sensitized recipients have the best chance to receive a kidney, and the waittime on dialysis. cross matches between new donor and recipient were performed centrally in our reference laboratory with cdc-tests. results: after 16 match runs, we found matching couples for 83/117 (71%) x + pairs, for 21/87 (24%) abo incompatible pairs with o recipients and for 29/38 (76%) abo incompatible pairs with non-o recipients. median pra of the 83 recipients in the x + group was 42% (2-100%). after 3 match runs chances for success became small. the overall success rate for abo incompatible and x + pairs in the dutch kidney exchange program after 4 years is 55%. however, the success rate for immunized patients in the x + group is significantly higher (71%) as our match program gives priority to those recipients with the smallest chance of finding a compatible donor in each match run. thus our kidney exchange program is especially suited for immunized patients. although paired kidney donation (pkd) program is an established method to overcome incompatibilities between kidney donor-recipient pairs (drp), significant proportion of the incompatible drp participating in such program could remain unmatched. domino-kidney transplantation (kt) in which altruistic living non-directed donor kidney (lndk) is offered to a pool of incompatible drp, and is used to initiate a chain of pkd transplants, could provide more opportunities of kidney transplantation to drp in pkd program. we introduce our experience of multicenter domino kt for the last 7 years sixteen hospitals participated in the domino-kidney transplantation between february, 2001 and july, 2007. 181 domino-kidney transplants were performed with 69 domino-kt chains initiated by altruistic lndk. 2-pair chains were 43, 3-pair chains 14, 4-pair chains 9, 5-pair chains 2, and 7-pair chains 1. the development of a multi-regional kidney paired donation program. using an optimization matching algorithm the new england program for kidney exchange (nepke) efficiently matches incompatible donor/recipient pairs. in 2006, the mid-atlantic paired exchange program (mapep) and other individual centers began sharing data with nepke. this is a report of the success of two regional programs working together to increase the probability of participants in both programs finding a compatible match. methods: incompatible pairs and non-directed donors (ndd) are referred to nepke through transplant centers. donor and recipient abo, hla and recipient hla antibody screening are entered into the computer database. utilizing the optimization program, searches for compatible matches are conducted every 30 to 45 days. the program identifies potential 2 and 3-way matches, ndd chains, and list exchange chains. following the determination of compatibility nepke notifies transplant centers involved of the potential match and centers accept or decline the offer. transplant centers notify their pairs and preliminary crossmatches are performed. a conference call is scheduled to coordinate simultaneous donor nephrectomies and recipient transplants. surgeons speak prior to incision to ensure simultaneous donation. results: from july 2005 to december 2007, 186 pairs and 10 ndds have entered nepke. during this time frame over one thousand possible matches were identified, with the majority of these matches involving the same pairs in multiple matches. after "optimizing" and eliminating multiple matches 13 two-way exchanges; 15 three-way exchanges; 7 three-way list chain exchanges; and 25 ndd chain matches were offered to transplant centers as possible matches. most common reason offers were declined include: positive crossmatch (21.6%); donor factors (20%), and recipient inactive or transplanted (20%). four offers occurred in mapep alone pairs, 47 in nepke, and 14 offers were cross-regional. three matches are pending for 9 additional transplants. one previous and one pending transplant are the result of cross-regional exchanges. five transplants performed and 6 pending involve ndd chains. one pending match involves a list exchange chain. conclusion: using a computerized optimization algorithm to match 2 and 3 way exchanges, ndd and list exchange chains has lead to a substantial increase in the number of kpd matches and transplants performed. cross-regional coordination is feasible and expands the number of transplants performed beyond the ability of individual exchange programs. as living donors become an increasingly important source of life-saving organs, there is growing concern about the lack of comprehensive research on donor outcomes. in addition to possible long term consequences, there is a risk that donors will experience complications during or following surgery. of the 13,000 living kidney donors in 2005-2006, none died during surgery, and 0.5% (n=68) needed blood transfusions during surgery. in the six weeks following donation, 3.9% (n=512) had at least one serious adverse event (sae): 1.7% (n=220) needed readmission following initial discharge, 0.6% (n=72) needed an interventional procedure, 0.5% (n=61) needed re-operation, 0.3% (n=41) had vascular complications, and 2.1% (n=274) had other complications. when all saes were considered in combination, the rate was 3.9%. because over 50% of ldr forms were submitted by transplant centers fewer than 6 weeks post-donation, all complication rates should be considered minimum estimates. one donor was reported to have died from donation-related causes within 6 weeks of donation. the number of living donor kidney transplants performed by a transplant center in 2005 -2006 ranged from 1 to 362 transplants. additionally, the risk of donor complications is not equal across transplant centers. for example, there was a significant correlation between the number of living donor kidney transplants performed at a transplant center and the percentage of that center's patients who were readmitted within 6 weeks of donation, with greater donor volume associated with a lower rate of readmission. living kidney donation is relatively safe, but prospective donors should be made aware that there is a non-trivial risk (3.9%) of short-term complications post-donation. as with many other major surgical procedures, complication rates are lower, on average, at institutions that perform a larger number of these procedures. we sought to determine if intensive screening improves detection of polyomaviral reactivation in asymptomatic patients and pre-emptive stepwise modification can improve outcome of polyomaviral nephropathy (pvn). methods:this is a prospective single center study. we randomly assigned de novo kt (cluster randomization) to intensive screening (is:n=648) and routine care (rc: n=260) for the detection of decoy cells. is was initiated at week-4 of kt and rc at the time of increase in serum creatinine. this was complemented with urine and blood nucleic acid testing. all patients had biopsies performed for detection of polomya nephritis (pvn). both groups were treated with pre-specified stepwise modification of it based on cell cytology and viremia (step 1: decrease dose of cellcept by 50%, step 2: decrease dose of tacrolimus by 50%, or switch to sirolimus therapy, step 3: discontinue cellcept). primary outcome included persistence of decoy cells/viremia following each step in modification of it every three months and secondary outcomes included acute rejection, graft function and graft loss. results: polyomaviral reactivation developed in 11.7% in is group and 40% had pvn without changes in serum creatinine. the estimated cumulative rate of primary outcome in the is versus rc groups, 3-months (51% vs. 79%) relative risk (rr), 0.47;95%ci,0.20-1.08;p=0.051); 6-months (20%vs.52%) (rr=0.67; 95% ci,0.38-1.20; p=0.009); and 12-months (2%vs.33%) (rr=0.88; 95% ci, 0.41-1.43; p=0.003). secondary outcomes: despite similar degrees of step-wise is modification, rate of acute rejection non-significant (p=0.25), but 25% of patients in rc loss the graft vs no graft loss in is group. patients who continue to remain on tacrolimus vs. those who were switched to sirolimus therapy had persistent viremia (or=10.25; 95%ci, 1.2-92.0; p=0.003). conclusion: is for poloymaviral reactivation allows early detection of pvn in the presence of stable graft function. stepwise modification in it resulted in early resolution of decoy cells and viremia in both groups, albeit slowly in rc group, and it did not prevent the graft loss in rc group. background: dna sequencing of the bk viral (bkv) genome non-coding control region (nccr) from individual patient isolates demonstrate divergent sequence and alterations in the arrangement of modularly conserved sequence blocks (p-q-r-s) ranging in size from 39 to 68 base pairs. aim: primary aim to molecularly clone and analyze patient-derived bk virus nccr sequence variants. our secondary aim is to determine if reporter gene constructs of patient-derived nccr variants differ in their promoter activity upon transfection into a mammalian cell line (vero) and a human primary tubular epithelial cell line. methods: bkv dna was amplified and sequenced from blood and urine samples of 18 renal transplant recipients. via sequence alignment, unique nccrs were determined. these sequences were pcr amplified and cloned into reporter plasmids containing the renilla luciferase gene. promoter activity was measured via luminometer 24 hours after transfection into vero cells and human tubular epithelial cells. results: variation in naturally occurring bkv nccr promoter regions exist as single basepair insertions and deletions, and insertions or deletions of partial sequence blocks (p-q-r-s). single basepair substitutions were most commonly seen (46% of analyzed samples). promoter activity within vero cells ranged from 188% to 39% as compared to nccr activity of an archetypal strain (wwb). low promoter activity (<50%) was seen in isolates with duplications of the p block and large deletions of the r block. conclusion: these sequence blocks are rich in regulatory elements and control the expression of both bkv structural and regulatory genes. variation in these cisacting eukaryotic transcriptional promoter binding sites corresponds to differential promoter activity in naturally occurring bkv isolates. current plans include repeating the promoter activity studies in human primary tubular epithelial cells. humoral and cellular immunity to polyomavirus bk large t and vp1 antigens after pediatric kidney transplantation. polyomavirus bk-associated nephropathy (bkvn) has emerged as a cause of graft failure after kidney transplantation (ktx). in a cohort of 37 pediatric renal recipients undergoing prospective three-monthly monitoring for bk by blood and urine q-pcr, we evaluated antibody response, measured by enzyme immunoassay using bk vlp, and cellular immune response, reported as frequency of ifnγ-secreting cells in a elispot assay after 9-day stimulation with bkv large t (lt) and vp1 peptides. we could not observe any influence of recipient pre-transplant bkv-specific igg or t-cell levels, which were generally low, on bkv infection after allografting. after transplantation, both specific igg levels, and frequency of bkv-specific t cells increased according to the degree of viral exposure. in detail, bkv-seropositive patients who never reactivate the virus (group1, n=10) did not show significant increase in igg levels (from a median od of 0.37 at month +1 to 0.39 at the end of follow-up), while patients with urinary shedding alone (group 2, n=14) or with viremia (group 3, n=13) increased from a median od of 0.3 to 1.2 (p=0.09), and 0.26 to 2.8 (p>0.0005). in the case of cellular immunity, vp1-specific t-cells increased in the three groups. conversely, lt-specific t-cells, which were high (median 71 sfu/10 5 cells) and remained unchanged throughout the follow-up period in group 1 patients, had a significant increase in recipients belonging to both group 2 and 3. interestingly, patients with urinary shedding who do not progress to viremia show a median 3-fold increase in lt-specific t-cell levels at peak viruria, compared to no increase observed in patients who develop viremia. the latter group mount a significant response to lt only after therapeutic reduction of immunosuppression. at peak viruria, viremic patients already show a 8-fold rise in specific igg compared to the 1.5 increase observed in group 2 recipients. our data suggest that inability to reach protective levels of bkv lt-directed t cells, rather than specific igg, predispose ktx recipients to bkv replication. introduction:the antibody response to human bkv virus (bkv) is incompletely characterized. antibody responses to the vp-1 protein have been detected in kidney transplant patients, but it is not known if these have virus neutralizing activity. methods:recombinant bk, jc, and sv40 virus like particles were used to produce a panel of 20 monoclonal antibodies. these antibodies were characterized for isotype and for ability to bind the respective antigens in elisa assays. to test neutralizing activity, bkv gardner strain (atcc# vr837) viral particles were incubated with the corresponding antibodies for 2 hours at 37 degrees c, and used to infect wi 38 cells. bkv infection was monitored by quantitative real time pcr using primers directed against the vp-1 gene. neutralizing activity was defined as greater than 95% inhibition of viral yield. results:the monoclonal antibodies were of the igg 2a or igg 2b class with the exception of one igg 1 and one igm antibody. all 12 anti-bkv monoclonal antibodies bound to bkv capsids in-vitro in elisa assays. this binding affinity was species specific, as only 1 antibody showed weak binding activity to jcv and sv40 capsids. neutralization of infectious bk virus was shown for 10/12 antibodies. denaturation of capsid proteins indicated that the monoclonal antibodies recognized primarily conformational epitopes, with only monoclonal antibody appearing to have a linear component. paucity of linear epitopes was further suggested by lack of reactivity of sera from bkv seropositive subjects in elisa assays based on genotype-specific short peptide sequences derived from the bkv vp-1 loop region. four monoclonal antibodies each generated from jcv capsids and sv40 capsids did not show any bkv neutralizing activity. conclusions: bkv vp-1 protein capsids contain species specific conformational epitopes which can elicit virus neutralizing and non-neutralizing antibody responses. measurement of these antibodies in renal transplant recipients may have diagnostic and prognostic applications. bkv specific monoclonal antibodies deserve further study as potential therapy of acute infections in the viremic phase. impact of immunosuppression reduction in bk viremic patients: 3 year follow-up. s. kuppachi, 1 a. guasch, 1 c. p. larsen, 1 k. e. kokko. 1 1 transplant center, emory university, atlanta, ga. background: development of bk nephropathy is a risk factor for allograft loss. bk viremia (bkv) precedes the development of bk nephropathy. it has been reported with 1-year follow-up that reduction of immunosuppression leads to control of bkv. here, we report our 3-year follow-up experience of bkv patients that were identified by a prospective screening protocol and managed by sequential reduction of immunosuppression. methods: all kidney or kidney-pancreas transplant recipients at emory university between 5/03-5/04 were screened prospectively for bkv by real time pcr during follow-up visits (1-6, 9, 12, 24, 36) . patients with a bk viral load of greater than 10,000 copies/ml blood received a kidney biopsy to screen for bk virus nephropathy by immunohistochemistry. bkv without nephropathy resulted in reduced immunosuppression by a 50% reduction in mycophenolate dose. bkv with nephropathy resulted in discontinuation of mycophenolate. all identified bk patients were monitored every 2-4 weeks until viral load was below 10,000 copies/ml. immunosuppression was further reduced if viral loads failed to decrease. results: 135 recipients were followed over a 36-month period. 24 patients had received simultaneous kidney and pancreas, 2 a liver and kidney and the rest kidney alone. 27 of 135 (20%) patients developed bkv within a year from time of transplantation. average time to diagnosis of bkv was 4.7 months. average dose of mycophenolate at 3 years was 1.3 g/d in the bkv negative population as compared to 0.56 g/d in the bkv positive population. average 12 hour trough blood level of tacrolimus at 3 years was 9.1 ng/ml in the bkv negative population as compared to 7.4 ng/ml in the bkv positive population. survival rates for both patient and organ were comparable at 3 years in bk viremic vs bk negative patients (100% vs 92%) and (88% vs 83%) respectively. while bk viremia is a historic risk factor for organ loss, prospective monitoring and reduction of immunosuppression is associated with comparable 3 year patient and organ survival to patients that never develop bk viremia. background: there are currently no bk virus (bkv) specific therapies available for clinical use. this study evaluates viral large t antigen as a potential target for drug development, since (a) this is a key molecule that participates in several different stages of viral replication, (b) has no homologous human protein, and (c) offers multiple functional domains for chemical binding, particularly the atp binding site, the dna binding site, the hexamerization surfaces, and the hinge region. methods: virtual screening and protein modeling techniques were applied to bkv large t antigen using a model developed from the known crystal structure of sv40 large t antigen. two different structural states of large t antigen (monomer and dimer structure) in three different states (with the nucleotide pocket empty, with bound adp and bound atp), were evaluated for a total of 6 large t antigen receptor conformations. results: a computational solvent mapping analysis of small molecular probes allowed identification of multiple functional sites, which represent potential drug binding pockets on the large t antigen molecule. it was possible to classify 1200 molecular conformations centered on the atp binding site, hexamerization surface and hinge region of the viral protein. we docked 1209 medium sized fragments (<100 da) to confirm the results obtained by computational solvent mapping, and further characterize chemical properties of the atp binding site. in another approach, known chemical structures of hsp90 and rho-kinase inhibitors were used to search compound databases and obtain a subset of 8294 compounds (mean size of 350 da) capable of docking large t antigen. cross-referencing the top solutions obtained by energy ranking, we were able to identify 50 compounds that bind large t antigen in all 6 conformational states. a subset of 5 compounds simultaneously binds the atp binding site, hexamerization surface and hinge region of bkv large t antigen. conclusions: virtual screening and three dimensional homology modeling technology has allowed us to identify compounds that can bind multiple sites on the large t antigen. these compounds are predicted to preferentially inhibit viral replication without the toxicity expected from simultaneous inhibition of host cell kinases. bk virus (bkv), a human polyomavirus, causes bkv nephritis, which often leads to graft loss after renal transplantation. currently, the only efficient therapy against bkv nephritis appears to be a reduction/change of immunosuppressive agents, and this may increase the inherent risk of rejection. since human renal proximal tubular epithelial cells (hrptec) represent a main natural target of bkv nephropathy, the analysis of bkv infection of hrptec is likely to provide necessary additional insight into bkv biology and contribute to the development of strategies for treatment of bkv nephritis. here we report the ability of 3-hydroxy-3-methyl-glutaryl coenzyme a (hmg-coa) reductase inhibitor pravastatin, which is routinely used to treat hypercholesterolemia, to repress bk virus entry pathways in hrptec and, correspondently, prevent bkv infection. the percentage of hrptec infected with bkv was assessed by immunofluorescent analysis in the absence and presence of pravastatin. both, the percentage of bkv infected cells and the intensity of bkv infection, assessed by western blotting using antibodies against large t antigen, were significantly decreased in hrptec treated with pravastatin. it is likely, that pravastatin's inhibitory effect is explained by depletion of caveolin-1, a critical element of caveolae. we demonstrate that bkv enters hrptec by caveolarmediated endocytosis and disruption of caveolin 1 mrna and protein inhibits bkv infection of hrptec. we provide evidence that pravastatin dramatically decreased caveolin-1 expression in hrptec and interfered with internalization of labeled bkv particles. our data suggest that pravastatin, acting via depletion of caveolin-1, prevented caveolar-dependent bkv internalization and repressed bkv infection of hrptec. our data represent the first report of inhibitory action of statins upon bkv infection. results: 74.6% of patients were caucasian, 12.2% hispanic, 8.9% african-american and 4.4% asian. overall 1-and 3-yr patient survivals were 85% and 77%. stratified by race, only african-american survivals differed from caucasian (3-year survivals of 71% vs. 77%; figure 1 ). compared to caucasians, african-american patients were younger (48yrs ±11.5 vs. 52yrs ±9.9), were more likely to be status 1 (10.3% vs. 5.1%), to have a serum creatinine ≥1.5mg/dl (37% vs. 31%), to receive an multi-organ transplant (8% vs. 5%), to have fulminant hepatic failure (12% vs. 7%), to have a higher meld score (22.9 ± 10.4 vs. 20.1 ±9.4) , to be in the icu (16% vs. 11%), and to be ventilated pre-transplant (8% vs. 5%); all p-values <0.001. after adjustment for each of these variables, race was still an independent predictor of mortality (p=0.01, hr: 1.22, ci: 1.048-1.425). multivariate analysis of the african-american group (n=1481) alone revealed that a bmi greater than 40 (p=0.001, hr: 2.5, ci: 1.49-4.17), a creatinine greater than 1.5 mg/dl (p=0.002, hr: 1.6, ci: 1.18-2.10), and icu admission pretransplant (p=0.018, hr: 1.5, ci: 1.07-2.17) are independent predictors of mortality in this subset of patients. conclusion: in the current meld era, race is still a predictor of worse outcomes, even after adjustment for multiple clinical variables. further work is necessary to elucidate why this disparity exists. the purpose of this study was to analyze the effects of successive pregnancies in female liver transplant recipients on newborn and maternal outcomes. data were collected from the national transplantation pregnancy registry via questionnaires, phone interviews and hospital records. analyses for linear trends (proportions and continuous variables) were done by chi square and least squares regression. there were 217 outcomes of 213 pregnancies, including twins. of the 125 liver recipients who had a first pregnancy, 61 had between one and four subsequent pregnancies. there were no significant differences in the variables analyzed as noted in the conclusions: successive pregnancies in liver transplant recipients are not associated with adverse fetal outcomes and/or increased maternal graft loss. female liver recipients with excellent allograft function without significant recurrent disease or chronic rejection who wish to have more than one pregnancy should not be discouraged to conceive. recorded and categorized in a blinded fashion. univariate, multivariate and survival analyses were performed. over a 4.5-year period (2002) (2003) (2004) (2005) (2006) (2007) , 148 olt recipients were randomized to receive a cca with biliary stent (n=76) or cca alone (n=72). patients with hepatic artery thrombosis (n=8; 5.7%) were excluded. there was no significant difference in demographic or graft-related variables. the mean age at transplant was 55 years, 82% were male, the mean meld was 21 and 23% had hepatitis c. the mean donor age was 46 years, 59% of donors were male, and the mean cold ischemia time was 8.7hrs. the only complication related to the biliary stent was one occlusion. the rate of overall bc in the stented patients was 24.7% vs. 32.8% in non-stented patients, (p=ns). however, stented patients had significantly less bc in the first 60-days post-olt (6.8% vs. 21.0%, p<0.02) and significantly less anastomotic leaks (2.4% vs. 9.6%, p<0.05). over the year following olt, stented patients also required less biliary therapeutic interventions (mean 1.8 vs. 0.9 interventions/patient, p<0.04) and fewer readmissions (mean 2.8 vs. 1.6 readmissions/patient, p<0.01). we also observed improved late graft survival (>6mo) in the stented group. intraoperative stenting of the cca at olt does not appear to reduce the long-term rate of bc but it decreases the incidence of biliary leaks and significantly improves many facets of early patient management. is background: long-term outcomes after retransplantation of the liver (re-olt) is inferior compared to primary olt. however, the survival benefit of re-olt based on model for end stage liver disease (meld) is not known. a single-center analysis of 421 adult patients who underwent re-olt between february 1984 to february 2007 was performed. survival benefits, at a given meld score, were calculated by comparing re-olt survival at 3 months to expected 3-month survival without retransplantation results: of 421 pts, 380 underwent re-olt, and 41 received 3 transplants. the figure shows re-olt survival benefit at any meld score with increased significance in patients with meld scores > 18. although meld scores 30-40 predicted the highest mortality after re-olt, they also demonstrated survival benefit. multivariate cox regression identified cold ischemia time >10 hrs (rr 1.8, p 0.001), meld 30-40 (rr 2.0, p 0.04), time from first olt (8 days -1 year, rr 1.8, p 0.02) and third transplant (rr 1.7, p 0.03) as independent predictors for mortality following re-olt. conclusions: re-olt should be considered in all patients even with low meld scores. although patients with high meld scores (30-40) exhibit poor survival outcomes, a survival benefit is achieved. survival benefit that considers both probability of death without re-olt and expected survival with re-olt, should be used for selection of retransplantation candidates. this study analyzed posttransplant complications, meld score, and donor ages and their effect on length of stay (los). methods: this irb approved retrospective review of our prospectively maintained database included 1427 liver transplant recipients transplanted between 1999-2006. los <14 days, 14-30d, and >30d were analyzed. primary analysis to look at los, meld, and donor age was performed using wilcoxon two-sample test. complication groups were analyzed using logistic regression and odds ratio estimates were determined. kaplan-meier for patient survival for the los groups was performed. univariate analysis: allograft dysfunction, vascular complication of liver allograft, intra-abdominal (other than liver), biliary, cardiac, pulmonary, neurologic, sepsis, renal, and endocrine were statistically significant (p<0.001) using fisher exact test. multivariate analysis: using logistic regression, significant complications were analyzed to determine the complications linked with los >14d and >30d. analysis of 1427 liver transplants demonstrated increasing los with increasing meld in each donor age group (50-60, 61-70) p<0.0001. for donor age >70, this relationship was not significant (p=0.2206). multivariate analysis using logistic regression determined odds ratio estimates for each complication resulting in los >14 days and >30 days. los >14 days and los >30 days were associated with different complications (as shown above). allograft dysfunction (including pnf) and renal complications were not significant factors in multivariate analysis. patient survival significantly decreased (p<0.001) with increased los >14 days. introduction: some patients with primary biliary cirrhosis (pbc) may require longterm corticosteroid (cs) therapy following liver transplantation (olt) due to recurrent inflammation in the graft. our center has attempted to minimize cs use in all of our olt recipients. we reviewed our experience in this cohort of patients to determine 1) patient outcome including recurrent disease and 2) long-term requirement for cs use in pbc patients. methods: from 1988 to 2006, 1,102 olts were performed in 1,032 adults at the university of colorado of which 70 patients (6.8 %) with pbc received 74 allografts. recurrence was defined by characteristic histologic changes on biopsy. bivariate and multivariate analyses were used to evaluate predictors of cs withdrawal. 13 potential predictors of cs discontinuation were considered: age, gender, bmi, race, presence of inflammatory bowel disease (ibd), type of graft (cadaver or living donor (ld), recurrence of aih, warm ischemia time, follow up time (time since transplant), and immunosuppressant (is). results: overall survival at 5 years was 85%. the 1, 5 and 10 year recurrence-free survival was 90, 72, and 54%, respectively. disease recurred in 18 patients (25.7%). of these 18 patients, none received a second transplant because of recurrent disease. cs was withdrawn in 73% of patients at time of review. independent predictors of cs discontinuation are age (>median) (p = 0.0029) and ld graft type (p=0.0018). conversely, cyclosporine (csa) (p=0.0012), female gender (p=0.0197), and bmi > 31 (p=0.0306) were negatively associated with cs withdraw. interestingly, cs withdrawal did not influence pbc recurrence. conclusions: 1) long-term outcomes in pbc patients are favorable and disease recurrence can be managed medically without abstracts re-transplantation. 2) using an aggressive cs minimization approach, almost 3/4 of the patients were cs-free at the time of last follow-up. 3) increasing age and ld grafts were associated with successful cs withdraw; while csa, female gender, and increasing bmi were associated with unsuccessful cs withdraw. incidence cholestatic disease (cd), either chronic rejection or recurrent primary sclerosing cholangitis (psc), post liver transplantation (lt) occurs in 5-35% of psc grafts. the study objectives were to evaluate the incidence and long-term outcome of cd. from 1985 to 2006, 141 grafts in 125 consecutive psc patients and 85 grafts in 79 concurrent alcoholic liver disease (ald) patients were compared. cd was diagnosed by biliary imaging and/or histology. median follow-up was 57 months. the groups were similar including meld score and cold ischemic time; however, roux-en-y biliary anastomosis was more common in the psc group (95% vs. 13%, p<0.01). the psc group had more cmv hepatitis (22% vs. 6%, p<0.01) and acute rejection (60% vs. 30%, p<0.01), and fewer biliary anastomotic strictures (7% vs. 16%, p<0.01). cd occurred in 38 psc grafts and 7 ald grafts (p<0.01). the incidence of cd was greater in the psc group (p=0.02, fig. 1 ). no significant risk factors for cd were identified. in the psc group the graft survival was lower in the cd group (p=0.04, fig. 2) ; however, patient survival at 10 and 15 years was not effected by cd: 73% and 67% vs 75% and 60% because the re-lt rate was greater in this group (26% vs. 6%, p<0.01). at 15 years, patient survival in the psc group was better than for ald (64% vs 22%, p<0.01). long-term outcome post lt for psc is good and better than for ald; however, cd continues to develop beyond the first 5 years with a prevalence of 43% at 15 years. graft loss in patients with cd is high, but with re-tx, patient survival is similar to non-cd patients. further studies to distinguish chronic rejection vs. recurrent psc may provide insight into the prevention of cd following lt for psc. discussion: our analysis showed that patients with aih have a worse long term survival compared to pbc and psc after ddlt. this may be explained by possibly more advanced disease at the time of presentation or more septic complications due to pretransplant salvage therapy with immunosuppressants. also,pbc had the worst relative outcome after ldlt in this group-possibly explained by the older age of the recipients. overall, ldlt offered better outcomes than ddlt in terms of survival in patients with aih,pbc and psc. this study highlights an important and previously unvisited aspect of transplantation for autoimmune and cholestatic liver diseases. inflammatory bowel disease course in patients transplanted for primary sclerosing cholangitis. ariana wallack, 1 joel s. levine, 2 lisa forman. 2 1 internal medicine, univ. of colorado hsc, aurora, co; 2 gastroenterology and hepatology, univ. of colorado hsc, aurora, co. the natural history of ibd following liver transplant (lt) for psc is unknown. prior studies have not shown factors consistently associated with disease activity, but were limited by small sample sizes. the aim of the study is to describe our experience with ibd post-lt in patients with psc and determine factors predictive of disease activity. a survey was mailed to 115 liver recipients transplanted for psc between 1988 and 2006 asking about medications, ibd activity and quality of life after lt. responses were linked to our lt database. results 88 (77%) recipients responded. 76% were male with a median of 82 (7-215) months since transplant. 93% were caucasian with a median transplant age of 46 (16-71) years. 16% developed recurrent psc post-lt. 74% had a diagnosis of ibd (63% uc). immunosuppression included tacrolimus in 84% and cyclosporine in 11%. 71% experienced at least one episode of acute rejection. 73% rated their quality of health 4-5 on a scale of 1-5. there was no significant difference in demographic variables between ibd and non-ibd cohorts. 66% of respondents had ibd pre-lt. of the 30 patients without pre-existing ibd, 7 developed ibd post-lt. of the de novo ibd cohort, 43% were male, median transplant age was 45 years (39-57), and 86% were caucasian. ibd developed in 71% at more than 3 years post-lt. there was no statistical difference between the de novo ibd cohort and those with pre-existing ibd except for ibd type (57% uc vs 88%, p=0.008). significantly more patients were not on any ibd medications post-lt compared to pre-lt (75% vs 91%, p=0.02). fewer post-lt patients were on aminosalicylates (63% vs 79%, p=0.03) and prednisone (14% vs 31%, p=0.03) compared to pre-lt. post-lt recipients developed fewer ibd flares requiring hospitalization (27% vs 45%, p=0.004). 38% reported improvement in ibd activity post-l. 23% and 33% reported no change and worsening activity, respectively. there was no significant difference between reported disease activity and immunosuppression, cmv status, rejection rate, recurrent psc, transplant age, gender or race. background: symptomatic cmv continues to be a significant problem. the costs associated with its development remain high without an optimal method for prevention. the aim of this study was to measure the pharmacoeconomic impact of implementing an abbreviated pre-emptive monitoring strategy versus valganciclovir (vgc) prophylaxis in a large teaching hospital. methods: costs for this analysis were based on a societal perspective, including drug, personnel, hospital, outpatient infusion and monitoring costs related to resources needed to perform pre-emptive monitoring and treatment of cmv related events. time per hr costs were nurse/data coordinator $45, physician $200, and pharmd $52. cmv pcr cost was $203. vgc cost per mg was calculated for prophylaxis, treatment and 30 days of consolidation as needed based on an awp ($0.09 per mg). estimated cost of cmv syndrome was based on cost of picc line placement, drug, personnel and supply costs based on 21 days of therapy and was estimated to be $11,885.50. inpatient admission cost per day were $3727. results: a total of 119 patients were included in this analysis. baseline and transplant demographics were well matched. table 1 displays the total direct and indirect costs accumulated for each group. cmv syndrome occurred in three patients for each group. there was no cmv disease in either group. 27% of patients in the pre-emptive group had dnaemia, but only 4 patients required oral anti-viral therapy. provider time and lab monitoring costs were significantly higher in the preemptive group, while direct medication cost was significantly higher in the prophylactic group. conclusions: frequency of disease severity and outcomes were equal in each group. although the overall costs between strategies is equivocal, allocation of resources to provide pre-emptive monitoring places the burden of disease prevention on the health care system versus the patient necessitating further abbreviation of these strategies. we propose a non-simultaneous form of kidney paired donation that starts with a living, non-directed donor (lnd). a paired donation matching algorithm was developed to allow for lnds to start potentially never-ending altruistic donor (nead) chains in addition to closed loops of 2-, 3-and 4-way exchanges. results: in july 2007, a lnd from michigan traveled 1500 miles to donate a kidney to a woman whom he had never met in arizona. the following week, the arizona recipient's husband donated one of his kidneys to a 32-year-old woman in ohio. the following month, the mother of this recipient traveled to a city 3 hours away to donate her kidney to a patient whose incompatible donor simultaneosly gave a kidney to the fourth patient in the chain. the incompatible donor for this fourth recipient is now slated to give her kidney to a patient in maryland. over the past 9 months, 60 transplant programs have partnered to perform 10 paired donation transplants. demonstrating the advantage of nead chains over classic paired donation, 8 of 10 transplants resulted from altruistic donor chains. conclusion: in order to fully realize the potential of the above approach, one must be willing to supplant two prevalent ideas: 1) that kidneys from altruistic donors should be given to the top candidate on the deceased donor waiting list, and 2) that paired exchanges must be done simultaneously. while there are certain pitfalls to using chains of donors as opposed to traditional "swaps" (i.e. the possibility that a donor could renege, or the accumulation of type ab donors who are not likely to be able to begin another chain), this proposed paradigm shift could result in a very significant increase in both the number and quality of paired donation kidney transplants. purpose: medical literature and national best practices correlate families' understanding of brain death with organ donation rates. analysis of hospital data demonstrated variability in family communication. although surgical residents frequently interact with family members of potential donors and play a critical role in the donation process, they receive no formal cst. we sought to determine if pre-training residents improved performances in cst involving explanation and notification of brain death to family members and aided efforts to achieve the national donation rate goal of 75%. methods: in collaboration with a regional organ procurement organization (opo), an educational model for end of life cst was developed. 17 surgical residents were divided into 2 groups. the first group (n=9) attended a 3-hour didactic session at the opo that included role-playing exercises explaining brain death to families. opo staff, trained to serve as family role-players and skills station coaches, debriefed residents after each simulation. the second group (n=8) received no specialized training. six weeks later both resident groups participated in formal videotaped family communication simulations. independent observers (hospital faculty and senior opo staff), blinded to training, evaluated residents' communication skills using a 12-parameter assessment tool. all residents reviewed their videotaped performances and evaluations, then repeated the simulations after six months. results: during the study period, the pre-trained resident group assessment scores increased by 25% (p=0.0001), while the untrained group increased by 39% (p<.0001). while evidence of improvement existed in both groups, pre-trained residents consistently scored higher when compared to untrained (80% vs. 71%, p=0.037). during this same time period, donation rates in the surgical intensive care unit (sicu) increased by 21%. conclusion: our educational model demonstrated effective training in communication skills during end of life discussions. although a direct relationship cannot be established, donation rates in the sicu increased after resident training. incorporation of this training program into resident education has the potential to improve donation rates and increase the number of organs available for transplantation. program. saverio mirarchi, 1 graeme n. forrest, 1 benjamin philosophe. 2 1 medicine, university of maryland, baltimore, md; 2 surgery, university of maryland, baltimore, md. objective: to improve the efficiency of care for solid organ transplant patients by having internists work in conjunction with transplant surgeons to manage transplant patients admitted to the hospital more than 30 days post organ transplant. this includes patients who have undergone kidney, pancreas, and liver transplants which our center transplants over 300 of these organs/year. in 2002, the organ transplant program was divided into a surgical transplant service (sts) and medical transplant hospitalist service (mths). the mths consists of four full time physicians, one part time physician, and one nurse practictioner with daily rounds from a transplant surgeon on a weekly rotating schedule. methods: we analyzed our data from the past five years since the inception of the mths at a large university medical center. the length of stay (los) for the mths and sts were compared to data from the previous combined program as well as data from the university health consortium (uhc). in addition, we looked at the cost of care to determine if there were any savings related to reduced los and adjusted for the combined salary of the mths. results: in the review period, the total admissions for both the mths and sts averaged 1450 admissions/year. over the past five years the mths showed a major decrease in the los index, defined as the ratio between the observed los and the expected los based on uhc data. this index dropped from 1.27 to 0.91 for patients on the mths. there was also a parallel drop in the sts los index from 1.53 to 1.05. this was associated with a significant cost savings for the hospital. on average, the program has realized an average savings of approximately $1,000,000 per year. when accounting for the combined salary for the mths group which is 625,000 dollars/year, the total savings over the 5 year period this amounts to $1.9 million. conclusion: the creation of a mths working within an organ transplant program at a large university center has been able to demonstrate a major decrease in los for transplant patients which has resulted in decreased costs and improved efficiency. this has also allowed more focused care for a complex medical population. we believe that our program can serve as a model for similar programs at other busy transplant sites. with increasing demand for kidney transplants(tx), more patients are opting to travel outside the us to obtain transplantation. we describe the characteristics and outcomes of 32 kidney tx recipients followed at our center who traveled abroad for a kidney tx between 1995 and 2007. methods: data were obtained via chart review. we compared demographics to all tx recipients at our center during the same period and compared post-tx outcomes to a cohort of patients transplanted at our center matched for age, race, tx year, dialysis time, prior tx, and donor type. median follow-up time was 487 days (range 31-3056). results: demographics are outlined in table. patients transplanted abroad were more likely to be asian and had shorter dialysis times. most patients were transplanted in china (44%) followed by iran (16%), the philippines (13%), and india (9%). living unrelated tx were most common. all patients were discharged on a calcineurin inhibitor, pred, and either mmf (90%), aza (7%), or rapamycin (3%). 7 patients received induction. only 4 patients received cmv prophylaxis. the median duration of hospitalization was 15 days. the median time post-tx to initial visit at our center was 35 days. 4 patients required urgent admission to hospital, 3 of whom lost their grafts. 17 patients ( graft survival at 1-year was 96% for both "tourists" and matched recipients at our center. median time to graft loss was 500 (31-2832) days. mean scr and rejection 1-year post tx was not significantly different between recipients transplanted abroad and at our center. conclusion: compared to patients transplanted locally, patients transplanted abroad had similar graft survival and renal function post tx, but had a high incidence of infectious complications. [background] the primary benefits anticipated following successful induction of allograft tolerance are avoidance of the complications of long-term immunosuppression and prevention of chronic rejection. we have previously reported successful induction of renal allograft tolerance in recipients of hla mismatched combined kidney and bone marrow transplantation (ckbmt). no evidence of chronic rejection has been observed in these recipients after immunosuppression-free periods of 1 to 4 years. in the current study, we have evaluated the longer-term economic impact of this approach. [method] the conditioning regimen for ckbmt included cyclophosphamide, thymic irradiation, anti-cd2 mab, and a calcineurin inhibitor, which was discontinued after 9-12 months. 18 stable renal transplant recipients receiving ongoing triple or double drug immunosuppressive therapy with compatible follow up times were compared with the four tolerant recipients. tolerant patients and stable patients on maintenance immunosuppression were also comparable with respect to their age, their original disease and donor-recipient histocompatibility. [results] the perioperative charges for ckbmt were approximately $90,000 higher than those for conventional living donor kidney transplantation. after 9-12 months, continuing medications in ckbmt recipients included only occasional over-thecounter analgesics. in contrast, the stable conventionally treated recipients were taking an average of 8 pills daily. these included treatments required for de novo diabetes (11%), hypertension (67%), hyper lipidemia (22%) and gastro-intestinal symptoms/ prophylaxis (78%) in addition to their maintenance immunosuppression. these needs resulted in annual maintenance charges of over $15,000/year/allograft recipient and do not include unmeasured costs related to issues such as quality of life or absences from employment. [conclusion] even with this admittedly expensive approach to tolerance induction, the overall medical costs for conventional kidney transplantation with ongoing immunosuppression and treatment for complications will exceed the cost of tolerance after approximately 5 years. increasing african american donation rates in a midwest metropolitan community. susan gunderson, 1 susan mau larson, 1 david m. radosevich, 2 clarence jones, 3 bill tendle, 3 tiffany scott. 1 1 lifesource, st. paul, mn; 2 transplant information services, university of minnesota, minneapolis, mn; 3 southside community health services, minneapolis, mn. purpose: african americans are underrepresented in deceased organ donation in this +2 million community. historically minimal educational outreach had occurred and this study was designed to understand the community's disposition toward donation and to increase support for donation. methods: television, newspaper, and radio advertising aired over a 24 month period beginning in 2004 using the nationally produced donate life-african american campaign as the primary intervention. other components included related faith-based and community outreach. the opo partnered with a minority focused community health clinic to survey african americans pre-and post-intervention. a mailed, selfadministered survey was sent to a sample drawn from organizational lists with a high likelihood of including african american households. for the evaluation, the sample was separated into 1) community members and 2) church members exposed to faithbased campaigns. results: african americans in this community have a sophisticated understanding of the importance of donation (average 12 of 15 knowledge questions scored correctly) in pre-intervention survey. in both community and church groups media exposure and donation knowledge increased after the media campaign (p<0.001 and p=0.004 respectively). similarly, donor designation rates for the entire population on the drivers licenses increased (33.0% versus 40.1%, p=0.119). among all african americans the propensity to donate was, however, unchanged following the campaign. propensity to donate increased (p=0.034) in the community sample whereas there was a reduced propensity to donation (p=0.092) in the church sample. during the study time period the opo also experienced significant increases in donation authorization rates among african-americans, increasing from 37% to 75%. summary: a media based campaign combined with grassroots outreach is an effective tool to increase knowledge and awareness. partnership between the opo and community and faith based leadership was a significant positive byproduct of the project and should be included in future outreach efforts. background: it has been hypothesized that the clinical benefits of anti-thymocyte globulin (atg) induction therapy do not completely result from immunodepletion, but may also result from induction of immunoregulatory t-cells (treg). in this prospective, controlled study we investigated the effect of atg-induction therapy on the frequency and phenotype of peripheral cd4 + foxp3 + cd127 -/low t-cells in kidney transplant patients. methods: after transplantation, 16 patients received atg-induction therapy (thymoglobulin ® ) and triple therapy consisting of tacrolimus, mmf and steroids. the control group (n=18) received triple therapy only. by flow cytometry, t-cells were analyzed for markers associated with immune regulation: cd25, foxp3 and cd127. within the foxp3 + t-cell population, the cd45ro (memory) and ccr7 (homing receptor) markers were characterized. results: pre-transplant levels of cd4 + foxp3 + cd127 -/low t-cells in all patients were 3-17% (median 6%) of cd4 + t-cells. one wk post atg induction therapy, no measurable numbers of treg were present. at 12 wks post atg-induction therapy, a higher proportion of the first detectable t-cells expressed foxp3 compared to the control-group (atg vs. control-group; 7 vs. 4%, median, respectively, p=0.03) and then returned to pre-transplant levels at 26 wks. this increased proportion of cd4 + foxp3 + cd127 -/ low t-cells resulted in a significantly higher foxp3 + /foxp3 neg ratio than in the controlgroup at 12 wks; 0.074 vs. 0.046, median, p=0.02) . at 26 wks, we found a decline in the proportion of naive foxp3 + treg (cd45ro neg ccr7 + pre-transplant vs. post-transplant; 21 vs. 9%, median, p=0.02) which was associated with a rise in the proportion of memory foxp3 + treg (cd45ro + pre-transplant vs. 26 wks post-transplant; 65 vs. 82%, p=0.02). moreover, the proportion of memory t-cells exceeded that in the control-group at 26 wks (atg vs. control-group; 82% vs. 67%, median, p=0.05) which was mainly due to an increase in the proportion of effector memory foxp3 + treg (cd45ro + ccr7 neg ). conclusion: after atg-induced immune cell depletion, a shift towards cd4 + foxp3 + cd127 -/low peripheral regulatory t-cells with the memory phenotype was measured in kidney transplant patients. this finding suggests that atg treatment triggers the generation of de novo peripheral regulatory t-cells by homeostatic proliferation. introduction in a prospective study, we investigated whether donor-specific regulatory cd4 + cd25 bright+ foxp3 + t cells develop in kidney transplant patients. methods we analyzed the percentage and function of peripheral regulatory cd4 + cd25 bright+ foxp3 + t cells of 51 patients before, 3, 6 and 12 months after kidney transplantation. the immune regulatory capacities of cd4 + cd25 bright+ foxp3 + t cells were assessed by their depletion from pbmc and reconstitution to cd25 neg/dim responder t cells at a 1:10 ratio in the mlr. in the first year after transplantation, peripheral cd4 + cd25 bright+ foxp3 + t cells decreased from 8,7% ± 3,0 pre-transplantation to 6,1% ± 1,8 at 12 months (p<0.001). while mlr reactivity to 3 rd party-ag (3 rd p) significantly improved (p<0.05), the reactivity against donor antigens remained low. functional analysis demonstrated potent donor-specific regulatory activities by cd4 + cd25 bright+ foxp3 + t cells after transplantation. depletion of cd4 + cd25 bright+ foxp3 + t cells from pbmc resulted into increased proliferation upon stimulation by donor antigens (p<0.01). upon reconstitution, the capacity of cd4 + cd25 bright+ foxp3 + t cells to control the proliferation of anti-donor reactive cd25 neg/dim t cells increased over time: from 58% (median) pre-transplant to 85% post-transplant at month 12 (p<0.01). moreover, the anti-donor regulatory activities by the cd4 + cd25 bright+ foxp3 + t cells were significantly more vigorous than those controlling 3 rd p-ag stimulated responder t cells (65%, p<0.05). the generation of potent donor-specific regulatory cd4 + cd25 bright+ foxp3 + t cells in the periphery of kidney transplant patients prevents the development of adequate alloreactivity. conclusions: these results indicated that anti-donor responses could be detected even in a significant proportion of "stable" long-term hla identical kidney transplant recipients. speculatively this may be the cause of late graft failures in this group of patients. expression klotho is a gene almost exclusively expressed in renal distal tubules and loss of expression is associated with accelerated aging. in various models of acute and chronic renal injury, and with normal aging, renal klotho expression has been found to decrease. increased donor age is associated with poorer long term allograft function. we hypothesized that klotho mrna expression in renal implant biopsies would correlate with donor kidney quality, as determined by donor chronologic age and peri-transplant renal injury. our recent unsupervised microarray analysis of 87 renal implant biopsies revealed a continuum of organ quality across all samples ranging from the best living donor (ld) kidneys at one end to the worst performing deceased donor (dd) kidneys at the other (am j transpl 2007, nov 16,epub). three predominant groups were identified of ld, dd1 kidneys with low risk (9.5%) of delayed graft function (dgf) and dd2 kidneys with high risk (35%) of dgf (p < 0.05). analysis of klotho gene expression among these groups also revealed a spectrum of expression from highest levels in ld to lowest in dd2 kidneys, especially those who developed dgf. differences were highly significant among ld vs dd kidneys (p < 0.001), although donor age was not different between these groups. klotho transcript levels were significantly different among kidneys that developed dgf compared to those with immediate graft function (igf) (p < 0.05). in conclusion, reduced klotho gene expression is associated with grafts at risk of poorer function and appears to be independent of donor age. decreases in klotho expression likely reflect other factors impacting the renal tissue, which may impact potential for repair and ultimately allograft function. anti renal allograft rejection episodes produce a stereotypical response in the allograft characterized by infiltration by cytotoxic t lymphocytes (ctl), potent ifng response by donor and recipient cells, and decreased transcripts associated with the epithelium. we previously identified pathogenesis based transcript sets (pbts) that reflect the disturbance in rejecting allografts (qcats -ctl, grits -ifng response, kts -decreased function, ajt 7:2712 , 2007 . we hypothesized that anti-rejection treatment would reverse the transcriptome changes of rejection more than histopathologic lesions. using microarray analysis we measured expression of these pbts in 18 antibody mediated rejection (abmr)(11 untreated, 7 treated) and 31 t-cell mediated rejection (tcmr) (25 untreated, 6 treated) biopsies, normalized to nephrectomy samples. histopathology scoring of the primary rejection lesions were analyzed; interstitial inflammation(i), tubulitis(t), intimal arteritis(v), and glomerulitis(g) (fig 1a) . of these, only i and t differentiated between abmr and treated abmr (p<0.05) yet none differentiated between tcmr and treated tcmr. pbts on the other hand differentiated treated from untreated for both abmr (p<0.04) and tcmr (p<0.005) with all 3 pbts (fig 1b/c) . the changes in transcript expression in treated cases were dramatic. particularly impressive was the consistent correction of the disturbances in pbt expression despite the heterogeneous treatment following abmr episodes. unlike abmr treatment, tcmr treatment always included steroids which may contribute to the more pronounced changes compared to abmr. the time of treatment before the biopsy, which also varied within the groups, did not affect the changes in pbt expression since values in treated cases approached those of nephrectomy samples. thus, histologic rejection lesions persist following anti-rejection treatment whereas transcript disturbances of rejection are greatly reduced compared to untreated cases. this study suggests that assessment of anti-rejection treatment is more sensitively monitored by transcript expression than histopathology. blood deciphering the mechanisms of tolerance and chronic immune-mediated rejection remains a major goal in transplantation. data in rodents suggests that toll-like-receptors (tlr), regulators of innate immune responses, play a role in determining graft outcome. however, few studies have focused on tlr in human kidney transplant recipients. we addressed this issue by analyzing the peripheral blood (n = 75) and graft biopsies (n = 26) of renal transplant patients and healthy volunteers. we analyzed, for the first time, the expression of tlr4 in pbmc from kidney recipients with contrasted situations: operational tolerance and chronic immune-mediated rejection (banff 2005), compared to patients with normal histology and stable graft function, non transplant patients with renal failure and healthy volunteers. we found that myd88 and tlr4 were significantly contrasted in the pbmc, and in particular in monocytes, of patients with chronic immune-mediated rejection vs. operational tolerance. chronic rejection patients had significantly increased tlr4 and myd88 compared to operationally tolerant patients, who resembled healthy volunteers and non transplant patients with renal failure. interestingly, analysis of tlr4 transcripts in graft biopsies from patients with normal histology or chronic immune-mediated rejection reflected the blood findings, with a significant increase of tlr4 in chronic immune-mediated rejection. thus, we provide data to support a link between tlr4 expression and long-term graft outcome. the role of tlr and their endogenous ligands in mediating allograft rejection or acceptance therefore warrants further investigation and could give rise to new strategies of therapeutic intervention. our results suggest that peripheral blood tlr4 shows potential as a biomarker of chronic immune-mediated rejection and that measuring blood tlr4 levels may help to identify patients experiencing chronic rejection who require a biopsy. moreover, our data suggest that absence of tlr signaling may be a feature of operational tolerance to kidney grafts. identification of immune identification of immunological tolerance is an important prerequisite in order to establish an individually-tailored approach to the post-transplant management of allograft recipients. it will also provide new insight into the mechanism underlying the balance between tolerance and rejection. here we present data from a multi-centre study aimed at identifying tolerance to renal allografts. we have collected samples from five selected groups of renal transplant recipients: drug-free tolerant patients that were functionally stable despite remaining immunosuppression-free for more than one year; functionally stable patients on minimal immunosuppression (<10 mg/ day prednisone); stable patients maintained with calcineurin inhibitors (cni); stable patients maintained on cni-free immunosuppression regimen; and patients showing signs of chronic rejection. a group of age and sex matched healthy volunteers was also included as control. several biomarkers and bioassays, were combined to provide an immunological 'fingerprint' of the tolerant state. immunophenotype showed a selective expansion of peripheral blood b and nk lymphocytes in drug-free tolerant patients. this group of patients was also characterized by the absence of anti-donor specific antibodies. the differential expression of several immune relevant genes and a high ratio of foxp3/ α-1,2-mannosidase expression in these patients was observed. tcr landscape analysis highlighted differences between the vβ repertoires of drug-free tolerant recipients and chronic rejection patients. additionally, direct pathway donor-specific hyporesponsiveness by ifnγ elispot and lack of indirect pathway anti-donor responses assessed by trans-vivo dth were detected in drug-free patients. the diagnostic capabilities of the combined results of several of the above mentioned biomarkers and bioassays are as follows: specificity 0.964, sensitivity of 0.933 and a positive predictive value of 82.4%. these biomarkers could be used to inform drug weaning protocols of kidney transplant recipients. bile acid aspiration stimulates lung allograft immunity. bile acids detected in the broncho alveolar lavage (bal) as a marker of aspiration has been associated in a dose dependent fashion to earlier development of bronchiolitis obliterans syndrome. we sought to study the relationship between bile acids and active immune molecules as detected in the bal. methods: bal collected prospectively from lung transplant recipients at routine surveillance bronchoscopies were assayed for bile acids. samples were then assayed by luminex for cytokines , and by elisa for pulmonary collectins (sp-a, sp-d). results were analyzed according to levels of bile acids as per roc testing for accuracy for bronchiolitis obliterans syndrome diagnosis (high levels ≥3.5 µmol/l). results: we prospectively examined 120 lung transplant recipients and a total of 271 bal samples were collected. in 114 no bile acids were detected, low levels were present in 110, and high levels were detected in 47 samples. samples with high bile acids had significantly greater innate (tnf-a, il-1b, il-12, il-10, il-6) and adaptive (ifn-g, il-2) cytokines as well as greater chemokines (mcp-1, il-8) compared to the other samples. in contrast pulmonary collectins sp-a and sp-d were significantly reduced in samples with high bile acids. the figures shows the median and interquartile range for each molecule according to bile acid levels. conclusion: bile acids detected in the bal as markers of aspiration stimulate the lung allograft immunity in a dose dependent fashion. in particular high levels of bile acids are associated with an impaired lung specific innate defense system provided by the pulmonary collectins and with a broncho-alveolar district "cytokine storm". tolerance/immune deviation iii the results of using ex vivo cd4+ t-cells converted dn t-cells in nod mouse models support the concept and the feasibility of potentially utilizing this novel cell-based therapeutic approach clinically for the treatment of autoimmune type i diabetes. horng-ren yang, 1 gouping jiang, 1 john j. fung, 1 shiguang qian, 1 lina lu. 1 1 immunology and general surgery, cleveland clinic, cleveland, oh. liver transplant tolerance was recognized by spontaneous acceptance of liver allograft in many species. the underlying mechanism remains unclear. interestingly, although liver allografts are accepted, hepatocyte transplants in the same combination are promptly rejected, indicating a crucial role of liver tissue cells in immune suppression. we have demonstrated a profound t cell inhibitory activity of hepatic stellate cells (hpsc), which are known to participating in repairing and fibrosis during liver injury. addition of activated (a) hpsc, but not quiescent hpsc, significantly inhibited allo-dc induced-t cell proliferative responses (mlr) in a dose dependent manner, which was associated with enhanced t cell apoptosis (tunel). neutralization of b7-h1 by anti-b7-h1 mab significantly reduced the hpsc-induced t cell apoptosis and reversed the inhibition of t cell proliferation, suggesting a key role of b7-h1. to evaluate this in vivo, balb/c islets (300) were co-transplanted with 3 x 10 5 activated hpsc (b6) into stz-induced diabetic b6 recipients. co-transplant with hpsc effectively protects islet allografts from rejection. this was associated with reduction of graft infiltrating t cells and enhancement of apoptotic activity. co-transplant with hpsc from b7-h1 -/livers markedly lost their islet graft protective capacity, associated with less apoptosis of infiltrating cells. to determine the subsets of apoptotic t cells, t cells were isolated from spleen, draining lymph nodes, and grafts for phenotype and function analyses. on pod 8, a marked reduction of graft infiltrating cd4 + (30.7 %) and cd8 + t cells (31.3 %) was seen in hpsc co-transplant group, as compared to islets alone, which was further progressed thereafter. cd8 t cells dropped ∼7 folds on pod 140. cd4 + / cd8 + ratio was increased from 0.5 at the early pod to 2.0 in the long-term survival grafts. adoptive transfer of cfse-labeled des transgenic t cells was used to track the response and fate of antigen-specific cd8 + t cells. the results showed active division of des + cells within the allograft in both the islet only and hpsc co-transplantation groups. however, accumulation of these des + cells was significantly lesser in the hpsc co-transplantation group as compared to that in the islet only group (1.9×10 3 vs. 10×10 3 cells per graft). these findings suggest that hpsc induce antigen-specific cd8 + t cell death, and may not inhibit their activation. donor-specific memory t cells are potent mediators of allograft rejection due to their ability to proliferate and give rise to cytotoxic and inflammatory cytokine-secreting effectors within hours of stimulation. furthermore, memory t cells have been shown to be relatively resistant to the effects of many tolerance-induction protocols, including blockade of the cd28 and cd154 pathways. while seminal studies have shown that donor-reactive memory cells, generated through pre-sensitization with donor tissue or infection with pathogens with cross-reactive epitopes, contribute to costimulation blockade-resistant rejection of fully mhc disparate allografts, the role of memory t cells specific for minor antigens in costimulation blockade-resistant rejection has not been well studied. we addressed the ability of memory t cells specific for a single donorderived class i epitope to mediate this process. tcr transgenic t cells (ot-i) specific for siinfekl/kb were adoptively transferred into naive b6 recipients, which were then infected with ovalbumin-(siinfekl) expressing listeria monocytogenes (lm-ova). at memory, mice received a skin graft expressing ovalbumin (mova), and therefore the siinfekl epitope. results showed that the transferred ot-i t cells proliferated in response to lm-ova, but not in response to a control infection with wild-type listeria (lm). at day 9, ot-i t cells comprised ∼15% of the total cd8+ t cell compartment. during memory, the siinfekl-specific cells comprised ∼1-2% of the total cd8+ t cell compartment. engraftment of mova skin on lm-ova memory recipients resulted in rejection with accelerated kinetics relative to lm infected controls (mst=13d vs 20d). following treatment with ctla-4 ig and anti-cd154, 9/10 lm-ova infected recipients experienced costimulation blockade-resistant rejection, while lm-infected controls went onto long-term graft survival (p<0.001). lm-ova-infected recipients also resisted the engraftment of mova-expressing donor bone marrow following a tolerance induction protocol containing busulfan, ctla-4 ig, and anti-cd154. these results suggest that memory t cells specific for a single surrogate minor antigen are sufficient to induce costimulation blockade-resistant rejection, and support the feasibility of using this model to study the specific requirements for donor-reactive memory t cell activation and tolerance induction during transplantation. the during an immune response, cd4 helper t cells can be instructed by non-antigenspecific signals to differentiate into functionally distinct subsets with mutually exclusive patterns of cytokine production. we find that ikaros, a zinc finger transcription factor required for lymphocyte development, is crucial for the development of polarized t helper subsets. in the absence of ikaros dna binding activity, cd4 t cells induced to undergo th2 differentiation in vitro or in vivo produce high levels of il-4, but fail to silence expression of ifn-gamma and il-17, cytokines that contribute to inflammatory disease processes such as autoimmunity and organ transplant rejection. similarly, ikaros is required for repression of il-4 and il-17 expression by th1 cells, and inhibition of ifn-gamma and il-2 production by th17 cells. our results show that ikaros controls the expression of transcription factors such as gata-3, c-maf, stat-6, and runx3, and in polarized th2 cells, ikaros inhibits ifn-gamma gene expression through direct repression of the t-bet locus. these studies place ikaros, a dna binding protein previously recognized only as a regulator of lymphocyte development, as a master regulator of peripheral t cell differentiation and function. introduction as the fastest growing subpopulation seeking organ transplantation is >65 yrs of age, it will be imperative to discern how aging impacts the acquisition of transplantation tolerance. prior work has demonstrated that viral infections induce the development of alloreactive t cells, which impede the induction of transplantation tolerance. in this study, we tested the hypothesis that aging alters host defense against viruses leading to the development of cross-reactive t cells, which impair transplantation tolerance induction. we first examined how aging modifies the function of plasmacytoid dcs (pdcs), as ifnα production by pdcs is essential for control of viral infections. using both in vitro and in vivo murine systems, we found that aged pdcs produced lower levels of ifnα in response to tlr9 activation with cpg sequences or herpes simplex (hsv)-2 virus (elisa). aged mice (18-20 mths) failed to the clear this virus as effectively as young (2-4 mths) mice. this was associated with increased liver inflammation, the release of systemic th17-skewing cytokines, il-6 and il-21, and augmented splenic il-17 levels in aged mice (elisa). prior to transplantation, unmanipulated aged mice (b6 or cba background) produced more donor-specific il-17 effector-memory t cells as compared to unmanipulated young mice (elispot). furthermore, mlr assays demonstrated that aged memory cd4+ t cells from unmanipulated mice produced significantly more il-17 in response to donor antigen compared to young t cells (elisa). to determine if aged recipients manifest an altered response to therapies that prolong allograft survival, aged and young b6 mice received balb/c skin allografts and perioperative anti-cd45 and anti-cd154. we found that aged mice rejected their allografts significantly faster (median survival 20 days) than young mice (mst, 76 days, p <0.01). similar results were noted when we employed perioperative treatment with anti-cd154 + dst and when we altered the donor-recipient (b6 to cba) strain combination. pre-treating aged mice with an anti-il-17 mab improved the efficacy of the graft-prolonging therapy compared to aged mice that received control mab (p = 0.04). conclusion our results suggest that impaired control of viral infections with aging leads to the generation of alloreactive il-17 producing t cells that impair therapies that may induce transplantation tolerance. prevention of type 1 diabetes by thymus genetic modification with protective mhc class ii molecules. jesus r. paez-cortez, 1 michela donnarumma, 1 chaorui tian, 1 john iacomini. 1 1 transplantation research center, boston, ma. introduction. susceptibility to type 1 diabetes is determined by multiple genetic factors, among the strongest of which is the inheritance of at-risk genes that lead to disease development. here we examined whether diabetes can be prevented by providing protective mhc class ii genes through directly infecting the thymus of diabetes prone nod mice. methods. after direct exposition of the thymus, lentiviruses encoding control (phage-cmv-dsred-ires-zsgreen-w) or protective mhc class ii iaβ d (phage-cmv-iaβ d -ires-zsgreen-w) genes were injected in a single thymic lobe of 3 to 4 week old female euglycemic nod mice. gene expression was determined by microscopic examination at different time points after injection and blood glucose levels were monitored weekly to examine whether this approach prevents the development of diabetes. the presence of diabetogenic cd8 + t cells were detected by flow cytometry via tetramer staining of splenocytes. pancreatic islet integrity and insulin production was assessed by immunohistochemistry. results. viral gene expression was observed exclusively in thymic epithelial cells beginning at 5 days post-injection in both groups. nod mice injected with control lentivirus developed diabetes by 22 weeks post injection, similar to non-treated animals (18-20 weeks). in contrast, phage-cmv-iaβ d -ires-zsgreen-w injected animals remained normoglycemic at 12 months post-injection. diabetogenic cd8 + t cell population were not detected in splenocytes of iaβ d injected animals using mhc class i tetramers. islet integrity and insulin production was preserved in the treated group, in marked contrast to controls, which exhibited characteristic lymphocytic infiltration of islet cells and low insulin storage. conclusions. thymic genetic modification with protective a mhc class ii iaβ d molecule can be used to prevent diabetes in nod mice. central deletion of diabetogenic t cell populations may be involved in prevention of autoimmunity in these animals. role of invariant nkt cells in liver sinusoidal endothelial cell-induced immunosuppression of t cells with indirect allospecificity. masayuki shishida, hideki ohdan, yuka tanaka, masataka banshodani, yuka igarashi, toshimasa asahara. surgery, hiroshima university, hiroshima, japan. we have reported that liver sinusoidal endothelial cells (lsecs) endocytose portally injected allogeneic splenocytes and can negatively regulate t cells with indirect allospecificity via the fas/fasl pathway. as a result of in vitro transmigration across the lsecs from balb/c mice treated with a portal injection (pi) of b6 mhc class iideficient (c2d) splenocytes, the naive balb/c cd4 + t cells lost their responsiveness to the stimulus of balb/c splenic antigen presenting cells (apcs) that endocytosed the donor-type alloantigens. however, they maintained a normal response to the stimulus of balb/c apcs that endocytosed third-party c3h alloantigens. in the present study, we examined whether invariant nkt (inkt) cells influence the ability of lsecs to endocytose irradiated allogeneic cells. balb/c wild-type (wt) mice or balb/c cd1d-deficient (cd1d -/-) mice that lacked inkt cells were portally injected with 30×10 6 irradiated b6 c2d splenocytes labeled with pkh-26. only 3.56 ± 3.19% lsecs endocytosed the labeled splenocytes in the balb/c cd1d -/mice at 12 h after pi, whereas 16.82 ± 2.69% lsecs endocytosed the labeled splenocytes in the wt control mice (p < 0.001, n = 4 each). when balb/c wt mice intraperitoneally received 4 µg α-galcer, before pi of b6 c2d splenocytes, the expression of mhc class ii on lsecs and endocytic activity of lsecs were enhanced. thus, we found that the endocytic activity of lsecs was regulated by the inkt cells. intraportal adoptive transfer of lsecs isolated from balb/c wt mice, treated with a pi of b6 c2d splenocytes, into balb/c mice significantly prolonged the survival of subsequently transplanted heart allografts (n = 4) as compared to the adoptive transfer of lsecs isolated from balb/c cd1d -/mice, treated similarly, into the balb/c mice (n = 4). however, intraportal adoptive transfer of lsecs isolated from balb/c wt mice, which received 4 µg α-galcer intraperitoneally prior to pi of b6 c2d splenocytes, into balb/c mice did not result in a further prolonging of the effect (n = 4). these findings indicate that inkt cells are required for such lsec-induced immunosuppression of t cells with indirect allospecificity; however, α-galcer-induced activation of inkt cells does not promote such suppressive effects on these t cells. in conclusion, naive inkt cells play a pivotal role in the lsec-induced immunosuppression of t cells with indirect allospecificity. notwithstanding the considerable amounts of in-vitro data supporting the nonimmunogenicity and immunomodulatory effects of mesenchymal stem cells (msc), scanty and conflicting data are available on their in-vivo immunomodulatory capacities. in this study we formally investigated whether msc had immunomodulatory properties in solid organ transplantation, using a semi-allogeneic heterotopic heart transplant mouse model, and studied the underlying mechanism(s). msc, isolated from bone marrow by adherence, were depleted from cd45+cd11b+ cells before injection. bone marrow-induced hematopoietic mixed chimerism is the most robust mechanism for the induction of transplantation tolerance. however, immunogenicity of bone marrow cells requires harsh immunosuppressive regimens that can lead to severe sideeffects including death. here, we examined whether embryonic stem (es) cells can be successfully coaxed to form hematopoietic progenitor cells (hpc) which potentially could be less immunogenic than bone marrow cells. here, we transduced es cells with hoxb4, a hematopoietic transcription factor that confers self-renewal properties to hematopoietic cells and differentiated them into hematopoietic cells. transduced cells had a 100-1000fold greater proliferation capacity than controls. at the end of the differentiation procedure, most cultures were >80 % cd45 + . hpcs were purified using immunomagnetic bead separation. the separated cells were further characterized for leukocyte markers and showed a high percentage of cd34, cd117, cd31 and low class i, but no class ii expression. further, they poorly express co-stimulatory molecules such as cd80 and cd86. when transplanted in rag2 -/γ c _/_ mice, hpcs fully reconstituted bone marrow, forming multi-lineage hematopoietic cells. to now determine whether these cells engraft in allogenic recipients, the cells were transplanted in syngeneic and allogenic mrl mice. all transplanted animals became chimeric (n>30), reaching 20-30 % after 28 days. thereafter donor cells declined as a result of out-competition by resident bone marrow cells. this pattern was identical in both syngeneic and allogenic recipients. unexpectedly, allogenic chimeric mice became tolerant to donor-type cardiac allografts as monitored over 100 days. grafts showed no mononuclear cell infiltration or signs of chronic rejection. interestingly, the t cells in tolerant animals showed responses to mrl alloantigen similar to that of controls, suggesting that our protocol was likely non-deletional, but could involve regulatory t cells. indeed, when stained for cd25 + foxp3 + cells, the allografts showed a high percentage of these cells, but not in controls, confirming our hypothesis. thus, these data show for the first time the potential of es-derived hpcs to regulate engraftment of allografts, providing an alternative approach for the induction of transplantation tolerance. we had previously shown that a20 is part of the regulatory atheroprotective response of endothelial (ec) and smooth muscle (smc) cells to injury. a20 is a nf-κb dependent gene with potent anti-inflammatory effects in ec and smc, through blockade of nf-κb. a20 also serves an anti-proliferative function in smc and opposite anti-apoptotic or pro-apoptotic functions in ec and neointimal smc. based on these functions, a20 would be a good candidate to prevent transplant arteriosclerosis (ta) and chronic rejection in vascularized organ grafts. this is supported by a20 expression in ec and smc correlating with the absence of ta in rat kidney allografts and long-term functioning human kidney allografts. fully mismatched c57bl/6 (h2 b ) and balb/c (h2 d ), were used as donors and recipients of an aortic to carotid allograft. in this combination, ta lesions start at 4 weeks and become occlusive by 8 weeks when left without immunosuppression. a20 expression in the graft was achieved by recombinant adenoviral (rad) mediated gene transfer prior to retrieval. control mice were infused with saline or control rad beta-galactosidase. the grafts were harvested at 4 weeks and analyzed for ta lesions by measuring intima to media ratios (i/m) and for markers of inflammation and of the immune response by immunohistochemistry. a20 expressing vessels were significantly protected from intimal hyperplasia with i/m reaching 0.4± 0.09 as compared to saline (1.62±0.17) and beta-gal (1.86±0.06) treated vessels. this effect of a20 did not associate with a decrease in infiltrating cd3, cd4 or cd8 t cells in a20 vessels as compared to controls. a possible modification of the phenotype of these t cells (t-regs vs. effector t cells) is being explored. rather, protection from ta correlated with increased expression of endothelial and inducible nitric oxide synthases (nos) in ec and smc of a20 expressing vessels, suggesting that this effect was, at least in part, related to increased in situ production of nitric oxide. in conclusion, we present the first direct evidence that expression in the vessel wall of the anti-inflammatory and atheroprotective protein a20 prevents transplant arteriosclerosis through a mechanism implicating increased expression of nos. carbon monoxide inhalation reverses established chronic allograft nephropathy through the no pathway. g. faleo, a. nakao, j. kohmoto, r. sugimoto, k. tomiyama, a. ikeda, m. a. nalesnik, d. b. stolz, n. murase. thomas e starzl transplantation institute, university of pittsburgh, pittsburgh, pa. chronic allograft nephropathy (can) is the most common cause of graft loss; however an established therapeutic strategy in preventing/treating can is not yet available. we have previously shown that carbon monoxide (co) effectively inhibits can development. here, we examine the mechanisms of co in overturning can through vascular endothelial cell protection. methods: orthotopic kidney transplantation (ktx) was performed in lewis to binephrectomized bn rats under brief tacrolimus (0.5 mg/kg, d0-6, im). by d60 after ktx, bn developed can with decreased creatinine clearance (ccr, 0.58 ±0.1 ml/min), significant proteinuria (92.8 ±30.5 mg/24h), and increased banff scores for intimal arteritis, interstitial fibrosis, and tubular atrophy. recipients were then treated with inhaled co 20 ppm from d60 to d150. results: inhaled co effectively reversed the severity of can and markedly improved renal function at d90 (table) and recipient survival (>150d vs. 81d air control). co treatment resulted in reduced cytokine mrna levels (tnf-α, ifn-γ) and improved banff scores compared to untreated controls. in untreated allografts, cd31 expression on peritubular capillaries (ptc) was markedly diminished, while co-treated grafts showed normal cd31 expression, suggesting significant improvement in maintaining ptc integrity with co. interestingly, enos and inos protein expression was significantly upregulated in untreated grafts, while it was maintained at steady levels in co-treated grafts. immunohistochemistry revealed enos expression on vascular endothelial cells while inos on infiltrates. further, serum nitrate/nitrite levels were significantly higher in untreated than in co-treated recipients. elevated levels of mda, a marker for oxidative stress, in air control group were accordingly reduced in co-treated grafts. renal cortical blood flow data showed a better perfusion in co-treated group at 90d (69. chronic allograft vasculopathy (cav) is a component of chronic rejection and a major cause of graft loss. non-immunologic factors and indirect allorecognition participate in the pathogenesis of cav. new therapies with tolerogenic dendritic cells (dcs) are based on in situ-delivery of alloag to "quiescent" dcs of the recipient's lymphoid organs via apoptotic cells, vesicles or particles. we have shown that the ability of apoptotic cells to deliver alloag and an inhibitory signal to dcs down-regulates the indirect alloresponse and prolongs allograft survival in mice. aims: to test if targeting of recipient's dcs in situ with donor apoptotic cells ameliorates cav by down-regulating indirect pathway allo-immunity. methods: we performed functional aortic (abdominal) transplantation in mice [balb/c→c57bl/6 (b6)]. b6 mice were injected i.v. with 10 7 balb/c uvb-induced early apoptotic splenocytes (d-7). sixty days later, grafts were evaluated in sections with h&e, vangieson's (elastic fibers) and masson's (collagen) techniques. cfse-labeled 1h3.1 tcrtg cd4 t-cells specific for ia b (b6) loaded with ieα 52-68 (balb/c) were used to evaluate the indirect pathway t-cell response. results: pkh67 + balb/c apoptotic cells injected (i.v.) in b6 mice were captured by splenic cd8and cd8α + dcs, but not plasmacytoid dcs. splenic dcs with apoptotic cells remained quiescent in vivo (mhc-i/ii lo , cd80/86 lo , icosl + , pdl-1/2 + ) and were unable to up-regulate mhc-ii and cd86 upon culture with gm-csf. injection of donor apoptotic cells induced defective activation and deletion of indirect pathway 1h3.1 cd4 t-cells. therapy with donor apoptotic splenocytes reduced intimal thickness in aortic allografts (100±21 vs.196±28mm in controls; p<0.001 ) and proliferation of α-smooth muscle cells and collagen deposition. the effect of apoptotic cells was allospecific, superior than that of cells alive, and depended on the physical properties of apoptotic cells, since necrotic cells did not achieve the effect. treatment with donor apoptotic cells decreased significantly the indirect pathway t-cell response (assessed by elispot for ifn-γ) and reduced the level of circulating alloab. conclusion: in situ-targeting of recipient's dcs with (early) apoptotic cells carrying donor alloag is a novel approach to prevent cav by down-regulating the indirect pathway alloresponse. the antifibrotic agent, pirfenidone, has direct inhibitory effects on t cell activation, proliferation, and cytokine and chemokine production, leading to suppression of host alloresponses. gary a. visner, 1 fengzhi liu, 1 hanzhong liu, 1 liqing wang, 2 wayne w. hancock. 2 1 medicine, children's hospital boston, boston, ma; 2 pathology, children's hospital of philadelphia, philadelphia, pa. there is an urgent need to develop new therapies effective against the fibrotic and other complications of chronic allograft rejection. while pirfenidone (pfd) is an established anti-fibrotic agent, we previously showed that pfd treatment reduced acute rejection in a rat lung transplant model suggesting that it might have direct immune modulating properties. accordingly, in this study, we tested the effects of pfd on t cell responses. we first evaluated whether pfd alters t cell proliferation and cytokine release in response to t cell receptor (tcr) activation in vitro. since pfd can inhibit tgf-β by mononuclear cell fractions, we also examined whether pfd affects the suppressive effects of regulatory t cells (cd4+cd25+). the effects of pfd on alloantigen-induced t cell proliferation in vivo were then assessed by adoptive transfer of cfse-labeled t cells across a parent->f1 mhc mismatch, as well as by using a murine heterotopic cardiac allograft model (balb/c->c57bl/6). pfd was found to significantly inhibit tcr-stimulated cd4+ t proliferation in vitro (p<0.01), whereas cd8+ t cell proliferation was not significantly affected. while the beneficial effects of pfd were not associated with increased cd4+ t cell apoptosis, pfd use inhibited tcr-induced production of multiple cytokines and chemokines, including . interestingly, there was no change on tgf-β production by purified t cells, and pfd also had no effect on the suppressive properties of naturally occurring regulatory t cells. similar to the in vitro studies, pfd inhibited allo-antigen-induced t cell proliferation in vivo (parent->f1 model), and showed synergistic effects with low dose rapamycin in this model. lastly, though pfd alone did not affect the tempo of acute cardiac allograft rejection across a full mhc mismatch, use of pfd plus a subtherapeutic regimen of rapamycin significantly prolonged allograft survival (p<0.05), decreased mononuclear cell infiltration and prevented development to chronic rejection, including arteriosclerosis and myocardial fibrosis. we conclude that pfd may be an important new agent in transplantation, with particular relevance to combating chronic rejection by inhibiting both fibroproliferative and alloimmune responses. we have previously reported studies in miniature swine showing that transplantation (tx) of prevascularized donor islets as part of composite islet-kidney (i-k) reversed diabetic hyperglycemia across fully allogeneic barriers, while free islets did not. in order to test the potential clinical applicability of this strategy, we have extended it to a fully allogeneic nonhuman primate model. methods: two diabetic baboons received composite iks and one diabetic baboon received free islets across fully allogeneic barriers. (1) i-k preparation in donors: two i-ks were prepared by isolating islets from 60% partial pancreatectomies and injecting them under the autologous renal capsule, allowing for vascularization before allogeneic tx. these i-ks were harvested at days 203 and 102 for allogeneic ik tx. (2) induction of insulin-dependent diabetes (iddm) and allogeneic i-k or free islet tx: all recipients received streptozotocin at 2000 mg/m 2 x (body surface area). iddm was induced successfully in two animals, while one animal required total pancreatectomy to induce iddm. after confirming iddm by fasting blood sugar (fbs) >300mg/dl for 3 consecutive days, either i-ks or free islets were transplanted (single donor to each recipient) with atg (day -3) followed by mmf and low-dose tacrolimus. free islets were injected into the liver through the ileocolic vein. islet function was assessed by fbs and renal function was assessed by serum creatinine. immunologic status was examined by cml/mlr assays. results: all three recipients had strong ctl/mlr responses to donors pretx, indicative of a fully allogeneic combination. fbs decreased immediately after i-k tx and no insulin therapy was required throughout the experimental period (days 225 and 279). bs levels averaged 82.2+/-15.6 mg/dl in the first baboon and 125.4+/-42.7 mg/dl in the second. normal creatinine levels (<1.0mg/dl) were maintained by these life-supporting ik grafts. in contrast, the recipient of allogeneic free islets had unstable bs levels and required insulin from day 60 (bs 400 at day 60) conclusions: life-supporting i-ks from single donors achieved glucose regulation without insulin therapy and maintained normal renal function. these results demonstrate the feasibility of composite i-k tx in a non-human primate allogeneic model, with possible clinical applicability for the cure of diabetic nephropathy. donor cell infusion without immunosuppression as a novel therapy for induction of donor-specific tolerance in islet cell transplantation. xunrong luo, 1 kathryn pothoven, 1 derrick mccarthy, 2 matthew degutes, 2 aaron martin, 2 xiaomin zhang, 3 guliang xia, 3 dixon kaufman, 3 stephen miller. 2 1 medicine, northwestern university; 2 microbiology and immunology, northwestern university; 3 surgery, northwestern university, chicago, il. background in autoimmune models, peptide-pulsed splenic antigen presenting cells that are chemically fixed with ethylcarbodiimide (ecdi) have been used as a powerful and safe method to induce antigen specific t cell tolerance. ecdi fixed donor cells for allo-antigen specific transplant tolerance has not been well studied. material and methods c57bl/6 mice were rendered diabetic by stz. kidney subcapsular allogeneic islet transplant was performed 10 days after diabetes stabilization (day 0). 1x10 8 ecditreated donor splenocytes were injected i.v. either once (day -7) or twice (day -7 and day +1). animals were analyzed for graft outcome. results control mice receiving islet graft alone rejected the graft between day 11 to day 18 (mst = 14 days, n=8). mice receiving one dose of ecdi-treated donor splenocytes (on day -7) showed similar graft survival as controls (mst = 15 days, n=5). in contrast, mice receiving 2 doses of ecdi-treated donor splenocytes (on day -7 and day +1) showed significant prolongation of graft survival with 72.7% functional grafts at day 60 (n=11), and some remained functional >100 days. this protection is donor-specific as mice receiving ecdi-treated sjl cells rejected balb/c islet grafts as controls (mst = 18 days, n=4). immunohistochemistry of protected grafts showed positive insulin staining within well-defined islet architecture. peri-islet infiltrates were composed of cd4+, cd8+, and cd11c+ cells, with occasional foxp3+ cells. anti-donor antibody production (igg1,g2a,b, g3) was completely abolished in long-term graft survivers. this tolerance was undisturbed by anti-cd25 antibody treatment during maintenance stage, but could not be established if treatment was given around the time of the first donor cell infusion. in addition, lack of pd-l1 also impaired tolerance induction evidenced by using pd-l1 -/as recpients. conclusion 1. multiple infusions of ecdi-treated donor splenocytes significantly prolonged allo-graft survival in the islet transplant model. 2. the protective effect is donor-specific and is dependent on regulatory t cells as well as the pd-l1 signaling pathway. therapy with ecdi-treated donor cells may emerge to be a novel and potent agent for induction of donor-specific transplant tolerance. mice. rebecca stokes, 1 k. cheng, 1 c. scott, 1 w. hawthorne, 2 p. o'connell, 2 j. e. gunton. 1 1 garvan institute, darlinghurst, nsw, australia; 2 nptu, westmead, nsw, australia. the aim was to investigate the effects of increasing hif-1α protein in human islets upon islet-transplant outcomes. hif-1α is a transcription factor which co-ordinates a program of cellular responses to stressors including hypoxia. in other cell-types hif-1α improves survival following hypoxic-challenge. hif-1α functions as a heterodimer with arnt which we have shown to be important for normal β-cell function (1) . islet transplantation subjects islets to hypoxia. it is thought up to 70% of islets die within 1 week of transplantation and this is at least partly due to hypoxia. the role of hif-1α in β-cell function is unknown. we hypothesized that increasing levels of the protective factor hif-1α in islets before transplantation would improve survival and engraftment and thus improve islet transplant outcomes. isolated human pancreatic islets from 9 separate donors were cultured overnight in control media, or media supplemented with desferrioxamine (dfo), a small molecular stimulator of hif-1α protein. islets were transplanted into diabetic scid mice. there were 3 transplant groups, with mice receiving: 1.supra-physiological-mass transplant of 2000 control-cultured ieq (islet equivalents) 2.minimal-mass-transplant of 600 control-cultured ieq, or 3.minimal-mass-transplant of 600 ieq cultured with dfo. for each human donor, at least 1 of each of the 3 transplant groups was performed to avoid the confounder of inter-donor variability. recipients of 2000 control ieq cured in 42% of cases. minimal-mass-transplantation was ineffective: 600 ieq cured 0% mice at 28-days. however, minimal-mass-transplant of dfo treated islets had 53% success (p<0.001 vs group 2 and p=ns vs 2000-control-ieq). blood glucose levels were markedly improved in the 600 dfo treated group compared to 600 ieq control group (p<0.0001) and equivalent to 2000 control ieq transplants (p=ns). this data demonstrates increasing hif-1α in human islets prior to transplantation markedly improves islet transplant outcomes. hif-1α and dfo may have a therapeutic role in human islet transplantation. long-term disappearance of neovascularization of transplanted islets. eba hathout, nathaniel chan, annie tan, john chrisler, john hough, naoaki sakata, john mace, ricardo peverini, richard chinnock, lawrence sowers, andre obenaus. loma linda university, loma linda, ca. we recently reported an in vivo time-line for neovascularization of transplanted islets using dynamic contrast enhanced (dce) magnetic resonance imaging (mri) over a 14-day period. however, vascularization of transplanted islets must be maintained for extended periods to provide long-term function. in this dataset, we investigated whether vascularization was maintained in transplanted feridex-labeled syngeneic murine subcapsular islets (400 ieq per kidney) using dce imaging on an 11.7t mr scanner and subsequent immunohistochemistry over 180 days. sub-capsular transplants could be visualized at post-transplant days 3 and 14 using t2 weighted imaging. however, the islets could not be seen on mri at post-transplant day 180. injection of the contrast agent gadolinium (gd)-dtpa for dce at 3, 14 and 28 days showed increased signal in the transplant area. at 180 days, there was no change in signal intensity after contrast injection during dce. immunohistochemistry confirmed mri and dce findings. these results suggest that islet neovascularization occurs early after transplantation but is likely not maintained for the 180-day duration of our experiments. this work was supported by nih/niddk grant # 1r01dk077541. a) t2 imaging at day 3 clearly identifies iron-labeled islets (arrows) in the subcapsular region. no iron-labeled islets are observed at day 180 (arrows). b) dce imaging for neovascularization of transplanted islets in the subcapsular region demonstrates a temporal decline in signal intensity. oleanolic acid, a natural triterpenoid, significantly improves islet survival and function following transplantation. n. angaswamy, 1 d. saini, 1 s. ramachandran, 1 n. benshoff, 1 w. liu, 1 n. desai, 1 w. chapman, 1 t. mohanakumar. 1, 2 1 surg, wusm; 2 path & immunol, washington univ sch med, st. louis, mo. oleanolic acid (oa), a triterpenoid in medicinal herbs, is an integral part of normal human diet. oa has anti-oxidant, anti-inflammatory properties (inhibits inos & cox2) & lowers plasma glucose levels. we hypothesis that these properties of oa will prevent early islet cell loss following transplantation & also benefit long term function of allograft. c57bl/6 mice, made diabetic by streptozotocin (200mg/kg) were transplanted with 500 balb/c islets (isolated by collagenase digestion) under kidney capsule. oa (0.5mg/day) was administered i.p. in 100 µl of pbs (with 6m dmso) or pbs-dmso as vehicle control daily from day -1 onwards. blood glucose was monitored daily. immunohistochemical analyses of grafts were performed for cd4 & cd8 markers. cellular immune responses to donor antigens & cytokines produced by cells and in sera were measured using elispot & luminex assays. effect of oa on function of transplant with suboptimal dose of islets (100-250) was also analyzed. optimal dose of islets (500) transplanted into diabetic bl/6 mice administered with oa significantly reduced time taken to reverse diabetes following transplantation (<2±1 vs 4±2 days, p=0.003). further, oa treatment reversed diabetes even with suboptimal dose (200) of islets while untreated animals did not achieve normoglycemia. as expected, control diabetic mice rejected on 6±2 days whereas, oa administration alone prolonged islet allograft survival to 23±3 days (p<0001). oa treatment resulted in >3 fold increase in serum kc, il-10 & vegf (p<0.0003) & 2 fold decrease in mcp-1, ip-10 & il-4 (p<0.005) in luminex assay. stimulation of splenocytes from oa treated mice with donor balb/c cells resulted in significantly reduced ifng (4.5 fold), il-4 (3.5), il-2 (2.3) & il-17 (4). in addition, proliferation in mlr was also reduced 2.5 fold. immunohistochemical analysis of grafts showed significant reduction in cellular infiltration in oa treated animals with reduction in both cd4 and cd8 t cells. daily administration of oa markedly improved islet engraftment & function with reversal of diabetes even when suboptimal dose of islet were transplanted. further, oa treatment allowed significant long term survival of allograft with no other immunosuppression. we demonstrate that prevention of inflammatory signaling cascades by oa resulted in marked reduction of cellular infiltration into graft allowing long term function of allograft. endoplasmic reticulum stress may be an important cause of cell loss after human islet isolation. soon hyang park, 1 michel tremblay, 2 steven paraskevas. 1 1 surgery, mcgill university health center, montreal, qc, canada; 2 mcgill cancer center, mcgill university, montreal, qc, canada. purpose: to evaluate the presence of endoplasmic reticulum (er) stress, induced by conditions to which islets are subjected during isolation (ischemia, nutrient deprivation, thermal stress and cytokine release) in human islets and to determine if this leads to the unfolded protein response (upr), which could alter cell survival. methods: human islets were purified from cadaveric pancreata by collagenase dissociation and continuous density gradient purification. islet preparations were cultured in serum-free medium and sampled at the end of isolation and daily thereafter. total mrna was purified and gene expression evaluated by rt-pcr. activity in upr signaling pathways was evaluated by immunoblot. apoptosis was measured by a caspase-3 activity assay. representative trends observed in >5 isolations are described. results: following isolation, a rapid increase in upr signaling was observed in the perk and ire-1 modules of the upr. these include the phosphorylation of perk target eif2? and splicing of mrna for the transcription factor xbp-1. these changes occurred concurrently with a rapid spike in jnk activity and a rise in expression of the upr target gene chop. after these signals peaked, caspase-3 activity increased with time (apoptotic cells), as did expression of er chaperone bip (surviving cells). conclusion: we consistently observed upr activation in human islets. er stress and the upr may be one important and unrecognized cause of apoptosis in this context. current investigations focus on upr modification and determination of a causal relationship with apoptotic cell death. immunosuppression and the risk of renal transplant failure due to recurrent glomerulonephritis. atul mulay, 1 carl van walraven, 1 greg knoll. 1 1 the ottawa hospital, ottawa, on, canada. glomerulonephritis (gn) is the most common cause of end-stage renal disease among those who undergo kidney transplantation. recurrent gn is a major cause of kidney transplant failure. immunosuppressive medication is used to treat gn in the native kidney prior to the development of end-stage renal disease but the impact of different immunosuppression on recurrent gn post-transplantation is unknown. we used the united states renal data system to determine the association of routine post-transplantation immunosuppressant use with time to renal allograft failure due to recurrent gn. immunosuppressants were treated as time-varying covariates. the study-cohort included patients with kidney failure due to gn who received first kidney transplant between 1990 and 2003. the study cohort included 41,272 patients with a median follow-up of 51 months. ten-year overall graft survival (including death as graft loss) and death-censored graft survival was 56.2% and 70.5% respectively. use of cyclosporine (hazard ratio 0.93; 95% ci 0.62-1.41), tacrolimus (hazard ratio 1.03; 95% ci 0.67-1.60), azathioprine (hazard ratio 0.88; 95% ci 0.68-1.13) or mycophenolate mofetil (hazard ratio 1.10; 95% ci 0.85-1.40) was not associated with risk of graft failure due to recurrent gn after adjusting for important covariates. there was no difference of recurrent gn causing graft failure between cyclosporine and tacrolimus (p=0.4) or between azathioprine and mycophenolate mofetil (p=0.1). however, change in any immunosuppressant during follow-up was independently associated with graft loss due to recurrence (hr 1.31, 95% ci 1.07-1.60, p=0.01).when we restricted the analysis to patients who had no change in immunosuppression during follow-up we again found no association between any of the immunosuppressive medications and the risk of graft loss due to recurrent gn. despite the increased use of tacrolimus, cyclosporine and mycophenolate mofetil to treat gn in native kidney disease, the use of these medications following kidney transplantation had no impact on the risk of graft loss due to recurrent gn. glomerulosclerosis. junichiro sageshima, 1 gaetano ciancio, 1 alessia fornoni, 1 linda chen, 1 carolyn abitbol, 1 jayanthi chandar, 1 warren kupin, 1 giselle guerra, 1 david roth, 1 sherry shariatmadar, 1 gaston zilleruelo, 1 george w. burke iii. 1 1 university of miami miller school of medicine, miami, fl. background: disease recurrence is a major obstacle of kidney transplant for focal segmental glomerulosclerosis (fsgs). anti-cd20 antibody (rituximab) has been used for nephrotic syndrome of native kidney. the significant reduction of proteinuria in transplant recipients with fsgs recurrence was also reported after rituximab use for posttransplant lymphoma. we hypothesized that rituximab induction could alter the posttransplant course of fsgs recipients, particularly in those patients with rapid progression to end-stage renal disease who are higher risk of recurrence. methods: we compared the outcome of transplants for primary fsgs treated with and without rituximab. from jan. 2000 to dec. 2003 received renal allografts along with our "standard" immunosuppressive protocol, consisting of tacrolimus, mycophenolate, corticosteroids, antithymocyte globulin and/or daclizubab. from jan. 2004 to dec. 2007 received rituximab in addition to the "standard" immunosuppression. posttransplant proteinuria was treated with plasmapheresis (pp) and maintenance angiotensin blockade (ab). results: there was no adverse event related to rituximab infusion. the overall incidence of posttransplant proteinuria was significantly lower in recipients with rituximab induction (p < 0.05). four recipients treated with "standard" immunosuppression developed massive proteinuria (u-protein/creat. > 10) immediately following transplantation; they responded poorly to pp and ab. four other recipients had moderate proteinuria. in contrast to this, of the 18 patients induced with rituximab, only 2 had massive proteinuria and 4 had mild to moderate proteinuria which responded well to pp and ab. with a median follow-up of 26 months, there was no significant difference of graft survival between 2 groups (2-year survival: 81% without rituximab vs. 84% with rituximab). a half of the graft loss was related to non-compliance. conclusion: while the mechanism of action is unclear, our observation indicates that rituximab induction may decrease the incidence and severity of recurrence of fsgs following kidney transplantation. a larger-scale study is desirable to confirm this observation. mesangial chimerism in recurrent iga nephropathy. geoffrey talmon, 1 dylan miller. 1 1 department of pathology and laboratory medicine, mayo clinic, rochester, mn. background iga nephropathy (in) is the most common primary glomerulonephritis and nearly 50% of patients who undergo a renal transplant for in recur. data support that bone abstracts marrow-derived cells are capable differentiating into various mesenchymal cells within the kidney. the extent to which this phenomenon versus proliferation of resident mesenchymal cells is involved in populating mesangium is not well understood. the mesangial injury and/or hypercellularity seen in in provides a robust in vivo model for determining if this phenomenon is prevalent in human kidneys. design follow-up biopsies from male patients receiving female renal allografts for in that showed recurrent disease were selected. fluorescent in-situ hybridization and immunofluorescent staining was performed on unstained slides from the paraffinembedded tissue for smooth muscle actin, x, and y chromosome centromeres. cells within nonsclerotic glomeruli with triple positivity (y+) were assumed to be mesangial cells derived from the recipient. results four cases of recurrent in with nonsclerotic glomeruli were obtained, each displaying at least minimal mesangial proliferation by light microscopy (one "minimal", two "mild", one "moderate"). mesangial cells with y chromosome centromeric material were observed in each case (100%). between 6 and 34 mesangial cells were present in each glomerulus (mean 16.45) with no to three y+ cells seen (mean 1.8). these accounted for between 8% and 16% of mesangial cells in individual glomeruli. the ratio of y+ to total mesangial cells in each case ranged from 1:7.8. to 1:11.3 (mean 1:9.35 ). the case exhibiting minimal mesangial hypercellularity had a ratio of 1:9.6, those with mild had ratios of 1:7.8 and 1.8.7, and that with moderate 1:11.3. conclusions recipient-derived mesangial cells make up a fraction of the population of glomerular cells in renal allografts affected by recurrent in. although the number of cases is small, the number of recipient-derived cells does not seem to be directly related to the degree of mesangial hypercellularity seen by light microscopy. the consistent presence of these "colonizing" cells in patients with recurrent in does, however, suggest that there may be a role for targeted therapy directed against circulating recipient cells. in the mid 1970's, patients developing esrd secondary to systemic lupus (sle) were deemed to be poor transplant candidates because of concern for early recurrent lupus nephritis (rln) leading to allograft loss. subsequently, rln was considered an unusual complication of kidney transplantation, occurring in <4% of allografts. however, over the last decade, several reports have shown the frequency of rln to range from 8-30%. we sought to determine the frequency of rln at our center and to identify any clinical variables associated with rln. between 6/1977 between 6/ -11/2005 allografts in 166 patients with esrd due to sle functioned for more than 90 days after engraftment. immunosuppression consisted of azathioprine (aza), or cyclosporine (csa) and aza, or mycophenolate (mmf) and csa, or tacrolimus and mmf depending on the date of transplant. all received steroids. proteinuria was defined as 2+ on dipstick or urine protein/creatinine ratio >0.5. medical charts were reviewed. we found pathologic evidence of rln in 18 (24%) of 75 patients who underwent biopsy due to allograft dysfunction or proteinuria, comprising 10% of all patients transplanted for sle. characteristics of these patients are shown below: randomized studies have shown little or no increase in ar in kidney tx recips on p-free is; but concern remains about long-term outcome. we present 8-yr f/u of a protocol incorporating rapid (<6 days) discontinuation of p, with now over 1000 patients transplanted using this protocol. between 1/1999 between 1/ and 10/2007 between 1/ , 1003 adult tx recips were treated with thymoglobulin (5 doses)(extended in dgf), p (5 days), a cni, and either mmf or srl. of these, 658 were ld (260 lurd); 345 dd. of the 1003, 41% were female;88% white; mean recipient age was 47± 14 years and mean donor age was 39.7± 13.2 years. diabetes was present in 34.3% of the recipients and 12.5% of the transplants were retransplants. the peak pra was >10% in 20% of recipients; 14% had tx pra >10%. table 1 shows actuarial survival rates. graft survival rates were significantly better in ld vs dd transplants (p=0.002) and and acute rejection rates lower (p=0.04). compared to national data from srtr, overall outcomes were not significantly different. with mean follow-up of 3.5 years, a total of 110 (11%) recipients have died -the most common cause being cerebrovascular accident (14%) followed by malignancy (9%). there were only 5 (4.5%) patient deaths due to cardiac causes. of 195 (19.4%) graft losses, 86 (44%) were from dwf (death with function); 43 (10%) from cr/can. renal function has been stable with mean serum cr of 1.7 mg/dl at 3 and 5 years posttransplant with cretinine clearance of 63 and 60 respectively. at 5 years posttx, compared to ppretransplant values, recipients showed a 9.6% increase in weight, a 3% decrease in serum cholesterol, and a 10.8% decrease in serum lipid values. 84% of the kidney recips remain p-free; the most common reason for restarting p was acute rejection (ar). conclusion: short-term data suggests kidney tx recips do well with rapid discontinuation of p. our intermediate-term data suggests that patient and graft survival rates remain good and renal function remains stable. ongoing long-term follow-up is necessary. background. since 4/02 our program has employed a steroid-free, rapamycin and neoral maintenance immunosuppression regimen for kidney transplant recipients. prior to that time recipients were treated with prednisone, mmf and neoral. we noted a significant reduction in acute cellular rejection (acr) after implementing this regimen. this retrospective analysis was performed to examine the impact, if any, on the incidence, character, and outcomes of early ahr. results. this study includes 1611 consecutive kidney recipients transplanted between 1/99 and 12/06. there were 717 recipients in the prednisone, mmf, and neoral era (grp 1) and 894 in the steroid-free, rapamycin and neoral era (grp 2). recipient age, gender, african-american race, ab and dr mismatch, and frequency of pra>10% was not statistically significantly different between the 2 groups. however, 43% of grp 1 pts vs 63% of grp 2 pts received a living donor kidney due to a more recent volume increase in this procedure (p<0.001). there were a total 722 kidney biopsies in 466 pts performed in the first 6 months post-transplant; 478 in 287 pts when excluding pre-perfusion biopsies. nineteen percent (54/287) of these showed >50% peritubular capillary (ptc) c4d deposition. comparison of grp 1 to grp 2 demonstrated the following: 1) the incidence of clinical acute rejection in the first 6 months was 12.7% (91/717) in grp 1 and 5.6% (50/894) in grp 2 (p<0.001), 2) the overall incidence of a c4d+ biopsy was similar in the 2 groups ( conclusions. despite a significant reduction in the incidence of acute rejection using our newer, steroid-free immunosuppression protocol, there has been no reduction in the incidence of early (0-6 months) ahr evidenced by c4d+ kidney biopsy. however, the percentage of ahr unassociated with acr has significantly increased. the poor graft survival in pts with early ahr has not improved with our newer immunosuppression regimen. conclusions: four risk factors for ar were identified in the rdp study population: retx, aa race, age 18-50 (vs. >50) and pra >50 (vs. <50). four risk factors for gl were identified: pre-tx t1 dm, ar, dgf and dd tx (when ar and dgf omitted). these risk factors for ar and gl are the same as we observed in prednisone-containing protocols. additionally, many of these factors are not modifiable. identification of high risk groups allows for individualization of is. increasing lds and utilization of is protocols to decrease or minimize dgf and ar are goals for improving graft outcome. effect with the advent of more potent immunosuppression hla matching has been deemphasized in the allocation of deceased donor kidneys due to the limited impact on acute rejection and graft survival. an unforeseen consequence of poorer matching could be an increase in sensitization of patients in need for a repeat transplant. our study examined candidates listed in the us from 1988-2007 from the srtr database that were re-listed following loss of a primary kidney transplant (n=19,827). the primary outcome of the analysis was change in pra from the listing prior to recipient's initial transplant to the subsequent listing. absolute change in peak and current pra levels were examined in general linear models as well as the proportion of patients with a rise in pra level in a logistic model. results hla(a,b,dr)-matching in the primary transplant was strongly associated with change in pra level (p<.001, figure 1 ). among recipients with 6-hla mm, over 50% had a rise in pra at re-listing as compared to 25% of 0-hla mm recipients. younger recipient and donor age, males, deceased donor transplants and african american recipients were also significantly associated with elevation in pra. in addition, the effect was apparent stratified by primary donor type. while there might be a limited impact of hla matching on graft survival, many patients might be negatively impacted from poor hla matching from their first transplant when needing a second transplant. as high pra is one of the strongest risk factors for not getting transplanted, this should be taken into account when evaluating the impact of hla matching in kidney transplantation. this might be particularly important in younger patients and in patients with a long life expectancy in general because of the high likelihood of needing a second transplant during their lifetime. the were about 25% less likely to die or lose an ecd kidney from a donor aged 50-59 and 70% more likely with kidneys from donors older than 69. a similar trend was seen with older recipients, but the risks seemed to be lower. logistic regression indicates recipients older than age 65 were 5 times more likely to have a donor older than age 70 than recipients younger than 50, hypertension and creatinine >1.5 were less likely in older donor kidneys. interestingly, the use of these kidneys relative to the total number of ecd kidneys has decreased since 2002 (odds ratio=0.63, 0.53-0.76, p<0.001). conclusion: an increase in the supply of kidneys might be achieved with increased utilization from deceased donors older than age 70. outcomes were similar to those from donors age 60-69 in recipients that were older than age 50. outcomes , but approximates non-dcd survival thereafter. dcd listing for retransplantation and graft failure progressed continuously over 180 days versus 20 days in non-dcd. when retransplanted, dcd recipients waited longer and received higher risk allografts (p=0.039) more often from another region. more dcd recipients remain waiting for retransplantation with fewer removed for death, clinical deterioration, or improvement. conclusions: dcd utilization is impeded by early outcomes and a temporally different failure pattern that limits access to retransplantation. allocation policy that recognizes these limitations and increases access to retransplantaton is necessary for expansion of this donor population. orthotopic liver transplantation with allografts from dcd donors. roberto c. lopez-solis, 1 background: in the current era of liver transplantation, organ shortage continues to be a significant problem. the use of extended criteria allografts from donation after cardiac death (dcd) donors to increase transplantation rates is widely practiced. this study is a review of one of the largest single center experiences utilizing dcd donors in the world with a follow-up of almost 15 years. methods: from 03/01/1993 to 10/31/2007, 3,431 liver transplants were performed at our institution, 146 (4.2%) of which were liver allografts from dcd donors. patient and donor demographics, recipient and graft survival, and the incidence of primary non function, hepatic artery thrombosis, retransplantation, and bile duct complications were analyzed for this subset of recipients. results: kaplan meier analysis showed a 1-and 5-year patient and graft survival of 81% and 71%, and 71% and 59%, respectively. the mean age of recipients was 52 ± 10 years with an average meld score of 18.7 ± 9.3 (range, 6 to 40), and there were 101 male patients (69%). donor mean age was 36 ± 16 years and cold ischemia time was 646 ± 173 minutes. one hundred and three patients (70.5%) are alive and 24 (16.4%) underwent retransplantation. the incidence of primary non-function was 11.6% (17 patients) and hepatic artery thrombosis was 4.1% (6 subjects the fda warns against using sirolimus (srl) in liver transplants, reporting increased hepatic artery thrombosis (hat), excess mortality and graft loss when srl is used as initial immunosuppression (is) with calcineurin inhibitors. we report the largest experience to date of patients with srl used as initial is, assessing hepatic artery complications and survival outcomes. materials and method all 1554 olt pts from 1998-2007 were reviewed. those using srl as initial is were identified, and the remaining olt pts from that time period were used as controls. ultrasound assessed graft vascular status and any issues were verified by angiogram. . there were no significant difference in demographics variables, lt indication or pre-lt meld score between the two gr. mean follow-up was 6.8±3.9 months. all the enrolled pts were treated with abstracts an initial dose of cs of 2mg/kg/day, to target 100ng/ml for the first 10 days. pts were randomized on day 10 into one of the two following gr on a 2:1 basis. ev gr: initial dose of ev was 2 mg/day, to reach blood level of 8 ng/ml. the dose was increased on day 30, when cs was discontinued, in order to reach an ev blood level between 10 and 12 ng/ml. cs gr: after the 10 th post-operative day the dose of cs was adjusted to a target level of 250 ng/ml until day 30, then to 200 ng/ml until the end of month 6. all pts received basiliximab induction on day 0 and 5 after lt. pts were weaned off prednisone by 5 weeks. pt survival at 6 and 12 months was similar in the ev and cs gr (95.2% and 86.6% vs 87.5% and 87.5% respectively; p= ns). causes of death were sepsis (1), hcv recurrence (1), pulmonary embolism (1) in the ev gr, and sepsis (1), rupture of splenic artery aneurysm (1) the overall incidence of infection episodes was comparable between two gr (5.9% ev gr vs 12.5% cs gr; p=ns). cholesterol but not triglycerides increased in the ev gr compared with the cs gr (p<.05); ev dose reduction decreased such parameters without the need for statin implementation. conclusion ev monotherapy in de novo lt showed similar patient survival and incidence of morbidity compared to a cs immunosuppressive protocol. the primary endpoint was achieved inasmuch as renal function was statistically better in the ev gr. background: sir is a potent immunosuppressive agent that inhibits t-cell activation and proliferation. in lt recipients, sir has primarily been used as a renal-sparing agent, but its toxicity and tolerability in this population has not been well defined. aims: to identify the adverse effects and predictors of discontinuation of sir in lt recipients. methods: records from 327 adult lt recipients transplanted between 1/2000 and 12/2006 were reviewed. reasons for starting and discontinuing sir were captured, as were all significant adverse effects and laboratory abnormalities. factors predicting sir discontinuation in univariate analysis were further analyzed by multivariable logistic regression (mlr). results: mean age of the study group was 50 ± 11 years, and 75% were male. underlying liver disease was hcv ± alcohol in 56%, 24% had hepatocellular carcinoma, and 14% received living donor grafts. calcineurin inhibitors (cni) were started post-operatively in 91% (85% tacrolimus/15% cyclosporine), with or without mycophenolate and prednisone. 179 patients (54%) started sir a median of 15 days (iqr: 4-138) post-lt primarily for renal insufficiency (76%) or cni neurotoxicity (7%). sir was overlapped with tacrolimus and cyclosporine in 65% and 16%, respectively. prior to starting sir, total cholesterol was 139 ± 69 mg/dl, ldl-cholesterol 90 ± 49, triglycerides 193 ± 126. peak lipids after sir were 263 ± 97, 138 ± 81, and 475 ± 456 mg/dl, respectively, despite lipid-lowering therapy. serum creatinine was 2.05 ± 1.04 and 1.08 ± 0.5 mg/dl before and after sir, respectively. before sir, 70% of patients had no proteinuria, but only 36% had no proteinuria after sir. high range proteinuria (>300mg/dl) was noted in 3% before and 16% after sir. finally, sir was discontinued in total of 81 (45%) patients, for indications of cytopenias (20%), hyperlipidemia (19%), mouth ulcers (11%), sepsis (7.5%), skin reactions (7.5%), nephrotic syndrome (6%), gi intolerance (4%), pneumonitis/boop (2.5%), myopathy (2.5%), and combinations of above (20%). mlr failed to identify any pretreatment predictors of discontinuation. conclusions: immunosuppression with sir improves azotemia at the expense of considerable hematologic, metabolic, dermatologic, renal, pulmonary and muscle toxicity. considering the high incidence of proteinuria after sir treatment, the use of sir as a less nephrotoxic agent must be re-considered. and to identify the most effective protocol. peripheral blood was obtained from 4 ebvseronegative and 4 ebv-seropositive pediatric heart (h) tx patients. lcl vs dc-based methods were compared as follows: (i) lcl (ii) lcl + il-12 (iii) type-1 polarized dc (treated with il1-b, tnf-a, il-6 and ifn-g) loaded with mhc class i-restricted ebv-peptide pool (dc/pep.) and (iv) dc/pep. + il-12. the ebv-specific cd8 + t cell phenotype and function were screened using flow cytometry, ifng elispot and cytotoxicity assays. the yields and the functional activities of in vitro co-cultures differed based on the induction method employed, and on the ebv status of the patients tested. for the ebv-seropositive pediatric htx patients, all four methods resulted in the successful expansion of functional type-1 ebv-specific cd8 + t cells, suggesting that memory cd8 + t cell are readily reactivated in vitro. for the ebv-seronegative pediatric tx patients however, only the lcl + il-12 approach resulted in significant augmentation of type-1 ebv-specific ctls that were competent to secrete ifn-g (400±50/10 5 cells) and to kill (300±170 lu/10 7 cells) ebv + targets. we found that il-27 secreted by lcl (and not by dc) was critical in triggering expression of il-12rb2 on naive cd8 + t cells, and rendering these cells responsive to il-12p70. further addition of exogenous il-12p70 (which is generally not produced by lcl) proved to be essential for effective type-1 priming. however, blocking il-27 during ebv-priming has abolished il-12rb2 expression and subsequent ifng production. these results demonstrate that the inducible expression of il-12rb2 on naïve cd8+ t cells was dependent on il-27, and support the critical early role of ebv infected b cells in the in vivo priming of naïve precursors into potent ebv type-1 cd8 + t cell in children. serial ebv load monitoring in pediatric heart transplant patients (phtx) has identified a group of asymptomatic children that exhibit persistently high ebv loads in peripheral blood (≥16,000 copies/ml on at least 50% samples over a period of at least 6 months). these patients have a high rate of progression to late ptld. our goal is to characterize the deficiency of ebv-specific cd8 + t-cell immunity that allows this state to occur and be maintained. twenty-one stable ebv + phtx patients were categorized as follows: group 1 (n=6) no detectable viral load; group 2 (n=12) low viral load (≤16,000 copies/ ml); group 3 (n=4) high viral load (≥16,000 copies/ml). twelve healthy subjects were recruited as controls. flow cytometric analysis with hla-a2 ebv-tetramer (tmr) probes in conjunction with mabs against memory/activation markers was performed on peripheral blood cd8 + t cells, and their ebv-specific ifn-γ production was measured by elispot. ebv-"latent" specific cd8 + t cells in g2 patients were mostly cd62l + / cd45ro + (central memory) and expressed heterogeneous levels of pd-1 and high cd127 (il-7 receptor α), the ebv-lytic-specific cd8 + t cells were more frequent, and biased toward cd62l -/cd45ro + (effector memory) and cd62l -/cd45ra + (stable effector memory), corresponding to terminally differentiated memory compartments. this cell population also expressed heterogeneous levels of pd-1 and down-regulated cd127. in contrast, both ebv-lytic and -latent specific tmr + cd8 + t cells from g3 patients were homogeneously cd62l + /cd45ro + (effector memory), cd38 + and cd127 -, suggestive of "recently activated" phenotype. interestingly, although patients in groups g2 and g3 had high frequencies of ebv-specific tmr + cd8 + t cells (g2 0.65%±1.5, g3 1.9%±3.5, p=0.3), only g2 patients exhibited a direct correlation between tmr + cd8 + t cells and ebv-specific ifn-γ production. these results demonstrate that different levels of chronic ebv-antigenic pressure trigger significant differences in the phenotypic and functional features of ebv-specific cd8 + t cells from phtx, suggesting that the immunologic characterization of high ebv load carrier state is a combined "activated" phenotype with "exhausted" function of ebv-specific cd8 + t cells. ebv-encoded lmp1 indirectly activates the jak/stat pathway through induction of ifnγ. abstracts evidence of ptld. 15 children were enrolled at 5 sites. mean age at transplant 7.1 years, mean time to ptld 43 months . organs transplanted were lung 6, heart 5, kidney 4. all ptld were of b cell origin and expressed cd20 and all were ebv positive. histology was: polymorphic 11, monomorphic 3, hodgkins-like 1. 7 patients received 4 doses, 7 patients 8 doses and 1 patient 7 doses. treatment was associated with minimal side effects in 12, mild-moderate infusion related reactions in 2 and moderate reaction in 1. no patient had treatment discontinued because of side effects. twelve patients (80%) showed complete response after 4-8 doses, 2 had progressive disease and one had stable disease. at 24 months, 10 (67%) were alive with one graft loss (kidney) and none with residual disease. at latest follow-up (mean 60.4 months, 42-82), 10 remain alive with 1 further graft loss (lung) and no ptld. the 5 deaths occurred between 2.5 and 13 months and were associated with progressive disease (2), chronic rejection (2), and complications of elective surgery (1) . conclusions: these findings support prior registry data and suggest that rituximab without chemotherapy is a successful second line treatment in approximately two-thirds of children with refractory ptld. cancer after organ transplantation in france. jean michel rebibou, 1 fabienne pessione, 1 francois aubin, 1 bernard loty. 1 1 agence de la biomedecine, france. cancer prevention appears as a major challenge in transplantation. estimating cancer frequency is a major step toward designing prevention policy. we report on patterns of cancer incidence among 47000 transplantations registered in the french data base. ). the risk of lung cancer is higher for recipients of a thoracic organ and the risk of kidney cancer appeared higher for kidney recipients. ten year cumulative incidence was 8.6% for all cancer and transplantation types, 1.7% for nhl. multivariate analysis demonstrated that cancer risk increased with recipient age (p<10 -3 ), 10 year cumulative incidence was 15% for recipients older than 60 year. it was higher in male (p<10 -3 ) and in thoracic organ recipients when compared with kidney recipients (p<10 -3 ). cancer incidence did not vary according to the transplantation period (1995 ( -1999 ( vs 2000 ( -2005 .95 p=0.34) and nhl risk was significantly lower during the period 2000-2005 (rr=0.8, p=0.02). this work does not report any increase in cancer incidence among transplant recipients while cancer incidence increased in the general population. the observed decrease of nhl risk is of particular interest. the both costimulatory and co-inhibitory signals are delivered by b7 ligands through the cd28 family of receptors on t lymphocytes determining the ultimate immune responses. although b7-h4, a recently discovered member of the b7 family, is known to negatively regulate t cell immunity in autoimmunity and cancer, its role in transplantation rejection and tolerance has not been established. to study its role in physiologic rejection processes, we first treated b6 wt recipients of balb/c hearts with a blocking mab against b7-h4 or isotype igg control and found no difference in graft survival (mst 7 days, n=8 vs. 7, n=10). however, b7-h4 blockade resulted in accelerated allograft rejection in cd28 deficient b6 recipients (mst 9.5 vs. 19, n=8, p=0.003) , indicating that b7-h4 signaling can mediate negative regulation in the absence of cd28 costimulation. we next studied b7-1/b7-2 double deficient (dko) b6 recipients of balb/c heart allografts, as these mice are truly independent of cd28/ctla-4:b7 signals. while cardiac allografts were accepted in control dko recipients (mst>100, n=5), blocking b7-h4 precipitated rejection (mst 45, n=5, p=0 .01) demonstrating non-redundant functions of these two negative pathways. based on these results, we next evaluated the role of b7-h4 in acquired transplantation tolerance by blocking cd28:b7 using ctla4-ig. b7-h4 blockade abrogated prolongation of allograft survival by ctla4-ig (250 µg on day 2) in the fully mhc-mismatched cardiac allograft model (mst 13 vs. 46.5, n=8, p=0.0002) . we conclude that the novel b7-h4 molecule can regulate alloimmune responses independent of an intact cd28/ctla-4:b7 costimulatory pathway. the interplay between positive (stimulatory) and negative (regulatory) costimulatory signals is an important determinant of the outcome of the alloimmune response and could be exploited to induce tolerance. specific in a model of mhc-mismatched kidney allograft in the rat, treatment with anti-cd28 antibodies induced a form of tolerance independent of treg cells but associated with a two-fold accumulation of mdsc in the blood. to further characterize these cells, we analyzed their phenotype and mechanism of action by flow cytometry, western blotting and suppression assays. mdsc expressed cd80 and cd86, nkrp-1, cd172a (sirpa), cd11a, cd11b, his48 and for a fraction of them cd4 but did not express mhc class ii molecules. mdsc dose-dependently suppressed the proliferation of t cells in mlr and after stimulation with anti-cd3 + anti-cd28 antibodies. although detected in blood, bone marrow, spleen and lymph nodes, mdsc were only suppressive in blood and bone marrow. this suppression was lost after physical separation from the responding t cells by semi-permeable membranes in transwell assays, as well as after addition of l-nmma, a selective inhibitor of inducible nitric oxide synthase (inos), suggesting a role for no in the suppression. western blot analyses revealed that inos was expressed only after contact between mdsc and activated cd4 + cd25effector t cells and to a much lesser extent after contact with activated cd4 + cd25 high t reg cells. mdsc affected the viability of stimulated cfse-labeled effector t cells by blocking their proliferation but not their activation. in contrast, mdsc did not block the response of t reg cells stimulated by anti-cd3 + anti-cd28 antibodies. this selective suppression of effector but not of regulatory t cells was confirmed by cytokine profile analyses. in vivo, the expression of inos was higher in the blood of tolerant recipients, as well as in the graft, as compared with isografted recipients. in addition, the injection in stable tolerant animals of aminoguanidine, which inhibits inos, induced graft rejection within 3 weeks. in conclusion, these results suggest that mdsc, accumulated in the blood of tolerant recipients of kidney allografts, release high levels of no after contact with activated effector t cells and specifically control their proliferative response. liver non-parenchymal components inhibit dendritic cell differentiation and maturation. ching-chun hsieh, 1 horng-ren yang, 1 guoping jiang, 1 john j. fung, 1 shigunag qian, 1 lina lu. 1 1 immunology and general surgery, cleveland clinic, cleveland, oh. the inherent tolerogenicity of liver allografts could be due to comparatively large numbers of potentially tolerogeneic antigen presenting cells, in particular dendritic cells (dc). it is not clear whether the unique antigen presenting function of liver dc is intrinsic, or is altered by the microenvironmental factors within the liver. in the present study, we investigated the effect of hepatic stallet cells (hpsc), a unique tissue cells in the liver which were actively expanded in the liver allografts, on generation and function of bone marrow derived dc (bm dc). we hypothesize that liver hpsc may modulate immune response via inhibition of liver dendritic cells (dc) which are known of bone marrow (bm) origin. in this study, dc were propagated from b6 bm cells with gm-csf for 5 days. irradiated hpsc isolated from b6 mouse liver were added at the beginning into the culture at hpsc : bmdc progenitor ratio of 1:20. the differentiation, maturation and function of propagated dc were determined by characterizing their surface molecule expression and functions of instructing t cell activation / differentiation. the results showed that addition of hpsc markedly blocked the differentiation of dc from bm precursors (most cells remained in cd11b + cd11cprecursors stages). the incidence of cd11c + cells was 7% vs. 41.6% in normal bm dc culture (without hpsc). the presence of hpsc also prevented maturation of cd11c + dc, as evidenced by low expression of cd40, cd80 and cd86, but high of b7-h1. the inhibitory effect appeared to be mediated by soluble factor (s) produced by hpsc, since addition of the hpsc culture supernatant or transwell culture provided comparable inhibitory activity. culture of allogeneic cd4 + t cells with hpsc-dc elicited poor proliferative response in a 3d mlr assay, with low il-2 and ifn-γ production. three color staining of t cells stimulated by hpsc-dc showed that cd25 + foxp3 + t cells were preferentially expanded, suggesting that hpsc-dc were capable of inducting treg. in contrast, bm dc induced vigorous cd4 + t cells proliferation, most of activated cd4 + t cells were cd25 + foxp3associated with high levels of ifnγ and il-2 in the culture, indicating induction of th1 cells. in conclusion, liver tissue cells, such as hpsc markedly inhibit dc differentiation and maturation, suggesting that the tolerogenic property of liver dc may not be intrinsic, but is altered by the microenvironmental factors in the liver. . allograft rejection was also significantly accelerated when bm12 hearts were transplanted into cd28ko recipient (mst 14 days). to investigate the mechanisms of these findings, lymphocytes harvested at day 10 post-tx from spleens and regional lymph nodes were assessed by flow cytometry for phenotypic differentiation of the activation status, regulatory t cell markers and effector cell generation. the ratio of effector to regulatory cells was 1.4: 3.8: 5.2 in the wt, cd28ko and b7dko recipients, respectively. cytokine production was evaluated by elispot using donor specific antigen stimulation of recipient splenocytes. the mean ifn-γ production was 173 spots per 500,000 splenocytes in wt, 206 in cd28ko and 702 in b7dko. this study for the first time demonstrates the paradoxical role of b7-cd28 co-stimulation in fully allogeneic and class ii mismatched grafts and proposes a possible mechanism through the re-alignment of the effector to regulatory t cell ratio in favor of a highly alloreactive immune response. the major concern regarding kidney donation has been whether the occurrence of hyperfiltration, on the background of increasing prevalence of hypertension with aging and the decline in glomerular filtration rate (gfr) noted in some people as they get older, may put donors at a higher risk for progressive kidney disease. we have previously reported on donors > 20 years after donation and found them to incur no excessive risk of hypertension or kidney disease. herein, we report on renal and non-renal outcomes on the world's largest experience of kidney donors to date (n=302) who donated more than 35 years ago. methods: kidney donors were asked to fill out a survey detailing their medical history since donation and obtain a physical exam and laboratory testing by their local physician. results are expressed as mean±standard deviation (sd the graph below depicts the cross-sectional distribution of last serum creatinine available 35 years or more after donation regardless whether the donor is presently alive or not though the majority is from currently alive donors. conclusion: in the longest follow-up of kidney donors to date, this data indicates that 4-5 decades of living with one kidney has no serious adverse renal effects and a prevalence of hypertension that is probably similar to the general population considering the age of these donors. centers ) . before and after adjustment for comorbidity, 19 (7.8%) and 18 (7.5%) centers, respectively, met all criteria for review, but only 16 met criteria for review both before and after comorbidity adjustment. we conclude that failure to adjust for pre-existing recipient comorbidity results in grossly inaccurate estimation of expected gf per ktx center, more often resulting in expected gf that is too low. using data that are not adjusted for comorbidity to judge the quality of tx programs could encourage denial of access to high-risk patients. introduction: the purpose of our study was to examine temporal trends in the regionalization patterns and center volume-outcomes relationship for lung transplantation in the united states over the past decade. a retrospective analysis of all adult single-organ lung transplants included in the scientific registry of transplant recipients for three consecutive time periods between 1997 and 2006 was performed. for each time period, lung transplant centers were divided into three groups based on each center's annual volume of the procedure (low-volume group = 1-17 procedures per year, medium-volume group = 18-30 procedures per year, high-volume group = greater than 30 procedures per year). oneyear observed-to-expected patient death ratios were then calculated and compared for each group in each time period. a temporal analysis of the percentage of transplants being performed relative to center volume was also performed. statistical comparisons were made using chi square testing. results: a total of 12,603 lung transplant procedures were included in the analysis. in period 1, there was not a significant difference in the one-year observed-to-expected patient death ratio of low-volume lung transplant centers when compared to high-volume centers (ratio 1.15 for low-volume centers vs. 0.79 for high-volume centers, p = 0.07). by period 3, however, a significant relationship between center volume and outcomes had emerged (ratio 1.30 for low-volume centers vs. 0.79 for high-volume centers, p = 0.006). over this same time period, the percentage of lung transplants within the united states that are performed at low-volume centers has decreased significantly (from 43.3% of all lung transplants in period 1 to 22.7% in period 3, p < 0.0001), while the percentage being performed at high-volume centers has increased significantly (from 20.5% of all lung transplants in period 1 to 51.3% in period 3, p<0.0001). conclusions: a significant relationship between center volume and patient outcomes has emerged for lung transplantation over the past decade. at the same time, the percentage of these procedures being performed at high-volume centers has increased. these findings suggest that regionalization patterns for a given procedure may be influenced by the presence or absence of a volume-outcomes relationship for that procedure. liver transplantation (lt) has emerged as one of the few curative treatment modalities for patients with hepatocellular carcinoma (hcc). however, the increase in the incidence of hcc recurrence due to immunosuppressants administered after lt is a serious issue. we have recently proposed a novel strategy of adjuvant immunotherapy for preventing the recurrence of hcc after lt: intravenous administration of il-2-stimulated natural killer (nk) cells extracted from donor liver graft to liver transplant recipients. since the immunosuppressive regimen currently used after lt reduces the adaptive immune components but well maintains the innate components of cellular immunity, the augmentation of nk cells response might be a promising immunotherapeutic approach. we confirmed that the il-2-stimulated donor liver nk cells exhibited a significantly high level of trail and showed vigorous cytotoxicity against an hcc cell line without cytotoxicity against normal cells. after obtaining approval from the ethical committee of our institute, we successfully applied this therapy to 13 cirrhotic patients with hcc from january 2006. the average number of nk cells that had been administered to lt recipients at 4 days after lt was 304.2 ± 225.5 × 10 6 cells/body. the lt recipients were categorized as follows: (1) based on the milan criteria, 8 recipients met the criteria while 5 did not, and (2) based on the tnm stage, 2 recipients were categorized as pathological tnm stage i; 6, stage ii; 4, stage iii; and 1, stage iv. in our institute, the 2-year recurrence-free survival rates of the lt recipients treated with and without this therapy were 100% and 71.3%, respectively. kinetic studies revealed that in the early postoperative period, the peripheral blood obtained from the treated lt recipients exhibited a significant improvement in cytotoxicity against hcc cell line as compared to the untreated lt recipients (p < 0.01). furthermore, flow cytometric analyses revealed that the frequency of trail + nk cells increased remarkably in the peripheral blood of the treated lt recipients (p < 0.05). in conclusion, the administration of il-2-stimulated donor liver nk cells contributes to the promotion of host anticancer activity and has the potential to regulate hcc recurrence after lt. abstract# 500 vegf, a well-established angiogenesis factor, is expressed within allografts at high levels in association with acute and chronic rejection. in previous studies, we have reported that vegf possesses potent proinflammatory properties in part via its ability to mediate leukocyte trafficking into allografts. recently, we discovered that vegf mediates cd4+ and cd8+ t cell migration via interaction with its receptor kdr (also called vegfr2). blockade of t cell kdr significantly inhibits transendothelial migration. these observations suggest that kdr may be a novel t cell receptor for allogeneic lymphocyte recruitment. here, we first examined the expression of kdr on peripheral human cd4+ and cd8+ t cells by facs analysis. we found that ≤ 1% of circulating t cells express kdr, and its expression was at low levels on individual unactivated t cells. further, we found that the expression of kdr on t cells increased markedly following activation with mitogen and following interactions with activated allogeneic endothelial cells. induced expression of kdr on cd4+ and cd8+ t cells was at a similar level as that observed on endothelial cells. therefore, kdr appears to be selectively expressed on t cells, that traffick into allografts. to test the pathophysiological significance of these observations, we analyzed the expression of kdr in a total of 19 cardiac, and 5 renal, human allograft biopsies. we correlated the expression of kdr with cd3+ t cell infiltrates; and by double immunofluorescence staining, we determined co-expression of kdr on individual cd3+ t cell infiltrates. in cardiac allografts we found that kdr was expressed throughout the endomyocardium, and was most notable on endothelial cells in all biopsies examined. by grid counting of 3-4 areas of each biopsy, we found that the mean number of cd3+ t cell infiltrates ranged from 4 to 79 cells/hpf (x600 mag.). by double staining, we noted that kdr was expressed on 29 ± 4 % (mean±sem) of these cd3+ t cell infiltrates. similarly, we found that kdr was co-expressed on cd3+ t cells within renal allografts. while infiltrates were more focal, again 30 ± 2 % (mean±sem) of graft infiltrating t cells expressed kdr. collectively, these observations for the first time identify kdr as a novel receptor on allogeneic t cells. we suggest that intragraft vegf may interact with t cell kdr to facilitate homing and recruitment of allospecific lymphocytes into allografts. interaction of infiltrating cd8 + t cells and tissue cells in tolerant liver allografts: using tcr transgene approach. guoping jiang, 1 qiwei zhang, 1 horng-ren yang, 1 kathleen brown, 1 john j. fung, 1 lina lu, 1 shiguang qian. 1 1 immunology and general surgery, cleveland clinic, cleveland, oh. the liver allografts are accepted without requirement of immunosuppression in mice. the underlying mechanisms are not completely understood. we hypothesized that it resulted from an abortive t cell response within the liver due to hyporesponsiveness or apoptosis. to test this, we examined the activation and fate of allo-ag specific cd8 + t cells following liver transplants (ltx) compared with heart transplants (htx) that were acutely rejected. following transplantation [b10 (h2 b )→c3h (h2 k )], cfse labeled cd8 + t cells (10x10 6 ) from des tcr tg mice (h2k b specific tcr) were adoptively transferred into recipients. animals were sacrificed two days following des t cell administration for analyses of t cells in the grafts or draining lymph nodes (d-ln). host cd45 + leukocytes were quickly infiltrated following ltx. among cd3 population ∼48% were cd4 + and ∼52% cd8 + . cd8 + t cells were further increased thereafter in grafts and d-ln, associated with high inf-g production. cd8 + t cells in the liver grafts rapidly reduced to 36% by pod 14, and to 12% by pod 40. however, the incidence of cd4 + t cells remained high. cfse dilution assay and elispot showed an active division of des + t cells in liver allografts either on pod 7 or 40. these ag-specific cd8 + t cells functioned well evidenced by ifn-g production in response to allo-ag. however, compared to htx, the accumulation of des + cells in grafts was significantly lower in ltx [9.6% (17x10 4 /heart), and 0.59% (6.14×10 4 /liver)] on pod 7, and further dropped to 0.14% (3.14×10 4 /liver) on pod 40. the expended cohorts of adoptively transferred cells followed by their elimination suggested elimination of ag-specific cd8 + cells. to examine the role of liver environment, graft cd45non-parenchymal cells (npc) were isolated tested fro regulatory effect on des + t cell response. liver allografts showed significantly expansion of cd45 -npc, which were donor mhc class i + (h2b + ), b7-h1 + , trail + and low for cd40 and cd86. these cells did not inhibit des + t cell proliferation in response to b10 spleen stimulation, but significantly enhanced their death, which was dependent on b7-h1/trail. this was confirmed by using cd45 -npc from b7h1 -/or trail -/mice. in conclusion, activated t cells in liver grafts may stimulate tissue cells to express inhibitory and death inducing molecules, resulting in t cell death and graft acceptance. foxp3 + graft-infiltrating lymphocytes (gil) have been detected in the rejecting allografts of transplant patients. foxp3 is a marker for regulatory t cells (t reg ). published reports suggest that human foxp3 can be upregulated following tcr stimulation without induction of regulatory function. to investigate whether foxp3 + gil during acute rejection are t reg , we analyzed the phenotype of gil harvested from acutely-rejected non-human primate kidney allografts. methods: renal allografts with histologically-confirmed acute rejection were harvested at the time of necropsy from macaques that had undergone experimental transplantation. following digestion of kidney fragments using collagenase, gil were isolated using lymphocyte-separation media and cryopreserved. axillary lymph nodes (ax ln) were also isolated either at the time of necropsy or by biopsy. for seb-stimulated lymphocytes, ax ln cells were stimulated for 5 days in the presence of 25 ng/ml seb for 5 -6 days. to perform intracellular interferon-gamma (ifng) analysis, cells were stimulated with pma and ionomycin in the presence of brefeldin a for 5 -6 hours at 37°c. samples were prepared for flow cytometry by first staining for extracellular antigens. cells were then fixed and permablized (kit from ebiosciences) prior to intracellular staining for foxp3, ki67 and ifng. results: the percentage of foxp3 + in cd3 + gil was similar to that found in ax ln ( table 1 ). the majority of these cells were cd4 + . while 80% of the cd3 + /cd4 + /foxp3 + population of both ax ln and gil were cd25 + , a significantly higher frequency of cd39 + and ki67 + cells were found in gil (p < 0.01; student's t-test). simlar levels of cd39 and ki67 expression were found in seb stimulated lymphocytes. unlike the seb-stimulated lymphocytes, few ifng -producing cells were demonstrated following pma/ionomycin stimulation of gil. conclusion: our current data indicates that the majority of foxp3 + gil from acutely rejecting renal allografts are recently activated cd4 t cells that lack effector function. this suggests that foxp3 t cells within rejecting allografts may indeed be t reg . findings in mouse models of transplantation often fail to translate well in humans. three variables may account for the discrepancy: (1) evolutionary divergence between mice and humans, (2) influence of infection history on alloimmunity, and (3) use of highly inbred strains of laboratory mice. here, we investigated whether the use of inbred mouse strains skews the rejection phenotypes and their response to treatment due to decreased genetic diversity and/or fixation of undesirable genetic loci, known as inbreeding depression. we examined heterotopic cardiac allograft survival in outbred and inbred mouse populations in the presence or absence of immunosuppression. in the absence of immunosuppression, heart transplantation within or between outbred stocks of mice (n = 35) resulted in three distinct rejection phenotypes that resemble accelerated (1 -4 days), acute (8 -25 days), and chronic rejection (> 75 days), respectively. in contrast, all fully allogeneic grafts transplanted between inbred mice (n = 12) were rejected acutely (7 -13 days) as were historical controls (n > 50). the accelerated phenotype, present in 29% of outbred to outbred transplantations, was characterized by extensive hemorrhagic necrosis of the heart with thrombosis, neutrophil margination and neutrophilic arteritis, and did not correlate with donor:recipient mhc ii disparity. immunosuppression with t cell costimulation blockade did not prevent accelerated rejection (incidence = 27% in the treated group, n = 26) but did convert the acute rejection phenotype into longterm allograft survival in all groups studied. the same accelerated phenotype was observed if transplantation was performed from outbred to inbred mice (incidence = 39%; n = 23) but could not be duplicated if inbred to outbred transplantation was performed (n = 20). finally, c3 depletion with cobra venom factor abrogated the accelerated rejection phenotype in outbred to outbred transplantations (n = 16), suggesting a role for complement in the pathogenesis of this phenotype. in summary, our data (1) indicate that the use of outbred mouse stocks may uncover clinically-relevant rejection phenotypes not observed in inbred mouse strains, and (2) underscore the importance of the donor background in determining the phenotype of rejection. outbred mouse stocks may provide a platform to uncover mhc-unlinked genetic loci that play an important role in the outcome of solid organ transplantation. th17 are limited in their ability to reject allografts. elderly recipients represent the most rapidly growing segment of patients on the waiting list. however, little is known about age-dependent alterations of the immune response in organ transplantation. we examined age dependent t-cell functions in a transgenic mouse transplant model. effector t-cell phenotype, -function, cytokine production and regulatory t-cell function were analyzed in 3 and 18mths old b6 mice. in an in vivo transplant model, bl/6 nude mice were reconstituted with 2x10 6 young or old transgenic alloantigen-specific cd4 + tcells and engrafted with bm12 skin grafts. t-cell phenotype and cytokine secretion were sequentially analyzed in all lymphatic compartments. splenocytes of naïve old b6 mice contained significantly higher frequencies of t-cells with an effector/memory phenotype (cd4 + cd44 high cd62l low and cd8 + cd44h igh cd62l low; p<0.005). in vitro proliferation and ifnγ-production were significantly reduced in aged mice indicating an impaired t-cell response with increasing age as assessed by mlr (p<0.005) and elispot (p<0.001). in parallel, regulatory functions remained age-independent as alloantigen-specific cd4 + cd25 + foxp3 + t-cells isolated from sensitized old mice demonstrated a dose-dependent well preserved suppressor function. next, we tested the age-dependent alloantigen -specific cd4 + t-cell function in a transgenic skin transplant model: age did not significantly impact rejection kinetics (young vs. old: 10.1 vs. 14.3days, n.s.) however, t-cell migration and activation were significantly different: fewer numbers of activated cd4 + cd25 + and effector/memory phenotype t-cells (cd4 + cd44 high cd62l low ) were found in recipient spleens (p<0.05) and draining lymph nodes (dln) (p<0.05) after transfer of old t-cells. chemokine receptor staining revealed less cxcr3 + and ccr7 + t-cells in dln following the transfer of old t-cells (total cell numbers x104: cxcr3 + : 10.9±4.2 vs.0.95 ±0.2, ccr7 + : 4.7±1.0 vs. 0.35±0.2, p<0.05). this was paralleled by reduced intragraft t-cell infiltration as observed by immunohistochemistry. in summary, native elderly mice showed an increased frequency of effector memory t-cells but an overall impaired t-cell response. regulatory -t-cell function remained preserved. in vivo allospecific cd4+ t-cell activation and migration was impaired in elderly transplant recipients. the background: the sensitized transplant recipients may undergo an "accelerated" form of rejection, which is mediated by t cell-dependent mechanisms. these patients often experience increased rate of early rejection episodes, which are difficult to control with currently used immunosuppressive agents. methods: in our model of cardiac graft rejection in sensitized recipients, b6 mice are first challenged with b/c skin, followed 40-60 days later by b/c heart transplant (htx). unlike in naive hosts, htx rejection and alloreactive cd8 activation in this model are cd154 blockade-resistant. we first performed systemic analysis at the intragraft transcriptional level by microarray to identify disparities in local immune responses in naive vs. sensitized hosts. aiming to improve the efficacy of costimulation blockade in the sensitization settings, we then determined the role of cd4 t cells in costimulation blockade-resistant alloimmune response by using cd4 depleting (gk1.5) vs. cd4 blocking (yts177) ab, in conjunction with cd154 blockade (mr1). results: htx harvested from groups of naïve (day 4-6), sensitized (day 2-4), control ig or mr1 ab treated mice (n=3/gr) were subjected to microarray analysis. mr1 treatment suppressed htx expression of proinflammatory genes (il-1β, il-6, tnf-α), and t cell-targeted chemokines (rantes, mig, cxcl10) early after htx in naïve, but not sensitized recipients. five groups of sensitized mice treated at the time of htx with: (1) 5). ctl activation was determined by facs phenotyping at day 10 and 30. the simultaneous blockade of cd154 costimulation and cd4 help, but not a single blockade with mr1 ab or anti-cd4 ab, was required to inhibit peripheral alloreactive cd8 activation in sensitized mice. additionally, cd8 activation in the absence of cd4 help showed defective cytotoxic molecule profile, with suppressed perforin but upregulated granzyme b expression at the graft site. conclusion: cd154 blockade-resistant cd8 activation is critically dependent on cd4 t cells. this study provides novel immunological basis to study the potential synergy between adjunctive cd4 and cd154 targeted therapies to control accelerated graft rejection in sensitized hosts. mediators. g. einecke, l. g. hidalgo, p. f. halloran. department of medicine, university of alberta, edmonton, canada. the hallmark of t cell mediated rejection (tcmr) are interstitial inflammation and tubulitis. the mechanisms of tubulitis and epithelial deterioration during tcmr are unknown. we previously showed that tubulitis in mouse allografts is independent of cytotoxic molecules (gzma/b, prf) and is preceded by molecular changes with loss of epithelial genes, reflecting epithelial dedifferentiation. human tcmr is associated with loss of the same epithelial genes and re-expression of embryonic pathways (wnt, notch). we hypothesized that tcmr is mediated through soluble factors released by effector t cells or macrophages in the interstitium, and that supernatants of effector t cells would simulate these changes in epithelial cultures. we established an in vitro model in which cultured primary human renal epithelial cells are incubated with supernatants from effector t cell/monocyte co-cultures. the transcript changes in this model, analyzed by microarrays, closely simulated those in human and mouse tcmr (fig1), with loss of epithelial transcripts, activation of wnt/ notch pathways, and increased expression of ifng-inducible and injury-related transcripts previously defined in our mouse model. some of these changes were reproduced by incubation of epithelial cells with ifng or tgfb. the in vitro model identified additional epithelial transcript changes not previously identified in vivo (not affected by ifng or ischemic injury, not expressed in t cells, macrophages, b cells, or nk cells). expression of these transcripts (n = 305) was highly altered in human tcmr compared to nonrejecting biopsies of 177 human renal allograft biopsies and distinguished tcmr from antibody-mediated rejection in a hierarchical cluster analysis. thus we have established an in vitro model that closely simulates the epithelial events during human tcmr and confirms that these changes are independent of direct contact with inflammatory cells, supporting the hypothesis that interstitial effector t cells mediate allograft deterioration by soluble mediators. together with previous mouse and human data these results provide the first in vitro model of the epithelial consequences of tcmr. objective. c4 split product deposition to hla antigen-coated microparticles ([c4d] flowpra) was previously shown to be a specific marker of c4d-positive antibodymediated rejection (amr). the objective of this study was to assess the predictive value of [c4d]flowpra reactivity in a cohort of non-biopsied patients with stable graft function during the first year. methods. a total of 133 kidney transplant recipients were enrolled (inclusion criteria: functioning graft at 12 months; prospective collection of sera taken before and at 1-3, 6, and 12 months after transplantation). included patients were serially screened for humoral panel reactivity applying [igg] and [c4d]flowpra screening. results. fifty-four of the included 133 recipients had stable graft function within the first year and were not subjected to diagnostic renal biopsy. in this particular patient group, detection of complement-fixing hla reactivity tended to be less frequent than in the 79 patients with biopsied graft dysfunction (≥10% [c4d]flowpra before transplantation: 9% vs. 18% of recipients, p=0.2; ≥10% [c4d]flowpra after transplantation: 11% vs 24%, p=0.06). in line with our previous results, within the group of biopsied patients, pre-and/or post-tx [c4d]flowpra reactivity was tightly associated with the immunohistochemical detection of peritubular capillary c4d deposition (p=0.005) reflecting ongoing amr. remarkably, in initially stable patients, detectable [c4d] flowpra reactivity was not associated with inferior long-term outcomes. within this patient group, recipients with and without (pre-and/or post-transplant) c4d-fixing anti-hla reactivity did not differ with respect to 4 yr allograft survival (p=0.4), 4 yr serum creatinine levels (1.5 vs. 1.5 mg/dl; p=0.7), and proteinuria at 4 yrs (0.2 vs. 0.18 g/24h; p=0.8). similar results were obtained for a comparison of [igg]flowpra positive vs. negative subjects. conclusion. our data suggest that a considerable number of patients with initially stable graft function may have excellent long-term graft function despite serologically detectable levels of (complement-fixing) alloreactivity. for these antibody-positive recipients, a potential role of graft accommodation can be speculated. the immunoproteasome subunit beta 10 as a novel peripheral blood and intragraft biomarker of chronic antibody mediated allograft rejection in clinical transplantation. joanna ashton-chess, 1 in an attempt to identify non-invasive biomarkers of specific histological scarring, we compared publicly available gene sets derived from microarray studies of human renal transplant biopsies published in the literature with our own microarray data derived from studies of rat heart allografts. in this way we identified an immunoproteasome subunit (proteasome subunit beta 10 -psmb10) as a potentially interesting candidate. psmb10 is one of three members of the immunoproteasome that are induced by abstracts interferon gamma. messenger rna profiling in renal transplant biopsies (n = 52) with normal histology, interstitial fibrosis and tubular atrophy, calcineurin inhibitor toxicity, transplant glomerulopathy or chronic antibody-mediated rejection (banff 2005) revealed psmb10 to be strongly and significantly increased in chronic antibody mediated rejection vs. the other three histological diagnoses. receiver operator characteristic (roc) curve analysis showed that psmb10 mrna could diagnose chronic antibody-mediated rejection with an auc of 0.99, a sensitivity of 1.0 and a specificity of 0.95. moreover, psmb10 mrna was significantly increased in the pbmc (n = 24) of patients with chronic antibody-mediated rejection compared to those with normal histology. roc analyses revealed an impressive auc of 1.0 with all patients being correctly classified. similar results were also observed in a rat allograft model where psmb10 was significantly increased at day 100 post transplantation in both the heart allograft and the pbmc of animals presenting chronic transplant vasculopathy vs. syngeneic grafts. moreover, inhibition of the proteasome by administration of velcade® at 0.1 mg/kg every other day for the first 20 days post transplantation significantly and dose-dependently prolonged allograft survival (mst 31.7 days in velcade-treated vs. 6.3 days in untreated animals).together our data point towards psmb10 as a blood and intragraft biomarker of chronic antibody-mediated rejection as well as a potential therapeutic target. furthermore, our results suggest that using a threshold of psmb10 in the blood could help in guiding the decision to biopsy in the clinic. baff monitoring after b-cell depletion therapy for acute renal transplant rejection. valeriya zarkhin, 1 snehal mohile, 1 li li, 1 jonathan martin, 1 minnie sarwal. 1 1 pediatrics, stanford university, stanford, ca. introduction: the objective of this study was to investigate the interaction between b-cell activation factor of the tnf family (baff) level and circulating b-cell repopulation in pediatric patients with acute kidney transplant rejection treated with the b-cell-depleting agent rituximab. methods: 10 pediatric patients (3-23 yrs) with biopsy proven b-cell positive ar were treated with steroids and rituximab (4 x 375 mg/m 2 /dose/week). all patients were followed up for 12 months. peripheral blood cd19 cells and donor specific antibodies (dsa) were monitored monthly. serum level of baff was measured by elisa at ar, 1, 3, 6, and 12 months post-ar treatment and correlated with clinical outcomes. results: complete depletion of circulating and intragraft b-cells was observed with rituximab, with improvement in ar grade in all patients. the median time of peripheral b-cells repopulation was 5 months (range 3-12 months, fig. 1a) . no correlation was found between pre-treatment peripheral b-cell number and the b-cell repopulation time (r=0.59, p=0.09). baff levels rose significantly with b-cell depletion with maximum values at 3 months post-treatment (7.5 fold increase, p=0.0001) and returned to pre-treatment levels, with b-cell recovery, at 12 months (fig. 1b) . serum baff levels correlated positively with b-cell depletion >6 months (r=0.91, p=0.004, fig1b). a lack of depletion of dsa i, but not dsa ii correlated with higher baff levels (r=0.99, p=0.007). the timing of b-cell repopulation and depletion of dsa i may be dependant on serum baff level. anti-baff treatment may be considered in addition to rituximab or standard immunosuppressive treatment protocols in patients with persistent and/or antibody mediated rejection. the background: the development of donor specific hla antibodies (dsa) post-transplant has been associated with graft failure. we have shown in a longitudinal study that increases in dsa may precede rejection by months. this retrospective analysis evaluates changes in maintenance immunosuppression (mi) and appearance of dsa in stable transplant recipients. methods: sera from stable renal transplant recipients were collected at 4-6 month intervals and tested for the presence of dsa. the types and doses of immunosuppression were correlated with the appearance of dsa. two hundred eighty stable renal transplant recipients who received either a deceased or a living donor kidney were monitored post-transplantation for the development of dsa. patients have been followed for 1 to 7 years and had a minimum of 4 serum samples analyzed. all recipients received anti-lymphocyte induction therapy. maintenance immunosuppression (mi) consisted of a calcineurin inhibitor, prednisolone, and mycophenolic acid. hla single antigen beads analyzed in the luminex instrument were used to establish donor specificity of the antibodies. a chart review was undertaken to determine the doses of the mi posttransplantation. all mi was managed by the transplant team and changed according to clinical indications without regard to dsa. results: of the 280 patients monitored 37 developed dsa post-transplantation with a functioning graft. dsa was against hla-class ii antigens in 26 of 37 (70%); class i antigens in 8 of 37 (22%); and against both class i and ii antigens in 3 of 37 (8%). dsa against class ii was against dq in all except one case. in the majority of the recipients the appearance of dsa was preceded with dose reduction of the mi, either calcineurin inhibitor or mycophenolic acid or both. conclusions: our data show that dsa developed predominantly against hla-class ii antigens and that the appearance of dsa was often preceded with reduction of one or more of the mi. this data shows the importance of monitoring dsa with mi decreases in a stable allograft recipient. antibody production and antigen presentation are directly inhibited by mycophenolate mofetil. anat r. tambur, 1 joe leventhal, 1 nancy d. herrera, 1 joshua miller. 1 1 division of organ transplantation, northwestern university, chicago, il. immunosuppressive medications are primarily designed to target t cell proliferation. mycophenolate mofetil (mmf) exerts its effect by inhibiting de-novo synthesis of guanine, a dna building block. we, and others, have previously shown that mpa (active metabolite of mmf) affects the differentiation of monocytes into dendritic cells (dc). we further demonstrated that cell-surface receptors associated with antigen up-take and antigen-processing and presentation (cd83 and cd205) are down regulated when cells are matured in the presence of mpa. this phenotype translated into a decreased uptake of alloantigens and reduced stimulation of t cells. we concluded that mmf inhibits also cell functions requiring mrna synthesis. we now present data regarding the role of mpa in maturation and function of b-lineage cells. pbmcs from 10 subjects were cultured in the presence of cpg, il-2, il-10 and cd40l for 5 days to induce memory b and plasma cell maturation in-vitro. cultures were performed in the presence or absence of mpa (50 ugr/ul) for the length of the incubation period. in-vitro stimulation of b cells increased the memory population (cd19+ cd27+) from 2.8 +/-1.1% to 7 +/-2%. similarly, plasma cells were increased from 4.4 +/-1.3% to 6.4 +/-1.8%. the addition of mpa to the culture inhibited stimulation for both memory and plasma cells (4.7 +/-3% p=0.01; and 4.7 +/-2.3% p=ns, respectively). we have further analyzed the effects of mpa on antibody secretion using an elisa (measuring soluble antibodies) as well as a b-cell elispot assay (assessing the number of b cells that produce antibodies). while an expected increase in od values was observed for stimulated samples compared with non-stimulated samples, a significant decrease was observed when stimulation occurred in the presence of mpa. nonstimulated cells: 0.249 +/-0.28; stimulated cells: 0.391+/-0.33; mpa treated stimulated cells: 0.279+/-0.43; p<0.0005). the number of antibody producing cells was also significantly lowered when cultures were done in the presence of mpa (a mean of 106 cells were counted for the stimulated cells compared with 1-2 cells for non-stimulated and mpa-treated-stimulated cells). to our knowledge this is the first time where in-vitro experimental data document the inhibitory effect of mpa on b lineage cells and antibody secretion, although clinically known for some time. these results confirm our previous observations regarding the effects of mmf on non-proliferating immune cells. a non-allogeneic stimulus triggers the production of de novo hla and mica antibodies. luis e. morales-buenrostro, 1,2 lluvia a. marino-vazquez, 1 anh nguyen, 3 paul i. terasaki, 2 josefina alberu. 1 1 nephrology and transplantation., instituto nacional de ciencias medicas y nutricion salvador zubiran, mexico city, df, mexico; 2 terasaki foundation laboratory, los angeles, ca; 3 one lambda inc., canoga park, ca. background: in a previous study, we found that healthy people developed hla abs after immunization against hepatitis b virus. the aim of this prospective study was to establish if stimulation with influenza vaccine is capable of triggering the production of hla and mica abs. methods: we determined the presence of hla and mica abs (de novo and preformed abs) in 3 groups of patients vaccinated against influenza: a) 42 healthy adults, b) 40 esrd patients, and c) 25 tr. additionally, we followed 22 healthy unvaccinated people without exposure to sensitizing factor: d) control group. sera samples were collected at baseline (pre influenza shot), at 1 week, and monthly up 6 months after immunization. hla abs were assessed with labscreen single antigen beads for luminex. all samples of each patient were tested simultaneously. a luminescence value higher than 500 was considered positive only if it was 3 times the baseline value. we analyzed the data using chi square, one way anova test, and logistic regression. results: the table shows the types of abs in each group. interestingly, we found preformed abs across all four groups, including the control group (which is free of any known sensitizing factors). the proportion of de novo abs was higher in the group b and c. multivariate analysis shows that the only independent factor associated with development of de novo abs was the presence of preformed abs. we observed a nonspecific immunologic response triggered by external stimulus and was not necessarily associated to the vaccine in people previously sensitized. a introduction. decisions about the minimization and ultimate withdrawal of immunosuppression (is) would be facilitated by the identification of biomarkers associated with operational tolerance (ot). methods. as part of an itn/nih supported study tolerant kidney transplant recipients (off all is for > 1yr with stable function, n=22) were compared to recipients with stable function on is (sis, n=34), recipients with chronic allograft nephropathy (can, n=20), and healthy volunteers (hv, n=18). pbmc, whole blood total rna, and urine samples from each group were examined using flow cytometry, microarrays, and rt-pcr respectively. results. analysis of microarrays revealed significantly higher expression of b cell differentiation genes in tolerant recipients compared to the sis and can groups. consistent with this finding, tolerant recipients also displayed higher numbers of naïve b cells in peripheral blood and increased expression of cd20 in urine relative to the sis and can groups. no differences in treg or genes associated with regulatory cells were observed in tolerant recipients relative to other groups. these analyses failed to demonstrate significant differences between tolerant recipients and hvs although support vector machine learning methods suggested potential differences in a number of genes including nfat and calcineurin. finally, relative to tolerant patients, those with can showed decreased numbers of t and nk cells and expressed lower levels of genes associated with immune cell activation in peripheral blood. conclusions. differences in b cell numbers may be useful in identifying tolerant renal transplant recipients or those predisposed to developing tolerance and could potentially provide insights into the mechanisms of tolerance. erythrocyte development of antibodies (abs) to mismatched donor hla antigens has been associated with acute and chronic rejection. complement activation, and c4d deposition, has been correlated with humoral rejection of allografts. however, utility of c4d staining in ltx has been controversial. a recent study (arthritis rheum. 2004 nov; 50(11) :3596-604) shows a strong correlation between erythrocyte bound c4d (e-c4d) in diagnosis and monitoring of sle. goal of our study is to determine the utility of measuring e-c4d in the diagnosis of humoral rejection following human ltx. 26 ltx recipients were analyzed post-ltx for e-c4d using facs of rbcs incubated with anti-c4d (quidel) followed by fitc-goat anti-mouse.10 normals were also analyzed. the serum was analyzed for development of anti-hla abs by solid phase assays and for the presence of autoabs to kα1 tubulin and collagenv (elisa). biopsies from 11 patients were stained immunohistochemically for c3d deposition. summary of results are presented in table 1 . infection=2/10 ar=0/10 % e-c4d in normals-10.35%; mfi in normals-5.46; mfi=mean fluorescence intensity; ar=acute rejection; dsa=donor specific abs 16 out of 26 patients show significant increase in the % bound e-c4d (p<0.05) as compared to controls.11/16 had anti-hla and 13/16 had autoabs which were significantly different from those with low e-c4d (p= 0.005). staining of the biopsies showed c3d deposition in 6 recipients with increased e-c4d. all 4 patients with acute humoral rejection had elevated e-c4d. we conclude that there is a significant correlation between increase in % e-c4d in ltx recipients and development of abs to either hla antigens or auto-antigens during the post-ltx period. biopsies from patients with increased e-c4d showed deposition of c3d in the allografts. preliminary data suggest that measurement of e-c4d using a non-invasive method of flow cytometry may be of value in monitoring ltx patients for humoral rejection. innate immunity: chemokines, cytokines innate immunity is emerging as an important initiator and modulator of the adaptive immune response. in the setting of transplantation, ischemia-reperfusion injury and tissue trauma appear to potentiate the alloimmune response. one of the mechanisms through which the innate immune system modulates an adaptive immune response is dendritic cells (dc). in this study, we examined dc activation in a mouse model of skin transplantation by monitoring the expression of mhc ii and p40 chain of the proinflammatory cytokine il-12. the p40 expression was detected in live cells using the yet40 reporter mouse, in which a transgene for yellow fluorescent protein (yfp) was placed downstream of the endogenous il-12p40 gene, thus faithfully "reporting" p40 expression. skins from c57bl/6 or balb/c donors were grafted on the dorsal thorax of c57bl/6.yet40 mice. draining and non-draining lymph nodes (ln) were harvested at 24 hours and examined for yfp expression by fluorescent microscopy. a marked increase in the numbers of yfp-expressing dc was observed in draining ln in both syngeneic and allogeneic graft recipients. surprisingly, yfp-expressing dc also increased in nondraining ln when compared to non-transplanted controls. similarly, dc in draining and non-draining ln showed higher mhc ii expression than those in non-transplanted controls. upregulation of mhc ii was highest at 24 hours and decreased significantly by 48-72 hours. to assess whether dc activation in non-draining ln was functionally significant, we monitored the activation of adoptively transferred ovalbumin-specific ot-ii tcr transgenic t cells in response to footpad antigen challenge in mice with or without a syngeneic skin transplant on the contralateral upper thorax. otii t cells in transplanted animals proliferated approximately 5-to10-fold better and a higher percentage of the otii cells produced il-2 than those from non-transplanted animals. thus, local surgical trauma results in a widespread, time-limited, functional activation of dc that appears to act as a partial adjuvant for t cell responses. together, these data suggest that surgical trauma may incite a systemic barrier to transplantation via the activation of dc and therapeutic interventions that reduce the surgical trauma and dc activation may help to improve survival of transplanted grafts. tolerance of cardiac allografts: studies with cx3cr1-deficient mice. takaya murayama, 1 katsunori tanaka, 1 takuya ueno, 1 mollie jurewics, 1 guleria indira, 1 fiorina paolo, 1 paez jesus, 1 smith n. rex, 2 sayegh mohamed, 1 reza abdi. 1 1 transplantation research center, renal division, brigham and women's hospital, harvard medical school, boston, ma; 2 department of pathology, massachusetts general hospital, harvard medical school, boston, ma. although donor/tissue dendritic cells (ddc) have long been known to play a key role in mounting alloimmune responses, however, their generation, trafficking and role in tolerance have not rigorously examined. we have used b6.fvb-tg (itgax-dtr/ egfp) 57 mice which has a gfp linked to the cd11c promoter. using these mice as the donors of heart allograft transplantation provided us a unique model to study ddc posttransplantation. our trafficking data indicate there is a rapid migration of ddc into spleen (3 hours post transplantation) but not lymph nodes and that ddc were unexpectedly detected in the spleen of the recipients long after rejection of heart allografts suggesting ddc could escape from the immunosurveillance of host immune system. our data also show that ddc proliferate in the lymphoid tissue of the recipients and co-express class ii molecule of the recipients. we then show that cx3cr1 pathway regulates generation abstracts of heart tissue dc constitutively. as compared to wt hearts, cx3cr1 -/hearts contain lower number of dc and transplanting cx3cr1 -/donor hearts into wt balb/c mice led to significant prolongation of allograft survival without immunosuppression (mst of 8 vs. 17 days, respectively). increasing cx3cr1 -/heart dc by implanting donors with flt3-producing hybridoma cells has restored the time of rejection. unexpectedly, induction of long-term survival with anti-cd154 blockade (mr1) and ctla-4 ig (but not low dose rapamycine) was abrogated when cx3cr1 -/hearts were used as donors, with concomitant lesser tregs in the cx3cr1 -/heart allografts as compared to wt. furthermore, co-transplanting hearts from wt and cx3cr1 -/into the same recipient treated with mr1 resulted in significant prolongation of cx3cr1 -/heart allograft survival. depleting the ddc of heart donors prior to transplantation with diphtheria toxin also worsened markedly chronic rejection in the recipients at day 100 post-transplantation. our data indicate that, in contrast to the widely accepted dogma, the presence of donor dc in graft tissue is not only central to allograft rejection but also is necessary for the induction and maintenance of peripheral tolerance. local c5a interaction with c5ar on dcs modulates dc function, subsequently up-regulating allospecific t cell responses. qi peng, ke li, steven h. sacks, wuding zhou. mrc centre for transplantation, department of nephrology and transplantation, king's college london, guy's campus, london, united kingdom. the innate system of immunity plays an important role in ischemia-reperfusion injury and allograft rejection. the early stages of inflammatory processes are accompanied by complement activation. one biological consequence of this activation is the release of potent inflammatory anaphylatoxins, c3a and c5a, which have been reported to regulate a range of inflammatory responses. we previously reported that dcs express c3ar and c5ar, and c3a-c3ar interaction has a positive impact on murine bm dcs, in terms of activation phenotype and capacity for ag uptake and allostimulation. however, the role of c5a in modulating dc function remains unclear. the aim of this study is to investigate the role of local c5ar signalling in modulating murine bm dc function and subsequent regulation of the allospecific t cell response. we first evaluated if c5a-c5ar interaction could result from local expression of factors. our results showed that c5ar mrna was detected in wt dcs at different stage of dc culture by rt-pcr, and c5a was detected by elisa in the culture supernatants from different stages of dc culture. we next determined if c5a-c5ar interaction modulates dc function in allospecific t cell stimulation in vitro and in vivo. we found that bm dcs cultured from c5ar-/-mice or treated with c5ar antagonist (c5ara, w54011) exhibited a less activated phenotype (producing significantly less il-12 and more il-10, in response to lps stimulation); both c5ar-/-and antagonist-treated dcs (lps stimulated) showed reduced capacity to stimulate naïve alloreactive t cells, as measured by ifn-γ production and thymidine uptake. as regards interaction in vivo, following i.p. administration of the c5ara-treated dcs into allogeneic mice for 10 days, ex vivo mixed lymphocyte reaction showed that cd4+ t cells from those recipients have reduced thymidine uptake, but increased il-4 production compared to that with untreated dcs. conversely, dcs treated with c5ar agonist (c5a) exhibited a more activated phenotype (producing more il-12 and less il-10) and were more potent in allospecific t cell stimulation. our findings demonstrate that murine bm dcs can express c5ar and c5a can be generated locally; c5a-c5ar interaction up-regulates murine bmdc activation and their allostimulatory capacity. thus, targeting c5a-mediated signal may be able to prevent allograft injury. role of tnfα in early chemokine production and leukocyte infiltration into heart allografts. daisuke ishii, 1 austin d. schenck, 1 robert l. fairchild. 1 1 immunology, glickman urological and kidney institute., cleveland clinic, cleveland, oh. objectives: the acute phase cytokines il-6 and tnfα are produced early during inflammatory processes, including wound healing and ischemia/reperfusion. the goal of this study was to investigate the role of these cytokines in the induction of early chemokine production and leukocyte infiltration into heart allografts. methods: c57bl/6 (h-2b), balb/c (h-2d), and balb/c.il-6-/-mice received vascularized syngeneic or complete mhc mismatched, a/j (h-2a), cardiac grafts. grafts were retrieved at different time points and total rna and tissue protein were prepared and analyzed by quantitative rt/pcr and elisa to test expression levels of tnf-α, il-6, cxcl1/kc, cxcl2/mip-2, and ccl2/mcp-1 in the grafts. anti-tnfα mab (500 ug) was given at the time of transplantation with or without anti-cd154 mab (200 ug on days 1 and 2). infiltration of cd4+, cd8+ t cells, neutrophils and macrophages was assessed by flow cytometry and immunohistochemistry. donor-reactive t cell priming to ifn-g producing cells in the recipient spleen was measured by elispot. results: expression of tnfα and il-6 mrna reached an initial peak at 3 hrs post-transplant and a second peak at 9-12 hrs with equivalent levels in both iso-and allo-grafts. the neutrophil and macrophage chemoattractants cxcl1/kc, cxcl2/ mip-2 and ccl2/mcp-1 reached peak levels at 9 hrs post-transplant in both sets of grafts and then declined to background levels. il-6 deficiency in the recipient or the cardiac allograft did not prolong allograft survival. in untreated mice, heart allografts were rejected at 8.6 ± 0.6 days after transplantation. anti-tnfα mab decreased neutrophil and macrophage chemoattractant levels 50% at 9 hrs post-transplant and subsequent neutrophil, macrophage and cd8+ cell infiltration into the allografts as well as extended graft survival to 14.1 ± 0.8 days. anti-tnfα mab also decreased the number of donor-reactive ifn-g producing cd8 t cells almost 90% on day 7 post-transplant. whereas anti-cd154 mab prolonged survival to day 21, administration of anti-tnfα and anti-cd154 mab delayed rejection to day 32 and resulted in the long-term (> 80 days) survival of 40% of the heart allografts. conclusions; these data indicate that anti-tnfα antibodies can delay donorreactive cd8 t cell priming and leukocyte infiltration into heart allografts rejection. as a conjunctive therapy, tnfa antibodies can promote long-term survival of the allografts. introduction recent evidence indicates that inflammation impairs immune regulation, yet the mechanisms behind this effect are not clear, in particular in regards to transplantation tolerance. in this study, we investigated the role of inflammatory cytokines, il-6 and tnfα, in transplantation tolerance induction. we first examined the impact of il-6 + tnfα on in vitro t cell alloimmune responses. t cells that were stimulated by allogeneic apcs in conditioned media harvested from lps-activated dcs proliferated more than apc-stimulated t cells that were cultured in conditioned media derived from non-lps-activated dcs. the ability of cd4 and cd8 t cells to respond to allogeneic apcs in the lps-activated conditioned media was significantly impaired by the addition of either anti-il-6 or anti-tnfα mabs. the addition of both mabs further diminshed t cell proliferation, indicating that il-6 and tnfα synergize to augment in vitro t cell allostimulation. in support of these findings, we noted reduced t cell proliferation during the mlr when t cells were cultured in conditioned media derived from either il-6-/-or tnfα-/-lpsactivated dcs. furthermore, these diminished responses was restored by the addition of recombinant il-6 or tnfα, respectively. to examine the in vivo implications of these findings, we employed a murine skin allograft model, with recipients that were either b6 wild type, il-6-/-or tnfα-/-. these groups were transplanted with a balb/c skin graft and treated with (or without) perioperative costimulatory blockade (ctla4 ig and anti-cd154). in the absence of immune modulation, all groups rejected balb/c skin allografts at a similar tempo (<12 days). in the presence of costimulatory blockade, il-6-/-recipients (median survival time, mst = 22 days, p =0.05) rejected their allografts at a slower tempo compared to wt (mst = 16 days) or tnfα-/-recipients (mst = 18 days). however, this response in il-6-/-recipients was further delayed by administering a tnfα inhibiting mab (mst = 60 days), indicating that synergy between il-6 and tnfα occurs in vivo and prevents the ability of costimulatory blockade to delay the onset of allograft rejection. conclusions we conclude that synergy between il-6 and tnfα augments t cell alloimmune responses and impairs the effects of costimulatory blockade to delay allograft rejection. abstract# 529 background: calcineurin inhibitors (cni) are involved in the development of post transplant diabetes mellitus (ptdm). changes in insulin secretion and sensitivity are central mechanisms involved in the development of ptdm. in addition alterations in endothelial function seem to be involved. the present study investigated the effect of cni's on these factors. methods: in a predefined sub-study of a previously published randomized trial it was aimed to compare the effect of cni treatment (n=27) with complete cni-avoidance (n=27) on insulin secretion and sensitivity as well as endothelial function. an oral glucose tolerance test and endothelial function investigation with laser doppler flowmetry was performed in 44 patients, 10 weeks and 12 months following transplantation. results: insulin sensitivity differed already 10 weeks posttransplant and was significantly better after 12 months in patients never treated with cni drugs (p=0.043). endothelial function was significantly correlated with insulin sensitivity (n=27, r 2 =0.22, p=0.013) at 10 weeks posttransplant, but not after 12 months (p=0.54). insulin secretion tended to be higher in cni treated patients both at week 10 and month 12 (p=0.068). conclusions: findings in the present study indicate that long-term cni treatment reduce insulin sensitivity which was associated with impaired endothelial function. in response to this peripheral insulin resistance a tendency towards a compensatory increase in insulin secretion was seen. these effects combined may indicate a future risk for premature cardiovascular disease in cni treated renal transplant recipients, but this hypothesis needs further study. group(n) ktx ptx(kptx) ecd re-tx pra(>20%) race(aa) low immunologic risk alem (113) overall pt, ktx, and ptx survival are 96, 92, and 86% at 16 months median follow-up. actuarial survival rates, initial length of stay, delayed graft function, steroid free rates, major infection, and incidence of ptld (1 ratg pt) were similar for alem and ratg groups, but treated acute rejection (ar) occurred in 17(15%) alem pts compared to 29(27%) ratg pts (p=0.03) and biopsy proven rejection (bpar) in 13(12%) alem pts compared to 23(21%) ratg pts (p=0.04). only 1 alem bpar has occurred after 12 months. total daily mmf doses were similar for alem and ratg groups at 3 months (1567±457mg vs 1670±508mg). neupogen use was greater in the alem group, 26(23%), than in the ratg group (14(13%), p=0.03). excluding pak, chronic allograft nephropathy (can) was observed in 19(17%) alem pts and 27(25%) ratg pts. (p=0.14). conclusions: alem and ratg induction both provide excellent 1 and 2 yr pt, ktx, and ptx survival. alem is associated with lower acute rejection rates and perhaps less can, but requires increased neupogen administration to help maintain mmf dosing. thymoglobulin dosing intensity and density: effects on induction efficacy ruth-ann m. lee, 1 adele h. rike, 1 background: alemtuzumab (campath 1h) has been used as induction therapy for kidney transplant recipients with acute rejection rates reported by us and others of 7 to 20% at one year. the histologic type of rejection and the time frame for occurrence after treatment with alemtuzumab have not been well established. this study is a retrospective single center review of acute rejection episodes of kidney transplant recipients treated with alemtuzumab induction with respect to the kinetics and histologic patterns of acute allograft rejection. methods: from 11/01/03 to 10/31/06, 416 kidney transplants were done meeting the inclusion criteria for this review. all patients had negative t and b cell flow cytometric crossmatches and received induction therapy with alemtuzumab 30mg iv intra-operatively, methylprednisolone 500 -750 mg during the first 24 hours, and were then maintained on tacrolimus (target level 5-7) and mycophenolate mofetil without steroids. all episodes of biopsy-proven acute rejection (ar) were reviewed. patients in pre-transplant desensitization protocols or with documented non-adherence with medications were excluded from the analysis. results: a total of 44 of 416 patients (10.6%) experienced ar during the study period, with a mean follow up of 28 months (range 14-48 months). of the ar episodes, 19 (43%) occurred within the first 3 months post-transplant, 13 (30%) occurred between 3-6 months, 5 (11%) occurred between 6-9 months, 1 (2%) occurred between 9-12 months, and 6 (14%) occurred more than one year post-transplant. of the rejection episodes within the first 3 months post-transplant, 9/19 occurred within the first 30 days. histologic analysis showed that 9/19 rejection episodes (47%) within the first 3 months included an antibody mediated component (6/9 within the first 30 days.) in contrast, 3/25 rejection episodes (12%) which occurred greater than 3 months post-transplant were antibody mediated. of the 12 patients with antibody mediated rejection, only 3 patients had panel reactive antibody (pra) levels > 25% at the time of transplant. conclusion: this large experience with alemtuzumab induction therapy with a steroidfree maintenance protocol, demonstrates that the majority of rejection episodes occur within the first 6 months post-transplant, with the largest fraction in the first 3 months. a significant number of early rejection episodes are antibody mediated and occur in unsensitized recipients. in a randomized, international, multicenter study, comparing the use of thymoglobulin (tmg) and basiliximab (bas) in recipients at high risk for delayed graft function (dgf) or rejection, tmg was associated with less acute rejection (15.6 % vs 25.5%, p=0.02) and a lower triple endpoint (rejection, death or graft loss, 20.6% tmg vs 33.6% bas, p=0.02) but not a significantly lower quadruple endpoint including dgf. the purpose of this study was to compare the efficacy of tmg and bas for induction stratified by donor source: standard criteria donor (scd), extended criteria donor (ecd) or donor with hypertension (htn). methods: retrospective review of data collected in the original randomized trial. data-capture limitations necessitated defining ecd as donor age > 60 or donor age between 50 and 60 with both a donor history of htn and donor renal insufficiency (history of atn or creatinine above 2.5 mg/dl during the 24 hours prior to organ recovery/start of cold ischemia time). results: 75 recipients received ecd kidneys [tmg n=40 (28.4%), bas n=35 (25.6%), p=ns]. outcomes are presented below. there were no differences in the rates of dgf between the groups examined. conclusion: standard and non-htn donor recipients had a tremendous benefit of tmg compared to bas with less acute rejection and death. contrary to the perceived niche of tmg in ecd recipients, tmg has its most beneficial effect in scd recipients and recipients of donors without htn at risk for acute rejection or dgf. evaluation we have changed our immnosuppressive protocol in abo-incompatible kidney transplantations and attempted to determine whether the changes in agents have resulted in better outcomes. we used tacrolimus(fk), mycophenolate mofetil(mmf) and methylprednisolone(mp) in immunologically high risk patients between 2000 and 2007. moreover, we performed splenectomy at the time of the transplant surgery in 117 patients (group 1) with aboincompatibilities between 2000 ansd 2004, and administered rituximab as an alternative to splenectomy in 38 patients(group 2) with abo-incompatibilities between 2005 and 2007. in this study, we compared the graft survival rates as well as the incidence of acute rejection in these two treatment eras. the graft survival rate at one year was 94% in group 1 and 97% in group 2 (p=ns). the graft was lost in one case of the 38 cases of group 2 due to insufficient doses of the immunosuppressive drugs. the incidence rate of acute rejection was 15% (18/117) in group 1, and 11% (4/38) in group 2 (p<0.01). there were no significant differences in serum creatinine level one year after transplanatation between two groups(1.5±0.3 in group 1 vs.1.4±0.4mg/dl in group 2). no serious adverse events associated with rituximab or splenectomty were encountered in either groups. in abo-incomaptible kidney transplantation, rituximab under fk/mmf combination as an alternative to splenectomy seems to yield an excellent result in terms of the incidence rate of acute rejection. introduction: the choice of immunosuppression in the elderly kidney transplant recipient remains unclear. the objective of this study was to compare outcomes with different t cell-depleting induction agents in the elderly. method: all solitary kidney transplant recipients over the age of 60 years that received induction therapy with either alemtuzumab or thymoglobulin from 2002 to 2007 were included in this unos analysis. overall graft survival, the risk of graft loss, and the risk of rejection were compared using kaplan meier, cox proportional hazards, and logistic regression, respectively. results: patients receiving alemtuzumab had a significantly lower 3-year graft survival (70.8%) than patients receiving thymoglobulin (79.3%), p=0.02) (figure) after adjusting for other risk factors, alemtuzumab had a higher risk of graft loss compared to patients given purpose. the aim of this prospective randomized study was the comparison of efficacy and incidence of adverse events in two induction therapy regimens (atg versus basiliximab) in patients receiving a dual immunosuppression. methods. 120 recipients of first or second deceased donor kidney transplants were prospectively randomized to receive either atg (fresenius) or basiliximab (novartis) as induction therapy. dual immnosuppression consisted of tacrolimus (astellas) and methylprednisolone. cmv prophylaxis was not applied on a regular basis. statistical analysis was performed with fisher's exact or chi-squared test, anova or mann-whitney u test, kaplan meier curves and log-rank test. results. patient characteristics of populations treated with atg versus basiliximab were similar concerning average age (48 years), gender and dialysis time prior to transplantation (78 vs. 88 months). average donor age and cold ischemia were also comparable (43 vs. 41 years and 833 vs. 852 minutes). the actuarial 5-year patient survival for the atg subpopulation is 91,7 % in comparison to 85 % in the basiliximab group (n.s.). analyzing graft survival after 5 years, rates of 88,3 % in atg patients compared to 75 % in the basiliximab group can be observed (n.s.). the incidence of acute rejection episodes was similar in both groups (atg: n=20 vs. basiliximab: n=16). 18 (30%) patients in the atg group and 20 (33,3%) in the basiliximab group showed a delayed graft function. serum creatinine was not significantly different at 1 and 5 years (atg: 1,4±0,6 mg/dl and 1,4±0,8 mg/dl vs. basiliximab: 1,3±0,7 mg/dl and 1,5±0,9 mg/dl). patients in the atg group had a higher rate of cmv infections (n=13 vs. n=3; p=0,05), whereas patients treated with basiliximab had significantly more hematological complications like anaemia, leukopenia and thrombocytopenia. conclusion. comparing induction therapy with atg and basiliximab, our data shows similar patient and graft survival rates with slightly better results in the atg group. patients treated with atg had a higher rate of cmv infections but less hematological complications. predicting cardiovascular events (cvd) and the varied effects of immunosuppressive medication on cvd risk factors requires an understanding of how traditional and nontraditional risk factors impact cvd after kidney transplantation (ktx). single-center studies have generally lacked statistical power and generalizability. registry studies have lacked sufficient data on cvd risk factors. the port project is creating a multicenter international database of ktx recipients with the primary objective of developing risk prediction models for post-transplant cvd. as a preliminary data assessment, an analysis was done on 19,669 ktx from 1990-2007 from 11 transplant centers representing 5 european centers, 4 north american centers, and 2 centers from asia/oceania. all data were extracted from preexisting databases at each individual transplant center and processed into the consolidated port database. 1,131 major adverse cardiac events (mace) were identified, defined as non-fatal or fatal myocardial infarction, cardiac abstracts arrest, and sudden death. the mace-free survival curves by participating center are shown in the figure. in this preliminary analysis, the overall one-year cumulative incidence of mace was 2.2%, ranging from 0.2% to 4.4% across centers; and the five-year cumulative incidence was 5.8%, ranging from 0.6% to 13.5%. the prevalence of diabetes pre-transplant varied from 10% to 47% across centers. in cox proportional hazards models adjusted for age, gender, race, donor type, transplant center, and reported history of diabetes, hypertension (htn), and ami, patients with a reported history of ami had a 112% increased risk (79%-152%, p<0.0001) for mace. for the final analysis, the definition of mace will be expanded to include major revascularization events. the final port database will be used to develop and validate an equation to predict mace and other health outcomes of interest, after accounting for differential cvd risk factors internationally. abstracts to center. the most commonly used bp medications were beta-blockers, followed by ccbs, and both were used with the same frequency at 1 and 6 months. in contrast, the percentage of patients on an acei or arb more than doubled between 1 and 6 months, suggesting reluctance to use these agents early after tx (a practice not necessarily evidence-based); however, more than 70% of subjects did not receive acei/arb therapy even at 6 months. fewer than half of all patients received aspirin, including only 60% with dm and/or cvd. similarly, only half with dm and/or cvd received a statin at 1 and 6 months. these data indicate current management of ktx recipients fails to utilize optimal cvd risk reduction measures in a timely fashion, perhaps missing an opportunity to reduce long-term morbidity and mortality from cvd in this at-risk population. background: cardiovascular (cv) risk reduction has been a primary reason for pursuing early corticosteroid withdrawal (ecswd). to date, actual cardiovascular event data (cve) (rather than cv risk) has not been reported for ecswd. therefore, we analyzed and compared actual cv events (cve) and cv-related survival in ecswd (≤7 days) and chronic corticosteroid (ccs) pts. methods: cve and heart failure (hf) data were prospectively collected. cve were defined as sudden death, myocardial infarction, angina, and cerebrovascular accident/ transient ischemic attack. hf events were defined as pulmonary edema or hf diagnosis. conclusions: rtx recipients receiving ecswd experienced: 1) fewer cve and 2) a trend toward overall better pt survival. these differences in cve and pt survival do not present until at least 3 yrs ptx. and therefore require long term followup to become evident. abstracts immunohistochemistry was performed for cd4 + and cd8 + cells. results were compared with those of the patients on maintenance is (gr-is n=29) and the liver tissue from normal subjects (gr-normal n=11). results: the follow-up time in gr-tol was longer than that in gr-is.(gr-tol and gr-is: 121m and 52m, p<0.01) in gr-tol, typical features of neither acute nor chronic rejection were observed following banff criteria. the extent of graft fibrosis in gr-tol, however, was greater, than those in gr-is and gr-normal (gr-tol, gr-is and gr-normal ; 1.6, 0.9 and 0 (ishak's modified staging )(gr-tol vs. gr-is, gr-is vs.gr-normal p<0.01). each number of cd4 + and cd8 + cells in graft infiltrates was increased in gr-tol, compared with that in gr-normal, but equivalent with that in gr-is (cd4 + gr-tol, gr-is and gr-normal ;14.6,10.6 and 5.3 cells/field, gr-tol vs.gr-normal p<0.05, gr-tol vs.gr-is ns / cd8 + gr-tol, gr-is and gr-normal; 27.6, 26.3 and 10.5 cells/field gr-tol vs.gr-normal p<0.01, gr-tol vs.gr-is ns). conclusions+discussion : in tolerant graft after pediatric living-donor ltx, neither acute nor chronic rejection was observed, but fibrosis developed. because of the similar extents of cd4 + and cd8 + cells infiltrates and different follow up time between tolerant and immunosuppressed patients, it remains questionable whether fibrosis in tolerant graft is antigen-dependent. serial protocol biopsy before and after starting weaning is will detect fibrosis early, and observing whether reintroduction of maintenance is reverses fibrosis in that case will answer this question. operational tolerance may not always guarantee intact graft morphology. development of "operational tolerance" after pediatric liver background : in the setting of our pediatric living-donor liver transplantation (ltx), 15% of all the patients (significantly higher proportion, compared with those of other transplant centers) achieved complete withdrawal of immunosuppression (is), which is reffered to as "operational tolerance". nonetheless, some patients encountered rejection while they were undergoing weaning from is. it is,therefore,essential to identify and characterize the differences that will enable patients in these two distinct populations to be distinguished reliably. methods: the study groups consisted of group tolerance(gr-tol) in which 88 patients are successfully weaned off from is, and group rejection (gr-rej) in which 22 patients experienced clinically evident rejection during or after weaning process. the correlation between the clinical outcome (success or failure of weaning is) and following parameters was assessed ; donor/recipient age, donor/recipient gender, abo compatibility, hla mismatch, graft size, early (<1month) rejection episode and initial immunosuppression.results: there was no difference between gr-tol and gr-rej with respect to donor/recipient age (gr-tol and gr-rej;32y and 32y ns/35m and 26m ns), or donor/recipient gender (gr-tol and gr-rej (female);60% and 50% ns/60% and 73% ns). abo compatibility did not differ between the two groups(gr-tol and gr-rej (i dentical:compatible:incompatible);63%:27%:10%and 55%:27%:18% ns).the presence of hla-b mismatch was more frequent in gr-tol than that in gr-rej (gr-tol and gr-rej;95% and 76% p<0.05 ), while the presence of hla-a or dr mismatch did not affect success or failure of weaning is(hla-a gr-tol and gr-rej;73% and 76% ns, hla-dr ;83% and 82% ns). graft size did not differ between the two groups(gbwr gr-tol and gr-rej;2.9% and 3.2% ns). the patients in gr-rej experienced early rejection more frequently than those in gr-tol(gr-tol and gr-rej;17% and 50% p<0.05). mean trough level of tacrolimus within 7 days after ltx was compatible between the two groups (gr-tol and gr-rej;11ng/ml and 11ng/ml ns). conclusions: development of operational tolerance after pediatric ltx was associated with the absence of early rejection and the presence of hla-b mismatch between donors and recipients. auxiliary partial orthotopic liver transplantation (apolt) in children with fulminant hepatic failure. patients with fulminant liver failure (fhf) who undergo auxiliary partial orthotopic liver transplantation (apolt) have a chance to come off immunosuprresion (isp) when the native liver regenerates. it may be most beneficial for children with fhf; however, the literature regarding its use in children has been limited. from the beginning of the pediatric liver transplant program at our institution, 31 patients underwent liver transplantation for fhf. of those, 7 received apolt and the remaining standard liver transplantation (olt). seven children (age 8months to 8 years) who received apot (apolt group) were compared to matched control group of 11 patients (olt group). since apolt was offered routinely at out instituting since 2005, 6 of apolt cases were done since 2005. in apolt group, either left lateral segment or left lobe graft was used. recipients left lobe was removed in all cases. in olt group, 7 received whole liver graft and 4 received partial liver graft. all native livers showed submassive to massive necrosis at the time of transplant in pathology. all children (100%) in apolt group are currently alive with a median follow up of 761 days (range 116-4139 days) where 8 (72%) patients are alive in olt group (median follow up 1724 days). six of 7 children in apolt group (87%) showed native liver regeneration. first four apolt recipients (57%) are currently off isp with fully regenerated native liver. two of those patients developed complete atrophy of the graft liver, one underwent graft removal due to sepsis caused by severe rejection. one remaining patient who is off isp is displaying progressive atrophy of the transplant liver. incidence of acute rejection was 57% (4/7) in apolt group vs 30% (3/10) in olt group. other postoperative complications included hepatic artery thrombosis (hat) (n=1), bile leak (n=1), bilary structure (n=1) and bowel obstruction (n=1) in apolt group, hat (n=1), bile leak (n=2), bowel perforation (n=2), chylothorax (n=1) and aplastic anemia (n=2). median posttransplant length of stay was 15 days in apolt and 20days in olt group. conclusions: apolt was safely performed in children with fhf. significant proportion of recipients displayed native liver regeneration and came off immunosuppression. natural killer cell dysfunction in pediatric acute liver failure. nada yazigi, 1 greg tiao, 1 alexandra filipovich, 2 john bucuvalas. 1 in pediatric patients, indeterminate acute liver failure (alf) accounts for ∼50% of all cases, and carries a particularly poor prognosis without transplantation. evidence exists to suggest that acute liver failure may reflect a disproportionate immune response to a common stimulus. nk cells comprise a central component of the innate immune system. we hypothesized that nk cell dysfunction (innate or secondary to an antigenic insult) plays a pathologic role in indeterminate alf. we reviewed peripheral nk cell function in a series of 15 consecutive children cared for at cincinnati children's hospital, who met criteria for indeterminate alf as defined by the pediatric alf study group. peripheral blood testing was carried for nk cell number, cytolytic function and perforin and granzyme activity as part of our clinical alf protocol. seven of fifteen patients had nk cell dysfunction. only the severity of cholestasis was statistically higher in the nk cell dysfunction group. there was no statistical difference between the 2 groups with respect to age, inr, or peripheral blood cell counts. 3 of seven patients with nk cell dysfunction died in contrast to none of eight in the normal nk cell group. of the 3 patients with nk cell dysfunction who eventually received a liver transplant, 2 had severe early recurrence of chronic hepatitis in the graft at a year follow up. those outcomes are in sharp contrast to the group with no nk cell dysfunction where 7 patients needed transplantation, but all had no complications both on short or long term follow up. we documented nk cell dysfunction at the time of alf diagnosis in 7/15 pediatric patients with indeterminate alf. this subgroup of patients was found to have higher: mortality, risk of infections, as well as recurrent disease in the graft. our findings suggest that nk cell dysfunction is involved in the pathogenesis of indeterminate alf. as such, it could therefore be a prime target for therapeutic intervention, with goals to rescue the patient from liver failure and /or to improve post-transplantation outcomes. background hrs is a reversible renal failure which occurs in pts with advanced liver disease and portal hypertension and is characterized by a marked decrease in gfr and rpf in absence of other identifiable causes. vasodilation theory is currently the most accepted hypothesis to explain the pathogenesis. in decompensated cirrhotics, probability of developing hrs is 8-20%/yr and increases to 40% at 5 yrs. ideal treatment is ltx. however, there is an urgent need for effective alternative tx to increase surv chances for pts until ltx can be performed. interventions that have shown some promise are vasoconstrictors in splanchnic circulation and tips. main objective was to compare efficacy of two different regimens (albumin/terlipressin resp hes/terlipressin both w/ wo midotrine) against tips whereas grf was considered as primary efficacy endpoint. pts/tx dx of hrs was based on criteria, as proposed by international ascites club. only pts with esld on the waiting list for ltx were eligible to be enrolled. pts were assigned to tx arms and randomized w/wo midotrine. volume/vasoconstrictor tx lasted for 10d, mitodrine was continued; follow up for 90d. results iia (albumin/terlipressin); iib (hes/terlipressin); iic (tips) discussion combination of volume expansion/vasoconstriction improved effectively gfr in pts with hrs. use of albumin shows no advantage compared to (cost effective) hes. although a marked improvement was observed during iv-treatment, renal fct deteriorated upon treatment withdrawal whereas pts with continued mitodrine showed superior long term outcome. we analyze our single institution experience to quantify the long-term incidence of renal failure based on month 3 gfr compared to subsequent determinations. methods: this is an irb approved retrospective review of the prospectively maintained database of lt recipients. exclusion: patients on renal replacement therapy (rrt) at time of transplant, combined liver kidney, fulminant hepatic failure, and <1-year follow-up. gfrs (i 125 iothalamate glofil method) were measured at initial evaluation (ie), month 3 (m3), year 1 (y1), 2 (y2), 5 (y5), 10 (y10), and 15 (y15). patients were grouped by gfr >80 (g1), 60-80 (g2), and <60 (g3). ie and m3 gfr were used as starting points for longitudinal analyses. paired data analysis for ie, m3, y5 and y10 was also performed. renal failure was defined as gfr <30, received kidney transplant, on dialysis, or on kidney transplant list. results: 592 liver transplant patients were reviewed between 1985 and 1999. paired glofil data was available for the y5 and y10 analysis in 114 patients. m3 gfr correlated more with long-term renal function (p<0.0024). g1 demonstrated largest reductions in gfr over time. g2 and g3, when corrected for patients that got kidney transplantation and rrt, demonstrated progressive reduction in gfr. g3, g2, and g1 were statistically significant (p<0.001 wilcoxon two-sample test). this study clearly demonstrates progressive decline in gfr continuing out to 15 years after liver transplantation. m3 gfr correlates better with long-term renal function compared to ie gfr (not truly reflective of renal function at time of lt). if m3 gfr <60, data showed a high rate of renal failure in our paired data analysis by y5 (p<0.0024). by correcting for patients with renal failure, the previously reported stability in gfr between y1 and y5 is not seen. analysis of grouping demonstrates that g1 patients at m3 have lower incidence of renal failure >10 years after lt. g2 and g3 patients will be at higher risk for renal failure each year after transplantation. introduction: calcineurin inhibitors have demonstrated efficacy in liver transplantation. however, they have a potential to impair renal function. delayed tacrolimus (tac) administration may reduce the risk of renal dysfunction. methods: a prospective study included liver transplant pts randomised to delayed introduction of tac (day 5) + daclizumab (dac) (group a) or to immediate tac administration (group b). in both groups tac t0 was 10-20 ng/ml until week 4 and 5-15 ng/ml thereafter. mmf was given at 2 g/d for 2 months, and corticosteroids (cs) at standard doses. pts with a serum creatinine (scr) > 180 µmol/l at 12 hours (h12) were excluded. the primary endpoint was the rate of pts with a mean scr > 130 mmol/l at month 6. month 24 results are presented. results: 207 pts were randomised. baseline characteristics were similar. at month 6, mean tac t0 was 9.6 (group a) and 11.2 ng/ml (group b median follow-up post-tx was 8 years (5-21). most frequent tx indications were alcoholic (34%) and hcv (10%) cirrhosis. amdrd glomerular filtration rate (gfr) was < 60 ml/min/1,73m 2 in 11% (bt), 46% (1m), 48% (1y) and 55% (5y) of the patients. changes in gfr were then compared according to the immunosuppressive protocol: -group "cni+mmf" = a calcineurin inhibitor (cni) + mycophenolate mofetil (mmf). -group "cni" = a cni without mmf. in this group, some patients received only cni and some cni + azathioprine. there was no difference between those 2 sub-groups, neither on rf nor on cni doses. all those patients were thus pooled. in both groups, gfr decreased from bt: -14% in "cni+mmf" vs -25% in "cni" at 1m (p=0.06), -14% vs -29% at 1y (p=0.03), and -12% vs -33% at 5y (p=0.02). although their mean gfr bt was lower (86 vs 97 ml/min/1.73m 2 , p=0.0002), the decrease in rf in "cni+mmf" patients was less severe. nearly 50% of the patients had renal insufficiency in the 5 years following liver tx. the reduction in the gfr is less pronounced in patients treated with mmf even if they were significantly more at risk bt. except at 1m, there was no difference in cni doses between the 2 groups, suggesting that the sustained lower decrease in rf observed in "cni+mmf" may not be only explained by a cni dose reduction. acute rejection is a complex biologic process involving multiple cell types, cytokines and chemokines/chemokine receptors. we hypothesized that an mrna panel that included genes implicated in the anti-allograft response would distinguish allografts undergoing acute rejection from normal allografts with a high degree of accuracy. we tested this hypothesis by measuring levels of urinary cell mrna and peripheral blood cell mrna for cell surface proteins cd3, cd20, cd25, cd103, and ctla4; chemokines/ chemokine receptors ip10, mig, cxcr3; cytotoxic attack molecules granzyme b(gb) and perforin, and immunoregulators foxp3, tgf-beta1 and il-10. gene specific primer pairs and probes were used in pre-amplification enhanced real time quantitative pcr assays to measure mrna and transcripts for 18s rrna. for each cell source, we used logistic regression to identify a linear function of up to 5 log-transformed measures that would distinguish biopsies of ar patients from those of stable transplant patients. our study demonstrates that molecular signatures developed using urinary cell levels of just 5 genes (signature 1, urinary cell levels of mrna for ctla4, foxp3, gb, cd3, and mig), or a combination of 3 urinary and blood cell levels (signature 3, urinary cell level of ctla4 mrna and peripheral blood cell levels of cd103 and ctla4) differentiate ar from stable biopsies with 100% sensitivity and 100% specificity. blood cell levels alone are also informative, but less so. we conclude that molecular signatures, developed from noninvasively ascertained mrna profiles of urinary cells/ peripheral blood cells, predict acute rejection with extraordinary accuracy. clinical trials to validate the predictive value of these signatures are worthy of pursuit. we have described the association of cd20+ b cell infiltrates in renal transplant (tx) biopsies (bxs) with acute cellular rejection (acr) and tx dysfunction (dysfx). we have also found metabolically active plasma cells (pcs) staining for s6 ribosomal protein (s6rp) within these txs. herein, we report the significance of cd138+ pcs in rejection (rj) and evaluate the impact of cd20, cd138, and s6rp on long term tx fx by calculated creatinine clearance (crcl). we studied 46 tx bxs from 32 pediatric (ped) patients (pts) who were bxed for suspicion of rj from nov 2001 to nov 2004. pts were given daclizumab and maintained on prednisone, mycophenolate mofetil, tacrolimus or cyclosporine. immunohistochemical staining and quantification for cd20, cd138, s6rp, and c4d were performed under 500x light microscopy. bxs were classified by modified banff 97 criteria. crcl was followed 2 yr post-bx. cd138+ pcs were associated with c4d-negative acr (p=0.0002) but not antibody mediated rj (amr, p=0.82). roc analysis confirmed >5 cd138+ cells/hpf strongly associated with acr, yielding 89% sensitivity, 83% specificity, correctly classifying 84% and comprising total roc area 0.92 (95% ci 0.82, 1). higher cd138 counts at bx correlated with worse tx fx (fig 1) . a univariate regression model showed that cd20, cd138, s6rp and time were associated with a decline in tx fx at bx. multivariate model showed that cd20, cd138, and time had the main effects on crcl decline, with s6rp dropping out. all patients regardless of rj status had a 16 ml/min/1.73 m 2 crcl decline exerted by time (p=0.004). pts with cd20 had an additional sustained 19 ml/min/1.73 m 2 crcl decline seen 2yrs post-bx (p=0.03). pts with cd138 also had 19 ml/min/1.73 m 2 crcl decline at bx (p=0.03), but there was an interaction between time and cd138 that negated a sustained effect (p=0.04). this study identifies a numerical threshold of >5 cd138 cells/hpf that is associated with acr and tx dysfx. infiltrating cd20 cells had the greatest, sustained effect on tx dysfx. we conclude that cells of the b lineage, particularly cd20, play a key, but undefined, role in acr. intragraft there is now evidence that foxp3+ cells are not indicators of tolerance, since foxp3 is also increased during acute rejection. however, it is unknown whether foxp3+ cells are present during chronic antibody mediated rejection. moreover, the relative balance of regulatory, effector and cytotoxic pathways in chronic vs. acute injury has yet to be explored. here we addressed this issue. intragraft regulatory, effector and cytotoxic transcriptional profiles were analysed within renal transplant biopsies (n = 43) classified (banff 2005) as displaying normal histology, chronic calcineurin inhibitor toxicity (cnitox), chronic antibody mediated rejection (camr) and acute cellular rejection (acr). granzyme b, tbet and foxp3 mrna were measured by quantitative pcr and foxp3 -positive cells were additionally quantified in graft biopsies by immunohistochemistry. distinguishing mrna profiles were analyzed in the peripheral blood (n = 26). our data show that foxp3 mrna is increased not only in acr (p<0.0001) but also in camr (p<0.05). expression of foxp3 mrna correlated tightly with the density of foxp3 protein-positive cells by immunohistochemistry (spearman r = 0.71; p < 0.0001); foxp3+ cells were found in aggregates and within tubules. moreover, graft cytotoxic, effector and regulatory pathways were all found to be active in chronic as well as acute graft injury. significant increases in granzymze b, tbet and foxp3 mrna were observed in camr, cni-tox and acr compared to normal histology (p<0.0001, p<0.001 or p<0.05). however, differences in the relative contribution of each pathway were evident, with significant accumulation of foxp3 mrna predominating in acr and granzyme b predominating in camr. thus, camr can be distinguished from both acr and cni-tox by an unfavorable intragraft granzyme b/foxp3 mrna ratio (p< 0.05). interestingly, this ratio was reversed in the blood, suggesting different migratory patterns for regulatory and cytotoxic cells between the blood and the graft.our data thus confirm that intragraft and peripheral blood foxp3 accumulation is also a feature of camr of kidney grafts. moreover, camr can be distinguished from other graft injury types based on its intragraft or blood cytoxicity/regulatory profile. survival of solid organ grafts depends on life long immunosuppression which results in increased rates of infection and malignancy. induction of tolerance to allograft would represent the optimal solution for controlling both chronic rejection and side effects of immunosuppression. we previously showed that operational tolerance after kidney transplantation could occur in some patient. here, the potential of high throughput microarray technology allowed us to study the peripheral blood gene expression profile associated to operational tolerance and chronic rejection in a cohort of human kidney graft recipients (n=26). microarrays were used to compare the gene expression profile of pbmc from patients with chronic rejection and drug-free operationally tolerant recipients. results have been treated using a classical statistical and a non-statistical analysis based on the identification of key leader genes associated respectively to chronic rejection and operational tolerance, either as those mostly changing their expression or having the strongest interconnections. 343 differentially expressed genes were identified between operational tolerant patients and patients with chronic rejection. abstracts defined as missing > 10% of prescribed doses on mam and mems, and >2 sd among consecutive blood serum levels. results: participants were 28 transplant patients (m = 14.11 +3.38 years old, 75% male, 71.4% caucasian). on the mam, 67.9% of the patients acknowledged some non-adherence but minimized how many doses they missed. using mems technology, 90% had some non-adherence and specifically, 23.8% of the participants missed doses and only 56% of their doses were taken within the allowable time frame. using > 2sd criteria for blood serum levels, 42% of the participants were considered non-adherent. non-adherence worsened with years since transplant. more missed (r = .59, p = .001) and late doses (r = .59, p = 0.04) on the mam and >2 sd among blood serum levels (r = .83, p = .001) was associated with higher incidence of acute rejections. adherence data was examined for patients with documented acute rejections (n = 12). sensitivity and specificity of each detection method was also examined. the mam and sd detection methods each identified non-adherence in 67% of the patients with acute rejections; mems did not identify any additional non-adherent patients. only 25% of the patients with acute rejections were identified consistently by all three adherence detection methods; all patients with acute rejections were identified by at least one method. discussion: non-adherence worsening with time since transplant and was associated with acute rejections. since no single method of detecting adherence identified all the patients with acute rejections, multi-method adherence assessments should be used to accurately capture patients who are non-adherent. non na is a leading cause of allograft loss and results from multiple factors. locus of control (loc) and beliefs regarding health have been associated with adherence in other populations. 51 randomly chosen ktr's were interviewed using a confidential questionnaire administered by an outside investigator that included questions regarding loc, health beliefs and self-efficacy. the population was 55% female, 92% black, 15% hispanic, 71% deceased donor kidney, 47% diabetic, 49% greater than high school education, 36% employed, 30% married or cohabiting, 63% income <20k per year, 65% insured by medicaid. mean age 47.8±13.5 yrs, time on dialysis 72.8±51 mos, months since transplant 23.9±14.3, total meds 7.3±3.0. by pearson correlation, non-adherence (na), defined as "having missed doses of immunosuppression over the preceding 3 months", was not correlated with race, gender, income, type of insurance, age, marital status, type of txp, mos on dialysis, time since transplant, or number of medications. na was correlated with higher education level (r=0.4, p=0.006), current employment (r=0.3, p=0.05), and knowledge of most recent creatinine value (r=0.35, p=0.015). na was associated with concerns regarding prednisone (long term effects, dependency) r=0.33, p=0.018, feelings of greater personal control over illness (r=0.36, p=0.011), and inversely correlated with powerful others loc (feeling that one's health is dependent on other people), r=-0.31, p=0.029 and belief in the necessity of medication for maintenance of transplant health, r=-0.3, p=0.031. we conclude, in our population of inner-city patients: 1. na is not associated with standard demographic factors including income, race and gender. 2. contrary to findings in other populations, na is associated with higher education and current employment. 3. na is associated with knowledge about creatinine value, concern regarding long-term effects of prednisone and disbelief in the importance of transplant medications. 4. na is associated with feelings of personal control and feeling that powerful others (e.g. health care providers) are not of high importance in the outcome of illness. 5. education programs designed to address na in this population should be targeted towards altering negative beliefs regarding medications and stress the importance of partnering with the transplant team for optimal long-term outcome. purpose: the present study aimed to prospectively examine the relationships among nonadherence, health-related quality of life (hrqol), and family factors in adolescent kidney, liver, and heart transplant recipients. method: 68 adolescent transplant recipients aged 11 to 20 years (m = 15.8, sd = 2.5; 44% female; 57% kidney, 25% liver, 18% heart) and their parents participated. at baseline and 18-month follow-up assessments, adolescents and their parents independently completed phone interviews assessing self-/proxy-reported medication adherence, hrqol, and family cohesion and conflict. medical record reviews were conducted to obtain current medications, immunosuppressant drug assays, and clinical outcomes in the past year (i.e., rejection episodes, hospitalizations, graft loss). results: at baseline, adolescents classified as nonadherent based on self-report and tacrolimus standard deviation (sd) reported significantly lower general health perceptions (f(3, 64) = 3.63, p < .05), self-esteem (f = 4.58, p < .01), mental health (f = 3.09, p < .05), and behavior hrqol (f = 2.75, p < .05) compared to adolescents classified as adherent. similarly, parents of adolescents classified as nonadherent reported significantly lower physical functioning (f = 3.18, p < .05), self-esteem (f = 5.85, p < .01), and behavior hrqol (f = 2.82, p < .05) for their adolescents. family conflict was correlated with adolescent report of behavior (r = -.49, p < .01), physical functioning (r = -.33, p < .01), self-esteem (r = -.42, p < .01), and mental health hrqol (r = -.34, p < .01). family conflict was correlated with parent report of behavior (r = -.46, p < .01) and physical functioning (r = .24, p < .05). improvement and deterioration in hrqol from baseline to 18-month follow-up is currently being examined. it is expected that increased family conflict and decreased medication adherence will be associated with deteriorations in hrqol. the interrelationships between medication adherence, family conflict, and hrqol domains such as self-esteem and mental health suggest that interventions targeting these domains may result in improvements in medication adherence behavior. the use of cam in general is associated with non-disclosure by patients to physicians. 51 randomly chosen ktrs were interviewed using a confidential questionnaire administered by an outside investigator, including questions on cam usage, whether it was doctor-recommended, and whether the patient disclosed use. cam was defined as ingestion of herbal or other preparations, use of mind-body techniques or manipulation of the body for healing by someone not an allopathic medical provider. use of vitamins and spirituality were excluded. the population was 55% female, 92% black, 15% hispanic, 71% deceased donor kidney, 47% diabetic, 49% > high school education, 36% employed, 30% married or cohabiting, 63% income <20k per year, 65% insured by medicaid. mean age 47.8±13.5 yrs, time on dialysis 72.8±51 mos, months since transplant 23.9±14.3, total meds 7.3±3.0. 45% of patients (n=23) used cam. by pearson r, cam use was correlated with na to immunosuppressants, p= 0.041, r= 0.4, blood sugar-lowering medications, p= 0.003, r= 0.57, and cholesterol lowering medications, p= 0.0001, r=0.84, worries about long-term effects of medicines, p=0.02, r= 0.324, belief that doctors place too much trust in medication, p=0.032, r=0.3, and that natural remedies are safer than medicines, p= 0.034, r=0.3. cam use was inversely related to belief that health depends on allopathic medicines, p=0.014, r= -0.341, medicines protect from worsening disease, p=0.048, r= -0.28, and that following doctors orders is the best way to stay healthy, p= 0.04, r= -0.29, and that having a kidney transplant makes them feel happy, p= 0.005, r= -0.39. we conclude, in our population of inner-city patients: 1. use of cam is correlated with medication non-adherence. 2. patients who use cam are more worried about long term effects of medication, believe that natural remedies are safer than medications and that doctors place too much trust in medication. 3. patients who use cam do not believe that their health depends on allopathic medication, that medicines protect from worsening disease, or that following a doctor's orders is the best way to maintain optimal health. 4. patients who use cam are less happy with their kidney transplant. 5. disussing cam use and motivation for use is important in the transplant clinic and may alert the provider to possible risk for non-adherence. by multivariate cox analysis the risk of tg related to the presence of hla-iiab death censored graft loss occurred in 4.6% of patients without tg and in 38.4% of patients with tg (p<0.0001) hla-iiab are associated with higher risk of tg and reduced graft survival. furthermore, the risk of tg and its prognosis relate to the level of hla-iiab quantitated in a solid phase assay term survival of cardiac allografts in wild-type mice by alloantigen (alloag)-specific foxp3 + cd4 + cd25 + natural regulatory t (nt reg ) cells. guliang xia, 1 jie he methods: fresh naive cd4 + cd25 + nt reg were isolated from congeneic b6.pl mice via automacs and enriched for alloag specificity by in vitro culture with either anti-cd3/ cd28-coated dynabeads (d0-11), then donor bone marrow-derived dendritic cells 8% (d+16) for dc/beads-expanded nt reg , while total fold of expansion of nt reg remained similar (24.8∼30.2 for beads/dc-or 5.2∼7.1 for dc/beads-expansion) regardless of the presence or absence of tgf-β. introducing ra (100 nm) into bead/dc-based, tgf-β/ il-2-conditioned culture resulted in marginal improvement with 28.9% (d+18) nt reg being foxp3 + . in mlr assays, nt reg expanded with tgf-β/il-2 exerted more potent suppression than cells conditioned with il-2 alone. in vivo, beads/dc-expanded, tgf-β/ il-2-conditioned nt reg synergized with transient host t cell-depletion (anti-thy1.2 mab i.p. 50 µg at d-3 & 25 µg on d+2) in c57bl/6 mice to suppress balb/c heart allograft rejection with 57.1% (n=7) and 100% (n=10) allografts surviving over 100 days when 4x10 7 or 8x10 7 cells/mouse were injected immediately post-transplant, respectively. anti-thy1.2 treatment alone led to only 18.8% long-term survival. infused nt reg survived long-term (5.8 % circulating t cells (8x10 7 cell dose) or 1.2 % (4x10 7 cell dose) at d+7 post-transplant) and expressed high level foxp3 (40∼60%) in vivo. long-term surviving allografts showed characteristics of 'acquired immune privilege' with cellular infiltrates that were foxp3 + , tgf-β + , il-10 + and indoleamine 2,3-dioxygenase (ido) + , although signs of mild to moderate chronic rejection were still evident conclusion: t-bet deficiency results in up-regulation of il-17 expression in addition to th2 associated cytokines resulting in acceleration of chronic rejection despite profound deficiency of ifn-γ. t-bet deficiency may contribute to the alloimmune responses independent of ifn-γ by in situ hybridization, tir8 mrna level was higher (p<0.05) in cortical tubuli, glomeruli, perivascular and peritubular areas of kidney grafts at 1, 3 and 6-7 d post-tx, than in naive kidneys. to assess how local expression of tir8 affects the outcome of kidney grafts, we transplanted tir8 -/-b6x129 kidneys into dba/2 mice. most (83%) recipients of tir8 -/-kidneys rejected their grafts with a median survival of 9.5 d (n=12, p<0.05 vs wt) and had more severe graft dysfunction (bun levels) at day 1, 3 and 6-7 days post-tx, than recipients of a wt allograft (p<0.05) opticept trial: efficacy and safety of monitored mmf in combination with cni in renal transplantation at 12 months. r trough-based dose adjustments were made in the mmf cc arms. antibody induction and/or corticosteroids were administered according to center practice. primary endpoints were the proportion of patients with treatment failure (biopsy-proven acute rejection [bpar], graft loss, death), and mean percent change in calculated glomerular filtration rate (gfr; nankivell equation) at 12 months. safety endpoints were incidences of adverse events (aes) and serious aes baseline characteristics did not differ among treatment groups with living donors accounting for approximately 50% of grafts. 82% received tacrolimus (tac) and 18% cyclosporine (cya): cni doses and levels were significantly lower in group a. mmf doses were greater in cya-treated subjects in all groups cya treated patients and in group a (p=0.08); stability of renal function over time was greatest in group a. despite higher mmf doses in group a (p<0.001) at most time points, significantly fewer mmf withdrawals occurred in group a vs. groups b and c. conclusions: a concentration-controlled mmf and reduced level cni regimen is not inferior to that of fixed-dose mmf and standard-dose cni as regards bpar and other end points. this regimen facilitated higher mmf dosing without an overall increase in adverse effects, and with a trend toward preservation of kidney function versus standard-dose cni regimens comparison at one year of interstitial fibrosis (if) by automatic quantification in renal transplant recipients with cyclosporine (csa) discontinuation and sirolimus (srl) introduction introduction: we previsouly reported the clinical results of a multicentric study showing that csa conversion to srl at week (w) 12 is associated with a significant improvement in renal function. using routine renal biopsy (rb) performed at w52 during this study routine rb was performed at w52. for each rb, a section was imaged using a colour video camera and analyzed by a program of colour segmentation which automatically extracts green colour areas characteristic of if. results were expressed as percentage of if and grade according to banff classification. results: male donor gender was associated with higher if (30±2% vs. 23±2%, p =0.03). if was numericaly higher in patients who had experienced acute rejection (33±4%, n = 18 vs. 26±1%, n=99, p=0.06) there was a positive correlation between renal function and the percentage of if on rb (p=0.004). despite significant improvement of renal function at w52 in the srl group intent to treat (n=117) mean if (%) grade i (%) grade ii (%) grade iii (%) sirolimus (n=58) conclusion: despite significant improvement in renal function after csa to srl conversion at 3 months, we found no difference of if on rb at w52. the observed improvement of renal function may be due to a hemodynamic effect. a longer delay may be necessary to observe histological improvement. the higher if score than the one previously reported by others may be explained by the use of expanded criteria donors abstract# 528 effects of cni or mmf withdrawal on carotid intima media thickness in renal transplant recipients methods: we included 119 stable renal transplant patients on cni-based immunosuppression, including steroids (10 mg/d) and mmf (2g/d), who were randomized to mmf-withdrawal (group a: csa-auc 3000 ng*h/ml) or cni-withdrawal (group b: auc-mpa 75 µg*h/ml). patients were treated for traditional risk factors according to stringent predefined targets. ambulatory bloodpressure (abpm), lipids, estimated creatinine clearance (mdrd) and imt were measured at baseline and after 12 months. results: groups were comparable with respect to demographic characteristics, immunological profile, renal function, systolic and diastolic bloodpressure and lipids. mean duration of follow-up was 17.4±5.5 months. only 1 patient (1.3%) in group b and 3 patients (3.8%) in group c experienced acute rejection despite adequate exposure (p=0.31). imt did not change final renal function outcomes from the spare-the-nephron (stn) trial: mycophenolate mofetil (mmf)/sirolimus (srl) maintenance therapy and cni withdrawal in renal transplant recipients purpose: to compare the effect on renal function of maintenance immunosuppression with mmf and srl to that of mmf and a cni in renal allograft recipients. methods: in a 2-year open-label, prospective, randomized, controlled, multicenter study, 305 subjects maintained on mmf and a cni were randomized 30-180 days posttransplantation to either mmf (1-1.5 g bid) plus srl (2-10 mg followed by ≥ 2 mg/ day results: outcomes of the first 123 subjects receiving mmf/srl and 126 receiving mmf/cni (tac, n=98; csa, n=28) completing 1 year of follow-up will be reported here. final outcomes of all 305 subjects will be presented at the congress. mean time from transplant to randomization in both groups was 117 days. groups were similar at baseline for all reported renal function endpoints after 12 months of therapy, maintenance immunosuppression with mmf/ srl after cni withdrawal appears to preserve renal function when compared with a mmf/cni-containing regimen improved outcomes after de novo renal transplantation: 2-year results from the symphony study. h. ekberg, 1 h. tedesco-silva frei, 10 y. vanrenterghem, 11 p. daloze, 12 p. halloran at 2 years, the rate of uncensored graft loss was lowest in patients receiving tacrolimus (8% vs 10-13% in other groups; kaplan-meier estimates). gfr at the end of the core study was slightly better in the follow-up itt patients (65-70ml/ min) than in the core study itt patients (57-65ml/min), suggesting inclusion of betterperforming patients in the follow-up. renal function was generally stable over year 2. a slight improvement in gfr in the sirolimus group (+1.6ml/min) was observed, whereas the tacrolimus group still had superior gfr (69 vs 65-67ml/min in other groups). conclusions: in follow-up patients, renal function was stable during the second year and gfr differences were less marked than at 1 year a prospective randomized study of alemtuzumab vs rabbit anti-thymocyte globulin induction in kidney and pancreas transplantation gautreaux, 1 s. iskandar, 4 p. adams, 2 r. stratta. 1 1 surgery; 2 medicine; 3 pharmacy alemtuzumab (alem) and rabbit anti-thymocyte globulin (ratg) are the most commonly used t-cell depleting induction agents in kidney (k) and pancreas (p) transplantation (tx) expanded criteria donors (ecd) were included. results: between 2/1/05 and 8/24/07 225 pts enrolled and 222 pts were transplanted. of 222 pts, 180(81%) had ktx alone, 38(17%) kptx, and 4(2%) paktx. of 180 ktx alone, 152(84%) were deceased donor, and 61(34%) were ecds. recipient age, race, re-tx abstract# 535 purpose: to determine the impact of alginduction on long-term outcomes post-renal tx. methods: between 01/85 and 01/86, 123 consecutive adult pts received a deceased donor renal tx at a single institution results: the incidence of acute rejection was lower in gr.2 (28% vs. 75%, p<0.0001). the incidence of cmv infection was 10% in gr.1 and 18% in gr.2 (p=ns). the overall incidence of cancer was 22 abstract# 538 single-dose induction with rabbit anti-thymocyte globulin (ratg) safely improves renal allograft function and reduces chronic allograft nephropathy 1 clifford miles, 1 gerald groggel, 1 lucile wrenshall. 1 1 divisions of transplantation and nephrology we conducted a prospective, randomized trial in renal transplant recipients comparing two dosing protocols [single dose (6mg/kg) vs. divided doses (1.5mg/kg for 4 doses)] of rabbit anti-thymocyte globulin (ratg; thymoglobulin®). we present herein the results of the first 142 patients throughout the first 6 months post-transplantation, recipients of kidneys from non-marginal deceased donors derived the greatest benefit in renal function (egfr) from the single-dose regimen (p = 0.006). the incidence of chronic allograft nephropathy (can) was also lower in the single-dose group, in both clinically-indicated and protocol biopsies combined (p = 0.045) and in 12-month protocol biopsies alone high risk (race, pra) 8 (11%) in multivariable regression, allograft failure strongly predicted increased risk of subsequent cve. among listed candidates, receipt of a transplant was associated with significant time adjusted for baseline factors, cve after transplant predicted increased risk of subsequent mortality: hr 5.15 (ci 4.53-5.85) after is microalbuminuria post-renal transplantation is related to inflammation and cardiovascular risk our objective was to define the relationship between microalbuminuria and these risk factors in stable rtr. methods: over one year, we identified 223 stable rtr who were at least 2 months post-transplant and provided 3 successive urine albumin-to-creatinine ratio (acr) measurements, excluding those with recent illness and overt proteinuria. microalbuminuria was defined as averaged acr ≥ 2.0 in men and 2.8 in women (cda 2003). framingham-based traditional as well as novel cardiovascular risk factors associated with microalbuminuria were determined by univariate (p < 0.20), followed by stepwise backwards elimination (p >0.05) multivariate logistic regression analysis microalbuminuria did not correlate with prior acute rejection, delayed graft function, or any specific antihypertensive or immunosuppressive agents. conclusions: post-transplant microalbuminuria is highly prevalent and is associated with elevated crp, elevated bp, and smoking. its relationship to these other factors suggests that it reflects an inflammatory state in otherwise stable patients and thus may indicate graft and patient health the first year after kidney transplantation (tx) is associated with increased mortality relative to dialysis. early post-tx deaths are often cardiovascular (cv) and frequently occur after the first week post-tx. ctnt is a sensitive and specific maker of myocardial injury. in this study we investigated whether ctnt relates to early post-tx survival. methods: 396 patients received kidney tx from 9/2004 to 12/2006, 74% from living donors. ctnt was measured during the pre-tx workup and periodically while on the tx waiting list. 261 patients (64%) had a dobutamine stress echo (dse) and 103 (26%) had a coronary angiogram. the combined end point of the study was death or major cardiac events. survival was censored for graft loss. results: mean age was 51+12, 59% males. pre-tx ctnt level was elevated (>0.01 ng/ ml) in 56% of patients other dse derived parameters did not relate significantly to survival. ctnt further stratified the risk associated with other variables. thus, among patients with ef<55%, 2 year survival was 97%, 88% and 60% (p=0.0003) in patients with ctnt <0.01, 0.01-0.03 and >0.03, respectively. similarly, these ctnt ranges stratified risk in patients with low albumin conclusion: an elevated pre-tx ctnt is a strong and independent predictor of reduced early post-tx survival. ctnt allows stratification of risk in patients who have other risk factors such as low ef, low serum albumin and dialysis>2 years. in all patients, independent of any other variables, a normal ctnt was an excellent predictor (97%) of survival abstract# 545 validation of framingham risk assessment by actual cardiovascular event data in renal transplant recipients alloway, 2 michael cardi, 3 gautham mogilishetty, 2 shazad safdar excellent outcome after liver transplantation in children with cystic fibrosis some studies have reported benefits of liver transplantation (lt) in cf patients, but large outcome studies are not available. we report the outcomes of a large cohort of cf patients undergoing lt. methods: pre and post-lt patient characteristics, post-lt morbidity and mortality, and patient and graft survival were 2702 patients age < 18yr) received a 1 st isolated lt. 62 cf patients were listed for 1 st lt, neither waitlist deaths nor the probability of death from time of listing was different from non-cf. 42 (1.6%) cf patients underwent lt with an average followup of 3yrs (0-10yrs) average peld: 5.4 (86.5% had a peld < 10), median age: 11.9 yrs (0.7 -17.5) graft survival in cf patients was 85.0%, 81.2%, and 81.2% at 1, 3, and 5 yrs compared to 85.6%, 80.7%, and 78.1%. rejection rates were not different (50.6% cf vs 56.4% non-cf @ 5 yrs with 50% of these patients requiring dialysis. standardized height and weight scores showed no improvement over 2 years followup in the cf patients (height z -1.4 at tx to -1.4 at 2 yrs., weight z -1.2 to -1.5), but tended to improve in the non-cf group in addition, death rates from time of listing are not increased compared to non-cf patients. these data support lt as a treatment for cf liver disease, but studies investigating the lack of growth improvement and increased renal complications in these patients may further improve outcomes. abstracts full cni group. conclusion: compared to full cni, low cni/mmf a) allows renal function to recover in patients with impaired renal function at the time of ltx and b) preserves long term renal function. cni sparing in combination with mmf may become cellular islet autoimmunity influences clinical outcome of islet cell transplantation methods: twenty-one t1d patients received cultured islet cell grafts prepared from multiple donors and transplanted under anti-thymocyte globulin (atg) induction and tacrolimus plus mycophenolate mofetil (mmf) maintenance immunosuppression. immunity against auto-and alloantigens was measured before and during one year after transplantation. cellular auto-and alloreactivity was assessed by lymphocyte stimulation tests against autoantigens and cytotoxic t lymphocyte precursor assays, respectively. humoral reactivity was measured by auto-and alloantibodies. clinical outcome parameters remained blinded until their correlation with immunological parameters. results: all patients showed significant improvement of metabolic control and 13 out of 21 became insulin-independent. multivariate analyses showed that presence of cellular autoimmunity before and after transplantation was associated with delayed insulinindependence (p=0.001 and p=0.01, respectively) and lower circulating c-peptide levels during the first year after transplantation (p=0.002 and p=0.02, respectively). 7/8 patients without pre-existent t-cell autoreactivity became insulin-independent, versus 0/4 patients reactive to both islet autoantigens gad and ia-2 before transplantation. autoantibody levels and cellular alloreactivity were not associated with outcome. conclusions: cellular islet-specific autoimmunity affects clinical outcome of islet cell transplantation under atg-tacrolimus-mmf immunosuppression bmp-7 is downregulated & tgfβ1 to bmp-7 ratio favors emt during acute rejection of human renal allografts allospecific cd154+ t-cells predict rejection risk and measure immunosuppressive effect after abdominal organ transplantation in 100 recipients methods: allospecific cd154+t-cells were measured in <24 hours with polychromatic flow cytometry to identify rejectors (who had experienced acute cellular rejection within 60 days post-transplantation) in single mixed leukocyte responses (mlr) from 100 cross-sectional recipients-88 children with liver or intestine allografts, and 12 adults with renal allografts. where possible, results were correlated with proliferative alloresponses measured by cfse-dye dilution (n=45), allograft biopsies (n=46), and expression of ctla4, a negative t-cell costimulator, which antagonizes cd154-mediated effects (n=52). results: in the first 33 children, logistic regression identified donor-specific, memory cd154+ t-cytotoxic cells (tc) as enhanced among rejectors, compared with non-rejectors (408±231 vs 90±36 per 10,000 cells, p=0.003), relatively drug-resistant (r with drug levels =-0.5, p=ns), with greatest sensitivity/specificity (>93%) for rejectors noninvasively developed molecular signatures accurately predict acute rejection of human renal allografts greater emotional well-being (sf-36) and felt that their transplant interfered significantly less with various aspects of their life (iirs). conclusions: findings highlight the potential utility of assessing attachment style in transplant populations cni sparing in de novo renal transplantation: 3-year results from the symphony study one background: single center non-randomized results with steroid avoidance have shown patient and graft benefits. methods: 130 unsensitized, primary kidney recipients, 0-21 yrs of age, were enrolled from 12 us transplant programs (2004)(2005)(2006), in a prospective 1:1 randomized multicenter study of steroid-free (sf) vs. steroid-based (sb) immunosuppression with matched demographics. 24.1 % of sf and 27.5% of sb were african americans and 15.5% of sf vs. 8.7% of sb had esrd from fsgs. sf patients received extended (6 mo) vs. standard (2 mo) daclizumab induction in the sb group. patients in both arms received tacrolimus and mmf maintenance. protocol biopsies were performed at 0, 6, 12 and 24 mo, and for renal dysfunction. primary end-points were differences for standardized height scores and biopsy proven acute rejection (bpar) at 1 year. results at 1 year: 60 sf and 70 sb patients were enrolled; 10 sf and 16 sb were 0-5 yrs of age. patient survival was 100% in both arms. graft survival was similar (96.7% in sf vs. 98.6% in sb). intent to treat median delta height sds scores from baseline for different age groups were: 0.58 for sf and 0.48 for sb in the 0-5 yr old (p=0.80); 0.34 for sf and 0.34 for sb in the 6-12 yr old (p=0.86 protection of liver ischemia reperfusion injury by silencing of tnf-α and complement 3 genes. roberto hernandez-alejandro, 1 xusheng zhang, 2 dong chen, 2 xiufen zheng, 2 hongtao sun, 2 weihua liu, 2 marianne beduhn, 2 aminah shunnar, 2 motohiko suzuki, 2 norihiko kubo, 2 bertha garcia, 2 anthony jevnikar, 1,2 living kidney donation is rapidly increasing worldwide to offer a partial (?) solution for the numerous esrd wait-listed pts. in spite of properly followed guideline criteria conclusion: dcd donors are a viable source of liver allografts for transplantation. patients who receive dcd livers have outcomes comparable to subjects who receive grafts from brain dead donors. use of dcd livers from donors over 60 years of age is accompanied by a higher incidence of retransplantation and biliary complications. background: hypothermic machine perfusion (hmp) is in its infancy in liver transplantation (ltx). potential benefits include diminished reperfusion injury and improved early function. methods: the study was designed as a phase 1 trial of liver hmp. exclusion criteria included: multiple organ recipients, meld>35, icu patients, and patients >65 years of age. donor livers >65 years, biopsy with >30% macrosteatosis and dcd were also ineligible for hmp. seventeen patients were enrolled transplanted with livers that underwent hmp for 4-7 hours using dual centrifugal perfusion with vasosol solution at 4-6°c. patient, operative and early outcome variables were recorded. we compared outcomes to 17 matched cold stored (cs) controls from the same era. results: all 17 hmp grafts functioned immediately by usual clinical criteria with intraoperative bile production. results are summarized in table 1 . synergy between il-6 and tnfα promotes t cell alloreactivty and impairs the graft-prolonging effects of costimulatory blockade. hua shen, 1 bethany m. tesar, 1 wendy e. walker, 1 daniel r. goldstein. 1 1 internal medicine, yale university, new haven, ct. a novel role of th17 cells in allograft rejection and vasculopathy. francesca d'addio, 1 jesus paez-cortez, 1 m. javeed ansari, 1 laurie glimcher, 2 john iacomini, 1 mohamed sayegh, 1 xueli yuan. 1 1 transplantation research center, renal division, brigham and women's hospital, boston, ma; 2 harvard school of public health, boston, ma. introduction: transcription factor t-bet plays a crucial role in th1/th2 development. here, we investigated the role of t-bet in th17 differentiation and function of th17 cytokines in allograft rejection using an mhc class ii mismatched model of cardiac allograft vasculopathy. methods/results: cardiac allografts from bm12 mice were transplanted into wild-type as well as t-bet and ifn-γ deficient c57bl/6 recipients. t-bet-/-mice showed significantly accelerated allograft rejection (mst=14.75±0.96 days). however, as previously reported, all ifn-γ-/-and majority of the c57bl/6 mice accepted grafts for greater than 60 days. upon in vitro stimulation of recipient splenocytes by irradiated donor cells, t-bet-/-and inf-γ-/-lymphocytes produced significantly less inf-γ and more th2 cytokines. interestingly, production of the proinflammatory cytokines il-17 and il-6 was significantly higher in t-bet-/-(337±94.4 and 339±10.5 pg/ml) than c57bl/6 (135.1±68.3, 75.3±25.9pg/ml, p=0.0399 and 0.0001 compared to t-bet-/-) and inf-γ-/-(95.8±29.8, 20844.5 pg/ml, p=0.0135 and 0.0093 compared to t-bet-/-) mice. in vivo administration of il-17 neutralizing antibody (mab421) significantly prolonged survival of bm12 hearts (mst>30 days, p<0.01 compared to the 15.3±2.6 days of the control igg group) in t-bet-/-mice. immunofluorescence staining of bm12 hearts harvested from t-bet-/-recipients indicated that both cd4 and cd8 infiltrating lymphocytes produced il-17. however, t-bet-cd4 double knockout mice did not reject bm12 heart grafts, nor did the grafts exhibit chronic vasculopathy. in contrast, t-bet-cd8 double knockout mice rejected (mst:18.9±3.2 days). splenocytes from t-bet-cd4 knockouts produced significant lower il-6 (12.9±3.8) and il-17 (7.9±4.6 pg/ml) than observed in t-bet knockouts (940±176.3 and 210.2±73.4 pg/ml) and t-bet-cd8 knockouts (1240.5±215.6 and 113.1±61.2 pg/ml respectively) recipients when re-stimulated with donor cells, while there was no significant difference in inf-γ production. induction in the elderly transplant recipient: an analysis of the optn/ unos database. suphamai bunnapradist, 1 steven takemoto, 2 jagbir gill, 1 tariq shah. 2 1 medicine-nephrology, ucla, la, ca; 2 medicine-nephrology, national institute of transplantation, la, ca.we examined the incidence and mortality implications of cerebrovascular events (cve) after kidney transplant. we also compared variations in risk on the transplant waitlist and after allograft failure. methods: we used registry data from the us renal data system to retrospectively investigate ischemic stroke (is), hemorrhagic stroke (hs) and transient ischemic attacks (tia) among 29,614 adults who received kidney transplants in 1995-2002 with medicare as primary payer. patients with prior indications of cve in the registry were excluded. we ascertained events from billing claims, and estimated incidence of first events by the product-limit method. at-risk time was censored at: loss of medicare, 3yr transplant anniversary, non-cve death or end of study (12/31/2002). cox regression was used to identify independent correlates of cve, and to examine cve events as time-dependent mortality predictors. we estimated cve incidence after graft failure among patients without cve diagnoses prior to graft loss (n=2,599), and amongthe association between hyperuricemia at six months after kidney transplantation and the development of new cardiovascular disease, many studies have previously reported safe withdrawal of prednisone (pw) late after kidney transplantation (ktx). to determine the best immunosuppression regimen during the pw, we performed a prospective trial with stable ktx patients randomized to either csa or 's' based regimen. methods: all patients received antibody induction therapy at the time of rtx and maintained on csa, p and cellcept®. patients excluded if they had >1 acute rejection, >1 gm/d proteinuria or serum creatinine >2.5 mg/dl. 161 patients were enrolled and data presented for 141 patients with >52 weeks follow-up (f/u) with mean f/u of 112.2±29.2 weeks. no differences observed in baseline characteristics in both groups. all patients then randomized to either csa (n=69) or 's' (n=72) and cellcept® converted to equivalent dose of ms. csa dosed by c2 level (2-hour) with goal level of 600 ng/ml. sirolimus target level was 8 ng/ml. results: 5 patients withdrew from study, 12 patients on s returned to csa regimen because of side effects. 5 patients in the csa group and 1 patient in 's' group had ar (3 of them due to drug non-compliance). death censored graft survival was 100%. mean csa drug level acheived was 664±188 ng/ml and 's' drug level was 8.2±1 ng/ml. no significant differences noted in hematological values or bp measurements. csa ( purpose: the clinical significance of c4d positiviity in patients with acute rejection is well defined but its significance in stable graft function is undetermined. this study was performed to evaluate the clinical outcome of protocol biopsy-proven c4d positive renal transplants with stable graft function in the early posttransplantation period. methods: 151 renal allograft biopsies were included. protocol biopsies (n=79) were performed from stable allografts on day 14 posttransplantation, and indication biopsies (n=72) were performed from dysfunctioning allografts. incidence of c4d positivity was compared between protocol and indication biopsies. clinical characteristics, biopsy findings, graft function, acute rejection episodes, and graft survival rates were compared between the c4d-positive and c4d-negative grafts in each group. results: c4d deposition in protocol biopsies was detected in 4 of 79 biopsies (5.1%), whereas 9.7% (7 of 72 biopsies) in indication biopsies. the histological findings of c4d-positive protocol biopsies were minimal inflammation of tubulointerstitium. on the other hand, those of c4d-positive indication biopsies were various including acute humoral rejection, acute cellular rejection, acute tubular necrosis and calcineurin inhibitor toxicity. in the protocol biopsy group, graft function during 1 year after biopsy, acute rejection rate, and cumulative graft survival did not differ between the c4d-positive and c4d-negative grafts. all c4d-positive allografts maintained stable graft function without any antirejection therapy. in the indication biopsy group, graft function during 1 year after biopsy and acute rejection rate did not differ between the c4d-positive and c4d-negative grafts. however the cumulative graft survival rate was worse in the c4d-positive grafts than the c4d-negative ones (p=0.008). conclusion: c4d positivity associated with allograft dysfunction indicates a poor graft outcome. however, c4d-positive allografts with stable graft function in the early posttransplantation period take an indolent course. are methods: this was a retrospective single centre study reviewing all the adult patients who had a kidney transplant biopsy between april 2003 and october 2006 at guy's hospital. results: 45 patients had diffuse (>50%) c4d staining out of 228 who had kidney transplant biopsies in this time and had been followed up within the centre. of these 45 patients, 20 also had dsa prior or at the time of biopsy. the fall in egfr in this group a year post biopsy was greater than those with diffuse staining for c4d but no dsa. the mean change in egfr from the day of biopsy at a year was -3.1ml/min/1.73m2 (+/-12.1) in those with dsa compared with +17.7 ml/min/1.73m2 (+/-23.2) for those with c4d but without dsa. the changes in egfr from the pre-biopsy baseline at one year showed a fall in egfr in both groups but this was greater in those with dsa (-21.5 compared with -9.6 ml/min/1.73m2 ).of 179 patients who never had diffuse or focal c4d staining on biopsy, only 81 had had dsa tested. of these only 8 (10%) had a positive dsa result. from these eight, four had features of rejection and four did not. one person in each of these groups is dialysis dependant and one person in the rejection group has egfr <15 ml/min/1.73m2. although small numbers, this outcome appears to be worse than that of c4d negative patients with no dsa but features of rejection who in fact showed an improvement in egfr from the day of biopsy by 14.4 m l/min/1.73m2 or an improvement from their pre-biopsy baseline of 1.3 m l/min/1.73m2 at one year. conclusion: dsa is of additional value in evaluating risk of graft failure. this appears to be of value in those with and without diffuse c4d staining on biopsy. utility of post-transplantation flow cytometry crossmatching in predicting graft outcomes. michelle willicombe, 1 graham shirling, 2 ray fernando, 2 henry stephens, 2 paul sweny, 1 peter j. dupont. 1 1 department of renal medicine, royal free hospital, london, united kingdom; 2 histocompatibility laboratories, anthony nolan trust, london, united kingdom. de novo development of donor-specific anti-hla antibodies after renal transplantation may be associated with increased rejection and decreased graft survival. flow-cytometry crossmatches (fcxm) have been suggested as method of screening for development of donor-specific hla antibodies post-transplantation, but interpretation of crossmatch results can be confounded by antibodies directed against antigens other than hla. we assessed the impact of developing a positive fcxm post-transplantation on clinical outcomes in a cohort of live donor renal allograft recipients. methods: 29 patients were studied. 23/29 (79%) received tacrolimus-based and 6/29 (21%) ciclosporin-based immunosuppression. median follow-up was 24 months. all patients had negative complement-dependent cytotoxic (cdc) t cell crossmatches pretransplantation. 4/8 (50%) in the group with a positive fcxm had an acute rejection episode in the first 12 months compared with 10/21 (41%) in the group with a negative fcxm (p=ns). graft function at 12 months was not different between the groups (positive fcxm -median creatinine 137mmol/l; negative fcxm -median creatinine 141mmol/l; p=ns). 2/8 grafts (25%) were lost within the first year in the positive fcxm group compared with 1/21 (5%) in the group with negative post-transplant fcxm (p=ns). the development of a positive fcxm post-transplantation alone is not predictive of adverse clinical outcomes. this may be explained by the poor correlation between a positive fcxm and the presence of antibody directed against mismatched donor hla antigens. surveillance for development of donor-specific anti-hla antibodies after transplantation may be best performed using high-resolution bead technologies rather than fcxm. use of the fcxm alone, without establishing antibody profiles, is of limited predictive value. based upon the amount of antibody (ab) measured by the titer of donor specific hla antibodies, dsa, or the fluorescence intensity (fi) of the donor specific single antigen bead. it is unclear whether abs indentified by sensitive single antigen bead and solid phase assays (flow pra and luminex) correlate with and are predictive of a clinically relevant end-point (a + fcxm). we evaluated the pra, dsa, bead specific ag fi and fcxm reactivity of pre-transplant (pre-tx) sera from 219 recipients of a deceased donor renal allograft to determine whether amount of ab (measured by fi) predicts a (+) fcxm. patients with a (+) dsa, a (+) fcxm and pre-tx class i pra ≥ 80% (n = 103, mean pra of 91 ± 6%) when compared to patients with pre-tx pra < 80% (n = 58, mean pra 53 ± 21%) had comparable mean fis (7,407 ± 4,305 vs 7,483 ± 5,380) , fi ranges (1,000 -20,243 vs 1,552 -17,153 ) and median fis (5,681 vs 5,670). the class ii comparisons were of the same pattern. surprisingly, patients with a (+) dsa, a (-) fcxm and pre-tx class i pra ≥ 80% (n = 22, mean 90 ± 6%) compared to patients with pre-tx pra < 80% (n = 36, mean pra 50 ± 18%) had comparable mean fis (6,758 ± 4,841 vs 6,412 ± 3,844) , fi ranges (1,656 -16,596 vs 2,162 -11,637) we have recently showed that pre-treatment of the donor with epo causes a substantial reduction of the dysfunction and injury associated with the transplantation of kidneys recovered after cardiac death. 5-aminoisoquinolinone (5-aiq) a potent water soluble parp inhibitor has proven to reduce renal ischemia-reperfusion (i/r) injury. the aim of our study was to determine the effects in the graft and in the receptor of the pre-treatment of the donor with epo and treatment of the recipient with 5-aiq, in a porcine model of dcd kidney transplantation. material/methods:24 landrace pigs were killed by lethal injection; their kidneys were subjected to 30 min of warm ischemic time (wit) and then transplanted after 24 h of cold storage in celsior. in the pre-treated group, donors received a single dose of epo (1000 iu/kg) 30 min before cardiac arrest. in the treated group, recipients received a continuous dose of 5-aiq (5mg/kg/h) 10 minutes before reperfusion and maintained during 60 minutes. blood, urine and renal tissue samples were collected at the end of the experiment for biochemical, histological and immunohistochemistry (pars, inos and cox-2) evaluation. data analysis performed with graph pad prism statistical package; p<0.05 considered statistically significant. results:transplantation of kidneys from dcd resulted in: a significant rise of the levels of creatinine, n-acetil-b-d-glucosaminidase, glutathione-s-transferase, ast, ldh, alt, fractional excretion of na+, interleucin 1 and 6, malondialdehyde levels and myeloperoxidase activity (p<0.05); a significant reduction in urine flow and creatinine clearance, disturbances in the histological and imunohistochemistry pattern. administration of epo before ischemia and 5-aiq before reperfusion reduced significantly the biochemical (p<0.01), histological and imunohistochemical evidence of glomerular dysfunction and tubular injury. they also reduced systemic injury, inflammatory response and oxidative stress. conclusions:pre-treatment of the donor with epo and treatment of the recipient with 5-aiq causes a substantial reduction of the dysfunction and injury associated with the transplantation of kidneys recovered after cardiac death. in the hmp group perfusate ast levels strongly correlated with recipient peak ast by linear regression (p<0.001). conclusions: hmp of liver grafts provides safe and reliable preservation in our pilot series. perfusate ast may allow pretransplant prediction of reperfusion injury. a larger randomized trial will be necessary to demonstrate the magnitude of benefits of hmp over cs in ltx.purpose: liver discard rates have increased in a large, urban organ procurement organization from 8 % to 24 %. the reason is likely the result of increased transplant surgeon willingness to consider organs close to the margin of clinical acceptability. however, the costs associated with recovering these organs are high if the liver is discarded. we endeavored to determine whether a model based on pre-recovery data could predict liver discard introduction: we report our 6 years experience with the use of campath-1h (c1h) in adult liver transplantation. from december 2001 until july 2007 we administered c1h induction with low dose maintenance tacrolimus immunosuppression to 166 adult recipients of a liver allograft. most common primary diseases were laennec (n=60), cryptogenic cirrhosis (n=31) and autoimmune: psc (n=17), pbc (n=16) and aih (n=14). the first dose of c1h was administered immediately before (n=74) or after (n=92) the transplant procedure. follow up was until september, 2007. results: five year patient and graft survival was 90% and 85% respectively. there were 14 deaths due to stroke (n=2), chronic rejection (n=2), failure to thrive/pneumonia (n=3), sepsis (n=2), hepatic artery thrombosis, hcc, prostate cancer, graft lymphoma and non-compliance (one each). seven patients were retransplanted, for primary non function (n=2), portal vein thrombosis (n=1), hepatic artery thrombosis (n=1), hepatitis b (n=1) and chronic rejection (n=2). thirty six patients had biopsy proven rejection episodes: mild (n=28), moderate (n=9) or severe (n=3). the average tacrolimus 12 hour trough levels were 6.6, 5.2 ng/ml and 2.78 ng/ml for the 1rst, 3 nd and 5th year post-transplantation, respectively. there was no significant difference in the outcome of the transplant so far, between patients that received c1h before or after the transplant procedure. immunosuppression-related complications included a). opportunistic infections: most common were herpes zoster (n=19), cmv (n=3), and herpes simplex (n=3), b). neoplasms: skin cancer (n=3), kaposi sarcoma (n=1), lymphoma (n=2) and c). nephrotoxicity: five patients received a kidney graft for diabetic nephropathy (n=2), nephrotic syndrome (n=1) and calcineurin nephrotoxicity (n=2). conclusion: the use of c1h induction with half the usual dose of tacrolimus is an effective regimen in adult liver transplantation. the timing of c1h administration does not seem to affect the clinical outcome so far. a. david mayer, 1 james m. neuberger. 1 1 the liver unit, queen elizabeth hospital, birmingham, united kingdom. introduction: in the prospective respect study, primary liver transplant patients were randomised to 1 of 3 groups: a) standard-dose tacrolimus (target trough level > 10 ng/ml) for the 1 st month; b) 2 g mycophenolate mofetil (mmf) iv until at least day 5, 2 g po thereafter + reduced-dose tacrolimus (target trough level ≤ 8 ng/ml); and c) mmf as in group b + reduced-dose tacrolimus introduced on day 5 (target trough level ≤ 8 ng/ml) + daclizumab on days 1 and 7. steroids were given in all groups according to local centre protocol. results at 1 year showed that 2 g mmf + delayed and reduced tacrolimus + daclizumab is associated with significantly less impairment of renal function compared with standard treatment. here we present the results of the per protocol (pp) population. methods: the pp population, which was defined prior to the sub-group analysis, consisted of patients from the full analysis set who had no inclusion/exclusion criteria violation, had at least one creatinine clearance (crcl) value beyond 6 months, were treated according to the protocol and did not receive any prohibited medication during the first 14 days and for less than 1 week at any time during the study. a composite endpoint comprising freedom from renal dysfunction (≥ 20% decrease from baseline in calculated crcl), acute rejection, graft loss or death was also investigated. results: the full analysis set included 181, 168 and 168 patients, whereas the pp population only included 69, 67 and 67 patients in groups a, b and c, respectively. the mean difference in calculated crcl from baseline to 1 year was significantly smaller in group c compared with group a (-10.7 ml/min vs -22.0 ml/min, p = 0.038), but was not significantly different between groups a and b (-20.50 ml/min). the incidences of death (n = 2, 0, 1) and graft loss (n = 1, 0, 0) were similar in all 3 groups. the incidence of the composite endpoint at 1 year was in both the full analysis set and the pp population significantly lower in group c compared with group a (pp population: 70% vs 93%, p < 0.0001), but was not significantly different between groups a and b (85%). the pp analysis confirms the results from the full analysis set that 2 g mmf + delayed and reduced tacrolimus + daclizumab is associated with less impairment of renal function compared with standard treatment with no negative effect on death and graft loss. aims: post transplant lymphoproliferative disorder (ptld) is a serious complication of solid organ transplantation that is closely associated with epstein barr virus (ebv) infection. ebv + ptld lymphomas express several latent viral genes including latent membrane protein 1 (lmp1), a proven oncogene that is essential for human b cell transformation. lmp1 is able to activate erk, jnk, p38, nfκb and pi3k. the aim of this study is to determine whether lmp1 isolated from ptld tumors differs in signaling ability from lmp1 derived from the b.95 strain of ebv, originally isolated from a patient with infectious mononucleosis. methods: lmp1 variants isolated from a panel of ebv + ptld-associated b cell lines were cloned and sequenced. inducible chimeric constructs containing the lmp1 c-terminus and ngfr transmembrane domain were created for each tumor variant and expressed in the burkitts b lymphoma cell line bl41. lmp1 signaling in bl41 clones was induced by crosslinking of ngfr. activation of p38, erk, akt and jnk was assayed by western blotting (wb) with phospho-specific antibodies. nfκb activation was assayed by wb for iκb and cfos induction was analyzed by wb and the transam cfos binding assay. results: all three tumor variants of lmp1, as well as the b.95 lmp1 isoform, were able to induce p38 activation within 30min of ngfr crosslinking while akt and jnk were activated within 10min. all variants showed similar ability to activate nfκb. however, tumor lmp1 variants induced prolonged erk activation (up to 3hrs) while the b.95 lmp1 variant induced a transient response. cfos is induced only during the sustained phase of erk activation. indeed, the tumor variants of lmp1, but not b.95 lmp1, were able to induce cfos protein. similarly, cfos binding to the ap1 consensus site was only observed in tumor lmp1-induced nuclear lysates. two mutations in the c-terminus-aa212 (s vs g) and aa366 (t vs s) -are conserved in the tumor variants lmp1 compared to b.95 lmp1. point mutation of either of these amino acids from the b.95 to tumor variant version allowed for sustained activation of erk and subsequent cfos induction and binding to the ap1 site. conclusion: tumor-derived lmp1 has enhanced ability to induce the cfos oncogene and this property can be localized to two amino acids in the c terminus. these findings suggest that these specific amino acid residues of lmp1 are important in determining whether ebv infection is benign or results in ptld. the absence of interferon regulatory factor-3 (irf-3) confers protection against the liver ischemia and reperfusion injury through an il-10 independent pathway. elizabeth r. benjamin, 1 xiu-da shen, 1 feng gao, 1 yuan zhai, 1 genhong cheng, 1 ronald w. busuttil, 1 jerzy w. kupiec-weglinski. 1 1 surgery, dumont-ucla transplant center, los angeles, ca. toll-like receptor 4 (tlr4) mediated liver reperfusion damage after warm ischemia requires signaling through the myd88-independent, irf3-dependent pathway with cxcl-10 (ip-10) playing a central role in the injury development. studies using cxcl-10 ko mice have shown that these mice are protected through an il-10 dependent mechanism. we chose to investigate irf3, upstream of cxcl-10, to further characterize its role in the injury progression, and to better understand the involvement of il-10 in this pathway. methods: we used irf3 ko mice and their wt counterparts in a model of partial hepatic warm ischemia with 1, 2, and 6h of tissue reperfusion (n=2 ko, wt at 1 and 6h; n=3 ko, wt at 2h). wt bone marrow derived macrophages were generated and stimulated with lps to determine the kinetics of il-10 production. tissue was analyzed for histology and mrna levels were measured by qpcr. results: kinetic studies showed peak il-10 production at 1 and 2h post-reperfusion (pr). on pathology, irf3 ko mouse livers were protected from ir injury both early pr, at 1 and 2h, and at 6hrs pr when compared to wt. consistent with these data, il-6 mrna induction was decreased in irf3 ko, as compared with wt at 1, 2, and 6h. although il-10 induction was maintained in the cxcl-10 ko mice, the irf3 ko mice showed decreased levels of il-10 at 1h pr. by 2h, il-10 levels were normalized to wt. conclusion: irf3 ko mice are protected from liver ir injury with evidence of this protection as early as 1h pr. although cxcl-10 ko mice are protected from ir injury with maintained il-10 expression, the absence of the upstream molecule, irf3, confers protection in an il-10 independent manner. these data suggest a novel mechanism of ir injury mediated by irf3 in the liver. background: bone marrow (bm) transplantation may induce donor-specific tolerance to prevent rejection of allogeneic solid organs while maintaining immunity against infections and tumors. currently allogeneic bm transplantation is limited by donor t cell mediated graft-versus-host disease (gvhd), as well as a variable requirement for recipient marrow ablation and high numbers of donor bm cells. furthermore, sustained macro-chimerism has not yet been easily or predictably achieved in partially ablated patients or large animals. while rejection of allografts is mediated primarily by recipient t cells, recent studies have demonstrated the capacity of nk cells to reject allogeneic bm and to prevent long-term mixed chimerism. thus, nk cells represent a barrier to long term bm engraftment even with t cell tolerance. we have previously identified a novel type of regulatory t (treg) cell with a "double negative" (dn) phenotype (tcrab + cd3 + cd4 -cd8 -). dn-treg cells can effectively suppress anti-donor t and b cell responses and prolong graft survival in allo-and xenotransplantation models. we therefore tested the capacity of dn-treg to alter nk cell function. methods: c57bl/6 bm cells were i.v. injected into sub-lethally-irradiated (6.5 gy) cb6f1 (h-2b/d) in a "parent to f1" model, or into allo-disparate balb/c mice. bm cells were co-transplanted with various numbers of c57bl/6 dn-treg cells or cd4 + or cd8 + t cells as controls. recipient spleen cells were collected 7 days after to detect donor progenitors in a colony-forming-unit (cfu) assay. mice then received cardiac (n=8) or skin transplants (n=8) to confirm tolerance. we found that donor-derived dn-treg cells suppress nk cell-mediated allogeneic bm graft rejection in both "parent-to-f1" and fully mhcmismatched bm transplantation models. adoptive transfer of dn-treg cells with donor bm cells promoted the establishment of stable mixed chimerism and donor specific tolerance to bm donor cardiac and skin grafts (mst>160 days), without inducing gvhd in sub-lethally irradiated mice. perforin deficient dn-treg cells were unable to efficiently inhibit nk cell function, and donor bm did not engraft. these results demonstrate a potential approach to control innate immune responses and promote allogeneic bm engraftment and donor specific tolerance through the use of dn-treg cells.framingham risk score (frs) predicts cardiovascular (cv) risk in the general population, but may underestimate cv risk in kidney transplant (txp) patients (pts). frs has not previously been validated by prospective cardiovascular event (cve) data collection in kidney txp pts. the purpose of this study was to validate frs with actual observed cve data in kidney txp pts. methods: cve data was collected at routine intervals in our kidney txp pts and entered in a cardiovascular risk database. frs was calculated from baseline to 7 yrs posttransplant (ptx) individual frs factors of age, sex, smoking, diabetes mellitus (dm), high-density lipoprotein (hdl), total cholesterol (tc), and blood pressure (bp) were evaluated for their ability to predict acutal cve occurring after kidney txp. pts with coronary artery disease (cad) were excluded from the frs analysis. cve were defined as sudden death, myocardial infarction, angina, and cerebrovascular accident/transient ischemic attack. frs factors were evaluated by cox proportional hazards in univariate (uva) and multivariate (mva) models. rho kinase (rok) modulates calcium sensitivity of vascular smooth muscle cells and contributes to the regulation of peripheral vascular tone in man. in essential hypertension increased rok-activity contributes to the generation of vascular resistance. arterial hypertension is a common complication in renal transplant recipients. in this study we were interested in the role of rok for systemic hemodynamics in hypertensive renal transplant recipients (tx). we tested the specific inhibitor of rok fasudil. 10 tx and 10 matched control subjects (c) received either fasudil (1600 g/min) or placebo over a period of 30 minutes intravenously. peripheral blood pressure and heart rate were recorded every 5 min over a total of 120 minutes. measurements for pulse wave analysis (sphygmocor vt) were performed every 10 minutes during this period. statistics by anova for repeated measurements.compared to placebo fasudil significantly reduced peripheral mean arterial pressure p<0 .05; figure 1) and increased heart rate (+ 6.31.1 bpm, p<0.001) in tx but not in c. likewise, central systolic pressure(p=0.006; (figure 2), augmented pressure and augmentation index were decreased in tx only.we conclude that acute inhibition of rok by fasudil consistently and effectively lowers blood pressure in tx with a calcineurin inhibitor-based immunosuppression. interestingly, rok-inhibition also reduces central blood pressure and arterial stiffness in these patients. improvement of both these parameters has been linked to a reduction in cardiovascular morbidity and mortality in large trials. hence rok inhibition might prove beneficial for the treatment of hypertension in renal transplant recipients. use death with function causes half of late ktx failure, and cardiovascular disease (cvd) is the most common cause of death. chronic kidney disease (ckd) is a cvd risk equivalent, justifying aggressive risk reduction with blood pressure (bp) control, statins, aspirin, and use of angiotensin converting inhibitors (acei) and angiotensin receptor blockers (arb). dekaf is an nih-sponsored prospective observational study examining causes of ktx failure at 7 transplant centers in the us and canada, with current enrollment of over 1800 subjects. we examined the use of cardioprotective medications among patients transplanted after 10/1/05 with at least 6 mos follow-up, focusing on subgroups with preexisting diabetes (dm) and/or cvd. we conducted a retrospective cohort study to asses the prevalence and the predictors for the development of hyperuricemia at 6 months after kidney transplantation and the association between hyperuricemia and clinical outcomes including patient and graft survival, new cardiovascular events and chronic allograft nephropathy (can). adult patients who underwent kidney transplantation at mount sinai medical center between 1.1.2001-12.30.2004 were included. patients who died or lost the allograft within 6 months after transplantation were excluded from analysis. of the 307 patients with a functioning allograft at 6 months after transplantation, 163 patients (53%) had normal uric levels and 144 patients (47%) had hyperuricemia. after age, race, sex adjustment, receiving a cadaveric kidney, having an egfr<50 ml/min, and taking diuretics and cyclosporine were associated with a higher odds ratio of hyperuricemia. over a mean of 4.3 years of follow-up, 81 patients had one, or more, of the pooled outcomes; 40 had new cardiovascular events, 41 developed biopsy-proven can, 4 patients died, and 20 had graft failure. kaplan-meier survival curves demonstrated that the pooled outcomes of events occurred more frequently in hyperuricemic patients (figure, p < 0.001). due to association between low egfr and hyperuricemia, we analyzed the clinical outcomes in patients with low and normal egfr. while 44.7% of hyperuricemic patients with an egfr<50 ml/min had one of the pooled outcomes, it was 20.8% in patients with normal uric acid levels (p=0.038). among patients with an egfr ≥50 ml/min, 19.4% of normouricemic and 25% of hyperuricemic patients had one of the events. these results suggests an important association between hyperuricemia at 6 months after transplantation and the new cardiovascular events, biopsy-proven can, and graft loss in kidney transplant recipients with decreased allograft function. background: long-term survival after liver transplantation (lt) is now the rule rather than the exception. hence, assessment of outcomes for children after lt must consider not only the quantity, but also the quality, of life years survived and restored. aim: to examine key hrqol themes after pediatric lt raised by both recipients and their parent proxies, with evaluation by time (1-3 yrs, 3-5 yrs, 5-10 yrs, and >10 yrs) since lt. methods: semi-structured 1:1 item generation interviews were conducted in person with children (c) and parents (p) at time of ambulatory lt follow-up at 4 pediatric lt programs in canada and uk. all interviews were audio-taped, transcribed verbatim, and subjected to content analysis utilizing qsr nvivo 2.0 software for hrqol related theme generation. the participants interviewed were part of a larger research program aimed at developing a disease-specific instrument to assess hrqol for children after lt. results: data representing 91 (47% male) pediatric lt recipients was obtained from a total of 146 (62 c, 84 p) item generation interviews. median recipient age at lt was 2.4 (range, 0.05 to 17) yrs, for primary indications including biliary atresia (45%), fulminant liver failure (17.6%), metabolic liver disease (4,4%), malignancy (8.8%) and others (24.2%). median patient age at time of interview was 9.9 (range, 1.1 to 18.8) yrs. themes emerging at all time points post-lt included infection risks, limitations on physical activities, side effects from immunosuppression meds, educational supports, and ongoing bloodwork. themes identified within the medium (1-5 yrs) follow-up included worries about rejection episodes, need for future re-transplantation, school absenteeism, and altered sibling and family dynamics. the impact of living with a surgical scar was a more frequent theme with recipients >3 yrs from lt. as time from lt increased to >10 yrs, themes suggest a focus on normalization and health promoting behaviours, along with expressed desires to be like healthy peers. conclusions: unique hrqol themes emerged from item generation interviews not captured by currently available generic hrqol tools. hrqol themes identified after pediatric lt suggest the importance of considering time trajectories from lt, and a focus on elements of 'everyday life' apart from lt. the shortage of cadaveric donors has led many transplant centers to expand their criteria for accepting life-saving organs. utilization of donation after cardiac death (dcd) donors has been estimated to increase the number of cadaveric donors. we report our experience with a recently established dcd program at a pediatric hospital and the outcome with the transplanted grafts. methods: in 2005 a protocol for dcd was established at a free standing pediatric hospital. from 2005 to 2006 all patients undergoing withdrawal of care were evaluated for dcd. patients meeting criteria for dcd underwent withdrawal by the critical care team and organ retrieval was initiated if asystole was reached in less than 60 minutes. in addition, one dcd liver was imported and was included in the liver results. results: during the 2 year study period 7 patients (25% of total donors) underwent dcd resulting in 14 organs (11 kidneys and 3 livers) transplanted. the 7 cases had a mean donor age of 9 yrs (range 3-16), wit of 22 min (9-55), time sbp < 60 of 11 min (7-15), and time from asystole to aortic flush of 8 min (6) (7) (8) (9) (10) (11) (12) . four kidneys were transplanted locally with cit 5-14 hrs, no dgf, and one month creat 1.2-1.7. the remaining 7 kidneys were exported for transplant. four livers were transplanted locally with donor age 7 mth-16 yrs, wit 12-28 min, recipient age 14 mth-61 yrs, cit 6-15 hrs, ast peak 190-3630, ast day 7 33-44, inr peak 1.4-1.8, inr day 7 1.0-1.1, total bili one month 0.3-1.0, and graft survival 1.5-2.9 yrs. there were no vascular or biliary complications. conclusions: a protocol for dcd at a pediatric hospital increased the number of pediatric donors by 25%. liver and kidney grafts from pediatric dcd donors demonstrated excellent graft function and survival. liver retransplantation in children. a 21 year single centre experience. christophe bourdeaux, andrea brunati, magda janssen, jean-bernard otte, etienne sokal, raymond reding. pediatric liver transplant program, université catholique de louvain, saint-luc university clinics, brussels, belgium. when graft failure occurs in liver recipients, secondary transplantation represents the only chance of long-term survival. in such instance however, several surgical and immunological aspects should be carefully considered, with respect to their impact on final outcome.in the present study, the epidemiology and outcome of graft loss following primary pediatric liver transplantation (lt) were analysed, with the hypothesis that early retransplantation (relt) might be associated with lower immunologial risks when compared to late relt. between march 1984 and december 2005, 745 liver grafts were transplanted to 638 children at saint-luc university hospital, brussels. among them, a total of 90 children (14%) underwent 107 relt, and were categorized into two groups (early relt, n=58; late relt, n=32), according to the interval between both transplant procedures (< or > 30 days).ten-year patient survival rate was 85% in recipients with a single lt, versus 61% in recipients requiring relt (p=0.001). ten-year patient survival rates were 59% and 66% for early and late relt, respectively (p=0.423), the corresponding graft survival rates being 51% and 63% (p=0.231). along the successive eras, the rate of relt decreased from 17% to 10%, whereas progressive improvement of outcome post-relt was observed. no recurrence of chronic rejection (cr) was observed after relt for cr (0/19). two children developed a positive cross-match at relt (2/10, 20%), both retransplanted lately for cr secondary to immunosuppression withdrawal following a post-transplant lymphoproliferative disease.in summary, the current need for relt has been decreasing over years, with a parallel improvement of its outcome. the results presented could not evidence better results for early relt when compared to late relt. the latter did not seem to be associated with higher immunological risk, except for children with immunosuppression withdrawal following the first graft. the background: a serum conjugated bilirubin greater than 100 umol/l (cb100), in neonates who receive parenteral nutrition (pn), has been demonstrated to be a predictor of end-stage liver disease requiring transplantation. given the recent interest in the role of omega-6 lipids in the development of parenteral nutrition associated liver disease (pnald), we sought to examine in a multiple variable model the role of days of maximal lipid (>2.5 g/kg/day), in the development of this outcome. method: between 2003 and 2004, data were collected prospectively on all neonates undergoing an abdominal surgical procedure. univariate logistic regression models for the prediction of cb100 were developed with the following predictors: gestational age, percentile weight, percent predicted small bowel and colonic length, resection of the ileocecal valve, presence of a stoma, post-operative enteral tolerance, number of septic episodes, days of pn amino acid > 2.5g/kg/day, days of pn lipid > 2.5g/kg/day, and total days of pn. univariate predictors significant at the 0.2 level were entered into a backward stepwise multiple variable logistic regression. results: 152 infants received pn post-operatively, and 22 developed cb100. predictors that met criteria for consideration in the multiple variable model were: age (p=0.079), weight (p=0.059), small bowel length (p=0.001), presence of a stoma (p=0.163), proportion of enteral feeds post-operatively (p=0.049), days of pn amino acid > 2.5g/ kg/day (p=0.00), days of lipid > 2.5 g/kg/day (p=0.00), and total days of pn (p=0.00).the final multiple variable model which had a negative predictive value of 97.6% and positive predictive value of 52.6% is presented in the table below. our model suggests a key role of pn lipids and intercurrent septic events in the development of cb100 from pnald. these data may provide targets, such as careful line care, reduction in maximal lipid dose, or the use of alternate lipids such as omega-3 fatty acids, to prevent cb100 an identified marker for the need of subsequent liver transplantation in infants with pnald. terminal renal failure occurs in more than 10 % of liver transplant recipients after 10 years. we have previously shown that, beside renal toxicity of calcineurin inhibitors, renal lesions may be related to diabetes, arterial hypertension, accumulation of hydroxyethylstarch (elhoes), and the etiology of the liver disease. we made the hypothesis that these lesions may be already present at the time of liver transplantation (lt), a finding that could lead to adapt the perioperative management. this work investigated prospectively whether renal histopathological lesions were present before lt by performing systematically a renal biopsy by endovenous route in 60 candidates to lt with end-stage liver disease. these patients were 58 ± 10 years old, 46 males ; 10/60 had a diabetes, and 21 an arterial hypertension ; the liver disease was related to alcohol in 32 cases, hcv in 12 cases, hbv in 5 cases, and to a cholestatic disease in 7 cases. at the time of the pre-lt workup, the biochemical parameters were : child score10 ± 2, meld score 18 ± 4, prothrombin rate 50 ± 12, creatinin serum level 90 ± 6 umol/l, proteinuria 0.12 ± 0.04 g/24h. severe side effects related to the procedure were limited to 2 cases of macroscopic hematuria, lasting less than 24 hours. in 10 cases, the material obtained during the procedure did not allow the histological analysis. among the 50 samples available, 21 were considered as normal ; in 29 cases, lesions related to mesangial iga glomerulonephritis (14 cases), diabetic glomerulosclerosis (12 cases), elhoes accumulation (5 cases), thrombotic microangiopathy (1 case) were found, often associated ; in 5 cases, the lesions were severe and lead to combined kidney/liver transplantation in 2 cases. in conclusion, significant renal lesions are detectable in more than 50 % of the candidates to lt. interestingly, histological findings often combined lesions related to the liver disease and to an associated cause (diabetes, previous treatment by elhoes or interferon). results of histological analysis could help to decide either to perform a combined renal/liver transplantation, to adapt the immunosuppressive regimen, or to abandon the lt project. . because serum creatinine is one of the components of meld, liver candidates with renal insufficiency have been transplanted in increasing numbers, with some candidates receiving a kidney along with the liver transplant. we aimed to compare the liver graft outcomes for liver alone (lta) transplants with those from combined liver-kidney transplants (clkt). a propensity score analysis was used to reduce the impact of selection bias in the comparison of outcomes in the two groups. methods. demographics, clinical factors and outcomes on lta and clkt recipients from 3/1/02 to 12/31/06 (n=10,388) obtained from the optn database were used for the analysis. univariate post-transplant survival rates were estimated using kaplan-meier survival, and multivariable post-transplant outcomes were analyzed using a cox regression model with and without stratification by categories of the propensity score. the propensity score (probability of receiving a clkt) for each recipient was estimated using a logistic regression model. several donor and recipient factors were included in both the cox and logistic regression models. in this cohort, liver graft outcomes for clkt were significantly better than those for lta based on the multivariable analysis. the results were similar, although slightly less significant, when the model was adjusted for propensity. the superior outcomes of clkt may be due to unobserved differences between these groups of recipients, reflecting data not currently captured by the optn. effect of liver the tgfβ1 to bmp-7 ratio was higher in the ar group (median ratio: 1635) compared to recipients with stable graft function & normal biopsy (median ratio: 438, p=0.0002).our observations that bmp-7 is specifically down-regulated during an episode of ar and that the balance between tgfβ1 & bmp-7 is in favor of emt advance a mechanism for the deleterious impact of ar on the long-term outcome of human renal allografts. the non-statistical bioinformatic approach identified 68 leader genes which define the highest interaction genes derived from the 343 sam-gene list. an interaction map between the genes identified has been calculated. this network is formed around 2 majors clusters: a network of interleukins and a network of signal transduction which allow us the identification of key genes such as bank1, a negative modulator of cd40mediated akt activation, thereby preventing hyperactive b cell response in blood from patients with operational tolerance and il7r, a specific marker absent on potentially regulatory cd4 + cd25 +high t cells in blood from patients with chronic rejection. we have identified by a non-statistical analysis of the peripheral blood gene expression in human kidney recipients a cluster of genes which are strongly interconnected and which could be a starting point for further analysis of the molecular mechanisms of kidney graft operational tolerance and chronic rejection. fecal algorithm based on multiparameter mixed lymphocyte reaction assay for tailoring maintenance immunosuppressants after living donor liver transplantation. yuka tanaka, hideki ohdan, toshimasa asahara. department of surgery, hiroshima university, hiroshima, japan.background: no reliable immunological parameters exist for identifying liver allograft recipients in whom immunosuppressants can be safely withdrawn. for minimizing maintenance immunosuppressants, we established an algorithm determining anti-donor alloreactivity based on multiparameter mixed lymphocyte reaction (mlr) assay, wherein the number and phenotype of alloreactive precursorscan be quantified. we enrolled 68 adults undergoing living donor liver transplantation (lt). the initial immunosuppressive regimen comprised tacrolimus/cyclosporine and methylprednisolone, which were gradually tapered off by 6 months after lt. thereafter, therapeutic adjustments were determined by a policy of slow tapering off in the case of normal liver function. mlr assay was performed at 6 month intervals to monitor immune status. in this assay, cfse-labeled pbmcs from recipients were used as responders. irradiated donor and third-party pbmcs were used as stimulators. after coculture, the responder cells were stained with cd4 or cd8 mabs along with cd25 mab, followed by fcm analyses. the proliferation and cd25 expression of cd4 + and cd8 + t cell subsets in response to anti-donor and anti-third-party stimuli were analyzed; the immune status of lt patients was categorized as hypo-response, norm-response, or hyper-response for cd4 + t cells and as hyper-response for cd8 + t cells. of the 68 patients, 33 had normal liver function at >6 months after lt. we examined the fluctuation of immunosuppressants at 6 months after mlr in these patients. in patients whose immune status was categorized as hyper-response for cd4 + or cd8 + t cells (n=4), immunosuppressants had to be increased. in patients with norm-response immune status (n=7), immunosuppressant tapering was abandoned. immunosuppressant therapy was successfully tapered off in patients with hypo-response immune status (n=22). of 10 patients with hypo-response immune status at >3 years after lt, immunosuppressants were completely discontinued in 3. in these "operational tolerance" patients, the precursor frequency of anti-donor cd4 + t cells (mean=5.2±1.9%) was not reduced compared to that in non-tolerance patients, suggesting that donor-specific immune tolerance is maintained via inhibitory/ suppressive mechanisms rather than via clonal deletion. conclusion: multiparameter mlr assay can provide a clinically validated rule predicting the success of tailoring/weaning immunosuppression. psychological factors associated with non-adherence among adolescents before and after kidney transplant. nataliya zelikovsky. 1 1 dept. of pediatrics, div. of nephrology, the children's hospital of philadelphia, philadelphia, pa.purpose: little is known about psychosocial risk factors for poor adherence among pediatric transplant patients. identification of variables that impact illness management can guide targeted interventions to improve adherence. methods: a longitudinal study was conducted to determine whether quality of life (pedsql), family functioning (fad), and parent adjustment (pip) would predict adherence in adolescent transplant patients. psychological questionnaires were administered prior to and 12 months after the transplant. medical adherence measure (mam), a semi-structured interview was used to assess adherence. adherence was calculated as % missed and % late doses of those prescribed. results: 60 patients (m =14.32 years +2.18, 75% male, 63% caucasian) and their parents were evaluated at the time of listing for kidney transplant. the rate of non-adherence prior to transplant was high, with 90% of patients reporting some degree of nonadherence. of these patients, 37% missed and 28% took late > 10% of prescribed doses. on the quality of life measure, behavior issues were associated with missed (r=-.42, p=.01) and late doses (r=-.39, p<.05), and mental health issues were associated with late doses (r=-.38, p<.05). adolescent reports of problems in affective responsiveness among family members was associated with missed (r=.33, p=.04) and late (r=.51, p=.001) doses. missed doses were also associated with mother reports of difficulties with overall family functioning (r=.34, p<.05), communication (r=.41, p<.01) and role definitions (r=.33, p<.05) among family members. 49 of the families were re-evaluated one year after the kidney transplant. 65% had been on dialysis prior to transplant and 42% received living-related transplants. 58% of the patients reported some degree non-adherence post-transplant, and using more stringent criteria, 17% of patients reported missing and 27% reported taking late >10% of prescribed doses. worse quality of life such as limitations due to emotional problems (r=-.32, p<.05), behavioral problems (r=-.39, p<.01), and difficulties with family cohesion (r=-.32, p<.05) was related to worse adherence. discussion: adolescent quality of life in behavioral and emotional domains, and family functioning play a significant role in adherence both before and after transplant. programs to improve adherence among transplant patients should incorporate psychosocial supports and behavioral interventions to improve adjustment of patients and families. kidney transplantation leads to marked improvements in health, yet transplant (tx) recipients often have difficulty with sexual functioning, which can affect quality of life. specific sexual concerns of tx recipients remain under investigated. the purposes of this study were to 1) further establish the psychometric properties of the sexual concerns questionnaire (scq) including reliability and preliminary construct validity and 2) identify the sexual concerns of kidney tx recipients. the scq was answered by 390 kidney tx recipients who rated each item on a 0 (not at all) to 5 (extremely) scale. a cronbach's alpha correlation coefficient was calculated to determine the reliability of the scq. an alpha value of .83 was calculated for the questionnaire indicating it was reliable. exploratory factor analysis (efa) was performed to establish preliminary construct validity of the scq. as a result from the efa, 14 items were dropped and a 5 factor structure was accepted. examples of items and responses include question 1: "how difficult is it for your vagina to get or stay wet or moist?" (for women) and "how difficult is it for you to get or keep an erection?" (for men) and question 2 "how comfortable are you talking about sexual concerns with your doctors and nurses?"participants were also asked to indicate how important their sexuality was to them on a 0 (not at all) to 6 (extremely) rating scale. twenty-six percent of participants rated their sexuality as quite a bit important, 26% rated their sexuality as very important, and 20% rated their sexuality as extremely important. the findings provide evidence of a reliable questionnaire with evidence for preliminary construct validity. they also indicate that sexuality is an important issue for a majority of kidney tx recipients. a cross-sectional study of fatigue before and after liver transplantation. james r. rodrigue, 1 timothy antonellis, 1 p=0.78 # 1 (none) to 10 (extremely high); ♣ higher score = more fatigue, poorer sleep quality, or more mood disturbance; ¶ higher score = better qol one-third of pre-lt (32%) and post-lt (33%) patients reported severe fatigue. poor sleep quality was reported by 68% and 79% of pre-and post-lt patients, respectively. pre-lt fatigue was predicted by higher bmi (ß = 0.21) and meld (ß = 0.20), depression (ß = 0.35), and poor sleep quality (ß = 0.48), adj r 2 = 0.44, f = 10.72, p < 0.0001. post-lt fatigue was predicted by older age (ß = 0.19), tension-anxiety (ß = 0.27), anger-hostility (ß = 0.38), and poor sleep quality (ß = 0.18), adj r 2 = 0.38, f = 7.45, p < 0.0001. more fatigue was associated with lower sf-36 physical (r = -0.34) and mental (r = -0.44) qol. conclusions. fatigue and poor sleep quality are clinically significant problems for lt candidates and recipients. bmi, psychological functioning and sleep quality are modifiable variables that predict fatigue severity and should be targets of intervention when addressing fatigue symptoms. health literacy in kidney transplant recipients. elisa j. gordon, 1 michael s. wolf. 2 1 medicine, albany medical center, albany, ny; 2 medicine, northwestern university. background: in order to successfully manage the transplant long-term, kidney recipients must have a basic understanding of key transplant-related concepts and terms indicative of their condition and treatment to properly communicate with health care providers and manage their health. kidney recipients must also possess numeracy skills to enable proper medication-taking and to monitor serum creatinine levels, bodily temperature, etc. the objective of this study was to examine health literacy levels among kidney transplant recipients. methods: we surveyed 124 consecutive adult renal transplant recipients using the test of functional health literacy in adults (s-tofhla), and a modified version of the rapid estimate of adult literacy in medicine, called the "realm-transplant," which measured patients' knowledge of 69 kidney transplant-related terms that patients are expected to have familiarity with. open-ended and multiple choice questions assessed numeracy related to kidney survival. results: most kidney recipients (91%) had adequate health literacy (s-tofhla), but 81% were unfamiliar with at least 1 kidney transplant-related term (realm-t). patients who were less educated (p<0.0001), had lower income (p<0.002), and were single or without a partner (p=0.046) had significantly lower health literacy levels (s-tofhla). patients less familiar with transplant-related terms (realm-t) had less education (p<0.0001), lower income (p<0.0001), and were nonwhites (p=0.033). the five least familiar terms were: sensitization (50%), urethra (45%), trough level (41%), blood urea nitrogen (32%), and toxicity (31%). sixteen percent wanted more information about their transplant. numeracy levels varied: 21% knew the likelihood of 1-year survival; 29% knew that half of kidney recipients have problems with the transplant in the first 6 months; 86% knew the normal range of creatinine for kidney recipients; and 86% were aware of the risk of death within the first year of transplantation. conclusion: at this clinic, kidney transplant recipients generally had high levels of health literacy. however, most had difficulty recognizing frequently used transplantrelated terms, which could impede their understanding of health information and self-care management. greater efforts are needed to educate kidney recipients about transplant concepts, which may foster better self-care management, and ultimately transplant outcomes. abstracts by a single pathologist using the banff, cadi and cnit classifications. all indication biopsies with clinical acute rejection (ar; 23.3% for sf and 21.4% for sb) were excluded from this analysis. the histological and clinical parameters were assessed using multivariate generalized-estimating-equations statistical analysis. results: subclinical ar was present in 12.2% sf vs. 8.5% sb bx at 6 mo and 0% sf vs 5% sb bx at 12 mo (p=ns); borderline ar was seen in 2.4% sf vs. 8.5% sb bx at 6 mo and 10% sf vs. 5% sb bx at 12 mo (p=ns). despite the pristine condition of the kidneys at implantation, regardless of steroid exposure, there was a significant trend increase (p<0.0001) in chronic tubulo-interstitial damage; 35% of 6 mo bx and 53% of 12 mo bx demonstrated ifta; with moderate/severe changes (ifta grade 2-3) in 6.8% and 10% of 6 and 12 mo bx respectively. the prevalence of biopsies with ischemic glomerular changes (p<0.0001), tubular microcalcifications (p=0.009), vascular intimal thickening (p=0.0008) and the number of sclerosed glomeruli (p<0.0001) increased over the first year after transplantation, without any difference between the sb and sf group. a critical risk factor for ifta injury by multivariate analysis, independent of time after transplantation, was smaller recipient size. in this first ever serial histological analysis, embedded in a randomized multicenter pediatric study of steroid avoidance, we found significant progression of chronic graft injury in the first year post-transplantation in both study arms. small recipient size is the primary risk factor for tubulo-interstitial damage, likely related to vascular size discrepancies between recipient and the graft, resulting in chronic graft ischemia. in the 1-year symphony core study, a regimen with 2g mycophenolate mofetil (mmf) + low-dose tacrolimus (3-7 ng/ml) + daclizumab + steroids resulted in less acute rejections and better glomerular filtration rate (gfr) compared with 2g mmf + steroids and either standard-dose cyclosporine (csa), low-dose csa (50-100 ng/ml) + daclizumab or low-dose sirolimus (4-8 ng/ml) + daclizumab. methods: 960 patients participated in an optional follow-up of 2 years. gfr data from 79% of included patients (48% of the core itt population) were available at 3 years.here we present results in the itt population. results: at inclusion into follow-up 47%, 34% and 17% of patients received csa, tacrolimus or sirolimus, and at 3 years 37%, 31% and 14%, respectively. many follow-up patients had been switched to tacrolimus in the 1st year, including 24% of patients randomized to sirolimus. at 3 years 95% of patients were on mmf and 69% on steroids.over the 2nd and 3rd year all arms had a low rate of biopsy-proven acute rejection (bpar; 2-4%) and of graft loss (3-5%). low-dose tacrolimus remained clearly superior in terms of bpar (13% vs. 26%-38% in the other arms). uncensored 3-year graft survival was 89% with low-dose tacrolimus and low-dose csa, 87% with standarddose csa and 85% with low-dose sirolimus (p=0.19). patient survival was between 94% and 97% (p=0.52). in the four arms, the mean gfr change over the 2 nd and 3 rd year was between +1 and -3 ml/min and low-dose tacrolimus still had the highest gfr (69 vs 64-66 ml/min, p=0.15). observational follow-up results based on approximately half of the core symphony population indicate that during the 2nd and 3rd year renal function was stable, bpar and graft loss rates were low and many patients changed treatment regimen substantially. still, the itt arm with 2g mmf + low-dose tacrolimus + daclizumab + steroids was superior at 3 years with respect to renal function and graft loss but differences were less marked and statistically not significant. discovering histological evaluation of time zero donor kidney biopsies has not conclusively predicted graft outcome. we hypothesize that gene expression analysis could provide additional information to determine graft outcome in the first year of transplantation. to this end, we evaluated all 49 implantation biopsies obtained post reperfusion in 18 deceased donors (dd) and 31 living donors (ld) at our center. biopsies were evaluated and scored using banff criteria. low density real time pcr arrays were utilized to measure intragraft expression of 95 genes associated with programmed cell death, fibrosis, innate and adaptive immunity, and oxidative stress signaling. results of expression were defined as folds compared to a pool of 25 normal kidney biopsies. in dd, histological features of atn were more common (44%) than in ld grafts (6%; p<0.001), whereas arteriosclerosis was infrequent in both groups (6% and 15%, respectively), as well as the extent of glomerular sclerosis (0% and 2%). there was no association between these histological features and renal function at 1 year post transplant. not surprisingly, dd grafts displayed a pattern of gene expression remarkably different from ld, including an increased expression of complement protein c3 ( background: isa247 is a novel calcineurin inhibitor (cni), developed using a pharmacodynamic approach for use in autoimmune disease and solid organ transplantation. in moderate to severe plaque psoriasis, a canadian phase iii trial has demonstrated that isa247 is efficacious with minimal changes to renal function and a european trial is presently underway comparing isa247 to cyclosporine a (csa).in renal transplantation, a phase iia study comparing isa247 to csa in stable renal transplant recipients demonstrated isa247 to be efficacious and well tolerated. a phase iib study in de novo renal transplant patients comparing isa247 to tacrolimus is ongoing and final data will be available may 2008. we hypothesize that isa247 is non-inferior to tacrolimus in terms of efficacy.methods: this is a 6 month, randomized, multicenter, open-label, concentrationcontrolled study comparing three oral isa247 dosing groups (0.4, 0.6, or 0.8 mg/kg bid) to tacrolimus in 42 north american transplant centres. all cni's were titrated to target trough concentrations. inclusion criteria included males and (non-pregnant) females between the ages of 18-65 who were receiving a first deceased or living donor renal transplant. cold ischemia times were to be ≤ 24 hours, and peak panel reactive antibodies ≤ 30%. the primary efficacy parameter of the trial is non-inferiority (in at least one dose group) in biopsy proven acute rejection (bpar) at 6 months as compared to tacrolimus. secondary objectives include: renal function; pk/pd relationships; patient and graft survival; and proportion of patients with hypertension, hyperlipidemia or new onset diabetes mellitus (nodm).results: interim data, as previously presented at the 2007 atc, demonstrated that isa247 had rejection rates similar to tacrolimus (isa247 0.4 mg/kg bid 11%, isa247 0.6 mg/kg bid 8%, isa247 0.8 mg/kg bid 3%, tacrolimus 9%) and confirmed previous results indicating an improved safety profile. 334 patients have now been enrolled between january 2006 and june 2007, with an optional extension to 12 months added to the trial. a recent approval by both fda and health canada has allowed continued use of isa247 in these patients until commercialization. the six month final results will be available for presentation at atc 2008. key: cord-022888-dnsdg04n authors: nan title: poster sessions date: 2009-08-19 journal: eur j immunol doi: 10.1002/eji.200990224 sha: doc_id: 22888 cord_uid: dnsdg04n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx3, the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx3 facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th1-oriented adaptive response. actually, ptx3-deficient mice are highly susceptible to aspergillosis and develop th2 skewed responses; moreover, ptx3-deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx3, which does not show direct activity on fungal cells. finally, ptx3 alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx3-mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx3 amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx3-opsonised conidia, cd11b activation, internalization, recruitment to the phagocytic cup and cd11b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd11b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx3, fcgriia/cd32 mediates inside-out activation of cd11b and consequently phagocytosis of c3b-opsonised conidia. in vivo phagocytosis experiments performed with c1q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx3) and the cellular arm (fcgrs, cr3). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx3 is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx3 binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx3-/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd14, immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx3. we found high concentration of ptx3 in human colostrum (47.62 ± 13.5 ng/ml at day 1 post-delivery) compare to the one found in human serum ( x 2 ng/ml). the presence of ptx3 in human colostrum seems to be due to the secretion of ptx3 by human mammary gland since we report the production of ptx3 by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx3 production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx3 production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx3 supplying by breast feeding: (i) soluble ptx3 in colostrum (ii) hmc that can secrete ptx3 upon stimulation in the specific tissue, (iii) an increase of ptx3 production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx3 may participate to the beneficial role of breast feeding on the newborn health. a. m. baru 1 , j. stephani 2 , h. wagner 2 , t. sparwasser 1 1 twincore, institute for infection immunology, hannover, germany, 2 technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of 10 receptors in humans (tlr 1-10) and 12 in mice (tlr 1-9, 11-13). as transmembrane receptors, tlrs are expressed on the cell surface (tlr 1, 2, 4, 5, 6, (10) (11) (12) (13) and at endosomal membranes (tlr 3, 7, 8 and 9) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about 5 years later to this, toll-like receptor 9 (tlr9) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr9 as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr9 (hutlr9) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr9 (mutlr9) is also expressed on conventional dendritic cells (cdcs). consequently, tlr9 ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr9 (henceforth called as hut9 mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr9 knock-out background. we expect that hut9-mutlr -/mice mimic the human specific expression pattern of tlr9, i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr9. by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr9 ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c1q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin-12 is a heterodimeric cytokine consisting of the two subunits p35 and p40. the main inducers of il-12p40 are microbial components activating toll-like receptors with the magnitude of il-12p40 induction depending on the specific tlr engaged. differential induction of il-12p40 upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il-12p40 due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il-12p40 promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il-12p40 promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone 3 and 4 acetylation in specific regions of the proximal promoter region. acetylation of h4 showed a specific distribution pattern and occured mainly in regulatory elements within the il-12p40 promoter, whereas acetylation of h3 took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue 4 on h3 turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue 1 , w. ellmeier 1 1 medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi 1 , m. buxadé 1 , j. minguillón 1 , r. berga 1 , j. aramburu 1 , c. lópez-rodríguez 1 1 universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat5 is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat5 participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat5 is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat5, the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat5 could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat5 is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat5 is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat5. our work indicates that nfat5 is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr3, tlr4, tlr7 and tlr8 and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p50 and p65 nfxb subunits. interestingly, we observed that tlr3 stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr4, tlr7 and tlr8 stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl-2 expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr3, tlr4, tlr7 and tlr8. finally, in vivo tlr7 stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr4, tlr7 or tlr8 triggering can directly favor tumor development whereas tlr3 signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms-354825) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd8 + and cd4 + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr 4 ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd4 + and cd8 + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd4 + and cd8 + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: 1. innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr 4 stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . 2. adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni 1 , r. oatuni 1 , m. collini 2 , m. caccia 2 , p. castagnoli 3 , g. chirico 2 , f. granucci 1 1 university of milano-bicocca, biotechnology and bioscience, milan, italy, 2 university of milano-bicocca, physics, milan, italy, 3 singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin-2 (il-2) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il-2 production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca2+/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca2+ influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr4 engagement, depending instead exclusively on cd14. we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase-2 (cox-2) that, by generating prostaglandins (pgs), such as pge2, regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd14. given the essential involvement of cd14 in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd14 represents a major step towards the development of potential treatments with modes of action involving interference with cd14 functions. we have examined the interaction of cd55, a 80-kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd14. human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd14 antibody coupled to biotin visualised by qd-525-streptavidin and anti-cd55 antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd55 and cd14, suggesting a new functional role of cd55 as a member of a multimeric lps receptor complex. l. lundvall 1 , r.r. schumann 1 1 charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase-1 induction leads to an increased release of mature il-1b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d39) was established for assessing il-1b induction during this disease. the murine raw 264.7 cell line, the human astroglial u-87 mg and the murine microglial cell-line bv-2 were stimulated with the tlr4 ligand lps, the tlr2 ligand pam 2 cys, the nod2 ligand mdp, or the nod1 ligand c12-ie-dap, and, as control, atp alone or in combination. we assessed il-1b by elisa and caspase-1 and pro-il-1b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il-1b induction pathway. s. pneumoniae (d39) infected mice showed a significant increase in il-1b release after 24 hours. in vitro, an increase in il-1b levels after costimulation with lps or pam 2 cys, and mdp or c12-ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il-1b by tlr-and nlr-ligands was observed in raw cells, bv-2 cells, u-87 cells and primary astrocytes. active caspase-1 (p10) was induced by mdp or c12-ie-dap, corresponding with high il-1b responses when lps or pam 2 cys was added. sirna experiments show that a knock-down of nod2 leads to a diminished il-1b release after lps-and mdp-stimulation. the precursor forms of il-1b and caspase-1 seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il-1b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il-1b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il-1b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg 1 1 toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd3 and cd28 activation model system -tlr4 presence on cd4+ cells is found in mouse t cells, human t cells and jurkat cell lines. following cd3/cd28 activation for 48 hours we have identified a small but distinct populationof tlr4+ cells. further characterization indicates these cells to be cd4+cd25+ cells. further characterization of the expression and functional acitvity of the tlr4+ t cells indicates co-expression of tlr4 with md-2 indicating a functional tlr4 receptor. in addition lps activiation did not lead to upregulation of tlr4 expression in t-cells. the data indicate that tcr activation leads to tlr4 expression. the expression appears to be associated with cd4+cd25+ cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann 1 , d. kaul 1 , c. krüger 1 , f. zipp 1 , r. nitsch 2 , s. lehnardt 1 1 charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, 2 charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr7 plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna40) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr7 (and human tlr8) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr7 is expressed in these cells. incubation of microglia with all three of the above mentioned tlr7 ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il1-b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr7 knock out (ko) microglia by realbecause neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin-1 was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin-1 is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin-1-transfected plb985 cells, coronin-1 overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd11b labeling. concerning apoptosis, increased coronin-1 expression in dmf-differentiated plb985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin-1 significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or bid truncation suggesting that coronin-1 interfered with mitochondria-related events. to validate the prosurvival role of coronin-1 in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin-1 immunolabeling. we concluded that coronin-1 could constitute a potential target in resolving inflammation. p.-n. hsu 1 1 national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw264.7 macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p38 map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf-6 sirna and traf-6 decoy peptide, indicating this pathway is traf-6 dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b7-h1 and b7-dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il-6 was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il-2 and ifn-g which could not be overcome by restimulation with acd3. this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd4 t cells into t helper 17 (th17) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th17 differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th17 cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il-1 receptor-related protein st2 could be implicated in lps tolerance. the natural ligand of st2 was recently identified as interleukin-33 (il-33), a new member of the il-1 family. in this study, we investigated whether il-33 triggering of st2 was able to induce lps desensitization of mouse macrophages. we found that il-33 actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il-33 enhancing effect of lps response is mediated by the st2 receptor since it is not found in st2 ko mice. the biochemical consequences of il-33 pretreatment of mouse macrophages were investigated. our results show that il-33 increases the expression of the lps receptor components myeloid differentiation protein-2 (md2), cd14 and tlr-4 and the myeloid differentiation factor 88 (myd88) adaptor molecule. in addition, il-33 pretreatment of macrophages enhances the cytokine response to tlr-2 but not to tlr-3 ligands. thus, il-33 treatment preferentially affects the myd88-dependent pathway activated by the tlr. c. klotz 1 , b. lenz 1 , r. lucius 1 , s. hartmann 1 1 humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., 2008) . the cystatin effect was blocked by the application of anti-il-10 receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin-10 (il-10). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il-10 in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il-10 production after avcystatin stimulation. application of specific inhibitors revealed that the il-10 induction was p38 and erk dependent, and inhibitor titration indicated a higher sensitivity towards p38. western blotting experiments confirmed the phosphorylation of p38 and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il-10 is also regulated by the phosphatidylinositol-3-kinase (pi3k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il-10 and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose-7-phosphate (s-7p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose-5p and xylulose-5p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p38/jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s-7p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts 1 , y. morias 1 , p. de baetselier 1 , a. beschin 1 1 vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m1) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m1 activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m1 activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m1 in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd88, ifng, il-10, ccr2 and nf-kb to the development and/or recruitment of pathogenic m1 in the liver was investigated using knock-out mice or by delivering il-10 in infected mice. results: we established that cd11b+ly6c+cd11c+ tnf and inos producing dcs (tip-dcs) represent the major m1 liver subpopulation. tip-dcs differentiated in an ifng/myd88-dependent manner from cd11b+ly6c+ inflammatory monocytes in the liver of infected mice. ccr2 promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr2 ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il-10 enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il-10 in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p50 was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p50 and il-10 play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m1, in particular tip-dcs. the inflammatory activity of liver m1 is controlled by il-10 and/or p50 nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov 1 , j. driesen 1 , z. abdullah 2 , a. niño castro 1 , t. chakraborty 3 , m. krönke 4 , o. utermöhlen 4 , c. wickenhauser 5 , j.l. schultze 1 1 limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, 2 institute of molecular medicine and experimental immunology, bonn, germany, 3 institute of medical microbiology, university of giessen, giessen, germany, 4 institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, 5 institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine 2,3-dioxygenase, cyclooxygenase-2 and cd25 and production of prostaglandin e2 (pge 2 ) and interleukin 10. all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd25, expressed by regulatory myeloid cells, acts as an il-2 scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge 2 or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd25 + ido + cox-2 + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin 12 (il-12) and il-18 in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il-12 and il-18 or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il-12 and il-18. in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il-12 and il-18. therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel 1 , m. rodriguez gomez 2 , m. niedermeier 2 , y. talke 2 , n. göbel 2 , k. schmidbauer 2 , m. mack 2 1 unversity hospital regensburg, internal medicine ii, regensburg, germany, 2 university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il-4 and il-6. activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to 24h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp-1 cells infected with m. bovis bcg, the avirulent m. tuberculosis h37ra strain and the virulent m. tuberculosis h37rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva 1 , l.d. miteva 1 , s.a. stanilova 1 1 trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il-23 is a heterodimeric cytokine composed of a p19 subunit associated with the il-12/23p40 subunit. like il-12, il-23 is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il-12p40-related cytokines controls the appearance of normal th 1 and pathological th 17 mediated immune responses. in this study, we examined the dynamics of inducible il-12p40 and il-23p19 mrna expression and protein production in purified human monocytes and how jnk and p38 mapks inhibitors influenced il-12p40 and il-23 production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il-23p19 gene expression was higher than those observed for il-12p40 gene at all time-points. the level of il-23p19 mrna increased after 6 th h and reaching a maximum level at 9 th h (43.4 fold for c3bgp and 22.7 fold for lps). c3bgp and lps triggered il-12p40 gene transcription were almost equal at the 3 rd h (4.4 and 4.1 fold) and at 9 th h (7.8 and 7.9 fold, respectively) after stimulation. the higher level of il-12p40 gene expression was detected at 6 th h in lps compared to c3bgp stimulated monocytes (8.1 vs. 4.9 fold). however, il-12p40 and il-23 protein production was increased in the highest level after c3bgp stimulation. the inhibition of p38 led to the statistical significant augmentation of c3bgp stimulated il-12p40 production. the inhibition of the same map kinase enhanced lps stimulated il-12p40 production without significant difference. the inhibition of jnk and p38 mapks significantly decreased c3bgp stimulated il-23 production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c3bgp and lps to induce the expression of il-12p40 and il-23p19 mrnas in purified human monocytes. we showed that inhibition of p38 mapk down regulated il-23 and upregulated il-12p40 production in stimulated monocytes. we concluded that in human monocytes p38 map kinase activation has an opposite effect on the il-12p40 and il-23p19 expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd83, cd86 and cd40, and secrete il-12, thus acquiring the ability to induce proliferation and th1 polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd1c, bdca-3, and cd16. by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd16+ subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd16+ dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd16+ dc, including their survival and their ability to produce il-12p70. besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il-6, il-8 and mcp-1 involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg 1 , s. wolke 2 , a. brakhage 2 , m. gunzer 1 1 otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, 2 hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in 2004 nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and 2-photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to 3 hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor 7 (irf7), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr9. in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr9 to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens 1 , s. vander beken 1 , p. bogaert 1 , j. grooten 1 1 ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th1-driven immunological counterpart of the th2-driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd45 bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th1-and th2-driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd45.1 + ram remained constant in cell number for the first 2 days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day 4 of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker 1 , a. stojanovic 1 , a. cerwenka 1 1 german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr-1 + cd11b + f4/80 + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas 1 , f. marchesi 1 , m. fabbri 1 , s. schiarea 2 , c. chiabrando 2 , a. mantovani 1,3 , p. allavena 1 1 istituto clinico humanitas, rozzano, italy, 2 istituto mario negri, milano, italy, 3 università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m1 and m2. m1 macrophages act as a first line of defence against pathogens whereas m2 cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about 50 % of the monocytes differentiated after 5 days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd16, cd68 and low levels of mhc class ii. tc-macro produced il-10, il-6, tnf but not il-12, even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl2, cxcl8, ccl17 and cxcl10. the transcriptional profile of tc-macro revealed that several genes in line with an m2 polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g 100.000 kda). il-3 and il-6 were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl-2h3 rat mast cell line. we demonstrate that gr nuclear translocation begins within 5 minutes and completes after 30 minutes in dx treated rbl-2h3 cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within 5 minutes as non-genomic. studying gc-caused apoptosis, rbl-2h3 cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl-2h3 cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. 5 minutes of dx treatment inhibited ca 2+ -signaling in antigen stimulated rbl-2h3 cells in the concentration range of 100nm -10mm. moreover, 5 minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl-2h3 cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box 1 (hmgb1) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb1 is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin (1) . extracellular hmgb1 can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb1 exerts antibacterial functions in human adenoid and testis (2) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity (3). we asked whether hmgb1 from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for 40 or 120 minutes with 25 nm phorbol ester (pma), 100 ng/ml interleukin 8 (il-8), or 100 ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h2a-histone h2b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb1 in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb1 is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb1 localizes on nets. we hypothesize that net-bound hmgb1 might exert a direct antimicrobial function, or that nets might concentrate hmgb1 locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation (37°c, 3 h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor-1 induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr-1 and anti-vegfr-2 antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il-18. altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd1 molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd1a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd1a and cholesterol-dependent lipid rafts impact on cd1a surface expression and cd1a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd1-restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd1 group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd1a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd1 molecules in multiple sclerosis. we first analyzed cd1 expression on monocytes from 16 ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd1a was not expressed on ms patient monocytes, while the other members of the cd1 family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd1 molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd1 molecules in this context. c. ohnmacht 1 , d. voehringer 1 1 ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il-4 reporter (4get) mice as well as in vivo depletion of basophils are used to uncover their role during type 2 immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about 60h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th2 cells. these results demonstrate that basophils play a crucial role as effector cells in type 2 immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il-1beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water (10 mg and 100 mg daily) for 30 days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il-6 and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il-6 and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant 143038). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type-1 macrophages, mph1), and during resolution of inflammation (type-2 macrophages, mph2). methods: mph1 / 2 were generated by culturing cd14 + human monocytes for 6 days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph1 / 2 of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph1 / 2 were stimulated with lps and secreted il-6, il-8, il-10, il-12p40 and tnf were quantified by elisa. results: mph2 are relatively efficient phagocytes for apoptotic neutrophils whereas mph1 are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph1 and mph2 which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il-6, il-12 and tnf production is suppressed while il-10 secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp70 in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of 90 years old and older (long-livers). intracellular ros and hsp70 levels were registered by flow cytofluorimetry upon labeling with 2',7'-dichlorofluorescin diacetate (invitrogen) and anti-hsp70 antibody (brm-22, sigma), respectively. intracellular level of hsp70 was also estimated in neutrophils after heat shock (hs) performed at 43°c for 10 min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at 1-15 min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for 1 min at 42°c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp70 (hsp70 basal ) and ros level, both intracellular and extracellular. at the same time increased hsp70 level immediately after hs (hsp70 (0 min)) correlated negatively with intracellular ros (initial and after hs). the hsp70 increase value (hsp70 (0 min) -hsp70 basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp70 increase was registered in 60 min after hs (hsp70 (60 min) -hsp70 basal ). thus it was found that within this age group the alteration in hsp70 induced by hs in neutrophils but not basal hsp70 itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant #3303. d. goyeneche-patiño 1 , z. orinska 1 , f. mirghomizadeh 1 , s. bulfone-paus 1 1 forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd8 + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type 1 ifn. two novel genes, receptor transporter protein (rtp4) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp4 and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd88 and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd8 -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during 8 and 48 h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp4 and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine 2000. stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp4 in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd88 knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr 3 and 9, as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp4 and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd88 and trif and the pathways that they represent are not relevant in the expression of rtp4 and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano 1 , e. erba 2 , r. frapolli 2 , m. d'incalci 2 , a. anselmo 1 , s. pesce 1 , p. allavena 1 , a. mantovani 1, 3 1 humanitas clinical institute, rozzano, italy, 2 mario negri institute, milan, italy, 3 institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et-743) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl2, cxcl8 and the inflammatory protein pentraxin3 (ptx3) either at transcriptional and protein level, especially after tnfa/il1b stimulation. down-regulation of ccl2, cxcl8, ptx3 and also of il6 and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd31+ vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin-4 (il-4) is a key cytokine of the t helper 2 cell response. il-4 has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il-4 production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il-4 producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day 3-6 after birth, a small population of il-4 producing cells was observed in the absence of any stimuli. the il-4 + population was not detectable at 6 or 12 weeks of age. other cytokine producing cells (ifn-g, il-10) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il-4 + cell population. phenotyping of the neonatal il-4 + cells showed that they were ige + /mhcii -/cd4cells. the occurrence of cd4 + il-4 producing cells after pma stimulation increased slowly with age and did not reach adult levels by 12 weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il-4 at day 5 after birth. neonatal pbmc collected before colostrum uptake did not produce il-4 in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il-4 responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il-4 response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel 1 , d. voehringer 1 1 ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th1 cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat6-dependent manner when stimulated with the th2 cytokines il-4 or il-13. alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat6-dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat6-deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat6 in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat6 expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat6 expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat6-deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il-4 exposed macrophages from wild-type and stat6-deficient mice. candidate genes will be expressed using retroviral transfections of stat6-deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of 38 pleural effusions, including 15 malignant and 23 nonmalignant tumors. the following human malignant cell lines were tested: a549, ht29, hct116, sw60, mcf7, mda-mb231, jurkat and hl60. results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m1, and the tams isolated from the malignant effusions similar to m2. among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms4a4a and ms4a6a: highest trascriptional level after 18h of stimulation with 10-6m dexamethasone. ms4a murine genes are differently expressed respect to the human counterpart and only the homologs of ms4a6a (ms4a6b, 6c and 6d) have a similar regulation. finally, egfp-tagged ms4a4a, ms4a6a, and ms4a7 expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms4a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms4a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein-1 (mcp-1)/ccl2 and the gpcr ligand fmlp or the tlr4 agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il-1b or ifn-g, significantly and dose-dependently synergized with ccl2 in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin-8/cxcl8, but not of the ccl2 receptor ccr2 in monocytic cells. in turn, the induced cxcl8 synergized with ccl2 for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr1/cxcr2, because cxcl8 receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl2 intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, 2004) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for 20 min at 37°c with gentle shaking, followed by spinning down of pmns for 5 min, at 4°c and 500g. the supernatant was sedimented at 15000g for 10 min, 4°c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd56 has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd56 low cd33+ blood monocytes (mo) in healthy individuals and the increase in cd16+cd56+ blood mo in patients with inflammation has been reported. the functional activity of human cd56+ blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd56 expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd33 and activation markers such as hla-dr and trem-1. percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a 12.3 fold increase (p x 0.0001) in the total number of circulating cd56 low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd33 and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd56+ blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd33+ blood mo expressed increased levels of cd56 on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem-1+) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd56 on cd14 high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m1-and m2-polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m1 and m2 macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m1 type and were cd14 negative but cd80 and mhcii positive and produced high levels of il-8, tnfalpha and il-6 following lps stimulation. culture in the presence of mcsf resulted in induction of the m2 phenotype. these cells were cd14 positive with intermediate expression of cd80 and mhcii expression and produced high levels of il-10, il-6 and il-8 following lps stimulation. interestingly, already baseline il-8 production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m1-and m2-polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr4 ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl60 cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma (50 ng/ml) treatment for 48h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl60 cells expressed antigen receptors cd14, tlr2, tlr4 and cd68 , and displayed enhanced phagocytic activity and production of ros. expression of cd13, cd33 and cd15 was also maintained however the cells were hla-dr and cd1a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl60 cells to differentiate into macrophages in response to pma. hl60 may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta 1 , n. ferrer 2 , a. gómez 2 , j. gonzalo 2 , a. arbués 2 , a. anel 1 , c. martín 2 , j. pardo 1 , apoptosis, immunity and cancer 1 university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, 2 university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so2, a m. tuberculosis phop mutant strain that was shown (perez et al 2001) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al 2006) . in the present study, we compare the time course and phenotype of cell death induced by so2, bcg and wild type m. tuberculosis on the murine macrophage cell line j774 and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase-3 activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl2x4 deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type 1 receptor for vip (vipr1) gene is highly conserved through species and, in humans, is highly polymorphic. vipr1 has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from 53 blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr1 in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr1 after 3, 6, 9 and 12 h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g 50 %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from 53 donors that had been typed for the relevant vipr1 snps. the down-regulation of vipr1 correlates with the presence of a t at rs896 mapping in the 3'-end of the gene (p= 0.004). the vipr1 protein level was decreased about 40 % in monocytes of 3 subjects typed as t/t at rs896 whereas 3 subjects typed as c/c at rs896 maintained a high level of expression after 9h of lps treatment. the data show that different haplotypes of the vipr1 gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr1 vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska 1 , t. vavrochova 1 , d. filipp 1 , immunobiology 1 institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e7.5-13.5) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd14 in the early mouse embryo (me). facs analysis of cell suspension prepared from 10.5 day me showed that about 0.7-1 % of cells were positive for cd11b. these cells exclusively were also positive for cd14, tlr2, and cd45 antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days 7,5-13,5. reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e7,5), embryo-derived mhcii + macrophages start to appear in the embryo around day 13. multicolor facs analysis of cd11b, cd45, cd14, f4/80, tlr2, tlr4, c-kit and mhcii surface markers revealed differential expression of tlr2 and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd11b + tlr2 + cells isolated from the e10,5 embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il12/il23-dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies 16 presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and 11 have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in 9 out of these 11 patients mutations in several ifng pathway genes, other candidate genes are being investigated for the 2 other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp1. we did focus on the study of 2 genes which are considered as important pathogen recognition receptors of the innate immune system: tlr2 is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il-10 cytokine. using a case/household-contact cohort we did investigate polymorphisms of these 2 genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to 70-80 % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, 2009. hsp70 are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp70 have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp70 on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp70 (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp70 were used. exogenous hsp70 was used in concentration 1-10 ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio 1:1 and 2:1 directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp70 on their surface. results demonstrating effect of exogenous hsp70 on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp70 on amplitude of respiratory burst. the cells expressing surface hsp70 impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp70 might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp70 to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited 28 pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd14/cd86 and cd14/cd163 double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd14/86 subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd14/163 subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m2 polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević 1 , i. pilipović 1 , s. stanojević 1 , k. mitić 1 , k. radojević 1 , v. pešić 2 , g. leposavić 1,2 1 institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, 2 faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h 2 o 2 ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to 14-day-long propranolol treatment and measured both no and h 2 o 2 production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b 2 -and a 1 -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h 2 o 2 production. an increase in h 2 o 2 production in the presence of the a 1 -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h 2 o 2 production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b 2 -and a 1 -adrenoceptors on peritoneal macrophages (a stimulatory effect on b 2 -adrenoceptors and a suppressive effect on a 1 -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd14 ++ cd16 -('classical') and cd14 + cd16 + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd14 + cd16 + monocytes display higher tlr2 and -4 expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd14 ++ cd16monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr2 stimulation. methods: cord blood (n=8) and peripheral-blood (n=8) mononuclear cells were stimulated in vitro for 24hrs with peptidoglycan and subsequently analysed for cd14 and cd16 and intracellular il-12p70 and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il-12p70, both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il-12p70 was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il-12p70 (% positive cells and geomfi) was significantly higher for cd14 + cd16 + cells than for cd14 ++ cd16cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd14 ++ cd16cells were positive for tnf to a significantly higher extent than the cd14 + cd16 + cells. in particular the tnf response to tlr2 stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd14 ++ cd16monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a 16-year-old boy who admitted with complaints of seizures during the previous 2 months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g (50 mgr/m2/day, subcutaneously every other day) was started. after 2 months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for 3 months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf1 gene concerns the well-known gt deletion in the second exon of ncf1 gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = 45) subjected to moderate brain injury were randomized, and received either 1.5 mg/kg ubiquitin or vehicle (placebo) intravenously within 5 min after cci. levels of tnf-a, il-1b, il-6, il-10 and il-1 receptor antagonist were analyzed in brain tissue using real time rt-pcr at 4 and 72 hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at 24h and 7 days. data were analyzed with the mann-whitney u test and a two-tailed p x 0 .05 was considered significant. all cytokines were highly up-regulated 4 hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il-10 were significantly lower in the ubiquitin treated animals, whereas the levels of il-6 and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day 7. the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight (38) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while 18 were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p=0.02), tpp (p=0.00), osi (p = 0.00), mda (p = 0.00) were significantly raised but taa (p = 0.01) was significantly reduced in ptb patients compared with controls. the levels of mda (p = 0.04), neopterin (p=0.00) and tpp (p=0.00) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p=0.00), mda (p=0.00), neopterin (p=0.02), osi (p=0.00) were significantly reduced while taa (p=0.01) was significantly raised in c+m after 4 weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after 4 weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas 1 , e.m. cunha 1 , m.j. oliveira 1 1 icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles (20 nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to 5 minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage (70 %) of macrophages contained the metal particles than neutrophils (55 %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap-70 is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap-70 regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., 2006; sivalenka and jessberger, 2004) . swap-70-/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., 2002; sivalenka and jessberger, 2004; sivalenka et al., 2008) . crucial regulators of these processes are members of the rho family of small gtpases such as rac1 and rhoa. swap-70 interacts with rac1 in vitro and preferentially binds the active gtp-bound rac1. swap-70 supports the increase of active rac1 in vitro by a yet to be defined mechanism (shinohara et al., 2002) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap-70 and rac1. it was found that fulllength swap-70 preferentially binds to constitutively active rac1 (rac1q61l) but not to its dominant negative form (rac1t17n). binding assays with swap-70 truncated mutants showed interaction of swap-70's n-terminus with gtpgs rac1 or rac1 depleted of guanine nucleotide, whereas swap-70 central or c-terminal regions do not bind to any form of rac1. preliminary competitive-binding assays with overlapping 18mer peptides, spanning the entire swap-70 sequence, mapped the rac1 binding site near the n-terminus of swap-70. full-length swap-70 site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap-70 with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap-70. v. c. barbosa 1 , c. d. polli 1 , m.c. roque-barreira 1 , m.c. jamur 1 , c. oliver 1 , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl-2h3 cells were sensitized with ige anti-tnp and stimulated with dnp 48 -hsa or artinm. artinm binding to rbl-2h3 cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp-1 and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp-1 release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand 1 (psgl-1), b1 and b7-integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd44 (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il-1b, il-18 and tnf-a. herpes simplex virus 1 (hsv-1) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv1 infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il-18 and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv1. methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) (10ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv-1 for 18 h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot 2.0 software. results: in the first itraq experiment over 300 human proteins were identified in the hsv1 infected cell supernatants. from these proteins 119 had at least 3 fold increase after poly(i:c) + hsv1 infection compared to the uninfected cells. hsv1 infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of 2.7 fold increase in the protein amount in the poly(i:c) + hsv1 infected cell supernatant and a 2.6 fold increase in the hsv1 infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand 10, il-6, tnf-a induced protein 6, complement factor b, galectin-1 and mxa. at present, further experiments are on-going for more detailed analysis of the hsv1 infected macrophage secretome. h. p. prakash 1,2 1 german cancer research centre, translational immunology, heidelberg, germany, 2 max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein-1 (ciap-1) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap-1 and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal-3 (25 mg/ml) binding to monocytes, in the presence or absence of 10mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal3, laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal3 or rpmi and monocytes (1x10 5 ) were added into each insert. when necessary, hrgal3 was pre-incubated with 10mm lactose or sacarose. mcp-1 (100ng/ml) was used as positive control. we observed that hrgal-3 binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal-3 is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal-3, we observed that the association between these glycoproteins and hrgal-3 resulted in a 60 % increase in the number of migrating cells. both n-and c-terminal domains of hrgal-3 are involved in the association between laminin or fibronectin and hrgal-3, since the presence of lactose resulted in 50 % and 20 % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal-3 induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for 3 weeks in the presence of bmp4,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd14+cd45+ cells. the sorted cd14+cd45+ cells were further cultured for 7 -10 days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd14, m-csf receptor cd115 and the scavenger-receptor cd36, we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd14 and cd16 (fcgammariii) : the cd14lowcd16-and cd14+ cd16+. trancscriptional, phenotypic and functional assays suggest the alternative (m2) polarization of cd14+cd16+ embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m2 -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi 1 , p. kropf 1 1 imperial college london, immunology department, london, united kingdom the balance between t helper (th) 1 and th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th1 or th2 responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd4 + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg9vd2 t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f1-atpase) is expressed on many cell types and is a possible specific ligand for the vg9vd2 tcr. the present study aims at understanding the role of f1-atpase in antigen regognition. using video microscopy calcium imaging in single vg9vd2 t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f1-atpase. purified f1-atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg9vd2 cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f1-atpase. thus the f1-atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg9vd2 t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f1-atpase. by using purified f1-atpase and peptides derived from vg9vd2 tcr sequences, interaction sites between f1-atpase and tcr were identified on both ligands. based on these findings a generalized model for vg9vd2 t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg2d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg2d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg2d activation in vivo. by transiently overexpressing the nkg2d ligand rae-1-beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma5vdelta1 gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg2d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg2d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg2d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae1 induction. the primary systemic th2 response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg2d receptor suggest that different ones may play unique roles. a novel nkg2d-ligand, h60c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae-1 induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg2d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd1d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th1 (ifn-g, tnf) and th2 (il-4, il-13) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type 1 diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd4 -nk1.1 -inkt cells producing high levels of the pro-inflammatory cytokine il-17 has been identified (inkt17 cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt17 cells in type 1 diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il-17 as compared to c57bl/6 mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt17 cells present in nod mice were mainly cd4 -nk1.1 -, express the ror-g transcription factor and il-23 receptor, both molecules being usually associated with th17 commitment. we are currently analyzing, using co-transfer experiments, whether these inkt17 cells play a beneficial, a deleterious, or any role in the development of type 1 diabetes in nod mice. j. s. dodd 1 , r. muir 1 , s.s. affendi 1 , p.j. openshaw 1 1 imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd1d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd1d-deficient mice with poor nkt cell responses have inefficient induction of cd8 t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th2 immunity (increasing il-5 and il-10), promoting pulmonary eosinophilia and ablating cd8 t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd8 t cell activity (as measured by cd25 expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d4) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d7) weight loss by a cd8 t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd4 + ab t cells, are regarded as immature or t h 2 biased. vg9 + vd2 + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg9 + vd2 + t cells towards these activators. because il-23 is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg9 + vd2 + t cells. herein, we observed that zoledronate induced neonatal vg9 + vd2 + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h 1-like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h 2-like cytokines such as il-4 and il-5 were not induced. addition of il-23 to zoledronate selectively costimulated ifn-g production from neonatal vg9 + vd2 + t cells. furthermore, zoledronate/il-23 treatment resulted in neonatal vg9 + vd2 + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il-23 (il-23r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il-23 resulted in a further increase of t-bet expression in neonatal vg9 + vd2 + t cells. these changes in the expression of il-23r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il-23 treatment. of note, in contrast to adult peripheral blood vg9 + vd2 + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg9 + vd2 + t cells. altogether, these observations show that neonatal vg9 + vd2 + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il-23 is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e3 ligases such as hhv8 encoded k3, k5 or mhv68 encoded mk3. these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e3 ligases manipulate them. we discovered that both the cellular march and viral e3 ligases ubiquitinate cd1 molecules. however, whereas viral molecules inhibit cd1-antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd1 molecules. in contrast mhc class ii was only targeted by cellular and not by viral e3 ligases. furthermore cd1 molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e3 ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd2 negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht29 colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd2 neg clones in vivo, we generated hypodermal ht29 tumors in immunodeficient mice. concomitant injections of vd2 neg clones, in contrast to vd2 + cells, prevented the development of ht29 tumors. vd2 neg clones expressed chemokine c-c motif receptor 3 (ccr3) and migrated in vitro in response to chemokines secreted by ht29 cells, among which were the ccr3 ligands macrophage inflammatory protein (mip)-1d and monocyte chemoattractant protein (mcp)-4. more importantly, a systemic intraperitoneal (i. p.) treatment with vd2 neg clones delayed the growth of ht29 subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr3 antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd2 neg clones were not able to inhibit the growth of a431 hypodermal tumors. our findings suggest that cmv-specific vd2 neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht29 cells expressing the luciferase and realized orthotopic injection of ht29-luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint1, currently the only known determinant of a canonical iel compartment, that is selectively required for vg5vd1 + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint1 expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint1 function; for example, we demonstrate that the constitutive expression of wild-type skint1 fully restores detc development in a skint1 mutant mouse, but does not rescue normal detc function. thus, skint1 provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd1d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd19 + cd21 hi cd23 lo cd11c -) and splenic conventional dendritic cells (cdcs) (cd11c hi cd8a +/-cd11b +/-b220 -) from wt and cd1d -/mice as apcs for nkt cells from va14-ja18 transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd1d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd1d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il-4 by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il-4 producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h 2 response, whereas cdc-primed nkt cells rather favor a t h 1 response. objectives: il-18 is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il-18 give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd1d -/-) mice. we set out to investigate the activated b cells in il-18 injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il-18 (2 mg) for 10 days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day 14 was evaluated by flow cytometry and immunohistology. results: mice injected with il-18 developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd138 + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il-18 was increased in inkt cell deficient (cd1d -/-) mice, in contrast to published data. an increased response to il-18 was also observed in ja281 -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il-18 induced antibody responses. further characterization of the recruitment of b cells in il-18 injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il-18 induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd1d, are prone to autoantibody production and often involved in early immune responses. the il-18 induced antibody response in mzb deficient (cd19 -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il-18 induced antibody responses. we conclude that the role for inkt cells in il-18 induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c57bl/6 mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha 18-/-and cd1d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il-4. moreover, ifn-g production required the presentation of ehlppg by cd1d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il-12. similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd16 in cmv-infected individuals. cd16 is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that 71.9 ± 15.9 % of gamma-delta t cells from 21 cmv-infected ktr expressed cd16, when compared with only 19.8 ± 16.7% in 11 non cmv-infected ktr (p x 0.0005). similarly, 51.9 ± 25.7 % of gamma-delta t cells from 13 cmv-seropositive blood donors expressed cd16 compared to 27.1 ± 25.1 % in 15 cmv-seronegative donors (p x 0.01). cd16+ gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd16-dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il-12 and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd16 mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd107a on cd16+ gamma-delta t cell lines. cd16 is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd16+ gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a431 skin carcinoma cell line pre-incubated either with rituximab (anti-cd20) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd16-dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k5mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il-6, ifn-g, tnf-a, osm, ccl2, cxcl8, cxcl10, and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd40, cd86, hla-dr, and dc-sign, and lost cd14, ccr2, ccr5, and cxcr4. addition of further microbial stimuli (lps, peptidoglycan) induced ccr7 and enabled these inflammatory dcs to trigger antigenspecific cd4 + effector ab t cells expressing ifn-g and/or il-17. importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg9/vd2 t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg9/vd2 t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg9/vd2 t cells stimulated with hmb-pp in the presence of il-21 express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il-21 regulate expression of the b cell attracting chemokine cxcl13/bca-1, its receptor cxcr5, and co-stimulatory molecules involved in b cell help. purified peripheral vg9/vd2 t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to 4 days with and without hmb-pp, in the absence or presence of il-2 or il-21, or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl13 protein were detected in co-culture supernatants only when both il-21 and hmb-pp were provided, implying an il-21-dependent and tcr-dependent expression. vg9/vd2 t cells were confirmed as producers of cxcl13 by flow cytometry and immunofluorescence. under the same conditions, activated vg9/vd2 t cells expressed cd27, cd28, cd40l, cd70, icos and ox40. in contrast, neither cxcr5 nor ccr7 changed markedly by il-21 stimulation of peripheral vg9/vd2 t cells. conclusion: our findings confirm on the protein level that stimulation of vg9/vd2 t cells with hmb-pp and il-21 induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto 1 , m. emoto 1 1 gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)-12. although downmodulation of surface t cell receptor (tcr)/nkr-p1c (nk1.1) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il-12 stimulation. to determine whether failure to detect inkt cells after il-12 stimulation is caused by dissociation/internalization of tcr and/or nkr-p1c or by block of de-novo synthesis of these molecules, and to examine the role of il-12 in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p1c by inkt cells after stimulation with a-galcer or il-12, and influence of il-12 neutralization on down-modulation of stcr/snkr-p1c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il-12 neutralization. whereas s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of il-12, s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p1c remained unaffected by a-galcer or il-12 treatment, despite the down-modulation of ctcr and/or cnkr-p1c protein expression. in contrast, ctcr + cnkr-p1c + stcr -snkr-p1c -inkt cells and cnkr-p1c + snkr-p1c -inkt cells were detectable after in-vitro stimulation with a-galcer and il-12, respectively. our results indicate that tcr and nkr-p1c expression by inkt cells is differentially regulated by signaling through tcr and il-12r. they also suggest that il-12 participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p1c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg9-gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region 3 d1 (cdr3d1) and cdr3d2 repertoire, with a striking enrichment in a specific germline-encoded cdr3d1 sequence. differentiated gd t cells and the enriched cdr3d1 sequence were detected as early as after 21 weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd1-jd1 and vgi-jg1.3/2.3 rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd2-jd1 and vg9-jg1.2 selection was seen in pb tcrgd cells. detailed analysis of the cdr3 motifs revealed selection determinants in both vg9-jg1.2 (canonical length and cdr3 motif) and vd2-jd1 (minimal cdr3 length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚4) and tcrgd cb cells (6-7) to tcrgd pb cells (˚10 or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type 1 diabetes (t1d). we have previously shown that transgenic expression of a cd1d-restricted, va3.2-vb9 tcr in nod mice lead to an increase in cd1d-restricted type ii nkt cells (24abnkt cells), and prevention of the development of t1d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by 24abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc2.5 cd4+ t cells, in the presence or absence of selected cells from 24abnkt cell transgenic mice. results: in 24ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, 24abnkt cells expressed a high level of cxcr3 and a low level of ccr7 and cd62l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd4+ bdc2.5 t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from 24ab transgenic mice with bdc2.5 cd4+ cells resulted in the prevention of diabetes development. the protection from disease required a minor cd4+ subset of 24ab+ nkt cells, but was independent of cd25+ t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd1d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs44, induces in vitro inkt cell expansion and ifng and il-4 secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th2 profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd1d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il-10 with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr1 cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th17-type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th17 immune response is characterized by the secretion of il-17a and il-17f. the il-17 locus encodes the highly conserved il-17a and il-17f cytokines that are syntenic in 44kb distance to each other. besides cd4 + th17 and nkt cells, approximately 50 % of the il17a producers are gd t-cells. like cd4 + th17 cells, il-17 producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il-17 production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th17 cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il-17 or ifn-g. combined with the well known classification of il-17 producing gd t-cells along the markers cd27 and cd122, our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th17-type immune responses in vivo. in this context, the potential redundancy of il-17a and il-17f may complicate the analysis. so far, most studies were carried out with il-17a single-deficient or il-17f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il-17a and il-17f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg9vgd2 t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b1 and il-15 differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd2 tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp3) and, similarly to ab tregs, suppress the proliferation of anti-cd3/anti-cd28 stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd2 tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il-17 was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd11b in the liver following a-galcer treatment, numbers of cells lacking cd11b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma9/vdelta2 t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma9/vdelta2 t cells expansion upon stimulation with zoledronic acid (za) and interleukin-2 (il-2). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by 1) the bioinformatic analysis of gene expression profiling data 2) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in 43 patients (49 %) (responders, r), whereas 44 patients (51 %) were non-responders (nr). vgamma9/vdelta2 t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt2, whereas temra of r patients had an higher expression of the costimulatory molecule nkg2d. the proliferative response of vgamma9/vdelta2 t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, 82 % of r patients were m, whereas 77 % of um patients were nr (p x 0.001). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c57bl/6 mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il-12 p40 (clone r2-10f6; atcc) or anti-cd-1d (clone 1b1) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at 18 hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il-12p40, a component of both il-12 and il-23; pretreatment with anti-cd1d mab had no effect. il-12rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il-12 was not a critical factor. this suggestion is supported by the increased expression of il-23p19 and il-12p40 (but not il-12p35) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il-23 production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb1) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd27 expression. gd 27+ cells secrete interferon-g, while gd 27cells are capable of producing il-17. this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd27-defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine 123. cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to 40 % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚2% of gd 27+ cells but not at all in gd 27cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, 40-60 % of gd 27+ cells were positive for pgp activity, although their gd 27counterparts remained largely negative (p x 0.01). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd27. as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd 27+ and gd 27cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma9vdelta2 t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma9vdelta2 t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma9vdelta2 t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma9vdelta2 t cells from healthy donors were stimulated with different compounds (zol, ipp) for 24 hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma9vdelta2 t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma9vdelta2 t cells. moreover, mcp-2 depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma9vdelta2-mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma9vdelta2 tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma9vdelta2 t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg9vd2 t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg9vd2 t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg9vd2t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg9vd2 t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg9vd2 t cells and their effects on the viability of glioma cells, we expanded in vitro vg9vd2t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg9vd2t cell lines to kill three different glioma cell lines (t70, u251, u373) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg9vd2t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg9vd2 t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg9vd2 t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg9vd2 t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately 15 % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e7 protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd1d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e7-induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e7-expressing transplanted skin is dependent on interactions between populations of cd1d-expressing cd11c+/f480+ myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e7 graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il-2 and a-galcer. expression of cd1a, cd1d and the costimulatory molecules cd86 and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il-4) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd1d expression on the cell surface in comparison with control cells. in contrast cd1a expression was decreased. cd86 and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd1d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd1a, cd1b and cd1c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il-4 producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd1d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, 0.5-10 % of circulating lymphocytes express a vg9vd2 t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg9-enriched genes compared to conventional mhc-restricted cd8 + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf4 & crtam in gd and ab t cells respectively. because igsf4 binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd8 + ab t cells express crtam, and that engagement of igsf4 on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf4/crtam. we therefore sought to answer: 1. what is the function of igsf4/crtam on gd t cells? 2. how is the igsf4-crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf4 enrichment on resting gd t cells, with expression also detected on˚10 % of ab t cells. the properties of those cells are being examined. however, igsf4 generally correlates with markers of activation/antigen experience such as cd45ro. thus, igsf4 cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg9 + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf4 and crtam within 48 hours. however, engagement of igsf4 by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf4 may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg2d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf4 interactions are sufficient for cd8 t cells to kill gd t cells and/or vice versa. instead, crtam-igsf4 interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd3z is a 16 kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately 3-6 % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd2 tcr variable segment associated with the vg9 segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg9vd2 t cells without antigen presentation. in vitro stimulated vg9vd2 t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg9vd2 t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg9vd2 t cell anergy observed in hiv+ patients. to this aim, cd3z expression and ifn-g production by vg9vd2 t cells from hiv+ and hiv-subjects were analyzed. we show that vg9vd2 t cells from hiv-infected patients expressed lower level of cd3z compared with healthy donors. a direct correlation between cd3z expression and ifn-g production capability by vg9vd2 t cell was found. however, pkc activation by pma is able to restore cd3z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg9vd2 t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner 1 , m. swamy 1 , s.l. clarke 1 , a. hayday 1 1 king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd8aa or cd8 -cd4cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to 15 days. the cells are initially activated by plate-coated acd3 antibody and a cytokine cocktail and maintained further in medium containing low levels of il-2. after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg2d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il-6) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after 6 hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd94 in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg2 molecules in heterodimer with cd94, we screened the presence of these receptors on t cell subsets in bd. the expression of nkg2 a/c/d molecules on gd and cd8+ t cells were analyzed in 17 active and 9 inactive patients with bd and 21 healthy controls. expression of nkg2 molecules was evaluated on cd8+, gd t and cd56+ nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls (4.4 vs. 2.8 %, p=0.001). in addition to the increase of gd t cells, increased expression of activating nkg2c molecules was also observed on gd t cells (20 % vs. 11 %, p= 0.01). nkg2a expression on gd t cells was found to be higher than nkg2c expression in patients and controls; but nkg2a expression on the t cells was not statistically different in both groups (33.5 vs. 40 %). nkg2d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd8+ cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th1 and th2 cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg9/vd2+ lymphocytes. we have screened a panel of 26 lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg9/vd2+ cells, and selected two susceptible ("target") cell lines (over 60 % death in the assay) and two vg9/vd2+ resistant ("non-target") cell lines (under 20 % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs 697 (non-target), and validated the results by rt-qpcr quantification. results: we identified 37 commonly up-regulated and 50 commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp1, ifitm1 and prame, for example, are up-regulated, whereas cd274 and clec2d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg9/vd2+ target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd1d, in the rat. mice and rats have very similar cd1d and inkt tcr genes, with the exception of the va14 gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd1d revealed a very similar pattern of cd1d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il-4 production was 10-100 fold lower in the best responder rat strain (f344) compared to mouse (c57/bl6). since nkrp1a (rat homologue of mouse nk1.1) and tcr are not appropriate markers for rat inkt, cd1d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd1d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd1d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd1d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd1d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g9d2 t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g9d2 t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g9d2 t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g9d2 t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp-1. a significant (p x 0.01) reduction in intracellular bacterial numbers was observed (n=8) in the presence of pbmcs cultured with picostim+il-2 in comparison with pbmcs cultured with il-2 or media alone. picostim+il-2 caused significant expansion and activation of gd t cells following culture of pbmcs for 10-14 days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers 100-fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il-2, reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma9/vdelta2 (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than 5 % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: 1) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; 2) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); 3) to establish whether the same issues could be solved using a simplified protocol of dc generation. 1) dc were generated from cd14 + cells of healthy donors/mm patients; immaturedc on day 6 were induced to fully mature by incubation for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za. after 7 days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; 2) idc generated from cd14 + cells of hla-a*0201 + healthy donors/patients were pulsed with sv-peptide and stimulated for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za; after 2 rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd8 + t cells was determined by svpentamers staining; 3) the same experiments were performed both with dc generated following a standard protocol and a 48h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [6-3 h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd4 + cd25 + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin-2 on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp3 negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd1 subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg2d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd8+ have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n=8) and inactive (bdna) (n=20), versus healthy controls (hc) (n=30) and patients with recurrent oral ulcerations (ru) (n=14). to determine gd cytokine profile and surface markers treg-related in bd (n=9) and hc (n=11). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta1, vdelta2, cd8alpha, cd8beta, nkg2d, nkg2a and cd103. -intracellular expression of ctla-4, and foxp3. -intracellular expression of il-2, il-4, ifngamma, il-10 and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta2+ cells were significantly increased in ru. vdelta1+ and gdcd8+ lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg2d was slightly increased in gd from bda. -nkg2a expression by gdcd8+ was not different in bd versus hc. -most of gdcd8+ presented cd8alpha-alpha homodimers in bd and hc and were negative for cd103, foxp3 and ctla-4. gdcd8+ and gdcd8-subsets were (in bd and hc): -high ifngamma-producers without differences. -low il-2-producers: il2+ cells were lower in gdcd8+ than in gdcd8-. -low il-10-producers: il10+ cells were lower in gdcd8+ than in gdcd8-. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd8+ than in gdcd8--very low producers of il4 in most of cases. the hallmark in bd was the increase of gdcd8/vdelta1+. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg2a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd8+, except a lower percentage of il-2+ cells than in the gdcd8-subset. gdcd8+ from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd56 in humans. a subgroup, the invariant nkt cells (inkt), expresses the va24vb11 tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd3 + cd56 + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from 10 healthy umbilical cord blood samples and stimulated with ifn-g (50 ng/ml), anti-cd3 (25 ng/ml) and il-2 (500 ui/ml). these cells were cultured for 21 days and the expanded cd3 + cd56 + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd3 + c56 + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x 0,05 was considered significant. results: we could significantly expand cord blood cd3 + cd56 + nkt cells from 0,87±0,57 % to achieve an enrichment of 46,89±13,31 % (p=0,0002). table 1 shows the percentage (mean±sd,n=10) of phenotypic markers in cd3 + cd56 + cells at baseline (day 0) and after 21 days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb4 and vb8 families (p x 0,001). conclusion: our results show that cord blood-derived nkt cells are mainly cd8 + and cd94 + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb4 and vb8 families in these cells. l. marischen 1 , d. wesch 1 , p. rosenstiel 2 , a. till 2 , d. kabelitz 1 1 institute of immunology, kiel, germany, 2 institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod2. peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod2 gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp1 19 and pvama1 proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day 5 culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd69 + and cd25 + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day 3 after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd107a + (lysosomal associated membrane proteins: lamp-1) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th1 and th2 cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th1-like and th2-like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n=43) and healthy controls (n=13) were determined by flow cytometry using anti-cd3 and monoclonal antibody specific for the cdr3 loop of the invariant tcr a chain of inkt cells (clone:6b11). furthermore, after pma/ionomycin stimulation for 4 hours, intracellular ifng and il-4 cytokines were detected in cd4+cd8-, cd4-cd8-(dn), cd4-cd8+ and cd4+cd8+ subsets of inkt cells by five colour flow cytometry in patients with ad (n=10) and healthy controls (n=10). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x 0.01) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x 0.01). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r=0.726 and p x 0.001) and healthy controls (r=0.693 and p x 0.001). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il-4 level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x 0.05). the frequency, the number of inkt cells and the cytokine producing capacity of the cd4/cd8 inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il-4 that promotes the th2 differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting 37.5%, 16.5%, 12.1%, 7.9%, 7.9% and 7.5% of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells (1.8 %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd4 + nkt and cd4 -cd8 -nkt cells. nk1.1 + nkt cells may release large amounts of il-2, il-4, ifn-g and il-10 after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il-2 had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il-2. there was a significantly increase in the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd69 molecule existed in 68.95 % of the cells from large-scale selection. the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cell subsets in the giant lymphocytes were enhanced to 84.0 and 38.9 folds, respectively. under a light microscope at x400 magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of 5 fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than 1.0 and they are non-adherent cells. the differentiation pathway of the seb-activating cd8 + and tcrvb8 + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd8 + nkt cell and tcrvb8 + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl3 which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo-1am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h2begfp reporter mice. stimulation with anti-gd clone gl3 or anti-cd3 clone 2c11 elicited activation of gd t cells suggesting that tcr gd and cd3 molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl3 in vitro produced ccl4, ifng and tnfa. therefore, we were interested whether the ccl4 production of gd iiel influenced the homing of ccr5 cells such as lamina propria (lp) cd4 + foxp3 + cells (tregs). to test this, wt mice were i. p. treated with gl3 mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr5 + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr5 + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr5 expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il-4 production in g4 reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il-4 gene. in the absence of any manipulation gfp was expressed from the il-4 locus in populations of immature thymic nkt cells (predominantly cd4+cd44lotcrhi cells on a balb/c background, and cd4+cd44lonk1.1-on a c57bl/6 background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il-4 production was induced predominantly in cd4+ nkt cell subsets of the liver and spleen, and after i. n. administration, in cd4+ nkt cells of the airways. spontaneous and a-galcer-induced expression from the il-4 locus occurred in the absence of stat6 signalling, and did not require initial exposure to il-4 protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin-17 (il-17) plays an important role in neutrophil recruitment. herein, we investigated the role of il-17 receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c57bl/6 (wt) and il-17 receptor gene-deficient (il-17r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated 6 hours after surgery. the ability of il-17 mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il-17r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il-17 induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il-17r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il-17receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il-17 showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il-17 also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il-17 receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin-1 receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il-1r associated kinases to activate nfxb and p38 mitogen-activated protein kinase. the il-33-induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il-33 in different mast cell subsets. methods: different mast cells subsets (hmc-1, human cbmcs and murine bmmcs) were stimulated with il-33. the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk1/2, pakt, pnfxb, p38 and pjnk). additionally, we studied the signal transduction of il-33 in il-33r transfected hek293t cells. results: we found, that a tir family member, il-33r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il-33-induced cytokine production depends on c-kit transactivation. il-33r and il-1r accessory protein (il-1racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il-1r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il-33-induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il-33 in mast cells in absence of iger activation. (1) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase-3 activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease-1 (ho-1) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho-1 expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin 1 and 2. recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin 2 in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap1 small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap1 activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr2 shows a bimodal kinetic, with the first peak at 30''/1' and the second at 5' after stimulation. rna interference-mediated depletion of beta-arrestin 2 specifically inhibits the occurence of the second wave of rap1 activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin 2 is involved in rap1 activation and that the oscillations in the formation of rap1-gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c3g (rap1gef) and spa1 (rap1gap): preliminary results suggest that spa-1 has probably a role in the early activation peak. since this oscillatory chemokine-induced rap1 activation is present on other myeloid cell lines (hl60, 32d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin 2, localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh3 containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of 153 different sh3 domains that revealed over 20 putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)-3 and il-5 stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il-3 and il-5 receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il-3 , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn 328 are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: (1) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna-146 expression is induced by activation of the toll-like/interleukin-1 receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna-146a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna-146a transfected cells showed significantly reduced levels of the active proapoptotic caspases 8 and 3 (casp8/3). in line with this, mirna-146a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna-146a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna-146a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna-146a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il-15, il-7r a-chain and il-15r a -chain genes in hiv disease progression. methods: we studied 70 antiretroviral treated patients (progressors) and 71 long term non progressors (ltnp). we analyzed 2 snps in the il-15 gene, 5 snps in the il-7r gene and 3 snps in the il-15r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il-15r aa 182 and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il-7r and il-15r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd4 and cd8 t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art1, and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art1 in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art1 transcripts by rt-pcr analysis in human blood leukocytes. soluble art1, released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek293 cells with art1 and tnf resulted in modification of tnf at at least 2 distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r32, a site that has previously been implicated in binding to tnfr2. binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit225 than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art1 resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik 1 , t.v.poroshina 1 1 institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: 23 patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and 11 control men were assessed for slpi, tnf-alpha, il-8 and free tgf-b1 in ejaculate by elisas using stat-fax 303 plus. a 3 to 5 day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at -20 degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients (5127.571 ± 971.731 pg/ml, p x 0.001) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid (125.49 ± 17.9 pg/ml; p x 0.05). the il-8 concentration was elevated in all patients (366.7± 49.5 pg/ml; p x 0.05) in seminal fluid. free tgf-b1 was present in normal seminal plasma in high concentrations (346.4 ± 29.2 pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b1 concentrations were 36.2 ± 6.2 pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il-8 and free tgf-b1 are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi 1 , e. a. elgaaeid 1 1 faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th1 or th2 cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod2/card15 protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod2/card15 gene in cd. we performed a cases /controls study upon 75 cd patients and 90 healthy controls. this study suggests that in northen tunisian population, 3020insc mutation in nod2/card15 gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il-10 polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il-10 cytokine genetic profile in patients with ibd. we examined the contribution of il-10 gene promoter polymorphisms (-627 and -1117) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card15/nod2 gene mutations in cd presentation and location. in tunisian population, the 3020insc insertion in nod2/card15 gene is a marker of susceptibility to cd, while the a allele at position -627 in the il-10 promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il-10 gene polymorphisms and card15/nod2 gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il-2-dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il-2-driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il-2 molecule by ganglioside. but gangliosides apparently can also form complexes with il-2r; such complexes influence on the signal transduction through il-2r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il-2r subunits. in our work we use il-2-dependent cytotoxic t-cell murine line ctll-2. two different approaches for study of possible interaction types between exogenous gangliosides and il-2r subunits were applied: antibody staining of il-2r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with 125 i-dcp-gm1 followed by immunoprecipitation of il-2r subunits. the fluorescence intensity of the antibody-labeled il-2r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by 29.5 % after cells incubation with ganglioside gm1, and by 10.7 % after incubation with gm2. labeling of the cells with antibodies to the il-2r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm1 (2.8 %) or with gm2 (5.9 %). to determine the mode of this impressive masking influence of ganglioside gm1, photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm1 with subunits of il-2r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il-2r, but not for il-2r a-subunit. these results demonstrate that exogenous ganglioside gm1 can interact with a-and bsubunits of il-2r in different modes. interaction of il-2r b-subunit with ganglioside gm1 requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa 1 , j. bukur 1 , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak2 and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak2. results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak2 expression due to a gene deletion on chromosome 9, whereas the expression and functionality of other ifn-g signal transduction components like stat1 and jak1 were not affected. jak2 blockade by two jak2-specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak2 leading to a lack of jak2 expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak2 inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of 506 controls of the spanish population. methods: two il18 polymorphisms located on the il-18 promoter region, snps -607 a/c (rs1946518) and -137 g/c (rs187238) were genotyped using taqman snp genotyping assays. the functional il-18 gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il-18 -607 p=0,318. il18 -137 p=0,740) but may contribute to disease onset and aggressiveness: the genotype il-18 -607 cc genotype, which is associated with higher il-18 production, was significantly associated with higher tumor size (p=0,001), grade (p=0,030), t (p=0,001), m (p=0,012) and stage (p=0,002). the influence of the il-18 -103 gg genotype was less relevant, however was correlated with higher tumor size (p=0,036), grade (p=0,017), t (p=0,026) and stage (p=0,011). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il-18 polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il-18 gene (il-18 -607 and -137) may be associated with an worse prognosis of rcc. high levels of il-18 production can play an important role in grow, invasion and metastasis of renal cancer. interleukin-18 (il-18) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il-18 is its ability to induce the production of ifn-gamma in presence of il-12. moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l0. then it seems that il-18 has a crucial role in immunity against brucella infection. since the expression of il-18 can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il-18 gene polymorph isms and brucellosis. methods: a total of 188 patients with brucellosis and 77 healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il-18 polymorphisms at positions -656, -607, 137, +113, +127 and codon 35/3 using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il-18 polymorphisms at position -137 g/+113t/+127c (correlated with high production of il-18), codon 35/3c and -656g/-607c (correlated with higher production of il-18) were significantly higher in the healthy controls than the patients (p=0.000022, p=0.00185 and p=0.0441, respectively). discussion: as data revealed genotypes that have correlation with higher production of il-18 are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il-18 at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor 2 (ifngr2) mouse mutant. we have generated a mouse line carrying a conditional ifngr2 allele using the 2 loxp 2-flp recognition target (frt) approach. a targeting vector with 2 loxp sites flanking exons 4-6 and 2 frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat1 following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat1 activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd4 cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr2 on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez 1 , r. carretero 2 , p. saenz-lopez 2 , j. cantón 2 , j. carretero 2 , f. ruiz-cabello 2 , l.m. torres 1 , cts-143 1 hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, 2 hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and 130 normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca)12 allele of ifgn is significant more frequent in healthy control than in cin patient (p=0,030) in contrast homozygosis for (ca)12 allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p=0,030 and p=0,045 respectively). conclusions: our study suggest that ifng (ca)12 allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin-15 (il-15), a th1-related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il-15 single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: 190 patients with brucellosis and 78 healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il-15 (c267t, g367a, c13687a and a14035t) polymorph isms using pcr-rflp. results: at position 267, the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p=0.025 and p=0.042, respectively). at position 13687, the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p=0.050, p=0.015). discussion: as shown the frequency of cc genotype and c allele at position 267 were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position 13687 is lower in patients than the healthy controls and the frequency of c allele at position 13687 is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf-7 and skbr-3, as well as their ability to respond to several cytokines and to the g-1 gpr30 agonist. the mcf-7 and skbr-3 human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il-2ra, il-2rg, il-3/il-5/gm-csfr, il-4/il-13r, il-5ra, il-6ra, il-6r (gp130), il-7ra, il-10r, tnfr i, tnfr ii, ifngra, cxcr1, cxcr2, cxcr3, cxcr4, cxcr5. cells were incubated with il-10, ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr30 agonist. cytosolic ca 2+ concentrations [ca 2+ ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr-3 cells were found positive for a larger number of receptors than the mcf-7 cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf-7 cells with il-10 reduced the calcium response to g-1, while pretreatment with ifn-g and tnf-a potentiated the calcium response to g-1. tnf-b had no effect on mcf-7 g-1 stimulation. no direct effect on basal [ca 2+ ] i stimulation could be noticed when administering the cytokines alone. in skbr-3 cells, pretreatment with il-10 or tnf-b had no effect on basal [ca 2+ ] i and did not significantly alter the calcium response to g-1, while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g-1. pretreatment with tnf-a produced calcium oscillations and reduced the response to g-1. conclusions: mcf-7 and skbr-3 cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr30 stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd8a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd8a -pdcs and cdcs were infected with mcmv, whereas after 24h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd8 t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag1-deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag1 and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd8 t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg 1668, respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd8a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt3-l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f4/80 + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd8 t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr7 and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd11c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd8 t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd4 t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd4 t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd8 t cells in response to an adova infection due to an impaired cd4 t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than 500 passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il-12. to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp33 and vacv-gp33 expressing the gp33 epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p14 t cell receptor recognizing the gp33 epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p14ifnar -/mice. upon adoptive co-transfer of p14ifnar -/and p14ifnar wt/wt t cells and subsequent challenge with mva-gp33, a massive expansion of p14ifnar wt/wt t cells was observed, whereas p14ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp33 induced a rather similar expansion of ifnar competent and ifnar deficient p14 t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h5n1-specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h5n1 induced an exceptionally nf-kb dependent antiviral response. irf3 is essential part of this interferon-response of human endothelia. furthermore, we identified hmga1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h5n1. finally, nfatc4 was found to be a transcriptional regulator for specifically h5n1-induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h5n1 which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns1, delta-ns1 influenza virus (a recombinant influenza virus lacking the ns1 gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns1 during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting 2 days post-infection, reached its maximum at day 4, and triggered t cell priming in vivo. a direct comparison of delta-ns1 virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns1 protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns1 protein. thus, we propose that the virally encoded ns1 protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin 18 is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin 18 is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that 14-3-3 proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of 14-3-3 target proteins. thus, these results suggest that 14-3-3 proteins have a regulatory role in host defence against viral infections. i. wessels 1 , d. fleischer 1 , l. rink 1 , p. uciechowski 1 1 institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)-1b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il-1b expression is not elucidated, yet. it is known that the il-1b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il-1b when stimulated. b-cells which are il-1b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il-1b promoter and the impact of methylation on il-1b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl-60 cells were differentiated into monocytic cells after dihydroxyvitamine d 3 treatment. the monocytic phenotype was confirmed by flow cytometry. the il-1b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with 5-aza-2-deoxycytodine (aza) and changes in il-1b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl-60 cells, differentiated cells displayed upregulation of cd14 antigen and acquired the ability to express il-1b. by comparing the accessibilities of il-1b promoter we detected that the il-1b promoter was not accessible in undifferentiated hl-60 cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl-60 cells, demonstrating that the chromatin remodeling of the il-1b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il-1b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il-1b expression were found. our data indicate that the il-1b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il-1b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg 1 , g. wetzel 1 , h. arnold 1 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p65 family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il-1b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp2, 3, 4, 5 and 6 mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd88 was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. (2)). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd8 + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. 2008 aug 1;181 (3)). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. 2008 dec; 82 (24) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il-1b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory 4t1 breast cancer cells over expressing il-1b (4t1/il-1b) tumor cells, but rarely in untransfected 4t1 cells. secretion of il-1b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg2d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il-4ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used 130 healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array 5.0 having p x 10 -4 in two independent cohorts each consisting of 200 blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for 24h and we measured the levels of il-6, il-8, il-10, tnf-alfa and il1-ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed 150 snps with p x 1,0*10 -4 . to identify/replicate the association of cytokine production for these 150 we reanalysed these on a 200 cohort. a combined analysis revealed 10 snps with p x 9,1*10 -5 .these results are not genome wide significant. the 10 snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find 10 nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp60 have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp60 on adipocytes. for the first time we could show a hsp60-stimulated release of the proinflammatory cytokines il-6, cxcl1 and mcp-1 in a time-and concentration-dependent manner from murine 3t3-l1 adipocytes. analyses of hsp60-signalling in these adipocytes revealed that members of the mapk-family (erk1/2, p38) and the transcription factor nfkb are involved in hsp60-mediated induction of the mediators il-6, cxcl1 and mcp-1. binding-studies with fluorescence-labelled hsp60 demonstrated that the interaction of hsp60 with adipocytes exhibits basic features of a receptor-mediated binding. hsp60-binding to adipocytes was saturable and reached its maximum at 3.5 mm. binding was inhibitable only by the unlabelled ligand (52 %), but not by unrelated proteins, thereby proving the specificity of hsp60-binding. further analyses to characterize hsp60-receptor structures on adipocytes revealed the presence of toll-like receptor (tlr)4 on adipocytes. tlr4 has been found to be expressed on macrophages and to interact with hsp60, therefore suggesting tlr4 as a potential receptor candidate for hsp60 on adipocytes. in order to identify the responsible binding-epitope of hsp60 we investigated the effect of specific antibodies directed against different epitopes of the hsp60-molecule. incubation with antibodies directed against the n-terminus of hsp60 (aa1-200; 5-25 mg/ml) were capable of inhibiting the hsp60-binding to adipocytes (47-80 %) indicating that the n-terminal region of hsp60 is involved in receptor binding. our experiments demonstrate that hsp60 stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp60-mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf3. we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf3 knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with 3 % of fetal calf serum at 37°c and 5 % co2. bone marrow nonadherent cells were removed after 24 h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd10, cd29, cd73, cd21 and stro-1 and were negative for cd45. intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost 100 % of msc. interleukin-2, ifn-gamma and tnf (th1 cytokines) increased msc contractility, whereas il-10 (a th2 cytokine) decreased msc contractility. by immunofluorescence, we observed that il-2, ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il-10 decreased that incorporation. our results suggest that th1 and th2 cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca 2+ -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as 40 nm and 400 nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents (10 mm) at the 24-h culture interval reached the values of approximately 3 ng/ml and 2 ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of 2-6 h in rat pecs. the 24-h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p38 and erk1/2. it was not suppressed by the calcium chelating agents bapta-am and tmb-8. the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr 305/07/0061. pin1 is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin1 substrates and targeting sites. recent data show that pin1 interacts with apo-bec3g (a3g). the pin1/a3g interaction results in a reduced a3g expression and a diminished a3g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin1 gene (-842 g/c and -667 t/c) modulate pin1 expression; in particular, the -842 gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, 28; 69-74, 2007) . the -842 c/g and -667 t/c polymorphisms in the promoter of pin1 gene as well as pin1 protein levels were analyzed in 30 exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; 40 hiv-infected patients (hiv) and 40 healthy controls (hc). the genotype and allele distributions of the -842 snp was skewed in esn (genotype: p= 0.008; allele: p= 0.013). in particular esn showed a significantly lower frequency of the -842 gg genotype compared to hiv and hc (p=0.017 and p=0.019, respectively) and consequently a lower g allele frequency (p=0.026 and p=0.028, respectively). no significant differences were found for the -667 snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, 9-(r)[2-(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, 9-[2-(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. secretion of cytokines was determined after the 5-h culture by elisa. production of no was assayed at the interval of 24 h using griess reagent. approximately 300 compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. 9-[2-(phosphonomethoxy)ethlyl]-2,6-diaminopurine derivatives, c) 9-[2-hydroxy-3-(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) 9-heteroalkyl substituted 2-amino-6-guanidinopurines, and f) 2-amino-3-(purin-9-yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately 30 compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip-1a and cytokines tnf-a and il-10. although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n 6 -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as 2 to 5 mm. the remarkably enhanced secretion of chemokines was reached within 2-4 h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant 1m0508. host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and 10 million units of ifn-a treatment three times a week for 6 months was initiated. before and after treatment: percentages of the il-2 and ifn-g in cd4+ t cells were assessed to determine intracellular t helper cell 1 (th1) type cytokine expression. similarly, percentages of intracellular il-2 and ifn-g were detected to verify cytotoxic t cell 1 (tc1) type cytokine expression in cd8+ t cells. percentage of th2 and tc2 type cytokine expression, (il-4 and il-13) were determined in cd4+ and cd8+ t cells, respectively. six (50 %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th2 cells with respect to healthy controls before treatment. tc percentages, both tc1 and tc2, were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il-4 expression was higher and the percentages of th1 type cells were significantly low. il-13 expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency (29,8 %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects (6,6 % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt 1 , f. juengerkes 1 , b. schumak 1 , g. gielen 1 , j. kalff 2 , p. knolle 1 , b. holzmann 3 , a. limmer 1 1 university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, 2 university hospital bonn, department of surgery, bonn, germany, 3 department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il-12, the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh 1 1 rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il-17 and il-23 in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, 45 h. pylori-infected du patients (23 patients were positive for anti-caga antibody and 22 patients were negative for anti-caga antibody), 30 h. pylori-infected as carriers (15 subjects were positive for anti-caga antibody and 15 subjects were negative for anti-caga antibody) and 15 healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il-17 and il-23 was measured by elisa method. the mean serum levels of il-17 in total du patients (9.28 pg/ml ± 5.48) was significantly higher than those observed in total as subjects (5.19 pg/ml ± 3.75, p x 0.001) and healthy control group (3.55 pg/ml ± 3.76, p x 0.0001). in du group, it was found that the mean serum levels of il-17 in subjects with positive test for anti-caga (10.84 pg/ml ± 5.79) was significantly higher than those observed in subjects with negative test for anti-caga (7.65 pg/ml ± 4.74; p x 0.05). the mean serum levels of il-23 in du (8.66 pg/ml ± 8.41) and as groups (7.25 pg/ml ± 5.66) was significantly higher than those found in uninfected control group (3.64 pg/ml ± 3.36, p x 0.02 and p x 0.03, respectively). however, no significant difference was observed for mean serum levels of il-23 between du and as groups. moreover, in both du and as groups the mean serum levels of il-23 was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il-17 and il-23 in h. pylori-infected subjects as compared with control group. in du group the expression of il-17 influenced by the bacterial caga factor. a. aral 1,2 , a. atak 1 1 gazi university faculty of medicine, department of immunology, ankara, turkey, 2 gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il-6 levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il-6 elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il-6 levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the 48th hour and reached to maximum levels at the 1st week and decreased again at the 3rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the 1st week and started to decrease at the 3rd week. il-6 reached to its peak at the 3rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic 1 , m. jurisic 2 1 university of kragujevac, school of medicine, kragujevac, serbia, 2 university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in 43 radicular inflamed cysts and 15 odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd3, cd20 and cd68. tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x 0.05) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. 1) drawings blood from 20 patients and 20 healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. 2) detection of the gene expression of prolactin and tlr-2 in cd14+ peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p 0.05) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr-2 and peripheral prolactin expression in cd14+ monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u937 culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table-2 ). when tb household contacts and healthy controls were compared, cfp10 and esat6 seemed to be more useful than tst in tb contacts for displaying ltb (table-2) . although cfp10 spot numbers were much more than esat6 spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: 0,069)( table-3 ). both esat6 and cfp10 spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g 20 analytes immediately prior to liver transplantation and at sequential time points up to 90 days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip-10, il-6 and il-10. notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type 1 ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/1/68 (h3n2) (hk) differs from its putative avian precursor by 7 amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk-5aa-i62r, n81d, k92n, s193n, g144a, human (2-6); rhk-r2-l226q, s228g and rhk-7aa-i62r, n81d, k92n, s193n, g144a, l226q, s228g, avian (2-3)). among these variants, the double mutant rhk-r2 and the seven mutant (rhk-7aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l226q and s228g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about 50 pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip-10 and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r2 and rhk-7aa as compared to rhk and rhk-5aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il-8, shedded adhesion molecules (cd25, vcam-1, icam-1), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk-5aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of 98 newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il-10, and neutrophilic cd64 levels, procalcitonin and il-10 were measured by elisa technique while, neutrophilic cd64 by single colour flowcytometric technique. of these 49 "infected" infants, 16 had positive blood culture (subgroup ia: culture-positive sepsis), and 33 infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another 49 newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il-10, cd64, and crp. il-10 had the highest sensitivity and specificity, 92 % and 84 %, respectively, using cutoff n 17.3 pg/ml. for pct, the highest sensitivity and specificity, 65 % and 60 %, respectively, were at a cutoff value of n 36.4 pg/ml. neutrophilic cd64 had maximal sensitivity and specificity of 92 % and 71 %, respectively, at cutoff value of 2.6 %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il-10 and neutrophilic cd64, which together provided sensitivity and specificity of 95 % and 83 %, respectively, and npv 86 %. the combination of il-10 and crp had high sensitivity and moderate specificity, 93 % and 79 %, respectively. conclusions: il-10 and neutrophilic cd64 levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp3, naip5 or ipaf and the adaptor asc are involved in caspase-1 activation in response to bacterial infection, triggering the processing and secretion of il-1b and il-18. recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi3k, vps34 and can be inhibited with the pi3k inhibitors wortmannin and 3 methyladenine (3ma). in contrast, activation of akt, via class i pi3k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il-1b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with 3ma, wortmannin or an akt inhibitor. supernatants were analysed for il-1b by elisa. results: 3ma enhanced il-1b secretion by bmdc treated with the tlr3 and tlr4 ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il-1b secretion was greatly reduced in bmdc from nalp3 -/mice compared to wild type c57/bl6 controls. treatment with the akt inhibitor had no effect on lps-induced il-1b secretion by bmdc. tlr-dependent secretion of il-1a was also enhanced by treatment with 3ma. conclusions: these data demonstrate that il-1b secretion by bmdc in response to treatment with pi3k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp3 inflammasome. this response is limited to tlr3 and tlr4 agonists. inhibition of akt had no effect on lps-induced il-1b production, suggesting that the effect of wortmannin and 3ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi3k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr2 signaling by the inflammatory lipid mediator sphingosine 1-phosphate (s1p) through receptors 1 and 2 in human monocytes-macrophages, which could explain some of the s1p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s1p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s1p, and later analyzed by flow cytometry. a pharmacological analysis of the s1p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam-1 expression was increased following lps and s1p concomitant stimulation in both venous and arterial cells, suggesting that tlr4 and s1p receptors cooperate in the expression of icam-1. conversely, no cooperation was observed when tlr2 ligands were used. in order to elucidate which s1p receptor subtype was involved in the increase of icam-1 expression, we used a pharmacological approach with s1p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam-1 after lps and s1p challenge was significantly reduced by blocking s1p receptor 3 and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s1p receptors 2 and 3, which suggest that s1p receptor 1 might be involved in the effect. conclusions: altogether these data demonstrate that tlr4 and s1p receptors can interact to increase adhesion molecules such as icam-1 in human endothelial cells, and the s1p receptor subtype involved in the effect differs between arterial and venous cells. 15 with ssc without pah) and a pool of 12 sera of healthy controls (hc) were tested. results: in 1 dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with 7-10, 4-8 and 2-5 protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in 2 dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized 145±48, 127±26, 130±25 and 150 protein spots respectively. twenty one protein spots were recognized by more than 80 % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein 1 and peroxyredoxin 6 and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: 27 patients suffering from different diseases were enrolled in our study. 16 patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas 11 of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam-1 and vcam-1 were assesed by an enzyme immunoassay (elisa). results: 11 out of the 27 patients (40,7 %) had elevated levels of the intercellular adhesion molecule. 6 out of the 16 patients suffering from bone diseases (37,5%) had raised values (mean value 400 ng/ml) whereas 5 patients out of the 11 suffering from soft tissue diseases and diabetes (45,5 %) had raised values (mean value 735,5 ng/ml). reference value for icam-1 was 130-299 ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients (40 %) having increasd levels of icam-1. high icam-1 levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes (45,5 %) than in patients with bone diseases (37.5 %). mean values were found 735,5 ng/ml and 400 ng/ml accordingly. those findings verify the positive correlation between icam-1 and inflammatory diseases and tissue damage but not for vcam-1. colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: 16 cases of ulcerative colitis (urc), 16 adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, 33 tubular or tubulo-villous adenomas with low grade dysplasia, and 33 infiltrating adenocarcinomas. immunohistoobjectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd18 in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind 20 patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). 40 mg of pravastatina oral 2 hours they were administered before the procedure (group study, n=10) or placebo (group placebo, n=10), and control (group control, n=10). samples of outlying veined blood were extracted to the 24 hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd18 in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: 3 types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in 3 degrees: degree 0. without expression. degree 1. weak; degree 2. moderate; degree 3. intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree 0-1. the placebo group: mixed pattern, degree 1-2. group study (40 mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree 2-3. the percentage of cells that expressed cd 18 was greater in the group study (40 mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd18 answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam10 and adam17. we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam17 as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam17. these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam17 activity in the tissue. in the presence of the adam17 inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam17 as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam17-mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf-460/ vegf+405 is associated with rcc risk ( p= 0,017), metastases ( p=0,043), nuclear grade ( p=0,05), tumor stage ( p=0,029), and tumor size (p=0,04). on the other hand, the polymorphism vegf -2578 a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol 3,4,5-trisphosphate. therefore, pten is one of the main antagonists of the pi3-kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il-6 as well as il-8 levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il-6 as well as il-12 and il-23 mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th-17 t cells, we measured th-17 cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il-17 and a strong reduction of il-22 mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il-10 exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il-1ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il-10. based on our previous observation that support a direct role of il-10-activated stat3 in the enhancement of il-1ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il-1ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il-10. quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il-1ra promoter. crosslinked nuclear lysates were immunoprecipitated 30 and 60 min after il-10 addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il-1ra promoter. chip assays showed that the pol ii recruitment to the il-1ra promoter induced by lps is significantly increased by il-10, further strengthening the concept that the rapid enhancement of lpsinduced il-1ra gene expression by il-10 initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat3 immunoprecipitated dna showed statistically significant levels of stat3 binding to the il-1ra promoter only in cells stimulated with lps in the presence of il-10. surprisingly, anti-p65 and anti-p50 chip assays revealed enrichment of both p65 and p50 recruitment to the il-1ra promoter when il-10 was added to lps-stimulated cells, suggesting that il-10 enhances the recruitment of nf-kb to the il-1ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il-10-treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il-1ra promoter site is dependent on il-10-activated stat3, since it is greatly reduced when stat3 activation by il-10 is impaired. the molecular mechanism through which il-10-activated stat3 promotes the recruitment of nf-kb to the il-1ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il-15 acts as a specific negative transcriptional regulator of mouse mast cell protease-2 (mmcp-2). we examined the mechanisms underlying the repression of mmcp-2 gene expression. our data show that the "repressor" effects of il-15 on mmcp-2 promoter activity are still operating on the mmcp-2 591 bp long minimal promoter. moreover, il-15 deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy1. furthermore, chromatin immunoprecipitation revealed that il-15 promoted specific reciprocal recruitment of c/ebpb but not yy1 to the mmcp-2 promoter. finally, il-15 deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp-2. thus, we proposed that the expression of mmcp-2 and possibly other immunoregulatory genes may be regulated by il-15 through epigenetic modification and by balancing the content and binding of c/ ebpb and yy1 in mast cells. i. nagy 1 , k. filkor 1 , a. vörös 1 , l. kemény 2 , a. szász 1 1 bzaka, baygen, szeged, hungary, 2 university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir-203, mir-146a and mir-155, which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir-203 expression; in contrast, pgn re-stimulation had no further effect on mir-146a and mir-155 expression. next, we investigated the correlation between the expression of mir-203 and its two known direct targets: regulatory protein p63 and suppressor of cytokine signalling-3 (socs-3). although the gene-expression profile of neither p63 nor socs-3 changed, we found that the expression of mir-203 reversibly correlates with both p63 and socs-3 protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir-203 prior to pgn-treatment completely abolished both p63 and socs-3 down-regulation, revealing the involvement of mir-203 in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir-203 expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb1+/-had a lower incidence and burden of benign papillomas when compared to tgfb1+/+ animals. however, more scc developed in the tgfb1+/-mice. after acute and chronic promotion, tgfb1+/-skin showed a reduced proliferative response with no increase in epidermal tgfb1 or nuclear p-smad2 compared to tgfb1+/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb1 gene dosage. further, pharmacological inhibition of alk5 suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb1 are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb1+/-skin, tgfb1+/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb1+/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb1+/+ but not tgfb1+/-keratinocytes, indicating that tgfb1 switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb1 +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb1 acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of 132 patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp-1) and fibroblast (mrc-5) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of 132 ra patients and 82 controls the levels of survivin correlated to urokinase (upa) (r=0.46), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that 30/132 ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc-5 and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol 1,4,5-trisphosphate 3-kinase type b (itpkb) phosphorylates inositol (1, 4, 5) trisphosphate (ins(1,4,5)p 3 ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca 2+ responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h1 and h2 receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il-1b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il-1b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase-1 to generate the mature secreted active form. caspase-1 is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of 425 genes involved in splicing with an average 5-fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified 30 genes that significantly affect the production of il-1b by thp-1 cells after a 24h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il-1b secretion. tissue transglutaminase (tg2) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg2 in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg2 expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg2 can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg2 not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg2 inhibitor, in a transgenic mouse model cf and in the taz10 transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz10 tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg2 in generating inflammation in two very different pathologies. this work underlines the critical role of tg2 in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge 2 stimulated a-dc. preliminary results indicate no accumulation of hif-1alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif-1alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p38 but not erk1/2 or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt5a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il-12 and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt5a -but not wnt3a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt5a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt3a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp130 on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp130 in te. after infection with t. gondii, mice lacking neuronal gp130 (synapsin-cre gp130 fl/fl ) died significantly earlier in the chronic phase of infection than control gp130 fl/fl mice. death of synapsin-cre cre gp130 fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp130 fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp130-deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp130 expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp130 fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il-1b, il-6 and il-8 have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il-4, il-10 and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb1 further may generate more effective therapies. as tnfa mrna 3' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb1 suppresses tnfa protein production by upregulating the rnabinding protein fxr1, which can bind to tnfa mrna and inhibit translation. methods: using raw 264.7 cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb1, we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 3'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb1 and luciferase expression was quantified. cells treated with lps and tgfb1 were also examined for fxr1 expression using pcr and western blot. following fxr1 knockdown using sirna, the influence of tgfb1 and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb1. using the luciferase-tnf-3'utr vector we show that tgfb1 targets the 3'utr of tnfa. furthermore, tgfb1 and il-10 both upregulate fxr1 mrna and protein; and treatment with tgfb1 and lps can synergistically upregulate mrna expression, more than tgfb1 alone. following sirna inhibition of fxr1, tgfb1 can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp-1 and mmp-3 secretion from saecs, nhbes and fibroblasts to a peak of 2.5 +/-0.5 ng/ml, at 72 hours. interleukin-17 augmented comtb-stimulated up-regulation of mmp-1 and mmp-3 secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin-17 down-regulated mmp-9 secretion from saecs by 50 %. interleukin-22 up-regulated mmp-1 and mmp-3 secretion from fibroblasts but not from saecs. timp1 secretion from saecs was enhanced by interleukin-17 but there was no effect of interleukin-22. mmp up-regulation by interleukin-17 and comtb was inhibited by the pi3kinase inhibitor ly294002 and on western analysis akt (protein kinase b) was phosphorylated at 30 minutes. chemical inhibition of the p110d isoform of pi3kinase with ic87114 abrogated the il-17 and comtb driven secretion of mmp-3 from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome 10) accentuated mmp-3 secretion. these inhibitory effects were confirmed with sirna. mmp-3 up-regulation was secondary to increased gene expression with promoter activity peaking 24h after stimulation. in summary, interleukin-17 and interleukin-22 drive transcription dependent mmp-1 and mmp-3 secretion from airway epithelial cells and fibroblasts. interleukin-17 also increases timp but down-regulates mmp-9 gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi3kinase pathway is central in interleukin-17 driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto 1 , l.s. moreira 2 , e. gonzalez-rey 2 , m. delgado 1 1 institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, 2 university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within 15 years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper 1 response. cholesterol metabolism is regulated by factors such as pparg1 (proliferator activated receptor g), srb1 (a class b scavenger receptor), cd36 or abca1, that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th1 immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg1, srb1, cd36, and abca1. we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to 60 % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il-6 in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il-6 and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il-6, tnf-a, il-1b, il-8 and il-12, and the anti-inflammatory cytokine il-10, in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il-23 was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il-6 and il-10, and in some cases, of il-23. the maximal plasma levels of il-6 and il-10 were found at 24 hours and of il-23 between 48-72 hours. modest plasma levels of il-8 were also observed, with maximal production at various time points (4-24 hours). by contrast, production of tnf-a, il-1b and il-12 did not occur to a significant extent, while production of il-17 occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il-6, il-8, il-23 and il-10 also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il-6, il-10, il-8 and il-23, i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving 165 patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il-6, activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il-33 is a novel il-1 cytokine family member that is expressed as an intracellular precursor (pro-il-33) and is thought to be cleaved by caspase-1 to yield a mature bioactive form of the molecule (mat-il-33). to date however, evidence of cell-associated proteolytic processing and caspase-1 dependent secretion of mat-il-33 has not been reported. here we show that pro-il-33 but not mat-il-33 is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il-33 to the il-33r and also il-33r-dependent bioactivity of pro-il-33 on mast cells. we propose a previously unrecognized role for pro-il-33 as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il-23 p19 mrna expression in myeloid cells, and markedly increase secretion of il-23, but not il-12, by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il-23 promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop10. chromatin immunoprecipitation (chip) assays using anti-chop10 and isotype control mab were performed using nuclear lysates from u937 cells and il-23 promoter dna measured by qpcr. chop10 binding on the il-23 promoter was detected following stimulation of u937 cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il-23 promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop10 in il-23 gene transcription, u937 cells expressing shrna's specific for chop10 or non-specific gene target were tested for their ability to express il-23 following tlr and er stress stimulation. u937 expressing three independent shrna targets for chop10 exhibited significant reductions in il-23p19 mrna (up to 87 % reduction of the response to lps+tp) compared to u937 expressing a control shrna. chop10 shrna expression did not affect the expression of other lpsresponsive genes, including il-1, il-8, ccl3 and sod2. to identify if er stress induction of il-23 mediated by chop10 expression plays a role in a more physiological setting, we examined the role of chop10 in the induction of il-23p19 gene expression following chlamydia trachomatis (ct) infection. infection of u937 cells with live but not g-irradiated ct induced expression of er stress response genes, including chop10. u937 infected with live ct exhibited increased il-23p19 mrna expression compared to u937 infected with nonviable bacteria. chop10 silencing significantly reduced the ability of live ct to induce il-23p19mrna, confirming the important role of chop10 in this response. these data suggest that er stress induction of chop10 could contribute significantly to the pathogenesis of diseases in which il-23 plays an major role, through induction of il-17 and il-22 producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd45b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd45b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd45b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd45b maintained a sustained activation of p38 kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd45b-deficient mice showed only transient p38 activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd45b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd5 and cd69 were elevated on gadd45b-deficient thymocytes. thus, we provide evidence that gadd45b and a resulting persistent activation of p38 constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo 1 1 institut pasteur, paris, france, 2 monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il-7 is recognized as an essential factor for thymopoiesis, the nature of the thymic il-7 niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il-7 promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il7 transcripts (il-7 hi cells). il-7 hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl19, ccl25, cxcl12) and cytokines (il15) that are critical for normal thymopoiesis. in the adult thymus, il-7 hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il-7 hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il-7 levels. conversely, the frequency of il-7 hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il-7-expression by tecs. > together, our temporal-spatial analysis of il-7-expressing cells in the thymus suggests that thymic il-7 levels are dynamically regulated under distinct physiological conditions. this novel il-7 reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il-7 expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l5 and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl2-tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl2 and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl2-tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl2-tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg2 plasma cells and serum igg2 levels were˚5-10 fold increased. importantly, serum from young slc-tg;em-bcl2-tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in 2/3 and 6/6 cases, respectively. these values were increased when compared with control groups: 1/6 in fcgriib -/and 0/6 in bcl2-tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd27 + cd135 + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd117 hi cells, representing multipotent progenitors, or cd127 + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb2 and ephb3 expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b1 and/or ephrin b2, the ligands of ephb2 and ephb3 receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb1 or ephrinb2 genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb1/b2 double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd45+ cells in the cortex, increased proportion of k5+k8+ cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb1 and ephrinb2 in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink 1 , d. vanhecke 1 1 university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op9-dl1 cell culture system (1) . using this in vitro assay we obtain large numbers of human cycd3 + and cd4 + cd8 + double positive thymocytes starting from umbilical cord blood (ucb) derived cd34 + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il-7 and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd34 + cells upon notch signaling is cd7 followed by cd127. t cell specification is accompanied by the induction of cd1a and loss of cd34 on cd7 + cd127 + cells. these cd34 -cd1a + cd7 + cells become dependent on continuous il7 and notch signaling for sustained survival and further differentiation into cd4 + cd8ab + dp thymocytes. we found that flt3l is not essential for the differentiation of cd4 + cd8 + human thymocytes but that addition of exogenous flt3l in the co-cultures increases the number of cd34 + precursors and consequently result in higher yields of developing cd4 + cd8ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd3 + cd4 + cd8ab + dp subset in this in vitro assay suggesting that op9 stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn3 to dn4 stage, where bdnf and its receptor p75 are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga 1 , c. lópez-rodríguez 1 1 pompeu fabra university, barcelona, spain nfat5 is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat5 are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat5 participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat5 deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd8 + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat5-null mice are unable to mount cd4+-and cd8 + -immune responses. data from our laboratory indicate that nfat5-null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat5 during the development of t lymphocytes, we developed mouse models that delete nfat5 at early (lck-cre + /nfat5 flox/flox ) or late (cd4-cre + /nfat5 flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat5 is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat5 at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p53, is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues 58-88 of the proline-rich domain of p53. methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p53 null (p53-/-) and wild type (p53+/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p53-/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p53 has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p53 is not important for preventing thymic t-cell tumors. s. myrczek 1 , r. pardi 2 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, 2 vita-salute san raffaele university school of medicine, milano, italy jab1 is the catalytic subunit of the highly conserved cop9 signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab1 regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab1 regulates the activity of ap1 transciption factors. to date jab1 is thought to be essential for every cell type as jab1 knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab1. to investigate the function of jab1 in b cells we established a mouse strain deficient for jab1 selectively in b cells. mice with floxed alleles of jab1 kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb1-locus (m. reth, freiburg). ablation of jab1 expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b1 and b2 cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl2 under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab1-deficient, bcl2-transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab1 in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab1 when apoptosis is prevented. t. nitta 1 , s. murata 2 , k. tanaka 3 , y. takahama 1 1 university of tokushima, tokushima, japan, 2 university of tokyo, tokyo, japan, 3 rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd8 t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd8 t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd8 t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th1 and th2 antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd4+ t cells or functional deficits that selectively interfere with th1 or germinal centre responses. in this talk i will present data from some of the first 12 strains that have been identified including the first 5 strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn1 c-kit high (etp), dn2 and dn3 thymocyte populations was hybridised to affymetrix mouse 430a-2.0 genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors (85 out of 623 genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included 64 signal transducers (out of 590 genes) such as acvr1, bmpr1, fzd7, chemokine receptors cx3cr1, cxcr6 and integrins a2, a4, a9, ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata-3, tcf-1, notch-1, rag-1, rag-2 and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn2 stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn3 population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd8aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks 3 out of 4 of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e14 thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by 7-10 days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day 18 of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd34 + cd1ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged 1 month to 3 years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd34 + cd1precursors with recombinant wnt3a (100 ng/ml) or with licl (10 mm) for 12hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab (8e7) under conditions of phosphatase activity inhibition. wnt3a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il-15 and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st2 cells suplemented with il-7 and flt3l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt3a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt3a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il-7 and flt3l and modify the transcription factor profiles of cd34 + cd1thymocytes mainly increasing hes-1 and id3 expression levels. human th17 clones and circulating th17 cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th1 and th2 clones or circulating th1oriented t cells, respectively. accordingly, human th17 cells exhibited lower expression of clusterin, and higher bcl-2 expression and reduced apoptosis in the presence of tgf-beta, in comparison with th1 cells. umbilical cord blood naï ve cd161(+)cd4(+) t cells, which contain the precursors of human th17 cells, differentiated into il-17a-producing cells only in response to il-1beta plus il-23, even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd161(+) t cells but it increased the relative proportions of cd161(+) t cells differentiating into th17 cells in response to il-1beta plus il-23, whereas under the same conditions it inhibited both t-bet expression and th1 development. these data suggest that tgf-beta is not critical for the differentiation of human th17 cells, but indirectly favors their expansion because th17 cells are poorly susceptible to its suppressive effects. m. irla 1 , w. reith 1 1 university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd4(+) or cd8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd4(+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd40 on mtecs by cd40l induced on the positively selected cd4(+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd4(+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu 1 , i. ravens 1 , g. bernhardt 1 1 hannover medical school, institute of immunology, hannover, germany cd155 is originally identified as human poliovirus receptor (pvr) and as rodent tage4, which is overexpressed in rodent colon carcinoma. cd155 is also known as necl-5, a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd155 expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd155 is overexpressed in transformed cells and promotes the cell cycle. thus, cd155 seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd4 and cd8; the dn subset is further subdivided into four stages (dn1-4) by differential expression of cd44 and cd25. thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd4+ sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd8 lineage. here we show that the frequency of terminally matured cd8+ sp cells but not that of cd4+ sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd155, a selective deficiency of mature cd8+ sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd155 is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd8+ sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd8+ t cells. in cd155 deficient animals, a shift in the tcr repertoire displayed by the pool of cd8+ sp cells was found demonstrating that cd155 is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. 2005 , 202, 1599 . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht 33342, propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e9 (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd71 + ter119 + erythroid and cd45 + cd11b + myeloid cells are simultaneously cycling (s/g2/m) in the post-gastrulation mouse embryo (e10-12). the peak of lymphohematopoietic cell proliferation occurs at e13 in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence 24-48 hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e11-12 that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd40-cd154 interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd40, we hypothesised a role for cd40-cd154 interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd40 expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd154 message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd154-/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd154 on reconstitution of b cell numbers following depletion. we show that cd40 is expressed by pro-b cells, and these cells proliferate in response to cd40 signalling in vitro. pcr identified a source of cd154, negative for cd3eta, in the bm of wt mice showing this cd154 is not provided by activated re-circulating t cells. we have shown that when cd154 -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn1 (cd4 -/cd8 -/cd44 + /cd25 -) to the dp (cd4 + /cd8 + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn1 stage on. in the bone marrow we found yfp + /b220 + and yfp + /b220populations. thus these pta expression analyses show closely similar pattern to those observed with hucd25 preta-reporter transgenic mice (gounari f. et al. 2002 , martin et al. 2003 . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. 3, 489-496 (2002 ) martin c. h. et al., nat. immunol. 4, 866-873 (2003 . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz 1 , g. klein 1 1 zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm-332 which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x 70kda.mmp-19, a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm-332. in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp-19. by western blotting the zymogen and the activated form of mmp-19 can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm-332 and mmp-19 can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp-19 which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp-19, timp-2 and timp-3, are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp-19 plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of 15-20 % compared to only 5 % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd4/cd8 expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd4+cd8+ double positive (dp) tcrab low cells entering selection, and their cd4+cd8+ dp tcrab-immediate precedents followed by underrepresentation of the selected cd4+cd8+ dp tcrab high and the most mature cd4-cd8+ and, particularly, cd4+cd8-single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd4-cd8-double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd4+cd8-/cd4-cd8+ sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a 1 -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a 1 -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a 1 -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd4/ cd8/tcrab), in adult wistar rats subjected to 14-day-long treatment with a 1 -ar blocker urapidil (0.20mg/kg body weight/day s. c.). the a 1 -ar immunoreactivity was found in both thymocytes (mainly less mature cd3and cd3 low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed1-postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd4/ cd8/tcrab expression were observed. these changes comprised of an increase in the percentage of cd4+8+ tcrabthymocytes, which was accompanied by the reduction in that of cd4+8+ tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd4+8-tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd4-8+ tcrab high was reduced. in addition, the percentage of cd4+ t regulatory and cd161+tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a 1 -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a 1 -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around 4 weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c57bl/6 mice infected with m. avium (10 6 cfus, iv) were sacrificed at different time points after infection (5, 16 and 25 weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il-10) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at 16 weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at 24 weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il-10 in this organ. in the spleen, ifn-g reaches a peak of expression earlier (5 weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm29935 and ai067906 from nih and grant i-823 from the robert a. welch foundation to wtg, and by grants hl067256 and hl61897 from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. 2001) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c57bl/6 x balb/c)f1 mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f1 mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski 1 , s. lang 1 , m. stein 1 , t. winkler 1 1 friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h3ac) or methylation of h3 on lysine 4 (h3k4me2/3). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h3ac and h3k4me2/3) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar #3) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 5'rlm race at the ivar #3 element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo2 locus, is thought to be accessible only during the double-negative (dn) 1 and 2 thymocyte stages based on mrna expression, implying that translocations between lmo2 and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx1 (hox11), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed 1) to evaluate lmo2 and tlx1 breakpoint-site accessibility during thymocyte development; 2) to determine in which stage of development there is an increased chance for lmo2 or tlx1 translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo2 accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo2 and tlx1 loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn1 and dn2 development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn1, dn2, dn3 and immature single positive (isp) stages for both lmo2 and tlx1. conclusion: our findings show that both the lmo2 and tlx1 loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn1 and dn2 stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that 1 to 3 successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e10-12), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag2 -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under 6 months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd4 cell depletion. however, cd8 depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd8 t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd8 cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd8 t cells has important implications for vaccine development against neonatal infections. (2008) showed that irf8 knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf8 as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf8 in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf8 were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs17444416. conclusions: although recent findings indicated that irf8 function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf8 that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez 1 , t. boehm 1 1 max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn1 transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l1 expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd4 and cd8 t cells from tuberculosis patients highly express pd-1 when compared to healthy uninfected individuals. in addition, analysis of pd-1 expression in lung biopsies from tuberculosis patients revealed that pd-1 is expressed on cd4 and cd8 t cells confined to lung granulomatous lesions. finally, blocking of the pd-1/pd-l1 axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd-1/pd-l1 pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p100 to p52. among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt 1 aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly 1 wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th17 and th1 cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd4 + t cells, nfatc1 and c2 are predominantly expressed. nfatc1 is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc1/a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc1/c. as demonstrated by y2h screen and co-ips, nfatc1/c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc1/c -but not the unsumoylated nfatc1/a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc1 target gene interleukin-2. other lymphokines like ifng and il13 are reversely regulated. interestingly, ntreg cells which do not express il2 exerted only nfatc1/c, but no nfatc1/a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc1 from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc1 function. therefore, especially ntreg cells and anergized cd4 + t cells might be regulated by the long sumoylatable isoform nfatc1/c. lnk/sh2b3 and aps/sh2b2, two members of the lnk/sh2b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b-1 cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il-7 signalling in pre-b cells overexpression of lnk dramatically inhibits il-7-dependent growth demostrating that lnk negatively regulates il-7 pathways. furthermore, we showed that il-7 stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e3 ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav1. to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd3 complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd3 complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd4 + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh2 or sh3 domains known to mediate protein-protein interactions are key players in these processes. sly2 (sh3 domain protein expressed in lymphocytes 2) was identified as a putative adaptor protein containing a sh3 and a sam domain as well as a bipartite nls. sly2 belongs to a family of three molecules: sly1, sly2 and sash1.in humans, the sly2 gene is located on chromosome 21, in mice on chromosome 16. sly2 is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly2 protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly2 we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin3-associated polypeptide p30 (sap30) as a putative interaction partner of sly2. sap30 is a conserved member of the sin3a-hdac corepressor complex that contains histone deacetylase 1 (hdac1) and histone deacetylase 2 (hdac2) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected 293t cells. in addition, we could show a direct interaction between sly2 and hdac1. to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly2 increases the activity of hdac1 in whole cell lysates and, more precisely, in nuclear extracts of 293t cells. the interaction of sly2 with sap30 and hdac1 indicates a transcriptional function of this protein. within the sin3a-hdac corepressor complex sly2 might act as a switch for the activity of hdac1. cd46-cyt1 and cyt2 are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd19 (b cell marker) fused to the transmembrane and intracellular domain of cd46-cyt1 or cyt2 in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt1 coengagement controlled il-10 secretion, while cyt2 coligation inhibited ifng production. moreover, our preliminary data suggest that cd46-cyt2 inhibits the phosphorylation of several molecules known to be activated by cd46 stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd46 cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh2 domain and three sh3 domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut78 cells with gst fusion proteins containing full length nck, the three sh3 domains or the individual sh3 and sh2 domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp76 and cd3epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam68) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip55 once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g4, results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g4) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g4) degranulation as measured by cd107a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi3k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose-6-phosphate receptor which exhibits structural and functional similarity to the vps10p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose-6-phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla-4 (sctla-4) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla-4 in immune responses. using a specific anti-human soluble ctla-4 monoclonal antibody, jmw-3b3 that selectively binds the soluble isoform but not membrane bound ctla-4, or cleaved fragments of it, we demonstrate that sctla-4 plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla-4, secreting increased amounts of cytokines including interferon-g, il-17 and tnf-a, but lower amounts of il-10. soluble ctla-4 was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla-4 induced secretion of the immunoregulatory cytokine il-10 by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine 2,3 dioxygenase enzyme cascade was also initiated by sctla-4. it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla-4 it is crucial to t cell inhibition. membrane-bound ctla-4 exists as a homo-dimer on t cells but sctla-4 is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b7 ligands on antigen presenting cells. a third important observation from this study is that sctla-4 exists both in serum and culture supernatants as a natural 46kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla-4, concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il-10 dependent regulation is most critical, boosting sctla-4 secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin-1/efhd-2, in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin-1/efhd2 is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin-1/efhd-2 in the immature murine b cell line wehi231 enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin-1/efhd-2 impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g1 cell cycle arrest. to understand how swiprosin-1/efhd2 enhances pro-apoptotic bcr signals, we analyzed whether swiprosin-1/efhd2 is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin-1/efhd2 enhanced bcr-induced calcium flux in wehi231 cells, whereas shrna-mediated down-regulation of swiprosin-1/ efhd2 impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin-1/efhd2 interacts with phospholipase cg2 (plcg2) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg2 and swiprosin-1/efhd2 was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin-1/efhd2 silenced wehi231 cells with swiprosin-1/efhd-2 was inhibited by the syk inhibitor bay 61-3606. in analogy, swiprosin-1/efhd2 regulated syk activity positively. moreover, swiprosin-1/efhd2 re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg2 and of syk tyrosine residue 352, which is involved in syk activation. finally, reconstitution of swiprosin-1/efhd2 knock-down cells with swiprosin-1/efhd2 mutants revealed that the n-terminal putative sh3-binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi231 cells. interestingly, swiprosin-1/efhd2 re-expression in swiprosin-1/efhd2-silenced cells induced already in unstimulated cells raft partitioning of syk, plcg2 and the bcr, which was reversed after 2 min of bcr stimulation. in summary, swiprosin-1/efhd2 is an accelerator of proximal bcr signalling and acts through syk and plcg2 by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e3 ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc1 tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd28 co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p14 mice die within hours after a second challenge with p33 peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e3 ligase dead mice can spontaneously reject tc1 tumors. conclusion: cbl-b e3 ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e3 ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e3 ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd8 t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd8 + cd45ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip10 and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal 1) and co-stimulatory signals (signal 2), provided by beads coated with anti-cd3/cd28 antibodies. gene expression patterns were compared for cells stimulated with anti-cd3/cd28 beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip10, itac). the enhanced expression of granzyme-b, ifn-g, trail and ip10 were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd3-coated p815 cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal-3 to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd3 + foxp3 + and cd4 + cd25 high t cells. moreover, t cell receptor activation with combined anti-cd3 and anti-cd28 stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk-506, but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv-1 infection leads to immune dysfunction owing to a successive loss of the cd4 + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv-1 virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd4 and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk1/2. this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk1/2 directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap-1, nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk1/2 is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal 1 , t. brdicka 1 , v. horejsi 1 1 institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd45 are key regulators of src-family kinases in leukocytes. while cd45 is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh2 domain of csk binds phosphotyrosine 317 of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd25 and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd69 upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd25-csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd8 t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd8 t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie-2kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd8 t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd8 t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd8 t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd8 t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa-1 integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git2 in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi 1 , s. parusso 1 , b. frossi 1 , g. gri 1 , c. pucillo 1 1 university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il-6 -/-mcs and anti-il-6 receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il-6 derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd40 on naï ve b cells and the interaction of cd40 on b cell surface and cd40l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd40l e cd40 on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves 1 , n. bercovici 1 , a. caignard 1 1 inserm u567, paris, france inhibitory killer ig-like receptors (kir2dl1-2/3) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd8 + t cell subsets. these receptors suppress cd8+ t cell activation through recruitment of the src homology 2 domain-containing protein tyrosine phosphatase 1 (shp-1). to further analyse the yet largely unclear role of inhibitory kir receptors on cd4+t cells, kir2dl1 transfectants were obtained from a cd4 + t cell line and primary cells. the transfection of cd4 + t cells with kir2dl1 dramatically increased the t cell receptor (tcr)-induced production of il-2 independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir2dl1-itim phosphorylation, shp-2 recruitment, zap-70 and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp-2 and p-pkc-v but not of shp-1. in contrast, the kir2dl1/hla-cw4 interaction led to a strong synaptic accumulation of kir2dl1 and the recruitment of shp-1/2, inhibiting tcr-induced il-2 production. kir2dl1 may induce two opposite signaling outputs in cd4 + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir2dl1 receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg1 is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg1 still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg1 antibody during these conditions. the observed igg1 switching behaviour mimics that of b cells responding to lps and il-4, but is mediated by a different, stat6-independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana 1 , c. schwindling 1 , m. pasche 2 , c. junker 1 , c. kummerow 1 , u. becherer 2 , e.c. schwarz 1 , j. rettig 2 , m. hoth 1 1 saarland university, biophysics, homburg, germany, 2 saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai1 channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than 150 nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd4 + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd4 + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of 21 days. under tolerogenic conditions (ova alone), cd4 + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, 4-1bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd4 + t cell expansion and contraction kinetics in the early phase of the t cell response (days 1-6). in the late phase of the primary response (days 7-21), under immunizing conditions, the large majority of transgenic cd4 + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il-2, ifn-g, and il-17a, and only few ova-specific foxp3 + regulatory t cells ( x 10 %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd4 + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells (30 %) was substantially increased. on day 14, both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells (50 %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd4 + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il-17a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd8 t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd8 tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin-2, interferon-g and macrophage inflammatory protein-1 by cd8 tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd8 tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd8 tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog 35-55 protein. our results provide direct evidence that mc contribute to cd8-specific priming in eae and show that the tc proliferation failure is specific for cd8 tc from mog 35-55 -immunized w/w sh mice. the role of mc-cd8 tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd8 tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd8 tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd8 tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd28 engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd28 costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd3complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla-4, but not via any conventional motifs in this region. overexpression of trim augments ctla-4 surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla-4 localisation, mainly restricted to the tgn. ctla-4 vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla-4 trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla-4 expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla-4 transport to the cell surface. it is imperative to reveal the mechanisms by which ctla-4 is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd1 molecules, nkt cells are activated and release cytokines, including ifn-g, il-2 and il-4. nkt cells are efficiently recruited to the liver via cxcr6-dependent chemotaxis toward cxcl16 and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd8 t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd8 t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd8 t cell tolerisation via interaction with lsec. to this end we analysed cd1d expression on lsec and their ability to activate nkt cells by presentation of the cd1d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd1d, as agalcerpresenting-lsec were capable to induce tnf-a, il-2, il-4 and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd54 and b7-h1 on lsec. as naï ve cd8 t cell tolerisation by lsec critically depends on b7-h1, we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb2 in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg9, jg1.2, and vd2 gene products. they recognize nonpeptide antigens like (e)-4hydroxy-3-methylbut-2-enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd-1. one of the inhibitory receptors, pd-1, is a member of cd28/ctla-4 family and contains a single ig v-like domain in its extracellular region. pd-1 can bind to two b7 homologue molecules, pd-l1 and pd-l2. it has been reported that interaction of pd-1 with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd-1 is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with 2-methyl-3-butenyl-1-pyrophosphate plus il-2 to obtain gd t cells. pd-l1+ and pd-l1-human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l1 mabs, the pd-l1 extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd-1 in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd-1/pd-l1 interaction. results: gd t cells expressed pd-1 upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l1. we first examined whether or not the engagement of pd-1 receptor could modulate the cytotoxic activity of gd t cells. pd-l1-expressing tumor cells tempered cytotoxic activity of pd-1+ gd t cells, and cytokine production such as tnf-a was down-regulated by pd-1 engagement. in addition, inclusion of anti-pd-l1 mab reversed cytotoxic activity and cytokine production when pd-l1-expressing tumor cells were challenged by pd-1-expressing gd t cells. conclusion: pd-1 delivers inhibitory signals in gd t cells upon engagement with pd-l1. peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il-2 production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e3 ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e3 ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd4 + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova 323-339 peptide)-specific t cells from do11.10 tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e3 ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e3 ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler 1 , p. aichele 1 1 immh, university freiburg, immunology, freiburg, germany interleukin 12 (il-12) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il-12 has a direct influence on cd 8 + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal 1) and costimulation (signal 2). we analysed direct il-12 signaling to cd8 t -12 signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd8 t cells lacking il-12 signaling failed to up-regulate klrg1 and to down-regulate cd127 in the context of listeria but not viral infections. thus direct il-12 signaling to cd8 t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd8 + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd8 + t cells are tightly controlled in their effector functions by cd152 (ctla-4). we demonstrate that signals induced by cd152 reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd8 + t cells. for this novel function cd152 specifically represses the transcription factor eomes, but not t-bet. a cd152 mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd152-mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd8 + t cells differentiated in the absence of cd152 signaling could be demonstrated in vivo. the novel insights that cd152-mediated signal transduction in vivo indeed alters cd8 + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd8 + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p2 receptors. p2x 1-7 receptors open to non-selective ion channels, whereas p2y1, 2, 4, 6, 11-14 are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p2 receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr9 but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p2x7 antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca(2+)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek 1 , a. brouckova 1 , d. filipp 1 1 institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih3t3 cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y505flck. comparative 2-d gel analyses followed by ms/maldi identified rack1 as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih3t3 cells of ha-tagged rack1 with either a wild type lck or constitutively active y505flck revealed a significantly enhanced complex formation between y505flck and rack1 compared to that of wtlck. ectopic expression of y505flck with its domain-inactivating mutations showed that lck-rack1 interaction depends on functional sh2, sh3 and the c-terminal tail sequence of lck. lck-rack1 interaction is readily detectable also in primary cd4 + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack1 with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack1 co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack1 in the context of lck translocation to lr is further strengthen by the observation that rack1 is associated with elements of cytoskeleton. these results are the first to characterize rack1 as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin 21 (il-21) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd19+cd20+cd27-cd38-igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th2 response to alumova inducing large early germinal centres and massive plasma cell formation with more than 75 % of these switching to igg1. the plasma cells up-regulate cxcr4, but not cxcr3, a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th1-associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚5%), igg2a (˚30 %), igg2b (˚30 %) or igg1 (˚30 %). in addition to cxcr4, some 70 % of these plasma cells strongly express cxcr3. the induction of cxcr3 in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg2a switching during th1 responses. this is functionally significant for oti-dependent cxcr3 expression, as well as induction of switching to igg2a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg2b. t-bet is known to be induced in b cells exposed to ifng or tlr9 stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd88. objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd272) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd4+ t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd4+ t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla-4 pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa-1 and cd2 in the psmac of untransformed human t-cells. in marked contrast, tcr/cd3 accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl-1 regulates interclonal t cell competition during acute and chronic immune activation. we found p53-independent noxa gene induction and mcl-1 downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl-1, which was delayed in noxa -/cells. using ot-1 cells and altered peptide ligands we observed that the level of mcl-1 downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl-1 -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np366) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl-1 axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen 9 (ebag9) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag9 confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag9, which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g2-adaptin in t cells. both interactions suggested an involvement of ebag9 in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag9-/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag9-deficient ctls. these data imply a role for ebag9 in regulating the formation of mature ctl granules and identify ebag9 as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag9 defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag9-related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd8 t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd80/86 and il-12. in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd3/acd28 or pma/ionomycin could not significantly activate cd4 and cd8 t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca 2+ influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena 1 , s. carrasco 1 , i. merida 1 1 centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp1-ras-erk signal intensity is critical to determine the final cell outcome. rasgrp1 is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. 1. analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. 2. develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd8 cells expressing gfp-c1 domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c1 domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h4, cd278), a cd28-like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th2 biased responses and high production of the anti-inflammatory cytokine il-10, and was essential to the development of germinal centres. however, icos can also help in the il-21-dependent differentiation of inflammatory th17 cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd28 a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p85 subunit of class ia pi-3 kinases. these can complex with one of the three 110 kda catalytic subunits (p110a, p110b, and p110d) expressed by leukocytes that generate pip 3 affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi3k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi3 kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi3-k regulatory (p85a, p85b, p53a) and catalytic (p110a, p110b, and p110d) pi3-kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p85a g p53a g g p85b and p110a g p110d g g p110b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi3-kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd3-induced secretion of il-10 and il-4. during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap450, a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap450 construct that acts as a dominant negative, or sirna knockdown of endogenous akap450 expression in t cells prevents the correct organization of cd3z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa-1 localization was also analyzed to assess p-smac architecture and, interestingly, confocal 3d reconstruction revealed that lfa-1 ring was not clear in the akap450-disrupted cells. moreover, akap450 was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap450 decrease the phosphorylation of molecules such as lat, plcg1 and pkcv. these defective activation events as reflected in a reduction of il-2 production. together, our results underscore a key role for akap450 in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin85/cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh3 domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca2+ mobilization and cell activation involving the pi3k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc1d10c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr 1) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as 2) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. 3) we then studied the phenotype of carabin kd (shrna expressing) a20 b cells after bcr stimulation. 1) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t1 tot2 b cells and to follicular mature b cells. 2) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. 3) bcr simulation, but not lps stimulation, of carabin kd a20 b cells shows an acceleration of ras target erk1/2 phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk1/2 pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor 3 (tlr3) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd4 + t cells express tlr3 and respond to the well characterized synthetic tlr3 ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd4 + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr3. tlr3 stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd4 + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor 3 (irf3) as revealed by realtime-rcr analysis of ifn b and irf7, whose transcription depends on the activity of irf3. combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr3 signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd4 + t cells. this study was supported by dfg spp 1110 "innate immunity" (ka 502/8-3). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd3e cytoplasmic domain (cd3e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd3e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd3e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd3e cd and a fluorescent membrane dye (r18). with this assay, we show that the cd3e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd3e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd3e cd to lipid bicells. membrane binding by the cd3e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia 1 , y. ge 1 , u. quitsch 1 , p. beckhove 1 1 german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd4+ t cell help is believed to contribute to optimal cd8+ memory expansion via cd40l on cd4+ t cells binding cd40 on dendritic cells. however, a few reports suggest that cd40l-cd40 engagement may mediate direct cell-cell contacts between cd4+ and cd8+ t cells. in this study, we investigated the importance of cd4-cd8 co-operation and cd40l-cd40 interactions for t em proliferation. methods: we isolated human cd4+ and cd8+ t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd4+ and cd8+ populations were activated in vitro using anti-cd3/cd28 beads. proliferation was measured by [ 3 h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd40 or cd40l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd4+ or cd8+ t em cells, demonstrating that optimal t em expansion requires direct cd4-cd8 interactions. surprisingly, not only cd8+ but also cd4+ t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd4+ t em proliferation depends on signals from cd8+ t em cells. activation induced the expression of cd40 on both populations and cd40l on subsets of cd4+ and cd8+ t cells. blocking of cd40l on cd8+ t em cells impaired significantly cd4+ t em proliferation, which confirms that the improved expansive potential of cd4+ t em cells in mixed populations depends on cd40l co-stimulation by the cd8 t em subset. conclusions: our data demonstrate for the first time that activated cd8+ t em cells deliver help to the cd4+ t em subset via cd40l-cd40 signalling and may play an important role for cd4+ t em expansion upon stimulation. the t cell surface glycoprotein cd5, a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd5 localization upon t cell:apc conjugation. we have questioned which domains of cd5 mediate the localization within the is, and for this we have expressed cd5 mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd5 mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd5 by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd5 depends on sequences within the cytoplasmic domain, as a cd5 deletion mutant lacking most of the cytoplasmic tail, cd5.k384 stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd5 translocation was mapped within amino acids glu 418 and his 449 since the cd5.h449 stop mutant, just short of 22 aa is still able to translocate to the is, whereas cd5.e418 stop , that lacks 2 important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd5 translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu 418 -his 449 ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna-2 is missing, but lmp-1 is still expressed. using a hl derived cell line, we have shown that the cytokine il-4 can induce lmp-1 expression in vitro and can replace ebna-2. we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is 2, 3 or 4. a high affinity stat6 binding site is spaced by 4 nucleotides. we found three potential stat binding sites in the lmp-1 promoter, which we named lrs, tr and edl1. they were spaced by 4, 3 and 2 nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il-4-treated or non-treated kmh2-ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat6 binding site, or lrs-stat6. a stat6 complex binding to the gl-epsilon promoter and lrs-stat6 was induced by il-4. the specificity of the stat6 complex was shown by supershift experiments with anti-stat6, but not anti-stat5 antibodies. when gl-epsilon or lrs-stat6 was used as cold competitors in a 100-fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat6 contains a functional stat6 binding site. oligonucleotides, corresponding to lrs in which the stat6 site had been mutated, could not compete for stat6 binding. interestingly, the unlabeled lrs-tr with 3 nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by 2 nucleotides (lrs-edl1), it could not compete. thus, expression the transforming protein lmp-1 can be induced directly by the t cell derived cytokine il-4 in a stat6 dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp-1 and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd8 t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji 2007 (ji . 179:2250 . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd4 t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd4+ t cells primarily affect the proportion of cd4+ t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd40, cd80 and cd86 on parameters of cd4+ t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd40, a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd40l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd40 leads to an enhancement of ig and cytokine production. the current dogma postulates that these 2 signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd40l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd40l (rmcd40l) can increase proliferation induced by tlr3 (poly ic) and tlr4 (lps) agonists. by contrast, we never observed any synergy between rmcd40l and tlr1/2 (pam3csk4) or tlr2/6 (pam2csk4) agonists. to go further in the study of cd40l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd40l, named mini-cd40ls, based on a c 3 -symmetry core holding cd40-binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd40ls bind to immobilized human cd40 and compete with the binding of cd40l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd40l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd40l, mini-cd40ls synergize tlr4 (lps), tlr3 (poly ic) and tlr7/8 (r848) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd40ls and tlr 1/2 (pam3csk4), tlr 2/6 (pam2csk4) and tlr9 (odn 2395) agonists. synergy between cd40l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd40l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd8 + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd8 + t cell tolerance. we have defined a phenotypic profile for cd8 + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd25 and cd69, and high levels of expression of cd62l and ly6c. whereas, cd8 + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd25 and cd69, and loss of cd62l and ly6c expression. ly6c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly6c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly6c, expressed on naïve cd8 + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd8 + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for 3 weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to 60 days after infection. results: daily injection of paraoxon induced g 50 % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining 2 weeks of treatment. mice exposed to paraoxon exhibited g 80 % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with 80 % of mice surviving the infection compared to 20 % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th1 and th17 cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide 3-kinases (pi3k) constitute a family of enzymes that generate 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi3k are divided into two types: class ia p85/p110 heterodimers, which are activated by tyr kinases, and the class ib p110g (p110gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p110g deletion affects tcr-induced t cell stimulation. mice lacking p110g show a partial defect in t cell differentiation, activation and survival. p110g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd4+/cd8+ t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi3kg-specific inhibitor causes a reduction in the number of cd4+ memory t cells that mediate renal injury. similarly, pi3kg deletion in p65 pi3k transgenic mice also reduces the numbers of cd4+ memory t cells. there is therefore evidence that pi3kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi3kg regulates this process is not well understood. we studied the specific role of p110g in t cell activation. methods: we studied whether the tcr activates p110g and the consequences of interfering with p110g expression or function on t cell activation. results: we found that after tcr engagement, p110g interacts with and forms a complex with ga q/11 , lck and zap70. tcr stimulation activates p110g, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p110g controls rac1 activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p110g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p110g-/-t cells. our observations clarify the activation mechanism and mode of action of p110g in the control of t cell activation, confirming a crucial role for p110g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a4b2 and a7, expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c57bl/6j mice. they were stained with fluorescently labeled igm-, cd40-or cd23specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by 3 h-thymidine incorporation upon stimulation with anti-cd40 and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a7 nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a4 or b2 nachr subunits inhibited binding of igm-and cd23-specific antibodies but facilitated that of cd40-specific antibody. in contrast, antibody against a7 subunit prevented binding of anti-cd40 but not of anti-igm or anti-cd23 suggesting that a7 nachrs are located close to cd40, while a4b2 ones are close to bcr/cd23. consequently, anti-cd40-induced b lymphocyte proliferation was increased by mla much stronger than by a4b2-specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine-3. in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd40-mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a7 nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd4 + t h -lymphocytes to antigen presenting cells or of cd8 + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim1 and trpc3), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about 96 %. down-regulation of stim1 was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd4 + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, 2008) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be 300-600 pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein 70 (hsp70) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd8 + cytotoxic t-lymphocyte (ctl) and cd4 + t-helper cell (th1) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp70 with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k562 cells as well as targetindependent cytotoxicity. cd4 + cells exhibited a strong increase in proliferation after stimulation with hsp70, with rates of up to 29 %. in the presence of target cells, a 35-fold up-regulation of granzyme b mrna was observed after stimulation of cd4 + t-helper cells with hsp70 in combination with il-7, -12 and -15. the target cell-independent secretion of granzyme b by cd4 + cells was greatly augmented after stimulation with hsp70 plus il-2 or il-7, -12 and -15. in this study, we have shown that hsp70 is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd4 + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd3 + and cd8 + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp70 on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd40, which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd150 receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk1 overexpression in a model system was achieved by transfection. pjnk1/2 expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd150 on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk2, but not jnk1 activation. cd40 ligation on primary tonsillar b cells also resulted in jnk2 activation. however, bcr crosslinking did not affect the level of jnk1/2 phosphorylation. cd150-mediated jnk2 activation was independent from sh2d1a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd150 and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase 1 (hpk1) was associated with cd150 in primary b cells as well as in b cell lines. cd150-hpk1 association was independent from cd150 tyrosine phosphorylation and sh2d1a expression. overexpression of hpk1 in a model system significantly enhanced cd150mediated jnk2 phosphorylation. it is known that tnf family receptors such as cd30, cd40, rank trigger survival signals in hrs cells. we observed the expression of pjnk1/2 in hrs cells of primary classical hl. cd150 could be involved in sustained jnk2 activation in primary hrs cells, and this may reflect the role of cd150 receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk2 is activated via cd150 in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk1 is involved in cd150-mediated jnk2 activation. objectives: cd5 has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd5 upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd5 are involved in c-cbl phosphorylation and association. methods: el4 thymoma cell line was stably transfected with wild-type human cd5 or hcd5 cytoplasmic tail mutants: cd5.k384stop (maintaining only a pseudo itim); cd5.h449stop (lacking the distal s and y in the carboxy-terminal region); cd5. ¿ e418-l444stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y700 in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd3 in combination or not with anti-human cd5 biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py700) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x 0.05). in murine thymocytes, co-crosslinking of cd3 with cd5 induces an increase in c-cbl phosphorylation compared to cd3 alone. analysis of the el-4 transfectants showed that mutants cd5.k384stop and cd5.h449stop lost the ability to costimulate cd3-mediated phosphorylation of c-cbl. in contrast, cd5. ¿ e418-l444stop mutant, was able to efficiently costimulate cd3-mediated c-cbl phosphorylation, similarly to the hcd5wt. our results indicate that the absence of the pseudo itam in cd5 does not interfere with c-cbl phosphorylation in response to cd3 plus cd5 crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd5 appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd5 cytosplasmic tail, but rather, may indirectly associate with cd5 through the interaction with other sh2-sh3 domain-containing molecules, that may be recruited to cd5 through its carboxy-terminal region. l. kolly 1 , s. narayan 1 , j. tschopp 2 , a. so 1 , n. busso 1 1 chuv, rheumatology, lausanne, switzerland, 2 unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase-1 into inflammasomes and thus plays a key role in regulating capase-1-dependent il-1b and il-18 production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd4 + and cd8 + t cells were activated in vitro through anti-cd3 stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd3 ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd3 activated asc -/-t cells predominantly displayed a more th 2 phenotype, producing more il-10 (199 vs. 692 pg/ml; asc +/+ vs. asc -/-t cells respectively; p=0.0074) and less ifn-g (15,831 vs. 6 ,921 pg/ml; asc +/+ vs. asc -/-t cells respectively; p = 0.0021). when asc +/+ and asc -/-t cells were purified into cd4 + and cd8 + t cell fractions and activated individually using anti-cd3, no inhibition in proliferation was observed amongst activated asc -/-cd4 + and cd8 + t cells. interestingly, the activated asc -/-cd4 + t cell fraction produced significantly more il-10 when compared to activated asc -/-cd8 + t cells and asc +/+ cd4 + and cd8 + t cells (asc -/-cd4 + t cells = 380 pg/ml il-10; asc -/-cd8 + t cells = undetectable il-10; asc +/+ cd4 + t cells = 11 pg/ml il-10; asc +/+ cd8 + t cells = undetectable il-10). cd4 + and cd8 + t cell mixing experiments revealed that asc -/-cd4 + t cells are able to inhibit the proliferative ability of asc -/-cd8 + t cells, asc +/+ cd4 + and cd8 + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd4 + t cells. collectively, these results demonstrate that the absence of asc drives cd4 + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd4 + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge 1 and pge 2 suppress some t-cell functions including proliferation, activation and cytokine production. pge 2 signals through four types of gpcrs called the ep receptors. at low concentrations, pge 2 is believed to be necessary for t cell function, whereas at higher concentrations, pge 2 inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge 2 and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge 2 diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd4 + t cells with ep receptor antagonists was found to impair cell surface expression of cd71, cd69, cd25 and ox40 but not cd44. suppression of t cell proliferation by pge 2 has already been widely studied. however, blocking ep receptors in cd4 + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd4 + t cells to the chemokine sdf-1b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd44 + cd4 + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd4 + t cells. our results also suggest that considering pge2-mediated camp signaling in cd4 + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd4 + human t-cells was examined. oxidation affects several ca 2+ signalling pathways by altering the activity of ip 3 receptors, trp channels and store operated ca 2+ channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca 2+ signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd147 (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd147 knockout mouse possess enhanced mixed lymphocyte reactions and cd147 monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd147 signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd147 in jurkat t cells augments the secretion of the t cell growth-factor interleukin-2 (il-2) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il-2 promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd147, we identified the immunomodulatory sub-domain of cd147. supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd40 and il4 (simulates t cell-dependent activation). microarray assays identified about 104 mirnas in unstimulated b cells. 35 of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor-4 (irf-4). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf-4 transcripts differs from that observed for irf-4 protein abundance after b cell stimulation. further analysis identified the irf-4 transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk592 and the dfg forschergruppe for832. objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on 11 different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey 1 , f. hauck 2 , i. berberich 1 , f. berberich-siebelt 2 , gk 520 -immunomodulation 1 institute for virology and immunobiology, university of wuerzburg, würzburg, germany, 2 university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd4+ t lymphocytes, the transcription factor is predominantly expressed in t helper 2 (th2) compared to t helper 1 (th1) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g1 phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp-1 encoded by prdm1 is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp-1 is expressed in differentiated effector t cells where it is higher in th2 than th1 cells. the regulation of the blimp1 expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm1 promoter and activates blimp-1 expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp-1 in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp-1 in b cells using cre recombinase. moreover we found a new putative blimp-1 isoform lacking exon 2. currently, we analyze the expression of c/ebpb and blimp-1 in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening 53 cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd21low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd4 + t cells but not transitional b cells or cd8 + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein-1a (mip-1a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy2 revealed constitutively high background levels in cd21low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas 1 , v. courtois 1 , k. de luca 1 , r. sodoyer 1 1 sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il-2, il-4 or il-6 could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il-2, il-4 and il-6. furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a-136-0033-01835). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen-4 (ctla-4) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla-4 during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il3) in the tissue and the released eggs and first stage larvae (l1) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd3 and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla-4 during the infection, a neutralysing antibody (a-ctla-4; 4f10) was administered intraperitoneally (300 mg) two hours before subcutaneous infection with s. ratti il3. the in vivo neutralisation of ctla-4-signalling by applying a-ctla-4 during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th2 cytokines, such as il-4 and il-5 and a reduction of the proinflammatory cytokines ifn-g and il-17. the investigation of the humoral response showed a remarked increase of the igg1-titer in the serum during secondary infection in mice that had been treated with a-ctla-4 during primary infection. furthermore, the blockade of ctla-4 resulted in a diminished worm burden as indicated by reduced release of l1 in the faeces. these results suggest that the blockade of ctla-4 during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th2 response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg1 during secondary infection also reflects the induction of a potent th2 response. objectives: cd36 is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd36 is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd36 on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd36 expression. cd36 knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd36 ko compared to heterozygous mice. since reduced levels of cd36 are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd36 ko mice. after one injection of apoptotic cells, cd36 ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd36 on b-cells are involved in setting this threshold. conclusion: our data suggest that cd36 is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd152 acts as a major check-point of immune responses, but the mechanism by which cd152 controls peripheral t cell responses is unknown. the consequences of cd152 signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd152 in th1 cells reduced migration towards ccl4, cxcl12 and ccl19. crosslinking of cd152 together with cd3 and cd28 stimulation on activated th1 cells increased expression of the chemokine receptors ccr5 and ccr7, which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd152 expression and cd152-mediated migration-enhancing signals. importantly, migration of cd152 positive th1 lymphocytes in in vivo experiments increased, as compared to cd152 negative counterparts, showing that indeed cd152 orchestrates specific migration of selected th1 cells to sites of inflammation and antigenic challenge in vivo. these data show that cd152 signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd152 adds to the already significant role of cd152 in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd152 signaling on the longevity of cd28 null t cells. using a sensitive staining method for cd152, we show that human cd4 + cd28 null and cd8 + cd28 null t cells rapidly express surface cd152. serological inactivation of cd152 using specific fab fragments or blockade of cd152 ligands using ctla4ig in cd4 + cd28 null and cd8 + cd28 null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd152 crosslinking on activated cd28 null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd152 is mediated by pi3'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd152 act directly on activated cd28 null t lymphocytes and, due to its exclusive expression as a receptor for cd80/cd86 on cd28 null t cells, prevention of cd152 mediated signaling is likely a major target mechanism taking place during therapy with ctla4ig. objectives: cd45 is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd45 -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd148 (ptprj, dep-1) which acts as a positive regulator of sfk in cd45 -/-b cells and macrophages and can compensate for cd45 deficiency in these cells. indeed, combined deficiency of cd45 and cd148 in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd148 and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd148 -/mice were reported so far. however, in human t cells the role of cd148 may be different since naive human t cells express cd148 at a level comparable to b cells. using cd45 -/-/cd148 -/human t cell line (jurkat-derived js-7 cells) we tested the ability of cd148 to complement cd45 deficiency in t cells. we used retroviral transduction to express human cd45 or cd148 in js-7 cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd148 to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js-7 cells. expression of wild type cd45 or cd148 in js-7 cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd69 upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js-7 cells expressing cd148 when compared to control cells. finally, cd148 substrate trapping mutant expressed in js-7 cells interacted with lck in vivo suggesting that lck is a direct substrate of cd148 in js-7 cells. the results suggest a level of redundancy between cd45 and cd148 in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam1.6) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr 505 and tyr 394 we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native 2d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca2+ release-activated ca2+) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa-1 (leukocyte function-associated antigen 1) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa-1 prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al.2006) we describe a non-viral vector based approach (vockerodt et al. 2008) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in 9 independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd40 signaling cascade was conducted. after activating this particular signaling cascade (cd40) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. 2005 ). a rat thymic epithelial cell (tec) line (r-tnc.1) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc.1 cell line in vitro. it was found that a number of adhesion molecules, such as cd2, cd4, cd8, cd11a, cd18, cd54, cd90 was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc.1 line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il-2 activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc.1 cells. we found that both the adhesion (30 min and 3h) of activated thymoytes were partially blocked by mab to cd2 and cd8 molecules (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells). early adhesion was inhibited by mab to cd90, abtcr, mhc class i molecule (ifn-g stimulated r-tnc.1 cells) and cd4 molecule (ifn-g unstimulated r-tnc.1 cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc.1 cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay (6h), namely mab to cd2, cd4, cd8, cd90 molecule (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc.1 cells). our results also suggest the involvement of cd11a/cd18 dependent -cd54 independent pathway in adhesion and cd11a/cd18 dependent -cd54 dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc.1 cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin-27 (il-27), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il-27 favours naï ve cd4 t cell differentiation into th1 cells to the detriment of th17 or th2 differentiation. the il-27 receptor (il-27r) is a heterodimer composed of tccr, which confers ligand specificity, and gp130, a signal transducing chain that is utilized by several other cytokines. il-27 has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd8 t cells, but the potential impact of il-27 on human cd8 t cells has not been elucidated. our goal is to investigate the impact of il-27 on human cd8 t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il-27r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd8 than cd4 t cells expressing the complete surface il-27r (gp130+tccr). however, we detected high amounts of intracellular tccr in both, cd4 and cd8 t cells, but only polyclonal activation (anti-cd3) of cd8 t cells led to an actual increase of il-27r surface expression. purified cd8 t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il-27 activated stat1 and stat3 signalling with rapid kinetics in both cd8 and cd4 t cells, indicating the capacity of il-27 to signal through these molecules. addition of il-27 to anti-cd3 activated cd8 t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il-27 on human cd8 t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd-1/pd-l1 pathway is associated with production of the immunoregulatory cytokine il-10, the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l1 and pd1, as well as myelin basic protein (mbp)-stimulated il-10 production, pakt inhibition, and apoptosis (annexin v), in 50 ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); 24 had a diagnosis of stable disease (sms). results showed that: 1) pd-l1 -expressing cd14+ and cd19+ cells are reduced in ams compared to sms individuals (p=0.04); and 2) pd1 expression is increased in cd4+ t cells of sms individuals and is comparable on cd8+ t cells of ams and sms patients. this is associated with a significant decrease in il-10 production by mbp-stimulated cd14+ and cd19+ cells of ams patients (p=0.03). additionally, cd8+ anexin v+ (av+) cells were diminished and cd8+ pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd4+av+ and cd4+ pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l1/pd-1 pathway seen in ams patients result in a reduced mbp-specific il-10 production by cd14+ and cd19+ cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd8+ t. the pd1/pdl1 pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti-4-1bb in cd8 cells. this difference could be due to down regulation of cd28 by activated lymphocytes and possible preferential response of cd8 cells to anti-4-1bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin7 (stx7) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx7. rna interference technique was also used to down regulate stx7 expression in primary human ctls. results: we identified stx7 in ctls by pcr and immunocytochemistry. stx7 accumulates at the is after ctl/target cell contact. when stx7 function was blocked by overexpression of a dominant negative stx7 mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx7 is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the 20 years history of mouse t h 1 and t h 2 subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h 0, t h 1 or t h 2 phenotypes, based on their cytokine production (il-2, ifng or il-4). a comparative analysis of t-bet, ifng and il-4 mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca 2+response, membrane raft expression/organization, k + -and ca 2+ -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h 1 g t h 0 g t h 2, although the membrane cholesterol level (detected with anti-cholesterol ab, ac8) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h 1 cells upon activation than in t h 2 cells. t h 1 cells produced a more sustained calcium response with higher amplitude than t h 2 cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav1.2 and kv1.3 ion channels, major functional determinants of the sustained calcium influx. t h 2 cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd19-dynabeads. cd19 + isolated cells were facs sorted into cd19 + cd27naïve b or cd19 + cd27 + memory b cells. dna synthesis was measured by 3h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd19 + cd27naï ve b cells, of which bmp-6 and -7 were most efficient (40 % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd19 + cd27 + memory b cells by 40 -50 %. to induce differentiation, both naï ve and memory b cells were stimulated with cd40l and il-21. this increased the production of igm, iga and igg 10 -100-fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad1/5/8 in cd19 + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd40l/il-21-induced production of igs in mature human b cells. s. gutenberger 1 , k. warnatz 1 1 university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases 1 and 2 (erk 1/2). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd86 upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of 20 hd and 25 cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk1/2 phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk1/2. to increase the signal intracellular phosphatases were inhibited by h2o2. as markers of activation and initiation of proliferation, cd86 and ki67 expression were measured after 2 days of in vitro stimulation. k. theil 1 , p. aichele 1 1 immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd8 t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p14 cd8 t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b6-recipient mice and compared their expansion with wild-type (wt) p14 t cells after viral infection. we could demonstrate a severe impairment in the capacity of p14 t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p14 t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd8 t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p14 t cells into h8 mice. h8 mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp33-41. therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h8 mice are infected with lcmv8.7. this is a lcmv variant that has got a point mutation in gp33-41 and consequently cannot be recognized by the p14 t cells. s. frischbutter 1 , r. baumgrass 1 1 deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap1 which are important for expression of cytokines such as il-2, ifng and il17. it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl-10 was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl-10 phosphorylation but rather an inhibition of bcl-10 dephosphorylation. furthermore, calcineurin and bcl-10 co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl-10/malt1 signaling complex and dephosphorylates bcl-10 and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop 1 , j. lamoureux 1 , c. fortin 1 1 université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: 25 healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla-4 has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla-4 expression was higher after stimulation in t cells of elderly. there was differences between cd4 and cd8 t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd95l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh3 and ww domain proteins. since fasl surface expression is regulated by adam10-mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl2a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh3 domain proteins that potentially interact with the riped fasl prd, we used a sh3 domain phage display library containing all 288 sh3 domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx9 family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conseoptimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b7 family members on antigen-presenting cells with cd28 on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd28 co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound12, with classical gcs regarding their suppressive effect on cd28-costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd3 and anti-cd28, and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd28-costimulated human memory/effector cd4+ t cells by compound12 than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound12 was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound12 and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound12 than prednisolone. when evaluating possible mechanism for the increased activity of compound12 in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound12 which was not prevented by the partial gc receptor antagonist, ru-486, in vitro. moreover, in vivo we observed less induction of il-1beta and tnf-alpha by pre-treatment with compound12 than with prednisolone. our data indicate that the non-steroidal segra, compound12, may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b6 mice elicits robust cd8+ t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il-2+ cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa 224 -specific cells also express tnfa, but only about half of the ifng+ np 366 -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd8+ t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd8+ t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova 257-264 peptide shows a close correlation with division in vivo. early after antigen encounter (0-2 divisions) the vast majority of cells express only tnfa. after 3-4 divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions (4-6 divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd4+ t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd8+ t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd8+ t cells, expansion plays a very important role in the regulation of cd8+ t cell effector function. in addition to its chemo-attractant function, sdf-1a (stromal-cell derived factor-1a, cxcl12) has been described to costimulate cd4 + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd4 + t cells was increased by immobilized sdf-1a to a level similar to that obtained with the costimulatory molecule cd28. as visualized by real time confocal microscopy, t cells entering in contact with sdf-1a formed a tether and displayed an active scanning activity. since sdf-1a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd3 mabs, it is conceivable that the sdf-1a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf-1a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf-1a in the context of cd4 + t activation by antigen-presenting cells secreting sdf-1a. this study should help us to better define how sdf-1a modulates cd4 + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd4 + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd3 and anti-cd28 antibodies and cape for 2 days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by 3h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il-5 production and lymphoproliferation in cd4 + t cells stimulated by anti-cd3/cd28. cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd4 t cells express a variety of molecules on their surface, such as mhc-class ii, cd80, cd86, cd70, whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd4 t cells might induce t cell proliferation and differentiation from cd4 resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd4 t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after 5 days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd4 memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd25, cd30, and cd69. analysis of the cytokine profile of these cells revealed the differentiation of il-10-and ifn-g-double-producing cells in response to contact with th1 effector cells, and il-4-producing cells in response to contact with th2 effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd3/cd28 stimulation. whereas neutralization of ifn-g or il-4 during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd70, cd80, and cd86 could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to 64-85 %. conclusion: interaction of cd4 memory t cells with activated t cells resulted in the production of the cytokines il-4, il-10, and ifn-g. given the immunomodulatory capacity of il-4 and il-10, these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir 1 , s. thompson 1 , j.j. murphy 1 1 king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl1 leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il-2 and il-5 and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp36l1 by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp36l1 has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl1 differentiation was observed to be associated with downregulation of zfp36l1 protein. in an attempt to determine whether zfp36l1 downregulation was directly linked to bcl1 differentiation, a zfp36l1 shrna expressing lentivirus (psicor) was employed to knockdown zfp36l1 expression. this reagent downregulated zfp36l1 expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp36l1 shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp36l1 downregulation in promoting bcl1 plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of 32 patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all 32 isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd95 (fas, apo-1) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd3/cd28-triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo1 mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd95l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il-2 and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk1/2 phosphorylation, the expression of various activation markers, the il-2 production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb415) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb1,3galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd4+ and cd8+ t cells. in addition, mitogenic stimulus increase 3-fold the all binding to cd4+ t cells. previous studies in human pbmc showed that all binds to human cd4+ t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd4+ t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd3-dependent activation of purified cd4+ t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd25 and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd25 expression, but enhanced the anti-cd3-dependent proliferation and cd25 expression of purified cd4+ t cells. analisis of calcium influx showed that all enhanced anti-cd3 dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd4+ t cells by up-regulating t cell activation mediated by anti-cd3 stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in214609) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd3 complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd95 receptor. it has been previously demonstrated that cd95 ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp-76, slap-130 and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il-1r, il-2r, il-3r, il-7r, il-12r, il-18r, cd27, cd28, cdw137( 4-1bb), cd 95(fas) and cd178 (fasl) on hpbls in different cultured time, i. e. 0d, 1d, 3d, 5d, 7d, 9d, 11d and 14d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. 1. the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. 1) the expressions of membrane immune molecules before cultured. 2) expressions of the membrane molecules on hpbls during culturing. 5% fbs rpmi 1640 group (1640 group), il-2 group, pha group... (1) mtt assay. (2) proliferative times and growth curves of hpbls... 1. during cultured in vitro, there are expression changes of the il-1rs (il-1ra, il-2ra, il-2rg, il-3r, il-7r, il-12r, il-18r), co-stimulatory molecules (cd27, cd28, 4-1bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in 1640 group, il-2 group and pha group, but the rests are different. 2. our data also suggest that the hpbls cultured in cd3mcab+cd28mcab+il2+il1a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. 3. celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun 1 , r. tauber 1 1 zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p56lck and the ras/rac2 signalling pathway (1) followed by mitogen-activated protein kinases (2) and c-jun n-terminal kinase (1), which leads to an enhanced binding of l-selectin to soluble ligands (3). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues (4). here we show that the protein phosphatase 2a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp2a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp2a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer 1 , e. glasmacher 1 1 helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago1-4 genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm1 and rck or expression of dominant-negative gw182. interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd4 + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd8 + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd8 + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd8 + t-cell activation and differentiation. naïve cd62l hi cd44 lo cd8 + t cells were sorted and stimulated by anti-cd3 and anti-cd28 antibodies. results: firstly, we show that the activation and differentiation of cd8 + t cells require il-2 provided by activated cd4 + t cells at the initial priming stage after stimulation. secondly, this critical il-2 signal is delivered through il2rbg of cd8 + cells and is independent of il-2ra. besides promoting cell proliferation, il-2 stimulation increases the amount of ifng and granzyme b produced by cd8 + t cells. conclusion: therefore, our studies demonstrate that a full cd8 + t-cell response is elicited by a critical temporal function of il-2 released from cd4 + t cells, providing mechanistic insights into the regulation of cd8 + t cell activation and differentiation. most antigenic peptides recognized by cd8 t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a3168-176 is a tumor antigenic peptide presented by hla-a1 and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a3168-176 we setup an in vitro digestion assay using a 20-mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a3168-176 by tumor cells. by electroporating hla-a1 cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart-1/melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart-1/melan-a 26-35 specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart-1/melan-a 26-35 ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart-1 expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart-1/melan-a 26-35 epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart-1/melan-a 26-35 ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd8 + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd4 t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev 207: 293-313, 2005) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h2-m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity 11: 453-462, 1999) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide 84-103, whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from 84-103 peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the 84-103 epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, 2008), we found, after an initial induction at 8 hrs p. i., a strong inhibition of tapasin transcription at 24 hrs p. i. furthermore, also reduction of tap1 and tap2 transcription was observed contrasting to the elevated levels of erp57 and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß1, ß2 and ß5, which are replaced by their ifng-inducible counterparts ß1i, ß2i and ß5i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß5i is replaced by a thymus-specific subunit ß5t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß1i, ß2i, ß5i and ß5 in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß1,ß2 and ß5i (single intermediate proteasome), and the other comprises ß1i, ß2 and ß5i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent 10-20 % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about 50% of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a*0201) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a*0201 binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a*0201 from tapasin, but only ttp-ha dissociated hla-a*0201 from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl-721.220-a2 showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a*0201 from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a*0201. m. basler 1,2 , c. lauer 2 , m. groettrup 1,2 1 biotechnology institute thurgau, kreuzlingen, switzerland, 2 university of constance, division of immunology, department of biology, konstanz, germany two lmp7-dependent antigens have been described that relied on the 'structural presence' of lmp7 in the proteasome but not on the activity of lmp7. here we have investigated processing of the h-2d b -restricted uty 246-254 epitope of the male minor antigen uty reported to be lmp7-dependent. using splenocytes from lmp7 -/-, lmp2 -/and mecl-1 -/mice we found that the uty 246-254 epitope requires lmp7 and lmp2 but not mecl-1. curiously, a selective lmp2 inhibitor did not interfere with uty 246-254 presentation. objective: we investigated why the deletion but not the inhibition of lmp2 interferes with uty 246-254 presentation. we hypothesized that the 'structural' requirement for lmp2 is based on replacement of the caspase-like activity of b1 in the proteasome. methods: it was determined if t1a mutants of lmp2 and/or b1 can rescue the uty 246-254 epitope. we used a b1-selective inhibitor to determine if the inhibition of the caspase-like activity of b1 preserves the epitope. finally we determined by mass spectrometry if the uty 246-254 epitope embedded within a 25mer precursor peptide is differentially cleaved by lmp2-deficient and proficient immunoproteasomes in vitro. results: we found that t1a mutants of lmp2 and b1 rescue presentation of uty [246] [247] [248] [249] [250] [251] [252] [253] [254] . also inhibition of cells with a b1-selective inhibitor preserves uty [246] [247] [248] [249] [250] [251] [252] [253] [254] presentation. an aspartate in position 7 of the uty 246-254 sequence wmhhnmdli is preferentially used as a cleavage site by lmp2-deficient but not half as frequently by lmp2-proficient immunoproteasomes. the generation of the uty 246-254 epitope relies on the replacement of the caspase-like activity of b1 by lmp2 because the b1 activity destroys the uty 246-254 epitope. this is the first example for the 'structural' requirement of lmp2 for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp2 and perhaps also lmp7 and mecl-1 exert their function in antigen processing. a. linnemann 1 , a. musiol 2 , r. lindner 1 1 hannover medical school, cell biology, hannover, germany, 2 hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf6-regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf6) were overexpressed in 3t3 fibroblasts. we show that antibody-mediated oligomerization of mhc i in 3t3 fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf6: endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf6-regulated to a novel, arf6-independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv40 virus and since sv40 binding triggers mhc i oligomerization, this novel pathway may be involved in sv40 uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of 57 rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab3b, 3c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta2-microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab3b and rab3c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab3b or 3c positive vesicles. furthermore, the rab3b, 3c positive compartment were colocalizd with a fraction of rab27a at a juxtaposition of phagosomes. together these data demonstrate that rab3b and rab3c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over 90 % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over 80 viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd8+ t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf2a and bglf5 have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf1, has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens 2009). this represents a novel function for a virally-encoded gpcr. bilf1 is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf1 displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf1 is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf1 can hinder the recognition of virally-infected cells by cytotoxic cd8+ t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe 131 natural ligands, including two peptides derived from semenogelin-1, a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg1 is transcribed in thymus from both male and female individuals. finally, we detected the semg1 mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta1, beta2, beta5) and immuno-subunits (beta1i, beta2i, beta5i). deficiency in beta5i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta5i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta5i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta5i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta5i-deficient cells could be restored by treatment with d3t, which increases the amount of proteasomes independent of beta5i via induction of mixed proteasomes containing beta1i, beta2i and beta5. consequently, not the lack of the specific proteolytic activity of beta5i or immunoproteasomes, but the reduced proteasome quantity in beta5i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr2, 4 and 5, which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd4 + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd4 + t cells. the presence of exogenous tlr3 and tlr8 ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james 1 , i. bailey 1 , t. elliott 1 1 university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct26 colon carcinoma. this response is long lasting and mediated by both cd4 and cd8 t cells. importantly, the treg depleted ct26 specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a20, c26, bcl1, renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw11, which is h2-d d restricted. analysis of the generation of gsw11 in ct26 revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct26 cells substantially increased the amount of gsw11 present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct26 and immunised balb/c mice. greater than 80 % of mice injected with eraap kd ct26 were found to reject the tumour. analysis of t cell responses revealed the presence of gsw11-specific t cells, however, these responses were small (0.5-1 %). this compared to a much larger response to ct26 (˚5 %). preliminary results indicate that the majority of the t cell responses (non-gsw11-specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [1] . the di-allelic hla-a2 restricted minor histocompatibility antigen ha-1 locus codes for the highly immunogenic ha-1 his and the non-immunogenic ha-1 arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha-1 arg immunogenicity was the estimated numbers of cell surface presented copies i. e. 80/cell for ha-1 his and less than 5/ cell for ha-1 arg [2] . as ha-1 his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha-1 his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha-1 mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha-1 allelic peptides, we investigated the impact of the ha-1 his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha-1 his and ha-1 arg peptides were performed. moreover, the crystal structures of hla-a2 in complex with either ha-1 his , ha-1 arg or a ha-1 variant with a citrulline residue at position 3 were determined in order to obtain atomic level insights into the conformation of the hla-a2/ha-1 peptide complexes. our results exclude a role for antigen processing in preventing ha-1 arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha-1 arg allele in the hla-a2 peptide binding groove [3] . they provide a rationale for the lack of ha-1 arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd8 t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd8 t cell response. on the other hand, immunosubunit expression is not essential for development of cd8 t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp7 (ib5) and mecl-1 (ib2) develop a variety of autoimmune responses, including a latent form of t1d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd8 but not cd4 t cells. a cd8 t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose-6-phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd8 t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd8 t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, 162 autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr3 and dr4. analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type 1 (hsv-1) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd4 + and cd8 + t cells in hsv-1 immunity have yet to be clearly elucidated. to better understand the role of hsv-1-specific cd4 + t cells in immune control we have identified a 13 amino acid epitope derived from glycoprotein d of hsv-1. following flank infection, gd-specific cd4 + t cells were first detected in the draining brachial and axillary lymph nodes (ln) 5-days post-infection (pi), peaking at day 7 and declining thereafter. gd-specific cd4 + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day 6 pi and peaked at day 9. while hsv-specific t cells were first observed in the draining ln at day 5 pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as 2 days pi, with peak activity 4 days pi before declining to background by day 7. however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd4 + t cells was observed up to 23 days post-infection in the brachial ln. ex vivo analyses suggest that only cd11c + cells were involved in gd antigen presentation at days 2, 5 and 15 post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd8a + dcs can present the gd antigen to cd4 + t cells at day 2 pi, whereas by day 5 pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv-1 infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap1 and erap2 unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap1/erap2 in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp57. upon assembly of the heavy chain with b 2 m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp57. both crt and erp57 are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp57 to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp57 or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp57 must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp57 point mutant that fails to bind to cnx or crt was just as effective as wild type erp57 in normalizing rates of disulfide formation. we conclude that erp57 does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp57 point mutant, class i heavy chains, crt and the tapasin-erp57 disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp57 and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp57 have no effect on plc stability or class i surface expression, suggesting that erp57 plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies 5-11 % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb1*0405/dqb1*0401 haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr*0405 transgenic ab0 nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd4+ and cd8+ t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr*0405 transgenic mice (cd4+ t cell competent) develop aip even unprovoked, similar to ab0 nod mice (cd4+ t cell deficient), while hla-dr*0401, hla-dq8 or hla-dr*0405/dq8 (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr*0405 fails to protect from aip, likely due to defects in the induction of cd4+ regulatory t cells. our results identify hla-dr*0405 as a prominent risk factor for aip on the hla-drb1*0405/dqb1*0401 haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan 1 , c. britten 1 , h. overkleeft 2 , g. van der marel 2 , k. melief 1 , d. filippov 2 , f. ossendorp 1 1 leiden university medical center, section tumorimmunology, leiden, netherlands, 2 leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd8 and cd4 t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi 1,2 , x. hu 1,2 , a. kawanatachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping 8-mer and 10-mer epitopes (rypltfgwcf (nef138-10) and rypltfgw (nef138-8)) were found to be presented by hla-a*2402 and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y2f, y2w, t5c, f6l, w8r, f10r, f10y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef138-10 (wild type) and its four mostly common immune escape mutants (y2f, t5c, y2f&t5c, f6l), and also nef138-8 (wild type). we found that there was little difference between the nef138-10 (wild type) and nef138-10 (y2f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y2 or f2. interestingly the central bulge region of the peptide was becoming very flexible for the nef138-10 (t5c) and nef138-10 (y2f&t5c), which may affect the binding of peptide and the recognition of t cell receptor. for nef138-10 (f6l), the side chain of l6 was more flexible compared to the nef138-10 (wild type). alignment of the nef138-10 and nef138-8 showed that the nef138-8 became flat and the side chain of f6 was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef138-10 was featured, while the peptide nef138-8 was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef138-10 was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd8+ and cd4+ t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a2 restricted cmvpp65 derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a 2-3 log reduction of activation efficiency. this pattern was seen for 5/9 epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to 2-5 log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b7 restricted cmvpp65 rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t2 cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv 9-mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd8+ t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp100 and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp110, and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor-5 (fgf-5) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of 40 amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf-5 peptide was produced in vitro after incubation of proteasomes with a 49-amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf-5 spliced peptide by cells transfected with mutant constructs encoding fgf-5 proteins where the intervening segment was shortened from 40 amino acids to 30, 20 or 8 residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp100 peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b*4402, are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b*4405, appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b*4402 and b*4405 are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us3 protein. to address this hypothesis, us3 was stably coexpressed in b lymphoblastoid cell lines expressing hla-b*4402 or hla-b*4405. in the presence of us3, the surface expression of hla-b*4402 was substantially reduced whereas hla-b*4405 expression was relatively unaffected. although us3 was able to form complexes with both hla class i allotypes, only hla-b*4402 was retained intracellularly in an immature form whereas hla-b*4405 was transported to the cell surface. accordingly, in the presence of us3, hla-b*4405, but not hla-b*4402, constitutively presented a hla-b44 restricted alloantigen to reporter t cells, suggesting that us3 binds hla-b*4405 without interfering with peptide loading. us3 has been reported by others to bind the plc but surprisingly we have not detected such us3-plc complexes in our system. rather, in the presence of us3 we identified a pool of class i molecules distinct from the plc and only present in us3 expressing cells, implying that us3 may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa 1 , b. seliger 1 1 martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il12p70. since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for 24h with the gold standard of maturation (tnfa, il1b, il6 and pge2) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase-3 (lap3) had a more than 20-fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap1 and erap2 and of the immunoproteasome subunits lmp2 and lmp10 was enhanced between 2 and 5-fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr4 ligand mpla in comparison to the tlr-3 and 7/8 ligand polyi:c and r848. these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd4 + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il-4 induced surface expression of mature mhc class ii molecules and cd86. when ifn-g/il-4 primed pcmc were used as antigen presenting cells for cd4 + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd69 up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type 1-diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b15-23 to specific cd8+ t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd8 + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th1 immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd8+ t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd8+ tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd8+ t cell clones, faure, 2009, eur j immunol 39 (2): 380-90). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd8+ t cells after their in vitro differentiation into dc, 6 days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv-1 genome contains arfs within gag, pol and env genes encoding for a panel of hla-b*07 restricted epitopes. qprsdthvf (q9vf/5d) is one such epitope but its parental epitope qprsnthvf (q9vf/5n) has a significant higher frequency among hiv-1 isolates. strikingly, q9vf/5d-or q9vf/5n-specific ctls recognize apcs infected with hiv strains encoding for q9vf/5d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad8 ) encoding for q9vf/5n do not activate ctl responses raising the possibility that q9vf/5n epitope is not presented by infected cells. we asked whether introducing mutations within q9vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q9vf/5n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q9vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q9vf mhc-i presentation. we performed in vitro proteasomal digestions of 28mer peptides encompassing q9vf/5d or q9vf/5n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q9vf/5n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q9vf/5n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q9vf/5d or q9vf/5n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q9vf/5d peptide. we cloned and sequenced hiv-1 genomes from the three donors. surprisingly, out of 20 hiv proviral genomes isolated from pbmcs of q9vf/5d reactive donors, we could not find any virus bearing the q9vf/5d sequence. the isolated hiv sequences either encoded for q9vf/5n or had a stop codon within the epitope. in contrast, viruses encoding for q9vf/5d were isolated from pbmcs of the q9vf/ 5d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv-1 seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch 1 , y. wang 1 , d.c. tscharke 1 1 the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd8 + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd8 + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd8 + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h-2 bxd f 1 mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f 1 progeny, with some peptides being almost equally immunogenic in f 1 and inbred mice, while others elicit responses that are reduced by more than 90 % in f 1 mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f 1 mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f 1 mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd8 + t cells with a restricted vb usage in f 1 mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr2 agonist, s-[2,3-bispalmitoyiloxy-(2r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp-2), as an adjuvant for cross-priming against cellular and soluble antigens. malp-2 has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd8 + and cd8 -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr2/6 results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap1 expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap1) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to 90 % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, 50 and 10 % as compared to control rma cells, were injected s. c. in the flank of c57bl/6 syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to 45 days after injection. all mice injected with rma clone with a 50 % level of eraap expression developed a tumor and died within 23 days after injection. surprisingly, any animal injected with rma clone with a 10 % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd4 + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd4+ t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor (207 bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd20 therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor (2.5 bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg4 levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb1*0701-restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with 20-mer synthetic fviii peptides to stain epitope-specific cd4+ cells.cd4+ t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a2 domain: fviii 405-424 , fviii 421-440 and fviii 653-672 , as well as the c1 domain peptide fviii 2093-2112 , but not any c2 domain peptides. the c1 domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost.85:123-33, 2005 ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni 1 , s. albini 1 , m.z. limongi 1 , l. cifaldi 1 , d. fruci 1 1 ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p50 nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap1 and erap2, we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap1, erap2 and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap1 and erap2. this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p65 nf-kb subunit, we demonstrated that nf-kb is involved in erap1 and erap2 expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap1 and erap2 and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap1, erap2 and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch 1 , s. tenzer 1 , h. schild 1 , e. von stebut-borschitz 1 1 uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c57bl/6 mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th1 immunity, whereas ifng secretion of both cd4 + th1 and cd8 + tc1 cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il-12-dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd8 + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd8 + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm7 (lmp7 -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c57bl/6 mice. in addition, ex vivo co-cultures with infected dc from either lmp7 -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc1 cell ifng secretion and the dc restimulatory capacity of cd8 + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd8 + t cells, isolated from low dose infected c57bl/6 wildtype or lmp7 -/mice, were not detected. in summary, our data indicate that despite the fact that cd8 responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd8 + t cells against l. major is independent of the lmp7 subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a20, generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd4 + t cell epitope (84-103) from the g1 domain of aggrecan 10,000 times more efficiently than non-specific b cells and over 100 times more efficiently than the macrophage line j774. however, despite this highly efficient aggrecan capture, processing and presentation of the 84-103 epitope took at least 5 hours, comparable to the time required for presentation of aggrecan by j774. treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the 84-103 epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions 114, 115 and 116 at the peptide binding groove. position 116 also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b27 is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b27 subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b27 folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b*2705, peptide loading is relatively independent of this chaperone. we analyzed the effect of b27 subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b27 subtypes. the association of b27 heavy chain with tapasin was analyzed in c1r cells transfected with hla-b27 subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta-1, which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b27 peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me1 monoclonal antibody that recognizes b27properly folded b27/peptide complexes. the formation of fully assembled b27 molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc10, which recognizes mhc class i free heavy chains (hc), or with me1. maturation was analyzed by pulse-chase analysis, immunoprecipitation with me1 and treatment with endoglycosidase h (endo h). hla-b27 polymorphic positions other than 116, both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b27 subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd4 + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd8 + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c57bl/6 mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately 15 % of vacv global presentation in the context of h-2 b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd8+ and cd8-dc, which differ in their mhci antigen presentation capacities. cd8+ dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd8-dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd8+ and cd8-dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd8+ and cd8-dc was determined. surprisingly, cd8+ dc exhibit rapid surface mhci turnover compared to cd8-dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd8+ and cd8-dc were stabilized and no longer underwent rapid turnover. this suggests that cd8+ and cd8-dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd8+ and cd8-dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h-2kb loaded with the ova-derived peptide, siinfekl. cd8+ and cd8-dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd8+ dc, but not the cd8-dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd8+, and not cd8-dc. this data further validates the role for cd8+ dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp-1 cytosol using gelatinase b/mmp-9 as a model enzyme. in the first dimension, ion exchange chromatography separated the thp-1 proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp-9. in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp-9 on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp-1 cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp-9-cleaved or intact thp-1 cytosol. proliferation was assessed by measuring incorporated 3 hthymidine. results: 100-200 mmp-9 candidate substrates were isolated, of which 69 were identified, revealing many novel mmp-9 (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about 2/3 of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp-9 decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e3-19k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue 56 in hla-a11 and residue 93 in e3-19k are highly critical for association of both proteins. this defines a putative interaction surface between e3-19k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e3-19k and the class i antigen presentation pathway. moreover, because soluble e3-19k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap1 and tap2. tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap1 and tap2 were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap1 on tap2 was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap1 mutants suggested that the lack of tap1 protein expression is associated with a strong reduction of tap2 protein levels, which could be restored by tap1 gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap1 on tap2 different shrna plasmids specifically targeting tap1 or tap2, respectively, were stably transfected into constitutively tap1 and tap2 expressing hacat keratinocytes and colo794 melanoma cells. in both cell types the shrna-mediated tap1 and tap2 inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap1 caused not only an almost complete loss of tap1, but also a strong decrease of tap2 protein expression. in contrast, the tap2 knock down exhibited no influence on the tap1 expression. specific inhibition of the proteasome prevented the degradation of tap2 in the tap1 knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap1 on tap2 protein expression is not restricted to rare tap1 mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of 27 healthy volunteers was exposed twice to 1.5 minimal erythema dose of uv. blister roofs were then collected before and 1, 4 and 10 days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd14 + cd36 + and of a macrophagic cd14 -cd36 + cell subsets that emerge 1 and 4 days post exposition, respectively. more importantly whereas classical cd1a hi cd207 + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd1a low cd207and cd1a low cd207 + that emerged 1 and 4 days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd86 and hladr. finally, 10 days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey 1 , e. reeves 1 , t. elliott 1 , e. james 1 1 university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd4+ cd25+ regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct26, stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct26-derived cross-protective antigen, gsw11, which was found to be encoded within the ectotropic murine leukaemia virus (emv-1) envelope protein, gp70. this protein has previously been shown to encode ct26-specific cd8 and cd4 antigens, implicating it as a 'hot-spot' for ct26 tumour antigens. interestingly, we have identified a truncated version of gp70 which may be responsible for generation of gsw11. expression studies have revealed increased gp70 expression in ct26 compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw11) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p1 pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p4, p6 and p9 tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp2 molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp85-99, bound to hla-dp2 with high affinity (10-30 nm). binding studies of mbp85-99 derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f91a, f92a and k93a) were affected, and only by a 20-50 fold reduction in affinity for hla-dp2. the observation that none of the substitutions resulted in a complete loss of binding to hla-dp2 indicates that 1) hla-dp2 binding to peptides does not depend on a large hydrophobic residue accomodated in p1, or 2) mbp85-99 can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp2 molecule binds the immunodominant epitope in ms, mbp85-99, possibly in more than one register. additional studies are required to resolve the hla-dp2 peptide binding properties, and to determine whether expression of hla-dp2 affects the disease course in ms patients. results: depletion of mncs for cd14+ cells abrogated the tg-induced cytokine production and proliferation of cd4+ t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g 98 % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd4+ t cell proliferation was significantly reduced in cd14/cd19-depleted mnc cultures, as compared to cultures depleted for cd14+ monocytes alone. the same applied to the production of il-2, il-6 and tnf-a. production of ifn-g and il-10 was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa 1,2 , p. pala 1 , g. miiro 3 , j. todd 3 , p. kaleebu 1,2 , n. imami 2 , f. gotch 2,3 1 mrc uganda, basic science, entebbe, uganda, 2 imperial college, london, united kingdom, 3 mrc uganda, entebbe, uganda background: hiv-1-specific t-cell responses are preserved in hiv-1 infected individuals with non-progressing hiv-1 disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv-1 for g 8 years, maintaining cd4+ t-cell counts g 500, and with minimal cd4+ decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: 17 art naï ve hiv-1 infected patients from the entebbe cohort in uganda were recruited and stratified by cd4+ t-cell count, cd4+ decline slopes, and time of enrolment, into 2 groups -10 ltnp and 7 rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd4 t-cell count, and ifn-g, il-2 and il-4 elispot responses to pools of 22 to 34 peptides (18-mers overlapping by 10aa). peptides were based on consensus sequences of gag and nef from hiv-1 clades a1, a2 and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il-4 responses were significantly higher in the ltnp than in rp (p=0.02, ifn-g responses to gaga2 pool 1; p=0.04, il-4 responses to gaga1 pool 2). il-2 responses were low and not significantly different between ltnp and rp. there was a positive correlation between il-4 responses to gaga1 pool 2 and cd4 t-cell counts (r 2 =0.502, p=0.04), but no correlation between either il-4 or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv-1 specific responses were higher in ltnps than in rps confirming previous results. non-specific il-4 responses were high possibly reflecting baseline th2 responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her2/neuand her2/neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using 7-aad for dna analysis and specific antibodies directed against the proliferation marker ki-67, the m-phase specific phistone h3 as well as for the h-2l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her2/neuversus her2/neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g 0 /g 1 -, s-and g 2 /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g 0 /g 1 -phase, which continuously raised and peaked within the s-phase. however, h-2l d surface antigen expression was not altered in her2/neucells during cell cycle progression. in contrast to her2/neufibroblasts the her2/neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h-2l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd207) and ccr6 were cultured from cd34+ stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd34+ derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il-4-dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mø). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to 45 min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mø than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mø have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, 1999) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of 30 healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, 1992, 1999) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x 0,0001), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd4+, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, 2252 patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for 2207 of them. for 538 of these patients hla-drb1 data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p=0.02). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb-1a preparations, whereas no differences were shown for ifnb-1b. an association between hla-drb1*11 and nab-negativity was detected (p=0.028). the known associations between hla-drb1*15 and csf-positive ms and hla-drb1*04 and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb1 potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb-1a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb-1b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart-1 and gp100 more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni 1 , s. lorenzi 1 , f. mattei 1 , f. spadaro 1 , l. gabriele 1 1 istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd8 t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd8a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd8 t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd8 t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg7 thymoma line, we show that type i ifn promote the ability of cd8a + dc to capture apoptotic eg7 cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd8a + dc and the persistence of apoptotic eg7-cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg7-derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd8a + dc. as a result, eg7-loaded dc become competent at inducing ot-i cd8 t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd8a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. (2). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd8+ dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam3csk4, and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd8+ dc but not cd8-dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm1, which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a549 human lung cancer cell line. methods: dm1 induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a549 cells. we examined the morphological change using optical microscope. and gfp-lc3 punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm1 was showed high cytotoxicity on various cancer cell line, especially a549 cells. dm1 treated a549 cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc3 and beclin-1 protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc3 was increased concentration and time-dependently. beclin-1 also was increased. and then, we examined gfp-lc3 punctation on a549/ gfp-lc3 stable cells using confocal. a549 cells were formed gfp punctuation after dm 1 treatment. to confirm these data, we measured cell death ratio using 3-methyladenine (3-ma), an autophagocytosis inhibitor. after 3-ma treatment, dm1 induced cell death was decreased comparing with dm1 treatment. conclusion: dm1 did not induce apoptotic cell death. but dm1 showed several characteristics of autophagic cell death. taken together, dm1 induced autophagocytosis on a549 cells. these finding indicated that therapeutic potential of dm1 by triggering autophagic cell death. s. b. rasmussen 1 , s. r. paludan 1 1 university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr)9, which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr9 initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr9 recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv-1 infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv-1, we found that tlr9 dependent ifn regulatory factor 3 activation was abrogated. in addition, inhibition by 3-methyladenine of phosphoinositol 3-kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr9 dependent recognition. in the ongoing project we are examining how hsv-1 triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko 1 , i. berberich 1 , gk 520 -immunomodulation 1 universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl-2 family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a1 and its human homologue bfl-1 are anti-apoptotic members of the bcl-2 family. a1 is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a1. unless the c-terminal ends of other bcl-2 proteins the tail of a1 does not function as a strong membrane anchor. nevertheless, the last amino acids of a1 are important for the protein. in fact, the c-terminus of a1 serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a1 undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl-2 factor bimel results in increased stability of a1. this is due to reduced ubiquitination of a1 after binding of bimel. we conclude that the cterminus of a1/bfl-1 serves as a docking site for e3 ubiquitin ligase(s) that control the stability of a1 by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e3 ubiquitin ligase that interacted with a1 in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs-1 in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs-1 is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs-1 significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs-1 was manifest not only by the suppression of jaks acting upstream of p38 mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak 1/2 activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs1 which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs-1 overexpressing cells. the results strongly suggest that socs-1 acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl12/sdf-1a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl12 effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u-87, and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p53 and fas-l expression was carried out by western-blot. results: cxcl12 induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p53 pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p53-ser46 levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after 24h of tnf-a treatment. conclusions: cxcl12 induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p53function/levels. s. y. demiroglu 1 , r. dressel 1 1 universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp70 is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp70 on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp70 does not protect against cell death mediated by ctl. acute overexpression of hsp70 can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res 63: 8212) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp70 on granzyme b-induced apoptosis. methods: hsp70 was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp70 induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type 5. results: hsp70 did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp70 in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g1 peak measurements (p=0.01). in contrast, a partial protection of ge-tet cells was observed after acute hsp70 overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp70 does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp70 effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood 102:1788) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk1034 and dr394/2-3. the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr1 and tnfr2, both belonging to the tnf receptor superfamily. tnfr1-mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr2-associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr2, resulting in depletion of traf2 from the cytoplasm. here we confirm that tnfr2 induced depletion of cytosolic traf2 by the use of tnfr1-and tnfr2-specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf2 in the alternative nfkb pathway, we show that tnfr2 induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr2 and membrane tnf. j. c. morales 1 , d. ruiz-magaña 1 , d. carranza 1 , c. ruiz-ruiz 1 1 universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase-8. in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r2 and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp36l1 is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp36l1 may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl-2 protein is an important cell survival protein at different stages of b cell development. bcl-2 mrna also contains are regions that could possibly be targeted by zfp36l1 protein. in the present study, we have tested the ability of zfp36l1 protein to bind to a bcl-2 mrna are probe and to degrade bcl-2 mrna. recombinant bacterially expressed zfp36l1 protein was shown to bind specifically to a bcl-2 are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp36l1 also provided evidence that endogenous zfp36l1 in b cells could bind to the bcl-2 are. in order to examine whether zfp36l1 binding to bcl-2 are resulted in bcl-2 mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp36l1 knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl-2 mrna was expressed in both wild-type and knockout mefs. the half-life of bcl-2 mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp36l1 does play a role in degradation of bcl-2 mrna. overall, our data are consistent with a role for zfp36l1 in degradation of bcl-2 mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp1. since lmp1 is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp1 function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi3-kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te1) and lymphocytic (nc5) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes1 or tes2 (transforming effectors site) derived from the c-terminal intracellular part of lmp1, in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp1's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase-8. using this nc5 tumorigenic model in scid mice, we showed that induction of the dn lmp1-tes2 prevent development of tumours and mouse death. these dominant negative derived from lmp1 could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs1 and socs3 differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs1 exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p38mapk activities, while socs3 promoted apoptosis with an increase in p38 activities. in contrast, both socs1 and socs3 display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs1 or socs3 induced a slight decrease in g1 or s phase cells and a prominent increase in g2/m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g1/s transition and g2/m arrest induced by socs1 or socs3. socs1 promoted dna damage-induced p21 induction and g2/m cyclin b expression, while socs3 induced decrease in g1 cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein 90 (hsp90) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp90 has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp90 can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp90 is released during necrotic cell death and proinflammatory effects of extracellular hsp90 have been observed. thus, we asked whether apoptozing cells cleave hsp90 during apoptosis or how hsp90 is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp90 protein content. we observed that hsp90 is degraded during apoptosis resulting in the formation of a fragment of about 50 kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp90 (hsp90 alpha and hsp90 beta) we could show that the 55 kda fragment is formed after degradation of the alpha isoform of hsp90. further, we were able to show, that hsp90 cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp90 and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp90 was inhibited or if exogenous hsp90 was added. these results demonstrate that cleavage of hsp90 represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc3-i is processed to lc3-ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf-7 gfp-lc3 cells were therefore incubated in starvation medium for 3 hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl-2/beclin-1 control of autophagy. recently, jnk was shown to be necessary for beclin-1 upregulation, and jnk-mediated phosphorylation of bcl-2 is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf-7 cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol 3-angelate (pep005), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep005 provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb4 turned their response to pep005 from an increased to decreased rate of apoptosis. furthermore, our data show that pep005 inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl-1 and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p75, trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch11 and analysed by flow cytometry. expression of the caspase 8 inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch11 treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase 8 in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase 8 by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases 9 and 3. diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase-8 abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor 1 (tnf-r1), and tumor necrosis factor (tnf)-a receptor 2 (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid 1-o-octadecyl-2-o-methyl-rac-glycero-3-phosphocholine (et-18-och 3 ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd95 independent of its ligand fasl/cd95l. fas/cd95 is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin-1, an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl2-family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment (5-fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo 201, sw1116, lovo, caco-2, ht-29) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki-67, pcna, p53, bcl-2) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in 88 patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl-2, bax, caspase 3, 6, 9, nfkb, parp1/89kda, tnfr1/cd120a and cd95/fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl-2, bax, nfkb, parp1 was significantly lower and expression of caspases, tnfr1 as well as percentage of cd95 cells significantly higher as compared with control group. caspase 3 expression was significantly higher as compared with nfkb, parp1 and tnfr1. in comparison to the standard nutrition preoperative immunonutrition increased bcl-2 and nfkb expressions and decreased caspases and parp1 expressions. in addition, we found a significant down-regulation of bcl-2 expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega-3 fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x 0.01). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g1 phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x 0.007). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with 54 healthy volunteers receiving either la1 (10 9 cfu/day) or placebo, during 57 days prior to uv (2 × 1.5 med). blister roofs, liquid and skin biopsies were collected 1, 4 and 10 days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p53). while a similar decrease of lc for both groups was observed on day 1 after uv exposure compared to placebo, la-1 group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd1a low cd207 -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il-10 and a tendency to inhibit il-6 was observed in la-1 group compared to placebo. on day 4, la-1 group presented a greater recruitment of early lc precursors and a trend to increase cd1a low cd207 + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il-6, tnfa, il-8, and il-10) was observed in la1 group compared to placebo. we show that la1 limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la1 supplementation for photoprotection at a lower dose (1 log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn1 is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn1 appears to be expressed in all epithelial cells of the early thymic rudiment starting around e11.5. previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn1 in a nude (foxn1-deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn1. if true, it should be possible to target this cell type by use of foxn1-promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either 19 or 82 glutamine residues under the control of foxn1 promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn1 promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei 1,2 , y. toriumi 3 , k. kimura 4 1 european university viadrina, frankfurt (oder), germany, 2 shimane institute of health science, izumo, japan, 3 shimane university faculty of medicine, department of pediatrics, izumo, japan, 4 japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a 10-minute-rest period, followed by a 15-minute yoga exercise called asana, a 15-minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a 20-minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp2). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r=0.83, p x 0.02), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r=0.77, p x 0.05) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd4 (r=0.96, p=0.0001). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd4, within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper 2 response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd4 + cd25 + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox40l expressed on mcs and the constitutive expression of ox40 (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox40l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca 2+ responses and degranulation. the cross-talk between t reg cells and mcs through ox40-ox40l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th1 or th2 effector cells and the induction of antigen specific foxp3+ regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th2 cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th2 and treg subset, gata3 and foxp3 respectively, counter regulate each other (mantel y et al., 2008) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp3+ tregs and high gata3+ th2 cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost 100 million allergic patients are sensitized to the major birch pollen allergen bet v 1, which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v 1 recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v 1 molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g 90 %) of patients' ige binding to bet v 1 was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa 30-59 (p2) and aa 74-104 (p6) of bet v 1. cross-reactive ige epitopes between bet v 1 and related pollen allergens and plant food allergens involved primarily p2. p2 and p6 are not adjacent peptides in the bet v 1 sequence but define a surface-exposed patch on the three-dimensional structure of bet v 1. as determined by surface plasmon resonance, monoclonal antibody mab2 specific for p2 and mab12 specific for p6 showed high affinity, i. e., dissociation constants, k(d) = 8.35e-11 m and k(d) = 1.05e-9 m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p2 and anti-p6 antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v 1 allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th1 cells as well as tc1 cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th2 immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th2-mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th1/tc1 cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd4 + and cd8 + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd4 (gk1.5) or anti-cd8 (53.6.72) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr-1 monoclonal antibody rb6-8c5 or monoclonal antibody nimp-r14. one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd8 + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd4 + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd8 + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd8 + t cells, cd4 + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd4 + or cd8 + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th1 and tc1 cells operative in the elicitation of airway inflammation. conclusions: robust type 1 immune responses, although highly effective in the counter-regulation of local th2-mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h292. the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p 1, we were able to show that the major allergen of phleum pratense, phl p 1, although sharing molecular similarities with der p 1, does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p 1. chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il-6 and il-8 from nci-h292 cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p 1. in contrast to hdm extract grass pollen allergens induce the release of mcp-1 from respiratory epithelial cells, as well as moderate levels of il-12. detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr22) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, 3 distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): 7.2±5.7) and of il-13 (mean±sd: 1157±909 pg/ml), and reduced il-10 (mean±sd of il-10-spot forming units/2x10 5 cells (sfu): 912±510), compared to healthy subjects (3.4±2.7 si, p x 0.05; 355±396 pg/ml, p x 0.05; 1272±623 sfu, for proliferation, il-13 and il-10, respectively); children with non-ige mediated cma had a significant reduction of il-13 (192±362 pg/ml), compared to ige patients (p x 0.0004) and an increased, although not statistically significant, production of ifn-g (33.7±54.6 sfu) compared to control (9.5±9.5 sfu) and to ige-allergic patients (0.6±0.8 sfu). finally, tolerant patients showed reduced il-13 (636±1048 pg/ ml, p x 0.05) and proliferation (3.9±3.5 si), compared to acute ige-cma children. interestingly, the high level of il-10 observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il-4 was undetectable in all patients even blocking the il4-receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th2-like response with il-13 and proliferation is dominant in ige-mediated cma patients; by contrast a th1-skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang 1 , b. chua 1 , f.c. lew 1 , a. ho 1 , k. wong 1 , k.l. wong 1 , d. m. kemeny 1 1 national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd4 th2 t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th2 cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd8 t cells inhibits the induction of the th2 response. here we have investigated the effect of cd8 t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd8 t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of 3 airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd8 + t cells (36.7 %±4.1 % to 17.6 %±2.7 %). when ifn-gamma -/-ot-i cd8 t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced (39.6 %±5.1 %), suggesting an important role for cd8 t cell ifn-gamma. cd11c + cd103 + cd11blung dcs from cd8 transferred mice secreted higher levels (500pg/ml) of il-12p70 following ex-vivo stimulation as compared with animals that were not given cd8 t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd8 t cells can subsequently divert the local lung environment to one that favors th1 immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in 8 % of patients with hiv infection. this reaction is strongly associated with hla-b*5701 and appears to be mediated by cd8+ t cells producing inflammatory cytokines. we show that cd8+t cell responses can be primed in vitro, in normal blood donors who are hla-b*5701+, but not in non-b*5701+ donors. cd8 t cells, but not cd4 t cells, are expanded by abacavir pulsed autologous apc over a13-day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b*5701. similar responses were detected in abacavirhypersensitive hlab*5701+ patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b*5701 since they were undetectable using apc expressing closely related hla-b57 or b58 allotypes. responses to apc expressing mutants of hla-b*5701 demonstrated a crucial role for residue 116. isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b*5701 triggering cd8 t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b*5701 may be relevant to the role of other disease-associated class i allotypes such as hla-b27 and b51. a. jenckel 1 , s. bulfone-paus 1 , n. föger 1 1 research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin1a (coro1a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro1a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro1a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro1a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro1a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro1a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro1a during mast cell activation. cell fractionation experiments demonstrated that the association of coro1a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro1a phosphorylation. a functional correlation between coro1a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro1a. thus, coro1a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin1a (coro1a) deficient mice have demonstrated that coro1a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin1b (coro1b) is a closely related homolog of coro1a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro1b deficient mice and crossed them with coro1a deficient mice to obtain coro1a/coro1b double deficient mice. analysis of t lymphocytes from coro1a/coro1b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro1a/coro1b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro1a/1b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro1a/1b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn1 (also known as hacs1 or sly2) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology 3 (sh3) domain and a predicted bipartite nuclear localization signal. the samsn1 gene is located on mouse chromosome 16 and encodes a well conserved protein with 364 amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn1 in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn1 expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn1 in all tested hematopoietic cell types. the highest expression level of samsn1 mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd4+ and cd8+ t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly1 (hacs2) and sly3 (sash1) -were expressed only at a very low level in mast cells. the high level of samsn1 mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn1 in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn1 deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn1 deficient mast cells. in additional studies we are now investigating the effects of samsn1 deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn1 in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg1 antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., 2008). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg1 abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st8siai-v; st6galnac i-iv, st3gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg1 abs. results: we observed that the expression of st3gal i, iii and v coding genes was similar in both hybridomas, while the st3gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg1. in addiction, the expression levels of st8sia and st6galnac genes in the hybridoma producer of anaphylactic igg1 were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg1. conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg1 abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h 2 cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after 2 times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il-5 and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il-5 and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf1 mediates the differentiation into th1 cells by activating multiple genes which are independently crucial for the development of naive t cells into th1 cells. because irf1 is expressed in many different tissues, it can be considered as a master switch factor for th1 cell differentiation. methods: here, we tested mice deficient in irf1 in the murine acute asthma model to evaluate its importance in this th2 cell-mediated disease. the protocol setup was the following: 3 sensitizations s. c. with ova, followed by 3 challenges via ova inhalation and adoptively transferred wildtype cd8+ t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf1 deficient mice with the help of adoptively transferred cd8+ t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by 3.5 fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. 1) were also significantly higher in irf1 deficient mice. conclusion: interferon regulatory factor 1 plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd8+ t cells. mechanistically, a potential counterregulation of asthma by th1 cells is not available in irf1 knockout mice. together with our previous report that irf1 represents a susceptibility gene for allergy in the human, our data highlight irf1 as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c57bl/6 mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for 24hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk1-receptor and ppt1 mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd11c expressing apc substets characterized by cd4 and cd8 expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th-1 cytokine profile and increased levels of il-2 mrna. further the number of cd25 + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: 1) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase3, 2) asthma and ee are prototypic th2 diseases in which the cytokines il-4 and il-13 play a principal role through the activation of the transcription factor stat6, and 3) we have recently demonstrated that some chloromethyl ketones can downregulate stat6 by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat6, we analyzed its effect in the activation of stat6 by il-4 and il-13. we found that treatment of cells with ppis inhibited the ability of il-4 and il-13 to signal stat6 activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th2 mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied 12 asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd4 and cd25 high (treg)), that might inhibit the development of a th2 response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for 48 hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd69 or cd25 at 48 hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h4r. the level of intracellular ccl2 production in human lc was reduced after stimulation with h4r agonists and basal production could be restored when h4r was blocked with the specific antagonist jnj7777120. moreover histamine and a h4r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h4r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h4r in allergic skin diseases and encourage further exploration of the h4r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g1p3, an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g1p3 was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g1p3 which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd20 antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th2 to chronic th1-predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received 4 weekly intravenous infusions of rituximab at a dose 375 mg/m 2 body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g 5,000; g 6,000; g 19,000 mg/dl, respectively). all patients underwent prior treatment with omalizumab for 6 months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the 2 nd week after initiation of rituximab therapy up to 1 year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up 1 year), ige levels decreased from 5,512 to 1,500 mg/dl. the other two patients are in the 3 and 1-months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd20 antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) 1 and th2 cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either 1 %dncb, 25 %tma or to vehicle alone for 0.5-6h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il-2 (mean=131pg/ml, n=12, p x 0.001), il-17 (mean=69pg/ml, n=12, p x 0.001) and il-1a (683pg/ml, n=12, p x 0.001) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of 50ng/ear of murine recombinant il-2 or of the known regulator of lc migration; il-1b. interleukin-1b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after 4 (32 %) and 16h (31 %) (n=6, p x 0.001). in contrast, il-2 or control injections were without effect. however, il-2 administration caused an increase in cutaneous il-17 production (347pg/ml, n=12, p x 0.001) compared with control injection and naï ve tissue (19 and 63 pg/ml, n=12, ns). in addition, systemic treatment with anti-il-2 antibody failed to impact on lc migration provoked by dncb (33 % reduction; n=6, p x 0.001). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il-1a (987pg/ml, n=10, p x 0.001) and il-17 (248pg/ml, n=6, p x 0.01) expression was reduced to baseline levels by anti-il-2 treatment. these data demonstrate that il-2 is not involved in the regulation of lc migration, unlike il-1b and tnf-a. however, il-2 is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il-2 may influence lc and dermal dc maturation, via the expression of il-1a and il-17. allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey 2005), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p38-mapk-pathway related protein (p38prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p38 mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct-2005 -018681, www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, 15 % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il-1 receptor antagonist (il-1ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il-1b and of il-1-like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il-1-related genes by real-time pcr on mrna from blood cd14+ monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il-18, il-18bp, and caspase-1, both before and after therapy with kineret. il-1b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il-1b, tnfa, il-12) nor anti-inflammatory cytokines (il-10, tgfb) were detected. as expected, il-1ra was only detectable after therapy. il-18 was detectable in schnitzler's sera at higher levels than in controls (23.0 vs. 10.3 pm) and decreased after therapy (13.2 pm). the circulating il-18 inhibitor il-18bp was lower than in controls and not affected by therapy. thus, free il-18 levels were increased in schnitzler's patients as compared to controls (17.6 pm vs. 7.2 pm in controls) and decreased after therapy (9.9 pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il-1-related cytokines (il-1b, il-18), nor of the il-1/il-18-converting enzyme caspase-1 in blood monocytes. however, the high circulating levels of il-18 suggest an increased activity of caspase-1, as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around 20 % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr3 signal via myd88 to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd88 signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c57bl/6 myd88-deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd88-/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg1-titers in ova-treated myd88-/-mice compared to wildtype mice, although the nacl-treated myd88-/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over 15,000 species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies (6 mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = 4) or from non-infested contra-lateral sites. expression of mip-1a, igf-1, mcp-1 and ip-10 genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd3, cd4, cd8, cd21, mhc class ii, and p46 than that of holsteins. lymphocytes expressing wc1 and cd21 antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x 0.05). infested skin of nelores contained significantly more message for mip-1a, igf-1, mcp-1 and ip-10 than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th2 response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd3+ t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il-1 plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa-1 in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam-1/lfa-1 binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th1 than a th2 local response pattern by virtue of il-1 secretion and icam-1/lfa-1 interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th2 like micromilieu in acute towards a th1 dominated milieu in chronic lesions. the genotyping of ccl4l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi07/0329). psoriasis is an inflammatory dermatosis with 2 % prevalence among caucasians. hla-cw6 allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in -308 and -238 positions. strong association was found between polymorphisms in the -238 region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b27, and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to 40 years of age) were selected. hla-a -b -c -dr -dq alleles and tnf-238 and -308 snp were differentiated by pcr/ssp. analyzing the total group, 21 patients (30.5 %) were male, 48 (69.5 %) were female. in the male group, the mean age at disease onset was 16,3 years. severe forms were seen in this group (psoriatic arthritis in 4 cases and erythroderma in 2). seven patients (33,3 %) had a favorable evolution of the disease, but 14 (66,4 %) developed extensive psoriasis, covering over 30 % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw*06 allele was positive in 10 cases (47,62 %), tnf 238 g/a genotype was found in 5 (23,8 %) and tnf 308 g/a in 4 (19,1 %). in the female group, the mean age at disease onset was 14,6 years, one case of psoriatic arthritis and one of erythroderma. twenty-nine (60,4 %) had a favorable evolution of the disease and 19 (39,6 %) an unfavorable evolution. cw*06 allele was positive in 26 cases (54,2 %), tnf 238 g/a genotype was presented in 16 (33,3%) and tnf 308 g/a in 8 (16,6 %). severe disease was seen in male patients. there was no difference in frequency of cw*06 allele between male and female groups, but there was a tendency of significant difference in tnf 238 g/a genotype. we found that c57bl/6 mice were more susceptible than balb/c and dba/2 mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il-6 and mrp8/14, among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: 136 cases, which were studied retrospectively, included children (84 boys and 52 girls), aged 3-14 years, who had an anaphylactic reaction, out of the 915 that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food (44 %-particularly sea food and dried fruit), drugs (25 %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites (9 %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema (76 %), and gastrointestinal symptoms (37 %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g 100 was found in 26 out of the 47 severest cases (55,4 %), in which the adequate control was held, while in their vast majority (45 out of 47) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in 53,7 % (73 cases) there was a hereditary family history of atopy, while in 52 children (38 %) there was also a personal history of asthma. finally, at a great percentage (69 %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that 1)the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. 2) in particular, in many cases it is proved that there is a personal but also a family history of atopy. 3) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd86 we could observe dose dependent upregulation of programmed death ligand 1 (pdl-1) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a 60 year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since 35 years. in march 2006 a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters (120 mg) every second day. in april 2008 the patient presented himself in the consult with multiple livid papules with a diameter of 3 mm in the area of the auricle. the histological examination showed an hhv-8 positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th1 type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs1, socs3 and foxp3 in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n:4), the patients who showed recurrence or progression after treatment (non-responders, n:4) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n:7) were evaluated for socs1, socs3 ve foxp3 mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd3, cd4, cd25, foxp3, cd4 + cd25 high , cd4 + foxp3 + . expression of socs3 and foxp-3 mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs1 was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd25, foxp3, cd4+cd25 high , cd4 + foxp3 + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs1, socs3 and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs1 and socs3 molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of 62 silicosis patients, with mild (21), moderate (23) and severe (18) silicosis, 92 coal workers, exposed to inorganic dust containing fcs (exposed), and 43 healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than 0.05 was considered significant.neopterin level was significantly elevated in exposed (3,2±0,8ng/ml) compared to controls (1,8±1,3ng/ml; p=0,0001). moreover, the neopterin level in exposed was similar to silicosis patients (2,9±1,6ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls (81,9±22,7au vs 64,3±16,7au p=0,0001) and to silicosis patients (69,6±20,0au p=0,002). in contrast, igmcic was significantly elevated in silicosis than in exposed (70,2±18,8au vs 62,1±24,2au; p=0,03).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p=0,001 and 0,02 respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a*01, b*08, drb1*03 (these of good prognosis) *12/*13/*14/*15) or tbc (drb1*02/*05/*14/*16 and associations with dqa1*02/*03/*05) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a*01, b*08 and drb1*03 (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a*01, drb1*14. as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd4 + , cd25 ++ , foxp3 + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein 60 (hhsp60). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd4 + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il-17, il-6, il-8, tgf-b and ifn-g production prevailed, pointing to a th17/th1 weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp60. importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th17/th1 cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp60 might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices (1) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a 384 well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c3-complement, myeloid related protein 14 and two new proteins, integrin-ß4 and fibrinogen. data from more than 100 patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than 10 % and 16 %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least 3 mice per group with distinct cytokine pattern: th1 (c57bl/10, c57bl/6) and th2 (balb/c). muscular injury was performed by injection of bupivacaine. both th1-dominant strains presented more areas with regenerating myofibers and macrophages at 4 dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd3 bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at 4 dpi. balb/c mice showed increased collagen expression and decrease of mmp-9 activity associated with more mrna for tgf-b1. this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th1 cytokines) or myonecrosis and collagen deposition (th2 cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß1-adrenergic receptor (anti-ß1ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß1ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß1ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß1ec ii -specific rat monoclonal antibody (clone 13f6), and showed by elisa that it binds to the linearized ß1ec ii peptide. additionally, we confirmed with flow cytometry that 13f6 also binds the ß1ec ii in its native conformation, i. e. directly labeled circular ß1ec ii (dyl649-ß1ec ii ) peptide. moreover, we demonstrated activation of the ß1-adrenoreceptor by 13f6 using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß1ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß1ec ii -specific peptide-based therapy, by intravenously applying a circular ß1ec ii peptide in the dcm lewis rat model to neutralize the anti-ß1ec ii antibodies. while the peptide therapy strongly reduced the anti-ß1ec ii titers in the serum by up to 80 % and consecutively lead to clinical remission, elispot assays for the detection of ß1ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß1ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß1ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b3 or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca 614-629 epitope elicits autoreactive cd4 + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca 614-629 peptide. in a first step, hybridoma cells were generated by fusing bw5147 tcra -cd8lymphoma cells with myhca 614-629 -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca 614-629 -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle 1 , a. schwarting 1 , p.r. galle 1 1 university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase 3 (pr3), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr3 in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr3-positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il-8. pr3-mrna from the murine cancer cell line wehi-274 was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr3-promoter in the second intron of the mouse pr3 gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr3 transcript and its promoter indicates that the murine pr3 may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th1 mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd1d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th2 cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n=13) or pbs (n=11) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr4 activation, in the presence/ absence of och. subsequently we transferred 1.5x10 6 mdcs (n=11) or och-primed mdcs (n=11) (3 times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a 70.6 % reduction in plaque size compared to mice treated with mdcs (p x 0.05). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant 23.7 % (p x 0.05) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a 2 to 3-fold increase in these cells was detected 3 days after the last treatment with och-primed dcs (p x 0.05). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il-10. discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th2 phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c57bl/6 background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed 350 igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c57bl/6 and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr3 regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg2c were isolated only from fcgriib deficient mice, but not from c57bl/6 control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box 1 protein (hmgb1), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb1. we investigated if hmgb1-containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb1 remains associated with ncs released from late apoptotic cells in vitro. hmgb1-ncs complexes were detected also in the blood of patients with sle. hmgb1 containing ncs from apoptotic cells induced secretion of il-b, il-6, il-10, and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd88 and toll-like receptor 2. neither hmgb1-free ncs from living cells nor from apoptotic hmgb1-or hmgb1/2-deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb1 activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il-10 release by macrophages. immunizations with hmgb1-containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr2-dependent manner. in conclusion, hmgb1 in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher 1 , k.p. hoefig 1 , e. kremmer 1 , v. heissmeyer 1 stretches and helps in the selection of the correct splicing borders. a allele of (r61h) creates a strong binding site for a splicing enhancer protein srp40 according to bioinformatics. our findings indicate that, the putative branch point, r61h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank1. finally, we believe that bank1 delta 2 protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta 2 protein may contribute to the observed reduction in sle susceptibility. s. beermann 1 , r. seifert 1 , d. neumann 1 1 hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h 1 receptor (h 1 r), h 2 r, h 3 r, and h 4 r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd3 antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or 4-methylhistamine, a h 4 r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and 4-methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c57bl/6 mice induced by either a-cd3 antibodies or cpg-odn. this histamine effect was completely inhibited by the h 2 r-specific antagonist famotidine, while h 4 r-, h 1 r-, and h 3 /h 4 r-selective antagonists had no or only moderate effects. interestingly, the h 4 r-selective reagent jnj7777120, which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd3 induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h 4 r, jnj7777120 may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h 2 r and, to a much lesser extend, the h 4 r. using this assay system, cells obtained from control c57bl/6 mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega-3 fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of 20 ng/ml resolvin e1(rve1) on the release of tgfb, il-6 and il-17 in the culture of peripheral mononuclear cells (5x10 6 /ml) stimulated by phorbol ester (pma) (1nm), and the combination of pma and ionomycine (3 mg/ml) for 72 hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from 7 healthy subjects and from 10 patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = 0.010) and sjögren's (p=0.017) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve1 significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p=0.041) or pma+ionomycin (p=0.021), however rve1 was ineffective at reducing tgfb release in the sle and ss patients. rve1 caused a non-significant decrease in il-6 release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il-17 was not significantly modified by rve1 in any of the groups tested. the release of tgfb by 20 ng/ml of rve1 can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve1 tested, il-6 and il-17 release were not significantly affected in healthy or autoimmune patients. omega-3 fatty acid derived rve1 may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis 1 , a.k. tsirogianni 1 , m. herrmann 2 , h. m. moutsopoulos 1 , m.n. manoussakis 1 1 university of athens, dpt pathophysiology, athens, greece, 2 university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included 29 with primary ss (american-european criteria 2002) and 14 with sle (acr criteria 1997). age-and sex-matched healthy blood donors to the ss and sle groups (13 donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, 2007) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma (5 patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd68-staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p=0.0001). in ss patients, defective mb-phagocytosis involved only monocytes (p x 0.0001) and significantly correlated with the presence of extraglandular manifestations (p=0.02). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x 0.0001) and of ss (p=0.001). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: 30 healthy persons have been included in our study and 71 sle patients (66 women and 5 men) with various clinical signs (44 persons had 1 st degree of disease activity, 27 persons -2 nd degree of pathological process activity). 18 women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b 2glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in 36,6 %, aphl of igg class -in 45,1 % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x 2 =6,4; p x 0,02). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x 2 =3,9; p x 0,05). the increased levels of anti-ada were revealed in 11 of 18 women with hnp, and the combination of anti-ada and aphl (9/18) was found out more often than isolated anti-ada (2/18, x 2 =6,5; p x 0,02) or isolated aphl (3/18, x 2 =4,5; p x 0,05). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc1 and mdc2, and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc1, mdc2 and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from 31 pss patients fulfilling the american european consensus group criteria (aecc) and 28 gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p=0,0002) and mdc2 (p x 0,0001) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd5+ b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin 21 (il-21) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd5+ b cells and increased il-21 levels. we therefore hypothesized that il-21 may have similar biological effects on cd5+ b cells in autoimmune diseases. here we demonstrate that the amount of il-21 in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to 14 % of cd5+ b cells in sle individuals expressed grb. in-vitro experiments revealed that il-21 was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor 9. importantly, il-21 significantly decreased the cd5+/cd5-b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd5+ b cell death. these results suggest that il-21-induced grb may play a regulatory role for cd5+ b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il-21 and grb levels in sle and showing that il-21 reduces the cd5+/ cd5-b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il-21 may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il-21 in sle and other autoimmune diseases. r. de palma 1 , e. d'aiuto 1 , s. vettori 1 , g. abbate 1 , g. valentini 1 1 second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd4+ t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il-1beta, il-6 and il-8, cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed 169 igg antibodies from single facs purified cd19+cd27+cd38+cd138+ bone marrow plasma cells of 3 patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran 1 , e. moazemi godarzi 1 , e. kamali sarvestani 1 , e. aflaki 1 1 shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin 6 (il-6) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il-6 gene at positions -572 g/c and -174 g/c have been described that are key regulators of il-6 gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il-6 gene polymorphism at -174 position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the -572 position significantly differed in sle patients and controls. at this position gg genotype was observed in 77.9 % of patients compared to 68.9 % in the control group (p x 0.014). the frequency of -572 g allele in patients (87.3 %) was also higher than in controls (83.2 %) (p=0.034). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. -174 polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x 0.04). at -572 polymorphism, a significant difference with regard to photosensitivity in male patients (p=0.04) was found. in conclusion, results of this study showed that -572 polymorphism plays an important role in susceptibility to sle and that -174 polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained 161 patients with ibd (89 with cd, 41 with uc, 31 with gluten sensitive enteropathy, gse) and 33 healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of 7 asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd4-ve cd8b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd4(+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling-1 (rgs-1) protein, we have now found substantial over-expression (10-100 fold) of rgs-1 in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs-1 decreases primary t cell responses to cxcl12 and ccl19, strongly implying that it may regulate t cell localisation. thus, rgs-1 may be a novel target for modulating t cells in ibd, consistent with which snps in rgs-1 have been associated with both coeliac disease and type 1 diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c57bl/6j mice were divided in control (c) and experimental (e) groups. c1-c3 received peanut protein immunization, animals of the control groups c4 were sham immunized, and control group c5 received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a 30 day peanut containing cd, the experimental group was divided into 5 groups (e1-e5). ova feeding began 7 days prior cd (e1) on day 0 (e2), 7 (e3), 14 (e4) and 21 (e5) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e1) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio 2 as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase 1 and secretion of bioactive il1-b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il1-b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying 5 % destrane sulphate sodium (dss) in the drinking water for 8 days to wildtype mice. when ultrafine nanoparticles were administered on day 6 and 7 by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il10 -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl 1 , n. schneiderhan-marra 1 , m. blum 2 , g. stein 2 , m. schmolz 2 , t. joos 1 1 nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, 2 edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [1] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco-2 cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of 24-well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h 1-associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h 2-like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il-17 production and t h 17 polarization and decreases recruitment of cd4 + cd25 + foxp3 + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd11c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd11c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h 17 and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen 1,2 , g. van assche 3 , p. rutgeerts 3 , a. liston 1 , j. l. ceuppens 2 1 k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, 2 k. u. leuven, experimental immunology lab, leuven, belgium, 3 k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp-1 allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced 3 types of colitis dss (th1/th17), tnbs (th1) and oxazolone (th2) in hp ko mice. neutralizing anti-il-17 mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th17/th1 cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. 1) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; 2) in dss but not in oxa colitis mice, il-17, ifn-g, tgf-b and il-6 were significantly increased (p x 0.01, dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x 0.05, ko vs wt). in tnbs colitis, we found elevated il-12 and ifn-g (p x 0.01, tnbs vs control). although not significant, il-17 was also somewhat upregulated; 3) in dss colitis we observed that il-23 enhanced differentiated th17 cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il-6 and il-21, more mln-t cells from hpko mice differentiated into th17 cells; 4) anti-il-17 mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il-17 showed reduced il-6, il-17 and ifn-g in both mln and lp (p x 0.05, anti-il-17 vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il-17, supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann 1 , g. talabér 1 , g. süt" o 2 , p. németh 1 , t. berki 1 1 university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, 2 university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that 2.2 million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd4+cd25hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th1 and th2 cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd4+ and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd4+ inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells (74.5 ± 3.5 %, mean ± sem), while cd4+ inkt cells ratio was moderate (25.4 ± 3.5 %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: 38.0 ± 7.1 %; cd: 34.0 ± 5.2 %, mean ± sem), while the percentage of cd4+ inkt cells was elevated (uc: 62.0 ± 7.1 %; cd: 65.9 ± 5.2 %, mean ± sem) in both disease groups. proportions of foxp3+ treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b1 gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b1 adenovirus (tgf-b1-exo). the t cell inhibitory function of tgf-b1-exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b1-exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b1-exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd4 + foxp3 + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b1-exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b1-exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd4 + foxp3 + regulatory t cells (treg) revealed that the relative numbers of cd4 + foxp3 + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b1 gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd4 + foxp3 + treg. tgf-b1-exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd8+ t-cells was similar in the four vaccinees as observed by ifng, il-2 production-profiles. however, the killing capacity of expanded cd8+ t-cells was distinct as observed by the competence to kill ns3-peptide presenting transfectants in vitro. as depicted in figure 1 , cd8+ t-cells cells from both vac1 (cleared ) and vac2 (chronic) produced il-2 and ifng after stimulation with ns3-peptide59. however, specific killing of the peptide loaded transfectants was only observed in vac 1, who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure 1 ] killing of ns3 peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd8+ t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd8 t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns3 from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr4-expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns3 might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns3 or eda-ns3 under the control of cmv promoter were prepared. recombinant ns3 and the fusion protein eda-ns3 were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a2 molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd40 agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns3 rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns3. results: immunization of mice with the plasmids expressing eda-ns3, but not ns3 alone, induced strong t cell responses against the main hla-a2 restricted cytotoxic t cell determinants from ns3. the recombinant eda-ns3 fusion protein, but not ns3, was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp-1 monocyte cell line. immunization of hhd mice with eda-ns3 fusion protein induced both cd4+ and cd8+ t cell responses against ns3 and, when immunized with poly(i:c) and anti-cd40 antibodies, was able to down-regulate the intrahepatic expression of hcv-ns3 rna. the recombinant eda-ns3 fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even 25 years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e2 protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd207+cd1a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd8+ responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to 3 years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd1a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd8+ t cells by mature dcs transduced with melan-a-recombinant human coronavirus 229e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin 2 (il2) and interleukin 15 (il15) will be involved, and fms-like tyrosin kinase 3 ligand (flt3l) will be expressed to modulate dendritic cells. il2 is known to enhance early t cell expansion and limits t cell overshoot, whereaes il15 guarantees survival of high affinity t cells during memory phase. on the other hand flt3l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik 1 , ag waisman 1 uniklinik mainz, 1. med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd8 t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il-10 production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd8 t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response 7 days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il-10 produced by b cells by conditional deletion of the il-10 gene in these cells. we found that b cell secreted il-10 has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb490 to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from 8 healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for 10 days in the presence of il-2 and il-7 with exposure to overlapping peptide pools of latent ebna-1 and lytic bzlf-1 antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd3, cd4, cd8, ccr7, cd45ra, il-2, tnfa, ifng and cd107a. the majority of ex vivo ebv-reactive cd4+ t cells as well as ebna-1-reactive cd8+ t cells were il-2 and tnfa-producing memory cells, the later being more frequent in bone marrow (cd4+, median, ebna-1: bm 0.69 %;pb 0.12 %; bzlf-1: bm 0.37 %;pb 0.01 %, p=0.039). after in vitro expansion a major subset of ebv-specific cd4+ and cd8+t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd4+ and cd8+ t cells retained, however, a tnfa single, tnfa/il-2 or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd4+ and cd8+ t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr7/cd45ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il-2 delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd4 + and cd8 + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd154 or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd4 + and cd8 + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd8 + and cd4 + t cells in peripheral blood with a hexon protein-spanning pool of synthetic 15-mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to 29-90 % in the t cell lines and the absolute numbers of both hexon-specific cd4+ and cd8+ t cells were 2 to 3 log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd8 + and cd4 + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype 5 (ad5) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad5 vaccine vectors expressing whv core protein was first examinated in c57bl6 mice. groups of mice were immunized with a dna prime-ad5 boost regimen or with dna and ad5 alone. ad5 was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd8+ t cell responses against peptide pools 1 and 3 in spleens of dna and dna-ad5 immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd8+ t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad5 very weak cd8+ t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad5 level of antibodies did not change. those antibodies were only igg2a subclass what indicates th1 t helper type of response. ad5-immunized mice had over 3-fold lower level of anti-whc: both igg2a and igg1 subtypes were detected. the weak response induced by ad5 may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m2e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal 2007) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m2e serum ab titers against peptide and native m2e. this correlated with a large number of m2-specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h1n1), which contains a variant m2e-sequence different in 3 amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x31 (h3n2) and the highly pathogenic pr/8 (h1n1) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m2e as a potential "universal" influenza vaccine. this research is supported by a nih t32 fellowship ca09171-32, the nih grant ai 46457 and a grant from the commonwealth of pa. l. yu 1 1 zhejiang university, zhangzhou, china interleukin-18 (il-18) is a cytokine produced by stimulated mononuclear macrophage system. in this report, 18-day-old chicken embryos were vaccined with the plasmid dna (pci-chil-18) encoding chicken interleukin-18 and the copy numbers of chil-18 in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil-18 in 18-day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil-18 could accelerate high concentrations of chil-18 in nonage peripheral blood, accelerate high expression of chil-18 in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil-18 could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil-18 (p x 0.05). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of 40nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd8 cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on 12 volunteers and a phase i study on 24 volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd8 cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv-1 neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) 120 and the transmembrane protein gp41. two sites on gp120 and gp41 are attractive targets for vaccine design: the epitope in the third hypervariable region (v3) is recognized by the human monoclonal antibody 447-52d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies 4e10 and 2f5. in order to elicit anti-hiv-1 neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp120-v3 region or the gp41-mper. the vlps are based on the acyltransferase component (e2 chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e2 chain self-assembles into a 24 nm protein scaffold resembling a vlp and that contains 60 copies of e2. efficient display and refolding of the v3 and mper regions in e2 vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the 5' of the gene coding for e2. the priming and boosting with a combination of vlps and specific hiv-1 envelope dna were used to immunize mice and rabbits. results: the v3-e2 and mper-e2 vlps were purified as stable 60mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of 447-52d, 4e10 and 2f5 antibodies to hiv-e2 monomers was confirmed by western blot. we obtained high titers of hiv-1 gp140-specific antibodies in mice immunized with a combination of vlps plus dna (hiv-1 sf162 gp160). these antibodies generated a low (20 %-27 %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e2 vlps plus dna elicited a low titer of hiv-1 gp140 specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp160 plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e2 vlps are able to display antigenic determinants of hiv and to induce high titers of hiv-1-specific antibodies. the e2 vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h5n1 whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h5n1 influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [1] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h5n1 whole virus wild-type vaccine, could induce an immune response and protect mice against h5n1 influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h5n1 antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h5n1 virus. the protective efficacy of the trivalent vaccine derived mainly from the h1n1 component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h5n1 virus, suggesting that antibodies are the main contributor to protection. h1n1 specific serum did not inhibit neuraminidase activity of h5n1 virus suggesting that protection was not mediated by neuraminidase n1-specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h5n1 whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h5n1 vaccine enhanced anti-h5n1 antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h5n1 vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h5n1 candidate vaccine. [1] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to 6 years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of 335 children in the first grade of primary school in the area of central and west macedonia. there were 234 greek and 101 foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following 1) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to 100%. 2) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). 3) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. 4) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type 1 diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om-85, a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd28 -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om-85. cytokine secretion, activation and proliferation of b cells and foxp3+ tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr2, tlr4 or the myd88 adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om-85. two synthetic tlr4 agonists used as adjuvant in human (om-174-dp and om-197-mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om-85-induced protection of diabetes required natural tregs, as nod-cd28 -/mice were not protected. remarkably, om-85 activated b cells and not t cells, promoting their proliferation and il-10 secretion, two phenomena that were tlr4-and myd88-dependent. om-174-dp and om-197-mp-ac two synthetic murine tlr4 agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd4 + cd25 + foxp3 + t cells and the proliferation of il-10 secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr4 signaling in the protective effect of om-85 on development of diabetes and show that two other tlr4 agonists induce proliferation of b cells and their secretion of il-10 as well as stimulation of regulatory cd4 + cd25 + foxp3 + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit 1 , a. legat 1 , s. thomas 1 , m. van mechelen 2 , p. hermand 2 , m. goldman 1 1 institute for medical immunology/université libre de bruxelles, gosselies, belgium, 2 glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide 4-phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor 4 (tlr4). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd14 (scd14) for its tlr4 ligand activity, we investigated the involvement of both forms of cd14 in the responses elicited by crx-527, a prototypical agp. first, we found that crx-527 efficiently induces nf-kb and irf3 activation in hek cells transfected with tlr4 and md-2 genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd14. likewise, crx-527 efficiently induces the synthesis of nf-kb and irf-3 dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd14 prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd14 respond to crx-527 but not to lps in serum-free medium. the addition of the soluble form of cd14 did not modify the levels of il12 and tnf produced by crx-527 stimulated dc but increased the levels of interferon-b (ifn-b). when scd14 was added to hek cells expressing tlr4/md-2, nf-kb activity was not modified but irf3 activity was increased in a dose-dependent manner in response to crx-527. we will further compare the responses induced by crx-527 in wild-type and cd14 deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h-2d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled 14 and 28 days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin-15 gene polymorph isms (c267t, g367a, c13687a and a14035t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [1] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n=14) have higher amount of both nonactivated (subtype infg+/cd69-, p=0.02) and activated (subtype infg+/cd69+, p=0.028) cbmc, producing ifn-g, as compared with newborns from urban mothers (n=79) exposure to pets and the risk of allergic symptoms during the first 2 years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc09/16 to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity (12-14 %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: 5 months -3 years (n=22), 4 -7 years (n=15) and 8 -12 (n=6) tb has the highest diagnostic value in children n 4 years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp-10 antigen is more sensitive for detection of tb-specific t cells compared to esat-6 antigen. 4. in children with tst 5-14 mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr2 and tlr4 in the development of spontaneous lupus disease by creating tlr2 or tlr4 deficient c57bl/6 lpr/lpr mice. methods: tlr2 and tlr4 deficient lupus prone mice have been generated by crossing c57/bl6-tlr2 -/-or c57/bl6-tlr4 -/-mice with c57/bl6 lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr2 or tlr4 deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr2 or tlr4 in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr4 deficient mice suggesting an important role of tlr4 in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr4 also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr4 pc14/13 expression of full length mcl-1 and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl-1, an anti-apoptotic protein. a splice variant of mcl-1 arises by removal of exon 2 and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl-1 (mcl-1l) and its splice variant (mcl-1s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl-1. method: neutrophils were isolated from children (diagnosed x 17 years) with jsle (n=14) and non-inflammatory conditions (control, n=14) and incubated with control serum, jsle serum alone or with jsle serum plus 20pg/ml gm-csf. quantitative real time pcr was used to assess mcl-1l and mcl-1s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl-1s to mcl-1l was also higher in jsle patients compared to controls (p x 0.05). the addition of gm-csf to jsle serum was associated with an increase in mcl-1l (1.66 ± 0.31) and a decrease in mcl-1s (2.56 ± 1.1) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl-1 compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl-1s, and less anti-apoptotic full length mcl-1 cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd4 + cyld ex7/8 t cells were transferred into rag1 -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex7/8 cd4 + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex7/8 mice are capable to control inflammatory responses. for this purpose cd4 + cd25 + cells were co-transferred with naïve wt cd4 + t cells into rag1 -/-recipients. interestingly, rag1 -/-recipients of cyld ex7/8 t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc17/16 the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a*03 in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb1*13, *01 and *0103 alleles, and a decreased allele frequency of drb1*15 in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb1*0103 is associated with the development of severe disease and positive association of cd with drb3*0301 and drb1*13. indeed, drb1*15 was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb1*07 frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb1*04 in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb1*15 were strongly increased with respect to cd and controls. however, the frequency of drb1*04 was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb1*15 is associated with uc in european, north american, japanese and korean populations methods: a total of 176 children were studied, (92 boys and 84 girls), up to 17 years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, 51 positive cases of children were found (29 %) with active infection : 28 boys (14 x 5 years of age, 12 5-10 years of age and 2 g 10 years of age) and 23 girls (10 x 5 years of age, 6 5-10 years of age and 7 g 10 years of age). pharyngitis was present in 47 children (92,2%), 39 had fever (76,5%) and 48 had lymphadenitis (94 %). the lab tests revealed leukocytosis up to 20.000 leukocytes in 29 cases (56,9 %) and leukocytosis g 20.000 in 9 cases (17,6 %). the most frequent complication documented was streptococcal superinfection in 13 children (25,5 %) and thrombocytopenia in 8 children (15,7 %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and 3) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il-13ra2, previously believed to be a decoy for il-13 only, is able to transmit a signal via il-13. our results support this and may suggest that il-13/ il-13ra2 signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il-4ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il-13 or il-13ra2 and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd4 + cd25 -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il-6 and il-1b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd4 + cd25 -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi-98, l. acidophilus ncfm tm and b. bifidum bi-504) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi-98 strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il-6 or il-1b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 1 1 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed 21 pediatric cd patients (13 active, 8 remission), 24 pediatric uc patients (17 active, 7 remission), and 37 age-matched non-ibd controls. nkg2d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x 0.05 was considered significant. results: nkg2d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd3+cd56+ nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg2d ligands on circulating monocytes is also observed. the dramatic increase of nkg2d+ lymphocytes, and the strong upregulation of nkg2d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg2d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc17/26 peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp3+ t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd4+ t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp7 knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß5i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp7 knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp7 knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp7 knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than 200 million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec-205 antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa 1-191) or hcv-ns3 (aa 1027-1218) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns3 and core we successfully optimized culture and protein purification conditions. briefly, ns3 was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec-205 to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec-205/ hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd01/2 mva-nef vaccination induces polyfunctional cd4 t-cells and increases the proliferative capacity of cd8 t-cells in hiv-1 infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv-1 protein nef (mva-nef) was safe in hiv-1 infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd4 t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il-2 and mip-1b, the expression of the activation marker cd154 and the differentiation marker cd45ra in nef-specific cd4 and cd8 t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il-2 and mip-1b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd4 t-cell response and a significant increase of polyfunctional nef-specific cd4 t-cells, simultaneously expressing ifn-g, il-2 and cd154. using the standard ics no increase of nef-specific cd8 t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd8 t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il-2 and cd154 expressing cd4 t-cells and the increase of proliferating cd8 t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd4 t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd8 t-cells in hiv-1 infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv-1 mediated membrane fusion and p24-detected replication. db81 was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with 25mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd4 + and cd8 + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns4 (1677-1756 aa) and rns5a (2061-2302 aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/2j mice were immunized intraperitoneally 2 times at a month interval with different doses (0.1 -2 mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns4 protein. immunization with rns5a-immunomax conjugate and rns5 in cfa (1.4 mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns5a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns5a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd01/21 degree of cross-genotype reactivity of hcv-specific cd8 t cells directed against ns3 the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd8 t cell response targeting hcv genotype 1 (gt1) and genotype 3 (gt3) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: 53 subjects (17 with gt1, 22 with gt3 and 14 anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns3 from gt1 or gt3. individual reactive peptides and the degree of cross-reactivity between the gt1 and gt3 variants were determined by ics. complete ns3 is sequenced from all viremic patients pd01/22 anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes 1, 4, 6 or 9 suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes 1, 4 or 9, but not 6 led to a significant reduction in viral loads. decreased splenic viral load after ifna1 treatment correlated with an expansion of activated fv-specific cd8 + t cells and nk cells in the spleen, whereas in ifna4-and ifna9-treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna2, 5 and 11 are under investigation pd01/23 elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd8 t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd8 t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd8 t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a*0201-or k b -restricted cd8 t cell responses by these dna vaccines differed. k b /ova 257-264 -and k b /s 190-197 -specific cd8 t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd4 + and cd8 + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least 16 healthy, randomly chosen blood donors were cultured for12 days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. 48 out of 72 tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than 50 % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of 5 peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, 3 hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd8 + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd8 + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il-12-exposed cd8 + t cells showed at least five-fold increase of survival rate in vivo than il-2-exposed cd8 + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd8 + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il-12-exposed cd8 + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd8 + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il-12 and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x 0.01) . there was a significant elevation of t ada and ada1 activities in iga deficient patients as compared to healthy individuals (p x 0.01) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd01/61 hiv-1 sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after 2-6 weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in 12 out of 17 patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, 10-day-old ross 308 broiler chickens divided randomly into 3 groups, 2 experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/79 strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/79 and h120 strains, via the drinking water at 10 days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, 7&14 days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd4 t cells from 25 patients with active tb and 28 patients with successfully treated tb were analysed for simultaneous expression of ifng and il2 at the single cell level using multi-colour flow-cytometry after 6 hours stimulation with ppd. moreover, cells were stimulated with esat-6 and cfp-10 receiver operator characteristics analysis revealed that a percentage of ifng /il2 dual positive cells x 56 % served as an accurate marker for active tb patients (specificity 100 %, sensitivity 65 %), while frequencies g 56 % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd03/7 necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in 1993 and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately 10 100 000 inhabitants) is 5.33 to 100 000. among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, 12 out of 14 children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently 6.12 to 100 000 inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of 1986 to 1993 led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd03/8 novel analogues of thalidomide inhibit cd80 expression and production of tnf-a, il-12, ifn-g, cxcl-9 this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta1-dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta1r7k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd80 and cd86. these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il-1a and il-1b, while il-6 levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il-1b production. using bmdc from nlrp3 -/-mice we examined il-1b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il-1b production is dependent on nlrp3. collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho-1 expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from 20 % in wild type (hmox1 +/+ ) mice to 87 % in ho 1 deficient (hmox1 -/-) mice. hmox1 -/-but not hmox1 +/+ mice developed end-stage multiorgan failure. mortality of hmox1 -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h 2 o 2 or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb1. similarly, circulating and cytoplasmic hmgb1 was increased in hmox1 -/-relative to hmox1 +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b-1,2-linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at 4-6 years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and 2-4 weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were 7.7 and 9.4 iu/ml and those of dtwp-pasteur were 8.2 and 8.6 iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were 30.2 eu/ml for dtwp-local and 47.9 eu/ml for dtwp-pasteur vaccines, respectively (p x 0.001). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd05/18 united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b:2 , a gram-negative coccobacillus. jrmt12, an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of 10 8 cfu of jrmt12. a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b:2 on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for 1 h before adding concanavalin-a (cona) pd06 -vaccination and immunotherapy against parasitic diseases pd06/1 evaluation of simian adenoviral vector adch63 expressing msp-1 as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype 5 (adhu5) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch63 is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp-1 and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm128 while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho-1 expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox1 locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho-1 is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp63 expression was confirmed by sds-page and elisa using monoclonal antibody against gp63. discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a5-180recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a5-180recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least 6 month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at -20°c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and 2 seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r=0.216, p=0.641) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il-17 also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il-17 on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il-17 to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il-17 and il-17-related inhibition of cfu-e growth were dependent on p38 mapk activity, since the p38 mapk inhibitor, sb203580, markedly downregulated il-17-induced activation of nos and reversed the growth inhibitory effects of il-17 on cfu-e. the in vivo stimulating effect of il-17 on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il-17 effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il-17 acts on bone marrow cells and also revealed a link between the il-17, no and hematopoiesis. further studies on il-17-mediated induction of both inos and enos methods: a total of 1785 blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of 68 samples (3,8 %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x 100 iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had 100 iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of 10 iu/l anti-hbs in all the blood units, which also goes for our study pd14/20 neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen 1,2 1 national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine 297 can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa-1a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa-1a iggs we monitored 12 children (9 boys and 3 girls) in ages from 3.2 to 17.2 years with average age of 8.9 years. in 11 of them all was diagnosed for the first time. 1 subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest (11 subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc3h1 gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. 2005, nature 435, 452-8). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 3'utr of icos, in which a 47bp sequence, containing a possible mir-101 binding site, was sufficient (yu et al. 2007, nature, 450, 299-303). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. 2000, j biol chem 275, 33655-62). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia-1-and ago2-deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r01 ai 42269. we have recently reported that 6-hydroxyl-1-methylindole-3-acetonitrile (6-hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw 264.7 macrophages. in this report, we investigated the effect of 6-hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin-8 (il-8), monocyte chemotactic protein-1 (mcp-1) in ht-29 human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed 6-hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated 6-hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. 6-hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p65. in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of 6-hma significantly reduced the mrna levels of il-8 and mcp-1 stimulated by tumor necrosis factor-a (tnf-a) in the ht-29 cells. pretreatment of 6-hma also significantly blocked the ixba degradation and nf-xb p65 nuclear translocation stimulated by tnf-a in the ht-29 cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of 6-hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, 6-hma could be new potential therapeutic agent for inflammatory bowel disease.cd4 serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd4 igg immune response in hiv-1-exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp120-binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd4 domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp120, reacted with the 2 external domains of soluble human cd4, in the absence of the target cells expressing the co-receptor ccr5. the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd4-gp120 complex and trigger the membrane fusion between effector (gp120 expressing) cells and target (ccr5 expressing) cells. thus, in the absence of ccr5 we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv-1 infections. a conventional protocol for the generation of monoclonal antibodies was used. db-81 (igg1, x), one of the anti-cd4 antibodies obtained, recognized preferentially cd4 complexed to gp120, as compared to cd4 alone, not competed for the gp120 binding site on cd4 and was specific for the second extracellular domain of cd4. g. röder 1 , l. geironson 2 , a. darabi 3 , m. harndahl 1 , c. schafer-nielsen 4 , k. skjödt 5 , s. buus 1 , k. paulsson 2 1 copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, 2 lund university, immunology bmc d14, lund, sweden, 3 lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, 4 schafer-n, copenhagen, denmark, 5 department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first 87 n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a*0201. to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn 1-87 /80 , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn /80 was demonstrated to be located on tapasin [40] [41] [42] [43] [44] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first 87 n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd8+ t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region 301-498 of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b*2705-restricted np-flu epitope (aa 383-391) was replaced by hla-b27 or hla-a2-restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard 51 cr release assay and through the ifn-g production. results: using hla-b27 or hla-a2 restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b27 molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a2 molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a2 restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b27 as a remarkable hla-class i molecule that, differently from hla-a2, can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b27 molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv-1)-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv1. consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p17-gag and p24 region was determined in vitro. 25mer peptides were digested with i20s and c20 proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv-1 p17-and p24-gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers 1 , g. koster 1 , o. landsverk 1 , f. skjeldal 1 , o. bakke 1 1 university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi3 kinases and thus ptdins(3)p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier 1 , l. visvabharathy 1 , j. fu 1 1 university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e3-19k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e3-19k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type 2 e3-19k and class i molecules.results: we showed that e3-19k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a11 molecules, with the mature form being more tightly associated. we also provided evidence that e3-19k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e3-19k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e3-19k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il-12, il-6, tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands 2 and 4, but also with the t-celldependent stimulus cd40l (cd154) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd40, cd83, and cd86 was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p38 and erk-1/2. strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd4+ t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th2 (il-4+) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th1 (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th1 / th2 balance and to identify potential t cell target antigens (ags), we analyzed circulating cd4+ t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il-4 expression in peripheral blood cd3+cd4+ lymphocytes were measured in 9 ccs patients and 7 healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il-4/ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il-4 and lower ifn-g intracellular expression were detected in cd4+ t cells from css patients, as compared to hs (il-4: 1.3±0.3 % vs. 0.50±0.21 %, p x 0.05; ifn-g: 14.2±4.5 % vs. 27.0±4.8 %, p x 0.025). similar results were obtained by elisa (il-4: 0.39±0.16 vs. 0.30±0.07 pg/ml, p x 0.05; ifn-g: 31.0±14.3 vs. 79.0±15.0 pg/ml, p x 0.05). elispot counts of hexavalent vaccine-stimulated cd4+ cells were positive for il-4 in 4/5 (80 %) css patients and in 5/5 (100 %) hs, and for ifn-g in 2/9 (22 %) css patients and 7/7 (100 %) hs. mpo stimulation determined significant ifn-g release in 5/8 (62 %) css patients, but not in hs (0/5) no il-4 response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd4+ cells from css patients show a th2-polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd4+ t cells, and responses towards it are instead th1-polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd4+ t cells in css. a. voigt 1 , e. opitz 1 , k. savvatis 2 , k. klingel 3 , k. stangl 1 , u. kuckelkorn 1 , p.-m. kloetzel 1 1 charité -universitätsmedizin berlin campus mitte, berlin, germany, 2 charite -campus benjamin franklin, berlin, germany, 3 universität tübingen, tübingen, germanymurine models of coxsackievirus b3 (cvb3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp2 of the stress-induced ip, were infected with 1x10e5 pfu cvb3 nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp7 as early as day 4 p. i. in lmp2-deficient mice. however, lmp2-deficiency was linked to less severe myocarditis at day 8 p. i. (he stain of cardiac tissue sections: wt 1.95 ± 0.20 vs. lmp2-deficiency 0.71 ± 0.06 (grade of myocarditis; scale 0-4; p x 0.001). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x 0.05), there was no difference in lmp2-deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb3 infection in lmp2-deficient mice. likewise, diastolic function was preserved in lmp2-deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp2-deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp2-immunosubunit function in regulatory processes of viral replication. absence of lmp2 confers host protection in enterovirus myocarditis. h. w. liao 1 , j. xu 1 , j.q. huang 1 1 sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype 2 (den-2) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr1 and tnfr1-2 were constantly very low whereas trailr2-4 decreased after den-2 infection. fasl was expressed at similar levels on huvecs throughout den-2 infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase 8, caspase 3 were also observed by western blot after by den-2 infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for 7 days into wis rat paw web tissue in saline containing 7.5x10 7 cells. group ii. s.epidermis was injected as in group 1 after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day 8.they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors 2,23 and 3,91 respectively (p x 0.05). immunohistochemical pictures showed increase in percentage of ox62 (migrating dendritic cell), mhc ii, his48 (granulocytes), ox7 (stem cells) and cd54 (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed1 (macrophages) and ox62 cells. popliteal lymph nodes contained evidently less of ox62, his48 and mhcii cells than in group i (p x 0.05). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega-3 fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega-3 fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega-3 fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega -3 fatty acids consumption. the results indicate that omega-3 fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega-3 fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega-3 fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density 1.24 and 202,000g, top 20 % layer contained lipoproteins only and 20-50 % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising 1 % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin-1. objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e2f2 mutation, and other affecting b cell apoptosis control, such as bcl-2 over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c57bl/6 (b6) genetic background. e2f2-/-bcl-2tg were obtained backcrossing e2f2-/-and e2f2+/+hbcl-2tg mice. e2f2-/-bcl-2tg, e2f2-/-, e2f2+/+hbcl-2tg and control mice (e2f2+/+) were followed up to 18mo-old. serologic studies: serum samples obtained at 3, 9 and 15 month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of 3, 9 and 15 mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h3]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl-2tg in b lymphocytes of e2f2-/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e2f2-/-bcl-2tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e2f2-/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e2f2 or the overexpression of a bcl-2 tg in the b6 genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor 9 (tlr9) abolishes the generation of antinucleosome igg2a and igg2b autoantibodies but exacerbates lupus disease. however, the tlr9-dependent tolerance mechanism is unknown. here we show that loss of tlr9 in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th1 and th17 t cells. transfer of a synthesized monoclonal polyreactive igm to tlr9 deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr9-dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n=115) (vitali et al. 2002) . ninety-seven of 115 (84 %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g 1 (fs+), while biopsies from 18/115 (16 %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in 27/115 (23 %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x 0.05). interleukin (il) 17, il-1ra, il-1beta, il-12p40, il-15, macrophage inflammatory protein (mip) 1alpha, mip-1beta, eotaxin, interferon (ifn) alpha, and il-4 levels were significantly increased in the gc+ patients (n=27) compared with the gc-patients (n=70). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il-4 found in these patients (gao et al. 2006) . increased titers of th17-associated cytokines, il-17, il-1beta and the il-23 subunit il-12p40, may indicate a higher activity of these cells in gc+ patients (nguyen et al. 2008) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il-6. the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg2. production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g 50 %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd88-dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd88-dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/-1 baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il-8. objectives: increased levels of il-18, an innate and inflammatory cytokine of the il-1 family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il-18 and other genes of the il-1/il-1r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from 48 patients and 32 healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il-18 and il-18bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il-18 are higher than in controls. serum il-18 correlates with disease activity and decreases upon remission. monocyte expression of the receptor il-18rb is increased and correlates with disease severity, while expression of tir8/sigirr (a down-regulatory receptor of the il-1r/il-18r family) is reduced. in mrl lpr/lpr mice, expression of il-18, caspase-1 and il-18rb genes precedes disease onset in lymph-nodes. in other organs, changes in il-1-related genes (il-33 and tigirr-1 up-regulation, tir8/sigirr down-regulation) occur after disease onset. free il-18 levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il-18 levels correlate with autoimmune lupus both in mice and humans. free il-18 may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il-18rb is a marker of pathology, suggesting increased il-18-dependent activation. both in mouse organs and human monocytes, tir8/sigirr expression decreases with disease, suggesting impaired control of il-18r activation. thus, il-18 may be involved in autoimmune lupus pathology, and il-18-related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f4 (amino acid (aa) 451-593) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f4 was recognized by all patients. fragment f1 (aa 5-30) was recognized by 9 of 34 dcssc patients. fragment f8 (aa 350-400) was recognized by 4 of 8 sle patients. analysis of clinical data revealed a significant difference between the f1 negative and f1 positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f1 and f8 could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd4 + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd4 + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril-2. by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd4 + t cells enriched in tregs were co-cultured with activated cd4 + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd4 + t cells anergy was also evaluated based on cbl-b, grail and foxp3 mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd4 + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd4 + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b-1 cells through targeting p110d by shrnas strategy. methods: we used the drugs, ly294002 and wortmannin, pan-specific inhibitors against pi3ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p110d. we then introduced either pan-specific inhibitors against all pi3ks or p110d-targeting shrnas into an sle-prone animal model, nzb/w f1 mice, for therapeutic purposes. the results suggested that pi3ks are not only important for the development of b-1 cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p110d. either pan-specific inhibitors against pi3ks or p110d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f1 mice. one inhibitor, ly294002, and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi3ks are critical for the maintenance of b-1 cell populations might shed light on future treating other diseases associated with b-1 cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool 1 , m. alarcón-riquelme 1 , s. kozyrev 1 1 institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank1 gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs10516487, r61h). we identified that bank 1 gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon 2 (delta 2). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank1 is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank1 isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs17266594), which is in complete ld with r61h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia1, which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a 57-year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti-2gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last 1year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow 1 1 cnrs 9021, strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed 18 patients, with minor clinical and/or biological manifestations of their disease, for at least 6 monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below 2. b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd19 surface protein for all patients. this cd19 lower expression is associated with cd45 lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf2 expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf2 lacking an exon in the c2-domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf2 in t-cells. smurf2 expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf2 expression was upregulated by tgf-beta stimulation in t-cells and smurf2 was markedly upregulated in tumor infiltrating cd4+ lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day 42, smurf2 transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf2 showed an upregulation of the tgfbrii and an activation of smad2 and 3 as compared to wild-type t-lymphocytes, which were previously described as typical smurf2 targets for degradation. in addition the transfection of smurf2 and a caga-luc plasmid into cos-cells for smad3-promotor studies yielded the same effect as shown by upregulation of the smad3 activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf2 splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf2 has beneficial effects on mucosal inflammation and tumor development. smurf2 thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat3 has important functions in cytokine signalling in a variety of tissues. however, the role of stat3 in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat3 activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat3 activation in dss colitis is dependent on il-22 rather than il-6. il-22 was secreted by colonic cd11c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat3 activity were highly susceptible to experimental colitis, indicating that epithelial stat3 regulates gut homeostasis. stat3 iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat3 regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il-22 and epithelial stat3 was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat3 activation regulates immune homeostasis in the gut by promoting il-22-dependent mucosal wound healing. stat3 seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd40 and tlr4 expression at day 3 and day 7 after infection. mycobacterial infection did not result in differential tlr2 expression as compared to uninfected cells. cd40 is involved in stimulating th1 pro-inflammatory responses, although map may interfere with cd40 signalling (1) . tlr4 signalling elicits anti-inflammatory responses, which can contribute to bacterial replication (2) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd40 and tlr4 receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd8 t cells by exogenous antigen leads to colitis. methods: eight million naïve cd8 + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h2-kb, were transferred i. v. into b6 mice. at day 0 and 4, mice were treated intra-rectally (i. r.) with 50 % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day 2 after the injection of ot-i cells. the phenotype of effector cells was evaluated at day 5 by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd8, the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd8 + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd8 + t cells. our study demonstrates that the local activation of antigen-specific cd8 t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b6 mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of 535 patients, (248 male and 287 female), with age average 69,7 years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x 2 and t-test programmes. results: in 372 patients (69,5 %,with age average 62,7 years of age) no antibodies were detected. on the contrary, in the remaining 163, 52 male and 111 female, (30,5 %, with age average 76,4 years of age), antibodies were detected. out of them, in 106 cases the results were strong positive (32 male and 74 female) and weak positive in 57 cases (20 male and 37 female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: 1) helicobacter pylori infection is relatively common in the general population (30,5%).2) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women).3) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova 1 , t. ulmannova 1 , k. stechova 1 , k. tesarova-flajsmanova 1 , v. stavikova 1 , j. nevoral 1 1 charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of 20 mothers whose infants were diagnosed with allergic colitis and 20 mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis (2-27 weeks, average 16.8 weeks of infant's age) or at the age of 12 weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il-4, il-6, il-10, il-17, il-23, ifn-gamma, tgf-beta1, egf and eotaxin. man-whitney u test was used for statistical analysis, p x 0.05 was considered statistically significant.results: il-10 as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x 0.001) in breast milk of mothers whose infants were suffering from allergic colitis (range 0-8.45 pg/ml, mean 2.1 pg/ml) than in control group (range 0-3.41 pg/ml, mean 0.35 pg/ml). higher concentrations of il-4 and lower concentration of tgf-beta1 were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. 8310-5. background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il-8 (murine homologs kc and mip-2) and its receptor cxcr2 are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated 12 h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at 2, 4, 16 and 22 h post-transfer. anti-kc antibody or its isotype control was administered at 20mg/mouse one hour before transfer followed by whole body and organ imaging 4 hours post-transfer. results: facs analysis revealed 80 % neutrophil purity, 35 % of which were cxcr2 + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, 4 h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than 10 15 bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model (1.5 % dss in drinking water for 1 week, followed by pure drinking water for 1 week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, 2 % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell 2004), treatment of commensal-depleted mice with the tlr4 ligand lps in drinking water protected against the lethality of 2 % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of 1.5 % they may become pathogenic and drive an intestinal inflammation. at 2 % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by 1.5 % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type 2 immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il-13 production which is an important pathological factor. neutralizing il-4 or il-13 prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th2 cytokines in colitis remain undefined, we used mice deficient in il-4/il-13 or the key receptor through which they signal, il-4ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il-4ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il-4/il-13 double deficient mice which were protected from colitis. removing il-13 production from il-4ra -/mice, by using il-4ra/il-13 double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il-13 mediates susceptibility in an il-4ra independent manor. recent evidence pc17/33 introduction: the activation of cd4 + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th1-like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th1 cytokines, such as il-5,-6 and il-13. however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc2 was found in uc and cd colonic tissue compared to control specimen. transmitted to the th2-mediated oxazolone-induced colitis model, nfatc2-production is significantly increased in both diseases, too. nfatc2 deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc2 deficient mice compared to control mice, which can be observed by tunel assays, caspase3 and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl-2 and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il-6, ifn-gamma, il-13 and il-17 by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc2 regulates il-23/il-17 in an indirect way. last, administration of il-6 blocked the protective effects of the nfatc2 deficiency in experimental colitis, suggesting that nfatc through il-6 signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc2 in colitis by controlling mucosal t cell activation in an il-6 dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd4cd45rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd4cd45rb high t cells also regulatory cd4cd45rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at 3 different time points during colitis progression. after 3 weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at 9 weeks. microrna was isolated from colons of mice in different stages of colitis progression (3, 6 and 9 weeks) and control mice that do not induce colitis (n=3 for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from 11 micrornas that demonstrated an induction during the development of disease we selected 4 micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd4cd45rb high transfer model. objective: the purpose of this clinical trial (id: nct00287677 of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for 6 months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in 3 groups: group a (n=8) receiving haart+ gh (for 6 months) + tt+hva/b vaccines (at month 6 post gh adminsitration); group b (n= 6) receiving haart+gh but not vaccines; and group c (hiv control group, n=7) with haart+vaccines (at month 6) but without gh. all patients are followed up 6 months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after 24 weeks of administaring hormone the absolute numbers of cd4 incresase from 562 ± 93 to 704 ± 112 cells per mm 3 (mean and sem; p x 0.0025). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd4 absolute numbers from 550±97 to 470±103 cells per mm 3 (p x 0.02). viral load remained undetectable in all patients. despite the increase in cd4 counts the percentage of recent thymic emigrants (as assessed by the expression of cd31) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv (80,000-160,000), hepatitis c virus (hcv; 2.3-4.7 million) and hepatitis b virus (hbv; 8-16 million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv-1 infected patients with g 350 cd4 + t-cell counts and plasma hiv-rna levels of x 1.7 log 10 copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from 4 timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g 50 and when the responses were g 2 times the standard deviation above the mean of replicate negative controls (mock electroporated dc). 16/17 patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in 4/16 patients before and 11/16 patients after vaccination. for rev, 7/16 patients showed a pre-existing rev specific response and 14/16 patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already 11/16 patients responding before and 16/16 patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in 13 out of 16 patients before vaccination and 16/16 patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus 71 (ev71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev71 is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev71 inactivated virus. methods: mice were immunized intraperitoneally with 10 mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after 0, 2, and 4 weeks. blood samples were collected at 0, 21, 35, and 45 days. the total serum anti-ev71 igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il-5 from splenocytes was also measured. results: immunization with ev71/ps-g showed that the anti-ev71 igg levels were significantly increased compared with ev71 alone or ev71/cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev71/ps-g group compared to ev71 alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev71/ps-g-immunized mice compared to those of ev71 or ev71/cfa/ifa-immunized mice, indicating a th1 cells response elicited by heat-inactivated ev71 vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd8+ t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd8+ t-cell responses against hcv-ns3, studying induction of il-2, ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il-2 and ifng production by cd8+ t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv-1-recognizing cd8 + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv-1 replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd8 + t cells by electroporation, and chose tcrs which were able to recognize the hla-a2 restricted hivpol-peptide iv9, or the hivgag-peptide sl9. results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il-2, tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd25, after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd4 + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd8 + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd8 + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt3l pretreated balb/c mice were incubated for 3 hrs with hcv ns5-coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g 80 % dcs and was used for immunization. dc expression of the maturation markers cd40, cd80 and cd86 was determined before and after ingestion of ns5-coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns5 expressing sp2/0 cells). results: in immunized animals, the ctl activity was increased 3-fold compared to mock immunized mice. accordingly, tumor challenge with ns5 expressing tumor cells showed a significant reduction in tumor growth. the number of cd4 + ifn-g + cells was increased g 3-fold and the number of cd4 + il-2 + increased g 5fold in the dc-ns5-bead immunization group. these results paralleled the proliferative response of splenocytes to ns5 protein obtained from immunized animals with the most significant response in the cd4+ population of dc-ns5-bead immunized animals. the use of ns5 coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th1 biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec-205 antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec-205 induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec-205 promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec-205 antibody can be developed. we screened the anti-dec-205 sequence computationally for putative hla dr4-restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec-205 antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec-205 as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih 1r21ai078800 a live oral vaccine based on human adenovirus (ad)4 has proved safe and effective in us military recruits for nearly 50 years. in these experiments, we have investigated whether replication-deficient ad4 can be an efficient potential vaccine carrier for oral vaccination. ad5 vectors were used throughout to provide a benchmark for efficacy. we generated novel ad4 and ad5 vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad4 than ad5. ad5 routinely infected and provided transgene expression in˚10 % of human cd4+ and cd8+ t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to 25 -30 % of cd8+ t cells present, showing that ad5 infected a surprisingly large proportion of t cells. in comparison, ad4 provided egfp expression in x 2 % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad4 and ad5 vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad4 and ad5 induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day 3 after dosing, and transgene expression being reduced below detectable levels by day 8. interestingly, when delivered together ad4 and ad5 vectors targeted the same subset of cells. together, these data show that ad4 is a viable alternative to ad5-based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies (20 recently commercialized; g 300 in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the 667 mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that 667-treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g 8 months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, 2005; gros et al, 2008; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd8 + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp60) of rhdv at two different locations: 1) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp-2), and 2) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp-306). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd8 + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp-2) was more immunogenic than insertion at position 306 for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp-2 at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr-2 ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd8+ t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd8+ t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np 147-155 specific cd8+ t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr-2 targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l929cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during 7 days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd2+, cd4+, cd8+, cd16+ t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by 1,5-2-fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns4 and ns5a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h5n1 influenza virus inhibits the ifn-g synthesis (mibayashi et al., 2007) and causes a decrease in cd4 + and cd8 + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., 2000) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a 10 amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions 129-131 (tat'kov c. i. et al., 2000) . deltaferon was i. p. administered to male non-inbred mice in doses of 2-40*10 4 iu once or twice at an interval of 48 hours, alone or in combination with double-stranded yeast rna preparation (5 mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., 1983) . results: when deltaferon was administered in doses of 2-20*10 4 iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of 2*10 4 iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon (2*10 4 iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd8 responses (tetramer+ hiv-1 gag p24 cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd8 responses after pla-p24 immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p24 were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas 1 , c. prego 2 , s. vicente 2 , m.j. alonso 2 , a. gonzález-fernández 1 1 university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, 2 university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer 188, with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with 1 or 2 immunizations to balb/c female mice (4 weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day 15 to 260 post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease (100 miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigenbackground: infection with human immunodeficiency virus type 1 (hiv-1) is characterized by dysfunction of hiv-1-specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p24 antigen from hiv-1 and the tlr3 ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p24 microcapsules containing p24 and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for 10 days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p24 peptides. intracellular staining of interferon-gamma (ifn-g), interleukin-2 (il-2) and cd107a after p24 stimulation was also performed. results: mddc from hiv(-) subjects, incubated for 24 hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p24 containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il-2, upon p24 peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd16b between our model cytokine il-2 and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p30gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il-2 dependent proliferation assays of il-2 variants. results: murine il-2 attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il-2 dependent cell line ht-2. we found that the membrane anchors comprising one and four ig-like domains (il-2::1iggpi and il-2::4iggpi) resulted in severely reduced vlp production by 293 producer cells and despite of an increased targeting of il-2::4iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il-2 fused to the minimal gpi-anchor acceptor sequence of hcd16b (il-2::gpi). il-2::2iggpi, however, showed comparable particle production and biological activity in vitro when compared to il-2::gpi. furthermore, il-2 fused to 2ig::gpi showed an increased capacity to co-stimulate primary p14 tcr transgenic t-cells specific for lcmv-gp 33-41 in the context of h2-d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. 2ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il-2::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f1816-b13 of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant 812079 & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild 1 , interdisciplinary transplant laboratory 1 charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects 2-25 % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of 2-3 months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with 6 different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from 2,5 month to 16 days. t cell lines are composed of cd8 and cd4 cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker 1 , m. assenmacher 1 , a. richter 1 1 miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp65-specific cd4 + and cd8 + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd45ro -cd25 -) cd4 + and cd8 + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp65 peptide pool and cd3-depleted autologous pbmc as feeder cells in the presence of il-2, il-7, and il-15. already 9-13 days after primary activation pp65 495-503 /a2-tetramer + cd8 + t cells were detectable for 3 hla-a2 + blood donors. to analyze cd8 + t cells having other specificities than for the peptide pp65 495-503 as well as probably primed cd4 + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to 3.9% of the cd8 + t cells and up to 3.8% of the cd4 + t cells after restimulation with pp65 peptide pool, but not with either irrelevant ie-1 peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for 20 days led to a 10 -100 fold expansion of pp65-specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd154 and cd137 only if restimulated with the pp65 peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il-2, indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd4 + and cd8 + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf 1 , k. mozdzanowska 1 , l. otvos 2 , j. erikson 1 1 the wistar institute, philadelphia, united states, 2 temple university, philadelphia, united statesthe influenza virus a matrix protein 2 ectodomain (m2e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m2e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine 2003; virology journal 2007), we generated a novel peptide and investigated its efficacy in inducing an anti-m2e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd8 + t cells, have been considered. clinical data confirm a crucial role for antiviral cd8 + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a*0201 and hla-b*0702 previously. considering other frequent hla alleles cmv specific cd8 + t cells were monitored longitudinally in 30 hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd8 + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd8 + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a*2402/pp65 (341-349) (7.6x10 6 cells/l) and hla-b*3501/pp65 (123-131) (4.4x10 6 cells/l) specific cd8+ t cells are significantly lower than those for hla-a*0101/pp50 (245-253) (17.2x10 6 cells/l) and hla-a*0201/pp65 (495-503) (13.2x10 6 cells/l) specific cd8 + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. (2,5 -50 mcg) . no adverse effects were indicated during trials (up to 25 month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after 4th immunization with 50 mcg of vichrepol.no differences were detected in cd4+ and cd8+ t cell counts and cd4+/cd8+ ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp70 or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp70 or gp70 alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb6f1 hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb6f1 mice with adenoviral nanoparticles expressing fusion proteins containing gp70 resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd4 + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd4 + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf01 exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted 90-99 % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf01 did not show any increase in their levels of ifn-gamma. conclusion: caf01 enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from 13 children aged 1-2 years old (6 boys and 7 girls) -group 1, and 11 children (6 boys and 5 girls) 6-7 years old -group 2 were isolated on a gradient of density before vaccination ( or revaccination) with priorix, 1 week, and 2 months after and incubated with cfse. then 2 million/ ml pbl were incubated in rpmi-1640 supplemented with 10 % fcs (the negative control), at presence of 5 mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing 5 % cî 2 at 37°c within 7 day. intensity of a fluorescence estimated on fl1 by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control 90 % pbl in both groups did not enter mitosis. in the positive control 90 % of cells have passed one and more mitoses. in group 1 measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in 2 months 15-25 % of lymphocytes demonstrated antigen-specific proliferation. in group 2, on the contrary, before the vaccination the most part of cells (75-80 %) has not entered division, but 20-25% of cells have passed 2 and more mitoses. in a week specific lymphocyte proliferation decreased and in 2 months it was increased up to 40-50 %. production of the interleukin (il) 6, ifn-g, tnf-a, il-4, il-1 was more informative than il-5, il-7, il-8, il-12. measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp120 cd4-binding site (cd4bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv-1 neutralizing polyclonal iggs directed against the cd4bs. the mabs were shown to react with an anti-cd4bs human neutralizing mab (b12), to elicit antibodies that recognize the gp120 molecule and an anti-hiv-1 neutralizing response in rabbits, confirming them as cd4bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp120 antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd4bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd4bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp120 titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b12 in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p1 and p2) reacted in elisa only with the cd4bs-directed igg fraction. the clones were also recognized in elisa by b12. p1 and p2-immunized rabbit sera showed a strong anti-gp120 titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb2 strain glycoproteins. in particular, 3/5 rabbits in the p1 group and 1/5 in the p2 group showed an 80 % hiv neutralization at dilutions ranging from 1:20 to 1:150. the immunoenzymatic assay used, allowed to detect a p1 and p2 reactivity in hiv-positive sera and was able to detect a b12 concentration equal to 10 ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines (17d and 17dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a 23-year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following 17d-204 vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd16, cd32 and cd64) along with increased levels of nkt-cells (cd3 + cd16 +/-cd56 +/-/cd3 + ratio) and activated t-cells (cd4 + and cd8 + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il-6, il-17, il-4, il-5 and il-10) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il-4 + ), cd8 + t-cells (il-4 + and il-5 + ) and b-lymphocytes (tnf-a + , il-4 + and il-10 + ). the analysis of cd4 + t-cells revealed a complex profile with increased frequency of il-12 + and ifn-g + and decreased percentage of tnf-a + , il-4 + and il-5 + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester 1 , h.-g. rammensee 1 , s. stevanović 1 1 eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd8 t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd8 t cell epitopes for the frequent mhc class i alleles a*01, a*02, and a*24 from the proteins pii (hexon), pviii, and e1a of adv strains ad2 and ad5 by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a 12-day recall stimulation of pbmcs from at least 16 healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd8 t cells. we could identify 27 new peptides eliciting ifn-g responses, several of which were confirmed as novel cd8 t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle (5 ara criteria) was diagnosed in a 40-year-old african female patient with hiv-1 (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd4 t-cell count x 200 cells/mm 3 . initiation of 500 mg mmf bid was associated with biological and clinical remission of sle and cd4 t-cell increase. no opportunistic infections or cancers were noted during a 3-year follow-up and the patient remained always naive to art. hiv-1-specific cd4 and cd8 t-cell responses were analyzed after 18 months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il-2 production following stimulation with a panel of 192 hiv-1-derived optimal epitopes (9/10-mers) covering various hiv regions and a pool of 105 hiv-1-derived peptides (15-mers overlapping by 11 aminoacids) encompassing the entire gag protein. all peptides are derived from hiv-1 consensus strain iiib. results: highly polyfunctional hiv-1 specific cd4 and cd8 t-cell responses against gag were detected. 11 epitope-specific cd8 t-cell responses were identified: except for one response restricted by hla a*23 and another one by hla cw*07, all the others were restricted by hla-b alleles and mostly by b*58 (n = 5). seven out of 11 responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il-2+) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv-1 specific cd4 and cd8 t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv-1 t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of 10 6 dl50/ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf-1 of 12-16 g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the 7 th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt-46767, cofaa and cgpi-20090305. . state of vaccine-induced measles immunity was determined by means of elisa in 1-3, 4-6 and 7-9 years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in 4-6 years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p 0.05) measles immunity and antibody level much lower (p p 0.05) than among children with mt conversion. in 7-9 years the comparison group kept decreased (p p 0.05) measles immunity, the majority (92±5.54 %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p 0.05) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p 0.05), the majority (83.3±4.1 %) of persons lost protected antibody level; among children with mt conversion in 1-3 years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p 0.05) to low values; among children with hyperergic mt igg level decreased (p p 0.05) and reached low (in 1-3 years), minimal protected (in 4-6 years) and lower than protected (in 7-9 years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il-17 producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein 65 (dnahsp65). methods: balb/c female mice were infected by intra-tracheal route with 10 5 h37rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp65 genetic vaccine was done at days 30, 40, 50 and 60 post-infection. each dose consisted of 100 micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd4+, cd8+ and gamma-delta t cells from lungs were determined 10 and 50 days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x 0.05 were considered significant (t test). results: at day 10 after the end of the immunotherapy, dnahsp65 treated mice exhibit increased numbers of absolute cd8+ and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il-17 producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp65 treated mice showed more ifn-gamma producing cells in both cd8+ and cd4+ cell populations. at day 50 after the end of the therapy, the main observation in mice which received dnahsp65 treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd4+ and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd8+ cells, together with a more frequency of gamma-delta t cells producing il-17. finally, the immunotherapeutic effects of dnahsp65 vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd8+ cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il-17, are the main effects of dnahsp65 immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il-17 and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as 60 loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of 3-5 log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput (96-well format), requires only 100 ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in 5 different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that 5 candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls 71 % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all 5 m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards 4 of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to 100 % in pb, 75 % in rx and 93 % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd40l, a co-stimulatory molecule preferentially expressed on activated cd4+ t cells, is the ligand of cd40. cd40-cd40l interaction induces the production of il-12 and the initiation of a th1-type immune response. several studies show that cd40l is required for the activation of macrophages and the maturation of dcs. moreover, cd40l enhances the capacity of cd8+ t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd40l. we have constructed the recombinant bcg strain expressing cd40l (rbcg2) by electroporation of bcg with pgfm11/signalsequenceag85b-cd40lec and an another recombinant strain with empty vector pgfm11 (rbcg1) as a control. the expression of cd40l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th1 type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd40l 2 weeks after vaccination but not at 4 and 8 weeks. rbcg2 seems to be more protective against paratuberculosis than rbcg1, but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd40l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il-1a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x 0.05) of tnfa, ifng and il-1a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x 0.05) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd40 mab and ag85a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd40 binding on cd40 transfected l929 fibroblasts. the conjugates were tested in vivo in wild type and cd4 + cell-depleted mice for the induction of specific anti-ag85a serum antibodies. splenocytes were challenged ex vivo with ag85a and were examined for their ability to produce th1-related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il-2. we developed a method to successfully crosslink anti-cd40 mab to ag85a. serum antibodies against ag85a were detected after immunisation with this conjugate vaccine in both wild type and cd4 + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag85a alone. production of two other th1-related cytokines, tnfa and il2, was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd40 conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd4 + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer 1 , r. burger 2 1 robert koch-institute, cellular immunology, berlin, germany, 2 robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd4-positive and cd8-positive t-lymphocytes. cd8-positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd8+ t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for 5 days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to 30 % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd4+ t-lymphocytes in vivo. similarly, the cd4-positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd4+ and the cd8+ subpopulation depended on the presence of apc. stimulation oft cd8+ cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd8-positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target 6 (esat-6) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, 83 iranian and afghan adults (38 patients with sputum smear and culture positive tuberculosis, 24 recovered patients during 6 months after full course of chemical treatment and 21 healthy individuals) were recruited to quantify the frequency of esat-6 and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of 4 spot forming unit ( g 20 spots per million), we found detectable response to esat-6 in almost 80 % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and 77 % of these individuals had detectable esat-6 specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in 14 %, 57% and 62 % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th1 immune response, against esat-6, in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish 1331 in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal 1 , p.k. upadhyay 1 1 national institute of immunology, pdc-1, delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl 2 solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c57bl/6 mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: 2.4 % alginate, 0.012 % pva concentration gives particles with size of 2-10 micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with 5 % trehelose and 3.5 % pvp (poly vinyl pyrollidone). there was 6 fold increase in proliferation index of spleenocytes and releases 600 pico-gram of interferon gamma after 4 week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by 10 % increase in cd86 and 10.5 % in ccr7 expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b100 is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b100s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b100s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b100s. by cell cycle analysis we determined that b100s is able to induce apoptosis in up to 80 % of jurkat and molt-4 t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b100s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b100s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase 9 is the first to be cleaved, followed by caspases 3 and 8, thus suggesting that b100s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b100s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b100s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd40, cd80 and cd86, and of the inflammatory cytokines il-6, il-23 and mcp-1. using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd4 t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th2 and th1/th17 skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd4 t cell responses. microcapsule based vaccination resulted in a marked induction of il-17 secreting th17 cells, without inducing strong th1 (cfa) or th2 (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg1, igg2c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th17 responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd4 + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd80, cd86 and cd40 and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il-6 and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr4-dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr4-defective c3h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il-1b secretion by dc, through its effects on caspase-1 processing, which is also tlr4-independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr4 signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc2461 (st11) or lactobacillus rhamnosus ncc4007 (lpr), each at 5x10 8 cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week 1 to week 7 of life. fresh feces were collected at 3, 5 and 7 weeks of life for determination of iga levels (assessed by elisa). pups were bled 2 and 4 weeks after immunization for determination of measles-specific igg1 and igg2a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st11-fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from 200 kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at 3:1 ration and washed 3 times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw264.7 by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of 24 extracts from 10 euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc6/propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that 15 up to 24 euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd3+ cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on 35 th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il-5 and ige serum levels were increased on animals from group i when compared to control.the enhancement on th2 immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd8+ ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd4+t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg2a and igg2b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren 1,2 , n. almqvist 2 , a. lönnqvist 1 , s. östman 1 , c. rask 1 , e. telemo 2 , a.e. wold 1 1 university of göteborg, department of clinical bacteriology, göteborg, sweden, 2 university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; 0.8 mg/ml) in the drinking water for 5 days and, 3 days later, 50 mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin-5 and interleukin-13. examination of gut sections from sea treated donor mice revealed increased density of cd8a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp21, proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna 1826, a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp21, or groel together with cpg-dna 1826 reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp21 together with cpg-dna 1826. the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp21 microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp21 microspheres induced protective ifng-secreting cd4 + t-cells and raised pulmonary pmp21-specific iga levels in vivo. also, pmp21 microspheres caused lower il-6 serum levels upon administration than the injection of pmp21 together with cpg-dna 1826, indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in 44 cases of s. aureus bacteraemia in iv drug users and 44 cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised 44 iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen 2006).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day 0) and four weeks thereafter (day 28). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter 2007) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st59 (spa-type t172, agr 1, and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p=0.01).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st59 strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st59 strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at 4 and 9 years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in 15 and 7 ds children, respectively. samples were taken before and 3-4 weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg 1 -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at 4 years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg 1 , igg 2 and igg 4 ). at 9 years, ds children had lower postvaccination geometric mean igg 4 anti-tt-titers only. post-vaccination igg 1 -anti-tt avidity levels were decreased in 8/15 and 4/7 ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. 2 kda; 30, 6 kda; 23, 9 kda; 19, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] 6 kda and 18, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 7 kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of 100 families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an 8-month period. sera were obtained from healthy subjects from the same community (2 months to 88 years of age). the highest incidence of salmonella-associated diarrhea, 74/1000, occurred in children under 5 years of age. the lowest incidence, 10/1000, was observed in the population aged 10 to 59. whereas serum from individuals ranging 15-70 years of age showed maximum igg1, igg3 and iga anti-s. typhimurium titres, children less than 5 years-old did not show detectable igg1 and igg3 titres and had weak igg2, iga and igm antibody levels; only their igg4 levels were comparable to those detected in adults. moreover, the levels of igg2 and igg3 antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs 07-18, a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses (67%), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a-1,6linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after 7-fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková 1 , s. bystrický 1 1 institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o135 using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd19, expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age 18-55 years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg1, igg2 and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present 5 years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after 5 to 7 days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg2 response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers 1:3200). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones (1:800 vs. 1:3200). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was 5. 85 fold of normal control). subcutaneous immunization gave a weaker stimulation: 1. 52 fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv 0032-06 and vega 2/7029/27 grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a-1,6-linked d-mannopyranose units and many branches composed of a-1,2 a-1,3 and/or b-1,2-linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant (6mg oligosaccharide per one conjugate dose) two times in 14 days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day 14 after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs (250g-350g) were treated orally with 50 mg respivax five consecutive days. after the last application, on days 3, 7, 10, 14, 28 and 42 six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on 4mm thick serial sections, stained with hemalauneosin. the populations of cd8, cd4 and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day 3 subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd8 positive cells, which number reached maximum at the end of the second week. on day 14 b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd4 and cd8 positive cells. on days 28 and 42 the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta 1 , s. majumdar 1 1 bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein-10 (ip-10) and interleukin-8 (il-8) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip-10 mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase2 (inos2) expression and was linked to the mapk signaling pathway via antagonistic regulation of p38mapk and erk1/2. further, ip-10 was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th1 cytokines and no. thus this study strongly demonstrates that ip-10, like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th1/th2 cytokines mediated by cd8+ t cells and activating cd4+ t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x 0.05) following immunization and after challenge with leishmania major. il-4 values were increased in all groups, but there was no statistical difference between the groups(p g 0.05) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x 0.05) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x 0.05) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x 0.05). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at 3 week intervals. three weeks after booster injection, 5×10 5 stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg2a/igg1was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type 1 immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin 10 (il-10), and cellular expression of cd4 and cd25. results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd4+ lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with 1,600 infective n. brasiliensis larvae on day 2 post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day 6 post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg1 and igg2a). this was independent of level of me supply. feeding regime did not affect levels of the type-2 cytokines il-4 and il-13. conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp63 is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp63 of l.major strain mrho/ir/75/er. methods: l.major promastigotes were grown in rpmi1640 supplemented with 10 % fcs. l.major rna extraction and cdna synthesis were carried out. gp63 gene segment was amplified by specific primers and cloned into ptz57r to construct ptz57r/gp63. the presence of gp63 into ptz57r was confirmed by pcr. then, ptz57r/gp63 was sent to determine the sequence of its nucleotides. after that the gp63 gene segment was sub-cloned into pet32 a (+) expression vector and transformed into e.coli bl21 (de3) plyss and gp63 protein was expressed in presence of 1mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of 100 % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, 2007) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including 50 volunteers. 10 male volunteers (18 and 45 years) for the adult group, and 40 children of both sexes (5-9 years) were enrolled. subjects received virosomal formulations containing 50 mg of ama 49-c1 (pev301t), an apical membrane antigen-1 derived synthetic phospatidylethanolamine (pe)-peptide conjugate and 10 ug of uk39 (pev302t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days 0, and 90. results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling 29 % of the treated mice to survive till 21 days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only 0.04 % of the amount fed and it remained in circulation in the blood only for 30 minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to 0.4 % ( of the amount fed but it's circulation was sustained till 6 hrs post feeding. under such conditions when 200mgm of curcumin bound to 300mgm of chitosan nano particles were fed one time daily for 10 days post infection to plasmodium yoelii infected mice 100 % of mice were cured and survived atleast for 100 days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma9vdelta2 t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il-10 responses, to epitopes on the dominant rbc autoantigen, anion exchanger-1 (ae-1, or band 3) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of 1 methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla-4 + regulatory t cells or soluble forms of ctla-4, may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =-0.81) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd8 + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b 1 (r medium =-0.74) and il-10 (r medium =-0.65) in isolated splenic hscs ex vivo. analysis of effector cd4 + t cells in spleen showed decrease of t h 1 cells quantity and simultaneous t h 2 cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd4 + cd25 + ctla-4 + -and cd4 + cd25 -il-10 + il-4regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h 1, t h 2 and regulatory t cells were correlated (r th1 =-0.74, r th2 =0.86 and r th3 =0.80 and r tr1 =0.68) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th3 =0.84 and r tr1 =0.76) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b1-and il-10-produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p38, jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin-17, t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il-17, as compared to non-infected controls. the infection also altered the effect of il-17 on mapk activation by preventing its stimulating effect on p38 mapk. moreover, in s.obvelata-infected animals il-17 markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il-17 induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. 2.-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from 1 ⁄2 to 1/64 with a normal serum pool. 3.-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r 2 g 0.98. the analytical range was from 1 g/l to 70 g/l. the coefficient of variation (cv) was x 10 % for [mc] n 1 g/dl, and x 20 % for [mc] x 1 g/dl. this procedure was successfully implemented to quantify the mc in 1100 serum samples between march 2008 and february, 2009. among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: 1. we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. 2. the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from 7 day and reached plateau level after 30 day of teratocarcinoma insertion. moreover, cd34 + cd38cells showed in spleen and main lymph nodes from 15 day and achieved plateau level after 60 day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at 22 day and had a maximum pick at 60 day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than 1,5 times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b 1 , il-10, pge 2 and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number =0.81 and r activity =0.92) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd40-ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd40l is cleaved to soluble (s)cd40l. we sought to examine the levels of scd40l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded 5 atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd40l -along with other products -were assayed by quantitative elisa. levels of il8, cd62p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd40l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l . to examine if scd40l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il-6 secretion. the il-6 concentration was consistently below 5-10 pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il-6. the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il-6 production over control (p x 0.05) at d2 after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l (p x 0.05). pre-incubation of b cells with cd40-blocking antibodies substantially abrogated il-6 secretion, unlike isotype-matched control. the partial blocking of cd40 binding on cd40 + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd40l (under investigation) and indicates a sustained role for pc-derived scd40l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd40l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule-1 (dnam-1) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from 30 aml patients before specific anti-leukemia therapy and 47 healthy donors. all results were analyzed statistically using spss version 15. results: aml patients under 65 years showed a significant reduction in the expression of dnam-1, nkp30 and nkp46 compared with age-matched controls. both healthy individuals and aml patients older than 65 years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp44 expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam-1 on nk cells and its ligand cd112 on aml blasts has been found in aml patients under 65 years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam-1 expression on aml patients and confirm previous reports showing a significant decrease of nkp30 and nkp46 on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel 1 , e. gorska 1 , u. demkow 1 , m. wasik 1 1 medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved 10 children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for 2 or 48 hours with or without dexamethasone in concentration 10 -6 m. analysis of: p-gp surface expression, p-gp function (rhodamine 123 test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was 14,52 %±11,32. after 48 hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine 123 efflux, which characterized p-gp activity, was 2,62 %±3,15. after 2 hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine 123 (4,31 %±4,03, p=0,015). average phi in lymphoblasts was 7,78±0,19. acidification of cells incubated 2 hours with dexamethasone was seen in 10-25 % percentage of cells 17) . rest of lymphoblasts showed alkalization (phi -8,00).the percentage of lymphoblasts in early stage of apoptosis after 48 hours incubation with dexamethasone (annexin-v test) was higher than in control cells (19,34 % vs 14,13 %; p=0,01). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd49d/cd29 integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd29 on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd38 and anti-cd29 (clon huts21) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures (18 hours). results: cd29 active form was expressed in the majority of normal and tumoral bm pc from healthy subjects (67.3±6.6, n=9) and mm patients in the early stages of the disease (32.6±7.5, n=17). in these cells, huts21 epitope was clearly upregulated by mn 2+ . in contrast, circulating pc were almost all huts21 negative, and levels did not significantly augment when these cells were exposed to mn 2+ . moreover, not only pb but also bm malignant cells from pcl patients were also huts21 negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index (2.35±0.9 brdu+ cells, n=3) in comparison to pc from mm patients in the first stages of disease (1.3±0.2 brdu+ cells, n=5). these results suggest that the active form of cd29 must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either 10 % fetal bovine serum (fbs) with and without fgf2,or 10 % human platelet lysate (hpl), 5 %hpl, (10 % fbs + 10 % hpl), (10 % fbs + 5 % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day 18). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p53 tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p53 dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: 1) ten cases with fa diagnosed on the basis of dna breakage analysis, 2) ten cases with aaa, and 3) ten normal control cases. the presence of p53 dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p53 dna was demonstrated in bone marrow of 90 % of children with fa, compared to 10 % in children with aaa (p x 0.001), while, no p53 dna was seen in normal control. a positive correlation between dna breakage and presence of p53 dna was seen in bone marrow from fa (p x 0.02, r0.81). the presence of p53 tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord-010119-t1x9gknd authors: nan title: abstract presentations from the aabb annual meeting san diego, ca ctober 7‐10, 2017 date: 2017-09-04 journal: transfusion doi: 10.1111/trf.14286 sha: doc_id: 10119 cord_uid: t1x9gknd nan background/case studies: zika virus (zikv) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (tt). rna persistence has been reported in whole blood (wb) long after clearance of viremia in plasma, raising concerns over the risk of tt with plasma based nucleic-acid amplification testing (nat). the dynamics of zikv persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of zikv infection. we sought to characterize the dynamics of infection through prospective enrollment of zikv rna1 blood donors. study design/method: donors identified through investigational zikv nat screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes. plasma and rbc were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks 1, 3, 6, 12 and 24 following index donations from 50 donors and detailed symptom questionnaires were administered at each study visit. blood compartments and body fluids were tested for zika rna by real time rt-pcr. plasma samples were tested for zika specific igm and igg antibodies results/finding: the percent of zikv rna1 samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table. plasma viremia declined rapidly after index donations whereas rbc-and wb-associated viral rna persisted for up to 3 months and peripheral blood mononuclear cell (pbmc) associated virus was detected intermittently at low levels and waning by 3 months. urine and saliva detection decreased significantly after 2 weeks and was undetectable by 3 months. of donors who were enrolled in the acute pre-seroconversion stage of infection 65% (15/23) developed multiple zikv related symptoms 1 week post index donation, compared to only 30% (7/27) for donors detected post-seroconversion. conclusion: zikv rna persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported. the persistence of zika rna in rbcs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress. wb testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women. iron status and novel risk factors for iron depletion in a diverse donor population bryan r. spencer* 1 , yuelong guo 2 , ritchard g. cable 3 , joseph e. kiss 4 , michael paul busch 5 , grier page 2 , stacy endres-dighe 2 , steve kleinman 6 , simone glynn 7 , alan mast 8 and for the nhlbi recipient epidemiology and donor evaluation study-iii (reds-iii) 9 . 1 american red cross, 2 rti international, 3 american red cross blood services, 4 blood systems inc., 5 blood systems research institute, 6 university of british columbia, 7 nih/ nhlbi, 8 blood research institute, 9 nhlbi background/case studies: blood centers and regulators in the united states (us) are evaluating strategies for minimizing iron depletion in blood donors. the logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors. studies donors have been conducted in predominantly caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy. study design/method: over 12,600 donors were enrolled from 4 us blood centers for ferritin testing. the study population was enriched for racial minorities [1600 african-american (aa), 1600 asian (as), 1000 hispanic (hisp)] and for "super donors" (1600, who had completed 101 donations in two years without low hemoglobin deferral). the minority donors and the remaining 6800 non-hispanic white (nhw) donors were an unselected population with no specific eligibility criteria. subjects completed questionnaires on risk factors for iron depletion. logistic regression was used to identify demographic and behavioral predictors of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml). results/findings: across all subjects, 19% had ais and 42% had lf, with a high degree of variability based on demographic factors and donation behavior. in models stratified by race, expected patterns common to all 4 groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > 50 years old. in models including all subjects, race was an independent predictor of both ais and lf controlling for age, sex, body weight, donation frequency, and other factors (table) . aa and as donors showed %20% decreased risk for ais compared to nhw, while hisp donors had 25% higher risk. daily use of exogenous iron reduced risk for lf and ais by 30 to 40%, respectively, while the estimated benefit from less-than-daily use was lower (5 to 19% protection). regular use of antacids was associated with a 20% or greater increment to risk. reported use of hormone supplements showed opposing effects in males and females. use of oral contraceptives or estrogen in females reduced risk by %15-20%, while males who reported current use of supplemental testosterone had twice the estimated risk for ais. conclusion: this large study confirms the high prevalence of lf and ais in us donors and the principal risk factors of age, sex, and donation frequency. the diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance. in developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population. data are reported as mean (6sd) *p < 0.05 compared to batf31/1, 200ul hod rbcs mfi, median fluorescence intensity background/case studies: during storage, red blood cells (rbcs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion. the proportion of cleared rbcs is correlated with storage duration, which may be responsible for the rapid clearance of up to 25% of transfused rbcs, reducing transfusion yield. it has been shown, using imaging flow cytometry that a subpopulation of morphologically altered rbcs accumulates during storage. the reduced surface area of these small rbcs (srbcs) suggests their rapid elimination by the spleen in the hours following transfusion. this hypothesis remains to be clarified, since the physiological mechanisms of rbc clearance remain to be precisely identified. study design/method: murine "young" and "old" rbcs (respectively on d1 and d14 of storage) were transfused into different models including splenectomized or macrophage-depleted mice. flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify rbcs retention in organs. the accumulation, during storage, and the posttransfusion disappearance of srbcs were analyzed by imaging flow cytometry. results/finding: using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration (62% on d14 vs 100% on d1 of storage). a clearance of the storage-damaged rbcs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized (74%) and macrophage-depleted (79%) groups. as in humans, we observed the accumulation of a subpopulation of small rbcs (mouse small rbc: msrbc) of reduced projected surface area with altered morphology. these msrbcs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than 50% at 2h post-transfusion. in contrast, in macrophage-depleted mice, msrbcs are kept in circulation at 2h posttransfusion. at 24h, these msrbcs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms. in control mice, storage-damaged rbcs are mostly retained in the spleen but also in the bone marrow (bm) . no retention is observed in the liver, kidney or lung. in macrophage-depleted mice, retention is decreased in the spleen and bm. conversely, elevated retention is observed in the bm of splenectomized mice, associated with a transient retention in the kidney and liver. conclusion: during storage of murine rbcs, damaged rbcs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms. they include, as observed in humans, a subpopulation of small rbcs which undergoes a rapid macrophage-mediated clearance. the increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors. age dependent relapsing and remitting autoimmune hemolytic anemia in a murine model andrea sut ling wong* 1 , amanda l richards 2 and krystalyn e hudson 2 . background/case studies: breakdown of tolerance to rbc antigens may result in development of pathogenic autoantibodies (autoab) and lead to autoimmune hemolytic anemia (aiha), a severe and sometimes fatal disease. aiha in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit. however, the mechanisms behind such observations are not understood. to gain insight into tolerance (or loss thereof) to an rbc autoantigen, we utilized the hod mouse, which expresses an rbc-specific triple fusion protein consisting of hen egg lysosyme (hel), ovalbumin (ova), and, duffy (hod). hod mice were bred to a transgenic mouse that expresses a t cell receptor specific for an ova peptide in hod presented by mhcii (otii mice). thus, hod 1 otii 1 mice are predisposed to have autoreactive cd4 1 t cells. study design/method: four cohorts of hodxotii f1 mice (16-48 mice/ cohort) were bled monthly for 15 months to assess for autoab production. peripheral rbcs were stained with anti-complement (c3) and mouse immunoglobulin ab. spleens were weighed and splenocytes were stained with anti-cd71 and ter119 to assess for the presence of rbc progenitors. statistical analysis between hod 1 otii 1 autoab 1 vs. hod 1 otii 1 autoabvs. hod -otii 1 was performed using kruskal-wallis test and corrected for multiple testing with dunn's test. results/finding: otii cd4 1 t cells were not deleted in the thymus of hod 1 otii 1 mice; rather, they matured to the periphery. despite these peripheral autoreactive t cells, no detectable autoab were observed in hod 1 otii 1 . however, as they aged, 15-50% of hod 1 otii 1 were positive for rbc autoab by 6 months. thereafter, $50% of the autoab 1 mice stopped producing autoab within two months after onset and remained autoab free throughout the study. in 3 of the 4 cohorts, 60-100% of autoab 1 mice were female. hod 1 otii 1 autoab 1 mice also had enlarged spleens compared to hod 1 otii 1 autoaband hod -otii 1 mice (0.42g vs. 0.21g and 0.14g, resp., p<0.04). this may due to rbc consumption, extramedullary erythropoiesis, or both. consistent with increased erythropoiesis, elevated numbers of rbc progenitors (cd71 hi ter119 inter ) were observed in the spleens of hod 1 otii 1 autoab 1 mice but not in hod 1 otii 1 autoaband hod -otii 1 (2.86% vs. 0.06% and 0.05% resp., p<0.03). moreover, autoab and c3 deposition were found (0.1-2% and 3-10%, resp.) on ter119 1 rbcs in all of the hod 1 otii 1 autoab 1 mice analyzed. conclusion: several features known to exist in human aiha were observed, including age-dependant autoab production, relapsing of autoimmunity after onset, and an increased frequency in females. this model may serve as an experimental system to investigate the mechanisms of aiha. b5-a01a reduction in neutrophil numbers is a risk factor for rbc alloimmunization amanda l richards 1 , christopher a tormey 2 and krystalyn e hudson* 1 . background/case studies: red blood cell (rbc) alloimmunization occurs in up to 10% of transfusion recipients (excluding abo and rhd). the underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not. patients who receive multiple rbc units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (mds) and sickle cell disease patients. however, despite chronic transfusions, some patients never develop rbc alloantibodies. it has been recently reported that poly (i:c)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (pmn) numbers. additionally, rbc transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by pmns. here, we test the hypothesis that pmns regulate rbc alloantibody generation. study design/method: mice: c57bl/6 (b6) mice were treated with pbs, or anti-ly6g to deplete pmns, followed by poly (i:c) to elicit inflammation, and finally a transfusion of allogeneic dio-labeled rbcs expressing a synthetic antigen, hod (hel-ova-duffy). multiple splenic cellular subsets were evaluated for dio fluorescence, an indirect measure of rbc consumption, at 18-24 hours post-transfusion. anti-hod alloantibody generation was assessed 14 days post-transfusion by flow cytometry. humans:retrospectively, mean white blood cell (wbc) and pmn counts were collected on chronically transfused mds patients at va connecticut healthcare. for alloimmunized patients (n55), wbc and pmn counts were assessed on the day of exposure to the alloimmunizing rbc unit, whereas counts were averaged for the entirety of rbc therapy for non-alloimmunized patients (n55). patients were matched for numbers of rbc transfusions. results/finding: mice: the mfi of anti-hod antibodies was significantly increased in pmn-depleted mice, compared to controls (3/3 experiments, p<0.01). while many control mice made no alloantibody (non-responders), all pmn-depleted mice made detectable anti-hod. pmn depletion also led to a significant reduction in dio1 leukocytes, suggesting a lack of compensatory mechanism(s) for rbc consumption. absence of pmns also shifted rbc consumption from macrophages to immune-stimulating dendritic cell subsets. flow cytometric analysis revealed that pmns with internalized rbcs upregulated expression of co-inhibitory molecules (e.g. pd-l1), compared to pmns (without internalized rbcs) from the same mouse; thus, pmns may regulate alloimmunization through antigen presentation and/or inhibitory signals. humans:. alloimmunized mds patients had a significant decrease in pmns, compared to non-alloimmunized (p<0.05); no significant differences were detected in mean wbc counts between the two arms. conclusion: these data demonstrate that in both murine and human settings, pmns may play a significant role in regulating rbc alloimmunization and may provide key insights into predicting which patients will become alloimmunized. b9-a03b cxcr5 1 pd1 1 and ccr7 1 expressions characterize responders to rbc immunization benoît vingert* 1,2,3 , marie tamagne 1,2,3 , sadaf pakdaman 1,2,3 , anoosha habibi 2,3,4 , philippe bierling 1,2,3,4,5 , rachid djoudi 1 and france pirenne 1,2,3,5 . 1 efs ile de france, 2 laboratory of excellence gr-ex, 3 imrb u955 -eq2, 4 ap-hp, 5 universit e paris est background/case studies: post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction. in human, mechanisms responsible of rbc alloimmunization are not fully defined. cd4 1 t cells are major for antibodies production. we have already shown in responder patients that the majority of anti-rbc cd4 1 t cells have a th17 profile. in contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating cd4 1 t cells with a cxcr5 1 pd1 1 phenotype. this phenotype is usually associated with the presence of tfh cells, specialized in the production of antibodies. it has been suggested that some of the activated circulating tfh could have a cxcr5 1 pd1 hi profile, with a differentiated expression of ccr7. ccr7 is essential for t cells domiciliation in lymph nodes where the encounter t and b cells is major for b cell differentiation and antibody production. others chemokines receptor like ccr6 and cxcr3 can also differentiate circulating tfh subpopulations. in this study, we were interested in the phenotype and function of these cxcr5 1 pd1 1 lymphocytes which were paradoxically highly represented in non-responder patients. study design/method: the membrane and functional phenotype of the circulating cxcr5 1 pd1 1 cells were compared in 2 groups of transfused sickle cell patients : alloimmunized (n514) and non-alloimmunized patients (n510). the analysis was also performed in non-transfused healthy controls (n512). all assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors results/finding: the cxcr5 1 pd1 hi subpopulation expression was identical between transfused groups and controls. ccr6 and cxcr3 expressions show no difference between the transfused groups or the controls. however, in non-responder patients, ccr7 expression was very strong independently of the expression of pd1. in the aim to determine the help of the circulating cxcr5 1 pd1 1 cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for 5 days with b cells, and in the presence of seb protein. the levels of antibodies after seb stimulation were identical with the cxcr5 1 pd1 1 subpopulations from transfused groups or controls. conclusion: the paradoxical presence of circulating cxcr5 1 pd1 1 cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response. however, in responder patients, the high expression of ccr7 on circulating cxcr5 1 pd1 1 cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses. in conclusion, the study of the cxcr5 1 pd1 1 profile and the ccr7 1 expression in these cells could help to differentiate responder and non-responder patients to rbc immunization. primed cd4 t cells to one rbc alloantigen can enhance subsequent alloimmunization seema r patel* 1 , ashley bennett 1 , kathryn girard-pierce 1 , connie arthur 1 , amanda mener 1 , patty zerra 1 , christopher a tormey 2 , jeanne hendrickson 3 and sean stowell 4 . 1 emory university, 2 yale-new haven hospital, 3 yale university, 4 emory university school of medicine background/case studies: while red blood cell (rbc) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. however, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion. however, how immunity to one rbc alloantigen primes immunization to a completely distinct alloantigen remains unknown. though cd4 t cell help classically occurs through direct recognition of a peptide that resides within a target b cell antigen, individuals who develop antibodies toward one rbc alloantigen experience increased rates of antibody formation against completely distinct rbc alloantigens. these observations suggest that cd4 t cells that respond to one alloantigen may directly facilitate immunity to a completely distinct rbc alloantigen. study design/method: b6 recipients were transfused with kel rbcs in the presence or absence of poly i:c (pic), followed by transfusion of hod rbcs, kel rbcs, rbcs expressing hod and kel (hod x kel), or a mixture of hod and kel rbcs (hod 1 kel). to examine the role of cd4 t cells, pic/kel primed b6 recipients were cd4 t cell depleted prior to transfusion. in addition, b6 recipients were adoptively transferred with cd4 t cells from na€ ıve or pic/kel primed donors, followed by transfusion of hod rbcs or (hod x kel) rbcs. anti-hod and anti-kel alloantibody formation was evaluated using indirect immunofluorescence staining. results/findings: kel rbc transfusion in the presence of pic (pic/kel) not only enhanced anti-kel antibody production through a cd4 t celldependent process, but this same priming event directly facilitated anti-hod antibody formation following subsequent (hod x kel) rbc transfusion (p < 0.001); pic/kel primed recipients transfused with (hod 1 kel) rbcs or hod rbcs alone failed to impact anti-hod antibody formation. the ability of immunity to kel to boost a humoral response to the hod antigen following (hod x kel) rbc transfusion required kel priming in the presence of pic. cd4 t cell depletion prevented pic/kel primed recipients from boosting an anti-hod antibody response (p < 0.0001) and transfer of cd4 t cells from pic/kel primed recipients likewise directly facilitated anti-hod antibody formation following a (hod x kel) rbc transfusion (p < 0.001). conclusion: these results demonstrate that cd4 t cells primed to one rbc alloantigen can directly enhance the immune response to a completely distinct rbc alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody background/case studies: platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood. immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of hla-matched platelets. still, many patients are refractory even after receiving hla-matched platelets. it was shown previously that cd8 t cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within 24 hours post transfusion in b celldeficient mmt recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of cd8 t cells prevents such clearance. since minor antigenic differences still exist between donor hla-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise hla-matched platelets. study design/method: to test whether minor antigens can stimulate cd8 t cell-dependent platelet clearance we examined platelet refractoriness using mova and oti transgenic mice. leukoreduced donor platelets from mova mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine mhc class i h2kb, were labelled in vivowith the fluorescent dye cfse and transfused into wildtype (wt, c57bl/6) mice or oti mice, whose cd8 t cell receptors recognize a specific ovalbumin peptide in the context of mhc class i h2kb. in some experiments oti mice were primed with mova or wt splenocytes one week prior to mova platelet transfusion, and in others wt mice were adoptively transferred with oti splenocytes 48 hours before mova platelet transfusion. platelet recovery was measured immediately after transfusion as well as after 2, 4, 8, 16, and 24 hours and on days 2-5. results/finding: transfusion of mova platelets into oti mice results in significant platelet clearance as compared to transfusion with wt platelets. clearance kinetics demonstrate platelet loss starting after 8 hours and peaking at 24 hours, and are similar whether oti mice are na€ ıve or previously primed with mova splenocytes. specifically, mova platelet recovery in oti recipients is 28-35% versus >60% in wt recipients at 24 hours (p<0.05), whereas transfusion of wt platelets into either oti or wt recipients is approximately 60% at 24 hours after transfusion. adoptive transfer of oti cd8 t cells into wt mice recapitulates the effect, with significant mova platelet clearance at 24 hours compared to wt platelet clearance (p<0.05). conclusion: this work extends the ability of cd8 t cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving hla-matched products. this study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of t cell responses may prove beneficial. background/case studies: alloimmunization against major histocompatibility (mhc) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants. we have previously demonstrated that pathogen reduction with riboflavin and uv light (uv1r) is effective both at rapidly killing donor white blood cells (wbcs) and at blocking their ability to stimulate an allogeneic response in vitro. furthermore, uv1r treatment of allogeneic platelet rich plasma (prp) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions. as cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by uv1r, as well as evaluate the immunogenicity of prp containing wbcs killed by other methods. study design/method: wbc-rich prp was prepared from c57bl/6 mouse blood and treated with uv1r, and wbcs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis. membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry. balb/c recipients were transfused with either uv1r treated wbc-rich prp, or uv1r treated wbc-poor prp either alone or with added untreated, apoptotic, or necrotic wbcs, all generated from allogeneic c57bl/6 donor blood. a second transfusion of untreated wbc-rich c57bl/6 prp was given 2 weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion. results/finding: uv1r treated wbcs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation. alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p<0.01) or necrotic (p<0.05) wbcs, but not those given uv1r treated or apoptotic wbcs. ex vivo cytokine responses to stimulation with c57bl/6 wbcs were reduced in recipients of either uv1r or apoptotic wbcs, and enhanced in recipients of untreated or necrotic wbcs. conclusion: the mechanism of wbc death following uv1r treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis. however, both uv1r treated and apoptotic wbcs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures. background/case studies: in mitochondria-less red blood cells (rbcs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of rbc storage lesion. oxygen saturation (so 2 ) of venous blood is generally assumed to be around 70-80% as measured from a central venous line. however, a recent investigation of so 2 levels in freshly prepared leukocyte-reduced red cell concentrates (lr-rccs) revealed unexpectedly wide so 2 distribution (mean 45.9%617.5% [yoshida et al. 2017; blood transfusion 15, 172] . the present study was undertaken to determine the distribution of so 2 in lr-rcc produced at a medium-size blood center using a novel non-invasive so 2 probe. additionally, quantitative metabolomics were carried out to examine the redox status of the stored rbc under various so 2 levels. study design/method: the so 2 from 977 units of lr-rcc were examined on five consecutive days representing 78% of the collected units during the period at a regional blood center where all the units were processed at room temperature within 8 hours of blood collection. so 2 was measured noninvasively through the pvc bag immediately prior to refrigeration by employing a resonance raman spectrometry (pendar microvascular oximeter a3u11; pendar technologies, cambridge ma). in addition to so 2 , process methods, rcc volumes, blood types, gender and process times were recorded for analyses. in a separate study, lr-rcc (n54) from human volunteers were stored in as-3 under normoxic, hyperoxic, or hypoxic conditions for up to 42 days (so 2 ranging from <3 to >95%) prior to uhplc-ms metabolomics analyses in presence of 13 c, 15 n or deuterated internal stable-isotope labeled standards for absolute quantitation. results/finding: measurements of so 2 carried out non-invasively at a blood center yielded a similar wide distribution as previous study from 497 units of lr-rcc procured and sampled invasively within 24 hours after blood collection [yoshida ibid]. the shape of the so 2 distribution appeared near normal with the mean of 47.0%621.0%, median 45.2%, range < 5% to > 95% and inter-quartile range (iqr) of 31.4%-61.9%. male donors showed higher so 2 compared to female donors (p<0.04). no correlations were observed between so 2 levels and processing time, donor age or blood types. metabolomics workflow indicated that lower so 2 levels ameliorate the energy and oxidative metabolic lesion. lower so 2 levels yielded higher rate of gsh synthesis, higher nadph concentration, higher gsh / gssg and nadph/nadp 1 ratios, lower supernatant urate consumption and lower purine oxidation. the surprisingly wide distribution of starting %so 2 levels was observed from lr-rcc manufactured at a blood center using 8-hour room background/case studies: cellular prion protein (prp c ) is a gpi-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (bm), and lymphoid tissue. prp c can be converted post-translationally into scrapie-prp (prp sc ), which is involved in the pathogenesis of neurodegenerative diseases including creutzfeldt-jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals. however, biological functions of prp c have yet to be conclusively elucidated. study design/method: in this study, prp c knockout mice (ko) are utilized to investigate the role of prp c in the hematopoietic system with controls of age and sex-matched prp c transgenic mice harboring a slightly augmented prp c expression. peripheral blood was examined by hematology analyzer to establish counts. bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages. histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy. results/finding: complete blood count (cbc) showed a significant increase of wbc in ko mice. closer analysis of wbc differential revealed that the elevated number of wbc in ko mice was due to lymphocytosis. specifically, ko mice had a 3-fold increase in the absolute lymphocyte count (ko 7.59 6 4.63 x10 9 /l vs. wt 2.90 6 1.32 x10 9 /l, p 5 0.0303), as well as a higher lymphocyte percentage compared to controls. ko mice also had a trend toward higher hemoglobin, rbc, and hematocrit compared to wt mice. additionally, platelet count in ko mice was higher than control mice. of interest, the mean platelet volume indicating platelet size was significantly increased in ko mice compared to controls (ko 6.00 6 0.29 fl vs. wt 5.24 6 0.56 fl, p50.0140). a comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of rbc and megakaryocyte in bm, and of lymphocytes in the thymus, spleen and lymph nodes. histological analysis of bm, thymus, spleen and lymph node tissue from ko and wt animals failed to show morphological differences between the two groups. conclusion: absence of prp c resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis. our findings indicate that prp c might be critical in the survival and trafficking of lymphocytes in peripheral blood. the molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies. potential role of cd81foxp31 regulatory t cells derived exosomes in their immune modulation yiming yang*, rufeng xie and jie yang. blood engineering laboratory, shanghai blood center background/case studies: exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process. human periphery blood cd8 1 foxp3 1 tregs cells are reported as more stable regulatory cells with greater inhibition effects. however, cd8 1 foxp3 1 tregs derived exosomes and their functions involved in cd8 1 tregs mediated immune-modulation were seldom reported. study design/method: cd8 1 t cells were freshly purified from pbmcs, cultured with anti-cd3/cd28 antibody packaged beads and il-2, and then polarized with tgf-b and rapamycin into cd8 1 foxp3 1 treg cells. the harvest cells were co-cultured with cd3/cd28 beads stimulating cd41cd25effector cells in the transwell plate. the supernatant derived from cd81 tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with peg. the harvest precipitation was resuspended in pbs and exosomes were analyzed by sem and nta. exosome surface marker cd63, cd81, tsg101 and other proteins expression were evaluated by flow cytometry and western blot. microrna was isolated with mircute mirna kit and mir-155, let-7b, let-7d were measured by qpcr. the precipitated exosomes were further purified by cd63 immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation. results/finding: as compared with direct contact co-culture, separated cd8 1 treg cells could suppress the proliferation of effector cells with a small decline (p>0.05), which means some non-contact factors involved in the cd8 1 treg mediated immune modulation. a total number of 4.57 6 0.52 3 10 8 /10 6 cells exosomes were harvest. electron microscope analysis demonstrated a kind of round-shaped membrane vesicle 50-150nm in diameter (145.16 6.7nm by nta). cd63 and cd81 were expressed on these background/case studies: regulatory t cells (tregs), containing cd4 1 and cd8 1 subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (aids). unfortunately, due to the instability of natural cd4 1 foxp3 1 regulatory t cells (ntregs) in inflammation conditions (including instability of foxp3, conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate cd81 regulatory t cells stability both in vitro and in vivo. in our previous works, we found that cd8 1 treg has an effective therapeutic function on cia mice. in this study we aim to investigate the stability of induced polyclonal human cd8 1 regulatory t cells in inflammation and transfusion. study design/method: human cd8 1 tregs were induced with tgf-b1 and rapamycin from cd8 1 t lymphocytes in vitro. collagen-induced arthritis (cia) mice were induced with type-two collagen as an autoimmune disease model. in vitro the stability of cd8 1 tregs when encountering with inflammation were test by foxp3 expression, th1 and th17 cells conversion in inflammations conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6) on day3, day6 and day9. in vivo, cd8 1 tregs were transfused into cia mice and then their survival in mice and foxp3 express were evaluated to reveal the stability of cd8 1 tregs in an inflammation condition model. additionally, we also investigate the stability maintenance of cd8 1 tregs when induced factor tgf-b1 and rapamycin were removed by testing the foxp3 expression on day3, day6 and day9. results/finding: ex vivo induced human cd8 1 treg were foxp3 1 (90.40 6 1.40%) and did not secret il17a (both in supernatant and % of cells). foxp3 express in cd8 1 tregs were maintained after induced factor tgf-b1 and rapamycin were removed on day3, day6 and day9. in vitro, foxp3, il2 and ifn-c expression has no significant difference when compared with controlled tregs on day3, day6 and day9 and did not secret il17a when encounter with inflammation conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6). in vivo, cd81 treg cells were transfused into cia mice on the peak of disease onset (35 days after the first collagen immunization, has inflammation condition in vivo) to test cd8 1 tregs survival. cd8 1 tregs were found in cia mice foot (27.4 6 2.03%), blood (4.55 6 1.03%) and spleen cells (1.90 6 0.05%) 72 hours after transfusion and their % of foxp3 1 were remained. conclusion: the results revealed that ex-vivo induced and expanded human cd8 1 tregs are stable in inflammation and transfusion and can maintain foxp3 expression when induced factor were removed, these make cd8 1 treg a novel and stable cell for potential cell therapy on aids. this research can provide some instructive reference and improve the utilization of blood components. tolerogenic dendritic cells induced by mtor suppression and control inflammation in chs model through s6k related proteins translation inhibition. li gao*. shanghai blood center background/case studies: tolerogenic dendritic cells (tdcs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (sle), rheumatoid arthritis (ra),et al. however, the traditional strategy base on the tdcs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tdc adaptive cell therapy in future clinical use. study design/method: human tdcs were derived from fresh purified monocytes from pbmncs isolated from buffy coat and induced by mtor inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated. the mature markers and endocytisis were detected by flow cytometry. the production of cytokines and chemokines was measured using elisa. mechanism investigation was analysis by real-time pcr and western blotting. contact hypersensitivity (chs) model, an atopic dermatitis animal model, was treated with tdcs induced via mtor suppression and analyzed by ear thickness and tissue leukocytes number calculating. results/finding: human tdcs treated with mtor inhibitors had a lower mature marker cd83/cd80/cd86 expression after tlr signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the lps absent group. moreover, mtor suppression extremely reduced the capacity of lps treated human dcs to stimulate autogenic na€ ıve t cell proliferation, which is one of the most important characteristics of tdc. beyond expectation, the common signal transduction pathway, mapk and nf-jb pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tdc when exposure to lps stimulation. however, the p70s6k and its downstreanm proteins, especially the protein s6, which controls the protein translation, were shown in charge of the tolerogenic induction mechanism. the data were also supporting the suggestion that rare difference on mrna transcription of the related functional proteins in tdcs induced by mtor inhibitors when exposure to lps stimulation from the non-induced cells, although there was more transcription of ido induced by mtor inhibitors. more important, edema responses of ears were clearly weakened in the chs model and recruited less leukocytes to the tissue when co-sensitized with mtor inhibitors or with tdcs induced by mtor inhibitors suggested that the tdc induced by mtor suppression were able to control hypersensitivity inflammation response in vivo. conclusion: accordingly, tdc induced by mtor suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mtor-s6 related protein translation inhibition. xiaoyun fu* 1,2 , mikayla anderson 1 and james c zimring 1,2 . 1 bloodworksnw research institute, 2 university of washington school of medicine background/case studies: red blood cells (rbcs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion. bioactive lipids generated during rbc storage have been implicated in certain adverse outcomes. recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (pufas) and their oxidized products (oxylipins) accumulate during rbc storage despite leukoreduction. to evaluate the extent of membrane lipid degradation and oxidation in stored rbc units among the donor population with different blood groups, we quantified pufas and lysophospholipids (lpls) in 405 leukoreduced rbc units. study design/method: rbc units from different donors were acquired and processed on day 43 (one day past their expiration). 35 bioactive lipids including 10 common fatty acids, 10 oxylipins, and 15 lpls were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (lc-ms/ms-mrm). total fatty acid concentrations of selected units were also analyzed. a one-way anova test was used to determine significant difference of analytes amongst the different blood groups. results/finding: we observed a wide distribution in concentration of major pufas in 405 stored rbc units. for example, arachidonic acid (aa) ranges from 0.5 -10.7 mm, linoleic acid (la) (1.4-28 mm), dihomo-c-linolenic acid (dgla) (0.1-0.8 mm), eicosapentaenoic acid (epa) (0.03-3.1 mm), docosahexaenoic acid (dha) (0.2-3.0 mm), and alpha-linolenic acid (ala) (0.06-2.3 mm). ten oxylipins including hetes, hodes, and dihomes, and 15 lpls including lpcs, lpss, and lpes all showed a large variation in concentration among donors. of 35 analytes quantified, 25 showed a significant difference in concentration among different blood types by one-way anova testing (fdr<0.05). the ab rh1 blood group consistently exhibited the lowest concentration of major pufas, while the o rh-blood group showed the highest, averaging a two-fold difference in concentration (o rh-/ab rh1). the fold increase of o rh-/o rh1 among pufas ranges from 1.3 to 2.1, suggesting the rh blood group, independent of the abo blood group, correlates with donor to donor variation in lipid metabolism. conclusion: the wide distribution in the concentration of bioactive lipids among 405 stored rbc units suggests that lipid degradation is highly donor-background/case studies: to ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality. in the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions. micrornas (mirnas) levels are modulated by these storage-related damages, which makes mirnas ideal candidates as potential biomarkers of quality monitoring. lately, nanoparticles have been widely studied and used for biosensing applications. the objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals. study design/method: gold nanoparticles (gnps) surrounded by a fluorescent silica shell were prepared using a wet chemistry method. mirna-223 was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions. custom rna and dna molecular beacons were designed and used as a probe for the specific detection of mirna-223 targets in pbs and human plasma. these fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-gnps using an edc/nhs cross-linking reaction. the hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence. results/finding: gnps (49 6 6 nm) surrounded by a thick fluorescent silica shell (22 6 2 nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability. conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis. the fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (r 2 rnaprobe 5 0.96 and r 2 dnaprobe 5 0.98) with mirna-223 concentration, down to a 10-nm limit of detection. hybridization assays in 1% human plasma appear to demonstrate denaturation of rna probes and targets. conclusion: biocompatible fluorescent gnps were prepared and used as tools for blood product characterization. the conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques. the functionalization of the probe is still being optimized. the fluorescence response of the molecular beacon was characterized for the detection of a model mirna target in pbs and in 1% human plasma. energy metabolism profile of erythrocytes during storage suping ren*, qun yu, yanbing wang, changlan li and yu wang. background/case studies: the moment the mature red blood cells (rbcs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues. red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell. glycolysis is the only energetic metabolic pathway for mature rbcs to obtain atp which is the energy for rbcs to maintain a number of vital cell functions. generally, the current methods used to measure rbcs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.xf technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, rbcs have no nucleus, mitochondria and other organelles, so application of the xf technology in erythrocytes and exploration of the assay conditions are necessary. 6.7 6 0.0 a 6.9 6 0.0 6.7 6 0.0 a 6.6 6 0.0 6.7 6 0.0 a 6.9 6 0.0 total atp,lm/ghb 7.6 6 0.3 a 5.2 6 0.3 7.3 6 0.2 a 5.5 6 0.3 7.3 6 0.4 a 4.9 6 0.5 extracellular lactate,mm 5 6 1 a 6 6 1 7 6 0 8 6 0 6 6 0 6 6 1 extracellular glucose,mm 32 6 1 a 50 6 3 2 9 6 1 a 38 6 1 2 5 6 0 a 28 6 1 extracellular na 1 ,mm 142 6 2 143 6 2 138 6 1 a 137 6 1 144 6 1 141 6 1 extracellular k 1 ,mm 1 6 0 a 4 6 0 1 6 0 a 5 6 0 1 6 0 a 4 6 0 a p<0.05, paired t-test b intercept blood system for red blood cells is not approved for commercial use. c this project has been funded in whole or in part with federal funds from the dhhs; aspr; barda; contract no. hhso100201600009c. background/case studies: pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer. in vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported. study design/method: swirling of distributed products was monitored one year before and one year after implementation of intercept pathogen inactivation. metabolic parameters like ph, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products. furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling. results/finding: in an experimental phase on a limited number of products (n56) to validate the intercept pathogen inactivation method, ph and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products. once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former. furthermore, the number of apheresis products with premature low swirling increased by 50% (63/12,492) compared to the previous year without pathogen inactivation (42/12, 931, p50.030, chisquare) . in contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased (2/15,286 vs 10/ 13,488). of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count (5.0x10 11 ) than unaffected controls (3.5x10 11 ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects. these signs included lower ph, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation. conclusion: the risk of increased storage lesion rates following intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above 5.0x10 11 . in vitro quality of single dose amotosalen/uva treated platelets in 35% plasma/65% pas-3 after 5 days of storage crystal stanley 1 , marguerite kelher 2 , nero evero 1 , melissa vongoetz 3 , betsy donnelly 3 and anna erickson* 3 . 1 belle bonfils memorial blood center, 2 university of colorado, 3 cerus corporation background/case studies: the interceptv r blood system for platelets is fda approved for the ex vivo preparation of pathogen-reduced amicus o apheresis platelet components (pc) in pas-3 to reduce the risk of tti, including sepsis, and to potentially reduce the risk of transfusion-associated gvhd. registration studies (clinicaltrials.gov nct02298842) are in progress to support approval of the trima o apheresis platform for collection of platelets components (pc) suspended in pas-3 and plasma. the objective of this study was to evaluate in vitro function of platelets suspended in 35% plasma/65% pas-3, collected using the trima platform, after treatment with the intercept blood system for platelets. study design/method: double dose apheresis pc, 7.5 6 0.6 310 11 platelets in 602 6 52 ml, were collected on the trima apheresis platform in 35% plasma/65% pas-3. a sample was taken from each donation prior to dividing the donation to produce intercept treated apheresis pc (t), using the small volume (sv) set, and an untreated control pc (c). input volumes for replicates, n56, were 302 6 26ml (t) and 300 6 27ml (c) with doses of 3.8 6 0.3 3 10 11 (t) and 3.7 6 0.3 3 10 11 (c). all pc were stored under the same conditions and evaluated on day 5 and day 7 for physical/metabolic characteristics. results/finding: on days 5 and 7 all t and c pc had ph228c !6.2. the dose recovery for t was 87%63%. on day 5, t had lower count, volume, dose, bicarbonate and glucose compared to c pc; however, parameters predictive of in vivo function (atp, morphology score, hsr, and esc) were equivalent between t and c (table 1) . conclusion: trima pc in 35% plasma/65% pas-3 treated with the inter-cept blood system for platelets using the sv set and stored for 5 days retained in vitro metabolic and functional properties consistent with in vivo functionality. induction of pluripotent stem cell-derived cardiomyocyte toxicity by supernatant of long term-stored red blood cells in vitro feng-yan fan 1,2 , yang yu 1 , li-ping sun 1 , shu-fang wang 1 , rui wang 1 , lei-ying zhang 1 and deqing wang* 1 . 1 the department of blood transfusion, the pla general hospital, 2 the department of blood transfusion, air force general hospital, pla background/case studies: recently, multi researches have reported that longer term-stored red blood cells(rbcs) units were associated with increased risks of clinically adverse events, especially in critically ill patients. however, other studies have concluded the negative results. whether rbcs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic. to study the adverse effects of longer term-stored rbcs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(ssrbcs), and study the possible mechanism. study design/methods: 1 five doses of leuko-reduced rbcs were prepared, and supernatant was isolated by centrifugation on d0, d14 and d35. we looked at the cardiotoxicity of ssrbcs on human-induced pluripotent stem cell-derived cardiomyocytes (hips-cms). hips-cms were treated with ssrbcs in 17% final volume simulating the large volume blood transfusion. using real-time cellular analysis (rtca) technology the beating of hips-cms was recorded in real time in detail. 2 levels of k and lactic acid (la) were tested using automatic biochemical analyzer.3 k and la solution with concentrations being consistent with ssrbcs were prepared and cocultured with hips-cms. we analyzed the cardiotoxicity of k and la solution on hips-cms. 4 treated hips-cms with d35 ssrbcs, d35 k and cell culture media for 48h. the nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence. total rna of hips-cms was isolated and mrna analysis microarray was implemented. screened for toxic effects related signaling pathways through bioinformatics analysis. results/findings: 1 d0 ssrbcs had no obvious influence on beating state of hips-cms-hips-cms treated with d14 ssrbcs stop beating, but beating patterns restored at 48h. hips-cms treated with d35 ssrbcs stop beating, and beating patterns did not restored at 48h. 2 levels of k and la in ssrbcs changed most obviously. 3 only d35 k solution made hips-cms stop beating and can restore in 48h; d0 k, d14 k and la solution did not influence the beating pattern in at the end of the treatment for 24h, hips-cms treated with d35 ssrbcs show obvious shrinkage. at the end of the treatments for 48h, cells treated with d35 k and d35 ssrbcs both show obvious shrinkage, the shrinkage in d35 ssrbcs group was more serious. the immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected. 5 gene expression array results show a total of 140 genes were differentially expressed in d35 ssrbcs group compared with naive group. there was no consistent separation within the d35 k and naive group. fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect. under the condition of simulating the large volume blood transfusion, ssrbcs of long term-stored rbcs have toxic effect on myocardial cells. 2 in addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects. 3 further study should be applied to signal pathways on ssrbcs induced cytotoxicity. 4 large volume transfusion of long term-stored rbcs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it. background/case studies: processing thawed, deglycerolized red cell concentrates (rcc) in a functionally closed system allows for a prolonged storage after thawing. thawed cells are better maintained in as-3 as compared to sagm. the presence of citrate in as-3 seems to be necessary to prevent hemolysis of thawed cells. during storage in as-3, atp and 2,3-dpg levels rapidly decline. recently developed additive solutions like pag3m and as-7 have shown to better maintain 2,3-dpg and atp levels during storage of normal, unfrozen, rcc. however, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells. we therefore designed pag3c in which the mannitol of pag3m was replaced by citrate. the aim of this study was to investigate the in vitroquality of thawed, deglycerolized rbc during storage at 2-68c in pag3c. study design/method: leukoreduced rcc (n56) in pag3c (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at 2-68c. on day 8, rccs were glycerolized using acp215 (haemonetics v r , braintree, ma) to a final concentration of 40% (w/v), frozen and stored for at least two weeks at -808c. after thawing and deglycerolization using acp215, rcc were resuspended in pag3c. during storage at 2-68c, stability (hemolysis), atp and 2,3-dpg levels were determined. results were compared with thawed rcc (prefreeze storage in sagm, n58) resuspended in or sagm (n54). results/finding: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels at day 8 as compared to storage in sagm, resp. 9.1 6 7.6 mmol/g hb and 1.9 6 0.7 mmol/g hb. hemolysis during post-thaw storage in pag3c remained below 0.8% for 35 days and was comparable with storage in as-3. in sagm, hemolysis remained below 0.8% for 7 days. during the first 2 weeks of post-thaw storage in pag3c, both atp and 2,3-dpg levels increased, followed by a gradual decline during prolonged storage. during the whole postthaw storage period, rccs in pag3c showed significantly higher atp and 2,3-dpg levels compared to as-3 or sagm. while in sagm and as-3, 2,3-dpg levels were undetectable after 7 days post-thaw storage, in pag3c, 2,3-dpg levels only decreased to 6.1 lmol/g hb after 35 days of storage. conclusion: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels. as compared to as-3, post-thaw storage in pag3c showed comparable hemolysis while atp and 2,3-dpg levels were much better maintained. based on a maximum allowed hemolysis of 0.8% and an atp content of >2.7mmol/g hb, thawed rcc can be stored at 2-68c for 35 days in pag3c. background/case studies: platelets (plts) are vital for effective treatment of hemorrhage. cold (48c, 4c) storage of plts in platelet additive solution (pas) is a promising alternative to conventional storage at room temperature (rt) due to a lower risk of bacterial concerns, preservation of plt function, and mitigation of plt activation. currently only 2 apheresis (ap) and pas systems are fda-approved for use in the us: trima and isoplate-pas (iso; terumo) and amicus and intersol (int; fenwal) . the goal of this study was to assess the adhesive function of long-term cold-stored plts collected by fda-approved ap/pas methods. study design/method: plts were collected (n54-5) in 65% iso using a trima or in 65% int using an amicus and stored for 15 days at rt and 4c. samples were tested on day 1 (baseline, bl), 5, 10, and 15 of storage to assess plt adhesion under shear flow (bioflux). acd vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (rbcs) for all bioflux runs. simulated whole blood was created by combining plts labeled with calcein-am with rbcs at 40% hct. labeled blood was perfused through microfluidic channels (fluxion) coated with 100 ug/ml type-1 collagen at 720s -1 shear rate. images were acquired every 30 sec for 10 min using a fluorescent microscope and % surface coverage was reported. data were analyzed using two-way anova and posthoc tukey test with significance at p<0.05. results/finding: both rt-int and rt-iso plts showed significantly decreased adhesion by day 5 of storage compared to bl (bl: 11.6 6 1.7%, rt: 4.9 6 1.2%; p<0.005). 4c-int samples showed no difference in adhesion at any timepoint compared to bl-int but significantly enhanced adhesion compared to both rt-int and rt-iso. in contrast, 4c-iso plts showed significant enhancement of surface coverage compared to bl-iso by day 5 (p50.03) and compared to 4c-int by day 10 (p<0.01). conclusion: our work suggests that 4c storage of plts collected with a trima ap system in iso for up to 15 days offers a significant enhancement in adhesive function compared to plts collected with an amicus system in int and stored at 4c. these results are surprising since both 4c-int and 4c-iso have been shown to express similar levels of cd62p, pac-1, and phosphatidylserine and may suggest differences in pas plt intracellular signaling. as expected, storage at 4c of plts collected on either platform demonstrated superior function to rt storage. a plt product with superior hemostatic function and a shelf-life 3x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster plt availability for trauma care in the us. table 1. comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after 2-week storage between unirradiated and irradiated groups (n 5 29) tang, is an anti-inflammatory agents and has a good safety records in clinic. it could reduce the severity of experimental autoimmune encephalomyelitis (eae), asthma, colitis, systemic lupus erythematosus(sle) and other immune diseases.however,its potential in inducing transfusion tolerance remains to be explored.the aims of our study are to find if baicalin could inhibit red blood cell (rbc) immunization and to elucidate the possible mechanism of yin-chen-tang in preventing hdn. study design/method: we used human red blood cells with adjuvant lipopolysaccharide (lps) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on rbc immunization. mice were divided into a normal control group, a human rbc transfused positive control group receiving human rbc and lps intravenously weekly for five weeks, a control group receiveing dexamethasone (10mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin (250mg/kg/day) intraperitonealy daily for five weeks. assessment of human rbc immunization was performed by measuring serum immunoglobulin g (igg) and immunoglobulin m (igm) against human rbc weekly. and the lymphocyte changes in spleen are also monitored by flow cytometry. results/finding: we found that baicalin treatment decreased serum igg but not igm production significantly since the second week, with a concomitant reduction in th17 cells and increase in cd4 regulatory t cells in both spleen and mesenteric lymph nodes. and there are no significant differences in the percentage of th1,th2,tfh and tfr cd4 subpopulation among all groups.in addition, baicalin treatment didn't decrease the size of spleen and the percentage of cd4 positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse. our results indicate that baicalin could inhibit rbc immunization especially igg production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events. in addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for yin-chen-tang as a widely used chinese herbal medicines in preventing hdn. comparison of immucor's pak plus and pak lx assays for the detection of human platelet alloantibodies randy m schuller* 1 , sarah kloss 1 , sara crew 1 and sandra j nance 2 . 1 american red cross, 2 american red cross and american rare donor program background/case studies: alloantibodies directed against human platelet membrane glycoproteins (gp) ia, iia, iib, iiia, ib, ix, iv, and cd109 have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait), post-transfusion purpura (ptp), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma. polymorphic epitopes on these gps give rise to 28 unique human platelet antigens (hpa). identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders. currently the only 510k fda approved test permits the identification of these hpa antibodies to the glycoprotein level. immucor has recently released pak lx, a research use only (ruo) assay in the united states that has the ability to identify hpa antibodies to a single nucleotide polymophism (snp). we compared the performance of pak lx to the fda approved immucor pakplus. study design/method: we compared pakplus and pak lx results from 40 plasma and serum clinical specimens. group 1 contained a single hpa alloantibody specificity with or without hla antibodies (n526). group 2 included 5 specimens with hla antibodies alone and group 3 consisted of 9 patient samples that were negative for both hpa and hla antibodies. pak lx utilizes a luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (hpa-1, hpa-2, hpa-3, hpa-4, hpa-5, gpiv) and hla class i. pakplus uses an elisa method and results can only be reported to the glycoprotein location (gpiib/iiia, gpia/ iia, gpib/ix, gpiv) along with hla class i. however, based upon the pattern of reactivity observed in the pakplus and pak lx assays it is possible to determine the most probable hpa antibody specificity to the hpa snp. results/finding: conclusion: when analyzing hpa antibody specificity, there is 100% concordance observed for hpa-1a, hpa-1b and hpa-5b antibodies. the pak-plus assay had difficulty discriminating hpa-5b from hpa-5a antibody when hpa-5a antibody was present (3 false positive samples) although the pak-plus signal od to cutoff od ratio was significantly higher for hpa-5a when compared to hpa-5b in these samples. the discordant hla class i antibody results between the assays was isolated to very weakly positive antibody (within 10% of the cut-off for pakplus and <2.0 adjusted ratio for pak lx). we conclude that pak lx is an easy to use platelet alloantibody screening method that has the ability to differentiate hpa antibodies to the allele level. histo-blood group antigen lewis y promotes cell migration via regulation of microtubule acetylation huijun zhu* and ping lu. shanghai blood center background/case studies: blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction. beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes. lewis y is a histo-blood group antigen belonging to abh family. ley consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology. ley is demonstrated to affect cell mirgration via various mechanisms. however. although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and ley. as microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of ley in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice. study design/methods: we first manipulate ley expression in breast cancer cells by overexpression or sirna knockdown of fucosyltransferases, and block ley activity in mda-mb231 cells using anti-ley antibody, to verify the effect of ley on cell migration. then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability. to establish the role of ley in cell migration via microtubule modification, we use hdac6 specific (tubacin) and nonspecific (tsa) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of fut1 overexpression on cell motility. results/findings: fut1 overexpression increases both ley expression and cell migration, while fut1 knockdown leads to the opposite. ley activity blockade by anti-ley antibody also significantly inhibits cell migration. western blot and immunostaining results show a-tubulin acetylation level is negatively related with ley expression. tubacin or tsa treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of fut1 overexpression in promoting cell migration is eliminated. conclusion: it can be concluded from the results above that ley can promote cell migration via regulation of a-tubulin acetylation, wherein ley may have interaction with deacetylase hdac6. as tumor promoter, hdac6 becomes the target of many anti-cancer drugs. we demonstrated the potential association of ley and hdac6 function in this study. many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to ley; they can even be components in a network with other important molecules and contribute to the destiny of diseases. transfusion of blood products is frequently needed by tumor patients. most attention is focused on the search of compatible blood for reducing transfusion reaction. however, it may lower the chance for the disease to advance to take account background/case studies: reducing the risk of bacterial contamination in platelet (plt) products is of great concern since plt storage occurs at room temperature (rt). pathogen reduction technologies (prt) were developed to inactivate pathogens prior to transfusion; however, studies have shown that prt may damage plts over the course of extended storage at rt resulting in a greater loss of function than what is normally concomitant with platelet storage lesion. storage of plts in platelet additive solution (pas) at 48c helps to preserve plt function and reduces the risk of contamination. in this study, we established the impact of prt performed after long-term coldstorage of plts in pas, instead of before storage, on plt function, mitochondrial respiration, and cell death parameters. study design/method: plt units were collected in pas (n53) and stored at 48c for up to 10 days. after this time period, the bag was treated using mirasol prt (riboflavin and uv). samples were obtained and tested on the day of collection (baseline, bl), pre-mirasol (pre), post-mirasol (post), and 30 minutes post-mirasol . aggregometry (adp, collagen, trap), rotem, flow cytometry (cd62p [p-selectin] , lactadherin [ps] , , and gpib), high-resolution respirometry (oroboros), and imaging flow cytometry (amnis) were used for analysis. data are reported as means6sem, and paired student's t-tests were used to determine statistical significance (p<0.05). results/finding: on day 10, p-selectin levels were significantly higher in pre than bl (p50.03). mirasol treatment caused a significant increase in pac-1 expression compared to pre (pre: 10.5 6 3.1%, post: 28.1 6 4.7%; p50.04), which remained after incubation. a significant drop in both collagen and trap aggregation was observed in post samples compared to pre, but adp aggregation response was preserved. no differences in p-selectin, gpib expression, and mitochondrial respiration were observed between pre and post samples. post-30 samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to pre and post. conclusion: prt treatment of plt units in pas after 10 day storage at 48c presents a unique alternative to prt treatment of plts prior to rt storage. in addition to providing a lower risk of bacterial contamination, 48c-stored pas plts may provide better preservation of hemostatic function than standard-of-care rt plts, even after mirasol prt treatment. however, we show here that mirasol prt of day 10 4c-stored pas plts followed by incubation (30 minutes or more) results in widespread cell damage and should be avoided. safety evaluation of lyophilized canine platelets in a model of coronary artery bypass graft (cabg) todd m. getz* 1 , arthur p. bode 1 , anne s hale 2 , michael stanton 3 , mark johnson 4 and g. michael fitzpatrick 3 . 1 cellphire, 2 bodevet, inc, 3 cellphire, 4 background/case studies: cellphire has completed a micro dose clinical safety trial using lyophilized human platelets. cellphire also evaluated the safety of lyophilized canine platelets (lcp) in comparison to liquid stored canine platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (cabg) in the canine. this safety study was in support of a future phase ii human clinical trial in cardiac patients. study design/method: three groups of eight mixed breed hounds underwent cabg to create an anastomosis and were administered lcps equal to 33, 10, and 3.3% of the total circulating platelet count (tcpc). one group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control (2-day old liquid-stored platelets). safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the 4 hr evaluation period. full necropsies with complete tissue analysis were also performed. efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (pt, aptt, fibrinogen, and teg). the results demonstrated that administration of the test article at doses up to 33% of the tcpc was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group. the mortality noted on study was considered to be related to the surgical model and not a result of test article administration. the results also demonstrated that administration of doses of 10% and 33% of the tcpc produced a significant decrease in blood loss. the lcps at 10% and 33% tcpc were as effective in mitigating blood loss as 2-day old liquid-stored platelets and trended towards being more effective. no appreciable differences in coagulation parameters were observed between groups. conclusion: the results of the study demonstrate that administration of lcp up to 33% of the tcpc was safe in a canine cabg model. the data also demonstrate that administration of lcp at doses of 10% and 33% of the tcpc reduced blood loss. these results suggest a starting dose above 3.3% tcpc may be required to achieve an effective dose in future human phase ii trials in cardiac patients. although the study was not powered for efficacy, these data indicate a level of safety, as 10% tcpc had similar efficacy signals as 33% tcpc with no observable severe adverse events. the starting effective dose may vary depending on the clinical indication. future studies will be required. this study was funded under barda contract hhso100201300021c. the study on pcr-ssp technique for the genotyping of cd36 329-330del.ac mutation and the genetic polymorphism of cd36 329-330del.ac in chinese population lilan li* and guoguang wu. nan-ning institute of transfusion medicine background/case studies: cd36 (platelet glycoprotein iv, scarb3) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in chinese population. except anti-hla, anti-cd36 is the most common antibody of clinically relevant platelet antibodies in chinese population, which is associated with the high frequency of cd36 deficiency in china. cd36 gene mutation is the main reason that leads to cd36 deficiency. cd36 329-330del.ac (frameshift at aa110) mutation is one of the cd36 mutations that causes cd36 deficiency. have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood. the recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions. this places an already highly vulnerable pediatric population at risk for acquiring preventable infections. the primary aim of this project was to determine mmr vaccination immunogenicity in patients chronically transfused with rbc. study design/method: medical charts were reviewed for vaccination and transfusion histories. mmr-specific antibodies were quantified in 28 pediatric patients who received both doses of the mmr vaccine at 12 and 18 months of age while they were on a chronic rbc transfusion program for sickle cell disease, b-thalassemia major, diamond-blackfan anemia or pyruvate kinase deficiency. there was no formal control group; long-term immunity rates in the literature are !90% for all mmr components. results/finding: table 1 shows immunogenicity to vaccine components. delays between vaccination and serology testing averaged 6.8 years (0.5 to 16.5 years). thirteen patients (46%) were chronically transfused at the time of serology. twenty-three patients (82%) seroconverted to at least one of the vaccine components. conclusion: to the best of our knowledge, this is the first study designed to measure the effect of rbc transfusions on mmr vaccine immunogenicity. although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the mmr vaccine in chronically transfused patients immunized prior to 6 months posttransfusion. weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post rbc transfusions is called for in chronically transfused infants. post-vaccination serology should be considered. cold stored uncrossmatched whole blood can be safely administered to pediatric trauma patients christine m leeper 1,2 , franklyn cladis 2 , richard saladino 2 , darrell triulzi 3 , barbara a gaines 2 and mark yazer* 1 . 1 university of pittsburgh, 2 children's hospital of pittsburgh of upmc, 3 institute for transfusion medicine background/case studies: the use of uncrossmatched cold stored whole blood (wb) is becoming increasingly popular in the initial resuscitation of trauma patients without a current abo group. wb has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood. this report details the initial use of wb in pediatric trauma patients. study design/method: pediatric trauma patients !3 years old and !15 kg with evidence of hemorrhagic shock were eligible to receive up to 20 cc/kg of cold stored, leukoreduced group o negative wb during their initial resuscitation. all wb units had a low titer of anti-a and -b (<50) to reduce the likelihood of hemolysis in non-group o recipients. biochemical markers of hemolysis were measured on the day of wb transfusion and the following two days. admission thromboelastograms were obtained and repeated as necessary during the resuscitation. after receipt of the maximum quantity of wb, conventional components were utilized. results/finding: in approximately 11 months, 15 trauma patients received wb: 7 group o and 8 group a recipients, 53% male, median (iqr) age was 11 (4.5-14) and 73% blunt trauma mechanism. patients were severelyinjured with a median (iqr) injury severity score of 36 (22-51) and 47% mortality rate. the median (iqr) quantity of wb transfused to group o recipients was 21.9 (14.8-24.3) ml/kg versus 13.4 (9-18) ml/kg to non-group o recipients. no transfusion reactions were reported. the mean 6 standard deviation haptoglobin concentrations for non-group o recipients was 51.3 6 14.4 mg/dl on day 0, 86.3 6 36.8 mg/dl on day 1, and 126.9 6 45.8 mg/dl on day 2; the corresponding haptoglobin concentrations for group o recipients were 51.4 6 38.0 mg/dl, 84.7 6 61.5 mg/dl, and 134.8 6 68.3 mg/dl, respectively (p>0.42 for all comparisons). similarly there were no significant differences in total bilirubin, ldh, creatinine, and potassium at any time point. regarding evaluation of cold platelet function, we compared the subset of patients who received wb but no warm platelets (n57) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n514). the mean 6 standard deviation platelet volume administered was 112 6 24 cc for whole blood recipients versus 147 6 68 cc for warm platelet recipients. when pre-and posttransfusion teg and platelet counts were analyzed, there was no difference in median platelet count or teg maximum amplitude (ma) between cold and warm platelet groups. conclusion: use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe. receipt of low titer group o wb did not lead to detectable hemolysis amongst the non-group o recipients. given this finding, the maximum quantity of wb per patients will be increased to 30 ml/kg. identification of red blood cell antibodies in human breast milk by novel adaptation of serological method philippe p pary*, alexis leonard, lauren hittson, naomi lc luban, deepika s darbari, yunchuan delores mo, cyril jacquot, valli criss and jennifer webb. children's national medical center background/case studies: human breast milk contains immunoglobulins that are present in maternal serum and secretions. data in mice has demonstrated the potential for kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking. a two week old infant with a history of rh-d hemolytic disease of the fetus and newborn (hdfn), previously treated with intravenous immunoglobulin and phototherapy, was referred for anemia and reticulocytosis. patient was o positive, positive direct antiglobulin test (dat) 41 with anti-human igg only, and a 31 positive antibody screen by gel method. antibody identification showed anti-d in both the plasma and eluate. patient was transfused o negative red cells and discharged. over several weeks, the patient returned twice for persistent anemia requiring additional transfusions. at eight weeks of age, evaluation showed a persistent dat igg reactivity concerning for continued antibody exposure. maternal breast milk was evaluated as a potential source. study design/method: based on similar properties of human breast milk and plasma, testing to identify igg antibodies using a stantard tube saline method was performed with a 60 minute 378c incubation, followed by 3 automated washes prior to the addition of anti-human igg reagent. as a control, breast milk from an o positive, antibody screen negative mother was used to assess for interference by milk proteins. antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method. antibody identification and titers were also performed when indicated. only freshly collected breast milk stored at room temperature for less than 3 days was found suitable for this technique. results/finding: the patient's mother showed plasma anti-d with a titer 4096 and the breast milk showed anti-d with a titer between 16 and 64. the patient had a consistent plasma anti-d titer of 8. the patient's mother chose to stop breast feeding after 8 weeks, and the patient's hemoglobin was improved at 12 and 16 weeks of age. using this method, we identified two additional cases of breast milk induced hemolysis: another anti-d and an anti-jka. conclusion: testing showed that it is possible to identify red cell igg antibodies in human breast milk using a standard tube saline method. we identified implicated antibodies in the breast milk received by infants with persistent anemia due to hdfn. breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding. cessation of breast feeding correlated with improved hemoglobin in affected infants. background/case studies: red blood cell (rbc) transfusion is lifesaving for patients with sickle cell disease (scd), but is commonly complicated by rbc alloimmunization. despite transfusion protocols serologically matching for c,e, and kell antigens, alloimmunization to rh antigens continues. scd patients often exhibit a hybrid rhd-ce-d gene which is often characterized by the production of a partial c antigen. it has been previously documented that 30% of c1 scd patients from the west indies and west and central africa are partial c and at (30%) risk for alloimmunization to the c antigen through transfusion of c1 rbcs. this study sought to determine the prevalence within a cohort of children with scd at a u.s comprehensive scd center. study design/method: rbc genotyping results performed on all scd patients using precisetype hea array (immucor, norcross, ga) at children's healthcare of atlanta were reviewed and compared to the serologic type for rh (c/c, e/e) antigens. the prevalence of c-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial c antigen based on the detection of the rhce*ce(733g,1006t) allele in the absence of an rhce gene encoding a conventional c antigen in trans, since this allele is commonly linked to the hybrid rhd*diiia-ce(4-7)-d gene which encodes the partial c antigen. review of the blood bank information system was performed to identify the number of c-antigen positive transfusion exposures and frequency of alloimmunization to the c antigen. results/finding: out of a total of 255 patients with genotype/rh phenotype data available, 78 (30.6%) were c antigen positive serologically. the allele frequency of rhce*ce(733g,1006t) was 0.071. in total, 15 (5.9%) patients possessed rhce*ce(733g, 1006t) in the absence of conventional c gene in trans. of the 78 c antigen positive patients, 15 individuals (19.2%) were predicted to be partial c based on four molecular profiles [rhce*ce(733g, 1006t)/rhce*ce:12; rhce*ce(733g, 1006t)/rh*ce:1; rhce*ce(733g, 1006t)/rh*ce(733g):1; rhce*ce(733g, 1006t)/rh*ce(733g, 1006t):1]. in these 15 partial c patients, no anti-c alloantibodies (or other rh antibodies) were detected after 60 transfusion exposures (57 c-antigen negative units; mean: 4, range: 0-36), likely from placement of a c-negative rbc restriction upon detection of the rhce*ce(733g, 1006t) allele. conclusion: this report confirms previous data of a high prevalence of the partial c antigen in scd patients historically typed as c-positive serologically, and demonstrates the benefits of rbc genotyping to prevent alloimmunization to a highly immunogenic rh antigen by identifying individuals who should receive c-negative blood. all patients with scd should have rbc genotyping performed for determination of their rbc phenotype, preferably prior to receiving transfusions. investigational detection of zika virus rna in us blood donors paula p sa a* 1 , megan l nguyen 1 , melanie c proctor 1 , david e krysztof 1 , gregory a foster 1 , erin k sash 1 , sandy s dickson 1 , joua yang 1 , jeffrey m linnen 2 , kui gao 3 , jaye p brodsky 4 and susan l stramer 1 . 1 american red cross, 2 grifols diagnostic solutions inc., 3 grifols diagnostic solutions, inc, 4 quality analytics, inc background/case studies: zika virus (zikv), an emerging flavivirus, is primarily transmitted by infected aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus. acute zikv infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders. the large proportion of asymptomatic cases, high numbers of returning travelers from zikv-active areas, severe clinical consequences to developing fetuses, the detection of rna in asymptomatic donors during the french polynesia epidemic, and 4 suspected cases of transfusion transmission in brazil led fda to release guidance documents to minimize the risk of zikv transmission via blood/ blood components. study design/method: investigational testing by mini-pool (mp)-nat using the procleix zika virus assay (tma) was implemented on collections from five presumed high-risk us states on 6/20/16 (fl, ga, sc, ms, al). following revised guidance on 8/26/16, testing was extended to all blood donations; conversion from mp-nat to individual donation (id)-nat was implemented in phases and completed on 12/12/16. travel history questions were discontinued on 1/23/17. confirmatory testing included repeat tma; in addition, rt-pcr, serology and red cell (rbc) tma were performed. estimates of viral loads were performed by end-point tma on plasma and rbcs. results/finding: as of 4/8/17, 2,288,855 donations were tested including 393,713 (17%) in 24,611 mps. no reactive donations were identified by mp-nat. of the 1,895,142 id-nat donations, 72 were initial reactive (ir) of which 8 (11%) confirmed positive (cp) by subsequent testing (cp rate of 1:286,107; positive predictive value of 11%; specificity of 99.997%). five (62%) cp donations were id-nat repeat reactive (rr); 3 (38%) donations were id-nat ir only, igm positive and rna positive in rbcs. cp donors resided in ma, tx, ca, ny, wv and 3 in fl, 2 of which were local transmissions. six donors had traveled to a zikv-active area returning to the us from 2 to 73 days prior to donation. two donors with a travel risk reported clinical symptoms; 6 cp donors (75%) remained asymptomatic. zikv rna was detected in rbcs from all cp index donations with estimated levels varying from less than 40 copies (c)/ml to about 8ê 5 c/ml. at the time of writing, the longest period of detection in rbcs was 91 days vs. 17 days in plasma from the same tma-rr donor. zikv rna levels in plasma were obtained from 1 ir and all rr donors, ranging from 12 to 2000 c/ml. study design/method: plasma from blood donors were screened by individual donation (id-nat) for the presence of zikv rna with the cobasv r zika test. id-nat1 samples were repeated in duplicate and further tested by a second nat to confirm infection and estimate vl, and for anti-zikv igm. simulated mps of 6 were prepared by diluting nat1 plasma 1:6 and tested to discriminate id-nat only detectable donations. nat yield samples for which simulated mp and conclusive igm results were available (n5308) were sorted into 4 categories corresponding to sequential stages of acute zikv infection: igm-/low vl; igm-/high vl; igm1/high vl; igm1/low vl. results/finding: of 52,942 donations collected april 3-december 31, 352 were reactive for zikv rna. igm-index donations had higher vls (mean 1.1 x 10 6 vs 8.3 x 10 4 iu/ml) and higher proportions of simulated mp-detectable results (93% vs 23%) than igm1 donations. the distribution by stage of infection was evaluated as the epidemic evolved. over the course of the epidemic, the rates of id-nat only detectible and igm1 donations increased (table 1) . conclusion: this study demonstrates how the viral and immunological profiles of zikv infection in the index donations shifted through the course of the 2016 pr epidemic. categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of mp vs id-nat screening likely correlate with vl and serological stages of infection. staging of infections also has implications for diagnostic testing and understanding the durations of zikv viral and immunological markers in blood and persistence of zikv in body fluids and tissues. cobasv r zika is not commercially available for blood screening. data generated under the cobasv r zika ind is preliminary and has not been reviewed by fda. this project has been funded in whole or in part with federal funds detection of zika virus rna in united states blood donations using cobas v r zika on the cobas v r 6800/8800 systems lisa lee pate* 1 , phillip c williamson 2 , michael paul busch 3 , susan rossmann 4 , scott jones 5 , ann butcher 1 , john duncan 1 , jean stanley 1 and susan a galel 1 . 1 roche molecular systems, inc., 2 creative testing solutions, 3 blood systems research institute, 4 gulf coast regional blood center -sugar land, 5 qualtex laboratories background/case studies: in february 2016, the us fda recommended that all blood donations in areas with active zika virus (zikv) transmission be tested with an fda approved nucleic acid test (nat) for zikv rna or treated with an fda approved pathogen reduction technology. the cobasv r zika test was approved under an investigational new drug application on march 30, 2016 and testing of puerto rico donations began on april 3, 2016. as a precautionary measure some blood centers in the us states also began nat testing for zikv. in august 2016, the fda recommended universal screening of all blood donations. the aim of this study is to describe the detection of zikv rna in blood donations collected in us states between april 3, 2016 -february 28, 2017 using the investigational cobasv r zika for use on the cobas v r 6800/8800 systems. study design/methods: donations were screened with cobasv r zika by individual donation testing. all initial reactive (ir) results were repeated in duplicate. supplemental testing included an alternative nat (altnat) assay which is less sensitive than cobasv r zika and serology testing for anti-zika igm and igg. reactive donors were invited to enroll in follow-up, which included cobasv r zika and serology testing. a donor was considered to be zika confirmed positive if at least one replicate of the repeat testing by cobasv r zika was reactive on index donation or follow-up, reactive by altnat on the index donation, or positive for anti-zika igm on index or follow-up. all ir donations were also retested at a 1:6 dilution to simulate mini-pool testing. results/findings: a total of 1,776,190 blood donations were screened using cobasv r zika. of 56 ir donations, 12 were repeat reactive (rr), 39 non-rr and 5 had no repeat testing. of the 12 rr donations, 7 were positive by altnat; 3 of these were igm positive. all 4 altnat negative donors were igm positive. one donor was alt-nat equivocal and igm negative. of the 5 rr donors that were not igm positive on index, 3 enrolled in follow-up and all seroconverted. of 39 non-rr donations, 38 were altnat negative and 1 is pending supplemental testing. 8/38 donors were igm positive on index. 30 donors were igm negative on index; 15/30 enrolled in follow-up; 14 remained igm negative and 1 was 1gm inconclusive. of 5 donations without repeat testing results, 2 met criteria for positive (1 was altnat positive, igm negative and 1 altnat negative, igm positive). 1 donation is pending additional testing. altogether, 22/56 ir donations met the criteria for true positive on the index donation. 9/22 (41%) true positive donations were reactive when retested in a simulated minipool. 16/22 were igm positive. conclusion: 0.001% of the 1,776,190 donations in us states screened for zikv rna were confirmed as true positives. cobas v r zika is not commercially available for blood screening use. using monte carlo simulation luiz amorim* 1 , marc germain 2 , gilles delage 3 , maria esther lopes 1 and yves gr egoire 3 . 1 hemorio, 2 hemaquebec, 3 h ema-qu ebec background/case studies: zika virus was implicated in very large and recent outbreaks, in french polynesia (2013) , and in brazil (2015/2016), which was followed by outbreaks in south america, central america and caribbean. four probable transfusion transmitted cases were reported in brazil; since 80% of zika cases are asymptomatic, the actual transfusion rates can be much higher than reported. in this study, we used a monte carlo simulation for risk estimation during the brazilian outbreak. study design/method: the data feeding the monte carlo simulation were collected from january 1 st , 2016 through november, 26 th , 2016, from brazil (the whole country) and for rio de janeiro state, one of the outbreak epicenters. the data came from brazilian epidemiologic bulletins and from brazilian blood donation figures. the risk assessment was performed separately for whole blood (wb) donation and for apheresis platelets (ap). the model took into account the following parameters: zika incidence in brazil and in rio; lognormal distribution symptomatic viremia (period: 5 days, with 99% of the values lower than 18 days); 20% of infected donors with symptoms lasting 2 days; 1.2 donation/donor/year for wb and 1.75 for ap. the formula for transfusion risk calculation was: incidence x infectious period x average donation number per donor per year (wb, x/y; aph, z/y) x (1 -proportion of refused donors) x (1proportion of discarded donations due to post donation -pd -information). results/finding: the table below shows the results. the estimated risk of transfusion transmitted zika is very important in brazil and in rio de janeiro, where it can attain 1:13,598, for apheresis platelets. the severe consequences of zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in brazil dengue (denv) arboviruses in the population are not available in brazil. the objective of this study was to assess the contemporaneous incidence of these agents in donors at 4 large geographically dispersed blood centers located in the southeast and northeast of brazil. study design/method: in the brazil public blood bank system, nat screening for hiv, hcv and hbv is performed on minipools (mp from 6 donations). the residual volume of mp plasma, 0.35 -0.45 ml, is routinely discarded. beginning in april 2016 each blood center saved $67 mps/week for retrospective testing using the triplex zikv, chikv, denv transcription mediated amplification (tma) assay developed by grifols/hologic. mps were shipped to the usa and batch tested at grifols. in the first two weeks (april 3-15) 3 mp6 were combined into pools of 18 donations; thereafter mp6 were tested without additional pooling. to estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and 95% confidence intervals (ci) calculated using the method developed by biggerstaff. results/finding: the triplex assay performance was shown to have very high sensitivity (95% limit of detection <20 copies/ml for zikv/chikv/ denvs) and to accurately discriminate each of the arboviruses. testing of the first 6 months of samples is complete for 6,292 mp, comprised of 37,752 donations collected from april 3 to october 9, 2016. a total of 77 pools were positive, with 76 detected between april-june 2016. the table summarizes the highest monthly estimated percent positive donors for each virus in each city. months with highest percent postive donors were april or may. at the peak over 0.6% of donors in belo horizonte and rio were viremic for zikv, whereas zika was not evident in donors in recife, but over 0.4% of donors in that city were viremic for chivk during the peak. conclusion: during the latter part of the arbovirus outbreak season in brazil in 2016, zikv, chikv, and denv were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in brazil were extensively exposed to viremic blood components. the use of donor mps for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections. universal zika screening for blood donors in singapore sally lam* 1 , sze sze chua 1 , mars stone 2 , michael paul busch 2 and ai leen ang 1 . 1 health sciences authority, blood services group, 2 blood systems research institute background/case studies: singapore reported its first locally transmitted zika case on 26 august 2016. the numbers rose rapidly to 386 cases by the end september, with eight clusters (hotspots) of cases island-wide. zika virus (zikv) shares the same mosquito vector, aedes aegypti, as the dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and guillan-barr e syndrome, which hastened singapore's blood services group (bsg) to look into securing the safety of blood supply from the zika threat. we aimed to assess the assay performance of usa-fda investigational (ind) procleix zikv nucleic acid technology (nat) assay for universal blood donation screening in singapore to prevent transfusion-transmitted zika infection. study design/method: all blood donations were screened for zika with the procleix zikv nat assay since 1 october 2016. zika nat reactive samples were tested at blood system research institute (bsri) for zika rna in plasma and red cells by pcr and for zika and dengue igm and igg antibodies. a zika confirmed case was defined by the presence of zika rna by pcr and/or zika antibodies. the analytical sensitivity was evaluated using 300 blinded frozen samples consisting of 25 replicates of 11 half log dilutions of the who international standard for zikv and 25 replicates of negative controls prepared by bsri. probit analysis was performed to determine the 50% and 95% limits of detection (lod) . clinical performance of the procleix zikv assay was also assessed with local patient samples obtained from institute of infectious disease and epidemiology, singapore and a 14 member blinded zikv reference panel from the usa-fda. results/finding: a total of 63,144 donations were screened from 1 october 2016 to 31 march 2017, with 1 false positive case and 1 zika confirmed donation detected. alternative zikv pcr tested positive in both the plasma and red cells with an estimated plasma viral load of 9.54x10 5 copies/ml. zika igm was negative in the index donation sample but present in the 10-day post-donation follow up sample.. the donor reported no clinical symptoms. the analytical sensitivity for the procleix zikv assay was determined to be 2.1 copies/ml at 50% lod and 10.0 copies/ml at 95% lod. the procleix zikv assay detected rna in 6 out of 9 patient samples and provided 85.7% agreement to the results of the usa-fda zikv reference material. conclusion: the investigational procleix zikv assay showed good analytical sensitivity and clinical performance, suitable for blood screening of zika infection especially in asymptomatic donor populations. bsg commenced universal zika nat screening by individual donation testing following the zika outbreak with 1 confirmed zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of 1 in 25,888 donations in singapore. background/case studies: a cap/aabb work group suggested that steps be taken to phase in rhdgenotyping for patients with a serologic weak d phenotype. weak d types 1, 2 and 3 express all the major rhd epitopes and these patients can be managed as rhd-positive, which may lead to a reduction in unnecessary rh immunoglobulin (rhig) administration and conservation of rhd-negative rbcs. study design/method: rhd genotyping was performed on all patient samples with weaker than expected or discrepant rhd typing results, utilizing a commercially available genotyping kit manufactured by immucor (rhd beadchip). initially, testing was performed at a reference lab while the rhd beadchip was validated and implemented at this institution. a serologic weak d phenotype is defined as weak to 21 reactivity on initial gel testing. if genotyping demonstrated weak d types 1, 2 or 3, the intent was to manage the patient as rhd-positive. if weak d types 1, 2 or 3 were not detected, the patient is considered at risk for alloimmunization and treated as rhdnegative. while rhd genotyping results were pending, rhd-negative rbcs were used and if pregnant, the patient was eligible for rhig. results were generally available in 2 to 4 weeks. results/finding: rhdgenotyping was performed on 22 patient samples over 15 months. of these 22 patient samples, 13 (59%) were weak d types 1 or 2. the remaining samples demonstrated a variety of alleles including known partial d variants (see table) . one patient identified as weak d type 1 required multiple transfusions over the study period, and refused rhd-positive rbcs. the remaining weak d types 1 and 2 patients have not received transfusions at this institution since they were genotyped. four of 4 obstetric weak d types 1 and 2 patients received rhig while genotyping was pending. conclusion: testing and management of patients with serologic weak d phenotypes is not standardized. rhd genotyping may lead to more consistent, personalized patient care and appropriate management of resources. in this 15 month study period 13 serologic weak d patients were identified who could be managed as rhd-positive, however this did not result in withholding any doses of rhig nor conservation of rhd-negative rbcs. genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies. these outcomes highlight the limitations of current genotyping processes, including long turn-around-time background/case studies: the rh blood group is highly immunogenic and the most clinically significant blood group secondary only to abo. currently, in the united states, blood donors who type rhd-negative by serology undergo weak-d testing to identify some weak and partial states of rhd expression. however, not all rhd expression can be detected serologically. it has been suggested that investigation of serologic rhd-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in rhd-negative recipients. study design/method: rhd genotyping of all serologic rhd-negative blood donors presenting to our blood donor center was implemented to identify units with altered rhd alleles that should be characterized as rhdpositive. repeat donations were not tested. initial serologic testing of blood donors was performed using 3 fda approved anti-d reagents. when reactivity with all 3 reagents was negative, rhd genotyping was performed using a commercially available genotyping kit manufactured by immucor (rhd beadchip). this assay detects over 80 rhd variant alleles and additional dna sequencing was performed in selected cases. to maximize efficiency samples were batched for testing; testing was generally performed once a month. if an rhd variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to rhd-positive recipients. results/finding: over a period of 8 months we tested 509 rhd-negative blood donors. there were 3 (0.6%) partial-d, 1 weak d (0.2%), and 3 (0.6%) del donors. in one donor sample a novel rhd allele was identified through dna sequencing (rhd*ivs5-46_42deltctc). the phenotype associated with this allele variant is unknown. investigation of previous donations from these 8 donors showed that 6 rhd-negative recipients received rbcs from 4 of these donors. five of these recipients underwent antibody screening after an average follow-up period of 5 months; anti-d was not detected in any sample (see table) . conclusion: serologic testing occasionally fails to identify some rhdpositive donor units, which could place rhd-negative recipients at risk for alloimmunization. dna-based testing can be used to identify donors who have the potential to sensitize rhd-negative individuals. in this limited study period a small number of serologic rhd-negative donors, whose genotype indicated potential to sensitize recipients, were found. however, review of recipient transfusion records indicated that prior exposure to these donors' rbcs did not lead to detectable immunization to date. future potential sensitizing events will be avoided by restricting these units to rhd-positive recipients. grifols diagnostic solutions labs, 5 grifols immunohematology center background/case studies: pregnant women with rhd variants may be candidates for rhig prophylaxis if molecular analysis reveals a genotype associated with possible anti-d formation. proposed testing algorithms advocate molecular characterization of weak d types but if a patient types as rhd-positive, no further action is proposed. women with partial d variants who may also be at risk of anti-d formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as d-positive but who may be candidates for rhig prophylaxis. our hospital is in an urban setting in which 63% of deliveries are to african-american patients. we initiated routine, full-gene rhdsequencing for obstetric patients whose serology demonstrated not only weak d, but also those who were categorized as "d1" with 31 reactivity to determine the prevalence of partial d patients in an ethnically-mixed population who may be at risk of anti-d formation. study design/methods: from october 2016 to march 2017, we performed routine d typing (neo, immucor) on 1875 obstetric specimens followed by rhd sequencing on samples with either a serologic weak d phenotype or anti-d testing strength of 31 using at least 1 antibody. solid phase and manual testing used the series 4 and series 5 reagents. four additional anti-d reagents manufactured by grifols (dg gel anti-d), quotient (anti-d blend), biorad (anti-d (rh1) blend), and ortho (bioclone anti-d) were also used for supplemental testing. rhd sequencing was performed by sanger methodology using routine clinical protocols. results/findings: rhd polymorphisms or variations were identified in all 13 samples. two of 13 (15.4%) were d1 with an rhd gene with only common, known intronic variants that is predicted to produce the "reference" rhd protein (ivs1-29c, rs2301153; ivs3 1 117c, rs28521909; and ivs3 1 124a, rs28562109). two (15.4%) were d1 and heterozygous for two apparently new rhd coding variations which we are confirming by further testing. four (30.8%) patients had rhd alleles with known potential to make anti-d (rhd*dol2, rhd*dar1.2, and 2 with weak d type 4.0). one had weak d type 96, which has uncertain susceptibility to alloimmunization and one was weak d type 1, which has not yet been associated with anti-d. interestingly, two (15.4%) had variable d expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning. one patient background/case studies: a weak d type 2 is a variant of the rhd protein that comprises an amino acid substitution located in the 12th transmembrane segment and expresses a reduced amount of the d antigen. this variant is known to be associated with the missense mutation c.1154 g>c which is the first nucleotide of the exon 9 of the rhd gene and thus could be implicated in exon 9 skipping when it is mutated. when performing ngs (next generation sequencing) analysis to fully genotype known patients, we identified an additional variant. study design/method: dna samples were studied by beadchip technology (immucor/bioarray solutions) and ngs using the sureselect human all exon v6 (agilent) and the nextseq500 platform. in silico analysis with different bioinformatic tools was used to predict splicing events. furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon 9 skipping of rhd gene. this study was completed by the comparative modeling between the wild type and the weak type 2 rhd proteins. results/finding: by a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of 10 patients previously characterized by beadchip technology. interestingly, 4 out of 10 carry the c.1154-31c>t intronic variation on the rhd gene, already described and associated with a del allele. among these last 4 patients, one has been previously characterized as rhd weak type 2 carrying the c.1154g>c (p.gly385ala). independently, sanger sequencing on 50 unrelated rhd weak type 2 samples pinpoint to a linkage disequilibrium between c.1154g>c (exac, maf 5 0.001145) and the c.1154-31c>t (exac, maf 5 0.2496). in silico analysis of both mutation located close to the splice acceptor site of the exon 9 does not predict a significant reduction of its strength score. with minigene vectors harboring rhd wildtype exon 9, mutant rhd c.1154g>c, mutant rhd c.1154-31c>t and double rhd mutants c.1154g>c plus c.1154-31c>t, we showed no influence on skipping of exon 9 due to these mutations. comparative modeling of rhd proteins pointed out an additional hydrophobic interaction on the rhd weak type 2 between ala385 (transmembrane helix 12) and val183 (transmembrane helix 6) hampering membrane insertion. conclusion: the c.1154-31c>t variation is always associated in cis with the missense mutation c.1154g>c on the allele rhd weak type 2. the c.1154-31c>t can be found alone on the rhd gene as a neutral polymorphism. we assess that these two mutations isolated or combined do not lead to abnormal rhd transcripts. our results clearly demonstrate that the weak d antigen reactivity observed with rhd type 2 red blood cells is due to the substitution of alanine at amino acid position 385 to glycine. topology of jk-weak or jk-negative single-nucleotide missense variants in the kidd protein glenn ramsey*. northwestern university background/case studies: the human urea transporter-b (hut-b) protein carrying the kidd blood group has 10 transmembrane (tm) and 2 tilted ureapore a-helices, a long extracellular connector segment, and 2 cytoplasmic segments at each end. numerous single-nucleotide missense variants (snmvs) weaken or abolish expression of jk a/b antigens determined at p.280. we mapped all reported jk-weak or jk-negative (jk-neg) snmvs onto the hut-b structure to explore topological correlates of jk antigen expression. study design/methods: jk*a and jk*b snmvs affecting jk expression were compiled from dbrbc and isbt registries, literature searches and 2010-2016 aabb, isbt and british blood transfusion society meeting abstracts. snmv locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian ut-b derived for analysis of ut function (levin ej, 2012) . results/finding: seven snmvs located within 1 amino acid (aa) from the exofacial or internal end of a tm helix are mostly weak variants (table) . all 3 at the exofacial ends (p.a93t, p.w240r, p.v333d) are jk-weak; the two jkneg exceptions p.g298e and p.g299e are at the internal end of the tm helix bearing jk a/b . four snmvs in the cytoplasmic n-terminal segment are mostly weak variants. in contrast, 13 snmvs within membrane helices are mostly jk-neg variants. three jk-weak snmvs (p.v10m, p.e44k, p.v76i) have been associated with allo-anti-jk a/b to the antigen on their alleles ("weak partial"). six of the 13 jk-neg variants are within 19 aa (p.270-p.299) of jk a/b at p.280. none of these snmvs are in the long extracellular connector region or the cytoplasmic c-terminal segment. jk-neg variants p.n289s and p.s291p are adjacent to p.288f and p.292l which line part of the urea transporter pore. conclusion: in the transporter-structured rhd and rhce proteins, snmvs with weak d, c, c, e or e expression are mostly within the rbc membrane, and non-canonical antigen-negative snmvs are unusual. in the structurally similar kidd hut-b, most jk-weak snmvs are at the ends of the tm helices or in the n-terminal cytoplasmic segment. among 13 jk-neg snmvs, most are in membrane helices. however, whether a variant appears jk-weak or jk-neg may depend on the extent of testing. next-generation sequencing may provide more complete structure-antigen correlations. background/case studies: the kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the slc14a1 gene, which encodes the urea transporter ut-b1. the kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine. the cause of the identical kidd-null phenotype with dominant inheritance [in(jk)] has not yet been defined, though it was first described in 1965. in contrast to recessively inherited kidd-null phenotype, this is not associated with mutations in the slc14a1 gene. the aims of the studies was to identify and characterize the causative gene for dominant kidd-null red blood cell phenotype (injk). jk-weak (bold)/ jk-neg expression n within 1 aa from tm a-helix end v76i, a93t, w171r, w240r, g298e, g299e, v333d* cytoplasmic n-terminal v10m, g40s, e44k, l45p in membrane tm and urea-pore a-helices r64w, r64q, g65d, i117t, a183v, l246r, a248t ‡, a270a §, l272f, n289s, s291p, t319m 2/10 * second nucleotide variant in this allele is synonymous (p.p196p). ‡reported as jk-neg but considered jk-weak by isbt. §near splice point. study design/method: we identified several families with dominant inheritance of the kidd-null phenotype in multiple kindreds in spain. we performed whole-genome linkage analysis, exome sequencing, expression (rt-pcr and western) analyses, and urea lysis using patients' cells. in addition, two probands underwent urine concentration tests. results/finding: using molecular approaches, we mapped the affected locus to a 5 mbp region in 19q13.11-13.2 with an lod score of 9.6. using deep sequencing, we identified a potential deleterious mutation in the znf850 gene, which deletes 84 bp resulting in loss of an entire zing finger domain. the identical del84-znf850 mutation is present in all affected individuals, and is absent from all controls tested (n>2000). in addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the znf850 locus, thus completely lacking the common allele, were identified. we also obtained dna from an unrelated injk individual reported from japan. in this individual, there was a similar, though not identical, znf850del84. none of the other potential genetic variants identified in the spanish kindreds was present in the dna from the injk individual from japan. consistent with the fact that the kidd antigen, encoded by the slc14a1gene, is a urea transporter that has been associated with renal function, we found that people with the znf850del84 in spain had an inability to concentrate their urine. conclusion: a predicted zinc finger deletion at znf850, prevalent in southern spain due to a founder mutation, leads to ut-b1 dysfunction and underlies the dominantly inherited kidd-null blood phenotype. the phenotype associates subnormal urine concentrating ability. in background/case studies: di-(2-ethylhexyl) phthalate (dehp) makes pvc film flexible and useful for blood products. during storage, dehp can leach from the bag film into solution and be metabolized. studies in rodents have suggested that exposure to dehp may be associated with adverse health effects, albeit at high dosages. attempts to find dehp alternatives for blood bags have been difficult due to the rbc membrane-stabilizing effect of dehp. bis(2-ethylhexyl) terephthalate (deht) a non-ortho-phthalate is structurally and functionally similar to dehp, but distinct from a metabolic and toxicological standpoint. deht can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor. the study objective was to evaluate the quality of fresh frozen plasma (ffp) stored in deht containers versus ffp stored in dehp containers at 30 days and 1 year. study design/methods: thirty-six wb units were collected into cpd solution, leukoreduced, centrifuged, and separated into rbc and plasma. abo identical plasma units were pooled together in groups of three. the 12 pools included 5 group a, 6 group o and 1 group ab. each plasma pool was weighed, mixed, sampled, divided into dehp and deht pairs, and frozen at less than -208c within 8 hours of collection. in vitro plasma testing (pt, aptt, factor v, factor viii, fibrinogen, protein c, and protein s) was done on day 0 (pool), day 30, and 1 year of storage. dehp and deht paired plasmas were thawed and tested at the same time. plasticizer concentrations were determined on day 0, day 30, and 1 year of ffp storage. dehp and deht and their monoesters were analyzed by liquid chromatography-mass spectrometry. internal standards were deuterated-dehp, mehp, deht and meht. the lower limits of quantification (lloq) were: dehp 5 2.9 ppm; mehp 5 0.3 ppm; deht 5 0.9 ppm; and meht 5 0.2 ppm. results/findings: mean and standard deviation (sd) for key clotting factors and plasticizer results are summarized in the table. there was no statistical difference in any plasma parameter between dehp and deht bags at the same time period. factor viii retained greater than 80% of its initial value. plasma stored in deht bags had an average plasticizer content 90% lower than that of the dehp bags. background/case studies: plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (rbcs) and/or crystalloid solutions. spray-dried plasma (spdp) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (ffp). the spray-drying process preserves coagulation proteins, but high molecular weight multimers (hmwm) of von willebrand factor (vwf) are decreased. the objective of this study was to compare spdp and ffp in reconstituted whole blood (rwb) to test the hypothesis that spdp is not inferior to ffp in facilitating platelet adhesion and thrombus formation. study design/method: under an irb-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at 100 g to separate rbcs from platelet-rich plasma (prp). prp was diluted 3-fold in pipes-saline with 1.4mm pge1 and centrifuged at 1900 g. the platelet pellet was resuspended in either spdp or ffp and recombined with the packed rbcs to create rwb with hematocrit of 34-40% and 150,000-250,000 platelets/ml. in addition, two rwb pairs were reconstituted with spdp diluted 1:1 (spdp50%) with plasma from a patient with type 3 vw disease (t3vwd). samples were fluorescently labeled with a gpiibiiia-specific antibody and the sample was flowed through a type i collagen-coated microchannel at a shear rate of 1600 s -1 for 180 seconds. still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (sa) along the length of the channel. ratio paired t-test was used to compare sa in samples reconstituted with spdp vs. ffp. the margin of noninferiority was 20% (spdp/ffp > 0.8). results/finding: six batches of spdp/ffp were evaluated using 17 subjects. there was no statistical difference between the spdp/ffp pairs (p50.7558). the mean ratio of spdp/ffp was 1.21 with a 95% ci of 0.84 -1.57. comparing spdp vs. spdp50%, there was no difference (median ratio 5 1.045, range: 0.95-1.14) in sa. two-way anova demonstrated that batch did not significantly affect ratio of sa in spdp vs. ffp. conclusion: spdp, despite a decrease of vwf hmwm, was not inferior to ffp in ability to support platelet adhesion and thrombus formation. on average, sa in samples reconstituted with spdp was 20% greater than in samples reconstituted with ffp. the lower limit of the 95 th % ci is a difference of 16%, which is less than the a priori determined margin of noninferiority of 20%. even with 50% dilution with t3vwd plasma, there was no reduction in platelet adhesion and thrombus formation in the spdp rwb samples. these data support the development of in-human studies to evaluate the efficacy and safety of spdp in preventing and reversing trauma-related coagulopathy. spray-dried plasma deficient in high molecular weight multimers of von willebrand factor retains hemostatic properties michael a. meledeo* 1 , qiyong peter liu 2 , grantham c. peltier 1 , ryan c. carney 2 , ashley s. taylor 1 , colby s. mcintosh 1 , james a. bynum 1 and andrew p cap 1 . 1 u.s. army institute of surgical research, 2 velico medical inc background/case studies: restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs. a single unit spray dried plasma (spdp) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von willebrand factor (vwf) and an increase in low molecular weight multimers. vwf is critical in platelet adhesion and thrombus formation. following work demonstrating enhanced function with use of glycine-based reconstitution solutions for spdp, this study examines two different spdp pretreatment conditions. study design/method: the samples were: (1) ffp; (2) ffp with 70mm glycine; (3) regular spdp without pretreatment (rspdp), rehydrated with glycine-hcl:glycine; (4) spdp pretreated with glycine-hcl (20mm); and (5) spdp pretreated with glycine-hcl:glycine (20mm:50mm; both pretreated were rehydrated in water). six donor-matched plasmas of each type were tested. vwf activity was measured by ristocetin cofactor assay. fibrin polymerization kinetics were analyzed by turbidimetry. thrombin generation (tg) was observed by thrombogram. chemistry was evaluated by i-stat. residual cell material was quantified by flow cytometry. coagulation properties were measured by thromboelastography (teg) in plasma and reconstructed whole blood (40% hct with 200 platelets/nl from typematched donors). platelet adhesion to collagen under shear was measured by bioflux. results/finding: pretreated spdp showed enhanced vwf activity over rspdp (p < .05). fibrin polymerization density was slightly diminished in rspdp vs. ffp (0.879 vs. 0.742 o.d., p < .01), but tg was unchanged. bicarbonate/base excess were lower in spdp samples vs. ffp (p < 0.001). residual cellular material (especially platelet-derived) was reduced threefold in rspdp vs. ffp (p < .01) and an additional twofold in pretreated spdps vs. rspdp (p < .05). teg results were unchanged in plasma-only samples; in reconstructed wb there was a reduction in amplitude (clot strength) in all spdp samples vs. ; p < 0.01). platelet adhesion was equivalent in pretreated spdps and ffp, while rspdp was improved vs. all other samples (71.53% surface coverage vs. 30.26-43.87%, p < .05). conclusion: spdp has a longer shelf life and easier storage requirements than ffp and was equivalent or superior to ffp in most of these in vitro assays. spdp pretreated with glycine solutions was similar to ffp in most assays and showed superior vwf activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated spdp. clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted. the interaction between red blood cell transfusion and lung injury: the influence of blood component manufacturing methods mathijs wirtz* 1 , anita tuip-de boer 1 , ruqayyah almizraq 2 , jason p. acker 3 , philip j. norris 4 , jennifer a muszynski 5 and nicole juffermans 1 . 1 academic medical center, 2 university of alberta, 3 canadian blood services, 4 blood systems research institute, 5 nationwide children's hospital background/case studies: red blood cell (rbc) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation. the causative factor is not known but may include residual cells or extracellular vesicles (evs) . in this study we investigated the functional effect of different manufacturing methods of rbc products on the response of pulmonary cells in an in vitro model of mechanical ventilation. study design/methods: groups of rbc products (whole blood filtered [wbf] , red cell filtered [rcf] , apheresis derived [ad] and whole blood derived [wbd]) were manufactured from 8 donors (blood type a or b). supernatants were prepared after 4-5 (fresh) and 41-42 days of storage (stored) for measurement of thrombin generation and ev analysis. a549 type ii alveolar cells were seeded onto flexible membranes and incubated with rbc supernatant. cells were subjected to 25% stretch using a cellstretcher. control cells were not stretched. after 24 hours, il-8 and il-6 production were measured. results/findings: both fresh and stored supernatants from ad products significantly increased pulmonary cell il-6 and il-8 production compared to incubation with other rbc products and non-incubated controls, which was further exacerbated by cell stretching. ad products also had significantly increased thrombin generating ability compared to other rbc products, as well as a significantly increased number of rbc-derived evs compared to rcf and wbd products (p<0.05) . incubation of stretched cells with stored wbf products resulted in higher il-8 production compared to other blood products and stretched controls. rcf products did not activate pulmonary cells, had an absence of tg and had low levels of evs compared to other products. conclusion: manufacturing methods markedly influence the interaction of rbc products with lung cells. ad products activate lung cells, which is further aggravated by cell stretching. this may in part be mediated by rbc-background/case studies: investigators previously demonstrated immunosuppressive effects of rbc supernatant on monocytes in vitro, with greater effects seen in response to older units. recent clinical data suggest that rbc manufacturing method may influence immunomodulatory potential, but this has not been directly measured. we used in vitro models to test the hypothesis that rbc supernatants obtained by different manufacturing methods will have differential effects on monocyte function. study design/method: rbc products were manufactured by 4 different methods from 5 individual donors, each: (whole blood filtration [wbf] , red cell filtration [rcf] , apheresis, and whole blood derived [wbd] ). rbc products were stored in sagm (wbf and rcf) or adsol-containing preservative solution (apheresis and wbd). supernatants were obtained after 4-5 days (fresh) and 41-42 days (expiry). monocytes were co-cultured in media plus 20% rbc supernatant or media only (control) followed by lps stimulation. experiments were performed in 5 replicates, each with a distinct monocyte donor. comparisons between groups by anova with dunnett's post-test for multiple comparisons. data are mean 6 sd of % of control values. results/finding: exposure to apheresis or wbd rbc supernatants suppressed monocyte lps-induced tnfa production capacity compared to controls (table 1) . this was true for fresh units and those at expiry. for monocytes exposed to rbc supernatant alone without lps, interleukin-8 production was higher after exposure to fresh wbf (248 6 115 % control, p 5 0.02) or wbd at expiry (292 6 111 % control, p 5 0.0005). conclusion: manufacturing method and/or storage solution significantly alters immunomodulatory effects of rbc supernatant on monocytes in vitro and may confound analyses of clinical effects of rbc storage duration, particularly within international multi-center studies. a magnetic levitation system to study the impact of donor gender, age and blood storage conditions on red blood cell density profile gozde durmus* 1 , alessandro tocchio 1 , anita howell 2 , kaushik sridhar 1 , jason p. acker 3 and utkan demirci 1 . 1 stanford university, 2 canadian blood services, centre for innovation, 3 background/case studies: the amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males. it is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population. it is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (rcc) units. as red blood cells (rbcs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells. study design/method: our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average rbc age and density of red cell units. we have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations. this label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of 10 -4 g/ml. first, to determine the effect of rcc storage on the density profile of rbcs, levitation and single-cell density profiles were measured at 7, 14, 21, 28, 35 and 42 days. in addition, to determine the effect of donor age and sex on the rbc density profile, blood samples from 24 volunteers with four different age and sex categories (male, 18-40 years; male, >60 years; female, 18-40 years; female, >60 years) were profiled. results/finding: first, we observed that the levitation and density profiles as well as morphology of rbcs within rcc units change significantly during storage. in addition, rbc density was significantly different between young (1.098 g/ml) and older female donors (1.109 g/ml) (p < 0.01). moreover, rbcs from young males (1.096 g/ml) were significantly less dense compared to rbcs profiled from older female donors (1.109 g/ml) (p < 0.05). conclusion: we have developed a magnetic levitation system for the point-of-care, real-time evaluation of rbc and red cell concentrate (rcc) quality. we envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes. cytokine production of pulmonary cells il-6 (pg/ml) il-8 (pg/ml) background/case studies: oxidation reduction potential (orp) or redox is the ratio of activity between oxidizers and reducers. redox imbalance caused by a higher production of reactive oxygen species (ros) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (os). while os can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease. in the present study we investigated changes in hemoglobin, free heme, and orp as red blood cells (rbc) age and the effects of red blood cell age on icu patient morbidity and mortality. study design/method: 120 icu patients were enrolled in this prospective observational trial investigating the effect of transfused rbc age on icu patient morbidity and mortality. all rbcs were pre-storage leukoreduced and abo identical. citrated blood samples were collected from each rbc unit prior to issue. the rbc supernatants were tested for free hemoglobin/ heme and orp. the patients were followed prospectively. results/finding: a total of 426 rbc units were transfused. patients and rbc characteristics are shown in the table. significant reductions were detected in orp values over storage duration (p<0.001). substantial correlations were also found between orp and free hemoglobin (p<0.05) and orp and free heme (p<0.05). interestingly, there was a statistically significant difference between the average orp values of the transfused rbc in patients who developed infection with higher orp values measured in rbc units given to patients who developed post-transfusion infections 132 6 10 vs 127 6 13 (p<0.05). no significant differences were observed between orp and patient mortality, hospital/icu days, or thrombosis. also, no correlations were detected between free heme/hemoglobin or rbc age and infection development. conclusion: these data demonstrate that older blood has lower orp values as well as increased free heme/hemoglobin. there were no differences in orp values between the different blood groups once rbc age was controlled for and there were no statistically significant differences in patient mortality associated with orp, free heme/hemoglobin, or rbc age. the decreased orp values observed in the older blood are likely attributable to the "storage lesion". higher transfused rbc orp values were associated with subsequent development of infection, and younger rbcs were found to have higher orp values. thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients. further studies are needed. background/case studies: no randomized trials in humans have addressed whether only exposure to red blood cells (rbcs) that have been stored for a long time is associated with harm. we explore the effect on inhospital mortality of transfusing rbcs stored for more than 35 days compared to rbcs stored for 7 days or less. study design/method: data from a multi-national randomized controlled trial were used for this exploratory analysis. the patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest rbcs in inventory or the oldest (standard issue) rbcs providing a large cohort of patients receiving rbcs with storage durations along the entire rbc storage continuum of 1 to 42 days. using a time dependent variable patient exposure was defined by the maximum storage duration of rbcs received. this was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to rbcs less than or equal to 7 days storage duration -reference group), medium age (at least 1 rbc of 8-35 days storage), and oldest (at least 1 rbc greater than 35 days storage). the primary outcome was all-cause in-hospital mortality. cause-specific cox regression models of in-hospital death assessed the effect of exposure of rbcs in each category to exclusive exposure to rbcs stored for 7 days or less. the effects of fixed and time-dependent confounders were dealt with through stratification and regression. sensitivity analyses were conducted with a) weekly partition with cut-points every 7 days, and b) a finer partition using cut-points every 3 days. results/finding: 24,726 patients receiving 90,530 rbcs were included in the analysis. exposure to rbcs stored for more than 35 days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest rbc units (stored for 7 days or less) after adjusting for several fixed and time-dependent potential confounders (hr 5 0.91; 95% ci: 0.72, 1.14; p 5 0.400). exposure to blood stored for at most 8-35 days yielded a similar hazard ratio (hr 5 0.90; 95% ci: 0.73, 1.10; p 5 0.295). in the sensitivity analyses using weekly partitions, exposure to rbcs stored for greater than 35 days compared to exclusive exposure to rbcs stored 7 days or less was not significant (hr 0.90; 95% ci 0.72, 1.14; p 5 0.381). the confidence intervals around the hazard ratios for the other 7-day intervals all include 1. similar findings were obtained with partitioning exposure data into 3 day intervals where exposure to rbcs stored for 40-42 days was not associated with increased risk of death compared with exclusive exposure to rbcs stored for 1-3 days (hr 0.82; 95% ci 0.37, 1.83; p 5 0.635). the confidence intervals around the hazard ratios for the other 3-day intervals all include 1. conclusion: individuals exposed to rbcs stored for more than 35 days were not at increased risk of in-hospital death compared to individuals exposed exclusively to rbcs stored for 7 days or less. transfusion of anaerobically stored red blood cells improves recovery in experimental rat hemorrhagic shock model alexander williams* 1 , cynthia walser 1 , tatsuro yoshida 2 , andrew dunham 2 and pedro cabrales 1 . 1 university of california san diego, 2 new health sciences inc. background/case studies: hemorrhagic shock (hs) severely decreases oxygen (o 2 ) delivery and induces cardiovascular collapse. in parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (rbcs) to restore blood volume, o 2 carrying capacity, and hemodynamic stability. the quality of the transfused rbcs determines the recovery from hs, and extent of clinical sequelae prompted by the hs. this study compares the ability to recover from hs with conventionally stored rbcs, anaerobically (o 2 saturation <10%) stored rbcs, or anaerobic/hypercapnic (o 2 saturation <10% and pco 2 (@378c) $70mmhg) stored rbcs. study design/method: packed red blood cells (prbcs) stored in as-3 after leukorfiltration were created from donor sprague-dawley rats. prbc units were randomly stored under either 1) conventional; 2) anaerobic; or 3) anaerobic/hypercapnic conditions. rats (150-200g) were hemorrhaged to 50% of blood volume, held in hypovolemia for 30 minutes, and resuscitated to recover blood pressure to 90% pre-hemorrhage with prbc stored for either 1 or 3 weeks. systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation. data were analyzed using two-way anova, followed by the appropriate post hoc analyses. 1(11%) neg patient showed short term response and 6(67%) patients showed progressive disease. at the neg group standard eval 1(11%) patient showed response and 3(33%) had progressive disease. 1(11%) neg patient had long term response compared to 11(21%) pos patients. at the pos short term eval 22(42%) patients showed response and 20(38%) patients had progressive disease. at the pos group standard eval, 20(38%) patients showed response and 6(11%) patients had progressive disease. overall, 28(53%) pos patients responded compared to 2(22%) neg. conclusion: there is a trend in lower response rate in patients with negative antibody screens compared to positive controls. these findings suggest that an anti-cd38 neutralizing substance could play a role in treatment response. alternatively, reduced cd38 expression may also contribute. the low response rates seen in both groups may result from biased selection. the need for repeat t&s and presumed repeat transfusions may be preselecting patients with more aggressive disease. also, only a small number of patients were suitable for review. a larger prospective study that controls for such variables is needed. a review of blood utilized during provider-activated and critical administration threshold-triggered massive transfusion events patrick ramos* and john hess. division of transfusion medicine, harborview medical center background/case studies: traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (rbcs) in a 24-hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event. to address these issues, a level i trauma center adopted the critical administration threshold (cat) as an additional indication for activating its massive transfusion protocol (mtp). this study reviewed blood utilized during massive transfusion events based upon whether the mtp was provider-activated versus cat-triggered. study design/method: all massive transfusion events between january and april 2017 were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused. the transfusion of three or more blood components in an hour defined cat. a massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the mtp or blood components were transfused at a rate that met cat criteria. the massive transfusion start time is based on either the time the provider activated the mtp or the time the first blood component was transfused, whichever came first. unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components. this information was tabulated to determine the monthly number of provider-activated mtps, cat-triggered mtps, and average blood component transfused per massive transfusion. conclusion: blood utilization is lower within the cat-triggered mtps even though it outnumbered provider-activated mtps. however, the mode for both groups suggests that most massive transfusion require less blood components than the average rate. using the mode provides an approximate 24% replacement of blood volume. this should be enough to counter the early signs and symptoms of hemorrhagic shock. though this study did not review the appropriateness of provider-activated mtps, using cat as an indicator ensures clinicians are prepared for a potential massive transfusion. further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused. the mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently. if this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused. the long term storage effect of 0.2m dithiothreitol on red cell antigen integrity in reagent red blood cells heike carrel* 1 , laurie sutor 1,2 , germ an leparc 3 , marjorie doty 3 and william crews 1 . 1 carter bloodcare, 2 ut southwestern medical center, 3 oneblood background/case studies: anti-cd38 drugs, such as daratumumab, pose a problem for the transfusion service. they may cause a number of false positives, including positive direct antiglobulin tests (dat), indirect antiglobulin tests (iat), and panreactivity in eluates. such results can prolong compatibility testing and delay delivery of blood products for patients. treating reagent red cells (rrbcs) with 0.2m dithiothreitol (dtt) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies. this study aimed to investigate the effect of dtt-treatment on rrbc antigen integrity over a 28 day period. study design/method: twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-d, -c, -e, -c, -e, -m, -s, -s, -fy a , -fy b , -jk a , and -jk b ), were tested against untreated and 0.2m dtttreated rrbcs (immucor panoscreen i, ii, iii; dtt from acros organics). dtt treatment of rrbcs was performed using the methodology described in the aabb technical manual (18 th edition). each of the 12 plasma aliquots was further separated into 28 aliquots and stored at -208c until day of use. fresh aliquots were thawed each day to avoid unintended antibody integrity degradation. a polyethylene glycol (immucor) enhancement technique was used and reactions were read at the iat phase. hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the haemonetics color comparator chart. serological antibody reaction strengths were observed and documented each day. (25) a monthly breakdown for both groups also displayed a downward trend in the average use of blood components. results/finding: there was noticeably more hemolysis with the dtttreated cells over time compared to the untreated cells. red cell antigens remained serologically detectable on the dtt-treated cells throughout the study, despite a greater degree of observed hemolysis. there was minimal difference in reactivity strength between untreated and dtt-treated cells for antigens not affected by dtt. in most instances, the dtt-treated cells reacted slightly more strongly. none of the antibodies produced reactivity strengths of less than 11 with the untreated or dtt-treated cells during the study. conclusion: long term storage of 0.2m dtt-treated rrbcs does not compromise antigen integrity. advance dtt-treatment and storage of a large aliquot of rrbcs may serve to increase efficiency in the transfusion service. background/case studies: monocyte monolayer assay (mma) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of rbcs for alloimmunized patients. the requirement for fresh auto/allogenic monocytes for mma is highly restrictive due to tedious processing of fresh peripheral blood (pb). our previous study described processing and cryopreservation of buffy-coat (bc) derived and fresh pb-monocytes for mma assay. the aim was to evaluate the functional properties of cryopreserved bc-monocytes as substitute for fresh pbmonocytes in mma in evaluation of previously reported clinically significant rbc alloantibodies. study design/methods: peripheral blood mononuclear cells (pbmcs) were isolated from buffy-coats (histopaque-10771), pooled, suspended in cryopreservation media (20% dmso; 1:1) and stored in liquid nitrogen. pbmc membrane integrity post-thaw was determined by trypan blue exclusion. pbmcs were cultured on poly-l-lysine-treated coverslips (378c, 5% co 2 , 1 h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (o1) rbcs sensitized with either anti-d (positive control), anti-scianna-2 (sc2) or anti-anwj or lipopolysaccharide stimulated for 2 h. aliquots of the sensitized rbcs were tested for opsonization by indirect antiglobulin test (iat). phagocytosis index (pi) was determined microscopically as the number of fully phagocytosed rbcs/100 monocytes. supernatants were analyzed for cytokines using luminex technique. results/findings: cryopreserved pbmcs showed 96.2 6 1% viability postthaw. we report no significant difference in phagocytosis of anti-d sensitized rbcs by cryopreserved monocytes vs fresh monocytes. we show a significant increase in tnf-a, il-1b, il-6, il-8, mip-a (p < 0.01), mip-b and gro (p < 0.05) secretion from cryopreserved bc monocytes vs both fresh bc and pb-monocytes. sc2-and anwj-sensitised rbcs resulted in a pi of 9.2 6 2% and 60.2 6 6.4% respectively vs anti-d sensitized rbcs (pi: 72 6 8.7%). a weak (11) reactivity by iat was observed for anti-anwj sensitized rbcs while anti-d sensitized rbcs resulted in 41 iat reactivity. these results correlated with previously reported results for clinical significance and mma when using freshly obtained autologous or healthy donor monocytes. conclusion: this study shows that cryopreservation preserved monocyte viability and phagocytosis function for mma. as previously reported with fresh monocytes mma assay, the two alloantibodies tested with cryopreserved bc monocytes were shown to have a phagocytic index of clinical significance (pi>5%). the use of cryopreserved bc-monocytes has the ability we describe antigen typing discrepancies in 5 patients, involving 3 antigens (c, jk a , s), revealed when serologic results differed from the phenotype predicted by dna testing. all 5 patients had 3-41 positive dat with anti-igg and warm autoantibodies identified in the plasma. investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents. study design/method: standard tube hemagglutination methods were used for antigen typing. rbcs were treated with edta glycine-acid (ega) using gamma ega kit. genomic dna was isolated from wbcs and hea precisetype performed. results/finding: the rbcs of patients 1 and 2 typed c-on initial testing with immucor gamma-clone anti-c, but were predicted c1 by hea precise-type. ega-treated rbcs gave 31 reactions with the same anti-c reagent. patient 1 rbcs gave variable reactivity (vw-11) with bio-rad seraclone and ortho bioclone anti-c. patient 2 rbcs gave 11 reactivity with all 3 anti-c reagents when incubated for the maximum incubation time allowed. patient 3 rbcs were jk(a1) with immucor gammaclone anti-jk a , which the manufacturer states is suitable for testing dat1rbcs, but predicted jk(a-) by hea. ega-treated rbcs tested jk(a-) with the same reagent. rbcs from patients 4 and 5 tested s1 with bio-rad seraclone anti-s (3-41), but predicted s-by hea. further testing with immucor gammaclone anti-s showed rbcs from both patients were s-. ega-treated rbcs from both were non-reactive with both anti-s reagents. conclusion: commercial monoclonal reagents are valuable resources, especially when phenotyping dat1 rbcs but not all manufacturers include reagent limitations regarding testing of dat1 rbcs. we describe 2 cases of false negative tests with monoclonal anti-c due to antigen blocking by igg, and 3 cases with false positive tests with anti-s (n52) and anti-jk a (n51) typing. false positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected. extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved. results concordant with dna testing were obtained with ega-treated rbcs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent. these cases caution the potential for both false negative and false positive results for samples with 3-41 positive dat and supports testing to dissociate igg from rbcs strongly dat1 before antigen typing. in addition, this report highlights the benefits of dna testing as part of the routine reference laboratory workup. background/case studies: sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions. accepted us clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management. to mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable. giblett, long ago, introduced a relative scale relating the rbc antigen immunogenicities to (an assumed) immunogenicity of "k" (http://bit.ly/2opqfew ). here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen. study design/method: we define immunogenicity, or sensitization risk, r, for any antigen ("ag") of interest, as the conditional probability of alloantibody ("ab") formation, given allo-exposure to ag, i.e. r :5 prob(ab|al-loexp), so that prob(ab) 5 prob(ab|alloexp)*prob(alloexp) and 0 r 1; rewriting prob(alloexp) 5 prob(recipient, "r", lacks ag)*prob(donor , "d" has ag); and estimating prob(ab) 5 nab/nr, nab denoting the number of ab in nr recipients, we obtain: nab/(nr*prob(r lacks ag)) 5 r * prob(d has ag), the left-hand side representing the observed sensitized fraction, u, i.e. the number of observed ab in relation to the number of recipients at risk. conclusion: several antigens, though corresponding antibodies may be rare (e.g. "jsa", "e", "u"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk. in conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available. our approach may be readily extended to additional rbc antigens and other antigen systems. background/case studies: aabb and fda require a 12 month deferral of donors with a tattoo applied using non-sterile needles or reusable ink. we review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink. we recently added two large states in which we collect blood to the acceptable states list (asl). we compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations. study design/method: we analyzed allogeneic interview responses to the screening question, "in the past 12 months have you had a tattoo?" and if 'yes', whether the tattoo was applied by a state regulated entity. blood centers in 2 states were selected for the analysis before and after state tattoo regulation. in state a, a comparison period of similar 3 months before (2/ 2015 -4/2015) and 3 months after (2/2016 -4/2016) was selected; for state b, a similar 4 months before (12/2015 -3/2016) and 4 months after (12/2016 -3/2017) was selected. frequency and rate of responses were compared in before and after periods. among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including hiv, hbv and hcv. results/finding: a higher proportion of donors presenting to give blood admitted to having a recent (<12 months) tattoo in the post period in both states. this increase occurred immediately following the addition of states a and b to the asl (data not shown). among those who responded yes to having a tattoo, in states a and b respectively, there was a 13-and 3-fold increase in accepted donors (table) . the absolute number of accepted donors with tattoos increased from 13 to 567 (state a) and 151 to 1,496 (state b), which annualized, represents a potential gain of 2,216 (state a) and 4,035 (state b) additional donations. all donors who had a tattoo in regulated states (asl) tested negative for hiv, hbv and hcv. conclusion: to counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base. the immediate rise in the number of donors reporting a tattoo following the addition of the 2 states may reflect a decline in self-deferrals based on having had a recent tattoo. we demonstrated an increase in the potential number of donations without compromising safety. background/case studies: transgender donors represent a small fraction of blood donors. determining their eligibility to donate has been challenging for blood centers. to assess behavioral risk, the donor is required to answer gender specific questions. the same is true when assessing trali risk where the donor is asked about a history of prior pregnancies. prior to the implementation of the fda's final rule, blood centers asked donors for their birth gender and determined eligibility based on that gender. if the donor changed their gender they were asked to answer both the male and female questions. the final rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender. in order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center. and tracked. donors were contacted to resolve any descrepancies. donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified. hla antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative. results/finding: from 2013 -2015 , there were 181 donors identified who had changed their gender from their birth gender; 121 female donors changed their gender to male and 60 male donors changed their gender to female. there were 7 (6%) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations. three of these donors were apheresis donors who had been tested for hla antibodies. one tested positive and the other two tested negative for hla antibodies. the four other donors were whole blood donors and had not been tested. an hla test was added to these donors' records so that the test could be performed the next time they presented to donate. conclusion: transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male. six percent of transgender males that we identified reported a prior history of pregnancy. at our center, when a donor requests a gender change from female to male, an hla test is requested for the next donation. first time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information. consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that hla antibody testing can be performed. effect of variable volume scale introduction in a large multi-site blood center ralph r vassallo*, marjorie d bravo and hany kamel. blood systems, inc. background/case studies: regulations allow whole blood donation [wbd] of up to 10.5 ml/kg or 15% of estimated blood volume [ebv] . traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the 15% limit. variable volume scales [vvs] can be programmed to vary unit volume (up to 550 ml) by donor ebv. this maximizes transfusable rbcs and plasma and recovered plasma [rp] volume. rp from wbds is a small but important source of derivatives and blood center cost recovery. we report the effect of introducing the hemoflow vvs on donor reaction rates and rp volume in a large blood center. compared to previous fixed settings, variable collection volumes were expected to decrease by 10 ml at ebvs <3.5l in donors !23 yo, but increase by 5-40 ml for all others. study design/method: donor vasovagal reaction [vvr] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [loc]) for successful wbds were obtained from the center's hemovigilance database for the 18 mos. before a 6 mo. phased implementation of the vvs, and the subsequent 24 mos. multivariable analysis [mva] by 6-mo. periods was performed in a model incorporating donor sex, age, first-time [ftd] vs. repeat status, ebv and donation site. both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse. results/finding: compared to the baseline period, a significant increase in prefaint reaction rates were noted in pre-implementation (impl) periods 1 & 2, continued during impl and post-impl periods 1 & 2, returning to the baseline rate in post-impl periods 3 & 4 (table) . more severe reactions showed an increasing trend that only became significant in post-impl periods 3 & 4. the mva showed the vvs as independent factor contributing to the increased prefaint and more severe reactions. however, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low ebv, ftd status and collection site (not shown). plasma unit volume increased an average of 3.8 ml during post-impl periods 1 & 2 from the temporally matched baseline & pre-impl period 1. conclusion: following an initial increase in mild vvrs during and immediately after implementation of the vvs, vvr rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend. the subsequent increase in prolonged/offsite reactions and loc after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the vvs. small but significant increments in rp volume improve derivative availability and offset the cost of the vvs. comparison of vasovagal and citrate reaction rates in donors according to type of apheresis procedure pierre robillard* 1 and yves gr egoire 2 . 1 hema-quebec, 2 h ema-qu ebec background/case studies: apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used. citrate reaction (cr) results from various degrees of hypocalcemia in donors. blood volumes taken from donors vary according to type of procedure and use of volume replacement. loss of blood volume is in part responsible for the occurrence of vasovagal reactions (vvr). this analysis was conducted to estimate the incidence of cr and vvr according to various types of apheresis procedures performed at our blood center. (yfv) were reported in some brazilian states -rio de janeiro, sao paulo, minas gerais and espírito santo, mainly. the vectors of those cases were mosquitoes from the haemagogus and sabethes genders, whose habitat is the tropical forests. since many brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the aedes mosquitoes. in order to minimize this risk, rio de janeiro health authorities decided to promote a mass vaccination in late march, 2017. the vaccine is produced with live and attenuated yfv, which can circulate for at least 4 weeks after vaccination. in some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases. due to that, brazilian blood regulation authority established a 4 week deferral period after yfv vaccination. this action could dramatically affect the availability of blood donors. this study shows the measures taken by rio de janeiro blood center to circumvent this risk and attract more donors. study design/method: the strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against yfv. there were no financial advantages to the donors, since yfv vaccine is completely free of charge for any brazilian citizen. the vaccine was administrated by trained nurses, in an office close to the donors session. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. the blood center annnounced just before the mass vacination campaign launching that it would vaccinate 600 people who came to the blood center to donate blood. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. results/finding: during the five days of campaign, we received 3,351 blood donors candidates; from those, 2,449 were accepted as a blood donor, after medical interview. the deferral rate was 26.9%. at the same period of the year 2016, there were 1,215 prospective donors, and 883 blood donations. the deferral rate was 27.3%. the "get vaccinated against yfv . . .but give blood before" campaign was able to attract, in a five day period, 1,566 additional donors, compared to 2016 same dates. that represents a 177.34% increase in the number of blood donations, without deferral rate increment. there was a slight increase in the proportion of first-time donors, from 42.7% in 2016 to 45.8% in 2017. conclusion: the strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against yfv. dose loss which must be accommodated when collecting plt donations to ensure the us plt dose of !3.0x10 11 is met. currently, triple set kits for pr are only approved in europe. plt loss, and adjusted apheresis targeting parameters may impact split rate (sr) or products per apheresis procedure. inventory suitable for pr without impacting us blood center srs warrants evaluation and optimization. study design/method: 1,000 apheresis collections from 4 centers with different srs were analyzed. a baseline sr for conventional pc was calculated assuming i) a minimum dose (allowing for production loss) of 3.1 x10 11 for single (s), 6.3x1011 11 for double (d), and 9.5x10 11 for triple (t) conventional pcs, ii) concentration and volume requirements from apheresis device manufacturer were used. for each collection, dose, volume, and concentration were assessed for pr kit compatibility, based on storage medium (pas or 100% plasma) assuming i) a minimum dose (allowing for production loss) of 3.5 x10 11 for s and 6.7x10 11 for d for pr units, ii) removing small quantities from units with excess volume or dose to meet pr specs., iii) if all or part of an out of parameter d or t collection could be divided into one or more kits for pr, eligible parts undergo pr, and the remainder treated conventionally, iv) collections unsuitable for pr specs. or would decrease sr if treated would be counted as conventional pcs. results/finding: conclusion: blood centers today can adopt pr for a significant percent of their current supply (as high as 99%) without affecting their sr. compatibly increases further by dividing t and large d donations. percent achievable depends on their current s, d, t proportion of collections and practices. changes to d and t collection parameters, optimized donation and counting accuracy, and volume reduction will improve pr compatibility further. individual analysis is warranted for each blood center. rbc 2rbc plt/p plt plt/rbc/p plt/rbc 2plt 2plt/rbc 2plt/p #donations 47990 63 1775 10832 968 476 67 1150 142 10252 citrate exposure (mls) 41-85 71 138 263 266 300 322 349 478 study design/method: a randomized (2:1), placebo-controlled, single blind, 15 subject, single-site study of ascending microdoses of autologous (apheresis-derived) thrombosomes was conducted. subjects were divided into 5 cohorts, receiving increasing doses, ranging from 1/1,000 -1/10 of the lowest effective dose found in the above rabbit model. cohorts 4 and 5 received the 1/10th dose, but cohort 5 received two 1/20th doses one hour apart. the primary end points were safety and tolerability. subjects were monitored in-hospital for 24hrs post infusion and followed for up to 60 days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests. results/findings: there were no serious adverse events (saes) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline. there were a total of 68 aes: 40 were treatment emergent (teae), of which 8 were treatment-related (6 thrombosomes and 2 control). all teaes were mild or moderate in severity. in cohorts 4 and 5, 3/4 thrombosomes subjects had treatment related adverse events. one cohort 4 subject developed an upper respiratory infection and elevated wbcs within 8 hours post infusion, which resolved by 24 hours, and an elevated d-dimer at 24 hours post infusion, which resolved by day 7. this subject also had an elevation of prothrombin fragment 1 1 2 at baseline, which increased post transfusion and peaked at 24 hours with resolution by day 14. one cohort 5 subject developed non-specific t-wave changes at 1 and 2 hours following her 2nd infusion that resolved by day 21 without clinical symptoms. troponin levels and echo stress tests were normal. ekgs were considered possibly a normal variant or related to placement of the ekg leads. another cohort 5 subject developed an igg platelet autoantibody on days 7-21, which was undetectable on days 42-60; there was no change in platelet counts. the thrombosomes autoantibody assay was positive at baseline, days 7-14, and negative on days 21-60. background/case studies: cryopreservation of platelets (plts) could extend the shelf life from 5-7 days to over two years. cryopreserved plts (cryoplts) appear to have a greater in vivo hemostatic effect than liquidstored plts. plts have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses. this study was designed to assess whether reconstituted cryo-plts carry out protein synthesis upon thawing and short term storage. study design/methods: apheresis plts were cryopreserved with 5% dmso and stored at 2808c. after thawing, the unit was reconstituted in thawed ffp spiked with either 500 lm puromycin (pm) or 250 nm biotinlabeled pm. plts were stored at room-temperature with agitation. samples were drawn immediately after reconstitution as well as after 2, 4 and 24 hours to assess pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by cd62p binding, phosphatidylserine exposure by annexin-v binding and microvesicle count in the supernatant. plt microvesicles (pmv) were prepared from the supernatant by ultracentrifugation. plts and pmv were lysed in a triton x-100containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads. results/findings: in vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of cd62p and phosphatidylserine. pmvs were generated during cryopreservation and the count increased by 11-fold during 24 hour storage. immunoblot analyses of the plts showed a 2-and 4-fold increase in pm incorporation after 4 and 24 hours of storage, respectively. massspectrometry revealed 23 unique proteins that were synthesized after 4 hours of storage, which was confirmed for gtpase and gtpase-regulatory proteins rac1, rap1 and rhogdi by immunoblot analyses. analyses of the pmv translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage. this finding suggests that a defined panel of proteins is packaged into pmvs upon freezing and thawing. additionally, the pmv translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-plt translatome, including the proteins rac1, rap1 and rhogdi. conclusion: this study has demonstrated that cryo-plts can synthesize proteins upon reconstitution in ffp and subsequent storage. discovery of a subset of these proteins in the pmv suggests their encapsulation, possibly in a selective manner. this observation provides novel insights into the capacity for protein synthesis in cryo-plts and the potential regulation of protein packaging into pmv. background/case studies: in 2015, the authors' hospital-based blood bank received variances from the fda and aabb for the use of cold stored platelets (csps) with a shelf life of 3 days. these group a csps, stored in a refrigerator in the emergency department, were used to support the trauma program for use in massively bleeding patients. the placement of the csps on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients. study design/methods: eight double unit csps were collected using the trima accelv r . two double csps were pathogen reduced using the inter-ceptv r pathogen reduction system. half of the csp pairs were subjected to flat storage in a refrigerator; the other half were loaded into a credov r 4-496 cooler with 2 units of ffp, 2 units of rbcs, and 1 unit of whole blood. three to 5ml of platelets were collected via syringe from each unit at 0 min (before storage in cooler or refrigerator) and after 0.5, 1, 3, 5, and 6 hours of storage for functional validation of platelets. the platelet count, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac-1 binding) were measured by coulter counter, 2 channel aggregometer, and digital flow cytometer. paired wilcoxon rank sum tests were used to analyze differences in degradation rates with p<0.05 deemed significant. conclusion: platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for 6 hours. therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival. c49-a03h molecular sieving: beyond genotyping ghazala hashmi 1 , reinhard klemm 2 and michael seul* 1,2 . 1 biomolecular analytics, 2 immunoinformatica background/case studies: more than a decade after its commercial introduction (hashmi2005 http://bit.ly/2ohlehe), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g. the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing. here, we introduce molecular sieving as an alternative to the current approach of managing special donor unit inventories. this novel process for dna analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns. study design/method: molecular sieving is a special format of leansequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding 401 rbc antigens in mns,rhce, lu,kel,fy,jk, di,yt,do and co, including the identification of 30 rhce alleles. molecular sieving extends these capabilities to the analysis of pools to attain large scale. thus, in one format of the process, 4*4*96 samples are accommodated in a single run. following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes. here, molecularsieving was used to identify suitable donor units for 135 sensitized sickle cell anemia ("sca") patients (tb1 in cas-tro2002, http://bit.ly/2oplxhr, excluding le and e(variant) and assuming 1 request per patient), presenting with up to 9 allo-antibodies ("ab") in multiple combinations. proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests. results/finding: sieving of only 1 =2 plate holding 4*48 candidate units from actual black donors, followed by profiling of 44 samples selected to enrich for "e neg" and "c neg" and "c-e-k-fya neg", produced assignments for 127 of 135 requests (594.1%), as indicated by colors, and shown in the row "assigned" below: thus, the number of assignments substantially exceeded the number of wells processed. moreover, the remaining 66 pooled samples produced 44 additional assignments to a second set of 135 requests, for a total of 171 assignments from only 92 wells. in another scenario, sieving of a full plate of 4*96 samples, produced $250 assignments for two successive batches of 271 requests from sca patients, a yield exceeding 2.5x. sieving alone typically fills 65-75% of requests of moderate complexity ( 5 ab). conclusion: molecularsieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies. molecular sieving for identifying red blood cells with special phenotype attributes kristopher fernandez 1 , monica kalvelage 2 , ghazala hashmi* 1 and michael seul 1 . 1 biomolecular analytics, 2 lifeshare blood centers background/case studies: providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization. the allelic diversity of the predominantly black patient population, especially at the rh locus which encodes a variety of "partial" phenotypes further complicates the problem (chou2013 http://bit. ly/2ppvfeq ). study design/method: molecular sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including 30 at the rhce locus) that encode 401 mns, rh, lu, kel, fy, jk, di, yt, do and co antigens. based on sieving, samples may be grouped by molecular attribute patterns ranging from single "ag2" (e.g. e2,c2,e2,c2) to specific combinations of "ag2" (e.g. c2e2k2fya2 and c2e2jsa2) or combinations of alleles such as those encoding partial rh phenotypes. sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns. genomic dna from 384 (predominantly) black donors, independently genotyped by one of two commercial methods were provided by lbc. at bmx, 96 pools were prepared prior to amplification, and analyzed by a novel leansequencing method. results/finding: all pool genotypes were consistent with available individual sample genotypes. antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous. illustrative of the former pattern type are these: appropriate pool queries revealed that sieving alone identified, among the 124 c2 samples, 24 that were also v2 and vs2 and, among the e2 samples, 12 that were also negative for any partial_e phenotype. illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence. by segregating het pools into subpopulations, we were able to select 6 specific "ee" pools of which 4 were demonstrated (in subsequent profiling) to contain an e-sample. we also identified pools "het" for alleles indicating the possible presence of a rare donor, for example yta|b (8 pools), co a|b (8) and others. conclusion: molecularsieving of a single 96-well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "ag-neg" patterns including "partial:" rh phenotypes and combinations of c2, e2 and jsa2. these samples are thus confirmed "ag neg" and available for assignment. sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand. "antigen-neg" pattern partial_c partial _c, _e samples available after sieving 124 132 100 100 360 208 192 40 28 92 52 background/case studies: sequence information generated from next generation sequencing (ngs) is often computationally phased using haplotype-phasing algorithms. utilizing experimentally derived haplotype information improves this prediction, as routinely used in hla typing. among the 36 blood group systems, however, experimentally derived haplotypes are known for short genes only, such as icam4 (landsteiner-wiener) and ackr1 (duffy). for longer genes, such as abo of >20 kb, most haplotypes are only statistically derived. we recently established a large dataset of long ermap haplotypes, which code for the scianna blood group system. study design/methods: the nucleotide sequence of >21 kb each was used for all physically confirmed 48 ermap alleles that we previously published. full-length sequences were aligned and variant sites were extracted manually. the bayesian coalescent algorithm implemented in beast v1.8.3 was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny. results/findings: we found at least 4 clades representing clusters of 5 to 11 alleles. for each clade, one observed allele was identified as the ancestral allele for its cluster of alleles. using the 4 alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant. conclusion: we explored the phylogenetic structure and evolutionary events underlying the origin of different ermap alleles and predict ancestral alleles. in the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles. the probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence. the alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of ngs data. the new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode group o. rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of a and b and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression. abo genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate abo genotypes with phenotypes. background/case studies: evolutionarily related abo and gbgt1 genes encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the biosynthesis of a and b, and forssman (fors1) oligosaccharide antigens responsible for the abo and fors blood group systems, respectively. human at and bt possess leuglygly and metglyala, respectively, at codons 266-268, and these tripeptides are important in determining the sugar specificity of enzymes, n-acetyl-d-galactosamine (galnac) for at and galactose for bt. functional fss possess gly-glyala at the corresponding codons, and exhibit galnac specificity. it has been recently shown that human at gained weak fs activity when the leu-glygly was substituted by glyglyala, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates. study design/methods: we have searched for additional mechanisms that might enable human at to express fors1. a variety of amino acid substitution constructs of human at were prepared. additionally, exon deletion constructs of at mrna transcripts were also prepared. dna from those expression constructs was transfected into cos1(b3galnt1) cells, and cell-surface expression of fors1 antigen was immunologically monitored with a monoclonal anti-fors1 antibody. results/findings: we found that met69thr/ser substitutions also conferred human at with weak fs activity. we also found that the deletion of exon 3 or 4 of human at transcripts bestowed weak fs activity. because altered rna splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of fors1 antigen on certain cancer cells and tumors in forssman antigen-negative human species. furthermore, the co-introduction of one of those changes together with the glyglyala substitution synergistically conferred strong fs activity, in addition to strong at and bt activities. conclusion: the substitution of the glyglyala tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme. met69thr/ ser or exon 3/4 deletion may alter the intra-glogi localization of the enzyme. and those mechanisms function in synergy. the overlapping usage of acceptors by glycosyltransferases encoded by abo and gbgt1 genes is reminiscent of common ancestral origin of alpha 1,3-gal(nac) transferase genes. the finding that at can synthesize fors1 implicates that the boundary between abo and fors systems may not be as strict as was previously delineated due to the crosstalk in-between. rh typing is required by the fda and fact/aabb for identity testing. since most antibodies in cb plasma are maternal in origin, the abo/rh phenotype relies only on the red cell typing. a and b antigens are not fully developed at birth, presenting about one third of a or b antigen expression levels compared to adult cells. this can result in indeterminate abo results for some cb products. we evaluated the use of dna-based methods for abo typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results. study design/methods: a total of 29,308 cb units (cbu) were typed for abo/rh (beckman coulter pk system blood grouping and phenotyping) during the period 7/1/2008 -4/1/2017. abo genotyping targeting specific snps for groups a, a2, b, o1, and o2 and, if needed, gene sequencing was conducted in cases with indeterminate results, and in 4 cbu that were provided for transplantation with abo discrepancy found at the transplant center. results/findings: sixty-two (0.21%) cb samples had no reportable abo/ rh phenotype on initial testing, and therefore the cbu could not be used clinically. molecular abo/rh typing resolved all but one. all cases were heterozygous (a/o, b/o, or a/b); in 53% the predicted abo phenotype was a rh neg (table 1a ). the predominant donor race was caucasian (65%). four cbu with abo discrepancy were also evaluated by genotyping (table 1b) . in 3 of those, abo typing performed at the hospital on the day of transplant differed from that reported by the cb bank; the fourth was identified by posttransplant abo typing of the recipient. molecular genotyping resolved the discrepancies. cbu identity was always verified by confirmatory hla typing. conclusion: there is currently no fda approved dna-based abo assay. however, abo genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" cbu that could not be used otherwise. further, genotyping can help resolve abo discrepancies. abstract cobas v r hev for use on the cobasv r 6800/8800 systems is a qualitative pcr test for the detection of hev rna in human plasma. the purpose of this study was to evaluate the prevalence of hev rna among us blood donations collected in the midwest, a region reported to have a higher prevalence of hev infection, and the eastern us. study design/methods: 30,695 fresh and 20,029 frozen edta plasma samples from american red cross donors, collected from february 2015-2016, were de-identified and screened by individual donation testing (id-nat) using cobasv r hev for use on the cobasv r 8800 system under a research protocol. samples were primarily from midwestern and eastern regions of the us. samples reactive on cobasv r hev were further tested by an alternate hev nat, hev rna quantitation, hev genotyping, and for hev antibodies. results/findings: of 50,724 valid results, a total of 3 donations were reactive on cobasv r hev and all were confirmed positive. the confirmed donations were from a 65-year old male in indiana, a 21-year old male in california, and a 55-year old female in kentucky. all 3 donations were positive by hemi-nested pcr and alternative hev nat; however, only the kentucky donation had a high level of hev rna (1440 iu/ml), and was strongly positive for both igm and igg hev antibodies. the indiana donation was genotyped as 3a, the california donation genotype 3b, and no genotype determined for the kentucky donation (see table) . the clinical specificity for the cobasv r hev test in id-nat was 100% (95% exact ci: 99.993% to 100%). conclusion: based on the 3 confirmed-positive donations of 50,724 tested, the hev prevalence was 0.006% (95% exact ci: 0.001% to 0.017%) with a detection rate of 1:16,667 (95% ci, 1:588-1:100,000). to date, no cases of tt-hev have been documented in the us. however, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted hev. background/case studies: monitoring the epidemiology of ttis within the donor population is critical to provide an ongoing assessment of infection risks associated with fda policy changes such as the msm deferral criteria. ttims is a multi-center, federally-funded program intended to derive hbv, hcv and hiv prevalence, incidence, viral genotypes, and donor risk factors for greater than 50% of blood collected in the us. ttims is supported by two distinct coordinating centers (laboratory and risk factor, lrcc, and donation database, ddcc). here we report 13 months of prevalence along with demographic trends from the ddcc. study design/methods: four blood providers and their respective testing laboratories participated. standardized consensus-positive (cp) monitoring definitions were established for donor test results for hbv, hcv and hiv. these results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set. rates of nucleic acid test (nat) yield (seronegative) and concordant positives (serologic plus nat positives) were combined to comprise cps, were computed overall for donors and donations and by demographic, geographic and temporal characteristics. where appropriate, rates were compared for differences using 95% confidence intervals. this analysis contains data from 9/1/15-9/30/16. results/findings: among 7,578,462 donations reported (16.2% from firsttime and 83.8% from repeat donors), there were respectively 483, 1489 and 194 cp results for hbv, hcv and hiv with corresponding rates of 6.37,19.63 and 2.56 per 100,000 (pht) donations. prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of 23:1, 24:1 and 5.4:1 for hbv, hcv and hiv. rates (pht) among males were higher than among females for all markers (hbv 8.3 vs 4.2; hcv 23.5 vs 15.2; hiv 3.9 vs 1.0). in general, higher rates for all markers were seen among minority donors, those in the 25-39-year age group (also 18-24 year for hiv), and those from the southeast (and south central for hiv and hcv, and southwest for hbv). no trends were noted over time when 3-month periods were compared. conclusion: data from 4 major us blood systems were successfully combined and are a baseline for monitoring purposes. demographic trends are similar to those observed in other donor studies and generally agree with community trends. changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population. screening donated blood from babesia endemic regions of the united states using a transcription-mediated amplification assay on a fully automated system vanessa bres* 1 , melanie c proctor 2 , deanna self 1 , monique portugal 1 , adrian gurrola 1 , laura tonnetti 2 , sonia bakkour 3 , cheryl lobo 4 , michael paul busch 3 , susan l stramer 2 and jeffrey m linnen 1 . 1 grifols diagnostic solutions inc., 2 american red cross, 3 blood systems research institute, 4 new york blood center background/case studies: the procleix v r babesia assay on the procleix panther v r system is a qualitative in vitro nucleic acid test currently under development. the assay, which is based on transcription-mediated amplification (tma), detects four clinically relevant babesia species (b. microti, b. divergens, b. duncani, and b. venatorum) in human whole blood specimens. this test is intended to screen blood donations individually and in pools of up to 16 donations. whole blood samples are lysed and then pooled on the automated procleix xpress v r system prior to testing on the procleix panther system. these studies evaluated the preliminary analytical and clinical performance of the procleix babesia assay on the panther system. study design/method: analytical sensitivity was determined by diluting in vitro synthesized rna transcripts for the four babesia species. fresh b. microti-infected hamster whole blood, cryopreserved b. duncani-infected hamster whole blood and fresh b. divergens-infected human erythrocytes were tested to determine the limit of detection (lod) of parasites/ml (p/ml) by probit analysis. clinical sensitivity and specificity were determined by screening 32,274 unlinked whole blood donations collected from august 25 th 2016 to april 7 th 2017 in the northeastern united states. initial reactive donations were confirmed by repeat testing, pcr, and/or igg immunofluorescence assay (ifa). reactive individual donor lysates were tested in pools of 16. results/finding: the procleix babesia assay detected all four babesia species with a 95% lod ranging from 7.10-13.51 copies/ml. the preliminary 95% lod in parasites/ml ranged from 0.64-3.61 p/ml for b. microti (n59), from 0.92-1.52 p/ml for b. duncani (n52), and from 0.62-4.95 p/ml for b. divergens (n52). of the 32,274 donations screened, 17 initial reactive and 14 confirmed positive donations were identified for specificity of 99.991% (95%ci: 99.972-99.997%). of the confirmed positive specimens, 8 were reactive by both ifa and pcr, 5 by ifa only and 1 by pcr only. all confirmed positive samples were reactive in lysate pools of 16. donors of reactive donations resided in ct (11), nj (1), nh (1) and me (1) for an overall incidence of 1:2,305, and 1:1,433 in ct. conclusion: the procleix babesia assay on the procleix panther system demonstrated high clinical specificity and sensitivity and detected all four babesia species with similar sensitivity. all confirmed positive donations were also detected in pools of 16 thus demonstrating the effectiveness of pooled lysate screening. conclusion: use of the lag avidity assay shows that in both first-time and repeat hiv-positive us blood donors, newly-acquired (i.e., incident) hiv infections are more frequent in younger donors. the use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type. epidemiology of hepatitis b virus, hepatitis c virus and human immunodeficiency virus in united states blood donors lauren a crowder* 1 , whitney r steele 1 , ed p notari 1 , james haynes 1 , roger y dodd 2 and susan l stramer 1 . 1 american red cross, 2 american red cross (retired) background/case studies: from 2004 -2012 , the prevalence of hbv and hcv in us blood donors decreased, while hiv rates remained constant. however, incidence has not been recently calculated. here we report the prevalence, incidence and residual risk (rr) of hbv, hcv, and hiv in a large us blood system from 2008-2015. study design/methods: prevalence was calculated in 2-year intervals. incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (py). rr was calculated using the window periods of 18.5, 7.4 and 9.1 days for hbv, hcv and hiv, respectively. linear regressions were calculated with p<0.05 (*) as significant. results/findings: from 1/1/08-12/31/15, there were more than 48 million donations from 13,204,447 donors (51.4% female, 33% first-time (ft), 81.4% caucasian). there were significant decreases in donation prevalence for hbv and hcv (p50.014 and 0.044), but no significant decrease in hiv during the 8 years (see table for f and r 2 values). a significant decrease was seen in ft donor prevalence for hbv and hcv (p50.026 and 0.042). prevalent ft donors were significantly more likely to be male (68.3% -hbv, 59.8% -hcv, 79.7% -hiv; p<0.001). incidence for all agents declined (significant only for hbv; p50.035). the decrease in hcv incidence was not significant, but there were fewer incident donors in the last 2-year period (74 in 2012-2013 vs. 19 in 2014-2015) . hcv incident donors in 2014-2015 were more likely to be male (79.0% vs 46.0% in 2012-2013, p<0.001) and were younger (84.2% vs. 67.6% in 2012-2013 <40 years, p50.011). overall, incident donors were more likely to be caucasian males (p<0.01). rrs for all 3 agents decreased over time with rrs in 2014-2015 of 1 in 1,565,000; 1 in 2,680,000; and 1 in 2,435,000 for hbv, hcv and hiv, respectively. conclusion: prevalence, incidence and rr of hbv, hcv and hiv have generally decreased within this blood system over the 8-year time frame. as donor screening and deferral regulations evolve, it is important to monitor these risks. it is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates. furthermore, in 2015, mayv was isolated from a patient in haiti, suggesting the virus is already circulating in the caribbean. the extent of mayv transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for mayv emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion. study design/method: platelet components (pc) prepared in pas were spiked with mayv and treated with amotosalen and uva illumination. samples were collected pre-uva and post-uva illumination for infectious titer determination. as-5 rbcs were spiked with mayv, mixed with glutathione (gsh)/processing solution, dosed with 200lm amustaline, and incubated for 18hrs at room temperature. samples were collected prior to the addition of amustaline (pre-treatment) and following the 3hr incubation (post-treatment) to determine infectious titers. infectious titers for all samples were determined by plaque assay on vero76 cells. the extent of inactivation was determined by comparing the infectious titers (plaque forming units (pfu)/ml) in pre-vs. post-treatment samples. results/finding: mayv was inactivated to the limit of detection in both pc and rbcs. in platelets, >6.9 log 10 , or >6.2 log 10 pfu/ml, inactivation of mayv was achieved. in rbcs, inactivation of mayv was >6.2 log 10 , or >5.5 log 10 pfu/ml. conclusion: this study demonstrates robust inactivation of mayv by both amotosalen/uva treatment in pc and amustaline/gsh treatment in rbcs. these systems are efficient at inactivating alphaviruses that have demonstrated or have the potential for transfusion-transmission, including mayv, chikv and rrv. prt offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple alphaviruses are epidemic or endemic, and testing is not feasible. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). thrombotic thrombocytopenic purpura with high adamts-13 inhibitor may represent a distinct disease subset in response to therapy based on immature platelet count (a-ipc) dynamics hamza n gokozan* 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues. early therapeutic plasma exchange (tpe) initiation has improved survival. absolute immature platelet count (a-ipc) has been found to aid in diagnosis and follow-up of ttp patients. a-ipc changes in response to therapy in patients with low adamts13 activity and high inhibitor have not been analyzed in a patient cohort. we analyzed a-ipc response to therapy in five patients with adamts13 deficiency and high inhibitor at a large tertiary academic medical center. study design/method: patients had adamts13 activity of <5% and high inhibitor (1.4-8). mean age of cohort 22.8 years (range 17-64). four patients were female and one was male. patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean 15.2 x 10 9 /l, range 9 -27 x 10 9 /l) and low a-ipc (mean 1.5 x 10 9 /l, range 0.5 -3.6 x 10 9 /l). patients were initiated on daily tpe and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab 375 mg/m 2 (4 patients) and cyclophosphamide 400 mg/m 2 (one patient). tpe continued until platelet count reached 150 x 10 9 /l for at least two consecutive days. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: patients responded rapidly to daily tpe (mean of 2.4 days [range 1-4 days]) when they achieved a three-fold increase in a-ipc from baseline (mean 11.1 x 10 9 /l, range 2.2 -25.3 x 10 9 /l) and a rapid improvement in platelet count. however, this improvement in platelet count was not accompanied by expected decreases in a-ipc, suggestive of recovery from disease. all patients experienced platelet (mean 217.6 x 10 9 /l, range 200 -294 x 10 9 /l) and a-ipc (mean 19.4 x 10 9 /l, range 13 -28.5 x 10 9 /l) decreases that occurred concurrently while receiving daily tpe so that after a mean of 11.6 days (range 8-14 days) mean platelet count was 65.4 x 10 9 /l (range 14 176 x 10 9 /l) and mean a-ipc 3.2 x 10 9 /l (range 0.7 -6.6 x 10 9 /l). patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with tpe after a mean of 20.8 days of a-ipc and platelet count instability. a-ipc trended to levels indicative of restoration of a negative feedback after this time. conclusion: rapid decreases in platelet counts after a good response in ttp patients may raise suspicion for presence of high adamts13 inhibitor. patients with a high inhibitor have similar a-ipc dynamics during which initial high a-ipc production is followed by unexpected decreases in a-ipc concurrent with platelet counts. recovery occurs once negative feedback between platelet and a-ipc production is re-established. patients with a high inhibitor may represent a distinct subset of ttp as suggested by a-ipc responses. benchmarking the centralized urgent plasma exchange service for patients admitted with a diagnosis of thrombotic thrombocytopenic purpura at a multi-hospital healthcare system jansen n seheult* 1 , michelle n stram 1 , joan sevcik 2 , alesia kaplan 2,3 and joseph e. kiss 2,3 . 1 department of pathology, university of pittsburgh medical center, 2 blood systems inc., 3 university of pittsburgh background/case studies: consensus guidelines recommend that therapeutic plasma exchange (tpe) must be started as early as possible and within 4-8 hours after the diagnosis of thrombotic thrombocytopenic purpura (ttp) has been made; however, there are limited data documenting actual practice. there are several operational facets of delivering a centralized urgent tpe program in a multi-hospital healthcare system, including: central venous (cv) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure. this study analyzes the time elapsed between major steps from diagnosis to initiation of tpe in patients admitted with ttp. study design/method: a retrospective review of the electronic medical record and laboratory information systems from january 1, 2013 to november 1, 2016 was conducted to identify all ttp patients undergoing urgent tpe. demographics, comorbidities, and other pertinent laboratory tests (such as adamts-13 activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients. temporal data for tpe request, cv access placement, plasma product release (which usually happens after cv access), arrival of tpe team and initiation of the procedure were extracted from procedure notes and the blood bank information system. descriptive and summary statistics were generated using stata version 14 (statacorp, tx). group comparisons were made based on hospital location, level of care and history of ttp using a wilcoxon rank-sum test. results/finding: of the 96 ttp patients identified, 22 were excluded due to missing temporal data for important variables. the majority (85%) of patients were treated at central academic centers, with the remainder being treated at peripheral sites. fifteen patients (20%) had a prior history of ttp and 26% had severe adamts13 deficiency on admission. the median time from tpe request to initiation was 5.6 hours (interquartile range: 4.7-7.2 hours). there were non-significant trends to shorter time intervals from request to cv access and request to tpe initiation in patients admitted to the intensive care unit (icu) versus non-icu patients (table 1) . treatment was not started within an 8-hour window in 13 patients; the median time to cv access was significantly longer in these patients (5.8 vs 2.47 hours, p<0.001). two of these patients had a prior history of ttp and only four patients had severe adamts-13 deficiency. the majority (more than 70%) of the time interval between tpe request and tpe initiation was spent obtaining cv access and plasma products. there were no significant differences in time intervals comparing patients with a new diagnosis of ttp versus patients with recurrent/ relapsed disease (table 1) or between patients treated at a central academic center versus a peripheral hospital. conclusion: the consensus 4-8 hour target window from tpe request to initiation appears feasible for a centralized tpe program servicing a multi-48a transfusion 2017 vol. 57 supplement s3 hospital healthcare system. addressing limitations in availability of cv access would likely yield the greatest improvement in timeliness of urgent tpe. cytoreductive therapy for cellular hyperviscosity: utility of cytapheresis treatment for chronic myelogenous leukemia and essential thrombocythemia. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (cml-at) or essential thrombocythemia (et) may improve short-term outcomes. however, no randomized controlled trial (rct) assessing the efficacy of cytapheresis treatment in this group of pts has been performed. study design/method: from january, 2006 through january, 2017, we performed cytapheresis (cy) treatments (txs) for 123 pts with either cml-at or et, and clinical and/or laboratory evidence of cellular hyperviscosity. 84 pts (68%) had cml-at and received 319 leukapheresis (lp) txs; 39 pts (32%) had et and received 124 thrombocytapheresis (tc) txs. cml-at pts presented with median wbc 398 x 10 9 /l (range 193-689 x 10 9 /l), of which 63% had blast percent >75% or blast count >100 x 10 9 /l. median age was 42 years (8-79 years); 62% were male. cns symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates. 22% of cml-at pts had no sxs of lks; 40% pts had sxs of either cns or pulm lks (1 sxs), and 38% pts had sxs of both cns and pulm lks (2 sxs). et pts presented with median platelet (plt) count of: 1738 x 10 9 /l (642-3510 x 10 9 /l)and 71% pts had sxs of thrombosis (evidence of cva or tia, mi, or dvt). median age was 66 years (31-89 years); 58% pts were male. results/finding: all pts received a course of cy tx with following objectives: 1) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and 2) stabilizing cml-at pts for induction chemotherapy (ind chemo). wbc (or plt ct) tx goals were: wbc count (ct) <100 x 10 9 /l for cml-at pts, and plt ct <450 x 10 9 /l for symptomatic et pts and <750 x 10 9 /l for asymptomatic et pts. cml-at pts received median of 3 lp txs (mean 3.9 txs/pt; range 2-8 txs). et pts underwent median of 2 tc txs (mean 3.4 txs/pt; 1-7 txs). outcomes were evaluated by percentage of pts who: 1) reached wbc (or plt ct) tx goal, and 2) received ind chemo. "improved" outcome was defined as pts who reached their wbc (or plt ct) tx goal during cy tx; "stabilized" were pts who achieved >50% reduction in wbc (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither. in cml-at cohort, 76% pts improved, 21% pts stabilized; and 3% pts worsened. in et cohort, 85% improved, 14% stabilized, and 1% were unchanged. for cml-at pts, median final wbc ct 5 96 x 10 9 /l (range 66-307 x 10 9 /l); 94% pts received ind chemo. for et pts, median final plt ct 5 705 x 10 9 /l (263-1087 x 10 9 /l); 95% pts had resolution of thrombotic 49a transfusion 2017 vol. 57 supplement s3 symptoms. 4% of cml-at pts and 0% of et pts expired within 1-4 days after course of cy tx. of 3 expired pts, 2 pts had both blast crisis and sxs of cns/ pulm lks; 1 pt had intracranial hemorrhage or cva; and 2 pts were hypotensive, intubated, or unable to tolerate ind chemo. conclusion: pts with cml-at or et and evidence of impending thrombosis may benefit from cytoreductive therapy. a limited number of cytapheresis treatments (median 2-3 txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes. a rct to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful. background/case studies: partial normal saline replacement during plasma exchange procedures is common practice. benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations. however, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions. the goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution. study design/method: a four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used 100% albumin or 80% albumin-20% normal saline (80/20) as replacement. patients who received plasma entirely or partially as replacement were excluded. the procedure type ordered (100% albumin vs 80/20), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases. repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where 100% albumin was used versus those that used 80/20. covariates included were fluid types, age and gender. odds ratios (or) and 95% confidence intervals (ci) were used as a measure of risk. we used the term significant for a two-sided p-value < 0.05. results/finding: during the study period, 3650 procedures were documented for 414 subjects (46% female), age range 0-93 years, of which 2,470 (67.7%) received 80/20. the type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity. replacement with 100% albumin had a significantly lower risk of having either event than by using 80/20, [p50.002, or (ci): 0.40(0.22, 0.72)] , and also had a significantly lower risk of causing hypotension [p50.023, or (ci):0.45 (0.22, 0.89)] in addition to a lower risk of causing citrate toxicity [p50.042, or (ci): 0. 24 (0.06, 0.95)]. age had a significant effect on having a hypotensive event [p50.04, or (ci):1.1 (1.0, 1.1)] but no effect on citrate toxicity or the combined outcome. gender had no effect on frequency of any event. conclusion: partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure. age also increases the risk of hypotension. use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients. background/case studies: therapeutic apheresis (ta) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (aes). there are few studies published on aes associated with ta but they lack uniformity of data. moreover, there is no common database in the united states (us) to report ta-associated aes. we evaluated the annual incidence rates of aes associated with ta at a large tertiary academic medical center over a 10 year period and compared it to published literature. study design/method: we conducted a 10-year retrospective study of ta procedures performed and aes were classified according to criteria described in table 1 . during the study period, ta were performed using cobe spectra (software versions 4.7 and 6.1) and since 2013 the spectra optia apheresis system (version 8.0). literature search was conducted for data published on aes associated with ta. four studies from us and 13 non-us studies (canada, europe and japan) were analyzed. trend for ae rates from 2007-16 was also analyzed. statistical analysis was performed using chi square and spearman rho tests. results/finding: the overall ae incidence was 6.9% (396 of 5,684 procedures) during 10 year period. frequency of aes associated with therapeutic plasma exchange (tpe) was significantly higher (8.5%, p<0.00001) compared to other ta procedures. we found significant correlation between number of tpe and aes (spearman rho 0.7, p50.002) over the 10 years and significant down trend of moderate and severe aes with a spearman rho of -0.64 (p50.04) and -0.83 (p50.003) respectively. there were no fatalities during the study period. majority of aes were grade i (60%) and grade ii (28%): 32/5684 (0.6%) procedures were not completed due to aes. comparison of aes [6.9% (396/5,684)] to both european [11.2% (n513, 12, 256/ 109, 842) ] and other us studies [13.6% (n54, 860/6,324)] showed a statistically significant difference (p<0.0001). conclusion: overall incidence of aes was significantly lower than current published literature. incidence of aes published in other countries is significantly lower than rates published in us. differences in incidence of aes in literature emphasizes need for uniform reporting and stratification of aes and development of a common database to report ta-associated aes. we propose a grading rationale in order to standardize reporting of ae (table 1) . variations in biochemical markers of bone metabolism during plateletpheresis: impact of socio-demographic and lifestyle factors? markus dettke*. akh vienna university hospital background/case studies: plateletpheresis is associated with short-term variations in biochemical markers of bone turnover. socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health. in the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors. study design/method: altogether 200 platelet donors with a donation activity of up to 150 platelet donations participated in the study. after a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism. blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (oc) and the bone resorption marker cross-linked telopeptides of type i collagen (ctx), among other parameters. the effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors. results/finding: plateletpheresis resulted in an increase in the serum levels of the bone resorption marker ctx and the bone formation marker oc. both parameters returned to base levels within 2 hours after the end of the collection. multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in ctx or oc. there was no association between individual donor career or the number of previous donation and the observed increase in bone turnover. the only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis. increase in serum ctx, showed an inverse correlation to changes of serum ionized calcium. continuous iv supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for ctx compared to oc. conclusion: the amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism. known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover. transfusion with optimized blood products versus transfusion with standard products in a trauma-transfusion rat model mathijs wirtz* 1 , jordy jurgens 1 , jacoline buchner-doeven 1 , joris roelofs 1 , philip spinella 2 , jennifer a muszynski 3 , carel goslings 1 and nicole juffermans 1 . 1 academic medical center, 2 washington university school of medicine, 3 nationwide children's hospital background/case studies: transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products. we hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction. study design/methods: blood products were prepared from syngeneic rat blood according to blood bank standards. soluble mediators were removed from red blood cells (14 days old) and platelets (5 days old) by washing. plasma was filtered through a 0.22um filter. rats ($350 grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $30% of their estimated blood volume, which was calculated to be 57ml/kg. hemorrhage continued until a mean arterial pressure of 40mmhg was reached. rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham. blood samples were taken up to 4h after trauma to assess biochemistry and coagulation status. ex vivo whole blood stimulation tests with lps were performed after sacrifice, and organ damage was assessed by histopathology. blood products were sampled to assess for biochemical changes. comparisons between groups was done by anova and dunnett's post-test for multiple comparisons. results/findings: filtering or washing of blood products significantly stabilized ph, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products. both resuscitation groups received an average of 17ml/kg of blood products in a 1:1:1 ratio. however, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, asat and alat. the coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care. immune response to lps was decreased following trauma compared to healthy controls but did not differ between groups. conclusion: filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response. these results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model. safety and efficacy of tranexamic acid during cardiovascular surgery: a single center before-and-after study takuma maeda* and shigeki miyata. national cerebral and cardiovascular center background/case studies: tranexamic acid (txa), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of txa is effective in reducing blood loss after cardiovascular surgery. however, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes. consequently, we stopped using txa in april 2013, which enabled us to conduct a before-and-after study. the present study aimed to examine the association between txa and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model. we also assessed the association between txa and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and 30-day mortality). study design/method: this single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between january 2008 and july 2015 (n53535). because of missing data on patient characteristics, 257 patients were excluded. the incidence of adverse effects associated with txa and other clinical outcomes were evaluated before (january 2008 to march 2013, n51987) and after (april 2013 to july 2015, n51291) using a propensity score model. we estimated propensity scores using a logistic regression model for txa use as a function of 18 baseline variable, generating 969 pairs of patients who received or did not receive txa. we also evaluated the adverse effects of txa using segmental regression analysis. results/finding: propensity-matched analysis showed that seizures were more common (8.7% vs 3.7%, p<0.001) and ventilation time was longer (15 h vs 13 h, p50.04) significantly in the txa group than in the non-txa group. in contrast, transfusion volume and blood loss were significantly lower in the txa group than in the non-txa group (2000 ml vs 2200 ml, p50.009; and 1265 ml vs 1460 ml, p<0.001, respectively). however, 30-day mortality was not statistically different between the groups (1.6% vs 1.4%, p50.82). none of the other outcomes were significantly different. segmental regression analysis yielded similar results. conclusion: even though txa may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality. the use of txa is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of japanese society. it seems to be advantageous to use txa because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time. sustained impact of blood management strategies in orthopedics: continuous quality improvement linda levinus* and michele deeney. new england baptist hospital background/case studies: transfusions are one of the most over-utilized treatments performed in any hospital setting (choosing wisely campaign, april 2014, www.choosingwisely.org/societies/american-association-of-bloodbanks). costs and risks associated with transfusions are high and may have a significant impact on patient safety. in our institution we perform over 12,000 joint replacements and spine surgeries per year, making transfusion-associated costs very high. since our last formal evaluation of the metrics used post implementation of patient blood management (pbm) strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians. the objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard table 1 ). the data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through fy16. length of stay has also shown a continued reduction, which is an indicator that the pbm strategies implemented have not compromised quality outcomes. further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our pbm program. going forward, these practices, along with investigating use of additional pbm strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics. safety and efficacy of 4-factor prothrombin complex concentrate: a retrospective review of outcomes at an academic hospital stephanie jalaba*, hollie benson, nan zhang, jill adamski and theresa kinard. mayo clinic arizona background/case studies: 4-factor prothrombin complex concentrate (pcc) contains factors ii, vii, ix, x, proteins c and s and is used for reversal of vitamin k antagonists in acute major bleeding or urgent, invasive procedures. occasionally, it is used off-label when plasma is not optimal for achieving hemostasis. this study compares the efficacy of on-label and off-label use of pcc in correcting coagulation parameters and reducing allogeneic blood transfusion. study design/methods: a retrospective chart review was performed for pcc use at our institution in 2015. marginal modeling (gee method) was used to account for within patient correlation and assess changes in lab values and products transfused. logistic regression (gee method) was used to evaluate potential risk factors for unsuccessful hemostasis (uh5 rate of transfusion after pcc ! rate before pcc) or thrombotic complications. results/findings: the reduction in pt (p5.005) and ptt (p5.05) was significantly greater in on-label than off-label use. interestingly, transfusion reduction in rbc (p5.03) and plasma (p5.04) after off-label use was significantly greater than on-label use. 20 cases, both on-label and off-label, with uh were associated with cell saver, acute normovolemic hemodilution (anh), or cardiopulmonary bypass (cpb). the odds of having uh were 5.5 times (p5.0072) more with cell saver or anh, and 5.3 (p5.0130) times more with cpb. post-pcc thromboses were identified in 6 cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin k, factor viia, or extracorporeal support. background/case studies: when a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (rbc) at all times by always having an in-date type and screen specimen. per current aabb standards, this necessitates a new sample every 3 days. this can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a picc line may be placed. in order to mitigate these issues, we chose to extend the type and screen specimen to expire after 7 days in patients without rbc alloantibodies other than passively acquired anti-d due to rh immune globulin administration. study design/method: patients expected to have an antenatal hospitalization of at least 4 days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to 7 days. the transfusion service medical director reviews the case and gives final approval. we observed only 1 patient did not have an in-date specimen when the extended out-dating was requested. thirty-eight (38) patients were in-patients continuously until delivery. five patients were discharged prior to delivery-1 moved to another state, 1 was admitted later at another local hospital, and three were readmitted for later deliveries. the mean interval from approval to delivery was 17 days (range 0-63). six (6) patients delivered within 3 days of approval. after approval, the mean number of additional specimens per patient was 2.1 (range, 0-9). no patient required transfusion prior to delivery. five patients received transfusion of at least 1 rbc at the time of delivery, and none had evidence of transfusion reaction. conclusion: since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe. with only 6 patients delivering within 3 days of approval for extended specimens, 37 patients avoided collection of at least 1 specimen each, and 16 patients avoided at least 4 collections each. since new antibodies are not detectable for at least 10 days after immunization, even longer extension of pre-transfusion specimen out-date may be considered. although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to 7 days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient. iron metabolism in critically ill patients developing anemia of inflammation margit boshuizen* 1,2 , jan m. binnekade 1 , benjamin nota 2 , pieter r tuinman 3 , kirsten van de groep 4 , olaf l cremer 4 , janneke horn 1 , marcus j schultz 1 , robin van bruggen 2 and nicole p juffermans 1 . 1 academic medical center, 2 sanquin research and landsteiner laboratory, 3 vu university medical center, 4 university medical center utrecht background/case studies: anemia due to inflammatory processes (anemia of inflammation, ai) frequently occurs in critically ill patients. in ai, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis. knowledge on changes in iron metabolism during the course of ai is limited, hampering the development of strategies to counteract ai. this study aimed to investigate the dynamics of parameters of iron metabolism during the development of ai in critically ill patients. study design/methods: a case control study was performed in 2 tertiary icus in the netherlands comparing 30 patients who developed ai during icu stay with 3 control groups: 30 non-anemic patients with sepsis, 30 non-anemic patients without sepsis, and 10 patients with anemia due to acute blood loss. patients were matched on age and sex. a linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time. results/findings: in patients with ai, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) . ferritin and hepcidin were increased in ai compared to controls. in the course of ai development, erythroferrone decreased. differences in iron metabolism between groups were not influenced by disease severity. patients with ai differed from patients with anemia due to acute blood loss, the latter was characterized by high iron (15.4 vs. 2.9 mmol/l, p<0.001) and transferrin saturation (53 vs. 9 %, p<0.001), and low ferritin (104 vs. 645 mg/l, p<0.001). conclusion: in critically ill patients with ai, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy. iron metabolism in ai is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that ai is not solely determined by severity of inflammation. iron metabolism in ai patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic icu patients should take the cause of anemia into account. clinical oral abstract session: novel approaches to processing and assessing cell therapy products a paradigm shift in stem cell isolation and storage jeffrey drew*. cells4life group llp background/case studies: widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells. the recommended therapeutic cell dose is approximately 2.5 x 10 7 cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals. cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (wcf). however, all current methods result in significant loss of the wcf, some up to 65%, whilst leaving 25% of the starting volume of erythrocytes. additionally, there is an almost total loss of potentially important, low abundance cellular subsets. the use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery. study design/methods: we have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy. on combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within 30 minutes. the wcf remains in solution and can be easily separated from the erythrocyte sediment. the wcf can then be concentrated by gentle centrifugation into a small volume containing less than 1% of the original erythrocyte content. the addition of dmso for cryogenic storage and controlled freezing using standard procedures then completes this simple process. results/findings: we have clearly demonstrated that this method allows almost the entire wcf to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined. in addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately 65% of the cd341 fraction post separation and freeze thaw (table 1) . possibly more important, the cfu assay results reproducibly yield higher counts of cfu-gm, cfu-gemm and bfu colonies (table 1) which is a strong indicator that this method will improve patient outcomes. in addition, our separation method isolates and preserves the megakaryocyte-like cells (cd451cd611) and early projenitor cells expressing oct4 and nanog (markers for vsels) which are two examples of cellular subsets usually lost using current separation techniques. conclusion: these results demonstrate that our method achieves: 1. routine recovery of the wcf at levels higher than current methods, independent of volume. 2. higher percentage recoveries of all cell types tested than can be achieved with existing methods. 3. markedly higher post-thaw recovery of viable nucleated cells than any current methodology. 4. almost complete removal of hematocrit. as a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units. therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods. effects of implementation of an absolute lymphocyte count target, in addition to cd341 target, for hematopoietic progenitor cell collection edwin a burgstaler*, luis f porrata, dennis a gastineau, eapen k jacob and jeffrey l winters. mayo clinic background/case studies: lymphoma patients receiving >0.5x10 9 lymphocytes(lymph)/kg during peripheral blood stem cell transplant have superior survival. in addition to a cd341 cell target of 4.0x10 6 /kg, a lymph target was also implemented. fifty patients before (no alc) and after (alc) implementation were retrospectively evaluated. study design/method: lymph and cd341 yields, number of collections, lymph target reached, and days to engraftment were examined. mobilization was g-csf (g) or g-csf 1 plerixafor (g1pl). consecutive no alc and alc procedures were examined. the mann-whitney and chi square tests were used for statistical comparison, p< 0.05 considered significant. results/finding: 110 no alc and 159 alc collections occurred among the 50 patients. fenwal amicus was used for 91% of the no alc and 99% of the alc collections (terumobct spectra optia cmnc used for remaining). diagnosis was 5 hodgkin's and 45 non-hodgkin's lymphoma (no alc); 7 hodgkin's and 43 non-hodgkin's lymphoma (alc). pre procedure wbc and lymph counts were significantly higher for no alc (wbc 49.3, lymph 2.0x10 9 /l) than alc (wbc 39.1, lymph 1.2x10 9 /l). equivalent whole blood (corrected for ac) was processed for no alc (16.4l) and alc (17.1l). for alc group, extra collections beyond cd341 target were: 0 days: 24%, 1 day: 36%, 2 days: 22%, 3 days: 16%, and 5 days: 2%. significantly more patients were mobilized with g1pl in no alc group (n581) than alc group (n560) and 42 collections in alc group had mobilization discontinued after cd341 cell target reached. there was no significant difference in g (13.2x10 9 lymph) compared to g1pl mobilized collections (13.0x10 9 lymph); both were significantly higher than the collections where mobilization had been discontinued (5.9 x10 9 lymph). days to wbc engraftment (13.5 no alc vs 13.0 alc) and platelet engraftment (13.0 no alc vs 12.0 alc) were not significantly different. median number of collections for no alc (2) and alc (3) were not significantly different. data (medians) in the table. conclusion: not all patients achieved the 0.5x10 9 lymph/kg or even the 0.3x10 9 lymph/kg targets. implementation of a lymph target increased patients obtaining 0.5x10 9 lymph/kg from 40% to 54%. only 12% had <0.3x10 9 lymph/ kg. discontinuation of mobilization once cd341 cell target was reached significantly reduced lymph yield. the median increase of one collection per patient following implementation was less than had been expected. extended preprocessing storage impairs cord blood hematopoietic stem cell activity suria jahan* 1,2 and nicolas pineault 2,3 . 1 canadian blood services, 2 university ottawa, 3 canadian blood services, centre for innovation background/case studies: large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (ucb) unit. current net-cord-fact standards specify that units can be stored for almost 48 hours at room temperature (rt) as long as units are cryopreserved by 48-hours post-collection. the impact of such delay on hematopoietic stem cell (hsc) function is unclear since most studies have not used transplantation assays that measure hsc key properties and activities. we hypothesized that such processing delay reduces the engraftment activities of ucb units. we set out to measure the loss in engraftment activities associated with preprocessing storage. study design/method: ucb units (n53) were split with one half processed immediately (baseline 8-12 hours) and the second after 43 hours storage at rt. ucb were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use. viability was assessed post-thaw, and thawed ucb buffy coat cells were transplanted into nsg mice. serial transplantation was used to test the self-renewal and differentiation activities of hsc, while limiting dilution (ld) assay and poisson statistic were used to estimate the frequency of scid repopulating cells (src) in thawed units. results/finding: storage before processing had no significant impact on the recovery of viable post-thaw cd451 cells and cd341 cell (n53). primary nsg mice were transplanted with a ucb cell dose that contained a total of 7,500 annexinv neg viable cd341 cells. the latter was done to avoid any bias towards one group or another. short term platelets (190 vs. 140 hplt/ml, p50.06) and leucocytes (1.2% vs. 0.2% hcd451, p<0.02) engraftment at 4-weeks were significantly reduced in stored mice vs. baseline (n53), and similar results were observed long-term at 16-weeks. long-term human bone marrow (bm) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups. bm cells from primary mice were transplanted into secondary recipients and human engraftment investigated 3 months post-transplant. strikingly, the frequency of human cd451 bm cells was 10-fold greater in baseline vs. stored mice (p<0.01, n52). hence, storage at rt of ucb units is associated with a deficit in engraftment activity likely due to a loss in hsc activity and/or numbers. to distinct between both possibilities, the net number of src in baseline and stored samples for two units were calculated by ld transplantation assay. the net number of src measured 22-weeks post-transplants were reduced by 30% in unit 1, and by 80% in unit 2. conclusion: prolonged preprocessing rt storage significantly impairs the engraftment activities of ucb units. the reduced engraftment in secondary transplants coupled with the results from the ld assays suggest that this engraftment deficit origins from loss of hsc numbers. our results stress the importance of rapid ucb processing to avoid loss of engraftment activity. acoustic microfluidic separation of blood components charles lissandrello, ryan dubay, kenneth kotz and jason fiering*. draper background/case studies: new cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing. while apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process. continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood. it has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density. meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells. it has been shown previously that acoustic separation can separate blood components including purification of lymphocytes. however, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges. in contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with rbc and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel. study design/method: acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($1mm). this results in an acoustic force across the channel that drives cells toward the axial center stream. because the force increases with a cell's size and density, lymphocytes experience a weaker force than rbcs and other classes of wbcs. thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet. likewise, platelets can by separated from lymphocytes. initial and output cell counts are measured by a standard hematology analyzer. results/finding: in our acoustic system, lymphocyte purity (% of total wbcs) was enriched up to 97%, using leukapheresis product as the starting material. this enrichment was achieved in a single pass through the device (residence time of 1sec). total lymphocyte recovery was 43% and monocyte concentration was reduced 76%. furthermore, in a two-pass process platelets were reduced by 75%. in a 12-fold parallel system we tested rbc separation from plasma and achieved 90% separation at 72ml/hr. conclusion: acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels. such disposable devices are suitable for scale up to clinical bioprocessing systems. lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument. background/case studies: the use of natural killer (nk) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies. nk cells have been used at our institution for the past 15 years. most patients have a reaction with nk cell infusion with some reactions being quite severe. we retrospectively analyzed the reactions associated with nk cell infusions to help address why some patients have more severe reactions than others. study design/method: retrospective chart review of nk cell infusions performed at our institution from 9 clinical protocols from 2008-2016. an infusion reaction was defined as any symptom from the time of nk cell infusion up to 4 hours afterwards. a severe reaction was defined as any symptom with grade 3 or higher severity (graded on common terminology criteria for adverse events-ctcae). preliminary data was analyzed using r 3.3.1. two major endpoints of interest were: 1) infusion reaction with any symptom and 2) severe infusion reaction. to numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (iqr) were used. a wilcoxon test was performed to test the association between the continuous variables and our end points. a chi-square test was used to test the association between categorical variables and our endpoints of interest. results/finding: there were a total of 127 nk cell infusions. there were 119 (94%) patients with an infusion reaction of any symptom and there were 37 (29%) patients with a severe reaction. infusion rate (ml/min) was similar among those with any reaction (median52.55, p50.42) and those with severe reaction (median52.52, p5 0.42). infusion rate (ml/min/kg) was also similar among those with any reaction (median50.03, p50.43) and those with severe reaction (median50.03, p50.15 respectively). incubation of nk cell product overnight in il-2 vs il-15 had similar reaction rates for those with any symptom (88% had reaction with il-2, 86% had reaction with il-15, p50.94) and those with severe reaction (28% had severe reaction with il-2, 24% had severe reaction with il-15, p50.80). patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the nk cell product (median52.44 x 107) versus those without (median51.92 x 107, p50.02). conclusion: our preliminary data analysis reveals that a higher number of monocytes in the nk cell product may contribute to severe infusion reactions, causing patients to have a grade 3 or higher symptom. limitations to this study include this was a retrospective review at a single institution. a streamlined mixed lymphocyte reaction (mlr) assay for evaluation of human mesenchymal stem cell immunomodulation activity christopher p delavan 1 , maryanne c herzig* 1 , barbara a christy 1 , james a. bynum 2 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research background/case studies: mesenchymal stem cells (msc) have been investigated for treatment of acute respiratory distress syndrome (ards), graft versus host disease (gvhd), wound healing and trauma. a consensus is building that the immunomodulation by mscs is key to their therapeutic potential. mscs suppress peripheral blood mononuclear cells (pbmc) proliferation in vitro, suggesting a correlation for suppressing pbmc inflammatory responses in vivo. current mixed lymphocyte reaction (mlr) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated pbmcs in the presence of mitotically inactive mscs. in the study detailed here, mscs are analyzed in a direct co-culture with pbmcs using a luminescent atp assay. study design/method: blood was obtained from an in house blood bank and pbmcs were separated by centrifugation over ficoll-paque in leuco-sep tubes as specified by the manufacturer. the pooled donor pbmcs were stored at -80. mscs derived from bone marrow, adipose tissue or umbilical cord (bm-msc, ad-msc, uc-msc, respectively) or human umbilical cord endothelial cells (huvec) were serially diluted starting at 50-60,000 cells/ well and cultured in 96 well plates for 4-48 h in their respective medias. on day 0, mscs were washed, resuspended in pbmc media and incubated with or without 150,000 freshly thawed pbmcs/well, in the presence or absence of phytohemagglutinin a (pha, 0-5 lg/ml). proliferation of both mscs and pbmcs was assessed in triplicate wells by quantitation of atp levels using the bioluminescent reagent cell titer-glo (promega). results/finding: pbmc proliferation in response to pha gave a robust atp signal by 72 h, with >6 fold increase over control pbmcs. no increase in atp response or proliferation was seen in the absence of pha. co-culture with mscs inhibited pbmc proliferation dependent upon msc passage, source, msc media additive. intra-assay variance of triplicate samples was 10.0%. inter-assay variation of msc preps run under identical conditions was 7.5%. inhibition of pbmc proliferation was graded from 0-100% over the range msc concentrations therefore an ec50 of msc cell number resulting in 50% suppression of pbmc could be determined for each msc prep. this ec50 however was dependent upon pbmc donor pool. conclusion: direct co-culture of live mscs with freshly thawed pbmcs give a robust determination of immunosuppression by mscs. graded responses can be determined, allowing comparison of potency between msc preparations. this streamlined assay can be performed within 72 h, without irradiating cells and with minimal equipment outlay. background/case studies: a high prevalence of iron depletion (id) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute 10% or more of the us blood supply. differences between donors 16-18 years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion. study design/method: donors aged 16-49 were eligible for ferritin testing if they donated at a high school (hs) blood drive at the start of the 2015/16 academic year at two blood centers. samples from return donations over the remainder of the school year were also tested. the prevalence of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml) were estimated for 16, 17, 18 and 19-49yo groups separately for both genders. linkage to operational databases established first-time (ft) vs repeat (rpt) donor status. linear regression analysis tested for differences in natural log of enrollment ferritin values by age. multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors. results/finding: a total of 4265 donors contributed 6219 donations. donors were evenly split by gender, 66% were ft donors, and 87% were 16-18yo. ft and rpt 16-18yo donors had on average lower ferritin values at enrollment (p<.0001), and a greater percentage were iron-depleted than donors 19-49yo (table) . in repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both ais and lf. controlling for these covariates, donors aged 16-18 have sharply higher risk for iron depletion than donors 19-49yo. odds for lf were 4 to 6 times greater in the younger donors, and for ais were 3-to 4fold higher. preliminary statistical models indicate 16yo donors may have greater risk for lf than 17 or 18yo by 4 to 5 percentage points, controlling for other factors (p5.06). conclusion: the prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in 16-18yo donors than in adult controls. logistic regression analysis confirms lower age as an independent risk factor for iron depletion. blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors. mitigation of iron deficiency in young donors -a preliminary report ralph r vassallo*, marjorie d bravo, mary townsend and hany kamel. blood systems, inc. background/case studies: iron deficiency is observed in blood donors who meet regulatory hemoglobin (hb) requirements for blood donation. frequent donations result in negative iron balance and eventually lead to anemia. young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs. study design/method: serum ferritin testing was implemented on 12/19/ 2016 by a large blood collector. testing was performed on successful 16-18 y/o whole blood and apheresis donations. low ferritin (lf) was defined as a value < 20 ng/ml in females (f) and < 30 ng/ml in males (m). donors with low ferritin were notified of deferral from red blood cell (rbc) donations (12 months for f and 6 months for m) and counseled to take 18-28 mg of elemental iron daily for 60 days. for m and f, a ferritin < 12 ng/ml indicated absent iron stores (ais) and < 26 ng/ml indicated iron deficient erythropoiesis (ide). ferritin levels ! 20 ng/ml in f and ! 30 ng/ml in m were considered as indicating an iron-replete state. conclusion: ferritin testing of young donors identified individuals with lf who would benefit from risk mitigation, e.g., delaying subsequent rbc donations and/or taking iron supplements. lf is more common in f than in m donors. lf is more prevalent in m and f donors with any rbc donations in the prior 24 months. an appreciable number of donors with no rbc donations in the prior 24 months presented with lf. these data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for m than for f, e.g., universal iron replacement in teen male donors may not be warranted above a certain hb value. ferritin blood screening in minor or young adult donors jennifer l ritter* 1 , joan williams 1 , michelle humphries 1 , nancy haubert 1 , ben reynolds 1 , michael phillips 1 , randall spizman 1 , ralph r vassallo 2 , hany kamel 2 , sally caglioti 1 , german leparc 1,3 and phillip c williamson 1 . abstract completely investigated. the adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors. 1 new studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors. 2 study design/methods: over 28,000 serum samples from donors aged 16, 17 and 18 years were analyzed for ferritin levels using the beckman coulter au680 instrument and reagent kit. the anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. immune complexes formed in solution scatter light in proportion to their size, shape and concentration. the decrease in light intensity is measured spectrophotometrically. 3 results/findings: background/case studies: the risk of cardiovascular (cv) disease in adults can often be identified during adolescent years. the presence of even borderline levels of multiple risk factors increases the likelihood of a cv event. our blood program routinely provides a total non-fasting cholesterol (tc) and blood pressure (bp) measurement for all blood donors. we added glycated hemoglobin (hba1c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among 21,007 adolescents (ages 16-19; 61.5% female) who donated blood from 2015 to 2016. study design/method: abnormal risk factor levels were defined as hba1c ! 5.7%, sbp/dbp ! 120/80 mm hg and tc !170mg/dl, as suggested by the american heart association for adolescents. the presence of isolated risk factors was defined as one single abnormal risk factor per individual. clustering of risk factors was defined as the presence of 2 or more abnormal risk factors in the same individual. donor sex was recorded at the time of donation. results/finding: table 1 shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort. overall, 11,283 (53.7%) adolescents had at least one abnormal risk factor (61.8% of males, 48.6% of females). of these, 8,709 adolescents had isolated abnormal risk factors, and 2,574 adolescents had clustering risk factors. higher proportions of males were in the abnormal bp alone, background/case studies: pre-donation determination of hemoglobin (hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality. however, a variety of hb testing strategies are used across blood services to satisfy this selection criterion. this study aimed to identify how hb screening practices vary across blood donation services and to what extent they influence deferral rates for low hb. study design/method: an online survey was performed among members of the biomedical excellence for safer transfusion (best) collaborative. additionally, data from literature were used to extend the dataset. the survey involved a detailed assessment of hb screening practices, numbers of donations and low hb deferrals for male and female donors separately. multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, hb cutoffs (high/low with high defined as !13.5 g/dl for men and !12.5 g/dl for women), iron monitoring (y/n), iron supplements (y/n providing or prescribing), and geographical location on deferral rates due to low hb. results/finding: data were included from 52 blood services worldwide and complete data were available for 25 blood services. deferral percentages for low hb varied from 0.01% to 8.81% among male donors and 0.03% to 46.73% among female donors. hb deferral rates were notably higher in asian blood services. overall, iron monitoring was associated with 53% lower hb deferral rates in men (95% confidence interval [ci] 11% to 75%) and 61% lower rates in women (95%ci 15% to 82%). iron supplementation was associated to 57% lower hb deferral rates among women (95%ci 22% to 76%) but there was no evidence of such an effect among men (p50.680). each one-week increase in minimum donation intervals resulted in 8% lower hb deferral rates among women (95%ci 1% to 14%) but not among men (p50.454). at the 5% level of significance, higher hb cutoffs do not appear to have an effect among men or women. conclusion: the variation in hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in hb screening and deferral practices. mitigation strategies should consider the variable response among men and women. these insights can help improve both blood service efficiency and donor care. were: characteristics of donors (age, sex, size, weight, region); hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the 5 previous years and the lifetime). data were analyzed using logistic regression stratified by sex. results/finding: 9.15% of all candidates for wb donation were deferred in continental france in 2015. deferral was significantly more frequent in women (11.16%) than in men (7.29%), due to anemia in 24.41% of deferred women and 9.79% of deferred men. plotting mean hb recovery against time showed mean recovery times ranging from 20 to 30 weeks. analysis (table) identified 3 main factors associated with a higher likelihood of hb recovery: higher logarithm of time since previous donation, lower levels of hb at previous donation, higher number of blood donations in the 5 previous years. conclusion: the 3 main factors associated with higher likelihood of hb recovery after wb donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors. mean times required for hb recovery were long enough to require further studies to assess interdonation intervals in france. background/case studies: red blood cell (rbc) transfusion has been related to thrombo-embolic events. microvesicles in the rbc product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration. study design/method: we investigated whether transfusion of rbcs containing microvesicles promotes coagulation in human recipients. as transfusion is mostly administered to ill patients, we used a model of mild endotoxemia. eighteen healthy volunteers were randomized to receive either saline, 2 days stored or 35 days stored autologous rbc transfusion two hours after infusion of lipopolysaccharide (lps, from e.coli, 2 ng/kg). blood was sampled every 2 hours up to 8 hours after lps infusion. results/finding: lps resulted in a mild increase in thrombin generation. during storage, the total number of microvesicles increased from 1.4e108 (iqr 8.3e107-1.9e108) /ml in the fresh product to 1.7e110 (iqr 7.9e109-2.3e110/ml; p<0.01) in the stored product (p <0.001), which were mostly rbc derived vesicles. after transfusion, microvesicles from stored rbc products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within 6 hours. however, infusion of stored rbc microvesicles did not augment thrombin generation. levels of d-dimer and thrombin-antithrombin complex were also unaffected. conclusion: transfusion of autologous rbcs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia. background/case studies: transfusion-associated circulatory overload (taco) is characterized by hydrostatic pulmonary edema related to blood transfusion. we sought to examine contemporary risk factors and outcomes for taco during a period where patient blood manaement has led to declines in blood utilization. study design/methods: at four academic hospitals, cases of taco were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity. taco incidence was calculated, and clinical characteristics were compared with control patients. odds ratios (or) were calculated using multivariable logistic regression. hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression. results/findings: 200 cases of taco and 405 matched controls were enrolled from 20,845 transfused patients who received 128,263 blood components from may 2015 until july 2016. taco incidence was 1 case per 100 patients transfused. in addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of taco: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (see table) . compared to controls, taco cases were more likely to require mechanical ventilation (71% vs. 49%; p < 0.001), experienced longer intensive care (4 vs. 3 days; p50.04) and hospital length of stay following transfusion (10 vs. 7 days; p< 0.001), and had higher mortality (21% vs. 11%; p50.02). conclusion: the incidence of taco was lower than what has been reported by prior active surveillance studies. despite declines in its incidence and the number of blood components transfused per case, taco remains a complication of transfusion with significant associated morbidity and mortality. in addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with taco after controlling for other covariates in the model. additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for taco. background/case studies: the residual risk of bacterial contamination of single-donor apheresis platelets (ap) was recently addressed by the march 2016 fda draft guidance to enhance the safety of platelet transfusion. this document also describes an existing pathway for ap outdate extension from 5 to 7 days using an fda cleared rapid test (rt). our hospital based transfusion service has used this rt to enhance the safety of ap transfusion since july 2008 and to routinely extend ap outdate to day 7 since february 2016. this study reports a 103 month experience of secondary screening of ap using a rt. study design/methods: all ap were obtained from our hospital-based donor center or one of four external suppliers. ap were screened by culture based methods post-collection and prior to entry into our inventory. from july 2008-january 2016, ap underwent rt on day 4. day 6 and 7 units were transfused with physician approval when deemed medically necessary. any units remaining in inventory on day 8 had a second rt performed. from february 2016-january 2017, ap underwent rt on day 5 with routine outdate extension to 7 days by performing a second rt on day 6 and a third rt on day 7, as per manufacturer instructions. any positive rts were repeated in triplicate. repeat rt positive units were quarantined and cultured to identify true positives. false positives (fp) were defined as repeat rt negative (type 1) or repeat rt positive with negative confirmatory culture (type 2). all rt results were reviewed during both study periods. ap transfusion and outdate rates were also summarized. results/findings: since july 2008, 20,010 ap were entered into inventory. of these, 11,840 (59%) were transfused prior to rt testing. the remaining 8170 (41%) underwent rt on day 4 or day 5. of these 43 (0.5%) were rt positive (29 type 1 fp, returned to inventory; 14 type 2 fp, discarded), leaving a total available inventory of 8156 units tested by rt. of these, 5631 (28% of original inventory) were transfused before the end of day 5 and the remaining 2525 (13% of original inventory) reached a day 5 outdate. a total of 1561 (8% of original inventory) were transfused on day 6 or day 7. of these, 768 underwent a second rt on day 6 (2 rt positives; 1 fp type one and 1 fp type 2) and 233 underwent a third rt on day 7 (no positive results). a total of 964 (5% of original inventory) outdated on day 7. of these, 754 underwent a second rt on day 8 (no positive results). conclusion: to date we have performed 9925 rts on ap at our hospital. no true positives have been identified. use of rt over the study period decreased our outdate rate from a predicted 13% to only 5%. a total of 1522 ap have been tested twice by rt (768 on day 5 and 6; 754 on day 4 and 8) with 2 (0.1%) positive results, both of which were deemed fp by repeat testing or culture. a total of 233 units have been tested 3 times (day 5, day 6 and day 7) with no additional positives identified. we have not yet identified any units with an initial negative rt result that subsequently converted to a true positive. there is a low fp rate which should also be expected when performing repeat testing on the same unit. these data suggest that the yield for repeating the rt every 24 hours, as currently specified by the manufacturer instructions, is quite low. additional studies are needed to clarify how rt can optimally be used to enhance detection of ap bacterial contamination. survival of trypanosoma cruzi in human blood components laura tonnetti*, aaron thorp and susan l stramer. american red cross background/case studies: trypanosoma cruzi, the agent of chagas disease, is associated with 8 to 10 million infections worldwide, mostly in latin america. despite the extensive immigration from endemic areas, only 5 cases of transfusion-transmission (tt) t. cruzi have been reported in the us, before blood donor screening was implemented in 2007. contributing factors to the low number of tt cases are a possible association between parasite lineage and tt, and high numbers of unreported cases. platelets are almost exclusively involved in t. cruzi tt cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (rbcs). we investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in tt of t. cruzi. study design/method: whole blood (wb) units were spiked with t. cruzi trypomastigotes to a final concentration between 10-10,000 parasites/ml. each parasite concentration in wb was tested x2. an aliquot of contaminated wb was used to prepare hemocultures to detect live parasites before preparation of components. rbcs were separated and half of the components leukoreduced (lr) by filtration. platelets and plasma were separated, along with one aliquot of plasma collected before lr. rbcs were stored at 48c for up to 42 days; platelets were stored at 228c (rt) under agitation for 5 days and plasma was frozen at -208c. aliquots for culture were removed weekly from rbcs, daily from platelets and after 30 days from frozen plasma. all samples were cultured in liver infusion tryptose (lit) media at 278c for detection of live parasites for up to 16 weeks. results/finding: hemocultures from spiked-wb were positive at all concentration of parasites. lr'd and non-lr'd rbcs cultured before storage were positive at all concentrations. after storage at 48c, rbcs from all units spiked with 10,000 parasites/ml were positive for up to 21 days; all further times yielded negative results. at lower concentrations, only non-lr'd rbcs spiked with 1000 parasites/ml were positive for up to 7 days. plasma samples cultured before freezing were positive at the highest concentration in one non-lr'd sample, while all others were negative. platelets obtained from wb spiked with 10,000 and 1000 parasites/ml were positive up to 5 days at rt. no parasites were observed in plasma or platelets prior to storage at lower concentrations. molecular analysis to determine the presence of parasite dna in each component is on-going. conclusion: platelet storage conditions offer a suitable environment for t. cruzi survival; however, high concentrations of parasites also survived in rbcs at 48c for up to 3 weeks. leukoreduction offers partial protection, while freezing conditions appears unsuitable for t. cruzi survival. hemovigilance monitoring of platelet septic transfusion reactions (str) after treatment with intercept tm pathogen reduction or large volume, delayed bact/alert tm bacterial culture screening richard benjamin* 1 , marion lanteri 2 and larry corash 1 . 1 cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: amotosalen/ultraviolet a (uva) light (inter-cept tm blood system, cerus corporation) pathogen reduction (pr) and delayed, large volume, bacterial culture with the bact/alert tm system (dlvbc) (biomerieux, inc) represent respective best-in-class systems to reduce the risk of str associated with platelet concentrates (pc). where implemented, hemoviligance (hv) programs continue to receive reports of suspected str, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure. study design/methods: united kingdom (2006 -2015 ), french (2006 -2015 , swiss (2011 -2015 ), and belgium(2009 -2015 hv reports, and cerus corporation's adverse event records were reviewed to assess the residual risk and imputability of str with amotosalen/uva-treated or dlvbc-screened pc. results/findings: approximately 1.35 million dlvbc-screened were issued with a 7 day outdate after release into inventory 3 days after collection, and $2.3 million amotosalen/uva-treated pc were released into inventory on day 1 or 2, with a 5 to 7 day shelf-life. no septic fatalities were reported with either technology. the french, belgium and swiss hv programs monitored >2.83 million conventional, non-dlvbc-screened pc and recorded 58 str and 9 fatalities. concurrently, zero definite and 2 possible str were reported with 607,871 amotosalen/uva-treated pc, significantly fewer than with conventional pc (table 1 ) (20.5 str per million vs. 0.0 per million, p<0.001). one definite, 1 possible, 7 undetermined/indeterminate non-fatal str and 5 contaminated "near miss" pc were reported with 1.35 million dlvbc-screened pc between 2010 and 2015, for a reduced falsenegative rate compared with the prior five years (3.7 str per million vs. 16.3 per million, p <0.05). hv programs highlight a major weakness when reporting str. stringent criteria are used to determine definite imputability, including evidence of patient infection, pc contamination and irrefutable evidence of a donor source, with confirmation of strain identity. reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis. some of these cases are almost certainly due to bacterial contamination of pc, suggesting that the actual rates of sepsis are considerably higher than that reported by hv programs. conclusion: best-in-class pathogen reduction and bacterial culture systems reduce str risk, although underreporting and inadequate clinical data may result in underestimation of the true rates. pathogen reduction of background/case studies: despite extant mitigation measures (e.g. diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the united states (us). consequently, the us food and drug administration has recommended adoption of additional measures such as point of release testing (port) and/or pathogen reduction to safeguard against transfusionassociated sepsis. however, port poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention. we evaluated a second bacterial culture to contend with residual risk. study design/method: phased implementation of secondary bacterial culture testing (bact/alert tm ,biomerieux, inc., durham, nc) was initiated in october 2016 for all platelets received at our institution. at time of receipt at the blood bank (day 3 post collection), products were sampled using a sterile connection device (tscd tm , terumo, elkton, md) and a sampling kit (sam-plok tm sampling kit, 10 ml, itl biomedical, malaysia). five mls of product was transferred aseptically to bact/alert bpa (aerobic) culture bottles using the same sampling device. inoculated culture bottles were loaded into the bact/alert incubator modules and incubated at 35c for three days. results/finding: a total of 9473/11,066 (85.6%) platelet products were successfully cultured (934/1373 [68.03%] and 1842/1912 [96.3%] in october 2016 and march 2017 respectively). over the 6-month period, two true positive cultures were obtained (incidence of 1 in 4736 platelet products). the cultures grew acinetobacter species (case a) and coagulase negative staphylococcus species (case b); both positive results were obtained four days following collection. repeat testing of cases a and b grew the same organisms identified in the initial cultures. there was a co-component in our inventory (case a) with negative initial and repeat cultures. none of the products were released for transfusion. the initial post-collection product cultures remained negative at the collection facility. over the same time period, no false positives were detected. implementation required hiring one additional dedicated fte; the total cost (technologist time, equipment and related supplies) was calculated to be $us16.83 per product tested. the cost per averted case was $us79,707. conclusion: we demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions. this presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions. importantly, it offers a viable alternative to port in high volume institutions where logistic (e.g. time and personnel) constraints impede practical adoption of port. an increase in cases of blood culture positive transfusion reactions (bcptr) was noted at our hospital; bcptr was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction. we sought to characterize the risk and clinical presentation of bcptr at our institution. study design/method: an analysis was conducted of all reported transfusion reactions at johns hopkins hospital (jhh) between january 2009 and december 2016. the data, extracted from hemovigilance records, were evaluated to determine the incidence of bcptr; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction. bacterial culture results were evaluated for both patients and associated blood products (i.e. partially transfused or residual product in blood bag). results/finding: in the 7-year study period, a total of 3280 transfusions reactions were reported, 18 of which were bcptr (0.55% of transfusion reactions). of the 18 bcptr, 15 (83%) were associated with apheresis platelets, 2 (11%) with red blood cells, and 1 (6%) with plasma. recipient diagnoses spanned hematologic/oncology (n512), renal (n53), cardiac (n51), autoimmune (n51), and obstetrics (n51). an organism was identified in both the blood product and recipient in 10 (56%) cases; in 6 (33%) cases an organism was grown in the blood product but not the recipient; and in 2 (11%) cases an organism was isolated from the recipient only, due to inability to culture the product. the transfusion recipients in 5 of the 6 cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion. symptoms of bcptrs included fever (83%), chills (67%), nausea and vomiting (50%), pain (27%) and dyspnea (22%). blood pressure (bp) decreased in 22%, increased in 17%; 61% of reported bcptrs had no change in bp. conclusion: the signs and symptoms of bcptrs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting. furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions. hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions. furthermore, excessively stringent criteria (cdc/nhsn blood safety surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics. clinical oral abstract session: immonohematology and genetics --sickle cell disease and beyond blindspots and cross-reactivities of anti-human globulin specific for igg subtypes heather howie 1 , jenna lebedev 1 , linda kapp 1 , xiaohong wang 1 , meghan delaney 2 , lay see er 1 and james c zimring* 3 . 1 bloodworksnw research institute, 2 bloodworks nw, 3 university of washington school of medicine background/case studies: there are four different subclasses of human igg (igg1-igg4), each with different effector function. essentially all existing data on the effect of igg subclass on hemolytic transfusion reactions and hdfn, were generated using ahg specific for igg subclasses. in recent decades, it has become appreciated that there are at least 29 natural human variants of igg. in this study, the reactivity of igg specific ahg was tested against all 29 known variants. study design/methods: the heavy and light chain variable regions of an anti-k1 monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known 29 igg variants. plasmids were expressed by co-transfection into cho cells. the resulting panel of antibodies were pre-incubated with k11 rbcs and were then subjected to testing with currently available igg subtype specific ahg (monoclonal ahgs from southern biotech and sanquin, polyclonal ahgs from sanquin and the bindingsite). all testing was carried out by flow cytometry. results/findings: polyclonal reagents against igg2, igg3, and igg4 had cross-reactivity with variants found in other igg subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table 1). titrations of the ahgs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected. however, cross-reactivity could be neutralized by pre-incubating ahg with the crossrecognized igg forms (against a third party antigen); the remaining reactivity recognized the intended igg subtype without detectable cross-reactivity. no cross-reactivity was detected for polyclonal anti-igg1 or for any of the monoclonal ahgs tested. monoclonal anti-igg3 had a blindspot for igg3-04, due to the shorter hinge region on igg3-04. no blindspots were detected in other monoclonal or polyclonal ahg. conclusion: the relative quantitation of different igg subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology. herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots. as such, the existing data regarding igg subtype biology may have some inaccuracies as a result of these defects in igg specific ahg. genotype matching for pediatric sickle cell disease patients nancy robitaille* 1 , yves dominique pastore 1 and maryse st-louis 2 . 1 chu sainte-justine, 2 hema-quebec background/case studies: among the different treatment modalities available for sickle cell disease (scd), blood transfusion is frequently used. however, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for c, e and kell antigens. this is partly explained by different antigen frequency among caucasian blood donors and african-american recipients, and by variants in the rh blood group of people of african-descent. blood group genotyping has been proposed as a potential way to alleviate this problem. the scd cohort of a pediatric academic hospital was genotyped for rhd, rhce and fy genes. the primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar rh variants could be identified. study design/methods: since 2008, our local blood provider intensified recruitment of african-descent blood donors. these donors were phenotyped and genotyped for clinically relevant antigens by different means: genomelab snp stream, laboratory-developped assays and idcorext. as of 2014, 205 scd children were genotyped by sequencing rhd, rhce and fy cdnas after obtaining informed consent. extended red blood cell phenotypes were done at diagnosis at the hospital. patients' genotypes were compared to h ema-qu ebec's donor database to attribute blood donors to specific patients. results/findings: from diagnosis until september 2016, 117 (57%) patients had been transfused and 14 had antibodies with known blood group antigen specificity: anti-c, anti-e (2), anti-hrb, anti-fya, anti-jka, anti-jkb (2), anti-s, anti-m, anti-sc2, anti-leb (2). seventeen patients (8.3%) were either d2 or partial d. rhce results showed that 163 patients expressed a normal c antigen and 32 expressed partial c. as for e antigen, 163 had a normal antigen, 38 bore a partial antigen and 3 were weakly expressed. fy(a2b2) phenotype was found in 182 (89%) patients. a total of 2606 genotyped blood donors of african-descent were available. the table below indicates the compatibility with these donors. conclusion: this study shows that several patients have rhce variants difficult to match, even with available genotyped blood donors from their community. although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs. the continued effort put towards recruitment and pheno/genotyping should improve the situation. using genetic markers to select responders and non-responders sickle cell disease (scd) patients for transfusion with rh haplotype matching red blood cell (rbc) units tamires delfino dos santos 1 , emilia sippert 1 , mayra dorigan de macedo 1 , sheila fatima perecin menegati 1 and lilian castilho* 1,2 . 1 hemocentro unicamp, 2 university of campinas background/case studies: rbc alloimmunization has been associated with several factors and with individual characteristics of each patient. we recently found that tnfa-308a, il1b-511t cytokine polymorphisms, rhag 808g>a and hla-drb1*15 alleles may predict a good responder phenotype (sippert et al, transfusion 2017) and that rhag 808a and hla-drb*15 alleles are closely linked to rh alloimmunization. based on this and considering the challenge to fulfill the transfusion needs of the patients with rh variants, we used these genetic markers to select responders and nonresponders scd patients for transfusion with rh haplotype matching rbc units and evaluated the risk of alloimmunization. study design/method: our study included 96 non-alloimmunized patients with scd, homozygous for hbs, receiving a range of 5-289 rbc units. rbc antigen phenotypes of each patient and history of rbc antibodies were obtained from the medical records and transfusion service computerized database. rbc genotyping was performed using whea, wrhd and wrhce beadchip arrays (bioarray solutions, immucor) in accordance with the manufacturer's instructions. cytokine gene polymorphisms (tnfa-308g>a, il1b-511c>t) and the rhag 808g>a gene polymorphism were analysed by pcr-rflp and taqman assays. hla class ii genotyping was performed using pcr-sso. results/finding: among 96 non-alloimmunized patients, 21 were homozygous or compound heterozygous for rh variant alleles. from those, 6 had rhag 808a and/or hla-drb*15 alleles and at least one cytokine polymorphism (tnfa-308a or ilb1-511t) associated with risk of alloimmunization and were transfused with extended and rh haplotype matching rbc units. the other 15 patients with no risk factors associated with rbc alloimmunization were considered non-responders and were not transfused with extended and rh matching units. all patients were followed for one year and did not develop rbc antibodies. conclusion: these findings contributed to the development of a transfusion strategy for non-alloimmunized scd patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder scd patients, allowing blood with high level of compatibility to be five discrepant samples required sequencing. id core xt identified three rhce*cear samples encoding a partial c, and a partial e (predicted phenotype: vweak, vs-) and 2 were confirmed by sequencing. the third sample was found to be rhce*cevs.01,rhce*cebi on sequencing (predicted phenotype v1,vs1). the 3 samples were typed as v1 (or ce s ) and vs1 (or e s ) by hea. in addition, id core xt accurately identified rhce*ce[712g]in 2 samples. this snp has been linked to various allelic variants affecting c and e antigenic expression. both samples were predicted to be c1 by hea. conclusion: blood group genotyping platforms vary depending on the specific snps that are included in each assay. such variations may be clinically significant when genotyping is used as a tool for providing matched blood. discrepancies leading to differences in the predicted phenotype could affect unit selection. despite the discrepancies between the 2 methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes. background/case studies: over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (waiha) on the basis of dat scores of agefractionated rbcs: type i waiha, comprising 80% of patients, showed increased binding of autoantibodies to aged rbc, whereas type ii waiha autoantibodies (20% of patients) bound young and old rbcs with no apparent prejudice. band-3 is a ubiquitously expressed rbc transmembrane protein which plays a vital role in maintenance of rbc structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with waiha. band-3 is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal rbc senescence, which causes band-3 to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band-3 aggregates forming new epitopes which are recognized by natural igg autoantibodies causing phagocytosis and destruction of senescent rbcs. type i waiha has been postulated to be caused by an exacerbation of normal rbc senescence. study design/methods: in an effort to confirm and characterize the two waiha subtypes we age-fractionated whole blood samples from 22 patients with waiha on discontinuous percollv r gradients and looked for differences in dat results between less (young rbcs) and more dense (aged rbcs) fractions, fractionation patterns and band-3 tyrosine phosphorylation. results/findings: we confirm that two distinct types of waiha can be identified based on autoantibody reactivity with the youngest and oldest autologous rbcs. further, comparing 5 type i and 5 type ii patients, we found that type i is characterized by 5 percollv r fractions (similar to healthy storage-matched controls) but increased band-3 tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of rbc development. type ii patients were characterized by 3-4 percollv r fractions, lacking the fraction containing the oldest rbcs, and showed a complete lack of, or dramatic decrease in, band-3 tyrosine phosphorylation compared to healthy storage-matched controls. conclusion: these results confirm the two distinct types of waiha. in type i waiha, the increased binding of autoantibodies to older rbcs coupled with increased tyrosine phosphorylation of band-3 suggests that rbcs from type i patients are aging faster than rbcs from normal healthy controls; this may represent an accelerated and pathogenic form of normal rbc senescence. in contrast, type ii waiha where autoantibodies bind strongly to either young or old rbcs coupled with a lack of fractionated bands that represent the oldest rbcs and a dramatic diminution in tyrosine phosphorylation of band 3 suggests faster destruction of rbcs, consistent with the early published data, and metabolic changes that could affect rbc function. microbial pathogen primary sequence correlates with blood group antigen immunogenicity ian baine* 1 , burak bahar 1 , jeanne hendrickson 2 , krystalyn e hudson 3 and christopher a tormey 1 . 1 yale-new haven hospital, 2 yale university, 3 background/case studies: it is known that specific groups of patients immunologically respond more readily than others to rbc antigens. while rbc antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved. studies have shown that there is significant primary sequence identity between common rbc antigens and microbes, and that cross-reactivity is possible between antigens in experimental models. we hypothesize that responder populations may be immunologically primed to form rbc alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity. study design/method: we performed peptide homology searches of the most immunogenic rbc antigens, based on previously published antigenicity findings. thirteen amino acid peptides containing the polymorphic residues of k, jk a , lu a , e, c, m, c, fy a , and s antigens were queried for identity with microbial peptides using the blast database (blastp, pam30 abstract algorithm, e value51x10 -6 , word size5 6, gap costs: existence59 exten-sion51). search results were restricted to bacteria and fungi, with a selective threshold of >80% identity set for inclusion criteria. to corroborate with observed patient data, we also examined preceding cultures from 162 alloimmunized patients to explore agreement between specific pathogens and rbc alloantibodies. results/finding: significant peptide identity was found between rbc antigens and pathogenic organisms including b. fragilis, p. aeruginosa, candida spp. among others. linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when fy a (an outlier) was removed (b5-0.0017, r 2 50.624 & p50.0197); that is, lower immunogenicity antigens were associated with larger number of kingdoms. k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (c), moderate (e, c, s, m) and high (k, jk a , lu a , fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence. of 162 alloimmunized patients reviewed, 105 were culture-positive. of these, 76% of the anti-c/c group (13 of 17 patients) and 16% of the anti-k group (7 of 43 patients) had microbe-antibody agreement. remaining microbe-rbc antibody agreements ranged from 0 -11.1%. overall, 21.9% (23 of 105 patients) demonstrated agreement. interestingly, we observed a particularly strong agreement between infection with klebsiella species and anti-k, despite the lack of >80% sequence identity. while 27.6% (29 of 105) patients reviewed had positive cultures for klebsiella species, 62.1% of these (18 of 29 patients) demonstrated an anti-k. conclusion: our study highlights the potential connection between microbial infection and rbc alloimmunization, based on shared epitopes. we speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between rbc antigen immunogenicity and prevalence of microorganisms. longitudinal studies of microbial carriage (or acute microbial infection) and rbc alloimmune responses in larger patient cohorts may be informative. background/case studies: thromboelastogram (teg) has been incorporated into many hospital armories to manage transfusions during cardiovascular (cv) surgeries. some institutions use well-defined protocols for teg utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative). on the other hand, at some institutions teg utilization is driven mainly by clinical judgment. when teg is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before teg is performed. there is no published literature on how pre-teg transfusions impact teg results and guide further transfusion requirements during cv surgeries. in this study, we have tried to address this issue. study design/method: we retrospectively reviewed 109 tegs performed on 76 patients undergoing cv surgeries at our institution from jan 1 to dec 31, 2016. no specific teg protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period. only the first teg performed during surgery was included in the analysis. we excluded the patients that received only red blood cell (rbc) transfusions during the surgery because rbc transfusions are usually not based on teg results. for the 56 tegs analyzed, teg results were divided into three categories: "normal" (reaction time (r), kinetics (k), angle (a), maximum amplitude (ma), and lysis at 30 minutes (min) all within reference range), "hypocoagulable" (r>10 min, k>3 min, a<53 degrees, ma<50 mm) and "hypercoagulable" (r<5 min, k<1 min, a>72 degrees, ma>70 mm). fisher's exact tests and z-scores for two population proportions were used to identify statistically significant differences in teg results and blood product utilization. results/finding: out of 56 tegs analyzed, 37 patients (66%) received pre-teg transfusions. we found significantly fewer hypocoagulable teg results in pre-teg transfused patients than nontransfused patients (8% vs. 32%, p50.02). the data also reflected a trend suggesting that there may be more normal teg results in pre-teg transfused patients compared with nontransfused (32% vs. 11%, p50.07). there was no statistically significant difference in transfusions after obtaining teg results in both groups. however, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-teg compared to nontransfused (100% versus 33%, p50.06). conclusion: pre-teg transfusions impact teg results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during cv surgeries. the decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-teg transfusions may be due to more clinical significant bleeding in these patients to begin with. background/case studies: orthotopic liver transplantation (olt) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion. in this setting, cell salvage autotransfusion (cs) is been used as an alternative to decrease allogeneic red blood cell transfusion. however, as long as some studies have shown that cs in olt decreases allogeneic blood transfusion, others reported that cs presented little benefit or might have been associated with increased blood loss through fibrinolysis. in this study, we evaluate cs efficacy in reducing allogeneic blood transfusion in the intraoperative period. study design/method: we retrospectively evaluated data from 670 liver transplants, performed from 2011 to 2015 in a single-center. patients were divided in two groups: one with cell salvage (cs) and another without cs (ncs). study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups. cs was used in all liver transplant recipients but patients with malignancy and sepsis. blood transfusions were indicated based on clinical and hemodynamic criteria. clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and model for end-stage liver disease (meld) score. statistical analyses were performed using t-test, chi-square test, mann whitney test. results/finding: in this study period, 670 olts were performed. a total of 345 patients was submitted to cs. the median age was 51 years (range 10-78 yo). cirrhosis caused by chronic hepatitis c virus infection was the main etiology of liver disease. hepatocellular carcinoma (hcc) was found in 31,6% of the patients. the average meld score was 29,6 6 9,4 and it was slightly higher in the cs group (31,3 vs 27,9, p<0,001) . there was no statistically significant difference in other variables such as body weight, height and cold ischemic time. the mean salvaged blood volume was 8856 6 4503 ml and mean reinfused blood volume was 914 6909 ml. allogeneic blood transfusion was required in 71,8% patients in the cs group, compared to 46,7% patients in the ncs group. however, average red blood cells (rbc) and fresh frozen plasma (ffp) units transfused were lower in the cs group. the threshold for rbc transfusion was significantly lower in the cs group (2,4 units vs 3,39 units, p<0,001 background/case studies: hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsaddin en.cite.data. massive transfusion protocols (mtp) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geaddin en.cite.data. blood product wastage benchmarks are loosely established, and data on wastage associated with mtps especially sparse. with a redesign of mtp and obstetric massive transfusion protocols (obp) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols. study design/method: following institutional review board approval, a retrospective study on blood product wastage associated with the mtp and obp between july 2015-december 2016 was performed. data on numbers of products dispensed and wasted were manually collected from transfusion service paper and electronic records and an automated data report from the electronic medical record. results/finding: the mtp resulted in higher total number of wasted products than the obp (27 and 15 products, respectively) however, obp wastage occurred more frequently in the 18 month period. this reflects automatic thawing of cryoprecipitate in the first round of deployed products in the opb. mtp-trauma activations contributed higher wastage than non-trauma activations (23 versus 4 products). this is skewed by one month when 20 products were wasted due to expiration of product on the floor. cooler-related issues (6) and products dwelling too long out of a controlled environment (5) were common reasons reported for wastage. the overall product wastage rates for mtp: trauma, mtp: nontrauma, and obp were 1.7%, 0.3%, and 2.3%, respectively, with a total exsanguination protocol waste rate of 1.33%. the difference between the overall proportion of waste between the mtp and obp protocols was insignificant (p50.176). conclusion: wastage associated with both protocols was low and there is no statistical difference between mtp versus obp wastage. coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods. better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols. a 17 year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group o, rh negative (d-) red blood cells (rbcs) through a rapid infuser during resuscitation. transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample. the current fda guidance and aabb standard of two samples for determination of blood type to prevent cases wrong blood in tube (wbit) or electronic identification systems do not always catch or clarify these errors. study design/methods: patient was tested by manual tube method. two different technologists using two different reagent racks performed initial testing with matching results. results/findings: two samples were collected during resuscitation from the patient and typed as o d-. patient was transfused with 5 units of o d-rbcs before stabilizing. two days later another sample was collected and typed as o rh positive (d1) with mixed field being seen on the anti-d. a weak d testing was performed to see if the negative result with anti-d could be strengthened through incubation. both original samples still resulted as d(table a) . after consulting the patient care team it was discovered the samples were collected above the iv site after one unit had been completed and while the second unit was being transfused. it was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate. the transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected. conclusion: the initial samples were collected above the iv site and were contaminated with the d-blood product being rapidly transfused during resuscitation. the samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the iv site. because both samples were collected while the unit was being transfused, contamination was in both. use of a handheld barcode system would not have caught this error because the patient had been correctly identified. future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen. facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site. impact of cell saver usage during solid organ transplants at a major institution holly ross* 1 , edward smith 2 , thomas brown 2 , foeks jeremy 2 , metcalf suzanne 2 , james johnson 2 , peter davis 2 , karafa sw badjie 1 and abba zubair 1 . 1 department of laboratory medicine and pathology, transfusion medicine, mayo clinic, 2 department of anesthesia, mayo clinic background/case studies: our institution performs an average of 398 solid organ transplants (sots) yearly. transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (rbc) transfusions annually. even the best practices for allogeneic transfusion are not without risk. transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization. the advent of intraoperative blood recovery has reduced the need for allogeneic donor rbcs during surgeries expected to bleed heavily. with the cell saverv r (haemoneticsv r , braintree, ma), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support. this study sought to examine the amount of allogeneic donor rbc units saved during sots through the use of the cell saver for intraoperative blood recovery. study design/methods: data was collected for sots which utilized the cell saver. these included liver, liver/kidney combination, lung, and heart transplants. data a 29 y.o. female was admitted to the trauma department after a motor vehicle collision (mvc) and transfused 2 o(1) rbc units from the kiosk. her blood type was determined as o(-) with a negative rbc antibody screen (as). she was transfused 10 more units of o(-) rbc. two months later, a repeat as identified two new rbc alloantibodies, anti-d and anti-e. the anti-d formation resulted from the 2 o(1) rbc transfused from the kiosk, but the source of the anti-e was undetermined since e antigen is expressed in 1% of rh(-) individuals. the trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a 29 y.o. female patient should not have received o(1) rbcs. study design/method: an investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (ems). results/finding: the trauma unit was busy with 10 admissions during the 5 hours preceding the patient's arrival. the chart review found the following physical attributes; patient was overweight (107 kg) with obvious facial deformities from the mvc, that compromised age assessment. it was determined that the kiosk was fully stocked with both o(-) and o(1) rbc units. one clinical provider recalls that the patient identification (id) might have been unknown. review of the ems communication states "patient is a 50 y.o. female." conclusion: use of visual examination to determine age was significant in the selection of o(1) rbc for this patient. the trauma staff proposed and implemented a change in policy to prevent future incidents. any female patient that arrives without id or written confirmation of age will be transfused o(-) uncrossmatched rbc until a blood type can be determined. after being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution. this is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched rbc transfusions. rate of abo/rh confirmation in outpatient pelvic organ prolapse surgery alexis r peedin*, taylor brueseke, yara park and jay s raval. university of north carolina background/case studies: approximately 375,000 surgeries for urinary incontinence or pelvic organ prolapse (pop) are performed annually. for abdominal pelvic floor disorder (pfd) surgeries, transfusion rates historically range from 6-16%, whereas transfusion rates for vaginal and robotic pfd surgeries range from 0.2-1.6% and 0.3-1.4%, respectively. since the implementation of college of american pathologists (cap) requirements for abo/ rh confirmation, approximately 15% of patients who receive a transfusion in our hospital required a second abo/rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative type & screen (t&s). the primary objective of our study was to assess the rate of abo/rh confirmation in women who underwent outpatient pop surgery. study design/method: this was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing pop surgical repair from may 2015 -may 2016 in our academic tertiary care institution. among this sample, patients were excluded if their first t&s was drawn before our institution implemented the abo/rh confirmation requirement. fisher's exact test was used, and statistical significance was defined as p<0.05. results/finding: we identified 66 patients for analysis, of whom 65 (98.5%) had a preoperative t&s ordered. two (3.1%) of these 65 patients had positive antibody screens; one patient had an anti-k and one had a warm-reacting autoantibody. fifty-nine (90.8%) of the 65 patients required a second abo/rh specimen per hospital protocol; 51 (86.4%) of these actually had a second specimen drawn. in patients for whom abo/rh confirmation was indicated, there were no differences between those who did and did not have abo/rh confirmed when comparing age, body mass index (bmi), pre-operative hemoglobin (hgb), or surgical approach (table 1) . no abo/rh discrepancies were identified. one patient received 1 unit of red cells after abdominal pop surgery. conclusion: the rate of requiring abo/rh confirmation before pop surgery was markedly higher than that seen in all patients receiving transfusions at our institution (90.8% vs. 15%, respectively). because the vast majority of women undergoing vaginal or robotic pop surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative t&s for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent abo/rh confirmation. volume reduction of red cells to reduce transfusion-associated adverse events related to hyperkalemia maressa t pollen*, laura knicks, linda van tol and c. michael knudson. background/case studies: one attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia. this problem is exacerbated in conditions of massive transfusion and in patients with renal failure. washing rbcs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time. here, we estimate the amount of potassium that is removed by volume reduction of red cell units. we also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia. study design/method: expired or over temperature units (n527) that had been removed from inventory were utilized for these studies. each unit was weighed and a volume reduction procedure was performed. the supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays. for all formulas, weight was converted to volume using a specific gravity of 1.05 g/ml. the hematocrit (hct) of the volume reduced rbc was measured using a sysmex xs-1000i instrument. the percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (rbc hct x rbc volume) by the total supernatant prior to the procedure (residual supernatant 1 removed supernatant). the remaining free potassium (meq) was calculated as the (concentration of potassium in the supernatant (mmol/l) x the estimated red blood cell residual supernatant volume. to simulate the process that would occur in the setting of a massive transfusion protocol (mtp), 5 units were subjected to the volume reduction while recording the time needed to process all 5 units. this was performed twice for a total of 10 units processed in this manner. results/finding: the volume reduction procedure reduced the supernatant volume by an average of 72% (range 49%-87%). in units between 21 and 42 days (n510), the estimated mean residual k1 was 1.89 meq (range 0.61 to 2.21). in the two mock mtp trials, the time to complete the procedure was approximately 50 minutes and we estimate an additional 5-10 minutes would be required to modify and issue the units in our lis/emr. conclusion: a manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells. this procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia. the procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion. processing, cryopreservation, and non-specialized hospital collection. preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (ns) alone, ns plus antibiotic/antimycotic (ab/am) and a dry container. prolonged exposure to ab/am solution retarded outgrowth of mscs, but control of microbial growth in cultured tissue samples was needed. these findings were used to construct a validation study. study design/methods: a validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth. collected uc tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal (3) and caesarian (2) births. uc collections were divided into 3 segments to test 3 conditions. segment explants were placed on 0.1% gelatin-coated gridded tissue culture plates (32 explants per plate) in enriched medium specified for msc outgrowth containing antibiotic only with an endpoint of 21 days. growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants. one segment (fresh control) was dissected and planted without further processing. the 2 remaining tissue segments were soaked in (ab/am) saline solution for 1 hr and 24 hrs at 48c, respectively. tissue segments were frozen in cryo bags with a proprietary 10% dmso/large molecular weight sugar solution. background/case studies: it has been the practice in our institution to process 3 or 4 times the total blood volume (bv) of the patient, up to a maximum of 25 liters (l) per procedure, to obtain peripheral blood cd341 stem cells. as a consequence, a patient often would need to spend 6 hours or more on the machine. it would be desirable to be able to specify the exact volume of blood to process to achieve the desired cd341 cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage. study design/methods: our institution recently implemented the new spectra optia cmnc collection protocol, a continuous flow and continuous collection procedure that uses the automated interface management (aim) system to precisely manage the separation interface. an analysis of our 2016 collection data suggested a highly reliable collection process, so a prediction algorithm (pa) based on the linear regression between the patient's cd341 pre-count and cd341 yield, normalized per liter of blood processed, was derived utilizing the patient's cd341 pre-count, the patient's weight in kilograms (kg), and the target cd341 dose/kg. this pa calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral cd341 stem cells. the initial equation was modified to add an additional 15% to the predicted volume, to account for the natural variability of the process. this pa was then tested prospectively in the clinical setting. results/findings: in 8 patients, representing both allogeneic and autologous donors, the average blood volume processed was 14.8 l. the range was 4.9 l -21.6 l. the target dose was achieved in all patients. our previous practice for these 8 patients would have required, assuming a standard 4 bv procedure, processing an average of up to 28 l per patient, with a range of 20-62 l. to quantify how well the new pa works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected cd341 yield. the result was a high correlation between these two ratios (r 2 5 0.92), indicating that the algorithm produces very consistent results. conclusion: the predictability of our collection process during the time period analyzed was a robust r 2 5 0.92, confirming the findings in the first data analysis. the blood volumes processed and patient time on the machine decreased substantially, with some patients only needing 2 hours or less to achieve their target dose. nurses and lab medical technologists have seen a dramatic change in their workflow. the number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day. all in all, implementation of this pa has produced huge increases in patient and provider satisfaction. important factors that likely contributed to the success of the protocol included the precision and consistency of the aim system of the apheresis device, as well as the small number of nurses (1-2) who performed the procedures, resulting in less variability. the economic impact of this pa has not been quantified, but might be an interesting area for future studies. background/case studies: zarziov r , a biosimilar granulocyte colonystimulating factor (g-csf) has recently been introduced into clinical practice. its use has stimulated a certain debate regarding their possible less efficacy and security on cd341 mobilization. the aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as g-csf is used. study design/method: we retrospectively evaluated autologous mobilization processes performed between june 2015 and march 2016. patients (n525) evaluated were diagnosed with malignant lymphoma (n515), multiple myeloma (n59) and primary amyloidosis (n51) and were mobilized according to standard protocols. collection cd341 cellularity target was established ! 2x10e6/kg. two groups, good and bad mobilizers, have been determined. predictors of unsuccessful mobilization were defined by >65 years old, previous fludarabine, lenalidomide, or bendamustine treatments or !2 previous regimens, present peripheral cytopenias, active disease and previous mobilization failure. mann-whitney u test was used to compare means and comparisons of medians were performed by the median test. cd341 count was performed according ishage protocol. adverse events (ae) were analysed according to ctcae v4.0. results/finding: the media (range) general collection parameters were: cd341 (day 5) 27.50/ml (4.5-157.5/ml), blood volume processed 23204ml (9718-39618ml) and 4.96 (2-7.30) exchanged volemias. seventeen patients were considered bad mobilizers, 7 needed plerixafor and 5 had to undergone a collection procedure twice. there were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (sd) ); p50.071]. there were no significant differences on mobilization characteristics and product cellularity between both groups. five mobilization ae were observed [muscle pain (n52), fever (n52) and flu syndrome; all grade 1]. two patients could not undergo hematopoietic stem cell transplantation due low cd341 cellularity. conclusion: there are differences between products collected from the good mobilizer (rich in gm and cd34) versus poor mobilizer (with plerixafor) rich in cn and cmn. the mobilization with zarziov r could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting. background/case studies: mesenchymal stem cells (mscs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma. however, mscs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common. before applying mscs in a preclinical animal model, we sought to determine the procoagulant properties of rat mscs in vitro. study design/methods: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured and passaged using dmem medium with 20% fetal bovine serum. bmsc and amsc at passage 2-5 were used in this study. the tissue factor expression of mscs was determined by immunohistochemistry. citrated whole blood collected from normal rats was treated with rat bmscs and amscs at low, medium and high doses (1.5 3 10 4 /ml, 7 3 10 4 /ml and 1.5 3 10 5 /ml respectively). the prothrombin time (pt), coagulation properties and platelet aggregation (response to adp, collagen and par4) were measured by hemostasis analyzer, rotational thromboelastometry (rotem) and impedance aggregometry (multiplate) respectively within 30min and 2hr after incubation. results/finding: tissue factor was significantly expressed among both bmsc and amsc at all passages in vitro. bmsc and amsc at any dose and time of treatment neither shortened nor elongated pt in whole blood. however, both bmsc and amsc significantly shortened the clotting time (ct) (none: 334 6 35 seconds, versus low, medium and high doses of amsc (145 6 2, 111 6 6, and 75 6 12 seconds), and bmsc (155 6 2, 90 6 10, 80 6 7.0 seconds), p<0.05), clot formation time (cft, p<0.05) and increased alpha angle (p<0.05) by natem measurement, but did not significantly affect the ct, cft and alpha angle by extem. maximum clot firmness (mcf) and fibrinolytic index were not affected by mscs. there was no significant impact of both bmsc and amsc on platelet aggregation simulated by adp, collagen and par4. no significant differences of hemostatic and platelet function were found between the treatments of bmsc and amsc. conclusion: consistent with reports from human derived msc, both rat bmsc and amsc significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment. however, mscs had no direct impact on platelet aggregation in vitro. as considering the procoagulant capability of mscs, future study will be necessary to determine the optimal dose and safety of using mscs for systemic application in vivo. comparison of the terumo bct mnc and cmnc protocols for peripheral blood stem cell collections lindsey westbrook* 1 , neil bagamasbad 2 , reynold dilag 2 , melissa nasser 2 , nicole bauer 2 , jennifer wheeler 3 and mary berg 1 . 1 department of pathology, university of colorado -anschutz medical campus, 2 department of medicine, division of hematology, university of colorado hospital, 3 scientific support, terumo bct background/case studies: terumo bct recently offered a new method of peripheral blood stem cell (pbsc) collection using the spectra optia, an apheresis instrument. the new protocol, continuous mononuclear cell collection (cmnc) collects cells continuously as opposed to the older protocol, the mononuclear cell collection (mnc) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from mnc within a cell separation chamber. our institution has used both protocols and the purpose of this study was to compare pbsc product characteristics and run times between the cmnc and the mnc protocols. study design/method: a retrospective review and comparison of parameters from 120 collection procedures using the mnc protocol and 173 collection procedures using the cmnc protocol was done using the t-test. data from patients/donors (including 36 allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (cd34)-positive (cd341) throughput, cd341 collection efficiency (ce%), platelet loss 71a transfusion per total blood volume processed (plt loss/tbv), and collection product characteristics were included in the analysis. results/finding: numerical results are summarized in the table. the mnc and cmnc donor groups included 14 and 22 allogeneic donors, respectively. donor weight was not significantly different between the two groups. pre-procedure wbc values were also similar between the two groups. run time was found to be significantly shorter using the cmnc protocol compared to the mnc protocol. product volume was also significantly lower in the cmnc group compared to the mnc group. although the volume was lower, the cmnc product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the mnc product. the cd341 throughput was significantly higher in the cmnc group than the mnc group. the cd341 ce% was found to be slightly increased in the cmnc group, though not significantly. the platelet loss was not significantly different between the protocols when normalized for total blood volume. product hematocrit (hct%) was significantly higher using the cmnc protocol; however, the red blood cell volume never exceeded 20 ml due to the lower product volume with the cmnc protocol. the cmnc protocol collects a smaller volume of a purer product when compared to the mnc protocol with comparable platelet and red blood cell loss. staff members who perform apheresis procedures are pleased by the shorter run time. background/case studies: hematopoietic stem cell (hsc) donors and their recipients need not have a matching blood type. eventually, the hsc recipient will become the blood type of the hsc donor. this scenario can become quite a conundrum if the hsc recipient becomes a patient in need of an organ transplant. in order for a patient to receive a donor organ, the patient and donor's blood type and hla typing must be compatible. study design/methods: blood type was determined using gel test cards. hla typing was determined by using sequence-specific oligonucleotide (sso), sequence-specific primer (ssp), and sequence based typing (sbt) technologies. hsc sources were bone marrow and umbilical cord blood. results/findings: patient #1, originally typed as an a2, had 1 bone marrow donor and 2 cord blood transplants. one of the cord blood transplants successfully engrafted. the engrafted unit was from a type o donor. patient #1 is now typing as type o. patient #2 was originally typed as a2 and received a bone marrow transplant from a type b donor. patient #2 is now front-typing as a b and backtyping as an ab. since the patient's abo front and back-type do not match, a note must be made, that when confirming abo during crossmatch, the abo will not match. the patient now has an hla and abo identical kidney match (his father who is a type b). previously, the patient and his father were abo incompatible. the abo and hla results on both patient #1 and patient #2 indicate that the hsc transplants have engrafted. results also indicate that the abo and hla now match that of the donor and differ from the recipient's original abo and hla type. due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. both patients will be entered into the unet system according to their "new" abo and hla types, as unos regulations require patients to be listed as per the results of two separate abo typing tests. the patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial. background/case studies: mesenchymal stem cells (msc) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma. mscs express tissue factor (tf) that activate the clotting cascade and interfere hemostasis. hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including mscs. in this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat msc in vitro. study design/method: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured using dmem medium with 20% fetal bovine serum under either normoxia (20% o 2 ) or hypoxia (3.5% o 2 ). msc growth curves were measured by cell counter. the tf expression was determined by immunohistochemistry. cd90/cd29 and cd45 were measured as positive and negative markers of msc respectively by flow cytometry. the citrated rat whole blood was treated with msc (1.5 3 10 5 /ml) either from normoxia or hypoxia. the coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (rotem). results/finding: hypoxia potentiated the growth of bmsc by 15%, but depressed the growth of amsc by 30% at day 5 in comparison to normoxia. both bmsc and amsc equally expressed cd90 and cd29 but not cd45 under any culture condition. tissue factor was significantly expressed among bmscs and amscs from both normoxia and hypoxia. whole blood treated with bmscs and amscs from normoxia significantly shortened the clotting time (ct: 468 6 64 (control), versus 170 6 13 (bmsc), and 195 6 60 (amsc) seconds) by natem. hypoxia also significantly shortened ct (165 6 20 (bmsc), 169 6 50 (amsc) seconds, p<0.05 as compared to control), but the changes in ct were not significantly different between bmscs and amscs. maximum clot firmness (mcf) and fibrinolytic index did not change after treatment with bmsc and amsc regardless of the normoxia or hypoxia conditions. conclusion: tissue factor is constitutively expressed in rat bmscs and amscs. adjustment of the msc culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of msc (bmsc and amsc). this study also suggests that the procoagulant properties will not be affected if mscs are recruited into injured tissues with hypoxic environments. future study will be necessary to determine the optimal dose msc and whether it is safe to use mscs for systemic application in trauma. effect of double-end cryopreservation on gene-transduced human hematopoietic stem and progenitor cells sandeep k srivastava*, jiaqiang ren, steven highfill, narda theobald, suksee deravin, andre larochelle, david f stroncek and sandhya r panch. national institutes of health background/case studies: current early-phase clinical gene therapy trials use freshly collected or cryopreserved cd341 cells as the starting fraction prior to gene manipulation. following gene-transduction and culture, the end product is infused fresh into recipients. for wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (dec) of cd341 cells during manufacture (i.e. immediately post-collection and again, post-gene modification). dec helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion. our objective was to study the effects of dec on gene transduced mobilized peripheral blood (mpb) cd341 cells. study design/method: cryopreserved cd341 cells from 2 healthy adult donors were thawed and transduced (tr) in retronectin coated tissue culture bags with an ef1-alpha-yfp lentivirus (2.5% concentration) and media (x-vivo-10, human serum albumin(hsa), 100 ng/ml each of cytokines (scf, tpo and flt3-l) over 2 days. untransduced (utr) cells were cultured as controls. tr and utr fractions were re-cryopreserved. a standard freeze-mix of 5% dmso, 6% pentastarch, hsa, plasma-lyte a was used for cryopreservation. viability, hematopoietic stem cell (hsc) (cd34 1 cd38 -cd45ra -cd90 1 cd49f 1 cells) phenotyping and cfu assays were done following first thaw (pt1), post-transduction (ptxn) and second cryopreservation-thaw (pt2). results/finding: tnc recovery decreased gradually in the donor samples at each step. transduction efficiency, cd34%, cfus were similar before and after pt2. hscs ranged from 824 to 1655 cells/10 6 cd341 cells in the pt2-tr arm compared to a range of 286 to 1416 /10 6 cd341 cells after pt1. viability, % cd341 and cfus were lower in the tr compared to the utr arm. this difference was not altered after pt2 (table) . conclusion: dec of mpb human cd341 cells decreases tnc recovery, but has minimal effects on cd341 cell phenotype, transduction efficiency and cell function. hsc numbers were within acceptable range after recryopreservation. lower viability and cd34% in the tr arm compared to the utr arm is likely due to vector toxicity. this was unaffected by recryopreservation. additional studies to assess dec mediated changes on cd341 cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in nod/scid mice will inform clinical trials. background/case studies: autologous peripheral blood stem cell (pbsc) transplantation has been used as a powerful resource during the treatment of some hematological malignancies. cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy. in some cases, pbsc are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial. our work presents retrospective data on pbsc infusion after long-term storage. study design/method: all products were harvested after patient mobilization with g-csf by apheresis with cobe spectra v r . flow cytometry analysis of cd341 cells was performed prior to cryopreservation. the cryoprotective solution was freshly prepared by addition of 20% hydroxyethyl starch, 18% human serum albumin and 10% dmso at final concentration. pbsc were cryopreserved by direct immersion on -808c mechanical freezer (dump freeze) and stored until transplantation. post-thaw viability was determined from stored cryotube samples by trypan blue exclusion minutes prior to infusion. cells were thawed and infused on bedside. engraftment was defined as the first day of 03 consecutive days of neutrophil count >0.5 x10 9 /l and platelet count >20 x10 9 /l after 07 days. with g-csf for four days and patients with g-csf for five days with use of mozobil when cd341 was below 10 x 10 6 cells/l on the fourth day. hpc collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients. all procedures were realized based on a prediction algorithm using pre-cd341 on the day of the collection and estimating wbc liters to be processed to obtain sufficient stem cells for the transplant. this algorithm was designed using linear regression of peripheral blood cd341 on the day of the collection versus collected cd341 per liter of blood processed. there was no distinction between patients and donors, once the efficiency coefficient was used for both. collected material was sent to analysis and total cd341 was calculated. final laboratory count of cd341 per kilogram was compared with the number predicted by the algorithm with spearman's correlation to evaluate whether the formula is effective. calculations were made using ibm spss 23 software. results/findings: among patients collecting hpc for autologous transplantation, 69,23% needed only one day of hpc harvesting, while 25,64% needed two days and 5,13% needed three or more days. our collection efficiency (ce) and standard error of the mean (sem) was 49 1-2,91%. after comparing predicted values with cd341 collected in the final product, we found a very strong correlation of 0.873 (p<0.01) for patients and a strong correlation 0.653 for healthy donors (p<0.01). conclusion: the use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate wb liters to process and avoid unnecessary procedures in both patients and healthy donors. this study evaluated the phenotypic characteristics of uc-mscs derived from fresh and cryopreserved cord tissues (ct), as described in isct's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; !95% cd90, cd105, cd73 and 2% cd14, cd19, cd34, cd45, hla-dr) study design/method: umbilical cord tissue (n510) was washed, blood vessels removed, cut into 0.5-3mm pieces, and washed twice in saline. fresh tissue was immersed in 0.9% saline for same day culture, while frozen tissue was cryopreserved for at least 24 hours prior to culture. for colony forming unit (cfu) testing tissue was plated directly in a 25cm 2 tissue culture flask following a wash in pbs with antibiotic/antimycotic. the tissue was allowed to adhere for 10 minutes prior to the addition of cell culture media. media was changed several times a week. cells were passed when robust colony growth was observed and in subsequent cultures >80% confluence. all cells were tested on an msc flow panel at passage 2 just prior to confluence. results/finding: both fresh and cryopreserved tissue showed excellent colony forming capabilities. average time for cellular emergence of 8 days (fresh 5 7.8, frozen 5 8.1), and 13 days (total) for the msc's to reach passage 1 (fresh 5 12.6, frozen 13.4). all cells were ready for flow analysis in approximately 3 weeks time. there was no statistical difference between fresh and frozen tissue in their colony emergence (p 5 0.81), or their growth rates (p > 0.05 for all). flow cytometry showed average !95% for positive markers and 2% negative markers. there was no statistical difference between fresh and frozen flow result (p > 0.05). conclusion: uc-msc's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology. flow cytometry analysis showed strong msc phenotype in both fresh and frozen samples. the data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain msc colonies. studies have shown that hsct improves survival and disease-free survival rates when compared to conventional chemotherapy treatments. the increase in the number of hscts over the last years has demanded quality and safety improvements of cell processing and cryopreservation services. cell recovery and viability are crucial parameters to assess ucb quality as a viable hsct graft source. study design/method: twenty-five ucb units cryopreserved for periods of 2 up to 11 years (2004 to 2017) were analyzed. units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using dmso (dimethylsulfoxide) cryoprotectant with 10% concentration. informed consent and the unit discard terms for all units were obtained. units were thawed in a 378 c water bath and 0.5ml aliquots were diluted at a 1:1 proportion with 5% human albumin solution and plasmin were prepared, enabling dmso stabilization and concentration reduction. the following analysis were performed: nucleated cell count (tnc) in an automated hematologic counter and cell viability using flow citometry. post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using 7-aad marker through flow cytometric analysis. results/finding: ucb storage period was 7.24 years (mean) and cell recovery was 86.31% (mean). there was no statistically significant correlation between storage period and post-processing cell recovery (p 5 0.11). post-thaw cell viability of 63.13% (mean) showed no statistically significant correlation with unit storage period (p 5 0.07). post thaw cell viability results are within parameters defined in other studies. background/case studies: umbilical cord (uc) tissue is a rich source of mesenchymal stem cells (mscs) that can be collected noninvasively at birth and stored for potential future use. as such, a growing number of stem cell banks have established uc storage programs based on mounting preclinical evidence of its therapeutic potential. however, little has been reported on the ability to isolate msc-like cells from uc tissue after extended periods of cryopreservation. this work describes and characterizes the isolation of mscs from uc tissue cryopreserved as a composite material at a family stem cell bank for 5 years. study design/method: donated uc units from consenting mothers were evaluated. units had been cryopreserved as composite tissue pieces in ln 2 vapor in a dmso-based cryoprotectant for 5yrs. (5.49 6 0.431; n54). units were rapidly thawed and rinsed in dpbs, then 25 pieces were excised from each using a biopsy punch. pieces from each unit were explanted in a 5x5 grid pattern in msc-supportive medium and incubated for 7 days, after which the tissue was discarded and media exchanged. cells were isolated on the 14 th day, counted, and subcultured for two passages. at the end of each passage, cells were collected, counted and population doubling time was calculated. isolated cells from each unit were also evaluated for msc immunomarkers. results/finding: small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a 100% success rate. cells were positive for the msc markers cd73, cd90, and cd105 (98.8 6 0.7%, 98.7 6 0.6%, and 97.8 6 0.6%, respectively) and negative for the hematopoietic markers cd34/45 (1.1 6 0.7%). passage 1 and passage 2 doubling times were 1.92 6 0.47 days and 2.07 6 0.43 days, respectively, which are in line with values reported for mscs isolated from fresh uc tissue. conclusion: due to their immature status, ease of collection, and potential therapeutic value, uc mscs are an appealing candidate for future clinical 75a transfusion 2017 vol. 57 supplement s3 research and treatment. the present work demonstrates that the long-term cryopreservation of uc tissue does not disrupt the ability to isolate functional mscs from the tissue at a later date. importantly, growth characteristics of isolated mscs appear to be comparable to those reported for mscs from fresh uc tissue. based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion. these results support the feasibility of storage of uc as a composite material for future potential cell isolation and expansion to clinically relevant doses. large volume leukapheresis with spectra optia cmnc protocol in adult and pediatric patients: performance and determination of cd34 yield prediction algorithm ines bojanic* 1 , nelly besson 2 , ivana vidovic 1 and branka golubic cepulic 1 . 1 department of transfusion medicine and transplantation biology, university hospital centre zagreb, 2 terumo bct background/case studies: large volume leukapheresis (lvl) have shown to enhance cd341cell yield collected. this study evaluated performance and safety of the spectra optia cmnc protocol (version 11) in adult and pediatric lvl. a prediction algorithm for cd341cell yield was also tested. study design/method: we evaluated retrospectively 67 lvl performed in 46 adult patients, and 14 lvl in 11 pediatric patients treated in uhc zagreb from march 2016 till september 2016. mobilization regimen combined chemotherapy and filgrastim; 2 poor mobilizes received plerixafor additionally. a combination of acd-a and heparin was used as anticoagulant (acd-a:whole blood ratio 1:24). in patients weighting 25kg (n59), a rbc prime was performed. cd34, lymphocyte(ly) and monocyte(mo) collection efficiencies (ces) were calculated. a customized prediction algorithm was determined on linear regression between pre-cd341cell count and cd341cells collected / blood volume processed. prediction accuracy was evaluated by comparing predicted cd34 values to real cd34 yield. results are presented as median (iqr). results/finding: in both groups, cd34, ly and mo ces were high. target cd34 dose was successfully reached in 1 procedure in 30 (65,2%)adults and in 9 (81.8%) children. all procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in 5 (7.5%) adults, while all pediatric apheresis went uneventful. no bleeding episodes occurred, and no transfusion was needed. product and procedure characteristics* a high correlation between precd341cells and cd341cells collected/ blood volume was observed in both groups (r 2 50.97 and 0.83 in adults and children respectively, p<0.0001) suggesting cd34 yield could be predicted based on precd341cells and blood volume to process. linear regression equations served as prediction algorithm. the high correlation between predicted cd34 yield and observed cd34 yield (r 2 50.95 and 0.82 in adults and children respectively, p<0.001) showed accuracy of the algorithm. implementation of the algorithm could have allowed sparing a median of 10.1(8.9-12.9)l of blood in 20 adult procedures, and 5.9(3.5-7.8)l in 7 pediatric procedures. conclusion: lvl performed using spectra optia cmnc protocol is safe and efficient in adults and in low body weight children. high cd34, ly and mo ce1 were observed in both groups. implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort. mesenchymal stem cell therapy in steroid refractory graft-versus-host disease (gvhd) emese molnar* 1 , aniko barta 2 , arpad batai 2 , zoltan csukly 2 , zita farkas 2 , laszlo gopcsa 2 , gabor tatai background/case studies: steroid refractory acute graft-versus-host disease (gvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (hsct). more experience accumulates in the immunomodulatory effect of mesenchymal stem cell (msc) infusion in numerous immunopathological disorders -such as gvhd -and signals. mscs have a hlarestrictive and non-immunogenic nature. study design/method: we have evaluated the efficacy of msc transfusions in cases of acute gvhd refractory to conventional immunosuppressive treatment. the patients with steroid-resistant gvhd had received third-party mscs (derived from wharton's jelly and bone marrow) 4 times per case weekly at a dose of 1 million cells/kg. clinical response was assessed 28 days after administering the first dose. complete remission was defined as the complete disappearance of symptoms. partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition. results/finding: in all 12 patients had received 13 cycles of msctreatment (4 dose per cycle). the median age was 47 years old (19-56) with a male/female ratio of 1:2. distribution of the original malignancies (n): acute myeloid leukemia: 6; acute lymphoblastic leukemia: 2; myelofibrosis: 1; myelodysplastic syndrome: 1; multiple myeloma: 1; t-cell lymphoma: 1. nine patients had undergone allogeneic hsct with matched unrelated donors, the other three had stem cells derived from hla-identic relatives. the first episode of gvhd after hsct was started on the median 63rd day (7-455). the involved organs were skin (2), gut (4), skin and gut combined (7) and even lung in 3 cases. the median time of msc's first infusion was 274 days after the stem cell transplantation (hsct) and 165 (19-1974) days after the first episode of gvhd. 4 of the 13 cycles of msc-treatment led to complete remission (30.8%) and 7 resulted inpartial remission (53.8%). conclusion: we have evaluated msc-therapy as an effective treatment of gvhd in the majority of the observed cases with 83% overall cumulative response rate. the application of third-party mscs offers a promising alternative in the therapy of gvhd and other gvhd-associated complications after hsct. further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety. background/case studies: stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team. whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment. a typical goal for most adult procedures is 2 million cd341 cells/kg. if a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal. given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections. measuring a patient's cd341 cells/ml in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the 2 million cells/kg goal. the ideal minimum cd341 cells/ml that will lead to successful harvest has not been conclusively identified. study design/methods: we analyzed the collection data from 55 patients to evaluate the predictive value of the cd341 cells/ml level. data was collected over 6 months from every patient who underwent a stem cell collection. four patients were allogenic donors and 51 were autologous donors. the patients' weight, diagnosis, and pre-procedure cd341 cells/ml level were all collected. the run time, amount of volume processed, and the absolute viable cd341 cells collected were recorded. the collection efficiency and the cd341 cells/kg were calculated for each patient. results/findings: our data showed a strong linear correlation between pre-procedure cd341 cells/ml and post-procedure cd341 cells/kg (r50.95). any patient who had a pre-procedure cd341 cells/ml count of 29 or greater had a collection of at least 2 million cells/kg. any patient who had a pre-procedure cd341 cells/ml count of 16 or less collected less than 2 conclusion: the pre-procedure cd341 cells/ml level in the peripheral blood has a very strong predictive value for the post-procedure cd341 cells/kg level. to confidently know that a patient will be able to produce the desired 2 million cells/kg, a pre-procedure cd341 cells/ml count of at least 29 should be obtained. for any patient with a count below 16, they should be counseled that their collection is likely to take at least a second day and a second procedure. further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between 16 and 29 cd341 cells/ml should be conducted. heidi elmoazzen 1 , antonio giulivi 1 , michael halpenny* 1 , lisa martin 1 , donna perron 1 , chris bredeson 2 , lin yang 1 , locksley mcgann 1 , paul birch 1 and jason p. acker 1 . 1 canadian blood services, 2 ottawa hospital background/case studies: a critical aspect of hematopoietic progenitor cell processing is the cryopreservation method. our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing dmso (5% final concentration) and hes (hydroxyethyl starch). pentastarch (hes source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor. this required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality. study design/method: the validation study consisted of 3 phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant. phase i -samples from four different cryoprotectant formulations were tested for tnc, cd34, viability and cfu at three points during manufacturing (fresh, post processing and post thaw). phase ii -mock hpc, apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations. phase iii -five clinical transplants were performed with hpc, apheresis products cryopreserved using the recommended replacement (hetastarch). results/finding: phase i -results indicate that aliquots cryopreserved in 5% dmso and 1.7% hes (hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (cfu). phase ii -the majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations. phase iii -transplants performed resulted in a mean engraftment time of 12.6 days for anc500 with no adverse patient reactions observed. engraftment times using the new hetastarch formula were compared to the previous engraftment times with no significant difference. conclusion: a change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality. in addition, maintaining the current 5% dmso final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation. this study demonstrated the acceptability of the hetastarch formulation using 5% dmso and 1.7% hetastarch to replace pentastarch in the cryoprotectant formulation used for cryopreservation of hpc, apheresis products. background/case studies: autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (pbscs). traditional mobilization regimens include granulocyte colony stimulating factor (g-csf) with or without chemotherapy, but have failure rates ranging from 5% to 40%. plerixafor is an adjunct agent used to improve mobilization in many clinical settings. however, its high cost is a significant concern. the manufacturer-recommended dose is 0.24 mg/kg, therefore patients weighing >100 kg would require a second vial, thus doubling the drug cost. in 2013 we implemented a policy of capping plerixafor at 24 mg for patients weighing >100 kg. this retrospective study compares the mobilization of patients >100kg who received capped doses (2013) (2014) (2015) (2016) , with historical control patients (2010-2013) who received full or uncapped doses. study design/method: patients weighing >100 kg with crcl >50ml/min who received capped and full doses of plerixafor were identified in the pharmacy database. electronic medical records were used to collect baseline characteristics and cell collection data. results/findings: a total of 47 and 40 consecutive patients were included in the capped and full dosing groups, respectively. they showed comparable baseline distributions of age, weight, gender and diagnoses. plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups. in the capped dosing group, fewer patients received chemomobilization or plerixafor upfront. when compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of cd341/cells kg and achieved a comparable collection success rate. the strategy dose capping plerixafor at 24 mg for patients >100 kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half. mean and range of %cd34 in peripheral blood were calculated. the data show that in the non-hispanic group, the youngest donors (<30yrs) have a higher pre-apheresis %cd34 level than any of the other groups, reaching statistical significance when comparing the %cd34 pre-apheresis between the youngest group (<30 yrs) and the oldest group (>540 yrs). hispanic donors show statistically similar %cd34 pre-apheresis levels over all age groups. moreover, the hispanic older age group (>540yrs) had a statistically higher %cd34 pre-apheresis level than the non-hispanic older age group. conclusion: in this analysis of 121 sequential unrelated pbsc donors, hispanic donors maintain a similar pre-apheresis %cd34 level even as the donor ages, while non-hispanic donors show a decreasing pre-apheresis %cd34 level as they age. if proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups. this small data set would suggest that people of hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age. further studies of larger cohorts are needed to validate this observation. if proven, this has far reaching implications within the stem cell research and therapy arena. background/case studies: an update in hpc apheresis collection software led to higher collection volume in the organization's human progenitor cell (hpc) products without a corresponding increase in total cellular counts. incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger dmso load to the recipient, often resulting in the need to transfuse over several days. the objectives of this study were to develop suitable mock hpc (mhpc) products and evaluate the effectiveness of the biosafe pericell volume reduction technology on white blood cell (wbc) recovery and viability. study design/method: hpc products are not readily available for development. mhpc were created from whole blood buffy coats (bcs). fresh abo compatible bcs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells. the mhpc products were then diluted in plasma to produce an appropriate concentration and volume. hpc collection data from last 3 years was analyzed to determine the 95 th percentile, median and 5 th percentile values for both hpc volume and wbc concentration. six mhpc products were tested; three high wbc (234 x 10 6 cell / ml) and three low wbc (114 x 10 6 cells / ml) concentrations, each at high (505 ml), low (265 ml) and median (355 ml) volumes. each unit was processed sequentially from high, median and low volumes. hence, the highest mhpc volume was processed for volume reduction first with a sepax 2 (pericell protocol, cs.430.1 kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth. one additional mock product was prepared for a reproducibility study and was volume reduced three times. wbc concentration and 7-aad viability was determined before and after each volume reduction. a control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time. results/finding: mock hpc products had a mean starting 7-aad viability of 76 6 8% [range 64-85]% and a hematocrit of 14 6 5% [9-19] which is well below the maximum allowable limit of the pericell. no significant differences in wbc recovery or change in viability were seen between the 6 mhpc products. aggregate data showed that the mean wbc recovery of the volume reduction process was 97 6 8% [64-105] with a 3 6 3% [-2-11] change in viability. the recovery protocol used to salvage product after each volume reduction gave a recovery of 99 6 4 [92, 104] % and a change in 7-aad viability of 2 6 2 [0, 11] % from the input product. the method was found to have a cv of 2.0%. the change in wbc concentration and wbc viability of the test products was not significantly different from the unprocessed control samples. conclusion: mock bc products are a suitable alternative where hpc products are not available for development and are a good use of product otherwise directed for rejection and disposal. the volume reduction protocol evaluated had minimal impact on the wbc concentration and wbc viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with hpcs and will facilitate transfusion of hpc products into the recipient. the protocol is now in use with patient hpc products and engraftment kinetics will be tracked in a postimplementation study. validating 78a transfusion clinical assessment. in the first phase, 2 cryopreserved pbsc products were tested. two aliquots were thawed simultaneously for each product: one was passed through a pre-set infusion pump and a second control aliquot was drained by gravity. each aliquot was tested for baseline total nucleated cell (tnc) count and viability, and for final tnc recovery, trypan blue (tb) viability, cd34 7-aad viability, and potency (cfu). the effect of longterm exposure to dmso was assessed by visually inspecting the product for aggregates and measuring viability up to 3 hours post thaw. the second in vivo phase included use of an infusion pump for 10 consecutive autologous patients, with comparison of infusion and transplant outcomes to 18 previous infusions by gravity drip. comparison variables included infusion rate, adverse events (ae), and engraftment time. results/finding: no significant differences were observed between infusion pump and drip for the 2 products tested in vitro, including tnc recovery, cell viabilities, and potency. for both methods tnc tb viability decreased by more than 20% within 1 hour, while cd341 cell viability remained stable up to 3 hours post thaw. small aggregates appeared after 1 hour for both methods and increased by a similar rate over time. comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables. engraftment time was similar for both groups. anc days to engraftment for pump and drip were 10.8 6 1.3 and 11.6 6 1.0, respectively (p-value50.075). platelet days to engraftment for pump and drip were 17.9 6 2.2 and 20.2 6 5.0, respectively (p-value50.207). infusion rates were slightly higher for the pump group. for control patients, 2 required transfer of products to syringes due to slow infusion rate and 2 others experienced allergic and hypotension infusion adverse events. conclusion: no significant in vitro or clinical differences were observed between thawed pbscs infused by gravity or an infusion pump. these results demonstrate that the use of a pump for pbsc infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times. donor racial distribution among the 278 zikv ineligible cbus was: caucasian 52%, asian 9%, black/aa 20%, and multi-race 21%. racial distribution of all clinical cbu donors was caucasian 49%, asian 15%, black/aa 20%, and multi-race 17%, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel. there were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility. conclusion: our study indicates that currently the leading risk factor for ineligible cb donors is potential exposure to zikv: 78% of all ineligible cbus and 21% of all banked cbus in the study period. we anticipate the number of cases to decrease following maternal education and travel warnings. recognizing the importance of zikv in public health, and its potential transmission via hct/p products, an fda approved screening test for hct/ p donors becomes a timely necessity. acknowledgments: funded by zimmer biomet, a zimmer biomet company, ibgrl red cell reference and nhsbt reagents background/case studies: during storage, red blood cells (rbcs) become less deformable, deplete 2,3-diphosphoglycerate (2,3-dpg) and adenosine triphosphate (atp), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium. longer rbc storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation. a sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (citra labs, llc, braintree, ma), is approved by the u.s. food and drug administration for the rejuvenation of stored rbcs. the solution acts by restoring 2,3-dpg and atp in stored rbcs to levels equivalent to those in the circulation. the aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored rbcs. study design/method: a 10 ml aliquot was removed from abo/rh grouped, leucocyte depleted rbc units (n 5 20), which were stored in sagm for 22 days, to act as untreated controls. the remainder of each unit ($270 ml) underwent treatment with the rejuvenation solution (50ml, 60minutes at 37 o c), followed by cell washing twice in sagm ('manual' centrifuge-based process). to represent current transfusion laboratory practice, units were crossmatched against plasma from 39 random donors, using both diamed gel column and glass tube technique. phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on rbc surface antigens (a, b, d, c, c, e, e, k, m, n, s, s, p 1 , lu a , k, kp a , kp b , le a , le b , fy a , fy b , jk a , and jk b ), including whether it exposed crypt antigens (t, tn, tk*, th, tx*, and cad). crossmatch and phenotype agglutination scores observed for the untreated and treated rbcs were then compared. results/finding: crossmatch findings were defined as compatible, suitable, and incompatible. the study identified no difference between the crossmatch reaction profiles of untreated and treated rbcs. furthermore, no difference was observed in the phenotypic state between untreated and treated rbcs. conclusion: treatment of 22 day old stored rbcs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples. background/case studies: cryopreserved platelet production is burgeoning worldwide. currently, there are no automated platelet cryopreservation methods. by contrast, red blood cell cryopreservation using the acp 215 (haemonetics corp., baintree, ma) has automated the processing within a closed system, increased labour productivity and provided high quality blood components. purpose: to automate platelet cryopreservation procedure. study design/method: apheresis platelet concentrates (pc) were collected on the trima accel system. platelet counts were performed using an abx micros 60. pc were centrifuged at 1250g in a sorvall rc3c1 centrifuge (sorvall, usa) for 10 min. the combination cryoprotectant dmso1dextran (cryosure dex40, germany) was used for pc cryopreservation. cryopreserved pc (cpc) were frozen and stored in a kelvinator chest freezer. cpc were thawed at 37 degrees c (barkey plasmatherm) for 10 min. cpc osmolality was measured with an osmomat 030 osmometer. results/finding: staged platelet cryopreservation technology has been developed. platelets were cryopreserved in a closed system (patent no.: ru 169287 u1). during the first stage, cpc were spun to separate a plateletrich plasma (prp) fraction from platelet-poor plasma (ppp). the second step was to resuspend the prp by adding a combination of dmso1dextran (cryosure dex40) , as a cryoprotectant, to obtain a final concentration of 5% dmso in the platelet suspension. the injectomat mc agilia and npbi compomixer m3 were instrumental in automating that phase. pc to be frozen had an osmolality of no less than 1500 mosm/l. prp and ppp were frozen at a cooling rate of 1-38c/min and stored at -85 0 in the chest freezer for up to 24 months. pre-transfusion defrosted platelets were also processed in a closed system (patent no.: ru 167874 u1). our transfer set made it possible to automate platelet resuspension in plasma through the agency of the exadrop v r . post-thaw prp was resuspended in plasma, which lowered the osmolality to 380 mosm/l. freeze-thaw recovery of platelets was 80% or more of the original population. defrosted pc were stored at 20-24 0 with continuous gentle stirring from a helmer platelet agitator for no longer than 4 hours before transfusion. it took no more than 30 min to cryopreserve pc and process pre-transfusion thawed platelets. the automated processing accounted for the bulk of the time (over 20 min). conclusion: the automated technique developed reduced the workload while offering reproducibility of the procedure and high cpc quality. the use of closed systems ruled out bacterial contamination. employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring. bacterial detection in leukoreduced apheresis platelets on day 4 and day 5 evelyn c. oyler*. suncoast blood bank background/case studies: the recently published fda draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to 7 days. this evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets. study design/method: a large community blood center and transfusion service collects leukoreduced apheresis platelets (lrap) using amicus separator system (fenwal, lake zurich, il) and trima accel system (terumo bct, lakewood, co). previously-cultured lrap units were sampled on day 4 for secondary culture using bact/alert (biomerieux, durham, nc) and rapid bacterial tests using bactx (immunetics, boston, ma) and pgd (verax, marlborough, ma). if lrap unit is still available, it is also sampled and tested for rapid testing on day 5. a total of 60 lrap units were tested over a 3-month period: 50 were cultured and rapid tested on day 4; 10 were rapid tested on day 5. the rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use. results/finding: of the lrap units evaluated for this study, there were 59 true negatives (tn) and 1 false positive (fp) on day 1 when tested by bact/alert, with 60 tns on day 4. bactx testing results showed 50 tns on day 4 and 10 tns on day 5. testing using the pgd kit showed 50 tns on day 4; and 8 tns and 2 fps on day 5. fp results were confirmed by performing a secondary culture, which were found to be negative. bactx requires a specific analyzer and 30 minutes are required for result interpretation. there is no instrument requirement for pgd and reactions can be read within 20 minutes. conclusion: the results of this evaluation makes pgd the best fit for this blood center based transfusion service. pgd offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for lrap in 100% plasma and lrap in pas/plasma. its ease of use allows for testing of lrap on day 4 and day 5 during the night shift to be accomplished without additional staffing and allows to extend outdate to 7day storage of lrap. change in growth factor content of human serum for use as eye drops during frozen storage for 1 year jos lorinser 1 , pieter f van der meer 1 , hans van der heiden 2 and dirk de korte* 1 . 1 department of product and process development, sanquin blood bank, 2 mu-drop background/case studies: growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome. stability of growth factors during frozen storage in mini containers (140 ml) is unknown. if these products can be stored at -188c it will be feasible to store this product in 3-star household freezers, making the product available for patients in need of serum eye drops. the purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at -188c or <-25 to -358c packed in a new micro dose device for single use as eye drops. study design/method: serum produced from 500 ml whole blood donations from non-remunerated healthy donors was quickly frozen. after frozen storage at <-258c for 3-12 months and controlled thawing, six different sera were used to fill a large number of mini (140 ll) containers, which were refrozen and stored at either -188c or <-258c. during storage at 3 months intervals, samples were tested for several growth factors, using magpixv r luminex multiplex assays and compared to control samples stored at <-808c. growth factors tested were pdgf-aa&ab/bb, tgf-ß1/2/3, vegf, 80a transfusion 2017 vol. 57 supplement s3 egf, fgf2. the study was a fact-finding study, without preset acceptance criteria. results/finding: pdgf-ab/bb and tgf-ß1 were the most abundant growth factors, on average 35, resp. 40 ng/ml. also pdgf-aa was detected at relatively high concentration in human serum, on average 11 ng/ml. tgf-ß2, egf and vegf were detected at relatively low values, resp. 3 ng/ml, 0.5 ng/ml and 0.3 ng/ml. average levels of fgf2 and tgf-ß3 were close to detection limit (< 0.2 ng/ml). the controls stored at <-808c showed for all growth factors close to 100% of the initial values in samples at t50 (moment of filling mini containers). for serum stored at <-258c for up to 12 months, most factors showed less than 2 % decrease, except for pdgf-aa and tgf-ß2, showing 6% resp. 3% lower values. for serum stored at -188c the values for tgf-ß1, egf and vegf were stable, whereas pdgf-ab/bb, pdgf-aa and tgf-ß2 showed a decrease of resp. 9, 17 and 3%. conclusion: human serum eye drops can be stored in the new micro dose device at -188c (3-star household freezers) or <-258c (professional freezers) for at least one year after preparation without large decreases in growth factor content. the maximum decrease was found for pdgf-aa in serum stored at -188c. it is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness. further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond 1 year. ruqayyah almizraq* 1 , heather inglis 2 , phillip norris 2,3 , jennifer a muszynski 4 , nicole juffermans 5 , jelena holovati 1 and jason p. acker 1,6 . 1 university of alberta, 2 blood systems research institute, 3 university of california, san francisco, 4 nationwide children's hospital, 5 academic medical center, 6 canadian blood services background/case studies: different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components. the aim was to identify, quantify and characterize residual cells and extracellular vesicles (evs) in stored rbc products produced by different blood manufacturing methods. study design/methods: thirty-two rbc units produced using whole blood filtration (wbf), red cell filtration (rcf), apheresis, and whole blood derived (wbd) methods were examined (n58 per method). residual platelets and white blood cells (wbcs) were measured on day 5 using flow cytometer (fc). on storage day 5 and 42, number and cell of origin/surface markers of evs were assessed with fc, and concentration and size-profile of evs were examined using tunable resistive plus sensing (trps). results/findings: on day 5, apheresis and wbd units had significantly greater residual platelets in comparisons to rcf (vs: apheresis p<0.01, wbd p<0.05) and wbf (vs: apheresis p<0.0001, wbd p<0.01) methods. while rcf units yielded the lowest count of platelet-evs (cd41a1) on day 5 and 42, the highest number of platelet-evs were in apheresis (day 5) and in wbd (day 42). similarly, there was significant difference among methods in the number of wbc-evs (cd31, cd141, cd161, cd191, cd66b1) and rcf contained the smallest concentration. moreover, both trps and fc showed an increase in the total number of evs on day 42 vs day 5 in all of the processing methods. noteworthy, trps showed that the number of small evs/exosomes (< 200 nm) was greater than large evs (! 200 nm) in all of the products on day 5 and 42, and the highest level of evs < 200 nm were in apheresis units. trps results also showed a significant difference in the evs size-profile amongst all rbc products (p<0.05). conclusion: this study shows that the method of manufacturing significantly affects rbc and non-rbcs evs characteristics throughout storage, which has the potential to impact quality and safety of rbc products. the differences in the evs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences. coagulation and complement assays in whole blood stored at 48 centigrade maryanne c herzig* 1 , crystal lafleur 2 , chriselda g fedyk 1 , sherrill j. slichter 3 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research, 3 university of washington background/case studies: whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least 2 weeks of storage at 48c without agitation. it may be possible to extend the preservation of platelet function by agitating wb. in order to more fully characterize the quality of wb stored at 48c with or without agitation, we evaluated complement activation as a marker of inflammatory potential. study design/method: subjects donated one unit of wb collected in cpd-a2 (citrate phosphate dextrose anticoagulant with adenine). the wb was not leukoreduced nor was it separated into components. units were stored under refrigerated conditions for 10, 12, 15, or 22 days after collection. units were stored for 12 days without agitation. units stored for 10, 15 or 22 days were agitated during storage with a model 400 hybridization incubator at 48c set for end over end rotation at 2-3 rpms. at the appropriate time point, platelet free plasma was obtained from the wb sample and stored at -808c. the frozen plasma was analyzed by elisa assays to determine: thrombinantithrombin complex (tat) as a marker of coagulation; soluble cd40l as a measure of platelet activation and granule release; plasmin anti-plasmin complex (pap) as a marker of fibrinolysis; plasminogen activator inhibitor (pai-1) as another fibrinolytic measure; and complement activation markers c3a, c4d, c5a and c5b-9. data was analyzed by one way repeated measure anova. results/finding: only 49 6 12% of the platelets were recovered in units stored for 12 days without agitation. these levels did not meet fda requirements of 5.5 x 10 10 platelets per wb unit. subsequently, wb was agitated and platelet recovery was 71-76%. no difference was seen in elisa analysis for agitated or non-agitated samples. no change was seen in tat or pap levels between t0 (day of collection) and t10, 12, 15, or 22 measurements. significant elevations of pai-1 and scd40l indicate activation of platelets and inhibition of fibrinolysis (p<0.001). activated complement peptides c3a, c5a, and c4d were all elevated over time (p<0.001) while sc5d-9 was not. however, only c3a and c4d levels at t22 were above normal reference ranges at 1.30 and 1.41 times maximum reference, respectively. conclusion: whole blood agitation appeared necessary to recover platelets at or above fda requirements. whole blood stored at 48c for 10-22 days did show some activation of complement proteins. in contrast to studies in stored red blood cells with elevations of sc5d-9 reported, wb showed elevation of c3a, 5a and c4d and not sc5d-9. complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life. meredith lummer* 1 and christian todd 2 . 1 cerus corporation, 2 community blood serivces background/case studies: the interceptv r blood system for platelets (cerus, concord ca) is used for the pathogen reduction (pr) of platelet collections, and replaces irradiation, cmv testing, bacterial culture and point of issue bacterial testing. to better understand pr compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly 1.1x10 10 6 5.6x10 9 9.8x10 10 6 5.6x10 10 310 6 330 430 6 440 900 6 260 28000 6 33000 3 6 3 186 31 30 6 22 110 6 97 rcf 1.9x10 10 6 7.4x10 9 4.2x10 10 6 1.1x10 10 13 6 4 316 14 530 6 160 5100 6 2000 3 6 3 96 7 176 7 346 11 apheresis 2.4x10 10 6 2.0x10 10 1.0x10 11 6 6.1x10 10 520 6 320 700 6 310 2200 6 1900 9800 6 4100 14 6 17 7 6 5 466 15 120 6 24 wbd 6.4x10 9 6 3.1x10 9 4.6x10 10 6 1.5x10 10 350 6 140 760 6 360 1000 6 180 4400 6 2400 3 6 2 426 23 57 6 24 120 6 56 platelet collections must meet specific volume, concentration, and dose ranges to qualify for intercept pr. changes made to apheresis devices included adding the following 4 collection targets: 4.4x10 11 in 350ml, 6.6x10 11 in 400ml, 6.8x10 11 in 400ml, and 7.0x10 11 in 400ml. study design/methods: four months of collections were retrospectively analyzed. platelet collections were evaluated to determine eligibility for pr treatment, and all products meeting pr processing specifications (unless intended for an hla matched recipient at a hospital not able to accept pr products) underwent pr treatment regardless of potential impact to split rate. a minimum post-treatment dose of 3.0x10 11 or 6.0x10 11 was required to classify collections as singles or doubles respectively. volume/dose mitigation (removal of volume to increase the number of products eligible for pr) was not utilized during this study. thus units were treated conventionally if volume, dose, and/or concentration did not meet pr specifications without further manipulation. results/findings: 64% of all single and double collections were eligible for and underwent pr treatment. split rate for single and double collections was 1.34. conclusion: it is possible to treat 64% of single and double platelet donations with intercept pr at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices. platelet collections that fall outside of the specifications for pr are processed and distributed as conventional products. strategies to increase eligibility toward 100% while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation. further evaluation is needed to determine the additional quantity of pr eligible products resulting from such changes. monique p gelderman* 1 , andrey skripchenko 1 , fei xu 1 , ying li 1 , stephen j wagner 2 , pamela h whitley 3 and jaroslav g vostal 1 . 1 fda/cber/ obrr/dbcd/lch, 2 american red cross holland laboratory, 3 american red cross mid-atlantic research facility background/case studies: platelets (plts) stored at room temperature (rt) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur. storing plts at cold temperature (4-6 o c [ct]) limits bacterial growth but results in rapid clearance upon transfusion. the development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies. thus, an animal model of plt circulation that could predict performance of human plts in human volunteers would positively impact the development of alternate storage conditions. study design/method: we designed an immunodeficient (scid) mouse model to evaluate recovery of human plts and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new plt storage condition: thermocycling plts (11 hrs ct: 1 hr 37 o c [tc]). autologous apheresis plts stored for 7-days at rt, tc and ct were radiolabeled and infused into healthy human volunteers (n59) and the same non-labeled plts were also infused into mice (n590). blood samples from humans and mice were collected over time to generate survival and clearance curves of the plts in circulation. flow cytometry was used to detect and analyze the human plts in the mouse samples to generate such curves; counts <5% were considered background. results/finding: the mean recoveries of infused plts were 51.2 6 16.7% for rt, 37.7 6 12.3% for tc and 23.1 6 8.8% for ct in humans. in mice, mean recoveries of the same plts were 24.9 6 10.3% for rt, 19.1 6 9.8% for ct and 16.2 6 6.9 for ct (mean6sd). to compare performance of the plts in humans and mice we expressed all recoveries as a percentage of the rt recoveries. in humans tc was $74% and ct was $45% of rt. in mice tc was $76% and ct was $64% of rt. the area under the survival curve (auc) was calculated for the individual mouse study and human trial data sets. the results of both auc were normalized to 100% for rt plts. human tc plts had 26% auc while ct plts had 11% auc compared to rt plts in humans. in comparison, the same tc plts had 39% auc and ct plts had 26% auc of the rt auc in the mice. the calculated ratios of the auc between the tc plts and ct plts of the human data set and mouse model data set are 2.4 and 1.5, respectively. conclusion: the scid mouse model differentiates between rt plts and ct plts similar to humans based on auc and plt recovery data. however, the mouse model cannot differentiate between ct plts and tc plts as occurs in humans. even though the mouse model cannot differentiate between ct plts and tc plts, it may still be a useful tool to screen other novel storage conditions for human plts. converting the component manufacturing from a manual process to automation nicole peters* 1 and geeta paranjape 1,2 . 1 coastal bend blood center, 2 carter blood care background/case studies: initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes. our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation. the compomat g5 was evaluated using a multi-team approach including component manufacturing staff, equipment management, qa, regulatory affairs and it. study design/method: after a comprehensive evaluation, the team decided to purchase the compomat g5 with the compomaster net software for data management. implementation was planned for a november 2014 go-live. . to centralize processing, new work counters were installed. fresenius kabi installed the compomat g5s and compomaster in june 2014. training and validations were successfully completed and a full launch occurred mid-march 2015. device and sop training was performed. training qualification checklists were completed for each technician with a required number of successful units processed and completed december 2014. validation was completed and signed off in march of 2015. manufacturing data was collected using the compomaster net data management system and our quality control software for platelet (plt) parameters, including plt count, plt weight, and plt yield from before implementation (bi) and after implementing (ai) of the compomat g5 system. data points were collected from 210 units bi and 302 units ai. results/finding: upon initial implementation, staff training and use, the compomat g5 was found to be easy. plt weight spread was reduced from an average of 22gm to an average of 15 gm. actual plt weights were reduced from an average of 63gm to 59gm, resulting in an average increase in recovered plasma of 3.78ml per unit. plt count on average increased from a count of 1435 to 1506 (10 3 /mm 3 ) with a negligible change in plt yield. conclusion: plt weight spread was reduced by 31.8% after implementation of the compomat g5 and our plt concentrations increased on average by 5%. we were able to consistently produce a smaller volume plt (average 59 gm), which gave us 3.78ml more plasma per unit for recovered plasma. the team intends to review a dryer cryo as a next step for potential additional plasma yields for recovered plasma. deglycerolization of manually glycerolized, frozen rccs using a closed system cell processor anita howell 1 , angela hill 1 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (rccs), many rare rccs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory. a study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (rbc) in vitro quality. as the closed cell processor uses a fixed centrifuge bowl for deglycerolization and rbc resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product. study design/methods: 13 abo/rh matched lr sagm rccs were pooled and split to produce 6 large (354 ml) and 6 small (244 ml) rccs. the rccs were stored to 14 d and glycerolized manually by mixing 400 ml of glycerol with the rcc in a 2000 ml freezing bag. units were frozen at -658c for ! 72 h before being removed from frozen storage and thawed in a 378c water bath. 3 large rccs and 3 small rccs were deglycerolized using the organization's current procedure on the cobe 2991 cell processor prior to re-suspension in 0.9% saline, 0.2% dextrose. the remaining rccs were transferred into a 1l bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of 75 6 5%, and deglycerolized in a 275 ml centrifuge bowl on the acp-215 with re-suspension in as-3. rbc quality was tested at 24 6 2 h post-deglycerolization. results/findings: large rccs had significantly higher hemoglobin per unit (cobe: p50.006, acp215: p50.007) and lower cell recovery (cobe: p50.002, acp215: p<0.001) post-deglycerolization than smaller rccs on both cell processors. large rccs deglycerolized on the cobe 2991 had higher hemolysis (p<0.001) and supernatant potassium (p50.001) than did small volume rccs. large cobe 2991 rccs had higher hematocrits (p50.033), hemoglobin (p50.006), and recovery (p50.001) than did large acp-215 rccs. however, all cobe 2991 rccs had higher (p<0.001) hemolysis (0.99 6 0.24 %) levels than did acp-215 rccs (0.31 6 0.02 %). cobe 2991 rccs failed to meet regulatory hemolysis standards of 0.8%. conclusion: addition of a 400 ml bolus dose of glycerol to rccs of different volumes results in different concentrations of glycerol in the frozen rcc product and may lead to differences in frozen rcc quality. additionally, the size of the rcc impacts quality for rccs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits. use of the closed cell processor with resuspension in as-3 and storage for 24 6 2 h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in rccs glycerolized manually. the acp-215 cell processor can therefore be used to deglycerolize rccs glycerolized using a manual, open system glycerolization method. background/case studies: washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions. platelet washing process is time-consuming which may delay transfusion. this study was conducted to evaluate the manual platelet washing method (mm) using 0.9% saline and centrifugation and the semi-automated washing method (sam) using the cobe 2991blood cell processor. study design/method: in this study, 20 units of single donor platelets were evaluated (10 washed using the mm and 10 washed using the sam. the collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate. the platelet counts were measured on the sysmex exn and the total plasma protein samples were measured on the roche cobas 6000. results/finding: table 1 shows that the average platelet recovery for the sam (92%) was significantly higher compared to the mm (82%). the mm had a slightly higher average protein removal compared to the sam. no platelet clumps were observed in either the mm or the sam. it was observed that the hands-on time for the mm took 10-15 minutes longer than the sam. background/case studies: the interceptv r blood system for platelets is currently licensed for pathogen reduction (pr) of amicus platelets in inter-sol (pas-3) for input platelet doses of 2.9 to 8.0 3 10 11 platelets in 255 to 420 ml of 47 to 68% plasma and 32-53% pas. a new platelet processing set was designed with three storage containers (ts) to process apheresis platelet components in pas-3 containing doses of 6.0 to 12.0 3 10 11 platelets in a volume of 420 to 650 ml. study design/methods: apheresis pcs (amicus v r ) were collected in 35% plasma and 65% pas-3. one study was performed at the nominal dose (9.2 -10.0 x10 11 platelets), volume (558 -629 ml) in 65% pas/35% plasma using single donor apheresis collections. two studies were performed to evaluate the high dose and high volume condition (9.7 -11.8 x 10 11 platelets in 593 -659 ml) using either single or pooled donations. input pcs (n520) were treated with the intercept ts set by the end of day 1 post collection; the incubation time in the compound adsorption device (cad) container ranged from 4 to 16 hours and the intercept treated pcs were stored in 3 containers (n560). day 5 and 7 post-donation pcs were evaluated using a panel of in vitro platelet function assays results/findings: in vitro function data for apheresis pcs in pas-3 treated in the intercept ts set demonstrated acceptable in vitro function (table 1 ). all intercept treated pcs had ph(228c) !6.2. platelet dose and volume recovery post-treatment ranged from 82% to 99% and 88% to 92%, respectively. conclusion: pathogen reduced platelet components processed using the intercept ts set from either single or pooled apheresis donations maintained acceptable in vitro quality through 7 days of storage. intercept blood system for platelets ts set is currently not approved for use in the us. background/case studies: the possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern. while current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats. the pathogen reduction technology (prt) represents a proactive strategy to further reduce transfusion-transmitted infectious risk. however, the scientific community broadly agrees over the fact that prt has negative impacts on the product's quality markers. this study aims at evaluating the impacts of the mirasol prt on platelet (plt) quality and plt processing. study design/method: two abo-compatible platelet concentrates (pcs) containing 100% plasma obtained from either apheresis or sagm whole blood (wb)-derived processing were paired, pooled and then split into two equal units. one unit was used as a non-treated control (ctrl) (n56). riboflavin was added to the other pc unit and then exposed to uv light according to the manufacturer's instructions for the mirasol prt (teru-mobct) (test) (n56). numerous in-vitro quality markers (plt concentration, atp, po2, pco2, ph, glucose, lactate, sodium, and potassium) were measured for both mirasol-treated and non-treated pcs on days 1, 3, 5 and 7 for apheresis pcs, and on days 2, 3, 5 and 7 for wb-derived pcs. two flow cytometry assays were used to evaluate cd62p expression with and without thrombin activation, and to measure the percent annexin vpositive plt. transfusion 2017 vol. 57 supplement s3 results/finding: platelet recovery was 92 6 5% and 81 6 10% for apheresis and wb-derived pcs, respectively. mirasol-treated pcs showed higher levels of annexin v-positive cells (3% 6 1 (test), vs. 1.7% 6 0.5 (ctl) on day 5) and a higher rate of cd62p expression than control pc units (58% 6 7 (test), vs. 23% 6 6 (ctl)) on day 5). the mirasol treatment generates changes in ph, glucose and lactate for pcs during storage. conclusion: the mirasol treatment induces a loss in the net number of plts/unit and elevated platelet activation. changes in ph, glucose and lactate suggest that prt affects plt metabolism. finally, prt has numerous impacts on logistic, storage and processing time constraints of blood bank operations. nevertheless, the mirasol prt is routinely used in europe with acceptable clinical outcomes. evaluation of a test method to detect bacterial contamination in platelets; bactx tm assay ji hye park sexton* 1 , lorraine blagg 2 , christi e marshall 1 , herman woodson 1 , sean erony 1 , krishna patel 1 and eric gehrie 3 . 1 the johns hopkins hospital, 2 johns hopkins hospital transfusion medicine dept, 3 johns hopkins university school of medicine background/case studies: bacterial contamination of platelets (plts) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality. therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical. the bactx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria. here, we report an analysis of the bactx assay at our hospital. study design/method: we aimed to determine the sensitivity and specificity of the bactx assay. 340 intact leukoreduced apheresis plt (lrap) units were tested by bactx at storage day 4. as a control, each intact lrap was also cultured by an automated bacterial detection system (bact culture) on storage day 3. the results of the bactx test were compared to the results of the bact culture system. results/finding: a total of 340 lrap were tested. 335 lraps initially tested negative by bactx, while 5 lraps initially tested positive by bactx. all 5 initial positive bactx tests were negative when subjected to repeat testing. in contrast, all lraps tested negative with the bact culture system. the specificity of the bactx test was 98.5%. we did not have any true positive test results; therefore, the sensitivity of the bactx could not be determined. conclusion: this is a small study of only 340 platelet units. the expected rate of bacterial contamination of platelets is less than 1 per 2000 units. the 1.5% initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the bactx assay. in vitro quality of rejuvenated and washed cpd/as-1 and cp2d/as-3 rbc alan d. gray* 1 , matt landrigan 2 , pamela whitley 3 , michael wellington 3 , sherrie sawyer 3 , shalene hanley 3 , emily rondeau 4 , louise herschel 4 , neeta rugg 5 , patricia a.r. brunker 3 , shawnagay nestheide 5 , jose cancelas-perez 5 , larry dumont 6 and zbigniew m. szczepiorkowski 7 . and 2,3-dpg to fresh levels. the objective was to demonstrate that in vitro quality measures are maintained for rbc when stored for >24 hours after treatment with an fda approved rejuvenation solution. study design/method: whole blood (530-550 ml) was collected and processed at 3 sites into leukocyte-reduced rbc (a total of n563 cpd/ as-1 and n564 cp2d/as-3). 50 ml of rejuvenation solution (citra labs) was added to each rbc on day 35 (d-35), incubated for 60 minutes with agitation at 378c water bath (helmer dh4), washed (haemonetics acp215), and stored in as-3 at 1-6 oc for 7 days (d-36 through d-42). in vitro recovery (%) was calculated and hemolysis, atp, and 2,3-dpg were determined on day 0, d-35, d-35 after rejuvenation and washing (postrjv), d-36, d-38, d-40, and d-42. all units were cultured on d-35 postrjv and on d-42, and then concentrated by centrifugation on d-42. results/finding: in vitro rbc recoveries were 95.7% and 95.5% (as-1 and as-3, respectively) and no bacterial growth was observed. hemolysis on d-42 was maintained <1% in 58/63 (92%) as-1 units and 63/64 (98.4%) as-3 units. all as-1 and as-3 units (100%) had hemolysis <1% following concentration by centrifugation. morphology score was reduced to 77% (as-1) and 74% (as-3) by d-35, restored after rejuvenation (91%, 92%, respectively) and maintained through d-42 (>90%). atp was restored and maintained above fresh levels after rejuvenation. 2,3-dpg was restored above fresh levels and was maintained !80% of fresh levels through d-38. all values were significantly different compared to d-35 except as noted (p<0.001, paired ttest) ( table 1) . conclusion: rejuvenation of stored rbc restores atp and 2,3-dpg above fresh values and morphology to near fresh levels while maintaining improved in vitro rbc quality measures through d-42 when compared to nonrejuvenated rbc on d-35. this study is funded by zimmer biomet. storage >24 hours is not fda approved for use at the time of this publication. liposomes and rejuvenation: new approach for improving quality of stored red blood cells luciana da silveira cavalcante 1 , jason p. acker* 1,2 and jelena holovati 1 . background/case studies: liposomes have been shown to minimize rbc membrane damage occurring during 42-day hypothermic storage (hs), while rejuvenation solutions have been shown to restore rbc metabolism by maintaining atp and 2,3-dpg levels. this study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored rbcs. study design/methods: five leukoreduced packed rbc units obtained were pooled and split. the units produced were segregated into four experimental groups: sham control (s), liposome-treated (l), rejuvesol-treated (r) and liposome 1 rejuvesol-treated (l1r). the prbcs were incubated for 1 h at 378c with hepes-nacl (sham), liposomes (dopc:chol, 7:3 mol%, 2 mm lipid), rejuvesol or liposomes plus rejuvesol. the in vitro quality was accessed by hemolysis, deformability, aggregation, atp and 2,3-dpg at day 42 hs. results/findings: hemolysis was significantly decreased in all treatments compared to sham control (0.60 6 0.06%): l (0.53 6 0.01%, p50.042), r (0.43 6 0.02%, p50.004), l1r (0.48 6 0.06%, p50.020). ektacytometry analysis showed an increase in maximum elongation (ei max ) in r (0.55 6 0.01, p50.010) and l1r (0.55 6 0.01, p50.010) treatments compared to s (0.53 6 0.01) but not l (0.53 6 0.01, p50.936). rbc rigidity (kei) increased in all treatments compared to sham (1.19 6 0.07): l (1.28 6 0.06, p50.025), r (1.44 6 0.17, p50.010) and r1l (1.44 6 0.06, p50.004). aggregation amplitude was significantly increased by r treatment only (24.07 6 1.67 au vs. 19.12 6 1.38 au, p50.004). atp levels were significantly higher in all treatments compared to sham (1.64 6 0.14 mmol/g hb): l (2.00 6 0.21 mmol/g hb, p50.010), r (4.70 6 1.20 mmol/g hb, p50.004), l1r (5.00 6 1.56 mmol/g hb, p50.004). the levels of 2,3-dpg were no longer detectable in s and l treatments at day 42. the combined treatment was comparable to r (2.38 6 3.26 mmol/g hb vs. 2.62 6 2.20 mmol/g hb, p50.868). conclusion: both rejuvenation and liposome treatments improved the quality of stored rbcs compared to sham control. the combined treatment (l1r) did not have a greater impact in improving in vitro quality of stored rbcs compared to rejuvenation alone. step toward a unique and adaptable thermoregulation system lucie boyer 1 , eric ducas 1 , patricia landry 1 , nathalie dussault 1 , jacques bernier 1 , danny brouard* 1 and anne maltais 2 . 1 h ema-qu ebec, 2 institut de technologie des emballages et du g enie alimentaire background/case studies: h ema-quebec (hq) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area. in collaboration with the institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the 500-ml whole blood leukotrap rc system (haemonetics corp.). the objective is to design a packaging system for the rapid cooling (t < 108c) of one to six 500-ml whole blood units (wbu) within 8h from collection. moreover, the insulating and thermoregulation system must maintain the internal temperature of wbu between 18c and 108c for 24h under extreme external conditions (-308c to 408c), including the initial blood cooling period. study design/method: the proposed packaging design is based on an external coroplast box containing six vacuum insulated panels (vip) for increased insulating efficiency. preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned 58c phase change material (pcm). the number of pcm, their position and conditioning were optimized and tested in order to meet the expected performance criteria. preconditioned pcm were stored into vip boxes for 24h at 20-248c before each test to mimic a worst-case scenario for remote blood drives. for the experimental testing, 500-ml wb bags were filled with 555 ml saline 0.9% at t 5 308c to mimic freshly collected wb. probes were positioned inside the saline-filled bags to monitor temperature profiles of wbu under extreme winter (-308c) and summer (408c) conditions. shipping boxes were filled with either one or six bags (n5 2). results/finding: the results showed that the thermoregulation box prototype is able to cool wbu bags under 108c in 4.55 6 0.62h and maintain their internal temperature between 18c and 108c for 24h with final values ranging between 6.38c and 9.38c for the extreme summer scenario. similar results were obtained for the extreme winter scenario; units reached the 108c threshold value in 2.4 6 0.2h and the bags' internal temperatures were within the acceptable range for 24h. conclusion: the insulating and thermoregulation system met hq performance criteria. preliminary results showed that pcm could be conditioned at temperatures higher than -138c without any significant impact on the system performances. hq is currently validating the shipping box prototype performances. additionally, we are working on reducing the pcm conditioning time to optimize logistic operations. as this packaging has many advantages in terms of durability, price and convenience, hq intends to evaluate this system for the packaging and transport of other lines of blood products. stuart weisberg* 1 , christopher c. c silliman 2 , beth shaz 1 , marguerite kelher 2 and claudia s. cohn 3 . 1 new york blood center, 2 bonfils blood center, 3 department of laboratory medicine and pathology, university of minnesota background/case studies: platelets collected and stored in platelet additive solution (pas) reduce recipient exposure to donor plasma components. to better define the effects of pas on platelet supernatant composition, we compared total protein, isohemagglutinin titers, hla antibodies and in vitro neutrophil (pmn) priming activity in supernatants of pas-c platelets to plasma platelets. study design/methods: apheresis platelets from group o blood donors were collected into either 100% donor plasma (n550) or 65% pas-3 / 35% donor plasma (n550). within 12 hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-a and anti-b titer, and pmn priming activity within the total and lipid extractable fractions. all samples were screened for hla antibodies. screen-positive samples were tested using luminex single bead assays for antibody strength and specificity. soluble cd40 ligand (scd40l) was measured using solid-phase elisa. results/findings: supernatants of pas-c platelets had significantly lower total protein concentration, anti-a and anti-b titers compared to plasma platelets. there was no significant difference in the number of hla-antibody screen positive pas-c (3/50 products) compared to plasma platelets (2/50 products); however, the hla-antibody screen-positive supernatants of pas-86a transfusion 2017 vol. 57 supplement s3 abstract c platelets had fewer hla specificities (2 specificities) compared to those of the plasma platelets (18 specificities). pmn priming activity was significantly increased in the supernatant of pas-c platelets. the lipid extractable fraction was not affected; however scd40l levels were increased in the supernatant of pas-c compared to plasma platelets (table 1) . conclusion: decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of pas-c platelets. decreased anti-a and anti-b titers may prevent hemolysis from minor abo mismatch. lower hla-antibody specificities may mitigate transfusion related acute lung injury (trali). increased pmn priming by pas-c platelets is likely due to platelet membrane release of scd40l and not bioactive lipids. although scd40l has been associated with trali, only pmn priming with lipid -not cytokine -agents has been causally linked with trali. the mechanism and clinical impact of increased scd40l in pas-c platelets remain to be elucidated. background/case studies: current guidelines require a reduction of residual white blood cells (rwbc) below 5x10 6 wbc in us and 1x10 6 wbc in europe, per unit. the established reference method for testing rwbc in platelet (plt) and red blood cell (rbc) products is flow cytometry. alternative technologies have been developed including hemocytometry and microfluorometry. study design/methods: this study compared performance and workflow efficiency of the facsvia, a flow cytometer with a simplified workflow and automated loader to the adam automatic microscopic cell counter based on imaging technology. nonfiltered whole blood (wb) samples, apheresis platelet units (n52) and leukoreduced (lr) rbc units (n52) were used to generate spiked samples. apheresis platelets and lr rbc were filtered to deplete wbcs and were used as a diluent. nonfiltered wb samples were the source of wbcs to prepare a sample of 1000 wbc/ul. the spiked samples of 5, 12.5, 5, 25, 50 and 100 wbc/ul were prepared from the source sample of 1000 wbc/ul and filtered platelet and rbc units. to evaluate linearity, wbc concentrations (0, 12.5, 5, 25, 50, 100 wbc/ul) were measured using adam and facsvia. samples were stained and run in triplicate on each analyzer. data was analyzed using linear regression. the results were proportional to the wbc concentration in the spiked samples. reproducibility of the two systems was measured by running spiked samples (0, 5, 25, 50 wbc/ul). 10 tubes of each sample were stained and run per system. the %cv and %diff were calculated. a batch of 20 samples (plt and rbc) were run on both analyzers, repeated for 5 days. workflow efficiency was assessed observationally by measuring the time of tasks performed. tasks recorded were instrument qc, assay controls and sample testing and analysis. results/findings: the wbc concentration results for plt and rbc samples on facsvia correlated well with adam (r-plt50.996, slope50.972), (r-rbc50.999, slope50.992). the %diff-plt at 5, 25, 50 wbc/ul were 7.8, 4.7 and 10, respectively. the %diff-rbc at 5, 25, 50 wbc/ul were 10.8, 3.2 and 14.7, respectively. the average total testing time was similar on both instruments; 89 min for the facsvia and 92 min for the adam. of the total testing time, adam required continuous hands-on time, while facsvia demonstrated 62% (56 of 89 min) hands-off time. conclusion: both instruments showed comparable precision, linearity and accuracy. while the average total testing time was similar on both instruments, facsvia offered a significant workflow efficiency advantage. users saved an average hands-on time of 56 minutes that could be used on other tasks. platelet rich plasma and quality control: is there a role for the blood bank? claudia s. cohn* 1 and mickey koh 2 . 1 department of laboratory medicine and pathology, university of minnesota, 2 st george's hospital and medical school background/case studies: autologous platelet-rich plasma (aprp) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases. prp isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aprp is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment. thus, the consistency and quality of aprp is questionable and the lower yielding prp may have decreased efficacy. study design/methods: a survey was designed to assess aprp manufacture, usage and quality control (qc) measures taken prior to its use. a survey was developed with input from content experts. the survey was sent to members of best and isbt. survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown. a total of 62 completed and partially completed surveys were received. results/findings: responses came from 13 countries, but the majority of responses came from the united states (us). of the respondents, 35% reported aprp use in their hospital. aprp was used predominantly for outpatients, though >40% of hospitals also used aprp in the in-patient setting. in most hospitals, aprp was used by 1-5 mds; however, 3 hospitals had >10 mds using aprp. the aprp was used for orthopedics, wound/incision repair, rheumatology and other indications. in the us the aprp was manufactured outside of the blood bank, while outside the us aprp was isolated by blood bank personnel. nearly all the aprp manufacturing was done with no quality control (qc) measures (97%); however, 3 respondents assessed the final product prior to release. these qc measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing. in some cases, if the aprp failed qc it could still be used, pending an md's approval. in the 3 hospitals conducting qc on the final aprp, the testing was done by the blood bank. a subset of respondents from african nations also used allogeneic prp (allprp). in contrast to the patterns of use with aprp, allprp was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'. the allprp was manufactured in the blood bank or the donor center with no qc other than a regular check of the centrifuge used to isolate the prp fraction. conclusion: prp is used in hospitals throughout the world for a wide variety of indications. the blood bank is involved in its manufacture in some countries, but in the us aprp is made outside of the blood bank. quality control of aprp production and the final product is not done in most hospitals. to improve the consistency and efficacy of prp, more stringent qc measures need to be in place. background/case studies: the morphology of donated red blood cells (rbc) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (ps), and decreased intracellular atp. these changes have been associated with increased rbc clearance within hours of transfusion. analysis of morphological alterations of stored rbc with imaging flow cytometry (ifc) has identified a subpopulation of small rbc that accumulates upon storage. this rbc subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (roussel, dussiot et al, 2017) . some of the storage alterations are reversible when the rbc metabolism is reestablished. as such, treatment with a rejuvenation solution (citra labs) before transfusion is expected to restore some of the rbc properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion. study design/methods: a multi-parametric analysis of rbc alterations was performed to evaluate the effect of rejuvenation on rbcs stored in sagm (n56) under blood bank conditions at day 3 (d3), at day 42 (d42), after rejuvenation (r), and after rejuvenation and washing (rw). morphological alterations of stored rbcs were evaluated with ifc (imagestream x mark ii, amnis v r ). results/finding: rejuvenation increased the level of intracellular atp, confirming the metabolic effect of this process. population distribution as per rbc projected surface area measured by ifc depicted a well-demarcated subpopulation of small rbc that increased with storage from 2.1-8.8% at d3 to 8.3-68.1% at d42. rejuvenation markedly reduced this storage-induced spherocytic shift (1.7-29.3%) and partially restored rbc morphology, an effect confirmed by differential interference contrast microscopy. the restoration effect of the rejuvenation process did not correct the storage-related loss of rbc elongation but was associated with a decrease in ps exposure (table) . conclusion: our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation. the impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation. red cell concentrate volume and manufacturing method impact post-thaw quality in cryopreserved products processed using a closed cell processor anita howell 1 , angela hill 1 , tracey turner 2 , april xu 2 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: the blood service uses both top/top with whole blood filtration (wbf) and top/bottom with red cell filtration (rcf) methods to prepare cpd/sagm lr red cell concentrates (rccs). mean volume (ml) is higher in wbf units (314 6 15) than in rcf units (275 6 16), with similar hematocrits. a closed system cell processor is currently being implemented for cryopreservation of rccs. post-deglycerolization re-suspension in as-3 additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size. the impact of the resulting variation in hematocrit on post-thaw in vitrorbc quality was evaluated to ensure that regulatory standards can still be met for rccs at the extreme edge of the input volume range. study design/methods: 12 small rcf (252-263 ml) and 12 large wbf (322-353 ml) rccs were stored for 21 d before being glycerolized and frozen at -658c for !72 h. large rccs whose red cell mass exceeded the capacity of the 275 ml deglycerolization centrifuge bowl were volume reduced prior to glycerolization. rccs were thawed in a 378c water bath, deglycerolized and re-suspended in as-3. rccs were stored 14 d and then tested for in vitrorbc quality. results/finding: small rcf rccs had lower (p<0.05) hematocrit, specific gravity, hemoglobin per unit, supernatant k 1 and na 1 concentration, deformability (ei max ), and higher (p<0.001) recovery than did large wbf units. no significant differences in hemolysis, atp, 2,3-dpg, p50, rbc indices, rbc morphology, or residual glycerol were seen between groups. the majority of units met acceptance criteria (table 1) , however 8 of 12 large wbf units had rbc recoveries < 80% due to pre-glycerolization volume reduction, and 2 of the small rcf units had hemoglobin values < 35 g per unit. when the recovery and hemoglobin failure rates are analyzed against the organization's rcc production volume distribution, the mean recovery is projected to be well above 80% and the hemoglobin failure rate would be below 10% of units tested; compliant with regulatory standards. conclusion: the differences between groups in the cryopreserved rcc physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass. the lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality. 0.03 6 .02 0.33 6 .28 0.83 6 .3 0.32 6 .14 elongation index (30pa) 0.602 6 .008 0.585 6 .017 0.580 6 .017 0.578 6 .017 this study is funded by zimmer biomet. (hasan 1994) . the objective was to determine the effect of rbc rejuvenation on rbc oxygen release capacity (orc) and estimated oxygen consumption (vo 2 ) after simulating a single unit transfusion of either standard or rejuvenated rbc stored for 42 days. study design/method: oxygen dissociation curves (odc) (hemox analyzer, tcs scientific) were generated from fifty-two (52) rbc units (leukocyte-reduced), cpd/as-1 or cp2d/as-3, on day 0, day 42, and after rejuvenation and washing (pw). the odc for each sample was used to determine orc (ml o 2 /g hb) and total releasable oxygen (tro) of the unit (ml o 2 ). orc was determined by assessing the change in % o 2 saturation from 100 mm hg po 2 (e.g., lung) to 40 mm hg po 2 (e.g., venous blood) multiplied by 1.34 ml o 2 /g hb (li 2016). a simulated baseline pretransfusion vo 2 of 115 ml o 2 /min was estimated using the day 0 orc and assuming a 7 g/dl transfusion trigger with a cardiac output of 5 l/min and 5 l blood volume. paired student's t tests were used for comparative statistical analyses. results/finding: rbc rejuvenation on day 42 restored orc and tro to levels greater than day 0 ( table 1) . orc of the rejuvenated unit was 1.5 6 0.2 times and 3.4 6 0.5 times greater than rbc on day 0 and day 42, respectively (p<0.001). vo 2 increased after a simulated single unit transfusion of rbc (day 0, day 42, and pw) by 19.3%, 8.9%, and 28.8% over the pre transfusion vo 2 , respectively (p<0.001). conclusion: these results suggest a transfusion with rejuvenated rbcs has the potential to release 3.3 times the volume of o 2 compared to standard, untreated rbcs stored for 42 days. inferior oxygen delivery to tissues (vo 2 max) has been observed during exercise in healthy human volunteers after transfusion of two autologous rbc units stored for 42 days vs 7 days which seem dependent on genetic variability and storage time (bennett-guerrero 2017). therefore, transfusion practices to correct anemia may be less effective than intended due to the variable orc of standard stored rbc units. transfusion strategies should consider whether the use of rbc with increased orc may be physiologically advantageous. disclosure: this study was funded by zimmer biomet. rejuvenation solution as an adjunct storage solution maintains physiological hemoglobin oxygen affinity during rbc unit storage andrea ansari* 1 , jay srinivasan 1 , gustaaf de ridder 2 , alan d. gray 3 , matt landrigan 4 , keaton charles stoner 5 , angela crabtree 6 , jessica poisson 7 and ian welsby 8 . 1 duke university school of medicine, 2 duke health pathology, 3 citra labs, a zimmer biomet company, 4 zimmer biomet, 5 duke university, 6 department of pathology, durham veterans affairs medical center, 7 duke university hospital, 8 duke university medical center background/case studies: deleterious changes develop during the storage of packed red blood cells (rbcs) collectively called the "storage lesion". these include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of atp, snitrosohemoglobin, vasodilatory capacity, and cell surface ps expression, and depleted 2,3-diphosphoglycerate (2,3-dpg). the loss of 2,3-dpg increases the oxygen affinity of hemoglobin, resulting in lower p50 (partial pressure of oxygen at 50% hemoglobin saturation). decreased p50 may negatively impact the ability for transfused rbcs to release oxygen to peripheral tissues. an fda-approved rejuvenation solution (citra labs) can restore normal levels of atp and 2,3-dpg, normalizing membrane function and oxygen affinity, respectively. this process requires incubation at 378c for an hour, an impractical step in time-sensitive situations, followed by washing of the rbcs. we tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of rbcs. study design/method: eight units of group a1, leukoreduced prbc stored in as-1 were obtained from our local blood center. after 3 days of storage, units were divided into 4 separate aliquots: control (ctl), wash (w), standard rejuvenation (sr), and cold rejuvenation (cr). the rejuvenation solution (50ml) was added to the cr group, and all groups were then stored for another 12 days at 1-68c. on day 15 of storage, the sr group was incubated for 1 hour at 378c with rejuvenation solution, after which the w, sr, and cr groups were separately washed on a c.a.t.s v r (fresenius kabi) using the high quality wash setting. hemoglobin p50 was measured by tonometry using a hemox analyzer (tcs scientific). deformability (elongation index or ei) was measured by ektacytometry (lorrca mechatronics). supernatant plasma free hemoglobin (pfhb) was measured using visiblelight spectrophotometry. cell surface ps expression (ps1) was measured by annexin v flow cytometry. all group results were compared using nonparametric wilcoxon signed-rank tests with a 5 0.05. results/finding: significant differences in p50 were noticed between all groups (table 1) . ei, ps1, and pfhb did not differ between groups. conclusion: cold rejuvenation prevents the increased oxygen affinity (lower p50) seen over 15 days of rbc storage without adverse effects on deformability or hemolysis. this offers an alternative to incubated rejuvenation to provide clinicians with ready access to rbcs with a high/normal p50 that may better release oxygen to the tissues. cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function. reactive antibodies and other inflammatory agents in rbcs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (trali), anaphylaxis, and even death. in this study, a multifunctional bead-based filter was evaluated for removal of k 1 , along with free hemoglobin (hb) and other prbc contaminants that can contribute to transfusion related adverse events. study design/method: ten leukocyte-reduced prbc (300ml) units stored in as-1, obtained from a regional blood donor center at expiration (42 6 2 days), were passed by gravity through sorbent-devices containing 50 ml of multifunctional polymer bead, at a flow rate of 20 ml/min. supernatants were analyzed for k 1 removal as well as free hb, antibodies and cytokines (27-plex, biorad). rbcs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively. results/finding: filtration of the aged prbc units through the sorbent device reduced [k 1 ] from 54.2 6 5.0 to 1.98 6 1.3 meq/l; equivalent to an 84.6% reduction. free hb was reduced by 96.3% from 2.5 6 1.0 to 0.39 6 0.2 mg/ml. antibodies, specifically igg, iga, and igm decreased from 9.91 6 3.1 to 2.40 6 1.1 mg/ml (77.7%), 0.48 6 0.1 to 0.25 6 0.01 mg/ml (48.9%), and 0.73 6 0.2 to 0.49 6 0.1 mg/ml (31.5%), respectively. inflammatory cytokines were significantly reduced, specifically: ip-10 from 144.27 6 16.2 to 18.43 6 2.7 pg/ml (87.2%), mip-1b from 37.37 6 5.7 to 7.23 6 2.5 pg/ml (80.7%), and pdgf from 1348.3 6 291.9 to 77.91 6 22 pg/ml (94.2%). filtration had no significant impact on cell surface markers of rbc viability (<0.1% decrease) or sensitivity to osmotic changes. values listed represent mean 6 sem (p < 0.01 for all analytes tested). a paired ttest was used to assess significance. conclusion: the sorbent filter was highly effective in reducing the levels of extracellular k 1 as well as free hb, antibodies, and cytokines from prbcs without impact on rbc viability or integrity. this study demonstrates the viability of a multifunctional sorbent filter for removal of k 1 along with other detrimental components from stored prbcs that can readily be incorporated into transfusion practices to minimize adverse effects. background/case studies: platelets carry no rh antigens, but residual red blood cell (rbc) in platelet products can immunize d negative recipients if the donor is d positive. current recommendation is to give rh immunoglobulin (rhig) to rh negative patient if they receive rh positive platelet unit to avoid potential alloimmunization to d antigen. a recent study has shown a very low frequency (1.5%) of d alloimmunization when a rh mismatch platelet is transfused. restricting d negative patients to receive only d negative platelets could create shortage and cause inventory challenges. higher yields of platelets with minimum to none residual rbcs are obtained with new generations of apheresis machines. as a consequence, the need for prophylactic rh immunoglobulin (rhig) may be unnecessary with the use of apheresis derived platelets. the accurate determination of residual rbc in a platelet unit is important for patient safety to prevent rh alloimmunization. hemocytometer is considered the gold standard for cell counting. however, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers. currently there are no standardization and/or guidelines to advise what system to use for rbc quantification in platelet products. study design/method: we designed this study to quantify the residual rbc in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods. we measured the amount of red blood cells per microliter in 50 apheresis and 50 whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, sysmex (sysmex america, lincolnshire, il) and advia 2120 (siemens healthcare diagnostics, tarrytown ny). the whole blood derived platelet units were produced using acrodose tm system technology. we conducted non-parametric permutation test based on 10000 permutations to compare sysmex and advia between apheresis and whole blood derived groups. abstract collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. study design/method: the addition of a rejuvenation solution to stored red blood cells (rbcs) has been shown to increase atp and 2,3-dpg profiles to fresh levels. the objective was to compare 50% hemoglobin-oxygen saturation (p50) and morphology profiles of fresh(day of collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. results/finding: in vitro rbc recovery (overall) was 97.2 6 2.2%. hemolysis (%) was similar on day 42 before and after dry-air incubation with the rejuvenation solution (0.34 6 0.14% vs 0.35 6 0.14%). percent hemolysis (%) decreased after washing (0.24 6 0.07%) and was maintained below <1% for all units during storage for 24hr (0.51 6 0.19%). average atp and 2,3-dpg were restored above the average fresh values. the morphology score decreased $25% by day 42, which was restored to near fresh values following rejuvenation and washing and storage 24hr (93.7% and 95.1%, respectively). rbc oxygen affinity, as assessed by p50, was restored above fresh values. all values were significantly different compared to day 42 (p<0.001, paired t-test) ( table 1) . conclusion: rbc morphology was restored to near fresh and average atp, 2,3-dpg, and p50 were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process. rbc morphology, atp and 2,3-dpg were maintained during storage 24hr. rejuvenation of refrigerated rbcs may offer avenues to improve rbc quality prior to transfusion. vandi ly*, dimath alyemni, warren r korn, matthew j brune and julie katz karp. thomas jefferson university hospital background/case studies: blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana. therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion. donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, d9tetrahydrocannabinol (thc) and 11-oh-d9-tetrahydrocannabinol (11-oh-thc). study design/method: de-identified donor plasma segments were sequestered and stored frozen until time of testing. testing for thc and 11-oh-thc was performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) based on a method modified from lacroix and saussereau. in summary, this method used dabsyl chloride derivatization of thc and 11-oh-thc to produce samples for lc-ms/ms analysis. lc used a c18 column. post-column detection by ms/ms used positive ion electrospray with q1:q3 ion pairs of m/z 5 605.3:225.3 (internal standard (is), d3-thc), m/ z 5 602.2:225.2 (thc), and m/z 5 618.3:256.1 (11-oh-thc). quantitative results for thc and 11-oh-thc were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from 0-50 ng/ ml for both thc and 11-oh-thc. limits of quantitation, defined as 5 standard deviations above background, were 0.7 ng/ml for thc and 7 ng/ml for 11-oh-thc. results/finding: a total of 424 donor plasma samples were tested for thc and 11-oh-thc. no samples tested positive for either thc or 11-oh-thc. theoretical calculations according to statistics of a poisson distribution indicated that there would be a 50% probability of one or more positives at a prevalence of 0.16% positive samples, and a 95% probability of one or more positives at a prevalence of 0.71% positive samples. results thus indicated a boundary of prevalence of the presence of active thc-metabolites in plasma samples to be less than 1% among this donor population. standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than 12 hours for post-exposure detection of thc and/or 11-oh-thc in plasma. conclusion: testing of 424 donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives. statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than 1%. probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics. elisabeth maurer-spurej* 1 , ruqayyah almizraq 2 , daniel millar 3 and jason p. acker 4 . 1 university of british columbia, 2 university of alberta, 3 lightintegra technology inc., 4 canadian blood services background/case studies: the controversy around the quality and clinical impact of aged red blood cell concentrates (rcc) is ongoing. current studies are limited by the lack of quality measures suitable for routine screening of rcc. based on evidence that fragments called microparticles (mp) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of mp screening to characterize the effect of rcc production methods and storage. study design/method: red blood cell concentrates were prepared by whole blood filtration (wbf; top/top) or red cell filtration (rcf; top/bottom) methods, centrifuged to prepare a supernatant and tested for mp content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, atp and red cell deformability on days 7, 21, and 42 of storage. one rcf rcc was tested on days 1, 5, 14, 21, and 43 and six 10 ml aliquots were stored in parallel and tested on days 14, 21, and 43. all samples were tested for mp content and compared to the other quality indicators. results/finding: mp content showed a linear increase with storage time with statistically significant differences between days 14, 21 and 43 (p<0.001) and correlated with supernatant hemoglobin, and inversely with atp or rbc elasticity. both mp testing methods agreed with respect to total mp content. starting levels of the quality indicators varied between donations, preparation methods (wbf rcc contained much higher levels of mp), and storage time. mp content in the 6 aliquots were consistent at each time point but statistically higher than in the original rcc on and after day 21 of storage. conclusion: mp content correlates with measures of hemolysis and other rbc quality indicators and could be implemented as a routine screening tool. differences in mp content between donors, processes and age could be monitored and used to inform component production decisions. measuring mp content would allow 100% screening of rcc products in studies and pragmatic qc initiatives which are needed to settle the controversy about the clinical effect of rcc age. single donor spray-dried plasma: the future of plasma therapy? qiyong peter liu*, jihae sohn, ryan c. carney, sruthi sundaram and mark a popovsky. velico medical inc background/case studies: frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time. spray-dried plasma (odp, on demand plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care. the objective of this study is to determine if odp can be consistently manufactured at a blood center with key proteins and coagulation function comparable to ffp. study design/methods: units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into odp using velico's spray dryer. odp (n 5 60) and paired ffp aliquots were stored for 31-33 days at 2-68c and -188c, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance. the volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted odp and ffp for direct comparison. results/findings: compared to ffp, odp had ! 80% levels of functional clotting factors (fibrinogen, factors ii, v, vii, viii, ix, x, xi and xii), plasminogen, and protease inhibitors (antithrombin iii, protein c, protein s; plasmin, c1 esterase and alpha 1-proteniase inhibitors). the level of factor xiii in odp was slightly lower, about 70% of ffp by both activity and antigen assays. odp was identical to ffp in the levels of albumin, immunoglobulins (iga, igg and igm), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor xiii. the levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments i1ii and ddimer) and complement (c3a and c5a) activation in odp remained similar to ffp. odp was equivalent to ffp when assessed by aptt, pt and thrombelastography. transfusion 2017 vol. 57 supplement s3 abstract spray-drying fragmented a substantial number of high molecular weight von willebrand factor (vwf) multimers into smaller ones, leading to a net increase of vwf multimers in odp. the size re-distribution reduced the vwf ristocetin cofactor activity (vwf:rco) to 62% in odp relative to ffp, but had no impact on vwf antigen and factor viii function (stabilized by vwf). vwf-specific studies have shown that odp retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by meledeo et al/us army institute of surgical research and bercovitz et al/blood center of wisconsin). conclusion: odp can be manufactured at a blood center with a quality comparable to that of ffp. future studies will determine if the product is bioequivalent to ffp and comparable in safety and efficacy. background/case studies: the collection time of whole blood is, according to european guidelines, limited to 15 minutes. in addition, donations with collection times between 12 and 15 minutes should not be used for preparation of platelet (plt) concentrates (pc) because of the chance of too much activation of plt. it seems justified to re-evaluate the quality of plt from these donations because new generations collection systems and mixers were introduced, including a more efficient needle. the aim of this study was to investigate the in vitro quality of pc prepared from 12-15 minutes buffy coats (bc) with the aim to prevent unnecessary discarding of bc and to simplify the total blood bank process. study design/method: single-donor pc (spc, n56) were prepared from one 12-15 minutes bc and 60 ml of autologous plasma in a 600 ml pvc-dehp container. as a reference, spc from donations with collection times of <12 minutes were prepared (n55). in addition, pc were prepared from 5 bc, of which at least 4 bc were from 12-15 minutes donations (n55). after pooling of the bc, 300 ml of pas-e was added and a standard pooling set with a pvc-bthc storage container was used for storage of pc. all pc were stored for 8 days at 22 6 28c and sampled at regular intervals for determination of the in vitro quality. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg), using kaolin as an activator, was applied for assessment of the overall clotting capacity. values are expressed as mean6 sd. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or non-normal distributed data respectively. results/finding: volume (67 6 5 vs. 66 6 16 ml) and platelet content (74 6 11 vs. 71 6 15 x10 9 ) were similar in both groups. at the end of storage, both groups showed comparable in vitro quality (day 8, ph(378c): 6.84 6 0.16 vs 6.83 6 0.17, other data not shown). no differences in aggregation response after stimulation with arachidonic acid, adp or collagen were measured. teg parameters in both groups were also comparable. the five-donor pc fulfilled all requirements of european guidelines, aside from occurrence of small aggregates at day 6 and/or 8 in 2/5 pc (possibly because sometimes ab0 incompatibility was accepted). on day 8, plt showed low cd62p expression (17.1 6 1.8%) and phosphatidylserine exposure (annexin v binding, 8.9 6 1.9%). hypotonic shock response of platelets was comparable with historical data. conclusion: single-pc in plasma as well as five-donor pc in pas-e, prepared from 12-15 minutes whole blood donations had a normal composition and showed good in vitro quality during 8 day storage. to substantiate that the exclusion of 12-15 minutes donations for pc preparation could be stopped, further studies will be performed. the effects of a pneumatic tube system on red blood cell units amy mata* 1 , jessie miller 1 , ranee marie wannarka-farlinger 1 , sandra bryant 1 , scott a hammel 1 , sherry stern 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: the use of pneumatic tube systems (pts) has become commonplace in many healthcare facilities throughout the world. the purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care. a downfall of ptss is that they have the potential to play a role in increased hemolysis. while several studies have been published on the effects of ptss on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (rbc). the objective of this study was twofold: to determine if the pts that is in use at our facility contributes to an increase in hemolysis of rbc units and to evaluate how the pts system affects red cell microparticle (rmp) levels. study design/method: forty-one units of as-3 rbcs, 20 irradiated and 21 non-irradiated, were selected for the study. the units varied in age, ranging from 2 to 42 days old. specimens were obtained from each unit both prior to and after being transported through the pts, which runs underground and spans the length of a mile and a half. specimens were spun down and the plasma supernatant was removed. all specimens were evaluated for plasma hemoglobin (hgb), potassium (k), hemolysis index (hi), and rmps. the wilcoxon signed-rank test and p value were used to compare the pre and post values. additional statistical analysis was performed to compare the values after adjusting for age and irradiation. results/finding: after sending the rbc units through the pts, hgb, hi, and rmps were statistically (p< 0.05) higher than before. when adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and hi. the k values did not significantly change. rmps significantly increased, but only if the units were irradiated (p50.02). (table) conclusion: the use of a pts provides an effective means to transport blood products; however, it can contribute to biological changes within rbc units. it is uncertain at this time how those changes can affect the outcome of patients who receive these products. each pts system is different in its specifications and should be validated prior to being used to transport blood products. validation of factor viii levels of thawed fresh frozen plasma after 5 days of storage pei lun karen lim* 1 , erma sofia sumardi 1 , isamar eduardo ancheta 1 , susan lim 2 , christina yip 1 , lip kun tan 2 and shir ying lee 3 . 1 national university hospital singapore, 2 national university hospital, 3 national university hospital, singapore background/case studies: plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure. fresh frozen plasma (ffp) has to be placed in the freezer within 8 hours of processing and stored at -188c or colder in order to preserve its coagulation factors. thawed ffp has an expiration period of 24 hours hence to reduce wastage, this study aims to investigate factor viii (fviii) activity in thawed plasma stored for 5 days and kept at 1 to 68c. fviii was chosen as it is an important coagulation factor in correcting coagulopathies. arbitrary fviii level acceptance limit was set as not less than 50 iu/dl. study design/method: randomly selected units of ffp (n510) were measured for fviii concentration based on clotting assay (stav r -deficient 92a transfusion 2017 vol. 57 supplement s3 viii diagnostica stago). fviii levels were measured at five time points: prefreezing, 0, 24, 72 and 120 hours post-thawing. ffp were thawed using helmer plasma thawer (helmer scientific) at 30 to 378c for 35 minutes. an aliquot of thawed ffp from each unit was removed and measured for fviii before refrigeration (0 hours post-thaw). thawed plasma (tp) units were kept in a refrigerator at 1 to 68c for 5 days for subsequent testing. results/finding: results obtained were listed in table 1 . units 7 to 9 were not tested for fviii at post thaw-24 hour due to operational issues. the overall fviii concentration decreased at an average of 13% from pre-freezing to post thaw 0 hour. after further storage of tp post thaw-24 hour and -72 hour, residual fviii level remain to be above 50 iu/dl except unit 10 which had a lower initial fviii concentration. at post thaw-120 hour, 7 out of 10 units tested had residual fviii activity within the pre-set standard of 50 iu/ dl. the average decline from 0-hour post-thaw to 24-hour, 72-hour and 120hour post-thaw was 36.5%, 42.7% and 47.9% respectively. there was no observed trend of any blood group having higher or lower pre-freezing fviii and this is likely due to small sample size. conclusion: decrease of coagulation factor such as fviii in ffp is expected due to its diminishing stability. nevertheless, our data showed that majority of the tp retained at least 50 iu/dl of fviii. typically patients with factor levels below 30 iu/dl may start to show abnormal coagulation profile. while tp is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding. further study extending to measurement of other labile factor such as fv may add value to the validation study. validation of the pathogen reduction method using amotosalen/ uva: comparing pathogen-reduced pooled prp-platelets and conventional single prp platelets for quality and bacterial inactivation efficacy lubna ahmed almenawi 1 , ayman mohamad sabri 1 , ali abdullah alajeafi 1 , ashwaq hasan alhekri 1 , saleem bin mahfouz 1 , ali hasan alkhodari 1 , rawya saeed shealy 1 , marcus picard-maureau* 2 and hussain bana almalki 1 . 1 king abdulaziz hospital and oncology center, 2 cerus europe bv background/case studies: the growing number of transfusiontransmitted infectious (tti) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in saudi arabia. while for a limited number of these pathogens tti risk can be reduced using blood screening assays, alternative solutions are anticipated. pathogen reduction (pr) technology was identified as a potential solution. validation of amotosalen/uva photochemical treatment in our blood center was performed by comparing the platelet component (pc) quality of the standard "control" single-donor prp-concentrate in 100% plasma over a 5 day storage period and the new "test" pathogen-reduced, pooled (pools of 5) prp pc in 100% plasma over a 7 day storage period. the efficacy of the bacterial inactivation was also assessed in our setting. study design/method: the quality parameters of 4 leucoreduced test pcs were assessed at day 7 of storage and compared to leucoreduced control pc at day 5 of storage. the test pcs were pathogen-reduced with the intercept blood system (cerus corporation, concord, u.s.a.) at day 0; the process was completed by day 1 post-collection. samples were taken daily for quality analysis from test and control pc until day 5 and day 7, respectively. for bacterial spiking, additional pc were spiked with each receiving 4 ml of 1 mcfarland ($ 1.2x10 9 cfu) s. aureus, s. epidermidis, e. coli, p. aeruginosa or s. viridans, respectively, to challenge pr efficacy. results/finding: the average platelet loss in the test pc post pr treatment was 4.7% 6 2.0, the total average platelet loss at day 7 was 11.2% 62.8. the average platelet loss in the control units at day 5 was 9.5% 61.4. the average ph of the test units at day 7 was 6.64 60.04 and in the same range as the control pc, ph 5 6.89 60.09. glucose concentration in test pc at day 7 (13.8 63.0 mmol/l) was lower than in the day 5 control units (18.32 61.06 mmol/l). lactate levels increased during the course of storage; lactate levels at days 5 and 7 were outside the range of the assay (>15 mmol/l). cultures inoculated with pathogen reduced, bacterially spiked units were negative after 7 days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth. conclusion: the quality parameters of the pathogen reduced test pc were within specifications and comparable to the conventional control pc. the high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/uva pathogen reduction is safe and efficient to enhance pc transfusion safety. keaton charles stoner* 1 , jay srinivasan 2 , jessica poisson 3 and ian welsby 4 . 1 duke university, 2 duke university school of medicine, 3 duke university hospital, 4 duke university medical center background/case studies: the coagulation cascade relies on a complex interaction between proteins known as clotting factors. cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients. however, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation. my study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation. study design/method: the duke proteomics core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry transfusion developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses. a tissue factor-activated test on the rotemv r delta hemostasis analyzer (extem) was performed on each condition. for each source, dose-response curves for clotting time (ct), alpha angle, and maximum clot formation (mcf) were generated using linear regression models. inter-source unit variability was determined by anova and tukey's hsd post-hoc analysis (rstudio inc.). results/finding: lc-ms/ms identified 256 proteins in cryo; of the 10 most abundant, only fibrinogen was relevant to coagulation. notably, the american red cross (arc) single donor source had the steepest slope for mcf (4.44 mm/dose), indicating a greater per dose potency than the other sources. the arc single donor source had the highest mean mcf across all dosing levels, but also the highest standard deviations and response variability. the arc single donor source was significantly more potent than the australian source. conclusion: paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for ct, mcf, and alpha angle can provide physicians with more information regarding cryo dosing. future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy. viral inactivation and enrichment of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers from fresh frozen plasma (ffp)using, "vips plasma, virus inactivation treatment system". background/case studies: the solvent/detergent (sd) process used for plasma can safely inactivate all lipid-enveloped viruses. the method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact. this study is done to assess viral inactivation and, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers enrichment capacity of, "vips plasma, virus inactivation treatment system". study design/method: "vips plasma, virus inactivation treatment system" comprise of interconnected bag system where the s/d reagents are removed by filtration and the final products subjected to bacterial (0á2 lm) filtration. cryoprecipitate mini-pools (400 6 20 ml) were subjected to doublestage s/d viral inactivation, followed by one oil extraction and a filtration on a s/d and phthalate [di(2-ethylhexyl) phthalate (dehp)] adsorption device and a 0á2 lm filter. the initial and the final products were compared for visual appearance, blood cell count, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers. initial and final products were also checked for hiv, hbv, hcv, dengue, malaria and bacterial contaminations. results/finding: our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers were well conserved (table 1) . kit ensured bacterial sterility (table 3 ) and most importantly, final product was free of hbv, hcv and hiv (table 2) . conclusion: it's the first time, "vips plasma, virus inactivation treatment system", is used in south asia for product enrichment and viral inactivation. results showed effective product enrichment and viral inactivation in our conditions. but further investigation is needed to characterize functional activity of the enrich component. irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries. background/case studies: buffy coats (bc) from donors who used pain medication like aspirin and ibuprofen up to 4 days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (nsaids) is inhibition of platelet (plt) aggregation. these nsaids inhibit the enzyme cyclooxygenase-1, thereby blocking synthesis of thromboxane a 2 from arachidonic acid. however, the quality of platelet concentrates (pc), prepared from this bc is not known. the aim of the study was to investigate the in vitro quality of pc prepared from nsaid-bc and autologous plasma during storage. study design/method: single-donor pc (spc, n518) were prepared from a nsaid-bc and 60 ml of autologous plasma. information about the type of pain medication was extracted from the anamneses form. the spc were stored for 8 days at 22 6 28c and sampled at regular intervals. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg, kaolin) was applied for assessment of the overall clotting capacity. spc in plasma from normal controls (n55) were investigated as a reference. values are expressed as mean6sd or as median & iqr. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or nonnormal distributed data respectively. results/finding: volume (69 6 4 vs. 66 6 16 ml) and plt content (67 6 14 vs. 71 6 15x10 9 ) were similar in both groups. on day 8, both groups showed comparable ph and changes in plt content (data not shown). phosphatidylserine exposure on day 8 was significant higher in a subset of donors who had used ibuprofen (n55). aggregation tests with arachidonic acid revealed in general a low or absent response for spc with aspirin (0,0-30, p<0.05), diclofenac (31,1-76) and naproxen (0,0-24, p<0.05), compared to normal controls (76, . no differences were detected in aggregation with adp or collagen. with teg, slightly longer r-times (initiation phase) were measured on day 1 in spc with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen). these differences disappeared during storage. conclusion: storage properties of spc prepared from nsaid-bc were comparable with spc from normal controls. main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen. plt from donors who used ibuprofen showed little or no deviations. this is most likely caused by the fast (<24 hour) disappearance of ibuprofen from the blood circulation and the reversible binding to plt. the use of bc from donors who used ibuprofen will be further investigated in a 'worst case' (pc in plasma) and 'best case' (pc in additive solution) scenario. the effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to plt. background/case studies: previously it was shown that donors could be classified as having platelets (plt) with good, average or poor storage properties [bontekoe, transfusion, 2014] . a main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of ph during storage of 'poor' plt concentrates (pc). this might be caused by a different functionality of the plt mitochondria and there are indications that donors with a history of 'poor' pcs are more likely to have health issues, pointing towards metabolic syndrome and type 2 diabetes (t2d). because of the strong rise of people with t2d in the dutch population, the aim of this study was to characterize plt from whole blood donors diagnosed for t2d, but accepted as donor. study design/method: twelve whole blood donors with t2d, not using insulin, were selected and buffy coat (bc) and plasma were, after overnight hold, used for preparation of a single-donor pc (spc). an equivalent number of spc was prepared from age and sex matched control donors, derived from the same collection sessions. spc were stored for 8 days at 22 6 28c and sampled on day 1, 4 or 5 and 8. the diabetic marker hba1c was determined in red cells and cholesterol and triglyceride levels in plasma. from both groups 3 'good' (ph day8 >6.6) and 3 'poor' (ph day8 <6.3) storing spc were selected and analysed in more detail. results/finding: donors were of age 57 6 10 year and primarily men (75%). donors with t2d had a higher mean bmi (30.3 6 4.6 vs.25.4 6 3.4 kg/m 2 ) and higher hba1c than controls. the spc of both groups had the same volume (70 6 5 vs 726 2 ml) and plt content (71 6 9 vs 73 6 11x10 9 ) but on day 1 glucose concentration was higher in the diabetic group (20.5 6 1.7 vs 18.9 6 1.4 mm, p<0.05). on day 8, the average in vitro quality was comparable in both groups (data not shown). when combining 94a transfusion 2017 vol. 57 supplement s3 the selected 'good' and 'poor' storing plt from both groups, a large difference in lactate production was observed (0.14 6 0.04 vs 0.36 6 0.03 mmol/ day/10 11 plt). the 'poor' plt showed a faster decline of the mitochondrial membrane potential (as measured with jc-1) during storage than 'good' plt. remarkably, a difference in triglyceride levels was detected on day 1 ('poor':2.2 6 0.7 vs 'good':1.1 6 0.2, p<0.01). conclusion: bc from donors with t2d who did not use insulin and fulfilled all donor criteria, were comparable with bc from age and sex matched controls, and seem suitable for preparation of pc. when selecting the 'good' and 'poor' storing plt from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria. metabolic syndrome and t2d are still suspected as health issues involved in 'poor' storage of plt because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored pcs. whole blood leukoreduction failures --following manufacturer's instructions may not be enough karen klinker*, nancy m. dunbar and zbigniew m. szczepiorkowski. background/case studies: our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation. the manufacturer recommends minimum wait time of 30 minutes prior to filtration. anecdotally, the vendor states waiting an hour improves the leukoreduction. we experienced leukoreduction failures in january and february of 2017 detected by our routine qc. we initiated an investigation as to the cause of these unexpected failures. study design/method: for each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past. hemoglobin s determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin s positive. results/finding: a relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure. we found that shorter wait times increased the percentage of leukoreduction failures (see table 1). all units that failed had wait times less than one hour. a similar trend was noticed for the previous year. the investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure. staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues. platelet counts on the donors involved were available and none were outside of the normal range. various lot numbers of the collection sets were involved, and no donors were repeat failures. conclusion: in our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures. we extended our minimum wait time to 60 minutes based on our data. we have not experienced any leukoreduction failures after this change. absolute immature platelet count in diagnostic algorithm and management of pediatric thrombotic microangiopathy hamza n gokozan* 1,2 , katharine a downes 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: prior studies highlighted the utility of absolute immature platelet count (a-ipc) and a-ipc ratio once therapeutic plasma exchange (tpe) is initiated to differentiate thrombotic thrombocytopenic purpura (ttp) from other thrombotic microangiopathies. this can be helpful to determine those who may benefit from prompt initiation of tpe when tests such as adamts13 are not readily available. we report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for ttp in which a-ipc measurement was clinically useful. study design/methods: previously healthy 12 month old unvaccinated girl presented with history of diarrhea for 5 days which was bloody at onset, accompanied by fever and dehydration. laboratory results showed: white blood cell count: 32 x 10 9 /l, platelets: 62 x 10 9 /l, bun: 77mg/dl, creatinine: 2.4mg/dl, lactate dehydrogenase 1940 u/l. hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis. peripheral blood smear revealed schistocytes. on third day of hospitalization, platelet count decreased to 44 x 10 9 /l, adamts13 sample was sent out and tpe was initiated for clinical suspicion of ttp versus hemolytic uremic syndrome, atypical versus shiga-toxin mediated. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/findings: platelet count began to increase prior to tpe initiation (74 x 10 9 /l and a-ipc of 4.7 x 10 9 /l). two consecutive tpe were completed which resulted in a platelet count decrease to 54 x 10 9 /l and a-ipc of 5.1 x 10 9 /l. a-ipc ratio was 1.1 below the ratio of 3 which has been reported for ttp patients. similarly a-ipc count was not below 5 x 10 9 /l threshold reported in setting of ttp with severe adamts13 deficiency. at this time stool culture obtained prior to start of tpe came back positive for e. coli o157:h7 toxin. testing of c3, c4, factor h, factor h autoantibody, factor i and factor b were normal. adamts13 activity was 93%. patient was treated for the infection and platelet count improved within 10 days to 315 x 10 9 /l, with resolution of her renal failure: bun: 42 mg/dl, creatinine: 0.65 mg/dl. no additional seizures were observed during follow-up. conclusion: measurement of a-ipc can be used to aid clinical decisions in pediatric patients suspected of ttp especially when adamts13 testing and those for other etiologies are still pending. tpe did not seem to have a significant effect in a-ipc but decreased platelet counts in this patient. a-ipc is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time. background/case studies: thrombotic thrombocytopenic purpura (ttp) is a thrombotic microangiopathy characterized by low adamts13 activity. many patients with severe autoantibody-mediated adamts13 deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years. it is unclear if disease course and characteristics of recurrent/relapsed ttp may be different from that seen at initial presentation. since absolute immature platelet counts (a-ipc) have been shown to be useful in the diagnosis and to follow response to therapy of ttp patients, we proceeded to evaluate if a-ipc pattern was different in relapsed verse initial presentation. study design/methods: our study cohort consisted of three patients (two female and one male) with acquired ttp (adamts13 activity <5%) who underwent daily therapeutic plasma exchange (tpe). clinical course and laboratory values were reviewed. platelet count (plt), immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were analyzed during treatment course. a-ipc values at presentation and peak, a-ipc peak time (days), and plt count recovery time (days) were compared between initial onset and relapse episode for each patient. a-ipc percent change in relapse episodes compared to initial presentation was calculated. results/findings: all patients had an increased %-ipf, and decreased a-ipc and plt count at presentation in both initial and recurrent episodes. once tpe treatment was initiated, a-ipc rapidly increased and reached a peak value 2-3 days prior to plt count recovery, consistent with that previously described in ttp patients. however, compared to first onset, recurrent episodes featured lower a-ipc at presentation (results shown as percent decrease, column 1), increased peak a-ipc value (results shown as percent increase, column 2), delayed a-ipc peak, and delayed plt recovery (table 1) . moreover, recurrent episodes required more procedures compared to initial presentation (table 1) . conclusion: recurrent/relapsed ttp demonstrate lower a-ipc at presentation and a delayed and increased a-ipc peak value in response to tpe compared to initial presentation. a longer treatment course was observed in recurrent patients. future studies of more relapsed ttp patients are needed. donors undergoing frequent plateletpheresis and its effect on the hematological parameters sweta nayak*, poonam coshic and r.m pandey. all india institute of medical sciences background/case studies: frequent plateletpheresis donors are assets for the blood banks. the well-being of these donors has been a matter of concern. in our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure. we also try to compare the effect 3 cell separators used for plateletpheresis on the post donation hematological parameters. study design/method: the study was conducted during february 2016 to march 2017 on all the repeat plateletpheresis donors coming to the department of transfusion medicine for the 2 nd time within a month of the first plateletpheresis. the values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated. the plateletpheresis were done either on hemonetics mcs1 separator (hemonetics corporation, braintree, massachusetts, usa), fresinius separator (com.tec), dn (fresinius hemocare gmbh, bad homburg v.d.h, germany) and gambro trima accel, software version 5.0 after taking consent from the donors. the target collection of each procedure was a dose of 3 x 10 11 platelets in 200-250 ml of plasma. to compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at 2 consecutive donations within 7 days were considered. data was analyzed by stata 14. within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or wilcoxon rank sum test. the comparison among the cell separators was done by kruskal-wallis test or one way anova. results/finding: of the 98 donors, 35 repeated the plateletpheresis within a week (group i) and 63 underwent 2 nd plateletpheresis within 8-30 days (group ii). no significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the 2 groups (p50.025). though above the eligibility cutoff of 1.5 lakhs/ml, platelet counts were lower than baseline in group i donors whereas it was higher at 2 nd plateletpheresis in group ii donors. there were 49 donors who presented to us for the 3 rd time for plateletpheresis with a mean gap between 1 st and 3 rd plateletpheresis being 46 days. no significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p50.000). plateletpheresis through all the 3 cell separators had similar effects on the hematological parameters. conclusion: there was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis. post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis. efficacy of therapeutic plasma exchange on angiotensin ii type 1 receptor antibodies in two kidney transplant recipients chisa yamada*, silas p. norman, milagros samaniego and laura cooling. background/case studies: some kidney transplant recipients develop antibody mediated rejection (amr) without detected hla donor specific antibodies (dsas) in sera. in recent years, angiotensin ii type-1 receptor antibody (at1rab) has been reported to cause amr, especially refractory amr, possibly by contraction of renal arteries. at our institution, therapeutic plasma exchange (tpe) followed by ivig every other day has been applied to reduce at1rabs in kidney transplant recipients, and we here report efficacy of tpe treatments in two cases. study design/methods: two kidney transplant recipients who received tpe treatment followed by ivig to decreased at1r ab are reviewed. results/findings: case 1: the patient is a currently 43-year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age 22, and a second deceased donor transplant due to a rejection of the transplanted kidney at age 38. three years post-transplant, her creatinine (cr) started to rise from 0.7 to 1.35 mg/dl and a biopsy showed banff criteria grade 2 amr, grade 2a t-cell mediated rejection (tcmr) and grade 3 interstitial fibrosis and tubular atrophy. hla dsa had been negative in serum, but high level at1rab was identified at >40 u/ ml (high: >17 u/ml, intermediate: 12-17 u/ml, negative: <12 u/ml). she received 6 tpe treatments every other day and started losartan. after a course of tpe, at1rab decreased to 32 u/ml and histology showed improvement of amr and tcmr, however, cr kept increasing slowly to 1.9 ml/dl. in one month, her at1rab increased again to >40 u/ml, therefore, she received 3 more tpe treatments with a decrease in her at1rab to 16 u/ml. although at1rab level increased slightly to 20 u/ml after 3 months, her cr has been stable at 1.3-1.6 ml/dl. case 2: the patient is a 25-year-old mean 1/-se -54.71/-12.9% * 183.1%1/-12.8%* * p<0.05 96a female with malignant hypertension who received a deceased donor kidney transplant at age 24. her cr started to rise 2 weeks post-transplant from 1.4 to 2.68 mg/dl without detectable hla dsa. although biopsy showed no amr or tcmr, there was focally severe arteriopathy. she was found to have high at1rab level at 18 u/ml. she received 6 tpe procedures every other day and at1rab decreased to 8 u/ml with a decrease of cr to 1.98 mg/dl and improved arteriopathy in histology. because her at1rab level slightly increased to 12 u/ml over the next 2 weeks, she started weekly tpe treatment. after 5 weekly tpe, tpe treatment was stopped because her at1rab level remained relatively unchanged. her cr has been stable at around 1.5 ml/dl to date. conclusion: we present 2 kidney transplant recipients who received tpe treatments for high at1rab levels. a course of tpe procedures followed by ivig every other day was effective to decrease at1rab levels; however, weekly tpe had no effect on reducing at1rabs. tpe treatment may be also beneficial to improve histological amr and clinical kidney function. experience in management of thyroid storm by plasmapheresis tatiana belousova*, vanya jaitly, brian castillo, hlaing tint, kimberly klein and yu bai. university of texas health sciences center at houston background/case studies: thyroid storm (ts) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with graves' disease. clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately. we report two cases where ts with severe cardiac complications was managed by plasmapheresis (plex) with excellent effect. study design/method: a 36 year old man (patient a) with a medical history of hyperthyroidism present with ts complicated with cardiogenic shock [ejection fraction (ef) < 10%], renal and hepatic dysfunction as well as coagulopathy. patient was persistent tachycardic while being intubated, sedated and requiring tandem heart support. a 33 year old man (patient b) with a medical history of hypothyroidism (on synthroid for 2 years), end stage renal disease and non-ischemic cardiomyopathy (ef of 20-25%) presented for evaluation of dual kidney-heart transplant. he subsequently developed ts with multiorgan failure. standard steroid medication treatment showed little response. results/finding: both patients underwent urgent plex along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised. a 1-1.5 plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed. both patients showed significant clinical improvement within 12 hours of the procedure completion. their total t4, t3 and free t4 levels trended to normal or near normal range within 24 hours (table) . in addition, the plex effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated plex unnecessary. both patients had total thyroidectomy 3-4 weeks after the event with great clinical outcome. conclusion: our cases demonstrate that plex is a safe, effective treatment option in managing ts patient with severe cardiac dysfunction. the procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications. extracorporeal photopheresis in s ezary syndrome treatment: hospital-based blood bank experience sandra ortega s anchez* 1 , laura martínez molina 2 , cristina muniesa montserrat 2 , octavio servitje bedate 2 , silvia cosano navarro 1 and maria isabel gonz alez medina 1 . 1 banc de sang i teixits, 2 dermatology service. background/case studies: extracorporeal photopheresis (ecp) is an immunomodulatory therapy widely used since 30 years in cutaneous t cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last 20 years, also used in graft versus host disease treatment. the use of ecp in cutaneous t cell lymphoma (ctcl), mycosis fungoides (mf) and s ezary syndrome (ss) in their erytrodermic form are recently categorized by the american society for a pheresis (asfa) 2016, as first line treatment alone or in combination with other therapies, with a strong recommendation: grade ib, category 1. since mf and ss are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression. the objective of this observational study is to assess outcomes of 10 patients diagnosed with ss and compare them in their first evaluation once the 20 th procedure is been performed. study design/method: ecp is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( 8-methoxypsoralen, 8-mop) and uva light, and subsequent reinfusion of the treated cells which are now induced to apoptosis. volume treated varies from 1.5 to 2 total body volume (tbv) and the schedule for ss disease is one cycle (two daily ecp procedures) twice per month. the venous access was peripheral in all cases except in 2 where central catheter was needed. the procedures were performed with optia or amicus devices for the aphaeresis and external uva irradiation for off-line system (in 7/10 patients) and with online system (therakos) just in 3. main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ecp treatment. results/finding: global response rate is 77'7% (partial remission 66.6% and complete remission 11.1% with maintained response). no severe side effects related with the procedure were found. the patient outcomes analyzed are similar to results in published literature. conclusion: cases treated in our hospital confirm the efficacy of ecp in ss treatment, with a good safety profile. another great advantage of ecp is the relative lack of immune suppression. many questions remain still unanswered about ecp: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results. all these questions and more make prospective studies necessary to be performed. : 3835 u/l) requiring transfusions, mild thrombocytopenia (144 x 10 9 /l), acute kidney injury (bun 175 mg/dl, creatinine 2.51 mg/dl). by the third hospitalization day hgb improved to 10 g/dl, however with worsening thrombocytopenia (16 x 10 9 /l) that led to clinical concern for ttp. peripheral smear showed many red cell fragments. patient was transfused with platelets day prior to first tpe. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: four tpe in five days were performed (hospital days 6-10). platelet count and a-ipc improved to 52 x 10 9 /l and 6.6 x 10 9 /l respectively just prior to first tpe. response to four tpe led to a decrease in both platelet count (30 x 10 9 /l) and a-ipc 1.98 x 10 9 /l. these dynamics did not resemble those which had been described for ttp patients with adamts13 deficiency. adamts13 obtained prior to tpe initiation was resulted at this time and was 67%. no causative organism or toxin was identified after urine, blood, and stool examination and culture. based on these results, tpe was discontinued which led to an immediate increase in a-ipc (2.64 x 10 9 /l) that preceded platelet count increase to 80 x 10 9 /l three days later when patient was discharged. other laboratory values at this time were ldh of 635 u/l, hgb: 11.2 g/dl in the setting of recovery of renal function. conclusion: timely diagnosis of ttp is essential to start of tpe. a-ipc dynamics differ in ttp compared to other thrombotic microangiopathies. in our patient a-ipc failed to improve despite tpe and improved once procedures were discontinued and were followed by increases in platelet counts three days later. when ttp is not the causative etiology, a-ipc can help adjust therapy and lead to clinical improvement. further research is needed to characterize immature platelet dynamics in non-ttp microangiopathies. infection and its role in the clinical course of idiopathic thrombotic thrombocytopenic purpura associated with severe adamts13 deficiency eiman hussein* 1 and jun teruya 2 . 1 department of clinical pathology, cairo university, 2 texas children's hospital background/case studies: ttp is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient adamts13. since the introduction of therapeutic plasma exchange (tpe) as a treatment modality for ttp, its prognosis has improved dramatically. nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes. despite the notable progress that has been made with studies that emphasized the pivotal role of adamts13, the epidemiology of ttp remains uncertain. previous studies have suggested that many factors appear to influence its pathogenesis. some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course. one of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against adamts13. the aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic ttp. study design/method: patients with idiopathic ttp who underwent tpe from january 2008 through march 2017 were studied. sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered. we only included patients with adamts13 activity of less than 10%. data on infections that occurred at or within a week prior to the development of ttp were analyzed. results/finding: thirty-two patients were categorized as idiopathic ttp with severe adamts13 deficiency. eight patients (25%) were associated with suspected bacterial infection. four of the 8 patients (50%) showed acute relapse coincident with bacterial infections. central line associated staphylococcus aureus infections occurred in three patients and acinetobacter urinary tract infection was reported in one patient. one patient had symptoms of respiratory infection before the development of ttp, on his initial as well as his relapsing episode. refractoriness to treatment was demonstrated in 3 patients. it was associated with dental abscess in one patient. the other two were associated with mycoplasma pneumonia. tpe sessions were continued in all refractory patients until their death. conclusion: in patients with idiopathic ttp refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes. sandra satoe kayano*, marcos paulo colella, rafaela guerra maciel, ingrid priscila ribeiro paes ferraz and rafael colella. a c camargo cancer center background/case studies: therapeutic leukapheresis (tl) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion. leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis. the objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than 10 kilograms using a single apheresis procedure. study design/method: in october 2015 and june 2016, two children with possible leukemia were submitted to tl procedure. they were 6 and 9 months old, and weighted 7,0 and 9,1 kilograms. central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system. the device was primed with 285 ml of abo, rh and kell compatible, leukocyte-reduced, irradiated, 64% hematocrit packed rbcs, and the anticoagulant used was acd-a plus heparin (750 ml of acd-a and 7,500 units of heparin), at a blood to anticoagulant ratio of 25:1. a complete blood count was determined before and after apheresis. the room was heated to avoid hypothermia, and ionized calcium was measured every 30 minutes to prevent hypocalcemia. during the collection, changes in blood pressure, oxygen saturation and heart rate were observed. net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume. when the procedure was completed, the blood that filled the apheresis tubing was discarded. the patients were in the intensive care unit (icu) under the supervision of a pediatric physician and icu nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner. results/finding: the white blood cell (wbc) in blood was counted immediately before apheresis in both subjects, and were 120.000 and 150.000/ mm 3 . the formula "collection pump flow 5 0,0003 x inlet flow x preapheresis wbc count" was used with the goal of removing up to 3 x 10 9 leukocytes/ml. a single leukapheresis procedure was performed with 2 total blood volume processed per patient. immediately after the 2-hour procedures, wbc count were 74.000 and 92.000 wbc/mm3, and 12-hour post tl, wbc count were respectively 45.000 and 70.000/mm3. net fluid balance was zero in both procedures, and the patients required no transfusion. conclusion: tl was safe and efficient. experience with leukodepletion in infants is limited, and a procedure in children weighing 10 kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection. however, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases. background/case studies: nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures. data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice. the korean society for apheresis (ksfa) has launched an online web based registry system for apheresis procedures since 2006. we report the data from the year 2016. study design/method: the registry is consisted of two sub-registries. one addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures. data is registered by voluntarily participating hospitals in korea. results/finding: a total of 13,302 apheresis procedures were performed at 37 hospitals. therapeutic plasmapheresis was the most frequent procedure (50.4%) followed by autologous peripheral blood stem cell (pbsc) collection (23.9%), allogeneic pbsc collection (11.0%), donor leukapheresis (4.0%), and therapeutic leukapheresis (3.9%). cobe spectra (37.4%) and amicus (16.8%) were the most widely distributed instruments. centrifugation was the dominant technique (92.2%) for therapeutic plasmapheresis. detailed information was given for 4,199 therapeutic plasmapheresis procedures performed on 786 patients (some items were not completely filled out). spectra optia (42.7%) and cobe spectra (26.6%) were the most frequently used instruments for therapeutic plasmapheresis. fresh frozen plasma (ffp) was used most frequently (47.2%) as the replacement fluid followed by 5% albumin (26.3%), 4% albumin (13.3%), and 5% albumin 1 ffp (11.1%). most of the procedures were performed for 1 plasma volume (72.4%). acd (88.4%) and heparin (11.5%) were used for anticoagulation. central venous catheter (91.9%) was the dominant type of vascular access. major clinical indications were desensitization for abo incompatible renal transplantation (24.1%), antibody mediated rejection in renal transplantation (19.9%), thrombotic microangiopathy (11.5%), desensitization for abo compatible renal transplantation (4.7%), neuromyelitis optica spectrum disorders (4.6%), and hyperviscosity in monoclonal gammopathies (4.6%). adverse reactions were observed in 8.5% of the procedures. allergic reaction (55.2%), hypocalcemic symptom (20.4%), and hypotension (6.9%) were frequently reported. therapeutic effect was achieved in 86.5% of the patients. our apheresis registry has been well run for 10 years. recent data reflects the increase of abo incompatible transplantation in korea. revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region. plasma exchange may not always be necessary in patients with severe hypertriglyceridemia and acute pancreatitis. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: hypertriglyceridemic pancreatitis (hp) is characterized by severe hypertriglyceridemia (shtg: triglyceride >1000-2000 mg/dl), acute pancreatitis (ap), and absence of other causes. hp is a potentially fatal complication of acute pancreatitis with an incidence of $18 deaths/100,000 cases/year. complications of shtg include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy. we report on the effective use of plasma exchange (pe) to treat patients (pts) with hp refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions). study design/method: we reviewed the medical records of 41 pts who were diagnosed with hp from january, 2009 through january, 2017, and referred for immunotherapy evaluation. 27/41 (66%) pts received conventional therapy (ct) and pe (pe group), and 14/41 (34%) pts received ct alone (ct group). mean age was 36 years (range 16-79), and 56% were female. baseline mean triglyceride level (normal <150 mg/dl) for pe group was 6,728 mg/dl (4,652-12,486) versus 3,142 mg/dl (1,697-5,120) for ct group. baseline mean lipase level (normal <393 u/l) for pe group was 1,798 u/l (797-2,745) versus 923 u/l (472-1,796) for ct group. results/finding: all pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving 2-3 medications. 24/27 (89%) of pe group and 11/14 (79%) of ct group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis. 20/27 (75%) of pe group and 6/14 (43%) of ct group received heparin therapy to stimulate lipoprotein lipase release. the pe group underwent an average of 2.85 pe treatments (txs) (median of 2, range 1-4 daily txs) using 5% albumin; 7/27 (26%) required ffp to treat dilutional coagulopathy. in most cases, we did not perform pe txs when baseline triglyceride levels were <3000-4000 mg/dl and lipase <950-1375 u/l (2.5-3.5 x upper limit of normal). mean triglyceride levels after 2 pe txs were 1,976 mg/dl (627-3,968) for pe group (mean decrease 72%); mean triglyceride levels after 48 additional hours of ongoing ct were 1,576 mg/dl (487-2,873) for ct group (mean decrease 50%). while the pe group achieved a greater mean decrease in triglyceride levels after 2 pe txs (compared to the ct group after 48 hours of ct), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p>0.05). limitations of the retrospective cohort study include lack of long-term follow-up. conclusion: this small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia. it suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms. randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis. role of plasma replacement in therapeutic plasma exchange for hypertriglyceridemia: a single patient study geoffrey wool* and angela treml. university of chicago background/case studies: our apheresis service performs chronic therapeutic plasma exchanges (tpe) for a 47-year-old man with a chronic history of hypertriglyceridemia >1000 mg/dl, diabetes mellitus type ii, and chronic abdominal pain. his abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing. targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia. hypertriglyceridemic pancreatitis is a category iii indication for tpe by asfa 2016 guidelines, in a patient unresponsive to optimal medical management. asfa 2016 guidelines for this disorder state that "some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (tg) removal. no direct comparisons of replacement fluids have been reported". there are three apheresis physicians on our service and use of partial plasma replacement has been variable. we undertook a retrospective study of the efficacy of partial plasma replacement in this patient. study design/method: we have performed 39 tpe on this patient. we performed a chart review to capture replacement fluid use and pre-and post-tg levels, if drawn. tpe was performed using spectra optia (terumo, lakewood, co) exchanging approximately one plasma volume, using entirely 5% albumin for exchange fluid (100% albumin procedures) or partial plasma replacement (2-3 units of thawed plasma). twenty-six tpe had pre-and post-procedure tg values available. we determined the percent tg reduction achieved by the tpe. we also determined the daily rate of tg increase until the next tpe appointment (to assess any long-term effects of plasma preventing tg rebound). significance was assessed by student's t-test (one-tailed, heteroscedastic). results/finding: twelve tpe were performed with partial plasma replacement, while 27 were performed with 100% albumin replacement. table shows that partial plasma replacement was associated with significantly greater % tg reduction. the rate of subsequent daily tg increase was also lower with partial plasma replacement, but this did not meet significance. one mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional iv diphenhydramine. conclusion: we have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in tpe for hypertriglyceridemia. in this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % tg reduction, but not with prevention of post-tpe tg rebound. safety and efficacy of local albumin replacement for therapeutic plasma exchange phandee watanaboonyongcharoen* 1,2 , metha apiwattanakul 3 , sompis santipong 2 , jutaluk jaipian 2 , jettawan siriaksorn 2 and ponlapat rojnuckarin 1 . 1 chulalongkorn university, 2 king chulalongkorn memorial hospital, 3 prasat neurological institute background/case studies: therapeutic plasma exchange (tpe) with albumin replacement has been used to treat a variety of diseases. however, there had been rising cost and supply shortage of imported albumin in our country. to solve the problem, our national blood centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin. the objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for tpe. study design/method: all tpes using local albumin as a replacement from two tertiary care hospitals performed from june 2016 through february 2017 were included. complete blood count and serum calcium were tested before tpe. serum albumin was tested before and after tpe. local albumin is available as a 20% solution. before using, it was diluted to a 4% albumin concentration with normal saline. all the patients were hospitalized and received oral calcium before tpe to prevent hypocalcemia. the adverse effects were recorded. results/finding: the total of 156 tpes in 38 patients were included as shown in the table. neurologic disorders were the most common indication for tpe, followed by autoimmune diseases. the median total plasma volume was 3,000 (range 1,750-4,200) ml. although the corrected calcium level was low (<8 mg/dl) in 3.2% (5/156) before the procedure, no clinical manifestation of hypocalcemia was detected. adverse effects were observed during the tpe procedure in 2 patients. the first patient had 2 events of mild symptomatic hypotension. he previously took angiotensin converting enzyme inhibitor. the second patient complained nausea after finishing tpe. all reactions were mild. the incidence of adverse effects was 1.9% (3/ 156). in 2014, the incidence of tpe adverse effects was 1.6% (2/125) when commercial albumin was used. the difference was not statistically different (p 5 1.000). median serum albumin levels pre-tpe and post-tpe were 3.6 (1.9-4.4) and 3.9 (2.4-5.0) g/dl. the increase in serum albumin after tpe was statistically significant (p<0.001). eighty-two percent of pre-tpe serum albumin levels were lower than 4.0 g/dl explaining the rises of albumin after the procedures. we demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing tpes. safety, efficancy and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: experience from a private outpatient collection facility within the eu markus dettke*. akh vienna university hospital, cyto-care.eu background/case studies: within the eu the collection of mononuclear cells (mnc) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. we report about the challenges to perform the leukapheresis procedure (la) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. study design/method: we reviewed the records of altogether 60 outpatients who underwent a total of 100 la procedure at cyto-care, a private held medical practice/ certified cell collection facility located in vienna, austria. all patients participated in various industry-sponsored clinical p i-iii trials; the study sponsors were responsible for the manufacturing of the active cell product. disease entities were mainly prostatic cancer (75%) and ovarian cancer (20%). based on differences in the study protocols la was performed either one-time (41%), two-times (27%) or three-times (32%), with an interval of at least 2 weeks between repeated collections. results/finding: all patients successfully completed the apheresis course. because of poor venous access, 3 out of 60 patients (5%) required a shortterm femoral catheter insertion. there were no serious side effects in patients who required a femoral catheter, or in patients with repeated la procedures. side effects of the la procedure mainly consisted on mild hypocalcaemia-related symptoms in 16% of patients. a follow-up survey one week after completion of the la revealed no infectious complications, and no patient required hospitalization. median cell yield collected per single apheresis was 1.4x10 10 wbc consisting of 1.1x10 10 mnc. mnc cell yields remained stable even in repeated la collections. all cell products were successful transformed into an active cellular product. analysis of the cost structure showed that the total cost of care was 32% lower in the setting of a private collection center compared to hospital-based apheresis centers. conclusion: leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. this service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products. although typically associated with monoclonal gammopathies (e.g. waldenstrom's macroglobulinemia and multiple myeloma), hvs has rarely been reported in patients with disorders of immune system such as rheumatoid disease, sjogren's syndrome, hiv and igg4-related diseases. therapeutic plasma exchange (tpe) is indicated in hvs due to monoclonal gammopathy (asfa category 1 indication). however, there are limited data for the utility of tpe in hvs due to polyclonal gammopathy. study design/methods: a 70 year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months. fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns). pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-ro antibodies. serum rheumatoid factor was markedly elevated to 57,000 iu/mls (ref. range < 35) and anti-cyclic citrulline peptide antibody was elevated to 34,339 units (ref. range < 20) . serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes. serum igg, igm and iga were 4610, 2890 and 1320 mg/dl respectively. a cryoglobulin screen was negative. serum free kappa to lambda ratio was 1.74. peripheral blood flow cytometry did not identify any monoclonal bcell population. plasma viscosity was noted to be 8.5 centipoise (cp) at admission (ref. range 1.6 -1.9). pet-ct imaging was negative. the patient was treated with high dose steroids; a single tpe procedure was performed using the following parameters: volume treated -1 total plasma volume; replacement fluid -5% albumin and normal saline in a 50:50 ratio; replacement fluid volume: 110% of the total volume processed. the procedure was tolerated without complication. results/findings: immediately post-tpe her plasma viscosity level dropped to 2.4 cp. serum igg, igm and iga levels decreased to 2040, 1510 and 672 mg/dl respectively. her rf had decreased to 19,900 iu/ml. the patient reported subjective improvement in strength. she subsequently received two infusions of rituximab separated by two weeks. her plasma viscosity has remained less than 3 cp since tpe. conclusion: polycolonal gammomathy (e.g. secondary to ra) is a rare cause of hvs. tpe can provide transient relief of symptoms in unusual cases of hvs and may facilitate therapy to prevent recurrent hvs episodes. therapeutic plasma exchange in neuromyelitis optica spectrum disorders -experience from tertiary care centre in north india ratti ram sharma*, rekha hans, satya prakash, naveen sankhyan and neelam marwaha. postgraduate institute of medical education and research background/case studies: neuromyelitis optica spectrum disorder (nmosd) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis. tpe is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase. study design/method: to assess the efficacy of plasma exchange in patients of nmosd not responding to high dose intravenous steroids. we did a retrospective review of tpe records for patients with nmosd over a period of three years (jan 2013 -dec 2016). tpe was done using, cobe spectra (terumo bct, lakewood co. usa), replacing one to one and half patient plasma volume with 5% human serum albumin or fresh frozen plasma on alternate days. the improvement in clinical signs and symptoms was recorded after each tpe procedure and at the end of the therapy. adverse reactions if any were also recorded results/finding: eleven patients of nmosd between 4 to 35 years age (m: f; 1:2) underwent 62 tpe procedures with an average of 5.6 per patient. all the patients were on high dose immunosuppressant therapy without much clinical improvement. three (27%) patients had only visual symptoms, 5 (46%) had both visual as well as muscular symptoms whereas 3 (27%) patients had muscular symptoms only. three (27%) out of the seven tested, were positive for aqp4-igg. all the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients. post exchange recovery in the muscle power was observed in 8 patients with grade-1, in 1 patient, and by grade-2, in seven. adverse events were observed in 8% (5/62) of the procedures with allergic reactions to replacement fluid as most common event (n-3) followed by hypotension (n-2). follow up was available in 55% (6/11) of patients and are doing well on immunosuppressive therapy. one patient died due to respiratory failure after 3 months and another had relapse for which he underwent second tpe cycle and continue to do well. conclusion: tpe is a safe and effective adjunct therapy to high dose immunosuppression in nmosd. trima accel software upgrade from 6.0 to 6.4 for platelet collections rachel m beck*, kimberly j duffy, sandra bryant, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: terumobct released trima accel software version 6.4 as an enhancement to allow for the collection of platelets (plt) with platelet additive solution (pas) and provide additional improvements to increase overall reliability. additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates. this software was expected to function similarly to version 6.0. the objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected. study design/methods: prior to 1/16/2016, plt collections were performed on nine trima accel machines operating with version 6.0. upgrading and validating all nine machines to version 6.4 occurred from 1/16/2016 to 4/30/ 2016. the trimas were programmed with the same plt configurations both before and after software update. platelet collection data from version 6.0 (5/1/2015 to 9/30/2015) was compared to version 6.4 (5/1/2016 to 9/30/ 2016). incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period. generalized estimating equations (gee) were used to assess differences between plt collections with version 6.0 and 6.4, adjusting for multiple visits per donor, with significance defined as p-value < 0.05. results/findings: following the upgrade to version 6.4, staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products. version 6.4 of the trima accel showed a statistically significant increase in possible leukocyte contamination from 3% to 5% of collections as compared with version 6.0. both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version 6.4. conclusion: due to fda limitations not allowing for the implementation of trima accel pas plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time. subsequently, the version 6.4 software is no longer required. with the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version 6.4 currently does not provide added value over version 6.0 for plt collection. pulmonary and neurologic symptoms due to leukostasis. therapeutic leukocytaphersis (tl) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis. tl procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density. there are two programs in the cell separator, a mononuclear (mnc)program which has greater centrifuge speed and efficiency for the collection of mncs and a polymorphonuclear (pmn)cell program with lower centrifuge speed for the collection of pmns. hydroxyethyl starch(hes) is preferred for the collections of granulocytes for transfusion from healthy donors. use of hes facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection. though use of hes in tl was not associated with adverse events with its use as a volume expander (pagano) its use in tl varies and no reports are available on the efficiency of leukodepletion using hes for tl. study design/method: we received a request for leukoreduction in 32 yearold lady with chronic myelogenous leukemia (cml) who had a good response to imatinib. she is 30 weeks pregnant with an increased wbc count due to the discontinuation of imatinib. we performed tl with the cobe spectra using a replacement fluid of 500 ml 5% albumin. wbc counts were monitored pre and post tl in the patient and in the collected product. we modified the collection based on these results using the mnc program with acd-a or the pmn program with acd-a . as leukodepetion was not adequate with these programs we elected to use hes after discussion with the patient and her physician. tl was performed using 500 ml of hes with citrate and the pmn program. wbc pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated. results/finding: the efficiency of % wbc depletion was calculated by product wbc to patient wbc based on blood volume and also pre to post wbc the patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures conclusion: therapeutic leukocytapheresis in cml patients is safe and more effective in reducing the wbc count with the use of 500 ml of hydroxyethyl starch with anticoagulant. post procedure patient wbc counts sometimes may not provide the data on the efficiency of leucodepletion. background/case studies: early recognition of hypertriglyceridemia (htg) in the setting of acute pancreatitis (ap) is critical to initiate effective therapy. the role of plasmapheresis as an early/adjuvant approach in acute htg-induced pancreatitis is controversial. currently, there are no consensus guidelines in optimal therapy and is asfa category iii. reported here is a case where the tg level as well as clinical symptoms improved after one therapeutic plasma exchange (tpe). study design/method: a 45 years old male with history of hypertension, htg, and diabetes mellitus (dm) presented to our emergency department with excruciating abdominal pain. the patient was diagnosed with htg at 20 years old. he was treated initially with diet and lifestyle modification. however, his clinical course has been compromised after developing pancreatitis with 3 acute episodes requiring prolong hospital admission of approximately 2 months each which were successfully treated medically. however, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency. since the first episode of pancreatitis, he was then medically managed with fenofibrate, lovaza, lisinopril, levemir and novolog. during evaluation on current admission, he was found to have a tg level of 4980 mg/dl, lipase 92 u/l, glucose 250 mg/dl, bicarbonate 24 mmol/l, anion gap 12. ct findings were consistent with ap without evidence of necrosis and stable pancreatic pseudocyst. medical therapy was started with omega 3 fatty acid, fibrate, statin, hydration as well as pain control. statin therapy was suspended on day 3 of hospitalization, because he was noted to have elevated liver function tests (lft) and tpe was requested and started on day 3 after admission. results/finding: the patient tg decreased by 52% (2365 mg/dl) with medical therapy, followed by additional 67% (767 mg/dl) after one volume of tpe. his symptoms significantly improved and was discharged with medical treatment on day 6 after admission. compared to previous episodes, his hospital stay was significantly decreased. tg levels remained below 1000 mg/dl at 20 days follow up after discharge. conclusion: early tpe may be of value in treating patients with elevated tg associated with recurrent pancreatitis. plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety. background/case studies: from 2009 to 2013, a national blood donor center in southeast asia conducted a program to monitor the ferritin levels of platelet blood donors. the aim of this study was to explore the trend of changes in ferritin. study design/method: in this study, we collected 5,129 cases whose ferritin levels have been monitored more than twice with an interval of detection in 150-160 days. the collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay. inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over 50 lg/l. and the upper limit was set to be 244 lg/ l in male and 158 lg/l in female as described in manufactures insert. the impact on ferritin from gender, age, and the blood donation frequency were examined with anova test. the blood donations frequency was categorized into five groups: 0 times, 1 to 3 times, 4 to 6 times, 7 to 9 times and more than 10 times. the high frequency (more than 10 times group) blood donors were analyzed ferritin changes in longitudinal data. results/finding: there were 5,129 donors included in the study, of which 4,944 were male (96.4%) and 185 were female (3.6%). the mean ferritin was 82.0 lg/l in male (95% ci: 80.7-83.2 lg/l) and 66.5 lg/l in female (95% ci: 60.9-72.0 lg/l). the result of anova indicates that the group with the highest frequency (more than 10 times) has the significant lowest ferritin level (p<0.05). the average change of ferritin if donation over 10 times would up to 13.4 and 14.1 lg/l in younger and elder 50 y/o male and 18 and 23 lg/l in female. and then for high frequency (half a year more than 10 times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin. the average change about ferritin in high frequencies donors (over 10 times in 150$160 days) was reduced from 21.5 lg/l in the first period to 4.1 lg/l in the third period (1 period5150$160 days). along with the more and more period, the decline of ferritin decreased. conclusion: this analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors. but the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down. a rare case of blood donation precipitating acute delirium joseph griggs* 1 , mary townsend 2 and lizabeth rosenbaum 3 . 1 university of new mexico hospital, 2 blood systems, inc., 3 blood systems inc. background/case studies: we report a case of whole blood (wb) donation that precipitated a transient agitated delirium. a 22 year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation. approximately 10 minutes after an uncomplicated wb donation, the donor had an observed, brief loss of consciousness in the post-donation area. no fall or injury was seen. shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding. the donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained. he ultimately was sedated and intubated, and transferred to the intensive care unit. study design/method: an extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and pcr-based studies for multiple organisms including west nile, herpes, hiv, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse. radiographic imaging was performed including a chest x-ray, and a ct and mri of head and spine. in addition, an eeg was performed. the inpatient neurology and psychiatry services were consulted for this patient. results/finding: after the sedation was discontinued, the patient was successfully extubated and rapidly improved. he completely returned to baseline within 24 hours of onset of the event. laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse. radiographic imaging and eeg studies showed no abnormalities. in addition, infectious disease marker testing performed by the blood center laboratory was negative. investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers. a week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes. conclusion: to our knowledge, this is the first report of blood donation precipitating a transient acute delirium. at the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient. the health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor. however, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed. although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood. a targeted approach to increasing the african american blood donor pool arnethea sutton* 1 , william korzun 1 , teresa nadder 1 , susan roseff 2 and elizabeth ripley 1 . 1 virginia commonwealth university, 2 virginia commonwealth university medical center background/case studies: a continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from african american donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations. one population in particular, african americans, only account for 1% of blood donors in the united states. literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community. study design/method: african americans in richmond and norfolk, virginia were recruited through churches and local universities. the study's aims were to develop, implement, and assess a targeted educational approach incorporating the theory of planned behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate african americans non-donors to attempt to donate blood. participants attended a 1-hour educational session where they were educated on the importance of red blood cell donations from african americans. participants completed three surveys -one before the session, one directly after the session and one, two months after the session. a two-proportion z-test was used to compare the known proportion of african americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables. results/finding: a total of 142 subjects were included in the data analysis. sixteen percent of the study participants presented to donate as a result of attending the educational session. this resulted in a statistically significantly higher proportion of african americans presenting to donate than the current proportion in the areas of the state where this study was conducted. results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor. the educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session. conclusion: this study shows that a targeted educational program can change attitudes toward blood donations in african americans resulting in an increase in new blood donors. additional studies are needed to see if this behavior will continue and whether african americans can influence their community to increase awareness and motivation for life-long blood donation. were from female basic trainees conclusion: the significant increase in hemoglobin deferrals at basic training site a from 2015 to 2016 could be a result of a change in the blood drive timing of the training schedule of that location. in 2015, basic trainees at site a were scheduled at day 57 of 70. in january 2016, the blood drive date changed to day 60 of 70. the extra three days in the basic training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population. at basic training site b, the significant increase from 2015 to 2016 of hemoglobin deferrals can be attributed to a larger male population presenting at this site for basic training. additionally, the percentage of female recruits donating at the blood drives decreased in 2016. these observations support the hypothesis that the increase in hemoglobin deferrals in 2016 resulted from the implementation of the male hemoglobin standard change from 12.5 to 13.0 g/dl at basic training site b. when planning for blood drives at basic training site b, screening of an additional 24% of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard. blood donation in the donor with spinal cord injury joan-ramon grífols* 1 , eva alonso 1 , oscar bascuñana 1 , monica romero 1 , teresa vich 1 , elena castaño 1 , laura carbonell 1 , eva palomas 1 , saray almerge 1 , francesc carpio 1 and xavier curia 2 . 1 banc de sang i teixits, 2 institut guttmann background/case studies: donation of blood components (bc) in donors with spinal cord injuries (sci) is poorly studied. paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of bc donation. the literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their sci it's obvious. in daily practice these potential donors are often rejected for donation with no specific criteria related to their sci. the objectives of this study are to establish the selection criteria for bc donation in people with sci based on medical criteria. to evaluate the rate of adverse donation blood reactions of these donors against a donor control group without sci. study design/method: our organization regularly organizes a donation campaign at a rehabilitation center for patients with sci. in this campaign some donors with sci as donors without (professionals of the center, relatives, etc.) donate blood. from january 2015 to december 2016 we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had sci and number and typology of adverse reactions to the donation detected in both groups. donors with sci higher than t5 due to the high risk of autonomic dysreflexia were excluded for donation. donors with sci below t5 and less than one year of evolution were set as temporary exclusion criteria. the presence of neurogenic bladder was not considered a reason for exclusion. results/finding: in the analyzed period, 219 donors came to give blood, of these, 15 (7%) were excluded for donation for various reasons. two of the donors excluded suffered sci higher than t5 excluding them due their high risk of dysreflexia. another one donor excluded suffered sci lower than t5 but his hemoglobin levels were lower than our selection criteria. of the 204 donors selected for donation 16 (7.8%) had sci lower than t5 and t6. adverse reactions to donation (1.4%) were recorded in our haemovigilance program, none of them in donors with sci. conclusion: according to our experience donors with sci lower than t5 have not had any type of adverse reaction to the blood donation. there should be selection / exclusion criteria based on the donor's paralytic conditions. the vagal syndrome that could appear as a complication to the donation in these sci donors should be approached differently to the usual protocols that we use. blood donor center's experience with changing from manual to automated blood pressures kimberly j duffy*, sandra bryant, audrey e traun, kristine i borth, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: blood pressure (bp) is important for determining the health and suitability of blood donors. the manual method of reading bp can result in variability due to minor variances in the way staff perform the manual procedure. automated bp devices are able to reduce the variability in bp determination. in december of 2013, automated bp devices were validated and replaced the manual bp method in our blood donor center. the objective of this retrospective study is to determine if the change from a manual to an automated bp process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed. study design/methods: data for the manual bp process was accumulated for an 11 month period from january 2013 to november 2013. the same information was assembled for the automated bp process for the 11 month period of january 2014 to november 2014. the automated bp process implemented in mid-december 2013; so the december data for both 2013 and 2014 has been excluded from the study. bp, bp deferrals, reactions, donor weights and demographics were evaluated for each time period. a donor may be included multiple times in each year and could be in both sets of data. generalized estimating equations were used to assess differences between automated and manual bp with significance defined as p < 0.05. results/findings: significantly more people were deferred using automated bp compared to manual bp readings (p50.006). both systolic and diastolic bp measured significantly higher by automated bp method than by manual method. although donors in the automated bp group experienced fewer reactions than those in the manual bp group, the reduction was not large enough to reach statistical significance. even after adjusting for gender, weight and age at donation, bp deferrals, systolic and diastolic bps all remained significantly higher (all p < 0.03) with the automated bp while and reactions remained non-significantly lower (p 5 0.086). conclusion: automated bp devices have improved convenience for both staff and donors. with a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated bp devices may play a minor role in the safety of blood donors. for the purpose of this study, only the hemoglobin values that were below 12.5 g/dl will be compared as a surrogate for deferral. to adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet a and lancet b, using the appropriate distribution for the data type, defining statistical significance as p-value < 0.05. results/findings: the average hgb was slightly lower with lancet b but there was a larger change with the number of donors under 12.5. statistically more visits with hgb less than 12.5 g/dl used lancet b than lancet a. additionally, fewer first time donors were seen during the lancet b time than during the lancet a time. after adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under 12.5 was 16.5% higher with lancet b than with lancet a. conclusion: donor's hgb was slightly lower with lancet b than lancet a, but not clinically different. slightly more lancet bs were used per visit than lancet as. in addition, more hgb deferrals were obtained using lancet b than lancet a. even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet b than lancet a. the slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels. prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended. blood donors' acceptance and response towards implementation of automatic appointment booking yi lin ang*, ching lian toh and william choon hong sim. health science authority background/case studies: with surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management. disliking the obligation imposed by appointments, singaporean donors generally prefer "walk-ins" as opposed to appointment bookings. blood services group (bsg) singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling. this paper aims to study donors' level of acceptance towards this initiative. study design/method: to determine the donors' acceptance rate, data was collected from 1 january to 31 march 2017. after completing their donation, donors were automatically given the next earliest eligible date for their next donation. those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (donor-care) to make changes to the appointment offered. a reminder will be sent to their phone via sms and/or email to their account three days before the appointment date. data was collected from donor-care and was used to measure the number of appointments made and declined over the three months period. donors who declined appointment scheduling were verbally interviewed for their reasons. results/finding: a total of 6680 donors who has donated blood in the blood bank's main branch were used as the baseline for this study. 85% of donors (n55678) accepted automatic appointment booking, whereas some donors (n51002) were not comfortable with it. 77% of those who declined still preferred walk-ins (n5771) based on their own time schedule, the rest decided that variable situations (n5112), donation frequency (n569) and choice of preferred donation locations (n550) were reasons for declining automatic appointment booking. prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through donor-care was 19%. a comparison was made and found that this study shown a significant increase of acceptance rate by 66%. conclusion: generally, the results were positive and the automatic appointment booking system enabled bsg to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention. bsg is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate. currently bsg has 4 collection centers, each managing its own appointment system. the eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site. 4) , 3.28 0.4940 4 1 poisson distribution, 2 normal distribution, 3 logistic distribution, 4 lognormal distribution 105a transfusion (p>0.05) in donor and reference populations except in younger (20-44 yrs) male donors (p<0.0021; donor 4.9%, reference 10.0%). mean donor sbp, dbp, and pulse were 125 6 14.7 mmhg, 75.1 6 9.6 mmhg, and 75.9 6 11.2 bpm, respectively. screening blood pressure levels consistent with hypertension (29.4% male; 16.6% female) in the 20-44 year donor group, significantly (p<0.0001) higher than the reference population (11.2% male; 8.7% female). no differences were observed in the 45-64 year groups. conclusion: normal source donor demographic and physiologic characteristics often paralleled those of the reference usa populations. however there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in 20-49 year old females. developing blood donor educational materials gay wehrli* 1 , susan rossmann 2 , louis m. katz 3 and dan a waxman 4 . 1 university of virginia health system, 2 gulf coast regional blood center -sugar land, 3 americas blood centers, 4 indiana blood center background/case studies: donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating. donor education (de) materials must address mandates set forth by regulatory agencies. these materials must be accessible and understandable by the general population. the goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized de materials. this study was irb approved as an exempt protocol. study design/method: we developed a de document written at an 8 th grade comprehension level. a convenience sample of volunteers was identified for this two-part study. a focus group (fg) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page de document. the quiz was followed by a group discussion for feedback. the preand post-quiz contained the same 10 multiple choice questions with single best answers including the option to answer, "i don't know." the de document was revised based upon the fg feedback and quiz results. the revised, 3.5 page, de document was then tested using the same pre-and post-quiz during individual interviews (ii). results/finding: demographics and quiz results are summarized in table 1. results from the fg and ii revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain hiv testing. post-quizzes from the ii group revealed an improvement in knowledge acquisition for all four areas. feedback from both groups reiterated that the document was too long. conclusion: developing de materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level (8 th grade). testing de materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population. the next steps for this group will be to pilot the further revised, two-page de document at donation sites. effect analysis of the 'rh(-) blood supply program' establishment hyesung han*, deokja oh, buja hur and chulyong kim. korean red cross blood services background/case studies: the rh(-) blood supply program was developed in 2011 for the purpose of prompt and stable blood supply. based on the computerized system, the program operates the emergency contact/ communication. this program has 4 major functions such as the request of the emergency blood, the recruitment and management of the rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program. the aim of the research is to validate the effect of rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the rh(-) blood supply program. study design/method: researchers investigated the database from 2011 to 2016 after the rh(-) blood supply program was developed. investigators analyzed and compared the recruitment and blood donation of the rh(-) blood donors for the emergency blood donation and securing the blood supply upon request. results/finding: the data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from 5.6% to 21.4% in 2011 and 2016, respectively. also, the actual participation rate of rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from 26.8% in 2011 to 57% in 2016. moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request. conclusion: the result showed that the rh(-) blood supply program was effective for the recruitment/management of the rh(-) blood donors for the emergency blood donation. this system contributes to recruiting and managing rh(-) blood donors who pledge to donate blood and securing rh(-) blood in emergency situation . the institution that needs to meet the demand of rare blood type could possibly use the rh(-) blood supply program which leads to securing special type blood. hanwei chen*. wuhan blood center background/case studies: in china, volunteer blood donors can donate platelets by apheresis (ap) up to 24 times per year. however, the awareness and knowledge of ap donation is much lower than whole blood donation among the chinese population. there are approximately 1.3 million doses of ap transfused within 1.375 billion people each year in china; it is one challenge to recruit new ap donors and retention them as frequency ap donors in china. study design/method: one stratified recruitment and retention strategy established and applicate at wuhan blood center since 2006. firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; secondly, group message for permanent ap donors and had not donated with an interval of more than 180 days in low inventory. thirdly, specific recruiter telephone for those ap donors who had donated aps for more than 4 times and had not donated for more than 90 days or less than 4 times with an interval of more than 60 days from the last donation; the last one is preparing one letter of thanks for those ap donors who gave more than 8 times annually which advise them to voluntarily come to the blood center for ap donation when they were available. results/finding: over the past decade, the overall donation time of ap donors increased by 7.46 times from 5550 to 41420 and the doses of ap increased by 7.41 times from 7363 to 54553 within 10 years. the aps collected fulfilled the clinical needs. according to the donation frequency, ap donors were divided into 5 groups: those who donated ap once, those who donated 2-4 times, 5-9 times, 10-29 times, and those who donated more than 30 times, respectively. it was found that the number of permanent ap donors who donated ap more than 30 times was only 965 (2.1%), but they denoted a total of 76432 doses of ap (29.2%) from 2006 to 2016. conclusion: aps increased at a rapid and steady pace in wuhan blood center from 2006 to 2016, which not only met the clinical needs but also were supplied to other region outside wuhan. and in addition, the permanent ap donors who gained more attention donated the greatest percentage of platelets. in conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region. years were evaluated at 4 sites on 2 consecutive donations for finger stick (fs) hemoglobin (hb) per site policy. venous (ven) and capillary (cap) zpp and ven ferritin (fer) were performed per manufacturers' direction. donors were assessed for subclinical iron deficiency using ranges (fer <26 ng/ml and zpp levels >100 umol/mol heme) at 3 hb levels. participants completed an online survey between donations to collect data on symptoms of anemia. univariate linear regression analysis was used to determine relationship between tests. results/finding: subclinical iron deficiency was present among first-time and repeat blood donors at all 3 hb levels with both genders and all age groups. (table) there was a highly significant correlation between fs zpp and ven zpp 87.8% (r50.937) at first and 86.5% (r50.93) at second donations. at first donation when compared to fs hb, only 10.4% (r50.323) of variation could be explained by variation in fs zpp, 12.3 % (r50.35) by ven zpp and 9.4% (r50.307) by ven fer. at second donation, when compared to fs hb, only 9% (r50.30) of variation could be explained by variation in fs zpp, 14.4% (r50.38) by ven zpp and 20.1% (r50.448) by ven fer. for each donation, variation among tests (fs hb, ven fer, ven zpp and fs zpp) was significant (p<0.001) suggesting strong evidence against correlation. 55% (181) responded to the survey of which 4% (13) reported not feeling well after donation. it should be noted that noted that 1% (3) female study participants reported feeling unwell after the first donation and had ferritin levels below 26ng/ml but the zpp levels were less than 100 umol/mol heme. of the 3% (10) male participants that reported not feeling well none had ferritin levels below 26 ng/ml nor ven or fs zpp levels above 100 umol/mol heme. conclusion: subclinical iron deficiency was present at all hemoglobin levels. there was insufficient correlation with fs hb and ven fer to support use of fs or ven zpp analysis as measurement of iron stores for blood donors. symptoms reported by study participants were not consistent with laboratory results. the minimum male hb was raised from 12.5 to 13.0 gm/dl. fda imposed specific vs ranges for acceptable pulse (p) and blood pressure (bp), removing center-by-center discretion. a survey of members of america's blood centers (abc) was performed to assess the impact on donor deferrals resulting from these changes. study design/method: online survey software (surveygizmo, boulder, co) was used to solicit collections and deferral information from 59 blood centers over two intervals, july-dec. 2015 and july-dec. 2016 (i.e., before and after the implementation deadline for the final rule respectively). information on deferral at presentations for whole blood (wb) donations and apheresis platelet (ap) donations was requested for hb thresholds and vs. the information was stratified by gender (male5m, female5f), and abo type. statistical analysis included t-tests for numerical and chi-square for categorical data (minitab 17.0, chicago il). p <.05 was considered significant. results/findings: data were provided by 40 of 59 centers invited, representing 2,420,886 and 2,945,802 wb donations and 272,094 and 319,161 ap donations in aggregate during the two intervals respectively. gender and abo distributions appeared representative of the us donor base. among m wb donors the rate of deferral rose from 1.5% to 2.9% in the two intervals among aggregated donation attempts (p<.001), and for m ap from 1.8 to 3.5% (p<.001). the mean "by center" deferral rates (table) were similar to that and significant (p<.001). mean by center hb deferral rates among f donations during the two intervals were 11.6 and 11.9% (p50.241) for wb, 11.8 and 13.0% (p5.041) for ap, respectively, absent any change in their acceptable hb thresholds. data on vs deferrals were much sparser. for p deferrals, only 12 centers could provide specific high vs. low vs. irregular pulse deferrals; 27 provided only a summary (i.e total pulse deferrals), and 1 could provide none. for bp, 8 provided detail (high vs. low), 28 summary and 4 none. p deferrals increased in the successive intervals among f wb donors from a center mean of 0.57 to 1.49% (p5.018) and for m wb donors from 0.78 to 1.16% (p5.006). where details were available, high and irregular pulses were responsible for most of the changes for both genders. bp deferrals were not significantly increased among wb donors, regardless of gender. the data sets and deferral rates re: vs in ap donors were quite small, possibly reflecting culling during their prior donation experience. conclusion: substantial additional donor deferrals attended the increased hb thresholds for m in the final rule, for both wb and ap. changes were more modest among female donors, consistent with the absence of changes in allowable hb levels. modest but significant changes attended more stringent requirements for vs, though data limitations restrict this aspect of the analysis. background/case studies: diabetes mellitus is reaching potentially epidemic proportions in india. given the disease is now highly visible across all sections of society within india, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years. due to its ease of use, several studies have found that hba1c testing can identify patients in the community who might otherwise go undiagnosed. we took an initiative to find out the incidence of diabetes by random blood sugar (rbs) measurement among indian blood donors and measure the hba1c levels among those with rbs >180 mg/dl study design/methods: a prospective study was done at department of transfusion medicine and department of biochemistry from 1 st march 2017 to 31 st march 2017. total of 1,861 blood donors were tested for rbs. those with rbs > 180 mg/dl were further tested for hba1c by gold standard hplc method using variant ii biorad. blood donors with >180 mg/dl rbs and hba1c > 6.5% were advised to consult a physician for further evaluation. results/findings: of the 1,861 donors tested, 44 (2.36%) donors showed a rbs of > 180 mg/dl. forty two (95.45%) were males and 2 (4.54%) females with a mean age of 40.55 years (26-56 years). of these, 14 (31.81%) were known case of type-ii diabetes mellitus (dm) on oral medications and were excluded. of the remaining 30, 8 (26.66%) of them had a family history of dm. of these 30 donors, 8 donors did not give a consent for testing for hba1c. among the 22 donors tested for hba1c levels, 16 (72.72%) had hba1c > 6.5%. all the 16 donors were counselled and referred to a physician for further management. the overall incidence of donors having dm in the population is 0.87% (16 of 1839 donors). conclusion: screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society. incidence of low ferritin levels in regular male blood donors with acceptable hemoglobin levels in singapore ramir alcantara* 1 , hwee huang tan 1 and ai leen ang 2 . 1 health sciences authority blood services group, 2 health sciences authority, blood services group background/case studies: iron deficiency is a known complication of regular blood donation. in order to protect the donor's health and prevent iron deficiency, aabb increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from 12.5 to 13.0 g/dl last may 2016. the current minimum acceptable hemoglobin for male donors in singapore is 12.5 g/dl. the aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels (12.5-12.9) and in donors with hemoglobin 13 g/dl and above. study design/method: during a 4 month period, serum ferritin testing was performed on 350 regular male whole blood and 250 regular male apheresis donors who made at least 3 donations in the last two years with an acceptable hemoglobin level. the donors were divided into groups according to donation type and hemoglobin range; group a (whole blood with hemoglobin 12.5-12.9) group b (whole blood with hemoglobin !13, group c (apheresis with hemoglobin 12.5-12.9) and group d (apheresis with hemoglobin !13). the serum ferritin levels of the four donor groups were compared and analyzed. a ferritin level below 30 ug/l is considered low and levels below <12 ug/l are considered having absent iron stores. results/findings: 55.1% of donors in the study have ferritin levels below 30 ug/l. there were more donors with low ferritin in group a compared to group b, 80% and 53% respectively (p<0.05). in apheresis donors, low ferritin rates were higher in group c donors compared with group d, 49% and 30% respectively (p50.001876). ferritin results for the 4 groups can be seen in table 1. conclusion: more than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or 54.3% have absent iron stores. donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after 4 months or more. since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented. due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin 12.5-12.9 g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in singapore be increased to 13.0 g/dl. other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media. background/case studies: safe blood is a crucial and irreplaceable component in the medical management of many diseases. the voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than 15 % of the demand of the blood in pakistan. motivation among the youth, particularly students, is essential to make voluntary blood movement more successful. to assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in pakistan study design/method: a cross sectional prospective study was conducted among 600 students from different universities and colleges of karachi. a well-structured and pre-tested questionnaire, in english, was used to access the knowledge, attitudes and practices about voluntary blood donation. a scoring mechanism was used to understand overall knowledge level. obtained data was analyzed. results/finding: the sample population consisted of 54% male and 46% female students in the age group of 18-28 years. only 65 % of the students have heard about voluntary blood donation and 28 % of the students have given blood once in their lifetime and among them 19 % are blood donors at the moment. 42 % of the participants believed that there is a specific reason why they don't donate blood and 59 % believed that there is a risk involved for the donors, when donating blood. 80 % students wanted to promote voluntary blood donation. fear and lack of awareness on blood donation are the reasons for not donating blood. students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends. (21); miscellaneous effects were reported in 23 courses. side effects led to interruption of supplementation in 55 instances. ferritin levels (mgt6sd) at entry into the program and at the last visit were 48.9 6 2 and 65.4 6 1.7 mg/l in participants, vs 64.1 6 2.2 and 56.3 6 2.2 mg/l in controls. the positive impact of iron supplementation on ferritin levels was observed only in those who took !75% of the tablets. ferritin levels<26mg/l were found in 4,8% of participants and 14.7% of controls. deferral for low hemoglobin was below 1% in both groups. conclusion: an iron supplementation program in a drbcd program is feasible.however, when taking into account acceptance to participate and compliance with supplementation, only 50% of donors obtain full benefit from such a program. using an iron preparation which is better tolerated may increase compliance. background/case studies: hereditary hemochromatosis (hh) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under 21cfr630.10 and the collection is a physician-ordered therapeutic phlebotomy. blood collections establishments do not need an exception or alternative under § 640.120 to make a collection under this provision if the requirements set forth in § 630.15(a)(2) are met. the objective is to describe current hh donors and long-term contributions of to our hospital-based donor center and hospital blood supply. study design/method: in 2001, an irb protocol was approved for the enrollment and therapeutic phlebotomy of hh patients/subjects. this required filing an fda variance to permit hh donor blood for use in our allogeneic supply without disease labeling. the frequency of therapeutic bleeds are guided by routine clinical assessment, mcv/hemoglobin, serum ferritin, and transferrin % saturation monitoring. serum ferritin levels of 50 -75 ng/ ml are targeted for maintenance phlebotomy. operationally, a custom, computerized database application is employed to ease phlebotomy management. results/finding: since inception, the cumulative number of hh subjects enrolled in the hemochromatosis protocol reached 547, of whom 365 (67%) are c282y homozygotes. without active recruitment, accrual rate is about 7 per quarter, with 69% of subjects qualifying as allogeneic donors. the mean current age is 59.7 years, 65% male, 96% caucasian. the majority of hh donors (276 of an active cohort of 318) are in the maintenance phase of therapy with an average of 2.6 donations/year and a 4% deferral rate. over the last 5 years, hh donors contributed approximately 8-11% of the hospital's allogeneic blood supply, averaging 475 whole blood units for transfusion per year. moreover, hh donor's whole blood (wb) donations provided 30-40% of blood for in vitro research at our institution with an average of 180 wb research donations/year. there have been no hh donor-derived transfusion-transmitted infections over 16 years. since 5/23/16, with an increase in male hgb deferral threshold to 13g/dl, there has been only 1 hh male deferral from blood donation. conclusion: a simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from hh subjects was established. blood donated by hh donors remains an important resource at our hospital. hh donors benefit from careful medical follow-up of their iron status. this mutually beneficial relationship is feasible and sustainable. testing for accuracy of non-invasive blood hemoglobin methodology in a blood donor setting michele walker*, sharon garcia and mythili ram. gulf coast regional blood center background/case studies: the objective of the study was to assess the accuracy of hemoglobin (hb) levels measured on the orsense nbm-200 non-invasive occlusion spectroscopy device by comparing them to hb levels measured on venous samples with a laboratory hematology analyzer. in addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method. study design/method: study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (sd) of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results. staff were provided training on the use of the nbm-200 non-invasive occlusion spectroscopy device. over a span of 7 days, 200 eligible blood donors, both male and female, were first screened by the nbm-200 non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method. a venous sample was collected from each of the 200 blood donors for the performance of hb measurement on the sysmex hematology analyzer within 1-3 hours of collecting the venous samples. results/finding: the sd of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results was not to exceed 1.1g/dl. the hb measurements obtained from the nbm-200 and the sysmex hematology analyzer were analyzed using the statistical software minitab and the sd of the difference was reported to be 0.978 g/dl. the precision of the nbm-200 yielded a co-efficient of variation of .02 g/dl and a standard deviation of .33 g/dl. conclusion: the operators found the nbm-200 easy to install, maintain, and operate with minimal training. the nbm-200 non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results. it was comparable to the capillary finger stick method and deemed suitable for screening donors. donors were satisfied with the process and appreciated the safe, painless methodology. ronel swanevelder 1 , ravi reddy 1 , dhuly chowdhury 2 , don brambilla 2 and edward l. murphy* 3 . 1 sanbs, 2 rti international, 3 ucsf/bsri background/case studies: to maintain an adequate blood supply, south african blood centers need to collect more blood from their majority black african population. success in recruiting first-time black blood donors has been tempered by lower suboptimal return rates. study design/method: we performed a prospective cohort study of firsttime, black blood donors donating during a four-month period in 2014 and followed them for one year. within 56 days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone. questions used 4-point likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (muthivhi et al. 2015) . linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year. results/finding: we included 2,902 first-time black donors with median age 23 and female predominance (59%). within one year, 1,786 donors (62%) attempted at least one additional donation. when likert scales were analyzed as an ordinal variable (45 strongly agree to 15 strongly disagree), donor return was associated with the following motivators "blood donation is an easy way to make a difference" (odds ratio for each likert increment (or) 5 1.16, 95% ci 1.06-1.28), "i donated in response to adverts/campaigns on the radio, tv or newspapers" (or51.11, 95% ci 1.00-1.23). responses to altruism-associated statements were not associated with return. among deterrents, donors were less likely to donate if they agreed with the statement "i am afraid of the sight of blood" (or50.83, 95% ci 0.72-0.96) and "i wasn't treated well by the <blood center> staff" (or50.85, 95% ci 0.74-0.97). surprisingly, donors were more likely to return if they agreed with the statement "i was afraid of finding out about my hiv status" (or51.19, 95% ci 1.03-1.37). a secondary analysis treating the likert scales as 4-level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "if i give blood then blood will be available when i need it" and "i don't know where the nearest blood collection point is" were more likely to return. conclusion: this novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation. it is interesting that self-esteem and marketing predicted return better than altruism. fear and poor customer experience are recognized deterrents which could be addressed. we plan to use these data to construct black donor recruitment interventions which may be tested using randomized trial designs. willingness to donate blood during the summer christopher d bernard 1 , ramya ghantasala 1 , obhijit d hazarika 1 , nicole leonard 1 , cori a polonski 1 , zachary b wunrow 1 , michelle heleba 2 , jan k carney 1 and mark k fung* 1 . 1 university of vermont larner college of medicine, 2 american red cross blood services background/case studies: each year donation rates fall in the summer months straining blood banks' capacities to meet local demands. in hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits. study design/method: an anonymous 16 question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region. questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation. results/finding: a total of 292 surveys were received. survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "too busy" (27.5 %) and "traveling is a time for me to relax." (30.6 %). of the respondents who travel in the summer, very few reported donating while traveling (3.4 %). summer donation rates between summertime travelers (36.5 %) and non-travelers (36.4 %) were essentially equivalent. the most preferred methods of scheduling appointments were via the regional blood donor center website (45.6 %) and phone (28.4%). willingness to use a regional blood donation smartphone app was highest among respondents ages of 18 to 34 (45-55%) and lowest among ages 55 and older (13-15%). of respondents with no prior knowledge of summer seasonal shortages (22 %), 2/3rds indicated newfound motivation to donate. background/case studies: viral infections (adenovirus, ebv, cmv, bk, hhv6, and rsv etc.) have been implicated as major contributors to posttransplant morbidity and mortality in hematopoietic stem cell transplantation (hsct) from unrelated donors. investigators have shown that in-vitro expanded virus specific cytotoxic t lymphocytes (ctls) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the hsct setting in recent clinical trials. present clinical trials have shown that ctls can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (pbmcs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin-4(il-4) and il7. others have used banked third party epstein barr virus (ebv)-specific ctls generated from third party ebv-seropositive blood donors with encouraging results. study design/methods: eligible and consented blood donors were tested for cmv antibodies by serology. cmv-seropositive whole blood (wb) units underwent buffy coats processing from non-leucocyte reduced wb units collected in fenwal triple blood-packs tm that underwent 2 hard spins at 3800 rpm for 7 minutes with separation after each spin on a compomatev r g5. plasma and buffy coat was separated from red cells after the first spin. the second spin lead to the separation of the buffy coat from plasma. the buffy coats were submitted to the gmp stem cell lab for processing of cytomegalovirus-specific ctls. hla typing at high resolution for hla-a/-b/-drb1 loci was obtained for all donors. results/findings: forty five eligible healthy blood volunteers (13m [29%]: 32 [71%] f); median age 42 years (range 21-70) donated a unit (500 ml) blood from which buffy coats (average volume 56 ml) were processed. the buffy coat process was previously validated on 20 wb units. the mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures 1 and 2. all of the buffy coats received by the gmp stem cell lab were adequate in cell numbers to be processed. the processing of buffy coats from whole blood is a viable option for the concentration of pbmcs specifically for production of viral specific ctls as third party off the shelf products as well as use in other research projects that require pbmcs from healthy adults. background/case studies: the goal of this presentation is to describe the journey and challenges towards tjc, patient blood management (pbm) certification. transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy. providence holy cross medical center (phcmc), as the providence california region alpha site, has embarked on this journey. our goals are pbm certification and reduction of the number of unnecessary transfusions by 20% within 12 months of the program launch while improving patient outcomes. this paper will discuss our journey toward certification and the various hurdles being overcome. study design/method: tjc, aabb, and the society for the advancement of blood management have served as our primary resources for identifying current evidence-based transfusion practices and management methods. we needed to identify our organizational gaps in data gathering and analysis. then we could determine baseline performance and set improvement targets. from our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff. we took the following steps to develop our pbm program: formed an interdisciplinary pbm team consisting of physicians, nurses, blood bank staff, and data analysts constructed a report on rbc transfusions to help identify outliers and opportunities background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and rbc allocation before each surgery. the extent to which hospitals have an msbos and its design was explored in this survey. study design/methods: the survey was designed, piloted and refined by members of the best collaborative and invited colleagues. it was then encoded in online survey software and the link distributed to best members and colleagues who were encouraged to respond and to further distribute it. the survey was open for 34 days. results/findings: there were 158 completed responses, of which 73 (46%) indicated that their hospital had an msbos and 85 (54%) did not. the majority of hospitals without an msbos were academic centers (36/85, 42%) from oceania (26/85, 31%) or europe (23/85, 27%), had between 500-999 beds (30/85, 35%); the majority of these hospitals transfused between 1,001-4,999 rbcs (21/85, 25%) per year. 15/85 (18%) are going to implement an msbos in 2017. of those with an msbos, the majority 23/73 (32%) were from north america. the majority were academic hospitals (39/ 73, 53%) with 500-999 beds (43/73, 59%) that transfused !20,000 rbc units per year (21/73, 29%) offering a wide range of surgical services. on average there were 207 6 577 procedures listed in the msbos'. the msbos recommended no pre-transfusion testing for a mean of 30% of the procedures listed, a pre-operative type and screen for 38%, crossmatching rbc units for 28%, and for 4% of procedures a different recommendation was made. most (32/73, 44%) of the msbos' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only 5/73 (7%) of msbos' were created solely by using procedure-specific data, and most (35/73, 48%) do not use patient-specific data in making a testing recommendation. most msbos' are updated less frequently than annually (30/73, 41%), and the hospital transfusion committee is often (39/73, 53%) involved in updating it. the msbos' are generally available electronically in both the operating rooms and in the blood banks. it was the opinion of the majority of respondents (30%) that the msbos was used regularly by only a limited number of surgeons and anesthesiologists, 23% of respondents felt that it was regularly used by all surgeons and anesthesiologists; 10% felt that it was not used at all at their hospital, 36% did not respond. conclusion: an msbos was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used. however, 18% of the hospitals currently without one indicated that it would be implemented in 2017 suggesting that these hospitals perceive the value of having one in place. implementing and following an msbos can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing. blood management -one hospital system experience leana serrano rahman*, mallika gupta, susan solometo, ronald walsh and joan uehlinger. montefiore medical center background/case studies: our system, a pioneer aco, is a 1490-bed tertiary-care referral center dedicated to serving patients from across the new york city area and beyond. the comprising four hospitals see 93,000 hospital admissions and nearly 300,000 emergency department visits annually. we have active programs in high risk ob, stem cell transplant, solid organ transplant (heart, liver, and kidney), ct surgery, ecmo, oncology and critical care. transfusion medicine plays a key role in the support of these services. blood product spending in 2015 was approximately $15.8m. in nov. 2015, an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures. the vice president-sponsored multidisciplinary committee was composed of representatives of: surgery, anesthesia, blood bank, pediatrics, perfusion, cardiothoracic surgery, critical care, medicine, and emergency department. study design/method: first important step: "know your numbers"-although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information. baseline numbers were imperative to the committee's ability to effect change. a home grown one time only report revealed which services and clinicians were the highest volume users. the initial plan was to target their use with education. an initial goal was set to reduce expenditure by $1.2m. the journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization. utilization was analyzed using a home grown crystal report "transfused patients by location". this report was further compared to utilization patterns (2014 and 2015), by "dollars spent" and "total units per patient" by the project manager using excel. key initiatives developed by the committee 1. development of evidence based transfusion triggers. 2. education on evidence based transfusion triggers across multiple campuses, specialties and resident programs 3. clinical information system (cis) "soft stops" when ordering blood products outside guidelines. rbc order set defaulting to "1" unit instead of "2" units. 4. updated guidelines posted to easy to find internal intranet spots results/finding: despite higher patient volumes and a more complicated patient mix in 2016, we were still able to reduced blood product expenditures by $933,874 when compared to 2015. conclusion: in spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management. cord blood pathway to reduce iatrogenic blood loss in neonatal intensive care patients tracy shachner* 1 , anna w rains 2 and christopher t clark 3 . 1 university of tennessee graduate school of medicine, 2 univeristy of tennessee medical center, 3 univeristy of tennessee graduate school of medicine background/case studies: anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population. methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia. at a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established. the cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (nicu), preventing need for additional blood draw. the blood tubes are saved for 1 week post-delivery, with cost of $1.10 per delivery tray for sterile tubes. with an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age. study design/method: labor and delivery data from our facility in 2016 was analyzed, and the gestational age and birth weight of all infants transferred to the nicu was collected. from this data, we were able to calculate the total blood volume of these infants using medcalc 3000 system. by using the blood volume values, and assigning a value of 1.5 ml as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established. transfusion results/finding: in 2016, there was a total of 3,331 infants delivered at our facility. out of all the deliveries, 487 (14%) infants were transferred to the nicu. of those infants, 27% received at least one red blood cell transfusion and 7% received at least one platelet transfusion. of the 487 infants transferred to the nicu, 98 (20%) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to 1% (which we considered to be significant), had the cord blood pathway not been in effect. the percentage of blood volume preserved in these infants ranged from 1.0% all the way up to 3.9%. in those 98 infants, the birth weight ranged from 400-1650 grams, and the gestational age ranged from 22 weeks to 36 weeks and 4 days. conclusion: the established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of nicu infants who are most susceptible to iatrogenic anemia. the infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than 2500 grams). development of a standardized response team for massive hemorrhage events outside of an operating room setting james burner* 1 , shannon davis 2 , suzan new 2 , vaishali patel 2 and oren guttman 2 . 1 university of texas southwestern medical center, 2 ut southwestern medical center background/case studies: managing a massive transfusion protocol (mtp) in an operating room (or) is a relatively frequent occurrence with team members well trained in their specific roles. however, in the event of mtp activation outside of an or, sufficient and/or appropriately trained individuals may not be present. this can lead to a scene of confusion and chaos with potential for patient harm. study design/method: a failure mode effects analysis was performed to develop a standardized process for managing mtp outside of an or setting. with participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's mtp was analyzed for potential errors. the results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-or patient. results/finding: code hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring). our code hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (emr) coordinator. their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during mtp and enable enhanced closed loop task performance. the hemorrhage coordinator establishes reliable 1:1 communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination. the emr coordinator enters all orders into the emr, sends/communicates laboratory results and ensures blood products are available to the resuscitation team. the primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices). additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments). conclusion: the code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an or setting. future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation. background/case studies: preoperative anemia affects up to 50% of surgical patients and increases the risk of red blood cell (rbc) transfusion. both preoperative anemia and perioperative rbc transfusion are associated with increased risk of adverse outcomes following surgery. preoperative treatment of anemia includes oral and intravenous (i.v.) iron and erythroid stimulating agents (esa) such as erythropoietin (epo); however, the optimal treatment strategy for preoperative anemia remains to be established. our objectives were to evaluate the efficacy and safety of esa and iron therapy based on their effects on the prevalence of rbc transfusions and adverse thrombotic events. study design/method: we searched the cochrane central register of controlled trials, medline and embase from inception to july 2016; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings. no language restrictions were applied. we included randomized controlled trials in which adult patients undergoing surgery received either an esa and/or iron before surgery, versus iron or no intervention. three authors independently reviewed the studies and extracted data from included trials. risk of bias was assessed for all included studies. where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models. our primary outcome was the number of patients transfused with red blood cells. secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism). results/finding: a total of 79 randomized controlled trials (8, conclusion: amongst patients undergoing surgery, the administration of an esa in addition to oral or i.v. iron was associated with a reduction in patients requiring rbc transfusion. intravenous iron was less effective at reducing rbc transfusion. neither treatment was associated with any clear increase in risk of adverse thrombotic events. additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia. evidence based blood therapeutics scott neeley* 1 and stephanie rogers 2 . 1 dignity health st joseph's medical center, 2 dignity health background/case studies: over 12 million units of packed red blood cells (prbc) are transfused annually in the united states and there is no clinical basis for as many as half of these transfusions. no randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence. study design/methods: a system wide goal was launched across 39 hospitals to decrease the number of prbc transfusions given to clinically stable patients with hemoglobin (hgb) levels >5 7.0 g/dl. the numerator consisted of all prbc units transfused to patients with a hgb of 7.0 g/dl or greater prior to transfusion and the denominator consisted of all prbc units transfused. exclusions included cardiac surgery, nursery, nicu, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which 4 or more prbc units were transfused in one episode. data was extracted directly from the electronic medical record and hospitals received patient level detail every month for all prbc units transfused to patients with a hgb of 7.0 g/dl or higher prior to transfusion. an extensive educational campaign re: evidence-based transfusion practice was launched for physicians and nurses, including the development of a blood therapeutics toolkit, development of standardized dignity health blood therapeutics guidelines, a one day blood therapeutics advanced training symposium, on-site visits to 21 hospitals including 16 cme presentations, online physician and nursing educational videos, communication tools including infographics and "7 is the new 10" buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers. additionally, the ehr powerplans were revised to ensure available selections for "transfusion indication" (required field) were aligned with evidence based guidelines. facilties were encouraged to develop multi-disciplinary blood therapeutics committees to review all transfusions given to patients with pre-transfusion hgb >5 7.0 g/dl on a routine basis, providing feedback to providers whose transfusions were deemed not in accordance with current evidence-based guidelines. results/findings: from fy2015 to fytd2017, there was a 26% reduction in prbc units transfused to patients with hgb >5 7.0 g/dl, starting at a baseline of 67% down to 41%. this represents an fy17 annualized savings of $9.732m, from a baseline of 82 units per 1,000 patients days down to an average 71 units and approximately 2,000 fewer units transfused per month. conclusion: blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk. transfusions to stable, non-bleeding patients with hgb levels >5 7.0 g/dl are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm. furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of prbc should be transfused rather than two. three af studies (sdm5-0.258) reduced rbc units and two studies decreased the percentage of patients transfused (or5 0.700). forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (or 50.264) and rbc units transfused (sdm5-0.553). qualitative/meta-analyses were translated into recommendations by an expert panel and approved by the lmbp workgroup for reducing rbc transfusion. recommendations are: early assessment and effective am; rt, hb alerts in cpoe/cds; reduction of blood loss and af assessing the percentage of patients and rbc units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement. conclusion: conclusion: the lmbp a-6 method led to evidence-based recommendations for reducing transfusion. critical laboratory support is needed to achieve continuous quality and patient safety. background/case studies: reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management. the use of electronic decision support tools such as best practice alerts (bpas) to enforce red blood cell (rbc) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse. the tools in use to date have not provided a dose of rbcs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of rbc given. a therapeutic hemoglobin/hematocrit (hgb/hct) targeted approach to rbc indications/ orders allows for the calculation of a dose of rbcs to achieve the desired target and could further reduce the use of rbc units. our group has developed a computer algorithm to calculate rbc dose based on patient specific data drawn from the electronic medical record (emr) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice. this study describes our initial experience with the use of this algorithm in non-surgical rbc transfusion. study design/method: the blood utilization calculator (buc) is a mathematical formula that draws patient specific information including index hgb/ hct and calculates a dose in number of units of rbcs to transfuse in order to achieve a selected target hgb/hct. hgb/hct target based indications for rbc transfusion were designed and used as the basis for rbc order set with in the ethe buc was embedded within the emrs rbc order set to provide a recommended transfusion dose in number of units when any nonsurgical rbc indication was selected. the target hgb/hct for these indications was 7g/dl/21% or 8g/dl/24%. the number of rbc units ordered and transfused were tracked prospectively for each of the orderable indications. comparison of units transfused per month before and after the buc implementation was performed using student's t-test. results/finding: historically, the three non-surgical rbc indications represented approximately 42% of the total rbc transfused. prior to the buc the mean number of non-surgical rbc units transfused was 590 1 24 units/ month. after the first 5 months of buc activation the mean number of units was 439 1 50 units/month a reduction of 151 units/month or 26% of nonsurgical blood use (p50.003 by t-test). non-surgical rbc use now represents approximately 29% of the total rbc use hospital wide a 13% reduction. this change represents a significant cost savings in rbcs over time. conclusion: the use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical rbc transfusion rate providing enhanced patient blood management and potential cost savings. implementation of patient blood management at a community hospital -30 month report card richard gammon*. oneblood, inc. background/case studies: a collaboration between blood center between (bc) as consultant and three hospital (4001 beds) healthcare system (hcs) to implement a patient blood management (pbm) program was undertaken. this is a review of the first 30 months. study design/method: during year one pbm working group was established. achievements included physician engagement programs, creation of transfusion committee and providing nursing education. auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved. in year two, it created best practice alerts (bpa) when an order did not meet transfusion threshold criteria. bpa showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (rbc)) and allowed ordering physician to cancel order after review. a blood administration video was created. it was mandatory that all physicians granted privileges complete within six months. low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (wg) to address knowledge and practice gaps. in year three, as historically at this hcs very few jehovah's witness patients (jwp) presented, pbm wg was involved with implementation of a bloodless medicine program. all steps of care were addressed including identifying jwp at registration, creating 115a transfusion special arm bands, forming a bloodless medicine physician group, implementing nursing bpa in the electronic medical record, creating advanced directives and marketing to the public. results/finding: the following were monitored for compliance (2q14 vs. 1q17): present and completed consents (66 vs. 94%), present and completed nursing flow sheets (19 vs. 96%), transfusion thresholds supported (73 vs. 100%), discharge instructions provided (17 vs. 86%); (3q15 vs.1q17) vital sign compliance (39% vs.71%). jwp increased from 27 to 225 (04/16-03/17). cost savings were realized by decreased utilization and implementation of bpa. (table 2016 -1q17) conclusion: pbm implementation at a hcs is a continuous and multiyear process. even with a robust program challenges such as vital sign compliance remain. improving patient outcomes in the golden-hour beatrice lebeuf*. medical city plano background/case studies: in emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival. nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging. rapid administration of blood products is vital to the survival of these patients. we implemented bloodtrack emerge (haemonetics, braintree, ma) in our trauma emergency department (ed) as part of a quality improvement initiative to more efficiently provide group o rbcs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource. study design/methods: we treat approximately 30-40 trauma patients monthly. an assessment of our current blood supply chain revealed a multistep, manual process that took about 8 minutes to prepare and physically transport a cooler from the blood bank to the ed. coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not. this practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only 10 percent of coolers fully used. it also consumed valuable staff time as technologists typically made 20-45 trips per month from the blood bank to the ed. plus, there was no effective way to maintain traceability, control access to coolers or monitor usage. results/findings: since our november 2016 implementation, bloodtrack emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the medical city plano's verification as a level 1 trauma center. rather than preparing coolers of blood in case they may be needed in emergency situations, bloodtrack emerge provides ed staff ready access to emergency units whenever they're actually needed -and frees up an estimated 6-10 hours of tech time per month during which they can perform other tasks. audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response. plus, by stocking emergency blood supplies in the ed, the blood bank isn't unnecessarily tying up group o rbc units. today, the blood bank stocks and maintains 2-4 units of group o rhd negative, 4 units of o rhd positive, and 4 units of group a thawed plasma/ liquid plasma in bloodtrack emerge. conclusion: implementing bloodtrack emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations. background/case studies: platelets are a limited resource for which the benefits of transfusion must be weighed against the risks. in 2015, the aabb published platelet transfusion guidelines to assist providers. at our academic medical center, a computer provider order entry (cpoe) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions. discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g. count < 10 k/ml [prophylaxis]) with the option to add a free-text comment. the order is placed and data is stored for later review. study design/method: override platelet orders placed from june 2015-october 2016 were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department. one of five "codes" was assigned to each order: i-indicated or ni-not indicated (based on institutional/aabb guidelines); nmi-need more information; p-protocol (e.g. liver transplant), and nic-non-indication comments (e.g. reserve for or). free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides. results/finding: over a 17-month period, 1,270 cpoe override platelet orders occurred. the percentages of code assignments by month are provided in table 01 below. overall, 532 (42%) were assigned as not indicated (ni). the top keywords assigned to free-text comments were "platelet count less than. . ." (325), "active bleeding" (303), "platelet count of . . ." (173), and "downtrend" (92), many with specified platelet count goals. certain platelet count goals and reasons for transfusion (e.g. "downtrend," "anticipate drop," or "per service,") are not included in institutional or aabb guidelines. of note, 618 (49%) of overrides were placed by hematology-oncology providers. conclusion: a majority of override platelet orders were determined to not be indicated based on institutional and aabb guidelines. of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions. it is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns. this review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on hematology-oncology) in order to improve blood product utilization practices. background/case studies: early diagnosis of iron deficiency anemia (ida) by clinical laboratories (cl), with effective prevention and treatment in primary care may have an impact on packed red blood cell (prbc) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers. they all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population. results are described after implementing a process to prevent ida, its early detection and treatment for years 2014-2016. study design/methods: performance measure after educational and organizational intervention. setting: public integrated healthcare system located in north africa bordering morocco, isolated by 207 km sea distance to nearest continental spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production. cl involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments. process: guidelines for first step cl diagnosis of ida and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose). transfusion was avoided for stable ida patients without active bleeding or coronary heart disease, with a safety hemoglobin (hb) threshold of 5,5g/dl. severely anemic patients were closely followed to asses hb increase and referred for etiology studies when hb> 9 g/dl. background/case studies: bedside nurses are critical in safeguarding the delivery of appropriate patient care. more recently, nurses have also begun to play an important role in patient blood management (pbm) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside. the goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice. study design/method: a short electronic survey (12 questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion. the survey was distributed to all registered nurses via email from floor leaders. responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift. results/finding: there were a total of 32 complete responses (16%). the nurses had a range of experience from less than one year to forty years. ninety percent stated they discussed transfusion necessity with patients, 81% with physicians, and of these, 59% reported doing so proactively before an order was placed. ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only 3% responded that they would suggest product was not needed. nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of 7-8 g/dl (56%), platelet count of 20-50,000 (38%), and inr of greater than 2.0 (69%). conclusion: this study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity. the limited number of survey responses suggests a discomfort with their level of education in transfusion practice. this, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of pbm programs. background/case studies: the use of red blood cell per 1,000 inhabitants may vary 3 folds between european countries, revealing that there may be substantial room for blood optimization strategies. patient blood management (pbm) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes. the objective of our study was to assess the effect of a nationwide pbm program on public health in portugal. study design/method: the first phase of this research project involved a group of 18 key opinion leaders (kol) in a stated preference inquiry to assess the relative value of specific pbm strategies, grouped in pbm pillars, to highlight the need for strategy prioritization in the implementation of a nationwide pbm policy. adaptive conjoint analysis techniques were used to elicit kol preferences. in the second phase a decision analysis model was used to estimate the impact of pbm implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy. model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from portuguese national health databases and literature review. the public health value of pbm implementation in portugal derives from the comparison of two scenarios: "current clinical practice" and "with pbm implementation". results/finding: kol elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred pbm strategies (14.4% and 14.0%), for the remaining strategies weights varied between 7.0% and 10.6%. we estimate that 384,704 patients would be eligible for pbm strategies in one year time horizon, resulting in 594 premature death avoided (3.8% reduction) corresponding to a gain of approximately 1,500 life years and a reduction of 3,660 (6.0%) disability adjusted life years (daly) relative to the current clinical practice. a decrease of 233,141 in-hospital days is expected mainly due to a 8.4% reduction in hospital length of stay and a 37.3% reduction in 30-day readmission rate. in this population the overall transfusion rate could decrease to 4.3% from the current 8.7% (51.2% reduction) implying 17,202 blood transfusion avoided and 65,214 red blood cells units spared. conclusion: we anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources. results/finding: 237 adult liver transplants were performed during the evaluation period. preoperative hemoglobin, creatinine, meld score, spontaneous bacterial peritonitis (sbp), preoperative hemodialysis, gender, and portal vein thrombosis (pvt) gave the strongest model predicting rbc usage. if the model predicted <1250ml of rbcs, all cases with 0ml transfused were captured and only 7.8% of the time >1250ml were used. if 1250-2000ml rbcs were predicted to be transfused, >2000ml were used 25% of the time. if predicted usage was >2000ml, 53% of the time it exceeded 2000ml. conclusion: a model using specific preoperative factors can be used to predict intraoperative rbc usage. patients at risk for >1250ml of rbc transfusion can be identified with reasonable accuracy using this model at our institution. use of this model might help improve preparation and utilization of the blood bank. review of blood ordering practice for elective surgeries in a maternity hospital qi raymond fu*. kk women's and children's hospital background/case studies: pre-operative over-ordering of blood is common, resulting in waste of blood bank resources. blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients. the cross-match to transfusion (ct) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service. according to the american association of blood banks (aabb), a ct ratio of less than 2.0 is favorable, and anything above indicates over-ordering and cross-matching of blood. to achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital. study design/methods: data on elective surgeries requiring blood for standby was collected retrospectively over a 3 month period (jan to mar 2017). details of total blood cross-matched, issued, transfused and returned were analyzed along with the ct ratio. results/findings: during the 3 month period, there were 274 patients undergoing obstetrics and gynecology procedures requiring blood on standby. a total of 494 units of blood were requested. 154 units were crossmatched, of which 138 units were sent to the operating theatre (ot). only 33.3% of blood issued to ot were transfused (n546) while the rest were unutilized. the observed ct ratio was 3.35. conclusion: although only 31% of total blood requested was crossmatched, the ct ratio remains above the recommended guideline of !2.0, with almost 70% of cross-matched blood unutilized. there is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload. establishing and adhering to a maximum surgical blood order schedule (msbos) could help in conserving blood and prevent over-ordering of blood. background/case studies: total knee arthroplasty (tka) is a major orthopaedic procedure with increased perioperative blood loss. this perioperative blood loss could be more significant in patients undergoing bilateral tka in a single stage. the increased blood loss in bilateral tka often requires blood transfusion which results in high post-operative morbidities. study design/methods: in this retrospective study 35 patients who received tranexamic acid (txa) (study group) and 31 patients who did not receive txa during surgery (control) were evaluated for blood loss and transfusion requirement. the study group received a single bolus dose of txa 1gm iv before tourniquet deflation on first side knee. statistical background/case studies: blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks. one strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products. we investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior. study design/method: this retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital. each time a crossmatch for packed red blood cells (prbcs) was ordered via the hospital's electronic ordering system, an indication (e.g. "hemodynamically stable with hemoglobin < 7.0 g/dl") must be selected. if the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated. ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications. an alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion results/finding: over seven months, there were 1732 unique alert encounters. of these, 1531 (88.4%) led to a crossmatch being ordered while 201 (11.6%) led to the order being canceled. providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds (7.0 g/dl) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds (8.0 or 9.0 g/dl background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins. with improved patient data management systems it is now possible to create an msbos based on actual red blood cell (rbc) utilization data on a per-patient basis. this study investigated the transfusion patterns at 4 academic hospitals with data-derived msbos. study design/method: the 4 hospitals were in 2 groups, with one shared msbos for each group. three of these hospitals were large academic centers while one was a children's hospital. at each center the msbos recommended no pre-transfusion testing if 5% of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (t&s) if 5-24% of the patients had been transfused, and a crossmatch of the median number of rbcs transfused if !25% of the patients had been transfused. data were collected at each center over a 1 month period between january to march 2017 and included a maximum of 400 cases per hospital during that one month to ensure equal representation between centers results/finding: between these 4 centers there were a total of 1599 cases analyzed. some of the more frequently performed surgeries included orthopedics (23% of cases), general surgery (16%) and cardiac surgery (11%). there were 1362 t&s ordered for these cases, of which 5 were positive for antibodies on the day of surgery. of all the t&s ordered, 52% were ordered in accord with the msbos recommendation, 26% were ordered when the msbos did not recommend one, and in 0.2% a t&s was not ordered when the msbos recommended one. background/case studies: peripartum blood transfusion is more common in south africa than in the usa and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio 5 6.12 for prenatal hemoglobin (hgb) 8-8.9). we therefore analyzed the etiology and characteristics of antenatal anemia according to hiv status at a large hospital with a hiv prevalence of 29% among obstetric patients. study design/method: we studied a sample of anemic (hgb<10.0 g/dl) pregnant women who were referred to an antenatal anemia clinic at a large hospital in south africa. clinical information was abstracted and blood was sent for laboratory studies. t-tests were used to compare continuous variables between groups. results/findings: a total of 301 women were enrolled, with median age 27 (interquartile range 23-32) years, median gravida 2 / para 1 and median gestational age 28 weeks. mean hgb before referral was 7.5 g/dl and most were already taking oral iron therapy. a total of 169 women were hiv positive with mean cd41 lymphocytes counts of 394 cells/ul; 29 (12%) of hiv positive subjects were on anti-retroviral therapy (art) prior to the pregnancy and 156 (92%) were on art during the current pregnancy. iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in 292 (97%) of women. there was concurrent chronic disease (n52), infection (n52), vitamin b12 deficiency (n52) and antenatal hemorrhage (n56); 10 had other/unknown/missing causes of anemia. there were few pregnancy related complications. hiv positive women had higher levels of c-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and rbc folate than hiv negative women (table) . conclusion: iron deficiency is the overwhelming cause of antenatal anemia among south african pregnant women. compared to hiv-negative women, hiv-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate. a high proportion of hiv positive women were receiving art, consistent with national guidelines. future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion. background/case studies: a 2 month old boy presented to our institution after a 1 month hospitalization in japan. he was admitted there, several weeks after his unremarkable term birth to an ab rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count. he was found to be anemic and thrombocytopenic and required multiple transfusions. also, he had a diffuse, scaling, erythematous rash over his inner thighs. study design/method: initial workup was suspicous for an allergic/necrotizing enterocolitis. the patient had an elevated ldh and potassium, and concern was raised for leukemia with possible tumor lysis syndrome. a sample sent to our blood bank showed an anti-e, with a positive dat (igg and complement), and was positive for e, e, and c antigens. concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the japanese hospital; both possibilities were excluded. further workup revealed no infection or hematologic proliferation. biopsy of his rash showed spongiotic dermatitis. his clinical course deteriorated, and he developed hepatomegaly and jaundice. a concern for wiskott-aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated ige (15270 ku/l; rr: 0-2.9). anti-platelet antibodies were identified. three days after admission, testing was sent for genetic alterations of foxp3, while a japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of foxp31 cd41 lymphocytes. the majority of these indications are seen in adults and for which a reported plasma wastage is $1.8%. fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population. during the utilization review process at our primary pediatric institution, we noted a mean wastage of 8.2% over the last 5 years. with recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (mtp) outcomes, our facility decided to implement the use of thawed plasma and benchmark mtp plasma wastage. study design/method: blood utilization review revealed an increase in the overall percentage of plasma wastage from 2014 to 2016, with a peak of 11.6% (range 3.2%-11.6%). a single cause could not be readily identified prompting us to query children's hospital association (cha), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for 2016 mtps. results/finding: in 2016, mtp was activated 28 times. in 6 cases the patient did not receive any blood product and in 11 cases plasma was already available at the time of rbc allocation/issue. this left 11 cases to evaluate. the median time to plasma availability was 29 minutes (range 4 minutes -61 minutes). the mean plasma wastage for mtp activations was 32% (range 0-100%). of the 9 cha replies, 3 were using thawed plasma and their wastage was </5 5%. conclusion: increasing clinical evidence supports the prompt transfusion of plasma in the setting of mtp to decrease morbidity and mortality. our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of 29 minutes. our implementation began with thawing a single unit of plasma for "level 1 neuro trauma" alerts, and we are now keeping a single unit of ab plasma thawed at all times. plasma is now available at the same time as rbcs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are 9.5 %) to a level consistent with external benchmarking. prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the trauma team. ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice. background/case studies: bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (hsct). granulocyte transfusions (gtx) have been used for over 40 years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children. to contribute to this discussion we demonstrated 5 cases of gtx in pediatric patients with age from 8 months to 16 years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing hsct and chemotherapy. study design/method: donors: data concerning a total of 56 granulocytes donations from 2015 to 2016 were collected from 43 health donors (21 male/ 22 female). donors had matched blood type and serology status for cytomegalovirus when the patient was negative. granulocytes were mobilized with a single dose of rhu-g-csf (filgrastim) subcutaneously, 5mcg/kg/dose (donors up to 70kg, maximum of 600mcg) and oral dexamethasone (8mg), 12h prior to apheresis. apheresis was performed using cobe spectra v r or spectra optia v r with citrate as anticoagulant. all products were irradiated with 40gy. results/finding s: granulocytes number increased 5 times from basal levels and the median of cells collected were 4.89 x10 10 (range 1.09-9.85). in general donors reported no significant adverse reactions. from 56 donations, only in 4 was reported light paresthesia during the procedure. patients: all patients were medicated prior to transfusions with diphenhydramine and acetaminophen. the product was transfused within 24 hours after collection, with efforts to transfuse as soon as possible. details about the transfusions are described on table 1 . adverse reactions for patients were minor and not frequent (4 in 66 gtx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that gtx is well tolerated. conclusion: our study provides further evidence that gtx transfusions can be safely performed on pediatric patients. irina kumukova* 1 , elena kurnikova 1 and pavel trakhtman 2 . 1 russian institute for paediatric haematology, oncology and immunology, 2 instituteffor pediatric hematology, oncology and immunology background/case studies: infections cause major mortality among persons with prolonged neutropenia related to hsct and chemotherapy and among patients with granulocyte disorder. the granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics. we want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections study design/method: we analyzed the retrospective data for the period 04/2012-02/2016. donors underwent granulocyte mobilization with g-csf in a dose 3-5 mg/kg s.c. 12-16 hrs prior to donation; granulocytes were collected with cobe spectra, caridianbct. the analysis was performed through microsoft excel, nonparametric mann-whitney's and spearman tests results/finding: was performed 169 granulocytes collections in 153 donors (82 f, 71 m), ages 18 to 58. the mean increment of wbc after stimulation was 22,6x10 9/l (9,2-41,9 x10 9/l; f-23,2 x10 9/l; m-21.9 x10 9/l); in the age groups 18-39 years (n5121) and over 40 years (n532), the mean increment of wbc was respectively 22,2 x10 9/l (9,2-41,9x10 9/l) and 24,02x10 9/l (12,6-35 x10 9/l). the mean volume of treated blood was 566, a50,05) . the mean number of total wbc in the product was 38,43x10 9/l (13.38-77x10 9/ l; f-34,8x10 9/l, m-42,7x10 9/l). only three donors (1,96%) had apheresisrelated adverse effects the analysis included 244 granulocyte transfusions in 35 cases of infectious complications in 32 patients. the mean number of transfusions was 6.9 (1-60). each patient received transfusions from mean 5 donors (1-32); the granulocyte transfusions started at the mean 8 days (2-30) after infection; the mean dose of wbc on the transfusion was 11.6x10 8/kg (2-30.7x10 8/kg). wbc increment was able to estimate after 144 transfusions. wbc increment was recorded after 96 transfusions (66.7%); the mean increment was 1,12x10 9/l (0,01-11,38 x10 9/l). efficacy was determined by the number of patients who survived an infection episode and amounted to 80.6%. in the cases of severe sepsis, the efficacy of transfusions was 76.2%. the frequency of post-transfusion reactions was 22%, (n 5 8): febrile nonhemolytic reactions 8,3% (n 5 3); pulmonary complications 11,1% (n 5 4); the anti-hla antibodies formation 2,7% (n 5 1) conclusion: no significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline wbc and their increment after stimulation. the mean total wbc in the collected product and the mean volume of treated blood from men and women were significantly different. perhaps the reason for lower cell products from women is to treat smaller blood volume. the lack of increment wbc after transfusion is not equivalent to lack of its efficacy. we found a significant association between transfused dose and increment of wbc. we deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics cp150 hemolytic disease of the newborn due to anti-rh17 keegan barry-holson* 1 and jennifer andrews 2 . 1 stanford university, dept of pathology, 2 stanford university, dept of pathology & pediatrics background/case studies: anti-rh17 is a rare antibody against a high incidence red blood cell (rbc) antigen (ag) present in people with common rh phenotypes. this ag is missing in individuals who are rh null or have rh deletion phenotypes, including d--/d--. d--individuals strongly express d and lack c, c, e, and e ags. the clinical relevance of anti-rh17 has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (hdn). we present a rare case of hdn due to anti-rh17 alloimmunization during pregnancy in a d--mother. study design/methods: an o-positive full term infant was born to an opositive g3p1 > 2 mother with a negative 1 st trimester antibody screen and no prior transfusions. she had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the 1 st trimester. father's blood type is unknown but presumably he has rh antigens. the infant was transferred to our institution at 6 hours of life because he was found to have anemia (hemoglobin 12.0 g/dl), severe hyperbilirubinemia (total bilirubin (t bili) 9.0 mg/dl), reticulocytosis (8%) and a positive direct antiglobulin test (igg 21). he was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for hdn. he was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis. t bili rose to a maximum of 16.7mg/dl on day 8 of life and phototherapy was restarted. his t bili subsequently stabilized and he was discharged home and followed in clinic. meanwhile, his mother donated blood given there were no compatible red blood cells available in the united states via rare donor query. nine days after discharge, he was readmitted for worsening anemia (hemoglobin 6.3 g/dl) and was given steroids and washed maternal red blood cells. he was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia. results/findings: at delivery, the mother's antibody screen was positive and anti-rh17 was identified; no other alloantibodies were detected. antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement. maternal serum was pan reactive against panel cells and non-reactive against d--cells. anti-rh17 sera did not react against maternal rbcs. phenotyping of the mother revealed that she was d1 c-e-c-e-. molecular testing confirmed her d--genotype; molecular beadchip test yielded no type due to low signal for e, e, v and vs ags. genotyping for rh variant and targeted genomic rhce testing failed to detect several rhce exons. father was unavailable for further testing. conclusion: we report a rare case of hdn due to anti-rh17 antibody in a d --mother. we hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population. these studies have important implications for genetic counseling for mother's sisters. management of severe autoimmune hemolytic anemia: a case report of an infant treated with manual whole blood exchange with rapid clinical improvement yunchuan delores mo* 1 , cyril jacquot 1 , valli criss 1 , philippe p pary 1 , jay greenberg 1 , naomi lc luban 1 and edward cc wong 2 . 1 children's national medical center, 2 quest diagnostics background/case studies: management of severe autoimmune hemolytic anemia (aiha) presenting with life-threatening anemia is challenging, particularly in the pediatric population. mortality rates in aiha are typically low; however, in children, the rate may be as high as 4-11%. although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (wbex) to successfully treat aiha in older children and adults refractory to first line treatment. to our knowledge, this is the first case report in which an infant with severe aiha has been successfully treated with manual wbex in an acute care setting. study design/methods: case report format. results/findings: a 2 month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (hb)/hematocrit (hct) of 1.6 g/dl/4.9%. wbc counts (19 x 10 9 /l) were mildly elevated and platelet counts (410 x 10 9 /l) were within normal limits. her history was notable for upper respiratory tract infection 6 days prior to the onset of anemia. laboratory studies on admission showed hyperbilirubinemia (total 7.1 mg/dl, direct 1.4 mg/dl), normal ldh (318 u/l), and undetectable haptoglobin (<7 mg/dl) indicative of ongoing hemolysis. clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis. she was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access. the patient's blood group was o, rh (d)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin (3-41 reactivity) with positive autocontrol. dat was 41 positive for anti-igg and negative for c3 despite a positive cold antibody screen. the patient weighed 6.9 kg with an estimated total blood volume of 620 ml. she initially received simple transfusions totaling 20 ml/kg of least incompatible group o rh(d)-negative rbcs with no incremental response. manual wbex was then performed with 463 ml of reconstituted whole blood consisting of o, rh(d)-negative rbcs and ab fresh frozen plasma (ffp) to an hct of 40%, utilizing the central venous catheter. no adverse events took place over the course of the 2 hour exchange. her one hour post-exchange hb was 9.5 g/ dl and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin (21). after initiation of steroid therapy (methylprednisolone, 2 mg/kg/day), she continued to improve clinically. one week later, the patient was discharged home with a hb of 11 g/dl. one month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal igm and iga levels with markedly elevated igg levels (3168 mg/dl). at a subsequent follow-up visit 3 months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids. conclusion: we demonstrate a case of severe neonatal aiha successfully treated with manual wbex. the main advantages of wbex include removal of both autologous rbcs and plasma as well as infusion of allogeneic rbcs. in this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation. in summary, manual wbex is a potentially safe procedure that may be performed in young children with severe aiha. abstract operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury. clerical and serologic investigations revealed no cause for hemolysis. mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered. study design/methods: in vitro simulated transfusions were performed via syringe. measurements included hematocrit (hct), free hemoglobin, and visual hemolysis index. washed and unwashed red blood cells (rbcs) were tested with or without a one-way valve, using a 24 or 16 gauge (g) intravenous (iv) catheter. each one-way valve was used to test three identical samples. constant pressure was applied manually (rapidly, 1.431/-0.49 ml/ second) or with a mechanical syringe pump (slowly, 2 ml/min). a subset of the manual transfusions was timed. control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter. results/findings: the one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed rbcs (see table) . with the 24g catheter, the mean change in hct was -3.531/-0.69% with the one-way valve and 0.221/-0.13% without (p<0.00001). comparing the one-way valves tested, differences in hemolysis were observed (change in hct; p<0.0001). during rapid manual transfusion with a 24g catheter and unwashed rbcs, hemolysis was greater for samples that took longer to transfuse 4.5ml when using a one-way valve (change in hct versus time: r5-0.75, p<0.0001) compared to a significantly different (p50.0085) slight increase in hemolysis for samples that took less time to transfuse 4.5ml when not using a one-way valve (change in hct versus time: r50.58, p50.23). correlations between time and hemolysis were similar, but insignificant using 24g with washed rbcs and the 16g iv catheter. conclusion: mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve. during rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis. background/case studies: gerbich (ge) antigens expressed on glycophorin c are present in 99.9% of the population. ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). ge antibodies also suppress erythropoiesis resulting in late-onset anemia. we report a case of hdfn due to anti-ge3. study design/methods: a woman of paraguayan origin with prior terminated pregnancies presented at 24 weeks gestation with passive anti-d and an anti-ge3 titer of 256. she was d-and ge:-2,-3, 4 by antigen typing. her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at 37 weeks. cord blood was dat positive for igg; the eluate confirmed anti-d and anti-ge3. the birth hemoglobin (hgb) was 12.6 g/dl, reticulocyte (retic) was 8.6%, bilirubin (bili) was 2.8 mg/dl; the infant was discharged. on day 7 of life, the infant was referred to pediatric hematology for lethargy and poor feeding, with hgb 7.6 g/dl, retic 2.6%, and bili 6.6 mg/dl. ge3-blood was not available from the blood center or rare donor registry. the mother was b rh-and baby was b rh1. obstetrics had to authorize maternal blood donation due to her hgb of 10.9 g/dl. maternal blood collection and rbc washing was expedited and the infant received 40ml of maternal rbcs within 24 hours, at which time his hgb was 6.1 g/dl. post-transfusion hgb was 10.8 g/dl. one week later, the infant was symptomatic with hgb 7.1 g/dl, retic 1.0%, bili 2.1 mg/dl. a 2 nd aliquot of 60ml washed maternal cells was transfused. two weeks thereafter, the infant had hgb 7.8 g/dl, retic 0.7%, anti-ge3 titer 8, and needed another transfusion. the maternal blood stored for just 3 weeks had hemolyzed necessitating a 2nd maternal donation for baby's 3 rd transfusion. at 6 weeks, the infant's anti-ge3 titer was 2, hgb 9.2 g/dl, retic 1.7%; no transfusion was necessary. at 8 weeks of life, hgb was 10.2 g/dl, retic was 3.3%, and the baby was thriving. results/findings: serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-ge3 hdfn. molecular analysis revealed that the mother was homozygous ge3-negative ge*01.-03, the father had homozygous wild type ge*01, and the infant was heterozygous ge*01/ge*01.-03. conclusion: the infant had hdfn due to antibodies to the high prevalence ge3 antigen. the continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-ge3. hemolysis of stored maternal blood was consistent with the absence of glycophorin c. this case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of hdfn resulted in a successful neonatal outcome. background/case studies: patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes. in pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice. in addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult. because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution. we designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions. study design/methods: a data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders. we focused on rbc orders given the tripicu randomized clinical trial results (1) supporting a hemoglobin trigger of 7 g/dl in stable critically ill children and ffp since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (inr) values without bleeding. results/findings: in 2016, 14, 247 rbc orders occurred and the top three patient groups were: 34% in congenital heart disease patients, 25% in hematology/oncology patients and 14% in neonates in the neonatal intensive care unit (nicu). average hemoglobin of every patient was 9.85 g/dl as measured in the 72 hours prior to rbc order placement. in 2016, 3105 ffp orders occurred and the top three patient groups were: 46% in neonates in the nicu, 28% in congenital heart disease patients and 13% in pediatric intensive care patients. average inr of every patient was 2.09 as measured in the 72 hours prior to ffp order placement. conclusion: we have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital. this serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses. background/case studies: bacterial contamination of plts remains an ongoing threat to transfusion recipients. recently, a psoralen-based pr technology that reduces the replication potential of pathogens in stored plts was fda approved. we describe our approach to phasing pr-plts into our inventory, including preliminary results of an ongoing qa study of neonatal and pediatric (peds) recipients of pr-plts. study design/methods: before the arrival of pr-plt, we undertook an educational campaign for hospital administrators, it staff, laboratory staff, clerical/clinical aides, nurses, and physicians. we also contacted risk management and the hospital ethics committee. phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based pr-plt product. shortly following the arrival of pr-plt, we introduced day 5 bacterial "safety measure" testing of our conventional (c-plt) supply. a peds qa study monitored plt utilization and adverse transfusion event reporting relating to both pr-and c-plt transfusions. this study evaluated neonates (0-4 months of age), infants (>4-12 months of age) and children (>12 months-18 years of age) who received at least one transfusion of pr-plts. results/findings: risk management and the ethics committee agreed that both pr-plts and bacteria tested c-plts would be the hospital standard of care. pr-plts were phased in and transfused to patients based on abo compatibility and expiration date, per routine, without regard for patient age or medical condition. after 4 months, pr-plt represented 30% of our platelet inventory (average daily plt inventory: 45 units). we encountered no complications with the pr platelet phase-in, either from a clinical, informatics or logistical perspective. due to the dual inventory, many peds patients in all age groups were transfused with both pr-and c-plts (table) . two potential transfusion reactions (trs) were reported over the study period in teenage recipients, one associated with a c-plt and the other with a pr-plt. in both cases, the symptoms were ultimately attributed to an underlying medical condition. no rashes were observed among 16 transfused neonates (0-4 m) who received any pr-plts and phototherapy. background/case studies: packed red blood cell (prbc) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children. however, evidence shows that hemoglobin (hgb) in prbcs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased 2,3 dpg levels. standardization of prbc transfusion practices in this population and the scientific evidence on which current practice is based is limited. additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events. study design/method: medical records of 60 pediatric patients receiving prbc transfusion over a 12 month period were retrospectively reviewed. a total of 44 patients were identified as receiving allogeneic prbc transfusion. 16 patients who received autologous blood (cell salvage) were excluded. patient characteristics, length of stay, prbc transfusion volume, pre-and post-transfusion hgb, and adverse events were collected. results/finding: the average pre-transfusion hgb was 10.6 g/dl with post-transfusion hgb rising to 14.5 g/dl. the mean prbc volume transfused was 46.3 ml using a dose of 15ml/kg for all patients. complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (nec), and death (table) . conclusion: evidence based transfusion guidelines are lacking in neonates and infants. a typical dose of 10-15 ml/kg in a 2 kg patient, for instance, would translate into 3 full prbc units (about 1000 ml) in an average size adult. the current standard dose of 10-15 ml/kg yields very high increases in hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity. additionally, many of these patients received volume reduced products which delivers a higher hgb concentration per transfusion. dosing should be based on goal hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear. pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only 3 case reports suggesting this association. however, each of these cases is confounded by other findings in addition to a mycoplasma infection. we describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a mycoplasma infection without a detectable cold agglutinin. study design/methods: the patient is a 7month-old, previously healthy female infant who presented to the hospital with a 1-week history of fever, fatigue, decreased appetite, and pallor. she was only treated with acetaminophen. she also developed clear rhinorrhea the day before hospital admission. at the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of 2.7 g/dl and 9.1%, respectively, platelets of 635,000, and a reticulocyte count of 10.3%. all other elements of the complete blood count were within the normal reference range for age. a complete metabolic panel revealed no abnormalities except for a total bilirubin of 4.9 mg/dl with a direct fraction of 0.43 mg/dl. a filmarray respiratory panel (biofire diagnostics; salt lake city, ut) detected mycoplasma pneumoniae, while all other pathogens (19 total) were non-detectable. the patient was started on a 5-day course of azithromycin (zithromax). results/findings: prior to rbc transfusion, blood bank evaluation revealed that the patient was o-positive and had a stronglyreactive antibody screen. further testing demonstrated an antibody reactive with all reagent red blood cells. the dat was strongly reactive for igg but very weakly reactive for c3. an eluate was reactive with all reagent red cells tested. finally, a cold agglutinin study was negative with undiluted serum. in addition to starting azithromycin, the patient was given iv methylprednisolone. during her 8-day hospital course, the patient received 2 rbc transfusions on the day of admission and several rbc transfusions thereafter (see table 1 ). despite transfusion, her hemolytic process persisted, so she was infused with a dose of iv immunoglobulin on hospital day 6. her hemoglobin rose to 8.4 g/dl on hospital day 7 and increased to 9.5 g/dl on hospital day 8. at that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next 2 weeks. she was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to 11.2 g/dl on day 57 after her hospital admission) with no recurrence of her hemolytic process. conclusion: m. pneumoniae infection is a typical cause of cad and has only rarely been associated with warm autoimmune hemolytic anemia. our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant. with the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a m. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. ) and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia. the difficulty in eliminating the cord blood testing is the neonatologists' reliance of using abo incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing. currently the ts requires all positive dat tests to be communicated to the nursing staff immediately. given that the dat strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the ts staff may also consider notifying nursing staff only for those patients whose dat is 3 or 41. platelet and leukocyte immunohematology, testing and genetics table 1 . of 53 pairs, 7 pairs were complete match (2/2), 26 pairs were partial match (1/2), 20 pairs were complete mismatch (0/2). the matching rate of hla-dpb1 in our study is 13%. conclusion: the matching rate of hla-dpb1 in 10/10 hla matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of hla-dpb1 in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between hla-dpb1 and unrelated hematopoietic stem cell transplantation. this work was sponsored by national science foundation of china (81401732) background/case studies: thrombotic thrombocytopenic purpura (ttp) is caused by severely reduced activity of the von willebrand factor-cleaving protease adamts13. therapeutic plasma exchange (tpe) as well as immunosuppression minimize the morbidity and potential mortality of this presentation. absolute immature platelet counts (a-ipc) have been shown to help diagnose and follow ttp patients' responses to therapy. we report the case of a man with relapsing ttp, low adamts13 with high inhibitor, treated with mycophenolate mofetil in which a-ipc-indicated an unexpected response to therapy. study design/method: a 56 year old male with a 7-year history of ttp, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing ttp. patient had been treated in prior admissions with tpe, prednisolone, rituximab, and cyclophosphamide with clinical improvement. he was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, adamts13 <5% and inhibitor of 3.6. on day of admission platelet count was 95 x 10 9 /l which decreased within five days to 14 x 10 9 /l leading to initiation of daily tpe along with mycophenolate mofetil discontinuation just prior to tpe start. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. abstract results/finding: a-ipc and platelet count were 1 x 10 9 /l and 14 x 10 9 /l respectively. counts improved rapidly post-tpe initiation and after one tpe his a-ipc tripled to 3.2 x 10 9 /l achieving the ratio of 3 previously shown to be diagnostic of ttp. on day 5 his a-ipc and platelet counts had improved to 7.5 x 10 9 /l and 218 x 10 9 /l respectively. absence of anti-pf4 antibodies ruled out heparin-induced thrombocytopenia at this time. on day 6 he had an unexpected decrease in both a-ipc and platelet count to 4.8 x 10 9 /l and 132 x 10 9 /l respectively, worsening by day 8 to 1.7 x 10 9 /l and 40 x 10 9 /l respectively despite daily tpe. patient received 25 additional tpes that failed to improve a-ipc or platelets which on day 32 were 0.4 x 10 9 /l and 13 x 10 9 /l respectively. a-ipc had remained at this level for 16 days suggesting that the observed decrease was irreversible. adamts13 activity remained <5% low with a high inhibitor. patient's clinical condition continued to deteriorate and family placed patient on comfort care. conclusion: ttp patients have low a-ipc and plt counts at presentation, with the former improving first post-tpe initiation. despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low a-ipc. in the setting of ttp, or relapsing ttp use of immunosuppression should be closely followed and a-ipc may aid in establishing early if therapy is affecting platelet production. application of luminex bead technology to detect hpa-1a, hpa-3a, and hpa-5a antibodies su-dan tao*, ying liu, yan-min he, ji he and fa-ming zhu. blood center of zhejiang province, key laboratory of blood safety research, ministry of health background/case studies: detection of antibodies against human platelet antigens (hpas) is crucial for patients' refractory to platelet transfusion therapy. in the text, luminex bead coupled with anti-gpiib/iiia and anti-gpia/iia monoclonal antibody was implied to detect hpa-1a, hpa-3a, and hpa-5a antibodies, and the sensitivity of luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (maipa) assay. study design/method: monoclonal antibodies p2 and gi9, specific for platelet glycoproteins gpiib/iiia and gpia/iia, were separately coupled to luminex xmap beads. four standard sera, containing anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b respectively, were bought from nibsc; three negative sera without hpa antibodies were prepared from ab type blood donors. platelets (containing hpa-1aa, hpa-3ab and hpa-5aa) were collected and reacted with anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins. the beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a luminex100. the hpa-1a serum was diluted to 10 serial dilutions (from neat to 1/502) to test the sensitivities of maipa and luminex beads assay. the two methods were then used to test five blinded samples which were collected from fmait patients. results/finding: luminex bead technology showed that the mfi values of hpa-1a, hpa-3a, hpa-5a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( .08 vs 37.05), which implied that the luminex bead technology could specifically identify negative and positive sera of anti-hpa-1a, anti-hpa-3a, anti-hpa-5a. furthermore, because the platelet was hpa-5aa, the hpa-5b serum did not react with the coupled beads with mfi was comparable to negative control (286.59 vs 127.25). the sera were re-tested by maipa and the results of which were comparable to luminex bead technology, illustrating that detecting hpa antibodies by luminex beads technology was successful. the sensitivity of luminex bead assay and maipa to detect anti-hpa-1a was 1/128 (0.78iu/ml) and 1/64 (1.56iu/ml), respectively. no cross-reactivity was observed with the samples containing hla, abo or other platelet antibodies. all results of five blinded samples tested by luminex assay showed that four sera were positive for gpiib/iiia antibodies which were consistent with maipa results. conclusion: the luminex beads coupled with gpiib/iiia and gpia/iia monoclonal antibodies could be successfully used to detect hpa-1a, hpa-3a and hpa-5a antibodies via the epitopes on platelet glycoproteins. the sensitivity of luminex technology was higher than maipa technology. (ahus) is a thrombotic microangiopathy (tma) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin. the literature suggests the presence of pathogenic mutations in complement proteins in 50% of cases of ahus. there is a lack of well-defined recommendations regarding testing for genetic ahus. complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs. we reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population. study design/method: we performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from 1 january 2014 to 31 december 2016. clinical history was solicited by genetic counselors. cases were classified by the authors as primary ahus (tma and renal failure without identifiable cause), secondary tma (tma and renal failure with identifiable cause previously associated with tma) or non-tma. the test panel identified variants in complement proteins (cfh, cfi, mcp, factor b, c3, c4bp, thbd, dgke, cfhr3, cfhr1, cfhr4 and cfhr5) that were classified as vus (variances of uncertain significance), pathogenic or benign by the american college of medical genetics. chi square analysis/fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary ahus versus secondary tma. independent sample t-test was used to compare differences in continuous variables between primary ahus and secondary tma. results/finding: of 134 patients tested, pathogenic mutations were detected in 13% (18/134) and vus in 35% (47/134). 20% (27/134) of patients did not fulfill criteria for tma; no pathogenic mutations were found in this group and 9 (33%) had vus. 31% (42/134) of patients had primary ahus; of these, 28% (12/42) had pathogenic mutations and 40% (17/42) had vus. 48% (65/134) of patients had secondary tma; of these, 9% (6/65) had pathogenic mutations and 32% (21/65) had vus. in patients with pathogenic mutations, 39% (7/18) were children, 22.5% (4/18) had a positive family history of ahus and 28% (5/18) had recurrent disease. patients with primary ahus had a significantly lower age at presentation (22 6 18 vs. 33 6 20 yrs; p-value: 0.005) and a higher proportion of pathogenic mutations (28% vs. 9% p-value: 0.009) compared to patients with secondary tma. gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups. conclusion: we found a lower frequency of patients with pathogenic mutations compared to reported literature. our data suggests that patients with secondary tma should be carefully evaluated prior to ordering genetic testing and those without tma should not undergo this test. counting of platelets in platelet concentrates on hematology analyzers pentraxl80 and sysmex xn9000 compared with a flow cytometric method farshid ezligini 1 , kjersti roen eriksen 1 , annette vetlesen 1 , thomas larsen titze 1 and geir hetland* 1,2 . 1 oslo university hospital, 2 university of oslo background/case studies: hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (plt) concentrates (pcs). a flow cytometric method for counting of plt in pcs has been developed as validation tool (van der meer et al, transfusion 2012). therefore, it is pertinent to evaluate plt counting in bcs on hematology analyzers with this validation method in a flow cytometer. study design/methods: samples from ten apheresis pcs and 33 buffy coat-derived pcs were subjected to plt counting on hematology analyzers pentraxl80 (horiba abx, montpelier, france) and xn9000 sysmex toa (kobe, japan) (both impedance score), and additionally, diluted and stained with anti-cd41a fitc in truecount tubes (bd biosciences)(internal bead standard) for measuring in a gallios flow cytometer (beckman coulter, indianapolis in, usa). results were analyzed by paired samples test and shown in bland-altmann plots. results/findings: mean plt values x10 9 /l 6 sd were 819 6118, (<) 1106 6137, (<) and 1195 6176 for counting by sysmex toa, pentraxl80, and the gallios flow cytometer, respectively. sysmex count was the very lowest 129a transfusion 2017 vol. 57 supplement s3 abstract (31.4% less than for flow cytometry), but all plt counts were significantly different (p<0.001), although least so (7.4%) between pentra and flow cytometry. conclusion: as validated by the flow cytometric method, pentraxl80 seems suitable for routine quality control of pcs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting. the much lower plt count on sysmex may reflect its optimization for plt counting in whole blood rather than in pcs. fast, precise & easy hpa typing with real-time pcr jonathan downing 1 , arishma lata 1 , roland russnak 2 , zachary antovich 2 , heather dunckley 1 and thierry viard* 2 . 1 new zealand blood service, 2 linkage biosciences background/case studies: the interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis. human platelet antigens (hpa) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible hpa. thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders. the new zealand blood service performs hpa typing on a pool of platelet donors to provide compatible transfusions where the need arises. the molecular basis of most hpas has been characterized as generally caused by a single-nucleotide polymorphism (snp). hpa typing has typically been performed using pcr-ssp, a method that utilizes time-consuming post pcr analysis steps. the aim of this study was to evaluate the use of real-time pcr-based techniques in a transfusion laboratory setting. study design/method: we evaluated a commercially available solution which consists of 24 reactions that identify both variants of 12 relevant snps located within hpa genes (hpa-1 through hpa-11, and hpa 15). genomic dna purified from 48 blood samples, previously genotyped for hpa-1,-2,-3,-4,-5 and -15 by our in house pcr-ssp method were used in this study as validation samples. results/finding: results of the validation samples were 100% concordant with typing obtained by pcr-ssp. the real-time pcr approach overcomes the major challenges of hpa molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time. the analysis is facilitated by a software which generates the results. with less than 10 minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately 90 minutes. further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced. the real-time pcr approach with automated analysis was implemented by the new zealand blood service tissue typing laboratory in late 2016 and to date has tested 749 dna samples from 400 blood donors (349 donors were tested in duplicate). concordance between the sample replicates was 100%. there were 24 occasions where the assay had to be repeated, giving a repeat rate of 3.2%. occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required. this occurred most commonly with the hpa-3 (4.7%) and hpa-5 (1.2%) assays. conclusion: real-time pcr with automated analysis provides an effective, robust an accurate method for molecular hpa genotyping. with its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting. genetic variation of cd36 antigen deficiency expression in jiangsu chinese han population qing chen* 1 , jianyu xiao 1 and chengyin huang 2 . 1 jiangsu province blood center, 2 jiangsu province blood center background/case studies: cd36 has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-caucasian. cd36 deficiency varies widely among different ethnic populations, with the frequency of 3-11% in asians and 2.4% of african americans, respectively. however, there is little information on the molecular basis of individuals with cd36 deficiency in jiangsu chinese han population. study design/method: to investigate platelet cd36 expression levels and to determine the molecular basis of cd36 deficiency on the platelet surface of the han population in jiangsu region. cd36 expression levels on platelets were detected by flow cytometry among 243 blood donors in jiangsu region. donors without cd36 antigen expression on their platelet surface were further to be determined the expression of cd36 antigen on their peripheral blood monocyte cells. the coding exons of cd36 gene and adjacent introns were amplified and sequenced in cd36 deficient individuals. results/finding: among these 243 blood donors, cd36-deficient and cd36-expression individuals were 2.47% (6/243) and 97.53% (237/243), respectively. the frequencies of type i and type ii cd36 deficiency among the study population were 0.41% (1/243) and 2.06 % (5/243), respectively. among 237 individual with platelet cd36 expression, according to mean fluorescence intensity (mfi) value, 45, 141 and 51 individuals showed low, moderate and high expression levels of cd36, respectively, and their mfis were 1725.9 6 343.6, 3876.1 6 788.5 and 8431.6 6 529.9 (p<0.05), respectively. the type i cd36 deficiency individual were heterozygous for 1200-13a>g and 430-14c>g, respectively. among type ii cd36 deficiency individuals, two harbored a t insertion at position 560 in exon 6 which caused frameshift at codon 187; one has a t>c exchange at position 538 in exon 6 which resulted in a tryptophan to arginine substitution at codon 180; one has a a insertion before the 17th bp of the start codon atg in the promoter region; one were heterozygous for 748 1 2t>c and 1006 1 2t>g, respectively. conclusion: platelet cd36 surface expression levels were diversified in the jiangsu chinese han population. the frequency of the type ii cd36 deficiency was higher than that in type i. the study findings indicated that the frequency of cd36 deficiency in the chinese population is slightly lower than that in other asian countries. background/case studies: cd36-deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders. the frequency of platelet cd36-deficient individuals widely varies among ethnic groups, with 3% to 11% in japanese, 8% in sub-saharan africans, 2.4% in african americans, and 0.3% in caucasians. although some studies of cd36 deficiency are focused on the asian populations, relatively little information has been reported in the chinese population. here we investigated the cd36 expression on platelets in large samples of the eastern chinese donors. study design/methods: peripheral blood samples were collected from 1282 unrelated platelet-apheresis donors in the eastern china. the expression of cd36 antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (fitc-anti-cd36 and peanti-cd41). the isotype control (fitc-mouse igg) was also analyzed to calculate a reference range of cd36-nagtive phenotype. for those donors with cd36-negative platelets, cd36 antigen expression on monocytes was analyzed further to distinguish between cd36 type i and type ii deficiency. flow cytometric parameters were statistically analyzed by mann-whitney test. the work was supported by national natural science foundation of china (81570170) and zhejiang high-level innovative health talents. results/findings: the mfi (mean fluorescence intensity) of platelet cd36 in all 1282 samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype. on account of this limitation, we classified the cd36 phenotypes using the (mean 1 3sd) of the background mfi observed in isotype controls. forty-three samples were detected as cd36 deficiency on platelet, in which one sample was cd36 negative both on platelet and monocyte. the frequency of cd36 type i and type ii deficiency in the eastern chinese donors was 0.08% and 3.3%, respectively. the average mfi of cd36 deficiency samples was significantly lower than cd36 positive samples (15.2 6 7.9 vs 79.8 6 37.8, p< 0.0001). conclusion: the frequency of platelet cd36 deficiency in the eastern chinese donors was close to japanese and african americans. it means that the possibility of cd36 antibody occurred by pregnancy and transfusion in this population is existed. it is useful to find and register cd36-deficient donors by large-samples screening for potential immune thrombocytopenia patients with cd36 antibody. background/case studies: cd36 (gpiv, chromosome 7q11.2) is an 88 kda glycoprotein expressed on multiple cell types including platelets (plts), monocytes (mono), & erythroblasts. although rare among whites, cd36 deficiency (cd36-n) is observed in 3-10% of africans (t1264g) & is classified as either type i (cd36-n plt, cd36-n mono) or type ii (cd36-n plt, cd361 mono). an acquired type ii cd36-n phenotype can also be observed in the setting of myelodysplastic syndrome (mds). type 1 cd36-n individuals can develop anti-cd36 alloantibodies with plt refractoriness & neonatal alloimmune thrombocytopenia. we report a case of profound plt refractoriness caused by anti-cd36 in a patient with newly diagnosed mds. study design/method: hla antibody testing was performed with a commercial bead-based fluorescent assay. cd36 phenotyping (plt, mono) of patient & family members was performed by flow cytometry (fc). cd36 staining of bone marrow was performed by immunohistochemistry. plt crossmatching (plt-xm) was performed by the american red cross. pltspecific alloantibody testing & cd36 dna sequencing were performed at a commercial reference laboratory. results/finding: the patient was an 80 year-old, group o1 african-american male who presented with blurry vision & lightheadedness. complete blood count findings were significant for hemoglobin 4.4 g/dl & plt count 5k/ml. bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, 5-10% blasts & a complex karyotype with del(7)(q22q34) consistent with mds. plt refractory work-up was initiated after repeated plt transfusion failures with corrected count increments (ccis) < 5. hla antibody testing was negative (class i panel reactive antibody (pra)50%). the patient was plt-xm-incompatible with most donors (10/14). a trial of 4 group o, plt-xm-compatible plts was unsuccessful (cci 1). subsequent testing for plt-specific alloantibodies identified anti-cd36. fc-phenotyping showed no cd36 on patient's mono or plt, consistent with type i cd36-n. preliminary dna results show that the patient is heterozygous for t1264g. because cd36-n apheresis plt were unavailable from blood suppliers, the patient's 3 children & grandson were screened as possible donors: all showed normal cd36 expression on plts. trial of eltrombopag & romiplostim was attempted with no improvement in plt count. repeat hla antibody testing (day 16) demonstrated new class i alloantibodies (pra 5 55%) in response to transfusion (21 apheresis plts, 5 rbcs). given his plt refractoriness & poor prognosis, the patient opted for hospice. conclusion: we describe a patient with cd36-n & severe plt refractoriness in the setting of new mds, and 7q-chromosomal abnormalities. the absence of cd36 on plt & mono support congenital type 1 cd36-n although a contribution by the patient's underlying mds cannot be excluded. rapid platelet donor classification: hla & hpa profiles by "leansequencing" without dna purification dipika patel 1 , kristopher fernandez* 1 , eric senaldi 2 , pascal george 2 , michael seul 1 and ghazala hashmi 1 . 1 biomolecular analytics, 2 central jersey blood center background/case studies: prophylactic platelet transfusion is the standard of care for managing thrombocytopenia. in the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy. while the benefits of customizing transfusion therapy have long been recognized (gmur1978 http://bit.ly/2q51heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of dna analysis. to address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined hla class i and hpa signature without dna purification using a novel "leansequencing" process. study design/method: under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $100 adult donors who had made ! 6 donations in the previous 12 months. samples (labeled with study barcodes) were shipped weekly to biomolecular analytics ("bmx") for preparation of "crude extracts" for leansequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps. briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for 2, 3, 4, 5, 6, 7, 8, 9, 11, 15 and for hla class 1 (a,b,c) , the latter using a proprietary design that limits analysis to informative alleles in the hla sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software. a subset of crude extracts was purified and analyzed side by side with positive and negative controls. results/finding: crude extracts from buccal swabs produced viable profiles for hpa as well as hla class i with significant savings in time-to-result. as an illustration, the table reports allele frequencies for platelet-antigens ("hpa") that are consistent with a predominantly caucasian or hispanic platelet donor population (http://bit.ly/2pdplf8) in hw equilibrium. similarly, hla-class i haplotype frequencies were determined. conclusion: leansequencing lends itself to the rapid determination of hla-class i and hpa signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project. the process could be readily implemented to another site using the elements and process developed. the "pool & plex" process and the early donor recruitment enables economies of scale for matched donor procurement. the serological characteristics and heritage background of a novel hla allele, hla-a *26:82 chuan-fu zhu*, yong-hong song, xiang-min nie and wen-ben qiao. blood center of shandong province background/case studies: there are 16,429 hla alleles documented according to the imgt / hla sequence database in janury2017, and more than 80% of them were identified in the last 10 years. besides sequences many of the novel hla alleles have not been analyzed their serological reactivities. hla-a *26:82 allele was fist detected in our laboratory during our hla typing for china bone marrow donor program(cmdp). for further study, the serological characteristics and heritage investigation were performed. study design/methods: the routine hla tying for the potential donors from cmdp were performed by bi-allelic sequence-based typing method,using a commercial kit (rose europe gmbh, frankfurt, germany). in the case of no full matched hla typing results, group specific hlassure-se sbt typing kit (texas biogene inc., taipei, taiwan) was employed to identify the nucleotide sequences of the novel allele. fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele. the hla serological specificity was indicated by one lambda(asn72d)hla kit. results/findings: no full matched result was obtained at hla-a locus in hla typing for a donor,which suggested the possible existence of a novel allele. the latter nanalysis indicated that the proband have a nove1 nucleotide sequences at hla-a locus, the new sequences was most close to those of hla-a *26:01:01:01, but 1 nucleotide substitution in exon 4, by nt 746 c-a (codon225 acc-aac), which resulted in one aminoacid substitution ,thr-asn. the novel hla-a allele was officially named as hla-a background/case studies: anti-d is a frequent cause of hemolytic disease of the fetus and newborn (hdfn). as a rule, immunization occurs in d negative pregnant women, but occasionally anti-d is also observed in carriers of d variants. currently, maternal plasma analysis for determination of the fetal rhd status became an exciting new tool for the management of d-negative pregnant women, but one of the challenges in non invasive fetal rhdgenotyping is the presence of d variants in the pregnant women. we present a case of a 13 year-old pregnant woman typed as ab1, who delivered a baby affected by severe hdfn. the newborn was typed as b1 and presented a positive direct antiglobulin test (dat) with an anti-d identified in the eluate. the baby was treated by exchange transfusion and the mother's sample was investigated. study design/method: serologic testing was done by hemagglutination in gel cards. genomic dna was extracted from whole blood by spin column and all rhd exons were sequenced by sanger sequence method. results/finding: the mother's rbcs reacted 41 with the four monoclonal anti-d used (igm clones p3x61 and rum 1 and the blends clones th281ms26 and d1751d415) and were typed as c-c1e-e1. an anti-d was identified in her serum. molecular analysis showed the 410c>t and 455a>c in exon 3, the snp 509t>c changes in exon 4 and the 667t>g nucleotide change in exon 5. the set of snps found is similar to the molecular background of dol3, except for 455a>c change. conclusion: this novel set of snps found in this mother is related to a novel rhd allele leading to a partial d antigen involved in the production of an anti-d that can cause severe hdfn. this finding shows the need to elucidate the clinical significance of different rhd genotypes in various ethnic backgrounds. the and erytra v r (the routine reference platform) was performed. a total of 1089 immuno hematological tests (465 abo/d grouping (including 33 newborn samples), 12 extended erythrocytic phenotype, 562 antibody screening, 14 antibody identification, 16 dat) and 20 crossmatches were performed on patient's whole blood samples. the erytra eflexis v r performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations. concordance between systems was assessed and discrepancies were analyzed. the following performance metrics were assessed: time to first result (ttfr), turn-around time (tat) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system. for the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. a threshold for in vitrodetection of anti-d gamma globulin was also determined. v r analyzer and the reference method were obtained in 99.2% of the abo/d tests (n5265), 99,7% of the antibody screening tests (n5377), 88,8% of the antibody identification tests (n59) and 100% of the dat tests (n510). there were 4 discrepancies (2 abo/d for the same patient, 1 for antibody screening and 1 antibody identification: in both cases, the erytra eflexis v r could conclude whereas erytra could not due to a poor reaction. use of the stat mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than 10 min). detection threshold of the d antibody was assessed at 2.5 ng/ml (0.0125 ui/ml) whereas the french recommendations are 20 ng/ml. the possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation. the impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility. v r results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory. erytra eflexis v r meets both the requirements for french regulatory in immunohematology and for iso 15189 accreditation. background/case studies: kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (hdfn). we report a case of hdfn secondary to anti-kpb that resulted in multiple intrauterine transfusions of kp (b-) donor cells and hemolytic anemia upon birth. case: a 31 year old g5p3 presented during her fifth pregnancy with anti-kpb with an initial titer measured of 64. by history, the anti-kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated. the patient's antibody titers peaked at 16 during the fourth pregnancy which resulted in a healthy male without anemia or jaundice. . in the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery doppler exams (1.7 moms) peaking at 27 weeks. this resulted in three intrauterine transfusions. due to potential labor and the finding of reversed diastolic flow on middle cerebral artery doppler studies, a finding that has been associated with impending intrauterine fetal demise, caesarean delivery was performed at 35 weeks gestation. the baby boy required phototherapy for hyperbilirubinemia. the indirect bilirubin at birth was 3.4 mg/dl with 13.6 g/dl hemoglobin. the baby typed as o positive, kp (b1) with a micro positive dat. the antibody workup revealed an anti-kpb. continued hemolysis required one more transfusion at 6 weeks of age. the positive dat and passively acquired anti-kpb were no longer detected by 8 weeks of age. his hemoglobin recovered to 9.0 g/dl with an indirect bilirubin of 1.4 mg/dl at 9 weeks of age. all clinical signs of hemolytic anemia were resolved. study design/method: serologic testing included peg iat by tube methods. acid elution was performed using immucor gamma elu-kit ii. molecular testing was performed using immucor bio-array hea platform. results/finding: antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies. a new weakly reacting anti-s was detected on the day of the delivery. the baby typed as s positive however the anti-s was not detected in an eluate prepared from the baby's red cells. all of the intrauterine transfusion units were s negative. conclusion: to our knowledge only five case reports have been described for anti-kpb which resulted in moderate to severe hdfn. pregnant mothers with anti-kpb detected should be monitored closely. background/case studies: in some clinical cases, the c3d-specific dat may be too insensitive to detect low, but significant levels of c3d, or it may be inconclusive due to spontaneous red cell (rbc) aggregation. further, the dat is not well suited to quantify the number of immunoprotein molecules on rbcs, since a "1111" reaction corresponds to about 500 molecules/ cell. a number of flow cytometric methods for the detection of rbc-bound c3d have been published. however, these are mainly designed to quantify the fraction of rbcs with c3d-sensitization. the aim of this study is to present a flow cytometric method for the quantification of the level of rbcbound c3d. study design/method: ten microliters (ul) of 1:80 (after documenting experimentally that this amount ensured maximum binding of anti-c3d) mouse monoclonal anti-human anti-c3d (abcam, clone 7c10) were added to 5 ul of a 2.5% rbc suspension. after incubation for 60 minutes at 4c, samples were washed x3, and 25 ul of 1:10 diluted anti-mouse-f(ab)2-pe (ro480, dako) were added. after incubation at 4c, samples were washed and resuspended before being acquired on a flow cytometer (becton dickinson facscanto ii). to enable calibration of fluorescence signals in antibody binding capacity (abc), a calibration standard (dako qifikit) stained with ro480 was run in parallel with all experiments. background fluorescence (in abc) was subtracted to yield net abc values corresponding to specific staining with anti-c3d. the assay, in parallel with our routine dat (dc-screening i, id-card, gel card, biorad) was applied to a series of a1 rbcs stained with 10 levels (2fold dilution, 1:1 -1:512) of o serum with high titer anti-a. to estimate the normal range of rbc-bound c3d, edta-stabilized samples from 4 healthy donors were tested. finally, the assay was applied to a sample from a patient with clinical aiha with an inconclusive dat due to unspecific dat polyreactivity. results/finding: the correlation of the net level of rbc-bound c3d (values ranging from 0 to 3,393 abc) with level of 0-serum dilution (used to sensitize a1 rbcs) proved to be highly linear (logarithmic vs. logarithmic plot; r2 5 0.97, p < 0.0001). compared with dc-screening 1, the sensitivity of the flow cytometric assay was superior. it detected c3d sensitization at least 4 dilution steps further. the median normal level of rbc-bound c3d was 11 abc (range 7-20 abc, n54). the assay enabled demonstration of specific c3d-sensitization in the patient; the level of rbc-bound c3d in the sample was significantly elevated (1,907 abc). conclusion: the presented flow cytometric assay is capable of quantifying the level of rbc-bound with a high degree of linearity and analytical sensitivity. further, it is capable of quantifying the level of rbc-bound c3d in dat polyreactive samples. background/case studies: abo blood group system of red blood cells (rbcs) consists of a and b oligosaccharide antigens and anti-a and anti-b antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(landsteiner's law). because of the expression of those antigens on some epithelial and endothelial cells in the body, the abo matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation. in spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of abo. forssman (fors) system is another rbc blood group system which consists in a glycosylation polymorphism specified by the gbgt1 gene. in humans, the abo and gbgt1 genetic loci are located on chromosome 9q34, and the functional alleles encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the last biosynthetic steps of a and b, and forssman (fors1) oligosaccharide antigens. the molecular genetic bases for allelism of those two systems in humans have been well-elucidated. the abo and gbgt1 genes are also present in some other species in addition to humans. however, the presence/absence and functionality/non-functionality are species-dependent. molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown. study design/methods: utilizing genomic information available from gen-bank and ensembl databases, the gene maps of the chromosomal region surrounding the abo and gbgt1 genes have been constructed of 88 vertebrate species. results/findings: extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations. however, numerous differences were also identified. these include chromosomal rearrangements, as well as the insertions and amplifications of specific genes. interestingly, the abo and gbgt1 genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution. conclusion: genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments. therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes. alloimmunization despite phenotype matching in a patient with sickle cell disease and a complex rhce genotype jessica kneib* and emily coberly. university of missouri health care background/case studies: red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease. sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least c, e, and k1 antigens to reduce this risk. unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial rhce variants. study design/method: a 14 year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion. transfusion 2017 vol. 57 supplement s3 the patient's blood type was o positive and her red cells had been previously phenotyped as c-, c1, e-, e1 and k1-. an antibody screen was positive, and antibodies against c and e antigens were identified in the plasma. the patient had only received phenotypically matched units negative for c and e antigens for all previous transfusions at our institution, based on her known red blood cell phenotype. blood samples were sent to a reference laboratory for molecular testing to look for partial rhce variants that might explain the antibody development. results/finding: molecular testing was performed to reveal the presence of two different partial rhce alleles, resulting in a predicted phenotype of d1, c-, e-, partial c1, partial e1. the probable rhce genotype, rhce*ce-jal/rhce*ce733g, results in partial expression of both c and e antigens. in addition to the known risk of alloimmunization against the absent c and e antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens. based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody. conclusion: although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial rhce genotypes. in this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against c, c, e, and e antigens. as the patient had already made alloantibodies against c and e antigens, it was determined that she would require units that were molecularly matched to her rhce variants for all future transfusions. this case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial rhce variants. altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant woman and her newborn carolina bonet bub* 1 , maria giselda aravechia 1 , thiago costa 1 , marilia sirianni 1 , eduardo bastos 1 , leandro santos 1 , lilian castilho 2 and jos e kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp background/case studies: rhd*weak d type 2 is a variant commonly found in caucasians associated with a weak d phenotype. as previously reported (vege et al, transfusion 2007) the c.1154g>c change (p.g385a), which characterizes the rhd*weak d type 2 allele is a splicing variant that induces skipping of the whole rhd exon 9. we report an altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant women and her newborn with weak d expression. study design/method: the d antigen expression was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. rhd genotyping was performed with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. results/finding: both pregnant women and newborn samples were phenotyped as d1 w c-c1e1e1. the samples showed weak hemagglutination reactions (11/21) with all anti-d clones used. rhd beadchip results showed the ls* signal indicating a possible deletion of exon 9 in both dna samples. sequencing showed the c.1154g>c change and the intronic c.1154-8t>a and c.1154-31t>c substitutions, which are associated to the rhd*weak d type 2 allele. conclusion: our results showed that c.1154g>c associated with c.1154-8t>a and c.1154-31t>c variations had probably a functional impact on splicing inducing exclusion of exon 9 in both dna from mother and newborn. this finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-d igg prophylaxis for women with weak d type 2. background/case studies: sickle cell disease (scd) patients require red blood cell (rbc) transfusions to minimize disease-specific symptomatology. previous studies have shown that more than 50% of children with scd receive at least one rbc transfusion in their lifetime. both simple transfusions and erythrocytapheresis are associated with increased risk of rbc alloimmunization. published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies. therefore, we looked at the alloimmunization rates of pediatric patients with scd in the unites states (us) and other countries. study design/method: a literature search was performed for studies published on alloimmunization rates of scd pediatric patients including hbss, sickle beta-thalassemia and hbsc. we evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per 100 transfused units across world literature and compared them using chi-square analysis. results/finding: fourteen studies reporting data to derive alloimmunization rates of pediatric scd patients were found. these included eleven us studies with 1,057 patients and 3 studies from other regions (brazil, egypt and france) with 641 patients. majority of patients included in the studies had hbss disease. patients received either episodic, chronic simple transfusions or erythrocytapheresis. age range for the us studies was 0 to 26 years and for the other countries 0 to 20 years. available data from 5 us studies included a total of 91 alloantibodies, the most frequent of which were antibodies to c, e, kell, m, s and kidd antigens (18.7%, 16.5%, 15.4%, 7.7%, 7.7% and 6.5% respectively). alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per 100 transfused units in other studies. we evaluated rates using both approaches as per available data. us had an alloimmunization rate of 16.5 % (14.1 to 19.2, 95% ci) vs. 9.4% for non-us studies (7.3-11.8, 95% ci) (p50.0008) and 134a transfusion 2017 vol. 57 supplement s3 more alloantibodies per transfused patient (0.25 vs. 0.096, p50.0001). similarly, the number of alloantibodies per 100 transfused units in the us, evaluated from five studies, was higher compared to a large french patient cohort (0á68 vs. 0.33, p50á0005). average number of rbc units transfused per patient in the us was also higher compared to data from france (77 vs. 45, p50.0001). conclusion: despite limited studies available to compare alloimmunization rates in pediatric scd patients in the us and other countries, the overall rates are higher in the us. though no definitive reasons could be concluded from the available data, limiting the number of rbc exposures, i.e. units transfused in non-critical conditions could lead to lower alloimmunization rates. results/findings: a post-transfusion sample was referred to the irl for a trxn investigation. there were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum. abo/rh and crossmatches using lo-ion tm were repeated on the pre-and post-transfusion samples with no discrepancies. the post-transfusion dat was positive with a negative eluate. the hospital requested another unit before the investigation was complete. antibody identification on the post transfusion sample with lo-ion tm was negative. suspecting a weak antibody, additional investigation using peg tm on both samples revealed an anti-c. no additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using peg tm . conclusion: the patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-c in the patient's plasma/serum against c antigen on the transfused cells. anti-c was not detected by our routine antibody identification techniques. the mma confirmed anti-e, -m and -c were clinically significant. laura bailey* 1 , melissa grohotolsky 2 , lisa deblass 1 , bala carver 1 and kip kuttner 2 . 1 health network laboratories, 2 miller keystone blood center background/case studies: the en a antigen is a high prevalence antigen in the mns blood group system. the antigens of the mns system are carried on glycophorin a (gpa) and glycophorin b (gpb). anti-en a is a rare immune igm/igg antibody made by individuals who lack all or part of the gpa protein. anti-en a has been implicated in fatal htr and hdfn. the en(a-)phenotype can result from either a rare deletion of the gpa protein or the even rarer m k phenotype. because individuals with the m k phenotype lack both the gpa and gpb protein their red blood cells type as m-, n-, s-, s-, u-, en(a-), wr(b-) and have reduced sialic acid. study design/method: 23 year old white mennonite female g1,p0 presented to her midwife for prenatal care with the intent of home delivery. she had a positive antibody screen by solid phase at the hospital transfusion service. an antibody identification panel was done in gel. testing for antibodies against selected cells (u-and u var ) in tube with peg enhancement and phenotyping was done. based on mns phenotype, anti-en a was suspected. the specimen was referred to an immunohematology reference laboratory (irl). the testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration. following identification of anti-en a by the irl the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home. the midwife was also advised to consider autologous blood donation and /or testing of siblings. results/finding: testing by the hospital blood bank demonstrated positive reactivity in the antibody screen. the gel antibody panel ahg phase resulted in 21 panagglutination and a negative autocontrol, suggesting a high prevalence antibody. the phenotype was performed and determined to be m-, n-, s-, s-, u -. outdated u variant reagent cells reacted in peg igg phase ruling out anti-u. anti-en a was suspected and the sample was referred to the irl. allogeneic adsorptions were performed to rule out antibodies to common red cell antigens. lack of reactivity on a ficin panel eliminated the presence of anti-u,-wr b . phenotyping with unlicensed anti-u was negative and unlicensed glycine soja demonstrated 11 reactivity, suggesting that the patient is en(a-). the patient's phenotype is consistent with the m k phenotype. based on the lack of reactivity on the ficin panel, the antibody was identified as anti-en a fs. since anti-en a is extremely rare, this specificity could not be confirmed due to the lack of en(a-) cells and appropriate antisera. the baseline antibody titer was 2 at igg phase without enhancement. conclusion: this case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital. studies performed after the patient was transferred closer to home confirmed the anti-en a (fs) and genotyping was performed. three titers were performed for the remainder of the pregnancy and held at 2. although anti-en a has been implicated in hdfn, a healthy infant was delivered without complications. this patient should be monitored closely through future pregnancies. autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery. background/case studies: a 15 year old caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the immunohematology reference laboratory (irl) for antibody identification and rbc genotyping. initial serologic testing by the referring facility and the irl demonstrated anti-d, anti-c and/or anti-g specificity with a positive auto control and igg dat. anti-g has an anti-d, -c specificity and is most frequently found in rr individuals exposed to r'r cells. the g antigen is present on rbcs expressing either rhd and/or c and very rarely on d-c-g1 (r g r) cells. both rhce*c and rhd genes encode ser103 which determines g expression. rare rhd variant antigens lacking ser103 are g-. study design/methods: serologic evaluation included tube testing using gamma lo-ion tm (immucor, inc., norcross, ga) enhancement, elution studies (gamma elu-kitv r ii (immucor, inc.)), edta glycine acid treatment (gamma ega tm kit (immucor, inc.)), allogeneic adsorptions with papain treated intact rbcs, reagent and patient-derived rbcs and antisera. molecular testing was performed with bioarray precise type ivd hea assay (immucor, inc.). results/findings: molecular testing revealed an rhce*ce genotype (with a c-e1c1e-predicted phenotype) and an otherwise unremarkable rbc typing report. serologically, the antibody(ies) demonstrated an anti-d, -c, -g specificity in the serum and eluate using r o r, r 2 r 2 , r'r, r g r and rr cells. this patient is predicted to be r 2 r 2 (dce/dce) therefore, anti-c is possible but an allogeneic anti-d or -g is exceptionally unlikely. allogenic adsorptions using papain treated r o r and r'r cells excluded anti-c and anti-d, leaving anti-g as the only explanation of the initial findings. reactivity with the patient's ega treated (dat negative) cells against the "neat" serum, eluate and anti-g antisera confirmed auto anti-g. conclusion: warm autoantibodies are common findings and often have an rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used. this anti-g had no reactivity with g-cells. the differentiation of anti-g from anti-d and anti-c is generally academic as transfusion recommendations are the same: provide rhd-, c-units. it is relevant and clinically important to determine the presence or absence of anti-d in rhd negative women of childbearing age who present with an anti-g specificity. if anti-d is 135a transfusion 2017 vol. 57 supplement s3 excluded these women should receive rhig as part of their prenatal care. in this case differentiating anti-d, -c from an auto anti-g was necessary to provide transfusion recommendations. providing rhd-and c-units to give serologically compatible rbcs could result in formation of an allogeneic anti-e. automated eluates: comparison of solid-phase red cell adherence and gel automated eluate testing jayanna slayten* 1 , christa voliva 1 , kathy fletcher 1 , heather vaught 2 and tracie ingle 1 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: acid eluates (elu kit ii. immucor. norcross, ga) are to be tested via tube iat method in parallel with the recovered last wash per the manufacturer's package insert. finck et al (immunohematology 2011; 27:1-5) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (id-mts.igg card. ortho clinical diagnostics. raritan, nj) and automated solid-phase red cell adherence systems (echo. immucor. norcross, ga). our study looked to compare the use of the automated gel microcolumn analyzer (vision, ortho clinical diagnostics. raritan, nj) to the solid-phase red cell adherence analyzer (echo, immucor. norcross, ga) for the testing of acid eluates in a regional midwestern transfusion service. study design/methods: twenty patient samples, less than 7 days from collection and drawn in edta, were used to prepare acid eluates (elu-kit ii. immucor. norcross, ga) while retaining the last wash to be tested in parallel. two samples were >21 dat positive, 2 were weakly dat positive and 16 were dat negative. the prepared eluates were observed for color (bluegreen/bg, blue-brown/bb, blue-purple/bp), and the ph was documented for the prepared eluate (whatman 6.0-8.1ph. whatman international. maidstone, england). the prepared eluates and last washes were tested on the vision and echo against an antibody screen. if the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity. prior to the eluates being tested on the automated platforms, they were spun for 5 minutes twice to remove any rbc debris which could cause false positive reactions. results/findings: the eluates prepared ranged in color: 5 bb, 14 bg and 1 bp. the ph of all eluates ranged from 6.9-8.1 with the highest percentage of eluates at a ph of 8.1 (35%). sixteen of the 20 eluates tested yielded the same results in both automation platforms (concordance of 80%). four eluates with different results are summarized in table 1 . conclusion: the study demonstrated that both analyzers may be used for eluate investigations. both methods yielded apparent false positive results on samples which were initially dat negative. the echo was more sensitive, yielding false positive results (3) when the vision was negative, while the vision was false positive with one eluate with echo negative. there was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or ph of the eluate. a larger study may be able to better elucidate the apparent false positive results noted in this study between echo and vision eluate study. background/case studies: blood agglutination observed by landsteiner in 1900 led to the discovery of human blood groups. in the abo system >200 alleles have been described. the glycosyltransferase encoded by most results in weakened expression of a or b or the null (group o) phenotype. as testing methods and reagents improve, donors may appear to change their abo type. here we describe a frequent group o blood donor (38 units over 17 years) who is actually a w . study design/methods: donations were tested with the pk7300 instrument (beckman coulter inc.). routine forward and reverse abo testing was used to investigate the discrepancy. molecular studies were performed by dna sequencing of abo introns 1,2 and 4 and exons 6 and 7. specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by pcr. the template used is genomic dna extracted from whole blood collected in edta. pcr-amplified exons are subjected to bidirectional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results/findings: serologic results are shown in table 1 . tests with anti-a, -a1, -b anti-a,b were negative as were the a2 cells in reverse testing. the results of dna sequencing of abo introns/exons are shown in table 2 . the significant changes were found in exons 6 and 7. in exon 6 there was a nucleotide (nt) deletion of 261g which resulted in a shortened transcript due to a stop codon, and another nt substitution lead to the amino acid change gly117ala. mutations in exon 7 included a nt substitution causing a pro156-leu change and a nt deletion 1061c resulting in shortened transcript. conclusion: serologic testing of the donor plasma with a2 cells was nonreactive revealing the abo discrepancy. molecular testing confirmed the donor genotype is heterozygote a/o [abo*o.01.01/abo*aw.02] which predicts a w phenotype. normally, donor rbcs are tested with anti-a and -b and the reverse type confirmed by testing the with a1 and b cells. this abo discrepancy was caused by the presence of anti-a1 in the plasma causing the forward and reverse type to be interpreted as group o. according to fda guidelines, the donor is technically group a, and as such all donations need to be labeled as group a. the donor was contacted and instructed to cease donating blood for transfusion. if donations continue, the unit labeled group a would likely test as group o at the transfusion facility resulting in an fda reportable error. there are numerous reports in the literature of the relative insusceptibility of a2 cells to destruction by anti-a, however, there is one hemolytic transfusion reaction to a x blood transfused to a patient with a potent anti-a titer >1:1000. (schmidt, nacarrow et al. 1959) . a review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion. 136a transfusion 2017 vol. 57 supplement s3 extraction of gdna from edta-anticoagulated whole blood from pilot tubes derived from the unit. dna extraction from whole blood is performed on up to 96 blood tubes using the biorobot universal system (qiagen). there is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature. we set out to assess if blood up to 15 days post collection yielded suitable gdna for downstream rbc genotyping. study design/method: 92 edta blood tubes collected from random blood donors were used to extract dna from 200 microliters of whole blood on day 5, 12 and 15 days post collection. blood samples were stored at 2-8c before and after extraction. tubes were brought to room temperature and rocked before loading on the biorobot. extraction was performed using the mdx blood minikit (qiagen). resulting dna samples were assessed for gdna yield and absorbance a260/a280 using a nanodrop 2000 (thermo scientific). the extracted gdna was tested using precisetype hea molecular beadchip ("hea", immucor) and failure rates on both the biorobot and the hea were assessed. results/finding: all three extractions were successful with no invalids (result50) on the biorobot universal report. no evidence of visible clots or splatter during extraction was noted by the technologist. out of the 92 samples, 20 samples were chosen at random and concentrations were measured using nanodrop for each of the extracted plates. dna concentrations ranged from 10.8 to 62.6 ng/ul. all readings with the exception of 1 (10.8ng/ ul) had concentrations >5 15ng/ul. interestingly, the one that was <15ng/ ul on day 5, yielded >515ng/ul on day 12 and 15 post collection. over the next 3 months, 67 sets of 92 samples were extracted and tested by hea. eighty-three (1.3%) failed extraction and 82 (1.3%) failed hea. none of the samples that failed extraction were 12 or 15 days post collection; none of those that failed hea were 15 days post collection; 3.7% were >10 <15 days post collection. conclusion: based on these results it can be concluded that edta blood tubes up to 15 days post collection can be used as a source of gdna for rbc genotyping without negatively effecting the concentration of the resulting dna samples and the validity of the resulting genotyping. case study: investigation of persistent negative antibody screens on patients receiving daratumumab raeann thomas 1 , carlos villa 1 , rachel davis-rauser* 1 , helen carpenter 1 and vrunda patel 2 . 1 university of pennsylvania, 2 hospital of the university of pennsylvania background/case studies: daratumumab is an anti-cd38 monoclonal antibody therapy that received fda approval for treatment of multiple myeloma in 2015. communications suggest all patients receiving therapy would have a positive antibody screen because cd38 is a common antigen expressed on red blood cells. currently, 154 patients have been treated with daratumumab at a large academic medical center. a wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens. while there are several potential causes, neutralization of anti-cd38 antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity. study design/method: samples received were drawn as a standard of care. indirect antiglobulin testing was performed using solid phase red cell adherence and gel. neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma. a dilution control was made by adding saline to each positive patient's plasma. samples were incubated for 1 hour at room temperature and antibody screens were repeated. serial two-fold dilutions were also tested to determine if the neutralization could be titered. testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity. results/finding: all control samples remained positive. positive/negative samples were negative in solid phase testing across all patient combinations at 1:1 dilutions. variable reactivity was observed in gel. serial dilutions showed that neutralization for 2 negative patients was observed up to a 1:4 dilution. conclusion: results suggest that patients' plasma may have a substance that neutralizes the antibodies. there is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab. additional studies are necessary to uncover how this correlates to patient outcomes. further studies using a standardized daratumumabspiked sample will be conducted. background/case studies: the mns blood group is a red cell antigen system located on glycophorin a (gypa) and glycophorin b (gypb). individuals lacking gypa or both gypa and gypb on their red blood cells may develop a rare antibody against the en (a) antigen. the en (a) antigen is a highprevalence antigen, located on gypa. we present a case with a rare red cell phenotype and alloimmunization to the en (a) antigen. a 28 y/o g1p0 at approximately 23 weeks gestation was discovered to have an anti-en (a) antibody in her plasma on a prenatal type and screen. this was worrisome for both mother and fetus, as the en (a) antibody is of igg isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn) [1, 2] . further testing with red cell antisera revealed that the patient lacked m, n, s, s, and u antigens. a multiplex, allele-specific, pcr platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the m, n, s, s, and u antigens. these findings were consistent with a null phenotype for both gypa and gypb antigens, i.e. m (k) m (k) phenotype. the patient's husband and father of her unborn baby demonstrated a m1n-s1s1 phenotype by the same serological and molecular means. given the exceedingly rare incidence of en (a-) individuals (positive frequency >99.9), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [1, 2] . however, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). the consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers. if transfusions were required for the mother or fetus, our options were to either search for rare units lacking the en(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype. at term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route. the delivery was without event. no transfusions were necessary antepartum or postpartum. study design/methods: n/a results/findings: n/a conclusion: anti-en(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman. providing this patient with rare en(a) negative red cells via national or international blood donor registries would have been an arduous task if needed. this patient had many compatible family members available and willing to donate blood. the m(k) null allele (s) within this family is likely due to a genetic recombination among the gypa and gype genes rather than a mutation in both the gypa and gypb genes [3] . this results in the absence of glycophorins a and b and the constitutive antigens of the mns blood group system. our patient was exposed to the en(a) present on glycophorin a on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization. in conclusion, this case report demonstrates a clinical approach in identifying a rare anti-en(a) antibody in a prenatal sample. the clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it. background/case studies: transfusions are essential for patients with scd and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (rbc) alloantibodies and autoantibodies complicates transfusion therapy in such patients. routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and scd. nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations. the molecular understanding of blood groups has enabled the design of assays 137a transfusion 2017 vol. 57 supplement s3 that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units dna typed. based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during 3 years for patients with scd and thalassemia. study design/method: blood group genotypes were determined in 67 dna samples from chronically transfused patients with scd, in 65 patients with thalassemia and in 3000 dna samples from blood donors. laboratory developed tests (ldts), hea beadchip tm , rhd beadchip tm , rhce bead-chip tm , and sequencing were used to determine the genotypes among patients and donors. molecular matching was performed in 3 levels: (1) rh and k matching; (2) extended matching and (3) extended matching including rh variants. we considered the total of red blood cell units requested for each patient and a number of 2 donations per year for the compatible donors. results/finding: according to the patients needs we performed molecular matching for 100% of our thalassemic and scd patients at level 1, 90% for scd patients and 70% for patients with thalassemia at level 2 and 30% for patients with scd and 90% for patients with thalassemia at level 3. the patients were transfused with a median of 36.4 rbc units. after three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients. conclusion: molecular matching has shown clinical benefits to the patients with scd and thalassemia, contributing significantly to reduce the rates of alloimmunization to 5-10% with c e k matching and <1% with extended matching. improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hb levels and reduction in the % of hbs in scd patients, better in vivo rbc survival and diminished frequency of transfusions. allahna lilly elahie* and sandra fazari. hamilton regional laboratory medicine program background/case studies: the ideal manual backup method for an automated antibody detection system is an important choice. currently, our backup method is saline tube (6 drops plasma, 30 minutes incubation). the change to either a low ionic strength solution (liss) or polyethylene glycol (peg) method would reduce incubation time to 10 minutes and specimen volume to 2 drops, both important laboratory considerations. objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of peg and liss, and to determine the most appropriate manual backup method for the existing automated solid phase system. study design/method: a total of 202 specimens were compared utilizing: automated solid phase red cell adherence assay (sprca) with manual tube peg and liss, some samples were not sufficient quantity to test in liss. identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions. calculations were based upon comparison to sprca. results/findings: a total of 164 clinically significant antibodies were detected using sprca technique, as well as 9 warm autoantibodies and 97 nonspecific reactions. peg demonstrated the highest sensitivity and lowest specificity while liss was least sensitive and most specific for clinically significant antibodies. for warm autoantibodies, liss was more sensitive than peg with both being 100% specific. both reduced the detection of nonspecific reactions. while peg had more nonspecific reactions (30 versus 13), it identified more clinically significant antibodies (129) than liss (93). (table) conclusion: ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one. peg was selected as the backup manual method even though peg has a higher sensitivity to nonspecific reactions. this study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions. comparison of thiol reagents in denaturing cd38 on rbcs patricia a arndt* 1 , anthony salazar 2 and regina m. leger 1 . 1 american red cross blood services, 2 long beach memorial medical center background/case studies: monoclonal anti-cd38, e.g., daratumumab (dara), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (iats) due to expression of cd38 on red blood cells (rbcs). this serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies. one popular method is to denature the cd38 antigen by treatment of rbcs with thiol reagents, e.g., dithiothreitol (dtt) or 2aminoethylisothiouronium bromide (aet). chapuy et al described (2015) and validated (2016) 2), and 6% aet (ph 8.0) as per the aabb technical manual, 17th ed. these treated and untreated rbcs were stored in alsevers at 4c and tested on days 1, 2, 4, 5 and 8 by two methods: 1) polyethylene glycol (peg) iat using plasma from two myeloma patients who had received dara (plasmas from 8 total dara patients were tested with reactivity 5 1-31), and 2) flow cytometry using phycoerythrin (pe)labeled anti-cd38. rbcs were also tested on days 1 and 5 or 8 with a serum containing anti-k by peg iat. results/findings: the 0.2m dtt in ph 8.0 pbs had a final ph of 7.3 and the ph of the commercial 0.2m dtt was 6.5. results are in table 1 ; flow cytometry results from days 2, 4 and 5 (data not shown) were similar to those from days 1 and 8. rbcs treated with 0.2m dtt (both sources) or aet were nonreactive with anti-k and plasma from all dara patients and gave very low results (% positive events) with pe anti-cd38 by flow cytometry for up to 8 days after treatment. rbcs treated with 0.01m dtt reacted similarly to untreated rbcs with anti-k and dara plasmas, and showed only some weakening (10-30%) of reactivity with pe anti-cd38. background/case studies: clinically significant hemolytic disease of the fetus and the newborn (hdn) is often caused by feto-maternal rhd incompatibility. with the discovery 1997 of cell-free fetal dna (ccfdna) in maternal plasma, it became possible to determine the rhd genotype of the fetus using non-invasive techniques. however, the reliability of the non-invasive prenatal rhd test (nip rhd) is dependent on sufficient amounts of cffdna in the maternal plasma sample. recent studies show that the fraction of ccfdna in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (bmi). thus, high maternal bmi, may impair the validity of nip rhd. the aim of this study was to examine the effect of maternal bmi on the correctness of nip rhd and the correlation of maternal bmi with fraction of ccfdna to total free dna in the sample. study design/method: measurements of body height and weight of pregnant rhd negative women in gestational week 12 were obtained from patient records and used for the calculation of maternal bmi. data on bmi were combined with the results from nip rhd (real-time pcr targeting rhd exon 5 and 10) and sample fraction of ccfdna (measured as threshold cycle [ct] value of rhd) to total free dna (measured as ct of ccr5) in gestational week 25. the correctness of nip rhd was determined by correlation with postnatal serological rhd determination. results/finding: a total of 1618 pregnant women were included. nip rhd was positive in 987/1618 (61%), negative in 582/1618 (36%) and inconclusive in 49/1618 (3.0%). compared to the postnatal rhd type, 9/987 (0.1%) of nip rhd results were false positive (fp) and 4/582 (0.7%) were false negative. in 5/49 (10%) of inconclusive nip rhd, the postnatal rhd type was positive. mean bmi (n51618) at gestational week 12 was 25.3 (10-and 90-percentiles: 20.0 -32.4). there was no difference in mean bmi between individuals who tested inconclusive or false negative by nip rhd compared to the remainder (p50,71). the fraction of ccfdna was calculated for 150 randomly selected nip rhd true positive cases. median ccfdna ratio was 5.47 (the distribution had a highly positive skew, 10-and 90-percentiles: 0.64 -27.2). there was no statistical correlation between bmi and fraction of ccfdna to total free dna (r2 50,012; p50.49). conclusion: neither the correctness of nip rhd test result nor the fraction of cffdna to total free dna appear to be correlated to maternal bmi with regard to maternal plasma samples drawn in the 25th gestational week. delayed hemolytic transfusion reaction due to anti-lan antibody: a case report. adla dh angelina*, suneeti sapatnekar and suzanne bakdash. cleveland clinic background/case studies: lan is a high-prevalence antigen and the sole member of the lan blood group system. anti-lan is a very rare igg antibody, with conflicting information regarding its clinical significance and potential for hemolysis. we report a case of delayed hemolysis in a patient with anti-lan antibody. study design/method: the patient's medical record and available literature were reviewed. results/finding: an 83 year old man, o-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy. the antibody screen and panel were panreactive by multiple test methods (gel, liss, peg) with negative autocontrols and dat and a saline antibody titer of 1, suggestive of an antibody to a high-frequency antigen. anti-lan antibody was identified by a reference laboratory. only 1 in 20,000 donors are lan-, but two frozen rbc units were locally available and transfused postoperatively. the patient's siblings were tested; one o-positive, lan-sibling was identified. nine months later, the patient was admitted for surgical management of metastases. at this time, the antibody screen was weakly reactive with 1 cell and new antibodies were ruled out. blood conservation measures were instituted, including limited blood draws and cell salvage for surgery. due to bleeding during and after surgery, 4 lan-rbc units were transfused over 4 days, including rare donor units and units from the sibling donor. another surgical procedure was then performed; by post-operative day 2, the patient had symptomatic anemia with hemoglobin (hb) 5.3 g/dl and serially increasing troponin. no lan-rbc units were available. four rbc units untested for lan were transfused without adverse event; the units were presumed lan1 but crossmatch compatible and phenotypically matched for the patient's other antigens. a post-transfusion hb of 10.2 g/dl was maintained for 4 days. the antibody screen was negative on day 3 post-transfusion, but strongly panreactive on day 6, with a positive dat (igg 21, c3 11) and anti-lan antibody identified in the plasma and eluate. there was also evidence of extra-vascular hemolysis, including a progressive decrease in hb from 10.9 g/dl on day 5 to 7.4 g/dl by day 8 with no bleeding identified, and increase in total bilirubin and ldh (peak 2.4 mg/dl and 304 u/l on day 7) with normal haptoglobin. the patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection. a lan-rbc unit was transfused on day 8 with good response (hb 8.1 g/dl). the patient remained stable and was discharged to a skilled nursing facility 6 days later. conclusion: transfusion of lan1 rbcs caused a resurgence of anti-lan antibody and a delayed hemolytic transfusion reaction 6 days after transfusion. the rarity of lan-units may require a patient with anti-lan to be transfused with lan1 units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion. delayed serologic transfusion reaction caused by auto-anti-f. karen yunker* 1 , andrea gerner 1 , lynne stewart 1 , carol sostok 1 , mollie bell 2 and gregory r halverson 2 . 1 st. elizabeth healthcare, 2 hoxworth blood center background/case studies: anti-f was first described in 1953 by rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused. the f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the 6th antigen assigned to the rh blood group system (isbt rh6). it is capable of causing significant transfusion reactions and mild hdfn. we report in this case a 56 year old caucasian male, admitted for evaluation of suspected t-cell lymphoma, who appears to have had a delayed serologic transfusion reaction (dstr) due to auto anti-f. abstract study design/method: antibody screen and compatibility testing was performed by automated solid phase (echo and neo, lmmucor, inc). red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions. molecular genotyping was performed using the bloodchip assay (grifols, san marcos tx). elution studies were performed using the elu-kit ii (lmmucor, inc.) results/finding: the initial antibody screen (as) was negative and the patient was transfused 1 unit o-rbcs. two weeks later the patient received an additional o-rbc. within 4 days the hgb had decreased from 8.3 to 7.1 g/dl, the as and direct antiglobulin test (dat) were now positive, and ounits were incompatible. anti-f was identified in the patient's plasma and eluate. three additional units were requested for transfusion. due to the rarity of o-f-rbcs, the patient was transfused 3 r 1 r 1 (dce/dce) rbcs with no reported complications. the patient was discharged to follow up in clinic. molecular genotyping showed the patient was rhd deleted (rho* del) and had normal rhce (rhce*ce/rhce*ce) genes which predict a d-c-e-c1e1f1 phenotype. the rh phenotype and as was repeated on a sample collected 18 days later. the c typing was micro positive, mixed field only after 5 minute incubation. the other rh antigens were not mixed filed, and the as was non reactive. however, the dat was weakly positive with anti-lgg. no elution study was performed. conclusion: the expected post 24 hour hgb increment from the receipt of a standard unit of blood should be near 1 g/dl (or 3% hct.) throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation. the first transfuion resulted in a 0.9 g/dl increase, and the second unit was only 0.5 g/dl. the last transfusion of 3 units increased by only 1.2 g/dl. less than three weeks later, the rhc antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of 3 r 1 r 1 units was nearly complete. in a case from 1989, ohto and kariyone (transf. 1989; vol29, no. 3) reported a 51 cr ásurvival study of f1 rbcs in a patient with anti-f. they showed that the initital survival of f1 cells was fairly normal, however, after 18 days, there was a sudden increase of red cell destruction, and by day 27 all f1 cells were cleared from the circulation. it is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood. this patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity. background/case studies: use of dithiothreitol (dtt) treated reagent red cells (rrbc) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by daratumumab, an anti-cd38 drug for treatment of multiple myeloma. daily preparation of dtt-treated rrbc for testing of individual patients is burdensome for the laboratory and may delay patient care. we evaluated the effectiveness of batch-prepared dtt-treated rrbc, stored up to 9 days after treatment, in antibody detection tests. study design/methods: in-date rrbc (ortho clinical diagnostics, raritan nj) were selected based on phenotype to match the antisera to be tested. rrbc were treated with 0.2m dtt (sigma-aldrich, st. louis mo) and stored in reagent red cell diluent. rrbc were tested with commercial antisera (ortho clinical diagnostics, raritan nj and immucor, norcross ga) per the manufacturer's instructions for specificities from the rh, duffy, kidd and mns blood groups (see table 1 ). patient source antibodies (anti-d, anti-c) were also tested. testing was performed before dtt treatment, on the day of dtt treatment and up to 9 days following the dtt treatment of rrbc. reactions were graded using standard serological grading of 0 (negative) to 41 (positive) reaction strength. stored dtt-treated rrbc were also observed for hemolysis during the storage period. results/findings: see table 1 for a summary of results. commercial monoclonal and human source antisera, and patient source antibody, were reactive with the dtt-treated rrbc throughout the storage period. reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested. mild to moderate hemolysis was noted in the dtttreated rrbc's during the storage period. conclusion: dtt-treated rrbc showed adequate reactivity with various red cell antisera after storage for up to 9 days. this suggests that dtttreated reagent red cells can be stored for at least 9 days and used for the detection of alloantibodies with minimal effect on detection ability. batch preparation and storage of dtt-treated rrbc can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-cd38 therapy. interference: more than just kell? marilyn stewart*, angela treml and geoffrey wool. university of chicago background/case studies: daratumumab (dara) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine blood bank antibody screening tests. dara is an igg monoclonal antibody that binds cd38 that is present on the red cell surface. at the university of chicago blood bank, we have seen many patients treated with dara and were showing this interfering reactivity. it has been well described that cd38 is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as dtt. we performed a validation of dtt-treatment of reagent rbc to abrogate dara interference. study design/methods: the validation was done to prove that dtt treated red cells could be used to screen patients receiving dara and still detect clinically significant allo-antibodies. screening cells and panel cells selected for dtt treatment were those rbc homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma. several patients that had received the dara drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not dara treatment). reagent screening cells and panel cells were treated with 0.2m dtt prepared using the sop from judd's methods in immunohematology and the aabb technical manual. the treated cells were preserved between testing episodes using alsever's solution, stored at abstract 2-5c, and observed for hemolysis (none was seen) for up to 21 days. all immunohematology testing using dtt-treated cells was performed using gel methodology. untreated and dtt treated cells were tested with anti k before any patient testing was done. the untreated cells reacted 2-41 with the anti k, and the treated cells were negative. these controls were run and tested each time dtt treatment was done. thirty eight patient samples, including six dara patient samples were tested. results/finding: of the six patients who had dara interference in their untreated antibody screens, all samples had negative reactions with the dtt treated cells except one patient, which had weak reactions in one cell. this specimen was repeated three times and all repeats had weak positive reactions in the same cell. this sample was sent to the arc reference lab for dtt treatment and all clinically significant antibodies were ruled out. patients with allo-antibodies present in their plasma did react with the dtt treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-e antibodies in 4 patients. plasma from these four patients with a nascent anti -e all showed no reactivity with dtt treated cells. plasma from fourteen patients with a long history of anti-e (greater than 6 months) did react with the dtt treated cells. conclusion: dtt treatment eliminates dara interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-e. because of the negative testing with some of the alloanti e antibodies, dara-treated patients at ucm will be given both kell and e negative blood if they have immunohematology testing performed using dtt reagent cells. mahboubeh rahmani* 1 , monique scott 2 , garcia curtis 2 , ellice wong 2 , alexa j siddon 1 and christopher a tormey 1 . 1 yale-new haven hospital, 2 va connecticut healthcare background/case studies: benign ethnic neutropenia (ben) seen in approximately 25% to 50% of persons of african descent is characterized by neutrophil count of <1.5x10 9 /l with no obvious cause and no increased susceptibility to infection or any other adverse effect. at present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis. in this study, we investigated whether duffy (fy) blood group phenotyping would be a potentially useful modality to help identify patients with ben; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting. study design/method: cases included patients clinically diagnosed with ben; and controls were chosen randomly from the pools of patients from whom a cbc and type and screen were checked for any other reason. cases and controls were tested for the rbc antigens fy a and fy b phenotype using serologic methods. the fy phenotype, absolute neutrophil count (anc), white blood cell (wbc), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record. where appropriate, data were compared statistically using the mann-whitney u test with significance set at p<0.05. results/finding: subjects who were clinically identified as having probable ben included 7 patients (mean age 48.7; all self-identified as african-american; 6/7 were male) and controls included 50 patients (mean age 68.5; 10 self-identified as african american; (50/50 male). all of the cases (100%) diagnosed with ben had fy(a-b-) phenotype. mean anc (1.95x10 3 /ul) and wbc counts (4.04x10 3 /ul) were significantly lower in the cases with ben and fy(a-b-) phenotype (p50.0008 and 0.001, respectively) compared with controls (mean anc 5 5.46x10 3 /ul ; mean wbc count 5 8.14x10 3 /ul). there was no significant difference in mean platelet counts (161x10 3 /ul vs 213x10 3 /ul; p50.2301) or mean hemoglobin levels (12.4 g/dl vs 11.7 g/dl; p50.6031) between the two groups. none of the patients with ben had an accompanying marrow-suppressive hematologic disorder based on record review; however, 18 subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome. conclusion: testing for fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of ben. further studies regarding fy phenotyping comparing controls with neutropenia for any reason to our ben population are in progress to better determine the positive predictive value. these tests were compared to the provue for concordance. additional samples tested with anti-igg,-c3d were correlated against tube testing for the dat and antigen typing for: c, c, e, e, and k. results/findings: the ih-1000 had 100% concordance for all blood grouping assays. for ahg assays, the ih-1000 detected an anti-jka1e, anti-fya 1 warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the provue. the ih-1000 identified one additional anti-e not identified on the provue. discrepancies were also noted with the non-cord dat results. five samples were positive on the ih-1000 with anti-igg,-c3d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube. the table below summarizes the results. conclusion: this study demonstrated that the ih-1000 analyzer and associated ih-system tm gel cards are equivalent to the ortho provue. with random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the ih-1000 is an ideal immunohematology system for the hospital transfusion service environment. chris elliott*, susan barnes, fiona lisle, debra smith and whitehouse natalie. background/case studies: the erytra eflexisv r (grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using dg gelv r technology. erytra eflexisv r analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large uk acute hospital transfusion laboratory. study design/methods: a comparison study was performed between the erytra eflexisv r and erytra (our routine system providing the reference platform). a total of 2944 tests were performed on 1,214 adult patient samples and 208 donor red cell units. erytrav r eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations. concordance between systems was assessed and discrepancies analyzed. time to first result (ttfr), overall turn-around time (tat) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded. for ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. fault recognition and messaging was assessed by simulating failures e.g. reagent absence. results/findings: blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested. concordant results between the erytra eflexisv r analyzer and reference method were obtained in 99.9% of samples tested. there were 4 discrepancies, all antibody screening (2 false positives, 1 failure to detect a very weak prophylactic anti d and 1 positive reaction not detected on the erytra but panels on both systems suggested a genuine anti cw). ttfr and tat depended significantly on a number of factors including; number and variety of tests requested and whether the stat functions were activated. the analyser seemed to prioritise antibody screening prioritization of the group, especially for stat samples, was considered preferable the laboratory team found the software easy to use with some improvements over existing ertyra software. physical design of the analyser was considered good with easy access to almost all areas. probe changing was quick and simple. while the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased. conclusion: results showed the erytra eflexisv r offered a robust automated solution for routine transfusion testing. the device could comfortably deal with a medium laboratory (processing 80-100 group and screens per day). it is very flexible being able to deliver grouping, antibody screening and identification, dat, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features. this allows a compact design with maximum flexibility without compromising on turnaround times cp201 evaluation of two monoclonal anti-e as reagents for the detection of the rh e antigen and its variants gregory a. denomme* 1 , kathleen bensing 1 , michael schanen 1 , cindy piefer 1 , randall w. velliquette 2 , christine lomas-francis 2 and connie m. westhoff 2 . 1 immunohematology reference laboratory, versiti/bloodcenter of wisconsin, 2 immunohematology and genomics laboratory, new york blood center background/case studies: monoclonal antibodies are used as reagents for automated and manual phenotyping. false negative phenotypings have implications for variant antigens; e.g. altered c antigen mistyped as a cblood unit stimulating anti-c in a c-recipient. the development of new 7/0 7/0 7/0 cecf 1/1 2/0 2/0 rhce*ce or rhce*ce compound heterozygotes ce254g 1 ce733g or ce48c,733g or ces or ceti 6/0 6/0 6/0 ce733g 1 ce48c,712g or 48c,733g 4/0 4/0 4/0 ce733g 1 ces or cemo or ceek or ceek(var) or cern 8/0 8/0 8/0 ce48c,733g 1 ce48c,712g or cemo or ceti 2/0 2/0 2/0 ce48c,712g/ce254g/733g; ces/ceti; cear/ceek; ceek/cejal; cemo/cebi 7/0 7/0 7/0 total 106/8 110/4 113/2 142a transfusion 2017 vol. 57 supplement s3 reagents should include an evaluation of antigen variants to confirm fidelity. we evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed rh e variants. study design/method: two monoclonal anti-e clones, rd9/4 and rd12/2, and a licensed comparator anti-e (p3gd5121ms63), all from diagast (loos, france), were evaluated. rbc samples were either recovered from frozen storage (n 5 42) or edta blood from donors (n 5 72) and were tested using a manual tube method or on a pk7300 automated platform. a score 6 (11) or greater was deemed acceptable for manual tube and a positive call for automated testing. results were tabulated by complexity of rhce*ce alleles (table 1) . results/finding: the specificity of the monoclonal anti-e were confirmed using common rhce haplotypes: r 1 r 1 , r 2 r 2 , r 1 r, and rr. twenty-one different rhce*ce alleles were included in the extensive panel: 40 were rhce*ce that were in trans to rhce*ce; 16 were various rhce*ce plus rhce*ce48c compound heterozygotes; 31 were rhce*ce or rhce*ce homozygotes; 27 were various rhce*ce and rhce*ce compound heterozygotes. the comparator reagent was negative or unacceptably weak for 6 rhce*ce alleles in trans to rhce*ce (rhce*cear, rhce*cemo, rhce*-cejal, rhce*cehar), with 1 rhce*cear/rhce*ce48c compound heterozygote, and with 1 rhce*cecf homozygote. rhce*cear, rhce*cemo, rhce*cejal, and 1 of 2 rhce*cecf homozygotes were detected using the comparator reagent. rd9/4 and rd12/2 failed with 4 and 1 e variants, respectively (table 1) . failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the rd9/4 clone. none of the reagents detected e antigen variant expressed on 1 example of rhce*cehar/rhce*ce. conclusion: rd9/4 and rd12/2 anti-e reacted with more e variants than the comparator reagent. the e antigen encoded by rhce*jal and rhce*ar is not always detected when in trans to rhce*ce. however, double-dose expression was detected suggesting that the 3 monoclonal reagents bind weakly to the respective altered e antigen epitopes. the e antigen encoded by rhce*cehar continues to be a challenge to detect. meihong liu*, teresita mercado, orieji illoh, maria rios and zhugong liu. obrr, cber, fda background/case studies: extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (rbcs) that match those of the recipient. this is especially important in the management of chronically-transfused patients and patients with rbc alloantibodies. several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes. targeted next-generation sequencing (ngs) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples. we developed and evaluated targeted ngs assays using two different target enrichment platforms for extended blood group genotyping. study design/method: two custom design platforms sureselect and halo-plex were used independently for preparation of probes that target the entire genes of 19 blood group genes associated with the expression of 56 blood group antigens from 17 blood group systems. we used the illumina's hiseq 2000/2500 system to perform next generation sequencing first on sureselect-enriched genes from 16 dna reference samples with average target design coverage of 97.5%, and then on haloplex-enriched genes from 32 dna reference samples with average target design coverage above 97.0%. twelve samples were enriched and sequenced in both methods to allow a direct comparison. all reference samples were previously characterized for 38 blood group genetic variants in these 19 genes using taqman snp assay and sanger sequencing assay. serological data were also available for these samples. the ngs data were analyzed by clc genomic workbench. sequencing variants were detected and annotated using dbsnp database. blood group genotype calls by the two targeted ngs methods were compared with the reference results. results/finding: for the two targeted ngs methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of ngs, sequencing coverage, and genotype concordance. a higher percentage of the haloplex reads (80.54%) were mapped to the target regions relative to the sureselect reads (29.23%). the mean sequence coverage depth of the targeted bases was around 200x for sureselect method and 300x for haloplex method. some exons, such as rhd exons 4 and 8, 10, rhce exon 10, ermap exons 5 and 12, cd55 exons 10 and 11, cr1 gene (most exons) and gypb exon 5, are consistently covered with less than 10x coverage by both sureselect and haloplex targeted ngs methods. both methods detected rhd gene deletion in a few representative samples. the genotype call concordance on 38 blood group genetic variants was assessed by comparing ngs results to taqman genotyping and sanger sequencing results, and more than 90% concordance was obtained for both targeted ngs methods. incorrect calls were restricted to four complex blood group genes: mns, rhd, rhce and abo, and involved mainly heterozygous variants and indels. conclusion: using two targeted ngs methods, we have correctly detected more than 90% blood group genetic variants in 19 selected genes. evidence rhce*cehar does not encode for rh34 (hr b ) antigen debra j bailey* 1 , trina horn 2 , paul mansfield 2 , najmi qazi 1 , pamela nickle 2 , jessica keller 2 , margaret a keller 2 and jan r hamilton 1 . background/case studies: the rhce gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete. new information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation. the rhce*cehar allele was first described in 1996 and has a phenotype of c2e2c1e1 w f1 w , g2, hr 0 1 w , hr2, hr s 2, rh:33, rh:50 with a partial d antigen expression. we describe new information regarding an rh haplotype that includes an rhce*cehar allele and its apparent rh:234 (hr b 2) expression. study design/method: rbc typing was performed by standard tube methods with polyclonal and monoclonal antisera. antibody identification studies were performed by standard tube hemagglutination methods by published techniques. molecular immunohematology testing was performed on genomic dna extracted from whole blood and included hea, rhd and rhce beadchips (immucor) and pcr-rflp analysis for rhce c.254c>g and rhd c.1136c>t. results/finding: a sample from an african american female with a history of an anti-e and anti-k was evaluated for unexpected antibodies. her red cell serologic rh phenotype on an untransfused sample was d1c1e2c1e1. her plasma contained an alloanti-s and an antibody that reacted strongly with all random e2k2s2 reagent red cells except her own. the unidentified reactivity persisted following ficin and dtt pretreatment of reagent red cells. only d22 and dc2 red cells were non-reactive in initial tests. differential adsorption studies excluded antibodies to all other common antigens and hr b except e, s and k. when subsequent examples of e2s2k2 red cells homozygous for the rhd*diiia-ce(4-7)-d, rhce*-ce48c,733g,1006t haplotype (i.e., r' s /r' s ) and rhd*diiia, rhce*-ce48c,733g,1006t/ rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t (i.e., bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-hr b . the patient's red cell antigen genotype identified the following probable rh haplotypes: rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t. additional antigen typing of the patient's red cells with unlicensed antisera indicated an hr1 (2 of 2 sources) and hr b 2 (2 of 3 sources) phenotype. conclusion: the rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t haplotype is one of the rh haplotypes expressed by the original hr b 2 individual bastiaan. the hr b antigen status of red cells of individuals with the rhce*-cehar allele has not been described. we report an individual with the probable rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t rh haplotypes and production of alloanti-hr b . the specificity of the alloantibody produced and the red cell hr b serologic antigen type supports the conclusion the variant allele rhce*cehar does not encode for the hr b antigen. excluding clinically significant alloantibodies in the presence of interfering antibodies with high-titer, low-avidity characteristics. background/case studies: high-titer, low-avidity antibodies (htla) are a group of clinically insignificant antibodies (ab) directed against highprevalence red cell antigens. they interfere with the exclusion of clinically significant red cell ab and crossmatch testing, leading to long work-ups and potential transfusion delays. we often use automated solid phase red cell adherence assay antibody panels (sp) when htla interference is seen by other methods, and undertook this study to determine its efficacy. study design/methods: a search of the laboratory information system database was conducted for patients with htla between 1/1/2006 and 3/ 31/2017. all patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant ab (rule out). results/findings: over approximately 11 years, 81 patients had htla established at least once by titration studies. serological investigations on a total of 118 samples using a combination of gel, sp, and peg and liss tube methods, and occasional dtt and ficin panels, found that htla interference noted most frequently in gel (primary method) was, indeed, less often seen with sp. however, the proportion of cases achieving rule out on sp was no greater than that with peg testing (table) . for samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory. reference testing on 20 samples was successful in rule out in 60% of cases. in an additional 12 patient samples, with negative antibody screens, htla were identified upon work-up for incompatible crossmatches. conclusion: sp is useful in avoiding interference from htla, but this conclusion is limited because sp was performed in only 40% of samples, and the inability to use select cell panels with sp made it difficult to complete rule out on samples containing multiple ab. peg testing was available for 71% of samples, and was at least as effective. further, manual testing allowed flexibility in selecting test cells when other ab were present. both sp and peg testing may be used alone or in combination to avoid interference due to htla, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations. background/case studies: the dau family of rhd alleles is characterized by c.1136c>t (p.thr379met). the dau0 allele harbors only this change, is not associated with depressed or altered d antigen expression, and is the ancestral allele from which other dau alleles are purported to have evolved. srivastava et al (transfusion 2016, 56:2520) recently summarized serologic characteristics and associated anti-d alloimmunization for 18 dau family alleles. we investigated two samples with the c.1136c>t change referred with weak d antigen expression. study design/method: serologic testing was performed by standard tube methods using licensed anti-d reagents and the albaclone partial rhd typing kit. genomic dna was isolated from wbcs and used in manual and array assays and for amplification and sequencing rhd. results/finding: sample 1 was from a 17 yo multiracial female. her rbcs reacted 11 s at immediate spin (is) and 31 in iat with immucor gammaclone and series 4 and 5, and mi1 at is and 41 in iat with ortho bioclone anti-d. rbcs did not react with 2 of 12 (lhm 174/102 & 57/17) anti-d in the partial d typing kit. this pattern did not match any of the defined partial d epitope patterns. rhd beadchip found no changes but rflp detected c.1136c>t characteristic of dau. rhd sequencing confirmed c.1136c>t and identified two adjacent changes, c.787g>t and c.788g>t (c.787_788delinstt), in exon 5 encoding p.gly263leu. sample 2 rbcs reacted 1w at iat with both ortho bioclone and quotient albaclone delta, but were non-reactive with immucor gamma-clone, series 4 and 5, and quotient albaclone blend and alpha anti-d. papain treated rbcs were 11s in iat with ortho bioclone. these results suggested a d el like phenotype. rhd beadchip found no changes but rflp detected c.1136c>t. sequencing confirmed c.1136c>t and found a new c.761c>t change (p.ser254-leu) in exon 5. the c.761t has not been reported, but c.761g encodes a stop codon (p.ser254stop) in japanese (vox sang 2015, 109:359). conclusion: we report two new alleles: rhd with c.787_788delinstt (p.gly263leu) and rhd with c.761c>t (p.ser254leu), both also carrying the c.1136c>t (p.thr379met) characteristic of the african dau cluster. d antigen associated with p.263leu is a partial d antigen with a novel epitope pattern. the p.254leu change is associated with a del-like phenotype, the first observed to our knowledge for a dau allele, and d antigen on the rbcs is not detected in iat by 5/7 commercial anti-d. the rhd nucleotide changes reported here are not in dbsnp database. this study brings the dau family of alleles to 21. the number and diversity of alleles in the dau cluster supports that the c.1136c>t change is a major ancestral african background allele (wagner et al, blood 2002,100:306). tae eun kim*. krc btri background/case studies: there have been the cases of anti-d alloimmunization caused by the transfusion of serologically d negative blood component. by analysis of genotype of the blood component, all of them were confirmed as asian type del. for that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay. we established the algorithm for the genetic analysis of rhdin blood donors. in this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test. study design/method: from september 2016 to present day we got 130 samples of repeated blood donors who are known to be d negative, c positive and/or e negative from 15 blood centers. we obtained the consent for the test from all of the donors who provided samples. as a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (pcr-ssp) for the region of promoter, exon 4, exon 7 and exon 10 in rhd gene. based on the results of pcr-ssp, we discriminated the results into total rhd deletion, rhd-ce-d hybrid and rhd variant. when the results were discriminated to be rhd variant, we additionally analyzed the sequence of exon 9 to confirm the existence of c.1227g>a and c.1222t>a variations. for the sample with indeterminate results, we performed sequencing for the full region of exon. when the result was confirmed to be rhd deletion or rhd-ce-d hybrid, the blood components were regarded as rhd negative. when the result was confirmed to be rhdvariant, the blood components were regarded as rhd positive. blood components were not supplied until the final results were obtained. results/finding: for the 130 sample, we identified 71 cases (54.6%) of total rhd deletion, 18 cases (13.8%) of rhd-ce-d hybrid, and 41 cases (31.5%) of rhd variant. 39 of rhd variant were determined to be asian type del with c.1227g>a variation. 2 cases of rhdvariant were regarded to be unknown variation. conclusion: the frequency of rhd variant in this study was 10 % higher than that of the general d negative donors not considering rhce phenotype in a previous study. for that reason, we considered that the genetic analysis of rhd targeting the donors of d negative, c positive and/or e negative is more efficient approach to identify rhd variant and better way to improve blood safety in the transfusion medicine related with rhd negative blood donors. lei fang tsai*, ping chun wu, shu hui feng, yi wen tsai, ming hung chen and shun chung pai. taipei blood center, taiwan blood services foundation background/case studies: certain abo subgroups or physiologic conditions may lead to mixed-field agglutination on abo typing among blood donors. the b 3 phenotype was found to be the most common subgroup in taiwanese. however, it is hard to distinguish the b 3 phenotype from other b subtypes also with mixed-field agglutination using routine serology without the genotype. this study aimed to evaluate if flow cytometric method could alternatively differentiate different b subtypes with mixed-field agglutination rather than using molecular genotyping. study design/method: blood samples from 30 taiwanese blood donors exhibiting known common abo phenotypes were included to establish normal flow cytometric patterns and genotyped. blood samples (n552) from b subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping. flow cytometric method was performed by facscalibur flow cytometry using the gamma-clone anti-a and -b. for genotyping, exon 6 and exon 7 of the abo gene were amplified and sequenced. the abo*b3.03 allele was confirmed by pcr-rflp analysis. results/finding: among 52 subjects with b 3 or ab 3 phenotypes, 47 were genotyped as abo*b3.03. the abo*b3.03 group performed similar characteristic flow cytometric pattern and the profile was reproducible over time. the pattern showed the main population of cells expressed no b antigen, while a percentage (37.81 6 6.62) of the rbcs exhibited b antigen levels diminishing with increasing of fluorescence. other 5 subjects with b 3 or ab 3 subjects, genotyped as abo*b3.06(n51), abo*bw.03(n51), abo*bw.11(n51), abo*bw.12(n51) and abo*bw.29(n51), displayed flow patterns differed from the abo*b3.03 group. the abo*bw.03, abo*bw.11 and abo*b3.06 subjects also showed a main population of cells expressed no b antigen and, however, less percentage of rbcs exhibited b antigen levels (<10% in abo*bw.03 and abo*bw.11 subjects and <20% in abo*b3.06 subject). both abo*bw.12 and abo*bw.29 displayed a wedge-shaped pattern. conclusion: the flow cytometric method for the detection of b antigens on rbc might be useful in discriminating between b subtypes with mixed-field agglutination, especially abo*b3.03 genotype. this approach could assist the serological abo subgrouping in clinical reference laboratory. frequencies and specificities of "solid-phase only" detected erythrocyte antibodies: is solid phase testing worth the headache? karen finegan*, karen gray, jill adamski, theresa kinard and qun lu. background/case studies: an effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system. the data available from parallel testing on solid-phase, gel, and peg performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care. study design/methods: throughout 2016, the transfusion service used automated solid-phase red blood cell (rbc) adherence as the primary method for antibody screening and identification. all solid-phase antibody screen positive samples were re-tested using both gel column agglutination and peg method manually in order to determine which method should be used for antiglobulin phase crossmatch of rbc products. all antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed. results/findings: a total of 398 patients were positive on solid-phase antibody screen and re-tested on gel and peg antibody screen. in 12% (n549) patients antibody reactivity observed in solid phase only and the concurrent gel and peg testing were completely negative. of them clinically significant rbc alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in 22% (n511), 4% (n52), and 74% (n536) of the cases, respectively. rbc alloantibodies identified in solid-phase only included anti-e (n54), anti-jka (n53), anti-k (n52), anti-jkb (n51), both anti-e and anti-c (n51) (see table 1 ). conclusion: solid-phase only rbc antibodies are clinically important in a significant portion of cases (roughly 1 in 4 cases). workup for solid-phase only antibodies is not "unnecessary" workload. transfusion of corresponding antigen negative rbcs to these patients prevented possible hemolytic transfusion reactions. full-length nucleotide sequence of ackr1 alleles encoding duffy (fy) antigens in africans of ethiopia qinan yin*, kshitij srivastava, addisalem taye-makuria and willy a flegel. background/case studies: the human ackr1 gene (previously known as darc), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the duffy blood group antigens (fy). the duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites plasmodium vivax and p. knowlesi. the study of fy variants in the low altitude and tropical gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time. in the present study, we determined the full length nucleotide sequence of the ackr1 gene encoding fy antigens in donors from ethiopia's southwestern gambela region. study design/method: edta-anticoagulated whole blood was collected from study 60 volunteers in the gambela region (nct01282021). the whole ackr1 gene was amplified in one reaction covering 12,125 base pairs (bp). this primary amplicon was re-amplified using nested primers covering 5782 nucleotides. nucleotide sequence was obtained by 14 sequencing reactions and manually annotated using ncbi refseq ng_011626.2. the sequencing covered 1008 bp of both exons, 480 bp of intron, 2101 bp of 5'-flanking region, 947 bp of 5'-utr, 53 bp of 3'-utr and 1092 bp of 3'-flanking region and encompassed all the 470 variations present in dbsnp and nhlbi esp databases. results/finding: among the 60 samples, a total of 15 snps, including one novel snp in 5'-utr were observed. 4 snps occurred in the exons, 5 in 5'and 3'flanking region, 4 in 5'-utr and 2 in the intron. all 60 individuals carried the snp indicative of the common fy:2 phenotype; while 58 individuals were homozygous and 1 was heterozygous for the gata box mutation. no splice site mutation was detected. as 46 individuals were observed as being homozygous or heterozygous for 1 snp, we could unambiguously assign 8 distinct alleles. in the remaining 14 individuals with 2 or more heterozygous snps, allele specific pcr is required to identify the alleles. conclusion: we sequenced more than 5.5 kb of the ackr1 gene and identified at least 8 different alleles. the present study found that the vast majority of alleles (117/120) in the gambela population as defined by 15 snps, were similar to the clinically relevant fy*02n.01 allele, which in turn is defined by only 2 snps at positions c.1-67t>c and c.125g>a. out of the remaining 3 alleles, 2 were similar to fy*02 with the fy(b1) phenotype and 1 was similar to fy*02w.01 with the fy x phenotype. the high frequency of fy*02n.01 (95%) in this study is similar to other studies conducted in western, central and south-eastern regions from gambia to mozambique (95%-100%). a more detailed analysis, including other regions of ethiopia, will be useful to support transfusion care in the us for ethiopian-americans, the majority of whom may be of mixed ethiopian ethnical background. judith aeschlimann*, sunitha vege, christine lomas-francis and connie m. westhoff. immunohematology and genomics laboratory, new york blood center background/case studies: the homology, proximity, and inverted orientation of rhd and rhce on the chromosome favor gene conversion events. regions of rhd are transferred into rhce and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens. rhd*diiia-ce(4-7)-d is the most common hybrid and is found in african blacks. it arose by conversion of exons 4-7 of rhce*ces into rhd*diiia and no longer encodes d antigen, rather (somewhat confusingly) encodes partial c antigen from the rhd locus. this hybrid allele is in cis to rhce*-ces, together known as the r's haplotype. we investigated atypical rh genotyping results in three samples; two associated with weak d typing and one patient with sickle cell disease (scd). study design/method: serologic testing was by standard methods. genomic dna was isolated from wbcs. all samples were investigated by hea precisetype, rhd and rhce beadchip, rflp, and rh-cdna sequencing. snp-specific sequencing was used to establish linkage/phasing. results/finding: sample 1 (male) and sample 2 (multiracial female), both c1c2e2e1 (presumed r1r1), presented with weaker than expected d typing; 11 is and 31/41 at iat. rhd beadchip identified the common african rhd*diiia-ce(4-7)-d hybrid encoding partial c antigen with apparent conventional rhd in trans. these results did not provide an explanation for weak d antigen. hea indicated rhce*ce /ce, concordant with the rh phenotype, but c.733c>g and c.1006g>t (heterozygous) was also detected. as rhce*ce with 733g and 1006t has not been reported, rh-cdna analysis was done. transcripts from the rhce locus included one conventional rhce*ce in trans to rhce*ces with exons 2 and 3 replaced with rhd*diiia, and from the rhd locus, one conventional rhd and the hybrid rhd* diiia-ce(4-7)-d were found in both samples. sample 3 (scd male), d1c2e2c1e1, by rh beadchip had one conventional rhd and rhd*diii type 8, and rhce*ce733g/ces. as rhd*diiia type 8 has never been found with either of these rhce alleles, rh-cdna analysis was performed. transcripts representing a unique conversion event at the rhd locus, specifically rhce*ce(48c) exons 1 and 2 had replaced those exons in the common hybrid rhd*diiia-ce(4-7)-d and expression of partial c antigen was lost these unique hybrid alleles have been deposited as genbank#: ky926711and ky926710. we report two different and novel complex rh rearrangements: two samples thought to be r1r1 had a unique rhce locus representing a gene conversion into rhce*ces, designated rhce*ces-diiia(2-3)-ce. in kind, a sample genotyped as diii type 8 rather had a novel rhd locus representing a gene conversion into the common hybrid, designated as rhd*ce48c(1-2)-diiia(3)-ces(4-7)-d . these represent novel events on the r's haplotype that can confound rh genotyping interpretations. interestingly, samples 2 and 3 have weaker than expected d antigen typing, despite the presence of a conventional rhd with rhce*ce [r1 haplotype (dce)]. it is important to further investigate samples with unconventional results when interpreting rh genotypes. high-frequency antibodies anti-lu(b-) and anti-yt(a-) in a multi-transfused patient: a case study nadia baillargeon*, carole ethier, cynthia parent, jessica constanzo-yanez, maryse st-louis, marie-claire chevrier and andre lebrun. hema-quebec background/case studies: a 81-year-old caucasian female was referred to our immunohematology reference laboratory (irl) for serological investigation. she was diagnosed with anemia, renal failure and cardiac history. her hemoglobin level was recorded at 81g/l. her pregnancy history was not provided. she had received 5 units of packed red blood cells (rbcs) in the past including 1 unit within the last 3 months. none of the transfused unit was phenotypically-matched. the referring hospital obtained panreactivity in gel with liss-suspended rbcs and ficin-treated rbcs and negative direct antiglobulin test (dat) and autocontrol (at). study design/methods: abo/rh, dat and antibody identification were performed by h ema-qu ebec's irl according to approved techniques. in addition to liss-suspended rbcs and papain-treated rbcs, trypsin-treated and chemical-treated reagent rbcs such as dithiothreitol (dtt) were tested. alloadsorption were done using papain-treated allogeneic rbcs (r 1 r 1 , r 2 r 2 , rr). id core xt platform (progenika biopharm / grifols, vizcaya, spain) was used to analyse 29 polymorphisms which determine 37 antigens including carthright and lutheran blood groups. pcr-ssp (sequence specific primer) and pcr-rflp (restriction fragment length polymorphism) were also performed to verify the absence of the high frequency antigens yt a and lu b . sibling samples were also requested to conduct a family study. results/findings: initial serologic testing showed strongly reactive panels in gel with liss suspended rbcs, papain-treated rbcs as well as trypsintreated rbcs and dtt-treated rbcs but negative at in each media leading to a probable antibody directed against high-frequency antigen. alloadsorption procedure allowed the identification of an anti-jk a . a select panel of high frequency antigens absent in caucasian population was tested. the patient's sera react weakly with one jk(a-), lu(a-b-) reagent cell. in the meantime, genotyping results confirm the probable phenotype of the patient as jk(a-) lu(b-) yt(a-). additional testing in gel using trypsin and dtt differential effects on antigens lu(b) and yt(a) were performed to confirm antibody specificities. no rbcs unit jk(a-) lu(b-) yt(a-) were available for transfusion. selected units were jk(a-) and lu(b-) as alloanti-yt a are known to cause none to moderate transfusion reactions. her daughter' sample were types as yt(a1) and lu(b1). conclusion: serological study showed the presence of an anti-jk a in addition to two antibodies directed against high prevalence antigen namely anti-lu b and anti-yt a . the association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination. background/case studies: the kel blood group system, consisting of 36 antigens encoded by the kel gene, is organized into 19 exons. there are approximately 30 kel alleles associated with a kell null phenotype (k 0 ) in which no kell antigens are expressed, and 12 alleles associated with a kell mod phenotype (k mod ). individuals with the k mod phenotype express very weak amounts of antigen on the surface of the rbc, and expression levels vary based on the allele present. here we describe the molecular and serologic testing that was performed in the case of a 53 year-old hispanic male blood donor whose rbcs phenotyped k-k-js(b-) kp(b-). study design/method: the blood donor was phenotyped for k, k, kp b and js b antigens using standard tube agglutination methods. adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (american national red cross). genomic dna (gdna) was isolated from an edta blood tube using standard techniques. dna was genotyped for human erythrocyte antigens using the precisetype tm hea molecular beadchip (immucor). exons 10, 11, 12,13 and 14 and flanking intron sequences were amplified and sequenced. total rna was extracted using rneasy lipid tissue mini kit (qiagen) and kel cdna was amplified and the resulting pcr product was subjected to sanger sequence analysis and aligned using sequencher (genecodes). results/finding: precisetype tm hea molecular beadchip testing predicted the sample to be k-k1 kp(a-b1) js(a-b1). kel-cdna sequence analysis was performed and detected a single transcript species with c.578c, c.841c, 1790t, and missing the sequences corresponding to exons 11, 12 and 13. amplification of the exons from gdna did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact. cdna analysis was repeated and the same aberrant transcript was detected. adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after 37c incubation at the peg-igg-agt phase. conclusion: here we describe a donor homozygous for a novel kel*02 allele. this donor was presumed to have a k 0 phenotype based on serology, but after molecular testing, has been reclassified as a k mod phenotype with extremely weak expression of k. the discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells. the 11 variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations. in contrast, the allele reported here is associated aberrant mrna transcript. we propose that this allele be named kel*02m.12. here we report a case of a possible novel b subgroup observed in a pregnant black female. the patient specimen was referred to our reference laboratory to investigate a possible abo discrepancy. the referring facility reported the patient's red blood cells were nonreactive with reagent anti-a and anti-b and the patient's plasma was reactive with a cells, but nonreactive with b cells using automated gel methodology. study design/methods: serological testing of the patient's red blood cells was performed using routine and enhancement methods. molecular testing by pcr-rflp was performed to determine the patient's genetic abo typing and predicted abo phenotype. results/findings: serological testing of the patient's red blood cells is summarized in table 1 ; similar results were obtained with multiple sources of antisera. enzyme treatment failed to enhance reactivity. patient sera strongly agglutinated a1 and a2 cells, but failed to agglutinate multiple sources of b cells at all phases of testing. molecular testing by pcr-rflp resulted in an uncommon banding pattern and indicated the presence of c.261 deleted g, characteristic of o alleles, c.467t, characteristic of a2 and some uncommon o alleles, and c.703a and c.1096a, characteristic of b alleles. genomic sequencing of exons 6 and 7 confirmed the presence of an o allele, abo*o09 261del g, 318t, 467t), and the presence of a b allele (297g, 526g, 657t, 703a, 796a, 803c, and 930a), but did not reveal any changes associated with previously reported weak subgroups of b. conclusion: while serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone. additional abo gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , diana gazito 2 , afonso cortez 2 , lilian castilho 4 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: after the elucidation of the molecular basis of vel, molecular tools have been used to explain the reduced expression of vel antigen in different populations. negative or weak reactions are generally related to the 17-bp deletion in smim1 in homozygous or heterozygous status. however, other nucleotide changes have been described to reduce the vel expression, as for example, the major a allele of the snp rs1175550 located in the second intron of the gene, a regulatory region in erythroblasts. this study aimed to characterize the genetic changes related to atypical vel expression in a brazilian population. study design/method: a total of 400 blood donor samples from the southeast region of brazil were typed for vel with an anti-vel serum from our inventory in gel-iat. samples typed as vel-negative were further analyzed by adsorption-elution. molecular study was performed in samples with negative results, in samples reacting 21 and in samples with reactivity of 31. dna was isolated from peripheral blood and smim1 was sequenced. results/finding: from 400 donor samples studied, 4 were serologically vel negative by gel-iat but positive by adsorption-elution, 158 presented a 21 reaction and the remained samples showed a reactivity of 31. genotyping results showed that the 4 samples with negative results and 5 of 26 samples that presented 21 reaction were heterozygous for the 17 bp deletion and presented the a allele rs1175550 in homozygous status. from the 21 of 26 remaining samples with reactivity of 21, 19 (90%) had the a allele of rs1175550 and 14 (66.7%) had the a allele of rs6673829. in contrast, in the 16 samples with stronger reactions we found the a allele of rs1175550 in 5 (31.25%) samples and the a allele of rs6673829 in 3 (18.75%) samples. conclusion: the molecular changes rs6673829 and rs1175550 are located in intron 2 distancing 38 nucleotides. this study reinforces the association of the a allele of rs1175550 with reduction of vel expression and suggests the involvement of a new rs6673829 change in vel expression. in conclusion, the several patterns of vel expression found in different populations can be influenced by different molecular changes. background/case studies: the d antigen is the most immunogenic antigen after abo. consequences of misclassification of the d-antigen in patients or donors can be severe. some persons inherit mutations resulting in quantitative reductions of d antigen on the cell surface (weak d), some inherit rh haplotypes that result in biochemical effects that reduce the availability of the d antigens to reagent anti-sera (ceppellini effect), and others inherit d genes which are qualitatively different than wild type d. these latter individuals are often not identified until after they have formed anti-d. we hypothesize that some of these persons at risk of forming anti-d might be uncovered if they have weak and/or disparate d typing results with reagents that recognize different epitopes of the d antigen. study design/methods: all testing was performed using microtiter-well direct agglutination on the galileoneo or galileoecho (immucor, norcross,ga). any specimen that did not react as 0 (rh negative), or !31 on the neo or !21 on the echo (rh positive) for both series 4 and series 5 anti-d antisera were included. patients with discrepant historical types also were evaluated. any specimens meeting the inclusion criteria were tested on the neo, echo, and by saline tube method using series 4 and series 5 anti-d antisera. genotyping was performed from whole blood samples sent to immucor genotyping laboratory in warren, nj using an algorithm of: rhd beadchip, rhdxp (prototype assay), rhd zygosity, and rhce beadchip. results/findings: 80 patients met inclusion criteria for molecular testing for the d antigen. weak or rhd variants were identified in 51 of 80 (63.7%) of the samples. ceppellini effect (i.e. c in trans to rhd) resulting in weak d reactivity was seen in 16 of 80 (20%) of samples. 67 of 80 (83.8%) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm. 40 of 80 (50%) of tested samples had results indicating weak/variant d proteins with the potential to cause alloimmunization to the d antigen. the remaining 13 of 80 (17.3%) samples did not have identified genetic cause for the weak and/or discrepant d test results and were presumptively classified as wild type d. conclusion: transfusion services that use the galileoneo or galileoecho to perform rh typing should consider molecular testing of patients whose rh typing results are discrepant, or positive but <31 on the galileoneo or positive but <21 on the galileoecho, as about half of these patients can develop anti-d. this is particularly relevant for females of child-bearing potential where avoidance of d-positive transfusions and administration of rhig during pregnancy is prudent until their d typing can be confirmed by molecular testing. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , rosangela person 2 , lilian castilho 4 , afonso cortez 2 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: rhd and rhce, are major protein constituents of red blood cell membrane, composing a complex together with rhag. many variant rh proteins have been described and most of them affect the integration of rh proteins in the membrane. d antigen expression can be affected by several molecular changes and also by the rhcehaplotypes. the present study investigated the score of reactivity of samples presenting a strong reduction in d expression. study design/method: a total of 108 samples were included in the study, being 69 previously genotyped as rhd*dar1.2, 37 rhd*dar3.1 and 2 rhd*dau6. the samples were phenotyped in neov r (immucor) to d, c/c and e/e antigens by direct agglutinationin microplate. results obtained in neov r were expressed in a score from 0 -99 corresponding to the reaction intensity. zygosity assay was performed by a multiplex real-time quantitative pcr using a set of rhd-specific primers in rhd exon 10. rhce genotyping was performed by pcr-rflp and ssp-pcr. the presence of a d-cehybridexon 3 was identified by amultiplex pcr. sequencing and identification of rhce variants were also performed when necessary. results/finding: zygosity results showed that 10 of 108 samples (5 dar1.2, 4 dar3.1 and 1 dau6) had 2 rhd genes, were phenotyped as c1e-c1e1 and genotyped as rhce*ce/rhcece. rhd and rhce genotyping in these 10 samples showed the presence of the d-ce-d s hybrid gene. rhce variants investigated in 2 dar1.2 samples showed the rhce*-cear/ce s genotype, in 2 dar3.1 samples the rhce*cevs.02/ce s genotype and in the dau6 sample the rhce*ce s /ce genotype. table 1 describes the differences found in the reactivity of d among the samples carrying the (c)ce s allele and in the samples homozygous for rhce*ce. the results showed that the presence of rhce*(c)ce s significantly reduces the expression of d antigen, probably due to the expression of the partial c partial antigen in trans to rhd. additionally, the samples with reduction on d expression carrying rhce variant alelles phenotype can be useful to provide compatible blood to some patients with rarerh variant alleles. background/case studies: drugs are known to interfere with routine blood bank testing. a novel monoclonal humanized 5f9 antibody (hu5f9-g4) that binds human cd47 has been entered into clinical trials for patients with acute myeloid leukemia, non-hodgkin lymphoma and solid tumors. we describe two cases of patients treated with hu5f9-g4 (anti-cd47) who had abo discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma. study design/method: this is a retrospective review of two cases with immunohematology work-up showing abo discrepancy and plasma panagglutinin. the first case is of a 69 year old female with progressive follicular lymphoma who was enrolled in phase 1b/2 trial of hu5f9 g4 in combination with rituximab designed for patients with relapsed/refractory b cell nhl. she had no prior transfusion history and her historical blood type was not known. two rbc units were requested in anticipation for a surgical procedure. the second case is of a 48 year old male with refractory diffuse large b cell lymphoma enrolled in hu5f9-g4 clinical trial. his historical blood type was a rh d positive with a negative antibody screen. he received three rbc units within the past month prior to testing and receiving the anti-cd47 therapy. results/finding: the abo typing in the first case showed a discrepancy between the forward typing (41 with anti-a, non-reactive with anti-b) and the reverse typing (31 with both a 1 cells and b cells). rhd typing was positive. the extended reagent rbc panel tested with the patient's serum reacted with all cells tested at the immediate spin (is) phase (11 to 41), at liss-37c (11 to 31), at liss-polyspecific ahg (m1), and at peg-anti-igg (m1 to 11). plasma reactivity at is persisted with dtt or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or rest adsorption. repeat testing, which avoided the is and 37c readings, was non-reactive in the antihuman globulin (ahg) phase using both liss and peg enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens. the direct antiglobulin test (dat) and autocontrol were negative. the rbc units issued to the patient were crossmatch compatible at 37 o c ahg phase. the abo typing of the second case performed after anti-cd47 administration showed a discrepancy between the forward (41 with anti-a) and the reverse (41 with both a 1 and b cells). rhd typing was positive. the antibody screen performed in solid phase technology was positive with all reagent red cells. his plasma reacted with all reagent red cells at is (21), at 37c in liss (21), and liss-polyspecific ahg (m1). the dat and autocontrol were negative. his genotype was determined to be a1/o and full rbc phenotype by dna analysis was obtained. repeat testing which avoided the is phase did not show reactivity at peg-ahg excluding all alloantibodies directed toward common red blood cell antigens. conclusion: anti-cd47 therapy interferes with blood bank testing by causing abo discrepancies and panagglutinin reactivity in the plasma at is, 37c liss, but not at ahg phase using gamma-clone anti-igg, unlike the anti-cd38 interference. knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood. background/case studies: a middle-aged male with discrepant abo typing results was investigated. initial forward typing was group o but no anti-b was seen in the reverse typing. an unexpected 31 reaction was noted with an anti-a,b reagent. genotyping surprisingly showed abo*o.01.01/ o.01.01, consistent with group o. after initial testing at the referring center, samples were sent for extended analysis. study design/method: standard serological methods and flow cytometry were used. a panel of abo reagents (n523) and lectins were tested with both native and papain-treated red blood cells (rbcs). lewis phenotyping was performed, as was genetic testing for abo, gbgt1 and a4galt. papain-treated patient rbcs were used to screen donor plasmas (n578) and two reactive plasmas were dtt-treated. results/finding: positive reactions were obtained with 3 polyclonal anti-a,b and a monoclonal anti-b (clone g1/2) when tested with the patient's papain-treated rbcs. a panel of lectins gave negative results. genetic testing confirmed the predicted group o and ruled out the presence of fors1 or nor antigens. the patient was le(a-b1) and thus a secretor. a positive crossmatch was seen with 47% of group o plasmas, while no reactivity was obtained with a or b plasmas. dtt treatment of crossmatchpositive plasmas indicated the antibody to be mainly of igg type. this was confirmed by positive flow cytometry cross match using anti-human igg secondary antibodies. reactivity remained after b-zyme treatment, thus excluding the normal (type 1 or 2) b antigen to be the underlying reason. inhibition with lewis substance significantly decreased reactivity. enzyme activity assay showed the patient's plasma to contain a fully functional b glycosyltransferase. on the suspicion that the patient had non-erythroid cells producing breactive type 1 chains, a sample from a hematopoietic stem cell transplant (hsct) patient (group b secretor receiving group o donor cells) was included as a control and gave the same type of reactions. conclusion: the medical history of the patient was queried and he had indeed undergone an hsct $15 years earlier. the reactions are likely due to uptake of recipient-derived ble b (type 1) antigen (isbt no. 007006), which is the dominant lewis antigen in the recipient's original blood group, b le(a-b1). interestingly, b-zyme did not affect ble b . anti-ble b is not simply anti-b plus anti-le b but an inseparable and rarely reported specificity, which appears to be common among group o donors. the phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups. background/case studies: the provuev r and visionv r (ortho scientific, raritan new jersey) automated analyzer use mts-gel tm card technology to perform immunohematology testing. benefits of automated testing include improved efficiency and enhanced reliability. after eight years of using the provuev r our transfusion medicine service switched to the ortho visionv r analyzer in january of 2017. shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?") results. additionally, some test columns were noted to be visually negative but called positive (11) by the analyzer. the objective of this study was to investigate the cause of "?" and apparent false positive results on visionv r three-cell antibody screens. study design/methods: with assistance from ortho diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?" results, and the gel card lot numbers used for testing from 1/29/2017 to 3/31/2017. reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results. investigation also included review of daily qc records, instrument maintenance, instrument diagnostics, and camera calibration. results/findings: of 19,647 columns run as part of antibody screens, 1,633 (8.3%) columns generated "?" results. assuming 30 seconds of technologist time per "?", we estimate that 13.6 hours were needed to resolve and update these results. among all potential causes investigated, only the gel card lot number was associated with the number of "?" generated (table 1) . in 26 cases, all three columns were visually negative but the analyzer reported 11 reactivity with 1 of 3 cells. all cases had mts-gel tm antibody identification panels performed, 25 of 26 also had a mts-gel tm ficin panel. the yield for the 51 panels performed was two routine panels with weak reactivity against hla1 cells, and four ficin panels with weak reactivity with no apparent specificity. fourteen patients coincidentally had a subsequent type and screen; 13 were negative. one patient newly demonstrated anti-jka. fifty percent (13/26) of visually negative but analyzer positive samples were tested with gel card lot number 3, 38% (10/26) with lot 1, and 12% (3/ 26) with lot 2. conclusion: the incidence of "?" and visually negative analyzer positive results is dependent on the specific lot of mts-gel tm cards used. the difference between the lots is being investigated by ortho diagnostics, and remains to be explained. to avoid unnecessary waste of technologist time and other resources, with assistance from ortho diagnostics, we have results/finding: of the methods evaluated, the dtt method proved the most useful for mitigating dara interference. cord cells were effective but in limited supply and alloadsorption was ineffective. of the three different dtt methods evaluated, the tube method initially failed which led to re-evaluation with the addition of liss (passed). the gc method was the most sensitive method. following release of dara, samples from 28 patients (137 cross-match samples, 301 units issued) were tested using both liss tube and gc iat methods. despite dtt treatment, the gc method remained positive by iat in 16/28 patients. further testing was performed in 9/16. eight were tested for the presence of antibodies at 188c and confirmed in 8/8. rouleaux formation was observed in 5/9 patients, 4/5 had reactivity detectable at 188c. no transfusion reactions have been reported to date nor has alloantibody formation been observed to date. conclusion: as previously reported, the dtt method was the most useful for mitigating dara interference. the observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the liss tube iat. this may be due to the washing phase in this technique which dissipates rouleaux formation. reactivity due to cold reactive antibodies can be eliminated by performance at strict 378c. our practice is now to use both dtt iat methods on initial patient referral, if residual reactivity in gc is observed use liss tube in preference thereafter in these patients. a further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude dara use if suspected. wendy beres* 1 , sandra nance 2 , david moolten 3 and p. dayand borge 3 . (2):47-53). our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels. this 2.5 year retrospective study was performed to assess levels of rbc-bound igg, iga and igm in normal donors. study design/method: residual edta-anticoagulated aliquots from 150 random allogeneic and 20 autologous blood donors were sequestered and tested per institutional review board approved protocol. the samples were tested by fc with fluorescein isothiocyanate (fitc)-labeled anti-human igg and iga (jackson immunoresearch lab, west grove, pa) or fitc-labeled anti-human igm (life technologies, carlsbad, ca) at optimized dilutions in dulbecco's pbs containing 0.6% bsa. the becton dickinson facscalibur tm or facscan tm (san jose, ca) fc analyzed 50k rbcs from each sample. edta-anticoagulated samples and ig coated control rbcs were tested to determine fc settings and control for validity and cross-reactivity. controls reacted as expected. there were less autologous donors tested and with a mean age of 62 these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured. despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated rbc-bound iga, igg, and igm levels. this emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing. long range pcr reveals the genetic basis of an antibody in pregnancy to a high prevalence mns antigen judith aeschlimann* 1 , anna burgos 1 , virginia lew 2 , sunitha vege 1 , susan veneman 3 , christopher j gresens 3 , jonathan hughes 3 and connie m. westhoff 1 . 1 immunohematology and genomics laboratory, new york blood center, 2 blood centers of the pacific, 3 bloodsource background/case studies: recombination events have generated many gypa and gypb hybrids giving rise to glycophorin (gp) variants that express low-prevalence antigens (e.g. mia, miny, mur). in rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g. enkt, eneh, enav). complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes. we investigated samples from a pregnant asian (hmong) woman with an antibody to an unidentified highprevalence mns antigen, and samples from her sister. study design/method: standard methods were used for rbc typing with licensed and unlicensed reagents and for antibody identification. dna was isolated from wbcs, and hea precisetype, exon-specific amplification and sequencing gypb exons 1-6, and long range sequencing of exon 2-6 (5.4kb amplicon) were performed. snp-specific primers were used to associate changes (phasing) to specific alleles. results/finding: rbcs of the pregnant proband typed s-s-(gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high background/case studies: the rhd antigen is clinically significant and immunogenic and therefore individuals who develop anti-d are at risk of haemolytic transfusion reaction. rhd polymorphism shows substantial ethnic variability and at least 100 rhd variants associated with weak d alleles have been reported. in this study, we report two new rhd alleles in brazilian blood donors associated with weak d antigen expression. study design/method: the d status was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. most common weak d and partial d alleles were investigated by allele specific (as) pcr and with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. in order to determine rhd allelic combinations, we also performed rh-cdna cloning and sequencing. background/case studies: donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (rbc) genotyping panel. when serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely. investigation of the discrepancy often leads to identification of variant antigens. it is known that the set of gyp variants associated with expression of the st a antigen can also be associated with n typing discrepancies in m1n-individuals (meyer et al. br j haematol. 2016; 174:624-36) . the st a allele, also described as gyp*401, is a hybrid gypb-gypatranscript with the crossover in intron 3. we sought to investigate five n typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel. background/case studies: a sample from a 24 years old pregnant, african american female g1p0 was sent to the blood bank for abo/rh and antibody screen. the sample was analyzed using the provue analyzer (ortho diagnostics). the patient was typed as o pos with no reverse type discrepancy. a retype of the same sample was performed using tube method with biorad reagents per hospital policy due to no previous abo/rh history on file. the retype showed that the patient was a subgroup with anti-a 1 antibody present in the plasma. the sample was referred for genotyping, with the suspicion of a 3 like phenotype. genetic testing did not support the serological findings of a 3 subgroup and a new abo allele, abo*784c that has never been reported in correlation with an a 3 like subgroup was detected study design/method: the patient rbcs were typed with anti-a 1 (immucor) and anti-a,b (biorad and grifols dg gel). an anti-a 1 antibody work up was performed using three different lots of a 1 cells and three lots of a 2 cells, as well as a type o screening cell and auto control . the tubes were read at is and also incubated at rt and 48c for 30min. the patient 's initial antibody screen using ortho gel was negative. conclusion: the patient delivered a healthy baby boy at 35 weeks of gestation. the baby cord was sent to the laboratory. the baby serological type showed an a 3 b phenotype and it was referred for genetic testing. the baby rbcs showed the same abo*784c found in the mother. the previously reported abo*784a allele encoded an aspartic acid to asparagine change at position p.256 consistent with an a weak phenotype. also, at least five other alleles encoding an a 3 phenoytpe consisted of polymorphisms at positions c.745 through c.871, giving special characteristics to this new and unreported abo allele. from the data collected, it can be concluded that this a3/ aweak phenotype is encoded by the variant allele abo*784c. this highlights the clinical relevance of confirming the serology of abo subgroups by molecular methods. philip berardi*, jacqueline cote, gwen clarke, vito scalia, robert liwski and mindy goldman. canadian blood services background/case studies: elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens. the commonly used single nucleotide polymorphism (snp) arrays require nucleic acid isolation which is typically achieved by extracting genomic dna from whole blood. this method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location. dna extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction. canadian blood services (cbs) has performed large scale dna extraction and hla genotyping for the onematch stem cell and marrow registry using buccal swabs since 2007; buccal swabs are also used by other unrelated donor stem cell registries, such as the us nmpd. we sought to assess the accuracy and reliability of using dna extracted from buccal swabs in predicting blood group antigen expression. study design/method: we performed parallel red cell genotyping on an automated typing platform, the progenika/grifols idcorext assay (progenika biopharma-grifols, bizkaia, spain) using dna extracted from blood and buccal tissue from volunteers. for antigen systems with available serologic reagents, we also compared results with serologic typing. we evaluated three different methods of dna extraction and performed testing regardless of dna yield or purity. two buccal swabs (puritan medical products, guilford, maine) were used for each test. swabs were stored at room temperature, and dna extraction was performed within six days of collection. in the initial phase of the study, buccal swab samples (n 5 15) were processed with the automated biorobot m48 robot using the magattract dna mini m48 extraction method (qiagen, venlo, the netherlands). extracted dna had a mean concentration and purity of 38.1ng/ml and 1.83 respectively. in the second phase of the study (n 5 39), dna extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the qiaamp dna mini kit, using either manual or an automated qiacube robotic workstation (qiagen, venlo, the netherlands). results/finding: the manufacturer's recommended analytical range for dna concentration was 20-80ng/ll and the recommended purity was an absorbance ratio of 1.63-2.1 (a260/280) for use of the id corext platform. dna extraction from buccal swab samples did not meet these specifications in several cases. however, in most cases, a lower concentration of dna was adequate for prediction of phenotype. the dombrock system was the most susceptible to failure of interpretation in the samples with a low dna concentration, with "no call" results reported. there was 100% concordance in genotyping results when source dna was extracted from whole blood or buccal tissue; there was also 100% concordance between predicted phenotype and serologic testing results. conclusion: this study supports the use of genomic dna extracted from buccal tissue on the id corext for predicting rbc phenotype with high accuracy. extraction methods may require optimization to achieve dna yields within the recommended analytical range of the assay. performance evaluation of id rhd xt, a genotyping assay for the detection of high-prevalence rhd negative and weak d types araitz molano 1 , izaskun apraiz 1 , maría azcarate 2 , miguel angel vesga 2 , montserrat rubia 2 , mercedes piedrabuena 2 , fernando puente 3 , barbera veldhuisen 4 , ellen van der schoot 4 and m onica l opez* 1 . 1 progenika biopharma, a grifols company, 2 centro vasco de transfusi on y tejidos humanos, 3 banco de sangre y tejidos de arag on, 4 sanquin blood supply research background/case studies: it is well established that weak d 1, 2 and 3 phenotypes are not at risk for forming allo-anti-d, whereas a few weak d and all partial d and negative phenotypes are. routine serologic d typing does not distinguish among them, consequently rhd genotyping is recommended, especially in patients. id rhd xt (progenika, grifols) is a qualitative, pcr/luminex v r xmap hybridization-based genotyping test for the identification of the following rhd gene allelic variants: rhd*weak d type 1, rhd*weak d type 2, rhd*weak d type 3, rhd deletion, rhd*pseudogene and rhd*diiia-ce(3-7)-d and itgb3 gene: hpa1a and hpa1b, in genomic dna extracted from whole blood specimens. in this study the performance of id rhd xt genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for rh and hpa-1 blood group typing. study design/method: a cohort of 1000 previously serotyped samples for d antigen obtained from three european blood centers were analyzed with id rhd xt at progenika. samples were distributed as recommended by the annex of the common technical specifications 2009/108/ce for a ivd product of list a (!10% clinical samples, >2% neonatal specimens and !2% weak d donors). for the intended use of the product, weak d serotyped donors were enriched (n5160, 16%). commercial serology tests for d antigen predicted phenotype and bi-directional-sequencing (bds) for weak d type confirmation and hpa-1 predicted phenotype were used for comparison. transfusion results/finding: no system failure, 100% call rate and no inconclusive results were obtained. discrepancies were found for d antigen between serology and id rhd xt predicted phenotype results, although a 100% concordance was obtained when analyzed by bds, considering id rhd xt result correct. concordance between id rhd xt and bds results for the weak d type was 100%. the following id rhd xt predicted phenotype results were obtained: d negative (n5361), no amplification variant (n515), weak d type 1 (n522), weak d type 1 heterozygous (n51), weak d type 2 (n532), weak d type 2 heterozygous (n51), weak d type 3 (n534), weak d type 3 heterozygous (n51), weak d types 1, 2 or 3 not detected (n5533). regarding hpa-1 blood group, the predicted phenotype results obtained by id rhd xt were 100% concordant with bds results: hpa-1a positive (n5157) and hpa-1a negative (n56), hpa-1b positive (n546) and hpa-1b negative (n5117). conclusion: id rhd xt genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence rhd negative and weak d types (100% specificity and 100% sensitivity for d antigen, hpa-1a and hpa-1b antigens). that makes it a useful tool for the implementation of the rhdgenotyping recommendation on patient blood transfusion and anti-d prophylaxis. background/case studies: scd patients form red blood cell (rbc) antibodies at higher rates than other transfused populations. multiple predictors of alloimmunization have been reported but not well replicated in large scd cohorts. we investigated the clinical, laboratory and genetic predictors of alloimmunization. study design/method: a large scd cohort was established in brazil to investigate disease outcomes. at participating sites, patients are currently transfused with abo/d/cc/ee/kell matched rbcs prophylactically and extended phenotypically matched rbc after first antibody forms. policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study. transfused subjects with 11 rbc alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and t-test or wilcoxon rank-sum tests as appropriate to compare continuous variables. backward elimination multivariable logistic modeling was used to generate odds ratios (or) and identify independent predictors of alloimmunization using results of univariate analyses. all subjects had peripheral blood whole genome snp typing performed using an affymetrix array, which included enhanced content for blood related snps. genome wide association (gwa) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization. a p value <50.05 (clinical analysis) or <5x10 -8 (gwa) was considered statistically significant. results/finding: of the 2795 cohort patients, 2272 (81.3%) transfused subjects were included with 129 alloimmunized children <18 years (11.0% of 1172) and 224 alloimmunized adults (20.4% of 1100). in multivariable logistic regression models, age (or 4.2, p50.009, for age 501 compared to 0-4), gender (or 1.3, p50.04, for female compared to male), transfusion history (or 3.5, p<0.0001, for 811 transfusions compared to 1-5), site, hemolysis (or 1.3, p50.05, for log transformed lactate dehydrogenase) and presence of autoimmune disorders (or 4.5, p<0.0001) were independent predictors of alloimmunization. gwa identified a single snp of unclear biologic significance associated with alloimmunization (eefsec gene responsible for incorporation of selenocysteine into proteins). conclusion: rbc alloimmunization is primarily driven by transfusion burden in this scd cohort. hemolysis remained significantly associated with alloimmunization after controlling for transfusions. presence of an autoimmune disease was also associated with rbc alloimmunization, indicating more systemic immune dysregulation may be present in scd patients who develop rbc alloantibodies. however, the gwa did not identify snps in immunoregulatory genes significantly associated with rbc antibody formation in the study population. background/case studies: physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests. to reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing. mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage. there has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice. study design/methods: transfusion service tested sample pairs from 32 newborns less than 2,000 gr birth weight. one of the samples was collected from the newborn as a heel stick sample, the other from the placenta. the following tests were performed on the sample pairs: abo, rh, antibody screen and direct antiglobulin test with igg (dat). results/findings: abo, rh and dat tests were performed on 32 sample pairs. dat test was negative on 30 sample pairs and two were positive. there was 100% concordance with the abo, rh and dat tests performed on these sample pairs. antibody screen was performed on 32 placental blood samples and 29 heel stick samples. twenty eight sample pairs were negative with the antibody screening test. there was one positive heel stick sample, which was also positive using the placental sample. one heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample. this antibody which was detected only in the placental sample was a passive anti-d mother received during pregnancy. this discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody. conclusion: this study shows that placental blood sample is not inferior to heel stick sample regarding abo, rh and dat testing. based on this comparison study placental blood can be used for pre-transfusion testing for < 2,000 g birth weight newborns. o-(7.4%), ab1(6.8%), b-(2.7%), and a-(0.6%). among the tested donors, 89.2% were d positive with r1r being the most common rh phenotype. in the kell blood group system, 4.5% of the donors were k positive, while the k antigen was found to be 99.4%. the most common phenotype in the duffy blood group system was fy(a-b-), while the fy(a1b1) was found at a higher frequency compared to what has been reported in the black population. (table) the commonest phenotypes for the kidd and mns blood group systems were jk(a1b1) and m1n-s1s1 at 47% and 22.6% respectively. the le a 1 and le b 1 alleles were seen in 21.7% and 67.3% of donors respectively, while lu b -phenotype was found in 3.3% of the donors. the frequencies of the rare phenotypes jk(a-b-), le(a1b1) and lu(a-b-) were 0.3% , 0.3% and 2.7% respectively, while the m1n-s-s-and m-n1s-s-phenotypes were not found. the frequency of the p1 antigen was found to be at 78.9% similar to what has been reported in caucasians. conclusion: this is the first study to examine the frequencies of rbc blood group phenotypes among the omani blood donors. the results show higher frequencies of the rare null phenotypes fy(a-b-), jk(a-b-) and lu(a-b-) compared to what has been reported in caucasians. the frequencies of the duffy blood group system resemble what has been reported in the black population. this data is helpful in understanding the influence of the arab ethnic background on the rbc blood group systems and warrants large genotype-phenotype studies in the region. quantitation of anti-d in serum using flow cytometry amanda whitelonis*, izekial butler, karen leighton, scott jones and anand srinivasan. qualtex laboratories background/case studies: rh(d) antibodies (anti-d) are developed in rhnegative individuals when exposed to d antigens. this scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients. quantitation of anti-d in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn. quantitation of anti-d is also performed in quality control operations of organ procurement organizations and plasma fractionators. it is a common practice to report the strength of anti-d in serum as antibody titer values but quality control operations require a quantitative value. we have developed a screening assay using flow cytometry to quantitate anti-d in serum. study design/method: we have developed a method to quantitate anti-d in serum using flow cytometry, by modifying the protocols of christensson et al., and hilden et al.. red blood cells from rh-positive blood samples were washed three times in phosphate-buffered saline (pbs) at ph 7.2 and the supernatant was discharged. a dilution buffer containing 2% human serum albumin (v/v) in phosphate buffered saline was prepared. serum samples or who anti-d standards, suspended in dilution buffer were mixed with 10ll of washed red cells. the cell suspensions were incubated for 30 min at 378c. following incubation, fitc-labeled anti-igg diluted in buffer was added and the mixture was incubated for an additional 15 min at 228c. the samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals. green fluorescence was collected using a band-pass filter set for 515-548nm. events were recorded at a frequency of 1000 cells. results/finding: multiple dilutions of who anti-d reference standard were tested against rh-positive red blood cells from five different donors. the reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition. after optimizing these factors, a linear regression was calculated to establish the standard curve. the fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of rh(d) antigens in reference standard. serum from thirty rh(d)-immunized volunteers were analyzed for concentration of anti-d and the results were benchmarked with antibody titer values. conclusion: based on our study, we conclude that the quantitation of rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-d antibodies in serum. the method is reproducible and advantageous over reporting antibody titer values. the operations of this platform can be translated to a well-plate based high-throughput flow cytometry. sarah k harm* 1 , mark yazer 2 , nancy m. dunbar 3 and biomedical excellence for safer transfusion (best) collaborative 1 . 1 university of vermont medical center, 2 university of pittsburgh, 3 dartmouth-hitchcock medical center background/case studies: the use of emergency issued group a plasma and uncrossmatched group o whole blood (wb) in patients without a valid abo group is becoming increasingly common in the usa. it is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established. this study was designed to determine the rate of high titer donors using a titer threshold of 50. study design/methods: three academic hospitals that routinely issue group a plasma units for emergency issue participated in this study. before issuing this plasma to patients, a 1:50 dilution of the donor's plasma in saline was produced and added to group b reagent red blood cells (rbc). if any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group a or o recipients. at these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added. at one center samples were taken from the plasma of group o wb units and the same procedure was followed using a1 and b reagent rbcs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the wb unit was considered high titer and it was then centrifuged into an rbc unit for transfusion while the plasma and platelet components were discarded. two centers provided plasma testing data for a 4-year and 5-year period, respectively. one center provided plasma and wb testing data for a 1-year period. results/findings: in total there were 7106 group a plasma units tested and 654 (9.2%) had a high titer anti-b. the range of high titer group a plasma units between these three centers was 7.4%-12.5%. of the 1778 wb units tested, 388 (21.8%) units had a high titer; 221/1778 (12.4%) of the units had a high titer anti-a, 61/1778 (3.4%) had a high titer anti-b, and 106/1778 (6%) had high titers of both anti-a and anti-b. background/case studies: dithiothreiol (dtt) is a sulfhydryl reagent that denatures selective blood group antigens. reagent red blood cells (rbcs) treated with 0.2m dtt is used as a tool in identifying antibodies to high frequency antigens. recently, dtt has become widely used in destroying cd38 on reagent rbcs and render them free from plasma anti-cd38 drug interference. procedures for the preparation of 0.2m dtt has been published advocating the use of buffered saline at different ph levels. in this study, an effect of ph on 0.2m dtt treatment time is investigated. study design/methods: non-buffered saline (nbs, thermo fischer scientific inc, middletown, va), used in the preparation of 0.2m dtt, was adjusted to ph7.16, ph 7.56, ph 7.96 using sodium phosphate dibasis (sigma aldrich, saint louis, mo). reagent rbcs (immucor, norcross, ga)(n53) were treated with the 0.2m dtt solutions in parallel by mixing 1:4 ratio of packed rbcs to 0.2m dtt solution followed by incubation at 378c. for up to 45 minutes during treatment, the expression of k antigens was measured every 5 minutes by tube method using 2 different sources of anti-k. to assure uniformity, all reactions were graded by the same investigator. each reaction grade (in each rbc and each antiserum) is converted into a semiquantitive score and an average score was calculated every 5 minutes for each ph level. the reduction in average scores between different phs were also calculated at every 5 minutes to measure the impact of 0.2m dtt reagent ph on the rate of k antigen destruction. results/findings: the expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by !11) after 15 minutes of dtt treatment at ph 7.96; 15 minutes at ph7.56 and 20 minutes at ph7.16. complete loss of k expression was seen after 25 minutes of dtt treatment at ph7.96; 35 minutes at ph7.56 and 35 minutes at ph 7.16. the reactivity patterns of k antigen tested with 2 sources of anti-k correlate with each other. the reductions in average scores were seen between 15 to 30 minutes range of dtt treatment time when ph 7.16 was raised to ph7.56; 15 to 30minutes range when ph7.56 was raised to ph7.96; and 15 to 30 minutes range when ph7.16 was raised to ph7.96. conclusion: the use of higher ph buffered saline may shorten the treatment time it takes to weaken or destroy k antigen. based on the comparison of reaction scores between different ph levels, the ph levels did not have an impact on dtt treatment up to 15 minutes and/or beyond 35 minutes of incubation. the ph of the 0.2m dtt reagent relative to the treatment time is a factor to consider during the validation of dtt-treatment process and qualification of 0.2m dtt reagent in a laboratory. background/case studies: data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the united states. the aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental united states. study design/ method: the aim of this retrospective study was to evaluate a cohort of prenatal patients (n 5 756,221) drawn between july 1, 2013 and june 30, 2014. these patients were divided into united states census bureau regional and divisional categories according to their place of residence. prenatal blood work was collected which included an abo, rh(d) and a screen for unexpected alloantibodies. samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification. results/ finding: in total, 11,647 patients were found to possess clinically significant red cell antibodies for an overall incidence of 1.5 percent. the three most commonly encountered antibodies were anti-d (n 5 7639) 63.1%, anti-m (n 5 1288) 0.6%, and anti-e (n 5 1227) with a frequency of 10.1%. a total of 455 (3.9%) prenatal women were found to possess two or more antibodies. in general, the combination of anti-d and anti-c proved to be the most common, with 182 instances (40.0%) followed by anti-e and anti-c with 79 (17.4%), and anti-c, anti-e with 26 (5.7%). of the multiple antibodies identified, 435 (95.6%) included at least one antibody from the rh blood group. the south region had the largest number of antibodies identified with 7474 or 61.8% of the total. the west had 2111 (17.4%), the midwest 1538 (12.7%) and the northeast with 979 (8.1%). a contingency table, using the two-sided fisher's exact test, was performed comparing the northeast, south, midwest and west regions. the p value of anti-d was calculated to determine nonrandom associations and values of 0.05 and below was deemed significant from a region-to-region perspective. with regard to anti-d, the pacific division comprised of california, oregon, washington, and alaska, had p values below the 0.05 thresholds when compared against seven of the eight other divisions. the west south central division (texas, oklahoma, arkansas, and louisiana) did not show statistically significant results when compared against the pacific division (p 5 0.17). conclusion: depending upon the antibody, statistically significant variations between geographical regions and divisions within the united states were observed. this relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations. reduction in repeat testing using gel technology amy mata* 1 , lindsy rich 1 , sherry stern 1 , sharon wangen 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: our institution currently uses the immucor neo (immucor, inc., norcross, georgia) to perform abo/rh and antibody screen (absc) testing utilizing solid phase technology. when results are unable to be obtained from the immucor neo, testing is repeated on the manual testing bench using tube agglutination. this repeat testing can lead to significant expenses including reagents, supplies, and technologist time. it was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard. a side-by-side evaluation was performed between the immucor neo and the ortho vision (ortho clinical diagnostics, rochester ny) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed. the evaluation looked at abo/rh and absc testing as those are the only tests that are currently automated in our laboratory. study design/method: thirty specimens that were processed on the immucor neo and resulted in no type determined (ntd) for abo/rh testing were selected to be tested on the ortho vision. twenty-three specimens that were processed on the immucor neo and produced positive results for absc testing were selected to be tested on the ortho vision. all specimens were edta tubes and were collected within the previous 4 days. the timeframe between when the specimen was tested on the immucor neo and the ortho vision was 1 to 2 days. results/finding: of the 30 ntd specimens from the immucor neo, 8 resulted in valid abo/rh typings on the ortho vision. three results were flagged indicating possible extra reactivity. upon performing a visual review of all 3 results, it was determined that there was no reactivity and a valid result was present. the other 19 samples required manual tube testing to interpret the abo/rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis. of the 23 absc specimens that were resulted out as positive on the immucor neo, 11 specimens produced a negative result on the ortho vision and were confirmed to be negative with manual tube testing using peg as the enhancement media. one specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative. nine specimens that were positive on the immucor neo were also positive on the ortho vision. one specimen proved to be an anti-m that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out. all showed discrepant results with monoclonal anti-c reagents, with a similar pattern of reactivity: 3-41 with ms24 (n515), 1-31 s with ms23 (n59), no reaction with ms273, dgc02, p3x255 (n514). 14 samples tested with a polyclonal anti-c showed a 1-31 reactivity. 3 d1c1e1c1e1 cases tested with a polyclonal and monoclonal anti-e (ms16, ms21, ms62, ms63) showed no weakened reactivity. rhce sequencing (genomic dna or cdna) showed a c.286g>a mutation in exon 2, predicted to encode the p.gly96ser substitution. for 2 apparent r 1 r 2 donors, a f-negative type allowed the prediction of a rhce*ce286a/rhce*ce genotype. altogether, our results are consistent with the presence of a very likely rhce*ce286a allele (c and e in cis) in all samples. 3 d1c1e1c1e1 individuals were reactive 11 s with the original source of anti-rh55, slightly weaker when compared to rhce*ce286a/rhce*ce rbc samples available from our cryobank (21). conclusion: our results confirm that the c.286g>a mutation alters the conformational properties of the rhce protein, either on a ce or ce background, and encodes the low-prevalence locr antigen (rh55). the locr reactivity appears to be rather similar when coded by rhce*ce286a or rhce*ce286a alleles. this was quite an unexpected finding, since the p.gly96ser substitution is close to the critical amino-acid for c/c expression (p.pro103ser). none of our 15 cases made anti-c and/or anti-e but few were subject to a potential alloimmunization background. however, as rhce*-ce286a was reported to code for a partial c (rh:-26), we consider that rhce*ce286a likely encodes partial c and e, this being also supported by the predicted localization of the p.gly96ser change on the second extracellular loop of the rhce protein. background/case studies: weak d genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a rhd typing discrepancy, or a serological weak d phenotype, to determine if they carried the weak d genotypes 1, 2 or 3. the purpose of this study was to analyze the underlying rhd genotypes of the patient samples received for weak d genotyping since published recommendations, in particular those found to not carry the weak d 1, 2, or 3 genotypes. study design/methods: between 9/2015 and 2/2017 50 samples were received for weak d genotyping. testing was performed using pcr-rflp targeting the sequence variants in the rhd gene that have been previously defined. samples that did not have weak d types 1, 2, or 3 genotypes, but 156a transfusion 2017 vol. 57 supplement s3 had evidence of rhd genetic sequences in exon 7 and/or intron 4 in preliminary testing were evaluated by sanger sequencing for rhd and rhce exons 1-10 to determine the underlying rh genotype. when provided, the patient's ethnicity and presence of anti-d was recorded. results/findings: the majority of the samples were from obstetrical patients (62%) followed by transfusion patients (28%); 10% had no clinical indication provided. 34 samples (68%) were found to be weak d type 1, 2, or 3 (24, 6, and 4 samples, respectively). 5 samples (10%) appear to be genetically rhd negative. genetic sequencing was performed on 11 samples; 9 had rhd genetic variants that were not weak d types 1, 2, or 3 (table) . all of these variant rhd samples also showed some variation in the rhce gene. two samples (4%) had wild type rhd alleles; further evaluation is ongoing. conclusion: most samples tested by weak d genotyping were found to be weak d types 1-3. of the 11 samples that had evidence of an rhd gene and did not carry the known weak d types 1-3 polymorphisms, 9 (82%) of were found to have other rhd variants, and 2 (18%) did not have underlying genetic variation detected in the rhd gene. the majority of the non weak d types 1-3 variants were dar alleles, which are often associated with anti-d production. background/case studies: rhd genotyping has been recommended to guide transfusion of d-negative rbcs and administration of rh immunoglobulin to patients with discordant or weaker than expected d typing, particularly for females and ob patients . the recommendation is based on observational evidence, primarily from europe (flegel 2006, curr opin hematol13:476) , that individuals with weak d types 1, 2, and 3 are not at risk for clinically significant anti-d. the implications and utility of this approach for the diverse u.s. population are not yet clear. here we report 15 months experience with rhd genotyping on 352 samples referred with discrepant or weak d typing investigated from january 2016 to april 2017. study design/method: serologic testing was performed by standard tube agglutination with licensed reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assays and rhd sequencing for some. ethnicity was known for 153 samples (53.3% caucasian, 32.2% african american/african, 6.6% multiracial, 4.6% hispanic, 2% asian, and 1.3% other). results/finding: rhd genotyping identified weak d types 1, 2, and 3 in 155/352 (44%) and alleles known to encode partial d phenotypes in 168/ 352 (47.7%) (table) . uncommon or rare weak d alleles including types 6, 15, 40, 42, 45, 51, 57 (n52), 59, 61, 78, 91 , and 119 were found in 13 (3.7%). the partial d alleles found were diverse, but the largest number included partial rhd*d 4.0 (n562) and *dar ( conclusion: in a multiracial cohort of 352 individuals with weaker than expected d typing 44% were due to weak d types 1, 2, or 3 and would not be considered at risk of clinical significant anti-d, but for 56% there is potential or unknown risk. these studies are important to gain insight into the prevalence of specific alleles in the u.s. multiethnic population and to continue to evaluate and refine rhd genotyping for clinical practice. cp242 rhd*07.02 allele causes discrepant genotyping results for rhce small c sabine scholz* 1 , sandra schneider 1 , sabrina k€ onig 1 , susanne helmig 1 and vicky van sandt 2 . 1 inno-train diagnostik gmbh, 2 rode kruis vlaanderen background/case studies: in the human rh blood group system the c, c, e, e and d antigens are expressed by the two highly homologous genes rhce and rhd. after d, c is the most immunogenic rh antigen. the difference between c (307c) and c (307t) is caused by the snp on position 307 on the rhce gene. the rhd*07.02 allele (also known as rhd cat vii type 2) carries the snp 307t>c on the rhd gene and additionally the snp 329t>c. this rhd*07.02 allele has been described to partially express rhc on the d polypeptide (faas, transfusion, 2001) . aims: genotyping was performed to clarify the cause of the weak c expression. serology of a patient sample (male, 81938) indicated a partial c phenotype with a cde. study design/method: rhd and rhce phenotyping was done by accredited routine protocols (monoclonal ab id card: diaclon rh subgroups, seraclone anti-c). genotyping was performed with a taqman probe assay (rbc-fluogene veryfy, inno-train diagnostik gmbh), sso (rbc-lifecodes, gen-probe inc.), in-house ssp-pcr (hila, rode kruis-vlaanderen) and commercial ssp-pcr (rbc-ready gene cde, inno-train diagnostik gmbh). sanger sequencing of the rhd gene was performed using an inhouse method (inno-train diagnostik gmbh). results/finding: discrepant genotyping results were generated by different test systems: the taqman probe based assay showed in repetition a ccee genotype, while the sso system rbc-lifecodes predicted in repetition a ccee phenotype. in ssp-pcr the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. the parallel analysis of the rhd gene with rbc-ready gene cde test system revealed a variant d cat vii rhd allele. sequencing of the dna sample identified two snps on one of the rhd alleles (307t>c, 329t>c) confirming a rhd*07.02 and one rhd*01 allele. hispanic female in preparation for surgery resulted in variable reactivity and weakly positive d reactions when using microtiter-well agglutination versus tube testing. determination of whether the d antigen expression represented a weak d or a variant d could not be resolved by serologic testing alone. here we report the characterization of a novel rhd gene mutation identified by rhd gene sequencing. study design/method: serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms galileo neo and galileo echo (immucor, norcross,ga) and by standard tube testing using the immucor series 4 and 5 anti-d reagents. further immunohematologic evaluation was performed by standard tube testing (immediate spin -is, and indirect antiglobulin -iat) using orthobioclone, immucor gammaclone, immucor series 4 and series 5, and albaclone anti-d reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assay (immucor, bioarray) and rhd sequencing. results/finding: rbc reactivity is summarized in the table. dna testing detected a hybrid rhesus box associated with the rhd gene deletion, indicating the patient was hemizygous for rhd. rflp assay and rhd beadchip did not identify any changes. rhd gene sequencing identified a new c.463a>g change in exon 3 encoding an amino acid change p.met155val. the predicted location of this change is within the fourth transmembrane segment of the rhd protein. conclusion: we identified a novel rhd allele with c.463a>g (p.met155val) change in exon 3. several snps, deletions, and insertions have been reported with changes in exon 3. phenotypes of these genetic variations result in rh negative, weak d types, and variant d. since this change has not been previously identified, we are unable to determine if this confers a risk of anti-d alloimmunization, but the rhd c.463a>g snp results in serologically weak phenotypic expression of d antigen when tested by microtiterwell agglutination on the neo/echo platforms. in this case the combination of microtiter-well agglutination and dna sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays. serologic and molecular detection of an antibody to a high incidence antigen in patient with history of chronic transfusions georgia spanos* 1 , juan merayo-rodriguez 2 , christopher lough 1 and nancy eckert 3 . 1 lifesouth community blood centers, 2 life south community blood centers, 3 lifesouth community blood center-headquarters background/case studies: the jo a antigen is one of three high incidence antigens in the dombrock system. the prevalence of this antigen is 100% in most populations and greater than 99% in the black population. the jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with dithiothreitol (dtt), w.a.r.m. tm (immucor) and zzap treatment. anti-jo a is an igg antibody that demonstrates at ahg phase. hemolytic transfusion reactions to the jo a antigen vary from none to moderate/severe. hemolytic disease of the fetus and newborn (hdfn) has not been observed with any antibody associated in the dombrock system. there are two common phenotypes present in the black population:hy negative/ jo a negative and hy weakly expressed/jo a negative. study design/methods: an antibody identification and red blood cell (rbc) units were requested for an o positive, 57 year old, african-american female with a history of sickle cell disease and no history of pregnancy. the patient was not recently transfused, however, had a history of chronic transfusions. last reported transfusion was three years prior to the current specimen. there were no known rbc antibodies at the time of the request. facility reports that the patient's hemoglobin(g/dl)/hematocrit(%) (hgb/hct) is 6.4/ 18.8 and does not appear to be in sickle cell crisis. a request for phenotypically matched units, as per hospital policy, for c, e, k and s was received by our immunohematology reference laboratory (irl). results/findings: anti-jo a was detected in patient plasma reacting with liss and peg (tube method) and manual gel-iat. the antibody was resistant when tested with dtt treated red cells. in-house frozen reagent rbcs negative for the jo a antigen (positive for hy) were used to serologically prove the presence of the antibody to this high incidence antigen. an allogenic peg adsorption was performed to rule out other common clinically significant antibodies. anti-kp a was identified using this adsorbed plasma. further testing with molecular genotyping (grifols idcore xt ) confirmed the patient's genotyping as antigen negative for the jo a , kp a and positive for hy. conclusion: molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout florida, georgia and alabama. staff is able to search our database for any combination of antigen negative phenotypes using the internal 510(k) blood establishment computer software (becs) integrated blood bank information system (ibbis). this enabled us to locate one refrigerated and three cryogenically preserved jo a negative rbc units. we found 251 eligible blood donors that could be recruited via an automatically generated call list. the request for rbcs was cancelled. patient's clinical symptoms improved without transfusion and repeat hgb/hct increased to 7.1/21. the patient's sibling is historically negative for the jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf. in order to continue having blood components available to meet all our patient's needs, irl staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion. rbcs of two females whose samples were referred for rhd genotyping with previously reported alleles for which serologic reactivity had never been investigated. study design/method: serologic testing was performed by automated analyzer, galileo echo and neo (immucor, norcross, ga), and by standard tube testing with licensed anti-d reagents and the albaclone advanced partial rhd typing kit. genomic dna isolated from wbcs was used for immucor rhd beadchip assay, pcr-rflp, and rhd sequencing. results/finding: patient 1 was a 29 yo female, c2e2c1e1, whose rbcs reacted 11 by echo and 31 by neo with anti-d4, and '?' with anti-d5. testing with d4 and d5 by tube gave 21 and 11 w on initial spin (is) respectively and 41 by indirect antiglobulin test (iat). rbcs were non-reactive at is with ortho bioclone and biorad seraclone, and 1 w with immucor gammaclone anti-d, and all were 21 at iat. rbcs did not react with two (lhm 174/102 & 57/17) of 12 anti-d in the alba partial d kit. this pattern did not match any partial d identified by these clones. rhd beadchip detected an inactive rhd pseudogene in trans to rhd. gene sequencing confirmed the presence of the pseudogene, but rhd had a c.780c>a change encoding p.his260gln. patient 2 was a 20 yo pregnant female, c2e2c1e1, whose rbc were 1 w at is and 31 at iat with immucor series 4 and 5 and gammaclone, and moderately reactive, 21 is and 41 iat, with alba alpha, alba blend and delta anti-d. rbcs did not react with two (lhm 174/102 & 170/45) anti-d in the partial d kit with no known partial d pattern. dna testing predicted she was rhd hemizygous and rhd beadchip detected markers for rhd*dar but exon 2 gave low signal (ls). sequencing found a hybrid dar with ce-specific nucleotides in exon 2 from c.150 to c.203 encoding amino acid changes p.ile60leu and ser68asn. conclusion: we found two previously reported rare alleles: rhd with a c.780c>a (p.his260gln), previously found in france (lefloch et al. genbank ku363612), and rhd*dar with part of exon 2 replaced by rhce, reported in sub-sahara africa (granier et al. transfusion 53:3009) designated rhd*dar(ce2:v505v-s68n) with an allele frequency of 0.002 to 0.016. blood samples were not available to test for alterations in d expression for either allele. we provide serologic evidence that these alleles, found in two females evaluated by rhd genotyping, inform transfusion and rh immune globulin prophylaxis, as they encode partial d phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-d. background/case studies: hu5f9-g4 is a human monoclonal igg4 antibody recognizing cd47 that is in clinical trials to treat hematologic or solid malignancies. cd47 is a transmembrane glycoprotein that binds to signalregulatory protein a (sirpa) on macrophages and functions to regulate phagocytosis. blocking cd47 is thought to enhance phagocytosis and promote anti-tumor responses. cd47 is also highly expressed on rbcs, and the purpose of this study was to evaluate anti-cd47 drug interference in blood bank testing. study design/method: serologic testing was performed by standard methods. serial samples (n57) from 2 patients were tested over the course of 1 month treatment. plasma was tested at immediate spin (is) and by iat with r2r2, rr, d--, rh mod and rh null rbcs, as cd47 expression levels vary depending on rh phenotype. dtt and enzyme treated rbcs were also tested. both immucor gamma-clone anti-igg (does not detect igg4) and ortho bioclone anti-igg (total igg) were used. for titration plasma was diluted in pbs. allo-adsorptions were performed with papain treated rr rbcs and eluates were made using gamma elu-kit ii. results/finding: anti-cd47 was observed in plasma as soon as 1 hour post infusion. plasma reacted 31 to 41 at is and 41 with all panel cells in peg iat using ortho anti-igg. d--, rh mod and rh null rbcs were nonreactive at is and weaker (31 and 21) in peg iat with ortho reagent. reactivity with all panel cells by ortho igg gel card was 31. in contrast, iat reactivity using gamma-clone anti-igg was only 1 w to 11, and this reactivity was confirmed to be carry-over agglutination. d--, rh mod and rh null were non-reactive in peg iat using gamma-clone anti-igg . the anti-cd47 titer was 1 at is and peg iat with gamma-clone anti-igg, but was ! 256 with ortho anti-igg. plasma reacted with dtt, trypsin, papain, a-chymotrypsin or w.a.r.m. treated rbcs. somewhat unexpected, autocontrols were negative and dats were non-reactive or microscopic only. acid eluates (n54) were 31 reactive with ortho, and non-reactive with gamma-clone anti-igg. plasma reactivity was removed after 4x allo-adsorption with papain treated rr cells, but in some samples low level (micro-11) reactivity remained. peg adsorption was invalid due to precipitation/complexing of antibody. robust plasma reactivity interferring in abo reverse typing was observed, and weak spontaneous agglutination of the rbcs in the abo forward and rh typing. conclusion: hu5f9-g4 anti-cd47 therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but abo and rh typing. high levels of cd47 expression on rbcs results in plasma agglutination at is, mimicking reactivity observed with igm antibodies although hu5f9-g4 is igg4. reactivity was observed in all phases and with all test methods. cd47 is not cleaved from rbcs by dtt, trypsin, papain/ ficin, dtt with ficin (w.a.r.m.) or a-chymotrypsin, and treatment of rbcs with these does not mitigate interference. numerous adsorptions with papain treated rr rbcs were required to remove anti-cd47 reactivity from plasma. use of immucor gamma-clone anti-igg, which does not detect igg4, can mitigate interference in iat although carryover reactivity may be observed. due to blocking by anti-cd47 on the patient rbcs, dat and autocontrols were weak or non-reactive; however eluates prepared from the dat1 rbcs were strong and pan-reactive using ortho anti-igg. background/case studies: a caucasian woman with history of a caesarean section and a rbc tx in 1985. in august 2016, she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions. five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation. ab screening was now positive, with an antibody reacting with all panel cells detected. because of the urgent need for rbc tx, two weakly cross-match positive rh1k matched units were transfused with a warning of possible alloantibodies. the patient got acute hemolysis. study design/method: a gel technique was used in the hospital transfusion laboratory. in addition, various antibody identification panels and special serological and genotyping methods were used in the reference laboratory. kel sequencing was done by the international immunohematology center. results/finding: the hospital transfusion laboratory results were o rhd neg, dat neg, and the ab identification was 21 with untreated and 31 with enzyme-treated cells, with weakly positive autocontrols. a sample was submitted to the reference laboratory for additional investigation. dat was weakly positive, while ab identification results were similar to the hospital results. different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups. after pk, vel neg, jk:-3 etc. had been excluded, k-phenotyping revealed a k 0 -phenotype. a total of 38 silencing mutations are known for the kel gene and the genotyping kits used did not recognize these. the anti-ku antibody reacts with all cells apart from the k 0 -phenotype. the presence of dtt-sensitive anti-ku was confirmed with dtt-treated panel cells. anti-ku may cause immediate and delayed hemolytic transfusion reactions. samples were taken from the patient's two siblings and daughter. kel sequencing revealed kel*02n.19 with c.2023t encoding p.675ter (reported in an individual from austria in 2007). there are two known k 0 -patients in our country, both homozygous for c.2023t. the daughter was a c.2023t heterozygote, while the siblings did not have this variant. a new operation is necessary but no k 0 -donors are available in our country. with the help of the isbt rare donor working party, a k 0 o rhd neg donor was found in japan and one unit was delivered to us for use in the next operation. conclusion: an alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct coombs is positive. a combined serological and genotyping approach offers the best solution for problematic antibody cases. compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor. transfusion strategy for the serologic weak d phenotype in tunisia based on rhd alleles and rh haplotypes mouna ouchari* 1 , kshitij srivastava 2 , houda romdhane 3 , saloua jemni yacoub 4 and willy albert flegel 1 . 1 nih, 2 dtm/cc/nih, 3 regional blood transfusion center sousse, 4 regional blood transfusion center sousse, tunisia background/case studies: d antigen variants have been studied molecularly in many arab populations, including gaza, tunisia, egypt and libya, 159a transfusion 2017 vol. 57 supplement s3 since 2009. the tunisian population has the largest known prevalence of weak d type 4.0 alleles, occurring in 1 of 105 rh haplotypes, compared to 1 in 6,060 or less in europe. a systematic study was missing for samples with the serologic weak d phenotype routinely found in blood donor and patient testing in tunisia. the study was designed to obtain data on weak d type 4.0 in a population known to harbor the greatest prevalence of such allele worldwide. study design/methods: a total of 13,431 random blood donors were serologically screened for the d antigen using 3 routine techniques. samples with weak reactivity were tested with a panel of 6 monoclonal anti-d (partial rhd-typing set) to identify partial d phenotypes. the rhd gene was sequenced in all samples with serologic weak d phenotype. the rhce gene was also tested molecularly by either direct sequencing or using the rhce beadchip kit to ascertain the rhce allele linked to the rhd allele. results/findings: a total of 67 discrepant samples (0.5%) were observed and expressed the serologic weak d phenotype. among them, 60 carried an allele of the weak d type 4 cluster (89.6%), of which 53 samples (88.3%) showed the weak d type 4.0 allele. only 1 sample each was found for the weak d types 1, 3 and 100 and the dvii, while 3 samples showed the consensus rhd sequence. no mutation in any of the 10 rhd exons was detected in another 3 samples. the molecular analysis of the rhce gene showed that 59 out of 67 samples with serologic weak d phenotype (88.06%) had a variant rhce allele and the most common associations were: weak d type 4.0 linked to rhce*cevs.04.01; weak d type 4.2.2 with cear; and weak d type 4.1 to rhce*cevs.02, while the other rhd alleles were linked to one of the common rhce alleles. conclusion: almost 90% of the weak d phenotypes in tunisia were caused by alleles of the weak d type 4 cluster, of which 88% represented the weak d type 4.0 allele. based on established rh haplotypes for variant rhd and rhce alleles and the lack of adverse clinical reports in tunisia, we recommend d positive transfusions for patients and no rhig administration for pregnant women with weak d type 4.0 in tunisia. we propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-d immunization would occur in tunisia associated with weak d type 4.0 phenotype. there is a possibility that the rhce*cevs.04.01 allele, typically associated in tunisian individuals, may protect from allo-anti-d immunizaton and other rhce alleles, such as rhce*ce more often associated in individuals of other ethnic groups, may not. however, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations. martha rae combs* 1 , heather simmons 1 , christine lomas-francis 2 , gayane shakarian 2 , sunitha vege 2 , lauren hutelmyer 3 , sandra nance 4 , jessica poisson 1 , nicholas bandarenko 1 and connie m. westhoff 2 . 1 duke university hospital, 2 immunohematology and genomics laboratory, new york blood center, 3 arc pennjersey, 4 american red cross, immunohematology reference laboratory, biomedical services background/case studies: plasma from a transfused, a1, 2 year old white female, post liver transplant with rbc aplasia, reacted at rt and in peg iat with all rbc samples tested except her own. study design/method: standard hemagglutination methods were used for antibody id and antigen typing. acid eluates were prepared using gamma elu-kit ii (immucor). genomic dna was isolated from wbcs and used for hea precisetype array and kel and sc gene sequencing. samples from the proband and her mother were tested, as applicable. results/finding: the patient's dat was negative. her plasma reacted with 0.2m dtt-treated and papain-treated rbcs, all available rbc samples lacking high-prevalence antigens, and with phenotypically similar rbc samples [c2, k2, fy(a2),s2]. reactivity was detected to a titer of 64; it was not removed by prewarm technique or by 4x peg alloadsorption. the adsorbed plasma reacted with 0.2m dtt-treated rbcs. extensive rbc phenotype results were unremarkable except for the following: k2, k2, js(b2), kp(a2b2) and sc:21,23. her plasma reacted with k o , mcleod, sc:21,22 rbc samples and dtt-treated sc:21 rbcs at rt and peg iat but her diluted plasma and pretransfusion eluate showed relative kp b specificity. the patient was transfused 4 aliquots of crossmatch incompatible kp(b2), s2 rbcs. her post-transfusion dat was 21 with anti-igg, 11 with anti-c3d. the eluate reacted with all rbc samples except 1 kp(b2) sample. she tolerated additional aliquots from 4 phenotypically similar rbcs untested for high-prevalence kell or scianna antigens. the hea precise-type predicted k2, k1, kp(a2b1), js(a2b1) and sc:1,22, discordant with her rbc phenotype. kel gene sequencing identified a homozygous change, c.1481a>t (p.glu494val) (kel*02.10) encoding the low prevalence antigen, ul a , but no changes associated with lack of kell system antigens; however, her rbcs typed ul(a2). sc sequencing found heterozygosity for a 5'-2g>a change (rs12124733, 24 to 30% prevalence) and conventional sc*01, predicting sc:1,22,3. kel and sc results on the mother were kel*02/kel*02.10, heterozygous for the sc change 5'-2g>a, and her rbcs typed k2k1 kp(a2b1), sc31, ula1, consistent with dna predictions. plasma collected 7 months later was nonreactive at rt and in peg iat. her rbcs were dat2 and now typed k1, kp(a2b1), ul(a1) sc11 and sc31,concordant with predicted kell and sc phenotypes. conclusion: we report an example of kell and scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the kel system. to our knowledge, this is the first report of a ul a kel*02.10 homozygote. the rbcs may lack a high-prevalence antigen antithetical to ul a . without dna testing and gene sequencing, the patient would be presumed to have kell null and sc null phenotypes, a search for k o , and/or sc:21,23 rbc units would be performed and we would not have been prompted to re-type her rbcs when the dat was negative. background/case studies: anti-jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction. in nearly all cases, this antibody is identified in the context of a phenotypically homozygous jkb patient, jk(a-b1). other scenarios are quite rare. we present two cases of anti-jka in which this phenotype was not observed. study design/method: patient a is a 55-year-old multiparous female with no known transfusion history. her blood typed as o positive with a positive antibody screen, negative dat, and a clearly identified anti-jka in plasma. the patient phenotyped as jk(a-b-). genotyping revealed the presence of the jk*b allele, but not the jk*a allele. complete sequencing of the jk gene showed an intron 5 polymorphism in homozygosity. specifically, the patient showed a jk*b(ivs5-1a)genotype, associated with a jkb null phenotype. anti jk3 was not identified. the conclusion was an allo-anti-jka in a jk null patient. the patient did not receive any transfusions. patient b is a multiply transfused 64 old female. her blood typed as a positive with a positive dat and antibody screen. both the plasma and eluate revealed an anti-jka. despite the recent transfusion, the patient phenotyped as jk(a1) 41 and jk(b1) 21. genotyping showed the presence of both jk*a and jk*balleles. whole gene sequencing was not performed. there was no hematologic or biochemical evidence of hemolysis. the patient was considered to have an auto-anti-jka and jka negative cells used for transfusion. results/findings: patients a and b both developed anti-jka while having uncommon phenotypes/genotypes. conclusion: it is common for jk null patients to develop anti-jk3. however, we speculate that expression of the kidd glycoprotein with the jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-jk3 or anti-jkb. auto-anti-jka is usually reported in the context of an active hemolytic process, but patient b illustrates an auto-anti-jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for rh epitopes. these rare cases of anti-jka require phenotypic and genetic analysis for the jkb epitope and jk*b allele respectively, and in more complex cases whole gene sequencing. background/case studies: donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories. package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification. these outcomes may go unrecognized unless serological results are available for comparison. study design/method: a routine blood donor, self-identified as african american, was selected for red blood cell genotyping. dna was extracted and genotyping was performed using two commercial platforms 160a transfusion 2017 vol. 57 supplement s3 (precisetype, bioarray, warren nj; idcore xt , grifols, emeryville, ca). genotype results were compared to historical serological results. discrepancies were resolved by sanger sequencing (grifols ih, san marcos, tx). results/finding: genotyping results showed variants in both the duffy (fy) and kell (kel) blood group systems. the donor's genotype was concordant on both platforms, fy*a/fy*b_gata, kp*a/kp*a, for a predicted phenotype: fy(a1b-); kp(a1b-). when genotype results were compared to historical serology, it was noted that the donor previously typed fy(a-) on 3 separate donations. no previous kpa or kpb serotyping was available. sequencing of fy exon 2 revealed a 287g>a mutation, fy01*n.04, known to silence fya. sequencing of kel exons 1-19 exposed a silent polymorphism in exon 8, 846g>c. this polymorphism causes a dropout artifact yielding a false negative kpb interpretation. conclusion: the discrepant fy*a result, as well as the unlikely kp(b-) type prompted the request for sequencing. the rare fy01*n.04 mutation has been reported in people of caucasian descent. this is the first example of this fy mutation identified in this regional population. the kpb antigen is present in nearly 100% of all populations. however, kp(b-) is most frequently seen in people of caucasian descent. to date, 64 self-identified african american donors have been genotyped as kp*a/kp*b at this blood center. given the diversity of regional heterogeneity, it is feasible to identify a kp(b-) donor, self-reporting as african american. red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization. donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types. in this case, a donor selected for genotyping based on ethnicity was initially thought to have 2 genetic variants not previously reported in those of african descent. only 1 was proven to be present. this case acts as a reminder that genotype limitations must be considered even when using licensed methodologies. this case report presents two group o pediatric patients who had been on enteral feeds and had absent/weak anti-b that became strong over time in patient 1. study design/methods: patient 1 was a 7 year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at 3 years of age. anti-b changed from undetectable/weak to strong at the age of 7 years. patient 2 was a 17 month-old female with a metabolic urea cycle disorder who underwent a liver transplant. anti-b was 0/11. both patients were on total parenteral nutrition (tpn) since birth and had strong anti-a and normal immunoglobulin testing. abo typing with enhancing techniques is presented in table 1 . results/findings: both patients typed as group o on forward typing. anti-a was strong in both patients. anti-b varied in strength in patient 1 with 0-11 reactions up to 7 years of age. thereafter, abo typing showed mainly strong anti-b. patient 2 had 0/11 anti-b. conclusion: intestinal bacteria stimulates production of anti-a and -b. unexpected changes in anti-b that caused abo discrepancies are reported here for 2 children on long-term tpn. patient 1 had absent/weak anti-b since birth up to 7 years of age, then developed strong anti-b with no change in feeding regiment and medications. patient 2 had consistently strong anti-a and absent/weak anti-b. these findings support the notion that normal colonization of the gut is important in the development of anti-a and -b and suggests that microflora of the gut in patients on prolonged tpn is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet. difference in strength of anti-a and-b could be due to stronger a than b antigen expression on gut bacteria. results/finding: a daratumumab protocol was established that incorporated use of the cord panel. multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, dat and genotype. after daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication. repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication. the cord panel ruled out underlying alloantibodies. selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity. conclusion: the cord panel was used 28 times over a five month period to rule out underlying alloantibodies. tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution. the daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner. teresa gorey* 1 and elizabeth hart 2 . 1 brigham and women's faulkner hospital, 2 university of massachusetts-dartmouth background/case studies: the purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies. several antibody detection methods (polyethylene glycol (peg), liss, and albumin) are routinely used in small transfusion services. the utility of peg is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure. the code of federal regulations, title 42, cfr part 493.1271(a), states the manufacturer's instructions are followed when testing for unexpected antibodies. the package insert for gamma peg tm (immucor inc., norcross, ga), states that negative reactions may be examined with an optical aid. based on these directions, our institutional policy is to confirm all negative reactions using the microscope. study design/method: a one-year retrospective document review was performed on all patient samples in which a positive antibody screen (absc) triggered the antibody identification (abid) to be performed in 2016. a total of 232 samples were evaluated. each abid was subcategorized; (1) as being a new antibody for our facility or in the patient's shared electronic health record within the partnersv r healthcare system and (2) whether a microscopic absc result triggered the abid. also, patients with known antibodies were grouped according to a microscopic absc result. a comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant. results/finding: a total of 83 abids were performed on new patient samples. of the new abid samples, 29 (35%) had microscopic absc results. for the previously known antibody patients, there were 35 which accounted for 15% of the total abids performed. when reviewing the total abid workups, a total of 64 (28%) of the abscs had microscopic results which resulted in an abid being performed. the antibodies identified in the 29 new antibody samples were: conclusion: a total of 86% of the new antibodies identified based on a microscopic absc were clinically insignificant. the manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results. due to the results of this study, a decision will be made to: (1) discontinue the use of the microscope, (2) switch to a peg manufacturer whose directions indicate to observe macroscopically for agglutination, or (3) define the use of the agglutination viewer as the optical aid. decreasing the number of abids will save time and money while providing potential rbcs for transfusion in a timely and efficient manner. anton") has a prevalence greater than 99% in all populations. hereditary absence of anwj has only been described once (in a single family). however, red cell expression of anwj may be markedly decreased to near undetectable levels in blood donors of the in(lu) (or "dominant lutheran inhibitor") phenotype. similarly, anti-anwj antibody formation is rare, with only 10 cases reported in the literature. the antibody developed in the context of hereditary absence of anwj (i.e., a true alloantibody) in only one of the cases. in the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient anwj antigen suppression. most of the reported cases lacked clinical or laboratory evidence of hemolysis. however, in the most recently reported case, involving a 56-yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after rbc transfusions, necessitating transfusion support with anwj-negative and in(lu) rbcs. the case was also unique in that the anti-anwj resulted in a direct antiglobulin test (dat) that was positive for complement only, rather than igg like all previous cases in which the dat was performed and was positive. study design/method: a 59-year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (ahtr) with development of a panagglutinin on indirect antiglobulin test (iat) screens. prior to identifying the specificity of the panreactive antibody, the patient received 10 rbc transfusions and showed signs of hemolysis with six of them. the first three transfusions were prior to her positive iat and were electronically crossmatched. the next seven transfusions were incompatible by antihuman globulin (ahg) phase crossmatch, but were extended phenomatched for clinically significant antigens. the patient's ahtr signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and ldh. the dat, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for igg only (1-21), and negative with anti-c3b, c3d reagent. the antibody showed a peak gel-igg iat titer of 32. results/finding: the antibody was identified as having anwj specificity. the patient's pre-transfusion sample showed weak anwj expression (w1), altogether suggesting an auto-anti-anwj. monocyte monolayer assay testing using the patient's plasma and rbcs from the ahtr-implicated units yielded monocyte indices ranging from 33 to 83%, consistent with the clinical hemolysis observed. given the patient's group o, rh d negative blood type and continuing transfusion dependence, in order to avoid further ahtrs, international collaboration was necessary in order to procure and provision group o, rh d negative rbcs that were also serologically negative for anwj. the patient was successfully transfused three such units without further incident. conclusion: this is the second documented case of anti-anwj in a patient with aplastic anemia and, overall, the third anti-anwj case associated with ahtr. this case also underscores the importance of international collaboration. cold auto-antibody 12 anti-p 1 4 anti-m 2 anti-sd a 6 anti-le b 1 anti-jk a 1 anti-k 1 anti-e 1 anti-c 1 results/finding: three hundred and ninety weak d genotypes have been determined to this day with frequencies of 21% (type 1), 5% (type 2), 9% (type 3), 25% (type 42) and 40% other than 1, 2, 3 or 42. further investigation was conducted to determine the molecular identity of the «others». out of 157 samples, 119 (75%) were confirmed to be legitimate serological weak or partial d, mainly deletions of exon 5 or both exons 4 and 5. a surprising amount of 38 samples were discovered to be normal rhd. conclusion: along with sandler et al. (2015) data, our findings highlight the difficulties hospitals face in interpreting serological weak d. trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point. altogether, our findings allow to share the frequency of weak d types 1, 2, 3 and 42 obtained in serological weak d, 45 years old quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical d typing. were classified as fnhtr. taco incidence was 0,6%. no trali happened in the period. prophylaxis were used in 98% of patients. conclusion: fnhtr is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions. fnhtr occurred 3 times less than allergic reactions. this might be explained by universal leukoreduction and universal prophylaxis adopted at our institution. further studies are necessary to evaluated the benefit of this approach. that cannot be associated with a specific rbc unit or were deemed unrelated to transfusion, 358 rbc transfusion aes were analyzed. chi-square test and logistic regression were used to compare the ae incidences among transfusion groups. results/finding: univariate and multivariate logistic analyses showed that irradiated rbcs were associated with a significantly increased incidence of transfusion-related aes (p <0.05). there was a significant difference in febrile non-hemolytic transfusion reaction (fnhtr) (0.27% vs 0.11%, p <0.001) or aes with a non-allergic type inflammation etiology (0.30% vs 0.14%, p <0.001) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated rbcs and non-irradiated rbcs. in contrast, the incidences of allergic aes (0.028% vs 0.024%, p 5 0.614) were similar between these two groups. the incidences of inflammation aes after transfusion of irradiated rbcs that were stored for 1, 2, 3, and 4 weeks were 0.25%, 0.32%, 0.39% and 0.41%, respectively (p 5 0.084, logistic regression) but there was a significant difference in the incidence of inflammation aes caused by irradiated rbcs stored for a week (0.25%) and longer than a week (0.35%) (p < 0.05). conclusion: irradiated rbcs associated with a higher incidence of transfusion inflammation aes compared to non-irradiated rbcs and this risk increased when rbcs were stored longer than 1 week after irradiation. while it is likely the patient population is a factor in ae caused by irradiated rbcs, it is also possible that rbc radiation damage, as shown in previous studies, contributed to this increased ae incidence. a list of patients with one of these icd10 codes was generated. the emr was searched to find the clinical scenario in which trali was mentioned. these patients' records were then searched within our laboratory information system (copath), to determine if they had a transfusion reaction reported to our transfusion medicine service. results/finding: the search of our electronic medical record found 11 patients from 2011-2016, who had trali mentioned in their chart as a diagnosis or possible/likely diagnosis. one patient was excluded from our study because trali was mentioned as a past medical history from an outside hospital. only the patients who had trali listed as a diagnosis or possible diagnosis were included in this study. these 10 patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress. the clinical teams caring for these patients were either giving a diagnosis of trali or considering trali as a possible diagnosis. of these 10 cases, only 2 of them were reported to our transfusion medicine service as transfusion reactions. of the reported cases, one was determined to be trali and the other one was consistent with taco. eight out of those 10 cases were never reported. background/case studies: despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (dhtr). this transfusion reaction is seen in as many as 1 out of 100 transfused products. therapeutic plasma exchange (tpe) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe tpe for clinical management after profound hemolysis. study design/method: case review of a patient was performed after diagnosis and treatment of severe dhtr. results/finding: a man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria". he had a known history of anti-d and anti-c, and was transfused two units of crossmatch compatible rbcs seven days prior during a previous admission. readmission hemoglobin (hb) was 8.7 g/dl but declined to 7.3 g/dl the next day. an antibody screen was consistent with anti-d, anti-c, and direct antiglobulin test (dat) was negative. he received three units of crossmatch compatible rbcs over days 2 and 3 with poor responses. on day 4, routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from 1.0 mg/dl to 1.8 mg/dl (reference 0.8-1.3 mg/dl), and lactate dehydrogenase was above reportable linearity, >2500 u/l (reference 122-222 u/l). testing revealed additional anti-e, anti-jkb, dat c31, plasma free hb 64.4 mg/dl (reference 1-15.2 mg/dl), and hemoglobinuria. four of five transfused rbc units were jk(b1), one of which was also e1. one volume tpe was performed to remove free hb on days 5, 6, and 7 using fresh frozen plasma as replacement fluid for haptoglobin supplementation. creatinine peaked at 3.7 mg/dl on day 13, decreased to 2.3 mg/dl before discharge on day results/findings: twenty three cases were identified, of which 20 had medical records available for analysis. ten (50%) patients were male, the mean age was 50.4 years (range 24-76 years), 15 (75%) had an underlying hematologic malignancy or bone marrow disorder, and 3 (15%) had a history of coronary artery disease (cad). the implicated units included 14 (70%) red blood cells and 6 (30%) platelets; 17 (85%) patients received a single unit, and 3 (15%) received two or more within the previous 6 hours; the mean volume transfused was 153.3 ml (range 20-280 ml). the mean time to onset of chest pain was 92.15 minutes (sd 85 minutes), with 90% of patients presenting within 2.5 hours and 100% within 6 hours of starting the transfusion. chest pain was present as the only symptom in 35% of the cases, and for the other cases the accompanying symptoms included dyspnea (30%), fever (25%), back pain (20%), and hypo-and hypertension (10%). a post-transfusion chest x-ray was performed in 65% of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (taco). electrocardiogram was performed in 70% of cases and showed no findings to suggest acute ischemia. three (15%) patients had a minimal increase in their troponin levels, although 1 had a history of chronically elevated troponin due to stress cardiomyopathy. fourteen (70%) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than 10 minutes in the majority of patients (90%). no cases resulted in new admission to the icu or procedure cancelation. conclusion: chest pain associated with transfusion was infrequent, but several such cases were identified during the review period. this symptom is not a diagnostic criterion for any of the other hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain. larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies. background/case studies: thrombotic microangiopathy (tma) in children is most commonly seen in the form of hemolytic uremic syndrome (hus). however, tma may be seen in the presence of streptococcus pneumoniae (spn). the action of bacterial neuraminidase of spn results in exposure of the normally "hidden" thomsen-freidenreich antigen (t-antigen) found on erythrocytes and other tissues. ultimately, this may lead to spn induced hemolytic uremic syndrome (phus) with subsequent hemolysis and end organ damage by naturally occurring anti-t antibodies against the exposed t antigen. specific lectins or anti-sera can confirm exposure of the t antigens in phus. alternatively, phus can be identified by minor crossmatch incompatibility resulting from agglutination of exposed t antigens on recipient's erythrocytes to anti-t antibodies in the plasma portion of blood products. we present a case of suspected phus that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical hus (ahus). study design/method: a 6 months old boy presented with respiratory failure. he was found to have blood cultures positive for spn as well as hemolytic anemia, thrombocytopenia, and acute renal failure. he was shiga toxin negative and had normal levels of adamts 13. based on the findings, the clinical team was concerned for phus. therefore, he received washed erythrocytes. for his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product. as a result, a minor crossmatching was suggested and performed to determine if t activation was present. results/finding: minor crossmatch was performed with patient's erythrocytes and plasma of abo-identical platelets to be transfused. no agglutination was seen at immediate spin, 37 degree, or anti-human globulin phase. check cells were found to be 21. these findings were conveyed to the clinical team and platelets were issued without washing. due to the lack of identification of t activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with ahus. the patient was then treated with eculizumab with clinical and laboratory improvement. we present a case clinically consistent with phus. confirmation of this diagnosis is done with lectins or anti-sera that are not readily available. an alternative means of identifying phus is by minor crossmatch incompatibility. by demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of ahus with appropriate management. background/case studies: orthotopic liver transplantation (olt) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding. we report a case of massive transfusion in an olt patient necessitating an abo blood group switch (from o1 to a1) to sustain transfusion support and minimum group o rbc inventories. study design/methods: type & screen (ts, gel) and anti-a titers (tube) were performed using routine methods. a chart review was performed for pertinent medical and laboratory findings. results/findings: the patient was a 63-year-old o1 man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha-1 antitrypsin deficiency who presented for olt (donor o1). during olt, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy. he required rapid high volume rbc and plasma support, which strained hospital inventories. after receiving 46 units of o1 rbcs and 26 units of o1 plasma with ongoing severe hemorrhage, he was switched to group a products. ten units of a1 plasma were transfused to wash out anti-a antibody prior to transfusion of a1 rbcs. due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for 7 hours post-transplant. the patient's total estimated blood loss was >20l. he received a total of 71 units of rbcs (including 23 a1), 63 units of plasma (including 37 a1), 6 units of cryoprecipitate, and 9 units of platelets. towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two rbc units he received were o1. on postoperative day (pod) 1, a ts showed predominantly a1 rbcs with trace o1 rbcs, as well as very low anti-a igm and igg titers (table 1) . he received two additional o1 rbc units (1 each on pod 4 and pod 12) with increasing o1 rbcs on ts and rising anti-a titers. his blood type was unequivocally o1 by pod 13. the patient showed recovery of liver synthetic function on pod 1 (factor 5 activity 5 58%) complicated by cholestasis. conclusion: this study shows successful switching of a group o patient to group a in the setting of rapid hemorrhage and massive transfusion. by pod 13, the patient had reverted to o1 with recovery of anti-a titers. at 3 months post-olt, the patient is alive with signs of improving biliary graft function. a new rfid transfusion safety system anna millan* 1 , alfred mingo 1 , maria isabel gonzalez 1 , antoni mena 2 and juan pedro benitez 2 . 1 bst, 2 at-biotech background/case studies: a new transfusion safety system (tss), based on processes and technologies, especially, identification by radio frequency (rfid), is currently implemented in two hospitals, a general one (h1) and an oncology center (h2). the tss is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (nm) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (pse) and the blood components administration (bca) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (tis). the tss allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of nm. study design/method: retrospective analysis of 2016 transfusion activity in both h1 and h2 shows 7970 pse and 12572 bca, out of 13163 and 20676 respectively, since the tss deployment in 2015. retrospective analysis and classification of 6700 security events has been done. results/finding: activity results for both hospitals are shown in the table below. the safety events have been classified in pretransfusion sample extraction (pse), blood component assignment (bcas) in the transfusion service and blood component administration (bca) near misses (nm). for h1, nm related to pse accounted for 42.39% of all, being the mistake in concordance between patient identification and prescription order the most frequent (52.03%). the nm detected in bcas were 12.1% of all and mostly (74.52%) occur when the patient information in the tis does not match the one registered in the tss. the nm detected in bca are 45.51% of all and mostly (36%) the systems detects a not assigned bracelet. for h2, nm related to pse accounted for the 47.49% of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent (65.84%). the nm detected in bcas accounts for 24.48% of all and in 69.08% occurs when the patient information in the tis does not match with the one registered in the tss. the nm detected in bca are 28.02% of all and in 65.61% of them the blood components were assigned to another patient. (1, 3, 4, 5, 8, 9, 12, 14, 19, 23, 26, 51, 56, 68) were analyzed via a commercially available elisa. comparison of adequate response to ppv23, defined as ! 2 mcg/ml for >7 serotypes, was perform based on alloimmunization status. statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests. results/findings: pre-vaccine sp titers were available in 72 patients (alloimmunized, 15); pre-and post-vaccine titers were available for 19 patients (alloimmunized, 6). of the 72 patients, 25 were on chronic transfusions, 24 were on hydroxyurea, 11 were surgical splenectomized, 58 patients had no history of surgical splenectomy or status was unknown. forty-four patients had a previous history of ppv23 in the previous 10 years; 9/44 also reported previous history 13-valent sp conjugate vaccine within the last 5 years. baseline pre-vaccination titers (n572) showed no difference between alloimmunized and non-alloimmunized patients (all p-values >0.13). in the group with pre-and post-vaccination (n519) titers available, 11 out of 13 (85%) non alloimmunized patients had an adequate response versus 4 out of 6 in the alloimmunized group (67%, p 5ns background/case studies: blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance. to safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (tres) as they occur from "vein-to-vein", i.e. from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time. multiple over-lapping error documentation processes are needed to capture and report tres from within and outside of blood bank (bb). we present a comprehensive error management program along with data on five years of benchmarking tres at a large academic medical center. study design/method: tres were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations. in addition, tres as observed and reported by providers and clinical staff (i.e. blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the bb and hospital quality through the veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm. all serious errors were reviewed daily and summation of tres was discussed on a monthly basis. mapping tres within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from 2012 to 2016). patient harm events recorded within the veritas system from january to july 2016 were investigated in depth. transfusion reactions were excluded in this analysis. results/finding: an average of 114 tres per month and 1300 per year were found over five years. 81% of tres are associated with pre-bb activities, 10% occur within bb, and 9% are post-bb events. sample collection and handling represent 80% of total tres. most tres (96%) were reported by bb staff, 4% were reported by non-bb staff. patient harm analysis revealed an average of four level 0 (near miss), three level 1 (no known harm), and 0.3 level 2 (patient harm) per month. no deaths related to tre were detected over the seven month january to july 2016 period. patient harm was associated with tres occurring in the bb (17%) and post-bb (83%). these events were reported externally (78%) and by bb staff (22%). conclusion: although most tres were detected in the pre-bb phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm. the tres causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-bb and bb phases. these results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-bb phase aimed at reduction of waste associated with sample collection and handling, and the post-bb and bb phases aimed at improving tre detection and decreasing patient harm. background/case studies: uncrossmatched red blood cells (rbc) and emergency issued platelets (plt), plasma and cryoprecipitate (cryo) are lifesaving in a bleeding patient without a valid type and screen. collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed. this study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued. study design/methods: a list of patients on whom blood products had been emergently issued between january 1, 2013 and march 28, 2017 was obtained from the blood bank at a regional maternity care hospital. patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded. the total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within 6 hours of the first emergently issued products were enumerated. apheresis plt units were multiplied by 5 and added to the number of individual whole blood plts; apheresis plasma units were multiplied by 2 and added to the number of whole blood plasma units. results/findings: seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified. average age was 31. the majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted. that plt wastage was higher than the other products was likely due to the 4-hour post-pooling room temperature shelf life. keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank. . patient blood platelets were higher before prophylactic than therapeutic transfusions (18[10 9 /l vs. 14[10 9 /l, p50.029). there were no significant differences in the frequency of effective therapeutic (55% vs. 72%, p50.1) and prophylactic (63% vs. 54%, p50.09) transfusions between the prcs and 25gypcs. we did not find significant differences between prcs and 25gypcs in cci1 after prophylactic (16.0 6 7.1 vs. 19.2 6 8.7) and therapeutic (11.3 6 9.0 vs. 11.8 6 5.8) transfusions, in cc24 after prophylactic (20.0 6 9.2 vs. 22.5 6 12.8) and therapeutic (13.3 6 8.9 vs. 13.9 6 8.) transfusions. there were no significant differences between prcs and 25gypcs also in ma1 after prophylactic (62.2 6 8.5 vs. 60 6 8.5, p50.7) and therapeutic (61.3 6 9.9 vs. 60.9 6 12.7, p50.08) pc transfusions. reduction of the severity of bleeds was obtained in 78 (86%) of the 91 cases after prpc transfusions and in 51 (84%) of 61 cases after 25gypc transfusions. there were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, 3 and 2 cases). background/case studies: an 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in 2006. it enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components. an error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in 2014 under the category 'errors relating to information technology'. this error was the incorrect use of the emergency transfusion process for non-emergency transfusions. the standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag. the emergency transfusion option is only intended for use with 'emergency group o rhd negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label. the emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion. it was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient. study design/method: this center worked with the software supplier to develop a solution which corrects the weakness in the process. the revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion. the use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not. results/finding: there were 593 units administered using the emergency transfusion process in the 3 months before the change was implemented. it was found that 51/593 (9%) units were non-emergency units administered incorrectly without a bedside compatibility check. following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month (2530 components administered), 109/2530 (4%) were emergency units which were administered correctly. users of the system reported the revised process was quicker, safer and unified with other functions on the device. conclusion: the improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check. this report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety. background/case studies: recent recommendations indicate one red blood cell (rbc) unit should be transfused at a time with reassessment after each transfusion to determine the need for more. however, the practices of canadian transfusion medicine (tm) experts and what constitutes a reassessment are unknown. therefore, we conducted a survey of tm experts across canada to gather information on their practices and criteria for reassessment. study design/method: tm experts were identified and contact information obtained from the canadian national advisory committee (nac) and from contacting least one tm expert per province. each respondent was assigned a unique study id after consenting to the survey, allowing for anonymity on analysis. the survey contained demographics, general practice questions, and questions regarding transfusion in: 1) a stable anemic inpatient, 2) a stable anemic inpatient to be discharged, and 3) an asymptomatic post-operative inpatient. results/finding: we identified 67 canadian tm experts: 48 (71.6%) provided a response and most had a primary place of practice in a laboratory setting (38/48; 79.2%). for a stable, non-bleeding, anemic inpatient, 87.5% of respondents recommended transfusing one rbc unit, then reassessing. recommendations were more variable in outpatient settings, with 31.2% generally recommending transfusing two rbc units then reassessing. recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period (1-2 hours), a repeat hemoglobin >18 hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings. lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds. with an asymptomatic patient to be discharged, fewer respondents chose to transfuse (38.1%) compared to an inpatient potentially symptomatic due to anemia (72.1%). none of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down. conclusion: tm experts generally recommend transfusing one unit at a time in stable inpatients. assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause. "top-up" transfusions were not recommended. these recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices. background/case studies: current evaluation of red blood cell (rbc) post transfusion recovery is based on ex vivo labeling of stored rbcs with radioactive chromium-51 ( 51 cr). this method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple rbc populations in the recipient. rbc labeling with s-nhs-biotin (bio-rbcs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused rbcs in vivo. the purpose of this study was to scale up and optimize the biotinylation procedures to the current good manufacturing practice (gmp) environment. study design/method: packed rbc units (n514) were divided into two 150ml aliquots, which were labeled with selected concentrations of s-nhsbiotin (3 and 30 lg/ml) in a cgmp closed system (average bio-rbcs hematocrit of 38.4 6 1.6%). optimization of labeling efficacy was determined by flow cytometric analysis of bio-rbcs using fluorochrome-conjugated streptavidin (sa). approximately 2 million rbcs were measured in triplicate. quantum simply cellular beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, mesf) and infer number of biotin molecules per rbc. the lower limit of detection was determined for rbc labeled with varying amounts of biotin. product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after rbc biotin labeling. results/finding: investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled rbcs revealed that 561nm excitation of phycoerythrin (pe)-sa and high laser power (150mw) provided the best separation between the two bio-rbc populations, and between labeled and unlabeled rbcs. labeling with 3lg/ml of biotin resulted in $50,000 mesf/rbc, and were detectable among unlabeled rbc at a lower limit of detection (lld, 95% ci) of 1 in 380,000 (0.0003%). the lld95 for rbc labeled with biotin at 30lg/ ml was $ 1 in 1 million (0.0001%). biotinylation was not associated with increased levels of hemolysis (0.40 6 0.22% before labeling versus 0.34 6 0.12% after labeling; p50.09) or bacterial contamination. conclusion: the resulting manufacturing process produces large volumes (150ml/transfusion) of bio-rbcs with low risk of contamination or hemolysis. the flow cytometry assay can detect bio-rbc in unlabeled blood at very low frequency. we plan to use this technology to study the impact of donor characteristic on rbc storage stability and post-transfusion survival. background/case studies: blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs. hemoglobin-based oxygen carriers (hbocs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available. since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer-250 (hboc-201) effects on coagulation parameters alongside freeze-dried plasma (fdp) in an in vitro model of hemorrhage/resuscitation. study design/method: whole blood (wb) was collected from healthy donors under an approved institutional standard operating procedure. in the first study (limited resuscitation), samples were: (1) wb, (2) wb110% hboc volume (model of two units in an adult), (3) wb110% fdp, and (4) wb110% hboc110% fdp. samples (5)-(8) simulated autoresuscitation by adding 25% plasmalyte to 1-4. susceptibility to lysis was tested with 75ng/ml tissue plasminogen activator (tpa). follow-up studies were performed with severe resuscitation simulations of 50%, 60%, 75%, and 100% volume replacement with hboc and/or fdp, with or without prior 25% plasmalyte dilution. coagulation parameters were obtained with a coagulation analyzer and thromboelastography (teg). rbcs/hemoglobin were measured on a hematology analyzer. thrombin generation was quantified by thrombogram. platelet aggregation was measured in multiplate and adhesion to collagen under shear in bioflux. viscosity was evaluated by rheology. results/finding: a limited resuscitation model with hboc and/or fdp had no effects on fibrinogen, pt, aptt, ph, hct, or hemoglobin. in teg, wb, wb1hboc, and wb1hboc1fdp had reduced clot strength with dilution and tpa. there was increased susceptibility to tpa-induced lysis between wb and wb1hboc in autodilution simulation (mean lysis 4.79% vs. 16.36%; p<.05). hboc and fdp had no statistically significant impact on thrombin generation. no effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow. hboc (10%) did not significantly change viscosity. severe resuscitation simulations had increased pt/ptt and reduced clot strength, particularly in hboc-only resuscitation; however, even 75% hboc volume replacement produced clots with acceptable teg parameters. conclusion: in a limited resuscitation model with hboc-201, there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting. considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of hboc1plasma as initial resuscitation of hemorrhagic shock. therapy in patients with acute major bleeding. while published literature has largely focused on the efficacy and safety of pcc, actual usage practices are less characterized. our aim was to describe the pcc usage practices within a tertiary care center. study design/method: we conducted a retrospective review of the electronic medical records of patients who received pcc between its addition to our institution's formulary in 8/2013 and 2/2015. we compiled information about the usage of pcc in these patients. descriptive statistics were generated with microsoft excel. results/finding: of 81 patients, 24 were on warfarin. pcc was most frequently prescribed for hemorrhage due to surgery (43%). pcc was given for warfarin reversal in 31% of cases. a subset of patients received plasma within 2 hours prior to pcc (40%) or 24 hours after (47%). pcc was most frequently ordered in the or/perioperative service (25%). conclusion: the majority of pcc usage was "off-label" in terms of being prescribed for indications other than warfarin reversal. the most frequent indication was hemorrhage due to surgery, and pcc was most often ordered in the or/perioperative service. although guidelines recommend the use of pcc as a plasma alternative, plasma was administered within hours of pcc in a notable subset of patients. background/case studies: in emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently. however, in some cases, poor communication and lack of clear expectations between the blood bank and patient care areas can lead to frustration and delays in the timely provision of blood products. an incident prompted an appraisal of our emergency release protocol (erp), which revealed gaps in communications and expectations by both the blood bank and nursing personnel. thus, it is imperative that there is a standardized er protocol with clear communications for both the blood bank and nursing personnel. reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (tat) for our er protocol. study design/method: in 2016, several meetings were conducted with stakeholders (critical care units (icu), emergency department (ed), internal medicine, interventional radiology (ir) etc.) in an effort to identify process gaps, improve communications, and expectations for er episodes. the goals was to design a process for emergent blood product request and release in life threatening situations that will; 1) simplify and expedite the process; 2) improve communication and expectations to decrease tat; 3) improve patient safety and meet compliance. in order to achieve these goals, a series of activities were conducted. these included meetings with all stakeholders to ensure process improvement meet the needs intended. a series of training sessions with nurse educators in icu, ed, ir and surgery managers were conducted. during the meetings, communication goals, and expectations were defined and agreed upon. training sessions included powerpoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow. the impact on the current process was analyzed and, as a result, led to the revision of the current sop, addition of pre-labeled emergency pack blood (4 units of o neg rbc's) and implementation of an electronic emergency blood order set. results/finding: in the ten months post implementation of our improved, standardized er pack protocol, a total of 61 er episodes were received. the average tat from order to delivery at the bedside was reduced by 50% (7.0 minutes compared to 14 minutes previously), while the compliance rate for er orders and physician documentation was 100% (61/61), with no current wastage of blood products. conclusion: the implementation of the improved standardized er protocol significantly improved communications and expectations, decreased tat and delays in transfusions while ensuring patient safety and compliance to regulatory requirements. background/case studies: massive transfusion (mt) in the trauma setting has been extensively studied. yet, the literature in non-trauma areas, especially oncology is rather sparse. the following study was conducted to understand the background and outcomes of mt in cancer patients. study design/methods: this was a single center retrospective study performed at a large cancer center between february 2016 -february 2017. mt was defined as the transfusion of ! 10 rbc units in a 24-hour period. the following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (mtp) was activated, and survival at 30 days. results/findings: thirty mts occurred during a one year period. a total of 192,441 blood products were transfused during that time period. gender distribution was 21/30 (70%) males, and the average age of all patients was 68 with a range of 21 to 70 years of age. surgical patients accounted for 26/30 (86.7%) mts, and 4/30 (13.3%) were critical care patients. tumor categories included carcinomas (14/30), sarcomas (13/30), leukemias (2/30) and lymphoma (1/30). resection of tumor followed by complex reconstruction was the cause of the majority of mts. metastatic renal cancer (6/30) was the most common disease seen followed by sacral chordoma (4/30). mtps were activated in only 8/30 (26.7%) cases. thirty-day survival was seen in 25/30 (83.3%) patients. only 1 of 5 mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by gi hemorrhage (3/ 5) or perisplenic hematoma (1/5). the overall ratio of rbc:ffp in the entire patients ( background/case studies: plasma is a straw-colored supernatant of blood that is used for type and screen (t&s) and crossmatch. in the analytic phase of testing, plasma is examined prior to processing. plasma occasionally becomes discolored, interfering with crossmatch procedures. timely identification of the etiology allows for corrective actions and minimizes delay in transfusion. study design/method: during the analytic phase of blood bank testing, samples were evaluated for t&s and crossmatch; this identified three samples with discolored plasma. we present a series of cases that illustrate the testing process. results/finding: a 54-year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy. a preoperative t&s specimen contained bright green plasma. review of her preoperative case revealed exposure to intravenous methylene blue. this dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects. alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use. although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique. a specimen drawn one week later contained clear plasma. a 26-year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies. administration of a synthetic blood product resulted in dark maroon colored plasma. the most common cause of a dark red color is hemolysis of the sample, which is usually discarded. in this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples. with this in mind, the sample was not discarded and testing was completed by tube method. a 70-year-old woman admitted with acute stroke was treated with a thrombolytic. her t&s revealed cloudy white plasma that could not be used for the crossmatch procedure. common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol. although an etiology could not be identified a repeat specimen drawn several hours later was clear. conclusion: these cases highlight the importance of an appropriate evaluation of discolored plasma. once a discolored sample is identified, a repeat sample is required to confirm the change in color. in the first two cases, the discoloration persisted, prompting further clinical investigation. once the etiology was identified, need for further testing and eligibility for further transfusion was determined. testing by tube method could be performed in two cases. in the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay. decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists. caleb wei-shin cheng* 1,2 , rebecca ross 2 , christopher a tormey 1,2,3 and amit gokhale 1,2 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: daratumumab (dara) is a igg1 monoclonal antibody therapy that specifically targets cd38, a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma. dara interferes with blood bank testing as it binds to cd38 expressed on red blood cells, causing pan reactivity. the dara interference can be overcome with the use of dithiothreitol (dtt) treated reagent red blood cells. to minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a dara protocol in our blood bank. the purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during dara treatment at our institution. study design/method: all dara patients' antibody workups were completed using dtt pre-treated reagent red blood cells. if the antibody screen was negative, k antigen negative rbc products are provided. if an antibody is identified, k negative along with that particular antigen negative blood is provided. our electronic medical record (emr) was searched for patients who received dara over the past eight months. study subjects were examined to see if they had pre-existing alloantibodies before dara treatment and whether they formed new alloantibodies during dara treatment. the age, gender, type and screen pre-dara treatment, type and screen post-dara, intervening blood transfusions, and the date of first dara treatment was recorded. results/finding: overall, 54 subjects were identified for analysis. their mean age was 67.8 years, with 29 male and 25 female subjects; all were diagnosed with multiple myeloma. we found an alloimmunization rate of 0% (0/54) prior to administration of dara. of these patients, 22 were transfused with red blood cells (rbcs) after initiation of dara therapy. following our testing/matching protocol, none of these (0%; 0/22) patients formed a confirmed, new alloantibody during dara treatment; each of these patients underwent at least one follow-up screen after their first rbc unit. we also found no complications in providing crossmatch compatible units to any of the 22 patients. conclusion: to our knowledge, this is the largest case series reporting on results of overcoming dara interference with blood bank type and screen testing. the protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving dara therapy. it is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted. background/case studies:a multi-facility transfusion service began stocking liquid plasma in september of 2015 for use in massive transfusion and trauma situations. due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use. a policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented. this study evaluated the effects of that policy on inr values of plasma recipients. study design/methods:a retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (ffp) and liquid plasma (lqp) in changing inr values of recipients. all plasma units transfused within the facility from september 1, 2015 through april 7, 2017 were identified. the following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion inr values, and whether or not vitamin k was administered. patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin k. the change in inr for each recipient was calculated, along with the average change in inr for each group. background/case studies: in gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation. transfusion could generally be avoided in those without haemodynamic instability. the oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia. besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (hb). the present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in hong kong. study design/methods: anonymous data of gynaeological patients age 60 was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge. all transfusion episodes associated with surgical operations during same admission are excluded. results/findings: in 2016, 2,523 unique women receiving a total of 5,889 units of red cells (rc) in 2,906 transfusion episodes were identified. their median age was 45 (range 11 -60). the distribution of pre-and post-transfusion hb and units of rc transfused were summarized below: in this cohort, pre-and post-transfusion hb were absent in 46 (1.6%) and 283 (9.7%). 635 (21.9%) transfusion episodes were associated with the use of 3 units or more rc. as a result, 1385 (47.7%) episodes resulted in a post transfusion hb ! 9g/dl. parenteral iron or tranexamic acid was uncommon during hospitalization and was given 2 (<0.1%) and 116 (4.6%) women respectively. upon discharge, 442 (15.0%), 84 (3.2%) and 1,994 (65.8%) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively. however, neither were given to 386 (16.0%) women. conclusion: in the present study, it is observed that 49.7% transfusion episodes were given at hb ! 7g/dl. a substantial number of episodes (71.7%) were transfused with multiple units and resulted in almost half having a post transfusion hb level (! 9g/dl). for iron replenishment and bleeding control, up to 16.0% transfused women were not given iron or tranexamic acid at discharge. the results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved. it is recommended that in-depth education and training should be provided for a better gynaecological patient blood management. background/case studies: granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection. however, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions. we report a case of granulocyte transfusion therapy following chimeric antigen receptor t-cell (car-t) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed b-cell acute lymphoblastic leukemia (b-all). study design/method: granulocytes (0.8-1.3x10 10 per unit) were collected from abo-identical unstimulated donors at a regional blood center. each unit was irradiated with 25 gy and transfused over 3-4 hours within 24 hours after the time of collection. the patient's response and laboratory data were reviewed in the medical record. conclusion: this data suggests that a diagnosis of aml is associated with anti-hla antibodies. an increased frequency of blood group a in patients with aml has been reported, but here no statistically significant difference between abo blood group frequencies was found in any category except the patient's with hla antibodies. blood group b has a significant association with hla alloimmunization in the studied patients. it has been reported in a large study of female blood donors that no difference in hla antibody frequency was observed based on abo blood group at centers using the flow-based assay. although the reasons for the higher rate of group b blood type among patients with anti-hla antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially aml. females with aml who are blood group b appear to be most likely to have hla alloimmunization among patients with hematologic malignancies. implementation of electronic solution to reduce risk of mistransfusion in a regional transfusion service debra lane* 1 , lee grabner 1 , brenda herdman 2 , robert fallis 1 , amin kabani 3 and charles musuka 3 . 1 canadian blood services, 2 kenora rainy-river regional laboratory program, 3 diagnostic services manitoba background/case studies: patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion. the rate of mistransfusion has remained unchanged in over 50 years. attempts have been made to reduce mistransfusion including barrier devices, barcoding and rfid. within a regional background/case studies: the role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (rbc) units is receiving increased attention. however, the impact of donor characteristics on efficacy of rbc transfusion has not been studied in largescale donor-recipient outcomes databases. study design/methods: we conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between 2008 and 2011 before patient blood management initiatives. linkage was performed between blood donor characteristics and hospitalized rbc transfusion recipients who received a single rbc unit. studied exposures for this analysis were blood donor sex and age in addition to rbc storage age. the wilcoxon test was used to examine changes in hemoglobin level following rbc transfusion, and , and 38% were male. recipients of rbc's from male and female donors had similar pre-transfusion hemoglobin levels (8.7 g/dl; p50.94); however, transfusion recipients of male donor rbc units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female rbc units (9.9 vs 9.8 g/dl; 1.2 vs. 1.1 g/dl; both p50.02). female recipients had a larger rise in hemoglobin per rbc unit compared to male recipients (1.3 g/dl vs. 1.0 g/dl; p<0.001). female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p50.01). rbc storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p50.53 and p50.32, respectively. conclusion: rbc units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes. this suggests that the dose of hemoglobin is lower in female than male rbc units. this analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and rbc efficacy, hemolysis and other donor-component-recipient interactions. background/case studies: people who identify as jehovah's witnesses (jw) comprise less than 1% of the population of the united states. however, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings. the degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted. however, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider. as such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution. study design/method: the electronic medical record (emr) utilized in this study in an institutionally modified version of epic ea best practices alert (bpa) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (jehovah's witness) discrete data fields. the alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override. data on the triggers are automatically collected through the emr systems and generated into a report by informatics personnel. results/finding: the available data covers triggers in the two month postimplementation of the bpa. the bpa triggered 33 times in total, affecting 15 patients and 21 users. stratified by location, the majority of triggers occurred in the perioperative areas (18 times) and the liver icu (6 times) with a minority occurring on the regular hospital floors and emergency department. nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected. orders that triggered the bpa included type & screens, human albumin 25% iv solution, human albumin 5% iv solution, immune globulin (human) solution. conclusion: despite the limited and very preliminary data, the user action findings seem to indicate that the bpa is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders. given the limited types of orders that the bpa is triggering on, the pattern suggests that the bpa is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many jw patients. despite these positive initial findings, this is an ongoing study to track the efficacy of the bpa and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal. intervention to address inappropriate cryoprecipitate-ahf orders at a tertiary medical center sirisha kundrapu* 1,2 , mahmut akgul 1,2 , hollie m reeves 1,2 , robert w maitta 1,2 , marcie pokorny 2 , anne capetillo 2 and katharine a downes 1,2 . background/case studies: although introduced for the management of hemophilia a, now cryoprecipitate is primarily indicated for low fibrinogen levels. at our institution the transfusion medicine service (tms) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders. we aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders. study design/method: we conducted a 7-month retrospective study (january-july 2016) of adult cryoprecipitate order quality assurance forms. the reference range for fibrinogen was 200-400 mg/dl with critical value of 50mg/dl. cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the tms. during the study period, tms evaluated orders for appropriateness of dosing and agreement with estimated required doses. post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels. statistical analysis was performed using chi-square and t-tests. results/finding: there were 301 adult (>18 years) orders reviewed by tms out of which 299 were approved. of the 299 approved orders, 136 (45.5%) were in agreement with tms's estimated dose. of 163 (54.5%) orders that were not in agreement with the tms's estimate, 142 (47%) were underestimated and 21 (7%) were overestimated. seventeen of 299 orders had no post-transfusion fibrinogen levels. without intervention, there would have been a median deficit of 23.6 mg/dl (range 0.3 to 124 mg/dl) and a median excess of 13.3 mg/dl (range 0.6 to 155 mg/dl) of fibrinogen from the target. median difference between target and actual post-transfusion fibrinogen level was 12 mg/dl above target, which is significantly higher with intervention than without (which could have been 12 mg/dl below the target; p<0.0001). median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention (11 vs. 2.7 mg/dl, p50.07). median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention (15 vs. -23.5 mg/dl, p<0.0001). seven of 299 (17) 40 (24) orders were for critically low fibrinogen (<50 mg/dl) and 4 of these were under-estimated requests and reached target fibrinogen with tms's estimate and approval of required units to be transfused. overall most frequent orders were 10 and 5 units (59.5% and 25%) i.e. 2 and 1 pools and the most frequent orders in the disagreement group were 10, 1, 5 and 2 units (33%, 20%, 17% and 16%). there is a significant difference between agreement and disagreement groups based on clinical service ordering the units (table) . conclusion: intervention by tms to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels. further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage. patient characteristics, medical records, vitamin k administration, and adverse events, were collected (table) . results/finding: the average pre-transfusion inr was 2.36 and posttransfusion was 1.91. only 22% of patients had their inr corrected to 1.5, while 28% had no change, or had increased inr. (table) . the majority (67%) of patients received 2 units of plasma. the mean plasma dose was 6 ml/kg. there were 4 transfusion reactions reported, 1 non-hemolytic and 3 transfusion associated circulatory overload reactions in which 1 required admission to the icu. two patients experienced bleeding during ir procedures (tips) and 1 developed a hematoma (tunneled central line). the median of inr correction in this study was 1.9 with no relationship to the number of units of plasma transfused and/or if vitamin k was administered. this study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when inr is 1.9. randomized trials are needed to assess whether the inr is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit. 3 of the 111 patients experienced bleeding complications indicating that inr of 1.9 may be considered safe in some lower risk procedures. current practices may provide little or no benefit, with substantial risk of life threatening complications. background/case studies: group ab plasma, which lacks anti-a and anti-b antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available. approximately 4% of the population is group ab, which limits the available inventory of group ab plasma. of group ab population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (trali). this makes type ab plasma a limited resource. our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known. group o individuals make up 45 % of the population and have no a or b antigens on their cells. group a is the second most prevalent blood group in the us population (40%) and has no b antigen on their cells. so, group a plasma is compatible with both group o and a patients, approximately 85% of the patient population. before patient's blood type is known, type o red cell units are transfused with a plasma, which decreases the chance of hemolysis. to conserve ab plasma, we instituted a policy effective july 1, 2014 as follows: 2 units of group a plasma and 3 units of group ab plasma is provided for the massive transfusion protocol (mtp) along with 5 units of o negative rbc until patient blood type is known. study design/method: this prospective study is designed to monitor the use of group a plasma in mtps at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma. direct antiglobulin test (dat) is performed if patient received incompatible plasma. if dat is positive, lactate dehydrogenase (ldh), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction. results/finding: we reviewed 235 mtps at our institution between july 2014 and march 2017. twenty patients (8.5 %) were transfused with incompatible group a plasma (5 group ab and 15 group b patients). five patients died due to severe injury, and follow-up testing of these patients could not be performed. the remaining 15 patients had negative dat, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis. none of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion. conclusion: our study adds more evidence of the safety of group a plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known. based on this and other recently published studies, starting in april 2017, our institute will provide only group a plasma for emergency release and mtp cases before the patient blood type is known. average ( background/case studies: in 2013, bonfils immunohematology reference lab (irl) sent out approximately 245 special platelets for patients with hla antibodies. by 2016, hla platelet orders increased dramatically and the irl sent out over 650 special platelet products. the purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time. study design/methods: bonfils blood center has over 10,000 donors in the database with historical hla typing. however, only approximately 3500 of those donors actively donate. in the denver area, one of the most common hla types is a1 a2 b7 b8. only 81 of the 10,000 donors have this type (0.81%). therefore, to fill an hla platelet order request for a common hla type, only 28 donors in the system would be a perfect hla match. with that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order. after a donor is recruited and donates, it takes at least two days to fill an order. for a less uncommon hla type like a9 a11 b17 b35, there is only 1 out of 10,000 donors (0.01%) that match perfectly. in those cases, there are no donors to recruit to fill such an order. in some complicated cases, the irl was provided with an hla antibody list or panel reactive antibody test (pra). in order to find product for these patients, lists of platelets in inventory with corresponding hla types were printed. if a patient had an antibody to a1 for example, all of the a1 positive platelets were crossed off the list. this cross-out process would continue manually until the only platelets on the list were the ones positive for hla antigens to which the patient did not have antibodies. these platelets are pra matched to the patient. in order to automate this process a report linked to the donor database was created to find both pra platelets in inventory and donors for recruitment. the blood center medical director began suggesting that hospital clients order a pra for each patient with platelet refractoriness. the pra test is fast and it is a definitive method to discern hla antibody mediated refractoriness from platelet refractoriness due to other causes. results/findings: in all but the most complicated cases with rare hla patient phenotypes, it was much easier to find a pra patient matched platelet on the shelf than an hla match donor. in 2012, approximately 27% of these special order platelets were pra matched and the remaining 73% were hla matched by donor recruitment. by 2017, approximately 59% of special platelets sent are pra matched. this change resulted in a 2.2 fold increase of finding product in inventory to fill orders quickly. conclusion: developing a system to provide pra matched platelets is a faster alternative to finding hla matched platelets thus contributing to better patient care. background/case studies: in urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (mtp) is critical to the timely delivery of these products. each mtp pack at ucm contains 6 packed red blood cells (prbcs), 4 fresh frozen plasma (ffp) units, and 1 plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in labor and delivery (l&d) or if one is requested. at ucm, blood products are generally transported through the pneumatic tube system (pts). we undertook a review of our mtp issuing practices and efficiency patterns over the last three and half years. study design/method: the electronic archives of the blood bank laboratory information system and electronic medical record at our institution were queried for patients who had mtp activations. the archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on. results/finding: between august 2013 to march 2017 mtps were activated at ucm, of which 251 orders could be traced to the origin: 118 on inpatient floor (including icus), 58 in the operating rooms, 46 in the emergency department, 25 in labor and delivery, and 4 in other procedure rooms. of the 2207 prbcs that were issued, 1406 were transfused (64% utilized); of the 1446 units of ffp that were issued, 901 were transfused (62% utilized); of the 359 platelet packs that were issued, 246 were transfused (69% utilized); of the 64 units of cryoprecipitate that were issued, 49 were transfused (77% utilized). since march 2016, the time of first product issue after the initiation of an mtp has also been tracked. of the 84 events that fall within this time period, 39 (46%), had the first product issued in 5 minutes or less. another 31 (37%) were issued between 5-10 minutes, resulting in over 80% of patients being issued their first blood product within the first 10 minutes. only 15 of 84 (17%) events had an initial time greater than 10 minutes and none were greater than 21 minutes. conclusion: the majority of our activations currently come from inpatient floors (primarily icus). as our institution anticipates the introduction of an adult level 1 trauma center, we anticipate this balance will shift. in addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during mtp activations ($60-70%). again, we anticipate utilization rate of issued mtp products to increase with the introduction of a new adult trauma center. we have recently begun tracking time to last product issued during an mtp, but cannot report on that variable at this time. overall, our data show that our transfusion service is generally performing adequately to issue the first product within 10 minutes of mtp protocol activation. this data only reflects time to issue in the pts; patient care areas can experience additional minutes delay in pts delivery and arrival of product at bedside. we must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient mtp care. mehreen yasin* 1 , shailesh macwan 1 , arline stein 1 , jane fischman 1 , nancy nikolis 1 , matthew bank 1 , lennart logdberg 1 , alexander indrikovs 2 , sherry shariatmadar 1 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: massive bleeding is generally defined as any patient who requires 1 blood volume replacement within 24 hours and/or receives transfusion of greater than or equal to 4 units in one hour with 177a transfusion 2017 vol. 57 supplement s3 ongoing bleeding. our mtp was officially implemented in 2013 in preparation for an initial verification as a level 1 trauma center by acs. our mtp has the following packages: 1st pack has a ratio of 4:4:1 (rbcs, plasma & platelets) and subsequent packs a ratio of 6:6:1. our mtp also includes prothrombin time (pt), activated partial thromboplastin time (aptt) and fibrinogen testing after each pack is transfused. this data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies. however, attempts to supplement mtp packs with cryoprecipitate (cryo) and prothrombin complex concentrate (pcc) were challenging to accomplish in a timely manner. study design/methods: due to challenges in timely supplementation of mtp packages with cryo and pcc, the protocol was modified in march 2016 to add cryo and pcc at a defined point in the mtp (cryo is included in the 3rd pack and pcc in the 4th pack). in order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received >20 rbc at our institution as these massively hemorrhaging patients would receive pcc based on our current protocol. we reviewed the blood products received by these patients and their available laboratory data. results/findings: we had 8 patients who received >20 rbc in 2015 and 2016. mtp had been activated for all patients and all patients received between 0.5 to 1 unit of plasma for each rbc unit transfused. despite receiving these ratios of blood products, all patients had elevations of their pt >16 seconds and many had elevations of the aptt and fibrinogen levels less than our institution's target of 200 mg/dl (table 1) . as anticipated, improvement in the coagulation parameters was noted with cryo and pcc supplementation. conclusion: our data on massively hemorrhaging patients supports a role for supplementation of our mtp with cryo and pcc in patients who require transfusion of >20 rbc. our current protocol with the addition of cryo and pcc at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients. background/case studies: red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (ta-pls). however, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype. a living donor liver segment transplant resulted in a case of ta-pls with donor-derived anti-d that had the potential for causing a clinically significant hemolytic event. the donor's plasma contained anti-d. anti-d was absent in the recipient's pre-transplant plasma, but present in the recipient's 5-day and 114-day post-transplant plasma. although these findings established a diagnosis of ta-pls, hemolysis did not occur because the recipient's blood group phenotype was d-. the conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of pls and limits capturing the true scope of the syndrome. study design/method: to determine the standard of practice for detecting and diagnosing ta-pls, a retrospective 10-year pubmed search for peerreviewed english-language journal articles was conducted using key words "passenger lymphocyte syndrome." cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies. results/finding: of 63 published cases (31 reports) of ta-pls, 8 (4 reports) were stem cell and 55 (27 reports) were organ transplants. all 8(100%) stem cell transplants and 52 (95%) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying ta-pls associated with hemolysis. of the 4 reports of stem cell ta-pls, 3 actively screened for antibodies in the immediate post-transplant period, and of the 27 reports of organ ta-pls, 1 actively screened for antibodies. these screens detected 5 cases of stem cell ta-pls before hemolysis became apparent and 2 cases of organ ta-pls with antibodies without hemolysis. it can be inferred that ta-pls is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely. conclusion: a new category of "non-hemolytic ta-pls" is recommended to capture otherwise undiagnosed cases where ta-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case. to ensure including the full scope of ta-pls, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated. occult hemolytic anemia due to anti-mur in a patient receiving blood from a region with a prominent asian donor population jean oak* 1 , rosario mallari 2 , marc de asis 2 , elaine shu 3 , jonathan hughes 4 and tho pham 1,3 . 1 stanford university, 2 stanford health care, 3 stanford blood center, 4 bloodsource background/case studies: mur antigen is present in 7-10% of individuals in southeast asia, taiwan, and parts of southeastern china, but is rare elsewhere. antibodies against mur antigens are clinically significant, hence many countries in asia routinely screen for it while other countries, including the us, does not include mur in the standard screen. we describe a case of an occult anti-mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large asian donor population. 41 year old hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia. initial antibody screen and dat were negative, and the patient received 1-2 rbc units every 1-2 weeks to maintain a hemoglobin (hb) level of 8 g/dl. the patient remained stable for 5 months when his hb level acutely dropped to 6.6 g/dl. the antibody screen remained negative for an additional 2 months when it became positive for anti-jka and anti-mur. donor ethnicity data was available for 30 of the 33 rbc units he received. 3 units were from an asian donor, and a unit transfused 13 days prior to the hb drop was from a caucasian/chinese donor. study design/method: we reviewed the ethnicity data of 64,495 donors at a hospital-associated blood center located in a region where asians comprise approximately 30% of the population. results/finding: 6.6% of donors identified as chinese, vietnamese, filipino, or other southeastern asian. these donors account for 5245 of 37933 (13.8%) rbc collections. conclusion: identification of anti-mur in this patient was triggered by the presence of a concurrent anti-jka alloantibody. since over 10% of the rbc supply in the local blood center was collected from chinese or southeast asian donors, chronically transfused patients are at risk of developing anti-mur-mediated hemolysis that could be missed on a standard screen. this finding raises a possible need for blood banks located in regions with a prominent asian population to implement screening for anti-mur. brian adkins* 1 , princess maynie 1 , carol chandler 1 , shelia garret 1 and pampee young 2 . 1 vanderbilt, 2 vanderbilt university medical center, department of pathology, microbiology and immunology background/case studies: antibody titration is a testing modality vital to both obstetric and transplant services. manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive. in fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method. the orthovision automated analyzers offers automated titering of patient plasma using gel technology. although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing. the higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation. moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision. [3] [4] [5] there is minimal information on the comparisons of tube titers to orthovision automated titers or assessment of the reproducibility of this automated method. study design/method: rh and non rh minor rbc antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ortho vision analyzers to assess precision and inter-instrument reproducibility. results/finding: a total of 26 samples have been analyzed (table) , 17 rh and 9 non-rh antigens. titers via automated testing on orthovision resulted in a mean titration being 2.77 (range 1-7) times higher. the average fold change for rhd/c/e antibody titers were 3.2, whereas the average fold change for non rh titers was 1.03 (range 1-2). the range for anti d titers was particularly variable, 2-7, whereas for c/e, it was 1-3. the overall reproducibility/precision of the automated analyzer was $90%. to correlate the 178a transfusion 2017 vol. 57 supplement s3 increased titers observed with some classes of antibodies, particularly anti d, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values. conclusion: automated titration of antibodies using the orthovision analyzers resulted in highly reproducible results between different instruments using the same sample. however, the automated analyzers consistently yielded higher values, particularly with rh d, with results $3 times higher than in manual tube testing. interestingly, the difference in titers of non rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small. in order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus. platelet additive solution reduces the isoagglutinin titer in apheresis platelet units maxim tynuv*, elizabeth j furlong and willy a flegel. dtm/cc/nih background/case studies: isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-a and/or anti-b may cause a hemolytic transfusion reaction (htr) in a recipient with cognate antigen. apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of htr due to plasma incompatibility if given based on short outdate and not abo type. at our facility testing is performed on all apheresis platelets with a cutoff titer of 250. units above the cutoff are marked as "high titer" and only given to abo plasma-compatible recipients or washed with saline to reduce plasma. however, washing platelets is a time consuming process that results in a loss of up to 33% of the platelets. platelet additive solution (pas) is used as an alternative collection and storage solution, replacing approximately 65% of donor plasma in the final product. the goal of this study was to determine what affect pas has on isoagglutinin titers and whether using pas could lead to a revision of one facility's procedure for management out of group platelet transfusions. study design/method: isoagglutinin titers of whole blood edta samples were compared to the final apheresis platelet unit collected in pas (intersol, fresenius kabi, lake zurich, il). using two-fold dilution steps, plasma was tested with pooled red cells (equal mix 0.8% suspension of a1 and b cells, ortho, raritan, nj) in a gel matrix test (mts buffered card, ortho, raritan, nj) with 15 min incubation (room temperature) prior to centrifugation (mts ortho workstation). fifty two donors were group o, 32 group a, and 16 group b. results/finding: of the 100 whole blood edta samples tested, 26 (25 group o and 1group b) exceeded a high titer threshold of 250. when the pas samples of these 26 donors were tested, only one (group o) exceeded the same threshold. pas specimens showed a consistent two-fold decrease in titer compared with whole blood specimens. nearly half of the group o donors exceeded a titer of 250 when whole blood specimens were tested. conclusion: only one sample from apheresis platelets collected in pas exceeded our clinically applied titer threshold of 250, a 96% decrease from the number of whole blood specimens exceeding the threshold. testing the platelet bag collected in pas instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed. furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using pas or not. other components. the majority of blood components in israel are collected and distributed by magen david adom (mda), from 2 main locations. several hospitals in israel also collect platelets in-house. as part of an effort to understand plt utilization, a nationwide survey of plt transfusion and expiration was conducted. study design/methods: data on the disposition of all plt units, acquired from mda and collected in-house, during the calendar year 2016 was requested from all hospitals in israel. the number of plt distributed to hospitals by mda was also collected. plt wastage was defined as the sum of plt that were returned and not reissued from the hospital blood banks and plt that expired on blood bank shelves. results/findings: sixteen of the 27(59%) hospitals in israel, along with mda, participated in the survey, listed as a to p. the results are presented in the table along with each hospital's distance from the 2 mda facilities. for some hospitals, the sum of transfused and wasted plt was slightly less than the number of plt supplied by mda; this is likely due to the small number of plt that had not either been transfused or expired by the time the data collection period ended. three of the largest hospitals (c, b and a) collected plt in-house in addition to acquiring units from mda. these 3 hospitals had a lower overall rate of wastage including their own donations than the other 13 hospitals that did not collect in-house plt. the other 13 hospitals had wastage rates ranging between 9-54%. no correlation was apparent between the hospital's distance from the mda facility or its number of beds and the plt wastage rate. conclusion: there is considerable platelet wastage in israel. large hospitals in israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals. factors known to affect plt utilization and wastage such as patient diagnosis mix, policies about how plt are issued and accepted back into hospital inventory, plt inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates. possible immune-mediated hemolysis due to platelet transfusion masked by underlying hemolysis in a patient with blast crisis sirisha kundrapu* 1,2 , christopher j gresens 3 , anne capetillo 2 , hollie m reeves 1,2 and katharine a downes 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center, 3 bloodsource background/case studies: transfusion-related hemolysis with abomismatched platelets is rare with a reported incidence of <0.1%. most commonly in such cases group o platelets having high titer anti-a result in clinically significant hemolysis when transfused to a group a or ab recipient. we present a patient with a possible hemolytic reaction following transfusion of abo mismatched platelets presenting in the setting of underlying disease associated hemolysis. study design/method: a 58-year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (sdp). two hours post transfusion he developed chills, rigors, and increased blood pressure (117/65 mm hg to 205/89 mm hg) followed by hematuria (500 ml). chills and rigors resolved; blood pressure stabilized after 15 min with diphenhydramine, solumedrol, and acetaminophen. negative. patient abo group, rh (d) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies. laboratory indicators of hemolysis are summarized in table. notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels. despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-a. this revealed donor anti-a titer results of 256 (igm) and 1,024 (igg); donor was deferred from future platelet donations. conclusion: while the post-transfusion sample had no visible hemolysis and a negative dat, increased total/ indirect bilirubin after transfusion and high titer donor anti-a are supportive of immune mediated hemolytic transfusion reaction. the key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-a. ana paula hitomi yokoyama* 1 , leila patricia de sousa fontenele 1 , isabel nagle reis 1 , carolina bonet bub 1 , araci sakashita 1 , raffael zamper 1 , cristiane nakazawa 1 , tatiane almeida omura paula 1 , patricia silva batista 1 , marcio dias almeida 1 , fernanda loureiro de andrade orsi 2 and jose mauro kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp-universidade estadual de campinas background/case studies: orthotopic liver transplantation (olt) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease. despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements. peri and intraoperative transfusion of red blood cells (rbc) have been previously reported as major predictors of post -operative mortality . identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in olt. we conducted a single center retrospective analysis of 671 cases of olt performed between 2011 and 2015 in brazil in order to identify predictive factors for red blood cell transfusion study design/method: a retrospective analysis in a single institution was performed, and charts of 671 consecutive patients submitted to liver transplantation between 2011 and 2016 were reviewed. the following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (meld), duration of warm and cold ischemia. categorical variables were analysed using pearson chi-square test. continuous variables were analysed using t-student test. a forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant. results/finding: in univariate analysis, female patients, absence of hepatocellular carcinoma (hcc), primary diagnosis, corrected meld and warm ischemia time were significantly associated with consumption of rbc use in the intraoperative period. multivariate logistic regression of these factors showed that female patients (or 1,726 -95% ci: 1,147-2,597, p:0,009), absence of hcc (or 0,295 -95% ci:0,199-0,437, p:0,0), cirrhosis of any cause (or 4,161 -95% ci 1,816-9,534 -p:0,001), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (or 5,236 95%ic 2,212-12,394) and retransplantation due to primary non function of the graft (or 5,791 95%ci 1,33-4,25,206, p: 0,019) were independently associated with rbc transfusion requirements. conclusion: in this study, female patients, absence of hcc, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with rbc consumption in intraoperative period. determination of rbc transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies. prevalence of high-titer anti-a1/b in group o platelet products. charles k. childers* 1 , mark destree 2 , ashley rose 2 and theresa nester 3,4 . 1 madigan army medical center, 2 bloodworks northwest, 3 bloodworks nw, 4 dept of laboratory medicine, university of washington background/case studies: with platelet substitution policies, minor aboincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply. however, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high anti-a1 or anti-b titers, typically in a group o donor. one method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention. the percentage of high titer anti-a1/b in group o platelet products is presented from a large regional blood center collected over 10-12 months. data from both pre-storage pooled platelet units (pspp) and apheresis derived platelet units (aplt) is shown. study design/method: platelet component samples were collected in 2 ml edta sample tubes. a single 1:150 dilution of plasma was prepared using a hamilton microlab 600 series dilutor using 2235.0 ml saline diluent and 15.0 ml platelet component sample. using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of a1 or b red blood cell reagent. reaction tubes were centrifuged immediately in a serological centrifuge at 3175 rpm for 20 seconds. reactions were read using a lighted agglutination reader. the presence of macroscopic agglutination (weak or greater) with either the a1 cells or b cells was recorded as a positive reaction, indicative of a high titer anti-a1 or anti-b. retesting of samples was performed to confirm high titers. results/finding: the above results indicate that, when a titer cut-off of 150 is used, approximately 3% of group o apheresis platelets will have a high titer, most commonly with anti-a1. less than half of a percent of pspp units will have a high titer. testing units for the titer can help to change abo out-of-group platelet substitution policies. in our example, the bloodworks transfusion service was able to change from a policy of volume reducing any group o apheresis platelets being issued to a group a or ab patient, to giving high titer products to only group o patients. the subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their 5 day outdate. after 10 months of testing pspp units and verifying that the products rarely had a high titer (0.28%), the blood center stopped performing this testing for pspp units. rh1) ] started complaining of worsening back pain two and half hours after receiving one unit of rbc for a drop in hematocrit to 19% (from 24% on the previous day). his hematocrit did not increase (18%), and over the ensuing 12 hours, he became anuric and jaundiced. clerical checks confirmed that his forward type was a positive, which was also the type of the rbc unit transfused, but revealed anti-a at a titer of 8 in his plasma. furthermore, the direct antiglobulin tests (dat) were positive for c3 in the pre-and post-transfusion blood samples. anti-a was not detected in his plasma collected three days earlier, however. although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (ldh) and total and indirect bilirubin results/finding: the positive dat in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the a rbc unit. in the setting of recent abo-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-a by proliferation of donor lymphocytes, or pls. we performed an emergent rbc exchange using o rbcs with a goal hematocrit of 24% while reducing the number of a rbcs in his circulation by approximately 70%. his pain improved rapidly thereafter, and he had complete recovery of renal function. conclusion: pls should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in abo-mismatched hpc transplant recipients, especially when the hpc source is from peripheral blood. as in our patient, it usually takes 7-14 days for antibodies to develop and they are short-lived (3-5 weeks). due to the severity of his manifestations, we performed an emergent rbc exchange successfully. furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for abo-mismatched transplant recipients, which has since been remediated electronically background/case studies: group o rhd negative (oneg) red blood cells (rbcs) are a precious resource. to conserve the oneg inventory while minimizing the risk of rhd alloimmunization in oneg females of childbearing age, transfusion services may automatically provide group o rhd positive (opos) rbcs to rhd negative males and/or rhd negative postmenopausal females during bleeding emergencies. despite these conservation strategies, shortages of oneg rbcs occur. the goal of this study was to determine how the utilization of oneg rbcs can be optimized using agebased opos switching for routine transfusions in oneg patients. study design/methods: recipient age and abo/rhd group were obtained for all allogeneic rbc transfusions during the 2016 calendar year from 9 hospitals. an additional hospital* provided data for august-december 2016. rbc transfusions in patients <1 year of age, and in patients whose age and/ or abo group were unknown, were excluded from analysis. the abo/rhd group of each rbc unit was compared to that of the recipient to determine the number of oneg rbcs transfused to all patients, the number of rbcs transfused to oneg patients and the number of oneg rbcs transfused to oneg patients. the number of oneg rbcs transfused specifically to oneg patients >/5 70 years was also determined. results/findings: see table 1. the fraction of all transfused rbcs that were oneg ranged from 5-14% (row f). the percentage of oneg rbcs transfused to oneg patients ranged from 37-89% (row g); thus, non-oneg patients received 11-63% of the oneg units transfused (row h). hospitals differed widely in the practice of issuing oneg rbcs to oneg patients (68%-100%; row i). overall use of oneg rbcs could have been reduced by 10%-39% if opos units had been given to all oneg patients >/ 5 70 years old (row j). conclusion: during times of oneg shortage, age based opos switching rules may be applied for routine transfusions. this would help to ensure the availability of oneg rbc units for oneg females of childbearing age. rasha eldeeb mohammed* 1 , nehad mohammed 2 , marwa aly 2 and nashwa fahmy 2 . 1 national blood transfusion services, 2 nbts background/case studies: sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients. splenectomy has been shown to increase human leucocyte antigen immunization. the aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans. study design/method: this study was conducted on 206 multitransfused patients who received blood transfusion chronically at our central blood center. they were 129 thalassemia patients (128 bthalassemia patients, one patients with a thalassemia), 10 sickle cell anemia patients and 6 immune hemolytic anemia patients (4 auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients). 29 oncology patients, 32 chronic diseases patients. history and demographic data were documented. all the patients who received blood are examined for the presence of the spleen.our patients were subjected to direct & reverse blood grouping (abo& rh) tests, alloantibody screening and detection. results/finding: statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients. the study revealed that: 32 out of 48 (67%) alloimmunized patients and 16 out of48(33%) non alloimunized patients(p<0.001) . statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form conclusion: patients who had splenectomy had a higher alloimmunization rate removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components. restrictive transfusion triggers rather than specific evidence. therefore, two systematic reviews of a) rbc transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and b) to identify studies comparing strategies were performed. study design/method: methods medline, embase, cinahl, web of science, national guideline clearinghouse, and the trip database were searched from inception to june 2016. screening and data abstraction were done independently by two assessors. for review a, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles. for review b, the primary outcome was rbc utilization. secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality. meta-analysis was done using the mantel haenszel random effects model. results/finding: review a identified 136 articles for data abstraction, where 48 articles were transfusion guidelines. there were 12 guidelines (25%) that made a recommendation, 11 for a single unit and 1 for multiple unit transfusion strategy (table 1) . review b identified 3 retrospective cohort studies that were eligible and data abstraction was performed. all utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period. meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (or 9.4, 95% ci 5.02-17.60), although heterogeneity was high (i 2 597%). conclusion: our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of rbcs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies. additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used. guidelines groups should review research in this area to determine if a recommendation can be made. background/case studies: platelets made with platelet additive solution c (pas c) and treated for pathogen reduction (pr) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma. with the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use. the literature from europe has shown that platelet and red cell use does not increase when pr and pas products are used. evaluation of rbc use at our institution has shown no change in the number of products transfused per patient per month. we are evaluating whether the mixed inventory has led to more platelet transfusions. study design/method: we looked at occasions when patients received all of their platelet transfusions on a single day. by doing this we were able to exclude refractory patients from the analysis. the information obtained from routine quality management audits of transfusions between december 2016 and february 2017 was used for this analysis. the information included the ordering service, product release time, product code, pre and post counts. statistical analysis was performed using minitab. results/finding: during the 3 months, 1723 units of platelets were transfused to 238 recipients. over the 3 months, a median of 4 units was given to each patient with a range of 1 to 69. the overall distribution of products used was 58% plasma, 24% pr, 7% pas f and 11% pas c. thirty percent of patients (n572) received all of their products on a single day. single units were given to 54 patients while 14, 3 and 1 received 2, 3, and 4 units respectively. the distribution by product type was 56% plasma, 25% pr, 13% pas c and 4% pas f. this same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the 3 month period (p5 1.00). the distribution by service was different for the groups receiving multiple units. for single units the distribution was 44% hematologic malignancy, 22% infusion clinic (nos), 13% solid tumor medicine, 11% surgery, and 9% pediatrics. for those receiving multiple units the distribution was 50% surgery and 16% each for solid tumor, hematology and infusion (nos). the chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a p value of 0.022. conclusion: the distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the 3 month period. the patient's clinical service was a better predictor of the use of multiple products than the type of product given. this suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario. the effect of red blood cell transfusion on iron metabolism in critically ill patients margit boshuizen* 1,2 , yvemarie b.o. somsen 2 , maike e. van hezel 2 , marleen straat 2 , robin van bruggen 1 and nicole p juffermans 2 . 1 sanquin research and landsteiner laboratory, 2 academic medical center background/case studies: anemia of inflammation (ai) has a high prevalence in critically ill patients. in ai, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis. ai is treated by red blood cell (rbc) transfusions. it is known that rbc transfusions increase iron level in neonates and thalassemia patients, but the effect of rbc transfusion on iron metabolism during inflammatory processes is unknown. since one unit of rbcs contains 220 mg of iron and 25% of the rbcs are cleared by macrophages within 1 hour following transfusion, rbc transfusion could increase iron levels and iron availability for erythropoiesis. we investigated the effect of rbc transfusion on iron metabolism in icu patients, and additionally compared the effect in septic patients to non-septic patients. study design/method: in a prospective cohort study in 52 icu patients who received one rbc transfusion, different iron parameters were measured before and 24 hours after transfusion, to determine the effect of a rbc transfusion over a period of time. next, the impact of a rbc transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed. plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and il-6 levels were determined. results/finding: in this cohort, serum iron levels were low and did not change following transfusion (4.1 vs. 4.3 mmol/l, p50.69). also, the transfusion had no effect on transferrin saturation (12 vs. 13 %, p50.13), ferritin (531.0 vs. 599.0 mg/l, p50.74) and il-6 levels (35.0 vs. 25.5 pg/ml, p50.09). hepcidin levels increased in these icu patients after rbc transfusion (223 vs 332 ng/ml, p50.01). in septic patients, rbc transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (-2.7 vs. 3.7 % change, p50.05). other iron parameters did not differ between septic and non-septic patients. conclusion: transfusion of one unit of rbcs does not increase iron levels in icu patients. the increase of hepcidin suggests rbc transfusion induced upregulation of hepcidin, despite the absence of a significant increase in il-6 or plasma iron levels. this increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis. in sepsis, rbc transfusion decreases haptoglobin levels, suggestive of hemolysis. in conclusion, rbc transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion. the effects of pas and pr on platelet use barbara mendez, judith delmonte, elizabeth mccabe and joanne becker*. roswell park cancer institute background/case studies: with the anticipated release of the fda guidance: bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion, the use of pathogen reduced platelets (pr) which are often produced from products made with platelet additive solution (pas) may become more common. our institution has been transfusing platelets made with additive solutions since 2011 and pathogen reduced platelets have been available since 2016. in our data validating pas and pr, the post counts from transfusion of pas-c and pr products have been statistically lower than platelets in all plasma (pp) or pas f products. our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused. study design/method: the data was obtained from the routine quality reports produced for the blood utilization committee at our facility between 2012 and 2016. during this time pas c, pas f and pr went from 13% to 40% of all platelet products given. all recipients had an oncology diagnosis. the data collected included the service, unit number and product code. the number of unique recipients was determined monthly. the data was converted to plt/month/recipient for analysis. statistical analysis was performed using the two sample t-test results/finding: the data was normalized to plt/recipient/month. in 2011 patients received an average of 5.41 units/recipient/month and in 2016 the average was 5.39 units/recipient/month. the intervening data points for 2013, 2014, and 2015 were 5.92, 5.66, and 5.92 respectively. the 5 year average was 5.66. the slope of the graph for all 5 points was y5 -0.004 15.672. the two sample t-test showed that the plt/recipient/month from 2012 to 2016 was not statistically different with a p value of 0.81. conclusion: the implementation of pas and pr platelets in the oncology environment has not increased in the number of platelet transfusions given. in additional analysis, the red cell use has decreased (data not shown). this can be interpreted as indicating that patients have not had increased episodes of bleeding. although the post platelet count from pas/pr platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given. background/case studies: it is reported that the incidence of alloimmunization in aml patients is unrelated to the number of transfusions the patient receives and most patients who have hla antibodies do not exhibit platelet refractoriness. many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests. recent data in leukemia and hematopoietic stem cell (hsct) recipients transfused exclusively with leukoreduced products show that 4% to 8% develop alloimmune platelet refractoriness. objective: to determine an improvement in platelet count with the match grade and/or the abo blood group of the hla matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction. study design/method: clinically documented platelet refractory patients, who received hla matched irradiated sda platelets with their hla typings for hla-a/-b and hla antibody identification were reviewed. there were two strategies utilized, the hla strategy (matching recipient and donor hla-a/ -b types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those hlas to which the patient had antibodies) strategy. statistical analysis: a one sample t-test using minitab 17 statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least 5 k/ul. the analysis revealed that the mean of 9.35 k/ul (n584) had a 95 percent lower bound confidence interval platelet increment of 7 k/ul (p<50.001) results/findings: 123 (median 4 range [1-43]) hla matched leucoreduced irradiated sda platelets were transfused to 17 (6m/11f) patients, median age 60 years (range 27-83). 15/17 (88%) patients showing broad alloimmunization to hla class i/class ii antigens. 2/17(12%) patients had anti-hpa antibodies (gp iib/iiia and gp iib/iiia and gp ia/iia). the majority 16/17 (94%) had a diagnosis of hematologic malignancy (aml/mds/mpn/ cmml/mm); 9/11 (81%) female patients had prior exposure via pregnancy and 4/11 (24%) had a history of hsct. 63 (51%) platelets were abo identical-platelet increment median 7 k/ul (range -14 to 61), 53 (43%) were abo compatible -platelet increment median of 2k/ul (range -10 to 46) and 7(6%) were abo incompatible with platelet increments median 8k/ul ( range -10 to 32). platelet counts were performed within 24 hours in 73 (57%) transfusions. the hla match grade of the transfused platelets were as follows: the use of massive transfusion protocol (mtp) in a community hospital rohini patel* 1 , renee leblanc 2 , dongfu xie 2 , alice cabe 1 and yanyun wu 2 . 1 overlake hospital, 2 bloodworks northwest the use of massive transfusion protocol (mtp) in a community hospital background/case studies: the establishment and use of massive transfusion protocol (mtp) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding. however, it is not well established if the use of mtp also has value in small hospitals and community hospitals, and how mtp is used in fig. 1 these settings, such as indication for mtp, blood products used, and the outcomes of these patients. study design/method: retrospective review of transfusion data from a community hospital with a bed size of about 350 for 3 years (from 2014 to 2016) was performed. patients with mtp requested are included in this study. results/finding: please see the table below for the summary of data. notably, patients with gi bleed and ob bleed are the two most common indications for mtp, and 68 % of patients survived with the support of mtp. in one case, no blood product was used. the establishment and readiness of mtp can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals. in these settings, mtp is most commonly used for patients with massive gi bleed and ob bleed. if the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible. included in the mns system, and residing on glycophorin b (gpb), the u antigen is absent in less than 0.25% of the black population. those with altered forms of gpb, known as u variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). this case illustrates the balance between the need to transfuse and avoiding complications thereof. a 32-year-old ghanaian woman with scd and history of chronic transfusion presented with diffuse pain and a hemoglobin value of 6.4 g/dl (baseline 9-10 g/dl). she is known to be e, c, k, fya, jkb, s, s negative, u variant, and has anti-e, c and u antibodies. there were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible u variant units. the decision to transfuse was made. the patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was 5.9 g/dl. a transfusion reaction work-up was ordered. post-transfusion serum sample was negative for hemolysis and no new antibodies were identified. the post-transfusion dat was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin (8.8 to 6.2 mg/dl). two additional u variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to 6.2 g/dl. the patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline. study design/methods: molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services. conventional methods were used to monitor the patient's condition pre and posttransfusion. results/findings: each donor unit came from a different donor but all were the same gpb genotype as the patient. the patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case. conclusion: transfusion of u variant red cells to a u variant patient should be undertaken with great caution due to epitope and antibody heterogeneity. this case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, scd patient with anti-u. sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff. background/case studies: patients with decompensated waiha may require transfusion with red blood cell (rbc) products that are cross-match incompatible due free autoantibodies. the feasibility of blood transfusions in waiha patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused rbcs may be destroyed more rapidly in patients with active hemolysis. to study the actual vs. theoretical risk of increased hemolysis in waiha patients, we investigated the post-transfusion (post-tfn) hematocrit (hct) change in waiha patients who were transfused compatible rbcs compared to those who received li blood. we further hypothesized that a post-tfn hct would be inversely related to the degree of ahg-phase incompatibility. study design/method: we reviewed all transfusions to patients in our quaternary-care hospital with a history of waiha from october 2015 to march 2017. patient hcts were ordered by prescribing physicians for clinical purposes. a transfusion episode was defined as all units released in the interval before a post-tfn cbc. ahg-phase crossmatch was tube tested in saline per clinical procedure. transfusion medicine physicians determined the release of least-incompatible units. statistical tests were performed with statcalc (epiinfo, cdc) and www.socscistatistics.com. results/finding: there were 139 rbc products transfused to 40 waiha patients. twenty-three (57.5%) patients received at least 1 incompatible unit. the mean age was 51.4 years (range 4-93 yrs) with 50% women. ethnic composition was 55% african-american, 40% caucasian, and 5% patients of mixed/other ethnicity. one hundred fourteen (82%) of these products were released as li products and 25 (18%) were compatible. ninetythree (81.6%) of the li product transfusions had a post-tfn hct change of <3% whereas only 14 (56%) of the compatible product transfusions resulted in a post-tfn hct change of <3% (p50.0092, v 2 (1), exact methods). the mean hct increase in the compatible group was 1.83% per unit vs. a slightly lesser per-unit increase of 1.71% in the li group (p50.82, t-test, 2-tailed) within the li group, there was no difference in the per-unit hct change according to strength of incompatibility (table) . strength of ahg incompatibility was not available for 12 units. units that were 31 incompatible had a lower mean post-tfn hct rise compared to all other li units (1.49% vs. 2.15%); however, this difference was not statistically significant (p50.38). conclusion: the post-tfn hct change for transfusions of li units to patients with waiha was less than the expected 3% per unit more frequently than it was for waiha patients who received compatible products (81.6% vs. 56%). however, likely due to our small sample size, the mean differences were not statistically significant. interestingly, there was no difference in the per-unit post-tfn hct according to differing strengths of incompatibility in our sample, although the mean increase for the 31 li products was less than all other li products combined. the increase was unexpectedly low for weaklyincompatible units, which we are further studying. future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications. transfusion management was summarised by individual hospital, type and total cases. in-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of rbcs in the first 4-hours from mt onset) was calculated with 95% and 99.8% control limits to indicate potential outliers. data were analyzed using statistical software (stata). results/finding: there were 5482 mt cases from 25 hospitals (17 tertiarylevel, 6 smaller/medium sized acute-care and 2 specialist women's). number of mt cases per hospital ranged from 5 to 721. patient median age was 65 years (iqr 49, 76), 62% were male and 73% required admission to intensive care. the most common clinical groups were cardiac surgery (21% cases), trauma (20%) and gastrointestinal hemorrhage (13%); however there was marked variation between hospitals. ratios of transfused products, analyzed according to bleeding context, varied between hospital types. the pooled average adjusted in-hospital mortality for the 17 tertiary-level hospitals was 21% (range 13% to 33%) and 16/17 (94%) were within the 95% control limit. cb that required !10 rbcs within 24-hours of mt onset occurred in 40% of cases. comparison of transfusion management for this subset of mt cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals (67% versus 78%; p50.03). conclusion: patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable. results are made available to participating hospitals in the anz-mtr to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation. data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research. background/case studies: in hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer. with this, infusion fluids, including blood products, are administered under pressure. this is done because veins of trauma patients are often not suitable for infusion of fluids. suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes. there is no information available concerning transfusion of platelets under pressure via a bone needle. the aim of the study was to investigate the effects of warming and administration of a platelet concentrate (pc) under pressure via a bone needle on the in vitro quality of platelets. study design/method: pools of 5 bcs and 280 ml of platelet additive solution iii (pasiii) were used to produce pcs (n55). pcs were stored on a flatbed agitator (60 cycles/min) in a temperature-controlled cabinet at 22 6 28c for 4-7 days. to mimic hospital conditions, pcs were warmed using a blood warmer and transfused via a bone needle to a transfer bag. on the pcs a pressure of 300 mm hg was applied. using clamps, a flow velocity of 90-120 ml/minute was realized. platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters. results/finding: due to priming of the transfusion disposable with saline, the pcs were diluted 10-30%, resulting in a significantly increased pc volume and decreased platelet concentration after simulated transfusion. because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion. after simulated transfusion, the pcs still fulfilled the requirements for platelet concentration (0.8-1.6x10 11 /l) and number (>250x10 9 /unit). simulated transfusion had no effect on the percentages of cd62p and annexin v positive cells, indicating no activation or induction of apoptosis. ph was not influenced by simulated transfusion. due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion. conclusion: warming and simulated transfusion of pcs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets. transfusion of warmed pcs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets. it is recommended to study the in vivo effects in a limited clinical study. alesia kaplan* 1,2 , joan sevcik 2 and joseph e. kiss 1,2 . 1 university of pittsburgh, 2 blood systems inc. background/case studies: low titer a plasma has been safely used as a substitute for ab plasma in trauma patients. low inventories of ab plasma can cause a delay in life saving therapeutic plasma exchange (tpe) procedures in ab patients needing plasma replacement. here, 2 ab non-bleeding patients are presented who safely received ab and low titer a plasma for tpe. one ab patient who received ab plasma only was used as control to compare hemolysis laboratory data over tpe course. study design/method: a retrospective review of tpe procedures for 3 patients was conducted from medical records. number of procedures, volume replaced, total number of plasma units, number of a plasma units, quantity of a plasma and hemolysis laboratory data were recorded. average quantity (ml) for a plasma and % of a plasma out of total volume of plasma used were calculated. all a plasma units were low anti-b titer units. in the laboratory, plasma dilution 1:50 is prepared and tested with reagent b cells. if agglutination is not observed, the unit is labeled as "low titer anti-b". hemolysis laboratory data was traced with linear graphs and trends were compared between patient 1 and 2 and 3 (control). results/finding: all 3 patients were ab blood type. patient 1, a 57 year old female with recurrent adamts13 deficient ttp, received 2 courses of tpe (total 12 tpe procedures) for relapse and exacerbation. ten out of 12 procedures were performed with ab and a plasma (average 916 ml of a plasma or 24% of total plasma volume for 10 tpe procedures). patient 2, a 27 year old female with thrombocytopenia, schistocytes and presumed ttp, received a total of 12 tpe procedures. four out of 12 procedures were performed with ab and a plasma (average 1210.5 ml of a plasma or 48% of total plasma volume for 4 tpe procedures). patient 3, a 33 year old female with adamts13 deficient ttp who served as a control, received a total of 10 procedures with ab plasma only. haptoglobin, ldh, hemoglobin and total bilirubin were graphed and compared between 3 patients. the trends of hemolysis laboratory data for patient 1 and 2 were comparable with patient 3. all 3 patients had negative dat. only patient 3 received 2 rbc transfusions. all 3 patients had a favorable clinical outcome with tpe treatments and adequate platelet recovery. conclusion: in this study, tpe was effectively performed without evidence of increased hemolysis using up to 48% of low titer a plasma. this approach can reduce strains on limited supplies of ab plasma while providing a vital treatment alternative for ab patients undergoing tpe who require plasma replacement. when cd36 negative platelet unit is not available for a patient with anti-cd36 antibodies sameer khatri* 1 , charles harmon 1 , brian r curtis 2 and chisa yamada 1 . background/case studies: refractoriness to platelet (plt) transfusion can be caused by antibodies (abs) against human leukocyte antigen (hla) class i antigens (ags) or less frequently against plt specific ags (psas). glycoprotein iv (cd36) is one of the identified plt surface ags and deficiency is rare, but found in asians (3-11%), sub-saharan africans (7-8%) and also in some people from mediterranean descent. two types of cd36 deficiency have been described. type 1 deficiency is the complete lack of cd36 on both plts and monocyte-macrophages whereas type 2 deficiency lacks cd36 on plts with variable expression (12-99%) on monocytemacrophages. transfusing plts in a patient with cd36 deficiency is challenging given the rarity of cd36 negative phenotype and risk of further immunization when giving ag non-matched platelets. study design/method: a patient with cd36 negative phenotype who received multiple plt units was reviewed in the electronic medical record. results/finding: a 21 year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple plt and rbc transfusions. he received more than 20 units of apheresis plt units over a 2 week period without any significant increase in plt count. cross-match compatible plt unit found in 1 of 32 units and hla matched units were tried without success. at that point, a cd36 ab was identified in the serum and the patient's type 1 cd36 deficiency was confirmed by flow cytometry. his hla class i panel reactive ab (pra) was 95% due to multiple plt transfusions, although all abs were low levels. the patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in plt increase. following three doses of ivig, he received a cd-36 negative (but blood type different and hla 187a transfusion 2017 vol. 57 supplement s3 unmatched) plt unit from his relative with only a slight increase in plt count. however, he started to respond to cd36 non-tested apheresis plts after receiving a fourth ivig and two rituximab infusions. since then, he has received ivig every 2 weeks. other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia. the mean corrected count increments (cci) when post-transfusion plt count was available are shown in table. with desensitization therapy, his cd36 antibody positive reactivity in serial dilutions has reduced from 1:32 to 1:2 dilutions and his hla class i pra has decreased to 37%. he is currently receiving 2 apheresis plt units twice a week and rbc units periodically. his bone marrow (bm) has been slowly recovering evidenced by increased wbc count from zero to up to 1.0 k/ml and slow increase of reticulocyte counts. current plan is rbc/plt transfusion support until bm recovers or a haplo-identical transplant if bm recovery fails. conclusion: we report a case with anti-cd36 abs that received multiple plt transfusions. this case demonstrates that decreasing ab level with immunomodulation can be an alternative option for successful plt transfusion when compatible plts are not available for patients with rare or multiple abs to plts. table: mean available cci for plt transfusions a blood center's experience screening donations for babesia microti using enzyme-linked immunoassay methodology nancy van buren*, jed gorlin, vanessa reynolds and deborah anderson. background/case studies: our blood center, located in an area considered to be moderately endemic for babesia microti, implemented universal screening of red cell collections from minnesota and wisconsin under an investigation new drug (ind) study in oct 2015 utilizing the immunetics investigational enzyme-linked immunoassay (elisa) performed by creative testing solutions (cts). this test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational ifa/pcr test combination. study design/methods: we performed a retrospective analysis of our screening test results and deferral rates for 2016 to evaluate for seasonality, donor abo bias, deferral rates, and outcomes of lookback investigations. since an opt-out of this research test was originally offered, we report donor opt-out rates. results/findings: from jan through dec 2016, 101,854 blood donations were screened for b microti by immunetics elisa. of those, 267 (0.26%) were positive. the percent of positive donations was evaluated monthly revealing a variable reaction rate between 0.08% and 0.42%. no patient babesia transmission has been reported since implementing this test, but we only had 4 documented babesia ttd cases from 2007-2017. donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in minnesota and wisconsin. test performance characteristics were analyzed by abo group with no demonstrable differences in positive rates. the opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test. of 267 positive test results, 160 lookback investigations were initiated representing 59% of positive donations. lookbacks were only performed when there was a donation within 12 months of the new positive screening test, according to ind protocol. no confirmatory testing was performed per ind protocol or for donor counseling, so the true positive rate is unknown. in the prior ind trial, up to 80% were unlikely to transmit infection in our region, i.e. were pcr and blood smear negative. although a small number of antibody positive, pcr negative donors may be actively infected, no transfusion-transmitted babesia infections were identified by lookback investigations. notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup. overall, donor deferral represented 0.25% loss of eligible donors during this follow-up period. deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility. conclusion: testing for b microti may help improve blood safety, particularly in endemic regions. although only 0.25% of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection. a direct test capable of detecting babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal. a reinstatement protocol for donors who test positive should also be considered. nonetheless, the current method of screening is inexpensive compared to pcr-based methods. background/case studies: human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens. they establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter. some studies suggest they are ubiquitous, present in >90% of the human population, and that immune surveillance is required to control the level of the virus load. study design/methods: we have developed a quantitative dna pcr assay for the most conserved region of the anellovirus genome that detects all known genotypes of the virus. we used this assay to examine viral loads in the plasma of us blood donors and transplant recipients pre-transplant and three months post-transplant. results/findings: for 53 blood donors, 51 were positive with an average load of 1.49x10 2 copies/ml of plasma, a median value of 80.5 copies/ml of plasma, ranging from 0 to 1.87x10 3 copies/ml. pre-transplant viral loads were similar. for 41 transplant candidates, 40 were positive with an average of 3.70x10 2 copies/ml of plasma, a median value of 88 copies/ml of plasma, ranging from 0 to 1.18x10 5 copies/ml. post-transplant viral loads were remarkably different. for 94 transplant recipients, all were positive with an average of 3.14x10 5 copies/ml of plasma, a median value of 1.25x10 5 copies/ml of plasma, ranging from 0 to 4.6x10 7 copies/ml. conclusion: these results validate the pcr assay that was developed and confirm that detectable viral loads of around 100-200 copies were present in >90% of the blood donors surveyed. in addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least 2 orders of magnitude above that of non-immunosuppressed individuals. background/case studies: the screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens. furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance. the development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as dengue virus (denv), west nile virus (wnv), zika virus (zikv), yellow fever virus (yfv), usutu virus (usuv) and chikungunya virus (chikv). study design/method: an innovative diagnostic approach combining generic rt-pcr amplification and identification on low cost microarrays has been developed. we have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific mean cci pre-ivig: all plt (17) 0.2 post-ivig: all plt (41) 5.5 post-ivig: cd36-negative plt from relative (1) 0.8 post-ivig: single donor apheresis (23) 4.3 post-ivig: cross-match compatible (15) 6.1 post-ivig: flow cross-match compatible plt (2) 12.6 188a transfusion 2017 vol. 57 supplement s3 detection of the viral genomes. analytical performances of the test were evaluated on viral standards and on clinical samples: denv (1/2/3/4), wnv, zikv and chikv. forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls. we have designed two sets of degenerated primers for the generic rt-pcr amplification of all flaviviruses and for chikv. biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate. after addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader. results/finding: one original generic probe for denv and specific probes designed for each of the four denv serotype, wnv, the two zikv lineages and for chikv, were validated. the use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows 100% of specificity, with no false positive results on the 40 control samples, and no cross reactions. using viral reference standards, we have observed sensitivities of 1 tcid 50 /ml for denv-1, denv-3 and chikv and of 10 tcid 50 /ml for denv-2, denv-4 and zikv. finally, the first results obtained on 110 denv(1), 69 zikv(1) and 50 chikv(1) clinical samples show 85%, 87% and 96% correlation respectively between our approach and commercial or in house real time rt-pcr methods. conclusion: this innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses. this methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections. babesia microti serological testing with pooled samples: a feasibility study laura tonnetti*, aaron thorp, letitia dixon and susan l stramer. background/case studies: blood donation screening for babesia microti, a tick-borne intraerythrocytic parasite endemic in the northeast and upper midwest us, is performed under an investigational study using nucleic acid and immunofluorescence assays (ifa). however, ifa is a time consuming and labor intensive procedure. with the possibility of an fda licensed screening assay(s) in the near future, we investigated if b. microti testing by ifa in pools of plasma or serum could be a feasible screening approach. study design/method: to test if the increased amount of plasma or serum interferes with background fluorescence, pools of 4, 8, 16 and 32 were prepared from 192 plasma or 192 serum samples determined to b. microti-negative by individual ifa screening. the pools were tested by ifa with or without a blocking step using bovine serum albumin (bsa) and goat serum to minimize background fluorescence. potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from 1:128 to 1:1024 (2-fold dilutions) in the pools. results/finding: non-specific fluorescence was visible in pools of 16 or higher and was not eliminated by the addition of a blocking step. pools of 4 or 8 samples did not show significant increased background. there was no difference between testing of pooled serum or plasma samples. when one single positive sample was included in the pools of 4 or 8 samples, the pool tested positive and the final titer was the same as the positive sample tested individually. when two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer. conclusion: this study represents a proof of concept that serological testing for b. microti by ifa in pools of up to 8 plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test. background/case studies: the rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components. the bact/alert virtuo* is an advanced, next generation system with improved automation, connectivity and with data management systems. the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert (bta) bpa (aerobic) and bpn (anaerobic) bottles. as plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for virtuo studies. study design/method: human plasma (thawed and pooled) and saline controls were seeded with $100 cfu/ml of 12 organisms associated with platelet contamination and incubated at room temperature for 18-24 hours. colony counts were performed initially and after incubation. plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher (1 1 log) than the colony count of the seeded saline after incubation. results/finding: the serially diluted strains and all bioball tm strains except p. aeruginosa, nctc 12924, were determined to be plasma resistant. the bioball tm p. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into 4 ml of leukocyte reduced apheresis platelets (lrap) and inoculated into bta bpa bottles and loaded into the bta 3d and virtuo the organism was recovered 100% . conclusion: results confirm that previously tested organisms and additional strains are plasma resistant with the exception of p. aeruginosa, nctc 12924. however, the bpa bottles still recover p. aeruginosa in the presence of lrap. bpa/bpn bottles inoculated with select organisms from this panel in the presence of 4ml lrap demonstrated 100% recovery when loaded onto the virtuo and 3d ( table 1) . further studies may be required to determine if higher test volumes of lrap could affect the recovery of plasma sensitive strains. * virtuo is not fda cleared for platelet testing a. notoscriptus, identified as a major urban vector of rrv, is also capable of transmitting dengue virus 1-4, and has recently been found in los angeles, illustrating an expansion in range. with the growing geographical distribution of aedes species mosquitoes, the potential for rrv to enter local transmission cycles outside of australia is significant. in 2014, a probable transfusiontransmission (tt) was confirmed as the cause for an rrv infection in australia, validating the reality that rrv tt can occur. rrv morbidity leads to clinical manifestations that are similar to chikv infection, with varying degrees of arthralgia, which can become debilitating. various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional tt in endemic areas and could mask the spread of the disease globally. study design/method: platelet concentrates (pc) prepared in pas were inoculated with rrv, amotosalen was added to final concentration of 150 mm and the units were treated with uva light. pre-and post-treatment illumination samples were collected for titration. as-5 rbc units were contaminated with rrv, mixed with processing solution/glutathione (gsh) and treated with amustaline at a final 200 mm concentration. pre-and post-treatment samples were removed prior to amustaline treatment and 3hrs after amustaline addition, respectively, for titration by plaque assay on vero76 cells. log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples. results/finding: inactivation of rrv was achieved to the limit of detection in pc and rbc. in pc, >5.1 log 10 or log 10 /ml of rrv was achieved, with >5.5 log 10 or >5.2 log 10 /ml of rrv inactivated in rbc. conclusion: these studies illustrate that amotosalen/uva and amustaline/ gsh treatments are effective at inactivating rrv in pc and rbc, respectively. these data corroborate previous results achieved with other alphaviruses, including chikv and mayaro virus which are inactivated at high titers in pc and rbc, demonstrating the ability for these systems to mitigate tt potential and maintain safe blood component availability in endemic areas. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). background/case studies: the interceptv r blood system for platelets is designed to inactivate pathogens and contaminating leukocytes. this photochemical treatment process utilizes amotosalen and low energy ultraviolet a (uva) light. the current available sets include small volume (sv; 255-325 ml), large volume (lv; 300-390 ml) and dual storage containers (ds; 300-420 ml) designed to treat platelet doses between 2.9 and 8.0x10 11 . the new triple storage (ts) set was designed to expand the dose range to 12.0x10 11 and the maximum volume to 650 ml, generating either 2 or 3 doses of pathogen reduced platelet components (pc). the objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in pas, or 100% plasma using ts set. study design/methods: for each experiment, a platelet pool was prepared either in 47% plasma/53% pas or 100% plasma with a final volume of $650 ml and a dose of 9-12 3 10 11 platelets. these conditions represent inactivation using the lowest amotosalen concentration (135 mm) and highest concentration of platelets. platelet units were inoculated with high titers of viruses, or bacteria and treated. control (pre-uva) and test (post-uva) samples were serially diluted and cultured. plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays. log reduction was calculated as the difference between the log 10 titers in control (pre-uva) and test (post-uva) samples. conclusion dromedary camels were identified to be the reservoir of mers cov, transmission to humans occurs through direct and indirect contact. mers cov has been detected with high genomic titers of 6-10 logs in respiratory secretions of mers patients, and with lower genomic titers of 4-5 logs in blood. the presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas. the high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude mers cov contamination of blood products. pathogen reduction with amotosalen/uva technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of amotosalen/uva treated blood products. the aim of the study is the assessment of the mers cov inactivation efficacy in human plasma with amotosalen/uva pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units. pre-uva titer post-uva titer log 10 reduction/ml (log 10 /ml) 47%plasma/ 53% pas e .coli 6.0 <-1.0 >6.0 e. cloacae 6.4 <-1.0 >6.4 k. pneumoniae 6.6 <20.1 >6.5 s. aureus 6.7 <-1.0 >6.7 blue tongue virus 4.9 <-1.0 >4.9 bovine viral diarrhea virus 4.6 <-1.0 >4.6 adenovirus-5 1 3.9 <-0.6 >3.9 100%plasma k. pneumoniae 1 6.5 -0.5 >6.5 s. aureus 1 6.2 <-0.7 >6.2 adenovirus-5 1 4.5 <-0.6 >4.5 1 n53 190a transfusion 2017 vol. 57 supplement s3 abstract study design/method: four therapeutic human plasma units were spiked with a fully characterized mers cov clinical isolate followed by pathogen inactivation with amotosalen/uva (intercept blood system, cerus corporation) at four different days. pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-pcr. samples post pathogen reduction have been passaged 3 times up to 9 days, assessing the infectious titer and genomic titer every 3 rd day to exclude the presence of low-titer infectious particles. results/finding: all viral particles in the plasma units were completely inactivated with an average efficacy of !5.8 log infectious titer. no viral replication was observed after 9 days of passaging post inactivation. the genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer. conclusion: amotosalen alone had a slight effect on the infectious titer while amotosalen/uva effectively inactivated all infectious mers cov viral particles in the plasma units with an inactivation efficacy above 5 logs infectious titer, giving evidence for improved blood safety of amotosalen/uva treated plasma in mers cov endemic regions. estimating the prevalence and incidence in a national blood service in taiwan for hcv eradication program yun-yuan chen* 1 , jen-wei chen 1 , chi-ling chen 2 , sheng-nan lu 3 and pei-jer chen 2 . 1 department of research, head office, taiwan blood services foundation, 2 graduate institute of clinical medicine, college of medicine, national taiwan university, 3 division of hepatogastroenterology, department of internal medicine, kaohsiung chang gung memorial hospital background/case studies: world health organization (who) has set a goal to eliminate hcv by 2030, and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness. this study aimed to evaluate the prevalence and incidence of hcv infection in taiwan. study design/method: in taiwan, anti-hcv (since 1992) and 8-sample mini-pools triplex nucleic acid test of hcv, hbv and hiv (since 2013) have been used in the routine blood screening. prevalence of anti-hcv and hcv rna were estimated in the first-time donors during 1999-2016 and 2013-2016, respectively. age-standardized prevalence and its 95% confidence interval (95% ci) were calculated with adjustment of who world standard population 2000-2025. for the incidence study, donors who have donated blood two or more times during 2013-2016 and who were without a history of anti-hcv positive before the follow-up period were included. the incidence and its 95% confidence interval was estimated from the number of new hcv rna positive cases divided by the person-years of follow-up. results/finding: the crude prevalence of anti-hcv in the first-time donors was dramatically decreased from 15.2 per 1,000 donors (95% ci: 14.8-15.7) to 4.0 per 1,000 (95% ci: 3.7-4.3) during 1999-2016, and the agestandardized prevalence was also decreased from 27.0 per 1,000 donors (95% ci: 25.6-28.4) to 7.7 per 1,000 (95% ci: 6.9-8.5). the agestandardized prevalence of anti-hcv was generally higher in female donors before 2015, but it was significantly higher in male donors at 2016 (p-value50.03). a total of 1,036 hcv rna positive cases, 1.9% of them were anti-hcv negative, identified from 579,286 first-time donors during 2013-2016, and the crude and age-standardized prevalence of hcv rna was 1.8 per 1,000 (95% ci: 1.7-1.9) and 5.0 per 1,000 (95% ci: 4.3-5.7), respectively. crude prevalence of hcv rna was significantly higher in female donors (p value <0.0001), but no significant difference was found after age standardization (p value50.93). both the prevalence of anti-hcv and hcv rna were increased with age (p for trend<0.0001). in the incidence study, a total of 68 new hcv rna positive cases, 23.5% of them were anti-hcv negative, found from 1,202,165 donors followed for 2,415,668 person-years. the incidence of hcv rna was 2.8 per 100,000 person-years (95% ci: 2.2-3.5), and no significant difference was observed between both genders (p-value50.41) and between age groups (p for trend 0.37). conclusion: the prevalence of hcv infection has been dramatically decreased by 71.5% during 1999-2013. it becomes significantly higher in male donors and that needs to monitor in the future. incidence of hcv rna is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics. dalia ashour* 1 , sahar muhmmad 2 and dalia el dewi 2 . 1 national blood transfusion services, 2 azhar university background/case studies: blood safety is a challenge in egypt because of the high prevalence of hcv and hbv. nucleic acid amplification test (nat) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion. the primary benefit of nat is the ability to reduce residual risk of infectious wp donations. the estimated reduction of the wp utilizing nat for hcv is 70-12 days, hiv from 22 to 11 days, and hbv from 25-30 days. study design/method: this cross sectional study was conducted in national blood transfusion center (giza, egypt) from 2012 to 2015, the total number of donor samples to be screened is 178685, the age of the donors ranged from 18 to 50 years, and they were of both sexes (m: f 5 3:1).screening by nat ulterio assay (grifols diagnostics; formerly novartis diagnostics) was done in parallel with eia testing for hbsag, hcv-ab and hiv ag/ ab. using individual donation nat (id-nat). multiplex nat yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample. statistical analysis chi-square (v2) test was used to measure the association between two qualitative variables. results/finding: nat screening detected a total of 75 nat yield donations among 178685 (0.04%) seronegative donors. among these 75 nat yields cases, 53 (0.03%) were reactive for hbv, 20 (0.011%) were reactive for hcv and 2 (0.001%) were reactive for hiv-1. we stratified the age of the donors into 3 groups; group a (18 -28 years), group b (29 -39 years) and group c (40 -50 years). the prevalence of nat yield to the three viruses was significantly higher in either group b or c, compared to group a (p 5 0.0089; with 95% confidence interval (ci) 5 0.0085 -0.0520 & p 5 0.0247; with 95% ci 5 0.0025 -0.0534 respectively). prevalence of nat-hbv; was significantly higher in age group b, as compared with group a (p 5 0.0224; with 95% ci 5 0.0032 -0.0413). on the other hand, there was no statistically significant difference between groups c and a and between groups b and c. comparing groups b and c combined with group a found a significantly higher prevalence of hbv in the former (p 5 0.0335; with 95% ci 5 0.0015 -0.0352). nat-hcv; did not differ significantly between the three groups (p 5 0.3222; with 95% ci 5-0.0089 to 0.0161 between groups a and b & p 5 0.1340; with 95% ci 5 -0.0055 to 0.0270 between groups a and c & p 5 0.4277; with 95% ci 5 -0.0080 to 0.0215 between groups b and c). nat-hiv; did not also differ significantly between the three groups (p 5 0.3801; with 95% ci 5-0.0077 to 0.0077 between groups a and b & p 5 0.3172; with 95% ci 5 -0.0056 to 0.0077 between groups b and c). in either group a and c, no nat-hiv detected. nat yield to the three viruses was significantly higher in males than in females (p 5 0.0013; with 95% ci 5 0.0136 to 0.0507). nat hbv was significantly higher in males (p 5 0.002; with 95% ci5 0.0103 -0.0413), but the prevalence of either hcv or hiv did not differ significantly between males and females (p 5 0.3835; with 95% ci 5 -0.0077 -0.0145 & p 5 0.2751; with 95% ci 5 -0.0044 -0.0066; respectively). conclusion: in this study the nat yield of 75 in 178685 assumes more significance when one considers the fact that single donation is used for generating 3 components that can be used by 3 recipients. hence, in effect the nat yield becomes 3 times that is, 225 in 178685. saving 225 recipients from tti out of 178685 (0.13%) is indeed very significant. results/finding: of the 303,569 donors who were tested by our donor center, 1,386 (0.460%) were repeat reactive. a seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring. there was a single case of transfusion transmitted babesiosis reported from our center during this period. a patient who was transfused with two units of packed red blood cells (rbcs) from two donors in the beginning of july presented in august for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis. the donors were called back, however one of them could not be tracked. samples were sent to the state for further testing: an immunofluoresence assay was performed (combination of igg, igm and iga). the test was positive at 1:128 titer. the screening elia s/co of this donor was 0.2782. both donors were indefinitely deferred as blood donors. conclusion: our data confirm a decreased risk in transfusion transmission with the use of a screening assay. prior to implementation of the screening there were 6-10 transfusion transmitted babesia cases per year from 2008-2015 (table 2 ). in the 11 months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed (1 in 303,569 donors tested). thus the babesia eia screening test effectively prevents ttb. however, there was a substantial loss of donors due to being screen positive. four years of experience with id-nat at a tertiary care centre in north india: implications for transfusion transmission and donor screening. jasmeet singh*, amarjit kaur, gurpreet kaur, rajesh kumar and sonia gupta. dayanand medical college and hospital background/case studies: transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe. blood safety is a formidable task especially in a high population country like india. newer technologies like id-nat equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods. this study aims at examining the effect of id-nat as an additional test on the safety of blood supply. study design/method: a retrospective observational study was conducted to analyze the data of 4 years of additional nat testing at blood bank, dmch, ludhiana from september 2012 to december 2016. results/finding: results 1.73% (2041 of 118021) units were initially nat reactive. these units were further tested, of which 90.98% were discriminated (70 hiv, 1051 hcv, 726 hbv and 10 co-infections). the remaining 6.71% (137) were repeat non-reactive and 1.91% (39) could not be discriminated. overall, nat yield rate was one in 837, whereas virus-specific nat yield rates were one in 59,010 for hiv, one in 1873 for hcv, one in 1639 for hbv and one in 29,505 for hbv/hcv coinfections, respectively. conclusion: id-nat screening of all blood donations at our institution over past 4 years has increased the screening sensitivities to check viral load and prevented transmission of 141 probable transfusion transmitted viral infections. assuming 100 % component preparation it saved 423 transfusion recipients from harm. implementation of nat along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers. min xu 1,2 , wei mao 3 , tao he 3 , yashan yang 1,2 , zhan gao 1,2 , chunhong zhang 3 , hongmei liao 3 , jingxing wang 1,2 and miao he* 1,2 . 1 institute of blood transfusion, chinese academy of medical sciences & peking union medical college, 2 sichuan blood safety and blood substitute international science and technology cooperation base, 3 chongqing blood center background/case studies: many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients. with more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations. therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety. study design/method: pooled plasma sample were collected from 5,000 voluntary blood donors from chongqing, china. total dna and rna were extracted and amplified with random primers pcr respectively in order to construct a 250pe library to peform deep sequencing by illumina miseq. all reads were trimmed to remove low quality bases and adapter sequences. the fully overlapping paired-end reads passing the quality filter were concatenated using pear. we classified the final reads using kraken and a kraken database made from complete refseq bacterial, archaeal and viral genomes, along with the grch37 human genome. the unclassified reads by kraken were aligned to ncbi nt database using blastn with cut-off evalue as 1e -5 . the best alignment hits were used to classify the reads. krona was used to generate all taxonomic distribution plots. finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience. abstract results/finding: 1.23 gb raw data with 2,450,046 reads were generated in the dna library. meanwhile, 1.98gb raw data with 3,967,242 reads were generated in the rna library. after cleaning the human background, 211 reads from bacteria, 98 reads from viruses, and 341 reads from parasites were identified (table 1) . no hazardous viruses were identified as potential threats to blood safety. except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as plasmodium sp. and leishmania infantum (table 1) . conclusion: the investigation has revealed the metagenomics of the qualified blood donations in chongqing, china. the results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety. the displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety. however, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens. the validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens. background/case studies: the caribbean has become an endemic region for several emerging viruses in the last decade. after a chikungunya outbreak in 2015 most recently zika was shown to be endemic on the caribbean island of curacao. to effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply. pathogen reduction (pr) is considered an important new approach with potential benefits. the introduction and experience of use of pr platelets in the dutch caribbean over a period of one year is presented. study design/method: pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (mirasol prt, terumo, belgium) was introduced. all thrombocyte concentrates provided to the general hospitals on the dutch caribbean islands of curaçao, bonaire and sint maarten were pr and data collected over the period of 1 february 2016 to 1 february 2017. thrombocyte concentrates are prepared out of 4 single donation units by the buffycoat method. results/finding: over the period 260 platelet concentrates were provided to adult and pediatric patients. these included patients on the intensive care and neonatal intensive care departments. no adverse events were reported and the cci for each transfusion was within the expected outcome. introduction of pr had minimal impact on the logistics of thrombocyte concentrate preparation and availability. furthermore no transfusion related bacterial contaminations were reported. conclusion: pr of platelet concentrates seems viable and safe for use in a small scale caribbean setting with endemicity for emerging viruses like chikungunya and zika. it offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability. michael phillips* 1 , germ an leparc 2 , phillip c williamson 1 , lani palmer 1 , ben reynolds 1 , maria noedel 1 and lindsey houghton 1 . 1 creative testing solutions, 2 oneblood background/case studies: due to the risk of travel and sexually transmitted zika infections, the food and drug administration issued a guidance document on february 16, 2016 recommending that blood centers in puerto rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by march 1, 2016. with the high incidence of zika virus (zikv) in puerto rico and uncertainty of the impact to the continental u.s. blood supply, there was intense pressure to implement a donor screening test for zikv. the project was initiated on february 16, 2016 and included clinical trial requirements, client onboarding and laboratory operations. stakeholders consisted of clients, the manufacturer, institutional review boards (irb), informational technology (client and lab based), the food and drug administration (fda), the centers for disease control cdc, and the florida department of health. clinical trial requirements included development of instrument and assay validations, sop creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling. client onboarding began with confidentiality agreements between the client and the sponsor. a zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities. the complexity of the project increased when mosquito borne zika transmission was identified in two counties in florida. this required zikv testing to be performed on collections in both florida and puerto rico. the zikv-nat is performed in singlet, unlike the mpx and wnv assays which are run in minipools. this had a significant impact on instrument capacity. despite these obstacles and the changing regulatory requirements, the zikv screening test was implemented within six weeks. study design/method: one metric used to measure client service levels is our ability to meet established upload time goals for individual clients. the percentage of samples released on time is evaluated daily with a running monthly total. our upload time goals were negatively impacted from july through september due to the unexpected increase in zikv testing, the requirement to perform testing in singlets and the resulting instrument capacity issues. additional instruments were sourced in october and operations stabilized. conclusion: on february 16, 2016, the project to implement a zikv ind test was initiated. six weeks later, testing was performed on the first batch of samples. despite the changing regulatory requirements over time, the implementation was extremely successful. initiating a new ind testing within 6 weeks is unprecedented and required exceptional collaboration between all participants and stakeholders. background/case studies: plasmodium falciparum (pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in africa. in 2015, who reported $212 million new cases worldwide, resulting in >400,000 deaths. malaria prevalence is highest in sub-saharan africa, home to 90% of all infections accounting for 92% of mortalities. both the incidence and prevalence of malaria in africa significantly increase the potential for transfusion-transmission (tt), with little to no screening of products in developing countries. the objective of this study was to evaluate the inactivation of pf in whole blood (wb) using a system specifically developed for the realities of the developing world and in support of the swiss red cross humanitarian foundation for whole blood pathogen inactivation for africa. the inability to consistently supply blood components leads to routine wb transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust wb pathogen inactivation system is desirable. the approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication. the process includes addition of 0.2 mm amustaline and 2 mm glutathione (gsh) and a 24h at room temperature (rt) incubation after which the treated wb unit is suitable for storage up to 7 days at rt. study design/method: for each experiment, a wb unit was spiked with ring-stage pf-infected red blood cells (irbc). a pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed 24h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation. these samples were serially diluted in flasks containing medium with 5% fresh rbcs. the diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting irbc in blood smears and by flow cytometry. pretreatment cultures were terminated after reaching >1% parasitemia, while no residual pf was detected in post-treatment cultures. log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples. results/finding: robust inactivation of pf in wb was achieved to the limit of detection, at >7.5 log 10 or >6.0 log 10 /ml. conclusion: pf was inactivated to the limit of detection in wb after treatment with amustaline/gsh, illustrating that the system has potential to mitigate the risk for pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on wb transfusion. (this system for wb is not approved for commercial use). increased patient safety and improved inventory management with 7 day apheresis platelets nancy m. dunbar* and zbigniew m. szczepiorkowski. background/case studies: a pathway currently exists for apheresis platelet (ap) outdate extension from 5 to 7 days using an fda cleared rapid test (rt). in february 2016, our hospital based transfusion service implemented the use of rt on day 5, 6 and 7 to routinely extend ap shelf life to 7 days. prior to this, we tested aps by rt on day 4 and transfused day 6 or day 7 units with physician approval when deemed medically necessary. this report describes changes observed in transfusion practice and platelet inventory management one year following routine use of 7 day platelets. study design/methods: data were obtained for two 12-month study periods: october 2014-september 2015 (pre-implementation) and february 2016-january 2017 (post-implementation). the interval transition period was intentionally excluded. for each study period, we determined the total number of aps transfused, rt status on the day of transfusion, total number of rts performed, expired ap units, and aps obtained from suppliers using ad-hoc ordering. we also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index. results/findings: data are shown in table 1 . the number of ap transfusions increased by 7% post-implementation, comparable to a 4% increase in inpatient admissions and an 11% increase in surgical volumes. the hospital length of stay and case mix index were similar for both periods. the average number of platelet transfusions per patient was not statistically different (3.16 pre; 3.12 post, p50.91). the number of rts performed increased by 130%. the percentage of transfused units tested at least once by rt prior to transfusion increased by 21% (p<0.0001). the outdate rate decreased from 5% to 2% (p<0.0001). ad-hoc ordering decreased from 21% to 9% (p<0.0001). conclusion: use of an approved rt for routine ap outdate extension to day 7 was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion. increased cost of rt was offset by reduced ap waste and less frequent need for ad-hoc ordering. sheila o'brien* 1 , vito scalia 1 , carla osiowy 2 , michael carpenter 2 , anton andonov 2 and margaret fearon 1 . 1 canadian blood services, 2 public health agency of canada background/case studies: the rates of hepatitis b (hbv) and hepatitis c virus (hcv) positive donations are low (6.6 and 4.9 per 100,000 donations, respectively) and most are among first time donors. we aimed to determine the frequency of various genotypes of hbv and hcv in canadian blood donors confirmed positive for hbv and hcv. study design/methods: in 2011 the roche multiplex assay (hcv/hiv/ hbv) was implemented in minipools of 6 units. hcv nat was in place since 1999 (using minipools of 24) but this is the first time donors have been screened by hbv nat. hbsag, anti-hbc and anti-hcv were tested using the abbott prism assay. confirmatory testing for hbsag was by the prism neutralization assay. anti-hcv repeat reactivity was confirmed by the inno-lia hcv score line immunoassay. since march 2016 all samples testing hbv nat positive, or confirmed positive for hbsag and all hcv nat positive or anti-hcv confirmed positive samples were considered positive and samples were sent to phac for sequencing. a sample from each positive donation was aliquoted and frozen at -20 o c. genotyping was carried out by sequence and phylogenetic analysis of the hbv surface antigen coding region. hcv viral rna was extracted and subjected to reverse transcription and pcr amplification in the 5' ntr-e1 and ns5b regions. sanger sequencing of these regions represents approximately 15% of the genome. results/findings: all confirmed positive donations were whole blood donations. there were 42 hbv positive donations. of these, 37 had tested hbv nat positive. genotypes were 8 type a, 6 b, 4 c, 17 d and 2 e. there were 5 samples hbv nat negative but hbsag positive (2 were anti-hbc reactive). of these, 4 could not be sequenced and one was genotype a (also anti-hbc reactive). there were 30 samples considered hcv positive. of these, 17 samples were hcv nat positive. genotypes were 5 type 1a, 3 1b, 3 2c, 2 2b and 4 3a. there were also 13 samples hcv nat negative but anti-hcv positive. none of these could be sequenced. conclusion: the first 8 months of molecular surveillance show a range of genotypes for hbv and hcv for samples identified as nat positive. to date no samples that were nat negative anti-hcv reactive could be sequenced, however one nat negative sample that was positive for hbsag and anti-hbc reactive was hbv genotype a. surveillance over a longer period is background/case studies: the bact/alert 3d microbial detection system (bta 3d) is currently fda cleared for the quality control testing of leukocyte reduced apheresis platelets (lraps). the bact/alert virtuo microbial detection system (virtuo) (biom erieux, st. louis, mo) is a new generation of bact/alert instrumentation. the underlying colorimetric technology used in previous generations of bact/alert is used in the vir-tuo and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection. the objective of this study was to compare the performance of the virtuo and bact/alert 3d (bta 3d) instruments, using bact/alert bpa (aerobic) and bact/alert bpn (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (lraps).* study design/method: the study was performed at two institutions, one in the us and the other in the uk. aliquots of lraps were seeded with low levels (1-20 cfu/ml) of 11 bacterial species commonly associated with platelet contamination, and 20 replicates (10 per instrument) of 4 ml aliquots per bottle were inoculated into bpa and bpn bottles. one set of bottles was loaded into bta 3d and the other into virtuo and incubated until signaled positive by the instruments or for up to 7 days. overall detection rates and time to detection of bacterial contaminants between instruments were compared. additionally 98 bottles were tested in each instrument (lraps only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations. background/case studies: the implementation of nucleic acid testing (nat) blood screening is still a challenge in resource-limited countries. at the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited. a higher prevalence of infections is observed in relation to developed countries. as a consequence, more incident cases of infections can be expected. in our country, some hospital blood banks could not afford nat due to high costs, but belong to a net that centralizes nat in a reference blood center. the process to consolidate small blood banks in regional blood centers, which will be able to implement nat, is not yet complete. although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved. the aims were to compare the prevalence of hiv, hcv and hbv by nat screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the nat yield rates for hiv, hcv and hbv in a period of three and a half year experience. study design/method: a regional blood donor center (rbdc) has centralized nat screening from centers in different regions of the country due to since august 2013. this process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results. when a window period was suspected, the nat screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm nat results. this rbdc have also been developed a 100% voluntary donor program since 2011 and is the only center in the country that has achieved this goal. results/findings: a total of 264,343blood donations were studied from august 2013 to december 2016. in the rbdc, where only voluntary blood donations are recruited, the prevalence was 18 per 100,000 donations for hiv (ic95% 8-34:100,000); 14 per 100,000 for hbv (ic95% 7-29:100,000) and 18 per 100,000 for hcv (ic95% 8-34:100,000). in all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was 89 per 100,000 donations for hiv (ic95% 77-103:100,000); 70 per 100,000 for hbv (ic95% 59-83:100,000) and 78 per 100,000 for hcv (ic95% 66-91:100,000). window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving nat yield rates of 1: 66,086 for hbv; 1: 132,172 for hiv and 1: 264,343 for hcv. conclusion: the hiv, hbv and hcv prevalence was lower in a center where the tasks to sustain a voluntary blood donor program were developed. nat yield rates could be reduced in the region if this program could completely be applied in all centers. mechanisms leading to obi include various factors such as imperfect host's immune response and viral variation factors. this study was to determine the viral loads of obi under currently recruitment and screening among blood donors in five blood services of zhejiang province, china. study design/method: before donation, the donors were screened and precluded with hbsag preliminary test positive and alt level abnormal. following, the samples were detected for hbsag twice using different elisa reagents and hbv dna using tma or qt-pcr techniques. then, the samples with hbv dna positive and elisa negative were tested for the viral loads using taqman technique in cobas s201 system. hbv s region was also sequenced. results/finding: 234 obi were found in the 230,000 donations. in the viral loads assay, 43 samples were negative and 104 samples' viral loads were lower 20iu/ml. the mean viral loads was 1.85 6 0.41 (log10) iu/ml in other 87 samples,while the mean viral loads with hbsag1/hbv dna1 samples was 2.38 6 0.83 (log10) iu/ml. 60 samples of obis have analyzed the hbv genotype, which b was the most prevalent subtype (69.0%) and the other was hbv c genotype(31.0%). compared the samples with hbsag1/hbv dna1 ,we found two obi samples carrying with 318t>c mutation, which could cause an amino acid s55f. conclusion: in this study, the viral loads of obi infection in donors was much low than hbsag1/hbv dna1, and some unique variation was identified in the obi individuals. 195a transfusion in general population. screening of blood donors for hbv in india is primarily based upon detection of hepatitis b surface antigen (hbsag) in donor's sera. the current study was undertaken to determine the prevalence of occult hbv infection (obi) in voluntary blood donors and to analyze the burden of hbv window period donations. study design/method: this is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (nabh) accredited apex blood bank, located in maharashtra state, india. monolisa hbsag ultra (bio-rad, france)sandwich type elisa using monoclonal and polyclonal antibodies was used for hbsag detection in donor's sera. all the elisa non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (mpx-pcr) by cobasv r taqscreen mpx test. the donors which were found to be positive for hbv dna were followed upat 15 th days, 1month, 3 months& 6 months by monolisa hbsag ultra (bio-rad, france) to analyze interval of window period and to delineate the window period donations (wpd) & true obi. background/case studies: occult hepatitis b infection (obi) is characterized by hepatitis b virus (hbv) dna-positive, but hbv surface antigen (hbsag) -negative. since may 2015, we have been testing apheresis donors for hbv nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection. the number of apheresis collections increased significantly year by year, however, data on hepatitis b virus marker rates among these donors continue to be lacking. the aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of obi among apheresis donors in a region of central china. study design/method: apheresis donors' data from may 2015 to dec 2016 was retrospectively analyzed. all samples were tested for hbsag, hbv dna, and other markers. nucleic acids testing (nat) was performed on the roche cobas s201 platform using pools of 6 serologically negative samples and any pools positive would undergo nat again individually. hbsag negative, but hbv dna positive were further tested for hbv dna quantitative pcr, antibody to hepatitis b surface antigen (hbsab), antibody to hepatitis b core antigen (hbcab), hepatitis b e antigen (hbeag) and antibody to hepatitis b e (hbeab). results/finding: in the evaluation, 68547 seronegative donations were screened by nat and a total of 20 hbv dna-reactive/hbsag-negative donors were detected. no hiv rna -reactive or hcv rna -reactive sample was detected. complete serologic screening of the index donations indicated that the majority of these donors had an occult hbv infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations. age distribution of the age group 31-55 years old showed a large proportion, who accounted for 80% of reported infections. most of the hbv dna cases (about 80.0%) reached senior high school education. the average hbsag dna positive rate was 0.029% (20/ 68547). incidence among apheresis donors in this period for hbsag dna were 2.91/10000. these estimates were comparable to those among repeat whole blood donors. we developed pathogen reduced (pr) cryo derived from ffp and pf24 with 5 day stability at 228c. study design/method: six replicates of type-matched pools of whole blood derived (wbd) and apheresis (aph) plasma were split to produce conventional control (225 610 ml) and test components (625 ml 625 ml). test components were pr with amotosalen and uva light. aph and wbd ffp were produced by freezing plasma within 8 hr and wbd pf24 within 24 hr. cryo was manufactured according to site sops and frozen at -308c (test 62 6 2 ml, control 22 6 2 ml ). test and control cryos were thawed at 378c, and characterized immediately post thaw (t50), and after 5 d storage at 228c and tested for fb and fviii function, thromboelastography (rotem) and thrombin generation (cat). results/finding: pr cryo retained sufficient fb and fviii activity post thaw and over 5d at 228c (table) for hemostatic capacity. rotem (extem) showed retention of fibrin formation (a angle) and clot quality (mcf) (table) . thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (etp), and time to peak (tt) despite lower fviii levels. these parameters were maintained through 5d storage at 228c. conclusion: pr cryo can be processed from 3 plasma sources, including pf24, and stored at rt for 5 days. pr plasma provides adequate levels of fb with hemostatic capacity equivalent to control as demonstrated by rotem and cat. use of pf24 with stability over 5 days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection. cryo produced with psoralen-treated (pr) plasma is not approved for use in the us. performance of a new automated alinity s assay for antibodies to t. cruzi darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , lynne fleischmann 1 , mirjana sarac 3 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics, 3 abbott gmbh & co. kg background/case studies: the parasite, trypanosoma cruzi (t. cruzi), is the cause of chagas disease which is endemic to the americas and infects 6-8 million people. in order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-t. cruzi assays with good specificity and sensitivity. in nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of the new automated chemiluminescence immunoassay for the detection of antibodies to t. cruzi was evaluated on the alinity s automated platform and compared to another onmarket chemiluminescent immunoassay. precision was assessed over 20 days using a panel of positive and negative samples. sensitivity was evaluated on 407 presumed antibody positive specimens and specificity was evaluated on 7621 random blood donor samples. results/finding: precision was 7% cv or less for positive samples over 20 days. the overall specificity in a blood donor population was 99.99% (7620/ 7621). sensitivity was 100.00% for 407 presumed antibody positive samples. conclusion: these results indicated that the new automated alinity s chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-t. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries. performance of a new automated alinity s assay for hepatitis b surface antigen and hepatitis b surface antigen confirmatory randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , lynn martin 1 , daniela kaleve 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: despite the development of sensitive nat methods, blood transfusion in many parts of the world relies on serologic screening for hepatitis b surface antigen (hbsag) to prevent transfusion transmitted hbv infection. sensitive hbsag assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection and confirmation of hbsag was evaluated on a next generation automated platform, abbott alinity s. precision was assessed over 20 days. sensitivity was evaluated using 511 known positive samples, 30 commercially available seroconversion panels, the who standard, 23 hbsag mutants, and 94 hbsag genotyped specimens (a through h). specificity was evaluated on random blood and plasmapheresis donors. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.98% (7998/8000). sensitivity was 100% for 511 presumed positive samples. sensitivity was 100% for all genotypes. 100% of the mutants were detected vs 83% for the comparator assay. seroconversion detection was equivalent to the comparator assay with 157 reactive samples detected with the alinity s assay and 154 reactive samples detected by the comparator assay. analytical sensitivity ranged from 0.015 to 0.016 iu/ml. the alinity s hbsag confirmatory assay confirmed all known positive hbsag specimens, including 3 hbsag mutant samples that were not confirmed by the comparator hbsag confirmatory assay. conclusion: the new automated alinity s hbsag assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay. however, the alinity s hbsag assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants. performance of a new automated alinity s immunoassay assay for hiv darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , kevin callear 1 , susan sullivan 1 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics background/case studies: blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus types 1 and 2 (anti-hiv-1/2). blood centers require very high throughput anti-hiv-1/2 assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen. study design/method: the performance of the new chemiluminescence combination immunoassay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen was evaluated on the abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors. sensitivity was evaluated using presumed positive samples for hiv-1, hiv-2 and hiv group o antibodies and hiv-1 p24 antigen. seroconversion sensitivity was evaluated with 41 commercial seroconversion panels. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.96% (8082/8085). sensitivity was 100% for 813 presumed antibody positive samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. also, sensitivity was 100% for 102 antigen positive viral isolate samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 135 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s hiv ag/ab combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay. performance of a new automated alinity s immunoassay for the detection of anti-hbc antibodies randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , joyce siregar 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: in countries with a low prevalence of hepatitis b, blood donations are commonly screened to detect the presence of antibodies to hepatitis b core antigen (anti-hbc) alongside hbsag and hbv nat to detect donors with occult hepatitis b infections (obi). blood centers require anti-hbc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-hbc on the alinity s system. study design/method: the performance of a new chemiluminescence anti-hbc assay for the detection of anti-hbc antibodies was evaluated on the next generation automated abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors. sensitivity was evaluated using specimens characterized as anti-hbc positive by means of serologic methods. analytical sensitivity was assessed using the who 1st international standard. seroconversion sensitivity was evaluated using 10 commercial seroconversion panels. results/finding: precision was less than 6% cv for positive samples over 20 days. the blood donor specificity was 99.93% (6946/6951). sensitivity was 100% for 500 samples presumed to be anti-hbc positive. analytical sensitivity results on the alinity s anti-hbc assay ranged from 0.57 to 0.62 iu/ml. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 134 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s anti-hbc assay provided good performance in specificity, sensitivity and precision versus the comparator assay. performance of a new automated alinity s immunoassay for the detection of htlv i and htlv ii antibodies melanie anderson* 1 , anton vanweert 2 , ed bakker 2 , mark paradowski 1 , jane bryant 1 , tuan bui 1 , joyce siregar 1 , george chen 1 , george schlauder 3 and gregg williams 1 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott diagnostics background/case studies: in endemic countries, universal blood screening is necessary to prevent transfusion transmitted htlv infections (anti-htlv i/htlv ii). in non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high htlv prevalence. blood centers require high throughput anti-htlv i/htlv ii assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against htlv-i/ii antibodies for the alinity s system. study design/method: precision was assessed over 20 days using htlv i and htlv ii positive samples. specificity was evaluated using 8,001 blood donor specimens from europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 500 preselected htlv i and htlv ii positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using the mp diagnostic htlv blot 2.4. results/finding: imprecision was less than 7.0% for positive samples over 20 days. clinical sensitivity was 100.00% (500/500) on preselected htlv i and htlv ii positive samples. the specificity was 99.98% (7,999/8,001) on a blood donor population and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new alinity s automated htlv i/ ii assay provided very good performance in specificity, sensitivity, and precision. sensitivity and specificity were comparable to the comparator assay. claudia ramirez 1 , michel garcia* 2 , fernando palomino 3 and guillermo orjuela-falla 1 . 1 national blood bank colombian red cross, 2 universidad del rosario, 3 fuats background/case studies: current hepatitis c virus (hcv) supplemental testing algorithm for blood donations in colombia, requires that an immunoblot assay be performed on every hcv enzyme immunoassay (eia) repeatreactive sample. a higher proportion of indeterminate (ind) results by immunoblot assays has been documented for non-us donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors. this work aimed to establish the distribution of immunoblot results in colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots. study design/method: in total, 387 anti-hcv-reactive donor samples (signal-to-cutoff (s/co) ratio greater than 1.0; abbott architect i2000sr) underwent supplemental testing by immunoblot (either chiron riba hcv 3.0 sia or hcv blot 3.0 test, mp diagnostics). negative (neg), indeterminate (ind) and positive (pos) blot results were grouped by s/co ranges as follows: 1-4.99, 5-9.99, >10. band detection and intensity were independently analyzed for indeterminate and positive results. results/finding: immunoblot results were negative in 57.9% (224/387) of samples, indeterminate in 30.7% (119/387) and were positive in 11.4% (44/ 387). a direct relationship was observed between positive immunoblot and increased s/co. the proportion of ind results were higher in the s/co group 5-9.99 (63.2%) compared with the 1-4.99 (29.9%). in samples with indeterminate results, ns3_2 was the most frequent band detected (52,9%). in contrast, the most frequent band in the group of positive results was core (93,2%). only one sample from the indeterminate group (0.8%) had a strong band intensity (31), compared with 10 samples from the positive group (22.7%). conclusion: the proportion of indeterminate immunoblot results in this sample of colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of us donors. the high proportion of ind results found in the s/co group (5-9.99) suggests that the optimal s/co ratio for predicting a confirmed anti-hcv result in this population should be higher than the one recommended by the cdc for us population (>5). overall, these results suggest that the supplemental testing algorithm for blood donations in colombia could be improved not only by using high s/co ratios as an alternative to immunoblot, but also by introducing hcv genomic assays instead of immunoblots, at least for samples with intermediate s/co ratios. ns3_1 and ns3_2 cross-reactivity in colombian population warrants further investigation. performance of the alinity s immunoassay for the detection of syphilis antibodies melanie anderson* 1 , ivanka mihaljevic 2 , manuela miletic 2 , miljana stojic vidovic 2 , irena jukic 2 , jane bryant 1 , mark paradowski 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 croatian institute of transfusion medicine, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: blood donations are commonly screened for syphilis in order to detect the presence of antibodies to the bacterium treponema pallidum. in addition, continued pressures on laboratory operations demand that the full panel of ttid assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to t. pallidum. study design/method: performance of the new automated chemiluminescence immunoassay for the detection of antibodies to treponema pallidum was evaluated on the alinity s system. precision was assessed over 20 days using positive samples. specificity was evaluated on samples obtained from 9,101 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 514 preselected positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm with 3 confirmatory assays, inno-lia tm syphilis score, and mikrogen recomline treponema igg and igm blots. results/finding: imprecision was less than 6.0% cv for positive samples over 20 days. clinical sensitivity was 100.00% (514/514) on preselected syphilis positive samples. the specificity was 99.97% (9,063/9,066) for blood donor specimens and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new automated alinity s syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay. performance of the new automated alinity s assay for anti-hcv melanie anderson* 1 , ed bakker 2 , anton vanweert 2 , jane bryant 1 , mark paradowski 1 , tuan bui 1 , lynn martin 1 , george chen 1 , gregg williams 1 and george schlauder 3 . 1 abbott laboratories, 2 sanguin diagnostics, 3 background/case studies: serological screening for antibodies to hepatitis c virus (hcv) often in conjunction with nucleic acid testing (nat) is used worldwide to prevent transfusion transmitted hcv infections. while nat provides improved sensitivity and detection of hcv in the pre-seroconversion window, serological testing provides continued detection of hcv in infected individuals and individuals with resolved infections with no detectable hcv rna. blood and plasma centers require very high throughput anti-hcv assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection of antibodies to hcv was evaluated on the alinity s system. precision was assessed over 20 days evaluating positive samples. sensitivity was evaluated using 501 preselected positive samples and 30 seroconversion panels. specificity was evaluated on samples obtained from 8,113 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the inno-lia tm hcv score and nat/hcv discriminatory nat assays. results/finding: imprecision was less than 7.0% cv for positive samples over 20 days. overall clinical sensitivity was 100% on 501 preselected anti-hcv positive samples. seroconversion sensitivity was better than the comparator as evidenced by the new anti-hcv assay identifying 5 more bleeds than the comparator assay. the specificity was 99.99% (8,111/8,112) for blood donor specimens and 98. 98% (194/196) background/case studies: zika virus (zikv), which has been outbroken in south america and the united states since middle of 2015, was declared as the public health emergency of international concern by who in feb 2016. in addition to mosquito, zikv can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion. the potential for transfusion-transmitted zika virus was shown in french polynesia where 2.8% of asymptomatic blood donors tested were positive for zika virus rna using nucleic acid test (nat). several case reports have confirmed that zikv can be transmitted by transfusion. it has been shown that among blood donors, 73.8% of the zikv infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in micronesia was approximately 1:5 to 1:6. thus zikv has raised a great challenge to transfusion safety. measures should be taken to prevent transfusion-transmitted zikv, including temporary deferral of blood donors in epidemic locations, donor self-reporting of zikv symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, nat of blood donations, and pathogen inactivation of blood products. in this study, we evaluated zikv inactivation in plasma by using methylene blue photochemical treatment (mbpt). study design/methods: plasma units from randomly selected healthy donors were collected and spiked with zikv. samples were added by mb at a final concentration of 1lm and assayed after illumination with visible light from both sides for 5, 15, and 30min. viral infectivity and zikv rna loads (reverse transcription pcr) were measured in spiked plasma before and after mbpt and confirmed using repetitive passages in cell culture. control was zikv spiked plasma without photochemical treatment. results/findings: zikv titer of control sample was 4.5 log 50% tissue culture infectious dose (tcid 50 )/ml. no viral infectivity was detected after mb photochemical inactivation treatment for 5min, 15min or 30min and the losses of the infectivity were further demonstrated by 3 repetitive passages of cell culture. meanwhile, zikv rna loads decreased significantly during the initial 5min of treatment whereby ct-value jumped from 18.25 (control) to 25.50 (mbpt for 5min) (table 1) . conclusion: it showed that mb photochemical treatment could effectively inactivate zikv in plasma. rna lesions were induced during mbpt process so that nucleic acid reverse transcription and amplification were inhibited. mbpt is proved to be an efficient method to prevent plasma transfusiontransmitted zikv infections. gilles delage* 1 , margaret fearon 2 , susan l stramer 3 , megan l nguyen 3 , france bernier 1 , sheila o'brien 2 , vito scalia 2 , sakina smith 3 , yves gr egoire 1 and boris hogema 4 . 1 h ema-qu ebec, 2 canadian blood services, 3 american red cross, 4 sanquin background/case studies: hepatitis e virus (hev) is known to be transfusion-transmissible. as part of the risk assessment for this infection, a study was carried out in 14,000 canadian blood donors in 2013. in a subset of 4,000 donor samples the seroprevalence was 5.9%. however, no donor samples were positive for hev by an in-house nucleic acid test (hev-nat). since that study suggested exposure to hev in canada but used an hev-nat with a limit of detection of 250 iu/ml, a larger study was performed using a more sensitive hev-nat assay. study design/method: donors were informed about the study in the predonation reading materials. linked samples from approximately 50,000 canadian whole blood donors including 30,000 from canadian blood services (cbs) and 20,000 from h ema-qu ebec (hq) were collected. clinics were selected to ensure representative sampling of the donor population. all 199a transfusion 2017 vol. 57 supplement s3 donations with available plasma samples were tested by individual donation nat at the american red cross laboratory in gaithersburg, md, using the cobas v r hev test (95% lod 18.6 iu/ml, 95% ci 15.9-25.6) for use on the cobas v r 6800/8800 system. this test is not currently approved in canada or the usa, but is available as a ce marked test. all nat-reactive donors are questioned concerning risk factors for recent hev infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate nat, viral load, genotyping and igm/igg serology), are notified by letter, and deferred from donating for 6 months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous 6 months are destroyed. recipients will be traced in the event of any products transfused in the previous 6 months. results/finding: as of april 10, 2017, 9 of 39,834 (19,395 cbs, 20,439 hq) tested samples with valid results have been found hev-nat reactive: 8 donors have been confirmed by further testing to date. confirmation is pending in 1 donor. of the 9 donors, 7 were from quebec, and one each from nova scotia and alberta (7 male, 2 female). ages ranged from 21 to 70 years. only two donors reported non-specific symptoms (fatigue). in terms of risk factors: 6 ate pork (including 3 who ate pork liver), 4 ate shellfish, 2 ate venison, and 3 drank well water. one donor had no identifiable risk factor. viral loads ranged from 3 to 151 iu/ml, of which 2 were <10, 3 were 10-50, and 3 were >50 iu/ml; 2 were anti-hev igm positive and 4 anti-hev igg positive at index (wantai assay). conclusion: the prevalence rate of acute hev infection in this donor population appears to be around 1/4400. the data from this study will contribute to the ongoing risk assessment of transfusion-transmitted hev infection in canada. prevalence of malaria parasite in donated blood at nakasero blood bank, uganda gerald nsubuga* and musiisi ezra. uganda blood transfusion service background/case studies: introduction infectivity of donated blood with malaria is a significant health problem facing humanity. in uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current uganda national blood transfusion service (ubts) guidelines by the ministry of health. as a result, the proportion of donated blood that is infected with malaria is largely unknown. malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about 300 to 500 million cases and 2 to 3 million death occur per year. however the study aimed at determining the prevalence of malaria parasites in donated blood at nakasero blood bank, kampala, uganda study design/method: a cross sectional study was carried out in nakasero blood bank, kampala, uganda in four hundred and seventy randomly selected donor samples at the blood bank between june and august 2014. both thin and thick glass stained blood smears of 417 blood samples with giemsa was examined using microscope. results/finding: of the 417 donated blood samples, 17 (4.1%) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of plasmodium in relation to sex, age and blood group (p>0.05), majority of the blood donors that tested positive belonged to blood group o (64.71%). the prevalence of malaria parasite in the study was 4.1%. regardless of the prevalence, the presence of malaria parasite (plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in uganda. the ministry of health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia. using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities. components. the bact/alert virtuo* (virtuo) is an advanced, next generation system with improved automation, connectivity, and with data management systems. most importantly, the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert bpa (aerobic) and bpn (anaerobic) bottles. bpa and bpn bottles were tested on virtuo and bact/ alert 3d (bta 3d) to evaluate repeatability to detect growth in seeded leukocyte reduced apheresis platelets (lrap) without platelet additive solution (pas), throughout platelet shelf life (3, 4 and 5 days after collection). study design/method: pooled lrap were seeded with low levels of 6 organisms commonly associated with platelet contamination at 3, 4 and 5 days post collection. the seeded lrap were inoculated into bpa and bpn bottles on 10 different days (not consecutive) alternating between 2 teams of 2 people each. seeded bottles were loaded into a virtuo and a bta 3d and incubated until declared positive or negative (up to 7 days). additionally, bpa and bpn bottles inoculated with 4 ml of unseeded lrap were tested on the virtuo and the bta 3d (120 and 40 bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by lrap results/finding: the repeatability of the virtuo to detect organisms in lrap was demonstrated by a recovery rate of seeded bottles of 99.9% for the virtuo and 99.5% for the bta 3d. the virtuo demonstrated an average improved ttd of 3.2 hours, when compared to the bta 3d in the presence of 4 ml lrap platelets. the lrap did not cause false positives. additionally, the age of the lrap units (within 5 day expiry),did not impact the ttd when seeded with organism background/case studies: zika virus (zikv) is an emerging flavivirus that is transmitted by the aedes aegypti mosquito and sometimes a. albopictus mosquito. most infections are asymptomatic. zikv nucleic acid testing (nat) became a required test for blood donors per the fda guidance entitled, "revised recommendations for reducing the risk of zika virus transmission by blood and blood components". based on our geographical location, implementation of this testing began 12 weeks after this guidance was issued. we performed zikv nat for donors of whole blood and blood components under an investigational new drug (ind) study (sponsored by hologic, inc.). we performed a retrospective analysis on all nat results as there is a potential to defer donation due to false positive screening results. study design/method: donors that consented to donate blood and be tested for the zikv were obtained from three blood banks in colorado and nebraska. nat was performed using the procleix virus assay which is a qualitative in vitro nucleic acid assay system that detects zikv rna in plasma specimens. the assay was performed on the automated procleix panther system. all testing was performed according to the manufacturer package insert. results/findings: in the event of a reactive result, donors would be retested by nat in addition to other testing (igm antibody testing, neutralization test). donors are deferred for 120 days barring continued zikv testing and nonreactive results. a total of 2,485 donors were screened for zikv. all donors screened for zikv were nonreactive by nat. no invalid test results were obtained. in addition the number of failed test runs due to instrument or assay issues were experienced were quite low (1.0%). this data indicates that both the assay and instrument are robust. there was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our blood bank customers. conclusion: the reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results. this screening is important to continue to ensure blood safety in the united states. robust inactivation of the yellow fever virus 17d strain can be achieved using amotosalen and uva light for pathogen reduction treatment (prt) of platelet components andrew laughhunn 1 , felicia santa maria 1 , yvette girard 1 , peter bringmann 1 , marion lanteri* 2 and adonis stassinopoulos 2 . 1 microbiology department, cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: yellow fever virus (yfv) is known to cause explosive outbreaks, such as the one in angola in 2015. the rapidly increasing number of infections in brazil, with hundreds of fatalities since december 2016, is of concern. yfv is a flavivirus transmitted by aedes mosquitoes and could spread, like zika virus, to other parts of the americas where the vector is endemic. with no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the 17d-yfv strain. yfv vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects. in addition, transfusion transmission (tt) of live attenuated yfv has been reported with severe clinical outcomes, especially in immunosuppressed patients. in order to prevent tt by yfv vaccine strain, the aabb recommends a 2 weekperiod deferral after yfv vaccination. yfv outbreaks and vaccination campaigns may therefore reduce blood availability. this pilot study evaluated the ability to inactivate 17d-yfv using amotosalen (s-59) and uva light prt of platelet components (pc). study design/method: pc in 65%pas (n53) or 100% plasma (n51) were spiked with high titers of 17d-yfv and treated with s-59/uva prt. samples were taken pre-and post-uva illumination and infectious titers were determined, by plaque assay using vero76 cells. the extent of inactivation was quantified by comparing titers before and after inactivation. results/finding: pre-prt infectious titers were 4.71 6 0.7 log 10 pfu/ml for pc in 65% plasma and 5.19 log 10 pfu/ml for pc in 100% plasma while titers in post-prt samples were <-0.7 6 0.0 log 10 pfu/ml for pc in 65% plasma and <-0.7 log 10 pfu/ml for pc in 100% plasma. inactivation to the limit of detection of >5.41 6 0.7 log 10 or inactivation of >4.71 6 0.7 log 10 pfu/ml was achieved for pc in 65% plasma. inactivation to the limit of background/case studies: the use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients. this high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. although abbott's alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels. study design/methods: the purpose of this study was to determine if the eight developed abbott alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin. for each of the alinity s assays evaluated (hiv ag/ab combo, htlv i/ii, anti-hcv, chagas, hbsag, anti-hbc, syphilis, and cmv igg), samples spiked with a concentration of biotin at approximately 1000 ng/ml were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples. two samples, one negative and one positive, were tested with all assays, except the hiv and htlv assays, which each tested two positive samples (1 hiv-1 antibody and 1 hiv-1 p24 antigen, and 1 htlv-i antibody and 1 htlv-ii antibody, respectively). results/findings: for the negative samples, the sample to cutoff (s/co) differences between the biotin spiked and control were 0.00 for hcv, hbc, syphilis, cmv igg, and chagas, 0.01 for hiv ag/ab and htlv i/ii, and 0.03 for hbsag. for the positive samples, the mean s/co % differences between the biotin spiked and control were 0.00 % (antibody sample) and 0.36% (antigen sample) for hiv ag/ab combo; 0.90% (htlv i antibody sample) and 0.32% (htlv ii antibody sample); -1.43% for anti-hcv, -2.52% for chagas, -0.71% for hbsag, -0.37% for anti-hbc, -1.62% for syphilis, and -0.59% for cmv igg. conclusion: eight abbott alinity s assays were evaluated to determine if they were susceptible to biotin interference. these results indicate that the eight alinity s assays do not show susceptibility to biotin interference at an approximate concentration of 1000 ng/ml. robustness of the abbott prism methods to biotin interference c fischer 1 , r schneider 2 , w leonard 2 , m cobb 3 , g schlauder 3 , g williams 3 , m zuske 2 m janulis* 2 . 1 transfusion medicine, abbott diagnostics, wiesbaden, germany, 2 add diagnostics, 3 transfusion medicine, abbott laboratories, chicago, united states background/case studies: the use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses. the increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. the purpose of this study was to identify any abbott prism assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin. after a comprehensive review of abbott's current on market prism assays, no assays were identified that utilize biotin-streptavidin capture; however, 3 assays were identified for subsequent testing as they contain biotin in their assay design. background/case studies: bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies. while bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth. based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from 10 to 100 colony forming units (cfus)/bag (i.e. 0.03 to 0.3 cfu/ml). one major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of 10 8 -10 9 cfu/ml over the 5 days product shelf-life. moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test. the aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates. the adaptability of the process with the blood transfusion services requirements was of major concern. hence, attention was focused on an easy to automate technique able to deliver results on day 2 after collection. study design/method: a large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps. an original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay. as recommended, 24 hours (day 1) after collection a sampling volume of spiked platelets (0.1-1 cfu/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format. an immunoassay was performed for the detection of the captured bacteria. results/finding: this approach was tested on a panel of 25 bacterial strains involved in transfusion reactions. the pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection. the full test developed in this study combining a pre-analytical culture step followed by an there are many stakeholders are involved in hcv eradication program, including government authority such as centers for disease control and prevention, national health insurance and health promotion administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, npos and academia. results/finding: tbsf is a private nationwide single blood services program in taiwan, and performs anti-hcv screening test and nat confirmatory test on every collected blood, which is a large-scale population screening of hcv in taiwan because of its high blood donation rate (7.5%). tbsf confirmed positive test result of repeated blood donors, and can identify hcv rna seroconversion cases as recently-infected hepatitis patients. those infected patients would be referred to physician for further medical care and deferred permanently by tbsf to secure blood safety. by interviewing the newly-infected cases, the risk factors of hcv patients can be studied and then help identifying and eliminating sources of hcv infection. tbsf also contribute to health education by teaching our donors being aware of potential risks of hcv infection and keep monitoring every parameters of hcv epidemiology to evaluate the efficacy of hcv eradication program. conclusion: in hcv eradication program, tbsf can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation. thus, among all stakeholders, tbsf is particularly important and can play a pivotal role in eradicating hcv by 2030 in taiwan. the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates ute gravemann 1 , frank tolksdorf 2 , wiebke handke 1 , thomas h. m€ uller 1 and axel seltsam* 1 . 1 german red cross blood service nstob, 2 maco pharma international, gmbh background/case studies: the theraflex uv-platelets system (macopharma) is a uvc-based pathogen inactivation system for platelet concentrates (pcs). inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans. previous studies with the first set of bacteria species of the who international repository of platelet transfusion relevant bacterial reference strains revealed a high inactivation capacity for clinically relevant bacteria. aim of the current study was to investigate the bacteria inactivation efficacy of the theraflex uv-platelets system for enterobacter cloacae, pseudomonas fluorescens, staphylococcus aureus and streptococcus bovis which have recently been added to the who international repository. study design/method: pcs were produced from 5 buffy coats using the additive solution ssp1 (macopharma) with a residual plasma content of 35%. for inactivation kinetics, pcs (n53) were spiked with bacteria to a final concentration of approx.10 6 colony forming units (cfu)/ml and irradiated with increasing doses until the full uvc dose was achieved. samples were taken for the bacterial titer determination after each irradiation step. for sterilization studies, two pcs were pooled and inoculated with bacteria to a final concentration of approximately 0.3 cfu/ml. bacteria were allowed to grow for 6 h in the pcs at 22 6 28c under agitation. after splitting, one pc remained untreated (growth control) while the other one was uvc-treated. after storage for seven days, samples were taken from both bags for sterility testing by bactalert (biomerieux) and for determination of the bacterial titer in the untreated control units. results/finding: bacteria in pcs were inactivated in a dose-dependent manner by treatment using the theraflex uv-platelets system. mean log 10 reduction factors ranged from 6 to 7 for enterobacter cloacae (6.3 6 0.6, pei-b-p-43), pseudomonas fluorescens (7.1 6 0.4, pei-b-p-77), staphylococcus aureus (6.6 6 0.4, pei-b-p-63), and streptococcus bovis (7.0 6 0.3, pei-b-p-61). pcs (n512 for each species) spiked with these different bacteria species were efficiently sterilized (12 out of 12). treated pcs remained sterile during storage for 7 days, while bacteria in non-treated pcs grew to high titers of 10 6 -10 8 cfu/ml. the theraflex uv-platelets system efficiently inactivates a broad range of different bacteria species, including the who reference strains. sterility is maintained over a storage period of 7 days. these results suggest that the uvc-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions. transfusion transmissible infections among blood donors and strategy on direct laboratory testing cost of blood screening at national blood bank center, addis ababa, ethiopia abraham zewoldie*. national blood bank service background/case studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (ttis). hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv) and syphilis are the most serious infections transmitted during blood transfusion. serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of ttis. knowing the current prevalence of ttis among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup. study design/method: a retrospective analysis of blood donors' record covering the period from july 1, 2008 to july 30, 2013 was conducted. the data was collected from the nation al blood bank (nbb) center donor data base. in addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs. data was first exported to spss version 16 software for analysis. data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables. p values less than 0.05 were considered significant. results/finding: a total of 173, 207 consecutive blood donors were screened between 2008 and 2013. the overall seroprevalence rate of hbv, hiv, hcv and syphilis of blood donors was 5.0%, 1.6%, 1.4% and 0.1% respectively. the hiv-hbv co-infection was higher among blood donors 135(41.79%) followed by hbv-hcv co-infection whish accounts about 103(31.89%). significantly increased sero-prevalence of ttis was observed in among family replacement donors, factory workers, daily labors and the age group of 26-35. in this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was 746,773.9 ethiopian birr. conclusion: a significant percentage of the blood donors harbor ttis. the nbb center should work on voluntary blood donor mobilization and develop culture of voluntarism. the direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm. thus, the new strategy can be implemented to make screening of ttis cost effective in nbb center. transfusion transmitted malaria in a 14 month old infant patricia davenport* 1 , geeta paranjape 1 and laurie sutor 1,2 . 1 carter bloodcare, 2 ut southwestern medical center background/case studies: in 2017 at a large pediatric hospital, a 14 month old infant was supported for 31 days by extracorporeal membrane oxygenation (ecmo). over this time 113 blood products were transfused. about 10 days after end of ecmo support, a routine blood smear examination revealed inclusions in some of the patient's red cells. the patient had also been having intermittent fever. malaria was confirmed by pcr as plasmodium ovale (p. ovale). because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products. study design/method: the investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote. we identified 27 donors of red cell products. each donor was contacted and was asked four questions. additional questions were asked for clarification if needed. based on donor response, risk for active malaria infection was assessed. we also considered areas where p. ovale is, or is not found. donors identified as having possible risk were tested for antibodies and parasitic dna. results/finding: the five donors who had been ill all had common cold or bronchitis like symptoms. donors who traveled went only to non-risk areas. three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the u.s. as adults. it was discovered that one of these three did not meet all donor criteria. the donor had failed to disclose that he had not completed 3 years stay in the u.s. after emigrating from cameroon, an area endemic for p. ovale. he had not travelled anywhere after coming to the united states in october 2014 and answered "no" to travel. antibody tests on this donor were positive for p. ovale and p. falciparum, but pcr tests were negative. another possible at-risk donor, a former resident of iran was tested and was pcr and antibody negative. the third donor has not yet been tested but the country of residence does not have p. ovale malaria. conclusion: while it could not be definitively proven that the donor with antibodies to p. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment. transfusion-transmitted babesiosis outside an endemic area: a case report german felix leparc*. oneblood background/case studies: an 81 y.o. male patient was admitted to the emergency room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically. during this admission, he received a total of 13 units of red blood cells. approximately 4 weeks later, he was re-admitted due to another episode of gi bleed manifested by melena. as part of his routine evaluation, a cbc was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with babesia sp. study design/method: upon notification of a suspected case of transfusion-transmitted babesiosis, lookback of all donors involved in prior transfusion event was initiated. results/finding: to confirm the presumptive diagnosis of babesiosis, pcr was performed and babesia microti dna was detected. an evaluation of the patient's risk factors revealed that prior to the gi bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery. no travel history to the us midwest, and while he travelled to new england two years ago he did not spend time outdoors. he was splenectomized in his mid 20's. donor lookback identified a donor who lived in new london county, connecticut but spent the winter season in central florida, where the blood donation (double rbc collected by apheresis) took place. he had never been diagnosed with babesiosis, but participated regularly in outdoor activities in connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it). upon testing, he was found to be negative for b. microti on pcr as well as igm antibodies, but had igg antibody titers of 1:256. the recipient of the other rbc unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed. during phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative. conclusion: while transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case. once licensed assays for babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration. transfusion-transmitted stenotrophomonas maltophilia from a red cell unit: a case report ashley c gamayo* 1 , andrea j linscott 2 and donny dumani 3 . background/case studies: transfusion-transmitted bacterial infections (ttbi) are rare, but serious complications of blood product transfusions. from 2011-2015, 8% of 173 transfusion-associated fatalities reported to the fda were attributed to bacterial contamination. red cell units are rarely implicated in severe and fatal ttbi. when present, contaminants are often gram-negative rod (gnr) bacteria with psychrophilic properties. we present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with stenotrophomonas maltophilia (s. maltophilia). study design/methods: a 27-year-old female with sickle cell disease was admitted to the hospital for possible pain crises. pre-transfusion blood and urine cultures collected on day 1 of hospitalization showed no growth after five days. on day 3, the patient required a blood transfusion for which she was issued a cmv-safe, irradiated, hbs-negative, crossmatched, o-negative red cell unit. the 318 ml unit had been aliquoted via sterile connecting device 12 days prior for a pediatric patient. all 26 ml of the pediatric aliquot were transfused without adverse effects. the patient's pretransfusion temperature was 37.18c. within 45 minutes of starting the transfusion, the patient's temperature increased to 39.38c and subsequently reached a maximum of 39.58c. the transfusion was stopped and the blood bank notified immediately. gram stain of the remainder of the transfused component revealed gnr bacteria. blood was collected from the patient for culture and antibiotic treatment initiated. results/findings: initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test. blood cultures from both the patient post-transfusion and the implicated red cell unit grew gnr bacteria identified as s. maltophilia. further microbial testing revealed the cultured pathogen was able to proliferate at 4-68c; a finding not characteristically observed in s. maltophilia. conclusion: this is the first definitive case of ttbi with s. maltophilia. this bacterium is a globally emerging gnr that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients. contamination was unlikely due to an asymptomatic donor. there was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component. the patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified. the patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization. the transfusion reaction was classified as definitive, severe tti of definite imputability. validation of commercial immunoassays for detecting hbsag and hiv antibodies in production pools karen leighton, izekial butler and scott jones*. qualtex laboratories background/case studies: plasma fractionators test plasma production pools for hbsag and hiv antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling. the european medicines agency (ema) has published guidelines for the validation of immunoassays for the detection of hbsag and hiv antibodies in production pools. the aim was to validate commercial immunoassays for the testing of production pools for hbsag and hiv antibodies utilizing the ema guidelines. study design/method: a lower calculated cutoff value for the abbott prism hbsag and hiv o plus assays was determined by calculating the mean signal-to-cutoff ratio (s/co) plus 3 standard deviations of four different types of plasma production pool samples. the calculated cutoff values were utilized for the rest of the validation. the detection limit was determined by testing in triplicate, serial dilutions of who hbsag and hiv antibody standards diluted in plasma. a normalized detection limit was calculated for the hbsag assay using production pools containing low, typical and high anti-hbsag titers. intra-assay variability was determined by testing a minimum of 6 determinations of a low positive control in 1 run. inter-assay variability was determined by testing at least 3 representative negative production pool samples, at least 1 low positive sample (about 3 s/co) and a titration series of who standard spiked into plasma production samples. runs were performed on six separate days using two different instruments and two different lots of assay reagents. results/finding: the lower calculated cutoff values for the hbsag and anti-hiv assays were both below the manufacturer cutoffs of 1.00 and were 0.72 and 0.48 respectively. the hbsag assay detection limit was 0.065 iu/ ml for source plasma and 0.120 iu/ml for recovered plasma samples. the normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at 15-25 0 c where all samples were still reactive for hbsag. the anti-hiv lowest positive dilution for all replicates varied between 1:10,000 to 1: 1,250,000 depending on subtype and group. the % cv of the s/co values of the replicates of the intra-assay variability validation were less than 5% for both assays. the %cv of the s/co values of the panel of samples of the inter-assay variability validation were less than 14%. conclusion: a lower calculated cutoff value could be determined for commercially available immunoassays for hbsag and anti-hiv. these immunoassays could meet all of the recommendations in ema validation guidelines. the abbott prism hbsag and hiv o plus assays can be utilized to test production pool samples. was performed on 6 donors (3-17 days after the index donation) -3 donors in the follow up study and 3 tested by the doh. no donors tested by the doh participated in the follow up study. follow up testing was negative for all 6 donors. denv antibodies were negative in 9 donations and equivocal in 1. our initial reactive rate is higher than that reported to date for the procleix zikv tma of 1 per 23, 342 [p. williamson, et al transfusion, in press] . conclusion: universal testing under ind was successfully implemented and incorporated into blood center operations. we have noted an initial reactive other demographics that should be analyzed for their potential to be used to predict cmv seroconversion rate include gender, age, race, ethnicity or a combination of these. background/case studies : growing the geographic footprint has been a priority for the organization since 2014. over a four year period, the organization doubled the number of blood centers, with continued growth expected. with the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk. the internal audit function was centralized in late 2011, for which the program consisted of 2 types of audits, an operational compliance audit and a support systems compliance audit. each type was performed twice per year at each main center. this model was no longer serving the changing organization. study design/methods: lean six sigma concepts were applied to this project. survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program. this information was the primary input to the swot analysis (strengths, weaknesses, opportunities, and threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design. potential solutions were placed into a pugh matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection. each potential solution was compared to criteria for evaluation and selection of the best solution. results/findings: the program was re-designed to perform internal audits annually as a single, team-based comprehensive audit. remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings. a formula was created to determine on-site audit time that included adjustable risk factors. the audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs. the team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns. conclusion: the comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing quality or increasing compliance risk. external inspection performance has achieved record performance levels the past year. diversity of auditor skills led to a stronger skill presence, which was consistently applied across system. auditing is more efficient and effective. stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system. auditors and auditees have increased in knowledge, and the internal quality audit program has improved. background/case studies: in many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma). banc de sang i teixits (bst), adopted the fully automated reveos system (terumo bct inc, lakewood, co) few years ago to manufacture blood components. in june 2016, bst started a validation of new blood bags manufactured by terumo bct with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter. study design/method: to perform this validation, 300 blood donations were used under different conditions (see table below ). the current filter evaluated for the platelet pool (lrf-xl, haemonetics corporation) was compared to a new filter (terumo bct inc.). the new blood bags were manufactured using a new vinyl supplier. a portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (qc) tests (routine qc performed at bst following european directorate for quality of medicines & healthcare; cytokine analysis, such as p-selectin and platelets recovery through the filter). results/finding: the results are very similar between both bags, current and new one, as well as filters. all the analysis done to evaluate the quality of the blood components were similar in all conditions. also, it was shown a better performance on platelets pools, when they came from bags centrifuged with 60 ml of plasma, vs. 30 ml of plasma and additive solution. conclusion: these new bags and filter have shown a similar behavior when using them for manufacturing blood donations with reveos system in our blood bank. regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools. no issues should be found if they are implemented in routine use. it's planned to start this implementation during this year, 2017; so then there will be larger results in order to have a proper procedure qualification. conclusion: patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood. it is clear that some requests for exquisitely rare types are not able to be filled with current donors. molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years. it is recognized that commercial platforms do not include many of these making these rare types even more challenging to find. consideration should be given to testing more donors of all ethnicities to identify more rare donors. recommendation #1: updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation. updating our educational materials will likely have a minor impact. recommendation #2: implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency. initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over 120, 000 donors. the recommendation to limit the number of donations would have a substantial impact. for this analysis, the focus was on 16-18 year olds and premenopausal women (ages 19-55) donors. on average, 16-18 year olds donate 1.3 times a year and premenopausal women donate 1.49 times a year. if both of these groups were limited to donating once a year, a total of 4,845 donations from 16-18 year olds and 9,272 donations from premenopausal donors would not be collected. conclusion: after analyzing the impact of the aabb association bulletin #17-02, the bulletin will have a significant impact on both donors and our local blood supply. more than half of donors would receive either ferritin testing or iron supplementation. if the only measure employed is limiting the number of times a donor could donate for 16-18 year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals. background/case studies: transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians. these deficiencies may be due to the frequency and type of education. the majority of medical students in the united states receive four or fewer hours of transfusion medicine education. the transfusion medicine academic award group published educational content guidelines for medical school, residency and fellowships. however, the frequency and educational methods remain poorly evaluated and with little guidance. we investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners. study design/method: three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications. the simulation group received all educational activities within a 2.5 hour inperson, high-fidelity live session. the hybrid group received some educational component online and also attended an in-person high-fidelity simulation session. the online only group received all educational materials online, including a pre-recorded-video simulation session. the learners were second year medical students enrolled at one institution. the same faculty members taught all live sessions and developed all online materials ensuring the content was the same. a pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications. the educational session was evaluated by the likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding). results/finding: 97% (101/104) of the simulation group students improved their post-test scores and had an average likert scale rating of 4.1 (very good). 89% (63/71) of hybrid group students improved their post-test scores and had an average likert scale rating of 4.2 (very good). 89% (90/101) of online only students improved their post-test scores and had an average likert scale rating of 3.0 (good). the average changes in scores were statistically significant within all training groups (p value < 0.0001). additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p<0.0001) and the hybrid group (p<0.0001). conclusion: our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum. the high-fidelity simulation curriculum is also preferred over the online only education as indicated by the likert survey results. aaron j wyble*, yeon mi kim and barbara j bryant. university of texas medical branch background/case studies: diagnostic management teams (dmts) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care. dmts employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations. the interpretations must be of moderate to high complexity in order to be clinically valuable. recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care. the timeliness of the dmt reporting is vital to patient management. the inherent design of a dmt also provides an educational opportunity for trainees at academic centers. study design/method: the transfusion medicine service at a large university-based academic medical center implemented a dmt in 2016. all cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from july 2016 through january 2017 were evaluated by transfusion medicine residents. the electronic medical record (emr) of each patient was also reviewed to determine relevant clinical history. all significant findings were presented at the transfusion medicine dmt conferences. the dmt was comprised of physicians from transfusion medicine, hematology/oncology, anesthesiology, transfusion service technical staff as well as visiting clinical staff from surgery, obstetrics and gynecology, transplant services, and pediatrics. the dmt integrated the clinical and laboratory data to formulate relevant interpretations and recommendations. the final dmt reports were placed into the emr for access by health care providers. financial benefits of a transfusion medicine dmt were also evaluated. results/finding: in a 7-month period, 504 cases of complex antibody identification workups (65%), transfusion reactions (2%), consultations for blood component utilization (6%), and deviations from standard operating procedures and massive transfusion protocols (27%) were presented at the transfusion medicine dmt conferences. the placement of dmt narratives in the emr as progress notes and laboratory reports provided informative and timely communications. residents participating in dmts demonstrated improved clinical and laboratory correlation skills. as a result, resident competency in transfusion medicine was enhanced. over $68,000 of revenue was generated utilizing the standard professional component cpt codes. conclusion: dmts encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services. additional benefits of a dmt program include resident, clinician, and technical staff education and the generation of revenue for the institution. streamlining a blood center and hospital transfusion service supply-chain with an informatics vendor-managed inventory solution hamilton c. tsang* 1 , david lancaster 2 , dianne geary 2 , robert scott 1 , anh thu nguyen 1 , adam garcia 2 , raina shankar 1 , leslie buchanan 1 and tho pham 2 . 1 stanford health care, 2 stanford blood center background/case studies: inventory management is both a major challenge and an integral part of hospital transfusion service (hts) and blood centers (bc) operations. the current process at our institution involves twice-per-day shipments from the bc to the hts, with each shipment predicated upon current stock levels at hts. manually obtaining inventory levels for each product is time-consuming. the manual determination is also errorprone. we aim to enhance inventory management operations by developing an informatics solution to (1) streamline the ordering process to accurately reflect inventory status and transfusion practices and (2) re-allocate valuable hts tech time. study design/method: at our hts, the general inventory accounts for over 50 product categories broken down by component, blood type, irradiated status, and cmv-serology status. we therefore sought to establish an electronic method to reliably infer the general inventory level. since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g. special antigen units, cross-matched units), over a 5-month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory. once we had a reliable electronic method to determine inventory levels, we implemented a 3-month pilot period. we analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: (1) the ratio of units transfused per week to the number stocked (t:s), (2) the number of products ordered as stat, and (3) the number of expired products. we created in-house programs on visual basic for applications (microsoft, redmond, wa) for both the calibration and pilot periods. 2486 lines of code were written for both programs, including 2 class modules and 34 distinct subroutines. results/finding: during the pilot period, we investigated our system's noninferiority. the average weekly t:s ratio for cryoprecipitate, plasma, and rbc, respectively, were 1.03, 1.21, and 1.48 before the pilot period compared with 0.88, 1.17, and 1.40 during the pilot period. these differences did not reach statistical significance (p50.28). we also monitored the number of stat ordered products before and during the pilot period, which were 27 and 31 stat units per week, respectively (p50.86). lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were 226 and 196 units, respectively (p 5 0.28). an estimated 7 hours per week of technologist time was reallocated to other tasks once the electronic census was adopted. this translates to 0.175 fte and $18,200 per year saved from labor costs per year if permanently adopted. conclusion: we created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering. our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same t:s ratio, and not increasing the number of expired products. this is achieved while freeing up over 360 hours of staff time per year. future directions include full automation with involvement from hts informatics department. transfusion practice improvement: gaining traction through the use of a provincial transfusion quality improvement plan denise evanovitch* 1 , yulia lin 2 , troy thompson 1 , allison collins 1 and sheena scheuermann 1 . 1 ontario regional blood coordinating network, 2 sunnybrook health sciences centre background/case studies: a provincial regional blood coordinating network (prbcn) held a "quality focus day" (qfd) in 2014 to explore transfusion quality indicators to be included in a province wide quality improvement plan (qip). the plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use. the following recommendations were made: select a blood component that most hospitals could monitor display progress in a public forum so that hospitals could compare themselves to peers strike a province-wide transfusion qip committee to guide the development of the plan, supporting resources and ongoing improvement initiatives study design/method: a provincial transfusion quality improvement plan (ptqip) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient. there was further collaboration with other organizations such as the provincial health quality division, choosing wisely after the launch, an informal survey indicated that 74 of the province's 158 hospitals were interested or had already adopted portions of the ptqip. to further assist hospitals in advancing their qips, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success. both hospital and provincial reports can be generated from the tracking tool. a more formal survey conducted in 2017 indicated that 93% plan to implement or already have implemented the ptqip and 43% of the respondents already have put prospective order screening by technologists in place. conclusion: helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives. taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses. background/case studies: military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as pre-hospital transfusion (pht). helicopter emergency medical services (hems) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications. there is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a pht program. herein, we report our experience as a large hospital system embarking on the development of a multi-state pht service. study design/method: in october 2016 a work group was formed to establish pht services for the hems providing care to over thirty regional hospitals. composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: federal/state regulations; inventory structure/management; product storage/testing; tracking/traceability; emergency release protocol; and staff training. while there are no specific regulations governing pht, the regulations pertaining to blood product storage, validation, and monitoring apply. the fda, aabb, and state agencies were each consulted to ensure compliance with all directives. results/finding: the largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state hems. similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites. the system's fda licensed blood supplier was deemed responsible for product consignment and transport between the four hems sites. the blood inventory at each site was designed to contain: group o positive rbcs, group a low anti-b titer liquid plasma, and four-factor prothrombin complex concentrate. a military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed. validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan. staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the hems medical director with additional oversight provided by transfusion medicine physicians. conclusion: our work group successfully identified the challenges associated with a multi-state pht helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training. our pht service will go live in 2017. publishing this experience may benefit future sites as they launch similar pht initiatives. blood transfusion during humanitarian emergencies yetmgeta e. abdella* 1 , rana hajjeh 1 and cees th. smit sibinga 2 . 1 world health organization regional office for the eastern mediterranean, 2 international quality management (iqm) consulting background/case studies: more than 76 million people are affected by humanitarian emergencies in the eastern mediterranean region of the world health organization (who), where some of the most affected countries in the world are located. in these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances. humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex. despite these obvious needs, across the region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies. study design/method: we searched pubmed and index medicus for the who eastern mediterranean region for data on availability and safety of blood transfusion in humanitarian emergencies. we conducted a structured survey of blood transfusion services (bts) in all countries in the region to identify the following: type of humanitarian emergencies between 2006 and 2016; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges. additional information was collected during a regional consultation (eastern mediterranean region) held in may 2016 in tunisia. results/finding: we found 24 publications on disaster from five countries in the region and 16 publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the region. however, none dealt with the questions of availability and safety of blood transfusion during emergencies. twelve countries (54.5%) responded to the survey. armed conflicts and terrorism are the commonest types of emergencies with estimated 10-85% of the injured requiring blood transfusion. nine countries have emergency preparedness and response plans for bts. potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors. seven of the responding countries keep an emergency blood stock. collaboration between the different stakeholders exists in seven countries. lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified. conclusion: there is a need to integrate bts in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise. (63, 85, and 108 for 2014-2016) . the number of collections per registered trt donor varied significantly, ranging from 0 to 12 therapeutic draws/donor per year. excluding those that didn't present for a therapeutic blood collection, the average number of trt collections/donor per year decreased from 3.8 to 2.8 between 2014 and 2016. conclusion: our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on trt referred for therapeutic phlebotomy due to elevated hemoglobin values from 2014 through 2016. it is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on trt, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per trt donor decreased during this timeframe. the percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral. our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population. as it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than 56 days, is sufficient to mitigate the risks of testosterone therapy. hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements. approaches involving the use of a vein illumination device in a blood donor center sara matheson*, kimberly j duffy, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge. unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration. in a blood donor center, the primary selection of a vein is done by palpation within the antecubital area. prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed. vein illuminator (vi) devices are available to aid in visual display of potentially suitable veins. such a device was made available to staff in march of 2010. after an initial testing and instructional period, the vi has since not been used by staff. the objective of this study is to discover reasons why staff does not use the vi to identify potentially suitable veins. study design/method: a staff survey was developed and distributed to staff in march 2017 to inquire about usage of the vi and obtain feedback about the device. at the time that the survey was sent, the device had been available for several years. the survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback. results/finding: the survey had a 77% response rate (n533). of these, 78.8% have never or very rarely utilized the vi. self-reported reasons for low utilization focused on two dominant themes. first, that the device is not needed and second that it doesn't accurately show veins. 87.9% of respondents are aware of where the vi is stored and a more accessible location to share the device was not identified. although 93.9% of respondents have been provided training on using the vi, the group was mixed regarding their comfort level in using the device independently. only 48% of the group was willing to try vi. conclusion: infiltration and incomplete collection account for approximate 3% (770 units/year) of qualified blood donors, yet vi does not appear to be a viable solution for our blood donor program. there seems to be both an opportunity and challenge with vi implementation. the opportunity is to create critical awareness of problems with vein cannulation. the challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation. benefits of converting from mcs1 to alyx penny schroeder* and elizabeth parker. indiana blood center background/case studies: in 2015, apheresis red cells (arc) represented 4.7 % of total red cell collections at our center. hae mcs1 ln8150 was utilized to collect arc. due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies. study design/method: fresenius kabi demonstrated the fenwal alyx technology as well as the business case to the primary stakeholders. all implicated departments were involved in the initial impact assessment. a multidepartment kick off meeting was held and project team formed. due to product demands, the decision was made to validate arc and plasma apheresis. the primary departments affected were blood collection and production. fresenius kabi provided sample validation plans, sops, training and training materials for use. four mobile-carts were purchased for easy transportation of alyx and quick-connect feet for installation on mobile buses. the lead trainer and the bc technical administrator traveled to an affiliate blood center to observe their alyx program and identify best practices. a team of blood collection trainers and preceptors were the initial group trained and validation performed. this team also served as the subject matter experts and field preceptors. fresenius kabi returned for advanced alyx operator training. the training plan targeted previous mcs1 operators first and then operators new to apheresis with a training goal of 30% of mobile staff. validation of the 12 alyx began 06/01/16 and took approximately 45 days to complete. during this time, fresenius kabi conducted alyx education and apheresis recruitment training to all collection and recruitment staff. the mcs1 machines were removed from service 07/08/16. alyx go-live occurred 07/13/16. additional operator training continued through september 2016. results/finding: due to ease of mobility and use of alyx, reduced procedure time compared to mcs1 and donor conversion training we increase components collected. alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles. this decreased kit cost by $19.21 each providing an estimated annual savings of $239,000. conclusion: with the multiple alyx donation types we were able to increase our collection of right type procedures by approximately 2.5% and decrease our kit costs by 22%. with alyx the collection plasma on mobile blood drives is now possible. due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from october 2016-march 2017. background/case studies: high frequency of donation is a risk factor for iron deficiency. because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk. minimum hemoglobin (hb) has long been the same for males and females at 125g/l, but for males this falls below the normal limit. as a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum hb increased to ! 130 g/l) and for females (minimum interdonation interval increased from 56 to 84 days). the longer interdonation interval in females was gradually implemented, starting with donor messaging in october 2016, changes in rebooking of donation appointments in december 2016 and culminating with eprogesa criteria changes on march 5, 2017. both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in hb deferral rates in female donors. we aimed to assess the impact of these changes on hb deferral rates. study design/method: percentages of hb deferrals were calculated as the number of donation attempts that resulted in hb deferral divided by the number of successful donations plus hb deferrals multiplied by 100. percentages were calculated for male and female donors before and after changes were made. results/finding: the percentage of hb deferrals increased in male donors from 0.89% in the 3 weeks pre-implementation to 2.16% in the 3 weeks post-implementation of the change in the hb criterion. hb deferral rates for female donors were 12.6% in september, 12.0 % in october/november, and 9.9 % from december to march, 2017. conclusion: hb deferral was more frequent in male donors after the minimum hb was increased to 130 g/l. the gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased hb deferrals. a longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention. in the past 8 years, 59,223 blood products, derived from 10,509 procedures, were distributed to 185 different investigators in over 200 laboratories. whole blood was the most common product (45.2%), followed by unmanipulated mononuclear cell collections (28.6%), and elutriated monocytes or lymphocytes (19.8%). less common requests included platelets (2.5%), plasma (2.5%) and granulocytes (0.8%). adverse donor reactions were infrequent (0.33% of procedures). conclusion: we report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank. research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion. hospital-based blood donor center's experience with implementing platelet pathogen reduction system kimberly j duffy*, mary m benike, james r stubbs and justin d kreuter. background/case studies: the safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats. the recent fda approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers. in order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications. the objective of this study was to determine the optimal collection settings to enable 100% collection of pathogen reduced platelets while limiting the loss of products. study design/methods: the collection instrument evaluated for this study has fda approval for platelets suspended in 100% plasma. the corresponding pathogen reduction system used for the study has 3 kits with 3 different collection specifications. all apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer. the yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device. in order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections. staff was instructed to collect the highest available yield per donor. after collection, volume, platelet yield, and concentration data was obtained. this data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than 6.8 x10 11 , thus meeting the criteria for a double product. results/findings: a higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than 1800 x 10 3 /ml was more likely to be outside the specification of the pathogen reduction kit. the platelet concentration target of 1867 x 10 3 / ml results in discarding products and was quickly removed from instrument settings. collections with a platelet yield as low as of 3.5 x10 11 and platelet concentration of 1167 x10 3 /ml were more likely to produce a product that was not within the specification of the pathogen reduction kit. abstract conclusion: the loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise. the goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications. final collection instrument configurations are platelet yield of 7.0 x10 11 and 6.8 x10 11 at the volume of 400 mls and platelet yield of 4.2 x10 11 , 4.0 x10 11 , and 3.5 x10 11 at the volume of 300 mls. moving from subjective to objective donor eligibility screening platforms: a blood center's journey angela dirr* 1 and steve cihura 2 . 1 bonfils blood center, 2 bbc / bsi background/case studies: in 2012, the device used by bonfils blood center to determine donor hemoglobin and donor eligibility was reaching its end of life, and bbc needed to define a path forward for a reliable replacement device. study design/method: bbc evaluated 3 devices with the following criteria in mind: 1) device disposable costs, 2) reagents/controls/quality control, 3) objective hgb/hct measurement, 4) portability and durability for a mobile environment, 5) ease of use, 6) donor experience, 7) battery life, 8) validation requirements plans, 9) blood center suitability, and 10) ability to link to becs. multiple departments including donor care, equipment management and validation, quality, and regulatory affairs were involved in the evaluation and product selection. bbc tested 50 donors per each device at both a fixed and a mobile site. bbc also considered donor feedback for the choice of replacement technology. the project started february 2013 with a targeted implementation date of july 2013. after creating necessary sops and adopting existing sops, bbc successfully completed the validation of the devices, and chose the compolab technology from fresenius kabi as the new device for bbc blood bank. results/finding: the compolab was selected as it met project scope and selection criteria. it was important for bbc to reduce paperwork and daily tasks. the compolab eliminates daily qc reducing paperwork, time and improves error management. after converting to the new technology, bbc donor deferral rates increased by approximately 15%. as a consequence to this increase, bbc conducted reminder training with bbc staff to ensure proper sampling technique and higher sample quality. over time, bbc deferral rates stabilized to 4.59% in 2014 and 4.29% in 2015. during this time period, bbc also successfully recruited new blood donors to bbc program, which may have contributed to an increase in deferral rates. in 2016, the deferral rate increased again, probably due in part to the fda final rule "requirements for blood and blood components intended for transfusion or for further manufacturing use", which went into effect in may 2016. conclusion: during the evaluation for new equipment, bbc learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business. after comprehensive evaluation of multiple donor eligibility screening platforms, the compolab device was selected at bbc facility. it met the majority of all aspects of the project scope and qualifying criteria. bbc also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates. flowmetry on platelet apheresis. tetsu yamamoto* 1 , ayumi araki 1 , hiromi sanyoshi 1 , hiromi kanai 1 , hiroya kikuchi 2 , katsushi tsukada 2 and kazuhide mure 2 . 1 hokkaido red cross blood center, 2 japanese red cross hokkaido blood center background/case studies: vasovagal reaction (vvr) is known to be the most common adverse reaction to blood collection, but effective measures for preventing vvr have not yet been developed. effective timing of interventions during apheresis donations in particular should hold the key to predicting vvr, but no research has been done on the topic. study design/methods: this study investigated the potential to predict vvr from fluctuations in peripheral blood flow measured by laser doppler flowmetry in platelet apheresis donors, a population highly likely to experience vvr. data were collected from 354 individuals who donated platelets during the 6-month period between february and august 2015, and data from the 30 donors who experienced vvr were analyzed. to calculate the level for issuing vvr alert, the percent decrease in blood flow (dbf) and the percent decrease in heart rate (dhr) were calculated, the time from alert to vvr was estimated for three dbf levels, and the detection performance of each alert level was calculated. results/findings: eight of the 156 men (5.1%) and 22 of the 198 women (11.1%) experienced vvr. one donor did not experience vvr during blood collection, but had a delayed reaction while resting afterward. mean maximum dbf in the 30 donors in the vvr group was 64.7 6 13.7%, which was significantly higher than the 25.6 6 11.7% in the non-vvr group. at a maximum dbf threshold of 45%, sensitivity for discriminating between vvr and non-vvr donors was 93.3% and accuracy was 94.4%. when 45% dbf was used as the alert level, alerts were issued for 44 donors, including 25 in the vvr group. therefore sensitivity for predicting vvr was 83.3% and specificity was 94.1%. mean time from alert to diagnosis in the vvr group was 4.03 6 4.35 minutes, and accuracy of the alert was 56.8%. some of the vvr could not be predicted even the value of maximum dbf exceeded 45%. the reason was supposed to be the difference of donor susceptibility on dbf. conclusion: we investigated whether vvr in platelet apheresis donors can be prevented by prediction and found that it is possible to predict vvr early enough before onset to intervene by monitoring dbf in real time during blood collection using laser doppler flowmetry. future research must also investigate whether the incidence of vvr can actually be reduced by interventions such as adjusting extracorporeal circulation. the risks of alloimmunization in sickle cell patients using c, e, k negative blood: experience of a hospital apheresis and transfusion service grace banez sese* 1,2 , salam abdus 3 and shabrina shah 3 . 1 inova blood donor services, 2 inova fairfax medical campus, 3 inova fairfax medical campus transfusion services background/case studies: red blood cell (rbc) transfusion is often a lifesaving measure for patients with sickle cell disease (scd). it is critical in the management of scd complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome. a wellrecognized complication of chronic transfusion in scd patients is alloimmunization to rbc antigens. to prevent alloimmunization, transfusion with rbcs negative for c, e, and k antigens has been advocated. this has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions. we report a summary of our three year experience with the prophylactic transfusion of rbc units negative for c, e, k antigens for scd patients during red blood cell exchange transfusions (rbcx). study design/method: retrospective review of 10 scd patients with a history of stroke, refractory sickle pain crisis and priapism was done. rbcx was performed every 4 to 8 weeks from december 2013 to march 2017. blood bank work-up used the mts gel method for antibody screen and identification. our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner. results/finding: a total of 10 patients, 3 females and 7 males, who underwent a total of 178 rbcx from october 2013 to march 2017, using an average number of 7 rbc units per rbcx. rbc units negative for c, e, and k antigens were used during rbcx for 8 patients. two patients positive for c antigen underwent rbcx, using e and k antigen negative rbc units. review of the antibody screen test results performed prior to each of the 178 rce showed that no new clinically significant alloantibodies were formed after exposure to multiple rbc units. conclusion: although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for scd patients, studies suggest that the standard of care for transfusion of all patients with scd is to provide rbc negative for c, e, and k antigens. this ability to find these rare units is also affected by the characteristics of one's institution and blood supplier. it is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units. the approach by our institution to transfuse rbc units negative for c, e, k or study design/method: venous blood specimens of 168 healthy volunteers were collected before blood donation and after blood donation immediately, 1 day, 1 week, 4 weeks, and 12 weeks among men and 16 weeks among women. immunoglobulin g (igg), immunoglobulin m ( igm) , immunoglobulin a ( iga)and complement component 3 ( c3) , red blood cell (rbc), white blood cell count ( wbc) , hemoglobin (hb), hematocrit (hct), and serum iron (fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality. results/finding: the level of igg slightly decreased after blood donated immediately, iga and c3 decreased significantly but still within their normal ranges, igm did not change after blood donation. the level of iga significantly decreased at 12 weeks among men and 16 weeks among women, while c3 significantly increased at the same time period. igg, rbc, hb, hct and fe started to recover 1 week after blood donated and reached their levels before blood donated within 12 weeks among men and 16 weeks among women. conclusion: the biomarkers mutually changed over the course of 12 weeks among men and 16 weeks among women. donating 400 ml blood will not significantly affect overall blood quality. utilizing amicus dxt relay data managment solution to increase platelet split rate and improve amicus productivity janelle wilhelm* and jennifer kaluza. memorial blood centers background/case studies: with the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base. we also faced the challenge of managing multiple collection sites in multiple states. the decision was made to implement amicus dxt relay data management solution to provide us insight into procedure details to make data driven decisions. day to day variability previously dipped as low 40% split forcing reactive planning. study design/method: incorporate dxt to strategically plan our day to day operations. dxt reports were monitored by management and with the fresenius kabi team for productivity by site, phlebotomist and device. reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis. this allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction. reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate. results/finding: the monthly dxt report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate 15 percent and increased concurrent plasma collections by 24 percent. with utilization of the dxt reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than 62 percent split. phlebotomist qns rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall qns rate to consistently below 3 percent. conclusion: dxt was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers. dxt provides invaluable tools for the operational supervisors to monitor their staff and improve productivity at their multiple sites. next step is to develop the plan for implementation of paperless documentation with dxt and healthcare-id. the ability to immediately review data directly from amicus was key in the productivity improvements realized. evaluating the impact of a background/case studies: as blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety. it is a prerequisite for all registered nurses (rns) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice. aim: the aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program. the results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme. study design/method: all rns from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in october 2015. after which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions. the same questionnaire was administered to the rns one year later in september 2016 for post-training programme evaluation. individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation. results/finding: in 2015 and 2016, a total number 1,097 rns and 965 rns completed the questionnaires, giving a response rate of 78.5% and 67.4% respectively. the overall mean score in 2015 was 6.24 points (range 0 to 8). the mean score in 2016 was 6.57 points (range 2 to 8). the percentage of rns having perfect scores of 8 increased from 8.8% in 2015 to 20.5% in 2016. table i below shows the results for each question item. the implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst rns. further training may be needed in the preparation of blood sets and management of fever. background/case studies: clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain. this global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses. study design/methods: a descriptive 'ex-post facto' survey design was used; 264 purposively selected blood prescribing clinicians and nurses from 60 hospitals in 13 countries of the 4 human development index (hdi) groups (low, medium, high, and very high) participated. three research questions were answered, while seven null hypotheses were tested at .05 level of significance. descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -pearson product-moment correlation coefficient (ppmc), analysis of variance (anova), were used to analyse the hypotheses. results/findings: quality of clinical use of blood was positively and significantly correlated with levels of awareness (r5 .137; p5.03; df5262) and accessibility (r5.184; p5.01; df5262) to e-continuous learning among blood prescribing clinicians/nurses. there was significant difference in levels of awareness [f(3,260) conclusion: today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood. the higher the hdi level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs. there is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion. accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the low and medium hdi countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors. the results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes. background/case studies: transfusion medicine (tm) didactic teaching materials for pathology residents are not widely available to share among residency training programs. the advancing blood knowledge (abo) leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (cop). educational theorist etienne wenger defined cops as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly. study design/method: as a pilot project, 7 junior faculty co-investigators from 5 west coast institutions each had 2 months to create a 30 minute powerpoint presentation on a fundamental tm topic, after which 2 other members had 2 months to review and edit. therefore, each member created 1 and reviewed 2 presentations (three total steps). during each step, members wrote 2 multiple-choice questions for those particular topics. in the end, each topic would have 6 quiz questions to assess learning. at completion, 7 evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents. three methods were planned to measure effectiveness of these materials: 1) pre and postlecture abo leaders exam using the questions made for each topic to assess learning; 2) pre and post-lecture 20 question validated examination (best collaborative) to assess learning; 3) resident in-service examination trends specific to tm. results/finding: six presentations were developed as 6 of the 7 abo leaders members continue to participate in this cop for tm education. abo leaders and best pre-test results are shown in tables 1 and 2. abo leaders pre-test data could not be obtained for institution b, and 3 trainees declined to participate in the examinations at institution a. challenges experienced by the cop have included heterogeneity between institutionsõ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all 6 presentations during the defined institution-specific teaching period. post-test results will be included when assessments are complete. conclusion: despite logistical and organizational challenges, it is feasible to create a multicenter cop for tm education. the impact of such a group on resident learning will be assessed and plans for growth will be evaluated. background/case studies: the traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases. the use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine. our goal is to use and evaluate the relevance of this approach in resident education. study design/method: a clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation. during a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics. each resident completed a 10 question preand post-test on topics related to the vignette. several meetings for review and adaptation of the script, topic discussion, and rehearsals were held. there were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance. during the skit, each resident presented at least one major transfusion management teaching point. results/finding: the educational activity, including the 40 minute vignette performance and the 20 minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis. all performers significantly improved their scores in the post-test (mean 95 1 4%) when compared to the pre-test scores (mean 67 1 26%) ttest p<0.017. during the vignette discussion, residents together identified all the intended non-conformances and answered related questions. residents in the audience actively participated in the post skit discussion and 90% reported a satisfactory learning experience. conclusion: dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents. with specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember. the skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care. hannele sareneva*, susanna sainio, inna sareneva, tiia kivipuro and taru jaske. finnish red cross blood service background/case studies: the finnish red cross blood service (frcbs) is the nationwide blood service provider in finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers. the frcbs serves as the national blood group reference laboratory and provides a wide range of other laboratory services e.g. tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation. frcbs also performs antenatal blood group and rbc antibody tests covering whole country. as a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services. study design/methods: we have performed customer surveys to healthcare professionals to assemble the needs for education. based on these results and continuous feedback frcbs provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * handbook for blood products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians. for every education we collect numerical feedback as following: "how did the education responded your expectations" and "can you utilize the knowledge in practice". we also inquire "how likely you would recommend the training for your colleges" indicating net promoter score (nps). results/findings: more than 350 healthcare professionals participate training days at frcbs annually. in addition our experts give tens of lectures at hospitals across finland. feedback from educations has been very good, varying between 8.3 to 9.4 (in the range of 4-10). nps varies between 83 and 98. according to customer surveys frcbs provides appropriate education to healthcare professionals. this score has increased 2011-2016 from 8.4 to 9.0. conclusion: feedback, nps scores and surveys ensure that education and training program of frcbs responses to customer needs. hospitals can utilize annual courses of frcbs in their own initiation programs. together with clinical contact persons in hospitals our aim is to ensure patient blood management (pbm) and to optimize use of blood products. we also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students. educational outreach and effect on reporting septic transfusion reactions kathleen m grima* 1 , anne eder 2 , beth a. dy 1 and mary o'neill 1 . 1 american red cross, 2 georgetown university background/case studies: hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center. about 1 in 100,000 apheresis platelet donations are implicated in septic transfusion reactions (strs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions. a large blood center designed an educational outreach program to increase awareness of strs and assessed its effect on the rate of str reporting to its national hemovigilance program. study design/method: in dec. 2015, a large blood center developed a web based course on strs for cme/ceu credit. letters were sent to 2,300 hospital customers about recognizing and reporting strs, and alerting them to the availability of the course. blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting strs, using the online educational content. the physicians tracked their interactions. the blood center's national hemovigilance program compared the number of strs reported in the 12 months before and after launching the educational outreach. results/finding: the web based course was completed by more than 700 participants; 117 were physicians. based on a review of the evaluations, the course was highly valued with 93% of participants rating it excellent or very good. the blood center physicians gave over 200 presentations to hospital customers. reporting of suspected strs in 2016 increased by 23% compared to the prior year. the increased reporting came from 2 specific regions. the total number of strs that met the hemovigilance definitions for definite (culture-confirmed) and probable strs in the nationwide system increased but did not change significantly compared to the previous years. the educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of strs. while the number of reports of suspected strs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed strs across the national blood system. this finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside. more targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine. implementation of subscription-based cgmp e-learning laurie mcgraw*, courtney saphier, helene belton, sallie bittner and ward scott. gulf coast regional blood center background/case studies: previous cgmp e-learning courses we developed required 30-45 minutes for learners to complete. while feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules. at the same time, a shift in design trends suggest that subscription-based learning is more effective (thalheimer, 2014.) subscription-based e-learning utilizes 5-7 minute modules, delivered at regular intervals. this changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind. study design/method: we began developing cgmp subscription-based elearning in 2016 by selecting our first five series topics: equipment, personnel, labeling, sops, and records. the first topic, equipment, was divided into modules on selection, validation, calibration, quality control, and maintenance. these modules, and pre-and post-quizzes for the equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system. the pre-quiz was assigned to employees in june 2016, with a new equipment module assigned each month for the following five months. the series concluded in december 2016 with the post-quiz. results/finding: using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four kirkpatrick levels. while our previous cgmp courses received good ratings from learners, the equipment series received the highest rating of 3.5 on a 4-point scale. of employees who completed all versions of our cgmp courses, the majority preferred the equipment series over all previous courses combined. comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with 21 positive and 1 negative comment. level 2: learning the average score of users increased 13% from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees. a two-sample t-test determined the result to be statistically significant with a t-critical value of 1.647 and a t-stat value of 5.641. level 4: results while equipment-related errors decreased by 20% after training, there is not enough data to demonstrate a statistical significance. conclusion: our level 1 and 2 evaluation data validated that the subscription approach was effective. knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements. as a result, we are continuing development of the remaining series. background/case studies: the interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams. we hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in october 2013, a multidisciplinary educational meeting called friday blood conference (fbc) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice. during these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion. study design/method: a survey was sent to fbc participants (n5151) to retrospectively capture the effect of fbc on interdepartmental collaboration. the survey was structured to obtain formative feedback using the published interprofessional collaborative practice competencies (icpc) as a guide. these core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care. results/finding: our survey response rate was 35%. of those, 96% endorse that fbc creates a climate of respect within our transfusion practice, 94% believe it has improved communication between work units, and 98% feel that fbc leads to increased understanding of interdepartmental processes. notably, laboratory scientists and transfusion nurses have the highest attendance rate. furthermore, those attending via the online broadcast report the lowest satisfaction, with only 56% responding positively. the main reasons individuals attend fbc are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care. suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of fbc for later viewing. conclusion: the application of icpc in transfusion medicine was an effective lens to assess the value of interprofessional collaboration. although there is room for improvement, the results support that fbc has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice. novel approach to curriculum development: demystifying transfusion medicine ritcha saxena* and ananya saxena. all saints university school of medicine background/case studies: transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel. transfusion carries considerable advantages as well as risks. consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps. and the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine. study design/methods: 41 students of undergraduate semester 3 and 59 students of semester 4 participated in the study. self-directed learning resources combined with modules of interactive instruction were implemented in a tbl course design. five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students. the students' reaction to tbl in transfusion medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity. the participants were first assessed with readiness assurance testing (rat) to guarantee that they understood the concepts and their application followed by case study based test questions. results/findings: students' reaction to tbl was primarily positive, with 86% of students giving a positive feedback. evaluation through readiness assurance testing (rat) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study. students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications. anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation. conclusion: our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians. tbl is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students. results/finding: open house attendees were given tours of the bb, led by a bb attending, bb residents, bb supervisor, or bb quality coordinator. the patient blood management nurse was also in attendance to answer attendee questions and educate about patient blood management. light refreshments were offered to the attendees in the bb break room. the first bb open house was held on wednesday, 12/7/16 from 9-11am. there were 17 attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in facilities management and the university office of admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the university office of admissions. the second bb open house was held on thursday, 4/20/17 from 7-11am. there were 14 attendees, including 2 regular blood donors (who were also employees in the office of international affairs and supply chain, respectively), a hematology/oncology fellow, and 11 surgical residents. background/case studies: simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (scd) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells. it is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure. given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks. study design/method: a web application was generated (www.phamcalcs.com). the performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data. the performance of the automated and depletion rbcx calculators was validated using the terumo bct (lakewood, co) calculator up to a fraction cells remaining (fcr) 50% as patients with fcr 50% may benefit from delaying the procedure for performance in the future. validation process included (1) a deming regression to globally assess the predicted vs. actual results and (2) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |d| 15%. validation was performed for hematocrit (hct) and hemoglobin s (hgbs) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion rbc exchange. results/finding: see table 1 background/case studies: with the focus on new technologies the modern medicine requires more expenses. despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment. the issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency. in russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs. as a multidisciplinary approach, pbm optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution. however, the prosperous implementation of pbm also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist. study design/method: at the medical simulation centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion. the main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods. during 2 days (18 academic hours) the trainees can attend 6 lectures, discuss the methodical materials, participate in 3 seminars, 2 interactive clinical discussions, a master class and a game that presents the modelling of working processes. since initiating the project in june, 2016 with the group capacity 220a transfusion 2017 vol. 57 supplement s3 of up to 35 people the number of medical specialists who have attended the training is nearly 450. results/finding: the medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards conclusion: the launch of the program "guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of patient blood management and reach the compliance in practice. the effect of emergent situation drills on technologist teamwork and comfort levels abigail neils*, raeanne stensgard, rebecca wren, elisabeth greer, amy mata and camille van buskirk. mayo clinic rochester background/case studies: teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously. in an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (esd). the esd were based on common emergent situations encountered in the lab and were run once per month per shift. the main goal of esd was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (sop) deviations. study design/method: prior to esd implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations. one year post esd implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations. the surveys asked employees to rate satisfaction and comfort level on a grading scale of 1-100; 1 being least satisfied/comfortable and 100 being most satisfied/comfortable. the pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey. in addition, unplanned sop deviations related to emergent situations were counted for one year before and one year after esd implementation. results/findings: out of 35 total technologists, 31 technologists took the pre esd survey and 25 technologists took the one year post esd implementation survey. table 1 shows the lab averages from the pre and post surveys as well as the percent difference. out of the employees who responded to the post survey, 19 (76.0%) answered "true" to the statement "esd have improved my comfort level with emergent situations." in the year prior to esd implementation there were 14 unplanned sop deviations; in the year after esd implementation there were only 5 deviations. conclusion: all but one area increased in comfort level post esd implementation. also most technologists agreed that the esd helped improve their overall comfort level with emergent situations. the goal of implementing esd has been met based on the unplanned sop deviation decrease and technologist satisfaction increase; therefore esd were deemed effective. monthly esd will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels. therapeutic background/case studies: category i indications for red blood cell exchange (rbc exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention. as described in the first installment of this series about therapeutic plasma exchange (tpe), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population. rbc exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood. nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care. providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery. a previous project explaining tpe to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including tpe and rbc exchange. study design/method: in collaboration with a child life specialist, an ageappropriate story-driven explanation of the rbc exchange procedure was adapted from a previously implemented project related to tpe. artwork was produced with the aid of a medical illustrator to complement the story-line. results/finding: the story board addresses why rbc exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure. the idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy. the booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use. conclusion: using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety. this second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies. transfusion safety officer resource manual leonor de biasio*. it is also intended to be utilized by hospitals that do not have a formal tso position but which have delegated the responsibilities to other staff. the resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood. the resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the tso role. turning on pathogen reduction: a case of flipping the switch kassandra poffenberger*, darla wendt, jennifer vrieze and james r stubbs. mayo clinic background/case studies: a critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care. with the implementation of pathogen reduction technology (prt) using interceptv r blood system for platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections. our institution collects the majority of its blood products and supplements inventory from a major blood collection center. it was crucial for the component laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors. our approach in introducing prt for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory. study design/method: it was essential to prioritize who would be trained first. collections occur monday through friday from 0600 to 1600. the first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by cerus deployment team. the second group was those who would process platelets on weekends and evening hours without direct management support. the last group was the technologists who would be working during normal hours with direct management support. prt processing for platelets in 100% plasma is broken up in to two days. on day 0 platelets are treated with amotosalen and placed in a compound adsorption device to remove residual amotosalen for 12-24 hours. on day 1 products are removed from the cad and modified into final product codes and labeled. each technologist was trained one on one, over a one week period. the trainers alternated training processing days for day 0 and day 1. in the weeks following training it was important that each technologist rotated back thru prt processing to maintain proficiency. results/finding: 13 of 18 employees were trained in a two month time period. prior to "flipping the switch" the daily average of products collected was 21. for the two month training period the daily average rose to 23. conclusion: our "flip the switch" training plan for implementing prt platelets in 100% plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process. it was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts. technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory. accepted depending on each individual's conscience. due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care. we describe the process of identifying jw in our hospital and communicating treatment needs to staff. study design/methods: proper treatment of jw requires the ability to identify the patient and his/her needs. when a jw is admitted to our hospital, our electronic medical record (emr) triggers several processes based on the patient's listed religion. one process creates an order that reminds caretakers to complete the declining blood consent (dbc) with the patient. the dbc contains language declining mabf and reviews the mibf with the patient to identify any that would be accepted. the emr order regarding the dbc provides educational links that include a bloodless policy, step-by-step instructions on obtaining the dbc, and information on alternatives to transfusion. a second emr process triggers a stop-gate to prevent the completion of any mabf order or mibf order for a product that the patient has declined. a third enrolls patients in the minimal blood volume labs protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws. additionally, at registration, a bloodless packet is added to the patient's paper chart. this packet contains the dbc, a glossary of dbc terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart. these steps remind the caretakers of the patient's special requests. finally, the patient blood management (pbm) department receives emr developed reports which identify jw presenting to the hospital. these patients are followed by the pbm nurses and medical director during the duration of care. treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed. results/findings: nearly 100% of jw that enter our hospital have a dbc completed. this has resulted in increased education of the medical staff. in addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients. conclusion: our hospital has found success by using an education-based team-oriented approach involving emr, pbm, and caretakers when caring for the jw patient. this approach has set up a foundation for treating other bloodless medicine patients. background/case studies: transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers. this process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes. cost management has a great impact on technical processes guiding decisions upon supplies and technical staff. activity-based costing (abc) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions. we demonstrate how an effective abc approach can result in financial savings without compromising process quality in a mid-size transfusion service. study design/method: materials costs can represent as far as 90% of an activity. in 2015 we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock. purchases were performed on demand. at the end of 2015 we performed a supply inventory on all departments to plan future purchases and control residual stocks. in 2016, we implemented annual purchases and satellite storages were supplied only to replenish programmed stock. cost drivers were defined upon activities on standard operational procedures (sops) resulting in a 2016 cost estimate. technical staff was involved in cost driver calculations to indicate possible changes to sops, supplier and deliveries. to minimize seasonal fluctuations we compared last quarter 2015 (q4/15) with last quarter 2016 (q4/16). in this work we present activity data from blood collections to illustrate abc method. results/finding: in q4/15 1756 blood bags were used compared to 1998 in q4/16, demonstrating an activity "13.78%. price negotiation resulted in 12.58% readjustment. both indicated an estimated cost "28.10% with a possible impact of over us$ 35,000. we have identified a real cost #2.31% in q4/16, representing an overall #14.89% and us$ 3,716.72 (r$12,235.16) savings. conclusion: economy had deteriorated in our country in 2016 with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical. even with adverse economy, abc showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes. cost drivers calculations demanded review of sops and suppliers by technical staff resulting in optimization of activities. also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly. automated verification of immunohematology results and the impact to donor testing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh title: automated verification of immunohematology results and the impact to donor testing background/case studies: staffing challenges in today's blood banks require instrumentation with minimal operator intervention. technology advances have developed where every immunohematology result does not necessarily require operator visual review. this study evaluates the impact of automated result verification on the bio-rad ih-1000 tm immunohematology system through the ih-com tm data management system (dms) for donor processing laboratories. study design/method: a multi-center study was performed on donor samples as shown in table a evaluating two of the most commonly used ih-system gel cards available in the us. workflow data was analyzed using process modellar app (ipad). this study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17, statistical the transfusion team collaborated with multiple user groups to educate them regarding the new processes. a gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options. the use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus. results/finding: user groups requested additional training sessions as questions arose regarding use of the ehr for blood ordering. because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood. department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order rh immune globulin, a cord blood workup, etc. conclusion: leadership was challenged to provide a stable and positive environment during a complex set of changes. the simultaneous hospital move/merger and implementation of a new ehr constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance. training is essential to the success for a scope of change this big and should not be minimized. while training was thorough prior to the move, gaps were nonetheless discovered following the move. abstract conclusion: strategic development partners funding and support based on newly developed government strategy on blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in ethiopia. even though the identified positive impacts mentioned are achieved, the bts remains with multiple challenges and needs continuity of funding and more partner support and government commitment. pilot implementation of a comprehensive hybrid performance management system at national blood service zimbabwe blessing mukwada*, judith j parirewa and tonderai mapako. national blood service zimbabwe background/case studies: the national blood service zimbabwe (nbsz) introduced its first performance management system (pms) in 2006. in the 2015-2018 nbsz strategic plan it was noted that the current pms lacked objectivitety and there was no relationship between perfomance and remuneration. in order to revise the pms, the nbsz set up a three membered committee at the executive management level to spearhead the revamp of the nbsz pms. the aim of the new pms was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan. in this paper, we share how nbsz revamped and implemented its new hybrid pms that derived its inputs from established pmss and nbsz monitoring and evaluation (m&e) process that have been linked together. one-selected departmental results for one quarter are shared to demonstrate how the system works. study design/method: pms committee developed and shared with executive management a pms conceptual and implementation framework. consultations including field visits were done on three established pms to assess suitability for nbsz adoption. a hybrid pms was adopted for nbsz and a pilot application for one quarter on selected department was done. review of policy, procedures to including hybrid pms templates and forms were done. pms committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new pms, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools. throughout the process risk assessment were done. results/finding: the nbsz hybrid pms is based on five levels of planning namely strategic, departmental, branch, sectional and individual. the fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels. the levels of accountability were properly defined for each level of planning. a weighted overall integrated individual scorecard (iis) is determined based on 60% individual and 40% for the other four levels (10% for each). the bonus (%) is calculated based on the iis as follows; category a: 100% (iis >575%), category b: 75% (iis: 50 -<75%), category c: 50% (iis: 25-<50%) and category d: 0% (iis < 25%). on the pilot implementation, the individual scores for 12 staff ranged from 71% to 100%. the iis were 76% to 81%. the number of staff in each bonus categories were 11, 92% (category a) and 1, 8% (category b). conclusion: the new hybrid pms was generally accepted by all staff and it was easily implemented at various staff levels. this provides a basis for the full implementation of the new pms and this simplified pms can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision. rare donor engagement with american rare donor program (ardp) margaret c manigly* 1 , deborah r fludd 1 and sandra j nance 2 . background/case studies: rare donors are defined as a blood type occurring in less than 1 in 1000 people in a given population. these donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor. once found, if the facility is a member of the american rare donor program (by being an aabb accredited or american red cross accredited irl), the donor is registered in the ardp database as a rare donor. in 2016, there were 65,801 active rare donors in the ardp. with the mobility of the population in the usa, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need. in addition, when recruitment is needed for a patient need, correct contact information on the donor is required. study design/method: the ardp procedure for ardp members requires that donors be contacted every six months to ensure that ardp (or the facility) has their latest contact information. the timing is determined by the postal service time limit of six months to forward mail to a new address. this contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate. this contact is achieved by ardp sending a contact card by postal mail twice yearly to all donors for whom the ardp has address demographics. results/finding: the ardp reports on the information obtained from the contact cards returned in the ardp annual activity report to the ardp members at the aabb annual meeting. of the 6398 (9.7% of total active donors) returned contact cards alerting ardp of changes in calendar year 2016, 355 (5.5%) were donors moving from one ardp facility to another, 1369 (21.4%) were donors no longer eligible to donate, and an additional 4324 (68.4%) were address changes. other changes were 115 (1.8%) reactivated donors and 235 (3.5%) donors who we were notified were deceased, or did not want to be listed in the ardp. in 2016, 5390 new rare donors were submitted to ardp for registration. the number of donors that could potentially be lost to follow-up in 2016 was 4709 (355 1 4324), which would be 87.4% of the new donors submitted. conclusion: with nearly a 10% response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ardp contact card, and inform ardp of address changes and changes in their health status that affects their ability to donate. this is evidence of the importance of the card in ensuring correct donor contact information. in 2016, 4709 donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move. the ardp contact card is effective in retaining the relationship with the ardp registered donors and keeps the address information of rare donors current. workflow comparison of two gel analyzers in a large transfusion service j peter pelletier* 1 , barbara j bachman 2 , mike leamy 2 , susan olson 2 and candace williams 2 . 1 university of florida college of medicine, 2 bio-rad laboratories background/case studies: vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market. the purpose of this study was to evaluate the provue (ortho clinical diagnostics) against the ih-1000 (bio-rad laboratories, inc.) in a large volume transfusion service using lean process flow. study design/method: twenty-two (22) runs of one to six (6) samples per run were observed for two ortho provues alternating testing at a large transfusion service performing 153,000 types, screens, type & screens (t&s) annually. the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. regardless of quality or speed metrics evaluated, the ih-1000 demonstrated a significant reduction (improvement) in process steps and associated times when compared against the ortho provue (p < 0.001). ih-1000 process steps and time studies addressed in the table below did not account for the ih-1000 reagent storage capacity. in reality, the improvements would be greater than what was displayed here in a real-life operation. evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the ih-1000 (77% reduction, a difference of 43 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the ortho provue for a large volume transfusion service. workflow comparison of two high volume, high throughput analyzers aaron samson* 1 , kimberly monnin 1 , barbara j bachman 2 , kyla warren 2 , susan olson 2 and candace williams 2 . 1 clinical pathology labs, 2 bio-rad laboratories background/case studies: few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities. the purpose of this study was to evaluate the galileo v r neo (immucor) against the ih-1000 tm (bio-rad laboratories, inc.) using lean process flow. study design/method: a total of 12 separate test runs of 72 or 144 samples per run were observed over a three day period on the galileo neo at a reference laboratory annually performing approximately 211,500 type & screens (t&s). the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/finding: detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (table, part a) . time studies focused on operator time, analyzer time, and maintenance time (table, part b). regardless of quality or speed metrics evaluated, the ih-1000 demonstrated significant reduction (improvement) in process steps and associated times when compared against the galileo neo (p < 0.001). evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the ih-1000 (difference of 120 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the galileo neo for high volume/high throughput testing facilities. workflow impact of automated result verification for patient and donor blood typing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh background/case studies: immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety. to alleviate these challenges, the ih-1000 tm instrument and complementary ih-com tm data management system (dms) were designed to provide lean automation to enhance blood testing facility workflow. the purpose of this study was to focus on the lean functionality of automated result verification on the ih-1000 and ih-com dms and determine its impact on workflow. study design/methods: internal and external studies using the ih-1000 with the ih-com dms were performed with patient and donor samples. assays included abo/rh blood grouping and antibody screening (abs) as shown in table a . workflow data was analyzed using process modellar app (ipad). the evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17. statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/findings: using automated result verification, only 0.93% out of 6,339 samples evaluated for abo/rh testing would require visual verification, resulting in a 98% reduction in operator touchpoints (p < 0.001) and a labor saving of 444 minutes (7:01 hh:mm) for abo/rh testing. for 8,750 antibody screens, automatic validation of results would result in 99.5% reduction in operator touchpoints (p < 0.001) and a labor savings of 378 minutes (6:18 hh:mm). no false positive or false negative typing results or false negative screenings occurred with results auto-verification. (rbc) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management. over the past 2 years a steady increase in demand for o neg rbc compared to other blood types has been observed at our blood center. utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits. despite heightening awareness, percent o neg rbc sales continued to rise by 1% annually and peaked at 16% in mid 2016. to better understand this increased demand a survey was conducted to gather insight for improved utilization. we speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of o neg rbc sales. study design/methods: a tie tag was designed as a survey tool and attached to each o neg rbc distributed to hospital customers for an 8week period in late 2016. hospital transfusion service staff were asked to record the final disposition of the o neg rbc (transfused, wasted, returned) on the tie tag. information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers. completed tags were returned to the blood center. customers are allowed to return rbc units with greater than 10 day shelf life remaining. units with tie tags attached were in hospital inventories for up to 3 months due to the shelf life of rbc. return rates and percent of net sales (gross sales minus returns) by abo/rh type were tracked monthly before, during and after the survey. results/findings: participation was 100% of the 56 hospitals surveyed. mean percent o neg rbc gross sales for a 3 month period before, during, and after the survey was 16.6%, 16.0% and 16.5%, respectively. mean percent o neg net sales during the 3-month survey fell to 13.5% compared to an average of 15.4% in the 3 months prior. during the 3-month survey period o neg rbc monthly return rate increased to an average of 28.4% compared to an average of 23.0% in the 3 months prior. for the 3 months after the survey the average o neg rbc return rate further increased to 28.9% while mean percent o neg rbc net sales trended slightly upward to 14.1%. when customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported. conclusion: during and after the survey percent o neg rbc gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged. however, during the same period the increase in o neg rbc return rate and corresponding decline in percent net sales suggests improved o neg rbc utilization. increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent o neg rbc net sales. tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving o neg rbc utilization. acoustophoretic separation of platelets from whole blood: a relevant and practical alternative to centrifugation pierre bohec* 1 , jeremie gachelin 1 , veronique ollivier 2 , thibaut mutin 1 , xavier telot 1 , benoit ho tin noe 2 and sandra sanfilippo 1 . 1 aenitis technologies, 2 hôpital bichat, inserm u1148 background/case studies: shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates. transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions. aims: here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets. study design/method: whole blood was obtained from 14 donors and fractionated using an acoustic-based device. platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples. quality of isolated platelets was evaluated using the surface expression of two activation markers (p-selectin, pac1) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation. platelets isolated using a soft-spin protocol, were used as inactivated control. results/finding: fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than 80 % (p< 0.001) and a purity of platelets close to 91.0 %. we did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol. conclusion: this acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings. automation in blood bank processing: where we go? robert fernandez, lluis puig, pilar ortiz, joan ovejo, nuria martinez, elena valdivia and susana g gomez*. banc de sang i teixits background/case studies: nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production. at banc de sang i teixits (bst), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over 250.000 blood donations for an area with more than 7 million people. study design/method: the automation of blood donations process, bst has done different changes on the equipment. in 2005, orbisac (terumo bct) was the equipment to obtain buffy coats and from this product, we got platelets concentrates. it was in 2007, when we moved from this equipment to atreus 2c (terumo bct), to get red blood cells, buffy coat and fresh frozen plasma. then we did some updated on atreus; in 2011 we changed to atreus 3c (terumo bct) and finally in 2013, we moved to reveos system (terumo bct). since the changes in 2007, our blood components were red cell concentrate, plasma, platelets and a leukocyte residue. while all these changes in processing equipment, we added also some automation in our registration (donation id, weight and temperature) and labeling steps, implementing two homemade robots. and finally, to get better results and more efficacy in our production, in 2009, bst incorporated an engineer to introduce lean manufacturing methods. these methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure. results/finding: once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations. this evaluation was done for processes during 2008 and 2016. conclusion: with these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way. we encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary. in a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank. background/case studies: the laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of remote blood allocation devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload. as part of this initiative, bloodtrack haemobanks (hb) (haemonetics, braintree, ma) were installed and interfaced to the existing safetrace tx (haemonetics, braintree, ma) laboratory information system. one hb was installed in the methodist hospital (rmh) campus which includes a busy outpatient infusion therapy center (itc). study design/method: an assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff. frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times. for the itc, with a daily outpatient volume of 140-160 patients and an average, round-trip travel time of approximately 15-minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects. itc nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care. about 30% of patients in the itc have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow. often for patients in the itc, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood. these inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the itc. results/finding: hb devices allow nursing staff to access red blood cells (rbc) for the majority of their patients at the point of care. since implementing in november 2015, the hb has significantly improved the turnaround time of rbc issue -from 15-minutes to less than 60-seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window. prior to hb implementation, blood bank staff at rmh were issuing approximately 750 rbc per month out of the window for non-surgical patients. this has been reduced to approximately 300 rbc per month, a 60% average monthly reduction. conclusion: having the hb located in the itc has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders. the use of hb devices has not resulted in a reduction in blood bank fte, but rather a shift in workload; from issuing products to monitoring inventory and restocking. consists of registered paramedics that are pararescue specialists and helicopter personnel. when in combat, the squadron conducts personnel recovery operations and rescues downed airmen. when stationed in the us, they mitigate in state emergencies and perform aeromedical evacuations. in 2015, they supported a civilian medical emergency and the patient needed a transfusion in the field. they procured blood products from a distant air force base with adjacent medical facility. at the debriefing, members of the 131 st rescue squadron (131 rqs) decided to find a local civilian blood supplier. the master sergeant contacted our blood center and set up a contract for blood supply. study design/method: blood center representatives met with the 131 rqs master sergeant in january 2016. we asked what 131 rqs's order and delivery expectations were. he said sporadic use and the blood order would be 2 rbcs. we wrote a procedure for consignment and packaging, using standard blood transport boxes. we developed a communication template for staff to anticipate the 131 rqs needs. staff was trained based on data from january 2016 meeting. we contacted the 131 rqs in september 2016 to perform a trial run. at that time, we learned the master sergeant was shipped out to military theater. we invited his replacement to the blood center. this pararescue senior airman had just returned from syria and was assigned to civilian duty. he had no prior knowledge of the 131 rqs association with a civilian blood center. based on his field experiences, he changed the blood order from 2 to 6 rbcs. he introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions. we rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers. the blood center and 131 rqs performed a mock run on october 31, and we felt prepared for any future events. results/finding: on november 11, 2016, the 131 rqs was deployed to a civilian aeromedical evacuation. we anticipated a 6 rbc order. the actual order was 7 rbcs and 4 ffp. staff was preparing frozen ffp to ship, as was their norm for filling hospital orders. realizing that they could not thaw plasma in flight, we contacted the 131 rqs and offered liquid plasma instead, which they accepted. product was consigned and picked up at 4:30am by the 131 rqs. the patient was transfused in the field and then taken to a nearby hospital. at our joint debriefing on november 28 th , we established a maximum blood order of 10 rbc and 4 liquid plasma, noting future orders may request fewer products, yet meet the preferred 2 rbc; 1 plasma transfusion ratio. conclusion: military personnel are adapted to instantly adjust to an ever changing environment. regulated blood centers are not as adaptable. with clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the air national guard. (table 1 ). the highest mean fib concentration was 535 mg/donor unit; lowest mean fib concentration was 264 mg/donor unit. all sites had a mean fib concentration at least 100 mg/donor unit above the fda minimum requirement of 150 mg/donor unit. fifteen of 17 blood centers completed the manufacturing process survey. one used a leukocyte reduction filter with ahf destined plasma. all blood centers manufactured single donor cryoprecipitate; 12 manufactured pooled donor cryoprecipitate. most froze plasma in a -188c or colder blood bank freezer. one froze plasma using dry ice, and one used a blast freezer. two blood centers method of thawing frozen plasma took longer than 10 hours. conclusion: blood centers consistently met the overall fib minimum requirement with a mean of 345 mg/donor unit, over double the fda requirement. however there is variability in fib levels amongst blood centers. in general, manufacturing processes were similar with a few exceptions. blood centers should inform their hospital customers of their average fib level in cryoprecipitate in order to most appropriately care for patients receiving this product. compliance & productivity improvement via engineered-staffing/ scheduling calculator application (app) mary deck, mark angelelli and kevin lee*. american red cross background/case studies: the healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure. applications of basic industrial engineering tools, coupled with lean-six sigma techniques such as time study analysis, bottleneck elimination & process standardization to transfusion reduce variation has been transformed into an application (for short "app"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking. study design/methods: a time study analysis offers valuable data about the process requirements. once this baseline has been established, translating the data into a user-friendly app would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations. important concepts such as lean-pull production system, bottleneck elimination, work-load balancing together with basic development of the app using ms excel software will be demonstrated. results/findings: successful rollouts and implementations of the staffing/ scheduling calculator app across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard. the app interactive-based approach, programmed via a commonly used software, ms excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e. match incoming volume/work content to staffing availability). the staffing/scheduling calculator app has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking. conclusion: besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the staffing/ scheduling calculator app will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization. all coolers were prepared in a walk-in refrigerator. two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with rbc units on top of the ice. a quality-controlled thermometer was placed on top of the rbc units. a control thermometer was place at the interface between the ice and the rbc units in one large and one medium cooler. the start temperature was recorded and then the temperature was recorded every 15 minutes for a 12 hour period or until the temperature exceeded 68c. results/finding: the temperature recorded from the thermometer on top of the units in all five coolers reached >68c in 75 minutes as shown in table 1 . the control thermometer recorded temperatures maintained at 1-68c for the entire 12 hour observation period in both the large and medium cooler. conclusion: when units are placed on top of the ice in a cooler, the temperature is not reliably maintained at 1-68c for more than 45 minutes. these data support a policy of wasting units that are returned to the blood bank with rbc units on top of the ice. background/case studies: an fda draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (pc) via pathogen reduction (pr) or secondary rapid testing (rt). hospitals must understand the cost implications that may result. our objective was to create an interactive model to analyze the budget impact for different pc types across the range of existing us hospitals. study design/methods: an excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to 27 us hospital transfusion service directors. the model was reviewed and refined by a panel of seven transfusion medicine physicians. the model allows base-case assumptions to be overwritten with values specific to the institution. three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (ae), shelflife, and reimbursement for a hospital that purchases all of its pcs: 100% conventional (c-pc), 100% pr-pc, and mix of 75% c-pc/25% pr-pc. the model predicts a modest ($4%) cost increase for pr-pc compared to c-pc depending on the degree of pr conversion; this takes into account cost offsets such as elimination of bd and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement. the effective pc shelf-life is potentially increased with pr due to elimination of bd, and is dependent on nat turnaround time. benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses. future iterations of this model will also enable hospitals to consider scenarios in which rt is used. this model can serve as an important tool for hospitals considering pr adoption. in january 2011. a report was created to identify donors previously classified as rare according to the american rare donor program (ardp) criteria. donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or iga deficient. the new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate. a database was created to track the letters sent to rare donors. in august 2013, inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (rbc) collected to minimize unit age at transfusion. the inventory reduction occurred in phases and was completed by january 2015. a study was performed to determine the impact of the inventory reduction on the number of rare donor donations. study design/methods: the total number of allogeneic rbc donations, rare donor donations, and number of rare donor letters sent was analyzed from 2010 to 2016 (see table) . the percentage of rare donor donations per year was calculated. background/case studies: blood centers (clients) often carry low inventory of blood and blood components. laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products. in order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to clients. in 2016, zkv-nat testing was implement for travel deferral donors (july), followed by universal individual donor screening in september and november in response to the fda recommendations for "reducing the risk of zkv transmission by blood and blood components". per the fda guidance we implemented mandatory zkv testing for clients with proximity to areas with locally acquired mosquito-borne cases of zkv within 4 weeks (sept. phase 1) and nationwide within 12 weeks (nov. phase 2). zkv testing is performed on individual samples, unlike all other nat tests that are performed in minipools (16-donations). therefore zkv testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study. study design/method: within two regional testing labs, participating in the same clinical trial, lab 1 had 86% and lab 2 had 77% of clients requiring universal zkv testing. we evaluated a 12-month test result upload performance period to determine the impact of zkv test implementation. results/finding: during 2016, lab 1 upload time performance ranged from 92% to 94.2% from january to july; upload time performance fell between august through november, returning to 94.2% performance in december. lab 2 upload time performance ranged from 91.4% to 95.3% january to august. performance fell september through december 83.3% -88.5%. lab 1 experienced a low of 75% upload time performance during phase 1 when there was a rapid implementation; 69% clients required zkv nat. improved performance was observed during phase 2, with a 16% increase in zkv clients. for lab 2: phase 1 experienced a modest decline of upload performance ranging from 83.3% to 88.5% with 33.3% of clients implementing zkv nat. performance was 87.1% in phase 2, when an additional 43.3% of clients implemented zkv testing. conclusion: with an unprecedented rapid implementation of zkv testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric. enhanced platelet bacterial screening in an eight-hospital system robin larson* 1 and colleen a. aronson 2 . 1 advocate lutheran general hospital, 2 acl laboratories/ advocate hospitals background/case studies: in response to two platelet-related septic transfusion reactions and the draft fda guidance released in march 2016 regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the verax pgd enhanced platelet bacterial screening test in 6 of the 8 hospital transfusion services. the 2 sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier. the 6 sites which implemented the verax pgd test perform testing on all day 4 and day 5 platelets to be issued for transfusion. this abstract summarizes the data collected for the first 5 weeks of testing. study design/methods: platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day 4, day 5, and those that were tested twice. inventory reports were reviewed for platelets issued on day 2 or day 3 that did not require testing, and for the total number of platelets issued over the time period studied. results/findings: in the month of february (1 week of performing the test), 48.1% of all platelets issued by the 6 participating transfusion services were day 2 or day 3 platelets. in march that number dropped to 29.9%. it is expected that this number will level off at some percentage at or below 29.0% with further data collection. in february 25.9% of platelets were tested twice prior to final issue from the transfusion services. in march conclusion: the percent of platelets issued fresh (day 2 or day 3) will likely level off at some number at or below 29.9% due to inventory management from both the blood supplier and the individual transfusion services. testing platelets twice is undesirable. ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing. in addition, 5 of the 6 sites performing testing are level 1 trauma centers and need to have tested platelets available at all times. this will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed 100%. as the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease. background/case studies: in order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(pcs) for providing a reliable source for clinical application. to speed up the storage research of pooled pcs in china, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for pcs and the quality changes during storage in pvc-bthc blood bags. study design/method: pcs were prepared from 400 ml virus free whole blood by platelet-rich plasma (prp) method. five or six bags of abomatched pcs were pooled and filtered with leukocytes filter for pcs(n57). the swirling phenomenon, ph, automatic blood count, platelet aggregation, hypotonic shock response (hsr), the extent of shape change(esc), cd62p expression, atp level in platelet, glucose and lactate concentration were detected before and after filtering, and on days1, 3, 5 and 7 of storage, respectively. results/finding: the platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was (86.7 6 1.6)%, relative change rate of hsr was (3.87 6 12.75)%, the residual leukocytes were (0.15 6 0.15)310 6 . the ph, hsr, and the cd62p expression of pooled platelet concentrates before and after filtering were (7.00 6 0.17) vs (7.06 6 0.16), (66.96 6 12.35)% vs (63.22 6 8.26)% and (28.94 6 14.25) % vs (31.60 6 16.77)%. there is significant change for wbc after filtering (p<0.01). during storage in pvc-bthc blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table 1. conclusion: storage in pvc-bthc blood bags for five days, the quality of pooled pcs met the requirements of chinese standards (gb 18469-2012) . it can be a complementary source for apheresis platelets supplement in china. evaluation of samplokv r segment sampler to obtain and measure samples from blood component tubing segments abbejane blair*. ajblair laboratory consulting background/case studies: current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination. itl biomedical has developed samplokv r segment sampler (ss), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments. ss was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure. study design/method: ss obtains fluid samples from sealed tubing segments into a needleless syringe. it consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device. a needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the ss chambers and, gently pushed onto the needles to pierce each end of the segment. the sample from the segment is then withdrawn into the syringe. the study was performed at rhode island blood center (providence, ri) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback. two lengths of tubing segments were filled to contain sample volumes of 500ml and 1000ml. two users then evaluated the ss tubing segment types with 500ml or 1000ml samples for a total of 10 data points. samples were collected into the attached 1ml or 3ml syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube. results were tabulated as pass or fail. results/finding: a total of 10 ss were evaluated by two users. all samples were successfully collected and transferred into tubes. insertion of the segment edge requires observation to ensure placement onto the needles. any air bubbles collected into the syringe could easily be moved to the top by background/case studies: the management of platelet inventory is crucial due to a number of factors including the 5 day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g. platelet collection kits, testing, product processing). the use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste. the objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (fte) allocation. study design/method: in january 2015, a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day. the tool uses daily historical transfusion data from the last five weeks. additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate. the number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends. the effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and fte information for a one year period prior to the implementation of platelet inventory management to one year period following implementation. results/finding: by implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs. the staffing adjustments and targeted collections have lowered fte and outdate cost by 22%. the platelet outdate rates dropped after implementing the platelet inventory tool from 14% (1324 units) to 11% (874 units); a 21% decrease. fte was able to be monitored closely with the donor schedule and lowered from a yearly average of 7 fte to 6.2 fte, lowering fte by 11%. conclusion: considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections. staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets. given these positive results, we are beginning to develop a similar tool for our whole blood collections. identifying opportunities to right-size hospital inventory using compotrace radio frequency id inventory management system nanci fredrich* 1 , jaclyn mckay 1 , jennifer curnes 1 and rowena punzalan 1,2 . 1 bloodcenter of wisconsin, 2 children's hospital of wisconsin background/case studies: the ability to track inventory of blood components in real time is challenging for both hospital transfusion services (ts) and blood centers (bc) using current blood bank information systems (bbis). in addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain. a pilot was designed to track and monitor all blood components from distribution at the bc to issue in a hospital ts using fresenius kabi compotrace radio frequency id (rfid) enabled inventory management system. the objectives were to determine feasibility of the compotrace system and analyze compotrace data for real-time usage and optimal inventory levels. study design/method: a 3 month pilot was conducted at a pediatric hospital and its bc using both bbis and compotrace systems to track all adult-size blood components. staff were trained on use of compotrace system. upon receipt of order from pilot hospital, bc staff applied rfid tags to all component bags and scanned components into the compotrace system. components were transported and delivered to ts following established procedures. upon receipt at the hospital, components were scanned into inventory using both the ts bbis and compotrace systems. dual scanning of components occurred upon issue to or return from floor, component modification or return to bc. products for emergency use or at time of high demand were not rfid-scanned. a priori, the pilot would stop if the compo-trace system hampered current workflow, component issue was delayed or if ts errors increased. no inventory changes were made during the pilot. results/findings: real-time data from compotrace system provided actual usage for all blood components including component disposal and shipment to and from bc. average daily rbc inventory levels and usage for selected blood types is shown in table. lessons learned related to equipment and workflow: (1) use of smaller irradiation canister may damage rfid tag, which was resolved by relocating tag, (2) ts workflow and stat orders challenged consistent use of dual processes to track component status. however no increase in ts errors or delay in issue of components occurred. conclusion: use of rfid to track blood components from bc to final disposition is feasible. real-time data from compotrace system identified optimal inventory levels for rbc at the pilot ts. use of real-time rfid to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory. background/case studies: physicians expect blood to be available at all times. following a national appeal in july 2016 for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (ts) recognized a potentially dire situation given the institution's patient acuity. our hospital-based ts supports a full range of services: a level i trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services. a regional donor center supplies our blood products. to insure appropriate response to patient needs, the ts created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages. the approach is described herein. study design/method: at the direction of the transfusion committee (tc), ts directors presented the concern for impending shortages to the hospital quality directors (qd) committee. the qd committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional health care quality (hcq) activities. the qd recommended creation of a multidisciplinary team: "the blood shortage task force (bstf)", analogous to an existing task force started for management of drug shortages. results/finding: with hcq and tc support, the ts created the bstf and blood shortage management algorithm (bsma). standing members of the bstf include ts medical director (chair), senior vice president (svp) of hcq, svps of clinical services director of regulatory affairs, legal counsel, and representatives from ethics, social work, pharmacy, patient referrals, and communications. ad hoc members include those whose patients would be most impacted by the specific shortage. the bsma designed by the bstf provides a framework for ts's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the bstf. trigger criteria include: marked ts concern; essential product; high likelihood of inventory depletion; broad patient impact. once convened, the bstf is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care. conclusion: faced with the potential for limited blood supply, the ts reached beyond the laboratory and engaged the tc and members of hcq to assemble a robust, multidisciplinary task force. this resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients. abstract background/case studies: cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants. the collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within 8-hours after collection. this tight 8hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products. study design/methods: the american red cross (arc), in partnership with researchers from the georgia institute of technology (gt), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate. after reviewing blood collecting and processing schedules, collection locations, and other factors, arc-cryo subject matter experts together with gt researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model. results/findings: to facilitate implementation, a decision support tool (dst) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized. the implementation of the dst led to an increase in the number of whole blood units satisfying the tight 8-hour completion time constraint for cryo production (capacity expansion). in particular, during the 4 th -quarter of 2016, a blood processing region was able to process about 1000 more cryo units/month (an increase of 20%) at a slightly lower collection cost (cost avoidance), resulting in an approximately 40% reduction in the per unit collection cost for cryo. conclusion: by utilizing operations research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved. this interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a finalist on the 2017-the franz edelman award, recognizing outstanding achievements and practices in operations research. inventory management and transfusion practice before and after 7-day apheresis platelets sarah k harm*. university of vermont medical center background/case studies: the shelf life of apheresis platelet (ap) units stored in plasma may be extended from 5 to 7 days in the usa using an fda cleared rapid test (rt). in august 2016, our hospital based transfusion service began using a rt on day 6 and 7 to routinely extend ap shelf life to 7 days. this report describes changes in platelet inventory management and transfusion practice six months following routine use of 7-day ap. study design/methods: data were obtained for two study periods: september 2015-february 2016 (pre-implementation) and september 2016-february 2017 (post-implementation). the study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region. the transition period from 5-day to 7-day ap inventory was excluded. the following data was collected for each study period: the total number of ap transfusion recipients, ap units transfused, expired ap units, ap units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay. results/findings: data are shown in the table. the number of ap transfusions decreased by 3% post-implementation while inpatient admissions and surgical volume increased by 1% and 3%, respectively. the hospital length of stay was similar for both periods. ap inventory decreased by 36% post-implementation and the outdate rate decreased from 29% to 15% (p<0.0001). ad-hoc ordering was not statistically different between study periods (p50.10). the average number of ap transfusions per patient between pre-and post-implementation periods was not statistically different (2.1 and 1.9, respectively, p50.65). furthermore, a new "rejection threshold" for lipaemic products will be implemented. this threshold represents the tg concentration above which viral marker testing for donor screening will be affected. in kcbb abbott's prism assays are used for: hbsag, anti-hcv ab, anti-hiv ab, anti-htlvi/ii . results/finding: using data management system and file records in kcbb as regard discarding blood components due to lipaemia during the last five years (2012) (2013) (2014) (2015) (2016) , it was demonstrated that number of discarded rbcs due to lipaemia during the whole period was 4892 units. number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was 8546, 24, and 2 units respectively. the mean number of discarded rbc units of the five years of the study exceeds 50% of the tested ones. literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations. by reviewing sample requirements for viral marker testing in kcbb, the accepted level for tg in blood samples is below 1000 mg/dl, and so the rejection threshold for lipaemia is level equal to or more than 1000 mg/dl. conclusion: many blood product units are discarded needlessly in kcbb due to lipaemia in the last five years (including 4892 rbcs, 8546 plasma products and 24 apheresis platelet units). in an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from 200 mg/dl to 1000 mg/dl which does not affect blood safety. a follow up study is recommended after applying the new threshold to evaluate the new policy. logistical management of the incorporation of pathogen reduced single donor platelets (pr-sdp) into inventory at a u.s. tertiary care medical center eric gehrie* 1,2 , rebecca ross 3 , debra mraz 3 , anne baker 3 , zenna neal 3 , melanie champion 3 and edward l. snyder 2,3 . 1 johns hopkins university school of medicine, 2 yale university, 3 yale-new haven hospital background/case studies: the approval of pr-sdp by the fda provided an opportunity to improve the safety of our platelet inventory across all patient demographics. we outline our approach and address issues we faced during the first 4 months of pr-sdp availability. study design/methods: our nursing education team provided presentations to the nursing and clinical unit support staff. a company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards. presentations to physicians were made by the blood bank medical staff. information technology personnel created a new product type in the blood bank computer system, tested the abo/rh truth tables, and ensured that billing codes were in place. the necessity for transiently supporting a dual inventory of pr-sdp and conventional platelets led to consultation with the ethics committee and risk management, to confirm that pr-sdp and conventional platelets (c-plts) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care. we chose to not gamma irradiate any unit of pr-sdp, consistent with the package insert. results/findings: the ethics committee and risk management confirmed that informed consent was not needed for transfusion of pr-sdp. pr-sdp available from our blood supplier incremented monthly. over the first four months of pr-sdp availability, 777 pr-sdp were transfused at our hospital (out of a total of 3286 platelets transfused). after 4 months of scale-up, pr-sdp were approximately 30% of inventory. questions received during the nursing and medical conferences related to: the risk of bacterial contamination with c-plts vs. pr-sdp; toxicology of the pr process; scanning pr-sdp labels into the electronic medical record; and the need to irradiate pr-sdp. our use of a "safety measure" addressed concern over bacterial contamination of c-plts. published pr-sdp toxicology data comparing the content of psoralens in food products such as grapefruit ($12 mg per 100g) to the content in pr-sdp (<1 ng per ml) addressed toxicology concerns. nursing/it allayed concern over scanning issues with a simple demonstration. finally, we ensured that all parties were aware that fda did not require irradiation of pr-sdp. presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization. company personnel did not present at medical or nursing conferences per institutional policy. no background/case studies: ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations. in september 2017, the apheresis collection process (acp) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (dpd). the process review has led to several changes, including the substitution of the pre-donation platelet (plt) count measurement before donation type allocation, in favor of the use of the donor's past donation records. multiple processing steps were eliminated, and the evaluation of plt concentration as a function of time, deduced from complete blood count (cbc) measurements, allowed the centralization of the analysis at the qc department. finally, introducing the concept of non-optimal donations has led to an increase in the proportion of dpd. study design/method: at the donation centers, whole blood (wb) from 10 donors was collected in k 3 edta tubes. plt concentrations were determined at the qc department using the coulter act 5 diff hematology analyzer (beckman coulter). sample tubes were stored at 20-248c and measured at 24, 48 and 72 hours post-collection. single platelet donations (spd) or dpd were collected using the trima accel. units were pooled and split in elp (extended life platelet, terumo bct) storage bags to mimic spd (250 ml; n58) or dpd units (500 ml; n54). plt pools were stored at 20-248c under mild agitation for seven days except for dpd, which were split in two 250-ml bags after 18 6 1 h. samples were taken on days 1 and 7. ph, po 2 and pco 2 , hypotonic shock response (hsr), extent of shape change (esc), cd62p expression, atp content, lactate and glucose concentrations were used as in vitroquality markers. results/finding: plt concentration as a function of time, determined from wb cbc measurements, showed no significant difference at 24h (247 6 32 pltx10 9 /l), 48h (247 6 27 pltx10 9 /l) and 72h (247 6 32 pltx10 9 /l) postdonation. dpd can be stored in the same collection bag for 24h after donation without any significant impact on plt quality markers. plt concentrations were within the manufacturer's acceptable limits (1141-1526 pltx10 9 / l) before splitting. on day 1, lactate and pco 2 concentrations increased, and po 2 decreased in dpd. however, these values normalize to those of control units at the expiration day. conclusion: this project was approved by health canada and implemented in our organization in march 2017. there are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality. post-implantation efficiency data will be compared to the targeted 12% increase in the targeted number of plt units per donation ratio. phased implementation of pathogen-reduced platelets in a health system elizabeth s. allen* 1 , colleen vincent 2 and patricia kopko 1 . 1 university of california -san diego, 2 american red cross background/case studies: pathogen reduced platelets (prp) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex. early evidence shows only 40-45% of double platelet collections meet requirements for pathogen reduction treatment. 1 blood centers need hospitals to implement prp to start manufacturing, but hospitals may not wish to use prp until they can provide the product to all patients. scaling up manufacturing at the blood center and phasing in prp across patient populations meets both parties' needs. we evaluated this strategy at our university health system (transfusion volume: 9,500 apheresis platelets annually), which includes two hospitals (750 inpatient beds) and an outpatient cancer center. study design/method: before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained. live training was provided for nurses in the outpatient cancer center (week 0) and the bone marrow transplant (bmt) ward (week 6). an e-mail communication explained the change to all physicians and nurses. in phase 1, we implemented prp in the outpatient cancer center. these patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur. in phase 2, we expanded usage to include the inpatient bmt ward. in phase 3, we lifted all restrictions so prp could be used throughout the health system, with the goal to reach 100% prp within 6 months. results/finding: in phase 1 (weeks 1-6), we requested 31 prp products weekly, based on typical usage in the outpatient cancer center. our blood supplier provided an average of 23 prp weekly (range 9-33), and prp constituted 44% of platelet transfusions in the cancer center. in week 2, excess prp inventory required use of prp in the inpatient bmt ward ahead of schedule, a practice which continued throughout phase 1. in phase 2 (weeks 7-8), we formally expanded issuing of prp to include the inpatient bmt ward and requested 91 prp products weekly. our blood supplier provided an average of 57 prp weekly (range 44-69), and prp constituted 53% of platelet transfusions in the phased-in areas. in phase 3 (weeks 9-10), we began issuing prp throughout the health system. our supplier provided an average of 70 prp weekly (range 61-78), and prp constituted 43% of all platelet transfusions. scaling-up is ongoing. conclusion: phased implementation of prp by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing. background/case studies: maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals. our health system comprises 21 hospitals including smaller community hospitals (sch) and larger tertiary care medical centers (tcmc). for several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage. this encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of 2 apheresis platelet (ap) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost. study design/methods: a "round robin" (rr) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (rbc) standing orders. an efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals. platelets are transferred at the time of rbc standing order delivery based on a predetermined route schedule. each day, 2 ap are delivered to the sch and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver. these platelets typically have a 48 hour shelf life remaining. the same process occurs at the next sch on the route. all retrieved platelets from the sch are delivered to the tcmc which is the last stop on the route. thus, the sch has adequate number of units available for regular transfusion and massive transfusion protocol. results/findings: review of our rr process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an ap inventory for patients requiring urgent platelet transfusion. an additional benefit was further decrease in ap waste (table 1 ) resulting in a cost savings of $50k. an additional cost savings of approximately $25k was noted due to decreased cost of emergent platelet transportation. conclusion: our novel rr process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of ap waste at our health system from our previous platelet sharing process. we anticipate additional decreases in ap waste as 237a transfusion 2017 vol. 57 supplement s3 we further streamline our process. with the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste. post implementation adjustments of our pathogen reduction process jacqueline carlson* 1 , james r stubbs 1 , scott a hammel 1 and manish gandhi 2 . 1 mayo clinic, 2 mayo clinic-rochester background/case studies: the implementation of pathogen reduction for apheresis platelets using cerusv r intercept system for apheresis platelets was a substantial endeavor encompassing many different areas. as with any process change, adjustments and modifications can occur along the way. after implementing 100% pathogen reduction technology (prt) for apheresis platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products. study design/method: our prt validation consisted of 100 apheresis platelet products. each product was tested pre-processing for white blood cell (wbc) content and platelet yield, along with post processing platelet yield. this data was used to calculate our yield and volume retention during processing. we anticipated products with preprocessing yields of 3.0, 3.1, and 3.3 x10 11 may end up below a 3.0 in the final storage bag and would need a post-processing sample to ensure the product met criteria at !3.0x10 11 platelets. results/finding: during the validation, we discovered one collection was not leukoreduced and two collections started at a 3.4 yield but ended with a yield below 3.0. these two discoveries led to adjustments in our prt platelet process. with the wbc failure, we reviewed the wbc count on the sysmex xe-2100d preprocessing report to see if it would alert us to a potential wbc failure. the review discovered that 99 of 100 results were 0.00 or 0.01x10 3 / mcl with the exception being the wbc failure with a count of 0.29. further monitoring of the wbc counts discovered a result of 0.04 which was tested on the adam r-wbc for wbc count and determined to not be leukoreduced. we decided all sysmex wbc results from the pre-processing sysmex report would be reviewed prior to processing and a wbc result of 0.03 will be tested on the adam to confirm a leukoreduced product. we also discovered 2 of 4 (50%) of the 3.4 preprocessing yields products ended with a post processing yield <3.0. we decided to increase the yields requiring post processing samples to include the 3.4. conclusion: we are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of !3.0x10 6 platelets and to have enough data to accurately determine our volume and yield loss during processing background/case studies: the university of kentucky medical center (ukmc), a large academic hospital with level i trauma center, is supplied with blood products by the kentucky blood center (kbc) on a consignment agreement-based contract. ukmc is kbc's largest consumer of blood products. as platelet usage can vary widely day to day platelet usage projections are provided to kbc by the ukmc blood bank, thereby allowing kbc to act accordingly with a given day's stock (i.e. import vs export). daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs. this process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy. study design/method: daily platelet projection forms from 9/16-12/16 were retrospectively reviewed and compared to actual usage data over that same time. the prediction system used in the ukmc bb up to that time (estimated clinical need 1 6) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage. the prediction system was subsequently changed based on this data in 2017; the revised prediction method (estimated clinical need 1 11) was then evaluated retrospectively using the same data sources covering 1/17-4/17 and then compared to the prior method. results/finding: the average number of platelets transfused from 9/16-12/ 16 was 18.2 u/d with a standard deviation of 5.3 u/d; the predicted amount was 13.7 u/d. the difference between the predicted amount and the number of units used was -4.5 u/d. 79% days (23d/month) were under-predicted (average: 6 u/d). 17% of days (10) were under-predicted by !10 u (average: 12 u; max: 15 u (4x)). the average number of platelets used from 1/17-4/17 was 17.5 u/d with a standard deviation of 4.4 u/d; the predicted amount was 18.3 u/d. the difference between the predicted and units used was a 10.8 u/d. 38% days (11d/ month) were under-predicted (average: 3.5 u/d). one day (1%) over this period was under-predicted !10 u (11 u). conclusion: review of clinical platelet usage over this time identified a relatively stable average daily clinical demand. adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages (17% a 1%), reduced the number of days under-predicted (79% a 38%), and decreased the discrepancy on those under-predicted days (5.9 u a 3.5 u). these improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining kbc's supply flexibility or severely limiting ukmc clinical settings. rapid implementation of zika virus (zkv) nat blood donor screening joan dunn williams* 1 , maria noedel 1 , nancy haubert 1 , kenneth hudson 1 , larry morgan 1 , robert shaw 1 , tracy fickett 1 , jamie jue 1 , valerie winkelman 1 , sally caglioti 1 , german leparc 2,3 and phillip c williamson 1 . background/case studies: on 08/26/16, fda issued a guidance document for "reducing the risk of zkv transmission by blood and blood components". in response, a plan was implemented for mandatory zkv testing for all clients with locally acquired mosquito-borne cases of zkv within 4 weeks; nationwide in 12 weeks. this organization performs testing for clients (blood centers, hospitals) across the country. we report on 1 of 2 manufacturers' (sponsor) provided investigational new drug (ind) protocols. a single project management (pmo) system was used to control all required processes. study design/method: project focus included: clinical trial requirements, client onboarding, lab operations (labs). our objectives were to implement zkv testing for 44 clients within 4 weeks, and an additional 21 clients within 12 weeks. to minimize the impact to labs a staggered implementation was used with tracked/streamlined communications from stakeholders: vendors, institutional review board (irb), it (client and lab based), client services and labs. results/finding: clinical trial requirements increased the complexity of implementing an unlicensed test. documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting. multiple irb documents were required. to ensure accuracy in ind commitments a principle investigator was assigned to labs with client sub-investigators. deliverables were multiple including client requirements, vendor responsibilities and labs. client onboarding included confidentiality agreements between client and sponsor. an immediate zkv based webinar provided materials and understanding of sponsor protocol, lab test system, and client/donor based responsibilities. to facilitate and ensure effective communication, twice weekly conference calls were held. clients sent questions which were facilitated by labs and directed to sponsor. specific to clients were irb documents, it updates/validation for zkv test ordering and result receipt. labs were multifaceted: vendor instruments, assay materials, package inserts, staff training. assessments included: zkv sample volume, throughput, instrument capability/capacity. work requirements included vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting. all clients were provided with zkv testing within required timeframes. conclusion: the success in meeting a rapid implementation of zkv testing was largely due to a centralized pmo system which provided a controlled process for sponsor, client, vendor and labs. within lessons learned strength was found in a multi-client onboarding process. a weakness was in understanding instrument test volume capacity throughput which was exceeded during the 4-week period but overcome during the 12-week cycle. red blood cells baby units traceability and discard in kuwait central blood bank and five hospitals marwa moemen al deeb* 1 , hala samuel boules 1 , fatemah saleh al matroud 1 , rabab hussien ali dashti 1 , hanan alawadhi 2 and reem al radwan 3 . 1 kuwait central blood bank, 2 kuwait central blood bank, 3 kuwait central blood bank background/case studies: ill children are more likely to receive red blood cells (rbcs) transfusion than any other patient age group. rbcs are the component most often transfused during neonatal period. small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk. traceability is the ability to trace each individual unit from donor to recipient or disposal. blood component should be fully traceable from collection to final disposition. the kuwait central blood bank (kcbb), is preparing baby units and distributing it to all hospitals all over the country. kcbb, being accredited by the american association of blood banks (aabb), is following the aabb's regulations in tracing every component. study design/method: this is a retrospective study to assess final deposition and the percentage of discard of prepared packed rbcs baby units in the kcbb and five hospital blood banks (hbb). also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products. methods: a total of 3000 rbcs baby units were randomly chosen to be traced to their final deposition from the year 2012 till 2016. half of them (1500 units) were traced in kcbb. tables showing the numbers of the chosen units were distributed to the five governmental hbb (60 units for each year of the study period). results/finding: preliminary results show that the tracing of rbcs baby units in the kcbb is 100% efficient. results from other hospitals are under process. statistical analysis of the traceability will be done as soon as the data is collected. the study will analyze the usage of the baby units in different departments and the percentage of discarded units. the traceability of rbcs baby units in the kcbb is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing. most of the kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels. the percentage of discard of the baby units in the hospitals is very high. this may be due to the practice of using fresh blood (<5 days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure. the creation of a national policy for using rbcs baby units is highly recommended to reduce the discard of such units. we also calculated the number of false positive results. the study traced all products through mid-march 2017. results/findings: a total of 339 products were tested. fifteen units (4%) had a false positive result and could not have their life span extended. of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations. cross-reactive antibodies were identified in all 15 by the vendor and none were true positives by re-culture. of the 324 units that were successfully tested, 200 were tested again on day 6 for use on day 7(62%). there were 166 platelets transfused (51%) and 158 expired after day 7 (49%). the cost to test the products including controls was $12,970 and our calculated cost to produce 324 products would be $77,436. if we had needed to import products to meet needs, the cost would be roughly $91,300 without shipping costs which are estimated at $14,815.50. we averaged 40 expired platelet products per month (range 6-67) before verax testing and 26 (range 9-40) after implementation. conclusion: using verax point-of-care testing saved 166 platelet products from discard. the cost savings were $93,145.50 from importing and $64,466 from producing a replacement for those 324 products. the average discard rate per month went from 40 to 26 after verax implementation. extending platelet shelf life to 7 days more than paid for the cost of testing and ensured products were available for patients who needed them. secure text messaging in transfusion medicine: can texting decrease wastage? melanie estrella* and elsie lee. george washington university hospital background/case studies: secure text messaging in hospital settings allows for quick, easy, and hipaa compliant communication between members of patient care teams. it works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications. secure texting has potential to be a useful management tool in transfusion medicine in reducing blood product wastage. for example, it provides a relatively low-burden means for busy clinicians to provide feedback to the transfusion service about scenarios of potential wastage. this information can be used to identify areas in which management strategies could be developed. it also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage. the goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage. it is hoped that the results will identify secure texting as a useful management tool in transfusion medicine. study design/method: wastage records that were investigated without the assistance of secure texting from july to december 2016 were reviewed to identify the most common scenarios of preventable blood product wastage. wastage records from january to april 2017 were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician. results/finding: for 2016 data, 129 units were investigated without the use of secure texting. of these, 118 units were identified as preventable wastage, and 11 wasted units were considered beyond the control of the clinician. the categories for preventable wastage were defined as follows: 1) product not released after procedure/ or when patient stabilized (42) 2) product returned outside of appropriate temperature range (40) 3) clinician unaware product was assigned (36). thus far in 2017, wastage records have identified 31 units of preventable wastage. secure texting was used by a transfusion service physician to investigate. twelve responses provided useful feedback for future management strategies, 11 responses thanked the transfusion service for the information, and in 8 instances, the message was read with no reply. conclusion: secure text messaging has the potential to improve communication in transfusion medicine. it is easy to use, hipaa compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the transfusion service. sequence of reagent adding for cryopreservation freezing solution guoling chen*, xu zhao, andrew tiss, sasha turner, devin emerson, manijeh shemirani, sharon novak, david garvin, john eng and wanxing cui. medstar georgetown university hospital background/case studies: dimethyl sulfoxide (dmso), plasmalyte-a (plas-a), human serum albumin (hsa) are widely used to prepare cryopreservation freezing solution. some use autologous plasma instead of plas-a and hsa. this study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution. study design/methods: materials: 99.9% dmso, plas-a, 25% hsa, autologous plasma extracted. containers: transfer pack (bag) and polystyrene tubes. the freezing solution recipe used in this study is (volume ratio) 99.9%dmso: plas-a : 25%hsa51:2:2. plas-a and hsa are kept at room temperature (20-258c, rt) and refrigerated at 48c, plasma at rt (to simulate the end-of-centrifuge temperature), dmso at rt (due to high freezing point 18.58c). different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken. total 14 tests. at least 10 minutes cooling after dmso, before adding the next reagent. see table: (1) after directly adding 99.9% dmso alone to bag, the bag turned from transparent to white, so dmso should not add first. (2) in tube, autologous plasma first, dmso next, powder-like precipitates. (3) in tube, dmso first, hsa next, precipitated instantly, a layered appearance. (4) & (5) in tube, plas-a first, then hsa, dmso at last, precipitates formed; rt plas-a and hsa combination formed a thicker precipitate than those kept at 48c. (6)&(7) in tube, hsa first, dmso next: precipitation formed heavily, sculpture shape. precipitation in the 48c group is slightly milder/slower than rt group. so hsa should not be added first. (8)&(9) trace of hsa(<1ml) was mixed into the plas-a bag (500ml). in tube, such "hsa-contaminated" plas-a was added first, then dmso, small fragments of precipitates formed, so dmso should not add last. background/case studies: maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals. however, daily blood product use is difficult to anticipate. platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store. due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration. sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage. study design/method: we have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive 29-month period. using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors. these include day of week, number of abnormal cbc, location-specific hospital census data, and other less important factors. we exploited this relationship to develop a mathematical model to guide collection and ordering strategy. results/finding: this model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below 10 in our model. compared with historical expiration rates during the same period, our model reduces the expiration rate from 10.5% to 3.2%. with an annual platelet usage of approximately 13,000 units, this reduction equates to approximately 950 units saved from expiration annually. depending on platelet pricing in different regions, this accounts for annual savings between $450,000 to $650,000, per institution. conclusion: to our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand. thawed plasma implementation: signficant cost savings and decreased plasma wastage morvarid moayeri* 1 , russell thorsen 1 , rosaline ma 1 , antonio g insigne 1 , amy decourten 1 , florence panganiban 1 , patricia mckean 1 , cyril jacquot 2 , sara bakhtary 1 and ashok nambiar 1 . 1 ucsf health, 2 children's national medical center background/case studies: plasma (ffp, pf24, pf24rt24) stored at 1-6c outdates 24 hours after thawing. if collected in a functionally closed system, it may be relabeled as thawed plasma (tp), extending expiration to 5 days from the thaw date. although coagulation factor levels decrease over this period, they remain above hemostatic levels. as tp can be safely used for the vast majority of patients requiring coagulation support, we implemented use of tp in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage. study design/methods: the massive transfusion protocol at our instiution already allowed the use of group ab tp. following a review of literature and practice at other large centers, the transfusion committee extended the approval of tp to all patients. neonates (<4 months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which we also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units. conclusion: in many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited (24 hr) shelf-life. tp has an extended shelf-life, and can be used interchangeably with ffp and pf24 for most patients. implementing tp in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource. the merging of immunohematology reference lab's (irl) inventories-using technology to create advanced search functions alexander delk 1 and richard gammon* 2 . 1 oneblood, 2 oneblood, inc. background/case studies: immunohematology reference labs (irls) must maintain diverse inventory of antisera to aid in antibody identification, antigen type rbc units, and meet regulatory requirements. when our current organization was established, two irl sites had independent inventory management systems. although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (ifus) were not. our irl developed a synergistic method to organize and store antisera coupled with in-house designed custom excel spreadsheet to organize and search antisera and view ifus. study design/method: a list of similarities and differences was constructed. best practices of both methods were identified. we determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening. sequential lab assigned numbers were given to antiserum for each category: s (rare sera) and b (bulk sera). a dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth. antisera assigned to a box remained in that box, but may be moved within the box. the box itself may be moved among freezers. to track boxes, location and movement within the box, a custom excel spreadsheet was created. its location tracking feature allowed for two different storage methods to function in one spreadsheet. the spreadsheet had a tab for s and b antisera categories. abo group, desired and unwanted antibodies filters allowed quick search for appropriate antisera. the spreadsheet also had hyperlinks to scanned ifus. results/finding: sequential lab s and b numbers were assigned to new additions using a dynamic/static storage system. an excel spreadsheet with scanned ifus (hyperlinks) was used. pre-merger systems, it took on average 5-8 minutes to choose an antiserum and obtain the appropriate ifu. post-merger system was reduced to on average 2-5 minutes. (table) conclusion: the merging of two irl's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked ifus. this process saved valuable technologist time and organized the antisera more efficiently. abstract continue to flash until they are removed from shelf and their status updated in our database. 'units allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < 4 hours. the xm/hla platelets tab provides patient names and status of units allocated. a 'trxn/xmplat' tab lists pending transfusion reactions and platelet cross match reports. dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from 34% to 14%). conclusion: using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service. the dashboard is stable, customizable and requires little maintenance. initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products. over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage. use of deglycerolized red blood cells for hospital transfusion service inventory management ronnie l. hill*, jason corley and lizabeth ostiguin. us army background/case studies: regional blood shortages have been documented across the united states during the winter holiday timeframe. deglycerolized red blood cells (drbcs) have been shown to be an effective alternative though more expensive to manufacture. this study looks into the fiscal and inventory efficacy of using drbcs to meet the needs of transfusion services during times of blood shortage. study design/method: on three separate occasions, a medium sized dod donor center used its frozen blood inventory to produce type o drbcs to meet the needs of two regional transfusion services. all frozen red cells were manufactured by an offsite facility with the haemonetics acp-215 using the low glycerol (40%) freezing method and frozen at -658c within six days of collection. thawing occurred in a 328c water bath in the following order: 7 o positive and 1 o negative on 3 january 2017; 7 o positive and 1 o negative on 7 february 2017; and 8 o negative on 22 february 2017. deglycerolization occurred on site using the acp-215 with all units passing internal qc requirements. drbcs were shipped the same day to a hospital transfusion service, allowing for 13 days of shelf life prior to expiration. results/finding: during the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type o red blood cells (rbcs). o positive rbcs were only available through nbe at $240-280 and had the limitation of arrival on the next business day. collection and processing time of liquid rbcs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling. drbcs cost the dod on average $400 per unit to produce and distribute. drbcs have a shorter shelf life, 14 days versus the 211 days for other rbcs, but are washed during deglycerolization and thus produce fewer transfusion reactions. one tech can operate up to four acp-215's and deglycerolize four units at a time. in january and february 2017, it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment. conclusion: while not as readily available as traditional rbcs, drbcs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory. collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe. based on this, drbcs can be ready faster than freshly collected units of blood. there is an increased cost associated with manufacturing frbcs which is compensated for by the longer available shelf life of 10 years. having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type o red blood cells for hospital transfusion services. validation of a human anti-tetanus toxoid immunoglobulin assay performed on the abbott c8000 izekial butler* 1 , karen leighton 1 , scott jones 1 and rachel beddard 2 . 1 qualtex laboratories, 2 biobridge global background/case studies: plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools. this testing serves as a quality control test and helps estimate the antibody potency of the product. the binding site, human anti-tetanus toxoid immunoglobulin liquid reagent kit is for use on a turbidimetric analyzer. the aim was to optimize and validate the human anti-tetanus toxoid immunoglobulin liquid reagent kit for use on a photometric analyzer. study design/method: experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-tetanus toxoid immunoglobulin kit utilizing the abbott c8000 instrument. precision of the new assay parameters was determined by testing 10 replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run. the panel samples were created by spiking appropriate amount of a who tetanus antibody standard into sodium citrate plasma. accuracy was determined by testing a series of samples ranging from 1 iu/ml to 60 iu/ml of tetanus antibody. the samples for the accuracy study were created by diluting an appropriate amount of a who tetanus antibody standard with sample diluent from the reagent kit. linearity regression was determined by using the accuracy study values within the range of 2.0 to 45.0 iu/ml. stability of samples was determined by testing samples stored at 2-8 0 c and -20 0 c in triplicate at various time intervals. results/finding: the %cv for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from 1.2855 to 1.3142. so, precision was acceptable since the %cv for all samples tested was 5%. the mean values for the samples tested in the accuracy study were all 610% of the expected value which was much lower than the acceptance criteria which was 615% of the expected value. the linearity of the assay was acceptable with a r2 ! 99.0%. the linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration. the sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to 14 days at 2-8 0 c and up to 1 month at -20 0 c. conclusion: the data presented shows the successful optimization of the human anti-tetanus immunoglobulin reagent kit for use on a photometric analyzer. validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for 14 days at 2-8 0 c and stored up to one month at -20 0 c. a deep dive audit of intravenous immunoglobulin use for immune thrombocytopenia: is its use inappropriate? jiajia liu*. university of toronto background/case studies: intravenous immunoglobulin (ivig) is a generally safe and effective therapy for immune thrombocytopenia (itp) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated. due to concerns regarding adverse effects, cost and resource availability, an ivig request form was implemented in our jurisdiction in 2010 to track utilization and appropriateness. a recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of ivig was broadly inappropriate (shih et al, 2017) . as such, we aimed to conduct an extensive chart review of patients who received ivig for itp at our institution to assess appropriateness of use. study design/method: we conducted a retrospective chart review of all patients with itp who received ivig in our institution from april 1, 2014 to march 31, 2015. local research ethics board approval was obtained. results/finding: 40 patients received ivig for itp at smh over the study period for a total of 76 unique ivig infusions. the most common indications for ivig within currently accepted guidelines were: active bleeding (13, 17%), pre-operative or antepartum care (22, 29%), a platelet count of less than 10 and contraindication to corticosteroids (8, 11%). additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy. indications that fell outside of guidelines included: use of ivig as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic itp despite a platelet count between 30-50 x 109/l. 6 patients received ivig for a likely diagnosis itp while 245a transfusion being investigated for alternative explanations for thrombocytopenia. three patients were refractory to all other therapy for itp and were dependent on regular ivig infusions. 18/76 (24%) of infusions consisted of 2g/kg over 2 days; the remainder of infusions consisted of 1g/kg. of those who received 2g/kg,3 of patients (17%) had evidence of partial remission after a first 1g/kg dose. ivig was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines. conclusion: we found, at our institution, that use of ivig for itp was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations. we believe that ivig remains an important treatment for itp particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing. detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate ivig use in multiple settings. we believe that there is an opportunity to promote administration of a single 1g/kg dose to minimize unnecessary utilization of ivig amongst hematologists who manage itp. a process for improving crossmatch bench ergonomics janet dornfeld*, sheng-chung cheng, ann eggebrecht, beth greer, savannahsue rondeau, brian rognholt and beth taylor. mayo clinic background/case studies: a mission of our institution is to reduce the risk of work-related injuries. accordingly, each year an ergonomic survey is undertaken as a component of a general department of laboratory medicine and pathology safety audit. our 2016 survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches. study design/methods: a seven item ergonomics survey of the working environment was sent to 32 staff members in early february of 2017. twenty-two technologists responded for a 69% response rate. the below table below reports the survey items and responses. results/findings: the most problematic area was the available workspace. of the respondents, 81% indicated that workspace size was insufficient and 71% that the chairs at the fixed height benches were problematic. problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height. our laboratory lean team operational support group was tasked to aid with the bench redesign and to choose products for improving the workspace. our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space. the configuration of the new workspace was guided by the survey findings. adjustable height workstations were recommended to replace our fixed height bench. we worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work. the benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface. the opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing. conclusion: the survey was effective in identifying working areas for improvement. employee comments have been positive for the new workstations. an effectiveness assessment will follow, using the original survey, to assess the success of the project. a retrospective study of emergency department initiated type and screen testing: were patients transfused after testing? sandra lamm* 1 , neil bangs 1 and kimberly sanford 2 . 1 vcu health system, 2 virginia commonwealth university background/case studies: type and screen (t&s) testing is often ordered on patients presenting in the emergency department (ed). if the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation. if the patient does not need a transfusion of red blood cells (rbcs), the testing and second phlebotomy is an inefficient use of resources and time. study design/method: as part of a performance improvement initiative in transfusion medicine, we performed a retrospective study of all t&s orders that were initiated in the ed from 1/1/2015 to 6/30/2015 to determine if testing was subsequently followed by transfusion of blood products. patients were stratified by ed department, time from t&s draw (tsd) to transfusion (<4 hours, > 4 hours < 24 hours), and if a second sample was required. results/finding: a total of 3144 t&s orders were initiated from the ed in this time period. 2787 (88.7%) patients were not subsequently transfused any type of blood product within 4 hours of tsd and 2584 (82.2%) patients were not subsequently transfused any type of blood product within 24 hours of tsd. a total of 2119 (67.3%) patients required a second sample. of these patients requiring a second sample, 1960 (92.5%) were not subsequently transfused any type of blood product within 4 hours of tsd and 1886 (89%) were not subsequently transfused any type of blood product within 24 hours of tsd. conclusion: routine ordering of t&s testing is not an efficient use of resources and time as many patients are not subsequently transfused. ultimately unnecessary t&s and second sample collection and testing for those patients not subsequently transfused within 24 hours of tsd amounted to an estimated $699,706 in unnecessary patient charges and approximately 628.7 nursing hours for phlebotomies in a six month period. anti-d from alloimmunization versus rh immune globulin: detective work in the blood bank and transfusion medicine services (bbtms) margaret diguardo* 1 , debra berry 1 , yunchuan delores mo 2 and gay wehrli 1 . 1 university of virginia health system, 2 children's national medical center background/case studies: the institute for healthcare improvement triple aim incorporates enhancing patient satisfaction by providing high quality, safe care. towards these goals the bbtms is charged with communicating to obstetric physicians (obs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk. thus, when anti-d is detected in a female of childbearing age, it is critical to determine whether this represents rh immune globulin (rhig) or alloimmunization (alloanti-d). review of a patient's electronic health record (ehr) helps quickly identify rhig administration, but if this documentation is missing, then it is easy to assume presence of alloanti-d. rhd alloimmunization impacts mom, fetus, newborn and future pregnancies. therefore, without a national, comprehensive health information exchange (hie) system, it is imperative to investigate beyond the on-site ehr whether a patient received rhig at an outside hospital (oh). we report an irb approved (exempt) case series where detective work revealed rhig administration at ohs. study design/method: over a two month time period, anti-d was identified in four pregnant women. review of their ehrs did not reveal a history of abstract rhig administration; nor did subsequent direct communication with their obstetricians (ob) reveal a history of rhig. based on each patient's home address, the bbtms of any nearby ohs were contacted as was a primary care physician if listed in the ehr. results/finding: investigations beyond the ehr and obs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites. through phone calls to the bbtms of ohs, a history of one or more rhig administrations within the preceding three months was found for each patient. our bbtms records and ehr were amended to reflect the presence of a passive anti-d due to rhig, rather than alloanti-d. the changes were also directly communicated with the ob caring for each patient. conclusion: when a new anti-d is identified in a pregnant female, investigation is required to determine whether it is passive rhig versus alloanti-d. when neither the ehr patient history or ob reveal a rhig history, it remains in the patient's best interest to investigate further. through phone calls to oh we revealed a history of rhig administration in four patients. finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent rhig administrations when indicated, at our institution. future strategies for avoiding similar situations include expanding our national hie for critical information such as bbtm history and allergy history and expanding use of wallet-size patient identification cards with rhig and alloantibody histories. auditing massive transfusion protocol colleen a. aronson* 1 , elizabeth halperin 2 , sharon breining 2 and mona papari 3 . 1 acl laboratories/ advocate hospitals, 2 advocate health care, 3 itxm background/case studies: a large midwest hospital system with 5 level i trauma sites evaluated how to audit the massive transfusion protocol (mtp). the possibility of real time audits is impractical due to the unpredictability of these events. a search of the internet found an example from new zealand for post process evaluation. this was shared with a team as a starting point and then adjusted for system specific priorities. to start the audit, the initiation of the mtp needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders. study design/method: the transfusion service (ts) was determined to be the source of truth for all of the mtp events. a tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage. this was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event. the focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (surgery (or), emergency (ed), labor and delivery (l&d), etc.), type of event, use of tranexamic acid (txa), calcium chloride (cacl), temperature monitoring and pre/ post lab results. a trial was started and 3 months of data were evaluated that contained 29 events. results/finding: there was an equal number of events that were initiated in the ed and the or (12). male patients were involved 69% of the time and 31% of time the patients expired. trauma of some type was the majority of the cause but 13.8% of the cases involved gi bleed and only 6.9% were obstetric cases (see chart). the lowest hemoglobin (hgb) was found to average 7.1 with the post hgb average of 9.7. ratios of 1:1 for red blood cells (rbc) to plasma as well as rbc to platelets (plt) and cryoprecipitate (cryo) were also determined with a target of 4:1. it was found that the rbc: plasma was 1.9:1, rbc: plt was 5.9:1 and rbc to cryo was 7.4:1. use of txa was only 24.1% and cacl was utilized in 58.6% of cases. conclusion: although this data is for a short period of time it has pointed out several opportunities for improvement. the use of mtp in gi cases was not previously understood but opens up a new group of people for which education and understanding of the mtp process is needed. the low use of txa needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the mtp process. the product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement. the process will now be expanded to the level ii trauma sites in the system and routine evaluation will be shared with all sites. automated report significantly reduces turnaround time for rbc antibody alert jessica l dillon* 1 , jody a barna 1 , donald e ulinski 1 and nancy m. dunbar 2 . 1 dartmouth hitchcock medical center, 2 dartmouth-hitchcock medical center background/case studies: clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion. at our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (emr). an interpretative comment is also entered by the transfusion medicine service (tms) physician after the antibody work-up has been reviewed (this may be up to 2 weeks after the antibody is identified). this comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units. since clinicians may not always review these results, the tms physician also simultaneously adds an "allergy to red blood cells" alert in the patient emr at the time the interpretive comment is entered. study design/methods: in july 2016, we implemented an automated report to reduce the turnaround time (tat) for entry of the allergy alert. the report contains all detected red cell antibodies in the prior 24 hours and is provided to the tms physician during daily morning rounds (monday through friday) for manual entry of allergy alerts. this study describes a three month comparison both before and after the automated report intervention, to evaluate the tat for allergy alert entry into the emr. age ( abstract results/findings: between august 2015 and november 2015 (pre-implementation) , newly identified clinically significant antibodies were resulted for 56 patients compared to 51 patients between the months of august 2016 and november 2016 (post-implementation). the tat for allergy alert entry for both periods is shown in table 1 . we observed that 57% of allergy comments were performed within 24 hours in the post-implementation period versus only 30% pre-intervention (p50.0067). using the new process, nearly all of the alerts were entered into the emr within 72 hours of antibody resulting and none of the entries were missed. conclusion: there was a significant improvement in the tat for allergy comment entry following implementation of an automated report. this project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification. blood bank verbal tool implementation for cardiovascular surgery rita louie* 1 , shailesh macwan 1 , nancy nikolis 1 , arline stein 1 , janelle richardson 1 , manju bagu 1 , lennart logdberg 1 , alexander indrikovs 2 , vishesh chhibber 1 and sherry shariatmadar 1 . 1 north shore university hospital, 2 northwell health background/case studies: our institution is a tertiary care facility performing over 1500 cardiovascular surgeries (cvs) in 2016, an increase of 117% after the healthcare system cvs integration in 2015. transfusion support of these patients includes preoperative preparation of prbcs according to a maximum surgical blood order schedule. additional blood components are issued as orders are placed. until december 2016, the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication. after 2 reported events in q3 2016 that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements. in collaboration with cvs, the blood bank implemented a new workflow process to enhance communication with the cvs team, reduce turnaround time and improve patient safety. study design/method: 1. open discussions and collaboration between blood bank and cvs nursing teams 2. mapping the process using flowcharts for additional blood orders from cvs. 3. identify bottlenecks and brainstorm solutions. 4. a verbal cvs order process and form was implemented to improve communication between cvs and blood bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders. a read back was also documented for verification of the order. 5. the blood bank staff immediately processes this order while waiting for the written order to arrive. upon receipt of the written order the blood is issued to the or. 6. follow plan-do-check-act. the transfusion safety officer reviews each order for the following parameters: number/type of products, turn around times (tat), wastage/returned products and overall efficacy since implementation of this process. results/finding: a significant improvement was noted in communication and tat after implementation of the process described above. for the period 12/23/16-4/7/17 the blood bank has received 327 verbal orders with varying product combinations. the table below represents average turn-around times to issue blood products: conclusion: the introduction of the verbal order tool for cvs has streamlined the blood ordering process leading to increased efficiency and lower tat. effective communication between the or team and transfusion service is the key to timely provision of blood products for these critical patients. challenge of blood type testing for multiply transfused sickle cell disease patients jayanna slayten* 1 , tracie ingle 1 and heather vaught 2 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: we report our midwestern, university transfusion service challenge of obtaining the correct blood types in rbc exchanged sickle cell disease (scd) patients tested by our primary testing method, solid-phase red cell adherence analyzer echo (immucor. norcross, ga). the echo operation manual in chapter 12-6 and appendix d it states: "warning: the galileo echo cannot reliably detect hemagglutination reactions that are graded as 11 or less in tube methodology. the galileo echo does not generate as interpretation of mixed-field. such a mixed-field reaction will be interpreted as positive, negative, or equivocal." we report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused scd patients. study design/method: two scd when initially tested by the echo as o, d negative; however, each patient was historically o, d positive. both patients had received a rbc exchange transfusion with 8-11 o, d negative red blood cells over 30 days previously. repeat testing of the samples was completed by the vision (ortho clinical diagnostics. raritan, nj), neo (immucor. norcross, ga), and by standard abo/rh manual testing (anti-a, anti-b, anti-d series 4, anti-d series 5, a1 cell and b cells. immucor. norcross, ga). the repeat testing was compared to verify the patient's abo/rh typing and the results were entered into the computer system to allow for assigning the patient's abo/rh typing and electronic crossmatch. results/finding: table 1 summarizes the initial and repeat testing with the two patient samples. although the echo failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of o, d negative blood with the detection of mixed-field in the d typing or by failing to interpret the abo/rh as not type determined (ntd). the vision and manual abo/rh typing yielded the easiest mixed-field to interpret macroscopically. conclusion: our results agree with the findings of summers et al (trans-fusion 2009; 49:1672 -1677 who reported the challenge detection of mixed-field with the use of the echo compared to improved detected with automated gel column agglutination. when the samples were tested by multiple automated and manual abo/rh methods, the expected mixed-field was detected. the failure of the echo to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the abo/rh when there is not a historical abo/rh to compare. to avoid this risk, it may be appropriate to re-type first time scd patients by other methods rather than the echo to avoid this challenge. consistent with summers do not account for regional distribution. many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on odr and bsr, but we hypothesized that these are crude key performance indicators (kpis) requiring redevelopment. study design/method: kpi redevelopment occurred in a large tertiary care hospital blood bank in canada, responsible for 75% and 20% of transfusions in the region and province respectively. rbc supply, inventory, and disposition data were retrospectively assessed from february 2014-june 2015 as the baseline period. a "demand-driven inventory planning policy" (ddip) was instituted to assess and implement the optimal rbc reorder quantity based on the difference between the historical maximum and minimum rbc inventories during weekdays; that would not lead to blood shortages. shelflife inventory (sli) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (abt) and received (abr). iterative simulation modeling (r statistical software) was then performed to optimize sli in a post-implementation period from june 2015-october 2016. results/finding: modeling predicted observed rbc disposition. through simulation, optimization of sli was found to occur by optimizing a set of kpis for each abo blood group (table 1) . this led to a reduction in observed overall sli (7.2 6 1.8 days vs 6.0 6 1.5 days, p<0.01) and odr (0.9% vs 0.5%). the bsr was not significantly increased during the postimplementation period. conclusion: optimization using simulation modeling of multiple factors other than bsr and odr led to further efficiency gains in a large tertiary care hospital blood bank. hospital blood banks should use an integrative approach with a set of kpis to optimize the supply chain. this approach requires validation in other blood banks and jurisdictions. (6)) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing. the hospital must maintain records of this lookback notification as part of the patient's medical record. paper records of lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost. to facilitate the lookback process and reduce paper documentation we sought to use the electronic medical record (emr) to perform and document notifications. study design/method: representatives from transfusion medicine (tm) and information technology (it) worked together to define minimum and optimal emr solutions. minimally, a completed paper packet could be scanned into the emr. this solution had no advantages in terms of ease of use, process control, or transparency. desired optimal functionality includes the ability to send letters in the emr, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received. the emr system at our institution, epic (epic systems corp., verona, wi), has a function called "letters" with the capacity to do all these tasks. a series of five templates were developed: hiv and hcv letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters. templates are opened within the patient's emr and demographic information is automatically populated by epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g. rbcs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender. the completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic in basket as well as in the patient's emr. physicians may electronically complete and return the response form within epic, or print it and return the form by fax. results/finding: between january 2014 and december 2016 thirty-five (35) notifications were sent to physicians using epic letters and of those, fourteen (14) responded to the epic notification and five (5) used the provided electronic response form. for these cases the time to mail or handdeliver paper notifications was avoided. the remaining 21 cases required follow-up paper notification, but the electronic letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician. conclusion: lookback notifications within the emr makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors. secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters. evaluation of ordering practice in the operating rooms and its impact on product wastage alexandra budhai* 1 , denden benabdessadek 2 , annu george 2 and alexandra jimenez 2 . 1 westchester medical center, 2 new york blood center background/case studies: blood product wastage is an issue that many hospitals aim to address. the or was identified to have the highest rate of wastage within our hospital. in this study, we assessed the appropriateness of the product order and utilization by the or to understand its impact on wastage. study design/methods: data on product orders, issue, and return for two months were analyzed. the hospital cpoe and product requisition forms were used to collect this data. the surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (msbos). trends for inappropriate orders for products by physicians were evaluated. results/findings: a total of 941 orders were reviewed. approximately, 30% of these products were issued to the or. we found that the physician orders were within the guidelines of the msbos for most cases (89%), but of the issued products, all were returned to the blood bank in 40% of cases. we observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal. in addition, all of the products ordered for c-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases. conclusion: the data analyzed demonstrates that the majority of surgeons are adhering to our institutional msbos guidelines. it was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible. our analysis revealed that the hospital's msbos does allow for an excess in blood ordering for some surgical procedures. the msbos should be updated to reduce the suggested maximum product order. in general, the data does not imply that the blood product wastage in the or is due to the ordering practices of the surgeons. a larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties. background/case studies: the visionv r and visionv r max (ortho diagnostics, raritan new jersey) are id-mts tm gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> 50 type and screens (t&s) per day] blood banks 1 , respectively. our laboratory which serves a large 1278-bed multispecialty academic hospital and receives 275-300 t&s specimens per day needed to replace three provue analyzers prior to the availability of the visionv r max. we implemented three visionv r analyzers to work with our existing neov r and echov r (immucor inc, norcross georgia). a recent multicenter field application trial of the visionv r reported a mean turnaround time (tat) for t&s and abo, rh typing (abo/rh) of 32.2 6 4.5 and 27.5 6 5.6 minutes 2 , respectively. the objective of this study was to determine visionv r tats under routine daily high-volume practice. study design/methods: one visionv r was in operation during a five-week period (phase i), and then two additional analyzers were brought into service (phase ii). tats are defined as the time when the order is received by the instrument to when the test is completed and available for review. three-cell screen and abo/rh tats, and number of visionv r antibody panels were collected for a nine-week period. the tat for the screen was used as the tat for the t&s because the screen is the rate determining step. all testing was performed using in-service analyzers on routine patient samples by trained technologists. samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time. results/findings: under the high volume conditions of our laboratory with three visionv r analyzers, the mean t&s tat was 30% longer and had a larger standard deviation (s.d.) than the published trial result of 32.2 6 4.5. transfusion 2017 vol. 57 supplement s3 abstract during phase i visionv r 1 performed 263 panels. during phase ii visionv r 1 performed 351 of the 361 visionv r panels. conclusion: our visionv r analyzers are used under high volume conditions more suitable for the visionv r max. when balanced with the testing menu, including ability to perform select cell panels, our tats using three analyzers were satisfactory. the large standard deviation indicates that opportunities remain for improving tats through workflow improvement. from west nile virus to the emergence of zika virus: a nationwide survey of how regulators are keeping the blood supply safe and available falisha atwell* 1 , john roback 2 , ronald arkin 1 , michael bartlett 1 , robert geiger 1 and jaxk reeves 1 . 1 university of georgia, 2 emory hospital background/case studies: with the emergence of zikv in the united states, it is important to assess the fda's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases. this research compares the responsiveness of the fda during west nile virus (wnv) and zika virus (zikv) outbreaks to evaluate our current preparedness. study design/methods: the literature review was conducted to analyze fda's response time during the wnv crisis and determine if it was effective and efficient. the research survey was performed to determine if the donor history questionnaire (dhq) adequately screens donors for zikv as the sole preventive method (as per the february 2016 guidance for industry: recommendations for donor screening, deferral and product management to reduce the risk of transfusion-transmission of zika virus) and to determine if the current regulatory practices (including the august 2016 guidance for industry: revised recommendations for reducing the risk of zika virus transmission by blood and blood components) are perceived to be effective and efficient in the face of the current zikv outbreak. survey monkey was used and participation was anonymous. over 4,000 emails and web-links were sent to members of aabb, scabb, seabb, and personal network with a 10% target response rate. participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter. results/findings: the literature review revealed that the fda's response was slow during the wnv outbreak, while the zikv response is efficient thus far. a total of 317 participants responded to the survey (7.94% response rate). statistically, participant agreement with fda's decisions was performed by "t" test (with n-15317-15316 df) of the null hypothesis that the mean50 vs. the alternative that the true mean is> 0. overall participants had favorable opinions of the fda's decisions. statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way anova models were used with likert-scale question responses as if they were continuous. the f-statistic and p-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable. there were no significant differences in the years of experience and profession variables for participants. region was determined to be unreliable due to undefined states for each region listed. conclusion: the research revealed that industry experts conclude that the current system of dhq and fda guidance documents, if issued timely, are adequate. background/case studies: when evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab. there are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time. study design/methods: the study involved a current state to a future state comparison of testing processes with an instrument ortho provuev r (pv) and manual testing vs. an instrument ortho visionv r (ov). data collection methods included direct observation, time studies, and interviews. the pv bench performs type & screens (ts) on the pv and manual abid/selected cell panels in the gel test. all other testing; cord blood(cb), dat, unit confirm(uc), patient type confirm(pc) and crossmatch(xm), etc. are done manually in tube. the future state incorporated the ov. ts, abid and uc were evaluated in both states. cycle time(ct) was averaged based on 3 run cycles. ct was comprised of 3 metrics; instrument time(it), standby time(st) and labor time(lt). st may be comprised of 2 components, time that could be utilized as "walkaway" time or vigilant time (vt) which requires operator presence but not operator action. for automated instruments, vt for each cycle was measured as instrument access unavailable. instrument daily maintenance (dm) ct was evaluated as well. similarly, timing of manual tube test processes used these metrics. for repetitive activities within a process, such as uc or xm, a time per individual process was captured and then multiplied per unit. results/findings: table 1 provides details about the metrics of current state and future state processes. tube based test timing is as follows: pc (2:50), xm (32:23), cb (13:18) and dat (10:00). by implementing the future state, an average $1.3 min. lt and vt is saved on each sample loaded for ts equating to a 73% labor reduction over the current state. a 19% improvement in tat on the ts was achieved in the future state. moving from manual abid to automated processing resulted in a 59% lt reduction. on average, a 38 min. continuous walk-away time is achieved for each automated abid. uc had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics. conclusion: based on the metrics evaluated and compared between current state and future state, the ov has demonstrated improvement in lab operations to both the labor required and result tat delivery. opportunity exists to automate workflows on other tests that are still manually performed. background/case studies: high throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $100 type and screen samples per day. the erytrav r (grifols) is a fully-automated walkaway analyzer utilizing 8-column gel cards for pretransfusion testing. the blood bank validated and implemented the use of erytrav r for abo/d typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform. the blood bank also validated automation of donor unit retypes. the instrument has bidirectional interface to the blood bank lab information system (lis), hcll tm (hemocare life line, mediware). instrument validation and implementation were done in conjunction with the software version upgrade of hcll tm and an interface system change to maestro tm . study design/method: correlation testing of the erytrav r results with the manual tube testing (peg iat; reference method) was performed on 100 patient samples for abo/d typing and antibody screening; of which at least 10 had a positive antibody screen. out of the 10, 5 had known antibody specificities. forty-two rbc units were also tested for abo/d confirmation; of which 17 were d(-) and 25 were d(1). calculations of concordance, sensitivity, and specificity were performed. precision studies were also done. interface testing of erytrav r , hcll and the hospital's information system using the maestro tm interface system was performed and validated. results/finding: concordant results between both methods were obtained in all of the 100 patient and 42 donor samples tested (100% concordance). all 10 samples with positive antibody screens were obtained by both methods. all clinically significant antibodies were detected by both systems. erytrav r gave 100% sensitivity and specificity. the precision studies showed that both methods gave the same type and screen results for 5 samples at 3 different testing events. after validation of the lis upgrade and interface system change, a bidirectional interface with hcll tm was established. the instrument has been operational in our lab for over 3 months. conclusion: erytrav r was found to be reliable and accurate and can handle the high workload of our lab. users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable. the validation of the the instrument is straightforward. the major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring lis upgrade and migration of the data integration system. a post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential. implementation of a system-wide platelet inventory report optimizes platelet utilization and reduces unit wastage elly landolfi* 1 , craig fletcher 2 and peter millward 1 . 1 beaumont hospital, 2 beaumont health system background/case studies: a sufficient number of blood components should be available to meet routine and emergent hospital needs. this must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short 5-day shelf-life. we report the results of a quality improvement project implementing a custom computerized platelet inventory report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates. the report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location. all system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier. study design/method: the study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data. the report went live october 2014 and quality data was reviewed from august 2013 to december 2016. the collected data was then analyzed using descriptive statistical methods. results/finding: data from 2016 indicates platelet wastage comprised 1% of total received platelets and 79% of these wasted platelet units were due to expiration. other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility. the mean of monthly wasted platelet units 12 months preimplementation of the report was 13 units, compared to 11 units 12 months post-implementation and 5 units 24 months post-implementation. wastage rates improved from 6% (wasted yearly platelets/total received yearly platelet units) in 2014, the year of report implementation, to post-implementation rates of 3% in 2015 and 1% in 2016 (see table) . importantly, this occurred despite a greater than 30% increase in platelet inventory between 2014 and 2016 and resulted in cost savings of over $60,000 in this period. conclusion: study limitations included restricting data collection to one campus. the option to transfer expiring platelet units to another blood bank was available to all 4 participating sister hospitals. it would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices. aside from lowering platelet wastage within 2 years of implementation, additional benefits to the report included facilitating ordering from the blood supplier. cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units. we have shown achievement of this end is facilitated by a customized daily platelet inventory report -an efficacious and easily adaptable tool with demonstrable gains. valerie halling* 1 , lisa marie button 2 , lori scanlan-hanson 2 , karen koch 2 , janet finley 2 , deepi goyal 2 and camille van buskirk 3 . 1 mayo clinic-rochester, 2 mayo clinic, 3 mayo clinic rochester background/case studies: transfusions in the emergency department of a level i trauma center were ordered using a handwritten order form. the transfusion lab's (tl) management team and medical director met with emergency department (ed) leadership and it resources in 2014 to define the needs of a successful electronic blood transfusion system. the handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected. the potential error sources included clerical errors involving the patient's name or medical record number (mrn), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (stat or routine), or not including the prescriber call-back information. the number of ed reported transfusion related events in 2013 and 2014 were 63/1187 (events/ed transfusions 2013-2014). study design/method: electronic ordering for the ed was implemented march 31 st 2015. any transfusion orders generated from the ed are now electronic, unless in the case of electronic downtime. the system electronically fills in the patient's name and mrn, controls for the type of blood product being ordered, requires an order priority and provides service contact information. it was designed to accommodate transfusion ordering needs for adults, pediatric patients <35kg and pediatric patients >35kg. 252a transfusion orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate. the electronic system was designed so that an order cannot be submitted unless all critical fields are completed. results/finding: the electronic ordering system has been in place for 2 years (april 2015 -march 2017), and during that time there was 1 instance of blood being ordered for an unintended patient 0.09% (1/1081). this was because a previous patient's medical record was accessed rather than the intended patient's medical record. there have been no instances of clerical errors (name misspelled or mrn transposition etc.), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order. electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance. prior to electronic order implementation, in 2013, 17/651 (2.61%) units were transfused in the ed but not charted in the patient's medical record. in 2014, 18/536 (3.36%) transfusions were not charted. however, in 2016, the first full year of electronic transfusion order capability, only 4/462 (0.87%) transfusions were not charted in the patient's medical record. conclusion: electronic ordering in the ed has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians. it allowed the order to be processed more quickly by tl, resulting in a faster turnaround time. improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against. implementation of blood bank automated attendant lok tse*, gerald motta and maria aguad. brigham and women's hospital background/case studies: the blood bank receives numerous nonemergent phone calls on a daily basis. these calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred. the hospital is categorized as a level 1 trauma center, with over 700 inpatient beds and over 50 operating rooms. a proposal to implement a blood bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (mtp) and emergency release of blood products. study design/method: the first step was to categorize the types of phone calls received by the blood bank by creating a phone log. data were collected and analyzed for four weeks. the blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls. it was very important to maintain patient safety and quality of service at the same time. the automated attendant consist of: option 1 (urgent) for trauma, emergency release, mtp and obstetric hemorrhage emergency release; option 2 (verbal) for verbal orders and coolers set up; and option 3 (staff) to speak with staff member. instructions were also given for specimen inquiry and product availability in the hospital information system. results/finding: the data in table 1 showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries). most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability. there was an overall decrease in phone calls by 68% with the implementation of an automated attendant. conclusion: with the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders. the decrease in phone calls freed up team members to perform other critical tasks in the department. improved detection of wrong blood in tube errors: implementation of a two-sample blood type verification process ariana king* 1 , steven zibrat 1 , geoffrey wool 2 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: our organization used a blood bank identification (bbid) band system for pre-transfusion testing and detection of wrong blood in tube errors (wbit). additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available. the bbid bands were prone to clerical errors and excessive specimen rejections, and believed to miss some wbit errors. in 2015, blood bank accounted for 48% of all rejected clinical laboratory samples, yet comprised only 5% of total laboratory volume; 88% of rejected blood bank samples were due to bbid band issues. the wbit error rate detected by bbid-based system was 0.006%. study design/method: a multidisciplinary workgroup was formed to review data and best practices. the decision was made to discontinue bbid bands and implement a two-sample verification process, in keeping with standards. a new laboratory test order was created in the emr system and embedded into the existing t&s order. providers are prompted to order the abo verification test only when no previous abo/rh typing results are found. education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically. the new process went live in september 2016. results/finding: in the five months following implementation, four wbit errors were detected with the second sample. these may have been missed using the bbid band system. improved detection revealed a wbit error rate of 0.128%, three times the national average of 0.043%. under the new system, rejected blood bank samples decreased from an average of 50% to 28% of all rejected laboratory samples, a 43% decrease. implementation of the new process produced a net savings of $55.8k. conclusion: replacing the bbid band system with two-sample verification successfully improved our ability to detect wbit errors among patients who lacked historical blood bank results. additionally, discontinuation of the bbid system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization. next steps are for blood bank and laboratory quality leaders to partner with nursing leadership to drive down wbit error incidence. 253a addendum with the final culture results. we used a student's t test to determine whether there was a statistically significant difference in the mean tat for result addendum entry in the post-implementation period compared to the pre-implementation period. results/findings: in the pre-implementation period, we cultured 19 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-78 days (mean 19 days, sd 20). in the postimplementation period, we cultured 22 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-12 days (mean 7 days, sd 2; p50.0082). there were no positive cultures during either study period. conclusion: our study demonstrates that tat for documentation can improve with the use of information technology to notify the transfusion medicine physician when results are available for documentation in a patient's emr. improved turnaround time of type and screen samples michaelene hultman* 1 , marcus holme 1 , johnathan bakst 1 , gunta musa 1 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: the primary test performed in the blood bank with regard to pre-transfusion testing is the type and screen (tys). the current target for this institution's blood bank is an 80 minute turnaround time (tat). in april of 2016, the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method. the average number of outliers increased 104%. tat analysis of a representative one week sampling per month showed an increase in outliers from 28 per month to 57 per month. average monthly tys samples performed is 2758. these numbers did not improve even upon returning to the original facilities. study design/method: two ortho clinical diagnostics visionv r analyzers (raritan, n.j.) were purchased for the blood bank. the instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests. batch testing was eliminated allowing samples to be run as received. the results were auto interpreted, and transmitted to the laboratory information system (lis) based on predetermined rules. only results in need of manual review or interpretation were held back. final verification of results was performed by the technologist within the lis. reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run. key quality indicators including tat continued to be monitored throughout implementation. data was monitored for significant changes and improvements in patient care. the go-live date was 12/20/ 2016. results/finding: the average number of outliers decreased 61% from 57 per month to 22. further benefits include a reduction in the number of technologists needed to perform tys testing. additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $134,000 for fiscal year 2017. conclusion: the use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in tat over manual gel method. improvements in the timely reporting of final product culture results in the patient's emr. barbara a. hewitt*. dartmouth hitchcock medical center background/case studies: in certain transfusion reactions it is required that a culture of the returned blood product be performed. these cultures are reported in our cerner operating system but those results do not cross over to the patient's emr . the finalized product culture results are entered into the patient's emr as an addendum to the transfusion reaction clinical note. a review of the transfusion reaction database revealed that there were occasions when the final product culture results were not entered into the patient's emr in a timely manner. it is important to the patient's care for the transfusion medicine service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health. this information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility. study design/methods: a review of a specific 7 month period revealed that the timeframe in which the finalized product culture results were entered into the patient's emr ranged from 0-65 days with a mean of 13.64 days. it was determined that this was not in the interest of improving patient care. in collaboration with laboratory information services a report was created in which once product culture results were finalized an email would be generated notifying the medical director and the transfusion safety officer that results were available. results/findings: data was collected for 7 months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's emr improved to a range of 0-7 days with a mean of 2 days. conclusion: improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions. this process can be made easier when the correct tools are used. omer ilyas* 1 and randy levine 2 . 1 northwell health, 2 lenox hill hospital background/case studies: transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening graft versus host disease (gvhd). after noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank. study design/method: the project was separated into three parts. in the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit. the variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank. all patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood. the second part of this project was an educational intervention. residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor. residents were also instructed to order irradiated blood for all patients on the oncology unit. in the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful. results/finding: pre-intervention, 67 units were transfused on the oncology floor with 38 units (57%) requiring irradiation and only 22 of those 38 units (57%) ordered as irradiated. since the blood bank occasionally issues irradiated blood without a specific order, 9 additional irradiated units were issued (31/67; 46%). post-intervention, 29 units were transfused on the oncology floor with 15 units (52%) requiring irradiation and all 15 of those units (100%) ordered irradiated specifically to prevent gvhd. eight additional irradiated units were ordered with no requirement for irradiation; thus 23 of the 29 (79%) total units were ordered as irradiated. again, 4 additional irradiated units were issued (27/29;93%) without a specific order by the blood bank. the results are summarized in the accompanying table. conclusion: this quality improvement project demonstrates that educational intervention can succeed in changing clinical practices. continued monitoring of ordering practices will ensure that compliance continues. we plan to expand the quality improvement project to other settings, including the emergency department and surgical floors. we expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events. samantha ngamsuntikul* 1 , charlotte van dyke 2 , dina garza van hoose 2 and rachel beddard 1 . 1 biobridge global, 2 south texas blood and tissue center background/case studies: at our blood center, apheresis platelets and red cells are collected on trima accels and double red cells on haemonetics 8150s. in addition to routine quality control (qc), qc is performed for instrument flags on collection instruments. quality control for apheresis platelets includes: volume variance and rwbc; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass. study design/methods: during the period of january 1, 2016 to april 19, 2017, 2,097 total collections were flagged for additional qc by our trima accels and haemonetics 8150 instruments. quality control at our center is tracked by our quality control software management system, hematerra's hemacomply which allows the ability to track and retrieve this information. the majority of products flagged for instrument qc pass and are released for distribution. a small percentage, however do fail qc leading to loss of product. quality control data can be retrieved and monitored for trends using a quality control software management system. background/case studies: in 2015, the centers for medicare & medicaid services (cms) rolled out a plan for implementing iqcp (individualized quality control plan) as a new quality control option based on a risk management plan for clia laboratories performing non-waived testing. this plan was meant for clia approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples. study design/method: clia clinical laboratories can either follow traditional clinical clia qc requirements according to the regulations or implement iqcp. while we perfrom traditional qc assessments on all the tests we perfrom on our cellular products we did decide to implement the iqcp program within in our quality control laboratory. we followed the iqcp process for assessing some of our qc tests used to assess the safety, purity and potency of our cell based products. one test in particular where we applied this tool was in the review of our qc sterility testing method and found it to be a very useful in improving the overall process. the tool walks you through three process requirements: 1) risk assessment, 2) quality control plan and 3) quality assessment for the preanalytical, analytical and post analytical phases of testing. abstract conclusion: the integration of the iqcp into the quality control laboratory was determined to be a success. the iqcp tool was successful in identifying gaps within the sterility testing process. this tool will be used on additional quality control tests and manufacturing processes. the implementation of the iqcp program ensure regulatory qc requirements appropriate for testing performed. we were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error. objective performance of massive transfusion protocols at a single institution gustaaf de ridder* 1 , rachel jug 1 , kimberly ingersoll 1 , nicholas bandarenko 2 , nicole guinn 3 and jessica poisson 2 . 1 duke health pathology, 2 duke university hospital, 3 duke health anesthesiology background/case studies: hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients. using a balanced 6:6:1 transfusion ratio (tr) for massive resuscitation is recommended based on trauma data. objective performance during massive transfusion protocol (mtp) activations is poorly studied and there may be differences based on site or medical service of mtp initiation. with the impending release of a unified, redesigned exsanguination protocol (exp) at our institution, we established baseline performance characteristics for our existing mtp and obstetric massive transfusion protocol (obp). study design/method: following institutional review board approval, we performed a retrospective study on blood product utilization and outcomes of mtp and obp activations from july 2015-december 2016. data were manually collected from transfusion service paper records, electronic (safe-trace) records, and an automated data report from the electronic medical record (epic). conclusion: we observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation. trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells. los and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion. we have identified an opportunity for improvement in mtp transfusion ratios observed in trauma cases, the specific environment from which the 6:6:1 ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious. patient identification improvement strategy to help reduce unacceptable specimens arline stein* 1 , nancy nikolis 1 , linda benison 1 , ruthmire thelusca 1 , renee liberty 1 , sherry shariatmadar 1 , alexander indrikovs 2 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: our blood bank (bb) processes approximately 60,000 specimens per year. bb specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation. in such cases, a new specimen is requested to be drawn as per protocol. our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the bb supervisor for completion. following completion, the report was sent to the nurse manager of the patient care unit (pcu) for follow-up and investigation with the staff members involved. this process was cumbersome, taking a few days before the staff member of the pcu was alerted to the deviation in protocol. at times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services. study design/method: in june 2016, a patient identification improvement strategy was implemented jointly by the department of nursing and the bb to address mislabeled, unlabeled and unsigned specimens as part of a patient safety initiative. currently, following this strategy, when an unacceptable specimen is received, the nurse manager (nm) of the pcu is immediately notified by bb staff. the nm promptly initiates a debrief process with the staff involved in drawing the specimen. a debrief form (tool) was created to guide the discussion. this process is followed 24/7. the nm will also engage other available staff in a huddle to review the incident and reinforce the policy. the debrief form is then submitted to hospital qa and the bb with preventative actions included. we believe in using the just culture model to help us understand the reasons why the staff did not label the specimen according to policy. just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care. results/finding: the table below represents the percentage of unacceptable specimens identified by the bb since the second quarter of 2016. the implementation of this new process has led to a decrease in the number of unacceptable specimens up to 30% quarterly following its implementation. the opportunity for direct intervention by the nm with the staff involved has risen from 75% to 96%, due to the immediate debrief process. abstract conclusion: the patient identification improvement strategy allows for real time engagement of the bb and pcu staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection. the heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience. platelet transfusion practices among pediatric oncology patients: a single institutional experience nicole m crews* 1,2 , morgan rockwell 2 , joseph hagan 1 , jun teruya 1,2 and shiu-ki hui 2 . 1 texas children's hospital, 2 baylor college of medicine background/case studies: despite advances in adult platelet transfusion (ptx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses. therefore, ptx are commonly guided by local institutional recommendations (ir). the aim of this study was to determine the degree of adherence of ptx practice to ir at a pediatric tertiary institution. study design/method: retrospective review of ptx practices including transfusion thresholds, responses and dosages were collected. platelet counts within 24 hours pre and post transfusion were evaluated. patients (0-18 years) receiving prophylactic ptx from july to december 2016 admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included. for prevention of volume overload, the ir for ptx were < 10ml/kg for patients < 35kg and one apheresis unit (au) for patients >35kg; therefore, patients were separated into 2 groups: < 35 kg and > 35 kg. a significant proportion of orders for both < 35 kg and > 35 kg did not meet patient platelet threshold criteria (p<0.001). conclusion: ptx threshold above ir for both groups were 31 ( 35 kg) and 48% (> 35 kg). most common reason for above ir threshold was an invasive procedure or low molecular weight heparin therapy. greater than 10% of ptx dosage in both groups were above ir, however the platelet response did not increase significantly (p>0.05) with a higher dose vs. ir dose. this study demonstrated that there were still considerable deviations from ir in ptx practice among pediatric oncological patients. in addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events. each institution should conduct a quality assurance review to determine ptx practice. pre-surgical sample process improvement to enhance patient safety and compliance lisa marie button*, stephanie saathoff, jered luedke, benjamin colvin, umalkair amare and james r stubbs. mayo clinic background/case studies: our institution provides the option for presurgical samples (pss) to be drawn up to 56 days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding 3 months from the date of pss collection. when pss patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the pss draw to the day of surgery/possible transfusion. study design/method: an electronic fix was designed that was applied to the surgical intake process. a new set of questions was added to the a.m. admit questionnaire that must be completed prior to the patient's surgical procedure. the questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank. the blood bank techs review the alert and inactivate the patient's pss based on the new transfusion/pregnany information. one year post-implementation of the electronic fix, transfusion lab performed a retrospective review of all pss alerts generated during a three-month period. results/finding: the results of the review were analyzed and are displayed in the table. it was determined that only 2.1% of patients with a pss alert had an active sample requiring inactivation. conclusion: implementing an electronic solution that requires documentation about pss eligibility upon return for surgery has resulted in an estimated 3220 (805x4) pss alerts in the blood bank each year. of these alerts, it is estimated that approximately 68 patients (17x4) per year are identified as no longer eligible for pss status. once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and pss status. while the benefit of having fewer false positive pss alerts (97.9%) was recognized as an ideal future state, it was not compatible with the institution's current it project of implementing a new electronic medical record (emr) system. the safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new emr with the intention of targeting only patients with an active pss in the blood bank, rather than all surgical patients. weill cornell medicine, 7 columbia university school of medicine background/case studies: blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays. risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation. given the complexity and high-risk nature of blood ordering a proactive risk assessment (pra) for blood product ordering using the fmea methodology was conducted. the goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering study design/method: to evaluate the electronic blood ordering redesign process, a pra was completed using the fmea methodology. the team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the risk priority number (rpn). all rpns with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode. results/finding: the group scored the identified failure modes by categories used in root cause analyses. the electronic blood order process has internal logic and alerts that improve communication and reduce the risk score. several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the rbc order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system. a transfusion history will be available to providers when ordering blood products to further reduce communication risks. categories for failure modes included clinical,communication,equipment people,process and system. the average overall failure mode rpn was reduced by 29% with the communication category average rpn having the greatest reduction of 72%. conclusion: an fmea of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration. accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood. reducing turn around time for type and screens in the blood bank kimberly ouellette* 1 , karen king 1 and joseph sweeney 2 . 1 rhode island hospital, 2 lifespan academic medical center background/case studies: expeditious turn-around times (tat) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner. the blood bank at rhode island hospital, a level 1 trauma center and teaching hospital associated with brown university, was originally designed to accommodate tube testing by all technologists. the original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third. as technology changed, the blood bank adopted first the manual gel station and then the automated gel system (ortho provuev r ) but did not adapt the space. the second section of the blood bank contained the manual and subsequently automated gel stations with no other changes. the process of sample receipt through completion of testing and issuing of units remained segmented and inefficient. the average tat for type and screens was 80 6 32 minutes. study design/method: the blood bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology. the first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory. a wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt. the third section remained, but was repurposed for teaching medical technology students and residents. in addition to the remodel, the blood bank retired the ortho provuev r for the ortho visionv r , which is considered a true continuous feed machine. although the inter-device tat is not significantly different (30 minutes for the provuev r and 28 for the visionv r ) the visionv r is built with a scheduler that effectively handles the system and processes samples efficiently. the visionv r is also equipped with two centrifuges to process samples, which further reduces tat when multiple samples are onboard. a bi-directional interface was designed to allow for test orders (type and screens) to go to the visionv r and test results to go directly from the visionv r to the lis without the need to manually order the tests or transmit the results. data on tat were collected and analyzed using independent t tests and chi square. results/finding: the mean tat pre-and post-reconfiguration and implementation of the ortho visionv r and a fraction of samples with tat over 90 minutes are shown in the table. the results show a reduction in tat by 14 minutes with a 20% reduction of tat greater than 90 minutes. conclusion: a combination of new technology and space remodeling can lead to a significant reduction of tat of testing in the blood bank. caleb wei-shin cheng* 1,2 , lorna orengo 3 , monique scott 3 and christopher a tormey 1,2,3 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: the type and screen (t&s) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients. despite this, erroneous blood product administration may occur as much as 1 in 19,000 blood transfusions. to prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management. hemolysis rates for t&s specimens are reported to be as high as 25% prior to interventions, but may potentially be reduced to as little as 1.5%. however, there is little published data on non-hemolysis-related type and screen rejections. an initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of <5%, with a particular emphasis on non-hemolysisassociated forms of rejection. study design/method: a root cause analysis (rca) was performed over the preceding 6 months to obtain a baseline understanding of the errors involved. t&s submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously. when a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection. following rca, an intervention was created to resolve the most common issues documented that resulted in rejection. approval for the intervention was granted by the department chair, transfusion committee, forms committee, and the medical executive committee. after implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention. results/finding: over the study period, the t&s rejection rate averaged 8.2%. reasons for specimen rejection were divided into 5 groups: 1) hemolysis, 2) blood bank witness collection form errors, 3) quantity not sufficient, abstract 4) duplicate sample, and 5) specimen tube labelling errors. the highest percentage of rejections was due to improperly-filled witness forms (table 1) . after multiple form redesigns and approval by appropriate committees the new form was implemented. preliminary data collected thus far demonstrates a 6.8% rejection rate with only 1 rejection relating to witness form errors. conclusion: rejected t&s specimens are an impediment to safe clinical care as it may delay medical/surgical management. rejection rates could be reduced through simplification of blood bank specimen collection forms. care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error. future quality initiatives will aim to simplify complex healthcare processes without compromising patient care. reduction of failed whole blood donor testing runs on the roche cobas s 201 system christopher shahan* 1 , christina dejesus 1 , mosi mccall 1 , fallon hampton 1 , tangi herring 1 , judy davis 1 , anjali patel 1 , sonya gomillion 1 and bonnie maltby 2 . 1 qualtex laboratories, 2 qualtex laboratories background/case studies: as part of our quality control program, we track the number of technician related failed runs observed on the roche cobas s 201 system. this system is used to test whole blood donor samples for human immunodeficiency virus (hiv) rna, hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and west nile virus (wnv) rna. technician related failed testing runs can cause the laboratory to report results outside of the contractual 10-12 hour turnaround time. failed runs also cause retesting which increases reagent utilization for the system. currently 42% of whole blood donor testing turnaround time delays are due to issues and failed runs on the s 201 system and we have 3 technician related failures per week. a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s 201 system. study design/method: the number of technician related failed runs on the s 201 system were tracked from 10/11/2015 thru 12/11/2016. a pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures. the 5 whys were performed to determine root causes of technician related failed runs. a gemba walk was performed on all of the lab testing processes to help identify areas for improvement. the process improvement team talked, met, observed, and worked directly with staff that operate the s 201 system. roche was also contacted to provide guidance on how to help decrease technician related failures. results/finding: the main root cause determined was that there was no current process flow map for whole blood donor testing using the s 201 system. counter measures implemented included creating a two phase process map. one phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples. roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s 201 system. after counter measures were implemented, the number of technician related failed runs decreased from 3 to 1.2 failures per week, which was a 58% decrease. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s 201 system. this lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by 58%. patients who were transfused for pre-transfusion hgb >7g/dl with resulting post-transfusion hgb >9g/dl were reviewed. demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by 11 volunteer internal medicine residents. group discussion for each case ensued before determination of transfusion appropriateness occurred. principal investigator/attending physician then made final determination of appropriateness of rbc transfusion. results/finding: 265 patient charts were reviewed. 91 were excluded for bleeding and cardiovascular instability. 106/174 (60.9%) were determined to be transfused inappropriately. there was no difference in appropriateness of transfusion with respect to age or sex. patients with solid tumors (76.19% vs 55.73%, p50.0181) and anemia of chronic disease (76.47% vs 54.1%, p50.006) were more likely to be inappropriately transfused. patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb 7.7 g/dl vs 7.3 g/dl, p<0.0001). inappropriately transfused patients also had higher median post-transfusion hemoglobin (9.9 g/dl vs 9.4 g/dl, p<0.0001). moreover, lab evalutions revealed association with lower folate levels (median 8.1 nmol/l vs 15.7 nmol/l, p50.029). 29/106 (27.3%) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between. 7/64 providers were responsible for 32.3% of all inappropriate transfusions. 1/68 appropriately-transfused patients experienced an fnhtr. 3 deaths unrelated to transfusion occurred (1 in appropriate, 2 in inappropriate group). conclusion: physicians in training are interested in promulgating optimal rbc transfusion practice. this study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion. beyond cpoe, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions. successful implementation of a blood bank information system in a small-scale caribbean blood bank: a structured step-wise approach. luigi sille* 1 , willem martin smid 2 and ashley john duits 1 . 1 red cross blood bank foundation, 2 sanquin consulting services background/case studies: an important tool for complying with gmp quality standards is the effective use of a blood bank information system (bis). validation and implementation of a bis is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment. . small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention. for the introduction of a bis at the blood bank of the dutch caribbean island of curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting. study design/method: the red cross blood bank foundation curaçao is the sole provider of labile blood components for the dutch caribbean islands of curaçao, bonaire and sint maarten. after selection of the bis provider for implementation isbt and bcsh guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures. these procedures were meant to evaluate and validate the features of a bis (e-delphyn, hemasoft america, miami, usa) before introduction. the outcome of the approach was entered in worksheets that were evaluated by the implementation team and management. from this the implementation plan was designed and implemented. an external auditor (sanquin consulting services, amsterdam, netherlands) was invited to evaluate the implementation and validation plan and its practical implementation. the evaluation was performed according to risk assessment of critical process steps. results/finding: based on the isbt and bcsh guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a bis was designed. comparison of the current processes and procedures were compared to the bis characteristics making use of 8 worksheets. with these worksheets the existing gaps with the bis procedures were carefully described. these gaps and the appropriate procedural changes for bis or blood bank were effectuated. the worksheets also provided the basis for staff training in a separate training environment before bis introduction. during the early validation phase all procedures and processes were audited by an external auditor. with the feedback of the expert several improvements were added for the validation and subsequent implementation processes. conclusion: with the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a bis in a small scale caribbean blood bank. the program as designed seems well suited for small scale blood banks contemplating introduction of a bis. time and cost savings through implementation of a remote blood fridge jessica peters* 1 , dee dee cassidy 1 , jed b gorlin 1,2 and nancy l van buren 1,2 . 1 hennepin county medical center, 2 innovative blood resources background/case studies: rapid delivery of emergency release group o red blood cells (rbc) are vital to patient care. commercial remote blood fridge packages are available but have large upfront and maintenance costs. we implemented a remote blood fridge directly in our emergency department (ed) using an under counter fridge requiring id access, and a selfdeveloped ios application that scans, tracks and real-time alerts transfusion service (ts) to products used and to whom they were dispensed. prior to ed fridge implementation, rbc units were verbally requested and an ed blood runner would pick up and return the cooler. given that our ed is located in a separate building from the ts, this meant 10 or more minutes may be required for transit of units often released in less than 5 minutes. the net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required. implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release rbcs more quickly, with the observed benefit of decreasing wasted staff time. study design/method: the remote blood fridge was implemented in july 2016. data for rbc requests in coolers, rbc returns and rbc transfusions from the ed was collected and compared. baseline data included january 2015-june 2016, and post change included august 2016-december 2016. july 2016 data was excluded as it included both the pre and post processes. results/finding: baseline data shows that the ed requested an average of 99 rbc/month in coolers. post change this dropped to 62 rbc/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge. baseline data also shows that an average of 54 rbc/month were returned (55%). post change, the average rbc/ month returned was 27 (44%), this represents an absolute 50% reduction in number of returned products. each rbc dispensed and returned takes approximately 20 minutes to complete paperwork and transport, therefore this change saved an average of 540 minutes per month. it was also noted that the average rbc/month transfused was 44 for baseline and 57 post change. this confirms that the decreased requests and returns were not due to decreased patient volume or severity. the fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of 15-20 minutes (estimated) was reduced to 3-5 minutes. conclusion: implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient. this change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns. implementing a blood fridge can also be done at a reduced cost through homegrown processes. transfusions are everyday procedures and over 250 patent-applications have been filed related to "transfusion medicine" and over 400 related to "transfusion alarm", during the last 30 years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions. the aim of this contribution is to present a developed low-cost real-time individual intravenous blood-transfusion monitoring system, based on the internet of things. study design/method: the designed system is based on a commercially available pan-tilt-zoom (ptz) camera, employing an 1/4 inch color cmos sensor, providing effectively 1.0 mp, a 3.6 mm lens, ir-cut, day/night minimum illumination 0.1 lux/f 1 and 808 viewing angle. the camera is focused on the droplets and acts as vis/ir detector with a 24 hz sampling-rate. custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g. clotting, blood 260a transfusion 2017 vol. 57 supplement s3 abstract or other suspensions depletion etc.) and enables, if necessary, wide-angle image-capturing. the video image-audio settings provide for compression h.264, video frame rate (fps) 1-30/s, refresh rate 50 hz and audio input, through bidirectional built-in microphone. the acquires an ip-address, the connection mode is wireless, the network interface is wi-fi/802.11/b/g, the supported protocols include dhcp, tcp/ip, upnp, http, smtp and p2p is provided. typical 5v power-supply, sized 165x125x101mm and weighing 370 g. client software is required. the ir range is 10-15 m; ir-cut filters, remote access, dual stream, motion detection, day/night and ir night vision distance of 10 m are offered. two-way radio-link is provided, as well as, trans-flash (tf) recording and storage on a 32 gb sd-card. pan/tilt-horizontal 355 o and pan/tilt-vertical 90 o movements can be performed. the system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as nibp, ecg, and spo 2 , if present. results/finding: the system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation. conclusion: the system can measure infusion-speed with a deviation lower than 6 3 %. the developed iot-system takes advantage of the existing hospital wi-fi networked environment and offers a low-cost solution, under 20 $ for each monitoring-set. it allows for even multi-platform (ios, android, windows) smart-phone, short-range connectivity, for up to 4 participants, for example nurse, physician etc. two potential approaches for the quality control of bact/alertv r culture media using various bioball tm organism preparations patricia rule*, michelle keener and christine crawford. biomerieux inc. background/case studies: the bact/alertv r bpa and bpn culture bottles are used with the bact/alert microbial detection system for rapid screening and detection of microbial contamination in leukocyte reduced apheresis platelets (lrap). recent changes in the clia quality control guidelines and aabb accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth. a study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel. study design/method: the general protocol consisted of three replicates each of each organism inoculated into two lots each of bpa and bpn by two different analyst. the study was two part in that aspergillus brasiliensis, candida albicans, bacillus subtilis subsp. spizizenii, pseudomonas aeruginosa, escherichia coli, clostridium sporogenes, staphylococcus aureus and streptococcus pyogenes were prepared from bioball singleshot (30 cfu), multishot 550 cfu or highdose 10k organism preparations at a low level (< 50 cfu) and evaluated on the same day of preparation as method validation. the second part of the study utilized escherichia coli and staphylococcus aureus prepared and frozen at a higher level and then evaluted over a 14 day study as a stream line approach to routine quality control testing of the bact/alert culture bottles. inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point. inoculated bpa and bpn bottles were loaded into the bact/alert microbial detection system at 36 c for automatic monitoring of growth. negative bpa and bpn bottles were included in duplicate at each day of testing. results/finding: escherichia coli, staphylococcus aureus, streptoococcus pyogenes and bacillus subtilis subsp. spizizenii were positive in both the bpa and bpn culture bottles. the aerobic aspergillus brasiliensis, candida albicans, and pseudomonas aeruginosa grew and were reported positive in only the bpa aerobic culture bottle as expected. while the obligate anaerobe, clostridium sporogenes was positive only in the anaerobic bpn culture bottles. bacterial cultures were positive in the bact/alert bpa and bpn bottles < 1 days and the fungal organisms in < 2 days. the overall agreement was 99.8 % in 698 bottles tested here. no significant differences were observed in the time to detection between the different lots or between the different analyst. conclusion: the bioball prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of bact/alert bpa and bpn culture media. the method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory. use of an electronic patient identification system for blood banking specimen labeling found to be superior over historical armband approaches annie newton* 1 , diane schafer 2 , debra brown 1 , jesse cox 3 , scott koepsell 3 and sara shunkwiler 3 . 1 nebraska medicine, 2 the nebraska medical center, 3 university of nebraska medical center background/case studies: anticipating the implementation of the new (30 th addition) aabb standard concerning the confirmation of patient abo blood typing of type and screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands. continued use of the armbands would require a second sample for abo confirmation of patients that did not have a historical blood type on file. concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction. moreover, 2 nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern. study design/method: current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well. an in-depth evaluation, including a failure modes and effects analysis (fmea) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart. an alternate process for specimen labeling and abo confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system. extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process. alerts were congruently built into the electronic health record (ehr) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use. results/finding: within days of implementing the new process (september 11, 2016), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling 4 in 6 months post implementation compared to 144 in the 6 months prior. in addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential 2 nd specimen collections needed for abo confirmation. conclusion: use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for 2 nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction. background/case studies: based on a few small randomised controlled trials (rcts) performed in the late '90s and in early 2000, intravenous immunoglobulin (ivig) use has been suggested as a potential treatment to avoid exchange transfusion (et) for rh hemolytic disease of the newborn (hdn). this treatment modality is now routinely used for rh-hdn and has been extended to hdn caused by abo incompatibility or by other red blood cell antibodies. however, larger rcts performed since 2010 have shown that prophylactic ivig did not reduce the need for et, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions. the primary objective of this study was to describe the usage of ivig for hdn at a tertiary academic referral hospital. study design/methods: a retrospective chart review was performed of all neonates who received ivig for hdn in the neonatal intensive care unit (nicu) from january 1, 2005 to june 30, 2016. data collected included patient demographics features and diagnosis, indications for ivig, neonatal laboratory results, treatment details, adverse events and patient outcomes. results/findings: ninety-seven neonates received ivig during the study period: 57% were female and 7% were less than 34 weeks of gestational age. none had co-existing g6pd deficiency, pyruvate kinase deficiency or spherocytosis. all neonates received phototherapy prior to ivig treatment. indications for ivig were abo-hdn (41%) and rhesus-hdn (59%). antibodies most often implicated in rh-hdn were anti-d (22/57), anti-d and anti-c (22/57) and anti-c (5/57). sixteen infants with rh-hdn had received intrauterine transfusions. the mean cumulative dose of ivig was 1 g/kg (range from 0,3 g/kg to 3,8 g/kg). neonates received one to four ivig administrations. table 1 shows the number of patients receiving ivig during two time periods. three adverse reactions were noted during ivig administration: cutaneous rash, hypotension and fever. of all neonates, 14 required an et for rh-hdn and 3 for abo-hdn. forty-five (46%) patients needed top-up transfusions during hospitalisation and until three months of age: 8 with abo-hdn and 37 with rh-hdn. the mean number of transfusions was three (range:1 to 7). conclusion: although initially described for rh-hdn, abo-hdn is now one of the most frequent indications for ivig in neonates. the optimal use of ivig in abo-hdn needs to be better characterized. our study shows a wide variation of ivig dosing and a significant proportion of neonates requiring top-up transfusions. further research is required to evaluate whether anemia in abo-hdn might be exacerbated by hemolysis from ivig isohemagglutinins and if it is dose-dependent. background/case studies: background: one of the most serious adverse reactions to transfusion is the development of graft versus host disease. symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia. the clinical course is rapid with an over 90% case fatality rate. the patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of hla-matched cellular blood products and the recipients of cellular blood products donated by blood relatives. the basic etiology of ta-gvhd is the inability of the transfusion recipient to mount an effective immune response against donor t-lymphocytes. treatment options for ta-gvhd are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication. study design/methods: most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components. however, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components. following a "near-miss" situation, our hospital information department developed a 4-part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components. results/findings: our blood products ordering system has been redesigned to include 3 specific queries to identify those patients who required irradiated cellular blood products. first, physicians have been notified to include the need for blood product irradiation in the patient problems list. once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the problem list is modified by the clinical staff. second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list. finally our system will automatically default to request irradiation on all cellular products ordered for children less than 4 months of age to comply with local irradiation policies. conclusion: we believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm. we believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate. using lab information system and a dynamic dashboard for labeling and tracking coolers russell thorsen, rosaline ma, peter suslow, gina giannarelli, sara bakhtary, ashok nambiar and morvarid moayeri*. ucsf health background/case studies: our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ors, icus, cath-lab, etc. coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites. a robot that can hold one cooler delivers products to locations not served by the pneumatic tube. on average, 30 coolers are issued every day. cooler set-up is a multi-step, labor-intensive process. transfusion service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage. we developed a lab information system (lis)-based solution to manage cooler labelling and tracking more efficiently. study design/method: nine cooler test batteries were built; the batteries for rbc, plasma, platelet and cryoprecipitate (2 each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and rbcs with variable expiration times) is slightly different. the second battery in each pair was built to avoid duplicate test cancellation by lis when a second cooler (for same component type) is being set up for the same patient. each battery consists of tests capturing the following information: cooler location, cooler id, number of units issued, and expiration. custom barcodes representing each test battery and 45 different locations can be scanned from a 'quick-pick list', avoiding need for manual entry. when coolers are returned, a final entry is made in the test battery, updating lis. a dynamic cooler tracking dashboard with live-feed from lis displays data captured in the test battery. elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in lis) is captured automatically. color codes alert users to coolers that have less than 1 hour before expiration. a flashing alert pops up for coolers that have expired. results/finding: we replaced our manual process (hand-write patient information and expiration time on 2 separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel lis-driven labeling and tracking system. each time a cooler is set up, a test battery is ordered and resulted in lis by scanning the related custom barcodes. a single lis-generated label is printed and attached to each cooler. cooler expiration is defaulted to 10 hours (per our current cooler validation) from the time the test battery is ordered during cooler set up. techs pay attention to expiration of each product they place in a cooler. if an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown. color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction. conclusion: using lis for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient. these tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking. improving cryoprecipitate collection operations using operations research and analytics-based methods american red cross, 2 georgia institute of technology ap72 reduction in unnecessary use of type o-negative rbcs in a level i bellevue hospital-nyulmc our hospital is a level i trauma center serving a diverse predominately non-caucasian population. historically we stocked our trauma blood bank monitored refrigerator with 2 o-negative rbcs. trauma requested that we stock additional rbcs to be able to initiate a mtp for multiple patients at the same time. believing that most of our trauma patients are male, elderly, or rh-positive, we agreed add 2 type o-positive rbcs to the stock. rules for determining which units to use were established. o-positive rbcs are to be given to 1a) all adult males (am), 1b) women of non-childbearing age (wncba), and 1c) if both o-negative rbcs were used but not yet restocked, and o-negative rbcs are to be given to 2a) women of childbearing age (wcba) and 2b) children until the patient's aborh type are determined. we sought to assess the impact of this change on our usage and purchases of o-negative rbcs. study design/method: all patients issued emergency release trauma rbcs following the addition of o-positive rbcs were assessed 3%) would have needed to be o-negative. the addition of o-positive rbcs to our trauma refrigerator will enable us to markedly reduce our purchases of o-negative rbcs. ap73 saving apheresis platelets through use of verax point of care testing jennifer rhamy* 1 and rebecca wride 2 . 1 st. mary's regional blood donor center, 2 st. mary's regional medical center background/case studies: our rural hospital-based blood center serves 17 hospitals and a diverse patient population including acute trauma. because of the varying need for platelet products (varies between 0 and 8 per day in 2017), we investigated the use of the verax point-of-care test to better manage our valuable inventory barrett lawson 2 and jun teruya 1,2 . 1 texas children's hospital, 2 baylor college of medicine ap127 vision titers --easier or problematic? (table 1) . results/findings: post intercept, t had volumes of 261-320 ml, with 98 6 4% hemoglobin (hb) recovery. t had 10-fold less extracellular protein than c. after 35 days of storage t had higher atp and na 1 than c while lactate and hemolysis were lower. hct, ph, k 1 and glucose were equivalent between t and c on d35. d35 hemolysis for t was 0.08-0.31%, while for c it was 0.10-0.57%. t and c atp was >2mmol/g hb, the level of atp associated with effective rbc viability, throughout storage (table 1) . hematocrit (hct, %) 58.5 6 2.5* 56.3 6 1.7 60.8 6 2.7 61.8 6 2.9 hemoglobin (g/unit) 59 6 7 6 0 6 4 not measured hemolysis (%) 0.02 6 0.01* 0.06 6 0.04 0.16 6 0.07* 0.29 6 0.14 ph (378c) 6.9 6 0.1* 7.3 6 0.1 6.7 6 0.1 6.6 6 0.1 total atp (mmol/g hb) 7.7 6 0.5* 5.0 6 0.4 4.6 6 0.6* 3.9 6 0.5 k1 (mm) 1.5 6 0.3* 5.7 6 2.5 53. total tested total plts issued feb 87 121 64 48 24 185 181 mar 226 516 342 276 133 858 757 totals 352 730 471 375 174 1201 table: 2. resident reports to the intranet "drop box" increased from 53.7% to 69.3% to 100%, each over 2 month time spans. conclusion: safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time. safe ordering prevented recurrent allergic reactions in our patient population. the tso plays a pivotal role in ensuring the full circle of communication occurs. processes that integrated the pathology resident improved with pdsa cycles and impacted the quality and timeliness of hand off. finally, the data provided from the residents enabled efficient participation in hemovigilance. decreasing results/finding: the main root cause determined was that there was no standard work process. sops were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow. counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the batch release department. specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing. after counter measures were implemented, the number of exceptions decreased from 17.4 per day or 10,370 dpmo to 6.3 per day or 3,539 dpmo. this is a statistically significant difference since the p-value calculated was 0.002. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory. this approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process. background/case studies: our blood bank processes approximately 4,400 specimens per month. since 1998, the requirement of having a second blood type on record was met by:1. utilizing the historical blood type and the current specimen, or 2. having second type performed on same specimen by different technologist, and 3. each type and screen specimen signed by 2 staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.to comply with the aabb standards 29 th edition, #5.16.2.2 a decision was made to change our practices. we considered challenges encountered at other hospitals and collaborated with nursing and it to create a streamlined and safe process. in april 2016, the second specimen procedure was implemented addressing the following: ii. extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively. results/finding:1. there was a minor adjustment period with more phone calls made to blood bank to explain the process. 2. there was minimal impact on turn around times for release of components. 3. aborh retype workload decreased from 1500 to 950 (35% to 20% of t&s volume) per month. 4. unnecessary blood draws minimized, improving patient experience. 5. no emergency release requests due to absence of a second specimen. the second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services. overall feedback from staff on the process has been positive. our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution. background/case studies: the hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome. with this in mind, aabb and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as 15 to 48%. computerized provider order entry (cpoe), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment. the objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion. additionally, factors associated with inappropriate transfusion were examined. study design/method: in our 926 bed hospital, a retrospective chart review was performed (07/01/15-12/31/15) on hospitalized internal medicine patients. cpoe with hospital guidelines for rbc transfusions were in place. transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies. charts for background/case studies: our midwestern university-based transfusion service (ts) evaluated the appropriateness the automated platform vision (ortho clinical diagnostics. raritan, nj) for prenatal titration studies. it has been established from previous publications that the micro-column assay, of which the vision is based, may lead to higher titer results compared to standard tube titrations. this study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective. study design/method: twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the vision. the samples were manually tested with a standard two-fold serial dilution. the titer was reported as the last tube to demonstrate a 11 reaction by macroscopic observation. the titer studies were then repeated using the vision. the results of the manual and automated processes were compared and categorized as "< 2 grade" or "> 2 grade" difference between endpoints. this analysis is similar to the acceptable ranges used for evaluating college of american pathologists (cap) proficiency survey challenges. a cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer. results/finding: table 1 demonstrates a summary of the samples tested by manual titer study and vision titration method. the vision titer results (mean, median, and mode) were higher than the manual tube titer results. less than half of the samples (48%) were > 2 titer results higher, while the majority was 2 titer results different (52%). the cost analysis is summarized in table 2 . the indirect cost (labor) was significantly lower with the use of the vision. the reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the vision completing the titration as part of the profile of the titer study of the analyzer. conclusion: with an estimated 41% decrease in the cost of a vision titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank. however, the vision demonstrated the expected increase in titer results compared to manual tube titer results. this would impact the critical values currently utilized. an impact assessment for clinical staff would be necessary to adequately implement the change in method. consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values. in addition, as part of changing to the vision an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to vision titer results which would be higher and may be interpreted as a significant change for clinical care of the patient. what is the best practice for testing residual white blood cells in blood components for monthly routine quality control? janja pajk*. general hospital celje background/case studies: we wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components. our aim was to validate the adam device for counting thne number of residual white blood cells (wbc) in leucocyte depleted and in non-leucocyte depleted blood components (bc) and to compare with standard counting method by microscopy in fuchs rosenthal chamber (frc) used in ghc and with flow cytometry (fc). study design/method: after 23 samples of red blood cells (rbc), platelets (plt) and fresh frozen plasma (ffp) (leucocyte depleted in top and top (t/ t) bags and non-leucocyte depleted in top and bottom (t/b) bags) were stained with propidium iodide (pi) on r-slides; adam -rwbc device was messured fluorescent images of stained wbc nucleus. data were analised by image analysis software and later compared with results of testing samples in frc by microscopy and with fc.15 samples of bc were microscopic tested in frc at department of laboratory medicine in ghc; another 8 samples were measured with fc in ucc maribor. results/finding: 15 samples (6 rbc, 3 plt, 3 ffp-all leucocyte depleted and 3 non-leucocyte depleted ffp) were tested in triplicates on adam and with frc once.coefficient of variation of (kv%) of samples measured on adam for leucocyte depleted bc varied for: rbc from 44,10 -173,21; plt from 86,60 -100,00; ffp from 86,60 -173,21; and for non-leucocyte depleted ffp from 0,69 -8,85 (table 1) . 8 samples (2 rbc, 2 plt, 2 ffp -all leucocyte depleted and 2 nonleucocyte depleted ffp) were tested in triplicates on adam and with fc once.kv% of samples measured on adam for leucocyte depleted bc varied for: rbc from 91, 56; plt from 78, 21, ffp from 66, 21 ; and for non-leucocyte depleted ffp from 11,17 -15,91 (table 2) .high percentage of kv was noticed in samples with low numbers of wbc (in leucocyte depleted bc; low percentage of kv in non-leucocyte depleted ffp, with higher amount of wbc was observed. conclusion: all samples tested with adam met expected criteria for wbc in bc in european union (less than 1x10 9 /unit for leucocyte depleted or 1x10 6 / unit for non-leucocyte depleted) and were comparable with those tested with fc; the correlation with microscopy in frc was worse.with use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.from january 2017 we changed our protocol for testing residual wbc in bc with adam device and we advise it as the best practice for monthly routine quality control. key: cord-010092-uftc8inx authors: nan title: abstract of 29th regional congress of the isbt date: 2019-06-07 journal: vox sang doi: 10.1111/vox.12792 sha: doc_id: 10092 cord_uid: uftc8inx nan in the fin de siecle was heavily concentrated in vienna. freud, boltzmann, schr€ odinger and mach might be the first names to find, whenever one cites austrian scientists. but more related to transfusion are the noble prize winners max perutz and karl landsteiner. landsteiner 0 s fate illustrates the brain drain beginning in the early 30 s escalating in 1939 with the "anschluss", which lead to the forced emigration of many scientists. a loss which was not regenerated in the post war years and was further aggravated by dubious and often undisclosed relations and scandals in the nazi-era. all together this leads to a severe loss of credibility and productivity of universities across decades. opening university access in the early 80 s and intensive historical work-up of scandals transformed the austrian universities to open and effective scientific institutions driving innovation in the country. austria has achieved a great economic deal in recent decades, which was accelerated by the eu membership in 1995. as a result of strong long-term economic performance, the country's gross domestic product (gdp) per capita is the eighth highest among oecd countries and fourth in the eu28. levels of poverty and income inequality are both below the oecd average. investment in research and development (r&d) increased since the eu accession, when austria's r&d intensity (aggregate r&d expenditure as a percentage of gdp) was well below the oecd average and significantly far lower than switzerland -a country to which austria prefers comparison. the eu target of 3% r&d intensity was first met in 2014 and is 2018 the sixth highest among oecd countries and the second highest in the eu28. austria showed the second highest increase in r&d intensity of all oecd countries, exceeded only by korea. the rapid expansion was matched by a similar increase in human resources and scientific output of universities. austrian science in quantum mechanics, quantum communication and information is world renown. vienna is a major biotech hub, as is linz in mathematics and mechatronics and graz in automotive and production technologies. austria has been a net resource recipient in the horizon 2020 and the preceding 7th framework programme. small and mediumsized enterprises show a high propensity to co-operate with universities and other research organisations and more and more included in scientific grant schemes. vienna is the largest student city in the german-speaking world and consistently ranks among the top cities in the world on quality-of-life indices. as austria possesses globally recognised cultural attractions ranging from famed salzburg festival to the vienna new year concert its inhabitants are not aware of the progress made in r&d and how thriving innovation is going on in their country. they still love to show their cultural heritage and events and impress the world with some kind of eternal sound of music. patients with refractory b-cell malignancies as non-hodgkin lymphomas (nhl) resistant to standard therapies have a dismal prognosis. the outcome is even poorer in patients relapsing after autologous stem cell transplantation. most of these patients do not qualify for an allogeneic hematopoietic cell transplantation (hct) due to refractory disease, lack of a suitable allogeneic donor, higher age or cumulative toxicity of previous chemotherapy. despite patients undergoing allogeneic hct normally profit from a graft-versus -lymphoma effect, overall survival in patients with nhl after hct remains short. a similar situation can be observed for patients with acute lymphoblastic leukemia (all). therefore novel treatment modalities are urgently needed. chimeric antigen receptor (car)-t cells, a new class of cellular immunotherapy involving ex vivo genetic modification of t cells to incorporate an engineered car have been used in clinical trials. in the majority of studies b-cell malignancies treated with cd19 targeting car-t cells have been analyzed. austria had the advantage to participate in two international trials in the past and is currently involved in further car-t studies. recently, results from cd19 directed car-t cell trials with an increased follow-up of patients led to fda (food and drug administration) and ema (european medicines agency) approval of tisagenlecleucel and axicabtagene ciloleucel. common adverse events (aes) include cytokine release syndrome and neurological toxicity, which may require admission to an intensive care unit, b cell aplasia and hemophagocytic lymphohistiocytosis. these aes are manageable when treated by an appropriately trained team following established algorithm. in this presentation, results of four large phase ii cd19car-t cell trials for patients with nhl and all and focus on aes is summarized. preoperative anemia is a known risk factor for increased perioperative morbidity and mortality in patients undergoing major surgery. previous studies have not only shown higher in-hospital mortality, but also an increased hospital length of stay, greater postoperative admission rates to intensive care and prolonged use of mechanical ventilation and intensive care resources in patients with anemia compared to those with normal preoperative hemoglobin concentrations. about 30% of patients scheduled for major surgery suffer from preoperative anemia. this figure is even higher in patients requiring orthotopic liver transplantation, where up to 75% of all patients are diagnosed with anemia prior to surgery. transfusion of packed red blood cells (prbcs) is commonly used to correct anemic hemoglobin values. however, transfusion of prbcs has been associated with increased morbidity and mortality in patients undergoing cardiac, orthopedic, and abdominal surgery. additionally, transfusion of prbcs is associated with a greater incidence of postoperative acute kidney injury in patients undergoing orthotopic liver transplantation. as preoperative anemia might increase the perioperative use of prbcs, negative effects observed after prbc transfusions might even be augmented. data on the influence of preoperative anemia on morbidity and mortality after orthotopic liver transplantation are limited. thus, we retrospectively analyzed the association of preoperative anemia and mortality in adult patients undergoing orthotopic liver transplantation at our institution. in addition, we examined the influence of anemia on perioperative parameters such as transfusion requirements, surgical complications, early allograft dysfunction, acute kidney injury, and the need for renal replacement therapy. based on the results obtained in the retrospective analysis, an ongoing prospective randomized clinical trial was initiated. the two suspensive treatments in sickle cell disease (scd) are hydroxycarbamide, inducing the production of the functional hbf, normally repressed at birth, and red blood cell (rbc) transfusion, a critical component of scd management. however, rbc transfusion is not without risk. repeat exposure to allogeneic rbcs can result in the development of rbc alloantibodies which can make it difficult to find compatible rbcs for future transfusions. however, the main concern of alloimmunization is the development of hemolytic transfusion reaction, with in the most severe cases, hyperhemolysis, leading to multi organ failure and death in 4% of the cases. the prevention of this life threatening condition must be based on risk factors. however, although some risk factors, such as alloimmunization, have been identified, much of the mechanism underlying dhtr remains a mystery, particularly in severe cases presenting hyperhemolysis. here we will describe the current and future development to prevent and treat this severe syndrome in order to decrease exposure to transfusion in scd but also improve red blood cell quality, some new products are developed. oxidative damage is one of the parameter that could be diminished. some work is also ongoing to prevent filter blockage during leucodepletion of precious rbcs units from afro-caribbean donors carrying the sickle cell trait. finally, in countries with higher risks of transmission of infectious disease, treatment of red blood cell units against infectious agents can be discussed. the only current curative treatment of scd is hematopoietic stem cell transplantation (hcs). however, the occurrence, frequency, and effects of immune hematologic complications in hcs remain and will be discussed. finally, gene therapy is a real hope as a definitive curative treatment. clinical trials are ongoing in france and will be discussed as well as the remaining place of transfusion in this therapeutic. in the context of the chronic myeloid leukemia (cml), we have hypothesized that quiescent leukemic hematopoietic stem cells (hsc) compartment, escaping to the current tyrosine kinase inhibitors (tkis) treatment, in part associated in the molecular relapse, may be targeted by cart-cells immunotherapy. gene expression profiling studies have established that a cell surface biomarker il-1rap is expressed by the leukemic but not by the normal cd34 + /cd38-hsc. this talk will focus of the whole process of development of a cart-cells starting from recombinant il-1rap protein mice immunization to produce a specific monoclonal antibody (mab), to the proof of concept demonstration, before moving into the clinic. we produced and selected a specific anti-il-1rap mab (#a3c3 clone, diaclone sa, besanc ßon, france). after molecular characterization of antigen-binding domain, nucleotide sequences were fused with 3rd generation t cell activation coding sequences and cloned as a single chain into a lentiviral backbone comprising a safety switch suicide gene icasp9 (inducible caspase 9) and a monitoring/selection cell surface marker δcd19. we demonstrated in-vitro and in an in-vivo xenograft murine model that il-1rap car t cells can be activated in the presence of il-1rap+ cell lines or primary cml cells, secrete pro-inflammatory cytokines, degranulate and specifically killing them. we also demonstrated that multi-tkis treatment over a 4-year period does not affect transduction efficiency of cml patient t-cells by il-1rap car vector and that autologous cart-cells are able to target il-1rap+ leukemic primary hsc. "off-tumor-on target" toxicity prediction, by studying il-1rap expression on a tissue macroarray comprising 30 normal human tissues (3 donors) , with #a3c3, detected various il-1rap intensity staining in only few tissues. regarding the healthy hematopoietic system, #a3c3 flow cytometry staining did not detect hematopoietic cells, except monocytes that express poorly il-1rap. as expected, monocyte subpopulation is targeted by autologous il-1rap cart cells (ratio e: t = 1:1), but at a lower level that il-1rap cml cell line. in-vivo investigation of specific toxicities of autologous il-1rap cart-cells against hsc and/or immune cells on a human-cd34 + cord blood cell engrafted/nog murine model, but also by an in-vitro cd34 + colony forming unit assay didn't reveal any significant toxicities in immunocompetent cell subpopulations, suggesting that healthy cd34 + hsc are not affected. finally, to overcome potential toxicity, functionality of the icasp9/ rimiducid â safety switch was demonstrated in-vitro but also in-vivo in a nsg tumor xenograft model, showing that, when activate, the system is able to eliminate more than 90% of cart-cells, after exposure to ap1903. in conclusion, based on cml model, we demonstrated that il-1rap is an interesting target for cart-cell immunotherapy, with a limited "on target, off tumor" predictable toxicity. next step will be the up-scaling of the process in order to match with the use in human regarding also the regulatory requirements. this strategy may be applied, in the future, in other hematological malignancies. mortality ranges from 20 to 30% for trauma victims with severe bleeding and is largely dependent on the transfusion therapy from which they can benefit. the nature of this therapy has an impact on prognosis with a halving of mortality when the plasma/prbc ratio is greater than ½ and a decrease of about 20% when the proportion of platelets transfused is close to that of whole blood. the speed with which such therapy is actually administered has a major impact as well with an increase in mortality of 5% for each minute of delay in making the entire therapy available. this can be explained mainly by the fact that the probability of death of these patients is greater within minutes of their admission to hospital with a median time to death of 2 h after admission. to allow plasma, platelets and prbc to be made available in a timely manner, north american trauma centers have mandated that trauma centers have massive transfusion packs at the patient's bedside within 15 min. to further simplify and speed up logistics from distribution to transfusion, several trauma centers now use whole blood stored at 4°c. this return of an "old" product is largely inspired by military experience where whole blood is mainly used "warm" immediately after collection with compelling evidence of its effectiveness. its return to civilian practice requires the ability to deleukocyte it while preserving platelets and to store it while maintaining their hemostatic functions. good quality data shows this is achievable and several clinical studies are planned to begin in the coming months. in france, the french blood establishment and the french army are cooperating to initiate the prospective randomized non-inferiority storhm trial (sang total pour la r eanimation des h emorragies massives) which will be comparing whole blood to separate blood components in an 1/1/1 ratio in severely bleeding trauma patients. the primary endpoint will be a thromboelastographic parameter (maximum amplitude) assessed at the 6th hour after admission. secondary endpoints will include early and overall mortality, lactate clearance (reflection of the effectiveness of resuscitation) and 24 h organ failure. this trial will be recruiting 200 patients in 6 french trauma centers and is planned to be initiated second half of 2019. local/neighbours day: innovation in germany 1c-03-01 mesenchymal stromal cells for regenerative therapy bm-msc / asc obtained from these protocols have been characterized in detail in preclinical evaluations. manufacturing licenses for msc and asc and a platelet-derived growth factor concentrate have been obtained and they have been explored in several clinical trials for treatment of bone defects (ortho-ct1: eudract number: 2011-005441-13; ortho-ct2: eudract number: 2012-002010-39; maxillo1: eudract number: 2012-003139-50) . we will summarize results of completed clinical trials which confirmed feasibility and safety of autologous msc /asc treatment and provided evidence for efficacy (gjerde et al, stem cell res. 2018 introduction: in vitro produced megakaryocytes (mks) may serve as source to produce platelets (plts) ex vivo or in vivo. we have established a strategy to differentiate mks from induced pluripotent stem cells (ipscs) in bioreactors. this study aimed at the large-scale production of mks using microcarriers to increase the mk yield and to characterize their phenotype and functionality after irradiation as a method to decrease possible safety concerns associated to the ipsc origin. methods: ipscs were cultured in an aggregate form in presence or absence of microcarriers using 50 ml stirred flasks. cells were differentiated into mks using tpo, scf and il-3 in apel2 medium for a period of 22 days. non-irradiated or irradiated ipsc-derived mks were analysed for polyploidy, phenotype and proplt production using flow cytometry and fluorescence microscopy. also, plt-production was investigated in vivo. non-irradiated or irradiated mks were transfused to nod/ scid/il-2rcc-/-mice and blood was analyzed for human plts. results: differentiation of mks in presence of microcarriers resulted in an 8-fold increase of mks per ipsc in comparison to only aggregates. this resulted in mean of total mk harvest of 18.7 ae 6.8 9 10 7 in microcarrier-assisted bioreactors in comparison to 4.9 ae 1.3 9 10 7 mks collected from bioreactors containing only aggregates. interestingly, mks produced in microcarrier-assisted bioreactors showed higher proplt formation capacity than mks derived from only aggregates bioreactors. mk phenotype and dna content was comparable between mks derived from both types of bioreactors. irradiation of mks did not affect their phenotype and capability to form proplts or plts after transfusion into nod/scid/il-2rcc -/mice. conclusion: microcarriers showed to significantly increase the yield of ipsc-derived mks in stirred bioreactors to clinically relevant numbers. this may facilitate the use of ipsc-derived mk for ex vivo production of plts, direct transfusion or for innovative mk-based regenerative therapies. although the rosetta stone, found by the troops of napoleon in egypt near the city of rosetta (rashid) contains only a small amount of text in three languages it was key in deciphering hieroglyphs. the rosetta mission tried to achieve something similar: by looking at a tiny body its goal was to decipher the origin of the solar system, planets including earth and life. after more than 12 years the rosetta spacecraft softly crashlanded on comet churyumov-gerasimenko on september 30, 2016 . it has travelled billions of kilometers, just to study a small (4 km diameter), black boulder named 67p/ churyumov-gerasimenko. the results of this mission now seem to fully justify the time and money spent in the last decades on this endeavor. where are we from? where are we going? are we alone in the universe? these are some of the big questions. in this talk i will show which answers we got from rosetta and comet chury. we follow the pathway of the material which makes up our solar system from a dark cloud to the solar nebula and finally to planets and life. i will show that indeed we are the result of stardust and that what happened here may happen elsewhere in the universe. cells, tissues and entire organs can collectively be seen as "living drugs". genetically unaltered cells are routinely used in clinical practice to treat diseases as diverse as anemia, bleeding disorders, leukemia and organ failure. ground-breaking advances in genetic and genome engineering technologies are propelling cell therapies to the frontline of medical research and practice. the hematopoietic system is particularly amenable to genetic engineering because specific cell types can be purified based on the expression of specific surface proteins and the ability to culture and expand cells ex vivo. the recent unprecedented clinical success of killer t cells reprogrammed by chimeric antigen receptors (cars) to attack cd19 expressing tumor cells demonstrates the power of immunotherapy with genetically engineered immune cells. however, given the rapid development of novel genome engineering and synthetic biology tools we are likely only at the beginning of a new era of engineered cellular therapies. i will present recent progress in immune cell reprogramming, gene correction, safety aspects and remaining challenges such as manufacturing. 1d-04-03 cell free nucleic acids (cfna) circulate in the plasma of all individuals and are thought to be released by host and foreign cells into the circulation. after fractionation by centrifugation, cfnas can be extracted from the supernatant of whole blood samples or manufactured blood products. these dna or rna sequences can be of human, bacterial, viral or fungal origin. most of them are human double stranded dnas. research on cfnas is increasing, thanks to technological advancements in molecular biology. some of their results are already implemented in clinical practice in the areas of prenatal diagnosis, oncology and infectious diseases. the latter investigation focuses on the exploration of non-human cfnas, the field of metagenomics. high throughput sequencing associated with bioinformatics, the so-called new generation sequencing (ngs), has sped up the investigations of non-human cfnas. this tool provides the opportunity to classify cfnas into a human or non-human category, and then to identify them. it is thus possible to explore simultaneously the whole landscape of bacterial, viral and fungal populations. presently, ngs of human blood has already proven its feasibility and its value in identifying emerging viruses or investigating clinical cases of fever of unknown etiology. ngs of cfnas is also particularly effective in analyzing the different genotypes of a virus in case of a co-infection (e.g. hepatitis c virus). studying cfnas with the new molecular technologies is therefore of great importance in transfusion medicine, especially regarding security and clinical transfusion reactions. first, transfusion transmitted infections are the most feared adverse complications. second, febrile non-hemolytic transfusion reactions are also the most frequently reported adverse events in hemovigilance systems and their physiological mechanism -if only one-remains not clearly elucidated. investigating cfnas could thus improve our understanding and strategy aiming at reducing those two clinical adverse events. surveying comprehensively the composition of circulating infectious agents in a blood product by ngs technology could be very interesting for investigating a severe febrile transfusion reaction. moreover, when the costs of analysis will be reduced, it might be possible to screen prospectively and regularly the whole metagenomics of asymptomatic blood donors, in addition to the classical epidemiological surveillance. for instance, in a study testing a ngs method on manufactured fresh frozen plasmas, an astrovirus (mlb2) has been identified. finally, it is the responsibility of transfusion physicians implicated in the manufacturing of blood products to ensure that cfnas within a blood product do not have a clinical impact on the innate immunity of the recipients. according to recent research in vitro, cfnas purified from blood products can induce the transcription of inflammatory cytokines by mononuclear cells. as nonhuman cfna have an effect on toll-like receptors (tlr-linked inflammatory pathways), it would be also relevant to insure that donor's cfnas have no significant effect on the immune system of the recipient. in conclusion, cfnas are very diverse molecules contaminating blood products. technological progress makes now their investigation more available. besides being useful markers of infection in asymptomatic donors, their impact on the recipients' immunity should be further investigated. an active life and regular training is part of a healthy life style and for many this includes participation in endurance exercise competition at different levels. thus, it is highly relevant to know how a blood donation affects exercise performance and how close this can done to an endurance competition. endurance exercise performance is determined by many factors, but three of the primary are maximal oxygen uptake, the relative load that an individual can sustain over time and finally the efficiency of movement in the given discipline. over the years, a number of studies have sampled blood volumes ranging from 450-1200 ml and applied different methodological approaches to measure maximal oxygen uptake over a recovery period ranging between 3-28 days. overall, the general finding is a reduction in blood haemoglobin and an attenuated maximal oxygen uptake as well endurance performance after blood donation. in normal to well-trained men maximal oxygen uptake and performance was normalized after two weeks in one study after a normal blood donation (450/470 ml), but remained attenuated after four weeks in another study, despite the change in blood haemoglobin concentration was similar and the design and methodology also similar in the two studies. in addition to maximal oxygen uptake the relative load that can be tolerated during exercise is probably also attenuated, through a decreased arterio-venous oxygen extraction, but the available data is very limited. the first part of this talk will highlight the major findings and discuss some of the methodological issues that complicate interpretation and conclusions. there are sex differences in circulating blood volume, haemoglobin concentration, haematocrit and hormone levels and thus it is entirely possible that there is a sex difference in the effect of blood donation on physical performance and the recovery after blood donation. in addition to the basic physiological sex differences, there is also a higher prevalence of iron deficiency in premenopausal women, physically active women and women donating blood. therefore, we studied the influence of a standard 450 ml blood donation on maximal oxygen uptake and endurance performance and the subsequent recovery in physically active women. we observed that in iron sufficient women blood haemoglobin concentration and maximal oxygen uptake were back to baseline 28 days after blood donation, but endurance performance was normalized already after 14 days. the second part of this talk will discuss the sex differences in the effect of blood donation on maximal oxygen uptake and endurance performance. overall, the available data suggest that, with a careful conservative approach, 4 -5 weeks are needed after a normal blood donation to be fully recovered to participate in endurance exercise competitions. more than one in ten attempts to donate blood result in a temporary deferral, due to concerns about the impact of the donation on the donor or recipient. there is well established evidence that temporary deferrals impact negatively on donors, with a large proportion of those deferred failing to return at the end of the deferral period. this presentation provides an overview of deferrals from the donor perspective, describing the likelihood of receiving a deferral for different donor subgroups. the impact of temporary deferrals on the future donation of donors, considering both short-term and longer-term donation patterns, will also be reviewed, outlining which donors are at highest risk of non-return following a deferral, and what is known about the accumulative impact of multiple deferrals on donors. several hypotheses have been proposed to account for the strong negative impact of deferrals on donor behaviour, and there is preliminary evidence of psychological factors, such as emotional reactions, predicting intention to return. research is also beginning to emerge on the effectiveness of tailored interventions to mitigate the impact of deferrals on donor behaviour. the evidence for these preventative interventions, and for strategies to reactive donors once they have lapsed post-deferral, will be reviewed. recommendations for blood centres will be made, as well as suggestions for future research to address continuing gaps in knowledge. in his influential study "the gift relationship" (1970) , richard titmuss coined the idea of voluntary, non-paid, blood donation being the gift of life for a fellow citizen. this metaphor has been powerful in mobilizing donors (busby 2004) . it conveys a direct relationship between blood donation and patients' vitality, as well as a difference between gains and costs. as the gift of life, blood donation is seen to symbolize pure altruism and promoting solidarity between strangers. but can we apply the metaphor as successfully into donating blood for research? we asked a group of finnish blood donors what they would think if the frc blood service invited them to give a blood sample and personal information for research. the blood donors were usually willing to contribute to research for the public benefit, because they saw great potential in science to create solutions to help patients in the future. however, based on our interview data and previous research, we suggest that the analogy between gift of life and donation for research did not work all the way. the metaphor fails to address donors' questions on new types of relationships, interests and risks related to the use of personal data for research. left unanswered these could discourage donating for research. hence, we argue that the gift of life metaphor is not applicable to donor recruitment at the research context. in this presentation we wish to look for a better metaphor for donation for research that blood services collecting research data could apply. academy day: transfusion challenges in patients with sickle cell disease 2a-02-01 immunohaematological features of patients with sickle cell disease (lfa) should be considered as polymorphic antigens in the african population and these lfa are not present in most commercial panels. the situation is even more complicated when recipients lack high-frequency antigens, the most common ones being hr-, hr b -, sec-, u neg , u+ var , js(b-), (hy-), and jo(a-). finally, there is a high rh diversity among people of african descent. because they harbor variant alleles and/or partial rh antigens, they are at risk of developing alloantibodies. in this setting, screening for partial rh antigens makes sense. the figures illustrating this diversity vary with the approach used. one of them is to take into consideration rhd or rhce*ce variant alleles. in several american studies, their prevalence was estimated to be 29-36% and 53-72%, respectively. other teams take into consideration d, c and e partial antigens. their prevalence was estimated to be 8.4-14%, 12.5-27.7%, 3.3-3.5%, respectively, and the alloimmunization rates were 17.6%, 14.3-30%, 7.1%, respectively. as a result of these phenotype discrepancies, scd patients are more likely to be alloimmunized. an overall immunization rate of 2-6% is commonly admitted in the general population. depending on the unit selection policy and/or the study design, the immunization rate in scd patients varies from 7% to 76%, the highest figures being established when an abo/rh1-only matching policy is implemented. in a meta-analysis of 24 publications, the overall alloimmunization rates were around 20%. alloimmunization is thought to be enhanced by an inflammatory state, which is often present in scd patients. they are more prone to develop a new alloantibody. using a stochastic modeling of alloimmunization, they have a 61% increased risk of producing additional antibodies versus 30% in the general population. autoantibodies have been identified as a risk factor of alloimmunization. as a result, scd patients often have complex mixtures of allo and autoantibodies. rh antibodies and those considered as irregular natural antibodies are present in a significant proportion. another characteristic of the antibodies in scd patients is their evanescence; up to 70% of alloantibodies become undetectable within a few years of their initial development. relatedly, about a third of dhtrs are reported to happen in patients with no previous history of immunization. in addition, a third of patients will not develop an antibody after a dhtr. identifying patients at risk of developing a dhtr is key to managing them properly. alloimmunization is a serious risk of red blood cell transfusion in patients with sickle cell disease (scd) and can result in severe (delayed) haemolytic transfusion reactions, exacerbation of clinical symptoms and life-threatening hyperhaemolysis. once alloimmunized, the presence of alloantibodies in the patients' blood further complicates pretransfusion testing and hampers the selection of compatible blood products. numerous studies have shown that scd patients have a relatively high risk of alloimmunization as compared to the 'general' population. this is not only explained by the large number of transfusions given but also by the increased exposure to foreign antigens as a result of differences in the antigen make-up of the scd recipients and the blood donor population. other factors involved in the immune response such as age at first transfusion, inflammatory state, hla typing are under investigation and are starting to unravel. because blood transfusion is still one of the main treatment modalities for scd and some patients have a life-long transfusion dependency it is important to minimize the alloimmunization rate. theoretically, complete matching for all relevant blood group antigens would prevent alloimmunization. this however, is only possible when all donors are comprehensively. matching strategies should be developed to minimize alloimmunization while balancing patients' need and donor availability and is cost effective. to develop a (preventive) matching strategy some factors need to be established; 1) which antibody specificities are clinically relevant 2) which antigens are most immunogenic 3) what is the availability of specific antigen typings in the donor population 4) how should recipients (and donors) be typed, phenotypically and/or genotypically and to what extent. the latter is especially important in scd patients since they are of african descent and the prevalence of genetic variations in this population is relatively high. rhd and rhce variants are common and can remain undetected when serological typing is used but can be discovered with high resolution molecular typing. patients with partial rh phenotypes are at risk for alloimmunization. apart from special rh phenotypes in individuals from african descent, the fy(a-b-) phenotype related to the gata-box mutation in the fyb allele and the u-or u var phenotype resulting from genetic variations in the mns alleles are also common. several studies have shown that in scd patients antibodies directed against rhd, rhe, rhc and k are most frequently found when unmatched transfusions are given. preventive matching for these antigens has proven successful in reducing alloimmunisation. extended matching for all rh antigens fy(a), jk(a) and jk(b) can further decrease the alloimmunisation rate. currently, different countries have preventive matching strategies in place for this vulnerable patient group. as genotyping is more and more available and within reach, optimal antigen typing approaches for patients and donors, combining serology and genetics are being developed. in this lecture several aspects of antigen typing approaches and preventive matching strategies that will most benefit scd patients of will be discussed. artificial intelligence has become a buzzword that will appear about anywhere in the media. we can forget that ai, or the subfield in this computer science field machine learning, has been around for over 50 years. improvements in computing power, abundance of data, progress in computer science, and the arrival of affordable cloud solutions have now brought it to our daily lives. also in health care news about ai has become omnipresent. and some landmark papers have come out on algorithms outperforming (teams of) physicians in the diagnosis of all kinds of skin disease, eye disease from retina scans, and detect cancer in ct scans. however, little of these solutions have actually shown up in our clinical practice yet. in anaesthesia, we worked with the first algorithm to come to anaesthesia practice; hypotension during surgery is associated strongly with poor outcome like myocardial ischemia, surgical complications, renal failure and even mortality. we worked on a machine-learning trained algorithm that predicts hypotensive events using the arterial blood pressure curve up to 15-30 min before the actual event. to get fda and ce approval, however, mere mathematic validation is required. this can be achieved on retrospective datasets. in reality, we need more before we can use these algorithms to support our decision-making; after internal (retrospective) and external (prospective but passive use) validation steps, clinical (i.e. rct validation is needed. moreover, we will need to assess the economic impact too. ultimately this tool has now reached clinical practice and is starting to help us go from reactive to more proactive hemodynamic management. like this, we have started to work on machine-learning tools to predict the incidence of specific types of patients coming into a&e and predicting infections after surgery. we will discuss our approach, essentials to start with machine learning, practical learnings. we will also discuss a first project design to use machine learning in managing bleeding patients to get the best therapy advice for blood product use like plasma, fibrinogen et cetera. how can we start using this tool in unison with our existing tools to improve science and clinical practice in our respective (bio)medical fields? thrombocytopenia is a very common hematological abnormality found in newborns, especially in preterm neonates. two subgroups can be distinguished: early thrombocytopenia, occurring within the first 72 h of life, and late thrombocytopenia, occurring after the first 72 h of life. early thrombocytopenia is associated with intrauterine growth restriction, whereas late thrombocytopenia is caused mainly by sepsis and necrotizing enterocolitis. platelet transfusions are the hallmark of the treatment of neonatal thrombocytopenia. most of these transfusions are prophylactic, which means they are given in the absence of bleeding. however, the efficacy of these transfusions in preventing bleeding has never been proven. in addition, risks of platelet transfusion seem to be more pronounced in preterm neonates. because of lack of data, platelet transfusion guidelines differ widely between countries. in a recent randomized controlled trial (planet-2/matisse study) among preterm infants with severe thrombocytopenia, we found that those randomly assigned to receive platelet transfusions at a platelet-count threshold of 50 9 10 9 /l had a significantly higher rate of death or major bleeding within 28 days after randomization than those who received platelet transfusions at a platelet-count threshold of 25 9 10 9 /l. this presentation summarizes the current understanding of etiology and management of neonatal thrombocytopenia. transfusion-associated circulatory overload (taco) is a severe transfusion adverse reaction that is associated with increased mortality and morbidity. the incidence of taco in adults varies from 1% to 8%, but is probably underdiagnosed and underreported. the incidence in the pediatric population is undetermined. taco usually occurs in patients who receive a large volume of blood product over a short period of time. it is more common in patients with known risk factors such as cardiovascular disease, renal failure, and older or younger age (> 60 years or < 3 years). hospitalised patients and intensive care patients are also more at risk. the typical presentation of taco is respiratory distress (dyspnea, tachypnea) occurring within 12 h of a blood transfusion. associated signs and symptoms are hypoxia, hypertension, tachycardia, positive fluid balance, high central venous pressure, and acute or worsening pulmonary edema on chest x-ray. echocardiography and measurement of brain natriuretic peptide (bnp) or its n-terminal prohormone (nt-probnp) is helpful for diagnosis. several definition criteria have been proposed for taco, but none are adapted for children, particularly critically-ill children who are more at risk. this is probably the main reason why taco is even more underdiagnosed and underreported in the pediatric population. in a recent study, we compared the incidence of taco in a pediatric intensive care unit using the international society of blood transfusion (isbt) criteria, with two different ways of defining abnormal values: 1) using normal pediatric values published in the nelson textbook of pediatrics; and 2) using patients as their own controls and comparing pre-and post-transfusion values with either a 10% or 20% difference threshold. we monitored for taco up to 24 h post-transfusion. a total of 136 patients were included. taco incidence varied from 1.5% to 76%, depending on the definition used. with such wide variability, we conclude that a more operational definition of taco is needed in pediatrics, particularly for critically-ill children. differential diagnosis from other dyspnea-associated transfusion adverse reactions (e.g. transfusion-associated lung injury, anaphylaxis) is important because treatment differs, as do guidelines to the blood bank. treatment for taco is similar to that of any other cardiogenic pulmonary edema: oxygen, diuresis, ventilatory support. prevention is possible by avoiding unnecessary transfusions, transfusing only the necessary amount of blood product, avoiding rapid transfusions, and using diuretics. background: the risk and importance of transfusion-transmitted hepatitis e virus (tt-hev) infections by contaminated blood is currently a controversial discussed topic in transfusion medicine. in particular, the infectious dose is a not finally determined quantity. the different countries have chosen different approaches to deal with this pathogen. one central question is the need of individual nat screening (id) versus minipool nat screening (mp) approaches to identify all relevant viremias in blood donors. aims: comparison and evaluation of the available screening strategies in relation to the infectious dose to minimize the risk of tt-hev infections. methods: we systematically reviewed the presently known cases of tt-hev infections and available routine nat-screening assays. furthermore, blood donation screening strategies for hev ehev in effect in the european union were compared. we also describe our own experiences of hev screening utilising an id-nat-based donor screening algorithm compared to mp-nat in pools of 96 samples. from november 2017 to january 2018, a total of 10,141 blood donations were screened for the presence of hev rna using a mp-nat (in house, realstar hev rt-pcr kit) and an id-nat (cobas 6800 platform). results: the review of the literature revealed a significant variation regarding the infectious dose causing hepatitis e. in the systematic case review, all components with a viral load (vl) greater than 5.00e+04 iu caused infection (definitive infectious dose (difd) . the lowest infectious dose resulting in tt-hev infection observed in general was 7.05e+03 iu (minimal infectious dose (mifd). the infectious dose of the different blood products is mainly influenced by the remaining plasma content. our data comparing the two different hev screening algorithms revealed eight hev rna positive donations using a mp-nat (incidence 1:1,268) , whereas 17 hev rna positive donations were identified by id-nat (incidence 1:597); all id-nat only positive donations had vl < 25 iu/ml. summary/conclusions: taken into account the current knowledge on the required mifd, the difd, and the analytical sensitivities of the screening methods, we extrapolated the detection probability of hev-rna positive blood donors using different test strategies (nat assay, id vs. minipool with different pool sizes). we also considered the amount of plasma in the different blood products and calculated the infectious doses needed to be detected. only id testing would be sufficient to detect the minimum vl in the donor to avoid tt-hev infections based on the currently known mifd, but a highly sensitive mp-nat should be adequate as a routine screening assay to identify high viremic donors and avoid tt-hev infections based on the difd. we have also determined that the incidence of hev infection was approximately 50 % higher if id-nat was used. however, vl were below 25 iu/ml and will most likely not result in tt-hev infection taken into account the currently known mifd or difd. the clinical relevance of and need of identification of these low level hev positive donors still require further investigation. in the last 25 years several pathogen inactivation (pi) technologies have been developed to be applied to blood components. technologies for inactivating pathogens in plasma and platelets are available in the european union, and some others are currently under development. the first pi technology introduced in the market was for plasma, and was based on the addition of methylene blue and the illumination with light (theraflex mb-plasma, macopharma and gr ıfols). for platelets and plasma two technologies are licensed, one is based on the addition of amotosalen and the illumination with ultraviolet light (uv) (intercept â , cerus) and the other one combines the addition of riboflavin (vitamin b 2 ) and the illumination with uv light (mirasol â , terumo bct). currently another technology for platelet inactivation, based on the illumination with uvc light and strong agitation is under development (theraflex, macopharma). for red blood cells one technology based on the addition of one molecule (amustaline, cerus) is being developed. the mechanism of action, and the spectrum and level of inactivation of pathogens varies among the different technologies. in addition, the number of studies with clinically relevant endpoints and the number of patients included in the studies is not homogeneous. there is published evidence for most of them that show that the treated blood components are safe and efficacious for the patients although, for treated platelet concentrates some decrease in the posttransfusion recovery and survival of the transfused platelets occur, with differences between the different technologies. however, cumulative experience on the use in routine, for some of the technologies for almost 20 years, support the concept of the safety and efficacy of the blood components treated with pathogen inactivation technologies without a significant increase in utilization. the use of pathogen inactivation for blood components is not widespread. differences in epidemiology between countries, infectious risk perception, concerns about potential adverse effects associated with its use and economical considerations might explain the differences observed in its implementation. the history of the p1 and p k antigens is complicated and sometimes confusing because of several changes to the nomenclature. the association between the antigens and their genetic home has raised many questions as well as the longstanding enigma regarding the molecular mechanism underlying the common p 1 and p 2 phenotypes. the system (isbt no. 003) currently includes three different antigens, p1, p k and nor. the p1 antigen was discovered already in 1927 by landsteiner and levine while p k and nor were described in 1951 and 1982, respectively. as for the abo system, naturally-occurring antibodies of igm and/or igg classes can be formed against the missing p1/p k carbohydrate structures. anti-p1 is usually a weak and cold-reactive antibody very rarely implicated in hemolytic transfusion reaction (htr) or hemolytic disease of the fetus and newborn (hdfn). however, some antibodies against p1 have been reported to react at 37°c, bind complement and cause both immediate and delayed htrs. the p k antibodies can cause htr and anti-nor is regarded as a polyagglutinin with unknown clinical significance. a higher frequency of miscarriage is seen in women with the rare phenotypes p and p 1 k /p 2 k . the rbc of the fetus as well as of the newborn express low amounts of p1, p and p k antigens but the placenta shows high expression and is consequently a possible target of the antibodies and the cause of the miscarriages. the p k and p1 antigens have wide tissue distributions and can act as host receptors for various pathogens and toxins. furthermore, altered expression of p k antigen has been described in several cancer forms. a longstanding question has been why individuals with p phenotype not only lack p k and p expression but also p1. recently it was clarified that the same a4galtencoded galactosyltransferase synthesizes both the p1, p k and nor antigens and in addition the p 1 and p 2 phenotypes was confirmed to be caused by transcriptional regulation. transcription factors bind selectively to the p 1 allele in the 5'-regulatory region of a4galt, which enhance transcription of the gene. it has been debated whether the p k and p1 antigens exist on glycoproteins in the human rbc membrane or if glycolipids are the only membrane components carrying these epitopes. a recent publication shows that the p1 antigen can be detected on human rbc glycoproteins and thus glycosphingolipids can no longer be considered as the sole carriers of the antigens. the blood group system which started out with one antigen, p1, has now gained two more members namely p k and nor. step by step the biochemical and genetic basis underlying the antigens expressed in this system has been revealed but still many questions remain to be solved. neither gata1 nor klf1 represent a blood group system but mutations in the genes encoding these transcription factors (tfs) have been shown to result in simultaneous altered expression of blood group antigens in certain rare blood group phenotypes. in particular, mutations in the klf1 gene are responsible for the dominantly inherited in(lu) phenotype, commonly referred to as lu(a-b-) because of the gross reduction in lutheran antigens expression. red cells from in(lu) individuals, though, have also weakened expression of other blood group antigens, like the high-incidence antigen anwj, the antigens of the indian blood group system (cd44) and p1, among others. since the first description of klf1 variants associated with the in(lu) phenotype, many other variants of this gene have been reported with an impact on blood group antigen expression and they are listed on the klf1 table of blood group alleles. other than klf1, a mutated gata1 gene has also been found associated with the x-linked form of the lutheran-mod phenotype and has likewise been registered in the gata1 allele table. besides the effect of tf variants on blood group antigen expression, there are transcription factor-binding site polymorphisms in regulatory regions of blood group genes, which also have an impact on the expression of the encoded antigens in red cells. the first example of such type of polymorphisms was described in 1995, when the disruption of a gata motif in the ackr1 gene promoter was found to abolish erythroid gene expression in fy(a-b-) individuals of african descent. the impact of mutations affecting gata1 binding sites has also been described in some abo subgroups, like the am and bm phenotypes. a regulatory element with gata binding sites in the first intron of the abo gene has been found to be altered in individuals with these phenotypes, either by deletion or by a point mutation disrupting the gata motif. recent findings have also revealed that xga expression on red cells is dependent on gata1 binding to a control element located 3.7 kb upstream of the xg gene. a single nucleotide polymorphism (snp) within this region was shown to correlate very well with the expected distribution of the xga negative phenotype in different populations. further work has demonstrated that this g>c snp disrupts a gata1 binding site and consequently abolishes erythrocyte xg expression. overall, these investigations have allowed to elucidate the underlying genetic basis for xga expression and have made xga genotyping possible. similar to xga, the p1 antigen has been known for a long time to be determined by the a4galt gene but the molecular basis underlying the common p1/p2 phenotypes has remained elusive till recently. several cis-regulatory snps had been identified in non-coding sequences around exon 2a, which showed a very good correlation with p1 antigen expression. interestingly, potential binding sites for several hematopoietic tfs were identified in the same region. finally, recent investigations have demonstrated the role of the runx1 tf in the expression of p1 antigen, by selective binding to a regulatory site present in p1 but not in p2 alleles. to summarize, variation in blood group antigen expression may result from mutations or polymorphisms in the regulatory region of blood group genes. recent reports have unravelled the molecular mechanisms underlying the expression of p1 and xga blood group antigens, which involves tf binding to allele-specific regulatory elements. similar mechanisms may also regulate antigen expression in other blood group systems. 2c-06-03 clinical immunology, copenhagen university hospital, copenhagen, denmark since the discovery of cell-free fetal dna (cffdna) in pregnant women's blood, the development of noninvasive prenatal testing (nipt) has provided new diagnostic applications in prenatal care. in transfusion medicine and clinical immunology, cffdna is extracted from maternal plasma to predict fetal blood groups with the purpose of 1) guiding targeted rh prophylaxis in non-immunized rhd negative women and 2) assessing the risk of hemolytic disease of the fetus and newborn (hdfn) in immunized women. i will give an overview of noninvasive prenatal testing of fetal blood groups. based on the literature, i will summarize the current experience with noninvasive prenatal testing of fetal rhd and other blood groups. for rhd negative pregnant women, routine clinical testing is available in several countries world-wide to assess the risk of hdfn in d immunized women, and routine testing to guide rh prophylaxis is now implemented as nationwide service in 6-7 european countries. noninvasive prenatal testing for fetal rhd is highly accurate with sensitivities of 99.9%, as reported from clinical programs. in general, the sensitivity is challenged be low quantities of cffdna, especially in early pregnancy. the specificity is challenged by the polymorphic rh blood group system, where careful attention is needed to navigate among the many rhd variants. rhd variants may complicate cffdna analysis and interpretation of results, especially in populations with mixed ethnicities. despite these challenges, fetal rhd testing is very feasible when implemented with careful attention to these issues. for blood groups that are determined by snps, such as kel or rhc, the main challenge has been interference from the maternal dna when analyzing the fetal dna which has resulted in low accuracy and lower sensitivity, when using qpcr. with the application of more novel techniques such as next generation sequencing and droplet digital pcr, accurate noninvasive prediction of these fetal blood groups has been demonstrated. the success of predicting fetal rhd and its successful clinical implementation into national programs should encourage wide-spread use of cell-free dna based analysis. future work on noninvasive prenatal testing of fetal blood groups determined by snps may consolidate the application for cell-free dna testing for such targets, including human platelet antigens. at isbt, the newly formed cfdna subgroup of the red cell immunogenetics and blood group terminology working party will work to facilitate clinical applications, implementation and evaluation of cell-free dna testing. blood banks in most of the nordic countries all share a vein-to-vein approach which in short means that the collection of blood, the preparation of blood components, testing/release and storing is served by a single actor. on top of that recipient and donor blood grouping, crossmatching, delivery, registration of transfusion and of any complications is usually handled in a single blood banking information system (bbis). this means that blood banks in the nordics are traditionally operated by a single vendor. the needs for process control in a single vendor bbis, the present solutions, unsolved challenges and untapped possibilities of streamlining processes have been scrutinized with the intention to describe separate processes and to acquire best of breed or best of suite it-systems. the aim for integrations, rather than building an integrated it-system, to support the need for a vein-to-vein process is a precondition in the nordic countries. with multiple it-systems supporting isolated processes, we intend to facilitate development in these and furthermore increase the flexibility in the whole process. we set out to reveal any existing knowledge in the literature on it vendor strategies for blood banks, but we didn't succeed in identifying any relevant literature. however, a systematic literature study on vendor strategies when choosing health it was based on the prisma method, and identified 837 studies, but only 10 was eligible for full text review and 5 met the inclusion criteria. even this broader literature study reveals very little evidence. two studies find single vendor strategies poor and conclude "best of suite" solutions to be optimal. one study was not able to correlate vendor strategies to the investigated productivity, but concludes that best of suite and best of breed strategies requires larger organizational changes than a single vendor strategy. in summary, the existing research is contradictory. this paper adds basic knowledge for breaking down the process control of blood banking in smaller processes. this adds the possibility for identifying best of breed or best of suite vendors, instead of relying on single vendor it solutions. furthermore it is a call for more research in the field of vendor selection strategies which this study didn't succeed in identifying. 2d-07-02 applying drones to supply blood to remote areas: rwanda's experience biomedical services, rwanda-ministry of health-national blood services, kigali, rwanda background: in rwanda, blood transfusion services started in 1976. during the 1994 genocide against the tutsis almost all the socioeconomic fabric of rwanda was destroyed as well as its health infrastructure. the healthcare system was suffering in its aftermath, and there were health inequalities between urban and rural areas, including access to blood for transfusion. from 1995, the government started to rebuild all courses of life including the health system and the blood service in particular. the national center for blood transfusion (ncbt) was then mandated to provide safe, effective and adequate blood and blood components to all patients in need. this was pivotal in achieving health related mdgs 4, 5 and 6. today, rwanda has an ambitious vision to put all 12 million citizens within 30 minutes of any essential medical product. while every second matters in emergency management, the use of drones was the perfect solution to many of the last mile challenges that have been traditionally difficult to overcome. it is impossible to forecast accurately down to the need of a single patient. the government has provided an easy solution by centralizing supply and providing on-demand, emergency medical deliveries by drone. doctors are now empowered to provide the quality care with all the supplies on hand, patients can now be treated close to home, and we eliminate waste from potential overstocking when health workers know that they have a quick and reliable source of supply. description: in 2016, the government of rwanda started to operate the world's first national drone delivery program for blood and other lifesaving medical products. these drones can carry two to six units of blood at a time and deliver in 15 -45 minutes depending on a hospital's location. the average duration was between 2 -4 hours round trip with the vehicle system, before the use of drones. drones currently deliver blood to 25 health facilities throughout the country and are set to reach 90% of transfusing health facilities outside kigali by the end of the year. within the first year, healthcare workers saved an average of 3.1 hours per delivery and a total of 10,115 hours of lost time on road pick up they could instead dedicate to patient care. by march 2019, over 11,000 deliveries have been made, with 30% of those being emergency deliveries. a total of more than 19,500 blood units have been delivered. in february 2018, zipline obtained the highest rating from the health facilities being served in a performance evaluation conducted by the national center for blood transfusion. when a doctor or medical staffer needs blood, they place an order through the hemovigilance order portal. they are then sent a confirmation message saying a drone is on its way. the drone flies to the health facility at up to 100 km/h. when it is within five minutes of the destination, the medical staffer receives a notification. the drone then drops the package, attached to a parachute, into a special drop zone. conclusion: supply is not a developing country problem, it is a global issue. rwanda was just the first one to recognize the potential of this technology and decided to do something about it first and fast, to ensure access to universal access to all blood products. 2d-07-03 scottish national blood transfusion service, edinburgh, united kingdom supporting the provision of viable transfusion services in remote/rural areas is more than just a geographical challenge. limited qualified blood bank resource; small throughput volumes; increased regulation are only three of the additional factors combining to threaten safe and sustainable transfusion service delivery. inventory management and out of hours service provision were identified as essential areas where it was thought that technology, in the form of a remotely controlled blood fridge, could provide a key element of the overall solution. a radio frequency identification (rfid) fridge racking system was installed in a standard blood bank fridge and connected to the laboratory information management system (lims) common to both the remote and central blood bank. the central blood bank was enabled to test patient samples from the remote laboratory, identify components located in the remote fridge suitable for the patient and allow correct component issue, even when qualified staff were unavailable in the remote laboratory. testing has concluded that installation of remote fridge management can play a major role in helping to maintain a remote inventory permitting patient compatible components to be issued. by sustaining transfusion services in remote communities we can avoid transportation of patients who require transfusion support to locations miles from home. antibodies to hna-1b, fcgriiib and hna-2 have been reported, too. neonatal alloimmune neutropenia results from maternal antibodies transferred transplacentally to the fetus and is caused by all known hna-antibody specificities, i.e. hna-1a, -1b, -1c, -1d, hna-2, hna-3a, -3b, hna-4a, -4b and hna-5a specificity. hna and hla antibodies can induce mild febrile transfusion reactions and trali. since the introduction of the male only plasma strategy, in many countries the trali incidence decreased but it is still one of the most common causes of severe transfusion reactions. especially hna-3a antibody containing plasma from female donors is responsible for severe or even fatal reactions. but also hna-1a, -1b, hna-2 and hla class i and class ii antibodies were reported. the latter activate monocytes to secrete soluble factors that act on the primed neutrophils in the narrow lung capillaries. laboratory testing: laboratory work-up requires the knowledge of the patient's clinical condition and the methods that are appropriate to detect the relevant antibodies. the classical granulocyte agglutination test (gat) in combination with the granulocyte indirect immunofluorescence test (gift) can detect nearly all relevant antibodies. hna-1, -2, -4, -5 and hla class i antibodies are clearly detectable in the gift while hna-3 antibodies strongly agglutinate neutrophils in the gat. the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) test detects all hnaantibodies except for hna-3 with high glycoprotein specificity and sensitivity but is time consuming and requires highly skilled personnel. for trali diagnostics laboratory testing is completed by methods like the indirect lymphocyte immunofluorescence test (lift) or elisa for hla class i antibodies and hla class ii specific elisas. since several years fluorescent bead based assays (luminex) enable faster and more automated hna antibody detection but to date not all specificities, especially hna-3, can be reliably detected so that still the classical gift and gat have to complete the methodological spectrum. serological typing today is mostly reduced to the determination of hna-2 in the gift because the molecular reason for the hna-2-null phenotype is not completely understood. establishing only one pcr-asp reaction for the main cd177*787a>t polymorphism would comprise the risk to miss other molecular causes. however, for all other hna allelotyping by pcr methods is the first choice. summary/conclusions: granulocyte serology still today is widely based on a variety of manual methods and will be reserved to specialized laboratories as it requires experienced laboratory staff and profound knowledge of granulocyte immunobiology. 2d-08-02 norwegian national unit of platelet immunology, laboratory medicine, university hospital of north norway, tromso, norway maternal alloantibodies against antigens on human platelets can cause severe thrombocytopenia and bleeding in fetus or newborn, identified as fetal/neonatal alloimmune thrombocytopenia (fnait) . although most cases the thrombocytopenia is selfresolving within the two first weeks of life, some infants present bleeding symptoms and thus require platelet transfusion. a set of laboratory analyses are required to confirm the fnait diagnosis. in addition to guiding compatible platelets to the affected newborn, the correct diagnosis will be valuable to assess the risk of fnait in subsequent pregnancies. in addition, platelet alloantibodies may also complicate platelet transfusions by immune-mediated platelet refractoriness, and require proper identification of the patient's antibody specificities prior to selection of donor platelets. the algorithm for laboratory investigations include both serological and molecular assays, and depend on the objective and timing: whether there is an urgent need for platelet transfusion, follow-up of a pregnancy with known risk, or to do full-scale laboratory testing to confirm diagnosis. molecular genotyping should include all hpa systems relevant for the local population (in caucasians hpa-1, -2, -3, -5 and -15), preferably with optional extended panels for systems for low frequency populations due to immigration/mobility and for less frequently seen alloantibodies (hpa-4, -6 to -11 are most commonly included). serological testing of antibody binding to platelets is often initially tested by flow cytometry analysis (direct test and/or cross-match). however, the detection of platelet-specific antibodies is often complicated by the presence of anti-hla class i antibodies and thus require sensitive platelet glycoprotein-specific assays. serological testing for platelet-specific antibodies includes as a minimum panels of antigens on gpiib/iiia, gpib/ix, gpia/iia and cd109 and preferably additional targets for populations with asian/african origin. several methods are available; i.e. bead-based assays and elisa based methods. however, most reference laboratories perform variants of the monoclonal antibody immobilization of platelet antigens assay (maipa), as reported by the 19th international platelet immunology workshop of isbt (2018) . the investigations also include measurement of the anti-hpa-1a by quantitative maipa if present, as this is reported to potentially predict the severity of fnait. for pregnancies with known risk of fnait, there are methods available to perform non-invasive prenatal typing from maternal plasma. the most feasible and so far appropriate for routine testing is fetal hpa-1 typing with quantitative pcr or by melting curve analysis. other sophisticated, yet resource-demanding techniques have also recently been reported -importantly also for typing of other hpa-systems. 2d-08-03 molecular basis of hna-2 expression justus liebig university, institute for clinical immunology and transfusion medicine, giessen, germany human neutrophil antigen 2 (hna-2) is a neutrophil-specific antigen located on gpi-anchored glycoprotein cd177 (also known as nb1). hna-2 is absent on the neutrophil surface of 3-5% of the healthy individuals that divided the population to hna-2 positive and hna-2 null individuals. exposure of hna-2 null individuals to hna-2 positive neutrophils during pregnancy, after transfusion or transplantation, induces immunization against hna-2 and consequently the production of iso-antibodies. the hna-2 iso-antibodies are involved in the mechanism of neonatal alloimmune neutropenia (nain), transfusion-related acute lung injury (trali) and graft failure following bone marrow transplantation. presence of cd177 on a neutrophil surface of hna-2 positive individuals follows a bimodal expression that categorizes the circulating neutrophils to hna-2 positive and negative subsets. the cd177 gene contains 9 exons encoding a protein of 437 amino acids. the lack of hna-2 (in hna-2 null individuals) is associated with the presence of a missense mutation, cd177*c.787a>t in exon 7 of cd177 gene inducing a premature stop codon in codon 263. this mutation alone or in combination with cd177*c.997delg has been introduced as the main reason for the absence of cd177 in hna-2 null individuals. a pseudogene (cd177p1) highly homologous to exons 4-9 of cd177 gene is located downstream of the cd177 gene. conversion of exon 7 of cd177p1 into cd177 gene is responsible for the generation of cd177*c.787a>t missense mutation. in addition, the heterozygosity or homozygosity of cd177*c.787a>t is accounted for regulation of hna-2 negative and positive neutrophils subpopulations. genotyping has revealed the hna-2 null individuals, heterozygous for cd177*c.787a>t mutation without cd177*c.997delg, indicating the presence of a complementary mechanism regulating cd177 expression. newly in hna-2 null individuals and individuals with atypical cd177 expression a cd177*1291g>a polymorphism in combination with cd177*787a>t is described. altogether these data indicate a complex compound mechanism(s) for regulation of cd177 expression on the neutrophil surface. this presentation will summarize recent findings on cd177 expression and highlights the potential genotyping methods for genetic assessment cd177 expression of donors and patients. blood products ordered, transfusion start and end times, whether patients experienced a reaction to the transfusion as well as vitals measurements taken before and during the transfusion, including temperature, oxygen level, blood pressure and heart rate. for the validation process, 100 transfusion nursing notes were sampled and reviewers assessed the accuracy of the information regarding 1) blood product ordered, 2) whether the patient experienced a reaction, and 3) the start and end times of the transfusion. for each of these fields across all 100 sampled notes, the claritynlp tool reproduced these data points with 100 percent accuracy. in addition, the tool supplied transfusion end times for numerous structured records that were missing this key data point. summary/conclusions: claritynlp can very efficiently digest a large number of transfusion nursing notes simultaneously and also does an excellent job of extracting the main characteristics of a transfusion, which can be used in partnership with structured data to produce a more accurate and more complete picture of patient transfusions. immunohematology and genomics, new york blood center, new york, united states antibody-based typing, with a positive result reflected in agglutination of the red cells (rbcs), has served the profession for nearly a century enabling safe and effective transfusion therapy. the power of rbc typing by serologic methods lies in the availability of standardized antibody reagents which target many of the specificities of significance for transfusion, and the ability to directly detect antigen expression on the rbcs. hemagglutination has historically been relatively inexpensive, particularly for abo and rhd as the most important blood groups in most populations. serologic rbc typing is reliable, requires no sophisticated equipment, is generally straightforward to perform, and is fast requiring less than 1 h to results. hence, antibody-based testing has been considered the "gold standard" for blood group typing. with the age of genomics, dna-based genotyping is increasingly being used as an alternative to antibody-based methods. most antigens are associated with single nucleotide changes (snps) in the respective genes. genotyping has been validated by comparison with antibody-based typing and has been shown to be highly correlated. the power of genotyping of rbcs lies in the ability to test for antigens for which there are no serologic reagents, and to type numerous antigens in a single assay using automated dna-arrays. this increases accuracy and weak antigen expression can be revealed. fresh rbc samples are not required for dna extraction, and there is no interference from transfused rbcs or igg bound to the patient's rbcs. dnabased typing is economical in that it provides much more information, but testing requires special equipment, training, and 24-h turn around. what then is the best approach to use? will serologic typing be replaced by dnabased typing? indeed, genotyping will increasingly be used in the practice of transfusion medicine, especially with the growth of whole genome sequencing (wgs). however, because serologic typing for abo and rhd is fast and accurate and often relied on for sample identification, genotyping will not be the sole means for routine typing. genomic sequencing approaches will certainly reveal unrecognized changes and genetic variability in rbc membrane proteins, but not all variation will be immunogenic. a genetic polymorphism must be associated with antibody production to be considered a blood group antigen. the importance of an antibody, and antibody reactivity, then will continue to be the central defining principal in transfusion. as two sides of a coin, both are key to safe and effective transfusion therapy. since the mid-1980s, research in the molecular basis of structural and functional aspects of proteins carrying and producing the antigens has led to an upgraded and modern understanding of blood group variation. most commonly, single nucleotide polymorphism (snp) based basic molecular typing techniques were utilized to test new findings on a smaller subset of samples, and resulted in concordant sero-and genotyping results in general. however, quite commonly a small number of all samples delivered discrepant results, triggering consecutive rounds of analysis and resolution, finally resulting in a better knowledge with respect to the underlying blood group variation. such rounds of repetitions represented the synergistic incremental process of learning, learning for serologists and molecular biologists. inheritance of public, presence of high frequency, or low frequency, or partial antigens and notice of weakly expressed, or almost undetectable antigens marked the path of incremental learning and may best be exemplified by discoveries within the blood group systems abo, rhd and kell. naming for pheno-and genotypes coevolved alongside the permanent discovery of new antigens. at present, antigens and their antithetic counterparts (if tested and if existent), are more commonly reported independently, as exemplarily shown for the following kell phenotype consisting of three antithetic antigen-couples: kk, kp (a+b+), js(a+b+). alternatively, the same phenotype could be stated as: kel:-1,2,3,4, (5),6,7. genotypes, on the other hand, rather mirror the actual biological background, e.g. display the two parental alleles (or "haplotypes") present in an individuals' sample. genotype of the above mentioned example would read: kel*02.03/ 02.06 (italicized). in an idealized diagnostic environment and for most blood group systems encoded by proteins, every single blood group allele would be defined by its full genomic sequence, derived from one parental chromosome (including "some" 5'and 3'-untranslated regions). thereby, every such "ideal allele" would fully declare presence or absence of all its public, low-and high-frequency antigens and possess its "ideal name". by trend, biallelic snps and their immediate relation to antithetic antigen couples might have distracted from the originally intended meaning of "blood group alleles", more recently. finally, genotypes only dependent on (ideal) allele names, and considering mendelian inheritance patterns (dominant, recessive), would allow for fully comprehensive phenotype predictions. more recently, blood group serology, e.g. the "second side of the coin", seems to gain momentum. since the advent of whole genome sequencing and access to many more than 1000 human genomes, it seems that dozens of new blood group alleles are discovered, almost on a daily basis. beside the challenge of naming this multitude of alleles, respective discoveries are frequently made in samples lacking any phenotypic blood group pre-values. clear procedures will be needed to address naming and analyzing the phenotypes resulting from previously unknown alleles. as a consequence, questions asked 30 years ago have changed: today, molecular biologists looking at hundreds of newly discovered blood group alleles find themselves not being asked by serologists any more: "can you confirm my serology?", but instead, pose their question to the experts for the blood group phenotype: "can you confirm my genotype?" 3a-s02-03 background: the screening of blood donors and returning travelers from active transmission areas have highlighted the importance of diagnosis of acute arboviral infections. in the context of co-infections and similar clinical signs in endemic zones, the differential diagnosis of arboviruses is essential to discriminate the causative agent. the detection of viral nucleic acids in serum or plasma provides a definitive diagnosis, however, in most instances, viremia is transient within less than two weeks after the onset of clinical illness. in addition, the cross reactivity due to the high degree of structural and sequence homology between zikv and other flaviviruses is a significant concern. the combination of molecular (identification of viral genomes) and immunological assays (detection of the immune response) is a key challenge to follow the natural history of these infections and to improve the patient management and the epidemiological surveillance. aims: in this context, we have developed an innovative platform based on agglutination of superparamagnetic nanoparticles (npmag) covalently grafted either with nucleic or proteic probes to face the continuing emergence of arboviruses. methods: dengue (denv) and zika (zikv) viruses are selected as models in this study. a pan-flavivirus rt-pcr is used for the molecular assay to amplify the viral genomes. then, biotinylated viral amplicons are captured specifically on complementary original polythiolated probes coated on npmag. for the immunological assay, npmag are grafted with viral ns1 proteins to capture anti-denv or anti-zikv antibodies potentially present in the plasma samples. both tests are performed in disposable cuvettes in a homogeneous format. a magnetic field generated by an electromagnet is applied to the reaction medium to align the npmag into chains to enhance the capture of the targets between two npmag. aggregates formed are detected when the field is turned off. the optical density is measured in real-time at 650 nm during several cycles of magnetization / relaxation. results: in this study, molecular analytical performances were evaluated on human samples from blood donors with no history of infections as negative controls, on viral standards and on clinical samples. using viral references standards, we have observed sensitivities of 10 -10 2 tcid 50 /ml for zikv and denv (serotypes 1/2/3/4) after a detection phase of around 5 min. the first results obtained on 16 zikv (+) clinical samples previously tested by commercial real-time pcr (ct < 36, altona) showed an 88 % correlation between the two detection methods. no false positive results or cross reactions were observed. concerning immunological assays, commercial human plasma from donors tested positive for denv or zikv antibodies were detected positive with our innovative approach in less than 10 min (sampling + detection) instead of 2 h with classical elisas. further assays on clinical samples are planned to confirm these preliminary results. summary/conclusions: this innovative strategy combining molecular and immunoassays on the same analytical platform offers new opportunities for rapid blood testing to improve the surveillance and the prevention of arboviral infections. background: zika virus (zikv) caused a dramatic epidemic in puerto rico (pr) during 2016, with up to~2% of blood donors reactive for zikv rna in id-nat testing at the peak in june 2016. aims: perform a serosurvey for anti-zikv igg using six panels of 500 donor specimens each collected in march 2015, at the beginning, peak and end of the 2016 epidemic, and from march 2017 and april 2018. methods: we employed a commercially available zikv igg elisa antibody (ab) assay based on the zikv ns1 antigen from bio-techne to characterize zikv seroprevalence in the 6 9 500 cross-sectional sample sets (anonymized with selected demographic information). results: 500 pr donor samples collected in april 2015 were initially evaluated using the manufacturer supplied cut-off to confirm that the zikv ab results were largely negative (3 positive, 3 equivocal) despite the high dengue virus seroprevalence (>90%) in pr that could potentially lead to false positive zikv ab results. we then used this dataset, together with known positives collected 1-3 months postdetection from zikv nat yield donors, to set a population-specific cut-off based on receiver operating characteristic (roc) curve analysis. this cut-off yielded sensitivity and specificity values of > 99%, and an area under the curve (auc) of 0.999, demonstrating a highly accurate assay. we used this new cut-off to calculate final rates of seroreactivity in the additional 5 sample sets (2500 samples) and estimate seasonal incidence. rates of reactivity, together with mean net od for only the reactives (shown in parentheses), were calculated for each sample set: background: sex hormone intake in blood donors occurs in three demographic groups: premenopausal women who take contraceptive drugs (progestins with or without estrogens), postmenopausal women who receive estrogen replacement therapies that may be combined with progestins, and testosterone therapies in men. we hypothesized that sex hormone therapies may modulate the quality of red blood cell (rbc) products via alterations of rbc function and predisposition to hemolysis during cold storage. aims: the objectives of this study were to evaluate the association between sex hormone intake and rbc measurements of hemolysis, and to examine possible mechanisms by which sex hormones interact with rbcs. methods: self-reported sex hormone intake and menstrual status were evaluated in 6,636 female blood donors from the national heart, lung and blood institute's rbc-omics study. the associations between hormone intake and donor scores of spontaneous storage, osmotic, or oxidative hemolysis were determined in all women and by menstrual state. the interactions between sex hormones and rbcs were determined by sex hormone (progesterone, 17b-estradiol, or testosterone) potency to inhibit calcium influx or hemolysis during incubations or cold storage. the calcium fluorophore, fluo-3am, was used to define rbc calcium influx in response to treatments with sex hormones or drugs that modulate transient receptor potential cation (trpc) channel activity including hyp9 (a selective trpc6 activator). results: sex hormone intake by menstrual status was higher in premenopausal women (25.3%) than in postmenopausal women (7.4%). female hormone intake was significantly (all p < 0.0001) associated with reduced storage hemolysis in all females (0.32 ae 0.16% versus 0.35 ae 0.29% in controls), enhanced susceptibility to oxidative hemolysis (37.9 ae 9.3% versus 35.8 ae 9.9% in controls), and reduced osmotic hemolysis in postmenopausal women (23.1 ae 10.2% versus 26.8 ae 12.0% in controls). in vitro, supraphysiological levels of progesterone (10 or 20 lmol/l), but not 17b-estradiol or testosterone, inhibited spontaneous or hyp9-induced calcium influx into rbcs, and were associated with lower spontaneous hemolysis after 30 day cold storage (0.95 ae 0.18% versus 1.85 ae 0.35%, progesterone 10 lmol/l versus solvent control (dimethyl sulfoxide, 0.1%), p < 0.0001). co-incubations (2.5 h, 37°c) of rbcs in the presence of progesterone and a trpc6 activator (hyp9, 25 lmol/l) suggested that progesterone protected against hyp9-induced hemolysis (1.45 ae 0.13% and 1.01 ae 0.09% versus 2.63 ae 0.19%; hyp9 + progesterone at 10 or 20 lmol/l versus hyp9 alone, p < 0.05 by one-way anova). summary/conclusions: this study revealed that sex hormone intake in blood donors is capable of modulating rbc predisposition to hemolysis and led us to propose new mechanistic pathways by which progesterone regulates calcium influx and hemolysis in human rbcs. pre-and postmenopausal women respond differently to hormone intake and its effects on rbc responses to osmotic or oxidative stress. progesterone modulates calcium influx into rbcs via a mechanism that may involve interactions with membrane trpc6 channels, activation of which is associated with pre-hemolytic events such as senescence and eryptosis. 3a-s05-01 international cooperation, swiss red cross, wabern, switzerland background: red cross and red crescent societies were playing an important role in setting up blood transfusion establishments in many low resource countries. by the mid-1970s, the red cross was active in the national blood programs in approximately 95% of countriesmostly in blood donor recruitment and education. today, major organizational developments in blood transfusion services were made in high income settings, where nearly 50% of all worldwide donations take place (home to only 17% of the population). who data shows that the median annual blood donation rate in high-income countries is 3.21% of the population compared to 0.46 % in low-income countries. the factors for this low turnout are multilayered, but it is well-known that most resource poor settings suffer from a low rate of regular donors and challenges to set-up and financially sustain a national blood donor program. the red cross and red crescent (rc) societies assume a key role by reaching and retaining donors from the communities and contribute significantly to better safety and availability of blood. partnerships and international collaboration, such as the swiss red cross (src) program, are aiming to strengthen national structures to improve blood safety and to face today's epidemiological, demographical, and technological challenges. aims: the present work aims to review the role, the mandate and the impact of rc societies in improving blood safety through systematic "voluntary non-remunerated regular blood donor" (vnrbd) programming and international partnerships. methods: data and evidence is drawn from the src international cooperation projects over the last 30 years, more specifically partnering with three rc societies, and the data from the global advisory panel (gap) of the ifrc including their 2018 global mapping. results: the promotion of vnrbd has been a specific objective in all src supported programs. through the engagement of the rc society, the training of volunteers and partnering with the health authorities, the projects significantly increased the blood donor rate by recruiting and retaining donors from the communities. for example, the rc societies increased total donations by 25 % in lebanon; vnrbds by 71% annually in kirgizstan, and from practically zero to 3'588 in south sudan. the importance of rc societies was also underlined in the 2018 published global mapping of gap, which showed that 36 (19%) of them provide level a (full blood service), 75 (39%) are level b (systematic blood donor recruitment) and 47 (25%) are level c (vnrbd blood promotion) blood services. gap has also commenced a new three year vnrbd support program aimed at establishing tools and materials for national societies. summary/conclusions: the red cross / red crescent movement has a unique mandate and position in improving global blood safety at all levels; with its huge network of volunteers, even blood donors in remote communities can be reached and retained. rc societies in low resource settings with a level b or level c role should further capitalize on partnerships with local and international actors to leverage technical assistance and funding for their activities. background: it is essential to motivate and encourage the public to donate blood and be eager to help saving lives, in order to maintain safe and adequate national blood supply. aims: "technical assistance for recruitment of future blood donors (europeaid/ 132420/d/ser/tr)" project aimed to avoid problems in supplying the safest blood to contribute to the improvement of public health by (i) increasing the knowledge of primary and secondary school students regarding blood donation, (ii) creating sensitivity in school principals and teachers regarding voluntary non-remunerated blood donation (vnrd), (iii) motivating family members of the students for blood donation and (iv) creating public awareness through media. methods: an effective coordination is established between ministry of health (moh), ministry of national education (mone) and turkish red crescent (trc). the existing curricula and textbooks of primary and secondary schools were reviewed and revised, and corresponding materials on the importance of blood donation were created. the human resources capacity of moh, mone and trc to support raising awareness on blood donation were developed. to raise public awareness on blood donation nationwide, education and recruitment campaigns on were organized in 500 pilot schools. additionally, media and public relation campaigns on blood donation were organized throughout the country. results: (1) existing curricula and textbooks relevant to promoting blood donation were reviewed, revised and reported to the board of education of mone. (2) corresponding educational materials for students and teachers were developed and distributed. (3) blood donation clubs were established in 500 pilot schools. (4) trainings were conducted for 688 personnel of moh and trc on blood donation regarding their responsibilities. (5) cascade trainings were conducted for 3218 personnel of transfusion centers and 4399 school principals in 81 provinces. (6) information seminars were delivered to 251.475 students and 15.739 teachers and family members of students during school campaigns. (7) four animation films on blood donation were produced and broadcasted on the national tv channel (trt). (8) three different computer games targeting different age groups were developed and uploaded to the web portal of the project and distributed to the pilot schools. (9) media spots were produced and broadcasted 3.851 times in 33 different tv and radio channels. (10) billboard posters and brochures were prepared and distributed to 81 provinces for raising public awareness. (11) advertisements about the project and the importance of vnrd were displayed 386 times on national and local newspapers, 1.117 times on online news, and broadcasted on 6 national tv channels. (12) during the campaigns, 28.310 units of whole blood were collected in pilot schools. (13) visibility kits to recruit future blood donors are prepared and distributed throughout the project activities. (14) awareness and knowledge level of students and their teachers/parents on the importance of blood donation are increased to 32.07 % and 35.99 % respectively, assessed through pretest and posttest. voluntary non-remunerated donation rate of national demand increased from 73.66% to 82.54 in two years. summary/conclusions: training and campaign programmes successfully increased the knowledge on blood donation. to achieve national self-sufficient safe blood supply, efforts for recruitment should be continued. background: despite 70% of pakistan's population being under 29 years, only 10% of blood supplies come from voluntary donors while remaining blood is collected from 'family replacement donors'. in pakistan the system has outsourced the mobilization of blood donors to the patient families. as a result many people reach out to their networks including on facebook to locate blood donors. there are thousands of posts each month in pakistan seeking blood donors on facebook. to facilitate needy families, the global social media giant facebook launched a special blood donation feature for pakistan in collaboration with sbtp, pakistan. the feature makes it easier for people to sign up to become blood donors and helps connect these voluntary donors with people and organizations in need of blood. similar features have been launched by facebook in india, bangladesh and brazil to address the problem of blood shortages in those countries. however, among these four countries pakistan has unique position because of the existence of a national counterpart, sbtp which can facilitate facebook in promoting its feature and provide the feedback on the impact of this innovative effort for continuous improvement of the feature. aims: to promote voluntary blood donations and blood safety in pakistan through facebook. methods: the facebook and sbtp teams launched a pilot to study the impact and effectiveness of the facebook blood donation feature as a tool of community engagement. a six months plan has been chalked out to measure the impact of this tool in five selected blood centres. a checklist called "p0 checklist" was shared with these blood centres to fulfill some basic requirements for an official blood bank page including a display picture, cover photo, contact information, directions, etc. regular skype meetings are held between the teams of sbtp, facebook (san francisco and singapore) and the blood centres to monitor the progress of the pilot and generate feedback. results: the facebook blood donation feature has recorded remarkable success with over one million signups within few months in pakistan. the blood centres participating in the pakistan study have experienced enhancement in the voluntary blood donations trend with 3-10 walk-in donors and an average of more than 20 telephonic queries regarding voluntary blood donation per month in each center. the trend is gradually surging as the feature is being refined on the basis of feedback received. the pilot will end in june 2019. the statistics generated since january 2019 are very encouraging and underscore the importance of social media in reaching out to the untapped potential blood donors. the study will be used to plan an effective nationwide strategy to increase donor mobilization, recruitment and retention. background: in our region, an increasing number of patients of african or asian origin with sickle cell disease (scd) or transfusion dependent thalassemia (tdt) require red blood cell (rbc) transfusions, and many have rbc alloantibodies. selecting optimally matched rbc units for these patients is essential for preventing not only acute hemolysis but also further alloimmunisation. beside antigen-matching for abo, rh d, c, c, e, e and k, patients with scd and tdt should ideally receive rbc units matched also for m, s, s, fya, fyb, jka and jkb (extended phenotype). this is the policy at our center, which currently provides rbc products to 31 patients with hemoglobinopathies. because the vast majority of our blood donors are caucasians, the selection of matched rbc units for patients of different ethnic origin can be difficult. therefore, expanding the number of available african and asian blood donors is becoming increasingly necessary. aims: hereby the recruitment strategy of non-caucasian blood donors introduced at our center is described and the results obtained during six years are reported. methods: since 01.01.2013, whenever a first-time blood donor of non-caucasian origin is registered, an alert is entered in the donor file to trigger the determination of the extended rbc phenotype along with routine testing. rbc antigen determination is performed in our laboratory with serologic methods. in selected cases (i.e. suspected rhd or rhce variant), samples are sent for molecular analysis (ssp pcr). rare rbc phenotypes relative to ethnicity are, among others, fy(a-b-), s-s-, lu(b-) and those with uncommon rh phenotypes. if a rare rbc phenotype is detected, a coded comment is entered in the donor data and the donor is listed in the national rare donor file. results: from 01.01.2013 until 01.03.2019, an extended determination of rbc antigens was performed in 352 subjects presenting for blood donation. twenty-nine rare donors (8%) were identified and included in the rare donor file: 25 fy(a-b-), 1 lu (a-b-), 1 lu(b-), 1 fy(a-b-) and s-, 1 ccddee (r'r'). overall, these 29 donors provided 105 rbc units (range . to date, all donors are still active and 14 are reserved for dedicated donations. the internal price of rbc antigen testing per donor is approximately 100. -chf, resulting in a total financial effort of around 35,200.-chf in the time since the project was started. summary/conclusions: in our experience, a "passive" recruitment of non-caucasian blood donors based on ethnicity has an overall low efficiency from a logistic and financial point of view. moreover, african and asian blood donors may require investigations for hemoglobin variants and serology for malaria in addition to routine testing. nevertheless, a targeted determination of extended rbc antigen phenotype does allow the identification of persons with rare phenotypes. currently, measures for the active recruitment of potentially rare blood donors are being implemented at our center. after a pilot phase, a project for a nationwide recruitment strategy will be elaborated. a further goal is to build a national registry of patients with hemoglobinopathies requiring transfusions. blood transfusion is an essential treatment. transfusion safety consists of several components. although all are important, ion richer countries the order of priority is typically: 1.) avoidance of transfusion transmittable infections; 2.) quality of the blood product with a strong focus on component therapy; 3.) prevention of severe transfusion reactions; 4.) avoidance of clerical errors; 3.) sufficient availability of blood. the keynote of this lecture will be that the order of priorities on transfusion safety should probably be different in resource limited environments. 1.) sufficient availability of blood and proper utilisation; 2.) avoidance of transfusion transmittable infections; 3.) avoidance of clerical errors; 4.) prevention of severe transfusion reactions; 5.) quality of the blood product. most important, in regions with limited resources patients suffer from under-transfusion because not enough blood is available. all efforts should be made to reduce wastage of the available blood either by inappropriate storage, handling, or nonindicated transfusions. in addition, prevalence and number of pathogens transmittable by transfusion is much higher than in the richer parts of the world. this is aggravated by the fact that rejection of blood donors based on their history is problematic when the blood bank is empty. the aim to develop centralized national blood services with few sites manufacturing cost effective (low personnel costs) high-quality blood products, which are distributed to regional hospitals is not matching the reality of infrastructure, governmental support, and functionality. in healthcare systems with limited resources usually available personnel (hands) is not a problem, while reagents, equipment and even electricity are precious resources. the lecture will propose to focus on staff training and education, establishing local hospital-based transfusion services, which provide the blood products for the region, based on donor recruitment campaigns adjusted to the available technology, local culture, including replacement donor programs, with retention of safe donors as highest priority. fractionation of blood into components had been standard in transfusion medicine, but recently whole blood has experienced revival for patients with acute blood loss. given main transfusion indications such as postpartum hemorrhage, or severe trauma, in regions with limited infrastructure, whole blood might be the more appropriate product. most patients requiring transfusion in these regions are younger and volume overload by whole blood is not a major issue. in addition, frequent electricity failures do not allow prolonged storage of plasma at -20°c (this is therefore mostly wasted), although this issue can be overcome by solar powered freezers. ideally whole blood should be pathogen inactivated for which two methods are currently available. to reduce frequencies of acute hemolytic transfusion reactions again education and training to minimize clerical errors in the transfusion process are most important. extended testing for other rhesus antigens and k beside abo and rh-d in transfusion dependent hemoglobinopathy patients may help to reduce delayed hemolytic transfusion reactions. currently a leukodepleted pathogen-inactivated whole blood product might mostly serve the needs for blood transfusion in regions with limited infrastructure . the developed world should invest research efforts to develop such a product available at affordable costs. background: in modern transfusion medicine, serological investigations for blood cell antigens are complemented by genotyping arrays and pcr assays. whilst these platforms are informative in the majority of reference investigations, they are limited in their ability to define nucleotide variants associated with rare antigens and unable to detect novel variants potentially affecting antigenicity. next-generation sequencing is increasingly being employed in reference settings, providing information that cannot be obtained through these methods. whole genome and whole exome sequencing have been successfully employed in many investigations of novel and rare antigens, however concerns remain regarding the collection of genomic data unrelated to reference investigations, and reporting clinically significant incidental findings. these concerns can be addressed through the use of targeted sequencing panels. we report on the design, testing and efficacy of a panel providing a comprehensive genotyping profile for red cell, platelet and neutrophil antigens in a single test. aims: design a customised targeted exome sequencing panel for red cell, platelet and neutrophil antigen genes, and benchmark the efficacy against a commercial medical sequencing panel (illumina trusight one -tso). -test the panel and in-house genotype prediction script on sequence outputs from 51 samples with known red cell, platelet and neutrophil genotypes and phenotypes and determine whether predictions are concordant. methods: the panel was designed with 2654 probes covering exons of 64 genes associated with red cell, platelet and neutrophil antigens. using illumina nextera rapid capture technology, 51 samples were tested over five sequencing runs, on standard and micro chemistries, to determine optimal sample plexity per standard run, and the efficacy of smaller flow cells for lower throughput applications. an in-house python script was used to predict star-allele genotypes based on variants listed in isbt and embl databases. these predictions were compared to results from serology, snp array and previous tso data. results: coverage consistently averaged > 2509, with 94% of target at a quality of q30. optimal sample plexity for a standard run was determined to be 14 samples, allowing for sufficient coverage of all clinically significant variants. for red cell samples with previous typing data (excluding rh structural variants), the script correctly predicted 99.5% of snp based red cell genotypes. script predictions were 100% concordant for platelet genotypes, and four of five neutrophil antigen genotypes. hna2 genotypes defined by cd177 could not be reliably determined. the increased target coverage of the panel allowed for detection of a clinically significant heterozygous variant in scianna system, previously undetected by the tso panel due to extremely low coverage. additionally, a variant defining a potentially novel null allele was detected in the p1pk system. summary/conclusions: the panel demonstrates considerably higher coverage, quality and throughput compared to the tso and allows for detection of variants previously overlooked due to low sequencing coverage. up to 14 samples can be reliably sequenced in a single run. our script correctly predicts over 99% of snp based alleles; however, rh structural variants require further manual analysis. background: to ensure the safety of a transfusion it is critical to identify the blood type of both donor and recipient. serological methods for typing abo, rh and kel use monoclonal antibodies, however, typing reagents for rare blood groups are expensive, unavailable or unreliable. dna-based identification of human blood groups has been used to overcome these limitations and its application has reduced rates of alloimmunisation in chronically transfused patients. however, to date, the cost per sample has prevented the universal application of dna-based donor typing aims: to achieve universal adoption of dna based donor typing, the blood transfusion genomics consortium (bgc) set out to develop an affordable dna based platform, capable of typing all red cell antigens, hla class i and ii and human platelet antigens. methods: the uk biobank axiom array, previously used to type 600,000 uk citizens, was redesigned for donor typing using three approaches: i) mining transfusion medicine knowledge, e.g. isbt allele tables; ii) inclusion of loci associated with donor health; iii) extraction of all coding variants in relevant genes with a frequency > 1:20,000 identified in large-scale sequencing data. samples from nhsbt and sanquin blood donors (n = 7,871) were used for performance assessment. red cell and platelet antigens for each donor were inferred from genotypes using the bloodtyper algorithm and concordance with clinical serological typing results assessed. results: concordance between genotypic and serological typing results was 99.9% for 95,022 comparisons; 29 of the 89 discrepancies were serologically negative and genotypically positive for a given antigen (k/k, fy[a/b], lu[a/b]). in all cases dna variants known to modify or weaken antigen expression were detected, displaying the power of genotyping to detect variant 'weak-antigen' expression. across 48 antigens for which serology was available, genotyping provided a 3.6-fold increase in the number of typing results available per donor (47.9 vs 13.2). furthermore, genotyping provided data on an additional 224 clinically relevant antigens, allowing identification of antigen-negative donors and blood group identification for which antibodies are not commercially available. the power of a genotyped panel of donors to support patient management was demonstrated by a retrospective analysis of clinical cases referred to nhsbt. from 3,146 patient referrals with > 3 alloantibodies between 2014 and 2018, 772 unique alloantibody profiles were identified. we found that there was a 2.6-fold greater likelihood of finding o negative compatible donors for these patients when using genotyping data from the 4,721 nhsbt donors. importantly, the number of alloantibody combinations for which no compatible antigen-negative donor could be identified fell from 293 to 246, representing an additional 164 patients that could be provided with directly compatible blood using the same donors. summary/conclusions: through the bgc efforts an affordable fully automated genotyping platform, including the processes for quality assurance and data analysis, has been developed. furthermore, we have demonstrated the real-world benefits more extensive donor characterisation can provide when selecting blood for patients with multiple antibodies. the results of this international collaboration provide opportunities to introduce fully-automated genotype-based donor typing in a safe and cost-efficient manner in blood supply organisations. 3c-s06-04 biobank performs whole-genome sequencing (wgs) from individuals nation-wide. these data are suitable for allele frequency analysis to demonstrate gene expression and genetic profile in our population, also to estimate the significance of each antigen in transfusion practice. aims: we aim to provide and verify population-based blood group antigen profile using wgs and dna samples from taiwan biobank. methods: a near 1500 wgs and demographic data were analyzed. annotations of blood group antigen were performed according to variants from isbt allele tables, including 2 transcription factors; variants for the lewis system were obtained from previous studies. annotations of blood group variants were verified by 4 dna samples with targeted sequencing on illumina miseq, and specific variants were verified by 40 dna samples with the commercial genotyping kit or sanger sequencing. allele frequencies from wgs analysis were compared with population serology data using two-proportion z test. results: population-wide blood group antigens were analyzed, revealed in-depth antigen expression profiles in all systems (except ch/rg). the antigen frequencies from wgs were similar compared with published serology data, except for the antigens and possible explanations listed as follow, 1) m, n: insufficient sequencing reads, 2) c, c: identical rhce exon 2 with rhd exon 2 for c allele, 3) mur: insufficient read length/depth for gypa/gypb hybridization calling and individuals from high prevalence of mur antigen in aboriginal tribes were not enrolled. blood group antigen predictions and variants from wgs were accord to dna verification. furthermore, 13 systems shown no genetic variations, predicting uniform antigen expression in our population, and we can manage transfusion with minimum concerns for antigen mismatch in these systems. moreover, we found weak and null alleles in our population for blood group systems that we had previously no knowledge of, such as lan, jr, and vel. these variants were helped to identify a patient with anti-jr a carrying homozygous jr a null alleles. summary/conclusions: taiwan biobank wgs is suitable for full blood group antigen profile determination with few adjustments required for specific antigens. the population antigen allele frequency provides valuable insight to antigen significance in transfusion practice, matching strategy for our patients, and estimation of the likelihood to obtain for specific antigen negative blood from mass population. also, the genetic variants revealed in this study can help us to locate rare donors, and to integrate variations into routine donor blood group screening to provide suitable blood at a low cost efficiently. background: providing adequate amounts of safe, appropriately matched blood to meet the demands of an expanding and aging patient population presents a challenging global problem. for these reasons, sustainable in vitro sources of red cells may offer a desirable alternative to reliance on donor blood. the first stable immortalized early adult erythroblast cell line, bel-a2, has been shown to differentiate efficiently into mature, functional reticulocytes (trakarnsanga et al., nat commun. 8:14750, 2017) and consequently could provide a readily available tool for diagnostic use and proof of principle for future therapeutic use. aims: at ibgrl, next generation whole exome sequencing (wes) has been used previously to accurately predict blood group phenotype in a number of blood group systems, including abo, rh and mns (tilley & thornton, transfusion medicine 27 (suppl.2) :42, 2017). here we have used it to analyse and document bel-a2 blood group-related genotypes and predict blood group phenotypes. additional genes involved in cell-growth and enucleation were also analysed in order to further elucidate the characteristics of the bel-a2 cell-line. methods: bel-a2 cells (day 196) were cultured in expansion medium and genomic dna (gdna) was isolated from 1 9 10 6 cells on day 5. for wes, gdna libraries were prepared using nextera â rapid capture exome enrichment and sequenced on illumina â miseq. sequence alignments for 41 genes encoding all 36 known blood group systems and 12 further genes encoding transcription factors and cell enucleation-associated proteins were visualised using integrative genomics viewer, whilst illumina â variant studio was used to identify observed mutations. mutations in coding regions were used to determine bel-a2 genotype and predicted phenotype. results: good coverage of most of the selected genes was achieved. alignment of homologous blood group genes including rhd/rhce, gypa/gypb and c4a/c4b was problematic and additional analysis of coverage of these genes was required for accurate interpretation. despite a number of polymorphisms observed across the tested genes, bel-a2 did not express any novel or rare blood group antigens. genotyping results predicted a common antigenic profile, in agreement with previous serological and genotyping results where available. although a number of missense single nucleotide variations were detected in analysed genes, including cr1, cdan1 and tmx4, these were common polymorphic variants and unlikely to be of any functional significance. summary/conclusions: wes was used to determine bel-a2 genotype in relation to blood group genes and selected genes encoding transcription factors and proteins associated with cell enucleation. wes allowed accurate prediction of blood group phenotypes, showing full concordance with available serological data (trakarnsanga et al, 2017) . a small number of mutations were identified which are of unknown significance and require further work to determine any potential phenotypic effects. this complete record of the bel-a2 blood group-related exome will enable reliable gene editing strategies for future diagnostic and therapeutic purposes. additionally, knowledge of the full cell line exome will allow analysis of any emerging genes of interest and provide better insight into the mechanisms of erythroid differentiation and enucleation. background: emerging evidence, especially in neonates, has shown potential harm associated with liberal platelet transfusion strategies. very little evidence exists regarding optimal platelet transfusion thresholds in critically ill children. randomized controlled trials may be difficult due to lack of equipoise from providers. if regional variation in practice exists, comparative effectiveness studies may be an alternative approach. aims: to describe regional variation in platelet transfusion practices in critically ill children. methods: secondary analysis of a prospective, observational study. subjects were grouped according to region (north america, europe, middle east, asia and oceania) and nation. transfusions were analyzed as prophylactic (given to prevent bleeding) or therapeutic (given to treat bleeding). the primary outcome was the total platelet count (tpc) prior to transfusion. sub-groups analyses were performed in children with an underlying oncologic diagnosis and those supported by extracorporeal life support (ecls). the dosing and processing of the platelet transfusions were analyzed as secondary outcomes. results: five hundred and forty-nine children from 16 countries were enrolled (67% in north america, 17% in europe, 7% in oceania, 5% in asia, and 4% in the middle east). overall, the median (iqr) tpc prior to prophylactic transfusions (n = 360) differed significantly on a regional basis (p = 0.04) and ranged from 12 (8-41) x10 9 cells/l in the middle east to 45 (20-66) x10 9 cells/l in asia. the median tpc prior to prophylactic transfusions did not significantly differ between countries (p = 0.08), nor did the tpc prior to therapeutic transfusions (n = 189) differ on either a regional (p = 0.16) or national (p = 0.57) basis. for children supported by ecls (n = 90), there were no regional (p = 0.06) or national (p = 0.40) differences for prophylactic transfusions. however, significant differences in the tpc prior to therapeutic transfusions were observed on both a regional (p = 0.02) and national (0.04) basis with the middle east, in particular israel, transfusing at the lowest median (iqr) tpc [28 (16-81) x10 9 cells/l]. for children with an underlying oncologic diagnosis (n = 233), no differences were seen in the tpc for prophylactic transfusions (n = 175) on a regional (p = 0.19) or national (p = 0.20) basis. nor were differences seen in the tpc prior to therapeutic transfusions on a regional (0.86) or national (p = 0.33) basis. there was significant variability in the dosing of platelet transfusions on both a regional (p < 0.001) and national basis (p < 0.001). the median (iqr) dose based on volume ranged from 8.4 (5.0-11.2) ml/kg in north america to 12.6 (9.8-16.0) ml/kg in europe. the vast majority of transfusions were leukoreduced and irradiated but significant variation exists in storage duration on both a regional (p < 0.001) and national (p < 0.001) basis. summary/conclusions: regional and national variation exists in platelet transfusion practices among critically ill children, especially in those given therapeutic transfusions while supported by ecls. considering this variation, comparative effectiveness studies may be an appropriate approach to gain evidence to optimize platelet transfusion thresholds. background: the optimal threshold for prophylactic platelet (plts) transfusion in pediatric patients with cancer is still controversial and current clinical practice comes from studies on adults and on inpatient setting. the international guidelines (icmtg, 2015) recommend, for all age patients, a prophylactic platelet transfusion when plts count is ≤ 10 9 10 11 /l and a platelet dose of 1.1 9 10 11 per square meter (sm) of body-surface area (bsa) in inpatient and 2.2 9 10 11 /sm in outpatient setting. aims: in january 2018 we started in our children's hospital a prospective protocol in order to evaluate the impact on bleeding risk of current clinical practice of prophylactic platelet transfusion in inpatients and outpatients onco-haematological patients. methods: bsa was calculated from age-standardized weight. inpatients received a dose per transfusion of 1.1 9 10 11 /sm and outpatients a dose per transfusion of 2.2 9 10 11 /sm. platelets were transfused when the count was ≤ 10 9 10 11 /l or in presence of bleeding signs; pediatric aliquots were obtained from buffy coat derived pooled platelet concentrates or apheresis platelet concentrates, according disponibility. results: from january 2018 to december 2018 a total of 9839 platelet pediatric aliquots were transfused: 5534 (56.2%) were obtained from apheresis platelet concentrates and 4305 (43.8%) from buffy-coat-derived pooled platelet concentrates. the majority of platelets pediatric aliquots (7505-76.3%) were transfused to onco-hematological patients undergoing hematopoietic stem cells transplant (hsct) or conventional chemotherapy. among them, 6365 aliquots were transfused in inpatient setting: 1675 (17%) in the hematology unit, 2772 (28.2%) in the oncology unit and 1918 (19.5%) in hsct unit. a total of 1140 (11.5%) aliquots were transfused in outpatient setting: 840 (8.5%) to patients affected by hematological malignancies and 300 (3%) to patients with solid tumors. five major bleeding events (who grade ≥ 3) were observed during the study period and all of them occurred in hospitalized patients. two patients with solid neoplasm developed a who grade 3 bleeding event. two patients with hematologic malignancies and a patient with neuroblastoma (n = 3, 1.2%) developed intracranial bleeding (who grade 4). the platelet count at the time of the event was 22 9 10 9 /l, 25 9 10 9 /l and 8 9 10 9 /l, respectively. summary/conclusions: our results showed the efficacy, in onco-hematological pediatric patients, of a prophylactic platelet transfusion protocol based on international guidelines: a very low incidence of who grade 4 bleeding has been observed in inpatients setting only (1.2% vs 1% of plado trial, sj slichter, nejm, 2010) , while in outpatients setting the double platelet dose prevents the major bleeding event (who grade ≥ 3) occurrence. background: the problem of blood-borne infections remains relevant in transfusion medicine. pathogen reduction technologies (prt) provide a preventive approach to a wide range of transfusion-transmitted infectious diseases. to date, prt widely used for platelet concentrates and blood plasma, however, the use of these technologies for the treatment of red blood cell-containing blood products undergo research. aims: the aim of our study was to evaluate the safety and efficacy of transfusions of pathogen-reduced (test group) red blood cell suspensions (rbcs) and compare these data with gamma-irradiated rbcs (control group). methods: the technology based on the combined action of riboflavin and ultraviolet (mirasol prt, terumo bct, belgium) was used to reduce pathogens in whole blood. subsequently, the rbcs of the test group were derived from pathogenreduced whole blood. the control rbcs were irradiated at the gammacell 3000 elite (best theratronics, canada) at a dose of 25 gray. all rbcs were used for transfusion for 14 days from the harvest day. 70 pediatric patients with various oncological and hematological diseases were randomized to 2 groups of 35 members in each group. the test group of patients received transfusions of a pathogen-reduced rbcs; the control group received transfusions of a gamma-irradiated rbcs. the next day after transfusion were assessed hemoglobin and hematocrit increment, the level of potassium and haptoglobin in the patients' serum, the frequency and severity of transfusion reactions. 3-5 days after the transfusion, the direct antiglobulin test (dat) was performed and after 14-21 days the indirect antiglobulin test (iat) was performed. the interval to the next need for transfusion was also evaluated. results: the increase in hemoglobin and hematocrit (p = 0.2), as well as the concentration of potassium (p = 0.44) and haptoglobin (p = 0.25) in the patients' serum after the transfusion did not differ between groups. none of the patients in both groups had hyperkalemia after transfusion. in each group, two patients had febrile non-hemolytic transfusion reactions of comparable severity (p = 1). all dat and iat tests were negative in both groups. the interval between transfusions were not significantly different between groups (p = 0.39). only in the test group was found the correlation between the increase in the hemoglobin and hematocrit values with the volume of transfusion, with the dose and the adjusted dose of hemoglobin obtained for the transfusion on body weight. and in this group was found inverse correlation between the hemoglobin and hematocrit increment with the level of hemolysis in the rbcs. summary/conclusions: we found that the clinical efficacy and safety of rbcs of the compared groups did not differ. there was no evidence of immune elimination and allo-sensitization caused by pathogen-reduced rbcs. according to our data, the spectrum of efficiency and safety indicators of pathogen-reduced rbcs is no worse than that of gamma-irradiated rbcs, provided that rbcs is used for 14 days of storage. the founded correlation suggests that the efficiency of pathogen-reduced rbcs transfusions is more dependent on the characteristics of the rbcs. background: patient blood management (pbm) programs are expanding at an international level. a recent nationally representative study from united states observed pediatric age group as the only age group showing lack of objective evidence of pbm initiatives (goel et al, jama 2018) . aims: this study aims to identify trends in peri-operative blood utilization in children undergoing elective and non-elective surgeries over 5 years duration from 2012 to 2016. methods: using 5 years data (2012) (2013) (2014) (2015) (2016) perioperative transfusions decreased steadily per year from 6.4% in 2012 to 5.5% (14% cumulative decline) in 2016 for children of all ages (or 0.966; 95% ci 0.957-0.976; p trend < 0.001). the cumulative change in elective procedures was 9.2% versus 27.2% decrease in urgent/emergent procedures (p trend < 0.001). summary/conclusions: in this large prospective registry study of > 350,000 children undergoing elective/non-elective surgeries, a statistically significant decrease in utilization of peri-operative rbc transfusions was seen across 5 years from 2012 through 2016 with more significant decrease in urgent/emergent procedures than elective procedures while these findings need evaluation for non-surgical indications of transfusion, these results may provide first evidence of peri-operative pediatric patient blood management strategies being implemented to optimize transfusions in pediatric population. adverse events -tti, immune interactions and risk 3c-s08-01 transfusion-transmitted infections (tti) are a long-standing and well recognized concern in medicine, which is tackled on the highest level to guarantee the safety of the transfusion procedure for all stake holders. these include the recipient patients, the donating volunteers, the health care workers involved, and their respective contact persons. accordingly, current national and international guidelines including expert societies and the who provide medical, technical, and legal frameworks, which are the basis for the standard operating procedures. nevertheless, there are important challenges, which render tti a "moving target", and reflect the dynamics in three main areas. first, a change in the type and number of recipient patients with past or ongoing immunomodulatory / immunodeficiency component (examples being hiv/aids, sot, allogenic hct, monoclonal antibody therapies, small molecule inhibitors). second, changing exposure to known agents in donors due to global travel, migration and displacement, as well as environmental/climate change. third, discovery and diagnostics of old and new agents with their known or presumed impact as tti. these aspects will require careful review of data and studies, and judicious discussion of the potential action such as selection versus close monitoring to keep tti rates as low as possible, to deliver maximal safety of patients and stakeholders. background: the implementation of nucleic acid testing (nat) and the development of sensitive and specific serologic assays to detect hbsag and anti-hbc antibodies significantly reduced the risk of hbv transfusion-transmission. the apparent redundant testing for two direct viral markers prompted debates on maintaining hbsag screening, particularly in low endemic countries where blood donations are screened for anti-hbc. however, frequencies of 2-20% of hbsag-confirmed positive/nat negative donations have been reported depending on the sensitivity limit of the molecular assays used. the nature of this discrepancy between hbsag and dna remains largely unknown and it is essential to evaluate any potential negative impact on blood safety before considering removing hbsag testing. aims: the prevalence in blood donors and the molecular mechanisms responsible for a persistent undetectable or barely detectable level of viral replication in the presence of a sustained hbsag production were investigated in a collaborative study including five laboratories/blood centers in europe and south africa. discrepancy between viral dna and hbsag levels suggested the presence of mutations that may negatively affect hbv replication and/or infectious viral particle production. methods: donor samples from france, south africa, poland, and croatia were selected for having hbsag levels ≥ 100 iu/ml and being id-nat (procleix-ultrio plus tm [95% lod: 3 iu/ml]) non-reactive/non-repeatable reactive (nr/nrr) with undetectable viral load (vl) or < 6 iu/ml (n = 44) or nat repeat reactive (rr) with vl < 6 iu/ml (n = 32). french samples initially tested nat nr/nrr with procleix-ultrio (lod 95%: 11 iu/ml) were retested with ultrio plus prior inclusion in the study. hbv dna load was quantified (cobas taqman hbv [loq: 6 iu/ml]). hbv dna was purified from 5 to 12 ml of plasma after ultracentrifugation. the whole hbv genome, pre-s/s, precore/core and bcp regions were amplified and sequenced. results: following viral concentration, hbv dna presence was confirmed in 79% of all samples with undetectable or vl < 6 iu/ml. hbv genotypes were a1 (33.3%), a2 (18.4%), a3 (3.3%), b (3.3%), c (1.7%), d (25%), and e (15%). all samples were anti-hbc positive and 73% of ultrio-negative samples tested positive with ultrio plus. unusual 1-2 nt insertions/deletions identified in bcp regulatory elements (tata boxes, pginr, epsilon domain) suggest altered viral replication. amino acid substitutions (n = 16) or deletions (n = 4) at positions reported involved in nucleocapsid formation, particle envelopment and virion formation were observed in the core protein of 30 samples. the replicative properties of the bcp and core variants are currently evaluated in vitro as a surrogate model for direct infectivity testing. preliminary results indicate that the variants tested so far have replicative capabilities similar to those of control viruses. analysis of pol, s, and hbx proteins is ongoing. summary/conclusions: these data confirmed the presence of extremely low level of circulating dna-containing viral particles in id-nat non-reactive or nonrepeated reactive blood donations with concomitant high hbsag levels and anti-hbc reactivity. despite the presence of mutations in the viral genomes potentially affecting virion production, preliminary data indicate that some of the viruses in plasma retain the ability to replicate in vitro and to constitute a potential infectious risk. 3c-s08-03 background: in switzerland highly sensitive nucleic acid screening in an individual donation format for hepatitis b virus (hbv id-nat) and hepatitis b surface antigen (hbsag) detection is mandatorily performed (guidelines of swiss transfusion src, switzerland). since 1998, hbv (hb) vaccination is recommended in switzerland for children and adolescents until the age of 15 and for adults belonging to known risk groups. aims: to highlight that low anti-hbs titers several years following hbv vaccination still confer protection and enable the host immune system to clear hbv dna without development of serologic markers of disease. methods: a retrospective donor interview was conducted to complete information not covered by the questions included in the standard donor questionnaire. routine hbv serological donor screening was performed on a quadriga system (diasorin, former siemens) with the enzygnost hbsag assay (diasorin, former siemens). further hbv tests were performed on the abbott architect i1000 analyser (hbsag neutralisation, hbeag, anti-hbc igg/igm, anti-hbc igm, anti-hbe and anti-hbs). routine id-nat screening for hiv/hcv/hbv was performed with the roche cobas mpx test on a roche cobas 8800 platform. hbv id-nat positive samples were confirmed with a quantitative hbv nat assay (abbott). background: hepatitis b core-related antigen (hbcrag) is a structural antigen of hbv, consisting in hbcag, hbeag and the p22cr precore protein. quantitative hbcrag measurement is a sensitive marker of viral replication reflecting the cccdna content and persistence of disease. hbcrag positivity was found to be a significant risk factor of hbv reactivation in hbsag-, anti-hbc+, hbv dna-patients (occult hbv infection, obi) undergoing immunosuppressive therapy. aims: no data about hbcrag status in apparently healthy subject with obi are available. the aim of this study was to analyse this marker in our cohort of obi blood donors. methods: hbcrag was measured in 69 blood donors confirmed to be carriers of obi (hbsag-, hbv dna+). of them, 59/69 (85.5%) donors were anti-hbc positive, and 10 (14.5%) negative. 18 donors had both anti-hbc and anti-hbe reactivities. a group of 11 young blood donors vaccinated for hbv infection (hbsag-, hbv dna-, anti-hbc-), and 9 patients with chronic hbv infection (hbsag+, hbv dna+) were used as negative and positive controls group, respectively. serum hbcrag was measured using a chemiluminescent enzyme immunoassay on the lumipulse g600 automated analyzer (fujirebio, tokyo, japan). the lower limit of detection (lod) of the quantitative assay is 2 logu/ml and the lower limit of quantification (loq) is > 3 logu/ml, due to nonlinearity results between 2 and 3 logu/ml. levels of hbcrag were tested in the three groups and analysed in comparison to the presence of anti-hbc and anti-hbe. statistical analysis was performed by the ibm statistics spss 19.0.0. results: all donors in the negative control group had undetectable hbcrag levels, whereas all patients in the positive control group have detectable hbcrag (mean value: 3.0 logu/ml, range 2.0-4.9), confirming that individuals without prior exposure to hbv would not have detectable hbcrag. hbcrag was detectable in 34/69 obi donors (49.3%), with a mean value of 2.21 logu/ml (range 2.0-3.20). hbcrag could be measured only in 2 obi donors (3.0 and 3.2 logu/ml), being below the loq of the test in the majority of obi (32/34). considering the presence of anti-hbc, hbcrag was detected in 29/59 (49.1%) anti-hbc+ and in 5/10 (50%) anti-hbc-obi, with no significant difference in their mean levels (2.21 ae 0.32 vs 2.14 ae 0.26; p = 0.44). interestingly, the presence of anti-hbe (18/62) was independently associated with higher hbcrag levels (2.38 ae 0.42 vs 2.14 ae 0.22; p = 0.03). summary/conclusions: identification of donors with obi is critical to prevent the risk of hbv transfusion-transmission. being hbcrag associated with the cccdna content and replication, our results suggest that the presence of hbcrag, even if not quantifiable, could be useful marker to confirm the occult infection status, even in anti-hbc negative donors. the association between hbcrag, anti-hbc and anti-hbe could also be a useful marker to identify obi donors with a higher risk of hbv reactivation. 3c-s08-05 hc group. human peripheral blood mononuclear cells (pbmcs) from blood donors were stimulated with hbv polypeptides pool in vitro. t cell proliferation assays (cfse) was used to detecting t cell proliferation, enzyme-linked immunospot assay (elispot) was used to detecting the frequency of hbv-specific ifn-c secreted t cells. spss 20.0 statistical analysis software was used for statistical analysis. the measurement data of normal distribution were tested by two independent samples t test; and the comparison between multiple groups was analyzed by one-way anova. mann-whitney u test was used for comparison between non-normal data sets. p < 0.05 was considered statistically significant. results: 1. proliferation characteristics of t cells. the proliferation of cd4 + t lymphocytes was mainly stimulated by specific hbv polypeptide pool, and the proliferation rates of obi group and chb group were significantly higher than those of hc group (3.0%, 3.3% vs. 1.7%), with significant difference (3.0% vs. 1.7%, p = 0.016, 3.3% vs 1.7%, p < 0.001). 2. the frequency of specific ifn-c secreted t cells. the response intensity of the obi group (25 sfc/10 6 pbmcs) and chb group (25 sfc/10 6 pbmcs) was higher than that of the hc group (5 sfc/10 6 pbmcs) under the stimulation of hbv polypeptide pool, and the positive rate of t cell response to the stimulation of hbv polypeptide pool was the highest in the obi group (64.0%). summary/conclusions: both obi and chb had higher rates of hbv-specific t effector cell proliferation and ifn-c secretion than the healthy control group. compared with the chb group, obi group had a higher positive rate of t cell response, which may be one of the causes of host immunity resulting in obi. further studies on other immune factors are required. background: western blood transfusion practices are currently changing due to various drivers such as blood management policies, ongoing technological developments, and new therapeutic options. in the netherlands, as in many high-income countries, these have resulted in a diminishing trend of red blood cells. therefore, it is important for blood bank management to anticipate the future demand of blood products for the sake of medium and long term decision making. to support this decision making, we have employed scenario development, which is used in many other sectors (such as finance and transportation) and can also be applied to blood transfusion. building upon a prior literature review and semi-structured interviews of international experts, we gathered experts together for scenario sessions to assess the opportunities and threats for sanquin's medium-term (15-20 years) strategy using an online platform and face-to-face discussions. aims: to assess for opportunities, threats, and the organizational implications thereof for the medium-term future of sanquin, the dutch national blood bank. methods: twenty-one multidisciplinary experts in blood transfusion agreed to participate and were separated into two groups for half-day interactive sessions. using an iterative process through an online platform, experts brainstormed opportunities and threats for sanquin, which were categorized into themes. these themes were ranked according to importance and certainty, and through consensus, experts chose two themes with high impact and high uncertainty. for these chosen themes, specific actions for the blood bank were listed to mitigate and/or enhance the threat or opportunity. discussions were ample throughout. results: with regards to opportunities and threats for sanquin's medium term strategy, experts brainstormed many ideas and categorized them under 10 themes: political context/ changing legislation, novel products and alternative applications, donors, international markets, commercialization, digitalization, change in perceptions, research, demand, and organizational structure. after ranking for importance and certainty, six themes were chosen: change in perceptions, international markets, political context (opportunities), demand (opportunities), research (vulnerabilities), and donor (vulnerabilities). for each of these themes experts provided specific actions for the organizations to mitigate threats or stimulate opportunities accordingly. these actions included increased transparency and improved communication with the (donor) public, lobbying in political spheres, increased activities in educational institutes and large funding organizations, and creating and collaborating on novel blood products on an international level, to name a few. summary/conclusions: these results show that mapping and assessing a blood bank's future using a multi-disciplinary group of experts is conducive as an effective means of collection a diverse range of opportunities and threats. this provides an opportunity for blood bank management to become proactive towards these potential opportunities and threats and possibly evolve future strategies for the organization. showed that iron-deficient female blood donors were more likely to have depressive symptoms than non-iron deficient female blood donors. among participants with depressive symptoms, females with low plasma ferritin levels had significantly increased odds for reporting a "feeling of lacking energy and strength" (or = 2.11; 95% ci: 1.03-4.31). as it is known that blood donors are at an increased risk of iron deficiency, it is important to determine whether those genetically predisposed to lower plasma ferritin levels have a higher risk of experiencing the tiredness/lack of energy symptom. aims: to investigate whether there is an association between polygenic risk scores (prss) based on plasma ferritin levels and the tiredness/lack of energy symptom in blood donors. methods: the dbds is an ongoing nationwide blood donor cohort, of which genome-wide genotype data are available for 72,000 participants. genotyping was performed using the infinium global screening array (illumina â ) and imputation was achieved based on a scandinavian reference genome. ferritin prss, based on an icelandic ferritin gwas (n = 150,000), were calculated for all dbds participants. 12,903 female donors were available for the analysis. data on depressive symptoms were obtained using the validated major depression inventory scale (mdi), a selfreport mood questionnaire, which assesses the presence of 10 depressive symptoms. a donor was classified as "tired" if they responded "all the time" or "most of the time" to the question "how often do you feel that you lacked energy and strength?". logistic regression analysis was performed, adjusting for age. for generating the quantile plots, the participants were distributed evenly into six quantiles based on their prs, whereby quantile 1 contained the donors with the lowest prss (genetically predisposed to lower ferritin levels) and was set as the reference quantile with or = 1 from the age-adjusted regression analysis (tiredness~quantile). results: prss in females ranged between -0.42 and 0.50 (mean 0.01). a total of 12,523 female donors were classified as "not tired" and 380 (2.9%) were classified as "tired". no significant difference in ferritin prs was found between "tired" and "not tired" female donors (tired mean prs: 0.00; not tired mean prs: 0.01). an age-adjusted logistic regression model found this to be insignificant (or: 0.74, 95% ci: 0.33-1.66), p = 0.47). to visualise the lack of association, a quantile plot was created, separating the female donors into six equal quantiles based on their prs. no clear trend was observed; donors with the highest prss (in quantile 6) had or = 1.02 (p = 0.93) of being tired when compared to those in quantile 1 (or set as 1). summary/conclusions: no significant association was found between the ferritin prss of female blood donors and the tiredness/lack of energy symptom. further studies are needed to understand the effect of blood donation versus genetic constitution on tiredness among female iron-deficient blood donors. background: antiretroviral therapy (art) is critical for the control of clinical progression of human immunodeficiency virus (hiv) infections. however, the outcome of art could be limited by drug resistance-associated mutations (drms), even lead to the transmission of drug-resistant hiv to treatment na€ ıve patients such as blood donors, which is a huge concern to art. drms surveillance in hiv infected groups is strongly recommended by world health organization. characteristics of genetic diversity and drms of hiv among blood donors may provide comprehensive data to monitor viral evolution and optimize art, play important roles in blood safety. aims: limited data concerning the epidemic of hiv-1 subtypes and drms of blood donors is available in china. this study is to investigate genetic characteristics and drms of hiv-1 infected blood donors. methods: from 2016-2018, 177 blood donations collected from 24 blood centers, covering almost the whole of china, were confirmed as hiv-1 positive by national centers for clinical laboratories using abbott realtime hiv-1 assay or cobas taq-man hiv-1 test, version 2.0. then hiv-1 gag (973 bp, hxb2: 1074-2044), pol genes (1454 bp, hxb2: 2068-3521) (encoding the whole protease (pr) and a part of reverse transcriptase (rt)) was sequenced after viral rna extraction and amplification. hiv-1 subtype based on gag and pr-rt regions was determined by comprehensive analyses of los alamos hiv blast tool, rega hiv-1 subtyping tool, phylogenetic trees and online jphmm program. drms analysis was performed in the stanford hiv drug resistance database. results: among 177 donations, gag and pr-rt regions of 160 samples were sequenced successfully. the distribution of hiv-1 genotype was as follows: crf_07bc = 66 (38.8%), crf_01ae = 58 (36.3%), b = 9 (5.6%), crf_08bc = 2 (1.3%), crf55_01b = 4 (2.5%), crf59_01b = 1 (0.6%), crf65_cpx = 1 (0.6%), crf67_01b = 2 (1.3%), crf79_0107 = 1 (0.6 %), crf85_bc = 1 (0.6 %), urf_0107 = 6 (3.8%) and urf = 9 (5.6%). 26 of 160 hiv-1 isolates were identified to have drms. there were 4 (15.4%, 4/26) protease inhibitors (pi) accessory drms, 3 pi major drms and 20 (76.9%, 20/26) non-nucleoside reverse transcriptase inhibitors (nnrti) drms. most of blood donors with drms were crf01_ae and crf07_bc (61.5%, 16/26). 3 of 4 pi accessory drms were q58e. the pi major drms included m46l, m46i and n88s. n88s could result in hlr to atazanavir (atv) and nfv, llr to indinavir (idv) and saquinavir (sqv). v179d/e is main nnrti drm (70.0%, 14/20). a combination of v179d and k103r among two samples acted synergistically to reduce efavirenz (efv) and nevirapine (nvp) susceptibility. furthermore, two blood donors with k103n mutation in reverse transcriptase gene had high level-resistance to efv and nvp. summary/conclusions: overall, the most prevalent subtypes among blood donors in the study were crf07_bc (38.8%), crf01_ae (36.3%). besides, other rare crfs and several urf_0107 and urfs were also found in these hiv-1 isolates, which suggested the epidemic of hiv has been shifted from high risk populations into general populations, including blood donors in china. drms were observed in 16.3% donors in the study, which may result in resistance to pis and nnrtis, especially the hiv-1 variants with n88s mutation in pr gene and k103n mutation in rt gene. in summary, our findings indicate that increasing diversity of hiv-1 in blood donors and remind us the necessity of timely genotypic drug resistance monitoring and molecular epidemiology surveillance of hiv-1 among blood donors. background: labeling of platelets is required to measure the recovery and survival of transfused platelets in vivo. currently a radioactive method is used to label platelets. however, its' application is limited, due to safety issues and the inability to isolate transfused platelets out of the circulation. biotin-labeling of platelets is an attractive non-radioactive option, however, no validated protocol to biotinylate platelets is currently available for clinical purposes. aims: the aim of this study is to develop a simple, standardized, reproducible method to label platelets with biotin as a non-radioactive alternative to trace transfused platelets in vivo. methods: six pooled buffy coats derived platelet concentrates (pcs) stored in 100% plasma were biotinylated at day 1 and day 7 of storage. to distinguish the effect of the processing steps from the effects of biotin incubation, 'sham' samples were processed. for the biotinylation procedure, 50 ml of pcs was washed twice and incubated with 5 mg/l biotin, dissolved in phosphate buffered saline-pas-e (1:9), for 30 min. stability of the biotin labeled platelets after irradiation was tested. annexin v and cd62p expression were assessed as measures of platelet activation. applicability of this method to other platelet products was assessed in three pooled pcs stored in 65% pas-e and three single donor apheresis pcs. results: the method was reproducible performed in a closed system. after biotinylation, 98.4% ae 0.9% of platelets were labeled. platelet counts, ph and 'swirling' were within the range accepted by the dutch blood bank for standard platelet products. the number of annexin v positive cells was not significantly altered by the biotinylation procedure in both fresh and stored platelets. in contrast, cd62p expression was increased in biotinylated platelets 48.4% iqr(41.7-56.2%) compared to the control samples 12.3% iqr(9.5-12.7%) on day 1 of storage. however, biotinylated platelets were not more activated compared to sham samples 50% iqr(41.7-56.2%). thus only the procedural steps led to increased cd62p expression and not the biotin label itself. all samples showed maximal response to thrombin receptor-activating peptide. for platelets labeled at day 7, a similar pattern was observed. irradiation of biotin labeled platelets did not alter the stability of the biotin label nor cell quality. furthermore this method is also applicable to pooled pcs stored in pas-e and apheresis pcs, with similar patterns in annexin v and cd62p expression. summary/conclusions: we developed a standardized and reproducible protocol according to good practice guidelines (gpg) standards, for biotin-labeling of platelets for clinical purposes. the procedural steps, which are similar to the steps used for production of hyperconcentrated platelet products, led to an increased cd62p expression, but did not alter the annexin v expression. this method can be applied as non-radioactive alternative to trace and recover transfused platelets in vivo. blocking activity over the prototypic chs4 insulator in cell lines and substantially reducing genotoxicity in a c-retroviral vector-mediated carcinogenesis mouse model. in contrast to chs4, these insulators are small-sized (119-284 bp vs 1.2 kb) and can be easily accommodated in gt vectors without detrimentally affecting vector titers. aims: we aimed to test whether a1, one of the newly discovered cis, could reduce vector-mediated genotoxicity in the challenging context of sin-lvs, by insulating a therapeutic globin-vector. methods: we tested the genotoxicity effect in the il-3-dependent 32d cells, which upon transduction with oncogenic vectors become il-3-independent, leading to transformation. 32d cells were transduced with sin-lvs: the b-globin-τνs9.3.55-, the insulated b-globin-a1-tns9.3.55and the oncogenic sffv-gfp-vector. transduced cells were expanded in 10% il-3 and transduction efficiency was determined by vector copy number (vcn). transduced 32d cells were seeded in methylcellulose with 10% or 0-1% il-3 to detect the il-3-independent and potentially transformed clones. the il-3-independent clones were further expanded in 10% il-3 and infused in partially myeloablated and il-3-treated c3h/hej mice. wbc analysis, blood smears and bone marrow(bm) cytospins were performed. results: the a1 insulator did not negatively affect vector titers (τνs9.3.55, a1-tns9.3.55, sffv-gfp: 2.8, 1.8, 2.5x10^8 iu/ml, respectively). 32d cells were successfully transduced with all vectors (%vcn positive colonies: 40-100%) and expanded up to 400-fold. the a1-insulator decreased the number of il-3-independent colonies by 78-93% over the uninsulated vectors. the uninsulated vector-transduced, il-3-independent colonies, were greatly expanded in culture with 10% il-3 over the a1-transduced colonies (sffv, τνs9.3.55, a1-tns9.3.55: 48, 40, 10 fold change, respectively). il-3 independence as a transformation event was confirmed in vivo by the development of overt leukemia (hyperleukocytosis, splenomegaly, bm-and extramedullary site-infiltration) in mice transplanted with the il-3-independent and expanded colonies. summary/conclusions: under forced oncogenic conditions, the a1 insulator effectively protected a therapeutic vector from vector-mediated genotoxicity. a1 may serve as a safety feature in the construction of globin-sin-lvs. background: novel rare nucleotide substitutions are frequently identified in rhd, the gene encoding the immunogenic d antigen of the clinically-relevant rh blood group system, resulting in d variant phenotype. so far, it has been commonly accepted that substitutions of amino acids located either in a transmembrane or intracellular domain of the rhd protein induce weak d phenotype, i.e. reduced d antigen density at the surface of red blood cells. recently we showed by functional analysis using a "minigene splicing assay" (msa) that a decrease in d antigen expression may be due also to alteration of cellular splicing. aims: here we pay attention to the general disruption of this mechanism and the related phenotypic consequences in novel and previously reported single-nucleotide variations in rhd. we then sought to characterize functionally by msa novel candidate splicing variants in rhd. then we extended the project by studying prospectively all single-nucleotide variations reported in rhd exons, in order to assess globally the correlation between in silico prediction and functional analysis and to gain insights into the reliability of bioinformatics tools in line with the available phenotypic and/or clinical data. methods: seventeen novel or uncharacterized rhd variations, including missense, synonymous and intronic substitutions, were selected for functional analysis by msa in human cell models. a second set, including 46 missense variants reported in rhd exons 6 and 7, was further analyzed. functional data were compared with an algorithm derived from the quepasa method and tools available in the alamut suite. a published 3d protein model was used to visualize the location of missense amino acid substitutions and to assess potentially their respective phenotypic consequence. results: a novel "universal" minigene was validated and used successfully to characterize eleven novel splicing variants. those variants include six intronic and four missense substitutions close to the consensus dinucleotide splice sites, as well as the c.1065c>t synonymous variation associated with a weak d phenotype, which creates a de novo splice site. very interestingly, c.1154g>t (gly385val; d-negative) disrupts totally normal splicing, while c.1154g>c (gly385ala; weak d) and c.1154g>a (gly385asp; d-negative) only partially alter the mechanism. further visualization of amino acid changes in a 3d model suggests that gly385asp, but not gly385ala, dramatically impair rhd protein structure/folding. subsequently the global analysis of mutations in rhd exons 6 and 7 by msa showed that inclusion of whole exon sequence in the mature transcript is significantly reduced in 15/46 (32.6%) variants, which correlates well with the quepasa-like prediction (sensibility = 0.93, specificity = 0.94). additionally, while normal exon inclusion is affected by c.1012c>g (weak d type 70), the associated leu338val substitution does not seem to be deleterious to the protein. summary/conclusions: on the basis of our functional data, this work shows that splicing disruption in the presence of rhd variants is a common and general mechanism that may act independently or synergistically with alteration of protein structure through amino acid substitutions, resulting in a weak d phenotype. it also illustrates the potency of combining functional tests and in silico tools towards the phenotypic/clinical interpretation of rare variants. background/aims: monetary and non-monetary incentives may support blood services in recruiting blood donors but have also been criticized for violating ethical principles and threatening blood safety by attracting donors with a high risk for infectious diseases. although incentives for blood donors have been discussed extensively over the past decades, empirical research on this topic remains limited. the aim of this study was to describe attitudes towards incentives for blood donors in europe and show donor return rates of compensated and non-compensated blood donors in south-west germany. methods: first, we present results of a secondary analysis of the eurobarometer, a nationally representative survey in all member states of the european union. in 2014, participants were asked to evaluate eight potential incentives for blood donations as acceptable or unacceptable. these incentives were refreshments (e.g. coffee), physical check-ups (e.g. blood pressure), free (testing) laboratory parameters, free medical treatment, complimentary items (e.g. first aid kits), monetary travel reimbursements, additional cash reimbursements, and release from work. second, we conducted a retrospective analysis of donor return patterns of 6.210 compensated and 68.205 non-compensated donors who started donating blood at mobile and fixed donation sites. compensated donors received either 25 eur as a regular reimbursement for their expenses (at a fixed donation site), in accordance with the german transfusion law, or a singular free entrance for an amusement park (at a mobile donation site). these compensated donors were compared with noncompensated donors who started either at a fixed or mobile donation site. chisquare statistics were used to test for differences in regular donor status after 24, 36, 48 and 72 months between compensated and non-compensated first-time donors. results: among german participants of the eurobarometer, physical check-ups (51.9%), refreshments (50.0%) and free (testing) laboratory parameters (38.3%) showed the highest acceptance as an incentive for blood donors. travel reimbursements and free medical treatment were rated as acceptable by 28.6% and 25.3%, respectively. the lowest acceptance was for release from work (17.8%), complementary items (13.9%) and additional cash reimbursement (11.8%). interestingly, the acceptance of potential incentives varies considerably across europe. in south-west germany, donor return of first-time donors differed significantly by type of compensation. among compensated first-time donors, who received 25 eur as a monetary reimbursement, the proportion of regular donors after 48 months (19.9%) was significantly higher than among comparable non-compensated donors (9.2%). however, a non-monetary compensation (free entrance) did not increase donor return rates. conclusion: the eurobarometer survey indicates that in most european countries monetary incentives are only accepted by a small minority. refreshments, checkups, free (testing) laboratory parameters and free medical treatment were most popular as incentives for blood donors. however, results of our four non-randomized donor samples from south-west germany suggest that monetary compensation may increase the likelihood of donors returning to fixed donation sites. regular monetary reward may therefore help to recruit regular donors especially in urban settings. incidentally, non-monetary compensation by a free entrance, however, may not affect donor return. background: previous research showed that whole blood (wb) donors that are temporarily deferred on-site are at higher risk of lapsing, yet very little studies have focused on differentiating the effects that different deferral reasons (e.g., travel, hemoglobin [hb]) may have on donor lapse. in addition, donor experience (i.e., firsttime or repeat donor) has also previously been found to affect donor lapse, yet novice (1-5 prior donations) and reactivated donors (returning after years of not donating) may respond differently. finally, it is currently unclear how and why different deferral reasons and donor experience interact in influencing donor lapse. aims: our aims were to understand 1) how deferral reasons and donor experience jointly affect donor lapse, and 2) why donors may lapse after temporary deferral. methods: a mixed methods approach was used. first, we used sanquin's donor database for a quantitative analysis of return behavior of all dutch wb donors between 2013 and 2015 (n = 343,564). the first wb donation for each donor was identified as the target donation. lapse was defined as non-return within a followup period of two years after the target donation. target donations included 25% new donors, 41% novice donors, 30% experienced donors, and 4% reactivated donors. deferral reasons included travel, hb, medical short-term (<28 days duration), medical long-term (>28 days duration), and miscellaneous. next, we interviewed 31 temporarily deferred donors to understand the deferral process from their perspective. semi-structured interviews were used to understand how these donors cognitively and emotionally experienced on-site temporary deferral. we analyzed the interviews (using the framework approach, cf. hillgrove et al., bmc public health, 2012) to identify key topics and underlying themes. results: of target donations, 11% were deferred, mostly for travel (40%), medical short-term (23%), and hb (19%). survival and time-to-events methods showed that the different deferral reasons and donor experience levels differentially impacted donor return or lapse. importantly, experience and deferral interacted in influencing return (rate). for instance, deferred new donors were more likely to lapse than eligible or experienced donors (ors < 0.75, p's<0.001). even though deferral also affected return of experienced donors, this effect was smaller or even non-existent for certain deferral reasons (e.g., travel-and hb-related deferrals). qualitative results showed that almost all donors experienced temporary deferral as disappointing, particularly when it was unexpected (e.g., first-time deferral). not all donors (fully) understood the aims of deferral or how to prevent on-site deferral. donor beliefs about why deferral would lead to lapse were related to recurring deferrals, (mistakenly) interpreting deferral as permanent, or feeling all the effort did not pay off. summary/conclusions: reasons for temporary deferral differently impact risks of donor lapse at different levels of donor experience. for new donors all reasons for deferral are related to higher risks of lapse, whereas some reasons for deferral seem not to affect lapse among more experienced donors. unexpected or recurring deferrals may explain why donors lapse after temporary deferral. blood banks may tackle disappointment after deferral by explicitly showing that the donor is still valued, for instance by using personalized communication or offering an alternative good deed. background: blood donors experience a temporary reduction in their hemoglobin (hb) value after whole blood donation. in the netherlands, the hb value is measured before each donation, and a too low hb value (cut-off values: 8.4 mmol/l (135 g/l) for men and 7.8 mmol/l (125 g/l) for women) leads to a deferral for donation, in order to prevent iron deficiency and anaemia. the minimum interval between two donations is internationally set at 8 weeks, but over time donors exhibit iron deficiency so that blood donors are temporarily deferred from donation each year. in the us 35% -75% of deferrals are due to low hb, especially in women (editorial, transfusion, 2012) . due to the recovery process after each donation and the unobserved heterogeneity of donors, advanced statistical methods are needed to model the longitudinal data of hb values of blood donors. aims: to estimate the shape and duration of the recovery process of hb until the hb value has returned to its pre-donation level, to assess whether one can distinguish between donors with fast and/or slow recovery of their hb level and to predict future hb values. methods: the study is based on data of the donor insight study, which was a prospective cohort study performed by sanquin in the netherlands from 2007 to 2016. we employed three statistical models for the hb value: (i) a mixed-effects models, (ii) a latent-class mixed effects model, and (iii) a latent-class mixed-effects transition model. in each model, a flexible function was used to model the recovery process after donation. the latent classes identify groups of donors with fast or slow recovery times, and donors whose recovery time increases with the number of donations. the transition effect accounts for possible state dependence in the observed data. all models were estimated in a bayesian way, using data of a sample of 1000 new entrant donors (500 males and 500 females). prior information from the clinical literature (boulton, vox sanguinis 2004) about the recovery process three days after blood donation was incorporated into the analysis since these values were not identified in the observed data. results: the results show that the latent-class mixed-effects transition model fits the data best. we also found that the recovery process shows a concave process (initially fast followed by slower recovery). the estimated recovery time is much longer than the current minimum interval of 58 days between donations. namely, depending on the subgroup that the donor belonged to, males showed a recovery time of 100 to 419 days, while the estimated recovery time for females varies between 54 to 503 days. these results suggest that an increase of this interval may be warranted. summary/conclusions: the analysis shows the usefulness of the sophisticated statistical models that make use of historical information to model complex processes in time, in this case the hb trajectory over time across repeated donations. in addition, our results suggest a (much) longer time lag between subsequent donations to avoid anemia. background: complications of blood donation are known to reduce donors' return for future donation. the episode study (experience success in donation) showed that water drinking shortly before donation had an effect of 23% reduction of selfreported vasovagal reactions (vvr) in younger novice whole blood donors (wiersum-osselton, transfusion, 2019) . aims: in this study we analysed the return for a subsequent donation of the donors participating in the episode study. this was a predefined secondary outcome of the episode study. methods: the episode study was conducted in young (<30 years) whole blood donors making their first, second, third or fourth donation in geographically selected collection centres. the study interventions were: 330 ml water drink, 500 ml water drink or squeezing a ball (placebo intervention) during the wait after the screening interview and before phlebotomy, and a control group without intervention. participating donors were sent an online questionnaire about their experience within a week following their donation attempt. in the netherlands donors are usually invited for blood donation in accordance with hospitals' needs; the aim is to invite eligible donors at least once a year. donors were included in the return analysis if they had received at least one invitation within 400 days after the index donation and we analysed their return for a donation attempt within 421 days. associations with the interventions and donors' donation status, gender and reported symptoms at their index donation were analysed by calculating return percentage of eligible donors and by binomial logistic regression. results: out of the 8199 episode participants who had received an invitation, 6538 (79.7%) returned within the study period. there was no difference in donor return between the two water groups. the likelihood of return was significantly increased in both water and placebo intervention donors compared to the questionnaire group (or 1.2, 95% ci 1.06-1.4 and 1.3, 1.12-1.5 respectively). return was slightly lower in women (or 0.88, ) and lower in first-time donors (or 0.86, 0.77-0.96) than after a 2 nd -4 th donation. a staff-recorded or self-reported vvr at the index donation reduced donor return (or 0.47, 95% ci 0.37-0.60 and or 0.53, 0.46-0.61 respectively). other symptoms following donation were also associated with a lower return percentage. summary/conclusions: in this cohort of younger new and novice blood donors, 79.7% returned for a subsequent donation. a vvr (either staff-recorded or selfreported) reduced donor return. donors who received a study intervention, either water or placebo, were more likely to return, whether or not they had suffered a vvr. it is conceivable that the mere fact of study participation could also have increased donor return, even in de questionnaire group; this will be examined in the total population of target group donors. background: the contribution of older blood donors to the blood supply is substantial. in australia, donors aged > 50 years contributed 41% of all donations made in 2017. however, with ageing, the general health status of older donors changes relatively faster, thus progressively affecting their ability to donate. an indepth understanding of the relationship between older donors' health status, future donation patterns, and risk of iron-deficiency could be of a great value to inform the blood service to predict the number of future donations, and manage the risk of iron-deficiency. aims: to understand the relationship between self-reported health, blood donation patterns, and the management of identified iron-deficiency in older blood donors. methods: we linked the sax institute's 45 and up study baseline data collected between 2006 and 2009 to the blood service donation records, inpatient records, and medicare records*. the data-linkage was conducted by centre for health record linkage. using these linked data, we examined the relationship between health, donation patterns, and iron-deficiency and its management. results: we followed up 22,058 active whole blood donors for 200,403 eligible person-years (average age at recruitment 56.4 years, 56.3% female, average follow up 9.1 years per-person). after adjusting for the effect of age, sex, body-mass index, education, non-english language spoken at home, country of birth, smoking, physical activity, regular use of multivitamins, alcohol consumption at enrolment, and total number of whole blood donations in the 2 years prior to enrolment, participants with better self-reported health at recruitment showed significantly higher rates of donation. excellent, very good, good, and fair/poor health status donors made 1066 (95% ci 1056-1075), 987 (981-993), 900 (891-909), and 710 (690-730) donations per 1000 person-years, respectively. iron-deficiency was identified in 8.9% of donors in the study (n = 1964, 95% ci 8.5-9.3) . sixty percent of those with iron deficiency (n = 1,175, 95% ci 57.6-62.0) visited their general practitioner (gp) within 60 days of the identification of irondeficiency, and 48.4% (95% ci 45.5-51.3) of those visiting gp underwent further iron status examination and monitoring. after adjusting for several potential confounders including the total number of donations made during the follow-up period, excellent self-reported health status was independently associated with lower risk of iron-deficiency (p for trend = 0.004). summary/conclusions: information on self-reported health status can be an effective indicator to estimate the future donation yield of an older blood donor panel, and risk of developing iron-deficiency. donors with better self-reported health had a higher number of future whole blood donations and a lower risk of iron-deficiency. donors referred to gps for management of their iron status utilised the health services as expected, however there is an opportunity to improve their contact with their gps. * medicare records was provided by australian government department of human services. anaemia is a major public health issue, affecting 25% of the population worldwide according to the world health organization. iron deficiency is responsible for approximately half of all cases globally, with other causes including anaemia of chronic disease, other nutritional deficiencies, haemoglobinopathies, renal impairment, malignancy and bone marrow disease. in the elderly, where anaemia is even more common, the cause is frequently multifactorial. anaemia is associated with increased mortality, decreased cognitive and physical function, depressive symptoms and fatigue, particularly in older adults. poor outcomes have also been reported in anaemic patients with underlying comorbidities such as cardiac and renal disease, and cancer. within a hospital setting, anaemia is highly prevalent. preoperative anaemia, affecting up to 30% of patients, is associated with poor clinical outcomes including higher in-hospital mortality, longer length of stay and higher icu admission rates. anaemia management requires a proactive and multi-faceted approach, typically involving a multi-disciplinary team in which the transfusion practitioner plays a vital role. this includes screening of high-risk patients and pre-admission clinics to identify and manage patients at high risk of peri-operative anaemia. implementation of patient blood management (pbm) guideline recommendations has been shown to be effective to prevent and optimally manage anaemia within the community and hospital settings. the transfusion practitioner has key roles in the coordination, monitoring and auditing of pbm programs. active patient involvement and engagement of all members of the multidisciplinary team, including primary care clinicians, are also key to enhance the success of such programs. tp01-02 the role of the transfusion practitioner in anaemia assessment and management: processes, tips and resources for creating background: patient blood management (pbm) is an evidenced based integrated multi-disciplinary approach aimed to improve clinical outcomes by effectively managing and conserving the patient's own blood, thus reducing unnecessary exposure to transfusion. pbm has the patient as the central focus with the aim being to improve their outcomes and include them in the process. pbm includes three pillars: 1) optimising the patient's own blood, 2) minimising blood loss and 3) optimising a patient's physiological tolerance of anaemia. delayed assessment/management of anaemia contributes to increased health costs and unnecessary blood transfusions, and transfusion has been recognised to be associated with increase morbidity and mortality. the term transfusion practitioner (tp) includes those known as transfusion nurses, transfusion safety officers, haemovigilance officers, or patient blood management (pbm) coordinators. a key aspect of the role is driving and influencing clinical blood management activities to help align practice to internationally recognised guidelines and standards, including pbm. aim: to demonstrate the tp role in anaemia assessment & management and discuss strategies, processes, tips and resources for creating organisational and cultural change to implement pbm. context: literature outlines the importance of a multidisciplinary team to implement pbm related changes, and tps play a fundamental role within these teams to support 'buy in'. tps are seen as enablers, pulling resources together, engaging with those involved, providing education and facilitating change. they are often the ones to conduct audits, collating data and evaluating outcomes. approaches to implement pbm should be tailored to suit individual organisations. the authors will outline different approaches, highlighting where the tp can support or lead activities. one approach to anaemia assessment is to undertake an audit, examples of available tools will be shown. with this data, the tp along with the pbm team can explore options for corrective action. these could include interventions such as developing a pathway where all or a specific group of patients are assessed and or treated either at a preoperative clinic, or with their local general practitioner; through to more complicated strategies such as establishing anaemia clinics. the skills of the tp are a valuable asset to analyse clinical specialties/patient mix who should be targeted to achieve best outcomes, they know the organisation and as such are well placed to help develop a process/concept that will suit, and they can provide education and support to promote and embed these practices. conclusion: appropriate assessment and management of anaemia requires a multidisciplinary approach. the tp plays an active and crucial role in this team. examples of processes, tips and resources to support change and embed a pbm culture across the clinical spectrum will be shared. 3d-s11-01 department of hematology and central hematology laboratory, inselspital bern, bern, switzerland immune haemolytic anaemia (iha) is characterized by an increased breakdown of red blood cells (rbcs) due to allo-and/or autoantibodies directed to rbc antigens with or without complement activation. clinical and laboratory signs of haemolysis in concert with the presence of a positive direct antiglobulin test characterize iha. alloantibodies formed during pregnancy and/or after prior transfusions may cause acute or delayed haemolytic transfusion reaction after transfusion of a rbc product incompatible with the specificity of the alloantibody. autoantibodies to rbcs reduce the survival of endogenous and hamper the recovery of donor rbcs after transfusion. lymphoproliferative disease, autoimmune disease, infection or drugs often cause autoantibodies to rbc, but frequently no obvious cause can be identified. besides the antigen specificity, the isotype critically determines the biological activity of rbc antibodies in vivo. the isotype defines the affinity to fc-gamma receptors on cells of the reticuloendothelial system as well as the capacity to activate the classical pathway of complement, igm being the most effective. antibody-mediated complement activation results in the opsonisation of rbc with c3bc/c3d with subsequent complement receptor-mediated removal by phagocytes (extravascular haemolysis). occasionally, complement activation proceeds via the activation of c5 to the formation and insertion of the membrane attack complex resulting in intravascular haemolysis. there is growing evidence that the innate immune system plays an important role in the pathogenesis of iha. the process of complement-mediated haemolysis results in systemic inflammation, which contributes to morbidity and mortality of patients suffering from iha. complement activation results in the release of anaphylatoxins, which are strongly vasoactive and mediate chemotaxis, inflammation and formation of radical oxygen species. release of cell-free haemoglobin and cell-free haeme upon haemolysis induces endothelial cell activation, no-depletion, cytotoxicity, ros formation and neutrophil activation. natural plasma scavengers, such as haptoglobin and hemopexin complex with their target molecules, cell-free haemoglobin and haeme, with subsequent removal of the complexes via cd163 and cd91-mediated phagocytosis. although being positive acute phase proteins due to consumption the plasma scavengers become exhausted during chronic haemolysis thereby failing to prevent the adverse biological effects of cell-free haemoglobin and haeme in the circulation. inducible haeme oxygenase-1 (ho-1) is an efficient cellular scavenger by breaking down haeme into biliverdin with subsequent formation of bilirubin, co and ferrous iron with subsequent oxidation to ferric iron and storage by the ferritin h chain. ho-1 has an established role in the systemic protection from systemic inflammation induced by haemolytic and non-haemolytic diseases. the lecture will emphasise the role of innate immunity with a special focus on different plasma-and cellular systems involved in the pathogenesis of systemic inflammation in patients suffering from iha. 3d-s11-02 understanding erythrocyte clearance c roussel, p amireault, p ndour and p buffet research and teaching, institut national de la transfusion sanguine, paris, france the clearance of erythrocytes is essential in physiology, disease and transfusion. elimination of erythrocytes altered because of senescence or pathological processes is expected to protect the microcirculation from obstruction by adhesive or rigid erythrocytes. it also contributes to the harmful consequences of anemia and hemolysis in hereditary and acquired red blood cells diseases as well as in conditions associated with auto-or allo-immunization. immunobiology has explored in great details antibody-mediated clearance of erythrocytes but conventional approaches may not be fully operational to explain delayed hemolytic transfusion reactions. some important clearance processes are independent from the recognition of molecules or antigens on the erythrocyte surface. increased erythrocyte stiffness triggers their clearance in hereditary spherocytosis, malaria and possibly also in the context of autoimmune anemia. knowns and unknowns on the mechanisms and sites of erythrocyte clearance will be presented based on a critical review of old and recent contributions. 3d-s11-03 cardiovascular and endocrine-metabolic diseases and aging, istituto superiore di sanit a, rome, italy existing literature indicates that red blood cells (rbcs), beyond gas transport, exert a complex role in human physiology, being involved in many functions essential to maintain ion, metabolic and immunological homeostasis. rbcs display an immunomodulatory activity on adaptive immune cells by promoting t cell growth and survival and inhibiting activation-induced cell death. the balance between cell death and survival controls t cell homeostasis and anomalies in this balance account for diseases linked to excessive or faulty t cell growth. rbcs are able to modulate innate immunity by binding endogenous molecules such as chemokines and mitochondria-derived dna, as well as external agents such as pathogens. rbcs can also directly modulate innate immune cell activation or tolerance by controlling the maturation of the circulating pro-inflammatory subset of dendritic cells (dcs). these cells are potent inducers of primary antigen-specific t cell responses, produce tnf-a when stimulated by lps and are the principal il-12p70-producing cells among leukocytes. the pro-inflammatory capacity of circulating dcs is controlled by rbcs that are able to inhibit their maturation and il-12 production. in diseases characterized by local th1 inflammatory response such as psoriasis vulgaris and rheumatoid arthritis, pro-inflammatory dcs play a role in the induction and perpetuation of inflammation. collectively, literature data indicate that rbcs exert important modulatory functions that may result in immune activation or quiescence, depending on the environmental conditions. when rbcs encounter a microenvironment characterized by an intense production of ros, the rbc defenses get overwhelmed or are unable to counteract the new pro-oxidant status and become themselves a source of ros, which cause the generation of senescent signals on rbcs. the major feature of oxidized rbcs is the clustering and/or the breakdown of band 3. other features are the complexation of hb with spectrin, the loss of glycophorin a, the externalization of phosphatidylserine and the reduction of the "marker of self" integrin-associated protein cd47. a similar senescence phenotype has been documented in rbcs during the storage period. oxidized, senescent or stored rbcs, due to surface antigen modification and to the release of pro-inflammatory molecules, fail to control immune cell homeostasis thus contributing to the perpetuation of inflammation and to the pathogenesis of immune-mediated diseases associated to oxidative stress, such as autoimmune diseases and atherosclerosis. our research group demonstrated that rbcs from patients with carotid atherosclerosis presented a senescent phenotype similar to that acquired by rbcs from healthy subjects following to in vitro oxidation. oxidized erythrocytes fail to control t lymphocytes apoptosis and lipopolysaccharide-induced monocyte-derived dc maturation, thus representing dangerous signals for adaptive and innate immunity and contributing to the pathogenesis of atherosclerosis. in conclusion, the crosstalk between rbcs and the immune system represents a mechanism to maintain immunological homeostasis. however, in high oxidative stress conditions, that can take place during a prolonged storage period or in particular diseases, rbcs can acquire a pro-oxidant behaviour and lose their functional and homeostatic features. by interfering in immune system homeostasis, rbcs become a potential tool that can be manipulated to improve or reverse pathological situations characterized by anomalies in the control of adaptive and innate immunity. transfusion therapy remains an important treatment modality for patients with sickle cell disease (scd). transfusions are given to lower the percentage of circulating sickle rbcs, and to decrease blood viscosity and have been shown in clinical trials to reduce the risk of stroke by 90%. however, many indications for transfusion in scd remain controversial partly due to insufficient randomized clinical trials data and in part because of our limited understanding of the complex pathologic networks leading to diverse disease complications in scd despite the common single mutation. similarly, we have incomplete mechanistic understanding of why chronic transfusion protocols must be continued for those indications supported by clinical data. the beneficial effects of transfusion therapy in scd need to also be weighed against potential transfusion risks including alloimmunization associated with lifethreatening delayed transfusion reactions, increased iron stores associated with increased oxidative stress and exposure to infectious agents. we believe that a deeper understanding of the benefits as well as harmful effects of transfusions is crucial to optimize our current transfusion therapy protocols in scd. this knowledge may provide highly needed guidance, which is currently lacking, for expansion or limiting existing indications for chronic transfusions in scd. 3d-s12-02 treatment of thalassaemia department of pediatric hematology, ege university, faculty of medicine, bornova/ izmir, turkey thalassaemia is a devastating blood disease with a significant worldwide burden. annually, 60,000 children are born with a major thalassemia. life-time rbcc transfusions and iron chelation remain standard of care treatment in thalassaemia. transfusion therapy still account for significant iron overload related morbidity and mortality despite chelation therapy which is associated with poor adherence, safety concerns and varied efficacy. higher risk for transfusion transmitted infections (ttis) exists for thalassemia patients whose transfusion exposure sustains lifelong. although, the risk of transmission for traditional viruses is exceedingly rare in the modern era, emerging infectious diseases continue to be recognized as potential threats to transfusion safety. the inadequacy of blood safety points to the necessity for an additional layer of security for the blood supply in the developing world. pathogen reduction technologies for rbcc may imply a proactive, more generalized approach against new and re-emerging pathogens in the developed world and may be an ultimate safeguard for transfusion safety in the developing countries. rbc alloimmunization may become a major challenge in thalassaemia management. prevention is the key reducing the burden of alloimmunization. while the recommendation is to transfuse thalassaemic patients with c/c,e/e,kell compatible blood, it is not universally practiced. extended molecular rbc typing may be an appropriate adjunctive test in addition to serological typing before embarking on transfusion therapy. if a complete rbc antigen profile has not yet been performed in an alloimmunized patient, genotyping is the only option for accurate detection of rbc antigens that may guide the antibody identification. allogeneic stem cell transplantation (a-sct) is the only available curative therapy in children with hla matched sibling which is available to approximately 20% patients. in the absence of msd, mud transplant with high compatibility criteria has still limited experience. mismatch related, cord blood and haploidentical donor scts are considered experimental. a-sct carries a substantial risk of saes and mortality, both increasing with recipient age and disease severity. dfs is 88% in paediatric and 65% in adults. gene therapy for correction of the a-globin chain imbalance overcomes the problems of donor availability and immunologic complications associated with a-sct. multicenter clinical studies on gene addition therapy by using self-inactivating lentiviral vector are currently underway. recently, gene editing by either gene disruption or gene correction emerged as a potential alternative to gene addition therapy in beta-thalassaemia. a new era of novel therapeutics is unfolding in thalassemia management. several targets have been identified that can improve alpha/beta chains imbalance, ineffective erythropoiesis, or iron dysregulation and a number of those now have agents in preclinical and clinical development. hydroxyurea may improve globin chain imbalance and be beneficial for reducing or omitting transfusion requirement in selected group of patients. ruxolitinib has shown the limited effect on pretransfusion haemoglobin and reduction in transfusion needs, but allowed steady decrease in spleen volume that may serve for avoiding splenectomy in beta thalassaemia. luspatercept may restore normal erythroid differentiation and improves anaemia and hepcidin mimetics or tmprss6 inhibitors may modulates ineffective erythropoiesis by iron restriction and improves anaemia and organ iron loading. background: thalassaemia major (particularly b-type) and sickle cell disease (scd) are the commonest clinically important haemoglobinopathies, representing major sources of morbidity. recommended therapy is regular transfusion of safe, good quality blood, and monitoring of related complications. thalassaemia international federation (tif) guidelines, in place since 1987, include strategies for precautionary measures and use of scientific progress in detection, inactivation and elimination of transfusion transmissible pathogens. antigen-matching strategies to avoid alloimmunization against rbc antigens and other measures including haemovigilance are key components for safe blood, alongside voluntary, non-remunerated blood donation and laboratory quality assurance programmes. aims: we present the contribution of tif and the greek experience in ensuring safety and availability of blood for thalassaemia patients applying internationally accepted standards and recommendations. methods: tif -a non-profit, patient-driven organization with 204 national thalassemia associations in 62 countries -promotes national control programmes for prevention and management contributing to the achievement of final cure. the main working methods are provision of education, expert support, networking, communications and projects to support improvements in the quality of health, social and other care. in greece, technical standards for blood donor selection and testing are applied in compliance with directive 2004/33/ec as well as haemovigilance programmes and traceability procedures for recording adverse reactions and events associated with the transfusion of rbcs (directive 2005/61/ec). pre-transfusion and transfusion measures recommended by the council of europe are applied. in particular, measures for transfusion of "the right blood at the right time for the right patient", leucodepletion, rbc washing and accurate cross-matching and antigen and antibody screening for an extended matching policy are practised. fresh (up to 15 days old) rbcs are used. molecular testing for abo and rh d is performed in cases with blood group discrepancies. haemovigilance in greece covers 95% of total blood supply. data on ttis in 3,067 patients with thalassaemia and scd-thalassaemia in 2010-2017 are analysed. results: tti prevalence in thalassaemia syndromes was: hbv 1.8% (occult type 1.3%), hcv 54%, hiv 0.3%, htlv 0.8%, wnv 0.5% and hev 3%. most frequent adverse reactions in 2015-17 were allergic (incidence 1:854), non-haemolytic febrile reactions 1:2,631, "other" 1:5,883, alloimmunisation 1:6,667, taco 1:100,013, tad 1:50,006, tt-hev 1:100,013. hyperhaemolysis was diagnosed in two scd patients, delayed haemolytic transfusion reaction in one thalassaemia intermedia patient. trends in 2010-2017 show reduced incidence of alloimmunisation against rbcs. rates of allergic and pyrexial ars remained stable. no major abo incompatibility case was reported and no fatal transfusion reaction of transfusion has been recorded. summary/conclusions: blood safety in transfusion has significantly improved in high and upper-middle income but unfortunately not in lower and low income countries. blood shortages and lack of stringent protective measures for thalassaemia patients is the reality for many developing countries. tif focuses particular attention on the provision of support and the promotion of initiatives promoting the safety and adequacy of blooda key component of the lifelong management of patients with transfusion-dependent thalassaemia. background: b thalassemia is the most common group of hereditary hemoglobinopathy diseases. affected people with major thalassemia are dependent on regular blood transfusion which leads to iron overload. hepcidin is a peptide and an important regulator of iron homeostasis. expression of this hormone is influenced by polymorphisms within the hepcidin gene, hamp. aims: this study aimed to analyze the association of three polymorphisms in promoter of hamp, rs10421768, rs117345431, and rs142126068 with iron overload in major b thalassemia patients who do not respond to iron chelating therapy. materials and methods: a total of 102 samples from major b thalassemia patients were collected. genomic dna was extracted and sequenced for snps rs10421768, rs117345431, and rs142126068. statistical analysis was performed on ibm*spss* statistic 23 using independent t test and fisher test. results: our analysis revealed statistically significantly difference between the level of cardiac iron concentration and c.-582a>g variant (p = 0.02). for rs117345431 statistical analysis was on the edge of significant relationship between minor allele and serum ferritin (p = 0.058). all samples were homozygous for allele t of rs142126068. summary/conclusions: different factors affect iron overload in thalassemia. our findings and others emphasize the role of hepcidin polymorphism as a key component in iron homeostasis. ten to twenty years ago, countries in south eastern africa faced the peak of the devasting hiv/aids epidemic leading to an up to 20 years drop in general life expectancy. with the burden of hiv/aids falling mainly on the economically active population of young and medium-aged adults, the epidemic endangered social and economic stability in nations most heavily affected. today, despite aids still being a major cause of death in south eastern africa, the epidemic has become an example of public health gains that can be achieved through programmatic, evidencebased approaches that are endorsed by globally aligned policy and funding strategies. based on his work from lesotho, where one out of four among adults is infected by hiv, niklaus labhardt will take the auditors through the history of hiv programs in south eastern africa and show how innovative, pragmatic and evidence based implementation brought the region to a stage where the goal to end the aids epidemic by 2030 might be in reach. background: in france, the deferral for men who have sex with men (msm) was reduced from permanent to 12 months in july 2016. since this change has not impacted the residual risk (rr) of undetected hiv among blood donations, the ministry of health is considering a greater access of blood donation to msm. two scenarios have been studied: s1. deferral of msm during the 4 months preceding the donation; s2. deferral of msm who have had more than one sexual partner in the 4 months preceding the donation, similarly to all other blood donors in france. aims: to assess the impact of these two scenarios on the hiv rr estimated over the period july 2016-december 2017 which is the baseline rr with the current 12month deferral for msm. methods: baseline hiv rr was calculated with the classical incidence-window-period method, where hiv incidence was derived from a detuned assay (eia-ri) detecting recent infections (≤180 days) since all hiv-1 antibodies positive blood donations are tested with this test. the assessment of the impact of both scenarios on the baseline hiv rr was based on (i) data obtained from surveys among msm in the general population and in blood donors (compliance survey), to estimate the number of additional msm who would give blood in each scenario, and on (ii) hiv incidence estimate among these additional donors. this incidence was estimated: for s1, from msm blood donors with the current deferral policy (12 months) and for s2, from monogamous msm of the general population. results: from july 2016 to december 2017, 8/28 (29%) hiv-1 positive blood donors tested with the eia-ri were identified as recently infected, allowing to estimate the baseline hiv rr at 0.16 in 1 million donations [95% ci: 0.04-0.34], or 1 in 6,380,000 donations. for s1, the number of additional msm donors was estimated at 733 and the number of additional hiv positive donations at 0.09, resulting in an hiv rr of 0.16 in 1 million donations [95% ci: 0.04-0.35] or 1 in 6,300,000 donations. for s2, the number of additional msm donors was estimated at 3,103 and the number of additional hiv positive donations at 4.92, resulting in an hiv rr of 0.23 in 1 million donations [95% ci: 0.05-0.56] or 1 in 4,300,000 donations. sensitivity analysis shows that if both the number of msm and the hiv incidence were multiplied by 1.5, the risk would be 1 in 6,225,000 donations for s1, and 1 in 3,000,000 for s2. summary/conclusions: for both scenarios, the hiv rr remains very low. for s1 (4-month deferral), the risk is identical to the baseline rr and is very robust to variations in the model parameters. for s2 (no more than one sexual partner, 4 months), the risk is 1.5 higher than the point estimate of the baseline rr and sensitivity analysis shows that this estimate is less robust than for s1, since the risk could be 2 times higher than the baseline rr. for both scenarios, there was a modest increase in eligible msm donating. 3d-s13-03 background: recruiting safe blood donors amongst the largest hiv-positive population in the world is a major challenge for south african blood transfusion services. south african donor deferral criteria and deferral periods for perceived high risk activities have evolved over time, but current risk factors for infection have not been formally assessed. in addition, most studies have reported risk factors for prevalent hiv infection whereas risk behaviours for incident infection are more informative as donations with these infections could occur during the window periods of available screening assays. aims: to identify the demographic and behavioural risk factors associated with incident hiv infection among blood donors in south africa. methods: we conducted a case-control study with incident hiv-infected blood donors compared to infectious marker negative controls. incident hiv cases and controls seronegative for hiv, hepatitis b and c viruses and syphilis were accrued from a donor pool covering 8 of 9 provinces in south africa. controls were frequency matched at a 3:1 ratio to cases on race, age and geography. incident hivinfections were hiv rna positive by individual donation nucleic acid amplification testing (id-nat; procleix, grifols) but antibody (ab) negative (prism, abbott) as well as those rna+/ab+ donors with recently-acquired hiv based on limiting antigen avidity (lag) assay results with normalized optical density values of < 1.5. eligible cases and controls completed a confidential audio computer assisted structured interview (acasi) on motivations for blood donation and behavioural factors, including behaviours in the 6 months before donation. frequencies and measures of statistical association for risk behaviours comparing cases and controls are reported after adjusting for multiple comparisons. results: from november 2014 to january 2018, we enrolled 323 incident hiv cases and 877 controls; 202 (62.5%) cases and 544 (62%) controls were ≤ 29 years old. there were significantly more female cases 230 (71.2%) than female controls 439 (50.1%) (p < 0.0001). significant hiv risk factors (all p < 0.0001) reported within the 6-months before donation included: having a primary sex partner who is male; reporting increasing numbers of male sexual partners for both females and males; frequency of vaginal sex; frequency of vaginal sex without condoms; use of methods to clean, dry, or tighten one's anus before sex; and having visited a traditional healer for medical care. lack of medical aid (private health insurance) and reports of injury or accident with blood loss were also associated with an incident hiv infection. summary/conclusions: our study has identified a set of novel, putative risk factors for incident hiv infection among south african blood donors while confirming a number of previously known sexual risk behaviours. not having private health insurance and being injured may be markers of socio-economic context that place individuals at higher risk rather than behaviours that directly increase hiv transmission risk. the detection of risk behaviours by acasi in donors who passed predonation questionnaires and interviews suggests that acasi has the potential to improve risk behaviour identification. background: in france from 2000 to 2016, among male blood donors (mbds) found hiv-1 positive at blood donation screening, 31% did not disclose any risk factor for hiv infection during post-donation interviews, while 38% reported having sex with men (msm), and 24% and 7 % reported heterosexual sex (hts) and other risk factors, respectively. aims: in order to gain new insights into the risk factors for hiv-1 infection in mbds, we performed an hiv-1 genetic network analysis, including hiv-1 positive mbds and patients included in the french primary hiv infection anrs co6 primo cohort (pc). methods: 284 mbds, who donated blood between 2000 and 2016, and 1340 pcs, included between 1998 and 2014, were studied. epidemiological data were collected by the french blood service (efs) upon blood donation or post-donation interviews for mbds, and upon inclusion for cps. viral strains were sequenced and genotyped in pol gene, and a recent infection assay was performed to date infection in mbds (recent: <6 months). a partial transmission network was computed based on tamura-nei 93 nucleotidic distance (threshold for hiv-1 s/t b = 1.5%; for non-b s/ t = 0.8%) and assortative mixing was evaluated for mbds epidemiological data, including risk factors for hiv infection (msm, hts, others and unknown). selfreported data were then compared to assortativity-enhanced data. results: hiv-1 strains from 81 mbds and 126 pcs were linked into 54 clusters including at least one mbd. primo-only clusters were excluded from the analysis. compared to mbds who did not cluster, those found linked to the network were younger (30 vs. 36 year-old; p < 0.01) and were more likely to have a recent infection (48% vs. 34%; p = 0.03). assortative mixing indexes showed that paired individuals were more likely to live in the same area (p < 0.001) and to have the same risk factor for hiv infection (p < 0.001) compared to a random distribution. imputing msm risk factor to non-msm individuals paired with msm changed the distribution of risk factors as follows: msm: 38% vs. 49%, hts: 24% vs. 21%, other: 7% vs. 6% and unknown: 31 vs. 24%. summary/conclusions: after validating the assortativity of risk factors between paired individuals, and imputing msm risk factor to individuals self-reported as non-msm (including those with no identified risk factor), up to 18% (31/176) of mbds could be reclassified as msm. this is a worst-case scenario, as the network analysis does not exclude the possibility of one or several persons between two paired individuals (missing link). altogether, these results could help reevaluate the hiv residual risk linked to msm mbds, especially in the frame of the evolution of blood donor deferral criteria. background: although most individuals remain asymptomatic, htlv infection can lead to adult t-cell leukaemia/lymphoma (atll) and htlv-1 associated myelopathy (ham). the serious nature of these diseases, evidence of transmission via non-leucodepleted blood, and concern about a high prevalence among donors originating from endemic areas led to the uk blood services introducing universal blood donation screening in 2002. monitoring through routine surveillance commenced and htlvinfected donors were invited to participate in the htlv national register cohort study to assess disease progression. these data together with evidence from lookback to previously untested donations and cost-effectiveness analysis were reviewed by an expert working group in 2013 and 2015. aims: to describe the epidemiology of htlv among uk blood donors and evidence of disease progression from long term follow up of asymptomatic donors. methods: uk blood donations screened, and infected donors identified are reported to a national surveillance scheme. these donors are contacted, their results explained and information about clinical history and possible sources of infection are collected. where appropriate, htlv-infected donors are consented to the register, with participants completing a baseline questionnaire about their health, flagged in registries for cancer or death, and followed up about every 2-3 years. results: in the uk 2002 -2017, 254 htlv-infected donors were identified. prevalence among new donors was steady around 5/100 000 donations. prevalence among repeat donors peaked in 2002 (2.7/100 000 donations), with most in previously untested. from 2004 to 2016, prevalence of 0.07 per 100,000 donations (average of one positive/year) was recorded. in 2017, prevalence among new donors increased to 9.9/100,000 donations (17 positives), with increased numbers associated with asian ethnicity and coinciding with an increase in collections from bame groups. overall, most were women (182/254, 72%), uk-born (125/254;49%) and htlv-1 infections (228/254;90%). mean age was 43 years. almost all positive donations were from previously untested donors (240/254), with seroconversion within a year of previous donation confirmed for only 5 of the 14 previously tested donors. typically, infections were associated with endemic countries (including caribbean region, west africa, iran, india and japan), acquired through breast feeding or from their heterosexual partner originating from these countries. interestingly, three were thought to have been infected through self-flagellation. a total of 114 htlv-positive asymptomatic blood donors have already been recruited to the htlv national register, and during over 800-person years follow-up, none had developed atll or ham. summary/conclusions: over 16 years of testing, few seroconverters were identified, suggesting very little ongoing transmission among uk blood donors. the lack of disease among the cohort study was also reassuring, although it is likely too early to detect associated symptoms of a slow progressing disease. recruitment to this unique dataset continues, also outside of the blood donation setting. as a result of these surveillance data, evidence from lookback, and cost-effective analysis, in 2017 nhsbt ceased to test donations from previously tested donors unless the donation was being used to manufacture a non-leucodepleted component. finland lies in northern europe between the 60°and 70°n latitude. the length of the country is 1150 km and width 550 km. by surface area it is the fifth largest country in eu. the population of the country is 5.5 million resulting in the lowest population density in eu (15.8 inhabitants/km3). the whole country is inhabited, although most of the population is packed in the south. the climate of finland is influenced mainly by its latitude, but the warm waters of the gulf stream and the north atlantic drift current also play a role. due to finland's northern location, winter is the longest season. the southern portions of the country are snow-covered about three or four months of the year, and the northern regions for about seven months. long distances, low population density and the extreme climate give logistical challenges. it is estimated that these logistical costs can be as much as 10-20% of gdp in finland. the finnish red cross blood service (frc bs) has been the nationwide blood service provider in finland since 1948. frc bs collects annually about 200 000 whole blood units of which 50 % are collected in 10 fixed sites and 50 % in mobile sessions around the country. central activities (donor recruitment, medical support, production, testing, supply chain management, digital services and administration) are located in helsinki. management of transfusion is highly dependent on the logistical arrangements from blood donation sites to the central facilities and from the central inventory to the hospitals. the logistics is outsourced to three major partners all of whom have their roots in nationwide public transportation and logistics services. posti ltd is a state owned company having its roots in the national postal and telecom office. today it is the leading postal and logistics service company having the widest network coverage in finland. blood units collected at different fixed sites and mobile sessions are transported overnight by posti ltd to the frc bs central facilities by 8 am on the day following the blood donation. posti ltd is also used for the regular deliveries of blood products to the hospitals. the other important partner is matkahuolto ltd, which was founded in the 1930s to maintain bus stations and to serve as a common marketing company for the bus and coach services in finland. it maintains a nation-wide package delivery system based on the scheduled bus route network. matkahuolto ltd is used to transport donor testing samples from the donation sites to the central laboratory. by this arrangement it is possible to obtain most of the donor samples to the laboratory around midnight, which significantly speeds up the completion of laboratory results. the third logistics partner is jetpak finland ltd, which operates the air freight for the national flight company finnair. blood transfusion services can be managed centrally in a large sparsely populated country in a manner that is of high quality, safe and cost effective. however, the supply chain has to be planned carefully. background: elearning is a divisive topic. it is often criticised as an inferior form of education while simultaneously being promoted as a means to provide education to large numbers of people in a consistent, cost-effective manner. bloodsafe elearning australia (bea) is a government-funded blood transfusion education program that commenced in 2007 and provides courses in clinical transfusion practice and patient blood management (pbm) including: -clinical transfusion practice (4 courses) -pbm: general (7 courses) -pbm: medical (6 courses) -pbm: acute care and surgical (4 courses) -pbm: obstetrics and maternity (3 courses) -pbm: neonates and paediatrics (7 courses) aims: to determine the engagement, outcomes and impact of learning of bloodsafe elearning australia courses. methods: a retrospective analysis of user registrations, course completion records, course evaluation data and red cell usage in australia to determine learner demographics, and the impact on acquisition of knowledge and application to clinical practice. results: in the period from 1 july 2007 to 31 january 2019: -489,600 people registered as learners -1,072,299 courses were completed -these learners came from 182 countries, with 11,141 (2.3%) of them from outside of australia. analysis by profession shows that: -82.4% are nurses and/or midwives -11.2% are medical -6.4% are laboratory, anaesthetic technicians or other. analysis of user evaluation data (n = 2,885) from 1 april 2015 to 31 january 2018 shows that these courses have a positive impact, with 88.7% of respondents stating they gained additional knowledge, 65.2% able to make changes to clinical practice, and 88.2% reporting that these changes will improve patient safety and outcomes. analysis of international participants shows greater benefits with 93.5% gaining knowledge, 76.4% able to change their clinical practice and 91.9% believing this will improve patient outcomes. analysis of red cell usage in australia shows that since 2012 there has been a 21.1% reduction in red cells issued. this has been achieved through a number of pbm activities including development of guidelines, research and audits, education, waste reduction strategies, and promotional campaigns. bloodsafe elearning australia courses on pbm were released in 2012 and are one part of this pbm activity, and it is notable that these courses have the widest reach as they are undertaken by a large proportion of doctors, nurses and midwives in australia who are not directly involved with the blood sector. stakeholder feedback shows that the program provides credible, consistent education that is cost-effective, reduces duplication, is 'best-practice' elearning, is readily accessible, and allows institutions to focus on the development of practical transfusion skills. summary/conclusions: this analysis shows that elearning is a well-accepted, wellutilised form of education for healthcare workers to learn about clinical transfusion practice and patient blood management, and learners gain knowledge that can change their clinical practice and improve patient outcomes. it is also likely that these courses have contributed to better utilisation of a scarce, freely-donated resource. this approach has global reach and availability, and is a cost-effective model for improving transfusion practice in the developing world by providing education for millions of healthcare workers. 3d-s14-03 prospective platelet auditing: analysis of trainee compliance with guidelines pathology, columbia university, new york, united states background: apheresis platelets are a component product with high cost and limited supply. furthermore, there is a potential for severe transfusion reactions associated with this product such as transfusion related lung injury (trali), and sepsis due to bacterial contamination. therefore, transfusion guideline compliance is closely monitored by many centers. this quality assurance analysis describes our experience with prospective platelet auditing performed by physicians in pathology residency training. aims: this study aims to evaluate the ability of physicians in training to perform prospective auditing and compare policy compliance for different levels of experience. methods: this is a quality assurance analysis of a prospective platelet audit program for a 12-month period (january 2018-december 2018). the blood bank paged the on call physician any time an order was placed for a patient with a platelet count of > 50,000/ll, ≥2 doses of platelets with no interim repeat count, or an unknown platelet count. audit records created by physician trainees in their first post graduate year (pgy1) were compared to subsequent years (pgy > 1). information collected included the total number of doses requiring approval, number of products approved, training year for the approving physician, and transfusion indication. cost analyses assumed $500 for a dose of platelets. descriptive statistics and comparative analysis using a pearson's chi-square were used with a difference of p < 0.05 considered statistically significant. results: there were 1446 platelet doses requiring approval with 670 (46%) routed to the pgy1 group and 776 (54%) to the pgy > 1 group. there were 847 (59%) ordered doses that were in compliance with hospital transfusion policy and 599 (41%) that were not in compliance with hospital policy. of the 847 appropriately ordered doses, the pgy1 group declined release of 7 necessitating the clinical team to insist upon release without approval, and there were zero such instances in the pgy > 1 group. when paged by the blood bank, pgy1 physicians approved product release not in compliance with policy for 191/670 (29%) doses while pgy > 1 physicians approved not indicated products for 79/776 (10%) of doses (p < 0.01). products not indicated by hospital policy were held from release by pgy1 physicians for 113/670 (17%) doses and 216/776 (28%) doses by pgy > 1 physicians (p < 0.01). the ordered doses not in compliance with hospital policy had an estimated cost of $299,500. of this cost, there was a calculated $164,500 savings of products not released due to prospective auditing. there was an additional potential savings of $135,000 for products not indicated but released ($95,500 from the pgy1 and $39,500 from the pgy > 1 group). summary/conclusions: despite a higher number of requests being routed to the more senior pgy > 1 group, there were a disproportionately higher number of out of compliance platelet orders being released by the pgy1 group in addition to withholding needed products on several occasions. potential mitigation strategies for this could include a closer level of oversight for pgy1 physicians, and the potential monetary savings could justify a hiring a dedicated patient blood management team or quality assurance manager to monitor compliance and provide feedback to clinicians. 3d-s14-04 what can we learn from how adverse events are detected? norwegian directorate of health, oslo, norway background: the primary aim of reporting systems, such as haemovigilance systems, should be learning and improvement and to identify risk areas, not simply counting errors. to understand and learn from adverse events the description of how, where and why they occur, and how they are detected, is important. to support our understanding, we use a predetermined classification that is required for reporting to eu, supplemented by classification suggested by ihn, who and ourselves. in 2015 we started asking the blood establishments what steps they would take to prevent recurrence of the event, and we added a simple classification to tell how the adverse event had been detected. aims: this study aims to analyze how different types of adverse events reported to the haemovigilance system were detected, whether the current quality management systems used in norwegian blood establishments had effective barriers and whether new barriers should be considered. methods: adverse events reported to the norwegian haemovigilance system in 2017 and 2018 were analyzed with focus on how the adverse event had been detected. in all cases classification had been performed by the reporter of adverse events and were confirmed, or reclassified if necessary, by the haemovigilance team before analysis. for analysis based on classification we used powerbi (microsoft). results: a total of 188 adverse events were reported from norwegian blood establishments. all had been classified according to how the adverse event had been detected. twenty (10.6 %) adverse events were detected because of alarms or warnings from it-systems or equipment. routine checks by blood establishment staff detected 39 (20.7 %) events and formal internal or external reviews detected one event. seven (3.7 %) events were detected because the donor became ill shortly after donation, but the illness was not caused by the donation. sixty-four percent of events were detected in a way that did not fit our present classification and hence were classified as "other". twelve out of 16 wrong blood in tube were detected by an alarm from the it-system or routine check, as were six of 22 events related to blood ordering, two of seven errors in testing, six of 17 events where incorrect blood had been transfused, and eight of 64 events related to donor selection. in 80 reports human error was listed as the cause of the event and 27 of these were detected by alarms or routine checks. summary/conclusions: detection of adverse events by alarms or routine checks are highly efficient when the blood establishment has historic data to check against, as exemplified with wrong blood in tube or a patient require irradiated blood components. when no historical data exists or when the quality management systems do not require routine checks, events are usually detected by chance. further analysis is needed to see if and where the quality management systems should be improved. the wide variety of adverse events can make it difficult to select which area to prioritize in the improvement work. results: the hb measurements from the finger prick were on average 1.2 g/l (0.9%) higher than from the venous blood samples. the range of the difference was -21 -+22 g/l. these results were used in order to add novel information to determine the measuring uncertainty of hb measurement in frcbs. in 1.4 % (12/845) of the donors in this study the venous hemoglobin measurements were below the cut-off point of donor eligibility. in those measurements the difference of the finger prick and venous hemoglobin measurement was at most + 9 g/l. 92 % of the hemoglobin results from the finger prick were in the range ae10 g/l compared to the venous hemoglobin results. 63 % of the results from the finger prick were between ae5 g/l (the precision of the device) compared to venous hemoglobin results. in 13 cases the difference between finger prick and venous measurements was outside 2 standard deviations from the mean i.e. 2.5 % from the bottom (n = 9) or top (n = 4) of distribution. systematic errors were seen in some nurse's results both towards too low or too high hb result in the finger prick measurement and some nurses had random errors in both directions. the batch of cuvettes, donors' age, gender or the time of sampling were not detected to have an impact on the difference between finger prick and venous hb measurements in this study. summary/conclusions: the results of the poc measurements compared to the cell counter were in agreement with published data and with manufacturers' information on the device. the practical skill test is a workable way to develop competence and operations to measure the hemoglobin from the finger prick. it offers an opportunity to give personal feedback to nurses concerning their personal performance in the use of the current hb measurement technic. it also provided data on the accuracy of the poc method in the everyday donor selection process. background: whole blood donation has frequently been related to iron deficiency. a blood donor loses per donation about 8% (men) to 81% (menstruating women) of iron stores. to replenish the iron lost by blood donation in a donation interval of 56 days, a donor needs to absorb 4.5 mg iron per day. this amount exceeds the reported maximal amount of absorbed iron of 3-4 mg/day, eventually leading to iron deficiency, with consequences such as donor deferral and possibly iron deficiency-related symptoms (decreased physical endurance, fatigue, pica, restless legs syndrome, and cognitive functions). since hb levels do not reflect donors' true iron status, measuring ferritin is a better way to detect low iron stores in whole blood donors. studies from usa and denmark showed that on the introduction of ferritin measurement with either extension of donation intervals or iron supplementation in case of low iron stores, deferral percentages for low hb declined in both male and female donors. aims: to gain more insight in iron status of whole blood donors during their donor career, how this affects donor health and which measures may prevent low iron stores in donors. methods: in the netherlands, sanquin blood bank is currently implementing a policy with ferritin-guided donation intervals. in brief, ferritin levels are measured in all new donors and in repeat donors every 5th donation or in case of an hb below the deferral threshold. donation intervals are extended if ferritin levels are < 15 lg/ l, or ≥ 15 and ≤ 30 lg/l (for 12 and 6 months respectively). we anticipate that routine ferritin measurement will ultimately result in a lower prevalence of iron deficiency, less hb deferrals and improved donor retention. this will be further evaluated in a stepped wedge cluster-randomized trial 'find'em', which may also identify subgroups of donors prone to develop (symptoms of) iron deficiency. in addition, implementing ferritin screening may lead to a decreased donor availability. for this purpose, we modeled the impact of the implementation of our ferritin deferral policy on donor availability over time, which provides insight for both the expected size of the impact of the ferritin deferral policy and the time and rate at which this impact is expected to occur. this allows the blood bank to timely plan actions to counterbalance possible donor shortage and ensure an adequate blood supply. lastly, iron supplementation can be an alternative measure instead of donation deferral. as the used and recommended dosage of iron supplementation varies widely across blood services, sanquin is planning to start a new study in whole blood donors to gain evidence on the dosage and frequency of iron supplementation and its effect on ferritin and hemoglobin levels and donor health. results: the before-mentioned studies are ongoing and results will be expected from 2020 onwards. summary/conclusions: iron deficiency is a frequent side effect of whole blood donation. to prevent iron deficiency and its consequences, like donation deferral and health issues, more evidence-based insight in iron management of whole blood donors is being generated. 3d-s15-02 superdonors -genetic risk profile and risk of low hemoglobin deferral background: no reliable method exists for stratifying new blood donors into those who can maintain sufficient hemoglobin (hb) levels and those who will be deferred because of a low hb (<7.8 mmol/l [<12.57 g/dl] for women and < 8.4 mmol/l [<13.54 g/dl] for men). polygenic risk scores (prss) have shown great promise in predicting complex disease risk. prss could also prove useful for identification of donors genetically predisposed to low hb levels, and, thus, to an increased risk of deferral. aims: the objective of the study was to evaluate the association between prs (modelled to predict hb level as a quantitative trait) and risk of deferral as a binary outcome. methods: the danish blood donor study (dbds) is an ongoing nationwide blood donor cohort since 2010 with more than 110,000 participants. extensive genotyping has been performed on approximately 72,000 dbds participants using the infinium global screening array (illumina â ) and extended by use of imputing based on the pan-scandinavian reference genome. based on hb and genetic data on more than 150,000 icelandic individuals (an independent discovery cohort), we constructed 9 different weighted prss for individuals from dbds. information on the donors' whole blood donations following inclusion into dbds unto end of 2017 was obtained from a nationwide donation database, scandat. the best predictor of hb among the nine prss was chosen and used in all subsequent analyses. we performed multilevel mixed-effects linear regression analysis with hb as outcome, and prs as factorized explanatory variable with cutoffs at 5, 10, 25, 40, 60, 75, 90 , and 95 th percentiles, respectively. moreover, the models had a two-level clustering on donor id and donation site and an id-specific random intercept; and further adjusted for: sex(binary), age(continuous), year of donation(factorized), and time since last donation (continuous). lastly, risk of deferral was evaluated in random effects logit models with similar covariables and clustering structure. results: mean number of donations per donor after dbds inclusion was 6.9 donations. generally, we observed a statistically significant positive association between prs(hb) and current hb levels. compared with donors in the 40-60 prs percentile group, donors below the 5 th percentile had lower (-0.23 hb mmol/l (95% ci: -0.25; -0.21)) and donors above the 95 th percentile higher (+0.19 hb mmol/l (95% ci: 0.18; 0.21) hb levels. in the random effects logit models we observed a marked increase in deferral risk with decreasing prs percentile strata. with the 40-60 prs percentile stratum as reference, donors below the 5 th percentile and donors above the 95 th percentile had odds ratios of deferral of or = 2.58 (95% ci: 2.27; 2.93) and or = 0.46 (95% ci: 0.39; 0.54), respectively. summary/conclusions: we found a statistically significant positive association between prs(hb) and hb levels and a markedly increased risk of deferral with decreasing prs(hb). from a scientific point of view, it is unsurprising that a genetic score for hb from an independent cohort is associated with hb in another cohort. however, from a practical perspective, prss may be the first step in a personalized donation approach to donors and their risk of deferral. background: individually calibrated inter-donation intervals for repeat blood donors have the potential to minimize the risk of iron related adverse outcomes (e.g., hemoglobin deferral or collecting a donation from a donor with low or absent iron stores) without unduly impacting the donated blood supply. machine learning has shown promise for personalized clinical risk assessment. aims: our aim is to use machine learning to develop donor-specific, personalized inter-donation intervals that minimize the risk of adverse outcomes while maintaining or improving the adequacy of the donated blood supply. methods: using a public use dataset from the reds-ii donor iron status evaluation (rise) study (cable, transfusion, 2012) we defined donor profiles with physiological measures including hemoglobin, ferritin and soluble transferrin receptor along with questionnaire responses regarding diet, reproductive health indicators, and demographics. we used these profiles (58 features, 3,162 donations from 1,025 repeat donors) and the time until the next donation attempt to predict iron-related outcomes of the next donation attempt. possible outcomes were no adverse outcome, hemoglobin deferral, low-iron donation (ferritin < 20 ng/ml for women and < 30 ng/ml for men), or absent-iron donation (ferritin < 12 ng/ml for men and women). we trained multiple machine learning models on 2,543 of the donations and selected the model with the best performance (lowest cross-entropy loss in cross validation). we assessed the best model's performance on a hold-out test set of 620 donations, which were not used to train or select the model. we then used our model to generate risk estimates for these 620 test donors as a function of days since their last donation, which varied from 56 days to 250 days. to show individual variation, we generated graphical representations of individual donors' risk over time. results: ferritin, log ferritin, body iron, and time since last donation were most useful for predicting iron-related adverse outcomes at the next donation attempt. the estimated risk of adverse outcomes at the next donation attempt varied considerably across donors. as expected, the risk of adverse outcomes 250 days after the last donation was lower than the risk 56 days after the last donation for most donors (risk of hemoglobin deferral decreased for 84% of donors; risk for low-iron donation decreased for 61%; and risk for absent-iron donation decreased for 94%). summary/conclusions: the risk of iron-related adverse outcomes as a function of time since last donation varies considerably between donors. machine learning models trained on relevant donor profiles can effectively estimate how an individual's risk will change over time. individual risk estimates could allow blood centers to protect highrisk repeat donors while continuing to allow more frequent collections from low-risk donors. further study is needed to ensure this approach works well for donor classes that are not well-represented by the rise dataset, to assess risk prediction outside of the physiological measures collected in the rise study, and to determine the viability of assigning an optimal inter-donation interval to a first-time donor using this approach. background: iron depletion is common among repeat blood donors, who contribute a large proportion of the blood supply in many countries. exogenous iron from multivitamins with iron or iron-only supplements helps prevent donation-induced iron depletion, but whether dietary iron protects against iron depletion in repeat donors has not been rigorously evaluated. available data from the reds-ii rise study in the us (cable, transfusion, 2011) and from the danish blood donor study (rigas, transfusion, 2014) suggest minor impact of dietary iron consumption on blood donor iron status in multivariable regression models. both studies, however, analyzed food items singly, such as beef or fish, rather than in aggregate, so precision was limited. aims: to evaluate whether a composite measure of dietary heme iron consumption, weighted for frequency and iron content, was associated with incident iron depletion among repeat blood donors. methods: a re-analysis of the rise cohort was undertaken to test the hypothesis that reported levels of animal protein consumption was associated with lower risk for incident iron depletion among repeat blood donors. the six blood centers participating in rise enrolled first-time and frequent donors for 15-24 month follow-up of donation frequency and iron status. a brief checklist of 8 food categories was administered at baseline to assess frequency of consumption of several categories of animal protein that are rich in heme iron, the biochemical form of iron most readily absorbed. an iron composite score (ics) weighted for frequency and heme iron content was derived and subjects were grouped into tertiles (thirds) of ics. iron status was assayed at enrollment and study completion and at roughly one-third of donation visits in between. modified poisson regression with generalized estimating equations was used to generate risk ratios controlling for donation frequency and other covariates. results: of 2425 enrolled donors, 1406 were iron replete at baseline and completed the food checklist. the median value of the ics for each tertile (lowest to highest) was 7. 2, 13.1, and 22 .0 mg of heme iron weekly. these values are equivalent to approximately 3, 6, and 9 servings of beef per week, or alternately twice as many servings of chicken or pork. across 2236 follow-up visits with iron outcomes assayed, almost 33% of donor visits were associated with intermediate iron depletion (serum ferritin < 26 ng/ml) and 8.5% with complete depletion of iron stores, representing serum ferritin < 12 ng/ml. after controlling for demographic factors and donation frequency, the lowest tertile of ics was associated with a greater than 2fold higher risk for complete iron depletion during all follow-up visits (rr 2.40, 95% ci 1.51, 3.81, compared to the highest tertile). summary/conclusions: in this longitudinal evaluation of dietary iron and iron status, blood donors with low intake of heme iron had an elevated risk for developing advanced iron depletion. these results suggest that blood centers should continue to recommend iron-rich diets to repeat blood donors. background: blood donors lose approximately 250 mg of iron with every blood donation. as a result, frequent blood donors are at risk of iron deficiency and low hemoglobin (hb) levels, which may affect their health and eligibility to donate. lifestyle behaviors such as dietary iron intake and physical activity, may influence iron stores and thereby hb levels. gaining insight into associations between lifestyle behaviors and hb levels is valuable for blood supply organizations, as lifestyle behaviors can potentially be considered to prevent hb deferrals. examining the mediating role of ferritin, a measure reflecting iron stores, in these associations will help to gain insight into whether iron stores could be the limiting or enabling factor that links lifestyle behaviors to hb recovery after donation. aims: to investigate associations between lifestyle behaviors (dietary heme and non-heme iron intake and physical activity) and hb levels, and whether ferritin mediates these associations. methods: donor insight-iii (dis-iii) is a dutch cohort study of blood and plasma donors and included 2,552 donors. participants who were pregnant, had hemochromatosis, used iron supplements/medication, got a hysterectomy or bilateral oophorectomy were excluded (n = 292). hb levels were measured in edta whole blood samples using a hematology analyzer (xt-2000, sysmex, japan) and ferritin was measured in plasma from lithium heparin tubes (architect ci8200, abbott laboratories, u.s.a.). dietary heme and non-heme iron intake (grams/day) were assessed using a food frequency questionnaire adapted to measure iron intake. moderate-tovigorous physical activity (mvpa, minutes/day) was assessed using the international physical activity questionnaire (ipaq)-short form. results: in total, 2,260 (1,186 female) participants were included. donors with higher intakes of heme iron had significantly higher hb levels (regression coefficient (b) (95% confidence interval (95% ci)) in men and women respectively: 0.147 (0.069 to 0.225) and 0.094 (0.013 to 0.175) mmol/l), independent of age, smoking, menstruation, number of donations in previous two years, donation interval, sedentary behavior, the other lifestyle variable (i.e. (non-)heme iron intake or mvpa), and initial hb level. non-heme iron intake was negatively associated with hb levels (-0.015 (-0.026 to -0.004) and -0.018 (-0.032 to -0.005) mmol/l for men and women respectively). ferritin mediated associations between dietary iron intake and hb levels (indirect effect in men and women respectively: 0.073 (0.046 to 0.107) and 0.065 (0.031 to 0.100) lg/l for heme and -0.003 (-0.008 to 0.000) and -0.007 (-0.013 to -0.002) for non-heme). more mvpa was negatively associated with hb levels in men only (-0.004 (-0.008 to -0.001)), which was not mediated by ferritin. summary/conclusions: in conclusion, higher heme and lower non-heme iron consumption are associated with higher hb levels in donors via higher ferritin levels, indicating that donors with high heme iron consumption may be more capable of maintaining iron stores to recover hb levels after blood donation. more mvpa was associated with lower hb levels, although effect sizes were small, independent of ferritin. taking a donor's lifestyle behaviors into account may be useful in preventing low hb levels in blood donors. immune thrombocytopenia (itp) is still diagnosed by exclusion of other causes for thrombocytopenia. sensitive and specific detection of platelet autoantibodies may support the clinical diagnosis and prevent misdiagnosis of itp. for example, the direct monoclonal antibody immobilization of platelet antigens (maipa) assay, performed with in vivo sensitized patient platelets, offers platelet glycoprotein specific autoantibody detection with high accuracy. a drawback is that low platelet counts demand a large blood sample to have sufficient patient platelets available for analysis. circulating platelet autoantibodies are more difficult to detect by maipa; and may demand more sensitive detection platforms, such as those using surface plasmon resonance. in general, the presence of anti-gpiib/iiia, anti-gpib/ix and anti-gpv platelet autoantibodies is investigated. all these antibody specificities have been found in patients with itp. in itp, platelet autoantibody-mediated destruction via the spleen has been proposed; but also other mechanisms leading to low platelet counts in itp may play a role. inhibition of megakaryocytopoiesis by autoantibodies or by t cells has been suggested. in mice, gpib-directed antibodies induce loss of platelet-sugar epitopes, inducing hepatocyte-medicated platelet destruction. platelet autoantibodies can cause complement activation, which may contribute to platelet autoantibodymediated destruction. interestingly, we recently found that lack of detectable platelet autoantibodies is correlated with non-responsiveness to rituximab (cd20 moab) treatment in itp patients. in children with newly diagnosed and often transient itp, platelet autoantibodies of igg class or not often found, but of igm class are present for short duration. in conclusion, testing for platelet autoantibody characteristics and their pathologic effect may be helpful in establishing the diagnosis of itp and in choosing the best individualized therapy for itp patients. 4a-s16-02 thrombopoietin receptor agonist (tpo-ra) treatment raises platelet counts and induces immunomodulation in immune thrombocytopenia (itp) jw semple 1 , r aslam 2 , e speck 2 , j rebetz 1 and r kapur 1 1 lund university, lund, sweden 2 st. michael's hospital, toronto, canada background: itp is an autoimmune bleeding disorder in which autoantibodies and/ or autoreactive t cells target the destruction of platelets and megakaryocytes in the spleen and bone marrow. several therapeutic options e.g. corticosteroids, intravenous immunoglobulins (ivig), rituximab and splenectomy are available for patients but inadequate efficacy, side effects and/or expense can make them undesirable. for the last 10 years, tpo-ra e.g. romiplostim and eltrombopag have made a substantial contribution to the treatment of itp patient's refractory to first-line treatments. of interest, approximately 30% of patients that are tapered from tpo-ra therapy show a sustained response (e.g. a stable higher platelet count than before treatment). the mechanism of how tpo-ra induce these sustained responses is unknown. aims: to analyze the efficacy and immunomodulatory properties of a murine tpo-ra (amp4, amgen) in a well-established murine model of itp that demonstrates both antibody-and t cell-mediated thrombocytopenia (chow l et al., blood 2010) . methods: platelet glycoprotein (gp) iiia (cd61) knockout (ko) mice were immunized with cd61 + platelets and itp was initiated by the transfer of their splenocytes into mice with severe combined immunodeficiency (scid). the scid mice were treated with either placebo or tpo-ra weekly and platelet counts and serum anti-platelet antibodies were measured weekly. results: in an initial pilot dose escalation study, control na€ ıve scid mice treated with a single subcutaneous bolus of different concentrations of murine tpo-ra (1, 10 and 20 ug/kg) had significantly higher platelet counts by 72 h post infusion. in addition, compared with untreated mice, bone marrow histology revealed significantly increased numbers of megakaryocytes. maximal platelet count increases were observed with the highest tpo-ra dose and this dose was chosen to treat scid mice suffering from itp. when scid mice were treated with weekly injections of tpo-ra, platelet counts began to increase after 2 weeks and were fully rescued to control levels after 3 weeks post splenocyte transfer. of interest, compared with non-treated itp mice, serum igg anti-platelet antibody production in the tpo-treated mice was significantly reduced starting from two weeks post splenocyte infusion. summary/conclusions: these results suggest that murine tpo-ra is not only an efficacious therapy for murine itp but also induces immunomodulation indicative of immunosuppression. thus, this model may be able to elucidate the mechanism of how tpo-ra's induced immunosuppression in patients with itp. background: desialylation, the loss of sialic acid content on platelets (plts) glycoproteins (gps) was recently identified to contribute in immune thrombocytopenia (itp). however, the potential impact of autoantibodies (aabs) on megakaryocyte sialylation remains unclear. aims: to investigate the effect of itp aabs on plts and megakaryocytes (mks) sialylation and the subsequent impact on plt survival. methods: aabs from well-characterised itp patients induced gp-modifications were tested using a lectin binding assay. after incubation of mks or plts with itp or control sera, glycan changes were analysed by flow cytometry (fc). to investigate the impact of desialylation on plts life-span, the nod/scid mouse model was used. results: 112 itp sera were investigated in this study. 35 (31%) sera induced a significant increase in rca signal on plt surface compared to control sera from healthy donors (rca-mean fold increase (rca-fi): 3.23, range: 1.76-13.61, p = 0.0001). in addition, 23 (21%) sera caused higher ecl binding to test plts (ecl-fi: 2.31, range: 1.54-5.7, p = 0.0001). injection of desialylating aabs resulted in accelerated clearance of human plts from the circulation of the nod/scid mice which was significantly reduced by a specific neuraminidase inhibitor that prevents background: autoimmune hemolytic anemia (aiha) is a rare autoimmune disease characterised by hemolysis associated with the presence of immunoglobulins (igg, igm, or iga) and/or components of complement system on red blood cells (rbcs), which is usually demonstrated by a positive direct antiglobulin test (dat). depending on the presence of an underlying disorder, aiha can be subdivided into primary and secondary and, by the temperature at which autoantibodies bind optimally to rbcs, into warm antibody aiha (waiha), mixed aiha (including both warm igg and cold igm antibodies), cold agglutinin disease (cad), paroxysmal cold hemoglobinuria (pch) and dat negative aiha. a frequent finding in immunohematology is the presence autoantibodies on rbcs without clinical symptoms of hemolysis that may later develop. aims: the aim of this study was to analyse serologic findings and transfusion support in patients with aiha and also to analyse dat positive patients without clinical symptoms. methods: we included data for all adult patients with aiha and dat positive patients without clinical symptoms diagnosed and/or treated at the university hospital centre (uhc) zagreb, croatia in the period between 2014 and 2018. the diagnosis of aiha was defined by anemia with features of hemolysis (elevated bilirubin and/ or elevated lactate dehydrogenase and/or low haptoglobin level) and a positive dat. results: the data from 64 patients (52% women) meeting the inclusion criteria was analysed. the mean age at the time of aiha was 63 years (range 22-89 years). the mean hg level at diagnosis was 68.60 g/l. dat results were positive mostly with igg+c3d (59%) or igg (31%). most patients had warm aiha (70%). other types of aiha diagnosed were mixed aiha (15%), cad (11%), pch (1.5%) and dat negative aiha (1.5%). in 6 cases alloantibodies were detected with autoantibodies in the patient's plasma. patients were treated with corticosteroids as 1st line therapy and some with intravenous immunoglobulins (ivig). in severe or refractory patients rituximab and/or splenectomy was applied. a total of 80% of patients were transfused at a mean hemoglobin level of 67.88 g/l. during this period we detected 136 dat positive patients without clinical symptoms. summary/conclusions: most patients from our study were diagnosed with warm type of aiha, followed by mixed type aiha and cad. on the other hand, pch and dat negative aiha were very rare, which is in concordance with relevant literature. most patients were transfused despite therapy used, which is not desirable in patients with aiha and should be better controlled, especially in moderate cases of anemias, where this is rarely necessary. a significant number of patients that were dat positive without clinical symptoms may later develop aiha and should be closely monitored. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february 2017, there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to diagnosis, investigation and management of patients with aiha in english nhs trusts. methods: a survey of diagnostic and management practice was designed, piloted and disseminated to clinical transfusion leads in all english acute nhs trusts from november 2017 to march 2018. completion was by a consultant haematologist treating patients with aiha but a response that represented a departmental consensus was encouraged. results: responses represented 42% (58/137) of english acute trusts. median number of adults with aiha diagnosed annually was 4-6. in the preceding 5 years, 31% (18/58) recalled at least one patient who had died due to aiha. although 7% (4/57) undertook a bone marrow biopsy in all patients, 93% required additional features, mainly: neoplasia, age over 60 or being treatment-refractory. for patients with suspected drug-induced immune haemolysis, 59% (34/58) would not organise confirmatory tests, either because it was considered unnecessary (29/34), or because clinicians were unsure how to access tests (5/34). when determining aiha subtype, 29% (17/58) indicated there were no circumstances in which they would undertake cold antibody testing (antibody titre and/or thermal amplitude), with 12 considering this unnecessary and 5 unsure how to access tests. in 4 clinical scenarios of patients with aiha and dat positive to c3d ae igg ae cold associated symptoms, up to 87% (47/54) of respondents would not test for cold antibodies. for first line treatment of primary warm aiha, mean duration of prednisolone 1 mg/kg given before judging the patient refractory and reducing the dose was 3.5 weeks (sd 1.70, range 1-19 weeks). second line treatment of choice was rituximab for 82% (45/55) of respondents and splenectomy for 5%. intravenous immunoglobulin and splenectomy were the most cited rescue therapies. for primary cold haemagglutinin disease (chad), first line treatment was rituximab-based for 88% (49/56) but single agent steroid for 9%. we also explored the potential for future audit and research. 64% (37/58) of respondents were able to identify patients with aiha who previously required transfusion. 96% (55/57) of respondents would consider supporting a registry of patients with aiha requiring transfusion. the key questions that respondents thought a registry should address were: morbidity and mortality, treatment response, and differences in the diagnosis and treatment of aiha subtypes. there was uncertainty over access to cold and drug-induced antibody tests and clinicians do not always conduct bshrecommended cold antibody tests for aiha with c3d positive dat. initial treatment of primary warm aiha and chad broadly matched bsh guidelines although 44% (25/57) would continue prednisolone at 1 mg/kg beyond the recommended 21 days before starting a taper, with greater toxicity risk. summary/conclusions: the findings support the need for a range of research initiatives, including creation of an aiha registry. preoperative anemia is common and is associated with adverse outcomes in the peri-operative period. preoperative anemia also increases the risk of allogeneic blood transfusions, which may lead to increased perioperative mortality, increased hospital length of stay and infections. diagnosis and treatment of anemia is one of the tenets of patient blood management (pbm), along with reduction in unnecessary transfusions and diagnostic phlebotomy, as well as use of hemostatic agents to reduce bleeding among many others. effective pbm is multi-disciplinary, multi-modal, timely, individualized and patient-centered. early referral to pbm and multi-modal pbm interventions are associated with greater improvement in pre-operative hemoglobin. pbm has been shown to reduce transfusions and cost, while system-wide, multi-modal programs may also be associated with improvement in mortality. using examples from our local research and practice, i will discuss three aspects of pbm. iron and erythropoiesis stimulating agents (esa) are effective, safe and used extensively in management of pre-operative anemia. previous studies have questioned whether esa leads to increased risk of thrombosis, however, recent systematic reviews do not support these concerns. another pbm approach is to reduce bleeding during surgery by using hemostatic agents such as tranexamic acid (txa). txa reduces transfusion requirements in knee and hip arthroplasty, and is safe, widely available and relatively cheap. txa is effective in both anemic and non-anemic patients, making it an attractive universal pbm strategy. finally, recommendations and evidence-based guidelines on pbm exist, including the most recent international guidelines developed by the pbm international consensus conference. however, knowledge translation in pbm has been a problem and a number of barriers to its implementation have been identified. these include perceived or actual lack of expertise, time, and resources, as well as lack of physician and patient engagement. one way to address patient engagement is education through character driven animation and we are currently trying this approach. 4a-s17-02 low vs . high hemoglobin trigger for transfusion in vascular surgery (tv): a randomized clinical feasibility trial (the tv trial) background: current guidelines advocate to limit red-cell transfusion during surgery, but the feasibility and safety of such strategy remains unclear as the majority of evidence is based on postoperative stable patients. aims: we assessed the effects of a protocol aiming to restrict red-cell transfusion during elective vascular surgery. methods: fifty-eight patients scheduled for lower limb-bypass or open surgery of abdominal aortic aneurysm were randomized to a low-trigger (hemoglobin < 8.0 g/ dl, 5 mmol/l) vs. high-trigger (hemoglobin < 9.7 g/dl, 6 mmol/l) for red-cell transfusion throughout hospitalization. intraoperative change in cerebral-and muscle tissue oxygenation was assessed by near-infrared spectroscopy. we used a nationwide registry to collect data on death and major cardiovascular events, which encompassed (1) severe adverse transfusion reaction, (2) acute myocardial infarction, (3) stroke, (4) new-onset renal replacement therapy, (5) vascular reoperation, and (6) amputation of the lower limb. results: the primary outcome, mean hemoglobin within 15 days of surgery, was significantly lower in the low-trigger group: 9.46 g/dl vs. 10.33 g/dl in the hightrigger group (mean difference 0.87 g/dl; p = 0.022, longitudinal analysis) as were units of red-cells transfused ( background: controlled non-hemato-oncological studies have consistently demonstrated a single-unit red blood cell (rbc) transfusion policy as well as a stringent hemoglobin (hb) rbc transfusion threshold to be safe and reduce blood product utilization. yet, it is unclear whether these conclusions also apply to the hemato-oncological patient population. aims: to quantify reduction of rbc blood product utilization by the introduction of a restrictive single-unit hb-triggered rbc transfusion policy among the inpatient hemato-oncological population. methods: under the liberal transfusion protocol, applied up till november 1, 2017, standard double-unit rbc transfusion was indicated with a hb threshold ≤ 8.1 g/dl and/or anemia-related symptoms. following this date, the restrictive transfusion protocol was introduced involving a lowering of threshold to 7.3 g/dl and single-unit transfusion. for patients with an asa-score of ii-iii and iv, a hb threshold of respectively ≤ 8.1 g/dl and ≤ 9.7 g/dl applied. we evaluated rbc blood product utilization over a 6 month period starting december 1, 2016 (liberal protocol) and december 1, 2017 (restrictive protocol) in all hemato-oncological patients admitted for chemotherapeutic treatment including hematopoietic stem cell transplantation (hsct) with an expected duration of neutropenia of ≥ 7 days. analysis of categorical and continuous data was performed using the chi-square and mann-whitney test, respectively. results: during both observational periods, 137 patients were admitted who in total received 164 therapy cycles, including 56 acute myeloid leukemia (aml) induction cycles and 69 autologous hscts. distribution of indications of admittance, median age, duration of hospitalization and duration of neutropenia did not differ between both periods. during the restrictive period, in 303/402 (75.4%) of transfusions the assigned hb trigger was adhered to. the percentage of single-unit transfusion episodes increased from 29/112 (29.0%) to 81/111(73.0%) with the introduction of the restrictive protocol. overall, rbc blood product utilization per admittance did not reduce under the restrictive protocol (cumulative number of transfused rbc units 4.0 (interquartile range (iqr) 2.0-8.0) during the liberal versus 2.5 (iqr 0.0-9.0) during the restrictive period (p = 0.36)). however, rbc blood product utilization per neutropenic day demonstrated a trend towards reduction: 0.25 (iqr 0.11-0.33) versus 0.15 (iqr 0.00-0.32) units per day during the liberal versus restrictive period, respectively (p = 0.06). this reduction was mainly attributed to autologous hscts during which rbc blood product utilization decreased from 2.0 (iqr 0.0-2.0) to 0.0 (iqr 0.0-1.5) units (p = 0.06), corresponding to a reduction from 0.13 (iqr 0.00-0.21) to 0.0 (iqr 0.0-0.13) (p = 0.01) units per neutropenic day. moreover, 14/38 (36.8%) patients during the liberal versus 21/31 (67.7%) during the restrictive period did not require rbc transfusion during admittance. consequently, stringent hb thresholds as compared to single-unit transfusions seem to more strongly impact rbc blood product utilization. summary/conclusions: a hb-triggered single-unit transfusion policy results in a strong reduction of rbc blood product utilization in the setting of autologous hsct. no utilization reduction was observed among other hemato-oncological inpatient populations receiving intensive chemotherapy. further improvement of protocol adherence rates could potentially increase the benefit of this blood saving strategy. 4a-s17-04 assessment of hb content of packed red cells (prbc): is it time to label each unit with hb content? r jain 1 , n marwaha 2 and s sachdev 2 1 transfusion medicine, aiims, new delhi 2 transfusion medicine, pgimer, chandigarh, india background: in the current era of evidence based medicine and individualized care of patients, rbc transfusion continues to be administered on the basis of conventional wisdom and the notion of an average benefit per unit. the existing blood transfusion practice based on the "number of units transfused" ignores the fact that the total hb varies markedly among the individual rbc units. aims: the present study was aimed at estimating the hb content in packed red cell unit prepared by three different protocols from 350 ml and 450 ml whole blood collection in three types of blood donors: replacement blood donor (rd), first time voluntary donor (ftvd) and regular voluntary blood donor (rtvd). methods: a total of 900 prospective blood donors were included in this study. three hundred whole blood collections were performed in each of the three groups of donors (rd, ftvd, and rtvd). within each group 100 collections were done in double 350 ml, triple 450 ml and quadruple 450 ml blood bags respectively. a pre-donation venous sample was drawn from sample collection pouch for analysis in hematology analyzer as reference method for hb concentration of donor. the hb content of packed red cell units were estimated after collection of representative sample from the blood unit. volume of prbc unit was estimated by the formula of weight of blood in prbc divided by specific gravity. the hb content in unit was estimated by the formula: hb content in unit = hb value of the prbc unit (g/dl) 9 volume of prbc unit (dl). results: in this study the hb concentration (g/dl) was comparable among three types of blood donors except that rtvd had lower hb values when compared to rd (p = 0.007). hemoglobin concentration of prbc ranged from 14.2-29.6 g/dl; mean hb was 21.02 ae 2.90 g/dl. net hb content of prbc bag was lower in prbc prepared from rd as compared to ftvd (p = 0.0001) and rtvd (p = 0.008). the hb content of prbc units prepared from 450 ml collection ranged from 35.19-87.36 g and from 350 ml collection ranged from 30.77-65.78 g. we observed a wide range of net hb content in the prbc units and the correlation coefficient showed the strongest association of net hb content of the prbc unit with the overall volume of prbc (r = 0.730, p = 0.0001).higher volume prbcs have more hb content. volume of prbc bags in the study ranged from 155 ml to 370 ml (including both 350 and 450 ml collections). summary/conclusions: the present study shows that labelling hb content of the prbc unit help in better inventory management for patients. the hb content may help in decision making for release of units for paediatric/low weight versus adults/ higher weight patients. adopting a policy of optimizing dosage of rbc transfusion could have the potential to significantly improve rbc utilization and decrease patient exposure to allogenic blood. this would help further in the clinical transfusion practices based on evidence. 4a-s17-05 nv more, p desai, s rajadhyaksha, a navkudkar and n deshpande transfusion medicine, tata memorial centre, homi bhabha national institute, mumbai, india background: red blood cell (rbc) transfusion is an important medical therapy benefiting the patient in a wide spectrum of clinical setting. critically ill intensive care unit patients in particular, as well as medical and hemato-oncology patients, are among the largest group of the user of rbc. periodic review of blood components usage is essential to assess the blood utilization pattern in any hospital or health care set up. our institute is a 639 bedded tertiary care oncology centre with approximately 18,000 to 20,000 rbc transfusions annually. these transfusions are required in various stages of patient treatment like chemotherapy, radiotherapy, surgical and palliative care and there are established guidelines by the institute to be followed by clinicians. aims: to study clinical practices of rbc transfusions based on indications and to evaluate appropriateness of rbc utilization practices at the institute. methods: this was a prospective observational study, started after approval from institutional ethics committee. total of 4413 rbc transfusion events in adult patients over a period of four months were included and analyzed as per institutional guidelines for their appropriateness. details of transfusion events in form of pre transfusion hemoglobin, indication of transfusion, type of request, number of unit requested and issued, time of issue, site of transfusion and adverse reactions, etc were obtained from department of transfusion medicine records. overall statistical analysis was descriptive using spss software. chi-square test in cross tables was applied to see the relationship between different variables and considered significant if p-value was < 0.05. results: total 4413 rbc transfusion events for 2012 patients were analyzed. there were 1877 (43%) events in 628 patients of medical oncology and 2536 (57%) in 1384 patients of surgical oncology. maximum transfusions were received by patients in age group of 41 to 60 years (47%). total 83% of transfusion events were appropriate as per institutional guidelines. all transfusions administered in operation theatre were found to be appropriate with p value < 0.05. inappropriateness was more 53%(396/735) and significant in daycare setup (p < 0.05). anemia was the most common indication of rbc transfusion observed in 90% of events (3971/4413). total 62% rbc transfusions were given as planned and 38% as urgent transfusions. most common adverse transfusion event observed was allergic reaction in 0.3% of total transfusion reactions. summary/conclusions: clinical practice of rbc transfusions in our hospital was largely found to be appropriate and rational with adherence to institutional guidelines. blood utilization audits should be conducted regularly by transfusion services and results should be discussed with clinician for ensuring judicious use of the scarce resource. the concept of transfusion safety officer (tso) can be introduced for better coordination between clinicians and blood transfusion services to improve practices. 4a-s18-01 paul-ehrlich-institut, langen, germany on a global scale, blood services are quite diverse in regard to aspects like organisational structure, regulatory background, donor populations, donation rates or pathogen epidemiology. the world health organization (who) recognizes blood and blood products as essential medicines and provides guidance to member states for various aspects like blood regulation, best practices in blood collection and transfusion, or screening parameters. more recently a who guideline on residual risk of transfusion associated infections has been established which may facilitate decision-making for the most appropriate screening algorithms. it emphasizes the need for regional evaluation of screening assays and regulatory control of blood-associated ivds. background: babesia, a protozoan parasite that infects red blood cells, is a leading infectious cause of mortality in u.s. transfusion recipients. babesia is usually transmitted through the bite of an infected tick but may be transfusion transmitted (tt) or transmitted from mother to child during pregnancy. babesiosis is a world-wide disease; the ticks that carry babesia have a global distribution. babesiosis has been reported throughout europe and in canada, korea, india, and japan. prospective testing of blood donations in endemic areas of the u.s. revealed 0.38% of donors were positive for babesia dna or antibodies (moritz, nejm, 2016) aims: -to report results of ongoing babesia clinical trial -to explain significance of babesia as a tt infection methods: in cobas â babesia for use on the cobas â 6800/8800 systems, is a qualitative polymerase chain reaction nucleic acid amplification test, developed to detect in whole blood (wb) donor samples the 4 babesia species that cause human disease: b. microti, b. duncani, b. divergens, and b. venatorum. testing began in october 2017 under a u.s. fda-approved investigational new drug application. wb was collected into a proprietary medium that lysed red blood cells and stabilized babesia rna and dna. donations were collected in states with high, low, and no babesia endemicity and screened as individual blood donor (idt) samples. reactive index donations were retested in simulated minipools of 6 (mp6), plus 3 idt replicates with cobas â babesia. reactive index donations were also tested with 2 validated alternate babesia nat and for b. microti igm and igg antibodies. donors with reactive results were invited to enroll in a follow-up study to test for additional evidence of infection. results: to date, 256,802 valid donations have been screened with cobas â babesia, and 15 (0.006%) were reactive. 13 of 15 (87%) initially-reactive donations were confirmed to be positive for babesia with a positive alternate nat or serology result. 1 of 13 (8%) confirmed-positive donations was collected in a state with low babesia endemicity (pennsylvania), and 1 (8%) was collected in a state where babesia is not considered endemic (iowa). 11 of 13 (85%) confirmed positive donations were collected in states with high endemicity. 8 of 13 (62%) confirmed babesia-positive donations were detected in late fall or winter. all 13 (100%) confirmed babesia-positive donations were reactive in mp6. serology results are available for 12 of 13 confirmed-positive donations: at index, 6 of 12 confirmed babesia-positive donations were only igg-positive, while none were only igm-positive; 3 were positive for both igg and igm. 3 of the confirmed-babesia positive donations were negative for both igg and igm antibodies. cobas â babesia showed an overall specificity of 99.999% (256,787/256,789; 95% exact ci: 99.997%>100%). summary/conclusions: the cobas â babesia test successfully identified 13 babesiapositive donations, including 3 confirmed-positive donations with no igm or igg reactivity. 2 donations were collected in states considered low-or non-endemic for babesia. 8 confirmed-positive donations were collected outside of the summer babesia season, when most clinical cases occur. screening with cobas â babesia continues in several laboratories. cobas â babesia is not fda licensed or available commercially. background: babesiosis in humans is caused by the erythrocytic protozoan parasite, babesia microti which is transmitted by tick bites, but is also transfusion transmitted. although frequently asymptomatic or presenting with flu-like symptoms in a normal host, if immunocompromised infection can lead to severe complications and death. b. microti is endemic in the north eastern/upper midwest united states where partial testing of donations has been implemented. in canada, a 2013 study of~14,000 donors did not identify any b. microti antibody-positive samples, suggesting low risk at that time, but risk should be monitored. aims: to evaluate the prevalence of b. microti-positive donations in potentially atrisk areas in canada. methods: between july and november 2018, 50,752 blood donor samples were selected from sites near the us border. minipools were tested for b. microti nucleic acid by transcription mediated amplification (tma) using the procleix â babesia assay on the panther â system with individual testing on reactive pools. reactive donations were also tested by b. microti-specific: american red cross (arc) igg immunofluorescence assay [ifa] and imugen ifa/pcr. a subset of 14,758 tma-negative samples, primarily from the province of manitoba and eastwards to nova scotia, were tested for b. microti antibody using the arc ifa and if positive, the imugen ifa/pcr. donor age, sex, donation status, residential location and collection site location were recorded. donors who tested reactive/positive were informed, deferred and asked about risk factors (possible tick exposure and travel within canada, the usa and elsewhere, history of symptoms) and a follow-up sample was requested for supplemental testing (tma, arc ifa). reactive donations were removed from inventory. results: the 50,752 donor samples were proportional to collections in target geographic regions. age group, sex and donation status were also similar to the donor base in the collection areas. one sample from winnipeg, manitoba was tma reactive and antibody positive on supplementary testing. the donor did not remember symptoms or spending time in wooded areas. he visited the city of fargo, north dakota, usa. the subset of 14,758 samples tested for antibody were also proportional to collections in the targeted areas. four antibody-positive samples were identified from mid-september to october 1, all in south western ontario near lake erie. none were tma reactive. three were interviewed and none remembered any symptoms, any likely tick exposure, or relevant travel within canada or the usa. summary/conclusions: this is the largest b. microti prevalence study in canada. the results indicate very low prevalence with only 1 tma-confirmed-positive donation of 50,752 tested. the donor was from the only region in canada where one autochthonous human case has been reported and active tick surveillance identified b. microti positive tick populations. seropositive donations in south western ontario may suggest low prevalence in that region, but interpretation is less certain due to lack of corroborating supplementary results or case history. given the close proximity to the us border, forgotten us travel should not be ruled out. 4a-s18-04 background: the protozoan parasite toxoplasma gondii is prevalent in animals and humans worldwide. wild and domestic felids are the definitive hosts, and homoeothermic animals serve as the intermediate ones. after primary infection, the parasite persists lifelong within latent tissue cysts. transmission is by ingestion of undercooked or raw meat infected with cysts, by ingestion of food or water contaminated with oocysts, or transplacentally. however, it can also be acquired by blood transfusion and organ transplantation. toxoplasmosis can be a severe disease in immunosuppressed people and neonates whose mothers have acquired primary infection during pregnancy. aims: there is no information about the specific epidemiology of t. gondii infection in blood donors in portugal. therefore, we sought to determine the seroprevalence of t. gondii and associated risk factors in the population of blood donors in portugal. methods: between september 2015 and july 2017, 520 blood donors who attended the portuguese blood and transplantation institute blood banks located in oporto, coimbra and lisbon, and also at regional blood collection meetings, were invited to participate in the study. a written informed consent was obtained and a questionnaire about socio-demographic and behavioural variables was answered. sera were assessed for igg antibodies to t. gondii by a modified agglutination test (mat) commercial kit (toxo-screen da â biom erieux, lyon, france). results: of the 520 blood donors (mean age 38.55 ae 11.14; range 18-65 years old), 38.1% were positive for antibodies to t. gondii. when questioned about toxoplasmosis, almost half the blood donors did not have any knowledge about the disease. the centre of portugal had the highest seroprevalence (55.1%) followed by the north (37.2%) and the south (25.3%). blood donors living in rural areas had a significantly higher seroprevalence (p = 0.001) than those living in urban areas. seroprevalence increased with age, with the highest seroprevalence (60.2%) found in the age group of 46-55 years old (multiple logistic regression [mlr]: or = 7.68; ci: 4.08-14.46; p < 0.001), and decreased with educational level (p < 0.001). engaging in soil-related activities (gardening or agriculture) was significantly related to t. gondii seropositivity (p = 0.02). regarding water consumption, untreated sources (even though including mineral and tap water) was confirmed as a risk factor (mlr: or = 2.72; ci: 1.27-5.86; p = 0.001). other behavioural and eating characteristics, including cats in the household, eating raw or undercooked meat, processed pork products, or not washing raw fruit and vegetables before eating, were not associated with t. gondii infection. summary/conclusions: the risk of t. gondii transmission through blood transfusion is low, and serologic testing of antibodies, with exclusion of blood donors, appears not to be feasible. immunosuppressed individuals, organ transplant patients and pregnant women, should receive t. gondii antibody-negative blood components for transfusion. this study explored the epidemiology of t. gondii in portugal thus providing useful information on the seroprevalence and potential risk factors for t. gondii transmission. information regarding toxoplasmosis and its prevention could be promoted by medical and public health authorities among blood donors, and also the general population, when addressing policies, and designing screening programs, for monitoring and controlling infection and disease in portugal. 4a-s18-05 who is syphilis testing excluding? c reynolds 1 , c pearson 2 , k davison 2 and s brailsford 1 1 nhsbt/phe epidemiology unit, nhs blood and transplant 2 nhsbt/phe epidemiology unit, public health england, london, united kingdom background: screening for treponemal antibodies to detect syphilis in blood donors has been in place in england since the 1940s. there have been no reported syphilis transfusion transmissions in england since records began in part due to sensitivity of the organism to cold storage. since we have specific tests in place for other sexually transmitted infections such as hiv and hepatitis b virus (hbv), the utility of syphilis screening is often questioned. however, it may be a useful proxy for higher risk behaviours particularly following shortening of deferrals for higher risk sexual behaviours from 12 to 3 months in november 2017 and against a background of increasing infectious syphilis in the general population. aims: here we describe the epidemiology of recently-acquired syphilis in blood donors in england compared with hiv and acute hbv infection between 2009 and 2018. methods: monthly donation testing results are collected from the nhs blood and transplant (nhsbt) screening centres and reference laboratory. the demographics, possible sources of infection, and compliance to donor selection in confirmed positive donors are collected by proforma at post-test discussion with the nhsbt clinical team. recent syphilis is classified as igm positive and/or recent history including a negative donation within 12 months for regular donors. results: between 2009 and 2018 there were 153 recent syphilis cases, 121 hiv and 32 acute hbv infections identified by donation screening. recent syphilis rates per 100,000 donations increased from 0.66 to 1.64 whereas hiv decreased from 1.04 to 0.19 with less than 5 positive donations in 2018. acute hbv rates rose slightly from 0.28 to 0.38 in 2018. males outweighed females accounting for 71.9%, 63.6% and 59.4% of cases of recent syphilis, hiv and acute hbv respectively. nearly a quarter of cases of recent syphilis and hiv were seen in donors below 25 years old. of the male donors with recent syphilis, 58.2% reported sex between men and women (sbmw), 19.1% sex between men (sbm) and 22.7% did not report a risk. this contrasted with hiv where 45.5% of male donors reported sbm, just 2.6% not reporting a risk. overall 19, 32 and 3 males with recent syphilis, hiv or acute hbv respectively were non-compliant to the sbm deferral in place at the time of donation. in 2018, 26 donors with recent syphilis aged 23-65 years (median 36 years) were excluded from the donor pool, including 3 non-compliant to the sbm deferral. there were fewer than 5 hiv cases identified in 2018, all over 40 years old, all compliant, reporting sbmw. of the 6 hbv acute cases in 2018, 5 were male, all but one in the 45 and over age-group. summary/conclusions: over the 10 year period demographics of recent syphilis cases appeared similar to hiv with highest rates in young males, albeit lower proportions reporting sbm. following the switch to a 3 month deferral, hiv case detection continued at low level, while syphilis screening continued to exclude higher numbers spanning all age-groups, potentially at risk of other sexually transmitted infections, including non-compliant donors. background: globally, an estimated 113 million blood donations are given annually. in the blood service we are obliged to monitor donor health and ensure that blood donation is safe. in recent years, large-scale blood donor cohort studies in several countries have increased our knowledge on health effects of blood donation. health concerns relate both to immediate side effects like fainting and to possible long-term health issues related to repeated blood or plasma donation. the studies have provided us with data that can now help us introduce an evidence-based individualised donor care -a parallel to personalised medicine. individualised donor care in the management of iron depletion: studies have shown that a large percentage of our frequent whole blood donors, especially young women, are iron depleted. iron depletion is a strong predictor of deferral for low haemoglobin but has also been associated with e.g. restless legs syndrome and lower birth weight in children of frequent donors. the risk of iron deficiency can be mitigated by ferritin-guided prolongation of interdonation intervals or by iron supplementation. prolongation of interdonation intervals can challenge our inventories. iron supplementation, on the other hand, may give gastrointestinal side effects and other effects have been proposed as well, e.g. the masking of malignant disease and increased iron availability with subsequent risk of infection. in a large study we found that iron supplementation is not associated with increased risk of infection. what is the optimal balance between iron supplementation and prolongation of interdonation intervals? a growing number of blood services have implemented various flavours of iron management regimens generating more results. moreover, genetic studies in e.g. the uk, us, holland, and denmark can help us to find donors at high risk of iron depletion or low haemoglobin. we can use all these data in a big data approach in the pursuit of an individualised risk assessment model. other risks for blood donors: the presentation will also cover other risks associated with donation. new studies identify predictors of fainting after blood donation and also new interventions to prevent fainting. the global demand for plasma derived medicinal products has increased severalfold the last 10 years. plasma donors are bled up to 104 times per year in the us. very little is known about the health effects of frequent plasma donation. we know that immunoglobulin levels decrease with frequent donation but how does this affect health? summary/conclusions: the precautionary principle mitigates risk through early intervention prior to evidence. we tolerate next to no risk of transfusion-transmitted infectious diseases. the health of the blood donors, however, has not been protected similarly. we owe to our whole blood and plasma donors to investigate health effects of blood donation and ensure their safety. while the first attempts may not be perfect, we now have the tools to construct models for individualised donor care. background: in 2014, the isbt, aabb, ihn and eba jointly issued the standard for surveillance of complications related to blood donation which categorized donor adverse events (dae) into 6 categories (16 subcategories) defined by specific criteria. severity and imputability were briefly described but were optional. subsequent validation of these categories demonstrated consistency in categorizing reactions, but wide variation in assignment of severity. in 2018, with international input, the aabb donor biovigilance committee developed a severity grading tool (sgt) using a recognized medical adverse event grading system in which neutral grades replace subjective terms (mild, moderate, severe). aims: a large us blood collection establishment (bce) applied the draft sgt to assess its use in real cases of dae. methods: we performed retrospective analysis of all allogeneic and apheresis needle-in donations between 1/1/2017 to 9/30/2018. severity grading was assigned based on criteria defined by the sgt. database review of dae was performed, and each event was assigned a grade based on the type of outside medical care (omc), and on specific key search terms. search terms for omc included emergency room, emergency medical response, urgent care, healthcare professional, and hospital admission. additional specific key search terms included fracture, concussion, laceration, dental injury, surgery, and hospitalization. since duration and activities of daily living (adl) limitations were not captured in our dae database, cases in our dae claims' database were reviewed. case files of events classified as grade 2 or higher were individually evaluated by a physician for grading accuracy. results: in 1,511,758 needle-in collections, 31,320 dae were graded for severity. the majority (16,143, 51.5%) were vasovagal reactions (vvr), followed by 8,570 apheresis-related (27.4%), 6,572 needle-related (21.0%) and 35 allergic (0.1%) events. the majority of dae were grade 1 accounting for 98.6% of all dae, followed by grade 2 (1.2%), and grade 3 (0.1%). there were 2 grade 4 and no grade 5 dae. among the vvr, 98.1%, 1.6%, 0.2% and 0.01% were grade 1, 2, 3, and 4 respectively. grade 3 vvrs included 14 concussions, 11 fractures, 1 dental injury, and 2 pre-faint and 8 fainting events requiring hospitalization for work-up. two grade 4 vvrs involved falls resulting in intracranial hemorrhage requiring immediate medical intervention. for allergic and apheresis dae, there were only 6 and 5 grade 2 reactions respectively, and no grade 3 or 4 events. needle-related dae included 98.3% grade 1, 1.6% grade 2, 0.1% grade 3, and no grade 4 events. of the six grade 3 needle-related dae, 4 were nerve irritations lasting > 6 months, and 2 were dvts requiring hospitalization. summary/conclusions: the sgt provided consistent assignment of severity for the majority of dae, based on outside medical care and specific key search terms. assignment of severity based on impact on activities of daily living or on duration of injury/condition requires tracking over time making such assignments more difficult; modification of our dae tracking database and claims database to capture adl and duration should improve severity assignment for such cases. background: the international haemovigilance network (ihn) has collected aggregate data on complications of whole blood and apheresis donations from member national haemovigilance systems (hvs) since 2006. aims: we analysed the data collected in 2006-2016 in order to learn from the data and consider future improvement of data collection. methods: national hvs entered annual data on donor complications in the passwordprotected "istare" (international surveillance of transfusion adverse reactions and events) online database. from 2008 the donor complication spreadsheet allowed entry of separate data for whole blood donation (wbd) and apheresis, but also provided an option for entering data for all donation types. annual numbers of whole blood and apheresis donations were also collected. the harmonised international standard definitions were implemented in 2015. reactions were captured according to severity level (mild, moderate, severe) but without distinction between donor sex or first time vs repeat donation. extracted data were used to calculate national and aggregate donor complication rates (generally per 1000 donations). results: twenty-four hvs provided figures for donations and donor complications for one or more years (median years per country was 7, iqr 2-8). the total number of country years (cy) was 138, covering 155 million donations. the overall complication rate was 6.3/1000 donations and the median country rate was 3.2 complications/1000 donations (iqr 1.1-10.1). rates were generally consistent within a hvs from year to year but showed considerable variation between hvs; this was also the case for reactions classed as severe. not all countries differentiated between mild and moderate reactions and some reported all reactions under a single severity level. vasovagal reactions were the most commonly reported complication: overall 4.6/ 1000 donations, median country rate 3.1/1000 donations (iqr 0.6-7.7). rare and apheresis-related types of complications such as generalized allergic reaction (0.10 per 100,000, 40 cy), and major blood vessel injury (category available since 2015; overall 0.12 per 100,000, 6 cy) were only reported occasionally. eighteen of the hvs provided separate data for complications of whole blood and apheresis donations in one or more years (total 89 cy, 101.6 million wbd and 26.3 million aphereses, total 128 million donations). for these hvs the median rate of vasovagal reactions was 3.4/1000 wbd (iqr 1.0-9.1) and 1.5/1000 apheresis procedures (1.0-4.2) . reported haematoma rates were higher for apheresis than for wbd: the median per hvs was 0.39/1000 wbd (iqr 0.31-1.2) vs 4.2/1000 aphereses (0.69-5.6); rates of arm pain and/or nerve injury (not separated in 2006-2013) also tended to be higher: median 0.09/1000 with wbd, iqr 0.03-0.34, vs 0.39/1000 with apheresis, 0.05-0.57. summary/conclusions: international reporting allows hvs to study rates of blood donation complications, to distinguish between wbd and apheresis complication rates and capture information about very rare events. variability of reporting and of severity assessment between countries impairs the feasibility of comparisons between hvs. work is needed to improve harmonisation of classification of donation complications and severity assessment for data comparison and research. background: to prevent iron related hb loss, screening with ferritin testing was implemented in stockholm county (approx. 52000 registered blood donors) during a two-year roll-out. iron supplementation is offered to blood donors but has not prevented hb deferrals resulting in 8000 control visits per year. ferritin testing is hypothesized to increase iron compliance. aims: implementation of ferritin testing for surveillance of iron levels for the entire blood donor population with specific attention to new donors, women returning after pregnancy, donors with low hb and at return visit after low hb. yearly testing of plasma and platelet donors. methods: ferritin testing, following a staff education program, was implemented for applicant donors, donors with low hb, women after pregnancy, apheresis donors, followed by screening of registered blood donors per donation site. after initial screening, donors will be tested at each 4 th (women) or 8 th (men) donation, and with yearly testing of young adult blood donors below 25 years. six nurses were educated to process ferritin and blood count results. donors with aberrant ferritin were contacted by letter. results: establishment of cut-off levels and algorithms for ferritin testing and iron treatment was evidence based but met practical limitations such as number of analyses and results that could be processed per week, limitations in liss set-up, blood demand contra preferred cut-offs, iron supplementation compliance. for applicant donors, hb testing show that 20% of female and 9% of male applicants cannot be registered because of low hb (125 and 135 mg/l respectively). adding ferritin testing, a preferred cut-off level of 30 lg/ml (male reference level), would result in additionally 24% female and 1.3% male applicant donor loss. as this would threat the blood demand, cut-off was set to 15 lg/ml for women, above the female reference 10 lg/ml, with an acceptable 6% loss of female applicant donors. for registered blood donors, 4000 mg of extra iron tablets were offered at low ferritin (10-29 lg/ml). this was sometimes combined with prolonged intervals and often repeated before ferritin was restored above 30 lg/ml. donors with ferritin below 10 lg/ml (in 0.6% applicant donors, 0.8% registered donors) or above 600 lg/ml (0.5% applicant donors, 0.1% registered donors) were deferred and recommended to see their physician. for hb deferral, the interval was prolonged from 3 to 5 months, irrespective of ferritin levels. this, together with iron supplementation, resulted in an increase from 50% to 70% approved hb at return. the team of nurses processing ferritin and blood count results (1½ nurse fulltime weekdays) reacted to approximately 40 donor results daily, representing 20% of test results. summary/conclusions: many female donor applicants have suboptimal ferritin levels although they meet required hb for donation. iron treatment was added to retain donors with low ferritin as only prolonged intervals may danger the blood supply. for implementation of ferritin testing, it is necessary to have a well-functioning and agile organization to create and apply algorithms for testing, extension of intervals and iron treatment. background: since november 2017 a new donor screening regime is introduced in the netherlands where serum ferritin levels in whole blood donors are measured periodically to further control potential iron deficiency in donors. donor deferral thresholds are set at 30 and 15 ng/ml, and donors are deferred for six and twelve months respectively if ferritin levels are below these values. as limited information is available on ferritin recovery in whole-blood donors, the policy is introduced in parts such that adaptations to the implementation may be considered based on intermediate results and the impact of the measure on donor well-being can be evaluated. aims: to assess the effect of donor deferral on donor ferritin levels. methods: ferritin levels are measured in new donors and at every fifth donation in repeat donors. donors with ferritin levels below the indicated thresholds are deferred and ferritin is re-evaluated at their return for donation after six or twelve months. the policy allows estimating long term trends in ferritin levels post donation in repeat donors. as ferritin levels are measured in all new donors a reference distribution of ferritin levels in healthy individuals is obtained as well. results: among repeat donors 46% (44% of 16,433 male donors, and 48% of 13,525 female donors) have ferritin levels below 30 ng/ml and are deferred for their next donation. furthermore, the distributions of ferritin levels in repeat male and female donors are similar and each has an average ferritin level of 43 ng/ml. in contrast, we found that only 27% of new female donors (n = 13,283) and 1.7% of new male donors (n = 6,334) have a ferritin levels below 30 ng/ml. the average ferritin level in new donors was 148 ng/ml for males and 60 ng/ml for females. comparing the ferritin levels in new and repeat donors, a reduction in average ferritin levels between 1.5 and 2.0 was observed in female donors and between 3.2 and 4.4 in male donors. both ratios increased with donor age. at the end of december 2018 2884 donors with low ferritin levels returned for donation after six or twelve months deferral. repeat ferritin measurements show that on average the ferritin levels in female donors increased by 13 ng/ml per year whereas average ferritin levels in male donors increased by 34 ng/ml per year. summary/conclusions: in line with earlier findings in literature our results show that repeat donations substantially reduce ferritin levels in repeat donors. these range from 1.5 to 2.0 in female and from 3.2 to 4.4 in male donors, who generally have higher ferritin levels. deferral of donors with low ferritin levels seems to be effective in increasing ferritin levels in donors, however, further monitoring of follow-up in repeat donors is warranted to see whether the proposed scheme allows for sufficient donor recovery over time. there are~7000 different rare diseases and the genes for half have been identified. approximately 3.5 million uk citizens experience premature ill-health because of a rare disease. a conclusive diagnosis is generally not reached and on average the diagnostic odyssey lasts 2.2 years. the main aims of the 100,000 genomes project are to reduce the diagnostic delay by embedding whole genome sequencing (wgs) to accredited standards in the care path of patients with undiagnosed rare diseases. the project started in 2013 and dna samples from 100,000 nhs patients and their close relatives have been analysed by wgs. here we review the results from the nihr bioresource pilot study for the 100,000 genomes project comprising phenotype and genotype data from 13,037 individuals recruited at 83 hospitals using approved eligibility criteria for 15 rare disease domains. we determined the population structure including ethnicity and relatedness estimation, high level phenotypes collected using human phenotype ontology (hpo) terms and quality control and summary metrics for samples and variants. the sequence resource contains over 165 million unique variants in the 10,258 genetically independent samples, with 47% of variants previously unobserved in other large scale publicly available genome datasets. we summarise the curation of gene lists and pertinent findings in 2,000 unique diagnostic-grade genes for the 15 domains. over 1,000 reports assigning pathogenic or likely pathogenic causal variants have been issued with diagnostic yields varying between domains from 0.5% to 55%, while the proportion of novel causal variants ranged between 25% and 73%. we show the power of the bayesian association test, bevimed, to recapitulate decades of clinical genetics discoveries and by identifying >30 novel genes and novel diseasecausing variants in the non-coding space of the genome. we show how typing data for all red cell, hpa and hla class antigens can be extracted from wgs data. we mined the data from the 100,000 genomes project and similar sequence resources to re-version the probe content of the uk biobank axiom array. we genotyped 7588 donors from england and the netherlands with this new array and observed a 99.92% concordance when comparing 92,387 blood centredetermined antigen typing results with genotype-determined ones. for the 48 red cell and hpa antigens that were available for 7,473 donors, the array typing provided a 3.6-fold increase in typing results per donor (13.2 vs 47.9) and 38 rare donors were identified. using the genotyping data we identified 2.6 times more compatible units among this cohort of donors when blood demand was modelled using referral data from 3,146 english patients with more than three red cell alloantibodies. in conclusion the 100,000 genomes project has shown the feasibility of using wgs across a universal healthcare system to deliver a diagnosis for patients with rare diseases. based on these results the nhs has commissioned the analysis of another 500,000 dna samples from patients with cancer and rare disease. with analysis of dna by wgs and arrays becoming part of routine clinical care, blood services must develop competencies to extract transfusion and transplant relevant information from clinical-grade genotyping data. next-generation sequencing (ngs) enables the sequencing of thousands of genes, the exomes, and even entire genomes by single experiments at a reasonable price. there have also been advances in cytometry: use of antibodies with different fluorescence tags enables simultaneous monitoring of the expression of dozens of antigens. however, immunological methods cannot detect every variant discovered by ngs. genome sequencing reveals not only the exome but also the regulatory elements of transcription/translation, such as promoters and enhancers. rna sequencing determines which genes and spliced transcripts are expressed. it is amazing to realize how much this novel technology has been contributing to the better understanding of various biological phenomena. since the initial cloning of the human blood group a transferase cdnas in the early 1990s, we have been studying the abo genes, a and b glycosyltransferases, and a and b oligosaccharide antigens. various scientific disciplines including genetics, immunohematology, biochemistry, enzymology, and glycobiology have been applied to their study. we have made several important scientific contributions. we demonstrated the central dogma of abo: the a and b alleles at the abo genetic locus encode a and b transferases, which synthesize a and b antigens, respectively. we elucidated the allelic basis of the abo system. we found 4 amino acid substitutions between a and b transferases and inactivating mutations in o alleles. we became the first who succeeded in the abo genotyping, discriminating the aa and ao genotypes, as well as the bb and bo, which was impossible by the immunological approach. we have taken a simple experimental strategy: preparation of eukaryotic expression constructs of a/b transferases and their derivatives, dna transfection to human hela cells or their sublines, and immunological detection of the a/b antigen and/or biochemical examination of the enzymatic activity. we used this to show that the codons 266 and 268 are crucial in determining the sugar specificities of galnac/galactose of a/b transferases. we also identified mutations in several subgroup alleles causing restricted substrate use and diminished transferase activity. we also showed that cis-ab and b(a) alleles specifying the expression of both a and b antigens by single alleles encode a-b transferase chimeras. since then, other scientists have characterized more than 250 abo alleles. recent human genome sequencings have identified many more single nucleotide polymorphism variations. the genome sequences of many species are also available. taking advantage of those sequences and associated information, we have expanded our research to include evolutionarily related a1,3-gal(nac) transferases and their genes and scaled it up from the genetic to genomic level. in this talk, i would like to present the followings. 1: our elucidation of the molecular genetic basis of the abo blood group system (as requested by the organizer); 2: identification of novel abo alleles by others; 3: more snp data from genome sequences and potential problems for abo genotyping; 4: findings obtained from analysis of abo genes from other species; bacteria, vertebrates, to primates; 5: a1,3-gal(nac) transferases and their genes and the crosstalk between a transferase and forssman glycolipid synthase (fs); and 6: the potential causes of generation of abo polymorphism and of species variations of the gbgt1 gene specifying the fors polymorphism. in recent years, there has been a concerted effort to improve our understanding of the quality and effectiveness of transfused blood components. the expanding use of large datasets built from electronic health records allows the investigation of potential benefits or adverse outcomes associated with transfusion therapy. together with data collected on blood donors and components, these datasets permit an evaluation of the effect of donor and blood component factors on transfusion recipient outcomes. large linked donor-component recipient datasets provide the power to study exposures relevant to transfusion efficacy and safety, many of which may not otherwise be amenable to study for practicality or sample size reasons. analysis of these large blood banking-transfusion medicine datasets allow for characterization of the populations under study and provide an evidence base for future clinical studies. knowledge generated from linked analyses has the potential to change the way donors are selected and how components are processed, stored and allocated. however, unrecognized confounding and biased statistical methods continue to be limitations in the study of transfusion exposures and patient outcomes. results of observational studies of blood donor demographics, storage age, and transfusion practice have been conflicting. this review will summarize statistical and methodological challenges in the analysis of linked blood donor, component, and transfusion recipient outcomes. 4c-s20-01 a large deletion spanning xg xg and gyg2 gyg2 constitutes a genetic basis of the xg null phenotype, underlying anti-xg a production background: the xg blood group system comprises the homologous antigens xg a and cd99. the cd99 gene resides within pseudoautosomal region 1 on the short arms of the sex chromosomes and thus mimics autosomal inheritance. xg, on the other hand, is x-linked and straddles the pseudoautosomal boundary; a truncated pseudogene composed of only the first 3 exons remains on the y chromosome and therefore males carry a sole full-length copy of xg. this phenomenon manifests as asymmetric frequencies of the xg(a+) phenotype between the sexes: roughly 11% of women and 34% of men are xg(aà). also, whilst xg a immunization is rare, the vast majority of all anti-xg a makers reported are men. recently, we reported that the rs311103c variant disrupts a gata motif between xg and cd99. this abolishes erythroid xg a expression and causes the common xg(a-) red cell phenotype. however, rare individuals who produce anti-xg a cannot be accounted for by this finding. we hypothesized that a structural defect in the xg coding region causes the true xg null phenotype underlying anti-xg a production. aims: we undertook to determine a genetic explanation for anti-xg a production. methods: genomic dna (gdna) was extracted from two whole blood samples and cell-free dna (cfdna) from 13 archived plasma samples from donors producing anti-xg a ; one cfdna sample was from a female donor and the rest from males. polymerase chain reaction (pcr) experiments, sanger sequencing, and database searches were performed to identify and confirm the deletion. aliquots of gdna from four males reported to carry a similar deletion in the 1000 genomes project were also tested. results: in one gdna sample, exon-specific pcr identified a deletion involving part of xg and the downstream gene gyg2. database searches indicated that the most likely deletion was the infrequent genomic structural variant esv2662319 reported in the 1000 genomes project. further analyses with a short (714 bp) and a long (3555 bp) pcr amplicon across the suspected breakpoint determined that this deletion was approximately 114 kb and corresponded well with esv2662319. this finding was confirmed in the second gdna sample. given the rarity of anti-xg a producers, we decided to test for the same deletion in cfdna extracted from old archived plasma samples. of the 13 cfdna samples, poor quality in four samples prevented amplification even from control reactions and one was contaminated with bacterial dna. in the remaining nine samples, eight could be amplified for the deletion-specific 714-bp short amplicon while one was negative for the deletion. sanger sequencing of the amplicons revealed a heterogeneous repetitive dna element, ltr6b, hinting at a previously-reported recombination event. this deletion was not detected in the samples from the 1000 genomes project which reiterates the previously identified deficiency in data interpretation and reporting for deletions. summary/conclusions: a large deletion disrupting the xg and gyg2 genes accounts for the xg null phenotype underlying the majority (10 of 11) of anti-xg a makers. one sample remained unexplained, indicating further heterogeneity to be explored. our data help to explain why anti-xg a production is rare and has primarily been reported in men. background: s and s antigens encoded by gypb differ by one nucleotide (nt), c.143c>t, p.thr48met. two different genetic backgrounds are associated with silencing of s antigen and a u+ w phenotype. these include the nt change c.230c>t (p.thr77met) causing partial exon skipping and designated gybp*03n.01 (gypb*ny) and c.270 + 5g>t, an intron change causing complete skipping of exon 5, designated gypb*03n.03 (gypb*p2). aims: samples from three individuals, a previously transfused african american sickle cell patient (p1), a blood donor of unknown ethnicity (p2), and an african american patient (p3) (lapadat r. 2014 aabb abstract) were investigated for discrepant serologic and molecular results when determining s and s phenotype. methods: standard methods were used for rbc typing with licensed s and s reagents and rbcs from donor p2 were also tested with monoclonal and polyclonal anti-s and anti-s. dna was isolated from wbcs and hea precisetype performed on p1 and p2. p1 was also tested by gypb*s/s as-pcr, exon 5 pcr-rflp for c.270 + 5g>t and as-pcr for c.230c>t. p3 was tested for gypb*s/s and c.230 c>t and c.270 + 5g>t changes by a real-time pcr-fluorogenic 5' nuclease taqman chemistry. for all, gypb exons 1-6 were amplified and sanger sequenced and aligned to consensus using clustal x. results: rbcs of all three probands typed s-and strongly s+ while dna testing indicated c.143t/c (gypb*s/s). assay for the two common gypb*s silenced alleles, c.230c>t and c.270 + 5g>t, indicated all three samples had both silencing mutations previously reported to be independently associated with a sàu+ w phenotype. hea precisetype could not interpret this novel allele combination and indicated gypb*s as pv (possible variant). samples were confirmed to be heterozygous for c.143c/t, c.230c/t and c.270 + 5g/t by exon specific sequencing and as-pcr, pcr-rflp and real-time pcr. by long range sequencing of gypb, all three were heterozygous c.59t/g and c.60a/g (p.20leu/trp), c.67a/t (p.23thr/ser), c.71a/g and c.72g/t (p.24glu/gly), c.143c/t (s/s), c.208g/t (p.70val/leu), c.230c/t (p.77thr/met), and c.270 + 5g/t. all samples were also c.251g/g (p.84ser) and heterozygous for several previously recognized silent changes in exon 2, c.87t/c, c.96t/c and c.102a/g. summary/conclusions: we report a novel silenced gypb*s allele that can confound gypb genotyping interpretation. the allele was found in three probands associated with a sàs+ phenotype. in these samples, two changes previously reported to be inherited independently and both associated with silencing of s antigen are carried on the same allele. dna-based testing could not rule out that c.230t or c.270 + 5t are separate and that gypb*s was also silenced. robust s+ rbc typing indicates both changes are on gypb*s. gene sequencing confirms the c.270 + 5t change is on a gypb*03n.02 [gyp*he(ny)] background. c.230c>t (rs79492560) and c.270 + 5g>t (rs139511876) have a frequency of 0.0053 respectively 0.032 in the african population (exac). although we identified 3 samples, the frequency of this novel allele is unknown. background: the lutheran blood group system currently consists of 25 antigens. these antigens are of low immunogenicity and may cause mild-to-moderate transfusion reactions and hemolytic disease of the fetus and newborn. the activation of lu-glycoprotein/bcam on red blood cells (rbcs) and its interaction with laminin-5a is thought to play a role in vaso-occlusion in sickle cell disease and other hematological disorders. the two glycoprotein isoforms lu-glycoprotein and bcam are encoded by the bcam gene which consists of 15 exons located on chromosome 19q13.2. a number of rare lutheran phenotypes have been previously recorded in israel, including lu:-6,9 observed among iranian jews, lu:-20 in one thalassemia patient and one case of lu:-21. in this report, a previously transfused pregnant arab patient with b-thalassemia intermedia was investigated because she presented with an antibody to an unknown high frequency antigen (hfa), potentially related to the lutheran system. aims: to characterize a novel lutheran antigen through serological and molecular investigation of a patient with a lutheran related antibody. methods: initially, the red cell phenotype and the presence of a lutheran related antibody in the serum of the patient were detected by standard serological techniques, utilizing enzyme treated and chemically modified cells and rare cells and sera from the nbgrl collection. further serological investigations were carried out using standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were performed using soluble recombinant lu protein (srlu). eluates were prepared using acid elution method (gamma elu-kit ii). genomic dna was isolated from whole blood and all exons of the bcam gene were amplified by pcr and directly sequenced by sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: the patient's plasma reacted with all cells tested, except for three examples of in(lu) cells and cells treated with 2-aminoethylisothiouronium bromide, trypsin and a-chymotrypsin. inhibition studies with srlu protein showed complete inhibition of the antibody, thereby confirming the antibody to be directed toward an epitope on the lu-glycoprotein. in addition, testing of inhibited plasma revealed the presence of underlying anti-e and anti-fy a . an eluate was prepared to isolate the patient's lu-related antibody and this eluate was found to be incompatible with examples of lu:-5, lu:-6, lu:-8, lu:-13, lu:-21, lu:-22, and lu:-23 cells, whereas in(lu) were compatible. results of serological typing of the patient's cells, for lu system hfas, could not be conclusively determined due to the patient having been recently transfused. however, results suggested (through absence of mixed field reactivity) the patient's cells to be lu: -1,2,8,12,13,-19,21 . bcam sequence analysis confirmed the patient to be lu*02, lu*18 and revealed a novel homozygous mutation c.1351a>c in exon 11, encoding p.lys451gln in the lutheran glycoprotein. summary/conclusions: a novel homozygous mutation c.1351a>c (p.lys451gln) in exon 11 of bcam was identified in a patient with an antibody to a lutheran hfa. serological and genetic evidence presented here indicates discovery of a novel antigen of the lutheran blood group system, which we propose to name lura. background: lutheran glycoprotein and basal cell adhesion molecule antigen b-cam are two isoforms of a type i membrane glycoprotein residing on red cell surfaces. both isoforms are adhesion molecules with the main function of laminin binding, and both carry antigens of the lutheran blood group system (lu). the system currently comprises 25 antigens, all encoded by mutations in the alternatively spliced single gene bcam located on chromosome 19. currently, isbt lists 20 high incidence antigens in the system. aims: we report a case study of an individual with an unidentified alloantibody to high incidence antigen present in her plasma. samples from the patient and her family were investigated. we provide here serological and molecular evidence for a novel high incidence antigen of the lutheran blood group system. methods: serological investigations were performed by standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were completed with soluble recombinant lu (srlu) protein. genomic dna was isolated from whole blood of the patient and her family members; all the exons of the bcam gene were amplified by pcr and analysed by direct sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: presence of a lu-related antibody in the patient's plasma was confirmed, reacting moderate strength by liss iat with untreated and papain treated cells. cells from the patient's mother, father and two siblings were all incompatible with her plasma, though weaker than panel cells, reflecting dosage. only in(lu) cells were compatible with patient's plasma. the antibody was successfully inhibited with srlu protein, thereby confirming the epitope recognised by the antibody resides on the lutheran glycoprotein. the patient's cells were found to be lu: -1, 2, 3, 5, 6, 8, 13, 20, 21 . bcam sequencing revealed a novel homozygous mutation c.121g>a in exon 2, encoding p.val41met in the lu glycoprotein. the c.121g>a change appears to be an extremely rare mutation, listed in gnomad database with a frequency of 3.98 9 10 -6 and with no known homozygous examples. homology model of the novel lutheran glycoprotein was subjected to all-atom molecular dynamics calculations to analyse potential conformational changes. summary/conclusions: we report serological and genetic evidence for a novel antigen of the lutheran system, which we propose to name lunu. the evidence will be submitted to the isbt red cell immunogenetics and blood group terminology working party for consideration for allocation of antigen status. the absence of this high incidence antigen arises from a rare single amino acid change p.val41met in the lutheran glycoprotein and the presence of anti-lunu in the patients' plasma was presumed to have been made in response to previous pregnancy. on native, papain-treated (diagast) and trypsin-treated (sigma) rbcs. genomic dna was extracted from peripheral blood cells by an automated method, amplified by sema7a exon-specific primers and sequenced. results: the proband was a 32-year-old female patient of moroccan origin, group a, d+c+e-c+e+, k-, without transfusion history. she was hospitalized at 27 weeks gestation for a blighted ovum requiring a manual vacuum aspiration, with a significant hemorrhage risk. a rbc antibody screening was performed by a first laboratory. the antibody reacted 1 + by iat on all native reagent rbcs, with negative autocontrols, but was nonreactive on papain-and trypsin-treated cells. an anti-ge2 was initially suspected, due to the pattern of reactivity and ethnic background. new blood samples were referred to our national immunohematology reference laboratory. the antibody showed the same profile. anti-ge2 and anti-ch1 could be ruled out. the serum was nonreactive with two jmh:-1 and positive with two jmh:-3 samples. the patient was found to be jmh1 positive. in addition, a soluble recombinant jmh protein (jmh imusyn/inno-train) fully abolished the reactivity of the panagglutinating antibody. the antibody was an igg4. overall, these results were consistent with a probable jmh variant and prompted us to perform sema7a sequencing. three nucleotide changes were found, in homozygous state: a rare nonsynonymous change in exon 7, c.709g>a (p.asp237asn, rs140707085, maf < 0.01, sift score = 1); a common synonymous change in exon 12, c.1545a>g (p.gln515gln, rs741761, maf = 0.5); a rare non-synonymous change in exon 14, c.1865g>a (p.arg622his, rs140128092, maf < 0.01, sift score = 0.36). the analysis of surface accessibility of asp 237 and arg 622 using the 3d structure of sema7a (rcsb pdb-3nvq https://www.rcsb.org/structure/3nvq) showed that only arg 622 was predicted to be an exposed-epitope. interestingly, all other reported jmh variant phenotypes correspond to an arginine substitution. of note, we retrospectively found another individual of algerian ancestry (pregnant woman) with a pan-agglutinating igg4 antibody showing a similar pattern of reactivity, and with the same three changes in sema7a. we unfortunately could not perform a cross-compatibility testing with the proband (no material left and unsuccessful contact). summary/conclusions: serological and molecular studies allowed us to provide evidence for a novel high-prevalence antigen in the jmh blood group system, very likely encoded by the p.arg622his substitution in sema7a. we propose to provisionally assign the name jmh7 for this antigen. interestingly, our two unrelated jmh:-7 individuals were from north african ancestry. background: the abo system was discovered almost 120 years ago and the underlying structures later elucidated as carbohydrates carried by glycoproteins and glycolipids. the terminal trisaccharides galnaca3(fuca2)gal and gala3(fuca2)gal constitute the clinically important a and b epitopes, respectively. clausen et al. (pnas, 1985) showed that the a antigen could be extended to a repetitive glycolipid a epitope, galnaca3(fuca2)galb3galnaca3(fuca2)galb4glcnac-r. however, extended forms of b antigen have not been described. we encountered two related situations with unexplained serological reactivity. firstly, enzyme-conversion to group o treatment of group b (b-eco) red blood cells (rbcs) with a3-specific gh110 family exogalactosidase (bzyme) abolishes b antigens as detected by hemagglutination and flow cytometry with all monoclonal anti-b tested. despite this, 40% of group o plasmas have been reported to give positive crossmatch results with b-eco rbcs. secondly, plasmas from ab and b individuals of the globoside-deficient p k phenotype contain anti-p and anti-px2 but react stronger with bpp-rbc than with app/opp-rbc. based on these findings, we hypothesized the presence of a bzyme-resistant, b-related glycolipid. aims: to identify the molecular basis of the enigmatic serological observations outlined above. methods: plasma and eluates from an a 1 b individual with the p1 k phenotype were investigated by hemagglutination and flow cytometry, as were eluates from b p 1 k and o plasma. rbc membrane glycolipids were extracted from two batches of pooled, expired group b-rbc units (frozen 500-litre reference preparation and confirmatory preparation from 24 freshly collected units). native or enzyme-treated glycolipid fractions were analysed by liquid chromatography electrospray ionizationmass spectrometry (lc-esi/ms) and immunostaining of thin layer chromatography (tlc) plates. antigen expression in the h+bà human erythroleukemia (hel) cell line was analysed by flow cytometry following overexpression of selected glycosyltransferases. results: anti-p-depleted eluates made from a 1 b p 1 k plasma contained anti-px2 and antibodies of unknown specificity that reacted stronger with native or papaintreated bpp-rbcs compared to app/opp-rbcs. anti-px2 was removed by adsorption onto opp-rbcs but reactivity (here designated anti-extb) remained against b/bpp/b-eco rbcs. lc-esi/ms of glycolipid fractions from group b units revealed an unknown hexnac-hex-(fuc-)hex-4hexnac-hex-4hex heptasaccharide. upon b-nacetylhexosaminidase treatment of this candidate structure, a group b type 2 hexasaccharide was produced, demonstrating that the terminal hexnac of the hexnac-gala3(fuca2)galb4glcnacb3galb4glc heptasaccharide was b-linked. since the discovery of the anti-platelet effects of aspirin platelets have been a major therapeutic target for pharmaceutical companies and also a very profitable target. however, the effectiveness of aspirin has also been a challenge as it is an inexpensive drug and any new agent needs to show clear benefit over aspirin. furthermore the risk of bleeding from anti-platelet agents, especially cerebral bleeds, has also presented challenges. in the 1990's orally active gpiib/iiia antagonists were considered to be the 'super aspirin' but clinical trials showed increased mortality and ultimately this class of drugs was relegated to iv use only in high-risk patients. gpib/ix/v antagonists were also a promising drug target but no agent made it to market. the real breakthrough was the discovery of the p2y12 antagonist clopidogrel which, in conjunction with aspirin, proved to be very effective at preventing thrombotic events and as a result it became the biggest selling drug in the world at the time. with clopidogrel now offpatent the combination of aspirin and clopidogrel is a formidable challenge to any new agent both in efficacy terms and pharmacoeonomic terms. so is there a future for new anti-platelet agents? with the growing awareness of the role of platelets in inflammation and an understanding of how the immune activation of platelets differs from the classical haemostatic activation of platelets it is now possible to develop novel anti-platelet agents that target inflammation without compromising haemostasis. it is here that we should look for the next generation of anti-platelet agent. 4c-s21-02 university hospitals of geneva, geneva, switzerland platelet function defects, either congenital or acquired, are associated with increased bleeding risk, particularly in a perioperative setting. the use of platelet function assays is therefore tempting in order to tailor transfusion and limit platelet transfusion to those bleeding patients with impaired platelet function, as assessed by those assays. however, the current guidelines provide only weak recommendations supporting the routine use of these assays. indeed, there are numerous platelet function assays on the market that differ in their method of evaluation of platelet function and agreement between their results is at best moderate. the threshold values beyond which procedure-associated bleeding risk becomes worrisome is not standardized. moreover, observational studies addressing the predictive value of platelet function testing in perioperative or spontaneous bleeding are not consistent. finally, management trials with randomized patients assessing the benefit of platelet function testing are scarce. more recent data identified selected situations where platelet function testing may be useful though. i will review the different platelet function assays as well as selected clinical studies addressing the impact of platelet function testing to improve bleeding and transfusion-related outcomes. the latest recommendation will be addressed too. background: platelet refractoriness complicates the provision of platelet transfusions in management of thrombocytopenia in oncology patients. platelet refractoriness poses challenge due to alloimmunization to hla and human platelet antigens and is associated with adverse clinical outcomes. aims: a prospective study was undertaken to analyse result of platelet compatibility with post-transfusion platelet count increment and to ascertain presence of platelet antibodies as causative factor in platelet refractory oncology patients. pulmonary complication after blood transfusion is the leading cause of transfusionrelated morbidity and mortality, with an incidence reported between 0.05 -15% of all transfused patients. the most important transfusion related pulmonary complications are transfusion associated circulatory overload (taco), transfusion related acute lung injury (trali) and transfusion associated dyspnea (tad). in this presentation the recent changes in the international definitions will be presented and discussed. furthermore, insights in the different underlying pathophysiologic mechanisms will be highlighted. in the past decades only for trali prevention strategies have successfully been designed and implemented. currently no evidencebased treatment strategy is available for any of these life-threatening syndromes. insight in the pathogenesis of pulmonary complications after transfusion should pave the way for future prevention and treatment studies. the issue of the impact of iron overload / toxicity on the hematopoietic stem transplantation (hct) outcome has been firstly addressed in the field of transfusion dependent thalassemia. today the concept has been extended to other diseases characterized by periods of variable duration of transfusion dependence such as myelodysplastic syndrome (mds) and myeloproliferative diseases. patients requiring regular blood transfusions certainly develop iron overload leading to tissues and organ damage. iron burden before transplant significantly impacts outcome and long-life posttransplant. it is well known that iron overload is deleterious for organs such as liver, heart and endocrine glands and it has been postulated could also increases the risk of infections and severe graft versus host disease early after hct. recent preclinical data has shown how increased production of reactive oxygen species (ros) resulting under iron overload condition, could impair the stem cells clonality capacity, proliferation and maturation. also, microenvironment cells could be affected through this mechanism. for this reason, iron overload is becoming an important issue also in the engraftment period early post-transplant. high baseline ferritin levels before hct have been shown to negatively influence clinical outcome, but nowadays, ferritin is considered a steady and not biologically active form of iron, while free iron forms as non -transferrin bound iron (ntbi) and labile plasma iron (lpi) are considered the main trigger of cell damage more representative of the dynamic tissue damage. the scientific community is moving the iron disease from a "bulky" disease, such as classically in thalassemia (based on quantitative iron parameters as ferritin, red blood cell transfusion number, mri) to a "toxic" disease (based on active and dynamic biological markers as ntbi/lpi). at this time in all the studies published on hct setting, only the correlation between direct or indirect estimates of iron overload (mainly serum ferritin) and outcome parameters has been explored, while the duration of exposure to toxic iron species has not been taken into account. the first study that explored the lpi role in relationship with outcome was published by wermke and colleagues in malignancies. they investigated the predictive value of both stored (mri-derived liver iron content) and non-transferrin-bound-iron, defined as enhanced labile plasma iron (elpi) on post-transplantation outcomes in patients with acute myeloid leukemia or mds. their prospective, observational all-ive study showed that patients who had raised elpi concentration at baseline, also had significantly increased incidence of non-relapse mortality at day 100 (33%) compared with those who had normal elpi at baseline (7%) (p = 0.00034). reinterpreting transplant predictive factors in the light of the current advances in understanding iron homeostasis further supports the concept that the key to successful transplantation is regular and life-long chelation therapy to consistently suppress tissue reactive iron species and prevent tissue damage in the years before hct. in transfusion medicine, the role of donor sex was long considered to be limited to the increased risk of trali observed after transfusions from female donors. this risk has been shown to be limited to female donors with a history of pregnancy and to plasma rich products (i.e. excluding red blood cell products, typically containing < 50 ml plasma). until, in 2011, we found that sex-mismatched red blood cell transfusions were associated with increased recipient mortality. since then, several other studies have confirmed these findings, but some studies also did not find an association. all of these studies relied on the analyses of routinely collected health care data, which was not primarily intended to be used for research. as a result, analyses are complex and often difficult to properly appraise based on published descriptions. therefore, the discussion about possible reasons for these discordant findings has largely focused on the methodological approaches of the different studies. other potential explanations include differences in donor or patient populations, production methods, or storage time of blood products. the different potential explanations are expected to be associated with different underlying biological mechanisms. therefore, further delineating which donor, patient, and product characteristics modify the observed association could provide more insight into the underlying mechanism. in 2017, we observed that only transfusions from female donors with previous pregnancies were associated with increased mortality and only in male recipients under 50 years. this leads us to postulate that pregnancy induced long term changes in the female immune system are transferred during red cell transfusion, with negative consequences for young male recipients. the low amount of plasma present in red cell products further lead us to assume a cellular component, like passenger leukocytes, to be involved. it has been shown that micro-chimerism of passenger leukocytes can persist for decades after transfusion, even of leuko-reduced blood products, suggesting long term immune-modulation could play a role. we hypothesized that passenger leukocytes would die during storage of blood products and the negative effect of ever-pregnant female donors, on the survival of young male red cell recipients, would therefore be attenuated by increased storage time. however, our data seem to indicate the opposite. the risk of death was increased over three-fold for young male recipients of old (>24 days storage) red cells from ever-pregnant donors, compared to for young male recipients of fresh (<10 days storage) red cells from ever-pregnant donors (3-year cumulative incidence of death 15.4% versus 4.8%). the negative control group (i.e. young male recipients of red cells from male donors) showed a much weaker association of mortality with storage time (i.e. 7.2% versus 4.7%). these findings seem to falsify our hypothesis that mortality could be caused by passenger leukocytes, establishing long term immune-modulatory effects. another potential mechanism that has been suggested could be the presence of cellfree dna in transfused blood products. this cell-free dna increases during storage. however, more research is needed both to establish if cell-free dna can also be linked to previously pregnant blood donors and by which mechanism it could negatively affect young male transfusion recipients. clinical trials (cts), the gems in clinical research for generating robust evidence in medicine and public health, are costly and complicated undertakings. in resource limited setting like sub-saharan africa (ssa) where the health systems are sub-optimal and where capacity for research is limited, the conducting of cts can be a daunting challenge. the challenges of undertaking cts in rls may be categorized based on the occurrence of the bottleneck(s) in relation to the ethics and regulatory approval process: pre-approval: protocol development: in order to develop a context-specific protocol which is subsequently subjected to an ethics and regulatory approval process, investigators need to review and ensure that the protocol is pragmatic and feasible with respect to implementation. this results into a time-consuming reiterative process of reality-checking the protocol. site selection: in light of the limited research infrastructure, investigators in rls and their developed world partners spend considerable time reviewing and selecting suitable sites for participation in the anticipated protocol for the cts. suitable sites are usually very few and with competing on-going studies. approval: institutional review board (irb) approval: the irb approval process can be quite lengthy (6-9 months) with considerable unpredictability in the periods between the initial and subsequent irb reviews. national regulatory approval: the requirements by national regulators are unusually innumerable with limited flexibility to accommodate specific cts. post-approval: the key post-approval challenges for cts implementation in rls are attaining appropriate participant enrolment and maintaining high retention rates. specifically, for participant enrollment, the challenge may be unforeseen competing cts targeting the same participant pool or community perspectives that may discourage participants from getting screened for the cts. retention may also be a challenge particularly where participants view enrollment as a chance to access healthcare services may therefore not have any incentive to keep in a study after the initial study visits. in conclusion, cts are complex undertakings wherever they are conducted but are doubly challenging in rls like sub-saharan africa. the bottlenecks at the preapproval, approval and post-approval stages are considerable. nevertheless, it is rewarding to perform ctus in rls given that the data generated therein is highly valued by national regulators and may hasten the registration process for medical products. background: interest in an appropriate and effective whole blood (wb) pathogen reduction technology (prt) is growing, especially in sub-saharan africa where the residual risk of transfusion-transmitted infections (ttis) remains unacceptably high and wb is still frequently used. cerus corporation, manufacturer of the intercept tm blood system, and swiss transfusion src are collaborating on a clinical development program to adapt intercept prt using amustaline (s-303) and glutathione (gsh) for red blood cells (rbcs) into an appropriate prt for wb in resource-limited settings in africa. treatment with amustaline/gsh has been shown to inactivate a broad spectrum of transfusion-transmissible pathogens in rbcs. studies with amustaline/gsh in wb have shown effectiveness against a duck hepatitis b virus (>5.3 log reduction) and plasmodium falciparum (>7.5 log reduction), with future studies planned. a wb prt system with amustaline/gsh also has the potential benefit of minimal electricity requirements. aims: to describe the safety and clinical objectives for a phase 1 clinical trial using the amustaline/gsh prt system for wb in africa, and describe research and development efforts to adapt the intercept prt system for rbcs into a robust and appropriate wb system for settings with high burdens of tti and limited resources. methods: the protocol for a phase 1 clinical trial using pathogen-reduced wb treated with amustaline/gsh in an african country is presented, as are current research and development activities related to the development of a prt system for wb. results: in the planned phase 1 clinical trial in africa, 20 clinically stable patients with anemia who require wb transfusion will be randomized into two study arms at a large medical center in a sub-saharan african country. enrolled patients will receive one unit of non-leucocyte-reduced wb treated with amustaline/gsh, or a unit of untreated control wb or rbcs. the primary safety endpoint will be the incidence of high-imputability transfusion reactions (swissmedic ≥grade 2) within the first 24 hours of transfusion. data will also be collected on all adverse events and transfusion reactions (all grades) and the development of treatment-emergent antibodies to pathogen-reduced wb or auto-antibodies within 59 (ae3) days of the study transfusion. clinical efficacy will be characterized by hemoglobin increment 24 hours after transfusion adjusted to hemoglobin dose and body weight. summary/conclusions: a prt system for wb is being developed based on the intercept prt for rbcs that is in advanced development in europe and the united states. intercept-treated rbcs have met efficacy and safety endpoints in phase 3 clinical trials. the amustaline/gsh prt system used to treat intercept rbcs has demonstrated effective inactivation against a broad spectrum of agents that may result in ttis. a phase 1 clinical trial using an adapted prt system for wb in africa is the first step in a clinical development program that includes additional pathogen inactivation efficacy studies and improvements to the wb prt implementation process. together, these developments and evaluations represent progress toward a realistic and appropriate prt for wb in africa and other resource-limited settings. background: in australia, demand for plasma-derived products has increased dramatically, and there is a need to increase plasma collections. first-time donor retention, including the rate at which first-time donors return, is a pressing issue. a quick return is optimal as this increases the overall plasma yield and is associated with long-term retention. however, we lack evidence of effective interventions to encourage first-time donors, particularly those donating plasma, to return and to establish a higher frequency donation routine. working from schultz's (2014) framework, this intervention study was based upon insights from interviews with first-time plasmapheresis donors. participants identified barriers such as time and lack of knowledge about plasmapheresis. facilitators included being able to help more people and to donate more frequently than allowed with whole blood. participants generally favoured donating at a frequency of every 4 weeks. aims: the aim of this study was to test the effectiveness of three intervention conditions compared with the business-as-usual (bau) procedure on the proportion of donors returning to donate plasma and the number of plasma donations. we report on the data from 2 months post-donation. methods: donors were randomly assigned to one of four study conditions. in conditions 1 and 2, donors received an email one day after their initial donation. in the first condition, donors received the bau 'thankyou' email. donors in the second condition received an alternative email with content derived from the interview study. donors in the remaining conditions received either the bau email (condition 3) or the revised email (condition 4) coupled with a telephone call. the phone call was scripted to provide additional information about plasma, including how often plasma can be donated, a suggestion to donate every 4 weeks, and a prompt to forward-book appointments. results: the final sample (n = 6788) comprised 3859 women (57%) and 2929 men (43%) aged 18-70 (mean = 32). after two months 37.2% of donors returned to donate plasma at least once. after controlling for gender, age, and blood group, donors in each of the intervention conditions were more likely to return to donate plasma than were donors in the bau condition. the greatest effect was found between donors randomized to condition 4 (revised email + phone call), or = 1.305, ci = 1.128-1.510, and bau. donors assigned to the two telephone conditions (condition 3 and 4) donated plasma at a higher frequency than bau. summary/conclusions: this study tested the effectiveness of interventions designed to encourage first-time plasma donors to return to donate plasma and to establish a routine of donation. early indicators suggest that the evidence-based email and phone call elements are more effective than bau in bringing donors back to donate plasma, and the revised email combined with a phone call had the greatest positive effect on short-term plasma yield. background: healthy individuals with hereditary hemochromatosis (hh defined as hyperferritinemia and homozygous p.c282y mutation), but also carriers of other hfe mutations (p.c282y/p.h63d or homozygous h63d) with elevated serum ferritin (sf) are accepted as blood donors, if allowed by local regulations and if eligibility is fulfilled. generally, blood components are released for transfusion at normal sf levels (< 200 ng/ml in females, < 300 ng/ml in males). aims: prospective, two-center, randomized study comparing the efficacy and tolerability of double-erythrocyte apheresis (2rbcaph) and whole blood phlebotomy (wbph) for iron depletion in asymptomatic subjects with hh or hyperferritinemia and other hfe mutations in the setting of routine blood donation. methods: eligibility criteria included age ≥ 18-60 years, total blood volume ≥ 5 l, bmi < 35 kg/m 2 , hb ≥ 140 g/l, elevated sf levels and no end organ damage due to iron overload. 2rbcaph (360 ml rbc) were scheduled every 14 days and wbph (450 ml) every 7 days until sf was < 100 ng/ml. a complete blood count and sf were measured at baseline, at every visit and at follow up 8 weeks after completion of the study. adverse events were systematically recorded. the treatment effect was tested by poisson regression, with gender, hfe mutation, bmi and baseline sf as covariates. results: 30 subjects (5 females; mean age 47 years) were randomized to wbph (n = 16; 1 female) or 2rbcaph (n = 14; 4 females). hfe mutations were p.c282/ p.c282y in 17 subjects, p.c282y/p.h63d in 9, and p.h63d/p.h63d in 4. at baseline, mean hb was 149 g/l (sd 7.8) and median sf was 504 ng/ml (iqr 406-620 ng/ml). 222 procedures (wbph n = 146, 2rbcaph n = 76) were completed; 9 were interrupted (local hematoma, insufficient flow); 35 (16 wbph, 19 2rbcaph) were postponed because of low hb and 15 for non medical reasons. there were 2 drop-outs in the wbph arm due to depression and poor compliance, respectively. anemia (hb < 130 g/l in males, <120 g/l in females) occurred after 15 visits in 8 wbph subjects and after 5 visits in 3 2rbcaph subjects. fatigue was reported after 37 phlebotomies and 31 aphereses. only 5 participants (17%) completed the study per protocol. 136 blood components (94 rbc concentrates and 42 plasma units) for transfusion were obtained. overall, a median of 7.5 wbph (iqr 6.2-9.8) was needed to reach sf < 100 ng/ml, corresponding to 1.8 times of 2rbcaph (median 4.0, iqr 3.0-5.8) (p = 0.0001). analyzing separately p.c282/p.c282y and p.c282y/p.h63d carriers, the relation wbph to 2rbcaph was 1.6 and 1.8, respectively. treatment arm and hfe mutation were the covariates with significant effect on the primary endpoint (p = 0.0001 and 0.007, respectively). summary/conclusions: 2rbcaph is more efficient than wbph for iron depletion in healthy subjects with hh or other hfe mutations and moderate hyperferritinemia. intensive treatment schedules, generally recommended for hh, are difficult to keep because of hb drop and compliance. less intensive treatment in asymptomatic individuals with hh and their inclusion in blood donation would avoid negative effects on quality of life and benefit blood collection centers in the long term. background: serum ferritin (sf) measurements in whole blood (wb) donors demonstrated that female sex and intensity of donation are major risk factors for iron deficiency. approximately 200 ml red blood cells (rbc) and 200-240 mg iron are lost with wb donation. double unit rbc (2rbc) collections of 360 ml (ca. 40 ml less than the rbc amount of two wb donations) lead to a loss of about 400 mg iron. in switzerland, the maximal allowed donation frequency for male donors is once every 6 months for 2rbc and once every 3 months for wb donation. aims: to describe and compare the course of hemoglobin (hb) and sf in male subjects donating wb and 2rbc at our institution. methods: we included 294 wb and 151 2rbc donors (n = 445) who donated with the maximal allowed donation frequency over 48 months between 2008 and 2013, yielding 4,704 wb and 1,208 2rbc donations. we excluded subjects with hyperferritinemia and known hfe mutations. hb limits were 135 g/l for wb and 140 g/l for 2rbc donation. with 2rbc apheresis 360 ml rbc were collected. sf was measured on a predonation serum sample; hb was determined from finger prick samples. the donors received no iron substitution. we used generalized estimating equation models for hb and sf trajectories. results: mean age at the first blood donation was 53 (wb) and 48 years (2rbc), respectively. at the first donation, mean hb was 153 g/l (sd 13) in wb and 159 g/l (sd 8) in 2rbc donors; mean sf was 44 (sd 52) and 73 lg/l (sd 56), respectively. on average, hb and sf were higher in 2rbc donors (5.1 g/l and 26 lg/l, respectively; p < 0.001). there were 137 subjects with sf < 30 lg/l in wb and 19 in 2rbc group, and 85 with sf < 50 lg/l (but > 30 lg/l) and 40, respectively. in 2rbc donors, between the first and the last donation, mean hb declined from 159 g/l to 157 g/l (p < 0.05) and mean sf from 73 lg/l to 66 lg/l (ns). in wb donors, mean hb dropped from 153 g/l to 152 g/l (p < 0.05) and sf from 44 lg/l to 35 lg/l (p < 0.001). similar results were found when adjusting for age and season. hb values dropped from baseline until the 11 th donation for wb donors and until the 4 th donation for 2rbc donors with an upward trend thereafter. in both groups, no hb value below the limits of blood donation and no anemia were observed. sf reached a nadir at the 4 th donation in both wb and 2rbc donors (37 lg/l and 60 lg/l) and increased thereafter in 2rbc donors. in wb donors, sf followed a parabolic trend that peaked at the 10 th donation, and then declined until the last donation. summary/conclusions: the maximal allowed blood donation frequency for wb and 2rbc male donors in switzerland is not only protective for the development of anemia, but also for deferral of blood donors because of low hb. this was observed even in subjects with low sf at baseline. background: granulocyte concentrate transfusion is a potentially lifesaving option for patients without functional neutrophils. however, recent studies have failed to demonstrate the anticipated clinical effectiveness of this procedure. granulocyte concentrates are manufactured using sedimentation agents to separate granulocytes from red blood cells and enhance granulocyte collection efficiency. high-molecularweight hydroxyethyl starch (hes) is most commonly used for this. however, authorities recently restricted the use of hes due to its unfavorable risk-benefit-profile. modified fluid gelatin (mfg) is an already used alternative sedimentation agent. as the granulocyte product contains these substances, any impact of the sedimentation agent on granulocyte function may affect the clinical effectiveness of granulocyte transfusion. aims: we tested the hypothesis that mfg is not inferior to hes in terms of the functionality and viability of granulocytes. methods: granulocytes from ten healthy donors were isolated, aliquoted and incubated in parallel for 2 hours with either 0% (control), 7.5%, 15% or 30% mfg (gelafundin 4%, b. braun melsungen ag) or hes (hespan 6%/450/0.7, b. braun medical inc.), respectively, and granulocyte migration, chemotaxis, reactive oxygen species (ros) production, neutrophil extracellular trap formation (netosis), antigen expression of cd11b, cd62l and cd66b, and viability were subsequently investigated in vitro. testing was performed using live cell imaging of the cells embedded into a collagen i matrix for parallel testing of migration, ros production and netosis. in addition, flow cytometric (facs) analysis was utilized for surface marker expression, viability and respiratory burst measurement. results: granulocyte migration decreased in a dose-dependent manner in response to hes and mfg. relative to the controls, all three concentrations of hes lowered migration distances (p < 0.001 respectively), whereas only the higher concentrations (15% and 30%) of mfg showed lower relative migration distances (p < 0.001 respectively). track straightness was reduced with both sedimentation agents at 15% and 30% to the same extent (p < 0.001 respectively). hes resulted in lower cd11b expression (p = 0.028) and higher cd62l expression (p = 0.007) compared to the controls, whereas the differences for cd66b did not reach statistical significance. mfg did not affect the expression of any investigated surface antigen mediating endothelial adhesion and transmigration in comparison to the controls. no significant differences in the timing of ros production or netosis, or in neutrophil viability or respiratory burst were observed. summary/conclusions: these results indicate that mfg is not inferior to hes in terms of granulocyte phenotype and function in vitro when used at equal concentrations, and that potential impairment of granulocyte function can occur with hes. background: plateletpheresis donation leads to a well-known transient decrease of donor's platelets. the question of long-term effects raised with the development of regular donations by some donors in order to satisfy a growing demand. a seminal work (lazarus, transfusion, 2001) stated that there is a sustained thrombopenia in frequent plateletpheresis donors, correlated with the total number of donations. aims: french regulation authorizes up to 12 plateletpheresis donations per year, with a minimum 4 weeks interval between them. we tried to evaluate the risk of sustained thrombopenia under these conditions. methods: we retrieved all plateletpheresis donations occurring between 01/01/2014 and 08/31/2018 from the french civilian blood donors' base and then selected a cohort of donors with at least 24 donations during that period. in order to minimize measurement errors, platelet counts analysed were means of three consecutive donations, i.e. 8 measures for each donor. results: the cohort includes 2,186 donors (384 women and 1,802 men). mean platelet counts fluctuate between 276.4 and 278.6 platelets/ml. analysis of variance does not show any statistically significant difference (f = 0.462), even taking donor's sex or age in consideration. there is no difference if we consider the total duration of the 24 donations, either. donors with the lowest first counts show a significant rise in subsequent measures and donors with the highest counts show a decrease trend, exhibiting a classical regression toward the mean. summary/conclusions: plateletpheresis french regulation does not seem to be at risk of sustained donor thrombopenia. this conclusion is in agreement with recent literature data. the primary biological role of the human leukocyte antigen (hla) system is the regulation of the immune response to foreign antigens. because of this role, hla genes and molecules have an important role in transplantation, etiology of many autoimmune, non-autoimmune and infection diseases, but also in transfusion medicine. an increasing probability of an hla non-compatible blood products, tissues or organs exists due to the extremely high polymorphism of hla genes, with more than 20,000 described alleles to date, and their different frequency distribution in various worldwide populations. the hla system, originally discovered as a result of a transfusion reaction in the 1950s, can cause detrimental immune reactions in transfusion therapy. hla antibodies present in the patient are responsible for some of these reactions, while in other cases hla antibodies or hla reactive cells present in the transfused product are accountable for the immunoreactivity. hla antibodies form as a result of exposure to foreign hla antigens during pregnancy, transplantations and blood transfusions and can cause platelet immune refractoriness, febrile transfusion reaction, transfusion-related acute lung injury, and transfusion associated graft versus host disease. in order to avoid or reduce the development of these transfusion-related events, hla antibody negative or compatible products should be used. almost all existing methods presently used for molecular typing of hla polymorphisms are based on polymerase chain reaction, but with different resolution levels (low resolution -two digits or high resolution -four digits). in addition to providing a more precise detection of polymorphisms at hla classical loci (e.g. hla-a, -b, -c, -drb1, -dqb1), molecular methods can also determine polymorphisms at hla loci which previously could not be typed by serology (e.g. hla-drb3, -drb4, -drb5, -dqa1, -dpa1). the most commonly used method for the detection of hla antibodies was until recently complement-dependent cytotoxicity (cdc) technique, but it is increasingly being replaced by a more sensitive, solid phase based method (luminex technology). in conclusion, an accurate and precise determination of both hla gene polymorphism and hla antibodies presence is essential for the safe and efficient administration of transfusion products. background: in only a minority of pregnancies complicated with anti-hpa1a antibodies serious fetal/neonatal disease develops. the difficulty in predicting which mothers should be treated with ivig hampers implementation of fnait screening. we found that fc-core fucosylation and galactosylation are highly variable in anti-hpa1a igg, and that these glycan features strongly affect binding to fccriiia receptor. the level of fc-core fucosylation of anti-hpa 1a alloantibodies was found to correlate with platelet count and outcome of the newborn, suggesting that antibodyspecific fucosylation might serve as a biomarker in fnait screening. however, at present the fc-glycosylation pattern can only be determined by complicated methods involving purification of the antigen-specific igg, and analyzing trypticly released -igg-derived-glycopeptides by tandem liquid chromatography-mass-spectrometry (ms) techniques. these methods, although powerful, are not yet suited for high throughput clinical screening. aims: our aim was to provide a simplified method to quantify the biological activity of anti-hpa-1a antibodies, and possibly other alloantibodies against blood cells. methods: here we explored if cellular surface plasmon resonance (spr) imaging can replace ms, resulting in less complicated handling of patient sera and donorantigen-bearing cells. the strength of the binding of platelets to fccr on spr sensor was monitored under flow. the spr sensor was equipped with both wt fccriiia (sensitive to fc-glycosylation status) and mutant fccriiia-n162a (insensitive to fcglycosylation status). in addition, the biosensor was prepared with anti-platelet cd61 (c17) and anti-igg to calibrate the number of injected platelet as well as to quantify igg-opsonization. the quality of the anti-hpa 1a glycosylation was monitored as the ratio of the binding of opsonized platelets to the wt and the mutant n162a-fccriiia. platelets opsonized with recombinant glycoengineered anti-platelet antibodies with different levels of fc-fucosylation were used as standards. for validation, 166 plasma samples with anti-hpa1a antibodies, already analyzed by mass spectrometry and with known clinical outcome were tested (sonneveld, bjh, 2016) . results: we found that the ratio between the binding to the wt fccriiia and to the mutant n162a-fccriiia correlated with the level of fucosylation of the hpa1a antibodies, as measured by mass-spectrometry (r = à0.524; p < 0.0001). overall, a similar predictive value for disease severity was obtained as we previously reported for this retrospective cohort. in addition, quantitative information on antibody concentration can also be extracted using the fccriiia-n162a receptor as sensor on the chip, while anti-igg gave aspecific signals, presumably because it recognized cytophilic platelet-fccriia-bound antibodies as well. summary/conclusions: in conclusion, the combined use of wt and mutant fccriiia in a label free spr assay provides both quantitative and qualitative information of platelet bound anti-hpa 1a antibodies, which circumvents the need for purification of specific antibodies and laborious mass spectrometric analysis. this approach might be generally applicable to determine the biological activity of cell bound antibodies not only for anti-hpa1a in fnait, but also for anti-rhd alloantibodies in hdfn or anti-platelet antibodies in itp. background: immunization against the human platelet hpa-1a alloantigen is the most common cause of severe fetal and neonatal alloimmune thrombocytopenia (fnait) in otherwise healthy term newborns. the screening for hpa-1a antigen in pregnant women is an important tool for identification of pregnant women at risk of having a fetus/neonate with fnait. any targeted intervention depends on efficient screening methods as well as sensitive and specific methods for detection of anti-hpa-1a. within the framework of the polish-norwegian project (prevfnait) we have performed hpa-1a screening program in poland. aims: our aim was to assess the frequency anti-hpa-1a antibody detection and the clinical outcome of newborns identified through the study. women who joined the program due to the fnait in the previous child or in the current newborn are not analyzed in this study. methods: hpa-1a screening of 24 244 pregnant women in 8-40 gestational weeks was performed by facs phenotyping or rq-pcr genotyping at ihtm in warsaw. hpa-1a negative/hpa-1b/1b women were tested for hla drb3*01:01 and for anti-hpa-1a antibodies by maipa (followed up at week 17-20, 28, 32, 38-40 and 6 weeks after delivery). if anti-hpa-1a were detected, quantitative maipa was performed. all hpa-1a negative women were contacted for information concerning the newborn. if the baby had thrombocytopenia and anti-hpa-1a were not detected by maipa, the look back samples were tested retrospectively by paklx test (immucor). results: 554 hpa-1a negative women were identified (2.3%). anti-hpa-1a was antibodies were detected by maipa in 44 women (two delivered tweens). in addition, anti-hpa-1a antibodies were later detected by paklx in further 3 women who delivered baby with severe thrombocytopenia and/or ich. total number of immunized mothers was 47 (8.5%). they delivered 49 babies; 35 were boys. three women were treated by ivig: two by 4 and 8 injections since 33 th and 26 th gw respectively. the anti-hpa-1a concentration in the 1 st one was 0.4; 0.1; 5.88 iu/ml in 17, 28, 32 gw respectively and in the 2 nd <0.05 iu/ml in all examined samples. the decision on treatment was based on the low plt count~50 g/l in the fetus in cordocentesis. their newborns (one delivered tweens) were healthy. the 3 rd treated woman entered the program in 35 gw (anti-hpa-1a concentration was high 22.64 iu/ml). she obtained one injection of ivig. her baby was born with mild thrombocytopenia with no ich. severe fnait occurred in 5/49 newborns: in 3 with anti-hpa-1a detected in paklx only and in 2 with antibody concentration in maipa -1 st : 6.86/12.91/ 23.36 at 28/32/38 th gw respectively; 2 nd : 8.85/17.58 at 32/38 th gw respectively. ich was observed in all of them; plt count was < 50x10 9 in four, 56 9 10 9 / in one. summary/conclusions: 1/ the severe thrombocytopenia due to anti-hpa-1a alloimmunisation in our prospective study occurred in 2/10 000 pregnancies 2/ the paklx could improve anti-hpa detection in the screening program and should be considered as an additional diagnostic test, if maipa result is negative 3/ the hpa-1a alloimmunisation frequency is higher in pregnancies with male than female fetus. background: foeto-maternal platelet alloimmunization (fmpai) is mainly characterized by foetal and / or neonatal thrombocytopenia (fnait), sometimes revealed by intracranial hemorrhage (ich) or even by foetal death in utero (fdiu). the experience of the pnil milwaukee (usa) reported in 2014 that the diagnosis of alloimmunization was carried in only 33% of neonatal thrombocytopenia cases with a clinical symptomatology highly suggestive of an alloimmune etiology. aims: the aim of this two-year study was i) to determine the frequency of platelet incompatibilities in fnait, ich and fdiu and ii) to evaluate the frequency of detectable platelet alloantibodies (alloab) and their specificity in cases of incompatibility. methods: platelet genotyping was performed by hpa beadchip genotyping kit (bioarray solutions, immucor, warren, nj). serology investigation was carried out by 3 different methods: complete maipa kit (apdia bvba, turnhout, belgium), pack lxtm assay (immucor gti diagnostics, waukesha, wi) and « in house » maipa. all 2017 and 2018 data were collected using the laboratory information management system. results: 544 patient files were analyzed. no incompatibility is demonstrated in hpa-1 to -9, -11 and -15 systems in 19.3% (n = 105). hpa-1 and / or 3 and / or 5 incompatibilities were found in 271 cases (49.8%), hpa-2 and / or 15 in 87 cases (16%). platelet alloimmunization was globally confirmed in only 10.6% of the cases. 58 platelet alloabs were identified regardless of clinical manifestations: 24 anti-hpa-1a (41.4%), 20 anti-hpa-5b (34.5%), 8 anti-cd109 (13.8%), 2 anti-hpa-5a and anti-hpa-15b (3.4% respectively) and 1 anti-hpa-1b and anti-cd36 (1.7% respectively). 40 alloabs were found in the context of neonatal thrombocytopenia, 6 in ich and 10 in fdiu, and 1 in a follow-up of pregnancy. even if no anti-hpa-3 alloab could be identified, the incompatibility in this system was highly associated with fnait, ich and fdiu (n = 55, n = 32 and n = 17 on 114 cases). summary/conclusions: this study strongly confirmed the known immunogenicity of some hpa systems and highlighted overall the severity of hpa-3 and hpa-5 incompatibilities. the definite diagnosis of fmpai is difficult to make due to the present technical difficulties in the detection of antibodies against the hpa-3 and hpa-15 systems. however, our results suggest that special attention should be paid to the management of pregnancies with these incompatibilities due to the frequency of severe foetal/neonatal adverse events. background: fetal and neonatal alloimmune thrombocytopenia (fnait) is a potentially life threatening disease caused by maternal alloantibody formation against fetal human platelet antigens (hpas), of which anti-hpa-1a is accountable for the fast majority of the cases. population-based screening for fnait has been topic of debate for over decades. logistically as well as financially, the major challenge of such a screening is the typing of pregnant women to recognize the 2% hpa-1a negative women. at present, hpa-1a typing is mostly done by genotyping. for costeffective implementation of anti-hpa-1a screening there is need for a high-throughput, quick and low-cost phenotyping assay. aims: the aim was to develop a high-throughput, quick and low-cost phenotyping assay in order to identify hpa-1a negative pregnant women. methods: an automated sandwich elisa was developed to perform hpa-1a phenotyping using a murine monoclonal anti-gpiiia as coating antibody and horseradishperoxidase-conjugated recombinant igg1 anti-hpa-1a as detecting antibody. to ensure the applicability for high-throughput testing in a potential screening setting, 20 ll of the uppermost plasma of 3 -6 days-old stored edta anticoagulated blood tubes was used, without first swirling or spinning them. in two phases, samples of pregnant women were tested and compared to an allelic discrimination polymerase chain reaction assay as golden standard. in the first phase, samples from unselected consecutive pregnant women were tested. the second phase was part of a prospective screening study in pregnancy and confirmatory genotyping was restricted to samples with an arbitrary set od < 0.500 in the hpa-1a elisa. the developed elisa was optimized to require no additional handling (swirling or spinning) of stored tubes. during phase i, 506 consecutive samples were tested. in phase ii, the hpa-1a elisa was performed in another 62,171 consecutive samples, with confirmatory q-pcr in 1,825. the two phases combined, samples from in total 1,585 hpa-1a negative and 823 hpa-1a positive pregnant women were genotyped. the assay reached a 100% sensitivity with a cut-off od between 0.075 and 0.200, leading to a specificity of 99.6%. summary/conclusions: a quick, low-cost and reliable assay for hpa-1a phenotyping was developed that can be used in a population-based screening setting to select samples that has to be tested for the presence of anti-hpa1a antibodies. because plasma from non-mixed or spinned tubes of three to six day-old samples can be used, this assay is applicable to settings with suboptimal conditions. background: cytomegalovirus (cmv) sero-prevalence in ireland is lower than that which is reported in many other european countries. a study of 1047 pregnant women in 2002 found that 30.4% of irish women were cmv seropositive in comparison to 56% from western europe and 92% eastern europe and 97% from africa. an internal study carried out by the irish blood transfusion service (ibts) in 2010 indicated the rate of cmv seropositivity in irish blood donors was 21.97%. therefore a significant proportion of the irish donor and recipient population are susceptible to primary cmv. this is of particular concern for patients for certain at-risk groups such as very-low birthweight cmv seronegative neonates, cmv seronegative patients undergoing transplantation and other cmv seronegative immunocompromised patients. this results in a demand for the provision of cmv sero-negative blood components. in 2018 the ibts evaluated the abbott alinity s cmv igg assay as a replacement for the cmv mastazyme eia (total ab eia). aims: to assess the performance of the abbott alinity s cmv igg screening assay in comparison to the cmv mastazyme eia (total ab eia). methods: diagnostic sensitivity was determined by testing 48 confirmed cmv igg positive donors from an external laboratory. sensitivity was assessed using three seroconversion panels (n = 54). analytical sensitivity was calculated using linear regression analysis of the who first international standard for anti-cmv igg. diagnostic specificity was determined by testing 6127 donors. further evaluation of discordant results was carried out using the architect anti-cmv igg and igm assays and vidas anti-cmv igg and igm assays. results: the diagnostic sensitivity of the alinity s anti-cmv igg assay was determined to be 100%. the seroconversion sensitivity reported 42 out of 54 samples reactive. the analytical sensitivity of the alinity s cmv igg assay was determined to be 1.12 iu/ml. the validation reported 65 discordant results from 6127 donor samples tested with both the alinity s cmv igg assay and the current mastazyme total assay. 60 discordant results were observed (alinity s anti-cmv igg positive/mastazyme total negative). further testing of these samples classified 27 discordant results as positive, 12 as negative and 21 as indeterminate. 5 discordant results were observed (alinity s anti-cmv igg negative/mastazyme total positive). further testing classified these samples as negative. overall the diagnostic specificity was determined to be 99.80%. summary/conclusions: both the seroconversion and analytical sensitivities are comparable between the alinity s cmv igg assay, the cmv mastazyme total ab assay, the architect cmv igg assay and the vidas igg assay. the slight variations can be attributed to the individual assay cut-off definitions, which can vary greatly between cmv assays. it must be noted that the determination of the diagnostic specificity (99.80%) does not include indeterminate discordant results. further testing will be carried out to try to characterize all discordant samples in collaboration with abbott. this evaluation did not identify any donors with isolated confirmed cmv igm antibodies in a pool of 6127 donors. based on this evaluation the abbott alinity s cmv igg assay is a suitable replacement to the mastazyme total ab assay for blood donor screening. background: africa has a unique set of challenges regarding safe blood transfusion. two of the largest contributing factors are: 1) the most common disease states in sub-saharan africa (ssa) require large amounts of blood as lifesaving interventions e.g. malaria, 2) the highest burden of infectious diseases transmissible through transfusion (tapko, toure, & sambo, 2014) is found in ssa. this has often led to the binary donor base that exists in ssa, consisting of voluntary non-remunerated blood donors (vnbd) and family or replacement donors (frd) as transfusion centres are unable to supply the demand when relying only on vnbd. voluntary non-remunerated donors are the safest blood donors as they have no incentive (other than altruistic motives) and are not under social pressure to donate, both factors that may induce individuals knowing or suspecting themselves to be infected with a blood-borne agent to donate blood. nucleic acid testing (nat) in conjunction with serological testing is the gold standard for testing, however, the vast distances and high temperatures of africa makes transport of traditional plasma samples a logistical challenge. many publications evaluating the stability, suitability, and ease of use of dried blood spots (dbs) for nat have been published. generally, results have been shown to be comparable to traditional plasma samples. dbs is being used successfully in the early infant diagnosis (eid) programs for hiv by means of pcr testing, especially in africa. aims: 1. to demonstrate that dbs and/or dried plasma spot (dps) testing is suitable for blood donor screening and can make nat testing more widely available in africa 2. to determine the diagnostic sensitivity and specificity of testing dps and dbs samples, in comparison to testing of plasma samples. methods: 900 negative new donor samples and 100 confirmed positive donor samples, as defined by routine blood safety screening done at western cape blood service, were screened using a dried blood spot kit. after routine testing was completed, one dbs sample and one dps sample for each blood donor were prepared and analysed with the ultrio elite assay on the panther analyser. summary/conclusions: dbs/dps can be used as a sample for screening blood donors as the invalid rate was 0.56%, and only found on dbs samples. logistically dbs/dps is well suited for the resource-poor countries as samples are: -easy to obtain (fingerpick samples could be used.) -transport is simplified as samples will not leak or haemolyse due to high temperatures. -samples can be stored at room temperature dbs/dps demonstrated acceptable specificity. the ultrio elite performed well with regards to hiv and hcv sensitivity. sensitivity with regard to hbv was not as high but this could be due to very low and erratic viral loads. background: sanquin blood supply is responsible for the blood transfusion services in the netherlands. at the national screening laboratory sanquin (nss) annually more than 750.000 blood and plasma donations are tested, on average 3.000 samples per day. for more than 10 years, infection serology testing was performed using the prism (abbott diagnostics), but since mid of july 2018, serological testing for the hbsag, hiv ag/ab, anti-hcv and anti-hbc is done with abbott's alinity s system. aims: to compare the numbers of initially and repeatedly reactive results of whole blood and plasma donation samples and the rate of non-specific results leading to deferral of donations and donors for prism and alinity s assays using data from 6 months before and 5 months after implementation of the alinity s systems at nss. methods: initial and repeat reactive rate of the assays run by either prism (hbsag, hiv o plus, hcv) or alinity s (hbsag, hiv ag/ab combo, anti-hcv,) were calculated for january to june 2018 (prism) and august to december 2018 (alinity s). due to the lack of a true confirmatory method for anti-hbc, we only compared the rate of repeatedly reactive results for prism hbc and alinity s anti-hbc. results: the rate of repeat reactive results for prism (p) and alinity s (a) assays were as follows: 1) hbsag p 0.01 % (42/390.736) versus a 0.03 % (87/322.394); 2) hiv p 0.07 % (262/390.735) versus a 0.06 % (201/322.470); 3) anti-hcv p 0.11 % (427/390.737) versus a 0.03 % (109/322.476). the rate of anti-hbc reactive samples was not significantly different between prism (0.39 %) and alinity s (0.42%). over the study period, the rate of initially reactive samples for the three main screening assays (hbsag, hiv, hcv) was also comparable between alinity s (0.39 %) and prism assays (0.34 %), mainly attributable to a rather high number of initially reactive alinity s hiv ag/ab results. this was due to initial issues with blood collection tubes that were resolved. as a result in december, the rate of initially reactive samples decreased to 0.16 %, which was significantly lower than for the three prism assays (0.33%). summary/conclusions: the introduction of the alinity s assays lead to a decrease of the average repeat reactive test results (hbsag, hiv, hcv) by 0.17 % as compared to the prism, mainly due to a lower false reactive rate of the alinity s anti-hcv assay. this will be further investigated for first time and multiple time donors. with the implementation of the alinity s at sanquin we aimed to improve not only the operational efficiency but also to further minimize unjustified disapproval of donors. these first data show that the low initial and repeat reactive rates of the alinity s assays indeed have a positive impact on unnecessary deferrals of donations and donors. background: in blood banks, testing all blood donations for markers of infectious diseases plays an important role in maintaining the safety of blood transfusions. mandatory serological testing in switzerland is performed for anti-hcv, hiv ag/ab, hbsag and syphilis. highly specific and sensitive tests with corresponding automation are essential for this purpose. aims: a comparative study was carried out to evaluate the usability of the newly launched alinity s system (abbott) and the specificity of the infectious disease parameters hbsag, anti-hcv, hiv combo and syphilis (abbott) with the currently used elisa methods on the quadriga befree system (all diasorin, formerly siemens healthcare diagnostics). methods: the study took place at the interregional blood transfusion service in berne, switzerland. the specificity of the parameters was studied on 2,748 blood donor sera from both first time and repeat donors. the samples were tested first on the quadriga be free system with enzygnost hbsag 6.0, enzygnost anti-hcv 4.0, and enzygnost hiv integral 4 assays and on the pk7300 with the newbio-pk tpha assay (newmarket biomedical). all samples were retested on the same day with hbsag, anti-hcv, hiv combo and syphilis on the alinity s. initial reactive samples were repeated in duplicate. discriminatory tests were carried out for repeatedly reactive samples using alternative screening tests and neutralisation (for hbsag) on an abbott architect i1000 system and immunoblots (hiv-, hcv-, syphilis-inno-lia, fujirebio). for all samples, results from our routine individual donation nucleic acid testing (hcv, hiv, hbv, roche cobas 8800 system) were available. results: based on the results from testing 2,748 blood donations, the observed specificities of alinity s assays (a) and enzygnost assays ( summary/conclusions: the alinity s system was easy to use by the operators after a very short introductory training and provides good operational efficiency such as high throughput even when selective testing for samples is needed. the observed specificity of abbott alinity s versus siemens enzygnost assays is comparable in a blood donor screening setting. unfortunately, we were not able to analyse statistically the specificity data due to the insufficient number of donor samples tested in parallel. it is worth mentioning that around 90% of the samples included in the study derived from repeat donors who had been previously tested with the enzygnost assays but were "first time donors" for the alinity s assays. all four assays from both systems exhibit a very good specificity and are highly suitable and practicable for routine blood donor screening. background: effective screening for transfusion-transmissible infections is essential to ensure safe blood transfusions. the world health organization recommends mandatory serological testing of blood donations for human immunodeficiency virus (hiv), hepatitis b (hbv)/c (hcv), and syphilis. due to increasing demands on clinical laboratories, there is a need for reliable and accurate automated blood screening tests. the fully automated cobas e 801 analyser can be used with elecsys â infectious disease parameters to screen donor blood samples. aims: to compare the performance of elecsys â infectious disease parameters on the cobas e 801 analyser (roche diagnostics) with other commercially available assays for routine first-time blood donor screening. methods: we provide results from etablissement franc ßais du sang (montpellier), a blood bank which participated in a large, multicentre study of the cobas e 801 analyser. the following infectious disease marker assays were compared: hiv, elecsys â hiv duo versus prism hiv o plus; hcv, elecsys â anti-hcv ii versus prism hcv; hbv surface antigen (hbsag), elecsys â hbsag ii versus prism hbsag; hbv core antigen antibodies (anti-hbc), elecsys â anti-hbc ii versus prism hbcore; syphilis, elecsys â syphilis versus newbio pk tpha assay. specificity was tested using residual fresh serum samples from unselected first-time blood donors, and calculated according to assay package inserts and site-specific cutoffs. samples were tested using comparator assays, then retested the same day using elecsys â assays. initially reactive samples were repeated in duplicate; confirmatory tests were conducted on repeatedly reactive samples. confirmatory tests: hiv, nucleic acid testing (nat), architect hiv ag/ab and inno-lia â hiv i/ii score assays; hcv, nat, archi-tect hcv and inno-lia â hcv score assays; hbsag, nat, architect hbsag and elecsys â /prism hbsag confirmatory assays; anti-hbc, nat, hbsag, anti-hbs, and architect anti-hbc assays; syphilis, architect syphilis tp and inno-lia â syphilis score assays. sensitivity was tested using 30 preselected, anonymised, positive, citrate-phosphate-dextrose-plasma samples (plasmatec laboratory products) and compared with archived data for comparator assays. sensitivity was calculated according to the final nat result. results: across all infectious disease markers, specificity to detect repeatedly reactive samples using elecsys â versus comparator assays was similar (99.81-100.00% versus 99.79-99.98%; n ≥ 5195). in specificity analyses, there were 14 discrepant results for hiv testing, 27 for hcv, two for hbsag, eight for anti-hbc, and five for syphilis. sensitivity of the elecsys â hiv duo assay (83.33%; 95% ci 65. 28-94.36) was higher than the prism hiv o plus assay (76.67%; 95% ci 57.72-90.07), but the difference was not statistically significant. sensitivities of elecsys â and comparator assays were the same for hcv (85.19%; 95% ci 67.33-95.97), hbsag (70.00%; 95% ci 50.60-85.27), anti-hbc (100.00%; 95% ci 85.75-100.00), and syphilis (100.00%; 95% ci 88.43-100.00); three hcv and six anti-hbc samples were classified negative/ indeterminate and excluded from the analyses. in sensitivity analyses, there were two discrepant results for hiv testing, three for hcv, and five for anti-hbc. summary/conclusions: elecsys â infectious disease parameters on the cobas e 801 analyser demonstrate high specificity/sensitivity for screening first-time blood donor samples, with similar clinical performance to other commercially available assays. background: individual plasma and serum specimens from whole blood or plasmapheresis donors are tested for absence of infectious agents by serological assays prior to use for transfusion or production of blood derived therapeutics. the department of plasma analytics (pa), takeda (austria), and haema ag, grifols (germany), both labs with high throughput and a high level of automation, were seeking for alternatives to replace their current serological test systems (abbott prism next). aims: to allow a direct comparison of the two final candidate analyzers alinity s (abbott) and cobas e801 (roche diagnostics gmbh), a side by side evaluation was carried out by the pa and haema with support from abbott and roche (provision of instruments and reagents). the aim was to compare assay specificities as well as handling and performance of the instruments. the outcome should be used to better understand potential specificity differences and practical handling aspects (throughput, etc.) of a next generation serological analyzer. methods: the two candidate instruments were installed in the pa. from march to june 2018, close to 10,000 aliquots from routine preselected repeat donors, provided by haema, were run on both study instruments in parallel. plasma samples were tested for hbs antigen (ag), hcv antibody (ab), hiv ag/ab, and partially for syphilis ab. serum samples were additionally tested on hbc ab. samples with repeat reactive results ("rr", two reactive results out of three tests) not confirmed by confirmatory tests were counted as false reactive. the necessary sample size was calculated based on a one-sided comparison of proportions with the aim to detect potential specificity differences (a = 10%) in the size of those specified by the manufacturers' instructions. two different lots were tested for the three main assays. results: out of 7,389 plasma and 2,242 serum samples, 41 test results representing 37 individual donations were found rr on one or both instruments. two samples were confirmed positive (1 9 hbsag, 1 9 hcv), two others were indeterminate. the sample containing low level antibodies against hcv was pcr negative and only detected by the roche system. the percentage of false reactive results for the five assays on the two systems were (alinity s/e801): hbs ag: 0.04/0.03 % in a total of 9590/9589 samples tested; hcv ab: 0.01/0.09 % in 9620/9585, p < 10%; hiv ag/ab: 0.03/0.09% in 9362/9329, p < 10%; syphilis ab: 0.1/0.07% in 2971/2987; hbc: 0/0% in 1531/1549. no significant difference was found between the calculated specificities in our study and the manufacturers' data. a potential influence of sample matrix and kit lots was assessed. a trend towards more false reactive results in serum vs plasma was found for nearly all assays. no clear-cut statistical difference was seen between lots. summary/conclusions: the study results are in line with the manufacturers' specificity data, showing that the alinity s hcv ab and hiv ag/ab assay show a slightly higher specificity in a population of plasma and serum samples from repeat donors prescreened by prism. a possible influence on the test specificity by the sample matrix was detected but needs further investigation. the possibility to edit complex genomes in a targeted fashion has not only revolutionized basic research but biotechnological and therapeutic applications as well. with the rapid development of genome editing tools, in particular zinc-finger nucleases (zfns), transcription activator-like effector nucleases (talens), and the crispr-cas system, a wide range of therapeutic options have beenand will bedeveloped at an unprecedented speed. therapeutic genome editing in hematopoietic cells enable new interventions in the blood and immune system, including novel approaches to treat immunological disorders, infectious diseases, and cancer. we have developed gmp-compliant protocols to manufacture gene edited cd34 + hematopoietic stem and precursor cells (hspcs) as well as chimeric antigen receptor (car) t cells, with the final goal to provide novel cell therapies for patients suffering from primary immunodeficiencies, chronic infection with human immunodeficiency virus type 1 (hiv-1), and some tumor entities. despite great success in improving their specificity, engineered designer nucleases can induce genotoxic side effects by introducing mutations or chromosomal aberrations. we have established novel genome-wide assays that enable us to detect chromosomal aberrations induced not only by off-target activity but also by on-target activity, such as micro-aberrations and translocations, with unparalleled sensitivity. in toto, our developed protocols allow us to achieve genome editing in hematopoietic cells with high efficiency and to assess the genotoxic risk associated with the expression of crispr-cas nucleases and talens in clinically relevant human cells, so forming the basis for planned phase i/ii clinical studies. adoptive t cell therapy (act) has proven a potent means to treat blood-borne tumors and solid tumors. adoptive cell therapies include t cells that are genetically engineered with tumor specific t cell receptors (tcrs), or with chimeric antigen receptors (cars). in addition, tumor infiltrating cells (tils) can be isolated from tumor lesions, which are then expanded and reprogrammed in vitro prior to transfusion into the patient. the anti-tumoral efficacy of act products depends on several parameters, including the capacity of cd8 + t cells to produce cytokines, chemokines and granzymes, a feature that is critical for effective anti-tumoral responses. here i will discuss our efforts to develop and improve act products for future clinical use. i will present pre-clinical work on developing til therapy for non-small cell lung cancers. in addition, i will show that human cd8 + t cells can be divided into different subsets, and that only one of those subsets is highly cytotoxic. this finding may help improve the quality of genetically engineered t cell products, like tcr and car t cell products. background: the baltic states -estonia, latvia and lithuania have a lot in common. we are located side by side, share the baltic sea as a gate to the west, and more importantly, a common history. we were members of the ussr and suffered 50 years of soviet occupation. we held hands in a 600 km long human . . .chain" across the three states to express our mutual support, and later on, even joined the european union on the very same day -june 1 st , 2004. the three differ a bit in size, population and more in the languages spoken in each one, but that does not explain why the path towards voluntary unpaid donation varies as it does. aim: the aim is to describe the journey towards voluntary non-remunerated blood donation in the baltic states after regaining independence from the soviet union. methods: the information was collected from published and unpublished memories, annual reports and written interviews with latvian and lithuanian colleagues. results: in soviet times, all orders came from moscow and quality control was conducted from the capital city of latvia, riga. donors were mostly paid and given an extra vacation day. big factories were the best places to collect blood and people were queuing to donate. in 1991, the soviet union fell apart and the baltic states suddenly got the freedom and responsibility to decide. in estonia the first edition of "guidelines for the preparation, use and quality assurance of blood components" was taken as guidance in 1992. a lot of advice came from finnish colleagues. in 1997, it was decided to move towards non-paid voluntary donations. the process took 6 years. the first couple of years were economically difficult for the reborn state, as money had less value than food. instead of cash, donors were given rapeseed oil, sugar and pasta, for example. as the situation improved, food items were replaced by small symbolic gifts that carry sentimental value. it has been this way for more than 15 years by now. in lithuania, the process started later, the first program for developing a framework for voluntary non-remunerated donations being carried out in 2006-2015. it resulted in 51% of the donations being unpaid. the second program initiated in 2016 is still ongoing, aiming towards 100% non-remunerated donations by 2020. by the end of 2018, they had reached 99.2%. in the beginning, the main obstacle was a private blood center creating unfair market conditions. in latvia, monetary compensation for blood donations still exists, but the younger generation has been encouraged to donate blood for free and some results can already be seen. summary/conclusions: a common starting point does not guarantee the same results, at least not at the exact same time. examining the circumstances leading to the different outcomes could benefit countries yet to start moving towards non-remunerated donations as well as those considering the opposite. haemoglobin (hb) was as expected significantly different between women and men (meanaesem: 13.8 ae 0.01 vs 15.5 ae 0.01 g/dl; p < 0.000). percentage of females with low hb < 12.5 g/dl were 7.1%, 4.9%, 5.2%, 6.7% and 4.2%, percentage of males with hb < 13.5 g/dl were 0.5%, 0.6%, 0.7%, 1.7% and 1.2% for the age groups 1-5 respectively. ferritin values were higher in males compared to females (median; 25 th -75 th %>tile: 51;31-79 vs 157;106-231 lg/l; p < 0.000) and in older age groups compared to younger age groups (median; range in age groups 1-5 in females: 40;3-702, 52;6-619, 60;6-480, 60;8-725, 98; 10-604 and in males: 99;8-661, 161;18-1080, 206;15-1090, 220;26-1430, 221;33-981 respectively) . percentage of females with ferritin ≤ 15 lg/l were 8.6%, 3.8%, 3.7%, 6.7% and 2.0%, while percentage of males with ferritin ≤ 15 lg/l were 0.2%, 0.0%, 0.1%, 0.0% and 0.0% for the age groups 1-5 respectively. white blood cell counts (wbc) were slightly higher in females compared to males (meanaesem: 7.2 ae 0.02 vs 6.6 ae 0.03; p < 0.000). percentage of females with wbc > 12x10 9 /l were 1.8%, 1.3%, 1.7%, 1.4% and 0.3%, while percentage of males with wbc > 12x10 9 /l were 1.4%, 0.9%, 0.5%, 1.2% and 0.9% for the age groups 1-5 respectively. none had wbc < 2x10 9 /l. platelet counts (plt) were higher in females compared to males (meanaesem: 257 ae 0.6 vs 222 ae 0.6; p < 0.000).percentage of females with plt < 150x10 9 /l were 1.0%, 1.5%, 2.2%, 2.4% and 1.0%, while percentage of males with plt < 150x10 9 /l were 2.6%, 3.6%, 2.7%, 2.4% and 1.6% for the age groups 1-5 respectively. among the low plt counts most were caused by edta-dependent pseudothrombocytopenia. extreme deviations from normality were seldom and referred to gps for further investigations. summary/conclusions: first time donors are young with 75% younger than 30 years of age and the female/male ratio was 58/42. of the 16 583 first time donors with data on ferritin available, 14% had low ferritin (≤ 30 lg/l). the typical male first time donors neither had low hb nor low ferritin, even with a significantly lower ferritin in younger donors. in female first time donors the prevalence of low hb (6%<12.5 g/dl) and low iron stores (23%≤30 lg/l) is high. in all, while all first time donors are highly appreciated, campaigns could target the male population to even out the gender imbalance. blood centers must be aware of the higher prevalence of low iron stores in the youngest donors. background: the aim of assessing suitability of prospective blood donors is protection of their health and the safety of transfused patients. selection process is not always effective in obtaining all relevant information from blood donors in a timely manner. for several reasons, some risks remain undetected or they are disclosed at a future donation(s). therefore, recording and management of post-donation information (pdi) are of great importance for improvement of transfusion safety, donor counselling and education as well as overall improvement of the selection process. aims: the aim of the study was to present results of pdi management at croatian institute of transfusion medicine (citm) and the effect of education activities on their trends. methods: we have analyzed reports on pdi recorded in two-year period (2017-2018), according to the types of information obtained, age and sex of blood donors, total number of their donations preceding pdi, and the time of receiving the information. the effect of an information leaflet on pdi launched in november 2017 was assessed by comparing results in two study years. results: a total of 491 pdi were recorded: 266 in 2017 (1/396 donations) and 225 in 2018 (1/452 donations) with the following distribution: nonsexual risk as tattoo and piercing (17.5%), surgical procedures (17.1%), travel history (16.1%), infections/ contact (15.3%), other medical reasons (13.4%), endoscopy/invasive diagnostic procedures (12.2%), malignancy (4.3%), autoimmune diseases (3.7%) and sexual risks (0.4%). majority (76.4%) were late pdi, revealed on the future donation(s): 58.7% on the first next donation, 28.9% on the second and 12.3% after more than 2 subsequent donations. the mean age of blood donors associated with pdi was 40 ae 12 years (median 39 years), while the mean age of all donors in 2017/2018 was 38 years (median 37 years). of all pdi, 81.1% were related to male donors (84% in total pool of citm donors). using chi-square test there were no significant difference between female and male donors in total pdi frequency and in their distribution to early and late pdi (p > 0.05). the median number of all donations preceding pdi was 6 for female donors and 19 for male donors. implementation of education leaflet for blood donors resulted in 15.4% reduction of pdi in 2018 compared with 2017 (p > 0.05). the effect is more pronounced (p < 0.05) when comparing second and first half of 2018 (-25.6%). reduction is observed in all types of pdi with the exception of infections/contact (because they are mostly early pdi) and malignant diseases. the share of early pdi increased from 21.1% in 2017 to 26.7% in 2018, which may suggest better awareness of blood donors on the importance to inform blood bank on changes in their health status. summary/conclusions: our study points to the importance of systematic recording and management of pdi, including education of blood donors about the need of providing all relevant facts related to their health and the safety of donated blood in a timely manner. we are planning further improvements by providing information on this topic on posters and screens on donation sites. background: currently, the transfer of data between organizations and/or computer systems is very limited, and where present is typically proprietary. in the absence of a standardized reference format individual organizations and vendors attempting to integrate disparate databases must develop unique solutions. aggregation of information from multiple sources is complex and costly, constituting a significant barrier to effective analysis of data to improve practice and inform policy. aims: to standardize the definitions and facilitate integration of key data items used in blood donation and transfusion. we report here on an initial effort to map internationally harmonized critical steps in the blood collection/donation process in order to test the approach. methods: through a collaborative process of serial conference calls and correspondence, an informal multi-national consortium of experts across the transfusion industry are attempting to create a vocabulary with sufficiently precise definitions to be usable by automated systems and that can be the foundation of a blood collection/transfusion medicine common data model (cdm), using the following steps: -define the scope of activity to be addressed and segment into key processes. -identify the set of data elements in each segment that are common to all systems. -review and consider existing standards and definitions for each data element. -develop draft definitions for each data element. -release draft to public domain for critical review and refinement with long-term goal of gaining widespread endorsement. results: a standardized approach to blood donation was mapped through identification of common pathways and core mappable data elements. denominator data associated with donor characteristics and blood collection was selected as the first segment to address. a dictionary (or vocabulary) of common terms has been created and will be presented for international comment. summary/conclusions: developing an international consensus on the core elements and their definitions across the transfusion chain is critical for data integration and automation efforts. the expected benefits of this endeavor include that it allows the establishment of algorithms to automate reporting and thus reduce hands-on staff time; reduces time and resources needed to integrate new databases; allows systems to continue to use existing concepts and definitions internally while also providing data output in a standardized format; supports the ability to consistently analyse, interpret and present information regardless of the data source; establishes data definitions against which new systems can be developed; helps to improve comparability of results by providing a common data model for researchers and policy makers; improves confidence in data integrity and reliability of the derived information as a © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 basis for rational decision making; and reduces data gathering effort and cost thus improving opportunities for more efficient/complex data analysis. standardizing the transfusion medicine dataset is the first step in achieving the automation of data transfer and analysis needed globally to drive patient safety, research innovation, and best business practices. further steps must address the precise methods of data exchange, identification of responsible entities for maintenance and further development, and engagement of computer system developers. red blood cell (rbc) alloantibodies develop in a subset of individuals following exposure to non-self rbcs through transfusion, pregnancy, or other activities; these antibodies can lead to difficulty locating compatible rbcs, acute or delayed hemolytic transfusion reactions, or hemolytic disease of the newborn. alloimmunization is underestimated due in part to antibody evanescence, the random nature of posttransfusion antibody screens, fragmented medical care, and the lack of widespread antibody registries. factors that influence who will develop detectable alloantibodies are not well understood. transfusion burden is one risk factor for alloimmunization, though many highly transfused individuals never form alloantibodies despite exposure to many rbc units (and many non-self abo blood group antigens). individuals with sickle cell disease (scd) and myelodysplastic syndrome (mds) are more likely to form rbc alloantibodies than most other patient populations. individuals with rheumatologic and other forms of autoimmunity, though not chronically transfused, are also at higher than average risk of forming rbc alloantibodies. inflammation, in a broad sense, is one common thread among these diagnoses associated with high prevalence rates of rbc alloimmunization. reductionist murine models support some types of inflammation (including viral-like stimuli) around the time of rbc exposure as being associated with an increased likelihood of alloantibody formation. strategies other than transfusion avoidance or extended antigen matching beyond abo/ rh would be beneficial to prevent new rbc alloantibody formation, especially in patients at highest risk. background: the unique genetic makeup of the omani population makes them rich in the genetic blood disorder. 45 % of omani populations are àa/àa gene carriers, 44% àa/aa, and 11% of the population are aa/aa. around 10 % of omani nationals carry the gene for hbs, and 2 -3 % carry the gene for b-thalassaemia. recent statistics show that there are around 400 patients with thalassaemia major and 3000 with scd in oman. the other rbc abnormality that is common in oman is g6pd deficiency which is found in 28 % of males and 12 % of females. omanis are known to have the highest frequency of a thalassaemia and g6pd reported so far in any race. although blood transfusion is one of the supporting treatments of scd, it can cause some serious complications for the patients. alloimmunization of red blood cells is one of the consequences of blood transfusion. alloimmunisation of the rbcs can cause haemolytic transfusion reactions and may trigger hyperhaemolysis, in which transfused and patient's own rbcs are destroyed. alloantibodies can cause delay in the process of transfusion, it can be costly and time consuming. high number of patients developing alloantibodies may indicate a major difference in the patient and donor population. it may also indicate lack of a controlled, generalised sickle patients management policy. in oman the decision of transfusing scd patient is left to physicians attending the patient. aims: this study is aimed to highlight the increasing number of alloimmunised sickle cell patients. in the royal hospital we get 40 new cases of sickle patient with alloantibodies each year. the acknowledgement of these cases may help in is assessing the current practice of transfusing scd patients, or will help to define the donor and patient population difference. methods: 418 patients were recruited in the royal hospital for this study. edta blood samples were taken for antibody screening test and in the positive cases antibody was identified, all tests done by capture technique using immucor neo machine. results: of the 418 scd patients, 49% of the patients were male and 51% female, mean age was 17 years, in the range of 1-72 years. 38 % of the scd cases were positive for the alloantibodies, 72% were female and 28% were male, the age range was from 6-68 years. 78% of the positive were scd, 19% s trait and 3% were s/ bthal. most of the patients developed one antibody, however cases of multiples antibodies were also detected. 51% of the patients were with single alloantibody, 30 % of them with two antibodies, 10 % with three antibodies, 7 % with four antibodies and 2 % with five antibodies. the majority of the cases were igg against rh antigens anti-e is being the majority 23%, followed by anti-d 13%, anti-k 17%, anti-c 14%, anti-c 8%, anti-jk a 5%, anti-jk b 5%, anti-fy a 5%, anti-e 3%, anti-s 3%, antis 1%, anti-kp a 0.7%, anti-fy b 0.3% and igm being 2%. summary/conclusions: rbc alloimmunisation rate is high in oman majority of the patient affected are female. interestingly sickle trait patients were also transfused and 19% of them developed alloantibodies. the practice of transfusing rh and kell matching blood unit is implemented four years ago and still high alloimmunization percentage is achieved. background: in ghana, routine pre-transfusion investigations for patients with sickle cell disease (scd) involve only abo-d typing and immediate spin crossmatch, without screening for irregular rbc antibodies aims: determine the prevalence and specificities of and risk factors for rbc alloantibodies in multi-transfused patients with scd methods: in 2018, a cross-sectional study in multi-transfused patients with scd, from two tertiary hospitals in ghana was performed. participants' data on demography, transfusion and medical history were recorded. antibody screening and identification tests were done at sanquin, the netherlands, with standard serology using liss as enhancer and with papain treated rbc panel cells ('enzyme only'). characterization of rhd genes was done by multiplex ligase amplification assay. logistic regression was used to determine the association of patient characteristics, i.e. sex, age at enrollment (continuous), age at first transfusion (categorized as ≤ 1, 2-5, 6-9 and ≥10), previous pregnancy, number of transfused units (2, 3-5 and 6-10 and > 10), and years after last transfusion (<1, 1-2, 2-5, >5y) with presence of alloantibodies results: 226 patients (100 males and 126 females, median age 17 years, range 1.7-66) were included. the median number of transfusions was 3 (range 2-40). the median years after last transfusion was 2 (range 2 weeks-55.5 years). in 56 patients, anti-rbc antibodies were detected. in 14 of them the antibodies were weakly reactive with enzyme treated cells only or pan-reactive, possibly some of them representing autoantibodies or antibodies against high frequency antigens. in seven patients enzyme-only anti-le a was demonstrated, likely naturally occurring antibodies. thus, in at least 34 patients (15.0%) alloimmunization was demonstrated or suspected; in 11 patients the alloantibodies were 'enzyme only'. besides, the 36 alloantibodies of known specificity (8 anti-d, 2 anti-d+c, 16 anti-e, 1 anti-c, 3 anti-e, 1 anti-k, 1 anti-s, 1 anti-le a , 1 anti-go a ), three antibodies reactive only with fy(a-b-) cells and two antibodies of yet unidentified specificity were detected. in six d-patients (3 had been pregnant) anti-d (together with anti-c in two patients) was found. in three out of four d+ patients with anti-d, an rhd variant gene was demonstrated (2 dau-alleles and 1 diii type 4 or diva-2). logistic regression revealed that none of the risk factors analysed was associated with the presence of antibodies in the 34 patients. immunobiology -red cell alloimmunity 65 fifty-eight patients, had experienced an adverse reaction during or shortly after transfusion (12 patients had dark urine). adverse reactions were associated with the number of units received (or 1.71 (95% ci, 1.25-2.33; p = 0.001), but not with the presence of antibodies (p > 0.7) summary/conclusions: in at least 15% of multi-transfused patients with scd alloimmunization could be demonstrated, mainly (80%) directed against rh antigens. the enzyme only reactivity, coupled with absence of antibodies in seven of 12 patients with probable haemolytic reaction and known evanescence of especially non rh antibodies suggest possible low titre and disappearance of some clinically relevant antibodies. given the high immunization rate together with the high frequency of adverse transfusion reactions, pre-transfusion screening for rbc antibodies should be considered for patients with scd. background: rh blood group system and mainly antigen d is one of the most immunogenic, diverse and clinically important protein-based blood group. antibody anti-d may induce hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. anti-d prophylaxes become ineffective if an anti-d immunization has occurred. approximately 1% of the d+ population carries rhd alleles associated with reduced d antigen expression. qualitative variants, in which some epitopes are lacking and can produce anti-d antibody, are usually termed partial d. by contrast, d weak is commonly defined as a quantitative variant that have all d epitopes and should not make anti-d. del is a very weak form of d antigen and cannot be detected by routine serological tests. because some of del individuals have already developed an anti-d antibody whereas others did not this group contains both qualitative and quantitative changes. aims: investigation was prompted by finding discrepant results in typing of d antigen in a pregnant woman /3 rd pregnancy, 1 st delivery, 2 abortions in 1 st trimester/. routine serological techniques detected d negativity and the presence of antibody allo-anti-d in clinically significant titre. the non-invasive testing of d status of the foetus from maternal peripheral blood was indicated, but this was not applicable due to presence of the rhd gene in the woman's dna sample isolated from buccal swab. our aim was to investigate the discrepancy and determine the underlying rhd genotype. methods: blood samples, dna from peripheral blood and buccal swab of the pregnant woman were investigated. routine blood grouping and antibody testing were performed by column agglutination. two anti-d sera (id-diaclon anti-d igg (cell line esd1) by biorad and anti-d duo igm+igg, clone: th28 + ms26 by immucor) were used for adsorption/elution test for identification of del phenotype. initial rhd genotyping was performed by rt-pcr (exons 5,7,10) with the dna from buccal swab; further resolution was performed using pcr-ssp (fluogene; inno-train diagnostik gmbh); sequencing was performed by sanger analysis (inno-train diagnostik gmbh). results: genotype was identified as rhd positive by ce-certified pcr-ssp kits (fluogene). sanger sequencing of rhd from exon 1 to 9 revealed presence of a nucleotide deletion in position c.147dela, which is specific for allele rhd*01el.04. this nucleotide change results in the amino acid change p.val50leufs*5 causing the del phenotype. presence of antigen d was proved by adsorption/elution technique. titre of the anti-d was rising during the pregnancy to the 2000 level two weeks before the delivery. the newborn was delivered by s.c. without a sign of hemolytic disease. blood grouping of the newborn revealed blood group a, d negative, dat negative, testing for del was not performed. summary/conclusions: the case reported here shows that females with rhd*01el.04 allele are able develop strong anti-d immunization, so this type of del phenotype belongs to the "partial del subgroup". presence of variant rhd gene in mother disabling antenatal fetal genotyping from maternal blood by current methods requires a more attentive approach to care for such pregnancies. supported by mh cz-dro uhkt 00023736 and rvo-vfn 64165. 5a-s29-05 ea scharberg 1 , s rothenberger 1 , a st€ urtzel 1 , n gillhuber 1 , s seyboth 1 , e richter 1 , g rink 2 and p bugert 2 1 institute for transfusion medicine and immunohematology, drk-bsd ba-w€ u-he, baden-baden 2 institute for transfusion medicine and immunology, heidelberg university, medical faculty mannheim, mannheim, germany background: rb a (di6) is a low prevalence antigen of the diego blood group system. it has been found in few families only. the clinical significance of anti-rb a is unknown so far. the slc4a1*c.1643c>t (p.pro548leu; isbt allele name: di*02.06) allele is the molecular basis of the rb a antigen. in the gnomad database this gene variant was found in only one of 125,742 sequenced genomes (allele frequency: 0.000004). aims: to prove the frequency of the allele in our population and gain an rb a positive donor we performed a molecular screening for di6 in 1,700 blood donors. after our antibody screening test accidentally contained an rb a positive test cell we found out that anti-rb a is a very common antibody specificity. the frequency of the antibody in patients and blood donors was proved. methods: for the molecular screening of the blood donors we developed a pcr-ssp method. the antibody screening test in 3,652 patients and in 964 blood donors was performed in the gel technique (biorad ahg id-cards) using a 3 cell screening panel (drk-bsd src) including an rb a positive test cell. positive reactions with the rb a positive cell were confirmed by an additional rb a positive test cell of different source. additional antibodies were excluded or identified in the same method using an antibody identification panel (drk-bsd irc). results: the molecular screening for the di*02.06 allele in 1,700 blood donors revealed no single positive individual. within the first 2 weeks of usage of our antibody screening test which accidentally contained the rb a positive test cell 84 patients with anti-rb a were found. it was 2.3% of 3,652 patients tested in 21 laboratories in different parts of germany. some laboratories stopped using the rb a positive lot to avoid expensive and time consuming identification and conformation tests. in 18 of 964 randomly tested blood donors (1.9%) anti-anti-rb a was also present. summary/conclusions: despite the very low frequency of the di*02.06 allele, anti-rb a is a very frequent unexpected antibody in patients and blood donors in germany. it is obviously naturally occurring and is even more frequent than anti-wr a and anti-vw we found in previous studies in around 1% of patients and donors. 5a-s30-01 national blood center, ministry of health and sports, yangon, myanmar hemovigilance which detects every event not only for patient' reactions and donor's complications but also incidents and near misses definitely improve quality of blood transfusion services especially for those situations where implementation of all the standards in one time is not possible. healthcare system in myanmar is still in the stage of requiring priority for clinical professions and has limited resources for supportive roles. supportive services including transfusion service are still not a center of interest from prioritization of health care system. blood transfusion service has been practiced in myanmar since 1935. real essence of transfusion service is hidden behind laboratory practice and transfusion is regarded as part of laboratory investigation. hospital laboratories take care of testing of blood donated by replacement donors. this kind of transfusion services under laboratory umbrella is still being practiced in myanmar except national blood center (nbc) which was established in 2003 in accordance with blood and blood product law. this law was formulated cohesively with who strategies of blood safety. in 2002, who global data-based study sent questionnaires for assessment of safety status of transfusion service. nbc noticed that there was no data which can support corrective actions for safety. from that time onward, active retrospective review of existing data and introduction of records, prospective finding of process errors and any events from hospital blood banks were recorded and taking into actions at local level. cost of every unit of blood is supported by government. in 2017, national blood and blood product committee was established. the steering committee is working hard to get cooperation from every service by aiming to prevent those undesirable events before establishment of national level policy, standards and guidelines for sustainable service quality. in conclusion, by using essence of hemovigilance as a tool, quality of transfusion service can be improved step by step to fulfil the gap in spite of limited resources. the system started in local, extends to regional level by getting agreement of importance from hemovigilance results and is finally approaching to national level endorsement. background: erroneous transfusion of abo-incompatible(aboi) blood almost always reflects a preventable breakdown in transfusion protocols and standard operating procedures and can have disastrous consequences, with significant morbidity and mortality. these incidents need to be investigated in a systematic manner to identify system vulnerabilities to mitigate risks and improve patient safety. since 2016, reporters to shot have been asked to score(0-10) the extent to which the cause of incidents can be attributed to key factors: staff, environmental, organisational and government/regulatory which helps recognise the key factors identified whilst investigating these incidents. aims: to understand why unintentional transfusion of aboi blood components continue to happen despite standard procedures and national guidance available. methods: retrospective analysis of unintentional transfusion of aboi blood components reported to shot between 2010-2017(inclusive) was done to identify common themes and recognise areas of improvement. information provided using the shot human factors investigation tool (hfit) between 2016-2017 was reviewed to understand more about why the errors occurred. results: sixty-seven unintentional aboi transfusions were reported between 2010-2017; majority (56/67, 83.6%) were red cell transfusions but aboi plasma (9/67) and platelet transfusions (2/67) were also seen. most errors occurred in the clinical area (45/67, 67%), and could have been detected at point of administration. in 21(31%) cases, the error could not have be detected at the point of administration with a primary laboratory error in 10/21(48%) incidents. reviewing data from hfit for cases in 2016-2017 (13 aboi cases), the total score for staff culpability was 100, compared to a total score of 99 for all the other three organisational and system factors. this disparity is most obvious for the 4 aboi red cell cases, all of which scored the maximum 10 for staff culpability, i.e. 40/40 compared to 8/120 as the combined total score given to the other factors. in the preceding years (2010 to 2015), there were no hf scores available; however, the emphasis on staff-related culpability is demonstrated by 37 cases that included an outcome of the local case review and 14 (37.8%) mentioned staff-related retraining or disciplinary procedures. the risk of haemolysis and serious harm is more likely with aboi red cells than with other components with 2/56(4%) that resulted in death, 14/ 56(25%) major morbidity and 40/56(71%) no or minor adverse reaction. of these cases, one resulted in conviction for manslaughter and at least two staff dismissals. summary/conclusions: transfusion never events continue to occur, and it is evident that investigations into such incidents focus mainly on staff failings and do not consistently identify system wide changes that need to be incorporated to address prevalent issues. national recommendations and a safety alert to 'use a bedside checklist' immediately prior to administration were issued between 2015-2018 to support prevention of such errors but never events continue to persist. current approach is ineffective because it often leads to apportioning blame, rather than understanding the often-complicated and multidimensional factors contributing to the error. this must be replaced by a holistic approach which addresses local work pressures and embraces advances in automated technology like electronic prescribing and barcode scanning. of the confirmed trars, n = 27 were possibly related to treatment, n = 2 trars were probable, and n = 2 were definitely related to treatment; n = 37 trars were grade 1, n = 4 were grade 2, and none were grade 4. in recipients of conventional wb, there were n = 13 (1.12%) ars, n = 32 (2.75%) fnhtrs, n = 1 (0.09%) taco, n = 0 trali, and n = 16 (1.38%) unclassified transfusion reactions. of the confirmed trars, n = 55 were possibly related to treatment, n = 1 trar was probable, and n = 8 were definitely related to treatment; n = 54 trars were grade 1, n = 1 was grade 2 and n = 1 was grade 4. there were 21 mirasoltreated wb transfusions in pregnant women and 2 trars (9.5%), both grade 1 and probably related. there were 80 transfusions of mirasol-treated wb and 84 transfusions of conventional wb in patients < 18 years old resulting in n = 7 (8.33%) trars in recipients of mirasol-treated wb and n = 10 (11.90%) in recipients of conventional wb. summary/conclusions: timely data reporting of trars and expanding the hv infrastructure has helped to improve the hv system in ghana. of 2181 wb transfusions in routine use in ghana, there were 4.02% trars in recipients of mirasol-treated wb and 5.59% in recipients of conventional wb. additionally, mirasol-treated wb was safely transfused in pregnant women and pediatric patients. haematology, monash health, melbourne, australia background: transfusion-associated graft-versus-host disease (ta-gvhd) is rare and usually fatal. it can be prevented by provision of irradiated blood products to at-risk individuals, such as those receiving nucleoside analogues, alemtuzumab, bendamustine or with hodgkin lymphoma (hl). duration of risk is uncertain, so ensuring these individuals correctly receive lifelong irradiated blood components, as currently recommended by anzsbt and bsh guidelines, is challenging. in australia, platelets are routinely irradiated, but red blood cells (rbc) are not. aims: to determine whether patients receiving fludarabine, cladribine, bendamustine, alemtuzumab, or dacarbazine (for hl), appropriately received irradiated rbcs. secondary outcomes included rates of ta-gvhd after unintended exposure to non-irradiated components, factors influencing correct issue of irradiated rbcs such as transfusion management plans, and provision of adequate clinical information on blood requests. methods: we performed a retrospective audit to identify patients receiving therapies indicating risk for ta-gvhd using pharmacy dispensing records from january 2008 to october 2018 at monash health, a multi-campus university hospital in melbourne, australia. diagnosis, treatment dates, group and hold (g&h) requests, rbc transfusions, and follow-up information were sourced from laboratory and medical records. results: we identified 310 patients who received fludarabine (n = 52, 17%), bendamustine (n = 29, 9%), cladribine (n = 17, 5%), dacarbazine for hl (n = 164, 53%) and alemtuzumab (n = 48, 15%). the median age of patients was 46 years (range 8-92) and 171 (55%) were male. median follow-up was 30 months (range 0-132). post-exposure, 42 patients (14%) received transfusions with 33% correctly receiving irradiated rbcs. the remaining 28, all from haematology/oncology, received a total of 192 unirradiated rbcs. in 8 patients, this was rectified on subsequent transfusions. there were no cases of ta-gvhd at median follow-up of 14.5 months (range 0-75) from first rbc transfusion. after medication administration, 99 patients had g&h requests after a median of 3 months (range 0-129). only 23% of requests had sufficient clinical information to prompt irradiation, such as hl or medication details, and only 20% asked for irradiated components. preventive strategies have now been employed. transfusion management plans for haematology patients were implemented in march 2017. for audited patients, these were written from 38 days prior to 104 days after medication exposure. two were written following inadvertent unirradiated rbc transfusion. patients identified in this audit will have a laboratory flag generated and prospectively, pharmacy dispensing records will be sent to blood bank to identify at-risk patients. our hospital is transitioning to electronic medical records (emr). an alert will be generated in emr when ordering transfusions if there has been exposure to these medications. however, clinical awareness and documentation remain vital. additional measures include patient education, alert cards, and ongoing collaboration with medical staff to encourage transfusion planning. summary/conclusions: recognition of patients at risk for ta-gvhd remains low, even among haematology units. we are making progress on ensuring provision of lifelong irradiated blood components in patients exposed to nucleoside analogues or alemtuzumab, as well as hl patients. implementation of an emr and additional strategies in this domain is important to prevent ta-gvhd. background: blood transfusion is considered an essential element in the management of patients globally. it might be risky and transfusion related adverse reactions may occur with the less adherence of transfusion policies. standard guidelines regarding the screening of blood for infectious disease, genuine need of transfusion and abo compatibility are followed and monitored drastically. however, patient assessment during transfusion especially at patient bedside and post transfusion is also equally important. aims: we are a newly established hospital and are working towards the best possible management of patients. in this regard to minimize the transfusion errors and to highlight if any lacking being practiced during transfusion, we conducted this study to observe the compliance rate of documentation of transfusion form by the healthcare staff and also to observe the compliance of line of action taken in case of occurrence of transfusion reactions. methods: this was a observational study conducted at nibd and bmt, pechs campus from february 2018 to february 2019. ethical approval was obtained prior to the study. transfusion form for each transfusion was filled. the form provided information on documentation of blood product receiver name, employee identity number, date and time of receiving blood product, patient name, medical record number on units, on patient's wrist band and on transfusion form. abo compatibility on the unit and on form, medical record number from wrist band, name and employee identity number of two healthcare staff started transfusion, transfusion start and completion time. time, temperature, blood pressure, pulse and initials of staff at the time of order, onset, after 15 minutes and at the completion of transfusion were also included. transfusion reaction form was also filled by the healthcare staff. data was analyzed by using spss version 23.0. results: a total of 500 transfusions forms were analyzed. over all compliance rate was 18%. out of 500, 115(23%) forms were available in source notes and of 115, 88 (76%) were partially and completely filled. higher compliance was seen in the initial months of hospital establishment than later months (p-value = 0.000). highest non compliance was seen in documentation of initials of duty doctors on transfusion form at the completion of transfusion(3%) and highest compliance was seen in documentation of name by healthcare nursing staff at the start of transfusion(89%). a total of 02(0.4%) adverse events were reported from red blood cells and platelets. mean time of start of symptoms was 2 hours and 30 minutes for red blood cells and for platelets it was 1 hour and 13 minutes. transfusion was instantly stopped as the symptoms appeared with no delay of time and actions were taken to resolve the reactions. time of appearance of symptoms and time of start of medication were documented and error free. all blood bags were returned to the blood bank and discarded after 6 hours as per the policy of hospital. summary/conclusions: the study was conducted to highlight the scarce practices that are being implemented by healthcare staff in context of documentation and reporting of transfusion reactions at our hospital. stringent actions should be taken for the adherence of compliance by healthcare staff to avoid morbidities and mortalities. we believe that it will also be helpful to provide baseline information in the process of preparation of a national guidelines and protocol on blood transfusion procedures. 5a-s31-01 as buser, a holbro and l infanti regional blood transfusion service, swiss red cross, basel, basel, switzerland to make blood supply safer, pathogen inactivation (pi) technologies have been developed. they are based on photochemical (amotosalen/uva or riboflavin/ uv) or uv-c light treatment to reduce potential pathogens in blood components. this gain of safety might however be offset by "off target" effects of these technologies. in virtually all clinical platelet transfusion trials, it has been demonstrated that post transfusion increments with pi platelet (plt) components are lower as compared to conventional components, indicating different biological behaviour such as survival/ clearance of nontreated and treated plt. published studies have also suggested shorter survival of platelets in vivo in animal studies. additionally, data of the rates of alloimmunization and refractoriness after transfusion of pi platelets are show discrepant results. animal studies suggest a reduction of the rates of alloimmunization when transfusing (leukoreduced) pi plt as compare to conventional plt. in the clinical setting, published data, including very recent reports, showed different rates of hla class i and ii alloimmunization with the two currently available photochemical based pi technologies. while pi of plt components surely benefit patients regarding pathogen safety, the impact of potential off target effects possibly impairing efficacy of pi plt transfusions need more investigation. background: brucellosis is an endemic disease and still a major health problem in saudi arabia. ministry of health in saudi arabia listed brucellosis as a notifiable disease due to its endemicity. in the last ten years, the incidence has decreased significantly to approximately 15 cases per 100,000 but is still higher than that in developed countries. human-to human transmission is extremely rare including breast feeding, transplacental, sexually and blood transfusion. five cases of brucellosis through blood transfusion have been reported in the literature. brucella transmission through blood transfusion is likely underreporting due to the long incubation time of 2-4 weeks (range, 5 days to 5 months),vagueness of clinical presentation and lack of hemovigilance systems in endemic areas. (allohsct) and 130 (4.6%) autologous (autohsct) hsct patients, with mean corrected count increments (cci) of 8.7 9 10 3 , 8.0 9 10 3 and 7.2 9 10 3 , respectively. mean cci decreased in a linear fashion between day ≤ 2 and day 7 pcs (9.0 9 10 3 , 9.1 9 10 3 and 8.5 9 10 3 at ≤ 2 days; 6.7 9 10 3 , 5.8 9 10 3 and 4.9 9 10 3 at 7 days, respectively), although the number of pc transfused on day 7 to autohsct patients was small (n = 71). background: nipah virus (niv) is a paramyxovirus (genus henipavirus) that emerged in the late 1990s in malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in bangladesh and india. niv infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of niv contaminated foods. nipah virus (niv) belongs to the list of pathogens identified by the who to have the potential for a global pandemic. aims: this study aimed to investigate the efficacy of the theraflex uv-platelets system to inactivate niv in platelet concentrates (pcs). the theraflex uv-platelets system (macopharma) uses uvc light without the need of any additional photoactive compound. methods: plasma reduced pcs from 4 bcs (35% plasma in additive solution ssp+) were spiked with virus suspension (10% v/v). pcs (n = 2, 375 ml) were then uvcirradiated on the macotronic uv machine (macopharma) and samples were taken after spiking (load and hold sample) and after illumination with different light doses (0.05, 0.1, 0.15 and 0.2 (standard) j/cm 2 )). the titre of the niv (malaysia) was determined as tissue culture infective dose (tcid 50 ) by endpoint titration in microtitre plate assays on vero 76 cells (atcc â crl-1587 tm ). the results of the infectivity assay demonstrated that uvc irradiation dosedependently inactivated niv. after spiking a niv titer of 6.2 (bag no. 1) and 6.5 (bag no. 2) log 10 tcid 50 /ml was received in the pcs. at a uvc dose of 0.10 j/cm 2 and higher niv was inactivated down to the detection limit of the system (1.9 log 10 tcid 50 / ml), resulting in log 10 reduction factors of ≥4.3 (bag no. 1) and ≥4.6 (bag no. 2). summary/conclusions: our results demonstrate that the theraflex uv-platelets procedure is an effective technology to inactivate niv in contaminated pcs. vs. 364 ae 20.6 9 10e11 platelets/unit, p < 0.001), whereas the platelet content of apheresis pc did not change (305 ae 9.9 vs. 300, ae16.1, p = 0.57). summary/conclusions: pathogen reduction resulted in the transfusion of older pc on average, but without altering the number of pc ordered or the use of pc per patient. pathogen reduction has improved pc stock management without an increase in platelet demand, despite lower platelet content of buffy coat pc after pr implementation. donors and donation -donor adherence -are we doing the right thing? the transfusion procedure is the last step in a multi-process supply chain. the task of matching supply with demand requires donor managers to consider average consumption rates on a weekly or monthly basis, but to also have insight into variability in order distribution and possible attribute (blood groups) requirements. since hospitals and blood banks are usually not deeply interwoven and often only ex-post data is available, forecasting methods should be implemented. a thorough analysis of order pattern to set weekly target inventories and safety levels is required to close the information gap. a collection plan needs to identify possible bottlenecks which can be prevented through the planning of inter-shipping, changes in message urgency and building of reserve donor pools. constant analysis of collection and mobilization kpis allows donor managers to implement the rolling-wave planning approach and continually adapt to changing requirements, unexpected events and overall systematic variability. the variability happens on the demand side, as order quantities and their attributes, such as blood group distribution, are subject to change. however, also the supply is subject to significant variation, as donor response rates, attrition, deferrals and overall availability of donors are not constant. the data was collected with the face-to-face interview method right after the donation. 1478 first-time donors has attended to the study in 18 regional blood centres in 29 cities in turkey. the survey included items in accordance with the standard tpb predictors of attitude, self-efficacy, and intention. self-identity, anticipated regret, donation anxiety, paraphernalia anxiety, personal moral norm, descriptive norm, satisfaction, motivation also assessed for the first-time donors. the relation between the predictors and intention confirmed with correlation analyses. the predictors' distribution analysed by multiple linear regression. a number of goodness-of-fit indices were calculated and examined for each tested models (ibm, amos spss). the results of goodnessof-fit tests for proposed model provided a better fit to the data than these models (cmin/df = 14). moreover, this result indicated that the fit between the proposed model and the data could be improved with further modifications with the inclusion of paths between motivation and attitude, self-identity and intention. moreover, inclusion the paths between donation anxiety and intention and between self-efficacy and attitude, on contrary to recent analyses suggesting opposite paths. evaluation of goodness-of-fit tests showed good result for revised model with a value of cmin/df = 3.55, close to perfect fit. the revised model revealed that attitude was the strongest positive direct predictor of intention followed by personal moral norm, self-identity, motivation and anticipated regret (path coefficients: 0.47, 0.28. 0.19, 0.17, and 0.5, respectively). donation anxiety was the negative direct predictor of intention (-0.05). satisfaction was the strongest positive indirect predictor of intention via attitude and followed by self-efficacy (0.90 and 0.08). paraphernalia anxiety was the negative indirect predictor of intention (-0.03). descriptive norm did not show any significance. our model accounted for 75.3% of the variance in intention. summary/conclusions: these findings suggest several potential avenues for enhancing donor retention. the results obtained with this study provide important data from the standpoint of donor retention, which should be, implemented in the future strategies of turkish red crescent. background: transpose-transfusion and transplantation: protection and selection of donors, is a european consortium project, including partners from 16 countries, reviewing donor selection and protection policies for substances of human origin (soho).one of the main issues in the current donor selection system, which transpose aims to tackle, is that for many, if not most criteria, is not evidence based. the transpose consortium therefore tries to re-assess selection criteria, revised them where needed and provide recommendations as evidence-based as possible. transpose additionally adds to the current european directorate for the quality of medicines & healthcare (edqm) guidelines by emphasizing donor safety. aims: the aim is to compare existing donor eligibility criteria throughout europe, and to compile a list of risks to consider, with evidence-or consensus-based deferral criteria to provide more uniform donor screening criteria. methods: there are three horizontal work-packages (wps); wp1 coordination, wp2 dissemination, and wp3 evaluation of the project, and four technical ones with specific deliverables and milestones to be regularly produced: -wp4 inventory of donor selection & protection practices; -wp5 development of risk-based guidelines for donor selection and protection; -wp6 development of a standard donor health questionnaire (dhq); -wp7 training course/workshop on the use of the guiding principles, guidelines and the dhq. the transpose project launched in september 2017 and will complete in spring 2020. wp4 has completed its work in october, wp5 will complete its work in june 2019, and wp6 and wp7 have recently commenced. results: with the use of the deliverables created by wp4, we have created an indepth inventory of current practices in donor selection and protection, including overview of similarities and differences across european countries and across soho types. there is an agreement amongst experts that existing guidelines are often based on the precautionary principle rather than on risk assessment. consequently, in the development of wp5's guidelines for donor selection and protection, we now make an effort to also emphasize donor safety, in a more evidence-based way via the use of risk-based assessments. this will result in a standardized dhq with a common trunk and more in -depth questions per soho. summary/conclusions: the impact of the outcomes of transpose will be threefold. first, outcomes are expected to be of help in revising donor selection and protection related eu directives. second, the set of guiding principles and donor selection & protection guidelines will facilitate eu member states to take a next step in implementing donor selection and protection policies in a consistent and clear-cut way to the benefit of both donors and recipients of soho. third, a standard donor health questionnaire with carefully guided local/regional/national adjustments will become available per soho which can be used widely and will consequently enable comparisons of the prevalence of certain risks and risky behaviours throughout europe. background: transpose-transfusion and transplantation protection and selection of donors is a european consortium project, including partners from 16 countries, that reviews donor selection and protection policies for blood, plasma, tissues, assisted reproductive technology (art) and stem cells (together soho). donor selection criteria (dsc) in europe are based on eu-directives, guidelines and countries' own additional criteria. literature shows that particular criteria are outdated or not risk-based, often leading to unnecessary donor deferral or an underestimation of risks for donors. aims: to 1) provide a comprehensive inventory of current systems for selection and protection of donors and donations, 2) critically review them and 3) recommend an over-arching donor health questionnaire (dhq) including all necessary criteria currently used by different eu-member states (eu-ms). methods: in-depth semi-structured interviews with key stakeholders in blood collection were conducted to identify main topics for improvement in the current dsc. these formed the basis for a survey sent to professionals from collection institutions of all soho to get feedback on current systems from as many eu-ms organisations as possible. questionnaires were sent to a total of 163 experts (40 blood; 40 plasma; 27 tissues; 47 stem cells; 9 art) and 39 (24%) completed questionnaires were received. where information was lacking, additional experts were asked to recommend upon dsc. results: for blood and plasma donation four main areas of concern in dsc were identified: risk-based selection, adaptability, flexibility and consistency. the stakeholders agreed that dsc are often outdated and lack evidence, hence leading to unnecessary deferral of donors and underestimated risks for donors. they suggested to base dsc on group risk-assessment (risk-based selection) and on conducting more research to achieve standardized risk perceptions and evidence-based deferrals, either for safety of recipient or donors. criteria could be made more detailed to fit specific groups to defer less donors (adaptability). furthermore, implementing criteria was considered easy, but abolish criteria when not regarded as a risk anymore seems almost impossible (flexibility). additionally, deferral periods are perceived too long, seen as both negative, i.e. jeopardizing donor return intention and positive, i.e. no risk for safety (consistency). changing legislation into guidance was an often-mentioned suggestion to improve dsc. specific feedback on plasma donations revealed that many whole blood topics are not applicable to plasma-only donors, e.g. parasite infections such as malaria (no deferral needed); travel history (no deferral needed), and recent bacterial and viral infections (deferral periods currently too long). a clear need for more research on plasma collection-related issues was identified. summary/conclusions: dsc are perceived redundant on a substantial number of aspects by most stakeholders. besides achieving the goal of save and sufficient soho for patients, many regulations could be improved to diminish deferrals and decrease donor risks. transpose will add to reviewing, improving and harmonising these regulations and criteria. furthermore, transpose will provide suggestions to improve directives and guidelines and a dhq, focusing on both donor health protection and safety of donations, but also removing deferral criteria that are not relevant (anymore), and offer a future research agenda to make dsc more evidence-based. background: transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of 24 stakeholders from both not-for-profit and private blood collecting organizations as well as researchers and officials. the project aims to create new evidencebased donor selection criteria as well as guiding principles for risk assessment of threats to the safety of all substances of human origin (soho) except solid organs. as part of this, an inventory of current donation-related risks was performed, including an investigation of both type and number of adverse events reported. aims: we here aim to present an overview of reported adverse events in plasma and whole blood donation in europe and to compare this to the anticipated risks rated by transpose stakeholders. methods: national or local data on adverse reactions from the years 2014-2017, both serious and mild, in whole blood and plasma donors was collected from the relevant stakeholders (eighteen and nineteen respectively). stakeholders were also asked to grade the most important anticipated donor risks according to severity, level of evidence and prevalence. we then compared the relevant risk categories as evaluated by the stakeholders with the categories of the provided data, as well as the heterogeneity of category numbers. results: thirteen stakeholders provided data on adverse events during whole blood donation in a given year, including in total thirty-three different categories of adverse events, ranging from only one unspecified reaction to seventeen different categories, with an average of nine categories per stakeholder. the most frequently used categories were hematoma (included by 77%), arterial puncture (77%) and nerve damage (54%). vasovagal reactions were also frequently included (75%); however, this was being done variably as vasovagal reactions unspecified, and acute and/or delayed vasovagal reactions. only one stakeholder reported iron deficiency. for plasma donation, seven stakeholders provided data on adverse events. a total of twenty-seven different categories were reported, ranging from one to seventeen per stakeholder, with an average of nine. the most frequently reported adverse events were hematoma (86%), citrate reactions (86%) and arm pains and nerve damage (both 57%, respectively). anticipated risks in blood donation were rated by nine stakeholders rating iron deficiency, vasovagal reactions and hematomas the greatest risks to donors. for plasmapheresis, six stakeholders rated vasovagal reactions, hematomas and citrate reactions as highest risk. summary/conclusions: as shown, categories used to describe adverse events in blood donation vary tremendously across europe, with some countries only being able to provide total numbers of adverse events without further specification. furthermore, there is a gap between perceived high donor risks and reported adverse reaction categories in donor vigilance for whole blood, as reports on iron deficiency are virtually absent despite being considered the most significant risk. our findings show the need for international collaboration on creating an international standardized donor vigilance system, to gather more insight into donor risks to protect the health of donors. plenary session -a glimpse of the future pl-03-01 modern transplantation medicine has made significant progress within the last decades due to a better immunological understanding of rejection and advances in immunosuppression. however, the severe side effects of long-term, typically lifelong, immunosuppression and the shortage of donor organs remain the major restrictions in transplantation. the idea behind all research to improve transplant outcome has always been the modification of the recipient's immune system to ideally induce a specific tolerance towards the donor's graft. in fact, the immunological blindness of the recipient towards the donor's graft is achieved by a general reduction of the immune system's competence and represents a major burden for transplant patients. the idea of invisible organs is an entirely different approach to solve the problem: instead of inducing an immunological blindness of the recipient's immune system an immunological invisibility of the donor's organ is created. this is achieved by genetically engineering the transplant to eliminate the organ's immunogenicity defined by the gene products of the major histocompatibility complex (mhc) and minor histocompatibility antigens. in addition to manipulating the expression of mhc genes required for immune recognition, immune cloaking strategies are used to evade immune rejection. these approaches take advantage of creating an immunosuppressive environment and expressing immune suppressive molecules by immunomodulatory transgenes. mhc engineering and immune cloaking in an entire organ is achieved during ex vivo perfusion by lentiviral transduction of gene expression modifiers and transgenes to induce a permanent immunological invisibility of the organ. importantly, mhc engineering also prevents the presentation of minor histocompatibility antigens, which usually are not possible to match between donor and recipient, but which trigger potent immune responses and graft rejection. eliminating the targets of cellular and humoral rejection as well as creating an allograft-specific immune environment through immune cloaking camouflages the organ and equips it with a powerful set of defense weaponry. immune-engineering of transplants achieved during the inevitable ex vivo period of the allograft after explantation without the need to accept off-target effects allows keeping the recipient's immune system fully functional and capable to combat infections and cancer. in pre-clinical in vivo studies from rodents to minipigs a clear survival advantage of ex vivo engineered transplants could be demonstrated. this approach has the potential of eliminating the burden of organ rejection and immunosuppression, thereby sustainably increasing transplant survival, organ availability and quality of life. gene editing for sickle cell disease: re-expression of the fetal c-globin genes (hbg1/2) could be a universal strategy to ameliorate the severe b-globin disorders sickle cell disease (scd) and b-thalassemia by induction of fetal hemoglobin (hbf, a 2 c 2 ). we have previously identified bcl11a erythroid enhancer sequences, marked by hbf-associated common genetic variants, that are required for repression of hbf in adult-stage erythroid cells but dispensable in non-erythroid cells. recently we have optimized conditions for selection-free on-target crispr-cas9 editing in human hscs as a nearly complete reaction without detectable genotoxicity or deleterious impact on stem cell function. we demonstrate that cas9:sgrna ribonucleoprotein (rnp) mediated cleavage at core sequences of the + 58 bcl11a erythroid enhancer results in highly penetrant disruption of gata1 binding motif, reduction of bcl11a expression, and induction of fetal c-globin. erythroid progeny of edited engrafting scd hscs express therapeutic levels of hbf and resist sickling, while those from b-thalassemia patients show restored globin chain balance. moreover we find that hscs preferentially undergo nonhomologous as compared to microhomology mediated end-joining repair. nhej-based bcl11a enhancer editing approaching complete allelic disruption in hscs appears to be a feasible therapeutic strategy to produce durable hbf induction. in this presentation, i will compare and contrast bcl11a enhancer editing to other autologous curative gene therapy and gene editing approaches at various stages of clinical and pre-clinical evaluation. oxygen is vital for life. without oxygen death is assured for aerobic organisms. although everybody knows this fact a lot of medical acts forget to take care of it, leading to a lot of potential troubles. indeed, during cell respiration the glucose oxidation by oxygen gives carbon dioxide, water and energy. this energy also called atp is necessary for cellular metabolism and consequently for life. we have identified an extracellular hemoglobin coming from a marine worm, called arenicola marina, which is able to deliver oxygen to this animal living in the intertidal areas on the atlantic coast in france between the north sea and biarritz. this molecule called m101 was developed in the medical device named hemo 2 life â . we have showed that this product was very efficient to protect organs before transplantation. a multi centers clinical trial performed under the supervision of pr. le meur from the chu of brest, on 60 patients waiting kidney grafts showed a delay graft function reduced roughly by three between the two kidneys harvested on the same donor with and without hemo 2 life â and grafted on recipients. in 2018, a world first was realized in france by the pr. lantieri to georges pompidou hospital in paris, france. indeed, it was the first time that a patient received a second graft face. this surgery was realized with hemo 2 life â and showed a very nice result according the pr lantieri, the anastomosis were very easy and no edema was observed. furthermore, we have developed dressing incorporating m101 making a product called hemhealing â . preclinical data on diabetic mice showed an increase of healing process. hemoxycarrier â , a therapeutical oxygen carrier, is also in progress of development in order to address ischemic diseases such as the sickle cells disease, myocardiac infraction and stroke. this universal oxygen carrier without blood typing, which is the ancestor of our red blood cell containing hemoglobin showed that it is able to deliver oxygen at different biological levels, cellular, tissues and organs and could address a multitude of medical applications. background: main goal of transfusion is saving life and/or improve the health status of human by "safe blood" which needs regular, voluntary, unpaid blood donors. donor recruitment is being more sensitive and challenging part of the blood supply system in actual global socio-economic conditions. achievement to enough voluntary non-remunerated blood donation (vnrbd) can be established by an efficient donor recruitment. efficiency of the donor recruitment has still close relation with blood donor recruiter although there are so many new tools. occupational specifications, rights and responsibilities of blood donor recruiter have wide range differences between countries which cannot be explained completely by the specific conditions of each country. also, a concrete document which has an international consensus was not existing on this subject. turkish blood foundation (tbf) has been organizing an international workshop since 2012; anatolian blood days (abd). "who is a blood donor recruiter?" was the topic of abd-vii at 9-11 march 2018. aims: main aim of the workshop was to check and evaluate the existing systems of the participant countries. than create a model for clearly defining occupational specifications, responsibilities and rights of blood donor recruiter. methods: experts from 25 countries participated in the workshop. those countries are albania, algeria, bosnia-herzegovina, estonia, france, germany, hungary, india, kazakhstan, lithuania, macedonia, montenegro, oman, portugal, qatar, romania, russia, saudi arabia, serbia, slovenia, sri lanka, tajikistan, turkey, uganda, uzbekistan. these countries reflect almost all religious, ethnical, social, cultural and economic situations of the world. a questionnaire which was analyzing existing systems at participant countries sent before the workshop. after country presentations 4 different discussion groups were organized. below listed topics were announced at final declaration. results: donor recruiter: 1. should have university degree preferably in marketing and business administration field. 2. should have a certificate and/or professional experience in public relation 3. should have efficient skill in conversation, sociability, independence, self-confidence, reliability, resilience and conscientiousness as well as to work in a team 4. should get a special training which includes not only social topics such as public relation, marketing, etc. and medical topics related bb&tm before practicing alone as a donor recruiter 5. should be a permanent staff 6. should have basic salary and performance bonus might be given 7. is eligible to monitor and modify mobile team working period at blood drive 8. should participate the mobile blood drive which he/she has organized 9. should participate the group who will create promotional materials for national blood service 10. number at each blood establishment should be defined based on annual blood collection such as 5 staffs for 50,000 whole blood collection annually in germany. summary/conclusions: in conclusion; both donor recruitment and retention are not easy tasks to undergo while public are aging, and birth rates are decreasing all around the world. dedicated blood donor recruiter whose occupational specifications, rights and responsibilities are clearly defined will be the corner stone of the success for providing enough safe blood for transfusion. ct smit sibinga 1 and j emmanuel 2 background: africa is a large continent with 55 independent states and a total population of 1,275,710,034 (february 2018) . healthcare policies and strategies are developed through who's advocacy, guidance, and support from hq in geneva and the 2 who regional offices; eastern mediterranean regional office (emro) supporting 8 arabic speaking countries and the african regional office (afro) responsible for 47 sub-saharan countries. population distribution is approximately 40.6% urban. there are a large number of different local dialects and languages spoken. the main languages spoken are english, french, portuguese, spanish and arabic. countries are mainly classified by undp as being of low and medium human development index the africa society for blood transfusion (afsbt) has members in most countries, advocates for the development of sustainable and effective blood services, and has developed a stepwise 3 level accreditation program. in 2016 emro held a consensus meeting developing a "strategic framework for blood safety and availability for 2016-2025" with a set of priority interventions focusing on leadership and governance, cooperation and collaboration, provision of safe blood and blood products, appropriate clinical use of blood, and quality system management. in 2001 all 54 member states of the african union (au) countries, in abuja, nigeria, pledged that national budget for health should be at least 15% of the national fiscal budget. in 2013 ministers of health of who member states endorsed that blood and blood products be included in the essential medicines list; these endorsements and who's universal health coverage (uhc), have yet to be fully implemented. aims: to analyze (gap-analysis) to what extend countries in africa implement the world health assembly resolution wha63.12 on availability, safety and quality of blood products, which urges governments to ensure safe, accessible, affordable and available supplies of blood and components from voluntary non-remunerated blood donors, which meet clinical transfusion requirements and achieve national self-sufficiency, following who guidelines and recommendations. methods: to provide an overview of the current status of the blood supply in africa strengths and weaknesses, data from who's 2016 global status report on blood safety and availability were analyzed and used. the study has been descriptive and explorative. results: the 2016 report identified a number of areas requiring attention; principle amongst these were -inadequate funding; -lack of governance and leadership; -ineffective public education on blood donation; -absence of capacity building for clinicians on rational use of blood; -lack of haemovigilance and implementation of quality management systems; -the need for regulatory or oversight mechanisms. summary/conclusions: national authorities should address areas requiring attention if progress towards ensuring a sustainable safe and sufficient supply of blood products is to be achieved. key is the commitment and support of national governments, which should implement resolutions and recommendations agreed by ministers of health at wha and african union. background: the core function of the blood donation testing (bdt) laboratory is to screen every unit of blood collected from a donor for blood group type and infectious disease markers to ensure safety of the national blood supply prior to transfusion. the lab operates daily on two work shifts, comprising of 4 staff on the morning (am) shift (from 8:00 to 17:30) and 5 staff on the afternoon (pm) shift (from 13:00 to 22:00) on weekdays and 3 staff on the am shift and 5 staff on the pm shift on weekends. bdt lab has a staff strength of 15 to be rostered for the 2 work shifts. each staff is on a five-day work week and has to work 11 pm shift and 9 am shift per month on average. the higher number of pm shift leads to staff feedback that they do not get sufficient time in the evenings for family and social or leisure activities. a lean six sigma project was initiated to review the work rostering to improve the work-life balance of the staff. aims: the project aims to reduce the number of staff working on the pm shift without affecting the downstream processes and continues to meet the timely release of blood supply to the hospitals. methods: lean six sigma tools were used to study the bdt lab workflow process and to identify factors that contributes to the higher number of pm shifts that the staff has to take on. data on the turn-around time and the man-effort required for each screening tests performed was analyzed. a survey was also conducted to understand the preference of the staff on the acceptable number of pm shifts per month. results: the main contributing factor for more staff required to perform the pm shift is due to majority of the daily donation samples being received only in the evening. as this factor is beyond the control of the bdt lab, redeploying work from the pm shift to the am shift was eventually selected as the solution to reduce the number of staff needed for the pm shift. the screening test that was shifted was determined based on the test system that has the shortest turn-around-time and is able to allow continuous release of results. at the same time, most of the staff must be trained for that test system. a trial on the new roster involving 5 staff on am shift and 4 staff on pm shift was conducted. the total number of pm shift per month was reduced from 124 to 112 using the re-defined process. the 10% reduction translates to fewer number of pm shifts that the staff has to undertake and was able to meet the staff's expectation. summary/conclusions: with the adoption of the new process workflow, bdt lab was able to reduce the number of pm shifts that the staff needs to be rostered using evidence based process improvement method. most importantly, the lab has a satisfied team of staff with better work-life balance. background: preparedness of blood transfusion services for emergencies and crisis situations is an important issue concerned with patient and transfusion safety. aims: having an experience of delay in blood component supply in an emergency situation due to partial interruption of hospital information system (his), it is aimed to create a crisis kit and constitute an alternative work flow for emergency in crisis situations. methods: it is stipulated that the failure of his which is normally conducts all process for transfusion would be disabled in a disaster or crisis situation. a brain storm was made on possible challenges associated with disability of his during transfusion emergencies. according to the scenarios a kit was developed for the process management of transfusion emergencies. results: a flow chart was designed in proper with transfusion emergency definitions of who and instructions were written to explain the flow chart. all forms categorized with different colour codes are designed to fill with handwriting. the kit consists of flow chart and instructions, analysis request forms (blue coloured), blood component request forms (pink coloured), proceeding forms (green coloured), pens and blood sample tubes with edta were put into a plastic folder labelled as transfusion emergency disaster & crisis (tedc) kit. additionally, the kit is placed in a sealed clear plastic bag and delivered to all inpatient and intensive care units of pediatrics and pediatric surgery. a training programme concerned with transfusion emergency situations and usage of the tedc kit was developed for health care workers involved in blood transfusion process. pre and post-assessment tests were developed for the evaluation of effectiveness of the training programme. summary/conclusions: it's challenging to improve the response capacity of blood transfusion services during emergencies and crisis situations. abstract withdrawn. background: india is a developing country having 2760 licensed blood banks majority have manual documentation which causes inaccuracies and errors in blood bank activities. monitoring is a herculean task. computerisation is the need of the hour but this goal involves many hurdles and challenges aims: the aim of this study is to discuss the challenges faced during computerisation of blood bank activities and the remedial solutions for it methods: department of transfusion medicine, king george medical university, lucknow is one of the biggest blood bank of the country with annual collection of 70,000 blood units. two years ago, the blood bank worked on totally manual system. computerisation involved challenges associated with hardware and software installation and personnel training. hardware was installed in two phases. initially hp system but later shifted to apple imac due to frequent breakdowns. with hp server. software installation (easy software) involved erratic internet connectivity hence changed to lan. customisation involved radical changes according to our needs. at times we had to change our way of working to suit the software. biometrics linking, medical registration, cash id generation, donation, serological crossmatch, automated blood grouping, labelling, chemiluminescence & nat testing, blood component preparation and camps were all included with challenges at every level. remedial actions were taken from small to big. training of the staff was the most essential part of the implementation of computerisation who initially showed considerable resistance and at time faking ignorance due to apprehension that their mistakes will be highlighted and they will be penalised for it. it was a herculean task in creating their password protected identity and enforcing them to use it. gradually the staff realised that computerisation made their task easier as it cut down on paper work and repetition and also prevented serious mistakes from happening. hard copies at certain essential areas were still maintained to continue work in the event of major breakdown of computer results: computerisation aided us in regulating the movement of the donor which at times was repetitive due to manual entry. transfer of data ensured a safe supply and the mistakes could be retraced very easily. implementation which included installation, training and enforcement took a period of 6 months. after overcoming all the challenges we minimised hard copies to 6 registers and started taking printouts of the other necessary details. the turnover time for the employees due to computerisation decreased by 20%. waiting time for attendant decreased by 10%. traceability of all the units became 100%.supervision of the activities being carried out was 90% accurate. identification of the donors was easy due to biometrics which included thumb impression and iris scanning. the decision making time for donors decreased by 50% thus making the system more efficient. summary/conclusions: manual to computerisation involves involvement from source to supply and it is essential to anticipate the challenges and be prepared for solutions in order to make its implementation successful p-007 ct smit sibinga 1 , y abdella 2 and f konings 2 1 iqm consulting, zuidhorn, netherlands 2 who eastern mediterranean office, cairo, egypt background: who defined essential medicines (ems) as medicinal products that satisfy health-care needs of the majority of a population. they should be available at all times, in adequate amounts, in appropriate dosage forms, with assured quality and affordability. in 2013 blood and blood products (whole blood, red cells, platelets, plasma, and plasma-derived products) were added to the who model list of ems. appropriate and effective regulatory framework (legislations, regulations, etc.) and a functioning regulatory authority (ra) is crucial for management of blood products as ems. however, particularly in the less developed world, these prerequisites have barely been implemented. aims: to analyze and advise on existing legislation and regulations. existing legislative instruments of the 22 member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. methods: existing legislative instruments of the 22 member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. results: various formal legislative documents of only 9/22 countries are put in force by governments [1960 (egypt) till 2017 (pakistan -sindh)]. most are detailed descriptions of ra, operational establishments, and specific requirements. however, none of these legislations complies with who and eu recommended formats and contents, and will not support effective regulatory oversight to promote and enhance quality, safety and availability of these ems. summary/conclusions: government should provide effective leadership and governance in developing a national blood system (nbs, fully integrated into the national health-care system. essential functions of a nbs include an appropriate regulatory framework with legislations, regulations and other non-legislative instruments administered by a ra. these documents should spell out principles and cadres, standard setting, and organization of the blood system to ensure an adequate supply of blood and blood products and safe clinical transfusion for which a model was designed. the structure of nbs will depend on organization and level of development of the health-care system. however, all critical activities within nbs should be coordinated nationally to promote uniform standards, economies-of-scale, consistency in staff competency, quality and safety of these ems, and best transfusion practices. key is formulation of an appropriate regulatory framework administered by a ra responsible for regulating the vein-to-vein chain in the preparation and use of these ems. background: the capacity of blood transfusion service to provide adequate supplies of blood components is the issue of concern for health providers worldwide; longer term observations of trends in this respect are therefore of crucial value. aims: the study aim was analysis of some basic activities of the polish blood transfusion service in 2008-2017 including organizational changes, numbers of donors, donations and blood components as well as activities directed at increasing their safety. methods: retrospective analysis of data supplied by the regional blood transfusion centers (btcs). results: in the discussed period, blood and blood components were collected in 21 regional btcs and local collection sites as well as during mobile collections. although the number of local collection sites decreased from 170 in 2008 to 133 in 2017 in favor of mobile collections, which increased from 8,672 to 13,189, the former is still the number one location for donating blood. on average 47.36% of all donations were performed in local collection sites. the total number of blood donors both at the beginning and the end of the discussed period was similar (583,908 in 2008 and 588,184 in 2017); over 99% of all donors were non-remunerated. however, the number of first-time donors dropped significantly (from 237,658 in 2008 to 143,038 in 2017). the total number of donations increased from 1,076,655 in 2008 to 1,249,655 in 2017; most frequent were whole blood collections (from 1,016,411 in 2008 to 1,171,302 in 2017) . some blood components (mostly plasma and platelet concentrates) were also collected by apheresis. most frequently prepared blood components were red blood cell concentrates -rbcs (996,678-1,154,239 units per year), fresh frozen plasma -ffp (1,090,369-1,289,021 units) and platelet concentrates -pcs (81,692-129,143 units, with significant increasing tendency). additional processing methods such as leukocyte depletion and irradiation were more frequently applied to pcs (52-57.6% in respective years irradiated, 75.18-92.07% leukocyte-depleted), than to rbcs (4.46-8.46% irradiated, 7.65-20.7% leukocyte-depleted). in 2010, the pathogen reduction technologies in plasma and the pcs were implemented. up to date however the use of these technologies is limited in most btcs. in 2017 approximately 11.5% of pcs and 8% ffp units issued for transfusion were subjected to pathogen reduction technologies. summary/conclusions: our study data may contribute to the assessment of some long-term tendencies observed in polish blood transfusion service and may serve practical-benchmarking. this in turn may prove beneficial to the transfusion community as a whole. background: in poland 80% of hospitals depend on blood for the treatment of patients; over 1.5 mln units of blood components are annually transfused. it is therefore purposeful to expand the knowledge on factors impacting on blood transfusion service (bts). the institute of hematology and transfusion medicine (ihtm) as competent authority is responsible for collection of data related to the activity of all polish blood transfusion centers (bcts). this data is exploited to a much lesser degree than the recently available statistical methods and data processing tools would allow. moreover, survey of research in the field of public health indicates a negligible share of issues related to bts. it seemed therefore necessary to "fill in the gap" with true assessment of performance of the polish bcts for improvement of bts activity. 1 st stage of our investigation refers to collection, merging of data from different sources, their unification and preparation (big data) for further analysis to be performed using multidimensional statistical analysis and data mining methods. aims: assessment of the activity of the polish btcs over the 20 year-period in two stages. goals at 1 st stage: 1. data digitalization; scanning of paper documents. 2. development of a uniform template for collecting digital data from various sources. 3. standardization, unification and quality improvement of available data: filling in missing data, elimination of errors, duplicate records etc, that may distort the outcome of analyzes. 4. selection of data for analysis. methods: digitalization and big data methods for processing various types of data: a) stored in paper form (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) , b) digital stored in two file types (.doc and .xls, for the years 2005-2010 and 2011-2017, respectively). for each data-type, a separate excel file model was created. the models were then merged into one analytical table with data processing methods. results: 1. 1344 pages of paper documents were scanned. 2. models developed for data from 3 different sources: a. paper-data were rewritten and ascribed to its model; outcome -3 tables, 272 columns, 10,400 rows. b. .doc and .xls. filesdata were ascribed to 2 other models; outcome -6 tables, 1656 columns, 788 rows. 3. the 3 models were merged into 1 analytical table to create a 588 mb database (comparable to approx. 784 min of music). 4. the data was subjected to standardization, unification and quality improvement: filling in missing data, elimination of errors and duplicate records that may distort the results of analyzes. 5. selection of data for analysis at 2 nd stage. summary/conclusions: the 1 st stage provided a set of selected data for analysis in the 2 nd stage which will rely on multidimensional statistical analysis and data mining methods. the outcome of such analysis will contribute to optimal realization of objectives: a) gaining in-depth knowledge about the fundamental phenomena that shape polish bts, b) identification of potential changes bcts, c) development of overall guidelines for change management. aimed to touch untouched or less touched topics of bb&tm. so far 8 workshops were organized and each year around 30 countries were participated from almost all religious, ethnical, social, cultural and economic situations of the world. 6. supported realization of major changes in bb&tm in turkey; a convincing medical individuals and agencies mainly moh to give the deserved consideration to bb&tm b encouraging the recognition and establishment of national blood program c issuing a new blood act and numerous necessary bylaws, etc. d creating an appropriate standard donor questionnaire form e changing blood transfusion practice from %96 whole blood (at 1996) to 5%. f changing donated blood screening criteria; while anti-hcv screening became obligatory malaria, screening cancelled g preparing national guidelines h promoting haemovigilance nurse post i promoting patient blood management 7. around 11,000 blood bankers attended national courses, 7,000 attended national congresses, 18,000 attended nationwide symposiums. summary/conclusions: bbtst can be accepted as a sample how a scientific nongovernmental organization may give a very positive impact on developing and progressing bb&tm activities with close collaboration moh and other related organizations abstract withdrawn. background: globally there is growing investment in information technology (it) in business. this similar trend has been observed in blood establishment computerized systems (becs). the it investment can be high hence it decisions need to be properly informed. the africa society for blood transfusion (afsbt) encourages the use of its in african blood services as this optimize quality blood services, thereby improving patient's outcomes. afsbt established in 2016 an it working group (afsbt itwg) with the support of the swiss red cross (src) to spearhead the it standards among member blood services. a number of priority it thematic areas were identified. these includes it governance which focuses on creation (strategic alignment) and preservation (risk management) of business value. there is absence of published literature on how a structured it governance framework can be implemented in a resource constrained setting. a review of the national blood service zimbabwe (nbsz) it governance was done based on published it governance framework. aims: to explore how a structured it governance can be developed, implemented and monitored in a resource constrained setting. methods: a published mit-cisr framework which has six components was used to assess the strength, gaps and opportunities of the it governance. results: nbsz has been implementing an evolving structured it governance system. in terms of service strategy and organization there is a well-established it function which is reflected in the nbsz strategic plans. this ensures it annual budgetary support, which averages 4.3% of the total budget. the it governance arrangements are such that decision rights are assigned to different it staff (executives, it specialist, and users). a range of it solutions have been embedded within the nbsz operations such as becs, financial, donor mobile application, social media, temperature monitoring, and human resources. the business performance goals are defined and are congruent across the various business units. it organization and desirable behaviors are documented in the ict policies and procedures and were needed remedial actions are available through the code of conduct. the it metrics are included within the nbsz monitoring and evaluation system which use a four colored traffic lighting reporting system. it was noted that the it accountabilities are undesirably tilted to the it specialist only hence some ict projects tend to have delayed deliverables. the it governance mechanisms are supported with tools such as service level agreements and established communication approaches. simple excel based solutions are used to track critical performance metrics such as on the interactive blood supply management status, which averages 122.8% (2018) based on a 5-day projected stocking and supply levels. nbsz need to properly document the return on investments on all these ict initiatives, which is estimated (2016/2017) to be at 3.2% of annual savings. summary/conclusions: blood services in resource constrained settings can implement a properly structured it governance and this will ensure maximum return on it investments. the nbsz approach will be shared and further developed in the afsbt itwg to support other blood services in improving their it governance. haematology/blood transfusion, alfred health, melbourne, australia background: in october 2018, an integrated electronic medical record (emr) was implemented at an australian metropolitan multi-campus heath service using cerner millennium tm , aiming to achieve himss (healthcare information and management systems society) level 6. prior to implementation, large numbers of blood specimens were collected from patients unnecessarily and sent to pathology without a test request attached (no blood test requested -ntr). these specimens required additional processing in the laboratory. electronic specimen collection using cerner specimen collect tm allowed streamlining of specimen processing by eliminating paper requests. as part of the new workflow, individual specimen labels are printed with the specified blood test and correct tube type. this helps prevent the practice of collecting additional specimens due to uncertainty of the collection requirements. aims: • to quantify the expected reduction in ntr specimens following introduction of electronic specimen collection, and outline the benefits • to determine the impact on collection errors and wrong blood in tube (wbit) events methods: data was obtained directly from cerner millennium tm using a ccl (cerner command language) query which is run monthly by pathology it staff. this data includes all specimens registered for the month with indication of rejected specimens, wbit & ntr samples. 'rejected specimens' includes incomplete specimen and/or request certification, unlabelled specimens/requests and mismatched specimens. further information about wbit events was collated from riskman reports and staff interviews. results: data from the 12 months prior to emr implementation was compared with 3 months post. ntr numbers reduced from 4220/month to 2019/month (52% reduction), freeing up more storage space in fridges. rejected specimens due to inadequate patient request labelling reduced from a mean of 27/mth to 6/mth. wbit numbers have increased slightly: before having median 1 (range 0-2), after with median 2 (range 0-3). although it was hoped that wbit incidence may reduce with the new emr, 4 of the post implementation 6 wbits involved electronic specimen collection. departure from planned protocols involving a lack of working printers, causing staff to print patient labels away from the patient's bedside, as well as multiple patient labels printing on individual printers appear to be a main cause of the emr wbits. summary/conclusions: emr implementation has led to a reduction in ntr, and rejected specimens due to inadequate request labelling, as well as increased storage space in laboratory refrigerators. associated benefits include: • decreased financial costs of the wasted equipment • decreased staff time collecting and processing unusable specimens • decreased environmental impact of manufacture and disposal of unused specimens • decreased potential of iatrogenic anaemia work in preventing the occurrence of further wbits is ongoing, by ensuring that label printers are in working order, are in plentiful supply and easily accessible to staff; and also ensuring positive patient identification and blood collection by the patient's bedside remains a priority. jm mustaffa 1 , k teo 2 , s tsai 1 , p heng 1 , r sagun 1 and m wong 1 1 laboratory medicine 2 khoo teck puat hospital, singapore, singapore background: khoo teck puat hospital (ktph) is a 761-bed general and acute care hospital, opened in 2010, serving more than 800,000 people living in the northern sector of singapore. the blood bank of ktph department of laboratory medicine provides specimen testing and blood transfusion services for ktph as well as the neighbouring yishun community hospital (ych), one of the largest community hospitals in singapore providing intermediate care for recuperating patients including rehabilitative services. the process of ordering transfusion-related test requests in both hospitals is through printed forms. aims: in line with the hospital directive to move towards electronic patient management, the ktph blood bank intended to implement an electronic type and screen (e-t&s) system. the goal of the project is to ensure zero patient identification errors and maintain full traceability and accountability for the blood collection process in all transfusion-related testing. another aim of the system is to reduce repeated venepunctures when specimens are rejected due to missing essential patient information on the printed forms by implementing mandatory fields in the e-t&s form. methods: the e-t&s was implemented in phases. phase 1: an online version of the printed form was signed electronically by the ordering doctor and a witness within the electronic medical record system, sunrise clinical manager (scm) system with the doctor counter-checking by signing on the specimen label to ensure correct patient identification. phase 2: the ordering doctor is not required to sign on the specimen label since fingerprint biometrics are required for the electronic signin. phase 3: elimination of the witness step for blood collection. specimen collection and rejection data from 2016 to 2018 was analysed. specimen rejection rate was presented as percentage of rejected specimens (mislabelled, unlabelled and clerical errors) over total specimen count for each month. results: between january 2016 and march 2017, before the implementation of the e-t&s phase 1, the average rejection rate for blood bank specimens was 0.16% and 1.14% for identification and clerical errors respectively. during phases 1 and 2 of implementation, rejection rate increased due to unfamiliarity to the new work processes. by february 2018, with the implementation of the final phase of the e-t&s system the specimen rejection rate was 0.38% and 0.12% for identification and clerical errors respectively. rejected specimens were mostly from the few locations that had to use paper requisition due to workflow or infrastructure limitations. summary/conclusions: the e-t&s system was implemented successfully in ktph. full traceability and accountability of the blood collection process was maintained with the fully electronic system. the adoption of electronic documentation has also reduced the number of preventable repeated venepunctures that were due to incomplete order information on the printed forms. future developments in technology and full implementation of e-t&s system in all hospital locations may make zero patient identification error achievable and ensure transfusion safety in all patients in the near future. background: blood component administration represents a critical phase due to the possible occurrence of errors during the different steps from the identification of the patient to the infusion of the product. error occurrence can be reduced by the implementation of validated information systems. we tested the scweb â system at the bedside in a transfusion outpatient clinic. aims: the aim of the study is to validate a system designed to assist and to control blood administration step by step using electronic devices to ensure traceability and documentation of the process methods: the scweb â system is based on it monitored checklists which guide the personnel to follow the procedure, according to best practices; the system must initially be activated by the operator which is recognized by an auto-signing system based on bluetooth low energy which avoids the operator having to identify himself/herself beforehand. appropriate privacy protection is provided. thereafter the system takes up the task to give instructions and to verify the adherence, by asking an active confirmation of the proper fulfillment of the activities; a continuous registration and documentation is made by the system. standards and specifications for each step of the procedure have been configured on scweb â system to track in detail operator and patient identification, presence of informed consent to transfusion, blood pressure, pulse and temperature recording, vein access, verification of the blood unit. an alarm has been set after 15 min, to ensure the control of patient's conditions. for each step, an active confirmation of the action is required and nurse and doctor direct involvement must be actively confirmed on the device by both operators. the system has been tested at the bedside on 30 patients admitted to the outpatient clinic for 45 red cell concentrate transfusion; compliance of the personnel and organizational impact has been recorded. results: the system required a very short training: ease of scweb â system allows its implementation without negative impact on organization of transfusion outpatient clinic and without difficulties by operators (nurses and doctors), who appreciated the help given by the it check system. the registration of the electronic check list offered a reliable tool for the traceability of the transfusion procedure, also granting a paperless and timely available documentation of the entire process through a registration in electronic format of all the operator's action in every single phase of the transfusion process. when prescribed, confirmation of the checklist was only possible in the presence and with the active confirmation of two operators (doctor and nurse). summary/conclusions: the scweb â system is useful as a barrier against the mismatch of transfusion (preventive measure), as a traceability and documentation measure and as a tool for training of personnel in blood transfusion administration; it avoids paper registration during the transfusion process, due to the timely registration of the activities performed by operators recognized by the system thanks to the bluetooth low energy auto-signing device. the scweb â system will be connected to the transfusion data management system, to monitor all the process from the arrival of the unit from the blood bank. background: he blood banks aims at reducing cost and increasing customer satisfaction by providing quality in service. the quality in service can be attained by streamlining the processes and restructuring the supply chain of the organization by implementing it tools. aims: aim is to understand the complex flow of information and processes within the supply chain of the blood bank. the requirement of such a study is a part of the integrated erp modeling for the integrated functioning of a blood bank. methods: he approach used to understand and map the sequence of processes, and the work responsibilities of each process and the operational decisions involved at each step is process mapping and data flow diagrams for front end system modeling and analysis. the processes are mapped and represented in a schematic diagram. dfd (data flow diagram) are constructed for representing the system. a context diagram is also constructed for understanding the entities interacting with the system. the emr systems aim at replacing (or supporting) the paper based medical records. the whole model of the system is divided into two parts-front end and back end. the front end design and analysis is done using epc (event-driven process chains), resource views, data flow diagram for data view. reporting was on donor selection, finance and collection of blood bag, blood collection process, component preparation, blood testing and blood distribution results: process mapping using event driven process chain generated a whole view of the processes involved. the resource view gave an organizational structure and the personnel involved. the data view using context diagram and data flow diagram gives a flow of data and amount of data involved. this framework can be used for business process reengineering for the blood banks by conducting a time study and removing non value added activities. data view helps analyze redundant data in each process. it also helps in staff training and orientation within the department. summary/conclusions: a systematic overview presented in this paper facilitates in removal of non value added processes, duplication of data, bottlenecks, reduction of cycle time and thereby improving service quality in blood banks. background: the transfusion of blood components, one of the most prevalent interventions in clinical practice is a major expenditure item in healthcare services which tend to increase in recent years. aims: it is intended to investigate the impact of transfusion associated costs to hospital costs in pediatric intensive care unit (picu) patients. methods: during a year period (january 2017-december 2017) 76 patients, 40 females and 36 males receiving transfusion with blood components along the stay in picu were included in the study. transfusion associated costs and total costs for healthcare services for children treated in picu was collected by using hospital information system (his). statistical analysis of data was performed by spss software (version 22.0, spss inc., chicago, il, usa). mann-whitney u test and kruskal-wallis test was performed for comparison of independent categoric variables and numeric data; chi-square analysis was performed for comparison of two numeric variables and spearman correlation analysis was performed for associations. results: the median age of patients was 12.0 months (interquantile range-iqr 26). the median length of stay was 16 days (iqr 30). in total 400 blood components were transfused in which of 227 red blood cell concentrates, 114 apheresis platelet concentrates, 6 granulocyte concentrates, 51 fresh frozen plasmas, and 1 cryoprecipitate and 1 whole blood. the ratios of transfusion associated expenditures to hospital costs were categorized in intervals of percentages as < 5%, 5-10%, 11-15% and > 15%. most of the patients (63.2%) were ranked in the lowest interval. the medians for hospital cost and transfusion associated cost were 5478.76 euros (iqr = 11280.02) and 130.57 euros (iqr = 354.86), respectively. a significant strong positive correlation between numbers of transfusions and hospitalization cost of picu was detected (r: 0.674, p < 0.01). while it was found a significant weak positive correlation between transfusion associated cost and hospital cost (r: 0.247, p = 0.032) there was also a significant weak positive correlation between the age and transfusion associated cost (p = 0.048, r: 0.227). a significant difference was found between the patients with and without hematological malignancies (p < 0.01) for transfusion associated cost. the reason why pediatric dosages are mostly prefer is that the hospital provides healthcare for only children and splitting of the blood components was common in the hospital. but unexpectedly a significant increase on the transfusion associated costs which is related to split blood components was detected (p < 0.05). summary/conclusions: studies on the economics of blood transfusion have been conducted mostly in patients who require chronic or multiple transfusions. picus, specialized facilities that provide care for patients with severe life-threatening diseases are major departments often necessitate multiple transfusions. there are many variables to evaluate the impact of transfusion associated cost to hospital cost in picu patients, but the major factors are underlying conditions, admitting diagnoses and transfusion strategies. although there are unexpected data in our study demonstrated the increasing impact on transfusion associated cost originated from blood components split for pediatric usage no significant relationship was determined to explain this situation. further studies on the economics of blood transfusions have to be carried out to clarify the variables of transfusion associated costs. background: approximately 55.7% of the transfused blood component is packed red cell (prc). over ordering of prc unit is a common practice and excessive pretransfusion testing was being wasteful of resources and have adverse consequences on cost. high crossmatch to transfusion (c/t) ratio as quality indicator of blood bank implies that crossmatches were performed unnecessarily. aims: the aims of this study were to evaluate the cost effectiveness of strategies for limiting the number of pretransfusion testing of ordering prc. methods: all prc units who ordered from dr. hasan sadikin hospital from january 2016 to december 2018 were collected in this retrospective study. number of ordering prc unit, completed pretransfusion testing of ordering prc units, and prc units that were transfused were recorded. restrictive pretransfusion testing strategies were done based on the hemoglobin level and diagnosis as transfusion indication criteria. cost effectiveness was measured by multiplying the unit cost of pretransfusion testing and number of prc unit. results: out of total 177,370 ordered prc unit, 166,910 (94.1%) were subjected to pretransfusion testing and 63.1% (105,260) of ordering prc unit which are pretransfusion testing were transfused. this means that 5.9% (10,460) of ordering prc unit were not subjected to pretransfusion test. this showed savings of 1,098,300,000 rupiah. c/t ratio was 1.6 which demonstrate a good ordering pattern. however, 16.4% (27,451) of completed pretransfusion testing of ordering prc unit were not transfused, leading to blood bank loss of 2,882,355,000 rupiah. summary/conclusions: strategies for limiting the number of pretransfusion testing on the good c/t ratio was still associated with saving cost effective background: blood is a precious resource for saving patient lives. the purpose of blood and blood component therapy is to provide suitable and safe blood products to achieve best clinical outcomes. nurses have an important role in ensuring safe blood transfusion. it is crucial for nurses to have sufficient knowledge about blood donation and collection, storage, component preparation, possible adverse effects of blood transfusion and necessary management and care. aims: the aim of this study was to assess the impact of an educational intervention on knowledge and awareness of nurses regarding blood transfusion services and practices. methods: the baseline study to assess the knowledge and awareness regarding blood transfusion services and practices of the nurses posted at various areas of the hospital including wards, operation theatres and critical care areas was carried out at our institute hospital which is a tertiary care teaching centre. the nurses were then sensitized and educated regarding blood transfusion services and practices during their day to day activities by referring them to the blood transfusion guidelines of the institute. subsequently, a self-developed questionnaire which was used for the baseline assessment of knowledge and awareness of the nurses was again used to reassess them. a total of 19 questions were included in the questionnaire pertaining to: general awareness (two questions), blood donation (two questions), testing and blood component preparation related (two questions), storage of blood/blood components (two questions) and pre-transfusion checks and bed side transfusion practices (eleven questions). fifty nurses each were included for both the baseline as well as post-sensitization assessment. for different category of questions, the correct response rates were compared with those obtained in the baseline study using mann-whitney test. the entire study duration was spread over a period of three months (december, 2014 to february, 2015 . results: the overall mean percentage of 'correct' responses for 19 questions in the baseline study was 61.75%, whereas post sensitization it was 77.21%. the mean percentage increase in general awareness related questions was 21.49%, 13.95% for storage of blood/blood components related questions, 17.37% for pre-transfusion checks and bedside transfusion practices related questions, 21.33% for testing and blood component preparation related questions and 27.27% for blood donation related questions. the percentage increase in correct response was found to be statically significant for each of the five categories of questions. the overall mean percentage increase in correct response rate was also statistically significant (p < 0.001). summary/conclusions: this study revealed that after sensitization and educational intervention there was a significant improvement in the knowledge and awareness of nurses regarding the blood transfusion services and practices. abstract withdrawn. background: tact, introduced in the uk in 2014 to support managers, provides resource-saving, continual, 'real-time' monitoring of knowledge-based competency of staff in transfusion laboratories. tact is available online 24/7, complementing existing practical competency schemes and external quality assessment. multiple variations on a standard pre-transfusion testing scenario are generated using constrained randomisation; logic rules for automatic assessment of sample acceptance, abo/d, antibody screen and identification (as/id), and component issue are based on bsh guidance. during 2018, tact was offered internationally to transfusion laboratory managers to trial, and saw uptake in five countries. the core tact programme, based upon uk guidelines, is under review for programming conversion, to be customisable for the international community. aims: to assess the feasibility of tact programming conversion to meet the requirements of country-specific pre-transfusion testing guidelines, and to direct future programming in line with feedback from international users. methods: guidelines from 3/5 international users were obtained and translated where necessary. these were compared against the core assessment elements of current tact programming. international users were approached for their feedback on the current version of tact, as it compared to their local policies and practices. results: the following criteria were cross-referenced: specification of transfusion request forms, sample label acceptance criteria, reagents used for abo/d and as/id, resolution of grouping anomalies, alloantibody confirmation/exclusion, and selection criteria of blood components for transfusion-dependent patients and women of child-bearing potential. apparent differences included:-australia:--selection of red cells for patients with immune anti-d. greece:--inclusion of the name of the patient's father on the transfusion request. italy:--testing of all new patients with an anti-a,b reagent and two different monoclonal anti-d reagents. international users in the same three countries supplied feedback. this included suggestions for:-greater complexity of cases presented, provision of patient history, inclusion of follow-on tests e.g. phenotyping and cells for antibody confirmation/exclusion, broader range of reaction strength grading, and official professional cpd credits. the following differences were noted:-nomenclature used, the format and content of the request form, use of english abbreviations of patient clinical details, and the availability, provision and specification of blood components. summary/conclusions: this analysis has shown very few instances where the current tact iteration differs from the guidelines reviewed, and that it is feasible to expand the use of tact on a more international basis. the current iteration of tact has been developed to represent an abbreviated scope of pre-transfusion testing practices, which can be applied to laboratory practice outside of the uk without difficulty. further work is required to enable international users to configure tact such that the system represents all laboratory practice on an international basis. aims: these courses provide education to clinicians on patient blood management and safe transfusion in neonatal and paediatric settings in order to improve patient outcomes and increase awareness of the national patient blood management guidelines. this analysis aimed to investigate the uptake, practical use, and perceived value of the courses by learners. methods: a retrospective analysis of course completion statistics and course evaluation data. results: there have been 2,376 paediatric and neonatal courses completed from 6 march 2018 to 28 february 2019 with 89.6% of learners being nurses and/or midwives. analysis of course evaluation data (n = 33) showed that these courses: -provide knowledge (96.9%) -improve patient safety and outcomes (84.9%) -result in change to clinical practice (69.9%) -are relevant to clinical practice (70.9%) -are easy to use (67.7%) -are readily accessible (58.1%). examples that learners provided of how they can apply this learning to their clinical practice include: -"[i am now] more aware of special requirements for neonatal blood transfusion" -"[i] feel more confident especially when talking with parents" -"[i will now be] checking the patient's blood results and will speak up for unnecessary blood sampling" -"[it's good that] when there is ambiguity in clinical practice [this is] very well shown by explanation from experts in the field" -"we don't do a lot of transfusions [and this is] a reminder that transfusions are not always the first answer to the baby's clinical picture". summary/conclusions: analysis of course and user evaluation data demonstrates that these courses are being used by nurses and doctors working in the neonatal and paediatric settings and that they provide knowledge of pbm that can be applied to clinical practice, thereby contributing to improved patient care. background: blood transfusion is a high-risk clinical activity that must be mastered both theoretically and practically in order to guarantee the required result without any incident or complications. the mastery of transfusion knowledge among nurses represents a very important link in the transfusion chain. the objective of this work is to compare the theoretical and practical knowledge of transfusion among two groups of nurses divided according to their seniority. aims: this is a cross-sectional descriptive study conducted over a period of 1 month [1 st april-30 th april] 2017. we selected two groups of care staff: the 1 st group consists of 50 students at the end of their training at the higher institute of nursing sciences. the 2 nd is made up of 50 nurses working in 5 university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of 15 simple or multiple choice questions, 7 were related to theoretical knowledge of labile blood products and 8 to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. methods: this is a cross-sectional descriptive study conducted over a period of 1 month [1 st april-30 th april] 2017. we selected two groups of care staff: the 1 st group consists of 50 students at the end of their training at the higher institute of nursing sciences. the 2 nd is made up of 50 nurses working in 5 university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of 15 simple or multiple choice questions, 7 were related to theoretical knowledge of labile blood products and 8 to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. results: the participation rate in the survey was 100%. the 2 nd group participants had an average seniority of 9 years . more than half of them (64%) had seniority of less than 10 years. only 12% had more than 20 years of experience. the rate of correct answers for all items combined was 44.4% for students versus 42.5% for practicing nurses. the theoretical knowledge part was more mastered in the 1 st group than that of practicing nurses (44.8% vs 33.4% of correct answers). on the other hand, the control of the transfusion act was better in 2 nd group (44% vs 50.5%). the overall "dangerous" response rate was 47% for students and 41.7% for practicing nurses. false practical knowledge was more common in group 1 (59.5% vs. 41.5%). summary/conclusions: the theoretical as well as the practical knowledge remains not well mastered by the care staff. our study highlighted the best theoretical mastery for young students and practical for practicing nurses. this could be explained by the freshness of knowledge in the first group and the daily practice in the second group. background: the european commission (ec) directive 2004/33/ec on blood donor selection criteria is 15 years old. in the meantime, knowledge on risks related to blood donor selection has progressed and challenged several obligatory rules. transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of more than 24 stakeholders from both not-for-profit and private organizations providing substances of human origin (soho). the project aims to provide evidence-based donor selection criteria and guiding principles for risk assessment of threats to the safety of soho. as part of this work, an inventory of current blood donor selection criteria in europe and an evaluation of the evidence behind current practice was performed by experts working on this project. aims: to identify the gap between the ec directive 2004/33/ec on whole blood donor selection criteria and a current evaluation of the clinical relevance of the criteria based on scientific literature by a panel of european experts within the transpose project. methods: in 2018, we performed an inventory of blood donor and transfusion recipient risks in participating european countries. project members were asked to provide the existing donor selection criteria related to these risks and to carry out a risk-based evaluation for each of them. the evaluation was based on the available scientific literature and on a risk assessment template based on the abo risk-based decision-making framework, developed by transpose. all risks with divergent assessments within the panel were resolved through discussion; in all cases an expert consensus was established. subsequently we compared the results with the content of the ec directive 2004/33/ec for every risk, thereby identifying discrepancies and missing items in the directive. results: the panel identified 64 risks considered to be significant, distributed between donors and recipients. for 35/64 (55%) of them the expert evaluation deviated from the content of the ec directive, or the ec directive provided no information about the decision making. in particular, a discrepancy was observed for 9/20 criteria concerning general health and medication, 12/22 for transfusion transmissible infections, 10/13 for high-risk behaviour and travel, and 4/9 for other diseases. summary/conclusions: our results highlight a significant gap between the whole blood donor selection criteria stated in the ec directive 2004/33/ec and the scientific evaluation performed by a panel of transpose participating experts. this gap includes both new risks not addressed in the ec directive and addressed risks that are however evaluated differently. this involves both blood donor and transfusion recipient safety, and various medical and epidemiological topics covering several aspects of the blood donation criteria. we strongly recommend a change in the european legislation, allowing a procedure to guarantee that blood donor selection criteria are updated regularly within the framework of the european institutions, to keep aligned with scientific progress, epidemiology and changes in medical practice, in order to enable an updated risk-and evidence-based framework for donor selection criteria. the risk-assessment method elaborated in the transpose project is a valuable instrument for this purpose. background: the brazilian health regulatory agency -anvisa has developed the method for assessment of potential risk in hemotherapy services (marpsh) which is based on the data collected during the inspections of blood services carried out by regulatory authorities. using marpsh any blood service can be classified in one of 5 possible potential risk categories: high, medium-high, medium, medium-low and low risk. each category represents a different potential risk level, according to the proportion of compliance with the established regulatory requirements. marpsh has been used since 2007, showing a trend of risk reduction on blood services evaluated all over the country. aims: this work aims to describe the utilization of marpsh as a tool for an integrated risk management model. also, it shows the main results obtained after 10 years of data monitoring and coordination of regulatory actions and policies by anvisa, targeting quality and safety of blood products. methods: the utilization of marpsh follows a network risk management model since the inspections are carried out by decentralized organs in all 27 states and some municipalities. the inspectors fulfill a standardized inspection guide containing the regulatory requirements, where each item is associated with a level of risk, varying from i to iii as the risk increases. at the end of the inspection, after a statistical calculation, the service is categorized. this classification gives an estimate of its quality profile, guiding the adoption of suitable measures for risk management by local authorities and services. these data are send to the states (if realized by municipalities) and to anvisa that perform consolidation in a national level. either states or anvisa use data to coordinate risk management measures in a broader spectrum. data are continuously monitored by anvisa as part of its strategical panel of indicators. anvisa follows up specially blood services in high and medium-high risk with the aim of helping or complementing local authorities' actions. additionally, anvisa periodically sends this information to the brazilian ministry of health and local governmental organs from brazilian national blood system that also support actions to improve quality in their blood services networks. results: since 2007, when the assessment covered 109 blood services, marpsh reached 1218 blood services in 2017 (57% of the blood services registered) what corresponded to almost 100% of the inspection cover in this year. over this period (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) it is possible to notice a dramatic decreasing in trend for proportion of blood services classified as high and medium-high risk, varying from 26% to 9%. summary/conclusions: marpsh generates data necessary to the categorization of blood services into five levels of potential risk. as a result, the regulatory actions are applied by local organs with the purpose of reducing or eliminating risks involved in the production and use of blood components. data have shown a significant risk reduction over 10 years of marpsh's utilization. additionally, monitoring of this data at a national level has been permitting appropriate planning and prioritization of integrated strategies directed to risk management and strengthening of blood services in brazil. background: sub-saharan africa has the highest need of blood transfusion in the world, mainly for childbearing women and children suffering from malaria. meeting basic quality and operational requirements to provide patients with safe blood remains a challenge in these settings. aims: the aim was to identify and prioritize potential hazards for patients in blood bank practices in the democratic republic of the congo (drc). we focused on two subsets: (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products, using failure mode effect analysis (fmea). methods: two risk analysis workshops were organized at the national blood transfusion centre in kinshasa, the democratic republic of the congo. in both workshops, a multidisciplinary team was invited to represent different hospitals and profiles in transfusion management in drc: quality coordinators (n = 2), training coordinator (n = 1), medical doctor for donor selection (n = 2), hemovigilance officer (n = 1), laboratory technicians performing donor sampling, blood qualification and production (n = 7), biomedical scientists (n = 3), microbiologist (n = 1), clinical biologist (n = 1), nurse (n = 3). the principle of fmea was applied, which implies identification of possible hazards (failures) in a process flow, followed by scoring each hazard according to their impact, probability, detection and feasibility. in the both workshops, participants were guided by an external facilitator who guaranteed common understanding of the methodology. main focus of the risk analysis was potential harm to the transfused patient in the process of (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products. all ideas were written on coloured cards and mapped on a chart according to their impact, probability, detection and feasibility score. hazards were ranked according to their final risk score by multiplying these four scores. results: in the process of sensibilisation and selection of donors, the three hazards with the highest final score for impact, probability, detection and feasibility were: (i) a paid donor is recruited after sensibilisation by family members of the patient, (ii) donor selection staff approves a non-eligible donor for blood donation because of personal/financial motivation, (iii) blood donors are not correctly informed about blood donation during sensibilisation by family members of patients. in the flow of qualification of blood products, highest scores were made for: (i) no double check for validation and sorting of qualified blood products, (ii) stock-out of reagents, (iii) no check for match between registered test result and tested blood tube. regarding production of blood products, the top three consisted of: (i) transport time between blood collection and processing is >8 h, (ii) storage of qualified and quarantined blood products in the same fridge (some sites only), (iii) power cuts. summary/conclusions: the risk analysis resulted in three prioritized hazards in the process of donor selection/sensibilisation and blood product qualification/production. some are very specific to the sub-saharan african setting and have been described before (power cut, family and paid donors, stock rupture,. . .). an action plan needs to be put in place to reduce their final risk score. the risk analysis needs to be continued for the remaining blood transfusion flows. background: 19.3 million of germany's population, so just under a quarter of residents, have a migration background. the majority of these has roots in regions where the population has a distribution pattern of blood group and hla-antigens that differs considerably from the predominant one in the german population. sufficient supply of these individuals with red blood cell (rbc) and platelet concentrates (tc) will continue to be a major challenge in the future, as blood donors with compatible blood group antigens are dramatically underrepresented in the local donor pools. many migrants suffer from severe hematological disorders such as b-thalassemia or sickle cell disease and will not only need compatible blood transfusions, but an allogeneic stem cell transplantation in the foreseeable future. as healthy family donors often are not available, at present suitable stem cell donors with a similar genetic background can only be found in international donor registries. aims: this project was initiated to recruit new donors with a migration background for blood donation and to increase the number of blood stem cell donors among this group. methods: serological extended blood group phenotyping was performed by automated gel card technique (fa. grifols, erytra) and included ab0, rh (ccdee), kk, fy (ab), jk(ab), lu(ab), m, n, s, s. hla typing for hla-a, -b, -c, -dr, -dq, and -dp was performed by next generation sequencing. allele frequencies were analysed using genepop version 4.2; the rare and very rare alleles were defined according to the allele frequency database (www.allelefrequencies.net) rbc genotyping using next generation sequencing is currently being established and will include additional antigens with the most frequent distribution pattern differences between migrant and resident populations according to literature. results: so far, more than 8800 blood donors with a migration background have been recruited for a blood donation in this project. amongst this group, over 1000 blood donors from more than 20 non-european countries enrolled as potential stem cell donors. an initial evaluation of the data revealed a very similar distribution of blood groups compared to the current blood donor population in north rhine-westphalia. of 600 migrant donors, ten fy(a-b-) donors were identified, which corresponds to a percentage of 1.6%. amongst 509 hla-typed potential stem cell donors, we found 28 (5.5%) with rare and very rare alleles. summary/conclusions: blood donors with rare blood group and hla phenotypes (e.g. null types such as fy(a-b-)), are in demand for adequate medical care of people with a migration background. the technological development of blood group determination by next generation sequencing will significantly improve the supply for all blood transfusion recipients in germany. this project is funded by the european development fund 2014-2020 (erdf) and the european union. background: mortality due to uncontrolled haemorrhage following trauma is the most important cause of potentially preventable deaths. trauma care systems in low and middle income countries like india, are still in developing phase. also, the role of blood component therapy in improving patient outcomes has been mostly derived from combat settings. application of these protocols in an urban setup has not been well established and marked variation in practice exists. hence this study aims to identify the key components of transfusion practices to optimize the transfusion protocol in trauma settings. aims: to study the current transfusion practices in severely injured trauma patients, admitted to the red area/resuscitation bay after initial triage in ed methods: this prospective observational study was conducted over a period of 1 year starting from june 2017 to may 2018 at the department of transfusion medicine in collaboration with emergency department at jpnatc, aiims, new delhi. the study included severely injured patients (iss ≥ 16) that were admitted within 24 h to the red area/resuscitation bay after triage. data collected included the demographics, injury, laboratory and transfusion details for these patients results: during the study period 885 patients (83.5% males) were enrolled. mean iss scores was 21.89 . mean time to hospital admission after injury was 9:03 (iqr 3.38-13:48) hours. mean time to first rbc transfusion following admission was 2:09 (iqr 0:27-2:45) hours. approximately 49.3% (436) patients were in shock (sbp < 90 mm hg &/or pulse rate > 110/min). whereas, 160 (18.1%) patients were coagulopathic (pt ≥ 1.5 times of normal). during initial 24 h of admission, these patients were transfused with 2929 (69.7%) rbc, 1986 (51.8%) ffp, 2327 (42.9%) rdp and 384 (81.5%) cryoprecipitate of total blood components utilized for these patients. massive transfusion (defined as transfusion of ≥ 5 units/4 h) was given to 190 (21.4%) patients. summary/conclusions: significant quantity of blood components were required during initial resuscitation in severely injured patients. pre-hospital transfusion can significantly reduce the time to transfusion. further studies are needed to assess utility of pre hospital transfusion in severely injured patients. background: allogenic stem cell transplantation recipients are known to be the main consumers of platelet concentrates (pc). the geneva university hospital is one of the three allogeneic hematopoietic stem cell transplantation (hsct) centers in switzerland. since the blood center is also part of the hospital, data of pc consumption are easily available. as needs rose steadily since several years, with an average increase of 9% per year, pc supply is a serious concern for our center. aims: in this study we tried to evaluate if any pre-transplant indicator could help to forecast the number of pc needed after an allogeneic hematopoietic stem cell transplantation. methods: this observational retrospective study was conducted in geneva hospital on 78 patients suffering from various inherited or acquired disorders of the hematopoietic system who were treated by hsct in 2016. pc consumption was examined from january 2016 to december 2018. the five indicators were: gender, stem cell source (bone marrow (bm) vs peripheral blood stem cell (pbsc)), donor type (hla matched (10-8/10) vs haploidentical), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient. results: data for a total of 78 patients aged from 3 to 74 years were analyzed; 48 (62%) were male and 30 (38%) female; 35 (45%) were cmv-negative and 43 (55%) were cmv-positive. out of a total of 78 transplants, 14 (17.9%) were haploidentical and 64 (82.1%) hla-matched. according to the stem cell source, bm was transplanted in 22 cases (28.2%), and pbsc in 56 cases (71.8%). two patients also received a cd34 + stem cell boost. our analysis showed that, with a mean follow-up of 652 days, the number of pc transfused to our patients treated by hsct ranged from 0 to 383 units, with an average of 37 and a median of 15, illustrating a high variability. the results indicated that gender, stem cell source (bm vs pbsc), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient do not have any statistical impact on platelet consumption. however, we observed a tendency of an increased need for platelet transfusion when patients were cmv positive. our results also showed a statistically significant (p = 0.034) higher number of pc transfused for patients treated with a haploidentical (89) versus hla-matched (26) transplant. summary/conclusions: this study points out the high variability of platelet consumption after hsct, which limits the forecast of platelet production needed to support allogeneic hsct recipients. a larger cohort would be required to confirm a potentially higher platelet consumption in cmv positive patients, and to consolidate our results showing a higher pc consumption for patients treated with haploidentical transplant. abstract withdrawn. background: historically at our institution, a minimum of four red blood cell (rbc) units were crossmatched for all cardiac surgery cases regardless of surgical case-type or patient characteristics. two rbc units were packed in validated blood product coolers and brought to the operating room (or); the balance of crossmatched units remained in the blood bank. a retrospective review revealed that very few rbcs were transfused (2016: 20% (371/1813), 2017: 19% (322/1742)). moreover, approximately 8 products were wasted each month as a direct result of this practice. thus, we recognized an opportunity to improve inventory management in terms of personnel activities and blood component utilization. aims: the goal of this study was to reduce advance preparation of coolers in cardiac surgery cases without compromising patient care and safety. we limited our intervention to those patients who were eligible for electronic crossmatch. we maintained the aforementioned historical practice for those patients with history of and/ or those who currently demonstrated clinically significant red blood cell alloantibodies. methods: a multidisciplinary group consisting of representatives from the blood bank, cardiac surgery, cardiac nursing, cardiac anesthesia and surgery quality department was assembled in october 2017 to determine whether a modification of practice was reasonable and safe. group members evaluated site specific society of thoracic surgery (sts) cardiac surgical data between july 2014 and december 2016 to establish intraoperative red cell transfusion rates classified by type and urgency of surgery. the group's main goal was to discontinue preparation of default coolers for patients eligible for electronic crossmatch who were scheduled for all types of non-emergency cardiac surgery cases in which ≤ 25% of historical cases required at least one red cell transfusion. additionally, team members simulated the multiple protocols by which red blood cells could be prepared and delivered to the or and estimated the time for each scenario. results: review of sts data showed that the following cases met the criteria of ≤ 25%: elective primary coronary artery bypass graft (cabg), urgent primary cabg, elective mitral valve repairs, and elective aortic valve replacements. simulation showed that, in patients eligible for electronic crossmatch, preparation from receipt of order to completion of unit packing for delivery took 2.5 min using the pneumatic tube system (maximum of 2 units per tube) and 4.5 min using delivery of a cooler using a human courier. summary/conclusions: based on the simulation results, and with consensus agreement from the multidisciplinary group, default cooler preparation for elective primary cabg, urgent primary cabg, elective mvr, and elective avr was discontinued in december 2017. one year following implementation of the change in policy 940 rbc units were issued to the or (a 54% reduction); 38% (361) were transfused, compared to 19% in 2017. wastage rates decreased from 8 products a month to 1 per month on average. summary/conclusions: the most obvious drawback of pabd is the higher cost in running the program in comparison with collection of allogeneic blood in the areas of additional patient attention and clerical input in labeling, separate storage and so on. in this audit, 34% of the autologous blood components were not transfused into the intended recipients and wasted; in this context, the pabd program could not be considered as a cost-effective approach in protecting blood safety. background: the national blood service zimbabwe (nbsz)'s blood supply management status (bsms) is an integral process of ensuring the availability of a safe and sufficient blood supply provision. nbsz introduced a new daily blood bank statement with improved metrics from 1 may 2018. the new analytics approach focuses on three interactive components of the blood bank statement; the available stock, quarantine stock (as per the desired 5-days stocks level), and the demand versus supply. it is imperative to have a closely monitored blood supply chain because blood has limited shelf life with uncertainties in both supply and demand. the 'blood-for-free' proclamation by the government of zimbabwe in july 2018 set more pressure on the blood demand. these metric-based analytics seek to assess if the nbsz's improved blood bank statement is a realistic model for the bsms. aims: to assess the use of the interactive metrics in monitoring the blood supply management status. methods: a prospective cross-sectional study was conducted. a total of 704 daily blood bank statements which were submitted between may and december 2018 from each of the five branches were analyzed. the bsms which is calculated as the average of the three interactive measures of quarantine stock, available stock and demand versus supply was determined. sub-analysis of branches was done to determine individual branch performance. analysis by month was done to assess seasonal variations. findings and recommendations were shared among key stakeholders to validate the bsms methodology. results: overall the quarantine stock average was 130.4% (sd +/-101.6), the available stock was 142.3%: (sd +/-118.8) and the demand versus supply was at 95.6% (sd +/-11.3).the overall bsms was 122.8%; (sd +/-77.2) for the study period. gweru and masvingo nearly supplied all the demanded blood with 99.2%, overall bsms of 118.1% and 99.7%, overall bsms of 144.3% respectively. bulawayo supplied 98.3% of the blood demanded with an overall bsms of 99.1%. mutare supplied 97.8% with a bsms of 180.1% and harare 85.6% and a bsms of 78.0%. there were monthly variations but the service could supply above 90% of the blood demand. in the month of may the service met 91.6% of the demand and a bsms of 87.8%. in november and december it supplied 92.6%, bsms of 100.8% and 92.8%, bsms 131.8% respectively. august also had a below average supply of 93%, bsms -98.2%. june, october and september recorded above the average values; 97.3%, bsms of 126.8% and 98.7%, with a bsms of 117.2% respectively. summary/conclusions: the overall bsms performance was satisfactory and it was noted that branches capacitated according to demand. the new interactive analytics approach is appropriate for showing the blood bank status and assessing the performance of the branches. this new approach has optimized the decision-making process in blood supply management. the metrics are tracked using excel based model hence this approach is suitable for resource constrained settings with limited ict infrastructure . st vincent's hospital melbourne (svhm), a tertiary hospital supporting medicine, surgery and non-major trauma emergency and itu services implemented a mtp in 2008. subsequent mtp reassessment has led to implementation of regular multi-disciplinary review of all mts to identify areas for improvement in transfusion and other aspects of support for critically bleeding patients. aims: to implement a systematic service-wide stakeholder review of mt events at svhm aiming to identify deficiencies and implement improvements in mt management. methods: a multi-disciplinary mt review team was established as a subcommittee of the hospital transfusion committee (tc) to update the organisational mtp in 2016 and subsequently continued to meet quarterly as the mt review subcommittee (mtrs) of the tc, systematically reviewing all aspects of mts at svhm. instances where 4 or more red cell units are transfused in <4 h are identified from the laboratory information system and reviewed by the mtrs which includes representatives from accident and emergency, intensive care, operating suite (os) and transfusion laboratory staff; the head of the patient's treating unit is also invited to contribute. reviews include: demographics, clinical details, comorbidities, time from patient arrival to pre-transfusion specimen collection/receipt, time from blood request to release/transfusion, regularity of full blood examination (fbe)/coagulation (coag) testing, timing of blood component transfusion, total component provision/ratios, component waste, patient outcome, and communication between various clinical areas and also the laboratory. a discussion summary with actions/ recommendations is provided to the tc and some cases referred to the hospital mortality/clinical review committee. results: cases reviewed: 65 from 10 treating units including cardiothoracic surgery (18) hepatobiliary/gastrointestinal/colorectal surgery (24), vascular surgery (6), neurosurgery (4), orthopaedic surgery (3), endocrine (2) and "other" (encompassing general surgery, urology, general medicine and oncology -8). areas for monitoring/improvement identified: transfusion documentation, regularity of fbe/coag specimen submission, reducing time between patient arrival and specimen collection, reducing specimen transport time, interfacing point of care bloodgas analysers to the central pathology result management system as well as component management/waste reduction and the introduction of viscoelastometry assessment in the os. 18 of 65 reviewed cases involved the transfusion of emergency uncrossmatched o rhd negative red cell units. the appropriateness of the use of this precious resource is also reviewed by the mtrs. summary/conclusions: the svhm mtrs meets regularly to review mt events and formalise multidisciplinary collaboration in identifying possible improvements to support these often critically ill patients. matters highlighted include communication issues, delays in specimen delivery and blood component waste minimisation. areas for further work include minimising delay between mt events and review, and formalisation of key performance indicators for mts. background: the use of radio frequency identification (rfid) technology to manage the blood supply chain is recognized as a major enhancement to the operations of blood banks and hospital transfusion services. to facilitate optimal blood supply management, it is crucial to guarantee the integrity of rfid tags throughout the transfusion chain. since rfid tags can be affixed to blood products very early in the process, these tags undergo the same process-steps as the blood products themselves (e.g. centrifugation, label printing, shock-freezing and irradiation). aims: the goal of this study was to validate the mechanical and functional resistance of biolog-id rfid tags through different blood related processes: centrifugation, label printing, shock-freezing, intensive reading at à40°c, and irradiation. biolog-id tags are passive hf (13.56 mhz) tags. they are compliant with is0 15693, iso 18000-3 and follow the guidelines for the use of rfid technology in transfusion medicine (vox sanguinis, 2010). methods: biolog-id tags were evaluated using a series of rfid encoding and reading tests. before each of the processing steps, each tag was encoded with donation number, site id, product code, blood group and expiry date. the data was encoded using the isbt 128 format. the different processing steps and conditions tested were: -centrifugation: quintuple whole blood bags, filled with 450 ml water. centrifugation at 4,500 rpm for 10 min. 270 tags processed, 15 tags per kit affixed at different positions. -shock-freezing at à80°c: shock-freezer (angelantoni, sf40), 50 units processed, reading immediately after removal from shock freezer. water, 30 tags irradiated at 30 gy and 30 tags at 50 gy results: all biolog-id tags were encoded and read with a 100% success rate in all series of tests. summary/conclusions: biolog-id rfid tags can be encoded and read through common processes used throughout the blood transfusion chain. their mechanical and functional integrity is not affected by centrifugation, shock-freezing, intensive reading at à40°c, printing, eto sterilization and irradiation. background: the provisioning of compatible red blood cells by international cooperation is presented. the units were meant for an 18-year old female, with homozygous sickle cell disease (scd) and multiple complications. patients' blood group was a positive with anti-c, -e, -wr a and an antibody to a high prevalence antigen in the rh system, anti-hr b possibly combined with anti-hr b (rh34). the antibody was not reactive with rh null , -d-or hr b negative cells. the donor center put out an international request for group a or o, rh null or -d-units lacking wr a and possibly k, fy a , jk a , wr a , do a and s (the latter antigens for prophylactic matching). the patient sample had been genotyped for rhd and rhce using mlpa and sanger sequencing and the patient was found to carry rhd*01/rhd*03n.01 and rhce*cevs.01/ rhce*cevs.03. aims: the request was sent to the american rare donor program (ardp). the ardp working with the american red cross national molecular laboratory, used the rh genotype information to identify donors carrying the same or similar rh variant alleles using the rh allele matching approach described previously (keller et al. transfusion 2013 53(2s):174a). methods: a recent blood sample was used to confirm anti-hr b ; no anti-hr b was detected. the patient rhd and rhce alleles were used to build punnett squares for both genes with donors carrying the same and similar alleles that would be predicted to be compatible. tier 1 donors are those predicted to carry the same combination of rhd and rhce alleles as the patient. tier 2 donors are those predicted to be homozygous for one of the allele combinations carried by the patient. tier 3 donors are those predicted to carry alleles similar (but not identical) to those carried by the patient, with similar predicted phenotype. the database of donors in the ardp carrying rh variant alleles was queried against the alleles in the patient-specific punnett square. results: donors of group a or o and matched for rh alleles were identified as follows: 102 tier 1, 357 tier 2 and 100 tier 3 donors. after the clinical team agreed to drop one or more of the prophylactic antigen matches, one tier 2 unit lacking s and jk a was identified at the american red cross. while the request was being processed, the patient experienced a sickle cell crisis, red cell aplasia and recurrent aiha and her hemoglobin level dropped from 7 to 1.9 g/dl. at that time, she was transfused the only compatible units available -2 of the rare -dphenotype and her hb increased to 3.8 g/dl and eventually to 7 g/dl. the tier 2 rh allele matched unit was shipped to amsterdam where it was frozen, and reserved for the transfusion care of this patient. summary/conclusions: this case illustrates how rh allele matched blood can be found for a highly rh alloimmunized patient, and can avoid use of the exquisitely rare -d-or rh null blood. background: blood transfusion has been a complicated and high-risky clinical procedure. any error could cause serious injuries to patients. to better assure the procedure safety. aims: we enhanced and built a blood transfusion database platform and develop inventory management strategies to better guarantee the patient transfusion safety. methods: we designed six new features of the platform (1) assuring the patient identification with barcode techniques; (2) designing a structured order entry; (3) proactively reminding the physicians with patient's previous blood transfusion reaction with related precautions including the use of leukoreduction filter; (4) automatically reminding physicians the happening of reaction and suggesting relevant test; (5) building a complete traceability log system; and (6) supporting data analysis. the blood transfusion safety team includes medical technologists, nurses, physicians, system analysts, and blood transporter and the whole process is electronic management. the new blood transfusion platform integrated the workflow, reduced the incidence of abnormal blood samples collected (0% after implementation, p < 0.01), reduced the time of call for medical technologists with blood component preparation and improved the achievement rate of emergency 30-min blood crossmatch (98.1% after implementation, p < 0.05). the barcode correctly identified patients and monitored the entire transfusion process to reduce the error rate of blood component supply (0% after implementation, p < 0.01). summary/conclusions: after the transdisciplinary team approach with e-monitoring and a better design of clinical decision support module with barcode technology, blood transfusion database platform improve the blood supply efficiency and assure blood transfusion safety. background: in the modern world, terrorist acts are characterized by a multiplicity of combined injuries to a large number of victims. qualified medical care is urgently required for a large number of patients in one locality at the same time. it leads to increase in emergency demand for blood components, mostly red blood cells. the desire to donate blood to the victims is a natural manifestation of society's solidarity in response to tragic events. however, donor activity and patient needs do not always correlate. aims: to analyze the donor activity during the terrorist attacks. methods: a retrospective analysis of donation activity in periods of terrorist attacks in moscow (2004 moscow ( -2011 . the average daily blood donations' number (dbdn) before ta compared with the number of donations in day after ta and with the dbdn during 7 days after ta. also the number of delivered rbc units (d-rbcu) daily before ta and daily in 7 days after were compared. results: in 2004-2011, 5 terrible ta occurred in moscow: 141 people died and more than 629 were injured. with the explosion in subway in 02/2004 42 people died, 250 were injured. the number of d-rbcus increased by 10% on ta-day, and by 30% during next 7 days. dbdn in the 1st day after ta increased 6,7 times, and in the next 7 days -1,6 times. second explosion in subway in 08/2004 resulted in 10 died, 50 injured. the number of d-rbcus increased by 80% on ta-day, and by 0% during 7 days. dbdn in the 1st day after ta increased 1,1 times, and in the next 7 days -2,2 times. in 2006 (explosion on market) resulted in 14 died, 61 injured. d-rbcus delivery increased by 20% on ta-day, and by 10% during 7 days. dbdn in the 1st day increased 2,0 times, but decreased to 0,6 times during the next week. with subway explosion in 2010 40 people died, 88 were injured. the number of d-rbcus increased by 10% on ta-day, and by 0% during 7 days. dbdn in the 1st day after ta increased 6,5 times, and in the next 7 days -1,6 times. with the explosion in airport in 2011 35 people died, 180 were injured. rbcus delivery increased by 50% on ta-day, and by 40% during next 7 days. dbdn in the 1st day after ta increased 6,1 times, and in the next 7 days -2,0 times. summary/conclusions: an increase in donor activity is observed already the next day after ta and usually lasts for 7 days, but does not correlate with the number of victims. the rbcs' delivery from blood bank increases in all cases on the day of the ta. therefore, the guarantee for patients is the maintenance of rbcs' stock, including cryopreserved ones. it is also necessary to promptly send excess of red blood cells harvested at the peak of activity to the cryobank. background: rh system is the major blood group system besides abo system. even after proper blood grouping and cross matching there is a possibility of alloimmunisation in recipients against the rh or minor blood group antigens like kell, mnss, duffy etc. in medical colleges which cannot bear the financial burden of complete phenotyping of patient and donor, implementation of rh & kell phenotypes match blood transfusion can play a major role in preventing alloimmunisation and adverse events in multitransfusion patients aims: to evaluate the efficacy of rh & kell phenotyping as a cost effective measure instead of extended phenotyping in multitransfused patients methods: study was carried out in the department of transfusion medicine, one of the biggest blood bank of the country with annual collection of 70,000 blood units. 2000 patients of thalassemia, aplastic anemia and leukemia were taken who required multiple transfusions. complete phenotyping was done initially of all the patients before transfusion. 1000 patients were taken as control and the other 1000 were taken as cases. 2482 blood units of healthy donors were chosen (2442 were males and 40 were females). in all the donor units, identification of rh & kell phenotyping was done by the antigen antibody agglutination test by the erythrocyte magnetize technology on fully automated immunohaematology analyzer qwalys. these blood units were transfused to 1000 patients who had been selected as cases. in the control group, patients were transfused blood units which were not phenotyped for rh & kell but gel crossmatching was done. follow-up was done on these patients for transfusion reactions and at the end of six months they were evaluated for any alloimmunisation. results: at the end of 6 months, no reactions were reported in cases receiving rh & kell phenotype blood and no alloimmunisation was seen on repeat phenotyping. the control group on the other hand reported reactions in 5 cases (0.5%) and phenotype at the end of three months showed alloimmunisation with 'e' antibody. the phenotypic frequencies of rh & kell blood groups in the population were comparable with other published studies. amongst the rh antigens (e) was the most common (99.44%) followed by d (95.45%), c (89.65%), c (53.1%) and e (17.65%). thus 'e' was the most common and e was the least common of all the rh types. background: the prevalence of a particular blood group has an uneven distribution in different geographic areas and is largely determined by the national composition of the population. moscow is one of the largest city of europe with population of 12.5 million. the understanding of prevalence of red blood cells antigens (rbc-ag) among the population has great importance for blood banking planning. aims: to determine frequency and distribution patterns of transfusion-significant rbc-ag among donors in the moscow region. methods: the results of immunohematological studies on ab0, rhesus and kell systems were analyzed retrospectively in 352362 blood donors for 14 years (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) in moscow. data collection and processing was carried out using the regional information system for transfusiology. rbc-ag detection (ab0, rh, kell) systems was performed using microplate method (automatic immunohematological analyzer "galileo neo" (immucor, inc., usa)) and "ih-1000" (bio-rad laboratories, usa) with diagnostic cards. results: the most frequent blood group is a (ii) 35.8%, 0 (i) blood group 34.3%, b (iii) 21.3%, ab (iv) 8.6% (n = 352362). rh(d+) was established as positive in the presence of antigen d and as rh(d-) negative in its absence. donors with weak variants of antigen d (du) were determined as rh (d+) positive. the ratio of rh (d+) and rh (d-) was 82.8% and 17.2%, respectively. donor's phenotype detection was routinely conducted from the 2013 year, therefore the number of donors was 152883. the most common phenotype among donors ccdee (32.61%), the second in frequency ccdee (19.33%), the third in frequency rhesus negative phenotype ccddee (15.42%) in the studied population. the ccdee and ccdee phenotypes were 14.04% and 12.17%, respectively. the most rare are ccdee (2.65%), ccdee (1.86%), ccddee (1.54%). other options: ccddee, ccdee, ccdee, ccddee, ccddee, ccdee, ccddee were detected in single cases and amounted to a total of 0.37% (n = 152883). cw antigen was tested in 104230 donors and was detected in 5.28%. cw is most commonly found in donors with ccddee phenotypes (2.36%), ccdee (2.03%) and ccdee (0.79%), with other variants of the data phenotype, the antigen was detected in 0.1% of the examined (n = 104230). antigen k was detected in 6.8% of donors, in 93.2% of this antigen is absent (n = 352362). summary/conclusions: the study of transfusion-relevant antigens distribution in population is necessary for building of effective and flexible model for blood service managing. a differentiated approach in choosing a strategy to form a long-term bank for storing blood components, taking into account the frequency of various antigen variants, contributes to improving the quality, accessibility and safety of medical care. of dislocated division of our blood establishment in orthopaedic hospital valdoltra (obv) in 2014 the number of outdated units at the hospital side dropped considerably. results: since 2008 when the issued number of red blood cell units (rbc) was 5170 the amount of issued units rose to 5678 in 2011 and then dropped more or less steadily to 4850 in 2018. in this period the hospitals' programmes rose for 10% in all areas. number of donated units declined from 6330 in 2011 to 4820 in 2018. after reorganization in 2009 the number of outdated units fell from 3% of stocked units to 1.5%. after setting a dislocated unit of ctdiz on obv location the number of discarded rbc fell from 397 to 41 in 2018. for transfusion specialist who is constantly in contact with the clinician in the hospital the most important day routine is when the stock availability is displayed. it happens 4 times a day; at 10 a.m. when the previews' day collection is released and another three times a day when the updates occur. the central base is led in ljubljana (the capital) and all centres are able to control and order the stock for the blood banking. blood wastage remained low and the traceability of the blood usage in south-western region remains high (99%). though it is not supported by an informational system the traceability form the blood bank to the patient is done on paper. this issue demands a big effort by the staff in blood bank and in hospitals. summary/conclusions: reorganization enabled better stock utilization and traceability of issued units. sometimes it is impossible to predict the peak demand of rbc especially during the summer season when the population of the area doubles and car accidents as well. transfusion specialist's effort in assuring the optimal blood stock represents the crucial daily routine. blood bank, grande international hospital, kathmandu, nepal background: voluntary non-remunerated blood donor consists of 78% blood donor's population in nepal. therefore demographic about the distribution of blood donors according to the age group is important to achieve 100% voluntary nonremunerated blood donors in nepal. aims: to explore the demographic distribution of the blood donor in different age group in the kathmandu nepal. methods: this is retrospective study conducted at nepal red cross society central blood transfusion service. data from january 2013 to january 2017 were collected from donor management software. the data includes socio demographic data. data has been process with spss version -17 results: during 4 years study period, total of 276,290 blood donation happened from both mobile blood collection and in-house blood collection. out of 276,290 collection, 48351 (17.5%) are from 18-24 age group; 106924 (38.7%) are from 25-31 age group 53324 (19.3%) are from 32-38 age group; 34536 (12.5%) are from group 39-45 age group; 23761 (8.6%) are from age group 46-52 and 9394 (3.4%) from age group above 53 respectively. summary/conclusions: the distribution of abo blood group varies regionally and from one population to another. in kathmandu, nepal 18-38 years age group is the most common age group encountered donating blood. the data generated in the present study and several other studies of different geographical region of india will be useful to health planners and future health challenges in the region. background: the information system on hemovigilance sihevi-ins©, coordinated by the national health institute was available in 2018 to all blood banks in the country. this software allows to centralize and record the identification data of a donor, its infectious and immunohematological tests, as well as the fractionation and final destination of each blood component obtained from a donor. aims: to describe the abo and rhd typing discrepancies in blood donors found in the blood group variables registered by each blood bank to sihevi-ins©. methods: retrospective analysis of the information registered by 80 of the 81 blood banks authorized nationwide between january and december 2018. results: sihevi-ins© received information of 854,462 accepted donors, 33% of them with more than one donation in the same blood bank in a period of 12 months. a total of 72 abo or rhd discrepancies were identified in 69 people, who made donations in 20 blood banks (estimated risk: one discrepancy per 11,876 accepted donors). five of the blood banks implicated in these discrepancies are hospital-based (annual average collection of 6,086 ae 3,785 units, representing 3.6% of the national collect). the remaining blood banks are distributors (average collection: 31,389 ae 25,704 units per year, representing 44% of the national collect). 75% of blood group typing discrepancies (n = 36) were related to the abo group. the most common discrepancy was between a typing group and ab typing group (67%) . in 25% of the cases, the same blood bank initially registered in the same donor, an o blood type donation and later an a blood type (n = 10) or b type (n = 4). rhd typing discrepancies account for 25% (n = 18) of the total. additionally, in three donors, a simultaneous discrepancy between abo and rhd typing was detected in the same blood bank. the results could be due to: a) failure in the warning mechanism before the release of the blood component; b) errors in typing the information of the donor registered in the system or c) failures in the identification of the donors at the time of selection. the above shows risk in the process of control of blood components release, which can impact patient safety unless abo and rhd typing blood groups are systematically verified before transfusion. summary/conclusions: despite blood banks have a verification and validation process through software to release blood components, flaws were detected. although sihevi-ins© is not a software to validate the information before the release of blood components, it was through this program that abo and rhd typing discrepancies were identified in donors who attended the same blood bank multiple times. this finding implies increasing the controls that should be used in each blood bank, to avoid lose traceability of the processes and to put at risk the life of the recipients. blood donor testing department, blood transfusion institute of nis, nis, serbia background: the ability to automate blood grouping and antibody detection procedures is a requirement for blood donor testing laboratories. mistakes in the sample identification and testing procedures could be prevented by testing on automated immuno-hematology systems. irregular antibody screening and abo/rhd grouping of blood donors are tests performed routinely in blood transfusion institute of nis. neo iris (immucor, usa) is a fully automated instrument for the abo and rh d grouping using microplate hemagglutination technique and antibody screening and identification using solid phase red cell adherence (sprca). aims: evaluation of the automated neo iris system for abo and d grouping and irregular antibody screening of blood donors in blood transfusion institute of nis. methods: during the evaluation period a total of 4417 edta-anticoagulated samples for abo and d forward and reverse grouping using microplate anti-s, 1 anti-s, 1 anti-jka and 6 anti-k. in one case ih-1000 failed to identify anti-c antibody in very low titer in sample with anti c+d antibody presence. in two samples (0.045%) false-positive result were observed both on ih-1000 system and neo iris and in two cases (0.09%)only on neo iris due to nonspecific reasons. summary/conclusions: abo/rhd grouping results obtained on neo iris system, using microplate method, have a good correlation with results on ih-1000 system as our routine column agglutination method. for antibody screening and identification neo iris showed high sensitivity for detection of clinically significant antibodies which is important step for increasing blood transfusion safety. background: continuing improvement of laboratory quality to provide accuracy test results for precise diagnosis and treatment is the mission of advanced laboratory. immunogenetic testing for histocompatibility including human leukocyte antigen (hla) typing, hla antibody detection and cytotoxicity test is critical for diagnosis and evaluation of transplantation and prognosis monitoring. in order to improve the quality of experiment competency, an external quality assurance schemes with review and education per year program was established and performed during the period from 2017 to 2018 in taiwan. aims: the proficiency testing (pt) held semiannually from 2017 to 2018 were reviewed to investigate the outcome of competency improvement of laboratories participated in the program. methods: the test items in the exercises were classified into 4 groups, hla genotyping (including pharmacogenetics hla typing), cytotoxicity test, hla and platelet antibody. the methods of hla genotyping include ssp (sequence specific primer), sso (sequence specific oligonucleotide), sbt (sequence based typing) and either ssp+sso or ssp+sbt were used, the methods of hla antibody including elisa, flow cytometry and luminex were used and the methods of platelet antibody including sprca and elisa were used. there are four shipments of exercise materials in two years and each shipment include two positive and one negative samples for antibody detection, two each of whole blood and serum for cytotoxicity of t and b cell and three whole blood for hla genotyping. aims: this study aims to survey transfusion related laboratory tests for the quality improvement of hospital's blood bank management. methods: we analyzed survey results of 11 kinds of routine work categories of 841 blood banks that were registered on korean association of external quality assessment service. blood bank worker voluntarily replied this electronic survey. the 11 categories were as follows: 1. characteristics of institution 2. the equipment of blood bank 3. the kinds of tube in blood bank 4. the present kinds of blood bank tests 5. abo and rh type tests 6. the cross-match tests 7. the irregular antibody tests 8. hemovigilance system 9. other blood bank tests 10. massive transfusion protocol 11. quality control issues we analyzed and compared each category data according to considering characteristics of hospitals. results: there were consensus and some differences of current blood bank tests. we presents the result of a pilot survey. especially the cross-match tests were divided by saline phase method added with irregular antibody tests or completion of 3rd step anti-human globulin phase according to institutional environment. automated typing machines or automated irregular antibody test devices were more increased in large-scale hospitals than small-scale hospitals. different kinds of tubes were used such as edta tube for abo and rh typing, plain tube for cross-match test. the retention segments of rbc were reserved for minimum 7 days. most blood bank were registered and regularly listed up transfusion events to korean hemovigilance system for safety transfusion. also, a lot of institution have none or underdeveloped massive transfusion protocol. more specific survey results will be analyzed in further poster presentation. summary/conclusions: this survey will show the current status of transfusion related blood bank test. this institutional blood bank comparison will be helpful to assess the currency of individual blood bank environments. abstract withdrawn. background: we know that quality management is a continuous process, involving implementation, maintenance and improvement. aims: our purpose is to show our experience in implementing the quality management system in the whole institution and our first steps in achieving the jacie accreditation in the stem cell collection facility in order to provide our patients and donors the best possible care. methods: the institute for transfusion medicine of the republic of macedonia (itm) is the main institution in charge of blood transfusion service (bts) in the whole country, which is the national unified system. the stem cell collection facility is a part of the itm. this facility is operational since 2001 year with 844 collections of stem cells (686 in patients and 159 collections in sibling donors) till now. we are obtaining the implementation and maintenance of qms through the establishing of the iso standardization for the whole institution (itm), as well as of implementing jacie standards in the stem cell collection facility. the two of our colleagues became the jacie inspectors and the standard operating procedures (sops) were developed, followed by regular meetings, trainings and self-evaluation of the personnel. we asked for the orientation visit from the independent jacie inspector in order to come one step closer to the jacie accreditation and to improve our overall qms. results: the institute for transfusion medicine of rm was a part of the ipa project "strengthening the blood supply system". this project aimed to ultimately bring the blood transfusion service to european union standards allowing the exchange of blood components and all other types of collaboration with other european union countries in future. the project put the basis for unification of blood transfusion standards and operating procedures in the whole country as well as set up essential education of blood transfusion personnel. although a lot of strengths were found during the orientation visit from jacie inspector, there are still a lot of areas for improvement. our strengths are motivated team and supportive institutional leadership including macedonian ministry of health. areas for improvement are: labeling of cellular therapy products and lack of laboratory for quality control. there is a national regulatory framework in place and who and world bank initiatives in macedonia which support quality in health care and accreditation. summary/conclusions: our institution has in plan to implement isbt 128 standards for labeling of cellular therapy products and to establish a laboratory for quality control of cellular therapy, as well as to meet all the requirements to become jacie accredited facility. working by standards, following the rules and regular self-evaluations will help us to maintain the strong quality management system. every institution will benefit from a quality management system that brings you into line with international standards. ensuring the quality of our services and products is essential to keep safe and strong blood transfusion service. background: implementation of robust quality assurance program is key to high performing blood establishments. quality control and quality assurance systems together constitute the key quality systems and are parts of quality management. effective and efficient quality control policies not only provide guidance that help to increase the reliability of results but also maintains the laboratory's consistence performance overtime. aims: therefore, we established a set of qc limit using historical data which can timely identify unexpected variation in the testing systems and trigger a review of test processes in blood screening laboratories as part of quality assurance system. methods: last two consecutive years (jan, 2017 to dec, 2018) qc data from archi-tect i2000sr (abbott laboratories, chicago) was extracted using abbottlink for philippines red cross tower national blood center total of 6532 data points (3523 data points in 2017 & 3009 data points in 2018) were obtained for 10 different qc levels for four serological blood screening assays (hiv combo, hbsag, anti-hcv, syphilis). the data was sorted for each assay/lot and qc level combination by year. qc limits were calculated using simple mean, standard deviation (sd) and coefficient of variation (cv%) and were validated and compared with manufacturer's recommendation. results: all the six positive quality control levels cv% (5.70-12.05) were within manufacturer's precision recommendation (within lab precision hbsag ≤ 10%, anti-hcv ≤ 15%, syphilis ≤ 15%, hiv ≤ 14%) in 2018. five out of six positive quality control levels cv% (5.72-15.80) showed within manufacturer's precision recommendation (within lab precision hbsag ≤ 10%, anti-hcv ≤ 15%, syphilis ≤ 15%, hiv ≤ 14%) in 2017 except syphilis tp positive control (15.8%). all four negative quality control levels showed the sd values within 0.009-0.41 in 2017 and 0.009-0.41 in 2018 respectively. summary/conclusions: excellent qc performance was observed in philippines red cross tower national blood center blood screening laboratory based on historical data and evidence-based laboratory qc limit for blood screening assays were established using historical data which takes into account total variation expected in a test system and offers a more robust and meaningful mechanism for setting control limits, for the first time. background: quality indicators (qi) in transfusion medicine (tm) are 'critically important aspects of transfusion medicine practice that are measured and utilized to gain insight for continuous quality improvement, into the degree to which the tm is capable of providing quality tm care, products or services for the aspect of practice measured following comparison of the measurement against acceptable local or international reference thresholds, benchmarks, standards, or practice guidelines'. the critical control point (ccp) selected for this study is 'administration techniques and monitoring of key elements'. this has been selected since the clinical fraternity plays a larger role in ensuring quality services in administration of blood components. there was a need to follow up compliance to standard protocol for bedside transfusion practices hence was decided to study the same with four selected quality indicators and introduce corrective measures if necessary. aims: 1. to assess the existing transfusion practices in the institute with specific quality indicators 2. to introduce corrective reforms to improve the existing practice 3. to assess the transfusion practices after interventions using the same quality indicators methods: to assess the existing transfusion practices in our centre, 295 transfusions were prospectively followed up with a structured checklist. the quality indicators used were (i) verification of blood components prior to transfusion (ii)initiation of transfusion within 30 min of release from the blood bank (iii) close observation of transfusions for the first 15 min (iv)completion of transfusion within the right time frame for each component. as a corrective measure, a transfusion monitoring format was designed which was distributed in every ward and the nursing officers were informed to monitor and document transfusions using that. in addition, the blood bank staff was made to call up the wards and ensure that the transfusions of every component had been initiated within 30 min of issue. transfusion practices were once again monitored by following up 306 transfusions using the same quality indicators. results: there was significant difference in all the four variables between the two phases. 57.3% transfusions were verified in phase i while 73.6% were verified in phase ii (p < 0.001). 69.5% transfusions were started within half an hour of issue while in the second phase, it rose to 83.1% (p < 0.001). 47.8% transfusions were observed in the first 15 min in phase i and 88.6% were observed in the second phase (p < 0.001). in phase i, 54.6% transfusions were completed within right time while the same in phase ii was 73.9% (p < 0.001). summary/conclusions: we recommend the following as quality indicators for bedside transfusion practices: background: antibody titration consists in performing antibody detection with selected red cells of different sample dilutions. the titer is reported as the reciprocal of the highest dilution that induces macroscopic agglutination. the usual applications of titration are prenatal studies and complex antibodies identification. some publications have demonstrated that more variation in antibody titer and titration score are noted upon repeat testing of the same sample when testing was performed in tubes as compared to repeat testing in gel. aims: to evaluate the efficacy of automated antibody titration versus manual method by using gel microcolumn technology. methods: edta-anticoagulated whole blood donors' and plasma frozen samples containing a known irregular (rh, kidd, duffy, mns, etc.) and regular (a1 & b) antibodies were selected. the titers of 126 samples were determined in parallel by using grifols analyzers (erytra and erytra eflexis) and compared versus grifols gel manual method by using grifols gel microcolumn technology and grifols red blood cell reagents. sixty of these also processed in parallel in erytra and erytra eflexis analyzers for comparison. for the precision study, 12 of these samples were tested in the 2 automated systems for 5 times (120 datapoints for each analyzer) on different testing days. the hands-on (manual intervention) average time required to complete a titration was measured (3 expert technicians) in 2 different sample workload (1 and 10 samples testing). these results were compared with the same number of independent titrations performed in grifols analyzers. for the walk-away time, 2 different sample workload (1 and 10 samples testing) were assessed in manual method (3 expert technicians) and compared to timings obtained when reproduced in grifols analyzers. results provided by analyzers were reviewed and compared to manual method. results: titer obtained by erytra or erytra eflexis was equivalent to the titer obtained manually (differences ≤ 1 titer: 73% ≤0.5 titer). the results proved that both instruments were equivalent in performing titration (differences ≤ 1 titer; 85% ≤0.5 titer). the precision results showed no difference between titers obtained through the 100% of the runs performed with the grifols analyzers (differences ≤ 1 titer: 75% ≤0.5 titer). the manual hands-on in automated system was reduced in a 15% compared to manual method for 1 sample. when the number of samples was increased (10 samples), the difference in hands-on in was even more reduced (65%). in addition, the walk-away was 46% higher in automated system compared to manual method. furthermore, when the number of samples was increased (10 samples), the walk-away difference was increased even more (78%). finally, automated system software demonstrated to increase the standardization of the test as all samples, results and reagents traceability were automatically managed. summary/conclusions: grifols gel system including erytra and erytra eflexis analyzers provided a scalable and efficient solution to perform standardized titrations in the immunohematology lab. the study proved that using grifols gel system, titrations can be run in an automated reliable way (less than one-fold differences versus manual gel), thus reducing at least 15% the hands-on, increasing at least 46% the walk-away, rising the standardization and automating all testing traceability. , and fourth case of use (results) scenarios, tasks were considered "very easy" by 30%>50% of users and "easy" and by 50-80% of users; 90%>100% of the users considered "sufficient" the design to ease the interaction; and 60%>80% of users never founding any situation of not knowing how to proceed. for the fifth case of use (user roles), 40% of users considered tasks "very easy" or "easy"; 40% of users considered "sufficient" the design to ease the interaction; and 40% of users never found any situation of not knowing how to proceed. for the sixth case of use (maintenance plan), 100% of users considered tasks considered "very easy" or "easy"; 90% of users never found any situation of not knowing how to proceed; and 100% of users considered the maintenance plan similar or better than other instruments. reliability analysis ( background: quality control procedures in blood group serology for reagents, techniques, personnel working and automated equipment are essential for the accuracy of the laboratory results. the observation of high number of uninterpreted results during blood donor grouping was a motive for investigation and possible targeting the problem. aims: to identify blood group interpretation problems by analyzing the testing results obtained with the commercial quality control samples routinely used during blood grouping. methods: a microplate (mp) system for performing abo and rhd, as well as rh phenotype and kell blood group determination with two automated analyzers techno (1 and 2) and correspondent two mp-readers lyra (1 and 2) using maestro software from diamed is currently in use for blood donor typing. three types of mp are being used such as: a, b, ab, dvi-, dvi+, ctl/a1, b profile for first time donors, then a, b, d ctl for repeat donors and finally, the c, c, e, e, k, ctl profile. the accuracy and safety of the blood grouping results is ensured by using the diamed q.c. system which consists of 4 + 2 tubes of whole blood and 2 tubes containing serum with known specific antibodies. we analyzed and compared the interpretation of the q.c. whole blood samples' results from both of the analyzers after a new optic camera was installed on the techno 1/lyra 1 system. ward to ward. methods: a prospective observational pilot study was done for around 140 prbc unit issues which were followed in real time for understanding the tat within blood bank & from ward to ward. as per the definitions, the areas where the times are documented perfectly are understood and considered for calculations. based on the conclusions of pilot study a monitoring form has been designed and utilised to monitor the tat within bb & wtw. the data is analysed monthly and an avg tat for bb & wtw is calculated. the common causes of delay in providing the blood components were analysed and strengthened to both reduce & control the tat. results: in pilot study, total wtw tat averaged to 90 min, with highest time taken 795 min, where there were additional processings like leukodepletion, irradiation, saline washing of red cells and holding the transfusion. lowest wtw tat was found to be 10 min where there was a prior information for crossmatch. after the surveillance form has been started, the average time taken for wtw tat came down to 70 min, maximum being 310 min (jan 2019), the areas where delay happened were identified as internal courier delays, technician delays, billing & other logistics delay. the concerned staff are put on regular training to maintain the tat. summary/conclusions: although ethically all the staff work for providing better care for patients, there will be few areas that delay the life supporting blood transfusion. monitoring using tat surveillance forms help in avoiding the delays and hence provide better & timely transfusion support. blood donation -blood donor recruitment p-059 hematological and physiological characteristics of regular blood donors with beta-thalassemia traits background: according to recent evidence, the physiological variability observed in the hematological characteristics of regular blood donors (linked -in certain caseswith genetic factors or the donor's lifestyle) may affect red blood cell (rbc) storage lesion. beta-thalassemia heterozygous (b-thal-het) blood donors represent a group of particular interest because of a) the high frequency of thalassemia mutations in specific geographical areas b) the physiology of the b-thal-het rbcs, which predisposes towards more effective management of storage-associated stress. aims: the goal of the present study was the comparative examination of the hematological and rbc physiological features of regular blood donors with or without beta-thalassemia traits before blood processing for transfusion purposes. methods: 204 healthy blood donors of greek origin (18-24 years old), who met the blood donation criteria were recruited in this study. plasma/serum (uric acid, electrolytes, extracellular hemoglobin, antioxidant capacity), cellular (rbc indices) and biological parameters (corpuscular fragility, proteasomal activity etc) were measured. the results were statistically analyzed and topologically represented in biological networks for both donor groups (+/-b-thal-het). significance was accepted at p < 0.05. results: b-thal-het represented 9% of the donor cohort. no differences in lifestyle (smoking, alcohol consumption, physical exercise) were observed between the two groups. nevertheless, regardless of sex and sex-dependent parameters (e.g. hct, hb concentration), b-thal-het demonstrated: a) reduced hct, mcv and mch (11% p = 0.039, 26% p = 0.008 and 30% p = 0.006, respectively) and b) increased rbc count (20%, p = 0.042) compared to the average donors. moreover, mpv platelet index was found slightly elevated (p = 0.053) and serum total protein concentration slightly reduced (p = 0.054) in the same group. a trend for higher plasma antioxidant capacity (p = 0.052) was evident in the group of b-thal-het, in addition to statistically significant lower levels of osmotic fragility (by 13%, p = 0.022) and hemolysis (by 41%, p = 0.001) compared to controls. finally, analysis of the three proteasome-associated enzymatic activities (n = 10 per group) in the rbc cytosol and the membrane, revealed similar levels in the two groups (p > 0.05). the b-thalhet and control biological networks showed insignificant variations in respect to the amount of connections and their hub profiles. however, differences were observed regarding the number or type of connections, or even their topology in the network, in the cluster of lipids (triglycerides, ldl etc), nitric oxide, clusterin, carbonylated plasma proteins and rbc osmotic fragility (correlated with the concentration of electrolytes selectively in b-thal-het donors) between the two groups. summary/conclusions: b-thal-het who meet the criteria for blood donation are a non-negligible sub-group of the total donor population in greece. they exhibit several similarities to the general cohort, but differ in fine characteristics of rbc physiology, including resistance to hemolysis and extracellular antioxidant capacity. the differential network profile of hematological and redox parameters may be important in respect to the subsequent blood processing and storage of b-thal-het erythrocytes for transfusion purposes. background: blood service in poland is based on voluntary and non-remunerated donations. regional blood donor centre in poznan as well as other regional centres (total of 23) are the only entities authorized to collect, process, store and distribute blood and its components to hospitals in the region of their activity but they are also responsible to provide sufficient amounts of blood and its components. regional blood donor centre in poznan is one of the largest blood centers in poland with the total number of donations exceeding 100,000 per year. in the recent years we have observed a growing popularity of tattoos among various age groups as well as among people registering to donate blood (first time and repeat donors) hence, it is critical to introduce suitable measures to ensure the safety of blood and its components. aims: the aim was to analyse the correlation between the increasing number of donors deferred from donating blood due to having tattoos made and the number of recorded confirmed hcv infections and the effect it may have on the safety of blood and its components. methods: the analysis was made using the data for the years 2010-2017 obtained from the computer system 'blood bank' which is in operation in regional blood centre in poznan, poland. we have analysed the total number of deferrals of donors due to recently acquired tattoo and the total number of recorded confirmed hepatitis c infections. we must note that the category of temporary deferrals due to tattoos is a broad one: it includes so called regular 'artistic' tattoos, permanent make-up procedures as well as medical tattoos. results: we have recorded a significant increase in number of deferrals due to tattoos from 400 in 2010 to 716 in 2017 (+79%). in the group of male donors this trend remained rather stable with a slight decrease: from 181 in 2011 to 151 in 2017 (à16.6%). in the group of female donors the growth was more prominent: from 219 in 2010 to 565 in 2017 (+158%). in terms of the recorded confirmed hcv infections a downward trend can be observed: from 63 in 2010 to 16 in 2017 (à74.6%). in the group of male donors from 43 in 2019 to 12 in 2017 (à72%), in the group of female donors from 20 in 2010 to 4 in 2017 (à80%). summary/conclusions: as we can conclude from the analysis the applied policy of temporary deferrals of donors with recently acquired tattoos (in the last 6 months) proves to be a reliable method of increasing the safety of blood and its components. nevertheless, the current conduct of the qualification of the donors which requires a 6 month deferral following the new tattoo must be complemented by various and numerous educational activities regarding the means of hcv transmission (and other bloodborne viruses such as hbv, hiv) and ways of protection from possible infections. special emphasis must be put on the group of female donors as the growth of deferrals was more prominent among them. at the same time it is vital to ensure for constant availability for all donors of well designed, concise educational materials (hard copies on the premises, articles, infographics, downloadables etc. on the website). background: a temporary deferral has a negative impact on donor retention, with many donors failing to return at the end of their deferral period. anecdotal evidence collected by the australian red cross blood service suggested that many donors do not know when they are eligible to return to donate, suggesting that a reminder message may be effective at promoting donor return once the deferral has ended. aims: the aim of this study is to evaluate the effectiveness of a reminder message on the return rates of deferred donors at the end of their deferral period. this reminder message notified donors that their deferral period was ending and encouraged them to make an appointment to donate. this study also aimed to determine the most effective time to send the message, message content, and mode of communication (sms vs email) in optimising donor retention post deferral. methods: three separate randomised controlled trials were conducted to answer these questions. data on donors' attempted return behaviour and subsequent deferrals, appointments and donations made one month after the deferral end date were collected and analysed. results: overall, 18.3% of donors who received a reminder message attempted to return compared to 12.8% of donors in the control group (p < 0.05). looking at each time point, donors who received the message 1 week before their deferral ended were 64% more likely to attempt to return compared to the control group (p < 0.05). the 1 week prior reminder message was particularly effective with males, with 30.3% attempting to return to donate, compared with 25.2% of females (p < 0.05). there were no significant differences in the return rates of donors who received the recipient versus non-recipient focused message, or donors who received the message via email or sms. summary/conclusions: a reminder message sent to deferred donors at the end of their deferral period is a simple, cost-effective way to promote donor retention, providing clear information regarding the date on which the donors can return to donate as well as a prompt to make an appointment background: our challenge is to provide 100% voluntary donation for safe blood, thus taking into account the current history of family donation, promotion of blood donation, level of awareness and voluntary donations from various institutions, the opinion of 1000 interviewees will give us a clearer idea of what we want to achieve and what needs to be improved in the future. aims: 1. provide 100% voluntary donation for safe blood. 2. establishing a special department within the national blood transfusion center responsible for marketing and promotion of voluntary blood donation. methods: this study was conducted as a combination of qualitative and quantitative methods. the study was a combination and identification of existing data, direct interviews with persons of different age groups, preparation and dissemination of questionnaires and analytical processing of the collected information. the study questionnaire with 60 questions in total was divided into 6 sections out of which 14 questions on blood practices were answered by all 1000 interviewees. 416 people answered 9 questions on the blood transfusion service. 5 questions on blood knowledge were answered by 270 people. 5 questions on the knowledge of the blood transfusion were answered by 220 people, 17 questions on blood donation were answered by 420 people and 10 questions on the communication channels were answered by 500 people. results: out of 1000 interviewees, 19% have never donated and did not intend to donate, due to the fact that most of them were afraid of needles and infections, while the smallest part didn't donate blood because it was not allowed by the religion, 40% did not donate, but expressed the readiness to donate in the future, 10% have donated voluntarily only once, 24% were family donors, 3% regular volunteer donors, and 4% have donated voluntarily several times and did not want to donate anymore. from those who have donated, 59% have donated for one of their relatives, 28% have donated for thalassemic children, 10% have donated to benefit free check-up and 3% have donated because it was valuable for their health. the question as to whether they would voluntarily donate again, 57% have answered yes, 22% no and 21% were still not sure. this means that donation of those who have donated once did not leave a positive impression, did not increase the desire to repeat the donation once again, rather it has restrained or made it unsafe for them to repeat donation. among the causes mentioned by the interviewees were bad conditions in the donation facilities, staff behavior, inadequate treatment, they did not feel good after donation and had hematoma at the venipuncture. summary/conclusions: based on the results obtained from the study, the national blood transfusion center needs the establishment of a genuine promotion department where there is a need for a transfusion doctor who should be an active part of it. the national blood transfusion center should build up and implement a rigorous retention policy for voluntary blood donors, as the study found out that around 60% of donors who have donated once would like to donate again. their attraction through a donor retention policy will surely lead to self-sufficiency with safe blood. the safe blood is a public good and for this reason it is the duty of all state instances, the media and non-governmental organizations to give their support in the promotion of voluntary blood donation. background: smoking, unhealthy diet, sedentary behavior and inability to maintain adequate exercise have significant consequences for several chronic disorders, including obesity. a balanced and equilibrate nutrition may prevent the negative consequences associated to the status of obesity. in italy, overweight and obesity is increasing with 3 adults of 10 overweight and 1 of 10 obese in 2017 with a higher frequency in the south. blood centers can play a public health role in obesity surveillance and interventions. aims: since the quality of life, self-reported by the patient, related to health and adequate quali-quantitative nutrition, are becoming necessary and relevant in the field of nutrition, we conducted a demographic study to evaluate the health status of the blood donors by monitoring the nutritional habits and lifestyle. methods: a descriptive cross-sectional face-to-face questionnaire was developed. it included a 41 item dietary assessment, reporting semi-quantitative food frequency, dietary behavior and questions on self-rated health status. normal weight was established with bmi < 25 kg/m 2 , overweight with a bmi ≥ 25 and < 30 kg/m 2 , and obesity with bmi ≥ 30 kg/m 2 . obesity prevalence was standardized by sex. donors were repeat blood donors, who had made at least 3 donations in the last 2 years, and were eligible to donate. results: of the 2468 blood donors enrolled between july 2017 and january 2018, 1390 were regular repeat donors, 1102 did not wish or chose not to respond at survey for several reasons (i.e. lack of time or privacy) and 288 accepted, of which 83 were deferred from blood donation and were excluded from the analysis. among the 205 included participants 68.3% (n = 140) were male, age ranged from 19-61 years with a mean age of 39.8 ae 11.1 sd and 31.7% (n = 65) were female age ranged from 20-62 years with a mean age of 37.7 ae 11.0 sd. data showed that donors followed mainly a mediterranean diet and had more awareness to lifestyle, women more than men, in comparison with general population. the prevalence of overweight was found 50.7% in men and 16.9% in women. our survey showed that 84.4% of the participants evaluated their health as "good", without gender difference (men, 86.4% vs women, 80.0%). besides, 14.6% reported their health as "very good". summary/conclusions: overweight and obesity are common among regular blood donors and it is more frequent in men than women. our preliminary data showed that women have a better knowledge of the nutritional properties of food and consequently adopt a more balanced and proper diet. furthermore, it is clear that they are aware about the relationship between lifestyle and health putting into practice their information. unfortunately, the survey structure, of observational nature, does not make it possible to establish whether women are more alert to health to participate more in donation programs or if, on the contrary, the status of regular donor could help the improvement of knowledge and healthy lifestyle. background: donor recruitment pose an ongoing challenge to blood banks worldwide. one approach to improve the effectiveness of donor recruitment is to target influencing factors. a yearly league is conducted at the sultan qaboos university (squ) to encourage university students and faculty to donate blood. during this, the colleges are evaluated based on different measures including the number of donors recruited from each college and the efforts made by the students in increasing the awareness of blood donation in the colleges and in the society via different means including the utilization of the social media. the whole competition is organized and ran by an independent group of students. aims: this study aims at studying the impact of the yearly squ college competition on the perception of blood donation among squ students. methods: a comprehensive anonymous voluntary survey was developed and used to assess perception of students aged 18-25 attending squ and other universities (non-squ) over a two years' period. analysis was performed using ibm spss statistics 22.0. categorized variables were presented in numbers with percentages and associations between the groups were analyzed using chi-square test. a p-value of < 0.05 was considered statistically significant. results: a total of 600 students were surveyed (300 squ, 300 non-squ). there was no statistical difference between squ and non-squ students with regard to past history of blood donation and the number of donations made. when comparing between both cohorts, 73% of the squ and 25% of non-squ students reported the university as the main source for information (p < 0.001), while 56% of squ and 45% of non-squ students reported that the social media was the main source respectively (p = 0.048). there was no statistical difference between male and female donors on their perception of level of self-knowledge on blood donation (p = 0.868). about 84% of the youth agreed that blood donation is one of the duties toward the community. squ students reported higher rates of respond to specific requests for blood donation (44.3% vs 29.7%, p < 0.001). squ students reported greater influence of peers (84% vs 60.7%, p < 0.001), personal knowledge (82% vs 74.7%, p = 0.029) and personal experience (79.3% vs 69%, p = 0.005) when compared to non-squ students. they also reported more feeling of commitment to the society (90.3% vs 78%, p < 0.001). squ students reported lower influence of parents (53% vs 65%, p = 0.005), lower rates of fear from needles (18% vs 32%, p < 0.001) and lower rates of fear from blood (16% vs 29%, p < 0.001). there was no difference between male and female genders in any of the discouraging factors. summary/conclusions: these results highlighted the positive impact and important rule of the youth in the promoting blood donations among themselves through this yearly college competition; in recruiting blood donors and in the dissemination of the knowledge of blood donation. distinct promotion strategies should be adopted to increased first time and repeated blood donation among the youth. we advocate for similar initiatives in encouraging blood donation and disseminate knowledge among individuals in the community. dubai blood donation center, dubai health authority, dubai, united arab emirates background: dubai is multicultural city in united arab emirates. only about 15% of the population consists of uae nationals with the rest comprising expatriates from various countries all over the world. approximately 85% of the expatriate population (and 71% of the emirate's total population) are asian, chiefly indian (51%) and pakistani (16%). dubai blood donation centre is the only centre providing blood donation services in dubai. arabic is the national and official language and english is used as a second language. in order to have good quality screening, it is important that blood donors understand the educational material and questionnaire properly. aims: dubai blood donation centre receives donors (nationals, residents and gcc card holders) from various countries. the aim of this study is to analyze the multinational profile of donors and to find out the need to add any third language to meet the customer needs and expectations. methods: a cross-sectional study of blood donors was conducted in dubai blood donation centre in 2019. the donors were asked about their country of origin, languages which they can read & understand and about the preferred mode of communication. results: a total of 1080 donors were surveyed and asked about the languages which they can read and understand and 1860 responses were obtained. the most common languages which can be read and understood by blood donors in dbdc are english (n = 760; 41%), arabic (n = 272; 14.6%), hindi (n = 268; 14.4%) and malayalam (n = 233; 12.3%). the donors come from different countries, most common 591 (54.7%) donors are indian and 73 (6.8%) are from uae. it was found that 45% donors can read and understand only one language. majority 884 (81.9%) donors can read and understand either of the official languages arabic or english. however, 196 (18.1%) donors can't read and understand these two official languages, the other common languages being hindi and malayalam. the donors were asked about the preferred mode of communication, 1290 responses were obtained. the most common mode of communication were sms and telephone (85% together). summary/conclusions: based on the above findings, it can be concluded that the blood donor profile in our centre is multinational which is a unique and almost similar to the population profile of dubai. as 18.1% donors can't read and understand arabic and english, so it has been decided that the educational material and questionnaire need to be prepared in one more language. hindi has been decided as the third language in the centre and donor questionnaire and educational materials in hindi will also be made available to the donors. further,the donors will be communicated through sms for routine messaging and disease notification while telephonic calls will be done only when the blood is urgently required. background: metabolic disorders (metds), including hypertension, dyslipidemia, hyperglycemia, and central obesity, are tightly associated with cardiovascular diseases and type 2 diabetes mellitus. due to the sedentary lifestyle and increased consumption of high-calorie diet in modern society, metds have become serious health problems worldwide. to have a better understanding and possible improvement on blood donors' health condition, we conducted a survey of the prevalence of metds among blood donors in a blood donation station located in the hsinchu science park in taiwan. participants with metds will be provided with health education materials about metabolic risk reduction, in order to prevent the development of future complications. aims: the aims of this study were to determine the prevalence of metabolic disorders among blood donors, and to calculate how much money would be paid to identify a case of hyperglycemia, hyperlipidemia, or undiagnosed diabetes. methods: this study was approved by the institutional review board of taiwan blood services foundation (tbsf). the body weight, body height, waist circumference (wc) and blood pressure (bp) of participants were measured. blood samples were obtained to determine the values of hemoglobin a1c (hba1c) background: the law on blood donation supports the development of the blood service and guarantees the protection of the donor's rights and the maintenance of health during blood donation in the russian federation. national criteria for donor selection for blood donation are used in the activities of blood service establishments and are aimed at ensuring the blood products safety. the study of the characteristics of blood donors allows to predict the development of blood service and to plan the volume of blood products for transfusion and plasma fractionation. aims: the aim of this work was to study the characteristics of whole blood and apheresis donors in the blood service in the russian federation. methods: indicators of activity in the blood service establishments in the russian federation in sectoral statistical observations over the period 2015-2017 and the calculation of indices characterizing the whole blood and apheresis donors were analyzed. data are presented according to the administrative division of russian federation into federal districts (fd). results: the proportion of whole blood donors was 83.7%, plasmapheresis donors -13.2%, blood cell apheresis, including plateletapheresis, donors -3.1%. for the period 2015-2017, the percentage of repeated and regular whole blood and apheresis donors increased from 66.2% to 72.2%. the percentage of first-time donors ranged from 27.8% to 33.8%. the largest proportion of plasmapheresis donors was observed in the volga fd (22.0%). about 28.9% of the total plasma was collected by apheresis from donors. the percentage of plateletapheresis donors increased from 1.9% to 2.3%. the largest percentage of plateletapheresis donors was observed in the central fd (2.9%), where a significant part of medical centers of cardiac surgery, hematology and bone marrow transplantation are located. the proportion of platelet concentrate collected by apheresis increased to 69.9% in 2017. actions to recruit young donors for blood donation and its components were regularly carried out in all federal districts. summary/conclusions: in the russian federation, the structure of donation is characterized by an increase in the proportion of plateletapheresis donors, stabilization of the percentage of plasmapheresis donors and an increase in the proportion of repeated and regular whole blood and apheresis donors. there are significant regional variations of donor's characteristics in the federal districts. background: shortage of blood supply despite continuous blood donation campaigns especially during local festive seasons has been a major issue in our country. thus, our faculty initiated blood donation drives in collaboration with national blood centre in order meet the demand for the blood requirements. however, the pre-donation deferral rate was relatively high among our young blood donors leading to loss of valuable blood units. understanding the causes of donor deferral provides direction on strategies for young donor recruitment and retention of future blood donation. aims: the aim of this study is to evaluate the young donor deferral pattern and to identify factors which could help in minimizing the preventable deferrals. methods: this is a retrospective study of voluntary young blood donors age between 20 to 23 years old recruited during mobile blood donation in faculty of medicine, universiti teknologi mara, malaysia. the study was conducted between january 2016 to december 2018. the data were retrieved from the official reports of each mobile blood donation. results: a total of 828 young blood donors had attended mobile blood donation during the study period. the overall pre-donation deferral rate is 25.6%. the main causes of deferral are low haemoglobin (hb) level (20.8%) followed by low blood pressure (16.0%), upper respiratory tract infection (11.3%) and sleep less than 5 h (9.4%). summary/conclusions: low haemoglobin and low blood pressure are the two common reasons for blood donation deferral among our young blood donors. in our study a significant proportion of deferrals are due to sleep less than 5 h whereby this could be prevented if the donors are aware of the donor selection criteria. strategies to mitigate preventable deferrals and improve blood donor retention particularly young blood donors as source of motivation for future blood donation are urged to avoid additional stress on the blood supply. background: in the modern world, donating blood has become a humane manner for saving of patients life. but there are barriers to blood donation which are designed to ensure both donor and blood recipients' safety. anemia is one of the most common health problems in the world. based on the who estimation, nearly a quarter of the world's population are suffering from anemia, its prevalence varies among the populations and age groups. the prevalence of anemia among men is 12.7% and in non-pregnant women is 30.2%. aims: the aim of this study was to determine the status of hemoglobin in volunteer blood donors referring to fars province blood transfusion service and to determine the demographic status of them during the last two years. our study included blood donors for all blood donors during the last two years. methods: the study is descriptive cross-sectional and our sampling was non-random and simple sampling method. all parameters related to the donors, including age, sex and type of donation were investigated and analyzed in spss software. results: the total number of referrals for blood donation was 454913. repeated blood donors was 44.8% of total population and had the highest number of referrals, followed by first and lapsed donors with 31.2% and 24% respectively. in terms of gender distribution, 6.2% were female and 93.8% were male. the highest rate of hemoglobin level less than 12.5 g/dl was found in first-time donors with 51.6% and the lowest prevalence was observed in lapsed donors, followed by repeated donors with 24.7%. 44.8% of the repeated blood donors have hemoglobin higher than 16.5. there was a significant difference between blood donation type and hemoglobin level. summary/conclusions: according to our findings, low hemoglobin levels are more common among first-time and female donors, and this requires a special training among these groups. because the high share of first time donors in blood supply and the positive impact of female donors on the blood safety, corrective action for that groups is recommended. finnish blood donor biobank j partanen, t wahlfors, m arvas, j clancy, k l€ ahteenm€ aki, e palokangas and n nikiforow background: the increasing need for large, well-characterized cohorts of healthy individuals for modern biomedical research, such as genomics or phenomics studies typically including tens or even hundreds of thousands of subjects, has posed the possibility of using blood services as an option for collecting samples and related data. the possibility to re-contact blood donors for repeated sampling or asking for additional data has further increased interest in collecting large biobanks from blood donors. there is also a need to study more thoroughly the effects of blood donation on donor health. aims: the first-phase goal is to recruit 50,000 blood donors with broad biobank consent for the finngen (https://www.finngen.fi/) project, a large publicprivate effort aiming to collect genome and health-related registry data of 10% (500,000) of the finnish population. (11.58%), dental examination 10(3.32%) and medication history 6(1.98%). permanent deferral namely, risk factor involving transfusion transmitted infections and chronic disease were 5(1.65%) and 8(2.64%) respectively. the prime cause of permanent deferral was risk factor involving transfusion transmitted infections while the temporary deferral was bed side hypertension. gender wise, the leading cause of donor deferral in male was bed side hypertension and anaemia was the major cause in female. summary/conclusions: the findings of the survey aid to evaluate the significant causes of blood donor deferral. this study suggests that the restrictive criteria can be used for blood donor selection. this will in turn increase the blood supply of tertiary care hospital. background: donor selection is the first step towards safe blood but retaining blood donors is also very important for the blood supply. donor questionnaire and the medical interview should provide optimal doctor deferral. aims: to evaluate deferral rate in blood donors in order to identify the main reasons and to target eventual corrective activities. methods: we analysed the data concerning blood donors who were registered in the period of three years (2016-2018). we used data from the information system e-delphyn. background: iron deficiency (id) in blood donors is an underestimated issue in many countries and may cause symptoms to blood donors even without anemia. id prevention is mainly based on the prevention of anemia in whole blood donors, which is done by deferring donors whose haemoglobin level is under defined threshold (120 g/l in women, 130 g/l in men in france). efs (french blood establishment) studies has observed that the rate of deferral for anemia is significantly higher in women than in men, either in french west indies (15.9% versus 3.4%) or in continental france (2.9% and 0.8%). assessing the prevalence of id is of great interest since strategies to counteract it must deal with donor health and self-supply. however, data on id are missing in france. aims: to estimate the prevalence of id in french whole blood donors and to identify risk factors associated with id. methods: this non-interventional, cross-sectional, multicenter study is performed in blood donors of efs and ctsa (blood center of the french military health service). all whole blood donors who met selection criteria are potentially included. donors coming for bloodletting and donors who refuse to participate to the study are excluded. no additional sample is taken for the study, ferritin is tested after blood qualification on surplus amount. samples are selected at random within all the geographical areas and all mobile blood drives and blood centers between march 11 and march 28, 2019. results: this study ferridon has been approved by ethical research committee. nine thousand (9000) whole blood donors will be included in efs centers in continental france. to have information on donors of afro-caribbean origin and comoros origin, 150 donations should be included in the french west indies and 120 in reunion island. additionally, 1500 whole blood donors will be included in ctsa centers. in this study, id is defined by ferritin lower than 26 ng/ml and iron overload is defined by ferritin higher than 300 ng/ml. all donors with iron deficiency or overload will received a letter advising to consult their general practitioner. weights will be calibrated on age, sex and geographical area so the sample will be representative of the french whole blood donors. estimation of id prevalence will take into account the weights and logistic regression model will be used to analyze risk factors associated with id. data will be analyzed during april and may 2019 to get result at the end of may. summary/conclusions: ferridon will be the first study on id in the french blood donors. considering the french health care system and diet, it will be interesting to compare those results to results from other countries. mostly this study will allow to consider various strategies dealing both with donor safety and self-supply. background: in portugal, with an aging population of around 10 million people, only 3.8% are blood donors. the country has a national center of blood supply and some central hospitals with a blood donation center. despite the growing practice of the excellent concepts of patient blood management, it is imperious to attract new donors. this need has been our inspiration to use new approaches towards people, in a constant work of promotion. aims: reach the majority of our local population using radio and telecommunication as well as social networks in an attempt to raise the number of new blood donors in a central hospital of the north of portugal. methods: active communication with the population of our reference area, via the social networks facebook tm and instagram tm , through educational digital posters and messenger service to answer any kind of questions. establish contact with radio and television stations as well as with the mayor of the city, journalists, schools, town hall deputies and celebrities, through email and telephone calls. design posters, flyers and public advertising to distribute in the city. results: through the social networks it has been possible to reach a population of dozens of thousands in our city, in a daily basis. the national and world donor days were celebrated with success, in our health facility, with city mayor and journalists, and also in three television stations with national broadcast, reaching millions of people. celebrities (sport, television, music, stand-up comedy, journalists and a magician) have accepted our challenge through videos or donating blood, appealing to blood donation and sponsoring our cause. these projects and continuous availability to innovate have given our hospital a self-sufficiency of 95% in 2018, instead of 80% in 2016, which implied receiving less blood unities from the national center of blood supply. our most recent project involves high schools, in an attempt to educate our next generation of donors, with meetings in the town hall with deputies and district school delegates. summary/conclusions: the aging population and the low percentage of blood donors are an important issue concerning public health. nevertheless, the good will and continuous advertising and educative work towards the population, appealing to the ethical and civil responsibility since young ages have shown to improve our capacity of response as a central hospital, increasing the auto-sufficiency of blood unities and the interest of younger donors. it is of the utmost importance to understand that this is a continuous and a hard work of the professional team of our hospital, involving countless calls, emails and hours to obtain some positive response, in an endless job. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . the mean interval between donations is shorter for former regular donors (4.9 months, p < 0.01) whereas donors with an interval of 9 to 12 months are more likely to be regular (aor 16; 95% ci 1.6-164). summary/conclusions: at the provincial blood transfusion centre of bukavu, the percentage of regular donors is low and there is a substantial loss of former regular donors. some factors identified to be linked to fidelity are unique to our study: female gender and a longer interdonation interval. other factors that are similar to those found elsewhere have a particular significance in our donor population which consists mainly of young people and people without income. efforts must be undertaken to ensure a supply from voluntary donations; recruitment strategies and target groups must be refined. future qualitative studies are needed to explain the various associated factors and better understand the motivations of regular and non-regular donors to improve donor retention. results: in kazakhstan, the proportion of donors is higher, especially primary. the number of blood and especially plasma donations is higher, which can be explained by the presence of several albumin and immunoglobulin production sites. increased evidence of blood transfusion rules, the development of a patient's blood management in combination with an increase in the quality of blood components cause a reduction in clinical need for red blood cells and plasma for transfusion. at the same time, the need for platelets is growing. it is difficult to assess the correctness of comparing the amount of banked whole blood. it is equally difficult to compare the number of received and distributed donor red blood cells and plasma: in russia they are measured in liters, and in kazakhstan in doses. with a certain degree of conditionality platelet extraction can be compared. in russia, they are counted in equivalent doses isolated from a dose of whole blood (at least 60 9 10 9 cells per dose), and in kazakhstanin therapeutic doses (at least 200 9 10 9 cells per dose). in 2017, the estimated consumption of platelets in kazakhstan exceeded the russian indicator by 28.0%. 86% of platelets in kazakhstan and 69.9% in russia are harvested by the apheresis method. inactivation of pathogens is performed in 92% of platelets in kazakhstan and in 15.4% in russia. pathogen inactivation with amotosalen allows us to abandon the examination of donors for cytomegalovirus and irradiation of platelet concentrate. the main modern trend in the use of cryoprecipitate is using it as a source of fibrinogen, a blood coagulation factor that is first depleted in coagulopathy associated with injury and massive bleeding. its production is growing in both countries, endowment in kazakhstan in 2017 exceeded the russian indicator by 141.4%. a significant plasma percentage in both countries does not pass quarantine due to repeated non-appearance of the donor and is subject to destruction. inactivation of pathogens is performed in 10% of plasma in kazakhstan and in 3.6% in russia. despite the instruction and selection of donors, laboratory screening of infection markers remains effective: russia more often identifies hiv and viral hepatitis c from potential donors, and viral hepatitis b and syphilis are detected in kazakhstan. 0.7% of donors in kazakhstan are exempted due to the results of multiplex screening of nucleic acids of three hemotransmissive viruses. summary/conclusions: the blood services of russia and kazakhstan perform tasks to provide medical organizations with effective blood components. in conditions of decreasing demand for red blood cells and plasma, it is advisable to focus on the efficiency of resource use and improving the quality of blood components produced. blood collection including apheresis p-091 finnish red cross blood service, helsinki, finland background: skin disinfectant must effectively reduce microbes from the arm of the donor. as a result of poor disinfecting microbes may be transferred from skin via venepuncture to the collected blood and contaminate the blood components. aims: to ensure the efficiency of the skin disinfectant used for donor arm disinfecting by validation. the validation has two criteria which the post-disinfection samples must achieve: 1. no bacteria growth near the puncture spot (result 0 cfu) in ≥ 90% of the samples. 2. total amount of bacteria on average is at most 2 cfu/25 cm 2 . at most 10% of samples are allowed to have 2-10 cfu/25 cm 2 . methods: microbiological samples were taken with contact plates from 30 voluntary persons' elbow folds before and after skin disinfection. the disinfecting was performed according to normal procedure by five nurses altogether with ethanol based disinfectant used in blood donation. on the sample plates an x was marked and this was directed to the puncture spot pointed by nurse. post-disinfection sample was taken at the moment the skin would be punctured with needle. results: the amount of bacteria varied from 20 to above 500 cfu/25 cm 2 in the pre-disinfection samples. disinfection reduced bacteria very well; the critical puncture spot was totally clean (0 cfu/25 cm 2 ) in 93.3% of the samples and 86.7% of the samples had 0 or only 1 cfu/25 cm 2 . the average number of bacteria after disinfection was 0,6 cfu/25 cm 2 and the maximum number was 4 cfu/25 cm 2 . most of the remaining bacteria were single colonies at the edges of the plates. summary/conclusions: both main criteria are fulfilled. the sub criteria of the second main criteria is also full filled if the not so critical colonies at the edges of the plates are not taken into account. the skin disinfectant in question is shown to be effective and can still be used in blood donation paying attention to thorough procedure performance according to the instructions and sufficient drying time of the disinfectant. background: research questions involving blood donation and recipient data often require advanced statistical methodologies. while such methodologies may appear in other medical research areas, specific tailor-made statistical tools and approaches are required for the analysis of blood-related data. these toolkits, which often require collaboration, are not always readily available to blood services, especially so in resource-limited settings. an international network of statisticians, epidemiologists and clinical researchers has been established for this purpose, which started with an invited session at the 2018 meetings of the international biometric society. aims: to exchange ideas, experience and knowledge to further improve the quality of blood sector research. methods: currently our network covers four major blood services and members from five different countries. the network has monthly conference calls about past and current research topics. we wish to extend this network further, and establish a subcommittee on statistical and epidemiological methodology with regular face-toface meetings at an international organization such as isbt. results: the monthly meetings have already demonstrated that the members share common problems and interests. for example, we are discussing techniques to analyze data with repeated measurements, e.g. eligibility haemoglobin tests, ways to assess the healthy donor effect, e.g. determining appropriate controls groups, and predictive models of blood supply and demand, e.g. stochastic processes and queuing models. the network also aims to organize training sessions in methodology either on site and/or by developing web lectures. summary/conclusions: an international network on statistical methodology for the analysis of blood donation and recipient data will improve the quality of research in the field of transfusion medicine research. expanding the network to include countries and blood services in research limited settings needs to be actively pursued. background: blood donor hemoglobin concentration (hb) is commonly measured from a skin-prick sample at the donation site, and low hb is the most common reason for temporary donor deferral. while a proportion of the deferrals do reflect true low hb, the skin-prick sample is prone to preanalytical error and variation resulting in false deferrals. aims: we assessed the applicability of a venous blood sample for second-line hb screening in blood donors failing the initial skin-prick test. methods: initial hb was measured from a skin-prick sample with the hemocue hb201 + (hemocue ab) point-of-care (poc) device. donors with hb < 125 g/l for females or < 135 g/l for males or with a decrease > 20 g/l from latest donation were included in the study. in the study group, a venous blood sample was collected for hb measurement with the poc device at the donation site. donation eligibility was based on this hb result. venous hb was also determined with a hematology analyzer (sysmex xn, sysmex co.). the blood service's current workflow served as the control group: two more skin-prick samples were collected and the donor's final hb and donation eligibility assessed with an algorithm based on all three skin-prick hb results. results: in the study (n = 305) and control (n = 331) groups, the proportion of male donors (27% and 26%) and the mean initial skin-prick hb (122 g/l and 123 g/l) were similar. significantly less donors were deferred from donation in the study group (40%) than in the control group (51%; chi-square test p = 0.004). the mean difference in venous hb with the poc device versus the hematology analyzer was à3 g/l (range à12 to + 6 g/l). two donors were incorrectly accepted based on venous sample poc result; however, in both, hb measured with the hematology analyzer was only 1 g/l below the limit of donation eligibility (124 g/l for a female and 134 g/l for a male). interestingly, a further 33 donors (27% of all deferred in the study group) would have been eligible for donation based on the hematology analyzer result. summary/conclusions: utilizing a venous blood sample for second-line screening of donors failing the initial skin-prick hb test significantly decreased low hb deferrals without compromising donor health. blood donors' and blood service nurses' reactions to the new workflow have been favorable. we conclude that valuable donations can be recovered and donor satisfaction increased by implementing a second-line hb screening model utilizing venous sample analysis at the donation site. background: there is a paucity of literature on haemoglobin (hb) reference values for adults above 60 years of age. this age group has been reported to use up to 50% the blood supply. some studies report a decline of mean hb with age, but others have found no change with age. conflicting findings of hb levels in the healthy elderly population may be associated with challenges in accessing data from healthy older adults, small sample sizes, selection bias and recent health population data. to donate blood, each individual is assessed as 'healthy' and must meet the minimum hb criteria. however, the hb criteria across countries vary and many blood collection services have an upper age limit for donors. as many populations around world are aging, restricting the upper age limit for blood donation may potentially affect the size of the donor pool and consequently the nation's blood supply. aims: to explore the hb levels of healthy older adults, through a multi-centre retrospective observational study of blood donors aged 60 years or older. methods: over a one-year period, hb values were collected from blood donors aged ≥ 60 years from blood centres of four countries. the estimated proportion of blood donors aged ≥ 60 years old for each country was 0.81% in south korea (sk); 2.57% in hong kong (hk), <3.18% indonesia (indo) and 9% in japan (jap). the minimum hb criteria varied between each country and ranged from 11.5-12.5 g/dl for women and 12.5-13.0 g/dl for men. hb levels were determined using point of care testing (hemocue, compolab, hemcontrol) or the xe-2100d sysmex dependant on the country of origin. statistical analysis of the mean, standard deviation and cumulative distribution of hb were determined by gender and age. background: medication usage is assessed to determine donor eligibility from the perspective of both recipient and donor safety. different time frames since last taken apply to different medications. assessment of medication use varies by jurisdiction, but most european centres use multiple questions. these often include a general question about recent medication use whereas the usa does not. at canadian blood services there are 6 medication questions on the donor history questionnaire (dhq), including any medication use in the last 3 days, vaccination and specific medications over different time frames (high teratogenicity medications). the name of each medication taken and reason for use are documented by staff at each donation attempt. assessment of the frequency with which this process occurs is the first step in improving efficiency of this aspect of donor screening. aims: to determine the percentages donors answering yes to medication questions by demographic variables. methods: all whole blood donors who completed the dhq (full length or abbreviated) in 2017 were included in the analysis. donors' answers to each of the 6 medication questions were extracted from the national epidemiology donor database, as well as sex and age. the number and percentage of donation attempts in which a donor answered yes to each medication question were calculated. donors who answered yes to any medication question were sorted by sex and by age group, the totals and percentages calculated. results: there were 975,067 donation attempts with a completed dhq. overall, 34% of donors answered yes to medications in the last 3 days, 8% to vaccination, and less than 0.1% to others (38% any). slightly more were female (42 vs 36%) of those who answered yes to any medication question, as well as by individual question. the percentage of donors answering yes to any medication question increased progressively in each age group from 24% of 17-19 year olds to 57% aged 60 + (p < 0.0001 for trend). summary/conclusions: more than one third of all donation attempts answer yes to a medication question and require further questioning and documentation. this is more common in older donors and follows a similar trend to general population medication use. comparison of ways of assessing medication use in different countries may help identify effective but more efficient approaches. in addition, the contribution to donor and recipient safety of assessing all medications should be assessed. blood center experience with trima accel 7 and tomes software j schreier 1 , a davison 1 , j gambarte 2 , y l opez 2 , c calonge 2 and e herranz 2 1 terumo bct, lakewood, united states 2 centro de transfusi on de la comunidad de madrid, madrid, spain background: in the madrid community, more than 4000 apheresis platelet collections were completed in 2018, of which almost 3000 were completed in the blood transfusion center and the remainder in several hospitals in the region. trima accel 7 was implemented to meet the productivity needs of the blood transfusion center while improving the donation experience. tomes (terumo operational medical equipment software), which enables bidirectional communication with trima accel devices, was used to connect and centrally manage all trima accel 7 devices with automated data capture and reporting. aims: the aim of this study was to evaluate operational improvements using trima accel 7 with tomes compared to trima accel version 6. methods: this was a retrospective study analyzing 1372 apheresis procedures on trima accel version 6 during the control period from 2 january 2018 to 10 september 2018 compared to 541 apheresis procedures on trima accel 7 during the test period from 11 september 2018 to 28 december 2018. this was not a paired study. operator interventions, and completed procedure rate comparisons, were analyzed using a 2-proportions test, whereas donor demographic data were analyzed using a 2-sample t-test. results: trima accel was used to collect single and double platelet products stored in platelet additive solution. operators selected either a single (target platelet yield = 3.0 or 3.5 9 10 11 ) or double (target platelet yield = 6.0 9 10 11 ) platelet donation based on desired procedure time not the maximum number of products that could have been collected per donor. no statistically significant differences were observed for donors in the test arm compared to donors in the control arm for total blood volume (control = 5148 ml, test = 5124 ml, p = 0.545), hematocrit (control = 43.7%, test = 43.6%, p = 0.233), or platelet pre-count (control = 249 9 10 3 / ll, test = 253 9 10 3 /ll, p = 0.091). females represented 21% of donors in the control arm compared to 23% of donors in the test arm. platelet split rate (platelet products per procedure) increased from 1.15 with trima accel version 6 to 1.18 with trima accel 7; procedure time decreased from 55.7 min to 53.3 min for single collections and from 63.0 min to 60.9 min for double collections with trima accel 7 (these differences were not statistically significant). the percentage of procedures that completed with no operator interventions due to access alerts increased from 62.4% to 84.8% (p < 0.001) and the rate of completed apheresis procedures increased from 89.8% to 92.4% (p = 0.083) with trima accel 7. manual transcription of data during the procedure was discontinued with the implementation of trima accel 7 with tomes. tomes captured procedural data and operator steps with barcode scanning and tracking of configured events. this information was transferred to tomes post procedure and printed as a final report. summary/conclusions: trima accel 7 significantly decreased operator interventions, and automated data capture with tomes eliminated manual transcription of data. both outcomes freed operators to complete other tasks and focus on donor well-being. background: the european committee (partial agreement) on blood transfusion (cd-p-ts) of the council of europe (coe) has appointed a working group (wg) to focus on issues with plasma supply management (psm). the task of the wg is, among others, to collect and analyze data in order to fill knowledge gaps concerning donor safety in plasmapheresis. in doing so, the working group will gather evidence base data to support the upcoming revision of the 19 th edition of coe's "guide to the preparation, use and quality assurance of blood components", the blood guide. an international survey was conducted sept-dec 2017, distributed to blood establishments (bes) by the cd-p-ts representatives to coe's member and observer states. the questionnaire included sections covering collection practices (volume and frequency), management of red cell loss, donor panel demographics and data on donor adverse events. aims: the aim of this study was to investigate whether collection practices following the recommendations published in the blood guide for maximal collection volumes and number of donations per year were indeed associated with higher levels of donor safety and improved donor base sustainability. methods: from the total of 36 respondents, 24 bes collected plasma for fractionation (pff) by apheresis and the study had a dataset covering 2,888,390 plasma donations in the latest fiscal year (lfy). the parameter used as marker of donor safety was the rate of immediate vasovagal reactions with loss of consciousness (vvr with loc) per 10,000 plasma collections. the parameter used as marker of donor base sustainability was the retention rate of donors, ie % donors active in the previous year returning to make a donation in the lfy. results: in the blood guide, the collection volume per apheresis is limited to 16% of the estimated total blood volume but maximally 750 ml, including anticoagulant. respondents had differing practices and scale of collection program 15 be were aligned or lower, and 9 be had higher collection volumes. altogether 14 reported the immediate vvr with loc rate, which mainly was lower than 10/10000 collections. there was a small trend towards reduced rate with larger collection volumes than allowed by the current blood guide. saline compensation during or after collection did not affect the rate of vvr with loc. no correlation was observed between the annual donor retention rate and the rate of vvr with loc or saline compensation practices, as reported by 16 respondents. the retention rate banded in the range of 50%>80% (mean = 60%, min = 25%, interquartile range = 14%, max = 90%). the association between maximum allowed yearly plasma collection (25 l) appears to be reasonably constant and showed no clear association with the donor retention rate. summary/conclusions: restricting the maximum collection limit according to the current blood guide was not associated with either lower vvr with loc or with higher donor retention rate. this study supports reassessment of current blood guide 0 s limits for collection volume of maximum of 750 ml per donation and 25 l per year per donor. methods: serum ferritin concentrations were established from sera stored at à30°c from repetitive platelet donors between 2014 and 2016, using architect â ferritin assay chemiluminescent microparticle immunoassay (cmia). the hematimetric parameters were evaluated in a total blood sample using the celldyn 1800 â . sixteen samples were obtained from women (age: 35.3 ae 12.4 years, range: 18-61) and 35 samples from men (age: 36.2 ae 9.4 years, range 20-61), corresponding to 55.2% and 37.2% of the total female and male repetitive donors of platelets by apheresis using trima accel â terumo-bct and amicus tm fresenius-kabi. the difference in the concentration of serum ferritin between the last and first donation was established, as well as the change in the predonation platelet count between the last and first event. results: in the study population, 43.8% of women and 40% of men performed repetitive donations of platelets by apheresis with an interval of less than three months. the change in ferritin concentration was evaluated according to the interval between donations. in women ferritin delta was à38.4 ae 52.2 ng/ml when the donations had an interval less than three months, vs 3.3 ae 13.0 ng/ml when the time between donations was higher (p = 0.046). in men the change in the ferritin levels was à38.5 ae 61.2 ng/ml with donation times less than three months vs © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 à3.3 ae 38.9 ng/ml with prolonged donation times (p = 0.042). in women, the change in platelet count was à2 ae 46.2 9 10 3 /ul, when the donations had an interval less than three months vs à22 ae 45.3 9 10 3 /ul when the time between donations was greater (p = 0.43). in men, the delta of the platelet count was à12 ae 37.0 9 10 3 /ul in donation times less than three months vs à13 ae 26.6 with higher donation times (p = 0.93). no correlation was found between the concentrations of serum ferritin and the platelet count (r = 0.05, q = 0.75 for males, and r = 0.34, q = 0.22 for females). summary/conclusions: the data obtained suggest that repetitive donation of platelets by apheresis with intervals between donations of less than three months, significantly reduce serum ferritin concentrations in women and men, although normal levels were maintained in both groups. there was no correlation with platelet count. therefore, it is proposed to develop prospective studies to establish the minimum time interval safety for platelet apheresis donor procedures. background: the demand for platelets concentrates is increasing continuously and becomes a challenge for the blood establishments. apheresis platelet collections may be a solution for this challenge. improving apheresis collection efficiency while maintaining blood donor safety is an important goal for the service du sang of the belgian red cross. aims: our establishment evaluated the improvements of the trima accel automated blood collection system version 7 (ta7) by comparing its routine performance with that of the previous software version 6.06 (ta6). methods: prospective, multi-site, controlled, non-randomized trial. apheresis collections were performed in three sfs sites: liege, mons and namur using the two trima software versions ta6 and ta7 sequentially. data were collected from december 2017 to april 2018 on ta6 and from june to july 2018 on ta7. simple and double doses of platelets (respectively 5.0 9 10 11 , 5.5 9 10 11 and 8.0 9 10 11 , 9.0 9 10 11 ) were collected in platelet additive solution (ssp+, macopharma) with concurrent plasma from the same cohort of donors in accordance to donor's eligibility and preferences. in order to maintain the same final platelets content in platelets concentrates, the trima accel's tool yield scaling factor (ysf) was subsequently adjusted from 1.06 (ta6) to 1 (ta7). platelet yield, duration of procedure, number of alarms requiring operators' interventions were recorded and evaluated. donor's hypocalcemia was avoided by giving preventively oral or intravenous calcium which was documented by the operators. results: five hundred ninety (590) collections with ta6 and 607 with ta7 were recorded, with 92% and 95% complete procedures respectively. mean duration of procedures was 70 min on ta6 against 72 min on ta7, p < 0.05. the mean alerts number per procedure on ta6 was 4.1 against 0.5 on ta7, p < 0.05 whereas the maximum alerts number per procedure was 57 and 12 respectively. on ta7, 76% procedures did not require operator's intervention against 40% on ta6,. with ta7 the inlet flowrate was automatically adjusted in 97.2% procedures. the inlet flowrate was increased in response to access pressure in 45.6% of procedures, for 3% of the procedures the inlet flowrate was decreased and for 48.6% of the procedures the inlet flowrate was increase and decreased on the same procedure by the ta7 autoflow system. summary/conclusions: ta7 with its autoflow function improves apheresis donors experience while decreasing operator' interventions through a significant reduction of access draw alerts. as expected from the trima accel platform, post-donation safety remains high. a weak increase in procedure duration was observed for the same platelet yield which may be resolved with further adjustments. background: trima accel system is an apheresis platform relying on continuous flow centrifugation to collect from a donor platelets, plasma or rbcs based on donor qualification. the latest software version -trima accel 7 (ta7) introduced the autoflow feature which allows for automated flow rate adjustments. moreover, ta7 leverages the mobilization capacity of the spleen increasing potential platelet productivity while maintaining high post-donation safety standards characteristic of trima accel. aims: the objective of this evaluation was to assess the impact of ta7 software by retrospective comparison of procedure data and potential for increased productivity with those of trima accel version 6 (ta6) in the same cohort of platelet donors. methods: eight hundred twenty one procedures, started on ta6 from 4th january 2016 to 10th october 2017 were compared to 995 procedures, started on ta7 from 18th october 2017 to 31 st december 2018. procedural data from the trima devices were captured using the cadence system (terumo bct, lakewood co). parameters investigated were the number of machine access pressure alerts per procedure, the potential for higher platelet yield collections and the actual collected yields within the same cohort of platelet donors. results: both donor populations (ta6 vs. ta7 respectively) were comparable and were characterized by: tbv -5207 vs. 5344 ml; platelet count pre-procedure -252 9 10³/ll vs. 252 9 10³/ll; hematocrit pre-procedure -44% vs. 44%. gender distribution was 11% female with ta6 vs. 2% with ta7. venous access pressure alerts were significantly improved by ta7 with an average of 0.2 alerts per procedure as compared to 2.0 with ta6, i.e. 92% decrease. this decrease went down to 91% if only male procedures were analyzed. the maximum number of pressure alerts went down by 84% from 90 alerts in one particular run in the ta6 cohort to 14 alerts in one ta7 procedure. procedure time for single platelet products was reduced from 51 to 49 min and for double platelet products from 59 to 54 min (ta6 and ta7 respectively). donor qualification possible was 52% of procedures yielding single products and 48% of procedures yielding double products with ta6. the percentage of procedures qualifying for doubles increased to 91% with ta7. in terms of split rates, i.e. how many platelet doses could be produced per apheresis collection, potential split rates increased from 1.48 to 1.91 from ta6 to ta7, respectively. in fact, the observed split rate rose modestly from 1.01 to 1.05, as shorter procedures were generally selected according to donors' preferences. summary/conclusions: in comparable donor populations, implementation of ta7 decreased the number of access pressure alerts significantly compared to previous trima versions. the average procedure duration was also found to be slightly reduced. implementation of ta7 has the potential to increase productivity significantly. the observed modest actual rise in split rate suggested that factors related to donor and inventory management will determine at which extent the potential of the new software will be used. donor compared experience on trima accel 7 to trima accel version 6 august 11 2017 to october 17 2017 or trima accel 7 during the test period from november 28 2017 to january 9 2018. this was not a paired study. donors completed the survey while recovering from the apheresis procedure in the cantina. results from the paper survey were transcribed into excel for analysis. results: 63 donors completed the survey during the control period whereas 86 donors completed the survey during the test period. the mean number of previous donations for the control period was 3.0 (min 1 max 6) and for the test period was 3.2 (min 0 max 7). there were no first time donors during the control period and 20 first time donors during the test period. 91% of donors rated their overall donation experience as good on trima accel 7 compared to 90% on trima accel version 6. zero (0) donor rated their experience on either trima accel device as poor. 100% of donors who responded to the question said they would donate on trima accel 7 again. summary/conclusions: no significant difference was observed in donor experience between trima accel version 6 and trima accel 7 as both versions receive high marks. background: the trend on growth of query for donor platelet concentrates is observed in russia for past few years. as reported by edqm in 2014, a higher number of the platelets was consumed compare to 2011 by 7.8%. patients' with hematological malignancies treatment requires platelet concentrates transfusions during chemotherapy, immunotherapy and hematopoietic stem cells transplantation. according to the data collected in national research center for hematology (nrch) in 2018, 635 (8.6%) of the 7,371 patients, treated within facility, received platelet transfusions as transfusion therapy. the total number transfused units is 14,292, which is 668 higher (by 4.9%) comparing to 2017. platelet concentrates production can be performed either by apheresis process or by pooling individual units recovered from the whole blood. taking into account that the nrch produces blood units for its own needs, the pooling is not suitable method for production because its implementation doesn't cover require for platelets and overproduces rbcs. that is why platelet concentrates in nrch are obtained by apheresis only. in summary, the growth on requirement for platelet concentrates and their safeness explains the need for a comparative study for effectiveness of platelet production using various apheresis systems. aims: the aim of the study is to compare effectiveness of platelet concentrate production using mcs + 9000 (haemonetics), upp and trima accel (terumo bct) version 6.0 protocols. methods: the data for 4868 protocols of platelet donations performed in 2018 were analyzed: 2773 on trima accel and 2095 on msc + 9000. all donors were voluntary and non-paid donors with previous experience of blood donations. the choice of the platelet collection device was random; analysis of the main characteristics of donors did not reveal any significant differences between the groups. the median age of the donor was 32 years old, height -175 cm, weight -76 kg, platelet count before donation -254 9 10 9 /l, hematocrit -42%. detailed data are presented in table 1 . student's t-test for unrelated sets was used for statistical analysis of the data. a value p of less than 0.05 was considered as significant. results: the data obtained showed significant difference (p < 0.01) between average number of platelets collected on trima accel (6.3 ae 1.06 9 10 11 /l) and on mcs + 9000 (5.09 ae 0.78 9 10 11 /l). while cost of consumables are comparable, trima accel demonstrated 23.7% higher efficiency. procedure duration also was comparable and averaged within 94 min for both devices. detailed data are presented in table 2 . it is crucial to mention that proportion of trima accel's donations was significantly increased in nrch during 2018 and reached 56, 7% in total (2017-19.4%). a flexible usage of trima accel's consumables for different procedures (regular platelet collection and collection in pas) allowed to change the pas/regular platelets collection ratio from 7.64% up to 43.7%. summary/conclusions: obtained results proved the effectiveness of the trima accel's use for platelets concentrates production. it allowed to increase the average count of platelets obtained for one procedure by 23.7% compared to mcs + 9000 while the cost of consumables and procedure duration are comparable. the donor's comfort during procedure did not affect either. in long terms increasing of number of platelets collected is reducing the cost of platelet concentrates production. abstract withdrawn. background: apheresis collected platelet concentrate is preferable in terms of reducing the risks of adverse reactions in platelet transfusion when compared to random donor platelet concentrates. aims: the aim of our study is to present our experience in collection donation of single donor platelets with apheresis. methods: this is a retrospective study performed in the institute for transfusion medicine from 2010 till 2019. all donors were fully informed on the donation procedure and signed an informed consent for donation. the optimal platelet count that we want to achieve was ≥ 3.0 9 10 11 equal to 12 random donor platelet doses. minimum preapheresis platelet count in donors requested to start the apheresis collection was 150.000/ll. platelet collection was performed using flow cell separators haemonetics mcs+ and trima accel. acid citrate dextrose formula a was used for anticoagulation. median precollection platelet count of donors was 273.000/ll, with range from 182.000/ll to 397.000/ll. male were 77% of the donors and females were 23%. the single procedure usually took 45-70 min. the median platelet count collected was 4.0 9 10 11 , range 2-6.5 9 10 11 . the median processed blood volume was 3215 ml and median used acd-a was 352 ml. mean total volume of collected product was 312 ml. the adverse effects included vein perforation and the numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and was very mild. summary/conclusions: the collected platelet count was more than the wanted optimum platelet count. the number of apheresis donors is increasing and we are working on expanding our voluntary platelet donors registry and increasing the number of typed donors in the registry. background: to determine value of hemoglobin in blood donors, there are some tools or methods used, such as: cyanmethemoglobin method that can detect of hemoglobin quantitative and methods cupric sulfate solutions (cuso4) can detect of hemoglobin qualitative. according to who (world health organization) to determine the level of hemoglobin in blood donors enough used cuso4 solutions with specific weight (y) 1.053 can to detect value of hemoglobin above or same with 12.5 gr/dl. but, cuso4 solution specific weight 1.053 can not to detect and elimination value of hemoglobin above 17 gr/dl or polycythemia sick. because it, central blood transfusion unit (utdp) as the central of blood service in indonesia to manufacture cupric sulfate solution (cuso4) with a specific weight (y) 1062 to detect value of hemoglobin below 17gr/ dl and determine value of hemoglobin above 17 gr/dl. because it, do the testing the accuracy of the solution cupric sulfate in detecting and eliminating donor with value of hemoglobin above 17gr/dl with the test of 35 samples. aims: to determine accuracy and effectiveness by blood donors unit in indonesia red cross to use cuso4 solution with specific weight 1.062 in to detect and elimination value of hemoglobin donors above 17 gr/dl. methods: used the method cyanmethemoglobin and cuso4 (y) 1.062 determination value of hemoglobin donor. test results were analyzed with spss software version 23 using nonparametric analysis wilcoxon test. results: this research testing the accuracy and effectiveness of using a cuso4 solution with specific weight (y): 1.062 in detecting and eliminating hemoglobin value donors above 17gr/dl. from data processing using spss with the wilcoxon test p value 0.02. summary/conclusions: it was found that the cuso4 solution (y): 1062 can detect hemoglobins value above 17gr/dl and more effective in checking the hemoglobin in blood donors. it can be seen from the data processing with spss version 23 with the wilcoxon test p value < 0.05. it is important to monitor the precise course by which repeated blood donation affects hb and the probability of low hb deferral. zinc protoporphyrin (zpp) is a functional indicator of body iron levels and is hypothesized to predict hb levels among blood donors. advanced statistical methods are necessary to properly analyze the longitudinal associations between zpp and hb in data with repeated donations per donor. aims: to determine whether predictions of future hb levels using current hb levels can be improved by taking zpp levels into account, and to illustrate the use of statistical models for repeated measurements of blood donors. methods: we used data from the zpp and iron in the netherlands cohort (zinc) study. we identified previous zpp levels (log-transformed) as the main predictor and adjusted for previous hb level, age, day and time of donation, donation history, bmi, blood volume and blood pressure. we used linear mixed models, which take into account missing data in the outcome and associations between repeated measurements, to investigate the longitudinal association between previous zpp and current hb levels. the longitudinal analysis with linear mixed models was contrasted with a simpler analysis based on the area under the receiver-operating-characteristic (roc) curve for the probability of low hb deferral. results: in total, 8194 whole blood donors (20,864 whole-blood donations) were included in the zinc study, 63% being female donors. previous zpp showed a statistically significant association (p < 0.001) with hb levels in females, but the size of the association was quite small (regression coefficient, b = à0.17, 95% confidence interval à0.22 to à0.11). the same was true for males, but the size of the association was even smaller. blood volume and age for women were significant secondary predictor variables; blood volume, age and donation interval for men. by comparison, the roc analyses showed relatively larger, but less statistically significant predictive effects of zpp on hb. summary/conclusions: zpp is a statistically significant predictor of hb levels, but the size of the effect after adjustment for previous hb and other variables is small. the results cast doubt on whether zpp is an effective predictive marker for hb level and low hb deferral, and suggest that zpp should not be included in prediction models for hb levels. by properly adjusting for associations between repeated measurements and by using all available data, longitudinal models provide less biased and more precise estimates than simpler cross-sectional analyses. background: finnish red cross blood service (frcbs) is a national blood service and is responsible for all blood collection and component production in finland. the highest age for blood donors was 66 years until the end of year 2013 and since beginning of 2014 donors between 66 and 70 years have been able to donate blood. blood donation after the age 66 is possible if the donor has donated within the last 24 months. the upper age limit was raised up based on adverse event data from frcbs donors up to 66 years and on published data from other blood establishments. aims: the aim of this study is to find out if the new policy from 2013 with upper age limit of 71 years is safe. therefore donor adverse event data was analyzed in order to evaluate if the blood donors older than 66 years have more adverse events compared to other donors. the most common donor adverse event for donors over 66 years, haematoma, was registered 211 times (0.05%). in the other age groups haematoma was registered 4408 times (0.56%) and the difference between the oldest age group compared to all other donors was statistically not significant (chi2, p = 0.012). vvrs with loc were registered 21 times (0.05%), vvrs without loc 25 times (0.06), and the total number of all daes was 293 (0.65%) in the age group 66 years or older. the respective numbers in the other age group were: 1152, 0.15%; 11055, 1.41%; 17550 (2.23%). the number of vvrs with and without loc, and total number of all daes in the age group 66 years and older was smaller than in the other groups and the difference between these groups was statistical significant (chi2, p < 0.0001). summary/conclusions: donors over the age of 66 years have less donor adverse events than other age groups. decision to raise the age limit from 66 to 71 seems proven to be right as the older age group has even less donor adverse events than other donors. background: deep vein thrombosis (dvt) of the donor's phlebotomy arm is a rare, but serious complication of blood donation that needs to be recognised and managed appropriately in a timely manner. post donation dvt will be classed as a 'serious adverse events of donation' (saeds)these are events that either result in donor death, hospitalisation, intervention or significant symptoms persisting for more than one-year post donation. aims: to review cases of dvt post donation reported in uk in 2016-2018 years and identify any common themes for improving practice methods: all data relating to saeds from the four uk blood services reported to shot in the last 3 years (2016-2018 inclusive) were reviewed to look for reports of dvt post donation results: a total of 135 saeds were reported in uk from approximately 5.8 million donations (whole blood and apheresis) collected during this period. three cases of upper limb dvt were reported during this time accounting for 2% of the saeds reported and rate of dvt of 1 in 1.9 million donations collected. -case 1: a regular male whole blood donor in his early 30s reported worsening arm pain 12 days following blood donation, had a painful venepuncture and a small bruise at site of donation. he was diagnosed to have an upper limb dvt extending to the subclavian and brachiocephalic vein and started on oral anticoagulants. no other contributory factor was obvious -case 2: a female donor in her mid-40s gave her sixth whole blood donation without event. 11 days after donation, she developed worsening arm pain in the donation arm and was diagnosed with an upper limb dvt and commenced oral anticoagulation. there was no other identifiable risk factor for the thrombosis -case 3: a female donor in her 20s developed pain, swelling, redness and itchiness in her donation arm and chest wall two days after donation. she also described prominent veins on the affected side compared to her other arm. she contacted the transfusion service one week after donation; by this time she was also breathless on minimal exertion. she was admitted to hospital and commenced on anticoagulant therapy. a diagnosis of dvt and associated pulmonary embolus was confirmed. the donor's only risk factor for thrombosis was use of the oral contraceptive pill. summary/conclusions: rare complications of blood donation, like dvt, can occur. superficial venous thrombosis may occasionally progress into the deeper veins of the donor's arm but dvt can also occur without signs and symptoms of superficial thrombosis. none of our patients had any overt evidence of superficial thrombosis. one patient in our series reported using oral contraceptive pill. no other risk factors for thrombosis was forthcoming. transfusion services should encourage donors to make early contact with the blood service if they experience arm complications so that they can be investigated and managed in a timely manner. staff dealing with such donors must recognise the possibility of this rare complication, explore other additional contributory factors and initiate prompt and appropriate management. background: voluntary blood donation is widely considered to be safe with very minimum chance of adverse reaction, which may occur during or after the end of phlebotomy procedure. aims: to find the adverse blood donor reaction among voluntary blood donors in tertiary care hospital in kathmandu methods: this is a prospective study done among voluntary blood donors at grande international hospital, kathmandu, nepal from february 2013 to march 2015. the outlines of reported and communicated adverse donor reaction were also collected after the blood donation from voluntary blood donors in different locations including outdoor and in-house blood donation drive results: in the present study 6,955 whole blood donors were included, during the period of 2 years, 105 (1.50%) adverse donor reactions were reported. majority 89 (84.76%) of adverse donor reactions were mild in nature such as, sweating; 27 (25.72%), light headedness; 19(18.09%), nausea and vomiting; 15(14.28), allergy and bruises; 11(10.47%), sore arm; 9(8.58%) and hematoma; 8(7.62%) while 16 (15.24%) were severe adverse reactions similarly, anaphylaxis; 11(10.49%), loss of consciousness; 3(2.85%) and convulsive syncope; 2(1.90%). markers of the adverse donor reaction were age, sex, pulse, weight, blood pressure and donation status. age and first time status were related with significantly higher risk of adverse reaction with 18-23 years old at higher risk compared to 24-55 years old. first time donors were at higher risk compared to repeated volunteer donors. summary/conclusions: the results of the study are helpful to identify and understand the complication of adverse donor reactions though the incidence of reactions in the blood donors is lower than in other studies. donor age and donation status were strong possibilities of complications. background: blood donors with pollen-induced allergy and asthma must often refrain from donation in pollen season despite medication, because of symptom severity or similarity to airways infection. extracts of the medicinal mushroom agaricus blazei murill (abm) given orally have been found to reduce ige anti-ovalbumin levels and ameliorate the skewed th1/th2 cytokine balance in mice sensitized to ovalbumin (takimoto, immunopharm immunotox, 2008; ellertsen & hetland, clin mol allergy, 2009). aims: the objective was now to examine whether supplementation with the abmbased extract that we used in the mouse model for allergy, could alleviate allergy and asthma in blood donors by reducing specific ige levels and basophil sensitization. methods: sixty donors at oslo blood bank with self-reported birch pollen allergy and/or asthma were recruited and randomized in a double-blinded, placebo-controlled study with oral supplementation for 7 weeks before the birch pollen season with the abm-based extract andosan tm (immunopharma, oslo, norway). this is a water extract of the bacidomycetes mushrooms abm (82%), hericeum erinaceus and grifola frondosa. the participants filled in questionnaires for allergic conjunctivitis & rhinitis, asthma and medication. serum ige (immunocap â , immunodiagnostics, sweden) and bet v 1-induced basophil activation in whole blood determined by cd63 expression in a flow cytometer (flow cast â , b€ uhlmann lab ag, switzerland), were analyzed before and after the pollen season. (trial record: nct03198455, clinical.trials.gov). results: there was significant reduction in allover allergy-related ailments and types of allergy medication used in the abm extract compared with placebo group during the pollen season and no side effects. also, abm treated asthmatics had fewer symptoms and used less medication than controls. in the abm group, serum levels of specific ige anti-bet v 1 and anti-t3, were significantly reduced during the pollen season as compared with levels in the placebo group. whereas the maximal allergen concentrations needed for eliciting basophil activation before the season changed significantly to lower concentrations (i.e. enhanced sensitization) after the season in the placebo group, these concentrations remained similar in the group given the mushroom extract. summary/conclusions: oral pre-seasonal supplementation with an abm-based extract for 2 months reduced general allergy ailments, asthma symptoms and medication in blood donors with birch pollen-induced allergy and asthma during the pollen season. this was due to reduced specific ige levels and basophils rendered less sensitive to allergen activation. the study suggests that supplementation with abm mushroom extract can have prophylactic effect on aeroallergen-induced allergy and asthma in blood donors. it may therefore reduce such ailments in affected blood donors and impact blood donations in the pollen season. results: dhv started in 20/9/2014. the data presented in this abstract is till 28/2/ 2019. data is collected from total of 114475 blood donors (86.67% male donors and 13.33% female donors). repeat donors accounted for 59.34% against 40.66% of first time donors. of the total number of donor adverse events recorded, 2.73% (2633) reported for male donors and 6.97% (1063) for female donors when the donor adverse events stratified age-wise, the highest incidence reported in age group 18-24 years (male 5.45% and female 13%). among age group 25-40 years, (male 2.4% and female 4.02%), whereas in age group 41-65 years, (male 1.52% and female 2.11%) data analysis of total 6226 reported and registered donor adverse events, are categorized as hyperventilation (864), sweating (1458), dizziness (pre-syncopal 3160), loss of consciousness (416), vomiting (108), convulsions (220), hematomas with re-bleed (255), nerve irritation (8) and off-site reactions (333). many donors showed multiple forms of reactions. summary/conclusions: evaluation of donor side effects helps to improve donation process and donor compliance. most frequently recorded reaction remains dizziness (pre-syncopal). our donor vigilance data show reactions occurred more frequently in younger age, female and first time donors. repeat donation and age are predictors for low rates of adverse events. participation in dhv implies an effort to improve donor care and safety infrastructure and a desire for national and international comparisons to determine best practices and also to look into effectiveness of risk reduction strategies and follow-up trends. pre-donation hydration was implemented as an interventional tool to test the effects of hydration on pre-syncopal reactions to blood donation, specifically targeting those at highest risk such as female, first-time, high school donors. the results are awaited. background: descriptions of deferral categories and a knowledge in the percentage of deferrals in each category are of value in formulating recruitment and retention strategies. this can also help in planning more efficient recruitment strategies and thereby assist in reducing the shortage in blood supply. aims: the aim of the study is to categorize all donors who were deferred during medical checkup and find out the donor deferral rate in dubai blood donation center from january 1 st 2016 to december 31 st 2018 and also to find out whether there is any yearly or seasonal trend in any of the categories of deferral criteria's which can aid in forecasting and managing donor pool. methods: a retrospective study of donors deferred during last three years from january 1 st 2016 to december 31 st 2018 was done in dubai blood donation centre. the donors deferred during pre-donation medical check-up were categorized into 8 categories including low hemoglobin, high and low bp, intake of antibiotic, fever and flu, taking other medications, travel history etc. the deferrals were analyzed monthly and yearly and then were compiled to find any yearly trend or seasonal trend in the donor deferral rate in any of the categories. the data were analyzed using the spss software and a p value of < 0.05 was considered significant. the assessment of donor suitability is in accordance with aabb standards and is consistently applied in every blood donation setting on each occasion of donation to all blood donors. results: during this study, 193,228 donors were registered from january 1 st 2016 to december 31 st 2018 and 41,037 (21.24%) donors were deferred. the common reasons of deferral were low hb, high bp, travel history, intake of antibiotics and cough/flu symptoms. there was a significant decrease in deferral rate from 24.07% (15302/63563) in 2016 to 20.79% (13180/63394)in 2017 and further to 18.74% (12419/66271) in 2018 (p < 0.05). the specific deferral rate due to low hb also significantly (p decreased during these three years (86/1000 in 2016, 68/1000 in 2017, 58/1000 in 2018), though no change was seen in the deferral due to other reasons. the reduction in the rate of deferral due to low hemoglobin may-be linked to the change in the staff performing the hemoglobin testing in dbdc (nurses instead of phlebotomist were assigned to perform hb estimation of donors). there was a seasonal variation in the deferral rate in all the three years-lowest in june (6/1000) and then increasing with a peak in october (19/1000) and plateauing till january. this pattern of deferral corroborated with the rate of deferral due to flu/fever and cough and antibiotics with an average of 4/1000 in june and increasing to 10/1000 in october (p < 0.05). summary/conclusions: staff competency is pertinent in accurately deferring donors. there is also a significant seasonal pattern in flu/fever and intake of antibiotics deferral rate that is reflected in the total donor deferral pattern. seasonal variation of specific category of donor deferral should be taken into account for donor recruitment and retention efforts. background: west nile virus (wnv) is a mosquito transmissible flavivirus. it has been shown (vogels 2017 thesis) that the common mosquito in the netherlands can transmit wnv in laboratory circumstances but presently does not lead to effective transmission. however, the number of outbreaks of wnv is increasing and moving from the eastern and southern european borders towards the traditionally more colder western and northern parts of europe. in order to prevent wnv transmission to blood transfusion recipients, dutch donors that travelled to regions with wnv risk are deferred for a period of 28 days for whole blood, platelet donations and quarantine plasma in order to exclude potentially infected asymptomatic donors. aims: to assess numbers of dutch donors who are deferred for travelling to wnv risk areas within europe, and the return after onsite and offsite deferrals of donors. methods: data from 2015 to 2018 on donation attempts and deferral were retrieved from eprogesa, the blood bank information system. onsite deferral is defined as a donation that was attempted in the deferral period or in the 7 days prior to deferral, all other deferrals are considered as offsite. a generalized estimating equation model was used to assess the association between onsite versus offsite wnv risk deferrals in 2015-2016 and subsequent return rates within two years (after which a donor is inactive according to domaine). results: in 2015-2018, 16,400 donation attempts led to onsite deferral for wnv risk; 87% at whole blood donation attempts, 13% at new donor examinations. in total 19,624 offsite deferrals could not be traced directly to a donation, but based on the next donation more than 90% were probably whole blood donors. the number of deferrals peaks each year during august, the major holiday period in the netherlands, and increased from 920 in august 2015 to 1711 in august 2018. this increase is probably caused by the expansion of wnv risk regions. the return rate of wnv deferred whole blood donors is slightly lower than for donors who are not deferred (92% versus 95%); for wnv deferred new donors the return rate is 75% (versus 87% for no deferral). thus wnv deferral resulted in approximately 400-500 extra lapsing donors during these 2 years. however wnv deferred donors, that are older (odds ratio (or) 1.03; 95% confidence interval (95% ci) 1.02-1.03), of male sex (or 1.16; 95% ci 1.03-1.31) and whole blood donors as opposed to new donors (or 2.01; 95% ci 1.70-2.38) were more likely to return to donate. there was no difference in return rate by offsite and onsite deferral. summary/conclusions: travel-related wnv deferrals are increasing with expanding risk regions, especially in the holiday season where the availability of donors is already low. although the numbers of donors who are permanently lost after wnv deferral are limited, the increasing numbers of lost donations make it important to consider alternatives to donor deferral such as wnv nat testing. background: low haemoglobin due to iron deficiency is increasingly recognized as a serious problem in many blood centers. donor education, iron supplementation, ferritin monitoring, and lengthening of inter-donation interval are currently the main mitigation measures. however, a number of factors in particular donor knowledge could impact their success. locally, iron supplementation programme was implemented since 2012 with target group of donors who have given blood within the last six months. aims: here we look at an online donor survey to gain insight on their view of the programme and knowledge. methods: donors with successful blood donation in the past six months would be given 14 days of one tablet of iron supplementation (100 mg elemental iron) since 2012. an electronic questionnaire was sent to blood donors in 2017 to assess their view on the programme and knowledge which focused on iron store and absorption, compliance and any side effects occurred. results: 3212 donors (male to female was 1:1.9) replied to the questionnaire. of them, 594 received iron supplementation (male to female was 1:1.6). most of the respondents (94%) had one or more donations in the preceding 12 months. of the 594 donors received iron tablets, only 246 (41%) took all; 69 (12%) took more than 50% but not all; 130 (22%) took some but less than 50% and 149 (25%) did not take any. gastrointestinal upset was reported in 113 (25%) donors and constipation seen in 193 (43%) among those who took at least some of the iron supplementation. most respondents answered correctly to the questions on the knowledge on iron store and absorption. when comparing those with better compliance (took more than 50%) to those who did not (took less than 50%), significantly more donors in the former knew vitamin c could enhance iron absorption (p < 0.05). on the other hand, no difference was seen when they were asked if 1) iron can only be absorbed from meat; 2) tea and coffee consumed during meal can enhance iron absorption; 3) everyone can take iron supplementation on their own; and 4) iron store in male is always more than female. summary/conclusions: the results suggested that there is definitely more room to enhance the blood donors' knowledge on iron store and absorption in order to improve the effectiveness of iron supplementation programme. besides, the side effects reported by the donors could be an important limiting factor that better alternatives should be explored and considered. background: vasovagal reactions (vvr) are a well-established deterrent to donor return. however, the correspondence between vvr experience and donor lapse is not perfect. in australia, for example, vvrs only reduce two-year return rates by 27% for whole blood donors and 22% for plasma donors. the elements of a vvr and the donor's interpretation of this event that protect against or encourage lapse have not yet been identified. aims: in this study we explored the views of donors on donating following a vvr, with a particular interest in their emotional reaction to the vvr, their understanding of what caused the reaction, and their intentions to return. methods: semi-structured telephone interviews were conducted with 36 whole blood and plasma donors who had a recent vvr experience. data were analysed using the framework approach. results: donors are generally motivated to give blood to help others and to positively impact on those in their communities. they anticipate feeling good after their donation but in contrast, for many, a vvr leaves them feeling anxious, embarrassed, and disappointed. for donors, the experience of a vvr negatively influences their perceived ability to donate successfully, and many fear it will happen again. however, this effect appears minimised among donors who at least partially attributed their reaction to their own behaviour, such as poor hydration. for donors already juggling multiple demands, a vvr may tip the balance with donating becoming too much of an effort and perceived risk. however, donors appeared more confident to return if they felt supported by staff or if they could donate with family or friends. summary/conclusions: this study provides valuable insight into the vvr experience, which will aid in the improvement of donor safety and retention. the findings highlight the need to improve communication at the time of and following a vvr, to educate donors on how to reduce their vvr risk, and to intervene to help donors maintain their perceived ability to give blood in order to maximise retention following a vvr. background: frequent blood donation depletes the iron stores of blood donors. iron depletion might have negative effects on the health of the general population, but its effect on the blood donor population is not well known. aims: to investigate the iron status of finnish blood donor population and how it relates to donor health, the finnish red cross blood service set up the findonor 10,000 study in 2015. we investigated whether there were changes in donors' selfrated health and if these possible changes could be associated with differences in iron biomarkers (ferritin and soluble transferrin receptor -stfr) or hemoglobin levels during the first study visit. methods: participants were recruited in three donation sites in the capital region of finland between may 2015 and december 2017. participants filled out an electronic questionnaire about their health and lifestyle at the donation site during their enrollment visit. participants were asked by letter to fill out the same questionnaire electronically during the summer 2018. we included the 1435 participants (596 men and 480 premenopausal and 359 postmenopausal women) who completed both health questionnaires. to evaluate self-rated health we used the well-validated single question: "how would you rate your health in general?". participants were able to evaluate their health status on a five-point scale: excellent, very good, good, moderate, and poor. iron biomarkers and venous hemoglobin were measured from blood samples collected at the first study visit. we first computed the odds-ratios of reporting poorer health depending on demographic group. we then compared iron biomarker and hemoglobin levels between donors who reported improved, similar or poorer health rating. results: donors who rated their health in the first questionnaire as moderate (n = 31), good (n = 483), very good (n = 684) or excellent (n = 237) health tended to report improved (58%), similar (63%), similar (55%) or poorer (55%) health ratings respectively in the second questionnaire. pre-menopausal women reported their health poorer in the second questionnaire compared to the first questionnaire more often than post-menopausal women (pre-menopausal 51%, post-menopausal women 38%), or = 1.37 95% ci 1.02-1.85). there were no differences between other groups. there were no significant differences in iron biomarkers levels (ferritin and stfr) or hemoglobin levels between donors whose health ratings were improved, similar or poorer. summary/conclusions: in this cohort, pre-menopausal women rated their health poorer at the end than at the beginning of the study more often than post-menopausal women. no association was found between changes in self-rated health and iron levels (ferritin, stfr) or hemoglobin levels. further studies about the factors relating to blood donors' self-rated health need to be carried out. background: in recent years, the blood donation business has made great achievements, but it still cannot avoid the occurrence of adverse reactions to blood donation which not only brings certain obstacles to the blood donation work, but also affects the enthusiasm of blood donors. aims: to understood the causes and other relevant factors of adverse reaction among blood donors, the information of blood donors at dai autonomous prefecture of xishuangbanna were analyzed in 2018. methods: the data of volunteers from january to december 2018 were analyzed. the causes of adverse reactions were classified, and the incidence of adverse reactions was compared in terms of gender, frequency, age and blood type of blood donors. results: there were 9081 blood donors in 2018, 69 (0.76%) of whom had adverse reactions and 9 causes were induced, among which mental stress was the most common factor that accounted for 78.26% (54 cases). there was no significant difference in the incidence of adverse reactions between men and female (p > 0.05). from the frequency of blood donation, the incidence in the first donor was significantly higher than that in the second donor (p < 0.01). when it comes to age, the incidence was different and the 18-25 age group was the highest (1.61%). among different blood group donations, there was no significant difference (p > 0.05). summary/conclusions: adverse reactions of blood donation is closely related to the psychological state and age of the blood donors. the staff of the blood center should further optimize the service, strengthen the communication and publicize the knowledge of blood donation. the ultimate goal is to increase the blood donation rate on the basis of reducing adverse reactions. background: blood loss due to repeated blood donation can lead to iron deficiency or anemia, but currently there is no management plan for the prevention of iron deficiency in korean blood donors. female and male donors are required to wait at least 8 weeks between blood donations in korea, which is the shortest period among all northeast asian countries. female and male donors are allowed to donate whole blood up to five times per year and platelets up to 24 times per year (if spaced more than 14 days apart for the latter) due to the chronic blood supply shortage. these facts induce concern about the impact of blood donations on the donors' iron status. aims: this study aimed to evaluate the effect of oral iron supplementation in repeat donors based solely on donation history. methods: the high-risk group included male donors with ≥4 whole blood donations or 16 plasmapheresis or plateletpheresis donations, and female donors with ≥3 whole blood donations or 12 component donations, both within the previous year. the control group consisted of first-time or reactivated (ft-ra) donors who had no history of blood donation in the past 2 years. the hemoglobin (hb) level, ferritin level, total iron binding capacity (tibc), transferrin saturation, and soluble transferrin receptor (stfr) of repeat donors at high risk for iron deficiency were compared to those of ft-ra donors. iron deficient erythropoiesis (ide) is defined as present if the log of the ratio of soluble transferrin receptor to ferritin was ≥ 2.07. the repeat donors took iron supplements for 4 weeks and the same tests were repeated after 2 and 4 weeks to evaluate their effects and the side effect and compliance was assessed. results: a total of 53 male and 57 female repeat donors were recruited, and 30 each male and female ft-ra donors were recruited to the control group. after 4week iron supplementation, among male donors, the prevalence of: low hb level (<13.0 g/dl) decreased from 24.5% to 1.9%; low ferritin level (<15.0 ng/ml) decreased from 58.5% to 3.8%; high tibc level (>380 lg/dl) decreased from 66.0% to 37.5%; low transferrin saturation (<20.0%) decreased from 58.5% to 35.4%; and ide (stfr/ferritin ≥ 2.07) decreased from 28.3% to 2.3%. among female donors, the percentage of: low hb level (<12.0 g/dl) decreased from 43.9% to 9.8%; low ferritin level (<15.0 ng/ml) decreased from 78.9% to 11.8%; high tibc level (>380 lg/dl) decreased from 68.4% to 24.5%; low transferrin saturation level (<20.0%) decreased from 70.2% to 22.4%; and ide (stfr/ferritin ≥ 2.07) decreased from 94.7% to 44.4%. in total, 15 male (28.3%) and 29 female (56.9%) blood donors reported undesirable side effects related to iron supplementation. a total of 38 male (71.7%) and 36 female (70.8%) blood donors were administered iron supplementations for 28 days. 89 participants (85.6%) answered that they were willing to take a complimentary iron supplementation. summary/conclusions: ferritin level, considered a reliable indicator of iron status, increased and ide decreased significantly after iron supplementation in female donor group, but not in male donor group, compared to the ferritin levels and ide of control donors. iron supplementation in repeat donors at a high risk of iron deficiency was shown to reduce their risk of iron deficiency or anemia irrespective of gender; however, 4-week oral iron supplement was not enough to restore iron storage level in the male donor group. background: c-reactive protein (crp) is an acute-phase protein and a non-specific maker of inflammation and tissue damage produced by the liver. several prospective epidemiologic studies have demonstrated that high-sensitivity c-reactive protein (hs-crp) is a predictor of future coronary events among apparently healthy men and women, hs-crp level greater than 3 mg/l has been independently associated with a 60% excess risk in incident of coronary heart disease (chd) as compared with levels less than 1 mg/l. frequent blood donation has been associated with a lower incidence of coronary artery disease (cad); however, there is a dearth of information on serum levels of crp in the nigerian donor population. aims: to investigate whether regular blood donation is associated with lower serum hs-crp level in nigerian blood donors. methods: a descriptive cross-sectional study carried out to measure serum levels of high sensitive c-reactive protein (hs-crp) and ferritin among blood donors attending the donors' clinic in lagos university teaching hospital (luth). subjects who did not meet criteria for blood donation were excluded. additional data on sociodemographic characteristics was collected using interviewer-administered questionnaire. serum ferritin was analysed using chemiluminescent microparticle immunoassay performed on the abbott architect ci4100 (abbott laboratories, abbott park, il, usa). serum concentration of hscrp was estimated by immunoturbidimetry method using analytical kits from erba diagnostics mannheim gmbh in semi-autoanalyzer (xl 180, erba mannheim). data was analysed using stata version 15 (stata corp) statistical software. results: in total of 313 blood donors, 278(90.8%) were males and 28(9.2%) were females, the mean age was 32.3 ae 9.3 years. two hundred and thirty four (74.8%) were first time donors and 79(25.2%) were regular donors, serum levels of hs-crp was slightly higher in regular donors compared to first time donors (0.95 ae 3.7 vs 0.35 ae 1.7 mg/l, p = 0.06) though the difference was not significant. serum levels of ferritin was significantly higher in first time donors compared to regular donors (87.56 ae 23.9 vs 46.44 ae 31.4 ng/ml, p = 0.02). interestingly, levels of serum hs-crp were significantly higher in male than female population (0.52 ae 2.4 vs 0.17 ae 0.2 mg/l, p < 0.03) and smokers than non-smokers (1.4 ae 4.6 mg/l vs 0.4 ae 1.9 mg/l, p = 0.02). correlation analysis showed no correlation between serum hscrp and serum ferritin levels in both categories of donors while there was a weak positive correlation between hs-crp levels and white blood cells among the first time donors. summary/conclusions: this present study did not reveal any decrease in baseline levels of serum hs-crp with regular blood donation; smoking status and gender were however associated with an increase in baseline hscrp. this finding suggests that hs-crp level might not be a useful marker of future coronary events in healthy blood donors in nigeria. background: because the blood donation removes 250 mg of iron from the donor, iron deficiency, frequently occurs in regular blood donors leading at a long term to the anemia. aims: to determine the effect of blood donations on ferritin levels in regular blood donors. methods: all prospective donors have been submitted to a physical examination and a health history assessment intended to ensure that the prospective donor is in a good general health and eligible to donate blood. the acceptance criteria are: • hemoglobin > or = 13.5 g/dl for male and > or = 12.5 g/dl for female • inter donation interval = 56 days • 5 donations/year for male and 3/year for female all eligible donors and deferred donors for all reasons except for low hemoglobin who accepted to enroll in this study and signed a consent. in addition to the medical exam, two samples have been collected one for cbc and another for ferritin. donation history, sex, age and weight have been documented. results: 1056 first time and regular donors accepted to enroll in this study. only 38 female donors (3.6%) participated to this study. 11.3% of the participants were first time donor. 21% of male and 26% of female frequent donors are iron deficient 173 out of 1018 male blood donors were iron deficient (17%) with serum ferritin < 30 ng/ml. 98.8% were repeat donors. 7 out of 38 female donors were iron deficient (18.4%) with serum ferritin < 13 ng/ ml, all were repeat donors. 19.2% of repeat donors were iron deficient 3/180 of the deplete donors were first time donors summary/conclusions: frequent blood donors have higher prevalence of iron deficiency than first time donors. female donors have a slightly higher prevalence of iron deficiency than male donors. prevalence of iron deficiency in abu dhabi donor population is lower than the published data. changes need to be done on: increase inter donation interval or restrict the total number of allowable donations in a 12-month period for whole blood and red cells modifying donor hemoglobin requirements testing for serum ferritin iron supplementation donor education abstract withdrawn. background: haemovigilance procedures aim to guarantee not only the safety of the recipients of blood and its components but the safety of the donors as well. every adverse reaction that occurs during the donation of blood or its components can potentially be a threat to the health of the donor which can subsequently lead to the decision of the donor to resign from donating blood. aims: the aim is to analyse the type and the frequency of occurrence of adverse reactions among the donors donating blood or its components independently of the method of the donation. methods: we have analysed the number of collected donations and the number of adverse reactions in the years 2014-2018 in the group donors of aged 18-24. we have specified following adverse reactions: vasovagal response without fainting, vasovagal response with fainting, vascular reactions (bruises) and other (e.g. allergic reaction to the anticoagulant, loss of blood pressure due to hypovolemia). the analysis was made using data obtained from computer system blood bank which is in operation in blood center in pozna n, poland. results: in years 2014-2018 the total number of 1904 adverse reactions among the donors was recorded which is 0.39% of the total number of collected donations. 50% of the adverse reactions occurred in the group of donors aged 18-24. vasovagal response without fainting was the most common adverse reaction in the total number of reactions and totalled 50.2% of all adverse reactions. in the group of donors ages 18-24 it totalled 27% of all adverse reactions. the second most common type of adverse reactions was vasovagal syncope that totalled 29.8%, in the analysed group of donors 15.96%. vascular reactions (bruises) totalled 10.2% of all adverse reaction, in the analysed group 5.6%. the remaining adverse reactions totalled 9.8%. summary/conclusions: 1. vasovagal reactions (with and without fainting) were proved to be 2 most common adverse reactions in the group of donors aged 18-24 i.e. in the groups of donors just starting to donate blood. it seems reasonable to continue with further research into the reasons for the occurrence of this psychosomatic reactions. 2. it seems beneficial to provide constant educational activities of young donors regarding the preparation for the process of donation of blood and its components (proper nourishment, hydration as well as planning the time for scheduled donation long enough for a safe and pleasant procedure. 3. it seems beneficial to provide constant training for the medical staff involved in the process of donation regarding active observation of donors, proper conduct in the situation when the adverse reactions occur during the blood donation, ways to minimize the fear of donors, effective communication with the donors (explaining the process of blood donation, proper behaviour after the donation e.g. avoiding physical exercise or straining the arm). blood products -blood processing, storage and release background: the accumulation of microvesicles (mvs) in rbc concentrates during storage may be responsible for clinical symptoms such as inflammation, coagulation, and immunization. aims: our aims was to determine whether any of cd molecules responsible for important functions are present on the microvesicles, and if their expression level is dependent on the storage period of rbc units. additionally, by using cytometric analysis and phagocytosis visualization in a confocal microscope, we examined the interactions of donor monocytes with erythrocyte microvesicles, depending on their time of storage. methods: erythrocyte microvesicles were isolated from "fresh" (2 nd day) and "old" (42 nd day) stored rbc units. qualitative and quantitative cytometric analysis of these membrane structures was performed using the annexin v-fitc, anti-cd235a-pe antibody, and calibrated beads. the microvesicles were also visualized under a confocal microscope. the expression of the molecules cd235a, cd44, cd47, cd55, cd59, and of phosphatidylserine was analysed using flow cytometry. measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. results: the analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0.5 lm in the "fresh" and "old" blood samples. we observed a statistically significant increase in the number of microvesicles in the "old" units (201 ae 139 mvs per 1 ll), as compared to the microvesicles in the "fresh" (67 ae 53 mvs per 1 ll). at day 2, the microvesicles had elevated expression levels of cd47 and reduced expression levels of phosphatidylserine. significant changes were also observed in the case of cd55 and cd59 molecules. the expression of these molecules of vesicles isolated from "fresh" rbcs was lower than in the case of 42-day vesicles. the phagocytosis index was significantly higher (28.44%) for the microvesicles isolated from 42-day stored rbcs than for microvesicles from the 2background: platelet concentrates (pcs) are conventionally stored at room temperature with a limited shelf-life of 5-7 days. alternative storage methods, such as cold storage and cryopreservation are attractive options due to the potential for extended storage, reduced bacterial growth and improved hemostatic function. cryopreservation of human pcs has been associated with formation of more microparticles and elevated procoagulant activity compared to liquid-stored (room temperature-and cold-stored) pcs. microparticles are submicron plasma membrane particles that have been postulated as potential mediators of adverse transfusion outcomes. similarities in the size and storage-related changes up to 7 days suggest that sheep may be a suitable model in which to investigate the effects of pc transfusion. previous research has established that room temperature stored sheep pcs contain fewer microparticles than human pcs. however, nothing is known of the effect of other storage conditions. aims: this study aimed to determine whether cold storage and cryopreservation contribute to variation in concentration and size of sheep platelet derived microparticles compared to conventionally stored sheep pcs. methods: sheep buffy coat derived pcs in 30% plasma/70% ssp+ were prepared with minor modifications to standard procedures for preparation of human pcs. sheep pcs were split into 3 units (n = 6 of each) on day 2 and stored either at room temperature (rt; 20-24°c with agitation) for 5 days, cold stored for 21 days (2-6°c no agitation) or cryopreserved (à80°c with the addition of 5-6% dimethyl sulfoxide) for 33-109 days and sampled post-thaw. platelet supernatant, prepared by double centrifugation, was stored at à80°c. the mean size and concentration of microparticles were measured using nanosight ns300 nanoparticle tracking analysis system (malvern instrument). results are mean ae standard deviation. storage associated changes overtime were determined using a one-way analysis of variance with bonferroni's post-test. paired t-tests were applied to determine the effect of cryopreservation. a p-value of < 0.05 was considered significant. results: at day 2, sheep pcs had a microparticle concentration of 3.77 9 10 10 ae0.84 9 10 10 microparticles/ml with a mean size of 156.6 ae16.7 nm. storage duration at rt sheep pcs was not associated with significant changes to microparticle concentration or size. cryopreservation of sheep pcs significantly increased the concentration (4.39 9 10 10 ae0.81 9 10 10 microparticles/ ml; p = 0.0087) and the mean size (170.9ae 20.5 nm; p = 0.0008) of microparticles post-thaw. the mean size and concentration of microparticles in the cold-stored pcs at day 21 was comparable to room temperature pcs stored for 5 days (165.4 ae17.5 nm vs. 163.6 ae20.9 nm; p = 0.4081 and 4.11 9 10 10 ae 0.54 9 10 10 microparticles/ml vs. 3.82 9 10 10 ae0.71 9 10 10 microparticles/ml; p = 0.16 respectively). summary/conclusions: cold storage of sheep pcs did not impact formation of microparticles over the 21 days storage period; however, cryopreservation increased microparticle concentration and the size post-thaw. further investigation is required to determine whether these findings are influence hemostatic function. a pre-clinical sheep model of cold-stored and cryopreserved pc transfusions can facilitate mechanistic studies and complement clinical trials. background: during storage, the properties of rbc in storage solution change ("storage lesion"). for instance, ph, atp and 2,3-dpg concentrations decrease upon prolonged storage. these changes can affect oxygen delivery by the cells. the capacity to deliver oxygen is defined as p50: the oxygen tension (po2) at which 50% of the hemoglobin is saturated with o2. an oxygen dissociation curve (odc) represents the non-linear relationship between saturated hemoglobin and po2. this relationship is dependent on temperature, ph, pco2 and 2,3-dpg. due to changes in these factors, the curve will shift along the x-axis. in whole blood, p50 is at a po2 of about 26 mm hg. not much is known about p50 of rbcs in storage solution, and the changes during storage. aims: to determine the oxygen dissociation of rbcs stored in standard red cell additive solution sagm and in pagggm (an experimental red cell additive solution, transfusion. 2010;50:2386-92). methods: rbcs were prepared in sagm (n = 3) or pagggm (n = 3). pagggm is designed to better maintain both atp and 2,3-dpg during storage. rbcs were stored at 2-6°c and sampled on day 1, 35 and 56 for (internal) ph, atp, 2,3-dpg and p50. p50 was determined by hemox analyzer (tcs scientific corp.). the principle of the hemox is based on the measurement of spectrophotometric properties of hemoglobin at different oxygen pressure. rbc samples were brought from oxygen-rich environment to oxygen-poor environment (2%) using n2 gas. p50 was determined from the obtained odc. results: the whole storage period, ph i of pagggm-rbcs was higher compared to sagm-rbcs. 2,3-dpg content of sagm-rbcs decreased during storage and was below the detection limit after day 14. 2,3-dpg content of the pagggm-rbcs increased the first days of storage and slowly decreased from day 7 on. at day 56, pagggm-rbcs still contained 2.3-dpg (2.2 lmol/g hb). p50 values decreased during storage from 26 mmhg at day 1 to 20 mmhg at day 56 for sagm-rbc and from 31 mmhg to 26 mmhg for pagggm-rbc. p50 values of pagggm-rbcs were higher during the entire storage period. summary/conclusions: during storage, the p50 decreased in all rbcs. the p50 was higher for the pagggm-rbcs during the whole storage period. the higher p50 in pagggm-rbcs seems to correlate with the higher 2,3-dpg content in these cells. background: in belgium 100% of the platelets are pathogen inactivated (pi) and legislation requires a minimum platelet content of 3.0 9 10 11 per platelet concentrates (pc). therefore routine pools are produced with 6 buffy-coats (bc). facing increased demand of pc and stable to slightly declining red blood cells (rbc) demand, production of whole blood (wb) derived platelets must be adapted to switch flexibly from 6 to 5 bc per pool. this dual pooling strategy should allow alignment between wb collection forecast, pc inventory, pc demand and pc production. aims: first develop a pooling procedure with 5 bc and 250 ml platelets additive solution (pas) instead of 280 ml for 6 bc, without changing the settings of our wb separators and platelets separators. maintain a content of ≥ 3.0 9 10 11 platelets with a ratio plasma/pas between 32 to 47% required for pi. after validation, deploy a dual pooling strategy (5 or 6 bc/pool). methods: wb is collected with top and bottom kit (composelect; fresenius kabi) and separated (macopress; macopharma) to produce 44 ml bc with 40% haematocrit (htc) and > 90% platelets recovery with average platelets content of 1 9 10 11 . 5 random bc are pooled with 250 ml or 6 bc are pooled with 280 ml of pas-e, platelets are then extracted on tacsi pl (terumo bct) and pc are treated for pi (intercept blood system; cerus). each pc is sampled and platelet content is determined (abx pentra xl 80; horiba). results: during the study 45594 bc were processed into 8225 pools (3756 (45.7%) with 5 bc and 4469 (54.3%) with 6 bc). before tacsi separation, bc mixture with pas-e had volumes of 421 ae 9 ml (5 bc) and 491 ae 8 ml (6 bc) with respectively htc of 19 ae 1% and 20 ae 2%. the plasma/ pas ratio was 37 ae 1% in both cases. tacsi separation was performed with one same program for both types of pools. after pi, platelets content of the pools was 3.8 9 10 11 ae 0.5 with 5 bc and 4.7 9 10 11 ae 0.5 with 6 bc (average ae standard deviation). pools below the limit of < 3.0 9 10 11 were 139/3756 (3.7%) with 5 bc and 1/4469 (0.02%) with 6 bc. the platelets concentrations (10 3 /ll) were 1395 ae 167 (5 bc) and 1491 ae 155 (6 bc). platelets recovery was 85% ae5 for 5 bc and 86% ae 6 for 6 bc. summary/conclusions: 45594 bc could theoretically produce 7599 pools of 6 bc or 9118 pools of 5 bc. this means a maximum potential gain of + 20% pc. in practice during shortage periods we switched from 6 to 5 bc when dictated by the actual inventory levels and hospital needs. the advantage of this dual pooling strategy was a gain in production capacity to cover these shortage periods (626 pc, +8%). the disadvantage of pooling randomly 5 bc is that 139 pools contained less than 3.0 9 10 11 platelets per pool potentially limiting their usage to low weight or paediatric patients. a preselection of the bc based on platelet count could optimize the 5 bc pooling procedure. background: apheresis-derived platelet concentrates (apcs) is a standard medical therapy indispensable to contrast bleeding or hemorrhage. however, bacterial infection caused by storage at room temperature (rt) still remains the major drawback. recently, we showed that cold-stored apcs are associated with better plt functionality but with accelerated clearance (haematologica 2018, pmid: 30115655). cold-induced apoptosis was identified as a potential mechanism of the shorter plt survival aims: to investigate the protective effect of apoptotic inhibitors during cold storage of apcs methods: apcs were collected and stored at rt and 4°c in the presence or in the absence of caspase-3 inhibitor. the phosphatidylserine exposure and the mitochondrial membrane potential (mmp) (tetramethylrhodamine ethyl ester perchlorate [tmre ] staining) were measured using flow cytometry. the protein expression was quantified by western blot results: a higher expression of the apoptotic marker phosphatidylserine was detected in cold-stored apc compared to rt (% apoptotic events meanaesem: 13 ae 1% vs. 5 ae 1% p = 0.018). to verify if the apoptotic signal, observed with phosphatidylserine, specifically involved the intrinsic pathway, the mmp was analyzed as a marker of alive cells. interestingly, after cold storage a decrease of the mmp was observed compared to rt indicating the activation of the intrinsic pathway (mean fluorescence intensity tmre meanaesem: 6.13 ae 1.89 vs. 18.53 ae 3.64, p = 0.038). accordingly, a decrease of the procaspase-3 level after cold storage was detected by western blot analysis. however, when plts were stored in the presence of caspase-3 inhibitor a significant rescue of the cold-stored cells viability was observed (tmre staining: % alive cells meanaesem: 74 ae 3% vs. 22 ae 3%, caspase 3 inhibitor vs. ionomycin, p = 0.005). this indicates that the activation of the apoptotic pathway, induced during cold storage, can be prevented using caspase inhibitor summary/conclusions: our results show that the reduction of cold-stored plt viability can be prevented by a specific caspase inhibitor. consequently, cold storage, associated with a better plt functionality, may become an efficient strategy for apc storage in combination with apoptotic inhibitors background: gamma-irradiation is used to treat red blood cell (rbc) concentrates (rccs) for patients who are immunosuppressed. this treatment is known to damage rbcs and to increase storage lesions. one of the causes of the storage lesions is the presence of oxygen. several studies have shown, based on different strategies to reduce o 2 , a reduction of storage lesions related to metabolism, protein modifications and cell morphology. aims: the present research work investigated the effect of gamma-irradiation on rccs stored under normal condition and hypoxia/hypocapnia. methods: saturation of o 2 (so 2 )-and abo-matched rccs from whole blood donations, leukoreduced and prepared in paggsm (macopharma, france) were pooled and split in two identical rccs within 24 h post-donation. one bag (treated) was submitted to oxygen and carbon dioxide adsorption (oxygen reduction bag, hemanext, usa) for 3 h on an orbital shaker (50 rpm) at 22°cae2 and then transferred to a storage bag impermeable to gas. the other one (control) was left as it is. the two bags were then stored at 4°c. a g-irradiation treatment (25 gy, gammacell 3000 elan, theratronics) was applied at day 2 or 14 and the rccs (expiry dates at day 16 or day 28, respectively) were stored until day 43. hematological parameters, glycolytic metabolites, extracellular potassium level, antioxidant power, morphology and deformability were measured. results: starting so 2 values were of 63.7%ae18.4 (n = 12) in control and of 20.8%ae9.8 (n = 12) in treated bags, and reached 90.8%ae9.1 and 6.6%ae5.9 at day 43, respectively. as expected, an increase in glycolysis rate was observed during deoxygenation without any influence from the irradiation. potassium levels were identical in treated and control, and reached around 70 mm at expiry with an irradiation-dependent kinetic release. antioxidant power and deformability were identical in both conditions. no difference in hemolysis was observed after irradiation on day 2 and the values stayed equivalent through end of storage (at day 16, hemolysis (control) = 0.37%ae0.24, hemolysis (treated) = 0.34%ae 0.15, p-value > 0.9999). when irradiated at day 14, hemolysis was lower (p-value = 0.033) in treated rccs at the end of storage (day 28, 0.67%ae0.16) compared to control (1.06%ae0.33). seven days post-irradiation, two-third of the control rccs were above the limit of 0.8% whereas all the treated rccs remain below the limit. quantification of microvesicles and morphological analysis confirmed these data. summary/conclusions: the storage under hypoxia has a beneficial effect on rbc storage thanks to a decrease in o 2 content and to an improvement of metabolism. this benefit provided equivalent storage when rccs were irradiated at day 2 and was an advantage when irradiated at day 14. importantly, the results show that combining irradiation with hypoxia/hypocapnia retained the improved hemolysis profile of o 2 depleted rbc. in summary, the reduction of o 2 level in rccs enables a better storage of rcc when a late irradiation is applied. background: in vitro blood circuit machines require a constant monitoring of blood flow rate which have to be maintained at a constant value. also, measuring the hematocrit of flowing blood in such machines is essential for performing real-time diagnostics. recently, acoustophoresis has emerged has a promising blood separation technology capable of replacing centrifugation for the preparation of platelet concentrate. to avoid damaging blood cells, the technique is used without infusing pumps thus increasing the need of flow monitoring. however, acoustophoresis chips performs at low flow rates, outside the range of available commercial flow meters. in addition, hematocrit measurement is of a particular interest for acoustophoresis since it is a direct indicator of the separation efficiency. aims: in this study, we present a straightforward doppler ultrasound system designed for measuring blood flow rate and hematocrit in an acoustophoresis chip [bohec et al, platelets, 2017] . we show that the stability of the in vitro environment can be used to obtain high level of accuracy of the doppler method using a basic and low-cost experimental set-up. this improvement allows a precise measurement of flow rates as low as 0.5 ml/min in sub-millimeter tubing. furthermore we evaluate the capability of the system to measure hematocrit of human blood samples coming from different donors. methods: the experimental set-up was constituted of an ultrasonic continuous wave doppler probe mounted on a 3d printed support. the accuracy of flow rate measurements between 0.5 ml/min and 1.5 ml/min was evaluated as well as the optimal measurement time. for 4 different blood bags, the relationship linking the total energy of doppler signals and hematocrit was derived. hematocrit in a range under 8% was estimated from doppler signals for each blood bag. results: the system is able to acquire exploitable doppler signals for the whole flow rate and hematocrit range. flow rate estimation from the signals shows a high accuracy with a mean measurement error under 3% for a measurement time of 2s. the mean error is still under 5% for a measurement time of 0.5s. hematocrit estimation from doppler signals shows a good linear correlation with reference measurements for bags 2, 3 and 4. hematocrit estimation for bag 1 diverges from reference for values above 6%. summary/conclusions: the proposed doppler ultrasound system is capable of measuring low blood flow rate in narrow medical tubing with a high accuracy. it is particularly suited for an acoustophoresis device but the versatility of the system makes it easily applicable to any in vitro blood circuit. we furthermore demonstrated that the system can be used for measuring hematocrit under 8% without additional developments. this finds interesting applications in blood sorting technologies but also demonstrates that doppler ultrasound is a potential simple and low cost method for measuring hematocrit of flowing blood in vitro. background: hereditary hemochromatosis (hh) is the most common genetic disorder in populations of northern european descent manifesting with high levels of storage iron (ferritin) in blood and tissues. the standard treatment is serial therapeutic phlebotomy to decrease iron overload. the collected blood is frequently discarded but some blood banks allow "healthy" hh patients to donate blood for patient use. red cell concentrates from hh donors have been reported safe for transfusion, but little or no data is available on platelet concentrates from hh donors, including the potential contribution of surplus iron to the "platelet storage lesion". aims: the aim of this study was to compare platelet quality, activation and aggregation over seven-day storage in platelet-rich plasma from patients with newly diagnosed hh and from healthy controls. methods: whole blood (450 ml) was drawn into compoflow blood bags containing cpd and sag-m from 10 healthy controls and 10 newly diagnosed hh patients. platelet-rich plasma (prp) was prepared from whole blood and split into four compo-flex bags each containing 20 ml prp (range 270-320 9 10 9 platelets/l). platelet quality tests were performed on days 0, 1, 3, 5 and 7 of storage. platelet aggregation was tested using a chrono-log aggregometer and four agonists (adp, arachidonic acid, collagen, and epinephrine). platelet expression of cd41, cd42b, and cd62p was measured with flow cytometry while ph and metabolites were measured with a blood gas analyzer. scd40l and scd62p in the supernatant were quantified using enzyme-linked immunosorbent assays. results: both hh and control groups included 7 males and 3 females. the mean age was significantly lower in the control group, 35 years (22-55 years), than in the hh group, 55.7 years (29-77 years) (p = 0.004) while ferritin levels were significantly higher in hh patients (median 847.5, range 498-1889) than in controls (median 45.8 ng/ml, range 9.28-97 ng/ml) (p < 0.001). in the hh group, 5 each had the c282y/c282y and c282y/h63d genotypes. results of prp quality control tests were comparable between the two study groups over seven days of storage (p < 0.05) with the exception of glucose (higher in hh patients on all time points, p < 0.05). platelet aggregation and the expression of activation markers (cd62p and cd42b) on platelets and in the supernatant (scd62p and cd40l) were comparable between hh and control prp units over all seven days of storage. the analysis revealed comparable and expected alterations in metabolic and platelet activation markers over seven-day storage in both groups. ph increased, glucose decreased, and lactate increased over time while cd42b expression decreased and cd62p increased. platelet aggregation responses decreased during storage but to a varying degree depending on the agonist, however, the decrease was comparable in cases and controls. summary/conclusions: these results suggest that high iron stores in hh do not adversely affect the quality of platelet units produced from hh patients. furthermore, the data also suggest that blood from hh patients, including platelets, can be donated for patient use. background: platelets are often shipped over long distances from collection centres to blood processing centres and subsequently to hospitals. platelet agitation facilitates oxygen transfer, thus promoting aerobic metabolism, and maintaining platelet ph. during shipment, platelets cannot be agitated continuously, which may promote anaerobic metabolism. previous studies have examined the effects of prolonged periods without agitation on apheresis platelets collected in plasma, but not platelets in platelet additive solution (pas). it is therefore important to determine whether platelet quality and function are maintained during prolonged transport or hold time in a shipper. aims: the aim of this study was to evaluate the effects of prolonged storage without agitation on the in vitro quality of apheresis platelets in pas. methods: triple dose apheresis platelets (n = 16) were collected using a trima accel platform in 40% plasma/60% pas (ssp+). after resting for 1 h, platelets were split equally into three components, packed into a shipper and transported immediately to the blood centre. upon arrival, one of the platelet components was removed (<6 h; t0), and the others remained within the shipper, without agitation. the second component was removed at 6 h post-collection (t6), and the third was removed at 23.5 h post-collection (rounded up to 24 h; t24). platelets were tested on day 1, 5 and 7 post-collection and in vitro quality and function were monitored. data were analysed using a two-way repeated measures anova, where a p-value of < 0.01 was considered significant. results: platelets held without agitation for 24 h consumed significantly more glucose than those removed at 6 h or immediately upon arrival (p < 0.0001), even on day 1 post-collection. this was accompanied by increased lactate production (p < 0.0001), indicating increased anaerobic glycolysis. consequently, the ph was significantly lower in t24 platelets (p < 0.0001), and on average it was 0.4 ph units lower than in platelets held in the shipper for 6 h or less. however, the ph remained above 6.9 in all components. mean platelet volume was also reduced in t24 platelets (p < 0.0001), suggesting acceleration of the platelet storage lesion. phosphatidylserine exposure, surface expression of cd62p and microparticle generation were significantly higher in the t24 platelets throughout the storage period (all p < 0.0001), suggesting platelet activation. release of scd62p was also increased in t24 platelets (p = 0.0006), whereas extended storage in a shipper did not affect release of rantes (p = 0.4488). adp-induced activation of glycoprotein iib/iiia, measured by pac-1 binding, was decreased in t24 platelets (p < 0.0001), indicating reduced platelet responsiveness to agonist stimulation. additionally aggregation in response to collagen (p = 0.0001) and adp (p = 0.003) were significantly lower in t24 platelets, suggesting a decrement in platelet function after prolonged storage without agitation. summary/conclusions: significant in vitro changes were observed in platelets held without agitation for 24 h. these results suggest that the length of time that platelets are held in a shipper should be minimised where possible. background: the shelf-life for platelet products has been restricted to 5 days. this very limited window of time is intended to sustain the quality of platelet and to reduce the risk of bacterial growth. we have recently demonstrated that in suitable platelet bags, the platelet product quality remains high after 5 days of storage. this was proved by examining in vitro, the quality parameters of platelets such as platelet concentration, glucose, ldh, and ph (alexopoulos k. et al., haema, 9, 2018) . our new target is to extend this research in 2 extra days of storage. we also want to determine if there is any bacterial development in this period. aims: the goal is to investigate the capability of storage period for platelet units, from 5 to 7 days. methods: in this study, platelets were collected from 11 normal blood donors in the blood bank department of general hospital of patras "agios andreas". a total of 450 ae 10 ml of whole blood was drawn into triple cpd/sag-m top-top bags blood container systems, lmb technologie (gmbh). the platelet concentrates were prepared by platelet rich plasma (prp) method and then they were placed in a platelet incubator with agitator (helmer pc 1200). samples were drawn aseptically with a needless access coupler (cair-lgl) on days 1, 5, and 7. platelet count was done by ceeldyn ruby (abbott all data shown are reported as mean ae standard deviation (sd). the swirling effect remained positive (+) during the seven days storage period. the bacterial screening was found negative. summary/conclusions: platelet concentration in the bag remained constant between day 5 and day 7, maintaining platelet yield. the decrease in glucose and increase in lactate, along with the decreased ph, show that the platelets remain metabolically active between days 5 and 7 of storage. the ph remained well within the acceptable range. no bacterial contamination was reported. thus, we conclude that platelet concentrates in these specific bags may be used with an extended shelf life of 7 days. further studies are needed with other platelet bags to confirm our hypothesis. abstract withdrawn. aims: we introduce rt-dc as a fast, robust and unbiased quality control tool for pc, rcc and hpsc. utilizing the interdependency between cell deformation and the molecular state of the cytoskeleton, we demonstrate that rt-dc is capable to assess the quality of blood products. methods: by rt-dc we assessed: i) platelets after storage at 4°c or room temperature (rt) over 10 days for 4 apheresis pcs in addition to standard in vitro platelet function assays; ii). red blood cells before and after gamma irradiation in addition to hemolysis; and iii) hpsc after cryopreservation with 5% or 10% dmso in addition to cell count, and in vitro viability. in addition we compared the regeneration time of patients' platelets and leukocytes after transplantation of hpsc products containing either 5% or 10% dmso. results: for pcs standard quality assurance tests did not show a major difference between 4°c and room temperature storage while rt-dc showed a highly significant difference between both start conditions (day 1-7, p < 0.001 and day 10, p < 0.02). for red cells, we found by rt-dc no impact of gamma irradiation with 30 gy over the entire storage period of 42 days assessing 3 different rcc. for hpsc, rt-dc showed that cryopreservation in liquid nitrogen resulted in a significant increase in deformation (0.052 for 5% dmso versus 0.034 for the control without dmso; p < 0.00004). however, this did not differ to high extent whether 5% or 10% dmso were used for cryopreservation (0.035 and 0.041, respectively; p < 0.02). hpsc viability was lower after cryopreservation using 10% dmso in comparison to using 5% dmso. overall, blood cell regeneration is comparable between 5% and 10% dmso. summary/conclusions: studying platelet and red blood cell concentrates as well as hematopoietic stem cells under different, clinically relevant, storage conditions our results demonstrate that intrinsic material properties reveal insights into cell function and allow to predict cellular state in a robust way and using small sample volumes. in order to offer more flexibility to the production process, the storage of bcs overnight (16 h) has been validated in our blood center. aims: the aim of the study was to assess the platelet quality in platelet concentrates derived from overnight stored buffy coats. methods: whole blood collected at day 0 was separated into plasma, bc and red cell concentrates either at day 0 or at day 1. bcs were then stored until the pooling step at 20°c without agitation and pcs were prepared at day 1 by pooling 5 isogroup bcs. seven "16 h-pcs" were prepared from bcs stored for 16 h (whole blood separation at day 0) and six "90 min-pcs" from bcs stored for 90 min (whole blood separation at day 1). standard quality control measurements were performed during the process and the storage. in addition, the quality of the platelets into the prepared pcs was assessed throughout the period of storage by measuring the hypotonic shock response (hsr) and by measuring by flow cytometry the proportions of platelets in apoptosis (marked with annexin v), of functional platelets (marked with cd42) and of activated platelets (marked with cd62 the changes observed during the 8-h storage period appear to be limited and compatible with a further pr process using a photochemical treatment (amotosalen and uva) with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the ipp pooling and leukodepletion set developed by kansuk. a storage period of 8 h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. methods: 32 dd-bc-pc were prepared with 8 bc and 280 ml of pas (intersol, fresenius kabi (germany) are sterile docked to the octopus harness and combined into a 600 ml pooling bag. the pool is centrifuged and the pc supernatant expressed through a bioflex cs leukodepletion filter into a temporary platelet storage container. the obtained dd-bc-pc were tested within 1 h of preparation and after storage for 8 h in the platelet storage container for volume, platelet content, residual leukocytes (wbc), plasma ratio and biological parameters, ph, po 2 , pco 2 , glucose, lactate, mpv, ldh, p-selectin and swirling. results: the platelet content of dd-bc-pc (n = 32) was on average 6.9 ae 0.3.10 11 in a volume of 393 ae 6 ml. the mean of plasma ratio was 42% [min: 39.3max: 43.9]. all pc contain <1.10 6 wbc [min: <lq; max: 0.38]. po 2 decreased from 166 ae 12 mmhg after separation (1 h) to 44 ae 18 mmhg after 8 h, ph (+22°c) and pco 2 was stable during the same time period. glucose decreased from 9.0 ae 0.3 mmol/l (1 h) to 8.7 ae 0.3 mmol/l (8 h) while lactate increased from 6.07 ae 0.46 mmol/l (1 h) to 6.57 ae 0.54 mmol/l (8 h). the vpm was stable with 7.6 ae 0.2 at 1 h and 8 h. ldh increased from 120.4 ae 8.6 u/l at 1 h and 125.8 ae 10.3 u/l at 8 h as p-selectin 45.7 ae 7.6 ng/ml at 1 h and 57.7 ae 7.0 ng/ ml. all units have maintained swirling. the changes observed during the 8-h storage period appear to be limited and compatible with a further pr process using a photochemical treatment with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the platelet pooling and leukodepletion set developed by fresenius. a storage period of 8 h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. background: the irish blood transfusion service (ibts) is the statutory body with the responsibility for ireland's national blood supply. in 2018 the ibts collected 130,183 whole blood units and performed 9,654 platelet apheresis procedures. blood is collected in three fixed and six mobile sites and is returned overnight in temperature controlled vans to a single processing site. in september 2017, the ibts implemented the tacsi automated system for the preparation of buffy-coat derived platelets as an alternative to the orbisac system. aims: the aim of this study is to compare operational aspects and quality data of buffy-coat derived platelets prepared by the tacsi (t-pcs) and orbisac (o-pcs) automated systems. methods: qc data of t-pcs and o-pcs from years 2016 to 2018 were analyzed. in 2016, the ibts prepared 5,609 orbisac platelet pools of which 5,477 were qc tested. in 2017 5,031 orbisac and 1,625 tacsi platelet pools were prepared and tested. in 2018 6,783 tacsi platelet pools were prepared of which 4671 were qc tested. qc data comprised pc volume, platelet content (10 9 /unit), residual leucocyte content (10 6 /unit) and ph at expiry. feedback from the processing laboratory operators was collected comparing operational aspects of preparation of t-pcs and o-pcs. results: from the 10,508 o-pcs tested in 2016 and 2017, mean pc volume was 292 ae 9 ml. initially the t-pcs mean volume was 273 ae 13 ml but more recently the volumes were increased by addition of a larger volume of additive solution to 303 ae 13 ml. platelet content (10 9 /unit) was significantly improved in t-pcs (384 ae 47) compared to o-pcs (356 ae 50) [p < 0.01] as well as residual leucocyte content (10 6 /unit) which was lower in t-pcs (0.05 ae 0.04) compared to o-pcs (0.1 ae 0.2) [p < 0.01]. ph at expiry was lower in t-pcs (7.32 ae 0.07) compared to 0-pcs (7.38 ae 0.06) but remained within the quality requirements. from an operational perspective with two tacsi devices, five operators produce approximately 48 t-pcs per hour compared to a lower throughput of 20 o-pcs per hour with five orbisac devices. the tacsi system is more compatible with batch processing which aligns with our staffing arrangements in routine production and has resulted in greater productivity since its introduction. with tacsi implementation, we reverted to manual pooling of buffy coats and new staff had to undergo training to achieve optimal platelet recovery. once staff were trained during validation, we observed a 10% increase in platelet recovery. processing laboratory staff indicated that tacsi devices were easier and simpler to maintain in comparison to orbisac, but highlighted that some improvements to tacsi system boxes could be made to make the system more robust. summary/conclusions: pcs prepared with tacsi and orbisac automated systems both met national and european quality requirements, however we observed a significant improvement in quality markers of pcs prepared with tacsi. processing staff reported easier maintenance and processing with tacsi compared to orbisac. throughput is higher with tacsi and there is greater additional capacity to increase production in the future if required. a perez aliaga 1 , f puente 1 , a aranda 1 , j domingo 1 , l call en 1 and a bah 2 1 banco de sangre y tejidos de arag on, aragon, spain 2 terumo bct europe n.v., zaventem, belgium background: the blood bank and tissues of arag on (bsta) is responsible for the collection, processing and distribution of blood in the region of aragon in spain. with five fixed and five mobile sites, the bsta collects and processes 44,000 whole blood units per year. to improve quality of processed units and working conditions of personnel, the bsta decided to implement the reveos automated system. validation studies were carried out in november 2012 and routine use started in july 2013. in parallel, the bsta initiated pathogen inactivation of platelet concentrates (pc) in november 2012 to improve transfusion safety. in november 2015, the mirasol prt system was implemented due to simplicity of this platform. aims: the aim of this abstract is to (i) describe the results of the validation studies carried out during reveos implementation (ii) present routine use data of products processed with reveos and (iii) provide information on mirasol implementation. methods: in 2012, 369 whole blood units were processed using reveos. quality parameters for red cell concentrates (rcc), such as: hemoglobin (hb), hematocrit (hct) and volume; plasma: volume and residual platelet content; and pcs: volume and yield, were analyzed. a control group consisting of units processed with a semiautomated platform was used as comparison. a survey was sent to the collection and processing staff for feedback on operational aspects of the use of reveos. from 2013 to 2018, qc data of rccs (n = 3025), plasma (n = 660) and pcs (n = 660) processed with reveos were collected. number of mirasol treated pcs and hemovigilance data from 2015 to 2018 were collected. results: volume, hb and hct of rccs processed with reveos were significantly higher compared to control rccs (volume: 296 ae 21 ml vs 251 ae 19 ml; hb: 59.9 ae 6.2 g/ unit vs 49.2 ae 7.8 g/unit; hct: 59.5 ae 2.7% vs 56.1 ae 6.3%). control plasma units had higher volume (280 ae 17 ml) and lower residual platelet content (9 ae 7 10 9 /l) compared to reveos plasma units (volume: 249 ae 23 ml; residual platelet content 19 ae 13 10 9 /l). for pcs, volume was higher for units processed with reveos compared to control (350 ae 20 ml vs 310 ae 10), but no statistical difference was observed in platelet content (reveos: 361 ae 60 9 10 9 /unit; control: 386 ae 80.10 9 /unit). we observed either an increase or stabilization in qc parameters of rccs, plasma and pcs during the 5 years routine use of reveos. for example, percentage of rccs units hemolyzed dropped from 4% to 0% between 2013 and 2018 and platelet content in pcs increased from 370 9 10 9 /unit (first six months in routine) to 390 9 10 9 /unit. plasma volume increased from 249 ml during validation to 267 ml in 2018. survey results from staff highlighted the user friendliness of the reveos device. number of mirasol treated pcs increased from 10% in 2015 to 59.7% in 2018 with no pathogen transmission nor serious adverse events reported following transfusion of these products. summary/conclusions: with implementation of two user-friendly systems, the reveos whole blood automation system and mirasol prt system, we observed an increased standardization of our processes enabling us to provide higher quality and safer blood products to hospitals and the patients they serve. background: white blood cells (wbc) may be regarded as contaminants in blood components and potentially reduce transfusion safety. there is currently enough evidence showing that wbc reduction through pre-storage filtration reduces the incidence of three major complications of allogeneic blood transfusions: febrile nonhemolytic transfusion reactions (fnhtr), refractoriness to random-donor platelet transfusions and transmission of cmv. leukodepletion is now mandatory in canada and several european countries. aims: the aim of this study was to evaluate leukoreduction performance and the quality of the resulting red blood cell concentrate (rbc) produced with new quadruple system top & bottom imuflex crc with an integrated red blood cells filter which was used in whole blood fractionation under routine conditions of san paolo asl blood bank methods: twenty-seven whole blood donations (450 ml) were collected with using quadruple blood bags top & bottom system with an integrated rbc filter (imuflex crc, terumo bct). centrifugation and processing (using t-ace ii + -terumo bct) of whole blood units followed the blood bank sop to obtain rbcs, plasma and buffy coat. rbcs were diluted with sagm (100 ml) and then filtered by gravity in average 2-3 h after blood collection to obtain leukoreduced rbc. average temperature during filtration was 26°c. samples were collected before and after leukoreduction for calculation of hemoglobin loss through the filtration process. filtration time was recorded. final hb, hematocrit (hct), residual platelets and white blood cells were determined after filtration using standard laboratory methods (blood cell counter) results: wb donations (n = 27) were performed. rbc characteristics post-filtration and separation were as follows: volume (266.8 ae 17.1 ml), hematocrit (57.8 ae 2.1%) hemoglobin (19.8 ae 1.1 g/dl) and hemoglobin per unit (53.0 ae 5.4 g); residual platelets (11.7 ae 2.2 x10³/ll); residual wbcs (0 à 0.004 9 10³/ll). the mean filtration time was 19 ae 3 min and no filter blocks were observed summary/conclusions: the usability and performance of the new quadruple system top & bottom imuflex â crc with integrated rbc filter was evaluated. all quality parameters complied with italian and european guidelines and requirements. in conclusion, the set was found to be a reliable and efficient collection and separation system that performs consistently background: novel devices for automated whole blood processing (reveos tm terumo bct) have been installed in regional center for blood donation and blood treatment in katowice to modernize and automate production of blood components. this system allows to separation of four whole blood units into four buffy coat depleted red blood cell concentrates (rbc), four units of plasma and four interim platelet concentrate (ipu) units in one automatic cycle. an ipu is the starting blood component for the production of pooled, leucoreduced platelet concentrates (lpc). residual leukocytes (wbc) are obtained as a byproduct and discarded. the use of this system shortens production time because it combines otherwise two separate steps: centrifugation and blood separation in press. aims: to evaluate the quality of the blood components obtained with the newly developed reveos nlr kits, which are a whole blood collection kits containing no leucocyte reduction filter for the rbcs. methods: blood [wb] was collected into nlr reveos set (terumo bct), and processed between 2 and 8 h from collection using the 3c protocol. quality control was performed on rbc, plasma and ipu. rbc were tested for hemoglobin (hb) content, hematocrit (ht), residual white blood cells (wbc), volume and hemolysis level on the last day (42 nd day) of storage. the content of wbc and platelet count (plt), protein, factor viii on the day of production and after one month of storage were determined in plasma. for ipu, platelet and leucocyte counts, volume and average platelet yield index (pyi) were displayed at the device after processing was recorded. furthermore, a comparison between fully automated and semi-automated whole blood processing was done, by comparing the amount of time necessary to fractionate 4 whole blood units using the two different processes. background: direct, single step automatic blood separation devices (reveos tm terumo bct) was installed in regional center in katowice in 2018. the system allows for full automation of whole blood fractionation. one of produced blood components is an interim platelet unit (ipu), containing most of the platelets and limited of white blood cells (wbcs). it is then pooled through a filter to form a therapeutic unit of leucoreduced platelet concentrate (lpc). a rough estimate of the platelet yield in the ipu, the so-called platelet yield index (pyi) is calculated and displayed by the device after the end of the separation phase. it is a useful tool to optimize selection of ipu for pools to produce lpc. aims: to determine the quality of polled lpc obtained on the basis of ipu from whole blood processed up to 8 h after collection and the usefulness of this method in daily practice. methods: ipus used for production of lpc were obtained using fully automated whole blood processing single step system -reveos tm (terumo bct). from the beginning of the application of the method, 1005 therapeutic units of lpcs were produced after overnight hold: 38 pools of 4 ipus and 966 of 5 ipus were made using platelet pooling set (terumo bct), using 250 ml ssp+ solution (macopharma). results: 42 therapeutic units of lpc were analyzed. the mean volume of lpc was 387 ae 13 ml, platelet content was 3.67 ae 0.46 x10 11 , and white blood cells (wbc) count was 0.15 ae 0.13 x10 6 , 100% all lpcs had wbc below 1 9 10 6 . ph measurements were made on the fifth day of storage, the ph was determined in 15 units of lpc and was 7.2 ae 0.04. summary/conclusions: the use of interim platelet units in daily practice increases the possibility of producing high quality blood components for patients and is a great alternative to the laborintensive manual methods of lpc's production. background: the development of cold-stored platelets has been hampered by the rapid removal of blood after transfusion by apoptosis and desialylation due to platelet storage lesions caused by refrigeration. previous studies have shown that antioxidants inhibited the activation and desialylation of cold-stored platelets for 5 days and also inhibited rapid elimination after transfusion in animal models. aims: it was tried whether in long-term (10 days or longer) storage conditions, antioxidants showed similar effects inhibiting the activation and desialylation of cold-stored platelets. methods: platelet-rich plasma (about 250 ml) were obtained from a healthy blood donors of type o blood group and divided into a 30 ml platelet storage bag manufactured by the manufacturer, and the conditions including room temperature and refrigerated condition and various kinds of antioxidants were prepared, (ph, glucose, lactic acid) and activation index (cd62p, gpiba (cd42b), annexin v), degree of reactive oxygen species and sialidase activity on the 7th, 10th and 12th day, and phagocytosis using the hepg2 cell line and platelet recovery after in vivo transfusion using scid mice were compared and compared. results: cold-stored platelets showed autoaggregation on the 2nd day of storage, and apparent aggregation on the fifth day of visual observation but not in the antioxidanttreated group. as expected, the ph, glucose and lactate levels of the cold platelets were maintained and the platelets at room temperature changed significantly. nac (n-acetylcysteine) -treated platelets were less active on day 12 and sialidase activity was maintained lower than that of control group on day 12. the degree of phagocytosis by the hepg2 cell line remained at the same level during the storage period, and the in vivo recovery rate in the animal model was superior to that of the antioxidanttreated cold platelets for 2 h and 24 h after transfusion. summary/conclusions: cold platelets could be developed as clinically effective agents for up to two weeks if appropriate preservatives were added, and antioxidants, especially n-acetylcysteine, were effective. background: the reveos 3c procedure produces the following three blood components for transfusion: plasma, rbcs, and a single interim platelet unit (ipu). whole blood is collected into the reveos blood bag set and held at room temperature a minimum of 2 h before processing on the device. after processing on the reveos device, the plasma product is ready to freeze. the rbc product is ready to combine with sag-m additive solution and leukoreduce using the integrated leukoreduction filter. the ipus are rested and agitated for the recommended time. for example, when whole blood is processed within 8 h of collection, the ipu is agitated overnight before pooling. because the ipu is made from one unit of whole blood, approximately 4 to 6 ipus are pooled together and leukoreduced by filtration prior to storage in a platelet storage bag. this leukoreduced platelet pool (4 units) can be supplied to the demand of hospitals like a platelet apheresis product. aims: to assess the usability of the reveos automated blood processing system to collect units of whole blood and to process the units on the reveos device, producing blood components that meet product quality specifications. methods: this study will evaluate approximately 19 units of whole blood using the reveos system and approximately 4 units of pooled platelet products. a total of 19 units of whole blood (wb) have been collected into the reveos blood bag set and then held at room temperature without the use of an active cooling system (or temperature control system), such as cooling plates, or in this case, ice packs. of the 19 units, 10 units have been processed on the reveos device using the fresh procedure (within 8 h of collection). nine units have been processed using the overnight wbhold procedure. all units have processed successfully without alarms, resulting in three components (rbcs, plasma, platelets). we also evaluated four platelet pools created from eighteen interim platelet units (ipus). one ipu has been discarded due to a 22-min whole blood collection time. the leukocyte count on all component using the automatic sysmex-xn 2000 cell blood counter. results: the red blood cells meet the criteria for hematocrit, hemoglobin and residual wbc counts are less than 1 million per unit. the plasma units meet the criteria for displayed volume accuracy compared to measured volume and also for factor viii levels. the average residual platelets in plasma levels meet the criteria. the platelet pools meet the acceptance criteria for platelet yield and volume. limited data (n = 4) suggests that this criteria will be easily met, provided that wb hold conditions are suitable for platelets. summary/conclusions: final results indicate that products produced from the reveos system meet or exceed product quality expectations. staff report that the reveos system is very easy to use. abstract withdrawn. background: thorough manual mixing of overnight-held whole blood (wb) units prior to centrifugation to separate red cell (rc) from platelet-rich-plasma (prp) is currently practised at the hong kong red cross blood transfusion service (hkrcbts). the mixing step is crucial to maximise platelet yield but laborious, and may cause elbow and wrist injuries in operators. a laboratory shaker, reax 20 (heidolph instruments, schwabach, germany), originally designed for mixing solvents, was modified to allow mixing source wb units by the supplier. to enhance the well-being of the operators in the context of occupation health and safety (oh&s) management, feasibility of the application of such blood mixer to replace the manual mixing steps in blood component preparation was explored. aims: to evaluate the in vitro quality control (qc) parameters of the blood components prepared with the use of the modified mixer. methods: fifty-eight wb collections, including 40 units in 450 ml quadruple bags (450q) with/without integrated leucofilter and 18 units in 350 ml quadruple bags (350q), were processed in accordance with hkrcbts' prevailing component processing protocols except using the motor-driven mixer in lieu of manual mixing. wb collections were divided into 2 groups, mixed at 2 revolutions per minute (rpm) for 2 min (n = 30) and 30 min (n = 28), then processed into rc, prp platelet and plasma components. in vitro qc assessments including haematocrit (hct), haemoglobulin (hb) and haemolysis at expiry for rc; absolute platelet count and ph for platelet concentrates (plt); volume and residual platelet count for plasma units were evaluated and compared. results: all components derived from both 450q and 350q processed with the blood mixer fulfilled the qc requirements stipulated by aabb standards and council of europe (coe) guidelines. there were no significant differences for all the qc parameters in rc and plasma components produced when mixed for 2 or 30 min. summary/conclusions: the overall performance of blood components processed with the motor-driven mixer met the acceptance criteria at the hkrcbts in accordance with the aabb and coe standards. higher platelet yield was observed with prolonged mixing for 30 min. however, prolonged mixing will much delay routine component processing. further evaluation on intermediate mixing time such as 3, 5 or 10 min may help determine an optimal mixing duration to maximise platelet yield without compromising processing workflow. to conclude, using the modified blood mixer to replace laborious manual mixing in components preparation is promising to improve the oh&s for blood component preparation operators. background: leukocyte reduction is widely used to reduce the risk of non-hemolytic febrile transfusion reactions and alloimmunization by multiple blood transfusions. around and after year 2000, universal leukocyte reduction was adopted in many developed countries. there are mainly two types of filter, one to filter whole blood and the other to filter red cell concentrate (rcc) used during pre-storage blood processing. aims: in this study, we conducted the filtration tests with sepacell tm rs2 for rcc in comparison with sepacell tm r-s11 and sepacell tm pure rc widely used in the world. background: the cardarelli hospital blood bank processes over 40,000 whole blood units yearly. in order to constantly cope with the huge demand for platelets from oncohematology and intensive care departments, a progressive review of the processing protocols for platelet concentrates (pc) from buffy-coat (bc) pools has recently been implemented to reduce the number of units assembled, from 5 (routine in italy) to 4. the 4-bc pc units have slightly lower volume and plasma content, factors that further limit the transfusion risk. in addition, the 4-bc assembly allows to facilitate the production of the pc units obtained from the same blood type, in order to minimize both the adverse reactions related to the abo/rh incompatibility and the amount of bcs not used. finally, the described innovation also impacts on the workload, with reduction of assembly, centrifugation and separation time. aims: the aim of this work is to define an optimized protocol for the preparation of pc units from 4-bc pools, so that the platelet yield is always higher than the minimum value required by current regulatory (2.0 9 10 11 cells/unit). background: blood is a basic component for human life and hence blood transfusion is an integral intervention in the treatment of patients. the need of blood transfusion is increasing with decreasing blood supply due to limited blood donations. conversely wastage of blood products is also a topic for discussion. to ensure the availability of blood products when they are needed, wastage of blood products should be controlled and minimized. aims: the study was designed to investigate the quantity and reasons of wastage of blood products at our hospital. methods: this was an observational study based on the statistical data available from blood bank department of nibd and bmt, pechs campus, karachi, pakistan. the study period was from february 2018 to february 2019. study was approved from institutional review board. for all the blood products wastage and reasons of wastage were evaluated. frequencies were calculated by using spss version 23.0 results: a total of 2880 blood products were available at our institute including 960 each of platelets, pack red cells and fresh frozen plasma(ffp). the overall wastage rate was 3.5%. pack red cells and platelets were fully consumed yet shortage of supply was observed. however, highest wastage was observed in ffp in our blood bank i.e. 102(10%). results of investigating the common causes of blood product wastage at the hospital revealed that it was highly associated with 02 common causes, including the expiry of unused products 60(59%) followed by broken bags 30(29%). summary/conclusions: this study showed over all diminutive wastage rate. ffp wastage was highest among all the blood products. wastage of blood products is a genuine issue in a hospital setup, strategies and plan of action should be discussed and implemented including enhanced collaboration with other centres to outsource the blood products or even donate at times, education and training of staff for better clinical use of blood products and ensure the availability when and where they are needed most. background: in the early 2000s, the blood service of the republic of kazakhstan functioned along the lines of the soviet period: donation of blood was approached to the place of its use (carried out at hospitals), screening of donor blood was carried out at one stage (enzyme immunoassay) on analyzers of semi-automatic type, there was no mechanism calculating the cost of blood components, the activity of blood centers was funded at the rate of 1 liter of whole blood. donation was not popular in society, household fears of infection were common when donated. aims: research objective is to study the main stages of reforming of blood services of the republic of kazakhstan (rok). the research methods are based on the system analysis of the condition of the rok's blood services based on the indicators of production activity for the period of 2010-2017. results: in order to effectively reform the blood services, the following steps were taken. the blood supply was centralized and optimized. blood collection at hospitals was terminated, more than 200 blood collection subjects were closed. the result was a more efficient use of technical, human and financial resources, as well as ensuring the high quality of the blood components produced. today, there are 18 blood centers in the republicone for each of the 18 administrative territorial units of the republic. the screening of donor blood for transfusion infection markers has been transferred into a two-step principleimmunological analysis and nat testing. a fully automated analytical equipment of a closed type is used for screening, unified for all blood centers of the republic. unification of the equipment choice allowed to provide centralized service maintenance of complex equipment and its uninterrupted operation. since 2012, the national reference laboratory has been operating in the blood service, which conducts an external assessment of the quality of laboratory research in blood centers, as well as research in controversial and complex cases. introduced pathogen inactivation of platelet concentrates in 100% of cases of platelets. erythrocytes are used only in the weighing solution and only after leukoreduction. the use of resource-saving blood harvesting technologies, as well as methods to further enhance the infectious and immunological safety of components, are expanding annually. the foundations for the development of applied scientific research in the field of transfusion medicine were laid, and an own school of transfusiologists was created. as a result of reforming the blood services, the mechanisms of reimbursement of blood services costs from the budget of the republic have been changed, uniform tariffs have been defined by type of blood components for all blood components suppliers in the country. summary/conclusions: the transformations made it possible to create a blood service in kazakhstan that corresponds to the best international practice and ensures the quality, efficacy and safety of transfusion therapy. further development of the blood service is aimed at deepening reforms, studying and developing cellular technologies, new properties of plasma blood components, and improving the clinical practice of using donor blood components. background: reconstituted platelet concentrate (rpc) is a component that contains platelets (thrombocytes) resuspended in plasma that is blood group compatible with the recipient. it is used when transfusion of platelet concentrate is necessary but the fresh platelet concentrates of the specific blood group are not available. group 0 rh-(negative) platelets resuspended in the ab group plasma are of universal use as such components can be used for all recipients independently of their blood group. in the area of activity of regional blood center in poznan rpc is used most often for patients after stem cell transplant incompatible with their blood group. aims: the aim of the analysis is to evaluate the loss in the number of platelets in the reconstituted platelet concentrates in order to determine their quality and usage. methods: we used for the analysis 30 units of pooled platelet concentrate derived from buffy coats and 30 units of pooled platelet concentrate derived from the apheresis. for the reconstitution group ab plasma was used. we investigated the number of platelets prior to and after the reconstitution. following measures were calculated: number of platelets in pooled platelet concentrates derived from buffy coats, number of platelets in pooled platelet concentrates derived from apheresis; average values and the standard deviation as well as the loss in number of platelets due to the reconstitution. the number of platelets was calculated with impedance method using horiba pentra xl 80 analyser. 1. pooled platelet concentrates derived from buffy coats the number of platelets before the reconstitution: 4.12 9 10 11 /unit ae 0.50 the number of platelets after the reconstitution: 3.40 9 10 11 /unit ae 0.50 the loss in the number of platelets: 17.5% 2. platelet concentrates derived from apheresis the number of platelets before the reconstitution: 3.68 9 10 11 /unit ae 0.55 the number of platelets after the reconstitution: 2.91 9 10 11 /unit ae 0.49 the loss in the number of platelets: 20.7% summary/conclusions: 1. reconstitution results in the loss of 17.5% of platelets in comparison to the initial value of pooled platelet concentrate derived from buffy coats. 2. the average number of platelets after the reconstitution is 3.4 9 10 11 /unit, which fulfils the quality requirements. 3. reconstitution results in the loss of 20.7% of platelets in comparison to the initial value of pooled platelet concentrate derived from apheresis. 4. the average number of platelets after the reconstitution is 2.9 9 10 11 /unit, which slightly deviates from the quality requirements. 5. the results that we obtained show that the process of reconstitution always results in the loss of number of platelets and that the quality of platelet concentrates that have not been reprocessed is always higher. because of that we recommend that the reconstitution should only be performed in special cases such as for patients after stem cell transplant incompatible with their blood group and that hospitals should be supplied with platelet concentrates derived from buffy coats and apheresis. methods: pf4 was extracted from human pcs by using immunoaffinity chromatography and specific anti-pf4 antibody on the column. hereafter, pf4 was purified with concentration tubes having 5 kda molecular weight cut-off and dialyzed via dialysis tubing with 3.5 kda cut-off. pf4 concentration was measured by bradford method. specific enzyme-linked immunosorbent assay (elisa) and dot blot analysis were used to determine the pf4 specificity. molecular weight of obtained pf4 was determined by polyacrylamide gel electrophoresis (page). results: our data confirmed clearly that separated pf4 had 30 kda molecular weight which is corresponding with a tetramer pf4. in addition, elisa showed that the pf4 qualified true antigenicity. dot blot analysis helped us to assure the specificity of pf4. summary/conclusions: we used and optimized a homemade protocol to isolate and purify the pf4. nevertheless, there was no any scientific report among the databases that utilized the similar purification method. recombinant pf4 is more feasible and ready-to-use, but its price may discourage the researchers to study the biology of pf4. unused human pcs and specific anti-pf4 antibody can considered as an alternative to separate the pf4 particularly in labs with the shortage of resources. background: whole blood is increasingly used in civilian hospitals as an alternative to blood components in trauma resuscitation with massive bleeding. however, the hemostatic effect of whole blood depends on the initial platelet content and decreases rapidly within the first 2 weeks of storage at 4°c. consequently the problem arises of how to provide wb to trauma centers without losing valuable low titer group o units if the units are not used within the expiry date of use. aims: to assess the in-vitro quality of red cell concentrates (rcc) produced from 7 days cold stored leucodepleted whole blood units filtered with a platelet-sparing whole blood filtration filter in order to save the rcc once the wb units has reached the expiry date. methods: 34 units of whole blood were leucodepleted within 24 h using a platelet sparing filter (imuflex wb-sp/terumo bct) and stored for 7 days at 4 ae 2°c. on the 8 th day of storage, units are centrifuged and the rcc is supplemented with sag-m additive solution for an additional storage of 35 days at 4 ae 2°c. in vitro parameters such as hemoglobin, hematocrit, residual leucocytes, platelet content, hemolysis, glucose, lactate, atp, 2.3 dpg were tested during storage. results: the imuflex wb-sp provided satisfying leucodepletion of the wb units (0.23 ae 0.2.10e6/u)) while maintaining 80% of the initial platelet content (85 ae 20.10e9/u). mean volume (464 ae 24 ml), hemoglobin content (54 ae 6 g/u) and hematocrit (36 ae 3%) was within conformance range. after 7 days of storage, 50% of the initial platelet content at collection is maintained and mean hemolysis is 0.27 ae 0.15%. rcc processing at day 8 was uneventful, without loss of hemoglobin or noticeable hemolysis. mean volume (266 ae 20 ml), hematocrit (56 ae 3%), hemolysis (0.18 ae 0.19%) were in conformance with mandatory standards at day 8. after 7 days of storage (i.e. day 14 after collection), rcc quality parameters were maintained with however an increase in hemolysis (0.34 ae 0.36%) which was above usual values routinely observed with rcc produced from day 1 processed units. 3 rcc out of 34 were above the maximum limit of 0.8% at day 14 after collection such increased hemolysis was more pronounced at day 28 after collection (0.70 ae 0.45%) with a proportion of rcc with a non-conforming hemolysis ratio above the accepted threshold (20% of the tested units). summary/conclusions: rcc units can be readily obtained from leucodepleted wb units with spared platelet contents after 7 days of cold storage. in vitro quality attributes of such rcc are within conforming ranges for hemoglobin, hematocrit, volume and hemolysis. hemolysis increases more rapidly than in standard rcc, thus limiting the possibility of use within 21 days after collection. additional studies are being initiated to reduce hemolysis during storage. g hetland 1,2 , f mahmood 3 , l nissen-meyer 1 and i nentwich 1 1 immunology and transfusion medicine, oslo university hospital 2 immunology, university of oslo, oslo 3 immunology and transfusion medicine, akershus university hospital, lørenskog, norway background: allergen-specific ige in donors' plasma can be transferred from blood donors to recipients via plasma-containing products and consequently sensitize recipient's effector cells bearing receptors for ige (mast cells, basophils, thrombocytes etc.). these cells can then degranulate after exposure to allergen. this can cause allergy symptoms of various degrees (johansson, allergy, 2005) . food allergen-specific ige antibodies, although causing mild or no symptoms in blood donors, can be of clinical importance to plasma recipients due to varying sensitization and activation grade of recipients' effector cells. aims: the aims of the study were 1) to assess sensitization profiles against an array of 55 allergens in 100 randomly selected blood donors and 2) to identify donors with high concentrations of food-specific ige antibodies that could bear risk of clinically important sensitization of plasma product recipients. methods: we tested serum samples from 100 blood donors randomly selected on one donation day outside the pollen season. we used 300 ll serum per sample in a multiplex ige line-blot enzymoimmunoassay for 55 analytes (euroline atopy screen, euroimmun, germany), which comprised 28 food allergen extracts and single allergens, 24 inhalant allergen extracts, 2 insect venom extracts and one carbohydrate allergen ccd. this high-throughput method enables scanning of 44 serum samples for ige against 55 allergens in less than 3 h. results are expressed as east (enzymoimmunoassay) classes: class 0 (no sensitization), 1-2 (weak sensitization), 3 (moderate sensitization), 4-5 (strong and very strong sensitization). results: east class, allergen, number of samples. food allergens: class 5none detected. class 4, sesame seed: 1. class 3, apple 1; hazelnut 2. class 2, apple 1; almond 2; hazelnut 1; sesame seed 1; soybean 1; cow milk alpha-lactalbumin 3. class 1: 26 donors weakly sensitized to one or more allergens as kiwi, apple, almond, hazelnut, sesame, soybean, wheat, mutton, beef, cow milk beta-lactoglobulin and alpha-lactalbumin. we did not find any donors sensitized to apricot, peanut, rice, rye, yeast, cow milk casein and carbohydrate allergen ccd. insect venoms: class 5, 4 and 3 none, class 2, wasp venom 7; honey bee 2. lower classes (0-1) not reported in this abstract. summary/conclusions: we found considerable sensitization to inhalant allergens but such sensitization probably represents low risk of clinical allergies due to passive transfer of antibodies. sensitization to insect venoms was negligible. we disclosed only one blood donor (1%) with high ige to sesame seed with a potential to transfer clinically relevant ige antibodies via plasma products. the need for a broad screening of donors for ige sensitization remains still questionable. background: the platelet storage lesion (psl) is one of the key factors that limit the platelet shelf-life to 5 days. the mechanisms mediating the psl are not well understood, but it is becoming increasingly evident that platelets from some donors do not store as well as others. similarly, assessment of many in vitro quality parameters has been used to predict transfusion outcomes, with varying degrees of success. donor attributes such as age, and body mass index (bmi) may contribute to these differences. aims: to characterise the variability in the quality and function of apheresis platelet donations and to examine associations between donor attributes and platelet characteristics. methods: apheresis platelets (n = 53) were collected, stored at 22°c with agitation, and tested at day 1, day 5 and 7 post-collection. vwf and fibrinogen binding (indicators of platelet adhesion; ae ristocetin stimulation), phosphatidylserine (ps) exposure (annexin v binding) and microparticles (cd61 + /annexin-v + ) were measured by flow cytometry. platelet aggregation was initiated with collagen, thrombin or arachidonic acid. thromboxane a 2 (txa 2 ) was measured by elisa. the proportion of procoagulant platelets (% coated platelets) was assessed by fibrinogen binding following collagen/thrombin stimulation. platelet-attached glycans were measured using flow cytometry and lectins ricinus communis agglutinin-1 (rca-1; detecting galactose-residues) and wheat germ agglutinin (wga; detecting sialic acid and nacetyl-d-glucosamine, glcnac-residues) ae stimulation with ristocetin. donation time, donor age, blood pressure (bp) and bmi were recorded. linear regression was performed using graphpad prism software. results: for many of the parameters measured, there was high inter-donor variability. platelets from subsets of donors showed up to 4-fold higher vwf and fibrinogen binding, formation of coated platelets and microparticle numbers. binding of lectins was also highly variable between the donors, indicating variability in sugar cleavage. following ristocetin stimulation, binding of all lectins increased, and again there was high variability in the responses, with up to 4-fold higher binding in platelets from a subset of donors. of particular interest, aggregation in response to arachidonic acid was observed in only 27% of platelet donations tested; although platelets from non-responders aggregated in response to the collagen and thrombin. txa 2 was also higher in the arachidonic acid responders (p = 0.027). there were few associations between platelet parameters and donor attributes, and these associations were typically weak. ph at day 5 was weakly associated with donor age (p = 0.009, r 2 =0.2613) but not bmi (p = 0.9143, r 2 <0.001), with a trend towards lower ph in younger donors. summary/conclusions: these data indicate that apheresis platelet products are highly variable. whilst donor characteristics may have some influence on the quality of the donated platelet product, there is a high level of inherent inter-donor biological variability. a much larger sample size is required to confirm these findings, and determine whether they have clinical implications. background: the frequency of beta thalassemia mutations, and thus, of beta thalassemia heterozygotes (b-thal-het) is particularly high in certain geographical regions. it has been reported that a significant proportion of donors (occasionally more than 10%) are b-thal-het that meet the criteria for blood donation (hb > 13.5 g/ dl). red blood cells (rbcs) of b-thal-het donors are characterized, in vivo, by particular geometry and redox status. despite sporadic indications that the rbc storage lesion may be milder in b-thal-het, targeted research on this donor group is still missing. aims: the aim of this study was to investigate whether b-thal-het rbcs storage at blood banks leads to a distinctive hemolytic, physiological and redox profile, thus, making b-thal-het a unique blood donor group. methods: blood samples from 10 healthy non-smoker donors (5 b-thal-het carriers and 5 controls) were analyzed before and after preparation and storage of leukoreduced packed rbc units in cpd/sagm at various time intervals. susceptibility in hemolysis (in the presence/absence of oxidative, mechanic and osmotic stimuli), redox status (lipid peroxidation, reactive oxygen species (ros) accumulation, antioxidant capacity), intracellular ca 2+ and proteasomal activities were determined. for statistical analysis, significance was accepted at p < 0.05. samples from the red cell units were collected aseptically, processed (dual centrifugation at 3,000 g for 15 min) and stored at à80°c. processed samples were thawed, and then analysed using the nanosight ns300 nanoparticle tracking analysis system (malvern instruments). samples from all time-points from each unit were analysed on the same day. data were analysed by one-way anova with bonferroni's multiple comparisons test. results: at d2, red cell units contained an average of 8.1 9 10 9 ae 4.0 9 10 9 mvs/ ml. the mean size of these mvs was 112.8 ae 10.5 nm and the mode size was 79.9 ae 10.3 nm. the concentration of mvs increased gradually throughout storage (p = 0.0276), reaching a maximum at d42 of 32.4 9 10 9 ae 1.8 9 10 9 mvs/ml. both the mean (p < 0.0001) and mode (p < 0.0001) size of the mvs increased during storage; however, this size increase primarily occurred in the first week of storage (d2 vs. d7: p < 0.0001 for both mean and mode). by d42, mean and mode size of mvs was 176.1 ae 4.8 nm and 171.8 ae 5.6 nm summary/conclusions: nanoparticle tracking analysis demonstrated the presence of mvs smaller than 400 nm in red cell units. both the concentration and size of mvs present in red cell units increased during the 42 days of routine storage. the concentration of these mvs was approximately 100-fold higher than we had previously detected using flow cytometry (aung, pathology, 2017) indicating the advantages of more sensitive techniques in characterisation of mvs. background: the lack of availability of sterile saline in a format suitable for use in blood centers for manual washing has led to an urgent need for blood services to consider alternative methods. for operational flexibility it would be desirable to be able to produce a washed rbc unit that had a shelf life longer than 24 h. aims: the aim of this study was to validate the manual method for washing rbcs using sagm solution both as wash and storage solution and to ascertain whether an extended storage period for washed rbcs may be feasible. methods: six 14 day old leukocyte depleted red blood cells (ld-rbc) and six 4 day old ld-rbcs were manually washed and stored in sagm, and half of the units were pre-stored irradiated (25 gy). a volume of 350 ml wash solution (sagm) was added to the ld-rbss by sterile connection. after mixing the units were centrifuged for 6.5 min at 2888 9 g at 4°c (hettich roto silenta 630 rs) before removing the supernatant using compomat g5 extractor. wash procedure was repeated twice using 450 ml sagm solution, and after removal of the last supernatant, 100 ml of sagm solution was added. all units were immediately measured for volume, haematocrit, albumin, iga, potassium, haemolysis, haemoglobin, ph, glucose and lactate and tested again after 24 h, 7 days and 14 days storage at 4 ae 2°c. results: all washed ld-rbcs met european specification for haematocrit (0.50-0.70) and all but one for hb content (≥ 40 g/unit). hemolysis increased during storage. the rate of hemolysis in irradiated ld-rbcs was greater over time than in nonirradiated units. all units, both irradiated and nonirradiated, met european specification for hemolysis (less than 0.8%) 14 days after washing. after wash, potassium levels were low and then increased during storage; increase was greater in irradiated than nonirradiated units. potassium concentration 14 days after washing and irradiation did not exceed those levels found at the end of shelf life (day 35) of standard ld-rbcs. ph decreased during storage due to the metabolic activity of red blood cells converting glucose to lactate. the ph level of the supernatant depends on the age of the unit and not on the irradiation. the glucose concentration of the supernatant after washing is high due to sagm solution. the concentration of glucose decreased and lactate increased due to the metabolic activity of red blood cells. there is currently no specification in europe or finland for iga in washed rbcs. aabb and american red cross rare donor program stipulate that level of iga should be less than 0.05 mg/dl (0.5 mg/l). our iga method 0 s lower limit for detection is 0.05 mg/l and all results were below this level. total albumin were well below finnish specification (<250 mg/unit). background: room temperature has been the standard storage condition for platelets since the 1970s, when it was shown that this improved in vivo survival compared to when stored at 4°c. however, storage at room temperature has several disadvantages, including risk for bacterial contamination and short outdating. recently, the interest in cold-stored platelets increased, especially for patients with a hemostatic need. using extensive analysis techniques, we evaluated the in vitro quality of cold stored platelets in additive solution. aims: investigation of the in vitro quality of platelets stored at 4-9°c in pas-e. methods: three experiments were performed, in which two platelet concentrates, prepared from 5 buffy coats and 300 ml of pas-e (pcs) were pooled and split in equal pcs. pcs were stored for 23 days at 4-9°c, one of each pair with agitation on a flatbed shaker and the other without agitation. various parameters were analyzed to study the in vitro quality during storage and compared to routine room temperature storage. results: during cold storage, the swirling phenomenon disappeared within one day. due to the lower temperature, metabolism of the platelets was lower as compared to room temperature storage. the metabolic conditions were acceptable with ph d1-d16: 7.05-6.77 with platelet count 400 9 10 9 /u and glucose still at 2 mm at least until 16 days of storage. platelet activation maintained acceptable levels with cd62p expression < 30%, while ps exposure increased rapidly; >20% after 6 days of storage. aggregation tests showed functional platelets until 13 days of storage. agitation during storage had no effect on any of the tested parameters. summary/conclusions: during storage of platelets at 4-9°c, the hematological parameters and ph met routine requirements, while swirling phenomenon disappeared already at the first day. the functionality of the platelets did not decrease during cold storage, indicating that the swirling phenomenon is not a good surrogate marker under these conditions. the strong increase of ps exposure might be involved in the observed short survival of cold-stored platelets. platelet concentrates stored at 4-9°c are potentially suitable as a hemostatic agent for patients with a bleeding in need of platelets, but more studies are needed. aims: the goal of the study is the reinforcement of platelet reserves for case of emergency events and increasing their availability for treatment, preferentially in patients with massive bleeding. methods: we performed a comparative study with cpp and fp in vitro. buffy coatderived pooled leukoreduced platelets 0 rhd negative were frozen in 5-6% dmso and stored at à80°c for 6 months. cpp were thawed at 37°c, then reconstituted in platelet additive solution ssp+ and compared to fp. we measured these parameters: platelet content, platelet concentration, platelet loss during preparation process, coagulation properties, volume, ph, dmso concentration, titres of anti-a and anti-b antibodies. results: the average platelet loss after the process of freezing and reconstitution was 24%. the amount of platelets and platelet concentration in unit was lower in cpp compared to fp, but high enough (amount 194 9 10 9 /unit, concentration 0.73 9 10 9 /unit). both types of plts (either pcc or fp) maintained an acceptable ph during storage. swirl was on value 3 in fp and on value 1 in cpp. the average plasma content in fp was 31% compare to 3.22% in cpp after reconstitution. measured titres of igm anti-a and anti-b antibodies were very low (0-1:2). cpp had faster clot initiation (rotem clotting time (ct) in cpp 69.2 s, fp 81.8 s). cpp contributes to a sufficient clot (rotem maximum clot firmness (mcf) in cpp 39.8 mm, fp 53.2 mm). summary/conclusions: our results shows, that cpp have higher procoagulation activity and simultaneously lower clot firmness. thawing and reconstitution of platelets are easy and fast processes if platelet additive solution is used. this method helps to increase the availability of platelets in emergency medicine. low plasma content in cpp enables their use as washed platelet product in specific groups of patients. methods: after donation, the whole blood was stored in room temperature overnight before separating next morning by reveos â system. seven abo compatible ipus, each with a target volume of 24 ml, were selected and then they were connected to the pooling set provided by terumo bct. prior to the pooling of ipus, 250 ml of additive solution (t-pas+ provided by terumo bct) was added and distributed evenly between the ipu bags. the pooling set was then kept 1 h on bench in room temperature followed by 1 h on agitator at 22 ae 2°c. after filtration, the pool might be manually adjusted if its volume exceeded the maximum of 420 ml to meet the requirements by the intercept tm blood system. the final products were two pathogen-reduced platelet units with a shelf life of 7 days. results: during validation of the method, 12 pathogen-reduced platelet units were controlled, in addition to the platelet count, for ph, glucose, po2, pco2 and lactate on day 2, 5 and 7 of storage. the platelet count was 2.27 ae 15 9 10 11 per unit on day 2. the ph value was 6.9 on day 2, 7.1 on day 5, and 6.9 on day 7. the glucose concentration decreased from 5.8 to 3.5 and 1.6 mmol/l on day 2, 5 and 7, respectively. the mean po2 level was 25.3, 25.0 and 28.7 kpa while the mean pco2 was 3.8, 1.7 and 1.8 kpa and the lactate concentration was 5.9, 9.6 and 12.9 mmol/l on day 2, 5 and 7, respectively. since routine implementation of the method in april 2017, regular quality controls showed an average of platelet count of 2.63 ae 34 9 10 11 (n = 161) with a volume of 204 ae 7 ml per unit. summary/conclusions: the validation of the method and the following two years of experience in routine shows that the pooling of 7 ipus processed in reveos â system meet the requirements needed for intercept tm ds processing set for pathogen reduction of platelets. the results from the quality controls of the final platelet units were in accordance with the local and eu guidelines. methods: data was analyzed from 11 published and unpublished clinical studies that performed both primary and secondary testing of platelets using the bta system. the studies included apheresis and whole blood derived buffy coat platelets and tested 4-10 ml sample volume per culture bottle. the 11 studies classified results based on aabb bulletin 04-07 definitions with some modifications. the following assumptions were made including: • data was summarized as total number of positive tests, observed by the total number of tests performed on each day post collection; • it was assumed that one test was performed per platelet unit; • all units eligible for secondary testing were negative by the primary test the data needed to demonstrate a benefit for the use of the bta 3d systems for detecting contamination that was not revealed by previous bacterial testing as well as clinical specificity. results: a total of 217,932 platelet units from the studies where secondary testing of platelets was performed were analyzed. platelets were tested on day 3, 4, or ≥ 6, and represented 8.9%, 44%, and 47% of the units tested, respectively. true positives were detected in 174 platelet units representing 0.08% of the total platelets tested. the majority were reported from platelets tested on day ≥ 6 with a total of 128. data showed the bta 3d system used for secondary testing detects the most prevalent contaminates reported, staphylococcus spp., in ≤27 h with the majority detected in ≤24 h after incubation, allowing for interdiction of the units prior to transfusion. instrument specificity was reported in 5 of the 11 studies for platelets tested at 3 days and ≥ 6 days with a total false positive rate of 0.27% (range of 0-1.1%). instrument sensitivity when used for secondary testing could not be determined since subculture of negative bottles is not performed during routine use. during previous validation testing of lrap and lrwbpc, 1,136 culture bottles were confirmed true negatives by subculture. summary/conclusions: data from the studies that tested platelets at 3 to 8 days post collection provided evidence that the bta 3d with bpa & bpn detects contaminants missed in previously tested platelet units. the data supports that the bact/alert 3d system is an effective safety measure for secondary testing of platelet products to extend platelet dating beyond day 3 and up to day 7 when testing is performed using the test parameters described in the bpa and bpn bottle ifus and according to the fda draft guidance. background: magnetic nanoparticles have recently shown great potential in nonradioactive labeling of platelets. platelet labeling efficiency is enhanced when particles are conjugated with proteins like human serum albumin (hsa). however, the optimal hsa density coated on particles and the uptake mechanism of single particles in platelets remain unclear. aims: we characterize the interaction between single particles and platelets and determine the optimal hsa amount required to coat particles. methods: ferucarbotran iron oxide nanoparticles were coated with hsa in different amounts (0.5-2 mg/ml) and we confirmed successful hsa coating by addition of a crosslinking hsa antibody (dynamic light scattering). we labeled platelets from pooled platelet concentrates with 2 mm ferucarbotran coated nanoparticles and analyzed labeled platelets for iron content (atomic absorption spectroscopy) and particle localization (transmission electron microscopy). single-molecule force spectroscopy was used to determine binding forces of nanoparticles to platelet compartments. we applied hsa-particles via linkers of different length (i.e. short~2 nm, medium 30 nm and long~100 nm) on the cantilever tip and let them interact with a platelet provided on a collagen surface. after interaction we determined the rupture force required for platelet retrieval. results: the iron content per platelet reached a maximum at 0.5-1.0 mg/ml hsa coated particles with 0.57 ae 0.36 and 0.54 ae 0.39 pg/platelet, respectively. however, the 1.0 mg/ml hsa coating resulted in~100-fold higher binding affinity to platelets than particles coated with 0.5 mg/ml hsa. depending on peg length between tip and particle, particles interacted differently with platelets as shown by one, two or three force distributions of 80, 220, and 360 pn, which correspond up to three different binding pathways, respectively. the results indicate that a particle can interact with three targets including platelet membrane, open canalicular system, and platelet granules. summary/conclusions: our results reveal mechanism of platelet-particle interaction on a single particle level and provide an optimal hsa concentration coated on particles to gain maximal platelet labeling efficiency. labelling of platelets by magnetic nanoparticles may substitute radioactive labeling. results: the activation/lesions on total platelets and small and medium-sized platelets platelet population was detected on storage day 3, by the increased expression of cd36. the percentage of cd36-positive cells among the population of large platelets did not change during storage. on the day 3, increased expression of cd42b and cd62p was detected, but only on large platelets. small and medium-sized platelets had increased cd62p expression only on day 5. the expression of cd42a on total platelets increased significantly on day 3, and stayed unchanged until day 5. the same pattern of cd42a expression was detected for small and medium-sized platelets, whereas on large platelets the expression continued to increase until the end of storage. a decreased percentage of cd41-positive cells was detected among the total platelets and populations of medium-sized and large platelets. the storage induced externalization of phosphatidylserine on total platelets or on any of the platelet populations was not detected. the levels of tgf-b and p-selectin in the pc-bc supernatants were unchanged during the 5-day storage period. increased annexin and pf4 concentrations were detected on day 5. the concentration of b-tg increased on day 3 of storage, and continued to rise until the end of storage. summary/conclusions: the evaluation of expression of activation markers on different platelet populations could be used as an additional analysis in quality control of platelet concentrates, and in the assessment of novel approaches to platelet concentrate manipulation i.e. for testing new additive solutions, cryoconservation protocols, and cryoprotectants. background: the primary goal of autologous blood transfusion is to reduce the risks related to allogeneic blood transfusion, including transfusion-associated graft-versus-host disease (ta-gvhd). although downward trends in rates of autologous blood transfusion have been reported in europe and the americas, it still plays a role in eliminating risks related to allogeneic blood transfusion in japan, especially ta-gvhd. since february 2009, the transfusion service in our hospital has managed 3 autologous blood conservation techniques and helped to prevent mistransfusion by employing a bar code-based electronic identification system. aims: the objective of this study was to determine the use of 3 types of autologous blood components in a single institution over an approximately 9-year period. methods: between february 2009 and december 2017, we retrospectively analyzed autologous blood transfusion, including perioperative autologous cell salvage (pacs), pre-operative autologous blood donation (pad), and acute normovolemic hemodilution (anh). we investigated the use of autologous blood components and the rate of complying with electronic pre-transfusion check at the bedside in the operating rooms. we also determined the adverse reactions to autologous blood transfusion, which were categorized according to the definitions proposed by the international society of blood transfusion (isbt) working party on haemovigilance. results: a total of 9,193 patients (9% of whom received operations) received blood transfusion, of which 5,080 (55%) received autologous blood transfusion alone, 2,101 (23%) both autologous and allogeneic blood transfusions, and 2,012 (22%) allogeneic blood transfusion alone. the rate of autologous blood transfusion among patients who received blood transfusion was 78%. pacs units were transfused to 5,830 patients (60%), pad units to 2,845 patients (30%), and anh units to 982 patients (10%), and multiple blood conservation techniques were used for one patient. the overall compliance rate with electronic pre-transfusion check at the bedside in autologous blood components was 98.6%. adverse reactions were observed only with pad transfusion and not pacs nor anh. the number and rate of adverse reactions to pad transfusion were 12 and 0.1%, respectively, of which the most common was febrile non-hemolytic transfusion reaction at 9 (75%), followed by allergic reactions at 2 (17%), and hypotensive transfusion reaction at 1 (8%). the severity of adverse reactions to pad transfusion was grade 1 (non-severe) in all cases, and no serious adverse reactions were observed. among pad units, the rate of adverse reactions to whole blood pad units was 0.55%, that to frozen pad units was 0.05%, and that to autologous ffp units was 0.10%. summary/conclusions: our observations indicate that the rate of autologous blood transfusion among patients who received blood transfusion was 78%, when all types of autologous blood conservation techniques were included. to accurately determine the rate of autologous blood transfusion in a hospital, it may be better for the transfusion service to manage all types of autologous blood conservation techniques. they are now clinically available as a blood product. the residual plasma level of this product, which is prepared using the automated cell processor acp215 (haemonetics corp.), is approximately 1%. recently, a retrospective multicenter study was reported that this product was effective and safety for prevention of recurrent or severe transfusion reactions. plt products are generally stored with continuous agitation to maintain their quality. the interrupted agitation of plt suspended in additive solution (plasma carryover: 3-40%) for up to 24 h was previously found to have only a slight impact on in vitro plt properties. however, in some small hospitals with no agitator, if the initiation of transfusion is delayed by an emergent change in a patient's condition, the interruption of agitation may be prolonged. aims: the aim of this study was to evaluate the effects the interruption of agitation for 48 h (shelf life of wpc in japan) on the in vitro qualities of plt. methods: leukoreduced apheresis platelet concentrates in 100% plasma were washed on day one after blood collection using the automated closed-system cell processor acp215 (n = 6). wpc, which were rested 1 h after preparing, were divided equally into control and test units using polyolefin containers on day one. control units were agitated from days one to seven. test units were stored without agitation from days one to three, and agitation was subsequently performed until day seven. both units were stored at 20-24°c. in vitro plt quality was examined on days one, three, four and seven. results: the plt concentration of prepared wpc was 110.9 ae 1.9 (910 10 /l) and the volumes of the control and test units after the division were 120 ae 11 and 123 ae 12 (ml). the ph values of the test units on day three were lower than those of the control units; however, the ph of both units were maintained at higher than 6.85 during the seven-day storage period. swirling was well maintained and no clumping was visible in both units during storage period. no significant differences were observed in plts concentrates, mpv, hsr, aggregation response. the pco 2 , po 2 , bicarbonate, glucose and lactate mean values of test units were slightly lower or higher than those of the control units on days three or four. the levels of cd62p expression were significantly higher in the test units than in the control units on days three (52.6% ae 6.0 vs 40.9 ae 5.7, p < 0.01); however, this difference decreased in a time-dependent manner after agitation resumed. the levels of cd42b expression of test units were relatively lower than those of the control units until day seven, but no significant difference between the two units. background: monitoring residual white blood cells (rwbcs) is a requirement for quality monitoring (qm) the production of leucocyte depleted blood components. although flow cytometry is widely used for monitoring rwbc, there are no widely accepted methods to accurately and consistently measure rrbcs in blood components. sysmex have developed a novel algorithm, termed the blood bank (bb) mode for their xn-series of haematology analysers which is specifically designed to quantitate the levels of rwbcs and rrbcs in blood components. aims: we have previously assessed the linearity, accuracy and reproducibility of the bb mode on spiked samples in an r+d lab. we sought to further assess the performance of the bb software in a routine, high throughput blood component manufacturing department. methods: units of plasma, platelets (pcs) and red cell concentrates (rccs) were produced according to standard uk specifications within nhs blood and transplant (nhsbt). qm of residual cells was tested using the bb mode whilst rwbc was additionally analysed by flow cytometry using bd leucocount kit. results: during a 2-month field trial over 10,000 data points were collected representing all types of manufactured component. for some pcs, bb mode results from some sample tubes that did not contain edta gave very high rwbc values, indicating a potential large number of ld failures. the results were significantly different from those obtained from pcs using edta samples (p < 0.0001) which did not show the same high values. for rccs or plasma, the range of results from plain and edta tubes were not significantly different (p ≥ 0.3246). the analyses of ld platelet and plasma concentrates by either bb mode or flow cytometry both show more than > 90% of ld components have less than 1 9 10 6 rwbc/unit. for ld failures (n = 14) there was a good correlation (r 2 =0.9848) between flow cytometry and bb mode measurements. spiking studies suggested that the limits of detection and quantitation of rrbc were around 6 and 8 rrbc/ll respectively. residual red cell counts from manufactured components showed a wide variation in their numbers between units. as expected platelet production methods also showed a significant difference (p < 0.0001) in rrbc contamination, with lower levels in apheresis platelets (median = 15 rbc/ll, n = 1820) compared to those produced from buffy coats (median = 783 rbc/ll, n = 77) in our hands, although the time taken to analyse samples is similar for flow cytometry and bb mode, considerable time can be saved on manual handling and the processing of samples for flow cytometry (approximately 2-3 h for 60-90 samples). summary/conclusions: we have been able to embed the sysmex bb mode into a routine production environment and confirm that its performance in spiked samples is mirrored in routine use. for platelets, sample collection in edta is essential. the bb mode offers an opportunity to reduce operator time compared to flow cytometry whilst gaining additional information on rrbc. abstract withdrawn. results: in our experiment, the typical size of a spectrin matrix section (l) was 60 to 220 nm (without oxidation). the heights (h) of dips were 4 to 10 nm. due to oxidation, the junctional complexes between spectrin and membrane proteins can rupture. only 10% to 15% of the spectrin surface has the same structure as in the control group. the values of l and h vary significantly depending on the intensity and time of exposure. we observed significant changes in the spectrin matrix after exposure to uv radiation in a model experiment. the local topological defects in the membrane arise from the action of oxidizing agents on the red blood cells. the mechanism of their appearance is connected mainly with the distortion of the spectrin matrix. as a result of oxidation processes, the spectrin molecules can be damaged. there is a transformation of tetramers to dimers. additionally, it can be easily seen with the afm, that spectrin network structure was essentially destroyed. most parts of the spectrin matrix have damaged structures with mesh breaks and dips after uv irradiation. also the results of network distortions in response to temperature changes were obtained. there are presented preliminary results of spectrin matrix change during long-term storage of prbc. summary/conclusions: atomic force microscopy in direct biophysics experiment allows to observe and to quantitatively measure the disturbances in the spectrin matrix nanostructure in response to oxidation processes in rbcs. these studies are important for the fundamental research of interactions of rbcs on the molecular level during redox processes and the consequences to their structure and function on the cellular level. this is important for the advancement of transfusion medicine, intensive care medicine, and molecular and radiation biology. methods: blood samples were taken from 8 donors during a prophylactic examination and collected with edta-filled microvettes (sarstedt ag and co., germany). all experiments were carried out in accordance with the institute guidelines and regulations. the polylysine-coated glass was used to perform the sedimentation method for formation of native rbc smears. it is important that any fixatives weren't used. the stiffness of rbc membranes was studied in native rbcs (control) and native cells after the application of modifiers: glutaraldehyde, hemin, zn 2+ (heavy metal ions). local stiffness was studied by atomic force spectroscopy (afs) (ntegra prima, (nt-mdt, russian federation). results: experimental kinetic curves i(z) were measured. nonlinear fitting method was used to determine the young's modulus. the experimental dependences of membrane bending were approximated by the hertz model to a depth up to 600 nm. the young's modulus e = 10 ae 5 kpa for control rbc. it was shown that some natural oxidants (hemin), membrane fixatives (glutaraldehyde) modifiers, heavy metal ions (zn 2+ ) significantly increased the absolute value of the young's modulus of rbc membranes up 2-3 times. the biophysical parameter hertz depth (h hz ) was determined for each curve. under the influence of modifiers the hertz depth h hz was changed from 200 nm to 600 nm. there are presented preliminary results during long-term storage of blood. summary/conclusions: the blood rheology is determined by rbc deformability, associated with membrane stiffness. the young's modulus can be used as a quantitative criterion to estimate the membrane state of a native cell. the results of the work can be used in clinical practice, in assessment of the quality of donor blood during storage before transfusion, in biophysical studies of rbc state. abstract withdrawn. immunohemotherapy, centro hospitalar universit ario são joão, epe, porto, portugal background: transfusion of blood and blood components is an essential resource in modern medicine. a proper use of human blood, being an irreplaceable resource, is necessary in order to achieve minimal wastage. blood wastage may occur for a number of reasons, like expiry date, haemolysis, seroreactivity or low volume. monitoring wastage of blood product during collection, testing and processing of blood is used as a quality indicator. aims: to determine the annual rate of discarded blood components due to expiry date in a portuguese university hospital blood bank (bb) from january 2016 to december 2018, in order to implement appropriate measures to minimize the number of discarded blood to a reasonable rate. methods: we retrospectively analysed the rates of blood components discarded after meeting their expiry date of a portuguese university hospital blood bank from january 1st 2016 to december 31st 2018. results: a total of 54,599 whole blood units and 3,166 apheresis platelets were collected during the study period. of the 66,289 blood components (packed red cells, whole blood pooled platelets and apheresis platelets) prepared during the study period, a total of 2,891 (4.4%) blood components were discarded, of those 75.4% due to expiry date. the rate of discarded packed red cells, according to this component production, decreased considerably over the years, in 2016 was 5.6%, in 2017 was 3.8% and 0.2% in 2018. similar tendency was shown in the pooled platelets for 2 consecutive years with 4.8% (2016) and 3.8% (2017), but with an increase in 2018 (7.7%). the rate of apheresis platelets had a more variable behaviour from 2016 to 2018 with rates of 1.3%, 0.6%, and 0.9% respectively. summary/conclusions: blood transfusion is an essential part of patient care. for this reason, the implementation of a quality system and continuous evaluation of all activities of the bb can help to achieve maximum quantity and quality of safe blood. we identified that date expiry was the main reason of discarded blood components, although there was a significant decrease in the rate of discarded packed red cells over these three years. properly implemented blood transfusion policies, donor screening and training staff as well as implementation of automation also helps to improve the process, reducing the discarding rates of blood and blood component. background: storage performance of platelets (plt) is associated with age of the donor. the risk for plt with poor storage performance, characterized by high lactate production and rapid acidification of a plt concentrate (pc), shows a positive correlation with age. we wished to explore whether high lactate production was associated with donor health issues. aims: to investigate high lactate production by stored platelets in relationship to donor health. methods: single-donor pc were collected by apheresis or prepared from 1 buffy coat and donors were evaluated who could be marked as 'rapid acidifiers'. in total, 31 apheresis pcs and 66 pcs from whole blood were included in four studies. information about donor health was obtained either from the blood bank information system or using questionnaires. in some donors, the lipid profile was measured from plasma, and the diabetes marker hba1c from red cells. triglyceride levels > 2.5 mmol/l and hba1c levels > 42 mmol/mol were defined as high. results: twenty two percent (21/97) of the donors were marked as 'rapid acidifiers' and 71% (15/21) of these donors had health issues. 'rapid acidifiers' were of age 57, 31-69 (median, range) years. three groups of donors can be distinguished: a) donors affected by metabolic syndrome, prediabetes and type 2 diabetes, indicated by high cholesterol and/or triglycerides, high hba1c and/or the use of medication to treat diabetes. b) donors affected by vascular diseases who reported or used medication to treat high blood pressure. c) "other" donors who used other medication to treat various other conditions. the remaining 'rapid acidifiers' (29%) did not have high triglyceride or hba1c levels and did not report health issues. summary/conclusions: pcs with rapid acidification by high lactate production are mainly collected from older donors with health issues. we postulate that high lactate production by stored plts is associated with health issues, and we will combine detailed donor information (health and behavior) with in vitro quality for a significant number of donations. background: the development of applied biotechnologies requires a search and creation of new methods of cells' functional completeness analysis. the instrumental assessment of platelets quality for the selection of the most effective donors, quality assessment of platelet concentrates for short and long-term storage and for the selection of platelets for cryopreservation is in demand in blood service. assessment of platelets morphofunctional status is possible using morphological studies of various platelet granules fractions (makarov, med. alfavit, 2012). among the biologically complete platelets there is a special population of cells, the so-called granule-rich platelets (grp). these cells contain the largest number of cytoplasmic granules (more than 10 visually distinct granules). it is established that grp have increased viability and functional activity. earlier we found a correlation between the grp level in blood plasma and shift of the redox potential value in blood plasma after cryodestruction of platelets (tsivadze et al., doklady physical chemistry, 2016). it was suggested that the shift of the redox potential may be partly due to the release of the low molecular weight antioxidants contained in functionally complete platelets outside the cell. in turn, the concentration of low molecular weight antioxidants can be estimated using the cyclic voltammetry. aims: the aim of the study was to estimate of cyclic voltammetry method possibilities for quality assessment of platelets. methods: the functionality of platelets was examined in platelet concentrate (pc) obtained by apheresis (1246.8 ae 90.1/ll). voltammetric analysis in pc before and after the platelets cryodestruction was carried out on platinum electrode in the potential range from à600 mv to +1100 mv using a potentiostat ipc pro l and saturated ag/agcl electrode as reference. for the morphofunctional analysis platelets were vitally stained with fluorochrome stains trypaflavin and acridine orange. microscopic examination of platelets was carried out using confocal microscope nikon eclipse 80i. the following parameters were evaluated: concentration and percentage of platelets with granules and concentration and percentage of grp. results: voltammetric studies in pc show that there are two oxidation peaks of low molecular weight antioxidants on voltammogram at potentials + 500 mv and + 800 mv. analysis of pc before and after the cells cryodestruction showed that changes in the height of oxidation peaks occur, indicating an increase of antioxidant content in blood plasma. at the same time a correlation between the changes in the height of oxidation peaks and the grp content in the sample was found. in samples with reduced initial grp content (less than 10%) after the cells cryodestruction significant changes in the height of oxidation peaks were not observed, regardless of the total number of cells in the pc. summary/conclusions: in conclusion, voltammetric analysis allows to indirectly estimate the population of functionally active platelets that in combination with other methods of analysis can serve to assess the quality of platelet products. background: determination of hemoglobin derivatives in blood is one of the most important studies in clinical laboratory diagnostics, especially during the storage of donor blood and its transfusion. concentration of hemoglobin derivatives can be changed during redox process. aims: to show the possibility of using non-linear fitting method to calculate concentrations of hemoglobin derivatives during reduction-oxidation processes. methods: for this we performed model biophysical experiment, in vitro. blood samples were collected into edta microvettes from 4 healthy donors (sarstedt ag and co., germany) during prophylactic examinations. all the donors gave their consent to participate in the study. a suspension of erythrocytes was prepared in pbs buffer with ph 7.4. we used ultraviolet (uv) irradiation of blood or nano 2 as oxidizing agent. the drug cytoflavin (stpf "polisan", russian federation) was used as an antioxidant. in our study we used digital spectrophotometer (unico 2800, usa) to measure the absorption and scattering of light (0.5 nm step). the method of nonlinear fitting was used to find the concentrations of hemoglobin derivatives. the empirical spectrum d l (k) exp was approximated by the theoretical curve d l (k l ) theor , which fits the experimental curve in the best way. under approximation the light absorption by different hemoglobin derivatives was considered in model. simultaneously effects of rayleigh light scattering on structures with size d<< k (coefficient s) and light scattering on particles with size d≥ k (coefficient k) were taken into account: d l (k l ) theor = e hbo2,l c hbo2 l+ e hb,l c hb l+ e methb,l c methb l+ e hbno,l c hbno l+ e methbno2 -,l c methbno2 -l+ e methbno,l c methbno l+k+s/k 4 l (1), where e h,l is the molar absorption coefficient for each hb h derivative at given wavelengths k l , c h is the concentration of the derivatives hb h , l is the thickness of the solution layer, d l (k l ) is the optical density of the substance, k and s are the parameters of the model. results: we determined the concentrations of hemoglobin derivatives without any additional chemicals in blood. there were measured experimental spectra for different agents action on blood. it was shown that concentration of methb increased after uv irradiation and nano 2 action (up to 90%). there were calculated c h for each hb h derivative. it was established that theoretical curves coincide with experimental data with good accuracy (r 2 = 0.98). incubation of rbcs with cytoflavin leads to reduction of methb to hbo 2 . summary/conclusions: the determination of hemoglobin derivative concentrations by the method of nonlinear fitting (without adding special chemical agents to blood) can be used for measurement of carboxyhemoglobin in blood during toxic state of organism. also it is important for assessment of rbcs quality before blood transfusion. background: the use of in line leukoreduction filters have been highly expanded in iranian blood transfusion centers within the last decade in order to provide sufficient leukoreduced blood fractions from healthy safe frequent blood donations to be supplied to the leukocyte sensitive patients. leukoflex lcr5, the dominant brand of such filters procured by iranian blood transfusion organization, is the most updated generation of the filters used around the world. aims: in this study, it is tried to recover the trapped leukocytes from this novel filter by different buffering systems and having optimized the elution mode, the cell differential of the viable recovered white blood cells were determined by flow cytometry. methods: having passed the routine virological tests, eight leukoflex lcr5 leukoreduction filters freshly used in tehran blood transfusion center were daily collected and each were back flushed by a self-designed mechanical system (a peristaltic pump, a triple junction with regulator part and an air pump) using various conditions and additives for pbs buffer at different phs in order to find the highest recovery yield for leukocytes. the optimized elute was characterized by flow cytometry for subcellular profile to be determined. results: it was illustrated that a system consisting of pbs (without cacl2 and mgcl2) in ph 7.2 containing 2 mm edta and 4%(w/w) dextran 40 without additive amounts of triton x100 was the most optimized buffering system for lcr5 filter back flushing. total cell content was also determined as 6.28*10 8 granulocytes, 4.27*10 8 lymphocytes and 0.8*10 8 monocytes using auto hemoanalysis and flow cytometric methods. summary/conclusions: in addition to partly compensating of the overhead expenses inflicted by application of leukoreduction filters on healthcare system, the results will assist blood organization system to be more classified in rational profile design, future cell therapy strategies and exceptional blood management. also, the recovered cells could be of significance in stem cell science, cellular interaction studies as well as novel molecular developments in drug discovery. vox sanguinis (2019) results: three lines of strategy are in place to pursue self-sufficiency of the largest number of pdmps. first strategy line: maximizing the yield of driving proteins, represented by immunoglobulins (ig) and albumin; this was assured by csl behring with a yield of 5.2 g ig (70% intravenous -privigenand 30% subcutaneous -hizentra) and 26 g albumin (alburex) per kg plasma fractionated, corresponding to 998.000 g ig and 4.992.000 g albumin; based on present demand, this represents 97% and 100% self-sufficiency, respectively, for naip regions. second strategy line: ensuring other products from plasma fractionation; the fractionator granted 6.000 g fibrinogen (riastap) and 6.000.000 iu vwf/fviii (haemate p) per year, which corresponds to the present demand of naip regions for both products, but it is under the full potential of plasma, thus providing a high margin of safety in case of increased demand (now the case of fibrinogen). third strategy line: exchanging cryoprecipitate, fibrinogen and vwf with italian regions whose plasma is fractionated by other companies to obtain prothrombin complex concentrates (pcc -kedcom) and antithrombin (atked) as to satisfy naip regions demand; this strategy allowed a supply of 5.000.000 iu at and 3.000.000 iu pcc, capable of ensuring self-sufficiency for naip regions until 2020. summary/conclusions: in italy, differentiation of plasma contract manufacturing among companies with different portfolios allowed naip regions to obtain a significant contribution to self-sufficiency from vnrd plasma for a variety of pdmps by different and complementary strategies consisting in maximizing the yield and the portfolio of proteins from the fractionator and exchanging products among regions for other pdmps at high demand but not included in the portfolio of a single fractionator. plasma check system: a valuable tool for plasma freezing validation and monitoring. background: the validation of plasma freezing processes may result problematic in the monitoring/control of critical process parameters (cpp). in 2017 in italy 923,944 litres of plasma were produced and frozen. aims: in order to assist plasma freezing validation and cpp monitoring, 151 of the 176 italian bes performing plasma freezing utilize the plasma check system (pcs), a system able to record, store and certify the temperature (t) detected at the core of "surrogate" bags during the entire freezing session, consistently with gmp requirements. pcs is patented and commercialized by expertmed srl, verona, italy (http://www.expertmed.it). methods: pcs consists of 3 parts: a) "surrogate" bags (check-bags) of 250 and 700 ml corresponding to the average standard volumes of the real products, containing a fluid validated to simulate the thermal behaviour of plasma; b) a mobile probe (cryo-med) positionable at the core of the check-bags; c) a dedicated software (memo-track). plasma freezing session data are tracked via barcode/rfid and can be consulted by the pcs that associates blast freezer code, operator code, cryo-med and check-bag. data on plasma freezing are stored in a shared folder and transferred to the be information system. the pcs can also be used to check and monitor the out-of-storage variations of core t of frozen plasma unit, i.e. during labelling and packaging procedures, thus allowing to establish optimal timeframes and operations and suitably validate these procedures. in the period 2016-2018, at the pievesestina be of emilia romagna region 210,871 plasma units were frozen so as to allow complete freezing within 60 0 to a temperature below à30°c, in 8,349 freezing sessions, using the pcs both for process validation, change control and for the systematic monitoring of core t at each freezing session. furthermore, at the bologna be 108 tests on the out-of-storage conditions of plasma units were carried out to revalidate the procedures of labelling and packaging. results: out of 8,349 freezing sessions carried out at the pievesestina be, 27 (0.3%) were detected to fail to reach à30°c at the core of the check-bags within 60 0 . of the latter, in most cases (70%) a technical error in the activation of the cryo-med was identified. in addition, the pcs was systematically utilized for periodical revalidation of the freezing procedures. the tests performed at the bologna be to validate the out-of-storage procedures of frozen plasma labelling and packaging allowed to modify the operating procedures in place so as to establish optimal timeframes and operations. this prompted corrective actions regarding: i) number of units to be taken out of storage sites at each labelling session (<24 units), ii) labelling time (<8 0 ), iii) optimal storage t (à40°c vs. à30°c), iv) optimal time between two openings of storage sites (>30 0 ). summary/conclusions: the pcs is a valuable system for plasma freezing validation and monitoring, as well as to perform monitoring and control of the whole pathway of frozen plasma in the be. it is a technologically advanced, easy-to-use and costeffective tool that can efficiently replace other traditional methods commonly used for the above-mentioned purposes. assessment of blood group matching quality using six sigma metrics background: six-sigma metrics provides a general methodology to evaluate a process performance on a sigma scale. implementation of six-sigma for quality assurance can benefit the health care sectors. one of the most important health care sectors is blood transfusion service. for that reason, maintaining a high quality in blood transfusion service is required. pathogen in activated plasma is one of the main products that are provided by the blood transfusion service. the process of producing pathogen inactivated plasma involves blood group matching step. the quality of this blood group matching is extremely significant for the delivery of plasma that satisfies the recipient need. aims: the aim of this study is to assess the quality of blood group matching of pooled plasma units using six sigma metrics, and to clarify the potential implementation of six sigma metrics as a quality management tool. methods: this retrospective study was conducted in the component preparation lab of kuwait central blood bank. the twelve months (january 2018 -december 2018) data of pooled fresh frozen plasma units were recruited and examined. the data was separated to data without double check (6 months) and data with double check (6 months). data statistics and analysis were conducted by the use of six sigma metrics. results: in a sample size of 6818 from the first six months a 38 mismatch was found which equals 5573 dpmo and 4.1 sigma metric. and in a sample size of 5854 from the second six months a 25 mismatch was found which equals 4277 dpmo and 4.2 sigma metric. out of the whole 12672 pooled units 63 were found to be mismatched. some of which were found to be discarded as abo discrepancy, broken, or expired. other was still available in the system, while the rest of the mismatched units were issued. summary/conclusions: using the six sigma principle the study presents a successful assessment of blood group matching quality. as a 4.1 sigma metrics obtained from the first 6 months, were shifted to a sigma metrics of 4.2 in the second 6 months, after the addition of a double-checking step to the blood group matching of pooled plasma process. the implementation of these metrics in our laboratory quality management has been shown to be very beneficial. in which six-sigma metrics were able to clarify the reduction in blood group matching errors. although six-sigma benefits in major quality improvements and helps to reach an error free laboratory services, yet it presents a new challenge to laboratory practitioners. currently, the hemophilia a patients treated with factor viii concentrated as the first line of therapy but it is more expensive and the supply is not sufficient so for now they have not used factor viii concentrate as prophylaxis therapy. for some cases, hemophilia patients in indonesia depend on subsidy from the world federation of hemophilia. the first handicapped concentrated case is just for therapy not for prophylaxis. big blood centers in indonesia produce routinely fresh frozen plasma (ffp) and cryoprecipitate-anti hemophilic factor (ahf) as replacement therapy for hemophilia a, but its content and safety of factor viii from 150 ml ffp need to be improved. nowadays, there is an available kit for producing minipool cryoprecipitate (mc) that has better safety and quality but it is available as liquid products, stored in very strict and specific temperature (à20°c). prophylaxis therapy for hemophilia patients needs a stable product, easy to use and convenient treatment for patients. aims: to analyze the content and safety of f viii with minipool cryoprecipitate (mc) and lyophilized mc for home therapy. methods: we produced 7 mc; 1 mc as the control, 3 mc were lyophilized with excipient and 3 mc without excipient. we analyzed the number of factor viii, the safety, and stability. we count the erythrocyte, leukocyte and platelet residual in mc using flow cytometry. we also measure the ph, osmolality, solubility to learn its stability after storage at 30 days at room temperature (30-32°c) and blood bank refrigerator temperature (2-8°c) at central blood transfusion services (cbts). results: we found the content f viii with excipient is higher (9.8 iu/ml) than without excipient (4.5 iu/ml) and the storage at blood bank refrigerator (2-8°c) is better than at room temperature (30-32°c) . in both group, there were no residual cells and bacterial found in mc. no significant difference in the ph, osmolality and solubility in both groups. summary/conclusions: the lyophilized mc with excipient stored at blood bank temperature (2-8°c) is better than room temperature. this experiment will be continued to know its stability in extended storage time. background: peptic ulcer disease (pud) is a multifactorial and complex disease, and it affects a wide range of people in the world. however, a perfect therapy for pud has not yet been available at present. therefore, we provided a novel therapeutic approach for pud patients and observed its effect in this study. aims: we provided a novel therapeutic approach for pud patients and observed its effect in this study. methods: in this randomized controlled trial, pud patients residing in chongqing were enrolled from 2016 to 2017. they were randomly assigned to two groups: (a) a control group used only rabeprazole, and (b) a platelet-rich plasma (prp) group that received a combined therapy of autologous platelet-rich plasma (aprp) and rabeprazole. the aggregation rate of aprp was measured via aggregation remote analyzer module. the therapeutic effect was assessed via the ulcer size and the symptom score. all data were recorded and analyzed statistically using spss. results: a total of 27 patients were included (12 patients as control group) and (15 patients as prp group) in the analysis. we found that the aggregation rate of aprp is not affected in ph 2.5 after treatment with pepsin. our results showed that there were no significant differences between the prp group and control group before the treatment, and there was also no significant difference in healing time between the two groups in different variables. however, regression analysis revealed that the healing time was 6.99 d less in the prp group than in the control group, and the patients with higher symptom scores in the initial examination need more time to heal in treatment. summary/conclusions: this study showed an encouraging preliminary result that aprp has a positive result in the peptic ulcer patients, and it seems to be a better choice for refractory pud patients. despite the further follow-up studies are needed to determine the duration of efficacy of aprp, the approach will be helpful for improving the pud treatment in clinical. background: the croatian institute of transfusion medicine (citm) collects, produces and distributes blood components in an area of 4.2 million habitants. annually, it collects about 100,000 whole blood and 3,500 apheresis donations. platelet concentrates (pcs) are more inclined to bacterial contamination due to storage conditions that favor bacterial replication. the citm decided to evaluate the mirasol pathogen reduction technology (prt) system as it offers the possibility to work with a non-toxic, non-mutagenic compound that upon uv illumination induce nucleic acid damage, reducing the risk of septic transfusion. aims: the study objective was to evaluate quality of pcs treated with the mirasol prt system for platelets and stored in tpas+ for 7 days at 22°c on a platelet shaker. methods: pcs were produced according to the citm's s.o.p., either through pooling of 4 bc with tpas+, "wbd", or through apheresis collection using two devices: the fresenius amicus, "ad" and haemonetics mcs+ system, "mcd". pcs were stored in 33% plasma and 65% pas and mcd were subsequently evaluated also in 38% of plasma and 62% pas. identical pcs were produced with a pool-split protocol to be prt-treated or serve as untreated control. pcs were treated with the mirasol system according to manufacturer's instructions. qc parameters, such as yield, ph and swirl were measured at days 1, 5 and 7. bacteria sterility test was performed at day 7 for a sample of all treated platelets. protein content of pcs produced routinely at the citm was determined to assess accuracy of plasma carry-over calculations for all processed pcs. results: mirasol-treated wbd (n = 10) and ad pcs (n = 8) stored in 33% plasma showed at day 7 an average ph ≥ 6.9; swirl ≥ 2.5 and yield = 3.3 9 10 11 . their untreated counterparts showed average values for ph ≥ 7:0, swirl ≥ 2.9 and yield 3.3-3.4 9 10 11 . mcd stored in 33% plasma (n = 6) that underwent prt showed at day 7 average values for ph = 6.7, swirl = 1.2 and yield = 3.05. control mcd showed average values for ph = 7.0, swirl = 2.8 and yield = 3.1 9 10 11 . mcd stored in 38% plasma (n = 8) that underwent prt showed average values for ph = 6.6., swirl = 1 and yield = 3.1. their untreated counterparts had average ph = 7.1, swirl = 2.9 and yield = 3.2. total protein content in pcs derived from wbd (n = 12), ad (n = 15) and mcd (n = 27) was 22 g/l, 20 g/l and 20 g/l, respectively. while the coefficient of variation of wbd and ad ranged from 3% to 4%, plasma products 129 respectively, the one of mcd reached 14%. all prt-pcs were negative for bacterial growth at day 7. summary/conclusions: mirasol treated wbd and ad produced according to citm current s.o.p. were quite similar to untreated controls at expiry, on day 7 and passed the requirements of the eu guidelines (19 th edition). quality of mcd units met eu criteria at day 5; swirl decreased significantly at day 7 which might be explained by the variability in plasma content of mcs+ -derived platelets, challenging the accurate calculation of illumination index for the mirasol treatment. all mirasol treated pcs showed minimal platelet loss at the end of storage. as the implementation of pr had to be cost-neutral it could only be implemented for~25% of the annual produced buffy coat platelet concentrates (bcp) (~40.000 bcp/year) and required a change in the bcp production method. the primary aim of the implementation was to offer increased blood safety to our most vulnerable patients. the secondary aim was to ensure that we built-up enough routine experience with pr to enable us to quickly ramp-up the production of pr-bcp to 100% if there were an outbreak of an emergent pathogen in the madrid region. aims: to verify if we could produce~25% pr-bcp without increasing the overall production cost (opc) for bcp. also evaluate the impact of pr on overall scrap rates of bcp, outdate rates and usage of other safety measures. methods: we compared opc for bcp between the pre-pr period (2017) . this cost was offset by substituting a semi-automated production method for bcp, which was used in 2017 to produce 28.7% of bcp-units. a manual double dose buffy coat production method (dd-bcp) in combination with pr enabled us to reduce the bcp-disposables cost by 89.2%. despite the moves from a semi-automated to a manual production method the overall scrap rates during production decreased in 2018 by 0.19%. the extension of max. storage time from 5 to 7 days for 24% of the bcp-units that were pr resulted in decreasing our overall outdating rates by 29% (versus 2017). this reduction in outdating rates reduced our opc in 2018 by 2.2%. in 2018 we gamma-irradiated 25.9% fewer bcp-units, but this had only a minimal impact on the opc. summary/conclusions: results of this study confirmed that we reached our initial objectives of producing~25% pr-bcp without increasing the overall production cost (opc) of bcp. it enabled us to offer increased blood safety to the most vulnerable patients. we built-up enough routine experience with pr so we could quickly rampup the production of pr-bcp to 100% if there were an outbreak of an emergent pathogen in the madrid region. background: irradiation of red cell units is undertaken to prevent transfusion-associated-graft-versus-host-disease (ta-gvhd) in immuno-compromised patients. while irradiators using radioactive c-ray sources are primarily found in blood establishments, they require regular recalibration and supplementary safety measures. xirradiation has been shown to have similar biological effectiveness to c-irradiation and does not require a radioactive source. there is international interest in moving away from gamma sources to reduce vulnerability to terrorism. although damaging, impacts of irradiation on red cells are well recognised. only a limited number of studies have compared red cell component quality following cand x-irradiation for both standard volume red cell concentrates (rcc) and neonatal red cell splits (rcs). aims: to compare the in vitro quality of rcc and rcs when subjected to cor xirradiation on day 14 of storage then stored for a further 14 days. rcs were also irradiated on day 5 of storage as that is most common practice in nhs blood and transplant (nhsbt). methods: four rcc were pooled and split into 4 arms on day 1 of storage, with 10 units in each arm. all units received an irradiation dose of 25.9-48.3 gy. two arms remained as standard volume rcc and were either cor x-irradiated on day 14 of storage. the other two arms were both split into 6 rcs on day 4 of storage before being irradiated on day 5 (early arm) or day 14 (late arm) of storage. for each replicate in these arms, 3 splits were c-irradiated and 3 splits x-irradiated. all arms were tested a day prior to irradiation and 1, 7 and 14 days post-irradiation for red cell quality parameters: haemolysis, intracellular atp and 2,3 dpg, supernatant potassium, glucose and lactate, ph and red cell microvesicle release. the rcc arms were sampled over storage; while for the rcs arms, 1 split was tested on each testing day post-irradiation. a 2-way anova was used to detect statistical differences over storage between cand x-irradiation for the same components. results: all components produced were within nhsbt specification for volume, haemoglobin and haematocrit. there were no significant differences in red cell in vitro quality parameters studied over storage between cand x-irradiated units, for standard volume arms or neonatal arms and whether rcs were irradiated early or late in storage. moreover, all arms were within haemolysis specification for the end of storage (>75% of units with < 0.8% haemolysis) and 100% of units had atp levels above the recommended minimum for acceptable post-transfusion survival (2.3 lmol/ghb). both haemolysis and potassium levels at the end of storage for the standard c-irradiated rcc were comparable to our laboratory's historic data for the same component. summary/conclusions: in summary, the storage quality of rcc and rcs post-xirradiation did not differ from c-irradiation in this study, providing reassurance that either method could be used in routine manufacturing. a pajares herraiz 1 , c coello de portugal 2 , m morales 3 , f solano 4 , c perez parrillas 5 , a rodriguez hidalgo 5 , t diaz rueda 5 and m flores 5 1 direccion, regional transfusion center toledo-guadalajara 2 transfusion service, toledo hospital complex, toledo 3 transfusion service, general university hospital of guadalajara, guadalajara 4 transfusion service, hospital nuestra señora del prado de talavera de la reina, talavera 5 regional transfusion center, regional transfusion center toledo-guadalajara, toledo, spain background: the regional transfusion center of toledo-guadalajara (rtc) manages the collection, processing and distribution of blood components for the hemotherapy area of castilla la mancha (spain) that serves 3 general hospitals (hospital complex of toledo (hct), university general hospital of guadalajara (ughg) and hospital nuestra señora del prado de talavera (nspt)) and the needs of 943,000 inhabitants. by also managing the hct transfusion service, it facilitates the handling of stocks. since 2008, rtc has initiated pathogen inactivation (pi) for a part of its platelet components(pc) with the intercept blood system (cerus) using a photochemical treatment with amotosalen and ultraviolet-a. this system allows the inactivation of a broad panel of pathogens and leukocytes, extending the shelf-life of the cp from 5 to 7 days. this affects the expiry and discards of this blood component, allows a better management of the inventory and has an influence on production costs. aims: the objective was to evaluate the influence of pi in the production of cp at rtc and the expiry in the hemotherapy area during the last 8 years divided into four periods ( results: pc were predominantly obtained from whole blood collections with 85% of bc platelets/15% of apheresis platelets. 77% of the available bc were used in production for period a and 88% for periods b, c and d. after wastes of approximately 1.9%, the distribution of pc was stable for the 4 periods studied. 7075 pc were distributed for period a, 7047 pc for b, 7467 pc for c and 7083 pc for d. the % of pi platelets with 7-day shelf life available in the 3 hospitals was limited to 13% during period a. it was then increased to 14.1%, 20.9% and 26% for periods b, c and d respectively. the percentage of wastes was stable at 0.2-0.3% but the discards due to expiry went down from 24.24% (period a) to stabilize at 10.9% in periods b and c and 10.3% in period d. in the 3 general hospitals the expiry went down from 20% to 5.89%(hct), 29.4% to 14.5% (ughg) and 33.36% to 17.68%(nspt) respectively. summary/conclusions: greater control of pc stocks through historical analysis and consumption projection, together with it tools and the use of pi pc with 7-day shelf life allowed reducing discards for expiry from 24.24% to 10.3% in the last period analyzed at rtc and the 3 major hospitals of the hemotherapy area. this has a great value in cost-reduction and improves inventory management and the efficiency of the processes. background: blood centers are faced with many challenges including availability of concentrate platelets as well as ensuring highest quality of the product. overcoming the shortage of platelet apheresis by using pooled platelet derived from whole blood units separated using automated standardized system, which can assist blood banks to meet the increase demand in platelets. the pathogen inactivation (pi) technology can improve the quality of the product by mitigating the risk of transfusion-transmitted diseases (ttd) and residual white cells, resulting in minimizing non -hemolytic transfusion reactions. however, the pathogen inactivation treatment must not impact the platelet quality and functionality significantly, as well as the patient safety. aims: evaluate the quality of pooled platelets derived from whole blood (five interim platelet units), separated using reveos automated blood processing system (terumo bct), pooled in 100% donor plasma and pathogen inactivated by amotosalen/uva technology. methods: five interim platelet units (ipus) produced with reveos device (terumo bct) from single whole blood donations, were pooled with a platelet pooling set (terumo bct) and leucodepleted with a lrf-xl filter (haemonetics). thirty pools have been included in this study, the units were treated using a large volume cerus intercept processing set for platelets according to the manufacturer's instructions and stored until day 5. the swirling was determined by visual inspection. the volume and yield content were assessed preinactivation and after treatment by pathogen inactivation with a cell counter (dxh-800, beckman coulter), rbc contamination was also measured preinactivation with a cell counter (beckman coulter), bacterial contamination was assessed by automated blood culture with a bact/ alert system (biomerieux). the ph of the platelet units was assessed with a phmeter (jenway), and residual amotosalen levels were assessed by an hplc assay. results: the impact of amotosalen/uva pathogen-inactivated pool platelet products quality were assessed. the pre and post-inactivation of the units showed a swirling score of 2-3. the average volume per unit of the pre-inactivation was 288 ml (278-302 ml) and post inactivation was 269 ml (254-284 ml), with average volume loss during inactivation was 36 ml (24-43 ml), corresponding to 13% (8-15%). the average platelet yield per unit pre-inactivation was 3.7 9 10 11 (3.0-4.4 9 10 11 ) and post inactivation 3.2 9 10 11 (2.8-3.9 9 10 11 ) with an average platelet loss of 13% (3-20%) . the average rbc contamination per unit (0.01-0.02 9 10 9 rbc/ml). the culture tests were negative, the average ph at day 1 was 7.4 (7.1-7.6), average ph at day 4/5 was 7.3 (7. 2-7.4 ). the average residual amotosalen concentration post treatment was 0.30 lm (0.23-0.35 lm). summary/conclusions: the quality of pathogen-inactivated pool platelets tested, met the criteria set by aabb guidelines. the volume and platelet loss were in acceptable range, in alignment with previously published data. a residual amotosalen concentration below 2 lm is considered safe and acceptable by french and german authorities. the evaluated data support the reasonable assurance of quality and effectiveness of the device when used in accordance with indication for use. background: the implementation of a pathogen inactivation process (pi) allows the redesign of processes to obtaining safe blood components by reducing the need for additional testing for pathogens detection, minimizing the residual risks (such as the infectious window period for those pathogens that are detected as usual), eliminates the need for selective tests (eg cytomegalovirus serology test) and complements gamma irradiation given its ability to inactivate white blood cells. in addition, the routine implementation of pi reduces the incidence of bacterial infection in recipients of blood components and allows blood services to proactively protect the blood supply against future emerging infections. aims: to verify the functional integrity and viability of platelet concentrates after being inactivated of any pathogenic agent, to be used as safe and functional components for transfusions. methods: a total of 106 independent platelet concentrates were studied. platelets are donated through a process called plateletpheresis according to the established norms, after the process, platelet concentrates were submitted to pi on the intercept blood system tm platform with uv-a illuminator; an immediate sampling of each donation of platelet concentrates was carried out taking a sample of 2 ml pre-inactivation and another sample post-inactivation (16 h after pi). the platelet viability of each sample was evaluated by demonstrating the cd62p expression marker by flow cytometry. once processed, platelet concentrates were released as safe components for donation. compiled the experimental data of the platelet count with platelet activation marker with respect to the total platelet, a comparative, nonparametric test of wilcoxon was carried out between two measurements (pre vs post) and the platelet viability after pi was determined. results: a total of 106 independent platelet concentrates were studied, where the average percentage of pre-inactivated platelets with expression of the cd62p marker, was 18%, while the percentage of functional platelets post inactivation was 19%, this result only shows that the functionality of the platelets is not being altered after the inactivation process. the wilcoxon test confirms that there is no significant difference between platelet activity pre-and post-inactivation, with a 95% confidence level. summary/conclusions: the process of photochemical treatment with amotosalen hydrochloride and long-wavelength ultraviolet light (uva) applied to platelet concentrates provides functional products without alterations in platelet function to be transfused. background: treatment of platelet concentrates (pcs) with pathogen reduction technologies is widely implemented in blood establishments to reduce the risk of bacterial contamination and to face the presence of new emerging agents in blood components. aims: the reduction of antioxidant power (aop) could be a quality control test to prove the complete viro-inactivation treatment. this evaluation has the goal to study the feasibility of the method from "abonnenc et al., transfusion, 2016" in another blood service, assessing the aop of platelet units treated by intercept technology. methods: the aop is expressed in edel value, one edel being equivalent to 1 lmol/l ascorbic acid. repeatability, intermediate precision and accuracy were determined. linearity was evaluated using the linear regression and the calculation of pearson's coefficient (r²). limit of quantification (loq) was determined by measuring aop using nacl samples to define the background. roc curves were used to determine a threshold to discriminate pcs before and after treatment. a distinction was realized between men and women and between apheresis (a) pc and buffy coats (bc) pcs. a one-year evaluation was assessed on pcs before and after treatment on the routine production. results: the coefficient of variation for the repeatability was less than 10%. for the intermediate precision, the coefficient of variation was less than 15%, but for the pcs after treatment, this result rose up to 21%. the r² value for the linearity was 97.7%. the detection limit corresponded to a result of 6 edel and the loq (equal to 10xsd) is 19 edel. concerning roc curves, the men apcs threshold was 58.5 edel compared to women apcs with 62.5 edel. the threshold for bcpc was 54 edel. all of these results had 100% of specificity. below this threshold, intercept treatment was considered to be executed. about the one-year experience on routine pcs production, 404 apcs (182 women and 222 men) and 263 bcpcs were tested. all of the bcpcs and women apcs were under the threshold after treatment. concerning men apcs, 14.0% of the pcs after treatment were not under the threshold. summary/conclusions: the device validation was satisfied. for the one-year evaluation and concerning men group apcs, the threshold found by abonnenc et al. was 89 edel. our study showed a threshold with 100% specificity and 90% sensitivity at 58.5 edel which is much lower. specificity was favored compared to sensitivity but the analysis should be revised to adapt the threshold to get higher sensitivity. this can lead to reduce the non-conformity and allows measuring the aop only after treatment. for women, our threshold was found at 62.5 edel compared to 66.5 edel for abonnenc et al. concerning sex in apcs, results were statistically lower in women group than men group before and after treatment. and for bcpcs, the two populations (before and after treatment) were very distinguishable and our threshold (54 edel) was lower than abonnenc threshold which was at 59.8 edel. in conclusion, edel threshold enables the segregation and depends on the preparation process adapted in each blood service. aims: this study has the goal of measuring antioxidant power (aop) level in plasma units treated by mb technology. the aim is to use such a test as a quality control assay for documenting the execution of pathogen inactivation treatments during the preparation of plasma units. methods: aop measurements were performed using a potentiostat electrochemical analyzer. a 3-ll volume of sample is deposited over the electrodes on a single-use microship. the aop is expressed in edel value, one edel being equivalent to 1 lmol/l ascorbic acid and reflects the redox status of the plasma units. different protocols were established to understand the role of mb, the illumination and the filtration on the aop variation measure: 1) complete treatment, 2) plasmas with mb without illumination, 3) plasmas without mb with illumination and 4) plasmas without mb without illumination. ten dosages on men donor samples, except for protocol 1 where n = 20, were realized during the viro-inactivation process, t1 corresponds to a dosage of plasmas before treatment, t2 the plasma after the mb dry tablet passage, t3 is the time after illumination and t4 corresponds to the final product (after filtration). results: in each protocol with mb, an increase was observed after addition of mb before illumination. after illumination, the edel values decreased for about less than 50%, which was expected because of the degradation of mb in its photoproducts during the illumination. in the series 1 and 3, the illumination seemed to have an effect by itself, with or without mb because the aop increased. the final filtration has the goal to eliminate the residual mb and its photoproducts. after this step, the aop values fell down. the series 2 was a confirmation of the efficacy of the filter to remove the mb as shown by the decreased aop in t4 (238 ae 16 edel at t3 and 209 ae 17 edel at t4). however, in the absence of mb (series 3 and 4), the results at t1 and t4 were not statistically different. summary/conclusions: the filtration decreases the aop rate, except when there was no mb. the results of non-complete viro-inactivation treatment allow concluding that the measure of aop rate may not indicate that the treatment was completed or not since significant differences before and after treatments were found in the non-complete treatment series. vox sanguinis (2019) background: the intercept blood system (ibs), a photochemical treatment with amotosalen and uva, is used to inactivate pathogens and leukocytes in plasma. the intercept tm plasma processing set (cerus bv, netherlands) was modified to incorporate plastic containers in non-pvc materials sourced from alternate suppliers and connecting parts and accessories in non-dehp pvc formulations, making the system dehp-free. the final storage container was modified with a higher contact surface with plasma to limit the thawing time. proportion of units with a fibrinogen concentration ≥ 2.00 g/l was 94% (>70% required). mean recovery fviii fibrinogen after ibs treatment and frozen storage were 75% and 90%, respectively. residual platelets were < 25 9 10 9 /l, leucocytes < 1 9 10 4 /l and red blood cells < 4 9 10 9 /l. all units had a protein content > 50 g/l. residual amotosalen was below 2 lm in all post-cad samples. the concentration of tat complexes was slightly reduced after treatment and frozen storage. concentrations of c3a and c5a were significantly reduced with the cad treatment. the plasma thawing time in a water bath at 37°c was consistently short (6-7 min). summary/conclusions: pathogen inactivated plasma units (ffp-a-ibs and ffp-wb-ibs) prepared with dehp free intercept processing sets retained in vitro characteristics which meet the quality standards for therapeutic plasma. the process did not activate coagulation or complement. reducing ffp thawing time from routine 8-10 to 6-7 min is an important benefit for emergency use. background: plasma coagulation factor concentrations usually differ for individual donors, therefore pooling of whole-blood derived plasma units moderates high or low coagulation factor concentrations and ensures transfusion of more standardized blood components. moreover, pooling contributes to dilution of reactive antibodies and may reduce the risk of non-hemolytic transfusion reactions and trali. additionally pathogen inactivation reduces the risk of transfusion-transmitted infections, and non-hemolytic transfusion reactions as well as gvhd through inactivation of residual lymphocytes. aims: assessment of the impact of plasma pooling and pathogen inactivation on the standardization of blood components and plasma quality. methods: the study included 5 experiments. for each experiment 5 male-donor, abo-compatible whole-blood derived plasma units (≥ 270 ml) were collected from different donors and pooled using the donopack optipool plasma pooling set (cerus europe b.v.). each of the 5-unit pools were split into 2 equal minipools which were subsequently treated with the in intercept blood system (cerus europe b.v.). then, each minipool was split into 3 (≥200 ml) therapeutic units. samples were collected before and after pooling as well as after inactivation to assess the coagulation factor content (fviii, fix, fibrinogen, vwf antigen using elisa) and coagulation time (aptt, pt). the study-analysis included samples from five pools from 5 single plasma units respectively ( background: biotin (bio) is an alternative to radioactive red blood cell (rbc) tracers which allows one to concurrently track in vivo multiple cell populations labeled at different bio densities. in american clinical trials, multi-labeled biorbc have been transfused in man to assess their survival (mock et al, transfusion, 2018) . in these studies, the different biorbc populations were monitored by ex vivo flow cytometry analysis using streptavidin. so far, the biotinylation reagents biosulfonhs was not complying with good manufacturing practices (gmp). moreover biorbc, with bio ≥ 18 lg/ml, have induced immunization of the recipient, in rare cases (schmidt et al, transfusion, 2017) . this represents an obstacle regarding the regulatory european authorities. aims: the aim of this study is to describe a procedure of biotinylation of rbc intended for clinical trials while refining the levels of bio ≤ 18 lg/ml. methods: sterile status is met throughout the process. rbc are taken from standard rbc concentrates and treated with biosulfonhs of gmp-grade (0 to 70 lg/ml) recently commercialized. washing buffer is of injectable-grade. biotinylation efficacy is controlled by flow cytometry with streptavidin conjugated to 2 different fluorochromes: phycoerythrin (pe) or brilliant violet (bv421). results: labeling with biosulfonhs of gmp-grade or non gmp-grade is comparable and 4 populations of rbc could be easily distinguished between themselves and from unlabeled blood cells. biosulfonhs (lg/ml): 0 (mfi 0.4), 4 (gmp mfi 12; non gmp mfi 9.5), 18 (gmp mfi 44; non gmp mfi 42), 70 (gmp mfi 174; non gmp mfi 180). streptavidin-bv421 brighter than streptavidin-pe is a promising tool because it amplifies by 1.7 the signal of fluorescence and allows a good differentiation of the 4 populations of rbc treated with only 0, 1, 4 and 18 lg/ml biosulfonhs. summary/conclusions: this preliminary study explores the feasibility of multilabeled biorbc production for clinical trials. the benefits of this approach are to overcome the need for non-radioactive tracers, to follow simultaneously various populations of rbc and consequently to limit the number of volunteers, and to reduce the risk of immunization using bio ≤ 18 lg/ml. background: rejuvenation is aiming to revert ageing-related disease development. heterochronic parabiosis studies revealed eotaxin in young and old murine blood as a regulator of brain aging and neurogenesis. umbilical cord blood (ucb)-borne factors including tissue inhibitor of metalloproteinases 2 (timp2) and neonatal immune cells also contributed to rejuvenation in animal models. human platelet lysate (hpl) is commonly used by us and others for highly efficient cell propagation in vitro (burnouf et al., biomaterials, 2016) . published data indicate only limited differences between adult and ucb-derived hpl, partly questioning enigmatic rejuvenation effects. aims: to verify candidate regenerative factors in neonatal blood products we compared protein contents of neonatal and adult plasma and platelets, respectively. methods: heparinized ucb samples (n = 9) were centrifuged within 3 h to collect neonatal platelet rich plasma. aliquots from apheresis platelet concentrates (n = 9) were used as adult counterpart. platelet concentration was adjusted to 7-10 9 10 11 / l. plasma supernatants and platelets were obtained by centrifugation and platelet pellets were re-suspended in saline. after two freeze/thaw cycles at à30°c/37°c for platelet lysis (npl; apl) the platelet fragments were removed by centrifugation. the protein content was analyzed with a proteome profiler tm array. nine samples of each group were pooled to avoid individual donor variations. a threshold of 50,000 au spot density was defined as cut-off. data were analyzed by graphpad prism 8 using two-way anova. results: semi-quantitative evaluation of 105 analytes per array revealed significant differences. in plasma samples and platelets 63 and 48 analytes were detected above cut-off, respectively. in neonatal plasma we found more highly prevalent proteins (>200,000 au spot density) compared to adult plasma (6/63 vs. 3/63). thirteen proteins were significantly elevated in neonatal plasma including growth/differentiation factor 15 (gdf 15), platelet derived growth factor aa (pdgf-aa) and serpin e1 (p < 0.0001). more highly prevalent proteins were detected in npl (10/48) compared to apl (1/48), and 20 proteins were significantly elevated including vascular cell adhesion molecule-1 (vcam-1), platelet factor 4 (pf4/cxcl4), epidermal growth factor and lipocalin-2 (p < 0.0001). in adult samples only 10 proteins were significantly higher in plasma and three proteins in apl compared to the neonatal groups (p < 0.05 to p < 0.0001). summary/conclusions: we detected significant differences in regenerative growth factor and cytokine contents of neonatal and adult plasma and platelet samples, respectively. additional experiments are underway to further characterize their impact in distinct functional readouts. background: the production and storage conditions of platelet (pl) products intended for transfusion are constantly evolving and need sometimes in vivo evaluations in clinical trials to ascertain whether the platelets have retained their ability to survive in the circulation. this requires that the transfused platelets can be distinguished from the recipient's circulating platelets. labeling of platelets with biotin (bio) affords to track in vivo and concurrently, multiple cell populations covered with various biotin densities as already described for red blood cells (mock, transfusion, 2018) . surprisingly, there is only one study describing the transfusion of human biopl (stohlawetz, transfusion, 1999) . so far, the biotinylation reagent bio-sulfonhs was not complying with good manufacturing practices (gmp), which represents an obstacle regarding the regulatory authorities. aims: the aims of this study are 1) to describe a procedure to label injectable human platelets with 2 densities of biotin, 2) to evaluate the impact of biotinylation on platelet functions, 3) to track human biopl in the circulation of the mouse. methods: platelets are taken from standard platelets concentrates and treated with 1.2 and 10 lg/ml biosulfonhs of gmp-grade, recently commercialized. main platelet functions are assessed in vitro. human biopl survival is evaluated in immunodeficient nsg-mice treated with liposome-clodronate to eliminate macrophages and to prevent rejection. circulating human biopl are detected ex vivo by flow cytometry with streptavidin phycoerythrin. results: using trap (60 lm), p-selectin externalization reveals a normal capacity of secretion for all biopl. gpiba and gpiibiiia expression is not affected by the biotinylation process. biopl have the ability to aggregate: using arachidonic acid (1 mm), amplitude of aggregation is 81.7 ae 2.5% (bio 0); 82.6 ae 2.3% (bio 1.2 lg/ ml); 79.2 ae 1.6% (bio 10 lg/ml). using collagen (2.5 lg/ml), amplitude of aggregation is 65.6 ae 4.2% (bio 0); 61.4 ae 4.0% (bio 1.2 lg/ml) 40.8 ae 6.6% (bio 10 lg/ml). the 2 biopl populations could be easily distinguished between themselves and from unlabeled blood cells in the mouse circulation during more than 48 h. after 4 h, the mean fluorescence intensities are 0.13 ae 0.02 for unlabeled circulating mouse platelets, 1.01 ae 0.03 and 8.2 ae 0.15 for circulating human biopl covered respectively with 1.2 and 10 lg/ml biotin. summary/conclusions: this labeling approach should be helpful to evaluate new platelet products in vivo and represents an alternative to radioactive tracers. it allows to follow simultaneously different platelet populations and consequently limits the number of volunteers in clinical trials. background: severe ocular surface diseases, dry eye syndrome, persistent and recurrent corneal epithelial defects and diabetic or neurotrophic keratopathy are mainly successfully cured by standard treatment protocols. however, not rarely does refractory to these usual treatments appear, especially with serious forms of disease. in military medical academy, autologous serum eye drops -auto seds and autologous platelet lysate -apl eye drops have been being applied in the treatment of ophthalmological patients in these categories, who were previously resistant to standard therapy. aims: to show the achieved results of therapeutic use of autologous blood products (auto seds and apl) in the treatment of ophthalmological patients who previously had not responded to conventional therapysingle center experience. methods: auto seds are prepared by taking autologous blood into tubes (bd vacutainer, cat, 10 ml) and apl in tubes with anticoagulants (greiner bio-one, acd-a, 9 ml). control on tti of every patient and sterility of every series has been conducted. before and after the treatment, subjective ocular discomfort (ocular surface disease index -osdi), objective parameters of the tear film (schirmer's test, rose bengal, tear breakup time -tbut) and measuring of epithelialization zone were analyzed. apl, obtained from platelet-rich plasma which had been frozen, unfrozen and diluted with nacl solution, up to 30%. auto seds were administered in the form of 20% eye drops. results: auto seds have been applied to 17 ophthalmological patients (10 men and 7 women), previously resistant to standard therapy. in total 44 treatments were performed (each lasted 20 days). for successful curing, one or two treatments per patient, in average, were applied. apl has been used multiple times to one patient with sj€ ogren syndrome and severe multiple tropical corneal changes. all ophthalmological patients had subjective improvements (the average pre and post treatment osdi scores were 71.3 and 19.6 respectively). also, objective progress was present in 83% of all patients (p < 0.001). summary/conclusions: the use of auto seds and apl in the treatment of ophthalmological patients, previously resistant to standard therapy, is in constant increase, because of its simplicity and low expenses. apl has turned out to be better than auto seds for patients with severe trophic changes, because apl contains larger amounts of the nerve growth factor, tgf-b, vegf and platelet derived growth factor. however, a larger number of clinical cases is needed for future conclusions. background: whole blood (wb) has recently regained favor in treatment of massively bleeding patients in military and civilian settings. 1 platelets (plts) are a vital component in clot formation. as a component of wb, it is critical that they maintain functionality throughout storage. red blood cells (rbcs) stored in hypoxic/ hypocapnic conditions preserve high level of 2,3-dpg while reducing storage lesions stemming from oxidative stress. 2, 3 on the other hand, effects of steady hypoxia (pco2~10-20 mmhg) on plts contained in leukoreduced wb is poorly characterized. aims: examine the effects of hypoxic conditions on plt function and microvesicle (mv) formation in wb stored hypoxically (h) and conventionally (c) for 3-week storage at 1-6°c. methods: 11 units of wb were collected at mayo clinic rochester blood donor center from normal healthy volunteers into 70 ml cp2d. wb was leukoreduced using plt-sparing filter (terumo wb-s) then split into control (c) and hypoxic (h). h-wb was processed by the oxygen-reduction bag (hemanext, lexington ma) and unit was stored in o2-free bag. 5 ml of wb were collected from each unit at day 1, weeks 1, 2, 3. plt counts, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation, nonactivated and agonists activated plt surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac-1 binding), and microvesicles (mv) were measured by coulter counter and digital flow cytometer. paired student t-tests were used to analyzed differences in degradation rates; significance: p < 0.05. results: h-plt counts declined to~60% by the o2-reduction process, while similar decline was observed after 1 week in c, and thereafter remained steady. plt activation (ps) increased over time (h >> c after processing; c increasing more rapidly during storage). p-selectin increased over time (h < c), while pac-1 showed large increase after 1 week, then remained steady (h << c). plt activation by trap or adp declined modestly over 3 weeks (~15%) while h-plt showed additional~10% reduction for all time points. collagen activation for c-plt increased after 1 week (74%) and gradually increased to 100% after 3 weeks (~20% reduction with h compared to c). plt-derived mv (cd61 and cd61/annexin v) increased~4-fold over storage time; day 0 mv levers were significantly higher for h, but subsequent increase rates were similar or lower. total number of plt-derived mv (cd42a) in wb supernatant increased 17-fold after 3 weeks for c, while h suppressed increase to 7-fold. (majority of the trends described above showed significant differences between h and c.) summary/conclusions: plts were activated over 3-week period when stored at 1-6°c in leukoreduced wb, accompanied by a modest loss of agonist-induced activation. oxygen reduction treatment initially activated h-plts, while subsequent increase in activation rates were suppressed compared to c-plts. wb plts retained activatability, and hypoxic condition showed only modest further reduction on the activatability. hypoxic wb may provide higher quality wb for trauma patients if the levels of initial plt activation can improved during oxygen reduction procedure. methods: after informed consent, eligible patients were randomized to either first receive autologous followed by allogeneic seds or first receive allogeneic followed by autologous seds. each sed treatment phase was one month, separated by one month of patient's standard treatment (wash out period) between sed treatment phases. the patients each donated 500 ml whole blood from which the autologous seds were prepared. allogeneic seds were prepared from blood from never-transfused male donors with blood group ab. all serum was diluted 1:1 by adding saline, and aliquoted in an eye drop dispensing system (meise, schalksm€ uhle, germany). at each visit, the osdi was determined using a validated questionnaire, with higher scores reflecting poorer outcomes. the results were analyzed intention-to-treat, and a random effects linear mixed model for cross-over design was used. results: in total, 19 patients were enrolled, 4 of whom were excluded because they failed the autologous blood donation. background: the following blood components for non-transfusional use (bcntu) are produced in our transfusional center (tc): 1) allogeneic platelet gel (pg), derived from buffy-coats (bc) and human cord blood platelet gel (cbpg); 2) autologous serum eye drops (sed). the creation of both types of platelet gel started in 2014 but only in 2017 we confirmed the process for daily production: these blood components are used to treat pediatric patients with epidermolysis bullosa. the sed, produced from 2018, is dedicated to treat patients with dry eye syndrome. aims: production and storage bcntu. methods: the whole process production of bcntu is traced on the transfusional informatic system (emonet-insielmercato), under the same conditions of another blood transfusional components. the process takes place in closed circuit using the laminar flow hood. 1) pg production starts from the bc resuspended in plasma that are not used for daily platelet concentrates, instead the cbpg is produced using cord blood units that are not used for hematopoietic transplant. both have a platelet concentration between 800-1200 10 3 /ll and negative blood cultures, required by the italian law; the units are frozen at à80°c and last 5-year. pg and cbpg must be activated with calcium gluconate or batroxobin to be used. 2) the ophthalmologist's patients, with dry eye syndrome, donate 120 ml of autologous blood; the serum is separated and after the dilution with a balanced saline solution (30%) are divided in 2 boxes containing 30 single-dose vials each: they are stored at à80°c and they last one year. negative blood culture was evaluated. results : background: candida albicans is the most common pathogen detected in fungal infections. aims: in this study, we aimed to evaluate the in vitro antifungal activity of volunteerderived platelet rich plasma (prp) against c. albicans atcc 10231 strain and the possible effects of certain chemokines, kinocidins that might play a role in this activity. methods: prp from nine volunteers were derived by using magellan prp â kit. 10% calcium gluconate was used to obtain autologous thrombin. c. albicans isolates with a final yeast concentration of 1 9 10 3 cfu/ml and 1 9 10 4 cfu/ml were inoculated on sabouraud dextrose agar at the 1 st , 2 nd , 4 th , 8 th and 16 th hours of incubation to reveal the antifungal activity of autologous thrombin-activated prp. the colonies were counted after 18-24 h of incubation at 30°c. chemokines and kinocidins (platelet factor-4, interleukin-8 and thymosin-b4) were also measured simultaneously by elisa method. results: compared with the pbs-control group, the prp-10 3 group showed that the antifungal activity was still going on at the 8 th hour. the difference in colony production between the two groups at 8 th hour was statistically significant (p < 0.05). it was observed that the antifungal activity continued at the 4 th hour, decreased at the 8 th hour in the group prp-10 4 group. although the same amount of prp was used and the same amount of chemokine and kinocidins were released in both groups, the concentration of c. albicans was considered to be important in the detection of more effective prp-10 3 group. although there was an increase in il-8 levels by hours in the two prp groups by elisa method, no antifungal effect was detected against c. albicans. it was observed that decrease in tmsb4 values results from the antifungal activity on the advancing hours in the prp groups. whereas pf-4 did not act an antifungal activity on prp-10 3 and prp-10 4 . summary/conclusions: even in our study group where the highest platelet counts were obtained at the lowest concentration, c. albicans reproduction could not completely eliminated as mentioned in the literature. repeated doses of prp applications, such as drugs used in patients, may have longer duration of action and even complete repression of reproductive outcomes. background: generally, blood is available in developed countries for transfusion. sometimes, transfused or previously pregnant patients form alloantibodies to red cell antigens and rarely, to antigens of high prevalence. this case focuses on a twoyear-old girl, of pakistani descent, diagnosed with neuroblastoma stage iv with anti-in b and -e. although the publications indicate that 4% of the pakistani, indian or iranian populations are in(b-), it was discovered that this blood type is exceedingly rare. an international search was required to ensure blood product availability for chemotherapy and autologous hematopoietic progenitor cell transplants aims: illustrate the response of the public to a powerful story of a child needing rare blood for treatment and international collaboration for provision of very rare units. methods case report: a two-year-old patient's sample was referred for antibody identification. the patient had received four transfusions (883 ml of red cells) in the preceding 10-day period. hgb level fluctuations were consistent with decreased transfused red cell survival. following the last transfusion of 225 ml, the hemoglobin decreased from 8.6 to 5.8. anti-in b , and a ficin-only reactive anti-e was identified in the serum and anti-in b in the eluate. the monocyte monolayer assay predicted the anti-in b to be clinically significant (68% reactivity). transfusion of antigen neg units once obtained, resulted in a stable transfusion response. although it was expected that in(b-) blood would be more easily sourced, only two donors in the usa were in (b-) e-. as 7-10 units of blood were requested for the post-transplant period, a national and international search was initiated, as was a robust media appeal to donors resulting in many donors for an intense domestic screening effort in the usa. the search of the who international rare donor panel by the international blood group reference laboratory revealed three known in(b-) e-donors; two british and one australian. they were contacted, recruited, collected and shipped to the usa with the work of the american rare donor program (ardp) staff and the isbt working party on rare donors (isbt wprd) members in each of the countries. results: the intense media coverage of oneblood (the florida blood center collaborating on treatment with the hospital) included online news outlets (youtube, facebook) resulted in over 27,000 responses from national and international potential donors to be tested for in b . isbt wprd members were sent the web information of potential donors identified in their countries by the ardp. over 3,500 samples from 75 blood centers and associated laboratories tested with anti-in b by oneblood. two new in(b-) donors were discovered (0.06%); but both typed e+, thus were not a match for the child. summary/conclusions: this intense media coverage and the overwhelming donor response was unprecedented in our experience. the coordination and cooperation among the numerous blood centers reflect the deep dedication of the blood banking community to the well-being of special patients in need. this case illustrates the response potential that a powerful story and a medical appeal for exquisitely rare blood utilizing social media and other online news outlets can generate. background: blood platelet units are generally stored in blood banks for 3-5 days, afterwards they are discarded. prepared infusible platelet membrane (ipm) from fresh or outdated human platelets correct the prolonged bleeding times in thrombocytopenic animals such as rabbits. infusible platelet membrane (ipm) as a platelet substitute may be the most feasible approach to reach the target market. our previous experiments have shown that ipm has a hemostatic efficacy to shorten bleeding time without any adverse effects in rabbits. aims: abnormal toxicity is the european pharmacopoeia standard for assessment of biological products which the test material is administered to the mice. in this study, abnormal toxicity of ipm was evaluated in experimental animal model such as mice to assure the safety of ipm without any evidence of serious toxicity. methods: in this experimental study, infusible platelet membrane (ipm) was prepared from outdated platelet concentrates. platelet concentrates were pooled, disrupted by freeze-thaw procedure, pasteurized for 20 h to inactivate the possible viral or bacterial contaminants with a sodium caprylate stabilizer, formulated by sucrose and human serum albumin and finally lyophilized. at first, the test for sterility is carried out under aseptic conditions for ipm vials and then we injected 0.5 ml of ipm (2 mg/kg) intravenously between 15 to 30 seconds into each 5 health mice, weighing 17-22 grams. these tests were performed according to eu pharmacopeia monographs. results: in the sterility test no evidence of microbial growth in our product is found. the abnormal toxicity test will be passed if none of animals die during 24 h after injection. if more than one animal dies, the preparation fails the test. if one of the animals just dies, the test is repeated. in our experiment all five mice were alive after 24 h of ipm injection. summary/conclusions: in this research the results showed that ipm as a platelet substitute is free of abnormal toxicity with adequate safety and it may be used in human clinical trial studies as a feasible approach to develop a platelet substitute in the future. however, further studies are required to confirm the different aspects of its safety as well. the success of such investigations may affect patients' care in transfusion medicine in the future. a substantial number of infants, especially premature infants, are unable to receive adequate amounts of their mothers' milk for a variety of reasons. the world health organization recommends that infants, especially preterm and ill infants are fed with quality-controlled donor milk if they cannot be fed with their own mother 0 s milk. due to the possible transmission of the human immunodeficiency virus many human milk banks closed in the 1980s, therefore the availability of donor milk has decreased. aims: we analyzed the processing of donor milk and the required laboratory tests to establish a human milk bank within our blood donation service in cooperation with the department of neonatology at the frankfurt university hospital. methods: based on the recommendations for promoting human milk banks in germany, austria, and switzerland (efcni) we evaluated the manufacturing steps and the quality controls require to establish a human milk bank. background: for patients suffering from severe ocular surface disorders treatment with blood derived serum eye drops (sed) is a highly effective therapy. autologous sed, prepared from the patient's own blood, is used preferably. for this approach we have more than 6 years of experience. if auto-sed cannot be manufactured due to medical reasons allogeneic sed present an alternative. since 2 years, the allogeneic approach is well established in our center. aims: retrospectively evaluation of our experience with allo-sed. methods: in germany manufacturing of allo-sed is only possible as an "individual healing attempt". for each patient experienced regular ab0-identical male donors without blood borne disease, who never received blood products and not taking any kind of medication are selected. additionally, donors must pass a questionnaire excluding any form of dry eye syndrome. allo-sed are manufactured directed for each individual patient according to the process for auto-sed in a closed system. patient files of our serum eye drops donors were screened for patients receiving an allogeneic treatment. data concerning indication for allo-sed, contraindication for phlebotomy, problems with donor selection and manufacturing, as well as serological and microbiological testing results were obtained. clinical results were evaluated © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 by ocular surface disease index (osdi) and patient's questionnaire, asking for subjective benefit, symptom reduction, possible side effects, consumption and comparison with artificial eye drops or, if applicable, with auto-sed. furthermore patients are undergoing regular ophthalmologic examination within a special consultation for dry eye syndrome at our hospital. results: 31 patients were identified receiving allogeneic sed, 15 patients had been treated autologous previously. in total, allogeneic sed have been produced 54 times since june 2017. indications were ocular gvhd (n = 15.48%), neurotrophic keratopathy (n = 9.29%), mucous membrane pemphigoid (n = 2.7%), sj€ ogren syndrome (n = 1.3%) and secondary keratoconjunctivitis sicca by virtue of chemotherapy, meige syndrome, rosacea, morbus bruton (n = 4.13%). contraindications for autologous donation were underlying disease (n = 19.61%), poor venous access (n = 12.38%), low haemoglobin (n = 8.25%), low body weight (n = 8.25%), very young age (n = 7.22%), circulatory disturbances (n = 3.10%) and lack of response to auto-sed (n = 2.7%). some patients presented more than one contraindication. manufacturing problems were: lipemic donor plasma (n = 2.4%), high donor haemoglobin (n = 2.4%) and unspecific positive serological findings (anti-hbs n = 2.4%). microbiological testing was sterile every time. as side effects one case of allergic reaction, suspected as serum protein allergy, appeared. clinical outcome can be considered equivalent to ased. subjectively, all patients benefited from the therapy and reported an alleviation of their symptoms. for some indications (highly active gvhd) allo-sed might even be the better option. summary/conclusions: considering our previous experience, allo-sed seem to be a safe and equally effective alternative to auto-sed for patients unable to donate blood. in case of urgent indication, timely supply can sometimes be difficult. to overcome this disadvantage licensing allo-sed as a new blood product with the possibility of production and storage in advance would be a desirable goal. in addition supply would become even safer by preparing allo-sed according to a quarantine principle like ffp. abstract withdrawn. background: vernal keratoconjunctivitis is a chronic, recurrent bilateral inflammation of the outer ocular layer. mostly affected are children and young people and the condition is more common in boys. the disease presents with eye pruritus (itching eye), photophobia (sensitivity to bright light), excessive tearing and foreign eye syndrome. severe cases manifest with diffusion of overgrown papillae usually of the upper eyelid, bursting of the connective tissue barriers and appearance of giant papillae that press on the cornea. corneal ulceration is a severe complication of vernal keratoconjunctivitis that may induce scarring, corneal neovascularization and occasionally perforation. treatment of keratoconjunctivitis mainly relies on steroids, mast cell stabilizers, antihistamines, immunosuppressive drugs (cyclosporine), artificial tears, contact lensdressing, cryotherapy and surgical papillae removal. we present the case of a 8 year-old girl with corneal ulceration who was applied artificial tears after traditional methods of treatment proved unsuccessful. aims: the aim was to share our experience on artificial tears therapy applied in ophthalmic disorders. methods: autologous blood (20 ml) was collected into disposable, sterile transfer bags used for routine blood component preparation (no anticoagulant) and incubated for 1 h at 37°c. the clot was then removed by centrifugation and the serum containing erythrocytes was press extracted. centrifugation was applied again to obtain serum free of cellular components. the serum was then divided into 0.3 ml segments (capsules)and the artificial tears applied to the left eye 8 9 daily. results: ulcer healing was reported after 4 weeks of therapy with artificial tears. the dosage was reduced to 4 9 daily. no recurrence of corneal ulceration was observed after subsequent 8 weeks. summary/conclusions: artificial tears are a safe and effective therapy for ophthalmic disorders in children. background: arv non-disclosure among hiv-positive donors who tested hiv antibody (ab) positive but rna negative (ab+/rna-), so-called false elite controllers, was previously described by our group in south africa, with > 80% of ab+/rnadonations since 2016 testing arv positive. the extent of undisclosed arv use at time of donation represents a significant risk to blood safety in a country with a growing treated hiv population. aims: to establish the prevalence of arv non-disclosure among four subgroups of hiv-positive donors in south africa along with demographic correlates of non-disclosure. methods: south african blood donors are screened by a self-administered questionnaire, which includes questions on current hiv status and arv use, followed by a semi-structured personal interview. specimens for hiv, hepatitis b and c testing are collected at time of donation. based on id-nat (procleix, grifols) and antibody (prism, abbott; western blot) testing, hiv-positive blood donations were classified as acute (ab-/rna+), recent (ab+/rna+, limiting antigen avidity [lag] odn ≤ 1.5), longstanding (ab+/rna+, lag odn > 1.5) and potential elite controller (ab+/rna-) cases. stored plasma from these donations were tested for four arv drugs using qualitative liquid chromatography-tandem mass spectrometry (detection limit 0.02 lg/ml). chi-square tests were used to assess associations of hiv case type, gender, ethnicity, age, donor type, and donor clinic (fixed, mobile) type with arv non-disclosure. results: during 2017, 1671 donors tested hiv-positive of whom 1315 had samples available that were tested for arvs. the overall prevalence of undisclosed arv use was 9.3% (n = 122) with efavirenz most frequently detected (115), followed by lopinavir (5) and nevirapine (2) . potential elite controller cases had the highest proportion of detectable arv (68/80; 85%) (p < 0.0001) followed by longstanding (37/741; 4.9%) and recent (17/366; 4.6%) infections. none of 82 acute hiv cases tested positive for arvs. there were no associations between arv use and gender or ethnicity. however, older (35 to 64 years) hiv-positive donors (49/305; 16.1%) were significantly more likely to test positive for arv than younger (15 to 34 years) donors (73/1010; 7.2%) (p < 0.0001). arv use was more frequent among first time (101/ 716; 14.1%) than in lapsed (13/277; 4.7%) or repeat (8/322; 2.5%) donors (p < 0.0001). donors at mobile clinics had significantly higher arv non-disclosure than donors at fixed sites (10.4% vs 5.0%; p = 0.0051). summary/conclusions: the 9.3% prevalence of undisclosed infection and arv use among hiv-positive south african blood donors is alarming. higher rates of nondisclosure among first-time donors was expected, but non-disclosure among repeat and lapsed donors suggests failure in donor education and assessment. the 4.7% prevalence among concordant ab+/rna+ cases may suggest sub-optimal viral suppression. lack of detection of arvs in acute cases should be qualified because the samples were not tested for tenofovir, the most common drug used in pre-exposure prophylaxis. donor motivation for non-disclosure of known hiv infection and arv use needs further investigation, since early arv initiation or infection while on prep could lead to low ab and rna levels, failure to detect hiv-infected donations and transfusion-transmission of hiv. blood bank, rotary blood bank, new delhi, india background: voluntary blood donation ensures safe blood transfusion. careful blood donor selection is of importance to provide safe blood to patients, although new methodologies have also been adopted by blood centers for blood safety and to minimize risk of transmitting infections through blood transfusion. the quality and the availability of blood components depend on the willingness to donate and reliability of the information given by the donors about their own health, including risk behaviour. blood donor history questionnaire is designed to evaluate donor's history in accordance with the guidelines laid down by the fda. donors, once deferred by the blood bank, will be less motivated to return for donation if he is not counseled effectively. it is important to reduce the number of deferrals by good donor comprehension and the centre should have a mechanism to recall temporarily deferred donors aims: the aim of the study is to analyse donor history and test results of those who donated blood with past history of jaundice. based on their history which suggested the type of viral infection they had, these donors were accepted or deferred. data was collected from voluntary blood donors who were screened for blood donation in the year 2018. methods: in this study, donor history was analysed with reference to history of jaundice. jaundice in donors after the age of 11 yrs, history of surgery, blood transfusion, body tattoos and acupuncture treatment within past one year of donation, history of multiple sex partners and related history and intravenous drug abuse history was taken into consideration. donors who revealed past history of jaundice were asked in detail about their illness and recovery. blood was donated by donors from whom the history of jaundice was elicited and it was understood that the type of virus which caused jaundice was not hepatitis b or c. those who could not give the correct history or were not sure of the cause of hepatitis, those individuals were deferred. aims: to assess the performance of this follow-up program in terms of donor participations, successful confirmed positivity rates, and potential reentry rates. methods: eligible donors were tested for hbsag, hcvab, hivab/ag, and tpab with two eias for each marker. samples reactive with at least one assay were tested further with electro-chemiluminescence assay (eca) and reactive samples were considered repeated reactive (rr). tpab reactive donations were re-tested with particle agglutination assay (tppa). samples eca or tppa non-reactive were considered non-repeatable reactive (nrr background: the blood donation service in suhl processes more than 160.000 samples annually from whole blood and apheresis donations, testing on average around 600 samples per day. for the last 5 years, serology screening was performed on the architect instruments (abbott) (arc), but will be changed to the alinity s system (aly) by middle of 2019. although the design of the aly assays is based on those of the arc assays, we undertook a thorough evaluation of the four mandatory screening assays detecting hbsag, hiv ag/ab, anti-hcv and anti-hbc. aims: to validate the 4 mandatory screening assays on the new aly system in our lab in terms of sensitivity and specificity, also including samples with known falsereactive results. determine the rate of false reactive results for hbsag, anti-hcv and anti-hiv that may lead to deferrals of donations and donors. methods: for sensitivity, we used known positive samples confirmed by immunoblot or nat. known unspecific positive samples for arc not confirmed by immunoblot or nat were testes for aly also. close to 2.000 unselected samples (edta plasma) from routine blood and apheresis donors were tested in parallel on both systems, arc and aly to determine the rate of initial and repeat reactive results. results: all known confirmed positive samples were identical detected by aly. samples with known unspecific reactive results were retested by aly with the following results: 19/33 anti-hcv, 19/21 hiv ag/ab and 04/22 hbsag were found reactive by aly to. one donation from an acute hiv infection in the early seroconversion period was detected by both methods in routine testing. there are no reactive results for aly not already known for arc. the specificity for the screening assays on aly versus arc assays were as follows: 1) hbsag aly 100. 00% (1994 00% ( / 1994 00% ( ) vs arc 99.95% (1993 00% ( /1994 ; 2) hiv aly and arc 99.95% (1992/1993); 3) anti-hcv aly 99. 90% (1994 90% ( /1996 90% ( ) vs arc 99.75% (1991 90% ( /1996 . the number of anti-hbc reactive samples did not differ between aly and arc. summary/conclusions: while the switch to the new system is mainly driven by operational efficiency, obviously, the high specificity of the alinity s assay will reduce unnecessary deferrals of donations and donors. abstract withdrawn. background: blood donor selection is the cornerstone for blood transfusion safety, designed to safeguard the health of both donors and recipients. donor safety is targeted by reducing the risk of complications associated with blood donation and transfusion safety by reducing the risk of transfusion-transmitted infections (tti) and other preventable transfusion reactions. there is always a compromise on blood donor safety as well as blood safety during outdoor mega blood donation drives due to various reasons, mainly due to more number of donations within a stipulated time. aims: to compare the blood donor selection patterns between in house blood donations and donations at mega blood donation drives and its influence on donor safety and blood safety in a tertiary care hospital in india. methods: a retro prospective study was done to audit and compare blood donor safety and blood safety over a period of 2 years from january 2016 to december 2017. blood donor safety was analyzed by two indicators: donor health questionnaire (dhq) monitoring and blood donor reaction rates and blood safety through tti positivity rates. (78) during mega blood donation drive. summary/conclusions: a good donor selection is a lengthy process which involves pre-donation information and advice: this is usually provided in a leaflet, especially about transfusion-transmitted infections (and the associated risk factors) and the potential risks of donation, filling of dhqs by the donor himself, donor interview: conducted by a qualified medical specialist trained in donor selection process and health assessment at the end of the interview to declare if the donor is eligible to give blood or deferred temporarily or permanently. it was observed that seroprevalence rates, number of donor reactions and incompletely filled dhqs were more among blood donations at mega blood donation drives when compared to blood donations during in house collections. this is mainly due huge number of blood donations with in a stipulated time where there is limited time spent on proper donor selection. stringent implementation of who strategy: "safe donor safe blood" is the only way for blood donor and transfusion safety. background: safety of blood transfusion is a great concern especially in crisis countries and during humanitarian emergencies. transfusion transmitted infections (ttis) are one of the major health problem in yemen that are associated with blood transfusion complications. aims: the aim of this study is to determine the prevalence of ttis among blood donors at national blood transfusion and researcher center (nbtrc this contributed to an additional reactivity of 0.09%, thereby total reactivity being 2.39%. 55% (22/40) of these were hcv reactive & 45% (18/40) for hbv. the nat yield was 1 in 1086 and the viral loads of nat reactives ranged from 1-9 x10 4 iu/ml for hcv & all the hbv yields had an extremely viral load of < 06 iu/ml. 12/40 nat reactive showed sero-conversion after 4-8 months with follow-up eclia screening, and 7 of these were hcv reactive and 4 hbv reactives. summary/conclusions: incidence rate indicate that the current risk of transfusion transmitted viral infections attributable to blood donation is relatively high in our country. parallel use of both serology and nat screening of donated blood in countries that have high seroprevalence can improve the blood safety. at our centre, by using best in class serology and nat technologies, we were would add an extra layer of safety to blood supply by interdicting samples from donor with recent infections. abstract withdrawn. abstract withdrawn. (1/190,298) . the both hiv-rna and hcv-rna detected donors by nat were identified in the window period. summary/conclusions: in this study, we found that nat could detect 283 infected cases with hbv-dna, hiv-rna and hcv-rna which were forgotten by serological methods therefore, nat is a sensitive screening method to detect low viral load and shorten the window period of the virus infection to ensure the safety of blood transfusions. service du sang, croix rouge de belgique, namur, belgium background: due to enhancement of kits specificity and machines throughput, roche elecsys â technology is a potential partner for blood donations screening laboratories. aims: the aim of the study was to assess the performance of the elecsys serology assays on a cobas e801 equipment for clinical specificity, analytical sensitivity and reproducibility. background: deceased donors are the primary source of organs and tissues for transplantation but the risk of infectious complications in the recipient is high and is the main cause of morbidity and mortality after transplantation. to minimize the risk of infections by organ or tissue transplantation, donors should be tested for anti-hiv-1/2, hbsag, anti-hbc, anti-hcv, and syphilis. further laboratory tests may be required depending on the history of the donor and on the tissue properties. certain grafts can be donated after circulatory death of the donor; however, the absence of the heartbeat may change dramatically the blood composition by e.g., haemolysis and proteolysis. this may have an impact on test performance and lead to false results. therefore, an assay validation is needed for testing of cadaveric samples. aims: a validation study was performed to demonstrate the suitability of elecsys hbsag ii, anti-hbc ii, anti-hcv ii, hiv combi pt, hiv duo, syphilis, htlv-i/ii, and chagas for the use in cadaveric samples from non-heart beating donors. methods: as the basis for validation, we followed the recommendations of the paul-ehrlich-institut (pei) "proposal for the validation of anti-hiv-1/2 or hiv ag/ ab combination assays, anti-hcv assays, hbsag and anti-hbc assays for use with cadaveric samples". comparison of spiked samples from living donors and cadaveric donors was used to demonstrate accuracy. to determine precision, two cadaveric specimens were tested in several replicates. acceptance criteria were implemented according to the pei recommendations. results: results were found to be within specifications requested by the pei recommendations for all tested assays summary/conclusions: the evaluated results support the extension of the use of these assays with cadaveric specimens. background: in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. aims: in this context, metagenomic analyses of viral content in blood donations collected in geographical zones recognized as "hotspot" for viral emergence represents a suitable approach without any a priori for the identification of a potential emerging viral risk that may compromise blood safety. methods: in the framework of a viral discovery program founded by the french national agency for medicine security (ansm in french), more than 900 plasma samples collected in 7 sub-saharan africa countries (2011) (2012) and the amazon region of brazil (2016) have already been analysed by metagenomics. results: although no viral sequence could be described as novel (i.e. new species or even a new genus), we unexpectedly identified a feline bocavirus in two donors from mauritania. a large diversity of known viruses that are not part of the regularly monitored agents were also observed, among which anelloviruses, hpgv-1 (formerly known as gbv-c), papillomaviruses, herpes viruses, parvovirus b19, chikungunya virus, enterovirus, and various small circular viruses (circo-, cycloand gemycircularviruses). while no significative differences was observed in the higher classification of detected virus (above families/genera) between africa and brazil, we observed variations at the sequence level allowing better resolution of the genetic diversity for several viruses (for example characterization of hpgv-1 genotypes). summary/conclusions: overall, the absence of novel viruses in blood samples collected across countries of two distant continents is reassuring regarding threats emergence. however, continuous monitoring of prospective blood banks should be continued. summary/conclusions: after the high peak observed in 2014 during the first period, this study shows that the decrease in the seroprevalence of viral markers is continuous over the next five years. the second period is marked by an irregular evolution of seroprevalence but with lower levels than the first period. the recruitment of new donors allows a quantitative increase in donations. however, improving the quality of blood products essential condition of transfusion safety is achieved through retention of recruited blood donors. background: in blood screening laboratories, samples may be transferred between automated serological and molecular instruments, and the potential for sample contamination is a serious risk to the integrity of nucleic acid testing (nat) results. the sensitive limit of detection (lod) for hiv and hcv nat assays combined with the high viral titers encountered in specimens from patients with acute infections presents a challenge for maintaining the sample integrity of negative specimens. at additional cost per test, this risk can be reduced with single-use filter pipette tips. aims: we evaluate the efficacy of applying induction heated washes to a non-disposable pipettor on serology instruments-alinity s, alinity i, and architect i2000sr (abbott diagnostics)-to preserve the integrity of samples transferred to a downstream molecular instrument, the m2000 realtime (abbott molecular diagnostics), which amplifies viral nucleic acid targets exponentially. methods: in this application of induction heating, the metallic pipettor warms under its own resistance to coil-induced electrical currents. by sweeping the pipettor through an induction coil, temperatures on the pipettor are elevated throughout its length. single donor high viral titer hiv genotypes a (5.78 log iu/ml), b (5.90 log iu/ml), c (5.62 log iu/ml), crf01 (5.61 log iu/ml), crf02 (6.19 log iu/ml), and urf (6.33 log iu/ml), as well as single donor high viral titer hcv genotypes 1a (6.84 log iu/ml), 1b (6.73 log iu/ml), 3a (6.64 log iu/ml), 2 (6.10 log iu/ml), 2q (6.65 log iu/ ml), and 4t (6.07 log iu/ml) were used as potential sources of contamination; these genotypes account for the majority of hiv and hcv infections worldwide. on serology instruments, one high viral titer hiv or hcv specimen and three consecutive susceptible negative samples (hiv/hcv rna negative human plasma, abbott molecular diagnostics) were tested on an hiv ag/ab combo or anti-hcv immunoassay (abbott diagnostics), and this schema was repeated four times per positive specimen. induction heated washes occurred between all samples processed on the serology instruments. the first susceptible negative from each testing block, with approximately 1 ml of residual sample volume, was then tested using the 0.6 ml abbott realtime hiv assay (lod 40 copies/ml) or 0.5 ml abbott realtime hcv assay (lod 12 iu/ml) and an hcv ag immunoassay (lod 1.24 fmol/l; abbott diagnostics). study acceptance criteria required that any susceptible negative sample had no detectable level of hiv or hcv rna. results: all first susceptible negative samples (n = 24 per platform per virus schema) run on alinity s, alinity i, and architect i2000sr using induction heated washes after a high viral titer hiv specimen or hcv specimen were hiv ag/ab combo nonreactive (< 0.20 s/co) and reported no detectable level of the hiv rna target, or were anti-hcv nonreactive (< 0.20 s/co) and reported no detectable level of the hcv rna or core antigen targets. summary/conclusions: while precautions should continue to be taken for samples run on molecular instruments, the integrity of samples originally tested on the alinity s, alinity i, and architect i2000sr was preserved for downstream molecular testing through the use of induction heated washes. aims: increasing the safety of blood and blood products -motivating the blood donors to be regular donors methods: national reporting system showed the high prevalence of ttis among first blood donors in compares with the regular donors. in 2015 per 2.083.914 donations, 54% % of confirmed positive hiv, 87% of hcv, and 93% of hbv cases has been reported among first blood donors. in the end of 2015 a national program named "pre-donation screening tests "has been developed and has been implemented in high prevalence provinces in whole country. based on this program, all first blood donors who accept in donation sites, if after donor selection process are eligible to donate blood, they refer to give just a blood sample for screening ttis tests. after 3 months, the invitation letters and smss send to the donors who have negative results for all screening ttis tests, and they can be eligible to donate blood after another donor selection process. in 2015, about 0.156% of all donations have been rejected because of at least one of hiv, hcv, or hbv confirmed positive results, while this reject rate in 2017 was 0.082%, which shows a significant decreasing the ttis prevalence among blood donation from 2015 to 2017. the prevalence of hiv, hcv, and hbv among donations has been decreased significantly in 2017 compared with the 2015. prevalence of hiv among donations reduce from 0.0032% in 2015 to 0.0024% in 2017, for hcv and hbv the same results have been experienced, respectively from 0.040% and 0.113% in 2015 reduce to 0.026% and 0.053% in 2017. it seems this applied study could effectively scale up the safety of national blood supplies. in addition this intervention could support iranian blood transfusion service to increase the proportion of regular blood donors from 80.19% in 2015 to 86.97% in 2017. it means that with increasing the regular blood donor population sizes, the safety of iranian blood and blood products will be more and more scaled up. summary/conclusions: evidence based reports show there is a high rate of prevalence of transfusion transmitted infections (ttis) among first blood donors. so an effective intervention which can reduce the risk of unsafe first blood donation can effectively increase the safety of blood and blood products. pre donation screening tests program in iran can support the national program to decrease the rate of ttis among blood donations from 0.0156% in 2015 to .082% in 20117. abstract withdrawn. abstract withdrawn. background: despite the universal application of viral inactivation and elimination technologies during the preparation of plasma-derived products, the exclusion of infectious donations before any other procedure remains the first essential step as well as the major determinant for the safety of untreated labile blood products. current selection and screening techniques have reduced the risk of viral transmission to very low levels, but there is still a very low but quantifiable risk of transmission through donations beyond routine detection, particularly during the " seroconversion window". "of an infection in a blood donor that is to say during the period when the recently infected donor has not yet developed a serological response. the level of residual risk, which must be as low as possible, is mainly conditioned by the rates of the infections concerned (hiv and hepatitis b virus (hbv) and c (hcv)) to blood donors. summary/conclusions: the evolution of serologic markers is generally satisfactory with continued regression, which has improved particularly for hiv. on the other hand, hepatitis b is still a concern because of its still high rate among new donors. it is desirable to initiate a regular donor vaccination program to protect against hepatitis b. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hcvab, havab igm and havab igg essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 61 samples were tested (19 for hcvab, 24 for havab igm and 18 for havab igg) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 31 essays we found the %cv hcvab ranged from 0 to 4.474%. 5 samples were tested for havab igm in a total of 55 essays and the %cv ranged from 0 to 11.475%. havab igg was tested in 3 samples during 33 essays and the %cv ranged from 0 to 4.861%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hcvab, havab igm and havab igg. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hbsag, hbsab, hbcab, hbeag and hbeab essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 85 samples were tested (10 for hbsag, 20 for hbsab, 18 for hbcab, 14 for hbeag and 23 for hbeab) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 30 essays we found the %cv hbsag ranged from 0-4.375%. 3 samples were tested for hbsab in a total of 33 essays and the % cv ranged from 0-5.196%. hbcab was tested in 3 samples during 32 essays and the %cv ranged from 0-2.811%. using 3 samples and a total of 28 essays we found the %cv hbeag ranged from 4.176-5.147%. 4 samples were tested for hbeab in a total of 29 essays and the %cv ranged from 0-4.444%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hbsag, hbsab, hbcab, hbeag and hbeab. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hivag/ab, syphilis and htlv i/ii essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 38 samples were tested (12 for hivag/ab, 14 for syphilis and 12 for htlv i/ii) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 31 essays we found the %cv hivag/ab ranged from 0 to 3.05%. 2 samples were tested for syphilis in a total of 22 essays and the %cv ranged from 0 to 1.481%. htlv i/ii was tested in 3 samples during 28 essays and the %cv ranged from 3.342 to 7.5%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hivag/ab, syphilis and htlv i/ii. , human immunodeficiency (hiv) and hepatitis c (hcv) viruses' infection in blood donors were 1.27%, 0.20% and 0.50% respectively. consecutive positive results for hbv were 9.20% (63/685), for hcv were 6.0% (16/266) and nil for hiv. there was no sample carry over in this. out of 79 consecutive reactive donors 69 were donated for same patients and 32 were related with infected patient which were statistically significant (p < 0.0001). summary/conclusions: among all tti reactive donors 7.4% (79/1061) were consecutive reactive. the reason for the same may be process related like sample carry over or reagent carry over or donor related. donor related reasons may be, one of the close relative is reactive for virus and that is transmitted to other family members. in our study 32 reactive donors either had close contacts with persons with history of infective disease or were their first degree family relatives. these findings were found statistically significant (p < 0.0001). this study recommends that in analysis of consecutive positive results in elisa along with looking for procedure/sample error, there is also a need to take retrospective history of donors for close contact with infected patient. background: screening for transfusion-transmitted infections (ttis) is critical in ensuring safety of blood products. transmission of infections through transfusion remains a major source of viral hepatitis especially hbv and hcv. the effectiveness of rapid immunochromatographic test (ict) devices for screening of blood is a concern and needs validation through advanced methods like chemiluminescence immunoassay (clia) and polymerase chain reaction (pcr). aims: the current study was conducted to evaluate the performance and screening effectiveness of commercially available rapid screening kits in comparison with clia and pcr. methods: this single centre, cross sectional study was conducted at the department of blood transfusion services, shaheed zulfiqar ali bhutto medical university, islamabad, from january -june 2018. a total of ten commercially available ict devices and one clia kit (liaisonr xl) were tested for their sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and accuracy using 100 positive and 100 negative samples each for hbv and hcv respectively, in comparison with the values determined by pcr. the ict kits included hightop, rightsign, wondfo, accu-chek, fastep, abon, immumed, insta-answer, biocheck and ctk. results: the sensitivities and specificities of ict kits for hbsag were 65% and 70% (hightop), 67% and 85% (rightsign), 62% and 73% (wondfo), 70% and 80% (accu-chek), 68% and 77% (fastep), 73% and 85% (abon), 77% and 83% (immumed), 80% and 90% (insta-answer), 67% and 81% (biocheck) and 72% and 83% (ctk) respectively. similarly the sensitivities and specificities of different ict kits for hcv were 69% and 80% (hightop), 76% and 83% (rightsign), 69% and 81% (wondfo), 78% and 79% (accu check), 68% and 68% (fastep), 63% and 73% (abon), 71% and 70% (immu-med), 79% and 68% (insta answer), 62% and 66% (biochek) and 69% and 78% (ctk) respectively. the sensitivity and specificity of diasorin liaison murex assay for both hbv and hcv were found to be 100%, when compared with pcr. the ppv, npv and accuracy were determined accordingly. summary/conclusions: rapid testing ict devices for both hbv and hcv available in pakistan were found to have a variable degree of sensitivity and specificity, when compared with pcr. comparatively expensive but quality methods are more reliable as compared to rapid devices. the data generated will help policy makers to prepare future plan of action and introduce the concept of quality control in blood centres. the analysis has shown that the population of blood donors also included people infected with syphilis. in reference to the number of the tested samples this number is quite significant. the analysis proved the increase in the number of syphilis infections among the blood donors which is consistent with the general trend in the population. summary/conclusions: we have proved that testing blood donors for the treponema pallidum infection increases the safety of recipients of blood and its components and that obligatory testing of donors is fully justified. , 1940 and 1945 , the use of third or fourth generation serological assays is mandatory for screening of blood donor units for hbv and hcv infection before transfusion. routinely, blood banks in india screen the units by the elisa testing. nat is not very common due to cost constraints. aims: the aim of this study is to determine the frequency and load of hbv dna and hcv rna in hbs and hcv reactive blood donors respectively, and hence it was intended to contribute to determining whether routine hbs and hcv screening of blood donors, using elisa method alone, provides any concrete benefits with regard to hbv and hcv risk reduction or whether the implementation of nat will be of great benefit to low-resource countries like india, which has high prevalence of hbv and hcv. abstract withdrawn. ,906 donors were routinely tested for hbv dna by using cobastaqscreen mpx-1 and mpx-2 (roche) or procleix ultrio and ultrio plus id (grifols) assays. obi was confirmed by repeat dna testing and by performing additional serological and molecular investigations on index and follow-up samples. anti-hbs concentrations were determined and anti-hbc antibodies were tested with three distinct commercial clia assays (anti-hbc elecsys roche, architect anti-hbc ii abbott, and hiscl anti-hbc assay sysmex). hbv pre-s/s, precore/core and bcp regions were pcramplified after viral particle concentration and viral amplicons were sequenced. results: 348 hbsag-/dna+ donors (1:1,462) including 294 confirmed obi were identified (1:1,731 prevalence). among obi donors, 160 (54.5%) tested anti-hbc+/anti-hbs-, 108 (36.7%) were anti-hbc+/anti-hbs+, 25 (8.5%) were anti-hbc-/anti-hbs+, and 1 anti-hbc-/anti-hbs-primary obi (0.3%). anti-hbc-/anti-hbs+ obi donors were significantly younger (mean: 21 years [range: 18-57 years]) than those with anti-hbc+/anti-hbs+ (mean: 44 years [range: 19-60 years]) and anti-hbc+/anti-hbs-(median: 45 years [range: 21-55 years]) profiles (p < 0.0001). hbv vaccination was documented for 18 (72%) of these donors and was reported in one donor but without definitive evidence. extremely low hbv dna loads (range: <10-155 iu/ml) were transiently detected in seven donors during follow up. genotypes identified were genotype b (n = 1/20) and genotype c (n = 19/20). preliminary analysis of core protein (n = 16) and bcp (n = 10) sequences showed no particular genetic feature that could be associated with altered antigenicity or core gene expression. follow-up was available for 15/ 25 anti-hbc-/anti-hbs+ donors (2-6 samples/donor; range: 2-56 months). anti-hbc remained undetectable with all clia assays in these donors except one. low transiently detectable levels of hbv dna were observed overtime with anti-hbs levels fluctuating between 12 and 1,000 iu/l. no significant difference in hla-a, -b (except hla-b*46 more frequently detected in anti-hbc negative obi), and -drb1*. summary/conclusions: overall, the 8.5% prevalence of anti-hbs-only in hbv dna positive obi carriers (1:20,355 of total donors) in dalian blood donors confirmed previous reports from south east asia. this phenomenon was not related to core antigenic variations but was significantly associated with younger age of carriers. a particular route and natural history of the infection may be considered. the hypothesis of acute-phase vaccine breakthrough was ruled out in 15/25 donors by the over 50 months stability of this serological profile. breakthrough in immunized donors may still be suspected. further studies are needed to evaluate the potential infectivity of anti-hbs-only/hbv dna+ obi carriers, and to characterize the potential viral and immunological mechanisms responsible for this unusual hbv infection profile. confirmatory laboratory, hungarian national blood transfusion service (hnbts), budapest, hungary background: vaccination against hepatitis b virus (hbv) is an effective tool to avoid the infection. in hungary, population born after 1986 is considered to be immunized, because inoculation has been mandatory for children as campaign vaccination since 2000. hbv vaccine is strongly recommended for healthcare workers, moreover trips to endemic countries and awareness of individuals could also be reasons of vaccination in immunologically na€ ıve age-groups. since the hbv vaccine contains surface antigen, a recent inoculation can cause reactivity of hbsag screening assays and positivity of confirmatory tests for several days resulting in deferral of donors from blood donation. the former regulation of hnbts, which was valid until december 31, 2018, allowed the re-entry of donors whose immunization records and negative hbv confirmation of the second blood samples proved that the previous vaccination had resulted in the hbsag confirmed positivity. aims: the aim of this study was to strengthen that vaccination against hbv before blood donation had resulted in the reactivity of hbsag screening and confirmatory assays between 2012 and 2018. background: in brazil, the introduction of nucleic acid tests (nat) for hbv-dna detection in the routine screening at public blood banks is relatively recent. at fundac ßão pr o-sangue/hemocentro de são paulo (fps-sp), about 130,000 blood donors are submitted to serological screening tests (hbv, hcv, hiv-1/2, chagas disease, syphilis and htlv-1/2) and nat for hiv, hcv and hbv per year. approximately 2% of the blood donations are discarded due to some reactive result; of these, the hbv discard rate was 0.65% in 2016. aims: our study aims to determine the potential infectious cases among samples that had one or more hbv-reactive screening results (anti-hbc, hbsag and mp-nat-hbv) and verify the different categories of hbv infection (acute, chronic, occult hepatitis b infection (obi) and immunological window). furthermore, to characterize the distribution of hbv genotypes, drug resistance and escape mutations and analyze the risk factors. methods: we carried out a cross-sectional study of roughly 74,000 donations from may 2016 to december 2016. hbv antibodies and antigen screening were performed using cmia kits architect â -abbott/hbsag and architect â -abbott/anti-hbc. nat screening was performed in minipools (mp) of six samples using kit nat hiv/hcv/ hbv -bio-manguinhos (sensitivity 95% lod 300 iu/ml for hbv). reagent samples (n = 407) that presented one or more hbv-reactive screening results (anti-hbc, hbsag and/or mp-nat-hbv), were submitted to individual nucleic acid extraction and "in house" quantitative real-time pcr-hbv (id-pcr-hbv) targeting the hbsag region (sensitivity 10-20 ui/ml). the hbv genotypes and mutation analyses were determined by direct sequencing of the hbv pol-gene/surface-gene and use of the online analysis tool geno2pheno [hbv] 2.0. socio-demographic and epidemiological data were also analyzed. financial support: fapesp 2017/16658-6. results: among the 407 hbv reactive samples, 377 were reactive for anti-hbc only (92.6%), 10 for hbsag only (2.5%) and 20 were reactive for both markers and hbv dna (4.9%). routine testing and id-pcr-hbv identified 20 (0.027%) samples of active infections that had all hbv reactive/positive tests results. no hbv dnayield samples or hbsagyield or obi were observed. viral loads for active infections samples ranged from 1.08e+01 to 2.45e+09 cop/ml (median, 2.12e+08cop/ml). hbv sub-genotypes a1, a2, c1, d3 and f1 were found in 70%, 5%, 5%, 15%, and 5% of the donors, respectively. no reverse transcriptase inhibitor-resistant variants were detected. escape mutations in small hbsag protein shb region were detected in 30% (6/20), with the following main substitutions 100c (3x), 120r, 123n and 144g. the mean age of donors with active hbv infection was 40 years, mostly donors were males (80%), mixed (40%) or white (40%) and had concluded high school (45%). summary/conclusions: discard rate due to isolated anti-hbc is high but no obi was found in the blood donor population studied. in addition, no case of immunological window for hbv or hbsagyield was detected. there was a predominance of subgenotype a1 and mutations associated with escape were found in 30% of hbv-dnapositive samples. continuous research and surveillance about hbv prevalence among blood donors are needed to maintain and evenly increase blood safety in brazil. background: screening for anti-hbcore antibodies in blood donors is considered to contribute significantly to blood safety, since it reveals donors with occult or probable occult hepatitis b, with variable results in molecular screening, due to very low viral load. however, universal anti-hbcore testing in blood donors, might exclude a considerable number of blood donors in countries with high hbv prevalence and even in countries with low to moderate prevalence, like greece. aims: the aim was to investigate the percentage of blood donors with natural hbv infection (confirmed positive anti-hbcore) or hbv immunization due to vaccination (anti-hbs+ only, due to vaccination) and predict the impact of generalized anti-hbcore testing. methods: during the period 15-30 november 2018, all blood donors were asked to give their consensus for additional screening for hepatitis b anti-hbcore and anti-hbs antibodies, besides the obligatory serological and molecular screening, the samples of few donors who disagreed, were not examined. all samples with repeated positive anti-hbcore results, were further examined for anti-hbcore igm and anti-hbe antibodies. furthermore, a new donor sample was requested, to confirm reactivity. the serology results were recorded in an excel spreadsheet. additional data, including age, sex, nationality, number of previous blood donations, abo blood group, family history of hbv infection, hbv vaccination, were also recorded and statistically evaluated. donors were informed of the positive results. results: a total of 620 edta samples were tested using the architect anti-hbcore, anti-hbs, nti-hbcore-m and anti-hbe assays (chemiluminescent microparticle immunoassay (cmia). repeated anti-hbcore(+) occurred in 24 (3.87%) samples, among which 7 (19%) were also anti-hbe(+), while anti-hbs was found > 200 m iu/ml in 15 (62.5%), between 100 and 200 miu/ml in 3 (12.5%), and < 100 miu/ml in 6 (25%). among anti-hbcore positive donors, 4/24 were foreigners (16.6%) and 20 were greeks, while foreigners consisted 6,29% (39/620) of donors examined. so, anti-hbcore was found positive in 10,25% of foreigners (4/39, all from countries with high prevalence for hepatitis b infection) and in 3,44% of greeks (20/581). in total, 285 (45.96%) samples had anti-hbs > 10 miu/ml (considered seroprotective for the donor). summary/conclusions: almost half of our blood donors (45.96%) were immunized, 261 by vaccination and 24 (3.87%) by natural infection. the incidence of natural infection was significantly higher in foreigners (10.25% versus 3,44%). if not all anti-hbcore+ donors, 25% with anti-hbs < 100 iu/ml, might be potentially infectious, especially for immunocompromised patients. if we choose to screen all blood donors for anti-hbcore and reject those with positive results, regardless of the anti-hbs levels, we would probably lose a significant number of donors and jeopardize blood sufficiency. alternatively, we could reject only those with anti-hbs < 100 or < 200, or choose to selectively screen pre-donation blood donors from countries with high prevalence of hbv infection. following this pilot study, the prevalence of immunization against hbv in large numbers of blood donors from various parts of greece, must be investigated, in order to decide whether to introduce such screening. aims: the aim of this study was to perform phylogenetic analysis of the donor samples with hcv found in the neighbouring villages to determine the nature of transmission. methods: altogether, approximately 2 million blood donor samples were screened with anti-hcv immunoassay (architect anti-hcv, abbott gmbh, wiesbaden, germany) and reactive results were confirmed with anti-hcv line-immunoassays (inno-lia hcv score, fujirebio europe, gent, belgium). based on lia positivity, in 20 samples an association of hcv infection was supposed, because the residence places of donors were in three neighbouring villages situated less than 20 km to each other. pcr was positive in 15 samples. from these samples, hcv sequencing and phylogenetic analysis were performed. fourteen hcv infected samples of general population and 11 of ivd users were also included into the study. results: phylogenetic analysis detected genetic relationship among the hcv virus sequences in donor samples. the most abundant was the 1a subtype, and it formed two different groups on the phylogenetic tree. according to their genetic distance, a more distant mutual ancestor could be supposed. two samples with 1b subtype originated from the same village, and their difference was only 2 nucleotides. three hcv from the ivd user control group showed close genetic relationship with the viruses detected in the donor samples. summary/conclusions: based on our phylogenetic analysis, hcv transmission in blood donors could be the consequence of the ivd use and the origin might be related to 2 or 3 primary human sources. during 2011 and 2014, a significant increase in the hcv seroprevalence among the ivd users was observed, which was approximately threefold in the rural areas of hungary. our recent findings highlight the importance of the proper donor selection, which can identify the typical signs of the ivd use. moreover, enhancing awareness of blood donors with education is a further significant issue in order to reduce the risk of transfusion transmitted infections. abstract withdrawn. background: in china, the residual risk of transfusion-transmitted hcv has been declining since screening of blood donors for anti-hcv and/or hcv nat from 1993. however, many high sensitivity reagent, using to test blood donors' samples, lead to false-positive results and donors loss. aims: this study intended to establish a donor reentry procedure for hcv screening reactive donors in china. methods: from march 2014 to december 2017, there were 2083 blood donor samples which were screened reactive or belonged to "grey zone" by elisa and/or reactive by nucleic acid test(nat) at the local blood centers were collected from 15 chinese blood centers. all these samples were sent to institute of blood transfusion (ibt) national reference laboratory where anti-hcv and hcv individual nucleic acid test (id-nat) were conducted. if the results were reactive for anti-hcv, then the samples were tested with a recombinant immunoblot assay (riba). results: based on this study, 1178 of 2083 donors in the study who could be classified into two categories for hcv status: 201(17.1%) true positive and 974 (82.9%) false positive. a total of 905 of 2083 donors lost to follow-up, their hcv status cannot be determined with certainty. based on these data, a reentry procedure for hcv screening reactive donors was proposed. summary/conclusions: based on our proposed donor reentry procedure for hcv screening reactive donors, a majority of screening false-positive donors (82.9%) can re-entry safely. abstract withdrawn. background: providing safe blood for transfusion in sub-saharan africa (ssa) is a particular challenge due to a combination of factors; limited resources and infrastructure, suboptimal diagnostics and a high prevalence of the major transfusion-transmissible infections (ttis). average seroprevalence data estimates from the ugandan and kenyan blood transfusion services (bts) for hepatitis c (hcv) currently stand at 6.6% and 0.9% respectively. between january and december 2016, in mbale (eastern uganda) the hcv prevalence amongst blood donors was an alarming 8.1%. with no provision or funds for confirmatory testing, the bts are unable to confirm or refute a diagnosis of active hcv. this results in large quantities of blood wastage, unnecessary anxiety in potential donors and high donor deferral rates limiting the donor pool. aims: we aim to determine the true prevalence of active hcv infection amongst seropositive donors in bts in uganda and kenya. in addition, we aim to compare the performance of locally used serodiagnostics and best available alternative tests and to examine the feasibility of cost-effective additional or alternative tests to help provide accurate results on the infectious status of blood. methods: 235 hcv seropositive blood samples from 3 bts study sites (kampala, mbale, mombasa) will be re-tested using the local serology screening test (abbott architect anti-hcv), an alternative who pre-qualified rapid antibody test (sd bioline) and a confirmatory test (hcv core antigen test). where there is discrepancy in the results or need for clarification, samples will be tested on the cepheid xpert platform by reverse-transcriptase pcr to obtain a quantitative rna result. s/co (specimen to cut-off) values for false positive samples (by screening serology) will be analysed and presented. pre-analytical factors (centrifugation speed, haemolysis check, time delay between collection and testing) will be controlled for and documented. results: pilot data from re-testing quarantined hcv seropositive donor blood (mbale bts) in uganda demonstrated that 45/50 seropositive blood (90%) was rna pcr negative. in december 2018, 156/249 (63%) of seropositive samples (by screening anti-hcv serology) in kampala bts had s/co values between 1.00-1.99 (1.00 is the cut-off indicating a positive sample). data from the re-testing of 235 seropositive samples as true representation of active hcv will be demonstrated and s/co values for the study period concomitantly with a retrospective analysis of january to december 2018. preanalytical factors, cost analysis comparisons of the diagnostic platforms coupled with costs of the donor deferral process in false positive cases will be presented. summary/conclusions: for the bts in ssa there are significant resource and financial implications, as repeat testing and donor deferral counselling is required. evaluating and introducing new and appropriate diagnostics and algorithms in the screening of hcv is crucial in improving the supply of safe blood transfusion services in east africa. background: in november 2017, the blood services of england, scotland and wales reduced donor deferral to three-months for commercial sex workers and individuals with higher risk sexual partners, including sex between men. the change was recommended after a detailed review by an external expert committee (sabto) which recommended that a shortened deferral of 3 months would allow detection of recently acquired infection and maintain residual risk (rr) at a tolerable level. recommendations were accepted by government but with a government commitment to explore a more individualised approach. aims: to assess the impact of a 3-month deferral on blood safety in terms of epidemiology of infections in blood donors and compliance with donor selection criteria, and to explore evidence required to develop a more individualised approach to donor selection policy methods: routine uk blood donation surveillance data for 2010-2018 (2018: preliminary) were reviewed. annual prevalence and incidence of hbv and hiv infection were estimated, with a poisson regression models to test for trends. incidence was calculated from donors seroconverting within 12-months, and/or microbiological and clinical evidence of recent infection. for donors positive in 2018, compliance with the 3-month deferral was determined. uk hemovigilance data were scrutinised for evidence of transfusion transmitted infections (tti) associated with newly eligible donors. results: from 2010 to 2018 among new donors, annual hiv prevalence decreased significantly by an average of 11.3% each year (p = 0.02) to 1.49/100,000 donations in 2018; no significant trend was observed for hbv. annual hiv incidence among repeat donors also decreased significantly by an average of 17.3% each year (p = 0.03) to 0.23/100,000-person years (pyrs) in 2018 (based on 2 seroconverters). there was no significant trend in hbv incidence over the study period, however between 2017 and 2018 incidence increased from 0.35/100,000 pyrs to 0.84/ 100,000/pyrs (based on 3 and 7 seroconverters respectively). with the information available to date, none of the 2018 seroconverting donors were non-compliant, and there was no reported confirmed ttis associated with the policy change. summary/conclusions: hiv prevalence and incidence has continued to decline. hbv incidence in repeat donors increased in 2018 although initial analysis suggests this is not associated with the policy change. monitoring continues, and residual risks will be re-estimated as data post-change accumulate. these data are reassuring, and therefore it is appropriate to scope the evidence for, and feasibility of, a more individualised approach to selection policy. a multidisciplinary steering group has been convened including representation from patient and stakeholder groups. gaps in knowledge are being defined, and a package of work is in development under the project of fair (for the assessment of individualised risk), using the abo rdf for guidance. background: permanent deferral of men who have sex with men (msm), established in the 1980s, primarily to minimise the risk of hiv transfusion-transmitted infections is increasingly challenged. accordingly, blood services in many countries have changed to time-based deferral. in canada, a 5-year deferral was implemented in 2013, reduced to 12-months in 2016; a 3-month deferral is now being considered. aims: to estimate the risk of undetected hiv among screened blood donations under a 3-month deferral since last sex between men. methods: the applied model combines features of previously published english and canadian models to estimate hiv risk under a 12-month deferral. three scenarios varying hiv incidence, prevalence and non-compliance under a 3-month deferral were modelled. assumed constants were the hiv nucleic acid window period, testing procedure error rate and assay sensitivity. model inputs were incidence under the current 12-month deferral, calculated as hiv positive donors with a previous negative within 12 months divided by number of person years, numbers of hiv positive donations, hiv positive msm, hiv msm incident cases and newly eligible msm donors (from donor surveillance and compliance surveys). the risk with a 3-month deferral was estimated for three scenarios, one determined "most likely", where msm donor non-compliance, hiv incidence and hiv positive donations do not change and msm newly eligible to donate are estimated from compliance surveys. this scenario is based on data from two sequential policy changes in canada. an "optimistic" scenario where non-compliance halves and a "pessimistic" scenario where msm hiv incidence, hiv positive donations, non-compliance and new msm donors double were also used. the median hiv residual risk was used as the final estimate. the uncertainty in this estimate was assessed with the 2.5th and 97.5th percentiles over the simulation (95% ci). results: incidence, per 100,000 donations, was estimated to be 0.15, 0.13 and 0.27 for the "most likely" "optimistic" and "pessimistic" scenarios, respectively. for the 3month deferral "most likely" scenario, hiv residual risk was predicted to be 1 in 32.6 million donations (95% ci: 1 in 217,692 million to 1 in 8.3 million). for the "optimistic" scenario, hiv residual risk was estimated to be 1 in 34.3 million donations (95% ci: 1 in 257,692 million to 1 in 9.1 million). finally, for the "pessimistic" scenario, hiv residual risk was estimated to be 1 in 16.0 million donations (95% ci: 1 in 34,091 million to 1 in 5.7 million). with these residual risk estimates, based on the number of donations in canada, one hiv infectious donation would be in inventory every 30 years for the "most likely" scenario, every 32 years for the "optimistic" scenario and every 15 years for the "pessimistic" scenario. summary/conclusions: the risks of hiv entering the blood supply in canada for a 3-month msm deferral are predicted to be very low for all modelled scenarios, including a "pessimistic" doubling of hiv incidence post change. background: safety of blood and blood products is a major concern in pakistan. the prevalence of transfusion transmitted infections among multi-transfused thalassaemia patients is high (above 25%). the hiv epidemic in pakistan is following the asian epidemic model where after establishment among the high risk groups, its transmission to general public is rapid. fear, stigma and ignorance have contributed heavily to hiv transmission in pakistan. the hiv detection among blood donors is on the rise and reports occur in media repeatedly. aims: to investigate the possible transmission of hiv through blood transfusion in punjab, pakistan and to highlight the steps being taken to reduce further transmission of infections methods: in september 2018, a report of hiv transmission through blood transfusion was reported in the media where a mother and her newborn acquired hiv after blood transfusion from a hiv positive donor (confirmed later). the case was referred to and investigated by the punjab blood transfusion authority (pbta). the pbta team took blood samples of both recipients (mother and her newborn) and the blood donor who was a family relative. the samples were tested by highly sensitive chemiluminescence immunoassay (clia). the clia results confirmed the presence of hiv in both recipients and the blood donor. due to maternal hiv antibodies transfer through the placenta, the infection status of the newborn was not re-confirmed as he died within two weeks. the donor informed that he had donated 22 times in the past few years. the pbta was able to trace only one earlier donation three months ago. the recipient (a female) was found, tested by clia and was found to be hiv positive. all these cases occurred in unlicensed private blood banks that were screening for hiv on rapid manual devices. the blood banks were sealed by the authority and infected cases were registered by the provincial aids control programme and are being treated. summary/conclusions: the main reasons for hiv spread through blood transfusion is the use of sub-standard rapid screening devices which are not evaluated and validated at a national level. in addition, the existing system relies on the family/replacement donors. the national safe blood transfusion programme, is implementing blood safety system reforms as recommended by who. under the reform agenda, the blood transfusion authorities have been made functional and grant licenses to only those blood banks with proper systems to ensure quality and safety of blood products. the programme is developing a national system for the evaluation, selection and validation of all assays used for screening of blood in close coordination with the drug regulatory authority of pakistan. to promote the culture of voluntary blood donations, the programme has taken concrete steps initiating with the formulation of a national blood donor policy, interaction with celebrities, celebration of world blood donor day and more recently the launch of blood donation feature through 'facebook'. the promotion of voluntary blood donation concept along with regulation of blood sector will reduce the risk of hiv transmission through blood transfusions in pakistan. mianyang blood center, mianyang 9 urumqi blood center, urumqi, china 10 rti international, rockville 11 national heart, lung, and blood institute, bethesda 12 stanford university, stanford, united states background: the incidence of hiv infections has increased substantially over the past decade in china, especially among young people, who represent nearly half of the chinese blood donor population. this upward trend in hiv infections underscores the importance of monitoring hiv prevalence and incidence in chinese blood donors. aims: to estimate hiv prevalence and incidence rate (ir) among chinese blood donors using blood donation data from five geographically-disperse blood centers in 2013-2016 participating in the recipient epidemiology and donor evaluation study-iii (reds-iii) china program. methods: western blot confirmatory testing was done on samples of blood donations reactive for hiv-1/2 on one or both rounds of routine elisa tests or positive by nucleic acid amplification testing (nat). multiple imputation was used for samples with missing confirmatory test results. hiv prevalence was calculated among first-time donors. to estimate hiv ir in first-time donors, single-well lag-avidity eia testing was conducted with first-time hiv recent (incident) infections defined as being infected within approximately 129 days based on avidity of hiv antibodies. a novel model was derived to estimate hiv ir among infrequent repeat donors who had provided only one donation in the 2013-2016 estimation interval. to derive an overall hiv ir for repeat donors, this estimate was combined with the classical-model ir estimated for repeat donors who had given at least 2 donations in the estimation interval. multivariable logistic regression model was used to examine factors associated with hiv infection. results: a total of 1,276,544 whole blood and apheresis platelet donations with postdonation screening results were collected at the five blood centers between 2013 and 2016, including 648,607 donations from first-time donors and 627, 937 donations from repeat donors. hiv prevalence among first-time donors was 68.04 per 100,000 donors (95% ci, 61.68-74.40). hiv ir was estimated to be 37.93 per 100,000 person-years (95% ci, 30.62-46.97) among first-time donors and 20.55 per 100,000 person-years (95% ci, 16.95-24.91) among repeat donors. hiv prevalence and ir varied across regions with an increasing trend observed at some blood centers. among first-time donors, being male, older than 25 years, minority ethnicity, less than college education, and certain occupations (commercial services, factory workers, retired, unemployed, or self-employed) were associated with positive hiv confirmatory testing results. summary/conclusions: although hiv prevalence and incidence remain low among chinese blood donors, it is important to monitor hiv epidemiology in blood donors on a continuous basis, especially among populations and regions of higher risk. further donor screening and education strategies need to be developed and evaluated to reduce these risks. the ir methods used in this study for first time donors as well as repeat donors who donate very infrequently is readily applicable to other countries who have similar donation patterns. background: in thailand, the national blood centre is responsible for blood donation service which includes follow-up and blood donor counseling in order to indicate the infection status, especially hiv-positive blood donors. currently, although the epidemic of hiv infection in thailand is in decline, the hiv-positive cases still have been found in blood donors screening. thus, monitoring of hiv infection status in blood donors and post-blood donor counseling are important for providing the hiv-positive infected donors lead to access the hiv treatment immediately. aims: to study the hiv follow-up cases on serological testing over 10 years for assessment of the hiv infection in thai blood donors. the retrospective analysis of hiv follow-up cases on serological testing (cmia, ics and western blot) was conducted during 2009 to 2018 at thai national blood centre. results: a total of 6,408,024 voluntary blood donations over 10 years, the repeated reactive results on hiv serological screening were 5,978 (0.093%) cases and only half of these hiv reactive donors returned to follow-up testing for ascertaining their hiv status. for hiv follow-up process, the hiv reactive screening donors must be followed for 6 months and tested by using the different three principles of hiv serological testing. a total of 5,978 hiv reactive results were separated to three patterns including hiv positive results, inconclusive results and negative results which the number of each group was 1,130 (35.7%) cases, 742 (23.5%) cases and 1,292 (40.8%) cases respectively. out of 1,130 hiv positive results, we found that 1,105 (97.8%) cases were positive with all hiv serological testing for the first-time follow-up and 25 (2.2%) cases were tested and become to positive results after follow-up more than one time. in 742 cases of inconclusive results, 539 (72.6%) cases were reactive only 1 or 2 testing(s) which these donors did not return to confirm again leading to temporarily deferred donors in blood donor system. in addition, 203 (27.4%) cases of inconclusive results could not conclude the hiv result although they were repeated several times. for the last pattern, 1,292 negative results cases showed 762 (59.0%) cases were negative results after follow-up over 6 months while 530 (41.0%) cases were inconclusive results before changing to negative results which almost cases of this group were reentry as blood donor after deferral period is over. summary/conclusions: the number of repeated reactive results on hiv screening was constant over 10 years of which returned to follow-up only half of hiv reactive donors leading to accumulation of temporarily deferred donors in blood donation system. hiv follow-up positive cases were informed and counseled immediately then referring to anonymous clinic for treatment. the problem and challenges of hiv follow-up were inconclusive results that were unclear and some of these did not return to retest lead to loss of re-entry donor who might be changed to negative result afterward. hence, the effective counseling and follow-up system need to be taken urgently to encourage the temporarily deferral donors returned to retest for reducing stigma of deferred donors in hiv follow-up cases. . we only analyzed the information that had non-reactive results for infectious markers reported by blood banks to sihevi-ins©, because they represent a risk for blood recipients. results: when loading the information of sivigila in sihevi-ins©, 241 donors were found (80% men); of these people 256 donations were obtained (97% whole blood). 166 donors (69%) had a reactive result for hiv being subsequently reported in sivigila. in addition, five of them were reactive simultaneously for hbv in blood banks and took on average 174 ae 83 days to be reported in sivigila. 25 donors (10.4%) had an hiv reactive result notified by sivigila and subsequently they were reactive in blood banks. this behavior may suggest an attempt to spread the disease. 20 donors (60% men) despite being initially reported in the sivigila database, presented a non-reactive result in a blood bank for hiv; one of them was reactive for syphilis and hbv and only one for hbv. this pattern may suggest false positive or negative results in one of the two databases analyzed. fourteen donors had negative test in blood banks for hiv and in a range of up to 6 months they were reactive by sivigila (93% of them donate whole blood). this conduct may suggest that accepted donations were in a window period and therefore warrant further investigation. considering that two blood components could be obtained on average from each donor, a potential risk is estimated for 28 recipients. summary/conclusions: the donors reported first in the blood banks through sihevi-ins© and later in sivigila allow to estimate an adequate orientation to the health services. the information from general epidemiological surveillance programs could improve the selection of donors and transfusion safety. background: it is assumed that bacterial contamination of blood products most often takes place during the donation process. the number of bacteria at this time point is estimated to be around 10-100 cfu per bag. little is known about the growth behavior of different bacteria species in whole blood (wb) units during storage and the distribution of bacteria to the different blood products. aims: aim of the current study was to determine the growth of different bacteria species in contaminated wb units and to study the distribution of the bacteria to the different blood components. methods: whole blood (n = 4-12 per species) was inoculated with approximately 10 cfu of different bacteria species (escherichia coli, klebsiella pneumoniae, pseudomonas fluorescens, staphylococcus aureus, staphylococcus epidermidis, streptococcus dysgalactiae, streptococcus pyogenes) and stored for 22 to 24 h at room temperature before centrifugation and separation into red blood cells (rbc), buffy coat (bc) and plasma. bcs from spiked wb were each pooled with 3 random bcs to prepare plasmareduced platelet concentrates (pc). samples were taken from wb after storage and from the blood products (rbc, bc, plasma and pc) right after preparation, and the bacterial titer was determined. sterility of pcs was tested by bact/alert after seven days of storage. results: bacterial growth in wb varied remarkably between donations and bacteria species. the highest titers in wb were detected for the streptococcus species, whereas staphylococcus aureus, staphylococcus epidermidis, escherichia coli and pseudomonas fluorescens did not multiply. bacteria preferably accumulated in the bcs during separation, reaching titers of up to 3.5 9 10 3 cfu/ml in bcs and up to 0.9 9 10 3 cfu/ml in the corresponding pcs right after preparation. in total, 33 out of 48 pcs tested positive for bacteria at the end of storage. the results were dependent on the species used: e.g., 6/6 pcs tested positive after spiking with streptococcus pyogenes, while only 1/8 pcs tested positive after spiking with escherichia coli. bacterial contamination of rbc and plasma units was much less frequent and associated with higher bacterial titers in the parental wb units. summary/conclusions: the growth and distribution of bacteria during processing of wb into the different blood products is species-dependent and remarkably varies between donations. results: both patients were male (69 yo and 65 yo) with a history of acute myelogenous leukemia status-post haploidentical stem cell transplant. the patients were thrombocytopenic and underwent simultaneous transfusion of irradiated, non-pr, day 4 platelets stored in platelet additive solution, from a single apheresis collection. the blood supplier's primary pre-release bacterial cultures were negative, and the on-site point of release secondary safety measure pan genera detection (pgd) testing was negative for both gram positive (gp) and gram negative (gn) organisms. both apheresis units also passed visual inspection prior to release from the blood bank. during transfusion, both patients displayed signs of septic transfusion reaction including rigors, fever, hypoxemia, tachypnea, tachycardia, and hypotension. transfusion reaction evaluations were initiated, and both patients were admitted to the medical intensive care unit and started on broad-spectrum antibiotics. gram stain of one platelet unit demonstrated gram negative rods (gnr) and gram positive cocci (gpc) in clusters, and the second platelet unit demonstrated gnr only. repeat secondary safety measure pgd testing of both units was negative for both gp and gn organisms. direct bacterial cultures of both platelet units grew both gnr and gpc identified as a. baumanii and s. saprophyticus after 18 h of incubation. colonies on the initial bacterial plates were too numerous to count (tntc), and subsequent re-plating of the platelet units showed: unit 1: a. baumanii tntc and s. saprophyticus with 8.5 9 10 4 cfu/ml unit 2: a. baumanii 6.6 9 10 7 cfu/ml and s. saprophyticus 2.2 9 10 6 cfu/ml blood cultures collected from both patients became positive within 8 h with gnr on gram stain, and both blood cultures ultimately grew both a. baumanii and s. saprophyticus. the primary pre-release cultures at the blood supplier remained negative. after days on antibiotics and pressors, both patients stabilized and were discharged home. the blood donor was interviewed, and he was well. no cultures were collected. summary/conclusions: this case documents failure of both primary pre-release bacterial testing and secondary on-site point of release pgd testing to identify two pathogenic organisms. a. baumanii and s. saprophyticus are unusual causes of septic transfusion reactions, and it is possible that these organisms have different limits of detection in the pgd assay compared to other organisms. rapid attention to clinical signs during transfusion and prompt initiation of antibiotics is critical for the management of septic transfusion reactions. we are currently evaluating ways to further reduce septic transfusion reactions, including increasing the utilization of pathogen reduced platelets. background: transfusion-associated infections due to the transmission of bacteria still represents a major risk in developed countries nowadays. despite the refrigerated storage of red blood cells (rbc), fatal reactions of patients receiving contaminated rbc units are repeatedly reported. in order to further increase the safety of blood transfusions, new strategies and measures have to be developed. in this context, transfusion-relevant bacteria reference strains can serve as a valuable tool for the validation, comparison and interpretation of these new developments. aims: conducting a collaborative study to establish the first repository for red blood cell transfusion-relevant bacteria reference strains. methods: six bacterial strains (serratia liquefaciens, serratia marcescens, pseudomonas fluorescens, listeria monocytogenes, yersinia enterocolitica a-105 and yersinia enterocolitica a-176) were distributed from the paul-ehrlich-institut to 17 laboratories in 10 countries for enumeration, identification and growth measurement in a 7-day interval for a total of 42 days after low-count spiking of rbc bags (10-25 colony-forming units (cfu)/rbc bag). results: except for s. marcescens, all other strains showed good-to-excellent growth in rbc. s. liquefaciens, p. fluorescens, y. enterocolitica a-105 and y. enterocolitica a-176 achieved >10 6 cfu/ml at day 14 and 10 9 cfu/ml at day 21. growth of l. monocytogenes was lower reaching a maximum concentration of >10 6 cfu/ml at day 35. in 9 out of 17 laboratories, s. marcescens showed no growth at all. summary/conclusions: five of the six tested strains showed robust growth in rbc independent of donor variability and are promising candidates to be adopted as official rbc transfusion-relevant reference strains by the world health organization. background: the samplok sampling kit (ssk), itl biomedical, is used by nhs blood and transplant (nhsbt) for sampling of platelet components (pc) for bacterial screening using the bact/alert 3d system. inoculation of bact/alert bottles is performed immediately after sampling. validation of delayed inoculation, with retention of the sample within the ssk devices, would allow a contingency for transport to other screening sites in the event of an incident that prevented testing at the sampling site. ssk maintain a closed system for sampling of pc and are compatible with standard blood collection bags. a graduated chamber (10 or 16 ml) ensures that only the required sample volume is collected and an integrated needle allows inoculation into bact/alert bottles. aims: the national bacteriology laboratory, nhsbt, evaluated the impact of prolonged storage of pc samples in ssk devices with regard to bacterial viability and detection. methods: four reference species were assessed: staphylococcus aureus (atcc 29523), streptococcus agalactiae (atcc 13813), escherichia coli (atcc 25922), clostridium perfringens (atcc 13124). a pool and split method was used with apheresis pc suspended in plasma. units were screened using bact/alert 3d prior to spiking to prove the absence of contamination. pc were spiked with a single species (range 4 9 100-2.8 9 103 cfu/ml) and tested on bact/alert with a 1 ml inoculation into anaerobic and aerobic bottles (positive control). enumeration was performed to confirm the bacterial concentration. each unit was sampled using two 10 ml ssk, which were held for a period of 6 h at 20-25°c. the process was repeated with a 24-h period. once elapsed, 8 ml of each ssk was inoculated into an aerobic and anaerobic bact/alert bottle, one ssk per atmosphere per species and the remaining sample was enumerated. all bottles were incubated on the bact/ alert system for a maximum of 7 days (36ae0.5°c) and subcultured if positive. results: positive controls had detectable growth by bact/alert, excluding aerobic bottles with c. perfringens. this was expected as it is an anaerobic organism. after the storage periods, all bottles had detectable growth by bact/alert. s. aureus showed an increase of 0.6-log10 after 6 h (2.0 9 102 to 8.9 9 102 cfu/ml) and 2.0-log10 after 24 h (3.1 9 101 cfu/ml to 3.4 9 103 cfu/ml). s. agalactiae increased by 0.7-log10 after 6 h (2.1 9 102 cfu/ml to 1.1 9 103 cfu/ml) and 0.09-log10 after 24 h (1.0 9 102 cfu/ml to 1.2 9 102 cfu/ml). c perfringens increased by 0.6-log10 after 6 h (1.3 9 102 cfu/ml to 5.0 9 102 cfu/ml) and 2.7-log10 after 24 h (4.0 9 100 cfu/ml and 2.2 9 103 cfu/ml). for e. coli, the concentration after 6 h was reduced by 0.28-log10 (2.8 9 103 cfu/ml and 1.5 9 103 cfu/ml), however this was possibly a result of inherent counting errors. at 24 h, an increase in growth by 2.7-log10 (1.5 9 102 to 7.9 9 104 cfu/ml) was obtained. summary/conclusions: storage of pc samples in ssk devices for up to 24 h does not have a negative effect on bacterial viability and detection using the bact/alert 3d system. background: the intercept tm blood system for platelets efficiently inactivates pathogens and leukocytes in platelet concentrates (pc). the system utilizes amotosalen and uva light and is available for the treatment of apheresis and whole blood (wb) derived platelets (mostly buffy coat pools) in europe in plasma or platelet additive solution (pas), and the treatment of apheresis platelets in the us (trima tm in 100% plasma or amicus tm for 65% intersol pas/35% plasma). acinetobacter baumanii and staphylococcus saprophyticus strains were isolated from a saline flush taken 7 h after successful and complete transfusion of an apheresis intercept-treated pc in 65% pas/35% plasma, from a patient with a suspected septic reaction that occurred 2 h post transfusion. bacterial isolates, and a sample of a gram stain-negative and culture-negative sister split pc were submitted for evaluation. we report here the in vitro inactivation of the fast growing, gram negative bacterium a. baumanii and slower growing gram positive s. saprophyticus. the sister unit was assessed for amotosalen break down products as an indication of successful inter-cept treatment. aims: the objective of the study was to evaluate bacterial inactivation of a. baumanii and s. saprophyticus in apheresis platelets, assessed immediately after treatment and with re-culture at the end of a 7 day shelf-life. methods: a double apheresis pc collected in 65% pas/35% plasma was split into three equal components, yielding approximately 250 ml of platelets/pc. a. baumanii and s. saprophyticus were grown in lb broth and each pc unit was inoculated with either bacterial strain or the combination of both strains, each at~6 log colony forming units/ml (cfu/ml). after inoculation, the contaminated units were treated in small volume (sv) intercept kits. samples were taken: pre and post-inactivation treatment, and at 3, 5 and 7 days of storage. the samples were analyzed by plating on lb plates (100 ll-10 ml). residual amotosalen levels were assessed by hplc. results: initial bacterial titers were 5.9-6.0 cfu/ml. following the inactivation treatment, no viable bacteria were detected by plate culture. no bacteria were detected after 3, 5 and 7 days of storage, corresponding to > 5.9 log 10 inactivation of both bacterial strains. performance of the intercept treatment process was confirmed in the sister pc unit as evidenced by levels of amotosalen and its byproducts characteristic of intercept treatment, as well as by review of the process documentation. summary/conclusions: amotosalen/uva effectively inactivated a. baumanii and s. saprophyticus individually and together below the limit of detection after 7 days storage. no bacteria in the sister pc by gram stain and culture, and the presence of amotosalen byproducts suggested that the pc collection involved in the septic reaction was sterile at the time of intercept treatment and was successfully illuminated. the possibility of only one-of-two split pc being contaminated due to biofilm formation is minimized in the intercept system which decants platelets into virgin storage bags at the end of treatment. contamination of the source pc container likely occurred after intercept treatment, possibly at the time of spiking for transfusion. background: studies in sub-saharan africa have documented bacterial contamination of blood products for transfusion varying between 0,3%>17,5%, up to 5000 times higher than in the north. published data from central africa are lacking. aims: the aim of this study was to determine the proportion of blood products contaminated with bacteria in three hospitals in the democratic republic of the congo (drc). to assure aseptic sampling, we used a closed system of sampling bags. in addition to the presence of contamination, we assessed semi-quantitative colony counts. methods: from july to december 2018, a total of 2411 blood products were sampled, of which 979 in hôpital provincial g en eral de r ef erence, kinshasa (hpgrk), 662 in hôpital p ediatrique kalembe lembe, kinshasa (hpkll) and 770 in hôpital saint-luc, kisantu (hslk). after compatibilization of blood products, 16 ml of blood was transferred from the primary blood bag to an attached sampling bag. sampling bags were sealed off, stored in the fridge and transported once daily to the bacteriology laboratory. using the adapter connected to the sampling bag, 4 ml of blood was inoculated in a blood culture (bactalertpf, biom erieux) and incubated at 35°c for 7 days. cultures were checked daily for signs of growth. in addition, 2 ml of blood was equally distributed on the cled and macconkey agar-coated sides of a dipslide (meus s.r.l.). dipslides were incubated 48 h for semi-quantitative culture, expressed as colony-formingunits (cfu) per ml. in case of blood culture growth, bacteria were identified and a second blood culture was inoculated to exclude contamination during processing. bacteria grown on semi-quantitative culture were also identified. results: a total of 1.6% (39/2411) of whole blood and red cell concentrates were contaminated with bacteria. in hpgrk, 2.7% (26/979) of blood products were contaminated, in hpkll 1.5% (10/662) and in hslk 0.4% (3/770) . the proportion of contaminated blood products was significantly higher in hpgrk compared to hslk (p = 0.00047). there was no significant difference between the other sites (p = 0.051 and p = 0.12). majority of isolated bacterial species were coagulase-negative staphylococcus spp./micrococcus spp. (46.7%) and bacillus spp. (33.3%). the remaining 20% of bacterial isolates were identified as non-fermentative gram-negative rods, klebsiella pneumoniae, staphylococcus aureus, mould, listeria spp., corynebacterium spp./coryneform bacteria. the concentration of all isolated bacteria was lower than 10³ cfu/ml, except for one coagulase-negative staphylococcus spp. found in hpgrk at 10³ cfu/ml. summary/conclusions: to our knowledge, we were the first to reach a sample size of more than 2000 blood products for bacterial culture and the first to use a closed system of sampling bags in sub-saharan africa, which guarantees aseptic sampling of blood cultures. this might explain the low bacterial contamination rate (1.6%) of blood products in three hospitals in drc compared to previous studies in other sub-saharan african countries. moreover, bacterial concentrations in the contaminated blood products were low (<10³ cfu/ml). the different proportions of contamination between study sites suggest that different environments and practices play a role in the risk for bacterial contamination. background: although the screening of the treponema pallidum (tp) is mandatory in blood donations, its necessity is controversial, because there have been no transmissions by blood products documented in the developed countries in the last few decades. aims: based on laboratory markers, active and early tp infected donors (aeid) were determined. the demographics of aeid and the frequency measures of cases were compared with that of early infected syphilis cases (syc) notified from the 18 to 65-year-old general population registered at the nphc. methods: altogether, 474,017 18 to 65-year-old donors were screened with anti-tp immunoassay (architect syphilis tp, abbott, wiesbaden, germany) between 2015 and 2017. reactive results were confirmed with immunoblots (viramed biotech ag, planegg, germany), which discriminated both specific anti-tp (igg, igm) and non-specific vdrl antibodies in five dilutions. meeting the criteria of anti-tp igg positivity with a vdrl titer of ≥ 1:8 and anti-tp igm positivity, donors were considered as aeid. they were stratified by age, gender and residence regions. the proportion of aeid (paeid) and syc (psyc) were calculated in first time (ft), and repeat tested (rt) donors and in the 18 to 65-year-old general population, respectively, in each year studied. the period prevalence (pp) of aeid and syc was estimated in the populations at risk 1,2 across 2015-2017. statistics: weighted proportions and one-way anova with tukey post-hoc test and z score test were applied. statistical significance was defined as p < 0.05. results: anti-tp reactivity was confirmed in 167 blood donors. aeid was proved in 85 cases with 36 ft and 49 rt donors. in that period, 1696 syc were notified. both in aeid and syc, the age group of 25-34 years with approximately 35% and 36% of individuals was the dominant. the proportion of men was 74% and 77% (p = 0.5) in the aeid and syc, respectively. paeid estimated in ft donors was significantly higher (0.020%; 0.023%; 0.032%, p < 0.0001) than that of rt donors (0.007%; 0.009%; 0.009%) and the proportion of syc (0.008%; 0.009%; 0.010%) in the general population. pp of aeid showed a roughly homogenous distribution in the 7 regions (0.011%-0.025%), however, pp of syc had a significant (0.058%; p < 0.0001) central hungary dominance in relation to the other regions (0.008%-0.016%). comparing the pp of aeid to syc, central hungary indicated a significant difference (0.025% vs. 0.058%, p < 0.0001), however, other regions showed no substantial differences. summary/conclusions: donors with anti-tp confirmed positivity are referred to the healthcare system. based on the laboratory markers tested, aeid could be separated and their demographical characteristics are pretty similar to that of syc notified from the general hungarian population. the proportion of early and active infection in ft donors is significantly higher than that of rt donors and the proportion of syc in the general population. given the considerable number of tp infection in background: quality assurance and safety of hematopoietic stem cells (hsc) with emphasis on prevention of bacterial and fungal contamination are the prerequisites for any transplantation procedure. bacterial contamination is of particular significance as it occurs relatively more frequently and bacteria are gradually gaining more antimicrobial resistance. aims: the aim was to determine the incidence rate of bacterial and fungal contamination during processing of transplantation material at the institute of hematology and transfusion medicine (ihtm) taking into account the hsc sourceperipheral blood (pbsc), bone marrow (bm) or cord blood (cb). methods: analysis involved both autologous and allogenic components collected at ihtm and other hospitals and dedicated for ihtm patients. in all, the analysis comprised 4135 donations, including 112 bm (2.70%), 3787 pbsc (91.60%) and 236 cb (5.70%) donations processed in our laboratory in the years 1996-2016. bm was collected in operating theatre-conditions, pbsc with cell separators -cs-3000 (baxter), cobe spectra (gambro) and trima accel (terumo bct) and cb was acquired from umbilical vein at obstetrics and gynaecology wards. aerobic and anaerobic bacteria contamination was determined at various incubation temperatures (room temperature and 35°c) using a variety of culture media. pbsc and bm were tested using 2 samples with trypcase-soy broth (tsb-t) and 1 with schaedler + vit k3 (biomerieux). cb was tested using bactec peds plus/f and bactec lytic/10/anaerobic/f (becton-dickinson). results: in the 1996-2016 period 42 contaminated products were found: 25 pbsc (0.66% of all tested pbsc units) and 17 cb (7.20% of all tested cb units). no infected bm products were determined. the overall percentage of contaminated products was estimated at 0,1%. in 2010, three (3) products were found contaminated with staphylococcus epidermidis; all came from one patient with central venous catheter and were collected on consecutive days. other products were contaminated mostly with staphylococcus epidermidis (61.36%). detailed results to be presented on the poster. summary/conclusions: according to ihtm policy no contaminated product is admitted to clinical use. the outcome of our study identifies processing experience of the staff as the main indicator of product quality. important is also proper assessment of donor health and condition of the injection site as products are usually collected from central venous catheter. the closed system is an additional safeguard against contamination during processing. the sample collecting procedure should help to avoid false positive results. background: syphilis is considered a global public health problem. the world health organization (who) estimates that there are annually around 12 million new cases of syphilis in the world, more than 90% occurring in developing countries. despite significant decrease in syphilis transfusion transmission. the recent increase in worldwide incidence associated with the risk of transmission through platelet concentrates (cp), which are stored at room temperature, have called attention to the potential residual risk of syphilis transmission by transfusion. between 2015 and 2017 we observed in our institution a significant increase of 24% in positivity of syphilis among blood donors from 0.62% in 2015 to 0.73% in 2016 and 0.77% in 2017 (p < 0.0000001). aims: to determine the prevalence of active syphilis in blood donors and characterize the serological profile of syphilis positive donors. methods: each positive sample in a treponemic chemiluminescence assay (cmia, abbott architect) performed during blood donor screening in 2017 was submitted to a treponemic elisa anti-treponema pallidum igm (euroimmun) and a non-treponemic test (antigen-omega diagnostics). samples with positive results for one or two of these tests (indicating recent syphilis) were submitted to a real-time pcr for syphilis. the inno-lia syphilis-fujirebio immunoblot test was also performed for samples that presented a positive result for elisa-igm alone. financial support: fapesp 2017/23028-9. results: among 123,851 samples screened in 2017, 958 (0.77%) presented a positive result for cmia -syphilis. of these, 626 (65.4%) were included in the study. a total of 106 samples (17%) showed vdrl+/igm+; 96 (15%) vdrl+/igmand 28 (4.5%) vdrl -/elisa igm+. the inno-lia syphilis test was performed as a confirmatory test in 28 (4.5%) samples that presented positive results for elisa igm and vdrl negative with 21 (3.35%) positive results, 1 (0.15%) undetermined and 6 (0.95%) negative. none of the 626 samples showed the presence of treponema dna by real-time pcr. the prevalence was 0.77%, the incidence was 0.1% in the year 2017, and the incidence in relation to the total positivity was 20.4%. both, prevalence and incidence were higher in men, white, not married, aging 18-29 years and high school educational level. we observed a 6% a-hbc seroprevalence in the elisa igm-syphilis positive samples and a prevalence of 1.5% htlv coinfection. summary/conclusions: we observed a significant increase in prevalence of syphilis in 2017 (0.77%) with an incidence of 20.4% of the total of cases initially positive in the cmia test. according to our data, we could identify a risk of syphilis transfusion transmission in blood banks that exclusively use the vdrl for donor screening, once we found 22 (3.5%) cases with negative vdrl and elisa igm and inno-lia positive. continuous monitoring of the profile of donors infected with syphilis at this time of reemergence of the disease is useful and important not just for blood banks, as it reflects the epidemiological situation of disease in community, and can contribute to the definition of health policies. background: transfusion related sepsis is a serious concern limiting platelet storage time to 5 days at room temperature. while most units are screened for bacterial contamination when collected, bacterial monitoring methods can take up to 7 days to detect contamination. thus, cold storage of platelets represents an attractive alternative for improving platelet safety. in this study, we assessed bacterial growth in platelets stored either at room-temperature (rt; 22°c) or refrigerated (cs; 4°c). aims: the aims of this study were to 1) assess the effect of storage temperature on platelet function and bacterial growth in "contaminated" platelet units, and 2) identify factors contributing to bacterial growth during rt storage. methods: apheresis platelets in plasma (plt) were obtained from healthy donors using the terumo trima accel automated blood collection system (terumo bct). fresh plasma (fp) was collected similarly. aliquots of plt or fp were transferred to ph safe minibags (blood cell storage, inc) and "contaminated" with acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus epidermidis, or pbs (uninfected control). minibags stored at rt were agitated using an orbital shaker set to 60 rpm while cs aliquots were stored under static conditions. bacterial growth was monitored daily through dilution plating. lactate levels were assessed by istat (abbott) cg4 + test cartridges. plasma glucose levels were assessed using blood glucose testing strips (germaine laboratories). platelet activation and aggregation were assessed on days 0, 1, 3, and 5 by flow cytometry and multiplate platelet aggregometry, respectively. results: bacterial growth progressed rapidly over the first 3-4 days post-collection in all plt aliquots stored at rt except those challenged with s. epidermidis. significant growth of s. epidermidis was not detected until day 4. bacterial numbers remained unchanged in refrigerated aliquots through day 5. rt storage resulted in significantly (p < 0.05) decreased platelet aggregation over time which was exacerbated by bacterial challenge. plt function was largely preserved with refrigeration regardless of challenge. bacterial growth was significantly reduced, or at least delayed, in fp. fp challenged with gram-negative pathogens exhibited a significant (p < 0.05) delay in bacterial growth at day 1. while growth of e. coli and p. aeruginosa recovered by day 2, growth of a. baumannii was significantly (p < 0.05) inhibited throughout. fp challenged with gram-positive pathogens exhibited significant (p < 0.05) reduction in bacterial growth relative to plt aliquots. bacterial growth correlated with plt lactate production. lactate levels in plts challenged with e. coli showed diminished significantly after day 3, indicative of lactate utilization. with exception of fp challenged with s. aureus, bacterial growth was restored in fp supplemented with lactic acid in all challenge groups. summary/conclusions: refrigeration preserved platelet function while both inhibiting bacterial growth and lactate production. conversely, the opposite was observed with rt storage. these data demonstrate that bacterial growth can be controlled through refrigeration without loss of function and rt storage may potentiate growth of certain bacterial strains through accelerated plt metabolism. background: bacterial contamination of peripheral blood progenitor cell (pbpc) for transfusion has been the cause of serious sepsis and life-threatening infections. however, a standard procedure or choice of test sample(s) has not been established to screen pbpc products for microbial contamination, because these products are not large enough to facilitate inoculation of the recommended volume for the automated blood culture systems. samples taken from by-product plasma and low volume pbpc product were cultured in routine sterility test. aims: to evaluate the residual risk of microbial contamination in pbpc products for transplantation, we cultured sufficient post-thaw inoculation volumes from pbpc products which were discarded for various reasons in our blood center. methods: in routine sterility test, a 20-ml sample of by-product plasma collected with pbpc product was inoculated into bact/alert bpa and bpn culture bottle (10 ml each) within 48 h after collection. the bottles were then placed in the bact/ alert system and incubated for at least 7 days or when a positive reaction was indicated by the automated liquid-media culture system. moreover, a 2-ml postthaw sample would be cultured before transplantation performed. in the residual risk investigation, discarded pbpc products were thawed, and then a 2-ml and a 20-ml aliquot were taken and cultured with the same method. all positive bottles were subcultured for bacterial isolation and identification. results: in september 2008 and march 2018, after maintaining in liquid nitrogen for 1 to 17 years, 205 pbpc products collected from 45 patients, which was preserved in a volume between 35 and 60 ml, were discarded. all of these products had been cultured negative in routine sterility tests with plasma samples. these 205 final products were thawed and cultured with both the 20-ml and the 2-ml aliquot. one of these pbpc products had the positive culture result with the 20-ml retested samples. nevertheless, the same pbpc product had the negative result with the 2-ml post-thaw pbpc sample and the 20-ml by-product plasma sample. propionibacterium acnes was isolated from the bpn positive bottle. summary/conclusions: the residual risk of microbial contamination in pbpc postthaw products still exist after routine sterility test with the plasma sample and the 2-ml volume of pbpc sample. the bacterium isolated from pbpc product was normal skin flora bacterium. an optimal screening method of pbpc products merits further study to increase the safety of the blood supply. background: hospital hygiene tools that serve as a proxy for assessment of microbial contamination are increasingly used. they include adenosine triphosphate (atp) bioluminescence and air particle counting. however, their use for microbial monitoring of blood banks remains underexplored. they could be of particular interest in a sub-saharan african setting (temperatures, dust) to circumvent bacterial culture and provide direct results usable for monitoring over time. aims: the aim of this study was (i) to quantify environmental bacteria in the air and on surfaces that are regularly in contact with blood products, and (ii) to evaluate atp bioluminescence techniques and particle counts as a predictor for bacterial contamination, in three blood banks in the democratic republic of the congo (drc). methods: samples were taken in three blood banks in the democratic republic of the congo: hôpital p ediatrique de kalembelembe (hpkll) (10 surfaces, 1 air), hôpital provincial g en eral de r ef erence (hpgrk) (24 surfaces, 2 air) and the national blood transfusion centre (cnts) (20 surfaces, 2 air). surfaces that are regularly in contact with blood products were selected (sealer, fridge, donor chair,. . ..). regular surfaces were sampled using rodac contact plates (23.7 cm²) containing cled and macconkey agar, irregular surfaces using swabs (nrsii, medicalwire). atp was measured on the same surface (pd30, kikkoman), expressed as relative light units (rlu) per 100 cm². air was sampled by active sampling (500 liter; spinair, iul) on cled and macconkey medium. in parallel, particles > 0.5 lm and > 5 lm were counted using a particle counter (14,15 liter; metone 227a). culture media were incubated for 48 h at 35°c before counting colony forming units (cfu). results: for regular surfaces, the median (range) viable bacterial count was 27 (6-60) cfu/rodac, 69 (6-103) cfu/rodac, 82 (3-132) cfu/rodac for hpkll, cnts and hpgrk, respectively. at hpkll, highest viable counts were found in the sink (plain growth) and cool boxes (43 and 60 cfu/rodac). in cnts the blood processing bench, the donor chair arm support and washing basin showed the highest counts (plain growth). whereas in hpgrk, most bacteria were found in a fridge (plain growth), blood bag trolley (plain growth) and manual separator (132 cfu/ rodac). gram-negative bacilli were isolated from water basins and sink in cnts and hpkll, but also surfaces close to donor chairs at hpgrk. the median (range) of atp per 100 cm² was 2.906 (778-26.497) rlu at hpkll, ) rlu at cnts and 35.235 (1.754-348 .052) at hpgrk. atp results and total viable count were not correlated (n = 49, p = 0.52). median (range) bacterial count in the air was 258 (210-375) cfu/500l for all sites together. there was no correlation found between total bacterial count and particles > 0.5 lm or > 5 lm (r = 0.7 and r = 0.6 respectively; p < 0.05; n = 5) summary/conclusions: total viable bacterial count of surfaces varies over blood bank sites. according to our results, atp and particle counts did not correlate with bacterial counts on surfaces and in the air, respectively. bacterial isolates from blood bank environments in drc need to be identified and seasonal variations need to be evaluated. background: the risk of transfusion-transmitted hepatitis e virus (tt-hev) infections in line with the question of the necessity of hev-nat screening of blood products is currently subject to an ongoing debate on the importance of timely introduction of hev screening of blood donors and the impact of blood safety. different countries have chosen different regulatory approaches. just recently, the german federal authorities have introduced mandatory testing of all therapeutic blood products beginning from january 1st 2020. however, we already decided to voluntarily test all our blood products since january 2015. aims: in this study, we present our results of a 100% screening of therapeutic blood products for hev rna including four years of active surveillance of hepatitis e infection among blood donors in germany. methods: from january 2015 to december 2018, a total of 386,307 allogenic blood donations from 69,956 individual german blood donors were screened in a minipool format of 96 samples of for the presence of hev rna (realstar hev rt-pcr kit, altona diagnostic technologies (adt), hamburg, germany). nucleic acids were extracted from 4.8 ml plasma using the chemagen msm-i extractor (viral 5k, perkin elmer chemagen gmbh). the 95% lod of the assay was determined to 4.66 iu/ml (447 iu/ml per single donation). the presence of hev-specific igm and igg antibodies was determined using the anti-hev igm/igg elisa (euroimmun, luebeck). hev rna concentrations were quantified using the first who international standard for hepatitis e virus rna for nat-based assays. all hev rna positive donors were deferred from donation for 3 months. follow-up samples were tested for the presence of hev rna and hev-specific antibodies. genotyping was performed by sequencing of the hypervariable region (hvr) and orf1. results: in total, 274 hev rna positive donors were identified. of these, 274 hev rna-positive donors, 216 were nat-only positive donations (78.83%, negative for anti-hev igm and anti-hev igg), three donors had a positive igm titer (1.09%), 30 donors showed reactive igm and igg titers (10.95%), 25 donors already had isolated igg titers (9.12%). median values of viral loads were approximately twice as high in index donations that were antibody negative. merely 62 donors showed elevated alt levels (22.63%), mostly within a double increase within the reference range (16.06%), only 6.57% of donors had even further elevated alt levels. significantly higher alt values were found in donors with a viral load > 1,000 iu/ml compared to the group with viral loads between 100 and 1000 iu/ml. available follow-up samples confirmed igg seroconversion for all donors, however we also observed long-term igm positivity in some donors. genotyping revealed genotype 3 in all cases. the month-dependent incidence ranges from 1:719 to 1:3,781 blood donations with a peak in june and july. summary/conclusions: the high number of identified hev rna positive donors emphasizes the need for hev-nat screening to increase the safety of blood products. this study further confirmed that hev infection is common in german blood donors. background: zika virus (zikv) is a mosquito-borne virus that has caused outbreaks in central and south america in february 2016, and has threatened the safety of blood transfusion globally. there is a high risk of zikv transmission by whole blood and blood components transfusion. it was reported that, zikv rna in infected patients plasma can only be detected within 1 to 2 weeks. however, in whole blood, zikv rna might present positive up to day 101 after the symptoms appear in some patients, even if the clinical symptoms disappeared with zikv rna negative in plasma. this phenomenon suggested that the presence of zika is associated with red blood cells (rbcs). moreover, another report showed that viral load in whole blood of type a west nile virus (wnv) patients was higher than type o, implying that the binding of virus to rbcs may be related to blood group glycoprotein. both of zikv and wnv are member of the flavivirus genus. the study is intended to explore whether zikv have the same adherence mechanism to rbcs as wnv. aims: to investigate the distribution of zikv in blood components and adherence of zikv to different blood types of rbcs in whole blood. methods: five units for each blood type of a, b, o and ab whole blood were randomly selected. each unit of 200 ml whole blood was divided into two half-unit. zikv was added to one half-unit in a certain proportion, and incubated at 37°c for 3 days. each component of whole blood was collected for viral load detection. in the other half-unit,rbcs were suspended in the same type pools of plasma with equal volume after the plasma removed from the whole blood after centrifugation. zikv was added with the same certain proportion, and then incubated at 37°c for 3 days. the whole blood samples and the upper plasma by centrifugation were collected detected for zikv rna. meanwhile, rbcs were washed and resuspended with normal saline followed by viral load detection. results: zika rna of these samples which extracted from whole blood, rbcs, and plasma were determined in a quantitative reverse transcription pcr, and viral rna of each component was all up to 10 9 copies/ml. although, zikv rna loads did not show significant difference in distribution between rbcs and their corresponding plasma components, zikv rna quantification were significantly higher than those in plasma (p < 0.05) in type o rbcs and lower than those in plasma (p < 0.05) in type ab rbcs. summary/conclusions: in our study, we detected high viral rna loads in rbcs. it was demonstrated that zikv adheres to erythrocyte in whole blood, and the blood type may have influence on the adherence. background: hong kong is not endemic for dengue virus (denv) with most of the documented cases being imported. the presence of sufficient number of mosquito vectors, aedes albopictus, in the territory has led to two self-limiting indigenous outbreaks affecting 20 residents from 2000 to 2014. during 14 august to 4 september 2018, another outbreak of 29 confirmed cases of autochthonous dengue fever were reported to the department of health, linked to two epidemiological clusters, one in lion rock park near wong tai sin (wts) district (19 cases) and the other in cheung chau, an outlying island (10 cases). aims: we assessed the risk of dengue transmission from blood donors during the 2018 outbreak using a simplified version of the probabilistic model developed by biggerstaff and petersen (b-p) and the european up-front risk assessment tool (eufrat) model (oei, transfusion, 2013) . methods: patient demographic and general population data were obtained from the centre for health protection and the department of census and statistics of the hong kong government for the number of 15-to 64-year-old patients in the 2018 outbreak and residents of the same age range in hong kong and wts district as at mid-2018 respectively (16-65 years old being the eligible age range for first time donation). to apply the b-p model, we estimated denv incidence among donors in hong kong territory and in wts with confirmed denv infection during 12 august to 8 september 2018 after correction for clinical:subclinical infections ratio, the mean length of asymptomatic viraemia and the probability of collecting blood from asymptomatic donors as described previously (seed, transfusion, 2009 ). to estimate the risk using eufrat model, outbreak and blood donation variables were entered into eufrat's web-based interface (https://eufrattool.ecdc.europa.eu/), which provided automatic calculation of risk-related output parameters. results: while using the b-p model, the estimated risk of collecting a denv viraemic donation was one in 778,000 (266,000-1,822,000) territory-wide for the 28-day study period but increased to one in 147,000 (50,000-345,000) in wts. similarly while applying the eufrat model, the risk of encountering a viraemic donor was 1 in 279,000 (118,000-752,000) territory-wide and 1 in 52,000 (21,000-188,000) in wts during the same period. the eufrat also predicted a territory-wide issue of 0.08 unit of denv-contaminated labile blood component during the outbreak period. summary/conclusions: like many mosquito-borne infections such as denv, the risk is characteristically localised and varies geographically and seasonally during outbreaks. the average predicted risk of collecting a denv-viraemic donation territory-wide is low at 1 in 778,000 during the 2018 outbreak based on the b-p model, which was generally considered as tolerable. however, the risk increased by 4 folds when blood donations were collected from wts residents, who had higher chances of paying visits to lion rock park in close proximity. it was then justifiable to institute risk mitigation policies such as geographically-based deferral and/or fresh component restriction, enhanced post-donation reporting, etc. to protect against blood safety. background: hev is a developing threat to blood safety following the reporting of several cases of transfusion transmission hev (tt-hev). transfusion-related hev infection has been reported in several countries but its true frequency is probably underestimated because it is often asymptomatic and testing of blood donors is infrequent. pakistan is classified as a highly endemic region; with sporadic cases of hev occurring throughout the year, mainly affecting the adult population. to the best of our knowledge, no studies have been reported from pakistan on the epidemiology of hev in blood donors. aims: to assess the epidemiology of the hev specific antibodies and serum alt levels in blood donors of capital twin cities of pakistan. methods: this cross sectional study was conducted from july 2017 to december 2017 at three blood banks in the capital twin cities (rawalpindi and islamabad) of pakistan. the blood donors were equally distributed across the three blood banks. only donors who tested negative for hiv, hbv and hcv were included in the study. serum alt levels were analyzed by using automated clinical chemistry analyzer (selctra pro m) using merck kits. all samples were tested for hev-specific antibodies (igm and igg) by using enzyme linked immunosorbent assay (elisa) kits (adaltis, italy). statistical analyses were performed using spss software version 21.0 (ibm). results: in our study population there were 445 (98.9%) males and 5 (1.1%) females. the mean age of recruited blood donors was 28.38 (sd ae 7.34), with a range of 18-55. younger donors were more common with a frequency of 18-27 year olds of 249 (55.3%). we found an overall hev igg prevalence of 17.5% and an hev igm prevalence of 9.1%. there were 10 (2.2%) blood donors who were positive for both igg and igm antibodies. our results revealed that the hev specific antibodies (igg, igm) prevalence increased with age. the mean value of serum alt was 33.8 (sd ae 41.0) with a range of 4-554 iu/l. the serum alt levels were elevated (>45 iu/l) in 73 (16.3%) blood donors. there was significant correlation (p=<0.001) between serum alt level and hev specific antibodies for igg and igm. summary/conclusions: this study shows that a significant proportion of blood donors at our blood centers have been infected with hev and may be able to cause tt-hev. as we have not yet measured hev rna, we have used igm antibodies as a proxy for donors who have active infection. hev is generally asymptomatic, so it is debatable whether mandatory hev screening in blood donors should be required. results of this pilot study show that there is a need to conduct a larger study at national level with highly sensitive assays before making screening for hev mandatory in pakistan. background: hepatitis e virus (hev) is a zoonotic virus. who estimates that there are 20 million hev infections, 3 million acute hev cases and 56 thousands hevrelated deaths worldwide each year. in recent years, the prevalence of hev in european and american countries has increased significantly. the survey results show that the positive rate of hev igg in blood donors is respectively 4.0% in new zealand, 13.5% in britain, 20.6% in denmark, 16% in the united states and 27.0% in the netherlands. hev has become a global public health concern. in addition to the food route of infection, several cases have been reported that hev can be transmitted via blood products. aims: to investigate the prevalence of hepatitis e virus (hev) infection among voluntary blood donors and potential impact on blood safety in guangzhou china. methods: blood samples from 5552 blood donors were collected from april 2017 to april 2008 at the guangzhou blood center and were tested for anti-hev igg antibody (hev igg), anti-hev igm antibody (hev igm) and hev antigen (hev ag)by enzyme linked immunosorbent assay (elisa). hev rna detection was performed on hev antigen positive samples by rt-pcr. the association of age, gender, ethnicity, occupation and alt with hev igg and igm were analyzed by chi-square test. multivariate logistic regression analysis was applied to identify the independent risk factors of hev infection. results: the positive rates of hev igg, igm and hev ag were 20.05% (1113/5552), 0.76% (42/5552) and 0.04% (2/5552), respectively. no positive hev rna was detected. age and ethnicity were independent risk factors for hev igg and hev igm. the rate of hev antibody increased significantly with age (igg or = 1.089, p < 0.001; igm or = 1.055, p < 0.001). donors who were zhuang minority (32.69%, 7.69%) showed higher anti-hev than those who were han ethnicity (19.89%, 0.70%), and the difference was statistically significant (igg or = 2.052, p = 0.023; igm or = 12.029, p < 0.001). in addition, we found that occupation was an independent risk factor for hev infection, where students showed the lowest anti-hev rate. summary/conclusions: the results indicate that the positive rate of hev antibody among blood donors in guangzhou is high, and the infection status differs in different populations. our study provides basic data for the estimation of risk of transfusion-transmitted hev. background: human cytomegalovirus (hcmv) belongs to the viral family of herpesviridae. it is an enveloped double-stranded dna virus, widely distributed in the human population (60-100% seropositive subjects worldwide) and cause of severe disease in immunocompromised patients and upon infection of the foetus. in normally healthy subjects, hcmv persists lifelong without clinical manifestation undergoing alternating phases of active viral replication and latency. since hcmv can be readily detected in blood, as free virus as well as associated to neutrophils and monocytes, hcmv transmission is a complication of blood transfusion. even though leukoreduction of blood products has been shown to significantly reduce the risk of hcmv transmission, higher inactivation standards may be required for high-risk, immunocompromised groups of patients. aims: in this study, murine macrophages infected with murine cytomegalovirus (mcmv) were used as a model to study the inactivation cell-associated cmv in human plasma using the theraflex mb-plasma system (macopharma). methods: mcmv expressing the green fluorescent protein was used to infect murine macrophages. infected macrophages were harvested 20 h after infection, washed and used for spiking of plasma. plasma units (n = 2, 290 ml) were spiked with infected cell suspension (10% v/v) and treated with the theraflex mb-plasma system according to the manufacturer's protocol using the macotronic-b2 illumination device (macopharma). samples were taken after spiking (load and hold sample), after illumination with different light doses (0 after addition of mb, 30, 60, 90 and 120 [standard] j/cm 2 ) and after blueflex filtration. mcmv titers were determined by endpoint titration and large volume plating on murine fibroblasts. infectious virus, which expressed gfp in infected cells, was detected using a fluorescence microscope. results: the results of infectivity assay showed that the treatment of human plasma by the theraflex mb-plasma system inactivated cell-associated mcmv in a dosedependent manner. after spiking with mcmv infected macrophages a mcmv titer of 4.2 (bag no. 1) and 4.5 (bag no. 2) log 10 tcid 50 /ml was achieved in the plasma units. in hold samples, a mcmv titer of 3.8 (bag no. 1 and bag no. 2) log 10 tcid 50 /ml was determined. the illumination step of the theraflex mb-plasma treatment procedure efficiently inactivated mcmv. already three-fourths of the standard light dose decreased infectivity of cell associated and remaining cell-free mcmv to infectivity levels below the limit of detection (≥ 2.9 log). further investigations would be needed to evaluate the log reduction capacity of the blueflex filtration step for cell-associated mcmv. summary/conclusions: the results with the murine model virus suggest that the theraflex mb-plasma system is an effective technology to inactivate cell-associated cmv in human plasma units. background: the use of pathogen inactivation (pi) technologies for platelet concentrates and plasma is slowly but steadily increasing. methods for treatment of red blood cells (rbcs), the most commonly used blood component, are still under development. aims: a novel approach for pi in rbc units employing uvc light was developed. methods: pi treatment was applied to full-scale rbc units after leukodepletion. the pi capacity of the uvc-based method was evaluated by bacteria and virus infectivity assays. a panel of in vitro assays to measure quality, metabolism, functional, morphologic, and blood group serology variables was applied to a pool-and-split approach in which pathogen-reduced rbcs were investigated in comparison to untreated rbcs. results: uvc treatment caused dose-dependent inactivation of bacteria and enveloped and non-enveloped viruses in rbc units. at a full dose, the mean log 10 reduction factors ranged from 4.2 (bacillus cereus) to 6.1 (serratia liquefaciens) for the tested bacteria, and from 3.1 (emcv) to ≥ 4.9 (vsv) for the tested viruses. uvc treatment did not alter rbc blood group antigen expression. quality of uvc-treated rbcs was maintained during storage, e.g. hemolysis in uvc-treated and untreated rbcs were well below 0.8% until day 35 of storage. summary/conclusions: the data obtained until now show that uvc irradiation is a potential new method for pi in rbcs and justify further development of this process. background: histo-blood abh antigens are the mayor allogeneic antigens in human and they are widely distributed in almost all tissues. the expression of a-1,2-fucosyltransferase (fuct2), encoded by fut2 gene, determines the secretor status of an individual. about 80% of caucasian population have a functional copy of fut2 (secretor gene) expressing abh blood group soluble antigens in organic fluids such as saliva and seminal plasma. this individuals are known as "secretors". soluble abh blood group antigens have been associated with several metabolic and infectious diseases as well as reproductive failures. the incidence of infertility related of both male and female factors continues to rise despite many advances in reproductive technologies. it is well known that abo antigens are expressed on sperm membrane and in seminal fluid of secretors as well as abo antibodies are present in cervical mucus. in previous studies we observed significant loss in progressive motility of spermatozoa of non-secretors compared to secretor ones caused by specific cervical mucus antibodies in abo-incompatible couples. in addition, sperm cells are haploid cells, so that a heterozygous individual has two sperm subpopulations, each expressing the corresponding allele. the specific antibody of cervical mucus will attack only its complementary sperm. aims: to evaluate the prevalence of secretor character in men belonging to fertile and infertile couples in order to investigate a possible association with reproductive success. methods: 126 samples of semen, 68 from infertile men and 58 from fertile controls were studied. comprehensive infertility evaluation was performed in all patients according to who 2010 criteria. secretor phenotype was evaluated in seminal plasma by inhibition of hemagglutination assay using saline erythrocyte suspensions, monoclonal antibodies anti-a, anti-b and lectin from ulex europaeus (anti-h). to distinguish between abo genes, genomic dna was extracted by an enzymatic digestion method. pcr was designed with two sets of oligonucleotides that allow to amplificate two different regions of the transferases without use of restriction enzymes. by comparison of bands of the pcr products, the individual genotype was determine. cervical mucus antibodies of their female partners were titrated with the corresponding red blood cells. results: results were analysed in both groups. in infertile couples with abo incompatibility, the frequency of non-secretor phenotype of male partners (76.9%) were significantly higher than those from fertile couples (21.6%) (p < 0.03) the results obtained by pcr in sperm cells correlated 100% with red cells phenotypes. summary/conclusions: incidence of infertility continues to increase. several factors have a negative impact on men's reproductive health. immunological implications are now being studied and considered as a cause of failure in sperm-egg interaction, even among normal gametes. secretor phenotype in male partners could help reproductive success by blocking cervical abo antibodies. furthermore, if the male is heterozygous, cervical mucus antibodies will only affect the corresponding sperm. we propose to evaluate abh antigen expression on sperm membrane and seminal plasma as well as abo antibodies in cervical mucus to contribute to the diagnosis and treatment of human infertility. background: the h blood group contains one antigen, the h antigen, which is present on virtually all red blood cells (rbc) and is the acceptor substrate of both a and b gene-specified glycosyltransferases. in blood group o the h antigen remains unmodified and therefore its rbcs show the highest and the rbcs of blood type ab the least amounts of h antigen. individuals with the so called bombay phenotype carry homozygous h null alleles (h | h) and do not produce any h antigen. the para-bombay phenotype retains some h antigen on rbcs either induced by a weakly active (h+ w | h+ w ) or completely silenced fut1 gene (h | h), mandatory linked with an active fut2 gene. aims: in this study, we aimed to develop an adapted flow cytometric method to quantify the relative amount of h substance present on rbcs in order to distinguish different abo phenotypes in routine diagnostics as well as to capture rare h-deficient phenotypes. methods: analyses were performed on a flow cytometer (facs canto ii, bd biosciences, ch) and measured with identical instrument settings. list mode data were evaluated and visualised using bd facsdiva software. rbcs were incubated with increasing concentrations of monoclonal anti-h antibodies (bric231-pe and a 1:1 mixture of bric231-pe/bric231, ibgrl, uk). after rinsing the cells with pbs, micro-aggregates were mechanically dissolved. rbcs from 29 blood donors with different abo phenotypes (o (5), a 1 (5), a 2 (5), b (5), a 1 b (5), a 2 b (4)) and 2 patients with genetically confirmed bombay and para-bombay phenotype were assessed. results: saturation of h antigen binding sites on type o rbcs was achieved only upon use of a 1:1 antibody mixture (bric231-pe/bric231) covering approx. half of the h-binding sites by unconjugated bric231. in contrast, non-o type rbcs reached saturation of h-binding sites using pure bric231-pe. rbcs coated with bric231-pe at saturation revealed a distinct pattern of mfi (mean fluorescence intensity) depending on the abo phenotype. in addition, mfis of rbcs upon staining with bric231-pe did discriminate bombay-and para-bombay type rbcs, respectively. summary/conclusions: adapted flow cytometry is able to distinguish variant expressions of rbcs h antigen. thus, our flow cytometric method may complement traditional serological and genetic analyses in routine abo phenotype cases or, more intriguing, when the bombay or para-bombay phenotype is suspected. it will be of interest to further prove this method by investigating additional rare h-deficient phenotype cases. s chen 1,2 , x xu 1,2 , x hong 1,2 , k ma 1,2 , j he 1,2 , j chen 1,2 and f zhu 1,2 1 blood centre of zhejiang province 2 zhejiang provincial key laboratory of blood safety research, hangzhou, china background: weakened a and b antigen expression results in abo typing discrepancies. h gene controls the development of h substance from which a and b antigens develop. depressed a and b antigen expression and strengthened h antigen expression are always simultaneously observed in abo subgroups. there are other possibilities for weak antigen expression of abo system such as leukemic change and pregnancy. it is undiscovered whether abnormal expressions of a, b and h antigen stand for abo subgroups in hemopathic patients. aims: the aim of this study is to explore the role of enhanced reactions with anti-h in direction to abo subgroups of hemopathic patients. methods: 109 samples from blood donors and hemopathic patients with nonconcordant abo typing by serological tests were collected after consent information. the agglutination strength of these rbcs with anti-h reagent was recorded. enhanced reactions were determined by comparison with the results from normal abo groups. the genomic dnas of 109 samples were extracted and genotyped for abo system. this work was sponsored by the medical science research foundation of zhejiang province (2018rc029). results: 69 samples in 80 blood donors showed increased expression of h antigen, of which 47 were identified as abo subgroups. there were 22 enhanced reactions in 29 hemopathic patients. however, 19 were finally confirmed as normal abo genotypes. no statistical significance (86.3% vs 75.9%, p > 0.05) in the frequency of strengthened h antigen expressions was observed between donors and hemopathic patients. the total number of subgroups is 50 and 3 respectively in blood donors and hemopathic patients. extremely significant statistical differences (68.1% vs 13.6%, p < 0.005) existed in the frequency of subgroups with enhanced h antigen, which meant the possibility of subgroups in hemopathic patients samples was less. summary/conclusions: the expression of h antigen is comparably enhanced in subgroups and hemopathies. but most of hemopathic patients with strengthened h antigen expression present normal abo genotypes. as a result, the enhanced reaction with anti-h is necessary but not sufficient for serological identification of abo subgroups in hemopathic patients. background: although the use of automated blood bank analyzer with the advantages of speed and efficiency has recently increased, the abo discrepancies in automated blood bank analyzer have caused the reporting delays of the results and increase of the task. aims: we analyzed the causes of abo discrepancies in automated blood bank analyzer and suggested a solution strategy based on the causes. methods: from november 2018 to january 2019, 55 cases (0.6%) of abo discrepancies among 9,550 abo blood type tests performed using the 15-min reaction mode of ih-500 in chonbuk national university hospital blood bank were identified. we compared the test results of 15-min mode with results of immediate mode using different red cell reagents, and analyzed the causes of discrepancies by performing additional tests such as microscopy, auto-control, antibody screening and identification, anti-a1 and abo genotyping. results: in the immediate reaction using different red cell reagents, 45 cases (81.8%) of discrepancies disappeared and 10 cases (18.2%) remained discrepancies. all abo discrepancies observed in the 15-min reaction were due to serum side causes, and one case (1.8%) was due to both of serum and red cells side cause. nonspecific response (28 cases, 50.9%), cold antibody (20 cases, 36.4%), rouleaux formation (3 cases, 5.5%), cis-ab (3 cases, 5.5%), and abo subtype (1 case, 1.8%) were analyzed as causes of discrepancies. one discrepancy due to cis-ab was disappeared in the immediate reaction using different red cell reagents, abo subtype was changed to totally different blood group, a. on the other hand, in cases of the discrepancy corrected by the immediate reaction using different red cell reagents, the intensity of the positive results still observed in immediate reaction was not different from the 15-min reaction. summary/conclusions: ih-500, an automated blood bank analyzer, was considered useful for automation of abo blood typing, and some observable abo discrepancies are expected to be mostly addressed by reexamining with immediate reaction mode using different red cell reagents. abstract withdrawn. background: abo blood group antigens mainly expressed on red blood cells, but along with that they also present on many organs and tissues like epithelia, platelets, vascular endothelia and neurons etc. the importance of abo antigens extends beyond transfusion medicine by association with various systemic diseases like cardiovascular diseases, gastric diseases, cancers, infectious diseases etc has been proven previously. previous researchers also tried to find out the involvement of abo antigens in neurological diseases like alzheimer's disease, parkinson diseases etc. but association with neurological tumours is less explored. aims: this study aimed to analyse the association of abo blood group antigens with neurological tumours. methods: a retrospective study in a tertiary care institute in india analysed the 2 years data from jan 2017 to dec 2018. the carcinoma patient's admitted in neurosurgical department during study period were included in our study. their diagnosis and abo blood groups were collected from hospital information system. data were analysed into microsoft excel 2016 and spss (version 22). results: during study period a total of 1970 patients with neurological tumours were admitted in our hospital. the blood group frequency of these patients were 20.91%, 37.51%, 32.74%, 8.83% for a, b, o and ab respectively. the common neurological tumours found in our study were glioma (33.55%) followed by pituitary adenoma (20.05%), meningioma (18.58%), schwannoma (8.98%), cavernoma (2.54%), neuroma (2.23%) and space occupying lesions (14.06%). the prevalence of abo antigens was almost similar in all neurological tumours except in neuroma. neuroma was found in 47.73% o group patients as compared to other blood groups which was found statistically significant (p < 0.05). summary/conclusions: in this study we tried to analyse the association of neurological tumours with abo blood groups antigens. we found there is no association of neurological tumours with abo blood groups because the prevalence on abo group in general population is almost similar in patient with neurological tumours except neuroma. neuroma group of tumours like neurofibroma, neuroblastoma, nerve tumours etc. were more common in o group of patients while in our population frequency of b blood group antigen (38.2%) is more common as compared to o blood group(33.4%). background: rhd and rhce represent homologous genes in head-to-head position on chromosome 1 (chr1, p36.11). they encode for the proteins rhd resp. rhce which compose together with rhesus associated glycoprotein (rhag), band 3 and ankyrin the ankyrin complex (ac) linking the red blood cell (rbc) membrane to aspectrin of rbc cytoskeleton (s.e. lux, blood, 2016). cooperatively, the proteins of ac are important for maturation and physiologic properties of rbcs. many proteins of the rbc membrane express blood group antigens on their extracellular surface and are therefore of concern in transfusion medicine. cepellini et al. described weakened hemagglutination reactions of rhd+ rbcs in the presence of an rhc+ antigen (cepellini et al, pnas, 1955) . we attempted to further elucidate the expression of rhd/rhag proteins in various rhce/rhce pheno-/genotypes using a sophisticated flow cytometry approach. aims: in this study, we investigated a flow cytometric method for measurement of the antigen-density of various rhce-phenotypes. methods: analysis was performed on a flow cytometer (facscanto ii, becton dickinson (bd)) using bd facsdiva software and identical instrument settings for all samples. optimized number of rbcs was incubated with saturating concentration of pe-conjugated anti-rhd antibodies brad-3/brad-5/fog-1 (ibgrl, bristol, uk). debris was excluded by rbc gating in fsc/ssc plot. quantibrite-pe beats (bd) were applied according to manufacturer's instruction to quantify the relative expression of rhd epitopes. in addition a representative number of samples from common phenotypes were assessed for expression of rhag using bric-69pe (ibgrl). results: a total of 146 samples from healthy blood donors with serologically defined rhcde phenotypes were included into this study (rr(21), r1r(20), r1r1(23), r2r(18), r0r(15), r1r2(27), r2r2 (22)). variant expression of rhd by different rhce phenotypes using brad-3-pe was shown. rhd was weakly expressed in presence of rhc antigen (cepellini effect). effect of rhd gene dose on rhd protein expression is mitigated by rhc/c genotypes. when only samples with molecularly confirmed phenotypes were assessed, the rhdce genotype predicts consistently the strength of rhd protein expression. outlier samples (3) were retrospectively genotyped and revealed rhdce genotypes as expected from the strength of rhd expression falsifying serological rhcde phenotypes. in contrast, rhe/e polymorphic site does not correlate with rhd expression. in addition, rhag protein is equally present across all rhcde phenotypes. similar results were obtained by using alternative anti-d antibodies such as brad-5-pe and fog-1-pe, although different antibody's avidity precludes quantitative comparison of antigen expression on rbcs. summary/conclusions: sophisticated facs methods reveal different expression of rhd on rbcs according to rhce/rhce phenotype/genotype. rhc/c polymorphic sites (c.48g>c, c.201a>g, c.203a>g of exon 1, exon 2 resp. and intron 2) are in linkage with rhd expression, confirming the observation by cepellini et al. in contrast, rhe/e (c.676c>g, exon 5) is not in linkage with rhd expression. based on epigenomic signature it is conceivable that altered transcription factor binding sites (tbs) of rhd mirrored by homologous rhc/c may cause variant rhd expression. rhe/e snp mirroring the homologous sequence of rhd in exon 5 is not recognised as tbs. in addition, although ac comprises all three rh proteins (rhd, rhce, rhag), their transcriptional regulations seem to be distinct. red cell reference laboratory, australian red cross blood service, perth, australia background: the rh26 antigen was first described when an antisera thought to contain a potent anti-c did not react with all c+ cells. these non-reacting c+ cells were classified as c+, rh:-26, and the antibody specificity anti-rh26. most polyclonal anti-c contain anti-c and anti-rh26. previous studies have shown 2 of 10 monoclonal anti-c reagents are actually anti-rh26. these reagents will not detect the c antigen where the red cells are rh:-26. aims: the australian red cross blood service investigated a phenotype discrepancy in a blood donor. the donor's historic phenotype c+ (r 1 r) was inconsistent with the current donation phenotype c-(r 1 r 1 ). we aimed to investigate the cause of the discrepancy so the donor could be assigned the correct phenotype, identify the root cause of the discrepancy and implement any corrective actions. methods: the donor's red cells were phenotyped with all available anti-c reagents as per the manufacturers product insert across both manual and automated testing platforms. following variable results and weaker reactions with some reagents, dna was extracted from the edta sample and was genotyped using immucor bioarray tm hea precise and rhce beadchip tm . targeted dna sequencing of rhd and rhce was also performed using the trusight tm one sequencing panel. a review of the historical phenotype results, including the testing platform and reagents used at the time was also performed. results: on the current sample the donor's red cells gave a 2 + reaction by tube with bio-rad seraclone â (2) [clone ms35] and immulab epiclone tm [clone anti-c reagents. the sample tested negative on the beckman coulter pk7300 using beckman coulter anti-c [clone 951] blood grouping reagent and tested positive (4) reaction on the immucor neo using immuclone â (1) anti-c [clone . immucor bioarray tm hea precise beadchip tm predicted a c+ phenotype and no variants were detected with the bioarray tm rhce beadchip tm . the trusight tm one sequencing panel genotyped the donor as rhd*01/*01n.01 and rhce*01.15/*02 with a predicted phenotype of c+, c+ w , d+, e-, e+, rh:55 (locr+), rh:-26. a review of the donor's historical records indicated the donor tested as c+ on the pk7200, which at the time was being used with an in-house bromelain preparation (sigma-aldrich) and diagast olymp pheno anti-c reagent [clone ms33]. summary/conclusions: results indicated the phenotype discrepancy was caused by the c+ rh:-26 variant associated with the rhce*01.15 allele. reagents containing clones ms-33 and ms35 correctly phenotyped the donor as c+, with the manual tube reagents showing a weaker reaction which may alert the operator to a possible variant which is important in the patient setting. the beckman coulter pk7300 and associated anti-c [clone 951] failed to detect the c antigen. this reagent appears not to detect the c antigen where it is associated with the rh:-26 phenotype, which is in contrast to the previous report by faas et al, transfusion, 1997 where it was demonstrated that clone 951 reacted with c+ rh:-26 bromelain treated red cells. abstract withdrawn. background: although serological rhd typing has always been challenging due to variation of techniques and variable sensitivity of anti-d reagents, most individuals are unequivocally typed as either rhd positive or rhd negative. however, variants of d (weak d and partial d phenotypes) may present typing difficulties. individuals with partial d (missing epitopes of the d antigen) must be typed as rhd negative as blood receivers, but as rhd positive, as blood donors. aims: the aim of our study was to evaluate the algorithm used since 2017 at ahepa university blood center, to resolve rhd typing problems among first time donors. methods: since automatic analyzers may type variants of rhd as rhd+, our practice is to routinely perform two different typing methods in first time donors: an automated microplate method on the neo analyzer (immucor) and the slide test, using a potent reagent (anti-d blend-immunodiagnostika). in case of negative, weak, slow or mixed-field reaction, further testing with an automated microplate weak d method [immucor-modified indirect antiglobulin (anti-igg) test] follows. the next step of the protocol consists of testing with the commercial id-partial rhd typing kit (bio-rad) comprising a panel of 6 monoclonal anti-d reagents, in an indirect coombs gel test assay. the patterns obtained with this kit can distinguish between d weak and partial d and can also differentiate between categories ii, iv, v, vi, vii dfr, dbt and dhar. the last step of our algorithm consists of molecular testing (immucor bioarray rhce and rhd beadchip assays) at the hellenic national blood transfusion center, in case of remaining uncertainty. results: we applied the above algorithm in 32 samples: a) by using the partial d kit, 23 samples were typed: four samples were characterized as "partial d" (3 dfr, 1diii) and 19 as "weak d". four of the weak d samples (all from women of reproductive age) were confirmed by molecular typing ("weak d type 1" three samples, "weak d type 4.0 or 4.3" one sample). b) the nine (9) remaining samples that showed atypical serological pattern, were sent for molecular testing, which characterized 2 samples as "weak d type 1", one sample as "weak d type 3" and another as "weak d type 11". results are pending for 5 samples. summary/conclusions: in our experience some partial rhds may be mistyped as rhd+ if the technologist does not inspect the pattern of the reactions and only takes into account the assignment by the automatic analyzer as d+ or d-. by use of our algorithm, serological characterization was sufficient to distinguish between weak d and partial d in 68,75% of cases. molecular typing was necessary in the rest. the integration of molecular techniques improves the quality and accuracy of d typing of blood donors. if applied to patients, it also allows administration of d positive blood without compromising safety to those carrying prevalent weak d types that have not been reported to produce anti-d. furthermore, it permits withholding rhig in case of pregnant women carrying such weak d types. background: rhd antigen is one of the most clinically significant blood group antigens. except d positive and d negative phenotypes, there are over 200 rhd variants, which represent as serologic weak d phenotypes (swd). patients with certain swd can make anti-d alloantibodies. by serology testing it is not possible to clearly distinguish among different swd. in croatian institute of transfusion medicine (citm) patients and pregnant females with swd are mostly reported as rhd negative and generally did not refer for confirmation, because molecular testing was not part of the algorithm. that remains the risk of shortages of rhd negative blood and overuse of anti-d immunoprophylaxis for pregnant females. according to uk guidelines patients with swd who are likely to require chronic transfusion support and females ≤ 50 years are treated as d negative and refer for confirmation of d type. people who are rhd genotyped as weak d type 1, 2 or 3 are not susceptible for rhd alloimmunisation. one study showed that in croatian population the most frequent variants are weak d type 1, 2 and 3. aims: the aim of this study is to estimate the prevalence of swd in patients and pregnant females and to find out serologic and molecular characteristics of swd referred for confirmation. methods: from 2013/01/01 to 2018/10/01 we analysed 119.845 samples of patients and pregnant females. rhd typing was performed by anti-d igm monoclonal reagents in direct agglutination micromethod on tango (bs232, bs226) (biorad, dreieich, germany), swing maestro [lmh 59/20 (ldm3) + 175-2 and th-28 + rum-1 + ldm1] and ih-1000 [lmh 59/20 (ldm3) + 175-2] (id-card, biorad, cressier, switzerland). cut-off value for tango was determined as ++ and for gel microtyping as +++. the samples with results below the cut-off were reported and treated as rhd negative, all except those which gave discrepant results at current testing or with historical data. these were sent to rhd genotyping for confirmation. dna extraction was done by qiaamp blood mini kit (qiagen, hilden, germany) and rhd genotyping by pcr-ssp kits ready geneweak d and ready genecde (inno-train, kronberg im taunus, germany). results: from 119.845 samples 300 (0,25%) were swd. 71/300 (24%) were referred to rhd genotyping. 55/71 (77%) samples were weak d type 1, 2 or 3, while 16/71 (23%) were weak d type 14 and partial d variants vii and va. serologic reactions with monoclonal igm anti-d reagents showed different pattern for weak d types 1, 2 and 3. clearly negative serologic reactions were given in 27/29 samples with bs 226 and bs 232, in 30/33 samples with lmh 59/20 (ldm3) + 175-2 and in 18/39 samples with th-28 + rum-1 + ldm1. summary/conclusions: the prevalence of swd in this study is rather low (0,25%). after rhd genotyping 77% of referred samples were finally reported as d positive. serologic determination of d variants is inconsistent and only rhd genotyping can resolve rhd status in swd. to define the permanent rhd status of swd female of childbearing potential and patients who are likely to be chronically transfused we will introduce rhd genotyping in the new algorithm. background: among all blood group systems, the antigens of the abo system are by far the most clinically significant. comes second in importance is the antigens of the rh system, which comprise d, c, e, c, and e antigens. another clinically relevant antigen is the k of the kell blood group system, which is known to be involved in both htr and hdfn. the distribution of the major blood group antigens, such as rh, and kell, is well-studied among populations of developing countries. in contrary, a relatively few studies have addressed their frequencies in saudi arabian population this is also the case in jazan province, where only two published studies have analysed the prevalence of abo and d antigens, while the frequency of other clinically important antigens, such as rh and kell antigens, is yet to be explored. aims: to determine the frequency of the following clinically relevant blood group antigens; rh(d, c, e, c, e) and k among saudi blood donors in king fahd central hospital in jazan province. methods: a retrospective, cross-sectional study was carried out in the blood bank of king fahd central hospital in jazan province. the red cell phenotyping records for blood donation of 3243 randomly selected saudi donors, who donated blood between january and june 2018, were reviewed to identify the prevalence for the following antigens: d, c, e, c, e and k. the hospital blood bank routinely performs rh/k1 phenotyping for all blood donation using either bio-rad or ortho diagnostic column agglutination technology (cat) platforms. phenotype frequencies were expressed as percentages. results: this study included a total of 3243 saudi voluntary as well as family replacement blood donors. the d antigen was found to be positive in 93.7%, while k antigen was positive in 11.1%. among other studied rh antigens, e was the most common (98.1%) followed by c (78.2%), c (70.3%) and e(26.9%). dce/dce (34.2%) and dce/dce (5.3%) were the most common phenotypes amongst d-positive and dnegative donors, respectively. surprisingly, dce/dce phenotype was significantly prevalent (11.9%) with almost 5 times higher frequency compared that reported in caucasians (2.0%). the rare phenotype dce/dce was found in 3 donors (0.09%), while dce/dce and dce/dce phenotypes were found in only one donor each. summary/conclusions: this study is the first to determine the frequency of rh and k antigens in saudi blood donors in jazan province. determination of the frequency of these clinically significant antigens in our geographical area will facilitate the selection of antigen-matched red cell units for transfusion in recipients with multiple alloantibodies. it will also help in the management of blood donation processes and planning the estimated need of blood stock of different blood group phenotypes to meet the patient's needs. abstract withdrawn. background: the gerbich (ge) blood group system includes several high-frequency antigens located on glycophorin c and d. with only few reports published on the clinical significance of antibodies directed against these antigens, it is unclear whether blood transfusions have to be antigen negative in the presence of an anti-ge antibody. the monocyte monolayer assay (mma) is an in-vitro method used to estimate the clinical significance of alloantibodies. aims: to illustrate the role of the monocyte monolayer assay (mma) in the transfusion management of a patient with an anti-ge alloantibody. methods: the clinical and transfusion history was retrospectively retrieved from the patient's medical records. serological investigations were performed by indirect antihuman globulin test. papain and trypsin treated cells were also used. the clinical significance of the antibody was assessed by mma. genomic dna was isolated from whole blood and the samples were further characterized by pcr. results: a 58-year-old male patient with lung cancer without previous transfusions was admitted (06/2009) for surgery. his hemoglobin was 13.2 g/l. an anti-ge antibody was detected and it was decided to transfuse ge-positive packed red blood cells (prbcs). however, no blood transfusion was needed. in july 2017, the patient was admitted for colon cancer surgery with a hemoglobin of 11,0 g/dl. the anti-ge alloantibody was still detectable and a ssp-pcr revealed the genotype ge*01.-03. an mma performed on the pre-transfusion sample revealed a monocyte index (mi) of 0.35% and the antibody was considered not to be clinically relevant. the mi was interpreted as following: 0-3% not significant; 3-5% inconclusive; >5% clinical significant. however, due to the clinical background of the patient it was decided to transfuse ge-negative prbcs, which were obtained from etablissement francais du sang (efs), paris, france. two days after surgery, the patient received 2 units of ge:-2,-3 prbcs without any transfusion reaction. one and a half year later (11/2018), peritoneal carcinomatosis, as a complication of colon cancer, was diagnosed. the patient's hemoglobin was 87 g/l and he had a passage disorder, symptoms of deterioration and an adynamia. based on the mma results from july 2017 indicating no clinical significance of the antibody, it was decided to transfuse ge-positive prbcs. in the following 16 days the patient received a total of 5 units of gepositive prbcs no immediate or delayed transfusion reaction were observed following these transfusions. two further mma's, performed on samples drawn on december 12 th and 16 th (12 days after transfusion of a total of three and two days after two further prbcs respectively), showed a mi of 0.8% und 1% respectively and the anti-ge antibody was considered still not to be clinically significant. summary/conclusions: we report the case of a patient with an anti-ge antibody transfused with ge-positive prbc. as ge-negative prbc are not available in switzerland and not easy to obtain internationally the mma can help in the decision on how to transfuse. in this case, the clinical course confirmed the mma-based prediction. transfusions of ge-positive prbcs were tolerated without signs or symptoms of immediate or delayed transfusion reactions. background: abo grouping discrepancies occur when the results of forward grouping are not corroborative to those of the reverse grouping. these may be due to weak subgroups of a and b, missing or weak abo antibodies or red cell alloantibodies. determination of correct abo blood group of a donor is essential for preventing abo incompatible transfusions and to avoid hemolytic transfusion reactions in the recipient. aims: to determine the frequency of abo discrepancies and their resolution to correctly identify the blood group of the donors. we also determined the frequency of 'weak d' positivity in rhd negative donors. methods: this was a retrospective study on donor samples collected from 1 st april, 2013 to 30 th september, 2015 (two and a half years). for discrepant samples, the abo and rhd grouping was repeated using tube technique using commercial antisera {anti-a, anti-b, anti-ab and anti-d (igm), anti-d blend (igm+igg), anti-h and anti-a1 lectins}. adsorption-elution testing was done for detecting weak subgroups of a and b. antibody screen (3-cell) and identification (11-cell) was done by gel technique (bio-rad, switzerland). 'weak d' testing in rhd negative donors was also performed by gel technique. antibody titration was done using tube technique. the donor details including name, age and the registration/unit number of the donation were also checked for all the discrepancies to avoid repetition while data analysis. results: we detected 104 (0.072%) abo discrepancies out of the total 144279 donor samples tested during the study period. out of these, 135043 (93.6%) were rhd positive. the most common cause of abo discrepancies was weak anti-b antibody (33/104; 31.73%), followed by weak anti-a antibody and weak subgroups of a (24/104 each; 23.07% each) and weak subgroups of b (5/104; 4.8%). the remaining 17.3% (18/104) discrepancies were due to agglutination with o cells in reverse grouping. the overall frequency of weak subgroups of a and b collectively was 0.02% ( background: detection of unexpected red blood cell (rbc) antibodies before transfusion is critical for prevention of hemolytic transfusion reaction. ideally, unexpected rbc antibody detection is carried out within 3 days after receiving a patient's sample. however, in some cases, retests could be performed after more than 3 days for evaluation of any transfusion reaction, quality control or research. therefore, it is necessary to determine the stability of antibodies after refrigeration or freezing for a certain period of time. aims: we carried out antibody identification test with fresh, refrigerated and frozen samples using automated analyzer ih-500 and manual tube methods to evaluate the stability of antibodies after storage and compare the results between the two methods methods: antibody identification tests were performed using ih-500 (bio-rad, 1785 cressier fr, switzerland) and manual tube methods. fifty samples showing positive results in antibody screening test by both methods were divided into three and tested immediately, 1 week after storage at 4°c and 1 month after storage at à20°c. the specificities and reactivities of antibodies at each storage state were recorded and compared between the two methods. results: specificities of antibodies identified were concordant between ih-500 and manual tube methods irrespective of the storage state. the results were as follows: anti-e/e+c, 15; anti-le a , 4; anti-di a , 4; anti-c+e, 3; anti-m, 3; anti-d, 2; anti-c, 1; anti-k, 1; anti-jk a , 1: anti-xg a , 1; unidentified antibody, 13; autoantibody, 2 cases. with regard to the changes in reactivity owing to storage, 26 (52%) samples (anti-e+c, 12; anti-m, 3; anti-di a , 3; anti-d, 2; anti-c+e, 2; anti-le a , 1; anti-c, 1: autoantibody, 1; unidentified antibody, 1) showed identical reactivities after 1 week and 1 month storage by both ih-500 and tube methods. however, 19 (38%) samples, comprising 12 unidentified antibodies, 3 anti-le a , 1 anti-c+e, 1 anti-e, 1 anti-e+c, and 1 autoantibody, showed decreased reactivities after storage in both methods. three samples, comprising anti-di a , anti-e+c and anti-k antibodies, showed increased reactivities after storage. one sample with anti-jk a showed increased reactivity only after 1 month storage, while one sample with anti-xg a showed decreased reactivity only after 1 month storage. higher reactivities were observed in all samples detected using the ih-500 analyzer than manual tube methods (p < 0.005, wilcoxon rank sum test). summary/conclusions: the specificities of unexpected antibodies detected by ih-500 and tube methods were the same in all storage states; however, reactivities were higher in ih-500 than in the tube method. twenty-six (52%) of 50 samples showed identical reactivities after 1 week refrigeration and 1 month freezing. nineteen (38%) samples showed decreased reactivities after storage; however, 12 (12/19, 63%) of them were nonspecific antibodies, unable to identify using commercial id panels. therefore, it is suggested that retests for evaluation of transfusion reaction, quality control or research could be reliably performed after more than 3 days, if stored appropriately in refrigerated or frozen states. abstract withdrawn. t gleich-nagel 1 , d huber-marcantonio 1 , n rufer 1 , g canellini 1 and c niederhauser 2 1 unit of transfusion medicine, interregional blood transfusion src, lausanne 2 laboratory diagnostics, interregional blood transfusion src, bern, switzerland background: a positive direct antiglobulin test (dat) is mainly found in patients with warm/cold autoantibodies or alloantibodies directed against transfused erythrocytes. the identification of antibodies fixed on red cells is important for the clinician, allowing the further evaluation of a patient's clinical situation including their current medication. in immunohematology the elution of a positive dat remains a tedious and expensive procedure. the blood transfusion service src (bts src) has derived a flow chart that indicates in which situation an elution of dat positive samples should be performed. in order to follow the bts src guidelines, it is mandatory to obtain additional data related to the patient's condition, such as haemolytic parameters and recent transfusion history. currently, our laboratory is not always able to apply the recommended flowchart, since information is often unavailable. aims: here, we performed a comparative study between the algorithm provided by bts src and our in-house strategy, which is based on the qualitative changes of a positive dat, without the need for additional patient and biological information. methods: details of dat positivity and the patient's transfusion history was taken from the software eprogesa (mak-system) and analysed in excel. we analysed a total of 3'753 dats and evaluated them for their positivity, whether an elution was performed or whether antibodies were detectable in the eluate. furthermore, we performed an additional analysis on those samples, that were derived from recently transfused patients (<14 days). results: a positive dat was found for igg and c3d in 421 out of 3'753 (11.2%) samples, a level similar to previous reports of positive dats for hospitalized patients. among these positive samples, 244 (57%) were eluted because of a qualitative change in their positivity according to our in-house algorithm. identification of warm autoantibodies or alloantibodies occurred in only 10.7% (26/244) of the cases. from the 161 patients transfused within the last 14 days and having a positive dat, 60 (14%) were eluted according to our in-house algorithm. the same samples would have been analysed if the swiss transfusion guidelines had been applied. however, this comparative study reveals a significant discrepancy in regards to overall sample numbers that should have been eluted according to the two algorithms (244 versus 60 samples). this is mainly due to the fact that the swiss transfusion based algorithm does not recommend an elution of positive dats from patients who did not receive a transfusion within the last 14-days, except if there is a significant clinical suspicion (e.g. haemolysis). summary/conclusions: this comparative study indicates that our elution-based algorithm was performed on all clinically relevant samples as recommended by the bts src guidelines. qualitative changes in the dat positivity represent our main parameter for selecting those samples to eluate. besides ensuring that no clinically relevant samples were missed, this strategy also led to a large number of unnecessary elution analyses. in conclusion, a significant reduction in the laboratory workload and economical savings arises if the relevant clinical information and patients history is known prior to laboratory analysis. background: novel anti-cd38 monoclonal antibodies, such as daratumumab (dara) and isatuximab, used in treatment of multiple myeloma, interfere with routine blood bank serologic tests. as part of the strategies to manage these patients, it is recommended to perform extended phenotyping to provide matched units (aabb association bulletin #16-02). many investigations have focused on the interference with iat for the screening and identification of underlying alloantibodies and how to overcome them, but less has been published on the potential interference with extended phenotyping techniques. aims: the purpose of this study is to compare different technologies to type the most important antigens in myeloma patients before and during the treatment with therapeutic anti-cd38 antibodies. vox sanguinis (2019) 114 (suppl. 1), 5-240 methods: edta-anticoagulated whole blood samples coming from 30 patients in different stages of treatment with daratumumab and 5 with isatuximab have been typed in parallel with dg gel microcolumn (grifols) and mdmulticard technology (grifols). the results are also compared with genotyping results obtained with id core xt (grifols). direct coombs, autocontrol and antibody screening has also been performed as complementary tests. results: the study provides that four patients had positive dat and/or ac before therapeutic cd38 antibodies treatment. in these cases, 6 of 7 negative antigens (fy / jk and/or s) turn to positive in gel technology but mdmulticard showed 100% agreement with genotype id core xt results. focusing in the data obtained during the treatment, 8 negative antigens were type as positive in gel technology (12% of the tests). mdmulticard agreed with genotype in 100% of the analyzed antigens. as complementary data, 13 of 66 patient-treated samples had dat or ac positive and 59 showed panagglutination. summary/conclusions: the results demonstrated that mdmulticard is an effective method to type cd38-directed cytolytic antibodies treated samples in addition to dat and or autocontrol positive samples. background: antibody titration is a semi-quantitative method to estimate the strength and concentration of antibodies present in plasma or serum sample. titration methodology should be validated together with clinical data to evaluate the relevance of the titer value in each application. the titer of an antibody depends on different parameters: the antibody concentration in the sample, the density of the corresponding antigen expressed on the red blood cells used, the affinity constant of the antibody-antigen and other parameters regarding the technique used (e.g. gel cards or tube test). gel cards technology reduces the intra and inter-laboratory variation in titration studies comparing with the tube technique. aims: to evaluate the suitability of dg gel coombs, dg gel anti-igg and dg gel neutral (grifols) for titrations using two sample volumes 25 ll and 50 ll. methods: twenty frozen plasma samples containing unexpected antibodies from different specificities (anti-jk a , -fy a , -k, -d, -e and -c) were titrated in dg gel coombs and dg gel anti-igg cards and 20 donor fresh plasmas with natural occurring antibodies (anti-a and -b) were titrated in dg gel coombs and dg gel neutral (saline technique). the titer of the antibodies was determined by testing two-fold dilutions of the plasma with selected red blood cells depending on the antibody tested. plasma samples were diluted in dg gel sol. selected red blood cells serascan diana, serigrup diana or donor blood were added into the card (50 ll at 0.8%). further, sample dilutions were dispensed into the card (25 ll or 50 ll). subsequently, cards were incubated 15 min, 37°c (coombs technique) and 15 min, 18-25°c (saline technique), centrifuged in dg spin and the results read. agglutination intensity was graded visually according to the instructions for use of dg gel cards. the reciprocal of the highest plasma dilution that gives macroscopic agglutination was interpreted as the titer. results: titers obtained with dg gel coombs and anti-igg (n = 80 titrations, titer ranged 0-256) were compared for each sample with unexpected antibodies. no differences were found between gel cards types (differences were ≤ 0.5 titer in the 98% of the cases). differences between dg gel coombs and neutral (saline technique) (n = 80 titrations, titer ranged 2-512) were observed when anti-a and -b antibodies were titrated using the same sample. the titer was similar or higher in coombs in comparison to the saline technique. coombs titers may be a mix of igm antibodies reacting at 37°c and igg antibodies. differences were > 1 titer in 35% of the comparisons and ≤ 1 titer in the rest of the cases (65%). comparing sample volumes of 25 ll and 50 ll in all cards (n = 160 titrations), higher titers were observed using 50 ll, as expected. differences were 1 titer in the 51% of the comparisons, <1 titer in 44% and > 1 titer in the 5% of the cases. background: autoimmune haemolytic anemias (aiha) are characterized by production of antibodies directed against red blood cells and destruction by the mononuclear phagocytic system or complement system. aiha observed in paediatrics is usually self-limiting and often precipitated by viral infections. in some, the condition is secondary to autoimmune diseases, drugs, infections or underlying primary immune deficiencies. appropriate immuno hematological evaluation to characterise the underlying autoantibody can help identify the type of aiha to aid in diagnosis & treatment of these cases. aims: retrospective analysis of immune-hematological evaluation, treatment and outcome of aiha in paediatrics. methods: patients aged 0-18 years, diagnosed with aiha, between april 2017-december 2018 (21 months) were included in this analysis. aiha was defined as positive direct coombs' test (dct) with anemia associated with corroborative evidence of haemolysis in the form of raised indirect hyperbilirubinemia, raised ldh, raised reticulocyte counts or red cell agglutination on peripheral smear. further monospecific dct and evaluation for the specificity of autoantibody was done for all patients using biorad gel cards and panel cells. steroids were given as first line in all; second line agents included cyclosporine and rituximab. red cell transfusion was given in those with severe anemia with cardiac decompensation. results: 10 patients were diagnosed during the study period with autoimmune haemolytic anemia. haemoglobin at presentation ranged from 2.5 to 9 grams/dl. the initial presentation was severe anemia in 7 children and mild-moderate anemia with thrombocytopenia (evan's syndrome) in 3. the trigger for haemolysis was infection in 4 children. rheumatological evaluation was performed for 5 children out of whom 2 were diagnosed as evolving lupus. primary immune deficiency evaluation was advised for 4 and one child was diagnosed as suffering from combined immunodeficiency. dat was positive in 9 out of 10 aiha patients as one of the infant had dat negative iga mediated aiha secondary to viral infection. two out of 9 dat positive cases had igg & c3d positivity on monoclonal dat testing whereas rest 7 had only igg coating the red cells. dat titration was more than 1:300 in 4 patients; where only 1 of these 4 patients had both igg1 and igg3 coating and rest 3 had only igg1. alloantibody screen was negative in all. specificity of autoantibody was found only in one case, which was against rh blood group antigen (anti e). all patients received prednisolone as the primary treatment. three children attained remission following a 4-6 weeks of steroids. in those who were steroid dependent, cyclosporine was used as the second line agent in 2 and rituximab was used in 3. out of these children 5 children are in sustained remission and off any medication, whereas the rest are on low dose steroids with cyclosporine. summary/conclusions: aiha is not an uncommon problem in children and can vary in its clinical severity. the proper diagnosis and management involves efficient immuno-hematological evaluation, as detailed characterization of the autoantibody coating the red cell is very important in guiding the clinician for management and prognosis. abstract withdrawn. background: drug-induced immune hemolytic anemia (diiha) is rare and has only been described once with dexchlorpheniramine (polaramine tm), an antihistaminic agent widely used in the treatment of a variety of allergic reactions. we report a case of diiha complicated with acute renal failure associated with antibodies to dexchlorpheniramine. a 64-year-old woman with no history of transfusion, was treated semimonthly with a combination of chemotherapy and targeted therapy for metastatic colorectal adenocarcinoma. her chemotherapy regimen consisted of oxaliplatin and 5-fu with leucovorin rescue (folfox). panitumumab (monoclonal antibody anti-egfr) was used as targeted therapy. premedication with dexchlorpheniramine iv was systematically given at the beginning of each cycle of treatment to reduce the risk of perfusion reactions mainly associated with panitumumab. the patient developed chills and febrile agranulocytosis during the first and second infusion respectively. the third infusion was not performed due to pyrexia, chills, general discomfort experienced by the patient at the beginning of chemotherapy. probabilistic antibiotherapy was administered and the patient recovered rapidly. during the next infusion (day 1), following premedication with dexchlorpheniramine, a more "impressive" reaction including all the above mentioned symptoms occured along with back pain and dark colored urine. the infusion was halted and no chemotherapy was delivered. bacterial infection at the implantable port was first thought to be the cause of this adverse event but was not confirmed. additional laboratory findings revealed biological signs of inflammation associated with iha and acute renal failure. the patient was treated with hemodialysis (day 5), two units of rbcs (day 6) and was discharged one week later in stable condition. dexchlorpheniramine was then suspected and samples collected on day 24 were sent for a diiha laboratory workup. aims: the aim of this study was to support a clinical diagnosis of diiha. methods: laboratory workup included direct and indirect antiglobulin tests (dat and iat). drug antibodies investigation was performed by incubating patient's serum and eluate from patient's rbcs in the presence of drug against normal donor rbcs that had not been previously treated with the drug (i.e., by the so-called "immune complex" method). control tests were performed in parallel. drug was diluted in pbs and tested at 1 and 5 mg/ml. results: dat was positive (anti-igg 2 + , anti-c3d 2 + ) and no unexpected rbcs antibodies were detected by iat in patient's serum and eluate without the in vitro addition of the drug. an antibody directed against untreated (titer 2) and enzymetreated (titer 8) normal donor rbcs was demonstrated only in patient's serum in the presence of the drug tested at 5 mg/ml by the gel method. the pool of normal sera did not react in the presence of the drug. summary/conclusions: the multi-drug treated patient described in this study was demonstrated to have dexchlorpheniramine dependent antibody detected by the "immune complex" method. the key to the diagnosis was the observation of positive dat with negative eluate tests which prompted a reexamination of the medications administered in temporal relationship with the hemolytic event. although rare, this case report should alert physicians to the need to investigate the possibility of dexchlorpheniramine induced hemolytic anemia in any patient who develop unexpected anemia after hematologic or oncologic procedures p-331 singapore, singapore, singapore background: daratumumab is a monoclonal antibody against cd38 used in the treatment of multiple myeloma and has been known to bind to cd38 on rbc's and interfere with indirect antiglobulin based serologic tests such as red cell antibody screens and crossmatch compatibility testing. in order to negate the interference of daratumumab, our reference laboratory follows the daratumumab protocol recommended by the aabb which uses dithiothreitol (dtt) treated reagent red cells in red cell antibody screening and identification test in patients known to have received daratumumab. aims: the objective of this study is to determine the impact of daratumumab in the turnaround time (tat) for red cell antibody screening and identification. methods: a retrospective review of the tat for red cell antibody screening and identification samples of patients known to be treated with daratumumab from october 2016 to december 2018 was performed. turnaround time is defined as the time the sample is received up the time the results were reported. the tat for routine red cell antibody screen and identifications were also reviewed during the same period and was compared with the tat of samples from patients treated with daratumumab. results: a total of 55 patients on daratumumab had samples sent to our reference laboratory for red cell antibody screen and identification during the study period. information on daratumumab treatment was not provided to the reference lab prior to the start of testing in 23 of the 55 patients while the use daratumumab was mentioned in the serology request form of the other 32 patients. the median tat for red cell antibody screen and identification is 212 min (range: 47-877) if information on daratumumab was provided prior to start of testing and 301 min (range: 133-1417) if information was not provided prior to testing. the median tat for routine testing is 198 min (range: 15-2245). using wilcoxon rank-sum test, turn-around time for antibody screening and identification for daratumumab treated patients was observed as statistically not significant when compared to routine samples (p value 0.72634). however, tat for serologic tests requests with appropriate medical history compared to the testing requests without relevant information was also observed to be significantly difference (p value 0.00694). summary/conclusions: there is no significant impact in the tat of red cell antibody screen and identification in patients known to receive daratumumab as compared to routine testing. however, there is a significant difference in the tat if information on daratumumab treatment is not provided prior to testing. this highlights the importance of providing the relevant medication information in the request form in order to prevent delays in testing and provision of blood to patients on daratumumab, which can result in improved organizational efficiency and have positive impact on cost and resource savings. background: daratumumab, an anti-cd38 monoclonal antibody, has been shown to be highly efficacious in the treatment of multiple myeloma (mm). cd38 is a glycoprotein highly expressed on plasma cells and, to a less extend, on the surface of red blood cells (rbc). when bound to cd38 on rbc, daratumumab interferes with the pretransfusion tests, with positive antibody screening and crossmatch. anti-cd38 interference is an important challenge as many mm patients will require blood transfusions during their treatment. dithiothreitol is a reducing reagent with multiple applications in blood bank testing. treatment of rbc with dithiothreitol irreversibly removes cell surface cd38 tertiary structure, avoiding the binding and testing interference by the anti-cd38 daratumumab. aims: to demonstrate the efficacy, safety and celerity of the protocol between the blood bank (bb) and haemato-oncology of our institutions, using just the crossmatch. methods: a retrospective research was used for the evaluation of the results obtained from the implemented protocol. this comprehends a previous contact by haemato-oncology that leads to a study of the patient before the beginning of daratumumab treatment, and consists in: abo/rhd grouping; rh and kell phenotyping, and other clinically significant antigens; antibody screening; and direct antiglobulin test. genotyping may be required for some patients who received previous blood transfusions. before the beginning of the therapy, a blood sample of the patient is sent to the bb to perform laboratory tests and frozen after. this frozen sample is used for crossmatching in patients that already started therapy, did not have a blood transfusion in between, and have a positive antibody screening and/or crossmatch. in further transfusions, in case of positive tests, the dithiothreitol-treated donor rbc is applied. the donor rbc antigens are always selected accordingly to patients negative clinically significant antigens, when transfusional support is needed. the laboratory tests are executed in gel column agglutination technique. results: since 2016, 32 patients were studied, from which 16 were transfused with 108 blood units, according to the protocol. there were no immunizations or adverse reactions to transfusion registered within the transfused patients, neither delay on the availability of blood units. patient blood sample collected and frozen prior to the beginning of the treatment, has shown to be a good strategy by reducing significantly the waiting time for the blood unit in the first transfusion. summary/conclusions: this protocol, which defines the communication among the involved professionals, has shown to be a secure and effective way of reducing interferences caused by daratumumab. it ensures the previous study of the patients and their transfusion with rbc respecting the patients negative clinically significant antigens. if not adopted, the mitigation measures described in this protocol, delays in the availability of the rbc requested and alloimmunizations, may and will possibly occur. a good communication between the bb and the haemato-oncology is crucial for a good time management when a transfusion is requested for these patients. three methods were used to resolve this dara interference. reagent rbc's were treated with dtt, which know to denature cd38 and then tested with patient plasma. allo-adsorption study was performed using a certain ratio of red cells to plasma. in addition, a selection of phenotyped cord cells were used as an antibody screening panel. results: dtt treatment of reagent red cells was successful at eliminating dara interference and allowing for the presence of underlying antibodies to be identified. in this case, underlying antibodies were not detected by using reagent dtt treated red cells or phenotyped cord cells. adsorption technique was ineffective at elimination the reactivity. summary/conclusions: dara is the first commercial fda-approved therapeutic monoclonal antibody used in treating multiple myeloma patients. • since cd38 is weakly expressed on normal red blood cells, dara attachment to red blood cells can interfere with pre-transfusion iat testing. • dtt treatment of reagent red blood cells and cord cells can abolish the interference of dara to test for the presence of underlying alloantibodies. • to prevent delays in issuing red blood cell units to patients, hospitals should send patient samples to be tested before receiving dara treatment to ensure that clinically significant alloantibodies are not being masked. background: antibody screening (as)is considered superior to antihuman globulin (ahg) cross match during pretransfusion compatibility testing. in spite of knowing the utility and superiority of as, it has not been adopted uniformly in india. therefore, scarce data is available from this subcontinent in terms of optimisation of red cell antibody detection during pretransfusion testing in form of "type and screen" aims: the main objective was to study the benefits of performing simultaneous antibody screening along with the blood grouping during the first hospital visit to the hospital. other objectives were to study the prevalence of clinically significant antibody among the indian population and to follow up the patients who were transfused antibody screen negative but cross match incompatible blood. we also studied some other relevant quality indicators related to efficiency of blood transfusion services methods: this prospective study was carried out at a tertiary healthcare centre in india between july 2014 and dec 2018 (54 months). the study protocol was submitted to institutional review board and permission was granted. blood grouping and as were done during patients' first hospital visit, which we called "type and screen". when the patient got admitted to the hospital and required blood transfusion, a blood request form was generated by the user and sent to blood bank. depending upon the results of antibody detection, further course of action was chosen. if patient was found to have no antibody, immediate spin test (ist) cross match compatible blood was issued and transfused. in such cases the procedure of ahg crossmatch testing was continued even after issue of blood. cases where ahg cross match test was found negative no further follow-up of the patient was done whereas when ahg cross match was found positive, patients were followed after the transfusion results: a total of 22888 patients were "type and screened". majority were from hemato-oncology, bmt, liver transplant, paediatric cardiac surgery, and medical icu units. clinically significant allo-antibody was detected in 145 patients and autoantibody was detected in 53 patients. alloantibody was detected mainly against rh and kell blood group systems. in diagnosed aiha cases, majority were in the form of warm aiha (58%) and 20% of aiha cases were having hidden single or multiple alloantibody. significantly higher proportion of patients in as positive group required blood transfusion than as negative group (45% vs 34%, p < 0.05). in both the groups, in planned cases, most of the time blood was issued immediately within the defined turnaround time except in 21 where either transfusion was delayed or surgery was postponed. it happened only in trauma or surgical bleed cases. expiry of blood was decreased significantly due to no usage of blood (1.2% vs. 5%, p < 0.05). during the period of study we obtained 10 cases where the ist cross match was compatible but the ahg cross match was incompatible. during follow up none of the cases demonstrated any sign of hemolysis summary/conclusions: in developing countries like us, optimization of as during pretransfusion testing increases operational efficiency and significantly decreases the expiry of blood. results: during the period when absc was performed on pk7300, 250,599 donation samples were tested and 4,895 (1.95%) were found absc positive. antibodies to red cells were identified in 250 donations out of 4,895 (5.11%) absc positive samples and in the rest, no irregular antibodies were detected. the prevalence rate for atypical antibody was 0.10%. the top 5 most frequent antibody specificities were: nonspecific cold antibodies (28.8%), anti-e (26.8%), anti-mi a (19.6%), anti-m (16.8%) and anti-le a (4.0%). a total of 225,124 donations were screened for atypical antibodies by ih-1000 and 2,275 (1.01%) were screened positive. among these, anti-red cell antibodies were identified in 1,239 samples (54.46%), which was significantly higher than those identified in pk7300 screened positive samples (p < 0.00001). the prevalence rate for atypical antibody as screened positive by ih-1000 and with confirmed red cell specificities was 0.55%, which was also significantly higher (p < 0.00001). the top 5 most frequent antibody specificities were: anti-mi a (55.3%), anti-m (21.1%), anti-le a (10.2%), anti-e (6.5%) and non-specific cold antibodies (3.6%). anti-fy b was detected in 7 cases, which would be missed detection by enzyme treated reagent cells on pk7300 system. summary/conclusions: the performance of the ih-1000 system using a 3-cell screening panel including one cell with mi(a+) expression and column agglutination technology with iat phase was superior in comparison with that of pk7300 in the context of higher sensitivity in detecting more true positive results and higher specificity in detecting more true negative and less false positive results. this has translated into the advantages of reduction in workload of reference laboratory in performing less antibody identifications in those false positive samples as well as enhanced transfusion safety by removing more irregular red cell antibody positive plasma-containing components from the issuable inventory, which may potentially lead to haemolytic transfusion reactions. the prevalence of irregular red cell antibodies of 0.55% in healthy blood donors in hong kong reflects more the true statistical figure. background: chronic red blood cell (rbc) transfusion is the upfront therapy for thalassaemia patients, however this therapy is featured by several adverse events including rbc alloimmunization. phenotype matched products transfusion policy can prevent alloantibody formation, but it makes routine transfusion more difficult for both the donor center and the transfusion service. a recent systematic review (franchini et al, blood transfus 2019) reported a rbcalloimmunization prevalence of 11.4%, with a higher incidence against rh and kell systems in thalassemia intermedia patients. aims: the aim of our retrospective study is to evaluate the rbc alloimmunization prevalence in thalassemia patients transfused in a single center over a 18 years period with limited phenotype matched rbc (rh and kell system antigens) units. methods: from 1990 to 2018 thalassaemia patients, with a minimum follow up of 1 year and transfused with more than > 10 rbc units, were included in our study. patients were studied for: blood group and rh / k phenotype determination, direct antiglobulin test (dat), irregular antibodies research (abirr). cross-match and detection of alloantibodies were performed using the indirect antiglobulin test by the column agglutination method. six-monthly dat and antibody screening were performed using the indirect antiglobulin test and enzymatic papain-treated rbc test. results: overall 57 patients (38 females, 19 males) were included in our retrospective analysis: 54 patients were affected by thalassaemia major and 3 by thalassaemia intermedia. rbc alloimmunization prevalence was 12.3% (7 patients): 3 patients were found to be positive for rbc alloantibodies, four with alloantibodies and autoantibodies. eleven alloantibodies were detected (1 anti-h, 1 anti-cw, 4 anti-e, 1 kpa, 1 anti-jka, 1 anti-jkb, 1 anti-m and 1 anti-lua). in 2 out of 3 alloimmunized patients we found an anti-e antibody reactive in enzymatic papain-treated rbc test only, in the third alloimmunized patient anti-kpa and anti-lua antibodies were detected, while in the remaining 4 patients, in which auto and alloantibodies were detected, a severe autoimmune hemolytic anaemia (aea) requiring therapy was diagnosed. in these cases the appearance of alloantibodies is concomitant with the presence of autoantibodies. among the 7 patients positive for alloantibodies, 6 were affected by major thalassemia and one by intermedia thalassaemia summary/conclusions: in our experience a limited phenotype matched rbc transfusion policy showed a rbc alloimmunization prevalence similar to literature data: 12.3% vs 11.4%; we didn't find higher alloimmunization prevalence in thalassemia intermedia patients may be due to the low patients number. we believe that introduction, in our department, of an extended-phenotype matched transfusion, including antigens of the main group systems (fy, jk, mns) and the main rare antigens (cw, kp, lu), could reduce the risk of red blood cell alloimmunization in thalassemia patients. abstract withdrawn. background: undoubtedly, preventing alloimmunization has an advantage over overcoming its consequences. however, the high cost of technical and organizational aspects of preventive measures requires their scientific substantiation confirmed by clinical and laboratory data. selection of donors of the rhesus (d, c) and kell (k) antigens for the red blood cell transfusions to hematological patients has been regulated in the russian federation since 1998. recipients with the phenotype c+c-transfuse red blood cell only with the same antigenic combination. for transfusions red blood cell obtained from k-negative donors are used. the compatibility of the donor and recipient with the antigens c, e, e, c w (rhesus system) and k (kell system) is additionally taken into account from april 2013. that is, transfuse red blood cell that do not contain antigens in the phenotype that are not in the recipient's phenotype. aims: to evaluate the efficiency of red blood cell donor selection using antigens of rhesus (c, c, e, e, c w ) and kell (k, k) systems for the prevention of the recipient alloimmunization. methods: immunohaematological studies using equipment and reagents of biorad (usa) were performed in 3642 patients of the hematology clinic. non-hodgkin lymphoma was diagnosed in 759 patients, acute leukemia in 600, multiple myeloma in 390, chronic lymphatic leukemia in 319, chronic myeloid leukemia in 218, aplastic anemia in 147, hemophilia in 105, myelodysplastic syndrome in 73, and other hematological diseases in 1031. the frequency of detection of antibodies to antigen c (0.25% vs 0.06%) and to antigen e (0.46% vs 0.12%) decreased four times. the frequency of detection of antibodies to the c w antigen has not changed significantly (0.10% vs 0.06%, respectively). selection of antigens c (rhesus) and k (kell) has been carried out in the clinic since 1998, therefore the immunization index for these antigens remained unchanged and amounted to 0.10% vs 0.12% for anti-c antibodies; 0.36% vs 0.36%for anti-k antibodies. alloantibodies to the antigens e (rhesus) and k (kell) were not detected for the entire observation period. summary/conclusions: research verified the effectiveness of alloimmunization prevention of recipients by selecting red blood cell for antigens c, c, e of the rhesus system and k (kell). the study concluded that selection of red blood cells for the antigens c w , e (rhesus) and k (kell) does not affect the level of alloimmunization of patients and is not clinically justified. background: in the russian federation, there is an order according to which patients requiring multiple transfusions, who are at high risk of immunological complications are to typed for red blood cell antigens: abo, d, c, c, e, e, cw, k, k. selection of erythrocyte-containing blood components is carried out taking into account the donorrecipient compatibility according to all the listed antigens. aims: analysis of results of immunological evaluation of patients of hematological clinic. methods: the study included 466 first time patients of hematology clinic in 2017-2018. typing of antigens of abo, rhesus, kell systems, screening and identification of antibodies were carried out using equipment and reagents from bio rad (usa). results: interpretation of results of immunohematological screening was complicated in 84 (18.0%) patients. the total number of complex cases was 96. the double population of red blood cell was most often determined in antigens of the rhesus system (10.9% of the total number of patients) as a result of previous transfusion therapy. of those, chimera for the antigen e was detected in 31 cases (60.8% of patients with the chimera for rhesus and kell antigens), cin 23 (45.1%), sin 15 (29.4%), e -5 (9.8%), cw -8 (15.7%), k -5 (9.8%). in such cases, donor red blood cells were chosen not carrying chimeric antigen for transfusions, in the presence of chimeras in both paired antigensred blood cell transfusion with the cc phenotype and / ee. chimera for abo antigens was detected in 0.6% of the examined individuals. the discrepancy between the direct and reverse blood grouping of the abo system in patients (1.1%) was due to a decrease in the production of antibodies -4 cases and the appearance of extra agglutinins -1 case. autoantibodies were detected in 3.9% of all patients, including 0.6% of patients, when they caused panagglutination phenomenon. upon detection of autoantibodies that complicate the individual selection of donors, transfused red blood cells that are compatible with antigens of abo, rhesus, kell, duffy, kidd, mns systems. alloantibodies were detected in 2.8% of patients, including specific anti-din 4 (0.86%), anti-dcin 2 (0.43%), anti-kin 1 (0.21%); antibodies of unidentified specificityin 2 (0.43%), polyspecificin 4 (0.86%). summary/conclusions: the complexity of interpreting immuno-hematological tests in hematological patients is due to intensive transfusion therapy, changes in red blood cell antigens and appearance of nonspecific antibodies due to underlying disease. red blood cell for transfusion in these patients should be selected taking into account the expanded red blood cell antigen profile. abstract withdrawn. background: blood transfusion is an essential part of therapy for many patients. although life-saving for many patients, blood transfusion is not without risk. the main goal of blood transfusion services is that transfused blood should be compatible with the patient. the clinical and serologic evaluation, which allows for the transfusion of the most compatible (or "least incompatible") blood, requires a joint effort between the clinician and the transfusion medicine physician. aims: root cause analysis of incompatible cross matches in patients. methods: in this prospective study, total of 3,49,497 crossmatches were performed over period of last four & half years, out of which 867 units were found incompatible by column agglutination method-cat in polyspecific (anti-igg+ c3d) gel media. a root cause analysis protocol was formulated to resolve incompatibility to ensure safe transfusion. results: on the evaluation of 3,49,497 crossmatches, only 867 units were found to be incompatible (0.14%). the major cause for incompatibility found in patients was aiha-(32.87%). other causes of incompatibility were infections (27.44%), multiple transfusions (17.41%), trauma (11.23%), evan's syndrome (4.15%), rh negative mother (3.57%), sca (2.99%) & incompatibility due to dat positive packed red cells (0.34%).the most common antibody found were anti-'c', anti-'s' & anti-'m'. summary/conclusions: the rca protocol involves a thorough evaluation of the patient's clinical condition and underlying pathology to identify the cause. a logical stepwise approach will enable provision of safe transfusion to the patient. background: antibodies to high-frequency antigens (hfas) are a transfusion hazard, as compatible blood is often very difficult to obtain. other clinically significant alloantibodies represent an additional transfusion risk. in patients treated with allogeneic bone marrow transplantation (bmt) recipient red cell alloantibodies may cause acute or delayed haemolysis of donor red blood cells (rbc) and contribute to morbidity and mortality. aims: the aim is to present the case of a patient with myelodysplastic syndrome (mds), multiple "common" alloantibodies and an additional alloantibody to a highfrequency antigen, treated with allogeneic bmt. methods: a forty-one-year-old caucasian patient with mds (raeb-1) was admitted to our hospital in january 2017 for unrelated allogeneic bmt. she previously received myeloablative conditioning therapy according to the flu / bu4 / atg protocol (5 days of 250 mg iv. fludarabine, 4 days of busulfan 792 mg iv, 2 days of 300 mg iv. antithymocyte globulin). the indirect antiglobulin test (iat), done in august and december of 2016, was negative. according to anamnestic data, the patient had two pregnancies. she received red cell transfusions during childbirth and platelets in december 2016. results: the patient's blood group was o rhd positive, iat positive. the donor blood group was a rhd positive, iat negative. phenotype of the recipient's rbcs, as well as the donor rbcs, was also determined. anti-e and -c w were found in the patient's plasma, but an additional alloantibody was suspected. the autocontrol was negative. column agglutination technology (cat) and tube technology were used to identify rbc antibodies. plasma was tested with pheno-matched rbcs, papain-and 0.2 m dithiothreitol-treated rbcs, as well as cord and autologous rbcs. adsorption and elution tests were done, excluding other "usual" clinically significant alloantibodies, and the patient received three incompatible (xm in iat, cat) yt(a+), e-, c w -, k-red cell units. the sample was urgently sent for an antibody investigation at the international blood group reference laboratory (bristol, uk). in the reference laboratory, anti-e, -c w and an alloantibody to a high-frequency antigen were confirmed, whose specificity was determined to be anti-yt a . anti-jk b was also suspected and later confirmed. before the patient was discharged from the hospital, she received eight more red cell units (yt(a+), e-, c w -, jk(b-)), during which she was serologically closely monitored. summary/conclusions: the results of the antibody investigation in this case study indicate the presence of multiple alloantibodies in a patient who has previously received immunosuppressive myeloablative conditioning therapy. in addition to the "common" alloantibodies (anti-e, -c w , -jk b ), an alloantibody to a high-frequency antigen (anti-yt a ) was detected in the patient. this patient was transfused with incompatible red cell units (yt(a+)) in an emergency, with no ill effects. although anti-yt a is rarely a clinically significant antibody, according to literature, it can cause immediate haemolytic transfusion reaction. additional risk were "common" clinically significant alloantibodies, especially anti-jk b , which was in this case extremely difficult to detect and had further complicated the selection of blood. background: the identification of an antibody against a high-incidence antigen always introduces a challenge due to the difficulty in finding compatible units of red blood cells (rbcs). in patients needing surgery it is important to minimize their transfusional needs by implementing patient blood management programs (pbm). tests that predict the clinical significance of antibodies, such as monocyte monolayer assay (mma) are also useful in guiding clinical decisions. kell blood group system contains highly immunogenic antigens. antibodies against these antigens are immunoglobulin g, and can cause severe hemolytic transfusion reactions and fetal anemia. results: case report we report the case of a 75-year-old female, with non-hodgkin lymphoma, chronic anemia and scoliosis with severe neurological compromise, proposed for lumbar spinal stabilization surgery. she had a total hip replacement surgery in 2004, with unknown transfusion history. her obstetric history was g6p4a2. the patient had no history of thromboembolic or hemorrhagic events. during pre-transfusional tests, she was typed as a 0 rr and had a positive antibody screening test. the identification studies were suggestive of an antibody against a highincidence antigen, so the surgery was delayed until clarification of these results. she was also referred to a pbm appointment where her hemoglobin was improved from 9.0 g/dl to 12.0 g/dl by administration of ferric carboxymaltose iv and darbepoetin sc. the patient was phenotyped as kp(a+b-) with anti-kpb, an antibody against a highincidence antigen (>99% prevalence worldwide). it is a rare antibody with variable reactivity, causing from none to moderate/delayed transfusion reactions. to access the clinical significance of this antibody, a mma was performed, resulting in a reactivity of 1.2%, suggesting no clinical relevance, however it could be altered after transfusion of kpb+ blood. in order to find compatible rbc's, several family members were phenotyped, however they were all positive to the kpb antigen. in portugal there were no 0 rr kp(b-) blood donors, as it is extremely rare, so we searched in the international rare donor panel (irdp) and two donors were found in spain. two units of compatible rbc's were requested prior to the surgery, which was performed successfully four months later without transfusional support. summary/conclusions: anti-kpb is a rare antibody that in some cases can cause hemolysis of the transfused kp(b+) red blood cells. the combination of kp(b-) and o rr, an extremely rare phenotype, presented a challenge in finding compatible rbcs. this case illustrates not only the complex transfusional and logistic problems that an antibody against a high-incidence antigen can pose, but also the importance of an efficient pbm programme to mitigate the transfusional needs in these patients. background: blood transfusion is an integral part of the supportive care for patients with sickle cell disease (scd). allo-immunization is a recognized complication to red blood cells transfusion (rbc) in those patients. this may result in difficulties in providing compatible blood, and may be associated with the risk of acute of delayed hemolytic transfusion reactions. aims: to describe transfusion management in a patient with scd who has multiple alloantibodies with difficulty in obtaining compatible blood, in order to highlight the importance and clinical consequences of this complication and suggest a possible management approach methods: an 18-years-old female patient with scd presented to our hospital with hemoglobin level of 4 g/dl secondary to acute splenic sequestration. she had a history of multiple previous admissions and many previous rbc transfusions. blood grouping and pre-transfusion compatibility testing were performed in addition to phenotyping of the patient's red cells. screening was done using column agglutination technique by automated machine (ortho; usa) and antibody identification was performed manually using commercial 11 cells identification panel. phenotyping for the patient was done using haemagglutination technique with mono-specific anti sera (bio-rad; switzerland). results: the patient was of group o rhd (positive). antibody screening was positive and antibody identification revealed probable anti-e and anti-fya with possible development of anti-k allo-antibodies, in addition to recent development of autoantibodies; giving pan-positive reactivity with the identification panels. phenotyping of the patient's rbcs was found to be r1r and k-negative. other masked allo-antibodies of undefined specificities were suspected and no compatible blood was found. the clinical condition warranted a blood transfusion, so least incompatible phenotypically matched rbc unit was released. the patient developed acute hemolytic transfusion reaction with drop of the hb level to 2.8 g/dl. despite screening hundreds of rbc units, no compatible units were identified, and no transfusion was given. the patient was managed conservatively using hydration, analgesics, hydroxyurea, erythropoietin, intravenous immune globulin (ivig), steroids, and rituximab. hb level increased to 8 g/dl in 2 weeks, and the patient was discharged from the hospital. the sample of the patient was sent to a reference lab (institut fur klinische chemie und laboratoriumsmedizin-regensburg -germany) for further investigations, clarifications and advice for compatible transfusion in case of need. the report of the reference lab revealed the development of additional anti-m and anti-s with confirmation of the presence of anti-fya, anti-k and warm auto-antibodies. phenotyping of rbcs was confirmed by molecular diagnostic testing done in the reference lab; as r1r, k-neg. summary/conclusions: finding compatible blood may be extremely difficult in patients with scd who develop multiple alloantibodies. it is therefore essential to perform an initial extended red cell phenotyping for the patients at diagnosis and to have on shelf ready phenotyped blood units for issuing to the patients, to minimize allo-immunization. transfusion may occasionally be avoided in allo-immunized patients, utilizing alternative options of treatment and reducing the risk of serious complications such as hemolytic transfusion reactions. background: red blood cell (rbc) antigens that are present on less than 1% of most populations are known as low incidence antigens and those present on more than 90% are known has high incidence antigens. the mns blood group system consists of 49 antigens carried on glycophorin a (gpa), glycophorin b (gpb) or on hybrids of these glycophorins. there are 35 low incidence and 10 high incidence antigens in the mns blood group system. an individual that is homozygous for gp.mur will be negative for the high incidence jenu (mns49) antigen. anti-jenu was first described in a thai patient with thalassemia where only 2 compatible units were found following screening of 3600 units. the jenu negative phenotype is a rare phenotype with an estimated frequency of 0.06%. a male patient with a history of previous transfusion presented with an anti-e and a weak auto antibody with no apparent specificity. a donor unit selected for cross match (group o rhd positive, c+e-c-e+, k-) was incompatible with a reaction grade of 4 + by column agglutination technology. the patient's sample and donor unit were referred to the red cell reference laboratory for investigation for a possible antibody to a low incidence antigen. aims: we aim to characterize the phenotype of the incompatible donor unit. methods: standard serological procedures were used to identify the antibody specificities in the patient's sample. blood group phenotyping of the patient and donor was performed by standard serological procedures. genotyping and zygosity testing was performed using polymerase chain reaction (pcr) high-resolution melting (hrm) assay. gp.mur is a gp(b-a-b) hybrid glycophorin resulting from a gene conversion event between gypa and gypb . this disruption to gpb impacts s expression. the donor was negative with anti-s moab (albaclone), positive with anti-s polyclonal (immulab) and negative with anti-s monoclonal antibody (immulab). this s and s phenotype was consistent with the previously reported examples of gp.mur homozygote jenu negative individuals. molecular testing was consistent with serology supporting gp.mur homozygosity and jenu negative phenotype. summary/conclusions: this donor has been added to our rare donor panel and their red cell donations are cryopreserved for future use in our rare donor frozen inventory. there is limited anti-jenu antiserum available to confirm the jenu negative phenotype. we currently rely on the serological profile of red cells presenting with the gp.mur phenotype, s negative and the discrepant s phenotyping to identify jenu negative donors. this case has highlighted the importance of following up unexplained serological incompatibilities. the development of a monoclonal antibody directed against jenu antigen would provide an opportunity to screen for suitable donors for this rare phenotype. background: molecules expressed on tumor cells are a target of interest for drug development by the use of monoclonal antibodies or blocking proteins. however these drugs have the potential to interfere in pretransfusion testing when the target molecule such as cd47 is also expressed on red blood cells (rbcs). recently, many drugs targeting cd47 have been developed but appropriate mitigation strategies and approach to selecting rbcs for safe transfusion is still an obstacle. aims: we describe a case of delayed hemolytic transfusion reaction (dhtr) by anti-jk a in a patient treated previously with cd47 targeted high affinity sirpa fusion protein alx148. methods: a 35-year-old woman diagnosed with nasal cavity squamous cell carcinoma was enrolled in an alx148 clinical trial. her blood type was group ab, rhd positive, and the antibody screening test was negative for the past 8 months. she had no previous transfusion history during the past two years. after two infusions of alx148, two units of apheresis platelets were requested for transfusion. the blood bank noticed that the antibody screening was positive and further investigation was proceeded. results: antibody screening showed trace positivity in both panel cells (i & ii) at room temperature (rt) and 37°c albumin phase, and 2 + at anti-human globulin (ahg) phase by tube method. the auto control was negative at rt and 37°c albumin phase, but 2 + at ahg phase. antibody screening (2 cells) and identification (11 cells) all showed 3 + at ahg phase using gel cards. direct antiglobulin test was 4 + for anti-igg and 3 + for anti-c3d using gel cards. two units of rbcs were requested for transfusion after hemoglobin decrease to 7.8 g/dl. rbc genotyping was unavailable at the moment. as her previous antibody screening was negative (anti-jk a not detectable), e-, c-, fy b -rbcs were given as a second best option, considering the phenotype distribution of major blood groups in the korean population. the hemoglobin level was well sustained between 11.1-12.0 g/dl but it decreased again to 9.2 g/dl twenty days after rbc transfusion. further laboratory investigation was consistent with a dhtr. the patient was no longer being given alx148, and antibody screening and dat decreased to 0-1+ reactivity. we presumed that antigen typing results would be reliable after chloroquine dissociation and cell washing using antisera that did not require ahg for testing. serologic phenotyping showed that the patient's cells were c+, e+, c+, e+, jk a -, jk b +, fy a +, fy b -, s-, s+, m+, n + . antibody identification using papainized panel cells revealed anti-jk a antibody. we concluded that the dhtr was due to anti-jk a , and jk a -, fy b -, s-rbcs were issued for further transfusion requests. the patient's hemoglobin level recovered to 13.6 g/ dl. the patient's genotype was later identified to be the same as serologic typing. summary/conclusions: communication with the physician and blood bank to perform adequate pretransfusion testing before administration of drugs targeted to cd47 is important to achieve safe transfusion for patients. serologic phenotyping using antisera which do not require ahg for testing can be used as a second option when genotyping is unavailable in a timely manner. background: transfusion is still a key treatment for sickle cell disease (scd) patients. as a result, these patients are much more exposed to transfusions' risks, the most feared one being a delayed hemolytic transfusion reaction (dhtr). we investigated a female scd pediatric patient with no known antibody, who was referred to us for a suspicion of two dhtrs. three transfusion episodes were reported (a total of four units collected from four donors). for the last transfusion, a premedication with rituximab was done. the patient was planned to undergo a bone marrow transplant with her brother as her donor. aims: to describe the molecular and serological workups needed to investigate a dhtr in a scd patient. methods: antibody identification and crossmatches were performed by iat gel testing with red blood cells/panels, which were used raw, papain-treated and trypsintreated. rbcs' phenotypes were determined by conventional techniques. semi-quantitative phenotypes were conducted by serial dilutions with a monoclonal anti-jk a (ms15/seraclone â ). dna was extracted using the magna pure compact instrument (roche). sequencing of jk exons 4-11 was carried out by in-house techniques. results: the antibody identification showed a very weak anti-jk a , which was only reactive on papain-treated rbcs. autologous control was also only positive in this technique. dat and the eluate were negative. as the patient had recently been transfused (less than four months earlier), on this first sample we were neither able to perform autologous adsorptions, nor verify her jk a /jk b phenotypes. in order to rule out the imputability of an anti-lfa in the dhtr outcome, crossmatches with her donors' rbcs were undertaken. three out of the four donors were tested. apart from the anti-jk a reactivity, none of them was reactive. because the patient had previously been phenotyped as jk(a+b+), her jk gene was sequenced. her genotype was determined as jk*01(28a,226a,303a,588g)/jk*02. to confirm this jk a variant allele, a family study was conducted. all her siblings were found to harbor the same genotype. her mother's and father's genotypes were jk*01(28a,226a,303a,588g)/ jk*01 and jk*02/jk*02, respectively. subsequently, autologous adsorptions were performed, which proved the anti-jk a to be an autoantibody. considering the weakness of this antibody, internal controls were used, in order to evaluate a possible dilution effect of this technique. finally, serial dilutions with the anti-jk a reagent showed a weakened jk a expression encoded by the jk*01(28a,226a,303a,588g) variant allele. this finding is consistent with the fact that the crossmatches between the proband's serum and her brother's rbcs were weaker than those performed with (jka+b+) rbcs. summary/conclusions: about a third of dhtrs are reported to happen in patients with no previous history of immunization. performing sensitive serological techniques in order to identify antibodies is necessary to select the most appropriate units. molecular work and extra serological testing can be useful to determine whether an antibody is an allo or autoantibody. even though in this case the anti-jk a was the only antibody identified, because it was proven to be an autoantibody, it is difficult to conclude if it was the cause of the dhtr. nevertheless, jk(a-b+) blood was issued, and no adverse events have been reported. luckily, the patient's bone marrow donor harbors the same variant allele. background: according to the aabb, a pre-transfusion sample must be obtained within 3 days of transfusion if a patient has been transfused or pregnant in the preceding 3 months. despite this safeguard, high risk patients (i.e. those recently transfused with a history of pregnancy or transfusion) may develop antibody during this 3 day window. to avoid issuing incompatible red blood cells (rbcs) to these patients, a new antibody screen (abs) sample should be drawn and tested shortly before anticipated transfusion. aims: we report a case of a 60 y/o man who presented to the ed (hospital day 0, hd0) with a post-fall intracranial hemorrhage and multiple fractures. anti-e and anti-jka were identified after admission on a new specimen prior to current specimen expiration (<3 days). methods: specimen #1 (s1) was sent on hd0 for type & abs (t&s) and crossmatch (xm) of 2 rbcs. abs and immediate spin xm were negative; there was no patient history. by hd9, he had 4 negative t&s specimens (hd0: s1; hd2: s2&3; hd6: s4) and had been transfused 4 rbcs (hd2: 2; hd5: 2) via electronic xm (exm). at 1730 hr on hd9, 2 rbcs were requested and could have been issued via exm since s4 was not expiring until midnight. however, given recent transfusions, bb staff first called the patient's nurse to review history. patient was uncommunicative, but had scars suggesting past trauma or surgery. s5 was requested and received at 1801 hr. results: s5 showed anti-e and anti-jk a in plasma and eluate. his hemoglobin/hematocrit (h/h) decreased from 10.2 (14.0-17.5 g/dl)/30.1 (41.5-50.4%) on hd6 to 6.9/ 20.6 on hd9. during this period, he underwent several surgeries without unexpected bleeding, documented jaundice or dark urine. two e-jk(a-) rbcs were transfused on hd9, which he tolerated well with an increase of hemoglobin from 6.9 g/dl to 8.6 g/ dl. he did well post transfusion with stable h/h between 8.1/24.2.0 to 8.5/25.3. he was discharged on hd19. repeat abs on s4 was negative. of the 4 rbcs transfused before s5, one was e+ and four jk(a+). the family reported that he was injured 20 years prior and had been admitted to 3 hospitals, but was unaware of transfusion. hospital #1 (h1) reported admissions 20 years ago (2 rbcs transfused) and 4 years ago; all abs were negative. h2 admission was 5 years ago with positive abs and inconclusive workup. h3 admission 4 years ago showed negative abs. summary/conclusions: the patient developed a significant antibody response in less than 3 days from the specimen collection, likely a secondary immune response to sensitization from a transfusion 20 years earlier. a new specimen was requested prior to transfusion even though the existing sample (which was abs negative) had not expired. this approach identified new antibodies, preventing transfusion of incompatible rbcs, and a potentially serious hemolytic transfusion reaction. this case suggests that for high-risk patients, abs more frequently than every 3 days may be beneficial. it is important to increase clinicians' and laboratorians' awareness of this issue. background: red cells with partial d antigen have historically been classified as such, based on the fact that the red cells type as d positive, but individuals make anti-d antibody when exposed to conventional d antigen. a definitive confirmation of the variant of d antigen is obtained after the rh d genotyping. aims: to present a case study of the patient's alloimmunisation with the present d partial antigen type dnb, most likely on previously received transfusions. methods: the patient's pretransfusion testing included the determination of the abo blood group and rhd type (id card diaclon abo/d dv+, dv-, reverse grouping, monoclonal antibodies, gel method), antiglobulin crossmatch, additional phenotyping (gel and tube methods), antibody screening, identification of the specificity of irregular anti-erythrocyte antibodies by commercially available red cell panels (id dia-panel bio rad gel method, panocell immucor, tube method) through an indirect antiglobulin test (iat) and enzymes. after routine rhd typing we continued further characterisation of the rhd antigen by serologic assay (bio-rad id-partial rhd typing),and finally by rhd antigen molecular genotyping (fluogene method on fluo vista machine). results: our patient is a 75 year old woman with a diagnosis of tu mammae who was preparing for total mastectomy surgery. she had a history of blood transfusions twenty years ago, and she also had two births. the blood group typing was: o, ccdee, k-, fy (a-b +), jk (a+b +), ss, mn, le (a+b -). the agglutination reactions that we tested with anti d serums were strong (4+). the compatibility test with rhd positive donated blood units was positive. the presence of anti-d and anti-fya antibodies in the serum of the patient was determined. we prepared one compatible blood unit, rhd negative and fya negative, for a surgery. interpretation of the id-partial rh d typing set indicated that this is a diii category of d partial antigen. a sample of blood of our patient was sent to the blood transfusion institute of serbia, where molecular typing of d antigen was performed and the presence of partial form of antigen d, dnb type, was found. summary/conclusions: rhd positive patients or donors with anti-d antibody presents in their serum should be tested for d genotyping. the recommendation for further transfusions of our patient with dnb d partial and her alloimmunisation is to prepare d negative, fya negative erythrocytic blood components, and as a possible blood donor it would be labeled as rh d positive. background: the jr blood group system consists of jr a (jr1), a high frequency antigen expressed by the abcg2 gene. the individuals with jr(a-) phenotype are mainly found in the japanese population and may develop anti-jr a when stimulated by blood transfusion or pregnancy. anti-jr a is a dangerous antibody for pregnancy, but also could cause mild or moderate neonatal jaundice. aims: to conduct the antibody specificity identification of the high frequency antibody in a pregnant woman with history of pregnancy but no transfusion. methods: abo, rhd and some special blood group antigens were identified by tube method in saline. antibody screening and blood group specific antibody identification were performed by indirect antiglobulin test (iat) in gel column. the reagent cells treated with trypsin, chymotrypsin and papain, were used to test the antiserum to obtain the characteristic of antibody reaction. the antibody titer in the patient's serum was detected. dna sample was extracted and 16 exons and adjacent intronic sequence of the abcg2 gene were sequenced. the sample of one family member was collected for testing. results: the blood groups of the patient were b, rhd(+), lu b (+) and kp b (+). the negative reaction of the serum reacted with all reagent cells were tested in saline, but positive (2+) in iat test, while the self-control was negative. the antiserums reacted strongly (4+ in iat test in gel card) with the papain-treated cells, but kept the same reaction strength (2+) with trypsin-and chymotrypsin-treated cells, which indicated the possible existence of anti-jr a . the titer of igg antibody in serum was 2. in cross matching test, the red blood cell of the patient's brother with the same abo and rhd blood group with the patient was successfully matched with the serum of the patient. the sequencing analysis of the abcg2 gene in the patient and her brother revealed one homozygous nonsense mutation in exon 4 (c.376c>t, p.gln126x). after the delivery of the pregnant women, no pathological jaundice was seen in the newborn. summary/conclusions: in the condition of the anti-jr a reagent was unavailable for the identification of jr a antigen in the patient, having an indication with anti-jr a by serological test, the alternative genotyping method was used. the most common silencing jr allele reported in asian population, especially in japanese population, was identified to indicate jr(a-) phenotype. immunohemotherapy, centro hospitalar vila nova de gaia/espinho, vila nova de gaia, portugal background: if the investigation of irregular/unexpected antibodies reveals a pattern in which all or most screen and panel cells are positive, with reactions in the same phase and with the same strength, along with a negative autocontrol, an irregular antibody to a high-prevalence antigen may be suspected. high-prevalence antigens are those that are present in almost all individuals (98% or more). fortunately, because these antigens do occur so frequently, it is not common to find a patient with an antibody to one of them. however, when it happens, it may become a troubling situation. aims: clinical case report of panagglutination in assessment of irregular antibodies. methods: collection of clinical data in scl ınico â and sibas â applications. results: woman, 76 years old, o rhd+, previously transfused with 4 red blood cells concentrates in 2006, was proposed to a correction surgery of a periprosthetic hip fracture. pretransfusion serologic tests were requested and irregular antibodies were detected (2+ in all the 3 screening cells). in order to identify the specificity of the antibody, a panel of 11 cells was tested; the result was considered inconclusive, due to positive reactions (2+) with all test cells in liss/coombs and atypical positivity with dragging in all cells in enzymatic environment. autocontrol and direct antiglobulin test were negative. it was decided to send two blood samples to the reference laboratory for a more complete immunohematological study. compatibilization of red blood cells to this patient was also requested. during the waiting period, haematopoiesis was optimized. although the patient did not present anaemia at admission, the analytical study revealed iron deficiency; therefore, supplementation with intravenous iron was performed. the reference laboratory also obtained a panreactive panel (2+ with all cells) in liss/coombs and weak positivity in papain. after allo-adsorption, the search for irregular antibodies was negative. an anti-yt a , apparently without clinical significance (negative igg1 and igg3) was then identified. transfusion was not needed either during or after the surgery, with a good recovery of the haemoglobin value in the postoperative period. summary/conclusions: yt a , which belongs to cartwright system, is a high-prevalence antigen in all populations. anti-yt a , an igg antibody stimulated by pregnancy or transfusion, is not as uncommon as we may think, which suggests that it is reasonably immunogenic. these antibodies are not generally considered clinically significant, but there are reported cases of acute and delayed haemolytic transfusion reactions in which anti-yt a has been implicated. therefore, although the described pattern of panagglutination in assessment of irregular antibodies may suggest the presence of an alloantibody directed against a highfrequency antigen, it is very important to confirm that hypothesis, recurring to a reference laboratory if necessary, to identify the antibody and to determine its clinical relevance. even if the identified antibody is associated with rare haemolytic transfusion reactions, it is crucial to optimize haematopoiesis when it is not an emergent procedure, in order to minimize transfusion and its associated risks. both for emergent and elective procedures, the creation of a national database of patients with already identified irregular antibodies would facilitate the administration of red cells concentrates without the implicated antigen. aims: to investigate the frequency and explore the genomic characterization of jk (a-b-) phenotype in blood donors in harbin, china. methods: all samples were screened for jk(a-b-) phenotype using a direct urea lysis test. and the results were confirmed with by iat using anti-jka and anti-jkb with a standard tube test. additionally, polymerase chain reaction amplification and sequence analysis of the jk gene were performed. results: from 80865 blood samples, four donors with jk (a-b-) were selected, at a frequency of 0.0049%. among these four samples available for sequencing jk gene, a total of two genotypes were discovered: heterozygote of ivs5-1g>a combining with heterozygote of 359g>a (gly120glu) and heterozygote of 896g>a (gly299glu) combining with heterozygote of 956c>t(thr319met). summary/conclusions: the frequency of jk(a-b-) phenotype in blood donors in harbin area was lower than the reported data from the populations in other areas of china and in finland, but higher than that in japan. ivs5-1g>a, 896g>a and 956c>t were common mutations in the before reports, while 359g>a was reported first time. in addition, it is an effective measure which establish the jk(a-b-) phenotype donors in this region, to solve the blood transfusion problem in patients with anti-jk3. background: blood types, indicating the type of blood group antigen expressed in the red blood cells, is determined by the type of allele at the blood group gene locus. therefore, when allele frequency of each blood group gene is determined, it is possible to predict the frequency of a specific blood type donor with a homozygous allele. it is also possible to estimate the proportion of donors within a particular blood type through combination of specific alleles. and because the ratio of blood group allele differs between ethnicity and race, this can be used as basic data for population genetics and anthropology. therefore, we present a study that examined the allele frequencies of 10 blood group systems in the korean population through blood group genotyping. aims: the purpose of this study is to determine the frequencies of blood group alleles in the korean population, to predict the proportion of homozygous donors, and to obtain the basic data of population genetics. methods: 5,213 blood donors from age 19 to 54 were recruited at korean red cross blood centers located nationwide. acquired samples were examined by blood group genotyping methods using the rbc genotyping system id core xt (progenika biopharma). for each donor, genotypes of 10 blood group systems, excluding abo and rhd, were identified. calculation of the frequencies of blood group alleles in the korean population was done. results: we conducted molecular genotyping of the rhce, kell, kidd, duffy, mnss, diego, dombrock, colton, cartwright, and lutheran blood group systems. the allele frequencies of these blood group systems in the korean population were estimated as follows. -rhce*ce 0.3%, rhce*ce 65.0%, rhce*ce 28.8%, rhce*ce 5.9% -kel*k_kpb_jsb allele 100% -jk*a allele 49.1%, jk*b allele 50.8%, jk*b_null allele 0.1% -fy*a allele 92.8%, fy*b allele 7.2% -gypa*m allele 51.1%, gypa*n allele 48.9% -gypb*s allele 5.1%, gypb*s allele 94.8%, gypb*mur allele 0.1% -di*a allele 4.7%, di*b allele 95.3% -do*a allele 10.1%, do*b allele 89.9% -co*a allele 100% -yt*a allele 100% -lu*b allele 100% summary/conclusions: the significance of this study is accumulation of data on the allele frequencies of blood group genes through highly accurate genotyping method in the east asia region. this enables the prediction of the proportion of donors with a combination of specific blood group alleles in the korean population, which accounts for a decent percentage of the population in this region. background: in donors from arabian countries only little is known about blood groups other than abo and rhesus. during the last years increased migration to central europe has put a focus on the question how to guarantee blood supply for patients from these countries, particularly because hemoglobinopathies with the need of regular blood support are more frequent in patients from that region. aims: blood group allele frequencies should be determined in individuals from syria, other arabian countries, and iran by molecular typing. methods: as most blood groups are defined by single nucleotide polymorphisms (snps) we introduced a maldi-tof ms assay to detect alleles encoding 16 blood groups including kk, fy (a/b), fy null , c w , jk(a/b), jo(a+/a-), lu(a/b), lu (8/14), ss, do (a/b), co(a/b), in(a/b), js(a/b), kp(a/b), rhce*c.697c>g, and rhce*c.733c>g. additional blood groups and polymorphisms like yt(a/b), s-s-u-, vel null , co null and rhce*c.667g>t were tested by pcr-ssp. a total of 1111 probands including 800 individuals from syria, 147 from iran, 123 from the arabian peninsula and 41 from northern african countries were included. results: 2% of the donors were homozygous for the fy null (fy*-67t>c, fy*02n.01) mutation, 16.2% carried the heterozygous mutation. 0.4% of the syrian probands were heterozygous for the do*350c>t mutation (both, do*jo1 and do*jo2; jo(a+/ a-)) that is virtually unknown in caucasian donors. 0.8% of the syrian donors heterozygously carried the kel*02.06 allele coding for js(a) (phenotype js(a+/ b+)) that is very rare in caucasians. however, no homozygous kel*02.06 carriers were identified. 1.8% of the syrian and 1.5% of all donors were negative for yt*a, which is definitely more frequent than in europeans. one donor from northern africa homozygously carried the gypb*c.251c>g, intron 5 + 5 g mutation, inducing the s-u+ w phenotype. 3.6% of all and 29.3% of northern african donors were heterozygous for the rhce*c.733c>g substitution, 0.3% of the syrian donors carried rhce*c.697c>g (heterozygously) and 0.004% of all donors were heterozygous for rhce*667g>t. heterozygosity for vel deficiency (vel*-01) was detected in 21 individuals (2%; 16 of them from syria) whereas only one syrian donor carried the homozygous mutation. none of the donors carried the aqp1*c.601delg (co*01n.06) mutation that induces the co null phenotype. summary/conclusions: the study provides a first overview on a number of different blood group alleles in blood donors from arabian countries. some blood group alleles that largely are lacking in europeans but had been described in african individuals are present in arabian populations at a somewhat lower frequency. in single cases it could be challenging to provide immunized arabian patients with compatible blood. methods: three unrelated individuals (2 blood donors and one pregnant woman) of polish origin who were typed as ab group with a very weak a antigen and normal b antigen expression were subjected to extended abo typing. in one case family studies were performed (blood samples from donor's mother, father and sister). sequencing analysis of this donor dna was performed three times (from two blood samples and buccal swab). serologic investigations were performed with standard methods: 1/gel cards diaclon id abo/d (anti-a: clone a5, anti-b: clone g1/2, anti-a,b: clone es131, es15+ birma 1+ es4; bio-rad) and diaclon id abd-confirmation for donors (anti-a: clone m297/628 = la-2; bio-rad); 2/tube techniques with: anti-a (birma 1; a-11h5, a1 s.pa1m095, c.9113d10), anti-b (lb-2, b-6f9, c.9621a8). genotyping was determined by rbc fluogene abo basic kit (inno-train, germany) and by sequencing of +7.21-kb site of abo gene to cover sequences ranging from the end of intron 1 to 3 0 utr of the abo gene. additionally sequence of exon 1 of the abo gene was analyzed. results: abo typing showed normal b and a very weak a antigens on rbcs of all three individuals (2 blood donors and one pregnant woman). the a antigen was detected by tube technique only using anti-a clones: birma 1 (1+ to 2+), a-11h5 (1+ to 2+) and c.9113d10 (weak+ to 1+); negative reaction of a antigen typing by gel cards was observed. the sera of all individuals contained anti-a1 antibodies. commercial pcr-ssp kit revealed three heterozygous a/b genotypes (absence of 1061delc typical for abo*a2 alleles). in all these individuals abo sequencing of 7.21-kb fragment confirmed the heterozygous genotype with 7 polymorphisms characteristic for abo*b.01 allele (297a>g; 526c>g; 657c>t; 703g>a; 796c>a; 803g>c; 930g>a) and detected a novel abo*a allele sequence with duplication-based insertion of 21 bp after 564 position (abo*a c.dup543_563; gcaggacgtgtccatgcgccg). as a consequence, the online protein translation predicts an in-frame duplication of seven amino acids after codon 188 (p.dup_182_188qdvsmrr), with synonymous change of the repeated codon 182 (cgc>cgg) and 188 (cgg>cgc) but both coding arginine (r). inheritance of abo*a c.dup543_563 allele was confirmed by family studies of one donor: his father and sister had a/b genotype associated with normal a and b antigens expression; his mother had normal a antigen expression. she carried abo*a1.01 allele and the same abo*a c.dup543_563 allele as a son. summary/conclusions: a novel a weak allele at the abo gene detected in three unrelated polish individuals is an in-frame insertion of seven amino acids to the wild-type glycosyltransferase a. the stability of the encoded protein may be affected causing the weak a phenotype. the inheritance of this mutation was confirmed in the family studies. background: since the cloning in 1990 of cdna corresponding to mrna transcribed at the blood group abo locus, polymorphisms and phenotype-genotype correlations have been reported by many investigators. although many subgroups have been explained at the genetic level, unresolved samples are still encountered in clinical practice. we report here the result of an abo investigation of a sample from a swedish blood donor that showed a very weak agglutination of rbcs with anti-a in routine forward typing. aims: to elucidate the genetic basis of the apparent weak a subgroup. methods: routine abo genotyping by pcr-asp and pcr-rflp including pcrbased analysis of the upstream cbf/nf-y-binding enhancer region was carried out. further genetic analysis was performed by dna sequencing of abo exons 1-7 (including 50 base pairs of the adjacent introns) and the proximal promoter. flow cytometric testing of rbcs was performed with monoclonal anti-a, anti-b and anti-h. results: the weak agglutination of erythrocytes with anti-a was accompanied by the expected lack of anti-a and anti-a1 in plasma. abo genotyping gave the genotype abo*a1.01/o2.01 usually consistent with normal expression of a antigen. enhancer analysis resulted in an amplification product corresponding to the expected single cbf/nf-y binding motif. flow cytometric testing of the sample showed a antigen expression with an almost chimeric pattern where the majority of the cells (approximately 85%) expressed the a antigen at a very low level, marginally distinguishable from the group o control. the remaining approximately 15% of the cells displayed an a antigen level ranging from normal to very weak. genomic abo sequencing showed an abo*a1.01-like allele except for a novel mutation located in intron 5, c.239+4g. the o 2 allele had an additional snp, c.689g>a, consistent with the abo*o2.03 allele variant summary/conclusions: a previously unreported variant, c.239+4a>g, likely effecting the 5 0 -donor splice site of intron 5 was found in an a weak sample. this type of mutations is expected to decrease mrna stability and/or cause skipping of the preceding exon in the mrna. however, small amounts of full-length enzyme might still be made, being able to give rise to the weak a antigen expression seen in this individual. interestingly, this mutation is very similar to the genetic variant underlying the weak a subgroup a finn . in this case, however, the c. 374+4a>g mutation is located in the 5 0 -end of intron 6 and is predicted to cause partial skipping of exon 6. strikingly, the a finn phenotype also results in a pseudochimeric pattern by flow cytometry but with only approximately 2% positively staining erythrocytes. due to the well documented lack of a-allele-derived mrna in peripheral blood, further transcript studies could not be undertaken. further studies are needed to investigate the exact mechanisms underlying the pseudochimeric pattern observed by flow cytometry in these two interesting genotypes/phenotypes abstract withdrawn. background: abo is the clinically most relevant blood group system in transfusion and transplantation medicine. abo genotyping is potentially useful in clarifying serologic blood grouping discrepancies. this scenario includes inherited subgroups alleles, temporary acquired variant abo phenotypes in disease or pregnancy, and chimerism due to exchange of progenitor cells early in fetal life or after blood progenitor cell transplantation. aims: to investigate the molecular basis for abo discrepancies detected in clinical samples, including donors and patients, sent to our reference laboratory during the past 3 years. methods: if routine abo grouping showed weak agglutination or forward vs reverse typing discrepancy, further abo typing studies were performed manually. adsorption-elution tests were also performed in some cases with polyclonal anti-a and anti-b to confirm whether a or b antigens were weakly expressed on the rbcs membrane. a pcr approach using sequence specific primers for a2, b, o1 and o2 alleles was used for initial genotype determination. the full abo coding region was analysed as previously described in selected samples for which abo discrepancy was still unexplained. allele specific fragments spanning exon 6, intron 6 and exon 7 were amplified using a forward primer targeting the 261g nucleotide (to exclude o1 alleles amplification) in combination with either b, a2 or a generic reverse primer. analysis was carried out by sanger sequencing. results: a total of 16 samples with suspected inherited abo subgroup alleles were selected for further molecular studies by sequence analysis. a subgroup alleles: in 8 out of 12 samples with suspected a subgroup alleles, the c.804insg insertion was detected corresponding to the abo*ael.01 allele. the abo*aw31.02-05 variant, a hybrid a1-o1v allele, was found in 2 cases. in 1 case we found the c.722g>c change, previously reported associated with weak a antigen expression. finally, a novel c.961c>g change was detected in an a2 allele. b(a) or cis-ab suspected alleles: the abo*b(a)04 variant carrying the c.640a>g change was found in 1 of 3 samples with bo1 genotype but a weak antigen expression. in the remaining 2 cases, a consensus b allele was detected, thus pointing to a potential chimerism as the cause of the results observed in abo grouping. finally, we have identified an abo*b01.02 allele carrying the nucleotidic change c.926a>g in the context of an abo phenotype vs genotype discrepancy. summary/conclusions: the sanger sequencing approach applied in this study have proved to be informative and helpful to determine the molecular basis of abo grouping discrepancies with suspected inherited subgroups. we found mutations, within exon 7 of the abo gene, in 14 out of 16 samples, including 2 novel alleles. chimerism was suspected in 2 cases of a antigen expression in samples with b1o1 genetic background carrying an apparent normal b allele. we are evaluating at the moment a deep sequencing approach by next generation sequencing to determine the presence of a small amount of a minor allele in the presence of a large surplus of the other two alleles. background: recently, the multiple pregnancy rate has been increasing due to advances in artificial fertilization including in vitro fertilization-embryo transfer. most dizygotic twins have dichorionic placenta, but 8% of them share the placenta. monochorionic dizygotic twins can have blood chimerism, leading to double rbc populations in routine abo serologic typing. recently, more sensitive and objective column agglutination tests with automated systems are being widely used. therefore, blood chimerisms in dizygotic twins can be detected more easily by routine abo blood typing. aims: we report congenital blood chimerism in monochorionic dizygotic twins of triplets, found incidentally during abo serological testing and confirmed by abo genotyping and str marker analysis. methods: a 20-year-old male (one of triplets) was admitted to the hospital for medical checkup. he did not have any history of transfusion or bone marrow transplantation. routine abo blood grouping test was performed using automated blood bank system ih-500; however, it showed abo discrepancy. the red blood cells showed double cell populations in a gel column with anti-a and anti-b. we carried out abo genotyping both from the blood and from a buccal swab. for the further evaluation, we performed abo serologic testing, abo genotyping, and str marker analysis in his family members. results: among the triplets, blood chimerism was demonstrated in the patient and his brother. they both showed a 3 b 3 phenotypes in the serologic test and tri-allelic abo genotypes in the blood, a102/b101/o01. however, in buccal swabs, the patient showed a102/o01 and his brother showed b101/o01. other members of the family (father, mother, and dizygotic sister) had regular abo blood types in the serologic test. we performed str analysis in the triplets and parents. eleven loci (d8s1179, d21s11, d7s820, csf1po, th01, d13s317, d16s539, d19s433, d18s51, d5s818, and fga) revealed more than one additional allele in the blood sample, apart from those in the buccal swabs. str marker analysis showed that his brother too had blood chimerism. summary/conclusions: we found blood chimerism in monochorionic dizygotic twins of triplets during routine abo blood typing, and this was confirmed by str analysis. as the application of assisted reproductive technology increases, the incidence of blood chimerism will also increase. blood chimerism can often create confusion during abo serologic typing and microchimerism can be overlooked in routine methods. therefore, it is helpful to use an automated blood bank system to improve sensitivity and blood chimerism should be considered if abo blood grouping reveals double populations. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial host. results: case #1 is a patient with an unclear abo phenotype: forward type b, reverse type ab. sequencing of genomic dna and cloned abo exon 7 detected variant c.29-5t>g in heterozygosity on an otherwise common a1 allele, and in trans an abo*b.01 allele. case #2 is a caucasian donor with an abo discrepancy: forward type aweak/o, reverse type a. sequencing also detected variant c.29-5t>g in heterozygosity on an a background, and in trans an abo*o.01.01 allele. given that this variant is located near the intron 1 splice acceptor site, abo*29-5g transcripts are postulated to undergo altered splicing, leading to an aweak phenotype. case #3 is a prenatal sickle-cell disease patient with an abo discrepancy: forward type aweak, reverse type a. dna sequencing detected variants c.467c>t (pro156leu) and c.709t>a (tyr237asn), both in heterozygosity on an otherwise common a1 allele, with an abo*o.01.01 allele in trans. thus, the data establish an association of allele abo*467t,709a with an aweak-like phenotype. case #4 is a donor with an abo typing discrepancy: forward type o, reverse type a. sequencing detected variant c.479c>g (pro160arg) in heterozygosity on an a2 background, and in trans an abo*o.01 allele. an interpretation of the data is that variant c.479c>g weakens the activity of the a2 transferase, with allele abo*a2(479g) encoding the aweak-like phenotype detected by serology. case #5 is a 9 year-old patient with an abo discrepancy: forward type o, reverse type ab. sequencing of genomic dna and cloned abo exon 7 detected variant c.803g>c (gly268ala) in heterozygosity on an a2 background, and in trans an abo*o.01.02 allele. the serology and molecular results suggest that allele abo*a2(803c) encodes a cisab weak phenotype. case #6 is a caucasian donor with an abo typing discrepancy: forward type o with a weak agglutination with anti-ab, reverse type o. dna sequencing detected variants c.739g>a (glu247lys) and c.871g>a (asp291asn), both in heterozygosity, in trans, and on a1 backgrounds. variant c.871g>a by itself constitutes allele abo*a3.01. the phenotype encoded by abo*739a is uncertain. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. results: case #1 is a 35 year-old pregnant female with an abo typing discrepancy: forward type o, reverse type a. pcr-rflp predicted abo*a1/abo*o1. sequencing detected variant c.107insg (val36gly>fs56ter) in heterozygosity on an otherwise common a1 allele, and in trans an abo*o.01.01 allele. it is unclear how the early truncation of the a1 transferase encoded by allele abo*107insg still allows for some residual enzyme activity, as suggested by the reverse a type. case #2 is a recently-transfused 72 year-old black patient with an unresolved abo type. sequencing detected variant c.297a>g (silent) in homozygosity and variant c.1049c>t (ala350val) in heterozygosity, both on an o1 background, with an abo*b.01 allele in trans. although variants c.297a>g and c.1049c>t are likely of no consequence to the abo phenotype of this patient, they are reported here as components of a new abo*o1(297g,1049t) allele. case #3 is a 40 year-old prenatal female with a rhd typing discrepancy. failure to yield an abo genotype on blood-chip (progenika), a genotyping microarray that interrogates polymorphic positions in rhd and abo, prompted dna sequencing. sequencing of genomic dna and cloned abo exon 7 detected variant c.436c>t (arg146cys) in heterozygosity on an abo*b allele background, and in trans an abo*o.01.01 allele. the phenotype encoded by allele abo*b(436t) is predicted to be b, as evidenced by forward typing on immucor neo and reverse manual typing. case #4 is a prenatal black patient with an abo typing discrepancy: forward type o in gel, a 2+ mixed field (mf) in tube. reverse type on a1 cells 1+ in gel, 0/1+ in tube. sequencing of genomic dna and cloned pcr products covering exons 6-7 detected variant c.784g>c (asp262his) in heterozygosity, and in trans an abo*o.01.09 allele. case #5 is the newborn baby of case #4, with a forward type a 1+ mf in gel, a 3+ mf in tube. sequencing of the baby's dna detected variant c.784g>c (asp262his) in heterozygosity, and in trans an abo*b.01 allele. from these results it is inferred that the phenotype encoded by allele abo*784c is a3-like. case #6 is an 18 year-old hispanic donor with an abo typing discrepancy: forward type a, reverse type o. sequencing of genomic dna and abo exons 5-6 and 6-7 detected variant c.979c>t (gln327ter) in heterozygosity, and in trans an abo*o.01.02 allele. the truncation of the a1 transferase at such a relatively late position is consistent with the retention of some enzyme activity, explaining the forward a type encoded by allele abo*979t. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. variants reported to date in the intron 1 enhancer include large deletions, small deletions and single-nucleotide substitutions. here we describe four new alleles with single-nucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. adsorption-elution by the heat elution method and testing for h and a substances in saliva were performed by following the procedures in the aabb technical manual. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. background: inactive alleles of the fut1 could be decreased or aborted the activity of the fucosyltransferase, which results in to form the bombay or para-bombay phenotype with weak or no h antigen expression on erythrocytes. now many para-bombay individuals have been found in the chinese population. according to names for h blood group alleles v5.1 170221 of red cell immunogenetics and blood group terminology working group of the isbt, 55 fut1 alleles were identified for bombay or para-bombay phenotype around the world. aims: the study was explored the distribution of fut1 alleles for the 19 chinese individuals with para-bombay phenotype. methods: the samples were come from the blood donors or the patients. the a, b, h antigens were determined using conventional serological method according to the manufacture's instruction. the sequences of the full coding region for fut1 was amplified, then amplicon was purified with enzymes digestion and used as template for sequencing bidirectionally. all nucleotide sequences obtained were analyzed and compared with standard fut1 sequence. results: nineteen chinese individuals with para-bombay phenotype were identified. ten of them were the donors and nine individuals were come from the hospital. the rbcs had a very weak agglutination reaction with anti-h in the most of the individuals. fut1 homozygous mutations were found in the 12 individuals and fut1 heterozygous changes were existed in 7 individuals after bidirectionally sequencing. .05%, 7.89%, 5.26%, 5.26%, 5.26%, 2.63%, 2.63%, 2.63% respectively in the 19 individuals with para-bombay phenotype. according to our previously reports, the fucosyltransferase activity of fut1*01n.06(c.551_552delag), fut1*01w.09 (c.658c>t) and fut1*01w.01 (c.293c>t) were abolished in vitro assay, while fut1 mrna levels of them had no effect compared with wild type. summary/conclusions: the fut1 mutations in the para-bombay individuals were various. the most common fut1 allele in the chinese individuals with para-bombay phenotype was fut1*01n.06(c.551_552delag). background: the regulatory mechanism of the abo gene is complicated and has been investigated extensively.variation in a antigen expression was recognized very early in the twentieth century and the a blood group was divided into a1 and a2. later the a blood group was subdivided further based on characteristic reactivity with human polyclonal antisera, i.e., strength of reactivity and presence of mixed field agglutination; presence of anti-a1, and whether a or h blood group substance was present in the saliva of secretor subjects. mutations critical for abo blood group phenotypes have predominantly been found in exons 6 and 7 of the abo gene, both of which encode the catalytic domain of abo glycosyltransferase. in our case report we show how mutation ranging from single nucleotide in the intron 1 enhancer element can alter the efficacy of enzyme and alter antigen expression. aims: this study aims to investigate the molecular basis of discrepant results of abo forward/reverse typing in blood donor. methods: the abo typing was performed using tube technique and column agglutination tests (bio-rad, grifols). standard tests were completed with adsorption-elution study using o plasma as a source of anti-a, and with saliva testing for presence of a and h substances. we performed quality control for these methods. abo group genotyping was performed using pcr with sequence-specific primer by commercial kit (abo-variant; bag healthcare, lich, germany). pcr-amplified exons and intron 1 enhancer were subject to bi-directional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results: standard serological forward tests identified blood group o, however, only anti-b iso-agglutinins were present. anti-a in adsorption-elution study was successfully adsorbed and eluted from the investigated cells. a and h substances were detected in saliva. abo genotyping using pcr-ssp indicated genotype o1v/a 1 . dna sequence analysis showed result abo*a01 (28+5859a), abo*o.01.02. the specimen was revealed as an a subgroup, probably a m with an unusual genetic variant in the intron region of the abo gene, the enhancer of the gene expression. summary/conclusions: we report the first case of abo*a01(28+5859a), the mutation located in the enhancer region of gene expression in allele a, that causes discrepant results not only in abo forward/reverse typing but also in molecular blood grouping tests. based on our serological findings, this subgroup is considered as a m . background: a chimera is a single organism composed of cells with distinct phenotypes and/or genotypes. several different types of chimeras are described: artificial, twin and dispermic. the artificial chimerism can be seen following hematopoietic stem cell transplantation, or more transiently following blood transfusion. the second type may also be inherited most commonly through blood exchange in utero between twins. dispermic chimerism is induced by the fertilization of two maternal eggs with two spermatozoa and their fusion into one body. this one is also called tetra-gametic chimerism. in transfusion medicine, chimeras are often detected when mixed field reactivity is observed in abo/d typing or, less commonly, when phenotyping for other blood group antigens. aims: this investigation was prompted by finding a double population of erythrocytes in a surgery patient with no transfusion history. our aim was to investigate the chimera and determine the underlying abo genotype of this patient. methods: routine blood grouping was performed by column agglutination. separation of the double cell populations was performed by differential agglutination with igm anti-d (immuclone, anti-d fast igm, clone: d175-2, immucor). initial abo genotyping was performed by pcr-ssp (fluogene; inno-train diagnostik gmbh); further resolution was performed using in-house pcr-asp and pcr-rflp methods. next generation sequencing (monotype abo; omixon using illumina sequencing platform) and sanger sequencing analysis were also performed. identification of reference alleles was investigated by fragment analysis of short tandem repeats (str) polymorphisms. results: double population was found in column agglutination in tests with anti-a and anti-ab, and subsequently when typing for d and c antigens, with approximately 85% of o d+c+ cells. the patient's genotype was identified as abo*o.01/*a by ce-certified pcr-ssp kit (fluogene). routine pcr-asp and pcr-rflp could not resolve the patient's genotype possible abo*a1/*o1 genotype was detected by pcr-rflp, but the pcr-asp analysis gave an apparent abo*a1 homozygote result. sanger sequencing of abo exons 6 and 7 also gave anomalous reactions: no abo*a allele was detected. homozygosity for c.261delg was observed as well as heterozygosity for c768c/a. this result therefore suggests the patient's genotype is abo*o.01/*o.01.26. next generation sequencing (omixon) revealed the same result. however, when pcr amplification of the cbf/nf-y enhancer vntr 3 0 -region was performed, possible heterozygosity was observed, i.e. a weak band representing a single copy, and one representing 4 copies of the enhancer region were present. presence of a single copy of the 43-bp cbf/nf-y enhancer vntr region is unique background: del is a very weak form of d antigen with low density expression of d antigen on the surface of red blood cell, which is generally typed as d-blood group as couldn't form agglutination in routine rhd blood group testing and could only be detected by the non-routine adsorption-elution test. in the east asian and southeast asian population, 9-30% of the individuals with serologically apparent d-phenotype are not these with truly d-phenotype, but del phenotype, which is very rare in caucasian and black ethic groups. and the rhd*01el.01 (rhd*1227a) is most prevalent (>99%) in del people in these regions, so the del carried this allele was commonly known as "asia type" del. in previous studies, no alloanti-d was observed in a large cohort of chinese "asia type" del pregnant women with d+ fetus to indicate no occurrence of alloanti-d immunization against d+ red cell in "asia type" del individuals. aims: to conduct genotyping analysis in the chinese patients having serologically apparent d-phenotype simultaneous with alloanti-d to confirm the existence of the "asia type" individuals to produce alloanti-d or not. methods: from 2017 to 2018, the blood sample of the patients or pregnant women identified with alloanti-d in our reference lab were collected. d antigen was confirmed again using the blend anti-d reagent (clone th-28/ms-26, igm/igg) by tube method in saline and indirect antiglobulin test (iat) in gel card. the zygosity of rhd gene was detected by hybrid rhesus box pcr with psti digestion. for the samples with d or dd genotypes obtained by rhd zygosity analysis, multiplex ligation-dependent probe amplification (mlpa) genotyping was conducted for rhd genotyping analysis. results: a total of 129 serologically apparent d-chinese patients (female, n = 128; male, n = 1) with alloanti-d were identified. different titers of alloanti-d from 1:2 to 1:4096 (≤1:16, n = 60; >1:16, n = 69) were detected including few cases with mixed antibodies (anti-d mixed with anti-c, n = 11; anti-d mixed anti-e, n = 5). serological rhd typing confirmed the serologically apparent d-phenotype. rhd*01n.01/01n.01 (homozygous rhd gene deletion) genotype was identified in majority of them (123/129, 95.3%) by rhd zygosity analysis, while rhd*01n.03/ 01n.01 genotype (n = 5) and rhd*01n.05/01n.01 genotype (n = 1) carried the rhd non-functional hybrid alleles were detected by mlpa. summary/conclusions: compared with the distribution of average 25% frequency of "asia type" del in serologically apparent dpopulation in guangzhou of china, no one case of "asia type" del was identified in the cohort of serologically apparent d-patients with alloanti-d in this study. this also provides evidence to confirm no occurrence of alloanti-d immunization in "asia type" del individuals. aims: a serologically rhd-negative donor was found to be rhd-positive in the routine rhd screen. to solve the discrepancy between serology and molecular screen, the sample was sequenced on dna and rna level. methods: phenotyping on id/iat-cards (bio-rad) was done using commercial anti-d antibodies. the adsorption-elution analysis was performed using an in-house pool of polyclonal anti-d antibodies. furthermore an antibody d-screen was performed (diagast). for rhd genotyping rh-type and partial d-type assays (bag health care) were carried out. the sample was further characterized by exon sequencing including flanking intronic regions. rna was extracted from whole blood, reverse transcribed and the cdna sequenced. for amplification and sequencing, both published (gassner, transfusion, 2005; legler, trans. med., 2001; richard, transfusion, 2007) and in-house primers were used. results: repeated phenotyping of the sample with commercial as well as, in-house anti-d antibodies confirmed the rhd negativity. in addition, the adsorption-elution analysis showed a negative result. however, genotyping, using commercially available kits, yielded a rhd positive result and no variants were detected. to investigate this discrepancy, all 10 rhd exons were sequenced. the sequencing data revealed the mutation c.148+2delt in the splice donor site of exon 1. to confirm the effect of the splice site mutation on transcription, rna from a fresh whole blood sample was analysed. as a positive control, gypb was amplified and sequenced from the same cdna. wild-type gypb (mns4) was found. with rhd specific primers, no product could be amplified. summary/conclusions: we present a serologically rhd negative case, that was identified as rhd positive by standard commercial genotyping kits. sequencing revealed the new splice site mutation c.148+2delt. rna sequencing yielded no detectable product. the donor was classified as rhd negative. this case of a discrepant result between serology and genetics shows the importance of a profound and highly sophisticated genetic investigation of conflicting laboratory results. j stettler, s lejon crottet, h hustinx, c von arx, f still, j graber, c niederhauser and c henny interregional blood transfusion src berne ltd., berne, switzerland background: one of the most immunogenic and clinically significant blood group antigens in transfusion medicine is the rhd antigen. variant rhd phenotypes with weakened or absent antigen expression pose a challenge for rhd status assignment in blood donors. to ensure patient safety, it is necessary to fully characterize these variants at the molecular level. aims: samples from two donors were investigated in our laboratory due to discrepancy in rhd typing. methods: rh blood group phenotyping was done by standard serological column agglutination testing (id-system, biorad). further rhd characterization was performed by an anti-d antibody panel containing 9 monoclonal antibodies (d-screen, diagast) and an adsorption-elution test using an in-house pool of polyclonal anti-d antibodies. molecular investigation was initially performed by ssp-pcr detecting common rhd variants (rbc-ready gene cde inno-train; rh-type bag health care). rhd sequencing was done on either dna or rna using published and inhouse primers for amplification and sequencing. results: by tube testing, the rbcs of donor 1 were predicted to be rh:-1,-2,-3,4,5. however, all ten exons of the rhd gene could be detected by routine genotyping. sequencing of rhd revealed a homozygous mutation c>g at position 1151 which is the second last nucleotide of exon 8 and thus might have an influence on exon splicing. by cdna analysis a transcript with a correctly spliced exon 8 was identified. the mutation c.1151c>g leads to the amino acid substitution t384r located in the twelfth transmembrane domain of rhd using the model of flegel (transfus apher sci., 2011) as reference. adsorption-elution testing using a pool of polyclonal anti-d showed a weak positive reaction, re-classifying the donor as rhd positive. this novel allele, rhd*1151g, could thus be categorized as a del allele. serological results displayed an almost normal rhd antigen expression for donor 2. further serological determination of the rhd antigen with 9 different antisera, however, showed a reaction pattern typically observed with a weak d variant. with commercial available kits no rhd variant could be detected. rhd sequencing revealed a novel homozygous mutation c.526g>c in exon 4. this mutation causes a p.a176p exchange in the sixth membrane-spanning domain of rhd. based on serological data, the donor is rhd positive and in case of transfusion the patient would be treated as rhd negative. summary/conclusions: here we report two novel rhd missense mutations c.1151c>g and c.526g>c harbouring an amino acid substitution within a transmembrane segment. the c.1151c>g variation displayed an unusual low rhd antigen reactivity and would have been mistyped as rhd negative without extensive genotypic testing. molecular analysis of variant c.1151c>g suggests that the t384r exchange causes a del phenotype rather than a miss splicing event. this was also confirmed by adsorption-elution testing. interestingly, variant c.526g>c could only be detected due to comprehensive serological and genetically investigation. background: the rh blood group system is highly polymorphic and one of the most clinically relevant systems in transfusion. actually d antigen is of critical importance due to its involvement in hemolytic transfusion reaction and hemolytic disease of the fetus and newborn. rhd gene variants are common in africans and mostly related to partial d phenotype. aims: rhd gene sequence was investigated in two african brazilian samples. we further attempted to take advantage of combining the molecular data and the available in silico tools for the functional interpretation of the variations, in order to get insights into the clinical phenotype that may be predicted a priori from genotyping. methods: sample #id1 is a d-negative donor self-declared as african descent. sample #id2 is a patient with sickle cell disease (scd) typed as d-positive with anti-d in his serum. serologic d typing was determined by manual gel test and by microplate in an automated instrument. sample #id1 was also submitted to adsorption/elution test. after genomic dna extraction, all ten rhd exons and flanking intronic regions from sample #id1 were pcr-amplified with rhd-specific primers and analyzed by sanger sequencing. sample #id2 was investigated by next-generation sequencing on the miniseq platform (illumina) by using a previously published, custom (selected blood group genes) ampliseq panel. a reported three-dimensional (3d) structural model of the rhd-rhd-rhag heterotrimer was used to visualize the position of variations and predict their putative functional/clinical effect. results: in sample #id1, a single nucleotide missense change, i.e. c.1151c>g in exon 8, was identified. this transversion is thought to replace a threonine by an arginine residue at amino acid position 384 (p.thr384arg) of the rhd protein. analysis in the 3d model clearly suggests a dramatic impact of the p.thr384arg substitution occurring in a functionally-critical, conserved motif in terms of interhelix interaction, which is supposed to be highly deleterious to the stability of the protein, and potentially impairs totally its expression at the red blood cell plasma membrane. this predicted functional effect is definitely in accordance with the d-negative phenotype reported in sample #id1. in sample #id2, the single c.325a>g transition was found in exon 2 leading to a threonine-to-alanine substitution at amino acid position 109 (p.thr109ala). amino acid 109 is located in rhd protein extracellular loop 2, and is thus thought to alter d antigen structure, resulting in a partial d phenotype. this hypothesis is in accordance with anti-d found in the serum of sample #id2. summary/conclusions: for the past years, due to the advent of next-generation sequencing and the subsequent identification of numerous rare variants, bioinformatics prediction and modelling tools have evolved and currently help physicians in diagnostics, clinical management and genetic counselling. we took advantage of some of those in silico methods to predict retrospectively the effect of two novel variant rhd alleles, including one d-negative and one partial d alleles. although phenotype and clinical symptoms remain definitely the standard determinants to assess the effect of genetic variations, use of those approaches may soon become valuable for guiding subsequent investigations in immunohaematology. abstract withdrawn. alleles of the weak d type 4 and diva cluster. in africans, the 16 most frequent were typically associated with alleles of the weak d type 4 (including dol and rhdpsi), diva and dau clusters with f223v occurring in > 10% of alleles; in addition the key mutations of weak d type 1 and dii and two inactivating mutations (c.1056_1057inst and c.1060delg) not reported in rhb were among the first 20 polymorphisms. in east asians, rhd(1227g>a) at 0.8% was most frequent, followed by dfv, weak d type 33, dbo-3, key mutations of diva and weak d type 4 cluster as well as rhce-like substitutions and the mutations of weak d type 25, type 15, type 66, rhd(a85v), dvl-1, weak d 119 and rhd(n135s). weak d type 151 and rhd(t148r) were frequent in south asia but not elsewhere. summary/conclusions: data from tgp and gnomad add relevantly to the knowledge on rhd alleles; tgd discloses linked intron polymorphisms, gnomad frequency data not biased by the likelihood of serologic detection. current typing strategies usually start with serology later complemented by molecular typing. in the future, molecular methods will gain importance and frequent alleles currently not distinguished from "standard rhd" may need a rational transfusion strategy. in this respect, the high frequency of weak d type 45 and type 33 in europeans was surprising, might warrant confirmation by alternative methods and should trigger discussion on rational transfusion strategies for these alleles. consistent with an r 0 haplotype and 1 probable dc-. two siblings that were abo compatible including the dc-sibling were incompatible at iat phase. reactivity could be completely adsorbed from the serum using r 1 r 1 , r 2 r 2 , and rr rbcs indicating the antibody is probably a single specificity. the donor returned in 2015 and 2017 to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. aims: the donor returned in 2015 and 2017 to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. methods: serologic testing was performed by manual tube testing using ahg in the indirect antiglobulin phase. rbc phenotyping was performed by standard tube hemagglutination testing from edta anticoagulated blood. rhd and rhce exons were sequenced using genomic dna and standard sanger dideoxy method with the bigdye terminator v3.1 cycle sequencing kit. sequence data was aligned to rhd_ng_007494.1. rhd zygosity was performed using pcr-rflp with mspi. background: according to recent findings in molecular immuno-hematology, rhd genotyping is strongly indicated in rhc+ and rhe+ donors classified in routine as d-negative. among these, one could find a non-negligible share of entirely new genetic alterations or even del alleles, which are often not identifiable with routine serological methods due to the low number of antigenic sites. aims: the present study reports the genotyping data of rhd on 201 rhc+ and rhe+ caucasian donors classified serologically as d-negative, all enrolled by a single transfusion center in italy methods: rhd serological typing was carried out in microplate direct agglutination tests (iris, immucor) by using 2 different anti-d igm clones (clone 1, dvi+: ldm1+esd1m; clone 2, dvi-: rum-1, th28) and 2 different anti-d igg clones (clone 1: ms26; clone 2: d415 1e4). all donors with d-negative results (n = 201, divided into 153 subjects with rhc+, 45 with rhe+ and 3 with both rhc+ and rhe+) were addressed to genotype analysis with rhd beadchip molecular test (immucor), pcr-ssp (bagene, inno-train) and/or rbc-fluogene (inno-train). the discrepant results between serology (d-neg) and molecular biology (wild-type or full-length rhd gene) were further investigated by bi-directional sequencing of the rhd coding regions. results: one-hundred donors have been analyzed retrospectively, as part of a pilot study. following the data obtained in this first phase, the analysis methods described above have been implemented in routine, allowing to include further 101 donors, studied prospectively. in 92.5% of donors (n = 186), the molecular analyses showed the complete deletion of the rhd locus, while in 15 cases (7.5%) a genetic status was found with "non-deleted" rhd. over all, bi-directional sequencing on these 15 donors revealed the presence of 9 negative and 4 weak-d variants. the list of rhd alleles we have identified at the molecular level is as follows: rhd*01n.82 (2 cases), rhd*01n.83 (1), rhd*01n.80 (1), rhd*01n.61 (1) , rhd*01n.05 (3), rhd*01el.08 (1), rhd*01el.18 (1), rhd*01el.17 (1), rhd*01w.54 (1) . moreover we found a donor with a lack of signal encompassing exons 1-5 of the rhd sequence (bioarray rhd beadchip), while 2 additional cases are currently under investigation. summary/conclusions: our study confirms that a non-negligible number of caucasian subjects, classified serologically as d-negative, present rhd gene alterations that differ from the common total deletion. in line with the literature data, we also found a frequency of about 1 in 50 cases (4 subjects out of 201), in which a donor re-classification as d-positive (weak d type) was necessary. hence, a wider use of molecular typing methods is desirable in order to achieve the correct genetic characterization and the appropriate phenotypic classification of "apparently" d-negative donors. background: without evidence of abnormal serological d antigen expression there will be no quest for weak d, partial d or d variant on the red blood cells. according to our blood donor registry we found that out of 43960 serologically typed donors, 85.5% were d+, 14.0% d-and 0.5% weak d. aims: to compare different weak serological reactions of the d antigen to the rhd genotyping. methods: molecular rhd typing using isolated dna and rbc-ready gene cde and rbc-ready gene d weak kits was performed in 22 blood donors, who were serologically typed as weak d using monoclonal blended igm/igg and dvi-and dvi+ anti-d reagents by slide and microplate (mp) technique respectfully, as well as by the antiglobulin test (iat) in gel and with the set of monoclonal partial d typing reagents (biorad). in addition, rhccee phenotyping and genotyping was also performed. results: all of the donors with serologically weak reactions were confirmed to be weak d variants by genotyping except one donor whose iat was false positive due to rbc autoantibodies. the frequency of d variant genotypes was as follows: 62% weak d type 1, 28% weak d type 3, one donor was typed as weak d type 14 and another one as weak d type 11. these weak d types were associated with different degrees of serologically determined weakness ranging from negative to weak positive reactions concerning slide and mp. all of them gave positive reaction ranging from 2+ to 4+ with iat, except for the weak d type 11 with the score of <1+ which gave negative reaction by slide and mp and inconclusive result with the set of monoclonal anti-d reagents for partial d typing. the percentage of donors, who, at serological typing were only found to be d positive in the iat was 19%. one of the weak d type 1 donors was negative with dvi-and positive with dvi+ reagent in the mp. the additional rh phenotype (genotype) was ccee in all of the donors except in the one who was genotyped as weak d type 14, as well as in the d negative donor, being ccee. summary/conclusions: further rhd genotyping is required to estimate the actual frequency of d variants in our blood donors. in practice, current serological methods are sufficient to detect almost all variant d phenotypes. there is a consensus that routine molecular d antigen screening in d negative donors in order to detect del variant when ddccee phenotyped red blood cell transfusion is practiced in all d negative patients does not seem to be cost-effective. background: rh null or rh mod -the so-called rh-deficiency phenotypes-are characterized by a null or severely reduced rh antigen expression (including d, c/c and e/ e), respectively. molecular genetic studies showed that these phenotypes are transmitted in an autosomal recessive manner. rh null phenotype originates from two different molecular events giving rise to the amorph type and the regulator type. the former is caused by homozygosity for silent genes at rhd and rhce loci, caused by inactivating mutations in rhce and deletion of rhd. on the other hand, the regulator rh null type as well as the rh mod phenotype are attributed to mutations in rhag gene when in homozygous state or when in heterozygosity with another rhag allele containing an inactivating mutation. a functional rhag is essential both for the correct rh complex assembly and rh antigen expression in the erythrocyte membrane. aims: the aim of this study was to investigate the molecular genetic basis of an argentinean proband with no detectable d, c, c, e and e antigens by standard serological techniques. methods: blood samples were collected from the proband, her parents and sister. the proband was a 14 year-old young woman with parameters of hemolytic anemia: low hemoglobin level (10 g/dl), reticulocytosis (14%), hyperbilirubinemia, increased ldh and marked spherocytosis. the d, c, c, e and e status was determined by standard serologic hemagglutination techniques using specific monoclonal antibodies. genomic dna was isolated using a modified salting-out method. dna samples were initially screened for the presence of intron 4 and the 3 0 untranslated region of the rhd gene using pcr strategies. rhc/c, and rhe/e alleles were studied by allele-specific pcrs to determine the rhce genotype. rhd zygosity was analyzed by pcr-rflp. rhd exon polymorphisms were studied by rhd exon scanning procedure based on pcr-ssp. rhag gene was investigated by exon-specific pcr amplification and sanger sequencing. results: no d, c, c, e and e antigens were detected in the proband's erythrocytes. the father and sister rh phenotype was: d+, c+, c+, e+, e+ whereas the mother rh phenotype was: d+, c+, c-, e-, e+. rh genotyping confirmed the rh phenotypes for all family members except for the proposita who genotyped rhd+, rhc+ and rhe+. all samples showed an homozygous status for the rhd gene and all rhd exons were detected by exon scanning. sequencing analysis revealed an homozygous c.920c>t mutation in rhag exon 6 in the proband whereas the rest of the family showed an heterozygous state in the same nucleotide position. the c.920c>t mutation is responsible for the p.ser307phe amino acid substitution predicted to be in the 10 th rhag glycoprotein transmembrane segment. summary/conclusions: this study described the molecular background responsible for an rh-deficiency phenotype in an argentinean proband. we identified the novel missense mutation c.920c>t in the rhag gene which results in the ser to phe single amino acid substitution that shows to be critical for rh antigen complex assembly within the erythrocyte membrane. further studies are being performed in order to determine whether the proband is rh null or rh mod . background: rh blood group system is the most immunogenetic blood group system and blood donor typing should account for all expressing antigens in order to prevent anti-d alloimmunization. aims: the objective of this prospective study was to investigate rhd alleles among blood donors who typed d-by serologic methods and positive for c and/or e. for this reason we developed an easy-to-perform dna-based screening method for the detection of rhd gene and positive samples were further characterized by two commercial pcr-ssp kits. methods: of 15692 individual blood donors within a 15 month period, 1688 (10.76%) typed as d-with standardized immunohematologic methods including the indirect antiglobulin test (iat). residual edta-anticoagulated blood samples were used to isolate genomic dna using the qiaamp dna blood kit (qiagen, germany) from 112 out of 151 (8.95%) c/e+ and serologically d-donors. all dna samples were tested individually for the presence of rhd-specific dna sequences in the rhd promoter, intron 4, exon 7 and exon 10 by a multiplex pcr-ssp method. the reaction was conducted in a final volume of 20 ll with primers that were applied as described by f. wagner et al. (bmc genetics, 2001 ) except antisense primer for exon 10 and the two primers amplifying an hgh gene fragment as internal control, designed by our laboratory. pcr products were visualized by electrophoresis on a 4% agarose gel with ethidium bromide staining. in case of a positive reaction the sample was analyzed by pcr-ssp d weak and pcr-ssp cde (inno-train, germany). results: out of 112 d-individuals analyzed, 101 were ddccee, 8 ddccee, 2 ddccee and one had a ddccee phenotype. molecular analysis showed that 104 (92.86%) were negative for all four rhd dna regions. among the other 8 samples, all of ddccee phenotype, three were found to be positive for rhd promoter, intron 4, exon 7 and exon 10, three for rhd promoter and exon 10, and two for exon 10 alone. further genotyping revealed five hybrid rhd-ce-d alleles [3 rhd-ce(2-9)-d and 2 rhd-ce(3-7)-d], one allele represented the del(m295i) genotype, while the remaining two samples did not show an allele that could be determined with the pcr-ssp kits. summary/conclusions: serotyping is the standard method to assign transfusion strategies but it is not always capable to correctly define all samples that show weak reactions in d. a rhd genotyping strategy is needed to confirm d-blood donors and thus to avoid anti-d immunizations. for these reasons we suggest the implementation of an easy and possible cost-effective method. background: more than 100 weak d types have been described to date. transfusion recipients with weak d type 1, 2, or 3 are not at risk for forming allo anti-d when exposed to conventional rh d-positive rbcs. molecular analysis of weak d offers a more reliable basis than serotyping to determine the prevalence of weak d types and optimal d transfusion strategies. background: the d antigen, which consists of a mosaic of epitopes, is determined in all the blood donors and patients. most people are either rhd-positive or rhdnegative, but there is a certain number of people who have a variation of the d antigen, which are called weak d, partial d and del phenotypes. aims: the objective is to use molecular methods to determine whether blood donors in republika srpska (with whom a serological weak d antigen has been detected) really have the weak d antigen. in addition, determine whether blood donors, who have been determined as persons who are rhd-negative, with the phenotypes c and/ or e, who have the rhd gene and d antigen on the erythrocyte membrane, so weak that it could not be determined by serological techniques. methods: blood samples were used from regular blood donors, who have been determined as persons with a weaker d antigen, as rhd-negative or as c and/or e positive (based on the agglutination strength) using serological techniques, the test tube method, the microplate method and the gel method. gp.mur was also modelled and shown to closely resemble the tertiary structure of glycophorin a. the predicted structure is 5 anti-parallel b sheets arranged in a "b barrel" also referred to as an ob-like-fold. the regions in which blood group antigens were identified in the predicted stable dimeric structure. summary/conclusions: ob-like-fold structures typically to bind oligonucleotides or oligosaccharides and are associated with cold shock proteins. further modelling is in progress to predict the structure of gpa/gpb heterodimers as a basis for understanding the presentation of blood group antigens. of interest, this finding is consistent with a previous report showing that this gpa binds to carbohydrates. this model serves as a foundation for future work regarding the properties of gpa, which includes identifying locations of specific interactions between gpa and other rbc surface proteins such as gpb and band 3, as well as identifying structural features of antigenic regions on gpa. . even though no significant differences were found among the groups studied, 4 haplotypes containing the mcc b and sl2 polymorphisms were identified in d samples but were not found in tb and l groups. summary/conclusions: this preliminary data obtained suggests that cr1 polymorphisms and haplotypes, especially those containing mcc b and sl2 snps, could be involved in the disease pathogenesis of tuberculosis and leprosy. the entrance of mycobacteria into macrophages is mediated by complement receptors that facilitate their uptake by host cells so the combined haplotypes could be enhancing parasite phagocytosis and inflammation. further studies are being carried out to establish whether cr1 polymorphisms are risk or protective factors and whether other genetic variations in this receptor are also involved. abstract withdrawn. background: the dombrock blood group system consists of two antithetical antigens, do a and do b , and three high-prevalence antigens, gregory (gy a ), holley (hy), and joseph (jo a ). the rare do null or gy(a-) phenotype lacks all dombrock antigens, and the do null alleles vary with both do*01 and do*02 backgrounds. here we report the molecular basis of a novel do null allele in a gy(a-) brazilian patient with anti-gy a . aims: case presentation: an alloantibody to a high-prevalence antigen was detected in the serum of a 42 year old woman from the northeast brazil with a history of 4 pregnancies but no history of previous transfusion. she required transfusion because of a schedule for total thyroidectomy surgery due to a large compressive nodular goiter. the antibody did not react with the autologous rbcs but reacted by the indirect antiglobulin test in liss with all panel rbcs and other rbc samples tested except with the gy(a-) phenotype. the corresponding antigen was resistant to treatment with papain but sensitive to dtt and trypsin. these results suggested that the antibody recognized an antigen in the dombrock blood group system. the purpose of this study was to identify the antibody specificity and to determine the molecular basis of the phenotype detected. methods: the red cells phenotype and the presence of the dombrock related antibody in the serum were detected by standard hemagglutination techniques. rbcs and antibodies were from our in-house collection of rare samples. genomic dna was prepared from peripheral blood of the patient. dombrock genotyping was performed by id-core xt platform (grifols, spain). the 3 exons of the do gene were amplified by pcr and directly sequenced. experimental immunohematology and diagnostic immunohematology 2 diagnostic immunohematology 3 experimental immunohematology, sanquin, amsterdam, netherlands background: typing of blood group antigens is essential to prevent transfusion reactions or haemolytic disease of the foetus and newborn. to date, the isbt recognises 360 blood group antigens. most antigens (322) belong to one of the 36 blood group systems. since the genetic basis of these systems is known, genotyping of these antigens is possible. the molecular background of 38 antigens is unknown and can only be determined serologically. one of these antigens is sd a (sid), first reported in 1967.~91% of the population carry sd a on erythrocytes, but this frequency might be higher since identification is difficult due to variability in expression. in 96% of individuals sd a is present in urine. cells with a high expression of sd a (cad/sda++) are used for detection of antibodies. recently, a 3-cells antibody detection panel of bio-rad contained a sda++ cell and many individuals with anti-sd a were detected. the b4galnt2 gene has been implicated in the synthesis of sd a . we collected individuals with and without anti-sd a to elucidate the genetic background of the antigen. aims: elucidation of the genetic basis responsible for loss of the sd a antigen on red blood cells. methods: routine diagnostics to identify antibodies in patients was performed using a bio-rad 3-cells panel, containing donor 626521 with high expression of sd a . additionally, 200 pregnant women were screened for anti-sd a . dna of eight samples with anti-sd a and eight samples without anti-sd a was isolated for further analysis. sanger sequencing was performed on b4galtnt2 exon 1-11. results: sequencing of b4galtnt2 revealed two homozygous mutations which are present in all eight individuals with anti-sd a , but not present in 6 controls. the remaining two controls are heterozygous for these mutations. the first mutation within exon 10, c.1396t>c (enst00000300404.2, rs7224888) changes a cysteine to arginine at position 466 of the protein. the second mutation in exon 11 c.1590a>g (rs16946912) does not change an amino acid. both snps have a maf of 0.11 and therefore we expect that 2.1% of the population is homozygous for the minor allele. genotyping of a large population of pregnant women and the serological detection of anti-sd a in women with a homozygous mutation is in progress. summary/conclusions: the high frequency antigen sd a has not been linked to a blood group system because the molecular basis for loss of the antigen has not been elucidated. the b4galtnt2 gene has been associated with sd a synthesis and therefore we analysed this gene for mutations in individuals with antibodies against sd a . a single homozygous mutation within exon 10 causing an amino acid change was found in all individuals with anti-sd a , and no individuals without antibodies were homozygous for this snp. from population studies we expected~4% sd a -negatives, but either this frequency is an overestimation because of difficulties to detect low expressed antigens or mutations in other genes are interfering with sd a synthesis. a larger study of individuals with homozygous mutations in b4galnt2 and linkage to sd a -negativity and presence of antibodies will be performed before sd a can be assigned to a new blood group system. abstract withdrawn. abstract withdrawn. background: erythrocyte duffy blood group antigen can scavenge chemokines in whole blood. duffy blood group gene consists of two major alleles: fy*a and fy*b. however, little is known regarding the association of duffy blood group polymorphisms with the red blood cell (rbc) chemokine scavenging. aims: the aim of this study was to determine the association of duffy blood group polymorphism with the rbc chemokine scavenging. methods: the duffy blood group were genotyped by 5ˊ-nuclease assay in healthy chinese han individuals, while erythrocyte chemokine scavenging function and duffy antigen expression from the same samples were measured using erythrocyte chemokine binding assays and quantitative flow cytometry respectively. results: rbc chemokine scavenging of cxcl8 was significantly lower in the individuals with the fy*a/fy*a genotype compared to those with fy*a/fy*b genotype (p = 0.016). similar result was also observed in rbc chemokine scavenging of ccl2 (p = 0.038). the expression of duffy antigen on rbc surface in the individuals with the fy*a/fy*a genotype was significantly higher compared to those with fy*a/ fy*b genotype (p = 0.021). summary/conclusions: duffy blood group polymorphism is associated with the differential rbc chemokine scavenging. it is probable that a change in duffy antigen structure caused by duffy blood group polymorphism is responsible for the differential rbc chemokine scavenging. background: individuals with p-phenotype can develop a naturally occurring anti-pp1pk and has clinical significance, causing hemolytic transfusion reactions or hemolytic disease of the fetus and newborn. finding and procuring blood units of pphenotype is a challenge because of its rarity throughout the world. therefore, acute normovolemic hemodilution (anh) can be an on hand tool in the perioperative successful management of patient with rare blood group. however, this approach has not been commonly used aims: n/a. methods: n/a. results: a 66-year-old korean woman was referred to samsung medical center for surgical management for gallbladder malignancy. her blood type was group a, d-positive. the patient had no known history of transfusion. however, antibody screening and identification test using the column agglutination method (bio-rad, cressier, switzerland) showed panagglutination with negative reactions to autologous red blood cells, indicating the presence of alloantibodies to high frequency antigens. the specimen obtained from the patient was sent to the central laboratory of the swiss red cross (bern, switzerland) and confirmed as anti-pp1pk. at first, the transfusion team of our hospital recommended the surgical team to postpone the surgery. however, anh was planned because postponing surgery was not preferred and the patient's preoperative hemoglobin was 12.9 g/dl. 800 ml of blood was withdrawn through a radial arterial catheter in two 400 ml blood bags containing citrate-phosphate-dextrose-a solution after anesthetic induction. equal volume of 6% hydroxyethyl starch solution was infused during the procedure. the patient underwent radical cholecystectomy and liver wedge resection with lymph node dissection, and two units of autologous blood were returned to the patient during surgery. she was then discharged 48 h later with a hemoglobin level of 12.3 g/dl. later, the family study was performed with the standard serologic method using the proband's plasma containing anti-pp1pk and sequencing of the a4galt gene, which were conducted according to the protocols by koda et al.(transfusion. 2002) . the proband and her brother were homozygous for c.1029dupc, indicating a rare p phenotype. summary/conclusions: we experienced that autologous blood transfusions via anh is an alternative to allogenic rbc blood transfusion in patients who have no blood available because of high alloimmunization antibodies against rare blood groups. " and the third sample as "gypb*s_gyp*[140a], gypb*s_null(ivs5 + 5t)" with a predicted phenotype: s-s+ mi a + and s+s-mi a +, respectively. the gypa specific primers used for discrepancy resolution detected the nucleotide substitution, gyp.c.140c>a, in gypa-b-a hybrid associated to gp.hut allele, thus confirming the id core xt result. the expression of mi a for one of these samples was confirmed using non-commercial anti-sera. hence, these three samples were not gp.mur but gp.hut phenotype. both alleles codify for the expression of mi a antigen since it is expressed on several hybrids between the usual forms of glycophorin a and b. two of these three gp.hut samples are african-american donors. gp.hut was reported in white people with a frequency about 0.06% and in thais with 0.04%. these three gp.hut cases found by id core xt in this study point to a higher frequency of this glycophorin variant and also to the presence in african american population. summary/conclusions: id core xt was able to detect two glycophorin phenotypes, gp.mur and gp.hut, which codify for the expression of mi a antigen. standard molecular methods should be implemented in pre-transfusion testing and obstetrical care routine to detect the most clinically relevant glycophorin variants in mns system. background: serf(+) is a high prevalence antigen in the cromer blood group system, which is encoded by a crom*12 allele. the lack of the serf antigen, serf(à) on red cells is caused by a single nucleotide polymorphism, c.647c>t in exon 5 of the decay-accelerating factor, daf gene. alloanti-serf has been found in thai pregnant woman with serf(à) and a serf(à) individual was found among thai blood donors. anti-serf is not a marketed product; hence, a molecular technique has to be implemented to genotype for the crom*12 allele among blood donors. aims: this study aimed to identify the crom*12 allele among thai blood donors leading to predicted serf(+) and serf(à) phenotypes. methods: dna samples obtained from 1,512 central thai blood donors were genotyped for serf allele detection using in-house pcr with sequence-specific primer (pcr-ssp) and confirmed by dna sequencing. results: the allele frequencies of crom*12(+) and crom*12(à) among 1,512 central thais were 0.992 (3,001/3,024) and 0.008 (23/3,024), respectively. the homozygous of crom*12(à/à) alleles was not found in this study. additionally, the pcr-ssp technique was validated by dna sequencing using randomly chosen 100 samples together with 23 heterozygous crom*12(+/à) samples and the results were in agreement. summary/conclusions: our results confirm a high frequency of the crom*12(+) allele in the thai population and their frequencies were similar to those formerly reported among thai blood donors. this study would be beneficial to predict the serf antigen from genotyping results due to unavailability of commercial antiserum. background: there is increasing interest in the use of molecular methods for predicting abo grouping. though nextgen and sanger sequencing have both been used to predict abo type, predicting abo type from buccal swab-derived dna and from deceased donors benefits from a quick and reliable method. besides a pcr-rflp that has been used by many labs for more than 25 years, there is a commercially-available research use only (ruo) kit, and both interrogate nucleotides associated with o1, o2, a2 and b with a representing the ancestral allele. aims: the aim of this report is to compare two low-resolution polymerase chain reaction (pcr)-based methods, for investigation of samples submitted to a reference molecular immunohematology laboratory for abo typing discrepancies. fifty-six peripheral blood samples were tested, 31 from patients and 25 from blood donors. methods: genomic dna was isolated from peripheral blood mononuclear cells. background: del is the weakest known d positive phenotype in the rh blood group system and detectable only by adsorption and elution tests. the rhd1227g>a change is an important marker for del phenotype in east asians. a rapid and efficient pcr method for rhd gene 1227 g>a genotyping is useful in east asian countries. aims: the aim of this study was to develop a method for rhd 1227g>a genotyping by using single-tube pcr with melting temperature(t m )-shift primers. methods: two allele-specific primer for rhd 1227g>a and a common primer were designed and synthesized. two gc-rich tails of different lengths were attached to 5 0 ends of the allele-specific pcr primers. single-tube pcr with t m -shift primers was carried out with the three primers. after pcr, melting curve analysis was performed. rhd 1227g>a could be genotyped by differences of the t m s of the pcr products. all of genotyping results were compared with those obtained from conventional pcr-ssp. for the discordant results, rhd exon 9 sequencing was performed to determine rhd 1227g>a genotype. results: a total of 84 samples were genotyped for rhd 1227g>a by pcr with t mshift primers. 32 samples were typed as 1227a+/g-, 22 samples were typed as 1227a-/g+, 6 samples were typed as 1227a+/g+ and 24 samples were typed as 1227a-/g-. two samples typed as 1227a+/g+ by pcr-ssp but 1227a+/g-by pcr with t m -shift primers were confirmed as 1227a+/g-by rhd exon 9 sequencing. summary/conclusions: the single-tube pcr with t m -shift primers for rhd 1227g>a genotyping is simple, rapid, accurate, and it is superior to conventional pcr-ssp. abstract withdrawn. background: the rh blood group system has numerous variant alleles, which may affect rh antigen expression, including rhd-rhce (d-ce) hybrid genes. these variant alleles are frequently found in people of african descent, and typically result in either d-negative (d-) phenotype, or partial d antigen expression, including silencing of high-frequency antigens and/or expression of low-frequency antigens. patients carrying those alleles are particularly at risk of alloimmunization, suggesting that their identification is important in diagnostics. quantitative multiplex polymerase chain reaction (pcr) of short fluorescent fragments (qmpsf) has proven successful for genotyping those dna samples carrying d-ce hybrid genes by assessing both qualitatively and quantitatively rhd and rhce gene exons. aims: the aim of this project was to genotype both rh genes in a cohort of brazilian patients with sickle cell disease (scd), which are known to be of african descent, by using the qmpsf approach and report hybrid gene variability in this population. methods: one-hundred fifteen dna samples were selected for the study and analyzed prospectively by the rhd-qmpsf and rhce-qmpsf approaches to investigate the copy number of all exons in both rh genes. genotypes were further confirmed or investigated by sanger sequencing and conventional pcr-rflp assays. results: in the 115 dna samples, 75 (65.2%) exhibited a "wild-type" profile by qmpsf analysis. hybrid genes involving exon 8, which is functionally not relevant as reported before, was found in 28 samples, including 11 and 17 samples carrying respectively rhd-ce(8)-d and rhce-d(8)-ce (two homozygous each). except two samples that require additional studies (1.7%), rhd zygosity was resolved successfully: 60 (n = 2 rhd gene copies; 52.2%), 49 (1; 42.6%) and 4 (0; 3.5%). clinically relevant, i.e. partial d, genotypes were identified in four hemizygous samples (4/115, 3.5%) carrying rhd*dau5, rhd*dv.2, a rhd*diiia-like allele, and a novel rhd*d-ce(7:g329h-y330s-n331i)-d allele, as confirmed by sequencing. other hybrid alleles, such as rhd*03n.01 and rhd*diiic, were also found in trans with a normal rhd*01 allele. in rhce, c/c genotype could be resolved. the rhce*ce16 (rhce*ce (48c)-d(9)-ce) allele, which is commonly cis-associated with rhd*ψ, was observed in four samples. however the clinically relevant polymorphisms in variant rhce alleles, such as those involved in cemo, cear, ceag, and ceti, were mostly identified by other standard methods. summary/conclusions: although most of the brazilian patients with scd investigated in this study did not carry rhd-rhce hybrid genes, qmpsf analysis has been shown to be an efficient tool in the whole genotyping process to investigate rh gene variation. as previously reported, it has been conclusive for characterization of rhd zygosity and identification of rare, as well as novel, variant alleles. additionally, our results show a large diversity of hybrid genes among the brazilian patients with scd. therefore, we suggest that qmpsf may be used as a complementary screening approach for assessing rh genotype in selected patients and donors. = 19) vs. non-bleeding (n = 15) patients. platelet, pmp and cp phenotype and function were evaluated by flow cytometry: activation and granule release were examined by antibodies against granulphysin (cd63), p-selectin (cd62p), activated gpiib/iiia (pac-1) and phosphatidylserine (ps) (lactadherin) unstimulated and adp, trap or collagen stimulated. coated platelets were identified as a highly granulated independent cell population appearing following collagen stimulation, gated on side scatter and gpiba (cd42b). normal healthy reference levels were available. results: the platelet count in bleeding (72 9 10 9 /l) and non-bleeding (68 9 10 9 /l) patients was comparable (p = 0,66). bleeding patients had a higher bat score compared to non-bleeding patients (10 vs. 2, p < 0,01). the proportion of cps was normal in all patients. however, in non-bleeding patients the proportion of ps+cps and per cell ps expression (mfi) (86,16% and 4,96mfi) were higher, compared to bleeding patients (64,08% and 2,44mfi, both p < 0,05), and the proportion of ps+cps correlated negatively with bat score (r 2 =0,26, p < 0,01). cd63 + cp was higher in non-bleeding (97,25% and 10,54mfi) compared to both bleeding patients (94,88% and 6,89mfi) and significantly higher than the reference level (88,44% and 5,36mfi, both p < 0,05). finally, the proportion of ps+pmps was normal in bleeding patients, but their pmps expressed higher than reference ps per cell, both unstimulated and for all agonist (134,12 mfi unstimulated vs 32,38 mfi reference, p < 0,01). summary/conclusions: patients with it exhibited different bleeding tendency despite comparable thrombocytopenia. in non-bleeding patients the proportion and per cell level of ps+ were higher, indicating that generation of cps with high ps expression is a critical factor determining bleeding phenotype. the finding of high pmp ps per cell level in bleeding patients could represent an inadequate compensation for lack of cp function, indicating that procoagulant pmps may be less important than cps for thrombocytopenic bleeding. quantification and characterization of cps may be a useful tool for future assessment of bleeding risk as well as a therapeutic target in it and other conditions with bleeding diathesis and/or thrombocytopenia. more studies investigating this field are warranted. background: alloantibodies against human platelet antigens (hpas) and human leukocyte antigen (hla) are implicated in several immune-mediated platelet disorders. detection of these antibodies is crucial in the diagnosis and management of these disorders. aims: to establish a method detecting hpa-1, hpa-2, hpa-3, hpa-5 and hla antibodies using luminex bead technology. methods: monoclonal antibodies specific for platelet glycoproteins and hla class i molecules were separately coupled to the luminex microbeads. positive anti-hpa-1a, anti-hpa-2b, anti-hpa-3a, anti-hpa-5a samples were used to validate the specificities of the luminex assay. the anti-hpa-1a, anti-hpa-3a standard samples were used to evaluate the sensitivities of the luminex assay by serial dilutions (from neat to 1/1024). results: 44 samples collected from patients or isbt platelet workshop were tested by the luminex assay. the results showed that luminex assay could detect antibodies against hpa-1a, hpa-2b, hpa-3a, or hpa-5a successfully from the known samples. the sensitivities of the luminex assay detecting anti-hpa-1a, and anti-hpa-3a were 1:512 and 1:64, respectively, using the standard samples. no cross-reactivity was observed in the samples containing multi-platelet antibodies, or mixture antibodies against hpa and hla. the results of 44 samples with platelet disorders were agreement with those of monoclonal antibody immobilization of platelet antigens (maipa) assay. summary/conclusions: luminex beads coupled with monoclonal antibodies could be successfully used to detect hpa and hla antibodies with high sensitivity. background: platelet transfusion is important in clinical treatment. the expression of abo antigen on platelet surface is differential, so it is usually need to ensure the consistency of the abo antigen in clinical transfusion. but in many cases, it is difficult to find the platelets that the abo blood type matched between the recipient and donor, and abo-incompatible platelet infusion is required in these cases. to data, the expression of abo antigens on platelets in normal blood group individuals is rarely reported in chinese population. aims: to understand the differential expression of abo antigen on platelet surface in population of zhejiang province, china. methods: total of 358 individuals with normal abo groups (101 group a, 100 group b and 101 group ab individuals, and 56 group o as negative control of abo antigens on platelets) were analyzed. the expression of abo antigens on platelets was determined by flow cytometry using monoclonal antibodies: fluorescein isothiocyanate (fitc)-conjugated mouse antihuman blood group a and pe-conjugated murine igg1 anti-b antibody (9431pe bgrl1). flow cytometric parameters were statistically analyzed by the mann-whitney test or the kruskal-wallis test to observe the difference in two or more groups using graphpad software v5.01. the correlation and regression analysis between a and b antigen in the platelets and rbcs were also performed by the software. population studies were reported as the mean and standard deviation (sd), and p values less than 0.05 were considered statistically significant. results: according to mfi values of abo antigens expression on platelets, the samples were divided into three groups: low expression (le), high expression(he) and moderate expression (me) according to the background mfi observed in group o samples. it was found that about 66.22% of the individuals had a weak expression of abo antigen on the platelet surface in zhejiang province. there was a significant difference in the intensity of antigen expression between these three different groups of the same blood group. for each blood group, there was a positive correlation between the intensity of abo antigen expressed on the platelet membrane and red blood cells of the individuals. results: 40 cases were found with antibody positive. among them, 6 cases (15%) were only anti-hla-i positive, 4 cases (10%) were only anti-hpa positive, 30 cases (75%) were both anti-hla-i and anti-hpa positive. 8 cases were found without anti-hla-i or anti-hpa. among the 34 cases with anti-hpa positive, the distributions of anti-gpiib/iiia, anti-gpia/iia, anti-gpib/ix, anti-gpiv were 73.5%, 61.2%, 50%, 44.1%, respectively., hla antibody positive rate in the female patients was higher than that in the male and hpa antibody positive rate in the female was lower than that in male, but there was no significance difference between them (p > 0.05). summary/conclusions: in ptr patients, the platelet antibody was mainly hla-i antibody combined with hpa antibody. background: human neutrophil antigens (hna) are polymorphic structures located on surface membrane of human neutrophils. alloantibodies against hna are implicated in a number of clinical conditions, including immune-mediated neutropenia and transfusion reactions. genotyping for human neutrophil antigen (hna) systems is an important in the diagnosis of disorders involving alloimmunization to hna. aims: the aim of this study was to investigate the hna allele frequencies among blood donors and hematological patients undergoing blood transfusions and to estimate possible hna incompatibilities and risk of hna alloimmunization. methods: a total of 303 blood donors and 302 hematological patients from the north-west region of the russian federation were recruited. dna samples were obtained and typed for hna-1, -3, -4 and -5 systems using polymerase chain reactions with sequence-specific primers (pcr-ssp). specific primers for hna were designed and the polymerase chain reaction amplification conditions were optimized. the v 2 test was used to test for the hardy-weinberg equilibrium for the hna systems. the probabilities of the incompatibility and the potential risk for alloimmunization against different hna systems after random transfusions were estimated based on the hna allele and genotype frequencies. results: in blood donors, the frequencies for the fcgr3b*01 (hna-1a), fcgr3b*02 (hna-1bd), and fcgr3b*03 (hna-1bc) alleles were 0.384, 0.584 and 0.032; for the slc44a2*1 (hna-3a) and slc44a2*2 (hna-3b) alleles, 0.804 and 0.196; for the itgam*1 (hna-4a) and itgam*2(hna-4b) alleles, 0.898 and 0.102; for the itgal*1 (hna-5a) and itgal*2 (hna-5b) alleles, 0.708 and 0.292, respectively. in hematological patients, the gene frequencies for hna-1a/1bd/bc, -3a/3b, -4a/4b, and -5a/5b were 0.376/0.588/0.036, 0.795/0.205, 0.887/0.113, and 0.699/0.301, respectively. no statistic significant difference between genotypes in these groups was observed. since the allele frequencies of hna -1, -3-5 for hematological patients and donors did not have statistically significant differences, possible hna incompatibilities and risk of hna alloimmunization were estimated based on the obtained data on the allele and genotype frequencies of hna in a group that combines donors and hematological patients (n = 605). the predicted risk of hna-1, -3, -4, -5 incompatibilities in this cohort were 34.9%, 26.9%, 19%, and 32.9%, respectively. the possible risk of hna-1a, -1bd, and -1bc alloimmunization were 0.233, 0.143, and 0.064, respectively; of hna-3a and -3b alloimmunization, 0.037 and 0.231; of hna-4a and -4b alloimmunization, 0.01 and 0.163; of hna-5a and -5b alloimmunization, 0.080 and 0.250, respectively. summary/conclusions: the information about hna gene frequencies can be used not only in blood services for detection and identification of hna alloantibodies in donors and assessment of alloimmunization risk but also for anthropological studies. background: non-invasive fetal rhd genotyping is performed using circulating cell-free fetal dna from maternal plasma sample and real-time polymerase chain reaction. this antenatal routine dna test is used to target rh-ig administration to prevent hemolytic disease of the newborn. aims: the aim of this study is to characterize maternal rhd variants responsible for indeterminate results during fetal rhd genotyping due to early amplification of at least one of the exons (5, 7 or 10) of the rhd gene. methods: 2004 samples were tested from 01/12/2017 to 01/12/2018 using free dna fetal kit â rhd. 44 samples (2,2%) yielded a premature signal for one or more exons of the rhd gene. after extraction of maternal cellular dna, the maternal rhd was characterized using rhd beadchip assay (immucor/bioarray). rhdiiia-ce(4-7)-d summary/conclusions: greater diversity is observed in the caucasian population rather than in the afro-caribbean. 65% of the identified variants are rhd negative alleles including alleles leading to partial rh2 antigen expression. unexpected alleles are found such as weak d type 1, 2, 5 or 11. these data underline the benefits of maternal rhd genotyping when abnormal early signals are detected during noninvasive fetal rhd genotyping. background: a considerable number of rhd alleles responsible for weak d phenotypes have been identified. serologic determination of these phenotypes is often doubtful and makes genetic analysis of rhd gene highly desirable in transfusion recipients and pregnant women. dna-based methods are useful for enhancing immunohematology typing in doubtful d phenotypes at pregnant women. aims: determination of the rhd gene in a cohort of pregnant women with doubtful d phenotypes. methods: determination of the rhd phenotyping was performed with microagglutination technique biorad and ortho diagnostic simultaneously. rhd genotyping was performed on 20 cases with d typing serological discrepancies with ready-to-use inno-train rbc-ready gene cde and rbc-ready gene d weak test kits based on polymerase chain reaction with sequence-specific priming (pcr-ssp) to unclear serologic findings. results: molecular analyses showed 16 of 20 (80%) pregnant women were rhd*weak d type 1 and not at risk for anti-d. rhd*weak d type 3 were typed in 3 cases (15%) and 1 case was rhd*weak partial 4.0 and potentially at risk for being alloimmunized producing anti-d allo-antibodies. summary/conclusions: appropriate classification of rhd phenotypes is recommended for correct indication of rhig in pregnant women. however, the serologic differences between rhd-negative and rhd-positive pregnant women is a real problem for unnecessary application of rhig prophylaxis in pregnant women with d variants. conclusion: antenatal rhig prophylaxis is useful in rhd negative pregnant women. with genotyping we found that 95% of serological doubtful rhd negative women was d variants that not produce anti d antibodies. in that cases those rhig prophylaxis was unnecessary and harmful as a product of human origin. on other hand there is a save up of a stock of rhig which is any way in deficit. is it time to think about cost benefit of rhig prophylaxis and genotyping in pregnant women. background: in may 2018, uk neqas (btlp) created an external quality assessment (eqa) sample designed to mimic a feto-maternal haemorrhage (fmh) bleed of 3 ml. all material used passed pre-acceptance serological testing; samples were dispatched to 298 participants in 17 countries. post-dispatch testing by flow cytometry (fc) using an anti-d marker showed a bleed volume of 1.1 ml so an investigation was initiated. aims: to determine the cause of the unexpectedly low bleed volume and what lessons could be learnt. methods: production methodology and results of pre-acceptance testing were reviewed. fc testing was repeated, plots examined, and the fmh scientific advisory group consulted for advice. further fc testing was performed at wbs using alternative markers, and the material used was investigated at ibgrl. participant results were examined to determine if the sample should be withdrawn from scoring. a questionnaire on how results were managed was sent to the 36 participants using fc with an anti-d marker. results: a material production methodology review showed no obvious cause of the erroneous in-house result. review of pre-acceptance testing images showed no issues, further d-typing of the cord showed 2 + reactions vs. two reagents by tube, cf. 4 + with two different reagents by column agglutination technology. repeat fc testing using the anti-d marker gave similar results; however, closer examination of the plots showed a left shift in the positive peak, indicating reduced fluorochrome binding, possibly due to reduced d antigen density on the cord cells. further fc testing at wbs demonstrated a marked reduction in fluorescence intensity with an anti-d marker. further investigation using an anti-hbf marker showed a bleed volume of 3.7 ml, indicating the correct proportion of cord material had been used during sample production. additional serology at ibgrl on the cord material showed reactions which were weaker than the control with 4/17 anti-d reagents. overall, the investigation supported the hypothesis that the cord material was d variant. a review of results submitted by participants mirrored the fc investigation and the sample was withdrawn from scoring, as the fc median result is used to calculate scores and the d variant cord was clearly affecting testing with an anti-d marker. the questionnaire showed that all 16 respondents examine fc plots and the gating used, but not all act on them before reporting results, and not all have a back-up plan for anti-d ig dosing in a similar situation. later sequencing of the d gene revealed the cord donor to be dvii which can have a lower than normal d antigen density. summary/conclusions: the use of a d variant cord in an eqa sample was not planned, but allowed uk neqas to highlight some important learning points: -thorough examination of fc plots is essential to avoid underestimation of fmh; a controlled procedure should be in place if modification of gates is required -access to cord/neonatal blood to allow serological investigation may be useful in a similar clinical situation -it is important to have a back-up plan for issuing anti-d ig in the event of an uninterpretable fmh result background: allo-antibodies against fetal blood group and platelet antigens produced by antigen-negative pregnant women can cause hemolytic disease of fetus and newborn (hdfn) and fetal and neonatal alloimmune thrombocytopenia (fnait). prediction of the fetus antigen status in immunized women is important for making decisions concerning further management of pregnancy. nipt is widely used for determination of fetal blood groups but determination of proper specificity in the real-time amplification of a single nucleotide polymorphism (snp), such as k or hpa-1a, requires modified protocols. droplet digital pcr (ddpcr) permits detection of low-grade fetal chimerism in maternal plasma dna with higher specificity using allelic discrimination pcr protocols. aims: to establish ddpcr protocols for non-invasive prenatal diagnostics (nipd) of clinically important blood group antigens. methods: dna was isolated from 133 plasma samples of pregnant women and donors with known genotypes (easymag, biomerieux). allelic discrimination protocols for determination of k/k (n = 29), s/s (n = 13), hpa-1a (n = 49), hpa-3 (n = 8), hpa-5 (n = 14), hpa-15 (n = 20) genotypes were performed using ddpcr method with droplet digital tm (biorad). the results of allelic discrimination performed using ddpcr were concordant with the already known phenotype/genotype of donors and pregnant women. ddpcr enabled the detection of 100-16,000 reads for total dna from plasma in tested samples. all fetal results were in agreement with antigen positive genotype of the neonates and the fetal chimerism was from 0,01% to 26,4% (one case was for advanced pregnancy -38 week of gestation). in 19/133 tested samples false positive results were detected at the level of 1 or 2 unspecific reads. summary/conclusions: the implementation of allelic discrimination protocols for ddpcr allowed detection of fetal-maternal incompatibility in k/k, s/s and hpa-1a, -3a/b, -5a/b, -15a/b antigens encoded by snp. background: in france, for pregnancies complicated by anti-d (rh1) and anti-c (rh4) allo-immunization, the tests currently used to quantitate maternal antibodies are tube method titration and continuous flow analysis determination of the antibodies concentration. recently, an automated assay was developed using the column agglutination technology on the ih-500 system (bio-rad â). aims: we wanted to evaluate the score, calculated from the agglutination profile of the antibodies on the ih-500 system, as a quantitative data to appreciate the level of maternal antibodies. methods: titers from 29 samples containing anti-d and 20 containing anti-c have been established using the semi-automated tube method performed since decades in our lab and the fully automated gel method on the ih-500 system. scores were calculated manually in both cases. antibodies concentrations were also determined for all samples by continuous flow analysis on our auto-analyzer device (evolution iii ams alliance). we looked for a possible correlation between anti-d and anti-c scores and the corresponding concentrations using the spearman correlation test. results: anti-d tube and gel scores were significantly correlated with the anti-d concentration values (p < 0.0001, r = 0.79 and p < 0.0001, r = 0.82 respectively). anti-c scores were also significantly correlated with anti-c concentration values (p < 0.0001) but gel scores have a better correlation coefficient than tube scores (r = 0.78 versus 0.55). it was easier to extrapolate gel score thresholds than tube score thresholds from the autoanalyzer values, with the aim of triggering fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery only for risk pregnancies. the determined gel score thresholds were 75 and 35, corresponding respectively to 5 ui/ml (250 uchp/ml) of anti-d and 7.5 ui/ml (500 uchp/ml) of anti-c. conclusions: calculating the score from the hemagglutination profile displayed by the ih-500 system provides added values compared to the sole reading of the titer. for anti-c immunization, gel scores are more discriminant than tube ones and better correlated to the concentration values established by continuous flow analysis. the proposed score thresholds to trigger fetal antenatal monitoring need, however, to be confirmed on more samples and to be clinically documented. background: hdnf is due to maternal igg alloantibodies directed against fetal antigens that cross the placenta during pregnancy, causing hemolysis in the fetus, anemia that can lead to edema, ascites, hydrops and, in some cases, death. the diagnosis and management of hdnf is based on maternal screening, and middle cerebral artery (mca) doppler monitoring. in severe hdnf intrauterine blood transfusions (iuts) and or exchange transfusion (et) after birth are necessary to correct anemia, to prevent and treat fetal hydrops. aims: we report eight years of experience in our immunohematology reference laboratory (irl) to highlight the importance of red cell antibody detection as a fundamental parameter to identify pregnancies with high fetal risk and to drive a correct treatment. methods: we report laboratory data from 250 pregnant women with a positive indirect antiglobulin test (iat) referred to our irl from january 2008 to december 2016. we performed antibody screening and identification by indirect antiglobulin test (iat) in microcolumn method with biovue system (ortho-clinical diagnostics, raritan, usa), and the title of antibodies in iat by tube method without additive. follow-up tests were also performed in the presence of significant red cell antibodies in order to check antibody title and begin clinical monitoring. threshold values were ≥ 1:8 for anti kell antibodies and ≥ 1:32 for other specificities. results: out of 250 women, 143 (57.2%) displayed clinically significant antibodies, 92 (36.8%) clinically insignificant antibodies and 15 (6%) natural antibodies of different specificities. among women with clinically significant antibodies the most frequent was anti-d (16.8%) also in combination with other rh antibodies (8.8%), while anti-k accounted for 10%, anti-e for 10% and antibodies against high-incidence antigens for 2.4%. anti-m and anti-le a antibodies were also found (16.8% and 8% respectively) but they were not clinically significant. among 143 women with clinically relevant antibodies, 37 showed a critic antibody title and they underwent gynecological and obstetric monitoring. 21 fetuses resulted affected by hdfn, displaying anti-d in 16 cases and anti-kell in 5. 11 fetuses with severe hdfn (anti-d in 7 and anti-kell in 4) required iuts, 2 were treated with et, 8 received red blood cells units at birth. summary/conclusions: the mother screening program led to important improvements in the outcomes of hdfn. the identification of women with clinically significant antibodies allowed an appropriate monitoring program and therapy. background: the hemolytic disease of the fetus and newborn (hdfn) is a severe disease, resulting from maternal erythrocyte alloantibodies directed against fetal erythrocytes. alloimmunization in pregnant women has been found to range from 0,4% to 2,7% worldwide. there are over 400 erythrocyte surface antigens, of which more than 43 have been reported to be associated with hdfn. although anti-rhesus d was once the major etiology of hdfn, the universal introduction of antenatal and postpartum rh immunoglobulin has resulted in a marked decrease in the prevalence of alloimmunization to the rhd antigen in pregnancy. consequently, alloantibodies other than anti-d emerged as an important cause of severe hdnf, in particular anti-k and anti-c. however, there are other antigens that have also been found to be associated with hdfn. aims: retrospective identification of erythrocyte antibodies in pregnant women in hospital de braga in 2016 and 2017. methods: this study was planned to assess the prevalence of erythrocyte antibodies responsible for alloimmunization, excluding abo-immunizations, in pregnant women attending the antenatal clinics of hospital braga during 2 years, from january 2016 to december 2017. in this study, we retrospectively evaluated the erythrocyte antibody screening results of pregnant women. women with positive erythrocyte antibody screening also underwent identification with gel card system following the manufacturer's instructions (diamed â ). the outcomes of infants, whose mother's indirect antiglobulin tests were found to be positive, were examined. direct antiglobulin tests, jaundice and phototherapy history, transfusion and mortality of the newborns were recorded. results: during the study period, 5982 pregnant women were attended in hospital de braga. the laboratory registered 100 positive erythrocyte antibody screening tests. the prevalence of positive erythrocyte antibody screening was 1,7%. anti-d was the most common antibody found (58,5%). anti-d prophylaxis given during pregnancy was responsible for 51 of 62 cases and maternal antibody titer levels did not exceed 8 among these cases. the prevalence of non-rhd immunization was 33%. anti-e (9,4%) was the most frequent alloantibody other than anti-d followed by anti-m (5,7%) and anti-c (4,7%). multiple maternal antibodies were found in 5 pregnant women. four women had 2 types of alloantibodies: anti-c and anti-e; anti-c and anti-d; anti-k and anti-cw; anti-e and a non-identified antibody. one pregnant had 3 types of alloantibodies: anti-d, anti-c and anti-e. of all cases of newborns whose mothers had a positive antibody screen tests, icterus occurred in 45% of them and phototherapy was given in 14%. summary/conclusions: the prevalence of positive erythrocyte antibody screening in hospital de braga was 1,7%. the erythrocyte antibody screening showed that anti-d was the most common antibody found (58,5%) in most of the cases because of anti-d prophylaxis. the prevalence of non-rhd immunization was 33%. the other most frequent alloantibodies were anti-e (9,4%), anti-m (5,7%) and anti-c (4,7%). an increasing prevalence of non-anti-d alloimmunization was found and there are currently no preventive strategies. in contrast to rhd alloimmunization, the main risk factor for non-anti-d alloimmunization is a previous transfusion therapy. thus, it is important to minimize the exposure of women to incompatible erythrocyte antigens through unnecessary transfusions when possible. background: the mns blood group system is one of the most complex blood group systems. although alloanti-m is a common antibody observed in pregnant women and could also be found in the serum of individuals who have not been exposed to m positive erythrocytes, it is rarely clinically significant and has been regarded as an unimportant antibody to cause hemolytic disease of the fetus and newborn (hdfn), especially in caucasian and black ethnic groups, for a long time. however, an increasing number of cases of severe hdfn resulting in fetal hydrops and recurrent abortion caused by alloanti-m have been reported mainly in the asian population, especially in the japanese and chinese populations. aims: to summarize the characters of serological testing in preterm twins newborns suffered with severe hdfn. methods: the blood sample of two newborns with severe hdfn and the mother, who had the history with three hydrops fetus, were collected. abo, rhd, rhce, and mn blood group typing of the twins newborn and their mother were performed in saline with tube or gel card. direct agglutination test (dat), elution test, antibody specificity identification and antibody titer detection were conducted by iat method in gel card. results: o, rhd(+), and ccee blood groups were identified both in the mother and the twins newborn. background: in france, since may 2018, the legislation does not promote anymore the use of the reference tube method for titration of anti-red blood cells antibodies. this opened the way to the use of newly developed automated anti-red blood cells antibodies quantitation by column agglutination technology. aims: we wanted to assess the performance of titration and scoring by the id-gel test on the ih-500 system (bio-radâ) and to compare it with the performance established for the reference tube method, used in our lab since decades. another objective of the study was to determine titer thresholds for the gel method, to trigger fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery. methods: an home-made internal quality control (iqc) prepared and calibrated using the international anti-d standard (01/572) was used to determine the intraassay and interassay imprecisions, regarding the score and the titer results. patients samples for testing were chosen during the 2-months assay period, regarding the specificity of the antibodies and the tube titer in order to cover a wide range of 2 have lower values. the highest differences (more than 2 to 3 dilutions higher) were seen for antibodies directed against rh system antigens. among the other specificities, anti-k (kel1) and anti-m (mns1) antibodies show the most samples with equal or lower titers compared to the tube method. conclusions: automated anti-red blood cell antibodies titration by column agglutination technology on ih-500 system shows better intra and interassay cvs compared to the tube method. it is explained by the fully automated process that includes the reading step. titer results are almost always higher with the gel technology. thus, it seems possible to safely extrapolate the titer thresholds defined for anti-red blood cells antibodies by the tube method to the gel method. however, based on future clinical studies and fetal/neonatal outcomes, it would probably be necessary to increase these thresholds, at least for anti-rh antibodies, in order to avoid heavy, expensive, stressful and useless monitoring of some pregnancies. results: the first case was a 1-day-old female infant, yellowish skin developed the next day after birth. her capillary bilirubin level was 13 mg/dl, the evidence favored neonatal hyperbilirubinemia and the clinical manifestation revealed hemolysis symptoms. her laboratory findings showed elevated reticulocytes (15.2%), ldh (1162 iu/l) and g6pd (25.3 u/ghb); dat (+/-), iat (-), anemia (hb 8.7 g/dl, hct 28%), and blood smear showed anisocytosis, spherocytes, and polychromatic rbc. her mother blood typed o, d positive, while her blood type was b, d positive and anti-b was found from her elution rbcs (3 + ). due to rarely severe anemia with abo incompatibility, maternal plasma was analysed for abo igg antibodies and showed high antibody a and b titre with 1:1024 and 1:2048. the female infant received one unit washed-prbcs for anemia and intensive phototherapy for hyperbilirubinemia. her clinical condition improved significantly, hb rose to 14.6, bilirubin level was within normal range, she was discharged. another 5-days-old male infant was our second case. on the third day after birth, yellowish skin discoloration developed and bilirubin level was 15 mg/dl. two days later, his transcutaneous bilirubin (tcb) measurement data was high and laboratory findings also showed raised reticulocytes (5.2%), dat (+/à), iat (à), hb 12. background: anti-indian b is a rare alloantibody against the high frequency antigen in b . individuals with the in:1,-2 phenotype (in(a+b-)) are observed with a frequency of < 0.1% in the indian population and have not been described in caucasians. the majority of anti-in b antibodies have been reported in individuals without previous transfusions, indicating the possibility of a naturally occurring antibody. anti-in b is considered clinically significant and haemolytic reactions after in b -incompatible transfusions have been reported. haemolytic disease of the foetus and newborn (hdfn) due to anti-in b has not been described. however, a positive direct antihuman globulin test (dat) may be observed. aims: to describe the challenges of managing a pregnancy and childbirth of a woman with an anti-in b . methods: serological investigations were performed by iat (tube and column agglutination). papain and trypsin treated cells were also utilised. soluble recombinant in blood group proteins (in-rbgp) (inno-train, germany) were used in neutralization tests. the clinical significance of the anti-in b antibody was determined by monocyte monolayer assay (mma). genomic dna was isolated from whole blood and the samples were further characterized by pcr amplification and sanger sequencing of exon 2 of cd44. results: in a 27-year-old pregnant (para 1) woman of indian origin without previous transfusions, an alloantibody of the specificity anti-in b with a titer of 1:2 was detected by iat (negative with papain-treated cells) at gestational week (gw) 32 and 36. the mma, performed in duplicate on samples taken at these dates, showed a mi of 0.5%/4.8% and 7.7%/6.3% respectively. the mi was interpreted as follows: 0-3% not relevant; 3-5% inconclusive; >5% clinical significant. the patient's parents were typed heterozygous, in:1,2 whereas her husband was homozygous, in:-1,2. due to the husbands phenotype, the fetus was predicted to be in b positive. doppler flow measurement of the peak systolic velocity in the middle cerebral artery of the foetus was normal. delivery took place at gw 41 without increased bleeding. the neonate presented no clinical manifestation of hdfn. neither the mother nor the baby required blood transfusions. summary/conclusions: we report the case of a pregnant woman of indian origin with an anti-in b alloantibody. the first mma, performed in gw 32, was inconclusive whereas the second mma, performed in gw 36, indicated that the antibody was clinically significant. if the mi-increase is only due to the pregnancy or has also a clinical significance, cannot be stated. in b negative blood components were not available and the patient's relatives were all in b positive. therefore, measures to avoid transfusions, including optimised peripartal management of haemostasis, was of utmost importance. with only few cases published, the risk of hdfn could not be excluded with certainty. an intrauterine investigation by doppler was performed to exclude relevant anaemia of the fetus. no transfusion was needed at delivery as there were no haemorrhagic complications. the neonate presented no clinical signs of hdfn. background: hemolytic disease of the fetus and newborn (hdfn) is a disease which if untreatedcan cause perinatal mortality and morbidity with a substantial risk for long-term sequela. in albania we lack of studies in this field. aims: the aim of this study is to determine the predictive value and the reliability of the "critical titre" during the evaluation of red cells alloantibodies ability to cause the hemolytic disease of fetus and newborn. methods: we conducted a descriptive, cross-sectional study. the data were collected in the university hospital for obstetrics and gynecology in albania. in the study were included 20 immunized pregnant woman for anti-d antibodies and their newborns which were affected from the hemolytic disease of fetus and newborn. the data belong to the period 2013 and 2018. results: the "critical titre" in our study was 8, meaning that this was the minimal value of the titre antibodies that could cause hemolytic disease of fetus and newborn. our study concluded that only 2 newborns were born without the hemolytic disease of fetus and newborn and the titre values were less than 4. moderate hemolytic disease of fetus and newborn were caused between the titre values 8-32. the summary/conclusions: the titre values of the mothers are a predictive option of the high risk of giving birth to a child with the hemolytic disease of fetus and newborn. it is recommended that in this cases the mother should be followed with doppler ultrasonography to measure the blood flow of the middle cerebral artery. also the doctors should recommend in pregnant women with positive coombs test not only the identification of the anti-d antibodies but also the identification of the other antibodies such as anti-e, anti-c, anti-k. background: rhd-negative pregnant women with allo-anti-d are at risk of having a fetus affected by haemolytic disease of the fetus and newborn (hdfn) where the fetus is rhd-positive. the rhd allele is highly polymorphic and many rhd variants give rise to an array of partial d phenotypes. the clinical significance for many partial d phenotypes is not well-established. rhd genotyping by non-invasive prenatal testing (nipt) to assess the fetal rhd status determines whether the fetus is at risk for hdfn. nipt tests also include strategies for detecting maternal rhd variants to provide for accurate reporting. however, the presence of a paternal rhd variant, while having the potential to confound nipt interpretation, is often not recognised. we report a "trio" family study triggered by a request for nipt for an rhd-negative pregnant mother, 16 weeks gestation, who presented with allo-anti-d and anti-jk a antibody. subsequent paternal and fetal rhd genotyping was conducted and revealed a novel variant rhd allele. aims: we aim to characterise the paternal rhd allele and review clinical case features. methods: rh phenotyping was performed by standard serological procedures. nipt tested for fetal rhd exons 4, 5 and 10. rhd genotyping on whole blood/cord blood dna was performed on the immucor bioarray rhd beadchip kit which predicts a rhd phenotypic variant of best fit. dna sequencing was performed using the illumina trusight one sequencing panel. copy number variation (cnv) analysis was used to assess the rhd exon structure and zygosity. results: the paternal red cells typed as group o rhd+c-c+e-e+, (ror). nipt genotyping detected fetal rhd signals for all 3 exons, predicting rhd-positive. no maternal rhd sequences were detected consistent with homozygosity for the rhd deleted haplotype. for both paternal and cord genomic dna (gdna), beadchip genotyping predicted a rhd variant "diiia/cehar". furthermore, signal drop out was observed at 3 nucleotide positions (c.1154, c.1193, c.1227) located in rhd exon 9 suggesting exon 9 was either deleted or rhce-replaced. paternal and cord gdna sequencing detected 4 out of 6 snps (c.186g>t, c.410c>t, c.455a>c, c.602c>g) associated with diiia phenotype plus 2 additional snps (c.604g>a, c.733g>c) on the rhd gene. both were rhd hemizygote by cnv analysis. no rhce variants were detected. clinical case features: the maternal anti-d quantitation increased from 5.3 iu/ml (16 weeks gestation) to 166 iu/ml (33 weeks gestation). the fetus required 3 intrauterine transfusions during the pregnancy to manage the hdfn. summary/conclusions: both father and fetus carry an rhd allele that does not align with alleles encoding diii phenotypes. this putative novel rhd variant allele comprises snps associated with diiia and with a possible exon 9 deletion/rhcereplaced. a similar allele was reported in literature, although based on sequence analysis only, with no phenotype data. the variant allele here encodes rhd-positive phenotype and we predict that there may be a loss of d-epitopes. notwithstanding, the clinical presentation shows that maternal anti-d against this rhd phenotype (presumed partial) is associated with a severe hdfn and that such rhd blood group phenotypes are of clinical significance for alloimmunised pregnancies. abstract withdrawn. background: cd109 is a glycosylphosphatidylinositol (gpi)-anchored protein with apparent molecular mass of 170 kda. in addition to being expressed on human plts, cd109 is expressed on activated t-cells, endothelial cells, cd34 + hematopoietic stem cells as well as on progenitor cells. in the chinese population, the calculated allele frequencies of hpa-15a and -15b are 0.505 and 0.495, respectively. based on these data, the risk of alloimmunization against hpa-15 alloantibodies due to incompatible plt transfusion or pregnancy is expected to occur in relatively high frequency. however, until today there is no report of hpa-15 alloimmunization in the chinese population. in this study, we analyzed sera from hydrop fetus cases by maipa technique and icfa. aims: to detect the anti-hpa 15b alloantibodies by maipa and icfa. methods: a 24-year-old mother, gravida 2/para 0. the mother in the first pregnancy was diagnosed hydrop fetus at pregnancy 33 weeks by ultrasound. in the second pregnancy, fetal hydrops was observed by ultrasound at pregnancy 24 weeks. the mother's irregular antibody test was negative. the maternal platelet specific antibodies and hla antibodies were negative. blood routine and morphological examination of fetal umbilical cord blood showed that plt count dropped to 4.7 9 10 10 /l, wbc count dropped to 2.96 9 10 10 /l, including neutrophil 37%, lymphocyte 16%, mononuclear 43%, eosinophil 2%, basophil 2%, red blood cells were normal, hb was 111 g/l. screening for hla and plt-specific antibodies was performed using a elisa-based plt antibody kit (pakplus, gti diagnostics) as recommended by the manufacturer. plt antibodies were detected by icfa and maipa.hpa genotyping was detected by cpr-ssp. results: the fetus's genotype was hpa-1a/a, -2a/a, -3a/a, -4a/a. -5a/a, 6a/a, 7a/a, 15a/b, naka (+) and the maternal was hpa-1a/a, -2a/b, -3a/a, -4a/a. -5a/a, 6a/a, 7a/ a, 15a/a, naka (+). the paternal genotype was hpa-1a/a, 2a/b, 3a/a, 4a/a, 5a/a, 6a/a, 7a/a, 15a/b, naka (+), which was the only incompatible antigen compared with the maternal hpa. samples were tested using the fresh plt panels consisting of hpa-15aa and -15bb homozygous donors. the reactivity of the negative control and the mother's sera with the plts from hpa-15a/a (donors 1), hpa-15a/b (donors 2) and hpa-15b/b (donors 3) donors by maipa. the mother's serum showed no reactivity against 15a/a plts, weak positive reactivity against 15a/b plts (od values 0.28), but strong reactivity against 15b/b plts (od values 0.38).this finding could be confirmed by one of the reference plt laboratories (japanese red cross kanto-koshinetsu block blood center, japan) using freshly isolated plts from hpa-15genotyped donors (anti-hpa-15b average value 9.8). summary/conclusions: in this study, we found anti-hpa-15b in a case of fnait (patient hpa-15aa, blood group o) using the maipa technique. we were able to detect the presence of hpa-15b alloantibody in one case of nait. background: fetal and neonatal alloimmune thrombocytopenia (fnait) occurs in 1:10000 live births in caucasians. serological and molecular human platelet antigens (hpa) genotyping tests are performed to investigate and conclude to fnait diagnosis. however, in few cases and particularly in case of suspicion of private platelet antigen, some specialized analyzes must be performed in the laboratory (lab). these analyzes can range from sanger or ngs sequencing to platelet serology with transfected cells. aims: the aim of our study was to explore where the frontier between research and care takes place in the field of platelet immunology through the prism of the fnait investigations carried out by the platelet immunology laboratories. methods: a two-part electronic survey have been sent to foreign platelet immunology experts (pie) from platelet immunobiology working party (piwp) members and espgi board members (n = 40). the first part focused on the lab practices and regulatory environment regarding to accreditation, contact with patient, informed consent and patient results. the second part stressed on the investigations performed to discover new platelet antigen and more precisely on the perceived status of these analyzes ( background: haemolytic disease of the fetus and newborn (hdfn) can occur when maternal red cell antibodies, directed against red cell antigens present on the fetal red blood cells, cross the placenta and enter the fetal circulation. in a "traditionally" conceived pregnancy, when hdfn occurs, it is as a result of maternal antibodies directed against fetal red cell antigens in the heterozygous state, whereby the antigen is inherited from the father only. with the advent of donor oocyte (do) in-vitro fertilisation (ivf), the addition of a third person into the reproductive equation allows for the possibility of a more severe form of hdfn when fetal red cell antigens are present in the homozygous state (one copy from father and one copy from donor) and maternal antibodies are directed against these. antigens expressed in the homozygous state will have more antigens sites per red blood cell and therefore are at an increased risk of red cell destruction from the maternal derived cognate antibodies. aims: to raise awareness of increased severity risk of hdfn in donor oocyte conceived pregnancies. methods: we describe two unusual cases of hdfn in our institution of two women whose pregnancy was induced using a donor oocyte and their offspring requiring transfusion support in the postnatal period to treat hdfn. results: the first is a case previously reported (doyle, quigley, fitzgerald et. al. transfusion medicine, 2014 ) of protracted hdfn due to anti-c, managed with phototherapy initially, then intervention with red cell top-up transfusion at 4 weeks post-delivery. the second is an unusual case of severe abo hdfn requiring exchange transfusion therapy (pre-publication). summary/conclusions: given the increased number of pregnancies conceived using do we recommend that antenatal guidelines are reviewed to create awareness regarding the potential increased risk of hdfn in do pregnancies complicated by allo-immunisation. critically, antenatal testing guidelines should highlight that the predicted outcomes associated with quantitation/titres can only be used when do has not been used to obtain the pregnancy. it is also essential that clinicians inform the blood transfusion laboratory when do has been used. abstract withdrawn. 19%) are deceased due to organ rejection, and 8/36 patients (22%) are deceased due to disease not related to rejection. summary/conclusions: the use of therapeutic plasma exchange for the treatment of antibody mediated rejection in solid organ transplant is safe and effective when used along with other treatment modalities. further studies will help determine whether it can be reproduced in larger cohorts and whether it is more effective in certain organs. background: extracorporeal photopheresis (ecp) is an important cellular therapy for the treatment of several (auto-)immune diseases such as graft-versus-host disease. the international standard for the ex vivo treatment of the leukapheresis product is the application of 8-methoxypsoralen (8-mop) and irradiation with uv-a light. however, the addition of 8-mop to the illumination bag is associated with a potential risk of contamination. aims: the basic principle of the ecp is the induction of apoptosis in the leukocytes. our aim was to find an alternative for the conventional apoptosis induction without the need of external substance application. the objective of the study was the investigation of the apoptosis levels and kinetics in leukocytes after treatment with 8-mop+uv-a compared to uv-c treatment without additional 8-mop. methods: we used an in vitro 72 h cell culture approach with human mononuclear cells from healthy blood donors. untreated control cells were compared with 0,2 lg/ ml 8-mop plus 2 j/cm 2 uv-a treated cells and 2 j/cm 2 (effective dose) uv-c treated cells. apoptosis in several leukocyte sub-populations was detected daily with annexin v and 7-aad flow cytometry standings. results: the apoptosis analysis of cd3 cd4 t-helper cells, cd3 cd8 cytotoxic tcells, cd19 b-cells, cd14 monocytes, cd3 neg cd56 nk-cells and cd3 cd56 nkt cells revealed no statistical differences in almost all of these cell types after treatment with 8-mop/uv-a or uv-c light. the apoptosis kinetic as well as the final apoptosis after 72 h were similar in both treatment groups. summary/conclusions: the addition of 8-mop to the photopheresis irradiation bag is a risk for potential infections. the main effect of the 8-mop/uv-a treatment is most probably the induction of apoptosis in the leukocytes. here, we provide information that this induction of apoptosis can also be achieved with uv-c irradiation without the need of 8-mop addition. the apoptosis patterns in most leukocyte subpopulations are very similar after treatment with uv-c compared with 8-mop/uv-a treatment. future in vivo studies are needed to prove the therapeutic effect of uv-c treated cells in the ecp setting. abstract withdrawn. background: therapeutic plasma exchange (tpe) is performed to remove the implicating substances from the plasma causing the disease. a periodic appraisal of tpe data is important to get insight into the procedural related effects and toxicities and overall outcome in order to have a guided future approach. aims: the purpose of this study is to observe the overall profile and outcome of the patients receiving the tpe in the medicine intensive care unit (micu) of a tertiary care hospital in south india. methods: a record based audit was conducted for the all the patients who were admitted to our tertiary care hospital of south india with 16 bedded micu and received tpe therapy between 1 june, 2016 and 31 december 2018. all the tpe procedures were performed using haemonetics multicomponents system (mcs) + ln9000 apheresis system based on intermittent flow centrifugation. we audited our tpe for: number of treatments, clinical indications, treatments prescribed and administered, any procedural or patient complications, and adherence to current best practice recommendations. results: sixty nine patients had undergone 269 tpe procedures. among them, thirty were female patients (43%). the median age 45 (13-75) years. guillain-barre syndrome (gbs) was the most common indication (72%) followed by cases of thrombotic thrombocytopenic purpura, diffuse alveolar hemorrhage, myasthenia gravis, autoimmune encephalitis and hypertriglyceridemia respectively. the tpe regimens received by patients in this icu were not always prescribed in accordance with current best practice recommendations. there were 48 (18%) episodes of patient related complications during the tpe treatments. in 8 (3%) procedures, technical error in the machine was encountered. summary/conclusions: the findings of this audit have identified differences between the current prescription recommendations for tpe and those applied. the infrequency of the therapy and the different indications may present a challenge for medicine intensive care clinicians to provide best care in all cases. background: microangiopathic hemolytic anaemia (maha) encompasses a spectrum of disorders characterised by widely disseminated thrombosis in small blood vessels resulting in formation of schistocytes and concomitant thrombocytopenia. plasma exchange (pe) needs to be considered as empirical and urgent life saving therapy in these disorders irrespective of waiting for specific testing like adamts 13 levels in thrombotic thrombocytopenic purpura (ttp) or complement levels or factor h antibodies in atypical hemolytic uremic syndrome (ahus). aims: to assess the efficacy and safety of plasma exchange in patients diagnosed as having microangiopathic hemolytic anaemias. methods: a retrospective analysis of all pe procedures performed in patients diagnosed as having maha was done over a period of 9 years (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . procedures were done on apheretic device (cobe spectra, terumo bct, lakewood co. usa). patients' pre and post procedural hematological and renal parameters were analyzed by applying paired t test. adverse event if any was recorded. results: pe was performed in 46 patients with diagnosis of maha (27-ahus, 16 -ttp, 1 each of post stem cell transplantation drug induced thrombotic microangiopathy (tma), post thyroidectomy tma and post-partum tma). the mean age of patient was 19.94 ae 19.58 years with m:f as 1.5:1. number of procedures per patient varied from 1 to 27. post pe recovery was observed within 10-14 days with statistically significant increase in mean platelet count from 40.05 ae 5.9 to 82.11 ae 12.10 9 10 9 /l (p = 0.000) and significant decline in mean lactate dehydrogenase level from 48.91 ae 34.73 to 10.98 ae 6.49 lkat/l (p = 0.000). there was also significant decline in mean percentage of schistocytes in peripheral smear from 5.44 ae 3.96% to 0.56 ae 0.89% (p = 0.000). the mean serum urea changed from 48.88 ae 24.56 to 24.50 ae 17.78 mmol/l and creatinine from 266.11 ae 142.59 to 173.09 ae 127.34 lmol/l (p = 0.000 and 0.001 respectively) with significant increase in urine output from 0.71 ae 0.53 to 1.06 ae 0.33 ml/kg/h (p = 0.000). adverse events were observed in 10 patients (21%), allergic reaction to replacement fluid (n = 6) being the commonest followed by hypotension (n = 2), rigors and chills (n = 2). overall survival rate at 6 months was 89%. summary/conclusions: pe had proven its safety and usefulness as life-saving first line treatment modality in maha. prompt and aggressive treatment helps in achieving early and complete remission in these patients. background: neuromyelitis optica (nmo) also known as devic's disease or devic's syndrome is a rare demyelinating disease of the central nervous system that most often results in selective involvement of the optic nerves (optic neuritis) and spinal cord (myelitis)and has female preponderance. neuromyelitis optica (nmo) attacks are poorly controlled by steroids and evolve in stepwise neurological impairments. assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. aims: to study the effect of tpe in neuromyelitis optica. methods: a 17 year old female in the medicine department, civil hospital, ahmedabad admitted with chief complains of weakness and numbness in the arms and legs, blurred vision, reduced sensation, difficulty in controlling bladder and bowels, uncontrollable vomiting and hiccups since 2-3 days in the medicine department, civil hospital, ahmedabad. attacks were treated with short courses of high doses of intravenous corticosteroid -methylprednisolone intravenous. but there was no clinical improvement. results: clinician advised for the trial of tpe in this patient. the procedure was performed by automated device with continuous flow centrifuge machine fresenius kabi-com.tec using double lumen femoral catheter. after obtaining informed consent from the relative of the patient, 6 cycles of tpe were performed on daily basis. after 6 cycles, both subjective and objective clinical response to tpe was estimated by three different sources (the patient, a transfusion medicine physician, and the treating neurologist). [1] for motor performance, patient was assessed on a disability scale (0 = healthy; 1 = minor symptoms; 2 = able to walk 5 meters without support; 3 = able to walk 5 meters with support; 4 = confined to bed or wheelchair; 5 = requiring assisted ventilation; 6 = dead).patient's motor performance was increased to scale 1(upper limb) and 2(lower limb) from scale 5, deep tendon reflexes were normal. visual function began to improve 1 week after the treatment. visual acuity was 6/6 after 4 weeks. summary/conclusions: assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. this suggests that tpe is beneficial in nmo patients during acute attack if there is no response to corticosteroid treatment. background: babesiosis is a tick borne infectious disease caused by the protozoa babesia. while most infections with babesia are asymptomatic, some patients present with a symptomatic infections and rarely this can be a severe life threatening illness. treatment is primarily with antibiotics but red cell exchange (rce) has been used in the more severe cases which are characterized by high grade parasitemia, evidence of severe hemolysis and or multi-organ failure involving the kidney, lung or liver. a threshold parasite level of 10% has arbitrarily been applied as an indication for rce, however, this threshold is not evidence based. aims: to report on patients with babesiosis and high grade parasitemia who were treated with antibiotics only without rce methods: data were collected from july 2014 to july 2018. a case was defined as a patient diagnosed with babesiosis for whom rce was requested on the basis of a parasitemia of > 10% but on clinical evaluation it was considered that rce could be withheld and the patient monitored awaiting response to antibiotics. results: three cases of severe babesiosis in which the use of rce was requested on the basis of a parasite level of greater than 10%, but was not performed. the rce was deferred on account of the good clinical state of the patient and the absence of renal failure. levels of parasite at diagnosis were 10.6%, 11% and 31%. all patients were followed daily until discharge. two of these patients had been splenectomized and each received a single unit of red blood cells during the hospitalization. the third patient had a long history of refractory lymphoma and was pancytopenic requiring multiple transfusions during the years before the diagnosis of babesiosis. she had transfusion transmitted babesiosis from a red blood cell transfused 46 days prior to the diagnosis. all three patients responded well to antibiotics and were discharged between 9-16 days with undetectable parasites. summary/conclusions: this small case series suggests that requests for rce solely on the basis of an arbitrary level of parasitemia should be questioned and the clinical state and evidence of organ failure considered in the decision to perform rce. abstract withdrawn. chronic transfusion program (ctp) remains the gold standard therapy for stroke prevention and for patients with a severe disease who have inadequate response to hydroxyurea treatment. aims: to evaluate the safety, efficacy and cost between scd patients on ctp that underwent both aet and partial manual exchange transfusion (pmet) procedures. methods: retrospective observational cohort study of patients with scd on ctp that have switched between pmet and aet. this study was carried out from 01/01/2017 to 31/12/2018 in a hospital in portugal. data on patient history, haematological values, duration of the procedure, intervals between them, adverse events as well as the cost of material and working hours were collected and compared between both procedures. results: a total of 6 patients met the inclusion criteria described. however, 1 patient was excluded from our study because of the lack of attendance to the ctp. during the study, we recorded 88 exchange procedures (42 pmet and 46 aet), both on peripheral venous access. from all those procedures the major concern was the poor venous access, which was the reason why 2 patients had returned to pmet. no major complication or alloimmunization was observed. the indications for ctp were cerebral vasculopathy (n = 2), stroke (n = 1) and recurrent vaso-occlusive crisis with multiorgan failure (n = 2). for both procedures, target values were to obtain a pre-exchange hbs level ≤ 30% for stroke and cerebral vasculopathy and ≤ 50-60% for other indications. the median hbs level before pmet was 42,6% (31,3-59,1) and 38,4% (18, 9) before aet. we documented a higher hbs level prior to the next procedure in 11,4% of patients (n = 10). despite that all patients remained stable without any major scd related event. both procedures were well tolerated and iron overload was well controlled (median ferritin level pmet: 1356,1 vs. aet: 1314,7 ng/ml). the duration of the exchange procedure was longer and the intervals between procedures were shorter with pmet (median pmet: 360 vs. aet: 60 min and pmet: 21 vs. aet: 24 weeks, respectively). annual rbc requirements per procedure were superior (median 2 vs. 4 units) and the overall costs related with aet were 2,2 times higher -18.180,93€ and 8.174,79€ aet and pmet, respectively (estimated cost per session aet: 790,48€ and pmet: 389,28€). summary/conclusions: our study shows, that the hbs level before both procedures, performed during the same interval, was similar. we verified that pmet has a comparable efficacy with aet in terms of preventing the development or progression of chronic complications and that the cost per procedure is significantly higher with aet. however, in a clinical situation where it is important to rapidly reduce the hbs level, and/or where the control of the target hbs is stricter so that the patients are clinically controlled without an increase in hospital visits, aet is preferred. we conclude that aet is more effective in the rapid reduction of hbs and ferritin levels, as well as being less time consuming. despite this, for the reasons described above, it is more cost-effective to maintain both aet and pmet procedures. background: erythrocytapheresis/red blood cell (rbc) exchange, involves removing of a large number of rbcs from the patient and returning the patient's plasma and platelets along with compatible allogenic donor rbcs. typical indication for rbc exchange is sickle cell disease and its related complications. however, one of the miscellaneous indications of rbc exchange is for the patients of methemoglobinemia who are refractory to treatment by methylene blue. acquired methemoglobinemia is more common than any genetic causes. acquired methemoglobinemia is caused by toxins that oxidize heme iron, notably nitrate and nitrite-containing compounds. for patients failing to respond to standard treatment with methylene blue or in whom its use is contraindicated; hyperbaric oxygen or rbc exchange is indicated aims: case reports on use of rbc exchange in methemoglobinemia are few and indications are based on anecdotal reports. methods: exchange was performed on the cell separator machine, com tec by fresenius kabi. results: we report a case of acquired methemoglobinemia where patient was admitted with peripheral capillary oxygen saturation (spo2) of 87% on air. the patient did not show improvement in spo2 level with effective emergency treatment of methylene blue. since, the patient was refractory to treatment with methylene blue, the decision was made by clinician to proceed with rbc exchange. the patient improved significantly after two cycles of one rbc volume automated rbc exchange, and was discharged with spo2 of 97% on air. summary/conclusions: automated rbc exchange can be used in patients of acquired methemoglobinemia successfully when methylene blue is ineffective, and may be superior to manual one. background: therapeutic plasma exchange (tpe) is known to disturb the ph and electrolyte status. patients with compromised liver functions may be at a higher risk of electrolyte imbalance due to metabolic abnormalities. aims: the aim of this study was to analyze the variation in ph, ionized calcium, sodium, potassium, and bicarbonate in liver disease patients undergoing tpe. methods: patients with liver disease undergoing tpe during the period from july 2016 to august 2017 were included in the study. data on patient demographics, details of the tpe procedure, blood gas analysis report and adverse effects of tpe (if any) were collected and analyzed. results: one hundred and seven procedures were done during the study period; of these 46 (43%) were done on the mcs plus (haemonetics corporation) and rest 61 (57%) were done on the spectra optia (terumo bct). the percentage change in ionized calcium, sodium, and potassium due to the procedure was found to be statistically significant (p = 0.000). the systolic (p = 0.010) and diastolic (0.001) blood pressure also changed significantly with the procedure. the predictors for the change in ionized calcium were found to be pre-procedure ionized calcium (p < 0.001), the age of the patient (p < 0.001) and the pre-procedure ph (p = 0.002). procedurerelated complications occurred during 30 procedures of which 13 complications (12.15%) were categorized as features of hypocalcemia. no association was found between hypocalcemic manifestations and pre-procedure calcium, change in calcium, age or gender of the patient. summary/conclusions: the tpe procedure in liver disease patients causes remarkable changes in ionized calcium, sodium, potassium and bicarbonate ions. the decrease in ionized calcium during the procedure is predicted by pre-procedure ionized calcium levels, ph and age of the patient. monitoring of these parameters and appropriate corrective measures are imperative to patient safety. background: therapeutic plasma exchange (tpe) in pediatric age group is technically demanding because of low blood volume, difficult venous access and poor cooperation of the patient during the procedure. we here present our experience of tpe in pediatric patients from our centre. aims: to assess the challenges during tpe in pediatric patients and formulate appropriate strategies. methods: we did retrospective analysis of all tpe procedures performed in pediatric patients over a period of 16 years (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . tpe procedures were done on two different apheretic devices (cs 3000 plus, fenwal usa and cobe spectra, terumo bct lakewood, colorado) daily or on alternate days depending on clinical condition of the patient. for all procedures, kit was primed with compatible packed red cells. adverse events during the procedure were noted and analyzed. results: a total of 356 tpe (range 1-22/patient with mean of 6.2 procedures) were performed for 55 pediatric patients with different indications like atypical hus (category i as per american society for apheresis (asfa) in total 44 patients, neuromyelitis optica (category ii) in 4 patients, rapid proliferative glomerulonephritis (category i), c3 glomerulopathy in 3 patients each and one patient of infective hemophagocytosis. the average age of patient population was 7.8 yrs (1.2-13 years) . the male:female ratio was 3:1 with an average weight of 25.5 kgs. adverse events were observed during 20 (5.61%) procedures. most commonly observed adverse events were allergic reaction to replacement fluid (1.4%) followed by hypotension (1.1%), line occlusion (0.8%), vasovagal, endotracheal tube blockage and symptomatic hypocalcemia was observed in one procedure each (0.28%).there was no corelation observed between physical parameters of patient with adverse events. all adverse events were managed as per departmental standard operating procedures (sops) and procedures were completed successfully except in one where the procedure was abandoned. no mortality was observed during the procedures. background: the hemoglobin (hb) content of packed red blood cell (prbc) units is heterogenous. the patient's blood volume is also variable which can be calculated based on the weight, height and body surface area (bsa) of the patient. the efficacy of a transfusion episode can be assessed if the hb content of prbc is known and the patient's post-transfusion hb increment is determined. aims: this prospective study was performed to compare the efficacy of the transfusion of prbcs based on hb content versus the standard transfusion practice in thalassemia major patients. we also determined the correlation between hb increment and the hb content of prbc units transfused. methods: a total of 160 registered thalassemia major patients of our institute were included in the study after excluding the patients who had allo-or auto-antibodies. the study was approved by the institute ethics committee. the enrolled patients were randomly divided into two groups: group i (n = 80)they received abo/rhd identical prbcs suspended in additive solution (saline, adenine, glucose, mannitol: sagm-prbcs) after determining its hb content (units with hb content ≥ 50 g); and group ii (n = 80)they received randomly selected abo/rhd identical sagm-prbcs. the hb estimation of the randomly selected units in group ii was blinded. following tests were done on pre-transfusion sample: hb estimation using the hematology analyzer (orion 60, ocean medical technologies, india), blood grouping using tube technique, anti-human globulin (ahg) crossmatch and direct antiglobulin test (dat) using gel technique (biorad, switzerland), antibody screening (abs) using a fully automated immunohematology analyzer (neo, immucor, usa). on the posttransfusion sample collected 1 h after transfusion, hb estimation and dat were performed. results: there was no significant difference among the patient characteristics of the two groups. the mean hb content of the sagm-prbc units was significantly higher (p = 0.000) in group i (mean ae standard deviation: 67.86 ae 8.07 g; range: 50.80-92.13 g) than group ii (60.92 ae 8.29 g; range: 40.86-86.76 g). the mean hb increment in group i patients (3.26 ae 0.83 g/dl) was significantly higher (p = 0.04) than the group ii patients (3.00 ae 0.76 g/dl). in both the groups i and ii, there was a significant negative correlation between hb increment and weight (p = 0.000 in groups i and ii), age (p = 0.001 for group i; p = 0.032 for group ii), body surface area (bsa) (p = 0.002 for group i; p = 0.000 for group ii) and blood volume (p = 0.006 for group i; p = 0.000 in group ii). in both the groups i and ii, there was a significant positive correlation between hb increment and hb dose adjusted for bsa and the hb dose adjusted for blood volume (p = 0.000 in both groups i and ii for both the parameters). summary/conclusions: the efficacy of transfusion is more when patients are transfused with sagm-prbcs having hb content of 50 g or more as compared to those who are transfused with randomly selected units. for optimal hb increment in thalassemia major patients, the transfusion strategy should be based on the hb content of the sagm-prbcs. background: in male transfusion recipients under 50 years of age, receiving red blood cells (rbcs) from an ever-pregnant blood donor has been associated with increased mortality, compared to receiving a product from a male donor. although it has been suggested that older units of rbcs could be associated with increased mortality, there are significant methodological challenges in these studies. other studies indicated the freshest units of rbcs could be associated with increased mortality among transfusion recipients. we hypothesize both the association between ever-pregnant donors, and fresh units, with mortality could be caused by passenger leukocytes in the transfused rbc units, which decay during storage. aims: to quantify modification of the effect of ever-pregnant donors on mortality in young male rbc transfusion recipients, by storage time. methods: data on transfusion recipients receiving their first-ever rbc transfusion in one of six major dutch hospitals between 20/03/2004 and 01/09/2015 was collected. for the current study, male transfusion recipients under 50 years receiving only transfusions from one donor sex exposure category were selected and followup was censored at three years after transfusion. differences in storage time between groups were estimated by linear regression, adjusted for total number of transfusions, patient age, blood group, transfusion year and month. in a single-unit, single-transfusion cohort, cumulative mortality was estimated separately for patients receiving transfusions from ever-pregnant or male donors and for 'fresh' (<10 days storage) or 'old' (>24 up to 36 days storage) rbcs. results: for recipients of only blood from male donors, the storage time of the freshest unit was 0.47 day shorter when comparing the 221 patients who died, to 1,623 patients who survived (ci: à1.41 to 0.48). for recipients of only blood from ever-pregnant donors, the storage time of the freshest unit was 0.64 day longer when comparing the 27 patients who died, to 101 patients who survived (ci: à2.53 to 3.80). in the single-transfusion cohort, 1,280 patients received a fresh rbc transfusion from a male donor, 52 of whom died; 138 patients received a fresh transfusion from an ever-pregnant female donor, 9 of whom died. 193 patients received an old transfusion from a male donor, 7 of whom died; 16 patients received an old transfusion from an ever-pregnant female, 2 of whom died. the 3-years cumulative incidence of death among young male recipients was 4.7% (confidence interval (ci): 3.6% to 6.2%) after a fresh transfusion from a male donor and 4.8% (ci: 2.2% to 10.4%) after a fresh transfusion from an ever-pregnant female donor. the 3-years cumulative incidence of death was 7.2% (ci: 3.8% to 13.6%) after an old transfusion from a male donor and 15.4% (ci: 4.1% to 48.8%) after an old transfusion from an everpregnant female donor. summary/conclusions: prolonged storage of rbcs from ever-pregnant donors was not associated with decreased mortality at 3 years. contrary to our expectations, our results indicate older units may potentiate the effect of ever-pregnant donors. however, due to limited sample size the observed differences were not statistically significant. background: according to the literature review, there was limited impact of premedication (antipyretics, antihistamines and steroids) before transfusion on the prevention of adverse transfusion reactions (atrs). however, the necessity of premedication remains controversial. the premedication before transfusion is still a common clinical practice in pacific-asian countries, along with the premedication rate ranging from 50 to 80%. in our previous investigation, we found that premedication rate was 92.5% in the outpatients in 2017, which was much higher than the reported rate in asia. aims: to investigate the incidence of atrs and decrease premedication rate without increasing the rate of atrs via education and evidence-based clinical practice. methods: the incidence of atrs from april to december, 2017 was retrospectively surveyed. evidence-based clinical practice was initiated since january, 2018. clinical data of the outpatients receiving transfusion therapy were requested and analyzed from january to september, 2018. the incidences of atrs and premedication rates in 2017 and 2018 were compared using chi-square test. a p value less than 0.05 was statistically significant. besides, feedback of the incidence of atrs and premedication rate was given quarterly to the clinicians during the investigation. results: from april, 2017 to september, 2018, a total of 5,018 blood units were transfused in the outpatients with 2,453 transfusion events. of these, 25 cases of atrs, including febrile nonhemolytic transfusion reactions (fnhtr) and minor allergic reactions were reported. the overall premedication rate in the outpatients was 92.5% in 2017, and was significantly decreased to 77.9% in 2018 (p < 0.001). it was reported that the incidences of atrs in 2017 and 2018 were 0.48% and 0.52% per unit, respectively. there was no remarkable difference between the incidence of atrs in 2017 and 2018 (p = 0.642). summary/conclusions: via education and evidence-based clinical practice, we successfully reduced premedication rate without increasing the rate of atrs in the outpatients. furthermore, introduction of computerized provider order entry (cpoe) and clinical decision support system (cdss) could be considered and be expected to prevent unnecessary premedication before transfusion, increasing the compliance with optimized transfusion strategies in the future. methods: a retrospective analysis was done over a period of one year to evaluate clinical efficacy of 19 granulocyte transfusions in 15 hemato-oncology patients with febrile neutropenia. mobilization of granulocyte donors was done as per standard protocol, which included subcutaneous injection of granulocyte colony stimulating factor (g-csf) 5-10 lg/kg and tablet dexamethasone 8 mg, 10-12 h prior to granulocyte harvest by apheresis. all granulocyte products were gamma irradiated before transfusion. patient parameters like white blood count (wbc), absolute neutrophil count (anc), hemoglobin and platelet count were recorded pre-and post-granulocyte transfusion. infection related mortality (irm) within 30 days of granulocyte transfusion was also recorded. results: minimum adequate granulocyte yield of 1 9 10 10 per unit was fulfilled in 90% of granulocyte harvests. clinical indications for granulocyte transfusions were fever, an absolute neutrophil count (anc) < 500/ll, evidence of bacterial and/or fungal infections (i.e. clinical signs of infection, positive cultures and radiological evidence) and unresponsiveness to appropriate antimicrobial therapy for at least 48 h. effects of clinical, microbiological and granulocyte transfusion related variables on infection-related mortality were investigated. the post transfusion anc (within 24 h) increased significantly (median value: 350/ll) as compared to baseline levels (median value: 40/ll) (p < 0.05). infection related mortality was observed in only 20% (3 out of 15) of patients. patients became afebrile within 2-4 days and culture negative within 3-6 days after granulocyte transfusion. for analysis purpose granulocyte transfusion episodes were grouped according to doses of granulocyte transfusions, based on european guidelines (standard dose: 1.5-3.0 9 10 8 cells/kg and high dose: >3.0 9 10 8 cells/kg background: hsa's blood services group (bsg) is singapore's national blood service. in 2016, we conducted our pilot national pbm audit to promote pbm practices. it was agreed that the audit would be performed annually with incorporation of a new indicator to continue promotion of pbm and sharing of good practices. aims: to provide an update on the second national pbm audit for 2017. results are compared to the pilot audit and summarized below. methods: we collected data on 3 performance indicators from 7 acute public care hospitals for 4 weeks each in march and august 2017 (the pilot audit covered 2 weeks in 2016). the performance indicators were: 1). percentage compliance to documentation of red blood cell transfusion indications 2). percentage of patients screened for pre-operative anaemia, 14 to 45 days before surgery 3). peri-operative transfusion rates (3 days before to 3 days after surgery) for 6 commonly performed surgeries: coronary artery bypass graft surgery (cabg), total knee replacement (tkr), total hip replacement (thr), nephrectomy, colectomy and hysterectomy. the first two indicators assess pbm efforts and were measured in the pilot audit. indicator 3) was added to the second audit to assess impact of pbm practices on transfusion in surgical patients. it was an appropriate time to incorporate this indicator as the hospitals would have been familiar with pbm since its introduction in 2012. results and recommendations were shared with the senior management and hospital transfusion committees of the participating hospitals. results: for indicator 1), 2 hospitals had a compliance of 57-61%, the remaining 5 had a compliance of 90-100%. all 7 hospitals incorporated electronic blood ordering but the usage was not compulsory in some. hospitals which mandated electronic ordering performed better as doctors could only order blood products after entering the transfusion indication. we saw compliance increase from 75% in 2016 to 100% in a hospital that had newly mandated electronic ordering. for indicator 2), results ranged from 30% to 92%. 2 hospitals made notable improvements when compared to 2016, achieving 83% and 88% respectively. they had implemented pre-operative workflows screening all elective surgical cases for anaemia at least 2 weeks before surgery. one hospital also started an outpatient intravenous iron service which reduced pre-operative anaemia rates. for indicator 3), mean number of transfused units for each surgery ranged from 1.5 to 2.8 units per patient, lowest being thr and highest being cabg. this suggests that some transfusions were potentially avoidable with more robust pbm practices. the rate of perioperative transfusions was highest for cabg at 46% and lowest for tkr at 7%. summary/conclusions: the annual national pbm audit increases pbm awareness, allowing hospitals to share and learn good practices and implement measurable improvements. based on this audit, a recommendation to mandate electronic ordering of blood products to improve adherence to red cell transfusion indications and implementing pre-operative workflows with consideration for intravenous iron support was made. this audit was more representative than the pilot, with a longer duration of data collection and incorporation of indicator 3) showing impact of pbm practices. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february 2017, there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to the diagnosis, investigation and management of patients with autoimmune haemolytic anaemia (aiha) in english nhs trusts. methods: we designed and distributed a survey to the clinical transfusion leads at all english nhs trusts between november 2017 and march 2018. the survey requested information on detailed, simulated clinical scenarios. the first simulated scenario described a young patient with active aiha 3 months after an allogeneic stem cell transplant, who has received multiple transfusions in the last 2 weeks and is hypotensive, tachycardic, with a falling haemoglobin (hb), currently 48 g/l. the second scenario describes a young man with a new diagnosis of warm aiha who has an initial hb of 104 g/l and returns to clinic at a 2-week interval with symptoms of fatigue. he is actively haemolysing and commenced on 1 mg/kg prednisolone. results: there was a 42% (58/137) response rate by trusts. faced with a 4-6 h delay for allo-adsorption studies, 68% (38/56) of respondents would instead transfuse acutely with abo, rh and k matched red cells negative for any previously detected alloantibodies, 7% (4/56) would transfuse with o rh d negative red cells and 25% (14/56) would wait for completion of allo-adsorption studies before transfusing. in this first scenario, a quarter of respondents appeared to delay a potentially lifesaving blood transfusion. 2017 british society of haematology guidelines recommend that when anaemia is life-threatening in the time required for full compatibility testing, abo, rh and k matched red cells should be transfused. in the 2017 serious hazards of transfusion (shot) report, the most serious and fatal of 95 cases of preventable delayed transfusion was a patient with aiha who died untransfused with an hb of 38 g/l, while awaiting alloadsorption studies. a key shot message was that if clinical harm to patients from withholding blood outweighs safety concerns over a possible delayed haemolytic transfusion reaction, emergency blood is essential and should be offered. the second scenario also identified considerable variation in transfusion practice. it can take several weeks for patients with aiha to respond to prednisolone so a transfusion threshold < 60 g/l after an hb fall of at least 40 g/l in the previous 2 weeks is perhaps overly conservative. summary/conclusions: the overall findings support a need for studies to explore barriers to uptake of guidelines, and to identify areas for further audit and research to guide safe and appropriate transfusion practice in aiha. background: balance between supply and demand of o d negative red cells remains a challenge for almost every blood service. with this re-audit, we wanted to collect objective and comprehensive information regarding usage of o d negative red cells supplied by nhs blood and transplant (nhsbt) to private and nhs hospitals in england. aims: the aim was to understand hospital practices, actual needs and possible avoidable usage of o d negative red cells. where possible, comparisons were made with two previous audits (2008) (2009) (2010) . methods: participating hospitals were asked to determine the fate of all group o d negative red cells they received between 14 th and 27 th may 2018 excluding substitutions and complete an organisational survey regarding activities, policies and stockholding practices with respect to o d negative blood. participating hospitals were asked to provide (if available) the prevalence (as a percentage) of o d negative patients in their population. this information, in conjunctions with hospital activities, will be used to estimate appropriate o d negative stockholding levels. background: o rhd-negative (neg) red blood cells (rbcs) are a precious resource, are often in short supply and transfusion of these units in emergency settings carries the potential risk of transfusion-related adverse outcomes such as haemolytic reaction due to minor blood group incompatibility. as such, their use should be closely monitored within health services. most recent australian guidelines (2008) for their use in emergency settings include pre-menopausal females of unknown blood group (mandatory indication) or while the blood group is being established; use should be limited to 2 or less units where possible before a switch to group-specific rbcs (acceptable indication). aims: audit of use of emergency uncrossmatched o rhd-neg rbcs against national guidelines in our institution (an australian tertiary metropolitan public hospital providing acute medical and surgical, emergency and critical care services). methods: use of emergency uncrossmatched o rhd-neg rbcs units over a six-year period was retrospectively reviewed. we collected information about rbcs transfused and discarded, adverse outcomes, patient characteristics, clinical indications and whether use met national guidelines or could have been avoided. results: 105 episodes of emergency uncrossmatched o rhd-neg rbcs were identified, encompassing transfusion of 241 rbc units to 103 patients and the discard of 2 rbcs (due to incorrect transport). of the 105 episodes, 78 episodes (74%) involved an eventual switch to group-specific rbcs (range of emergency units, 1-13 units). the main requester was the emergency department (53%). the most common clinical indication for transfusion was acute gastrointestinal bleeding (49%). of the 105 episodes, 28 episodes (27%) did not meet the guidelines for emergency use because > 2 units of emergency uncrossmatched o rhd-neg rbcs were issued. 2 episodes (2%) were flagged as potentially inappropriate as the patients were clinically stable according to documentation in the medical records. 30 episodes (29%) were identified as potentially preventable due to delay in pre-transfusion sample collection (defined as > 1 h elapsed between patient arrival and group and screen sample collection) in the setting of acute bleeding (6%), receipt of an unsuitable pretransfusion sample requiring sample recollection (5%), delay in pre-transfusion sample processing (4%), no valid pre-transfusion sample being available at the time of the bleeding episode despite having a planned elective procedure or being an inpatient with recent clinical bleeding (10%). only one patient was investigated for potential transfusion-related adverse outcome (1%) which was thought likely due to concurrent sepsis. summary/conclusions: over six years, 105 episodes utilising emergency uncrossmatched o rhd-neg rbcs were identified with 241 rbcs issued and 2 rbcs discarded. a significant proportion of episodes (29%) were potentially avoidable if there had been a valid pre-transfusion sample available in the transfusion laboratory at the time of the episode. efforts to minimise use of this precious resource are ongoing, and include feedback to clinical units regarding importance of valid pretransfusion samples prior to applicable invasive procedures and in bleeding patients, ongoing education to medical and nursing staff, and continuing audit of use of this blood component in the hospital haemovigilance programme. abstract withdrawn. abstract withdrawn. background: platelet transfusions are often given prophylactically to thrombocytopenic hematology patients. to which extent platelet function improves after transfusion, and how this improvement correlates with an increase in platelet count, is not well studied. flow cytometry has been used to evaluate platelet function after transfusion in a few studies and can be performed even at low platelet counts. rotational thromboelastometry (rotem) represents a more physiological measure of platelet function in whole blood that has not been extensively used in transfusion settings. we used these methods to investigate if platelet transfusion improves platelet function in hematology patients and if improvement correlates with increased platelet counts. aims: the aim was to evaluate the relationship between response to platelet transfusion, measured as corrected count increments (cci), and platelet function in thrombocytopenic patients with hematological disorders. methods: blood samples (sodium-citrate anticoagulated) were collected from unselected hematology patients receiving prophylactic platelet transfusions, after informed consent had been obtained. samples were taken at three time-points: within 1 h before transfusion, 1 h after and 14-24 h after transfusion (via a central venous catheter or a subcutaneous venous port). for each time-point, platelet response to adenosine diphosphate (adp) and thrombin receptor-activating peptide (trap-6) was assessed by flow cytometry by measuring p-selectin and pac-1 expression on single platelets. rotem analysis was also performed on all samples, using intem and extem reagents. results: an interim analysis was performed after inclusion of 22 patients. the mean platelet count before transfusion was 8 9 10 9 /l (range 2-34 9 10 9 /l). 1 h cci was 11 9 10 9 /l and 14-24 h cci was 6 9 10 9 /l, but response was highly variable. pselectin expression after stimulation with adp and trap was significantly higher at 1 h after and 14-24 h after transfusion compared to before transfusion (p < 0.05). pac-1 expression after stimulation with adp was significantly higher at 14-24 h after transfusion (p < 0.001), but not at 1 h after transfusion. in rotem, clot amplitude at 10 and 20 min (a10 and a20) as well as maximum clot firmness (mcf) improved after transfusion (p < 0.05). a significant correlation between absolute platelet count and p-selectin expression after trap and adp stimulation was found (r s =0.53 and 0.45 respectively, p < 0.001). absolute platelet count was also significantly correlated with mcf (r s =0.61, p < 0.001), where 84% of patients with a platelet count of more than 20 9 10 9 /l reached mcf values within the reference interval. summary/conclusions: platelet function generally improves after transfusion and was in our patient population correlated to the absolute platelet count, but was also seen at the single platelet level in flow cytometry. a post transfusion platelet count of more than 20 9 10 9 /l might be sufficient to significantly improve coagulation in heavily thrombocytopenic patients, but larger studies are needed to confirm this conclusion. abstract withdrawn. abstract withdrawn. background: sickle cell disease (scd) is a genetic disorder that is frequently referred to as a hypercoagulable state. hydroxyurea (hu) is known to decrease the frequency of vaso-occlusive complications and need for blood transfusions in severely affected individuals. although cross-sectional studies show that treatment with hu is associated with decreased coagulation activation, there are no prospective studies evaluating the effect of hu on coagulation activation. aims: to assess the effect of hu on markers of fibrinolysis (d-dimer) and endothelial activation (soluble vascular cell adhesion molecule-1 [soluble vcam-1]) in patients with scd in their non-crisis, "steady state." methods: patients, at least 10 years of age, with documented hbss or hbsb-thalassemia, eligible for treatment with hu were studied in this prospective, observational study. laboratory investigations were obtained at baseline, prior to commencement of therapy with hu, with repeat evaluations at three and six months of therapy. non-parametric test was applied to observe the association between hu therapy and the biomarkers of interest. results: twenty-five patients with scd (hbss: 15, hbsb thalassemia: 10) were enrolled (females: 15 [60%]), with a median age of 23 years (iqr: 11). following 6 months of hu, median values for wbc count (9.02 9 10 9 /l vs. 7.22 9 10 9 /l, p = 0.007) and d-dimer (1243.8 ng/ml vs. 830.2 ng/ml, p = 0.028) were significantly lower than baseline values, while the mean corpuscular volume (76.0 fl vs. 88.0 fl, p = 0.001) was significantly higher than the baseline value. no significant differences from baseline were observed in the median values for hemoglobin (9.1 g/ dl vs. 9.6 g/dl, p = 0.72), platelet count (250 10 9 /l vs. 196.5 10 9 /l, p = 0.71), lactate dehydrogenase (744 u/l vs. 567.5 u/l, p = 0.23) or soluble vcam-1 (567.8 ng/ ml vs. 526.4 ng/ml, p = 0.33) following 6 months of hu therapy. summary/conclusions: this exploratory study confirms that treatment with hu is associated with decreased coagulation activation in patients with scd, although no effect on endothelial activation was observed. by decreasing coagulation activation, hu may decrease the risk of thrombotic complications in scd. abstract withdrawn. abstract withdrawn. transfusion medicine, apollo gleneagles hospitals, kolkata, india background: reduction of immune responsiveness through blood transfusion has been documented by previous authors. breast cancer is considered as one of the commonest cancer globally and the second main cause of death in females transfusion of allogeneic blood in breast cancer surgery is variable and differences of transfusion incidence have been observed in the literature. where the maximum surgical blood ordering schedule (msbos) dictates cross matching and reservation of blood before surgery, factors deciding their utilization are varied and numerous. our hospital protocol guides that every patient planned for elective breast cancer surgery should routinely have a blood sample sent for reservation of one unit of compatible packed red blood cell (prbc) in the blood bank. aims: in this prospective study we aimed to audit the blood utilization in patients undergoing elective breast surgery and thereby optimize the blood ordering schedule, economic burden and loss of clinical resources. methods: the study included 478 confirmed breast cancer patients planned for elective breast surgeries from january 2012 to december 2017. patient and disease details like age, stage, tnm status, estrogen receptor (er) and progesterone receptor (pr) status, human epidermal growth factor receptor 2 (her -2) expression, triple negative breast cancer (tnbc) status, reproductive and treatment status were documented. patients were divided into younger group [≤40 years] and older group (>40 years). before surgery blood samples for compatibility testing were sent to blood bank for blood reservation. details of test, blood issue and blood transfusion were documented in the blood bank. approximate loss of time in minutes and wastage of resources in terms of money (inr) in the blood bank were noted. all results were calculated as mean ae sd and a 'p' value of < 0.05 was considered statistically significant. results: of the total 478 patients most underwent wide local excision of the breast and modified radical mastectomy. a total of 16 patients received 71 units of blood and blood components in all categories of surgeries. only 103 were younger women (≤40 years) with mean age of 31 years. non-transfused patients were significantly more than transfused ones (p < 0.05). frequency of blood transfusion was more in young patients (4.9%). seven (22.6%) of the total 31 stage iv patients received blood transfusions. frequency of blood transfusion was more in patients undergoing surgery after chemotherapy (8.8%). a significant loss of time and loss of revenue was observed. summary/conclusions: we conclude that routine compatibility test is not justified for all patients undergoing breast surgery. a more targeted approach is needed to reduce blood demand and associated cost to patient and blood transfusion services. background: blood transfusion guidelines are not only essential for the optimal use of blood products, but also help reduce transfusion-related adverse reactions and improve patient outcomes. the korean national transfusion guidelines were developed in 2009 and fully revised in 2016 by the korean centers for disease control and prevention and the korean society of blood transfusion. in our hospital, which is a 700-bed university hospital, a transfusion-indication data-entry program based on the national transfusion guidelines was developed in 2016. it was applied to the electronic medical record system and all transfusion orders, except emergencies, have been performed through this program since then. aims: we planned to record and analyze the reasons for transfusion in order to monitor blood product usage and provide feedback to clinicians. furthermore, we intended to contribute to patient safety through the appropriate use of blood products. methods: we classified transfusion-indications by the blood product requested and created a pop-up window listing these indications, which would appear at each regular transfusion order. indications for transfusion with each blood product were as follows: red blood cells (rbcs)acute blood loss, chronic disease (sub-classified as hb ≤ 7 g/dl, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, respiratory disease, age ≥ 65 years, age ≤ 6 months, chemotherapy), surgery/ procedure, transplantation and 'other'; platelets (plts)present bleeding, bleeding prevention (sub-classified as hematologic disease, solid tumor, peripheral blood stem cell transplantation, disseminated intravascular coagulopathy, infant), surgery/procedure, massive transfusion and 'other'; fresh frozen plasma (ffp)bleeding in coagulopathy, bleeding prevention in coagulopathy, massive transfusion, plasma exchange and 'other'. transfusion indications entered into the data-entry program from sep 2016 to feb 2018 were analyzed. results: the number of transfusion-indications analyzed was 16138 for rbcs, 11158 for plts and 6024 for ffps. the most common indications for transfusion were chronic disease for rbcs (7977/16138, 49.4%), bleeding prevention for plts (5726/11158, 51.3%) and 'other' for ffp (2180/6024, 36.2%). 'hb ≤ 7 g/dl' was the most frequent sub-indication of chronic disease (3570/7977, 44.8%), and hematologic disease was the most frequent sub-indication of bleeding prevention (3432/ 5726, 59.3%). many clinicians entered transfusion indication as 'other': rbcs (2866/ 16138, 17.5%), plts (856/11158, 7.7%) and ffp (2180/6024, 36.2%). however, the free-text supplied by the clinician when 'other' was selected, often corresponded to an indication already categorized in the transfusion-indication data-entry program; 82.9% of rbcs and 54% of plts. of the indications entered as 'other' in ffp, 80.3% were surgery/procedure-related. summary/conclusions: in our hospital, the release of blood products has been dependent on the data-entry of transfusion indications (except in emergencies) since sep 2016. transfusions of rbcs and plts were most common for chronic disease and bleeding prevention, respectively, but many cases entered as 'other' could have been categorized as existing indications in our data-entry program. therefore, we conclude that additional training is needed for clinicians regarding the determination of transfusion-indications and correct use of the transfusion-indication dataentry program, in order to use blood products more appropriately. methods: this was a prospective cohort designed study. subjects were children aged 1-18 years with indication of platelet transfusions in sardjito hospital yogyakarta indonesia. the patient samples were collected before and 1 h post-transfusion, the expression of cd62p on platelet was determined by flow cytometry method. results: there were 102 subjects who were divided into two groups. fifty-one subjects received non-leukodepleted pcs and the other fifty-one transfused by pre-storage leukodepleted pcs. the mean of pre-transfusion platelet cd62p for nonleukodepleted and leukodepleted groups were 26.2% and 27.7%, and the mean increase of post-transfusion platelet cd62p for non-leukodepleted was 10.1% and the mean decrease of leukodepleted groups was 3.3%. it was shown the increase of post-transfusion platelet cd62p for non-leukodepleted group, and it was significantly (p < 0.05) higher than in the leukodepleted groups. summary/conclusions: there was an increase of post-transfusion platelet cd62p expression in patients received non-leukodepleted, but a decrease in leukodepleted pc transfusions. background: preoperative anaemia is a common finding in patients undergoing surgery and often neglected in our country. aims: the objective of this study was to evaluate hb(values and the identification of cardiac patients who entered operation with anaemia. and also to study the correlation between hb values and the number of rbc (red blood cell) transfused unit methods: this is a retrospective, descriptive and analytical study. the data for this study was collected from the files in the statistic's service at qsut (university hospital center "mother teresa"). the object of our study were the files of 158 patients hospitalized in the period january -may 2018 in the cardiac surgery ward, which were subjected to cardiac surgery. from the files were collected data on age, gender, primary diagnosis, accompanying diseases. we also collected hb, rbc, htc (hematocrit), mcv (mean corpuscular volume), mch (mean corpuscular hemoglobin), mchc (mean corpuscular hemoglobin concentration). from the transfusion service at qsut and from the files were pulled out the transfused patients and the number of transfused units. results: based on the who definition for anemia (females < 12 g/dl and males < 13 g/dl), from the 158 patients included in the study, 61 (39%) were anaemic. from 117 males in the study, 40 (34%) of them were anaemic based on hb lab values, whereas from 41 women in the study anaemic were found to be 21 (51%) of them. from the 61 anaemic patients in the study, 35 (57.4%) of them with mild anaemia, 23 (37.7%) with moderate anaemia and 3 (4.9%) with severe anaemia. in the total of anaemic female 38.1% are under 65, while 61.9% are over/or 65 years old. in the total of anaemic males, 35% are under 65, while 65% are over/or 65 years old. it is noticed that most of them are with normochromic normocytic 67.2%, normocytic hypochromic anaemia 16.4%, hyperchromic microcytic anaemia 8.2%, macrocytic normochromic anaemia and macrocytic hypochromic anaemia respectively 1.6% and microcytic normochromic anaemia 4.9%. the average value of preoperative hb decreased from 12.8 g/dl before surgery to 10.3 g/dl after surgery, so there is a decrease of approximately 2.5 g/dl of hb value. in our 158 patients, 48% (76) were transfused and the remaining 52% (82) were not transfused. from 76 transfused patients 41 (54%) patients were anaemic. the correlation between the values of hb, rbc, htc and the number of transfusions shows that with the decrease of these values the number of transfused units increases. summary/conclusions: the diagnose of anaemia is underestimated before surgical intervention in our country and investigation of hb low values do not take the proper importance to find probable cause and correct it before surgical intervention. the lower the hb values, the greater the chance to be transfused and the number of rbc transfused units. failure to correct hb values before surgery results in unnecessary transfusions for the patients or which could have been avoided, eliminating also the risk of transfusion complications. background: alloimmunization after red blood cell transfusion is affected by various factors. it is known that the incidence of alloimmunization increases in certain diseases. extended red blood cell matching can be used to prevent the development of alloimmunization in diseases which the rate of alloimmunization is increased. in asia, extended red blood cell matching is not actively implemented. aims: we tried to investigate whether there is a difference in the disease categories between unexpected red blood cell antibody positive and negative groups. methods: from january, 2003 to december, 2017, the diseases of the patients who had undergone unexpected red blood cell antibody identification test at dong-a university hospital was examined through medical records. from january 2008 to december 2009, the diagnosis was made on patients who had two or more unexpected antibody screening tests. we analyzed the frequency difference of disease category between two groups. results: a total of 988 patients were performed with unexpected antibody identification tests. of 1896 patients who underwent more than 2 screening tests, 25 (1.3%) were positive. 1871 were consistently unexpected antibody negative. the patients with solid tumors (n = 375, 56.3%) and those with hematologic diseases (n = 112, 16.8%) had a higher incidence in unexpected antibody positive group. the patients with myeloid malignancy had a significantly higher frequency than lymphoid malignancy (p = 0.0002). the frequency of patients with liver cirrhosis was significantly higher in the unexpected antibody positive group (65/988, 6.6%) than in the negative group (77/1871, 4.1%) (p = 0.006). the incidence of non-hodgkin lymphoma was significantly higher in the unexpected antibody negative group (28/1871, 1.5%) than in the positive group (5/988, 0.5%) (p = 0.019). summary/conclusions: there was a difference in the distribution of diseases between unexpected antibody positive group and negative group. the patients with liver cirrhosis were more frequent in unexpected antibody positive group, suggesting that extended red blood cell matching would be considered. background: in hematological patients with multiple platelet transfusions (pc) often develop immune response to human leukocyte associated antigens (hla-i) and human platelet-specific associated antigens (hpa). besides, platelet associated immunoglobulins (paig) and complement components (pac) are found on platelet. this leads to increased platelet destruction and development of refractoriness to transfusions of donors' platelets. transfusion therapy using an individual selection of platelets and plasmapheresis, contribute, in the majority of cases, to the realization of efficient transfusion by pc. but, in difficult cases, there is a need to use intravenous immunoglobulin, which may promote the efficient transfusion of pc. aims: evaluate the algorithms of using the complex therapy of refractoriness to transfusions of donors' platelets with additional application of intravenous immunoglobulin (ivig). methods: in 2018 there were three female patients in the clinics of the centre for observation, age between 29 and 51 years (me = 39) with the ineffectiveness of complex therapy for overcoming refractoriness to transfusions of donors' platelets due to selection and plasmapheresis. the diagnoses were as follows: aplastic anaemia (aa)-2, acute myeloid leukemia (aml)-1. individual selection of platelets was carried out by the adhesion method on the solid phase (immucor "galileo neo"). paig and pac3/4 were evaluated by the method of flow cytometry (bd facscanto ii) by the method of double staining with cd41a. the density of fixed paig, pac was © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 evaluated by the median fluorescence intensity (mfi). the two patients with aa received ivig-igg therapy in the standard dose 0,4 g/kg per day, for 3 days. one patient with aml received ivig-iggam therapy in the standard dose 5,0 ml/kg/day for 3. results: under pressure of the complex therapy with the use of ivig in the standard dose there was are decrease in mfi over time in the case of two patients: aa-1 mfi-paigg reduced from 926 to 65; while the patient with aml: paiga reduced from 5506 to 187, and paigm from 1971 to 302, pac3 from 691 to 31, pac4 from 404 to 103. the patient with aa-2 over time, regardless of the treatment, there was an increase of mfi, but the effect of pc transfusions was achieved under pressure of complex therapy. under pressure of complex therapy all the 3 patients also reduced the frequency of reaction of alloantibodies when resorting to an individual selection and increasing the frequency of compatible couples "donor-recipient". summary/conclusions: delivery of complex therapy and the additional application of ivig enables an adequate transfusion therapy of pc, neutralize hemorrhagic syndrome and continue the treatment of the main disease. detection and monitoring of paig/pac during the development of refractoriness to transfusions of donors' platelets are additional markers for prescription of ivig therapy. anaesthesia, tan tong seng hospital, singapore, singapore background: blood transfusion is quite prevalent in paediatric cardiac surgical procedures. we hypothesized that the routine use of rotational thromboelastography (rotem) to guide transfusion decisions would reduce the overall proportion of patients receiving transfusions in paediatric cardiac surgery aims: the aim of the study was to find if the use of blood and blood products in pediatric cardiac surgical cases in a single centre is affected due to rotem. methods: sixty paediatric cardiac surgical patients undergoing cpb were included in this study. thirty patients (study group) were prospectively included and compared with thirty procedure and age-matched control patients (control group). in the study group, rotem, performed during cpb guided intraoperative transfusions. perioperative transfusions of blood and blood products, postoperative blood loss and hemoglobin levels were compared between the two groups. results: the patients in the control group received fewer transfusions of packed cells (60% vs 78%) and fresh frozen plasma (36% vs 84% p<o.oo1). more patients in the study group received platelets (72% vs 60%) and cryoprecipitate (50% vs 36%). the postoperative blood loss was lower in study group compared to the control group. the postoperative hemoglobin did not differ between the two groups. summary/conclusions: the results suggest that the routine use of rotem in paediatric cardiac surgery to guide transfusions is associated with reduced proportions of patients receiving transfusions and is evidence based practice. abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mycoplasma pneumonia (mp) infection has often been implicated in respiratory tract infections. although this agent has also been associated with other non-respiratory diseases, hemolytic anemia is the most common hematologic complication of mp infection. episodes of sudden onset of severe anemia are rarely seen in beta thalassemia. we report a 33-years-old female with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection aims: to report a case of patient with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection and how it was managed with minimal transfusion. methods: a 33-year-old female case of beta thalassemia major who developed autoimmune hemolytic anemia following mycoplasma infection. the patient is on monthly blood transfusion since the age of 3 years at dubai thalassemia center. she was admitted to the hospital with 5 days history of dizziness, severe low back and joint pain and her travel history indicated a four-week visit to pakistan. initial investigations showed a hemoglobin level of 7.8 g/dl the direct and indirect coombs test were positive. patient was transfused with two units leuko-depleted compatible prbc (o, rh +, negative for e and kell antigens.) and discharged next day. three weeks later, the patient was still with lower limbs pain and the hemoglobin was 8.5 g/dl with a positive dat with both igg+4 and c3+4, patient was not transfused as it was difficult to find matching blood; therefore she was given one week follow up. four days later, patient reported to the center with fever, generalized body pain and fatigue. she had significant drop in hemoglobin 5.6 g/dl, mcv 85.0, platelets count 216000, crp 3.6, retic count 2.18, blood culture no growth, total bilirubin 1.5, malaria parasite smear was negative, urine culture and microscopy were normal. mycoplasma pneumonia antibody showed positive with igg 57.1 and igm 21.3, she was started on azithromycin for 5 days and received 2 units prbc least incompatible leuko-depleted (o, rh +, negative for e and kell antigens.). after which her hemoglobin increased to 9.3 g/dl. she did not show any improvement in her condition over the following 2 weeks and repeat mycoplasma pneumonia antibody showed an increased titer, which denoted resistance to azithromycin; hence, we started her with second line of treatment levofloxacin for 10 days. screening for collagen disease were all negative apart from high esr of 130 mm/hr. patient remained afebrile and transfused with least incompatible available blood, post transfusion hb was 11.8 g/dl. three weeks later, during her routine follow up, she was doing well with esr dropping to 85 mm/hr and hemoglobin 10.2 g/dl, which indicated gradual settling of hemolysis, although the last dat remained positive. subsequent visits showed hb drop of 1.2 g/dl/week which is acceptable for transfusion dependant thalassemia. results: with conservative management patient condition improved. summary/conclusions: hemolytic anemia associated with mycoplasma pneumonia is usually self-limited and resolves spontaneously after mycoplasma infection elimination. packed red cell transfusion in transfusion dependent patient may be troublesome and can aggravate the condition; therefore it should be utilized and limited to significant symptomatic anaemia background: early plasma transfusion in women with severe postpartum hemorrhage is often instituted to prevent and treat coagulopathy, but whether maternal outcomes improve with this treatment is unclear. aims: to quantify the association of plasma transfusion within the first 60 min after diagnosing persistent postpartum hemorrhage and adverse maternal outcome. methods: we performed a retrospective cohort study of women with persistent postpartum hemorrhage, defined as postpartum hemorrhage refractory to first-line measures to control bleeding. women were enrolled from january 1, 2011 to january 1, 2013 in 61 dutch hospitals. in a time-dependent propensity score-matched analysis, women with transfusion of fresh frozen plasma within 60 min following the diagnosis of persistent postpartum hemorrhage were matched to women without plasma transfusion or plasma transfusion at a later time point during hemorrhage. plasma transfusion was not guided by coagulation tests. the main outcome measure was adverse maternal outcome, defined as a composite of mortality, hysterectomy and arterial embolization to control bleeding. we performed sensitivity analyses with initiation of plasma transfusion within 120 and within 180 min following diagnosis persistent postpartum hemorrhage, compared with other plasma transfusion strategies. results results: the cohort consisted of 1216 women with persistent postpartum hemorrhage, with uterine atony as cause of hemorrhage in 780 women (64.1%). in this cohort, seven maternal deaths (0.6%), 62 hysterectomies (5.1%) and 159 arterial embolizations (13.1%) were observed. plasma transfusion within 60 min in 114 women was matched on time-dependent propensity scores with 114 women with no or later plasma transfusion. rate of adverse maternal outcome was similar between women with and without early plasma transfusion, 21.2% versus 19.9%, adjusted odds ratio 1.09 (95% confidence interval 0.57-2.08). results of sensitivity analyses were comparable to the primary results. summary/conclusions: among women with persistent postpartum hemorrhage, initiation of plasma transfusion within 60 min following this diagnosis was not associated with improved maternal outcomes, compared with no or later plasma transfusion. whether other transfusion strategies or identification of women with high risk of coagulopathy will improve maternal outcomes remains to be studied. background: preoperative evaluation of increased risk for perioperative bleeding is essential for cardiac surgery patients. platelet aggregation, as a laboratory test for assessment of platelet function, is very efficient for optimal antiplatelet treatment and also for assessment of the increased risk of postoperative bleeding and perioperative application of allogenic red blood cell transfusions. aims: the aim of this study was to establish whether preoperative platelet function testing for the presence of residual effect of acetylsalicylic acid (asa) on platelet aggregation can be correlated with an increased risk for intraoperative bleeding during cardiac interventions. methods: a retrospective study included 60 patients who underwent surgical procedures (cabg ii and cabg iii) at the department of cardiovascular surgery, clinical center in ni s in period november 2017-july 2018. patients were previously taken asa at a dose of 100 mg per day, but therapy was discontinued 5 days before surgery. platelet aggregation was measured on the day of surgery using impedance aggregometer multiplate (multiplate platelet function analyzer, roche), using trap and aspi tests. patients were divided into two groups: control group (cg), which included patients with preserved function of platelets (aspi 790-1410 au*min), and examined group (eg), which included patients with inhibited platelet aggregation in aspi test (<790 au* min background: hemophilia b is an x-chromosome-linked inherited bleeding disorder resulting from reduced levels or an absence of factor ix clinically represented by recurrent prolonged bleeding. mutations can be inherited or acquired de novo. de novo mutations arise in about one-third of patients with haemophilia. the disease affects primarily males, but approximately 30% of carrier females have factor ix clotting activity lower than 40% and are at risk for bleeding. aims: case report of a hemophilia b carrier. methods: the clinical history of the patient was thoroughly studied by analyzing medical records and analytical results using the software available in the health institution and paper records. results: 45 year old woman, with no history of abnormal haemorrhage, healthy, up to 2004 when, after a dystocic delivery, presents with subcutaneous and ocular haemorrhage and dispersed bruises. a basic coagulation study is performed and a level of 20% fix is found. genetic analysis reveals a mutation in fix gene and she is diagnosed as a carrier of hemophilia b. except for a son, no other direct family members carry the mutation. in 2008, after total hysterectomy with bilateral adnexectomy due to an invasive carcinoma in the uterine cervix, the patient suffers a haemorrhagic shock with multiple transfusion support and around six months later undergoes an ureteronephrectomy also with the need for post-surgery transfusion support. the patient is referred to the immunohemotherapy department where she has regular appointments as an outpatient since 2009. in 2012, she requires surgery for the removal of a thyroglossal duct cyst, which is performed under prophylactic treatment with 2000 iu recombinant fix (rfix) daily, for four days, and up to 48 h after discharge maintaining basal levels of fix of 60% and with no major complications reported. in 2013, the patient is admitted to the er in haemodynamic shock and a history of black stools and syncope. at admittance, she receives a bolus of 4000 iu of rfix and requires severe transfusion support due to active bleeding. she is diagnosed with colonic angiodysplasia and undergoes surgery. daily doses of 3000 iu rfix are administered from day 1 (d1) and up to d7 maintaining fix levels around 60-80%. the patient is operated at d8 under the cover of 2000 iu of rfix, twice per day, and up to d14 after surgery reaching fix levels of around 80-110%. at d15 the dose was diminished to 2000 iu of rfix daily and at d17 she is discharged with no rfix replacement therapy necessary in ambulatory. since then, no complications or new occurrences have been reported. the patient is under prophylactic administration of rfix prior to minor invasive procedures (nodule biopsy, dental extractions and colonoscopies). summary/conclusions: hemophilia b carriers usually do not require any major health care for their daily routine or invasive procedures. however, in the case reported, basal fix levels conditioned any minor intervention to be performed. thus, this carrier must be considered as a moderate hemophilia b patient and mandatory prophylaxis with rfix must be performed prior to any invasive procedure. background: blood transfusion is a life saving procedure but transfusion associated complications are equally important that need to be addressed. with limited resources of blood donations and screening, excessive transfusions must be avoided to prevent risk and economic burden on patients. on the other hand discussion on appropriate use of blood product is a matter of debate in medical literature. it is recommended to restrict the liberal use of transfusion. aims: in this regard we aimed to observe the frequency of genuine and non genuine transfusion at our hospital and also to compare the outcomes of liberal and restrictive transfusions. methods: it was a cross sectional study carried out at nibd and bmt, pechs campus, karachi, pakistan, from february 2018 to january 2019. the study was approved from institutional review board. pack red cells (prc) transfusion in case of < 7 g/dl hemoglobin (hb) with cardiac history and < 7 g/dl hb with symptomatic anemia was considered genuine. platelets transfusion in case of sepsis and count < 50 9 10 9 /l, sepsis with bleeding and < 100 9 10 9 /l, patients on active chemotherapy without bleeding but platelet count < 10 9 10 9 /l and in patients presented with bleeding was considered genuine. patients who were presented with bleeding, deranged coagulation profile and with sepsis + bleeding considered genuine for fresh frozen plasma (ffp) transfusion. spss version 23.0 was used for descriptive and inferential statistics. results: a total of 250 transfusions were evaluated for genuine and non genuine transfusion. male predominance was high in our data 194(78%). out of 250, 134 (54%), 100(40%) and 16(6%) prcs, platelets and ffp were transfused respectively. out of 100 platelets, 10(10%) were platelet apheresis. 80/134 (60%) prcs and 22/90 (24%) platelets were non genuinely transfused as per the criteria described in methods. however, ffp and platelet apheresis were genuinely transfused. in non genuine group of platelet transfusion, all patients received more than 1 unit platelet and the increment of platelet count post transfusion was found statistically non significant (p = 0.121). thirty six (45%) of transfusions were done with more than 1 units of prcs in non genuine group. the mean hb level of patients received 1 unit and more than 1 units prcs was statistically same (p = 0.868) and it was found out that there was same increment in hb in both the groups post transfusion. (p = 0.867). summary/conclusions: we concluded that although blood transfusion is an integral procedure for saving life yet correct use of blood products is necessary in the era where blood donations are scarce and blood transfusion has its potential associated hazards. the approach towards the appropriate use of blood products will not only minimize the risk of transmission of infectious diseases but also will reduce the economic burden. longitudinal studies on large sample size are needed to validate this study. uncertain. an ovine model enables comparisons of; (a) invasive and non-invasive methods to measure oxygen delivery and utilisation at a tissue level, and of (b) the efficacy of various resuscitation fluids and transfusion protocols in treating haemorrhagic shock. aims: to develop an ovine model of haemorrhagic shock. methods: sheep (n = 4) were anaesthetised, ventilated and instrumented. instrumentation included blood flow, perfusion, and microdialysis probes placed in the brain, kidney, liver and skeletal muscle. haemodynamic, tissue oxygenation, tissue flow and respiratory data were recorded continuously by data capture software. non-invasive assessments, including brain and muscle oxygen saturation by nirs, and visualisation of sub-lingual microvascular perfusion, as well as arterial blood gas measurements and plasma/serum/urine collections were made at specific timepoints. after a baseline period (1 h) sheep were bled~40% of their estimated blood volume (estimated at 65 ml/kg) to a mean arterial blood pressure (map) of 30-40 mmhg. sheep were then resuscitated with either plasmalyte â (up to 20 ml/kg/ hr; n = 3) or transfused with sheep red cells (n = 1) to achieve a map > 65 mmhg. sheep were euthanised 4 h after resuscitation. data are presented as mean ae standard deviation. results: sheep were haemorrhaged an average of 931.7 ae 135.3 ml blood which combined with iatrogenic blood loss (~300 ml) corresponded to an average 40.2 ae 2.4% blood loss. two out of the four sheep met clinical criteria for haemorrhagic shock (map = 30-40 mmhg, lactate > 4 mm, svo 2 < 60%). across all four sheep the nadir map averaged 40.5 ae 13.1 mmhg, lactate peaked at 3.9 ae 1 mmol/ l, and nadir svo 2 was 41.3 ae 17.9%. all sheep survived to the end of the experimental protocol. summary/conclusions: these data demonstrate the successful induction of haemorrhagic shock in an ovine model. further experiments are planned to improve the protocol and to achieve 100% incidence of haemorrhagic shock, and then to compare invasive and non-invasive measures of oxygen delivery and utilisation as well as the efficacy of different resuscitation fluids and red cell transfusion. adverse events, including trali p-515 bilirubin were recorded within the 28-day period. the clinical parameters were compared against the reaction strength of the antibody reactions. the automated strength was measured by solid phase. the manual testing consisted of a 15-min incubation using liss and adding monospecific igg. the dat was performed manually by adding poly-specific igg and then testing with monospecific igg and c3d. the rh group and non-rh group had 11 and 10 cases performed manually, and results were 2+ or weaker further indicating the manual strength did not correlate with the clinical hemolysis. likewise, in 31/44 (70%) the dat was negative, and did not show any correlation with clinical hemolysis. however, when ldh and bilirubin were measured, the two parameters increased as the automated strength of the antibodies increased. summary/conclusions: most of the dshtr investigation was not associated with overt accelerated red cell destruction. a strong correlation was observed only between the automated immunohematology testing results and other laboratory markers of hemolysis. in our experience, the direct antiglobulin test and manual strength showed no correlation. background: numerous transfused patients present severe, sometimes critical clinical conditions. the occurrence of adverse transfusion reactions (atr) may induce deterioration in the clinical condition with a worsened clinical course and a lifethreatening or fatal outcome as is the case with nervous system impairment. in france, in 2017, out of 7,276 notified atrs, 113 (1.5%) and 6 (0.1%) were life-threatening and death respectively. aims: our aim was to evaluate the notified atrs with neurological signs that occurred in transfused patients over a period of six years and six months in hospitals in the auvergne rhône alpes area. the study included patients with reported atrs in hospitals in this area from january 1 st 2010 to june 30 th 2018. each atr was registered in the national haemovigilance database system. two signs observed at the time of the atr were analyzed: unconsciousness and convulsions. stroke was excluded. the type of atr, its severity, the blood product involved and its imputability were studied. results: during the period under study, 9,544 atr were reported, of which 29 included unconsciousness and/or convulsions (0.3%). of these 29 patients, 13 were females (44.8%) and 16 males (55.2%). unconsciousness alone was frequently observed (21 reports, 72.4%). convulsions were notified in 8 reports (27.6%) and were associated with unconsciousness in 2 of them. the diagnosis of seizure, with no other clinical signs, was established in 2 cases (6.9%). unconsciousness and/or convulsions were present in 8 allergic reactions (27.6%), 4 cases of transfusion-associated circulating overload (13.8%), 3 cases of suspected transfusion-transmitted bacterial infections and 2 hypertensive reactions. in allergic atrs, unconsciousness was notified in 7 cases and unconsciousness associated with convulsions in one. twelve atrs were severe (41.4%), 10 were life-threatening (34.5%) and in 4 cases, they resulted in the death of the recipient (13.8%). of the 8 allergic atrs, 4 were severe and 4 life-threatening. red blood cell concentrate was involved in 15 atrs (51.7%) and platelet concentrate in 9 (31.1%), including 5 cases with apheresis platelet concentrate and 4 cases with pooled platelet concentrate. fresh frozen plasma was involved in 5 atrs (17.2%). nevertheless, the imputability of the blood product was excluded or unlikely in 11 atrs (37.9%). in the 3 suspected transfusion-transmitted bacterial infections, the imputability of the transfusion was ultimately excluded after a negative result was obtained in the bacterial culture of the blood product. the imputability of the blood product was probable or possible in 7 and 9 atrs respectively, but was certain in only 2 atrs. summary/conclusions: unconsciousness and/or convulsions were rarely observed in atrs notified in transfused patients. nevertheless, the presence of these signs highlights the seriousness of the atr (26 ars, 89.7%). lastly, the imputability of the blood product was often excluded or unlikely. in the multivariate cox model for the effect of lpi on overall survival, adjusted for age and ipss-r category, elevated lpi levels were associated with inferior overall survival (hr 3.0, 95% ci 1.5-5.7, p = 0.001). this effect was most pronounced in the td-rs subgroup (hr 6.0, 95% ci 2.2-16.2, p < 0.001). similarly, elevated lpi levels were associated with inferior pfs (hr 3.4, 95% ci 1.9-6.3, p < 0.001) for the whole study population and the td-rs subgroup (hr 8.2, 95% ci 3.4-21.0, p < 0.001). in total 16 patients received iron chelation during the sample collection period (11 patients deferasirox, 5 patients desferrioxamine). lpi levels were normal in 14 out of the 17 samples collected during deferasirox treatment and in 2 out of 5 samples collected during desferrioxamine treatment. summary/conclusions: transfusion dependency is associated with the presence of toxic iron species and inferior overall and progression-free survival in lower-risk mds patients. in td-rs patients the effects were most pronounced indicating ineffective erythropoiesis leading to additional iron toxicity. background: post-transfusion immunomodulation has been reported to contribute to poor patient outcomes. clinically relevant transfusion models are needed to improve our understanding of underlying mechanisms. sheep transfusion models are of increasing importance in blood transfusion research as they provide several advantages over small animals, including their size, anatomy, physiology and similar blood volume compared to human. a current limitation of sheep transfusion models is the lack of characterisation of the sheep immune system. understanding the sheep immunology is necessary to advance sheep transfusion models, identify mechanisms that contribute to post-transfusion immunomodulation and facilitate the translation of findings into clinical settings. aims: to characterise the sheep leukocyte inflammatory responses to in vitro lipopolysaccharide (lps) challenge in edta and heparinized whole blood. methods: edta and heparinized sheep whole blood (n = 4 of each) was cultured with rpmi media (37°c, 5% co 2 ) alone or with the addition of lps (1-100 lg/ml; derived from escherichia coli 055: b5). the inflammatory response was assessed after 2 h (h), 6 h, 12 h, 24 h, 36 h and 48 h. supernatant was harvested at each time point and stored at à80°c. inflammatory cytokine/chemokine production was determined using sheep specific in-house elisa (il-1b, il-6, il-8 and il-10). twoway analysis of variance with bonferroni's post-test was used to measure the effect of incubation time and concentration compared to no lps matched samples. results: when edta was used as an anticoagulant, addition of lps resulted in production of sheep il-1b and il-10 but not il-6 or il-8. il-1b production was significantly increased following stimulation of 100 lg/ml lps for 6 h (p = 0.043) and declined following 36 h incubation. release of il-10 was significant 12 h post-lps stimulation with 100 lg/ml (p = 0.030) and reached a maximum at 24 h. the use of heparinized blood resulted in a different immune profile as all inflammatory markers tested were detected following stimulation with much lower concentrations of lps (1 lg/ml), although the incubation times differed. il-1b was significantly increased following 2 h incubation (p = 0.002), with increasing levels observed up to 24 h post-lps stimulation. il-8 production was evident from 6 h and reached significance at 24 h post-lps stimulation (p = 0.009). il-10 was significantly increased following stimulation of 5 lg/ml lps for 6 hr (p = 0.043) with lower concentrations of lps resulting in il-10 production at 24 h (p = 0.008). release of il-6 was significant after 6 h of 50 lg/ml lps stimulation (p = 0.046), with lower concentrations of lps resulting in il-6 production at 24 h (p = 0.015). in heparinized whole blood an lps concentration-dependent effect was evident for all cytokines. summary/conclusions: using a time-and concentration-approach our findings indicate that sheep are more tolerant and have a delayed response to lps stimulation compared to previous research using similar human in vitro whole blood culture models. in addition, data suggest that sheep have greater immune responses using heparin as anticoagulant for the collection of blood samples. improving our understanding of sheep immunology and development of relevant sheep transfusion models will provide a bridge between sheep models of transfusion and clinical settings. 1. rhdig inappropriately administered (unnecessary exposure) (n = 17, 40%) administered to: -rhd positive woman (n = 9) -rhd negative mother with rhd negative neonate (n = 5) -woman with immune anti d (n = 1) -administered in error (instead of other ig) (n = 2) 2rhdig delayed/omitted/wrong dose (risk of sensitisation to the d antigen) (n = 17, 40%) -omitted (n = 14) -delayed (n = 1) -inadequate dose (n = 2) 3administration without correct patient identification (n = 6, 14%) 4storage & handling (n = 3, 7%) failure to check the maternal and neonatal blood groups prior to administration was identified as a source of error. misinterpretation of blood results also led to women receiving product inappropriately. e.g. reading a negative antibody screen as the mother being rhd negative. patient identification was raised as an issue. rhdig is often stored in satellite blood fridges for easy access. collection from these areas did not always require confirmation against patient identifiers and there was no register of women who received product or link to the batch number to ensure traceability. two incidents involved the administration of rhdig when the prescription for other immunoglobulin products was not clear, leading to a child and a baby receiving rhdig instead of the intended immunoglobulin. summary/conclusions: these incidents indicate problems with the processes of appropriate identification of women who need rhdig, the use and interpretation of pathology tests and requirements for prescription and administration. these resulted in omitted and inappropriate doses of rhdig. blood matters has made a number of recommendations regarding rhdig administration: -all health professionals involved in rhdig administration should be appropriately trained in the use of rhdig -confirmation of the maternal rhd status is essential prior to prescription or administration -positive patient identification must be used prior to administration of rhdig -health services should consider regular auditing to identify areas for improvement relating to rhdig blood matters continues to work with maternity care providers to improve practice. centro comunitario de sangre y tejidos de asturias, oviedo 2 agencia gallega de sangre, organos y tejidos, galicia 3 banco de sangre y tejidos de cantabria, cantabria 4 banco de sangre de la rioja, la rioja 5 banco de sangre y tejidos de navarra, navarra 6 banco de sangre y tejidos de arag on, aragon 7 fundaci on de hemoterapia y hemodonaci on de castilla y le on, castilla y leon 8 fundaci on banco de sangre y tejidos de las islas baleares, islas baleares, spain 9 terumo bct europe nv, zaventem, belgium background: hemovigilance, a long-term monitoring process made mandatory by national and supranational regulations, begins with a systematic whole blood or blood component collection and ends with an examining period after transfusion of blood components into the patients. in spain, organized in 17 autonomous regions, the hemovigilance system is structured in three levels: (1) the local level comprised of transfusion centers and hospital based transfusion services that monitor and collect all transfusion related adverse events (ae) and level them up to (2) the regional hemovigilance coordinator, who communicates all the region's data to the (3) spanish ministry of health which issues an annual report and corresponds with european institutions. to ensure safer blood supply, pathogen reduction technology (prt) was approved and implementation started in spain in 2008. the mirasol prt system for platelets and plasma was introduced in 2010 and is currently being used in 10 of the 17 spanish regions. aims: to monitor the safety of the system, a passive hemovigilance study on mirasol treated products was initiated in the region of asturias and collaboration was extended to other regions (baleares, galicia, la rioja, cantabria, navarra, castilla y leon and aragon). methods: collected data included allergic and febrile reactions, trali and all other adverse event observed. severity of the event and level on imputability of the transfusion were also assessed using the who grading scale. hemovigilance data of mirasol treated products (platelets or m-pc and plasma or m-p) are included from 2012 to 2017 as blood centers started to apply the technology in routine. results: increase adoption of the mirasol system is observed between 2012, when 4,196 mirasol treated blood products were issued to hospitals and 2017 with 56,229 mirasol products issued. due to low number of transfusions of mirasol-treated blood components in 2012 and 2013, notification rates began to be analyzed in 2014, showing ae rates of 0.2%, similar to reports at the national level. stable transfusion reaction rates were observed with m-pc (around 0.23). rate of ae after transfusion of m-p is fluctuant between 0.06 and 0.17. this fluctuation could be due to the inconsistent numbers of m-p transfused from one year to the other. most of transfusion reactions (around 80%) were of grade i severity and grade ii level of imputability. allergic reactions accounted for most of the adverse events, with g&i > 2 reactions in 2016 and 2017 of respectively 0.18 and 0.07 no bacterial nor viral transfusion transmission was recorded on mirasol products during the study period (2012) (2013) (2014) (2015) (2016) (2017) . at the national level, nine cases of bacterial transfusion transmission (with g&i > 2) were reported. these transmissions were probably due to transfusion of non-pathogen reduced products. summary/conclusions: the observed notification rate of ae is similar to the national rate but allergic reactions with g&i > 2 is inferior with mirasol treated products. also, we found no reports of transfusion transmission infections nor cases of transfusion associated graft-vs-host disease, demonstrating safety of mirasol treated products. were attributed to human error (75%) with the lowest frequency in equipment failure (10%), compared to 21% and 29%, respectively, in the following three years. root cause analysis demonstrated failures in the quality management system including failures in administration, inadequate staffing for blood collection as well as in distribution and processing, and failures arising from institutional constraints and system failures in hospital management. high numbers of "other" aes (25%) in distribution and whole blood collection call for further investigation to indicate measures necessary for prevention and correction. errors related to incorrect blood component transfused (ibct) in 2007-2017 were 80 in 8,385,448 blood units (1/104,818) issued for transfusion. these resulted in 27 serious reactions (34%) (1 fatal, 11 life-threatening) . another 21 (26%) were related with ibct that did not cause a reaction. near misses (component not transfused) were 24 (40%) summary/conclusions: our data demonstrate increasing compliance with reporting requirements. questions about the initial factors for deviations in certain activities specifying failures in equipment and materials due to system as well as human errors, highlight the need for further specifications beyond "other" and "human error". background: the weakest link in the transfusion chain currently is the handling of blood components after their issue and the bedside blood administration practices. aims: to evaluate compliance with standard procedures for bedside blood transfusion practices by analysis of the "transfusion feedback forms" in a tertiary care multi-specialty hospital setting. methods: during the study period of 24 months, the transfusion feedback forms received from various clinical areas of the hospital were studied with special reference to the transfusion times. the data was categorized based on the patient's location as well as the time of transfusion, whether done in routine or emergency hours. results: 42,403 blood components were issued during the study period, while transfusion feedback forms for 37,816 components (89.1%) were received in the transfusion medicine department. delay in starting the transfusion (more than 30 min after issue) was observed in 2965 transfusion events (6.9%). the component transfusion time exceeded the permissible limits for 930 component (2.1%).the overall total permissible time for completion of components exceeded permissible limit in 2217 (5.2%) of transfusion events. the pediatric ward (23.9%), icu and ot complex (25.4%) were found to be the most non-compliant delay in transfusion, transfusion time and total transfusion time. amongst the 2965 delayed transfusions after issue, 2120 (71.5%) were during the routine hours i.e. between 7 am to 7 pm and 845 (28.5%) were in the non routine hours i.e. between 7 pm to 7 am. summary/conclusions: the audit of bedside blood transfusion practices has given us a good insight into various areas of noncompliances as well as the predominant locations in the hospital where the practices need to strengthened further. focused training program on safe blood administration practices for all staff involved in handling and transfusion of blood components is now planned to combat this issue. background: the international surveillance of transfusion adverse reactions (ars) and events (aes) (istare) of the international haemovigilance network (ihn) collects aggregate data from member national haemovigilance systems (hvs) in order to estimate the morbidity and mortality of blood transfusion in a holistic approach. the ultimate goal is to contribute to improving the safety of transfusion by close monitoring throughout the chain "from vein to vein". aims: we analyse recent istare data on suspected transfusion transmitted infections (sttis) for 2013-2016 in comparison to previous years of surveillance, [2006] [2007] [2008] [2009] [2010] [2011] [2012] methods: annual aggregate data from ihn member hvs on transfusion associated bacterial, viral and parasitic infections collected online in istare are analyzed by incidence in blood components (bcs) issued for transfusion, by severity and imputability as well as by blood component. ars with definite, probable or possible imputability were included in the analysis. trend analysis is performed to allow comparisons and to collect information on established and newly emerging infectious threats of blood transfusion. results: for 2013-2016 89 sets of annual aggregated data from 25 countries covering 85,521,393 bcs issued were analyzed. all ars totalled 76,907 and infectious ars amounted to 285 (0.4%). the overall incidence of the infectious ars was 0.33/ 100,000 units of bcs issued. bacterial infections were the most frequent (188, 66%), next viral (84, 29.5%) and then parasitic (13, 4.5%). serious were 46% and there were 11 fatalities (3.9%, incidence 0.013/100,000). nine deaths were attributed to sepsis and the other two were associated with non-malarial parasitic pathogens. one geotrichum clavatum fungal infection associated with apheresis platelets was reported as a free text comment. this very rarely recognized fungal pathogen caused a very severe infection in a patient but the route of transmission is inconclusive. the 84 viral sttis included 17 hbv (20%), 10 hcv (12%) and 2 hiv (2.5%). the 55 recorded as "other" (65.5%) including 24 cases of hev, one case of parvovirus b19, one cmv and one ebv. no case of tt-malaria was reported. other stt-pi were 15 (two fatal). the prevailing bcs were in general rbcs followed by platelets. comparison with corresponding data for 2006-2012 shows a consistent overall incidence in total sttis (0.33 vs 0.4/100,000). however, considerable differences were seen in separate categories, such as bacterial infections (significantly increased rate in 2013-2016, p < 0.001) and an almost doubled rate of parasitic infections (p < 0.001). compared to the earlier period, there were many fewer hbv infections (17 vs 160) and many more hev. a similar reduction in the rate of hcv and hiv was observed in 2013-2016 in comparison with previous years. this may be explained by the fact that nat testing for hcv/hiv/hbv has been implemented in many countries in the last decade. summary/conclusions: the infectious risk of transfusion overall remains very low. the rate of bacterial cases has increased and among other viral sttis the frequency of hev is increasing. the mortality of transfusion due to sttis is lower than in the previous period of surveillance. abstract withdrawn. background: one of the main aspects of haemovigilance system in hospitals is following of adverse events and reactions related to blood transfusions. aims: it was intended to analyse the adverse reactions related to transfusion of blood components in pediatric patients. methods: over a four year period (january 2015-december 2018), the haemovigilance records of all patients receiving blood transfusions procedures were reviewed and transfusion reactions were analysed. statistical analysis of data was performed by spss software (version 22.0, spss inc., chicago, il, usa). majority of blood components were provided by regional blood center organized by national red crescent society. but granulocytes collected by apheresis after donor mobilization and reconstituted whole blood for exchange transfusions were prepared in the transfusion center of the hospital. results: the median age of patients who developed transfusion reactions was 72 months (interquantile range-iqr 108). the median for the numbers of individual transfusions in children in a year was 12 (iqr 17). the median for the numbers of blood components individually transfused to patients was 18 (iqr 24). patients, anaphylactic transfusion reactions in 4 patients and transfusion-related lung injury (trali) in a patient. the overall incidence of transfusion reactions was estimated at a rate of 2.7 per 100000 units. summary/conclusions: it was reported that adverse effects related to blood transfusion, especially allergic reactions and fnhtrs are common in pediatric patients than adults. in a multinational study concerned about the transfusion reactions related with red cell concentrates, allergic transfusion reactions and fnhtrs were reported at a rate of 11 and in 100000 units and 26 in 100000 units, respectively. while the incidence of transfusion reactions in children was found 0.62% in a study from the u.s.a., the overall incidence of transfusion reactions in our study which was estimated at a rate of 2.7 per 100000 units represents a lower rate. hospital gran canaria dr. negr ın, gran canaria 6 hospital general universitario, ciudad real, spain 7 hospital nostra senior de meritxell, andorra, andorra 8 banco sangre y tejidos, santander 9 banc de sang i teixits, barcelona, spain 10 fundaci on hematol ogica colombia, bogot a, colombia 11 centro regional de transfusi on de almer ıa, almeria 12 complejo hospitalario de navarra, pamplona 13 fundaci on banc de sang i teixits illes balears, palma de mallorca 14 hospital de cabueñes, gij on, spain background: root analysis cause is defined as the cause of an error that, if it is treated, eliminates the repetition of the error aims: describe types of human and latent errors detected by a work group in the root analysis cause of transfusion incidents, analyze the concordance between the individual responses of the members and propose recommendations in order to improve transfusional safety. methods: in 2018 fifteen participants (nurses and hematologists dedicated to transfusion and component preparation) studied some incidents of administration of nonirradiated components and tried to approach the root causes by applying the classification of errors in mers-tm transfusion medicine. they transferred the answers to a questionnaire (simple or chain error, initial process affected, human and latent errors and measures derived from the analysis to correct the errors). the communication was made by mail and by the spanish transfusion society web forum, which contained the consultation documents. data and percentages are exposed for each type of error and the answers of the participants are tabulated. results: cases corresponded to 3 patients. two patients of 55 years of age diagnosed of acute myeloblastic leukemia (case 1 and 2) and chronic lymphatic leukemia (case 3). in one case, the hematologist of the transfusion service canceled an irradiation prescription; in another, a patient with fever was transfused in the emergency room without the irradiation requirement and it was later discovered that he had received a transplant of hemopoietic progenitors 1 month earlier; in the last case, neither the requesting doctor nor the laboratory technician nor the following doctor (prescriber) detected the alerts located in their respective computer applications. in all 3 cases, the story was judged as sufficient for analysis. the majority of reviewers (93%) diagnosed a chain of errors. there was agreement of 95% with respect to the initial process affected. the initial error was communication (42%), monitoring (45%) and compliance (62%), in cases 1, 2, and 3. 2-3 human errors were detected per case (average: 2.2, 2.7 and 2.4 errors respectively) and 2-3 latent errors per case (average: 1.8, 2.7 and 2.1, respectively). the latent errors most punctuated were: failures in the quality of the protocols (24%), in the transfer of important knowledge (22%), in the available technology (21%) and in the information to the patient (10%). all the participants contributed feasible measures of improvement according to root causes: 1) improve the quality and drafting of work procedures and their compliance, including procedures of effective communication between professionals, 2) train staff in knowledge important for safety, 3) communicate with computer application providers to improve the effectiveness and visibility of the alerts and 4) involve the patient with essential information to ensure transfusion safety. the measures were processed later as recommendations. summary/conclusions: the root analysis shows agreement between participants and allows the elaboration of useful recommendations to increase patient safety. this strategy can contribute to the comprehensive prevention of errors. background: in transfusion-associated circulatory overload (taco), pulmonary oedema develops primarily due to volume excess. data from the uk haemovigilance scheme, serious hazards of transfusion (shot) suggest that either the incidence of taco, or the recognition and reporting of taco, has increased over time. from 2007 to 2017, reports of taco increased from 6 to 92; deaths from 1 to 7, major morbidity from 3 to 20. known risk factors include pre-existing cardiac and/or renal dysfunction, low body weight, extremes of age (eg, <3 years, >60 years), concomitant fluid administration, positive fluid balance, peripheral oedema and hypoalbuminemia. in a small subset of cases reported to shot, taco developed following transfusion for severe anaemia in the absence of other risk factors. this may be an under-recognised independent risk-factor. aims: to raise awareness of severe anaemia as an under-recognised risk factor for taco and is potentially life-threatening transfusion. methods: cases of taco submitted to shot over the last 3 years were reviewed to identify cases where transfusion for severe anaemia was a key identifiable patient risk factor. results: the following are illustrative cases: -case 1: a patient in their 50s weighing 67 kg was prescribed six units of red cells for iron deficiency anaemia after being admitted with hb 37 g/l. the patient had no risk factors for taco except for profound anaemia. during transfusion of the fifth unit the patient became dyspnoeic, hypoxic and hypertensive. the patient recovered after diuretic therapy and had a post-transfusion hb level of 100 g/l. -case 2: a patient in their 50s presented with a 4-week history of weakness and dizziness and had felt unwell for 6 months. the hb was 34 g/l, ferritin 26 lg/l and ecg showed cardiac ischaemia. two units of red cells were transfused. after the second unit oxygen saturations fell despite supplemental oxygen, post-transfusion hb of 51 g/l. a third unit was transfused over 125 min and the hypoxia worsened with dyspnoea and crackles on chest auscultation. the chest x-ray showed an enlarged cardiac silhouette and pulmonary congestion. the patient improved with diuretics. -case 3: a patient in their 90s with severe megaloblastic anaemia, hb 41 g/l and peripheral oedema developed taco after transfusion of 3 units and recovered with diuretic therapy. summary/conclusions: chronic and acute anaemia are associated with compensatory cardiac changes irrespective of the aetiology of anaemia. this is further compounded by the underlying cause of anaemia particularly haematinic deficiencies (iron/b12 deficiency) that independently affect myocardial function. hyperdynamic circulation related to anaemia increases the load on the heart, causing myocardial ischaemia and hypoxia and if the anaemia is not corrected, eventually leads to heart failure. clinicians need to make an accurate diagnosis and avoid excessive transfusion in patients with severe anaemia with or without other additional risk factors. patients with chronic iron/folate/b12 deficiency without haemodynamic instability should be given the appropriate haematinic replacement. haematinic deficiency responds rapidly to appropriate vitamin/mineral. blood transfusions are to be given only when clinically indicated and even then, only the minimum volume needed for symptomatic relief transfused with consideration for diuretic therapy. methods: legal forms for reporting transfusion reactions were used in the retrospective analysis, which were adjusted by the department of quality assurance and quality control in the electronic form and distributed to clinics using blood components. clinicians were trained to report transfusion reactions through the hospital's transfusion board and through the "service for improvement of the quality and safety of health services" at the clinical center of sarajevo. analysis of the reported reactions in the institute include immunohematological and microbiological examination based on which the guidelines for further treatment with blood components are being made. users of registered services are obliged to report since 2015. results: total of 121,076 different blood components were applied in the period between 2015-2018. department for quality assurance and control has received 4 serious adverse reactions, 1 serious adverse event, 157 reports of transfusion reactions, of which 11 (7%) were inadequately filled, in the same period. from the above, 54 (34.39%) were transfusion reactions to erythrocyte blood components which were applied, 86 (54.77%) to platelet components and 17 (10.82%) were transfusion reactions after the application of fresh frozen plasma. the analysis has shown that the most frequent were febrile non-haemolysis reactions (88 or 56.05%), followed by allergic reactions (50 or 31.84%). two transfusion reactions (1.27%) were characterized as circulation overload. summary/conclusions: the frequency of serious adverse reactions and events was 0.004% (5 of 121,076) and 0.12% were reported transfusion reactions (157 of 121,076). with the establishment of the hospital transfusion board and with the increase of collaboration with the clinical center, significant progress has been made. it is necessary to increase awareness among clinicians in regards to the safe transfusion practice. reporting transfusion reactions should be a mandatory procedure, a path to the proper selection of blood components, monitoring adverse reactions, and for us, transfusiologist, guide to the safest, most efficient blood components. j garc ıa-gala, e martinez-revuelta, a caro-g omez, c castañ on-fern andez and i fern andez-rodriguez hospital universitario central de asturias, oviedo, spain background: elderly patients are the main group of transfusion recipients in our country. given their comorbidities are a risk group for the development complications related to transfusion. aims: analyze the incidence of adverse effects related to transfusion in the elderly population and to assess what factors may influence its appearance methods: transfusions were reviewed in patients > 70 years old. the variables analyzed were: sex, age, diagnosis/reason for transfusion, pre-transfusion hemoglobin (hb), number of transfused units, infusion rate and transfusion side effects, as well as the measures used to prevent or treat the transfusions. effects of circulatory overload results: a total of 93 patients were analyzed (48 women, 45 men), with a median age of 86 years (70-97). in total, 189 ch were transfused. 70 patients received 2 ch, 3 patients 3 ch, 10 patients received 1 ch. 10 patients were transfused at two different times. the median hb prior to transfusion was 7.6 g/dl. in the patients who received 2 ch was 7.5 g/dl, those who received 3 ch: 6 g/dl and those who received 1 ch: 7.75 g/dl. the infusion time could be estimated in 84% of the patients. in those who received 2 ch was 223.62 min; 249.33 min in those who received 3 ch and 109.75 min in those who received 1 ch. 13 patients (14% of the total) suffered some type of adverse effect related to the transfusion. in 9 patients there was an increase in posterior ta, in 2 an increase in hr, in 1 an episode of hypotension and in another one episode of acute respiratory failure. 70% of those who had an adverse effect were older than 85 years. patients with aht after transfusion, 75% received 2 ch and the remainder 1 ch. among their background, 66% had a history of ischemic heart disease. 33% also had a positive balance. the average previous bp was 137/65 mmhg and the subsequent one was 182/72 mmhg. 55% of patients did not receive diuretic treatment. in the case of the patient with acute respiratory failure was in oligoanuria, with positive balances. 2 ch was transfused in total. she was treated with oxygen therapy and with intensification of the diuretic treatment, recovering later. summary/conclusions: -patients > 85 years have a higher risk of suffering some type of adverse effect related to transfusion because they have pre-existing risk factors such as ischemic heart disease or heart failure. -we see that the risk of suffering some type of adverse effect in the elderly population is greater when we transfuse 2ch than 1ch. -we have appreciated that in those patients receiving 3ch, the infusion rate is higher. -the study highlights the lack of methods to prevent the development of circulatory overload. background: iron deficiency anemia is the commonest cause of anemia worldwide. weakness, fatigue, reduced physical activity and difficulty in concentration are the symptoms which are associated with its deficiency. the forefront treatment available is oral iron replacement therapy which is convenient, cost effective and has substantial outcome. another option is intravenous (i/v) iron when oral is not tolerable. despite of potential transfusion associated hazards and limited availability of blood due to shortage of voluntary blood donations, it is insisted by the patients prior to iron therapy. aims: the aim of conducting this study was to observe the impact of administration of oral iron, i/v iron and transfusion on hemoglobin levels in patients presented with iron deficiency anemia. methods: this was an observational study carried out at nibd and bmt, pechs campus, karachi, pakistan from february 2018 to december 2018. the study was approved by the institutional review board. diagnosed ida patients presented at our hospital were recruited for analysis who were given oral iron, i/v iron and transfusion for the correction of anemia. informed consent was taken from the participants. descriptive and inferential statistics was applied by using spss version 23.0. results: a total of 108 ida patients were analyzed in which 74(69%) were females and 34(31%) were males. the most common symptom in females and males was fatigue followed by body aches in females 12(16%) and pallor in males 10(29%). menorrhagia was present in 24(44%) of females of reproductive age. surgical history was present in 12(16%) of females while there was no surgical history in males. mean hemoglobin, mch and mcv of females at baseline was 8.0 ae 2.2, 60.5 ae 9.6, and 20 ae 8.3 while in males it was 7.8 ae 2.3, 63 ae 14.3 and 18.4 ae 6.1 respectively. sixty two (84%) females were advised oral and i/v iron and 12 (16%) received transfusion. however, in males 8(24%) received transfusion and 26 (76%) were advised oral and i/v iron therapy. it was observed that the increment of hemoglobin after oral/iv iron at average of 3 months follow up in males and females was same as that the transfusion (p > 0.05). summary/conclusions: in our society where blood donations are scarce especially voluntary blood donations that are considered to be the safest type of blood donation. we would like to draw attention towards the alternatives to correct anemia such as oral and i/v iron replacement therapy as our results revealed that there was no difference in the increment of hemoglobin between the two groups. we need to educate our society especially the older age adults and young women who are more vulnerable of getting ida to opt oral and i/v iron therapy. it will be cost effective, convenient and also has less risk than transfusion. cellular therapies -stem cell and tissue banking, including cord blood background: the differentiation of megakaryocytes plays an important role in the production of platelets. however, the underlying mechanisms regulating megakaryocytes differentiation have rarely been studied. aims: to identify candidate genes involved in megakaryocytes differentiation and investigate the potential regulatory mechanisms of megakaryocytes differentiation from human cord blood hematopoietic stem cells in vitro. methods: cb-derived cd34 + cells were isolated using density gradient centrifugation and magnetic activated cell sorting (macs). cultures were stimulated with only recombinant human tpo (100 ng/ml). after 12, 14 and 18 days, the mk fraction was selected by immunomagnetic sorting from the non-mk fraction using an anti-cd41a monoclonal antibody. rna-seq-derived gene expression data was performed on uncultured samples (day 0), cultured but unselected samples (day 7), and cultured, selected samples (day 12, 14 and 18) by using the next-generation sequencing (ngs) platform, and rq-pcr technology was used to verify the expression of transcription factors. results: the comparison of the transcriptome profiles among the five stages showed that a massive gene expression change occurred in megakaryocytes differentiation. a total of 65140 genes were detected, of which 16612 showed up-regulation and 48528 down-regulation. among these genes, 8 differentially expressed genes (degs) (fold change ≥ 10; false discovery rate < 0.05) were selected were further validated with rq-pcr, including gabre, cdhr1, wasf3, pkhd1l1, thbs1, pf4v1, lrrc32 and lgals12. the rq-pcr result indicated that the mrna expression level increased with the prolongation of culture time. however, pf4v1 mrna expression level was highest at day 14, lgals12 was highest at day 18. summary/conclusions: conclusion: our study identified a series of genes that may participate in the regulation of megakaryocytes differentiation. these results should serve as an important resource revealing the molecular basis of megakaryocytes differentiation and thrombocytopoiesis. preoperative anemia and blood transfusion requirement during hip and knee surgery rambam health care campus, haifa, israel background: blood transfusion (bt) is independently associated with increased morbidity, mortality and hospitalization length across different patient populations. due to bt-related risks, the concept of "patient blood management" (pbm) has been introduced to clinical practice. the three pbm pillars are: optimizing red cell mass, minimizing blood loss and optimizing physiological reserve. bt indications during orthopedic surgery include excessive bleeding or hemodynamic instability and not the hemoglobin (hb) level. in most clinical scenarios, a restrictive transfusion threshold (hb level: 7-8 g/dl) appears to be non-inferior to the liberal transfusion strategy in terms of blood use, morbidity and mortality. similar results are observed in highrisk patients after hip surgery. we hypothesize that preoperative anemia may lead to blood product overuse and its complications. aims: evaluating potential correlation between preoperative anemia and bt requirement during hip or knee surgery. methods: we reviewed medical files of patients who underwent hip or knee replacement surgery at rambam between 2011-2018. patients with hb level measurement within 90 days pre-surgery were included. receiving > 1 blood unit was considered a surgery complication and such patients were excluded. patient demographic and clinical data, including comorbidities, surgery type, length of hospital stay, were collected. we created a synthetic data cohort using mdclone healthcare data sandbox, an environment enabling fast data extraction and producing synthetic data for analysis that does not require irb approval. results: during the evaluated period, 2591 patients underwent hip or knee surgery; 231 were excluded from the analysis due to receiving > 1 blood unit. hb measurement within 90 days pre-surgery was available for 1202 patients. hip or knee surgery was performed in 588 (49%) and 614 (51%) patients, respectively. women comprised 60% (n = 356) of patients who underwent hip surgery. in the hip-surgery group, 14.5% of patients required bt, with this need being slightly higher among women (31.5% vs. 26.2%; p-value=ns). only 50 (8%) patients were transfused during knee surgery and this cohort was not further analyzed. patients receiving bt had a significantly lower mean hb level than those who didn't require it (11.93 g/dl versus 12.8 g/dl for women and 12.3 g/dl vs. 13.8 g/dl for men; p-value < 0.001). hospitalization was longer in transfused patients compared to non-transfused ones (mean 7.52 vs. 6.91 days, p-value = 0.018) and in patients with a low hb level (female < 12, male < 13.5) than in those with a high hb level, irrespective of receiving bt (p-values < 0.00045). patients with at least one of the following diagnoses: diabetes, renal failure, ischemic heart disease, were significantly more likely to have a lower preoperative hb level (p-value < 0.05). no other factors (e.g., patient's weight, rdw value or blood pressure) were predictive of transfusion need. the probability of a need for 1 blood unit was 0.43 in the hb 11 g/dl group and 0.15 in hb 13 g/ dl group (35%>reduction). summary/conclusions: anemia presence before elective hip surgery is a risk factor for bt requirement and longer hospitalization. diagnosis and management of anemia using timely pre-surgery consultations may minimize intraoperative bt, particularly in women and patients with comorbidities. real-patient data and prospective trials are warranted. abstract withdrawn. abstract withdrawn. background: cd36, known as platelet glycoprotein iv, belongs to type b scavenger receptor and is related to the pathogenesis of many diseases. type i cd36 deficiency was cd36 not expressed on platelets and monocytes. individuals with type i deficiency can produce homologous antibodies and cause related immune diseases. in recent years, it has been reported that cd36 deficient individuals cause fetal immune thrombocytopenia with fetal edema syndrome in asia. cd36 is not expressed in mature rbc, but exists in hematopoietic stem (progenitor) cells. anemia is an important cause of edema. in view of the phenomena of clinical and animal experiments, cd34 + hematopoietic stem (progenitor) cells were cultured in vitro to observe the effect of anti-cd36 monoclonal antibody on cd34 + hematopoietic stem (progenitor) cells. aims: to investigate the effect of anti-cd36 monoclonal antibody on proliferation and differentiation of human cd34 + hematopoietic stem (progenitor) cells in vitro. methods: choose 3 healthy full-term maternal women without various obstetric complications, take cord blood 20 ml. after density gradient centrifugation of ficoll cell separation solution, cd34 + hematopoietic stem (progenitor) cells were sorted by flow cytometry and cultured for 2-3 generations. mtt was used to examine the effect of anti-cd36 monoclonal antibody on the growth of hematopoietic stem (progenitor) cells. flow cytometry analysis was used to detect the apoptosis and cell cycle of cd34 + hematopoietic stem (progenitor) cells. the effect of anti-cd36 monoclonal antibody on the formation of cfu-e/bfu-e in hematopoietic stem (progenitor) cells was analysis by cfu-e/bfu-e account after 10-14 days culture. results: after umbilical cord blood was isolated by ficoll to obtain mononuclear cells, the hematopoietic stem (progenitor) cells of cd34 + were sorted by flow cytometry, and about 0.44% of cd34 + hematopoietic stem (progenitor) cells were isolated. different concentrations of anti-cd36 monoclonal antibody and hematopoietic stem (progenitor) cells were cultured in vitro. the od value of value (0.9 ae 0.15) of anti-cd36 monoclonal antibody group (2 mg/ml) was decreased than normal group (1.05 ae 0.12) (p < 0.05), and the od value (0.81 ae 0.11) was significantly decreased at the cd36 monoclonal antibody concentration of 32 mg/ml (p < 0.01). there was no significant difference between the hematopoietic stem (progenitor) cells culture group and the igg control group (p > 0.05). in the annexin v flow detection, the apoptotic rate of anti-cd36 monoclonal antibody group (2 mg/ml) was statistically increased than the normal group (p < 0.05). anti-cd36 monoclonal antibody significantly induced hematopoietic stem (progenitor) cells to undergo s phase cell reduction, g1 phase cells increased, and g1/s phase cell arrest occurred. the number of cfu-e/bfu-e clones in the normal group was 169 ae 12, the number of cfu-e/bfu-e clones in the control group was 172 ae 12, and the number of cfu-e/bfu-e clones in the anti-cd36 monoclonal antibody culture group was 85 ae 6. the number of colonies formed by hematopoietic stem (progenitor) cells in the anti-cd36 monoclonal antibody culture group was significantly lower than that of the other groups (p < 0.05). summary/conclusions: anti-cd36 monoclonal antibody can reduce the proliferation of human cd34 + hematopoietic stem (progenitor) cells and reduce the ability of erythroid differentiation. background: recently the new modern collection techniques were introduced in the apheresis procedures. cobe spectra system was replaced with spectra optia, and it was necessary to verify the efficiency of spectra optia in pbpc collections. aims: the aim of the study was to evaluate and optimize the new cmnc protocol spectra optia v. 11 (terumo) for pbpc collections in patients with haemato-oncological malignant diseases. methods: the results of 159 autologous pbpc collections were evaluated in: (a) well mobilized patients with precollection cd 34+ cells concentration in blood higher than 20/ll, (b) from only the first collections, which were performed either by the use cmnc spectra optia v. 11 or cobe spectra v. 6, v. 7, terumo (c) collections were performed in the standard and large volume leukapheresis regimen, lvl. engraftment data in 56 transplanted patients were assessed. results: standard collections were performed in 52 patients. the yield of cd 34+ cells was high, and no significant differences were found between the numbers of cd 34+ cells prepared from spectra optia 8,6 (1,3-41) 9 10 6 and cobe spectra 10,9 (1, 6 ) 9 10 6 /kg b. w. (a = 0,05; pval 0,619). the dependence of cd 34+ cell yield on the precollection concentration of cd 34+ cells in blood can be considered as linear with high correlation coefficients in cmnc spectra optia r = 0,95, and cobe spectra r = 0,93. lvl collections were performed in 107 of patients, and there were no significant differences between the numbers of cd 34+ cells prepared by cmnc spectra optia 10,9 (2-61,2)9 10 6 and cobe spectra 9,3 (2,4-86) 9 10 6 /kg b.w. (a = 0,05; pval 0,35). the relations between the precollection cd 34+ cells concentration in blood and the numbers of cd 34+ cells from collections can also be considered as linear with the correlation coefficients in cmnc spectra optia r = 0,93, and cobe spectra r = 0,78, respectively. in lvl, the median platelet loss was significantly lower in cmnc spectra optia (45%) than in cobe spectra (57%). a group of 12 patients was transplanted by means of pbpc prepared in the standard regimen. median time in the neutrophil reconstitution was in cmnc spectra optia as well as cobe spectra 11 days, while in platelets from cmnc 14 days, and from cobe spectra 12 days, respectively. the number of 44 patients obtained pbpc from lvl. the median time in neutrophils and platelets reconstitution was in cmnc spectra optia as well as cobe spectra the same, and corresponded with 11 and 13 days, respectively. summary/conclusions: cmnc protocol spectra optia is a modern, efficient and the safe system, which can be used for both standard and lvl procedures. in well mobilized patients the sufficient dose of cd 34+ cells for transplantation could be prepared from one standard or one lvl procedure. no serious adverse reaction have been observed. background: peripheral hematopoietic stem cells are collected from patients/donors after mobilization with g-scf. the aim of the collection is a fixed number of cd34 + cells/kg. this number depends on the pre-apheresis cd34 + number, the blood processed and the collection efficiency of the procedure. the aim should be to collect all the requested cells in 1 day, whenever possible. this is in order to reduce the dose of g-csf given to donor/patient and the resources used in the collection centre. the only parameter that can be adjusted is the volume of blood processed, if this is increased, the likelihood of collecting the requested amount of cells is increased, but only if the pre-apheresis cd34 number is high enough. therefore, you need to know, when it is feasible to increase the volume and thereby increasing the time of the procedure with the intention to collect all the requested cells in 1 day. it can also show if it is possible to reduce the volume of blood processed, thereby reducing the time of the procedure. aims: to develop an easy tool to calculate the volume of blood processed in order to collect the requested cells in 1 day. methods: the mean collection efficiency (ce) was calculated. ce is calculated as cd34 + cells collected/(pre-apheresis cd34 + number*processed volume)*100%. based on the mean ce, an excel sheet was generated to calculate the volume of blood that should be processed in order to collect all the requested cells. the excel sheet is designed so the user enters the pre-apheresis cd34 + number, patient weight and the requested number of cd34 + cells. the ce is fixed according to the mean ce calculated. the result is the volume of blood processed in order to collect the requested yield. based on that result, the apheresis machine will provide time for the procedure, thereby it is possible to evaluate if the collection can be finished in 1 day or not, e.g. by increasing the volume of blood processed. spectra optia â was used for all collections, cmnc for allogeneic donors and mnc for autologous patients. results: mean ce = 64% (n = 348). a ce of 40% was chosen as the cut-off for the cd34 calculation tool. using the cd34 calculation tool: allogeneic donors (n = 61): mean ce = 61%, mean blood volume processed = 3.3 9 tbv, mean time: 253 min, 37 donors were finished in 1 day collection (61%) autologous patients (n = 205): mean ce = 65%, mean blood volume processed = 2.4 9 tbv, mean time = 218 min, 173 patients were finished in 1 day (83%). the calculation failed in only 1 case (0.4%). in this case the volume of blood processed was reduced according to the calculation, but because of unexpected low ce, the requested number of cells was not achieved. summary/conclusions: the cd34 calculation tool based on an excel sheet has shown to be simple and easy to use in order to personalize the stem cell collection. immunotherapy products: blood product, pharmaceutical, or a new category all together? from 2001 till 2019. all donors were hla typed and matched; they were fully informed on the donation procedure and signed an informed consent for donation. minimum dose required to ensure successful and sustained engraftment was 2 9 10 6 /kg cd34 + cells and 2 9 10 8 /kg mono-nucleated cells (mnc). pbsc harvesting was performed with continuous flow cell separator baxter c53000, cobe spectra and spectra optia using conventional-volume apheresis processing the 2-2.5 total blood volumes per apheresis. a femoral catheter was used for harvesting and acid citrate dextrose formula a is used for anticoagulation. recombinant human granulocyte colony-stimulating factor (g-csf) is used to mobilize pbpc for collection. harvesting of pbsc is usually performed after 4 to 5 days of g-csf subcutaneous administration at a dose of 10 lg/kg body weight. results: all the donors were siblings of the patients treated at the university hematology hospital. there were 159 apheresis procedures performed in 106 healthy sibling donors. there were 75 males and 31 females, aged 20-55. one to two apheresis procedures were required to collect adequate graft. the single procedure usually took 3-4 h and the volume of collected stem cells was 50-220 ml. the needed number of mnc and cd34 + cells was successfully collected by 1.5 apheresis. there were 29 abo incompatible donors. procedures for mobilization and collection of pbpc from healthy donors are generally well tolerated. the only adverse effects of the apheresis procedure were bone pain as reaction of g-csf and numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and were very mild. the collected pbsc were used in allogeneic stem cell transplantation in patients with: acute myeloid leukemia -57 patients (53.7%), acute lymphoblastic leukemia -14 patients (13.2%), chronic myeloid leukemia -9 patients (8.5%), myeloproliferative disorders -8 patients (7.5%), myelofibrosis -5 patients (4.7%), severe aplastic anemia -5 patient (4.7%), non-hodgkin lymphoma -3 patient (2.8%), multiple myeloma -3 patient (2.8%), chronic lymphoblastic leukemia -1 patients (0.9), hodgkin disease -1 patient (0.9%). summary/conclusions: the apheresis collection of pbsc in healthy donors is an effective and safe procedure. we are developing our national stem cell donors registry as a part of bone marrow donors worldwide. in that way we hope we will help widen the world network of stem cell donors and enlarge the possibility for each patient to find the right match. background: leukocyte-removing filters for blood are being used widely as a universal leukocyte reduction policy is being adopted progressively throughout the world. filtration is one of the most effective methods for preventing various adverse transfusion effects caused by leukocytes included in blood components, such as febrile reaction, alloimmunization, and transmission of leukotropic viruses. aims: the goal was to evaluate whether the new domestic blood filter finecell, developed by kolon industries, gumi, korea, is appropriately suited to the international standards. and to reveal its efficacy and safety in the settlement of leukocyte reduction system in korea. methods: thirty-two units of packed red blood cells obtained from 400 ml whole blood collected from 32 healthy donors were used. this was done by analyzing the filtration time, residual leukocyte count, rbc recovery, and hemolysis rate during a storage period of 35 days after leukoreduction. results: the standards commonly used for the evaluation of leukocyte-removing filters are set by the food and drug administration of the usa and the council of europe. the results of our study satisfied these international standards. summary/conclusions: the newly developed domestic leukoreduction filter was, thus, found to be efficient and will contribute to the improvement of the quality of isolated blood components used in korea. faculty of science, humanities and education, technical university of liberec, liberec, czech republic background: as polymeric fibrous scaffold fabrication techniques strive to create structures that more closely replicate tentative extracellular matrix form and function, the need for increased scaffold bioactivity becomes more pronounced. the fibrous structure made from biocompatible and nontoxic polymers ensures mechanical stability, however cell proliferation requires further stimulation. platelet-rich plasma, which has been shown to contain over 300 bioactive molecules, has the potential to deliver a combination of growth factors (gfs) and cytokines capable of stimulating cellular activity. aims: the presented work deals with the preparation of nanofibrous materials with platelet growth factors incorporated into the internal fiber structure. polyvinyl alcohol (pva) was used for the preparation a material providing a progressive release of native gfs without need of subsequent crosslinking. methods: materials were prepared from pva (mw 125,000, 98% of hydrolysis) using electrospinning technology (nanospider tm 1ws500u). platelet lysate (pl) was prepared from thrombocyte rich solution (obtained from regional hospital in liberec, the concentration of 700-900 x10 6 plt/ml, freeze-thaw method with subsequent centrifugation). nanofibers were electrospun from 10% pva solution using water: ethanol (8:2) solvent system. materials with proteins were electrospun from solution containing 10% of pva and 10% of pl. morphological analysis was performed by scanning electron microscopy. protein release was monitored using spectrophotometry (bradford method) and chromatography. results: the prepared fibrous materials consisted of random oriented end-less fiber with smooth surface with minimal defects in structure. the morphology of materials was not altered by the addition of proteins. the average fiber diameter was: 340 ae 120 nm for pristine pva fibers and 350 ae 148 nm for pva with incorporated proteins (pva/pl). pva/pl layers contain 5-10 mg of protein per gram of pva. 60% of the proteins are released during the first day (burst release) followed by a gradual release of up to 2 weeks. summary/conclusions: nanofibrous pva-based nanofiber materials were prepared with native growth factors. the process used for the preparation of solutions and subsequent spinning does not affect the activity of the incorporated proteins, which are being gradually released. therefore, we believe that the developed material has great potential for use in tissue engineering e.g. to promote healing of chronic wounds. acknowledgements: supported by the czech health research council, project no nv18-01-00332. background: human a-defensins are small cationic peptides with antimicrobial and anticancer activity. up till now, six a-defensins have been described in humans. they include the human neutrophil peptides (hnp) 1, 2 and 3 which present in large amounts in neutrophil azurophilic granules and differed from each other only in the first amino acid. a fourth defensin, termed hnp-4, comprises less than 1% of the total defensins in neutrophils and has a distinct sequence from hnp1-3. the other two, human defensin 5 and 6, are synthesized mostly by intestinal paneth cells. neutrophil defensins (hnp 1-3) are 3.4 kda peptides that are characterized by three disulfide bridges. the pattern of disulfide bonds in the mature forms is crucial for the functional properties. due to this structural feature, synthesis of defensins using the chemical and recombinant approach presents quite a challenge. moreover, purification from the natural source can be very difficult because the large number of neutrophils is needed to obtain a sufficient amount of protein. in blood banks, leukocyte reduction filters are used to remove leukocytes from blood components in order to prevent adverse transfusion reactions. leukofilters contain high numbers of normal human cells and discard after use. aims: the aim of this study was to purify a-defensins from neutrophils trapped in leukocyte reduction filters. methods: blood bags from healthy blood donors were collected after written consent. all donors were screened for infectious diseases (hbv, hcv and hiv) and negative samples were included in the study. blood bags were filtered at 22°c by leukoflex lst-1 filters. the cells were extracted from the filters by back-flushing with cold phosphate buffered saline (pbs), ph 7.4, without mgcl2 and cacl2, containing 5 mm edta and 2.5% sucrose. the pbs was homogenized with the filter content and then collected in a sterile tube. neutrophils were separated from mononuclear cells by ficoll. isolated neutrophils resuspended in pbs 1x at a concentration of 1 9 10 7 cells/ml. for degranulation, cells were stimulated with 100 nm of formylmethionyl-leucyl-phenylalanine (fmlp) for 5 min followed by stimulation with 10 lm of cytochalasin b for 5 min. supernatant was collected by centrifugation at 200 9 g for 6 min. supernatant was incubated with mouse monoclonal antibody to hnp 1-3 and purification of hnp 1-3 was performed by lmacs protein g microbeads system. the presence of protein was confirmed by western blot. results: the presence of the 3.4 kda band was confirmed by western blot, which corresponded to the size of the a-defensins. summary/conclusions: the development of defensins as therapeutic products requires access to a steady supply of neutrophils. our results indicated that lst-1 filters are economical source for purifying a-defensins. anatomical sciences, abadan school of medical sciences, abadan, iran background: epigenetic reprogramming of terminally differentiated cell can modify somatic cells to a pluripotential state. there are several approaches that induce pluripotency in somatic cells. exposure the cells with the embryonic stem cell extract is an easy way, and some investigations were done on fibroblast cell line. however, its efficiency was low aims: the purpose of this study was to increase the number of reprogrammed granulosa cell as a full differentiated cell into pluripotential state methods: the human granulosa cells were cultured in the medium containing 5-aza-deoxycytidine and trichostatin a. then, the cells were exposed to mouse escs extract and co-cultured with mouse embryonic fibroblast in the presence of leukemia inhibitory factor (lif). alkaline phosphatase test and also immunohistochemistry staining for oct4, sox2 and nanog were performed after 24 and 72 h and 1 week results: the results indicated that after 24 h the granulosa cells were revealed a round and expanded morphology. the cells in all groups except in negative control, were showed alkaline phosphatase activity. the cells that were cultured in medium containing 5-aza-deoxycytidine and trichostatin a and exposed to the extract had the most numbers of alkaline phosphatase positive cells. immunocytochemistry showed the granulosa cells that were cultured in medium containing 5-aza-deoxycytidine and trichostatin a with extract expressed oct4 with weak intensity after 24 h. no expression of oct4, sox2 and nanog were observed in other groups at the same time. after 72 h, oct4, sox2 and nanog were over expressed in the cells that were treated with 5-aza-deoxycytidine, trichostatin a and extract. furthermore, there was high expression of oct4 in the granulosa cells that were cultured in medium containing dmso and exposed to the extract. after one week, the expression of oct4 and sox2 in the granulosa cells that were cultured in medium containing dmso and exposed to the extract was continued while its expression ceased in the other groups. the expression of nanog were ceased in all groups after one week summary/conclusions: present study revealed that the inhibitors of the methyl transferase (5-aza-deoxycytidine) and histone deacetylase (trichostatin a) could delete the epigenetic markers and improved cells reprogramming by administration of the extract abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mesenchymal stem cells (mscs) are adherent spindle shape cells expressing different surface markers. they show special criteria including, paracrine effects, differentiation to several tissue cells, migration, immunomodulatory and regenerative potentialities. mscs are isolated from different sources and employed as therapeutic tools to treat several diseases and injuries. however, some of mscs properties including secretion of growth factors and migration ability are controversial especially during remission or in presence of tumor. interestingly, msc-derived compartment could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. exosomes are kind of extracellular vesicles (evs) characterized via their size and releasing mechanism. usually they defined as less than 150 nm in diameter vesicles. they secreted from different cells and are also found in urine, blood, breast milk, cerebrospinal fluid and other body fluids. exosomes contain genetic material including dna, mrnas, micrornas (mirnas) and other biomolecules. mirnas are single stranded non-coding rnas transcribed from dna. immature mirnas are subjected to two known cleavages to modify to mature mirna that involve to either mrna degradation and gene expression process or cell-cell interaction and communication via secretion as the part of exosomes. aims: this study was aimed to discuss some aspects of exosomal micrornas derived from mscs in progression, diagnosis and treatment of some diseases. methods: different scientific data bases including pubmed, google scholar and scopus were used to find and review related articles. results: evs play important role either in intercellular communication related to pathological and physiological situation or intracellular communication, angiogenesis, immune system modulation and metastasis progression. mirnas could regulate expression of multiple mrnas then they play important role in different biological processes and contribute cell-cell interaction as well as influence in the progression of different disease. exosomal mirna-derived mscs are involved in cancer procession, tumor growth, angiogenesis and metastasis. they are used as diagnosis and therapeutic tools to treat different diseases such as renal failure, liver fibrosis, myocardial infarction. summary/conclusions: due to controversial aspect of using of intact mscs especially during remission or in presence of tumor, msc-derived exosome could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. aims: the aim of study try to use sybr green i based real-time pcr to identify homozygous, heterozygous, gene deletion or wild type for rhd exon 1, 5, 10 and 1227a. methods: for this study, we used real-time pcr with high resolution melting curve mode, and matrix mix containing sybr-green i were used for sequence specific primers of 1227 g>a and rhd exon 1, 5, 10. samples with rhd gene deletion homozygous/heterozygous, 1227 g>a heterozygous with rhd gene deletion and normal rhd, normal rhd homozygous/heterozygous and rhd1-rhce(2-9)-rhd10 homozygous/heterozygous were enrolled in our study. all samples were screened using rhd exon genotyping, sanger sequencing and rhesus box analysis. concentration and mass of dna samples were 1 in alleles of normal rhd/rhd gene deletion. the tm ratio of rhd exon 1 (87°c) to internal control (77°c) were 2.33 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 2.09-2.21 in alleles of rhd gene deletion/normal rhd, 2.53-2.66 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. the tm ratio of rhd exon 10 (81°c) to internal control (77°c) were 3.35 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 4.29-4.39 in alleles of rhd gene deletion/normal rhd, 4.67-6.10 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. the tm ratio of rhd exon 5 (81°c) to internal control (77°c) were 3.64 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 2.49 in alleles of rhd gene deletion/rhd1-rhce(2-9)-rhd10 3.47-3.59 in alleles of rhd gene deletion/normal rhd, 3.97-4.77 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. summary/conclusions: using the tm ratio of sequence specific primers to internal control is an effective way to detect the rhd gene deletion or rhd weak d types 1, 2 and 3 not detected") were tested with a method based on next generation sequencing (ngs) using the illumina miseq platform to detect a possible rhd variant not interrogated by id rhd xt. results: in total 583 dna samples were tested in 87 pools. fifteen (15) pools (181 samples) gave rhd deletion genotype and seventy two (72) pools (402 samples) resulted to the presence of rhd gene. the 72 positive pools were also analyzed individually. the genotype results obtained were: rhd exon 1 no amplification (1), rhd exon 6 and the genotype results obtained with id rhd xt (in pools and individually) were concordant with the results provided by the centers. hence, 100% accuracy was obtained using id rhd xt with dna pooled samples. the results of rh ngs for the samples with inconclusive results by id rhd xt showed rhd variants previously described: 1 sample rhd*93-94inst (del), 1 sample rhd*ivs3+1g (del), 9 samples rhd*weak d type 11 (partial d), 1 sample rhd*weak d type 5 (weak d), 1 sample rhd*weak d type 61 (weak d) and not described: 1 sample rhd*del 1-5 (unknown) summary/conclusions: id rhd xt is a high accurate tool for genotyping the most common rhd alleles associated to weak d and d negative phenotype in up to 20 pooled dna samples. use of rhd genotyping improve rhd typing in blood donations variant rhd alleles generate qualitative/quantitative alteration in serological expression of d antigen such as weak and partial rhd phenotypes which are clinically important in transfusion setting. population studies have shown varied distribution of the variant d alleles in caucasians, africans, east asians and indians. many countries have developed their own population-specific strategy for identifying d variants. our previous study in indian population showed absence of weak d type 1, 2, and 3 which are commonly found in caucasians d variant individuals. instead, a novel population-specific pattern i.e.~12-kilobase duplication event, including exon 3, was predominantly identified in 58.3% d variant samples. functional analyses showed that this genetic variation results in the expression of several transcripts, including a wild-type product. commercial genotyping assays available, mainly detect common d variants found in caucasians and africans, thus limiting its usefulness in india. hence, based on our findings we have designed a multiplex pcr assay specific for indian population that can be easily implemented at the laboratory level for genotyping variant rhd. aims: to characterize rhd variants using "indian-specific, rhd genotyping assay". methods: seventy samples referred to our laboratory for molecular characterization of rhd variants were included in this study. all rhd variant samples were serologically typed for results: out of the 70 rhd variants included in this study, 49 samples (70%) showed presence of indian specific allele i.e. exon 3 duplication. seventeen rhd variants samples showed presence of both exon 5 and 10. qmpsf analysis of these samples excluded involvement of rhd-rhce-rhd hybrids. sixty of the seventy d variant individual had r 1 r genotype this assay thus can be used routinely in indian laboratories to identify and characterize rhd variants. -128 non-invasive fetal kel1 genotypes from allo-immunized anti-kel1 women were done (34 positive confirmed fetuses, 6 undetermined, 16 positive non-confirmed, 22 negative non-confirmed and 50 negative confirmed). -190 non-invasive fetal rhc genotypes from allo-immunized anti-rh4 women were done non-invasive fetal rhe genotypes from allo-immunized anti-rh3 women were done (66 positive foetuses, 1 undetermined for 21,5% of the allo-immunized women, the pregnancy was compatible and no specific antenatal monitoring was necessary summary/conclusions: non-invasive fetal red blood cell genotype is a powerful tool to diagnose a feto-maternal red blood cells incompatibility and allows to legitimize a costly and heavy specific antenatal monitoring s purchla-szepioła 1 , m krzemienowska 2 , m pelc-kłopotowska 2 , m jurkowska 3 , m debska 4 , m uhrynowska 2 and e brojer the test developed by ihtm has been offered to clinicians and pregnant women since 2016 but it is not covered by the health care system. rhd nipt is not informative for mothers with rhd variant. in such cases further analysis of the molecular background is offered to exclude from immunoprophylaxis the women with weak rhd type 1, 2 and 3. aims: summary of a 3-year period of routine rhd nipt available at ihtm. methods: cffdna isolated using easymag, biomerieux from plasma of 366 pregnant women determined with standard serology as rhd-negative (in 8-38 week of gestation) was examined for the presence of exons 5 and 7 of rhd and ccr5 by realtime pcr using lc480ii (roche). maternal dna from whole blood was tested for identification of rhd variant using rbc fluogene rbc-dweak/variant (inno-train, germany) or the home-made protocol. results: in 123 cases the rhd gene was not detected in cffdna and the administration of rhig was not recommended. in seven cases ct-values for rhd and ccr5 indicated a maternal d variant (d ct ccr5-rhd >2); the genetic follow-up of six of them identified: rhd*01w.2 in 4 cases, rhd*01w.1 and rhd*15. in 235 cases the rhd nipd results indicated that a fetus is rhd positive and rhig administration was recommended it was recommended in the remaining 67% of mothers. in 1.9% cases with maternal d variant rhd nipt was not possible. however, in 5/6 such cases the test is unnecessary because follow up analysis revealed maternal rhd variant of the weak d type 1 and 2 in switzerland extended antigen-matching for duffy, kidd and mnsbesides rhesus and kell -is recommended for sickle cell disease (scd) patients. the ethnic diversity of red blood cell (rbc) antigen polymorphism engender that these patients are often transfused with rbcs from donors of african origin. this strategy, however, increases the likelihood of being exposed to certain low-prevalence antigens, such as rh23 (d w ), as these are almost exclusive to african populations. rh23 is encoded by several types of rhd*dv as well as by dau-5. anti-rh23 is associated with delayed hemolytic transfusion reactions (htr) aims: here we report a specific low-prevalence antibody newly formed by the same patient, meanwhile gravida 4, para 1, causing positive crossmatches with the rbcs of two of the four selected donors. subsequently, advanced serologic and genetic workup and close international collaboration enabled optimal patient care. methods: standard serological methods were used for antibody specification (biorad, cressier, ch and in-house). crossmatches were carried out by indirect antiglobulin test at 37°c. molecular typing of donors' and parental blood group antigens was performed by further serological analysis (institute national de transfusion sanguine, paris) revealed an anti-rh23 in addition to anti-fy 5 , anti-e and anti-jk a . genotyping of the two donors causing positive crossmatches presented heterozygosity for rhd*10.05 which encodes rh23. the newborn's phenotype was a r0r k-, fy(a-b+) and most likely rh23-and jk (a+b+), considering both maternal and paternal (a r0r, k-k+, fy(a-b+), jk(a+b-), rh23-) predicted phenotypes. the neonatal serum contained maternal anti-a1, anti-rh23 and anti-e. the direct antiglobulin test was positive but elution only showed nonspecific reactions with papain-treated cells. latter might have been caused by anti-fy 5 during her present pregnancy we were able to demonstrate that two positive crossmatches of two former compatible donors were caused by a new alloantibody against a low-prevalence antigen, namely anti-rh23, derived from several rh23 + rbc transfusions during the previous pregnancy. despite this challenging blood supply we were able to support the patient with a total of ten antigen compatible and crossmatch negative rbc units from french and swiss donors until delivery with increasing age, the relative number of women in the study population raise from 22% in the patients younger than 60 years to 58% in the patients older than 80 years. our study showed that cardiovascular diseases were the commonest indications for warfarin use in older patients with 59%. only 48,5% achieved target therapeutic range while the risk of thromboembolism and the subsequent need for proper anticoagulant therapy increases sharply with age, the bleeding risk rises as well. older patients are more sensitive to any given dose of warfarin and need a significantly lower total weekly dose. a well-informed patient provides one of the best defenses against bleeding complications. recent data demonstrate doacs advantages over warfarin, especially for older population: more predictable dosing, fewer drug interactions and reduced risk of intracranial bleeding although vast majority of fh cases are caused by mutations in ldl-r gene 17%-33% patients do not harbor genetic cause in the known loci. patients with homozygous/severe heterozygous fh are unresponsive (ldlc above 200 mg/dl with diet and drug therapy) and require additional extracorporeal therapy to reduce ldlc concentrations to prevent the development/progression of cad. ldl apheresis techniques remove apolipoprotein b-containing lipoproteins from blood and include heparin-induced extracorporeal ldl precipitation(help), immunoadsorption, dextran-sulfate adsorption methods: a 28y iraqi male visited cardiac-opd. ct coronary angiogram showed cad-dvd. he had multiple tendinous xanthomas and xanthelasmas. family history was significant for death of elder brother from coronary event at 30y, a sister with similar profile age 26y and one sister apparently normal. he was taking medical treatment for dyslipoproteinemia (ecosprin 75 mg od, ticagrelor 90 mg bd, rosuvastatin 40 mg od). despite dietary and medical treatment his dyslipoproteinemia was refractory. therefore cascade-filtration was planned with evaflux5a plasma fractionator. one procedure of cascade plasmapheresis was done on com.tec apheresis system (fresenius kabi, germany) separating patient's plasma as the first step and passing it through a pore sized based filter column 5a20 (evaflux, kawasumi, japan) as the second step. a total of 1.5x plasma volume(4470 ml) was processed. the patient was given continuous calcium infusion. the flow rate of 50 ml/min was maintained immunoglobulins) were not assessed. summary/conclusions: the procedure successfully met the requirement of reduction of cholesterol by 60%. the patient became responsive to the medical treatment. follow up of the patient up to a year has been uneventful with no additional procedure requirement actions included development of major haemorrhage protocols with improved communication and required instances of delayed transfusion to be reported to the uk national haemovigilance scheme (serious hazards of transfusion, shot) methods: delayed transfusion data have been collected from 2010. hospitals identify incidents and report them via an online database. mh may also result in avoidable or overtransfusion. reports are analysed and collated data published in the shot annual reports in july each year. results: the total number of reports of delayed transfusion has increased with time: 101, 95, 111 in the last 3 years. delayed transfusion in relation to mh was reported for 54 cases 2016-2018 contributing to death in 6 patients 7%) miscommunication was noted between clinical different teams, between emergency departments, porters and the transfusion laboratory, failure of bleeps, failure to communicate the urgency, failure to confirm the patient location. failures to follow mhp correctly occurred in 31/71 (43.7%): incorrect activation including failed alerts to porters, wrong contact telephone details and wrong components in the mhp packs most transfusions included red blood cells (median, 2 units); 14% of women were transfused with fresh frozen plasma (median, 2 units) and 2% with platelets. mean pre-and post-transfusion hemoglobin levels were 6.7 g/dl and 9.0 g/dl, respectively, representing an increment of 1.0 g/dl per rbc unit transfused (1.09 g/dl in soweto and 0.83 g/dl in durban). indications for transfusion included obstetric hemorrhage (31%), chronic anemia (29%), surgery or anesthesia (13%), other (9%) and not specified (17%). transfusion for chronic anemia (vs. hemorrhage) was associated with gestation ≥ 26 weeks (odds ratio = 7.07, 95% confidence interval 3.52-14.24). surgical blood loss was a common indication in trimester 1 (21%) that declined to 7% then 1% in trimesters 2 and 3. summary/conclusions: hemorrhagic complications accompanying spontaneous abortions and ectopic pregnancies in the first and second trimesters were the most common reasons for antenatal transfusion surveillance and analysis of blood transfusion reactions represents inseparable part of hemovigilance. aims: summarization of data on reported cases of transfusion reactions. methods: analysis of serious undesirable reactions to blood products administration in the czech republic (cr) during period 2016-2018. results: there were evaluated 1,281 688 of blood products administrations in 117,414 patients in the cr during defined three years period. announced 1,456 (0,11%) transfusion reactions including 50 severe transfusion reactions (20 9 adjudged with grade 3). the most frequent types of severe transfusion reactions: anaphylaxis 20, trali 7x, taco 5x, hcv 4x, hbc 2x, bacterial infection 1x, delayed hemolysis 1x. transfusion reactions incidence according to administered bp: red blood cells products: 882,017 administrations, 883 transfusion reactions (fnhtr 386x, allergy 227x, circulatory overload 66x, anaphylaxis 6x, trali 4x, hbv 2x, hcv 1x) platelets: 88,438 thrombocyte administrations, including 178 transfusion reactions (allergy 129x, fnhtr 30x, anaphylaxis 5x, circulatory overload 8x, delayed immune hemolysis 3x, acute circulatory overload 8x. granulocytes: 203 administrations, 2 transfusion reactions plasma: 293,801 administrations, 359 reactions reported (allergy 278, fnhr 25, circulatory overload 14, anaphylaxis 17x, trali 4x, hbv 3x, ards 1x. summary/conclusions: conclusions: comprehensive analysis and data processing help to appropriate prospective setting of blood products (bp) production and hemotherapy. concrete outputs from processed data triggered undermentioned changes in many departments in the cr: 1. plasma for clinical uses from male blood donors, 2. prestorage of leucocyte reduced blood products, 3. production of platelets in additive solutions, 4. implementation of pcr testing method for blood donors screening. background: skae's basic activities include epidemiological surveillance of all adverse events (aes) associated with deviations in the collecting, testing, processing, storage and distribution of blood and blood components that may affect quality and safety near misses" and "uneventful transfusion errors" are collected to identify preventable causes. incorrect blood component transfused (ibct) events are reported following ihn instructions. results: a total of 5 they were mainly associated with deviation in processing (22.4%) and attributed to equipment failure and materials (71%) whole blood collection, materials and distribution, as a result of product defect, equipment failure, human error and other. trend analysis showed a significantly increasing (p < .001) annual rate of total aes by 28% (95% confidence interval 26-30) ) 10% fibrinogen-depleted phpl or (3) 10% fibrinogen-depleted phpl plus heparin. internalization of fluoresceinamine-labeled heparin in stcs was investigated by flow cytometry and immunocytochemistry. all stromal cells were subjected to whole genome expression analysis (affymetrix human gene 2.1 st array) and data were analyzed using r/bioconductor and panther analysis tools. confirmative qrt-pcr was performed and protein levels of selected pathways were analyzed by a bead-based western blot system (digiwest â ). immunophenotyping, in vitro differentiation, longterm proliferation and colony forming units (cfu) assays were done for all cell types. results: in vitro exposure of heparin induced differential internalization and lysosomal accumulation in stromal cells, as well as regulation of distinct gene sets, both in a tissue-source dependent manner. affected signaling cascades were mainly involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis. the influence of heparin on protein expression and phosphorylation of four pathways (wnt, pdgf, notch and tgfbeta) was further analyzed, revealing most alterations in bm-stcs. independent of origin and medium composition, flow cytometry analysis revealed the characteristic fibroblastoid phenotype profile (cd73+/90+/105+ and cd14-/19-/34-/45-/hla-dr-). in addition a comparable osteogenic and adipogenic differentiation capacity was found summary/conclusions: internalization of heparin in lysosomes by stromal cells, differential gene and protein expression and phosphorylation changes were observed in a tissue-source dependent manner. however, stromal cell characteristics as immunophenotype pattern, long-term proliferation, clonogenicity and in vitro differentiation were unaffected, putatively by post-translational protein modifications. in this respect, application of porcine heparin is compatible with efficient manufacturing of stromal cell based medicinal products abo incompatibility may have no effect on the clinical outcome after allogeneic hematopoietic stem cell transplantation. however, it carries additional risks of hemolytic reactions, delayed red blood cell (rbc) engraftment and incidence of graft-versus patients were categorized according to abo compatible and mismatched groups; these were further sub-categorized into major, minor and bidirectional. direct coombs test (dct) was performed when hemolysis was suspected in the post-transplantation period along with serum lactate dehydrogenase (ldh) 5%) were male and 31(36.5%) female. mean age of abo matched and mismatched groups were (6.99 ae 4.6) years. most common indications for transplant included beta thalassemia major 53(60.2%), aplastic anemia in 25(28.4) and pure red cell aplasia 2(2.35%). source of stem cell was bone marrow in 45 and peripheral blood 40 patients abo matched while abo mismatched group comprised of 28(32.9%) patients with further subdivision into major (n = 13), minor (n = 13) and bidirectional in the post transplantation period, packed red blood cell and platelets were transfused in matched group (n = 401) and (n = 1837) comparably(n = 102) and (n = 480) in mismatched group. primary and secondary graft failure in matched group was 8.77% and 12.28% patients while in mismatched group graft failure was observed in 4(14.28%) patients respectively. positive dct in abo matched group in 1(1.75%) patient, whereas 2(7.14%) patients with major and minor abo mismatch group with raised ldh levels and deranged lfts were found. episodes of acute and chronic gvhd in abo compatible and incompatible groups were insignificant. overall survival in abo summary/conclusions: these results indicate that abo incompatibility does not seem to influence outcome of the patients undergoing allogeneic hematopoietic stem cell transplantation. careful monitoring of patients can help detect problems early and treat them efficiently, thus, reducing the number of life threatening events a picascia 1 , c sabia 1 , f cavalca 1 , g nicoletti 2 and c napoli 1 in our routine work with one lambda sab class ii reagents, we observed non-specific reactivity with some beads bearing dr4 and dr16 in patients without sensitizing events. this pattern was not confirmed by testing same sera with screening-and pra-beads suggesting non-specific reactions. aims: here, we sought to determine if fetal bovine serum (fbs) treatment would be effective in reducing/eliminating non-specific reactivity. methods: we tested 5 sera pre-treated with fbs from non-sensitized patients that showed the dr4/dr16 pattern. in particular, 5 ll of fbs was added to 95 ll of patient serum; incubated for 30 min at 37°c; centrifuged and subsequently tested in the sab assay. as controls, we treated sera from 5 patients with documented dsa including dr4/dr16 and 5 patients without hla antibodies. results: dr4/dr16 non-specific reactivity was eliminated or significantly reduced after fbs treatment. we found that patients with dr4 and dr16 dsa had no change in mfi values and additional reactivity was not observed in negative fbs treated sera transfusion medicine, national blood transfusion centre 2 transfusion medicine, national blood transfusion center 3 transfusion medicine, national blood transfusion centre, tirana, albania background: abo blood group, has been associated with many diseases, although the explanation for abo's blood group association and some illnesses is still unclear. aims: to find the distribution of cases by blood groups in patients with malignant pathology compared to donors in order to assess the presence of the abo blood group as an epidemiological indicator to identify populations exposed to different malignant pathologies methods: we conducted a case-control study. abo blood group and diagnosis of all patients have been studied. the control sample was collected from 17,992 healthy donors from which group a (36,8%), group o (41,7%), b (16,2%) and group ab (5,3%) resulted. the study was conducted in 3727 patients who have been transfused and submitted a request to determine the blood group at the blood bank at qsut during the period 2011-2017 results: among the 3727 patients, when all malignant pathologies were taken together, the highest frequency was seen in blood group a (39.7%), followed by 0 (39.5%), b (15.2%) and ab (5.4%). group a frequency was higher and o was lower compared to controls. a high incidence of blood group a is seen in: pancreatic cancer a (42%), in gastric cancer a (43%), colorectal cancer a (39, 6%), breast cancer a (41%), cervical cancer a (43%) and ovarian cancer a (42%) versus a (36.8%) in the control group. a high incidence of blood b is seen in multiple myeloma b (22%) and cervical cancer b (20%) versus b (16.2%) in the control group. blood group ab has a high incidence in malignant lymphoma ab (10%) versus (5.3%) in the control group summary/conclusions: it appears that individuals with blood groups a, b and ab are more at risk of developing malignant pathologies and individuals with blood group o are more protected. background: the high homology and opposite orientation of rh genes promote many rearrangements between them and generate a large number of rhd and rhce variants which can be inherited together. several studies have shown that those rh variants in patients with scd represent an additional risk for alloimmunization and delayed hemolytic transfusion reactions (dhtrs), but little clinical or biological evidence related to alloimmunization and dhtr are presented for all the rh variant alleles. it is well established that transfusion recipients with the most common weak d types 1, 2 and 3, are not at risk for forming alloanti-d when exposed to conventional rhd-positive rbcs. aims: we report here a case of a 12-year-old patient typed as weak d type 3, receiving d+ rbc units who presented anti-d in his plasma detected three weeks after the last transfusion. methods: rhd beadchip (immucor, nj, usa), was performed to identify the rhd variant allele associated with the weak expression of d. rhce genotyping was performed by laboratory developed tests. sequencing of rhd, rhce and rhag were performed to determine if there were other mutations that could explain the production of alloanti-d. serologic testing was by standard hemagglutination methods. the clinical significance of the antibody was evaluated by monocyte monolayer assay (mma). results: serological analysis showed a negative dat and the presence of anti-d in plasma (2+ by gel). anti-lw was ruled out. rhd genotyping revealed the patient was rhd*weak d type 3. rhce genotyping predicted the d+c+c+e-e+ phenotype. sequencing of rhd, rhce and rhag found no additional changes and confirmed the presence of rhd*weak d type 3. mma showed the anti-d was clinically significant (>5%). summary/conclusions: we report the production of alloanti-d in a scd patient with rhd*weak d type 3 allele. weak d type 3 patients are not considered to be at risk for allo anti-d but our results show that there are exceptions and that these anti-d can be associated with clinically significant rbc destruction. background: the mns blood group system is located on glycophorin a (gpa), glycophorin b (gpb) and hybrid glycophorins on the surface of the red blood cell (rbc). these glycoproteins are involved in complex structures interacting with other rbc surface proteins including the band 3/diego protein. the glycophorins are heavily glycosylated and contains multiple clinically significant blood group antigens. it has proved difficult to model the gpa extracellular structure due to its heavy glycosylation, and lack of homology with existing modelled proteins. aims: to develop an in silico model of gpa as a basis for improved predictions of structure function relationships methods: prediction of secondary structure and disorder: 1.1. predictprotein (https://predictprotein.org); 1.2. spider2 (http://sparks-lab.org/server/spider2/); 1.3. dsc (discrimination of protein secondary structure class): using an in-house implementation; 1.4. jpred4 (http://www.compbio.dundee.ac.uk/jpred4/); 1.5. raptorx (http://raptorx.uchicago.edu). prediction of secondary structure: 2.1. robetta (http://robetta.bakerlab.org/submit. jsp); 2.2. falcon (http://protein.ict.ac.cn/treethreader/); 2.3. itasser (https://zha nglab.ccmb.med.umich.edu/i-tasser/) threading methods to evaluate the quality of protein structures: 3.1. verify3d (http://servicesn.mbi.ucla.edu/verify3d/); 3.2. prosa (https://prosa.services.came.sb g.ac.at/prosa.php) protein-protein docking: 4.1. gramm-x protein-protein docking web server (http://vakser.compbio.ku.edu/resources/gramm/grammx/); 4.2. gramm (http://va kser.compbio.ku.edu/main/resources_gramm1.03.php) results: using in silico modelling we derived a stable tertiary glycosylated structure for gpa both as an individual protein and a homodimer. the hybrid glycophorin background: non-invasive prenatal testing of fetal antigen using cell-free fetal (cff) dna from maternal plasma of immunized women is widely implemented into clinical routine but the sensitivity and specificity of the method, especially for genotyping antigens encoded by single nucleotide polymorphisms such as k antigen, is limited by low proportion of cffdna in maternal plasma dna. according to literature reports detection of circulating tumour (ct)dna can be improved by selection of short ctdna fragments using automated electrophoresis methods. aims: the aim was to assess the feasibility for enrichment of cffdna fraction in maternal plasma dna by size selection using the pippin prep gel selection system. methods: plasma dna isolated using easymag (biomerieux) from rhd negative and k-negative pregnant women (n = 10) carrying fetuses with known genotype was loaded into 3% agarose gel casette (3% df marker q3, sage bioscience) and size selection of fraction was performed on a blue pippin tm (sage bioscience) with the elution from 45 min to 2 h 30 min of electrophoresis. results for real-time pcr detection of fetal rhd, kel*1 and ccr5 (as a marker of total plasma dna) in dna fraction after gel selection were compared to results obtained from non-processed plasma dna. results: the total dna level (measured by ccr5) was significantly lower in dna samples tested after gel selection (from 8.4 to 25.9geq/pcr) versus the level obtained from non-processed plasma dna (from 130 to 133geq/pcr). the results for fetal fraction (measured by rhd) from dna samples of rhd-negative pregnant women carrying rhd positive fetus tested after gel selection were from 1,5 to 6.6geq/pcr versus 10.8-11.2geq/pcr for non-processed plasma dna. results for kel*1 detection in plasma dna from k-negative pregnant women carrying k-negative fetus were kel*1-negative in dna samples tested after gel selection comparing to nonprocessed dna samples were false kel*1 positive amplification was observed. however, kel*1 detection in plasma dna from two k-negative pregnant women carrying k-positive fetus gave false kel*1-negative results in dna samples tested after gel selection comparing to non-processed dna samples were kel*1 positive genotype was obtained. the total dna level in samples from k-negative women was from 18.3 to 18.7geq ccr5/pcr after gel selection versus from 52 to 746geq ccr5/ pcr in non-processed dna samples. summary/conclusions: using the pippin prep gel selection system increases the proportion of cffdna fraction in total plasma dna by retaining long maternal dna fragments in the gel cassettes, but the protocol of gel separation dilutes the separated material decreasing the concentration of fetal dna and leading to false negative results of nipt. anti-rh1 quantification assay using ih-500 (bio-rad â ): promising results for monitoring rh:-1 pregnant women j beaud, h delaby, c toly-ndour, a mailloux and s huguet-jacquot centre national de r ef erence en h emobiologie p erinatale (cnrhp), hôpital saint-antoine, paris, francebackground: the generalization of immunoprophylaxis by anti-rh1 immunoglobulins since 1970 complicates the interpretation of the anti-red blood cell antibodies screening during pregnancy. to distinguish an alloantibody from a passive one, many laboratories in france use anti-rh1 microtitration. it is a column agglutination technology using red blood cells rh: 1, -2, -3,4,5 (r0r) . it permits to quantify low levels of anti-rh1 in comparison to a range of an anti-rh1 standard. performed since 1999 at the cnrhp and automated on evo clinical base tecan in 2008 (dilutions and distribution), anti-rh1 microtitration is well adapted to rh prophylaxis. aims: the aim of this study was to evaluate this technique on the ih-500 system from bio-rad â . methods: on ih-500, the reactivity of the bio-rad â reagents was compared with the cnrhp reagents (red blood cells r0r, anti-rh1 standard). the performances of the method were evaluated using three internal quality control (icq) (2 cnrhp home-made at 2 and 12 ng/ml and 1 bio-rad â at 12 ng/ml) on papainized r0r (plc) and native r0r (nlc). a comparison of results from patient sera ranging from 1.5 to 48 ng/ml was done between ih-500 and evo clinical base tecan. results: the results of the 3 qci are similar between the different reagents used. there is no significant difference between the 2 types of red blood cells except for the limit of detection: 1.5 ng/ml in plc -6 ng/ml in nlc. for the 3 qci, the intra and interassay imprecision based on the dilution degree show coefficients of variation between 0 and 15%, similar to those found with the evo clinical base. the correlation with the cnrhp technique performed on 50 samples in plc and 44 samples in nlc was satisfactory (deming plc: r2 = 0.80 y = 0.89x + 0.78 -nlc: r2 = 0.86 y = 1.08x-0.25). summary/conclusions: the anti-rh1 microtitration on the ih-500 offers similar performances to the method conducted at the cnrhp. the ih-500 allows automated reading of gel cards. however, it does not have a calculation or interpretation algorithm and does not directly give the concentration of anti-rh1. this final part remains manual and requires trained staff. background: haemolytic disease of the foetus and newborn (hdfn) due to maternal-foetal incompatibility has been perfectly framed for decades from the etiologic, pathogenetic and therapeutic point of view. the anti-d alloantibody is most frequently responsible for the most serious form of hdfn due to rhd incompatibility (rhdi hdfn). although immunoprophylaxis (ip) has reduced the number of cases of rhdi hdfn, this disease continues to occur and red blood cell alloimmunization still remains the most common cause of foetal anaemia. hdfn due to abo incompatibility (ab0i hdfn) is currently the most common neonatal haemolytic disease in the western world. however, only in about 1.5-2% of cases haemolytic disease demands transfusion support. aims: analysis hdfn from 2011 to 2018. methods: the hdfn's transfusional support is: intrauterine transfusion (iut) in the antenatal period; exchange transfusion (et) for severe hyperbilirubinaemia and neonatal transfusion of small volumes red cells for the newborn's late anaemia in the postnatal period. our policy for iut, for et and for the neonatal transfusion requires the use of a concentrated blood cells (ec) preferably group 0 rh negative (cde/cde) or negative for any red cell antigens if the mother has antibodies, fresh (<5 days), preferably cmv safe. for iut, the ec must be compatible with mother's plasma, must have hematocrit 70 + 5%, and irradiated. the unit for et must be compatible with the newborn's plasma, whit hematocrit 40% -60% and irradiated. the ec used in post-natal transfusions is usually divided into rates of 70 ml, hematocrit 55 ae 5%. results: in last 7 years, we calculated 59 neonates with hdfn (28 males and 31 females): 31 with rhdi hdfn, 19 with ab0i hdfn and 9 with hdfn due to incompatibility for other red blood cell antigens. we have produced 81 iut: 48 for our hospitalized patients and 33 for patients located in other hospitals. 25 of these 48 patients, who received iut, were immunized: 19 showed anti-d antibody and 6 antibodies different from anti-a and anti-b. 7, of the 31 infants with rhdi hdfn, were transfused in utero. 4 neonates on 59 (6.8%) have performed et: 1 with ab0i hdfn and 3 with rhdi hdfn; the latter had also been transfused in utero. 27 neonates on 59 were transfused after birth: 18 with rhdi hdfn, 3 with ab0i hdfn and 6 with hdfn due to incompatibility for other antigens. summary/conclusions: our case studies reflect the literature data. neonates with rhdi hdfn are the most numerous (52.7% of the total) and are those who have requested the highest blood supply both in the antenatal period (39.6%) that postnatal (9.6% performs et, 66.6% performs postnatal transfusions). neonates with aboi hdfn are 32.3%: nobody has received iut, only one has been subjected to et, and 11% has transfused after birth. patients with hdfn due to other antigens are 15%, have undergone iut 12.5% and were transfused after birth 22.2%. background: according to british guidelines on neonatal transfusion, since 2012 we shared with neonatologists a transfusion protocol for preterm babies. patients are anemic premature newborns with a gestational age ≤ 30 weeks and/or a birthweight lower than 1500 g, until 4 months of age. aims: reduce the incidence of iatrogenic anemia. methods: pre-transfusion tests were based on ab0 direct test, rh phenotype, direct and indirect antiglobulin test (dat, iat). a second blood sample was required for ab0/rh confirmation. blood transfusions were performed with 0 negative kell negative (0 cde/cde/kk) cmv negative irradiated erythrocyte concentrates (ec) of less than 5 days. ec were subdivided in 70 ml aliquots with a hematocrit of 55 ae 5%. according to the definition of "small volume transfusions", our protocol established that further four transfusions had to be delivered free of testing. after the fifth ec transfusion the supplementary release of ec was provided of type and screen (t&s) test with 72 h of validity. serological investigation and full compatibility testing were applied in the presence of a iat and/or dat positivity and in the case of mother alloimmunization. results: from october 2012 to the end of 2018, 694 premature newborns received 2328 ec transfusions within their first 4 months of life. in 51% of cases (n = 1186), transfusion requirement was comprised within the 'small volume transfusions'. another 44% of cases (n = 1035), requiring further ec administration, was requested of a blood sample for t&s determination and 5% (n = 107) for a cross-match test. in 79.4% of newborns (n = 551), being transfused within the " small volume transfusions", blood requirement of 1401 ec was fulfilled by the initial blood test (1102 blood samples). 13.3% of newborns (n = 92) received more than 5 transfusions (6-21; median = 8) accounting for 820 ec released and for this group 381 blood samples were required. summary/conclusions: with the exception of babies requiring crossmatch test, 381 blood tests were performed to sustain 643 infants transfused with 2221 units. the alternative option of omitting crossmatch test (otherwise suggested by italian directives), allowed a reduction of 75% of blood drawn without any adverse effect or incident reported. due to the relevance of anemia in premature babies, we suggest the application of this transfusion policy in all newborns in the first 4 months of life. background: glucose-6-phosphate dehydrogenase deficiency (g6pdd) is a sexlinked enzymopathy which is usually asymptomatic unless individuals are exposed to oxidative stress agents. the g6pd 202 genotype is the most common g6pd genotype in sub saharan africa (ssa). some studies have linked blood from g6pdd donors to poor outcome of a transfusion. however, there are no genetic screening programmes for blood donors in the region hence the contribution of g6pdd to the donor pool in the ssa setting had not been described.aims: this study aimed to describe the prevalence of g6pdd 202 genotype among donors in two regions in uganda. it also described the effect of g6pdd and the coinheritance of haemoglobin s and a-thalassaemia on the haematological quality of blood. methods: 3,255 blood samples from donor packs were utilized in a transfusion trial conducted in uganda, were genotyped for g6pd 202, haemoglobin s and a-thalassaemia. haemoglobin and haematocrit measurements for the donor units (packs) at the time of transfusion were used to describe the effect of g6pd 202 and co-inheritance with a-thalassaemia (n = 2,546) and haemoglobin s (n = 2,642) on the haematological quality of blood packs. a subset of 142 donor blood packs was utilized to determine the sensitivity and specificity of the carestarttm rapid diagnostic kit (rdt) for g6pdd. results: based on g6pd 202 genotyping, 10.3% (n = 274) of the blood samples used in the trial were deficient for g6pd enzyme while 5.3% (n = 142) were heterozygous. significant lower hemoglobin values were observed in red cell concentrates (p = 0.010) and whole blood (p = 0.009) donations of heterozygous g6pd 202 genotype. co-inheritance of g6pdd and a-thalassaemia resulted in significantly lower haemoglobin levels. the carestarttm rdt test was 83.3% sensitive and 0.8% specific for detecting donor blood packs with g6pdd. summary/conclusions: the prevalence of g6pdd among ugandan blood donors was similar to that in the general population. the heterozygous genotype resulted in lower haemoglobin concentration of the blood units. the use of carestarttm rdt for screening of stored blood units was not as efficient in this study hence further testing for the determination of g6pdd needs to be done on fresh samples from donors. transfusion medicine, jaypee hospital, noida, india background: during last two decade there has been a continuous remarkable improvement in desensitization therapy in high risk hla sensitized kidney recipients. in india there has been a tremendous increase in the number of kidney transplantations in patients having anti-hla antibodies (hla sensitized) with excellent success rate. aims: in present study, we are describing successful role of desensitization in 39 hla sensitized patients having preformed donor-specific hla antibody (dsa). methods: all patients were preconditioned with combined modality of a standard dose of rituximab, therapeutic plasma exchange (tpe) and low dose ivig. tpe was started using com. tec (fresenius kabi, germany) after 21 days of administration of rituximab. complement dependent cytotoxicity cross match (cdc-xm), luminex cross match with donor lysates (lm-xm, immucor inc., ga, usa) and flow cytometry cross match (fc-xm, bd facs canto ii).) was done in all cases. if any of the three tests was positive, single antigen bead assay (sab) was performed. desensitization therapy was given in all cases where dsa was detected. pretransplant tpe procedures were done until dsa (mfi < 1000) and cdc-xm became negative. cdcxm labeled positive at ≥ 10%. t-cell fcmx was considered positive above 42 mfi and b-cell fcmx was considered positive above 120 mfi. lmxm was considered positive above 500 mfi. sab was performed using lifecodes single antigen (lsa) class i and class ii kits (immucor, usa). if the specificity of anti-hla antibodies was against donor hla antigen(s) it was called as donor specific antibody (dsa). results: present study demonstrated the diagnostic and clinical superiority of adding fc-xm and lm-xm in pretransplant compatibility testing algorithm over cdc-xm. cdc-xm alone was not able to detect anti-hla antibody in 8 patients (20.5%). among the three pretransplant compatibility tests, fcxm demonstrated highest sensitivity. among the 39 cases initially screened 35 showed dsa positivity in sab. desensitization was done in those 35 cases only. in our study, sab was positive for class ii alone in 13(37%) while in remaining 22 (63%) cases it was positive for both class 1 and class ii. the number of pre transplant tpe procedures required was 6.7 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) . the mean number of post-transplant tpe sessions required was 0.7 (range, 1-6). during pretransplant and post transplant tpe procedures, five (14.3%) patients presented with allergic or hypotensive reactions which were managed conservatively. most of the patients were discharged after seven days of hospital stay whereas patients who required post-transplant tpe were discharged after a relatively longer hospital stay (mean-8.5, median-7 days). after three months, protocol biopsy was done in those cases only where post transplant tpe was required. protocol biopsy showed normal findings. in present study, the mean duration of follow up was approx 17 months with the longest duration of follow up of 36 months. summary/conclusions: in a country like india where there is a huge gap in the demand and supply of kidney and a large no. of patients waiting for a suitable organ, transplant across hla barrier could a good doable option. thorough pretransplant compatibility and tpe are essential tools to make these transplants program successful background: most transfusion-dependent chronically anemic patients are managed by simple red cell transfusions. however, some patients are not able to tolerate the additional volume associated with simple transfusions and are at a high risk of developing transfusion associated circulatory overload, if transfused with multiple red cell units. plasma-to red cell exchange (prx) is a modified procedure wherein an apheresis machine is used to remove patient's plasma, while simultaneously replacing with donor rbcs. this procedure allows for a rapid euvolemic transfusion of rbcs to patients that are severely anemic and intolerant to excess fluid volume. others as well as our group have previously described this procedure. we now summarize our institutions nearly seven years of experience performing this procedure on a routine basis. aims: to document patient experience with prx. methods: we performed a retrospective chart review of all patients that underwent prx at our institution in the last seven years. our protocol for prx has evolved during this period. currently, we perform the procedures using spectra optia (terumo bct, lakewood, co) machine using the plasma-exchange program and tubing set. if the patient's pre-procedure hematocrit (hct) is < 22%, we custom prime the tubing set with 5% albumin. the number of red cell units transfused to the patient depends on their pre-procedure hematocrit and is individualized to the patients. results: we have treated four patients with prx procedure. patient #1 is a 56-year-old transfusion-dependent male with beta-thalassemia major. the patient had experienced multiple congestive heart failure exacerbations secondary to simple transfusions and we consequently performed prx procedure, every 4 weeks, starting in 2012. the patient has completed 84 procedures with 3-4 units of washed red cells transfused to achieve a target hct goal of 45 to 57%. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient #2 was a 25-year-old female who had symptomatic anemia secondary to sickle cell disease (hb ss complicated by end-stage renal disease (esrd). she had progressively become intolerant to simple transfusions, including an episode of severe dyspnea, which required intubation. she underwent 9 prx procedures with 2-3 units of washed red cells. patient tolerated the procedures without any significant complications. however, during a different surgical procedure, she experienced cardiac arrest and subsequently passed away. patient #3 is a 33-year-old transfusion-dependent male with severe anemia secondary to sickle cell anemia (hb ss). he was intolerant to excess fluid because of esrd and congestive heart failure. he has undergone 38 prx procedures with 3-5 red cell units transfused to achieve a hct goal of 30%. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient #4 is a 48-year-old male with a sickle cell disorder (hb ss) complicated by esrd, heart failure and chronic hypoxemic respiratory failure. the patient has undergone two prx procedures with 4-5 red cell units. other than an episode of non-bloody emesis that was symptomatically treated, he tolerated both procedures. he continues to be managed on this regimen. however, the patient remains noncompliant with treatment. summary/conclusions: prx is a safe and efficient method to transfuse multiple red cell units to volume-intolerant anemic patients. background: transplanted organ failure due to antibody mediated rejection in abo-compatible organs is a serious complication with a bad prognosis. the goal treatment in these cases encompasses the following strategies: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. the 2016 american society for apheresis has assigned a category i to the use of therapeutic plasma exchange for the treatment of abo-compatible antibody mediated rejection in kidney, but a category iii to all other abo compatible organs: liver, lung, and heart. at our institution, a standardized approach for the use of therapeutic plasma exchange as a supportive intervention for abo-compatible immune mediated rejection, regardless of the organ type, has been in place since 2011. aims: a retrospective review was performed to evaluate our patient outcomes using therapeutic plasma exchange for the treatment of antibody mediated allograft rejection in abo-compatible solid organ transplantation. methods: patients used for the retrospective review were selected from an existing therapeutic apheresis list. the therapeutic plasma exchange protocol consists of: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. it is performed as follows: one plasma volume exchange is performed on days 1, 2, 3, 5, 7, 9 along with one or more of the above strategies followed by an ivig infusion. cases with allograft rejection in which plasmapheresis was not used were excluded. and t devos 7 aims: this study aimed to explore the possible causes of the decreased transfusion rate for all adult cardiac surgery patients. methods: data were collected from adult cardiac surgery patients during the mentioned time frame and were extracted from electronic patient files and a database of the department of cardiac surgery. a set of 63 variables was defined as possible confounders by a panel of experts. after discussion, 9 global variables (age, gender, duration of surgery, use of cpb (cardio-pulmonary bypass), american society of anesthesiologists (asa) risk score, type of surgery, urgency, attending cardiac surgeon and attending anesthesiologist) and 7 cpb-related variables (administration of cardioplegia yes/no (cpg), duration of cpb, circulatory arrest, hypothermia, duration of aortic cross-clamp, baseline hemoglobin and cpb-priming volume) were retained. negative binomial models for counts were used for data analysis. all analyses were performed with spss. results: 1018 patients were extracted from databases and further analyzed. the mean age of this group was 65,9 years (sd +/-14,1 years) and 67.3% of them were male. the mean duration of surgery was 256 min (sd +/-88,3 min). the decrease of perioperative rbc transfusion rate over four years was statistically significant (p < 0.001). in 2011, the mean use was 2,34 units per operation (sd +/-2,277), which changed to 1,38 units (sd +/-2,158) in 2014. three variables (urgency, attending cardiac surgeon, attending anesthesiologist) changed significantly over 4 years and were used in a multivariable model as confounders together with rbc transfusions and year. even after adjustment for these factors, the decrease in rbc transfusion rate was still statistically significant (p < 0.001). in the specific group of patients undergoing cardiac surgery with cpb (n = 640), the use of rbc was also significantly reduced (p < 0.001). in 2011, the mean use was 3,06 units per operation (sd +/-0,448) and this changed to 1,94 units (sd +/-0,167) in 2014. after correction for the 3 cpb variables that notably changed over the 4 years (cpg, priming volume and hypothermia) and the three previously defined confounders (urgency, attending cardiac surgeon and attending anesthesiologist) the reduction of rbc transfusions over 4 years still remained statistically significant (p < 0.001). summary/conclusions: our study shows evidence for a decreased rbc transfusion rate in adult patients undergoing cardiac surgery between 2011 and 2014. this tendency was also seen in the subgroup of patients undergoing surgery with cpb. possible explanations of the decrease are implementation of various established parts of patient blood management. however, a unique reason could not be identified in this study. background: growing worldwide demand for immunoglobulin products such as intravenous immunoglobulin (ivig) and subcutaneous immunoglobulin (scig) is driving plasma collection. patients with primary immunodeficiency (pid) or secondary immunodeficiency due to haematological malignancy or its treatment (sid) rely on these products to maintain therapeutic serum igg levels to minimise recurrent infection. efficacy of immunoglobulin replacement therapy (irt) in pid is well established but information on sid is limited. the different aetiologies of hypogammaglobulinaemia between pid and sid raised the question of whether sid patients on irt experience similar clinical and quality of life (qol) benefits as reported in pid patients. aims: to assess whether sid patients experience similar clinical and qol benefits while on irt as pid patients. methods: following ethics approval, data on dosage, serum igg trough levels and infection (bacterial, viral and fungal requiring treatment such as antibiotics) was collected from 14 adult pid and 13 adult sid patients from medical records and pathology reports, for their last 12 months of ivig and their first 12 months of scig. the starting and maintenance dose was 0.4 g/kg/month for ivig, transitioning immediately to 0.1 g/kg/week for scig without a washout period. a study specific questionnaire was developed to gather data on patient perceived side effects, treatment satisfaction and impact of irt on social/family life, work/study and their overall qol. paired t-test was used for parametric data and the wilcoxon signed-rank test for non-parametric data. results: sid patients were significantly older with a mean age of 62.9 years versus 43.4 years in pid patients (p = 0.007). a mean of three training session was required to reach competency in scig administration in both cohorts. there was a trend of reduced side effects on scig for pid and sid patients compared to ivig, with a significant reduction of headaches in the pid cohort (p = 0.041). the majority of patients experienced infusion site reactions, which were predominantly perceived as manageable. 55% of infections were respiratory tract infections. pid patients had slightly higher mean serum igg trough levels with scig (9.3 g/l) compared to ivig (8.4 g/l), and fewer infections on scig than ivig (mean annual infection rate of 1.64 vs 2.14 respectively). sid patients had higher mean serum igg trough levels on scig (8.4 g/l) than ivig (7.1 g/l) (p = 0.009) but experienced more infections while on scig versus ivig (mean annual infection rate of 2.15 vs 1.62 respectively). the number of hospitalisation due to infection decreased in both cohorts with scig. pid patients perceived that switching from ivig to scig improved their health and qol. in contrast sid patients perceived no improvements in health and qol. summary/conclusions: data from this pilot study suggests that the clinical and qol impact of irt in sid patients is different to that of pid patients. to support evidence based irt management and effective use of this limited and expensive blood product in sid, larger studies which account for different stages of malignancy and associated treatment regimes are required. background: there is an increasing platelet transfusion for treatment and prophylaxis of bleeding in patients with hematologic disorders and malignancies. because of limited resources, leukoreduced platelet concentrates is not yet implemented in most indonesian hospitals. in vitro platelet activation may cause morphology, functional, and ultrastructure changes. those changes will reduce the platelet viability, in vivo functions, and clinical efficacy. high platelet cd62p expression is the cause of faster platelet destruction in the reticuloendothelial systems. post-transfusion in vivo hemostatic efficacy can be determined by the measurement of corrected count increment (cci), recovery, and platelet cd62p expression. aims: to analyze the increase of platelet cd62p expression in patients of non-leukodepleted compared to pre-storage leukodepleted pc transfusion.background: haemorrhage is a leading cause of preventable death not only in the military trauma care, but also for civilian population suffering accidents or bleeding injuries in regions with low population density where health services should reach people in remote areas. resuscitation using blood products and limited infusion of normal saline improves survival for critically bleeding patients. nowadays there are hems programs (helicopter emergency medical system) carrying blood products around the world. the hems in castilla-la mancha, with physician and nurse, is the first out-of-hospital emergency service in spain that provides packed red blood cells (prbc) transfusion where the accidents happen without delaying the transport to the proper hospital for definitive treatment. this program has been developed between the blood center of ciudad real and the hems team ('gigante 2', emergency service castilla-la mancha). the goal of the designed protocol was to preserve the properties of the product to be transfused in out-of-hospital environment by hems teams. aims: to describe the process for out-of-hospital prbc transfusion in hems of ciudad-real. the protocol for out-of-hospital blood transfusion was developed according to criteria of medical indications and security, monitoring, and tracking of the product. methods: data for the observational retrospective study were collected from june 2014 to august 2018. the medical helicopter (ec 145t2) was provided with two prbc o rh(d) negative. the shock index was selected for the indication for transfusion according to the literature revised and as a simple rate to obtain out-of-hospital data. to achieve the feasibility and preservation of the prbc a prospective monitoring of volume was established, haematocrit, haemoglobin, leucocytes, coulter, hemolysis and microbiological culture. blood center established two groups: the case group for the prbc kept in the hems base and helicopter and the control group for the units remaining in the blood center with standardized blood conservation. for both groups, control and comparison of immediately obtained hematologic analyses, and 35 days after collection, were performed. the statistical analysis used spss 23.0 version (significance p < 0,05). results: 138 prbc samples were evaluated, 48,5% (67) from case group and 51,5% (71) from control group. analyses were tested day 1 and day 35 after collection. haemolysis was not observed. all cultures were negative. although significant differences were found between the parameter in the value of before-after in the value of the hematocrit, leukocytes and coulter, there are no differences between the prbc that flew and those conserved in the transfusion-service. all results comply with current legislation and blood transfusion standards. there have been administered 35 prbc transfusion to 28 patients during out-of-hospital advanced medical assistance. no post-transfusion reactions have been registered. prbc units have a 35-day rotation to allow the use of the units in the hospital after achieving their optimal status. summary/conclusions: the out-of-hospital transfusion protocol designed to transport blood (prbc) in the helicopter for hems has demonstrated to keep the standard conditions and properties of the product to be considered useful in the treatment for critically bleeding patients in the out-of-hospital. background: early and adequate treatment of major bleeding is important for survival and a good outcome. blood and plasma are given increasingly early including before hospital admission in trauma based on successes reported from combat experience. in 2010 the national patient safety agency issued a rapid response report requiring national health service hospitals in england to take actions to improve provision of blood in an emergency including provision of major haemorrhage protocols (mhp) and drills. the national reporting and learning scheme had identified reports of 11 deaths and 83 instances of harm due to delay over a 4-year period. aims: the aim of the study was to monitor the acid-base status of the patient by means of abg and to administer the blood component therapy based on teg results. methods: this study was a prospective observational study of 18 adult patients over a period of 7 months. serial monitoring of the abg in the intra-operative period was done. teg guided resuscitation was performed in all 18 cases. results: the abg analysis of all 18 patients showed decrease in the ph, increase in pco 2 , decrease in serum bicarbonate level and elevation in negative base excess. these components of metabolic acidosis can be attributed to massive transfusion. increased lactate, an independent parameter, which reflects poor tissue perfusion or shock and predicts need for massive transfusion was observed in all patients. all the 18 cases showed a decrease in ionized calcium levels which could be a result of citrate related toxicity. increased glucose was observed in all patients which may be due to increase in the catecholamine release as a response to haemorrhagic shock. electrolyte correction was given depending on results of the abg analysis wherever appropriate. two out of 18 cases showed an increase in r time indicating deficient coagulation factors, which was corrected with fresh frozen plasma (ffp). three cases showed elevation in k time indicating deficient fibrinogen levels, which was corrected by ffp. fresh frozen plasma was also given in 4 cases, which showed decrease in the alpha angle, indicating deficient fibrinogen, and cryoprecipitate was given in 2 cases. platelets were transfused in 5 patients showing a decrease in the maximum amplitude (ma), which indicates deficient platelets. summary/conclusions: teg as poc testing is an important tool in appropriate blood component therapy in massive transfusions. serial monitoring of abg helps in monitoring acid-base status of the patient and therefore is a guide in the correcting electrolyte level in patients undergoing massive transfusion. background: massive blood loss is encountered in various situations like trauma, major surgeries, gastrointestinal bleeds and obstetric haemorrhages. haemorrhage is an important cause of mortality and morbidity in massively bleeding patients. early recognition of haemorrhage and intervention is essential for survival. massive transfusion (mt) of blood is required to replenish blood losses and is a lifesaving treatment in these patients. a variety of haemostasis and pathophysiological changes occur during massive haemorrhage and massive transfusion. all of these changes contribute to the vicious cycle of progressive coagulopathy due to the 'lethal triad' of refractory coagulopathy, progressive hypothermia and persistent metabolic acidosis. aims: the aims of the study included understanding management of cases of massive blood transfusion in surgical patients, impact of mt of blood components on patient outcome, evaluating post-operative complications of massive transfusion and the development of institutional massive transfusion protocol (mtp).methods: this prospective observational study commenced after institutional ethics committee (iec) approval. it comprised of 40 adult surgical oncology patients who received massive transfusions and was conducted for a period of 7 months. every case of a massive transfusion was studied under the following headings (1) patient's details (2) patients base-line laboratory tests (3) resuscitation with transfusion (4) intra-operative laboratory tests (5) thromboelastography (teg) (6) post-operative complications (7) duration of stay in the hospital (8) 30 day mortality rate. results: complete blood count, serum electrolytes, arterial blood gases, coagulation profile and teg were used to monitor transfusion therapy in the intraoperative period. intraoperative laboratory parameters of patients showed dilutional coagulopathy, metabolic acidosis, hypocalcaemia, hypomagnesaemia, hyperkalaemia and hypokalaemia, increased lactates and glucose. electrolyte correction was done based on the derangement. the derangements were on a decreasing trend in the postoperative period and returned to baseline level by 2 nd or 3 rd post-operative day with no requirement of correction in the post-operative period. the post-operative outcomes were evaluated in terms of the surgical site infection (ssi) as per the centers for disease control (cdc) criteria, surgical complications as per modified clavien-dindo classification and respiratory complications. a total of 13 (32.5%) patients had ssi, 14 (35%) had surgical complications and 22 (55%) patients had respiratory complications. the length of the stay in the hospital was longer for patients who had postoperative complications. despite complications, owing to excellent peri-operative care, 38 (95%) patients were discharged alive. summary/conclusions: surgeries associated with massive transfusion are at an increased risk of ssi as well as morbidity and mortality. complications associated with rapid transfusions of blood, acute haemorrhage and associated risk of the surgery lead to a prolonged icu stay and increased length of stay in the hospital. a well-developed massive transfusion protocol optimizing the ratio and dose of the blood component therapy results in excellent patient outcome with minimal postoperative morbidity and mortality. background: despite the introduction of new oral anticoagulants (dabigatran, rivaroxaban, apixaban), vitamin k antagonists (vka), such as warfarin and acenocoumarol are still the most widely used oral anticoagulants for the treatment of non-valvular atrial fibrillation (nvaf). the use of vka must be regularly and often laboratory controlled in order to ensure the adequacy of therapy and to avoid subdosing or drug overdose. the most commonly used test for the control of oral anticoagulant therapy is the prothrombin time (pt), expressed in inr system, which provides an internationally standardized monitoring of the treatment. time in therapeutic range (ttr) represents a measure of the quality of the anticoagulant effect of vka and estimates a percentage of time a patient's inr is within the desired therapeutic. aims: the aim of this study was to evaluate of the effectiveness of vka therapy in patients with nvaf and to identify factors affecting the anticoagulation efficacy. methods: a retrospective study was conducted on a population of 725 outpatients with nvaf, treated with vka and followed in blood transfusion institute of ni s from january to december 2017. laboratory control of inr was done from capillary blood of patients on thrombotrack solo (axis shield, norway) and thrombostat (behnk elektronik, germany). targeted 15 ae 17.52%, but 49.72% of patients had a ttr less than 60%. patients were at high risk of thrombosis in 6.15% of time (inr < 1.5) and high risk of bleeding in 2.2% of time (inr > 4.5). the most significant independent factors affecting the quality of vka therapy are gender, arterial hypertension, diabetes mellitus and the use of amiodarone and antiplatelet drugs (aspirin, clopidogrel). summary/conclusions: the ttr is undoubtedly useful indicator of the effectiveness of vka treatment. the most important predictors of poorer efficacy of vka therapy are arterial hypertension, diabetes mellitus, patients' gender and the use of amiodarone and antiplatelet (aspirin, clopidogrel) drugs. to improve the quality of vka therapy, education of patient and better collaboration with them, as well as a successful teamwork with clinicians are also imperative. background: an estimated 1.9 million deaths per year result from haemorrhagic blood loss. at a cellular level, haemorrhagic shock develops when oxygen delivery is insufficient to meet oxygen requirements to maintain aerobic metabolism. successful resuscitation prevents further oxygen debt and repays the prior oxygen debt. this includes the administration of fluids and blood components (e.g. plasma, red cells and platelets). measurement of oxygen delivery and utilisation at a tissue level requires invasive monitoring not possible clinically, meaning that surrogate markers such as lactate and venous oxygen saturation (svo 2 ) are used instead. new technologies such as incident dark field imaging and near-infrared spectroscopy may offer a non-invasive alternative; however their utility in haemorrhagic shock remains background: transfusion-induced red cell alloimmunization is still a major challenge in transfusion practice. besides logistic problems for the transfusion laboratory, it may compromise available blood supply, and when undetected and unanticipated, it may risk haemolytic transfusion reactions. knowledge about risk factors can help to optimize preventive matching strategies. severe renal failure and subsequent renal replacement therapy influence the immune system and could therefore modulate the risk of alloantibody formation against foreign red cell antigens subsequent to transfusion. aims: this study aims to quantify the association between renal failure, according to its degree and its treatment with renal replacement modalities, and transfusioninduced red cell alloantibody formation. methods: we performed a multicenter case-control study within a source population of patients receiving their first and subsequent red cell transfusion between 2005 and 2015 in the netherlands (risk factors for alloimmunization after red cell transfusion, r-fact study). using a conditional multivariate logistic regression, we compared first-time transfusion-induced red cell alloantibody formers (n = 505) with two similarly exposed non-alloimmunized control recipients (n = 1010) during a five-week alloimmunization risk period. degree of renal function was categorized as: 'no renal failure' i.e. glomerular filtration rate (gfr) > 30 ml/min/1.73 m 2 , 'moderate renal failure' i.e. gfr ≥ 10-30 ml/min/1.73 m 2 during a continuous period of minimally seven days, 'severe renal failure' i.e. gfr < 10 ml/min/1.73 m 2 and/or use of any type of renal replacement therapy during at least one day of the alloimmunization risk period. odds ratios were interpreted and presented as relative risks (rr). adjusted rrs were conditioned on the matching variables and identified confounders. results: no renal failure was observed among 441 (87.3%) cases versus 838 (83.0%) controls; moderate renal failure among 24 (4.8%) cases versus 54 (5.3%) controls; and severe renal failure among 40 (7.9%) cases versus 108 (11.7%) controls. among the latter, 30 cases and 97 controls underwent renal replacement therapy. moderate renal failure and severe renal failure without renal replacement therapy were not significantly associated with red cell alloimmunization (adjusted rr 0.82, 95% ci 0.67-1.01 and adjusted rr 0.81, 95% ci 0.58-1.11, respectively). however, patients undergoing renal replacement therapy had a two-fold lower alloimmunization risk (adjusted rr 0.48, 95% ci 0.39-0.59) as compared to transfusion recipients without renal failure, unrelated to type and duration of renal replacement therapy. summary/conclusions: these findings suggest that patients undergoing renal replacement therapy have strongly diminished red cell alloimmunization risks. further research should confirm these results and elucidate the underlying pathophysiological protective mechanism. background: the ability of allogeneic hematopoietic stem cell transplantation(allo-hsct) to prevent relapse depends partly on donor natural killer (nk) cell alloreactivity. nk effector function depends on specific killer-cell immunoglobulin-like receptors (kir) and hla interactions. thus, it is important to identify optimal combinations of kir-hla genotypes in donors and recipients that could improve hematopoietic transplantation outcome. aims: to obtain the optimal combinations of inhibitory kir and its ligand between donor and recipient which is helpful for the guidance of selecting donors and recipients in hsct. methods: the pcr-sbt method was used for kir2dl1, kir2dl2/kir2dl3, kir3dl1, kir3dl2 and hla-a, -b, -c, -drb1, -dqb1 genotyping. 58 pairs of hla 10/10 identical donor/recipients matching samples were retrospectively analyzed. three different models of kir were established. there were kir-kir gene model, kirligand model and haploid model. in kir-ligand model, patients were divided into three groups: c1/c1 homozygote group (48 cases), c1/c2 heterozygote group (8 cases) and c2/c2 homozygote group (2 cases). according to the expression of 3dl1, 53 cases were 3dl1 positive and 5 cases were 3dl1 negative. there were 5 cases of bw4/bw4, 24 cases of bw4/bw6 and 24 cases of bw6/bw6 in the 3dl1 positive samples. according to the expression of a3/a11, they were divided into three groups: a3/a11 negative group (28 cases), a3/a11 heterozygous group (28 cases) and a11/ a11 homozygote (2 cases). according to kir genotyping, kir haploidentical group (38 cases) and kir haploid mismatched group (20 cases) were divided. the clinical data on neutrophil and platelet remodeling time, gvhd and os of 58 cases were statistically analyzed by the mann-whitney test or the kruskal-wallis test using graph-pad software v5.01. results: there was no significant difference in the time of neutrophil and platelet remodeling, the incidence of agvhd and the survival time after transplantation in the kir genotype model. in haplotype model, there was no significant difference in neutrophil and platelet remodeling time and survival time after transplantation. the incidence of agvhd was low when the kir haploid mismatched and kir3dl1 was positive. it was conducive to neutrophil and platelet remodeling when bw4/bw6 and a3/a11 was heterozygosity. summary/conclusions: it is important to establish the three different models of kir genotypes, haplotypes and receptor-ligand mismatches for analyzing the effect on the prognosis of allo-hsct. kir-ligand model plays an important role in hla unre-background: transfusion of platelet concentrates (pcs) has been associated with adverse outcomes including transfusion-related acute lung injury (trali). the underlying mechanism of trali has been related to the accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in pcs. current room temperature storage limits the shelf-life of conventional pcs to 5-7 days. alternative storage conditions, including cryopreservation, offers extended storage and a solution for blood banking logistics. cryopreservation of human pcs has been associated with higher concentrations of immunomodulatory mediators compared to room temperature stored pcs and it has been suggested that cryopreserved pcs may be immunomodulatory. to investigate the effects of cryopreserved pcs, a transfusion sheep model would be a beneficial approach. aims: to characterize immunomodulatory mediators in supernatants of sheep conventional and cryopreserved pc and to investigate whether storage duration and cryopreservation impacts the accumulation of these mediators. methods: buffy coat pooled sheep pcs (n = 3), prepared in 30% plasma/70% ssp+ with minor modifications to standard human procedures, were stored room temperature (rt) for 7 days (d) and sampled on d2, d5 and d7. cryopreserved sheep pcs (n = 3), prepared by the addition of 5-6% dimethyl sulfoxide, were stored at à80°c and sampled pre-freeze and post-thaw. supernatant was prepared at each time point with double centrifugation and stored at à80°c. concentrations of pro-inflammatory cytokines (interleukin (il)-6, il-1b, il-17a), anti-inflammatory cytokine il-10 and chemokines (il-8, monokine induced by gamma interferon (mig) and interferongamma induced protein (ip)-10) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of non-polar lipid mediators, such as arachidonic acid (aa), 12-hete and 15-hete were assessed in the stored sheep pc-and cryo-pc supernatant using commercial elisa. results shown as mean ae standard deviation. the effect of storage was determined at p < 0.05 using paired t-test. results: in rt stored sheep pc supernatant il-6, il-1b, il-17a, il-10, il-8, mig, ip-10, 12-hete and 15-hete were detected at d2, d5 and d7. storage duration significantly increased accumulation of ip-10 at d5 (253.4 ae 266.7 pg/ml compared to 569.7 ae 272.7 pg/ml, p = 0.008) and further increased at d7, and il-8 at d7 (3477 ae 937.4 pg/ml compared to 5092 ae 521.1 pg/ml, p = 0.0460). cryopreserved sheep pc supernatant pre-freeze and post-thaw contained equivalent or higher concentrations of il-6, il-1b, il-17a, il-10, il-8, mig, ip-10, 12-hete and 15-hete than rt stored d5 pcs. however, cryopreservation did not impact levels of any of the platelet derived mediators. summary/conclusions: several platelet-derived cytokines/chemokines, including high concentration of il-8 with neutrophil priming activity, and non-polar lipids were found in stored sheep pc supernatant. these immunomodulatory mediators may contribute to adverse outcomes associated with pc transfusion. storage at rt, but not cryopreservation was associated with increased accumulation of immunomodulatory mediators in sheep pcs. most importantly, similar to human pcs, sheep cryopreserved pcs contained at least if not higher concentrations of majority of cytokines as pcs stored at rt, therefore making sheep a suitable model in which to investigate immunomodulatory effects of cryopreserved pc transfusion. background: transfusion, despite being a lifesaving therapy, has been associated with adverse transfusion outcomes. transfusion related acute lung injury (trali) remains one of the leading causes of transfusion-related mortality. accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in blood products, including packed red blood cells (prbcs), have been implicated with the development of non-antibody mediated trali. however, how specific mediators contribute to the underlying mechanism is yet to be defined. during routine storage of human prbcs fewer than 10 cytokines/chemokines and several biologically active lipids have been identified. a sheep model of trali has successfully been developed using human prbc supernatant, however transfusing sheep prbc has not been investigated. to support the use of sheep prbc in the trali model and to better understand the precise mechanism, characterization of the potential mediators in sheep prbc is required. aims: to characterize immunomodulatory mediators in sheep prbc supernatants and to investigate whether storage duration impacts the accumulation of these mediators. methods: sheep prbcs (n = 5), prepared according to standard human procedures with minor modifications, were stored (2-6°c, 42 days (d) ) and sampled at d2 and d42. supernatant was prepared by double centrifugation and stored at à80°c. concentrations of pro-inflammatory cytokines (interleukin (il)-6, il-1b, il-17a), antiinflammatory cytokine il-10 and chemokines (il-8, monokine induced by gamma interferon (mig) and interferon-gamma induced protein (ip)-10) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of potential non-polar lipid mediators (arachidonic acid (aa), 12-hydroxyeicosatetraenoic acid (hete) and 15-hete) were assessed in the sheep prbc supernatant using commercial elisa. paired t-test was used to compare fresh and stored prbc supernatant (p < 0.05). results are mean ae standard deviation. results: at day 2, aa (75,142 ae 47,205 pg/ml), 12-hete (849.1 ae 235.6 pg/ml), 15-hete (87.6 ae 32.7 pg/ml) and il-1b (144.7 ae 198.9 pg/ml) were detectable in sheep prbcs supernatant. at day 42, storage duration significantly increased concentrations of aa (136,254 ae 60,433 pg/ml, p = 0.0425) and 15-hete (380.9 ae 116.3 pg/ml, p = 0.0022) in sheep prbcs supernatant. summary/conclusions: similar to reported findings of human prbcs, the predominant type of immunomodulatory mediators present in sheep prbcs were non-polar lipids. the concentration of these non-polar lipids increased during storage. these immunomodulatory mediators may contribute greatly to adverse outcomes associated with prbc transfusions. further investigation is required to determine whether stored sheep prbcs supernatant induce immunomodulation in sheep in vitro and in vivo transfusion models. background: dshtr incidence is reported as 1 in 2,500 transfusions, presenting days to months after the transfusion. the published data addressing the correlation between the strength of the antibodies detected after a dshtr has taken place and the corresponding clinical symptoms as measured by laboratory parameters that assess the presence of hemolysis is limited. aims: the aim of this study is to evaluate the correlation between the results of the dat, automated and manual antibody reactivity strength with the corresponding clinical parameters of hemoglobin, lactate dehydrogenase (ldh), bilirubin, and haptoglobin. methods: a dshtr is defined as discovering a new antibody within 28 days of a transfusion. for all positive antibody screens, a work-up is initiated consisting of identification panels, dats, antigen typing of the red cells transfused, and eluates at the discretion of the transfusion medicine physician. additional laboratory testing for hemolysis is requested when indicated. a retrospective review was conducted of patients who were identified as having a dshtr. levels of hemoglobin, ldh, and transfusion safety background: rhd immunoglobulin (rhdig) has been available for 50 years in australia. since its introduction for routine antenatal and postpartum prophylaxis, alloimmunisation has decreased from 16% to 0.2%, reducing the number of australian deaths from haemolytic disease of the newborn over a hundred-fold, to approximately 0.01 deaths per 1000. blood matters serious transfusion incident reporting (stir) system has been collecting transfusion incidents and adverse events across four australian jurisdictions since 2007. since january 2015, rhdig administration errors have been reported. aims: to understand incidents relating to the administration of rhdig and increase safety and awareness of risks. methods: health services registered with stir (n = 93) were notified of the inclusion of reporting rhdig incidents. when an incident is identified, the reporter sends an online notification to stir, prompting the appropriate investigation form to be sent for completion. the completed incident data are reviewed and validated by an expert group. data is de-identified and collated for reporting. results: during the period january 2015-december 2018, 45 reports were received; 43 reports were validated, with 2 reports excluded (reactions rather than administration errors). reports were categorised as below: background: following the nice transfusion guidelines, recommending offering iron before and after surgery to patients with iron-deficiency anaemia (ida), we worked collaboratively with the anaesthetic and pre-operative team to implement a clear and robust anaemia pathway for pre-operative haemoglobin (hb) optimisation. oral iron was started, where appropriate, and our anaemia pro-forma was sent for review in a virtual clinic to assess eligibility for intravenous iron. we performed a retrospective evaluation of the patients who received iv iron during the anaemia pathway. aims: the aim of this retrospective evaluation was to look at the cohort of patients who had received iv iron in 2017 and assess the effect of iv iron on haemoglobin levels for different defined groups. methods: we classified patients, as described in munting and klein, 2019, depending on their iron parameters as having either: -idaserum ferritin < 30 mg/l -chronic inflammation with idaserum ferritin 30-100 mg/l with transferrin % of < 20%/crp > 5 mg/l -anaemia of chronic inflammationserum ferritin > 100 with transferrin % of < 20%/crp > 5 mg/l patients were considered eligible for iv iron if the following criteria were met:1. an inadequate response to oral iron, or were unable to tolerate oral iron or the interval between diagnosis and surgery was short 2. the anaemia pro-forma was completed 3. hb was ≤ 120 g/l 4. they were classified as either having ida or chronic inflammation with ida or anaemia of chronic inflammation hb was measured prior and on average, 20 days following the iv iron infusion. we excluded patients who had their post iv iron follow up blood tests done after surgery. results: this retrospective evaluation included 80 patients. 48 patients were classified as having ida and 32 patients classified as having chronic inflammation with ida. those classified with ida had a mean hb of 96 g/l (55-120), a mean mcv of 77.4 fl and a mean serum ferritin of 16 lg/l. those with chronic inflammation with ida had a mean hb of 106 g/l (77-120), a mean mcv of 87.9 and a mean serum ferritin of 57 lg/l. follow-up hb was measured on average twenty days post iv iron infusion in both groups. the average hb post iv iron infusion in the ida group was 119 g/l (100-149) with an average increment of 23 g/l and in the group with chronic inflammation with ida the average post iv iron hb was 117 g/l (85-129) with an average increment of 11 g/l. summary/conclusions: in conclusion the group with ida had, on average, a lower starting hb that the group with chronic inflammation with ida and the average increment in hb 20 days post iv iron infusion was greater in the group with ida. however, the group with chronic inflammation with ida cases also responded to iv iron and therefore we strongly consider the use of iv iron in both groups. further studies to evaluate the ongoing effect of iv iron would help assess whether the same level of increment seen with ida can also been seen for the group with chronic inflammation with ida over a longer period and how long the increment was sustained. background: the expansion of personalized genomic medicine has led to the development of targeted therapeutic approaches for patients. one example is sipuleucel-t, an autologous cellular immunotherapy product used to treat prostate cancer manufactured from the patient's white blood cells. this study describes our experience with incorporating autologous cellular immunotherapy products into the workflow of the blood bank. the policies supporting the workflow are outlined and compliance with them is assessed. aims: this study aims to evaluate the process and method used to dispense and track the infusion of sipuleucel-t. methods: this is a retrospective analysis of the dispensation and administration of sipuleucel-t from january 2012-august 2018, which was handled exclusively by the blood bank. standard operating procedures and hospital policies were reviewed and compliance with these policies evaluated. included were patients who had the sipuleucel-t product dispensed and administered. information collected included the total number of products dispensed, patient age, adverse reactions/incidents, premedication, and patient outcome. descriptive statistics were used for data analysis. results: there were 154 products dispensed to 53 patients. the recipients were male patients diagnosed with prostate cancer with a mean age of 74 years. there were 3 doses (a complete course) administered to 50/53 (94%) recipients and a partial course (1-2 doses) administered to 3/50 (6%) recipients, for a total of 154 products. the blood bank workflow treated sipuleucel-t as a derivative in the computer system, listing the manufacturer (dendreon corporation) as the supplier. health care providers were instructed to follow the nursing policy for the administration of blood products and derivatives for the infusion of sipuleucel-t. this policy required documentation of the infusion in a transfusion nursing note and reporting adverse events to the blood bank as transfusion reactions. there were no adverse events reported to the blood bank, yet there were 5 adverse events described in provider notes; 2 of them necessitating transfer to the emergency department, and 1 requiring hospital admission. of the 154 infusions, 6 infusions were documented in a chemotherapy note rather than a transfusion note (4%), and 59 (38%) were documented as both a transfusion and a chemotherapy administration. there were 6 additional deviations from the blood product administration policy: 2 cases where the consent check was not performed, 1 case where the product was infused with ringer's lactate rather than normal saline, and 3 cases where the 2-person 3-way check erroneously indicated the product was irradiated. summary/conclusions: this study describes one approach to managing cellular therapy products as an extension to existing blood products dispensed by a blood bank. the findings demonstrate noncompliance with hospital policy with this new product as evidenced by failure to report adverse events and failure to follow hospital practices regarding administration. although sipuleucel-t is a product manufactured from an autologous blood product donation, the administration and perceptions of this product may be more similar to a pharmaceutical. as the field of immunotherapies derived from blood product donations continues to expand, these products may necessitate an entirely new approach to ensure proper management. abstract withdrawn. (ref 10310) . while the mnc procedure is fully automated, cmnc requires frequent interface checks to ensure the collection of the correct cell layer. at the rambam health care campus, a tertiary care center, solely the mnc procedure had been employed till 2017, at which point, the cmnc has been introduced for the use in patients with a white blood cell (wbc) count of ≥ 20,000/ll on the collection day. aims: the current study aimed to compare various parameters of peripheral blood stem cell (pbsc) collection, using the cmnc protocol in allogeneic donors and patients undergoing autologous stem cell (autosc) transplantation. additionally, data on autosc collection using mnc (n = 31) and cmnc (n = 88) procedures were compared. methods: data were retrospectively obtained from pbsc collection reports in 134 consecutive cmnc procedures, including 88 autologous and 46 allogeneic donors. the following comparisons were made: cmnc results of allogeneic versus autologous donors, a sub-analysis of cmnc results for autologous donors with a pb cd34 + count ≥20/ll versus allogeneic donors as well as mnc versus cmnc results in autologous donors. the collection efficiency-2 (ce-2) was defined as the total cd34 + amount in the collection bag divided by the amount of cd34 + cells in the pb processed by the collection apparatus. results: in the cmnc, the following parameters significantly differed between autologous and allogeneic donors: mean collection time (333 ae 59 and 289 ae 73 min, respectively; p = 0.001), the total blood volume processed (3.4 ae 0.8 and 2.4 ae 0.8, respectively; p = 0.001) and the final volume in the collection bag (337 ae 77 and 291 ae 84 ml; p = 0.001). the mean ce-2 in autologous versus allogeneic donors was 49 ae 24 and 66 ae 22, respectively (p = 0.003). using cmnc, the collection was effective in 94% of allogeneic and 63% of autologous donors. in autologous donors, a significantly lower collection bag volume (341 ae 84 and 396 ae 132, respectively; p < 0.01) and increased total wbc in the collection bag (263 ae 119 versus 149 ae 36, respectively; p < 0.000) were obtained using cmnc compared to mnc protocol. thirteen patients were treated with plerixafor due to a low pb cd34 + count following g-csf therapy; 7 of them achieved a cd34 count ≥20 and their collection was considered effective. summary/conclusions: the cmnc protocol is highly effective in terms of the cell yield in both allogeneic and autologous donors with a pb cd34 + count ≥ 20/ll. significantly superior collection results are obtained in allogeneic donors versus autologous ones. cmnc provides a significantly higher wbc and a lower final collection volume than mnc. similar total cd34 + cell counts are obtained with both methods. . tbv processed ranged from 1-4.8 tbv with mean of 2.6, average was 2.65 tbv for females and 2.74 tbv for males mean pre-apheresis cd34 + count was 92.89 cells/ll (range 33.14-152.64). mean postapheresis cd34 + count was 1402.3 cells/ll (559.02-2245.5). mean cd34 + cells x10 6 / kg recipients body weight was 7.5 (range: 2.77-12.33). our target yield was ≥3 9 10 6 cd34 + cells/kg body weight of the recipient and in only 2/34 (6%) cases, the yield was <3. 18/34 (52.9%) procedures were lvl and 16/34 (47.1%) were svl. summary/conclusions: most of our pbsc were done for haematological indications (85.3%) and the target dose was 3 9 10 6 cells/ll in single leukapheresis. in 32 cases (94%), target yield was achieved, only 2 cases had <3 but >2 yield. in our study donors <5 years have shown to mobilize better than the older children. hematocrit (hct) and weight showed correlation with cd34 + cell yield but they cannot be taken absolute predictors. wbc count cannot be taken as a predictor for cd34 yield as high wbc count did not convert into high cd34 yield or vice versa. high prepheresis cd34 + count gave higher postpheresis cd34 + count. large volume leukapheresis (lvl), >3 tbv gave higher yield as compared to standard volume leukapheresis (svl). blood volume processed related to prepheresis cd34 + count and/or the weight difference between the donor and recipient. other parameters like hematocrit, wbc count, age etc did not show correlation to the volume processed. in our study, younger age and prepheresis cd34 + count were found as the most relevant predictors for stem cell yield. background: allogeneic hematopoietic stem cell transplantation is an established therapy for many hematologic disorders. since the discoveries of the potential of peripheral blood stem cells (pbsc) in the hematopoietic reconstitution mid 1980s and early 1990s pbsc gradually replaced bone marrow as the preferred source of stem cells. the introduction of hematopoietic cytokines that can mobilize large number of progenitors into circulation accelerated pbsc usage. aims: the aim of our study is to present our 18 year experience with apheresis collecting of pbsc in donors. methods: this is a retrospective study performed in the institute for transfusion medicine of republic of macedonia and university hematology hospital for period background: obtaining unambiguous results of hla typing plays an important role in the transplantation of hematopoietic stem cells. appropriate selection of alleles in the level of hla between recipients and unrelated bone marrow donors reduces the risk of transplant rejection and graft-versus-host disease. new generation technology ensures the highest possible resolution and obtaining unambiguous genotyping results due to the high complexity of the hla system. currently, this is the selection method for obtaining hla test results at the high resolution level. aims: the aim of this study was to determine 11 hla loci (hla-a, -b, -c, drb1/3/ 4/5, dqb1, dpb1, dpa1, dqa1) in potential bone marrow donors from poland. the research included 18,500 potential bone marrow donors registered between 2017 and 2018. a novelty of this paper was that the amplification of all 11 hla loci was performed by using multiplex pcr primers in a single tube. that solution completely eliminated the need to pool amplicons. methods: the typing of the 11 hla loci (hla-a, -b, -c, drb1/3/4/5, dqb1, dpb1, dpa1, dqa1) of potential bone marrow donors was made by using the alltype tm ngs 11-loci amplification kit (one lambda). genomic dna was isolated from peripheral blood of 18,500 donors. hla genotypes were determined according to the manufacturer's protocol on the miseq illumina platform. the obtained sequencing data was evaluated by using the typestream tm visual ngs analysis software. results: the ngs method allowed to obtaining unambiguous results of genotyping of potential bone marrow donors, and also provided the identification of rare alleles, such as: c*12: 143, c*12: 30, c*05: 37, c*07: 151, drb1*11: 28, c*14: 04, b*51: 22, c*15: 13, dqb1*03: 12, drb1*14: 87, drb1*11:69. summary/conclusions: 1. new generation sequencing technology (ngs), which is based on pcr, ensures the highest possible resolution. 2. the ngs method allows to obtain more accurate sequencing results compared to the conventional methods. 3. the research has confirmed the superiority of the ngs method over conventional methods in obtaining unambiguous hla genotyping results at the high resolution level. background: the accurate results of hla typing are significant for ensuring the success rate of hematopoietic stem cell transplantation. currently, hla typing is mainly based on sanger sequencing, which has a high proportion of ambiguous combination results indicating potential errors for hla typing. it is necessary for finding a more accurate typing method to reduce the risk. next-generation sequencing (ngs) method could provide clonal sequencing of single molecules, which has been used for hla genotyping and improved the scope and precision of hla study. aims: to establish a full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology and be evaluated by classical sangersequencing method, which can effectively improve the accuracy of hla typing for donor and recipient in hematopoietic stem cell transplantation. methods: hla-i (hla-a, -b, -c) gene-specific primers were screened, and the amplification parameters were optimized to obtain full-length sequences of hla-i gene under the same condition. the sample library for the amplicon was prepared with transngs tn5 dna library prep kit and the sequencing step was carried out with illumina miseq platform according to the manufacturer' protocol. all the sequencing data in fastq format were analyzed by typestream visual software version 1.2.0(one lambda inc.)with the default setting. 94 cord blood samples were collected for hla typing with the mentioned above next-generation sequencing method in our study. in parallel, all the sample were also tested with the sanger sequencing method according to the previous study in our laboratory. results: 94 samples were successfully tested with two methods and the coincidence rate between two sequencing methods was 100%. with the next-generation sequencing method, the probability of ambiguous results among 94 samples in our study is 1.06%(1/94) for hla-a, 5.31% (5/94) for hla-b and 0% (0/94)for hla-c. however, the probability of ambiguous results with the sanger sequencing method is 96.8% for hla-a, 95.7% for hla-b, 100% for hla-c. summary/conclusions: the full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology was established, which could greatly reduce the probability of ambiguous results and effectively improve the accuracy of existing hla typing techniques. key: cord-005453-4057qib7 authors: nan title: the 45th annual meeting of the european society for blood and marrow transplantation: physicians – poster session date: 2019-07-03 journal: bone marrow transplant doi: 10.1038/s41409-019-0559-4 sha: doc_id: 5453 cord_uid: 4057qib7 nan background: allogeneic hematopoietic stem cell transplantation is routinely offered to patients with high-risk or advanced all in the hopes of improving outcomes. use of truly non-myeloablative (nma) conditioning reduces toxicity in other contexts but outcome data for all patients after nma transplants is lacking. we report the outcomes of 31 patients with all transplanted using a nma conditioning without t cell depletion. methods: first transplant patients between october 2006 and june 2018 were reviewed. these were consecutive patients until 2015 then only those considered unfit for fmc conditioning as per the ukall 2014 protocol. all patients were conditioned with fludarabine 25mg/m 2 /day for 5 days and cyclophosphamide 1g/m 2 /day for 2 days. short course mtx and ciclosporin were used for gvhd prophylaxis. standard supportive care was employed. thirty-one patients with a median age of 43 (23-67) met the criteria for this case review. 30 had b-all and 10 were philadelphia chromosome positive. 24 patients (77%) had high risk disease by standard diagnostic criteria. 27 (87%) were in first complete remission (cr1). matched sibling donors were used in 13 instances with the remaining being fully matched unrelated donors. 58% of patients had a hct-ci score of 0, 32% a score of 1 or 2 with 3 patients having a score of 3 or higher. median cd34 dose was 5.3 x 10 6 /kg (0.93-34.12) with a median cd3 dose of 2.13 x 10 8 / kg (0.12-7.37) results: trm was low at 7% at 1 year and 11% at 2 and 3 years respectively. no factors included in a univariate analysis (which included age, diagnosis, disease status, hct-ci, donor type, cmv risk and cell dose) significantly impacted trm. the incidence of classical acute (a) gvhd grade 2-4 and 3-4 was 18% and 8% by day 100 and 29% and 13% by day 180 if late onset agvhd is included. 24 out of 30 eligible patients developed chronic gvhd of any stage. relapse incidence was low (22% at 3 years in all patients, 17% in cr1 patients) and was not impacted by any pre-transplant factors including positive mrd post phase 2 induction (present in 6 patients). notably, in univariate analysis relapse was significantly lower in patients who developed chronic gvhd. background: allogeneic stem cell transplantation (allosct) is the treatment of choice for many patients (pts) suffering from acute myeloid leukemia (aml). the graft vs. leukemia effect (gvl), applied by immunocompetent cells of donor origin, is the most important effector mechanism for the eradication of leukemia, the presentation of leukemic or allospecific antigens by malignant blasts is regarded as a crucial trigger for an effective allogeneic immune response. conversely, insufficient stimulatory capacity by the leukemic blasts is thought to be a relevant escape mechanism from cellular immunotherapy (allosct or donor-lymphocyte infusion (dli)). the purpose was to test, whether the ability of malignant blasts to differentiate in vitro towards dendritic cells of leukemic origin (dc leu ) is associated with response to allosct or outcome after immunotherapy (second allosct or dli) for post-transplant relapse in aml. methods: leukemic blasts were isolated from peripheral blood (pb) or bone marrow (bm) samples of aml patients before allosct (n=47) or at relapse after allosct (n=22). a panel of 6 different assays was used to generate dc leu in vitro (5 of them containing gm-csf). finally, in vitro results were correlated with clinical characteristics and outcome of patients treated with donor lymphocyte infusion and/or allosct. results: dc leu could be generated in vitro from all 69 samples. when correlating proportions of dc-subtypes generated ex vivo with clinical data, significantly higher mean proportions of dc leu in the dc-fraction were found in responders vs. non-responders to immunotherapy (76.8% vs 58.8%,p=0.006, range:13%-99%). vice versa, the chance for response to immunotherapy was significantly higher, if a dc leu /dc ratio of >=50% could be reached in vivo (p=0.004). those patientswere characterized by a longer time to relapse (p=0.04) and by a higher probability for leukemia-free survival (p=0.005). similarly, generation of higher amounts (>8%, p=0.04) of dc leu in the mnc-fraction, and generation of more mature dc (>47% cd83+, p=0.03 using the best gm-csf containing assay) were associated with a longer time to relapse in the respective patients. moreover, overall survival was improved, if >70% dc leu /dc could be generated with the best gm-csf containing assay (p=0.048). conclusions: in vitro generation of dc/dc leu from leukemic blasts obtained in active stages of aml before allosct or at relapse post transplant were associated with clinical outcome. this observation supports a role of antigen presentation by leukemic cells for an allogeneic immune response in aml. disclosure: nothing to declare background: the role of autologous hematopoetic cell transplantation (hct) in the treatment of aml is not clear. trials in the past have shown that autologous hct consolidation lowers the risk of relapse, however the magnitude of this effect is limited . autologous hct is advocated in patients with aml with lower genetic risk in cr1.many of these patients will eventually relapse and will undergo reinduction followed by allogeneic hct in cr2. methods: the aims of this study is to analyze outcome of allogeneic hct performed in cr2 comparing patients with prior consolidation by autologous hct vs. patients with chemotherapy consolidation. primary outcome is non relapse mortality (nrm) of allogeneic hct in cr2 in patients with, or without prior autologous hct in cr1. secondary outcomes include leukemia free survival (lfs), relapse rate (ri), graft versus host disease free relapse free survival (grfs), overall survival (os), and treatment related toxicities. results: 2619 adult patients reigstered with the alwp of the ebmt with de novo aml were included, receiving a first allogeneic hct in cr2, in 2000 cr2, in -2017 or without (n=2202) prior autologous hct. patient and transplant characteristics are shown in the table. patient groups were not entirely comparable, patients with prior autologous hct were younger, had less often a favorable cytogenetic profile, had more commonly donors other than matched siblings and more often received reduced intensity conditioning (ric) as compared to mac conditioning. univariate outcomes are shown in the table with slightly higher nrm risks in patients with prior autologous hct consolidation. in multivariate analysis nrm risks in patients with prior autologous hct were 1.34 (1.07-1.67), p=0.01 after adjustment for patient age, cytogenetic risk category, year of transplant, donor type, conditioning intensity, sex matching, time from diagnosis to relapse and time from relapse to allogeneic hct as compared to patients with chemotherapy consolidation. similarly, risks of events in lfs and grfs were higher with prior autologous hct, 1.17 (1.01-1.35 ), p=0.03 and 1.18 (1.03-1.35 ) p= 0.02, respectively, risk of death was also higher 1.13 (0.974-1.32) p=0.1 but this was not statistically significant. conclusions: we may conclude that some of the advantages of potentially higher anti-leukemic activity of high dose chemotherapy and autologous hct when given to patients with aml in cr1(as was shown in a randomized trial by vellenga e et al with lower relapse and higher lfs by approximately 10% but no significant differences in overall survival) may be lost by higher toxicity of allogeneic hct in cr2 in case of subsequent relapse. background: although relapse is a major cause of mortality in patients receiving allogeneic hematopoietic cell transplantation (hct) for acute leukemia, limited and conflicting data exist on extramedullary relapse (emr). we aimed to describe the incidence, risk factors, outcomes and prognosis in relapsed hct recipients. methods: we retrospectively reviewed charts of consecutive allogeneic hct recipients transplanted in our center with the indication of acute leukemia (7/1990-7/ 2018). we recorded: age, gender, disease, previous extramedullary involvement, phase at transplant, type of transplant, donor, conditioning, graft-versus-host-disease (gvhd), infections, treatment-related mortality and relapse mortality. in patients with extramedullary relapses, additional data on clinical manifestations, imaging, cerebrospinal fluid testing, histopathology and management were additionally documented. incidence of isolated emr (iemr) and bone marrow relapse (bmr) was calculated using cumulative incidence (ci) analysis, with each and treatment-related mortality considered a competing risk. results: among 554 allohct recipients followed for 1.8 (0.04-27.75 ) years, 61 (11%) patients presented with emr. the majority of emrs involved the central nervous system (cns, 56%). isolated emr was observed in 38 patients at 9.5 (1.8-67.3) months. 10-year cumulative incidence (ci) of 10.5% for iemr was associated only with pre-transplant advanced disease phase (p< 0.001). bmr was observed in 149 patients at 9 (0.3-276 months), with a 10-year ci of 34.8%. in the multivariate analysis, bmr ci was independently associated with fungal infections (p< 0.001), pre-transplant disease phase (p< 0.001) and lines of treatment (p=0.042). 10-year trm of our whole cohort was 33.2%. the majority of iemr and bmr (75% and 81%, respectively) patients received systemic treatment combined with local radiation for iemr (26%) and donor lymphocyte infusions (dlis, 16% and 28% respectively) when feasible. extensive chronic gvhd was recorded in 47% of iemr and 48% of bmr patients. outcomes were poor in iemr, with 10-year overall survival (os) of 18.3%. favorable os in iemr was associated only with sibling donors (p=0.049) and not with other factors, such as treatment with dlis or presence of chronic gvhd. similarly poor outcomes (10year os of 19.1%) were observed in bmr. favorable os was independently associated only with the diagnosis of aml (p=0.050) and absence of bacterial infections (p=0.049). in the whole cohort, both iemr and bmr were independent unfavorable predictors of os (p< 0.001) along with extensive chronic gvhd (p=0.012). conclusions: in a large population with long-term follow-up, incidence of iemr was relatively high, developed at the late post-transplant period and associated only with disease phase at transplant. furthermore, iemr and bmr conferred similarly poor outcomes despite systemic treatment or extensive chronic gvhd. these independent predictors of survival highlight the unmet clinical need of novel approaches either as maintenance or treatment to reduce extramedullary or systemic relapse post allohct for acute leukemia. disclosure: no competing financial interest. impact of t-cell depletion on outcome in patients undergoing allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia background: after a diagnosis of acute myeloid leukemia (aml) the majority of patients (pts) who achieve complete remission (cr) eventually relapse, with only approximately 30% of pts maintaining cr for 3 years or longer. late relapses (after 3 years in cr) occur rarely (6-10%) in pts receiving hsct in cr1 and late effects are followed up by routine surveillance as well as preventative measures. the purpose of this study was to investigate long-term outcomes in pts with diagnosis of aml undergoing hsct at our institution in cr1. methods: a standardized follow-up of hsct-survivors is applied at our center. we analyzed 116 adult pts with aml in cr1 consecutively transplanted between january 2004 and december 2016 at our institution. a written consent was given for the use of medical records for research. a landmark analysis was adopted for patients in cr at 2-y after hsct (ltcr -long-term cr). results: ltcr was achieved after hsct in 91/116 patients (male 55, female 36) transplanted in cr1. the median follow-up was 6 years and the median age at transplant 52 years (r 20-72). the selected donor was a family haploidentical relative in 29 cases, an hla identical relative in 21, a match unrelated donor in 39 and a cordblood in 2. in this cohort of ltcr, the 5-year overall survival was 92% (95% ci 83-96). cumulative incidence of relapseevaluated in competing risk with transplant related mortality (trm) -and trm -evaluated in competing risk with relapse -were respectively 7% (95% ci 1-23) and 2% for the cr1 cohort. the event-free-survival (efs) was 91% (95% ci 83-95). the causes of death were relapse (6/10 pts), second cancer (3/10 pts) and sepsis (1/10 pts). the 5-year incidence of dyslipidemia -defined as cholesterol >/= 200 mg/dl, and/or ldl >/= 115 mg/dl, and/or triglycerides >/= 150 mg/dl or need for specific treatment -was 24%. the 5-year incidence of osteopenia / osteoporosisdefined as t-score lower than -1 and greater than -2.5 and t-score lower than 2.5 respectively -was 38%. the 5-year incidence of second cancer was 11%: 10 nonmelanoma skin cancer, 2 lung carcinoma, 3 cervical intraepithelial neoplasm, 1 thyroid cancer, 1 gastric cancer and 1 colon cancer. the 2-year incidence of chronic moderate-severe gvhd was 27% (95% ci 13-38), with the latest diagnosis performed on day 570. of note, 4/24 pts are still on active treatment at last follow-up. conclusions: relapse incidence is low for patient that reached ltcr: patients in cr1 at transplant can obtain excellent os and efs once reached the target of ltcr. a proactive long-term follow-up and strategy of counseling are essential to keep at best quality the survival advantage offered by hsct in patients with aml in cr1. disclosure: chiara bonini has research contract with intellia therapeutics. the other authors declare that they have no conflicts of interest. background: relapse, graft-versus-host disease (gvhd) and gvhd-associated mortality are major obstacles to success of transplantation from unrelated (mud) donors in children with acute leukemia (al). negative depletion of αβ t cells and cd19+ b lymphocytes, conserves the mature donor-derived natural killer cells and γδ t cells in the graft, may improve gvhd control, immune reconstitution and prevent the relapse. we present a retrospect analyses of a cohort of pts with al in cr transplanted from mud with depletion. methods: a total of 59 children with acute leukemia (34 aml, 25 all, 21 female, 38 male, median age 8,5y) underwent allo hsct from matched unrelated donor between june 2012 and july 2017. all pts were in complete remission (cr1=34, cr2=23, cr>2=2). all pts, except one, received treosulfan-based conditioning. either melphalan (n=56) or thiophosphamide (n=2) or etoposide (n=1) were added as a second agent. fludarabine was used in all pts. two types of gvhd prophylaxis were used: type 1 (n=35): hatg 50 mg/kg and post-hsct tacro/mtx (n=30) or without prophylaxis (n=5); type 2 (n=24): thymoglobulin(ratg) 5mg/kg, rituximab 200mg/ m 2 with either bortezomib on days +2, +5 (n=21) or tacro/ mtx (n=3) . aβ t cell depletion with clinimacs was used in all cases. the median dose of cd34+ cells was 9 x10 6 / kg, aβ t cells -15 x10 3 /kg. median time of follow-up for survivors was 5,3 years (range, 2, 3 -6,5) . results: primary engraftment was achieved in 100% pts., the median time to neutrophil and platelet recovery was 15 and 14 days, respectively. all evaluable pts achieved sustained complete donor chimerism by day +30. early (100 day) mortality was 3,4% (1pt -bacterial sepsis, 1pt -adv fulminant hepatitis), 5-years overall ptrm at 4 years was 13,5% (95%ci:7-26). six late trm events were due to: viral infection in 2 pts (cmv=1, adv+cmv=1), bacterial sepsis in 2 pts and 2 pts had bacterial and viral infection, all late deaths were associated with cgvhd and prolonged corticosteroid therapy. ci of acute gvhd grades ii-iv was 36% (95% ci: 25-50), acute gvhd grades iii-iv 3,7% (95% ci :1,5-14,5) . ci of cgvhd was 27%(95%ci:18-41). regimen 2 was more effective in prevention of agvhd ii-iv in comparison with regimen 1: 8% (95% ci: 2,2-30) vs 45,7%, respectively, p=0,04. all events with acute gvhd grades iii-iv had pts with regimen 1. ratg was also effective in prevention of cgvhd: ci at 4 years after hsct was 12,5% vs. 37%, respectively, p=0,04. cumulative incidence of relapse was 25% (95%ci: 14-50) without difference between ratg and hatg. event-free survival (efs) (event=death or relapse) at 4 years was 61% (95%ci: 48-73), overall survival 59%(95% ci:47-72), there were no difference between age and diagnosis. conclusions: we confirm that the depletion of tcrαβ +/cd19+ t lymphocytes from the graft ensures high engraftment rate. transplant-related mortality is caused by infections, mostly associated with cases of chronic gvhd. gvhd prophylaxis including ratg/rituximab/ bortezomib improves gvhd control in recipients of tcrαβ+/cd19+depleted grafts in comparison to hatg/ tacro/mtx apparently without loss of anti-leukemic activity. disclosure results: at baseline, r/r all with emd and lbl were diagnosed in 7 and 11 ino patients and 5 and 6 sc patients. median (range) age of the ino and sc patients was 55.5 (20-78) and 47.0 (28-64) years, with 8/18 (44.4%) and 8/11 (72.7%) males, respectively. the rate of cr/cri was significantly higher in the ino group (12/18 [66.7%] , 95% confidence interval [ci] : 41.0-86.7) compared with sc (2/11 [18.2%], 95% ci: 2.3-51.8; p=0.0144) (table) . allogeneic hematopoietic stem cell transplantation was carried out in 6/ 18 (33.3%) ino and 2/11 (18.2%) sc patients prior to any post-study induction therapy. the pfs hazard ratio [hr] was 0.502 (97.5% ci: 0.203-1.240; p=0.0410), with median pfs of 4.4 (95% ci: 1.9-7.1) months among ino and 1.6 (95% ci: 0.8-3.7) months in sc patients. the os hr was 0.661 (97.5% ci: 0.269-1.621; p=0.1478), with median os of 5.9 (95% ci: 3.4-9.4) months in ino versus 5.5 (95% ci: 2.1-6.7) months in sc patients (figure) . all patients had adverse events (aes). serious aes occurred in 10/18 (55.6%) ino and 5/11 (45.5%) sc patients; 4 (22.2%) ino and 0 sc patients had grade 5 ae. one (1/15, 6 .7%) patient in the ino group died from veno occlusive disease. conclusions: among r/r all patients with emd and lbl, improvement in remission rates, transplant rates, and progression free survival was shown in the ino group versus the sc group. although patient numbers were small and limited the ability for a robust comparison, these results support the use of ino in patients in this difficult to treat population with r/r all and emd or lbl. background: bcr-abl-targeted tyrosine kinase inhibitors (tki) revolutionized the outcome of patients inflicted with ph+ b-all. moreover, addition of tki may be relevant strategy for ph-like all patients. methods: we hypothesized that overcoming the bm microenvironment-mediated protection of all cells from tki-mediated apoptosis may further enhance the responsiveness to tki therapy. results: in vitro treatment of bcr-abl-positive all cell lines nalm1 and nalm20) with dasatinib resulted in significant dose-dependent cell growth inhibition, with ic50 of 10-15 nm (p< 0.01). furthermore, dasatinib exhibited significant growth suppression of bcr-abl -negative all cells (nalm6 and reh), with ic50 of 250 nm and 185 nm, respectively. however, when cocultured with bone marrow stromal cells (bmscs), dasatinib-mediated effect was abrogated in both ph-and ph+ all cells. furthermore, dasatinib treatment promoted significant upregulation of chemokine receptor cxcr4, on both mrna and cell surface levels. elevated cxcr4 expression was accompanied by increased responsiveness of all cells to cxcl12 stimulation, resulting in strong and sustained phosphorylation of erk1/2 and akt and increased adhesion capacity to bmscs. therefore, dasatinib-induced upregulation of cxcr4 promotes stroma-mediated survival advantage of all cells upon tki therapy. next, in order to overcome the cxcr4-mediated stromal protection, we choose to combine dasatinib with the histone deacetylase inhibitor panobinostat, for its known ability to deplete cxcr4 in aml cells. single-agent treatment with panobinostat demonstrated significant inhibition of ph-and ph+ all cell growth at low nanomolar concentrations (p< 0.01). importantly, combination of panobinostat with dasatinib synergized (ci< 0.5), effectively overcoming the protection provided by bmscs and inducing the apoptosis of ph-and ph+ all cells, as demonstrated by phosphatidylserine externalization, mitochondrial depolarization and dna fragmentation. furthermore, combining panobinostat with dasatinib significantly reduced cxcr4 surface levels in ph-and ph+ all cells. accordingly, cxcl12mediated responses, including erk1/2 and akt activation and adhesion to bmscs were significantly reduced upon combined panobinostat/dasatinib treatment. these data indicate that panobinostat effectively suppresses both basal and dasatinib-induced cxcr4 expression and function in all cells overcoming stroma-mediated resistance to dasatinib. to determine the molecular mechanism, we performed gene and protein expression analysis. panobinostat, alone or in combination with dasatinib, significantly down-regulated the protein levels of calcineurin, a serine-threonine protein phosphatase previously implicated in t-all and b-all pathogenesis, as well as of nfatc1, a critical effector of the calcineurin signaling cascade, and nfatc1-regulated target genes. it was previously found that calcineurin signaling positively regulates cxcr4 expression in t lymphocytes. additionally, cyclosporin a (csa) decreased both basal and dasatinib-induced cxcr4 surface levels in all cells, overcoming the protection of the bmscs which result in potentiation of the cytotoxic effect of dasatininb and panobinostat. combining csa with panobinostat resulted in deeper suppression of nfatc1-regulated target genes. we thus link the effect of panobinostat with calcineurin-dependent downregulation of cxcr4, blocking the ability of the leukemic cells to respond to cxcl12mediated stromal support. conclusions: taken together, our results identify calcineurin signaling pathway as a novel target of panobinostat in all cells and indicate that hdac inhibition with panobinostat may be effective strategy for facilitating the anti-leukemic activity of tki therapy. disclosure: nothing to disclose background: the treatment of relapsed/refractory acute lymphoblastic leukemia (rr-all) remains a clinical challenge with a generally dismal prognosis. allo-sct using a sequential conditioning ("flamsa"-like regimen) has shown promising results in relapsed/refractory aml, but little is known about the efficacy of this procedure in rr-all. methods: we identified 115 adult patients (45% females; median age: 38 y; range, 18-66) with all in primary refractory phase (26%) or in relapse (74%), allografted between 2000 and 2017 from a matched sibling (31%), matched unrelated (58%) or haploidentical donor (11%) at ebmt participating centers. almost half (49%) of the patients had t-all and 23% had a positive philadelphia chromosome. six patients (5%) underwent a previous autotransplant. karnofsky score was above 90 in 52% of patients. conditioning was myeloablative (mac) with high dose tbi in 30% of patients, reduced intensity (ric) including low dose tbi in 22%, or with chemotherapy alone in 48%. in vivo t cell depletion was performed in 77 cases (69%). most patients (74%) and about half of the donors (47%) were cmv positive. 14% of patients were males who received a graft from a female donor. the median follow-up was 37 (range, 13-111) months. results: overall, 14 patients (13%) failed to engraft, 18 (16%) died within 100 days after allo-sct without relapse, and 64 (56%) could achieve complete remission. at day 100, the cumulative incidences of grade ii-iv and grade iii-iv acute gvhd were 30% and 17%, respectively. the 2year cumulative incidences of chronic and extensive chronic gvhd were 25% and 11%, respectively. the 2-year relapse incidence (ri) and non-relapse mortality (nrm) were 45% and 41%, respectively. the 2-year leukemia free survival (lfs), overall survival (os) and gvhd relapsefree survival (grfs) were 14%, 17% and 12%, respectively. in a multivariable cox analysis, karnofosky score below 90 negatively affected ri, lfs, os and grfs. also, conditioning with chemotherapy alone, compared to tbibased conditioning, negatively affected relapse rates (hr=4.13; p=0.0006), lfs (hr=2.32; p=0.004) and os (hr=2.29; p=0.006). conclusions: allo-sct using a sequential conditioning regimen is proposed by different teams in rr-all, and could be an option, especially when considering a tbibased regimen. however, the overall 2-year lfs of 14% suggests that these patients still face extremely dismal outcomes, highlighting that other therapies (e.g. bite antibodies, inotuzumab, car t cells) need to be combined prior and/or after allo-sct in order to further improve outcome. disclosure: no conflict of interest, no funding received chemotherapy courses, only 2 pts were not treated: 1 pts for the worsening of the general status and the other for invasive fungal infection. results: forty-three pts (42%) were in complete remission (cr) and negative minimal residual disease (mrd) at the time of hsct; 16 pts were in active disease (16%), and 44 (42%) showed a morphological cr with positive mrd. 41pts (40%) developed chronic graft versus-host disease (cgvhd) as followed: 23pts (22%) mild, 17pts (16%) moderate, and only 1 sever grade respectfully. only 1 patient developed cgvhd after dli. the overall leukemia free survival (lfs) time was 16 months, the absence of cgvhd (hazard ratio -hr: 5,968; p = 0,01) and the pre-hsct disease status (hr 2,353; p = 0,028) were the most important factors on lfs. all pts treated with chemo-based regimens died due to progression or infective complications. 1 patient of aza/dli group is still alive with a extramedullary relapse; 2 pts treated with bl/dli are in cr. os was better for the dli group compared to the chemotherapy group (28 vs 2 months respectfully; p < 0,001). conclusions: dli after allo-hsct has exhibited definite anti-leukemic effects in post-transplant patients. bl and aza were reported to increase dli's graft vs-leukemia (gvl) effect. although cgvhd could be the most important protective factor against the relapse but it remains the main cause of morbidity. maximising the gvl effect without putting the patient at risk of gvhd still represents an unmet need. our data show that the combination of either bl or aza with dli infusion is safe and might represent an improvement in disease control in the early phase of relapse. disclosure: nothing to declare p018 increased detection of (leukemiaspecific) adaptive and innate immune-reactive cells under treatment of amldiseased rats and one therapy-refractory aml-patient with blastmodulating, clinically approved response modifiers (pg-e2,kit-k) or +pge1 (kit-m),patent 102014014993) convert myeloid blasts into dendritic cells of leukemic origin (dc leu ). after stimulation with dc leu , antileukemic tcells can be generated ex vivo. the compounds are approved for clinical use and are therefore attractive tools for immunotherapy in myeloid leukemia. methods: dc/dcleu-culture from rats'/patients' wholeblood (wb) with kits, mixed lymphocyte culture (mlc) of tcells with kit-treated blood, functional blast-cytotoxicity and leukemia-specificity assays (csa/elispot/degranulation/intracellular cytokine-assays). in addition flowcytometric evaluations of cellular and (leukemia-specific) lymphocyte compositions were performed from rats'/pts' blood in the course of the disease. results: 1) aml-diseased rats: each 3rats were treated with "i", "k" or "m" or were untreated (controls). a significant increase of dcleu could be detected in spleen/pb in kit-(esp. m) treated compared to untreated animals without induction of blasts' proliferation (ki67positivity): a significant reduction of blasts was seen with "m" (p=0.03/ 0.0001 in spleen/pb) and "i", but not "k". successful treatment correlated with an increase of cd62l+tcells, most likely representing tmem-cells, (p=0.07) and a reduction of cd4+treg (p=0.037). 2) 6 therapy-refractory aml-patients (during the course of decitabine/ld-aractreatment): kit-m was shown to ex vivo generate dcleu, activate immunereactive cells and mediate leukemia-specific/antileukemic response. activated or leukemia-specific lymphocytes were monitored in low proportions in active stages of the disease as well as of two patients during the further course of persisting disease. one of these patients (72 yo male), was offered an individual systemic salvage-treatment (kit-m, applied as continuous infusions) for refractory leukemia. after approval from the local ethical commitee,extensive information of the patient about the experimental nature of the treatment and obtaining his written informed consent. clinically the treatment was well tolerated and the patient improved clinically. neutrophils in wbc increased from 10% to 50%, thrombocytes reached 100 g/l after 24 days. after 4 weeks of treatment, the patient was discharged in good clinical conditions. 12 days later, progression of aml was seen with high blast counts in pb and bm. the patient developed severe sepsis and died few days later. immune monitoring showed (other than before treatment and in the patients without kit-m-treatment) a continuous increase of proliferating and non-naïve tcells, nk, cikand nkt-, th17 cells, bmem-cells and dc in pb. the production of ifnɣ producing t-, cik and nkt-cells was demonstrated, suggesting an in vivo production/activation of (potentially leukemia-specific) cells. immune stimulatory effects decreased after discontinuation of therapy. conclusions: treatment of wb as well as leukemically diseased organisms with blast-modulating kits (especially gm-csf and pge1) was well tolerated and induced clinical and immunological improvement (adaptive and innate immune system), whereas low counts of (leukemiaspecific) activated immune-reactive cells were found in non-kit-treated organisms. disclosure: nothing to declare p019 long-term outcomes after allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia with non-myeloablative and myeloablative conditioning: a single-center cohort study of 438 consecutive patients 1lars klingen gjaerde, niels smedegaard andersen 1 , lone smidstrup friis 1 , brian thomas kornblit 1 , søren lykke petersen 1 , ida schjødt 1 , henrik sengeløv 1 1 rigshospitalet, copenhagen, denmark background: since 2000, we have at our institution used a non-myeloablative (nma) conditioning regimen for older (>50 years) or significantly comorbid younger patients undergoing allogeneic hematopoietic stem cell transplantation (allo-hsct) for acute myeloid leukemia (aml). we aimed to compare the long-term outcomes of nma conditioned patients with myeloablative (ma) conditioned patients. methods: we studied 220 nma and 218 ma conditioned adult (>15 years) consecutive patients receiving their first allo-hsct for aml from 2000 to 2017 at rigshospitalet. nma conditioning consisted mainly of 2 gy total body irradiation (tbi) and fludarabine 90 mg/m 2 (95% of cases). ma conditioning consisted mainly of cyclophosphamide 120 mg/ kg and either 12 gy tbi (86% of cases) or busulfan 12.8 mg/ kg (5% of cases), or fludarabine 150 mg/m 2 and treosulfan 42 mg/m 2 (6% of cases). five percent and 19% of nma and ma conditioned patients, respectively, received anti-thymocyte globulin. patients were followed until death or end-of-followup on october 31 st , 2018. cumulative incidences with 95% confidence intervals (ci) of acute graft-versus-host disease (agvhd) grade ii-iv, chronic graft-versus-host disease (cgvhd), relapse and non-relapse mortality (nrm) were calculated and compared between nma and ma conditioned patients using gray's test with death as a competing risk (or relapse when comparing nrm). overall survival (os) was estimated by the kaplan-meier method. results: nma and ma conditioned patients were comparable when regarding sex (49% and 48% female, respectively) and donor (matched related donor in 34% and 36%, respectively), but differed, as expected by indication, with regards to age (median of 60 versus 42 years, respectively) and karnofsky score (< 90 in 18% and 11%, respectively). nma conditioned patients had generally a lower aml stage at transplant (1 st complete remission in 68% versus 49% of ma conditioned patients) and a lower aml cytogenetic risk (adverse risk in 17% versus 21% of ma conditioned patients). patients were followed for a total of 2090 person-years (median follow-up in surviving patients was 6.2 years). agvhd grade ii-iv occurred less frequently in nma conditioned patients (20% [ci: 15%-26%] versus 38% [ci: 32%-45%] in ma conditioned patients, p < 0.01), while cgvhd occurred in similar rates (50% [ci: 43%-56%] in nma conditioned patients and 51% [ci: 44%-58%] in ma conditioned patients, p = 0.77). there was a trend towards a higher relapse rate in nma conditioned patients (34% [ci: 28%-40%] versus 28% [ci: 22%-34%] in ma conditioned patients, p = 0.07), and nma conditioned patients had, however not with statistical significance, lower nrm (20% [ci: 14%-25%] versus 25% in ma conditioned patients, p = 0.27). os ( figure) was comparable, with 5-year os rates of 55% (ci: 48%-62%) in nma conditioned patients and 54% (ci: 47%-61%) in ma conditioned patients. conclusions: patients with aml undergoing allo-hsct with nma conditioning at our institution were older and frailer than ma conditioned patients, but their overall survival after transplantation was comparable. this might be explained by a generally lower aml stage and cytogenetic risk at transplant in nma conditioned patients. jedlickova 1 , saskia güller 1 , rosa toenges 1 , juliane steinmann 1 , hans martin 1 , hubert serve 1 , gesine bug 1 background: allogeneic hsct is urgently indicated in patients with aml in first complete hematologic remission (chr) after intensive chemotherapy with increasing or recurrent minimal residual disease (mrd). these patients are at high risk of hematologic relapse (hr) during preparation of their transplant and hsct with active aml was found associated with poor outcome. azacitidine has recently been shown to substantially delay or even prevent hr in >50% of patients (relaza2 trial, platzbecker et al., lancet oncology 2018) . we here present the outcome of a small cohort of consecutive patients with mrd-positive aml who received low dose cytarabine (ldarac) as bridging therapy prior to hsct. methods: mrd was assessed by quantitative polymerase chain reaction (qpcr) using mutated npm1 (n=5), runx1-runx1t1 (n=2), cbfb-myh11 (n=1) or kmt2a-ptd (n=1). mrd negativity was defined as ratio of oncogene to control gene (abl1) ≤0,01% while increased or recurrent mrd required a ratio >1% (shayegi et al., blood 2013) . primary endpoint of our retrospective analysis was progression to hr (≥5% bone marrow blasts or extramedullary disease); secondary endpoints were achievement of molecular remission prior to hsct, neutropenia g4 according to ctcae, thrombocytopenia g4, anemia ≥g3, admission to hospital, os and rfs. os and rfs were calculated from the first dose of ldarac. ldarac was self-administered subcutaneously by the patients at home at a flat dose of 20mg bid over 10 days and repeated after 4 weeks if necessary. results: between 12/2015 and 10/2018, nine patients (median age 55, range, 22-68 years) with low (n=7), intermediate (n=1) or high-risk cytogenetics (n=1) according to eln criteria 2017 were treated in continuous chr for increasing (n=2) or recurrent mrd (n=7) starting at a median of 260 (range, 154-651) days after the last consolidation therapy, i.e., duration of chr was >6 months in all pts. patients received one (n=4), two (n=2) or three cycles (n=3) of ldarac prior to hsct. in three patients, neutropenia g4 occurred and one patient needed platelet transfusion. all patients were managed in the outpatient setting. in eight out of nine patients (89%), hr was successfully prevented and 3 patients (33%) even became mrd negative prior to hsct. one patient (runx1-runx1t1 positive aml) progressed to hr after one cycle of ldarac and received salvage therapy with high-dose arac and mitoxantrone (ham) prior to hsct. all patients proceeded to hsct from a matched related (n=1), unrelated (n=7) or haploidentical donor (n=1) and are still alive (median follow-up of 666 days). conditioning regimens included fludarabine (flu)/melphalan (mel)/tbi (n=5), flu/mel (n=1), flu/tbi (n=1), flu/busulfan (bu)4 (n=1) and thiotepa/bu3/flu (n=1). after hsct, only the ldarac-refractory patient relapsed, resulting in a probability of rfs of 88% at 2 years. conclusions: our data suggest that a bridging therapy with up to three cycles of ldarac prior to hsct is feasible and was associated with favorable outcomes in patients with npm1-mutated or core binding factor aml and molecular relapse >6 months after achieving a first chr. the treatment has low costs, can be administered on an outpatient basis and is very well tolerated. clinical background: allogenic hematopoietic stem cell transplant (hsct) is the only curative treatment for all the patients with aml. high risk disease qualifies for upfront hsct irrespective of the presence of matched sibling donor (msd). in the absence of msd, haploidentical stem cell transplant is easier option with success rates as high as msd in a high volume transplant centre. we present our experience from a single centre. methods: we analyzed retrospective data of aml patients who have undergone hsct at our centre between january-2013 and august-2018. for msd transplant we used fludarabine + busulfan or fludarabine + melphalan conditioning regimen, in matched unrelated donor transplant (mud) regime used was fludarabine + busulfan + atg. we followed john hopkins's protocol for haploidentical hsct. cyclosporine + methotrexate was used as gvhd prophylaxis in msd and unrelated donor group and cyclophosphamide + tacrolimus + mycophenolate was used for haploidentical post-transplant. day 100 survival, overall survival (os), incidence of gvhd and cmv reactivation was computed. results: a total of 96 aml patients underwent hsct during the study period, the basic and clinical characteristics of the study patients are presented in table 1 . conditioning regime did not have significant impact on os. survival at day 100 was 78%. the os function and relapse free survival (rfs) function did not significantly differ between msd and haploidentical transplantation (68.3% vs 60.0%; p=0.225) and (68.3% vs 75.0%; p=0.760) (graph 1). disease status at latest follow up showed that 82% were in remission and 18% had relapsed. overall one year survival and five year survival in the entire cohort was 68% and 58% respectively. the average cost of msd transplant at our centre is inr 10,00,000 (€ 10000-12000), haploidentical transplant is inr 20,00,000 (€ 25000-27000) and mud transplant is inr 32,00,000 (€ 30,000 + 10000 for stem cell procurement). conclusions: our study showed comparable outcomes in msd and haploidentical transplant with respect to day100 survival, os, and rate of gvhd. in a developing country like india where patients are not covered under state health insurance, the additional cost of procurement of stem cells in a mud transplant would add to the financial burden to the patients. haploidentical transplant is a feasible option in case of non-availability of msd, due to ease of donor availability and strong motivation from the family donor to donate the stem cells. background: allogeneic stem cell transplantation (allo-hsct) is not indicated as consolidation of first complete remission (cr1) in favorable-risk acute myeloid leukemia (aml) bearing mutations in nucleophosmin (npm1) in the absence of flt3 internal tandem duplication (flt3-itd). nevertheless, a substantial proportion of patients eventually proceed to allo-hsct beyond cr1 or for chemoresistant minimal residual disease (mrd) while in cr1, which might compromise transplantation outcomes. the study aimed at examining the characteristics and results of allo-hsct in aml cases with mutated npm1 and wild-type flt3 (npm1mut/flt3wt), with special focus on molecular monitoring of mrd following transplantation. methods: from 11/2010 until 04/2018, 16 patients (women/men, 9/7) underwent allo-hsct for npm1mut/ flt3wt aml. at transplant, median age of patients was 44.5 years (range, 35-63) , and disease phase was cr1 (n=5), cr2 (n=9), or primary refractory (n=2). among the 13 patients who were transplanted in cr and had available molecular mrd assessments, 10 had detectable mutant npm1 transcripts by real-time quantitative pcr (rq-rcr). also, 4 patients fulfilled criteria of molecular relapse (increasing levels of npm1-mutated transcripts in two successive bone marrow samples), with mutant npm1 load of 386-4,900 transcripts/10,000 abl transcripts). the conditioning regimen was myeloablative in the majority of cases (n=14) or reduced-intensity (n=2). the type of donor varied, namely hla-identical sibling (n=6), matched unrelated (n=5), haploidentical relative (n=3), or double umbilical cord blood (n=2). results: engraftment was achieved in all cases, with a median time to absolute neutrophil count >500/ul of 16 days (range, 12-29) . among the 13 patients with posttransplant monitoring of mrd by rq-pcr, 9 exhibited a stable molecular remission whereas a rising level of npm1mutated transcripts was observed in 4 cases due to either hematologic (n=3) or molecular (n=1) relapse of disease. the cumulative incidences (cin) of hematologic relapse and non-relapse mortality (nrm) were 18.75% and 25% at 12 months, respectively. no events of relapse or nrm were encountered beyond 6 months from allo-hsct. out of 3 patients with hematologic relapse post transplant, 2 died of disease whereas one achieved a stable complete remission after withdrawal of immunosuppression. at a median follow-up time of 40 months (range, 14-89), 10/16 patients continue to be alive in cr. the estimated disease-free background: cpx-351 (vyxeos®) is an advanced liposomal encapsulation of cytarabine/daunorubicin at a synergistic 5:1 molar ratio. cpx-351 is approved by the us fda and ema for the treatment of adults with newly diagnosed, therapy-related aml or aml with myelodysplasia-related changes. methods: safety data were pooled from 5 studies of cpx-351 in adults aged 18-75 years with newly diagnosed or relapsed/refractory aml. cpx-351 induction consisted of 100 units/m 2 (cytarabine 100 mg/m 2 + daunorubicin 44 mg/m 2 ) on days 1, 3 , and 5 (second induction: days 1 and 3) . cpx-351 consolidation consisted of 65 or 100 units/m 2 (varying by study) on days 1 and 3. cpx-351 was evaluated against standard-of-care controls. results: baseline characteristics were generally balanced between cpx-351 (n=375) and controls (n=236); the majority of patients were aged ≥60 years (78%; 87%) and had secondary aml (55%; 72%). controls included 7+3 (n=192) and salvage therapy with mitoxantrone/etoposide/ cytarabine (n=23), idarubicin/cytarabine (n=8), other cytarabine-based chemotherapy (n=12), and mitoxantrone/ etoposide (n=1). the treatment-emergent adverse event (teae) profile of cpx-351 100 units/m 2 was comparable to induction controls, but associated with a greater proportion of patients with teaes, grade ≥3 teaes, and serious teaes during consolidation (table) . therefore, the cpx-351 consolidation dose was reduced to 65 units/m 2 in latter studies; this dose demonstrated an improved teae profile similar to consolidation controls. the most frequent system organ class was gastrointestinal disorders for both cpx-351 and controls; a lower incidence was reported for cpx-351 (90%) versus controls (95%), with this difference driven by the lower incidence of diarrhea for cpx-351 (46%) versus controls (66%). the most frequently reported grade ≥3 teaes were febrile neutropenia (cpx-351: 62%; controls: 59%), pneumonia (16%; 13%), hypoxia (10%; 11%), and bacteremia (10%; 3%). early mortality rates, both overall and by treatment period, appeared lower with cpx-351 versus controls at day 30 and day 60 ( table) ; the majority of early deaths were attributable to teaes. conclusions: across the 5 studies comprising the cpx-351 clinical development program, cpx-351 demonstrated a safety profile comparable to conventional chemotherapy in adults with newly diagnosed or relapsed/refractory aml. background: haploidentical hematopoietic stem cell transplantation (hsct) with post-transplantation cyclophosphamide (pgcy) marked improved clinical outcome. recent studies comparing allogeneic hsct using unrelated donors versus haplo donors in patients with acute leukemia have suggested equivalent outcomes. the depletion of tcells with pgcy was subsequently applied for unrelated hsct setting for patients with unrelated donor. methods: we performed a retrospective study on 90 patients with acute leukemia in order to compare the outcome after hla haploidentical (n=30) and unrelated hsct (n=60) with pgcy. the main characteristics of patients were similar in both groups. baseline disease were: 19 aml (63%) and 11 all (37%) for haplo group and 33 aml (55%) and 27 all (45%) for unrelated group. disease state at time of haplo and unrelated-hsct were following: 19 and 50 patients in cr1 (63% and 83%) and 11 and 10 non cr1 (37% and 17%). for aml recipients mainly received thiotepa, busulfan and fludarabine and for all recipients received tbi and etoposide conditioning. all patients who received pbsc graft were treated with rabbit antithymocyte globulin (atg) on days -2 and -1. results: at the time of analysis, the os and dfs did not differ between the haplo and unrelated groups (67% vs 63%, and 63% vs 56%). incidence of severe (grade [3] [4] acute gvhd was the same in two groups (10% versus 8%). recipients of haplo-hsct transplant were statistical significance less likely to experience disease relapse (3% vs 28%) and chronic gvhd (20% vs 47,5%). however, gvhd free relapse free survival (grfs) rate was slightly higher after haplo-hsct (77% vs 64%). addition, cumulative incidence of trm rate was higher after haplo-hsct (30% vs 15%).for haplo and unrelated groups who underwent hsct in cr1, the os were 84% and 67% versus 22% and 45% for those in non cr1. for aml, the os was same in two groups (haplo 63% versus unrelated 67%). however patients with all, the os was higher in haplo group compared with unrelated group (72% versus 59%). the impact of pretransplant disease state have a more powerfull effect on survival in the haplo-hsct setting (for aml cr1 77% versus non cr1 33% and for all cr1 100% versus non cr1 0%). viral reactivations were significant concern in both groups. conclusions: our retrospective analysis suggests largerly similar os and dfs with haplo versus unrelated transplants with pgcy for acute leukemia. our data indicate that haplo-hsct results in a lower incidence relapse and of chronic gvhd and higher grfs compared with unrelated hsct. in addtion, the pretransplant disease state have the important effect on the outcomes in both groups. allo-pbsc with atg can be used safely and effective as graft source in haplo-hsct with acceptable post-transplant outcomes and replaced bm in this settings. more statistical data for transplant related characteristics will be provided at the presentation.we emphasize that use the same pgcy gvhd prophylaxis for all types of allogeneic transplant. based on our results, we recommend haplo-hsct with pgcy against unrelated transplant for patients with acute leukemia. disclosure: disclosure of conflict of interest: none. excellent efficacy and tolerability of inotuzumab ozogamicin in b-cell all relapsed after allo-hsct background: donor lymphocyte infusion (dli) could be used prophylactically to reduce relapse after allogeneic hematopoietic stem cell transplantation for very high-risk leukemia/lymphoma without effective targeted therapy. to compare the safety and efficacy of prophylactic dli for prevention of relapse after allogeneic peripheral blood stem cell transplantation from haploidentical donors (hid-sct) and matched-sibling donors (msd-sct) in patients with very high-risk acute myeloid leukemia (aml), we performed a retrospective, observational cohort study enrolled in 21 hid-sct and 13 msd-sct recipients. methods: the very high-risk features were defined as: (i) in the non-remission (nr) state prior to transplantation, including primary induction failure, relapse untreated or refractory to reinduction chemotherapy, or untreated aml evolution from mds; (ii) achieving complete remission 1 with ≥3 cycles of induction of chemotherapy; (iii) carrying tp53, dnmt3a, tet2 or flt3-itd gene mutation. the scheduled time of the prophylactic dli was +30-60 days after transplantation for msd-sct recipients and +60-90 days for hid-sct recipients. the g-csfmobilized peripheral blood stem cells were infused to the recipient at a dose of 2×10 7 cd3 + cells/kg. csa was given at 2 mg/kg b.i.d from day -3 to day +90 (hid-sct) or to day +60 (msd-sct), and then tapered at 33% per month to be discontinued on day +150-180 (hid-sct) or on day +120-150 (msd-sct) unless graft-versus-host disease (gvhd) developed. if the patients received dli before day +90 (hid-sct) or day +60 (msd-sct), csa was given 8 weeks after dli in hid group and 4 weeks in msd group at a though concentration of 150-250 ng/ml for dliassociated gvhd prophylaxis, and then tapered and discontinued within 2 weeks unless gvhd developed. if gvhd occurred before the scheduled time of prophylactic dli, it would be delayed for 8 weeks when gvhd was well controlled. results: prophylactic dli was administered at a median of 71 (34-240) days for hid-sct recipients and 53 (35-97) days for msd-sct recipients (p=0.008), and both groups displayed similar baseline characteristics except for donor's gender distribution (table 1) . grade 2-4 acute graft-versushost disease (gvhd) at 100-day post-dli was higher in hid-sct group than that in msd-sct group (59.5% vs. 30.8%, p=0.05). grade 3-4 acute gvhd (17.5% vs. 7.7%), 1-year chronic gvhd (36.6% vs. 33.2%) and severe chronic gvhd (15.3% vs. 27.3%) were similar between two groups (p>0.05). one-year non-relapse mortality was higher in hid-sct group than that in msd-sct group with marginal significance (27.9% vs. 0.0%, p=0.061). one-year relapse rate was similar between hid-sct group and msd-sct group (21.6% vs. 36.5%, p>0.05). estimated 1-year overall survival (os, 55.1% vs. 83.9%) and relapsefree survival (rfs, 50.1% vs. 74.0%) rates were both similar between hid-sct group and msd-sct group (p>0.05). in multivariate analyses, non-remission status prior to transplant, poor-risk gene mutations and donor's age ≥ 48 years predicted a higher risk of relapse after dli. nonremission status prior to transplant predicted inferior os and rfs. patient's age ≥ 40 years also predicted an inferior os. conclusions: prophylactic dli after hid-sct demonstrated similar tolerance and efficacy for reducing relapse compared to that after msd-sct for very high-risk aml. disclosure: the authors declare no conflict of interest. prognostic impact of pre-transplant tim3 levels on transplant outcome in acute leukemia patients background: t cell immunoglobulin and mucin domaincontaining protein-3 (tim3), a negative regulator of t cells, is expressed on a variety of tumors including hematological malignancies like acute myeloid leukemia (aml) and some lymphoma types in which it was shown to be associated with an adverse prognosis. the aim of this study is to identify the prognostic impact of pre-transplant tim3 levels on early and late transplant related complications as well as post-transplant relapse and survival methods: a total of 177 hematopoietic stem cell transplantation (hsct) recipients with an initial diagnosis of acute leukemia [median age: 36(16-66) years; male/ female: 111/66] were included in the study. aml was the initial diagnosis in 99 patients (55.9%), acute lymphoblastic leukemia (all) in 74 patients (41.8%), mixed phenotype acute leukemia in 3 patients (1.7%) and blastic plasmacytoid dendritic cell neoplasm in 1 patient (0.6%). soluble tim-3 levels in pre-transplant serum samples were measured with enzyme linked immunosorbent assay (elisa). results: median pre-transplant tim3 level was 955.6 (65.8-3784.4) pg/ml in the whole cohort. pre-transplant tim3 levels were significantly higher in aml patients when compared to all [1063.7(409.5-3784.4) vs 831.4 (65.8-3254.4 ); p=0.01]. tim3 levels were significantly lower in patients with abnormal cytogenetics when compared to normal karyotype (p=0.017). cytogenetic abnormalities, including mainly a complex karyotype or chromosome 8 abnormalities, were more frequent in patients with low tim3 levels (p=0.053). pre-transplant tim3 levels were significantly higher in patients who developed post-transplant viral hemorrhagic cystitis (p=0.034). a positive correlation was demonstrated between tim3 levels and acute graft versus host disease (gvhd) grade (p=0.013; r=0.299). at a median follow-up of 14.6 (0.2-160.9) months, overall survival (os) was found to be better in low-tim3 group when compared to high-tim3 group, without statistical significance (%35.2 vs % 20.4; p>0.05) ( figure 1 ). probability of os was relatively better in both aml (42.6% vs 26.7%; p>0.05) and all patients (29.5% vs 19%; p>0.05) representing low pretransplant tim3 levels in the subgroup analysis conclusions: in this study, elevated levels of pretransplant tim3 levels in aml patients were compatible with the previous reports which had underlined an increased tim3 expression on aml stem cells. the possible association of tim3 expression with cytogenetic features should be confirmed with further studies as there is no adequate data except its relationship with flt3-itd mutational status. tim-3 is also expressed on exhausted t cells in patients with viral infections, including human immunodeficiency virus, hepatitis b and hepatitis c virus. it plays an essential role in the regulation of antiviral and antitumor immune responses which may be an explanation for the increased frequency of hemorrhagic cystitis in patients with higher tim-3 levels. the adverse prognostic impact of tim3 on gvhd and os was confirmed without statistical significance which may be related to small sample size. as tim3 has a wide spectrum of action in the tumor microenvironment including stimulatory and inhibitory activities, further clues are required to define the exact role of this molecule in the clinical course of allogeneic hsct in order to develop targeted therapeutic strategies clinical trial registry: n/a disclosure: nothing to declare p029 homozygous hla-c1 is associated with increased risk of relapse after hla-matched transplantation in recipients with acute lymphoid leukemia: a japanese national registry study background: after hematopoietic stem cell transplantation (hsct), the role of natural killer (nk) cells which express killer-cells immunoglobulin-like receptors (kirs) and recognize hla-class 1 ligands is important. kir2dl1 recognizes not hla-c asp80 (c1), but hla-c lys80 (c2) and has polymorphism based on the 245 th amino acid of the transmembrane domain. low frequency of c2 and high frequency of strong kir2dl1 are characteristics observed in japanese. by using large transplant database, we reported that homozygous hla-c1 (c1/c1) recipients displayed lower relapse rates than did c1/c2 recipients after hla-matched hsct for acute myeloid leukemia (aml; hr = .79, p = .006) or chronic myeloid leukemia (cml; hr = .48, p = .025). this effect seemed to be independent of acute graft-versus-host disease (agvhd) or cytomegalovirus reactivation occurrence (arima n et al bbmt 2018) . methods: relapse rates of japanese recipients who first underwent hla-matched hsct between 1996 and 2016 for the treatment of acute lymphoid leukemia (all) were compared between c1/c1 pairs and c1/c2 pairs, using data from japanese data center for hematopoietic cell transplantation and adjusting for transplant characteristics. cord blood transplantation was excluded. multivariable competing risk regression analyses were performed to evaluate relapses and relapse-free survival (rfs) was estimated using kaplan-meier method. results: after 61 recipients who did not achieve remission or experienced graft failure and 41 recipients not-expressing c1 were excluded, resting 2779 recipients aged 0-72 years (median, 31.2 years) were analyzed. the median follow-up period for survivors was 5.0 years. there were 2447 recipients expressing c1/c1 and 332 recipients expressing c1/c2, respectively. after hla-matched hsct, c1/c1 recipients had higher relapse rates than c1/ c2 recipients (hr = 1.55, p = .003), resulting in worse rfs among c1/c1 recipients (hr = 1.27, p = .034). the frequent relapse in c1/c1 recipients than in c1/c2 was noticeable among recipients with agvhd (hr = 1.89, p = .002), those without cytomegalovirus reactivation (hr = 1.84, p = .002), and those with ph-negative all (hr = 1.88, p = .001). conclusions: kir2dl1-positive nk cells may promote graft-versus-leukemia (gvl) in c1/c1 recipients with aml or cml but suppress gvl in c1/c1 recipients with all. one interpretation is that transplant-activated nk cells impair antigen-presenting cells or deprive cytotoxic tlymphocytes of their gvl effects on all cells. this hypothesis may be explained by the fact that agvhd was necessary for the recessive relapse in c1/c1 recipients with all. furthermore, ph-positive all cells sometimes mimic aml cells in terms of their frequent myeloid antigen expression and might be directly targeted by nk cells. it would be necessary to further clarify in vitro the character of nk cell-affecting in the transplant immunity against residual leukemia cells. disclosure: authors have nothing to declare. hematopoietic stem cell transplantation with sequential conditioning for children with relapsed/refractory acute leukemia nao yoshida 1 , kazuki matsumoto 1 , daiki yamashita 1 , yiqing zhu 1 , daichi sajiki 1 , ryo maemura 1 , hirotoshi sakaguchi 1 , asahito hama 1 1 children's medical center, japanese red cross nagoya first hospital, nagoya, japan background: patients with acute leukemia who fail to achieve complete remission show a dismal prognosis even with allogeneic hematopoietic stem cell transplantation (hsct). this study evaluated whether sequential conditioning approach that is cytoreductive chemotherapy applied shortly prior to the main conditioning followed by hsct can improve prognosis in such high-risk patients. methods: we retrospectively analyzed the outcomes of 90 children (median 8, range 0-18 years old) with primary refractory (n = 11) or refractory relapsed (n = 79) acute leukemia (aml n = 43, all n = 47) who received hsct in our department between 1990 and 2016. the stem cell source was related peripheral blood (pb) in 4 patients, related bone marrow in 31, unrelated bone marrow in 40, or unrelated cord blood in 15. the grafts were hla serologically matched (n = 63) or mismatched (n = 27) with the recipient. in total, 29 patients received the sequential conditioning approach. as cytoreductive chemotherapy, fludarabine/cytarabine/idarubicin/g-csf (flag-ida) was used in 12 patients, mitoxantrone or daunorubicin/cytarabin in 10, or other regimens in 7, and 6 of them were combined with gemtuzumab ozogamicin. without waiting for hematological recovery, the patients promptly underwent hsct; therefore, the median interval between cytoreductive chemotherapy and main conditioning was 11 days. the main conditioning regimens were total body irradiation-based myeloablative (n = 22), busulfan-based myeloablative (n = 4), or reduced intensity (n = 3). results: in 90 children with relapsed/refractory acute leukemia, the 5-year overall survival (os), leukemia-free survival (lfs), cumulative incidence of relapse (ri), and transplantation-related mortality (trm) were 24%, 21%, 53%, and 26%, respectively. in multivariate analysis, the use of sequential conditioning was identified as the most favorable factor for lfs (hazard ratio [hr] 0.37; p = 0.001), although there were no differences in the outcomes according to the types of cytoreductive chemotherapy or the main conditioning regimen. hla-matched donor (hr 0.46; p = 0.005) and pb blasts-negative at the beginning of conditioning (hr 0.49; p = 0.02) were also independently associated with better lfs. with sequential conditioning, leukemia burden prior to the hsct was significantly reduced; pb blasts became undetectable at the beginning of conditioning in 66% patients given the approach, while in 38% patients without the approach (p = 0.02). notably, the outcomes in the patients without pb blasts at the beginning of conditioning who received sequential conditioning were promising; the 5-year os and lfs reached 73% and 62% and the 5-year ri and trm were 33% and 5%, respectively. conclusions: our study reveals that hsct with sequential conditioning can be an effective and tolerable treatment option for children with relapsed/refractory acute leukemia. the treatment strategies that focus on the reduction of leukemia burden immediately prior to hsct may contribute to the induction of long-term remissions in patients with high-risk acute leukemia. disclosure: this research was funded by japanese red cross, nagoya 1st. hospital research grant nfrch18-0028. use of blinatumomab to achieve remission and consolidation with haploidentical transplant with cyclophosphamide post for the treatment of children with refractory acute lymphoblastic leukemia (all) background: most of patients with all in relapse or refractory to conventional treatment have only 30% possibilities to achieve long term remission. this report refers to the therapeutic efficacy and adverse events from the blinatumomab to achieve molecular remission in patients with pre-b cd19+ which lead to haploidentical with cyclophosphamide post transplant as a consolidation. methods: a pilot study was conducted in children with refractory all preb-cd19 +. as a strategy to achieved remission blinatumomab was used at a dose 10 μg/m2 for continous infusion of 48 hours, increasing the dose to 15 μg/m2 during 28 days, patients with a mrd of < 0.002 log, after 2 cycles received an haploidentical bone marrow transplant as a consolidation, the conditioning regimen was with total body irradiation scheme at 200 cgy/day/3 days, cyclophosphamide and etoposide. receiving prophylaxis for gvhd with cyclophosphamide. results: a total of 10 patients were included, seven of them achieved complete remission after 2 cycles of blinatumomab, one with partial remission (table 1) , these seven patients, six received an haploidentic transplant achieving graft in 6 of the transplanted patients. one patient had a bone marrow relapse in the first 6 months of the follow-up and 5 patients are free of disease with a follow-up to 20 months (figure 1). as a acute complication the 10 patients presented cytokine release syndrome, during the infusion of blinatumumab 10 patients presented tachycardia (table 3 ) and the 6 patients presented agvhd after hsct (5 grade i-ii and i grade iv). conclusions: allogeneic bone marrow transplant constitutes a treatment option on those patients that relapse or become refractory to treatment, one of the major problem is basically to identify a hla-identical donor, the alternative is an haploidentical donor. the most important factor to get these results is the disease status before transplant. the use of blinatumomab has proven to be effective in achieving remission in relapse acute linfoblastic leukemia pre-b cd 19+ or refractory to treatment. characteristics nº % male 4 40% median age at diagnosis, (range), years 9. 22 (7-12) status of disease 2 o + 3 relapse 6 60% refractory to primary or salvage therapy 4 40% complete remission after blinatumomab 7 70% partial remission after blinatumomab 1 10% active disease 2 20% [[p031 table] 1. table n°1 . demographic characteristics of patients undergoing blinatumomab (n=10)] disclosure: a. olaya-vargas, r. rivera-luna, y. melchor-vidal, h. salazar-rosales, g. lopez-hernandez, n. ramirez-uribe. we wish to confirm that there are no known conflicts of interest associated with this abstact, the only financial support was provided by mexican associations that helping children wiht cancer in a few patients. sequential high-dose chemotherapy reinduction followed by myeloablative allogeneic transplant for active acute myeloid leukemias methods: at our center, 27 relapsed/refractory aml patients were transplanted during chemo-induced neutropenia after high-dose salvage chemotherapy. median age at transplant was 52 years (range 21-62). patients suffered from de novo (n= 18/27, 67%) or secondary aml (n=9/ 27, 33%). genetic risk stratification was reported using stardardized groups proposed by the european leukemia net (eln) in 2010. favorable, intermediate i and ii and adverse risk category at diagnosis was observed in 1/27 (4%), 19/27 (70%), 7/27 patients (26%) respectively. all patients had active disease at the time of sequential therapy and median marrow blast count was 25% (range 7-88%). patients received a high-dose cytarabine based (mec in 17/ 27, 63%) regimen as salvage therapy. donors were haploidentical relatives for 15/27 (56%) patients, identical siblings and matched-unrelated for 6/27 patients (22%) and 6/27 (22%), respectively. a myeloablative conditioning was used to further implement anti-leukemic effects. conditioning, thiotepa-busulfan-fludarabine in 89% patients, was started at a median of 8 days (range [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] after the last day of chemotherapy. bone marrow and peripheral blood stem cells were used as graft source in 11/27 (41%) and 16/27 (59%) patients. graft-versus-host disease (gvhd) prophylaxis and supportive care were administered accordingly to each hsct platform. results: all patients engrafted. median day of neutrophil recovery was day +16 (range 12-23). median follow-up of survivors was 31 months (range 4-125). non relapse mortality and relapse incidences (nrm, ri) were 16% and 48% at 1 year and 16% and 58% at 3 years, respectively. overall cumulative incidences of acute and chronic gvhd were 48% and 43% at day +100 and + 400. one and 3 year overall survival (os) were 58% and 34%, while 1 and 3 year event-free survival (efs) were 35% and 26%. significant better os and efs were observed in patients with favorable-intermediate i-ii versus adverse risk score (1-3 years os 64% and 50% vs 43% and 0% p=0.022; 1-3 years efs 43% and 36% vs 14% and 0% p=0.013). adverse risk had a significant impact on os (hr 3.24, p=0.030) and efs (hr 3.33, p=0 .018) by univariate analysis and on ri (sdhr 3.02, p=0.031) by fine and gray test. conclusions: though small the patient cohort, our findings suggest that sequential therapy with a myeloablative hsct is feasible in treating relapsed/refractory aml. transplant-related toxicity was low (16%) and relapse was the major treatment-failure. however, even with this approach, patients with adverse cytogenetic features have a very dismal prognosis. for these patients, the use of new drugs before hsct and/or maintenance therapy after transplant is highly encouraged to improve outcomes. disclosure: alessandro busca: honoraria from gilead sciences, merck, pfizer pharmaceuticals and jazz background: in spite of satisfactory results of overall survival (os) after allohsct in 1 st and 2 nd cr aml, relapse free survival (rfs) and graft-versus-host-disease free/relapse free survival (grfs) require further improvement. the detection of mrd is one of the factors which influence on the outcome of allohsct in aml is unclear but identification is important to improve risk-adapted relapse prophylactic treatment after allohsct. aim. to evaluate outcomes of allohcst in 1 st and 2 nd cr pediatric aml depending on the level of mrd status before myeloablative (mac) or reduced intensity conditioning regimens (ric). methods: the data of 72 children with aml in 1 st and 2 nd cr underwent allohsct between 2008 and 2018 were analyzed. median age at the moment of allohcst was 8 years old (2-18). mrd negative status had 42 (58%) patients, 30 (42%) were mrd positive by flow cytometry. mac based on busulfan (16 mg/b.w.) received 27 (37%) patients, on treosulfan -7 (10%) patients. ric based on melphalan received 20 (28%) patients, based on busulfan (8 mg/b.w.) -18 (25%) patients. patients received prophylaxis of agvhd by atg 20 (28%) or ptcy -48 (66%) patients plus csa -23 (32%) or tacrolimus ± sirolimus -43 (60%) patients that depended on source of transplant (related, unrelated or haplo donor) . results: at the median follow up 3 years in the cohort of mrd positive patients os is 66% vs 72% in mrd negative (p>0,05). rfs is 56% vs 83% accordingly (p=0,01). graft-versus-host-disease free/relapse free survival (grfs) in mrd positive patients is 37% vs 51% in mrd negative (p>0,05). os, rfs, grfs in mrd positive patients after mac is 57%, 42%, 30% vs 75%, 68%, 43% after ric accordingly (p>0,05). os, rfs, grfs in mrd negative patients after mac is 75%, 85%, 55% vs 65%, 82%, 47% after ric accordingly (p>0,05). os, rfs in mrd negative patients with/without ptcy is 82%, 86% vs 42%, 78% (p>0,05); grfs is 62% vs 28% accordingly (p=0,042). os, rfs, grfs in mrd positive patients with/without ptcy is 66%, 55%, 54% vs 66%, 57%, 27% (p>0,05). conclusions: mrd status does not statistically significant affect on os that can be related to different approaches to the treatment of relapse after allohsct. mrd positive status statistically significant decreases rfs that underline the necessity of posttransplant therapy improvement. ric vs mac in all patients in first and second remission do not show statistically significant impact on os, rfs, grfs. ptcy significantly improves grfs in mrd negative patients. disclosure: none of the authors has anything to disclose. background: with increasing overall-survival (os) of lymphoma patients, higher incidences of therapy-related clonal bone marrow diseases, such as acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) are occuring. generally, the outcome is considered poor. allogeneic hematopoietic stem cell transplantation (allo hsct) often remains the only potentially curative treatment option. nonetheless, there is only little data available concerning this patient group. methods: we retrospectively collected data from 33 patients with therapy-related aml (taml) and mds (tmds) after treatment for hodgkin's lymphoma (hl; n=7 and n=2) or non-hodgkin's lymphoma (nhl; n=10 and n=14), who received an allo hsct between 2000 and 2018. median follow-up of surviving patients was 3.1 years (range 2.6 months-9.4 years). background: the prognosis of relapsed/refractory acute leukemia (r/r al) is poor and the treatment is challenging. in this setting, allogeneic stem cell transplantation (allo-sct) constitutes the only curative option although the high relapse rate and non-relapse mortality (nrm). the sequential conditioning regimen followed by allo-sct has been used for persistent disease and aims to improve disease control by intensified chemotherapy, thus conceding more time for the presumed graft-versus-leukemia effect to occur. methods: the clinical outcome of r/r al with the sequential conditioning regimen combining a chemotherapy rescue followed by ric allo-sct in our center is described. patients who underwent a sequential allo-sct from 2005 to 2017 are included. the primary endpoint was progression free survival (pfs) and overall survival (os) that were estimated by the kaplan-meier method. secondary endpoints were non-relapse mortality (nrm). background: recommendations of the 2017 european leukemia net (eln) for favorable-risk genetics (frg) acute myeloid leukemia (aml) favor consolidation over transplantation, although reviews suggest advantage of autologous stem cell transplant (asct) in event free survival. our objective was to compare the progression free survival (pfs) and overall survival (os) of normal karyotype npm1 mutated without flt3 itd or allelic ratio < 0.5 (npm1+) aml patients treated with consolidation chemotherapy alone (cc), asct or allogeneic stem cell transplant (allosct). methods: retrospective review of npm1+ frg-aml patients, treated in one institution (2008 to 2017) with the following induction regimens: cytarabine (ara-c) and vp-16 with daunorubicin (ade) or mitoxantrone (mice). consolidation regimens were ara-c with daunorubicin (ac-d), idarubicin, vp-16 and ara-c (mini-ice) or highdose ara-c (hidac). in asct, conditioning regimens were bucy or bvac and in allosct were bucy or flubu. pfs and os were calculated from the start of the last consolidation or stem cell infusion. results: a total of 39 patients were evaluated, with a median age of 53 years (y) (23-68y), 69% female, 95% with ecog performance status (ps) 0-1 and 36% with ageadjusted charlson comorbidity index (aacii) ≥2 at diagnosis. patients were treated with cc in 33% (n=13), asct in 36% (n=14) and allosct in 21% (n=12) of cases. there were no differences between groups for age, aacii, ps, leucocytes at diagnosis or extra-medullary disease. flt3-itd was more frequent in allosct group (64%) than cc (23%) or asct (8%; p=0.07). at induction, ade was used in 82% and mice in 18% of patients, with a complete remission (cr) rate of 95%. there were no differences between groups for induction regimen or cr. in cc group, consolidation regimens were 1 cycle (8%) and 2 cycles ac-d (61%) and 2 cycles mini-ice (31%). asct patients received consolidation with 1-2 cycles ac-d (78%) and 1 cycle mini-ice (22%), while allosct patients received 1-2 cycles ac-d (84%), 2 cycles hidac (8%) and no consolidation in 8%. [[p037 image] 1. figure 1 . pfs at 3y in cc, asct and allosct groups.] median follow-up was 39 months, pfs at 3y was 53% and os at 3y was 64%. pfs at 3y for asct group was superior then cc and allosct groups (61%, 51% and 44%, respectively; figure 1 ), although not statistically significant. os at 3y was statistically similar between groups, although inferior in allosct comparing to cc and asct (44%, 77% and 70%, respectively). conclusions: in this historical cohort review, although there was no advantage in os for asct in npm1+ frg aml, our data suggests that there might be a pfs improvement in asct over cc, which needs to be further addressed in prospective studies. disclosure: nothing to declare background: acute myeloid leukemia is a hematological malignant disease that motivates the persistent struggle in the scientific world to provide effective cure that can establish acceptable survival rates in this group of patients. autologous stem cell transplantation with myeloablative conditioning is still a powerful weapon that can be used against this entity methods: we have evaluated retrospectively patients with aml where autologous stem cell transplantation was performed in the period from 2000 till 2018. our group consisted of 94 patients; male patients 45 (47.8%), female patients 49 (52.2%). median age at diagnosis was 44 years (16-68). the average period from time of diagnosis to autologous sct was 7.05 months. results: in the majority of our group, we used myeloablative conditioning regimen with busulphan-cyclophosphamide, 60 patients (63.8%), in 2 patients (2.1%) we have added melphalan to bu-cy conditioning, in 22 (23.4%) patients we used beam conditioning and in the rest, 10 patients (10.6%) we used bam conditioning regimen. as auto graft we used peripheral blood stem cells (pbsc) in 78 patients (82.9%), and in 16 patients (17.1%) we used bone marrow. the main mobilising regimen for pbsc was g-csf + etoposide and it was performed in 44 patients (46.8%), and in the remaining 34 patients (36.1%) mobilising of pbsc was performed only with g-csf. the mean number od apheresis procedures done in our group was 1.55, and the mean number of collected mononuclear cells was 3.05x10 8 /kg tt. the mean time to engraftment was 12.8 days (9-23). the transplant related mortality (trm) was 2.1 %. the 5 year overall survival of our patients was 46.7 patients. the main reason for death was relapse of the primary disease(73%). 20 patients (21%)were treated with salvage chemotherapy regimen (flag-ida) because with the standard induction regimen 7+3 there was absence of adequate therapeutic response, or predominantly no complete remission was achieved. all patients were transplanted in complete remission conclusions: autologous stem cell transplantation could be an acceptable therapeutic solution for patients with aml as a consolidation therapy, where neither suitable compatible donor is available nor allogeneic stem cell transplantation could not be performed from various reasons depending on the bone marrow transplant unit disclosure: nothing to declare p040 prophylaxis dli alone may not prevent relapse of flt3-itd positive aml after allogeneic hct background: one of the most potent prognostic factors affecting outcomes in aml is the presence of cytogenetic and molecular markers which can guide the selection of post-remission therapies. recently, favorable outcomes of npm1 wt /flt3-itd neg /non-cebpa dm group after allogeneic hematopoietic cell transplantation (allo-hct) have been reported, that is similar to those of favorable risk by the eln risk classification. however, the role of allo-hct compared to consolidation chemotherapy has not yet been elucidated. methods: the data of 88 patients who were diagnosed with aml and received intensive induction therapy from 2015 march to 2017 july were included in the current study. to address the time dependence of the allo-hct, the simon and makuch method was used in the graphical representation and the mantel-byar test and andersen and gill methods for identifying risk factors for long-term survival. results: median age of the patients were 53 years (range 21-69), and 49 patients (56%) were male. npm1 mutation was detected in 14 patients (16%), and flt3-itd were none, low, and high ratio in 69 patients (78%), 9 (10%), and 10 (12%), respectively. the eln risk classification divided the patients into favorable, intermediate, and adverse risk group in 31 patients (35%), 38 (43%), and 19 (22%), respectively. npn1 and flt3-itd both negative group included 29 patients (33%). allo-hct was performed in 48 patients (55%). overall, complete response (cr) after induction therapy achieved in 63 patients (72%), and 7 patients (8%) were primary refractory disease. cr rates did not differ between npm1 wt /flt3-itd negative group (n=17/29, 58.6%) and other intermediate risk group (n=6/9, 66.7%; p=0.967) . with median follow-up duration of 12.9 months (range 1.3-39.0 months), one-year os rate were 100%, 83.5±6.9%, 56.1±12.8% in favorable, intermediate, and adverse risk group (p < 0.001). among intermediate risk group, os rate of npm1 wt /flt3-itd negative group was similar to other intermediate risk (p=0.403). allo-hct was performed in 11 patients of npm1 wt /flt3-itd negative group. one-year os rates did not differ between npm1 wt /flt3-itd negative and other for allogenic hematopoietic stem cell transplant (allo-hsct), as a strategy to prolong survival. methods: data from aml pts over 60 years, who underwent ric allo-hsct in our institution between september 2011 and september 2017, was retrospectively collected from clinical files to evaluate the overall survival (os) up to november 2018. we calculated the os using kaplan-meyer curves. results: we identified 15 pts, median age 62 y.o. (60-67) and median htc-i score 2. the median follow-up was 25 months. one patient (pt) had cml blast crisis and was on first major molecular remission. of the remaining 14 aml pts, 7 were in 1 st complete remission (cr), 4 in 2 nd cr and 1 with progressive disease (pd); the other 2 pts could be classified as mds according to 2016 who diagnostic criteria and were in cr1. donors (d) were: 3 matched unrelated (mud), 5 mismatched unrelated (mmud -9/10), 6 matched siblings and 1 haploidentical. thirteen pts were infused with peripheral blood hsc and 2 with bone marrow. conditionings were: flubcnumel in 5 unrelated donor (ud) pts and 4 siblings, flumel in 1 ud pt and 1 sibling, flubu in 2 ud pts and flutbi 2gy in 1 sibling and in the haploidentical. graft versus host disease (gvhd) prophylaxis was tacrolimus (tac) + mmf in 5 ud pts and 1 sibling, tac + mtx in 2 ud pts and cyclosporine (cya) + mmf in 1 ud pt and 5 siblings. all mmud pts had atg. ptcy was done in the haploidentical setting with tac + mmf. the median time to neutrophil and platelet engraftment for the whole cohort was 14 and 11 days, respectively. one pt with secondary engraftment failure required re-infusion of selected cd34+ cells. ten pts presented with mild acute skin gvhd. eleven pts had chronic gvhd, 2 classified as severe; 7 required systemic therapy, 5 of those beyond 1 year. the median time on immunosuppressants was 404 days. at 2 years the os was 63.5%. there were 6 deaths: 3 disease-related (2 relapses at 22 and 58 months and 1 pd at d+30), 2 infection complications (2 septic shock) and 1 to secondary neoplasia. other relevant complications were hypoxemic pneumonia in 5 pts, 1 urinary sepsis, cmv and ebv reactivation respectively in 8 and 4 pts; pulmonary and renal toxicity either in 2 pts. at end of follow-up, 9 pts were in remission, 8 without negative measurable residual disease (mrd), the other mrd negative pt died of septic shock and severe intestinal gvhd. conclusions: in this small cohort, chronic gvhd and infectious complications were major causes of morbidity but there were no treatment related deaths before d+100. pts maintaining or achieving mrd negativity after transplant had better survival. although with only 15 pts, these results suggest that allo-hsct is feasible as consolidation strategy in selected aml pts over 60 years. [[p043 image] 1. overall survival] disclosure: nothing to declare. background: hematopoietic stem cell transplantation (hsct) is the only curative option for fanconi anaemia (fa); an inherited disorder characterized by congenital anomalies, progressive bone marrow failure (bmf) and a predisposition to develop malignancies. methods: we retrospectively analysed the data of 27 consecutive patients that underwent hsct at this centre from 2001 till june 2018. the data was analysed for variables affecting the outcome in terms of overall survival (os). results: median age at diagnosis was 10 years (2-20 years). median age at transplant was 11.3 years (4-25 yrs). all patients at transplant were in aplastic phase. male to female ratio was 1.2:1. twenty-four (88.9%) patients had congenital anomalies along with bmf while 3 were phenotypically normal. twenty-three (85.2%) patients were 10/10 hla matched with siblings, 2 with parents and 2 with cousins. eleven (40.7%) patients had gender mismatch transplant. three patients had major and 6 had minor abo mismatch. background: paroxysmal nocturnal hemoglobinuria (pnh) is a rare clonal non-neoplastic hematopoietic stem cell disease whose incidence is 1.5-2.9 cases/million of individuals worldwide. disease characteristics and natural history have been mostly analyzed by multicenter, retrospective studies, with the limit of heterogeneous approaches. herein we report the incidence of severe complications and outcome in a real life setting scenario of pnh patients consecutively diagnosed and managed at our pnh referral center between january 1985 and june 2018. methods: patients received a homogeneous diagnostic and treatment approach according to the period of observation (availability of diagnostic tests and eculizumab). all patients treated with eculizumab received vaccination with conjugated anti-meningococcus acwyserotypes and, since 2016, conjugated anti-meningococcus b-serotype. in the event of any complication, patients could refer to dedicated hematology emergency rooms (er) 24 hours daily. the occurrence of renal failure and pulmonary hypertension was specifically evaluated. the renal function was studied according to the cockcroft-gault formula and the lung function was prospectively monitored by daytime-on exertion, nocturnal pulsoximetric profiles and complete spirometric tests, with dlco measurement. results: overall,48 pnh patients, median age 36 years (range 17-84), were analyzed. at diagnosis, 26 patients had classic pnh, 19 aplastic pnh and 3 an intermediate form. the cumulative incidences (ci) of thrombosis, and clonal hematologic neoplasm were 29%, and 6%, respectively. ci of pancytopenia in the 26 patients with classic pnh was 23%. one patient showed a spontaneous disappearance of the pnh clone. since 2005, eculizumab was administered in 28 patients. after eculizumab treatment 50% and 32% of patients reached hemoglobin level > 11g/dl and >8< 11g/ dl without transfusion, respectively, while 18% were nonresponsive. during eculizumab treatment no thrombotic event was observed while two severe infectious episodes (respiratory tract and urinary tract infection) were observed in only one of the 28 patients. extravascular hemolysis was demonstrated in 50% of patients. no patient showed a significant reduction of the renal function.out of 24 patients prospectively monitored for lung function no pathological alteration in any diurnal or nocturnal pulseoximetric test was observed. no patient showed obstructive or restrictive ventilatory deficiency, nor reduced dlco values. 30-years overall survival (os) was 90% (4 patients who died for non-pnh related reasons were censored at the last follow-up).a better os, even if not statistically significant,was associated to the absence of thrombotic events (90%vs70%), and the period of diagnosis (100% in 2006-2018, 91% in 1996-2005, 75% in 1985-1995) . conclusions: our study reports a better os and lower rate of severe complications in pnh compared to previous experiences. although renal failure and lung hypertension have been reported by other groups, we did not observe these complications along a prolonged follow-up. we can assume that the availability of a dedicated er service enabled an early diagnosis and prompt treatment in case of hemoglobinuria crises (reducing the risk or organ damage) or other complications. the use of eculizumab, together with improved supportive approaches, presumably accounts for the trend towards a better survival witnessed over the last decade in the management of pnh patients. disclosure: nothing to declare haploidentical and unrelated allogeneic stem cell transplantation in aplastic anemia:single center experience zafer gulbas 1 , elif birtas atesoglu 1 , meral sengezer 1 , i̇mran dora 1 , cigdem eren 1 , suat celik 1 , demet cekdemir 1 background: aplastic anemia is a syndrome of bone marrow failure characterized by peripheral pancytopenia and marrow hypoplasia. allogeneic stem cell transplantation from hlamatched sibling is performed in the firstline setting in young aplastic anemia patients and in elderly patients who are refractory to immunosuppressive treatment. but if the patient does not have a hla-matched sibling, allogeneic stem cell transplantation is performed from unrelated and haploidentical donors. in this study, we analyzaed and compared the results of aplastic anemia patients who had undergone allogeneic stem cell transplantation either from matched unrelated or haploidentical donors. methods: we collected and analyzed data of aplastic anemia patients who had undergone allogeneic stem cell transplantation from matched unrelated or haploidentical donor between 2011 and 2018. results: there were 10 patients who had received allogeneic stem cell transplantation from unrelated donors and there were 10 patients who had undergone haploidentical transplantation. but in 4 patients who had undergone haploidentical transplantation, engraftment failure had occurred and they were transplanted from different haploidentical donors fort he second time. so a total of 10 unrelated and 14 haploidentical transplants were performed. the median age of patients who had undergone unrelated transplantation was 29(16-55) and the median age of patients who had undergone unrelated transplantation was 22(19-61) . the results of the haploidentical and unrelated transplantations are shown in table 1 . the neutrophil and platelet engraftment times were significantly longer in haploidentical transplantations (p=0,006 and p=0,005, respectively). however, vod, gvhd and 100 day mortality rates were not different in the 2 groups. similarly overall survival (os) of the patients who had undergone haploidentical or unrelated transplantation were not significantly different (p=0,38) ( figure 1) . conclusions: although the number of patients are low in this study, we can conclude that urelated and haploidentical transplantation in aplastic anemia have comparable toxicity and efficacy. background: autologous stem cells transplantation (ahsct) is an effective treatment for very aggressive autoimmune diseases such as multiple sclerosis (ms). however, toxicity remains the major concern to a wide application of this approach. post transplant viral reactivations may induce severe complications and a rigorous monitoring of peripheral blood viral load for a prompt and effective therapy is required. a higher rate of ebv and cmv reactivation has been observed in patients affected by ms and conditioned with beam plus atg compared with a controlled group of lymphoma patients without atg in the conditioning regimen [1] . we report here the policy of our center about both monitoring and treatment of such side effect. methods: a series of 37 consecutive patients with ms, transplanted between 2014 and 2018 is included in this analysis. all patients were mobilized with cyclophosphamide 4g/sqm + g-csf and conditioned with beam plus rabbit atg (thymoglobulin©, 7.5mg/kg). monitoring of cmv/ebv dna on whole blood by quantitative pcr was performed after the engraftment, weekly for at least one month, then at longer intervals. pre-emptive treatment with valgancyclovir was started in case of cmv viral load ³1x10^4 copies/ml. in case of ebv assay between 1x10^4 and 1x10^5 copies/ml further determinations were performed and rituximab-based treatment was started if the viral copies exceeded 1x10 5 copies. patients received treatment in case of symptomatic disease for any value of the pcr of both viruses or ebv titer ³1x10^5 copies/ml. results: detectable dna for cmv was observed in 15/ 37 (40,5%) patients at a median time from transplant of 23 days (range 12-36) and 6/37 (16%) required pre-emptive treatment. all patients promptly responded to treatment within 2 weeks. ebv viral load was detectable in 19/37 patients (51,3%) at a median time of 22 days (range 12-52). one patient out of 19 started the treatment on first determination for high viral load (>1x10^6/ml); nine presented an ebv viral load over 1x10^4 copies/ml, three of them were treated thereafter for the persistent increase of the viral load (> 10 5 /ml). six patients spontaneously recovered the ebv reactivation. previous treatments were not predictive of any higher risk of viral reactivation. no impact on engraftment related to the reactivation was observed. conclusions: this policy shows that, despite a high rate of cmv and ebv reactivation, no grade iii-iv adverse events were observed, suggesting the key role of viral monitoring in these patients and the efficacy of the preemptive treatment. ebv reactivation at low titers should be monitored to identify those cases that could achieve a spontaneous resolution and avoid the induction of further immunosuppression by rituximab. these data confirm that patients diagnosed with ad undergoing autologous hsct need a more intense pattern of care than hematological patients. background: autoimmune diseases are chronic serious conditions that are often refractory to standard therapies. since 1996, autologous haematopoietic stem cell transplantation (hsct) has been a very promising alternative that has shown satisfactory long-term results. the aim of this study is to evaluate immune reconstitution and mortality following hsct in patients with autoimmune disease. methods: a retrospective study was conducted on patients with diagnosis of autoimmune diseases that underwent autologous hsct between july 2012 and january 2018 at a tertiary referral center in colombia, south america. descriptive statistics were used to analyze patient's demographic and clinic characteristics. results: seven patients were included, with a mean age of 37 years (range 26-57). five patients were female (71%). the indications for hsct were systemic sclerosis (n=3) , multiple sclerosis (n=2), and myasthenia gravis (n=2). the conditioning regimen administered in patients with systemic sclerosis was cyclophosphamide + human anti-t lymphocyte immunoglobulin, beam (carmustine, etoposide, cytarabine (ara-c), and melphalan) + human anti-t lymphocyte immunoglobulin in patients with multiple sclerosis, and cyclophosphamide + human anti-t lymphocyte immunoglobulin in myasthenia gravis. median time to myeloid engraftment (neutrophils>0.5×109/l) was 12 days post-transplantation, and platelet engraftment, defined as >20,000 platelets/mm 3 untransfused, was 11 days post-hsct. median time of hospitalization was 21 days (range 11-66); longer in-patient management was due to infectious complications. infectious complications included bacteremia caused by e. coli and pneumocystis pneumonia that resulted in septic shock and acute respiratory failure, respectively. evaluating t-cell immune reconstitution, none of the patients had reached normal cd4+ cell value after one year of hsct follow-up. four patients (57.1%) reached normal cd8+ cells value at 3 months post-hsct. regarding bcell immune reconstitution, 85.7% of the patients (6/7) had reached both igg and iga normal levels at one-month post-hsct, and four patients (57.1%) had achieved normal igm background: multiple sclerosis(ms) is an inflammatory disease caused by autoimmune reactivity of t cells against myelin. there is accumulating evidence of the efficacy of highdose chemotherapy followed by autologous haematopoietic stem cell transplantation(ahsct) in ms patients who failed response to standard immunotherapy, despite a variability in eligibility criteria, conditioning regimens and outcome. methods: we retrospectively reviewed ms patients submitted to ahsct in our centre (2014) (2015) (2016) (2017) (2018) . patient eligibility criteria were active relapsing remitting(rrms) or secondary-progressive ms (spms), with prior failure to treatment with disease-modifying therapies and evidence of disease activity (clinical relapse or new active lesions in magnetic resonance [mr] ). mobilization of cd34+cells to peripheral blood was performed with granulocyte colony-stimulating factor(g-csf 10μg/kg/day) and conditioning regimen according to beam protocol. the severity of ms disability was classified according to the expanded disability-status scale (edss) and ahsct toxicity was evaluated by ctcaev5.0. results: seven ms patients had undergone ahsct (4 female/3 male), with a median age at ahsct of 39.9 years (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) . median age at ms diagnosis was 31.6 years(27-36) and median time from diagnosis until ahsct was 6.8 years (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) . four patients (57.1%) had spms and 3(42.9%) had rrms, with 4(57.1%) having mr active lesions. pre-ahsct relapse rate per year was 2 (1) (2) (3) (4) . median baseline edss was 6.5(5.5-7.5). median number of previous dmts was 6 (3) (4) (5) (6) (7) (8) (9) . all patients had been treated with corticosteroids and copaxone, 5(71.4%) received rituximab, 6 (85.7%) natalizumab, 1 (14.3%) alemtuzumab, 2(28.6%) fampridine and 2(28.6%) fingolimod. all patients collected enough cd34+ cells in a single apheresis session. median number of cd34+ cells infused was 7.2±3.8x10^6/kg, for a mean dmso volume of 50.4 ±14.8 ml. median inpatient stay during ahsct was 28 days(21-47). all patients developed febrile neutropenia, one was admitted to intensive care unit due to sepsis. one patient developed an anaphylactic reaction to transfusion and another a self-limited encephalopathy. two (28.7%) patients had grade≥2 oral mucositis, and all had gastrointestinal toxicity (grade 2-3). median time until neutrophils>500/μl was 11(6-12) days, to platelets>20,000/μl was 9 days(5-12), and to engraftment was 13 days (12) (13) (14) . patients were transfused with a median of 1(0-2) unit for erythrocytes and 3(1-8) for platelets. there was no treatment-related mortality and no long term side effects have been observed so far. for a median post-ahsct follow-up of 8.7 months, no patient developed new lesions in mr, but 2(28.6%) experienced symptoms consistent with a clinical relapse, at a median time of 20.5(5-35) months, effectively rescued with corticosteroids. the absence of evidence of disease activity at 6-months was 71.4%. although there was no variation concerning edss punctuation, 4(57.1%) patients self-reported significant benefits, especially concerning limb strength and sphincter continence improving. conclusions: our real life results claim a stabilization effect of ms patients with highly active/progressive disease after ahsct, with no significant toxicity. the failure in reporting benefits in edss punctuation is probably due to a small sample size and short follow-up. more studies are needed to establish the best patient selection criteria and define the ideal time to include these patients in transplantation programs, as well as to evaluate its long term outcome. disclosure: nothing to declare. elena poponina 1 , elena butina 1 , anna yovdiy 1 , galina zaytseva 1 , natalia minaeva 1 , igor paramonov 1 background: reactivation of epstein-barr virus (ebv) represents a potentially life-threatening condition in approximately 30% of patients after allogeneic stem cell transplantation, with no specific treatment available. methods: we have previously developed a manufacturing protocol for the expansion of cytomegalovirus (cmv) and ebv-specific t cells by stimulation of g-csfmobilized stem cell grafts with defined peptide pools (gary, 2018) . this advanced therapeutic medicinal product is currently investigated in an ongoing phase i/iia trial (eudract number: 2012-004240-30) . however, the expansion of virus-specific t cells relies on a pre-existing virusspecific memory compartment in the stem cell donor. in virus-seronegative donors, no expansion can be achieved. we therefore aim to identify ebv peptide-specific t cell receptors (tcrs) that can be translated into off-the-shelf cell products for the treatment and prophylaxis of ebv infection and ebv-associated malignancies. leftover cells from five allogeneic stem cell grafts were expanded in vitro in the presence of hla-b35*01-restricted peptides (hpvgeadyfey from ebna1, eplpqgql-tay from bzlf1) associated with latent and lytic ebv infection. after expansion, single ebv-specific t cells were facs sorted using peptide-mhc (pmhc) tetramers, and individual tcr αand β-chain pairs were determined with single cell sequencing (han 2014 , penter 2018 . to confirm peptide specificity, dominant tcr pairs were transfected into a murine 58αβreporter t hybridoma cell line with nfat-driven gfp expression (siewert, 2012) . functional tcrαβ candidates were transduced into human peripheral background: lymphoid and myeloid acute leukemia are the most frequent cause of cancer related death in children. interactions between nkg2d receptor, expressed in cytotoxic immune cells, and its ligands (nkg2dl), that are upregulated in many types of tumor cells including leukemic blasts, are important for anti-tumor immune surveillance. nevertheless, tumor cells may develop immune scape strategies like ligand shedding, which reduces nkg2dl expression and may cause nkg2d receptor downregulation. engineering t lymphocytes with nkg2d car may overcome immune evasion and become an effective therapeutic strategy. methods: cd45ra -t cells were obtained by depletion of non-mobilized apheresis with cd45ra magnetic beads using clinimacs. nkg2d-car t cells were generated by lentiviral (nkg2d-41bb-cd3z) transduction of cd45ra -t cells with moi=2. the expression of nkg2d ligands was analyzed in peripheral blood or bone marrow samples from a total of 97 leukemia patients (aml=13, b-all=52 and t-all=19), at different status of the disease (diagnosis, remission, relapse/refractory), and in 10 different leukemia cell lines by qpcr and flow cytometry. cytotoxicity of nkg2d-car t cells against leukemia cells was evaluated by performing conventional-4 hours europium-tda assays. soluble nkg2dl (snkg2dl) concentration was measured in the sera of leukemia patients by elisa. to evaluate the effect of snkg2dl on nkg2d-car t cells, those were cultured in the presence or absence of different concentrations of snkg2dl for 7 days. one week later, cell proliferation and car downregulation were measured by flow cytometry using cell trace violet and nkg2d labeling, respectively. the production of ifn-g and tnf-a was measured in the supernatants by elisa. the effect on cytotoxicity was evaluated in a 2 hoursdegranulation assay by co-culturing snkg2dl pretreated nkg2d-car t cells against k562 cell line. results: nkg2d ligands were expressed in leukemia cell lines and leukemic blasts. nkg2dl expression changed with disease status with a trend to decrease at diagnosis and relapse/refractory compared to remission. nkg2d-car t cells were cytotoxic against 8/10 leukemia cell lines with a percentage of specific lysis over 50%. myeloid and t-all cell lines were more susceptible to nkg2d-car t cells (specific lysis ranging from 50-78%) compared to b-all cell lines (19-52%). physiological concentrations of snkg2dl caused an increase in nkg2d-car expression. however, supra-physiological levels of snkg2dl decreased nkg2d-car expression up to 5 times and increased cell proliferation up to 4 times. cd4+ subpopulation was more affected by downregulation, while proliferation had more impact on cd8+ subset. the effects of snkg2dl were dose-dependent and attenuated by il-2. conclusions: nkg2d-car t cells are cytotoxic against leukemia cells, specially aml and t-all, and thus could be a novel therapeutic approach for non-b leukemia, or those b-all that relapse with undetectable cd19 after cd19-car treatment. nkg2d-car expression may be downregulated only by supra-physiological levels of snkg2dl, although antitumor activity is not affected. il-2 softens the negative effects of snkg2dl inducing nkg2d expression, cell proliferation and cytokines production. the changes observed in nkg2dl surface expression at the different stages of the disease could be related to ligands release and immune escape. disclosure: nothing to declare denis-claude roy 1,2 , ines adassi 1,2 , céline leboeuf 1,2 , vibhuti p. dave 1,2 1 hôpital maisonneuve-rosemont research center, montréal, canada, 2 university of montréal, montréal, canada background: for patients with high-risk leukaemia, allogeneic haematopoietic stem cell transplantation is the only curative treatment. the presence of alloreactive t cells in the donor graft, however, leads to a high probability of developing graft-versus-host disease (gvhd) . t-cell depletion minimises the presence of gvhd-causing alloreactive cells, but often results in an increased incidence of infections and disease relapse. photodepletion treatment (pdt) can specifically deplete activated alloreactive t cells while conserving resting t cells. pdt-treated cells have been utilised after t-cell-depleted haploidentical transplant to help reduce infection and relapse. the efficacy and safety of such pdt-treated cells is currently under clinical investigation in a phase iii trial (hatcy, nct02999854; kiadis pharma). here the reactivity of pdt-treated donor t cells was assessed toward tumour-associated and viral antigenic peptides derived from wilm's tumour protein 1 (wt1p), preferentially expressed antigen in melanoma (pramep), and from cytomegalovirus and epstein-barr virus (cmv/ ebvp). methods: healthy donor (hla-a*0201) peripheral blood mononuclear cells (pbmcs) were co-cultured with irradiated pbmcs from another mismatched donor (1:1) in a 4-day mixed lymphocyte reaction. th9402, a photoactive rhodamine derivative, was added and cells were exposed to visible light to deplete the th9402-containing activated alloreactive cells. elimination of alloreactive cells post-pdt was assessed using cd25 and hla-dr as activation markers. an ex vivo expansion protocol was exploited to evaluate the impact of pdt on reactivity to tumour and viral antigenic peptides. post-pdt t cells were co-cultured with irradiated autologous monocyte-derived dendritic cells (10:1) pulsed with wt1p, pramep or cmv/ebvp. antigen-specific t cells were re-stimulated on days 7 and 14 with wt1p-or pramep-pulsed autologous pbmcs or with cmv/ebvp added directly to the culture. mhctetramer staining was performed on days 14 and 21; ifn-γ elispot was conducted on day 21. [[p057 image] 1. functional wt1-specific and viralspecific t cells can be expanded post-pdt] results: pdt resulted in a drastic decrease of cd25 and/ or hla-dr activation marker-expressing cd4+ and cd8 + t cells. pdt-treated cells showed a significant increase in background: acute lymphoblastic leukemia (all) is the most common childhood cancer and relapsed or refractory all is still difficult to treat. engineered t cells equipped with a synthetic chimeric antigen receptor (car) targeting cd19 have demonstrated remarkable efficacy to treat all. however natural killer (nk) cells are known for their target-independent cytotoxic potential without induction of cytokine release syndrome (crs) or graft-versus-hostdisease (gvhd), car-nk cells can overcome the persisting problem in the therapy with car t cells. as the use of viral vector generated car nk cells is limited by theire genotoxicity, cost and regulatory demands, we are developing an innovative protocol using non-viral sleeping beauty (sb) transposition of third party nk cells as a source to produce 'off the shelf' car-engineered cell products. methods: nk cells are isolated from peripheral blood mononuclear cells (pbmcs) using cd56 selection kits. they are successfully expanded ex vivo with il-15 cytokine stimulation under feeder-cell free conditions. after few days of expansion nk cells are electroporated using pmaxgfp. transfection efficiency and percent of living cells after electroporation is analyzed by flow cytometry. transposition based nucleofection using an sb100x mrna and a minicircles (mc) dna vector is performed at different time points after nk cell isolation. the transient mc-venus longtime expansion and the viability after sb100x based nucleofection is measured over two weeks. furthermore, α-retroviral (α-rv) cd19-car transduction of nk cells with different viral amounts (moi) is conducted and the cytotoxicity of the engineered cd19-car-nk cells against the cd19 positiv cell line supb15 is addressed. results: for an α-rv cd19-car transduction of maximal 1x10 4 nk cells we could show transduction efficiency of 68,96% for moi10. the α-rv cd19-car modified nk cells had a high killing activity against cd19 positiv supb15 cells (e:t ration 1:1 90,85%) compared to cd19 negativ k562 cell lines (e:t ration 1:1 16,42%) and the non-transduced nk cells (e:t ratio 1:1 9,03%). in first experiments with pmaxgfp vector based nucleofection, we could show an increasing efficiency of 55,9% 48h post electroporation with a only slightly decreas of living cells (21,5%) comparing to the non-electroporated nk cell viability. using sb100x mrna with mc-venus dna we electrotransfected 1x10 6 nk cells after fews days of cultivation and we reached 36,6% of transfected nk cells 24h post electroporation and a transient expression of mc-venus positive nk cells up to 54,6% efficiency with an increasing rate of live cells over 14 days after electroporation. conclusions: the sleeping beauty based nucleofection of nk cells is a very promising non-viral method to generate more easy, safer and higher amounts of genetically modified third party nk cells for therapy of all and has also a broad range of clinical applications. disclosure: winfried s. wels is an inventor on a patent describing chimeric antigen receptors with an optimized hinge region. axel schambach is an inventor on a patent describing alpharetroviral sin vectors. michael hudecek and zoltan ivics are inventors on patents related to sleeping beaut gene transfer technology. the remaining authors have nothing to disclose. background: mature immune cells from the stem cell graft are essential for the graft-versus-tumor (gvt) effect to eliminate residual malignant cells after hematopoietic stem cell transplantation (hsct), but donor cells are also involved in complications such as graft-versus-host disease (gvhd). methods: we performed a prospective study of the detailed graft composition in 102 recipients of peripheral blood stem cells (pbsc) or bone marrow (bm) in order to identify correlations to clinical outcomes, table 1. grafts were characterized with concentrations of t cells and nk cells together with a multi-color flow cytometry panel with staining for tcrαβ, tcrγδ, vδ1, vδ2, cd3, cd4, cd8, hla-dr, cd196, cd45ro, cd45ra, cd16, cd56, cd337 and cd314 for detailed immune phenotyping. cell contents in stem cell grafts were analyzed both as fractions of cd45 positive lymphocytes and as absolute concentrations converted to transplanted cells/ kg. fractions were evaluated in patients receiving both bm and pbsc (n=102), while concentrations (cells/kg) were only analyzed in patients transplanted with pbsc (n=88). table] 1. table 1] [[p059 image] 1. figure 1 ] results: we found, that patients transplanted with graft nk cell doses above the median of 27x10 6 /kg and fractions of nk cells out of lymphocytes above the median of 8.1% had significantly increased relapse-free-survival compared to patients transplanted with grafts containing nk cell doses below these values, figure 1 ; results stayed significant in multivariate analyses. relapse incidence was significantly lower in uni-and multivariate analyses in patients receiving grafts with high nk cell fractions compared with low fractions, p=0.01, with 1-year relapse rates of 8% versus 27% in patients transplanted with high versus low fractions of nk cells, p=0.01. peripheral blood concentrations of nk cells obtained from samples from 17 pbsc donors before g-csf mobilization were significantly correlated to graft concentrations-and fractions of nk cells.. analyses of graft contents of nkt cells showed that the incidence of grade ii-iv acute gvhd were significantly lower in patients background: extracorporeal photopheresis (ecp) is an immunomodulatory treatment that has shown efficacy in steroid refractory acute gvhd, but the mechanism of action is only partially understood. there is no clear relationship between the ecp-treated mononuclear cells (mnc) or lymphocyte numbers and response to ecp. the objective of the study was to analyse the relationship between the infused subpopulation cellularity and response. methods: 65 patients from 7 different centers with a total of 1008 ecp procedures were retrospectively analized. ecp procedures were performed from january-2011 to june-2017. all ecp procedures were performed with the off-line system. the response was defined as responder (complete and partial response) and non-responder. infused cell numbers for lymphocytes, monocytes and mononuclear cells (lym+mon, mnc) were calculated. for analytic purposes, the median number of cells infused per procedure until response (or until the median number of procedures until response for non-responding patients) and the cumulative number of cells infused until response (or until the median in non-responders) were calculated for all the subgroups. same procedures were performed with the number cells infused until day 30 of ecp. finally, the response and survival impact of infusing a number of cells above or below the median and in different tertiles was assessed until the median number of procedures needed to achieve a response. [[p060 image] 1. results: the median number of procedures until response was 3. we observed a trend towards a higher median number of monocytes per procedure and cumulative infused monocytes in responding patients (median number infused 19.0 vs 13.5 x10 6 /kg p=0.071, cumulative infused median number 71.5 vs 41 x10 6 /kg p=0.067) that was lost in the day 30 of treatment. there was also a trend toward higher median infused mnc until response for responders (54 vs 37 x10 6 /kg p=0.087). we observed no differences in the number of lymphocytes infused, but patients who received a number of lymphocytes per procedure over the first tertile (31 x10 6 /kg) presented higher response rates (75% vs 45%, p=0.0317). none of the other analysed parameters showed a significant impact in overall survival. conclusions: patients with acute gvhd who responded to ecp received higher numbers of monocytes and mnc in the early phase of the treatment (a median of the first 3 processes). also the patients who received higher numbers of lymphocytes in the first procedures achieved a higher response rate. these findings suggest the possibility that higher number of treated and infused cells could influence the response to ecp, but specifically designed prospective studies are need to asses this possibility. disclosure: nothing to declare background: the field of kidney transplantation has made enormous progress over the last decades towards being a standard treatment for patients with end-stage renal disease. however, administration of immunosuppressive drugs is still one of the major limitations of long-term allograft survival. therefore, strategies for induction of donorspecific tolerance are highly desirable. to this aim, a clinical phase i study with donor-derived modulated immune cells (mics) was conducted. methods: donor-derived mics were manufactured under gmp conditions. potency of mics was tested by different in vitro bio-assays. mics were administered to patients with an escalation from 1.5 x 10 6 mics/kg on day -2 (n=3, group a), to 1.5 x 10 8 mics/kg on day -2 (n=3, group b) or on day -7 (n=4, group c) before kidney transplantation accompanied by standard immunosuppressive medication post-transplantation. frequency of adverse events (ae) was assessed from day 30 until day 360 post-transplant. dynamic changes of various lymphocyte subsets in patients after mic therapy were detected by multicolor flow cytometry. donor-specific immunosuppression was assessed by measuring anti-donor antibodies and mixed lymphocyte reaction (mlr) against donor and thirdparty cells. results: in all kidney transplant recipients, we observed a median serum creatinine of 1.4 mg/dl at day 30 which remained stable until day 360 (median creatinine of 1.4 mg/ dl) without significant proteinuria. none of patients experienced rejection episode. 69 aes were observed while three aes being severe. most importantly, none of them was associated with mics transfusion. besides two infectious complications, no post-transplant positive cross match results against the donor or titers of de novo donorspecific antibodies were recorded. notably, immunosuppressive therapy could be reduced without signs of rejection in group c. after infusion, we observed a dramatic increase of cd19 + b cells up to a median of 300 cells/μl until day 30, followed by a reduction to 35 cells/μl on day 180 in group c. notably, regulatory b cells significantly increased from a median of 2% on day 30 to 20% on day 180. in parallel, the plasma il-10/tnf-α ratio increased from a median of 0.05 before cell therapy to 0.11 on day 180. after mic cell therapy recipient lymphocytes showed no or only minimal reactivity against irradiated donor pbmcs in vitro, while reactivity against 3 rd -party-donor pbmcs was not impaired. moreover, in vitro mics product demonstrated their immunomodulatory potency by inducing tolerogenic dendritic cells (tdcs) characterized by low expression of costimulatory (cd80, cd86) and maturation (cd83) as well as high expression of inhibitory marker cd103. functionally, tdcs could inhibit not only the release of ifn-γ but also the proliferation of cmv specific cd8 + t cells. moreover, mic-induced tdcs showed the capacity to inhibit donor-specific allo-reactive cd4 + and cd8 + t cell proliferation. conclusions: mic cell therapy modulates the immune system of kidney transplant recipients by increasing the ratio of regulatory b cells and facilitates the reduction of conventional immunosuppressive therapy without allograft injury or rejection episodes. therefore, mic cell therapy represents a promising strategy in transplantation medicine. we currently prepare a phase ii trial with mic cell therapy. disclosure: nothing to declare p062 genome editing of graft-derived t cells for postbackground: immunotherapy using car t cells has shown promising results to fight cancer. however, car-t cell production requires specialized infrastructure and operators, which implies high cost and centralized production. automated production of car-t cells in clinimacs prodigy device allows clinical-grade manufacturing of car t cells. methods: 100 million cd45ramemory t cells from healthy donors were cultured in texmacs supplemented with 100 iu/ml il-2. at day 0 cells were activated with t cell transact for 24h. at day one, activated cd45ramemory t cells were transduced with nkg2d-cd8tm-41bb-cd3z lentiviral vector at moi = 2. then, nkg2d-car t cells were expanded for 10-13 days. nkg2d-car t cell products were next harvested, counted and analyzed for viability, nkg2d-car expression and anti-tumor cytotoxicity. different quality tests including sterility, vector copy number, genetic stability, quantification of viral particles in the supernatant, myc/tert expression and endotoxin detection were performed. spare cells were cryopreserved either in autologous plasma and 10% dmso, m199 35% albumin and 9%dmso or hypothermosol. after 12 months, cryopreserved nkg2d-car t cell products were analyzed for viability, nkg2d-car expression and cytotoxicity. results: nkg2d-car memory t cells expanded up to 2076 ± 697 million with 77,8 ± 20% nkg2d-car expression and 76 ± 10% viability. harvested car t cells showed 90 ± 14% of specific lysis against jurkat cells and 31 ± 16% against 531mii osteosarcoma cell line. no microbiological contamination was observed in final car t cells products. vector copy number was ≤5 in all validations except for one. cgh and karyotype showed no genetic alterations. free viral particles were undetectable in the supernatants. no overexpression of myc/tert was found except for one validation. endotoxins were ≤0.25eu/ml. all cryopreserved nkg2d-car t cell products kept nkg2d-car expression one year after freezing. however, viability and cytotoxicity was best preserved using autologous plasma 10%dmso. conclusions: automated production of large-scale clinical-grade nkg2d-car t cells using clinimacs prodigy is feasible and reproducible, allowing a decentralized protocol to generate car t cells for clinical use. background: immune reconstitution (ir) is essential to control severe infections after hematopoietic stem cell transplantation (hsct). reconstitution of adaptive immunity may take up to 2 years to recover t-lymphocytes (lt). delay in early lt recovery increases the risk of relapse, viral infections and transplant related mortality. adoptive transfer of selected t cell subset with low alloreactivity potential is emerging as a strategy to improve ir. methods: depletion of cd45ra+ naive t cells, preserving cd45ro+ memory t cells could provide functional lymphocytes to protect against infection and leukemia relapse with low risk of graft versus host disease (gvhd). we present our experience with high-dose donor cd45ro + memory t cell as donor lymphocyte infusions (dli) to assess safety and outcome. a total of 58 dli of cd45ro+ after hsct was performed in cases of cmv/ebv reactivation (50%), mixed chimerism (26%), persistent lymphopenia (8,5%), graft rejection (3,5%) , relapse (3%) or to boost ir (7%). dli product was obtained performing a cd45ra depletion on donor leukapheresis product using the clinimacs® device. results: twenty-two pediatric patients, median age 11 years (range 3-18), with malignant (n=15) and nonmalignant diseases (7), received cd45ra+ (n=14), tcr alpha/beta (n=2) depleted grafts from haploidentical and cd45ra+ depleted grafts from match unrelated (n=6) and match related (n=2) donors. at a median of 97 days (range 15-462) after transplantation, patients received a total of 58 dli of cd45ro+ cells, median 2 (range 1-6), containing a median of 2.87x10 7 /kg (range 4.8x10 4 -2x10 8 /kg), cd3 +cd45ro+ 1.05x10 7 /kg (range 4.8x10 4 -1.09x10 8 /kg) and cd45ra+ cells 5 x10 2 /kg (range 0-9.8x10 4 /kg). all infusions were well-tolerated and did not develop or worsen gvhd. a total of 11/29 episodes of cmv/ebv viral reactivations decreased viral load, 4/29 cleared viral load and 5/29 showed a clinical improvement. a total of 4/5 patients with persistent lymphopenia there was a slightly increase in total lymphocyte count, but not to normal levels. prophylactic dli of cd45ro+ to boost ir increased lymphocyte count in 2 of 3 cases. none of the dli administered in cases of mixed chimerism, graft failure or relapse were effective in reverting those situations. conclusions: our preliminary data suggest that infusions of high dose cd45ro+ memory t cells are a safe adoptive immunotherapy strategy. efficacy has been observed in patients with lymphopenia and cmv/ebv reactivation, with no positive results in patients with mixed chimerism, graft failure and relapse. however, to determine the real efficacy of this strategy, prospective studies are required. disclosure: nothing to declare. background: increasing clinical trials have confirmed that chimeric antigen receptor t cells (car-t) targeting cd19 antigen (car-t-19) is a promising effective approach for the treatment of relapsed/refractory(r/r) b-cell lineage malignancies. considering cd19 is frequently expressed in large part of t(8;21) acute myeloid leukemia (aml) cells, we suppose that car-t-19 may be used as an approach to rescuing r/r t(8;21) aml patients. methods: both patients received lymphodepletion chemotherapy with decitabine 20mg/m 2 ×5d, fludarabine 30mg/m 2 ×3d and cyclophosphamide 300mg/m 2 ×3d (dac +fc). two days after chemotherapy, autologous/allogeneic cart-19 cells provided by the unicar-therapy biomedicine technology co.(shanghai, china) at a total dose of 5-10×10 6 cells per kilogram(kg) were infused dose escalation within 2 to 3 days. the research protocol was approved by the institutional review boards of the first affiliated hospital of soochow university and both patients gave written informed consent. results: both cases responded well with transient and reversible toxicities. case 1 presented with grade 1 cytokine release syndrome (crs), manifested by intermittent fever and chill from day 4 after car-t-19 infusion for half months associated with neutropenia. car-t cells expansion were observed in blood without obvious increase of cytokines. after infusion, case 1 achieved and maintained molecular complete remission (cr) for more than 10 months. case 2 presented with grade 3 crs manifested by continuous high fever, hypotension and grade 1 liver disfunction from day 1 after car-t-19 cell infusion for 1 week. obvious cytokines releasing (peak il-6 serum concentration 1774.5 pg/ml, peak crp serum concentration 367pg/ml) were detected which were associated with car-t-19 cell expansion in blood and no severe off-tumor effect was observed. after infusion, case 2 achieved hematological cr and cytogenetic cr and got 3 months disease free survival. conclusions: our report implicates that car-t-19 is a safe and promising approach to managing r/r t(8;21)aml with cd19 expression, and may provide a salvage treatment approach for all aml patients with cd19 expression and benefit a certain population with aml besides b-linage malignancies. clinical trial registry: na disclosure: nothing to declare. this work was supported by research grants from the national key r&d program of china (2016yfc0902800), national natural science foundation of china (81873443, 81270645, 81400155, 81500146) background: chimeric antigen receptor engineered t (car-t) cells have emerged as a powerful cellular therapy to treat malignant disease, which is currently revolutionizing field of cancer immunotherapy. a cryopreservation step postmanufacture is not only a logistical necessity for large scale cell manufacturing processes but also a mandatory request by regulatory authorities. in case relapse after 1 st car-t cell transplantation, a second application, maybe at a higher dose constitutes a therapeutical option. however, data concerning clinical grade car-t cell stability and functionality after months of cryopreservation have not been released by companies so far. to investigate the effect of cryopreservation on car-t cells, we performed this study. methods: different batches of cd19 car-t cells were manufactured according to gmp requirements at our institution. final car-t products were frozen at concentrations of 1 x 10 7 cells/ml (high batch) and 2 x 10 6 cells/ml (low batch) by a controlled freezing process with the biofreeze bv40 device and stored in liquid nitrogen tanks below -150°c until release. quality control tests for sterility, endotoxin and mycoplasma were performed for each batch according to european pharmacopoeia and united states pharmacopoeia guidelines. stability of cd19 car-t cells in terms of viability, recovery, transduction efficiency and functional capacity were determined by microscopy, multi-parametric flow cytometry as well as chromium-51 release tests following our sops. results: all the results of quality controls fully met the requirements of the regulatory authorities. stability results were highly robust and reproducible over time for all our gmp car-t batches. duration of cryopreservation (up to 90 days) had no negative influence on cell viability, recovery of viable cd19 car-t cells and transduction efficiency. however, the cell concentration for cryopreservation has a significant impact on the post-thawing viability (low batches vs. high batches: 96.33 ± 2.17 vs. 74.87 ± 8.68, p < 0.05) and recovery (low batches vs. high batches: 89.67 ± 6.76 vs. 74.90 ± 9.19, p < 0.05) of cryopreserved cd19 car-t cells, but not the transduction efficiency. moreover, we observed four transient side-effects of cryopreservation on the amount of cytokines released by car-t cells, the cytokine release on a per-cell basis, the multifunctionality of car-t cells and the killing capacity. of note, functional capacity of cryopreserved car-t cells after overnight resting was comparable or even enhanced for inf-γ and tnf-α release by cd4 + and cd8 + cd19 car-t cells when compared to fresh car-t cells. the multi-functionality of car-t cells could be preserved. furthermore, the killing capacity of cryopreserved cd19 car-t cells after overnight resting could reach the level of non-cryopreserved/fresh car-t cells. conclusions: cryopreservation up to 90 days has no harmful effect on transduction efficiency and functionalities of car-t cells. however, the cell number per milliliter freezing medium matters. dose over 1 x 10 7 cells/ml should be avoided. for the conduction of in vitro bio-assays to determine the function of car-t cells, an overnight resting process could mimic the situation after clinical application and eliminate the transient side-effects of cryopreservation to fully regain the functional potency of car-t cells. disclosure: nothing to declare background: dc and specific t-cells are important mediators of ctl-responses. we could already show that allogeneic donor-or autologous t-cells obtained from amlpatients can be stimulated by dc leu , resulting in a very efficient lysis of naive blasts. methods: chemokine-release (cxcl8, -9, -10, ccl2, -5, and il-12) was analysed by cytometric bead array in serum of aml/mds-pts as well as in supernatants from 5 different dc-generating-methods and correlated with pts' clinical course, dc-and t-cell-interactions as well as specific t-cell-reactions. the lytic activity of dcleu/blast -stimulated t-cells in mlc against naive blasts was quantified in a cytotoxicity assay. results: minimal differences in median chemokine-levels in pts' serum subdivided in subtypes were seen, but higher release of cxcl8, -9, -10 and lower release of ccl2 and -5 tendentially correlated with more favourable subtypes (< 50 years of age, < 80% blasts in pb). in persisting disease, a higher serum-release of ccl5 and at relapse a significantly higher ccl2-release were found compared to first diagnosis -pointing to a change of 'disease activity' on a chemokine level. whereas chemokine-levels in dc-culture supernatants compared to serum were variable, clear correlations with lateron (after stimulating t-cells with dcleu in mlc) improved antileukemic t-cell activity were seen: higher values of all chemokines in dc-culture supernatants always correlated with improved t-cells' antileukemic activity (compared to stimulation with blast-containing mnc as control) -whereas with respect to the corresponding serum values higher release of cxcl8, -9, and -10 but lower values of ccl5 and -2 correlated with higher probabilities to improve antileukemic activity of dcleu-stimulated (vs. blaststimulated) t-cells. predictive significant cut-off-values could be evaluated separating the groups compared. moreover, correlations with lateron achieved response to immunotherapy and occurrence of gvhd were seen: higher serum values of cxcl8, -9, -10 and ccl2 and lower values of ccl5 correlated with achieved response to immunotherapy. predictive cut-off-values could be evaluated separating the groups compared in 'responders' and 'non-responders'. higher levels of ccl2 and -5 but lower levels of cxcl8, -9, -10 correlated with occurrence of gvhd. conclusions: we conclude, that in aml-pts' serum higher values of cxcl8, -9, -10 and lower values of ccl5 and in part of ccl2 correlate with more favorable subtypes and improved antitumor'-reactive function. since in dcculture supernatants higher values of all chemokines correlated with improved antileukemic t-cell reactivity we conclude a change of functionality of ccl5 and -2 from an 'inflammatory' or 'tumor-promoting' to an 'antitumor'reactive function. this knowledge can contribute to develop immune-modifying strategies that promote antileukemic adaptive immune-responses. disclosure: nothing to declare background: allogeneic hematopoietic cell transplantation (allo-hct) is a curative treatment option for patients suffering from hematologic malignancies. infusion of donor lymphocytes (dlis) can induce sustained remission in case of minimal residual disease or relapse through potent graftversus-leukemia (gvl) effects, although graft-versus-host disease (gvhd) represents a common dose-limiting toxicity. as invariant natural killer t (inkt) cells are known to prevent gvhd while promoting beneficial anti-tumor effects, we investigated the role of inkt cells for successful dlis. methods: we analyzed dli samples by flow cytometry. inkt cells were identified by staining with pbs57-loaded cd1d tetramers. culture-expanded and purified dli-inkts were then tested against tumor cell lines and primary leukemia cells in an ex vivo tumor control model. tumor cell viability after coincubation with dli-inkts was measured by flow cytometry using 7-aad. results: inkt cells represent 0.05% (range 0.001-0.55%) of donor lymphocytes and can be expanded 300fold following a two-week protocol with a preferential expansion of cd4+ inkt cells. tumor cell lines such as jurkat were efficiently lysed after coincubation with dli-inkts. cd107a as a marker of degranulation was significantly upregulated on dli-inkts after stimulation by jurkat. in addition, increased concentrations of tnfα, ifn-γ, sfasl and perforin were measured after coincubation of dli-inkts with jurkat. we observed that tumor cell lysis correlated with the expression of the mhc-i-like molecule cd1d. consequently, adding a cd1d antibody to the coculture abrogated the dli-inktmediated kill of tumor cells. dli-inkts also efficiently lysed primary leukemia cells such as aml blasts: expression of cd1d on these aml blasts significantly correlated with dli-inkt-mediated tumor cell lysis (r 2 =0.7, p=0.03). conclusions: ex vivo expansion of dli-inkts and subsequent dli enrichment is an immunotherapeutic approach that could improve leukemia control and thus, prevent relapse after allo-hct without exacerbating gvhd. disclosure: nothing to declare. generation of antigen-specific cytotoxic t lymphocytes targeting wt1 using activated b cells sun ok yun 1 , kyung won baek 1 , hee young shin 1 , hyoung jin kang 1 1 seoul national university, seoul, korea, republic of background: the wilms tumor antigen 1 (wt1) is highly expressed in many malignancies including leukemia and targeting wt1 as a tumor associated antigen (taa) in cancer immunotherapy is attractive. in this study, we generated wt1-specific cytotoxic t lymphocytes to confirm if activated b cells can act as a cancer antigen presenting cell and induce ctls. methods: for the induction of ctls against wt1, activated b cells were used as an antigen presenting cells. b cells were isolated from pbmcs of normal healthy donors and activated with α-galactosylceramide (α-galcer) and nucleofected with wt1-coding plasmid dna. activated b cells were the cultured with pbmcs for 17days in vitro and harvested for assay. results: cells expanded about 3 times after 17 days of culture. we examined characteristic of wt1-specific ctls by their surface markers. wt1-specific ctls had more than 90% cd3+ marker, and ratio of cd8 to cd4 was 1.7-5.8. we also examined nkt cell markers to see if nkt cells were activated by il-15, a cytokine used in the induction of ctls, and the portion of nkt cells was about 2%. the ctls showed a decrease in naïve cell (cd62l+cd45ra +) and an increase in effector memory (cd62l+cd45ra-) and central memory (cd62l-cd45ra-) compared with non-stimulated pbmcs. subsequently, the ifn-γ elispot (enzyme-linked immunospot) assay was performed to confirm the response of the induced wt1-specific ctls to the wt1 antigen. when wt1-specific ctls encounters a target that does not have a wt1 antigen, it did not produce ifn-γ, but when it encounters a target cells loaded wt1 antigen, it responded to secrete ifn-γ. killing assays were also performed to determine the immunogenicity of induced ctls. the induced wt1 ctls was found to be killing more than 90% when the e:t ratio was 10:1 when the autologous pbmc met the target with wt1 pepmix. in addition, we found that wt1 ctls has killing activity when it encounters leukemia cell lines that express wt1 and matched hla-a*0201. conclusions: in this study, we can induce antigenspecific ctls that specifically react to wt1 using activated b cells as antigen-presenting cells. these observations confirmed that b cells activated by α-galcer can act as a taa presenting cell to induce taa specific ctls as viral antigen, such as pp65 and ie1, and consequently wt1specific ctls could be induced. moreover, ctls induced activated b cells had ability to recognize and kill the target cells expressing wt1 correctly. our results demonstrate that these in vitro expanded wt1-specific ctls using activated b cells can be a promising candidate for adoptive immunotherapy against cancer. disclosure: nothing to declare judith böhringer 1 , michael schumm 1 , christiane braun 1 , marina schmidt 1 , patrick schlegel 1 , christian seitz 1 , murat aktas 2 , georg rauser 2 , sandra karitzky 2 , peter lang 1 , rupert handgretinger 1 1 university children's hospital tübingen, tübingen, germany, 2 miltenyi biotec gmbh, bergisch gladbach, germany background: t cells with chimeric antigen receptors (cars) on their surface facilitate to target specific surface expressed antigens. research and clinical trials with cd19-car t cells show impressive remission induction rates and increased survival in heavily pretreated patients. therefore, car t cells are introduced as new potent cellular therapeutics in the clinical routine. in order to establish the manufacture of cd19-car t cells, validation runs with the fully automated clinimacs prodigy t cell transduction process have been performed using the miltenyi anti-cd19-car lentiviral vector. methods: unmobilized leukaphereses from 3 donors (2 x healthy, 1 x all) were used for the clinimacs prodigy t cell transduction process. leukocytes undergo a cd4 + / cd8 + t cell enrichment via magnetic beads, followed by stimulation with macs ® gmp t cell transact™, transduction with an anti-cd19-car lentiviral vector, expansion with il7 and il15, and final formulation to the cellular product. during and after the manufacture, facs analyses were performed as well as cytotoxicity assays after cd19-car t cell production. results: total volumes of leukaphereses were between 60 and 280 ml with 2.6 -4.0 x 10 9 total mononuclear cells. after enrichment 100x10 6 cd4 + / cd8 + t cells were transduced with anti-cd19-car lentiviral vector and were further expanded. cells were harvested on day 12. the final cell counts of the cellular products were 6.1, 7.2 and 5.0 x 10 9 mononuclear cells from two healthy volunteers and the all-patient, respectively. the transduction efficiency of the cd19-car t cells was 48.6%, 43.4% and 32.0% among viable cd3 + cells. the final count of car t cells was therefore 2.9, 3.0 and 1.5 x 10 9 cells. the final products exerted excellent cytolytic activity against cd19 + bcp-all cell line nalm-6. importantly, cd19-car t cells generated from the all patient demonstrated complete eradication of autologous blasts at 0.3 to 1 e:t ratio after 24 hours incubation. conclusions: the clinimacs prodigy t cell transduction process has been shown to run a fully-automated manufacturing process over 12 days without any deviations in a clean room environment on a single device. the user interaction was reduced to activities at only 3 days to set up the system and provide fresh medium and reagents. the transduction process yielded a high number of t cells with a high frequency of cd19-car t transduced cells. the results were comparable for both unmobilized leukaphereses from healthy donors and showed expected slightly lower results in the patient. finally, these results demonstrate that the clinimacs prodigy t cell transduction process is well suited to provide the clinical mb-cart19.1 r/r cd19+ bcm study with appropriate investigational medical products. disclosure background: allogeneic hematopoietic stem cell transplantation (allohct) is an effective strategy in the long term control of several hematologic diseases, however, patients could experience complications, as graft versus host disease (gvhd) and disease relapse. recently, the introduction of post-transplant cyclophosphamide (ptcy) allowed to significantly reduce gvhd, but disease relapse remains an important issue. donor-lymphocyte infusion (dli) is an established adoptive cell therapy for disease relapse after allohsct, but, in order to be efficient and safe, patients have to be off immunosuppression treatments and gvdh-free. here we report our data about efficacy and safety of dli infusion as treatment for disease relapse in patients who received peripheral blood stem cell transplantation (allopbsct) from hla-matched unrelated/related plus ptcy as gvdh prophylaxis in our clinical trial (nct 02300571). methods: we collected data from 13 patients, treated with ptcy (50 mg/kg/die, days +3+4), mofetil mycophenolate (mmf) and tacrolimus (t) as gvhd prophylaxis after allo-pbsct, who received dli infusions. they were treated between january 2013 and october 2018. we report data about overall response rate (orr), disease control rate (dcr), and dli-related mortality and morbidity. diagnosis were as follow: 5 had multiple myeloma, 3 had acute myeloid leukemia, 3 had acute lymphoblastic leukemia and 2 had lymphomas. all patients but one, who had chimerism loss, received dli because of disease relapse. results: median time between transplant and dli was 9 (range 3-87) months. median number of dli infusions was 2 (range 1-13). 10 patients (77%) received cyclophosphamide 300 mg/m2 preparative regimen the day before the cryopreserved dli infusions, while in the other 3 cases dli were associated with lenalidomide, ponatinib and 5-azacitidine. the overall response rate (orr) was 50%, while disease control rate (dcr) was achieved in 75%. the patient who received dli because of loss of chimerism converted it in full donor after 2 infusions. after dli treatment the incidence of acute gvhd grade i-iii was 54%, while was 46% for grade ii-iii and patients were started on short course of systemic immunosuppression treatments . none of these patients died because of dli adverse events. estimated 1-year overall survival was 77% with a limited follow-up length (6 months). conclusions: the infusion of non-manipulated lymphocytes from allogeneic donors is a valuable and safe strategy of treatment for patients relapsing after allopbsct with ptcy. ptcy showed high efficacy in gvhd prevention, allowing early discontinuation of immunosuppression drugs. because of this, we can reach the goal to transform transplant in a platform where we could add early dli infusions as a new strategy for disease control. clinical trial registry: nct 02300571 disclosure: nothing to declare p074 extracorporeal photopheresis in the treatment of refractory chronic gvhd: analysis of mononuclear cell infusion gillen oarbeascoa 1 , maria luisa lozano 2,3,4 , luisa maria guerra 5 , cristina amunarriz 6 , nuria revilla 2,3,4 , pastora iniesta 2,3,4 , cynthia acosta fleitas 5 , jose luis arroyo 6 , eva martinez revuelta 7 , andrea galego 8 , dolores hernandez-maraver 9 , mi kwon 1,10 , aurora viejo 9 , jose maria garcia gala 7 , concepcion andon saavedra 8 , jose luis diez-martin 1, 10, 11 background: extracorporeal photopheresis (ecp) is an immunomodulatory treatment that has shown efficacy in steroid refractory chronic gvhd, but the mechanism of action is only partially understood. in some studies, a correlation has been suggested between treated mononuclear cells (mnc) or lymphocytes and response to ecp. the objective of the study was to analyze the relationship between the infused cellularity and response in chronic gvhd. methods: 48 patients from 7 different centers with a total of 930 ecp procedures were retrospectively analyzed. ecp procedures were performed from january-2011 to june-2017. all ecp procedures were performed with the off-line system. the response was defined as responder (complete and partial response) and non-responder. infused cell numbers for lymphocytes, monocytes and mononuclear cells (lym+mon, mnc) were calculated. for analytic purposes, the median number of cells infused per procedure until response (or until the median number of procedures until response for non-responding patients) and the cumulative number of cells infused until response (or until the median in non-responders) were calculated for all the subgroups. same procedures were performed with the number cells infused until day 30 of ecp. finally, the response and survival impact of infusing a number of cells over or below the median and in different tertiles (t1, t2 and t3) was assessed until the median number of procedures needed to achieve a response. results: the median number of procedures until response was 3. we observed no differences in the median number of lymphocytes, monocytes or mncs infused until response or until day 30 between responding and non-responding patients. there were no differences in response if patients received lymphocytes or monocytes above or below the median number. nevertheless, patients that received a total absolute number of mncs above the median (64x108 cells) showed a trend towards a higher response rate (75% vs 61%, p=0.09). the patients that received a cumulative number of lymphocytes in the 3 first ecp procedures above the median showed improved overall survival (os) (2y os 85% vs 55%, p=0.024). patients that received a number of monocytes above the median showed a trend towards better survival (p=0.09), that was significant when the number of monocytes infused surpassed the first tertile (2y os 38% for t1, 79% for t2, 92% for t3, p=0.003). finally, the patients that received a cumulative number of mncs above the first tertile also showed improved survival (2y os 47% for t1, 74% for t2, 94% for t3, p=0.015). conclusions: there were no differences in the infused cellularity between responding and non-responding patients with chronic gvhd. at the same time, we found that except for a trend toward better response with higher mncs infused, there was no relationship between lymphocytes and monocytes with the response rate as other previous studies have suggested. however, even if there is no relationship with the response rate, the patients receiving the highest numbers of lymphocytes, monocytes and mncs in the cohort showed an improved survival, suggesting that larger quantities of cells could exhibit a protective effect. nevertheless, prospective studies that address this relationship are needed. disclosure: nothing to declare comparative analysis of the cytotoxic potential of cytokine-induced killer and natural killer cells for neuroblastoma therapy annekathrin heinze 1 , beatrice grebe 1 , eva mudry 1 , jochen früh 1 , bushra rais 1 , claudia cappel 1 , sabine hünecke 1 , eva rettinger 1 , thomas klingebiel 1 , peter bader 1 , evelyn ullrich 1,2 1 university hospital frankfurt, frankfurt, germany, 2 german cancer consortium (dktk) partner site:, frankfurt, germany background: neuroblastoma (nb) is the most common solid extracranial tumor in childhood. despite therapeutic progress, prognosis for high-risk nb is poor and innovative therapies are of medical need. therefore, we investigated the cytotoxic potential of interleukin (il)-activated natural killer (nk) cells compared to activated cytokine-induced killer (cik) cells against different human nb cell lines in vitro. methods: nk cells were isolated from peripheral blood mononuclear cells (pbmcs) using cd56 enrichment or cd3/cd19 depletion kits. they were successfully expanded ex vivo with different cytokine combinations such as il-2, il-15, il-18 and/or il-21 under feeder-cell free conditions. in contrast, cik cells were generated from pbmcs by ex vivo stimulation with interferon-γ, il-2, okt-3 and il-15. a comparative analysis of expansion rate, purity, phenotype and cytotoxic activity against different nb cell lines following different culturing protocols was performed. results: cd56 enriched nk cells showed a median expansion rate of 4.3-fold after 10 to 12 days in culture with a final frequency up to 99.0% nk cells and a median frequency of 0.5% cd3 + cd56 -t cells. in contrast, the starting cell product after cd3/cd19 depletion consisted of a median frequency of 43.5% nk cells that expanded significantly faster with 7.5-fold and also reached up to 98.6% purity without any relevant t cell contamination. cik cells expanded with a median rate of 30.8-fold and contained 3.3% nk, 84.2% t and 6.2% nk-like t cells. interestingly, nk cells, particularly after cd3/cd19, showed a significantly higher median cytotoxic capacity against nb cells depletion (46.6% for cd56 enrichment, 53.7% for cd3/cd19 depletion) compared to cik cells that induced 7.2% killing of nb cells with e:t ratio 5:1 in a 3 hours' co-incubation assay. interestingly, prolonging the ex vivo stimulation after cd3/cd19 depletion to 15 days enhanced the median expansion rate to 12.3-fold with a slightly reduced cytotoxic potential (40.9% for 11 days' ex vivo expansion, 31.1% for 15 days' ex vivo expansion, comparison of the same donors). the addition of an il21boost prior harvesting increased the expansion rate to median 12.6-fold (compared to 11.7-fold for the same donors) with an improved cytotoxicity of 51.5% (compared to 45.8%) . fortunately, all nk cell products showed a high viability and no relevant t or b cell contamination (median < 0.2%). interestingly, further optimization of the culturing procedure with use of another cell culture medium led to an improved median 24.4-fold (compared to 9.6-fold) nk cell expansion rate in 15 days, also resulting in comparable cytotoxicity of 52.5%. conclusions: nk and cik cell products may offer an innovative immune therapeutic option for patients with high-risk nb after allogenic stem cell transplantation. our study revealed that nk cells have a significantly higher cytotoxic potential to combat nb. interestingly, the use of il-15 expanded and il-21 activated nk cells developed from a cd3/19 depleted apheresis product is highly promising as additional immunotherapy in combination with haploidentical stem cell transplantation of children with nb. disclosure: nothing to declare. quantitative determination of donor allo-reactive t-cells in haploidentical donor-recipient pairs by enzymelinked immunospot (elispot) and mixed lymphocyte culture (mlc) assays background: t-cell alloreactivity is responsible not only for graft versus host disease and morbidity, associated with hematopoietic stem cell transplantation (hsct) but also for graft-versus-leukemia (gvl) activity. in this regard, monitoring and quantitation of alloreactive t-cells (allo-t) may potentially provide valuable information for individualized clinical management of transplant recipients. the aim of this study was the optimization of allo-т detection and comparison of the elispot and mlc assays. methods: allo-t were determined in 20 haploidentical donor-recipient pairs before hsct. donor mononuclear cells (mnc) served as effector cells (ec) . patient cd3depleted mnc were used as stimulatory cells (sc).the ratio ec:sc were 5:1 and 10:1. the frequency of allo-t in donor peripheral blood was tested in elispot assay and mlc. elispot provides the detection and quantitation of activated t-cells on the basis of cytokine secreted by each cell. the co-incubation time was 24 h for ifn-gamma and 48 h for il-2 detection. in mcl assay donor mnc were labeled with cfse and allo-t, proliferating in response to stimulation with alloantigens, were determined by flow cytometry on day 5. results: the median number of ifn-gamma producing allo-t per 300 000 donor mnc was 143,5 (2-1469; ec:sc ratio -5:1) and 75,0 (1-1440; ec:sc ratio -10:1). the median frequency of allo-t was 0,056% (0,00076 -0,538; ec:sc ratio -5:1) and 0,019% (0,00019 -0,632; ec:sc ratio -10:1) among lymphocytes. il-2-producing allo-t were less frequent in donor mnc in comparison with ifngamma-producing allo-t. the median number per 300 000 mnc was 7,5 (0,5-356; ec:sc ratio -5:1) and 6,0 (0-169; ec:sc ratio -10:1). the median frequency of il-2-allo-t was 0,0028% (0,0002 -0,130; ec:sc ratio -5:1) and 0,0022% (0 -0,0619; ec:sc ratio -10:1) among lymphocytes. the ec:sc ratio 10:1 is enough for stimulation of il-2 producing by mnc in elispot assay, but for optimal stimulation of ifn-gamma producing cells ec:sc ratio 5:1 is preferable. this suggests that allo-t are predominantly ifn-gamma producing cells. alloreactive proliferating t-clones were detected in mlc in 10 of 14 donor-recipient pairs on 5 day of cocultivation. median percentage of proliferating t-clones were 9,5% (2,1 -32,5; ec:sc ratio -5:1) and 7,6% (3,0 -24,1 ; ec:sc ratio -10:1) among lymphocytes. however, mlc assay only permit a qualitative analysis that confirmed the presence of alloreactive t-clones, giving no information on their frequency within the culture. results of elispot and mcl assay directly correlated. conclusions: allo-t were detected in 100,0% of assayed haploidentical donor-recipient pairs by elispot and only in 71,4% by mlc. this difference in detection is due to the fact that elispot allows to detect single cytokine secreting cell whereas mlc can reveal proliferating аllo-t clones. the analysis of allo-t in haploidentical donor-recipient pairs may provide rationale to manipulate the allo-immune response and to exploit the powerful ability of allo-t to control hematologic malignancies. disclosure: nothing to declare allogeneic mesenchymal stromal cell as rescue therapy in an infant with life-threatening respiratory syndrome due to a filamin a mutation background: cell-based therapy has gained attention in the respiratory system diseases and encouraging results are reported following mesenchymal stromal cells (mscs) administration. due to their capacity to produce and secrete a variety of paracrine factors and bioactive macromolecules, mscs became a key player in lung tissue injuries and function, reducing fibrosis, promoting the normal development of alveoli and pulmonary vessels. for the first time we used the msc infusions as rescue therapy in a pediatric patient with flna gene mutation and life-threatening respiratory syndrome. methods: a child with a new pathogenic variant of the flna gene c.7391_7403del; (p.val2464alafster5) at the age of 18 months, due to the serious and irreversible chronic respiratory failure and dismal prognosis, was treated with 4 intravenous infusions of allogeneic bone marrow (bm)-mscs at the dose of 1×10 6 mscs/kg body weight. bm-mscs were produced at "cell factory", fondazione irccs policlinico s. matteo, pavia,isolated and expanded ex vivo from healthy donor bm, following a previously reported protocol. premedication with antistaminic drug, 30 min before every infusion to avoid any potential reaction was performed. the evolution of the respiratory condition was detected. peripheral blood were collected before each msc treatment for treg and th17 monitoring. treg, defined as cd4+ cd127neg cd25+ cells expressing the forkhead box p3 (foxp3) transcription factor, and th17, defined as cd4+ cells expressing intracellular il-17, evaluation was performed by flow cytometry (facscanto; bd biosciences, san diego, ca) as previously reported, following standard procedures. results: no acute adverse events related to mscs infusion was recorded. during follow-up, patient maintained a good general condition and showed a regular growth. no systemic or respiratory infections occurred. after the second infusion, the child experienced a progressive improvement of his clinical respiratory condition, with a good adaptation to mechanical ventilation, in the absence of episodes of respiratory exacerbations. the baby maintains adequate volumes of exchange with substantial reduction of the inspiratory support. a reduction of trigger sensitivity was also obtained. thorax ct scan showed a recovery of the basal parenchyma bilaterally and the improvement of the anatomical-functional alignment and aerial penetration. after the first msc administration, an enrichment of treg and th17 percentage in peripheral blood, was observed. while, after the second msc infusion a significant increase in treg/th17 ratio was noted. conclusions: this report suggest that msc serial infusions are a promising therapy in aiding the respiratory failure, even in a pediatric patient with flna mutation. intravenous administrations of allogeneic mscs are feasible and safe without toxicity. our results suggest that to mitigate lung injury, mscs may act as regulators of treg and th17 balance. further investigations are upcoming to establish the useful of this therapeutic proposal in interstitial lung diseases in children. disclosure: nothig to declare p078 feasibility of il-15 stimulated donor nk cells manufacturing for early infusion in patients with high risk acute myeloid leukemia undergoing haploidentical transplantation background: nk cells provide a potent antitumor effect in the setting of manipulated haploidentical hematopoietic stem cell transplant (haplo-hsct). we propose a novel strategy to enhance the antitumor effect of allogeneic transplant through the infusion of nk cells stimulated with il-15 exvivo in adult high-risk acute myeloid leukemia (aml) patients undergoing unmanipulated haplo-hsct. the objective of this study was to provide efficiency and productivity data obtained in the manufactured cellular products infused. methods: selection criteria included patients with highrisk aml undergoing unmanipulated haplo-hsct. lymphoapheresis of the haploidentical donor was performed using spectra optia (terumo® bct) on days +6 and +13 after transplant. from the obtained product a double immunomagnetic cellular selection with clinimacs system (miltenyi biotec®) was performed in two steps: cd3+ depletion followed by positive cd56+ selection. the obtained an enriched cellular product of cd3-cd56+ nk cells was incubated with il-15 (10 ng/ml) between 12 and 18 hours at 37ºc and 5% co2 in gmp conditions. quality and microbiological controls were performed at the end of each manufacturing step. dxh cellular counters (beckman coulter®) and multiparametric flow cytometry were used for lymphocyte subpopulations and viability analysis (navios cytometer; beckman coulter®, conjugated monoclonal antibodies; miltenyi biotec®). the final product was infused intravenously to the patient on days +8 and +15 if manufacturing conditions were met (range of 0.5-100x10 6 nk/kg, purity ≥ 80%, viability≥ 70% and < 1x10 4 cd3+ cells/kg). if not, it was discarded. nk cell activation in the product was measured by the expression of cd25 and cd69. results: between november 2017 and april 2018, 3 patients were included in this ongoing trial. two products were manufactured for 2 of the patients, and only one for the first patient, due to transplant complications between first and second infusion. one product did not meet minimum viability criteria and was discarded. in the infused final products mean and sem of nk cell purity, recovery and viability were 83.7%±4.4, 30.9%±4 and 76.3%±17.4, respectively. log cd3+ depletion ranged between -5.48 and -6.03. median infused doses of nk cells and cd3+ cells per kg were 3.78x10 6 (2.8x10 6 -4.57x10 6 ) and 114 (87-532). complete manufacturing data of all 5 procedures are shown in table 1 background: cytokine-induced killer (cik) cells are a promising immunotherapeutic approach to combat relapse following allogeneic hematopoietic stem cell transplantation (hsct) for acute leukemia or myelodysplastic syndrome. to show safety and efficacy, a multicenter clinical study with 20 pediatric and 20 adult patients including up to eight cik cell applications with escalating doses is ongoing. methods: we favor single large scale cik cell generation with the aim to apply fresh cik cells and cryopreserve ready-for-use doses according to the study protocol in contrast to recurrent manufacturing. therefore cryopreserved cik cells were tested against freshly generated cik cells to approve equivalence. furthermore, an alternative medium supplement for cik cell culturing was investigated to avoid supply bottlenecks in ab-serum. results: fresh frozen plasma (ffp), platelet lysate (pl) and ab-serum in cik cell culture showed median expansion rates of 10-fold, whereas cultivation without medium additive resulted in significantly lower proliferation (p< 0.01). cik cell composition including t cells, nk like t cells and a minor part of nk cells was not significantly influenced by changing the medium additive. moreover, neither cytotoxicity against thp-1 cells nor cd25 expression on nk like t cells were significantly influenced by the different medium additives. for cik cell generation either ficollized peripheral blood (pb) or unstimulated leukapheresis (lp) products were utilized. with regard to repeated manufacturing within the clinical study, also cryopreserved lp and pbsc as starting material came into the focus of interest. comparing cik cell expansion rates, no significant differences for the entire cik cells and the subgroup of t cells were detected between the four starting materials. cryopreservation of cik cells had no significant effect on cik cell composition, cytotoxicity and cd25 expression on nk like t cells. a small, albeit not significant effect of cryopreservation on viability was detected, which was 86.1% before and 79.4% after freezing and thawing. conclusions: the challenge was an efficient time-, personal-and cost saving production of cik cells within the clinical study. introducing ffp enabled cik cell manufacturing for an increased patient cohort by avoiding supply bottlenecks in ab-serum. furthermore, cryopreservation allows the storage of ready-for-use cik cell doses fulfilling the demands of the clinical study. clinical trial registry: eudract number 2013-005446-11 disclosure: nothing to declare. automated generation of cd45ra depleted donor lymphocyte infusion (dli) with the clinimacs prodigy® cd45ra system 1, 2 methods: the current clinimacs cd45ra system was developed for graft engineering. up to 20x10e9 magnetically labeled cd45ra+ cells from leukapheresis products can be depleted from up to 50x10e9 white blood cells (wbc). we developed a new clinimacs prodigy® process in order to ease the procedure for routine-use, to reduce the specifications according to reported cell numbers for dli applications, and to enable the use of peripheral blood products with high amounts of red blood cells (rbc). the new system was tested by performance runs. an new fluorescent flow analysis protocol was developed. results: the resulting clinimacs prodigy pb-45ra system is an automated procedure with integrated labeling and washing steps. the new application software pb-45ra depletion enables to deplete up to 1.8x10e9 cd45ra + cells from up to 5x10e9 total wbc from peripheral blood products. a major difference of this process is the rbc removal option based on an integrated camera for cell pellet detection. the final cell product is provided in physiologic saline. verification runs with peripheral blood products (n=6 in total, n=3 with whole blood, n=3 with leukapheresis products) resulted in a mean depletion of 5.0 log (range 3.9 -5.7) for cd45ra + t cells in the cd45ra depleted product. viability of the target products was always above 96%, and mean wbc recovery was 66%. the mean process time was 3h23min (range 3h to 3h50min) without including the manual steps, i.e. tubing set installation and downstream analysis of blood products by flow cytometry. this data were in line with preceding evaluation runs (n=6), and results obtained in cooperation with an external beta test site. 3 the performance results were furthermore in line with results obtained on clinimacs plus instrument runs. for quality control of cd45ra depleted products we developed a flow cytometric analysis strategy for fast, accurate, and convenient analysis of even rare counts of remaining unwanted cells. it allows to determine naïve t cells at two different levels of subset staining. the minimum requirement for the flow cytometric analysis includes 4 colors to define viable cd3+cd45ra+ cells. for further evaluation of the naïve t cell subsets 3 additional colors are used to define viable cd3+cd45ro-cd95-cd62l+cd197+ cells. conclusions: the automated clinimacs prodigy pb-45ra system process is capable to deplete cd45ra+ cells efficiently from peripheral blood products within 4 hours. the new process is a fast, convenient, and regulatory compliant method for the preparation of ready-to-use cd45ra-depleted cell products for clinical applications. the submission to an european notified body for ce certification is an important next step. myeloablative conditioning regimen was preferred for 24 patients out of 25. gvhd prophylaxis regimens are csa +mtx: 20(%80), csa+mmf: 2(%8), csa only: 1(%4). atg was given 20 patients. despite been given gvhd prophylaxis 47(%27,1) patients out of 173 transplanted patients had gvhd features. of 47 patients, 25 had experienced steroid resistant gvhd after transplantation, including 17 (%68) grade 3 and 8 (%32) grade 4. ecp treatment was started mean 11 days after diagnosis of steroid resistant gvhd and 2 (%8) patients had complete response while 12 (%48) patients had partial and 11 (%44) patients had no response to ecp treatment on day 28. sixteen out of 25 patients had also received mesenchymal stem cell therapy as salvage therapy. only one patient had experienced hypocalcemic tetany, a complication of ecp procedure. thirteen patients had died and 12 were directly related with steroid resistant gvhd. other conditions like relapse of primary disease or pres syndrome also played role in death. conclusions: extracorporeal photopheresis is a reliable and effective second line treatment modality in steroid resistant gvhd. starting ecp sessions as soon as gvhd symptoms occur increases its effectivity. mesenchymal stem cell administration with ecp for 16 (%64) patients limits our study to reach o conclusion for efficacy of ecp itself. need for hemodialysis catheters, the prolonged sessions while adequate flow is not possible and catheter related infections are the lmitations for feasibility of ecp. disclosure: nothing to declare donor lymphocyte infusion administrations after allogeneic stem cell transplantations in pediatrics: a single center experience background: loss of chimerism is one of the major problems after allogeneic stem cell transplantation(sct). donor-lymphocyte infusions(dli) are used as a treatment after taper or stopping immunosuppression. in this study, dli experience in 20 patients with loss of chimerism after sct due to various benign and malign hematological diseases was presented. methods: between july 2015-august 2018, twenty patients, detected chimerism loss and received dli after sct were evaluated retrospectively. patients received myeloablative or reduced intensity conditioning, atg, cyclosporine a and methotrexate for gvhd prophylaxis. chimerism analyses were performed with short tandem repeat(str) method from peripheral blood. results below 95% were considered as mixed chimeric and below %5 were nonchimeric. when patients considered as mixed chimeric, immunosuppression therapy was ceased immediately and treated with dli. donor lymphocyte infusions were performed at two-week intervals with chimerism follow-up. student t, mann whitney u, ki kare tests and kaplan-meier analysis were used. results: between 1-16 ages (median 4), 7 female, 13 male patients were evaluated. the initial diagnoses were thalassemia major(10), aplastic anemia (3),all(4), aml (3) . dli initiation time was 119.65+-92.71 days after sct, total number of dli administrations were 3.5+-2.19. dose of dli was 1x10 5 -63.6x10 6 /kg (mean 20.5x10 6 /kg). nine patients' chimerism out of 20, fell below 95% at first month after transplant; 4 patients were nonchimeric, 2 of them were complet chimeric and 3 were mixed chimeric. eleven patients´chimerism were below 95% between 1-6 months after sct, 5 patients were nonchimeric and 6 were mixed chimeric. early mixed chimerism was found relevant with graft rejections (p=0.04). patients were followed up for 91-645 days. eight patients' chimerism increased after dli infusion and continued to decrease in 12 patients. after dli, acute gvhd has been seen in both group.the group with decreased chimerism after dli, dose was mean 12x10 6 ±23x10 6 /kg while the group with increased chimerism had dli dose mean 22x10 6 ±19x10 6 /kg. although the difference was not statistically significant, numerical value revealed significantly different. eventually10 patients out of 20 were mixed chimeric, 6 patients were complete chimeric and 4 were none. in thalassemic patients, 7 patients with thalassemia-trait donor were mixed chimeric, in 3 patients whose donors were normal, 2 of them were complete chimeric and one of them was nonchimeric.the difference was significant (p=0.02). the cd34 infusion doses revealed mean 6.61 ±5.06x10 6 /kg in mixed chimeric patients, 7.16±3.31x10 6 /kg in complete chimeric patients and 6.625±1.37x10 6 /kg in the patients with loss of chimerism. cd34 amount was seen high as numerical value in complete chimerics but no statistical significance was found. overall survival was 85%, disease-free survival was 25%. conclusions: we evaluated the efficacy of dl for patients with mixed chimerism in our patient group. we concluded that chimerism loss in patients with early decreased chimerism is similar to those in literature in spite if dli practices. dose and application frequency were greater in patients with increased chimerism. the small number and the heterogeneity of the patients limited our study. in this regard, studies with larger series and homogeneous groups are acquired. disclosure: nothing to declare phase i clinical trial of repeated administrations of bone-marrow derived mesenchymal stem cells in steroid-refractory chronic graft-versus-host disease patients nayoun kim 1 , young-woo jeon 2 , jae-deog jang 1 , keon-il im 1 , nak-gyun chung 2 , young-sun nam 1 , yunejin song 1 , jun-seok lee 1 , seok-goo cho (cghvd) is the most common long-term complication of allogenic hematopoietic stem cell transplantation which is associated with poor quality of life and increased risk of morbidity and mortality. currently, there is no standardized treatment available for patients who do not respond to steroids. as an alternative to immunosuppressive drugs, mesenchymal stem cells (mscs) have been used to treat and prevent steroidrefractory acute gvhd patients. these studies and reports have also provided a basis for using mscs in steroid refractory cgvhd patients. methods: to evaluate the safety and efficacy of repeatedinfusions of mscs, we enrolled ten severe steroid-refractory cgvhds patients. steroid refractory was defined as either no response to steroids lasting at least 4 weeks or progression of disease during treatment or tapering lasting at least 2 weeks. patients were intravenously administered with mscs produced from third-party bone marrow donors at a 2-week interval for a total of four doses. each dose contained 1x10 6 cells per kg body weight and all four doses consisted of mscs from the same donor and same passage. results: we enrolled ten patients (3 female/ 7 male, with a median age of 41.5(range 17-68). median of cgvhd affected organs was 3 (range 2-4) including the skin (n=4), eyes (n=8), oral cavity (n=9), lung (n=1), liver (n=2) and joints (n=6). all ten patients received their planned four doses of mscs, administering a total of 40 infusions. median time from initial cgvhd diagnosis to first msc treatment was 709 days (range 222-4413). msc infusions were well tolerated with no immediate or delayed toxicities. after 8 weeks of the first msc infusion, all ten patients showed partial response showing alleviation in clinical symptoms and increased quality of life. organ responses were seen in skin (n=2), eyes (n=5), oral cavity (n=8), liver (n=1), and joint(n=5). however, one patient died of progressive gvhd and one patient relapsed from primary disease. conclusions: repeated infusions of mscs was feasible and safe and may be an effective salvage therapy in patients with steroid-refractory cgvhd. further large-scale clinical studies with long-term follow up is needed in the future to determine the role of mscs in cgvhd. background: the majority of pregnant polish women (84%) have heard of cord blood banking. however, most doctors do not have sufficient knowledge about the possibility of using cord blood in order to respond to their potential concerns. only 16.5% of healthcare professionals were aware that cord blood could be used to treat haematological diseases. in order to make doctors aware of this issue and provide patients with the information they expect, we would like to present data on the use of cord blood stored in our blood bank for haematological and nonhaematological therapies. methods: the table presented below has been created using data from the general database of the polish stem cell bank, warsaw, poland. no data regarding umbilical cord blood data have been excluded. all patients were planned to be assessed on day 1, day 28, on discharge, 100 days after transplantation and 1, 2, 3, 4, and 5 years after transplantation, but in some cases, patients were lost from follow-up due to a persistent lack of reports from transplantation centres. results: in 32 cases, the therapeutic use of cb was transplantation (replacement of patients' own tissue); in 13 cases it was administration (infusion without destruction of patients' own tissue). thirty-three were administered as standard therapy and 13 as experimental therapy. conclusions: the survey study cited above, indicated low awareness of cord blood use among healthcare professionals. on the other hand, one study indicated that the expectations placed in cord blood banking may be unreasonable. as a private cord blood bank, we support recommendations which underline the importance of patient education. in poland, cord blood has been approved as standard therapy in approx. 80 diseases; most of them are rare, but polish law allows the use of cord blood for the siblings, parents and grandparents of a donor. additionally, 168 active and planned clinical trials throughout the world evaluate the therapeutic efficacy of cord blood in such areas as haematology, neurology, cardiology, diabetology, congenital paediatric disorders, ophthalmology, dermatology, gastroenterology, hiv infection, and the quality of life during aging. therefore, further indications may be expected in the future. background: cytokine release syndrome (crs) has been identified as a clinically significant, on target, off tumour side effect of the chimeric antigen receptor (car) t-cell therapies. it is clinically increased in interkeukin 6 and elevations in other cytokines, lactate dehydrogenase (ldh), c-reactive protein (crp), and ferritin. these side effects can include fever, fatigue, headache, encephalopathy, hypertension, tachycardia, coagulopathy, nausea, capillary leak and multi organ dysfunction. crs symptoms can appear as early as one day after infusion and can resolve quickly or last for weeks. it´s severity to be related to the disease burden prior to car t-cell therapy. methods: the bristol oncology and haematology centre will be providing car t-cell therapy to patients in early 2019. on collating from the leading consultant on the ward and fellow nursing team members it was apparent that an effective way at managing patients post car t-cell therapy side effects is to provide an educational and informative poster depicting a flow chart that will aid the practitioner to recognise and effectively treat/manage a patient with crs symptoms. results: none as of yet as this is a prospective tool ready for our first patient in early 2019 conclusions: through continuing reading and study days prior to the ward receiving its first car t-cell patient it is increasingly important that the entire multi disciplinary team recognise crs and understand the importance of early detection, careful monitoring and early intervention. background: allogeneic stem cell transplantation (allosct) is the only curative procedure for primary and secondary myelofibrosis (pmf, smf). elderly people are mainly affected, limiting the feasibility of intensive myeloablative chemotherapy regimens. the introduction of reduced-intensity conditioning (ric) made allosct feasible and effective for old patients. nevertheless, the incidence of pmf and smf is not negligible in young patients, theoretically able to tolerate also high-intensity therapy. very few data are available about the efficacy of ric-allosct in the particular setting of young-aged mf patients. methods: this study includes 56 myelofibrosis young patients (age < 55y) who received allosct between 2002 and 2016 at the university hospital hamburg/germany. four patients were previously splenectomized. patients mostly fall into intermediate risk groups according to dipss. four patients belonged to the high-risk triple-negative category (jak2/calr/mpl-). asxl1-mut was tested in 50 patients (pos: 17). in 96% graft source was pbsc, 2 patients received bmsc. only 30% of patients had a 10/10 hla-matched sibling, the others were transplanted from fully-matched (36%) or partially-matched (34%) unrelated donor. all transplants were conditioned according the ebmt protocol with busulfan (10 mg/kg po or 8 mg/kg iv), fludarabine (150 mg/m2), atlg (grafalon® neovii, germany) administered in 3 days at a dose of 20 mg/kg die for mud, 10 mg/kg die for mrd transplants, followed by cylosporina, and mycophenolate in the first 28 days. results: engraftment rate was 98%, median neutrophil engraftment time 15 days. platelet engraftment was reached by 51 patients (91%, median 19 days). four patients (7%) developed poor graft function, successfully treated with cd34+ selected pbsc-boost. after a median follow up of 8.6 years, estimated 5y-pfs and os were 68% and 82% respectively. dipss-risk and donor hla-matching resulted the only significant impacting factors on os. neither cytogenetic nor molecular abnormalities were significantly related to os. twenty-five patients (44%) experienced agvhd grade >1. c-gvhd was observed in 34 patients (65%), mostly (82%) beginning in the first 300 days after transplantation. cumulative incidence of trm was 7% at 1 year, with a plateau after the first year (5y trm = 12%). trm was observed only in patients with maximal grade (3) of marrow fibrosis. furthermore, trm never occurred in previously splenectomized patients (p=0.00), but no significant impact from splenectomy on os was observed (p=0.32). after transplant, 11 patients (20%) relapsed: 1 died without any treatment because of infection, 9 received dli (3 durable cr), 7 patients (6 after dli) underwent a second allosct, with long-term survival in 5 cases. conclusions: ric followed by allogeneic sct is a curative treatment approach for younger patients with myelofibrosis with a low nrm. the most important outcome-determining factor is donor hla-matching. interestingly, marrow high grade fibrosis showed to significantly impact trm. biological markers such as asxl1 mutation and cytogenetics, largely known as highly predictive for poor prognosis in the disease natural course, did not show any impact on survival, suggesting that patients harboring these abnormalities could get the greatest benefit from allosct. further data collection, and a prospective randomized trial are needed to confirm our conclusion. disclosure: nothing to declare p087 abstract already published. splenectomy as a risk factor for relapse after allogeneic hematopoietic stem cell transplantation in patients with myelofibrosis -retrospective cohort study background: splenectomy is a common procedure in patients (pts) with myelofibrosis (mf) performed to achieve improvement in blood cell counts and reduce b-symptoms. however, it has also been shown that splenectomy may adversely predispose to leukemic transformation in this setting. aim: to evaluate in a single-center retrospective analysis the long-term impact of pre-or post-transplant splenectomy on transplant outcome regarding overall survival and relapse risk. methods: this retrospective analysis comprises the data of 163 pts (93 male and 70 female) with primary (n=108) or secondary (n=55) mf after allo-hsct from hla-matched sibling (n=48) or unrelated (n=115) donors in our center between 1994 and 2018. the median age was 56 years (range, 22 to 75 years). a myeloablative conditioning regimen was performed in 142 pts, while 21 pts where treated with a reduced intensity conditioning. peripheral blood stem cells (n=154) or bone marrow (n=9) with a median of 7.0 x 10 6 cd34 + cells/kg bodyweight (bw) (range, 1.0 to 30) were transplanted. splenectomy was performed in 41 of 163 pts: 21 pts were splenectomized prior to and 20 pts after allo-hsct. relapse was diagnosed in 22 (14%) of 163 pts. the median duration to relapse after transplantion was 13 months (range, 3-99 months). results: the median duration of follow-up of this cohort was 28 months (range, 2-120 months), the 10-year overall survival (os) was 46%. 74 pts died, including 7 pts who relapsed and 67 pts who died of treatment related causes. the observed probability of relapse was significantly higher in splenectomized pts than in non-splenectomized pts: 37% versus 10% (relative risk (rr) 3.7, 95% ci, 1.5-9.4, p=0.007). at 10 years, the os was 50% in nonsplenectomised and 39% in splenectomised pts (p=0.35) (fig.1) . the relapse rate in splenectomised pts was independent of pre-(5 of 21 pts, 24%) or post-transplant (6 of 20 pts, 30%) treatment (rr 1.3, 95% ci, p=0.73) . conclusions: on the basis of our cohort, we could assert that pre-and post-allo-hsct splenectomy was equally and significantly associated with an increased relapse ratio in patients with mf, which also tends to negatively affect overall survival. [[p088 image] 1. figure 1 : the overall survival after allo-hsct in patients with myelofibrosis.] background: b-cell prolymphocytic leukemia (b-pll) is a very rare lymphoproliferative disorder. although allogeneic stem cell transplantation (allosct) could be a curative option, patients often do not qualify for this consolidation treatment due to an aggressive course of disease. in this case study, we report on three patients who failed ibrutinib therapy but achieved complete remission and even mrd negativity after treatment with the chimeric cd20-antibody rituximab, enabling them to undergo allosct. methods: clinical data and follow-up data were collected by chart review. results: all three patients (pt#1: male, 42 years; pt#2: female, 66 years; pt#3: female, 64 years) were referred with b-pll harboring highly complex aberrant karyotypes, including 17p abnormalities in pt#1 and pt#2. a tp53 mutation could be detected in pt#2 and pt#3. all three patients had symptomatic disease with rapidly increasing hyperleukocytosis and massive splenomegaly. two of them were treatment-naive and one relapsed after chemoimmunotherapy. all patients were put on ibrutinib 420mg. despite initial response to treatment, two patients developed progressive disease after 4 (pt#1) and 9 months (pt#2) on ibrutinib, whereas pt#3 remained in partial remission with persisting leukocytosis, precluding consolidating allosct as originally intended. in pt#1, ibrutinib was replaced by venetoclax, but without response. in order to control rapid lymphoproliferation, rituximab was added to venetoclax. grade 4 infusion reaction / tumor lysis syndrome (tls) (fever, tachycardia and hypotonia requiring intravenous vasopressors) followed each rituximab administration despite fractionating rituximab to small doses. however, continuation of rituximab (100mg/d over 10d) led to complete and durable clearance of hyperleukocytosis (from 250/nl to mrd negativity) despite venetoclax cessation. a similar pattern was observed in pt#2, who received rituximab while showing rapidly increasing leukocytosis upon ibrutinib. again, complete clearance of b-pll cells in the peripheral blood (from 148/nl to mrd negativity) occurred after initial grade 4 tls despite only modest cd20 expression on tumor cells in this patient. also, pt#3 achieved profound b-pll cell depletion (from 38/nl to a mrd rate of 1.1%) upon addition of rituximab to ibrutinib (without tls in this case). subsequently, all three patients were able to undergo allosct after conditioning with fludarabine and total body irradiation with 8 gy. pt#1 received stem cells from a hla-ident sibling donor, whereas pt#2 and pt#3 had unrelated donors (hla-ident and hla-matched respectively). with follow-up times of 17 and 11 months post-transplant, pt#1 and pt#2 are currently in ongoing mrd-negative remission. pt#1 developed an acute graft-versus-host disease (gvhd) of the liver (grade 3), nevertheless the clinical course was well controlled by immunosuppression. in pt#2 a chronic gvhd of the skin occurred. pt#3, who achieved mrd negativity after allosct, developed acute and chronic steroidrefractory gvhd of the skin and gastrointestinal tract. nine months post-transplant, gvhd deteriorated and after further complications the patient died of pneumonia 11 months post-transplant. conclusions: supplementary treatment with rituximab can induce deep remissions in patients with ibrutinibresistant, genetically poor-risk b-pll, thereby enabling them to undergo successful consolidation with allosct. a high risk of life-threatening infusion reactions / tls associated with the addition of rituximab has to be taken into account. background: there is little experience on the use of the newer targeted therapies in cll patients relapsed after allogeneic stem cell transplantation (allo-sct). against this background, we evaluate the safety and efficacy of the bcr inhibitors (bcri), ibrutinib and idelalisib, administered after allo-sct for the purpose of treating the cll relapse. methods: data from 11 cll pts who relapsed after sct, and were subsequently treated with ibrutinib (n=6), idelalisib (n=3) or both (n=2),were retrospectively collected in collaboration with the spanish group of cll (gellc) and the spanish group of stem cell transplantation (geth). results: transplant characteristics are summarized in table 1. eight patients received the bcri as the first salvage treatment after sct relapse, whereas 3 patients had received ≥2 prior lines of treatment. at the time of the onset of the bcri, 7 patients had rai 4 stage and 6 patients had a lymph node size ≥5 cm. del17p was present in 4 patients and del11q and complex karyotype in 2 patients, respectively. tp53 gene mutation was detected in 3 patients (all with del17p13). median time from sct to bcri therapy was 53.9 months, being shorter in patients treated with ibrutinib (n=8, median 51 months) than in those treated with idealisib (n=3, median 111 months). median time on ibrutinib and on idelalisib was 7.3 months (4. 1-18.8 ) and 6 months (3.0-23.4 ), respectively. the best overall response rate (orr) obtained with ibrutinib was 75% (1 cr, 4 pr, 1 pr+l) whereas it was of 40% for patients receiving idelalisib (1 cr, 1 pr+l). among the 8 patients treated with ibrutinib, 7 (87.5%) presented an adverse event (ae), being diarrhea (n=3), asthenia (n=2) and infections (n=5) the most frequent. hypertension was seen in 1 patient and none patient developed atrial fibrillation. five patients stopped ibrutinib treatment, due to toxicity (n=4) or progression (n=1). after ibrutinib discontinuation, 4 patients were newly treated with idelalisib (n=2) or venetoclax (n=2). all patients treated with idelalisib developed at least one ae, being diarrhea (n=3), pneumonitis (n=2) and neutropenia (n=2) the most common. four patients discontinued idelalisib because progression (n=3) or toxicity (n=1). venetoclax was given after idelalisib in 3 patients. although acute and/or chronic gvhd before bcri was documented in 7 (63.6%) and 5 (45.5%) patients, respectively, only one patient (treated with idelalisib) reactivated a mild chronic gvhd. none patient received infusion lymphocyte from donor after bcri and one patient underwent a second sct. with a median follow-up of 14.3 months (5.9-33.9) after the onset of the bcri treatment, 4 patients had died, all of them due to cll progression (3 richter´s transformation), whereas 5 patients remained in response (3 cr, 2 pr). the overall survival probability of the whole series at 12 months was 77.8% ±13.9%. conclusions: in our study, ibrutinib and idelalisib, administered in cll patients relapsed after sct did not increase the risk of gvhd reactivation but they show high incidence of adverse events. nevertheless, bcri offers a possibility of disease control in these patients with poor prognosis. further studies are needed to confirm these data. background: prior to the introduction of tyrosine kinase inhibitors (tki), median survival of chronic phase chronic myeloid leukemia (cp-cml) patients was approximately 60 months and the standard treatment with interferon-alpha resulted in complete cytogenetic responses in about 30% of the patients. autologous stem cell transplantation (auto-sct) was first attempted for patients in transformation in order to restore a second cp and was introduced secondarily in cp to try to prolong the response. the main rational for autografting in cp resides on the reduction of the tumor burden and the number of leukemic cells at risk of developing blastic transformation. nevertheless, auto-sct alone was not able to maintain a long-term remission. nowadays, tkis represent the state-of-the-art therapy for cml and the concept of auto-sct has only little interest while long-term follow-up and outcome in this setting are currently unknown. the aim of our study is to evaluate at a first time the longterm outcome of cml patients who received auto-sct in chronic phase, and to evaluate at a second time in a subgroup analysis, the outcome of those who received tki after having been auto-transplanted, mainly for disease progression/loss of response and/or to enhance disease response. methods: we found a total of 969 patients who received auto-sct for cp-cml in europe between years 1989 and 2004, 578 (60%) were males, median age at auto-sct was 47 years (range: 19-67), the median time between diagnosis and auto-sct was 19 months, stem cells source was peripheral blood in 84% of patients, most frequent conditioning regimen was busulfan 4mg/kg/day 4 days + 1 day of melphalan 140 mg/m² one day prior to the cells reinfusion. information about receiving tki post auto-sct was available only for 103 patients, first tki was imatinib for 89 (86%) patients, dasatinib for 8 (8%), nilotinib for 6 (5%) and ponatinib for one (1%) patient. results: after a median follow-up of 9.5 years (range: 1-27) from time of auto-sct for the whole population, the probability of overall survival (os) at 10 years was 50% (95% ci: 46-53); there was 540 (56%) patients who relapsed after a median time of 16 months after auto-sct. there was a total of 530 patients transplanted before the tki era and survived until the availability of tkis. when we performed a landmark analysis evaluating the outcome of patients who received auto-sct, survived until the tki era and received tki (n=103), the 10 years os probability of these patients from tki treatment was 70% (95% ci: 58-78). additional data requests will be sent to centers querying about prognosis, molecular responses, treatment and disease details. conclusions: we demonstrate here with these preliminary results that the introduction of tki has improved survival of cml patients. in addition, patients who received auto-sct, survived until the tki era and also received tki, had encouraging rates of long-term survival. an extensive analysis will be performed when additional data will be available and the study will be updated with more results. disclosure: nothing to declare a 35 year single center transplant experience in chronic myeloid leukemia background: allogeneic hematopoietic stem cell transplantation (hsct) has been considered for decades the only curative approach for patients with chronic myeloid leukemia (cml). in the tyrosine kinase inhibitors (tkis) era, hsct for cml has been reserved only to patients not achieving a cytogenetic remission or showing progressive disease after multiple tki treatment lines. however, a progressive improvement in the long-term survival has been obtained in the overall hsct population. the present study aimed at evaluating whether in cml patients transplanted at our center over a long time period -from 1983 to 2018 -the outcome improved over time. methods: 136 consecutive patients who underwent a transplant between 1983 and 1999 were compared to 43 patients who received the transplant between 2000 and 2018. overall survival (os), leukemia-free survival (lfs) and graft-leukemia-free survival (glfs) were estimated using the kaplan-meier method and the log-rank test was used to compare risk factors categories. results: of the 179 patients [median age 35 years (range7-66)], 148 (82.7%) were in 1 st or 2 nd chronic phase, 25 (13.9%) in accelerated phase and six (3.4%) in blast crisis. matched related donors and alternative donors (matched unrelated donors, cord blood or mismatched related donors) were used in 156 and 23 cases, respectively. as stem cell source, bone marrow was used in 142 patients, peripheral blood in 33 and umbilical cord blood in 4. tbibased conditioning regimens were used in 89 patients, while in the other 90 cases irradiation-free conditioning regimens were used. both in univariate and multivariate analysis, irradiationfree conditioning regimens (hr 1.8; 95%ci 1.1-3.0, p=.0014) and transplants performed in 1 st chronic phase (accelerate phase hr 2.1; 95%ci 1.2-3.8, p=.008 -2 nd chronic phase hr 4.9; 95%ci 2.3-10.3, p=.0001 -blast crisis hr 2.5; 95%ci 1.0-6.4, p< .05) were associated with a better os. patients transplanted before 2000 had a worse os (hr 6.5; 95%ci 2.7-15.5, p < .0001) and dfs (hr 2.2; 95%ci 1.0-4.8, p=.045). a trend for a worse glfs was observed in univariate analysis (hr 1.6; 95%ci 1.0-2.7, p=0.05), in the first period of observation. conclusions: our single center experience confirms that higher os, dfs and glfs are observed in cml patients allografted in more recent years. improvement of conditioning regimens, use of tbi-free conditioning regimens and supportive therapy, have presumably contributed to these results, together with the more recent strategy of close monitoring of minimal residual disease, and prompt use of tki or donor lymphocyte infusion in case of relapse. hsct is nowadays a safer therapeutic procedure in cml patients that should be considered timely in tki-resistant patients to avoid progression into a more advanced disease phase. disclosure: the authors declare no conflict of interest. reduced-intensity transplantation (rit) in patients with high-risk or advanced chronic lymphocytic leukemia in last 5 years: improvement of transplant outcomessingle centre experience . hct-ci ≥ 3 was in 20% of pts. source of stem cells was peripheral blood in 80% and bone marrow in 20% of pts. the median of infused cd 34+ cells was 5,4x10^6/ kg. the conditioning regimen consisted of fludarabine and melphalan (+atg in unrelated donor). gvhd prophylaxis were cyclosporine and methotrexate. results: all pts engrafted. none of 3 pts in cr before rit progressed at day +30 after rit and among 17 pts beyond cr before rit all of them achieved at least pr at day +30 after rit. 13 pts (65%) developed acute gvhd (2 pts grade iii-iv) and among 19 evaluable pts 10 (53%) of them developed chronic gvhd (6 mild, 2 moderate, 2 severe). with median follow-up 50 months (range 6-63 months) 15 pts (75%) are alive in cr. 3 pts (15%) relapsed or progressed 5, 19 and 21 months after rit and 2 of them died. last relapsed patient achieved next cr after ibrutinib. 3 pts (15%) died due to nrm. nrm till day +100 after rit was 5%. the estimated probabilities of 2-years cgrfs, pfs and os are 55%, 73% and 83%. conclusions: in spite of relatively small number of evaluated pts and retrospective type of analysis our data show that rit in pts with high-risk or advanced cll has achieved promising results (2-and 5-years pfs and os 73% and 55% resp. 83 and 57%) in recent years and these results are better than outcomes of our historical patient cohort from period 2010-2012 (2-and 5-years pfs 41% and 26% resp. os 56% and 35%, p=0.009) or ebmt published data of pts transplanted for cll in period 2000-2010 (2and 5-years pfs 46% and 35% resp. os 62% and 45%). current results of transplantation should be taken into account in our future decision-making process on indications for transplantation in pts with high-risk cll, of course also in the context of new or updated results of targeted cll treatment and its complications. disclosure methods: retrospective data and treatment outcomes were collected from the singapore childhood cancer registry (sccr). most children with cancer in singapore receive therapy at one of the two public paediatric cancer centers (kkh or nuh). a total of thirty two cases were diagnosed with cml and received treatment in either of these centers over a twenty year period (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . results: the age at diagnosis of the thirty two children ranged from 4 to 17 years (median 12.5years). six patients in the pre-tki era were treated with an upfront hsct. the remainder twenty six patients were initially started on a tki. of these 12/26 (46%) had a hsct at a median period of 22.5 months from diagnosis (range 5-43 months). the reason for hsct in ten out of the twelve children was due to high risk features i.e. accelerated/blastic phase/ no ccr/no cmr. the remaining two patients had a hsct due to parent and patient preference for attempt at upfront cure rather than the use of life-long and expensive tki therapy. non-compliance to tki therapy was a major finding in our teenage cohort. eleven of the eighteen transplants used a matched sibling donor. three patients had cord blood as their stem cell source. one patient had a single antigen mismatched related donor and three patients had a mismatched unrelated donor for their hsct. all patients except one had myeloablative conditioning with busulfan and cyclophosphamide. atg was added according to physician preference. one patient had cy/tbi conditioning because of pre-transplant lymphoid blast crisis. anti gvhd medications included cyclosporine/ methotrexate or tacrolimus and methylprednisolone in the cord transplant patients. six of the eighteen (33%) patients who had a hsct died. four died due to treatment related mortality (2 infections, 1 acute gvhd and 1 pulmonary fibrosis). one patient died due to an early relapse and one had a late relapse related mortality. for the pre-tki era, hsct related 5 and 10 year os was 83% and 67% respectively. post-tki era 5 and 10 year os was 75%. for the entire cohort, the 10 year os was 70%. conclusions: the post-tki era transplant outcomes from our two centers is comparable to leading centers in the world. outcomes for patients with mismatched unrelated donors was poor in our cohort. we recommend a haploidentical related donor transplant or an unrelated cord blood stem cell source for patients when a matched sibling or unrelated donor is not available. clinical trial registry: na disclosure: we have nothing to disclose. fludarabine, busulfan, and thiotepa may be a promising conditioning regimen for myelofibrosis patients undergoing allogeneic stem cell transplantation background: allogeneic stem-cell transplantation (sct) is a curative therapy for patients with myelofibrosis. however, recurrent disease and non-relapse mortality (nrm) are frequent causes of treatment failure. the optimal conditioning regimen for sct in this disease has not been defined. methods: we retrospectively analyzed transplantation outcomes of all adult patients given sct for myelofibrosis between 2003 and 2018 at a single large academic medical center. patients (n=59) were treated with several conditioning regimens that were grouped according to conditioning intensity. myeloablative conditioning (mac) included busulfan 12.8 mg/kg and cyclophosphamide 120 mg/kg (bucy, n=10), fludarabine and busulfan 12.8 mg/kg (flu/ bu4, n=9) and fludarabine and treosulfan 30-36 g/m 2 (flu/ treo, n=6). reduced-intensity conditioning included fludarabine and busulfan 6.4-8.0 mg/kg (flu/bu2, n=22). more recently we adopted the tbf regimen including fludarabine, busulfan 6.4-9.6 mg/kg and thiotepa 5-10 mg/ m2 (n=12). all patients were also given anti-thymocyte globulin during conditioning, irrespective of donor source. results: the median age was 59 years (interquartile range [iqr] 52-63). the majority of patients had documented splenomegaly (81%) and were not previously exposed to ruxolitinib (71%). donor type was an hla-matched sibling (41%), 10/10 (51%) or 9/10 (8%) matched unrelated donor. the dipps+ score distribution was intermediate-1 (16%), intermediate-2 (45%), or high (n=39%). the median followup was 3.2 years since the success of tyrosine kinase inhibitors (tkis), transplant-related mortality is considered too high to justify allogeneic hematopoietic stem cell transplantation (allohsct) as first-line treatment for chronic myeloid leukemia (cml) patients in chronic phase (cp). allohsct is currently considered for patients failing to at least 2 tkis or with disease in advanced phase. nevertheless, the optimal timing for transplant referral is still not well defined. methods: we performed a retrospective analysis on 23 consecutive patients with cml in cp receiving first transplants from an hla-identical sibling donor with partially t-cell depleted grafts from 1998 to 2016 at our center. partial t-cell depletion (ptd) consisted of in vitro alemtuzumab incubation of a part of the graft for infusion at day 0 while the rest, containing 100x10 6 cd3+ cells/kg was given as a t-cell add-back at day 1. donor lymphocyte infusions (dlis) were provided, in the absence of gvhd, in case of disease relapse or mixed chimerism. molecular monitoring was performed by 3-month bcr-abl1 rt-qpcr testing in peripheral blood during at least a 5-year period after hsct. thereafter, 3-month testing schedule was maintained where possible, or followed by a 6-month one. kaplan-meier method was employed to determine the probability of overall survival (os) and leukemia free survival (lfs) since allohsct. results: median age at hsct was 36 years (range, 18-58). all patients were in first cp but one who was in second cp. twelve patients were tki-naïve at hsct (1998 hsct ( -2001 , 4 patients had presented suboptimal response or/ and intolerance to imatinib (2002-2004 period) , while the last seven patients had presented suboptimal response or/ and intolerance to imatinib, dasatinib and nilotinib (2005-2016 period) . the time interval from diagnosis to transplant was < 12 months in 11/23 (48%) patients. 15 (65%) patients had an ebmt risk score of 0-2, while 8 (35%) patients of 3-4. the conditioning regimen was myeloablative for all but one patients. the stem cell source was pbsc for 22 patients and bone marrow for one. all patients engrafted. 14 patients presented molecular relapse and one patient hematological relapse with a median interval from transplant to relapse of 9 months (range, 5-70) . 17 patients received dlis (15 for relapse and 2 for mixed chimerism), while 3 patients in relapse also received tki. without prior administration of dli, 3(13%) patients presented grade ii agvhd and 2 patients moderate cgvhd. after dli, agvhd occurred in 3 and cgvhd in 4 patients. one patient died of disease progression 3 years after hsct and one of myocardial infarction 19 years after hsct. with a median follow-up of 14.4 years (range 2.3-20.6), 15-year os and lfs were 95%. at the time of the analysis 21/23 patients were alive and in major molecular response. conclusions: these results of excellent long-term survival and no transplant-related mortality suggest that ptcd improves the outcome of cp-cml patients transplanted from an identical sibling donor and they can be useful for deciding risk-adapted strategies. we believe that ptcd could allow earlier transplant referral of patients failing tkis and having an identical sibling donor. disclosure: nothing to declare single tertiary centre experience in allogeneic haematopoietic stem cell transplantation (allo-hsct) for primary and secondary myelofibrosis (mf) the only curative option for fit patients is allo-hsct. novel therapy is emerging but current recommendation is that eligible patients with life expectancy less than 5 years should be considered for allografting. methods: we retrospectively looked at the clinical features and outcomes of all allo-hsct for mf performed in our centre since 2010. results: 12 patients (10 male, 2 female) aged between 29-68 years old (median age 60) with intermediate-2 or high-risk mf as per the international prognostic scoring system (ipss) or dynamic ipps (dipps) were transplanted in our centre since 2010. 6 of them (50%) were diagnosed with pmf and the remaining 50% with secondary mf; 4 post-et and 2 post-pv mf. 2/12 of our patient group received a sibling allograft and 10/12 a matched unrelated donor allograft (70% received a 10/10 human leukocyte antigen (hla)-matched transplant, 20% a 9/10 hlamatched and 10% a 8/10 hla-matched graft). all patients received a reduced intensity conditioning (ric); 3/12 patients with fludarabine/ melphalan/ campath (fmc), 8/ 12 fludarabine/ busulphan/ atg (fbatg) and 1 fludarabine/ ara-c/ campath (flag/ campath); all received peripheral blood as source of hsc. engraftment occurred between day 13-48, with a median of d+17. one late graft rejection occurred. all patients were alive at d+100. 9 patients are currently alive; overall survival (os) is 75%. transplant related mortality (trm) was 16.6% at 1 year, 25% at 3 years. 1 patient died of graft versus host disease (gvhd) and 2 patients of septicaemia leading to multiorgan failure. acute gvhd grade ii skin occurred in 5 patients, grade iii and above in 2 patients. 5 patients have limited chronic gvhd. 2/12 patients received donor lymphocyte infusion (dli) for mixed chimerism (one of which had 2nd graft failure). out of these 2 patients 1 developed acute grade 4 gvhd and died. response rate: 6/9 alive patients i.e 66.6% exhibit no fibrosis in trephine biopsies, 1/9 alive patients had residual fibrosis but 100% donor chimerism, 1/9 alive patients had residual fibrosis with mixed donor chimerism, other patient non-assessable. conclusions: allo-hsct remains the only potentially curable option for myelofibrosis. in our centre which serves 1.1 million population, with 12 new cases per year, 12 patients were transplanted since 2010. our data suggest that close collaboration between mpn-treating haematologists and transplant physicians is required so that all suitable patients have a transplant assessment early in their disease course. novel molecular prognostic systems are likely to identify those best placed to benefit in future but this series currently supports allo-hsct survival and cure. (range, 105-4624) . dynamic international prognostic scoring system (dipss) score at the time of hct was intermediate-2 or high risk in 20 patients (95%), intermedate-1 in 1 patient. molecular evaluation was available in 14 out of 21: jak2 v617f mutation was detectable in 8 patients, mpl-w515k in 1 patient, carl in 1 patient. 4 patients were "triple negative" for driver mutations. cytogenetics information was available for 6 out of 21; among which 3 patients had complex karyotype, 1 trisomy 8 and 1 trisomy 9. 3 patients underwent splenectomy before hct. ruxolitinib was administered in 4 patients before hct. 10 (48%) patients received stem cells from an hla identical sibling, 9 (42%) from a matched unrelated donor and 2 (10%) from an haploidentical sibling. graft source was bone marrow in 7 patients (34%) and peripheral blood in 14 (66%). conditioning was myeloablative in 16 patients (76%), reduced intensity in 5 (24%). all patients engrafted. acute graft versus host disease was absent in 12 patients (57%), grade i-ii in 7 (34%), grade iii-iv in 2 (9%). in 18 evaluable patients chronic graft versus host disease was limited in 3 (17%), extensive in 4 (22%) and absent in 11 (61%). transplantrelated mortality at 180 days was 24%. main causes of death were: acute gvhd in 2 patients, chronic gvhd in 1, pancreatitis in 1, pulmonary aspergillosis in 1. relapse occurred in 7 patients and was the main cause of death in 4 of them. notably, 2 patients experienced late relapse after 6.6 and 17.6 years after hct. both of them are living while receiving ruxolitinib therapy. after a median follow up of 581 days (range, 38-11660), 10 out of 21 patients are alive. 7 of them (33%) are disease-free and 3 are living. the kaplan-meyer overall survival and disease-free survival at 10 years was 40% and 35%, respectively. conclusions: our experience confirms that hct is a valid option to achieve cure in one third of mf patients. two patients experienced very late (> 5 years) recurrence of mf. the rarity of this condition limits the amount of data and cases available for evaluation and study. life-long follow-up of all mf transplanted patients is warranted to better understand this rare event. disclosure: nothing to declare methods: а 37-years old female was diagnosed with jak2v617f-positive pmf, 46xx, ipss low risk, dipssplus intermediate -2 risk, subacute budd-chiari syndrome and portal vein thrombosis four years before allohsct. to reduce the splenomegaly and constitutional symptoms we performed pre-transplant ruxolitinib therapy 30 mg daily. after three months of therapy the patient achieved clinical improvement (eln criteria). contrast-enhanced computer tomography and magnetic resonance imaging showed enlarged intrahepatic collateral vessels and signs of portal vein thrombosis with cavernous transformation and multiple dilated collateral veins. gastroscopy documented enlarged esophageal veins. allogeneic stem cell transplantation was performed from 10/10 -hla matched unrelated donor with peripheral stem cells (6.1 x 10 9 сd34+ cells/kg). conditioning regimen consisted of fludarabine (180 mg/ m 2 ), busulfan (10 mg/kg p.o.). post-transplant cyclophosphamide was administered at 100 mg/kg at day +3, +4, and ruxolitinib 15 mg was used from d+5 till d+100 as graft versus host disease prophylaxis. results: starting d+1 the patient experienced eight episodes of ebv some of them with severe blood loss. to treat the bleeding episodes blackmore tube was placed six times with temporary effect. to place blackmore tube the patient was two times intubated and required mechanical ventilation. at d+18 leukocyte and neutrophil engraftment, full donor chimerism and molecular remission were achieved. platelet engraftment was documented only at d +42 and poor graft function was present due to cytomegalovirus reactivation (d+41) and parvovirus b19 reactivation (d+62). evb was stopped at d+95 only after two esophageal veins ligations, and two procedures of gastric veins sclerotherapy. soon (d+111) the patient achieved complete platelet recovery (more than 50x10 9 /l) and became red blood cells transfusion independent. at day + 180 complete remission was confirmed by splenomegaly resolution, regression of bone marrow fibrosis, full donor chimerism, jak2v617f-negative molecular status. cbc showed hb 130 g/l, platelets 73x10 9 /l, leucocytes 3,8x10 9 /l. ultrasound examination after transplant documented portal vein thrombosis recanalization. at day + 180 she developed mild (nih) chronic graft versus host disease with eyes and mouth involvement, which was managed with topical steroids. at d+958 after transplant the patient is alive in complete remission and has no recurrent bleedings. conclusions: splanchnic vein thrombosis can significantly complicate the course of allohsct in pmf. easy access to surgical, intensive care unit and endoscopic teams is required to make allohsct more feasible in this group of patients. disclosure all patients received treosulfan-based mac regimens, treosulfan(total dose, 36-42gms/m2) was given in combination with different conditioning drugs. the most commonly used regimen was treosulfan, fludarabine (150mgs/m2) and thiotepa(10mgs/kg) referred to as ftt that was used in 59%(n=55). serotherapy was given in 93% of patients(n=87), as either alemtuzumab or antithymocyte globulin in 82%(n=77) and 11%(n=10), respectively. post-transplant graft-versus-host disease (gvhd) prophylaxis was given in all patients, based mostly on ciclosporin. 46 patients(49%) received the transplant from identicalrelated donors, 46 patients(49%) received the transplant from matched-unrelated donors, and two patients(2%) had haploidentical transplants. 90% of the patients(n=85) were fully hla-matched. all stem cell sources were used as bone marrow in 59%(n=55), peripheral blood stem cells in 33%(n=31), and umbilical cord blood in 8%(n=8). this treosulfan-based conditioning was given as the 1 st transplant in 92%(n=85), and as the 2 nd transplant after the failure of a first procedure in 8%(n=9). two patients received treosulfan-based conditioned transplant twice. results: neutrophil engraftment and platelet engraftment occurred at a median of 13 days and 18 days respectively. chimerism was full donor in 55%(n=52), high donor in 18%(n=17), and mixed donor in 9%(n=8). gvhd developed in 43% of patients(n=40), with acute gvhd grade i/ii and grade iii/iv developed in 29%(n=27) and 2%(n=2), respectively. chronic gvhd grade i/ii and grade iii/iv developed in 13%(n=12) and 2%(n=2), respectively. all chronic gvhd were mild, limited, non-extensive, and resolved completely. none of our patients had persistent gvhd necessitating long-term systemic immunosuppression. mild vod occurred in 13%(n=12), and severe vod occurred in 2%(n=2). one of them died but was believed to be related to the underlying disease (wolman syndrome). viral reactivation occurred in 55% of patients(n=52), with cmv, ebv, and adenovirus reactivation was found in 34%, 21%, and 14%, respectively. five patients had invasive adenoviraemia that contributed to death in two of them. primary graft failure happened in two patients(2%) due to adenoviraemia. seven patients(7%) had secondary graft failure with autologous reconstitution. graft failure was significantly lower (p0.045) in the ftt group than other conditioning groups. at a median follow-up of 35 months (range, two-174 months), eleven patients(11.9%) died, with overall survival of 88.1%, and event-free survival of 80.9%. five patients died due to complications related to their original disease, while six patients died due to transplant-related causes (transplant-related mortality 6.5%). immune reconstitution in alive patients was achieved at a median of eight months. this time was significantly longer (p0.034) in ftt group. conclusions: this study demonstrates that treosulfan is a safe and effective conditioning drug that can achieve engraftment, with low rates of graft failure, transplantrelated mortality and morbidity, even if it is used twice in the same patient. disclosure: nothing to declare background: high-dose chemotherapy (hdc) followed by autologous stem cell transplantation (asct) is the treatment of choice for the patients with relapsed or high risk nhl. although the high-dose conditioning regimens commonly used in patients with non-hodgkin lymphoma (nhl) are beam (bcnu, etoposide, cytarabine, and melphalan), beac (bcnu, etoposide, cytarabine, and cyclophosphamide), survival of patients with nhl received above high-dose chemotherapy followed by asct was still unsatisfactory. methods: we prospectively evaluated the efficacy and toxicity of busulfan, etoposide, cytarabine and melphalan (bueam) including iv busulfan instead of bcnu of standard beam as a conditioning for asct in patients with nhl. the high-dose chemotherapy consisted of bu (3.2 mg/kg i.v. q.d. from day -6 to day -5), e (200 mg/m 2 i.v. b.i.d. on day -4 and day -3) a (1 g/m 2 i.v. q.d. on day -4 and day -3) and m (140 mg/m 2 i.v. q.d. on day -2) at 7 centers in korea. results: two hundred five patients were enrolled onto the study. main subgroup was diffuse large b cell lymphoma (n=104, 50.7%), t cell lymphomas (n=59, 29.8%), and nk/t cell lymphoma (n=22, 10.7%). upfront asct was performed in 160 patients (78.0%), and salvage asct in 45 patients (22.0%). the disease status of the patients before hdt/asct consisted of 133 patients (64.8%) with complete response and 72 patients (35.2%) with partial response. treatment related toxicities included nausea in 149 patients (72.7%), diarrhea in 127 patients (62.0%), anorexia in 107 patients (52.2%) and stomatitis in 97 patients (47.3%), which were grade i or ii in the majority of cases. the common grade iii toxicities were stomatitis (6.9%), diarrhea (5.9%), and anorexia (5.4%). there were no vod, and transplant-related mortality occurred in 4 patients (1.95 %), due to infection. one hundred fifty three patients (74.6%) achieved a complete response and 13 patients (6.3%) after asct, while 28 patients (13.7%) showed progressive disease. at a median follow-up duration of 38.6 months, the estimated 3-year overall survival and progression free survival for all patients was 74.5% and 56.6%, respectively. conclusions: the conditioning regimen of bueam for asct was well tolerated and seemed to be effective in patients with relapsed or high risk nhl. disclosure: none of declare background: allogeneic hematopoietic cell transplantation (hct) is potentially curative for high risk acute myeloid leukemia (aml) and myelodysplastic syndrome (mds), however both gvhd and disease relapse remain major challenges. we recently introduced a combination of posttransplant cyclophosphamide (ptcy) and atg (4.5 mg/kg) as graft-versus-host disease (gvhd) prophylaxis. the purpose of our study was to compare outcomes between ptcy/ atg and other gvhd prophylaxis regimens for high risk aml and mds. methods: we retrospectively investigated outcomes of 159 patients that underwent allogeneic hct between january 2014 and july 2017 for high risk aml (n=120, 75%) and mds (n=39, 25%). gvhd prophylaxis regimens were compared for overall survival (os), cumulative incidence of relapse (cir) and non-relapse mortality (nrm) in univariate and multivariable analysis. high risk aml was defined as secondary aml, therapy related aml, high risk cytogenetics (eln criteria) in cr1, good/ intermediate cytogenetic risk aml in cr2 and primary induction failure; high risk mds was defined as high/very high risk wpss score. results: median age of patients was 56 years (range 22-73 years). donors were matched related in 52 (33%) patients, matched unrelated in 89 (56%) patients and haploidentical in 18 (11%) patients. graft source was peripheral blood stem cells in 158 patients (99%). myeloablative conditioning was used in 54 patients (34%), reduced intensity regimens in 105 (66%) patients. ptcy combined with atg was used in 69 (43%) patients, other gvhd prophylaxis regimens were used in 90 (57%) patients. both donor and recipient were cmv negative in 18 (11%) patients. median follow-up of survivors was 29 months (range 14-56 months). univariate analysis demonstrated os of the entire cohort at 2 years was 49% (95%ci 41-57%), cir at 2 years was 22% (95%ci 16-29%) and nrm at 2 years was 32% (95%ci 25-39%). concerning gvhd prophylaxis regimen, 2-year os for ptcy/atg versus others was 46% (95%ci 33-58%) versus 51% (95%ci 40-61%) (p=0.87, figure) , 2-year cir for ptcy/atg versus other was 31% (95%ci 20-43%) versus 16% (95%ci 9-24%) (p=0.02) and 2-year nrm for ptcy versus other was 28% (95%ci 18-39%) versus 34% (95%ci 25-44%) (p=0.35). grade ii-iv acute gvhd was seen in 23% of ptcy/atg patients versus 59% using other regimens (p< 0.0001). chronic gvhd was observed in 20% of ptcy/atg patients versus 42% using other regimens (p=0.004). multivariable analysis for os confirmed that the gvhd prophylaxis regimen has no influence (p=0.19), while the predominant predictor of survival was age at hct (hr 1.03, 95%ci 1.01-1.05, p=0.01). for cir, the ptcy/atg combination had no influence compared to other gvhd prophylaxis regimens (p=0.6), while ric conditioning was the predominant predictor of relapse (hr 3.05 for ric, 95% p=0.01) . for nrm, the atg with ptcy combination demonstrated no significant difference (p=0.12), while age at hct was the predominant predictor (hr=1.04, 95%ci 1.01-1.07, p=0.02). conclusions: the ptcy/atg combination for gvhd prophylaxis has demonstrated on multivariable analysis similar os, cir and nrm with other previously used regimens at our center. a decrease in atg dose may potentially decrease the relapse rate while retaining the advantage of decreased gvhd. [ background: the combination of fludarabine with myeloablative doses of busulfan (fb4) represents a standard of care conditioning regimen before allogeneic transplantation in patients with myeloid malignancies (giralt, s.: the lancet oncology 2015). fb4 has potent antileukemic activity and is associated with low transplantrelated mortality and acute gvhd. however, early after transplantation (days 30-90), a proportion of patients may not convert to a full donor haemopoietic chimerism, particularly if anti-t lymphocyte globulin (atg) is used as gvhd prophylaxis (rambaldi a, et al.: the lancet oncology 2015) methods: we retrospective analyzed 104 patients who underwent an allogeneic stem cell transplantation after fb4 conditioning regimen at our hospital, from november 2007 to august 2018. the median age was 51 years (range 22-67) and diagnoses were aml 76%, mds 18% cml 5% mfi 1%). the disease status at transplantation was: cr1 in 59%, cr2 in 5% and active disease in 36% of patients. the stem cell source was represented by pbsc in more than 95% of cases and anti-t lymphocyte globulin (atg) was part of the conditioning regimen in more than 95% of cases at a dose of 5 mg/kg. the donor was a hla identical sibling (26%), a matched unrelated (65%) or mismatched (one allele or one antigen mismatched) unrelated, 9%. hematopoietic chimerism was molecularly evaluated by variable number of tandem repeats (vntr) on bone marrow (bm) mononuclear cells or peripheral blood (pb) t lymphocytes, purified by immunomagnetic positive selection (miltenyi, biotec). the analysis was performed at day 30, 60, 90, 180 and 360 after transplantation results: after 30, 60 and 90 days from transplantation, the proportion of patients with a full bm chimerism was 94%, 87% and 83%, respectively. at the same time points, the pb t cell chimerism was 47%, 65% and 69%. before day 100, 10 patients required the infusion of dli to treat a pending or overt hematologic relapse and 12 patients to convert the lymphoid chimerism from mixed (median 44%, range 0-76), to complete (successfully in 7 cases). after day 100, 13 additional patients required dli to treat disease relapse or progression and 8 patients to improve the chimeric status or the immune reconstitution. at 5 years, the overall survival is 64%, with a relapse and non-relapse mortality of 21 % and 11%, respectively ( figure 1 ). by uni and multivariable analysis, aml diagnosis and a mixed bm chimerism before day 100 were associated with relapse and overall survival while age > 50 was the only factor significantly associated with nrm. a mixed pb t-lymphoid chimerism before day 100 does not adversely impact on non-relapse mortality, cumulative incidence of relapse, leukemia-free and overall survival. conclusions: after fb4 and atg, a progressive increase of pb lymphoid donor chimerism develops gradually after transplantation, in most of cases without the need of dli. early mixed lymphoid chimerism does not compromise the main long-term clinical outcomes and may at least partially explain the low non-relapse mortality. an incomplete bm chimerism within the first 3 months strongly correlates with early disease progression or relapse. background: busulfan (bu) is widely used as a component of myeloablative conditioning regimen before hematopoietic stem cell transplantation (hsct) in children. bu has a narrow cumulative exposure window. the relation of bu exposure with toxicity is well established, but the link between the exposure and efs is not clear due to conflicting reports especially in pediatric patients. obtaining the ratio of bu to its metabolite i.e. metabolic ratio (mr) may serve as an indicator of bu gsh conjugating capacity of an individual, thus cumulative exposure of bu for a particular day that could be used along with auc as a marker to predict efs. the present investigation is aimed at evaluating the utility of bu mr to predict efs in children undergoing allogeneic hsct. methods: two different cohorts with children receiving bu in four times daily (qid, n=44) and once daily doses (qd, n=13) at st. justine's hospital, montreal were studied. bu and su levels were measured on day 3 of the conditioning regimen at the end of infusion (dose 9 in qid or dose 3 in qd dosing). efs was defined from the time of transplant until death, relapse, or rejection, whichever occurred first. a receiver-operator characteristic curve (roc) for bu mrs measured was plotted to show the trade-off in sensitivity vs. 1-specificity rates for efs, as the cut-off of the test was shifted from low to high. cutoff values were defined based on the youden´s j statistic (i.e. sensitivity+specificty-1). results: twenty-two males and 22 females aged from 0.1 to 19.9 years (mean±sd: 7.2 ± 5.7) from bu qid cohort had the mean mr of 5.9 (sd: 3.2). a cut off value of 4.9 in mr was chosen in roc analysis in this cohort, with better sensitivity (71 %) and specificity (70 %) for efs prediction (p=0.01, auc= 0.7 (95 % ci= 0.6-0.8). in qd cohort nine females, and four males aged between 0.4 and 15.8 years (6.7±5.1) had the mean mr of 29.3 (sd: 16.6). in roc analysis, a cut off value of 25.06 was chosen with better sensitivity (100 %) and specificity (100 %) for efs prediction (p=0.003; auc=1.0). conclusions background: treosulfan is an alkylating agent increasingly used prior to hematopoietic stem cell transplantation (hsct). the main objective of this study was to develop a population pharmacokinetic model of treosulfan in pediatric hsct recipients and to explore the effect of different covariates on treosulfan pharmacokinetics (pk). also, a limited sampling model (lsm) was developed. methods: in this multicentre study, 91 patients, receiving a dose of 10, 12 or 14 g/m 2 treosulfan a day, administered during 3 consecutive days, were enrolled. a population pharmacokinetic model was developed using nonlinear mixed effect modelling (nonmem version 7.3.0, using psn toolkit 4.7.0 and piraña version 2.9.7 as modelling environment). demographic factors, as well as laboratory parameters, were included as covariates. results: treosulfan pk was best described by a twocompartment model. a bodyweight-based allometric model improved the model more than a model incorporating body surface area (bsa). clearance (cl) and intercompartmental clearance parameters were 6.07 l/h/15.6kg (95%ci 5.46-6.68) and 2.15 l/h (95%ci 1.39-2.91). typical volumes of distribution of the central and peripheral compartments were 8.00 l/15.6kg (95%ci 6.88-9.12) and 2.05 l (95%ci 1.52-2.58). a model-based dosing table based on bodyweight is created to achieve a target exposure of 1540 mg*hr/l (table 1) , which was the median exposure of our population. estimated glomerular filtration rate (egfr) was shown to be the only parameter that significantly reduced interpatient variability in cl from 36.5% to 34.8%. a limited sampling model with 3 samples (taken at 1.5, 4 and 7 hours after start of infusion) accurately estimated pharmacokinetic parameters of treosulfan. conclusions: to the best of our knowledge, this is the largest cohort of pediatric patients treated with treosulfan used for a population pharmacokinetic study. we developed a two-compartment model with weight and egfr as covariates influencing treosulfan pk. recently we showed a relationship between treosulfan exposure and early toxicity. patients with an exposure >1650 mg*hr/l have an increased risk of developing grade 2 or higher mucositis and skin toxicity. another study in 87 pediatric patients with thalassemia major reported an association between treosulfan clearance (< 7.97 l/h/m 2 ) and poor overall survival. our model, together with the limited sampling strategy, can be used to adjust the dose, prior to or during treosulfan administration. ongoing studies conducted in different disease settings will determine if treosulfan exposure can influence patient outcome. subsequently, the optimal target exposure can then be established. background: autologous stem cell transplant (asct) is an effective treatment method for non-hodgkin lymphoma (nhl). until recently, carmustine, etoposide, cytarabine and melphalan (beam) was the most commonly used conditioning regimen. despite acceptable efficacy with beam, carmustine is associated with major pulmonary toxicity. for this reason, the aim of this study was to investigate the safety and efficacy of beb conditioning regimen for asct in nhl. methods: we conducted a prospective, multicenter, phase ii study for beb conditioning regimen for asct in nhl patients. a total of 33 patients were enrolled from 3 centers. they underwent asct with beb conditioning regimen (busulfan 3.2mg/kg for 3days, etoposide 400mg/ m 2 for 2days, bendamustine 200mg/m 2 for 2days) between 2016 and 2018. [[p111 image] 1. two year progression-free survival and overall survival.] results: the median age was 52 years (range 21-66) and 16 patients (48.5%) were men. the most common type was diffuse large b cell lymphoma (n=23, 69.7%) and more than half of patients (n=19, 57.6%) were classified as ipi score 3 or 4. eight patients (27.3%) had a history of relapse and 19 patients (57.6%) received more than 2 lines of chemotherapy before asct. most patients (n=27, 81.8%) were complete remission (cr) state at asct. a median number of 5.85x10 6 /kg cd34 cells were infused (range 2.0-18.6). all patients engrafted after a median time of 11 days (range 10-14). twelve patients (36.4%) experienced neutropenic fever and 16 patients (48.5%) had grade 3 toxicities during asct. however, no one had a documented infection, veno-occlusive disease, or treatment-related death. three months cr rate was 81.8%. during a median follow-up period of 10.2 month, 7 patients (21.2%) exhibited relapse or progression, while 1 patient (3.0%) died of the disease. the estimated 2-year pfs and os rate were 73.0% and 89.8%, respectively ( figure 1 ). conclusions: the beb conditioning regimens for asct is a feasible with tolerable toxicity in patients with nhl. disclosure: nothing to declare long-term report of total marrow or total lymphoid imrt in advanced leukemia, myeloma and lymphoma background: during the last three decades, total body irradiation (tbi) continues to play an important role in the conditioning regimens for patients undergoing stem-cell transplant (sct) for a wide variety of advanced hematological malignancies. however, tbi showed boundaries in dose limits for toxicity in allogenic and moreover in autologous stem cell transplantation. currently, the choice of conditioning regimen is based on the use of the least-toxic regimen to achieve the optimal therapeutic result. this report aims to assess the feasibility of a conditioning strategy based on high dose chemotherapy and whole-body radiotherapy focused on selective extensive tumor burden irradiation, both in allogeneic and autologous stem cell transplantation. methods: since december 2009, sixty-two patients (pts) have been irradiated by helical tomotherapy (ht) to extensive target before allogeneic or autologous transplantation. selected total marrow irradiation (tmi) schedules were planned to treat patients with high risk acute leukemia (all or aml) or multiple myeloma (mm) as a part of conditioning regimen. total lymphoid irradiation (tli) was planned for patients with refractory or relapsed (r/r) hodgkin (hd) or non-hodgkin lymphomas (nhl). results: tmi and tli allowed delivering therapeutic dose over extensive selected targets with wide reduction of toxicity to all the organs at risk (oars). the higher radiation doses rate to the oars is reduced from 30% to 70%. allogenic conditioning regimen was tli (4gy x 3fx) than fludarabine + endoxan for patients with hd (4 pts). tmi (4gy x 3fx) + fludarabine + melphalan for patients with mm (4 pts). tmi (4gy x 2 fx) + thiotepa + fludarabine + busulfan for advanced lam patients (4 pts). tmi as the boost (2-3gy) after conventional tbi was (12 gy in 6 bi-fractionated doses) by cyclophosphamide (18 pts). autologous preparation to sct consisted of tli (4gyx 3fx) followed by high-dose bendamustine and melphalan for patients older than 40 years and conventional feam (fotemustine, etoposide, cytarabine, and melphalan) for younger patients, in hd e nhl (20 pts). while tmi (4gy x 3 fx) plus melphalan was delivered for autologous sct in mm and lam (12 pts). no unexpected acute toxicity was found. in the allogenic setting, all the patients' engraftment was achieved in all patients. no acute graft versus host disease increasing was detected. within the autologous setting, only 33% developed grade 3/4 mucositis. none experienced grade 3/4 extra-hematological toxicity. outcomes of the specific disease will be reported. conclusions: the current report describes the clinical feasibility of using ht to deliver tmi or tli in the setting of autologous transplantation or during allogenic stem cell conditioning regimen, to allow all patients (old, fragile or with high tumor burden) to achieve an ablative regimen before sct. to our knowledge, this single institution experience describes data from one of the largest cohort of patients treated in europe since the development of this irradiation techniques. disclosure induction therapy in both groups of patients was based in polychemotherapy without the use of new drugs. case matching was performed according to age, clinical stage at diagnosis, and response to induction therapy. conditioning regimen consisted of iv bu at a dose of 3.2 mg/ kg once a day on days -5 to -3 followed by mel at a dose of -140 mg/m 2 on day -2 in the bumel group versus mel200 in the control group. maintenance therapy after transplant consisted of interferon and steroids in the majority of patients. results: the cut-off date for this update was june 30, 2018. after a median follow-up of 56 and 63 months in the bumel and mel200 groups respectively, 35 patients had relapsed in the bumel group and 82 patients in the control group. median pfs was 33 (95% ci, 25.4-48.3) months in the bumel and 24 (95% ci, 20.1-32.7) months in the mel200 group (p = 0.04) ( figure 1 ). in this update, 12 patients in the bumel group are in maintained response and 7 of them are in continuous cr (two with negative status for minimal residual disease) between 9 and 12 years after transplantation. ten-year os was not significantly different between both groups, being 41 (95% ci 30-58) months in the bumel and 29 (95% ci 18-47) months in the control group.transplant-related mortality was similar in both groups of patients (4% in the bumel and 2% in the mel200 group). regarding toxicity, bumel was associated with a higher incidence of mucositis and liver toxicity than the melphalan-only approach but no patient in our series developed sinusoidal occlusive syndrome and the hepatic toxicity observed was only grade i/ii. finally, no long-term side effects have been reported among bumel recipients. conclusions: this long-term follow-up analysis confirms that a therapeutic strategy including bumel as conditioning regimen beforeasct in patients with newly diagnosed mm is highly active and safe in these patients. [[p113 image] 1. figure 1 . progression free survival in the bumel (____) and control group (…… frequency of acute gvhd grade iii-iv [cc: 11%; ct: 17%; tt: 24%, p=0.057], and transplant-related mortality was higher in tt-carriers (cc:26%; ct:28%; tt:46%, p=0.02 cc&ct vs tt) . ta-tma, cmv infection/reactivation and cgvhd were also not different according to donor genotypes. fungal infections occurred more frequently as causes of death in carriers (cc: 9.7% vs. ct: 38.1% vs tt: 33.3%, p=0.022). conclusions: our results suggest that donor tgfb1 -1347c>t may exert an adverse influence on the outcome of myeloablative conditioning. our finding might be explained by the combination therapy of calcineurin and mtor inhibition in gvhd prophylaxis in myeloablative conditioning. disclosure: nothing to declare. treosulfan-based reduced intensity conditioning in hla-haploidentical transplantation using ptcy as gvhd prophylaxis in high-risk mds /aml of the elderly background: standard conditioning regimens prior to allogeneic hematopoietic stem cell transplantation (allo-hsct) are often associated with a considerable risk of severe adverse events, especially in elderly patients suffering from high-risk (hr) mds/aml. previous clinical studies have demonstrated feasibility of treosulfan-based reduced-intensity conditioning (ric) by stable engraftment, low non-relapse mortality (nrm), and favorable survival in elderly patients undergoing hla-matched related or unrelated allo-hsct (beelen et al, ash 2017 #0521). however, data for treosulfan-based conditioning in the t-cell-replete hlahaploidentical (haplo-hsct) setting in high-risk aml/mds patients are rare. here we report on the outcome of eleven patients treated with a treosulfan-based conditioning undergoing haplo-hsct using exclusively post-transplantation cyclophosphamide (ptcy) as gvhd prophylaxis. methods: eleven patients with high-risk (hr) aml (n=9)/mds (n=2) who underwent haplo-hsct using treosulfan for reduced intensity conditioning (ric) and ptcy as gvhd prophylaxis were retrospectively analyzed with respect to outcome and toxicity. all patients were >55 years old and transplanted between january 2016 and february 2018 at our institution. the majority of the patients (9/11) suffered from active disease at time of treatment initiation, only two patients presented in cr. all but one received sequential conditioning with cytoreductive chemotherapy using flamsa applied shortly prior to treosulfan-based ric (10g/m 2 over 3 days). a bone marrow graft was used in 9/11 patients. post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mmf. national cancer institute common terminology criteria for adverse events version 3.0 were used for nonhematologic toxicity assessment starting from sequential therapy initiation or conditioning until day +30. results: median age of the entire cohort was 63 years (range: 58-71). the hct-ci was ≥2 in eight pts (median hct-ci=2, range: 0-5). no graft rejection occurred. neutrophil and platelet engraftment were achieved in 100% and 91% of the patients at a median of 20 (16-23) and 26.5 (13-30) days, respectively. acute gvhd grade ii-iv occurred in 18% of the patients, exclusively involving the skin. no one developed severe (°iii-iv) acute gvhd. no patient died prior to haplo-hsct. severe nonhematologic regimen-related toxicities (°iii-iv) occurred in 2/11 patients, predominately affecting the gastrointestinal tract. no patient suffered from ≥two iii-iv°toxicities. all patients developed fever during treatment course, four with positive blood cultures. cmv reactivated in 6/7 patients at risk. no ebv reactivation or ptld occurred. six patients had clinical and radiological signs of pneumonia (probable invasive aspergillosis) without detection of aspergillus/antigen in the bronchoalveolar lavage. ci of nrm at day +180 was 0%. four patients relapsed within the first year after haplo-hsct, with two of them dying due to relapse. at last follow-up (dec 2018) 9/11 patients were alive. with a median follow-up of 5 months (2-31) estimated 1-year os and dfs were 80% and 59%, respectively. conclusions: treosulfan-based unmanipulated hlahaploidentical allo-grafting using ptcy as gvhd prophylaxis in hr mds and aml patients aged over 55 years is safe and well tolerated resulting in stable engraftment and a favorable toxicity profile. our preliminary data further show promising outcome with low nrm, no severe acute gvhd and favorable survival offering an attractive alternative in ric for haplo-hsct of the elderly. disclosure: nothing to declare comparison of outcomes of total body irradiation (tbi) vs non-tbi conditioning regimens in acute lymphoblastic leukemia for allogeneic transplantation background: in adult patients diagnosed acute lymphoblastic leukemia (all) long-term results are poor with intensive chemotherapy. allogeneic stem stem cell transplantation is the potential treatment that provides cure for these patients. myeloablative preparation regimens include total body irradiation (tbi̇)+ cyclophosphamide(cy) and busulfan + cyclophosphamide.in adult all patients wbi/cy widely used, but the toxicity rate is higher. the aim of this study is to compare the result and effect of the tbi/cy and busulfan/cy regimens in allogenic bone marrow transplantation in all patients. methods: between 1993 -2018 there were 137 all patients who underwent transplantation using myeloablative preparation regimen with or without addition tbi in the adult bone marrow transplantation units of medipol medical faculty, istanbul university istanbul medical faculty, sisli florence nightingale hospital, atakent acıbadem hospital adult bone marrow units . we analyzed overall survival (os), progression free survival (pfs), veno occlusive disease, acute and chronic graft versus disease development rates in these patients. results: demographic characteristics of patients summarized in table -1 there was no significant difference between groups in donor age, gender, stem cell source. it was observed that the relapse rate was not statistically significant in both group.there was no statistically significant difference between the patients who underwent myeloablative regimen and myeloablative regimen with tbi in relaps,death, os, pfs. (figure-1) [[p117 image] 1. figure 1 ] in terms of transplant complications there was also no respectable difference in development of vod and acute and chronic graft versus disease but vod was more common in the group that did not use tbi (p: 0.068) ( conclusions: although there are contradictory data in the literature, in our multicentre study, it was revealed that the addition of tbi in the myeloablative preparation regimen compared with myeloablative preparation regimen alone did not have a positive or negative effect on overall survival.we think that if we can prepare a good vod prophylaxis approches, we can give up tbi in future. disclosure (n=3) . for gvhd prophylaxis, cyclosporine a was given either alone (n=21), with mmf (n=13) or with methotrexate (n=1). the graft source was bone marrow (bm) in most cases (n=31), pbsc in seven cases, matched sibling cord +bm in two cases and one matched related cord. twentyfive of the donors were family donors and ten were unrelated. twenty-nine of the donors were 10/10 hla matched, six were 9/10 mismatched and one haploidentical. four patients had engraftment failure and required a second transplant, two of them were re-transplanted with cyclophosphamide and tbi, one with fludarabine, busulfan and campath, and one with no conditioning. thirty of the 35 patients are alive (86%). four patients died of transplant complications and one died of metastatic squamous cell carcinoma. eight survivors are mixed chimeras (81%-94% donor) and are all doing well, none of them developed any gvhd. nine patients developed acute gvhd, four of them with grade 3-4. seven of these patients later developed chronic gvhd, two of them have extensive disease. conclusions: our results show a high survival rate of 86%, with a low rate of engraftment failure and reasonable rates of gvhd. only one of our patients died of late effects of hsct for fa. mixed chimerism does not seem to present a problem. we conclude that reduced intensity fludarabine based conditioning regimens are a good treatment option for patients with fanconi anemia undergoing hsct. disclosure: nothing to declare total marrow irradiation + bendamustine as reducedtoxicity myeloablative conditioning prior to allohsct for younger patients with multiple myeloma background: the prognosis of patients with multiple myeloma (mm) has improved markedly over the last two decades. despite that, allohsct remains the only treatment option with curative potential. however, its use is limited due to high incidence of non-relapse mortality (nrm) after myeloablative conditioning while insufficient efficacy of reduced-intensity regimens. we developed a new protocol characterized by reduced toxicity while preserved myeloablative potential, based on the use of total marrow irradiation (tmi) in combination with bendamustine. the aim of this study was to evaluate its safety and efficacy in a singlecenter experience. methods: between years 2013-2018, mm patients below 55 years old were offered tandem auto-allohsct as part of first-line therapy. the decision was based on individual patient preferences after detailed description of potential risks. autohsct was preceded by melphalan 200 mg/m 2 iv. the conditioning prior to allohsct consisted of tmi performed using helical tomotherapy at the dose of 4 gy/d on days -3, -2, -1 (total 12 gy) and bendamustine 140-220 mg/m2/d iv. on days -5, -4 (total 280-440 mg/m2). the immunosuppressive therapy consisted of cyclosporine + methotrexate +/-atg. peripheral blood was used as a source of stem cells. results: the analysis included 18 patients (women -9, men -9). the median follow-up was 28 (4-68) months. the median age at allohsct was 44 (26 -53) years. the disease stage before allohsct was as follows: cr-6, vgpr-4, pr-6. patients were treated with hsct from either hlamatched siblings (n=7) or unrelated donors (n=11). the interval between autohsct and allohsct was 5 (4-23) months. all patients engrafted after allohsct with median time of neutrophil and platelet recovery of 14 and 12 days, respectively. one patient (6%) experienced grade 2 acute gvhd, while there were no cases of grade 3-4 acute gvhd. the incidence of mild, moderate and severe chronic gvhd was 17%, 0% and 6%, respectively. the rate of grade 3 non-hematological toxicities was 11%. one patient died of late bacterial infection. the incidence of trm was 5%. grade 4 adverse events were not reported. disease status 3 months after allohsct was: cr-10, vgpr-5, pr-3. the probability of os and pfs after 30 months was 94% (+/-6%) and 77% (+/-12%), respectively. the incidence of progression and trm was 17% and 6%, respectively. conclusions: allohsct using tmi 12gy + bendamustine conditioning protocol is characterized by good tolerance and low risk of gvhd. it may be used for younger patients with mm as part of tandem auto-allohsct strategy. encouraging results reported in this study should be confirmed in prospective clinical trials. disclosure: nothing to declare p120 comparison between two reduced intensity conditioning regimens in patients with a myeloid malignancy: a single center experience comparing fb2 with flumel background: hematopoietic stem cell transplantation (hsct) remains the only curative option for high-risk myeloid neoplasms. the optimal reduced-intensity conditioning (ric) is still debated. methods: a single-center retrospective analysis was conducted at our institution to compare two different ric regimens in adult patients transplanted for myeloid malignancy from 2001 to 2018. a total of 137 patients were analysed, 74 of them treated with busulfan-based (fludarabine 150 mg/m 2 , busulfan 6.4 mg/kg, fb2) and 63 with melphalan-based conditioning regimen (fludarabine 150 mg/m 2 ,melphalan 140 mg/m 2 , flumel). antithymocyte globulin (atg) was administered in all patients while no one received tbi. partial in vitro t-cell depletion was performed using alemtuzumab for low risk patients. results: the two groups were well balanced with a median age of 61 and 62 years in the fb2 and flumel group, respectively, and a median follow up of 46 months. the most frequent indication for transplant in both groups was aml (59.5 and 69.8% for fb2 group and flumel group, respectively) and the stem cell source was peripheral blood in 94.6 and 96.8% of patients. more patients in the first group had near to significant worst karnosfky status (< 90) at transplant compared to second (35. 1 vs 19%, p=0 .057) and more patients received a t-partial depleted graft (54.1 vs 33.3%, p= .028). the neutrophil engraftment was significantly shorter after flumel (15 vs 18 days, p < .01). the 3-year overall survival (os) and disease-free survival (dfs) were of 43.0 and 36.5%, respectively, after fb2 and 54.9 and 52.0% after flumel, respectively, and were not significantly different (p=.41 for os and .15 for dfs), with a karnofsky >= 90 being the only factor significantly associated in univariate analysis with better os and dfs (p=.02 for both). the cumulative incidence (ci) of grade 2 to 4 acute graft-versus-host disease (agvhd) was 16.2% after fb2 and 38.3% after flumel (p< .001) and was associated in multivariate analysis with both t depletion and ric type (p< .001 and .005, respectively). the ci of chronic gvhd at 3 years was 13.9% in fb2 and 22.1% in flumel group (p=.24) . the ci of non-relapse mortality at 3 years was 18.7% after fb2 and 29.6% after flumel (p=.11). the ci of relapse at 3 years was 44.8% for the first and 18.4% for the second group (p< .001) and was associated with conditioning regimen in multivariate analysis (p=.02). no difference in 3-years gvhd-free/ relapse-free survival (grfs) was observed between the two group (25.5% for fb2 and 37.6% for flumel, p=.48). conclusions: when comparing two ric regimens for myeloid neoplasms, we observed a higher incidence of agvhd after flumel whereas no statistical difference was noted for the cgvhd occurrence. while the toxicity appears to be higher after flumel, this result is counterbalanced by a higher proportion of relapse after fb2, accounting for no difference in os, dfs and grfs between the two groups. these findings could be partially explained by a larger proportion of patients receiving a partial t-depletion after fb2 ric, but a larger trial is needed to clarify this issue. disclosure: nothing to declare. once-daily vs 4-times daily intravenous busilvex in conditioning regimen before allogeneic stem cell transplantation for patients with myeloid malignancies: safety and efficacy background: busilvex (bu) is part of standard conditioning regimen before allogeneic stem cell transplantation (asct) for patients with myeloid malignancies and usually administered as an intravenous (iv) infusion 4-times daily. this study aimed to compare the saftey and efficacy of this schedule to a once-daily iv bu. we conducted a retrospective study in adult patients (≥18 years) with myeloid malignancies who received asct from hla-identical sibling donors between january 2011 and june 2018 following iv bu-based preparative regimens. graft-versus host disease (gvhd) prophylaxis consited of cyclosporine and short course of methotrexate. intravenous bu was administered 4-times daily (0.8 mg/kg every 6 hours x12 to 16 doses) or oncedaily in a 3-hour infusion (3.2 mg/kg x 3 to 4 days) since june 2015. results: ninty-nine patients were enrolled (54 men and 45 women). median age was 35 years (range, 18-50 y). the median time from diagnosis to asct was 5 months (range, 51days -7 years). diagnosis were acute myeloid leukemia (n=79, 80%), chronic myeloid leukemia (n=8, 8%), myelodysplasic syndrome (n=6, 6%), primitive myelofibrosis (n=4, 4%) and chronic myelomonocytic leukemia (n=2, 2%). thirty-seven (37.3%) patients had ebmt-score ≥2. sixty-five (65.6%) patients were transplanted in cr1, 5 (5%) beyond cr1 and 24 (24.2%) had active disease. conditioning regimens consisted of bu/cyclophosphamide in 86 patients (86.8%), bu/fludarabine in 13 patients (13.1%). four-times daily bu was given to 58 patients (58.5%, groupe1) and once-daily bu to 41 patients (41.5%, groupe 2). stem cell source were bm in 46 patients (46.5%) and pbsc in 53 patients (53.5%). globally, patients characteristics were well balanced between the two groups. the rates of severe complications were similar between the two groups with no statistically significant differences except oral mucositis (table1). non-relapse mortality (nrm) was comparable in the two groups (21% and 17% in groups 1 and 2, respectively, p=0.65). the relapse rate was 24% and 32%, respectively (p=0.4). after a median follow-up of 2 years (range, 5days -7years), the os was not significantly different between groups 1 and 2 : 64% vs 56% (p=0.09). however, the rfs was significantly better in the groupe 1 : 62% vs 56% (p=0.03). conclusions: once-daily iv bu regimen seems to be an efficient and safe alternative to the 4-times daily protocol. however, results should be interpreted with caution because the historical comparison and lack of bu pharmacokinetics studies. disclosure background: standard therapy of the most patients with juvenile myelomonocytic leukemia (jmml) is allogeneic hematopoietic stem cell transplantation (ahsct). the choice of optimal conditioning regimen for patients with jmml is crucial as well as long-term observation. we aimed to estimate the long-term follow-up and survival rates of patients with jmml after ahsct with the help of busulfan or treosulfan-based conditioning regimens. methods: thirty eight patients with jmml underwent ahsct in 2002-2018. we compared equal groups of patients received busulfan (n=19) and treosulfan-based (n=19) conditioning regimen. m:f=28:11. median of age at hsct was 2.5 (0.6-5). donor type: hla-related 10/10 -29% (n=11), hla-related 9/10 -2.6% (n=1), hlaunrelated 10/10 -39.4% (n=15), hla-unrelated 9/10 -15.8% (n=6), and haploidentical -13.2% (n=5). stem cell source: bm -55.3% (n=21), pbsc -26.3% (n=10), ucb -10,5% (n=4), and ucb+bm -7.9% (n=3) . disease status on hsct: cr -73.7% (n=28), refractory -26.3% (n=10). results: median follow-up 15.5 months (1-129 months) . the estimated 10-year overall survival (os) probability in patients received busulfan-based conditioning was 48,8 ±13,4% in comparison with 68,0±10,8% in patients with treosulfan-based regimen (р=0,458). event-free survival (efs) was 42,1±11,3% in group with busulfan-based regimen and 57,0±11,5% in patients with treosulfanbased conditioning (р=0,224). background: post-transplant relapse remains the leading cause of treatment failure in high risk (hr) acute myeloid leukemia (aml), myelodysplastic syndrome (mds), myeloproliferative neoplasia (mpns) receiving allogeneic hematopoietic cell transplantation (allo-hct), especially for patients with relapsed or refractory aml. recently, a sequential transplant approach, as developed by the munich group, comprising of intensive cytoreductive chemotherapy flamsa (fludarabine/amsacrine/cytarabine) to decrease leukemia cell burden shortly prior to conditioning regimen, has been successfully used for high-risk (hr) aml/mds with promising results. methods: we studied 48 patients (median age 53 years, range 26 -68) with hr aml (n=38), as defined by refractory, relapsed disease, secondary leukemia, or high/ very risk disease risk index risk, and hr mds (n=10) according to ipss-r, undergoing allo-hct using the sequential transplant approach in 2 institutions between january 2009 and october 2018. the sequential transplant approach combined a cytoreductive chemotherapy, which consisted of either flamsa (n=17), flag +/-ida (fludarabine/cytarabine/granulocyte colony stimulating factor /idarubicin) (n=23), or clo-arac (clofarabine/cytarabine) (n=8), followed by reduced (ric) (n=43) or myeloablative (mac) (n=5) conditioning regimen. all patients received peripheral blood stem cell from matched related donors (n=27) matched unrelated donors (n=14), or mismatched unrelated donors (n=7). post-grafting immunosuppression consisted of calcineurin inhibitor and mycophenolate mofetil in all patients. thymoglobulin was added for gvhd prophylaxis for unrelated donor transplant. results: the median time to neutrophil > 1000/μl was 10 days (range, 9-25) . with a median follow-up of 28.2 months (range, 1.4 to 103.1 months), the kaplan-meier estimate of leukemia-free (lfs) and overall survival (os) at 5 years were 45 % (95% ci, 8-30), 46% (95% ci, 8-30), respectively. patients receiving flag or clo-arac based sequential regimen showed a trend towards more favourable overall survival (os) as compared to patients given flamsa (5 year os: 53% vs 31%; p=0.236). at 2 years, the cumulative incidences of relapse and non-relapse mortality (nrm) were 46 % (95% ci, 31-60 %) and 15 % (95% ci, 7-21 %), respectively. in multivariate analysis, the type of sequential conditioning regimen did not show any significant impact on lfs, os, nrm or relapse. conclusions: sequential transplant conditioning with flamsa, flag or clo-arac followed by allo-hct is an effective strategy in overcoming the dismal prognosis of hr aml and mds, and enabling long-term disease free survival. more studies on effective strategies such as posttransplant maintenance therapy of prophylactic donor lymphocyte infusion, are needed to further eliminate the risk of relapse, without increasing risk of treatment related toxicity. disclosure: nothing to declare optimization of the blood sampling procedure for busulfan therapeutic drug monitoring (tdm) to optimize our sampling scheme (15 minutes, 1, 2, 3, 4, 6, 8 and 10 hours after the end of a 3-hour infusion), we reduced the number of blood samples collected, reducing nursing and laboratory staff time and increasing patient convenience. this study aims to show the performance of a simplified sampling protocol which includes the first 5 samples from the original protocol. methods: individual pk parameters were retrospectively estimated using 5 samples (simplified protocol) and were compared with those obtained after 8 samples (original protocol). individual pk parameter values for a one compartment model were estimated using a maximum likelihood estimation modelling algorithm (adapt 5.0) and the statistical analysis of the results was performed (statgraphics centurion xv). results based on the approved dosage recommendations, mean (sd) initial dose was 171.1(69.3) mg. after tdm, mean (sd) calculated dose at day 1 for the remaining days (to achieve the defined target cumulative auc) was 158,5 (72,6) mg obtained from the original protocol. according to the simplified protocol the result would be 159,8(73,7) mg. the median and the mean variation of the calculated dose were 0% and 1% (0-8%) between protocols. a strong relationship between the cl of the day 1-3 obtained from the original protocol and the simplified protocol is observed (r 2 =0.9985). this high correlation is also observed for patients with busulfan t 1/2 >3h (r 2 =0.9936), a population were the reduction of sampling could be more problematic. anova test for the log cl with the factors: patient, day of busulfan and type of sampling protocol was performed. sampling protocol was determined as non-statistically significant (p = 0.7248). conclusions: results suggest that both protocols are equivalent concerning to the busulfan cl estimation and calculated auc. variation between protocols regarding the calculated dose at day 1 for the remaining days to achieve the defined target cumulative auc is considered acceptable. we verified a strong relationship between busulfan cl obtained from both protocols and sampling protocol doesn't influence cl statistically. a reduced sampling collection of 5 determinations until 4 h after the end of the infusion is shown to be sufficient for the tdm of busulfan, so this was implemented in our centre in line with published data. disclosure: nothing to declare p125 impact of anti-thymocyte globulin doses in unrelated hematopoietic stem cell transplantation for patients with myeloid neoplasm background: anti-thymocyte globulin (atg) is widely used for the prophylaxis of graft-versus-host disease (gvhd) in hematopoietic stem cell transplantation (hsct). however, there is still controversy regarding the optimal dose of atg. therefore, we analyzed the impact of atg doses in unrelated hsct for patients with myeloid neoplasm. methods: this was a retrospective multi-center study that assessed the impact of atg doses on clinical outcomes in patients with acute myeloid leukemia (aml) or myelodysplastic syndrome (mds) undergoing an unrelated hsct. the patients who received peripheral blood stem cells (pbsc) transplantation after conditioning regimens containing i.v. busulfan (bu), fludarabine and rabbit atg between 2010 and 2017 were included in this study. results: a total of 96 patents, median age 45 years, with aml (n=74) or mds (n=22) were included in our analyses. 66 patients (69%) received a myeloablative regimen (i.v. bu>6.4 mg/kg). high-atg (atg 9 mg/kg), intermediate-atg (atg 4.5-5 mg/kg) and low-atg (atg 3 mg/kg) were given in 11, 49 and 36 patients, respectively. after a median follow-up of 23 months, the cumulative incidence of extensive chronic gvhd was 9.1% in the high-atg group, 13.8% in the intermediate-atg group and 29.7% in the low-atg group (p=0.31). conclusions: our study shows that the incidence of extensive chronic gvhd was similar regardless of the doses of atg after transplantation of pbsc from unrelated donor for patients with aml or mds. however, the rate of relapsefree survival and the rate of a composite end point chronic gvhd-free and relapse-free survival were significantly higher in the intermediate dose ( methods: we retrospectively retrieved data from the electronic medical records for consecutive patients aged 65 and older, who underwent an asct for lymphoma over the last 10 years at our institution. results: forty four patients ≥ 65 years old underwent asct between 1 aug 2008 and 31 aug 2018. twenty eight of them received a reduced-dose conditioning (median 25%, range 20%-33% dose reduction). the dose was reduced for 92% of patients ≥ 70 years old and for 53% of patients aged 65-69. the outcomes of the following three groups of patients were compared: a) age ≥ 65; without dose reduction, b) age 65-69; with dose reduction and c) age ≥ 70; with dose reduction (table 1). only one patient aged 70 received full-dose conditioning. there was no significant difference between the groups in the number of previous chemotherapy cycles (median 2, range 1-3). however, significantly more patients at the age of 65-69 were in complete remission (cr) pre-transplant in both full and reduced-dose conditioning groups (a and b). no significant intergroup differences were observed in the occurrence of complications (mucositis and infections), day 30 transplantrelated mortality (trm) or engraftment day. similarly, no significant differences were found either in the 1-year progression-free survival (pfs), which was 50%, 64% and 50%, or 1-year non-relapse mortality (nrm), which was 17%, 7% and 10%, respectively for groups a, b and c. the 1-year overall survival (os) tended to be higher in group b (85%), compared to groups a (66%) and c (70%). conclusions: beam/beac conditioning dose reduction was not found to adversely affect 1-year pfs and os rates. despite the fact that 2/3 of the patients in the age group ≥ 70 underwent asct in partial remission and had dose reduction, theier achieved trm, pfs and os rates were similar to those of patients aged 65-69. beam/beac conditioning at a 75%dose may be a suitable option for patients in their seventh decade requiring asct. this strategy should be further evaluated in prospective clinical trials. background: the transplant related mortality in autologous transplants for lymphoma and multiple myeloma, reported worldwide ranges from 0-5%. from 2004-2015, the trm at our center for these two diseases was approximately 20%. we introduced changes in mobilization schedule, conditioning regimens and drug dosages to determine whether these changes affected the transplant related mortality and overall survival. methods: from april 2004-december 2015, we used beam (bcnu: 300 mg/m 2 on day -6; etoposide 200 mg/ m 2 on days -5 to -2, cytarabine 200 mg/m 2 on days -5 to -2 and melphalan 140mg/m 2 on day -1 as conditioning chemotherapy for patients admitted in transplant unit for autologous transplants in hodgkin's and non-hodgkin's lymphoma. in patients with multiple myeloma high dose melphalan (200mg/m 2 ) was used. the mobilization protocol consisted of cyclophosphamide 1.5gm/m 2 followed by gcsf 5μgm/kg twice daily till stem cell collection was completed. from january 2016, we changed the beam protocol to bendaeam with dose modifications that included: bendamustine 150mg/m 2 on days -5 and -4, cytarabine 150mg/m 2 on days -5 to -2, etoposide 150mg/m 2 on days -5 to -2 and melphalan 100mg/m 2 on day -1. for multiple myeloma melphalan was reduced to 150mg/m 2 . we used only gcsf for mobilization of stem cells, which was continued till stem cell harvest was complete. response to treatment was evaluated by comparing trm and overall survival for two time periods: 2004-2015 and from 2016 till date. results: from april 2004 till december 2015, n=78 autologous transplants were performed. the male:female ratio was 2.4:1. fifty seven patients underwent transplant for lymphomas, n=16 for multiple myeloma and n=5 for other diagnosis. median age was 23±14.6 (2-64 years). the mean mnc was 4.7 × 10 8 ± 1.7/kg. engraftment was achieved in 80% of patients. the transplant related mortality was 19.5% and overall survival was 72% (follow up: 104 months). since january 2016 till march 2018 we have performed n=18 autologous transplants of which n=17 were males. fifteen transplants were performed for lymphomas (nhl:8, hd:7) and n=3 for multiple myeloma. median age was 24±15 (20 -64 years). the mean mononuclear cell count was 5.8 x 10 8 /kg and the mean cd34 count was 3.7 x 10 6 /kg. engraftment was achieved in all patients. the transplant related mortality was 0% and the overall survival was 83% (follow up 22 months). conclusions: we were able to reduce the autologous transplant related mortality to 0% by decreasing dosages of conditioning chemotherapy and changing the mobilization protocol. long follow-up is needed to determine late mortality and late relapse in comparison to standard chemotherapy dosages disclosure: nothing to declare risk and benefit of thiotepa based conditioning followed by autologous stem cell transplantation in high risk lymphomas 36 years (16-62) . stage (ann-arbor) at diagnosis of hd/dlbcl/pcnsl: stage ie n=0/ 0/10 (0/0/91%), stage ii n=7/2/0 (47/18/0%), stage iii n=2/ 0/1 (13/0/9%), stage iv n=6/9/0 (40/82/0%). median time from diagnosis to asct hd/dlbcl/pcnsl: 25/11/ 6 month (4-72). induction treatment in hd patients was abvd, in most dlbcl patients r-chop and in pcnsl patients high dose methotrexate and cytosin arabinoside. tumor status at asct hd/dlbcl/pcnsl: complete metabolic remission (cmr) n=4/1/11 (27/9/91%) and from pcnsl patient's n=8 (73%) were in first complete remission (cr1). type of stem cell graft was periferial blood stem cell in all case. conditioning: thiotepa (250mg/ m2 on days -9 to -7, busulphan 3,2mg/kg on days -6 to -4 and cyclophosphamide 60mg/kg on days -3 to -2 plus rituximab 500mg/m2 on day -10 in dlbcl and pcnsl. median follow up from asct 711 days . tumor stage at asct was defined with computer tomography with positron emission tomography (pet-ct). results: median time of engraftment was 10 days (9-14). thiotepa caused toxicoderma appeared at 9 (24%) patients. cytomegalovirus (cmv) reactivation was seen in 3 (8%) cases with low dna content (284,454,5500 copies/ml) and responded completely to oral valgancyclovir therapy. transplantation related mortality hd/dlbcl/pcnsl n= 2/3/1 (9/27/13%), in 4 cases bacterial sepsis and one systemic mycoses and one pulmonary fibrosis. incidence of long-lasting grade iii-iv thrombocytopenia and anaemia: n=15 (40,5%) and n=3 (8%), median time of duration from transplantation 71 days (31-720) and 35 days ( background: this study evaluated the efficacy and toxicity of intravenous busulfan and thiotepa as a conditioning regimen for autologous stem cell transplantation (asct) in patients with multiple myeloma (mm). methods: we retrospectively analyzed the data of 68 patients with mm who received the intravenous busulfan and thiotepa conditioning for asct between november 2016 and april 2018 in korea. results: the median time to transplant was 5.4 months, and 66 patients (97.1%) underwent asct within 12 months of the diagnosis. the overall response rate after asct was 95.6%, including 55.9% with complete response, 22.1% with very good partial response, and 17.6% with partial response. the most common severe non-hematologic toxicity (grade 3-4) was infection (44.1%). three patients (4.4%) developed venous-occlusive disease. one patient (1.5%) died due to severe pneumonia after asct. after a median follow-up of 13.0 months, the median progression-free survival (pfs) and overall survival (os) were not reached. conclusions: in conclusion, a conditioning regimen of intravenous busulfan and thiotepa was effective and tolerable. clinical trial registry: not applicable disclosure: the authors have declared no conflicts of interest. myeloablative haploidentical bone marrow transplantation with post-transplant cyclophosphamide in paediatric patients with haematological malignancies santanu sen 1 , sameer tulpule 1 background: haploidentical transplants have been shown to be safe and effective in treating haematological malignancies in the paediatric population. we have previously reported on our experience of using reduced intensity conditioning with post transplant cyclophosphamide in haploidentical patients. we herein report our experience of using a tbi based myeloablative conditioning to treat our first 8 patients with haematological malignancies. methods: 8 patients were enrolled in the study, 5 with relapsed acute lymphoblastic leukemia (all) and 3 with relapsed/resistant acute myeloid leukemia (aml). all aml patients had genetic markers of high risk disease and all all patients had very early relapses (either on therapy or within 6 months of stopping therapy). all patients were conditioned with an identical protocol using tbi-based myeloablative preparative regimen (fludarabine 30 mg/m 2 /d × 4 d and tbi 150 cgy bid on d −4 to −1 [total dose 1200 cgy]) followed by an infusion of unmanipulated peripheral blood stem cells from a haploidentical family donor. postgraft immunosuppression consisted of cyclophosphamide 50 mg/kg/day on days 3 and 4, mycophenolate mofetil through day 35, and tacrolimus through day 180. results: median time of neutrophil and platelet engraftment was 11 and 19 days, respectively. all patients achieved sustained complete donor chimerism by day +28. acute gvhd, grades ii-iv and iii-iv, was seen in 75% and 25%, respectively. disease progression occurred in 2 patients: 8 & 10 months after transplant and there was one death due to severe fungal infection. estimated twoyear survival and relapse were 75% and 24%, respectively. 2 patients had severe bk viremia and cmv reactivation occurred in 4 patients. all patients were successfully managed with appropriate supportive and antiviral therapy. conclusions: we report good outcome with a myeloablative conditioning in haploidentical transplants with excellent engraftment and hopefully a longer life expectancy. with small number of patients, it is difficult to state whether using a myeloablative conditioning would lead to better long term outcomes in this cohort of patients with very haematological malignancies, but we certainly showed that it is possible to achieve excellent early results. disclosure: nothing to declare fludarabine in combination with melphalan and atg can be the best conditioning for hematopoietic stem cell transplant of children with hemophagocytic lymphohistiocytosis methods: in this prospective study, we analyzed the outcome of two pediatric patients with hlh who had received hsct, using reduced-intensity conditioning (ric) regimen. they received the same ric regimen based on the use of fludarabine (30 mg/m 2 /day for 5 days) in combination with melphalan (70 mg/m 2 /day for 2 days) and horse antithymocyte globulin (atg 10 mg/kg/d for 4 days). cyclosporine and methotrexate were used as graft-vs.-host disease (gvhd) prophylaxis. results: a 2 months boy with primary hlh (fhl2) was transplanted from his mother and a 4 years girl with secondary hlh was transplanted from her brother. both of donors were hla match with their recipients. they were received 6 x 10 6 /kg and 10 x 10 6 /kg cd34 + cells from the harvested peripheral blood stem cells, respectively. they achieved full neutrophil and platelet recovery. the time to neutrophil recovery was 13 and 11 days, respectively. full chimerism was achieved for both of them. in addition, they was developed grade 3 and 2 of acute gvhd, respectively. gvhd was completely controlled with prednisolone. they are alive and in complete remission without any significant complications after 36 and 14 months, respectively. conclusions: it appears that fludarabine in combination with melphalan and atg may be the best conditioning regimen for hematopoietic stem cell transplant of children with hlh. due to a few number cases of this study, a study with sufficient sample size is required. disclosure background: hematopoietic cell transplant (hct) recipients often report depression and impaired quality of life (qol) before transplant. mixed evidence suggests depression may be a risk factor for greater mortality and worse qol. inconsistent findings may be due to the fact that previous studies have not evaluated antidepressant use. the aim of the study was to compare pre-transplant patientreported physical functioning and post-transplant overall survival (os) between four groups of hct recipients: 1) non-depressed/taking antidepressant (treated depression), 2) depressed/taking antidepressant (undertreated depression), 3) depressed/not taking an antidepressant (untreated depression), and 4) not depressed/not taking an antidepressant (control). it was hypothesized that physical functioning and os would be worse among patients with untreated and undertreated depression relative to those with treated depression and controls. methods: this retrospective case-control study included patients completing depression (phq-8) and quality of life (sf-12) questionnaires at pre-transplant. analyses were conducted separately for allogeneic and autologous recipients. results: participants (n=1,797) were 58% men, mean age 57 years (19-79), 39% allogeneic recipients. regarding depression and antidepressant use, 146 (21%) allogeneic patients were characterized as having treated depression, 47 (7%) as untreated depression, 49 (7%) as undertreated depression, and 461 (65%) as controls. hierarchical linear regression models indicated that after adjusting for significant univariate factors (performance status, disease status, and regimen intensity), allogeneic patients with treated depression (b=-2.58, 95% ci=-4.63, -0.54) reported better physical functioning than patients with undertreated depression (b=-6.06, 95% ci=-9.43, -2.70) and untreated depression ) but worse physical functioning than controls (p values <0.05). cox regression models indicated depression/antidepressant usage was not associated with os among allogeneic patients (p values>0.10).among autologous patients, 195 (17.82%) were characterized as having treated depression, 83 (7.59%) as untreated depression, 77 (7.04%) as undertreated depression, and 739 (67.55%) as controls. hierarchical linear regression models indicated that after controlling for significant univariate factors (gender, performance status, diagnosis, and disease status), autologous patients with treated depression (b=-2.97, 95% ci=-4.71, -1.23) reported better physical functioning than patients with undertreated depression (b=-8.63, 95% ci=-11.23, -6.03) and untreated depression (b=-8.62, 95% ci=11.15, -6.08), but worse physical functioning than controls (p values <0.05). cox regression models showed depression/antidepressant usage was associated with os (p values <0.05), with patients with treated depression demonstrating significantly worse os than other groups (p=0.05), but this association was no longer significant in multivariate analyses controlling for diagnosis and disease status (p=0.09). conclusions: patients with untreated or undertreated depression pre-transplant may benefit from depression screening and treatment to improve physical functioning. disclosure: hslj: consultant for redhill biopharma and janssen scientific affairs p133 eltrombopag (epag) induces a high percentage of responses in patients with post allo-hsct poor graft function (pgf) and no active gvhd lourdes aguirre 1 , aitziber lizardi 1 , pilar bachiller 1 , brigida esteban 1 , carmen gonzález 1 , nagore argoitia 1 , maría araiz 1 , aranzazu aguirre 1 , anunciación urquía 1 , carlos vallejo 1 background: persistent cytopenia is a life-threating complication after hsct. several causes can lead to this situation (viruses, gvhd, drugs, etc) . a specific entity is the one called "poor graft function (pgf)", which is diagnosed in pts with ≥2 cytopenias after day +30, in the presence of donor chimerism and the absence of gvhd or relapse. pgf is more frequent after alternative allo-hscts, such a haplo-identical, mismatched, or ucb. several therapeutic approaches for pgf, with poor results, have been tested. recently, epag has been shown to improve platelet counts in the post-allo-hsct setting. in this study, we analysed the efficacy of epag in pts with post-transplant persistent cytopenias. methods: the population analyzed includes all 175 pts who underwent allo-hsct from june 2015 through may 2018 in our unit. median age was 52 years (12-69). 102 were male (58.3%) and 73 female (41.7%). baseline diseases were: 69 aml, 39 lpd, 20 all, 18 mds, 15 mpd, 9 mm, and 5 bmf. donor was unrelated in 101 (54.3%) and was family in 74 (42.3%) (including 25 haplo-identical). conditioning was ric in 95 (54.3%) and intensive in 80 (45.7%). sc source was pb in 164 (93.7%) and bm in 11 (6.3%). median followup was 24 months (6-41). epag was initiated at some point during the first 6-month post-hsct period in 12 pts (6.9% of the series) due to thrombocytopenia (< 20000/mcl) plus, at least, one other cytopenia. patients characteristics shown in table 1. epag was started at 50 mg/day and escalated each 2 weeks to 75, 125 and 150 mg/day if platelet count was < 20000/mcl. global response was considered when, after epag, the patient needed no transfusions and reached the three of the following: platelets >50000/mcl, hgb >10 g/dl, and anc >1000/mcl. epag was tapered off in responders and discontinued if no response was reached after 16 weeks. results: at epag initiation, all the 12 pts had thrombocytopenia (< 20000/mcl), 10 had anemia (hgb < 10 g/dl), and 5 had neutropenia (anc < 1000/mcl). counts pre and post and response to epag are shown in table 2. among the 8 responders, all but one (who relapsed from thrombocytopenia and died from bleeding) were alive at analysis close (87.5%). among the 4 non-responders, three pts had gvhd-associated cytopenias, and finally died from infectious complications; the other patient relapsed from her aml, reached a new cr after treatment, and is alive and well 27 months afterwards. epag was tapered off and discontinued in 6/8 pts who responded; 2/8 responders are still on epag. epag was discontinued in the 4/4 pts who did not respond. rest of treatment details shown in table 2. conclusions: 1) epag worked striking well in subjects with pgf, an otherwise a life-threatening situation for patients. 2) epag induced impressive responses in platelets, but strong bilinear and trilinear responses were also seen. 3) epag did not improve gvhd-associated cytopenias. 4) to confirm these innovative and transcendent results, we have just initiated a multicenter prospective study on the role of epag for treatment of post-hsct pgf. 1232 * five out of the six urdt were mismatched ** the donor was a woman in the six cases: three sisters and three daughters background: hepatic vod/sos with multi-organ dysfunction (mod; typically, renal or pulmonary) may be associated with >80% mortality. defibrotide is approved for treating severe hepatic vod/sos post-hsct in patients aged >1 month in the eu, and for hepatic vod/sos with renal or pulmonary dysfunction post-hsct in the us. this analysis provides an overview of the safety results from 3 studies of patients with vod/sos, with or without mod, who received defibrotide 25 mg/kg/day. methods: safety data were pooled from patients with vod/sos post-hsct treated with defibrotide in a phase 3 trial (n=102) and a phase 2, randomized dose-finding trial (n=74 receiving 25 mg/kg/day). safety data for historical controls (hc) from the phase 3 study (n=32) also are provided. reported separately, due to differences in patient population and data monitoring protocol, are aes from the expanded-access program (t-ind) in patients with vod/ sos with and without mod (n=1000 post-hsct). vod/ sos was diagnosed by baltimore criteria/biopsy for the phase 2/3 studies; diagnosis by baltimore or modified seattle criteria was permitted in the t-ind. results: median patient age at hsct for the phase 2/ 3 studies was 24.0 years, 18.0 years for the hc, and 14.0 years for the t-ind. in the phase 2/3 studies defibrotide-treated group (n=176), 169 (96.0%) experienced aes; most common (>10%) were hypotension (36.9%), diarrhea (24.4%), and multi-organ failure (21.6%). treatment-related aes were at least possibly related to defibrotide (table) . any hemorrhage (an ae of special interest) occurred in 101 patients (57.4%); most commonly epistaxis (13.6%), gastrointestinal and pulmonary alveolar hemorrhage and hematuria (8.5% each), and conjunctival hemorrhage (6.3%). all 32 hc experienced an ae; most common (>25%) were hypotension (50.0%), tachycardia (43.8%), diarrhea (37.5%), nausea (31.3%), and pyrexia, agitation, and petechiae (28.1% each). any hemorrhage occurred in 24 patients (75.0%): most common (>10%) were petechiae (28.1%); hematuria, epistaxis, and pulmonary alveolar hemorrhage (15.6% each); and lip hemorrhage (12.5%). in the t-ind (n=1000), 385/512 patients with mod (75.2%) and 324/488 patients without mod (66.4%) had an ae; other than vod/sos and mod, most commonly (>10% in either subgroup) hypotension (15.2% and 8.4%, respectively). traes occurred in 210 patients (21.0%) ( table) . any treatment-emergent hemorrhage occurred in 166 patients with mod (32.4%) and 124 patients without mod (25.4%); most commonly (>5% in either subgroup) pulmonary hemorrhage (8.2% and 4.7%, respectively) and gastrointestinal hemorrhage (5.5% and 4.3%, respectively). conclusions: the incidence and type of aes were as expected in these critically ill patients. of the pooled patients, 96% had aes; 57.4% had a hemorrhage. all hcs had an ae, with 75.0% having a hemorrhage. in the t-ind, patients with mod had higher rates of aes. support: jazz pharmaceuticals event, n(%) phase 2/3 studies (n=176) disclosure: paul g. richardson has served on advisory committees and as a consultant, and has received research funding from jazz pharmaceuticals. angela r. smith and leslie lehmann have nothing to disclose. nancy a. kernan received grants from gentium during the conduct of the study, and her research was supported by the national cancer institute of the national institutes of health under award number p30 ca008748; the content is solely the responsibility of the author and does not necessarily represent the official views of the national institutes of health. she has a research grant from jazz pharmaceuticals. robert ryan and william tappe are employees of jazz pharmaceuticals and hold stock and/or stock options in jazz pharmaceuticals plc. stephan a. grupp has served on a steering committee and as a consultant to jazz pharmaceuticals. defibrotide for treatment of adults with hepatic vod/ sos with or without multiorgan failure after hematopoietic cell transplantation: results of a systematic review/meta-analysis background: although hematopoietic cell transplantation (hct), autologous or allogeneic, is potentially curable in various hematologic malignancies, the procedure is associated with serious and potentially life-threatening complications, among them veno-occlusive disease/sinusoidal obstructive syndrome (vod/sos) of the liver. several studies, prospective or retrospective, have reported outcomes of defibrotide, when used as prophylaxis or treatment, in a mixed population of adult and pediatric patients. in this systematic review/meta-analysis, we analyze outcomes of defibrotide when specifically used for treatment of adult patients with hepatic vod/sos with or without multiorgan failure. methods: a comprehensive search of 3 large databases (medline/pubmed, cochrane and embase) on november 2, 2018 identified 642 publications. analysis was restricted only to adult patients (defined as median age older than 16 years) who received defibrotide for treatment of vod/sos and were reported in prospective or retrospective (which included ≥ 5 patients) studies published in full manuscript form. there were no limitations based on language. data were extracted in relation to benefits [complete remission (cr) rate and overall survival (os)] and harms (hemorrhage, any site or organ-specific). a total of 15 studies (prospective=6; retrospective=9) with 1437 patients met inclusion criteria. results: the median year of publication of prospective studies was 2013 (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) and for retrospective ones 2016 (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) . the prescribed starting dose of defibrotide varied among studies ranging from 6.25 mg/kg/day to 80 mg/kg/day, mostly for a 21-day course. the pooled cr rate was 39% (95%ci=28-49%) for prospective and 54% (95%ci=39-69%) for retrospective studies. the pooled day +100 os rates were 43% (95%ci=37-48%) and 65% (95% ci=53-75%) for prospective and retrospective studies, respectively. the pooled rates of hemorrhage (any site) were 15% (95%ci=2-35%) for prospective and 21% (95% ci=4-43%) for retrospective studies. when analyzing organ-specific hemorrhage, 3 prospective studies (n=1091 patients) reported pooled rates of pulmonary alveolar (pa) hemorrhage of 2% (95%ci=1-3%) and of 5% (95%ci=3-7%) for gastrointestinal (gi) hemorrhage. only one retrospective study (n=14 patients) reported an incidence of pa hemorrhage of 7% (95%ci=0-34%) and a different study (n=14 patients) reported an incidence of gi hemorrhage of 14% (95%ci=2-43%). none of the 15 studies reported cerebral hemorrhage as a complication of defibrotide therapy. conclusions: this systematic review/meta-analysis confirms the efficacy of defibrotide for treatment of vod/sos with or without multiorgan failure, yielding cr rates of 39-54% and day +100 os rates of 43-65%. the purportedly higher pooled cr and os rates observed with retrospective (vs. prospective) studies are likely due to assignment-bias inherent to observational studies. moreover, although the pooled hemorrhage (any site) rates of 15-21% is considered proportionally significant, the pooled rates of pa and gi hemorrhage were ≤ 5%, in prospective studies. clinical trial registry: not applicable disclosure: m.a.k-d: consultancy for pharmacyclics m.m: received lectures honoraria and research support from jazz pharma efficacy and safety of defibrotide in the treatment of hepatic veno-occlusive disease/sinusoidal obstruction syndrome following hematopoietic stem cell transplantation: interim results from the defifrance study background: hepatic veno-occlusive disease/sinusoidal obstruction syndrome (vod/sos) is a potentially lifethreatening complication of conditioning for hematopoietic stem cell transplant (hsct) but may occur after nontransplant chemotherapy alone. vod/sos with multi-organ dysfunction (mod) may be associated with >80% mortality with supportive care alone. diagnosis of vod/sos was traditionally based on baltimore or modified seattle criteria; however, the ebmt recently published separate diagnostic criteria for adults and children. defibrotide is approved for treating severe hepatic vod/sos post-hsct in patients aged >1 month in the eu, and for hepatic vod/sos with renal or pulmonary dysfunction post-hsct in the usa. the goal of the defifrance study, requested by the french health authorities, is to collect real-world data on safety and efficacy in a broader patient population in france, including all indications. this is the first interim analysis of the largest current evaluation of defibrotide for the treatment of vod/sos in europe. methods: defifrance is an observational, multicenter, post-marketing study that includes any patient treated with defibrotide from hsct centers in france. this interim analysis is based on all patients treated with defibrotide, including those with severe and very severe post-hsct vod/sos. vod/sos was diagnosed using traditional criteria. day+100 survival, complete remission (cr; total serum bilirubin < 2 mg/dl and resolution of mod), and safety profile are reported. results: a total of 324 patients treated with defibrotide were included retrospectively and prospectively between july 2014 and october 2018 from 36 table] disclosure: mohamad mohty: has received honoraria and research funding from jazz pharmaceuticals, delphine lebon: nothing to disclose, ann berceanu: none, charlotte jubert: has received funding from jazz pharmaceuticals, ibrahim yakoub-agha: has received honoraria from jazz pharmaceuticals, stéphane girault: none, marie detrait: has received research funding from jazz pharmaceuticals, cécile pochon: none, fanny rialland: none, virginie gandemer: none, jean-hugues dalle: has received honoraria from jazz pharmaceuticals, régis peffault de latour: has received research grant / honoraria / board from pfizer, novartis, alexion; research grant amgen; and honoraria from jazz pharmaceuticals, david michonneau: has received honoraria from jazz pharmaceuticals, myriam labopin: has received honoraria from jazz pharmaceuticals, floriane delaval: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc, gerard michel: none, anne sirvent: none, laurence clement: none anne-lise menard: none, anne huynh: has received honoraria from jazz pharmaceuticals, virginie bouvatier: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc, raj hanvesakul: employee of jazz pharmaceuticals and holds stock and/ or stock options in jazz pharmaceuticals plc, zakaria medeghri: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc p137 incidence and predictors of severe cardiotoxicity in patients with severe aplastic anemia after haploidentical hematopoietic stem cell transplantation zheng-li xu 1 , lan-ping xu 1 , yuan-yuan zhang 1 , yi-fei cheng 1 , xiao-dong mo 1 , feng-rong wang 1 , yu-hong chen 1 , wei han 1 , chen-hua yan 1 , yu-qian sun 1 , ting-ting han 1 , yu wang 1 , xiao-hui zhang 1 , xiao-jun huang 1 1 peking university institute of hematology, peking university people's hospital, beijing, china background: severe cardiotoxicity after hematopoietic stem cell transplantation (hsct) is a rare but fatal complication. the aim of this study was to evaluate the frequency of severe cardiac complications and to assess the ability of various factors to predict these complications in patients with aplastic anemia after haploidentical transplantation., this is the first study evaluating the values of both clinical and imaging factors in the prediction of severe cardiotoxicity among saa patients after haploidentical transplantation. methods: a retrospective study was conducted in 216 consecutive aplastic anemia patients who received haploidentical transplantation from 2006 to 2017. all patients received a unified regimen including busulfan, cyclophosphamide (ctx) and antithymocyte globulin at our single center. results: a total of 12 (5.6%) patients developed grade iii or iv cardiac toxicity. patients with cardiotoxicity had significantly poorer overall survival (os) than those without cardiotoxicity (12.5% vs. 89.6%, p< 0.001). our multivariable model identified four independent adverse predictors of severe cardiotoxicity, including pre-transplant ecog score (≥2), abnormal st-t wave on 12-lead electrocardiogram (ecg), hyperlipemia and recalculated ctx dose (≥1.8 g/m2/d). a predictive risk model was refined as low risk (0-1 factor), intermediate risk (2 factors) and high risk (3-4 factors) . the respective incidences of severe cardiotoxicity were 50.0%, 6.0%, and 1.3% in the high-, intermediate-and low-risk groups (p< 0.001). the corresponding os rates were 49.0%, 80.4%, and 90.3% in the three groups (p< 0.001) at the last follow-up. conclusions: patients with high risk scores had the poorest outcomes and should be monitored closely. a reduced intensity conditioning might be recommended for these patients. disclosure: there are no conflicts of interest to declare. background: allogeneic stem-cell transplantation (allo-sct) is associated with significant transplant-related mortality (trm). acute renal failure (arf) is a frequent complication and usually presents early after the procedure, compromising its feasibility. the aim of this study is to analyse the incidence of arf, its risk factors and its potential impact on trm after allo-sct. methods: 422 patients were included (244 males [58%]; median age 43 years, range 16-67) treated with allo-sct consecutively between january 2001 and april 2012 in a single institution. patient characteristics are detailed in table 1. median follow-up was 1.8 years (range, 1.0-2.7). renal function was evaluated using creatinine and data was collected pre-transplant (baseline) and at the point when arf was developed after allo-sct. arf was evaluated using akin criteria, being akin-1 an increase 1.5-to 1.9-fold from baseline, akin-2 an increase 2.0-to 2.0-fold and akin-3 an increase ≥3-fold. chronic renal disease was evaluated one year after the date of arf using kdigo criteria. results: cumulative incidence of arf at 1 year was 63% (akin-1, 25%; akin-2, 27%; akin-3, 15%). in the multivariate analysis, arf (akin-1/2) was associated with: non-use of antithymocyte globulin in conditioning chemotherapy, p=0.02 (hr 2.3, 0.2 to 0.9) and development of severe agvhd, p=0.04 (hr= 1.5, 1 to 2.3). in patients with arf akin-3, the most important variables in the multivariate analysis were: use of methotrexate (mtx) plus cyclosporine vs mycophenolate mofetil plus cyclosporine as gvhd prophylaxis, p=0.009 (hr=1.9, 1.2 to 3.1); myeloablative conditioning vs reduced intensity, p=0.03 (hr=1.7, 1 to 2.8) and use of total irradiation therapy in conditioning, p=0.02 (hr=1.7, 1.1 to 2.8). trm at 1 year increased significantly according to akin: akin-1, 25%; akin 2, 35%; akin 3, 51%; p=0,003; hr=11.2. overall survival at 3 years according to akin was: akin 1, 52%, akin 2, 45% and akin 3, 29%; p=0,004 (figure 1). the incidence of chronic renal disease at 1 year after allo-sct according to arf was: no arf (8%), akin-1 (11%), akin-2 (15%) and akin-3 (16%); p=0.006. conclusions: arf is a frequent complication during the first year after allo-sct and is associated with several factors. arf akin-3 was associated with more intensive strategies received during conditioning, meanwhile akin-1/2 were related to development of gvhd. there is an association of arf (akin-1, 2 or 3) with development of chronic renal disease. background: the introduction of cellular therapies such as car-t and modalities of gvhd-prophylaxis with posttransplant/cyclophosphamide (ptcy) that increase the number of admission days have boosted the pressure of available beds in the bm-units. in this sense, our centre started an at-home allogeneic stem cell transplantation (allo-sct) program to follow aplasia from the d+1 until independent ambulatory patient. to evaluate the feasibility and safety of allosct, we compared two groups: allohsct/athome (ah-group) vs. allohsct/in-patient (ip-group). methods: we included 78 patients receiving allosct (january 2014-november 2018) in a single centre: 39 patients, ah-group and 39, ip-group. all patients received conditioning at the hospital. gvhd-prophylaxis consisted in tacrolimus (tk) plus mycophenolate (mpm) or methotrexate, or ptcy (d+3, d+4) plus tk (d+5). all patients received prophylaxis with levofloxacin, fluconazole and acyclovir. besides that, ah-group patients received prophylaxis with ceftriaxone 1g/24h iv or ertapenem 1g/ 24h iv, and aspergillus-prophylaxis with inhaled liposomal amphotericin-b or posaconazole during neutropenia. patients of ah-group since d+1 or d+6 (in ptcyprophylaxis) received a nurse visit at-home once daily. the visits by the physician were performed at the hospital and only during complication events. first-line therapy of neutropenic fever was meropenem 1 g/8h in both groups, using a portable infusion pump in ah-group. in this group, the absence of focal infection or signs of severe sepsis allowed returning home after the initiation of antibiotics. the platelets support was performed at-home and the red blood support at hospital. results: the median (range) age (years) of the series was 54 . the median follow-up of the series has been not achieved. the source of the sct was peripheral blood in all cases. we didn't find statistical differences between two groups (ah vs ip) in terms of age, diagnosis, type of donor, intensity of conditioning, gvhd-prophylaxis, toxicity (mucositis, acute renal injury, neutropenia and thrombocytopenia), agvhd, aspergilosis and trm. interestingly, a significant reduction of neutropenic fever was observed resulting the lower use of meropenem in the ah-group than ip-group. the admission median days were similar in the both groups and it represented 21-23 days the reduction in the total economic cost of the ah-group. the whole analysis of the results are detailed in table: in-patient group, conclusions: in our experience, at home allosct, including ptcy-gvhd prophylaxis, is a feasible and safe procedure reflected in similar trm and aspergillosis incidence. at-home allo-sct is associated with a significant lower risk of neutropenic fever than in-patient group, as well as a very low readmission rate. disclosure: gonzalo gutiérrez-garcía: honoraria from gilead. grant from jazz pharmaceutical and janssen. laura rosiñol: honoraria from takeda, janssen, amgen and celgene. the others author do not have any disclosures to declare. background: renal complications in sickle cell disease (scd) include episodes of acute kidney injury (aki), progressive chronic kidney disease (ckd) and hyperfiltration, defined by abnormally high glomerular filtration rates (gfrs). hematopoietic stem cell transplant (hsct) from an hla identical sibling donor is a well-established curative treatment for scd, but traditional myeloablative conditioning (mac) regimens pose risks of kidney injury due to intensive use of chemotherapeutic agents, infectious risks, and use of calcineurin inhibitors (cnis). aki and subsequent fluid overload (fo) are common in pediatric hsct with reported aki incidence of 21%-50% (kyung-nam koh et. al., 2017). we report renal outcomes in pediatric patients with scd who received hsct following a non-myeloablative conditioning (nma) regimen without cni exposure. methods: retrospective chart review describing renal outcomes in pediatric patients (18 years of age or younger) with scd (hbss) who underwent nma hsct in alberta, canada from july 2013 to february 2018. the nma regimen is illustrated in figure 1 . reported renal outcomes: 1) measured gfr (dtpa) pre-hsct, 2) aki (kdigo definition) post-hsct by reviewing all serum creatinine levels from pre-hsct to one month post-hsct, 3) %fo calculated: (max post hsct weight -baseline weight)/ baseline weight x 100 for the two first weeks post-hsct, and 4) estimated gfr (egfr) using the pediatric schwartz formula at last follow-up post-hsct, ckd defined as egfr < 60 ml/min/1.73 m 2 , mildly reduced gfr: 60-90ml/min/1.73 m 2 , and hyperfiltration: gfr ≥ 150 ml/ min/1.73 m 2 . [[p140 image] 1. results: eighteen patients (33% male, 3-18 years old at transplant) were included. most common pre-morbid events: vaso-occlusive crisis (n=17), acute chest syndrome (n=8), splenic sequestration (n=6), and cholelithiasis (n=4). median follow-up time: 27 months (range: 7 -62 months). all patients engrafted successfully with no acute or chronic gvhd. baseline measured gfrs were all > 60 ml/min/1.73 m 2 (range: 79-227) with mildly reduced gfr and hyperfiltration seen in one (5.6%) and 12 (66.7%) patients respectively. at baseline (pre-hsct), the only aki event was one transplant related aki secondary to delayed hemolytic reaction after exchange transfusion in preparation for transplant. post-hsct, there were no aki events. additionally, no substantial %fo post-hsct was observed. average %fo week one post-hsct: +0.01% (range: -4.2% -+1.0%) and week two post-hsct: +0.04% (range: -4.24% -+1.5%). post-hsct egfr remained > 90 ml/min/1.73 m 2 at last follow-up in all patients. hyperfiltration was present in 5 (27.8%) of the patients. conclusions: this is the first study describing stable kidney function in children with scd after the present nma hsct regimen with alemtuzumab/300 cgy total body irradiation (tbi) with prolonged post-hsct sirolimus. no episodes of aki or significant fluid overload were observed during the first month post-hsct, and no patient developed ckd during follow-up. further prospective studies are needed to confirm our findings and to determine if stable renal function persists during longer-term followup. disclosure: nothing to declare. lung microbiota in patients with idiopathic pneumonia syndrome (ips) after hct background: idiopathic pneumonia syndrome (ips) is a non-infectious pulmonary complication after hematopoietic cell transplantation (hct) and the etiology remains unknown. recent studies have reported that various diseases are associated with changes of microbiota. the aim of this study was to evaluate the lung microbiota in hct recipients with ips and identify microorganisms potentially associated with ips. methods: frozen bronchoalveolar lavage (bal) samples from hct recipients with ips (n=18) and research bal samples from asymptomatic hct recipients as controls (n=12) were retrospectively analyzed. all samples were negative for common viruses by quantitative pcr. sequencing libraries were made with 1ng of input dna per sample (nextera xt, illumina). samples were pooled and sequenced by hiseq 2000 to obtain 100-bp paired end data. sequence data analysis and read classification were performed with sunbeam and the quality control and read classification were performed using komplexity and kraken, which classifies bacterial, archeal, and viral genomes. we used sequence data of bronchoscope prewashes from a separate cohort as controls for environmental sources (n=24). bray-curtiss dissimilarity among samples was calculated using the vegan r packages. permanova and a two-sided wilcoxon rank sum test were used to compare between the study groups. results: bal samples started at a median of 22x10 6 raw read pairs per sample and reduced to 21x10 3 reads assignable to microbial taxa following quality control. the bacterial phyla proteobacteria and firmicutes were most abundant followed by bacteroidetes and actinobacteria in both bal and bronchoscope prewash samples. separation of bal and prewash microbiota using bray-curtiss dissimilarity plots showed that bal samples were distinguished by sequences assigned to staphylococcus, acidovorax, and bradyrhizobium species, while prewash samples were distinguished mostly by pseudomonas and elizabethkingia species, consistent with environmental sources (figure) . within bal samples, staphylococcus species were the main drivers of separation between ips cases and the controls (p=0.002, permanova, figure) . consistent with this, a linear discriminant analysis to identify taxa best distinguishing cases and controls identified staphylococcus, especially s. epidermidis, in ips cases with lactobacillus and streptococcus species in controls. we then compared relative abundances of s. epidermidis between all study groups. ips case samples were significantly enriched in s. epidermidis compared to control (p< 0.001, two-sided wilcoxon rank sum test) and prewash samples (p< 0.001). viruses were classified by category as human pathogens, non-human pathogens, and bacteriophages. torque teno viruses (ttv) was the most commonly detected virus among viruses that replicate on human cells, and there was a trend towards higher abundance in ips case samples than controls. conclusions: lung microbial sequences in hct recipients predominantly consisted of proteobacteria and firmicutes, and had considerable overlap with environmental background. patients with ips had significantly more staphylococcus sequences detected than asymptomatic hct patients. these results suggest that patients with acute lung injury post-hct show distinct patterns of lung microbiota, although heterogeneity of sample collection and processing cannot be excluded and no singular organism was uniquely associated with ips. a prospective study is required to confirm these findings and define the clinical significance of differences in abundance patterns. disclosure: nothing to declare p142 abstract withdrawn. romiplostim for the treatment of thrombocytopenia after allogeneic stem cell transplantation background: thrombocytopenia is a common complication after allogeneic stem cell transplantation (allo-hct). with variable possible causes, such as drug side effects, infections, poor graft function, graft vs host disease (gvhd) and immune mediated. the purpose of this study was to evaluate the efficacy of romiplostim, a thrombopoietin receptor agonist, in patients with prolonged thrombocytopenia with no obvious cause after allogeneic transplantation. methods: retrospective analysis of allo-hct patients who received romiplostim at a single bmt unit between november 2015 and november 2018. romiplostim was given because of prolonged (>3 weeks) thrombocytopenia (< 60,000 μl) that couldn't be explained by obvious causes such as administration of drugs (antibiotics/antivirals), infection or gvhd. all patients were in complete remission and had complete chimerism. response to romiplostim treatment was considered transfusion independence or plt>80.000/μl. results: in total, 19 patients (median 45 years, 19-67) received romiplostim. patients (10 male, 9 females) had aml (10 pts), all (8), mds (2) or hodgkin (1), received a myeloblative (busiphex-based: 16, tbi-based:1) or ric (2) conditioning and were transplanted from a sibling (5), vud (11) or haploidentical (3) donor with pbsc (16) or bm (3) . all patients revealed primary neutrophil (median 14 days, range 10-19) and >20.000/μl platelet (13 days, 7-31) engraftment. romiplostim was started at median day +104 (range 58-419) with a median dose 5 μg/kg (1) (2) (3) (4) (5) . the median platelet count before commencement of treatment with romiplostim was 24.000/μl (range 12.000-57.000) and 10 them (59%) were transfusion-dependent. in total 14/17 (82%) patients responded to romiplostim treatment. eight out of the 10 (80%) transfusion dependent patients responded to the administration of romiplostim. six out of the 7 patients (86%) who were transfusion independent at romiplostin initiation (plt median 28.000/μl, range 19.000-56.000) responded. the median duration of treatment was 74 days (15-253) and the median follow up from the commencement of romiplostim was 177 days (15-1080). three out of 17 (18%) patients experienced relapse of thrombocytopenia after discontinuation of romiplostim and re-initiation of romiplostim was commenced in all of them, of which 2 responded and 1 didn't. the administration of romiplostim was done on an external basis and was well tolerated by the patients. two patients experienced gvhd during romiplostim treatment (both patients transplanted from 7/8 unrelated donor, 25 and 42 days after initiation treatment with romiplostim). 3/19 patients interrupted romiplostim due to disease relapse. 11/19 patients receiving romiplostim are alive in complete remission and 8 died (3 due to relapse, and 5 due to trm). conclusions: we present high response rates to romiplostim in patients with prolonged thrombocytopenia after allogeneic transplantation. in this retrospective study there were no side effects from the administration of romiplostim. however, the administration of romiplostim after allo-hct should be controlled in prospective trials. disclosure we report a single-center analysis of 29 adult patients (median age 22 years, range 18-57, m/f 13/16), receiving tpo agonists for isolated severe thrombocytopenia (n=7) and spgf (n=22) after allo-hsct. primary diagnoses were aml (10), all (6), mds (7), pmf (4), mds/mpn (4), saa (4), cml (1), nhl (1) . severe pgf was defined as cytopenia in ≥ 2 lineages (platelet < 20 × 10 9 /l, anc < 0.5 × 10 9 /l, hemoglobin < 70 g/l any time after sustained engraftment), full or stable mixed donor chimerism > 90 % and no signs of relapse. median dose of romiplostim was 5 (range, 3-5) mcg/kg weekly, eltrombopag -50 (range, 50-150) mg/day. overall response (or) included cr (platelet ≥ 100 × 10 9 /l, anc ≥ 1.5 × 10 9 /l, and hemoglobin ≥ 100 g/l) and pr (platelet > 20× 10 9 /l, anc ≥ 0,5 × 10 9 /l, hemoglobin > 70 g/l). results: median time from pgf diagnosis to treatment with tpo agonists was 14 days (0-119), median treatment duration was 3 weeks (1-43). tpo agonists were well tolerated with no cases of grade iii-iv toxicity. tpo agonists were combined with rituximab (n=4), rituximab and dli (n=3) and hsc boost (n=1) in 8 (28 %) patients. a total of 14 (48 %) patients met criteria of response (cr: n=4, 14 %; pr: n=10, 34 %). combination therapy showed no difference in or compared to tpo agonists alone. or was not depended on the tpo agonist used nor the time to therapy initiation. median increase in anc in responders was 3.4 × 10 9 /l (0.8-6.0), in platelet count -48×10 9 /l (21-205). a total of 15 patients died due to relapse (n=2), gvhd iii-iv grade (n=3) and infection (n=10). two-year os from the start of tpo agonist therapy was 44 % (95 % ci, 25-62) with a significant difference between responders and non-responders: 71 % (95 % ci, 33-90) vs. 18 % (95 % ci, 3-40) (p=0,002). conclusions: this study showed promising results of tpo agonists for management of spgf. further studies are warranted to specify optimal timing and dosing regimen, predictors of response. [[p144 image] 1. two-year os in responders and nonresponders to tpo agonist therapy] disclosure: there are conflicts of interest to disclose p145 cytomegalovirus reactivation kinetics and peak titers as novel predictors of survival and relapse after allogeneic cell transplantation for hematologic malignancies saskia leserer 1 , evren bayraktar 1 , nikolaos tsachakis-mück 1 , michael koldehoff 1 , lara kasperidus 1 , esteban arrieta-bolanos 1 , mirko trilling 1 , katharina fleischhauer 1 , dietrich w. beelen 1 , amin t. turki 1 1 university hospital essen, essen, germany, background: after allogeneic hematopoietic cell transplantation (hct), human cytomegalovirus (cmv) reactivation associates with non-relapse mortality (nrm) but also with reduced relapse in patients with leukemia, as shown by numerous studies that evaluated cmv reactivation as a qualitative yes/no parameter in the first months posttransplant. we hypothesized that longitudinal quantitative assessment of cmv reactivation kinetics and virus loads might improve patient-specific clinical outcome associations. methods: this retrospective study included 705 patients with hct for hematologic malignancies treated between 01/2012 and 12/2017 at university hospital essen, germany. cmv titers were monitored weekly by quantitative pcr (qpcr); cmv reactivation was defined by a cutoff of >500 genome copies per ml. patients were included for analysis, if at least 5 measurements were available during the first 200 days after hct. in total, 11,508 samples were analyzed. subgroup analyses were performed according to the time of cmv reactivation (before/after +30d) or the cmv viremia titer (>100,000, 20,000 -100,000 and 500 -20,000 copies/ml). results: cmv reactivation was detected in 350 (median age 58 years; range 17-76 years) out of 705 patients. baseline characteristics (age, gender, underlying disease, transplant) of patients without cmv reactivation were comparable. cmv reactivation kinetics followed a gaussian normal distribution with a median first reactivation at +33d and peak titers at +47d. all except 1 patient reactivated before 100d, 40 % before +30d. overall survival (os) of the cmv reactivation group as a whole did not significantly differ from the non-reactivation group (34 vs. 38 months). however, in subgroup analyses os was significantly reduced in patients with very early (< +30d) compared to later reactivation (17 vs. 59 months, p=0.040). moreover and importantly, os was significantly reduced in patients with cmv reactivation at high titers of >100,000 copies/ml compared to those with lower titers ((10 vs. 45 months) p< 0.0001).cox regression analyses confirmed significantly reduced os for patients with cmv reactivation >100,000 copies/ml and < day +30 as compared to the other cohorts (hr 2.03, 95%ci 1.45-2.86, p< 0.0001 and hr 1.36, 95% ci 1.01-1.83, p=0.041) respectively). the nrm was consistently higher (hr 2.59; 95%ci, 1.69-3.97, p< 0.0001) for patients with cmv copies >100,000/ml. the risk of hematologic relapse was exclusively reduced in patients with a peak cmv viremia between 20,000 and 100,000 copies/ml (hr 0.55, 95% ci 0.32-0.95; p=0.033) as compared to patients without cmv reactivation. for other levels of cmv reactivation this effect was not observed. conclusions: our data showed that cmv reactivations before +30d or with high titers of >100,000 copies/ml associated with significantly reduced os, while cmv reactivations at intermediate titers between 20,000 and 100,000 copies/ml had a positive impact on relapse incidence. these findings underline the complexity of cmv reactivations after hct outcome, and support longitudinal evaluation of cmv titers and individualized quantitative kinetics models for risk assessment after hct to distinguish the advantageous from the detrimental aspects of cmv reactivation. disclosure: att has received lecture fees from jazz pharmaceuticals and travel subsidies from neovii biotech outside the submitted work. the other authors declare no competing financial interests within the submitted work. association of serum ferritin levels before start of conditioning with mortality after allosct -a prospective, non-interventional study of the ebmt transplant complication working party background: elevated serum ferritin levels occur due to iron overload or during inflammation and macrophage activation. a correlation of high serum ferritin levels with increased mortality after allosct has been suggested by several retrospective analyses as well as by two smaller prospective studies. methods: this international multicentric study aimed to study the association of ferritin serum levels before start of conditioning with allosct outcome. patients with acute leukemia, lymphoma or mds receiving a matched sibling allosct for the first time were considered for inclusion, regardless of conditioning. data were prospectively collected between 8/2014 and 2/2018. a comparison of outcomes between patients with high and low ferritin level was performed using univariate analysis and multivariate analysis using cause-specific cox model. variables included in the multivariate analyses were age, sex mismatch, diagnosis, disease status, karnofsky score, number of cd34 cells given, intensity of conditioning, type of gvhd prophylaxis, atg use, time from diagnosis to transplant, year of transplant and cmv status. results: twenty centers from 10 european countries reported data on 385 allosct recipients. patient characteristics are given in table 1 . the ferritin cut off point was determined at 1500μg/l (median of measured ferritin levels). overall survival of allosct recipients with ferritin levels above cut off measured before start of conditioning was significantly shorter ( figure 1a , univariate hr=2.3 ci=1.4-3.6 p=0.00041; multivariate hr=2.5, ci=1.5-4.1, p=0.0005). progression-free survival was also shorter ( figure 1b , univariate hr=2.1 ci=1.4-3.2 p=0.00014; multivariate hr=2.4, ci=1.6-3.8, p< 0.0001). excess mortality in the high ferritin group was due to both higher relapse incidence (univariate hr=1.7 ci=1-2.8 p=0.03; multivariate hr=2.2, ci=1.2-3.8, p=0.007) and increased non-relapse mortality (univariate hr=3.1 ci=1.5-6.3 p=0.002; multivariate hr=3.1, ci=1.5-6.4, p=0.002). non-relapse mortality was driven by significantly higher infection-related mortality in the high ferritin group (univariate hr=3.9 ci=1.6-9.7 p = 0.003; multivariate hr = 3.9, ci = 1.6-9.7 p = 0.003). acute and chronic gvhd incidence or severity were not associated to serum ferritin levels. conclusions: ferritin levels before start of conditioning can serve as routine laboratory biomarker to predict mortality after allosct. disclosure: the authors declare no confict of interest related to this study p147 prediction of reduced lung function and acute gvhd by surfactant protein d in allogeneic stem cell transplantation transplantation (hsct) and may progress to bronchiolitis obliterans that has a high mortality rate. surfactant protein d (sp-d) is an innate defense molecule involved in immune regulation at the epithelial surfaces, particularly in the lungs, and elevated levels have been associated with exacerbation of chronic obstructive pulmonary disease (copd). the aim of this study was to investigate, whether sp-d plasma levels and variants in the gene encoding sp-d may predict the development of reduced lung function after allogenic hsct. methods: we performed a population-based, singlecenter study of children (aged 6-18 years) treated with allogeneic hsct. the study consisted of 1) a prospective study of serial plasma sp-d levels and rs721917 genotypes in 55 patients during the first 6 months after hsct, and 2) a retrospective study of rs721917 genotypes within the sp-d gene in 247 patients transplanted between 1990-2017. pulmonary function tests were performed regularly as part of the clinical monitoring. results: at the day of graft infusion (day 0) sp-d levels were reduced compared to levels before start of treatment with conditioning chemotherapy, defined as baseline (615 ng/ml (quartiles 441-1132) at day 0 vs 771 ng/ml (542-1348) at baseline, p< 0.01). from day +7 sp-d levels increased and remained increased during the whole study period (771 ng/ml (542-1348) at baseline vs 1287 ng/ml (713-2549) at 6 months, p< 0.01). acute gvhd (agvhd) occurred in 25 patients, of those 17 patients with grade 2-4. high sp-d levels at day +14 were associated with the development of agvhd (1402 ng/ml (1244-2023) vs 839 ng/ml (523-1630), p< 0.01) ( fig. 1 ). the c/c genotype was associated with generally low sp-d levels and low fev1/fvc at all time intervals compared to the other genotypes, significantly 24-36 months post-hsct (p=0.02). there was no overall correlation between sp-d levels and lung function, but stratifying for genotype, high baseline sp-d levels were predictive for reduced fev1/fvc at 8-24 months in cc and tt homozygous individuals. conclusions: patients with a genotype causing low capacity for sp-d production are at increased risk of developing pulmonary impairment after hsct. in addition, our data lend support to other studies indicating that spd production may increase during inflammatory pulmonary disease, acting as a reactive, protective mechanism. further research is warranted to define the role of sp-d levels and genotypes as a prognostic tool for lung function and agvhd. [[p147 image] 1. background: allogeneic hematopoietic cell transplantation (allohct) means a long period of restricted mobility and a range of therapy related side effects on muscle function. in this context patients demonstrated a huge decline of physical capacity and muscle mass in particular, accompanied with a decrease of quality of life (qol). resistance training could maintain muscle mass but is limited by patientsb lood values (platelet-count) and well-being. whole body vibration (wbv) was shown to maintain muscle mass during bed rest and has less impact on blood pressure than conventional resistance exercises. furthermore it was also shown to be feasible in patients during high dose chemotherapy. therefore the aim of our study was to examine the effects of wbv during allohct on patients physical and functional performance as well as qol. methods: 43 patients receiving allohct were randomly allocated to either a wbv exercise group (ig) or an active control group (cg) doing stretching and mobilization. both groups exercised during the whole time of hospitalization for 5 times per week and underwent pre-, post-and followup-assessment. physical capacity was determined by maximum oxygen consumption (vo 2peak ) and maximum power (p max ) during cardiorespiratory exercise test and by maximum strength of the knee extensors and flexors (ex max , flex max ) during isokinetic strength test. functional performance was assessed by jumping height during counter movement jump (cmj) and time of chair rising test (crt) as well as power output during both tests. qol was assessed by questionnaires of the eortc. results: during allohsct vo 2peak and p max decreased in both groups but till follow-up an increase is seen in the ig (p=0.035; p=0.011). at day +180/follow-up a vo 2peak group difference is seen (p=0.034). ex max (p=0.003) and flex max (p=0.044) were only reduced in the cg during hospitalization. jumping height and power output decreased in the cg during hospitalization (p=0.005, p=0.039) and a difference between groups were seen in changes of jumping height from pre-to follow-up-assessment (p=0.033): increase in the ig and decrease in the cg. the ig showed a decrease in time from baseline to follow-up (p=0.022) in the crt and an increase of power output (p=0.009). qol decreased only in the cg during hospitalization (p=0.015) while during follow-up qol increased in both groups (ig: p=0.013; cg: p=0.037). in the cg physical functioning decreased during intervention (p=0.001) whereas an increase was seen in the ig from pre-to follow-upassessment (p=0.035). body image was significant worse in the cg compared to the ig at hospital discharge (p=0.007) as well as at follow-up measurement (p=0.030) where it got worse over time (p=0.036). conclusions: wbv was shown to maintain maximum strength, jumping performance and qol during allohct. although cardiorespiratory fitness could not be maintained by wbv during hospitalization, it seems in the follow up period till day + 180 that recovery of the cardiorespiratory system is enhanced by wbv carried out during allohst. nevertheless reasons for this changes in recovery have to be analyzed in further studies as well as treatment effects of wbv compared to conventional resistance training. disclosure: supported by a grant of the faculty of medicine and comprehensive cancer center freiburg respiratory virus infection within 1 year after of allo-sct is the significant risk factor of obstructive ventilatory disturbance kosei kageyama 1 , michiho ebihara 1 , mitsuhiro yuasa 1 , daisuke kaji 1 , aya nishida 1 , shinsuke takagi 1 , hisashi yamamoto 1 , go yamamoto 1 , yuki asano-mori 1 , naoyuki uchida 1 , atsushi wake 1 , akiko yoneyama 1 , shigeyoshi makino 1 , shuichi taniguchi 1 1 toranomon hospital, hematology, tokyo, japan, background: obstructive ventilatory disturbance (ovd) is one of the major life-threading complication at the chronic phase of allogeneic stem cell transplantation (allo-sct). bronchiolitis obliterans has been the most established etiology as a part of chronic graft-versus-host disease and major cause of late non-relapse mortality of allo-sct. but other etiologies impact on respiratory function after allo-sct and risk factor of ovd have not been well understood. methods: to address these issues, we retrospectively reviewed the medical record of 747 consecutive patients who first allo-sct at toranomon hospital between 2009 and 2017. to detect ovd, forced expiratory volume in 1 second (fev1.0) showed less than 80% of predicted in spirometry test was defined as positive. in the recipients who showed fev1.0 less than 80% in pre-transplant test, more than 20% reduction of fev1.0 was regarded as positive. nasopharyngeal swab of those who had upper respiratory tract symptoms were tested for the presence of respiratory viral antigens (adv, piv, and rsv). patients with ecog performance status of 4, had active infection at transplant were excluded from this analysis. the cases of early death or relapse before 30 days post-transplant, and the cases of graft failure were also excluded. results: the median age was 55 years (range, 16-74). underlying diseases were aml in 403, mds/mpd in 73, cml in 26, all in 82, atl in 19, hl in 12, nhl in 104, and others in 28. five hundred twenty-nine (71%) were not in remission at the time of transplant. five hundred eightythree patients (78%) were conditioned with myeloablative regimens, whereas 164 patients received reduced-intensity regimens. donor sources consisted of related peripheral blood /bone marrow (bm) (n=85), unrelated bm (153) forty-six developed ovd on median of 198 (60-804) days post-transplant. cumulative incidence of ovd was 6.4% in total population. in 490 recipients those who could spirometry, overall survival at 5 years was 73.2% in patients who developed ovd and was comparable with those who did not develop it (64.3%, p=0.486). in univariate analysis, disease status (cr/aa or noncr), recipient age (age< 55 or ≥55), prior autologous stem cell transplantation (yes or no), intensity of conditioning regimen (mac or ric), tbi dose (< 8 gy or ≥8 gy), busulfan dose (< 9.6mg/kg or ≥9.6mg/kg), donor source (cord blood or non-cord) had no impact on the incidence of ovd. patients who developed respiratory virus infection showed significantly higher incidence of ovd compared to those who did not developed it (12.4% vs 5.3%, p< 0.01). in multivariate analysis, respiratory virus infection was the only significant risk factor for the development of ovd (hr=2.43, 95% ci 1.30-4.57, p< 0.01). conclusions: respiratory virus infection within 1 year after allo-sct is the significant risk factor of ovd. disclosure: nothing to declare. background: metabolic syndrome (mets) is related to increased risk of cardiovascular disease and type-2 diabetes (dm-2) and usually seen in overweight individuals in the general population. we investigated mets and clinical risk factors two decades after hsct. methods: all male survivors treated with myeloablative allo-hsct during childhood (< 17 years) between 1980-2010 in denmark were invited to a follow-up study. mets was defined as the presence of at least three ncep atp iii criteria: fasting plasma triglyceride (tg) ≥1.7 mmol/l, high density lipoprotein (hdl) < 1.03 mmol/l or medical treatment of hyperlipidemia; fasting plasma glucose (fpg) ≥5.6 mmol/l; abdominal circumference (ac) >102 cm; bp ≥130 mmhg (systolic) / ≥85 mmhg (diastolic) or medical treatment for hypertension. patients with overt dm-2 were included into the mets group. furthermore, patients were examined for chronic graft-versus-host disease (cgvhd) by the nih-criteria at the time of follow-up and high sensitivity c-reactive protein (hscrp) was measured. the prevalence of mets was compared to a nordic reference group (hildrum et al. 2009) . results: we included 49 out of 97 eligible males (participation rate 51%) aged 18-44 years, median 29 years. median (range) follow-up was 21 (8-32) years. of these 49 males, 74% had a malignant diagnosis and 65% were treated with tbi-based conditioning. donors were matched siblings (n=21), matched relatives (n=3) or matched unrelated donors (n=25). mets was more prevalent (33%) in the young adult survivors compared to the prevalence reported for 20-39year-olds in the nordic reference (16 %). instead the prevalence was comparable to that reported for the 50-69year-olds (32%). of the components of mets, elevated tg (51%), hypertension (47%), and decreased hdl (40%) were frequent, while fpg was elevated in 16%. importantly, only 4% of those with mets had increased ac and mean bmi (23.8 kg/m 2 ) of the hsct survivors was within normal range in contrast to features of mets observed in the background population. having mets was significantly associated with tbi (rr = 7.9, 95%ci (1.1-55.3), p=0.004) as was the following single components of mets (mean in tbi group vs. mean in non-tbi group): elevated tg (2.34 mmol/l vs. 0.93 mmol/ l, p= 0.006), lower hdl (1.04 mmol/l vs. 1.38 mmol/l, p=0.001) and higher diastolic bp (80 mmhg vs. 72 mmhg, p=0.03). mets was only demonstrated in one patient who received non-tbi based conditioning. sixteen of 49 patients had cgvhd of which nine were moderate to severe cases, but cgvhd was not associated with mets. however, low-grade inflammation measured by hscrp was related to increased ac (rho=0.41, p=0.004) and tg (rho=0.34, p=0.028). conclusions: our results indicate that male long-term survivors of allo-hsct during childhood have a high risk of mets at an earlier age than the general population. the presence of mets despite normal bmi in several patients suggests unconventional etiologies like the effect of tbi and low-grade inflammation. disclosure: nothing to declare. results: this survey was completed by transplant directors (46%), transplant consultants (41%), nonconsultant grade physicians (8%), hsct clinical nurses specialists (cns) (3%) and other (2%) from 114 centres in 24 countries. 58% of the centres are adult-only, 21% paediatric-only and 21% treat adult and paediatric patients (mixed centres). 46% are located higher than 50 degrees latitude (northern countries) and 54% lower than this latitude (southern countries). at the time of the survey 84% were members of the european union (eu). measurement of serum vd is routinely performed in 47% of the centres prior and in 70% after allogeneic hsct. the main clinical indications are known osteopaenia/osteoporosis (86%), previous fracture (71%), treatment with steroids (68%), premature menopause (46%) and established menopause (32%). monitoring occurs every 3 months (39%), every 6 months (24%), once a year (18%) or at other time-points (19%). in this regard, seasonality is not taken into account in the majority of the centres (94%). local and national/international guidelines (nice) are only followed by 19% and 18% of the centres, respectively. the most common cut-off value of serum vd for commencing on replacement is 50 nmol/l (32%). northern countries tend to use values of ≥75 nmol/l whereas southern countries ≤50 nmol/l. 15% do not use cut-off values. following hsct, 83% of centres prescribe vd supplements to maintain calcium metabolism and bone health (92%), enhance immune reconstitution post-hsct (24%), gvhd prevention (17%), enhance immune-suppression to treat gvhd (10%), treat depression/fatigue (3%) and reduce relapse risk 2%. a "loading" dose is administrated in 30% (54% adult, 25% mixed and 22% paediatric), with a mean duration of 4 weeks . the median daily loading dose is 2,000 iu (286-20,000). the median "maintenance" daily dose is 800 iu (67-10,000). there are not remarkable differences between adult and paediatric centres or northern and southern countries. vd replacement is prescribed by transplant physicians (75%), family physicians (10%), endocrinologists (3%), cns (3%), others (4%) and in 5% of the centres, patients are advised to buy it over-the-counter. vd is prescribed combined with calcium carbonate in 52% and alone in 48% of the centres. it is eventually discontinued by 69% of the centres when therapeutical levels of vd are reached (69%), dexa scan returns to normal (12%) and symptomatic improvement (9%). conclusions: this survey has demonstrated discrepancies in monitoring and replacement of vd across ebmt allogeneic hsct programmes. although awareness has arisen over the last decade, there is still lack of evidence about the optimal levels of vd required for immunemodulation post-hsct. this survey emphasises the need for specific guidelines to harmonise the current management of vd deficiency in adult and paediatric hsct setting. disclosure background: the use of unmanipulated haploidentical sct (haplo-sct) with post-transplant cyclophosphamide (pt-cy) as gvhd prophylaxis has widely extended. primary and secondary graft failure are relatively uncommon complications. however, poor graft function (pgf) after haplo-sct with pt-cy has not been described thoroughly. the objective of this study is to describe characteristics, treatments and outcomes of patients with pgf after haplo-sct with pt-cy. methods: we retrospectively analyzed 132 haplo-sct with pt-cy consecutively performed between 2011 and 2017 in our centre. pgf was defined as either occurring after initial engraftment: persistent neutropenia (anc < 500/ul) with the need of at least 3 doses of g-csf and/or thrombocytopenia (platelets < 20.000/ul) with platelet transfusion dependence, with complete donor chimerism and without concurrent severe gvhd or disease relapse. results: nineteen patients were excluded from the analysis due to early mortality (death before day +30), primary graft failure (absence of neutrophil engraftment by day +28, with mixed chimerism) or secondary graft failure (development of severe cytopenias and mixed chimerism after initial achievement of neutrophil engraftment). thirty one patients (27,5%) were diagnosed with pgf. main characteristics of these patients are summarized in table 1 . twenty six patients (84%) presented with neutropenia and were treated with g-csf, while 5 patients (16%) only developed severe thrombocytopenia without neutropenia, and were treated only with platelet transfusion. twenty four patients (77,5%) had at least 1 cmv reactivation, 15 patients (48%) had 2 or more cmv reactivations and 21 patients (67%) received valganciclovir for cmv reactivation treatment. although most patients achieved adequate peripheral blood counts (pbc) with initial salvage therapy, 6 patients (19%) had persistent cytopenias in spite of g-csf, platelet transfusion, cmv reactivation resolution and myelotoxic drugs withdrawal. four of them were treated with a boost of cd34+ selected peripheral blood donor cells at a median of 170 days after . median cd34+ cells infused was 3,42 x10 6 /kg. these 4 patients achieved adequate pbc after salvage therapy and two developed gvhd. the other 2 patients were treated with increasing doses of thrombopoietin (tpo) receptor agonist (tra) eltrombopag. one patient started treatment 160 days after hsct with 25mg daily and increased dose to 125mg daily, with complete recovery of pbc 6 months after initiating tra. the second patient started treatment 110 days after hsct with 25mg daily and increased dose to 100mg daily, with complete recovery of pbc 2 months after initiating tra. twenty one patients (67%) with pgf diagnosis had long term survival. conclusions: poor graft function is a frequent complication after haplo-sct with cy-post. cmv reactivation and myelotoxic drugs could be the most relevant factors associated with development of this entity. although most patients recover pbc without specific therapies beyond g-csf and platelets transfusion, there is a small group of patients with persistent cytopenias. boost of cd34+ selected cells is effective in reverting this condition, with gvhd as main complication of this procedure. use of tra seems to be an interesting option for these patients, although more experience is needed to draw definitive conclusions. disclosure: nothing to declare. were also frequently observed. the high risk patients for anxiety (hads-a score ≥ 8) and depression (hads-d score ≥ 8) was found in 14.9% and 13.6%, respectively. 10.4% of patients was in high distress status (nccn dt score ≥ 4). we found that younger age (< 60 years) was significantly associated with poor quality of life score (fact-bmt) (p=0.001) and high risk of fatigue (p=0.008), anxiety (hads-a) (p=0.001), and depression (hads-d) (p=0.025). female sex was significantly related to lower physical well-being score and higher distress score (p= 0.046 and p=0.05, respectively). acute lymphoblast leukemia (all) survivors after allo-hct showed significantly worse quality of life score (fact-bmt) (p=0.006) and higher depression score (hads-d) (p=0.028) compared to those with other disease. chronic graft versus host disease (gvhd) and continuous immunosuppressant usage also have significant adverse impact on lower fact-bmt score (p=0.024 and p=0.033, respectively) and higher hads-d score (p=0.015 and p=0.019, respectively). but there was no significant difference in fact bmt, hads-a, hads-d, nccn dt according to donor type, conditioning intensity, anti-thymocyte globulin use, acute gvhd. smoking and alcohol drinking was continued in 7.5% and 17.9% of allo-hct survivors. 20.9% of survivors did not exercise regularly. regular health screening tests have been done only in 40 patients (59.7%). conclusions: allo-hct survivors over 2 years following allo-hct still have many physical and psychological symptoms. younger patients (< 60 years), female, all, chronic gvhd, and sustained use of immunosuppressant were significant risk factors for poor quality of life and anxiety. we need to build more active survivorship care plan after allo-hct especially for those patients. disclosure: all authors have nothing to declare. evaluation of the new ebmt criteria for the diagnosis of vod/sos in 693 consecutive transplant patients using an electronic patient record analysis system asha aggarwal 1 , nicola gray 1 , oliver lomas 1 , katalin balassa 1 , nadjoua maouche 1 , robert danby 1,2 , andy peniket 1 , grant vallance 1 background: veno-occlusive disease (vod), or sinusoidal obstruction syndrome (sos), is a recognised complication of haematopoietic stem cell transplantation. hepatic vasculature endothelial cells are damaged by conditioning chemotherapy, leading to venous occlusion and centrilobar necrosis. the ebmt criteria for diagnosis of vod are bilirubin >=34 with two of painful hepatomegaly, >5% weight gain and ascites. vod is often under-diagnosed, and as a result, treatment may be delayed. integrated electronic patient record (epr) systems are now widely used, and provide an opportunity to retrospectively audit practice to identify patients in whom vod may have been un-diagnosed or in whom treatment was delayed. in addition these systems have potential for alerting clinicians to the potential diagnosis of vod. methods: we have developed software to analyse the data downloaded from epr to identify patients in whom vod was a possible diagnosis according to the new ebmt criteria. in order to identify patients who may have had vod we first screened for patients with a bilirubin of >= 34 mmol/l (which is an absolute requirement for the clinical diagnosis of vod) within the first 50 days of transplantation. epr data was then used to assess whether patients had >5% weight gain. radiology reports were reviewed for patients who had bilirubin >= 34 mmol/l to ascertain if they revealed ascites or painful hepatomegaly. results: 652 patients underwent 693 transplant procedures (january 1st 2013 to july 31st 2018). 162 of all transplant patients (23.4%) were found to have a bilirubin of >= 34 mmol/l. 39 of 403 (9.6%) autograft patients and 123 of 249 (49.4%) allograft patients had an elevated bilirubin at this level. these 162 patients were assessed for evidence of 5% weight gain. this was the case in 30 patients overall-1% of autograft patients, 10.5% of allograft patients. seven patients (2 autograft and 5 allograft) had radiological evidence of ascites. two patients had a recording of painful hepatomegaly (both post allograft). overall our analysis identified 5 patients (0.7% overall) fulfilling the ebmt diagnostic criteria for classic early vod all of whom received defibrotide. all patients had received allogeneic transplants. we failed to identify any cases of late onset vod or any undiagnosed patients over this period. conclusions: this analysis enabled us to efficiently perform a complete audit of our practice to identify patients with vod. we would recommend using electronic patient records to retrospectively audit practice in this way. the tool that we have created for this analysis will be made freely available for public use and the details will be presented at the ebmt meeting. we now plan to extend the function of our epr system to provide alerts to clinicians when vod is a possible diagnosis and may lead to more rapid treatment of these patients. our data suggests that elevation of bilirubin and weight gain of > 5% will be the most frequently occurring criteria on which to base these alerts. disclosure: g.vallance has performed consultancy work for jazz pharmaceuticals. endothelial activation and stress index in predicting outcome of allogeneic stem cell transplantation-a retrospective cohort analysis zinaida peric 1 , tomislav taborsak 1 , nadira durakovic 1 , lana desnica 1 , alen ostojic 1 , ranka serventi-seiwerth 1 , radovan vrhovac 1 1 university hospital centre zagreb, zagreb, croatia background: endothelial dysfunction is a common pathophysiology of major complications after allo-sct, such as graft-versus-host disease, veno-occlusive disease, thrombotic microangiopathy and sepsis. endothelial activation and stress index (easix) is a simple score comprised of standard laboratory parameters (creatinine, ldh and thrombocytes) developed as a potential tool to predict allo-sct mortality by luft and colleagues. a recent validation of easix included three retrospective cohorts and showed that easix taken before start of conditioning can be used as an independent predictor of survival after allo-sct. methods: the aim of our study was to retrospectively evaluate pre-transplant easix in our cohort of consecutive patients who underwent allo-sct in the university hospital centre zagreb between 2012 and 2017. with the use of a cut-off used in the validation cohorts, we compared two groups of patients for overall survival (os) and transplantrelated mortality (trm). group comparisons were done using the log-rank test or gray test for competing risks outcomes. a multivariate analysis evaluated the association of os with relevant variables by using a cox's proportionalhazard regression model. results: our study group included 313 patients and comprised 180 males (57%) and 133 females (43%, with a median age of 48 years (range, 18 to 67 years) at the time of transplantation. the most frequent malignancies in our population were acute leukemia (196 patients, 63%) and myelodysplastic/myeloproliferative neoplasm (451 patients; 16%). the donor was an identical sibling for 106 patients (34%), matched unrelated donor for 176 patients (56%) and haploidentical for 31 patients (10%). 104 patients (33%) received a myeloablative conditioning regimen while 209 patients (67%) received a reduced-intensity conditioning regimen. with a median follow-up of 16 months (range, 12-60) for the whole study group, the os at 24 months was 60%, (95%ci 54-68) in the group of patients with low easix score and 43% (95% ci 33-56) in the group of patients with high easix score (p=0.004). this difference was mainly attributed to higher trm in the group with high easix score (32%, 95%ci 22-45 at 12 months) compared to the group with low easix score (18%, 95%ci 13-23 at 12 months) (p=0.009). in the multivariate analysis which included easix, patients' age, intensity of conditioning, diagnosis (lymphoid vs myeloid), status of the disease at transplant and type of the donor, worse os was independently associated only with older age of patients (hr 1.66; 95% ci, 1.07-2.59, p=0.02) and high easix score (hr 1.51; 95% ci, 1.01-2.24, p=0.04). conclusions: our retrospective data support previous data and suggest that easix could potentially serve as a valid tool for prediction of allo-sct outcomes. as a simple biomarker panel, easix could easily be implemented in clinical decision making in the field of allo-sct. these retrospective data need validation in a prospective study which is currently being conducted. clinical background: veno-occlusive disease (vod) is a potentially devastating complication that can occur after hematopoietic stem cell transplant (hsct) and in severe cases can lead to multi-organ failure. (mohty 2016) defibrotide has been proven to be effective to prevent and treat vod, and it is critical that clinicians are aware of how to diagnose and treat this serious complication of hsct. this study was conducted to determine if an online, simulation-based continuing medical education (cme) intervention could improve performance of hematologists/oncologists (hem/ onc) and advanced practice providers (nurse practitioners and physician assistants, apps) in the diagnosis and treatment of patients with vod. ( methods: a cme certified virtual patient simulation (vps) was made available via a website dedicated to continuous professional development. the vps consisted of 2 cases presented in a platform that allows clinicians to assess the patients and make diagnostic and therapeutic decisions supported by an extensive database of diagnostic and treatment possibilities, matching the scope and depth of actual practice. clinical decisions were analyzed using a sophisticated decision engine, and tailored clinical guidance (cg) employing up-to-date evidence-based and faculty recommendations was provided after each decision. one case was about vod and the other case was about acute myeloid leukemia (aml). decisions were collected post-cg and compared with each user's baseline (pre-cg) decisions using a 2-tailed paired t-test to determine p-values (p < .05 indicates significance). data were collected between 9/21/2017 and 11/7/2018. results: at the time of assessment, 115 hem/oncs and 409 apps had fulfilled the participation criteria for completing the vod case simulation. conclusions: this study demonstrates that vps that immersed and engaged clinicians in an authentic and practical learning experience improved evidence-based clinical decisions related to the management of vod. this vps increased the percentage of clinicians who utilized standardized criteria to diagnose vod and who ordered defibrotide and iv fluids for vod management. however, further education is needed to increase the competence and performance of clinicians, particularly apps, in these areas in order to positively impact patients. disclosure: nothing to declare. a nationwide retrospective study of hematopoietic stem cell transplantation in solid organ transplant recipients: on behalf of jshct, transplant complications working group background: the outcome of hematopoietic stem cell transplantation (hsct) in solid organ transplant remain unclear. to address this issue, we conducted a retrospective survey of the 404 japan society for hematopoietic stem cell transplantation centers. methods: to address this issue, we conducted a nationwide retrospective survey of the japan society for hematopoietic stem cell transplantation (jshct) centers. a first questionnaire was emailed to jshct centers requesting information on cases of hsct in sot recipient. patients' data about sot were collected by sending a second questionnaire to the centers with the patient. based on these reports, patients' data about hsct was identified in the japan transplant outcomes registry database by the transplant registry unified management program (trump), confirmed in 2017. results: of the 404 jshct centers, 238 responded to the survey (58.9%). 14 of the responding centers reported a total of 19 patients who had undergone sot from living donor, and subsequent hsct. they consist of three autologous hsct (auto-hsct) and 13 allogeneic hsct (allo-hsct). in auto-hsct, all patients had received liver transplant for hapatoblastoma. they achieved neutrophil engraftment at 30 days after hsct, and two of three patients were alive at one year after hsct. in allo-hsct (n=16), seven patients had received liver transplants, and nine patients had received kidney transplants. five patients received hsct from unrelated donor, and 11 patients received hsct from related donor; two donors were identical in sot. their stem cell sources were seven peripheral blood stem cell, six bone marrow, and three cord blood. all but one patients achieved neutrophil engraftment at 30 days after hsct. five-year overall survival (5yos) was 37.5%. while 5yos in patients with bone marrow failure (n=4) was 100%, that in patients with malignant disease (n=12) was 16.7%; all but one patients with malignant disease received allo-hsct in non-remission. seven of nine kidney-transplant recipients experienced dialysis, and three patients experienced renal rejection after hsct. on the contrary, no liver-transplant recipient experienced hepatic rejection. conclusions: in sot recipients, the outcome of allo-hsct for malignant disease was poor, partly due to disease status before allo-hsct. severe renal complications were common in kidney-transplant recipients, suggesting renal care with caution during and after allo-hsct. disclosure: this work was supported in part by the practical research project for allergic diseases and immunology (research technology of medical transplantation) from japan agency for medical research and development, amed. high incidence but low mortality of ebv related ptld after t-cell replete allo-peripheral blood hct with aggressive monitoring and without pre-emptive rituximab background: the aim of the study is to report the incidence and outcome of post-transplant lymphoproliferative disorder (ptld) in the setting of allogeneic peripheral blood hematopoietic stem cell transplantation (allo-hsct) combining post-transplant cyclophosphamide (ptcy) and anti-thymocyte globulin (atg) as graft versus host disease (gvhd) prophylaxis. methods: between october 2015 and may 2018, 195 adult patients diagnosed with hematological malignancies underwent a first t-cell replete allo-hsct in our center. all patients received a reduced intensity conditioning regimen with fludarabine, busulfan, and 200cgy of total body irradiation, combined with rabbit-atg, ptcy and cyclosporine (csa). ebv titres were monitored weekly by quantitative pcr in plasma samples. the cut-off value for test positivity was >600 copies of ebv dna/ml of plasma. last follow up was november 2018. median follow up for patients known to be alive was 19 months (range 5-35). results: patient information is summarized in table 1 . ebv reactivation was documented in 117 (60%) patients. median time to ebv reactivation and the diagnosis of presumed/proven (p/p)-ptld were 75 (16-326) days and 97 (54-306) days [3 (0-10) months], respectively. median time between first ebv reactivation to p/p-ptld was 21 (0-175) days. seventeen (14%) of the 117 patients developed p/p-ptld. median age was 55 years . two (12%) received mrd, 9 (53%) 10/10 mud, 1 (6%) 9/10 mud, and 5 (29%) haploidentical donor grafts. twelve (71%) were on therapeutic cyclosporine at diagnosis. pre-emptive therapy was not given to any case and only probable or proven ptld were given rituximab. treatment was based on reduction of the immunosuppression in 3 patients and with the addition of weekly rituximab 375 mg/ m 2 in 15 cases. fifteen (88%) achieved complete clinical responses with pcr negativity. two (12%) patients died secondary to ptld. conclusions: atg based conditioning is associated with increased viral reactivations. frequent ebv monitoring and pre-emptive treatment may lead to rapid disease control. further research is required to optimize monitoring and management strategies in allo-hsct recipients. disclosure: nothing to declare p160 acoustically enriched extracellular vesicles as potential markers for allogeneic hematopoietic stem cell transplantation complications hooi-ching lim 1 , robert palmason 2 , stig lenhoff 2 , thomas laurell 1 , stefan scheding 1,2 background: extracellular vesicles (evs) contain a number of condition-specific proteins, dna and rna types and might therefore be used for the early detection of posttransplant complications. however, traditional ev isolation (ultracentrifugation) is time consuming and requires large sample volumes thus making it difficult to perform longitudinal studies on larger patient cohorts. we therefore investigated whether recently-developed acoustic trapping could be applied to isolate evs from patient plasma for biomarker development. methods: plasma samples were collected from 10 consecutive patients before and up to 3 months after allogeneic hematopoietic stem cell transplantation. patients (age: 22-58 years) with high-risk or refractory/relapsed diseases were transplanted with mobilized pbsc from related (n=2) and unrelated donors (n=8) after standard conditioning. gvhd prophylaxis was cyclosporine and methotrexate. plasma samples were frozen and thawed for ev enrichment using a novel acoustofluidic-based technology (acoustic trapping). acoustic trapping uses ultrasound as a local λ/2 acoustic standing wave produced by a piezoelectric transducer over a capillary. first, 12 μm polystyrene beads are captured which serve as seeding particles. after washing, target particles (evs) are then captured ("trapped") in the acoustic field. a semi-automatic trapping device (acoutrap) was used to isolate evs from diluted plasma (1:2 in pbs). the number of evs and size distribution were analyzed by nanoparticle tracking analysis. mirna analysis was performed by qpcr.evs were enriched in duplicate from 50μl and 300μl of diluted plasma for nanoparticle tracking analysis and qpcr analysis, respectively. results: evs were successfully isolated from all plasma samples. a total of 89 plasma samples were processed. numbers of trapped evs ranged from 3.7x10 8 -5.5x10 9 before conditioning to 4.4x10 8 -1.5x10 10 per 50μl diluted plasma after transplantation. the maximum change in ev numbers in individual patients compared to pretransplantation values ranged from 2-fold to 7-fold. most patients showed slight increases in ev size after transplantation. eight of the patients showed signs of infection and received i.v. antibiotics. increased levels of evs (> 2-fold) were recorded in three patients during these episodes. furthermore, increased ev numbers were observed in a patient who required i.v. antiviral therapy for cmv reactivation. acute grade i gvhd was observed in five patients of which two had increased ev numbers (> 2-fold). one patient developed grade iv gvhd which was accompanied by a 4-fold increase in ev numbers. interestingly, progressively increasing ev numbers preceded the detection of early relapse in a pre-b all patient by three weeks. rna isolation from trapped evs yielded sufficient material for mirna profiling. here, first mirna profiling data demonstrated that mirnas were detected in ev samples (mir-103a, -23a, -30c, -142 and -451a) , and that acoustically enriched evs were not affected by hemolysis in contrast to the corresponding whole plasma samples (dcq of mir-23a and mir-451a). conclusions: acoustic trapping allows for efficient and rapid enrichment of evs from small volume plasma samples. trapped ev samples contain sufficient amounts of mirna for downstream analysis and are thus promising candidates for biomarker development in transplantation. disclosure: laurell and scheding are founders and board members of acousort ab, a lund-based biotech sme that develops particle and cell sorting methods based on ultrasound. the incidence, risk factors and outcomes of primary poor graft function after allogeneic hematopoietic stem cell transplantation fei gao 1,2 , jimin shi 1,2 , yi luo 1,2 , yamin tan 1,2 , xiaoyu lai 1,2 , jian yu 1,2 , he huang 1,2 , yanmin zhao 1, 2 background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative therapy for both hematologic malignancy and many other blood disease. while, primary poor graft function (pgf) is still a severe complication following hsct which lead to poor prognosis. up to now, the incidence and risk factors of pgf have not been totally revealed. methods: from january 2013 to december 2017, a total of 647 patients who received allo-hsct in our center were analyzed retrospectively. there were 9 males (47.4%) and 10 females (52.6%) with a median age of 36.21 years (21-49 years) . pgf was defined as persistent neutropenia (≤0.5×10 9 /l), thrombocytopenia (platelets≤20×10 9 /l), and/ or hemoglobin≤70 g/l after engraftment with hypocellular bone marrow and full donor chimerism, without concurrent graft-versus-host disease or disease relapse. incidence was calculated from all patients. of the 647 total patients, nineteen (2.94%) developed primary pgf. a 1:4 ratio of nested case control study using the good graft function (ggf) subjects transplanted in the same year with the same sex and age of ±5 years was carried out. results: data was analyzed by univariate and multivariate logistic regression, and univariate analysis identified disease species, the time from diagnosis to transplantation, disease states, myelofibrosis, splenomegaly, serum ferritin (sf) level, cmv infection, mononuclear and cd34+ cells in graft as potential risk factors (p <0.1) for pgf. multivariate analysis identified 3 elements as the independent risk factors (p <0.05), including cd34+ cells <5×10 5 / background: transplant survivors affected by cgvhd usually take one or more immunosuppressants, as well as prophylactic antimicrobials; use of multiple medication classes concurrently poses a risk for drug-drug interactions or amplified side-effects. the use of medications other than cgvhd-direct immunosuppressive therapies has not been well-characterized. this study aims to evaluate patterns of opioid analgesic use in a cohort of patients severely affected by cgvhd. methods: patients (n=335) with cgvhd were consecutively enrolled in a cross-sectional natural history study (nct00092235) from 10/2004-12/2016 at the nih. participants underwent a comprehensive evaluation including a detailed history and physical examination (including current medications), multidisciplinary evaluations, and laboratory and diagnostic testing. for this analysis, respondents were classified as receiving or not receiving an opioid analgesic. following the initial screening by univariate methods (n=335), multivariable logistic regression analysis (mlr) was used to identify a set of factors which could jointly impact opioid use. for mlr data were divided into a training (n=167 patients) and a validation set (n=168). results: study participants´median age was 48.5 years (19-75), 44% were female, 74% had severe cgvhd per nih scoring criteria, and 77% were currently receiving high or moderate levels of systemic immunosuppression. approximately one third (33%) were taking opioid analgesics (oa). based on the univariate screening results (p< =0.05), a set of 24 parameters was evaluated by univariate logistic regression in the 167-patient training set, and the following parameters retained their significance and were included in the mlr model: nih average score per organ, total lss, patient impression of severity, nih cgvhd severity, presence of skin erythema, karnofsky performance score (kps,) clinician's therapeutic intent, nih joint score, and with the presence of several cgvhd symptoms including rashes, mouth sores, avoidance of food, vomiting, weight loss, joint and muscle aches, joint limitation, energy loss, need for naps, fevers, anxiety. multivariable logistic regression identified kps < 77% as predictive of oa use, or 0.92, 95% ci 0.89-0.95. in the training set 56.4% of pts using opioids were correctly identified, 78.2% of those not taking opioids were identified, an overall fraction of correctly identified pts was 70.9% (95% ci 63.3 -77.7%), while in the testing set, 45.5% of those using opioids were correctly identified, and 67.9% of those not taking opioids were correctly identified, with overall 60.5% (95% ci: 52.6-68.0%) classification accuracy. conclusions: this study showed the burden of oa in this cgvhd cohort. lower kps was significantly associated with oa use, as well as self-reported symptoms and a more severe cgvhd disease, which could be of interest in the development of non-pharmaceutical interventions in this patient population. additional, prospective studies are needed to explore the indications for and effectiveness of oa in this population of survivors. disclosure: no conflict of interest to declare. rcts that tested an internet-based program and patientcentered survivorship care plans for hct survivors. patient and caregiver input is essential to inform the design and features for the mobile app platform so that it is usable and engaging for those it targets. methods: using a qualitative research design, we conducted telephone focus groups of adult patients and caregivers in the united states. adult (age >18 years at the time of study entry) hct recipients had to be at least oneyear post-hct to participate. participants had to be able to communicate in english, and could have received a hct for any diagnosis, and from any donor source or stem cell type. those who had multiple transplants were included. participants were asked to review printed and online visual presentations of the mobile app before the focus groups so they were prepared to discuss their responses to the materials during the call. focus groups were conducted to saturation, when no new qualitative content was offered. results: three focus groups were conducted with 22 total participants (20 patients, two caregivers/patient advocates). all patients received an allogeneic hct; average time since hct was 8 years (range: 2-22 years).the majority of participants were female (77.3%). participants had differing perspectives on the usefulness of the app to track follow-up appointments, lab values, and other health care plans. there was high interest in having the app tailored to meet specific needs of patients, including tracking information over time (e.g. test results, medications), and having health information available specific to their needs. to minimize duplication of information and data entry, participants recommended syncing the app with their calendars and online patient portals they already use. reasons provided for not using the app included perception that the materials repeated information already received, side effects such as graft-versus-host disease that restricted vision or motor skills, and lack of comfort with apps for some older participants. conclusions: many health technology and mobile apps are being created to improve patients' health and survivorship care. in this study, hct survivors and caregivers identified a variety of features that they would want in an app or website, in particular, features tailored to individual needs. health technologies provide an opportunity to improve survivorship care, but patients and caregivers should be engaged in the process of developing these tools to assure the technology fits their needs and will be used. given the effort required to maintain these technologies, they require testing for health benefits in rigorous clinical trials. clinical background: thanks to allogeneic stem cell transplantation (allo-hsct) patients suffering from hematologic malignancies have seen an increase in there life duration expectancy, but they are many side effets including decreasing in physical performance and in quality of life. the intensity of physical performance decrease is variable between patients, and today we did not know why. the aim of our study was first to characterize the physical performance of subjects less than 1 year following allo-hsct by the use of a cardiopulmonary exercise testing (cpet), and then to determine the predictive factors of exercise performance. methods: we did a retrospective analysis from 59 patients who had an allo-hsct at hematology department of toulouse-oncopole and cpet from 01/2015 to 09/2017. the cpet was performed using a cycle ergometer with o2 and co2 analyzer breath by breath, (masterscreen cpx carefusion, san diego, usa), a continuous 12-lead electrocardiogram, and a blood pressure monitoring. the protocol included a 2-min rest period, a 2-min warm-up of 20w pedaling followed by a 10w/min incremental phase, up to exhaustion, then a 2-min active recovery of 20w pedaling, then a 2-min passive recovery. three exercise markers were analysed: the peak of oxygen uptake (peak vo 2 ), the ve/vco 2 slope and the first ventilatory threshold (vt 1 ). data relative to conditioning regimen, short-term complications, impairment at cpet day, and physical activity since allo-hsct were gathered. results: after allo-hsct, nearly 3 over 4 patients reported fatigue, a half reported dyspnea, and 1 over 4 or more reported pain, muscular, neurological or psychological impairment. more than 60% of patients suffer from moderate or severe physical intolerance, particularly when myeloablative conditioning regimen was used. only 37% of patients followed rehabilitation sessions supervised by a physiotherapist, and non-supervised physical activity has been performed by 87% of patients. despite normal lung function tests and echocardiography findings in most patients, 80% had exercise intolerance (ei), 85% exercise deconditioning, and 55% had abnormal ventilatory efficiency. patients with moderate and severe impaired exercise capacity were significantly younger at diagnosis and at allo-hsct, such as patients with severe deconditioning conclusions: based on a retrospective study, we reported for the first time complete results from cpet and detailed clinical evaluation concerning deficiency and disability following first year after allo-hsct. these results confirm that exercise impairment is very frequent with more than a half of patients suffering from alterations of one or more of the three performance markers, despite being active. disclosure: nothing to declare demyelinating disorders: a paradigm of immunity disorders after hematopoietic stem cell transplantation background: neurologic complications are a major problem in patients who undergone hematopoietic stem cell transplantation (hsct). given the higher survival of transplanted patients, the burden of neurological complications is increasing in the last years. a significant reduction in overall survival was demonstrated in patients who developed neurological complication after hsct, irrespectively of the hematopoietic stem cell (hsc) source. neurologic disorders in transplanting setting comprise a wide variety of ethiologies including demyelinating disease, which are caused by immune and non-immune mechanisms. here, we analyzed the clinical presentation and the underlie ethiologies of patients developing hsct-related demyelinating disorders in order to give diagnostic and prognostic clues useful to manage these severe but treatable complications in the transplant setting. methods: a total of 45 patients of our department which developed neurological complications after hsct were consecutively collected and 33 (73%) of them, namely those having a diagnosis of a demyelinating disorder, were grouped and described according to the ethiologies of their neurological disorder. results: in 14/33 (42%) patients, an immune-mediated process was found, while 10/33 (30%) were diagnosed as having an infective etiology and 2/33 (6%) were supposed to have a demyelinating disorder caused by toxic exposition. a definitive etiologic diagnosis was not formulated in the remaining 7/33 (21%) patients. when patients who developed an immune-mediated demyelinating disorder (10/33) were compared to those in which a clear immune pathogenic mechanism was not detected (23/33), a higher incidence of acute graft-versus-host disease (agvhd) was detected in the former than in the latter (20% vs 8%). moreover, comparison of these two groups revealed that those with no evidence of immune-mediated process have a slight higher prevalence of t-cell depleted hsct thanthose with an immune-mediated demyelinating disorder (60% vs 50%). finally, a lymphoproliferative disorder pre-existing the hsct was detected in 5/14 (36%) patients with immune-mediated demyelinating disorder but only in 4/19 (21%) of those without evidence of immune-mediated processes. conclusions: demyelinating disorders may be responsible of near 40% of neurologic complications in the posttransplant setting and, among them, an immune-mediated process is likely to be involved in more than 40% of cases. our results suggest that the immune mechanism that underliesthe agvhd may also be involved in developing demyelinating disease in transplanted patients. it also may be possible that the lymphoproliferative disorder preexisting the hsct is a risk factor able to increase the risk to develop an immune-mediated demyelinating disorder in the post-transplanting setting. using a t-cell depleted hsct can increase the risk of immune-mediated disorders in at least a small fraction of transplanted patients. despite our results should be validated on a larger cohort of patients, we can speculate on the possible connections between the wide range of complex and still poorly defined immunity disorders which can influence the prognosis and course of transplanted patients. disclosure background: injury to the mucosal barrier and subsequent development of oral mucositis (om) is among the most common toxicities of allogeneic stem cell transplantation (sct). despite the high prevalence of om and its debilitating nature, prospective studies evaluating determinants of om are scarce. we therefore prospectively evaluated the occurrence of om following sct. risk factors for om and its implications short and long-term outcomes were assessed. methods: om was prospectively evaluated on a weekly basis in patients undergoing allogeneic hsct. the grade of om was determined based on the national cancer institute common toxicity criteria for adverse events (ctcae) scale (v. 4.0). severe om was defined as grade ii to iv. conditioning regimens were evaluated individually and according to intensity; myeloablative (mac), reduced intensity (ric) or reduced toxicity (rtc). the latter category included only patients receiving fludarabine and treosulfan at dose of 30-42 g/m2 (flu/treo). risk factors for the development of severe om were initially identified by a univariate analysis and then analyzed in a multivariate logistic regression model. association of om with peritransplant infectious complications, iv morphine consumption, hospitalization length, neutrophil engraftment, acute and chronic graft-versus-host disease (gvhd), non-relapse mortality (nrm) and overall survival were assessed in a univariate analysis. competing events were considered in analyzing engraftment, gvhd, and nrm. results: 115 patients who underwent an allogeneic sct between 2016 and 2017 were included. median follow-up was 316 days. leading indications for transplantation were acute myeloid leukemia (49%), lymphoma (16%), and myelodysplastic syndrome (15%). the majority of patients received an allograft from a matched sibling or unrelated donor (88%) and methotrexate gvhd prophylaxis (74%). the median time to om onset was 7 (interquartile range [iqr] 5-9) days. prevalence of grade ii-iv om was 60%. the median duration om was 10 [6-13] days, and iv morphine was administrated for a median of 5 [6-12] days for patients with grades iii-iv om (45%). in a univariate analysis a younger age (p=0.023), lower bmi (p=0.01), recent smoking history (p=0.08), recent antibiotics exposure (p=0.018), mac (p< 0.001), and use of methotrexate (p=0.009) were associated with an increased risk for grade ii-iv om. in a multivariable model the risk for grade ii-iv om was lower with rtc (i.e., flu/treo) vs. mac (odds ratio [or] 27.31; p< 0.001) and rtc vs ric (or 7.25; p=0.001), mycophenolate mofetil vs. methotrexate (or 3.43; p=0.027) and recent smoking (or 4.7; p=0.075). compared to lower grades, grade ii-iv om was associated with a longer hospitalization duration (median 29 days vs. 27 days; p=0.006), delayed neutrophil engraftment (median 14 vs. 12 days; p=-0.004), and more gastrointestinal related infections (10% vs. 0%; p=0.045). grade ii-iv om was not associated with increased risk of bloodstream infections, acute or chronic gvhd, non-relapse mortality, and increased mortality. conclusions: oral mucositis is prevalent among allogeneic-sct recipients. importantly, fludarabine-treosulfan, which is considered a myeloablative is associated with a markedly reduced risk for om. consequences of om include prolongation of hospitalization, delay in neutrophil engraftment, and a tendency for gastrointestinal infections, but does not increase the risk for gvhd and mortality. disclosure: nothing to declare background: the advent of recent diagnostic techniques for the assessment of iron overload (t2*-mri) and their systematic use as screening tools in the setting of secondary hemochromatosis have led to an increased awareness that focal nodular hyperplasia (fnh) represents a possible incidental finding after hematopoietic stem cell transplantation (hsct). methods: clinical and radiological features of patients undergoing hsct in a single pediatric institution have been retrospectively reviewed for fnh. in order to provide an estimate of the prevalence of fnh after hsct, we analysed all the t2*-mri scans performed during the last 5 years in our centre and recorded the number of patients with fnh (group a). in addition, data about patients incidentally diagnosed with fnh at abdominal imaging performed for different clinical indications have been collected (group b). results: eight out of 118 (7%) transplanted patients who underwent at least one t2*-mri scan from september 2013 to september 2018 were incidentally diagnosed with fnh. group b included 3 subjects with fnh incidentally found at ultrasound or non-t2* mri scans performed before 2013. overall, 11 transplanted patients (5 males, 45%), transplanted for al (9 cases) or bone marrow failure (2 cases) at a median age of 13.1±3.6 years, were diagnosed with fnh between 0.6 and 12.8 years after hsct, namely 3.2±2.6 years in group a and 7.0±4.1 years in group b. a variable degree of iron overload was demonstrated in all patient (lic: 230-340±50 microg/g; baseline serum ferritin: 685-3189 ng/ml). the potential risk factors for fnh are reported in table 1. in 8/11 patients, the radiological finding was pathognomonic; in 1/11 the diagnosis of fnh was confirmed histologically, while 2/11 subjects were labelled as "fnhlike", although a potential diagnosis of hepatic adenoma could not be ruled out. in 3/11 patients, fnh presented with an isolated lesion, while 8/11 had 2 to more than 10 hepatic nodules at diagnosis. the size of nodules at diagnosis ranged from 3 to 41 mm. in unenhanced mri scans, lesions were predominantly hyperintense on both t1-and t2weighted sequences. in dynamic studies with contrast medium, all lesions strongly enhanced during the arterial phase, with a variable degree of wash-out in the late venous scans. hepatic function tests were normal in all the enrolled patients at diagnosis of fnh. among the 9/11 patients for whom at least a follow-up scan was available, 1 presented a complete regression, 3 a reduction and 3 an increase in the size and/or number of lesions, while in 2 patients the nodules remained substantially unchanged after a mean radiological follow-up of 4.5±3.3 years. no malignant transformations were observed. conclusions: fnh represents a relatively frequent incidental finding after hsct. although a malignant transformation is rare, given the demonstrated variable evolution of the hepatic nodules, a radiological follow-up is highly recommended. disclosure: nothing to disclose. incidence and risk factors for hepatic sinusoidal obstruction syndrome after allogeneic transplantation: retrospective multicenter study of turkish hematology research and education group (threg), updated data methods: ten centers from turkey were enrolled in the study. we retrospectively evaluated the medical records of patients who were treated with allo-sct between january 2012 and december 2015. a baltimore criterion was used for assessment of hsos. four hundred twenty six (97.2%) of 438 patients who were treated with prophylaxis with defibrotide alone or one or more of the n-acetylcysteine, diuretics and heparin used defibrotide (10-25 mg/kg/day). results: the study included 1153 patients (687 males/ 466 females) with median age of 38 (15-71) years. the demographic and clinical characteristics of patients were summarized in table 1 seventy-three (84.9%) of patients with hsos were treated with defibrotide after diagnosis. the median time of starting defibrotide in these patients was 13.5 (2-29) days. thirty-seven (43%) of patients with hsos recovered completely and forty-nine (57%) of them died as a result of multi organ failure. the incidence of hsos-related mortality in allo-hsct cohort was found to be 4.2%. in univariate analysis, statistically significant associations were not found between hsos incidence and age/sex of recipient, type of conditioning regimen, stem cell source and type of gvhd prophylaxis. on the other hand donor type, engraftment status and prophylaxis for hsos were significantly associated with hsos development. hsos prophylaxis was significantly decreased hsos-associated mortality (p=0.001). conclusions: hsos still remains a serious lifethreatening complication of allo-sct. although the incidence is low, hsos is associated with increased 100-day non-relapse mortality. hsos prophylaxis especially with defibrotide, seems to reduce hsos associated mortality in high risk patients. disclosure: nothing to declare prophylaxis with defibrotide in adults at very high risk of veno-occlusive disease: results in 11 patients background: hepatic sinusoidal obstruction syndrome/ veno-occlusive disease (sos/vod) is a life threatening complication that can occur after hematopoietic stem cell transplantation (hsct). severe sos/vod rapidly evolves in multiple organ dysfunction syndrome (mods), associated with a mortality rate exceding 80%. precocity of defibrotide (df) treatment is the leading factor for efficacy. prophylactic use of df is recommended in children, but its value has not been validated in the adult population, although factors for individual risk assessment for vod are debated. we here present a real-world experience of df prophylaxis in adult patients at very high risk of sos/vod receiving allogeneic hsct. methods: from 2016 to 2018 we treated with prophylactic defibrotide and ursodeoxycholic acid (udca) 11 patients, median age 30 years (range 21-59). nine patients received allogeneic hsct for acute lymphoblastic leukemia (8 b-all and 1 t-all), one patient for severe aplastic anemia, one patient for primary myelofibrosis. they were all at high risk for sos/vod because of previous hepatotoxicity (3 patients), previous hsct (6 patients), double alkylating agent (5 patients) or previous treatment with inotuzomab ozogamicin (io; 7 patients). of the 7 patients treated with io, 6 received 2 cycles of io, and 1 received 1 cycle, with the last io dose administered a median 41.5 days before hsct (range 34-61 d). defibrotide was administered in 4 daily doses for a total dose of 25 mg/ kg per day and udca at the dose of 300 mg twice per day, starting from day -6 prior transplant. all patients received treosulfan-fludarabine based conditioning. in 5 patients thiotepa was added to the conditioning and in 2 patients a low dose 4gy tbi. gvhd prophylaxis included posttransplant cyclophosphamide, rapamycin and mycophenolate in all patients, except one patient with aplastic anemia receiving atg, rapamycin and mycophenolate. donor source was pbsc in all cases. seven patients received family haploidentical (mmrd) transplant, 1 patient a mrd transplant and 3 patients a mud transplant. results: the median duration of defibrotide therapy was 34 days (range 32-64 days). documented non-severe gastrointestinal bleeding occurred in 2 patients requiring defibrotide temporarily discontinuation, no other significant bleedings were experienced. four patients developed grade ii-iv acute gvhd and no transplant-associated thrombotic microangiopathy were diagnosed. overall, sos/vod occurred in 3/11 cases within 21 days after hsct (days 9, 10 and 13) and no late-onset sos/vod were diagnosed. sos/vod was very severe, causing mods and death in all 3 cases. all 3 patients were characterized by a common pattern of very high risk factors for sos/vod by prior hsct and salvage treatment for b-all with 2 cycles of io close to hsct. furthermore, they all received a fully myeloablative conditioning regimen with treosulfan and thiotepa and a mmrd transplant. conclusions: defibrotide prophylaxis was safe and well tolerated with no severe related complications. sos/vod occurred despite continuous df prophylaxis in 3/7 patients treated with inotuzomab ozogamicin close before undergoing 2 nd transplant. to reduce the incidence of severe vod, pre allo-hsct treatment with inotuzomab ozogamicin should prompt avoidance of other cumulative risk factors for vod, such as use of double alkylating agents. disclosure background: busulfan is the backbone of many preparative regimens administered to children undergoing allogeneic and autologous hematopoietic stem cell transplantation (hsct). among its many long-term adverse effects, busulfan can cause various degrees of pulmonary injury. although well described in adults, there are few large series exploring pulmonary toxicity of busulfan in children. we describe long-term pulmonary follow-up in a large group of children treated at a single center who had received high-dose busulfan and examine the relationship of systemic drug exposure and lung function over time. methods: all surviving children who had received highdose busulfan between 1993-2013 in the context of hsct at the schneider children´s medical center, were referred for serial pulmonary function monitoring (including spirometry, plethysmography and diffusing capacity for carbon monoxide [dlco] . pre-transplant testing was available for children who were old enough to perform the procedure. spirometry results were adjusted according to the revised global lung initiative formulas for age, gender, and height. pulmonary injury was defined as a z score below -1.96 for spirometry, or < 80% of predicted for the other parameters. busulfan levels were monitored following the second drug dose. all patients received busulfan in four daily doses. area under the curve (auc) calculations were performed by bayesian calculations. results: between 1993-2013, 263 patients aged 0-18 years were diagnosed with malignant or non-malignant diseases and treated with high-dose busulfan. of 130 shortterm survivors, 75 had at least one post-transplant pulmonary function evaluation. the mean age at treatment with busulfan was 7.9 years (range, 0.4-27 years). of these 75 children, 22 children had undergone autologous transplantation and 53 children had an allogeneic transplant. 7 of these patients eventually relapsed and 3 died. 26 children had one or more pulmonary risk factors before hsct -chest or upper abdomen radiation (13), chest wall tumors or lung metastasis (8), chest surgery (5), prior administration of pulmonary-toxic drug (3) or asthma (2). during follow-up (up to 14 years, median 5.5 years), fev1 and fvc spirometry tests both decreased significantly (p=0.002 and 0.001, respectively), while the decrease in dlco was not statistically significant. 35% of patients had abnormal pulmonary function tests and seven children had symptomatic disease which in two may have been manifestations of gvhd. interestingly, no correlation was found between busulfan auc, busulfan peak levels, the number of busulfan doses administered, the type of transplantation (autologous vs. allogeneic) or primary disease to pulmonary injury. even after censoring of children with pre-transplant pulmonary risk factors we noted a decrease of fev1 and fvc. conclusions: as in adults, pulmonary injury is observed in children treated with high-dose busulfan prior to hsct. no correlation was observed between busulfan auc and pulmonary injury. follow-up of children who receive this drug should include regular pulmonary monitoring, referral to a pulmonologist when subclinical pulmonary compromise is found, and counseling regarding measures that might prevent or ameliorate pulmonary damage. continued follow-up of this cohort of patients should inform our pretransplant patient information sessions, and the future use of busulfan in children. disclosure: nothing to declare background: transplant-associated thrombotic microangiopathy (ta-tma) is a specific complication of allogeneic hematopoietic stem cell transplantation (hsct). post-hsct tma has been attributed to the vascular endothelial damage caused by high-dose chemotherapy, calcineurin inhibitors (cnis), graft-versus-host disease (gvhd), infections. there is a little evidence published regarding the efficacy and factors influencing the outcome of withdrawal of cnis. methods: the analysis comprised a total of 54 patients, with diagnosed hematologic malignancy (aml (16), all (12), mds (4), hodgkin lymphoma (5), cml (3) and neuroblastoma (1) received allo-sct, from a matched related, unrelated or haploidentical donor between 2007 and 2018. patients were diagnosed with ta-tma based on cho criteria. the median age of patients was 23 (37 adults, 17 children). gvhd prophylaxis was performed with tacrolimus (tac) in 42, cyclosporine a(csa) in 11, combination tacrolimus+sirolimus (sir) in 12. 24 patients received atg and 26 ptcy. withdrawal of cnis was accompanied by administration of systemic steroids (21 patients) or substitution with sir after reaching levels of csa< 100 ng/ml or tac < 3 ng/ml in 13. the target concentration of sir was 3-9 ng/ml. in pediatric patients who received combination tac+sir, the tac was discontinued in one step while sir continued. median time to development tma was 31,5 days after allo-sct (range 1-408). median follow-up of surviving patients was 395 days. the primary outcome was overall survival (os) up to 2 years after development of ta-tma. results: the following significant predictors of 2-year os were identified: tac replacement with sir (p< 0,001), ptcy in prophylaxis (p< 0,001), acute gvhd (agvhd) grade 2-4 (p=0,029), previous sepsis (p=0,003), level of ldh in debut (p=0,001), combination sir+tac in prophylaxis (p=0,05), major ab0-mismatch (p=0,022), severity of cns symptoms (p< 0,001). there was no significant difference in os according to patients' age, sex, "salvage" disease status at transplantation, previous vod, viral (hhv 1,2,6, cmv, ebv) reactivations, count of cd34+ cells transfused, ldh level, shizocytes and creatinine in the debut of ta-tma. in the multivariate analysis replacement of cnis with sir (hr 0.27, 95%ci 0.08-0.96, p=0.018) and baseline ldh level (hr 1.01, 95%hr 1.00-1.01, p=0.029) were associated with survival differences. the cut off for ldh was 4xunl. agvhd grade 2-4 (hr 1.96, p=0, 081) and use of ptcy (hr 0.535, p=0.317) were not significant in the multivariate analysis (figure 1). ta-tma cases after ptcy were significantly less frequently associated with clinically significant agvhd (19% vs 68%, p< 0,001). the survival was higher after ptcy (74% vs 15%), but not significant due to sample size and other ta-tma factors. leading causes of death were: gvhd progression (11%), bacterial infection (11%), tma (17%) and other (19%) . conclusions: replacing tac by sir is an effective therapeutic strategy in a group of patients with debut of ta-tma at least after ptcy, where it is less likely to be associated with agvhd. there is a significant overlap of populations with ptcy prophylaxis and substitution with sir, thus the study is not powered to provide guidance for patients on conventional prophylaxis with ta-tma. [[p172 image] 1. disclosure: none of the authors has anything to disclose. donor-recipient ab0 mismatch effect on the allogeneic hematopoietic stem cell transplantation outcome: a single-center retrospective study background: because transmission of major histocompatibility complex and blood group system genes is independent from each other, approximately 40-50% of all allogeneic hematopoietic stem cell transplantations (allo-hsct) are realized crosswise the ab0-blood group boundary. however, due to the widespread expression of ab0 antigens on a variety of human tissues other than erythrocytes, ab0 incompatibility may have an impact on the outcome of allogeneic hsct that goes beyond the wellknown immune-hematological complications such as immediate hemolysis due to the presence of isoagglutinins and delayed hemolysis due to passenger b lymphocytes. here we aimed to assess the donor-recipient ab0 mismatch effect on the allo-hsct outcome, comprising non-relapse mortality (nrm), overall and relapse-free survival, posttransplant prc transfusion requirement, as well as relapse rate, incidence of graft-failure and acute gvhd. methods: clinical and laboratory data from 180 consecutive patients undergoing allogeneic hsct between 01/2008 and 06/2018 at the fondazione irccs ca' granda maggiore policlinico hospital in milan, italy, were retrospectively collected. kaplan meier estimates were used for the analysis of survival outcomes while nrm, relapse and acute gvhd cumulative incidences were investigated by competing risk analysis. results: the patient series included 105 ab0-match, 31 major ab0-mismatch, 34 minor ab0-mismatched and 10 bidirectionally ab0-mismatch transplants. indication for allo-hsct were mainly aml/mds (77 pts), all (34 pts) and t-nhl/ctcl (32 pts). mean overall survival for groups of patients undergoing ab0-identical, major ab0 mismatch and minor ab0 mismatch hsct were 66 months (95% ci [55 ;77]), 47 months (95% ci [28; 65) and 46 months (95% ci [31; 61]), respectively. nrm in the three groups were significantly different, with point estimates of 12%, 29% and 26% at 5 years, respectively, whereas no significant differences were observed for relapse rate and graft failure incidence. although not statistically different, incidence of acute grade iii-iv gvhd was twice as high in patients transplanted from minor ab0mismatched donors than in the ab0 identical group (16% vs 8%). following transplantation, prc transfusion requirement was significantly higher in the major ab0 mismatch then in the ab0-match transplanted patients (median 14 vs 7, p=0.01), with a marginal positive correlation between the anti-donor a/b igg titers measured prior hsct and the total number of prc transfused during the first year following transplantation. we observed only one case of prca occurring in a 50year-old 0+ woman who was transplanted from a 32-yearold male a+ hla-identical sibling using peripheral blood as the stem cell source following a myeloablative conditioning for aml in first complete remission. anti-a igg isoagglutinin titers prior to transplantation were 1:256. during the first year post transplantation, the patient required a total of 46 prc transfusions, with gradual resolution occurring only after introduction of danazole treatment. conclusions: in our patient cohort, both major and minor ab0 mismatch associated to a significantly higher nrm. major ab0 mismatch associated to a higher prc transfusion requirement. a more frequently occurring severe acute gvhd was also suggested in minor ab0-mismatch transplants. altogether, our results suggest that allo-hsct outcome may be significantly affected by ab0 blood group mismatch. disclosure: nothing to declare background: at-tma is a severe endothelial injury complication and it may involve the intestinal vasculature. intestinal tma could be fatal and missdiagnosed. clinical and pathological criteria to differentiate from intestinal gvhd are needed. the aim of this study was to analyze the incidence and histological characteristics of intestinal tma in patients diagnosed of systemic tma. methods: we analyzed the incidence of tma in 555 patients who underwent allo-hsct in our institution between january 2010-august 2018. tma diagnosis was based on ho criteria. we do a pathological review in 103 biopsies from 25 out of 52 patients in whom an endoscopy have been performed 30 days before and 60 days after the diagnosis of tma for suspicious of gvhd. review was performed by a pathologist expert in gvhd, who examined the biopsies in search of hystopathological features of gvhd, tma or viral infection. diagnosis of gastrointestinal gvhd was stablished according to mcdonald and sales criteria, while intestinal tma diagnosis was stablished by warren et al criteria. results: 52 out of 555 patients (9,4%) were diagnosed of tma. transplant characteristics and tma data of patients with systemic tma are shown in image. 47 out of 52 patients with tma (90%) had been diagnosed with prior/ simultaneous acute gvhd, 20 of them grade iii-iv, and 80% with gastrointestinal gvhd. intestinal tma have been reported only in 7 out of 25 patients (28%) at diagnosis, whereas when review based on warren criteria was performed, in 19 patients (76%) the pathologist found at least 1 of the criteria of endothelial damage and 48 % of the patients 3 or more warren criteria were founded. the most frequent features were endothelial cell swelling (n=16, 64%) and perivascular mucosal hemorrhage (n=15, 60%). review hystological features of biopsies are shown in table 3 of the image. regarding gvhd, it was found in 21 patients (84%) at diagnosis and in 23 (92%) at pathological review. with a median follow-up of 10 months (1-73) 32 patients of the 52 with systemic tma (62%) are dead. 9 of the deaths (41%) were related to tma (3 tma, 3 tma +gvhd, and 3 tma+infection). patients with 3 or more warren criteria in pathological review had poor outcome compared with patients less than 3 criteria (30% alive vs 51% at 12 months, p=0.9). conclusions: intestinal tma is a life-threatening underdiagnosed entity. only 7 patients of 25 patients were diagnosed of intestinal tma. we found that most of our patients had endothelial damage in the gastrointestinal biopsy pathological reviews. gvhd histological criteria were present in most of the patients, mainly histological grade i-ii. prognosis of these patients is poor and pathologist effords in diagnosed the entity is guarranted. disclosure: nothing to disclosure p175 strategies to reduce neutropenic fever and hospital readmission in multiple myeloma patients managed at home after autologous stem cell transplantation background: neutropenic fever (nf) is the most frequent cause of readmission in the outpatient autologous stem cell transplantation (asct) programs. in our at home model for multiple myeloma patients, we added primary prophylaxis with ceftriaxone, decreasing the incidence of fever during aplasia phase from 85% to 57.6%. the aim of this study was to analyze the addition of two strategies to reduce the non-infectious nf: withdrawal of g-csf and the addition of primary prophylaxis for engraftment syndrome with corticosteroids after asct. methods: between january 2002 and august 2018 111 myeloma patients were managed at-home since day +1 of asct. all were conditioned with mel200. all patients received prophylaxis with quinolone, fluconazole, aerolized pentamidine, low-dose acyclovir (hvs+), and ceftriaxone (since day +4). the patients were classified into 3 groups: group a (n=33; g-csf since day +7 without corticosteroid), group b (n=32; no g-csf and no corticosteroid), group c (n=46; no g-csf with prednisone 0.5 mg/kg/day since day +7 until granulocyte recovery). first-line therapy at home of nf was piperacillin-tazobactam 4.5 g/6h i.v. using a portable intermittent infusion pump. fever was an indication of immediate medical consultation and those patients presenting signs of focal infection or severe sepsis were admitted. other indications for readmission were: willingness of the patient or caregiver, uncontrolled nausea, vomiting or diarrhoea, and mucositis requiring total parenteral nutrition or i.v. morphics. results: the main characteristics of the patients and outcomes are shown in table 1. there were no differences between groups regarding age, gender, immunological subtype, response before asct, hct-ci, and cd34 cell dose infused. there were more patients with advanced disease (iss iii) in group c compared to group a (19.5% vs. 6.1%; p=0.003). the duration of neutropenia was longer in those groups that did not receive g-csf (a: 8 days, b: 11 days, c: 10 days; p< 0.01). comparing group a with group c, we observed that the incidence of nf and the readmissions rates were lower in group c (nf: 57.6% vs. 23.9%; p=0.002; relative risk reduction: 0.41, and number needed to treat 2.97; readmissions: 12.1% vs. 2.2%; p=0.07, respectively). the 10-day cumulative incidence of nf were 54.5% in group a, 40.6% in group b, and 23.9% in group c; p=0.009. the non-administration of g-csf with the addition of prophylactic corticosteroid did not modify the incidence and grade of mucositis, the first day and duration of fever, nor the number of bacterial infections documented. in the multivariate analysis, this combination (no g-csf with corticosteroid) maintained its protective effect for the development of nf and hospital readmission (or 0.07; p=0.001 and or 0.07; p=0.05, respectively). conclusions: the non-use of g-csf and the addition of prophylactic corticosteroid in mm patients managed at home after asct minimize the incidence of non-infectious fever and optimize hospital resources by reducing hospital readmissions. disclosure: nothing to declare. background: antibody titers to vaccine-preventable diseases decline during the 1-10 years after allogeneic hematopoietic stem cell transplantation (hsct) if the recipient is not revaccinated. it is therefore considered best practice to try to offer hsct recipients the same level of protection against all vaccine preventable diseases as the general population. few data in the literature are available concerning vaccine-related problems in hsct recipients. we performed a farmacovigilance evaluation in a cohort of allotransplanted patients followed in our clinic during a 1 year period. methods: from october 2017 to november 2018 we administered a list of recommended vaccines to 49 hsct recipients attending our routine out patient clinic who fulfilled the following criteria: cd4 t cells>200/μl, cd19 b cells>20/μl, anti-cd20 antibody infusion>6 months, ivig therapy>2 months, no active and severe graft-versus-host-disease (gvhd), no chemotherapy or biological therapeutic agents on going. vaccines suggested were influenza, pneumococcal conjugate (pcv13), polio (inactivated polio vaccine), diphteria, tetanus, acellular pertussis, hepatitis b, hepatitis a, haemophilus influenzae type b, meningococcal quadrivalent (mcv4), human papillomavirus, meningococcal b, measles-mumps-rubella (mmr), varicella. live vaccines (mmr and varicella) were not recommended before 2 years after hsct and in patients with chronic gvhd. all the patients were asked to take the list to the local health facilities in order to have the vaccines injected and a vaccination table arranged with the doses already received and those to receive. we checked the vaccination tables at each visit and monitored potential side effects and gvhd status at 3, 6, and 12 months after the first vaccine injection. results: twenty-nine out of 49 patients were evaluable (table 1), 16 without gvhd and 13 with chronic gvhd (5 mild, 4 moderate, 4 severe). median time after hsct was 34 months (16-240). median number of vaccines received was 8 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) . as regards patients without chronic gvhd, 2 out of 16 experienced fever after vaccine injections; 1 out of 16 developed transient mild reduction of platelet count; 1 patient reported headache and otalgia after vaccine injection, while another one transient joint pain; 1 out of 16 patients presented signs of mouth chronic gvhd (score 1 nih) and transaminase increase (grade 1 according to world health organization toxicity scale) 3 months after the first vaccine dose, so that cyclosporine dose had to be augmented. as regards patients with chronic gvhd, 4 out of 13 experienced fever after vaccine injections; 2 patients with mild chronic gvhd of the mouth presented hepatic flare two and three months after the first vaccine dose, respectively. in both cases a new increase of cyclosporin and methylprednisolone doses determined progressive normalization of liver enzymes. conclusions: these data show that vaccines were globally well tolerated in hsct recipients, even when they suffered from chronic gvhd. however, close monitoring is warranted in order to better evaluate possible vaccine side effects in this setting of patients. background: allogeneic hsct improves survival for aml patients over the age of 60 years of age when compared to chemotherapy alone. the haematopoietic stem cell transplantation comorbidity index (hct-ci) and ebmt score predict for non-relapse mortality and overall survival, yet little is known about whether qol is preserved in this patient group and whether hct-ci and other performance scores pre-bmt correlate with qol post allo-hsct. methods: we conducted a retrospective analysis of patients 60 years and older who underwent ric allo-hsct at the university hospital of wales, cardiff between september 2011 and december 2017 (n=41). hct-ci, karnofsky performance score (kps) and ebmt scores were calculated prior to transplant and qol measured using the fact bmt (version 4) questionnaire, which was completed at 3, 6 and 12 months post transplant. patients were grouped at the 3-, 6-and 12-month time points for each of the different performance indices, allowing group comparison against compound sub scores using the mann-whitney u test. results: 41 patients were included in this study, with median age 65 years (range 60-74). patient characteristics, including conditioning, donor type, pre-transplant hct-ci and kps scores are summarised in table 1. the 2 year and 5 year overall survival (os) for the patient cohort was 65.4% and 48.7% respectively. hct-ci of ≥3 vs 0 was significantly associated with poorer bmt-related qol domains at 3 months (p=0.035) and general qol domains at 6 months (p=0.025) post-transplant. while ebmt score showed no correlation with qol parameters, patients with kps of 100 vs ≤90 showed significant differences in both general (p=0.01) and bmt-related qol (p=0.04) at 3 months and in all qol domains at 6 months (symptomrelated qol p=0.05, general qol p=0.01, bmt-related qol p=0.01). importantly neither the hct-ci nor the kps pre-transplant predicted for qol at 12 months post transplant. conclusions: patient selection is key to ensuring maximum benefit from allo-hsct both in terms of overall survival but also with regards to qol and survivorship. we note that while patients with hct-ci ³3 or kps ≤90 had significantly poorer qol at 6 months post allo-hsct, qol was recovered by 12 months post transplant, with this significant difference no longer seen. our data shows that in selected aml patients over the age of 60 years with good performance status and low comorbidity index, a favourable outcome can be achieved with good qol maintained throughout the post transplant period. background: advances in allosct technology, supportive care, and use of reduced intensity conditioning regimens for older patients have led to significant improvements in longterm survival after transplant. the survivors have an elevated probability of late morbidity and mortality, including abnormalities in phosphocalcic metabolism and bone disease. rapid and progressive bone loss occurs within the first 6-12 months after transplant, and this is followed by a slow process of recovery, with bone loss persisting for 48 to 120 months. bone fractures can worsen the quality of life of allosct survivors, but the real burden of the disease is unknown. the objective of the study is to ascertain the prevalence of bone pathology and vertebral fractures early after transplant in our center. methods: this is a retrospective and observational study. forty-nine patients (25 male/24 female, median age 54y, range 19-69) that underwent allosct were included in the study in the period of 6 to 24 months after transplant (may 2016-december 2017). pre-and post-transplant risk factors associated with bone disease were recorded: age >65 years, female sex, menopause, hormone replacement therapy, previous treatment with steroids, previous fractures, weight < 40 kg, bmi < 20-25, low physical activity, low calcium intake, smoking, alcohol intake, and history of femoral fractures in parents. in all patients laboratory data (including serum calcium, 25-hydroxyvitamin d, and pth), lumbar and femoral bmd (dxa), and spinal x-ray were also evaluated. a vertebral fracture was defined as a reduction of >20% in the anterior, middle or posterior high of the vertebral body. results: we identified vertebral fractures in 12 (24%) patients. five patients had fractures prior to transplantation, and 7 patients presented "de novo" vertebral fractures following transplantation; therefore, the prevalence of "de novo" postransplant fractures was 7/49 (14%). most (85%) of these fractures were asymptomatic at the time of diagnosis. most patients (64%) with vertebral fractures had >3 pre-sct risk factors (median risk factors pre-sct 3, range 2-6), the most frequent being low calcium intake, steroid exposure, presence of previous fractures, and menopause. those patients with fractures and less than 3 risk factors pre-tph, added new risk factors after transplant, mainly steroid treatment. forty-four patients (90%) had vitamin d insufficiency (< 30ng/ml), 15 (32%) had osteopenia and 9 (18%) had osteoporosis. vitamin d insufficiency and bone disease were more frequent in women than in men (98% vs. 84% for vitamin d, 37% vs. 28% for osteopenia, 29% vs. 8% for osteoporosis, and 25% vs. 20% for vertebral fractures, respectively). conclusions: the prevalence of post-transplant bone disease and vertebral fractures in our series is high. most fractures appearing "de novo" after allosct were asymptomatic and were diagnosed by x-ray. patients who presented vertebral fractures frequently had more than 3 risk factors identified pre-sct. patients undergoing allosct should have their bone health assessed early in their treatment and, if indicated, should start preventative therapy to avoid bone loss and fractures. other measures such as physical exercise, vitamin d and calcium supplementation, and dxa and spinal x-ray at baseline and following transplantation are also highly recommended. disclosure: maría suárez-lledó received a grant from dkms-spain foundation. other authors have nothing to declare the use of g-csf in selected patients after autologous stem cell transplantation is associated with low incidence of engraftment syndrome background: the use of g-csf after autologous stem cell transplantation (asct) accelerates neutrophil recovery, however it has been related to an increased risk of engraftment syndrome (es) development in some studies. for this reason, we do not routinely prescribe g-csf after asct and we only use it in patients with significant complications (enterocolitis, severe sepsis, atrial fibrillation) after stem cell infusion. the main objective of this study is to evaluate the incidence of es in patients who receive asct for monoclonal gammopathies (mg), non-hodgkin lymphoma (nhl) and hodgkin lymphoma (hl) and receive g-csf only if needed. as secondary objectives we evaluate differences in the engraftment day as well as the length of inpatient stay. methods: we retrospectively analyzed patients with mg or lymphoma, who underwent asct conditioned with high dose melphalan (140-200 mg/m 2 ) or beam, respectively, between 2015 and 2017 in our center. specific clinical features for es according to spitzer and maiolino criteria were evaluated between 3 days before and 7 days after the engraftment. statistical analysis was performed with spss v. 15.0. results: thirty-one patients with mg and 34 patients with lymphoma were analyzed. median age at transplant was 56.8 years (48.7-65.4 ) and 41 patients (63.1%) were male. median prior lines of treatment in patients with gm or lymphoma were 1 (1-3) and 2 (1-5), respectively. table 1 shows patients´characteristics. mobilization with g-csf ± plerixafor was performed in 27 patients (36%) and chemotherapy + g-csf ± plerixafor in 38 patients (64%). median cd34 x 10 6 /kg cells infused was 3.6 (2.7-5.3). eleven patients (16.9%) received g-csf, 5 due to infection (2 enterocolitis, 1 listeriosis, 1 acute hepatitis, 1 septic shock) and 6 because of atrial fibrillation or fibrilloflutter. median time from sct to first day of g-csf was 5 days (5-7) and median time on g-csf treatment was 5 days (4-7). patients who received g-csf showed a short time to neutrophil engraftment (≥0.5x10 6 /l), 10 days vs. 13 days, p< 0.001 but longer duration of hospitalization, 18 days vs. 15 days, p =0.050. non-relapse mortality at day +30, +100 and +180 was 0%. es was diagnosed in 4 (6.2%) patients, 1 amyloidosis, 2 multiple myeloma and 1 plasmablastic lymphoma. there was not statistical difference in the incidence of es between patients who received g-csf (9.1%) and patients who did not (5.6%), p=0.533. analyzed by disease, es appeared in 1 of 6 patients who received g-csf in the lymphoma group (16.6%) but none of the 7 patients with mg that received g-csf developed it. we did not find statistical differences between patients who developed es and those who did not in age (49 years vs. 56 years, p=0. 314), length of hospitalization (19 days vs. 15 days, p=0.185) and the number of cd34 x 10 6 /kg cells infused (3.65 vs. 4.62, p=0.408) . conclusions: the use of g-csf in selected patients is associated with low incidence of es. our study confirms that the use of g-csf accelerates neutrophil recovery but it is unclear if it can increase the incidence of es, especially in patients with lymphoma. [[p180 image] 1. background: graft failure is one of the top-3 problems of allo-hsct (after gvhd and relapse). the problem of graft failure becomes more significant due to increasing number of allo-hsct with ric conditioning regimen from haploidentical and hla-mismatched unrelated donors. role of t cells in graft failure is well known. here we report an impact of t-memory cell subsets count before antithymocyte globulin (atg) administration on primary graft failure after allo-hsct. methods: sixteen patients with acute leukemia transplanted in national research center for hematology were included on this prospective study. all patients received horse atg at dose 10 mg/kg/day from day -4 to -1 before allo-hsct as gvhd prophylaxis and were balanced by other factors that could affect engraftment. detailed patients characteristics are listed in table 1 . peripheral blood samples were collected on day -4 before allo-hsct (before atg injection) in edta-tubes. flow cytometry analysis was performed on bd facs canto ii (becton dickinson, usa) to define t-memory subsets: t-naive and t-stem cell memory (tnv+scm) -cd45r0-ccr7+cd28+; t-central memory (tcm) -cd45r0+ccr7+cd28+; t-transitional memory (ttm) -cd45r0+ccr7-cd28+; t-effector memory (tem) -cd45r0+ccr7-cd28-; t-terminal effector (tte) -cd45r0-ccr7-cd28-, among cd4+ and cd8+ t-cells . sysmex xe-2100 was used to calculate absolute count of different t-cell subsets. mann-whitney u test was used for nonparametric data analysis between two groups. fisher's exact test was used for 2x2 tables. p-value less than 0.05 was considered statistically significant. results: an influence of t-memory cell subsets count before atg administration on primary graft failure is shown in figure 1 . according to our data high absolute number of cd4+ttm and cd4+tte is associated with primary graft failure. conclusions: based on these findings high absolute number of cd4+ttm and cd4+tte could be one of the prognostic factors of primary graft failure after allo-hsct. optimizing atg dose due to recipient absolute t-memory cell subsets count before atg administering may prevent graft failure and improve posttransplant results. background: upper gastrointestinal graft-versus-host disease (gi gvhd) has been an increasingly recognised entity following allogeneic stem cell transplantation (sct). budesonide, widely used in inflammatory bowel conditions, has also been found beneficial in gi gvhd. the major benefit of budesonide is attributable to its poor absorption and extensive first-pass metabolism via cytochrome p450 (cyp) 3a4, which translates to less systemic steroid-related effects. however, transplant patients are often exposed to multiple drugs, among which some agents act as cyp3a4 inhibitors and therefore can increase budesonide bioavailability and might lead to systemic toxicity. azole antifungal drugs are probably the most common concomitantly used cyp3a4 inhibitors in transplant recipients. methods: we reviewed allogeneic sct records for patients treated with oral budesonide for gi gvhd at our transplant centre between 2015 and 2018 retrospectively. the aim of the work was to assess the development of adrenal suppression with or without clinical features of iatrogenic cushing`s syndrome. the standard dose of budesonide was 3 mg three times a day. patients receiving prednisolone or other glucocorticosteroids and those with no available serum cortisol level measurements were excluded. results: our analyses identified four allogeneic sct patients in whom adrenal suppression was diagnosed with undetectable serum cortisol levels during oral budesonide treatment. of these patients two developed iatrogenic cushing`s syndrome and both patients were treated with cyp3a4 inhibitors concomitantly: 1. clarithromycin and fluconazole; 2. clarithomycin and voriconazole. the development was rapid (within 3 and 4 weeks). symptoms included morphological features such as moon face, high blood pressure, weight gain, peripheral oedema and proximal myopathy. symptoms resolved gradually following cessation of azole antifungal agents and on gradual weaning of budesonide. conclusions: although single agent budesonide treatment given for gi gvhd is rarely associated with systemic side effects, patients on azole antifungal drugs and macrolide antibiotics are at higher risk of systemic toxicity due to drug interactions. patients who are allergic to penicillin and receive macrolide-based prophylaxis can be especially vulnerable. to our knowledge the number of cases reported in literature about systemic effects of oral budesonide in transplant recipients is less than 10. our observation supports previous reports on the potential of oral budesonide to induce systemic effects. we therefore advise careful monitoring of patients treated with budesonide in combination with cyp3a4 inhibitors, including antimicrobial agents routinely used in sct. disclosure: none implemented strategies to overcome barriers in the establishment of a consolidated hematopoietic stem cell transplantation program in a developing country background: the national institute of medical sciences and nutrition "salvador zubiran" is a national health institute located in mexico city. although mexico is considered an upper-middle income country, more than 50% of the population lives in poverty without health care coverage and patients within this social stratum are referred to our institution. the first hematopoietic stem cell transplantation (hsct) in mexico was performed at our institution in 1980. from this year until 1997, hsct were sporadically performed (n=33), showing a poor overall survival (os) and high non-relapse mortality (nrm). these outcomes resulted from an unstructured hsct program, limitedresources, patient low socioeconomic status, and paucity of population-adapted procedures. in 1998, according to these results, a decision to establish a hsct program was made. therefore, in order to set up a successful hsct program, implementation of financial and medical strategies were necessary. the objectives of this study were to describe the barriers and implemented strategies for the establishment of a hsct program in mexico along with the outcomes of patients undergoing this procedure throughout the reorganization of the program. methods: this study is a health services research. barriers were detected based on the results of the hsct program from 1980-1997 (not shown). table 1 shows the financial, medical, and research strategies that were implemented for each barrier. results: from november 1998 to november 2018, 363 hsct have been performed in 322 patients at our institution. most hsct were autologous (n=213, 59%). forty one patients underwent 2 hsct. from the 322 patients, most were males (n=196, 61%) and the median age was 33.5 years (range, 15-65). the most frequent underlying diseases for auto-hsct were lymphomas (n=68, 36%), non-seminomatous germ cell tumors (n=42, 22%), and multiple myeloma (n=42, 22%). acute leukemias (n=41, 34%), aplastic anemia (n=25, 21%), and myelodysplastic syndromes (n=20, 17%) were the most frequent diagnosis for patients undergoing allo-hsct; and acute leukemia was the most frequent diagnosis for patients undergoing haploidentical hsct (n=10, 83%). acute and chronic gvhd were present in 25% (grades i-ii 89%) and 35% (limited 76%), respectively. for allo-hsct, 30, 100day, and 1-year nrm was 2.5%, 8%, and 12%, respectively; 30 and 100-day nrm in auto-hsct was 1.5%; 10year os was 63% and 56% for auto and allo-hsct, respectively. conclusions: future perspectives of the hsct program include the acquisition of funds for unrelated donors; to improve outcomes of patients undergoing haploidentical hsct, and to increase the number of in-patient rooms. we conclude that despite paucity of resources and other limitations, the implementation of financial, medical, and research strategies have shown that barriers can be effectively overcome in a developing country in order to establish a consolidated and nationally renowned hsct program, providing good outcomes for patients. disclosure: none of the authors have any conflict of interest to disclose. the effect of protective buffering on daily stress and relationship quality in dyads following hematopoietic stem cell transplantation: results from daily process methodology malgorzata sobczyk-kruszelnicka 1 , aleksandra kroemeke 2 , zuzanna kwissa-gajewska 2 , sebastian giebel 1 background: cancer-related support communication (e.g., protective buffering) may impact the risk for psychological and relationship distress in patients following hematopoietic stem cell transplantation (hsct) and their caregivers. previous studies have revealed that protective buffering (i.e., hiding one's concerns and denying one's worries) has mixed effects: is beneficial (for "protected" person), costly (especially for the person using it), or unrelated to dyadic wellbeing. there has been, however, little evidence linking dyadic protective buffering with distress using daily process methodology. we assessed (1) the relationship between daily protective buffering, and same-and next-day stress and relationship quality in patient-caregiver dyads following hsct and (2) whether similarity or complementarity in protective buffering between dyads is adaptive. methods: two hundred patients (after first autologous or allogeneic hsct) and their caregivers (spouse or another relative) independently completed measures of daily protective buffering, daily relationship quality, and daily stress for 28 consecutive evenings after patients´hospital discharge. actor-partner-interdependence model (i.e., both partners' and caregivers' reports regarding support communication and distress were studied) was used to test study hypotheses. results: for both patients and caregivers, multilevel structural equation modeling showed a significant positive relationship between daily protective buffering and sameday relationship quality. association of protective buffering with same-day stress level was negative. in next-day analyses, patient-reported protective buffering was related to patient's higher relationship quality, whereas caregiverreported protective buffering increased patient's daily stress. complementarity in protective buffering was related to higher immediate same-day relationship quality for both patients and caregivers, while benefits from similarity have delayed effects, although only in patients. conclusions: contrary to previous studies, protective buffering rather has a beneficial effect in dyads following hsct. protection of the partner and relationship against revealing negative emotions and powerlessness was not related to costs in both parties. the findings suggest that the effect of daily protective buffering in dyads following hsct depends on support timing (same-or next-day effect) and differs for both parties. patients seem to benefit the most from the similarity in protective buffering, while caregivers from complementarity. the "fit" between patient and caregiver in support communication ought to be taken into consideration in the practical approach. disclosure: nothing to declare. virus reactivation and low dose of cd34+ cell were associatied with secondary poor graft function within the first 100 days after allogeneic stem cell transplantation yuqian sun 1 , xiao-jun huang 1 background: secondary poof graft function (spgf) was defined as the secondary cytopenia after initial engraftment of hsct. it was shown to be associated with poor prognosis, however there are very few reports on the incidence, risk factors and outcomes of spgf. methods: patients who received transplantation from peking university people's hospitial during january, 2015 to december, 2015 were retrospectively reviewed if they fulfilled the following conditions: (1) diagnosed with acute leukemia or myelodysplastic syndrome; (2) received allo-sct from either matched sibling donor (msd) or haploidentical related donor (hid). pgf was defined as persistent neutropenia (≤0.5×10 9 /l), thrombocytopenia (platelets ≤20×10 9 /l), and/or hemoglobin ≤70 g/l for at least 3 consecutive days, transfusion-dependence, associated with hypoplastic-aplastic bone marrow (bm), and complete donor chimerism without concurrent graftversus-host disease (gvhd) or disease relapse. primary pgf was defined as the failure to achieve initial engraftment by days 28 after transplantation, while secondary pgf was defined as the fulfillment of the criteria after initial engraftment hsct. results: during january, 2015 to december, 2015, 564 patients who received transplantation from peking university people's hospitial were retrospectively reviewed. among the 490 patients who achieved initial engraftment, 28 patients developed spgf. the cumulative incidence of spgf on day 100 was 5.7%. the median time of secondary pgf was 54.5 (34-91) days after transplantation. low (< median) cd34+ cell dose (p=0.019, hr 3.07(95%ci, 1.207-7.813)), ebv reactivation (p=0.009, hr 3.648(95%ci, 1.382-9.629)) and cmv reactivation (p=0.003, hr 7.827 (95%ci, 2.002-30.602)) were identified as independent risk factors with spgf. there is no significant difference of pgf incidence in msd group and hid patients (p=0.44). the overall survival of patients with spgf at 1 year after transplantation was significantly poor than patients with ggf (50.5% versus 87.2%, p< 0.001). conclusions: in conclusion, spgf develop in 5.7% patients after allo-sct, especially in patients with cmv, ebv reactivation or infused with low dose of cd34+ cell. the prognosis of spgf is still poor due to lack of standard treatment. disclosure: there is no conflict of interet thiotepa with treosulfan and busulfan based conditioning are significantly more gonadotoxic than treosulfan previous studies suggest that busulfan results in long-term gonadal toxicity. no previous studies have compared gonadal toxicity outcomes after treatment with busulfan with treosulfan, a newer agent with similar marrow toxicity to busulfan but with reduced non-marrow toxcitiy. our aim was to determine whether there are differences in pubertal and fertility outcomes in paediatric patients treated with treosulfan compared with busulfan. methods: inclusion criteria were patients who had received either busulfan or treosulfan or treosulfan with thiotepa, only one hct and were aged 14 years and above in august 2018. eligible patients were reviewed in clinic as part of their routine follow-up, thus research ethical approval was not required. follice stimulating hormone, luteinising hormone, oestradiol, and pubertal history were noted. ovarian reserve was estimated in female patients by measuring serum anti-mullerian hormone (amh). male patients had serum testosterone measured and were also offered semen analysis. results: thirty-five patients met the inclusion criteria, of which twenty-five wanted to be reviewed (71%); seventeen females and eight males. mean age at hct was 13 years, mean age at review was 19 years and mean years since hct was 5 years. female patients treated with busulfan or treosulfan with thiotepa (n=14) had minimal amh and none of these patients were having regular periods. females treated with treosulfan (n=3) had normal amh and regular periods without needing hormone replacement. only four male patients opted for a semen analysis and all had significantly reduced sperm counts. conclusions: our results suggest that females treated with treosulfan have minimal (if any) reduction in ovarian reserve compared to other conditioning regimens which casue significant compromise. although this was a small study, and thus not suitable for statistical analysis, the clinical findings are marked. future studies should further investigate optimal doses of treosulfan that could be used to achieve bone marrow engraftment and limit long-term effects on fertility. disclosure background: autologous and allogenic hematopoietic stem cell transplantation (hsct) are potentially curative treatments for hematological malignancies. patients with related complications may need admission to the intensive care unit (icu) for specific therapy and organ support. mortality risk factors, supportive care and principal causes of admission in icu are described in our cohort of patients (pts). methods: we retrospectively studied 326 pts, 185 male, with a median age of 56,63 years (range: 18-73) who underwent allo-hsct in our center between july 2014 and october 2018. two hundred and twenty-seven(69,6%) pts received autologous hsct (auto-hct) and 99 (30,4%) allogenic hsct (allo-hsct); 50 from unrelated donor, 38 from identical sibling, and the remainder, mismatched related donor 11. twenty-three (7,1%) out of 326 pts were admitted in the icu in the transplant procedure admission. results: fifteen (65,2%) out of 23 pts were male with a median age of 55 years (range: 28-69). patients' baseline diseases were: multiple myeloma (34,8%), non-hodgkin´s lymphoma (26,1%), hodgkin´s lymphoma (8,7%), acute lymphoblastic leukemia (8,7%), myelodisplasic syndrome (8,7%), solid tumor (8,7) and acute myeloblastic leukemia (4,3%). fifteen (65,2%) pts received auto-hsct, 5 (21,7%) allo-hsct from unrelated donor, 2 (8,7%) allo-hsct from identical sibling, and the remainder haploidentical hsct (1) (4,3%). so, 6,6% of auto-hsct pts and 8% of allo-hsct were admitted in the icu. the median stay in the icu was 5 days (range: 1-30) and reasons for admission were: respiratory insufficiency (60,8%), septic shock (30,4%), renal insufficiency (4,3%) and multi-organic failure (4,3%). twenty-one (91.3%) pts required respiratory support with: nasal cannula or oxygen mask (c/m) (19%), non-invasive mechanical ventilation (nimv) (66,7%) and invasive mechanical ventilation (imv) (14,3%). fourteen (60%) pts needed inotropic agents for shock treatment. finally, 4 (4,5%) pts required substitutive renal therapy with hemodialysis or haemofiltration (hd/hf). eleven (47,8%) out of 23 pts died, 7 (63,6%) were male with a median age of 55 years (range: 24-64). ten of them (90,9%) needed imv and were treated with inotropic agents. all patients who required hd/hf (n=4) died. imv and treatment with inotropic agents were associated with icu mortality (or 6,5; p=0,03, or 7; p=0,008; respectively). conclusions: in our series of pts, 7,1% needed admission in the icu, presenting a mortality rate of 48% approximately. there were no differences in the prevalence of icu admission regarding hsct donor. main reason for admission was respiratory failure with imv requirement in 14,3% of pts. imv and treatment with inotropic agents were associated with icu mortality. an early identification of pts at risk of icu admission could have a beneficial impact on survival improvement disclosure: nothing to declare is there any association between thrombotic risk factors and veno-oclusive disease in childhood allogeneic hematopoietic stem cell transplantation? background: veno-oclusive disease (vod) is a major complication of hematopoietic stem cell transplantation (hsct). in some studies levels of fibrinolytic factors especially plasminogen activator inhibitor-1 (pai-1) level were found associated with vod. however, little is known about the relationship between thrombophilia risk factors and vod. in this study we aimed to investigate association of major thrombophilic gene mutations on vod in pediatric hsct patients. methods: we reviewed retrospectively 35 patients with vod who underwent hsct between 2010-2018 in ankara pediatrics and pediatric hematology-oncology training and education hospital, bone marrow transplantation unit, turkey. fifty-one patients who did not develop vod and transplanted during the study period were accepted as control group. we evaluated plasma homocysteine and lipoprotein a level, protein s and c activity and antigen levels and factor v g1691a mutation, prothrombin g20210a mutation, methylenetetrahydrofolatereductase (mthfr) c677t and a1298c mutations, plasminogen activator inhibitor-1 -675 4g/5g polymorphism before hsct. we also evaluated the patients' hospital files and noted the demographic values and complications of hsct. statistical investigations were done with spss statistics 17.0 for windows and p< 0.05 has been accepted as significant. results: there was no difference between control and vod groups as regard to age, sex, diagnosis, donor type, conditioning regimen, hsc source, and hla typing . there was no difference between the groups according to homocysteine, lipoprotein a, protein s and c activity and antigen levels. we did not find any relation between the genetic variations of thrombophilia and vod (table 1 ). in vod group there were 6 patients (17.1%) with acute graft versus host disease (agvhd) and in control group there were 7 (15.9%) patients with agvhd (p=0.046). febrile episodes were more frequent in vod group compared to the controls (respectively; n=30, 85.7% vs. n=23, 54.8%, p=0.006). 8-year overall survival was %77.1 in vod group and 100% in control group (p=0.001). disease free survival was also different between vod and control groups (respectively; 74.3% vs. 97.3%, p=0.001). conclusions: in literature there are recent studies showing higher pai-1 levels in patients with vod. however, in our study we did not find any relationship between congenital thrombophilia factors and vod. new studies with larger sample groups is necessary to better evaluate the association of congenital thrombophilia factors and vod. disclosure: nothing to declare p189 different strategies of chemotherapy-induced nausea and vomiting (cinv) prevention in hematological patients receiving an autologous hematopoietic stem cell transplantation: a single center experience ilaria cutini 1 , riccardo boncompagni 1 , chiara nozzoli 1 , antonella gozzini 1 , stefano guidi 1 , chiara innocenti 1 , massimo di gioia 1 , lorenzo tofani 1 , riccardo saccardi 1 background: despite the improvements of pharmacological control, cinv still represents a major problem in patient undergoing hematopoietic stem cell transplantation (hsct). we present here a comparison of two pharmacological strategies for preventing cinv in multiple myeloma (mm), hodgkin (hl), and non-hodgkin lymphoma (nhl) patients who received an autologous hsct in our institution. methods: from january 2015 to july 2018, we retrospectively analyzed 250 consecutive patients, median age 58 years (22-71yo) , diagnosed with mm, hl, and nhl, who underwent an autologous hsct following a melphalan 200 mg/sqm and beam/feam condition regimens, respectively. the first 122 patients received cinv prophylaxis with palonosetron i.v and dexamethasone 8 mg die (regimen a), whilst the following 128 were administered with fosaprepitant iv, ondansetron iv and dexamethasone 8 mg die (regimen b) both cinv prophylaxis was administered the day of melphalan infusion (day -1 form transplant). emesis breakthroughs were treated with alizapride and metoclopramide. nausea and vomiting were assessed through the ctcae 4.0 score system. categorical variables were compared with pearson chi-square test. results: the overall incidence of nausea was 78%, (55% grade 1, 41 % grade 2, and 4% grade 3, respectively). in regimen a was shown to be 80%, (56% grade1, 41% grade 2, and 3% grade 3, respectively) while in regimen b was 77% (54% grade 1, 41% grade 2, and 5% grade 3, respectively). pearson chi-square test did not show any differences between the 2 groups (p=0.679). the overall observed vomit was 32% (83% grade 1, 16% grade 2, and 1% grade 3). in regimen a it was (47% (84% grade 14% grade 2, and 2% grade 3), and 17% in regimen b (77% grade 1 and 23% grade 2). conditioning regimens didn't' have any significant impact on either nausea or vomit. patientsyounger then median (58 yrs), were reported to have higher incidence of both nausea, (p=0.028) not related to cinv treatment, and vomit (40% vs 24%, p=0.012). in multivariate analysis the overall incidence of nausea is related to age (younger patients have higher probability to develop nausea (or 2,282; p= 0,024) whilst the higher incidence of vomit is related to: regimen a (or 3.958; p< 0,001), previously reported nausea (or 4,506; p< 0,001), and no smoking habits (or 2,761; p=0,02). conclusions: both regimens are equally effective for nausea control however regimen b evidenced a better vomiting control. this finding is particularly relevant when the center policies include an early discharge program, therefore improving both patient's quality of life and procedure cost-effectiveness. clinical background: patients who underwent an allogeneic hematopoietic cell transplantation (hct) are challenged by medical, psychological and social complications. support groups might help hct-survivors to cope with these challenges. however, the existing literature about post-hct support groups is scarce. moreover, data on professionallyfacilitated support groups do not exist. the aim of this project was (1) to establish a professionally-facilitated support group and (2) to assess the discussed topics. methods: from 11/2013 until 6/2017 all patients who received an allogeneic hct at the adult stem cell transplantation program of the university clinic mannheim were invited to participate in a professionally-facilitated support group. additionally, spouses and life partners were invited. a theologian who is also a physician served as facilitator. he had no further function within the transplant team. the format of the group was unstructured without any rules regarding regular attendance. the facilitator did not provide topics or a curriculum. during the first year the group met every 14 days followed by a monthly schedule. from the fifth until the 39th meeting the attendance and the discussed topics were minuted by the facilitator. the content of the minutes was analysed by a combination of an inductive and a deductive approach. all participants provided their informed consent for the study. results: altogether 23 patients (female: n=10; male: n=13) and 10 spouses/life partners (female: n=9; male: n=1) participated. 13 patients (57%) and 6 spouses (60%) attended more than one meeting. among those who participated in ≥2 meetings the median time of participation was 16 months. the median count of participations was eight. 30% of the participants attended the meetings longer than one year, 9% longer than three years. there was no sex difference with respect to the frequency and the duration of participation. however, the frequency of participation decreased significantly the longer a participant was attending the meetings. during 35 group meetings the facilitator recorded 5138 thematically different contributions to the discussions divided in 37 distinct topics. these topics were grouped into 5 main categories [(a) medical topics, (b) private life and environment, (c) human relationships, (d) physical and mental condition and (e) the support group itself] and eight further categories [(1) compliance, (2) economic issues, (3) religion, (4) sexuality, (5) death and dying, (6) support and coping, (7) objectives and needs and (8) not otherwise specified issues] which could not be grouped in one of the main categories. the most frequent issues were medical topics (34%), human relationships (16%), physical and mental condition (15%), private life and environment (14%), financial issues (5%), the support group itself (4%), support and coping (4%) and objectives and needs (4%). noteworthy, death and dying (0.5%) were rare topics and sexuality was never mentioned. conclusions: to our knowledge, this is the first prospective and systematic analysis of a professionallyfacilitated support group for hct-survivors. these data might help to establish support groups and to identify psychosocial needs of patients and targets for specific support. disclosure: nothing to declare background: endothelial damage is associated with inflammatory complications that appear early after hsct, such as sinusoidal obstruction syndrome or acute gvhd. engraftment syndrome (es) is an inflammatory condition diagnosed by maiolino clinical score. potentially, es can exhibit high morbidity and mortality, especially after autologous-hsct in multiple myeloma (mm) patients since the introduction of new drugs such proteasome inhibitors and immunomodulatory drugs (imids). the objective of the present study was to evaluate if es is associated with endothelial dysfunction in patients with mm who underwent auto-hsct. methods: we included six patients with mm who received induction treatment including new drugs and consolidated their response with an autologous-hsct. we analysed comparatively the effect of incubating endothelial cells in vitro with serum samples from patients with es vs. no es. serum samples were collected before (pre), and after 5, 7, and 10 days from the transplant. an additional sample was collected at the es onset and at the discharge day (no es group). endothelial cells (hmec) in culture were exposed to media containing 20% of serum from each patient for 24h. cell growth was controlled morphologically. expression of the adhesion receptor icam-1 on the cell surface was analysed by immunofluorescence, and activation of the inflammation related p-38 mapk signalling pathway was evaluated by sds-page and western blot. results: exposure of hmec monolayers to sera from patients who developed es (onset day, n=4) resulted in an increased icam-1 expression on the cell surface, higher that the observed with sera from patients who did not develop es (discharge day, n=4) (26.4% of labelled area vs. 6.4%, respectively). in addition, in experiments with sera from patients not developing es, icam-1 expression on cells exposed to sera from day +10 was reduced with respect to the observed with sera from day +5, probably due to the corticosteroid used as a prophylaxis in our centre. this reduction was not observed in es patients. regarding phosphorylation of p-38, it was significantly higher in cells exposed to sera from es patients than in response to sera from patients who did not develop es. conclusions: the increase in the expression of the adhesion receptor icam-1 on the surface and the intracellular activation of p38mapk in endothelial cells exposed to sera from patients developing es indicates the existence of endothelial activation in association with es. interestingly, the prophylaxis of es with corticosteroid seems to be less effective in patients who developed es than in patients who did not develop this complication. these results need to be validated in a higher number of patients and modifications in additional markers of endothelial dysfunction should be investigated. disclosure: gonzalo gutiérrez-garcía: honoraria from gilead. grant from jazz pharmaceutical the other authors do not have any disclosure to comment. p192 association between uric acid levels before and after allogeneic haematopoetic stem cell transplant and transplant outcomes: a single centre experience background: uric acid (ua) is a known endogenous danger signal which activates the nod-like receptor protein (nlrp)3 inflasome.ua is released from injured cells during conditioning in allogeneic stem cell transplantation (hsct). a pre-clinical study has demonstrated that nlrp3 inflasome-mediated il-1 production regulates graft-versushost disease (gvhd). the ua role in inflammation and gvhd is unclear. there are discordant reports in the literature about a potential protective role of ua on gvhd after a hsct. methods: we performed a retrospective study to assess the association between serum ua levels pre-and posttable] 1. table 1 ] results: the characteristics of the 142 patients are shown in table 1 . median age was 52 years (range 15-69), and 80 patients (56%) were male. twenty-seven patients (19%) received low doses atg as part of gvhd prophylaxis. allopurinol was from the day before start of conditioning therapy until day 0. the median levels of ua were 4,8 mg/ dl before conditioning, 2,85 mg/dl at day 0, 3,1 mg/dl at day +7 and 3,2 mg/dl at day +14. there was no impact between the ua levels and os at any time of the hsct. ua levels at day +7 were associated with a higher ci relapse at 5 years (34% [95% ci, 20-49%] for ua level > 3,1 mg/dl, and 17% [95% ci, 8%-30%] for ua level ≤ 3,1 mg/dl [p= 0,046]). there was a trend for a higher ci of grade ii-iv agvhd for the subgroup of patients not treated with atg with ua < 4,8 mg/dl (48% vs 30%; p= 0,083) on day -8 and a higher nrm with ua < 2,85 on day 0 (50% vs 30%; p=0,080). conclusions: in our study the ua levels showed no impact on os, and only a tendency for ci of grades ii-iv agvhd grades ii-iv and nrm for the subgroup of patients not treated with atg. surprisingly, high levels of ua at day +7 of hsct were associated with a significant higher incidence of relapse. disclosure: dkms foundation, pi14/01971 (instituto carlos iii) and sgr288 (grc), generalitat de catalunya. background: veno-occlusive disease (vod) is an early, uncommon but serious complication of stem cell transplantation (sct) that is associated with high morbidity and mortality. defibrotide is the only licensed treatment for vod, and time to start of treatment (tst) affects outcomes. minor differences exist between the seattle, baltimore and classical ebmt (2016) criteria, which may trigger different start points for treatment. late onset vod (>21 days) is less recognised and we hypothesize, may have worse outcomes with longer time to diagnosis, and more limited treatment options across different healthcare systems. methods: electronic patient records from sept.2013 -oct.2018 at king´s bmt centre and pharmacy databases were reviewed, timepoint to clinical and bio-chemical manifestation of vod, diagnosis, tst, survival and longterm outcomes were analysed. results: 30 of the 532 patients(5.6%) who underwent an allogeneic sct, developed vod, including 2 paediatric cases. none of the autologous sct patients developed vod. the paediatric and autologous sct patients were not analysed any further. 28 adult patients (male=22;78.5%) developed vod at a median age of 56 years(range 26-72), of whom 21 developed < 21 days and 7 patients had late-onset vod as per ebmt criteria(range 22-93 days). 24 cases classed as severe and 4 as moderate vod. 10 patients received defibrotide at diagnosis, 7 patients within 3 days, 5 patients between 4-7 days, and 6 patients received treatment after 7 days. overall mortality for this cohort was 50%(14/28). 12/21 (57.1%) of patients with early onset vod and 2/7(28.5%) patients with late-onset vod died. of the 14 deaths, 10 died of liver failure and a further 2 patients had vod as a likely contributing factor in their deaths. 1 patient died with subarachnoid haemorrhage and 1 with relapsed disease. patients that received defibrotide after 7 days, 5/6 patients(83.3%) died, as compared to 3/5(60%) for treatments between 4-7 days, 6/17(35.2%) for treatments within 3 days. the lone surviving patient who received treatment after 7 days has severe chronic liver disease and it's complications. of the 21 patients who fit seattle criteria for early-onset vod, only 6 fit the baltimore or ebmt criteria for classical vod. 4 of these 6 patients met the baltimore criteria later than the seattle criteria were met(range = 2-9 days). conclusions: vod carries high morbidity and mortality, and beyond the known risk factors and with the caveat of limited numbers in this study, we strongly suspect this is further increased when time to definitive treatment with defibrotide is delayed, particularly beyond 7 days. nearly a quarter of cases with vod are late-onset as per classical ebmt criteria. however contrary to our hypothesis, their overall outcomes and mortality do not appear worse, with time to treatment again emerging as a strong predictive factor. conditioning treatment related factors, which play a stronger role in endothelial dysfunction in the hepato-portal circulation, may not be as much at play, perhaps for late-onset disease. uniformity in the use of diagnostic criteria, and high degree of vigilance, even beyond 21 days, leading to early treatments may improve outcomes in vod. disclosure: nothing to declare background: hsct-associated thrombotic microangiopathy (ta-tma) affects 10-30% of patients receiving an allogenic sct, with a high mortality up to 80-90% in severe cases. endothelial injury mediated by complement activation has been atribuited a major role in the pathogenesis, and blockade of c5 with eculizumab offers promising results. methods: we present our experience with 6 pediatric cases of ta-tma treated with eculizumab. the diagnosis of ta-tma was stablished attending to jodele et al criteria. clinical data were collected retrospectively from medical records. results: all cases were diagnosed between august 2016 and april 2018, with a median age of 11 years (2.5 -17) at time of diagnosis. primary disease was acute leukemia in 2 cases (1 all and 1 aml), severe aplastic anemia in 3, and primary immunodeficiency in 1. they received their first sct in all cases, 3 from mud and 3 from mmrd (cd45ra+ depleted haploidentical grafts), with mac regimen in 2 cases, and ric in 4 cases. 3 of them received calcineurin inhibitors (cyclosporine) as gvhd prophylaxis. all patients developed agvhd (grade 2 or higher in 3 cases). and 5 patients presented viral reactivation. hypertension was present in 4 cases at tma diagnosis, requiring 2 or more antihypertensive drugs in 3 of them. all patients had renal injury consisting of less-than-normal glomerular filtration rate (median of 41 (20-59)) and proteinuria, with urine protein-to-creatinine ratio higher tan 2 mg/mg in 2 cases (data not available in 2 patients). serum haptoglobin was decreased in just 2 cases at diagnosis, and schistocytes were detected in 3 patients. cutaneos signs were present in all cases, digestive symptoms in 2, neurological affection in 2, and notoriously all of them developed polyserositis. c3 and c4 were normal in all cases, with sc5b9 higher than 244 ng/ml in 2 patients and lower in 1 (data not available in 3 cases). all patients received defibrotide as treatment, and 4 cases received also rituximab, associated to therapeutical plasma exchange in 3. all of them received eculizumab, as first line in 2 cases (median of 40 days between diagnosis and eculizumab start). treatment was correctly monitorized with ch50 levels in 3 cases (not available quick enough in other 3). median number of doses needed in induction therapy was 8, and median interval between doses was 7 days. 2 patients required reduced interval and higher doses to maintain ch50 supressed. 2 patients did not respond, and died because of tma. 4 patients had hematological response, with chronic renal injury in 3 of them and resolution of acute renal failure in 1 case. nevertheless 1 patient responding to eculizumab died because of tma related complications, and 1 because of an invasive fungal infection. 2 patients are alive, with a median follow up of 6 months from treatment start. conclusions: our experience supports promising results of eculizumab based treatment for ta-tma, highlighting the importance of an early treatment and a careful therapy monitoring by ch50 supression. prospective studies are needed to achieve a better knowledge of this pathology and its treatment. disclosure: nothing to declare background: approximately 40-50% of allogeneic hematopoietic stem cell transplant (allo-hsct) are made with some sort of abo blood group system incompatibility. an hsct abo donor-recipient incompatibility implies risks of complications during the process of infusion as acute hemolytic anemia (ah), delayed graft and other later complications due to the presence of isohemaglutinins (pure red cell aplasia or passenger lymphocyte syndrome). also, abo incompatibility could impact on graft versus host disease (gvhd) incidence, and could be associated with not relapse mortality (nrm) and overall survival (os). there are not concluded evidence about the abo incompatibility impact, so the aim of this study was to identify complications and response associated with abo incompatibility in patients undergoing allogeneic hematopoietic stem cell transplantation. methods: a retrospective study was performed on patients who receive an allo-hsct between january 2014 and august 2018. two groups were performed according to the presences or not of abo incompatibility. demographic and clinical information was collected from physical and electronic medical records, and information was analyzed in spss v21 results: sixty-eight patients were enrolled in the study, 54% male, the median age was 34 years (19-61) with the following diagnoses: acute lymphoblastic leukemia 44%, acute myeloblastic leukemia 26.5%, granulocytic chronic leukemia 17.6%, myelodysplastic syndrome 4.4%, dendritic cell neoplasm 4.4%, aplastic anemia 2.9%. ninety-one percent of the patients received a transplant from an identical hla donor and 8.8% received a haploidentical transplant. fifty-two patients (76%) were abocompatibility (g1) and 16 patients (24%) had aboincompatibility (g2). none patient with aboincompatibility received a haploidentical transplant. the contrast between groups didn't show differences in fever, infections, bacterial isolation, presence and degree of acute or chronic gvhd and relapse of the disease. graft failure was 6%(g1) vs 20%(g2) (p=0.27), intermediate risk cmv serostatus 79%(g1) vs 87(g2) (p=0.35). the most relevant characteristics and complications are described in table 1. contrast analysis between g1 vs g2 showed that within the whole group there were 29 deaths (40% vs 50% respectively) (p=0.69), the overall survival 1-year was 74% vs 66% (p=0.58) with a median of 29 vs 34 months respectively; mortality associated with relapse was 68% vs 87% respectively, and mortality related with transplantation was 35% vs 13 % respectively. conclusions: abo incompatibility did not show association with complications related with the infusion, but there was a higher tendency of graft failure in the abo incompatibility group. it has no statistical significance, but it is important to expand its study. disclosure: none declared methods: retrospective data for 142 nhl patients who underwent asct between 1999 and 2017 was analysed. patients were identified using the swbmt database and data on mets was collected using paper and electronic hospital records. forty-eight patients were excluded due to loss of follow-up, inaccessible/incomplete records, or death. cause of death was not determined. the ncep-atpiii definition of mets was used. this requires ≥3 of 5 criteria to be met. a bmi of ≥30kg/m 2 and hba1c of ≥42mmol/l were used to replace central obesity and impaired fasting glucose, respectively. other criteria include triglycerides (tgs) ≥1.7mmol/l or treatment, high density lipoprotein cholesterol (hdl-c) < 1.0mmol/l (male), < 1.3mmol/l (female) or treatment and blood pressure >130mmhg systolic or >85mmhg diastolic, or treatment. results: the prevalence of mets in the cohort was 33% (n=31). eighty-two percent of patients (n=77) met one or more criterion for mets. twenty-seven percent (n=25) fulfilled only one criterion, 23% (n=22) fulfilled two criteria, 20% (n=19) three criteria, 12% (n=11) four criteria, and 1% (n=1) five criteria. the greatest prevalence of mets was in the 60+ age group, accounting for 17 out of 31 (55%) patients with mets. overall prevalence decreased with declining age ( table 1 ). the number of patients aged < 20 years was too small to make any judgement on risk. raised triglycerides was the criterion most frequently met (61/94 patients), followed by hypertension (48), raised bmi (26), low hdl-c (23) and an increased hba1c (15). conclusions: the prevalence of mets in our cohort (33%) was higher than the estimated worldwide prevalence of 25%, with the majority in the 60+ age category. this is in keeping with other post-transplant studies, which show an increase in prevalence of mets after transplantation. moreover, the overall prevalence of mets was greater in the older population, which could be associated with the cumulative effect of ageing on the decline of normal metabolic homeostatic mechanisms. background: acute renal failure (arf) is a frequent complication in the early post-allogeneic hematopoietic stem cell transplant (allohsct) period with either myeloablative (ma) or non-myeloablative (nma) conditioning regimens. the aim of this study was to compare the incidence of arf in both types of hsct and to evaluate its impact on overall survival (os) and non-relapse mortality (nrm). methods: all allosct performed in one center between 2010 and 2018 were included in this study. allohsct from cord blood and from haploidentical donors were excluded. the renal function and the incidence of the main complications after allosct from day 0 to day +90 were evaluated. arf was defined according to kdigo (kidney disease improving global outcomes) classification; the relative increase of serum creatinine levels was considered a marker of kidney damage. results: seventy-seven patients received a ma allohsct and 72 a nma allohsct. recipients of nma allohsct had a higher median age (61 years [range: 18-69] vs. 41 years , p< 0.001), higher frequency of arterial hypertension (29% vs. 6%, p< 0.001) and showed most frequently active disease at allosct (42% vs. 18%, p=0.002). in both groups the most frequent graft-versushost disease (gvhd) prophylaxis regimen was cyclosporine a and methotrexate. the median follow-up time was 2.3 years for the nma group and 3.6 years for the ma group. patients from the ma group had higher incidence of grade 3-4 mucositis (60% vs. 22%, p< 0.001) and acute gvhd of any grade (70% vs. 53%, p=0.029) than patients from the nma allohsct. the incidence of arf was similar in both groups (72% in nma and 71% in ma). in the nma group arterial hypertension (hr 2.05, p=0.018), obesity (hr 4.16, p< 0.001) and prior pneumonia (hr 3.19, < 0.001) were predisposing factors for arf by multivariate analysis, whereas any factor was identified in the ma group. arf had no impact on 2-year os in both groups (28% vs. 35% p=0.406 for the nma group and 45% vs. 40% p=0.623 for the ma group). however, worse os were observed in patients with grade 2-3 arf in the nma group (18% vs. 43%, p=0.048) and in patients with grade 3 arf in the ma group (25% vs. 59%, p=0.011). in turn, arf had no influence on nrm in the ma group but was associated with a trend for higher nrm in the nma group (59% vs. 35%, p=0.084). conclusions: arf is a frequent complication in patients receiving allohsct irrespective of the intensity of the conditioning regimen. moderate and severe arf had negative impact on os. disclosure: supported by grants from: asociación española contra el cáncer, aecc (gc16173697biga), instituto carlos iii (pi14/01971 fi), 2017-sgr288 (grc), cerca program from generalitat de catalunya, and "la caixa" foundation. treatment and risk factors of hepatic veno-occlusive disease after pediatric hematopoietic stem cell transplantation: a single-center experience barbaros sahin karagün 1 , ilgen sasmaz 2 , ali bülent antmen 1 background: defibrotide emerged as a promising treatment option for hepatic veno-occlusive disease, a significant cause of mortality in recipients of hsct. as vod diagnosis is quite difficult even with the recently introduced ebmt 2017 criteria, studies which report treatment outcomes and response to prophylaxis are required. our aim was to evaluate the efficacy of defibrotide prophylaxis in hsct recipients at our center. methods: a total of 236 transplants in 210 patients from january 2013 to july 2018 were included in this study. all patients had factors that increased the risk of vod and all received 25 mg/kg/day prophylaxis. patients' coagulation, renal and liver function test were monitored daily and all clinical findings and complaints were recorded. diagnoses were made via the ebmt 2017 vod criteria and patients who developed vod received treatment with increased df dose (40 mg/kg/day) and supportive interventions. after complete remission of vod findings, patients were returned to the prophylaxis dose. close follow-up of patients was performed until 100 days. results: in total, 17 patients developed vod (7.2%), none of the cases were severe (13 mild, 4 moderate). median age was 8.5 years and the most common clinical findings were weight increase, hepatomegaly, right upper quadrant pain and ascites development. in those with vod, treatment with 40 mg/kg/day df was initiated and average duration of treatment with this dosage was 7.4 (5-11) days. no adverse events were reported in any of the patients. conclusions: our findings are consistent with previous studies on this topic, and we believe that the use of df as a prophylactic agent for vod is beneficial for pediatric patients with risk factors. disclosure: the authors report no conflicts of interest in this work. background: several factors might influence outcome of allo-hsct. analysis of the impact of donor-receptor blood group-incompatibility have been performed in different series not always showing the same results. as a consequence, its clinical impact remains controversial. minormismatch is characterized by the ability of donor b lymphocytes to produce anti-recipient antibodies. in majormismatch cases, antibodies against donor antigens are present in the recipient. methods: 343 pts underwent allo-hsct between may 2011 and august 2018 in our center. median age was 52 years (range: 7-69). 193 pts were male (56.3%) and 150 female (43.7%). baseline diseases were: 138 aml, 76 lpd, 44 mds, 41 all, 21 mpd, 16 mm, and 7 bmf. donor was unrelated in 191, and related in 152 cases (including 36 haplo-identical). donor-recipient abo compatibility was as follows: 69 (20.1%) majormismatched (including 11 bidirectional), and 274 (79.9%) nonmajor-mismatched (including 68 minormismatched and 206 matched). donor-recipient rh compatibility was as follows: 50 (15.6%) major-mismatched, and 293 (84.4%) nonmajor-mismatched (including 34 minor-mismatched and 259 matched). the impact of donor-recipient abo and rh compatibility on transfusion needs (prbc and platelet concentrates) and survival by day +100 was analyzed. results: for the global series the median number transfusions by day +100 was: 4 (0-81) prbc and 4 (0-92) platelets concentrates. day +100 overall mortality was 9.3%. rh-incompatible and nonmajor abo incompatible cases showed no different results. however, major abomismatched cases needed more prbc transfusions (median: 6; range: 0-49) and more platelet transfusions (median: 7; range: 0-60), and had higher day +100 mortality (18.8%) (p < 0.05) (see table) . conclusions: our analysis showed: 1) donor-recipient rh-incompatibility, as well as minor aboincompatibility had no impact on prbc and platelet concentrates transfusion needs nor on 100-day mortality; 2) contrarily, donor-recipient major abo-incompatibility had a significant adverse impact on prbc and platelet concentrates transfusion needs and 100-day mortality. 3) donor-recipient rh-incompatibility and minor aboincompatibility.might be considered of marginal importance at the time to choose a potential donor. 4) donorrecipient major abo-incompatibility should probably be a factor to be considered, along with other features, to choose the best donor background: survivors of haematopoietic stem cell transplantation (hsct) are at significant risk of developing treatment-related complications, including cardiovascular risk factors such as arterial hypertension, that could eventually lead to cardiovascular disease. the aim of this study is to evaluate the incidence and risk factors of hypertension following hsct in a colombian population. methods: a retrospective cohort study was conducted to assess the incidence and risk factors of hypertension in 220 consecutive adult hsct recipients who underwent transplantation between 2009 and 2017 at a tertiary referral center in colombia, south america. blood pressure data, from two different measures, were collected at 7 time points: day of mobilization for autologous hsct and day 0 before infusion for allogeneic transplantation, day 7, and months 1, 3, 6 and 12 post-transplantation. hypertension was defined as having a systolic blood pressure >=140mmhg and/or a diastolic blood pressure >=90 mmhg. patients with history of arterial hypertension were excluded. results: one hundred and seventy-five patients were included, with a mean age of 44 years (range 15-67). ninety-one patients (52%) were male. one hundred and sixteen patients (66.3%) underwent autologous hsct and 59 (33.7%) allogeneic hsct. the most common indication for hsct was acute leukemia (26.3%), followed by non-hodgkin lymphoma (23.4%) and multiple myeloma (22.9%). twelve patients (6.9%) had medical history of type 2 diabetes mellitus (dm), 11 (6.3%) dyslipidemia, 24 (13.7%) alcohol consumption, and 25 (14.3%) tobacco smoking. only two of the patients with history of tobacco smoking were active smokers at time of transplantation. twenty-four patients (13.7%) had developed hypertension by the end of the first year post-hsct follow-up. two patients (8.3%) had systolic and diastolic, 12 (50%) had only systolic, and 10 (41.7%) had only diastolic hypertension. only one patient was hypertensive at more than two time points. the incidences of hypertension at each time point were 2.3% on day 7 post-hsct, 6.9% at first month, 9.8% at three months, 12.1% at 6 months, and 13.8% at one-year post-transplantation. allogeneic hsct (p< 0.01), therapy with calcineurin inhibitors (p< 0.01), pre-hsct fasting glucose levels (p< 0.05), acute gvhd (p< 0.05), chronic gvhd (p< 0.05), and media of diastolic blood pressure (p< 0.05) were significantly associated with the development of arterial hypertension. however, age, history of type 2 dm, history of tobacco consumption, volume of infusion, prophylactic treatment for gvhd with mycophenolate, chronic gvhd, serum creatinine level on day of hsct, and being overweight or obese at time of transplantation were not significantly associated with the development of hypertension. conclusions: arterial hypertension is a fairly common complication in hsct recipients. similar to findings reported in previous studies, association between allogeneic stem cell transplantation, therapy with calcineurin inhibitors, and acute and chronic gvhd, and post-hsct hypertension was found in the present cohort. further studies are needed to assess the link between hsct and developing long-term cardiovascular complications. disclosure: nothing to declare tramadol-based pain management of oral and esophageal mucositis in pediatric hsct recipients background: mucositis is one of the most common early hsct complications seen in about 70% transplant recipients with 20% of patients developing gr iii-iv mucositis. mucositis is characterized by painful gastrointestinal mucosa lesions impairing the solid and liquid foods intake and increased risk of infections, bleeding, and intestinal paresis. thus, it greatly decreases the quality of life of a transplant recipient. according to who recommendations, the moderate pain control in pediatric patient is based on the use of low-dose morphine. however, there are some factors such as genetic polymorphisms causing variable morphine pharmacokinetics in children, side effects, and social factors (caregivers' general unwillingness to use narcotic analgesics), which cause the need for alternative pain relief options in pediatric practice. tramadol, which has both opioid and non-opioid mechanisms of action, may be a feasible option in mild to moderate pain. it may be delivered via patient-controlled analgesia (pca), although there is no consensus on its optimal parameters in pediatric practice. methods: a total of 69 pediatric patients with a median age of 8 (range 2 to 18) years receiving an autologous or allogeneic hsct in our clinic as part of the treatment regimen for solid tumor (n=40), leukemia (n=24), acquired aplastic anemia (n=3) or inherited condition (n=2) were included. conditioning regimens were myeloablative (mac) in 54 and reduced-intensity (ric) in 15 patients. all patients had oral and/or esophageal mucositis accompanied by moderate pain. the pain severity was assessed using the scales corresponding to patient's age and varied from 3 to 6 points. the pain control was based on intravenous tramadol administration using patientcontrolled analgesia (pca) approach. the following pca parameters were used: loading dose of 0.5 mg / kg (not exceeding 25 mg), basal infusion rate of 0.25 mg / kg (not exceeding 12.5 mg), a bolus of 0.25 mg / kg (not exceeding 12.5 mg), lockout interval of 25 min. the maximal daily dose was 8 mg/kg/day. the pain control was considered adequate if a patient was satisfied or the basic and breakthrough pain score values were not higher than 3 and, accordingly. in case of inadequate pain control nsaids were added. non-responders were switched to morphine. all patients were divided into 2 groups based on conditioning regimen intensity. results: as a whole, 46% of patients did not require pain control measures escalation. the tramadol pain control rate was slightly higher for ric (n=9, 60%) compared to mac (n=23, 47%) recipients. in most cases the inadequate pain control was due to progressive mucosal lesions. the pca regimen used was characterized by very few complications. drowsiness was observed in 4 (7%) of patients, in all cases the patients also had anemia. there was only 1 (7%) patients with severe nausea requiring switching to morphine. conclusions: tramadol is an effective pain control option in transplant recipients with mild to moderate pain due to oral and esophageal mucositis without progressive mucosal lesions. the pca allows achieving a very low complication rate. therefore, this option may be considered for both mac and ric recipients. disclosure: no immune reconstitution of lymphocyte subsets after allogenic stem cell transplant (sct) and vaccination background: infectious diseases are a major cause of morbidity and mortality after allogenic stem cell transplant (sct). vaccines constitute an effective strategy to prevent infections but the optimal timing to start vaccinating is not well stablished. in order to individualize the early vaccination schedule, we studied the lymphocyte subsets involved in generating enough response to produce protective serological levels. methods: we studied retrospectively 20 patients that had undergone allogenic sct at our hospital. patient distribution -age range: 21-68 years-old; diagnosis: acute leukaemia/myelodysplastic syndrome/ chronic myeloid leukemia (16 patients), lymphoma (4 patients). analytic parameters: tcd4+, tcd8+, nk, total b and functional b lymphocyte subsets (naïve igd+cd27-, memory igd+cd27+ and igd-cd27+, and effectors cd27++cd38++). immunoglobulin levels (igg, iga, igm) and specific igg for pneumococcus, tetanus, hbv, chickenpox, measles, rubella and mumps. clinical parameters were collected from medical records. results: we distributed patients in two groups, based on the timing of lymphocyte analyses: -less than 12 months since sct (5 patients) no patient showed complete immune reconstitution, although 2 had enough t and functional b lymphocytes to generate response to vaccination. in these patients, vaccination for pneumococcus was completed and they generated sufficient protection antibody levels, despite being under immunosuppressive treatment. -more than 12 months since sct (15 patients) before the beginning of vaccination, we collected specific antibodies of 7 patients. we compared the serological status before and after sct and observed that protection against tetanus was the most frequently preserved (6 patients) and hbv the least frequent (2 patients). other than one patient treated with alemtuzumab, all patients in this group had minimum absolute count of tcd4 + (>200 cells/microl), tcd8+ (>200 cells/microl), nk (>100 cells/microl) and b cells (>100 cells/microl). we also observed presence of b effector and b memory cells, with predominance of igd-cd27+ memory cells. immunoglobulin levels were within the normal range. in this group, we registered vaccination in 13 patients. all of them were vaccinated against flu, and 11 against pneumococcus and hbv. the rest of vaccines administered were heterogeneous in type and timing. 6 patients were under immunosuppressive treatment at the time of vaccination and were able to generate enough specific antibodies for pneumococcus. conclusions: immune reconstitution was not completed 12 months after sct, although minimal immunological reconstitution was observed tcd4+ and no-switching memory b lymphocytes were the last ones to reach minimum normal values according to patient age. some patients maintain serological protection after allogenic sct. immunoglobulin levels were normal, suggesting no need for immunoglobulin administration to prevent infections. flu, pneumococcus and hbv vaccines were the most frequently administered. pneumococcus vaccination generated a much larger serological response than hbv. this seroconversion occurred in patients under immunosuppressive treatment. the analysis of lymphocyte t, nk, b total and b functional subsets could be useful when programming an early vaccination schedule after sct. completion of the vaccination schedule was heterogeneous despite giving specific indications. therefore a more rigorous supervision of the process may be required. background: the significant advances that have been achieved in the allogeneic transplantation (allohct) field, have resulted in better post-transplant outcome and therefore complications other than the graft vs. host disease (gvhd) or disease recurrence become increasingly important. the post transplant metabolic syndrome (pt-ms), which caused by several factors (i.e. immunosuppressive agents, chemo-radiotherapy, anti-viral, and biologic therapies) is a well known post transplant complication in pediatric allografted long-term survivors however, only few studies have evaluated the prevalence of the pt-ms in adults. in this retrospective study, we sought to evaluate the incidence, the risk factors and the impact of the pt-ms on the allosct outcome. methods: since 2011, 42 patients (25 males and 17 females) with adequate clinical and laboratory data and a minimum follow-up of 6 months were included in the study. their median age was 35.5 (17-62) years and after a myeloablative (n=28) or a reduced intensity (n=14) regimen they received either mobilized peripheral blood stem cells (n=34) or marrow graft (n=8), originated from full-matched siblings (n=35) or haploidentical donors (n=7). calcineurin inhibitors plus either short-term methotrexate or mycophenolate mofetil were given as gvhd prophylaxis. the diagnosis of pt-ms was based on the ncep-atpiii criteria; for patients with unknown data for abdominal circumference the body mass index (bmi) ≥25kg/m 2 was consider as a criterion for pt-ms diagnosis. the independent t-test, logistic regression analysis and logrank tests were used for the statistical analysis. results: twenty (47.6%) patients (12 males, 8 females) assessed to have pt-ms within the first 6 months following the allograft. seventeen diagnosed after the 1 st trimester post allosct and additional 3 patients after 2 nd trimester. sixteen out of 20 patients had elevated glucose and bmi>25kg/m 2 , 13/20 elevated triglycerides levels, 12/20 low hdl levels and 10/20 hypertension. four (20%) had already known history of ms before allosct (for 10 patients no data were available for ms diagnosis before allosct). interestingly, for 7/20(35%) patients who had diagnosed with pt-ms either in the 1 st or in the 2 nd trimester the syndrome was reversible and did not fulfill the criteria for pt-ms beyond 6 months post allosct. patients' gender, age, bmi, the type of conditioning regimen and gvhd co-existence evaluated as potential predisposing factors for pt-ms diagnosis. in univariate and multivariate analysis only the: bmi>25kg/m 2 and age>35 years were detected as significant risk factors (p< 0.03). the pt-ms did not affected negatively the survival or the nrm incidence post allosct conclusions: in our study, in agreement with other publications, we demonstrated that the pt-ms is not an uncommon complication post in the early post transplant period however, for a significant number of patients the syndrome was a reversible. for patients with high risk features (bmi>25kg/m 2 , age> 35 years, known history of diabetes-mellitus, dyslipidemia, hypertension) apart of close monitoring, specific diet and encouragement for adequate exercise might help to reduce the incidence and the severity of pt-ms. nevertheless, prospective and well design trials are warranted to determine the accurate incidence, severity and the impact of pt-ms on the allosct outcome. disclosure: no conflict ofinterest experience of a single center in the humanization of the hospitalization process: technology and team training impact on the qol of the patient and family maria claudia moreira 1 , marcia rejane 1 , marcia garnica 1 , andrea ribeiro 1 , paulo cesar dias 1 , ilza fellows 1 background: hematopoietic stem cell transplantation (hsct) is one of the most aggressive therapeutic modalities of internal medicine, making it a highly stressful experience for the patient and his family. the duration of hospitalization can be prolonged by several intercurrences, frequently generating anxiety in the patient and their caregiver, which may lead to confinement and reactive depression. interventions in the hospital environment, in addition to the continuous training of the multidisciplinary team, can have a positive impact in this process with improvement in the process of discharge and quality of life of the patient and his / her family. methods: the objective of this research was to evaluate the impact of a reformulation in the unit, completed in may 2018, which modified the facilities with availability of hermetic balconies in each room, with a view of an internal garden. there was also the addition of a screen in the corridor of the floor with images -technology known as videoowall, interconnected to motion sensors (kinects) that allow interaction between patients and families, besides facilitating physiotherapy and physical exercise. there was re-training of the multidisciplinary team with emphasis on the practice of humanization. the methodology consisted in the application of questionnaires of satisfaction to patients and their families during the period of hospitalization in a bone marrow transplant unit in the third quarter of 2018. the items evaluated ranged from the quality of the information provided by the medical team and nursing, to the cordiality and agility with which the patient and his patient were treated by the global team. the results were compared with a similar period of the same unit in the previous year and with the indices collected simultaneously in another unit of the same hospital (cardio-intensive). results: overall and segmental satisfaction scores in the various items surveyed were higher when compared to the previous period of the same unit and were also higher in those obtained in a high complexity unit of the same hospital, composed of patients submitted to mental and psychological stress similar to onco-hematologicos.a reports of "free speech" were also obtained anonymously, in order to guarantee the authenticity and free expression of the subjects analyzed. conclusions: the results obtained allowed the validation of the technical and professional team initiatives, bringing indicators that will allow better monitoring and support of these patients and their relatives in this difficult time of treatment. they served as an initial tool in the continuous process of humanization and stimulated the multidisciplinary team to continuously improve this process. disclosure background: pure red cell anemia (prca) is a rare complication of abo-incopatible hematopoetic stem cell transplantation characterized by anemia, reticulocytopenia and absence of erythroid precursors in patient's bone marrow. most patients with prca resolve spontaneously within months, however a small number of patients requires continued red blood cell (rbc) transfusions. the treatment of this complication is difficult and not standardized. different approaches has been used such as rituximab, donor lymphocytes, plasma exchange with different outcome. recently, a remarkable response to treatment with bortezomib has been described in a case of prca. methods: we reviewed 146 patients who received an allogeneic hematopoetic stem cell transplant (hct) between januar 2012 and august 2018 at our institution. sixty eight patients received a major abo-mismached hct. prca was defined as a completely absence of erythroid precursors on day +30 bone marrow puncture, with absence of donor red cells and the recipient requiring rbc transfusion. results: only one patient developed prca (1.5%). a 18 years old male received a myeloablative hla-matched abomismatched sibling donor transplant (brother, 12 years) for acute myeloid leukemia (aml), with t(8;21) cr1,mrd positive (runx1-runx1t1). the donor was blood type a rh positive and the patient 0 rh positive. the patient had no complication after transplant. the day +30 bone marrow puncture has shown only few erythroid precursors and day +100 puncture and biopsy no erythroid precursors, he had transfusion dependent anemia requiring a rbc transfusion every two weeks and retukulocytopenia. parvo virus and cytomegalovirus were negative. due to very high ferritin level (>4.000u/l) and increased luiver enzymes without signs of gvhd, the treatment with deferasirox has been started. the patient has achived cr1, mrd negative, and has evidence of complete chimerism. high titers of anti-a and anti-b issohemagglutinin was present.we started the treatment with rituximab 375mg/m2 weekly, 4 weeks, however without response. the pathogenesis of the prca is thought to be due to the recipients plasma cells, bortezomib, a proteasome inhibitor inducing apoptosis of plasma cells has been given s. c. 1,3mg/m2 two times weekly, for two weeks. the patient responded to the treatment two weeks later with increase in hb, which was 12,9 g/dl and increase in retikulocyte number. the patient has continued to be well at the last control. conclusions: prca aplasia is a rare but serious complication after abo-incompatible hct. bortezomib is an effective treatment for this complication if mediated by residual host isohemeagglutinins after hct and should be recommended as standard of care. clinical methods: this work is retrospective, observational, cross-sectional and analytical. it included all patients who received hsct at stem cell transplantation unit (utmo, by its spanish acronym) at solca-guayaquil, between the years 2009 -2016.we use the kaplan-meier method to analyze the survival rate between the autologous and allogeneic transplant. the information collected for this study was obtained from the database of the solcay institute and the review of the files of the patients included. results: at least, 150 patients have been undergoing to hsct between 2009-2016 years. according to the type of hsct, 42.1% received an autologous transplant and 57.9% received an allogeneic transplant, from which 79.3% were from a related donor. the main source of transplant was peripheral blood in 86.67%, followed by 12% obtained from umbilical cord blood and 1.33% by bone marrow aspiration. the most frequently reported pathologies were acute lymphoblastic leukemia (all) (34%), multiple myeloma (mm) (22%) and acute myeloid leukemia (aml) (13.33%). the overall survival was 68% (ic: 95%). the 82.53% of patients that were undergoing to autologous transplant have survive, meanwhile the patients that were undergoing allogeneic transplant only the 57.47% have survived (p< 0.05). the highest death rate occurred during the first year after hsct, and decreased considerably after that period. the main cause of mortality related to transplant (mrt) was the graft-versus-host disease (gvhd) (8%); however, the main cause of mortality in the study population (n=150) was relapse in 12.66% of the patients, presented more frequently in all. conclusions: the results showed that 68% of patients undergoing to hsct have survived. a high rate of deceased patients in this study, have died in the first year before the transplant (26.6 %%), due to relapse. the main cause of deceased in the study is not related to hsct, and was the relapse in 12% of patients, in compare the gvhd was the main cause of mrt (8%). we consider that hsct is a technique that is still under development in ecuador, but despite the short time it has been taking and the institutional and medical limitations present in the health field, has presented excellent results comparable to studies conducted in developed countries. [ background: pigmented epithelioid melanocytoma (pem, early known as 'аnimal type' melanoma) is a rare tumor with unpredictable clinical behavior and metastatic potential. pem generally has favorable prognosis. involvement of regional lymph nodes is not rare. extranodal and distant nodal metastases are extremely rare. we report about patient with fanconi anemia (fa) and pem with developed distant metastases in the early term after allogeneic hematopoietic stem cell transplantation (hsct). methods: 10-years old boy with fa was hospitalized for hsct. the blue-black painless nodulus 15х15 mm was noted on the left cheek. this lesion was observed from early childhood and during life only slightly increased in size. there were no distant and regional metastases on computerized tomography (ct) and scintigraphy with 99m tc. the nodulus and regional lymph nodes were radically removed before hsct. the resection margin was within the normal tissue. microscopically the derma and subcutaneous fat were infiltrated with epithelioid and spindle cells with total expression of s100, melana, mhb45, cyclind1. ki-67 expression level was 15-20%. histological structure was specific for pem. hsct with tcrαβ+/cd19+ graft depletion from match unrelated donor was performed. the conditioning regimen included total lymphoid irradiation 2gy, fludarabin 150 mg/ m 2 , cyclophosphamide 40 mg/kg, rabbit atg 5 mg/kg and rituximab 100 mg/m 2 . results: at +45 day after hsct was detected the tumor on the left cheek and parotid region with a histological structure identical to the primary lesion. on ct in s6 segment of the left lung was detected focus 20x20 mm with a cavity. invasive aspergillosis was suspected and empirical antifungal treatment was started. but in 15 days the lung lesion increased in size to 52x30x32 mm and penetrated in the bronchus. after bronchoscopy with biopsy, pem metastasis was histologically confirmed. moreover, the tumor on the face continued to grow. therapy with cobimetinib and vemurafenib was not effective and patient died from progression of pem on +98 day after hsct. conclusions: pem was early described as indolent tumor with rare distant metastasis and favorable prognosis. we suspect that pem may acquire an aggressive course in the absence of immunological control, especially in high immunocompromised patients after hsct. disclosure: nothing to declare p208 abstract withdrawn lidia gartcheva 1 , antoaneta mihova 1 , penka ganeva 1 , margarita guenova 1 , branimir spassov 1 background: the main objective of the study is to assess the dynamics of quantitative and qualitative changes in the parameters of the b cell population and the production of immunoglobulins in patients after autologous transplantation of hematopoietic stem cells in the course of recovery of the immune system. methods: 56 patients with hematological neoplasms undergoing autologous transplantation were included in the study: 30 women and 26 men, with an average age of 31 years. patients were diagnosed with lymphoma (n = 30), multiple myeloma (n=7), leukemia (n = 7) and solid tumors (n = 12). at the time of transplantation, 16 patients were in complete clinical remission or at least with very good partial response, 30 patients were in partial remission and 10 patients -with progression. all patients were evaluated in nine time points through 356 examinations by clinical-laboratory, flow cytometric and immunochemical methods. results: the percentage of cd19 (+) b cells reached the minimum values one month after transplantation then began to increase in the second month reaching a plateau around the mean values in the period 6-12 months after transplantation. the absolute number remained low during the entire period of observation. the amounts of igg and igm serum immunoglobulins gradually increased within the reference range throughout the entire period, while the iga level varied around the lower reference range. conclusions: implementation of an adequate humoral immune response is hampered by the reduction of circulating b cells, suppressed proliferative potential and functional deficits. restoration of b-cell function occurs over a period of 6 months to 2 years after autologous transplantation. clinical trial registry: no clinical trials disclosure: nothing to declare justyna background: allogeneic hematopoietic stem cells transplantation (allo-hsct) is a life-saving and well established therapy for wide range of diseases. however, it is still uncommon treatment for infants less than 12 months of age. the data about indications and outcome of allo-hsct in the youngest group of patients is sparse. the primary objective of this study was to assess the incidence, indications, post-hsct complications and general outcome of allo-hsct among infants not older than 12 months. latter sequelae of hsct such as physical and cognitive development were secondary aim of this study. methods: we retrospectively analyzed data of 63 patients who underwent allo-hsct before 1 year of age in department of pediatric hematology, oncology and bone marrow transplantation in wrocław during years 1999-2017. clinical and epidemiological features as well as indications for transplantation, early and late complications and general outcome were assessed. results: infants who underwent hsct in our department comprise 8.2% of all patients undergoing hsct in analyzed period of time. thirty-one (49.2%) patients received stem cells from matched unrelated donor (mud), 26 (41.3%) from mismatched (haploidentical) related donor (mmrd) and 6 (9.5%) from a sibling donor (msd). non-malignant disorders were indication for transplant in 49 (77.8%) patients and malignant diseases in 14 (22.2%) . acute graft versus host disease (agvhd) occurred in 33 (52%) infants, chronic graft versus host disease (cgvhd) in 15 (24%). majority of graft rejections were seen in infants transplanted from mmrd 7(63.6%), whereas the rest 4 (36.4%) was associated with mud. median follow-up in study cohort was 860 days, 1148 days for alive patients (range 72 days-19.5 yrs) and 72 days for those deceased (range 3 days-341 days). overall survival (os) in study cohort was 0.682 and transplant related mortality (trm) was 0.317. in children with malignancy 5 (35.2%) patients died comparing to 15 (30.6%) patients in non-malignant group respectively. main cause of death in analyzed group of infants was infection (60%). conclusions: 1. allo-hsct is rarely performed in children less than 12 months of age. 2. majority of those patients receive stem cells due to non-malignant disorder. 3. among youngest hsct recipients, haploidentical transplant are more common than in general pediatric transplant population. 4. graft rejection is a significant problem in infants transplanted from mmrd. disclosure: nothing to declare unusual non-infectious lung complication after allogeneic haematopoietic stem cell transplantation claudia lucia sossa melo 1,2 , manuel rosales 2 , francisco fernando naranjo junoy 1,2 , sara inés jiménez 1,2 , luis antonio salazar 1,2 , angela maría peña 1,2 , maría angélica chacón manosalva 3 , maria luna-gonzález 1 , claudia marcela chalela 1 , manuel ardila-báez 1 jirovecii infections, viral infections or nocardia. we describe the case of a patient with acute lymphoblastic leukemia (all) diagnosis with pap associated to a hsct and pulmonary pneumocystis. methods: a 55-year-old colombian female patient diagnosed with b-precursor all of high-risk in january 2017, positive philadelphia chromosome, positive bcr / abl in february 2017, infiltration to the central nervous system (cns), 2.53% of lymphoblasts, and karyotype without legible metaphases. refractory to induction according to the pethema protocol (vincristine, daunorubicin, prednisone, l-asparaginase) with presence of 13.9% blasts at the end of the induction. re-induction was performed with the flag-ida protocol (idarubicin, fludarabine, cytarabine) achieving complete remission, obtaining minimum residual disease (mrd) < 0.01. dasatinib was initiated by bcr / abl expression and cns involvement at the time of diagnosis. an allogeneic hsct was performed, from a male brother donor, with low intensity conditioning tt buflu and prophylaxis of graft-versus-host disease (gvhd) with tacrolimus and sirolimus. patient showed early posttransplant complications, given the reactivation of cytomegalovirus and hemorrhagic cystitis grade i due to adenovirus. late complications such us gvhd at the cutaneous level and subsequent hepatic and gastrointestinal involvement were seen too, for which immunosuppressive therapy was administered with high doses of systemic corticosteroid. results: patient was hospitalized on day +270 posttransplantation due to febrile neutropenia and respiratory symptoms, with normal chest ct, and ct of paranasal sinuses with acute pansinusitis, for which she received meropenem 1gr intravenously every 8 hours plus vancomycin 1gr intravenously every 12 hours during 14 days with symptom resolution. she remained hospitalized for cytopenias with normal bone marrow and 100% chimerism. on day +291 posttransplant she presented fever and leukocytosis, with acute respiratory failure with chest ct that showed bilateral alveolar occupation, "crazy-paving" pattern and frosted glass (see image), so diagnostic fibro-bronchoscopy was performed, reporting postoperatively for pneumocystis jirovecii. she received 21 days of trimethoprim-sulfamethoxazole, with a torpid evolution requiring mechanical ventilation and tracheostomy, persisting with hypoxemia. the report of cultures for fungi, mycobacteria, and respiratory panel of filmarray were negative. a pathology report was obtained with 30% neutrophils, as well as pas staining with acellular pink material and elevated serum ldh, with a diagnosis of secondary pap. the patient continued with poor general condition, refractory hypoxemia, high ventilatory parameters and hemodynamic instability, due to which she was not able to be a candidate for treatment with total pulmonary lavage; leading to multi-organ failure and later death. [[p211 image] 1. high resolution chest ct. sample opacification in frosted glass (a) and pattern ''crazypaving'' (b)] conclusions: the importance of considering the diagnosis of pap as a noninfectious pulmonary complication in patients with allogenic hsct despite its low incidence is recognized. disclosure: nothing to declare methods: once the project was approved by the clinical trials and ethics committee, 154 pairs of blood samples were drawn (154 from picc line and 154 from venepuncture) from 33 voluntary allo-hsct recipients who were receiving continuous infusion tacrolimus from february through august 2018. the pts had inserted a double-lumen polyurethane picc. tacrolimus was always administered through the red line, and the blood draw always performed through the purple line. all of the patients signed the informed consent. 22 were male and 11 women. median age was 55 years (30-68). 24 of the venepunctures were carried out in the arm where the picc was set, and the other 130 from the contralateral arm. a limited group of nurses performed the extractions of the samples. results: as shown in the table, tacrolimus trough levels determined in blood from venepuncture were similar to those in blood drawn through the picc (median: 10.7 vs 11.1 ng/ml). when comparing one by one in the individual patients, the differences were not significant, and changed the dosing prescription in no cases. conclusions: in our experience, there are not significant differences in tacrolimus levels draw from the picc line, compared with a peripheral site. so, in our opinion, if the line for tacrolimus infusion is properly identified and the one used for the sample draw is the alternative one, venepunctures to obtain sample from peripheral sites are not justified for tacrolimus levels measurements. background: patients undergoing a hsct may require icu admission due to transplant-related toxicities. the aim of this study was to analyse a single centre experience with hsct patients requiring icu admission and the factors affecting outcome. methods: we included all adult patients (age >=18) who had an allogeneic or autologous hsct during 2017 (d0 between 1-1-2017 to 31-12-2017) at st. george's hospital. data was retrospectively collected from patients' notes. icu outcome and 100-day survival were analysed. for those patients who were admitted to icu more than once, outcome was analysed from their last icu admission. results: 20 allograft patients were included. 14 were male, with a median age 47 years (range 23-68 years). 6 were female, with median age 61 years (range 51-67 years). diagnosis n (%) includes all 3 (15%), aml 7 (35%), acml 1 (5%), cmml 3 (15%), hl 1 (5%), mds 2 (10%), mds/mpn 1 (5%), fl 1 (5%), scd 1 (5%). sixteen (80%) patients received their first transplant, 4 (20%) received second transplant. eight (40%) patients had sibling donor, 12 patients (60%) had unrelated donor. sixteen (80%) patients had 10/10 matched donor, 2 (10%) patients had 9/ 10 matched donor, 2 (10%) patients had 8/10 matched donor. nineteen (95%) received reduced intensity conditioning (ric), one (5%) received myeloablative (ma) conditioning. majority of ric allo-hsct patients were conditioned with fludarabine, mephalan, campath (fmc). a small number were conditioned with busulfan, fludarabine and atg. the ma allo-hsct patient was conditioned with tbi, cyclophosphamide. gvhd prophylaxis was ciclosporin alone starting on day -1 with a target level of 150-250 ug/l for all ric and ciclosporin and methotrexate for the ma patients. two (10%) allograft patients were admitted to icu on three occasions. both patients were male, 58 and 68 years old. one had mmud allograft for mds/mpn. the other had 2 nd mud allograft for relapsed aml. the reasons for icu admission include sepsis, cardiac arrest and respiratory failure. the median duration of icu admission was 5 days (range 2-9). there were 2 deaths within 100 days of transplant. one patient died on day +11 during his second icu admission with multi organ failure (mof). one patient died after icu discharge on day +23 with relapsed disease, bronchopneumonia with disseminated fungal infection. icu mortality rate was 50%, and 100-day mortality rate was 10%. nineteen autologous patients were included (median age 61 (range 36-71 years)), 16 (84%) were myeloma patients who were conditioned with melphalan, 3 (16%) were lymphoma patients who were conditioned with beam. the icu admission was 0%. the 100-day mortality rate was 0%. conclusions: our centre's icu admission rate, icu mortality rate, cause of icu admission in allo-hsct patients and autologous patients is comparable to literature reports. autologous transplant is safe with no deaths and icu admissions despite an older age. the mortality rate for allo-hsct patients requiring icu admission remain high. all patients were appropriately referred to icu and there was no one who was denied icu admission. this analysis is being extended to preceding years. disclosure: nothing to declare liposomal doxorubicin for the treatment of iatrogenic kaposi sarcoma following hematopoietic stem cell transplantation background: iatrogenic kaposi's sarcoma (iks) represent a rare complication after hematopoietic stem cell transplantation (hsct), related to hhv-8 infection in hivnegative immunocompromised patients (pts). methods: we describe a case of iks occurred after an allogeneic hsct and we provide a review of the literature using pub med. results: a 70-year-old man, hiv-negative, received full hla-matched related hsct after a reduced intensity conditioning regimen for relapsed aml. gvhd prophylaxis was based on atg (fresenius 30 mg/kg), cyclosporine (cya) and methotrexate. no severe complication occurred in the first 100 days after transplant. shortly after cya withdrawal, he developed grade i acute gvhd. gvhd resolved after restarting cya. at fifth month after transplant, the patient developed several red and purple angiomatous plaque and nodules involving the skin of both lower limbs, right arm and the nose (figure 1). skin biopsy revealed multiple localizations of iks and positive hhv-8 viremia was detected in the peripheral blood. a visceral involvement was excluded. patient was treated with cya tapering and nine courses of liposomal doxorubicin 20 mg/m2 every 15 days, obtaining a negativity of hhv-8 viremia and partial response of the skin lesions. at last follow up, at 15 months after transplant, the patient was in complete remission (cr) for aml, cya-free without signs of gvhd recurrence and with his single stable residual iks lesion on his left limb, currently waiting for local radiotherapy. we found additional 18 iks published cases after hsct. most of post-hsct iks were secondary to an allogeneic-hsct (16 out of 19, 84.2%) and occurred in adult (78,9%) and male (68,4%) pts. median age at the time of iks diagnosis was 47.5 years (range 7-70). thirteen pts (68.4%) had mediterranean origin. the most frequent underlying disease was aml (47.4%). gvhd prophylaxis was primary based on calcineurin inhibitor. half of the pts developed gvhd and were treated with steroid and other immune suppressive drugs. median time between the hsct and the occurrence of iks was 8.5 months (range . cutaneous iks was the prevalent form of manifestation, however visceral involvement was reported in 7 pts (36.8%). in four cases (21.1%) an hhv-8 associated bm failure was report. immune suppression drugs tapering (36.8%) and chemotherapy (26.3%) were the most frequent actions taken after the diagnosis of iks. in most cases, liposomal doxorubicin was used as chemotherapy. cr rate was high, 63.2%, whereas progression disease occurred in 5 out 19 pts (26.3%), all of which had visceral involvement. in 3 pts (15.8%), iks was the cause of death. conclusions: withdrawn of immune suppression drugs and anthracycline based chemotherapy can represent a feasible treatment option for pts with iks after hsct. clinical background: acquired haemophilia a (aha) is an autoimmune disease caused by the spontaneous production of neutralizing immunoglobulin g (igg) autoantibodies (inhibitors) targeting endogenous fviii. treatment of these inhibitors presents additional challenges in a hematopoietic stem cell transplantation (hsct) recipient, because preservation of the graft that restores a normal hematopoiesis is critical. here we describe the management of a case of aha in an acute myeloid leukemia patient following hsct. methods: the clinical, laboratory and molecular aspects of a 56-year-old italian male who developed aha after allogenic bone marrow transplantation were collected and presented in order to show how we diagnose and manage this severe but rare complication within the special setting of hsct. results: a 56-years-old man with a flt-3 itd, npm-1, runx1-runx1t1 and cbfb-myh11 negative, not differentiated, chromosomally normal acute myeloid leukemia (aml) in third complete remission (cr) was submitted to a hematopoietic stem cell transplantation (hsct) from his haploidentical son. the conditioning regimen consisted of oncothiotepa, busulfan and fludarabine and was followed by the infusion of a t-cell depleted bone marrow graft. gvhd prophylaxis consisted of cyclosporine a (csa) and mycophenolate mophetyl (mmf). neutrophil engraftment occurred on day +24. recipient's autoimmunity was negative. at 27 months post-transplantation the patient received an antipneumococcal vaccination. fifteen days post-vaccination the patient was admitted to our in-patient ward due to general malaise, diffuse muscle and joint pains, cutaneous bleedings, oedemas, hyperchromic urines and constipation. physical examination revealed diffuse ecchymosis, swelling of deep muscles with a progressive functional disability due to hematomas and hemorrhagic suffusions of the tongue frenulum. and anti-factor viii inhibitors 6.46 bu/ml (high titers >5 bu/ml). thus, a diagnosis of acquired autoimmune haemophilia a was made and treatment with feiba combined with prednisone was started. patient's clinical conditions dramatically improved as he referred an improvement of movements and the resolution of joint and muscle pains despite the persistence of deep hematomas just after one day of treatment that had determined an increase of fviii:c value to 1.32% and an improvement of aptt to 47.06 seconds. on the following medical checks physical examination showed the progressive disappearance of deep muscle hematomas, and normal values of fviii:c. conclusions: aha is a rare but severe complication following hsct and it could appear years afterengraftment. a prompt diagnosis and an early treatment with feiba and corticosteroid are necessary to avoid life-threatening sequelae. the inclusion of the coagulation panel in the laboratory exams performed during the follow-up is advisable in order to early detect this life-threatening complication. disclosure: nothing to declare background: splanchnic thrombosis is an uncommon complication of myelofibrosis and a controindication to proceed to hematopoietic stem cell transplantation (hsct) due to the risk of additional vascular and endothelial complications. we present a patient with myelofibrosis (mf) that proceeded to hsct from an unrelated donor, despite splanchnic thrombosis unresolved after heparin treatment and unable to proceed to surgical treatment due to severe thrombocytemia. methods: a 67-year woman with mf secondary to essential thrombocythemia, with intermediate-2 score according dynamic international prognostic staging system (dipss) and with extreme splenomegaly (maximum diameter 30 cm), refractory to ruxolitinib, showed an extensive thrombosis of the portal and splenic veins, unresolved after 4-week heparin therapy, at the time of availability of an hla (8/8) and abo matched unrelated donor. she received a conditioning regimen including fludarabine and thiotepa and a gvhd prophylaxis with atg thymoglobuline, cyclosporine and methotrexate, followed by the reinfusion of 5.8x10 6 /kg cd34+ pbsc. at the time of transplant we were aware of an high risk of developing sos, on the basis of the older age of the recipient, the unrelated donor, the advanced stage of myelofibrosis and the ferritin serum level of 2.223 ng/mg. results: on day +9 after hsct sos complicated the aplasia phase, characterized by jaundice, ascites, weight gain, progressive increase in creatinine and bilirubin serum levels. an ultrasound of abdomen confirmed an unchanged thrombosis extension and the development of ascites. on day +10 the patient was categorized as very severe sos stage, according to ebmt severity criteria, because of doubling of bilirubin serum level in 48 hours and a 20% increase in comparison with her baseline weight. therefore, defibrotide was promptly started in association with diuretic therapy. the treatment was continued for 3 weeks and allowed gradual restoration of the water balance and normalization of bilirubin serum level. at the last follow-up, 6 months after hsct, the patient shows the persistence of a non-transfusion dependent anemia, platelets 80.000x10^3/ul, palpable spleen 4 cm below the rib, >95% allogeneic chimerism in the granulocytic compartment and 90% in the t lymphocyte compartment. splanchnic thrombosis is partially recanalized and replaced by collateral circles with cavernous aspects. the patient is on treatment with fondaparinux and has shown neither significant infectious episodes or acute or chronic gvhd. conclusions: we conclude that defibrotide treatment allowed to perform a successfull allogeneic transplant in a patient with mf associated with an overt picture of splanchnic thrombosis. background: hematopoietic stem cell transplantation (hsct) is associated with an increased incidence of secondary malignancies including skin cancer. squamous cell carcinoma (scc) is the most common type in patients who are receiving immunosuppressive therapy and chronic graft-versus-host disease (cgvhd) appears to be an important risk factor for its development. recent studies describe voriconazole exposure as an independent factor that may contribute to this increased risk as well. in our best knowledge, no cases of scc have been reported in pediatric allogeneic hsct to date. methods: we present a case report of a 9 year-old boy who developed a scc with high-risk features six years after undergoing hematopopoietic stem cell transplant. results: a 9 year-old boy with acute lymphoblastic leukemia (all) underwent a matched unrelated bone marrow transplant 7 years ago. he developed grade iv agvhd followed by extensive cgvhd with generalized scleroderma. he required intensive and continued immunosuppressive therapy and was on prolonged antifungal prophylaxis with voriconazole. in march 2017, he developed scc involving left temporal region that was completely excised. two months later, more lesions in scalp and nose were noted and intralesion treatment with methotrexate was started. however, an unfavorable evolution was noted and he was put on systemic treatment including cisplatin and cetuximab receiving the whole scheme from january to march 2018 and continuing only with cetuximab, ten doses in total, until may, for unaceptable and severe tubulopathy that required admission at the hospital in several ocassions. he achieved a very good partial response but progression was noted shortly in follow up. at this point, non curative therapeutic options were found and he was put on intralesion methotrexate and photodynamic theraphy in a weekly basis with palliative intention. unfortunately, tumor growth was fast and patient passed away in august 2018, fifteen months after squamous cell carcinoma diagnosis, due to tumoral progression. conclusions: 1) scc is a rare, non-previously described, secondary malignancy in children undergoing hsct. 2) high-risk features scc constitutes an aggresive disease with a median overall survival below 1 year. 3) cgvhd appears to be an important risk factor for its development. 4) voriconazole induced-photosensitivity might have played a role. 5) cisplatin based regimens +/-cetuximab are a therapeutic option in disseminated and/or high risk cases. as outcomes are unsatisfactory in these cases, alternative therapeutic options need to be explored. disclosure background: pregnancy is a rare event after allogeneic stem cell transplantation (sct) for acute leukemia. here we report, to the best of our knowledge, for the first time on a successful pregnancy after treosulfan-based conditioning. methods: a 29-year old woman was diagnosed with acute myeloid leukemia (aml) secondary to chronic myelomonocytic leukemia in july 2015. ovarian preservation was performed by leuprolide acetate depot injection prior to cytostatic chemotherapy. of note, no cryopreservation of oocytes or ovarian tissue was conducted. she received two cycles of chemotherapy consisting of idarubicine (12mg/m² on day 1-3) and cytarabine (1000mg/m² b.i.d. on days 1, 3, 5 and 7). due to secondary origin of aml sct was performed in first complete remission of aml after conditioning with treosulfan (14g/ m² days 3-5) and fludarabine (30mg/m² days 1-5). she received 4.94×10 6 cd34-positive cells per kilogram body weight from a hla-matched unrelated donor. results: follow-up bone marrow aspirates showed continuous complete remission of aml. seven months after sct she became pregnant, but decided for induced abortion. in january 2018, 25 months after hsct she became pregnant again and desired the child. medical examinations were performed monthly on an outpatient basis in stringent cooperation with the maternity clinic. the course of pregnancy was unremarkable, although she was hospitalized due to premature labor in the 34 th week of pregnancy. however, gynecological examination showed no clinical significant findings, so that section was planned and she could be discharged again. in the 38 th week of pregnancy she gave birth to a healthy girl (50cm, 2810g) by cesarean section. peripartum she developed hypoethesia of the left body half. neurological examination showed no abnormalities and she recovered immediately. there were no other postpartum complications. breastfeeding was established but additional food was necessary for a sufficient nutrition of the child. conclusions: this case of successful pregnancy following sct demonstrates that fertility can recover after treosulfan-based conditioning. however, detailed studies of ovarian function and fertility are necessary to gain more insight into the risk of premature ovarian failure. disclosure: nothing to declare. experimental stem cell transplantation p219 cd19-cart therapy before allo-hsct in children and adolescents patients who diagnosed r/r b-all with e2a-pbx1 background: b-all with e2a-pbx1 in children and adolescents is described with favourable prognosis. but there are more than 10% patients with e2a-pbx1 diagnosed as relapsed or refractory. the results of allo-hsct in children and adolescents with this group leukemia in our center was analyzed in order to understand the therapeutic effect of cd19-cart on the patients. methods: retrospective analysis, from june 1st, 2012 to july 31,2018, all children and adolescents diagnosed relapse or refractory b-all with e2a-pbx1 who received allo-hsct, total 30 cases. all patients was divided into two groups depending on whether or not accepted cd19-cart before allo-hsct. according to fcm-mrd and e2a-pbx1 level before allo-hsct, os lfs and cumulative recurrence rate were analyzed. r 3.2.0 was used as statistical analysis software. results conclusions: 1. for r/r b-all with e2a-pbx1 in children and adolescents, fcm-mrd pre-transplant hasn't obvious effect on the outcome of allo-hsct, while the level of e2a-pbx1 has obvious effect. the out come of e2a-pbx1 negative group was obviously better than positive group. 2. cd19-cart can obviously improve the os and lfs, it is mainly because of cd19-cart can makes more patients fusion to zero. 3. for r/r b-all with e2a-pbx1 in children and adolescents, if chemotherapy can't make the fusion to zero. it is suggested to accept cd19-cart therapy to make the fusion zero. it can improve the outcome of os and lfs. disclosure background: currently, hematopoietic stem cell transplantation (hsct) represents the only curative treatment for numerous hematopoietic malignancies like leukemias, immune deficiencies or metabolic diseases. cd34 serves a quality marker for stem cell grafts, which is not solely expressed on stem cells but also on a variety of progenitors. the role and the impact of these subpopulations remains unknown. we made use of our genetic barcode system to analyze the influence and contribution during reconstitution on a clonal level. methods: fluorescence activated cell sorting (facs) was used to sort hematopoietic stem and progenitor populations, namely hscs, mpps, cmps and clps, which were lentivirally transduced with our previously established bc32 barcoding system. after mixing the marked cells with bone marrow support, lethally irradiated recipient animals were and transplanted and monitored over 16 weeks. we focused on bone marrow, blood, spleen and thymus, on chosen endpoints (1w, 3w, 8w, 16w) and samples were used to analyze the contribution of the subpopulations during the reconstitution process based on fluorescent protein (fp) expression. to investigate the clonal contribution in different organs, we performed next generation sequencing (ngs) and frequencies of unique barcodes in a sample were analyzed by bioinformatical approaches. results: a maximum of 15% of cells expressed the encoded fps, which were mostly derived from the hscs and mpps. cmp-derived cells were only detected 1 week after transplantation in the myeloid compartment. cells derived from the clps were not detected at any time point. we analyzed the barcode content of the differently marked cells after next-generation-sequencing. in accordance with the facs data, the majority of the clones during the 16 weeks of observation are derived from hscs and mpps. cmp-derived clones were only contributing during the first weeks and clp-derived clones are barely detectable. we did not observe any major differences with regard to age of donor or recipient, despite the total number of clones is higher in the group, which received the "aged" graft, independently from the transduced cell population. conclusions: here we show the suitability of our highly complex multi-color barcode system to study the clonal contribution of hscs and three progenitor populations after hsct. our results will contribute to a better understanding how these different populations interact to support the establishment of a new hematopoietic system. emphasized by the variability in data of graft and recipient age, this comprehensive analysis gives rise to an impression to the necessity of personalized graft composition, by which treatment success could be influenced. disclosure: nothing to declare survival and fate of adipose derived mesenchymal stem cells in a rat brain injury model background: mesenchymal stem cells have been identified as promising candidates in the treatment of central nervous system (cns) injury through neurotrophic support and immunomodulation. adipose tissue is an attractive source of mesenchymal stromal/stem cells (ascs) for regenerative therapeutic applications because they can be harvested from autologous donors with minimally invasive methods, can be rapidly expanded ex vivo, show low immunogenicity if allogeneic, and can be used in autologous or heterologous settings. the present study examines the fate and effects of intracerebroventricularly (icv) transplanted ascs in a traumatic brain injury (tbi) model. methods: ascs were isolated from inguinal fat pad of adult wistar rats under sterile conditions and cultured according to standard procedures. ascs at passage 2 (2x10 5 cells) were seeded and transfected with sleeping beauty transposase and pt2 venus-neo r plasmids. selection with g418 antibiotic resulted in the generation of a homogeneous asc population which expressed fluorescent venus protein for several passages, phenotypic characterization showed that these cells were 99.6% double positive for cd44 and cd90 stem cell markers, verifying their mesenchymal origin. tbi was induced by stereotactic surgery under deep anaesthesia and subsequently icv transplantation of venus+ ascs was performed on adult wistar rats. normal ascs-transplanted and tbi-saline transplanted rats were used as controls. the proliferation, migration, survival and fate of transplanted ascs and their effect on injury restoration were examined six weeks post transplantation (pt). results: six weeks pt ascs expressed the fluorescence venus protein and therefore were identified in brain parenchyma. their presence into brain was also confirmed by masson trichrome staining, which revealed their collagen depositions. ascs were found in lesser numbers compared to those transplanted and exhibited no proliferative activity. ascs were found scattered distributed in brain as individual cells, and there were no aggregates of ascs or mass formation into lateral ventricles. extensive migration of ascs was mainly performed through white matter tracks in the corpus callosum and fimbria of hippocampus. six weeks pt ascs retained the characteristics of mesenchymal cells and did not differentiate into cells of neural lineage. ascs exhibited limited long-term survival, which is restricted in perivascular areas probably contributing to vascular formation. homing of ascs into peri-injured area was detected in half of the animals and achieved through the corpus callosum, as revealed by the collagen depositions, in this white matter track. transplanted ascs reduced the area of tbi cavity and did not enhance the astroglial scarring in peri-injured area. in tbi +ascs transplanted animals, the cortical injury site, showed a significantly smaller volume and lower % tissue loss compared to that of tbi+vehicle animals (1.90 ±0.38mm 3 and 12.25±2.83% respectively, versus 1.12 ±0.34mm 3 and 6.57±1.67%, p=0.015 and p=0.019 respectively). conclusions: considering the effects of ascs on inflammation and regeneration, we suggest that their transplantation after brain injury may promote host brain repair mechanisms. ascs transplantation may be beneficial in tbi, however some of its effects need careful and indepth evaluation. disclosure: nothing to declare xie-na cao 1 , yuan kong 1 , zhong-shi lyu 1,2 , qi wen 1 , min-min shi 1,2 , qian-yu sun 1 , yu-hong chen 1 , yu wang 1 , lan-ping xu 1 , xiao-hui zhang 1 , xiao-jun huang 1,2 background: poor graft function (pgf) remains a serious complication after allogeneic hematopoietic stem cell transplantation (allo-hsct). our previous work reported that abnormal bone marrow (bm) endothelial cells (ecs) were involved in the pathogenesis of pgf patients after allo-hsct (bbmt2013; bmt 2016; blood2016), but the explicit mechanism requires further clarification. autophagy is a self-degradative process responsible for the elimination of cytosolic components including proteins and damaged organelles. recent findings demonstrated that stimulation of autophagy could reduce oxidative status and angiogenic potential in ecsafter high-glucose exposure, from diabetic patients.however, little is known regarding the autophagy of bm ecs in pgf patients. therefore, the current study was performed to evaluate whether autophagy in bm ecs play a role in the pathogenesis of pgf. moreover, to investigate the effects of autophagic regulation on ecs and thereby regulating hematopoietic stem cell (hscs). methods: in the prospective case-control study, the autophagy levels were compared in bm ecs from pgf patients, and their matched good graft function (ggf) patients.the expression levels of autophagy-related markers (lc3, beclin1, and p62), and intracellular autophagosomes were detected by immunohistochemical staining, flow cytometry, western blot and transmission electron microscopy. subsequently, rapamycin (the autophagy activators) or hydroxychloroquine (hcq, the autophagy inhibitor) were administrated tothe 7-day cultivated bm ecs and human umbilical vein endothelial cells (huvecs), respectively.the autophagic vacuoleswere detected by monodansylcadaverine (mdc) staining assay. the bm ecsand huvecs were evaluated by cell counting, dii-ac-ldl and fitc-lectin-uea-1 double staining, migration, cell proliferation, and levels of reactive oxygen species (ros). to explore whether autophagy would affect the ability of bm ecs to support hscs in vitro, bm cd34+ cells from healthy donors were co-cultured with cultivated bm ecs and huvecs. colony-forming unit (cfu) and the apoptosis of co-cultured hscs were analyzed. results: the defective autophagy in bm ecs, characterized by decreased intracellular autophagosomes and autophagic vacuoles, decreased expression of lc3-ii and beclin1, and high level of p62, were observed in pgf patients compared with ggf patients. moreover, the coculture of bm cd34+ cells with bm ecs showed significant deficient cfu plating efficiency, and increased apoptosis of cd34+ cells in pgf patients. in vitro upregulation of autophagy by rapamycin quantitatively and functionally improved bm ecsand huvecs, which manifested as more dii-ac-ldl and fitc-lectin-uea-1 double stained cells, increased capacities of migration, lower levels of ros and apoptosis via regulating beclin1 pathway, whereas inhibition of autophagy by hcq aggravated the huvecs and bm ecs from pgf patients. furthermore, in vitro upregulation of autophagy by rapamycin significant improved cfu plating efficiency, and decreased apoptosis in bm hscs co-cultured with huvecs and bm ecs from pgf patients. conclusions: these findings suggest that defective autophagy in bm ecs may be involved in the pathogenesis of pgf. the effect of rapamycin in pgfpatients is potentially mediated by improving the dysfunctional bm ecsto support hscs. therefore, it would be of value to investigate whether upregulating of cytoprotective autophagy of bm ecs may ameliorate pgf, thereby providing a novel clinical intervention for pgf in the future. clinical background: heparanase (hpse) in an endoβ-glucuronidase that specifically cleaves the saccaride chains of heparan sulphate proteoglycans (hs), leading to a loss of integrity of the extracellular matrix and to release of hs-bound cytokines, chemokines, angiogenic and growth factors. hpse gene is polymorphic and includes approximately 300 snps. the combination of two snps, rs4693608 and rs4364254, are involved in the regulation of hpse expression with an inverse correlation between mrna expression and protein levels: gg-cc, gg-ct, gg-tt, ga-cc (low group) expressed high hpse concentration; ga-ct and ga-tt (median group) expressed intermediate hpse levels; aa-tt and aa-ct expressed low hpse concentration (high group). we studied hpse snps in the allogeneic stem cell transplantation (hsct) setting to evaluate a possible association with post-hsct outcomes. methods: we enrolled 228 patients submitted to hsct in our department since 2005 to 2016. for each couple recipient-donor, rs4693608 snp was genotyped using restriction fragment lenght polymorphism assay, whereas for rs4364254 snp an allele-specific polimerase chain reaction was applied. hpse genotype distribution was compared in different groups according to post-hsct outcome: graft-versus-host disease (gvhd), transplantrelated mortality (trm), overall survival (os), infectious complication and disease-free survival (dfs). statistical analysis was performed using ncss 10. results: distribution of rs4693608 snp was as follows: gg 16.7%, ga 49.8% and aa 33.5% among recipients and 17.6%, 53.3% and 29.1% among donors, respectively. hardy-weinberg equilibrium (hwe) was respected. distribution of rs4364254 snp was as follows: cc 15.1%, ct 37.8% and tt 37.8% among recipients and 15.3%, 36% and 48.7% among donors, respectively. rs4364254 snp distribution did not respect the hwe. an association was found between recipient rs4364254 snp and the cumulative incidence of agvhd among patients submitted to a reduced intensity conditioning (ric): 37.3% for tt genotype and 69% for ct or cc genotype (p=0.03). on the other hand, an association was identified between donor rs4693608/rs4364254 snps combination and the cumulative incidence of agvhd: 81.5% for low group donor, 48% for median group donor and 40.8% for high group donor (p=0.04). conversely, aa genotype for donor rs4693608 resulted independent risk factor for cgvhd de novo development (p=0.049, od 2.1) together to donor-recipient sex mismatch (female donor to male recipient vs. others: p=0.005, od 3.56) . considering cmv reactivation rate after hsct, an association was observed according to recipient rs4364254 snp: 82% for cc genotype, 63.5% for ct genotype and 57.1% for tt genotype (p=0.049). multivariate analysis confirmed recipient rs4364254 snp as independent risk factor for cmv reactivation after hsct (p=0.04, od 2.62) together with recipient cmv serostatus at transplant (positive vs. negative: p< 0.01, od 8.49). conclusions: hpse role was widely studied in the setting of inflammation, autoimmune diseases, hematological disease and tumor. however, it still remains debated the inducing or protective activity of hpse in the setting of gvhd. obviously, our results need to be confirmed in a validation cohort. clinical trial registry: na disclosure: nothing to declare novel protocol for autologous hsct in patients with high risk of complications: ambulatory chemomobilization and transplantation of fresh hematopoietic stem cells with backup storage background: autologous hematopoietic stem cell transplantation (ahsct) is standard of treatment in many patients with high risk of complications: dialysed patients, patients with heart and kidney amyloidosis or patients with systemic sclerosis. we introduced recently a novel protocol for ahsct: combination of ambulatory mobilization with very low doses of ara-c and g-csf connected with direct ahsct with fresh cells. this protocol allowed us to reduce the transplant risk in various patient groups traditionally connected with high risk of complications. in this work we summarize the experience in such high risk patients. methods: the prospectively collected database of patients after ahsct was searched for patients who underwent ahsct after chemomobilization with ara-c and transplantation with fresh cells and who fulfilled at least one study inclusion criteria: a) dependence on dialysis b) amyloidosis c) systemic sclerosis d) disqualification from transplantation at other centre due to the high risk of complications. there were together 19 patients selected for this analysis -9 with amyloidosis (6 with ≥ 2 organs involved), 9 dialysed, 2 with systemic sclerosis, 2 unfit at other centre. the database included prospectively recorded serious adverse events during the mobilization and transplantation. results: there were 20 transplantations performed in this group of patients. mortality was 0% at 100 days. all patients underwent successful ambulatory mobilization. all patients received mephalan conditioning with single infusion with median dose of 200mg/m2 (min 140, max 200). mean engraftment was 10.5 days for white blood cells and 12.7 days for plt over 20 g/l. the rate of complications was low with 7 cases of neutropenic fewer, 1 single bacterial culture with staphylococcus epidermidis without clinical signs of infection, median mucositis grade of 0.4 and without patients on parenteral nutrition. the median time of hospitalization was 19 days (min 14, max 29). conclusions: we present here novel protocol of transplantation combining chemomobilization and ahsct with fresh cells with excellent safety profile among most severely ill patients allowing for safe and efficient transplants. with this protocol we were able to overcome multiple risk factors and perform full intensity transplantation in very fragile patients. disclosure: nothing to declare single umbilical cord blood transplantation provides durable disease remission of advanced hematological malignancies in elderly patients background: although allogeneic hematopoietic stem cell transplantation (allo hsct) is potentially curative therapy in a variety of hematological malignancies, little has been reported of the outcome for elderly patients who are not in remission at transplantation. but it has been pointed out that recipient age alone can not be regarded as contraindication for allo hsct in the literature recently, supported by suitable donor, conditioning regimens and appropriate management of complications. we conducted a retrospective study of elderly patients who had advanced hematologic malignancies to elucidate the outcome of single umbilical cord blood transplantation (sucbt) in toranomon hospital kajigaya, japan. methods: we retrospectively investigated the outcomes of 19 patients aged over 65 who underwent their first ucbt from june 2013 to december 2017 in our medical center. results: diseases included acute myelogenous leukemia (n=12), myelodysplastic syndrome (n=3), adult t-cell leukemia/lymphoma (n=2), myelofibrosis (n=1) and chronic lymphocytic leukemia (n=1). the median age at transplantation was 69 years (range, 65-75) and follow-up for survivor post transplantation was 642 day (range, 391-767). all patients were not in complete remission (cr) at the time of transplantation. reduced intensity conditioning (ric) regimens were used in 10 patients. all patients received tacrolimus and mycophenolate mofetil as graftversus-host disease (gvhd) prophylaxis. all cases except 4 early death achieved neutrophil recovery at median 18 days (range, 13-28). at 1 year, overall survival (os) rate and disease free survival (dfs) were 31,6% (95% confidence interval (ci), 12.9-52.2). we performed univariate analysis to identify the factor that influenced os at 1 year, but no statistical significance was demonstrated at the age of transplantation (aged 65 to 69 vs. ≧70, 33.3% (95% ci, 10.3-58.8) vs. 42.9% (95% ci, 9.8-73.4), p=0.68). the cumulative incidence of non-relapse mortality (nrm) at 100 days was 47.4% (95% ci, 23.6-67.9%) and relapse at 1 year was 9.1% (95% ci, 0.0-24.6%). only two patients developed acute gvhd(ii-iv) and one developed severe gvhd at 49 days after transplantation. the main causes of death was infection (n=10), including sepsis (n=8) and viral encephalitis (n=2), followed by idiopathic pneumonia syndrome (n=2) and thrombotic microangiopathy (n=1) during the early phase of transplantation. in contrast, no patients died of recurrence. conclusions: although our report consisted relapsed/ refractory disease of elderly patients at the time of sucbt, durable remission and lower incidence of gvhd could be noteworthy compared with previous reports. further strategies to reduce the rate of nrm and longer duration of follow up would be warranted. disclosure background: pearson syndrome and kearns-sayre syndrome are metabolic disorders caused by a de-novo deletion in the mitochondrial dna (mtdna). allogeneic stem cell transplantation has shown to improve metabolic function in distal organs in several metabolic disorders, but bears significant morbidity and mortality, especially for patients with mitochondrial disorders. novel gene therapies may correct diseases rising from genomic dna mutations, but targeting the mitochondrial dna is complex. mitochondria are able to transfer into cells and between cells, as seen in preclinical models of mitochondrial and other metabolic disorders. here, we introduce a novel concept of mitochondrial augmentation therapy (mat) of autologous cd34+ cells in 4 children with mitochondrial deletion syndromes. methods: patients were treated under a compassionateuse program, approved by the sheba medical center irb and the israeli ministry of health. briefly, mobilization was performed using gcsf alone (n=1) or in addition to plerixafor (n=3) . cd34+ cells were isolated via miltenyi clinimacs system and co-cultured with maternal mitochondria, drawn from peripheral blood and confirmed nondeleted, for 24 hours, and re-infused to the patient without any conditioning. patients were followed for clinical and metabolic parameters. results: all four patients presented with different deletions in mitochondrial dna, and different baseline characteristics, and were treated at the age of 6.5, 7, 11 and 14 years. despite normal cbc, significant bone marrow hypocellularity was seen in 3 evaluated patients (20%, 30% and 50% cellularity at age 7, 11 and 14), which correlated with low colony forming unit capacity of patients and low yield of cd34+ mobilization in the leukapheresis product. patients received on average 2x10 6 enriched cells/kg (range, 1.1 -2.8), and the median enrichment of cd34+ cells was 135% (range, 103-162%). no infusion reactions occurred, and the only severe adverse events of this cellular therapy were leukapheresis-related anemia, hypokalemia, hypocalcemia and alkalosis, all resolved promptly with proper supplementation. follow-up duration is variable, ranging 5-22 months. we were able to show improvement in mitochondrial heteroplasmy (proportion of deleted mtdna of total mitochondrial dna) and in normal mtdna content, starting 1-5 months from cell therapy, which correlated with improved atp production in peripheral blood derived mononuclear cells. clinically, patients showed improvement in aerobic function and endurance (measured by the half-bruce protocol, sit-to-stand test and 6-minute walk test), muscle strength (hand-held dynamometry), and in quality of life, measured by the international pediatric metabolic disability scale. no metabolic crises occurred following cell infusion. conclusions: patients with deletion in mtdna have metabolic dysfunction, including poor bone marrow cellularity and function. hematopoietic stem cells in patients with mtdna deletions can be enriched with normal mitochondria, via mat, as first shown in our patients. this novel process is safe and results in increase in the normal mtdna in peripheral blood of patients, and in improved metabolic and clinical function. clinical trial registry: clinicaltrials.gov nct03384420 disclosure: moria blumkin, noa sher and natalie yivgi ohana -minovia therapeutics, employment p227 high cytotoxic efficiency of alpharetrovirally engineered cd19-specific chimeric antigen receptor natural killer cells for treatment of acute lymphoblastic leukemia stephan müller 1 , tobias bexte 1 , annekathrin heinze 1 , franziska schenk 2 , axel schambach 3 , winfried s. wels 4,5 , ute modlich 2 , evelyn ullrich 1, 5 background: autologous chimeric antigen receptormodified (car) t cells with specificity for cd19 showed potent antitumor efficacy in clinical trials regarding relapsed and refractory acute lymphoblastic leukemia (all). natural killer (nk) cells are cytotoxic lymphocytes that are capable to kill their targets in a non-specific manner and additionally do not cause gvhd. therefore, using cd19-car-nk cells exhibits several advantages, such as safety in clinical use, possible allogenic settings and the potential to also attack heterologous leukemia cells which lost cd19. previous approaches used cd19-car-nk cells pre-stimulated by feeder cells, bearing potential risks. thus, we focused on the optimization of generating cd19-car-nk cells by viral transduction under feeder-cell free conditions. methods: human nk cells were isolated from healthy donor peripheral blood mononuclear cells via cd56 negative selection. after a feeder-cell free expansion phase with interleukin 15, transductions were performed with an egfp or a cd19-car encoding vector at different multiplicities of infection (moi). to optimize gene modification different transduction enhancers (retronectin and vectofusin-1) and viral vector systems (lentiviral and alpharetroviral) were compared. finally, generated cd19-car-nk cells were tested in their ability to kill cd19positive and cd19-negative cell lines. results: nk cells transduced with a lentiviral egfp encoding vector or a lentiviral cd19-car vector using retronectin and vectofusin-1 showed similar transduction efficiencies for both transduction enhancers (egfp: retronectin moi 10: 12.9%; vectofusin-1 moi 10: 12.8%; cd19-car: retronectin moi 5: 10.7%, moi 10: 9.2%; vectofusin-1 moi 5: 11.3%, moi 10: 14.4%). the generated cd19-car-nk cells showed increased cytotoxic capacity against cd19-positive cells compared to nontransduced (nt) nk cells (72.7% vs. 23.6%, effector to target (e:t) ratio 1:1). both nk cell populations were equally efficient in killing cd19-negative cells (30.5% vs. 25.7%). alpharetroviral transduction of nk cells with an egfp encoding vector showed higher transduction rates with vectofusin-1 than with retronectin (retronectin moi 1: 5.3%, moi 5: 9.7%; vectofusin-1 moi 1: 55.3%, moi 5: 51.6%). further using vectofusin-1, similar transduction efficiencies could be achieved with an alpharetroviral cd19-car encoding vector (moi 1: 11.1%, moi 5: 49.2%, moi 10: 68.9%), outperforming the efficiencies of lentivirally generated cd19-car-nk cells in the same experiments (moi 1: 1.5%, moi 5: 8.4%, moi 10: 14.9%). additionally, alpharetroviral cd19-car-nk cells showed a higher cell killing activity against cd19-positive cells than lentiviral cd19-car-nk cells or nt-nk cells (90.5% vs. 62.5% vs. 9%, e:t ratio 1:1). interestingly, similar killing activities were achieved with an e:t ratio of 0.5:1 (88.9% vs. 58.3% vs. 10.3%) and alpharetroviral cd19-car-nk cells remained a stable cytotoxicity level at lower cell concentrations down to an e:t ratio of 0.1:1. all three nk cell populations were equally efficient in killing cd19negative cells (16.4% vs. 23.6% vs. 12.3%, e:t ratio 1:1). conclusions: cd19-car-nk cells can be successfully generated under feeder-cell free conditions using different transduction enhancers and viral vector systems. these data suggest the usage of vectofusin-1 in combination with alpharetroviral vectors to genetically modify nk cells to achieve sufficient amounts of transduced cells. these cd19-car-nk cells mediate high cytotoxicity and therefore may offer a new therapeutic option in the treatment of all. disclosure: axel schambach is an inventor on a patent describing alpharetroviral sin vectors. winfried s. wels is an inventor on a patent describing chimeric antigen receptors with an optimized hinge region. the remaining authors have nothing to disclose. graft-versus-host diseaseclinical walter spindelböck 1 , bianca huber-krassnitzer 1 , barbara uhl 1 , gregor gorkiewicz 1 , hildegard greinix 1 , christoph högenauer 1 , peter neumeister 1 background: steroid-refractory acute gastrointestinal (gi) graft-versus-host disease (agvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation (allo-hsct) associated with a high mortality rate. loss of intestinal bacterial diversity is thought to be associated with severity of gi-agvhd and an impaired intestinal microbiota with reduced diversity is an independent predictor of mortality. methods: the fecal microbiota transplantation (fmt) procedures were performed according to a protocol approved by the local ethical committee (29-027ex 16/17) after obtaining informed consent. donors were healthy adult subjects screened for potential infections by serologic and microbiologic tests according to local standards. donor stool was diluted with saline and homogenized to a volume of~250 ml fecal solution for instillation into the terminal ileum and caecum via colonoscope. microbiota sequencing analysis of 16s rdna was performed before fmts and afterwards at predefined timepoints. results: we report the outcome of nine patients refractory to 3-6 lines of immunosuppressive therapies with lower gi-stage iii (n=1) or iv (n=8) agvhd following repetitive fmts from a single donor. all patients had received an allo-hsct for mds (n=3) , aml (n=4), pmf (n=1) and mm (n=1) following a reduced intensity (n=5) or mac (n=4) conditioning regimen using pbsc as stem cell source. after an onset of lower gi agvhd between 11-465 days after allo-hsct, nine patients refractory to several lines of immunosuppressive therapies received 1-6 fmts (6 patients were treated with more than 2 fmts, in 3 patients fmt was only administered once or twice) mostly in weekly intervals. five patients achieved a clinical complete response with resolved diarrhea and no gastrointestinal complaints, and four of these could be discharged without gvhd symptoms. two patients (pr, nc) were discontinued after 2 or 3 fmts in pr or nc due to concomitant infections (metapneumoviral pneumonia, cmv gastroenteritis), the 2 other non-responders succumbed to gvhdrelated infectious complications. the establishment of donors' microbiota with the emergence of new taxa, an increase in bacterial richness/diversity, and the disappearance of the "enterococcus signature" were associated with disease control and response to fmt. except the possible transmission of adenovirus by fmt in one patient, no other immediate procedure-related infections or other side effects were observed. conclusions: restoration of dysbiosis by fmt might represent a promising novel therapeutic approach for a subset of patients with refractory lower gi-agvhd. vigorous donor screening for infectious disease is mandatory. clinical background: migration of allo-activated donor effector tcells from lymphoid tissues to target organs is an important step in acute graft versus host disease (gvhd). the sphingosine-1-phosphate-1 (s1p1) receptor plays a crucial role in lymphocyte trafficking. data from animal models suggest that pharmacological modulation of the s1p1 receptor reduces gvhd and improves mortality. we investigated this mode of action by using the secondgeneration s1p1 modulator krp203 for the prophylaxis of gvhd in a pilot clinical trial in patients undergoing allogeneic hsct. methods: a multi-centric, phase 1b, prospective, open label, two-part study was conducted to evaluate the safety, tolerability and pharmacokinetics of krp203 in patients undergoing allogeneic hsct for hematological malignancies. primary endpoint was safety. initial efficacy was explored based on the incidence of gvhd, mortality and relapse. part 1 was a single arm open label study to investigate the safety of 3 mg/day krp203 added to standard of care gvhd prophylaxis (csa/mtx) in 10 patients. part 2 was a randomized two-arm open label study to compare the safety, efficacy and pk of 3 mg/day of krp203 in combination with tacrolimus/mtx to 1 mg/day of krp203 in combination with csa/mtx in 13 patients. in both parts, treatment with krp203 was initiated 10 days before hsct and continued for an additional 100 days. patients were followed up for up to 2 years. results: 23 patients were included in the study. 16 of 23 patients completed the 110-day treatment with krp203 at the assigned doses. median duration of follow-up was 264 days (range 153 to 271 days). krp203 was safe and well tolerated. 11 serious adverse events (saes) suspected to be related to krp203 were observed. macular edema (n=3) and peripheral edema (n=1) as s1p related adverse events occurred and resolved without sequelae. of note, the incidence of macular edema in hsct recipients is unknown. neutrophil engraftment was confirmed in all patients with a median of 16 days (range 12 to 45 days). 5 of 23 patients presented with grade iii or iv acute gvhd (on days 50, 56, 68, 99 and 102) . no gvhd or infection related death occurred during the first 100 days. 100-day survival was 96%, with no death occurring during krp203 treatment. 1 death occurred on study day 90 due to lymphoma relapse. a second death occurred on study day 121 due to liver gvhd. four patients died in the follow-up period due to gastrointestinal gvhd (day 265), aspiration pneumonia (day 327) and relapse (day 533 and day 877). the kaplan-meier estimate of overall survival at 1 year was 0.75. when comparing the data from the two dose groups (1 and 3 mg krp203), no major differences in safety, engraftment, gvhd rate or mortality were observed. conclusions: this clinical trial was the first to test s1p modulation in this population. our data suggest that krp203 had no negative impact on engraftment and overall, was safe, and well tolerated. based on exploratory data, when comparing to matched historical mortality data, krp203 may have favorable effects on overall survival ( figure 1 ). background: uric acid is a danger signal contributing to inflammation. relevance to allosct has been demonstrated in preclinical models: the depletion of uric acid led to improved survival and reduced gvhd (j exp med. 2013 sep 23;210(10):1899-910). results of a clinical pilot trial suggested that peri-transplant uric acid depletion reduce acute gvhd incidence (bbmt 2014 may;20(5):730-4). methods: this international multicentric study aimed to study the association of uric acid serum levels before start of conditioning with allosct outcome. patients with acute leukemia, lymphoma or mds receiving a matched sibling allosct for the first time were considered for inclusion, regardless of conditionning. data were prospectively collected between 8/2014 and 2/2018. a comparison of outcomes between patients with high and low uric acid level was performed using univariate analysis and multivariate analysis using cause-specific cox model. variables included in the multivariate analyses were age, sex mismatch, diagnosis, disease status, karnofsky score, number of cd34 cells given, intensity of conditioning, type of gvhd prophylaxis, atg use, time from diagnosis to transplant, year of transplant and cmv status. results: twenty centers from 10 european countries reported data on 385 allosct recipients. patient characteristics are given in table 1 . the uric acid cut off point was determined at 4.3mg/dl (median of measured uric acid levels). overall survival (os) and progression free survival (pfs) of allosct recipients with uric acid levels above cut off measured before start of conditioning were significantly shorter ( figure 1a , os univariate hr=2.4 ci=1.6-3.7 p< 0.001; multivariate hr=2.8, ci=1.7-4.7, p< 0.0001) ( figure 1b , pfs univariate hr=2 ci=1.1-3.7 p=0.02; multivariate hr=2.7, ci=1.4-5, p=0.003). nonrelapse mortality was significantly increased in allosct recipients with high uric acid levels prior to start of conditioning (univariate hr=2 ci=1.1-3.7 p=0.018; multivariate hr=2.65, ci=1.41-5.01, p=0.003). in addition, there was a non-significant trend towards higher acute gvhd incidence (gvhd grades ii-iv univariate hr=1.2 ci=0.8-1.9 p=0.4; multivariate hr=1.5 ci=1-2.4, p=0.08) in allosct recipients with uric acid levels above cut off before transplantation. finally, the incidence of relapse after allosct was moderately increased in the cohort with higher uric acid levels (univariate hr=1.6 ci=1-2.5 p=0.09; multivariate hr=1.59, ci=1.02-2.49, p=0.04). conclusions: high uric acid levels before start of conditioning correlate with high mortality after allosct. our results can serve as rationale for clinical trials on depletion of uric acid during allosct. results: we found significant correlation between donors' ctla-4 +49a>g polymorphism and hsct outcome. genotype aa was present in 170 donors, ag in 183 donors and 44 donors was homozygous for g allele. recipients who received graft from g allele carrier donors showed significantly increased cumulative incidence of relapse (at 24 months aa: 20.7%, ag: 23.6% and gg: 34.3%; p=0.04). on contrary, the frequency of the acute gvhd grades iii-iv and cytomegalovirus (cmv) reactivation/disease decreased according to the presence of the g allele in the donor ctla-4 genotype [agvhd: aa: 20%, ag: 12%, gg: 5%; p= 0.014; cmv: aa: 24%, ag: 16%, gg: 9%; p= 0.039]. cumulative incidence of agvhd was also markedly decreased among patients with g allele carrier donors (at 100 days aa: 19.9%, ag: 10.4%, gg: 6.4%; p=0.01). donor genotype similarly influenced hsct outcome in mud donor and mac conditioning subgroups. overall survival (os) was not different in patient subgroups according to donor genotypes [os at 24 months: aa: 55.5 ±3.8%, ag: 54.4±3.7%, gg: 49.4±7.6%; p= 0.68]. we did not find any correlation between recipients' ctla-4 +49a>g polymorphism and hsct outcome. conclusions: several ctla-4 snps have previously been described to be associated with relapse rate, incidence of agvhd and os, but results are often contradictory in the publications. in our study, ctla-4 +49a>g polymorphism of hsct donors influenced risk of relapse, agvhd, cmv and cause of death, but not overall survival. the genotyping of ctla-4 +49a>g polymorphism in donors may help in the risk assessment process and the choice of personalised therapy. disclosure: nothing to declare. background: although steroids remain first-line therapy for the treatment of acute graft versus host disease (agvhd), response rates in patients with grade iii-iv disease are poor, with no apparent improvement in survival over the past 15 years. we performed a prospective, multicenter trial to assess the efficacy and safety of the combination of ruxolitinib and etanercept as a novel approach to treat grades iii-iv sr-agvhd . methods: forty malignant hematologic disease patients with grades iii-iv sr-agvhd after allo-sct from three centers in east china were enrolled from january 2017 to june 2018. ruxolitinib was initiated at a dose of 5-10 mg bid for 2 months, and then tapered gradually for another one month. etanercept was administrated at 25mg biw for 2-8 weeks. results: the median age of patients was 25 (range 15-59) years. at day 30 after the combination treatment, the overall response rate (orr) was 90% including 30 crs (75%) and 6 prs (15%). the median time to the optimal response was 13 (range 3-34) days. the incidences of cr per organ were 95.7%, 80.8%, and 80% for skin, liver, and gut, respectively. the agvhd relapse rate was analyzed for the patients who had achieved cr or pr and survived beyond 60 days. relapses in agvhd occurred in 9.38% (3/ 32) of responsive patients. the patients who received ruxolitinib within 14 days after agvhd onset have a significant higher cr rate that those with delayed ruxolitinib therapy (96.2% vs. 42.9%, p=0.001). and the patients without gut infections have a significant higher cr rate than infected cohort (92.6% vs. 46.2%, p=0.002). by logistic regression analysis, the time from agvhd to ruxolitinib (rr=4.17, p=0.011) and gut infection (rr=3.31, p=0.031) were independent predictors for incomplete response. thirteen patients (13/40, 32.5%) suffered from at least 1 infectious episode after the start of the combination therapy, and pulmonary infectious diseases was a frequent complication (9/40, 22.5%). iii-iv cytopenia and cmvreactivation were observed in 30% and 47.5% of patients. the 1-year overall survival (os) after initiation of the combination therapy were 76.8%. the 1-year nrm and relapse incidence was 17.9% and 19.9%, respectively. patients with complete response on day 30 had significantly higher os probability than non-cr patients (1-year os: 86.1% vs 48.0%, p=0.01). compared with the historical cohort of basiliximab and etanercept for sr-agvhd in our center (n=31), no significant difference was found on the baseline. although the orr in patients treated with ruxolitinib and etanercept is identical with the historical cohort, ruxolitinib group achieved rapider remissions in liver agvhd and gut agvhd than the historical cohort (gut agvhd: 11 days vs. 17 days, p=0.026; liver agvhd: 21 days vs. 28 days, p=0.039), thus, with regard to hospital stay after agvhd onset, the ruxolitinib cohort stayed shorter (median: 18 days vs. 29 days, p=0.005) than basiliximab cohort. conclusions: combined treatment with ruxolitinib and etanercept resulted in a rapid cr to visceral agvhd and meanwhile reserve graft anti-leukemia (gvl) effect as the relapse rate of primary disease is relatively lower. the various infection complications associated with ruxolitinib merit more attention. disclosure: nothing to declare background: graft-versus-host disease (gvhd) remains one of the main life-threatening complications after allo-hsct, especially in patients with non-malignant diseases. the standard gvhd prophylaxis strategy is mostly based on the use of calcineurin inhibitors alone or in combination with other immunosuppressive (is) post-transplant cyclophosphamide (ptcy) is effective gvhd prophylaxis optiont for adult patients (pts), but has limited data in children. methods: the study aim was to evaluate ptcy as gvhd prophylaxis in pediatric pts with inherited disorders undergoing allo-hsct. 96 pts, the most of them are pediatric age (median age -3 y.o., range 7 month -30 y.o.) with different types inherited disorders (β-thalassemia -10, bone marrow failure syndromes -26, storage diseases -46, primary immunodeficiencydisorders -14) were inrolled in retrospective study. donor type was: matched/mismatched unrelated (mud/mmud) -69, matched related donor (mrd)-15, haploidentical (haplo) -12. conditioning regimen was: myeloablative (mac) -43, reduce-intensity (ric) -53. graft sourse was: bone marrow (bm) -68, peripheral blood stem cells (pbsc) -26, combintions bm +pbsc/bm+cord blood -2. ptcy 50 mg/kg days +3, +4 based gvhd prophylaxis recived 33 pts., standart gvhd prophylaxis based on calcineurin inhibitors -63 pts. results: cumulative incidence (ci) of agvhd was 48%. grade 2-4, 3-4 agvhd were 40% and 22% respectively. ptcy based gvhd prophylaxis reduced ci of agvhd (35% vs 56%, p=0,025). another reduce ci of agvhd factors were mac (31% vs 59% in ric pts group, p=0,044), mrd (13% vs 44% in haplo group vs 56% in mud/mmud group, p=0,024), bm as a transplant source (44% vs 62% in pbsc group, p=0,05). in a multivariate analysis mac (hr 2,6 95%ci 1,7-6,, p=0,02), time from diagnosis to allo-hsct less then 22 month (hr 2,6 95%ci 1,1-6,2, p=0,03) were predictive for reducing ci agvhd. for agvhd 2-4 st. significant factor increase ci was female donor both in univariate (51% vs 34%, p=0,04) and multivariate analysis (hr 0,7 95%ci 0,2-0,8, p=0,02).5 years overall survival (os) was 60%. improving os factors were: transplant age younger then 5 y.o. (80% vs 35%, p=0,000), time from diagnosis to allo-hsct less then 22 month (72% vs 38%, p=0,000), engraftment (72% vs 22%, p=0,000). in a multivariate analysis only transplant age younger then 5 y.o. (hr 3, 3 95%ci 1, (4) (5) (6) (7) (8) p=0, 006) and engraftment (hr 0,3 95%ci 0,1-0,7, p=0,005) were predictive for os. conclusions: ptcy-based gvhd prophylaxis can be effective options for reduce risk of acute gvhd. using unrelated donors, bone marrow as transplant source and mac can reduce ci of gvhd. performing allo-hscr earlier from diagnos and in earlier age can improve os patients with inherited disorders background: diarrhea is a frequent complication after allo-sct. at onset it is often difficult to differentiate gi gvhd from other causes of enterocolitis. recently, non-invasive tests, such as fecal calprotectin (fc), have been validated as markers of gut inflammation in patients with inflammatory bowel disease, but only a few studies have been published regarding its use as a diagnostic marker in gi gvhd. methods: our aim in this study was to explore the levels of fc in allo-sct recipients with new-onset diarrhea. so far we have included 43 allo-sct recipients who developed acute diarrhea ≥ stage 2-4 at a median of 75 days (range:12-328) post allo-sct. stool samples were analyzed as soon as possible after the onset of diarrhea. fc levels were determined in addition to an extensive microbiological panel for infectious enterocolitis (including norovirus pcr and c. difficile associated diarrhea). endoscopies for histologic analysis were performed according to the treating physicians' discretion (n=15). results: patients characteristics are summarized in table 1 . median follow-up for survivors was 524 days (range:151-1834). twenty-eight patients (65%) were diagnosed of gi-gvhd. the additional causes of diarrhea were: drug-related enterotoxicity (n=6), viral enteritis (n=2), food intolerance (n=2), c.jejuni-enteritis (n=1), and non-specific causes (n=4). the concentration of fc was higher in patients with gi gvhd vs. other causes of diarrhea (544μg/g +/-71 vs. 58 μg/g +/-33, p= 0.03). patients who did not develop severe enterocolitis had normal to slightly raised calprotectin at the onset of diarrhea [< 100-150 in 16 out of 19 (89%) cases], including 100% (6/6) of patients with enterotoxic drug-related diarrhea. among the 28 patients with gi-gvhd, 13 (30.2%) were later found to be steroid-resistant. as shown in figure 1 , we found a significant association between high fc (≥400μg/g) and severe-refractory gvhd (hr 5.7, p=0.01). of note, high values of fc were also found in 3 patients with severe infectious enteritis (norovirus, adenovirus and c.jejuni infections), with baseline fc>800 μg/g, respectively. overall survival was 78% (ic95%:65-91) at 12 months. hypoalbuminemia and thrombocytopenia were the only variables linked to 1-yr os in univariate analysis, regardless of the cause of enterocolitis. conclusions: in the absence of standarized (and expensive) biomarker panels for analyzing and predicting gvhd onset and outcomes, the fc test may be an useful tool in the allo-sct setting. our initial results show that fc is helpful in predicting mild causes of diarrhea and to identify patients with a high probability of developing severe (and potentially steroid-refractory) gi gvhd, although high levels are also found in severe infectious enteritis. background: there is an urgent need for effective therapy for severe acute gvhd. results of gvhd therapies beyond 6 months are rarely reported. we here report a median follow-up of 4 years. we introduced mesenchymal stromal cells as therapy for severe acute gvhd, with a dramatic response in some, but not all patients. the placenta protects the fetus from the mothers haploidentical immune system during pregnancy. we found that maternal stromal cells from the fetal membrane, so called decidua stromal cells (dscs) were more immunosuppressive than other sources of stromal cells. methods: we treated 21 patients, median 49 years of age (range 1.6-72) for severe acute gvhd. all had biopsy proven gastro-intestinal gvhd. all were steroid refractory,11 after >7days or with progression and 10 after >3 days. we used an improved protocol where dscs were thawed and infused in a buffer with 5% albumin. dscs were given at a median dose of 1.2 (0.9-2.9) x 10 6 cells/kg and 2(1-6) doses, given one week apart. viability of frozen and thawed dscs was 95% (89-100) and cell passage was 4 (2-4). results: complete resolution of gvhd was seen in 11 patients and 10 had a partial response. the cumulative incidence of chronic gvhd was 52%. six had mild, 4 moderate and one severe nih overall gvhd severity scoring. nine patients died, 3 from relapse, 1 acute gvhd and septicemia, 1 zygomycetes infection, 1 liver insufficiency, 1 cerebral hemorrhage, 1multiorgan failure and 1 chronic gvhd with obstructive bronchiolitis. four years transplant related mortalliy was 28.6% and overall survival was 57%. survival was not significantly worse (p=0.33) than 66% for all 293 patients undergoing allogeneic hematopoietic cell transplantation during the same period 2012-2015. conclusions: to conclude, dscs seems to be a promising therapy for severe acute gvhd. randomized trials are under way. disclosure: nothing to declare p237 anti-apoptotic protein bcl-2 is upregulated in graftversus-host disease stem cell transplantation (allo-hsct) with 30-80% developing either acute or chronic gvhd. recently, bcl-2 inhibitor venetoclax was approved for treatment of chronic lymphocytic leukemia. induction of apoptosis and depletion of lymphocyte subpopulations e.g. follicular b-cells or cd4 + and cd8+ t-cells led to further exploration in autoimmune disease. methods: to establish expression levels of genes in the bcl-2 pathway, low-input rna sequencing was performed on t cells isolated from non-inflamed skin and peripheral blood of hsct recipients at 5 different time points before until 1 year after transplantation. furthermore, we analyzed blood, lung, gut and skin samples of 105 patients post allo-hsct with and without previously untreated acute or chronic gvhd by rt-pcr, flow cytometry and tissue immunofluorescence. [[p237 image] 1. bcl-2 is up-regulated in t and b lymphocytes of acute and chronic gvhd lesions.] results: rna-sequencing revealed that t cells upregulated bcl-2 upon conditioning treatment (day 0) and cells of patients who later developed gvhd failed to downregulate bcl-2 after transplantation (day+14, day+100). bcl-2 protein levels were elevated in overall leukocytes and pathogenic cell subsets including monocytes, cd8+ t lymphocytes and nkt cells showed significantly higher expression of bcl-2 in peripheral blood of gvhd patients as compared to healthy controls. these results could be recapitulated in tissue samples, where disease-promoting lymphocytes (t, b, nk, nkt) were numerically expanded and expressed bcl-2 in acute and chronic gvhd skin lesions. notably, non-pathogenic cell types such as keratinocytes did not exhibit increased bcl-2 expression compared to control samples from hsct recipients and healthy donors. while bcl-2 rna expression did not depend on type of conditioning (mac vs. ric) or gvhd grade, it correlated to disease severity and was significantly elevated in biopsies of patients with steroidrefractory gvhd. conclusions: we could show exclusive upregulation of bcl-2 in gvhd-mediating cell types in peripheral blood and tissue samples affected by gvhd, correlating to gvhd severity and response to first-line therapy. thus, bcl-2 inhibition may present a novel and urgently needed targeted therapy in treatment of steroid-refractory acute and chronic gvhd. disclosure: supported by a docmed fellowship od the austrain academy of sciences background: graft-versus-host disease (gvhd) represents a major contributor to morbidity and mortality in recipients of allogeneic hematopoietic cell transplants (hct). several therapeutic strategies exist for gvhd prophylaxis and include post-transplant cyclophosphamide (ptcy) and antithymocyte globulin (atg). while several groups have described the use of ptcy in younger patients, there is a paucity of data about the efficacy of ptcy in older individuals, particularly when combined with atg. we investigated the combined effect of ptcy with atg on transplant outcomes in older patients at princess margaret cancer centre, toronto, canada. methods: this retrospective study included all patients age ≥60 who underwent allogeneic hct for any indication at our centre between december 2013 and july 2017. overall survival (os) was calculated using kaplan-meier analysis and multivariable cox proportional hazards regression. cumulative incidence of relapse (cir) and non-relapse mortality (nrm) were calculated using competing risk regression (fine and gray method). incidences of acute (agvhd) and chronic (cgvhd) were compared using the fisher's exact test. results: of 133 patients, 84 (63%) were male. median age was 65 (range 60-74) and median follow-up among survivors was 28 months (range 6-60). acute myeloid leukaemia (aml) was the most common indication for hct (57 patients, 43%), followed by myelodysplastic syndrome (37 patients, 28%) and myelofibrosis (17 patients, 13%). eightyfour (63%) patients had a matched unrelated donor, 37 (28%) had a matched related donor and 12 (9%) had a haploidentical donor. one hundred twenty-five (94%) patients received reduced intensity conditioning. sixty-two (47%) patients received ptcy combined with atg (4.5 mg/kg) while 71 (53%) received other forms of gvhd prophylaxis. os at 2 years was 46% (95% confidence interval (ci) 37-54) in the entire cohort. patients who received ptcy with atg had a superior 2-year os compared with other gvhd prophylaxis regimens ( figure 1a ): 57% (95% ci 44-69) vs. 37% (95% ci 26-49), respectively (hr=0.6, 95% ci 0.4-0.9, p=0.02). the 2-year nrm for the entire cohort was 37% (95% ci, 29-46). patients who received ptcy with atg had a lower 2-year nrm compared to those who did not ( figure 1b ): 23% (95% ci 13-34) vs. 49% (95% ci 37-60), respectively (hr=0.4, 95% ci 0.2-0.7, p=0.002). the 2-year cir in the whole group was 24% (95% ci 17-32). use of ptcy with atg was associated with a modest increase in cir at two years ( figure 1c ): 35% (95% ci 22-49) vs. 16% (95% 8-25), respectively (hr=2.1, 95% ci 1.0-4.0, p=0.04). there was a trend toward lower incidence of grade ii-iv agvhd among patients who received ptcy with atg compared to those who did not: 15% vs. 30 % (p=0.06). the incidence of grade ii-iv cgvhd was lower in individuals who received ptcy with atg compared to those who did not: 26% vs. 45% (p=0.03). conclusions: in older hct recipients, use of ptcy combined with atg is associated with improved os, lower nrm, decreased risk of both agvhd and cgvhd and a modest increase in relapse risk. therefore the ptcy with atg combination represents an effective strategy for gvhd prophylaxis in older allogeneic hct recipients. disclosure: the authors have no conflict of interest to declare. outcome of severe graft versus host disease in pediatric patients with nonmalignant diseases after allogeneic bone marrow transplantation. a single center experience irina zaidman 1 , sigal grisariu 1 , batia avni 1 , ehud even-or 1 , bella shadur 1 , adeeb nasereddin 1 , polina stepensky 1 background: hematopoietic stem cell transplantation (hsct) remained the only curative option for many nonmalignant diseases in pediatric patients. survival after hsct has improved the last few years due to significant advancement in human leukocyte antigens (hla) typing techniques, less toxic conditioning regimens and better supportive care and resulted to 90% survival and cure in some non malignant diseases. graft-versus-host disease (gvhd) remains a major complication of hsct and leading cause of morbidity and mortality. prognosis of patients with high grade gvhd is dismal and survival rate varies between 25% to 55% in pediatric patients. methods: the retrospective study included patients with non malignant diseases who underwent allogeneic hsct at hadassah medical center from 2008 to 2018. the collected data included patient´s clinical data and transplant characteristics. the study was approved by the institutional helsinki committee. results: 182 children with nonmalignant diseases underwent 194 allogeneic bone marrow transplantations in hadassah university hospital during ten years period. fifty seven patients (31%) developed agvhd grade 1-4, twenty five of them (13.7%) grade 3-4. median age was 6.34 (range 0.37-17.7), most patients were males (17 males, 8 females). 9 patients underwent bmt from fully matched family members, 6 children were transplanted from matched unrelated donors and 10 from mismatched donors. twenty one of 25 patients with severe gvhd (83%) survived. four patients (17%) died from severe gvhd and complications of immunosuppressive treatment. 3 of 4 deceased patients were transplanted from mismatched donor, in 3 of 4 cases the age of donor was advanced, 2 of 4 patients developed severe gvhd and died after second hsct. all 4 patients were refractory to different treatment modalities. three of 4 patients died in 2012 and one in 2015, it was no death from severe gvhd in 12 patients that were transplanted and developed high grade gvhd after 2015. conclusions: the results of this study show a high survival rate of 83% in pediatric patients with non malignant diseases and severe gvhd. significant risk factors for mortality in our group included mismatched donor, advanced age of donor and second transplant. trend to better survival was observed after 2015. additional multicentral studies analyzed the outcomes of agvhd in pediatric patients with nonmalignant diseases are urgently required. background: chronic graft-versus-host disease (cgvhd) is a serious late complication after allogeneic hematopoietic stem cell transplantation (allohsct) with heterogeneous presentation and still poorly understood pathophysiology including inflammation and endothelial dysfunction. factor viii (fviii) and von willebrand factor (vwf) are coagulation factors but also known indicators of endothelial dysfunction and inflammation in different settings, and therefore could serve as interesting candidate biomarkers of cgvhd. methods: since 2013 patients after allohsct were assessed by the multidisciplinary cgvhd team at the university hospital center zagreb, croatia, using established nih cgvhd-related measurements. an extensive history, physical and laboratory evaluations were performed, including fviii, vwf:ag and vwf:ac analysis. descriptive statistic and non-parametric analyses were performed. variables that showed significant univariate correlations were used in multivariate logistic regression (mlr) to identify the most predictive for fviii, vwf:ag and vwf:ac in cgvhd patients. results: 70 cgvhd patients and 41 controls (subjects after allohsct without cgvhd) were analysed. median age of cgvhd patients was 42 (9-65) years, 50% females, 91.5% underwent allohsct for hematologic malignancies, 55.7% had myeloablative conditioning and 52.9% matched related donor. median time from hsct to study was 450.5 days and from cgvhd diagnosis to study 82 days. there were no demographic neither transplant related significant differences between cgvhd patients and controls beside stem cell source (peripheral blood 71.4% vs 51.2%, p=0.041) and history of acute gvhd (70.0% vs 22.0%, p< 0.001). majority of patients had moderate (52.9%) or severe (42.6%) nih global cgvhd score, 57.2% active cgvhd by clinician´s impression. median number of organs involved by cgvhd was 3 (1-6), and the most frequently involved organs were mouth, skin and eyes (52.0% each). cgvhd patients compared to controls had higher fviii levels (median 206 (52-453)% vs 182 (51-406)%, p=0.044, reference range 50-149%) and higher vwf:ag (median 261.6 (76.6-601)% vs 203.2 (51.9-600)%, p=0.030, reference range 50-160%), while vwf: ac showed a trend toward higher levels among patients (median 253.4 (54-601)% vs 178 (48.6-601)%, p=0.084, reference range 50-150%). patients had higher ggt (p=0.002), lower anticardiolipin igg (p=0.001) and igm (p=0.003), and lower albumin (p=0.018) than controls, without differences between other laboratory parameters. univariate analysis showed that among cgvhd patients higher fviii was associated with worse karnofsky score (ks) (p=0.031) and performance score (ps) (p=0.030), higher leukocytes (p=0.031), cholesterol (p=0.003), triglycerides, ast, alt, ggt, ldh, and lower albumin. higher vwf:ag and vwf:ac in cgvhd patients were associated with worse ks and ps (p< 0.001), with more active cgvhd (p< 0.001), worse nih cgvhd liver (p=0.042; p=0.039) and nih cgvhd mouth (p=0.012; p=0.009), higher total nih score (p=0.044; p=0.005), higher number organs involved (p=0.013; p=0.003), higher esr, monocytes, ddimers, ast, alt, ggt, ldh, triglycerides, β-2-microglobulin, ferritin, total proteins, iga and lower albumin. mlr analysis showed leukocytes (p=0.018) and cholesterol (p=0.010) as the strongest predictor of fviii (r2=49.8%; p< 0.001), while strongest predictor of vwf: ac was number of organs involved by cgvhd (r2=71.7%; p=0.031). conclusions: results of this study detected high fviii and vwf levels in cgvhd patients with possible reflections to cgvhd manifestations, what needs to be further confirmed in larger longitudinal studies. disclosure: this work was supported, in part, by the unity through knowledge fund project entitled "clinical and biological factors determining severity and activity of chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation", and also, in part, by the croatian science foundation project entitled "new biomarkers for chronic graft-versus-host disease". antonela samardzic -work financed by the croatian science fondations`"young researchers`career development project -training of doctoral students" background: thrombotic microangiopathy (tma) is a severe complication of allogeneic hematopoietic cell transplantation (hct) with multisystem involvement. a few recent reports have recognized evidence of tma in the intestinal vasculature (intestinal tma/itma) of patients with graft-versus-host disease (gvhd) with or without tma. we aimed to identify patients with itma and describe histological, clinical and prognostic features. methods: we prospectively evaluated available endoscopic samples (stomach and/or colon) from consecutive adult hct recipients for previously described histopathologic signs of itma (january 2017-september 2018). systemic tma was diagnosed according to the international working group criteria. we compared findings among 3 clinical groups: gvhd/systemic tma, gvhd/no systemic tma and no gvhd/no tma. results: we studied 20 patients, 5 classified as gvhd/ systemic tma, 11 gvhd/no systemic tma and 4 no gvhd/no tma. baseline transplant characteristics (age, donor, hla matching, conditioning) did not differ significantly among groups. histological features of itma, including loss of glands, total denudation of mucosa, apoptosis and detachment of endothelial cells, intraluminal fibrin, intraluminal microthrombi and mucosal hemorrhage were found in 6 patients. previously described features of intraluminal schistocytes were not observed in our patients. interestingly, loss of glands, total denudation of mucosa, apoptosis and detachment of endothelial cells were also found in patients with gvhd and no itma, suggesting that these features are not pathognomonic of itma. among 6 itma patients, two patients were classified in the clinical group of acute gvhd/systemic tma, while the other 4 patients had clinical and histopathological features of itma and severe grade iii-iv steroid-refractory acute gvhd (3 patients) or extensive chronic gvhd (1 patient) but no evidence of systemic tma. in the majority of patients (5/6), itma occurred during the early posttransplant period at 1.7 (0.9-4) months. clinical features (gastrointestinal bleeding, diarrhea, pain, nausea) presented no differences between patients with or without itma. prognosis was poor for patients with itma who suffered from a significantly higher mortality rate of 83% compared to the rest patient population (p=0.014). with a median follow-up of 11.1 (2.1-67.5) months, 1year overall survival probability (os) was 22.2 for itma, 55% for gvhd and 60% for systemic tma. unfavorable predictive factors for os were itma (p=0.048), hla mismatched donors (p=0.008) and gastro-intestinal bleeding (p=0.021). conclusions: intestinal tma has emerged as a novel distinct entity in patients with gvhd and/or systemic tma. distinct histological features may be useful in differential diagnosis of these severe hct complications. mortality rates higher than those of systemic tma highlight the need of proper recognition of itma that needs to be further studied in terms of diagnostic and therapeutic potential. disclosure: e.g. was supported by the european hematology association clinical research grant. the remaining authors declare no competing financial interest. the beneficial effects of thrombomodulin gene polymorphisms after hematopoietic stem cell transplantation background: chronic graft-versus-host disease (cgvhd) remains the major cause of late morbidity and mortality after allogeneic blood and marrow transplantation. treatment options for cgvhd, particularly its sclerotic forms remain limited. active hedgehog (hh) signaling was shown as a therapeutic target in both mouse and human cgvhd, with limited efficacy and significant toxicities described in a published clinical trial (defilipp, 2017). methods: adult patients with steroid refractory sclerodermatous cgvhd, defined as requiring >0.5 mg/kg/day of prednisone dose equivalent (pde), or need for second-or third-line therapy beyond corticosteroids and calcineurin inhibitors or sirolimus were eligible for this open label study of vismodegib, a first generation hh pathway inhibitor. primary endpoint was failure free survival, defined as absence of non-relapse mortality, no recurrent malignancy, steroid dose at 6 months =< 0.2 mg/kg/day of pde, and no addition of new systemic treatment. vismodegib was administered orally for 6-12 months, with dose reductions at development of toxicities. peripheral blood mononuclear cells were isolated from samples collected at treatment initiation and every three months thereafter. the immune profile of circulating b cells was analyzed by flow cytometry and t helper polarization by qrt-pcr of sortpurified cd4+ t cells. results: at the time of interim analysis, 6 patients were evaluated. 3 patients completed 6 months of treatment and five patients completed 3 months of treatment. therapy was discontinued in 3 patients prior to 6 months due to treatment-related (n=2) and unrelated (n=1) side effects. most patients experienced grade 2 toxicities (muscle cramps and dysgeusia), with only a single grade 3 toxicity (weight loss). 5 patients who completed 3 months of therapy demonstrated partial response, and overall, the primary endpoint was reached in 50% (3/6) of patients. in 2 patients who discontinued vismodegib, cgvhd worsened acutely after discontinuation. correlative analysis of immune cellular subsets in peripheral blood in 4 paired samples (pre-treatment and month 3 of therapy) documented modulation of b cell subsets pathogenic in cgvhd (pregerminal center and plasmablast-like b cells) and diminished t helper 2 polarization in cd4 t cells. conclusions: overall, use of vismodegib was associated with potential clinical efficacy in sclerodermatous cgvhd with possible mechanistic evidence arising in correlative studies. while side effects were common, further studies of hh inhibition in cgvhd are warranted. future studies should employ adjusted dosing regimens, along with supportive care interventions to offset side effects, and testing of novel hh inhibitors with enhanced safety profiles. clinical background: graft-versus-host disease (gvhd) results from recognition of host antigens by donor t cells following allogeneic hematopoietic stem cell transplantation (sct). we tested the hypothesis that somatic neomutations occurring after sct from donor and/or recipient dna may trigger gvhd. methods: we longitudinally analyzed both constitutive and somatic mutations by whole exome sequencing (wes) in 2 patients who received sct from a sex-matched hlaidentical sibling for npm1 mutated acute myeloid leukemia (pt#1) and jak2 v617f mutated primary myelofibrosis (pt#2). both patients were initially refractory to alloreactivity, i.e. not displaying any signs of gvhd, even after several donor lymphocyte infusions. acute gut gvhd finally occurred after a further dli preceded by a lymphodepleting chemotherapy. in pt#2, gvhd correlated with a graft-versus-tumor effect. wes was performed on dna from recipient saliva and donor pbmcs (germline samples) and from sequential post-sct pbmcs samples on a hiseq2500 illumina with 2x100bp paired-end reads at a mean depth of coverage of 190-210x. germline and somatic mutations were determined using in-house bioinformatic pipelines (named ewok from the curie institute and smaug from the henri mondor hospital), using briefly gatk as variant caller for germline samples, and a combination of 3 variant callers for matched normaltumor pairs. we adjusted parameters to detect somatic mutations at a minimal variant allelic frequency (vaf) of 5% compared to recipient and donor germline for all variations (minimal coverage = 8x for germline and 5x for tumor sample). results: wes allowed detecting somatic driver mutations explaining aml and pmf for both patients in the initial timepoint and all these driver mutations disappeared at the following timepoints. as expected, the somatic variant rate was 10x higher in pt#1 with aml than in pt#2 with pmf at each timepoint, except for the final gvhd timepoint. indeed, at this final point, the somatic variant rate dramatically decreased by 80% as compared to previous timepoints. by subtracting variants detected pre-and post-sct from those identified at the ultimate time-point of gvhd occurrence, we created 2 sets of 5 and 7 variants respectively for each patient (keeping only variants with at least 4 reads of mutated dna). these variants can be classified in 2 categories: (i) those with only with a slight increase at time of gvhd, i.e. ≤ 2-fold compared to highest previous vaf (lrrc43, or8u1, or10g9, alpp, frg1, frg2b and lilrb3 genes), and (ii) those with a significant increase at that time, i.e. > 2-fold compared to highest previous vaf (phf2, smpd1, ercc8 and krtap9-1 genes). none of the variants or genes involved was common between the 2 patients. ontology classification of mutated genes showed the implication of some of them in cell death, regulation of map kinase activity, mrna splicing and immune system process, making them good candidates for further studies. identification of variants appearing pre-gvh and turning off at time of gvhd is ongoing to unveil putative neoantigens that could trigger the alloreactive response. conclusions: using a comprehensive, pre-and post-sct, wes of donor/recipient pairs, we identified several neomutations from donor and/or recipient dna correlating with gvh/gvt effect development. disclosure results: a total of 169 patients experienced cgvhd, and mild, moderate, and severe cgvhd were observed in 66, 67, and 36 patients, respectively. the 2-year cumulative incidence of total cgvhd was 60.5% (95% ci, 54.7-66.3%), and the 2-year cumulative incidence of moderate to severe and severe cgvhd was 36.6% (95% ci, 30.9-42.3%) and 12.7% (95% ci, 8.8-16.6%), respectively. the patients who had 3 loci mismatched had a higher 2-year cumulative incidence of total cgvhd (66.2% vs. 54.5%, p=0.025) and moderate to severe cgvhd (42.3% vs. 30.6%, p=0.028) compared to those of the patients who had 1-2 loci mismatched. the patients who had maternal donors had a higher 2-year cumulative incidence of moderate to severe cgvhd (50.8% vs. 32.7%, p=0.018) compared to that of the patients who had other donors. the patients who had grade iii to iv acute graft-versus-host (agvhd) had a higher 2-year cumulative incidence of total cgvhd (91.7% vs. 50.0%, p< 0.001) and moderate to severe cgvhd (70.8% vs. 26 .3%, p< 0.001) compared to those of the patients without agvhd. in multivariate analysis, grade iii to iv agvhd was the only independent risk factor for total cgvhd (hr=2.6, 95%ci, 1.6-4.2; p< 0.001) and moderate to severe cgvhd (hr=3.8, 95%ci, 2.1-6.7; p< 0.001). in the model excluding agvhd, maternal donor was the risk factor for moderate to severe cgvhd (hr=1.5, 95%ci, 1.1-2.3; p=0.030). conclusions: we observe that severe agvhd was the most important risk factors for cgvhd after haplo-hsct, and further interventions should be considered in these patients to prevent severe cgvhd. disclosure: none of the authors have any potential financial conflict of interest related to this manuscript. background: extracorporeal photopheresis (ecp) has been successfully used for the treatment of graft-versus-host disease (gvhd). ecp therapy might restore the balance between effector and regulatory cells which is severely impaired in gvhd. nk cells are the first lymphocyte subset to be reconstituted after allogeneic hematopoietic stem cell transplantation (allo-hsct). as an important innate immune cell population, nk cells can temporally bridge the transient period of t-cell deficiency post allo-hsct, by protection from opportunistic infections and prevention of leukemic relapse by graft-versus-leukemia (gvl) effect. nk cells not only preserve homeostasis through targeted killing of allo-reactive t cells and thereby control gvhd but also enhance inflammation by secretion of tnf-α and ifn-γ and thereby promote gvhd. therefore, we investigated here the role of nk cells in gvhd patients under ecp therapy. methods: thirty four patients with steroid-refractory/ resistant agvhd ≥ ii°and moderate to severe cgvhd received ecp therapy which performed according to the guidelines. glucksberg and nih criteria were used for clinical staging of agvhd and cgvhd under ecp therapy, respectively. the comprehensive phenotypical analysis of nk cells was evaluated by multicolor flow cytometry. nk activity in terms of killing function, cytokine release capacity and proliferation function was monitored by chromium-51 release assay, intracellular cytokine staining and cfse staining, respectively. results: five different nk cell subsets were defined based on cd56 and cd16 expression. cd56 bri nk cells displayed an immature and activation profile with high expression of cd62l and nkg2d. agvhd patients had a higher frequency of cd56 bri nk cells when compared with hds and cgvhd patients, who were characterized by significant increase of the cd56 dim cd16 + and cd56 -cd16 + nk cell subsets with high expression of differentiation markers cd11b and cd57. of note, cd56 bri cd16 -nk cells could serve as a novel predictive biomarker for the response of agvhd patients to ecp treatment. in responding agvhd patients, an increase of cd56 bri nk cells was observed already during the early ecp treatment phase, suggesting immune reconstitution. after priming of the progenitors, ecp could differentiate immature cd56 bri nk cells into mature cd56 dim nk cells with reduction of cd62l on cd56 bri nk cells. moreover, cd56 dim nk cells could further be matured through upregulation of cd57 expression by ecp. notably, ecp therapy could shift the nk cells from a cytotoxic to a regulatory phenotype within the cd56 bri nk cells. in spite the immunomodulatory effect of ecp on nk cells, nk activity could be kept intact under ecp therapy. the killing activity of nk cells was stable as confirmed by a 51 cr release assay. ecp therapy had no negative effect on the quantity and quality of cytokine release by nk cells upon k562 stimulation. especially, the polyfunctionality of nk cells was not altered significantly by the ecp therapy. conclusions: nk cells play an important role in gvhd and could serve as a predictive cell population for the clinical response to ecp therapy. in the current study, ecp influenced the differentiation, maturation and education of nk cells ameliorating gvhd without comprising the antiviral immune defense and gvl effect. disclosure: the authors declare no competing financial interests, except the following: therakos mallinckrodt gave a financial support to as and ms for the documentation of the clinical course and for the analysis of immune cells of the patients, pw has honoraria and membership on advisory boards for sanofi-aventis. abstract withdrawn. cyclosporine levels >200µg/l on day 10 post-transplant was associated with significantly reduced acute graftversus-host disease following allogeneic hematopoietic stem cell transplantation monica bianchi 1 , dominik heim 1 , claudia lengerke 1 , martina kleber 1 , dimitrios tsakiris 1 , jakob passweg 1 , alexandar tzankov 2 , michael medinger 1 background: acute graft-versus-host disease (agvhd) remains a major complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). affected patients, especially with steroid-refractory agvhd, have a very poor prognosis. prophylaxis with cyclosporine a (csa) is the backbone of gvhd prevention in most conditioning regimens. methods: in a retrospective analysis of patients treated with allo-hsct, we correlated csa levels at the day of transplantation (day 0) and day +10 with the incidence of acute and chronic gvhd. we postulate that higher target csa levels >200μg/l will result in a lower incidence rate especially of agvhd after allo-hsct. results: we assessed 660 patients with either aml n=248, lymphoma/myeloma n=127, mds/mpn n=124, all n=79, cll n=36, cml n=23, or bone marrow failure n=22. in patients with clinically relevant agvhd grade ≥2, mean csa levels was lower on day 0 and day +10 (142±88 μg/l; and 183±64 μg/l; respectively) compared to patients without agvhd (156±81 μg/l; and 207±67 μg/l; respectively; day 0: p=0.003; day +10: p=7.57 x 10 -9 ). in patients with csa level < 200 μg/l, the incidence of agvhd was significantly more frequent compared to patients with csa levels >200 μg/l [(234/356; 66%) versus 91/248 (37%); p= 1.34 x 10 -12 ]. in patients with cgvhd, there was no significant difference between csa levels < 200 μg/l (128/330) compared to csa levels >200 μg/l (96/ 233; p=0.312). the optimal csa cut-off level for the prevention (i.e. roughly 50% incidence reduction) of agvhd was >201 μg/l at day 0 and >195 μg/l at day +10 ( figure 1 ) in a competing risk analysis, time to agvhd grade ≥2 (using death of other causes as competing risk) was associated with csa levels >200 μg/l on day 0 and on day 10, unrelated donors, myeloablative conditioning (mac), and for the diagnosis lymphoma/myeloma. conclusions: our data support close monitoring with active adjustments of csa dosing to maintain therapeutic csa levels above 195 μg/l in the first 10 days of allo-hcst to reduce agvhd. disclosure: noting to declare. liposomal cyclosporine a for inhalation (l-csa-i) to treat bronchiolitis obliterans syndrome: novel formulation with therapeutic potential for patients with bos following allo-hsct noreen roth henig 1 , emilie hofstetter 2 , dominik kappeler 3 , gerhard boerner 3 background: bronchiolitis obliterans syndrome (bos) is a rapidly progressive lung disease caused by t-cell mediated inflammation that leads to blockage of bronchioles, leading to respiratory failure and death shortly after diagnosis. approximately 4% to 10% of patients who undergo allogeneic hematopoietic stem cell transplant (allo-hsct) will develop bos, with 72-100% developing bos as a respiratory form chronic graft-vs-host disease (cgvhd) in addition to other signs of cgvhd. mean time to bos diagnosis ranges from 273 to 547 days post-transplant. the histopathology of bos after allo-hsct and lung transplantation is identical. early studies of l-csa-i for the prevention of bos in lung transplant recipients demonstrated therapeutic benefit. l-csa-i is a novel, liposomal formulation of cyclosporine administered via a pari investigational eflow â nebulizer which delivers a potent immunosuppressant to the site of disease. pharmacokinetics and tolerability of l-csa-i is presented. methods: retrospective review of two clinical studies of l-csa-i (isotonic, 4mg/ml) for bos associated with lung transplantation. both studies had a control arm and results reported here are for patients who received l-csa-i. subjects received 5mg (single lung transplant) and 10mg (double lung transplant) bid via inhalation. blood samples for pharmacokinetic analysis of cyclosporine a concentrations were collected before inhalation, immediately after inhalation, and thereafter in intervals of 15, 30, 60 min and 2, 4, 8 and 12 hours. local and general tolerability of l-csa-i was investigated. results: between the two studies, 85 subjects received either 5 or 10 mg bid of l-csa-i. pharmacokinetic models predict a constant drug level in the lung. maximum serum cyclosporine a concentration after inhalation was 57.42 ± 34.26 ng/ml. trough levels for up to 2-years of daily administration was 4-5 ng/ml with no evidence of accumulation following repeated exposure. tolerability data was assessed from 1068 patient-month exposure to l-csa-i. reported symptoms were: pharyngeal soreness 1%; cough 22%; dyspnoea 7%; and wheezing 1%. no subject discontinued due to intolerability. inhalation time is on average 9-13 min. conclusions: l-csa-i provides high and constant concentrations to the airways of the lungs and the site of bos. l-csa-i is well tolerated in lung transplant patients. use of l-csa-i instead of augmentation of systemic csa reduces the total drug exposure. a multicentre phase 2 safety and exploratory efficacy trial for the treatment of bos in allo-hsct recipients is underway. disclosure background: there are a number of biomarkers that predict non-relapse mortality (nrm), graft-versus-host disease (gvhd) and relapse incidence (ri) after conventional gvhd prophylaxis based on calcineurin inhibitors with or without antithymocyte globulin. currently there is limited data whether the conventional predictive biomarkers work with posttransplantation cyclophosphamide (ptcy) prophylaxis. methods: prospective single-center study in 2015-2016 enrolled 79 adult patients with acute leukemia in cr (34% with all, 66% with aml). 26 received matched related bone marrow (bm) graft with single-agent ptcy and 53 received unrelated peripheral blood stem cell graft (pbsc) with ptcy, tacrolimus and mmf. the grafts were studied by flow cytometry (facs aria ii, antibodies by miltenyi biotec). the following populations were analyzed: cd3, cd4, cd8, cd16cd56, nkt, inkt, treg, double-positive t-cells, double-negative t-cells, tcralpha/beta, tcr v11 memory cells. the crypreserved plasma from were analysed by elisa (commercial kits by ebioscience and critical diagnostics) for vegf a soluble tnf receptor (stnfr), il-8, il-6, soluble il-2 receptor, st2, il-17 and stnfr. the above mentioned biomarkers were tested in logistic regression with roc analysis, assays with auc>0.600 were selected for analysis in fyne-gray regression with competing risks. cut off levels were determined for significant parameters. results: median follow-up was 19 months (range 12-30). in the whole group overall survival (os) was 77%, eventfree survival (efs) 73%, grade ii-iv acute gvhd 13%, moderate and severe (m&s) chronic gvhd 20%, nrm 6%, mortality in patients with gvhd 0%, ri 20%. there was no difference between bm/related and pbsc/unrelated grafts in the incidence of gvhd, nrm and ri (p>0.11). the only significant predictor of acute gvhd were low levels of il-8 level on day+7 (p= 0.0490, 0% vs 15% with the cut off 40 pg/ml). m&s chronic gvhd was predicted only by the high percentage of inkt cells in the graft (p=0.0003, 31% vs 12% with the cut off 0.03%). there was a correlation between il-8 levels and number of nk cells in the graft (p=0.02). nrm was related to infectious complications, nonetheless high levels of vegf a on day 0 (p= 0.0458, 16% vs 0% with the cut off 100 ng/ml), st2 on day+30 (p= 0.0041, 11% vs 0% % with the cut off 40 ng/ml) and low percentage of cd16+cd56-cells in the graft (p=0.0215, 22% vs 2% with the cut off 1.3%). the identified biomarkers of nrm had no association with the pre-transplant crp and ferritin levels (p>0.4). the only significant parameter for ri was the level of cd34 cells in the graft (p=0.0434). none of the identified biomarkers significantly predicted overall survival (p>0.09). conclusions: in the related and unrelated grafts with ptcy the study of biomarkers has low clinical utility due to very low gvhd-related mortality. however st2 and vegf a can predict infection-related mortality. also the study verified previous observations that high level of il-8 is associated with reduced gvhd incidence after ptcy 1 and identified the importance of nk and inkt cells in the induction of tolerance with ptcy. references background: hematopoietic cell transplantation (hct) is the only curative approach for many hematological malignancies but life-threatening toxicities, such as graft-versushost disease (gvhd) and infections, still limit its fullpotential impact on the disease. strategies for keeping allohsct more effective and safe are needed in order to reduce morbidity while improving its immunological effect to control disease relapse. post-transplant cyclophosphamide (ptcy) has been demonstrated to improve acute gvhd (agvhd) and chronic gvhd (cgvhd) control in allogeneic bone-marrow hct from identical and haploidentical donor. the use of ptcy, after peripheral blood stem cell transplantation (allopbsct) from hla-matched unrelated/related donors, has been investigated by our group in a clinical trial (nct 02300571) and preliminary results were published last year. here we report updated efficacy and safety data about the expanded cohort of patients treated with ptcy followed by tacrolimus and mycophenolate mofetil (t/mmf). methods: we analysed data about 71 consecutive patients with high-risk hematologic malignancies received allopbsct from hla-matched unrelated/related donors between march 2011 and august 2018. gvhd prophylaxis was ptcy 50 mg/kg (days +3 +4), tacrolimus from day +5 and mmf from day +5 to day +28. primary objectives were cumulative incidence of agvhd and cgvhd. secondary objectives were event-free survival (efs), cgvhd-efs, overall survival (os) and non-relapse mortality (nrm). results: patients median age at transplant was 50 (range 19-74) years. 34 (48%) patients were transplanted in first complete response (cr), 16 (23%) patients in second/third cr, the others in disease control. a median dose of 7.0 (range 2-15) x 10^6 cd34/kg was infused. primary graft failure was observed in one patient. all patients were off mmf on day +28, the median day of tacrolimus discontinuation was 112 (range 39-467). eight out of 71 (11%) patients developed agvhd, 6 (8%) of them were grade ii-iii; median day of onset was day 68 (range . no grade iv was observed. no cases of late-onset agvhd were reported. cumulative incidence of cgvhd was 8% (6/ 71), median day of onset was 162 (range 140-268). systemic treatments were required, but all patients were able to discontinue immunosuppression (is). with a median follow-up of 22 (range 4-94) months, efs was 58%, cgvhd-efs was 52% and os was 72%. non-relapse mortality (nrm) was 3% (2/71): 2 patients died because of multidrug resistant bacteria septicemia. nowadays 41 patients are alive with no evidence of disease, being continuously off is and completely reintegrated in their normal daily life activities. conclusions: the updated reported results confirm, in a larger cohort of patients with a longer follow-up, that ptcy after pbsc-hct is highly active in agvhd and cgvhd prevention with extremely limited nrm. this strategy, not only allowed earlier discontinuation of immunosuppression, but also reduced the overall time of exposure to is for most of the patients. all these features might contribute, in the future, to transform hct into a safe immunologic platform that may be combined with advanced form of cellular therapies (car-tcells), aiming to increase safely the graftversus-tumor effect. clinical methods: pediatric patients (0-21 years) with nmd undergoing unrelated hct were eligible for this single center, phase i trial. following reduced intensity conditioning, abatacept (10 mg/kg iv on days -1, +5, +14, +28) was added to standard gvhd prophylaxis (cyclosporine, mycophenolate mofetil [mmf]). patients were followed for 2 years for standard hct outcomes. [[p257 image] 1. figure 1 results: since june 2014, 10 patients have been enrolled and transplanted (table 1 , excluding #7). donor source was bone marrow in all. with median follow-up of 3.5 years, 8 of 10 patients survive without disease. initial engraftment was successful in 9, at a median of 20 and 16 days, for neutrophils and platelets respectively. one patient (5) had secondary graft rejection in the setting of viral reactivation (cmv/ebv), with successful engraftment following a 2 nd unrelated hct. in 8 engrafted patients, myeloid (cd33) chimerism was 100% at all timepoints; t-lymphoid (cd3) chimerism was mixed but reached >/=95% (figure 1 ). one patient (7) with saa had primary graft rejection in the setting of inadequate tnc dose (0.5 x 10 8 /kg) and died from marrow aplasia/infection despite 2 nd hct. a second death from wilms' tumor occurred 17 months post successful hct, in a patient (1) with dba and constitutional chromosome abnormality. except patient 7, all patients received 4 doses of abatacept, which was well tolerated, with all severe adverse events expected for a hct population. cmv and ebv reactivation occurred in 3 patients each, with resolution using standard anti-viral therapy. one patient (6) was diagnosed with ebv-driven post-transplant lymphoproliferative disease, which responded to rituximab and immune suppression withdrawal. no patients developed severe acute (grade iii-iv) or chronic gvhd (table 1) , and no patients required systemic immune suppression at >1 year. conclusions: these preliminary data suggest that abatacept can be safely added to cyclosporine and mmf gvhd prophylaxis in pediatric patients with bone marrow failure undergoing unrelated donor hct, with encouraging rates of gvhd despite half of patients having a mismatched (7/8) donor. given the higher risk of graft rejection in this non-malignant cohort, rejection (in addition to gvhd) will be a primary focus in our subsequent multi-center, phase 2 trial. clinical trial registry: clinicaltrials. gvhd and may to have be separated from from toxicity to infectious complications in the early phase after allohsct. methods: from our files we identified 65 patients which had upper gastrointestinal tract endoscopy after allohsct in with biopsies were taken from the esophagus, stomach and duodenum simultaneously. of these patients 14 were excluded because of infection, reflux disease or drug toxity and the remaining 51 patients were included in our study. we evaluated the routine stained esophageal biopsies, applied a grading scheme and compared the histological findings with those within the stomach and duodenum, the endoscopic findings and the clinical course. results: in 32 of 51 biopsy samples of the esophagus, we identified histological features of acute gvhd, ranging from vacuolar degeneration (grade 1) and single-cell apoptosis (grade 2) to the formation of clefts (grade 3) and mucosa denudation in advanced cases (grade 4), resembling epithelial lesions in acute gvhd of the skin. these findings correlated with gvhd involving the stomach and duodenum and the clinical manifestations of gvhd in other organs. endoscopically patients with gvhd revealed signs of inflammation, ranging from erythema to ulceration in the more advanced cases, sometimes reminiscent of reflux or infection. clinically these patients had abdominal discomfort ranging from inappetence to nausea, accompanied by emesis or diarrhea and weight loss. conclusions: we have shown that acute esophageal gvhd occurs after allohsct and is correlated with acute gvhd in stomach and duodenum. it could be diagnosed and graded histologically. the endoscopic findings are signs of inflammation. our results may help to establish the histological diagnosis of acute gvhd using endoscopic biopsies from the esophagus and to explain the alterations observed in the esophageal mucosa in patients after allohsct. [ background: intestinal acute graft versus host disease (agvhd) is a major thread after allogenic hematological stem cell transplantation (allohsct), with a high mortality in patients which were refractory to steroid treatment in particular. recent papers point to a correlation of histological grading of intestinal gvhd and prognosis in patient after allohsct. however a comparison with clinical scores has not been performed so far. methods: in this analysis, retrospective data from 89 patients who underwent endoscopy due to clinical signs of agvhd (day +20 to +200 after allohsct) were evaluated. of each patient least 3 biopsies from different sites of the colon which were taken simultaneously. of each biopsy series the maximum histological grad of agvhd according to the lerner scheme was obtained and compared with the glucksberg stage of the lower gastro intestinal tract (gslgi) and the overall glucksberg grade (ogg). these three grades were compared for non-relaps related mortality using the log-rank test and for sensitivity to steroid treatment applying the receiver operating characteristic for the patients who received steroid treatment. for these patients the non-relaps related mortality for responder and non-responder were calculated using also log-rank test. results: the histological grade strongly correlated with the survival (p=0.009). a statistical significant correlation was also found for the gslgi (p=0.02), whereas the ogg revealed no significant correlation (p=0.09). non-relaps related mortality was mainly related to infection or sepsis (in 32/56 patients who died). -eighty-one of the patients received steroid therapy. the sensitivity to the steroid therapy correlated with each of the three scores (p< 0.0001) but was the strongest for gslgi (area under the curve (auc) 0.829), compared to ogg (auc 0.795) and the histological score (auc 0.691). the survival of the patients, which were sensitive to steroid treatment was significantly better than those of steroid refractory patients (p=0.005). conclusions: we found that histological and clinical grading in patients after allohsct with intestinal gvhd was correlated with survival and respond to steroid treatment. histological scoring may predict survival more precisely than ogg and gslgi but did not add substantial information to the prediction of treatment response. [ emerging evidences suggest that regulatory b cells (bregs) play essential roles in inflammation, autoimmune diseases and tumors. few data exist about the role of bregs in the contest of hematopoietic allogeneic stem cell transplantation (hsct). some authors have observed that bregs from patients with chronic graft-versus-host disease (cgvhd) were less frequent and less likely to produce il-10 than bregs as compared to healthy donors or patients without cgvhd. these findings suggest that bregs may be involved in cgvhd pathogenesis. the purpose of our study was to evaluate a possible role of b cell subsets on gvhd occurrence. methods: lymphocyte subset enumeration was performed by aquios cl flow cytometer (beckman coulter), a quantitative automated analyzer that performs two diagnostic panels: tetra-1 cd45-fitc/cd4-rd1/ cd8-ecd/cd3-pc5 and tetra-2 cd45-fitc/cd56 +cd16-rd1/cd19-ecd/cd3-pc5. b cell subsets (memory, mature and transitional b cells) on peripheral blood samples were analyzed by aquios designer software, a tool for the creation of user-defined applications. panel-1 cd19-fitc/cd10-pe/cd38-ecd/cd24-pc5/cd27-pc7 and panel-2 cd19-fitc/cd5-pe/cd38-ecd/cd24-pc5/ cd20-pc7 were specifically designed by beckman coulter for our center. the flow cytometric analysis was performed as follows: in donors and patients at basal level; on graft products and in patients at days +30, +60, +90, +120 after hsct. statistical significance was assessed with prism software (graphpad) by mann withney test. p < 0.05 was considered statistically significant. results: actually we enrolled 84 patients submitted to hsct in our center from november 2017. a preliminary statistical analysis was performed on 55 patients. stem cells source was peripheral blood (pb) in 27 cases and bone marrow (bm) in the others 28. the conditioning regimen was myeloablative in 26 patients and ric in 29 patients. agvhd was diagnosed in 22 patients (40%). no associations were found between b cell subsets in donors and patients at baseline and the occurrence of agvhd. however we found a higher median percentage of transitional b cells in graft products in patients without agvhd (9.6%, 4.5-26.7) compared to patients with agvhd (6.9%, 1.5-21.1) (p=0.02, fig 1a) . in addition, patients without agvhd showed a lower median percentage of memory b cells (24.2%, range 11.6-49) in graft product as compared to patients with agvhd (34.2%, range 10.3-68.15) (p = 0.03, fig.1b ). finally in the subgroup of patients receiving pb as stem cell source we observed a higher percentage of cd3+ lymphocytes in graft product in patients with agvhd (75%; range 68-80) compared to patients without agvhd (71%; range 62-78) (p=0.01). in the monitoring of b cells reconstitution we observed that cd19+ events did not appear before day +90 after hsct and these were b transitional immature events predominantly. conclusions: our data suggest a possible protective link between transitional b cells and agvhd development. these results data need to be confirmed in a larger cohort of patients. moreover, it will be interesting to evaluate the relationship between transitional b cells at day +90 and the occurrence of cgvhd. clinical background: agvhd is a major complication of allogeneic hematopoietic stem cell transplant (hsct) and a risk factor for post-hsct mortality. the objective of this analysis is to describe patients with agvhd who had a suboptimal response to corticosteroids. methods: patients who developed ibmtr severity index ii-iv agvhd after first hsct between 1/1/14 to 6/ 30/16 were included in an ongoing chart review at centers in the united states. patients who had ever participated in a gvhd prophylaxis trial or used jak inhibitors were excluded from the study. suboptimal response to corticosteroids was defined as use of additional systemic anti-gvhd therapy, inability to taper high-dose steroids (≥1 mg/ kg) by ≥25%, or tapered corticosteroids by ≥25% but not to < 10 mg/day. results: the analysis included 64 patients with suboptimal response to corticosteroids. mean age was 55 years; 66% were male. median time from transplant to agvhd diagnosis was 33 days. at the time of maximum agvhd grade, 33% of patients were grade ii and 66% were grade iii-iv; 58% had lower gi involvement, and 59% had ≥2 organs involved. from time of diagnosis to maximum agvhd grade, 52% of patients had new organ involvement or an increase in agvhd grade. median time from diagnosis to maximum grade was 5.5 days, and was 2.5 days for patients with lower gi involvement. systemic corticosteroids were initiated on the day of diagnosis for 73% of patients. average starting daily dose was 93 mg (1.1 mg/kg) for prednisone and 136 mg (1.6 mg/kg) for methylprednisolone. steroid dose was increased for 44% of patients during follow-up; 80% were unable to taper below 10 mg/day. among patients who received additional systemic anti-gvhd therapy (n=32), 41% increased their corticosteroid dose before initiation of additional anti-gvhd therapy. median time from initiation of corticosteroids to additional therapy was 16.5 days. frequently used therapies were mycophenyalate mofetil (25%), atg (16%), extracorporeal photophoresis (13%), tocilizumab (13%), etanercept (9%), and sirolimus (9%). agvhd recurred in 31% of patients and was managed by increasing corticosteroid dose in 70% of patients. 44% had any infection within first 100 days post-hsct. forty patients (63%) required hospital readmission(s); 40% had ≥2 readmissions within 100 days post-hsct, with a mean inpatient lengthof-stay of 28 days. relapse of underlying malignancy was reported for 13 (20%) patients. two-thirds (66%, n=40) patients died at a median of 87 (interquartile range (iqr): 44-180) days from agvhd diagnosis; a higher proportion (76%) of patients with maximum grade iii-iv agvhd died at a median of 63 (iqr: 41.5-186.5) days; majority (81%) of patients with lower gi agvhd died at a median of 68.5 (iqr: 44-137) days. conclusions: a majority of patients with agvhd who had suboptimal response to systemic corticosteroids had severe and rapidly progressing disease and resulted in a high mortality rate (66%); progression was more rapid and mortality increased for patients with lower gi involvement. most patients required readmission to the hospital with extended length-of-stay. an urgent need exists for effective and tolerable therapies that quickly resolve life-threatening agvhd in early stages of disease. disclosure results: median time to onset of bo from allohct was 6.9 months (range 0.6-31.2). previous acute gvhd in 52.2% (n = 24) [grades iii-iv 29.2% (n = 7)]. in 23.9% (n = 11) cgvhd had exclusive lung involvement, while the other 35 patients (76%) had other organs affected. at diagnosis of bo, 69.6% (n = 32) were under immunosuppressive treatment. 32.6% (n=15) of patients with bo received ecp as second-line treatment. median duration of treatment was 22 months (1.5-36.8 ) and time to response 9.9 months (1.9-30.2). median of sessions was 40 (4-108). evaluation of response was based on the evolution of fev1 measurement: 26.6% (n = 4) complete response; 40% (n = 6) partial response and 20% (n = 3) stable disease. one patient did not get any response and another was not evaluable. 66.6% of patients (n = 10) could reduce immunosuppression, and in one case it was completely discontinued. there is a trend for early separation between survival curves in favor of ecp ( figure 1 ). one patient had sepsis secondary to central venous catheter infection as complication related to ecp. conclusions: ecp has emerged as a promising treatment for bo after allohct. in our experience, ecp was effective to stabilize or improve the disease in many patients and allowed to taper esteroids with minimal associated complications. however, prospective studies and longer follow-up are needed to support these findings. disclosure: nothing to declare background: the key role of il-6 signaling in acute graft vs. host disease (agvhd) and cytokine release syndrome (crs) has evoked growing use of tocilizumab, an anti-il6 receptor (il6-r) antibody, in these settings. apart from regulation of t-and b-cell differentiation, immune cells migration to inflammatory sites and t-cell recruitment, il-6 complex with il6-r through gp130 upregulates production of fibrinogen (fg) and other acute phase proteins, including c-reactive protein (crp). methods: we retrospectively analyzed data of 7 patients treated with tocilizumab (8mg/kg) due to steroid-refractory (sr) agvhd and 4 patients because of crs. median age was 37 and 47 years, respectively. seven patients were transplanted from unrelated donors (mud/mmud) and 4 from sibling donors. eight patients received myeloablative and 3 reduced intensity conditioning regimen. analyzed data included concentrations of fg, crp, an incidence of infections at tocilizumab administration and in weeks following the infusion. results: stage ii agvhd was diagnosed in 1 patient, stage iii in 4, and stage iv in 2 patients. involvement of the gastrointestinal tract (gi) was observed in 85% of cases. the median fg concentration before tocilizumab administration was 2.56 g/l (range, 1.4-7) and crp 21 mg/dl (range, 1-260) and 60% of patients had an active infection. after infusion of the antibody, we observed a decline of fg and crp levels. the median level of fg was 1.02 g/l (range, 0.46-1.7) 7 -14 days after the tocilizumab infusion with no severe bleeding complications. a median crp value was 1.82 mg/dl (range, 0.1-36) despite confirmed infectious complications. three weeks after infusion of tocilizumab fg raised to the normal range in 85% of patients (fig 1) . five patients with sr agvhd achieved a complete response, and 2 had a partial response after tocilizumab therapy. [[p265 image] 1. fibrinogen levels in gvhd patients following tocilizumab infusion.] a group treated with tocilizumab due to crs had higher initial levels of fg 5.1 g/l (range, 4.1-6.6) and crp 143 mg/ dl (range, 80-227) before administration of the drug. reduced fg and crp levels from a baseline value were also observed in this group. however, concentrations were higher than in gvhd patients: fg 2.02 g/l (range 1.2-3.4) and crp 3.2 mg/dl (range 2.7-11). in all patients, a differential diagnosis of disseminated intravascular coagulation was excluded. conclusions: 1. fibrinogen declines after tocilizumab therapy due to its cytokine-regulated production in the liver. coagulation monitoring should be performed during the first 3 weeks after administration of the antibody to avoid serious bleeding complications. 2. crp concentrations remain low despite the presence of active infections following infusion of tocilizumab. crp fails as a marker of infection during 3 weeks following the therapy. 3. tocilizumab is an effective therapy in patients with agvhd, especially with the gi involvement. disclosure: nothing to declare vanishing bile ducts after allogenic hsct: is it really gvhd? antonio grasso 1 , lorenzo d'antiga 2 , aurelio sonzogni 2 , massimo gregori 3 , alessandra maestro 3 , roberto simeone 3 , natalia maximova 3 background: evaluation of liver gvhd was historically based by elevation of bilirubin levels and by reduction and degeneration of small bile ducts on histological samples of post-transplant liver biopsy. however, there is a lack of studies that compared histological finding of ductopenia between post-autologous hsct and post-allogenic hsct. studying severity of ductopenia following allogenic hsct, we aimed to demonstrate lack of correlation between ductopenia and clinical signs of liver gvhd. methods: we retrospectively collected a series of 72 allogeneic hsct performed from 2005 to 2017 in the institute burlo garofolo. all patients undergo percutaneous liver biopsy in most cases at three months, one year and three or more years after hsct. indications for biopsy were alteration noted at 2 weekly follow-up assessments of at least one clinical or laboratory marker of liver impairment or cholestasis. ductopenia was defined by number of portal tracts with no interlobular bile duct divided by the total number (severe if the ratio was less than 0.2). clinical gvhd was defined by nih consensus criteria results: our population involved 64% males and 36% females with oncological (70%) and non-oncological underlying disease (30%). clinical signs of liver gvhd were present in 18% of the patients (n=13), 8% with contextual intestinal involvement, 8% with cutaneous and intestinal involvement. 71 patients underwent biopsy at a mean time of 110 +/-32 days after hsct, 45 patients underwent a biopsy at 12 months after hsct and 35 patients after three or more years from hsct. results of biopsies are showed in table 1. no difference in incidence of ductopenia were found between liver gvhd group and no gvhd. table] 1. table 1 : incidence of ductopenia 3-6 months, 12 months and 3 or more years after hsct in total population, gvhd group and no-gvhd group] the group that not received chemotherapy prior the hsct had an overall incidence of ductopenia of 77% (severe ductopenia of 42%) statistically significative in comparison with the oncological underlying disease group (94% of ductopenia and 64% of severe ductopenia). furthermore, a little sub-group of 16 patients extrapolated from our population received liver biopsy before hsct for diagnostic assessments: of the 10 with an oncological underlying disease 70% already showed ductopenia, while no signs of ductopenia were found in the others with a nononcological disease. conclusions: there is no correlation between incidence of gvhd and histologically finding of ductopenia on liver biopsy. ductopenia may be caused in the first place by chemotherapy treatment received before hsct and myeloablative conditioning for hsct and it's not related with gvhd. this hypothesis is strengthened by the subgroup analysis of pre-hsct biopsy. background: second and third line therapies for steroid refractory acute graft versus host disease (agvhd) after allogeneic stem cell transplantation (asct) are still lacking. ruxolitinib, a selective januskinase 1/2 inhibitor could show high efficacy in agvhd, as well as extracorporeal photopheresis (ecp). here we report a single center experience of combining both therapeutic approaches in severe steroid refractory agvhd with additional analysis of immune status of these patients to elucidate direct effects of this treatment on immune response. methods: from june 2015 to february 2017, 18 patients (77.8% male, 22.2% female, median age: 58.5 years, r: 21-73) with steroid refractory agvhd of lower gi-tract after asct were treated with ruxolitinib and extracorporeal photopheresis as third, fourth or fifth line therapy. some patients showed additional agvhd of skin (n=7), liver (n=6) or upper gi-tract (n=2). all patients had an overall grade iii (50%) or iv agvhd (50%). steroid refractoriness was defined as no improvement in 7 days or aggravation after 5 days of steroid treatment.median start of ruxolitinib or ecp was day 86.5 after asct (r: 35-257). medianduration of ruxolitinib therapy was 59.8 days (r: 14-192) with a median start dosage of 20 mg per day (2 x 10 mg; r: 10-20 mg). all patients started with 2 ecp treatments per week with an individual reduction of treatment frequency. median number of ecp treatments was 20.5 (r: 2-71) with a median frequency of ecp therapy once a week (r: 0.5-2.1). cytomegalovirus (cmv) status of all patients and immune status of ten patients (lymphocyte count with cd4 + t helper lymphocyte and regulatory t cell count) were collected previously, after four weeks of starting combined treatment and four weeks after stopping the treatment. results: one-year estimated overall survival (os) of all patients was 50% with a median estimated os of 314 days. 3 patients died because of relapse of underlying disease, one of severe therapy refractory agvhd of lower gi tract and 5 due to infection complications in agvhd refractory setting. overall response was 55.5% (complete remission rate: 44.4%, partial remission rate: 11.1%). 72.2% (n=13) of the patients had cytopenia ctc i-iii during the treatment, no grade iv cytopenia was reported. cmv reactivation during ruxolitinib occured in 66.7% of cases (n=12). tapering of steroids could be performed rapidly with a medium reduction time of 1.75 days for reducing to half of the dosage.remarkably, regulatory t cells significantly increased during combined ruxolitinib/ecp treatment compared to regulatory t cell count before treatment (p=0.02) and after stopping treatment, regulatory t cell count decreased again (p=0.02, see figure) . significant changes in whole lymphocyte count or in cd4+ t helper cell count were not observed. conclusions: treatment of severe steroid refractory agvhd with ruxolitinib plus ecp could show a high complete remission rate of 44.4% with an one year os of 50%. detecting increased regulatory t cell count during the treatment underlines its direct effects on immune response and encourages to pursue this promising therapeutic approach. [ background: due to increased immunosuppression infections remain the main cause of death followed by higher risk of relapse in patients treated for acute graft versus host disease (agvhd) after allogeneic stem cell transplant (sct). here we report a single-centre experience with extracorporeal photopheresis (ecp) for acute gvhd that was introduced in order to reduce steroid treatment. comparison of overall survival (os) for patients on ecp and patients that received standard first line therapy for agvhd was performed. methods: we retrospectively analysed 62 patients (28 %) with acute gvhd grade ii-iv treated from january 2010 to october 2018 out of total 221 allogeneic sct in that period. all patients received calcineurin inhibitors or sirolimus while receiving steroid treatment for agvhd. twenty-five patients (40 %) received ecp with steroid lowering intent. we defined response as (1) reduction of steroid dose for at least 50% from baseline while not adding another immunosuppresive agent and (2) not repeating second steroid treatment if the ecp was started after lowering of steroids to prevent agvhd flare. we checked separately patient responsive and refractory/dependent to steroids. on average patients received ecp procedure once weekly. results: tapering of immunosuppressive therapy as defined was successful in 13 (52 %) out of 25 patients in ecp group. in a group of patients without ecp 14 (37 %) patients had steroid refractory or steroid dependent agvhd compared to 11 (44 %) patients in ecp group. four (16 %) patients with steroid refractory or dependent agvhd showed improvement in ecp group compared to only one (2,7%) in non ecp group. twenty (74 %) patients died due to infectious complication and 7 (26%) due to relapse in non ecp cohort. in ecp cohort 10 (77%) patients died due to infection and 3 (23%) due to relapse. median os was 5 months in non ecp group (r., 1-99) compared to 21 months (r., 2-76) in ecp group and os of 20 % at 5 years in non ecp compared to 35 % in ecp cohort was observed. patients with agvhd treated with ecp and faster steroid tapering had longer os compared to patients without ecp (p=0,03). conclusions: ecp enables successful tapering or withdrawal of steroid therapy in many patients, even in those who are steroid refractory or steroid dependent. reduction of immunosuppression leads to reduced incidence of infection and relapse which translates into a better overall survival. background: the curative potential of allogenic stem cell transplantation is hampered by graft-versus-host disease (gvhd). pre-clinical study showed an efficacity of jak1/2 inhibitor, ruxolitinib, in treatment of steroidrefractory gvhd. methods: we reported in this monocentric retrospective study, ruxolitinib response and follow up of 44 cases of chronic gvhd (cgvhd) not improved with standard immunosuppressive therapy. complete organ response (cr) was defined as the resolution of clinical manifestations of cgvhd in a specific organ. very good response partial (vgpr) was defined as an improvement of clinical manifestations of cgvhd with more than 50% decrease of corticosteroid, while a partial response (pr) was associated with less than 50% decrease of corticosteroid. treatment failure was defined by the absence of improvement of cgvhd, deterioration of cgvhd in any organ by at least one stage, the development of cgvhd manifestations in a previously unaffected organ, and the use of any additional agents to control the disease. results: median age at transplant was 52 years (range, 19-66). 59% of patients presented an acute myeloid leukemia. donor type was sibling (n=12), unrelated (n=24) or haploidentical (n=6).two patients benefited a cord blood transplant. patients received either myeloablative (64%) or reduced intensity (36%) conditioning regimens. stem cell source was peripheral blood for 75% of patients. patients presented mild (n=11), moderate (n=21) or severe (n=12) cgvhd according to nih score. median number of regimens prior to ruxolitinib was 1 (range, 0-6), among those corticosteroids (n=36). median follow-up after ruxolitinib was 18 months (range, 4-32). overall responses rate (orr) at 1 month was 84% with 34% cr, 6% vgpr and 43% pr ( figure 1 ). 13% of patients failed at 1 month after introduction of ruxolitinib. the rate of cr increased with time : 45% at 3 months (n=38), 53% at 6 months (n=30) and 65% at 12 months (n=23). but vgpr rate was rather stable at 3 months (24%), at 6 months (33%) and at 12 months (22%) vs 6% at 1 month. among the patients under steroids, 21 (58%) patients discontinued steroids. the 12-months overall survival (os) and diseasefree survival (dfs) after ruxolitinib was 89% (80%-99%, 95% ci) and 86% (76%-96%, 95% ci), respectively. severe cytopenia (grade 3 and 4) was observed in 12 patients. after introduction of ruxolitinib, 4 patients presented bacterial infections, 3 patients presented an invasive pulmonary aspergillosis and 1 patient developped a pneumocystis. cytomegalovirus reactivation requiring preemptive treatment was observed in 7 patients. no toxicities required withdrawal of ruxolitinib. [[p269 image] 1. figure 1 and partial response to mesenchymal stem cells (msc), as second-line therapy, varies from 15% to 82% in acute gvhd patients. we report our experience using mscs to treat refractory agvhd. methods: the study was a retrospective single center study. all data were collected from patients' files. twenty patients were enrolled (age ranging from 7 months to 19 years) between april 2014 and april 2018. results: five of these patients received reduced intensity conditioning and 15 patients received myeloablative regimens before hsct. one haploidentical, 1 autologous, 1 cord blood, 14 mud, 3 msd transplantations were performed. the patients were eligible if they developed grades ii-iv agvhd. all patients were treated with standard first-line treatment with corticosteroids and at least one second-line therapy. the definition of steroid resistant agvhd considered as either no response to steroid treatment lasting at least 7 days or progression during treatment of at least one grade within the first 72 hours. prophylactic treatment with calcineurin inhibitors continued at therapeutic dose level. totally, 77 doses of mscs were infused. the median dose of msc was 1.02 × 10 6 cells per kg body weight. the median duration between the diagnosis of agvhd and initiation of mscs therapy was 16 days (range: 4-241). the received msc doses ranged from one to seven. none of our patients had severe side-effects during infusions of mscs. overall, complete response (ocr) was obtained in 10 patients, partial response in 7 patients and no response (nr) was documented in 3 patients. in our study group, the complete response rates in liver, gastrointestinal, skin agvhd were 20%, 35%, 72% respectively. four patients (20%) died in 90 days after using mscs from complications of agvhd. eleven of 20 patients (55%) were still alive with a median follow-up of 558 days (range:171-989 days) after first mscs infusion. one year estimated probability of overall survival for patients achieving ocr and partial remission/no remission in 90th day of mscs were 87.5% and 20%, respectively. conclusions: in conclusion, mscs appears to be a safe and effective treatment option for pediatric patients with steroid refractory agvhd. disclosure: nothing to declare effect of extracorporeal photopheresis on production of serum elafin in chronic graft versus host disease arun alfred 1 , charlotte burton 1 , kathryn goddard 1 , nichloas matthews 1 background: extracorporeal photopheresis (ecp) is a second line therapy for steroid refractory, dependent or intolerant chronic gvhd (cgvhd). in order to guide ecp there is an unmet need for predictive and diagnostic biomarkers. elafin is a serine-protease inhibitor primarily produced by epithelial cells, particularly keratinocytes in inflammatory skin diseases and plasma and epidermal elafin have been identified as biomarkers of skin gvhd (1, 2) . since skin cgvhd is noted for a particularly high response rate to ecp, we conducted a study to investigate whether ecp affects the production of elafin. methods: serum samples were collected from 72 cgvhd patients (39 male /33 female; age range: 25-74) and 17 age-matched healthy controls (10 male / 7 female) before ecp and at 3 month intervals up to 1 year. patients had gvhd affecting skin (61/72), mucosal membranes (16/ 72), liver (14/72), joints (8/72), gut (17/72), eye (8/72), genital (4/72), and respiratory involvement (4/72). serum elafin was assessed by elisa (r&d systems). data were analysed using graphpad prism 6. statistical tests performed include 2-tailed mann-whitney, pearson's correlation test, and 2-way anova with repeat measures, as appropriate. results: chronic gvhd patients presenting for ecp had significantly elevated serum levels of elafin (p=0.0029; median of 22ng/ml, iqr 15-45ng/ml) compared to healthy controls (median of 14 ng/ml, iqr 9.6-18ng/ml).while 85% of patients had skin involvement, only 40% had elafin levels above the iqr of healthy controls. where disease scores were available (n=25) there were no significant correlations with modified rodnans (r=0.19) or nih bsa scores (r=0.37).sub-analysis was performed by grouping cgvhd patients according to quartiles of serum elafin at pre-ecp baseline. retrospective analysis of patients after 12 months of ecp (n=66) revealed that those with serum elafin levels in the upper quartile (elafin hi ) pre-ecp (min-max: 46-117ng/ml), showed a significant reduction after 3 months of therapy (p< 0.05; mean +/-sd :72ng/ml +/-26 ng/ml vs 53ng/ml +/-23ng/ml, respectively), which was sustained up to 12 months of ecp (p< 0.0001; mean +/-sd: 37 ng/ ml +/-20ng/ml). in contrast, patients with elafin levels below the upper quartile (elafin lo ) showed no significant change (mean +/-sd: 21ng/ml +/-11ng/ml vs 26ng/ml +/-20 ng/ml, respectively). of note, pre-ecp patients with elafin below the median received significantly more corticosteroid (cs) than those above, (p< 0.05; mean +-sd: 27+/-18mg/d vs 16+/-16mg/d, respectively), which was significantly reduced after 3 months of ecp (p< 0.001; to 13+/-10 mg/d), while cs dose was not significantly changed in elafin hi patients until 6 months (p< 0.05; mean +/-sd: 16mg/d +/-16mg/d vs 6mg/d +/-5.5mg/d, respectively). conclusions: consistent with recent data, we found that serum elafin is significantly elevated in a subset of cgvhd patients compared to healthy controls, but did not correlate with skin disease scores. ecp administration was associated with a reduction in serum elafin in the elafin hi subset. further, elafin lo and elafin hi patients tolerated different rates of ecp-mediated tapering of cs immunosuppression suggests pre-ecp elafin measurements may have predictive value. references : background: allogenic hematopoietic stem cell transplantation (hsct) is a potential curative treatment for many malignant and no malignant hematologic diseases, primary immunodeficiencies and some metabolic and deposit diseases in children. graft versus host disease (gvhd) is a major cause of morbidity and a leading cause of non-relapse mortality. corticosteroids are the standard first-line systemic treatment for both acute and chronic gvhd, whereas no second line option for corticosteroid-refractory patients is standardised. ruxolitinib is a potent inhibitor of jak 1/ 2 showing significant responses in refractory gvhd patients in recent reports. methods: we present two centres experience with ruxolitinib for gvhd treatment in pediatric patients. the study was conducted in two spanish pediatric hsct centres, hospital vall d'hebron (barcelona) and hospital universitario la paz (madrid). all patients receiving ruxolitinib since the drug was available were included for retrospective analysis. results: between march 2017 and december 2018 19 pediatric patients with acute or chronic gvhd with refractoriness to corticosteroids were treated with ruxolitinib, in 22 different episodes (one patient received it in 3 different moments, and one patient received it in 2). patient's sex at birth was female in 11 and male in 8 cases. median age at hsct was 10,94 years (0,79-18,11). primary disease was malignant in 11 patients and non malignant in 8. median time of gvhd diagnosis was 47,5 days (6-525). all gvhd episodes were treated with corticosteroids as first line, with maximum doses between 1-5 mg/kg/day (the main dose used was 2 mg/kg/day, 15/22 episodes). patients received a median number of 3,5 (2-7) previous lines of treatment including steroids before starting ruxolinib; they were extracorporeal photopheresis (19/22 episodes), sirolimus (7/22), mesenchymal cells (5/22) ruxolitinib initiation was indicated for acute gut refractory/steroid dependant gvhd in 9 episodes and chronic multisystemic in 7 episodes. other indications were chronic lung (3/22 episodes), chronic skin (2/22) and acute skin gvhd (1/22) . median post-hsct time of ruxolitinib start was 300 days. doses ranged between 2,5-10 mg/12h depending on age, weight, and tolerance (hematologic and liver toxicities). average duration of treatment was 186 days (11-616). complete response (cr) rate was 18,1%, global partial response (pr) 72,7%, and no response (nr) 9% (progression in one patient and recent treatment start in other patient). mean time to maximum response was 4 weeks. treatment stop cause was cr in 2 cases, infection in 4, liver toxicity in 1. no severe side effects directly related to ruxolitinib treatment were described. conclusions: ruxolitinib has been recently introduced as second line strategy for rescuing corticosteroid-refractory gvhd in pediatric patients. while results of randomized trials are lacking, we present our experience (two centres). the main indications for starting treatment were acute gut and chronic multisystemic gvhd. most patients achieved some grade of response (partial or complete), allowing stopping or tapering corticosteroids. toxicity profile appears to be acceptable. disclosure: nothing to declare. stability of tacrolimus concentration early after allogeneic hematopoietic stem cell transplantation reduces the risk of acute gvhd background: tacrolimus is used as an immunosuppressive drug after allogeneic hematopoietic stem cell transplantation (allo-hsct). it is well known that early concentration level of tacrolimus is correlated with the risk of acute graft versus host disease (agvhd), however, whether range of standard derivation (sd) of early tacrolimus concentration after allo-hsct also affect to the risk of agvhd still remains unknown. here, we investigate the correlation between the range of sd of early tacrolimus concentration after donor hematopoietic cells engraftment and the development of agvhd. methods: we retrospectively assessed 207 patients who underwent allo-hsct in our hospital from 2010-2017. all patients received standard gvhd prophylaxis by continuous intravenous (iv) tac with starting dose of 0.02 mg/ kg/day from 1 day before allo-hsct (day -1) and iv methotrexate on day 1, 3, 6 at dose of 10 mg/m 2 , 7 mg/m 2 , 7 mg/m 2 , respectively. tac dosage was adjusted to target the serum concentration of 8-12 ng/ml until at least day 30 and then tapered. to evaluate the sd of weekly tacrolimus concentration, the range of sd of tacrolimus concentration at day1-7 (week-1), day8-14(week-2), day15-21(week-3) and day22-28(week-4) were calculated. the difference of the range of sd between the 2 groups that develop or did not develop agvhd was compared by using mann-whitney u test. multivariate analysis was performed by using multiple logistic regression analysis. patients had given written consent allowing the use of medical records for research, in accordance with the declaration of helsinki, and the institutional review board approved the study. results: there were 114 males and 93 females and the median age was 45 years (range, 16 to 68 years). the risks of disease were low-standard in 141 and high in 66 pts. the number of donors were in 6 hla-identical sibling, 13 in hla-mismatched related donor, 81 in hla-matched unrelated donor and 107 in hla-mismatched unrelated donor. thirty-seven patients developed agvhd (grade i-ii; 28, gradeiii-iv; 9 patients). as a result, the wide range of sd at week-3 significantly increased the risk of agvhd (agvhd-group; 1.178±0.8159 ng/ml, agvhd+ group; 1.447±0.7359 ng/ml, p=0.02). multivariate analysis demonstrated that narrow range of sd of tacrolimus concentration at week-3 reduce the risk of agvhd (or=4.19; 95% ci: 1.59-14.80; p=0.005). there were no correlation between gender, age, disease status, hla with the development of agvhd. conclusions: the range of sd at week-3, an engraftment phase of donor hematopoietic cells, was significantly correlated with the development of agvhd. fine tuning of early tacrolimus concentration with narrow range of sd reduces the risk of agvhd, resulting in improvement of the overall survival after allo-hsct. disclosure: nothing to declair p274 ruxolitinib treatment for steroid-refractory graftversus-host disease han-seung park 1 , je-hwan lee 1 , jung-hee lee 1 , eun-ji choi 1 , miee seol 1 , young-shin lee 1 , young-ah kang 1 , mijin jeon 1 , kyoo-hyung lee 1 background: steroid-refractory graft versus-host disease (gvhd) is one of the most lethal complications after allogeneic hematopoietic cell transplantation. recent studies have shown that ruxolitinib, a janus kinase 1/2 inhibitor, is effective in patients suffering from gvhd. here, we report a retrospective result of ruxolitinib treatment for steroid-refractory gvhd. methods: all patients had received cyclosporine and a short course of methotrexate as gvhd prophylaxis. antithymocyte globulin was added for unrelated or mismatched familial donor hct. ruxolitinib 5 mg twice daily was added to immunosuppressive treatment in patients with steroid-refractory gvhd. results: a total of 27 patients with gvhd (acute, 8, including 3 patients with donor lymphocyte infusion [dli]related; and chronic, 19) were included in the analysis. all patients had grade 3/4 acute gvhd or severe chronic gvhd at the time of ruxolitinib treatment. six (75.0%) of 8 patients with acute gvhd responded to ruxolitinib, including 3 with complete response (cr). the median time to response was 13.5 days (range, 8-25) . nineteen patients received ruxolitinib for severe chronic gvhd, with the median of 3 involved organs (range 2-5). fourteen patients (73.7%) showed response to ruxolitinib, including 4 crs. the median time to response was 24 days (range, 12-138). five responders discontinued ruxolitinib and 9 patients are still on the agent. after a median follow-up duration of 8.7 months, 5 died (2 from relapse of disease, 3 from infection). the 1-year survival probability was 70.1%. eleven of 20 responders discontinued ruxolitinib. gvhd relapsed in 3 of 11 patients at 14, 35, and 149 days after ruxolitinib discontinuation. thrombocytopenia (12/27, grade3/4; 4) was the most common adverse event of ruxolitinib. during treatment, 4 with grade 3/4 infectious adverse events occurred; 2 pneumonias, 1 brain abscess, and 1 liver abscess. conclusions: ruxolitinib treatment seems to be effective for the treatment of steroid-refractory gvhd including long-standing chronic gvhd. the agent was well tolerated and relatively safe. disclosure: nothing to declare. il6-receptor antibody tocilizumab as salvage therapy in the treatment of severe chronic gvhd after stem cell transplantation: a retrospective analysis background: severe chronic graft-versus-host disease (cgvhd) remains the most relevant factor affecting survival and long-term quality of life after allogeneic hematopoietic stem cell transplantation (hct). besides corticosteroids there is no established therapy for cgvhd and many of the used immunosuppressive agents may lead to significant toxicity incl. infectious complications. tocilizumab (an il6-receptor antibody) has shown efficacy in acute gvhd and cgvhd. we retrospectively analyzed the efficacy and safety of patients having received tocilizumab for treatment of advanced cgvhd at our center between the years 2015 and 2018. methods: 9 patients with severe steroid refractory cgvhd and a median age of 50 years (range: 21-62 yrs) having received at least two prior lines of therapy for cgvhd (range: 2-8 regimens) were treated with tocilizumab for at least one cycle (q4w, dosage: 8 mg/kg iv, maximum: 800 mg) with a median number of 4 cycles (range: . nih consensus criteria grading for cgvhd and the immunosuppressive regimen were noted at the time of the first tocilizumab administration and after 3, 6 and 12 months of therapy. all patients received additional concomitant immunosuppressive agents already given at least 4 weeks without response before start of tocilizumab. no new immunosuppression (is) was added in parallel to tocilizumab and response assessment was stopped at start of any additional new is. all patients had received peripheral stem cell allografts. gvhd prophylaxis consisted of a calcineurin inhibitor in combination with methotrexate or mycophenolate and in case of unrelated donors atg was added. 8/9 patients had quiescent onset of cgvhd, one patient developed de novo cgvhd. the median number of days between hct and onset of cgvhd was 215 (89-545). the median number of days between hct and initiation of tocilizumab therapy was 1033 (510-1749) days. at cgvhd onset, 7/9 patients had mild cgvhd and 2/9 patients had moderate cgvhd. the thrombocyte count was < 100/nl in 5/9 patients. organs involved at initiation of tocilizumab therapy were skin (100%, all grade 3), eyes (78%), mouth (67%), fascia (67%), lungs (44%) and genitals (22%). 5/9 patients are still receiving tocilizumab at the time of analysis. results: as tocilizumab was given fairly recently in most patients, 6-and 12-month follow-up was only reached in 3/ 9 patients (33%). at three-month follow-up after initiation of tocilizumab therapy, 4/9 patients (44%) showed partial remission, 2/9 patients stable disease (22%), and 2/9 patients progressive disease (22%) of cgvhd. maximal response was partial remission (56%), stable disease (11%) and progressive disease (22%). 3 patients required subsequent new immunosuppressive treatment. one patient has not yet reached 3-month follow-up. during tocilizumab therapy none of the patients suffered recurrence of underlying malignancy. two patients developed significant respiratory infection and one patient developed soft tissue infection, all requiring antibiotic treatment and pausing of tocilizumab administration, hospital admission was not required. the os and rfs was 100% with median follow up of 8.5 months (range 2-12 months). conclusions: tocilizumab appears to be a promising treatment option in advanced cgvhd but further evaluation within a phase ii trial is required. disclosure: nothing to declare background: allogeneic hematopoietic stem cell transplantation (hsct) is for many patients suffering from aml the only curative treatment option. one major complication is graft versus host disease (gvhd), caused by donor immune cells attacking healthy tissue. regulatory t cells (treg) have been getting huge attention during the past years because of their important role in maintaining immune balance. here we collected peripheral blood samples from 11 patients at different time points after hsct to investigate immune-reconstitution of treg as predictive marker for the development of gvhd. methods: we collected blood samples from 11 patients in the course of allogeneic hsct prospectively once a week from d+7 up to d+200. all patients received conditioning regimen with fludarabine and melphalan, combined with alemtuzumab for t cell depletion. 9 patients developed acute gvhd in the later course. after isolation of pbmc`s we performed facs multicolor staining of t cell and nk cells. treg were identified as cd3 + cd4 + cd25 ++ foxp3 + , nk cells were characterized as cd3 neg cd56 + cd16 + and divided in nk cell subpopulation due to their expression of cd56 dim or cd56 high . results: 1. cd52 neg t cells: all patients developing acute gvhd in the later course showed significant elevated levels of cd4 + cd52 neg t cells, especially cd52 neg treg at d+50. 2. cd52 neg treg / cd8 + cd52 + t cells: one patient not developing acute gvhd showed lots of cd52 neg treg but missed cd8 + cd52 + effector t cells. we recently showed that cd8 + cd52 neg effector t cells are of impaired effector function. these data suggest that cd52 neg treg are only of relevance combined with functional cd8 + cd52 + effector t cells in the development of agvhd. 3. t cell marker: patients without agvhd showed elevated expression of garp on treg. garp was significantly higher expressed on cd52 + treg, indicating a better suppressive capacity of cd52 + treg. this was detected throughout from d+50 until d+200. tigit and ilt3 showed a heterogeneous expression profile without significant differences between the two groups. 4. nk cells: we detected a higher ratio of cd65 ++ /cd56 dim nk-cell population in patients without. we could also show that tigit is mainly expressed on cd56 dim nk cells. conclusions: we and others showed reconstitution of cd52 neg t cell subsets after alemtuzumab mediated t cell depletion -our data on effector t cells showed an impaired effector function for cd52 neg cd4 and cd8 t cells. recently we presented data on impaired suppressive capacity of cd52 neg treg and the association with acute gvhd retrospectively (wölfinger ebmt 2018, ash 2017). here we provide prospective data on patients after the use of alemtuzumab in the context of hsct: our preliminary data suggest that the total amount of cd52 neg-treg and the ratio of cd52 neg treg to cd8 + cd52 + treg on d+50 after allogenic hsct could predict agvhd. this data may be a basis for immune monitoring of patients at d+50 to evaluate their risk for agvhd and could lead to the use of prophylactic treg dli in the context of alemtuzumab mediated t cell depletion. disclosure: medac -travel support, novartis -consultancy fee, pfizer -consultancy fee, shireconsultancy fee background: multiple factors such as disease activity and severity, therapy and/or dietary habits can cause changes in nutritional status independently or by interacting with each other. presence of malnutrition or significant weight loss in chronic gvhd (cgvhd) patients was reported in literature up to 43%. the aim of this cross-sectional study was to identify factors that affect nutritional status in cgvhd patients. methods: nutritional status in patients with cgvhd treated at the university hospital center zagreb, croatia from 2015 to 2018 was assessed. anthropometric measurements (height, body weight (bw), body mass index (bmi)) and clinical validated tool patient-generated subjective global assessment (pg-sga) (where patients were categorized as well-nourished (pg-sga a), moderately malnourished (pg-sga b) or severely malnourished (pg-sga c)) were used. all patients were evaluated according to 2005 nih criteria for cgvhd diagnosis. descriptive and correlation analysis were preformed. results: in total, 44 adult cgvhd patients were included in the study, 23 women (52.3%), median age 47 (18-65) years, with mild cgvhd in 6 (13.64%), moderate in 24 (54.55%) and severe in 13 (29.55%) patients. according to the pg-sga rating 19 (43.18 %) patients had pg-sga a, 19 (43.18 %) pg-sga b and 6 (13.64%) had pg-sga c, giving a total malnutrition or risk of malnutrition prevalence of 56.82%. the mean bmi was 23.83±4.3 kg/m 2 with correlation to pg-sga rating (r=0.446, p=0.0024). malnutrition according to the bmi (defined as bmi< 21 kg/m 2 ) was found in 8 patients (18.18%). bw changes (10% or more in 6 months) were significant in 14 patients (31.82%). according to the pg-sga assessment tool, oral symptoms reported by 31 patient (70.45%) and decreased appetite reported by 12 patients (27.27%) were associated with oral cgvhd nih score (r=0.536, p=0.0002; r=0.441, p=0.0027) but not with bw or bmi. gastrointestinal (gi) symptoms assessed with sga, were generally mild with no correlation to gi cgvhd nih score. no significant association was found between nutritional status and other nih cgvhd scores. corticosteroid therapy present in 12 (27.27%) correlated with pg-sga rating (r=0.494, p=0.0006) but not with bw, bmi or appetite changes. in 23 patients (52.7%) with altered pg-sga rating, bmi, appetite and body weight changes, dietary counseling and oral nutritional supplementation were initiated. conclusions: oral symptoms, decreased appetite and corticosteroid therapy in our cgvhd patients were associated with altered nutritional status according to the pg-sga, but not with bmi. therefore, pg-sga might be a more sensitive tool in assessment of changes of the nutritional status and detection of patients at risk of malnutrition than bmi since it includes different factors like physical examination, presence of gi symptoms and corticosteroid therapy in its scoring system. nutritional counseling and support are important in cgvhd patients especially in presence of oral symptoms. disclosure: nothing to declare. background: sclerotic skin changes are common features in chronic graft versus host disease (cgvhd). one of the most challenging aspects in the diagnosis and management of sclerodermoid cgvhd (scgvhd) is the differentiation between reversible symptoms related to active cgvhd and nonreversible symptoms related to residual permanent damage such as long-standing fibrosis. although several candidate biomarkers of cgvhd inflammatory activity have been proposed, none of them are currently validated. therefore, there is a need for the development of more quantifiable and reproducible measurements tools to guide clinical decisions. we report our experience evaluating the usefulness of high-frequency ultrasonography (hfus) plus doppler ultrasound (doppler-us) and serum fibrosis biomarkers to determine the inflammatory activity of scgvhd. methods: we report 6 patients with scgvhd. hfus plus doppler-us were performed at diagnosis of scgvhd and at different time-points after treatment initiation. serum hyaluronic acid and pro-colagen-iii were measured as fibrosis biomarkers simultaneously with hfus and doppler-us. nih cgvhd 2014 consensus conference diagnosis criteria, scoring system, and response criteria were used to assess global and organ-specific cgvhd, and to measure overall response to therapy. abnormal ultrasound findings were defined as the presence of ≥ 1 of the following: hypoechogenic dermis, dermo-epidermal junction effacement, hypoechogenicity of septa and/or hyperechogenicity of lobules in hypodermis, hypoechogenic fascia, or myositis, for hfus; and, vessels thicker than 1mm in dermis and/or hypodermis, systolic pressure >10 cm/sec, and index of vascular resistance >0.75, for doppler-us. inflammatory activity was classified as mild, moderate and severe according to the severity of doppler-us findings. results: hfsu showed abnormal findings in all patients at diagnosis with no changes except in two patients along the treatment follow-up. inflammatory activity by doppler-us was observed in 5/6 patients at diagnosis (1 mild, 3 moderate, 1 severe). four patients responded to treatment (2 complete responses, cr, and 2 partial responses, pr), one presented clinical improvement less than pr, and one, progressive disease. all patients with clinical response had also a p-rom improvement or normalization. all patients achieving a response showed normalization (n=2) or improvement (n=2) of doppler-us findings. the patient with clinical improvement less than pr and the patient with progressive disease showed persistence of inflammatory doppler-us findings. most patients had normal or light increase of pro-collagen levels at diagnosis and no significant changes were observed during follow-up. levels of hyaluronic acid tended to be very high in patients with progressive scgvhd (patients 2 and 5) and tended to decrease or normalize in those who responded to therapy (patients 1, 2, and 3). conclusions: in this exploratory study, hfsu was a reliable method for evaluating sclerotic skin changes in scgvhd. doppler-us showed a good correlation with disease activity and response to treatment. serum hyaluronic acid levels might be a biomarker of disease activity that deserves further investigation. hfsu plus doppler-us is a useful, non-invasive, repeatable device in monitoring patients suffering from scgvhd. according to our results, doppler-us may be a more sensitive parameter than hfsu in assessment inflammatory activity of scgvhd. disclosure: maría suárez-lledó received a grant from dkms-spain foundation. other authors have nothing to declare post-transplant cyclophosphamide versus antithymocyte-globulin in hla-matched unrelated and haploidentical transplantation for hematologic malignancies background: post-transplant cyclophosphamide(ptcy) and antithymocyte-globulin(atg) are the most commonly used regimens for the prophylaxis of graft-versus host disease(gvhd). we compared these two regimens in hlamatched unrelated (mud) and haploidentical transplantation for hematologic malignancies. methods: we retrospectively analyzed the consecutive adult patients with hematologic malignancies who received mud and haploidentical transplantation at chungnam national university hospital between january 2013 and january 2018. patients who received second transplantation and had refractory disease were excluded. results: this study included 45 patients with median age of 54 (range, 18-71) years: 29 (64.4%) patients received mud transplant (8 and 21 patients in ptcy and atg group, respectively), and 16 (35.6%) patients received haploidentical transplant (11 and 5 patients in ptcy and atg group). graft source was peripheral blood stem cell in all patients. median follow-up duration was 15.5 months (range, 0.5-58.0). in mud transplant, the estimated 20-months survival rate were 85.7% in ptcy vs. 80.7% in atg (p=0.835), the 20-months relapse rate were 37.5% in ptcy vs. 35.0% in atg (p=0.663), the cumulative incidence of grade 2 to 4 acute gvhd were 25.0% in ptcy vs. 22.2% in atg (p=0.706), and the estimated 20-month extensive chronic gvhd rate were 25.0% in ptcy vs. 16.7% in atg (p=0.902). in haploidentical transplant, the estimated 20-months survival rate were 55.4% in ptcy vs. 40.0% in atg (p=0.936), the 20-months relapse rate were 42.9% in ptcy vs. 33.3% in atg (p=0.328), the cumulative incidence of grade 3 to 4 acute gvhd rate were 29.9% in ptcy vs. 33.3% in atg (p=0.686), and the estimated 12 month extensive chronic gvhd rate were 25.0% in ptcy vs. 50.0% in atg (p=0.575). patients receiving ptcy had significantly longer neutrophil engraftment time than those receiving atg in haploidentical transplant [median(range); 17.0(14.0-21.0) days vs. 14.0(13.0-16.0) days, p=0.005]. conclusions: ptcy might be a good option for the prophylaxis of gvhd in hla-matched unrelated transplant as well as haplo-identical transplant. disclosure: nothing to declare early fam therapy for post allo-hsct bronchiolitis obliterans syndrome background: bronchiolitis obliterans syndrome (bos) is a potential major complication after allogeneic hematopoietic stem cell transplantation (hsct). attributed to an allo-immune reaction against the small airways, bo is considered a pulmonary manifestation of chronic gvhd. reported incidence of bos ranges from 5 to 12%, and bos-attributed mortality as high as 30%-80%. a few years ago, a new therapeutic approach with fluticasone, azithromycin, and montelukast (fam) was described (norman bc, et al. bmt 2011) . our aim was to analyze the outcomes of pts who developed bos and were precociously treated with the fam scheme. methods: all the 209 allo-hsct performed in our center from january 2015 and july 2018 were included in the analysis. baseline diseases were: 69 aml, 49 lpd, 31 mds, 28 all, 16 mpd, 10 mm, and 6 bmf. day +100 and day +365 overall mortality were 9,1% and 24,4%, respectively. rest of characteristics of the series are shown in table. fam therapy was systematically started when any patient was first diagnosed with bo. results: eleven patients (5,3%) were diagnosed with bos. at diagnosis of bos, the pts exhibited a fev1 80% of predicted (median fev1: 74%; range; 54-86%) and/or a decline >10% from pre-hsct . at day +100, 6 pts had already the syndrome. two of them died before the end of the first year: one due to invasive zygomycosis (cns plus pulmonary) and the other to baseline disease progression. at day +365, 5 more pts had bos. two more pts with bos died at 15 and 17 months post-hsct due to baseline disease progression. at the close of the analysis, 7 of the 11 pts were alive. so, with a median follow-up of 29 months (range: 8-44), mortality and bos-attributable mortality of the pts with the complication were 36,4% and 9,1%, respectively. conclusions: 1) bos is an infrequent but very severe complication of allo-hsct; 2) bos seems to be less frequent in pts with prophylactic pre-transplant ratg or post-transplant cyclophosphamide, as well as in pts undergoing transplantation with bm (compared to pbsc). 3) early diagnosis and therapy are critical to minimize the bos-attributable mortality. disclosure background: donor lymphocyte infusion (dli) is an established treatment for patients with hematological malignancies relapsed after allogeneic hematopoietic stem cell transplantation (hsct). however, it is associated with an increased risk of graft-versus-host disease (gvhd) and modest anti-tumor activity. compared to the infusion of nonmobilized lymphocytes, granulocyte colony-stimulating factor (g-csf)-primed dli might induce a stronger anti-tumor effect and reduce the risk of infusion-induced gvhd. due to the limited experience of g-csf primed dli in patients relapsed after haploidentical hsct, we conducted a retrospective study of all patients at our hospital who received dli for the relapsed hematological diseases following related hla-matched or hla-haploidentical hsct. methods: the institutional research board approved the study. we identified 94 patients with hematological malignancies receiving dli following related allo-hsct at national taiwan university hospital between 1999 and 2018 aug. the infusate was obtained from the cryopreserved specimen, which had been collected and stored at multiple aliquots at the same time as the initial haploidentical peripheral stem cell graft. patients received dli for either hematological relapse, preemptive or prophylactic treatment. univariate and multivariate analysis was performed using cox proportional hazard regression model. results: for the 61 patients following related hlamatched and the 33 patients following hla-haploidentical hsct received 119 and 72 doses of dli, respectively. in comparison, the median cd3+ cell dosage of haplo-dli is significantly lower (p = 0.0224) than that of dli from sibling donors, with median cell dosage 0.45 × 10 7 /kg (range, 0.05-12 × 10 7 /kg) and 1.1 × 10 7 /kg (range, 0.04-9.78 × 10 7 /kg), respectively. the median time to dli from initial sibling hsct and haplo-hsct was 152 days (range, 13-3357 days) and 155 days (range, 13-946 days), respectively. overall, 12 (20%) of the 61 patients following sibling hsct developed grade 2-4 acute gvhd after dli, whereas 12 (36%) of the 33 patients receiving haplo-hsct developed grade 2-4 acute gvhd after dli (p=0.1460). importantly, for patients receiving dli with cd3+ cell dosage less than 1 × 10 7 /kg, there is no difference in the risk of developing grade 2-4 acute gvhd between patients receiving dli from sibling or haplo donors ( figure 1a) . interestingly, for patients receiving dli with cd3+ cell dosage more than or equal to 1× 10 7 /kg, 4 (50%) of the 8 patients following haplo-hsct developed grade 2-4 acute gvhd after dli, significantly more than 5 (14%) of the 37 patients following sibling hsct developed grade 2-4 acute gvhd after dli ( figure 1b) . the cumulative incidence of grade 2-4 acute gvhd at day 100 after haplo-hsct and sibling hsct were 50% (95% ci: 0.13 -0.79) and 13.5 % (95% ci: 0.05-0.27), respectively ( figure 1b , p = 0.0146). [[p282 image] 1. conclusions: our study shows that the administration of g-csf mobilized dli is feasible after haploidentical hsct for relapsed hematological malignancies. however, dli with cd3+ cell dosage more than or equal to 1× 10 7 /kg in patients receiving haplo-hsct is associated with significantly higher risk of developing acute gvhd than dli from the sibling donors. disclosure: the authors declare no competing financial interests. background: the fresenius phelix is a uva irradiation device used to photoactivate mnc collected on the amicus. the system is closed, utilizing a special mnc kit and modified instrument software. the preliminary results of a phase i safety trial involving three patients (12 treatments) with chronic graft vs. host disease are presented. methods: reasons for transplantation for the patients ages 37, 61 and 62 years were: acute myelogenous leukemia, myelodysplastic syndrome, and myelodysplastic syndrome with pnh. stem cell source was peripheral blood with a 10/10 match for all. each developed chronic skin gvhd. inclusion criteria included wbc and plt counts > 1000 and 25x10 9 /l, gfr > 30 ml/min/bsa, and ast 10-120 unit/l. exclusion criteria included active gi bleeding, nyha cardiac disease greater than grade iii, and the presence of light-sensitive diseases. amicus software 4.51 and phelix software 1.0 were used. settings included: 80 ml/min max draw rate, 2000 ml fixed cycle volume, 1.25 mg/kg/min citrate infusion rate, and 12:1 acd-a ratio. venous access was peripheral or subcutaneous port. target uva dose was 1.5 j/cm 2 and 8-methoxypsoralen dose was 3.4 ml. results: the following mean + sd procedure results were obtained: 2,341 + 14 ml whole blood with acd-a drawn, 191 + 3 ml acd-a used, 691 + 127 ml saline used, 91 + 5 minutes procedure time, and 4,881 + 419 ml total blood volume. minor alarms (n=4) on the amicus and no alarms on the phelix were encountered. all 14-day aerobic and anaerobic cultures were negative and mean endotoxin levels were 0.425 + 0.1752 eu/ml. mean pre/post cbc and plasma hemoglobin levels were: 12.5/11.8 wbc, 9.9/9.5 neutrophils, 0.06/0.05 basophils, 0.21/0.15 eosinophils, 1.06/1.03 lymphocytes, 1.23/1.06 monocytes, 314/293 platelets x 10 9 /l, 40/ 37% hct, 13.3/12.2 g/dl hgb, and 26.8/23.4 mg/dl plasma hemoglobin. plasma hemoglobin delta in the product was 0.00+0.001 grams and the subject was -0.15+0.70 grams. collected product hct. mean 1.95+0.255%. yields are in the table. adverse events included one each: acute respiratory failure, respiratory failure, muscular weakness, musculoskeletal discomfort, and peripheral swelling. three of four events occurred in one patient two weeks after the study procedure. none of the adverse events were considered related to the procedure or investigational product. the patient who experienced acute respiratory failure was removed from the study because of death due to pneumonia, felt to be unrelated to the procedure. conclusions: results indicate the new closed photopheresis system is capable of collecting sufficient mnc and irradiating the cells producing high lymphocyte apoptosis, with minimal alarms and adverse reactions. (21.4%)). 10 (23.8%) of the 42 patients also had acute gvhd of the skin or liver. 37 patients (88.09%) could be treated and controlled with methyl-prednislone monotherapy, 5 patients had steroid refractory gvhd of whom 3 patients (7.14%) could be salvaged with additional drugs (infliximab:2; tacrolimus:1); 2 patients (4.77%) had refractory acute gut gvhd and could not be salvaged despite more than three lines of therapy. at the time of reporting, 26 patients (61.9%) of the 42 are alive. 11 patients died due to transplant related mortality, while 5 patients developed relapsed disease. on binary logistic regression analysis, no baseline clinical or treatment related predictor (disease indication, disease status at transplant, transplant type, graft source, type of conditioning) could be identified for developing acute gvhd of the gastrointestinal system. conclusions: acute gvhd of the gastro-intestinal system is a significant cause for morbidity in allo-hct patients at our centre. further studies are warranted in our cohort, and a prospective analysis of gut microbiome analysis, faecal multi-drug resistance organism surveillance, conditioning related toxicity and antibiotic usage is ongoing. clinical trial registry: not applicable disclosure: the authors declare no potential conflicts of interest benefits and precautions of ruxolitinib in steroidrefractory acute gvhd background: corticosteroids are the standard first-line treatment option for patients with acute graft-versus host disease (gvhd), but approximately half of patients become refractory to steroids and require second-line treatment. ruxolitinib has the potential to treat gvhd in steroidrefractory (sr) patients based on retrospective clinical data. the ongoing prospective trials are currently enrolling patients to evaluate the therapeutic potential of ruxolitinib for gvhd. methods: we analyzed retrospectively clinical experience with ruxolitinib in patients (n=15) with grade 2~4 steroid-refractory acute gvhd patients compared with the control group not receiving ruxolitinib. in addition, immune status was evaluated about 6 weeks~8 weeks after the administration of ruxolitinib using flow-cytometry. ruxolitinib was used as a third option for sr gvhd, combined with previously used immunosuppressive drugs. and steroids were gradually decreased according to the symptoms and discontinued. patients received ruxolitinib 5 mg twice daily (bid), with increase to 10 mg bid if hematologic parameters are stable and no treatmentrelated toxicities. results: fifteen patients all were assessable for response. seven patients achieved a complete response, 5 had a partial response, and 3 had no response at 8 weeks after the first ruxolitinib dose. overall response rate was 75%. three were treatment failures. most adverse effects were manageable, except infectious complications. infectious complications were occurred in about 73% patients (n = 11), resulting in two deaths. common cause of infectious events included cytomegalovirus (n =5), herpes-zoster (n=2), epstein-barr virus (n=2), fungal infection (n = 2), pneumocystis jiroveci (n = 2), bacterial infections (n = 1), and pneumonia of unknown origin (n = 1). t cell counts tended to decreased in the group with ruxolitinib compared with the control group, especially cd4 cell counts. conclusions: ruxolitinib is effective in controlling sr gvhd and can lead to clinical benefits. however, we need to be aware of the infectious complications because ruxolitinib may lead to increased risk of opportunistic infections or reactivation of latent infections. in addition, common infectious complications are presumed to involve t cell dysfunction. clinical background: graft versus host disease (gvhd), being one of most common life-threatening complication post hsct, contributes significantly to morbidity and mortality. when affecting gastrointestinal tract (gi) it is the major cause of death in early period post hsct. due to widespread tissue involvement in most patients diagnosed with gi gvhd, surgical treatment is rarely considered. methods: among 972 allo-hsct performed in department of pediatric hematology, oncology and bone marrow transplantation in wroclaw, poland during years 1996-2018, 291 (29,9%) cases were diagnosed with gi gvhd. in this study we present 3 cases (1%) which were referred to and benefit from surgical approach. results: 1. male, 4 years old underwent hsct from matched unrelated donor (mud) due to chronic myelogenous leukemia (cml) and subsequent molecular relapse succesfully treated with donor lymphocyte infusion, followed by agvhd (skin and gut involvement, grade iv). extensive immunosuppression (steroids, mycofenolate mofetile, atg, okt3) resulted in significant resolution of agvhd symptoms. however aggravating severe abdominal pain and lack of gut movement suggesting bowel obstruction. due to presence of acute abdomen patient was immediately directed for laparotomy. resection of constricted bowel segment followed by 2 subsequent laparotomies for secondary obstruction provided complete resolution of abdominal symptoms. after 16 years of follow-up patient is alive and well. 2. eleven years old male was diagnosed with skin and gut grade iv agvhd on day +84 post mud-hsct performed due to acute myelogenous leukemia (aml). he received pronlonged immunosuppressive treatment including steroids, antibodies, msc and ecp which led to resolving of skin leasions and diarhoea. nevertheless patient was suffering from severe paroxysmal abdominal pain and incidentally vomiting. ct enterography showed partial small bowel constriction. after numerous surgical consultations, eventually on day+ 503 patient underwent laparotomy with constricted bowel resection. histopatological examination of resected tissue revealed moderate gvhd. immunosuppersion was tapered to low dose of steroids with ecp. for now, 2 years post hsct patient is alive, rarely experiencing mild abdominal cramps 3. fourteen years old female developed severe abdominal pain and high volume diarhoea on day +24 post mud-hsct performed for severe anaplastic anemia (saa). despite extensive immunosuppression (steroids, anti-tnf, anti-il2 antibodies) patient condition did not improved. through consistent stomach pain, suspected subileus confirmed by ct enterography, laparotomy was performed (day+158). resection of inflamated and obstructed bowel was made. microscopic evaluation confirmed prior gvhd diagnosis therefore immunosuppression including csa and tapered doses of steroids was continued. complete resolution of abdominal symptoms was almost immediately achieved post-surgery, however 2 months after recurrent abdominal cramps were observed and are now well controlled by pain killers. conclusions: commonly gi gvhd is diffused inflammatory process. however in some cases it may be localized and may lead to partial bowel constriction. in case of severe and prolonged stomach pain, despite of partial resolving of other gvhd symptoms, ileus should be considered. ct enterography may be useful for diagnosis confirmation. in those patients, surgical intervention may improve quality of life or even be a salvage approach. disclosure: nothing to disclose is there any impact of the uric acid levels during the preand early post-graft infusion period, on the gvhd occurence and allotransplant outcome? 36.8(16.7-61.6 ) years, who underwent allogeneic stem cell transplantation (allosct) from full-matched sibling donors for acute leukemia (n=31), very severe aplastic anemia/pnh (n=5), lymphoma (n=3), myelodysplastic/ myeloproliferative syndrome (n=3) . thirty-two patients were in remission at the time of allosct (cr1: 23, cr2: 7, beyond cr2: 2). for a better and more accurate assessment of the ua levels on the agvhd incidence, unlike to the other published studies which evaluated the ua levels only at day 0, we evaluated the ua levels in different time points during the the peri-transplant period (at the conditioning regimen initiation, and at days 0, +7 and +14). because the majority of our patients developed agvhd within the 15-30 days post-transplant, we did not incorporated in the study the of ua levels beyond the +14 day. we also investigated the effect of the ua on survival and the non-relapse mortality (nrm). the vast majority of patients received allopurinol from the 1 st day of conditioning regimen till day -1. the independent t-test, kaplan-meir method and logrank test were used in the statistical analysis. results: the median ua levels were 4.2, 2.5, 2.7 and 3.2 mg/dl at days -7, 0, +7 and +14 respectively. for the statistical analysis purposes, we grouped our patients as low-ua if they had values < 3 mg/dl or high-ua if they had >3 mg/dl. this threshold was chosen based on the ua values from all the collected samples (n=175). finally 16/ 42 (38%) patients developed agvhd; 14(33%) were assessed as gr ≥ii, while 7(16%) as gr iii-iv. the incidence of the agvhd gr ≥ii was similar (ranged from 30-35%) in both groups of patients (low-ua and high-ua) and for all the estimated time points (days -7, 0, +7, +14). we noticed a better 2-years overall survival for patients with low-ua (75% vs. 63%) however without any statistical significance. ten patients succumbed to nrm causes; 6/10 deaths attributed to gvhd complications. the nrm was assessed higher in the high-ua group (38% vs. 18%) but also this difference was not statistically significant. conclusions: though our study bears the limitations of the small number of patients and the retrospective origin, at least to our knowledge is the first which evaluates the impact of ua levels at different time points in the peritransplant period, on the agvhd incidence. in our study the ua levels did not influence the incidence or the severity of agvhd. the higher nrm rates for patients with ua>3.0 mg/dl merits further evaluation. definitely, the role of ua on the allosct outcome will be clarified through well designed prospective trials. disclosure results: five male patients (12%) had genital cgvhd manifestations presented by urethral stricture in 4/5 patients and phimosis requiring surgical treatment in one patient. all five patients had simultaneously cutaneous, oral, and/or ocular cgvhd manifestations. the first patient underwent urethroplasty of bulbomembranous part of urethra with termino-terminal anastomosis and urethroplasty of penile part of urethra with buccal mucosa autograft -bmg (dorsal onlay) that resulted in significant improvement of symptoms and normal miction afterwards. biopsy of the urethra showed mononuclear infiltration in lamina propria consistent with cgvhd. biopsy of the buccal mucosa was done prior to surgery and was negative for cgvhd involvement. the second patient underwent urethrotomy due to circular strictures, but symptoms reappeared again and he is now candidate for bmg. in two patients urethral dilatation was done, and the fifth patient presented with phimosis requiring circumcision, resulted in significant improvement of symptoms. conclusions: male genital cgvhd is an underrecognized and under-reported manifestation. patients after allo-hsct need to be actively asked about their genital symptoms and sexual function, especially if they are diagnosed with other mucocutaneous or ocular cgvhd. multidisciplinary approach, early recognition and frequent follow-up is necessary for timely start of treatment. new methods, such as bmg for cgvhd patients with urethral stricture seem promising and should be further investigated. disclosure: nothing to declare. p289 abstract withdrawn. heracles: a phase ii single-arm prospective study to assess the efficacy of fecal microbiota transfer in the treatment of steroid refractory gastro-intestinal agvhd post allo-hsct background: steroid-refractory acute graft-versus-host disease (sr-agvhd) is associated with an 80% mortality rate and reduced quality of life (qol). so far, there is no approved standard of care for agvhd second-line treatment. there is an urgent need to identify effective therapy for sr-agvhd to improve patients' outcomes. fecal microbiota transfer (fmt) might be beneficial to substantially improve the prognosis. higher gut microbial diversity is strongly associated with increased survival in gvhd patients. recent studies reported promising results of sr-agvhd patients treated with fmt. further evaluation to confirm the efficacy and safety of fmt for agvhd is warranted. the ongoing phase 2 study (heracles) investigates the efficacy of allogeneic fmt in the treatment of patients with sr-agvhd. heracles was launched after the odyssee study showed promising results in the reconstruction of gut microbiota diversity after induction chemotherapy with fmt in acute myeloid leukemia patients. we expect that fmt-based biotherapeutic drugs could be effective treatments to contain sr-agvhd, and thereby reduce the risk of life-threatening complications after allogeneic hsct. methods: heracles is a single-arm, multicenter prospective trial in 5 european countries. patients aged ≥18 years-old, who underwent allogeneic hematopoietic stem cell transplantation (allo-hsct) and developed a first episode of stage 3 or 4 agvhd with gut predominance resistant to a first-line steroid therapy are eligible for inclusion. main exclusion criteria comprise the use of other second-line gvhd therapy, patients with grade iv hyperacute gvhd, late onset agvhd, and overlap chronic gvhd and agvhd after donor lymphocyte infusion. patients receive a first maat013 enema within 5 days after sr diagnosis (v1) and 2 additional ones 1 week apart (v2/ v3) from each other. maat013 is a highly-diverse, microbiome-rich enema formulation obtained from pooled, rigorously screened faeces from healthy donors, manufactured with a standardized process using the signature maat microbiome restoration biotherapeutic (mmrb) platform. at inclusion (v1), before each dosing (v2,3), and 28 days post inclusion (v4), patients' faeces and blood are collected. safety monitoring will be performed with corresponding blood analyses. exploratory measures on faeces include characterization of gut microbiota composition and evolution, impact of maat013 on metabolism, and gut inflammation. immune system phenotyping will be performed by flow cytometry on peripheral blood mononuclear cells, and by elisa assay on plasma. patients' qol will be assessed using a standard, eq-5d-5l questionnaire. the primary objective is to assess the efficacy of maat013 by evaluating complete response (cr, according to modified glucksberg criteria) and very good partial response (vgpr, defined by martin et al., bbmt, 2009) 28 days post-inclusion (primary follow-up). secondary objectives include fmt safety assessment and evaluation of fmt impact on several endpoints, such as overall, relapsefree or gvhd-free survival and chronic gvhd evaluation, as well as multi-drug resistant bacteria carriage. patients will be followed-up until1 year after inclusion. overall, 32 patients are planned to be enrolled and treated, to assess overall response rates and maat013's safety profile. results background: anti-programmed cell death protein 1 (pd1) monoclonal antibodies can be used as "bridge to" a subsequent allogeneic hematopoietic stem cell transplantation (hsct) in patients with relapsed/refractory hodgkin´s lymphoma (hl). this strategy has been reported to be effective, but a frequent onset of steroid-refractory graft versus host disease (gvhd) was also reported. we report 3 clinical cases of patients affected by hl undergoing allogeneic hsct after having been treated with nivolumab. methods: the 3 patients of 48, 18 and 42 years respectively had advanced hl and had relapsed after a previous autologous (2) or allogeneic (1) hsct. they underwent a rescue therapy with 18, 12, 16 nivolumab cycles respectively, depending on the time of partial response achievement and the availability of a donor. two patients received a thiotepa-fludarabinecyclophosphamide conditioning, atg-based prophylaxis and pb cells from unrelated donors. the third patient received bm cells from an haploidentical donor using the "baltimora" nonmieloablative platform. results: at a follow-up of 11, 12, 15 months after hsct, respectively, all patients achieved and maintained a complete remission by pet-ct scans. all the 3 patients developed acute gvhd on day +32, +54 and +107, respectively. patient 1 progressed to grade iv acute gvhd with hepatic and intestinal involvement unresponsive to first line 2 mg/kg steroid therapy and second line etanercept plus extracorporeal photopheresis (ecp). third line therapy with ruxolitinib partially controlled the gvhd. gvhd onset in patients 2 and 3 was preceeded by a prolonged fever without microbiological findings. patient 2 developed hepatic grade ii gvhd with high transaminase levels, initially responsive to steroid therapy, then it progressed to gut requiring second line therapy with etanercept. patient 3 progressed to severe chronic gvhd with skin involvement and resulted unresponsive to steroids and ecp and it was partially controlled by ruxolitinib. immune reconstitution was delayed in all 3 patients: at 6 months post transplantation cd3 levels were 80/μl, 17/ μl and 127/μl and cd4 levels were 36/μl, 16/μl and 65/ μl respectively. only patient 2, that underwent haploidentical transplant and received post-trasplant cyclophosphamide (pt-cy), is off of immunosuppressive treatment at 11 months after hsct, without evidence of gvhd and no history of infections. out of the 2 patients receiving pbsc from unrelated donors and atg prophylaxis, patient 1 developed a disseminated fusariosis on day + 180 and died of cns fusarium localization 1 year after hsct, despite targeted antifungal therapy. patient 3 had pulmonary aspergillosis, sepsis by multidrug resistant psuedomonas aeruginosa and otomastoiditis: at +15 months after hsct, he is on ruxolitinib treatment with skin clinical partial response. conclusions: this case series confirms that nivolumab as "bridge to transplant" is effective in appropriately selected patients. however, risk of acute gvhd and delayed immune reconstitution may require a careful consideration at the moment of planning the transplant. a possible advantage of pt-cy gvhd platform and haploidentical donors should be addressed in larger studies. background: acute graft-versus-host disease (agvhd) is the most important complication after an allogeneic hematopoietic stem cell transplant (hsct). no standard secondline treatment has been established for the corticosteroid refractory agvhd. the anti-tnfα agents are a good option of treatment for these patients, especially when lower gi tract is involved. methods: from april 2010 to july 2017 we reviewed the outcome of 19 patients with steroid-refractory (sr) agvhd treated with etanercept as at least, second line treatment. etanercept dose was 25 mg twice a week for the first 4 weeks, followed by 4 weekly doses. results: median age was 52 years (range 15-69 years), and 12 patients (63%) were male. fourteen patients (74%) had a non-advanced disease status at hstc. eleven patients (59%) received a myeloablative conditioning, and the stem cell source was peripheral blood in 18 patients (95%). sixteen patients (84%) were 8/8 hla matched. the characteristics of the 19 patients, their agvhd stage previous to rescue treatment with etanercept and their outcome are shown in table 1 . seventeen patients (89%) had a classic agvhd while 2 had a late-onset agvhd. etanercept was given as a 2 nd , 3 rd and 4 th line in 3 (16%), 10 (53%) and 6 (31%) patients respectively. the median doses of etanercept administered were 7 (range 1-12), and just 4 patients (20%) completed the 12 doses planned treatment, of whom 3 were alive at 38, 24 and 18 months from the onset of rescue treatment. complications during etanercept treatment were: infection (n=9 [47%]: gram negative bacilli [n=6]), grade 2-4 neutropenia (n=8) and grade 3-4 thrombocytopenia (n=6). etanercept was indicated as a rescue treatment due to: progression after 3 days of agvhd treatment (n=1), no response after 7 days of treatment (n=11), no complete remission after 14 days of treatment (n=3) and relapse due to decrease corticosteroid doses (n=4). at the end of treatment 1 patient achieved a complete response and 3 patients a partial response, all of them are alive. these 4 patients received etanercept as a 2 nd (n=3) and 3th line (n=1) treatment, all of them had lower gi agvhd without any other organ significantly involved. causes of death were: agvhd with or without infection in 15 patients (78%) and leukemia relapse in 1 patient. conclusions: although if etanercept is an option for treatment of sr agvhd in some patients, their prognostic remains poor and more effective alternative strategies are needed. a prompt initiation of etanercept as a rescue treatment for sr agvhd is crucial to improve the prognosis. (58) 5(26) background: although both cyclosporine (csa) and tacrolimus are calcineurin inhibitors, csa is more widely used in pediatric hematopoetic stem cell transplantation (hsct) as a prophylactic drug for acute graft versus host disease (agvhd). there are some clinical experience but very few data about the clinical efficacy of conversion to tacrolimus. here, we present our single center data on this arguable topic. methods: this study involves the data of 71 pediatric hsct patients in medical park göztepe hospital between 2014-2018. all 71 patients had prophylactic csa therapy and for various reasons csa was converted to tacrolimus therapy. most of the patients had this conversion due to agvhd. as steroid is the first line therapy for agvhd, conversion to tacrolimus is done concurrently at the start of steroid therapy (within 72 hours after the start of steroid). and also, patients who had any other immunosupressive therapy for agvhd are excluded. response is defined as resolution of symptoms within 7 days after conversion. results: mean age of the study population is 130 months (5-266 months), male/female ratio is 1,3 (40/31), donor types are mud 48 patients (67%), mfd 18 patients (26%), haplo 5 patients (7%) and mean conversion time is 32 days (1-142 days) . the rationales for conversion are agvhd for 40 patients, unproper csa plasma levels for 7 patients, allergic reaction for 6 patients, nephrotoxicity for 6 patients, hepatotoxicity for 5 patients, severe headache for 2 patients, high arterial blood pressure for 2 patients and one each for refractory vomiting, autoimmune thyroiditis and visual disturbance. the subgroup analysis of agvhd patients reveals that mean conversion time for agvhd is 41 days (8-142 days) and there are only 11 responders whose agvhd resolve completely (%27) after conversion. all of the patients had proper tacrolimus levels after conversion due to unproper csa levels and also patients in allegic reaction, severe headache, visiual disturbance and refractory vomiting group responded to conversion completely but only one of the 6 patients in nephrotoxicity group responded and also 3 of the 5 patients in hepatoxicity group responded. the only one patient suffered from autoimmune thyroiditis did not respond to conversion. conclusions: in this study, it is obvious that there are response to conversion for some specific adverse effects of csa and tacrolimus is a good alternative for the patients who have unproper csa levels. conversely, the high percentage (%73) of non-responders shows that it is not feasible to make a conversion to tacrolimus for acute gvhd. disclosure: nothing to declare background: capillary leak syndrome is caused by the dysfunction of the vascular endothelial cells,and is characterized by weight gain,generalized edemas,unresponsive to diuretic treatment,and hypotension.it usually develops in the first 15 days post hsct.and it is of great difficuty to distinguish from other complications which are occured post the allo-hsct. to diagnose this complication at the early stage,it is very difficulty. methods: a 34-year-old man was admitted to ningbo first hospital for its abnormal in the peripheral blood .he was diagnosed with aml-m5 by the classical morphology and immunophenotype.cytogenetic evaluation showed a normal 46, xy(20).the patient achieved cr with induction therapy including idarubicin, cytarabine and etoposide. after consolidation therapy,an allo-hsct from hla identical related dornor(33-year-old male, donorrecipient matched by high resolution hla typing at hlaa, -b, -c, drb1, and dqb1, 10/10 matches) was performed.the recipient received conditioning with busulfan, 4 mg/kg/day injection for 3 days; cyclophosphamide, 50 mg/kg/day injection for 2 days; cytarabine, 2 g/m2/day injection for 1 day; semustine, 250 mg/m2/day orally for 1day; donor peripheral blood stem cells (pbsc:mnc: 6.43×109/l, cd34+:4.05×106/l) were mobilized, pheresed and administered to the recipient. gvhd prophylaxis consisted of traditional cyclosporine, short-course methotrexate (15 mg/ m2 at day +1, 10 mg/m2 at days +3, +6, and +11) and cyclosporin a injection 5mg/kg qer day was mot reduced untill the hematopoietic reconstitute sucessfully . on day +19, complete donor chimerism was acheieved. the csa was gradually reduced and tapered.on day +150,the patients was manifested with increasing in the time and volume of the faeces, he was diagnosed with ii°gvhd (gut).the standarded does of immunosuppressive drug including methylprednisolone and cyclosporin a was administrated. the immunosuppressive drug was gradually reduced when the gvhd was controlled.on day+271,the patient felt distress and the distress was not related to with the exercise,the temperature was normal,and he did not gain weight.there was no edema in the body.laboratory test including routine blood test,c-reactive protein,procalcitonin,blood gas analysis,cmvdna,ebvdna was normal. the ct scan shows that 1the pleural is filled up with water, and could not be enlarged promptly,2there is pericardial effusion in the body.pulmonary function test shows that 1 reduced function in ventilation and diffusion fuction.the laboratory test of the pleural effusion was normal,the blast cell was not detected in the pleural effusion,the cd34+ cell count was below the dectable level,the next generation sequencing for minimal residual disease shows that there was no gene mutation .thus, post capillary leak syndrome was considered .sirolimus was adopted and taken the place of cyclosporin a,immunoglobulin was adminstrated to reduce the edema. results: taking together comprehensively,the effusion in the pleural and cardiac was absorbed well. conclusions: occurance of capillary leak syndrome is rare,there is limited data about capillary leak syndrome. comprehensively,the mechanism of cls has not been totally identified.and there is no standard treatment to treat the complication.at present,the cls of this patient was absorbed well by administrating sirolimus,closely followup is needed. disclosure: nothing to declare graft-versus-host diseasepreclinical and animal models p295 short-term krp203 and posttransplant cyclophosphamide for graft-versus-host disease prophylaxis emi yokoyama 1 , daigo hashimoto 1 , takahide ara 1 , eko hayase 1 , takanori teshima 1 1 hokkaido university faculty of medicine, hematology, sapporo, japan background: post-transplant high-dose cyclophosphamide (ptcy) in combination with other immunosuppressants such as calcineurin-inhibitors (cis) has been increasingly used as gvhd prophylaxis after hla-haploidentical or matched hematopoietic stem cell transplantation (hsct). however,cis could hamper reconstitution of regulatory t cells (tregs) and tolerance induction after hsct, facilitating us to develop novel ci-free/ptcy-based gvhd prophylaxis. in the current study, we developed a novel gvhd prophylaxis in which ptcy was combined with short-term administration of krp203, a selective agonist of sphingosine-1-phosphate receptor type 1 (s1pr1), using murine models of mhc haploidentical bone marrow transplantation (bmt). methods: b6d2f1 (h-2 b/d ) recipients were lethally irradiated and transplanted with bone marrow cells and splenocytes from allogeneic b6 (h-2 b ) donors. cy at a dose of 50 mg/kg was intraperitoneally injected into the recipients on day +3, and krp at a dose of 1.0 mg/kg was orally administrated daily from day 0 to day +4 after bmt. donor t cells in the target organs and secondary lymphoid organs were evaluated by flow cytometric analysis. plasma levels of tnf-α were determined using cytometric beads array. to evaluate graft-versus-leukemia (gvl) effects, recipient mice were intravenously injected with luciferase-transduced p815 cells (p815-luc) on day 0, and in vivo bioluminescence imagingwas conducted weekly after bmt. results: severe gvhd was developed in allogeneic recipients and all mice died by day 50 after bmt.ptcy alone at a dose of 50 mg/kg significantly ameliorated gvhd and 30 % of ptcy-treated allogeneic recipients survived. oral administration of krp203 alone enhanced contraction of donor t cells in the lymph nodes and also ameliorated gvhd as has been previously shown with multi-s1pr agonist, fingolimod. next, we tested if shortterm krp203 on days 0 to +4 added to ptcy enhances anti-gvhd effects of ptcy. we found that survivals of ptcy+krp203 group were significantly prolonged compared to those of ptcy-alone group ( figure a) . plasma levels of tnf-a, clinical gvhd scores ( figure b) , and donor t-cell infiltration into the target organs such as the gut and skin were also significantly reduced in ptcy +krp203 group compared to ptcy-alone group (figure c and d) . unlike cis, addition of krp203 to ptcy promoted treg reconstitution after bmt. finally, bioluminescence imaging demonstrated that proliferation of p815-luc injected on day 0 was significantly delayed in ptcy +krp203-treated allogeneic recipients compared to control mice transplanted only with t-cell depleted bone marrow cells, suggesting that significant gvl effects persisted in ptcy+krp203-treated recipients. conclusions: a combination of short-term krp203 and ptcy is a promising novel calcineurin-free gvhd prophylaxis in mhc-haploidentical sct. we recently showed that donor inkt cells can be expanded ex vivo and that they are able to prevent activation and proliferation of alloreactive donor t cells while promoting efficient graft-versus-leukemia effects (schmid et al. 2018 ). however, the underlying mechanisms how human inkt cells induce immune tolerance after allogeneic hct are not fully understood. methods: monocyte-derived dendritic cells (dcs) were cultured in a mixed lymphocyte reaction with mhcmismatched t cells and culture-expanded inkt cells. tcell activation and proliferation was analyzed by multiparametric flow cytometry and released cytokines were measured via multiplex analysis. transwell assays and imaging flow cytometry were performed to elucidate cellcell interactions. bead-controlled flow cytometry-based cytotoxicity assays were used to evaluate dc apoptosis. apoptotic dcs were then purified by fluorescence-activated cell sorting to investigate their tolerogenic potential to prime regulatory t cells (tregs). results: the addition of inkt cells to mixed lymphocyte reactions resulted in a significantly reduced activation and proliferation of mhc-mismatched t cells. transwell assays and imaging flow cytometry revealed a cell contactdependent mechanism between inkt cells and dcs leading to apoptosis with increasing dna fragmentation of dcs over time. interestingly, various fluorescence-activated single cell sorted inkt-cell subsets were all able to induce apoptosis of host dcs. multiplex analysis revealed that dcs triggered inkt-cell release of cytotoxic factors like perforin, granzyme b and granulysin. blocking the inktcell receptor engagement with a cd1d antibody prevented inkt-cell degranulation as well as the subsequent induction of host dc apoptosis. inhibition of cytotoxic factors also abrogated apoptosis of dcs. in turn, sorted apoptotic dcs induced tolerogenic dcs characterized by a high expression of pd-l1 in mixed lymphocyte reactions. such tolerogenic dcs promoted the expansion of cd4 + cd25 + foxp3 + tregs and prevented activation and proliferation of mhcmismatched t cells. conclusions: we propose a novel mechanism how culture-expanded human inkt cells prevent gvhd after allogeneic hct. host dc apoptosis through donor inkt cells induces a tolerogenic immunoenvironment characterized by pd-l1 high dcs and expanding donor tregs inhibiting activation and expansion of alloreactive donor t cells. our findings pave the avenue for clinical translation of adoptively transferred culture-expanded inkt cells in humans. disclosure: nothing to declare results: vip-ko mice transplanted with allogeneic tcd bm alone had increased graft rejection with lower levels of donor chimerism and 33% day 75 survival compared with 73% survival of wt recipients. transplanting tcd bm plus 3 × 10e6 donor t cells from b10.br or balb/c donors in vip kio mice led to >95% donor chimerism and significantly increased gvhd-mortality compared with wt recipients, with 0% vs 40% survival in the b10.br-->b6 model (p< 0.01), and 0% vs 73% survival in the balb/c-->b6 model (p< 0.01). donor-derived t cells in vip-ko recipients had significantly higher th1 and th17 polarization, with higher rorγt in both cd4+ (p< 0.0001) and cd8+ (p< 0.0001) t cells, and higher frequencies of ifn-γ (p< 0.001), tnf-α (p< 0.05), and il2 (p< 0.01) in cd4+ and cd8+ t cells compared to wt recipients. b10.br-->b6 second allogeneic transplantation of radiation chimeras caused lethal gvhd mortality in vip-ko-->vip-ko and wt-->vip-ko mice, but not in wt-->wt or vip-ko-->wt b6 mice, demonstrating the protective effect of vip was due to synthesis by non-hematopoietic recipient cells. immunofluorescent imaging of allo-bmt recipients showed marked up-regulation of vip in lungs post-transplant and high vip production within neurons innervating the lungs. finally, we demonstrated that short-term administration of vip (10mcg/day) from day 0 to day 10 prevented gvhdmortality in vip-ko recipients transplanted with b10.br-->b6 mhc donor bm & t cells. conclusions: the absence of vip in recipient cells led to increased graft rejection in the absence of donor t cells and increased lethal gvhd when donor t cells were transplanted, indicating vip induced post-transplant regulates allo-reactivity of host graft-rejecting lymphocytes and donor gvhd-causing t cells. the protective effect of parenteral vip administration suggests vip-mimetics represent a novel approach to prevent and treat gvhd. these data also suggest a mechanism of action for the mitigation of gvhd by alpha-1 anti-trypsin (aat) whereby aat inhibits the proteolytic inactivation of endogenous vip. disclosure: dr. waller reports personal fees and other support from cambium medical technologies, grants from celldex, personal fees from kalytera, grants and personal fees from novartis, grants and non-financial support from pharmacyclics, and equity ownership in cerus corporation and chimerix outside the submitted work. in addition, dr. waller has intellectual property related to vip signaling that has been licensed to cambium oncology in which he holds equity. low-density neutrophils expansion is associated with acute graft versus host disease in allogeneic hematopoietic stem cell transplant patients background: low-density neutrophils (ldns) are distinguished from normal-density neutrophils (ndns) by their anomalous sedimentation within the mononuclear cell fraction after density gradient centrifugation of peripheral blood (pb). by analysing ldns and ndns from g-csfstimulated donors or lymphoma patients, we have previously demonstrated that, depending on physiopathological conditions, immature cd66b + cd10 -ldns can promote t cell survival and ifn-γ production, while mature cd66b + cd10 + ldns can exert immunosuppressive proprieties. aim of this study was to establish the frequency of cd66b + cd10and/or cd66b + cd10 + ldns in pb of allogeneic hematopoietic stem cell transplant (hsct) patients throughout immune reconstitution, and verify their potential correlation with acute graft versus host disease (agvhd). methods: patients undergoing hsct in our institution between december 2015 and june 2018 were prospectively enrolled in the study upon informed consent and after institutional board approval. criteria of inclusion were age ≥ 18 years and absence of rheumatologic or viral diseases. pb samples were collected at day +21, +42, +60, +90 and +180 after hsct and any time within day +180 in case of gvhd, before first-line therapy. eight healthy donors (hds) were enrolled as control. mononuclear, polymorphonuclear, and whole blood cells were analysed by flow cytometry after cd45 vioblue, cd16 apc-cy7, cd11b pe-cy7, cd10 pe, cd66b fitc staining. cd66b + ldns were expressed as percentage of cd45 + pb mononuclear cells (pbmcs) or cd45 + whole blood cells and were further characterized based on cd10 expression. cd66b + ndns, expressed as percentage of cd45 + whole blood cells, were also analysed for cd10 staining. results: 39 patients (m/f 25/14, median age 47) were enrolled in the study. patients received hsct from hlaidentical (13) or haploidentical (6) related and from hlaidentical unrelated (20) donors. after a median time of 33 (15-95) days, 13 patients developed grade ii-iv agvhd. no patients were receiving g-csf at agvhd onset. the scheduled assessments were interrupted in agvhd patients at the beginning of first-line treatment and in 4 patients relapsed of their primary malignancy. no patients developed de novo late-acute or chronic gvhd. starting from day +21 the frequency of ldns within cd45 + pbmcs was higher in all patients as compared to hds. the 25 patients that did not develop agvhd showed a decreasing frequency of cd66b + cd10 -ldns, with a progressive increase of cd66b + cd10 + ldns, from day +21 to +180. interestingly, patients with agvhd showed a significantly higher frequency of cd66b + cd10 -ldns as compared to patients without agvhd throughout the same time lapse (i.e. from day +21 to +90) (83.15 vs 50.8, p=0.027). consistently, patients with agvhd had a significantly lower frequency of cd66b + cd10 + ldns (12.1 vs 50.05, p=0.0014). the frequency of mature cd66b + cd10 + ndns was normal in all patients since day +21. conclusions: ldns are more represented in hsct patients than in hds, with a significant expansion of the cd66b + cd10subpopulation (with a parallel decrease of the cd66b + cd10 + subpopulation) in patients with agvhd as compared to those without agvhd. according to the previously demonstrated t cell activating function of cd66b + cd10 -ldns, it is tempting to speculate that the expansion of this subpopulation may contribute to agvhd development. disclosure: nothing to declare background: acute graft-versus-host disease (agvhd) is a major complication after allogeneic hematopoietic stem cell transplantation (allo-hsct) which has negative impact on the morbidity and mortality of the patients. accumulating evidences suggest that abnormalities of foxp3+ regulatory t (treg) cells contributed to the pathogenesis of gvhd, but the underlying molecular mechanisms still remain largely unknown. methods: in this study, we enrolled all the 40 patients treated with allogeneic hsct at the institute of hematology, chinese academy of medical sciences between 2016 and 2018,as well as 10 age-matched healthy adults as control samples. the ratio of tregs in pb and bm of healthy controls (hcs) and patients with and without agvhd was determined by flow cytometry. the transcription profile between tregs from patients with or without acute gvhd was measured,the pathway enrichment analyses were performed by the kyoto encyclopedia of genes and genomes (kegg) pathway database and geneset enrichment analysis (gsea).the expression of lkb1 at transcript levels and protein levels was measured by realtime pcr and analyzed by the nanopro1000tm system. a series of functional assays in vitro were performed to assess the function and stability of tregs from patients with and without agvhd.meanwhile, to assume the affect of lkb1 on gvhd outcome, we established a murine transplant model,which recipient balb/c animals were transplanted with the same amount of mixture made by bm, cd4 +cd25-tcon cells from c57bl/6 and cd4+ foxp3 yfp+ tregs from either foxp3crelkb1f/f or foxp3cre mice. results: in this study, we demonstrated that bm had decreased frequencies of tregs, accompanied with a reversed lower ratio of tregs frequencies between bm and pb in agvhd patients. meanwhile, the number and function of tregs in bone marrow also affected hematopoietic reconstitution. futhermore,to elucidate these mechanisms which regulate tregs homeostasis, we examined the role of lkb1 on tregs in patients with agvhd and in agvhd murine model. studies demonstrated that lkb1deficient tregs lost foxp3 expression and weaken suppressor function during agvhd. transcriptional profiling and pathway analysis revealed that nf-kb signaling activation and the impairment of a wide spectrum of immunosuppressive genes in agvhd tregs. further mice experiments suggested that cns2 methylation might lead to the instability of tregs in agvhd group. transplantation with marrow grafts from foxp3crelkb1f/fmice exacerbates gvhd lethality. conclusions: these studies indicate that lkb1 is a critical homeostatic regulator for tregs during agvhd. targeting of lkb1 therefore represents a novel therapeutic strategy that promote immune tolerance to mitigates the severity of agvhd. disclosure: national program on key basic research project (973 program) role of aryl hydrocarbon receptor in intestine after allogeneic hsct in mice won-sik lee 1 , soung-min lee 1 , sj-kil seo 1 1 inje university, busan paik hospital, hemato-oncology, busan, korea, republic of background: aryl hydrocarbon receptor (ahr) is a ligandactivated transcription factor that is activated by various small molecules from the diet, microorganisms, host metabolism, and xenobiotic toxic chemicals. the function of ahr has been demonstrated as a crucial regulator in intestinal homeostasis. here, we investigated the regulatory role of ahr in intestine of recipients after allogeneic hematopoietic cell transplantation in mice. methods: wild-type (wt) b6 (h-2 b ), ido -/-(h-2 b ) and ahr -/-(h-2 b ) mice were lethally irradiated and transplanted with 5 x 10 6 tcd-bm plus 2 x 10 6 t cells from balb/c donor mice. ahr activation in colon tissue of recipients was determined by the ahr target genes cyp1a1 and cyp1b1 expression using real-time pcr. the recipient mice were monitored every other day for survival and clinical score. histopathology and pathogenic effector cytokine levels in colon tissue were analyzed for evaluating ahr function. results: we observed that cyp1a1 was constitutively expressed in the colon tissue of naïve recipient mice. although the expression levels were increased by tbi conditioning, the additive up-regulation of its levels with donor t cell alloreactivity was not observed. in contrast, cyp1b1 expression was markedly induced in the colon tissue by donor t cell alloreactivity. we further observed that the cyp1b1 expression was significantly decreased in the colon of ido-/-recipients with donor t cell alloreactivity, but cyp1a1 was not changed. ido-/-and ahr-/recipient mice showed higher histopathological score for intestinal gvhd and increasing pathogenic cytokine levels in the colon compared with wt mice. conclusions: our results demonstrate that ahr-induced target gene profiles might be differently induced in intestine by ligand dependent manner after hsct, which affect intestinal gvhd. disclosure: nothing to declare. abstract already published. abstract withdrawn. in vitro platelet activation evaluation in allogeneic hematopoetic stem cell transplanted patients in response to haemostatic stimulation and cytomegalovirus stimulation (gvhd), complication of which one of the risk factor is cmv reactivation. the resultant inflammatory platelet response during the high-risk period of gvhd after allogeneic hsct remains unknown. our study aimed to characterize spontaneous platelet activation during the 2d and 3d months after allogeneic hsct, and in response to haemostatic stimulation and cmv stimulation. methods: we compared a group of healthy volunteers to a group of allogeneic hsct patients followed between the 30 th and the 90 th days after hsct. platelet activation was determined by the platelet surface expression of cd62p and cd63 using flow cytometer after stimulation by an haemostatic agent, thrombin-receptor activating peptid (trap) and after stimulation by cmv glycoprotein b. the inflammatory response was determined by the detection of immune mediators, rantes, cd62ps, pf4, cd40l and ccl3, using the elisa technique in the stimulated platelet supernatants. results: no platelet activation or molecules release were observed after stimulation by cmv glycoprotein b in both groups. rantes and cd62ps baseline levels are spontaneously higher in allogeneic hsc patients than in healthy volunteers. platelets from allogeneic hsct patients can be activated after haemostatic stimulation and release cd62ps and rantes. in this situation, platelets release more cd62ps, rantes and pf4 than platelets from healthy volunteers. conclusions: although no platelet activation was detected in response to cmv glycoprotein b stimulation, our study revealed a chronic platelet activation condition during the 2d and 3d months after allogeneic hsct with an haemostatic inducible hyper-responsiveness. this leads to the release of molecules with immune-modulating properties involved in the pathophysiology of gvhd. as we move further away from the hsct, that phenomenon seems to gradually weaken. clinical trial registry: nct03009708, fipalloc https://clinicaltrials.gov/ct2/show/nct03009708 disclosure: nothing to declare p305 efficient process and characteristics of umbilical cordderived mesenchymal stromal cells as a feasible source for anti-inflammatory therapy background: recently, umbilical cord (uc) has become attracted source of mesenchymal stromal cells (msc), because of abundant sources and ease of collection of fetal origin without invasive process for the donor and low immunogenicity with immunosuppressive ability and tissue repair potency. objectives of this study were to explorer the efficient and safe products and to evaluate the antiinflammatory potency of uc-mscs for the application of acute graft versus host disease (gvhd). methods: informed consent was obtained from mothers planning to have cesarean sections. uc tissue was cut and once cryopreserved. the safety assessment including infections and baby's health and development were done after 6 months of birth, and performed small-scale quality test of the frozen uc. then we initiated to isolate master uc-mscs from frozen-thawed uc by an improved explant method, which was passed for quality test. the master uc-mscs were cryopreserved once and thawed and expanded until p4. product cells were cryopreserved in original serum-free cryoprotectant dba-d solution. mixed lymphocyte reaction (mlr) assay co-cultured with uc-mscs was carried out using responder mononuclear cells (mnc) stained with cfse, and proliferation and cytokine secretion were analyzed by flowcytometry. results: uc-msc cultured showed significantly higher proliferation ability compared with those from bone marrow-derived mscs, and positive for cd105, cd73, cd90, and negative for cd45, hla-dr. cd80, and cd86 were negative even in the high concentration of ifn-γ, while bm-mscs became positive for hla-dr. pd-l2 was constitutively expressed in uc-msc, while pd-l1 was induced by the addition of ifn-γ. in mlr, responder t cell proliferation triggered by allogeneic dendritic cells was inhibited efficiently by 3rd party derived uc-mscs, in which was induced ido, pge2, hgf, and tgf-β analyzed by rt-pcr, and inhibited ifn-γ and tnf-α in the supernatant by cytokine beads array. uc-mscs migrated toward the tnf-α treated mnc and increased regulatory t cells incidence in peripheral mononuclear cells by the coculture. conclusions: these results demonstrated that cryopreserved uc are feasible and efficient source of mscs and frozen-thawed uc-mscs have high anti-inflammatory background: a new protocol is under development on the amicus separator that enables the device to perform ecp procedures. the amicus separator is used with a photoactivation device, disposable kit and 8-mop to provide ecp therapy in a closed system. the objective of this study was to evaluate the safety and performance of the investigational amicus ecp system in healthy human subjects. methods: an irb-approved written informed consent was obtained from 17 subjects (12 male, 5 female). the amicus ecp system processed either 500, 2000 or 4000 ml whole blood (n ≥ 5 per arm) using double-needle access and acd-a anticoagulation at a 12:1 wb:ac ratio. after mnc collection was completed, the subject was disconnected from the device. 8-mop (3.4 ml, 20 μg/ml) was injected directly into the collected mnc product and saline (approximately 200 ml total), which was photoactivated with 1-2 j/cm 2 uva light. post photoactivation, the amicus separator reinfused the treated mncs into a transfer pack. subject laboratory and safety parameters were evaluated; in vitro evaluations were performed on subject whole blood, collected mncs, treated mncs, and reinfused cells. lymphocyte and monocyte analysis were performed on samples purified using density gradient separation and cultured for up to 3 days post treatment. results: in 17 procedures, median (range) wb processed was 2016.0 (509 -4024) ml using 172.0 (51 -333) ml of acd-a. procedure time was 93.0 (66 -119) minutes, including photoactivation. no adverse events were reported. subjects' vital signs and hematology values were unremarkable and within expected values. the wbc count of the collected mncs was 13.50 (3.3 -30. 2) x10 3 /μl, comprised of 77.10 (47.9 -87.0) % lymphocytes, 15.50 (7.0 -36.8) % monocytes and 5.70 (2.9 -25.5) % granulocytes and platelet count was 94.0 (70 -169) background: transfusion of white blood cells (wbc) causes a number of transfusion reactions and complications, for example transfusion-associated graft versus host disease (tagvht), which still does not have effective treatment and is a fatal complication of transfusions. the only effective method of preventing tagvht is irradiation of blood components with ionizing radiation (x-ray or gamma radiation). but the use of ionizing radiation sources has a number of technical and material difficulties. the emergence of pathogen reduction technologies (prt) in blood components targeted by nucleic acids has opened the possibility of using these technologies as an alternative to irradiating of blood components. several prt demonstrated effective inactivation of wbc in platelet concentrates and blood plasma. so, determination of the influence of prt based on the combined effect of riboflavin (rf) and ultraviolet (uv) on the viability and proliferating potential of lymphocytes in whole blood is important. methods: samples of whole blood were obtained in 35 healthy volunteers. each sample was divided into three unequal parts: untreated control, gamma irradiated, and treated by rf and uv prt (mirasol, terumo bct inc.). mononuclear cells (mnc) were cfse stained, viability and proliferating activity were tested at intervals of 24 hours for 3 consecutive days by flow cytometry. statistical analysis was performed with xlstat 7.0. levels of significance were calculated by mann-whitney test, expressed as p-values (p< 0,05). results: the median viability of mnc after application of both methods of treatment was over 85,0% on day 0 and decreased to day 3 -median percentage of viable mnc were 84,0% (control group), 69,0% (after gamma irradiation) and 63,0% (rf/uv prt). the median of spontaneous proliferative activity on day 3 of untreated and gamma irradiated mnc did not differ (1,8% and 2,1% respectively, p< 0,05). phytohemaglutenin (pha) induced proliferation on day 3 in gamma-irradiated samples was significantly lower in comparison with control group (4,0% and 45,0% respectively, p< 0,01). in samples treated with rf/uv, spontaneous and stimulated proliferating cells was not detected. median percentage of proliferating mnc was less than 0,2%. the use of this prt on whole blood, as well as gamma irradiation, significantly reduces the viability of lymphocytes during storage for 3 days. conclusions: inactivation of wbc using rf/uv prt is a useful and very necessary bonus for a number of reasons. in one procedure two effects are achieved: infectious and immunological safety. the use of prt on whole blood gives the potential for obtaining pathogen-reduced and immunological safety components of blood, which reduces their material cost and staff loading. the use of rf/uv system does not have such complex security requirements and difficulties in servicing as the use of sources of ionizing radiation. the results demonstrate a promising potential for using this technology as an alternative to irradiation disclosure: nothing to declare p308 influence of patients´serum after allogeneic stem cell transplantation on t cell proliferation and treg function background: acute or chronic graft versus host disease (a/ cgvhd) is one of the major complications after allogeneic hematopoietic stem cell transplantation (ahsct). application of regulatory t cells (treg) as "immunosuppressive dli" to prevent or treat gvhd is investigated in clinical trials. here we ask the question, if there could be clinical conditions (e.g. cytokines or drug effects) limiting the efficacy of this approach. to face this problem we tested the influence of patients´serum on t cell proliferation and treg function. methods: lymphocytes from healthy donors were incubated with t cell medium (90% aim v + 10% serum + il2/okt3) containing serum from healthy donors or serum derived from patients after ahsct with or without gvhd (n=10). next we evaluated the suppressive function of treg by performing treg suppression assays, also comparing serum from patients suffering from gvhd versus serum obtained from healthy donors (n=8). proliferation of cfse stained t cells was measured after 5 days. to test the effect of immunosuppressive drugs on treg we performed treg suppression assays after incubation of treg with corticosteroids or tacrolimus or the combination of both drugs. results: serum of patients with acute or chronic gvhd had a negative effect on t cell proliferation. to avoid bias tests were performed with samples from patients without or only with low levels of immunosuppressive drugs. incubation with serum of patients without gvhd or with serum of healthy individuals showed no differences in t cell proliferation. treg from healthy donors showed a stronger antiproliferative capacity when incubated with serum derived from patients with gvhd. treg previously incubated with immunosuppressive drugs showed no decreased suppressive capacity. conclusions: components of serum from gvhd patients seem to have an antiproliferative effect on t lymphocytes itself. this fact might influence the clinical course of gvhd, but should not be a limiting factor for therapeutic application of treg dli. even the systemic treatment with immunosuppressive drugs e.g. corticosteroids or calcineurin-inhibitors should not diminish the treg application. in a next step we will analyze serum components responsible for this immunosuppressive effect with multi cytokine assays and proteomic analysis. the aim of our project is to develop new strategies to avoid gvhd and to optimize clinical settings for treg dli. disclosure background: hypercalcaemia can be very severe following stem cell transplant (sct) in some osteopetrosis patients. denosumab is a fully human monoclonal antibody that binds the cytokine rankl (receptor activator of nfκb ligand), an essential factor initiating bone turnover. rankl inhibition blocks osteoclast maturation, function and survival, thus reducing bone resorption. we describe the effective management of hypercalcaemia in a patient with rank mutation osteopetrosis who received a haploidentical sct. methods: our patient was diagnosed with osteopetrosis at 2 year of age with a defect in the tnfrsf11a gene which codes for rank and received a maternal haploidentical sct aged 4 years. the patients calcium levels were monitored regularly post sct. denosumab was administered for hypercalcaemia as per laboratory reports or clinical symptoms. the drug was diluted with water for injection to make 6mg/ml solution to facilitate subcutaneous administration. results: significant hypercalcaemia emerged on day +18 with a level of 3mmol/l and treated with hyper-hydration and diuretics. this was ineffective in reducing the hypercalcaemia; therefore denosumab was initiated on day +20 post-transplant. initial dosing was determined using the only available paediatric case report at 0.13 mg/kg. a repeated larger dose of 0.19mg/kg was given 4 days later due to an inadequate response (calcium decreased from 3.9mmol/l to 3.80mmol/l). the calcium decreased to 1.93mmol/l after this dose. four weeks later a third dose was required at 0.26mg/kg as the calcium level had increased to 4.1mmol/l. the dose was further increased to 0.32mg/kg for another four doses and then further increased to 0.65mg/kg for another 3 doses and repeated every 7 weeks. normalisation, but not excessive drop in calcium was achieved with these larger doses. over the 9 month follow up post-transplant there were three admissions lasting less than 24 hours for symptoms of hypercalcaemia. these were managed with denosumab administration and hyper-hydration. the remaining doses were given in an outpatient setting. conclusions: denosumab can be safely used as a first line agent in treating post stem cell transplant hypercalcemia in patients with osteopetrosis. a dose of 0.3mg/kg is required as an initial starting dose in order to control hypercalcemia. this is a new higher dose than previously suggested by the original report. denosumab can be effective even after dilution and safely given in children weighing less than 10kg. disclosure: nothing to declare methods: the clinical, laboratory and molecular aspects of this italian male patient who developed such a complication were collected and presented in order to discuss the origin, clinical outcome and management of this very rare post-transplant event. results: a 68-years-old man affected by a high-risk chromosomally abnormal, ph1-, mll-pro-b (egil b-i) all relapsed during maintenance treatment, nonresponsive to re-induction chemotherapy, in second complete remission (ii cr) after blinatumumab treatment received a female cb transplant. according to sorror's and ebmt scores he was considered a high-risk transplant. the patient and the cb unit were sex-mismatched, shared the same blood groups and were both cmv+/ebv+. he received a tbf conditioning regimen that was followed by the infusion of 0.54x10 5 /kg cd34+ cb cells. gvhd prophylaxis consisted of rabbit atg, cyclosporine a (csa) and mycophenolate mophetyl (mmf). neutrophil engraftment occurred on day +28, whereas platelets were never >20.000/μl. on day +67 a 2 cm bulged area became apparent on the left parietal region of the skull. an echotomography showed that the lesion adhered to the bone without infiltrating it and lacked blood vessels and suggested that it may be either a site of disease relapse or an area of infection. at the same time a bone marrow (bm) aspiration showed morphological cr confirmed by immune-phenotypic studies and x-y fish a complete chimera. since the patient was still febrile no biopsy was performed, but on day +94 the axial diameter of the lesion that on a ct scan showed the same appearance revealed by the previous echo-tomography increased to 4 cm. thus, the lesion was surgically removed and histological examination showed cd33+, cd14+/-, cd163+/-, cd45/lca+/-, cd21-, cd23-, cd35-, cd207-, and s100-neoplastic cells whose phenotype suggested a granulocytic sarcoma rather than a histiocytic sarcoma. immuno-chemistry confirmed this suggestion by showing a nuclear npm1 positivity. fish studies demonstrated that these neoplastic cells were of recipient's origin. a novel bm aspiration showed cr confirmed by immune-phenotypic studies and fish revealed a complete chimera. since the patient was still pancytopenic due to anti-cmv treatment, radiotherapy with 18gy in nine fractions were given and the lesion completely resolved. conclusions: a granulocytic sarcoma of recipient's origin occurring three months after a cb transplant is a very rare and unusual event. in order to explain such a complication we suggest that granulocytic sarcoma cells were dormant but already present at the time of pro-b all diagnosis and survived not only the initial all treatment but also the cb transplant conditioning regimen. we can't exclude that immune-suppressive treatments given early post-transplant might have promoted the outgrowth of these neoplastic cell population. disclosure: nothing to declare haemoglobinopathy and inborn errors of metabolism p311 abstract already published. addition of fludarabine on to anti-thymocyte globulin, busulfan and cyclophosphamide conditioning improves outcomes in low-risk matched-related bone marrow transplantation in children with severe thalassaemia flu-atg-bucy. atg dose was 4 mg/kg in all patients except patients with splenomegaly > 3 cm from costal margin and/or sex-mismatched/maternal donor in whom atg was increased to 7 mg/kg. all patients were younger than 15 years and had no hepatomegaly (liver ≤ 2 cm from costal margin) at bmt. results: actuarial overall survival (os) in the atg-bucy and flu-atg-bucy groups is 89% and 98%, thalassemia-free survival (tfs) 75% and 93%, gvhdfree and thalassaemia-free survival (gtfs) at a median follow up of 23.5 and 10.6 months was 72.6% and 93.3% months respectively, which is a significantly improved outcome by log-rank statistics (p=0.002) in the flu-atg-bucy group. there was no significant difference between the groups in pre-transplant characteristics and posttransplant complications except for the following: median cell dose more in 2 nd group with total nucleated cell dose of 8.7 vs 6.4 x 10 8 cell/kg with p< 0.0001; csa taper started later in the new protocol (184 day vs. 152 p=0.0005); median age at bmt (7.2 vs. 4.6 years, p=0.001); number of pre-bmt transfusions (p=0.01) and ferritin at bmt (2.214 vs. 1.599 ng/ml, p=0.002) were higher in the second group; day 30 and 60 chimerisms were also significantly higher in new protocol (p=0.02 and 0.03 respectively). there was a trend towards increased incidence of veno-occulsive disease (vod) and posterior reversible encephalopathy syndrome (pres) on the second group but this difference did not reach statistical significance. conclusions: adding fludarabine and targeted dose increase of atg in the standard bucy context seems to significantly improve outcomes of thalassaemia transplants without contributing to excessive gvhd or infectious complications. this protocol can be easily administered in low resource setting without major additional costs. clinical is an acquired clonal disorder of the hemopoietic stem cells for which the only curative treatment is allogeneic hematopoietic stem cell transplantation. however, there are still few reports on the outcomes of allogeneic hematopoietic stem cell transplantation (allo-hsct) in patients with pnh compared to paroxysmal nocturnal hemoglobinuria-aplastic anemia (pnh-aa) syndrome. our study aimed to compare the outcomes of allo-hsct for pnh with pnh-aa syndrome. methods: the clinical data of 46 pnh patients received allo-hsct (pnh = 16, pnh-aa = 30) in our center from july 2007 to june 2018 were analyzed retrospectively to compare the outcomes of pnh group with pnh-aa group. the clinical data including 28 male patients and 18 female patients, the median age was 29 years (range 6-54). all patients had received various treatments before transplantation such as steroids, androgens, cyclosporine (csa), antithymocyte globulin, and growth factors. the median interval from pnh diagnosis to hsct was 6 months (range 3-240). the conditioning regimen was modified bu/cybased regimen in haploidentical donors and unrelated donors, csa, mycophenolate mofetil (mmf) and shortterm methotrexate (mtx) were administered for graftversus host disease (gvhd) prophylaxis. patients with matched sibling donors were treated with the flu/cybased regimen and csa were administered for gvhd prophylaxis. results: there were no differences of baseline between the 2 groups (p>0.05) except gender and haploidentical donors. the median values of absolute nucleated cell counts were 10.58 (3.83-13.83 ) ×10 8 /kg in the pnh group and 10.81 (3.96-33.40 ) ×10 8 /kg in the pnh-aa group (p = 0.668). the median doses of cd34 + cells infused were 5.00 (3.14-8.42)×10 6 /kg and 3.57 (1.97-6.17)×10 6 /kg (p = 0.002), respectively. all patients attained complete engraftment, no patient occurred graft failure. the median time for myeloid engraftment were 11 (range, 7-14) days in the pnh group and 12 (range, 10-26) days in the pnh-aa group (p = 0.003). the median time for platelet engraftment were 13 (range, 11-16) days and 18 (range, 12-75) days (p = 0.002), respectively. with a median follow-up of 36 (4-132) months in the pnh group and 26 (4-75) months in the pnh-aa group (p = 0.428). in pnh and pnh-aa groups the incidences of grade i-iv acute graft-versus-host disease (agvhd) were 12.50% and 33.30% (p = 0.121), grade ii-iv agvhd were 6.25% and 20.00% (p = 0.209); chronic gvhd were 12.50% and 35.67% (p = 0.274), moderatesevere chronic gvhd were 0.00% and 14.39% (p = 0.146). in haplo-hsct and msd groups the incidences of infection were 37.50% (6/16) and 33.33% (10/30) (p = 0.777). no patient occurred early death and relapse. 3-year estimated overall survival (os) of pnh and pnh-aa groups were 100.0% ± 0.0% and 85.7% ± 6.6% (p = 0.141), gvhd-free and failure-free survival (gffs) were 100.0% ± 0.0%、78.7% ± 7.7% (p = 0.067). conclusions: the preliminary results indicated that allo-hsct is a feasible choice for pnh with favorable outcomes, time for myeloid and platelet engraftment in pnh group were faster than pnh-aa group. there were no differences in os and gffs between pnh group and pnh-aa group. disclosure: no disclosure pattern of calcineurin inhibitor-associated neurotoxicity in sickle cell disease patients receiving a stem cell transplantation background: allogeneic hsct with a msd represents currently the only curative option for sickle cell disease (scd), limited by a donor availability < 20%. neurotoxicity (nt) contributes significantly to hsct-associated morbidity and mortality. calcineurin-inhibitor (cni) associated nt ranges from 4.2%-28.8% (severe nt 4%-11%). the elevated incidence of nt in scd (around 30%) might be triggered by the systemic vasculopathy of scd, with the brain being the primary target. although both cyclosporine a (csa) and tacrolimus (fk506) have a proinflammatory effect, it is more pronounced in csa. infusion modalities also might impact (10.3% after bolus injections versus 3.3% after continuous infusion). methods: in a pilot study, we compared t-cell depleted haploidentical hsct (t-haplo hsct) with msd hsct in patients (pts) with advanced stage scd, using almost identical conditioning regimens. 32 pts (3-31 years; yrs) with homozygous scd or hbs 0/+ ß-thal were treated between 2012 and 2018. nine pts received a msd bone marrow graft, 23 pts received 24 t-haplo-hsct (1 second t-haplo due to graft rejection). immunosuppression consisted of either csa (6 msd, 16 t-haplo) or fk506 (3 msd, , in combination with mycophenolate mofetil (mmf). fk506 was administered as a 20-hours continuous infusion, csa as 4-hours bolus injections; both target level adjusted (csa: 100-120 ng/ml; fk506: 5-8 ng/ ml). duration of immunosuppression was >6 months in thaplo-sct and < 6 months in msd, depending on chimerism. results: cni-related nt was observed in 36.4%, severe nt (pres, visual disturbance, aphasia) in 21.2%. nt was more prevalent in msd (n=5, 55.5%) than in t-haplo (n=7, 29.2%). the incidence of nt was identical under csa (8/22; 36.4%) and fk506 (4/11; 36.4%), however the majority of severe nt (all pres) occurred with csa. complete recovery of nt was achieved in all pts either spontaneously or after switching to fk506/everolimus or withdrawal of fk506. moreover, 66.6% of pts with nt were >18 yrs, and 91.6% >12 yrs, suggesting an increased risk with age. only 37.5% of pts with pre-existing cerebrovascular disease experienced post-hsct nt. of note, 57.1% of pts with severe nt also developed mild acute gvhd. the overall (os) and disease-free survival (dfs) with a median follow-up of 17 months in t-haplo-hsct and 22 months in msd hsct was 91% vs. 100%, respectively. conclusions: our data confirm an elevated nt risk in scd pts following allo-hsct. importantly, the incidence of nt seems to be related to age (91% of pts with nt were >10 yrs), donor source (msd 55.5% vs. t-haplo 29.2%) and type of cni inhibitor where almost all severe nt (71.4%, particularly all pres) was observed under csa. continuous infusion of fk506 vs. bolus injections of csa might have levelled concentration peaks. the nt observed with csa could be the consequence of predominantly csarelated vascular toxicity inflicting pre-damaged vessels in scd. the mechanism of action could be related to other systemic endotheliopathies such as vod, tam and agvhd, which was observed in 57.1% of pts with severe nt, compared to an overall agvhd rate of 30%. disclosure: nothing to declare background: matched-related bone marrow transplantation (bmt) may cure over 80% of low-riskchildren with severe thalassemia (st) defined as a thalassemia syndrome with inability to keep a spontaneous hemoglobin > 7 g/dl. it is well known that patient status at the time of transplant is critical in predicting transplant outcome. liver size > 2 cm is an established adverse prognostic factor in terms of transplant-related mortality and, in our own experience,a spleen size > 3 cm from costal marginis associated with increase rejection rates (blood 2017 vol. 130 no. suppl 1 1944) . optimising liver and spleen size prior to transplant is likely to improve transplant outcomes. methods: we retrospectively reviewed the effectiveness of our strategy to reduce liver and spleen size pre-transplant using hydroxyurea, super-transfusion and intensive iron chelation. we considered liver size < 2 cm and spleen size less than 3 cm below costal margins as good risk features. liver biopsies were not performed thus pesaro risk classification could not be assigned. all transplant candidates were started on hydroxyurea for a minimum of 3 months and pre-transfusion haemoglobin was maintained > 7 gm/dl while on hydroxyurea. if the child had hepatospenomegaly at enrollmentand no improvement in liver and spleen size after an adequate trial of hydroxyurea (minimum of 3 months of treatment achieving maximum dose of 50 mg/kg day or tolerable haematological toxicity, i.e. neutrophil count between 1000 and 1500/μl and/or platelet count between 100.000 and 150.000/μl) patients were given a trial of supertransfusion maintaining haemoglobin above 12 g/dl) for a minimum of 3 months prior to declaring the patient as having failed downstaging. results: out of 119 transplants across 3 collaborating centers in india, 85 patients had no hepatosplenomegaly at enrolment and hence were not actively downstaged. twelve patients were excluded due to inadequate information on their records. all of the remaining 22 patients with enlarged liver and/or spleen were downstaged to low-risk features. all patients received adequate hydroxyurea trial among which seven (32%) patients required super transfusion in addition to maximal hydroxyurea. out of the 22 patients 18 (82%) were successfully down-staged with the above strategy and proceeded to transplant as low-risk patients. among the remaining 4 (25%) patients 3 had liver > 2 cm and one had a spleen > 3 cm only. there was significant improvement in liver and spleen size from the time of enrollment to transplant (p value 0.0004 and 0.0002 respectively by wilcoxon test for paired samples -two tailed) with median duration of downstaging of 9 months (range 2-27 months). there was no significant difference in overall survival (os) and disease-free survival (dfs) by log rank test between the downstaged group and those who did not have hepatosplenomegaly at enrollment (p value 0.59 0.64 respectively). conclusions: in the majority of children with thalassaemia and high transplant risk features liver and spleen size can be reduced pre-transplant using hydroxyurea and supertransfusions thereby decreasing transplant risk. disclosure: nothing to declare p316 abstract already published. abstract withdrawn. longitudinal analysis of the effect of hematopoietic cell transplantation on ocular disease in children with mucopolysaccharidosis i shows ongoing disease progression background: corneal clouding is seen in nearly all patients with mucopolysaccharidosis-1 (mps-1) causing visual impairment. hematopoietic cell transplantation (hct) is able to stabilize disease in many organs including the brain. however, residual disease in peripheral tissues is often described. therefore, the aim of this study was to determine the long-term effect of hct on ocular disease in mps-1 patients. methods: corneal clouding (grade 0-4) and visual acuity (decimal scale) were prospectively collected from all consecutive mps-1 patients treated with hct between 2003 and 2018 at the umc utrecht. the primary outcomes of interest, the effect of time on corneal clouding and visual acuity, were analyzed using a linear mixed model. the correlation between corneal clouding and visual acuity was analyzed with pearson's rho. other parameters studied were clinical phenotype, age at time of transplantation and hematological enzyme level after transplantation. other outcomes of interest analyzed included intra-ocular pressure, refraction, and macula and lens abnormalities. [[p318 image] 1. results: 24 successfully engrafted mps-1 patients were included (92% with >95% chimerism and normal enzyme levels after hct). corneal clouding stabilized during the first years after hct, but increased rapidly beyond three years (figure 1). other predictors for increased corneal clouding were age at time of transplantation (0.74, 95%ci 0.34:1.15; p=0.0026) and clinical phenotype (-1.02, 95%ci -0.18:-1.86; p=0.0335). visual acuity also worsened significantly over time (-0.03, 95%ci -0.06:-0.007; p=0.01). corneal clouding was strongly negatively correlated with visual acuity (ρ -0.60, p = 7.12e-11). conclusions: after initial stabilization, ongoing ocular disease is seen in mps-1 patients despite successful hct. this hallmarks the shortcomings of current standard therapies. new therapies that overcome the weak spots of current therapies are necessary to improve the late outcomes of these patients. clinical trial registry: n.a. disclosure: b.t.a.v.d.b. was supported by a research grant from the sylvia toth charity foundation, the hague, the netherlands, while working on this study. the sponsors of this study are public or nonprofit organizations that support science in general. they had no role in gathering, analyzing, or interpreting the data. all authors would like to thank all parents and patients for participating in this study. all authors state they have no competitive (financial) interests in this study. background: paroxysmal nocturnal hemoglobinuria (pnh) is an acquired clonal disorder of the hemopoietic stem cells for which the only curative treatment is allogeneic hematopoietic stem cell transplantation. haploidentical donor hematopoietic stem cell transplantation (haplo-hsct) is now increasingly applied as a curative therapy for patients with hematologic diseases. however, there are still few reports on the use of haplo-hsct for the treatment of pnh. our study aimed to compare the outcomes of haplo-hsct with matched-sibling donor transplantation (msd-hsct) for pnh. methods: the clinical data of 40 pnh patients received hsct (haplo-hsct = 25, msd-hsct = 15) in our center from july 2007 to may 2018 were analyzed retrospectively to compare the outcomes of haplo-hsct group with msd-hsct group. the clinical data including 23 male patients and 17 female patients, 13 classical pnh and 27 pnh-aa syndrome, the median age was 29 years (range 6-54). all patients had received various treatments before transplantation such as steroids, androgens, cyclosporine (csa), antithymocyte globulin, and growth factors. the median interval from pnh diagnosis to sct was 6 months (range 3-240). the conditioning regimen was modified bucybased regimen in haplo-hsct group, csa, mycophenolate mofetil (mmf) and short-term methotrexate (mtx) were administered for graft-versus host disease (gvhd) prophylaxis. patients with msd-hsct were treated with the flucy-based regimen and csa were administered for gvhd prophylaxis. results: there were no differences of gender, age, patients of pnh-aa and median time from diagnosis to transplantation between the 2 groups (på 0.05). the median values of absolute nucleated cell counts were 10.74 (4.80-22.86) ×10 8 /kg in the haplo-hsct group and 12.19 (5.14-17.25) ×10 8 /kg in the msd-hsct group (p = 0.866). the median doses of cd34 + cells infused were 3.57 (0.68-7.80) ×10 6 /kg and 4.00 (3.02-8.42) ×10 6 /kg (p = 0.151), respectively. all patients attained complete engraftment, no patient occurred graft failure. the median time for myeloid engraftment were 12 (range, 9-26) days in the haplo-hsct group and 11 (range, 7-15) days in the msd-hsct group (p = 0.065). the median time for platelet engraftment were 19 (range, 11-75) days and 13 (range, 11-25) days (p = 0.027), respectively. with a median followup of 26 (4-65) months in the haplo-hsct group and 36 (4-132) months in the msd-hsct group (p = 0.294). in haplo-hsct and msd-hsct groups the incidences of grade i-iv acute graft-versus-host disease (agvhd) were 32.00% and 20.00% (p = 0.343), grade ii-iv agvhd were 16.00%、13.33% (p = 0.759). chronic gvhd were 30.69% and 24.62% (p = 0.418), moderate-severe chronic gvhd were 12.73% and 7.14% (p = 0.522). in haplo-hsct and msd groups the incidences of infection were 32.00% (8/25) and 26.67% (4/15) (p = 1.000). no patient occurred early death and relapse. 3-year estimated overall survival (os) of haplo-hsct and msd-hsct groups were 86.5% ± 7.3% and 93.3% ± 6.4% (p = 0.520), gvhd-free and failure-free survival (gffs) were 78.3% ± 8.6% and 92.9% ± 6.9% (p = 0.250). conclusions: the preliminary results indicated that haplo-hsct is a feasible choice for pnh with favorable outcomes, haplo-hsct and msd-hsct had similar therapeutic efficacy. disclosure: no disclosure p320 pres in bmt for thalassemia major in india: lower incidence and limited impact background: posterior reversible encephalopathy syndrome (pres) is a relatively common complication seen after blood or marrow transplantation (bmt) for hemoglobinopathies with a reported frequency of 10-19%. pres has also been associated with poorer survival rates. severe hemoglobinopathies are one of the most frequent indications for bmt in the developing world, particularly in india. given the risk of rejection in multiply transfused patients and the need to minimize gvhd risk, immunosuppression post-bmt for these non-malignant conditions can be particularly intense and prolonged. we sought to measure the incidence and impact of pres in developing countries. methods: we analysed 194 successive transplants for thalassemia using protocol 1 (atg-bucy+csa/mmf or csa/mtx) maintaining cyclosporine a (csa) blood levels 100-150 ng/ml for 74 patients and protocol 2 (flu-atg-bucy+csa/mtx) maintaining higher csa levels post, i.e. 150-250 ng/ml for 120 patients from fully matched donors with g-csf-primed bone marrow. for 3 patients this was the second transplant from a different matched related donor. pres was confirmed with brain ct/mri for all patients. results: all recipients who had pres had sibling donors, 5 males and 2 females. age median 7.4 (iqr 5.6-8 years). the frequency of pres was 3.6%; disease free survival for patients who had pres was 100%. pres resolved completely in all. csa was switched to mmf in 5 patients who had received mtx and were on csa only at the time of pres occurrence, while csa was stopped but mmf continued in 1 patients taking csa/mmf combination and csa was continued for 1 patient. three patients with pres had grade 2 acute gvhd, 1 had grade 1 gvhd and none developed chronic gvhd. csa levels at the time of pres were a median of 133 ng/ml (iqr: 89 to194) with 1 patient having 419 ng/ml. three patients had pres while they were thrombocytopenic. hypertension stage 2 was observed in four patients, stage 1 in one patient, one patient was not hypertensive and in one patient blood pressure values were not available. two patients were on methylprednisolone 1 and 1.3 mg/kg/day and one was on dexamethasone 10 mg/m2/day. one patient was started on csa again after the pres episode and within 2 weeks had another one while on csa (level 30 ng(ml), methylprednisolone 1.5 mg/kg/day and ruxolitinib for gvhd. protocol 2 had statistically significant improvement in disease free survival from 67% to 91% (p< 0.001) with probability of occurrence of pres increasing from 1.4% to 5.0% (p = 0.17, see figure 1 ), yet had a benign course in all patients. conclusions: not stopping immunosuppression may have been the key factor which could explain why we have better outcomes with pres than what is reported. intensifying immunosuppression pre-bmt did lead to more pres, albeit not significantly, and yet it was quite manageable. even with addition of fludarabine our pres incidence is lower than previously reported. [[p320 image] 1. background: sickle-cell diseases (scd) are a group of genetic hemoglobin disorders marked by brain vasculopathy. allogeneic hematopoietic stem cell transplantation (hsct) is a curative option able to stop vascular disease progression. diffusion-tensor imaging (dti) is a magnetic resonance imaging (mri) technique sensitive to the brownian motion of water molecules and cellular environment. this microscopic quantitative technique is able to detect white matter (wm) alterations before a conventional mri. the aim of this study was to use dti to evaluate axonal damage and structural connectivity in the brain of patients with scd submitted to hla-identical sibling allogeneic hsct. methods: sixteen scd patients with no extensive vasculopathy detected by conventional mri (11 male, age range: 9 -33 years) and 17 age-matched healthy controls (10 male, age range: 6 -31 years) participated in this prospective study. mri acquisitions were performed in a 3t scanner two times for patients (before and 1-5 years after hsct) and at a single moment for controls. from dti acquisitions, fractional anisotropy (fa), mean (md), radial (rd) and axial diffusibility (ad) were calculated in the wm of the whole brain. structural connectivity was also analyzed, based on graph theory, obtaining efficiency, length path and clustering coefficients of the brain network. an anova test was applied to analyze fa differences among controls and patients, before and after hsct. a paired two-tailed t-test was used to determine statistical significance of changes in the fa, diffusivity mean values and network parameters before and after hsct. results: mean fa was lower in patients before hsct than controls (p = 0,038) and increased after hsct being not statistically different when compared to controls (controls = 0,3504; patients before hsct = 0,3328; patients after hsct = 0,3422; post hoc dunnett's test -error 0,03; anova test). when patients were compared before and after hsct, md and rd decrease after hsct (p = 0,038 and 0,047, respectively). on the other hand, fa increased (p = 0,044). after hsct, efficiency was higher (p = 0,023) and path length index was lower (p=0,027) than at study entry (table 1) . conclusions: this study indicates that, before hsct, patients with scd present axonal damage not detectable by conventional mri, when compared to healthy controls. we also suggest that hsct is able to promote axonal recovery and reorganization. partial diffusivity recovery could be associate to a still unidentified mechanism of myelin regeneration. in the future, longer follow up and comparisons with other forms of treatment are required. background: bmt is a well-established treatment modality for haemoglobinopathies, limited by the availability of related donors. unrelated transplantation has historically shown variable outcomes driven by gvhd and toxicity, and usually restricted to 10/10 matches, but the impact of reduced toxicity conditioning regimens is yet to be known. methods: from 2011 to 2018 twenty-five consecutive unrelated bone marrow transplants were conditioned with fludarabine 160 mg/m 2 , treosulfan 42 g/m 2 , thiotepa 10 mg/ kg and atg (thymoglobulin) 11.25 mg/kg if the source of stem cells was marrow (n = 21) or ptcy if pbsc (n = 4). endogenous haemopoiesis was suppressed pretransplantation for a minimum of 8 weeks. gvhd prophylaxis was provided with ciclosporin/sirolimus and mmf. thirteen patients were transplanted for b thalassaemia major, one of a thalassaemia major and 11 sickle cell disease. the median age was 8 years (2 -19). ten patients were 10/10 matched (7 thalassemia and 3 sickle) and 15 patients had a 9/10 match (7 thalassaemia and 8 sickle). the median cell dose was 3.88 x 10 8 tnc/kg (range 1.38 -13.3) and 5.22 x 10 6 cd34+/kg (range 1.10 -27.41). the median survival was 13.4 months (0.7 -68.3). patients with thalassaemia were pesaro class i or ii (pesaro class iii patients were intensively chelated pretransplantation to return to class i or ii). patients with sickle cell disease were transplanted for stroke or recurrent vaso-oclusive crises and/or acute chest syndrome not responding to hydroxycarbamide. results: all patients engrafted and achieved evidence of donor haemopoiesis on day +28 and achieved transfusionindependence and donor haematological values, but subsequently one 9/10 patient with thalassaemia suffered secondary graft failure on day +75 after macrophage activation syndrome. median neutrophil engraftment was 13 days (range 9 to 19) and 12 days (9 -22) for 10/10 and 9/ 10 patients respectively. patient with sickle cell disease had the platelet count maintained >50 x 10 9 /l at all times. the median platelet engraftment >50 x 10 9 /l was 31 days (range 21 to 53) and 40 days (range 15 to 86) 10/10 and 9/10 patients respectively. there were three deaths, all in the 9/10 matched group: two with thalassaemia (day +257 due to idiopathic pneumonia syndrome and day +102 due to mas) and one with scd (day +43 due to ips). there were different trends of complications seen by degree of matching that did not segregate otherwise by disease. conclusions: in conclusion, unrelated bmt for haemoglobinopathies with reduced toxicity regimens is feasible. whilst gvhd caused significant morbidity during the transplant period, other alloreactive/endothelial complications (vod, macrophage activation syndrome, idiopathic pneumonia syndrome) were only seen in the 9/10 transplants. disease-free survival, dependent on transplantrelated mortality, and lack of long-term toxicity, including chronic gvhd, are determined by the degree of matching. 10/10 matched transplants have excellent long-term outcomes with no chronic gvhd >18 months and can be considered for patients without a related donor; whereas 9/ 10 transplant have significant toxicity and mortality, warranting a haploidentical approach. disclosure: no conflict. long-term safety and efficacy of lentiglobin gene therapy in patients with transfusion-dependent β-thalassemia following completion of the phase 1/2 northstar study patients with transfusion-dependent β-thalassemia (tdt) may benefit from gene therapy involving β-globin gene addition to hematopoietic stem cells (hscs) enabling production of functional hemoglobin (hb). lentiglobin gene therapy contains autologous cd34+ hscs transduced ex vivo with the bb305 lentiviral vector encoding β-globin with a t87q substitution under transcriptional control of the encoding β-globin locus control region. the safety and efficacy of lentiglobin was evaluated in adults and adolescents with tdt in the 2-year phase 1/2 northstar study (hgb-204; nct01745120). methods: patients with tdt (≥ 100 ml/kg/year of red blood cells [rbcs] or ≥ 8 rbc transfusions/year) received g-csf and plerixafor for hsc mobilization. to generate drug product (dp), cd34+ hscs were transduced with the bb305 lentiviral vector. patients underwent single-agent, myeloablative busulfan conditioning, were infused with the dp, and were followed for safety and efficacy. results: eighteen patients have been treated in the completed northstar study. as of 14 september 2018, patients had a median follow-up of 38.9 (min -max: 29.3 -48.1) months. the median age at consent was 20 (min -max: 12 -35) years including 15 patients ≥ 18 years old. patients received a median cell dose of 8.1 (min -max: 5.2 -18.1) cd34+ cells x10 6 /kg with a median dp vector copy number (vcn) of 0.7 (min -max: 0.3 -1.5) vector copies/ diploid genome. the median liver iron content (lic) at baseline was 5.7 (min -max: 0.4 -26.4) mg fe/g dw. outcomes by age and baseline iron status will be presented. the median time to neutrophil and platelet engraftment was 18.5 (min -max: 14 -30) and 39.5 (min -max: 19 -191) days, respectively. four patients had platelet engraftment ≥ day 60 and four patients had platelet counts of ≤ 100x10 9 /l at month 12. none of these patients had ≥ grade 3 bleeding events post-lentiglobin infusion. transfusion independence (ti, defined as weighted average hb ≥ 9 g/dl without rbc transfusions for ≥ 12 months) was achieved in 8/10 patients with non-β 0 /β 0 genotypes and 3/8 patients with β 0 /β 0 genotypes. in patients who achieved ti, total hb at last visit was 9.1 -14.1 g/dl. lic increased from baseline in patients who achieved ti by a median of 55.6% and 12.5% at month 12 and 24 then decreased from baseline by a median of 9.2% and 44.4% at month 36 and 48, respectively. non-hematologic grade ≥ 3 adverse events post-infusion in ≥ 3 patients included stomatitis, febrile neutropenia, pharyngeal inflammation, and irregular menstruation. there was no transplant-related mortality, vector-mediated replication competent lentivirus, or clonal dominance. two patients experienced grade 3 serious veno-occlusive liver disease (table 1) . events resolved following treatment with defibrotide and were attributed to myeloablative conditioning. conclusions: in the northstar study, 80% of patients with tdt and non-β 0 /β 0 genotypes and 38% of patients with β 0 / β 0 genotypes achieved transfusion independence. the safety profile of lentiglobin remains consistent with myeloablative busulfan conditioning. longer time to platelet engraftment was observed in some patients, but no graft failure was reported. clinical background: sickle cell disease (scd) is an inherited hemoglobin disorder associated with high morbidity and mortality. currently, allogeneic hematopoietic stem cell transplantation (hsct) is the only curative therapy for scd. transplant outcomes with thiotepa, treosulfan and fludarabine (ttf) preparative regimen are encouraging but this regimen has not been directly compared to other preparative regimens in scd. we therefore planned to compare the event free probability for death, rejection and high grade acute graft versus host disease (agvhd) between ttf and busulfan and fludarabine (bf) regimens. methods: in this retrospectively cohort study, we included all patients with scd who received allogeneic hsct at our center or who were transplanted in other centers and referred to ours for follow up before day 100. patients were transplanted between july 2007 and december 2017. we used kaplan-meier curve to estimate the event free probability for death, rejection and high grade agvhd (grades 3-4). cox regression was used to assess the impact of the preparative regimen on these outcomes. results: a total of 61 patients were included with a median age of 20 years (interquartile range [iqr]: 14-26) and a median hemoglobin of 10 g/dl (iqr: 9-10). sixtytwo percent were males. the proportion of patients who had splenectomy, stroke and acute chest syndrome was 34%, 23% and 57% respectively. all patients received peripherally collected hematopoietic stem cells from a matched sibling donor with a median stem cell dose of 6 x 10 6 /kg (iqr: 5. 1-8.8 ). most patients, 95%, received cyclosporine or tacrolimus based agvhd prophylaxis. most patients received ttf (41%) or bf (53%) preparative regimens. all patients in the bf group received atg. the median follow-up time was 44 months (range: 2-127). four patients died during the follow-up period with an os of 93% (95% confidence interval [ci]: 87%-100%) at 5 years. the os was not different (hr 1.3, p = 0.82) between the ttf (91%) and the bf (94%) regimens. the probability of high grade agvhd free survival at day 100 was 91% (95% ci: 84-99) for all patients. this probability was 85% in the ttf group and 93% in the bf group and the difference was not statistically significant (hr 2.2, p = 0.39). the rejection free survival at 12 months was 93% (95% ci: 87-100) for all patients. no patients in the ttf group rejected while the rejection free survival at 12 months for the bf group was 90%. this was not statistically significant (p = 0.07). conclusions: in patients with scd undergoing allogeneic hsct from a matched sibling donor, the ttf preparative regimen is not associated with improved os, rejection free or high grade free agvhd survival when compared to the bf preparative regimen. larger studies are needed to confirm these findings. disclosure: nothing to declare. novel strategy for haploidentical hematopoietic stem cell transplant in sickle cell disease methods: 9 consecutive patients suffering from scd who underwent hhsct between jan 2018 till date were enrolled in the study. all 9 underwent autologous backup (target dose>5x10 6 /kg) followed by pre-transplant immune suppression (ptis) 2 cycles at 3 weekly intervals using fludarabine @30mg/m2/day(d1-d5) + cyclophosphami-de@1000mg/m2/day(d1) + dexamethasone@20mg/m2/ day(d1-d5) along with hypertransfusion (target hb 11-13gm/dl), hydroxyurea (20mg/kg/day) and azathioprine (2mg/kg/day) from day -60. the graft was mobilized using gcsf@10mcg/kg/day(d1-d5) + plerixafor@0.24mg/kg s/ c on d5 6-8 hours before the pbsch. conditioning included thiotepa 10mg/kg in two divided doses (d-7), fludarabine 30mg/m2 (d-6 to d-2), cyclophosphamide 14.5 mg/kg (d-5, d-4), tbi 2gy with thymic shielding (d-1), ratg (genzyme thymoglobulin 1.5 mg/kg (d-9 to d-7). gvhd prophylaxis included ptcy 50 mg/kg/day on d3 and 4, sirolimus (target levels 10-15ng/ml) (till 9-12 months post hsct) and mmf (till d35) starting from d5. results: the median age of patient's was 7 years (range 3-22 years). before transplantation all patients had repeated episodes of one or other complication warranting a transplant, non-responsive to hydroxyurea. six had maternal donors, 2 paternal and 1 sibling. median age of the donor was 41 years (range 19-51 years). all were dsa negative with a cutoff mfi of >2000 iu. all patients received 10x10 6 /kg cd34 cells irrespective of harvested dose which ranged from (10.13-34.82 x10 6 /kg). median cd3 dose was 16.59 x10 7 /kg (range 10.44-41.9 x10 7 /kg). all patients engrafted with median time to neutrophil engraftment 13 days (range 12-15 days) and median time to platelet engraftment 13 days (range 11-16 days). median duration of hospital stay was 30 days (range 24-35 days). one patient had cytokine release syndrome needing tocilizumab. five had engraftment syndrome treated with short course of steroids. two had cmv reactivation needing treatment with ganciclovir/valganciclovir. acute gvhd grade ii was seen in one patient. till date of analysis none had features compatible with chronic gvhd. of the 9 patients, 8 are alive without sickle cell disease with lansky/ karnofsky scores of 100. at median follow up of 164 days (range 61-271) the probabilities of survival, sca-free survival, and transplant-related mortality after transplant were 88.9%, 88.9%, and 11.1%, respectively. one patient died due to mdr klebsiella sepsis after being discharged initially while he was receiving iv ganciclovir on day care basis. he had full donor chimerism. none of the patient had primary or secondary graft failure. conclusions: pre-transplant immune suppression and upfront use of plerixafor for graft mobilization decreases the risk of graft failure and graft versus host disease leading to overall better survival in hhsct for sickle cell disease. disclosure: none. combined haematopoietic stem cell transplant and enzyme replacement therapy in wolman disease: outcomes and challenges jane kinsella 1 , denise bonney 1 , helen campbell 1 , robert wynn 1 , simon jones 1 background: infantile lysosomal acid lipase deficiencymore commonly known as wolman disease -is an autosomal recessive lysosomal storage disease, characterised by storage of cholesterol esters in the liver, spleen and gastrointestinal tract. these children present under the age of 6 months and traditionally had a poor prognosis, with almost all being dead by the age of 12 months. bone marrow transplant has been used to correct disease manifestations, but limited by high procedure-related mortality with the significant co-morbidities. the survival has changed over the past few years due to pharmacological enzyme replacement therapy but still presents challenges for these patients and their clinicians. in these children haematopoietic stem cell transplant we have offered bmt with enzyme replacement therapy, in certain specific circumstances. methods: four children with wolman disease being treated with enzyme replacement therapy, limited by alloantibody, or poor venous access, received treosulfan-based, myeloablative conditioning with serotherapy followed by a matched haematopoietic stem cell transplant: two family donors, one sibling donor and one unrelated donor. results: three of the four children survived transplant. they have continued to receive enzyme replacement therapy but at reduced dose and frequency with improved tolerability. they have continues to grow and develop. growth and gastrointestinal histology is improved for children having received transplant compared to those receiving enzyme replacement alone. monitoring of peripheral blood chimerism has shown a disease-associated engraftment defect, with mixed chimerism in the 3 surviving patient. conclusions: haemopoietic stem cell transplant is a suitable treatment option in children with wolman disease in whom receiving enzyme replacement therapy is not possible because of venous access, sensitisation or cost reasons. it improves their tolerability of the enzyme treatment and allows for a reduction in enzyme dose and frequency. however, the results of engraftment are not as good as expected for a transplant with myeloablative conditioning and a matched donor. an engraftment defect has been observed in lysosomal acid lipase deficient animal models. a further understanding of this poor engraftment in children with wolman disease is required as to determine whether the risks of transplant is beneficial in these patients and for the consideration of future treatment options including gene therapy. background: thalassemia major is the most common transfusion dependent hemolytic anemia in the world. the absent or reduced production of the β-chain of hemoglobin causes severe ineffective erythropoiesis, massive erythroid hyperplasia in the bone marrow and extramedullary hematopoesis occurs. patients require regular transfusion therapy lifelong. currently, the only proven curative treatment of thalassemia is allogeneic stem cell transplantation (sct). methods: we evaluated the immune reconstitution results of 23 patients at 1 year after hematopoetic stem cell transplantation at our pediatric bone marrow transplantation center between january 2015 and december 2018. all patients were not receiving any immunosuppressive treatment at least for 3 months and they have normal lymphocyte counts, immunoglobulin levels and transfusion independent. lymphocyte subtypes and chimerism percentages and the relationship with the donor type were evaluated at 1 year of transplantation. results: ages of transplantation was ranged between 1-17 years (median: 5 years). seven (30%) of them was male. matched unrelated donor type was chosen in 7 patients while others (16 patients) were transplanted from family matched donor (matched sibling: 9 patients, matched family: 7 patients). all patients received myeloablative conditioning regimen containing busulfan/treosulfan, cyclophosphamide, thiotepa and fludarabine. follow up time was between 12-47 months (mean: 24 ± 10 months). in 9 patients, whole bone marrow product was used while peripheral stem cell harvest in remaining patients. cd3 levels were found low in only 4 patients, in normal patients mean was 59% ± 14%. cd4 levels were severely low in 18 patients while cd8 in only 1 patient. cd8 levels were increased in total 13 patients in as compensatory. cd4/cd8 ratios were very low in all patients (range: 0.2-0.7). b cells (cd19+) were low in 3 patients while immunoglobulin levels were normal. chimerism values between 55-99% (mean: 93 ± 11%). donor and product types did not differ in cd3+ lymphocyte reconstitution at 1 year (p=0.15, p=042 respectively). all patients were alive and well at 1 year after transplantation. conclusions: after 1 year of transplantation, although patients are in well condition regarding to infection frequency and transfussion dependency, it was seen that their lymphocyte subtypes reconstitution could not be achieved enough as in normal children. we can conclude that low cd4+ cell levels were an expected finding in almost all patients. so, these patients may have a tendency to suffer serious bacterial and viral infections, and close follow up be required in terms of infections as long as cd4 levels continue to be low. immunoglobulin replacement therapy did not required even in patients with low b cell levels. disclosure: nothing to declare p328 phase 2 international, multicentre trial to assess haploidentical aß t-cell depleted stem cell transplantation in patients with sickle cell disease with no available sibling donor background: sickle cell disease (scd) is an inherited disorder with an estimate of 300,000 affected newborns per year worldwide. allogeneic hematopoietic stem cell transplantation (hsct) with a matched sibling donor (msd) is currently the curative standard of care for scd patients (pts). however, msd availability is < 20%. a t-cell depleted haploidentical hsct (t-haplo-hsct) from a relative, mostly a parent, expands the donor availability while exhibiting low gvhd rates and thus could offer cure to the remaining 80% of scd patients. in a pilot study, comparing t-haplo-hsct with msd hsct in advanced stage scd, using almost identical transplant regimens for both. the overall (os) and disease-free survival (dfs) was 90% vs. 100%, respectively. methods: these results led to the design of a clinical trial to assess tcd-haplo-hsct prospectively which aims to demonstrate that a hsct from a haploidentical relative is not inferior to a msd hsct with regard to major outcome parameter. this phase 2, prospective, stratified, open-label study is targeting enrollment of 212 patients aged 1-35 years with homozygous hbs disease or heterozygous hbsc or hbs 0/+ ß-thal suffering from severe or moderate scd related complications. inclusion criteria are clinically significant scd related complications such as stroke, silent crisis, pathological angio-mri, transcranial doppler (tcd) velocity >200 cm/s, 2 or more episodes of acute chest syndrome (acs) in a lifetime, chronic transfusion dependency, transfusion-refractory allo-immunization and others. pts fulfilling inclusion criteria will be stratified according to donor availability. pts with a msd will receive a bone marrow graft, pts requiring an alternative donor will be transplanted with an aß/cd19 depleted graft from a haploidentical family donor. the conditioning regimen for both groups will be identical with the exception that antithymoglobulin (atg-neovii ® ) is given upfront in thaplo-hsct versus day -3 to -1 in msd. chemotherapy consists of thiotepa, fludarabine and treosulfan. posttransplant immunosuppression will consist of mofetil mycophenolate and tacrolimus for a duration >6 months in t-haplo-hsct and < 6 months in msd, depending on chimerism. (eudract number: 2018-002652-33) results: primary efficacy endpoint: event free survival (efs). event is defined as incidence of acute gvhd, grade iii -iv, chronic gvhd, rejection (graft failure) or death (for any reason). key secondary endpoint(s) are os, dfs, graft failure, hematological and immunological reconstitution, quality of life (qol) assessment and fertility. the primary null hypothesis is: efs of scd patients treated with t-haplo-hsct is non-relevantly inferior to efs in the msd arm. conclusions: results will help to determine if an a/ß depleted t-haplo-hsct can be considered equivalent to msd hsct with regard to dfs, adverse events and safety, in order to offer this form of cure to the majority of patients with scd. disclosure: nothing to declare hit three birds with one stone: successful stem cell transplantation from one family donor to three siblings methods: in august 2016, three thalassemic siblings were admitted to hospital for stem cell transplantation from a full match donor, their 17 years old sister. the patients' general health conditions and specific health issues due to thalassemia were checked extensively. it was decided to perform first transplant to older sister whom the disease and transplant complications are expected more intense due to prolonged transfusion and chelation therapy. the oldest daughter of family, healthy, was planned to accompany her sisters in transplantation unit so parents can take care the others and organize this period for whole family. results: the 13 years old sibling was first admitted to bone marrow transplantation unit in july 2017. the conditioning regimen was busulfan, fludarabine, cyclophosphamide and thiotepa with antithymocyteglobulin(atg) and defibrotide prophylaxis was given. the healthy donor was admitted to hospital and received g-csf for 5 continuous days before harvesting. stem cells were collected peripherally on day 0 and viable cd34 + cells were 2211/ul. patient received 5,5 x10e6/kg stem cell and the other cell products were divided into 4 parts according to other recipients´weight. no infusion problems were recorded in stem cell transfusion. gvhd prophylaxis was given with cyclosporin and methotrexate. severe sinusoidal obstruction syndrome was observed and successfully managed with supportive therapy. neutrophils were engrafted +11. day, and platelets were on day 64. full blood chimerism results were %98 in day 30, %99 in day 60 and %98 in day 180 consecutively. after 3 months from first transplant the 4 years old sister was admitted to hospital on october 2017. same conditioning with defibrotide prophylaxis and gvhd prophylaxis were given and 6,8 x10e6/kg peripherally derived and previously frost stem cell was infused without any complications. mild sinusoidal obstruction syndrome was observed and managed with supportive therapy successfully. neutrophils were engrafted +11. day, and platelets were on day 16. full blood chimerism results were %99 in day 30, %99 in day 60 and %99 in day 180 consecutively. the third transplant was performed on january 2018 with the same conditioning and prophylaxis regimen. although defibrotide was used mild sos was observed and treated with supportive therapy with success. neutrophils were engrafted +10. day, and platelets were on day 27. full blood chimerism results were %99 in day 30, %99 in day 60 and %99 in day 180 consecutively. conclusions: the patients are being followed for over a year after first transplat, neither adverse nor gvhd symptoms were observed. we presented this case for being a unique example for match family donor transplant and the first successful example from one donor to three recipients. disclosure: nothing to declare results: our female patient admitted for anemia at 3 rd month of birth and was transfused every 4-5 months from 6 th months to 3.5 years of age. since investigations directed towards hemoglobinopathies or membrane defects like hereditary spherocytosis were unremarkable, she was not transfused for 6 years after the age of 3.5-years because hemoglobin level was constant over 7 g/dl. her bm examination showed erythroid hyperplasia and feature of dyserythropoiesis with a few binucleated erythroblasts. it was decided to follow-up the patient with a diagnosis of cda ii. after the age of 10-years, the need for transfusion started again for every 4 to 5 months which led the parents of our patient to request for bone marrow transplantation, however, the diagnosis was not definite, and because of the insufficient data for the transplantations for cda ii patients, it was decided to go on to follow-up. nevertheless, after 2 years, the frequency of transfusion gradually increased to every 2-3 weeks, and bone marrow transplantation was brought into question again. at that time, genetic examination was started and sec23b gene was analyzed by direct sequencing. hsct decision from her hla 10/10 matched brother, carrying sec23b mutation in heterozygous state, was taken. in the preparation regimen, busulfan (bu) at a myeloablative weight adjusted dose (4 days), 200 mg/kg cyclophosphamide (cy) (4 days), and 30 mg/kg antithymocyte globulin (atg fresenius) were used. graftversus-host disease (gvhd) prophylaxis was with cyclosporin a started on day -1 and short-term methotrexate on day +1,+3 and +6. she was transplanted with bm with a dose of total nucleated cells=7.1x10 8 /kg and cd34=3x10 6 / kg. neutrophile and platelet engraftment were achieved at +20 and +38, respectively. indeed, grade 4 hemorrhagic cystitis due to bk virus and a moderate veno-occlusive disease prolonged platelet transfusion days which concealed the exact engraftment day of platelet. the patient was discharged on day 45 with no more need for any transfusion and followed up as a complete chimeric with no type of gvhd since then. now, she is 20 years old, under regular surveillance at our transplant centre without any symptoms. conclusions: hsct data in cda ii patients is still insufficient, however based on data from tm patients with similar treatment approaches in td cda ii patients, it is seen that the hsct is reliable and effective. disclosure: nothing to declare hematopoietic stem cells background: the prognosis after frontline therapy in b-all patients have improved due to monoclonal antibodies (cd20, cd19, cd22) and approximately 90% of patients achieve complete remission. in relapsed and refractory (r/ r) b-all and also in mrd + outcomes are relatively poor. disease-free survival (dfs) in this cohort is 10-20%. in this cohort allo-hsct is indicated and complete remission before transplantation is crucial for prognosis. conventional chemotherapy is associated with high failure rate and significant toxicity. immunotherapy with monoclonal antibodies and car-t are more promising approaches. the aim was to evaluate the efficacy (frequency of responses, os, dfs) and toxicity, especially neurotoxicity and cytokinerelease syndrome, of a bispecific monoclonal antibody blinatumomab in patients both children and adults with persistence of minimal residual disease (mrd + ) or r/r b-all as a bridge to allo-hsct. methods: this study included 120 patients with high risk b-all blinatumomab treated in 2013-2018, among them 14 pts (12%) with t(9;22), 10 (8%) with t(4;11), with mll 11(9%), 84 pts (70%) who were refractory to previous chemotherapy, 66 (30%) after allo-hsct from deferent type of donors. median age was 21 y.o. (range 5m-71y.o), 55 children 0-18 y.o. (46%) and 65 adults >18 y.o. (54%). r/r all had 63 pts (52%), mrd + -57 pts (48%), median days of follow up were 227 (18-720). blinatumomab was applied as 28-day cycles followed by a 14-day off-period before the start of the following cycle. majority pts received one cycle (n=94, 78%). in r/r all group dose was of 9 mcg/d during the first 7 days and afterwards 28mcg/d. patients with weight less than 45 kg received 5 mcg/m 2 /d and 15mkg/m 2 /d accordingly. in mrd group dose was 15 mcg/m 2 /d. results: the frequency of responses to blinatumomab was higher in mrd + pts in comparison r/r all pts (85% vs 62 % p=0.007). in mrd + pts cr mrdwas achieved in 47 pts (82.5%), 10 pts (17.5%) were mrd+ after blinatumomab. two-year os in this group was 61%. twenty pts (34%) received allo-hsct. in rr all pts cr mrdwas achieved in 30 pts (48%), 9 pts (14%) were mrd+ after blinatumomab, 24 pts (38%) had no hematological response . two-year os in r/r all was 43%. fifteen pts (24%) received allo-hsct. os in cr mrdpatients who received allo-hsct was not significantly different in comparison with patients who received blinatumomab as a monotherapy (84% vs 71%, p=0.08). no significant differences in dfs were observed at two years in cr mrdpts depending status of the disease before therapy-mrd vs r/r (66% vs 59%). of the reported adverse events, febrile fever was the most common 91pts (76%), neutropenia 43 (35%), thrombocytopenia 46 (38%), infection 32 (26%), neurotoxicity 29 (24%), cytokine-release syndrome 8 (7%). all complications were reversible. conclusions: blinatumomab is effective option in patients with high risk b-all especially in the group with mrd persistence after previous chemotherapy and facilitates effective bridging to hsct. blinatumomab therapy is generally well tolerated. disclosure here we address the transcriptional regulation of differentiated cells from human embryonic stem cells (escs) using self-assembling peptide hydrogel without stromal cells, and compare with embryoid body (eb) culture system. methods: esc differentiation was induced in eb culture system or three-dimensional (3d) hydrogel culture system. the engraftment potential of differentiated cells was evaluated by flow cytometry. cd34 + cells from mobilized peripheral mononuclear blood cells (mpbmcs) or differentiated from escs at different times (day 7, day 10, day 14) were purified by fluorescent-activated cell sorting. sorted cells were captured on medium-sized microfluidic chips using the fluidigm c1 single cell auto prep system. sequencing was performed by hiseq x ten. results: self-assembling peptide hydrogel formed a 3d scaffold for cell culture, the pore diameter of which ranged from 50 to 200 nm. compared to eb culture system, escs in 3d culture system differentiated more potently. the differentiated cells from 3d system were short-term engrafted in the nog mice, and myeloid cells, b cells and t cells could all be detected in peripheral blood after transplantation. however, the engraftment was not obtained in differentiated cells from eb culture system. we obtained and analyzed 301 escs, 554 cd34 + cells from eb culture system, 440 cd34 + cells from 3d culture system, and 218 cd34 + cells from mpbmcs. the cells were divided into 11 cluters ( figure 1a ). in both differentiation systems, the cd34 + cells from day 7 were more heterogeneous than cd34 + cells from day 10 and day 14 ( figure 1b) . however, cd34 + cells from mpbmcs were more homogeneous, probably because the differentiated cd34 + cells contained several cell lineages, including hematopoietic cells, endothelial cells and mesenchymal cells. there is transcriptional overlap between individual cd34 + cells from eb and 3d culture systems. however, we found that cluster 3, which is composed mainly of cd34 + cells from 3d at day 14 and day 10, expressed similar level of several hematopoietic regulator as hsc, such as tal1, lmo2, erg ( figure 1c ). the cluster 6, which is almost the cd34 + cells from 3d at day 7, also expressed the highest gata2 among the clusters from differentiated cells ( figure 1c) . conclusions: our study demonstrates that 3d hydrogel culture system facilitates hematopoietic specification of escs. disclosure: nothing to declare higher cd34+ cell dose increases overall survival in the setting of dual t-lymphocyte suppression with atg and ptcy in matched related and unrelated donor allosct background: there is no consensus on the cd34+ donor cell numbers required for optimal outcomes in allogeneic stem cell transplant (allosct). there is controversy on the benefits or harm in higher cell dose for allosct. this study aims to evaluate the impact of cd34+ cell dose in allosct patients receiving reduced intensity conditioning (ric) combined with anti-thymoglobulin (atg) and posttransplant cyclophosphamide (ptcy) using related (mrd) and 10/10 and 9/10 matched unrelated donors (mud). methods: this is a single-centre retrospective analysis of 140 adult patients who received allosct for hematologic malignancies between october 2015 and may 2018. all received ric using fludarabine (30mg/m 2 /day: day -5 to -2), busulfan (3.2kg/m 2 /day: day -3 and -2) and total body irradiation (200 cgy: day -1). all patients also received rabbit-atg (4.5 mg/kg: day -3 to -1), ptcy (50mg/kg/day: day +3,+4) and cyclosporine (from day +5). unmanipulated peripheral blood stem cells were infused on day 0. analyses were done using 2 thresholds: (1) an arbitrary cd34+ cell dose of 6 x10 6 /kg (as this was our target dose) and (2) cell dose according to quartiles (< 5.67, 5.67-7.98, 7.99-11.34 and ≥ 11.35 x10 6 /kg). results: median cd34+ cell dose was 7.98 x10 6 /kg. median follow up was 19 months (range 5-35). median neutrophil engraftment was 16 (range 13-31) days and platelet engraftment was 21 (range 11-83) days. a cell dose greater than 6 x10 6 /kg was associated with an increased overall survival (os) at 1 year (71.3%; 95% ci, 62.3-80.3 vs 46.7%; 95% ci 30.3-63.1; p=0.018, figure 1 ). the higher dose was also associated with shorter platelet engraftment time (p=0.016, figure 2 ). there was no significant difference in neutrophil engraftment, nonrelapse mortality (nrm), relapse free survival (rfs), grade ii-iv acute graft versus host disease (agvhd) and moderate to severe chronic graft versus host disease (cgvhd), (table 1) . analyses using quartile cell dose thresholds showed a trend towards decreased os with a cell dose of < 5.67x10^6/kg, however this was not statistically significant ( figure 1 ). higher cd34+ cell doses were associated with shorter platelet engraftment time (p=0.005, figure 2 ). there was no significant difference in neutrophil engraftment, nrm, rfs, agvhd and cgvhd (table 2) . conclusions: cd34+ cell dose greater than 6 x10 6 /kg significantly increases overall survival in the setting of ric and dual t-lymphocyte suppression with atg and ptcy in mrd and mud allohsct. further studies in a larger number of patients and longer follow up are recommended to validate these findings. disclosure methods: fifty two adult patients were included. median cd34+ cells requested for infusion were 6x10^6/kg. all patients received the same ric regimen including fludarabine (30mg/m2/day day -5 to -2), busulfan (3.2kg/m2/day day -3 and -2), and total body irradiation (200 cgy) (day -1) combined with rabbit-atg (4.5 mg/kg: day -3 to -1), ptcy (50mg/kg/day: day +3,+4), and cyclosporine. unmanipulated peripheral blood stem cells were infused. last followup was november 2018. median follow-up was 13 months (range 5-26). median cell dose count infused was 9.83 cd34+/kg. we arbitrarily divided the cohort in two groups with cd34+ dose of >8x10^6 cd34/kg as cut-off point. results: findings are summarized in figure1. the infusion of more than 8x10^6 cd34/kg dose had a significant worse impact on overall survival (os) (p=0.022), relapse-free survival (rfs) (p=0.042) and cumulative incidence of acute gvhd (p=0.000). chronic gvhd could not be compared between the two cohorts due to the different median follow-up. conclusions: the infusion of a cd34+ cell dose count higher than 8x10^6 cells/kg had a significant adverse impact in overall survival and grade ii-iv acute gvhd in the setting of ric and dual t-lymphocyte suppression with atg and ptcy for haplohsct. disclosure: nothing to declare p335 long-term thymic activity and immune-reconstitution after haplo-identical allografting with post-transplant cyclophosphamide background: the use of post-transplant cyclophosphamide (ptcy) has expanded the application of t repleted haploidentical stem cell transplantation (haplo-hsct). in this setting, to investigate thymus role in longterm clinical outcomes, evaluation of immune reconstitution kinetics was performed. methods: twenty-nine patients (median age 53) were enrolled. blood samples were collected before conditioning and at 1, 3, 6, 12, 18, 24 months after haplo-hsct. analyses of cd4 and cd8 t-cell subsets by flow-cytometry were correlated by generalized linear models with real-time pcr (rt-pcr) quantification of signal joint t-cell receptor excision dna circles (sjtrecs), specific marker of naive t-cells thymopoiesis. a) naive; b) central; c) memory; and d) revertant cd4 and cd8 t-cells were defined as follows: a) cd45ra+cd62l+; b) cd45ro +cd62l+; c) cd45ro+cd27-; and d) cd45ra+/45ro +, respectively. sjtrecs rt.pcr was performed on genomic dna (100 ng) extracted from sorted cd4 and cd8 t-cells. results: a gradual increase in absolute numbers of all cd4 and cd8 t cell subsets and of sjtrecs copies from the first month up to 2 years post-transplant was observed ( figure 1) . however, at 2 years, cd4 and cd8 t-cell levels and sjtrecs levels were lower than those observed in healthy donors. sjtrecs kinetics was associated with the increase in cd4 naive t-cells (overall, p < 0.002). this correlation suggests that most of cd4 naive t-cells derives from thymic re-education of donor precursor stem cells, whereas cd8 naive t-cells undergo peripheral expansion after thymic production. furthermore, an increase in cd4 revertant memory t-cells was also significantly correlated with sjtrecs kinetic (p 0,041). central and effector memory t-cells showed a faster thymic-independent expansion in both cd4 and cd8 tcells. interestingly, sjtrecs levels and thymic dependent immune-reconstitution were higher in a cohort of 63 patients undergoing hsct from hla identical donors (manuscript in preparation). clinical outcomes and thymic function were correlated starting at 6 months after hsct. lower thymic output was significantly associated by multivariate analysis with low pre-transplant trecs values (p 0,002 and p < 0,001 in cd4 and cd8, respectively), moderate-severe chronic graft-versus-host disease (gvhd; p < 0,001 in cd8), and age (≥50 years, p 0,006 in cd8). conclusions: the thymus, despite age-dependent involution, substantially contributes to t-cell reconstitution after haplo-hsct. chronic gvhd and older age were significantly correlated with reduced thymic function. overall, lower production of sjtrecs after haplo-hsct as compared after hla identical sibling hsct may partly be due to a higher degree of "mismatching" of mhc molecules during thymic re-education. [[p335 image] 1. figure 1 ] background: the use of allogenic hematopoietic stem cell transplantation (hsct) in the treatment of adolescents and young adults (aya) with philadelphia negative all is decreasing with the adoption of pediatric inspired protocols to treat this age group and the incorporation of minimal residual disease assessment in the routine care of all patients. previously, its use was defined mainly by disease risk features at presentation. methods: a study on 209 aya (age 14-39 years), who underwent allogenic hsct at our institute for philadelphia negative all, between february 2005 and december 2015. all the studied patients received calgb based adult chemotherapy protocol for induction, and underwent a matched related donor (mrd) transplant with cy/tbi conditioning and mtx/csa as gvhd prophylaxis. the patients were eligible for allogeneic hsct, if they have a mrd plus one or more of the following risk factors: (1) age ˃ 30 years, (2) high presenting wbc count (>30 for b-all, >100 for t-all), (3) high risk immuno-phenotyping (pro-b, pro-t, early t, and mature t), (4) bulky splenomegaly or bulky lymphadenopathy, (5) high risk cytogenetics (4;11, 1;19, low hypodiploidy/near triploidy, complex), (6) cns involvement, (7) relapsed or refractory disease at d28 of induction. in this study, we investigated the impact of those different risk factors on the long term outcome of allogeneic hsct. results: the median os of our studied patients was not reached at 12.5 years, with a median dfs of 8.3 years (figure 1 ). in a univariate analysis, relapsed or refractory disease prior to transplant was the only independent risk factor for os and dfs (p-value= 0.36, and 0.01 respectively) (figure 2 ). in addition, patients who had 3 or more risk factors (41, 19.6%) prior to transplant had a significantly lower long term outcome compared to patients, who had one (100, 47.9%) or two risk factors (68, 32.5%) with a median os of 23 months, and a median dfs of only 11 months (p-value=0.32, and 0.009 respectively) ( figure 3) . conclusions: our results show that the long term outcomes of hsct in aya with philadelphia negative all treated on an adult type chemotherapy regimen, were significantly better in patients who showed a good response to initial therapy and a limited poor prognostic factors at presentation, with worsening of dfs as the number of poor prognostic features increase. we can conclude that, using this risk score can be helpful in predicting the outcome of allogenic hsct in aya with philadelphia negative all treated with adult type chemotherapy protocol. disclosure: no conflict of interest a prospective single center survey on donor-specific anti-hla antibodies and desensitization strategy in patients undergoing an allogeneic stem cell transplant background: in the setting of hematopoietic stem cell transplantation (hsct), considering the risk of poor engraftment or graft failure (gf), the detection of antibodies (ab) directed against donor specific hla loci (dsa) represents a contraindication to proceed with the same donor, suggesting the search of other donors. in many cases, there is not sufficient time to search for alternative donors and it is necessary to plan an immunosuppressive strategy to decrease the dsa level, thus reducing the risk of gf. to date, there is no consensus on desensitization standards to manage dsas in hsct. the aim of this study was to determine the incidence of anti-hla ab and dsas in hematologic patients candidate to an allogeneic hsct, and the efficacy of our desensitization protocol. here, we present an update of the results obtained with our strategy. methods: between august 2014 and september 2018, we prospectively screened for dsa 140 consecutive patients candidates to an allogeneic hsct. anti-hla ab research was carried out using the luminex bead assay (lifecode screen and lsa i/ii-immucor). the results were expressed as mean fluorescence intensity (mfi); mfi >1000 was considered positive. in case of a mismatched related donor, a flow cytometric crossmatch test (fcxm) was performed. if the patient had dsas and only one available donor, a desensitization strategy was employed, scheduled with rituximab on day -15, single-volume plasmapheresis procedures (pp), usually on day -9 and -8, intravenous immunoglobulins on day -7, infusion of hla selected platelets for dsa absorption in case of persistent antibodies directed against class i hla antigens. the aim of this schedule was to avoid interferences with chemotherapy and anti-t-cell globulins, infused during condition regimen results: since august 2014, 140 patients have been prospectively screened. thirty-three patients (23.6%) showed anti-hla ab and 9 of them (6.4%) had dsas: 6 were treated with the desensitization strategy, applied according to the mfi score and the fcxm result, and all of them obtained an engraftment; in 2 cases, an alternative donor was selected and in 1 case the research for an alternative donor is still underway. dsa detection was performed every 7 days after hsct for the first month and 60, 180 and 365 days following hsct. neither a dsa rebound nor other complications were observed during the follow-up. conclusions: our prospective analysis underlines the high frequency of anti-hla antibodies detection in hematologic patients, confirming the necessity to routinely evaluate the presence of dsas before an allogeneic mismatched hsct. our desensitization schedules based on the combination of pp, rituximab, ivig and platelet absorption proved successful in reducing dsas. we confirm the necessity of a prospective multicenter collaboration to better define the role of dsas against each hla locus and the critical mfi cut-off level associated with a higher risk of gf. transplant and transfusion specialists should joint to define a consensus for a standard desensitization strategy. disclosure the most frequent technique used for counterbalance partial incompatible hsct is cd34+ selection that is associated with sustained engraftment and effective reduction of t cells that minimizes gvhd. on the other hand, this approach could delay immune reconstitution and increase risk of viral and fungal infection. in mud setting the use of pbsc is the procedure that most centers have recently adopted. this implies the infusion of a relevant higher number of t cells 5 to 10 times more as compared with bone marrow (bm). since in our centre most part of our patients are primary immunodeficiencies, we applied a procedure to minimize the risk of severe gvhd infusing a controlled number of cd3 positive cells. methods: we report data about 91 paediatric patients who received 92 mud hsct (1 patient received 2 hsct) between 2001 and 2018 in the bmt unit of the children's hospital of brescia. patients received conditioning, according to the european group for bone marrow transplantation (ebmt) and the european society for immunodeficiencies (esid) guidelines. cd34+ selection has been realized by a milteny column with an ideal addback of cd3 positive cells of 30x10 6 /kg. stem cell source was bm in 62 cases and pbsc in 30 cases. results: median patients age at transplant was 2 years (range 2.6 months-17 years). the mean number of infused cells were: 12x10 6 /kg cd34+ and 36x10 6 /kg cd3+ in bm product, while 20x10 6 /kg cd34+ and 39x10 6 /kg cd3 + in pbsc. mean time for engraftment was day 15 post-hsct. as concerns acute gvhd overall incidence 64.1 % (59/92) of the children presented this complication, but only 11% (7/ 59) presented gvhd grade iii and none gvhd grade iv, while chronic gvhd presented in 7.6% (6 limited, 1 extensive/92). while acute gvhd incidence and severity weren't significantly different between bm recipients and pbsc recipients, the cases of chronic gvhd were prevalently in the latters. no major infections presented in the post-transplant period and immunological reconstitution both cellular and humoral was completed by 12 months. overall survival at 10 years is 75% (23/92). the results obtained show how it is possible control severity of gvhd if an addback of a controlled number of cd3+ lymphocytes. acute gvhd wasn't severe and only few children presented with limited chronic gvhd. the method allows to graft primary immunodeficiencies patients even with pbsc without infusing too many t cells. in fact, especially in very young children, the number could be excessive and risky. nevertheless in case of an oncohaematological patient, gvl effect is preserved. disclosure background: dc is a rare genetic disorder that results from a defective telomere length maintenance and is characterized by mucocutaneous features, bone marrow failure (bmf) and a high predisposition to cancer and pulmonary fibrosis. bmf remains the major cause of mortality and the hsct is the only definitive treatment to restore hematopoiesis but is limited by a high incidence of treatmentrelated mortality. methods: a retrospective analysis of 28 patients (pts) with dc who underwent hsct at the bone marrow transplantation unit in the clinical hospital of federal university of paraná, brazil, between july-1993 and november-2017. results: 15 boys and 13 girls, with a median age of 14y (3 -30y) received a hsct from a mds (n=7), mud (n=17) or mmrd (haploidentical, n=4). 27pts received bone marrow (bm) and 1pt received a cord blood unit (cbu). the median of tnc infused was 4,76x10 8 /kg (range 2,26-10,12x10 8 /kg) and in the cbu was 6,5x10 7 / kg. two pts received a myeloablative preparatory regimen with busulfan (bu) 12mg/kg + cyclophosphamide (cy) 120mg/kg or fludarabine (flu) 150mg/m 2 + antithymocyte globulin (atg). the remaining pts received a ric regimen with cy200mg/kg (n=5), flu150mg/m 2 + cy60mg/kg + atg5mg/kg (n=17), and flu150mg/m 2 + cy30 + tbi200rads (n=4, haplo). graft versus host disease (gvhd) prophylaxis consisted of cyclosporin (csa) and methotrexate or steroids (cbu) and post-transplant cy + csa + mycophenolate mofetil in the haploidentical transplants. 26 of 27 evaluable pts engrafted with a median time to neutrophil recovery of 20 days (range:13-36 days). one patient experienced primary graft failure (haplo) while second graft failure occurred in other 3pts. all these 4pts went a second hsct and 3 survived. acute gvhd grade ii-iv occurred in 6 of 26 pts at risk. moderate to severe chronic gvhd occurred in 6 pts with 5 cases occurring in pts who had previously presented acute gvhd. overall survival (os) was 53,6% at a median follow-up of 6y. the 5y os was slightly better in msd transplants compared to the others (54,5% x 52,9% p=0,053). causes of early death include adenovirus sepsis (n=1), toxicity to preparatory regimen and sepsis (n=2), primary graft failure (n=1). 13pts remain alive between 1-16y after hsct with a median fu of 8y. among them only 1pt has developed organ involvement by the underlying disease: hepatopulmonary syndrome (hps). 7pts died due to pulmonary fibrosis (n=1), liver fibrosis(n=1), gi bleeding(n=1), hps (n=2); cgvhd and sepsis(n=1), infection (n=1), and 2pts were lost to fu. conclusions: early mortality from bmf can be reduced by hsct, but late outcomes remain a consequence of the underlying disease. long term fu is essential in order to detect late complications related to the hsct procedure or the underlying disease. disclosure: nothing to declare single intra-bone cord-blood transplantation with a treosulfan-based regimen, atg-free and sirolimusbased gvhd prophylaxis: fast hematopoietic engraftment and immune-reconstitution in 20 patients background: cord blood transplants (cbt) require less stringent hla-matching, compared to peripheral blood stem cell or bone marrow. however, cbt has been associated with delayed engraftment and immune reconstitution, especially if in vivo t-cell depletion, such as antithymoglobulin (atg), is used. methods: from 2010 to 2018, 20 patients with high-risk diseases received intra-bone infusion of unwashed single cb unit with an atg-free gvhd prophylaxis; 7 were in active disease at cbt and 8 had received prior allogeneic stem cell transplantation. median age was 44 y [range (r) . conditioning regimen was myeloablative, with treosulfan and fludarabine in all, intensified with melphalan in 15 or with 4gy tbi in 4. hla matches was 4/6, 5/6, 6/6 in 12, 5 and 3 cases, respectively. gvhd prophylaxis included sirolimus and mycophenolic acid (mmf). results: after thawing, median cd45+ cells was 1.39 x 10 7 /kg [r 0.69-5.9], median cd34+ cells 0.08 x 10 6 /kg [r 0.04-0.23], and median cd3+ cells 3.05 x 10 6 /kg [r 1.29-5.9 ]. of the 16 evaluable patients all engrafted with a sustained full donor chimerism at day 100. median time to neutrophils (16/16, anc> 500/μl for 3 consecutive days) and platelet engraftment (14/ immune-reconstitution of cbt patients (tables 1a-b ) was compared with two cohorts of patients transplanted at our center from any adult donor with (81) or without (126 patients, including post-transplant cyclophosphamide cohort) atg in association with sirolimus and mmf. profiles of immune-reconstitution at day 90 -180 -365 showed a better cd4+ recovery at any time-point in both cbt and no-atg versus atg cohort, with no statistic significant difference in the first 2 cohorts. moreover, cd4 +/cd8+ ratio at any time point was better in the cbt cohort vs the no-atg cohort. b cell recovery was faster in the cbt cohort; immunoglobulin recovery was superimposable across different platforms. focusing on late events (>180 days from cbt), 3/12 pts experienced ebv reactivation, median time 586 days [362-655] treated with rituximab, and 1 experienced late hhv6 and cmv reactivation, both solved at last visit. sirolimus was withdrawn after a median of 188 days [r 64-394]. only 1 patient developed severe chronic gvhd, solved at last visit. overall, after a median follow-up of 330 days [r 9-1311], 11 pts are alive and well. conclusions: our data confirm that intra-bone cbt without in-vivo t-cell depletion is associated with fast hematopoietic engraftment and immune-reconstitution, with very low rate of chronic gvhd and late infective events. background: a promising improvement of hematopoietic stem cell transplantation (hsct) may lie in the transplantation of high numbers of pluripotent stem cells to minimize the time span between transplantation and immunological reconstitution. hence, an ex vivo platform is needed that supports hsc proliferation before application and, at the same time, the maintenance of pluripotency by diminishing hsc differentiation into lineage-specific progenitor cells. methods: to artificially model the natural hsc niche in vitro, we used 3d bone marrow (bm)-like scaffolds made of polydimethylsiloxane (pdms). these structures are based on a human long bone cross section as a representative of the bm. human cryoconserved hscs were cultured in distinct cultivation systems for 14 days under different conditions. cell counting and facs analyses at day 14 were conducted. for characterization of the cultivated hscs, we used antibodies against cd34 alone or in combination with antibodies against cd38, cd90, cd45ra and cd49f. results: for optimization of culture conditions for human hscs, a commercially available medium was supplemented with a panel of cytokines and valproic acid. we found a significant increase in the number of cd34+ hscs by simultaneously increasing their vitality using the 3d system compared with conventional 2d culturing. a further improvement was achieved by introducing a silicon oxidecovering of the 3d pdms structures, suggesting that hydrophilic surface properties offer superior attachment for semi-adherent hscs. for a more precise characterization of the cultivated hscs, we introduced a panel of facs markers reflecting the immaturity of the amplified hsc. surprisingly, with increasing immaturity of the cultivated hsc, non-covered 3d pdms revealed to be best suited for amplification: cell number of vital immature hscs was increased after cultivation on non-covered 3d pdms compared with silicon oxide-covered 3d pdms and the 2d system. conclusions: by establishing a 3d scaffold according to the human bm, we found a platform mimicking the natural niche of human hsc which is suitable to amplify human hscs in vitro and support their vitality, pluripotency and ability for self-renewal. [[p341 image] 1. with the introduction of sion covering of 3d pdms structures the maintenance of cd34+ hscs could be further improved. despite the better conservation of cd34 + hscs by using silicon oxide-covered 3d pdms, we found that immature human hscs obviously prefer more hydrophobic conditions found on non-covered 3d pdms. disclosure: nothing to declare. abstract already published. improvements in neutrophil engraftment following changes in freezing method background: in the setting of autologous haematopoietic progenitor cell (hpc) transplants for haematological disorders, peripheral blood stem cells are routinely collected via apheresis and cryopreserved. leicester royal infirmary had been using a controlled rate freezer (crf) to cryopreserve cellular therapy products up until 2017. in 2017 a literature review of cryopreservation techniques was undertaken, since the crf required replacement. this review found consistent evidence that cryopreservation using minus 80 o c is comparable to crf, and engraftment times should not be negatively affected by changing to a more simplified method of freezing. there would also be cost saving benefits from switching from a crf to minus 80 o c freezers. methods: as a result two minus 80 o c freezers were purchased following the acceptance of a preparation process dosier (ppd) which was prepared for the human tissue authority. validation was carried out, and from january 2018 stem cell laboratory at the lri switched from the crf method to minus 80 o c for cryopreservation of cellular therapy products. briefly, cells are frozen using 10% dmso (wak-chemie) in 20g/l human albumin solution (grifols) in cyrobags (origen biomedical, inc). the cells are transferred on cold packs to the minus 80 o c freezer. cells are packaged in between stainless steel heattransfer plates. the plates are placed within a bubble wrap bag, and are placed in a rack within the minus 80 o c freezer, which allows air to circulate freely around each bag. in one plate a el-usb-tc thermocouple data logger (thermosense) is inserted between the bag and stainless steel plate, to record the freezing profile. this data is downloaded after each run. after an overnight freeze at minus 80 o c, the cells are subsequently removed from the bubble wrap and plates, and are transferred to minus 150 o c freezers the following morning. results: a total of 40 patients have had autologous stem cells frozen using this method so far this year. in addition to engraftment data for neutrophils, post-freeze trypan blue viabilities were also compared to the previous year. during 2017 a total of 51 patients, who had all their cells cryopreserved, underwent 72 collections. the post freeze median viability was 90% (75-98%). a total of median neutrophil engraftment was 12.0 days, with a median cd34 dose infused of 4.0 x 10 6 /kg. during 2018 so far, 40 patients have undergone 57 collections. median viability is 95.5% (82-98%). subsequent median neutrophil engraftment is 11 days, with a median cd34 dose infused of 4.1 x 10 6 /kg. conclusions: ongoing savings of approximately £6400 per annum have been made by changing our procedure. the benefit of changing to a simplified method of freezing has also resulted in a reduction in staff working overtime. more importantly, this simplified cryopreservation method has resulted in an improvement in neutrophil engraftment times since changing the cryopreservation technique from the previous method using crf to mechanical freezing using a minus 80 o c freezer. disclosure: nothing to declare low doses of granulocytes in the apheresis product predict a better outcome of autologous hematopoietic stem cell transplantation in multiple myeloma patients background: high-dose chemotherapy with autologous stem-cell transplantation (asct) remains the standard consolidation therapy for multiple myeloma (mm). peripheral blood stem cell collection may be contaminated with large quantities of granulocytes and its consequences on the outcome of asct are still unclear. on the other hand, the effect of performing apheresis with high levels of monoclonal component (mc) on outcome is unknown. the objective of this study was to analyze the effect of total nucleated cells (tnc) and granulocytes count (considered as contaminating components of apheresis products) as well as the influence of mc in the apheresis product on outcome of asct in mm. methods: eighty-two patients diagnosed with mm were mobilized with filgrastim 10 μg/kg/day (plus plerixafor if insufficient mobilization of cd34+ cells on day 4). apheresis collection was performed with cmnc program by spectra optia cell separator. cd34+ count was carried out according to ishage protocol (target: ≥2x10e6 cd34 +/kg). subsequent cryopreservation were performed according to the local protocol. results: the medians (range) of collected cd34+, tnc and granulocytes were 4.69x10 6 /kg (1.69-15.54), 9.35x10 8 / kg (2.16-19.66) and 13.7x10 9 /kg (1.4-90.6), respectively. the medians (range) of infused cd34+, tnc and granulocytes were 2.99x10 6 /kg (0.89-10.93), 6.46x10 8 /kg (1.93-18.60) and 8.30x10 9 /kg (1.1-86.0), respectively. a successful collection after first line therapy was performed in 94% of patients. treatment for mm was continued after carrying out apheresis in 40% of patients, as per protocol. a significant reduction of mc was observed prior to asct, indicating a further improvement in responses after apheresis (p=0.005). an optimal response (cr or vgpr) at the time of apheresis was achieved in 45% of patients and a suboptimal response (partial or minimal response) was observed in the remaining 55%. undergoing apheresis in optimal response did not result in lower number of tnc or granulocytes in the harvest. the subtype of mc did not influence on the number of tnc and granulocytes in the product of apheresis. no differences in collected tnc and granulocytes were observed when plerixafor was used as mobilizing agent. the type of chemotherapy given prior the apheresis did not have influence on the characteristics of the apheresis product. a significant improvement in overall survival (os) probability (95%ci) was observed when low tnc (< 6.46x10 8 figure 1a ). lower incidence of relapse (p=0.044) ( figure 1b ) and non-relapse mortality (p=0.040) was observed in patients who received low granulocyte count in the graft. no significant correlation was observed between the time of engraftment and the number of tnc or granulocytes infused. similarly, no increase in the frequency of the engraftment syndrome was observed when higher number of tnc or granulocytes were infused. conclusions: in our series, low doses of granulocytes in the product of apheresis predicted a better outcome of asct in mm patients. the amount of mc at the time of apheresis did not have influence in the characteristics of the harvest. efforts for avoiding contamination in grafts are important for its impact on outcome of asct in mm patients. disclosure: dkms foundation, pi14/01971 (instituto carlos iii) and sgr288 (grc), generalitat de catalunya. background: our initial experience (from 2012 to 2017 years) with hematopoietic stem cell transplantation (hsct) from mud with tcrαβ+/cd19+ graft depletion in patients with cgd showed high rate of secondary graft dysfunction, the incident of graft rejection was 29%. to improve the outcome, we hypothesized that the use of plerixafor and g-csf as additional agents in conditioning regimen would offers advantages and better outcomes. this trial was registered at www.clinicaltrials.gov (nct03547830) methods: between april 2018 and september 2018, 5 patients with cgd underwent allogeneic hscts from mached unrelated donors with tcrαβ + / cd19 + graft. the conditioning regimen included -treosulfan 42 g / m2, fludarabine 150 mg / m2, thiotepa 10 mg / kg, g-csf 50 mcg / kg and plerixafor 720 mcg / kg. all patients received rabbit atg -timoglobulin® ("genzyme europe b.v.", the netherlands) 5 mg/kg for serotherapy. in all cases, tcrab +/cd19+ graft depletion was used with the immunemagnetic method (miltenyi biotec, bergisch gladbach, germany) according to the manufacturer's instructions. stem cell source was g-csf mobilized peripheral blood stem cells in all cases. posttransplant gvhd prophylaxis was not performed. minimal follow up was 90 days after hsct. results: neutrophil and platelet engraftment occurred at 12-17 days post-hsct in all patients. 3 patients had full donor chimerism in whole blood and in 2 cases we observed predominantly donor mixed chimerism at last follow up. all patients had full donor chimerism in cd15+ compartment and mixed chimerism in cd3+ lineage, but it stable without any sign of graft dysfunction. acute gvhd is verified in 1 of 5 cases and was limited to skin grade 2. all patients are alive for periods from 3 to 8 months post-hsct with good graft function without severe clinical problems. conclusions: we presented first experience of g-csf and plerixafor addition to conditioning regimen before hsct with tcrαβ+/cd19+ graft depletion in cgd patients . we suppose, the preliminary results are encouraging, as the frequency of primary and secondary graft dysfunction in patients from this group is not observed today, there are no significant toxic complications, as well as clinically severe manifestations of gvhd. currently, the recruitment of patients is continuing, and estimation of the rates of immune reconstitution and a more detail analyses will be evaluated later. background: introduction -it is believed that aboincompatibility is of minor importance in allogeneic stem cell transplantation (allo-sct) and that the clinical outcome is equivalent to abo-compatible sct. therefore, we performed a single center retrospective study to characterize the impact of abo-incompatibility on the outcome of haploidentical stem cell transplantation (haplo-sct). methods: this analysis included 27 consecutive patients who underwent haplo-sct for various hematological malignancies at our center between october 2010 and may 2018. we used our institutional database to evaluate details and characteristics of patients and transplant outcomes. results: demographic features of the patients and donors have been summarized in table 1 . all of the patients had advanced hematological disease with a high risk of relapse (22 patients with acute leukemia). out of 24 patients, early transplant-related mortality was seen in this cohort of 5 patients. the remaining 19 patients were followed in 1 and 42 months. donor type abo group switch was observed in a median of 45 days (30-60 days) after transplant. we were not able to show any statistical difference in terms of blood group switch between minor and major abo incompatible transplant. the median red blood cell (rbc) transfusions in the first 30 days for the abo compatible and incompatible transplants were median 4 units (range, 0-11) and median 4 units (range, 3-12) (p=0.6). no statistical difference was also encountered for the rbc transfusion need for stem cell source, peripheral blood vs bone marrow. a total of 15 patients were followed up for reticulocyte engraftment. the median time for reticulocyte engraftment was 19 days (range, 15-60) for all patients. reticulocyte engraftment was tended to be faster in minor abo-mismatched group (p=0.05) than major or abo-compatible ones. nineteen patients achieved independence from rbc support after a median time of 44 days (range, 11-61 days) in abocompatible patients, 25 days (range, 7-52 days) in minor abo-incompatibilityand 34 days (range, 20-57 days) in major abo-incompatibilitygroup, respectively (p>0.5). the engraftman kinetics due to major and minor aboincompatibilitytransplants were presented in table2. pure red cell aplasia was not developed in our cohort. conclusions: the present single center study provides new evidence for the importance of the abo system for erythrocyte recovery in haploidentic stem cell transplantation. it's important to note that, randomize prospective and larger studies are warranted. disclosure: nothing to declare low dose of anti-t lymphocyte globulin protects against severe forms of graft versus host disease in patients undergoing allogenic stem cell transplantation background: graft versus host disease (gvhd) is the most important complication after allogeneic stem cell transplantation (allosct). optimal dose of different anti-tlymphocyte globulin (atg) formulations in this setting has not been established yet. the aim of this study was to analyze the impact of a low dose of atg-fresenius (atg-f) in allosct outcomes. methods: we analyzed 57 adult patients who received an allosct for hematologic malignancies from october 2012 to march 2018. the gvhd prophylaxis included a total dose of 21 mg/kg (7 mg/kg on day -3, -2 and -1) of atg-f for patients who received a graft from peripheral blood, an unrelated donor; with a mismatch, and/or were older than 55 years; associated to a calcineurin inhibitor and mycophenolate mofetil/short course-methotrexate. statistical analysis was performed using spss v.22 and r software. results: median age was 57 years (18-70) and 66.7% of patients were males. seventy-four percent of patients underwent myeloablative conditioning. the stem cell source was peripheral blood in 51 patients (89.5%), 80% were from unrelated donors (28% mismatched). seventeen (29.8%) patients had high risk cmv status (d-/r+) (see image 1b). engraftment was observed in 54 patients (94.7%). primary graft failure occurred in 3 patients (2 myelofibrosis, 1 aml). twenty (39.2%) out of 51 evaluable patients developed grade 2-4 acute gvhd. the cumulative incidence of severe agvhd and moderatesevere chronic gvhd were 8.8% (95% ci, 3.2-17.9%) and 35.2% (95% ci 22.7-47.9%), only 5 (8.7%) patients developed severe cgvhd. twenty-nine patients (56.8%) discontinued immunosuppression before the first year of transplant. the median duration of immunosuppression for patients with moderate-severe cgvhd was 488 days (207-2046). at 2 years non-relapse mortality (nrm) was 21.9% (95% ci, 12.0-33.7%). thirty-nine (68%) patients developed relevant infectious complications. two (3%) patients died within the first 30 days due to gram negative blood stream infection. eleven (19.2%) had at least two episodes of cytomegalovirus (cmv) reactivation between day 30 and 100. three (5%) patients developed cmv gastrointestinal disease, 2 (3%) had probable invasive fungal infection and 1 (1.7%), post-transplant lymphoproliferative disorder associated to epstein barr virus. with a median follow up of 28 months for alive patients , the gvhd and relapse free survival (grfs) at one year, overall survival (os) and progression free survival (pfs) at two years were 47.6% (95% ci, 42.8-52.2%), 59% (95% ci, 54.5-63.2%) and 52% (95% ci, 46.9-56.5%), respectively. the relapse incidence at two years was 26.3% (95% ci 14.7-39.4%). complete remission at transplant was associated with better long term survival (80% at 2 years, p < 0.01). hla disparity did not affect os (see image 1a). conclusions: the use of low doses of atg-f is protective against severe forms of acute and chronic gvhd in a cohort with high prevalence of unrelated donors and a high median age. this strategy showed good results in grfs, os and pfs in a population at high risk for developing gvhd or relapse. disclosure: nothing to declare performance parameters of a ngs-product for chimerism monitoring -applicable in patients after hematopoietic stem cell transplantation methods: for this purpose, samples from patients with mixed chimerism (mc) with increasing amounts of recipient dna were analyzed and compared using realtime pcr of insertions/deletions (indels), fragment analysis of short-tandem repeats (str) and ngs of indels. results: whereas real-time pcr displayed excellent sensitivity down to 0,01 % mc, but poor precision above 20 %, fragment analysis exhibited good precision with limited sensitivity (> 2,5%). in contrast, ngs chimerism demonstrated good sensitivity, with a limit of detection (lod) of 0,1 % mc, and precision throughout the whole spectrum of patient/donor mixed chimerism. the ngs chimerism product (devyser chimerism) exhibited at least three (average eight) and at least two (average 5) informative genetic markers (indels), suitable for monitoring mixed chimerism of patients with their corresponding matched unrelated (60) or related (56) donor samples. in order to establish the performance of the separate techniques for determination of mixed chimerism on retrospective patient samples, a cohort of 27 patient monitoring samples (3-7 weeks post-hsct) with low (< 5%), intermediate or high mixed chimerism (> 20 %) were included and analyzed. dna from all monitoring samples was extracted from sorted cell fractions. the results show that although all evaluated techniques are suitable for monitoring patient/donor chimerism after allogeneic hematopoietic stem cell transplantation (hsct), only the ngs chimerism product exhibits high sensitivity (lod 0,1 %) and a broad dynamic range (detection range 0,1-100%) with good precision and accuracy throughout the whole spectrum of mixed chimerism (% patient/donor). in addition, the ngs chimerism product employ 24 non-population dependent highly informative genetic markers providing stable resolution power and thus suitable for monitoring mixed chimerism. disclosure: dan hauzenberger is medical adviser at devyser ab and shareholder in devyser holding gender distribution: male -59% (n=58), female -41% (n=41). age median -5,8 years old (8 months -17). stem cell source: bone marrow -80% (n=81), peripheral blood stem cells -19% (n=19). 82 patients (83%) received 10/10 matched unrelated donors hematopoetic stem cells transplantation and 17 patients (17%) -9/10 matched unrelated donors hematopoetic stem cells transplantation. differences in the antigen blood system: single group 41% (n=40), minor 32% (n=31), major 20% (n=18), mixed 11% (n=9). age of donor: 18-25 years old -25% (n=16), 26-35 -42% (n=27), 36-45 -23% (n=15), 46 and more11% (n=7). gender differences in donor/recipient: male/female 25% (n=24), female/male 18% (n=18), one sex 57% (n=56). we also took into account the impact of gender difference and cytomegalovirus serostatus in the donor/recipient pair. results: in 10/10 group the estimated probability of overall 2-years survive was 76% and in the 9/10 group 2-years survive was 63%. the increase in donor age of 10 years reduces the 2-years survive by 9-11% (p=0,117), however, the 2-years survive from donors over 46 years old was 100%. we have found no difference between 2-years survive in transplants from donors that are compatible/ incompatible with the antigen blood system, cytomegalovirus serostatus, or the gender differences in donor/ recipient. in the study of donor-related factors, we found the negative impact of an human leucincompatibility (9/10) on the incidence of chronic gvhd -29% (p = 0.019). the combination of cytomegalovirus positive serostatus of the donor and the negative status of the recipient increases the risk of primary graft rejection up to 50%, in comparison with others (p = 0.001). conclusions: our study showed the role of genetic matching on the hla system between the patient and the unrelated donor, and the donors age value. 10/10 transplants have better outcome and lower incidence of severe a. gvhd and ch. gvhd. younger donor increases 2-years survive, but there is a significant increase in 2-years survive if the donor is over 46 years old. disclosure: nothing to declare allogeneic stem cell transplantation in chronic myelomonocytic leukemia. a single center experience nine patients (45%) relapsed with a median of 6,3 months (1-87) with different strategies at this point: in all cases we modulated immunosuppression, in 2 cases as the unique strategy, in 2 cases with donor lymphocyte infusion (dli), in 3 cases we employed hypometilating agents (hma) and in 2 cases with intensive chemotherapy, reaching cr only in two patients, one of them after dli and the other one after hma and consolidation with a second asct. eleven patients (55%) died being the relapse the main cause (64%). transplant related mortality (trm) at +100 were 5% and global trm were 15%. in the last follow-up, 9 patients (45%) are still alive, 8 (89%) in cr and 1 (11%) in relapse situation. with a median follow-up of 58 months (5-138), the event free survival (efs) were 19 months (5-32) and the overall survival (os) were 56 months (0-116). we observed advantage in terms of os in those patients that reach cr at +100 post asct and in those who develop chronic gvchd (p=0.065 and p=0.012 respectively) conclusions: asct is still the only curative option despite the high relapse rates. to reach cr at +100 post asct and the development of chronic gvhd seems that they confer advantage in terms of os. the importance of knowing the molecular profile of the entities that we consider for asct. disclosure this study documents a first experience of a cell processing lab seeking to integrate process automation technology to wash and volume reduce products which can account for the initial material source volume variability, product characteristics, and number of bags. methods: here we report the pre-clinical assessment of the lab's initial work with the lovo cell processing system for a 5 product experience over 2 days with 1 machine. this study used products intended for destruction. the workflow used parallel and sequential processing schedule. after water-bath thawing, bags were sampled, weighed to determine volume, and subsequently connected to lovo or pooled into a transfer pack and then connected to lovo. the bags were then diluted 1:1 at 50 ml/min with lovo at +4-8c using 6% hydroxyethylstarch 130/0.4 (voluven, fresenius kabi) and processed using a 3 cycle procedure. after processing the bags were weighed for volume, sampled, and stored in a 4-8c refrigerator in their lovo final product bags. samples were assessed from t=0 to t=24 hours. results: cd34+ viability and absolute counts were determined using flow cytometry. processing duration and solution volume consumed was determined by the lovo's sensors and confirmed by the operator. data is presented as a percentage relative to the post-thaw values. note, the values presented are not total process yields. the results focus on the lovo processing step. conclusions: the operators easily integrated into the software to drive the machine. the machine demonstrated it's flexibility with a wide-range of volumes, cell-inputs, and number of bags. the lovo produced products which meet our specifications in a quick and reliable manner. further work on this platform will be performed to validate and qualify this system for production use. properties. the aim of this study was to evaluate prospectively the efficacy of a fbm protocol for the prevention of om in patients undergoing a hsct. methods: all patients consecutively who underwent a hsct at our center from 201x onwards received five weekly fbm sessions with a bidiodic laser (lumix 2®, prodent, italy), which simultaneously emitted at 650nm and 910nm with a power of 89mw and energy of 4j per point. the procedure started the day before the beginning of the conditioning regimen up to the tenth day post-transplant. the laser was applied in a defocused mode on each of the mucosal surfaces (12 areas). at each session, the morphine dose, the om level (according to the who scale) and pain through a numerical rating scale (nrs) were recorded. results: 27 consecutive patients (19 male/8 female) submitted to a hsct were analyzed. the median age was 44 years (range 4-66). eighteen patients had acute leukemia, 3 myelodysplastic syndromes, 6 lymphoproliferative diseases. the median number of treatment lines before hsct was 2 (range 1-5). at transplant, 13 patients had advanced disease. the myeloablative conditioning regimen mac (thyotepa, busulphan, fludarabine) was employed in 17 patients; the same conditioning, with a reduced dose of busulphan (ric), was infused in 10 patients. seven patients (26%) had no evidence of om. the incidence of grade ii-iv om was 65% in the group of patients receiving mac and the median duration 12 days (range 3-28); grade 4 om was observed, for 1 day, in 1 patient. in the ric group the incidence of om was 50%, the median duration 11 days (range 7-16); no patient had evidence of grade iv om. in the whole population, the maximum nrs value was 4. morphine administration was required in 23 patients, due to the occurrence of non-oral complications. conclusions: in our experience, prophylaxis with fbm to prevent or reduce om was safe and effective, compared to results of previous experiences reported in the literature, which used no prevention against this complication that negatively affects the quality of life of transplanted patients. further studies on a large series of are necessary to confirm our results. disclosure: nothing to declare background: cytogenetic abnormalities are an essential part of prognostic systems in myeloid malignancies before hematopoietic stem cell transplantation (hsct), however, their role in posttransplantation prognosis is unknown. the aim of this study was to assess the prognostic impact of genetic risk stratification of aml and mds patients on posttransplantation course, which could be an additional tool in making decisions regarding preemptive therapy. methods: a retrospective analysis covering patients treated with allo-hsct between 2012 and 2018. cytogenetic studies included karyotyping (c-and gbanding) and fluorescence in situ hybridization (fish). the number of analyzed cells exceeded european cytogenetics association guidelines (for each fish at least 600 interphase nuclei were analyzed). cytogenetic risk group in aml was assessed based on the eln 2017 criteria. patients with mds were stratified into three groups; favorable (good and very good prognostic score), intermediate, and adverse (poor and very poor) prognostic score according to ipss-r 2012. interestingly, the poorest survival was in patients with monosomy of chromosome 7, which was present in 6 patients of whom 5 succumbed to refractory disease, while all patients who had deletion of long arm of chromosome 7 (del 7q)-are alive at the time of writing of this report after a median follow-up of 34 months (21-73). relapse was diagnosed in 31 patients (31%), including; 13 (42%) with adverse, 15 (48%) with intermediate and 3 (10%) with favorable cytogenetic risk. among 12 patients with a complex karyotype and/or cytogenetic evolution prior hct: 8 patients (67%)relapsed, including 6 (50%)-who died. follow-up cytogenetic studies in relapse after transplantation were performed for 24 patients; 4 of them (17%) had clonal evolutions of the original karyotype with additional abnormalities-(50% died) and 7 (29%) had new aberrations in cells without primary changes (all died). in 18 patients (18%) (8 unfavorable, 8 intermediate group) cytogenetic relapse was diagnosed by fish analysis and they were treated with azacitidine (+/-dli) achieving cr (n=9, 50%), stabilization-(n-=3, 16%), transient response (n=1), while 5 deceased). conclusions: cytogenetic studies in patients after transplantation may facilitate assessment of mortality. karyotype may undergo cytogenetic evolution after allo-hsct. patients with monosomy of chromosome 7 seem to have a particularly poor prognosis. transplanted patients are vulnerable to new cytogenetic alterations. disclosure: nothing to declare methods: the primary end-points were the rate of complete response (cr), defined as no emesis and no nausea without rescue medications, for both acute (cr-24) and delayed (cr 25-120) cinv and rate of post-transplant complications until discharge. we prospectively analyzed 61 patients undergoing autologous (85%) and allogeneic (15%) stem cell transplantation and receiving cinv prophylaxis with nepa and dexamethasone (schedules shown in fig.1 ). in our series, 30 patients (49%) were female. patients median age was 55 years (20-74). the most frequent diagnosis were myeloma (46%) and lymphoma (39%), while 15% of patients were diagnosed with aml or mds. myeloma patients received one day hd-ct with melphalan (75% mel200/25% mel140). lymphoma patients were conditioned with feam (87,5%) or tt_flu_edx (12,5%) hd-ct. busulfan-based mds-ct regimen was offered to aml/mds patients. results: the incidence of cr-24 and cr 25-120 observed was 82% (50/61) and 47,5% (29/61), respectively. more than grade 1 nausea and vomiting (according to ctae-4), was reported in 36% (22/61) and 5% (3/61) of patients, respectively. female sex was associated with an increased risk of acute (hr 19,6; p 0,037 95% ci 7.980-2.191) but not delayed (hr 1,58; p 0,06 95% ci 200-2.042) cinv. similar rate of cr24 and cr25-120 was observed in one-day hd-ct (82% and 50%) compared to mds-ct (89% and 42,5%) group (pns). median lenght of stay was 23 days (15-51). no case of cardiotoxicity and no exitus was observed. the incidence of febrile neutropenia was 61% (70% fuo; 22% sepsis; 18% pneumonia). only one patient experienced an agvhd on day +9. neutrophil (>1000/mcl) and platelet (>50000/mcl) recovery occurred in median on day 11 (9-26) and on day 15 (4-45) respectively. conclusions: nepa seems to be safe and effective in preventing acute and delayed cinv in patients receiving both one day hd-ct and mds-ct as conditioning regimen for hsct. more studies are needed to define the better 5-ht3ra and nk-1ra combination and the better schedule in transplant setting. disclosure: "nothing to declare background: vod and ta-tma represent two early endothelial complications occurring after allogeneic stem cell transplantation (sct) sharing many pre-transplant risk factors. the aim of our study is to evaluate the impact of donor graft composition, engraftment kinetics and infections on the development of these endothelial complications (ec). methods: we retrospectively reviewed 55 consecutive sct recipients at our institu-tion between january 2015 and june 2018. the median age was 48 years (range 17-65). acute leukemia was diagnosed in 33 patients (60%). complete remission was documented in 63% of patients at transplant. donor source was from hla mismatched donor in 53% and from unrelated donor in 56% of the patients. hbv positive patients were 13% of the sample. conditioning regimen was busulfan based in 63% of patients. ursodeoxycholic acid and unfractionated heparin were given to all patients as vod prophylaxis. cyclosporine was used as gvhd prophylaxis. lymphocytic subpopulation analysis (cd3+, cd4+, cd8+ and cd16+/cd56 +) and cd34+ cells count on the donor graft were performed using bd facs cantoll. all patients had routine monitoring for ebv and cmv pcr, hemolysis tests, creatinine and electrolyte panels, proteinuria, complete blood count, blood pressure and schistocytes by direct examination until day +100. the fisher's exact test was used to compare categorical variables, while continuous variables were analyzed with anova test. diagnosis of vod and ta-tma were carried out by using ebmt and cho criteria respectively. results: the incidence of very severe vod and tma was 13% (7/55) and 14,5% (8/55) respectively. cmv reactivations with viral load over 30.000 cv/ ml was 27% and 5% in patients with and without ec, respectively (p 0,041; hr 2,97 p0,0055 95%ci 1,38-6,4). the median day to neutrophils (ns) engraftment (500/ml and 1000/ml) was 14 vs 17 and 14,5 vs 18 in vod/tma group vs control group (p0,03 and 0,027, respectively). more rapid neutrophils engraftment (ns >500/ml and ns>1000/ml within 13 days) was related to a higher risk of ec with a hr of 2,67 (p 0,015; 95%ci 1,2-5,9) and 2,94 (p 0,006; 95% ci 1,4-6,3). patients with ec received a donor graft with a higher median numbers (x10e6/kg) of cd3+ and cd8+ (p>0,05) and a lower numbers (x10e6/kg) of nk cells (p>0,05). patients who received a cd8+ cells count >12x10e6/kg and nk cells count < 2,5x10e6/kg presented a relative risk of ec of 2,37 (p 0,036; 95% ci 1,06-5,3) and 2,35 (p 0,004; 95% ci 1,02-5,4), respectively. there were no differences with respect to the other analyzed variables between patients who developed vod/tma compared with those who did not. (pic_01) conclusions: cmv viremia, early neutrophils engraftment and donor nk and cd8+ cells infused are associated with the risk of vod and tma. very few studies evaluated the link between these variables and the risk of developing such two complications. it could be interesting to investigate these relationships on larger series. clinical trial registry: not applicable disclosure: nothing to declare p358 when the last hope turns out to be just as good as best: haplosct following tbf conditioning, pt cyclophosphamide and tacrolimus as gvhd prophylaxis background: hematopoietic stem cell transplantation is an effective therapy for a variety of severe hematological diseases. in last decades, haploidentical sct (haplosct) followed by ptcy as gvhd prophylaxis has been reported as a valid alternative for patients who lack an hla matched donor. we therefore analysed outcomes with this. methods: 38 patients without hla-matched donor received a haplosct between 04/13 and 08/18. thiotepa 5 mg/kg (2 days), fludarabine 50 mg/m2 (3 days) and oral busulfan 1 mg/kg/6 h (3 days,with pkd dose adjustments) was used as conditioning regiment; in patients >55 years busulfan administration was limited to two days. gvhd prophylaxis consisted of cyclophosphamide 50 mg/kg on day +3 and +4, and tacrolimus as a continuous iv infusion from day +5. all patients received pbsc as the stem cell source. outcomes analysed were overall survival (os), progression free survival (pfs); cumulative incidences (ci) of gvhd, relapse and non-relapsed mortality (nrm). results: median ages was 54 (range 25-71). 53% male and 47% female. diagnoses were: aml 20 (53%), mds 7 (18%), all 5 (13%), hl and nhl 5 (13%), and 1 mm (3%). 37% (n 14) were transplanted in early disease status, while most cases (63%) were in advanced status, including, second/third cr (29%, n 11), one (3%) in aplasia without progressive disease and 23% (n 9) had active/progression disease, 8% (n 3) had stable disease; four cases were second alosct. thus, 50% of patients had a high or very high rdri and 50% had intermediate. ebmt score was ≤ 4 was in 58% of patients (n 22) . the donor was as son/ daughter in 50%, 29% a sibling and 21% a patient's mother. median time to neutrophil (0.5x10e9/l) and platelet (>20x10e9/l) recoveries were +20 and +27 days, respectively (g-csf was not used). only one patient had a primary graft failure attributed to anti-hla donor specific antibodies. median follow up in survivor is 22 months (range 2-63). overall survival is 81±6.5% (at 12 months) and 61±9% (at 63 months). pfs is 67±8% and 52±9% respectively. the cumulative incidence of relapse was 20% and 35%, respectively, while nrm is 13% at 63 months. day +120, grade 2-4 acute gvhd were 13%, while mild/ moderate and severe chronic gvhd were 6% and 3% respectively. ebmt ≤ 4 and first alosct were the only variables to clearly impact 60-months os in univariate analysis. a combined covariate of ebmt ≤ 4-no prior alosct vs other patients showed a 60 month os 81±13% vs 38±12% (p 0.001), pfs 65±13% vs 35±11% (p 0.014) and nrm 0% vs 29% (p 0.008) but without impact on relapse 34% vs 35% (p 0.68). conclusions: haplosct with an age-adapted tbf conditioning regimen, pbsc and ptcy followed by tacrolimus, led to very encouraging results, mainly in patients with a low ebmt score and as a first alosct. although formerly considered as a last alosct strategy, we now agree that the time has come to compare this strategy with hla mud (and even elderly sibling donors) in ongoing prospective randomized multinational trials. disclosure: nothing to disclosure background: autologous hematopoietic stem cell transplantation (autosct) for acute myeloid leukaemia (aml) is increasing becoming a viable option for an increasing number of patients due to limited availability of matched sibling or unrelated donor for allogeneic hematopoietic stem cell transplantation (allosct). we examined the relevant long-term outcomes in our local patient cohort. methods: we retrospectively reviewed the data for all autosct done for aml in our centre over a 17-years period between 1 st january 2001 until 31 st dec 2017 from our electronic record. patients with acute promyelocytic leukaemia (apml) were excluded from this analysis. patients were further stratified based on the number of high risk features present; not achieving complete remission (cr) following induction chemotherapy, high presenting total white cell count (wbc > 100 x 10 6 /ml, adverse cytogenetics (example: complex cytogenetics) and adverse molecular mutations (example: flt3-itd & mll gene arrangement). outcome data including mortality (overall survival (os) and non-relapse mortality (nrm)) and morbidity (leukaemia free survival (lfs)) were recorded and analysed. results: a total of 64 patients were identified. median age at diagnosis is 34-years old. the cohort comprised of 34 males and 30 females. the overall median os and median lfs is 3.9 years and 2.2 years respectively. the nrm is 1.6% (1/64). there was no difference in the median os and median lfs for the patients achieving cr following induction chemotherapy and those not in cr following induction chemotherapy; 4.4 years versus 3.9 years (log-rank, p=0.9) and 3.4 years versus 2.1 years (log-rank, p=0.9) respectively. the median os were statistically significant for patients with zero versus one and two and more high risk features present; 10.2 years versus 3.7 years versus 2.2 years (log-rank, p=0.4) respectively. however, the median lfs were not statistically significant for these three patient cohorts; 3.6 years versus 1.9 years versus 1.4 years (log-rank, p=0.7) respectively. conclusions: in our patient cohort, autosct appeared to be a feasible option for patents with aml without matched sibling or unrelated donor available. disclosure: none to declare methods: between 2014-2018, 16 thalassemic patientsunderwent hisct. the median age of patients was5 (1-8)years with male preponderance (n=10, 62.5%). across the gender and abo mismatch transplants were done in 43.7% and 25% of patients. stem cell source was bone marrow in 5 (31%) while peripheral blood in 11 (69%) of patients. mean stem cell dose was 5.6 ± 2.9 x 10 6 cells / kg and mean volume of product was 188 ± 60.58 ml. preparative regimen included anti-thymocyte globulin, busulfan, fludarabine and cyclophosphamide.graft versus host disease (gvhd) prophylaxis comprised of posttransplant cyclophosphamide on day +3 & +4 followed by tacrolimus and mycophenolate mofetil. patients were observed for hematopoietic recovery (neutrophil and platelet engraftment) and transplant related mortality including acute and chronic gvhd for skin, gut, liverand lungs, primary and secondary graft failure and infectious complications. results: nine(56.25%) of sixteenpatients were engrafted with full donor chimerism. twelve (75%) patients belonged to pesaro class i and 4 (25%) toclass ii patients. median time to neutrophil and plateletengraftment were13(11-20) and16(12-36)days respectively. average number of packed red cell and platelet transfusions were 4.35±7.54 and 20.8 ±19.18 respectively.primary graft failure was observed in 3 (19%) and secondary graft failure was observed in 4 (25%) patients. two patients received a second dose of stem cells and they engrafted at 20and 32 days of infusion respectively.2 of 3 patients with primary graft failure died, one with sepsis (day +23) and the other because of intracranial bleeding (day +21).acute gvhdof gut and skin (grade ii-iii) was observed in 2 patients each, within first 100 days post-transplant. none of the patients had grade iv gvhd. cytomegalovirus reactivation occurred in 50% of patients, all of them received pre-emptive therapy with intravenous ganciclovir. none of them developed cmv disease. invasive fungal infection was not observed in any of the patient. culture proven bacterialinfection was documented in 62% of patients requiring intravenous antibiotics during first 100 days post-transplant.overall survival and relapse free survival were 81.25 % and 56.25% over a median follow-up of 500 (21-1757) days. conclusions: haploidentical transplant is a suitable modality for thalassemic patients lacking a full matched donor in pakistan. in view of our results, we suggest that thalassemia patients should be offered hisctas an option for cure. clinical background: gemtuzumab ozogamicin (go) is an anti-cd33 monoclonal antibody with significant activity in de novo and relapsed/refractory (r/r) acute myeloid leukemia (aml). a relevant side effect consists of hepatotoxicity and especially sinusoidal obstruction syndrome (sos). the objective of this study was to analyze tolerability of go during the induction and reinduction therapy in patients with aml, and its possible impact on subsequent hematopoietic stem cell transplantation (hsct). methods: from 2004 to 2017, 24 patients who had received go in three hospitals were collected and their medical records were retrospectively reviewed. results: fourteen patients diagnosed with de novo aml received go (3mg/m 2 ) on day +1 in combination with standard chemotherapy (idarubicin and cytarabine, 3x7 schedule) as induction therapy. hyperbilirubinemia (bilirubin >1.5 unl) was detected in 4 patients and increase of aspartate aminotransferase (ast) (>2.5 unl) in 1. twelve patients achieved complete remission (cr) and one was refractory (1 not evaluated). in the r/r setting, 10 patients diagnosed with aml (n=8), biphenotypic acute leukemia (n=1) and acute promyelocytic leukemia (n=1) received go as 3rd or subsequent rescue therapy either as monotherapy (n=4) or in combination with cytotoxic chemotherapy (n=5). prior hsct was performed in 5 patients (autologous [n=2], allogeneic [n=3] ). rescue therapy was indicated for refractoriness (n=2), relapse (n=5), partial response (n=1) or absence of donor (n=2). four patients received 3 doses of go (3 mg/m 2 ) and 3 patients, one dose. hyperbilirubinemia (>1.5 unl) was observed in 1 patient and increase in ast (>2.5 unl) in 2 patients. seven patients achieved cr, 1 was refractory, 1 obtained partial response and 1 died early during induction). thirteen patients received subsequent hsct (autologous [n=4], allogeneic [n=9]) after go therapy (10 in the de novo aml and 3 in the r/r group). the reasons for not performing hsct in the remaining 11 patients were: low cytogenetic risk (n=1), active chronic graft versus host disease (gvhd) in previous hsct (n=1), early death during treatment (n=3), relapse (n=1), severe complications in rescue treatments (n=1), and unknown (n=4). the conditioning regimen was myeloablative (n=8), non-myeloablative (n=4) and sequential (n=1), and the donors were matched sibling (n=6) or unrelated (n=3) . cyclosporine, methotrexate and thymoglobulin were administered as gvhd prophylaxis in 7 patients and cyclosporine, mycophenolate and thymoglobulin in 3. hyperbilirubinemia was observed in 2 patients belonging to the de novo aml group. death after hsct occurred in 10 patients due to infection (n=5), relapse (n=3), gvhd (n=1) and traffic accident (n=1). three patients are currently alive in remission. no sos was observed in any patient. conclusions: in both de novo and r/r aml the administration of low dose go is feasible and does not have impact on subsequent hsct outcome. although some degree of hepatotoxicity was observed, no cases of sos were observed, either before or after hsct. disclosure: supported by grants from: asociación española contra el cáncer, aecc (gc16173697biga), instituto carlos iii (pi14/01971 fi), 2017-sgr288 (grc), cerca program from generalitat de catalunya, and "la caixa" foundation. outcome of allogeneic hematopoietic stem cell transplantation in patients with benign hematological disorders saqib ansari 1 , tahir shamsi 1 , uzma zaidi 1 , saima siddiqui 1 , tasneem farzana 1 background: allogeneic hematopoietic stem cell transplantation is a potentially curative treatment modality for hematological disorders. we evaluated the outcome of patients suffering from benign hematological disorders, including aplastic anemia, fanconi's anemia and thalassemia after matched related allogeneic transplantation. methods: all patients having hematological disorders including aplastic anemia (aa), beta thalassemia (btm), fanconi's anemia (fa) and severe combined immune deficiency disorder (scid) with hla identical related donors who underwent allogeneic transplantation were included. donors were given g-csf at a dose of 10 μg/ kg/day daily for four days prior to harvest. the conditioning regimens for thalassemia included cyclophosphamide (cy) + busulfan (bu) in 21 (20%), bu + cy + thiotepa in 4 (4%) and bu + cy + antithymocyte globulin (atg) in 81(76%). conditioning regimens for aplastic anemia included, fludarabine (flu) + cy in 38 (33%), flu + atg in 28 (25%) and cy in 38 (33%), cy+ atg in 9 (8%) patients. for fanconi's anemia flu + atg in 12 (52%), flu in 2 (9%), cy + atg in 4 (17%) and flu+ cy + atg in 5 (22%). flu + atg in 2(25%) and cy given in 4 (50%) and no conditioning regimen was offered to 2 (25%) patients with scid. results: a total of 250 allogeneic transplants were performed for benign hematological disorders including aa (n=113), btm (n=106), fa (n=23) and scid (n=8) from 2011 to july 2018. median age was 7.5 years (range 0.5 -48). across the gender and abo blood group transplants were 109 (43.6%) and 80 (32%). the median time to neutrophil and platelet recovery was 13 days (range: 9-46) and 18 (range: 10-35). primary and secondary graft failure was observed in 34 (13.6%) and 29 (11.6%). overall survival in aplastic anemia (63/113, 56%), beta thalassemia (82/106, 77%),fanconi's anemia (15/23, 65%) and severe combined immune deficiency disorder (6/8, 75%). eighty four patients expired (33.6%) among them 41 patients expired within 100 days post transplant main cause of deaths included sepsis (31%), multi organ failure (6%) and gut gvhd (5%) conclusions: in developing world scenario where non malignant disorders are leading cause of morbidity and mortality. bone marrow transplantation has been successfully implemented with better long term diseases free survival and quality of life. clinical background: hematopoietic cell transplantation (hct) remains the only curative therapy for many diseases, yet transplant survivors carry an unusually high burden of morbidities, primarily because of exposure to intense chemotherapy, radiation and /or gvhd. this study aimed to evaluate the burden of chronic diseases at the end of life after allogeneic-hct and to identify the disability-adjusted life years (daly). methods: the pubmed, medline, and ovid databases were queried utilizing specific mesh terminology (post, allo stem cell, hematopoietic, bone marrow, transplantation).we collected data on the impact of the hct on the variables affecting survivor's health in all aspects .the rates of late complications were compared to the risks in the general population (united states). results: a total of 7 studies fulfilled the selection criteria totaling to 6619 patients (table 1) . median os at 5-year mark varied widely between studies from 19% to 92%. majority of the patients at 10-year mark were found to have new comorbidities thereby indicating a huge burden of late effects at the end of life, though exact dalys could not be calculated due to incomplete data. hct survivors were found to have higher risk of premature arterial disease (pad) at 6.8% compared to the general population, however gvhd or the addition of tbi to the conditioning regimen were not found to be significantly associated with pad. regarding the risk of new cancers, the cumulative incidence of their development at 5 and 10 years was 1.71, and 3.61, respectively. increased risks compared with the general population were seen for some solid cancer including cancers of the lip: p=0.02, tonsils: p=0.05, oropharynx: p< 0.01, bone: p< 0.01, soft tissue: p< 0.01, and vulva: p=0.01. with respect to mental health, depression was prevalent in (10.8%) survivors, in whom (82.5%) were still on antidepressants at the last follow-up. cognitive impairment and other psychiatric disorders were found in (2.4%) and (2.7%) survivors, respectively. the most common cause of nrm in the first 5 years was gvhd. however, after 10 years, the leading cause of death in those conditioned with mac regimen was secondary cancer, but in the ric group, new cancers and gvhd contributed equally. conclusions: hct survivors remain at risk of significant complications which lead to premature death and their burden of comorbidities at the end of life is significantly more than that of general population. background: late onset hemorrhagic cystitis (hc) is a common complication of hematopoietic stem cell transplantation (hsct) frequently associated with reactivation of bk virus (bk-hc).there is no consensus as to the best therapy for bk-hc, and many different treatments have been reported. hyper baric oxygen therapy (hbot) is used as primary or adjuvant therapy in diverse clinical situations involving hypoxic injury to tissues and has been explored as a useful tool in treating bk -hc. we report our experience with hbot in combination with non-invasive supportive care in children and adolescents suffering from bk-hc following allogeneic hsct. methods: the computerized database of schneider children´s medical center of israel was reviewed for all patients aged 0 to 21 years who underwent hsct between january 2000 and june 2018 and developed bk-hc. hbot therapy consisted of 2 hours sessions at 2 atmospheres, with patients breathing oxygen by mask. parents accompanied patients during the treatment. results: fourteen patients with a variety of underlying diseases received (hbot) for treatment of bk-hc following hsct. the initial treatment for children with bk-hc at our center prior to 2008 included continuous bladder irrigation and intravesicular instillation of various medications. beginning in 2008, we adopted a non-invasive strategy that included the administration of oral anticholinergics (oxybutinin), systemic pain management, hyperhydration and the administration of weekly cidofovir with probenecid. hbot was administered to patients who failed the above regimen. with this protocol, the average time of starting hbot dropped from 13 (prior to 2008) to 9.8 days. the median onset of hc was 33 days post hsct. all patients were receiving immunosuppressive treatment at the onset of bk-hc. all patients suffered from macrohematuria with blood clots (grade iii cystitis), 12 (92.3%) experienced severe dysuria and 11 (84.6%) urgency. bk viruria was present in all patients, and concurrent bk viremia was detected in 80% of those who were tested. patients reported symptomatic improvement at a mean of 3.6 days following the initiation of hbot. no patient experienced serious adverse effects due to hbot, but two patients required insertion of tympanic ventilation tubes. eleven of our 13 patients (84.6%) experienced complete remission of bk-hc following hbot, with an overall response rate of 92.3% (12/13 patients).eight of our patients (61.5%) eventually succumbed due to either hsct complications or disease relapse. conclusions: hyperbaric oxygen therapy is a safe, effective, non-invasive and well tolerated treatment modality for bk -hc and should be considered for first line therapy for this complication of hsct. clinical background: every year, almost one thousand cases of hematological malignancies in pediatric population are reported in peru. allogeneic hematopoietic stem cell transplantation (allo-hsct) is an alternative strategy in many of these cases. only between 20-30% of the pediatric population that requires a hsct has a compatible human leukocyte antigen (hla) donor. the remaining 70% have to access international donor registries, extending the awaiting time and conditioning the progress of the disease. allo-hsct has the potential to help children with several hematological disorders with non-compatible hla donor. hla genotypically identical sibling donors are the best option when pursuing an hsct. nevertheless, patients' alternative sources of stem cells could be obtained from an haploidentical donor like one of their parents. the haploidentical transplantation program with macs was implemented in peru in 2016 to reduce the risk of graft-versus-host disease (gvhd), support the immune system reconstitution and to expand pool of donors. it allows patients to access a treatment that is efficient and safe, as shown in the depletion of positive tcrα/β+ and b cells procedures for allo-hsct. methods: the mobilized leukapheresis products (n=19) of haploidentical healthy donor was washed to remove platelets and preparations were performed according to miltenyi's clinimacs® manual for tcrα/β+ and cd19+ cell depletion. analysis of the initial leukapheresis product and tcrα/β + and cd19+ depleted graft (target and non-target product) was performed using flow cytometer. cells were analyzed for cd3+, cd45+, 7-aad, cd20+, tcrα/β+, tcrγ/δ +, cd34+ and cd133+ with fluorochrome-labeled antibodies from miltenyi biotec using a novocyte cytometer. the results obtained with the ex vivo t-cell depleted allo-hsct procedures show an overall survival (os) over 70% with an ic95% at the end of the first year with low incidence of gvhd. macs of tcrα/β+ and cd19+ cells are effective (logp 3.9 and logp 3.3, respectively) obtaining minimum levels of depleted lymphocytes within clinical established parameters for diverse pathologies. in addition, tcrα/β+ and cd19+ cell depletion does not significantly affect hematopoietic stem cell populations such as cd34+ and cd133+ cells or tcrγ/δ+ cell population. cd34+ and tcr γ/δ cells are highly recovered (93.15% and 88.76, respectively), which contributes with a better engraftment after allo-hsct. conclusions: peruvian results oscillated within european ranges with an os over 70%. our data suggests that the macs method is an efficient, effective and safe strategy for haploidentical hsct which has a remarkable cost-benefit ratio and makes it viable in countries of the latin american region with peruvian socio-economic characteristics. evaluation of genoresistance for viral reactivation treatment has been implemented as a strategy to improve os. better results are achieved in patients after allo-hsct with the validation of these tests in peru. preliminary pharmacoeconomic evaluations allow us to establish magnetic activated cell sorting (macs) as a promissory strategy compared to other alternatives for haploidentical hsct. it is necessary to increase the number of procedures in order to confirm efficacy and safety of macs in a larger population. disclosure: all authors have no conflicts of interest. abstract already published. micro-costing study of hematopoietic stem cell transplantation in two hospital institutions from southern of brazil background: hematopoietic stem cell transplantation (hsct) is a potentially curative treatment indicated for patients with onco-hematological, hereditary and immunological diseases. considering the increase of patients indicated to the hsct and the lack of knowledge about the costs resulting from this treatment, is important to identify and detail the resources consumed in each phase of hsct and provide knowledge to the public health brazilian system. we aimed measure the total cost of related hsct, based on micro-costing study of patients assisted in two hospitals in the south region of brazil. methods: hsct costs were estimated using the timedriven activity based costing method (tdabc), which measured cost of services / products based on actual consumption of resources. we collected data from medical records of 12 patients submitted to allogeneic hsct in 2017 from public and private (philanthropic) hospitals. we interviewed professionals involved in the tcth activities, we performed chrono-analysis and, we consulted financial and administrative systems reports of hospitals. in order to compare costs according to clinical complications observed in patients, we grouped into two ranges of complexity: low/ medium and high. the study was divided into stages: hsct processes mapping; costs measurement; and analysis of results. finally, the costs were compared: by activity, by resource and by hospital. this study was financed by psid-uhs, by an agreement signed between ministry of health and moinhos de vento hospital, through adjustment term number: 04 / 2014, and approved by research ethics committees. results: from the hsct processes mapping, the following steps were defined: (i) hospitalization; (ii) conditioning; (iii) transplantation; (iv) period of aplasia; (v) engraftment; (vi) observation; (vii) pre-and medical discharge. seven patients were classified in low / medium complexity level, with hospitalization median time of 41 days and an median cost of usd 66,278.29, whereas the other five patients, classified as high complexity, presented median time of 101 days and median cost of usd 300,367.13. the hsct costs evaluation identified that steps ii and iv presented greatest cost in high complexity patients. lower complexity patients presented, in steps ii and iv, median costs of usd 44,274.52 while in higher complexity usd 100,226.26. in addition, median costs of materials and drugs were usd 13,926.02 and usd 181,094.15 in lower and higher complexity patients. conclusions: tdabc method allowed the identification of the moment when patients consume the most resources. of all the hsct stages, periods of conditioning and aplasia presented higher costs, representing 76.90% of the total hospitalization value. in these stages, higher complexity patients presented three times higher the median cost. the resources that had the greatest impact were medicines and medical materials, costing 120 times more than lower complexity patients. conclusion: this study allowed a detailed identification of the hsct costs in patients with different complexity ranges in two hospitals from southern brazil. therefore, the identification of service demand regarding the clinical complexity, allows the generation of important information for the management of the best care in the health service. disclosure background: immunodeficiency due to lrba deficiency is characterized by hypogammaglobulinemia and autoimmunity. hemolytic anemia, lymphadenopathy, autoimmune hepatitis and, above all, autoimmune enteropathy are the fundamental characteristics of these patients together with the history of recurrent invasive infections.the therapy includes immunosuppressants, endovenous immunoglobulins. bone marrow transplantation is the final therapy of these patients, especially in the most serious cases and in recent years, also on the light of increasingly targeted conditioning regimes, it is associated with ever better prognosis. methods: we present the case of caterina, an 8-year-old patient with lrba deficiency, diagnosed at 5 years of age for a history of hypogammaglobulinemia, recurring invasive, bacterial and fungal infections and a picture of autoimmunity represented by ahia, myelitis (c3-c5), autoimmune hepatitis and enteropathy in treatment with abatacept and sirolimus. it also presents leptin deficiency lipodystrophy.she presented to our observation for ostemielitis in multiple outbreaks (tibia, femur and left knee) secondary to sepsis from mrsa. broad spectrum antibiotic therapy and curettage surgery were performed during hospitalization.after 5 months of broad-spectrum antibiotic therapy (daptomycin, rifampicin, ceftaroline, cotrimoxazole, levofloxacin, dalbavancin) there was resolution of the infections.because of the seriousness of the disease it is therefore decided to subject catherine to hematopoietic stem cell transplantation. results: therefore were performed bone marrow transplant from mud after conditioning at reduced intensity, delayed in 9 days, with treosulfan, fludarabine and thiotepa. prophylaxis for gvhd was performed with atg (5mg / kg / day), sirolimus (already practiced for enteropathy) and mmf. because of the hepatic picture, we also performed prophylaxis for vod with defibrotide for 21 days. transplantation was performed by peripheral stem cells with 18 x 10^6 cd34 / kg and 30 x 10^6 cd3 / kg.the patient had always presented good general conditions with engrafment of the pmn to the d + 13 and the plt to the d + 11. the chimerism at d + 20 was 100% donor both on pmn and on pbl. prophylaxis for gvhd was changed on d + 7 by replacing the sirolimus with tacrolimus for the appearance of grade i cutaneous gvhd . conclusions: we have successfully performed bone marrow transplantation in patients with lrba deficiency. the new antibiotic molecules, used to induce infectious remission, the new low-intensity regimens, the prevention of the most fearful complications (vod) have been the key to success in such complicated case. the high number of cd34 cells infused with a controlled number of cd3 were the key then of rapid engraftment with minimal gvhd readily controlled by the immunosuppressant. disclosure background: in the absence of hla-matched related donor, allogeneic stem cell transplantation from haploidentical donors are potential alternatives for patients with hematological malignencies with an indication to allogeneic stem cell transplantation. herein, we retrospectively assessed the outcome of haplo-sct for patients with refractory hematological malignancies. methods: this analysis included 27 consecutive patients who underwent haplo-sct for various hematological malignancies at our center between october 2010 and may 2018. we used our institutional database to evaluate details and characteristics of patients and transplant outcomes. results: demographic features of the patients and donors have been summarized in table 1 . all of the patients had advanced disease with a high risk of relapse. the majority of patients underwent haplo-sct from their parents. out of 24 patients, early transplant-related mortality was seen in this cohort of 5 patients. four patients treated with second haplo-sct and recovered hematopoiesis after second transplant. the remaining 19 patients were followed in a median of 4 months. donor type abo group switch was observed in a median of 45 days (30-60 days) after transplant. the median time for engraftment was 19 days (range, 15-60) for all patients. after the first transplant, 9 patients developed acute gvhd (37.5%) with 7 patients having grade ii-iii acute gvhd. five (18.5%) had chronic gvhd, none of them with extensive manifestation. the prepative regimen was relatively well tolerated with limited regimen-related toxicity. cmv reactivation occurred in 11 patients (40.7%) during the follow-up of the study. eight patients (29.6%) relapsed after a median of 132 days post transplant (range, 45-588 days). cr was achieved in 17 (63%) patients after haplo-sct. mean estimated 5-year os and pfs are 66.7%±0.9% and 92.3%±0.7%, respectively. conclusions: given the growing data on the similarity of outcomes after hla-matched and haploidentical sct, further studies are required to determine whether factors may be more important for donor selection than hlamatching. clinical trial registry: -disclosure: nothing to declare outcome of allogeneic stem cell transplantation for hodgkin and non-hodgkin lymphoma: single center experince from turkey ayşe uysal 1 , hale bülbül 2 , nur akad soyer 2 , mahmut tobu 2 , murat tombuloglu 2 , guray saydam 2 , filiz vural 2 background: allogeneic sct (allosct) is generally optionally treatment choice for young and fit patients with relapsed/refractory lymphoma who were heavily pre-treated and after the failure of autologous stem cell transplantation (asct). relapse after asct is associated with a poor prognosis and allosct is a potentially curative therapy for lymphomas which have relapsed after asct. methods: in this study, we evaluated 19 patients with hl and nhl who had treated with allo-hsct between november 2014 and december 2018 in ege university adult hematology transplantation unit. results: patients, disease and transplant characteristics were illustrated in table. histologic subtype of nhl was evaluated as t cell lymphoma (n=8;61,5%), mantle cell (n=2;15,4%), diffuse large b-cell lymphoma (n=2;15,4%) and b-cell lymphoma, unclassifiable, with features intermediate between dlbcl and classical hodgkin lymphoma (n=1;7,7%). all histologic subtype of hl was determined as nodular sclerosing. the median number of prior treatments before allo-hsct was 3 (range, 1-4). twelve (63,2%) patients had refractory disease, 3 (15,4%) patients were in complete remission and 4 (21,1%) patients were in partial remission before allo-hsct. the median time from diagnosis to allosct was 24 (range, 8-144) months. peripheral stem cell was used for stem cell source in all of them. total body irritation plus fludarabine plus cyclophosphamide and busulfan plus cyclophosphamide were preferred most frequently for conditioning as non-myeloablative and myeloablative, respectively. neutrophil engraftment was occurred median of 17 (range, 10-21) days. graft versus host disease (gvhd) prophylaxis was applied all of them and cyclosporine plus methotrexate was preferred most frequently (n=16;84,2%). gvhd was occurred in 68,4% of them (42,1% acute gvhd, 31,6% chronic gvhd and 7,7% both). veno-occlusive disease (vod) was occurred in 2 (10,5%) patients. transplant related death was observed in 5 (26,3%) patients. overall survival (os) and disease-free-survival (dfs) were evaluated as median 9 (range, 0-45) and 7 (range, 0-45) months, respectively. analyze of os and dfs was illustrated in figure. six patients are alive without disease. after a rapid tapering of immunosuopressive therapy was underwent a therapy with ponatinibat dose of 45mg/die results: afther a month of therapy we observed a rapid decrease in minimal residual disease on molecular assessment with an mmr of p190-bcr-abl/abl non detectable confirmed by bone marrow revaluations at days + 190, +222nd +244 after the salvage therapy. the patient has not had experienced of graft-versus-host disease, ponatinib treatment was well tolerated and considered safe with easily manageable side. conclusions: maybe in the era of tyrosine kinase inhibitors (tkis), philadelphia chromosome positive acute lymphoblastic leukemia (ph+ all) it could benefit from a combined treatment between transpalnt and tkis however more studies are needed to confirm these hypotheses. disclosure: nothing to declare immunodeficiency diseases and macrophage background: although a number of patients with hiv infection and hematological disease have successfully undergone allogeneic hsct together with combination anti-retroviral therapy (cart), short and long-term outcomes remain not well known. we report the largest spanish experience of hiv-infected adult patients with high-risk hematological malignancies with allogeneic hsct. methods: we retrospectively reviewed 22 hiv-positive patients who received allogeneic hsct between 1999 and 2018 in 5 spanish centers within geth (grupo español de trasplante hematopoyético y terapia celular). results: baseline and transplant characteristics of patients are shown in table 1 . median age was 44 years and 77% of the patients were men. the most frequent underlying malignancies were non-hodgkin lymphoma (9, 41%) and aml (7, 31%). in half of the patients an hlaidentical sibling was the donor; and in the other half, an alternative donor was used. peripheral blood was used as graft source in 86% of the transplants. at the time of hsct, all patients had been receiving suppressive cart for a median of 6 years and only 2 of them showed detectable plasma hiv rna, one of them because of poor adherence to cart together with the accumulation of multiple resistance mutations; and the other patient had detectable hiv rna at low levels (< 150 copies/ml). all patients received cart throughout the transplant procedure, being temporally stopped in two patients due to significant mucositis. after a median follow-up of 65 months (8-112), 5-year overall survival (os) and event-free survival (efs) were 46%. nrm was 14% at 12 months and relapse was 24% at 24 months. grade ii-iv agvhd rate was 40%, and moderate/severe cgvhd rate was 41% at 24 months. a significant proportion of patients (68%) showed infectious complications with viral infections as the most frequent cause. two patients had invasive aspergillosis and one patient presented disseminated tuberculosis. causes of death included infections (50%), relapse (43%) and toxicity (7%). among the 6 patients who died due to infections, 3 had severe chronic gvhd and were under immunosuppressive therapy. two patients showed severe toxicity related to drug interaction with anti-retroviral therapy. all survivors except one showed undetectable hiv load at last follow-up after hsct. conclusions: allogeneic hsct is an effective therapy for high-risk hematological malignancies in patients with hiv infection, providing long-term disease free survival together to long-term hiv suppression with cart. however, drug interactions with anti-retroviral agents, occurrence of gvhd, and frequent infectious complications account for a complex procedure in this population. selected hiv-infected patients with hematological malignancies should be considered for allo-hsct when indicated, in experienced centers, with a multidisciplinary care. disclosure background: primary immunodeficiencies (pid) are rare diseases often associated with genetic defects in the immune system, predisposing individuals to recurrent infections and increased risk of allergy, autoimmunity and malignancy. allogeneic haematopoietic stem cell transplantation (hsct) has been successfully used as a curative therapy for most severe forms of pid. because pid is a genetic disease, < 25% of these children will have a healthy, human leukocyte antigen (hla) matched sibling donor available, and umbilical cord blood grafts from unrelated donors are a suitable alternative cell source. we report the results of umbilical cord blood transplantation (ucbt) performed in 112 patients with pid between 2014 and 2018 at children's hospital of fudan university in china. methods: 112 patients included chronic granulomatous disease (cgd, n=39), severe combined immunodeficiency (scid, n=23), interleukin-10 receptor-a deficiency (il-10rad, n=27), wiskott-aldrich syndrome (was, n=7), leukocyte adhesion deficiency (lad, n=5), severe congenital neutropaenia (scn, n=3) and other immunodeficiencies (n=8). all patients were assessed by clinical immunologist to confirm clinical phenotype and genetic diagnosis. median age of 112 patients was 13 months (range, 2 to 144 months), and median body weight was 8.75 kg (range, 3.2 to 36 kg). all patients received a ≤3/10 hla alleles-mismatched cord blood unit, 16 were hla fully matched, 28 were 9/10 matched, 49 were 8/10 matched and 19 were 7/10 matched. median nucleated cells of the cord blood were 14.23x10 7 /kg (range, 3.55 to 51.5 x10 7 /kg), and median cd34+ cells were 3.86 x10 5 /kg (range, 0.73 to 30.27 x10 5 /kg). results: median follow-up time was 13 months (range, 1 to 57 months), the overall survival rate at 1 year for all patients was 74.6%, and was 83.7%, 78.3% and 60% for cgd, scid and il-10rad, respectively. 27 patients died, most deaths (19/27, 70.4%) occurred in +100 days after transplantation, the main cause of death was infection (24/ 27, 88.9%). 87/112 (77.7%) patients engrafted, median time of neutrophil engraftment was 24 days (range, 11 to 60 d), and median time of platelet engraftment was 36 days (range, 9 to 59 d). the cumulative incidence of grade 3-4 acute gvhd was 8.9%, and that of chronic gvhd was 10.7%. conclusions: unrelated ucbt should be considered for pid patients without an hla -matched sibling donor. effective control of infection before and after transplantation is important for improving survival. disclosure background: dedicator of cytokinesis (dock 8) deficiency causes a combined immune deficiency characterised by recurrent bacterial infections, susceptibility to viral infection, eczema, food allergies, vasculitis and increased risk of malignancy. due to the high morbidity and mortality of the disease hsct has been increasingly offered to patients as a potentially curative therapy 1 . methods: we retrospectively reviewed the outcomes of hsct for patients with dock 8 deficiency at great north children's hospital newcastle upon tyne between 2011 and 2018 (5 in published reference). results: ten patients with dock 8 deficiency were treated with hsct (median age 7.5y range 4.5-16.8y). median duration of follow up was 4.5years (range 1.3-6.5y). there were a range of donor sources (3 msd, 3 mud and 4 tcr ab/cd19+ depleted haploidentical), conditioning regimens (6 treo-flu, 4 treo-flu-thiotepa) and serotherapy (5 alemtuzumab, 3 atg+rituximab, 2 none). one patient who received a cd3+ tcr alpha beta/cd19+ depleted haploidentical transplant received add back t-cells with caspacide molecular safety switch (bellicum pharmaceuticals). skin only agvhd occurred in 3/10 patients (1x stage 1, 2x stage 3). no patients had cgvhd. overall survival was 60% (6/10). survival was comparable regardless of donor source. all deaths occurred within 13 months of transplant. the 4 patients who died had significant burden of disease pre-transplant: 1 patient had chronic liver failure secondary to cryptosporidial sclerosing cholangitis,1 had a cirrhotic liver secondary to cryptosporidium, cerebral vasculitis, an axillary aneurysm and aortic vasculitis requiring grafting of an ascending aortic aneurysm,1 was pn dependent for failure to thrive with a history of cryptosporidium infection and 1 had candida in a bal pre-transplant. causes of death in these patients were: respiratory failure (n=1), progressive encephalopathy (n=1), multi-organ failure with septic shock and encephalopathy (n=1) and multiorgan failure and septic shock after treatment for tma (n=1). two of these patients had reactivation of cryptosporidium prior to their death. pretransplant cryptosporidium was associated with mortality (graph 1). one patient who survived had suffered from stroke pretransplant. one suffered from a basilar artery aneurysm 7 years post-transplant at 19yo. at the time of latest follow up donor chimerism was 100% in 5/6 survivors and high level mixed in the other(100% cd3, 89% cd15 and 92% cd19). conclusions: this single centre study of hsct for patients is consistent with literature indicating that hsct is a potentially curative therapy for patients with dock 8 deficiency. the increased morbidity associated with cryptosporidial infection is likely to be a consequence of overall disease burden rather than an infection specific effect. this does however highlight the improved outcomes of transplant prior to development of multiple comorbidities and suggests that hsct should be considered early. it is unclear whether the late occurrence of vascular complications after transplant were caused by a manifestation of disease which is not corrected by transplant or a result of vascular injury sustained pre-transplant. reference methods: referred infants underwent testing for: immune phenotype (ab, gd, naïve and memory t cell, b cell and nk cell numbers); functional activity of t and nk cells; maternal engraftment; adenosine deaminase (ada) and purine nucleoside phosphorylase (pnp) enzyme activity; and genetic testing. those with a confirmed diagnosis of scid underwent either allogeneic hematopoietic stem cell transplant (hct) or (if eligible) gene therapy (gt). infants identified as having ada deficiency as the etiology of their scid received enzyme replacement therapy prior to proceeding to definitive therapy. results: twenty-three (66%) infants were confirmed to have scid. three (13%) of these infants had a family history of scid but would not have been identified without nbs. in addition, one infant, born prematurely at 28 weeks, was diagnosed as having pnp deficiency only after developing infections. this infant was identified by nbs but repeat testing at 32 weeks gestation was normal likely due to support of transient t cell production from exogenous enzyme provided by red cell transfusion. twelve infants with confirmed low trecs had a non-scid diagnosis: 5 with transient lymphopenia of infancy who normalized trec, immune phenotype and function, 1 with prenatal exposure to 6-mecaptopurine (6-mp), 3 genetically confirmed with digeorge syndrome, and 3 with prolonged lymphopenia. of the three with prolonged lymphopenia, two had recurrent infections: one ultimately diagnosed with ataxia telangiectasia and one with absent trec but near normal number of t cells, normal pha but no specific antigen responses, and absent b cells who will be undergoing transplant in the near future. the third continues with absent trec, short telomeres, low numbers of a/b t cells, presence of g/d t cells, vaccine responses and freedom from infection with no identified genetic etiology. in summary, 30% of the patients referred to msk with confirmed abnormal nbs for scid have a non-scid diagnosis. there is no uniform collection of data for these infants and the threshold trigger for repeat testing varies from state to state, so the incidence of significant non-scid disorders identified will also likely vary from state to state. although our institution specific experience is biased, as most infants had confirmation of a low number of trec prior to referral, the significant number of disorders in the non-scid cohort emphasizes the importance of full evaluations and follow-up for these infants. disclosure: none of these relate to the work being presented. susan prockop -research funding mesoblast and atara biotherapeutics. nancy kernan -research funding jaz pharmaceuticals. richard o´reilly research funding and royalties atara biotherapeutics. kevin curran consulting juno pharmaceuticals, novartis. j.j. boelens avrobio, magenta, chimerix and bluebird bio background: post-transplant autoimmune cytopenia (aic) is challenging and associated with substantial morbidity and mortality. we aimed to study the cumulative incidence (ci) of post-hct autoimmune cytopenia (aic) and its predictors in a cohort of children with primary immunodeficiency (pid). methods: in this retrospective study, we included 199 children with pid who underwent their first hsct with fludarabine(f)-treosulfan(t)±thiotepa(thio) at great north children's hospital from 2007-2017. main outcomes of interest were the ci of aic and its predictors. fine-and-grey regression models were used to analyse predictors of aic, considering death as a competing event. variables included were age at transplant (< 2.5 years vs >2.5 years), gender, diagnosis (scid vs immune-dysregulatory disorders vs other pids), pre-transplant aic, pre-and posttransplant respiratory virus, donor (mfd vs mud vs mmfd/mmud vs haploidentical donor), abo incompatibility, conditioning (ft vs ftthio), serotherapy (none vs alemtuzumab 0.3-0.6mg/kg vs alemtuzumab 0.9-1.0mg/kg vs atg), stem cell source (marrow vs pbsc vs cord vs exvivo t depleted pbsc), infused stem cell doses (tnc, cd34 and cd3), agvhd (none vs any agvhd), cgvhd (none vs any cgvhd), viral infections (cmv/adenovirus/ ebv/hhv6 viraemia), chimerism (full vs mixed chimerism (wb < 95%) within first year post-hct). impact of thymopoiesis using naïve t cell recovery was studied. results: median age at transplant was 2.4 years (range, 0.11-18.3 years). primary diagnoses were scid (22%), immune-dysregulatory disorders (28%) and other pids (50%). donors were mfd (21%), mud (48%), mmfd/ mmud (16%) and haploidentical parents (15%). stem cell sources were marrow (30%), unmanipulated pbsc (38%), ex-vivo t-depleted pbsc (23%) and cb (9%). 16% received additional thiotepa and 87% had csa/mmf as gvhd prophylaxis. median duration of follow-up of survivors was 2.9 years (range 0.2 to 10.2 years). 5-year os for the entire cohort was 81%. 6-month and 1-year ci of aic were 5% and 13%. of 21 developed aic, 17 (80%) had aiha, 3 (15%) had aiha±itp and 1 (5%) had aiha ±itp±ain. median onset of aic was 6.2 months post-hct (range 0.2-12.8 months). patients were treated with a median of 3 treatment modalities (range, 1-4). one (5%) had steroid, 8 (38%) had steroid+high-dose-ivig, 8 (38%) had steroid+high-dose-ivig+rituximab, 1 (5%) had steroid +high-dose-ivig+sirolimus and 3 (14%) had steroid +high-dose-ivig+rituximab+sirolimus. the median time to resolution in 18 (85%) who achieved remission after first aic was 6.5 months (range 1.4-28.6 months). 1 had one relapse and 2 had two relapses. 2 died after development of aic (1 aspergillus pneumonia; 1 multi-organ failure). of 19 (91%) surviving patients after aic, 4 had on-going aiha at median of follow-up 2.5 years post-hct (range 0.9-4.8 years). on univariate completing-risk analysis, age at transplant >2.5years (p=0.02) and pre-transplant aic (p=0.02) were associated with higher incidence of aic (figure 1a -ic). on fine-and-grey models, only age at transplant (hr 3.08, 95%ci 1.12-8.49, p=0.03) was independently associated with aic. of 157 with complete immune reconstitution data, naïve t cells >100cells/ml at 6 months post-hct was associated lower incidence of aic (hr 0.36, 95%ci 0.15-0.91, p=0.03) (figure 1d) conclusions: younger age and thymopoiesis were associated lower incidence of aic in children with pid after hct clinical background: human heme-oxygenase-1 (ho-1) deficiency has been reported to present with tetrad of anemia, nephritis, inflammation and asplenia and is fatal if not treated. its an auto-inflammatory disorder. macrophages/ monocytes express ho-1 and are engaged in recycling of red cells. human ho-1 deficiency results in intravascular hemolysis and severe damage to the endothelial system, kidneys, and other organs. transplantation of either healthy wild type macrophages or new macrophages produced by sct from healthy donor has been proven to be curative for ho-1 deficiency in mice. in 2018, we had reported first successful allogeneic sct for human ho-1 deficiency. here we report second successful non-myeloablative msd hsct for a child with ho-1 deficiency. methods: a 9-yr-old-girl presented with complaints of fever, anemia and severe hypertension. in the past, at the age of 7 years she was admitted for high fever for 1 month and needed blood transfusion for the first time for severe anemia and had high platelets and high ferritin. she was treated as macrophage activation syndrome with prednisolone alone and later cyclosporine was added. she had short stature, abnormal facies but normal development. hemoglobin 9 g/dl, urine for haemoglobin was positive, platelets 1055, 000/ul, ferritin 2568 mcg/l and urine albumin 4+ and urine rbc 20-30/hpf. ultrasound and ct scan abdomen showed asplenia. a diagnosis of ho-1 deficiency was suspected. mutation analysis showed homozygous missense mutations in exon2 (r44x) on chromosome 22q12, which would result in the absence of the functional ho-1 protein. both parents were carriers of this mutation. we managed her over next 6-years with prednisolone, hydroxyurea and mycophenolate mofetil (mmf). however she remained steroid dependent. hla-typing confirmed her healthy unaffected 13-year-old brother to be a fully matched donor. at the age of 16 years she was taken up for msd sct after taking informed consent. she weighed just 16 kg. we conditioned her with alemtuzumab-0.8mg/kg, fludarabine-160mg/m2, cyclophospamide-29mg/kg and total body irradiation 2 gray. we infused 9 million/kg peripheral blood stem cells from her brother. graft-vs.-host disease (gvhd) prophylaxis consisted of tacrolimus & mmf. results: she tolerated procedure very well. her entire hospital stay was uneventful and lowest platelet count recorded was 30,000/ul. her neutrophils engrafted on day +13 and she was discharged on day+19. his urine albumin was nil by day+7. she had no gvhd. her chimerism on day+22 showed 97 % donor cells, on day+60 was 98% and on day+180 was 98% donor. now he is day+210 post-sct and doing well. she has no evidence of hemolysis, proteinuria, hypertension, fever. she has normal ferritin and platelets. she has gained 3 cm height and 5 kg weight in last 7 months. she had no viral reactivation and her immune recovery at 6 months post sct is good. conclusions: non-myeoablative allogeneic msd sct is a curative treatment option for human ho-1 deficiency. disclosure: nil two decades of excellent transplant survival in children with chronic granulomatous disease: a report from a supraregional immunology transplant centre in europe background: haematopoietic stem cell transplantation (hsct) confers life-long curative therapy for chronic granulomatous disease (cgd). the ability of donor-derived neutrophils to replace recipient's defective neutrophils makes hsct a superior therapy compared to conventional standard of care using antimicrobial therapy. methods: we examined the outcome of children with cgd who received a first hsct at great north children's hospital from 1998 to 2017. outcomes included overall survival (os), event-free survival (es), toxicity endpoints, autoimmune disease, long-term survival and graft function. cox proportional-hazard models were used to analyse predictors of os and es. variables included for predictor analysis were age at transplant, donor, stem cell source, stem cell doses and conditioning. results: =55 children were included in this analysis. median age of transplant was 5.3 years (range, 0.6-18.0 years). 45 (82%) had x-linked and 10 (18%) autosomal recessive cgd. twenty (36%) had matched family donor, 31 (56%) had unrelated donor and 4 (8%) had parental haploidentical donor. prior to 2007, various conditioning regimens were used, with 21 (38%) patients undergoing conditioning with pharmacokinetic guided intravenous (iv) busulfan (bu) and iv cyclophosphamide with or without serotherapy. from 2007, the conditioning regimen was switched to flu-treosulfan-alemtuzumab with gvhd prophylaxis using ciclosporin (csa) and mycophenolate mofetil (mmf) for family and unrelated donors (n=24, 44%). flu-treosulfan-thiotepa-atg-rituximab was used for cd3 tcr alpha-beta cd19 depleted haploidentical grafts (n=4, 7%). ten (20%) patients had grade ii-iv acute gvhd while 5 had (9%) had grade iii-iv acute gvhd. none had chronic gvhd. the 5-year os for the entire cohort was 89% (95% ci, 67-95%) (figure 1 ). analysis by age at transplant revealed a 5-year os of 100% for children transplanted at < = 5 years of age and 81% (95%ci, 60-92%) for the children >5 years of age (p< 0.04) (figure 2) . the os was comparable between match family donor (88%, 95% ci, 61-97%) and unrelated donor transplant (89%, 71-95%) ( figure 3 ). all four haploidentical transplants were successful. the 5-year es for the entire cohort was 77% (95% ci 62-87%). none of the variables was associated with es. all seven patients with slipping chimerism received a successful second transplant. the five deaths were all due to transplantrelated complications (2 multi-organ failures; 1 pulmonary haemorrhage; 1 graft iv acute gvhd; 1 post-transplant lymphoproliferative disease). the median age at transplant of deceased patients was 10.0 years (range 8.4 to 18 years). the 1-year and 5-year cumulative incidence of autoimmune diseases were 9% and 12% respectively. three (5%) had immune cytopenia while 3 (5%) had autoimmune endocrinopathy (2 thyroid dysfunction; 1 type 1 diabetes mellitus). the median age of long-term survivors was 14 years (range, 2 to 36 years) with the median duration of follow-up of 6.5 years (range, 0.32 to 19.5 years). there was no late death in the entire cohort. the median donor myeloid chimerism was 100% (range 23 to 100%) conclusions: despite the limitations of a single centre study, our findings confirm that hsct is a safe and longlasting curative therapy for children with cgd disclosure: none non -medical challenges in the diagnosis and transplantation of patients with primary immune deficiency: an experience from a tertiary care center in india sagar bhattad 1 , stalin ramprakash 1 , raghuram cp 1 , chetan ginigeri 1 , fulvio porta 1,2 background: primary immune deficiencies (pid) are increasingly being recognized in several parts of india. despite being diagnosed, many patients fail to receive optimal care due to financial and social constraints. methods: case records of patients diagnosed and treated (including hematopoietic stem cell transplants) for pid diseases during feb 2018 -nov 2019 at aster cmi hospital, bangalore, india were analysed. factors leading to deferred or suboptimal care were assessed in detail. results: 65 patients with various pids were diagnosed during the study period (details in table). 35 of them warranted a hematopoietic stem cell transplant (hsct) as definitive curative treatment. a total of 7 children received 9 hsct. 2 of them died while 5 of them are alive and well. 17 children (13 with severe combined immune deficiency) died before a hsct could be carried out. 12 of them were critically ill at presentation, while 5 were stable but deferred further treatment citing financial and social constraints. 11 children needing transplant continue to remain on follow-up and have not been transplanted to date (4 of them have significant financial constraints, 3 families are not convinced about the need for transplant and 4 of them are being prepared for transplant). table: (scid -severe combined immune deficiency, vodi-veno-occlusive disease with immunodeficiency, cgd -chronic granulomatous disease, hlh -hemophagolymphohistiocytosis, was -wiskott aldrich syndrome, xlt -x linked thrombocytopenia, lad-leukocyte adhesion deficiency, msmd -mendelian susceptibility to mycobacterial disease, xla -x linked agammaglobulinemia, cvid -common variable immune deficiency, apeced -autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, cmcc -chronic mucocutaneous candidiasis, at -ataxia telangiectasia). conclusions: we present our experience from a developing country and discuss non-medical factors leading to suboptimal care in children with pid. only 20% children warranting hsct could be transplanted in our cohort. among those where hsct is potentially curative 48% of children died before hsct could be offered. transplants in developing countries pose unique challenges due to the absence of government funding and/or universal insurance coverage. in addition to delay in diagnosis and critical state of patients at admission, financial and social factors significantly contributed to poor outcome. disclosure: none the outcome of hematopoietic stem cell transplantation (hsct) in pediatric patients with hemophagocytic lymphohistiocytosis (hlh) in korea methods: the korea histiocytosis working party retrospectively collected nation-wide data from the patients diagnosed with hlh and underwent allogeneic hsct between 2009 and 2017. the clinical characteristics and treatment outcomes of the patients were analyzed. results: a total of 44 patients were enrolled. there were 31 patients with fhl (4 fhl2, 26 fhl3, and 1 fhl4), 7 infection associated hlh, and 6 secondary hlh of unknown cause. all the patients were treated with hlh-2004 protocol, and 30 patients achieved complete response (cr) after treatment for 8 weeks, while 14 did not. the main reasons for receiving transplantation were fhl in 26, reactivation in 17, and refractory disease in 1. the conditioning regimens were busulfan-based in 16 patients, fludarabine-based in 4, treosulfan-based in 7, and busulfan/fludarabine-based in 17. stem cell sources used for hsct were from peripheral blood in 36 patient, cord blood in 7, and bone marrow in 1. the donor types of hsct were unrelated donor in 33 patients and related in 11 (7 matched sibling donor, 4 haploidentical donor, 1 partially matched donor). the causes of death of 7 patients were disease reactivation/ progression in 3, acute gvhd with/without vod in 3, and graft failure in 1. five year overall survival rates were 82.4%, respectively. the disease status at the time of hsct was cr in 37 patients, and non-cr in 7. the 5-year survival rate of patients who received hsct in cr was 87% and 63% for patients transplanted while in non-cr status (p=0.046). patients who received hsct using peripheral blood stem cells had a better 5-year survival rate of 86% compared to 75% of patients who received cord blood stem cells, significantly. the presence of neurologic symptoms, disease status after intial 8 week therapy, conditioning regimen, and cd 34 positive cell count did not have statistically significant impact on survival. conclusions: hsct improved the survival of patients who had familial, or relapsed, or refractory hlh in the korean nation-wide hlh registry. these results are similar to other reports in the literature. the disease status at the time of hsct and the stem cell source of the transplant were the important prognostic factors that affected the survivals of the hlh patients who underwent hsct. clinical trial registry: no registry number. disclosure: to the best of our knowledge, the named authors have no conflict of interest, financial or otherwise p382 hematopoietic cell transplantation with reduced intensity conditioning regimen using fludarabine/ busulfan and fludarabine/melphalan for primary immunodeficiency diseases background: primary immunodeficiency disease (pid) is congenital disorders of innate or acquired immune system. hematopoietic cell transplantation (hct) was a treatment option for pids with life-threatening infections or immune dysregulations. reduced intensity conditioning (ric) was increasingly used to prevent complications in hct, but optimized regimens have not been established. we performed hct for pids with ric using fludarabine and busulfan (flubu) or melphalan (flumel) according to the guidelines of european society for immunodeficiencies (esid) / european society for blood and marrow transplantation (ebmt), and assessed the efficacy and safety of these ric. methods: from april 2004 to december 2017, 42 pid patients underwent ric-hct using flubu or flumel in tmdu were analyzed retrospectively. the auc of bu was set to 30 mg*hour/l for severe combined immunodeficiency disease (scid) and 60 mg*hour/l for non-scid. overall survival (os) was analyzed. results: the median age at hct was 2.0 (0.3-21) years old (35 male and 7 female patients). flubu was used for 23 patients (7 scid, 9 combined immunodeficiency disease (cid), 3 ectodermal dysplasia (eda), and 4 severe congenital neutropenia (scn)) and flumel was used for 19 patients (8 scid, 6 cid, 4 xiap deficiency, and 1 eda). anti-thymocyte globulin was used in 8 patients of flubu group and 7 patients of flumel group. cord blood in 21 and bone marrow in 21 was used for donor sources. matched donor was used for 7 and 8 patients in flubu and flumel groups (30.4 % and 42.1 %), respectively. median follow up period was 2.2 years. two years-os of all patients, flubu group patients and flumel group patients was 79.6 %, 89.8 % and 64.1 %, respectively. neutrophil engraftment was 92.9 %, 91.3 % and 94.7 % (all patients, flubu group and flumel group). in scid, all 7 patients in flubu group achieved engraftment and survived. seven out of 8 in flumel group achieved engraftment, but 1 patient had secondary graft failure and 3 patients died. in non-scid, 14 out of 16 in flubu group achieved engraftment, but 6 patients had secondary graft failure. all 11 non-scid patients in flumel group were engrafted and survived. two and 7 patients in flubu group and flumel group suffered from severe acute graft-versus-host disease (grade iii-iv). ten patients had hemophagocytic lymphohistiocytosis (hlh). viral reactivation or infection was observed in 20 patients, and resolved in all but one patient. conclusions: the ric-hct using flubu or flumel was advantagous for neutrophil engraftment, and flubu for scid and flumel for cid with immune dysregulation may be an effective opinion. flubu regimen needs to be improved for secondary graft failure in non-scid. prevention of hlh after transplantation using dexamethasone palmitate will be considered. disclosure: nothing to declare. survival after hematopoietic stem cell transplantation with tcrαβ/cd19 graft depletion in older children with primary immunodeficiencies background: tcrαβ/cd19 depletion is a graft engineering method that proved valuable in increasing the survival rate after hematopoietic stem cell transplantation (hsct) in patients with primary immunodeficiencies (pid). decreased survival rate in older patients with pid was previously reported after transplantations with nonmanipulated grafts. methods: 148 patients with various pid (excluding classic scid) who received allogenic hsct with tcrαβ/ cd19 graft depletion from 2012 to september 2018 in our center were analyzed. the median age at hsct was 3,5 years (range 0,43-17,63). patients were divided into 3 age groups: 0-6 years -95 patients, 6-12 years -30, 12-18 years -23. 112 patients received hsct from matched unrelated, 31 -haploidentical donors, 5 -siblings. conditioning regimens with 1-2 alkylating agents and antithymocyte globulin were used in all patients. 113 patient received short courses of various posttransplant immunosuppressants. median follow up after hsct is 1,95 years (range 0,04-6,3 years). results: overall survival (os) in 148 patients was 0,85 (95% ci 0,79-0,91). we observed similar os in the younger age groups: 0,89 (95% ci 0,83-0,96) in 0-6 years and 0,89 (95% ci 0,78-1,0) in 6-12 years of age. seven of 23 patients in older group (12-18 years of age) died, the os was only 0,65 (95% ci 0,43-0,86), p=0,065. all patients from older age group who died had combined pid: 1 -wiscott-aldrich syndrome, 1 -undefined pid, 1 -il2rg deficiency, 1 -dclre1c deficiency, 1 -nijmegen breakage syndrome (nbs), 1 -kabuki syndrome, 1 -icf syndrome. the median time of death after hsct was 0,64 years (range 0,26 -1,58). six of those had transplant-related mortality (trm). five patients had hsct-associated viral infections: 2 -cmv pneumonias, 3 -adv infections (1 -fulminant hepatitis, 2 -multiorgan). interestingly, 2 of them had prolonged history of disseminated viral infections (adv), with the reduction of viral load in blood and other fluids and tissues upon treatment. one patient with kabuki syndrome after hsct developed hhv8 associated kaposi sarcoma, was successfully treated. eventually all 3 patients with reduction of viral infection and sarcoma symptoms developed multiorgan failure with some clinical and laboratory evidences of endothelium cell damage syndrome. one patient with nbs died of high grade lymphoma progression. conclusions: hsct with tcrαβ/cd19 depletion demonstrates high survival rate in 148 patients with various pid. in our group patients' age older than 12 years predisposes to decreased posttransplant survival. patients with combined pid are at higher risks of posttransplant mortality. we conclude that at least in some of our patients with prolonged history of viral infections after hsct the cause of death could be multiorgan failure due to endothelium cell damage syndrome resulting from persistent inflammation and drug toxicity effects. disclosure background: chronic granulomatous disease (cgd) is a primary immunodeficiency (pid) caused by a mutation in 1 of the 5 subunits of the nicotinamide dinucleotide phosphate (nadph) oxidase, which leads to a reduction in the microbicidal activity of phagocytic cell. starting at an early age, cgd patients suffer from severe recurrent infections, as well as inflammatory events. allogeneic hematopoietic stem cell transplantation (hsct) is a curative option for cgd in patients with insufficient benefit from supportive care and prophylactic antibiotics. we reported a series of 39 patients with cgd who underwent unrelated umbilical cord blood transplantation (ucbt) at our center. methods: in this retrospective study, we observed a series of 39 consecutive ucbt performed at our center in children with cgd between 2015 and 2018.median age at transplantation was 17 months (range, 4 to 144 months), median body weight was 10 kg (range, 6 to 34 kg). 32/39 patients received a myeloablative conditioning regimen consisting of busulfan, fludarabine, cytarabine, cyclophosphamide and g-csf, 7/39 patients received another myeloablative conditioning regimen consisting of busulfan, fludarabine, cyclophosphamide and atg. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. results: engraftment occurred in 29/39 (74.4%) patients. 10/39 (25.6%) patients occurred graft failure, all of them received a myeloablative conditioning regimen without atg. median time to neutrophil and platelet engraftment were 24 (rang, 14-46) and 36.5 (rang, 15-51) days. 22/39 (56.4%) patients developed acute gvhd, with 4/39 (10.3%) episodes of grade iii-iv agvhd. chronic gvhd occurred in 3/29 patients (10.3%). at a median follow-up of 17 months (rang, 1 to 44 months), the overall survival rate was 83.7%, and event-free survival rate was 66.7%. conclusions: unrelated ucbt should be considered as potential curative methods in children with cgd. cgd patients who used myeloablative conditioning regimen with atg shows better graft and survival. disclosure: nothing to declare background: invasive fungal infections (ifi) remain a major cause of treatment-related morbidity and mortality in aml patients. although not uncommon, the presentation of unusually severe clinical features might be indicative for an underlying immunodeficiency. caspase-associated recruitment domain 9 (card9) is recognized to have a crucial role in effective antifungal response, leading to th1 and th17 differentiation and to the initiation of the inflammatory cytokine cascade. particularly interferon-gamma (ifng) increases macrophage activity. patients with homozygous card9 mutations are known to have a significantly increased susceptibility to life-threatening systemic candidiasis. however, some sequence variants may lead to increased ifi-susceptibility even in heterozygosity, e.g. under immunosuppression. ifn-γ has been described as an additive treatment option because of its immune stimulating effect on the leukocyte immune response in a situation of immunological "blindness". methods: here, we report the case of an 11-year old male with aml m6 with a severe systemic candida tropicalis infection, unresponsive to triple-antimycotic regimen, leading to multi-organ failure. he was discovered to bear a heterozygous card9 mutation, and ifn-γ immunotherapy leaded to complete response of all disseminated infections. results: the patient developed septic fever immediately after the first chemotherapy cycle. unexpectedly, candida tropicalis was confirmed in the blood culture within 24 hours. liposomal amphotericin b (ambisome ® ) was started immediately, however candida rapidly disseminated to lungs, liver, spleen, kidneys and cns despite extended antimycotic therapy with caspofungin, voriconazole and fluconazole. the patient was splenectomized due to massive infiltration ( figure 1 ). genetic testing for mycosis predisposition revealed a heterozygous mutation in the card9 gene, inherited from the father (c.809a>t(p.glu270val)). ifn-γ treatment was started (100 μg subcutaneously, 3 times per week), leading to an almost complete response of disseminated infections. due to the severe infection, chemotherapy had to be interrupted after one course. however, bone marrow remained in complete remission for almost one year. the patient experienced altogether two relapses requiring an unrelated allogeneic and a haploidentical hsct. under combined ifn-γ and ambisome/ fluconazole prophylaxis no further mycosis was observed despite extensive and prolonged immunosuppression. conclusions: ifi in aml patients are common, however an unusual presentation in presumably immune competent individuals should raise the suspicion for immunodeficiencies. in our case, an unexpected early candida-sepsis was completely unresponsive to an adequate multi-agent treatment. while ifn-γ is used in adults as an immune stimulatory cytokine, little data are available for children. to our knowledge, this is the first case of successful ifn-γ treatment of a pediatric aml patient with disseminated candida sepsis, bearing a card9 mutation. given the elevated mortality risk for ifi, and the apparently safe and well-tolerated application of ifn-γ, an adjuvant immunotherapy might be considered. further studies are needed to define the indication and duration of this kind of adjunctive immunotherapy. moreover, considering the wide heterogeneity of genetic mutations involved in ifi-susceptibility, genome-wide expression profiling might be useful for pediatric cancer patients, as the identification of specific immune pathways might help to identify individual ifisusceptibility in order to improve the outcome of those high-risk patients. [[p385 image] 1. background: chronic granulomatous disease (cgd) is curable by allogeneic hematopoetic stem cell transplant (hsct). recent reports of haploidentical donor hsct with with post transplant cyclophosphamide (ptcy) from family donors in pediatric primary immune deficiencies have shown encouraging results. however, it has not been reported in cgd. here we describe successful haploidentical hsct in a child with cgd with myeloablative conditioning and ptcy. a 3 year-old, male child diagnosed with cgd showed oxidative activity 0.3 % by dihydrorhodamine (dhr) test. he had no matched related or unrelated donor available so underwent haploidentical hsct after taking informed consent of the parents in may 2018. donor was his 5/10 hla matched healthy elder sister (oxidative activity 55 % by dhr). he has had multiple admissions for recurrent pneumonia prior to hsct. the conditioning was with rituximab 100 mg/m2 iv on day -8, thiotepa 10 mg/ kg/dose intravenous (iv) for 1 day (day-7), busulfan 3.2 mg/kg/dose daily iv for 4 days (day -6 to -3) and fludarabine 40 mg/m2/dose daily iv for 4 days (day -6 to -3) and rabbit anti-thymoglubulin (thymoglobulin) 1.5 mg/ kg/dose daily for 3 days (day-4 to -2). peripheral blood stem cells (8 million/kg cd34+ cells) were harvested from his sister and transfused to the patient on day 0. graft vs. host disease (gvhd) prophylaxis was with ptcy 50 mg/ kg on day+3 & 4, intravenous cyclosporine from day-3 (targeting levels 150-250 ng/ml) and mmf from day+5. results: his neutrophils engrafted on day+15 and platelets on day+17. chimerism on day+30, 100 and 6 months was fully donor. he developed no acute or chronic gvhd. at 6 months his lymphocyte counts showed cd4-280/ul, cd8-670/ul, cd19-15/ul and cd16/56-120/ul. his had no viral reactivation. he is disease free and gvhd free on day+190 post hsct and is on tapering doses of cyclosporine. his dhr test showed oxidative activity of 42 % on day +100. background: primary immune deficiencies (pid) are a functional disorder of inheritance immune system that increase predisposition to infectious disease in number and severity. the incidence is 1: 10,000 birth live; its immunological dysregulation may increase the predisposition of autoimmune diseases and malignancy, the latter being more frequent (4-25%). at present, the only curative treatment is hematopoietic stem cells transplant (hsct). methods: we describe all patients transplanted with primary immune deficiencies at instituto nacional de pediatria. the conditioning regimen depended on the type donor and pathology: myeloablative (41.07%), reduced intensity (37.5%) and non myeloablative (21.43%) without modification statistically significantly in overall survival. results: a total of 71 patients were included from 1998 to january/2017. severe combined immunodeficiency (scid) is the pathology most frequently transplanted ( figure 1) . seventy three percent have molecular diagnosis, and 44.83% have cases of family pid. the most used sources were umbilical cord blood (ucb) with 42.6% and peripheral blood (39.3%), however the trend of the source of obtaining it has been modified a few years ago (figure 2) . the median graft was 14 days for ucb, 16 days for bone marrow (bm) and 12 days for peripheral blood (pb) (figure 3 ) the main complications are infectious (bacterial 48.3% and viral 39.7%) and non-infectious as pre-graft syndrome (23.1%). conclusions: the overall survival was 54.1% survival according to pathology was: 100% chediak higashi syndrome, 68% scid, 66.7% griselli syndrome, 66.7% hyper igm syndrome, 60% was, 57% cgd, 50% hemophagocytic lymphohistiocytosis. disclosure: ramírez-uribe rosa maria nideshda, salazar-rosales haydeé, olaya-vargas alberto, lópez-hernández gerardo, del campo-martínez maria de los àngeles we wish to confirm that there are no known conflicts of interest associated with this abstact, the only financial support was provided by mexican associations that helping children wiht cancer in a few patients. long-term outcome following hematopoietic stem cell transplantation of wiskott-aldrich syndrome in a single institute mamoru honda 1,2 , yukayo terashita 1 , minako sugiyama 1 , yuko cho 1 , akihiro iguchi 1 background: wiskott-aldrich syndrome (was) is an xlinked disorder of hematopoietic cells, characterized by thrombocytopenia with small platelets, eczema, and immunodeficiency. hematopoietic stem cell transplantation (hsct) is the only curative treatment, and it is recommended to be performed as soon as was is diagnosed. myeloablative conditioning before hsct is recommended because there is a high risk of development of autoimmune disease in patients with mixed chimera after hsct. however, there are few reports about late complications such as pubertal development and eruption of teeth in patients with was receiving hsct. thus, we evaluated late complications in patients with was receiving hsct at hokkaido university hospital. methods: we reviewed medical records of 8 male patients with was who received hsct between 1995 and 2017. results: mean age at hsct was 1.4 (range, 0.6-6.8) years, and median follow-up time after hsct was 13.8 (range 2.9-23.4) years. conditioning regimen in all patients comprised busulfan at 4 mg/kg for 4 days and cyclophosphamide at 60 mg/kg for 2 days or 50 mg/kg for 4 days. additionally, anti-thymocyte globulin at 2.5 mg/kg/day for 1-2 days was administered in 6 patients. engraftment, normal platelet count, and complete chimera were confirmed in all patients. no patients showed complications such as severe chronic graft-versus-host disease, autoimmune disease, short stature (≤ -2.0 sd) and second malignancy. however, high ige level was observed in 4 patients. pubertal development has been confirmed in 6 patients. lack of complete eruption of permanent teeth has been observed in 3 patients who received hsct at age of < 2 years. conclusions: although this was a small-cohort study in a single institute, complete chimera has been achieved in all patients who received hsct with busulfan-based myeloablative conditioning. however, late complications such as male infertility and incomplete eruption of permanent teeth remain major problems. disclosure: nothing to declare methods: we performed unrelated umbilical cord blood transplantation (ucbt) in 5 consecutive children with lad-i. median age of 5 children was 13 months (range, 8 to 131 months), and median body weight was 13 kg (range, 8 to 24.3 kg). all patients received myeloablative conditioning regimen consisting of busulfan, fludarabine and cytarabine. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. all patients received a ≤2 hla alleles-mismatched cord unit, 1 was hla fully matched, 3 were 9/10 matched, 1 was 8/10 matched. median nucleated cells of the cord blood were 14.23x10 7 /kg (range, 4.6 to 20.62 x10 7 /kg), and median cd34+ cells were 3.87 x10 5 / kg (range, 1.95 to 5.77 x10 5 /kg). results: all patients engrafted, median time of neutrophil engraftment was 24 days (range, 13 to 28d), and median time of platelet engraftment was 33 days (range, 32 to 56d). median follow-up time was 13 months (range, 2 to 25 months), all 5 patients were alive with continuous completely donor engraftment, and achieved complete clinical remissions. 4/5 patients developed grade ii/iii acute graft-versus-host disease (gvhd), and 1/5 patients developed chronic gvhd with skin. conclusions: it is the first successful unrelated ubct for lad-i children in china. our data shows ucbt provided excellent outcome for patients with lad-i. disclosure: nothing to declare p390 excellent outcome using 'nktm' enriched hematopoietic stem cell transplants for patients with inborn errors of immunity results: majority of patients in the 2 cohorts had significant infective co-morbidities at the time of hsct with patients in the later cohort entering hsct earlier. patients in the later cohort were sicker at hsct. final engraftment occurred in all except 1 patient who received a hla mis-matched cord blood hsct. graft failures occurred in 6 patients (2 in earlier and 4 in later cohort); 3 of these patients received unmanipulated hscts from hla mis-matched unrelated donors (1 cb, 2 bm). second hsct were with same donors in 3 and different donors in 2 patients. no grade ii to iv acute gvhd or extensive gvhd occurred. one patient (cbt) died of infections/ non-engraftment. all 9 patients in the later cohort compared to 7 of 9 patients in earlier cohort are alive, engrafted and cured. performance status were 100% in all alive patients. of the 9 patients in the later cohort, 7 (6 hla mis-matched related and 1 hla matched related donors) received 'nktm' enriched hsct. in the 6 hla mis-matched 'nktm' enriched hscts, patients received high cd34+, cd3+cd45ro+ and nk cell doses, with median of 16.88 (range, 5.1 -36.5), 113.9 (range, 3.3 -232.6) and 89.0 (range, 22 -654) x10 6 /kg, respectively. no invasive infections occurred in these patients and immune reconstitution in t, b, nk compartments were complete at 1 year after hsct with cd4 > 200 by 6 months and tcrαb > 1500 by 1 year after hsct. background: viral infections contribute to significant morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-hsct), increasing both the human and the financial cost. antiviral agents are often ineffective or/ and associated with toxicity. methods: in view of t-cell anti-viral immunotherapy in greece, we evaluated the actual cost of conventional pharmacotherapy for cmv, ebv and bkv reactivations after allo-hsct, by calculating the costs of (i) the antiviral agents, (ii) the treatment (excluding transfusions) of antiviral drug primary toxicity (e.g. graft failure, cytopenias, renal or hepatic dysfunction) and secondary toxicity (e.g. leukopenia-associated bacterial infections), iii) the treatment (excluding transfusions, ie for bk cystitis) of infectionrelated complications, iv) the transfusions due to treatmentrelated toxicity (ie cytopenias) or infection-related complications (ie, bk cystitis), v) the inpatient or outpatient daily care. notwithstanding that blood and its products, as a common natural good, are provided free in our country, the costs related to blood and platelet collection, processing, storage, laboratory testing and infusions were included in our model. results: the treatment cost of cmv, ebv and bkv reactivations/infections for the first six months post allo-hsct was evaluated in 38/51 patients who reactivated viruses and were transplanted between 10/2015-11/2016 from matched related (17/51), matched unrelated (20/51), mismatched unrelated (8/51), haploidentical (5/51) and mismatched related donors (1/51). we detected 29 cmv, 35 ebv and 18 bkv infections/reactivations in 20, 32 and 17 patients respectively, with a mean of 2±0.4 infection per patient from all three viruses (1-7/patient). of note, 22/38 patients experienced reactivations from more than one virus, requiring repeated treatments with antiviral agents and/or rituximab. the cost of antiviral agents for all cmv, ebv and bkv reactivations/infections was 78,656€, 58,504€ and 11,331€ respectively (3,146€, 2,089€ and 1,416 €/patient, respectively). the treatment cost of toxicity related to antiviral drugs and infection-related complications was 70,358€ (4,309€/patient) excluding transfusions and 676,107€ (28,171€/patient) including transfusions. in particular, the cost of transfusions for bkv hemorrhagic cystitis reached 22,751€/patient. repeated (1-5) and/or prolonged (δm 38d, range 6-150d) hospitalizations were needed, up to a total of 745 and 159 days of inpatient hospitalization and short-term outpatient treatment, respectively. hospitalizations further increased the cost of inpatient and outpatient post-transplant care by 81,640€ and 8,000€ respectively (2,634€ and 500€/patient, respectively), onthe basis of a, rather underestimating the true cost, fixed, unified hospitalization fee (60€/day and 200 €/day). conclusions: overall, in a six-month study period, the treatment of cmv, ebv and bkv infections substantially increased the cost of post-transplant care by 956,283€ (27,322€/patient). the actual cost is undoubtedly higher as the hospitalization fee for transplant recipients is largely underestimated in greece. considering not only the hematopoietic but also the solid organ transplant recipients, the financial burden of antiviral treatment for national economies is enormous. given that antiviral pharmacotherapy is often associated with suboptimal efficacy, toxicity, development of drug resistance, reactivation recurrences and repeated hospitalizations, it is expected that a one-time treatment with multi-virus-specific t cells, able to expand in vivo and provide a long-lasting protection without significant toxicity, will serve as a powerful and costeffective treatment over conventional pharmacotherapy. disclosure: nothing to declare methods: this is a single-centre retrospective analysis of 293 consecutive patients who underwent tcd allo-hscts for myeloid malignancies between january 2012-june 2016. ebv-dna was monitored frequently on whole blood samples with standardised quantitative real-time pcr. serum protein electrophoresis was routinely tested with immunoglobulin subclasses identified by immunofixation electrophoresis. histological confirmation of ptld was based on standard who diagnostic criteria ('proven'), while those without biopsy were classed as 'probable' based on clinical & radiological criteria as defined by ecil-6 guidelines. results: majority of patients had aml(n-152/293) and mds(n-107/293) with a median age of 58 years(range . median follow up of survivors was 32 months(range 4-65). majority of patients(n-220/293;75%) developed ebv-r with a median time of 79 days[inter quartile range(iqr) 27-160 days] &higher cumulative incidence with atg(n-132) versus alemtuzumab(n-161)(p< 0.001). figure-1a shows schematic representation of ebv and ptld events (cumulative incidence of 6.8%(95%ci-4.0%-10.6%) at 12 months). significantly higher peak ebv dna viral load(evl) were noted in patients with ptld(p-< 0.001). development of post-hsct mg was observed in 29%(n-85/292). roc curve identified peak blood evl>150,000 copies/ml significantly correlated with risk of developing ptld (sensitivity-70.6%,specificity-79.4%;auc-0.82,p< 0.001). based on these estimates, subgroup of patients with no ebv-r(n-72/292), peak evl < 150,000(< 150k)copies/ ml(n-165/292) & >150,000(>150k)copies/ml(n-55/292) were categorised in 6 groups along patients with/without mg accordingly (groups 1-6;figure-1b). patients with ebv-r had significantly better os [5-year os of 52% vs 35%(no ebv-r);log-rank p< 0.001],with this survival benefit mainly driven by subgroup of patients with lower evl(< 150k)(p< 0.001). ptld patients had trend towards inferior 3-year os(15% vs 54%;p-0.051). patients with mg had a significantly better os irrespective of degree of evl (group 1-3,p< 0.001).we report a 'sweet spot' of low evl & presence of mg in these patients, with a clear survival advantage compared to those with no ebv-r and/or no mprotein (group-2 5-year os 62% vs 27% in group-6; hr-0.15;95%ci:0.06-0.34;p< 0.001;figure1b). overall cumulative incidence of relapse (cir) was 28%(95%ci:23-37) and non-relapse-mortality(nrm) of 24%(95%ci:18.6-30) at 5 years. multivariate analysis(mva) revealed absence of m-protein,high evl (>150k copies/ml) or no ebv-r and absence of any gvhd as significant factors for high cir. similarly, high evl or no ebv-r, absence of m-protein and itu admission were significant predictors of high nrm. conclusions: this study adds to our understanding of role of ebv viraemia & associated mg in tcd-hscts while highlighting its significant impact on risk of ptld, os, nrm & cir. low ebv burden and development of mg is protective with significantly better survival outcomes and we recommend pre-emptive approach of using rituximab for ebv-r /ptld is best employed at higher ebv burden (e.g. >150k copies/ml dna) in high risk patients and be prospectively evaluated in future studies. clinical trial registry: n/a disclosure: nothing to declare p394 impact of early candidemia on the long-term outcome of allogeneic hematopoietic stem cell transplant in non leukemic patients: an outcome analysis on behalf of idwp background: to assess the incidence of, and risk factors for, candida infection in the first 100 days post-allogeneic hematopoietic stem cell transplantation (hsct) and the impact on long-term survival. methods: outcome analysis of 50,188 patients, 61% male, median age 46 years (range 0-80), with diagnosis of hemoglobinopathies in 3176 (6.3%), bone marrow failure in 7626 (15.2%), lymphoma in 17743 (35.4%) and myelodysplastic/myeloproliferative disesases in 21643 (43.1%) patients who underwent hsct from 2000 to 2015: 420 with candidemia by day + 100, and 49,768 without candidemia. the incidence of 100-day candidemia was estimated by using the cumulative incidence method. the univariate and multivariate risk factor analysis for 100-day candidemia was performed with the cause-specific cox regression model. the occurrence of candidemia was analyzed as a timedependent covariate. the overall survival and non-relapse mortality after day +100 were assessed in a land-mark setting, this analysis was restricted to patients surviving to day +100 post transplant. [[p393 image] 1. figure 1a -1b] results: the incidence of candidemia by day +100 was 0.85% (95% c.i. 0.77-0.93) (420/50,188) and occurred at a median of 17 days post-hsct (range -7-100). considering the candidemia within 100-day from hsct as a time dependent covariate, a higher 100-day non-relapsemortality (nrm) (hr 3.47 (2.75-4.38), p < 0.0001), and a lower 100-day overall-survival (os) (hr 3.21, 95% ci 2.67-3.85), p< 0.0001) were obtained from the cox model for patients with candidemia. factors significantly associated with candidemia occurrence in the multivariate analysis were: gender female, increased age at hsct, bone marrow failure, lymphoma or myelodysplastic/myeloproliferative diagnosis, bone marrow or cord blood stem cell source, t-cell depletion, less recent year of hsct. among patients alive at day + 100, the 5-year nrm and os with and without candidemia were 28.2% vs. 18.8%, p < 0.0001, and 50.5% vs. 60.7%, p< 0.0001, respectively, after a median follow-up of 4.3 years (95% ci 4.3-4.4) (figure 1 ). in multivariate analysis, the occurrence of a candidemia episode within day + 100 was an independent risk factor for higher nrm, hr 1.52 (1.18-1.97), p=0.0013, and lower os, hr 1.30 (1.08-1.57), p=0.0061. conclusions: despite the general improvements in prophylaxis and treatment, the occurrence of early post-hsct candidemia had a negative impact on transplant outcome as showed previously in leukemic patients. abstract already published. carbapenem-resistant enterobacteriaceae colonizationimportance in the risk of cre bacteremia and mortality in stem cell transplant (hsct) and acute leukemia patients marcia garnica 1,2 , marco a f bellizze 1 , priscila g a de jesus 1 , rafaela r c gomes 1 , filipe m akamine 1 , alan j marçal 1 , luzinete co rangel 2 , andreia assis 2 , marcia rejane valentim 2 , angelo maiolino 1 background: spread of infections due to carbapenemresistant enterobacteriaceae (cre) is a worldwide phenomenon and has been associated with high mortality and clinical complications. gut translocation is the most important portal of entry of bacteria during neutropenia, and cre gut colonization is a possible risk factor for bacteremia during neutropenia. goals: in the present study, we describe the frequencies of cre colonization and analyzed its relationship with development of cre bacteremia and mortality in two different scenarios: stem cell transplant patients (hsct) and leukemia patients. methods: prospective cohorts of hsct (from 2012 to 2017) and leukemia patients (from 2016 to 2018). hsct patients were analyzed from conditioning until discharge (pre-engraphment phase) and leukemia patients from first induction chemotherapy until last intensification. if an hsct was performed in leukemia patient the patient was censored in leukemia cohort and included in hsct cohort. all patients had rectal swabs performed weekly during hospitalization for the identification of cre colonization. patients with at least one positive swab (cre colonization group) were compared to patients with no documentation of colonization (controls). the outcomes analyzed were bacteremia due to cre, and overall mortality. results: there were 493 hsct performed during the study (408 [83%] autologous and 85 [17%] allogeneic). multiple myeloma and non-hodgkin lymphoma were the most frequent baseline diseases (n=251; 51%, and n = 88; 18%), respectively. cre colonization was documented in 10% (n=50), and it was more frequent among allogeneic hsct and leukemia patients (p< 0.001 for both). cre colonized patients had longer hospitalization (25 vs. 20 days, p< 0,001), higher frequency of cre bacteremia (6% vs. 0.2%; p=0.004), and mortality (16% vs. 2.4%, p< 0,001) compared to non-colonized hsct. negative and positive predicted values for cre bacteremia were 6% and 99%, respectively. thirty-one patients were analyzed in leukemia cohort, accounting to 92 hospitalizations (median 3 hospitalizations per patient, ranging from 1 to 6). the median age was 58 years, and 90% aml vs. 10% all. cre colonization was documented in eight (26%), with a median time from leukemia diagnosis and colonization of 93 days (9 -503 days). cre bacteremia was documented only in colonized patients (25% vs. zero; p=0,046). all eight colonized patients were submitted to other cycles of chemotherapy after colonization, in one of them cre bacteremia relapsed. conclusions: a routine surveillance of cre colonization showed colonization frequencies from 10% to 25% in hsct and leukemia patients respectively and was effective to stratify cre bacteremia risk as the predictive negative value was over 95%. colonization had association with cre bacteremia and overall mortality. efforts to minimize risks for colonization and mortality are necessary. the information of surveillance can be a tool to improve adequacy in empirical febrile neutropenia therapy in hsct and leukemia patients. disclosure: nothing to declare background: the incidence of hepatitis b virus infection is high to 6.2% in asian population, so there is more and more attention to the risk of hepatitis b virus(hbv) reactivation in the hepatitis b core antibody positive patients during chemotherapy, anti-cd20 monoclonal antibody, hsct, or other intense immunosuppressive drug therapy (isdt). hepatitis b core antibody is associated with a significant risk of hbv reactivation in patients undergoing hsct. however, there are remain uncertain that the effect of anti-hbsag antibodies in hepatitis b virus reactivation among the hepatitis b core antibody positive patients undergo hsct. we aim to investigate the role of the anti-hbs and the necessity of anti virus in hepatitis b surface antigen(hbsag) negative, hepatitis b core antibody positive patients during hsct. methods: we enrolled 791 hematological malignant patients received hsct in our center from 2008 to 2016. we classified 665 hbsag negative and undetectable hbv dna patients into 4 groups as anti-hbc(-)anti-hbs(-) (n=189), anti-hbc(-)anti-hbs(+) (n=176), anti-hbc(+) anti-hbs(-) (n=49), and anti-hbc(+)anti-hbs(+) (n=251). results: hbv reactivation was identified in 16 patients (2.4%) after hsct. there was a significant difference in hbv reactivation rate in anti-hbc(+)anti-hbs(-) (12.2%) vs anti-hbc(+)anti-hbs(+) (2.8%) (p=0.01) and anti-hbc (+)anti-hbs(-) (12.2%) vs anti-hbc(-)anti-hbs(-) (0.0%) (p=0.000), anti-hbc(+)anti-hbs(-) (12.2%) vs anti-hbc(-) anti-hbs(+) (1.7%) (p=0.004), but not among anti-hbc(+) anti-hbs(+) (2.8%) and anti-hbc(-)anti-hbs(-) (0%) and anti-hbc(-)anti-hbs(+) (1.7%). whereas there were no difference according to the donor viral profile(p=0.774). the median time of hbv reactivation in hbsag negative patients accepted hsct was 645 (455-1957) days after hsct. all of the patients with hbv reactivation have been controlled with nucleos(t)ide analogues drugs, and 5 of them achieved reverse seroconversion which detect persistent anti-hbsag antibodies in their bodies. conclusions: the anti-hbsag antibodies negative and anti-hbc positive patients have the highest risk of hbv reactivation after hsct in resolved hbv patients. the anti-hbsag antibodies play a protective role in resolved hbv patients receiving hsct. we recommend not prophylactic anti hepatitis b virus in hbsag negativity and anti-hbsag antibodies positive patients following hematopoietic stem cell transplantation. disclosure: nothing to declare methods: 2230 allo-hscts performed between 1997 and 2016 for acquired bone marrow failure (70.6%) or hemoglobinopathies (29.4%), with bm±cb (75.8%) or pb±cb+bm (24.2%) as a stem cell source were included in this retrospective registry megafile idwp ebmt study. results: demographics: the median age of recipient was 17.7 years (range: 0.1-78), and 50.8% were children. 79.0% recipients and 75.4% donors were ebv-seropositive. 67.8% had hsct from a matched family donor, 4.6% from a mismatched family donor, and 27.6% from an unrelated donor. t-cell depletion was performed in vivo in 82.2%, and ex vivo in 6.6% patients. conditioning regimen was myeloablative in 63.7%, ric in 36.3%. median follow-up was 4.7 years (95% c.i. 4.3-5.0). transplant out-comes: ebv-seropositive recipients in comparison to ebv-seronegative recipients had lower os (85.4% vs 88.4%, p=0.035), and higher nrm (10.0% vs 6.4%, p=0.018). no other significant differences were found for: ri, rfs, and acute or chronic gvhd with respect to ebv pretransplant serostatus donor and/or recipient. multi-variate analysis: ebv serostatus as a risk factor did not reach significance, while a trend towards higher risk of development of cgvhd (hr=1.31; 95%ci 0.97-1.78; p=0.081) and better survival (hr=0.78; 95%ci 0.59-1.04; p=0.087) in allo-hsct from ebv-seropositive donors. allo-hsct in ebv-seropositive recipients had a trend towards lower risk of development of cgvhd (hr=0.75; 95%ci 0.56-1.02; p=0.066). when 4 subgroups (r-/d-, r-/ d+, r+/d-, r+/d+ ebv serology) were analyzed, the ebv serostatus had no significant impact on os, rfs, ri, trm and development of acute or chronic gvhd. conclusions: allo-hsct from ebv-seropositive vs ebv-seronegative donors are at 31% higher risk of chronic gvhd in patients with non-malignant hematological disorders undergoing allo-hsct, however this difference is non-significant in multivariate analysis. disclosure: nothing to declare. results: twenty-eight (30%) pts (19 male, 9 female) were tested positive (group 1) for subtype a (n=4, 14%), b (n=18, 64%) or a (h1n1) (n=6, 21%) while 67 (70%) pts (32 male, 35 female) were negative (group 2). vaccination rate in group 1 (32%) was significantly lower compared to group 2 (51%, p=0.002). the median time after transplantation (790 vs 565 days), t-cell counts (349 vs 296/ μl), bcell counts (162 vs 135/ μl), igg-level (8,3 vs 7,6 g/ l), proportion of immunosuppressed pts (75% vs 63%), male/ female ratio was not significantly different between groups 1 and 2. within group 1 influenza subtypes were similarly distributed in vaccinated and not vaccinated pts (a 11% vs 16%, b 67% vs 63%, a (h1n1) 22% vs 21%). pts. with subtype b infection had higher levels of t-(482 vs 274/ μl) and b-cells (232 vs 108/ μl) and a longer follow up from sct (1610 vs 470 days) compared to subtype a / a (h1n1) infection but differences were not significant. conclusions: influenza could be proven in one third of all tested pts. dominance of b and a (h1n1) pdm0e9 subtype occurrence corresponded to the flu epidemic dissemination in the german population. the most important protective factor for outpatient sct recipients was influenza vaccination. disclosure: nothing to declare background: cmv infection is one of the most frequent complications after haplo. some risk factors are well known but the best strategy (prophylactic or preemptive treatment) to mitigate this complication is not still well defined. the primary endpoint in our study is to describe incidence and risk factors to develop cmv infection or disease in haplo. as secondary objective we analyzed efficacytoxicity of treatment and cmv related mortality. methods: we analyzed 60 patients who underwent haplo in our center between may 2012 and may 2018. all of them received ptcy (d+3 and d+4), tacrolimus and mycophenolate as graft versus host disease prophylaxis. a preemptive therapy based on viral load was applied. treatment was started when >1000 ui/ml of cmv were detected in one determination or >500 ui/ml in two consecutive determinations. cmv analyses were made in plasma using cobas pcr technique® and positive viral load cut-off point was 137 ui/ml. the cmv viremia was determined weekly until d+100 and then every two weeks until immune reconstitution. results: the cmv infection and disease incidence at d +100 was 61.3% (38 episodes) and 19.4% (12 episodes), respectively. cmv disease was digestive (n=8), pulmonar (n=2), neurologic (n=1) and disseminated (n=1). the median time to first cmv infection was 39.5 days (20-151). thirty-six patients had at least one episode of cmv infection: 24 of them (66.7%) had one episode, 7 (19.4%) had two episodes and 5 (13.9%) had 3 or more episodes, respectively. only pre-transplantation cmv status was significantly associated with cmv infection (p< 0.001). risk factors are shown in image 1. the median viral load in first cmv infection and disease was 8314 ui/ml (542-51158) and 24842 ui/ml (271-126279), respectively (p=0.02).the median counts of cd4 lymphocytes at d+100 in cmv infection and disease were 262/mm3 and 120/mm3, respectively (p=0.09). preemptive therapy for the first 2 episodes of cmv infections (n=48) was valganciclovir (58,3%), ganciclovir (35.4%) or foscarnet (6.3%), reaching a complete viral load clearance in 77%, with a median time to response of 19.2 days (6-34) and a median treatment duration of 21 days (2-39). grade iii-iv toxicity (mainly hematologic) was observed in 55.6% (n=28), 30% (n=17) and 16.7% (n=3), respectively. three patients had an ul54 mutation, one of them with clinical and microbiological resistance to the 3 mentioned drugs. three patients (5%) had a graft failure secondary to cmv infections. five patients (8.3%) died as consequence of cmv infection: 3 before d+100 secondary to cmv disease (2 pulmonar, 1 disseminated) and 2 after d+365 due to graft failure and infectious complications. with a median follow up of 12.5 months, overall survival at 18 months for patients who had cmv infection was 58.9% compared to 66.5% for those who had no infection (p=0.08). conclusions: a high incidence of cmv infection in haplo with ptcy was shown in our series and it contributed to mortality in 8.3% of patients. only cmv status (d-/r+ and d+/r+) was significantly associated with higher risk of infection. identification of high risk patients and new prophylactic and treatment strategies may improve these results. disclosure: nothing to declare. methods: consecutive patients admitted at the sct unit between january-18 to november-18 were reviewed. only first admission was analysed. screening consisted of rectal and perineal swap on admission and weekly until discharge. in case of detection of mdro, patients were isolated and infection control strategies were applied. results: 67 patients were analysed, median age 53 years (18-70). 65% were male (n=44). median duration of hospitalization was 30 days(16-133). 293 swabs were performed, with a median of 4 swaps/patient (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) . patient characteristics are shown in table 1 . 60 patients (90%) spiked fever in a median of 9 days after admission . 13% (n=9) had previous documented mdro colonization. median neutrophil engraftment was 15 days (95%ci 14-15), in 24% patients (n=16) of patients had a positive screen: in 8 (50%) patients at baseline and in 8 (50%) patients were detected for the first time beyond baseline screen. cumulative incidence of colonization at 7 days was 10.4% (95%ci 3.2-17.6), at 15 days 18% (95%ci 8.2-27.8), and at 30 days 19.5% (9.9-29.1%) (figure 1 ). mdro identified were: 8 with extended-spectrum beta-lactamases producing e. coli (esbl-ec), 4 multidrug-resistant pseudomonas aeruginosa (mr-ps), 3 vancomycin resistant enterococci (vre) and 1 patient with carbapenemaseproducing (cp) citrobacter freundii. 7/16 colonized patients developed mdro infection (44%): 4 patients mr-ps, site of infection was 2 urinary tract infection (uti), 1 urethritis, 1 genital ulcer. two patients were treated with ceftolozane/ tazobactam, 1 with meropenem+amikacin;2 patients esbl-ec both uti treated with meropenem; 1 patient cpcitrobacter freundii uti treated with ceftazidime/avibactam. in 75% patients (12/16) antibiotic treatment at febrile episode was guided by positive screening. no mdro related icu admission or mortality was observed. in 76% patients (n=51) background: hepatitis e virus (hev) can cause chronic infection and liver cirrhosis in immunocompromised individuals. there is limited data on hev infections in patients undergoing allogeneic hematopoietic stem cell transplantation (hsct). the aim of this study was to investigate the frequency and clinical importance of hev in a swedish cohort of hsct recipients. methods: we analyzed serum samples from 262 hsct patients (241 adults and 21 children), collected 6 months after hsct. hev igg and igm were detected by elisa (dia.pro®), hev rna by reverse transcriptase pcr, and quantification of hev rna was performed by digital pcr. in all patients, who were positive for hev-rna and/or serology at 6 months, also samples collected at the time of hsct from both the patients and their donors were analyzed. in the hev rna positive patients, additional samples were analyzed to determine the duration of viremia. three hev rna negative controls were selected for each case of hev infection, matched for age, diagnosis, conditioning regimen and donor type. results: hev rna was detected in 8/262 (3.1%) patients. in three of the patients hev rna was positive during a period of 3-8 months, and two of these patients were infected already at the time of hsct. in five patients hev-rna was positive, at a low level, only at 6 months. 11/262 (4.2%) patients had detectable hev igg and/or igm, whereof eight patients were hev rna negative. in 4/8 (50%) patients with hev infection (hev rna positive) alanine aminotransferase (alt) was > 3 upper limit of normal (uln), in 1/8 (12.5%) patients > 1.5 uln, and in 3/8 (37.5%) patients alt was normal, at 6 months after hsct. bilirubin was elevated > 1.5 uln in 1/8 (12.5%) patients, and > 3 uln in no patient at 6 months after hsct. two patients died with ongoing signs of hepatitis and hev rna detected in blood. one of them developed acute liver failure, at the time interpreted as drug toxicity, and died of multi-organ failure. the other patient died of unrelated causes. the remaining six patients had cleared the infection at 7-24 (median 8.5) months after hsct. active gvhd was present at 6 months after hsct in 5/8 (62.5%) patients with hev infection, involving the liver in 3 of these patients. corticosteroid treatment was ongoing in 6/8 (75%) patients; the mean dose during the 14 preceding days was > 0.5 mg/kg in 1/8 (12.5%) patient, 0.25-0.5 mg/ kg in 3/8 (37.5%) patients, and < 0.25 mg/kg in 4/8 (50%) patients. hev infection correlated to elevated alt > 1.5 uln, or 8.3 p=0 .02) and > 3 uln, p=0 .02) at 6 months, but not at 3 months, after hsct, compared to hev rna negative controls. conclusions: hev infection was detected in 3.1% of patients tested at 6 months after hsct and was correlated to abnormal alt. spontaneous clearance was common but one patient died in acute liver failure, where hev may have contributed. hev infection is a differential diagnosis in patients with elevated alt 6 months after hsct. disclosure: nothing to declare monitoring of t-cell responses to viral-coded antigens in pediatric patients receiving tcrαβ-depleted haplo-hsct followed by bpx-501 cell administration background: αβ t-cell-depleted haplo-hsct is an effective option for children with hematological disorders in need of an allograft. however, recovery of adaptive immunity is impaired in these patients. thus, in order to accelerate immune reconstitution, we developed a novel approach based on post-transplant infusion of a titrated number of donor t cells, transduced with the suicide gene inducible-caspase-9, ic9 (bpx-501 cells, sponsor bellicum pharmaceuticals®; nct02065869). we previously reported on immune recovery of 108 children transplanted at our institution, showing that bpx-501 cells infused after αβ t-cell-depleted haplo-hsct expand in-vivo and persist over time, contributing to fasten adaptive immunity recovery (merli, ash2017). here, we report the results of lymphoproliferation assay to viral-encoded antigens to assess tcell function in patients transplanted with this approach. methods: we evaluated 142 children, 78 male and 64 female. median age at transplant was 5.7 years (range 0. [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . patients had either malignant (69 children) and nonmalignant (73) disorders. no patient was given any posttransplant graft-versus-host disease prophylaxis. nine children were enrolled in the phase i portion of the trial consisting of 3 cohorts receiving escalating doses of bpx-501 cells. the remaining 133 patients (phase ii portion) received the recommended dose of 1x10 6 bpx-501 cells/kg identified in phase i. bpx-501 cells were infused at a median of 17 days post-hsct (range 10-82). antigendriven activation of peripheral mononuclear cells was evaluated by lymphoproliferation assay with 3 h-thymidine pulsing at d+4 and harvesting at d+5. stimuli included pha or cmv, ebv and adv whole viral lysate. results are given as stimulation indexes (si, cpm stimulated sample/cpm unstimulated control). thresholds for positive response were arbitrarily set at si>3 for viral-encoded antigens and at si>5 for mitogenic stimulation with pha. fractions of responders are indicated in the figure. results: patients were analyzed from d+30 to d+720 post-hsct. pha responders (a) increased to 80%, while cmv (b), ebv (c) and adv (d) responders were 61%, 52% and 61% at 2 years after haplo-hsct. responses to ebv and adv antigens were slightly delayed but improved over time. responses to pha and to cmv (e,f) were analyzed in the cmv-reactivating and cmv-non reactivating groups (cmv-yes/cmv-no). significant differences in pha response were observed at d+60 and d+270. moreover, increased cmv responses were observed in cmvreactivators at d+270, d+360 and d+720, with approximately 100% of responders at d+720, as opposed to cmvnon reactivators which comprised 50% responders. neither primary disease, age nor tbi during the conditioning regimen influenced proliferative capacity of the two subgroups (not shown). conclusions: we showed a rapid recovery over time of t-cell function after αβ t-cell-depleted haplo-hsct followed by bpx-501 cells administration. when patients were grouped according to cmv reactivation (previously demonstrated as a strong driver of immune reconstitution), a significant difference in the number of responders among the patients experiencing viral reactivation was observed using the cmv lysate only but not the immunodominant pp65 protein (not shown), suggesting that other viral antigens account for increased t-cell responses. results of t-cell function after bpx administration complements the phenotypic data we already reported. clinical trial registry: nct02065869 disclosure: nothing to declare. background: cytomegalovirus (cmv) is associated with significant morbidity and mortality in allogeneic hematopoietic cell transplantation (allo-hct) patients (pts). cumulative incidence of cmv infection in high-risk patients such as cd34-selected or haploidentical hct have been reported as high as 61.8-84.5% and 53-81%, respectively. letermovir (ltv) was approved in 11/2017 for prophylaxis (ppx) in cmv-seropositive recipients (r+) of allo-hct. since 12/2017, ltv ppx was implemented at our center for both primary and secondary ppx. we report our real-world experience. methods: adult cmv r+ allo-hct pts who initiated ltv as primary and/or secondary prophylaxis were identified between 1/1/2018 and 6/30/18. cord blood transplants were excluded. the primary outcome was the incidence of clinically significant cmv infection (cmv viremia requiring preemptive treatment or cmv disease). pts were followed through 9/2018. results: 53 pts initiated ltv. 69.8% pts were at high risk for cmv reactivation and disease (primarily ex vivo t-cell depleted hct [n = 18; 34%] or haploidentical t-replete hct [n = 12; 22.6%]). the most common indication for hct was acute myeloid leukemia (n = 16; 30.1%) and the majority of patients received myeloablative conditioning (n = 34; 64.2%). 39 pts (73.5%) received ltv as primary ppx after hct, with a median day of ltv initiation of d+7 (range d+7─d+40). at ltv initiation, 34 pts had an undetectable cmv dna, and 4 had cmv < 137 iu/ml. clinically significant cmv infection requiring preemptive treatment occurred in 2 of 39 pts (5.1%). one patient was treated with valganciclovir (vgv) for persistent cmv < 137 iu/ml and received ltv as secondary ppx. a 2nd patient developed persistently detectable cmv (< 137 iu/ ml) and breakthrough cmv viremia with a mutation in ul56 at site c325yltv successfully treated with vgv. the median duration of primary ltv ppx was 116 days (54-221), with primary ppx continuing beyond 14 weeks post hct in 29 pts. the median additional follow-up in patients who discontinued ltv was 40 days (0-154), without clinically significant cmv infection to date. an additional 14pts (15 pts overall; 28.3%) received ltv as secondary ppx after cmv pre-emptive therapy. the median duration of secondary ltv ppx was 127 days (18-270), with no reactivation. ltv was not discontinued due to toxicity or intolerance in any patient. cmv outcomes are summarized in figure 1 . all-cause mortality for the 53 pts over the observational period was 11.3%. conclusions: primary ltv ppx significantly reduced cmv reactivation, and high-risk patients may benefit from extended prophylaxis. in patients who received preemptive therapy for cmv, use of secondary ppx showed no recurrent cmv reactivation. ltv is well tolerated. additional studies are needed to determine optimal ppx duration and to clarify role of secondary cmv ppx in high-risk allo-hct. the future standard of care will likely include extended primary ppx and secondary ppx and result in decreased morbidity and mortality associated with cmv. disclosure: andrew lin -nothing to declare, molly a. maloy -nothing to declare, valkal bhatt -nothing to declare, lauren derespiris -nothing to declare, meagan griffin -nothing to declare carmen lau -nothing to declare, anthony j. proli -nothing to declare, juliet barker -angiocrine bioscience , letermovir primary prophylaxis (pp) has been shown to reduce clinically significant cmv infection with a favorable safety profile. letermovir pp will improve the outcome of seropositive patients. however, patients who did not benefit from pp and experienced > 1 cmv episode (infection or disease) after hct may be candidate to secondary prophylaxis (sp). indeed half of them will have >1 recurrent episode after pre-emptive treatment (pet). letermovir is available since november 2017 as part of the french early access program for pp and sp. we report the outcome of patients who benefited from letermovir sp in the context of this program. methods: letermovir is granted, in a restrictive manner, by the french drug agency (ansm) on a case-by-case basis for prophylaxis of cmv episode, in cmv-seropositive adult allogeneic hct recipients. sp patients should have a negative baseline cmv pcr, have already experienced >1 cmv episode, in the context of a potentially harmful pet according to physicians. planned letermovir daily dose was 240 mg in case of concomitant cyclosporine and 480 mg otherwise. all patients were routinely screened by blood or plasma cmv pcr. results: between november 2017-july 2018, 57 patients received letermovir in the early access program, 22 for pp, and 35 for sp. among the 35 sp patients, 6 had previous cmv disease (gut: 5; cns: 1). mean age was 55±13 years, m/f ratio was 22/13. the sp cohort included one cord blood and 10 haplo-identical hct. main diagnoses were acute leukemia (46%) and myelodysplastic syndrome (26%). the conditioning regimen was myeloablative in 47% and included atg in 61%. based on available data (6 missing data, md), previous gvhd was present in 22 (76%) patients, and active at letermovir initiation in 17 (59%). thirty two (91%) patients were planned to receive immunosuppressants. donor's cmv serology was negative in 12/15 (80%) (20 md). at baseline, cmv pcr was detectable in 1/35 patients. letermovir was initiated a median of 195 days (iqr: 154-308) after transplant for a mean duration of 112 ± 47 days. only one (3%) patient developed cmv breakthrough. the median follow-up from letermovir initiation was 103 days. among the 57 patients exposed to letermovir prophylaxis, two patients permanently discontinued because of letermovir-related adverse events (acute gvhd and nephropathy for one, loss of appetite, pruritus, diarrhoea and weight loss for the other); two deaths occurred with no causal relationship to letermovir. data were consistent with the known safety profile of letermovir. conclusions: letermovir is or will be soonly available in most european countries for cmv prophylaxis in hct recipients. pending its routine use, letermovir used as sp was well tolerated and effective, with only 1/35 patients developing a breakthrough infection. in this high-risk population for cmv recurrence, letermovir may provide a safe bridge between pet and specific immune reconstitution, pending tapering or discontinuation of immunosuppressants. whether sp may improve survival deserves further studies. disclosure: thierry allavoine is a former employee of msd france, nathalie benard and amir guidoum are employees of msd france, marion masure is an employee of icta pm, sophie alain and catherine cordonnier have participated in advisory boards and have been members of the speaker bureau of msd. ibrahim yacoub-agha has received honoraria from msd, other authors: nothing to declare p407 real-world data on letermovir prophylaxis for cytomegalovirus reactivation after allogeneic hematopoietic cell transplantation: a single center experience patrick derigs 1 , maria-luisa schubert 1 , paul schnitzler 1 , carsten müller-tidow 1 , peter dreger 1 , michael schmitt 1 1 heidelberg university hospital, heidelberg, germany, background: reactivation of cytomegalovirus (cmv) still contributes substantially to morbidity and mortality after allogeneic hematopoietic cell transplantation (allohct). recently, letermovir became available as the first drug approved in europe for prophylaxis of cmv reactivation in seropositive patients who have undergone allohct. letermovir is neither myelo-nor nephrotoxic, and significantly reduced the incidence of cmv reactivation in a pivotal phase iii trial (nejm 2017; 377:2433) . therefore we adopted letermovir prophylaxis according to the label as standard policy in our institution: in seropositive recipients letermovir was initiated after engraftment and continued until day +100 or cmv reactivation. the aim of the present study was to investigate if the favorable trial results could be reproduced under real-world conditions. methods: the study cohort consisted of the first seropositive 35 patients who received letermovir prophylaxis at our institution (between march and august 2018). these were compared with a control cohort transplanted between august 2017 and march 2018 before the advent of letermovir. study and control cohorts were matched for cmv donor/recipient sero-status, underlying disease and donor type source of stem cells and application of atg. cmv viremia was monitored by a quantitative pcr twice a week during the inpatient period and weekly thereafter. patients reactivating cmv prior to engraftment were not considered as event in both groups. results: no major side effects of letermovir intake were observed. with altogether 5 reactivation events, the cumulative rate of cmv reactivation on day +100 was 14% (95%ci 1-45%) in the letermovir cohort and thus significantly lower than in the control group (20 events, 58% (95%ci 42-71%); hr 0.23 (0.10-0.51); p=0.0003). the median time to reactivation was 53 days for the control group and not reached for the letermovir group. the cumulative number of days on valganciclovir before d +100 was 151d for the 35 letermovir patients vs 689d for the 35 control patients. there were no hospitalizations for foscavir administration in the letermovir group compared to 5 hospitalizations in the control group. there were 2 deaths before d +100 in the letermovir group (one pd, one nrm) and 3 deaths in the control group (all pd). conclusions: this observational study confirms the safety and efficacy of letermovir for the prophylaxis of cmv reactivation in seropositive patients after allohct in a real-world setting. our results are in good concordance with the phase iii trial. although letermovir appeared to reduce the need for therapeutic valganciclovir and foscavir tremendously, larger samples with longer follow-up are needed to assess the impact of letermovir prophylaxis on non-relapse and overall mortality as well as on resource consumption. background: cmv viremia occurs in 40%-80% of cmv r + hct recipients. pet use has reduced the risk of cmv end-organ disease (eod) and associated mortality; however, pet use may lead to substantial antiviral use and healthcare resource utilization. limited real-world data are available on the outcomes of pet. therefore, we aimed to examine cmv outcomes (eod, resistance), cmv-related mortality by day (d)180 and healthcare resource utilization between pet and no-pet groups among cmv r+ recipients undergoing first hct. methods: we conducted a retrospective cohort study of adults, cmv r+ recipients of first peripheral blood or marrow allograft at mskcc identified from march 2013 through december 2017. data was extracted from electronic medical records and hct databases. cmv+ recipients were monitored weekly by quantitative pcr assay starting on d14 through d180 post hct. use of antiviral therapy for cmv viremia defined pet. high cmv risk (hr) comprised t-cell depleted (tcd) hct by cd34+-selection regardless of donor hla match or conventional hct from mismatched or haploidentical donors; low risk (lr) included conventional hct from matched related donors. cmv eod was scored by standard criteria. cmv resistance mutations were confirmed by sequencing (viracor-eurofins). length of stay (los) for hct admissions and readmissions were identified through d180. stratified analyses were performed to examine outcomes by pet use and cmv risk. background: in a phase iii randomized, double-blind, placebo-controlled study of cmv-seropositive post-hsct recipients, letermovir prophylaxis significantly reduced the incidence of clinically significant cmv infection through week 24. the objective of this research was to assess the impact of cmv prophylaxis on rates of rehospitalization in adult cmv seropositive allogeneic hsct recipients from the letermovir phase 3 clinical trial. methods: rehospitalization was recorded as an exploratory endpoint in the clinical trial at end of treatment (week14), time of primary endpoint (week24) and through an extended follow-up period (week48). cmv-related rehospitalization was assessed in the trial. prespecified analyses describe the observed rates of rehospitalization for the letermovir and placebo groups at the specified times. fine-gray cumulative incidence function(cif) regression models were used to explore the rate of all-cause, and cmv-related rehospitalization accounting for the competing risk of mortality. a multiple linear regression model was used to describe the cumulative length of stay (los) for all-cause rehospitalizations that occurred through week48 (excluding time of initial transplant stay). results: observed rates of all-cause rehospitalization were lower for the letermovir group compared to placebo at end of treatment (36.6%vs.47 conclusions: letermovir was shown to significantly reduce the rate of clinically significant cmv infection in a placebo-controlled randomized clinical trial. these analyses suggest that there is also a reduction in the rate and cumulative days of rehospitalization. this trial was not sufficiently powered to detect differences in this exploratory endpoint. nonetheless, these data provide valuable insights into the economic burden of cmv. real world data and findings from future clinical trials are needed to better understand the nature of the association between cmv and rehospitalizations. clinical methods: all consecutive patients with hematologic disorders who received hsct at our center between january 2013 and august 2018 were included. among the 278 evaluable patients, 172 received levofloxacin as antibacterial prophylaxis (group a) while 106 did not receive any fq prophylaxis (group b). baseline characteristics were similar in the two groups, except for the number of patients with advanced disease (34% in group a and 47% in group b, p 0,042). median duration of neutropenia was 16 days (range 9-44) in group a and 14 days (range 4-31) in group b. a positive rectal swab for carbapenem-resistant enterobacteriaceae (cre) was detected in 3 patients in group a and 8 patients in group b. results: overall, bsi was detected in 58 patients (20,9%), 29 (16,9%) in group a and 29 (27,4%) in group b (p=0,048). the median onset of bsi was 8 days post transplant (range 0-28), without significant differences between the two groups. in univariate analysis, fq prophylaxis (or 0,54; 95% ic 0,30-0,97) and bone marrow stem cell source (or 2,08; 95% ic 1,05-4,12) were significant factors associated with the risk of bsi. gramnegative bacteria accounted for 44,8% (n=13) of bsi in group a and 65,5% (n= 19) in group b, and gram-positive bacteria for 48,3% (n= 14) of bsi in group a versus 27,5% (n= 8) in group b, without statistically significant differences (p = 0,16). polymicrobic bsi were 6,9% (n=2) in group a and 6,9% (n=2) in group b. mdrgram negative bsi were detected in 4 patients (14%) in group a and in 6 patients (20,7%) in group b (overall, 2 cre, 7 esbl producing enterobacteriaceae and 1 mdr-pseudomonas). death attributable to bsi occurred in 6 of 58 patients (10,3%); 5 of these patients did not receive fq prophylaxis, but 2 of them had both a pre transplant kpc colonization and active disease at transplant. neither antibacterial prophylaxis (p = 0,98) nor bsi (p = 0,4) had a significant impact on overall survival (os). conclusions: the preliminary data of our study show that fq prophylaxis is associated with a reduced incidence of bsi, in particular gram-negative infections, with no impact on os. the limitations of our study may be the different group sizes and the retrospective nauture of the study. whether antibacterial prophylaxis should be avoided in the pre-engraftment period in still a matter of debate and needs to be evaluated in larger prospective studies. disclosure: nothing to disclose. gillen oarbeascoa 1 , nieves dorado 1,2 , laura solan 1,2 , rebeca bailen 1,2 , pascual balsalobre 1,2 , carolina martinez-laperche 1,2 , ismael buño 1,2 , javier anguita 1,2 , jose luis diez-martin 1,2,3 , mi kwon 1,2 1 hospital general universitario gregorio marañón, hematology, madrid, spain, 2 instituto de investigación sanitaria gregorio marañón, madrid, spain, 3 universidad complutense de madrid, madrid, spain background: incidence and outcome of invasive fungal infection (ifi) are not well characterized in the setting of peripheral blood, non-manipulated haploidentical stem cell transplantation with postransplant cyclophosphamide (haplosct). the aim of the study was to analyze incidence and risk factors of ifi in patients who underwent haplosct at our institution. methods: 132 consecutive patients who underwent peripheral blood haplosct with postransplant cyclophosphamide between 2011 and 2017 at our centre were reviewed. ifi was classified according to eortc definitions. proven and probable ifi were included. results: patients´characteristics are shown in table 1 . primary antifungal prophylaxis was performed with micafungin from day -1 until oral intake, followed by posaconazole until day +35. patients on steroid treatment for gvhd received prophylaxis with micafungin or posaconazole. 92% of patients obtained neutrophil engraftment. twenty-two episodes of ifi were observed in 20 patients: 10 proven and 12 probable, with a cumulative incidence of ifi of 17% at 500 days. most commonly isolated organism was aspergillus spp (n=5), followed by candida spp (n=4: 1 c. kruseii and 3 c. parapsilosis), and fusarium spp (n=2). isolated cases of inonotus spp, mucor spp and trichosporon ashii were observed. pulmonary involvement was the most frequent clinical presentation (n=10), followed by fungemia (n=5: 4 candidemia, 1 trichosporon ashii) and skin-pulmonary involvement (n=2). among patients with lung involvement, 10 showed probable ifi: 5 with elevated serum galactomannan and 3 positive galactomannan in bronchoalveolar lavage (bal). there were 2 patients without galactomannan, one with a positive bal culture for penicillum spp and the other with an aspergillus spp. median time to ifi diagnosis was 21 days. thirteen cases were diagnosed in the pre-engraftment period, 4 after engraftment and 5 cases after day +30. among patients with late ifi, median time to development was 220 days. all of them were associated with gvhd (3 grade iii-iv acute gvhd and 2 moderate/severe chronic gvhd). ifi outcome was favorable in 13 out of the 22 ifi. treatment was liposomal amphotericin b in 7 cases, voriconazole in 4 and combined treatment (amphotericin b and azole) in 6. there were 7 ifi related deaths, with a cumulative incidence of ifi related death of 6.4%. prior transplant (or 4.5, p< 0.01), particularly allohsct was associated to ifi development (or 8.2, p< 0.01). patients with previous allohsct presented ifi mainly from molds: 3 aspergillus, 2 fusarium, inonotus, trichosporon and mucor. there were also 2 candidemia episodes. no other factors were significantly associated to ifi occurrence. conclusions: in our experience, cumulative incidence of ifi in the setting of haplosct with posttransplant cyclophosphamide was similar than observed in previous studies in allosct. having received a previous sct, especially allosct, was the most significant factor related to ifi development. this high risk population should be closely monitored and could benefit from prophylaxis with azoles. disclosure: nothing to declare. methods: rsv infection was diagnosed in nasal wash (nw) or bronchoalveolar fluid (bal) by dfa (millipore, usa) or pcr (seeplex, seegene, kor). urti and lrti were defined according to ecil-4 guidelines. death from all causes was assessed within 90 days after rsv infection and was attributed to rsv if the patient had persistent or progressive rsv infection with respiratory failure at the time of death. neutropenia and lymphocytopenia were defined as an absolute neutrophil count (anc) < 500/ul and absolute lymphocyte count (alc) < 200/ul, respectively. results: median number of confirmed rsv infections per year was 12, ranging from 5 to 34. an outbreak of rsv was detected in 2017, possibly due to a lack of compliance with contact precautions in the unit. median patients' age was 26 years and time to rsv infection was day 80 (-11 to 1837). twenty-three patients (pts) had received an autologous transplantation (17.2%) and 111 were allogeneic hsct recipients (82,8%). median time to engraftment was 15 days, ranging from 10 to 27 days. at rsv diagnosis, 108 pts presented with urti (80.6%) and 26 with lrti (19.4) . surprisingly, around 18% of the auto hsct recipients had rsv pneumonia at diagnosis. variables significantly associated with lrti at diagnosis were mud hsct (no/ yes, or 0.42; ci95 0.20-0.89); anc < 500/ul (or 2.75; ci95 1.01-7.45); alc < 200/ul (or 3.25; ci95 1.12-9.45); and recent or pre-engraftment hsct (no/yes, or 0.38 ci9 0.14-0.98). among the 108 pts with urti at diagnosis, 19 progressed to lrti (17.6%). forty-four of the 134 pts died (32.8%) and mortality rate was significantly higher in pts with lrti in comparison with pts with urti (53.8% versus 27.8%, p=0.011). death was attributed to rsv in 11 of the 44 pts who died (25%). conclusions: autologous hsct recipients are also at risk of lrti caused by rsv. risk of rsv lrti is higher in mud hsct, infection acquired pre-engraftment or early after hsct, and low neutrophil and lymphocyte counts. continued education is necessary to sustain compliance to contact precautions in hsct units. disclosure background: measles is a life-threatening infection after allogeneic hct. due to the decreased coverage of vaccination in many countries, the disease reappears, increasing the risk of outbreaks worldwide. allogeneic hct recipients have been shown to be seropositive for measles in roughly 40-50% of the cases 3 years after transplant. however, these data were obtained before the 2000's from hct populations mainly conditioned with myeloablative (ma) regimens. our aim was to assess measles immunity before considering vaccination in a cohort of hct survivors including patients conditioned with reduced intensity (ric) or non-ma regimens. methods: allogeneic hct adult recipients who had not been vaccinated for measles since hct were routinely screened for measles immunity. measles igg titers were determined with a chemiluminescence immunoassay (liaison measles igg kit, liaison xl analyser, diasorin, italy). patients were considered to be seropositive if the igg titer was > 16.5 ua/ml. risk factors for seropositivity were analyzed. qualitative variables were described as numbers (%) and compared using the chi-2 test or fisher exact test as appropriate. quantitative variables were described as median or mean (range) and compared using the kruskall-wallis test. ors were estimated separately for factor yielding a p-value < 0.20 in the univariate analysis using logistic regression models. results: eighty-six patients, transplanted 1.5 to 38 years (mean: 13,5 years) ago, were included. the mean age was 53 years (range: 21-79), the sex ratio m/f: 0,5. the underlying diseases were acute leukemia: 49 (57%), myelodysplastic syndrome: 5 (6%), lymphoproliferative diseases: 17 (19.5%), myeloproliferative neoplasms: 11 (13%) and non-malignant diseases : 4 (4.5%). the hct was performed from an hla-identical donor in 52, an unrelated donor in 30, and a cord-blood in 4. conditioning regimen were ma in 48 (56%), ric in 20 (23%) and non-ma in 18 (21%) patients no patient had experienced measles or had received measles vaccination since transplant. fifty-seven of the 86 (66%) patients were seropositive for measles. measles seropositivity was not associated with conditioning regimen, patient age at transplant, patient age at time of assessment, donor age at transplant, lymphocyte count or gammaglobulin levels, or type of transplant (hlaid. vs others) measles vaccination before transplant or previous measles before transplant. the only parameters significantly associated to seropositivity were absence of previous gvhd (any type or severity, p=0,033 or 0,31 [0,10-0,94]), and absence of previous extensive chronic gvhd (or 0, 28 [0, 87] p0,027). conclusions: sixty-seven percent of allogeneic hct are seropositive for measles at a median of 7 years after hct before vaccination. the only risk factor strongly associated with seronegativity is extensive chronic gvhd. in patients background: cytomegalovirus (cmv) reactivation is a frequent complication after hematopoietic stem cell transplantation (hsct). extracellular vesicles (evs) have emerged as a promising new category of biological biomarkers in different scenarios, including inflammation, tissue damage, cancer and viral infections. we recently reported on the potential use of serum evs as biomarkers of agvhd (lia g. et al. leukemia (2018) 32, 765) . here, we investigated the potential correlation of cmv reactivation with plasma evs in post-transplant cyclophosphamide (ptcy) haploidentical-hsct (haplo-hsct). methods: plasma samples were collected after mononuclear cell separation at given time-points (pre-transplant, on day 0, 3, 7, 14, 21, 28, 35, 45, 60, 75 and 90 after haplo-hsct) and evs were extracted by a protamine-based precipitation method and their concentration and dimension were characterized by nano-tracking particle analysis (nanosight). after extraction, evs were analyzed by flowcytometry (guava easycyte flow cytometer) with a panel of 14 antibodies (cd44, cd138, cd146, krt18, cd120a, cd8, cd30, cd106, cd25, cd26, cd31, cd144, cd86, and cd140a). results: thirty-two patients with hematological malignancies underwent haplo-hsct between 2011 and 2015. cmv reactivation was observed in 20/32 (62,5%) and occurred at a median of 50 (range: 10-275) days after transplant. preliminary analysis (17/32 patients) showed that cd140a fluorescence (platelet-derived growth factor receptor-α or pdgfr-α), cd30 fluorescence (ki-1 antigen) and cd144 fluorescence (ve-cadherin) were associated with an increased risk of cmv reactivation (or 2.67 p=0.045; or 3.11 p=0.011; or 2.37 p=0.08), whereas cd31 (platelet endothelial cell adhesion molecule, pecam-1) concentration level was associated with a decreased risk of cmv reactivation (or 0.26, p=0.032). all these biomarkers showed a signal change before cmv reactivation (an increase with cd140a, cd30 and cd144, a reduction with cd31). (figure 1 ). conclusions: we observed a potential association of 4 evs membrane proteins with cmv reactivation: cd140a, cd30, cd144 and cd31. these proteins are crucial for endothelium and immune cells interaction. cmv can infect different cell types including endothelial cells (bentz gl. pnas 105 (14) 2008). moreover, cd140a (pdgfr-α) has been shown to function as an entry receptor for cmv expressing gh/gl/go complex (wu y. et al. plos pathog 13 (4) 2017). we plan to implement our analysis characterizing evs contents (mirnas) and will be applied to investigate other viral reactivations (e.g. epstein barr virus and human herpes virus 6). [[p414 image] 1. methods: to explore the value of cmv dna extracted from gi tissue for the diagnosis of cmv gastroenteritis, we retrospectively evaluated 71 patients, aged 17-67 (median 44.8 years) who received allo-hct from sibling(26), matched unrelated(40) or haploidentical donors(5), after receiving myeloablative (56) or reduced intensity conditioning(15). they all underwent endoscopy for gastrointestinal symptoms between 2012-2018. cmv dna from tissue samples and parallel blood samples were measured by q-pcr. positive cmv dna on the tissue was considered cmv gi infection.cmv gi disease was proven with the identification of cmv inclusion bodies or positive immunehistochemical staining using anti-cmv antibodies. results: overall, 91 endoscopic tests were performed (55 gastro-,36 colonoscopies) at a median of 73 days (iqr:145) post transplantation. symptoms included nausea, vomiting, diarrhea, abdominal pain and weight loss. cmv dna was positive in 41/91 tissue samples: median 536 copies/ml, range: 11-131x10^6. only half patients (22/41) had concurrent cmv viremia (plasma viral load>100c/ml). cmv gi infection was not correlated to the type of transplant, acute or chronic gvhd. gi cmv disease was documented by biopsy in 13 patients. cmv dna of the tissue, but not the plasma viral load, was a predictor of biopsy positivity (or: 1.6, 95%ci: 1.1-1.8, p=0.006). thirty-six out of 41 cmv dna positive patients received specific treatment for at least 10 days. symptoms resolved in 21/36 patients (60%) and the gi viral load was not a significant factor to predict cure. gi gvhd was diagnosed in 42/91 patients, among which 45%(19/42) with cmv dna positivity. median os was 453 days (95%ci: 297-608) for patients with cmv infection, similar to those without (median os: 890, 95%ci: 80-1699 days, p=ns). we studied separately endoscopies of the upper (55/91) or lower gi tract (36/91) . there was no significant relationship between cmv gastritis proven by biopsy and cmv dna levels in gastric tissue. however, the viral load of the colon was a predictor of cmv enteritis (or: 1.9, 95%ci: 1.9-3, p=0.007). the auroc of the q-pcr was 0.849 (95%ci: 0.659 to 1), the sensitivity was 85.7% and the specificity was 78.6% with a cutoff value of 370 copies/ml dna. conclusions: pathognomonic findings in the biopsy remain the gold standard for the diagnosis, especially for the upper gi tract. however, when the lower gi tract is involved, quantification of cmv viral load in the tissue may be a valuable tool to support the diagnosis. positivity of cmv dna of the gi tissue, in linearity to the cmv viremia, may guide to preemptive treatment for prevention of cmv disease . disclosure: nothing to declare background: clostridium difficile infection (cdi) is caused by cd overgrowth in antibiotic-disturbed intestinal microbiota. antibiotics targeting unselectively beneficial for t-regulatory cell formation strains of clostridiales may increase pro-inflammatory processes in the guts promoting or augmenting the development of graft vs. host disease (gvhd). the efficacy of cdi treatment has impact on the persistence of inflammation which might influence the alloreactive reactions. methods: we retrospectively and, from 2016, prospectively analyzed the data from 5 transplant centers concerning cdi occurrence, treatment efficacy, and gvhd development. the study included 77 patients with hematological malignancies who underwent allogeneic hematopoietic cell transplantation (allohct) between 2012-2018. results: median time to cdi was 14 days post-allohct with detection of both toxins a and b in 57% of cases. disturbance of intestinal microbiome was confirmed by a 59% rate of colonization with multidrug-resistant bacteria (mdrb). the cdi symptoms resolved with the negative toxins after the first line treatment in 76.6% of patients. the median time to remission and therapy duration was 8 and 10 days, respectively. fifteen therapeutic failures were observed after treatment with metronidazole (10), vancomycin (2) and a combination therapy (3) . eleven patients responded to second line treatment. thirty-seven (48%) patients died due to infections (17), relapses (10) and gvhd/infections (10). we noted recurrent cdi in 6 cases. eight patients died with active cdi. we observed occurrence or exacerbation of gvhd in 35 (44%) patients following cdi, including 25 cases with gut involvement (gi-gvhd). treatment with metronidazole and failure of the first line therapy increased the development or escalation of gi-gvhd (p= 0.03 and p< 0.001, respectively). the duration of cdi exceeding 10 days also had impact on the gi-gvhd incidence (p= 0.002). conclusions: 1. patients colonized with mdrb are at high risk of cdi. 2. high mortality due to infections and/or gvhd in patients with cdi. 3. due to lower efficacy and harmful immunomodulatory impact, metronidazole should not be the first line treatment in cdi post-hct. 4. emphasis must be put on fast cdi resolution to interrupt a vicious circle of the intestinal inflammatory processes. disclosure: nothing to declare establishing optimal preemptive cytomegalovirus therapy threshold post allogeneic hct in a patient population with high prevalence of seropositive status background: preemptive therapy (pet) for cytomegalovirus (cmv) reactivation post allogeneic hematopoietic stem cell transplantation (hct) was shown to decrease the incidence of cmv disease. however, the optimal pet threshold is unknown and there are significant toxicities associated with anti-cmv therapy. at our institution, we initiate pet at cmv dna titer above 1000 copies/ml (1560 iu/ml). our aim was to examine the efficacy of this approach including the incidence of spontaneous clearance in a population with high prevalence of cmv seropositive status. methods: after due irb approval, patients that underwent allogeneic hct were identified and records retrospectively extracted.cmv reactivation was defined as the first detectable viral titer post hct from plasma samples whereas clearance of viremia as the first date of two negative pcr values obtained at least 1 week apart. cmv monitoring was initiated post hct performed at least weekly during the first 100 days and every 2-4 weeks thereafter. a high sensitivity assay abbott realtime cmv was used with detection threshold of 20 copies/ml (31.2 iu/ml). analysis was computed using jmp v. 14.0.1 results: a. baseline characteristics: a total of 195 patients were identified and included with a median follow up of 18.1 (0.7-95.6) months. median age was 26 (14-63) years and 58% were male. indication for hct was for a malignant disorder in 77% of cases. the majority had a matched related donor (87%) and cmv igg was positive in both donor and recipient in 98% of cases. myeloablative conditioning was given to 117 (60%) and 109 (56%) received tbi. in vivo t-cell depletion was given to 76 (39%); atg in 39 (20%) and alemtuzumab in 37 (19%). b. cmv reactivation and pet: a total of 178 (91%) patients had a positive cmv pcr with median days to reactivation post hct of 17 ; 129 (66%) patients had peak cmv titer < 1000 copies/ml (low titer) whereas the remaining 49 (25%) had a peak titer ≥ 1000 copies/ml (high titer). patients with high titer were more likely to be older (p = 0.019), have malignant disease (p = 0.019), haploidentical or unrelated donor (p < 0.0001) and higher incidence of agvhd grade ii-iv (p = 0.003) as shown in the table. median peak titers for the low and high groups were 111 vs. 4638, respectively (p < 0.0001).120 (93%) patients with low titers cleared spontaneously with median time to clearance of 40 days (4-188), 1 (1%) received anti cmv therapy and the remaining died with active viremia (range 49-561 copies /ml) with active disease. one patient in the high titer group developed cmv disease. 2-year os and ci-nrm was 67.9% vs. 55.4% (p = 0.1) and 8% vs. 19.1% (p = 0.034) in the low and high titer groups, respectively. conclusions: cmv reactivation was high in this cohort however of low titer viremia in over 70%. a pet threshold of 1000 copies/ml (1560 iu/ml) appears desirable as it was associated with spontaneous clearance in almost all patients while minimizing treatment related toxicity. validation of these observations is warranted. background: the risk of pneumocystis pneumonia often warrants antifungal prophylaxis for recipients of blood and marrow or solid organ transplantation. however, complications such as myelosuppression, nephrotoxicity, and intolerance with the existing standard, trimethoprim/sulfamethoxazole (tmp/smx), may hinder or interrupt prophylaxis. rezafungin (rzf) is a novel echinocandin in development for prevention of invasive fungal disease caused by candida, aspergillus, and pneumocystis species in blood and marrow transplant patients. rzf has a favorable safety and tolerability profile and a low risk of drug-drug interactions. furthermore, the stability and pharmacokinetics of rzf allow for once-weekly dosing and broad distribution to the lung and other target organs. rzf was shown to prevent in vitro pneumocystis biofilm formation and to reduce the viability of mature biofilms. a previous prophylactic study was conducted using a broader range of rzf doses. in the current study, the efficacy of rzf was evaluated to better understand the minimum doses necessary to prevent pneumocystis growth in a mouse model. methods: c3h/hen mice were immunosuppressed (dexamethasone 4 mg/l in acidified drinking water) and then infected intranasally with p. murina (2 x 10 6 /50 μl). given the slow growth of p. murina, test agents were administered at the same time mice were inoculated to test for prophylactic efficacy. mice received intraperitoneal injections of either vehicle (control/steroid [c/s]), tmp/ smx 50/250 mg/kg/3x/week (wk), caspofungin 5 mg/kg once daily, or rzf 5 mg/kg or 0.5 mg/kg once daily, 1x, or 2x/wk. after a 6-week dosing period, mice were sacrificed and lung homogenates were processed for analysis to quantify the nuclei (trophic) and asci (cyst) forms of pneumocystis. prophylaxis efficacy was based on reduction of organism burden compared with c/s. nuclei and asci counts were log transformed and analyzed by anova; individual groups were compared by the student-newman-keuls t test. survival rates were compared using graphpad prism v5. results: all mice in the rzf groups had significantly reduced nuclei and asci burdens compared with the c/s group, and all but the lowest doses of rzf (0.5 mg/kg 1x or 2x/wk) worked as well as tmp/smx at reducing nuclei levels. similarly, all rzf groups except for the 0.5 mg/kg 1x/wk group showed reductions in asci levels comparable to that of tmp/smx. the survival rates were not statistically different between treatment groups. conclusions: rzf demonstrated potent in vivo efficacy for prophylaxis against pneumocystis in an in vivo mouse infection model at dose regimens much lower than the human equivalent phase 3 regimen. these data support the development of rzf for the prevention of invasive fungal infections including pneumocystis pneumonia. disclosure: melanie t. cushion: research funding (cidara therapeutics) taylor sandison: employee, stockholder (cidara therapeutics) alan ashbaugh: nothing to declare. yuhua ru 1,2 , ziling zhu 1,2 , yang xu 1,2 , suning chen 1,2 , xiaowen tang background: immunocompromising period following allogeneic hematopoietic stem cell transplantation (allo-hsct) may allow opportunistic pathogens to thrive and result in fatal complications. epstein-barr virus (ebv) infects more than 90% of chinese population, and its reactivation after hsct is one of the major concerns due to the increased risk of ebv diseases and post-transplant lymphoproliferative disease. with the development of infection prophylaxis and supportive care after hsct, demographic data on ebv reactivation post-hsct needs to be updated. methods: we retrospectively analyzed the data of patients who received allo-hsct between july 2011 and july 2014 in the first affiliated hospital of soochow university. quantitative pcr (q-pcr) was used to monitor ebv-dna load in peripheral blood dynamically. ganciclovir (pre-hsct) followed by acyclovir was given as viral prophylaxis. the treatment protocol for ebv reactivation consisted of tapering of immunosuppressive agents, antiviral agents (including ganciclovir and sodium phosphonatel), and rituximab for persistent positive patients. results: totally 890 cases from most of the provinces in china were enrolled (characterized in table 1 ), among whom ebv reactivation developed in 175 recipients. most reactivation events (95.4%) occurred in the first year post-hsct, with a peak of 113.8 incidence rates per 100 personyears at the second month. besides, more episodes of lateonset reactivation occurred in patients receiving grafts from haploidientical donors ( figure 1a ) . multivariate analyses revealed that the major impactors of ebv reactivation included atg as gvhd prophylaxis (p< 0.001), hlamismatched donor (p=0.001) and the appearance of chornic gvhd (p=0.042). cumulative incidence of ebv reactivation was low (2.9%) among 890 patients with no major risk factors, but increased to 11.7%, 27.3% or 41.9% with 1, 2, and 3 major risk factors, respectively ( figure 1b) . there was no statistical difference of overall survival between people with or without ebv reactivation (p=0.871). conclusions: we concluded that there are similar ebv reactivation impactors in chinese population compared to literatures, including atg use, hla-mismatched donor and the appearance of chronic gvhd. additionally, incidences of ebv reactivation increased significantly with the accumulation of risk factors. however, ebv reactivation had no impact on overall survival in current virus management protocol. disclosure: nothing to declare background: several studies have shown loss of diversity of the gut microbiome in association with significant gut injury following hematopoietic stem cell transplantation (hsct). prolonged broad spectrum antibiotic use further promotes loss of microbiome diversity and increases the risk of intestinal colonization by multi-drug-resistant (mdr) bacteria. aims of this study were to prospectively evaluate the overall changes in gut microbiome composition after hsct and differences in patients colonized by mdr bacteria and treated with carbapenems. methods: we performed a prospective observational study evaluating the gut microbiota of 20 hematological patients undergoing hsct, from admission (t0) through day +28 (t5). fecal microbiota was assessed by 16s amplicon-based sequencing. clinical, and microbiological data as well as fecal samples were collected every 7 th day from admission. results: one-hundred fecal samples were analyzed. overall, we found a progressive decrease of bacterial richness from t0 to t5, with a significant reduction of blautia, ruminococcus and dorea species, which are strictly associated with the production of short chain fatty acids (sca) (fig.1) . moreover, in the 30% (no.6) of patients who were colonized by esbl bacteria, we observed a significant reduction of clostridium spp and bifidobacterium species. as for antibiotic therapies, carbapenems were used as second line treatment of febrile neutropenia in 50% (no 9) of cases, usually associated with aminoglycosides. in patients treated with meropenem, a strong decline of blautia and ruminococcus species was observed. this finding suggests a correlation between carbapenem regimens and increase of pro-inflammatory bacterial strains in the gut. conclusions: our data support the hypothesis that loss of intestinal commensals that produce short-chain fatty acids may increase dysbiosis. moreover, for the first time we report significant and progressive alterations in the composition of blautia, ruminococcus and bifidobacterium species in patients treated with meropenem and colonized by esbl bacteria, respectively. our findings offer potential modifiable targets to reduce risk of colonization by mdr bacteria and to promote a carbapenem-sparing approach in the hsct setting. clinical background: cmv is associated with significant morbidity after allogeneic hematopoietic stem cell transplantation. strategies to prevent cmv-related complications include universal prophylaxis and preemptive therapy, more widely spread. antivirals used for cmv reactivation (cmv-r) produces major toxicities and costs. rate and characterization of cmv-r after haploidentical transplantation with post-transplant cyclophosphamide (haplo pt-cy) is scarce. our goal was to analyze cmv-r rate after haplo pt-cy, outcome, complications associated to therapy, and to identify risk factors. methods: one hundred haplo pt-cy transplants using peripheral blood as stem cell source performed between 2011 and 2016 in our center have been retrospectively reviewed. gvhd prophylaxis consisted of pt-cy 50 mg/ kg/day on days +3 and +4, mmf and csa from day +5 for all cases. cmv pcr was performed in a biweekly basis during admission for transplant and treatment, and weekly thereafter. cmv-r was considered with any cmv dna level by pcr assay above 100 copies/ml. prior four consecutive negative weekly pcrs were needed to consider a new reactivation episode. preemptive strategy was applied in all cases. data collected in relation to cmv-r included: cmv serostatus of donor/recipient (d/r), number of cmv reactivations, length of each reactivation, antiviral treatment used, need for admission to receive treatment and adverse events related to cmv reactivation and/or antiviral treatment. results: patients characteristics are summarized in table 1 . among 100 patients, 78 of them with positive cmv serology, 128 episodes of cmv-r were detected. seventysix patients (76%) had at least one cmv-r in a median of 30 days after transplant. none of them had cmv disease or die as a consequence of cmv-r. median duration was 22 days (15-37). valganciclovir or ganciclovir was used in 101 episodes (79%). foscarnet was used in 43 episodes (34%). six of the episodes occurred after initial discharge, and required re-admission for treatment, with a median length of hospitalization of 16 days (6-27). cytopenias requiring transfusion or g-csf support occurred in 36 episodes (36%) treated with ganciclovir or valganciclovir. three of them needed further cd34+ cells booster for graft rescue. mild acute renal failure and genital ulcers were found in 21 (49%) and 5 (11,6%) events treated with foscarnet, respectively. no cases of severe renal failure were observed. serological status different than negative/negative (n/n) (p 0.001) and older age (46 vs 38 years, p 0.02) were significantly associated with cmv-r. no relationship was observed with gender, disease, donor relationship, conditioning, gvhd or cells infused. more than 2 reactivations were more frequent among patients with grade ii-iv acute gvhd (agvhd) and moderate-severe chronic gvhd (cgvhd). conclusions: in our experience, rate of cmv-r after unmanipulated haplo pt-cy, using pbsc as stem cell source, is considerably high. a significant proportion of patients presented complications associated with cmv-r and its treatment. cmv serological status other than n/n and older age are associated with high risk of cmv-r. patients with grade ii-iv agvhd are at higher risk of multiple reactivations. this population could be benefited from primary prophylaxis, in order to decrease treatment´s complications, re-admissions and costs. disclosure: nothing to declare. impact of infectious events occurring during the first hundred days after hsct for hematological malignancy: a monocentric retrospective study over a five-year period marie-pierre ledoux 1 , célestine simand 1,2 , karin bilger 1 , annegret laplace 1 , bruno lioure 1 background: patients undergoing hematopoietic stem cells transplantation (hsct) for hematological malignancy often present with infectious events in the early stages of the procedure, some of which having a documented impact on the outcome of the graft. for instance, cytomegalovirus (cmv) has been shown by some authors to have a protecting effect against relapse, whose features remain to be elucidated. we conducted a retrospective monocentric study regarding the outcome in terms of graft versus host (gvhd), relapse and survival of 224 consecutive patients over a period of 5 years, whether they presented or not with an infectious event by day 100 among the following: cmv viremia, epstein-barr virus (ebv) viremia, human herpes virus 6 (hhv6) viremia, bk virus (bkv) viruria, bacterial bloodstream infection (bsi) or invasive fungal infection. results: a high proportion of cmv seropositive recipients underwent a viral reactivation of cmv by day 100 of the hsct: 81% if the donor is seronegative and 74% if the donor is seropositive. we observed that cmv wasn't associated with a lower relapse rate in our cohort, and data weren't sufficient to conclude firmly, but showed a trend towards a worse acute gvhd (hazard ratio hr 2.08, pvalue 0.06). no significant correlation was found for ebv viremia. occurring in 26% of our patients and mostly with an early timing, hhv6 strongly correlated with worse acute gvhd (hr 3.25, p-value < 0.001) but its impact on survival was not significant. bkv (27% of our patients) and bsi (46% of our patients) both correlated with poorer outcome in terms of overall survival (logrank < 0.001 and 0.014 respectively) although not significantly associated with relapse or acute gvhd. fungal infections were too rare events to draw any conclusion. conclusions: thus, contrary to many studies, we found no protection against relapse induced by cmv, although the trend for worse acute gvhd was obvious. the mechanisms behind this discordance could include early treatment, but remain to be studied. whether hhv6 is a cause rather than a consequence of acute gvhd or its treatment is debated, but the correlation is strong and the sequence of events suggests hhv6 might act as a trigger for gvhd. the association between bkv viruria and a higher mortality is in contrast with previous observations, and the lack for association with gvhd and relapse could suggest bkv is a surrogate for poor immune recovery and therefore other causes of non-relapse mortality. in addition to the direct lethal risk of bacteriemia, bsi also are a promoter of late non-relapse mortality through indirect toxicity. through the expansion of immune effectors they promote, one could assume that infectious events play a role in gvhd and gvl, and therefore have an interference with relapse. however, the association between each infectious event and outcome remains to be clarified to guide our prophylactic and therapeutic choices by a better understanding of the bright and dark sides of infectious events. disclosure background: rezafungin (rzf) is a novel echinocandin in phase 3 development for treatment of candidaemia and invasive candidiasis and for antifungal prophylaxis against invasive fungal diseases caused by candida, aspergillus, and pneumocystis in blood and marrow transplant patients. rzf is differentiated by stable, prolonged pharmacokinetics (pk) that allow for once-weekly dosing and a pk-pharmacodynamic (pd) profile correlating with efficacy. clinical in vivo evaluations of drug interaction potential were performed proactively to assess the risk of drug-drug interactions (ddis) with respect to the phase 3 dose of 400 mg once weekly and known pk exposure in healthy individuals. methods: this open-label study of 26 healthy inpatients assessed ddis between rzf (as perpetrator) and drugs known to have interactions with cyp enzymes and transporters (probe drugs): repaglinide (cyp2c8), metformin (oct/mate), rosuvastatin (bcrp/oatp), pitavastatin (oatp), caffeine (cyp1a2), efavirenz (cyp2b6), midazolam (cyp3a4), and digoxin (p-gp), as well as tacrolimus, a drug likely to be coadministered with rzf. an initial dose of rzf 600 mg was administered on the first dosing day, to approximate a steady state plasma concentration of multiple once-weekly 400-mg doses, followed by 2 once-weekly 400-mg doses on days 10 and 15. probe drug cocktails containing ≥2 drugs were administered, once before and once after rzf administration, on a schedule designed to allow for washout between doses and to limit interactions with other probe and test drugs. samples were analysed to determine respective drug concentrations in plasma (except for tacrolimus which was in whole blood) to characterize the pk profile of each analyte. area under curve (auc) and maximum concentration (c max ) were calculated from the plasma/blood concentration-time profiles by noncompartmental analysis. ln-transformed pk parameters were statistically analysed using an analysis of variance model. the ratio of geometric least squared means between each substrate drug when administered with and without rzf and corresponding 90% confidence intervals (cis) were calculated for lntransformed c max and auc. results: when rzf was given concomitantly with the probe drugs, six of nine substrates (metformin, pitavastatin, caffeine, efavirenz, midazolam, and digoxin) statistically demonstrated the absence of drug-drug interaction, as their 90% ci were all included within the default 80-125% noeffect boundary. three substrates had the upper (repaglinide and rosuvastatin) or lower (tacrolimus) bounds of their ci falling just outside of this range (figure 1 ), and these changes are considered unlikely to be clinically significant. conclusions: no meaningful pk interactions were observed between rzf and 9 drugs known to have ddis and/or likely to be coadministered with rzf. these findings provide evidence that no dose adjustment is expected when rzf is co-administered with these commonly used drugs, which stand in contrast with the ddi complications widely associated with azole antifungals. disclosure: voon ong: employee, stockholder (cidara therapeutics), michael boily: employee (altasciences), hong wong: employee (altasciences), taylor sandison: employee, stockholder (cidara therapeutics), shawn flanagan: employee, stockholder (cidara therapeutics) abstract withdrawn. background: cytomegalovirus (cmv) continues to cause morbidity following allogeneic hematopoietic stem cell transplantation (hsct). letermovir is a newly approved drug for cmv prophylaxis in cmv-seropositive allogeneic hsct recipients. however, there is a paucity of data for its efficacy in patients receiving in-vivo t-cell depletion (tcd). at weill cornell medical center, we perform in-vivo tcd with alemtuzumab for related and hla-identical unrelated transplants, and anti-thymocyte globulin for umbilical cord blood transplant supported by third party accessory cells (haplo-cord transplant).although these drugs reduce the frequency of graft-versus-host-disease (gvhd), they significantly delay t-cell immune reconstitution post hsct, and may cause higher rates of cmv reactivation. our historical rate of cmv reactivation in cmv seropositive recipients receiving high dose valacyclovir prophylaxis is approximately 35%. therefore, we implemented letermovir for cmv prophylaxis in february 2018. the primary aim of this study is to determine the incidence of cmv infection (defined as cmv viremia warranting treatment or development of end-organ disease) in tcd cmv seropositive allogeneic hsct patients who received letermovir prophylaxis. methods: this is a single center, retrospective cohort study to determine the incidence of cmv infection in adult, cmv-seropositive recipients receiving letermovir prophylaxis after in vivo tcd hsct with atg or alemtuzumab for gvhd prophylaxis. all included subjects were at least 100 days post-transplant. results: 31 allogeneic hsct transplant recipients met inclusion criteria. median age was 60 years, iqr [47, 67] and 48% were male. eight (26%) had a matched related donor, six (19%) had a matched unrelated donor, and 17 (55%) were haplo-cord transplants. their underlying malignancy and conditioning regimens are summarized in table 1 . 17 (55%) received atg and 14 (45%) received alemtuzumab for gvhd prophylaxis. median follow up time for survivors is 141 days, iqr [107, 187] . the incidence of cmv infection in the first 100 days post-transplant was 3% as only one patient reactivated with detectable cmv viremia. this same patient developed cmv pneumonitis with documented ul 56 resistance, and was successfully treated with ganciclovir. the incidence of cmv infection within the first 150 days post-transplant was 5% (1/19 patients) . six patients (19%) developed acute gvhd in the first 100 days, and one (3%) had relapse of their malignancy. five patients (16%) died within 100 days post-transplant, but none of these deaths were cmv related. background: infectious complications caused by endogenous adenovirus (adv) are common and associated with morbidity and mortality rates in patients after hematopoietic stem cell transplantation (hsct). adv infections occur in about 3% to 20% of hsct recipients, with significantly higher rates in pediatric patients. a better understanding of adenoviral-specific t-cells (advt) response in donors can serve as a basis to develop more effective strategies for antiviral therapy. methods: frequencies of cytomegalovirus (cmv)-and adv-specific t cells were determined by enzyme-linked immunospot (elispot) assays with adv5 hexon and cmvpp65 respectively in 80 health donors. we used 3x10 5 of mononuclear cells (mnc) per well in elispot assays. all donors were divided into 3 groups according to the number of spots per well (spw) as follows: high responders (hr) (≥50 spots; n=32), low responders (lr) (>10 and < 50 spots; n=39), nonresponders (nr) (≤10 spots; n=9). the average spot area of adv-and cmv-specific lymphocytes was calculated by immunospot® multiplate autocount™. cd45ra+ and cd45ro+ t-cells were generated by immunomagnetic negative selection. hla typing for class i and ii was performed by sequence specific oligonucleotides technology. statistical analysis was performed using graphpad prism v7.00 software. levels of significance were calculated by mann-whitney rank-sum test, expressed as p-values (p< 0.05). results: the median frequency per well of advts were 72 in hr group, 24 in lr group, 4 in nr group. the median spw of cmv-specific t cells in donors mnc were 147 and didn´t differ between 3 groups. antiviral activity may depend not only on the amount of advt but also on their ability to produce ifnγ. the average spot area for advt did not differ between hr, lr and nr groups and were 22,4, 23,1 and 19,1 mm 2 respectively. the median of the average spot area for anti-cmv t-lymphocytes was equal to 13,5 mm 2 . thus, the frequency of advt was lower than cmv-specific t-cells, but advt have the ability to produce more ifnγ per cell (p< 0.0001). in order to evaluate the distribution of the advt between naive and memory t cell compartments, we evaluated response to adv in preselected cd45ra+ and cd45ro+ fractions of t-cells in a group of 17 donors. the median frequency of advt in unfractionated mnc was 87; the median frequency of advt in cd45ra and cd45ro fractions were 2 and 73, respectively. the amounts of cd45ra and cd45ro tcells were normalized to their amounts in mnc. we evaluated the impact of hla-alleles on the anti-adv response of t-cells in different groups and found 2 association: hla-a*1 with hr group (p-value=0,0043; rr=1,954; 95% ci: 1,285 to 2,604) and hla-a*31 with lr group 0251; rr=1, 889; 95% ci: 1, 135 to 4, 426) . conclusions: in this study the frequency of donors with advt is 88,75% which corresponds to the reported frequency of adv-seropositive people (95%) in population of russia. advt are exclusively cd45ro-positive cells. the analysis of advt in potential hsct donors will allow to determine more accurately the amounts and functional activity of specific antiviral t-lymphocytes administered to patient and optimize antiviral therapy. disclosure: nothing to declare p429 abstract withdrawn. chemotaxis and exhaustion of γδ t cells in the allografts are associated with cmv reactivation after hematopoietic transplantation background: cytomegalovirus (cmv) reactivation and its related diseases remain the most common and serious complications in patients who underwentallogeneic hematopoietic stem cell transplantation (allohsct). we previously reported that the incidences of total and refractory cmv reactivation reached approximately 90% and 50% after haploidentical hsct. while majority of studies in the literatures focused on the adaptive cd8 + αβ t cellsand nk cells in anti-cmv immunity, increasing evidences highlighted the important role of γδt cells in this context. a progressive and prolonged expansion of vδ1 + t cells in response to cmv reactivation was observed after allohsct. the effect of vδ1 + t cells associated with cmv clearance has been reported in vitro and in vivo. in contrast to the reconstituted γδt cells post transplantation, whether the phenotypes of γδt subsets in allografts correlate to cmv reactivation in hsct recipients have not been documented. methods: the proportions and phenotypes of γδ t cells were detected inallografts those were unmanipulated g-csf-mobilized bone marrow (bm) and peripheral blood (pb) harvests from 20 donors for haplohsct. bm grafts were collected by aspiration on the fourth day of g-csf treatment (filgrastim, 5 μg/kg/day), and pb grafts were obtained on the fifth day by leukapheresis. immunophenotyping for γδ t-cell subpopulations, including the expression of cd3, cd4, cd8, tcrγδ, tcrvδ1, tcrvδ2, hla-dr, nkg2d, cxcr4, ccr5, pd1, ki67, ifnγ, tnfα, and il-17, was performed using flow cytometry. for detection of the intracellular cytokines, bm and pb grafts were pre-stimulated with 1x cell stimulation cocktail (500x, ebioscience). cmv dna in the peripheral blood of recipients was routinely monitored by quantitative pcr. the association of γδ t-cell contents in allografts with cmv reactivation in haplohsct recipients was analyzed using the mann-whitney u test and spearman test. all calculations were performed using spss 22.0 statistical software. results: we found that the proportions of total γδ t cells, and vδ1 and vδ2 subsets in both bm and pb grafts for cmv+ and cmv-recipients were comparable. neither the expression of hla-dr nor nkg2d in the allografts were significantly different in correlation to cmv reactivation after hsct. the productions of intracellular cytokines of γδ t subsets did not varied in bm and pb grafts for cmv+ and cmv-recipients. interestingly, the proportions of cxcr4+vδ1 and ccr5+vδ1 cells in bm grafts for cmv + recipients were significantly higher than those for cmvrecipients (p = 0.012 and 0.045, respectively). meanwhile, pma-stimulated ki67+vδ1 cells in bm grafts for cmv+ recipients were less than those for cmv-recipients (p = 0.039). in parallel, the concentration of pd1+vδ1 cells in pb grafts for cmv+ recipients were significantly higher than those for cmv-recipients (p = 0.023). conclusions: this study is the first to connect the chemotaxis and exhaustion of γδ t cells in grafts to the risk of cmv infection after allogeneic hsct. future studies should explore how the expressions of chemokines and exhaustion marker on the effector γδ t cells in allografts facilitate cmv replication and/or dissemination in the setting of hematopoietic transplantation. disclosure: all authors do not have conflicts of interest. this study is supported by the national natural science foundation of china (grants no.81770191 and no.81670167) results: incidence of ic was 2,9%: allo-hsct -3% (n=15), auto-hsct -2,7% (n=7). the etiology: c. parapsilosis 50%, c. albicans 27%, c. krusei 14%, candida tropicalis 5%, candida dubliniensis 4%. the most frequent underlying diseases was acute leukemia -45% (n=10). the median age was 8 y.o. [3 month -18 years] . the median day of onset of ic after allo-hsct was 63 , auto-hsct -12 [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . febrile fever was the main clinical symptom; septic syndrome develops in 32% cases. antifungal therapy was with echinocandins -23%, lipid ampho b -27%, azoles (fluconazole, voriconazole) -32%, without therapy (the early mortality) -18%. overall survival (os) at 30 days from diagnosis of invasive candidiasis was 50%. the central venous catheter (cvc) removal was the only factor significantly improved os (70% vs 33%, p=0,035). conclusions: incidence of invasive candidiasis in children after hematopoietic stem cell transplantation was 2.9%. the main etiology agent was c. parapsilosis. invasive candidiasis infections most often affect leukemia patients, developed later after allo-hsct than auto-hsct. overall survival at 30 days from the diagnosis was 50%. removing of cvc improved overall survival in children with invasive candida infections after hsct. disclosure: nothing to declare background: graft versus host disease (gvhd) and virusassociated enteropathy in allogeneic hematopoietic stem cell transplantation (allo-hsct) may cause severe quantitative and qualitative composition changes of intestinal microbiota, leading to the development of small intestinal bacterial overgrowth (sibo) on the background of immunodeficiency, which can have a negative impact on treatment effectiveness. the gold standard for diagnosis and the main criterion for sibo is the detection in the jejunum aspirate >10 5 /ml bacteria and/or the appearance of colonlike microbiota in small intestine. it is also acceptable to use an alternative non-invasive technique -hydrogen breath test, which could be especially important in patients with severe mucositis, grade iii-iv and thrombocytopenia grade iv. sibo diagnosis in the setting of the gastrointestinal tract damage and dysfunction in patients treated with allo-hsct is insufficiently studied. methods: the study included 7 patients with acute myeloid leukemia (n=3), acute lymphoblastic leukemia (n=1), myelodysplastic syndrome (n=1), non-hodgkin´s lymphoma (n=1), hurler syndrome (n=1), who underwent allo-hsct from an unrelated (n=4) and haploidentical donor (n=3) , and which were complicated by enteropathy development. in 2 cases, the enteropathy reason was a combination of intestinal gvhd and viral colitis (hhv-6), in 5 cases -viral colitis (hhv-6). all patients had esophagogastroduodenoscopy with species aspiration from descending part of the duodenum and feces collection, with further bacteria pcr identification. hydrogen breath test was performed also in which patients were treated with oral lactose 2 g/kg with subsequent hydrogen assessment after 30 and 60 minutes. the study was performed in the period from 20 to 741 days after allo-hsct. results: according to feces analysis data, colon microbiota composition significantly differed from the reference values. at the same time total bacterial mass of the duodenum was less in comparison with colon microbiota: 2e+08 (2e+7/8e+9) and 9e+10 (9e+7/3e+12), respectively (p< 0.004). quantitative composition of the duodenal microbiota was comparable to that of colon: lactobacillus spp. 4e+5 (1e+5/2e+7) > 1.5 e+5 (1e+5/4e+11), (p=0.48); bifidobacterium spp. 6e+6 (1e+05/3e+7) < 7.5 e+6 (2e+5/1e+9), (p=0.423); escherichia coli 7e+5 (1e+5/2e+6) > 5e+5 (4e+5/9e+5), (p=0.2); bacteroides fragilis group 6e+6 (0e+0/8e+9) > 5e+6 (2e+5/3e +12), (p=1.0); faecalibacterium prausnitzii 1e+6 (0e+0/ 1e+7) < 1.5 e+6 (1e+5/3e+10), (p=0.54), indicating the presence of sibo. in this case, the hydrogen breath test was completely uninformative: basal values -0.035 (0.01/0.06) ppm, hydrogen concentration in 30 minutes -0.04 (0.01/ 0.06) ppm, in 60 minutes -0.02 (0.01/0.07) ppm, which is less than in healthy volunteers. conclusions: quantitative composition of the duodenal and colon microbiota is similar in the case of intestinal gvhd and/or virus-associated enteropathy in allo-hsct patients, which may be of diagnostic value for sibo confirmation. the hydrogen breath test is an uninformative method for sibo identification in patients after allo-hsct. disclosure: nothing to declare johannes schulte 1 , patrick hundsdörfer 1 , sebastian voigt 1 background: adenovirus (adv) infections or reactivations frequently occur in the pediatric hematopoietic stem cell transplant (sct) setting and these infections contribute to increased morbidity and mortality. the nucleotide analog cidofovir might be effective in reducing adv load, however, nephrotoxicity is a considerable side effect. the new antiviral compound brincidofovir (bcv, cmx-001), a lipid-conjugate nucleotide analog with broad-spectrum antiviral activity in vitro, has been reported to be effective in cases where cidofovir treatment was unsuccessful. methods: data of eight pediatric patients undergoing sct for malignant and nonmalignant indications were analyzed. all patients were weekly monitored for adv viremia by pcr. in case two consecutive positive adv pcr results indicating a viral copy number > 1000/ml were documented, patients received a weekly dose of cidofovir. if no reduction of adv load was seen within two weeks after the commencement of treatment or side effects demanded cidofovir discontinuation, bcv was obtained through an emergency expanded access programme. results: eight pediatric patients developed adv viremia with maximum viral loads ranging between 9350 and 4430000 copies/ml. six patients had c type adv and two patients had non c type adv infections. five patients had viral co-infections: two had an additional cmv infection, one had an epstein-barr virus (ebv) and herpes simplex virus co-infection, one patient had an ebv co-infection and one patient had a bk virus co-infection. all eight patients initially received cidofovir, however, a substantial decrease in adv load could not be observed in any patient after a two-week administration course. except in one patient who had extensive intestinal graft-versus host disease (gvhd), adv infection was cleared in all patients within three weeks after the beginning of bcv treatment. in addition, all coinfections were cleared. no nephrotoxicity or other side effects were observed. conclusions: bcv was effective in all but one patient. oral bcv might not be effective in advanced upper gut gvhd, especially when applied via a gastric tube, yet this was observed in only one patient. eventually, without nephrotoxic side effects, bcv could be an useful alternative to cidofovir. disclosure: nothing to declare. background: the use of post-transplant high-dose cyclophosphamide (ptcy) has overcome the need for extensive depletion of t lymphocytes from haploidentical donor grafts, which traditionally resulted in severe and prolonged immunosuppression. however, reconstitution of cellular immunity may be delayed even after t cell replete haploidentical stem cell transplantation (haplo-sct) with ptcy. the study of the incidence and severity of viral reactivation is therefore relevant to the outcomes of haplo-sct with ptcy. methods: our study enrolled 42 patients (women/men, 19/23), who underwent t cell replete haplo-sct from 12/ 2013 to 10/2018 and achieved hematopoietic engraftment. median age at transplant was 53.5 years (range, 19-70) . the underlying disease was aml (n=17), all (n=9), mds (n=9), myelofibrosis (n=4), cml (n=2), or cll (n=1). the conditioning regimen was myeloablative (n=31), reduced-intensity (n=10) or non-myeloablative (n=1). peripheral blood was the graft source in the majority of cases (n=29) and bone marrow in the remaining (n=13). recipient/donor cytomegalovirus (cmv) serostatus was -/-(n=2), -/+ (n= 4), +/-(n=8), or +/+ (n=28). the combination of tacrolimus and mycophenolate mofetil was administered in addition to ptcy for prevention of graftversus-host disease. cmv, epstein-barr virus (ebv), and human herpesvirus-6 (hhv-6) reactivation was monitored by real-time quantitative pcr (rq-pcr) in blood twice weekly post haplo-sct. bk virus (bkv) reactivation was assessed by rq-pcr in urine and/or blood specimens in cases with symptoms suggestive of bkv-associated hemorrhagic cystitis (hc). results: with a median follow-up time of 25 months (range, , the cumulative incidences (cin) of relapse and non-relapse mortality (nrm) were 15.5% (95% ci, 6.2-28.6%) and 33.3% (95% ci, 18.9-48.2%) at 2 years, respectively. median disease-free (dfs) and overall survival (os) were 53.3% (95% ci, 39.5-71.9%) and 58.4% (95% ci, 44.6-76.5%) at 2 years, respectively. the cin of cmv reactivation/infection (>100 copies/ml) reached 75.7% (95% ci, 58.8-86.4%) at 3 months. cmv infection developed in 30 out of 40 patients who were at risk, whereas recurrent cmv reactivation was observed in 15 patients with a median number of 2 episodes (range, 2-6) per patient. the median total duration of antiviral therapy for cmv infection was 27 days (range, 14-199) . cmv disease (pneumonia) was documented in 2 patients. the cin of ebv reactivation (>1,000 copies/ml) was 50.9% (95% ci, 34.2-65.3%) at 12 months. no case of ebv-related post-transplant lymphoproliferative disorder was observed, however preemptive therapy with rituximab was required in 2 patients with rapidly increasing ebv viral load. hhv-6 reactivation (>1,000 copies/ml) was observed in 5 patients (cin, 12.3% at 6 months; 95% ci, 4.4-24.5%), with none of them requiring specific therapy. bkv-related hc occurred at a cin of 23.9% (95% ci, 12.3-37.7%) at 6 months. cystoscopy for bladder hemostasis was required in 4/11 and nephrostomy in 1/11 patients with hc. conclusions: despite preservation of non-alloreactive memory t cells, haplo-sct with ptcy is associated with substantial rates of viral reactivation (especially cmv and bkv) resulting in the need for prolonged antiviral therapy and considerable morbidity as well. therefore, strategies to prevent viral reactivation and disease are still warranted in haploidentical stem cell transplantation. disclosure: nothing to declare. background: adenovirus(adv) infections are a wellrecognised cause of morbidity and mortality in children and adults receiving an allogenic stem cell transplant(hsct).the reported incidence of adv infection is higher(6%-42%) in paediatric hsct than in adults(3-27%),but we currently lack accurate data of adv infection burden among adults.cidofovir has been extensively used as a pre-emptive anti-adenoviral therapy and is current standard of care.we present our single centre experience of adv incidence and outcomes with pre-emptive approach in adult patients receiving t-cell depleted(tcd) hscts for myeloid disorders. methods: this is a single-centre retrospective analysis of 332 consecutive hsct patients for myeloid disorders including aml, mds, mpn & aplastic anaemia between january 2012-june 2016 using atg or alemtuzumab based tcd.adv screening was performed in all patients with standardised real time quantitative pcr on weekly basis during standard risk period. figure 1a -1b] results: baseline characteristics (table1) of patients were similar across both cohorts with or without adv infection. overall 12.6%(n-42/332) patients were positive for adv dna on atleast one of the sanctuary sites(upper respiratory airway,blood,faeces,urine) and 45%(n-19/42) of these experienced disseminated infection(defined by adv in ≥2 sanctuary sites or rising adv dna copies in blood), while 2 developed typical adenoviral disease (pulmonary). among patients with disseminated infection,majority had adv in gastro-intestinal(42%),10.5% in genitourinary and 21% as both sanctuary site of infection,in addition to blood viraemia(85% of all cases).cumulative incidence of adv infection was 10.6%(95%ci:7.6-14.2%) at 12 months with median time of 108 days(iqr:19-304 days) to detect adv-dna post hsct.overall survival(os) at 5 years for whole cohort was 41%(95%ci:31-50;median os-57months) with no statistical difference between patients with disseminated adv infection vs those with none(log rank; p-0.68; fig-1a ). overall cumulative incidence of non-relapse mortality (nrm) was 23%(95%ci:18-28%) and relapse(cir) was 25.5%(95%ci:19-32%) at 5 years,but no statistical difference noted between patients with disseminated adv infection & those with none(nrm:p-0.31;cir: p-0.50;gray test).pre-emptive therapy with cidofovir (3mg/kg weekly iv infusion for 2 weeks and fortnightly thereafter until infection free) was required in 33%(14/42) of symptomatic adv infection patients and 52%(10/19) with disseminated infections.one patient required brincidofovir therapy for refractory disease,but one patient died due to severe sepsis, before adv specific therapy could be given.remaining patients were monitored and all self-recovered on cessation of immunosuppression.all patients treated with cidofovir developed renal impairment(defined by atleast >25% increase in baseline creatinine),however majority(73%) recovered their renal function near their baseline (fig-1b) . conclusions: adv infection remains a significant cause of morbidity in adult hsct patients, however pre-emptive management with cidofovir has improved os and nrm despite use of tcd conditioning.renal toxicity remains common with cidofovir but with use of intermediate doses, majority do recover their renal functions. clinical trial registry: n/a disclosure: nothing to declare background: publications on invasive fungal disease (ifd) in lymphoma patients are limited especially after allo-hsct. there are no data on outcome of allo-hsct in lymphoma patients with prior ifd. this study focuses on epidemiology of ifd before and after allo-hsct in children and adults with hodgkin's lymphoma (hl). methods: single center prospective study included 86 patients with classical r/r hl who received allo-hsct from 2002 to 2018. the median age was 27 (13-49) y.o., children (< 18 yo) -13%. allo-hsct from mud was performed in 45,4% (n=39), mrd -24,4% (n=21), mmud -15,1% (n=13), haplo -15,1% (n=13), with ric (100%) and predominantly ptcy-based gvhd prophylaxis (71%). primary antifungal prophylaxis was fluconazole in 85%, secondary -voriconazole (100%). eortc/msg 2008 criteria for diagnosis and bronchoscopy before allo-hsct in pts with ct-scan lung lesions were used. "active ifd" means ifd diagnosed just before hsct. median follow-up time was 12 months . results: incidence of ifd before allo-hsct was 12,8% (n=11). ifd prior to hsct were invasive aspergillosis (ia) with lungs involvement. antifungal therapy before allo-hsct was used in 81,8% pts with median duration -2 months. complete response to antifungal therapy was in 45,4% pts, partial response or stabilization -36,4%, and 18,2% pts had an "active ifd". after allo-hsct all pts received voriconazole as an antifungal therapy or secondary prophylaxis. cumulative incidence of relapse or progression of ia after allo-hsct was 18,2% with the median 49 day after hsct, which were successfully treated with voriconazole in post hsct period. incidence of ifd after allo-hsct for naïve patients was 17,6% (n=13/74). etiology of ifd after allo-hsct was ia -69%, invasive candidiasis (ic) -15%, mucormycosis -8% and 8% combined ifd caused by aspergillus fumigatus + rhizopus stolonifer. the median day of onset of ifd after allo-hsct was day+ 114 and was associated with post-hsct relapse of hl (p=0,04). the main site of infection were lungs (88%), the main clinical symptom -febrile fever (100%). antifungal therapy was used in all patients: voriconazole -59%, micafungin -17%, posaconazole -8%, lipid amphotericin b -8% and combination lipid amphotericin b with caspofungin -8%. overall survival (os) at 12 weeks from the diagnosis of ifd after allo-hsct was 80%. the 2-year os in children and adult with hl after allo-hsct was 73,3%. development of ifd after allo-hsct do not decrease the 2-year os rate (69,2% vs 74%, p=0,77). the impact of prior ifd on 2-year os in allo-hsct recipients was not statistically significant in all group (63,6% vs 74,7%, p=0,47) , and separately in children and adults. conclusions: incidence of ifd in children and adults with hodgkin's lymphoma before allo-hsct was 12,8%. incidence of ifd after allo-hsct in patients with hodgkin's lymphoma was 17,6%. the major etiology agents as before as after allo-hsct were aspergillus spp. ifd was a late complication after allo-hsct and associated with post-hsct relapse. despite the high incidence ifd before or after allo-hsct didn't influence the outcome in children and adults with hodgkin lymphoma. disclosure: nothing to declare our community has high cmv positive serostatus, which is a known risk for cmv infection or reactivation. we conducted a study to explore the incidence and outcome of cmv infection among post-hsct children. methods: medical records of pediatric patients (age ≤ 14 years) undergoing single allogeneic hsct from january 2014 to december 2016, at king faisal specialist hospital and research centre, riyadh, saudi arabia, were reviewed. all patients with active cmv infection or disease before and during transplant were excluded. a total of 307 patients were included in the study; 160 were female. median age at hsct was 5 years. recipient cmv serostatus was positive in 213 patients before hsct, and 232 donors were cmvpositive. the recipient-donor (r/d) serology was 63.7% r +/d+, 15.3% r+/d-, 13.7% r-/d+, and 7.3% r-/d-. indication for hsct was immune disorders 24.1%, hemoglobinopathies 20.8%, bone marrow failure 20.5%, malignant disorders 20.5%, histiocytic 8.1%, and metabolic disorders 5.9%. source of stem cells was bone marrow in 223, cord blood in 74 and peripheral blood stem cell in 10 cases. donor was matched related among 198, unrelated matched/mismatched in 74, haploidentical 24, and related with 1-antigen mismatch in 11. total body irradiation (tbi) based conditioning was used for 47 patients, while atg was used in 169 patients. results: out of a total of 307 patients, 101 patients developed cmv infection post-hsct (32.9%). incidence in female recipients was high (38.1% versus 27.2%, p-value 0.042). both recipient and donor cmv serology positive (42.5%) developed cmv infection (p-value < 0.001). however, no cmv infection in both recipient and donor negative group (r-/d-). the incidence of cmv infection post-hsct was high in patients received tbi based conditioning (22 out of 47, 46 .8%, p-value 0.027), and in haploidentical transplant with 66.7% (p-value 0.008). source of stem cells, myeloablative versus nonmyeloablative conditioning, atg use in conditioning and agvhd, did not exhibit significant association with cmv infection. in multivariable setting, when adjusted for primary indication for transplant, donor hla type, tbi based conditioning and recipient and donor cmv serology at transplant, haploidentical donor (odds ratio: 3.491, p-value: 0.041) and donor-recipient cmv sero-positivity (odds ratio: 2.228, p-value: 0.007) were found to be significant risk factors. cmv infection resolution rate was 83.2% (84). with a median follow-up time of 32.9 ±1.5 months from infusion, five-year overall survival of cmv infected group was lower (0.658±0.087) as compared to non-cmv infected (0.706±0.032, p-value: 0.285). conclusions: incidence of cmv infection post-hsct in our center is comparable to other centers. our data suggest that donor-recipient cmv positive serostatus, haploidentical donor, and use of tbi based conditioning necessitate close attention and surveillance. background: toxoplasmosis is a rare and underestimated complication following allogeneic stem cell transplantation (allo-sct) with an often fatal course. this is in part due to limited diagnostics relying mainly on imaging and detection of parasite dna by pcr. we present here eleven cases of toxoplasma disease following allo-sct. methods: we retrospectively analyzed 534 consecutive adult patients who received an allo-sct in our bone marrow transplant unit between july 2008 and july 2018. eleven (2%) of these patients were diagnosed of toxoplasma disease. the main characteristics of the patients are shown in table 1. all patients, except two cord blood, have received peripheral blood stem cells. fludarabine-based conditioning regimes were used in all patients. only the two cord blood patients received thymoglobulin in the conditioning. graft-versus-host disease (gvhd) prophylaxis consisted on tacrolimus plus mycophenolate mofetil in 5 (46%) patients and post-transplant cyclophosphamide followed by tacrolimus in 4 (36%). before the allo-sct was performed the igg/igm toxoplasma serology of the recipient and donor. we reviewed the absolute lymphocyte count (alc) and cd4 + lymphocyte count within four weeks prior to the diagnosis of toxoplasmosis, and if the patients took effective primary prophylaxis for this parasite. toxoplasma disease was defined as the presence of toxoplasma infection plus clinical, radiological or pathological evidence. toxoplasma disease was considered the main cause of death when no other major life-threatening infection or other potential fatal complication occurred immediately before death. results: median (range) age (years) of the eleven patients diagnosed with toxoplasma disease was 54 (19-69). for pancytopenia, no patient received trimethoprimsufmethoxazole (tmp-smz) but pentamidine for pneumocystis jirovecii-pneumonia (pcp) prophylaxis in 10 cases and atovaquone in one. toxoplasma serology pretransplant was positive (igg+/igm-) in ten of the eleven patients. all donors were seronegative (igg-/igm-) except two. toxoplasmosis was diagnosed a median (range) of 85 days (35-330) post allo-stc. the clinical presentations were as cerebral-encephalitis (n=4), chorioretinitis (n=3), pneumonitis (n=2) and disseminated toxoplasmosis (n=2). one case, patient and donor seronegative pre-transplant, was presented as a primary infection in form of chorioretinitis. all three patients with chorioretinitis were diagnosed after day +100 of allo-sct. at the time of toxoplasma disease, 8 of 11 (73%) of patients had an alc < 500 cells/μl and all of them with immunosuppressive therapy and corticosteroids for acute or chronic gvhd. we had cd4 + lymphocyte count only in four patients and in three of them was < 200 cells/μl. eight of the eleven (73%) patients died, with a median (range) of 21 days (9-65) since diagnosis of toxoplasmosis, and in 6 of them the toxoplasma disease was the main cause of death. conclusions: in our series, the incidence of toxoplasma disease after allo-sct is low and is related to high mortality, in accordance with what has been reported by other groups. positive pre-transplant serology and gvhd and its treatment were factors strongly related with toxoplasmosis. we encourage the use of tmp-smz instead of pentamidine for pcp-pneumonia prophylaxis in patients seropositive for toxoplasma gondii pre-transplant. clinical trial registry: data about its epidemiology in children are scarce. we retrospectively analyzed the incidence, the severity and the risk factors that contribute to the manifestation of this complication in a pediatric population. methods: during a 10-year period (january 2008 -june 2018) we performed in our center 376 allogeneic transplantations, 237 for malignant hematological diseases and 139 for non-malignant. the majority of our patients received myeloablative conditioning regimens. diagnostic criteria of hemorrhagic cystitis were the detection of the virus with pcr in urine samples and/or in blood samples, in combination with hematuria and lower urinary tract symptoms (dysuria, urinary frequency, urgency, suprapubic pain) that couldn't be attributed to any other reason. we defined the hemorrhagic cystitis as severe when one of the following factors was present: formation of clots and continuous bladder irrigation, obstructive uropathy with creatinine elevation or need for urological intervention. results: a total of 376 patients with median age 7,2 years (0,5-20) were studied. 37 children (10%, 95% ci, 7,2-13,3) manifested bk virus associated hemorrhagic cystitis with median age 12,9 years (5, [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 5) . onset of cystitis occurred at a median time of 27 days (day +-day +52) after transplantation. in 17 children cystitis was severe. the median duration of symptoms was 27 days (8-200). the median time of hospitalization for children with severe cystitis was 70 days (29-242) whereas for those who didn't manifest cystitis was 45 (25-184) . in 25 of the 37 patients we examined the presence of the virus not only in urine but also in blood samples. in 11 of them the test was positive and almost half of them (6) manifested severe cystitis. the risk factors that were examined were age, administration of antithymocyte globulin, type of disease, graft source, type of donor and the presence of acute graft versus host disease (agvhd). in multivariable analysis, independent risk factors for the manifestation of hemorrhagic cystitis were age > 6 years old (hr: 8,46, 95% ci, 2,86-25, p< 0,001), transplantation for malignant disease (hr: 2,26, 95% ci, 0,97-5,27, p=0,05) and the presence of agvhd (hr: 4,04, 95% ci, 2,01-8,1, p< 0,001). the overall survival of children with hemorrhagic cystitis was 46,3% vs 69,3% of those who didn't manifest this complication, but in multivariable analysis for survival cystitis wasn't a statistically significant risk factor. conclusions: according to our results, stem cell transplantation in children > 6 years old who suffer from a malignant disease and the presence of agvhd consist independent risk factors for the manifestation of bk virus associated hemorrhagic cystitis. the identification of the risk factors of this serious complication will contribute to better management of transplanted patients. further research through prospective trials can contribute to the better understanding of the pathophysiology of hemorrhagic cystitis and to the establishment of appropriate diagnostic and therapeutic guidelines. disclosure: nothing to declare p442 impact of natural killer cell reconstitution on outcomes in patients with early cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation background: cytomegalovirus (cmv) reactivation influences survival after allogeneic hematopoietic stem cell transplantation (sct) and induces natural killer (nk) cell expansion. we evaluated nk cell reconstitution and clinical outcomes following early cmv reactivation after sct. methods: lymphocyte subsets were measured by flow cytometry on day 100 in patients with hematologic malignancies undergoing sct between january 2009 and december 2017 at kanagawa cancer center, excluding patients with graft failure or death within 100 days. cmv reactivation was defined as initiation of preemptive cmv therapy following pp65 antigenemia surveillance. results: the subjects were 152 males and 94 females with a median age of 51 years (range: 18-69 years). the median follow-up period for survivors was 3.2 years (range: 0.8-9.6 years). there were 128 patients with acute myeloid leukemia, 64 with acute lymphoblastic leukemia, 32 with myelodysplastic syndromes, and 22 with other diseases. at transplantation, 166 patients were standard risk and 80 were high risk. myeloablative conditioning and reduced-intensity conditioning were employed in 99 and 147 patients, respectively. bone marrow transplantation, peripheral blood stem cell transplantation, and cord blood transplantation was performed in 98, 36, and 112 patients, respectively. cmv reactivation occurred in 141 patients (57%) at a median of 45 days (range: 15-93 days) after sct. grade ii-iv acute gvhd and chronic gvhd affected 89 patients (36%) and 80 patients (33%), respectively. among all patients, 5-year overall survival (os), cumulative nonrelapse mortality (nrm), and cumulative relapse (cir) rates were 66%, 10%, and 33%, respectively. in patients without cmv reactivation (cmvr-) versus patients with cmv reactivation (cmvr+), 5-year os, nrm, and cir were 79% vs. 55% (p < 0.001), 3% vs. 16% (p = 0.005), and 27% vs. 38% (p = 0.142), respectively. among all patients, the median level of cd3-cd56+ cells, cd16 +cd57cells, and cd16+cd57+ cells on day 100 was 236/μl (range: 19-2150/μl), 275/μl (9-2660/μl), and 98/μl (3-1767/μl), respectively. nk cell subsets showed no significant differences between cmvr-and cmvr+ patients. when patients were divided into low and high groups at the median level of each nk cell subset, cmvr+ patients with high cd3-cd56+, cd16+cd57-, or cd16+cd57+ cells showed significantly better 5-year os than those with low cells (72% vs. 35%, p < 0.001; 69% vs. 40%, p < 0.001; 66% vs. 45%, p = 0.001, respectively). high cd3-cd56+ cells were significantly associated with lower nrm (7% vs. 26%, p = 0.002), while high cd16+cd57-cells were significantly associated with lower cir (26% vs. 50%, p = 0.007). multivariate analysis confirmed these nk cell subsets as prognostic factors in cmvr+ patients. conclusions: nk cell reconstitution may contribute to improved transplantation outcomes in subgroups of cmvr + patients. disclosure: nothing to declare background: rezafungin (rzf) is a novel echinocandin in development for prevention of invasive fungal infections caused by candida, aspergillus, and pneumocystis spp. in patients at high risk of infection. rzf has demonstrated in vivo prophylaxis efficacy and low risk of drug-drug interactions. furthermore, the stability and pk profile of rzf allow for once-weekly dosing. rzf is also in development for treatment of candidemia and invasive candidiasis using a dosing regimen of rzf 400 mg followed by 200 mg once-weekly, which achieved >90% target attainment against candida. while lower doses might be useful to prevent candida and pneumocystis, invasive aspergillosis is a different challenge. we evaluated rzf dosing for prophylaxis against aspergillus fumigatus in blood and marrow transplant (bmt) patients using pk/pd simulations of the treatment dosing regimen. methods: a previous population pk model was refined using data from phase 1 and phase 2 trials of iv rzf (nonmem vers 7.2). stepwise forward selection (α = 0.01) and backward elimination (α = 0.001) were used to assess for relationships between interindividual pk variability and covariates, such as age, sex, bsa, albumin, liver and renal function markers, and infection status. the final model was validated by comparing model-based predictions to observed data. the model and demographic data from 100 bmt recipients at stanford medical center were used for monte carlo simulation (n=2,000) of expected rzf concentrationtime profiles in bmt patients receiving iv rzf 400 mg on week 1 followed by 200 mg weekly x 11. of the 100 patients included in the demographic dataset, 39 were female (mean values at baseline: age, 58 years [19-75 years]; weight, 69.8 kg [42-123 kg] ). the median (range) bsa in the demographic dataset was 1.84 m 2 (1.27-2.61), and albumin was 3.35 g/dl (1.8-4.43 g/dl) . free-drug concentration-time profiles were evaluated (97.4% human protein-binding) relative to the a. fumigatus minimal effective concentration required to inhibit 100% of isolates tested (mec 100 ; jmi 2014-2017 sentry international surveillance data). results: the population pk model was a linear, 4compartment model with zero order iv input. albumin, sex, infection status, and body surface area were statistically significant predictors of interindividual variability; clinical significance of these factors was not determined. the model provided precise, unbiased fits to the observed data (r 2 =0.993 observed vs individual-predicted concentrations). rzf plasma free-drug concentrations at weeks 1, 2, and 12 were above the a. fumigatus mec 100 (0.03 mg/l) for the entire dosing interval in 98.0%, 92.0%, and 90.2% of simulated patients, respectively, and in ≥99.8% for all 12 weeks based on the mec 90 (0.015 mg/l). conclusions: these data modelled from bmt patients support the rzf dosing regimen of 400 mg iv followed by 200 mg once-weekly for prophylaxis against a. fumigatus. current antifungal prophylaxis may be limited by toxicity, ddis, or patient factors such as mucositis. the pk of rzf and its spectrum, safety, tolerability, and lack of ddis may address current unmet needs in ifi prophylaxis for bmt and other immunocompromised patients. disclosure: janice brown: research funding, cidara therapeutics, elizabeth lakota: research funding, cidara therapeutics, shawn flanagan: employment, cidara therapeutics, taylor sandison: employment, cidara therapeutics, voon ong: employment, cidara therapeutics, christopher rubino: research funding, cidara therapeutics p444 is fungal prophylaxis necessary in non myeloablative peripheral blood stem cell allogeneic transplantation in the pre-engrafment period? julien vaidie 1 , jean-baptiste woillard 1 , stéphane girault 1 , marie-laure dardé 1 , arnaud jaccard 1 , daniel ajzenberg 1 , bernard bouteille 1 , pascal turlure 1 background: non myeloablative peripheral blood stem cell transplantation (pbsc), by limiting toxicity, can be proposed to elderly patients or patients with comorbidities. however, fungal infections remain a key issue that can negatively impact outcome, and increase duration and cost of hospitalization. systematic fungal prophylaxis have demonstrated benefits in outcome in the context of myeloablative conditioning but are not currently in reduced intensity conditioning allograft with pbsc. fluconazole prophylaxis is currently recommended in this situation (ecil). methods: primary objective of this retrospective study was to evaluate fungal infection incidence after allograft procedure in patients who received a non myeloablative allograft with pbsc in limoges university hospital between june 2009 and june 2018. patients received fludarabine 30 mg/m2/day between d-6 and d-2 before allograft and busulfan 3.2 mg/kg/day at d-4 and d-3. gvh prophylaxis consisted in rabbit anti-lymphocyte serum at the dose of 2.5 mg/kg at d-2 and d-1, and ciclosporin at the beginning dose of 3mg/kg per os twice a day. mycophenolate mofetil was adding for patients with hla-matched or mismatched unrelated donors. patients did not systematically receive antifungal prophylaxis during the neutropenic pre-engraftment period. when patients had fever during more than 72 hours, an empirical fungal treatment (caspofungine) was added to empirical antibiotics. as soon as neutropenic recovered and in the case of apyrexia without microbiologic documentation, antimicrobial treatments were stopped while in the case of microbiologic documentation, treatments were adjusted to germ in term of dosing and time of administration following recommendations. however, some patients received antifungal azole prophylaxis during the neutropenic pre-engraftment period in case of history of previous invasive aspergillosis (ia), or a nasal colonization by aspergillus. in post-engraftment period, posaconasole prophylaxis was administered for patients with systemic corticotherapy for acute graft-versus-host disease. results: 91 patients were evaluated (median [min-max] age of 60 [33-71] years). 20% of patients received an hlaidentical related donor, 69% an hla-matched related donor and 11% an hla-mismatched unrelated donor. the five years overall survival and survival without relapse or gvhd were 70% ic95 [59%-81%] and 62% respectively ic 95 [52%-73%]. the median time for neutrophil recovery was 15 days. 79 patients did not receive prophylaxis and only 12 patients received systematic fungal azole prophylaxis in the pre-engraftment period. two patients received an empirical treatment by caspofungine. only 1 ifi was documented during the neutropenic period : candida krusei in blood culture. in the post engraftment period, 37 patients with acute gvhd treated by corticotherapy received an antifungal prophylaxis by posaconazole and only 1 patient had a probable ia at day 100 despite prophylaxis by posaconasole. conclusions: except for patients with previous history of ifi, our results provide additional arguments against systematic fungal prophylaxis after reduced intensity conditioning with pbsc allogenic transplantation in the pre-engraftment period with a very low incidence of invasive fungal infections. in post-engrafment period, posaconazole prophylaxis is required for patient with gvhd treated by corticotherapy. disclosure methods: a simple, rapid and sensitive method using hplc with a diode-array detector (dad) was developed and validated for the quantification of letermovir in human serum using sorafenib as internal standard. after pretreating serum samples by liquid-liquid extraction with tert-butyl methyl ether, separation was achieved on a x-terra rp-18 column (dimension 150 x 2.1mm, 5μm) at 30 0 c using gradient elution with a mobile phase of 20 mm ammonium bicarbonate ph 7.8 (mobile phase solvent-a) and acetonitrile:20mm ammonium bicarbonate ph 9.2 (mobile phase solvent-b). samples were eluted at a flow rate of 0.3ml / min throughout the 20-minute run. uv wavelength mode was used, detection was at 260 nm. results: the calibration curve was linear (r > 0.99) in a concentration range of 25 -5000 ng / ml for letermovir. the hplc assay established for letermovir determination showed a high rate of accuracy and precision with an intraday variability of -9.21 to 12 % (accuracy) and 0.31 to 3.75 % (precision) and an interday variability of -2.6 to 6.9 % (accuracy) and 3.74 to 7.41 % (precision), respectively. 16 letermovir serum concentrations of 9 patients (8 male / 1 female, mean age 66.3 years) were determined in daily clinical practice. the mean concentration was 4752 ng / ml (median 4163 ng / ml, standard deviation 3925 ng / ml, range 89 -11452 ng / ml). conclusions: the newly developed hplc method is useful for the determination of letermovir concentrations. patient samples analyzed in a routine clinical setting demonstrated considerable interindividual variability. all measured concentrations were above the ec50 of letermovir. monitoring the concentration of letermovir could help to prevent over-or underexposure, especially in patients with polypharmacy which is frequent in allogeneic hematopoietic stem cell transplant recipients. disclosure background: the use of preemptive strategy (pet) has lowered the incidence of cmv disease in allo-sct to 5-10%. nonetheless the use of this strategy implies that more than 50% of seropositive patients will replicate cmv. several studies have shown that cmv replication is detrimental for patient survival although the viral load related to this bad outcome variates among studies. objective: to analyse thel impact of cmv replication in overall survival (os) in allo-hct patients. methods: to analyse the impact of cmv replication in os we perform a unicentric, retrospective study on 117 consecutive first allo-hsct patients transplanted between jan-2015 and oct-2018 with a median follow-up of 398 days (5-1266). all patients were monitored post-hct with real time pcr cobas-taqman® /cobas6800® (rtpcr) in plasma. the cut-off for inception of pet was 150 iu/ml. cmv mutations (ul54/ul97gene), were studied in plasma samples by sanger sequencing, median cmv viral load 3130 iu/ml (565-23900). results: patients (117): women/men (49%/51%), median age was 55 years (range 18-71). 101 identical allogeneic scts (86%), 16 haploidentical scts (14%). donors were related in 60 cases (51%) and 57 (49%) unrelated. progenitors source was 98% peripheral blood and 2% bone marrow. cmv status was (d+/r+) in 51%(n=60), (d+/r-) in 9%(n=11), (d-/r+) in 26% (n=31) and (d-/r-) in 11% (n=13), unknown 2 cases. positive pcrs were detected in 63 patients: one episode in 41 (65%); 2 episodes in 15(24%) and 3 to 5 episodes in 7 patients (11%). fifty-five patients (87%) received preemptive therapy. fourteen episodes (25%) were refractory/ probable refractory cmv infections (according to the criteria of chemaly r. cid 2018). a resistant mutation (ul54 gene) was detected in one patient with refractory infection patients that developed cmv infection had an inferior non-significant os at 3 years (61,3% vs 76,8% log-rank p 0,346). those patients that received pet for cmv had a significant inferior os compared with those that replicate cmv but didn't receive preemptive therapy (55,6% vs 100%, log rank p=0.04). os of patients that received pet was inferior compared with those without pet (with or without infection) (55,6% vs 79,9%, logrank p 0,05). no difference in survival was found for those patients treated pre-emptively that were refractory vs no refractory (53% vs 57,1%, log-rank p 0,51). conclusions: patients that received preemptive therapy had a significant inferior overall survival compared with those that didn´t replicate and those that replicate cmv but didn't receive preemptive therapy. this reinforce the relevance of prophylactic strategies for cmv with drugs with good safety profile like letermovir that in a randomised trial proved to decrease the need for preemptive therapy. disclosure: rafael, de la camara: has received grants from astellas, gilead, janssen, merck, novartis and pfizer clinical evaluation of stenotrophomonas maltophilia infection in allogeneic hematopoietic stem cell transplant recipients -retrospective single-center data analysis negative bacillus that causes severe infections associated with high morbidity and mortality in immunocompromised patients. the aim of our study was to determine incidence, characteristics and outcome of s. maltophilia infection in patients (pts) who underwent allogeneic hematopoietic stem cell transplantations (allo-hsct) in institute of hematology and transfusion medicine between october 2010 and november 2018. methods: we retrospectively evaluated incidence, clinical features and outcome of s. maltophilia infections in 338 consecutive patients with median age-42 years (range 19-71), who underwent allo-hsct from unrelated donors -238 (70.4%), matched sibling donors -87 (25.7%) and haploidentical donors -13 (3.9%) in our center. s. maltophilia was detected by culture-based microbiological tests. invasive infection was defined by isolation s. maltophilia from cultures in the presence of both clinical symptoms and signs of infection -blood stream infection (bsi), pneumonia with or without pulmonary haemorrhage. the only colonization status was defined as s. maltophilia culture-positive samples in the absence of infection symptoms. in vitro susceptibility tests to antibiotics were performed. results: 21 pts (6.2%) with median age-46 years (range 19-66) with s.maltophilia culture positive samples were identified. 19 (90.5%) underwent allo-hsct from unrelated donors, 1-from matched sibling donor and 1-from haploidentical donor. among them bsi developed in 7 pts (33.3%), pneumonia in 4 pts (19%) -with fulminant and fatal pulmonary hemorrhage in 3 pts (14.3%). all patients with pneumonia demonstrated bsi. positive sputum cultures were detected in 6 pts, in 4 pts hemoptysis was observed. the rest of isolated strains were identified as colonization (throat -in 3 pts, stool -in 9 pts). all 7 patients with invasive s. maltophilia infection before pathogen identification demonstrated persistent fever despite of the use of broadspectrum antibiotics (carbapenems, glycopeptides, aminoglycosides, colistin), prophylactic antifungals and antivirals. all of them received fluoroquinolone (ciprofloxacin) as a standard antibacterial prophylaxis before neutropenic fever occurred. all 3 patients (100%) with bsi, pneumonia and pulmonary hemorrhage died before engraftment (anc -0.0 g/l) -2 of them during 48-72 hours from the onset of a positive blood culture for s. maltophilia. the c-reactive protein (crp) concentration before identification of s. maltophilia invasive infection was > 26x-69x upper normal limits (unl). susceptibility to antibiotics of isolated strains from blood and sputum was respectively: 86% and 100% for ceftazidime, 100% and 100% for trimethoprim-sulfamethoxazole, 64% and 50% for levofloxacin; while 56% and 50% strains were resistant to ciprofloxacin. 1-year overal survival (os) and 2-y os for this group was 52.4 % and 47.6% respectively compared with 77.2% and 68.3% for group without s. maltophilia infection. conclusions: s. maltophilia invasive infections are associated with high morbidity and mortality in allo-hsct recipients especially in the period from conditioning therapy to engraftment. an exposure to broad-spectrum antibiotics in the treatment of neutropenic fever or confirmed bacteremia of other etiology is one of risk factors of breakthrough s. maltophilia infections. empiric therapy against s. maltophilia in selected patients in risk of such infection before pathogen identification may be lifesaving procedure. disclosure: nothing to declare. role of cmv reactivation following allogeneic stem cell transplantation in preventing relapses in patients with acute myeloid leukemia background: cytomegalovirus(cmv) reactivation is common in patients undergoing allogeneic stem cell transplantation. it has been shown recently that cmv reactivation is associated with reduced risks of relapse in patients undergoing allogeneic stem cell transplantation for aml. however the analysis of cibmtr data did not show any effect of cmv reactivation on relapse. with this background we conducted an analysis of patients suffering from aml who are undergoing allo-sct for their long term disease free survival with respect to cmv reactivation. methods: after obtaining permission from hospital medical records committee, we retrospectively analysed data from electronic medical records of patients undergoing allo-sct for aml at our center between january 2013 to august 2018. patients who underwent matched sibling, matched unrelated and partially matched allo sct were included. all patients underwent cmv monitoring with weekly pcr starting from the time of engraftment till d+100 following allo sct. value of ≥ 1000 copies/mcl was considered as cut off for initiation of treatment in matched sibling donor transplant but in unrelated donor or partially matched donor transplants, ≥ 500 copies/mcl was used as cut off for initiation of pre emptive therapy. results: total of 96 patients were included in study. median age was 28.01 ± 17.09years (2-69yrs). 60(62.5%), 30(31.3%) and 6(6.3%) patients underwent matched sibling, haplo (partially matched) and mud transplantation respectively. median follow up was 15 months(1-73months). (table 1) acute gvhd (grade2-4) was observed in 50(52.1%) of patients. cmv reactivation occurred in 70(72.9%) of patients. overall survival at last follow up was 63.5% (61/ 96patients). 17(17.7%) patients relapsed during follow up. relapse free survival at at last follow up was 61.5%. 64(90.1%) of 70 patients who had cmv reactivation didń t relapse, whereas 10(40%) of 25 patients who didn´t have cmv reactivation relapsed which was statistically strongly significant p < 0.001. (figure 1 ) similar results were seen in recently published paper from japanese society for hematopoietic cell transplantation (jshct) transplantation-related complication working group. conclusions: 1. cmv reactivation following allo sct had beneficial effect on preventing relapse in patients with aml. 2. probable immune activation resulting due to cmv reactivation may result in better graft versus leukemia effect preventing subsequent relapses. [ background: human herpesvirus 6 (hhv-6) causes lifethreating central nervous system disorders such as encephalitis after allogeneic hematopoietic stem cell transplantation (hsct). recent studies showed that cd134, a member of the tumor necrosis factor receptor superfamily, has been implicated as a specific receptor of hhv-6b, and that its expression levels in cd4-positive t cells after hsct could be related to the reactivation of hhv-6. real-time quantitative polymerase chain reaction analysis (qpcr) is the most commonly used method for detecting and evaluating hhv-6 reactivation after hsct, but more sensitive detection method is required. we recently developed a new monitoring method for hhv-6 reactivation using digital pcr (dpcr) which provides high sensitivity of detecting hhv-6 dna in clinical samples. in this prospective study, we evaluated the relationship between hhv-6 reactivation monitored by dpcr and expression of cd134 on cd4 + t cells before and after allogeneic hsct. methods: thirty-four patients who underwent allogeneic hsct for hematological diseases at keio university hospital (tokyo, japan) between january 2017 and march 2018 were consecutively enrolled into this study. peripheral blood samples of the patients were obtained before the conditioning (pre), the day of transplant (day 0), and weekly during the first month after transplantation (days 7, 14, 21, and 28) . hhv-6 viral load in plasma was quantitatively measured by dpcr. the primers and a probe of dpcr for hhv-6b were selected from immediate-early 1 (ie-1) protein transactivator region (u90). we evaluated the relationship between hhv-6 reactivation and the serial expression rates of cd134 in cd4 + t cells (cd134/cd4 ratio) measured by flow cytometry before and after hsct. results: median age of the patients was 51.5 years. onethird of patients received cord blood as a stem cell source. hhv-6 reactivation was detected in 23 patients (68%) with dpcr. a comparison of cd134/cd4 ratio between the patients with and without hhv-6 reactivation after hsct revealed that cd134/cd4 ratio was significantly higher in patients with hhv-6 reactivation than those without before conditioning ( in contrast, there was no such significant difference after transplant (days 7 to 28) . in multivariate analysis, higher cd134/cd4 ratio before conditioning (odds ratio (or) = 10.5, 95% confidence interval (ci): 1.3-85.1, p = 0.03) and stem cell source from human leukocyte antigen mismatched donor (including all cord blood transplantation cases) (or = 15.4, 95%ci: 2.0-121.0, p = 0.04) remained to be significantly associated with the incidence of hhv-6 reactivation. conclusions: higher cd134 expression rate in cd4 + t cells before hsct was associated with higher risk of hhv-6 reactivation, which could be a promising marker for predicting hhv-6 reactivation after allogeneic hsct. careful observation and monitoring may be needed in cd134 highly expressed patients. it is a subject of further research to clarify the role of cd134 + cd4 + t cell in hhv-6 reactivation. disclosure: nothing to declare. methods: criteria for the administration of ici (vistide) were grade iii-iv (clinically significant hematuria with clots) bk-related hemorrhagic cystitis after allo-hct which showed no improvement after symptomatic therapy with hyperhydration and bladder irrigation. cidofovir was diluted in 100 ml of normal saline and installed via a foley catheter which was blocked for 1 hour. not knowing the level of absorption of the drug we decided to give probenecid prophylaxis in all patients. ici was repeated weekly according to severity of symptoms. urine and plasma bkv viral loads were quantified by rq-pcr results: six patients (median 38 years, 29-51) received ici after allo-hct. patients had haematological malignancies (aml 2, all 3, mds 1), received busilfex-based myeloablative conditioning and a graft (pbsc 5, bm 1) from a 7/8 hla-matched (4pts), 8/8 (1pt) or haploidentical (1pt) donor. median time for the onset of bkv-hc after allo-hct were 53.5 days (range 32-117). all patients were under standard cyclosporine prophylaxis and none of the patients had any signs of acute gvhd at the time of onset of hc. the median pcr-bkv viral load at the onset of bkv-hc in urine and plasma were 5.75x10 8 (range 0.15x10 8 -59x10 8 ) and 141.5 (range 0-255), respectively. the median maximum pcr-bkv viral load in urine and plasma were 37.5x10 8 (range 4.7x10 8 -450x10 8 ) and 1.100 (range 0-400.000), respectively. five patients had impaired renal function (median egfr 49 ml/min, range 33-53) at first ici which was probably multifactorial. the median dose of intravesical cidofovir was 5 mg/kg (range 2.5-5 mg/kg) and a median number of 3.5 instillations (range 2-7) were given. in 5/6 cases symptoms of cystitis improved dramatically and hematuria resolved. virological response (at least 1 log reduction) was observed in all 5 cases. two patients experienced relapse of hemorrhagic cystitis and were retreated with ici which resulted in resolution of the symptoms and the hematuria. no deterioration of renal function of other systemic adverse effects were observed. after a median follow up of 190.5 days after transplantation (range 105-360), 3/6 patients are alive without cystitis symptomatology and 3 died (1 due to relapse and 2 due to trm). conclusions: in this retrospective study we propose that local therapy of bkv-hc with ici is safe and has high clinical and virological response rates. the administration of ici after allo-hct should be controlled in prospective randomized trials. disclosure: nothing to declare background: since cmv-preemptive therapy approach was implemented, cmv disease frequency is very low. however, cmv reactivation and the need of using nephrotoxic plus/less myelotoxic drugs is very frequent. in addition to the toxicity of the medications to avoid cmv disease, other potential adverse effects of cmv have been mentioned in medical literature. in this study, we wanted to estimate how recipient/donor serologic status influences the outcome of allo-hsct in our most recent series of patients. methods: the population analyzed for this report is the all 224 patients who underwent allo-hsct during the 4-year period from october 2014 september 2018 in our unit. median age at transplant was 52 years (range: 7-69). one hundred and thirty were male (58%) and 94 were female (42%). baseline diseases were: 83 aml, 51 lpd, 29 all, 29 mds, 14 mpd, 12 mm, and 6 bmf. donor was unrelated in 120 transplants (54,5%) and was family in 104 (45,5%) (including 34 haplo-identical). conditioning regimen was ric in 121 procedures (54%) and intensive in 103 (46%). stem cell source was pb in 210 (93,7%) and bm in 14 cases (6,3%). median follow-up was 23 months (range: 3-50). patient's and donor's cmv igg were positive in 188 (82,6%) and 92 (58,5%), respectively. recipient/donor serology was +/-(risk group 1) in 62 (27,7%), +/+ (risk group 2) in 126 (56,3%) , -/+ (risk group 3) in 6 (2,6%) y -/-(risk group 4) in 30 (13,4%). results: two pts underwent a second transplant before day +100 due to graft failure. overall mortalities (om) at days +100 and +365 of the rest of the series (222 pts) are shown in table. the highest risk group (recipient cmv + / donor cmv -) exhibited more than double om at day +100 and more than four times om at day +365, when compared with pts at lowest risk (recipient cmv -). those striking differences were mainly due to nrm. om for risk group ii (recipient cmv + / donor cmv +) was intermediate. conclusions: in our studied population, mainly adult patients, the combination of cmv-seropositive patient with a cmv-seronegative donor had a very clear adverse impact on hsct outcome. as a result, we considered that the election of a cmv-positive donor for a cmv-positive patient continues to be strongly advisable, whenever is possible. on the other hand, once letermovir has proved to be efficient and well-tolerated and has been licensed for prophylaxis of cmv in high risk recipients, this approach appears to be very attractive to try to avoid the adverse impact of recipient cmv-seropositivity, particularly when finally chosen donor is cmv negative. disclosure: nothing to declare an active surveillance and an early and individualized management is critical to avoid mortality from respiratory viral infections in allo-hsct recipients background: respiratory viral infections (rvis) are frequent among the general population. in transplant recipients, rvis are known to cause an important morbidity and potential mortality. for this reason and several others, as the need of preventing other pts from contagious or avoiding misdiagnosis with other infections processes, a high index of suspicion of vris is necessary. during the last few years, we have implemented an active and systematic surveillance policy orientedto early detection and management of rvis in the hsct recipients. methods: the population analyzed for this report is the 175 patients who underwent allo-hsct from january 2015 through march 2018 in our unit. median age at transplant was 51 years (range: 7-69). one hundred and four were male (59.4%) and 71 were female (40,6%). baseline diseases were: 64 aml, 38 lpd, 23 all, 19 mds, 15 mpd, 10 mm, and 6 bmf. donor was unrelated in 92 transplants (52.6%) and was family in 83 (47.4%) (including 29 haplo-identical). conditioning regimen was reduced in 91 procedures (52%) and intensive in 84 (48%).stem cell source was pb in 163 (93.1%) and bm in 12 pts (6.9%).median follow-up was 27 months (range: 8-46); at the close of the analysis, majority of the series (89.1%) had a follow-up superior to one year from hsct. a throat swab(ts) was taken from every patient with any, even minor, respiratory symptoms. the respiratorysamples were tested whith a complete pcr panel of human respiratory viruses: rhinovirus (rv), influenza a and b virus (iv-a, iv-b), parainfluenza virus (pivs 1-4), respiratory syncytial virus (rsv), metapneumovirus (mpv), coronavirus (cov), adenovirus (adv), and bocavirus (bov). results: day +100 overall mortality of the series was 8,6%. day +365 overall mortality was 25,1% (13,7% nonrelapse mortality -nrm-, and 11,4% progression/relapse mortality). causes of nrm reflected in table 1. no patients died due to rvis. from 1 st july 2016 through 30 th june 2018 (a 24-month period), 581 ts samples were obtained from 129 pts (73,7%).the median number of samples/patient was 3 (range: 1-17).a total of 162 (1-8) rvis episodes were diagnosed in 87 pts (49,7%).the median presentation of the first rvi was at the day + 226 (3-924) post-hsct. the viral distribution was: 47 rv (26.6%), 36 iv (20.3%), 33 piv (18.6%), 28 rsv (15.8%), 12 mpv (6.8%), 11 cov (6.2%), 9 adv (5.1%), and 1 bov (0.6%).there were 13 mixed (two or more viruses) rvi episodes. the temporary distribution of vri episodes is shown in figure 1 . conclusions: 1) symptomatic infections due to respiratory viruses are very frequent among the allo-hsct recipients. 2) a high level of suspicion, as well as an early and systematic screening and management policy, are critical to avoid potential attributable mortality and the nosocomial spread of rvis among the transplant recipients. 3) in our series, rhinovirus, parainfluenza and adenovirus might be detected at any moment of the year; the rest of the viruses showed a clear seasonal pattern (november to april). [[p452 image] 1. background: trimethoprim-sulfamethoxazole (tmp-smx) is the most suitable drug for prophylaxis against pneumocystis pneumonia and infections with toxoplasma after allogeneic haematopoietic stem cell transplantation (allo-hsct). allergic reactions or hypersensitivities, mainly exanthemas, occur in about 3-5 % of the patients, usually resulting in the use of alternative prophylactic drugs (e.g. pentamidine or atovaquone). it has been hypothesised that allergies might be cured with allo-hsct. methods: we conducted a retrospective chart review of patients with tmp-smx re-exposition after allo-hsct from december 2017 to september 2018. follow-up is current as of december 2018. results: six patients (f/m: 4/2, median age: 45 years, range: 25 -66 years) with a history of tmp-smx hypersensitivity prior to allo-hsct were re-exposed to tmp-smx after engraftment of a matched related (mrd, n=1) or matched unrelated (mud, n=5) donor. median time to re-exposition was 18.5 (range: 11-341) days after allo-hsct with one oral dose of tmp-smx. in four patients, tmp-smx was tolerated without any signs of hypersensitivity reactions and has been continued for a median of 231 days (range 87-315) until last followup. one patient (mud, re-exposition at d+341) experienced pruritus and erythema some hours after tablet intake. another patient (mud, re-exposition at d+18) developed an exanthema one day after re-exposition which was later diagnosed as a cutaneous gvhd. conclusions: re-exposition of tmp-smx in patients with prior hypersensitivity is feasible after allo-hsct. after successful re-exposition, patients can be treated with the best-studied drug for prophylaxis of infections with pneumocystis and toxoplasma. disclosure: nothing to declare brincidofovir for adenoviremia in paediatric hsct for primary immune deficiency background: reactivation of adenovirus is a severe complication of hsct associated with significant morbidity and mortality, particularly for children with primary immune deficiency (pid). the only drug currently licensed to treat adenovirus infection is cidofovir. brincidofovir is a lipidlinked derivative of cidofovir which has been shown to be a safe and effective alternative treatment to cidofovir. there is limited data describing the use of brincidofovir in patients undergoing hsct for primary immune deficiency. we reviewed all patients who received brincidofovir after undergoing hsct for primary immune deficiencies between 2016 and 2018 at the great north children's hospital, newcastle upon tyne, uk. results: of 78 patients transplanted for pid, 11 developed significant adenoviraemia (14%). all were treated with cidofovir initially but 6 were switched to brincidofovir because of a failure to respond or because of renal toxicity. of these, 4 resolved their adenoviraemia within 21 days of commencing treatment (figure 1). donor sources were tcr alpha/beta/cd19 depleted haplo-identical (n=4), tcr alpha/beta/cd19 depleted mmud (n=1) and 12/12 mud (n=1). patients were conditioned with treosulphan/fludarabine/thiotepa/atg/ rituximab (n=4), treosulphan/fludarabine/atg/rituximab (n=1) or treosulphan/fludarabine/alemtuzumab/ gcsf/plerixafor (n=1). occurrence of agvhd and treatment of agvhd are outlined in table 1. patient 5 died +111 days post-transplant of multi-organ failure, severe thrombotic microangiopathy and sepsis. although patient 2 initially responded to brincidofovir, reactivation occurred after cessation of treatment; severe diarrhoea precluded the reintroduction of brincidofovir and the adenoviraemia persisted with poor immune reconstitution. treatment with addback t cells was attempted however the patient died 194 days post-transplant after a cerebral haemorrhage. patient 3 had long-standing chronic diarrhoea which was thought not severe enough to warrant cessation of brincidofovir treatment. conclusions: the complete resolution of adenoviraemia in 4/6 patients who had previously failed to respond to prior therapy with cidofovir suggests that brincidofovir may be an effective treatment option for adenoviral reactivation post-hsct for pid. however, resolution of adenoviraemia is influenced by many other factors, including the adequacy of immune reconstitution, the degree of induced immune suppression and the presence of comorbidities such as gvhd. due to the small sample size it was difficult to assess the relative importance of these factors in this cohort. brincidofovir was well tolerated however its effectiveness may have been limited by poor gastrointestinal function in one patient (patient 3) and could not be used after a viral reactivation in another for the same reason. further studies of the use of brincidofovir in this specific cohort are needed to clarify the role and effectiveness of this treatment. background: there is a high prevalence of cmv seropositivity in algerian population. because of high morbidity and mortality in pts who underwent allo sct with cmv reactivation, effective surveillance and timely treatment using anti-viral therapy s required. the risk of cmv reactivation depends on the type of stem cell source, immunosuppression (is) and serological status of the donor/ recipient pair. methods: over a 24 months period (from 01/01/2016 to 31/12/2017), 254 pts underwent allo-hsct for malignant or non-malignant hematology diseases of which 246 pts are evaluated for this study. cmv reactivation was observed in 66 pts (26.82%) (aml: 33 pts, all: 8 pts, cml: 4 pts, multiple myeloma: 2 pt, nhl skin: 1 pt, primary myelofibrosis: 1 pt, aplastic anemia: 14 pts, fanconi anemia: 2 pts, β-thalassemia: 1 pt), with a median age of 30 years (4-54), sex ratio (m/f) of 1.75. allo-hsct done with sibling donors: 51 pts, haplo-identical donors: 14 pts and pheno-identical donor: 1 pt. all pts were treated by chemotherapy alone with myéloablative conditioning (mac) in 41 pts and reduced intensity (ric) in 25 pts. all pts received peripheral blood stem cells with an average rate of cd34 + cells: 7, 8.10 6 /kg (3.4-12.7). additional bone marrow graft was used in 7 pts that received a haploidentical graft without pt-cy. gvh prophylaxis associated cyclosporine (csa) and methotrexate (sibling and phenoidentical); csa-mtx-mmf or csa-mtx-cyclophosphamid (haplo-identical). before transplantation, donor/recipient pair is at high risk reactivation in 65 pts (98.4%). detection of cmv reactivation done by antigenaemia pp65 or by quantitative pcr weekly for the first 3 months and during an is treatment for acute or chronic gvhd. pre-emptive therapy is initiated by ganciclovir as soon as positivity of antigenaemia or increased viral load in pcr. results: a first reactivation occurred on average day 44 (16-83) in 66 pts (26.82%) of which 32 pts under corticotherapy for acute gvhd (27 pts), thrombotic micro-angiopathy (1 pt) and renal failure (4 pts) or due to a reinforced is for haplo-identical transplantation (5 pts). one pt with chronic gvhd presented a late reactivation 18 months after transplant. twenty pts presented a 2 nd reactivation on average day 135 (106-180) and 8 pts a 3 rd reactivation on average day 136 (130-143). pre-emptive treatment is introduced in the first episode by a viral dna polymerase inhibitor (ganciclovir: 22 pts; valganciclovir: 43 pts, foscarnet: 1 pt). the negativity of antigenaemia is observed on average at 5 days of treatment (3) (4) (5) (6) (7) . second line treatment was required in 14 pts (22%) due to resistance (12 pts), severe cytopenia (1pt) or renal failure (1 pt). the onset of severe cytopenia imposed a dose reduction (4 pts) or a therapeutic stop (6 pts) before 15 days. two pts received additional maintenance treatment for negativation delay. three pts (4.5%) died from cmv infections resistant to antiviral treatment (pneumonia: 2, colitis: 1). conclusions: cmv infection is a serious complication after allo-hsct. in the absence of vaccination, the systematic monitoring for cmv reactivation is strongly recommended for the establishment of a rapid and effective preemptive treatment. disclosure: nothing to declare p456 abstract withdrawn. results: in transplanted group, episodes of bkv reactivation occurred in 54 patients (42 %). in 26 cases only urine colonization (c) found before hsct. in this group in 10 patients (39 %) virus was transmitted from urine to the blood (b) . dysuria and/or hc were observed in 4/26 (15%) patients . all of them (100%) had urine and serum involvement. in 28 cases bkv replication was found after hsct (3 -cases detected in urine, 25 cases-bothserum and urine). dysuric syndromes and/or hc were found in 5/28 of cases (18%)-all in patients with serum and urine involvement. urinary tract was always first location of the virus. there was no case of isolated serum reactivation. the incidence of bk infection was higher in patients older than > 5 yrs (p< 0.05), transplanted from family donor (msd) (p< 0.05). mud recipients had more often both serum and urine reactivation (p< 0.05) than isolated urine involvement. sex, day of neutrophil recovery, conditioning regimen, or use of total body irradiation were not significant risk factors for bkv infection, or hc . six patients were treated with cidofovir (range 1-4 doses) with good response. there was no death due to evident bkv infection. conclusions: bkv reactivation remains one of the most frequent infectious complication in children undergoing allogeneic hsct. most of patients experienced mild infection and age < 5 years was the positive prognostic factor influencing its incidence. bkv monitoring and prompt treatment of hc resulted in excellent outcome. we observed surprisingly high rate of new bkv replication after hsct. disclosure: nothing to declare background: high-dose chemotherapy (hd-ct) and auto pbsct have been the standard therapy for multiple myeloma (mm) for more than two decades, despite a wide range of new therapeutic options. recurrent/refractory malignant lymphomas and recurrent/metastatic germ cell tumors (gct) also benefit from this intensive therapy. in comparison to allogeneic transplantation, this treatment is known for lower complication rates, e.g. infections. however previous studies have schown that treatment related toxicity may not be underestimated and depending on the conditioning regimen used. methods: we retrospectively analyzed 193 patients (225 cases) who underwent hd-ct plus auto pbsct between 2011 and 2017 in a single-center study. to anlyze the incidence of infections depending on the conditioning regimen, we formed the following categories based on the agiho: no infections, neutropenic fever, sepsis and severe sepsis. results: the median age in this analysis was 59 years; 74.2% were male. the most frequent diagnosis was mm (62.7%) receiving high dose melphalan (mel), followed by malignant lymphoma (26.7%) receiving beam (bcnu, etoposide cytarabine, melphalan) and relapsed/metastatic germ cell tumours (gct) (9.8%) receiving high dose carboplatin/etoposide (ce). 8% of all patients developed severe sepsis, 5 patients had to be ventilated and 2 patients died. sepsis was documented in 25.3% of all cases (57 cases). the majority of patients (58.2%, 131 cases) developed neutropenic fever and 8.4% (19 cases) didn´t have any infection complications. the beam conditioning regimen showed the highest tendency to result in a septic course (43.3%), followed by ce (27.3%) and mel (24.1%). the most commonly documented pathogen in 153 blood cultures was s. epidermidis (41.7%), followed by e. coli (18.5%) and s. mitis (9.9%). only in one blood culture we detected a multi-resistant pathogen (3mrgn e. coli). p. aeruginosa was detected in 4 blood cultures (2.6%), l. monocytogenes in 2 (1.3%) and s. aureus in 6 (4%). 77.8% of all patients developed diarrhea, only in 10.9% of these cases we could detect c. difficile. the conditioning regimen shows no significant effect on the incidence of c. difficile. the mean neutropenic period was 13.5 days in malignant lymphoma patients, followed by 10.8 in mm patients and 8.7 days in gct patients. the hospital discharge, calculated from the day of transplantation, was significantly different: for malignant lymphoma the mean was 20.8 days, for mm 19.6 days and for gct 14.5 days. conclusions: our data correspond to former published results by many groups. the beam regimen shows the highest infectious complication rate followed by ce and mel. the duration of neutropenia and hospital stay depends on the conditioning regimen. the type of infectious complication doesn't effect the progression free-and overall survival in our analysis. disclosure: nothing to declare. impact of donor and recipient cytomegalovirus serostatus on outcomes of unrelated allogeneic haematopoietic stem cell transplantation background: cytomegalovirus (cmv) is an important cause of morbidity and mortality in allogeneic haematopoietic stem cell transplant (hsct) patients. the aim of our study is to evaluate the outcomes of our cmv seropositive recipients who received grafts from seropositive unrelated donors (d+r+) compared with grafts from seronegative unrelated donors (d-r+). methods: this is a retrospective single center study on a series of cmv seropositive recipients who underwent hsct from unrelated donors between febuary 2012 to july 2018. a total of 96 patients were analyzed. their clinical course and laboratory results were reviewed for evidence of cmv reactivation and/or cmv disease. we defined cmv infection as detection of cmv reactivation or primary infection by antigenaemia or polymerase chain reaction (pcr) assays, but was not accompanied by signs and/or symptoms suggestive of a systemic disease. cmv disease occurred when cmv was isolated from any site in association with organspecific signs and/or symptoms. monitoring for cmv infection commenced upon engraftment (approximately day +14). peripheral blood samples were sent twice a week for cmv antigenaemia or cmv quantitative pcr. the duration of twice weekly monitoring was at least about 100 days. longer monitoring was performed in patients who experienced cmv infection after hsct. results: all patients received graft-versus-host-disease (gvhd) prophylaxis using anti-thymocyte globulin (atg) at 4.5mg/kg in addition to cyclosporin or tacrolimus. among the entire cohort of 96 patients, 72 (75%) had cmv infection, including 23 (82.1%) out of 28 patients from the d-r+ group and 49 (72%) out of 68 patients from the d+r + group. 16 patients (57.1%) from the d-r+ group and 19 patients (27.5%) from the d+r+ group had ≧2 cmv reactivation above the threshold for preemptive therapy respectively; p=0.007. 11 patients developed cmv disease, 6 (21.4%) from the d-r+ group and 5 (7.4%) from the d +r+ group. cmv resistance to both foscarnet and ganciclovir was detected in 3 patients (10.7%) from the d-r+ group but none from the d+r+ group. 2 patients died due to cmv disease, both were from d-r+ group. 2 year overall survival (os) were 60% versus 55% for d-r+ group and d+r+ group respectively; p=0.706. median survival was not reached at 2 years. 2 year non-relapse mortality (nrm) were 35% for d-r+ group and 24% for d +r+ group respectively; p=0.37. conclusions: the incidence of recurrent cmv infection was higher in the d-r+ group compared to the d+r+ group. there were no statistically significant differences between the 2 groups in terms of os and nrm. however, there was a trend towards higher nrm in the d-r+ group compared to d+r+ group. our findings suggest that for matched unrelated hsct, it may still be important to select a seropositive donor for a seropositive recipient. disclosure: none background: it´s known that some patients submitted to allogeneic stem cell transplantation (asct) could present a greater susceptibility to infection even when they are in long term complete remission or potentially cured. this fact is related to the dynamic of immunological recovery that is variable in every single patients and it is dependent from many factors: the haematological disease, the conditioning regimen, the age of patient and donor, the number of stem cell and lymphocytes infused, the anti-gvhd prophylaxis, the use of anti-thimoglobulins and others. in clinical practise we can observe patients who are potentially cured, who tapered and stopped the immunosuppressive treatment months or years ago and who are suddenly graved from opportunistic infections. the largest part of these infections is represented from varicella-zoster virus (vzv) cutaneous eruption. methods: in this report we retrospectively analysed a monocentric cohort of 38 patients submitted to asct for haematological malignancies from a median time of 30 months. all of them were free of disease. they stopped the immuno-suppressive treatment in a median time of 270 days after asct (range: 160-1642) and did not present later chronic gvhd needing treatment neither other moderate or severe chronic post transplant complications nor other diseases. prophylactic treatment with anti viral agents (acyclovir or valacyclovir) has been conducted simultaneously to immuno-suppressive treatment and for a period ranging between 1 to 6 months after its suspension. in this cohort of patients we considered the incidence of vzv eruption occurred after the suspension of the immunosuppressive treatment, and we analysed the immunological recovery in terms of lymphocytes sub-population after 6, 12, 18 and 24 months from asct. results: of these 38 patients considered, 10 developed at least one vzv manifestation. all the vzv presentation were cutaneous, we did not observe neurological, ophthalmic or visceral presentation. all the vzv manifestation occurred in patients who ended the anti-viral prophylaxis. median time of presentation was 575 days after asct (range: 174-1642) the remaining 28 patients did not present vzv manifestation nor other kind of opportunistic infection despite the absence of anti-viral prophylaxis. the analysis of lymphocyte sub-population after 6-12-18 and 24 months did not show a significant difference in b, t, t4, t8 and nk lymphocytes in the different post transplant period. conclusions: vzv reactivation seems not to be correlated with the number of the different lymphocyte subpopulations in the post transplant period. actually it is not possible to distinguish patients more suitable of vzv reactivation on the basis of lymphocyte sub-populations analysis, so anti-viral prophylaxis should be prolonged for a medium period after suspension of immuno-suppressive drugs. in absence of anti viral prophylaxis a careful clinical surveillance should be performed in order to treat early eventual vzv manifestations. disclosure background: infection and disease cytomegalovirus (cmv) are common problems in patients undergoing hematopoietic stem cell transplantation (hsct). cmv infection has a high overall seroprevalence, therefore, during the first 100days post-hsct, it is important to prevent reactivation of cmv. the international clinical recommendation is the use of ganciclovir as prophylaxis in hsct patients; however, the cost of this treatment is not accessible for our population. in this respect it has been used as an alternative valganciclovir because of its lower cost and oral administration. our study´s aim was to assess the response and safety of valganciclovir in comparison with ganciclovir to prevent viremia and cytomegalovirus disease in patients undergoing allogeneic hsct methods: a retrospective study was performed on patients who receive an hsct-allo between january 2014 and august 2018. participants were enrolled in two groups according to prophylaxis treatment: (a) ganciclovir 5mg/k once daily and (b) valganciclovir 450mg twice daily for 7 days pretransplant, at day +100; viremia was measured by pcr. demographic and clinical information was collected from medical records and furthermore analyzed in spss v21. results: sixty-eight patients were enrolled in the study, 54% male, the median age was 34 years (19-61) with the following diagnoses: acute lymphoblastic leukemia 44%, acute myeloblastic leukemia 26.5%, granulocytic chronic leukemia 17.6%, myelodysplastic syndrome 4.4%, dendritic cell neoplasia 4.4%, and aplastic anemia 2.9%. ninety-one percet of the patients received a transplant from an identical hla donor and 8.8% received a haploidentical transplant. thirty-four patients received ganciclovir (g1) and thirtyfour valganciclovir (g2). median age was 39 vs28 years (p=0.022), intermediate risk cmv 85% vs 76 (p=0.048), associated bacterial infections was 15%vs 32% (p=0.15), and fungal infections 6% vs9% respectively (p=0.5). the reactivation by cmv was presented in 21% vs 15% respectively (p=0.70). there were no significant differences in fever, bacterial isolation, dysfunction or graft failure, presence and degree of acute or chronic gvhd and relapse of the disease. the most relevant characteristics and complications are described in table 1. within the whole group there were 28 deaths, 53% in the ganciclovir group and 32% in valganciclovir group (p=0.14), overall survival 1-year was 66% vs 80% (p=0.58) respectively; in both groups 72% was associated with relapse and 28% associated with transplantation. conclusions: ganciclovir and valganciclovir were effective in preventing the reactivation of cmv, the only statistically significant difference was that the presentation of the disease appeared earlier in the valganciclovir group. no difference in toxicity between the groups was identified. disclosure: none declared background: invasive pulmonary aspergillosis (ipa) is a severe and serious complication that occurs in the immediate post-transplant period due to severe neutropenia or late usually following prolonged corticosteroid therapy during treatment of graft-versus-host disease (gvhd). the objective of this study is to analyze the epidemiological, diagnostic and evolutionary characteristics of this major complication over a period of 3 years. methods: from january 2015 to december 2017, 392 patients (pts) received an allogeneic hematopoietic stem cell transplantation (allo hsct) for malignant and nonmalignant haematological diseases. during the transplant procedure, anti-infectious prophylaxis consisted of pts isolation, digestive decontamination, fluconazole and aciclovir. secondary prophylaxis done for pts with prior history aspergillosis. during the follow-up, a standard chest x-ray is performed systematically at each control or in case of clinical signs a thoracic ct scan is requested from suspicion. the diagnosis of ipa is made according to the criteria of the eortc-msg based on the predisposing criteria of the host and clinico-radiological criteria (possible infection). galactomannan antigen and histopathology criteria are not common practice. results: a total of 29 ipa episodes (7%) were identified in 26 pts (aml: 15, aa: 5, all 3, cml 2, mm 1) of median age 36 (8-56) , sex ratio: 0.44. all of them had transplantation from a family donor (geno-identical: 22, haplo-identical: 4) with conditioning by chemotherapy alone and a graft of csp (24 pts) and peripheral stem cells-bone marrow (2 pts). all pts had at least one predisposing risk factor: antecedent of aspergillosis (3 pts), prolonged neutropenia> 10 d (5 pts), acute gvhd (8 pts), chronic gvhd (4 pts), prolonged corticosteroid therapy ≥ 0,3 mg /kg/day exceeding 21 days (12 pts). the diagnosis of api was possible on average at j172 (20-930) after appearance of clinical signs (in all cases) and evocative radiological in 25 cases (in 4 cases, the standard chest x-ray was normal). at the time of thoracic ct scan, 11 pts (37%) had characteristic signs: halo sign (6 pts), crescent sign (1 pt) and cavity (4 pts). other minor radiological signs are found in the other pts. empirical first-line antifungal therapy was started as monotherapy in 14 pts (voriconazole: 8, caspofungin: 6 pts) or in combination in 12 pts. a secondline treatment was required in 10 pts for failure after an average duration of 7 days . three pts presented a second episode after an average delay of 5 months (3) (4) (5) (6) with a favorable evolution of resumption of thetreatment. fourteen pts (54%) are alive with complete resolution after a median treatment time of 10 months (2-19). twelve pts (46%) died rapidly on average 14 days after diagnosis (ipa 11, relapse of his disease: 1) conclusions: ipa occurring after an allograft of allo-hsct is a severe complication with high mortality. it is essential, in each case, to identify the pts with risk factors, perform a thoracic ct-scan, send serum serology for apergillus galactomannan antigenand start specific treatment as soon as possible while waiting to be able to reinforce the diagnosis by direct examination or sputum or brochoalveolar lavage with aspiration. disclosure: nothing to declare background: cmv (cytomegalovirus) has a prevalence varying between 45-100%. its pathogenicity is relatively low in the general population, usually resulting in a selflimiting viral illness. in an immunosuppressed host, infection can lead to life threatening illness. disseminated cmv infection can manifest in a number of organs and is diagnosed using internationally accepted criteria. in the post solid organ and stem cell transplant (sct) setting, it is postulated that it is viral reactivation, rather than primary reinfection that leads to cmv viraemia. prevention of reactivation requires the presence of a competent immune system, mediated by t-cells. this accounts for the increased incidence in intensive and t-cell depleting sct conditioning regimens. despite improved outcomes following the introduction of cmv monitoring by pcr and pre-emptive treatments (current uk guidance), cmv pneumonitis still carries a high mortality. the use of cmv specific immunoglobulins (cmvig) for the treatment of this complication is generally not recommended post chemotherapy or sct in haematological cancers due to lack of evidence. however, cmvigs are widely used in the setting of cmv reactivation post solid organ transplants. we report the use of cmvig in patients with suspected cmv pneumonitis at a single uk centre. the aims of this retrospective study were to establish safety and review efficacy in this highly immunocompromised group of patients. methods: data was collected retrospectively on the use of cmvig in patients with haematological cancers post sct or chemotherapy alone between 2007 and 2017 at manchester royal infirmary, uk. all patients included had cmv positive pcr in blood (and or from bronchoscopy), as well as high resolution ct imaging evidence of cmv infection. the data was sourced from pharmacy database and crossreferenced with a departmental list. for each patient identified, case notes and prescriptions were sourced. data collected included patient baseline characteristics, timing of treatment, number of doses of cmvig and outcome. results: eight patients received cmvig for suspected cmv pneumonitis. seven patients were post sct and one patient was severely immunosuppressed with chemotherapy alone. median age was 40 years (range 16-68). the cmvig regimen used was 4ml/kg of cytotect ® on days 0, 2, 4 and 6, followed by 2ml/kg every four days until resolution of symptoms. there were no infusion related reactions observed. patients received a median of 4 doses of cmvig. four out of 8 patients responded to the treatment and showed full recovery but only 3 are alive and well to date. conclusions: this study shows that the use of cmvig is safe in the post-sct setting of acutely unwell patients with multi-organ failure. despite limitations of retrospective studies, there appears to be benefit for the use of cmvig in our patient population, with 50% of patients showing a full recovery from that episode. allogeneic sct plays a confounding role in the outcome of patients although the numbers in our study are small. there is clearly a need for better treatments of cmv pneumonitis. cmvig is a promising treatment but further studies are needed to identify the optimal dosing regimen and provide evidence of efficacy. disclosure: biotest-honaria p464 abstract withdrawn. background: the risk of fungal infection related to allogeneic transplantation is a well-known cause of morbidity and mortality. the main agents implicated are yeast during the neutropenic period and filamentous fungi after this period. methods: we decided to evaluate the effectiveness of a prophylactic regimen containing fluconazole since day -3. after discharge fluconazole was kept until day or 75 or switched to posaconazole in high-risk patients. patients with gvhd under steroids were kept under prophylaxis.the group of high risk patients was defined by one of the following variables: 1-non related donors 2-atg, campath or fludarabine in the conditioning 3-presence of gvhd with need of steroids above 0.5 mg/kg we have analyzed the patients submitted to allobmt during 2016 and 2017. all patients were first admitted to an isolation room with hepa filters.patients under secondary prophylaxis were excluded. breakthrough fungal infections during the first year and toxicity leading to discontinuation was evaluated. results: sixty six patients were included with 67 transplants. male/female ratio was 36/30. the age range was 0.6-64 yo with a median of 37. malignant (53) and nonmalignant (13)diagnosis were included. donor type was related (16) haploidentical (9) and non-related (42). the conditioning regimen includes atg in 21, campath in 5 and fludarabine in 41. fourteen patients were treated after discharge with fluconazole and 52 with posaconazole. three patients fluconazole were switched to micafungin for hepatic toxicity, two cases to amphotericin due to persistent fever and in one case to caspofungin for a proven fungal infection (candida parapsilosis in blood stream in day +18). after discharge and during the first year of follow-up a single case of possible fungal infection was diagnosed, in a patient with gvhd with a lung nodule. conclusions: during the neutropenic period after transplantation the main risk of fungal infection is associated with candidiasis. the greatest risk of aspergillosis occurs later and have a significant relation with gvhd. except for candida parapsilosis the main source of yeasts are the gi tract. the main source of aspergillus are aerosolized particles retained by hepa filters. in patients without a previous episode of fungal infection the main risk of filamentous fungi occurs only after discharge. we conclude that fluconazole alone or followed by posaconazole in high risk patients is a feasible and effective regimen for primary prophylaxis, in allogeneic transplantation. disclosure background: bk virus-associated hemorrhagic cystitis (bkv-hc) has emerged as a serious infection after hematopoietic stem cell transplantation (hsct). it is characterized by painful hematuria due to hemorrhagic inflammation of the urinary bladder mucosa, this causes significant morbidity, prolonged hospital care with extensive nursing requirements and increases in healthcare costs. the purpose of this study is to determine the incidence, risk factors, and duration of treatment in our center. methods: we performed a retrospective review of hsct patients at luis calvo mackenna children´s hospital in santiago, chile diagnosed with bkv-hc, from 1st january 2016 to 30th november 2018. we investigated the incidence, risk factors and duration of treatment of bkv-hc in paediatric patients undergoing hsct over a 35 months period. bkv-hc was defined as bk virus (bkv) detection in urine by pcr testing in association with clinical symptoms and hematuria grade 2 or higher. sixty-seven patients were trasplanted during this period. results: eleven patients were diagnosed with bkv-hc at our institution, only one with bk viremia. the cumulative incidence of bkv-hc in our series was 16%. all of them were treated with cidofovir. the median age at diagnosis was 9 years old (range: 3-13 y.o.). the median time from hsct to hemorrhagic cystitis (hc) was 40 days (range: 25-240 days), the median length of treatment was 9 weeks (range: 2-36). all patients received myeloablative conditioning regimens and used cyclophosphamide (100%); ten (90%) were unrelated cord blood transplant recipients and nine (81%) used antithymocyte globulin. a concomitant viral reactivation (cmv/vh6) was demonstrated in six (56%) patients. no patient died due to bkv-hc or its complications, but in the follow up three patients died, one in relapse and two of other post transplant´s complications. conclusions: bkv-hc is the result of a complex interaction between patient characteristics, donor type and conditioning regimen intensity. these patients experienced significant morbidity and prolonged treatment. in our cohort bkv-hc of all patients but one were transplanted with an unrelated umbilical cord blood unit, all of them received myeloablative conditioning regimen with cyclophosphamide and most of them received anti-thymocyte globulin. we also observed frequently co-existence of viral infections from herpes family as cmv and vh6. the main limitations of this work are its retrospective nature and it´s from a single center. more studies are necessary to better understand the epidemiology and risk factor associated with bkv-hc and the morbidities associated with its treatment. disclosure: nothing to declare how we manage hhv-6 reactivation in the posttransplant setting oscar borsani 1 , anna amelia colombo 1 , daniela caldera 1 , paolo bernasconi 1 1 university of pavia, san matteo hospital, pavia, italy background: hhv-6 encephalitis is a life-threatening complication in the post-transplant setting and it develops in about 1% of patients receiving traditional hsct. several risk factors were described. a differential diagnosis between hhv-6 encephalitis and other neurological complications is extremely important but often not-easy to achieve because of the highly heterogeneous clinical and radiological features and complexity of interpretation, especially in transplanted patients. here we described vignettes that represent and highlight distinct problems in the diagnosis and management of transplanted patients with suspected hhv-6 reactivation. methods: we collected the clinical, laboratory and radiological (electroencephalogram, brain mri and brain ct) data of transplanted patients who developed a neurological syndrome suspected for hhv-6 reactivation. hhv-6 was detected on serum and csf using rt-qpcr. results: 1) a 61-years-old patient developed a diffuse erythema and subsequent encephalitic syndrome following hsct. the brain mri revealed clear signs of limbic encephalitic and searching for hhv-6 on serum and csf revealed 150.000 copies/ml and 220.000 copies/ml respectively. an antiviral therapy was started but no clinical benefit was achieved. 2) a 59-years-old patient developed a typical neurological syndrome without brain mri findings of encephalitis and with no evidence of skin involvement. the lumbar puncture and csf analysis showed a total of 49.200 hhv-6 dna copies/ml. antiviral therapy with ganciclovir and foscarnet was promptly started with clinical improvement and a drastically reduction of hhv-6 dna on both csf and serum. a new brain mri revealed an acute limbic encephalitis. 3) a slight neurological syndrome consisting of confusion and amnesia developed in a 49-years-old-patient. brain mri findings were compatible with a wernicke syndrome, but no improvement of neurologic symptoms were obtained with thiamine supplementation. csf analysis did not revealed hhv-6 dna, which was detected at low copies number on serum analysis. a second brain mri was conclusive for limbic encephalitis, so an antiviral therapy with foscavir was started and radiological but not clinical improvement was noted. the patient died after few days. 4) in the last case we present a 52-years-old patient who developed a clinical picture of encephalopathy (i.e. amnesia, ataxia, drowsiness, weakness, depression) with rapid progression to coma after seventy-eight days from hsct. a brain mri showed a slight contrast enhancement in parietal-occipital regions. during the recovery phase from conditioning-induced cytopenia, an increasing in serum hhv-6 dna was detected. searching for hhv-6 dna on donor's follicles showed a chromosomally integrated hhv-6 (cihhv-6). cyclosporin a (csa) was interrupted and neurological improvement was observed in the following hours: a diagnosis of pres was made. conclusions: hhv-6 encephalitis should be suspected in transplanted patients with a clinical syndrome of encephalopathy. pcr detection of hhv-6 dna in csf associated with either typical brain mri abnormalities or a clinical diagnosis of nonspecific encephalopathy must lead to the urgent initiation of systemic antiviral treatment. if an increase of both serum hhv-6 dna and wbc is detected, a cihhv-6 should be confirmed. pres is an important differential diagnosis in transplanted patients which developed an encephalitic syndrome. disclosure: nothing to declare background: cytomegalovirus (cmv) infection is a major cause of morbidity and mortality after hematopoietic stem cell transplantation (hsct). it causes end-organ disease, multi-organ dysfunction syndrome, graft failure, increased susceptibility to infections and gvhd. greatest risk of cmv infection in a seropositive host is the reactivation of latent virus. methods: a prospective descriptive study performed at armed forces bone marrow transplant centre, rawalpindi, pakistan from dec 2016 to sep 2018. hundred consecutive patients who underwent hsct were followed with weekly cmv dna quantitative pcr from engraftment till day 100 for cmv reactivation. patients in whom cmv pcr showed more than 2000 copies/ml were treated with antiviral therapy. factors associated with cmv reactivation, outcome of antiviral therapy and effect of cmv on transplant outcome is studied. results: out of 100 cases, 82 were hla matched siblings, 15 were matched family donors and 3 were haploidentical transplants there were 66 males and 34 females. mean age was 11.9 ±8.9 years. fourty-two transplants were done in thalassemia, 35 in aplasia, 09 in leukemias and 14 in other hematological disorders and immune deficiencies. ninety-eight recipients and all the donors were cmv seropositive before hsct. cmv reactivation was seen in 81 patients and 42 of them had cmv viral load more than 2000 copies/ml and 39 patients had cmv viral load less than 2000 copies/ml. nineteen patients had no cmv reactivation. mean time to reactivation since transplant was 36±19 days. valganciclovir was given in 36 patients due to ease of administration and six patients were treated with ganciclovir during their hospital stay. only one patient had resistant disease. mean time to clear viremia was 20±12.8 days. the patients having viral load less than 2000 copies/ml, subsequently cleared cmv without any treatment. antiviral agents; ganciclovir and valganciclovir were equally effective for treating cmv infection with 99% efficacy, however, more adverse effects were seen with ganciclovir. myelosuppression i-iii was seen in 24% patients treated with valganciclovir and in 46% treated with valganciclovir. renal impairment i-ii was seen in 14% of valganciclovir and 20% of ganciclovir treated patients. steroid administration was strongly associated with cmv reactivation (p = 0.003). no statistically significant association was found with the use of atg, gvhd, underlying disease, abo or gender mismatch. os was 81.0 % and 93.1% in with and without cmv reactivation (p=0.1) and dfs was 83.3 % and 96.6 % in with and without cmv reactivation (p=0.06) conclusions: cmv reactivation was seen in 81% of the transplant recipients, this is higher compared to the western world due to high cmv seropositivity is this region. steroids administration in post-transplant period significantly increase the risk of cmv reactivation. preemptive therapy with valganciclovir effectively treats cmv reactivation with acceptable side effects. viral threshold for treatment should be decided considering the regional endemicity. cmv adversely affects the transplant outcome in terms of dfs and os. disclosure: no conflict of interest. acute nephritis requiring nephrectomy caused by adenovirus (hadv) and human polyomavirus bk (bkpyv) following allogeneic hematopoietic stem-cell transplantation in a patient with ph+ all background: adenovirus infection represents an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-hsct), with no established therapy. although different organs may be affected by disseminated hadv infections, kidney involvement has been rarely reported. co-infection of hadv and bkpyv are common complication in patients undergoing allo-hsct, but recent studies demonstrate that bkpyv may facilitate the replication of hadv and lead to elevated viremia with increased virulence and serious clinical consequences. here we report a case of an adult patient who required a monolateral nephrectomy due to hadv pyelonephritis as an early complication of allo-hsct for philadelphia-positive acute lymphoblastic leukemia (ph+ all). methods: in september 2014, an ethiopian gentleman was diagnosed with ph+ all at the age of 24 years. he was treated with polychemotherapy in association with the tyrosin kinase inhibitor imatinib mesylate achieving a complete remission (cr). one year later, due to disease relapse with cns involvement, he was started on vincristine and dexamethasone plus imatinib treatment and in april 2017 he was referred to our bmt center from ethiopia. upon confirmation of the p210 ph+ b-all diagnosis, therapy with the scr/abl dual inhibitor dasatinib associated to intrathecal chemotherapy was started and a salvage treatment with inotuzumab ozogamicin followed by an allogeneic hsct from a hla-identical brother was planned. having achieved a documented molecular cr disease status, in june 2017 the patient underwent allo-hsct following the fludarabine-melphalan reduced-intensity conditioning regimen. graft-versus-host prophylaxis included anti-thymocyte globulin, cyclosporine and mycophenolate mofetil results: on day +24 post-transplantation the patient developed macro-hematuria due to hemorrhagic cystitis and a ct scan unveiled a left pyelonephritis with marked kidney enlargement. kidney microbial investigations were all negative. at the same time, hadv viremia with very high copy number (>25000000 cp/ml) was documented and also elevated bkpyv (>25000000 cp/ml) viruria and viremia (6400 cp/ml). the genotyping of hadv evidenced serotype b11 mainly involved in infections of the urinary tract. treatment with cidofovir was immediately started; nonetheless, due to rapid clinical worsening despite maximal antibiotic therapy, on day +50 a left nephrectomy was performed, which led to a subsequent progressive resolution of the clinical symptoms and negativization of hadv and bkpyv viremia and viruria. pcr real time performed on the kidney tissue unveiled very high concentration of hadv copy number. conclusions: acute pyelonephritis due to disseminated hadv infection may represent a possible cause of severe complication following allo-hsct. monitoring of hadv copy number is helpful to evaluate infection severity and response to treatment. co-infection of hadv and bkpyv in immunocompromised patients should be always considered likely to worsen clinical course and outcome. disclosure: nothing to declare background: infection is a major cause of morbidity and mortality in patients (pts) receiving an allo-hsct. its severity is related primarily to the depth and duration of neutropenia. febrile neutropenia (fn) is defined as a neutrophil count below 500 cells/mm 3 and a fever ≥ 38.3°c at a single measurement or≥ 38°c 2 times at one hour intervals. the objective of our study is to analyze the epidemiological, clinical, biological characteristics of febrile episodes (fe) occurred in 136 pts who benefited an allo-csh over a period of 2 years. methods: from january 2016 to december 2017, 136 allo-hsct were performed in 136 pts including 106 sibling-hla identical, 26 haplo-identical and 04 phenoidentical for essentially acute leukemia (68 pts, 50%), acquired and congenital aplasia (45 pts, 33%). the median age is 22 years (3-59) and sex-ratio (m/f): 1.72. prophylaxis consisted on isolation sterile room with laminar flow, digestive decontamination, fluconazole and aciclovir. nine pts (6.6%) were infected at the time of hospitalization (cellulitis 03, pneumoniae 02, bacterial angina 01, veinitis 01, bronchial pneumonia 01, furuncle cutaneous 01) requiring treatment with antibiotics. conditioning regimen is myeloablative in all pts. anti-thymocyte globulin is used in 61 pts (44.8 %). peripheral blood stem cells (pbsc) are used in 117 pts (86%) with an average level of cd34+ cells: 7,5.10 6 /kg (2. 1-19.8) , bone marrow (bm) in 5 pts with a mean level of nucleated cells: 3.4 x 10 8 /kg (0.8-3.6) and the association of pbsc-bm in 14 pts (haplo-identical). at each fe, are practiced: chest x-ray, procalcitonin test, blood culture, microbiological study of urine and stool (if diarrhea). results: all pts showed aplasia with an average duration of 16 days (6-49), neutrophil engraftment was observed at day 18 (9-31). one hundred and twenty-nine pts (94.8%) presented 205 fe with an average of 1.5 per pt. eleven pts (8%) had 3 fe or more. forty nine (23,9 %) fe are clinically documented (digestive: 19, skin: 14, pulmonary: 11, urinary: 03, oto-rhino-laryngology: 02). the blood cultures are made at 195 fe, 136 fe are microbiologically documented (66 %): gram-positive bacteremia in 77% (mainly coagulase negative staphylococci) and gramnegative bacilli in 23% of cases. procalcitonin test performed during 157 fe: normal (32 cases), probable infection (90 cases), probable sepsis (15 cases), severe sepsis (19 cases) and septic shock (one case). empirical double antibiotic therapy is initiated in 120 pts without waiting for the results of the microbiological study. this association was sufficient in 15 pts (11 %). the transition to a second line was needed in 114 pts (83.8%) and third line in 82 pts (60%). antifungal is added in 36 cases (27%). eight pts benefited from g-csf. the evolution is favorable in 202 fe (98.5%), apyrexia obtained after an average of 3.3 days . three pts died (2%) by severe sepsis on a durable aplasia, 2 of which had a cellulitis before the conditioning. conclusions: fe increase morbidity and mortality in allo-hsct so prophylactic measures are essential. empirical antibiotics treatment has to be instituted very quickly in the absence of documentation. disclosure: nothing to declare p472 abstract withdrawn. atsushi satake 1 , masaaki hotta 1 , ryo saito 1 , akiko konishi 1 , hideaki yoshimura 1 , takahisa nakanishi 1 , shinya fujita 1 , tomoki ito 1 , kazuyoshi ishii 1 , shosaku nomura 1 1 kansai medical university, osaka, japan background: cytomegalovirus (cmv) infection remains a common complication after allogeneic hematopoietic stem cell transplantation (ahsct), which results in increased morbidity and mortality. letermovir is a novel anti cmv drug that inhibits the cmv-terminase complex. the purpose of this retrospective study is to elucidate the efficacy and safety of cmv prophylaxis with letermovir early after ahsct in clinical practice. methods: we retrospectively analyzed the incidence of cmv infection, cmv disease, preemptive therapy, adverse events through week 14 after ahsct, the rates of engraftment and overall survival. all patients underwent ahsct in our institution for hematopoietic malignancies between may 2018 and nov 2018. data collected in this study included patient's characteristics such as age, sex, disease status, donor source and cmv disease risk. cmv infection was evaluated by cmv antigenemia. this study was approved by the research ethics committee of the faculty of medicine, kansai medical university. results: thirteen patients (male 9, female 4) underwent ahsct and received cmv prophylaxis with letermovir. the median age was 54 years (range, 18-65 years). overall, 10 of 13 patients (76.9 %) were considered to be at high risk for cmv, including 5 patients (38.5%) with haploidentical donors, and 1 (7.7 %)with mismatched, unrelated donors. all patients began letermovir from day 0 after ahsct, and achieved engraftment (median 17, 13-22 days). no patient developed cmv disease and required preemptive therapy. one patient died of treatment-related mortality, and 2 patients died of acute gvhd. although one patient discontinued letermovir before day 100 after ahsct because letermovir was suspected to be a cause of persistent nausea, severe adverse events were not observed. conclusions: it is still unknown whether cmv prophylaxis with letermovir improves os and reduces trm; however, our data suggests that cmv infection is considerably inhibited by administration of letermovir early after ahsct. clinical trial registry: not applicable. disclosure: the authors declare noconflicts of interest for this study. background: cytomegalovirus (cmv) is cause of increased morbidity and mortality after transplantation of hematopoietic cells. the pathogenesis of cmv disease or infection is complex with multiple interactions with the immune system, mainly in acute and chronic graft-versus-host disease (gvhd). the aim of this study is to analyze the risk factors for the reactivation of cmv in patients undergoing allogeneic hematopoietic cell transplantation (hct). methods: prospective descriptive study of the risk factors for the reactivation of cmv in the described population. univariate and multivariate analysis of the predisposing factors were performed: donor graft, treatment with corticosteroids, use of antithymoglobin, serologic status, conditioning regimen and the presence of gvhd. results: during the period between august 2014 until january 2017, 71 patients were evaluated. 42.25% (n:30) had reactivation of cmv. average reactivation was 58 days post transplant. both (recipient and the donor) had positive cmv igg in 78.9%. in the univariate analysis, the reactivation of cmv was associated with haploidentical transplantation (p: <0.01), with the use of corticosteroids (p: <0.01) and gvhd (p: <0.01). in the multivariate analysis, the haploidentical transplant maintained its statistical significance in comparison with the related allogeneic transplant (p: 0.0012, or:7.07; ic95%:2.4-20.6) as well as the use of corticosteroids (p: 0.0014, or:9.25; ic95%:2.6-32.4). 100% of patients receiving corticosteroid treatment had grade ii / iii gvhd. the serologicac status, myeloablative conditioning regimen and the use of atg showed no statistically significant association. conclusions: in patients undergoing allogeneic transplantation, were found as risk factor to reactivation, those who received haploidentic transplantation and treatment with corticosteroids. another risk factor that showed greater reactivation was the presence of gvhd. disclosure: nothing to declare methods: a 37 y/o male was referred for allogeneic transplant following 3 cycles of induction therapy for aml with complex karyotype and axsl1 mutation having achieved complete remission following the first cycle of chemotherapy. his first induction cycle was complicated by a perianal myeloid sarcoma which became infected and required surgical drainage and formation of a defunctioning colostomy. results: following 2 allogeneic transplants, the first complicated by secondary graft failure and the second by primary graft failure he presented with two skin lesions, with a third lesion adjacent to his stoma developing shortly after admission. all lesions were erythematous with central necrosis and progressed rapidly in size over 48 hours. biopsy of the skin and para-stomal lesions revealed fungal mycelia, with culture subsequently identifying rhizopus oryzae. initial treatment was with liposomal amphotericin b 5mg/kg/day followed by dose escalation to 10mg/kg/day due to the development of new skin lesions. the patient had been taking posaconazole (tablet) prophylaxis since his first allogeneic transplant and peripheral blood drug levels checked at the time of admission were therapeutic confirming that this was a breakthrough fungal infection. consequently posaconazole was stopped and isavuconazole added to the treatment regimen. surgical assessment was undertaken but surgery was deferred on the basis of high risk due to the extent of the infection and the patient´s profound pancytopenia. the organism was tested for in vitro susceptibilitiy and found to be resistant to posaconazole (mic >16mg/l), with borderline resistance to isavuconazole (mic 16mg/l) and sensitive to amphotericin b (mic 0.25mg/l) (phe mycology reference laboratory, england). isavuconazole was therefore stopped and the patient was managed with liposomal amphotericin b along with daily granulocyte infusions. he underwent a third allogeneic transplant using a different unrelated donor and stable engraftment was achieved. post transplant there was initially an increase in the size of the para-stomal lesion, but no new skin lesions developed. following engraftment he underwent resection of the stomal lesion, with primary closure and re-siting of his stoma. amphotericin b was replaced by isavuconazole prophylaxis on discharge and he continues to make an excellent recovery. conclusions: whilst aspergillus species remain the most common cause of invasive fungal infections in allogeneic transplant patients, other species including the mucorales are seen, and generally associated with poorer outcomes. whilst there are standardised methodologies for susceptibility testing, fungi specific cut offs based on clinical outcomes are only available for a limited number of species/ antifungal agents. in this case, susceptibility testing demonstrated resistance to posaconazole which was consistent with the clinical presentation of invasive infection despite therapeutic levels of posaconazole. it is also worth noting that an estimated 45% of r. oryzae isolates in the uk are resistant to posaconazole. treatment with high dose amphotericin b resulted in improvement in small skin lesions with stabilisation of the larger stomal lesion until count recovery allowed surgical resection. background: total depletion of innate and adaptive immune cell populations occurs after intensive chemotherapy and hematopoietic stem cell transplantation (hsct). both t and b lymphocyte pools are restored slower that myelomonocytic populations. hsct patients are at high risk for bacterial and viral infections at early terms (< 100 days) post-transplant. the reconstitution of the immune system depends on the time required for stem cell recruitment, differentiation, expansion, maturation and release into the bloodstream. restoration terms for myeloid cells after hsct are usually defined as the 1st day with neutrophil count of ≥ 0.5 x 10^9/l with mean recovery terms of 12 to 20 days. high occurrence of cytomegalovirus (cmv) in hsct patients mostly result from reactivation of a latent virus acquired in early childhood. however, delayed immune reconstitution and subsequent infections such as cmv, adenovirus (adv) or herpes 6 (hhv-6) diseases are not unusual and still constitute a major cause of death in peru. methods: peruvian pediatric patients (n=22) diagnosed with aplastic anemia, mds, aml or all underwent a haploidentical hsct performed with the clinimacs device. patients treated were separated in two groups. the group of patients who received viral prophylaxis (ganciclovir) was compared to the group that did not receive any prophylaxis treatment. viral reactivation was confirmed by pcr test twice a week and clinical signs within 15 days after hsct. results: in the group that didn´t received prophylactic treatment, engraftment occurred close to day 10 post haplo-hsct and none of the patients developed gvhd (graft versus host disease). nevertheless, incidences of cmv, hhv-6 and bkv infections before day 15 post haplo-hsct were still high. an overall survival (os) over 65% with an ic 95% was reached at the end of the first year. on the other hand, the group of patients that received prophylaxis with ganciclovir did not developed gvhd and reached the engraftment close to day 10 with a very low viremia incidence after the first month post haplo-hsct. all viral reactivations were caused by cmv and the os was over 91% with an ic 95% at the end of the first year. previous prophylaxis to both the donor and the receptor with ganciclovir (5mg/kg) every 12 hours before and during the conditioning regimen has allowed a better control of viral reactivation. conclusions: the attempts to improve immune function and reduce nonrelapse mortality from infectious complications without increasing gvhd have focused on a partial t cell depleted graft, such as t cell depletion (tcr α/β). this graft retains a large numbers of effector cells, such as tcr γ/δ and natural killer cells. however, delayed immune reconstitution and subsequent infections are a big issue. a novel partial t cell depletion strategy such as depleted naïve t cells (cd45ra+ t cells) could enhance the recovery of immune function after haplo-hsct because donor pathogen memory t cells from the donor are retained. it is necessary to increase the studies and the database to set the scheme of previous prophylaxis to the recipient to contain the viral reactivation and to help a rapid immune reconstitution. disclosure: no conflict of interest is declared information was recovered from the medical records. results: thirty-four patients were included, of them with the following diagnoses: acute leukemia (27), granulocytic chronic leukemia (4), dendritic cell neoplasia (2), aplastic anemia (1). 80% of the patients received a transplant from an identical hla donor and 20% received a haploidentical transplant. mean age's patients was 32 years (19-57). prophylaxis with posaconazole was performed on 44% of the patients with identical hla and 86% on haploidentical group; the rest of the patients received fluconazole. the posaconazole group presented: fever 61%, mucositis gi-ii 94%, gastrointestinal toxicity gi-ii 67% (p= 0.006), hepatic toxicity 11%, kidney toxicity 17%, oral candidiasis 100%. during this period none of the patients presented invasive fungal infection in any group. there were 2 deceases, one on each group and none related to a fungal infection. the overall survival was of the 92% versus 93% on the posaconazole group and the fluconazole group respectively. conclusions: the prophylaxis with posaconazole and fluconazole is effective on the prevention of invasive fungal infection on the first 100 days. the toxicity was similar on both groups. posaconazole can be effective on the prevention of the haploidentical type. is necessary to continue following the patients with infection risk on a long-term period associated with the chronic gvhd. disclosure: none declared lymphoma these results open the question whether allo-sct should still be offered to these patients. methods: we aimed to define the role of allo-sct in refractory or relapsing after two lines de novo or transformed dlbcl patients, and its comparison with zuma-1 car-t cells trial (neelapu et al nejm 2017). we analyse long-term allo-sct results in 40 de novo (n=23) or transformed dlbcl (n=17) out of the 1000 allo-sct performed in our institution between october 1995 to october 2018. results: patients and transplant characteristics are summarized in table 1 . complete response (cr) at 100 days was 67,5% and 83% of them remain in cr at 12 months. with a median follow-up of 46 months, 5-year progression-free survival (pfs) was 54% and 5-year overall survival (os) 48%, with a 3-year transplant-related mortality of 18%. refractoriness at the time of the transplant was associated with a poorer prognosis, with only 2 out of 9 refractory patients being long term survivors (figure 1 ). similar results were reported for zuma-1 trial, with a best response of 55% cr retained in 79% of them at 12 months. with a median follow-up of 15 months, 18-months pfs was 41% and 18-months os 53%. patients characteristics did not differ in our series and zuma-1, except that all the patients in zuma-1 were refractory prior to therapy (table 1) . conclusions: although very few patients with de novo or transformed dlbcl are offering an allo-sct (4% of all allo-sct), this is a curative option in chemosensitive patients and with more mature data and longer follow-up than with car-t therapy; for these reasons, it should still be offer to these poor prognosis patients. moreover, almost all patients have now available donor, better graft-versus-host disease prophylaxis will decrease trm and morbidity, and new therapies will make more patients in sensitive disease before allo-sct. therefore, allo-sct and car-t cells are strategies to be discussed in every young patient with available donor. disclosure: honoraria as advisor or speaker from gilead ( methods: consecutive patients transplanted for hgbl (excluding burkitts lymphoma) between 2007-2017 in the ebmt database were included. data collected included age, sex, pathology subtype (hgbl (including subtypes), tfl, dhl), disease status at sct, conditioning (ma vs beam cam vs flu-mel-cam/atg), engraftment, day 100 outcome, trm, os and pfs and eligibility for emea licensed indication of car-t therapy. results: fifty patients (29m, 21f) with a median age of 47 at diagnosis and 50 at sct were included. the subtypes included hgbl (n=29), tfl (n=11) and dhl (n=10). indications for sct were: primary refractory (n=17), relapse < 12 months after primary treatment (n=12), previous autologous-sct (n=10) and dhl (n=11). the median lines of therapy was 4 (range 1 to 6). conditioning used was cytbi n=21, bu/cy n=1, etop/tbi n=1, flubucy n=1, beamcam n=7, fmc/t, n= 19. all patients engrafted with neutrophil >1.0 10 9 /l at median 15 days and platelets >20 10 9 /l at median 17days. the day 100 mortality was 8% (progressive disease 4%, nrm 4%) with a 5 year os of 56% and mortality due to progressive disease 22% and nrm 16%. disease subtype influenced outcome with an os for primary refractory hgbl, relapsed hgbl, tfl and dhl respectively of 53%, 58%, 57% and 89%. 23 patients were eligible for a licensed car-t product. conclusions: the outcome of these high risk hgbl patients have an acceptable os of 56%, with relapsed disease being the commonest cause of mortality. patients with dhl have a particularly good outcome in this series; recent evidence indicates that some of these patients with a non-immunoglobulin gene associated myc translocation could be managed more conservatively (ash sehn). the outcomes achieved with allogeneic-sct in this series will provide a baseline for outcome assessment with a cart program. disclosure: nothing to declare background: immune checkpoint inhibitors (ici) allow to achieve a durable remission in patients with resistant or refractory (r/r) classical hodgkin lymphoma. however, the information about optimal duration of therapy and the prognosis of the patients after ici cessation is limited (manson, blood 2018). therefore, the optimal role of hematopoietic sct (hsct) in this patient group is not defined. our aim was to determine remission duration in patients who discontinued ici monotherapy after achieving complete remission (cr). methods: this analysis included 20 patients (5 male/15 female) aged 19 to 47 (median 32 years) with r/r classical hodgkin lymphoma who were treated with nivolumab (3 mg/kg every 14 days) and achieved cr. response was assessed by positron-emission tomography/computed tomography (pet/ct) using lyric criteria every 3 month. after nivolumab therapy had been stopped the patients received no other treatment and disease was assessed every 3 months by pet/ct. median follow-up after therapy discontinuation was 20 (10-21) months. results: at the moment of therapy initiation 14 (70%) patients had stage 4 disease, 11 (55%) patients had progressive disease (pd), 4 (20%) patients had stable disease, 3 (15%) patients had partial remission and 2 (10%)complete remission; 12 (60%) patients had b-symptoms and ecog score >1. the median number of previous therapy lines was 5 (3) (4) (5) (6) (7) (8) (9) (10) . before nivolumab initiation high dose chemotherapy with autologous sct was performed in 9 patients (45%) and 8 (40%) received brentuximab vedotin. the median number of nivolumab cycles was 25 (18-30). cr was achieved after median of 6 (6-18) cycles. the median duration of therapy after achievement of cr was 7 (1-15) months. at the time of analysis, all patients were alive, 8 (40%) out of 20 patients relapsed after therapy discontinuation. the median progression-free survival (pfs) for the total group was not achieved. among patients with relapse, the median time before pd was 11 (5-20) months. after relapse all patients were retreated with nivolumab monotherapy or with chemotherapy combination. one patient achieved complete remission; 1 -partial remission; 1 -indeterminate response type 2. other patients are continuing the therapy and their response has not yet been evaluated. conclusions: while complete response was maintained in some patients at median follow up of 20 months after nivolumab therapy cessation, the pfs plateau was not reached. we report that patients with relapse after nivolumab discontinuation sustained sensitivity to nivolumab and achieved a response during retreatment with nivolumab monotherapy or with chemotherapy combination. in patients with unsatisfactory response to nivolumab retreatment, hsct option should be considered. disclosure: nothing to declare high dose chemotherapy with autologous stem cell transplantation in primary central nervous system lymphoma: data from the japan society for hematopoietic cell transplantation (jshct) registry center hospital, tokyo, japan, 4 national cancer institute, bethesda, md, united states, 5 okayama university hospital, okayama, japan, 6 kanazawa medical university, uchinada, japan, 7 kyoto university, kyoto, japan, 8 aomori prefectural central hospital, aomori, japan, 9 yamagata unversity school of medicine, yamagata, japan, 10 tenri hospital, tenri, japan, 11 hiroshima university, hiroshima, japan, 12 japanese data center for hematopoietic cell transplantation, nagoya, japan, 13 nagoya university graduate school of medicine, nagoya, japan, 14 shimane university hospital, izumo, japan background: high-dose chemotherapy (hdt) with autologous stem cell transplantation (asct) has been shown to improve prognosis of patients with central nervous system (cns) lymphoma. whereas the common regimen of hdt for pcnsl in the europe and the us is thiotepa-based regimen, e.g. bcnu-thiotepa, tbc (thiotepa-busulfan-cyclophosphamide), thiotepa-based regimen was only available before discontinuation of thiotepa in 2009 in japan. we report the results of asct for pcnsl from the japan society for hematopoietic cell transplantation (jshct) registry. methods: data from the jshct registry were retrospectively analyzed. 102 patients with pcnsl who received first hdt/asct between 2006 and 2015 were evaluated. distribution differences of clinical characteristics between groups were analyzed with fisher´s exact or mann-whitney u tests. overall survival (os) and progression free survival (pfs) were calculated using kaplan-meier method. two-group analysis of the cumulative incidence of relapse was conducted using the grey test. factors were analyzed in univariable analysis, and all factors with p≤.1 were retained in the multivariable model. all p values were 2 sided, and values were regarded statistically significant if p< .05. results: median age was 54 months (range 20-74) with 15 patients over 64 years of age; 38 males and 64 females. ecog-performance status (ps) at diagnosis was better (ps0-1) in 86 patients and poor (ps2-4) in 16 patients. serum lactate dehydrogenase (ldh) levels at diagnosis were elevated in 17 patients. karnofsky ps and cerebrospinal fluid (csf) protein concentration at diagnosis were not collected in the registry. 71 patients were in complete remission (cr), 21 patients were in partial response (pr), and 7 patients were stable disease (sd) or progressive disease (pd) at the time of hdt/asct. after hdt/asct, additional 20 patients achieved cr. with median follow-up period of 44 months, the 5-year os and pfs were 54.9% and 38.4%, respectively. the was no significant difference in os and pfs between upfront and salvage hdt/asct. since thiotepa, a key agent in hdt/asct for pcnsl, has been unavailable after the discontinuation in japan, the hdt regimens used were not uniform. thiotepa-containing hdt was received by 16 out of 32 patients before 2010, but by 2 out of 70 patients after 2011. thiotepa-containing hdt showed improved pfs (p=.019), lower relapse (p=.042) and a trend toward a survival benefit. in the multivariate analysis, non-complete remission at hdt/asct was an independent predictor for os (hr=2.40, 95%ci:1.25-4.58, p=.008) and thiotepacontaining hdt remained significant for pfs (hr=0.42, 95%ci:0.19-0.95, p=.038). [[p481 image] 1. os(a),pfs(b) in all patients (n=102) and cumulative incidence of relapse in cr patients (c; n=91)] conclusions: our results confirm the activity of thiotepacontaining regimen for hdt/asct in pcnsl patients. currently a pharmaceutical company re-develops thiotepa for new approval of hdt/asct in pediatric solid cancer and adult lymphoma in japan (japiccti-163433). further evaluation with the thiotepa by prospective clinical trials is warranted. disclosure background: t-cell non-hodgkin lymphomas (t-nhl) are rare diseases and they are associated with worse prognosis when compared to their b-cell counterparts. allogeneic stem cell transplantation (allo-sct) may have a curative potential for these patients due to the graft versus lymphoma effect. however, data is limited on the efficacy of allo-sct for these diseases. methods: we identified 53 patients (32% females; median age: 43 y; range,4-67) with t-nhl that underwent allo-sct at university hospital eppendorf between 1992 and 2017. twenty-one patients (underwent allo-sct from a matched sibling donor (msd) and 32 (60%) from a matched unrelated donor (mud). sixteen patients had ptcl (30%), n=8 (15%) anaplastic large-cell lymphoma (alcl), n=8 (15%) angioimmunoblastic large cell lymphoma, n=8 (15%) adult t-cell leukemia/lymphoma, n=5 (9%) hepatosplenic gamma/delta t-cell lymphoma, n=4 (8%) enteropathy associated t-cell lymphoma, n=2 (4%) tcell-prolymphocytic leukemia, and n=1 (2%) each extranodal t/nk-cell lymphoma, cutaneous t-cell lymphoma as underlying diagnosis. the median ann arbour stage at diagnosis was 4 (range, 1-4). ten patients (19%) had bone marrow involvement at diagnosis. all patients were heavily pretreated, 17 (32%) patients relapsed post autologous stem cell transplant (apsct) and one patient post allo-sct. fifteen patients (28%) were transplanted in complete remission (cr) (10 in 1 st cr, 2 in 2 nd cr), n=13 (25%) in partial remission (pr), and n=21 (40%) with advanced disease. most of the patients received myeloablative conditioning (91%). thirty-eight (72%) patients received total body irradiation based regimens and 15 (28%) received chemotherapy based regimens. twenty patients (38%) received anti-t-lymphocyte globulin (atlg neovii), and most patients (87%) received g-csf mobilized peripheral stem cells. results: overall, 49 patients (92%) had neutrophil engraftment (median days: 13; range,9-36) . at day 100, the cumulative incidences of grade ii-iv and grade iii-iv acute gvhd were 42% and 15%, respectively. after a median follow up of 12 months (range, 1-171) the cumulative incidences of chronic gvhd was 12% distributed evenly between limited and extensive. twenty nine patients (55%) achieved cr after allo-sct. median overall survival (os) and disease free (pfs) survival were 44 months and 12 months respectively. the 3 year os and pfs were 50% and 43% respectively. fourteen (26%; 95% ci [0.15-0.40]) deaths were due to non relapse mortality (nrm) and 15 patients (28%; 95% ci [0.17-0.42]) died due to disease progression. patients with a male donor had improved os compared to those with a female donor (3 year os male 56%, female 34%; p=0.038). patient gender, disease subtype, bone marrow involvement, type of allo-sct, donor, patient cmv status, abo incompatibility, disease stage at diagnosis, previous transplant, disease status at transplant, conditioning regimen, atg and stem cell source had no effect on os, pfs, nrm, and post transplant complications. conclusions: acknowledging the retrospective nature, our study shows that allo-sct induces high rates of complete remission, and may have a curative potential even in diseases relapsing post asct. however our findings need to be confirmed in larger prospective studies. disclosure: no funding, no conflict of interest p483 abstract already published. at-home autologous stem cell transplantation in lymphoma patients: clinical impact of non-g-csf administration post-transplant background: severe neutropenia remains the main cause of morbidity and mortality after autologous stem cell transplantation (asct). g-csf administration after asct is a common practice, performed to reduce the duration of neutropenia and its complications. in a previous work in patients with multiple myeloma managed at home after asct, we did not observe a deleterious clinical impact in those patients that did not receive g-csf post-transplant (martinez-cibrian n. et al, bmt 2016) . despite the fact that lymphoma patients receive a more intensive conditioning regimen that multiple myeloma patients, we hypothesized that the use of g-csf in lymphoma patients managed at home during the aplasia phase of asct does not provide a significant clinical benefit. methods: 93 lymphoma patients were managed at-home since day +1 of asct. between february 2010 and july 2016, 68 patients received at-home g-csf 5 μg/kg per day since day +7 until their anc reached 1x10 9 /l (g-csf group) and, since august 2016, 25 patients did not receive g-csf (non-g-csf group). all patients were conditioned with beam and received prophylaxis with a quinolone, fluconazole, aerosolized pentamidine and low-dose acyclovir (hvs+). in all cases we added primary prophylaxis with piperacillin-tazobactam 4.5 g/8h i.v., using a portable intermittent infusion pump (iip), from an absolute neutrophil count (anc) < 0.5x10 9 /l until the first day of fever or until attaining an anc of 1x10 9 /l. first-line therapy at home of neutropenic fever (nf) was refrigerated meropenem 1 g/8h i.v using a portable iip. fever was an indication of immediate visit to the hospital, and those patients presenting with focal infection or signs of severe sepsis were admitted. other indications for readmission were: willingness of the patient or caregiver; uncontrolled nausea, vomiting or diarrhea and mucositis requiring total parenteral nutrition or i.v. morphics. results: the main characteristics of the patients are shown in table 1. there were no differences between groups with respect to gender, diagnosis, stage of disease, comorbidity index (hct-ci), source of stem cells (peripheral blood) and cd34 + cell dose infused. the median (range) age (years) was 44 (19-71) in g-csf group and 52 in non-g-csf group (p=0.03). duration of neutropenia less than 0.5 x10 9 /l was significantly longer in non-g-csf group, with a median of 11 days (range 6-19), compared with 8 (range 6-17) in g-csf group (p < 0.0001). severe neutropenia, less than 0.1x10 9 /l, was also longer in the non-g-csf group (8 days (4-11) vs. 7 (5-13); p=0.014). no differences were observed in the time to platelet engraftment. g-csf post-transplant avoidance did not influence the incidence of neutropenic fever, the first day and duration of fever, the incidence and severity of oral mucositis, bacterial infections documented and number of readmissions. the median duration of the whole procedure at-home was 1 day shorter in the g-csf group (14 vs. 15 days; p=0.12). conclusions: the policy of not administering g-csf post-asct in our home-based program for lymphoma patients, that include intensive bacterial prophylaxis, did not have a deleterious impact on the main results reviewed, which suggests that elimination of its use can be achieved. disclosure the aim of this study was to analyze the spanish experience with patients diagnosed of nhl who received haplosct with pt-cy. methods: sixty patients who received haplosct with pt-cy in 17 spanish centers from 2012 to 2016 were analyzed. patients were followed-up until 2017. gvhd prophylaxis consisted in cyclophosphamide 50 mg/kg/d on days +3 and +4, and mmf and a calcineurin inhibitor from day +5. results: patients' characteristics are summarized on table 1 . median age of patients was 50, 75% male, and diagnosed from t cell lymphoma (37%). most of them didn´t achieve complete response prior to transplant (55%), but only 15% with active disease. up to 63% of patients had received previous transplant, from which 5% was an allogeneic transplantation. source of stem cells was mainly peripheral blood (90%), and reduced intensity conditioning was the preferred (82%) regimen. donors were 43% siblings (26), 38% offspring (23), and 18% parents (11). median neutrophil and platelet engraftment was 18 (16-21) and 28 (20-42) days, respectively. acute gvhd grade ii-iv rate was 55%, with only 6 patients developing grade iii-iv (10%). chronic gvhd rate was 17%, and only in 3 (5%) was extensive. median follow-up was 14 months. the 2-year overall survival and event free survival was 43% and 39%, respectively. the 2-year cumulative incidence of relapse was 14% and 2-year cumulative incidence of nrm was 22%. conclusions: relapsed/refractory nhl are aggressive entities with a fatal course in a short period of time. haplosct with pt-cy permit a new treatment option among these patients, with acceptable outcomes. more studies are needed with a larger cohort of patients and longer follow-up to confirm these results. disclosure: nothing to disclose. higher suv at pre-transplant and day 100 posttransplant pet scan both independently predict inferior survival in patients with diffuse large b cell lymphoma background: autologous stem cell transplant (auto-hct) can cure some patients with relapsed diffuse large b-cell lymphoma (dlbcl) but relapse occurs in about 50% of patients. while our center and others utilize routine surveillance imaging post-transplant, the utility in this setting is unclear. imaging is costly and exposes patients to radiation. novel interventions are now available for patients relapsing after auto-hct making early disease recognition crucial to intervene prior to clinical progression. hence, we studied impact of post-auto-hct surveillance (18)f-fdg-pet ct at day 100 on transplant outcomes. methods: we analyzed a cohort of 131 consecutive auto-hct recipients with relapsed/refractory dlbcl who then underwent pre-transplant pet/ct and surveillance pet ct at day 100 (interquartile range (iqr): 97-103 days) post-hct at the university of minnesota medical center. univariate analysis was performed to analyze pet parameters including deauville score (d), standardized uptake values (suv), total lesion glycolysis (tlg) and total metabolic tumor volume (tmtv) as predictors of relapse and survival after auto-hct. in addition, we assessed outcomes of patients with clinically versus radiographically detected relapsed dlbcl after auto-hct. other pre-hct factors analyzed included age, gender, conditioning regimen, performance status, consolidation radiation therapy, tmtv, suv, tlg. results: five-year cumulative incidence of relapse after auto-hct was 50% (95%ci 39 to 59) and overall survival (os) was 51% (95% ci 41 to 63). twelve (9%) relapsed prior to day 100. d-score for 91 patients with d100 pet/ct were d1 (22%), d2 (55%), d3 (0%), d4 (10%), d5 (13%) with median survival in years for d1, d2, d4 and d5 of 6.0, 6.8, 4.7, and 1.2, respectively. mean suv varied from 1.53 (d1) to 17.9 (d5). suv was predictive of relapse and os. risk of relapse increased with doubling of suv; 2-fold higher suv increased hr by 1.77 (95%ci 1.34-2.33; p= 0.01). mortality increased with doubling of suv in both pre-hct (2-fold increase in suv associated with hr 1.46 [95% ci 1.1 to 1.8]; p=0.01) as well as post-hct pet (hr 1.7 [95% ci 1.3 to 2.3]; p=0) irrespective of the bulk of tumor. in addition, risk of death was 4 times higher in d5 patients relative to d1 (hr 4.10 [95% ci =1.56 to 10.77]; p≤0.01). patients with d5 (n=12) had higher tmtv (137 cm 3 ) compared to d4 (n=9, tmtv 12.5 cm 3 ). the hazard ratio for death following relapse was 2-fold higher (hr 1.8 [95% ci 0.9 to 3.4]; p=0.08) if relapse was detected clinically versus only radiographically over a median follow-up time period of 3.3 years. other pretransplant patient and disease characteristics did not significantly influenced the outcomes. conclusions: in patients with r/r dlbcl undergoing auto-hct, surveillance pet/ct at day 100 identified patients with poor survival~1 year. higher suv in both pre-transplant as well as post-hct pet was predictive of increased mortality. these patients may benefit from novel treatments. [ there are concerns about the risks of nivolumab treatment before and after allo-hsct, due to the risk of heavy gvhd, thus the place of immune checkpoints inhibitors is not yet defined. this report include analysis of our center experience of nivolumab treatment in patients with r/r hl before and after allohsct. methods: we retrospectively evaluated the results of allohsct in 86 patients with r/r chl who had undergone transplant from 2002 to 2018. the analysis included patients received the flube conditioning and ptcy gvhd prophylaxis. in group a patients (n=20) received bridge therapy with nivоlumab and in group b patients (n=34) received bridge therapy with brentuximab vedotin or chemotherapy-based bridges. time from the last nivolumab administration to allohsct was at least 2 months. results: at the time of analysis, median follow-up was 12 (1-20) months for group a, and 15 (1-64) months for group b. there was no difference in two-year os (p=0,39) with significantly better efs (p=0,025) for group a versus group b: 95% and 95% vs 85,3% and 62% respectively. relapse incidence was 0% for group a versus 26,5% in group b (p=0,025). cumulative incidence of non-relapse mortality at 2 years was 5,0% and 13,8% in group a and group b, respectively (p=0,631). there was no difference in grade ii-iv (44% vs 27%, p=0.23) and grade iii-iv (22% vs 13%, p=0.3) agvhd, as well as extensive chronic gvhd (21% vs 28%, p=0,83) in groups a and b, respectively. ten patients with relapse after allohsct were treated with different doses (0,5-3 mg/kg) of nivolumab in cic725 center. at the median follow up of 16 mo (0,6-28) all patients remain alive. the objective response to therapy was assessed in 7 patients noted in all patients (100%), disregard the dose of the nivolumab: cr in 29%, and pr in 71%. the response was lost in four patients, which required nivolumab retreatment. none of the patients developed gvhd after nivolumab administration. in this analysis, there was also no correlation between dose of nivolumab and incidence and severity of adverse events. conclusions: allohsct in combination with immune checkpoints inhibitors is a good option for patients with r/r chl. consideration for immune-mediated toxicities and the potential for increased graft-versus-host disease remain important. early data suggest that nivolumab may be an efficient therapy in patients with r/r chl relapse after allo-hsct. further research needed. disclosure: the authors declare no conflicts of interest. background: transformation to diffuse large b-cell lymphoma (dlbcl) is considered to be one of the most unfavourable events of lymphoma natural history with poorer outcome as compared to de novo dlbcl (alonso-álvarez et al, bjh 2017). in patients suitable for salvage therapy, hematopoietic stem-cell transplantation (sct) could be an option, although its role is not well stablished. we analyse indication and outcome after transplant in transformed dlbcl at a single reference transplant unit. methods: out of 2565 total of transplants performed at our unit between 1995 and 2018 -1564 autologous and 1001 allogeneic-51 were dlbcl transformed from an indolent nhl. of them, 36 received an autologous sct (asct) and 15 an allogeneic sct (allo-sct). results: median age was 60 years old (range 40-69) and 52 (range 35-65) for patients receiving asct and allo-sct, respectively. all asct received beam as a conditioning regimen and most of the patients in the allo-sct group received a fludarabine/melphalan combination (73%). gvhd prophylaxis consisted on tacrolimus/sirolimus combination in 87% and calcioneurin plus methotrexate in 13%. regarding transplant disease status, 28 (78%) of the asct patients were transplanted in complete response (cr). in the allo-sct group, 11 (73%) patients had received three or more treatment lines before transplant and 13 patients (87%) had received a previous asct, being 11 (73%) in cr, 3 in partial response (pr) and 1 in progressive disease. transplant related mortality (trm) was 5.6% in the asct and 27% in the allo-sct group. overall survival (os) and progression-free-survival (pfs) at 25 months were 94% (os), 76% (pfs) for patients receiving asct and 63% (os) and 56% (pfs) for allo-sct. with a median follow up of 57 months for patients receiving an asct, 21 (58%) remain in cr. in the allo-sct group median follow up is 24 months for the whole group and 50 months for alive patients; 9 patients are alive and disease free and 6 have died, 4 due to trm (28%). regarding progression, 12 (33%) have progressed after autologous transplant and 2 after allo-sct. conclusions: indication for hematopoietic sct in transformed dlbcl is stablished in few patients. only 2% of the patients in our transplant unit receive a transplant due to transformed lymphoma, corresponding to a 2.3% of autologous activity and 1.5% of allogeneic activity. according to our results transplant should be considered a curative option. most of our patients were transplanted in cr, so new agents trying to reach best response before transplant should be considered. [[p488 image] 1. eva konirova 1 , antonin vitek 2 , marta krejci 3 , edgar faber 4 , katerina steinerova 5 , david belada 6 , jan novak 7 , juraj duras 8 , petr sedlacek 9 , veronika valkova 2 , andrea janikova 3 , ludek raida 4 , pavel jindra 5 , pavel zak 6 , tomas kozak 10 , marie trnkova 1 , michal karas 5 , marek trneny 1 management. however, differences in patient's characteristics as well as frequency of hsct indication in different lymphoma subtypes have been observed in the last decade. the aim of this study was retrospective analysis of hsct for lymphomas in czech republic. methods: data of adult patients transplanted between years 1993-2016 were retrospectively analyzed using ebmt database. results: between 1993 and 2016, 2816 autologous hsct (asct) were performed in 2651 patients (1511 men, 57%) with different lymphoma subtypes. the median age was 49 years (range 18-75). out of these, 2078 (78%) were patients with non-hodgkin lymphoma (nhl), 569 (21%) with hodgkin lymphoma (hl). the nhl group comprised of diffuse large b-cell lymphoma (dlbcl, 36%), follicular lymphoma (fl, 18%), mantle cell lymphoma (mcl, 16%) and t-nhl (9%). the frequency of asct in lymphomas increased from 1993 to 2000 and has been constant since 2000 (120-130 transplants per year). differences in frequency of asct were observed among lymphoma subtypes -decreasing numbers of dlbcl and fl and increasing numbers of t-nhl and mcl, with asct as part of the induction therapy. between 1996 and 2016 a total of 329 allogeneic hsct (allosct) were performed in 319 patients (200 men, 63%). median age was 46 years (range 19-66). out of these 257 (81%) were patients with nhl, 61 (18%) hl. the most common nhl subtypes were fl (27%), mcl (22%), t-nhl (22%) and dlbcl (16%). in the last 10 years the number of allosct for lymphoma is fluctuating around 20 per year. the median age at asct was significantly higher in the years 2010-2016 vs 1993-2000 [54.5 (18.1-74.8) vs. 40.9 (18.6-72.5), p < 0.0001, fig 1] , while the increase at allosct [46.5 (21.3-65.5) vs 41.6(20.3-64.5)] did not reach statistical significance (p=0.07). with median follow up for allosct, 5 y probability os for patient transplanted in the later period 2010-2016 was in relapsed dlbcl 25.7%, in fl 77.4%, in hl 51.0% and in mcl 45.7%, 5 y os for asct as part of first line therapy in the same period was in mcl 78.1 % and in t-nhl 48.2%. os was significantly better in all patients who underwent asct in the years 2010-2016 vs 1993-2000 (70.% vs. 57 .5%, p < 0.0001) and there was a trend towards better os in patients after allosct (with 44.7% vs 23.5%, p=0.1084) (fig 2) . conclusions: hsct remains important treatment modality for lymphomas in the era of targeted antibody and molecular therapy and we can transplant older patients due to better supportive treatment. acknowledgment: progress q28-9 uk from the czech ministry of education youth and sports disclosure: nothing to declare background: disease chemosensitivity to salvage treatment has been proven to be a major predictive factor for a favorable outcome after autologous stem cell transplantation (asct) for patients with refractory lymphomas. therefore the importance of effective and safe salvageregimens is indisputable. methods: we retrospectively compared the outcomes in terms of safety and efficacy, in 67 (hl:36, nhl:31) patients, with a median age of 34.5(16-75) years, who received as 1 st salvage either dicep [cyclophoshamide (1750 mg/m 2 ), etoposide (350mg/m 2 ), cisplatin (35 mg/ m 2 ), days 1-3, (n=23)] or the widely used regimen eshap (n=44). rituximab was additionally given to all cd-20 positive lymphoma patients. the statistical analysis based on the independent t-test, kaplan meir method and logrank test. results: the reason for salvage treatment was primary induction failure (pif, n=34), early relapse (< 12 months post induction-remission therapy n=14) and late relapsed disease (n=19). more specifically, 19/23 patients (83%) in the dicep-group, and 29/44 patients (65%) in the eshapgroup were assessed with pif or early relapsed disease, however this difference was not statistically significant. both regimens were well tolerated and no major organ toxicities were noticed. eleven patients (48%) from the dicep-group, while only 4(10%) from the eshap-group developed febrile infections. all patients were successfully managed with the appropriate treatment and only one, from the eshap-group, required for short period admission to the intensive care unit. after 1 cycle of dicep and 2 cycles of eshap the disease response was re-assessed by pet/ct scan. the overall response rate (>50% tumor reduction) was significantly superior for the dicep-regimen, reaching 92% (21/23 patients) vs. 64% (27/44 patients) for eshapregimen (p=0,003). eleven patients (48%) from the dicep-group and 14(30%) from the eshap-group achieved complete metabolic remission according to pet/ ct criteria (p=ns). the median hospitalization period was 20(5-25) days for the dicep-group compared to 10(10-19) days for the eshap-group. however, for the eshapgroup, an additional median of 20(6-33) hospitalization days were required, since 12 of the non-responders patients received a 2 nd salvage before asct. the mobilization and stem cell collection was successful for both groups, though significant higher number of cd34+ cells were collected in the dicep-group (17.2x10 3 /kg vs. 5.4x10 3 /kg, p=0,001). all but two patients (due to refractory disease) underwent asct. noticeably, the median period from 1 st salvage treatment to asct was significantly shorter for the dicepgroup (64 vs. 128 days, p=0,013), apparently because 12 non-responders patients from eshap-group treated with a 2 nd salvage. the 3-years overall and progression free survival were similar for dicep-and eshap-groups (95% vs. 88% and 70% vs 80% respectively). two heavily pretreated patients from the eshap-group developed secondary myelodysplastic syndrome post asct conclusions: in our series of patients both regimens proved to be safe. interestingly, despite the fact that more patients in dicep-group had poor risk disease the dicepregiment was significantly more effective, resulting thus in an earlier asct, less exposure to chemotherapeutic agents, that might led in less long-term toxicity. nevertheless, prospective trials with large series of patients are needed to define the role of dicep in the salvage treatment setting. disclosure: no conflict of interest background: although autologous hematopoietic stem cell transplantation (auto-hsct) is one of the best curative strategies for patients with chemosensitive t-cell lymphoma, major limitation remains a tumor contaminated graft-related relapse or residual disease after chemotherapy. several purging methods were introduced in auto-hsct for these limitations, however there are few studies of ex vivo purging of the autograft in lymphomas, especially t-cell lymphoma. therefore, we retrospectively analyzed 59 consecutive t-cell lymphoma patients receiving auto-hsct with/without ex vivo purging. methods: among them, 33 patients underwent autograft manipulation with ex vivo purging by cd34+ cells selection using a clinimacs device. results: with median follow-up duration of 42 months (range, 6-121 months), 3-year overall survival (os; 73.8% vs. 49.0%, p=0.017) and 3-year progression-free survival (pfs; 75.8% vs. 52.4%, p=0.039) in a purged and unpurged group, respectively. transplant-related mortality was observed in both groups (2 patients of a purged group and 1 patient of an unpurged group). neutrophil (10 vs. 9 days, p=0.240) and platelet (30 vs. 24 days, p=0.055) recovery were similar in both group and there was no engraftment failure. on subgroup analysis according to upfront and salvage auto-hsct, while survival outcomes were improved by stem cell purging in the upfront auto-hsct (os with p=0.039 and pfs with p=0.047), there were no different survival outcomes in salvage auto-hsct. the unmanageable late-infectious complications were few in both groups except for predominantly cytomegalovirus reactivation in a purged group (3 vs. 1 patient). conclusions: although cohort was a small number, ex vivo graft-purging method was feasible and safe in t-cell lymphomas. and this purging strategy observed the more favorable survival outcomes in the upfront auto-hsct than salvage setting. therefore, further randomized studies are needed to determine the firm efficacy of cd34+ purification with the large number of patients in auto-hsct for t cell-lymphomas. disclosure: nothing to declare nivolumab-based regimens in relapsed or refractory non hodgkin lymphomas: the role of hematopoietic stem cells transplantation methods: we analyzed data of 18 patients with r/r nhl, among them n11 with diffuse large b-cell lymphoma (dlbcl), n5 with primary mediastinal b-cell lymphoma (pmbcl), n1 with gray zone lymphoma (gzl) and n1 with gamma-delta peripheral t-cell lymphoma (ptcl), who received nivolumab-based regimens. the median age was 37 years (range, 18 -64 years). most of the patients n14 (78%) had a primary chemoresistant disease, the rest patients n4 (22%) had a relapse. the median of lines of prior therapy was 3 lines (range, 2-6 lines). all sixteen patients with dlbcl and pmbcl received 1 -3 cycles of nivolumab in combination with bendamustine, gemcitabine and rituximab (begern). the patient with gzl received 5 cycles of nivolumab in combination with brentuximab vedotin and epoch. and the patient with ptcl received 10 cycles of nivolumab monotherapy. results: at median follow up 8 months (3-16) objective response (or) after nivolumab-based regimens was noted in n10 (56%) patients, complete response (cr) and partial response (pr) in n9 (50%) and n1 (6%) patients, respectively. cr observed in n4 patients with dlbcl, n3 with pmbcl, n1 with gzl, n1 with ptcl. and pr observed in 1 patient with dlbcl. two responding patients with dlbcl underwent auto-hsct. and four responding patients (n1 dlbcl, n1 pmbcl, n1 gzl, n1 ptcl) received allogeneic hematopoietic stem cells transplantation (allo-hsct). the median duration of response for all n10 patients with or was 5 (range: 3-16 +) months. among n4 patients who achieved or without hsct, only n1 remain in cr. two patients who received auto-hsct had a relapse. one patient with dlbcl improved the response after allo-hsct from pr to cr, and all four patients with allo-hsct remain in cr. the probabilities of 1-year os and pfs rates were 49% and 31%, respectively. conclusions: nivolumab-based regimens can lead to an objective response in 56% patients with r/r nhl. however, the durability of response to therapy is not long. nivolumab-based regimens can be used as bridge to allo-hsct disclosure: there are no conflicts of interest to disclose background: patients with aggressive non-hodgkin lymphoma (nhl) who relapse after autologous stem cell transplantation have a dismal outcome and could benefit from radiotherapy, allogeneic stem cell transplantation or experimental treatments. systemic inflammatory parameters at diagnosis have demonstrated to be useful to predict lymphoma evolution. methods: we conducted a retrospective review of patients with aggressive nhl who underwent autologous stem cell transplantation (astc) to evaluate the relationship between ldh, β2-microglobulin, inflammatory parameters (lymphocyte (alc) and monocyte count (amc), ferritin or c-reactive protein) and imaging techniques before and on day +100 post-astc and progression free survival (pfs), as well as the role of residual disease directed radiotherapy (rt). results: one hundred and sixty patients with aggressive nhl received asct as consolidation treatment in our center between 2000 and 2017. the most common diagnosis was diffuse large b-cell lymphoma (dlbcl). one hundred and nine patients received upfront asct for high risk dlbcl (defined as age-adjusted ipi 2-3)(n=28) or for having received two or more lines to obtain first complete remission (n=28), for t-cell lymphoma (n=27) and for mantle cell lymphoma (n=26). the rest was performed in relapsed lymphomas. forty-seven patients (29%) relapsed and pfs was 110 months. pretransplant response was evaluated with ct scan in 66 patients (11 of this with partial remission (ct-pr) and 94 patients were evaluated with fdgpet/ct (27 were pretransplant positive (pet1); of these, 18 patients maintained positivity at day 100 after astc (pet2). pfs in patients with ct-pr was 46 months, in pet1 positive ones 99 months and in pet2 positive ones 21 months. univariate analysis showed pet2 positivity as the most accurate predictor of relapse (hr 3,13, p=0,004) followed by amc at day +100 (hr 1,002, p=0,13), albumin at day +100 (hr 0,93, p=0,03), ldh at day +100 (hr 1,002, p< 0,001) and pretransplant alc/amc ratio (hr 1,068 p=0,008). multivariate analysis only demonstrated an association with pet2 positivity (hr 7,78) p< 001 and ldh in day +100 (hr 1,008) p=0,003 with pfs. five and ten years overall survival were 61% and 37% in pet2 negative patients vs 25 and 5% in pet2 positive ones (p< 0,01). eight out of 19 patients with pet2 positivity did not relapse. salvage radiation therapy was used in 7 patients with positive residual mass and 5 of them did not relapse. two patients relapsed: one patient had residual mass and another had remote affectation from primary site and could be considered as progression before day +100. conclusions: post asct fdgpet/ ct is superior to conventional ct in predicting outcome in aggressive lymphoma after astc. pre and post asct systemic inflammatory parameters didn't help to improve the relapse risk prediction. addition of consolidative rt after astc has demonstrated improvement in pfs in patients with pet positivity. it would be neccesary to develop randomized trials to assess the role of rt in residual disease in advanced aggressive nhl with insufficient response to systemic treatment with pet response evaluation. disclosure: nothing to declare long term outcome of patients with lymphoid malignancy who underwent high dose chemotherapy followed autologous hematopoietic cell transplantation at a single institution over 20 years joanna romejko-jarosinska 1 , ewa paszkiewicz-kozik 1 , lukasz targonski 1 , lidia popławska 1 , jan walewski 1 background: high dose chemotherapy (hdt) and autologous hematopoietic cell transplantation (auto-hct) is a standard of care for relapsed/refractory lymphoma patients (pts) or it is used as a consolidation for myeloma and high risk lymphoma patients in first line treatment. we retrospectively evaluated long-term outcome including late effects and risk factors in patients with lymphoid malignancy who underwent auto-hct. methods: we collected data from 926 consecutive patients with hodgkin lymphoma (hl) (n=326), aggressive b lymphoma (dlbcl) (n=186), myeloma (n=199), indolent lymphoma (n=30), mantle cell lymphoma (n=122) and peripheral t cell lymphoma (n=63) who underwent auto-hct at our institution between 1997 and 2017. at transplant median (range) age was 46 (17-71) years, clinical stage iii/iv was found in 499 of lymphoma pts, complete remission, partial remission and stable/ progressive disease occurred in 462(50%), 397 (43%), 67 (7%) pts, respectively. beam regimen was used in 549 pts (59%), mel200 in 200 pts (23%) and other myeloablative regimens in 177 pts (18%). results 62%) ], respectively. partial remission or stable disease at transplant, clinical stage iii or iv, and age more than 60, were identified as risk factors associated with inferior os and pfs in univariate and multivariate analysis. histopathologic diagnosis was not a risk factor for os and pfs (p=ns). the outcome of patients who underwent auto-hct between 1997-2003 was inferior to the outcome of patients treated in 2004-2010 or 2011-2017. 5year os was 53%, 68%, 73%(p< 0.001) and 5 year pfs was 43%, 54%, 55% (p< 0.01), respectively. we recorded 37 (4%) cases of second primary cancer (18 solid tumors and 19 hematologic cancers). acute cardiotoxicity occurred in 5 patients from 1 to 15 years after transplant, and required heart transplant in 2 patients. 326 patients (35%) died. the main causes of death were progressive disease in 271 pts (89%), second primary malignancy in 22 pts (6.7%) treatment related mortality was 0.8% (8 pts), and mortality within 100 days was 22 (2,4%). [[p494 image] 1. pfs and os in patients underwent hdt and auto-hct 1997 -2003 , 2004 -2010 , 2011 -2017 conclusions: more than 45% of patients who underwent hdt and auto-hct had long term survival without progressive disease. older age, non-complete remission at transplant, advanced stage are associated with poor outcome. patients recently transplanted had a better outcome than patients transplanted before 2004. disclosure: nothing to declare outcomes after haploidentical and matched related hsct in lymphoma do not differ significantly: a single center study nadira durakovic 1,2 , zinaida perić 1,2 , lana desnica 2 , ranka serventi-seiwerth 2 , sandra bašić kinda 2 , ivo radman-livaja 2 , alen ostojić 2 , ante vulić 2 , dražen pulanić 1,2 , pavle rončević 2 , zorana grubić 2 , igor aurer 1,2 , radovan vrhovac 1,2 1 university of zagreb, school of medicine, internal medicine, zagreb, croatia, 2 uhc zagreb, zagreb, croatia background: allogeneic hsct still offers patients with relapsed/refractory lymphoma the best chance of long-term survival. in most such patients timing of hsct is crucial, therefore a related donor is preferred. we analyzed acute and chronic gvhd incidence, relapse and overall survival, but also time to immunosuppression (is) discontinuation and hematopoietic recovery comparing transplantation using haploidentical (haplo) and matched related donors (mrd) in single center in this indication. methods: in the time period between 5/2011 and 5/2018 at uhc zagreb, croatia, 10 mrd and 13 haplo transplantations in lymphoma were done, 15 for hodgkin and 8 for nhl. all patients transplanted from haploidentical donors received ptcy. data were computed using the r package. the probability of gvhd was calculated using the cumulative incidence method and subgroups were compared using the gray test. results: median age was 38 (19-62) years; 36 (19-62) in haplo and 41 (25-56) in mrd group. four patients were in pr and 9 in cr in haplo group, while in mrd group 5 patients were in cr and 5 in pr. in haplo group 12 patients (92%) received bone marrow (bm) and only 1 (8%) peripheral blood stem cells (pbsc). in mrd group all patients received pbsc. all patients in haplo group received nma ("baltimore") conditioning with ptcy while in mrd group 9 patients (90%) received flu-bu2atg, and only one received flutbi as conditioning protocol. in haplo group 85% patients were previously treated with autologus transplantation, 80% in mrd group. there was no significant difference in time to is discontinuation, 149 and 155 days in haplo and mrd group, respectively. patients after haplo recovered slower, recovering anc after 22.6 days (95% ci, 17.7-27.4) and 16.5 (95% ci, 13.9-19) (p=0.04) and recovering platelets after 34.6 days (95% ci, 15.5-53.7) and 10.8 (95% ci, 9.2-12.4) (p< 0.01) in haplo and mrd group. with a median follow up of 469 days, overall survival was 83% (95% ci, 63-100) in haplo and 80% (95% ci, 59-100) in mrd group. trm was 8% in haplo and 20% in mrd group. cumulative incidence of agvhd ii-iv was 31% (95% ci, 9-56) and 32% (95% ci, in haplo and mrd group, respectively (p=0.84). cumulative incidence of cgvhd requiring treatment was 22% (95% ci, and mrd 20% (95% ci, 0-62) in haplo and mrd group, respectively (p=0.46). all cases of cgvhd developed after dli. cumulative incidence of relapse was 32% (95% ci, 9-58) and 38% (95% ci, 7-72) for haplo and mrd group, respectively (p=0.77). conclusions: we found no significant difference in overall survival, relapse incidence, agvhd and cgvhd incidence between these two groups. hematopoietic recovery was slower after haploidentical transplantation, but it did not influence trm as it was higher after mrd. even though limited in number, this data contribute to the growing body of evidence that use of haploidentical donors, particularly in lymphoma setting, is as worthy as using matched related donors and should be at least second choice in donor selection, and in older patients (with older donors) probably the first one. disclosure: nothing to declare. adjuvant involved field radiotherapy post autologous stem cell transplantation for refractory/relapsed lymphomas results in favorable outcome with low toxicity: a single center experience background: involved field radiotherapy (ifrt) to previous bulky or localized residual disease, is a widely used treatment approach to minimize the risk of relapse post autologous stem cell transplantation (asct). however, the proper time for irradiation treatment remains controversial. adjuvant ifrt (adj-ifrt) in pre-asct period could cause undesirable toxicity which might delays or even cancel the asct resulting in increased risk of relapse, or could affect the marrow environmental and marrow niche resulting thus in impaired engraftment. on the other hand, the ajd-iftr in the early post-asct period, upon marrow recovery, offers a potential advantage by delivering irradiation after sufficient disease response, without affecting the engraftment. in this retrospective study we evaluated the safety and efficacy of the ifrt as adjuvant treatment in patients who had previously treated with asct for relapsed or refractory lymphomas. methods: twenty-three patients (hodgkin=14, non-hodgkin=9), aged of 34(16-76) years, underwent asct, for primary refractory (n=15) or relapsed (n=8) disease. patients who had bulky disease at the time of relapse or those with residual mass post salvage treatment, were considered as candidates for adj-ifrt, early (within 2-3 months) after documentation of autologous stem cells engraftment. all patients proceeded to asct with chemosensitive disease after a median of 2 lines of salvage therapy. at the time of asct 20 patients (80%) had residual disease while 4(20%) evaluated to be in complete remission. the preparative regimens were: single-agent melphalan (n=9), busulfan-etoposide-melphalan (n=7), beam (n=5) and bendamustin-etoposide-cytarabine-melphalan (n=3). filgrastim was given till neutrophills recovery, while prophylaxis against bacteria, fungus, viruses and pcp were administered till the completion of adj-ifrt. results: all patients engrafted promptly and successfully. no patient experienced any severe toxicity or active infection before adj-iftr. though our plan was to proceed with adj-ifrt within 3 months post asct, finally it was delivered after a median of 4.5 (2-7) months; the median radiation dose was 30(24-36) gy. ten patients received radiotherapy in the mediastinum, 9 in the abdomen/pelvis/ inguinal area 3 in the neck, and 1 in the left leg. the adj-ifrt was well tolerated. no patient experienced toxicity grade >3 and none required hospitalization. currently, after a median follow-up of 2(2-5) years, 19/23 patients are alive and well; the 5-years overall and progression free survival rates are 75% and 55% respectively. four patients died; 2 due to relapsed disease and 2 heavily pretreated patients due to secondary myelodyspalstic syndrome conclusions: in our study, the adj-ifrt in the early post transplant period demonstrated a safe and well-tolerated profile. taking into consideration the poor risk status of our patients (residual disease post salvage regimen or bulky disease at the time of relapse), the promising overall and progression free survival rates suggested that adj-ifrt post asct is also an effective approach. well designed trials are needed to clarify the role and the appropriate time of radiotherapy in the asct setting disclosure: no conflict of interest adverse prognostic impact of pre-transplant neutrophil/ lymphocyte ratio in lymphoproliferative disorders background: brentuximabvedotin(bv) is a chimeric anti cd30 igg1 antibody, conjugated to synthetic antitubulinmomomethylauristatin. bv is approved for the treatment of classical hodgkin lymphoma (hl) in relapse either after autologous stem cell transplantation (asct) or after two lines of combination chemotherapy in transplant ineligible patients. the aethera trial revealed increased pfs when bv is used as maintenance therapy for 16 cycles in high risk patients after asct. however, this schedule is associated with a high cost and significant toxicity particularly in term of peripheral neuropathy. our primary objective is to assess the efficacy of 4 cycles brentuximab as consolidation therapy after asct for relapsed/refractory (r/r) hl. secondary objectives include side effects, progression free survival (pfs), and overall survival (os). methods: this is a retrospective single center analysis approved by the irb of the american university of beirut medical center. we included in this study consecutive patients with r/r hl who underwent asct between 2014 and 2018, and received bv consolidation post-asct. results: we identified 18 consecutive adult patients with r/r hl treated with bv 1.8mg/kg iv every 3 weeks as consolidation therapy after asct. the indications for bv consolidation was primary refractory disease in 11 patients (61%), early relapse in 6 patients (33%) (after a median time of 10 months; range, 3-11) andextranodalinvolvement in one patient (6%). the median number of lines of therapy pre-asct was 3 (range, 2-5). the median time to bv initiation post-asct was 76 days (range, 35-188). patients received a median of 4 cycles (range, 3-4) of bv post-asct. after a median follow up of 30 months (range, 8-50), five (28%) patients relapsed after asct. the median time to relapse was 7 months (range, 4-21). median pfs and os were not reached. we did not observe any significant toxicities during or after therapy. conclusions: 4 cycles of bv consolidation after asct seem to be safe and effective in preventing relapse, however our findings need to be confirmed with larger prospective studies. chemotherapy or who progress after autohsct is poor. despite introduction of novel agents like brentuximab vedotin (bv) or nivolumab, allohsct appears the most effective treatment option with curative potential. the goal of this study was to evaluate efficacy of allohsct for hl, including patients pre-treated with novel agents. methods: between years 2012-2018, 45 patients (including 23 males) with hl were treated with allohsct in msc institute of oncology in gliwice, poland. median age was 32 (19-57) years. median lines of preceding chemotherapy was 4 (2-8); 34 (76%) patients had been pre-treated with autohsct, 24 (53%) with radiotherapy, 17 (38%) with bv, 7 (16%) -with nivolumab. disease status at allohsct was as follows: cr-19, pr-10, nr-16. patients were treated with hsct from either hla-matched sibling donor (msd, n=21), unrelated donor (urd, n=18) or haploidentical donor (n=6). conditioning was myeloablative in 21 (47%) cases. peripheral blood was used as a source of stem cells. results: all but one patient engrafted with median time of neutrophil recovery of 14 (9-20) days. the incidence of grade 2-4 and grade 3-4 acute gvhd was 16% and 11%, respectively, while the incidence of chronic gvhd was 43%. the probabilities of os and pfs at 2 years were 54% (+/-12%) and 55% (+/-8%), respectively. the incidences of progression and transplant-related mortality were 31% and 14%, respectively. the 2y pfs rates were 42% for msd, 72% for urd and 62% for haploidentical donors. in a univariate analysis pfs was affected by recipient gender (female -75%, male -38%, p=0.009) and disease status at allohsct (cr -78%, pr -47%, nr -38%, p=0.09). in a multivariate model the disease status other than cr was the only factor associated with increased risk of treatment failure (reverse pfs) -hr=3.13, 95%ci 1.45-6.76, p=0.004. neither donor type nor conditioning affected long-term outcome. conclusions: results of allohsct for patients with relapsed/refractory hl are determined by disease status at transplantation. efforts should be done to reduce tumour burden before transplantation, optimally to achieve cr. disclosure: nothing to declare background: brentuximab vedotin (bv), nivolumab and pembrolizumab have been assigned to chemorefractory hodgkin lymphoma treatment. impact of these agents on disease-free-survival after autologous stem cell transplantation (asct) remains under investigation. aim of the study is to compare bv-and nivolumab-treated patients with a control group. methods: clinical characteristics and outcomes of chemo refractory hodgkin lymphoma patients who underwent asct during 2011-2017. results: a total of 56 patients (33 men; 21 women, median age 37 years old, 19-65) were treated with bv: 24 pre-transplant, 30 post-transplant and 7 pre-and posttransplant. pre-transplant bv patients had primary refractory disease or early relapse in the majority (92%). post-transplant treatment occurred in the context of relapsed/refractory disease in 27 patients; 12 (40%) had an allogeneic stem cell transplant. among them, 6 had additional chemotherapy and 2 nivolumab, gaining a complete metabolic response. in the 15 rest of patients, change of treatment due to eventual bv failure occurred. bv was administered as a maintenance treatment in 10 patients. in six of them bv had already been administered pre-transplant as well. out of maintenance treatment patients, 2 relapsed and subsequently received nivolumab. two patients died due to prior chemotherapy complications, whereas 13 are currently on nivolumab treatment. pet-based response was available in 6 patients, 4 having a complete metabolic response (cmr) and 2 a partial metabolic response. stable disease was achieved by ctbased response in the rest patients. no major toxicities were observed. one patient presented with grade 2 asymptomatic hypothyroidism and one with grade 3 anemia attributed to non-inflammatory upper gastrointestinal blood loss. in total, 20 patients received anti-pd1 treatment, all post bv failure. with a median follow-up of 34.3 (1.5-202 .2) months, 5-year overall survival (os) was 65.9% in patients treated only with bv compared to 78.2% in patients treated with additional anti-pd1 treatment (p=0.356, figure) . median os for patients treated only with bv was 113.5 months, whereas median os has not been reached for patients that received anti-pd1 treatment. conclusions: bv pre or post-transplant and anti-pd1 treatment post-transplant after bv failure have outstanding results in chemo refractory lymphoma patients. treatment sequence in allogeneic transplantation eligible patients remains to be further studied. disclosure: nothing to declare background: allogeneic hematopoietic cell transplantation (allo-hct) with reduced-intensity conditioning (ric) has been used in heavily pretreated lymphoma patients with the promise of decreased treatment-related mortality. despite improvements in outcomes of patients with lymphoid neoplasms, several new agents emerge as potential therapies. therefore, we aimed to describe our long-term experience in patients with hodgkin (hl), non-hodgkin lymphomas (nhl) and chronic lymphocytic leukemia (cll) post allo-hct. methods: in this retrospective study, we enrolled consecutive patients who underwent allo-hct for lymphoid neoplasms in our institution (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) . results: in total, 50 patients (male:female=35:25) aged 36 (15-64) years, underwent allo-hct for hl (n=24), nhl (n=21) and cll (n=5). the majority of patients were diagnosed at stage iv (48%); 34% had bone marrow involvement and 66% had undergone autologous hct. most patients were heavily pretreated (median lines=4, range 1-11), 21 of them had received more than 4 treatment lines and at the time of transplantation only 14 had complete response, while 9 had partial response and 27 were refractory. according to disease-risk index (dri), patients were stratified at low (n=11, 23.4%), intermediate (n=12, 25.5%), high (n=20, 42.6%) or very high (n=4, 8.5%) category. among patients with hodgkin lymphoma, brentuximab vedotin was administrated in 7 and 4 of them were effectively bridged to allo-hct. all patients received ric, mainly fludarabine (150mg/ m 2 )-cyclophosphamide (2g/ m 2 ) in cll and nhl and thiotepa (10mg/kg)-fludarabine (120 mg/m 2 )-cyclophosphamide (60mg/kg) in hl from matched sibling (n=27), matched (n=15) or mismatched unrelated (n=8) donors. graft-versus-host disease (gvhd) prophylaxis consisted of cyclosporine or tacrolimus and mycophenolate mofetil or short-term methotrexate and additional low-dose antithymocyte globulin (5mg/kg) in unrelated donors. peripheral blood was the main cell source (48/50) and median number of cd34+ cells infused was 6.37x10 6 /kg (1.33-14.5) . two patients succumbed to advanced underlying disease before engraftment; while the other engrafted successfully. median time until neutrophil and platelet engraftment was 10 and 12 days respectively. eighteen patients (36.7%) developed acute gvhd (grade iii-iv, n=5), steroid sensitive in 10 (62.5%) and 11 relapsed. one-year cumulative incidence (ci) of extensive chronic gvhd was 78.2%, and 13 patients required more than one additional line of immunosuppression (range 1-5). ten patients presented cmv reactivation successfully treated with antiviral medication and 1 patient died from hsv7 encephalitis. with a median follow of 3 years (1-16 years), 10-year os was 40.4%, 10-year non-relapse mortality ci 23.4% and 10year dfs 32%. there was no difference in survival according to original disease (5-year os for nhl=60.2%, hl=46.7%, cll=31%%, p=0.67). multivariate analysis revealed high and very high dri as the single predicting factor of os (hr 9.69, ci 1.55-60.55, p=0.015), when assessing impact of disease, dri, prior treatment lines, gender and bone marrow infiltration at diagnosis. conclusions: our data suggest that ric allo-hct provides encouraging survival rates, potentially offering the chance of cure, with acceptable long-term mortality in selected high-risk patients with lymphoid neoplasms. dri that is mainly associated with disease stage at transplant independently affects survival. therefore, continued efforts are necessary for clinical application of novel agents aiming to lower disease stage pre-transplant. disclosure: nothing to declare results: six pts were identified, with a median age of 30 years at diagnosis: five with hl nodular sclerosis and 1 with lymphocyte depletion. the median number of therapeutic lines prior to allo-hsct was 5 [4-10]; four pts were previously treated with brentuximaband two pts had been submitted to high dose chemotherapy with autologous bone marrow support. at the time of allo-hsct, 4 pts had progressive disease (dp), 1 was in partial response and 1 in complete response (cr). five allo-hsct were performed with a related donor, 4 of wich were haploidentical (2 parents, 1 sibling and 1 descendant) and 1 with an unrelated donor (10/10). prophylaxis for gvhd was performed with tacrolimus and mycophenolate mofetil (with post-transplant cyclophosphamide in haploidentical allo-hsct). on day +100 evaluations, 5 pts had a cr and 1 patient (pt) had dp. the median time to relapse after allo-hsct was of 12 months. at the time of initiation of nivolumab, 5 pts were under steroid therapy for disease control, without other immunosuppressive therapy. the median time between allo-hsct and the beginning of nivolumab was 16 months. the initial dose was 1 mg / kg (associated with corticosteroid therapy), escalated up to 3 mg/kg biweekly, according to patient's tolerance. after the start of nivolumab, 3 patients, with previous gvhd manifestations, presented a worsening of the cutaneous gvhd, which required an escalation of immunosuppressive therapy. as toxicity, 1 pt had a grade 4 pneumonitis, 1 pt had a grade 4 encephalitis/hypophysitis, 1 pt had a grade 4 pancreatitis, 2 pts had headache (grade 1 and 3), 2 pts had a grade 2-3 cutaneous reation. with a median follow-up of 13 months since nivolumab treatment, the overall response rate was 100%: 1 pt obtained cr and 5 pts partial remission. nevertheless, there were 2 deaths after the onset of nivolumab: 1 pt at 4 monts with dp and another one due to acute myocardial infarction at 16 months. at the time of analysis, 3 pts maintained response under nivolumab treatment (median cycles 26) and 1 pt had therapy suspended because of toxicity. conclusions: these results demonstrate the high probability of achieving response with nivolumab treatment in patients with rr-hl relapsing after allo-hsct, but adverse events of grade 4 were frequent in this small group, and the treatment toxicity was significant. disclosure: nothing to declare background: intravascular large b-cell lymphoma (ivlbcl) is a rare form of large b-cell lymphoma with pathological findings of intravascular proliferation and/or sinusoidal involvement of lymphoma cells. according to their geographic distribution, ivlbcl could be dichotomized into asian and western variants. compared with the western variant, where skin involvement was common, the asian variant was reported to involve more frequently the liver, spleen and bone marrow, and hemophagocytic lymphohistiocytosis is more common in asian variant. diagnosis of ivlbl is still difficult because of the lack of overt lymphadenopathy and peripheral blood involvement. thus, timely diagnosis and immediate treatment remain as a challenge to improve outcomes for patients with the asian variant. therefore, we analyzed the clinical features and treatment outcomes of patients with the asian variant of ivlbcl. methods: we analyzed 46 patients who were diagnosed with ivlbcl between 2001 and 2018. all patients were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (r-chop). results: forty-six patients were diagnosed with ivlbcl, and their median age at diagnosis was 62 years (range: 34-82 years). male patients predominated (n=26, 57%), and b symptoms were present in 31 patients (78%). hepatomegaly and/or splenomegaly were observed in 26 patients (57%), whereas lymphadenopathy was less common (n = 22, 48%). bone marrow and liver were the most commonly involved extranodal organs (54%, and 48%, respectively) and were the most common sites of biopsy for diagnosis in this study. all patients received r-chop as a first-line treatment after diagnosis with a median number of six cycles (range one to eight). at the end of treatment, 31 patients achieved a complete response (cr), whereas eight patients showed progression. six patients died after the first or second cycle of r-chop, and the causes of death were treatment-related adverse events including cytopenia, infectious complications, and pulmonary hemorrhage. upfront asct was done for two patients including one patient with cns involvement at diagnosis, and these patients were still alive at the time of analysis without evidence of relapse. on the other hand, the outcome of six patients undergoing salvage asct after relapse was poor; thus, only one patient was alive. likewise, patients with disease progression at the end of treatment with r-chop showed dismal prognoses even after salvage chemotherapy except for one. at a median follow-up of 47.0 months (95% confidence interval, ci 25.0-69.0), the median overall survival was 45.0 months (95% ci 25.8-64.2). the treatment outcome of patients with the asian variant of ivlbcl is still not satisfactory. although upfront autologous stem cell transplantation might be effective for selected patients at high-risk of relapse, its role is still not clear, either. thus, further study should be warranted to develop more effective strategies for diagnosis and treatment. clinical trial registry: not applicable disclosure: nothing to disclare background: peripheral t-cell lymphomas (ptcls) are about 10% of non-hodgkin´s lymphomas usually with an aggressive clinical course and unfavorable prognosis.given their heterogeneity, consensus on the best first-line treatment and the role of autologous/allogeneic (asct/allosct) stem cell transplantation as consolidation is controversial. methods: we evaluated the overall survival (os), progression-free survival (pfs) and toxicities of a cohort of patients with ptcls submitted to asct/allosct intensification at our institution between january 2000 and july 2018. os was calculated from the date of diagnosis until death. pfs was measured from transplant until relapse, progressive disease or last follow-up. os and pfs rates were estimated using the kaplan-meier method and compared with the log-rank test. results: twenty-six patients were identified, 16 female (61%), median age was 46 years (range: 22 to 62). ninetytwo percent of patients presented with advanced-stage disease at diagnosis (ann arbor stage iii or iv), 38% with b symptoms. according to the 2016 who classification, histologic ptcl subtypes included angioimmunoblastic tcell lymphoma (n =12); ptcl not otherwise specified (n =6); anaplastic large cell lymphoma, alk-negative (n =5); anaplastic large cell lymphoma, alk-positive (n =1); nodal peripheral t-cell lymphoma with tfh phenotype (n =2). extranodal nk/t-cell lymphoma, nasal type and primary cutaneous subtypes were excluded. the ageadjusted ipi (aaipi) was low/intermediate low in 15 patients (57%) and intermediate high/high in 11 patients (43%). twenty-seven transplants were performed (19 asct, 8 allosct); 18 were consolidation in 1st response (16 asct and 2 allosct) with 16 in complete remission (cr) and 2 in partial remission (pr). nine transplants were performed as consolidation of 2 nd response (3 asct and 6 allosct) with 4 in cr and 5 pr. in 1 patient allosct was performed after asct, due to early relapse (< 12 months). beam regimen was used in asct as conditioning and flumel in allosct. all patients engrafted, the median time to leukocyte recovery > 1,000/μl was 14 days (range, 11 to 25). four of the 8 pts (50%), submitted to allosct had chronic graft versus host disease which was the most relevant complication of this analysis. considering the whole cohort, the median follow-up was 50.5 months (range, 2 to 217). the estimated 5-year os and pfs were 77% and 39%, respectively. seven patients relapsed (6 early) all after asct, there were no relapses after allosct, however, the results were not statistically significant between the allosct and asct groups; the 5-year os rates were 87% and 62% (p =0,16) and the 5year pfs rates were 85% and 59% (p =0,26) respectively. for the all patients treatment-related mortality (trm) was 3,7%; 7 patients died, 6 with progressive disease (asct) and 1 for hepatic toxicity (allosct) before d+100. conclusions: the results of this retrospective study, taking into account the adverse risk profile of the population, suggest that autologous/allogeneic stem cell transplantation as an effective and safe option for the consolidation of patients with ptcls. these results need to be validated in prospective studies, including a larger number of patients. disclosure background: autologous stem cell transplantation is used as consolidation therapy in relapsed lymphoma patients. however, outcome of lymphoma patients relapsing after autologous stem cell transplantation is poor and allogeneic stem cell transplantation which can be curative is used in the transplant eligible patients in this setting. besides, allogeneic stem cell transplantation can be an option before autologous stem cell transplantation in some high risk patients. in this study, we aimed to compare the survival rates of lymphoma patients older than 60 years of age and patients aged 50-60 who had undergone allogeneic transplantation in our center. methods: we collected the data of lymphoma patients older than 50 years of age who had undergone allogeneic transplantation in our center and analyzed the results by grouping them into 2, namely the ones between 50-60 years of age and the ones over 60 years of age. [[p506 image] 1. figure 1 results: there were 42 patients over the age of 50 who had undergone allogeneic stem cell trasplantation with the diagnosis of lmphoma between 2011 and 2018. 18 of these patients were over 60 years of age. 37 patients had non-hodgkin lymphoma and 5 patients had hodgkin lymphoma. the characteristics of the patients are summarized in table 1 . patients' comorbidity indexes were calculated with augmented hct-ci which includes patients' pretransplant ferritin, albümin and thrombocyte counts as a variable. no difference could be found between groups regarding neutrophil and platelet engraftment times and comorbidity indexes. however, acute graft versus rate and documented bacterial infection rate during the hospitalization period were higher in the 50-60 years age group (p=0,01). 100 day mortality rate and non-relapse mortality rate were not different between groups. more importantly, progression free survival(pfs) and overall survival(os) of patients in the 50-60 years age group and over 60 years of age group were not different (p=0,45) (figure 1) conclusions: in the present study, although the number of patients is low, we showed that lymphoma patients over 60 years of age have similar outcomes and transplant related toxicity as the patients between 50 to 60 years of age. pfs and os were very close in this study. we think that this may be due to low relapse rate in the patients and high mortality rate in relapsing patients. in conclusion, allogeneic stem cell transplantation which has a curative potential may be employed in transplant eligible elderly lymphoma patients disclosure: nothing to declare background: follicular lymphoma (fl) histologic transformation consist on the development of an aggressive lymphoma, usually a diffuse large b cell lymphoma (dlbcl). histological transformation has been considered to have poor prognosis. in pre-rituximab era median os ranged between 1 and 2 years, however, in recent series of patients treated with chemotherapy plus rituximab, the outcome of transformed fl has improved, especially in those that receive autologous stem cell transplantation (asct), who reach 5-year os up to 75% in some series. methods: we have retrospectively studied 19 consecutive patients undergoing asct for transformed fl between 2006 and 2015 in a tertiary center in the basque country, spain. patients were considered to have a transformed fl if they were diagnosed of a dlbcl and they have previous history of fl or histological evidence of a fl in another location. these patients were compared to a retrospective cohort of 38 dlbcl patients with high ipi or stage that received asct in first remission according to our institution strategy. pfs and os were calculated from the time of the asct. in the case of transformed fl, both relapses of the aggressive or indolent lymphoma were considered. survival analysis was performed with kaplan-meyer estimator results: a total of 19 transformed fl and 38 dlbcl patients were studied, with a median follow up of 41.4 and 51.7 months respectively. patient characteristics are described in table 1. 3-year pfs was 68% in transformed fl and 81% in dlbcl, and 3-year os was 81% and 84%, respectively (picture 1). there were no significant differences in pfs or os between this two groups (p = 0.44). in both groups all relapses occurred in the first three years after asct. among the patients with transformed fl 6 relapses were observed. five of them (83%) were aggressive relapses, while only one patient presented relapse as an indolent lymphoma (fl histological grade 3a with an aggressive clinical course). [[p507 image] 1. image 1: transformed fl and dlbcl pfs after asct] conclusions: in our experience, asct in transformed fl offers good results, similar to those in dlbcl. fl presents a natural course akin to that of dlbcl, with relapses occurring early and survival reaching a plateau. this data suggests that some patients with transformed fl can be cured after asct. disclosure: nothing to declare. safety and efficacy of intensive preconditioning regimen containing cladribine in autologous peripheral blood stem cell transplantation of refractory and relapsed young highly invasive lymphoma background: autologous peripheral blood stem cell transplantation (apbsct) is one of the main treatments for patients with non-hodgkin's lymphoma (nhl). effective and safe conditioning regimens can improve the cure rate of nhl. beam is the most common pretreatment scheme, but for refractory and relapsed young highly invasive lymphoma, especially for dual-expression dlbcl, pretreatment needs to be strengthened. studies have shown that the cladribine (clad)+gemcitabine (gem)+busulfan (bu) combination provides synergistic cytotoxicity in lymphoma cell lines.we evaluated the the safety and short-term efficacy of intensive preconditioning regimen containing cladribine (clad+gem+bu) for refractory and relapsed young highly invasive lymphoma undergoing apbsct. methods: ten patients with nhl received apbsct. ca)ctx+ ara-c) therapy followed by g-csf was used for pbsc mobilization. sevenr patients received conditioning regimens of beam(beam group): bcnu 300mg/ m 2 ·d -1 ×1d (-7d), vp16 100mg/m 2 · q12h×4d (-6d--3d), ara-c 200mg/m 2 ·q12h×4 d (-6d--3d), mel 140mg/m 2 ·d -1 ×1d (-2d). three patients received intensive preconditioning regimen containing cladribine (clgb group): clad 5mg/m 2 ·d -1 ×5d (-6d--2d), gem2500mg/m 2 ·d -1 ×2d (-6d, -2d), bu0.8mg/kg q6h×4d (-6d--3d). follow-up date expires on december 1, 2018. results: the age of 3 patients in clgb group was 41, 12 and 50 years, respectively. two patients were diagnosed as diffuse large b-cell lymphoma with double expression and one was diffuse large b-cell lymphoma with two recurrences. the patients of beam group were all high-risk, relapsed and refractory nhl.all patients were successfully engrafted after infusing apbsc. the average lowest leukocyte in clgb group and beam group were (0.023 ±0.023) ×10 9 /l vs (0.237±0.2033)×10 9 /l, respectively. the average lowest leukocyte in clgb group was lower than that in beam group. the average time to anc < 0.5×109/l in clgb group and beam group were 1.33d ±1.53d vs 3.57d±2.07d. the average time to anc≥0.5×10 9 /l in clgb group and beam group were 9.0d±1.0d vs 10.0d±2.6d; the average time to plt≥20×10 9 /l of clgb group was not different to that of beam group (8.0d±1.0d vs 9.6d±2.2d) the average time of neutropenia wasn't significantly different in two groups (8.33d±3.1d vs 8.0d±2.1d). the adverse reactions of gastrointestinal tract and oral mucosa were close in tow groups.vod, hemorrhagic cystitis, pretreatment-related interstitial pneumonia, liver and kidney dysfunction were not happened in tow groups. the rate of infectious fever was close in two groups (2/3 vs 4/7). the median followup period in beam group was 9 (1~19) months. in the beam group, a patient died 20 days after transplantation, because he was diagnosed with recurrent nkt cell lymphoma and intracranial infection caused by severe sinus infection. another case of beam group was diagnosed as double-expressed dlbcl, which relapsed 3 months after transplantation. the remaining patients in ebmt group survived disease-free. the follow-up time of 3 patients in clgb group were 2 months, 3 months and 9 months respectively. all patients survived without disease.however, the follow-up time is short and needs long-term follow-up. conclusions: the treatment of intensive preconditioning regimen containing cladribine (clgb) for refractory and relapsed young highly invasive lymphoma undergoing apbsct is safe. the time of hematopoietic reconstruction is short, and the adverse effects is tolerable for patients with refractory and relapsed young highly invasive lymphoma. the current short-term outcome is good, but the long-term effect need a longer time to follow-up. disclosure: this work was supported by national nature sciences found of china (81300417). there is no disclosure of conflict of interest.the all authors name: xiang-li chen, yu-zhu zang, wen-hui zhang, yin zhang, zhong-wen liu, ping-chong lei, jing yang, yu-qing chen, kai sun. background: small part of children with hodgkin disease (hd) demonstrate initial resistance to the standard and even "salvage" chemotherapy and need innovative drugs for the treatment. methods: a 15-year girl was diagnosed with classical hd (nodular sclerosis)corresponding to stage ii e b (fever >38°c) in april 2016.after two cycles of oepa (vincristine, etoposide, prednisone and doxorubicin) and next two cycles copp (cyclophosphamide, vincristine, prednisone and procarbazine) the patient again had progressive disease. as the patient achieved a partial response (pr) after "salvage"therapy with two cycles of igev (ifosfamide, gemcitabine, vinorelbine, and prednisone), she received auto-sct in february 2017 (patient status before auto-sct was pr). we used ccnu-containing conditioning regimen cem: lomustine (ccnu) 300 mg/m 2 + etoposide 1200 mg/m 2 + melphalan 180 mg/m 2 . at day +90 after auto-sct, the patient again demonstrated progression of the disease: pet/ct-examination showed mediastinal tumor mass enlargement with increased left lung nodule simultaneously to short metabolic activity. patient was under observation. at day +140 the disease had relapsed and progressed -examination by pet/ct demonstrated multifocal progressive disease with multiple pulmonary lesions and increased metabolism in comparison with the previous pet/ct scan. in july-october 2017, the patient had salvage chemotherapy with a combination of brentuximab vedotin (bv) (bv dhap (dexamethasone, cytarabine, cisplatin) + bv (without chemotherapy due to suspected invasive mycosis) + bv dhap), however, only partial pet-positive remission was achieved. because of many times relapsed and progressed disease pembrolizumab therapy was started in october 2017: 2 mg / kg every three weeks four cycles totally. toxic effects and serious complications during and after therapy by pembrolizumab were not observed. in february 2018, after pembrolizumab # 4, the patient showed complete metabolic remission of the disease by control pet-ct. in april 2018, the patient received haplo-sct with post-transplant hd-cyclophosphamide. we used conditioning modes with reduced toxicity (fludarabin 150mg/m 2 + treosulfan 42g/m 2 ), high doses of cy (50 mg / kg) on days +3 and +4. tacrolimus and mycophenolate mofetil started on day +5. mmf was terminated on day 35, tacrolimus -on day 180. patient did not have acute and chronic gvhd. results: at the moment the patient is alive and still in pet-negative cr with duration more than 9 mo. conclusions: pembrolizumab has demonstrated high activity against resistant hd even after post-auto-sct progression with good tolerability for the sick child. disclosure: nothing to declare p510 high dose chemotherapy followed by autolougous peripheral blood stem cell transplantation (asct) in diffuse large b cell lymphoma (dlbcl) median age is 38,9 years (14 to 67) and sex ratio (m/f) 1.14; ann arbor stage iii-iv: 104 pts. before hdt induction chemotherapy (chop, c2h2opa) was instituted and associated with rituximab in 100 pts (66,6%), 49 pts (32,7%) received more than 2 treatment lines and 16 pts (10,7%) received complementary radiotherapy. transplant disease status before hdt was complete remission (cr) in 93 pts, partial remission in 55 pts (rp) and disease progression in 2 pts. the delay from diagnosis to hdt is 11,7 months (4-103). the hdt protocols used are: tutshka : 87 pts, tutshka+vp16 : 42 pts, bam 12 (busulfan +cytarabine+melphalan) :16 pts et beam : 5 pts. all pts received, after thawing, mobilized pbsc obtained by g-csf mobilization (15μg/kg/d, 5 days) alone and froze in liquid nitrogen. the median rate cd34+ cells infused is 3,6 x 10 6 /kg (0. 87-17.36) . the median follow-up at 12/31/2017 is 67 months . results: the median time to graft (pnc > 0.5 x 10 9 /l) was 13 days (9-18). ten early deaths were observed including 8 infection (trm: 6,6%) and 3 in disease progression at 3 months. after 3 months of hdt 137 pts are assessable including 128 pts in cr (93,4%) and 9 pts in pr (6,6%). relapse was observed in 31 pts (22.6%) and it was earlier relapse in a period of 24 months in 15 pts (48%). deaths were among 39/150 pts (26%). persistent cr was achieved in 112/137 pts (81,8%) including 23/30 (76,6%) mlcl and 89/107 (83,1%) others dlbcl. the overall survival (os) and event free survival (efs) at 10 years are respectively 68% and 64%. the os and efs are better in patients who received rituximab in initial therapy : os (79% vs 54%; p< 0,001) et efs (79% vs 46%; p< 0,001). herein, we present one patient with refractory mcl, who were insensitive to chemotherapy and then experienced a dramatic improvement with ibrutinib mono-therapy but later developed ibrutinib resistance,ultimately resulting in the deterioration of disease and death. methods: we give the patient several examinations including ultrasound, bone marrow biopsy, lymph node biopsy, exome sequencing, sanger sequencing, and so on. for the treatment of lymphoma, the patient received chemotherapy, including 1 course of chop(cyclophosphamide 1.3g day 1, doxorubicin 40mg day 1,vinorelbine 40mg day 1, and dexamethasone 15mg from day 1 to 5) and 1 course of r-dhap (rituximab 600mg day0, cytarabine 2g q12 day 1, cisplatin 100mg day 1,dexamethasone 20mg from day 1 to 5)in succession.because of the failure to control disease progression, ibrutinib 560mg qd was used until the patient died. results: the 64-year-old man initially referred to our hospital for complaints of abdominal pain and distention over 2 months. ultrasound showed splenomegaly and multiple enlarged retroperitoneal lymph nodes.excisional biopsy conducted on the right neck lymph node revealed the presence of malignant cells.immunohistochemically, the neoplastic cells were positive for bcl2, bcl6,cd20, cd5, cd79a, cd43, ki-67(50%), sox11, cd21 (fdc) and cyclin d1 and negative for cd10, cd23 and cd3; fluorescence in situ hybridization(fish) showed igh/ ccnd1,t(11;14) 90%.thus a diagnosis of mcl was confirmed. 2 course of therapeutic chemotherapy were applied to the patient but he did not respond well.he suffered recurrent fever, thrombocytopenia, left abdominal pain, splenomegaly and multiple enlarged lymph nodes. then he received ibrutinib mono-therapy, and experienced a dramatic improvement as his body temperature was controlled, his hemogram became normal and his spleen and lymph node tapered.after about 6 months response of ibrutinib, the disease deteriorated rapidly and he died very soon. exome sequencing from the patient peripheral blood at this time detected one missense mutation in exon 5 of tp53 at nucleotide 524g>a, resulting in an argnine to histidine change at amino acid 175 (p.arg175his). but sanger sequencing of the patient bone marrow ffpe sample at the time of original diagnosis did not detect this mutation. conclusions: thus, our study reported a tp53 r175h mutation mcl patient who developed ibrutinib resistance and progressed aggresively, which may open new insight for future effort for alternative therapeutic strategies in ibrutinib-refractory mcl. disclosure: nothing to declare. minimal residual disease, tolerance, chimerism and immune reconstitution peripheral blood samples were obtained for routine analysis at several time points after hsct. all available blood samples between 0.5 and 2 years were used in the analysis. to assess changes in the cd19+b and cd19 +cd27+memory b cell counts over time while accounting for the correlation between the repeated measurements of each patient, we used linear mixed-effects models. wilcoxon rank test, kruskal-wallis test and linear regression were used for univariate analysis. results: at one year after hsct, univariate analysis showed that patients transplanted with a cb graft compared to bm and pbsc had a significantly higher absolute number of b cells (median bm= 501, median cb=1402, median pbsc=502 cells/μl, p=1.4e-5) and memory b cells (median bm= 26, median cb=75, median pbsc=23 cells/μl, . recipients with age under 5 years had significantly higher absolute numbers of b (median=919, median=473 cells/μl, p=2.9e-4) and memory b cells (median=54, median=22 cells/μl, p=2.7e-5) than above 5 years. increase in donor age was associated with a decreasing effect on b cell (r 2 = 0.35, p=5.9e-14) and memory b cell (r 2 = 0.25, p=2.1e-8) reconstitution as determined in regression analysis. following univariate analysis, we analysed these factors in a mixed effects model to assess the relation with differences in b cell or memory b cell numbers 0.5-2 years after hsct. in our analysis we found significant decreasing b cell and memory b cell numbers with increasing donor age corrected for recipient age and source (both p< 0.001). increasing recipient age also showed a significant decrease in b cell and memory b cell numbers (both p< 0.001) but there was no significant influence of donor source ( figure 1) . conclusions: b cell and memory b cell numbers after hsct are influenced by donor and recipient age but not by donor source. older donors and recipients show a decrease in b cells and memory b cells numbers 0.5-2 years after hsct. [[p512 image] 1. figure 1 . b cell development and donor age. green shows cb, red bm, blue pbsc at mean donor age.] 1 copenhagen university hospital rigshospitalet, copenhagen, denmark, 2 leiden university medical center, leiden, netherlands background: the outcome of allogeneic hsct is challenged by a delayed and long-lasting imbalanced t-cell reconstitution increasing the risk of acute gvhd, infections and disease progression. although the role of differentially and functionally distinct t-cell subsets in the development of complications has been addressed, little is known about the factors controlling their recovery. in this study, we investigated the impact of immuneregulating and homeostatic cytokines on the reconstitution of functionally distinct t-cell subsets and associated clinical outcomes. methods: we included 80 children undergoing allogeneic hsct for all (n=46) or aml (n=34) with a median age of 8.3 years (range: 0.8-17.8). donors were either mrd (n=21), mud (n=45) or mmud (n=14). bm (n=70) or pb (n=10) were used as stem cell source. conditioning regimens were based on tbi (n=25) or highdose chemotherapy alone (n=55) and included atg in 58 patients. thirty age-matched healthy children were included as controls. cytokines (il-7, il-15, il-18, scf, il-6, il-2 and tnfα) and active atg in plasma were longitudinally measured from before conditioning until 3 months after hsct (n=80) along with an extended phenotyping of t-cell maturation and differentiation by flow cytometry (n=41). results: the homeostatic cytokines il-7 and il-15 increased from pre-conditioning to peak 1-2 weeks post-hsct and gradually declined thereafter. il-6 levels were shortly elevated, while il-18 and scf remained relatively stable, and il-2 and tnf-α levels were below threshold of detection at all time points. the early rise of il-7 and il-15 was strongly associated with the degree of t-cell depletion by atg, while il-15 also correlated with markers of systemic inflammation. il-7 and il-15 levels were significantly higher in children treated with atg (p< 0.001) and correlated with both longer exposure to atg (p< 0.001) and increased levels of active atg (day +21: il-7: r=0.71, p< 0.0001; il-15: r=0.61, p< 0.0001), indicating that high levels of these cytokines reflected more pronounced t-cell depletion during lymphopenia. higher circulating levels of il-7 and il-15 were associated with a slow recovery of cd3+, cd4+ and cd8+ t-cell counts at day +30 and +60 post-hsct (p< 0.05), while the remaining cytokines did not correlate with immune reconstitution. looking into t-cell subpopulations, increased levels of il-7 and il-15 during the first month post-transplant were associated with lower numbers of naïve t cells and correlated with an increased proportion of cd4+ and cd8+ effector memory cells ( figure) . no differential effect of cytokines on polarization of cd4+ t cells into th1, th2, th17 cells or treg cells was found. in atg-treated patients, il-7 and il-15 levels at day +14 were significantly lower in patients developing acute gvhd grade ii-iv (p=0.0007 and p=0.0007, respectively). in the total cohort, increased il-7 levels were associated with a reactivation of ebv (p=0.040). conclusions: these findings suggest that quantification of il-7 and il-15 can be indicative for the degree of t-cell depletion during the first weeks after hsct and predictive of complications. overall, these results indicate that the lymphopenia-induced elevation of il-7 and il-15 is a major driver of the initial expansion of donor t-cells. background: mathematical kinetic models were adopted to study immune cell reconstitution after allogeneic hematopoietic stem cell transplantation (allo-hsct). the associations between acute graft-versus-host disease (agvhd), relapse and the immune cell reconstitution kinetic models were explored. methods: from june 1, 2011 to may 31, 2015, sixty-five patients with hematological malignancies after allo-hsct were recruited. peripheral blood was collected on +14 day, +28 day, +42 day and in +2 month, +3 month, +6 month, +9 month, +12 month, +18 month, +24 month. lymphocyte subsets were determined by flow cytometry, including in total t lymphocytes (cd3 + ), helper t cells (cd3 + cd4 + ), cytotoxic t cells (cd3 + cd8 + ), cd4/cd8 ratio, nature killer (nk) cells (cd3 -cd56 + ), nkt cells (cd3 + cd56 + ), b lymphocyte (cd19 + ), naive t cells (cd3 + hla-dr + ), static t cells (cd3 + hla-dr -), and regulatory t cells (cd4 + cd25 high foxp3 + ). mathematical kinetic models were calculated for immune cell reconstitution with spss. results: after allo-hsct, a logarithmic curve model was observed for cd3 + t cells. cubic curve models were observed for cd3 + cd4 + t cells, cd4 + cd25 high+ foxp3 + t cell, cd3 + hla-dr -t cells, cd3 + cd56 + nkt cells, cd19 + b cells. cd3 + cd8 + t cells, cd3 + hla-dr + t cells, and cd3 -cd56 + nk cells showed s type curve models. considering t cells were the major mediators for agvhd and graft-versusleukemia effect after allo-hsct. with established immune cell kinetic models, we found that different curve models were observed between patients with and without agvhd after allo-hsct. although the kinetic models were almost the same for leukemia-free and relapsed patients in the first 3 months after allo-hsct, significantly different kinetic curves could be observed thereafter. conclusions: the immune cell reconstitution showed different mathematical curve models after allo-hsct. kinetic reconstitution model of certain immune cell was associated with agvhd and relapse. hence, mathematical kinetic models of immune cell reconstitution may be potential indictor for predicting agvhd and relapse after allo-hsct. disclosure: nothing to declare lineage specific chimerism analysis in pediatric patients following allogeneic hematopoietic cell transplantation (hct background: the outcome of allogeneic hct is dependent on several variables that include patient age, disease and stage, cytoreduction, graft, graft manipulation, and graft versus host disease (gvhd) prophylaxis. one aspect of hct that remains poorly defined and studied is the donor/ host (d/h) chimerism post hct. since 2010, we followed patients with d/h lineage specific chimerism post hct. analyses were performed by short tandem repeat (str) polymorphism analysis at the american red cross blood services (philadelphia, pa). studies were performed on blood total leukocytes, myeloid/neutrophil cells, t-cells, bcells, and nk-cells. methods: in this retrospective study, the charts of 154 consecutive patients who underwent allogeneic hct between january 2010 to june 2015 on the pediatric bone marrow transplant service at mskcc were retrospectively reviewed. lineage specific donor chimerism post hct was studied including d/h chimerism trend, and factors with potential impact on chimerism including: age, disease, graft source, and t-cell depletion (tcd). preliminary analyzes performed on this cohort included wilcoxon rank test and cox proportional hazard analyses. results: 137 patients were selected based on the number of analyses. the median age was 11.3 years. patients had hematologic malignancies (n=95) or non-malignant hematologic diseases (n=27), or immune disorders (n=15). cytoreduction included tbi-(n=42), or chemotherapybased regiments (n=98). patients were recipients of t-cell depleted marrow or peripheral blood grafts (n=101), unmodified marrow or peripheral blood grafts (n=28) or cord blood grafts (n=8). full donor chimerism of myeloid cells, b-cells and nkcells, but not t-cells occurred early post-transplant. there was no difference in the percentage of total donor leukocytes at 3 months vs. 12 months post hsct (n=30), while the median of donor t-cell chimerism was 51% at 3 months and 91% at 12 months post hsct (p< 0.0001, n=42). figure 1 shows the impact of different factors including: (a) the use of tbi-or chemotherapy-based regimens, (b) age (< or > 3 years), and (c) type of graft (t-cell depleted vs unmodified vs cord blood). donor total leukocytes chimerism was significantly lower at 12 months as compared to 3 months for patients < 3 years of age (p=0.012). for most grafts, full donor chimerism of t-cells occurred early, while for t-cell depleted transplants, it took up to one year to complete. cord blood grafts were associated with high t-cell donor chimerism throughout the post-transplant period. there was a significant difference in the % donor t-cells at 3 and 12 months post hct when comparing t-cell depleted and unmodified grafts (p=0.015). conclusions: this preliminary analysis of lineage specific chimerism post-transplant showed that donor tcells may take one year to fully recover post-transplant, mostly following t-cell depleted grafts, without intervention. cord blood grafts were associated with high donor chimerism throughout the post-transplant period. lastly, factors associated with loss of donor chimerism posttransplant were younger age and non-malignant disorders. more in-depth analyses are being performed including the relation of chimerism and hct outcome. disclosure: eileen nicoletti -employee rocket pharmaceuticals, susan prockop -investigator atara biotherapeutics -research funding, susan prockop -mesoblast -research funding, nancy kernan -gentium -support; jazz pharmaceuticals -support, richard o´reilly -atara biotherapeutics -royalty, consultancy and research, jaap jan boelens -bluebird bio -consultancy, avrobio -consultancy; jaap jan boelens -chimerix -consultancy; magenta -consultancy background: the success of hematopoietic stem cell transplantation (hsct) lies with the ability of the engrafted immune system to remove residual leukemia cells via a graft-versus-leukemia effect. despite this, relapse remains the major cause of mortality among patients receiving hsct. one of the immune evasion mechanisms of leukemic cells to escape from donor t cell recognition in haplo-hsct is the genomic loss of the patient specific hla. it has been described in 20-30% of acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) relapses after haplo-hsct. the aim of this study was to analyze hla loss in a large cohort of patients who relapsed after t-cell replete haploidentical transplantation with posttransplant cyclophosphamide. methods: from december 2007 to september 2018, 180 patients with hematological malignancies who received a haplo-hsct were recruited. among them, 31 patients presented a relapse after haplo-hsct. hla typing was performed by real-time pcr using hla-kmr kit (gendx, netherlands).nine patients were excluded from the analysis because the kit employed did not include the recipientspecific hla. thus, a total of 22 relapse cases were analyzed. the analysis of chimerism was carried out using short tandem repeat pcr amplification (ampflstr sgm plus, thermo fisher, usa) with a sensitivity of 1%. results: genomic loss of the patient hla occurred in 6/ 22 patients (27%) ( table 1) . these patients presented different hematological neoplasms. interestingly, 4 patients presented lymphoid neoplasm (1 acute lymphoblastic leukemia (all-t), 1 dentritic cell leukemia (dcl) and 2 hodking´s lymphoma (hl)). hla loss relapses occurred later than classical relapses (370 vs.166 days). regarding the treatment received (table 1) , four patients were studied retrospectively. three of them were treated with donor lymphocyte infusions (dlis) + chemotherapy or other drugs at the time of the relapse. the other patient did not receive any treatment. in the end, all 4 patients died from disease progression. prospectively, we detected hla loss at relapse in other two patients. at the moment of relapse, the first case received brentuximab + haplo-hsct from alternative donor and the other case received daratumumab + haplo-hsct (pending). both patients are alive, the first one in complete remission (cr) and the second one in partial remission (pr). conclusions: the frequency of hla loss at relapse is similar in our cohort to what is described in the literature. hla loss has been identified in patients with lymphoid neoplasms, while this mechanism has not been previously described in such diseases. the analysis of this immune evasion mechanism should be implemented in the routine screening of patients transplanted from haploidentical donors in order to design effective rescue strategies. these treatments should not be based on dlis or second transplantation with the same donor, instead, alternative donors should be used. background: an adequate immune reconstitution (ir) is crucial to reduce transplant toxicity, relapse rate and mortality after allogeneic stem cell transplantation (allohsct). the aim of this, single center retrospective study was to investigate the correlation between the recovery of different lymphocyte subpopulations with the main transplant outcomes, including overall survival (os), disease free survival (dfs) and non-relapse mortality (nrm). methods: we analyzed the ir of 177 adult patients (aml n=62, all n=41, mds n= 24, nhl n=9, hd n=2, cll n=4, cml n=7, mm, n=11, mpn n=10) who underwent (allohsct) between january 2013 and march 2017. median age at transplant was 52 years (range 17. 3-71.3) with male/female ratio of 62%. donors were hlaidentical siblings (n=39, 22%), family haploidentical (n=11, 6%), matched unrelated (109, 62%), mismatched unrelated (n=11, 6%) and cord blood units (n=7, 4%). the stem cell source was the bone marrow (bm) in 30 patients (17%), the cord blood in 7 (4%) and g-csf mobilized peripheral blood in 140 (79%). the conditioning regimen was myeloablative in 99(56%) transplant, reduced intensity in 75 (42%) and immunosuppressive in 3 (2%). gvhd prophylaxis was based on calcineurin inhibitors in combination with methotrexate or mofetil mycophenolate. antilymphocytes immunoglobulins was used in 145 patients (81%) (anti thymocytes globulin, atg sanofi-genzyme in 114 or anti t-lymphocyte globulin, atlg -neovii biotech, in 31). the peripheral blood lymphocyte subsets (cd3+, cd3+cd4+, cd3+cd8+, cd19+ (b cells) and cd16+cd56+ (nk) were analyzed by flow cytometry at 1, 2, 3, 6, 12 and 24 months after hsct. post-transplant engraftment was molecularly determined by vntr analysis. results: as detailed in table 1 the proportion of full donor chimerism analyzed in the peripheral blood t lymphocytes improved progressively after transplantation and the same pattern was observed when the chimeric status was measured in bone marrow mononuclear cells. to favor the achievement of a full donor chimerism, dli were performed in 26 patients starting at the median of 96 days after transplant (range:34-447). with a median follow-up observation of 25 months (range 7-61), the one year os and nrm was 88% and 5%, respectively. at 6 months after allohsct, the achievement of values higher than 75, 175 and 65 /μl for cd4+, cd8+ and nk cells, respectively was significantly associated to a better os (figure 1 ), dfs (p = 0.05), and to a lower nrm (p< 0.001 for cd4+ and cd8+, p= 0.0034 for nk). a better lymphoid reconstitution was observed after the use of either a sibling or a haplo donor than a matched unrelated or cord blood donors. the use of atg was significantly associated with a delayed cd4+ recovery but with a faster nk cells reconstitution. conclusions: at six months after allohsct, recovery of cd4+ and nk cells predicts survival. monitoring of immune recovery may help to guide pre and post-transplant treatment strategies. days infections and disease control. several groups have demonstrated the independent prognostic value of different lymphocyte subpopulations in hsct outcomes. posttransplant cyclophosphamide (pt-cy) effectively prevents gvhd after hla-haploidentical hematopoietic stem cell transplantation (haplo). the use of pt-cy in hla matched related (mrd) or unrelated (mud) donors hsct, although less explored, has also been introduced. the aim of this study was to compare the early immune reconstitution after allogeneic hsct from haploidentical and hla-matched donors using pt-cy. methods: one hundred and sixty-four hsct performed in our center were analyzed: 125 haplo performed between 2011 and 2017 and 39 hsct from hla-identical donors (19 consecutive mrd sct performed with pt-cy between 2016 and 2017 and 20 mud sct with pt-cy between 2014 and 2017). pt-cy was administered at 50mg/kg/d in days +3 and +4 postransplant, followed by mmf 10mg/kg/ d and a calcineurin inhibitor (ciclosporina a or tacrolimus) from day +5 ahead. we retrospectively compared early immune reconstitution at day +30 and day +90 among these three populations. early ir was assessed through the analysis of lymphocyte subpopulations including total t lymphocytes cd3+, cd4 + and cd8+subpopulations, nk cd3-cd56+ cells, cd56 + bright immature subpopulation and total b cd19+ lymphocytes.. lymphocytes subpopulations were determined by multiparametric flow cytometry (fc500 and navios, beckman coulter®). results: all patients, but 1 mud and 1 haplo, received pb as stem cell source. 46 patients received prior transplant in haplo group. patient´s characteristics are shown in table 1. patients who received hsct from mrd showed the fastest ir, with statistically significant differences compared to haplo in almost all lymphocyte populations at day +30 (cd3+, cd4+, cd8+ and nk cells), and also in cd3+, cd8+ and b lymphocytes at day +90. comparison between haplo and mud hsct showed better ir among haplo, demonstrated by higher counts in cd3+,cd4+, cd8+ and nk cell counts at day +30. no differences were seen at day +90. (figure 1 ). percentage of immature cd56 bright nk cells was higher in mud hsct at +30, with no differences between haplo and mrd hsct. conclusions: in our cohort of patients with pt-cy based gvhd prophylaxis regimen, those who received hsct from mrd showed the earliest immune reconstitution compared to haplo and mud at day +30 and +90. haplo showed better ir compared to mud at day +30. nk maturation at day +30 was a little better among haplo and mrd hsct recipients than mud hsct patients. in our experience, using mostly pbsc as graft source, type of donor influenced early ir in pt-cy based hsct, background: cell-free dna (cfdna) isolated from plasma or serum has received increasing interest for diagnostic applications. however, the reported clinical usefulness of cfdna in patients undergoing allogeneic cell transplantation (hsct) is scarce. methods: the chimeric status both in peripheral blood and in cfdna obtained from plasma was investigated in 110 patients undergoing hsct. dna and rna were isolated from plasma within four hours of blood draw. patients were evaluated for chimerism at day +30, +100 and +365 post-transplant. a panel of seven microsatellites was amplified by pcr for chimerism detection and pcr products were analysed by capillary electrophoresis. for further cfdna characterization the micro rna (mirna) 200c was analysed using digital pcr. mutations frequently used for minimal residual disease assessment such as flt3-itd, npm1 and jak2 were also investigated in cfdna. results: the mean cfdna concentration in transplanted patients was 469 ng/ml, while in healthy donors used as control group (n=20) was 119 ng/ml. the mean cfdna concentration difference between both groups reached statistical significance (p= 0.0197). when analysing cfdna from transplanted patients and in the control group we could not detect dna fragments larger than 400 bp and the size range of the analysed fragments was between 80 and 200 bp. in 41 out of 110 patients a mixture of donor and recipient cfdna (mc) was detected. with the exception of three patients relapsing after transplant in which mc was detected both in peripheral blood and plasma in the rest of the patients (n=38) mc was detected only in plasma. the mean percentage of recipient cfdna in the plasma samples was 18% (range: 1-81%). all the patients with acute gvhd (agvhd) (grade: i-iv) (n=15) showed mc in plasma at least in one of the time-point tested. no significant difference was found in the mean recipient cfdna percentage in patients with agvhd grade i-ii when compared with grade iii-iv. meanwhile in the group of patients with chronic gvhd (n=42) mc in plasma was detected in 13 patients. in those patients with clinical improvement of agvhd (n=6) a decrease in the percentage of recipient cfdna was observed during treatment. in patients without improvement or even agvhd worsening (n=5) stable or increasing recipient cfdna percentage was detected. since recipient cfdna can be detected in patients without transplant-related complications we analysed the mirna 200c expression in all patients with recipient cfdna. a significant difference was found in the mirna 200c expression in patients with agvhd (mean mirna 200c: 9.7 mirna 200c copies/10 4 u6 copies) when compared with patients without gvhd (mean mirna 200c: 35.4 mirna copies/10 4 u6 copies). in those patients with extramedullary aml relapse (n=3) frequent mutations (flt3-itd, npm1) were only detected in the cfdna fraction. conclusions: longitudinal analysis of cfdna represents a useful complementary tool in particular for those patients with clinical complications after hsct. disclosure: nothing to declare comparison of the impact of atg/pt-cy-based and tcr αβ-depletion as gvhd prophylaxis regimens on the recovery of memory t-cell compartment background: over recent years haploidentical and hlamismatched donors have been increasingly adopted as a valid donor source. modern graft-versus-host disease (gvhd) prophylaxis regimens such as drug-based (antithymocyte globulin (atg), post-transplant cyclophosphamide (pt-cy)) or graft-manipulated (tcr αβ-depletion) demonstrate effective prevention of gvhd. here we report our data about an influence of different gvhd prophylaxis regimens after allo-hsct with pbsc as a graft source on cd8+ memory t-cells recovery. methods: our study comprised 32 leukemia patients who underwent allo-hsct with pbsc as a graft source in national research center for hematology, moscow, russia. detailed patients characteristics are presented in table 1 . peripheral blood samples were collected on day +30, +60 and +90 after allo-hsct. flow cytometry analysis was performed on bd facs canto ii (becton dickinson, usa) to define t-memory subsets: t-naive and t-stem cell memory (tnv+tscm) -cd8+cd45r0-ccr7 +cd28+; t-central memory (tcm) -cd8+cd45r0 +ccr7+cd28+; t-transitional memory (ttm) -cd8 +cd45r0+ccr7-cd28+; t-effector memory (tem) -cd8+cd45r0+ccr7-cd28-; t-terminal effector (tte) -cd8+cd45r0-ccr7-cd28-. sysmex xe-2100 was used to calculate absolute count of different t-memory cell subsets. mann-whitney u test was used for nonparametric data analysis. a p-value less than 0.05 was considered as significant. results: results of mann-whitney u test (calculated pvalues) to compare absolute number of t-memory cell subsets in terms of different gvhd prophylaxis regimens are presented in figure 1 . during all follow-up period the number of tnv+scm and tcm remains significantly reduced after atg+pt-cy or tcr αβ-depletion compared to atg-based immunosuppressive regimen. on day +30 we observe no difference in the number of tnv+scm and tcm cells after atg+pt-cy or tcr αβ-depletion. terminally differentiated cd8+ cells (ttm, tem, tte) count is significantly lowered in tcr αβ-depletion patients group in comparison to atg+pt-cy. nevertheless recovery of tnv+scm and tcm after pt-cy is faster than after tcr αβ-depletion. conclusions: according to our data the mechanism of pt-cy is seems to be more selective compared to tcr αβ-depletion due to its transient impact just on tnv+scm and tcm without affecting on the effector pool. through this it may lead to delayed reconstitution of adaptive immunity after tcr αβ-depletion compared to using pt-cy. clinical relevance of the quantitative characteristics of immune recovery in the context of different approaches to gvhd prevention remains to be established. background: immune effector cells, belonging to either innate or acquired immunity, play a key role on preventing disease relapse after allogeneic haematopoietic stem cell transplantation (hsct). most of known immune effector are cd3 + cd8 + t-cells and cd3 -cd56 + natural killer lymphocytes, while cd3 + cd4 + cells act as modulatory and regulatory cells. the early post-hsct ratio between these cellular subsets may be an indicator of graft vs-tumor (gvt) effect. methods: we retrospectively revised the immune recovery of 117 allogeneic hsct performed at our institution from 2013 to 2017, analysed on peripheral blood by multiparametric flow cytometry lymphocyte subpopulations panel. diagnosis were acute leukemias (69%), chronic myeloproliferative neoplasms (10%), lymphomas (10%), myelodysplastic syndromes (5%), multiple myeloma (3%) and severe aplastic anemia (3%). we established 2 early time-points of evaluation, 30 and 60 days from the graft infusion, to analyse the differences in disease free survival (dfs) and overall survival (os) between patients according to the cd3 + cd8 + x cd3 -cd56 + / cd3 + cd4 + ratio. results: median ratio at +60 days was of 0,5667. at this time point, patients who showed the ratio higher than the median had both a better dfs (median dfs time not reached vs 12 months; p = 0,018) ( figure 1 ) and os (median os time not reached vs 13 months; p = 0,014). likewise, ratio at +30 showed an advantage on dfs (p = 0,027), and not on os (p = 0,06). other factors possibly affecting both dfs and os were analysed in univariate analysis, such as the use of antithymocyte globulin (atg), conditioning regimen intensity, graft source, hla-matching and disease status at hsct, the latter being the only variable with a significantly detrimental impact on both os and dfs. disease status was confirmed an independent valriable associated with both dfs and os as well as +60 ratio both on dfs (hazard ratio [hr] -2,721; p = 0,015) and os (hr 2,627; p = 0,022). conclusions: our data show that cd3 + cd8 + x cd3 -cd56 + / cd3 + cd4 + ratio assessed at +60 is and independent predictor of transplant outcome, possibly representing a row indicator of anti-leukemic immune surveillance. the integration of this index with other known outcome predictors may help in improving the management of post-transplant phase. [[p524 image] 1. figure 1 background: allogeneic stem cell transplantation (alo-hsct) is a curative treatment but it is associated with lifethreatening complications. most deaths are due to relapse, graft versus host disease (gvhd) and infection. the pattern and quality of the immune reconstitution (ir) after transplantation may affect these outcomes. however, there are limited data on the association of the quality of the ir and either the development of gvhd and survival. methods: eighty-five patients who received a non t-cell depleted alo-hsct in our center from 2011 to 2014 were prospectively studied. most patients received hla-identical grafts. total cd4+ and cd8+ t cells, ccr7+cd4+ and ccr7+cd8+ (which include both naïve and central memory t cells) and naïve ccr7+cd62l+ t lymphocytes were quantified by flow cytometry. data were collected at days +30, +60, +90, +180 and +360 after alo-hsct. the association between ir and the gvhd was studied through an anova. for the multivariate analysis, a logistic regression was performed including those confusing clinical variables that were significant in the univariate analysis (p≤0.10). the study of overall survival (os) versus ir was performed with a cox regression model. results: total cd3+ t lymphocytes reached normal numbers within the first two months. median t cd8+ count was 263 cells/ul after one month, which is within the normal range. conversely, it took nearly one year to get normal counts of cd4+ t cells (542 cells/ul). the only two clinical parameters conditioning a worse recovery of the cd4+ t cells were the previous alosensitization of the donor and the sex, being female donor and male recipient the worst combination for the ir. no parameters influenced the quality of the reconstitution of cd8+ t cells. of note, the age or the hla status did not influence the quality of the ir. when the patients were divided into gvhd and no gvhd, we found no differences in the recovery of either the proportion or absolute count of every t cell subpopulation, including total t cells as well as naïve/central memory t cells, both cd4+ and cd8+. finally, a multivariant analysis confirmed that the absolute counts of cd4+ccr7+ t cells at day +90 as well as the absolute counts of both cd4+ccr7+ t cells and naïve cd4+ccr7+cd62l+ at day +180 were associated with better os. conclusions: in conclusion, neither the development of gvhd nor other relevant parameters seem to play a determinant role in the quality of the ir. to our knowledge this is the first study which demonstrate a clear association between the recovery of naïve cd4+ t cells measured by flow cytometry and the os. disclosure: nothing to declare p526 abstract already published. azacitidine (aza) for prophylaxis or pre-emptive therapy for myeloid neoplasms after allogeneic stem cell transplantation whom 21 were treated prophylactically and 11 preemptively. median age was 55 years [range, 15-69] and all patients had a diagnosis of aml or high-risk mds. prophylactic treatment consisted of aza 32 mg/m 2 for 5 days in cycles of 28 days. in the pre-emptive setting, 5 patients received 75 mg/m 2 for 7 days per cycle and 6 patients 75 mg/m 2 for 5 days per cycle. a median of 6 cycles [range, [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] was administered in the prophylactic group and of 4 cycles [range, 4-22] in the pre-emptive group. ten patients also received at least one dli after the third aza cycle: 8 patients in the prophylactic group and 2 patients in the pre-emptive one. results: during follow-up, 10 patients had significant delays in treatment plan due to transitory mild complications. however, 15% of patients (n=5) presented infectious complications requiring hospitalisation and 40% of patients (n=10 in the prophylaxis group and 3 in the pre-emptive group) presented some form of gvhd. in patients who developed gvhd, 3 had to discontinue treatment (all in the prophylaxis group); also 8 patients discontinued treatment due to disease progression. the overall drop-out rate was 37.5% (n=12). survival was analysed from initiation of treatment with aza and median follow-up was 16 months. one-year efs was 95% in the prophylaxis group, with only one patient relapsing and no deaths. in the pre-emptive group, the 1-year efs was 45% and the median efs was 6 months; 1-year os was 70% and median os was 24 months. conclusions: we conclude that post-transplant aza treatment is a well-tolerated therapy, but the incidence of side effects remains discordant in the literature. results in the prophylaxis group are excellent, but patients with positive minimal residual disease treated pre-emptively had a lower outcome with only stabilisation of the disease. randomised prospective trials are needed to define patients who would benefit the most from this treatment and at what timing, dosage and duration of treatment. disclosure: nothing to declare. abstract already published. results: all subjects experienced hematopoietic engraftment at a median of 17 days (range 14-20) and demonstrated full donor myeloid chimerism. m-mdsc and pmn-mdsc recovery peaked at a median of 22 days posttransplant. the median peak absolute m-mdsc count was 9,828 cell/ml (range 9,180-41,120/ml) representing a range of 1.5% to 4.8% of pbmcs. the pmn-mdsc peak was more robust, with a median absolute peak of 103,730 cells/ml (range 11,330-676,800/ml) representing a median of 25.3% of pbmcs (range 10. 3-44.2%) . of note, the one patient who developed severe, life-threatening gvhd had the lowest absolute and relative pmn-mdsc recovery (11,330 cells/ml and 10.3% of total pbmcs). recovery of m-and pmn-mdscs occurred at a similar tempo and magnitude in two recipients of standard gvhd prophylaxis (tacrolimus/methotrexate). however, while mdscs isolated from ptcy recipients exhibited clear t-cell suppressive capacity, those from the comparison patients did not (see figure) . conclusions: mdscs recover rapidly and robustly after allohct using ptcy as gvhd prophylaxis, and may play a role in mitigating gvhd risk by mediating t-cell suppression. this may be a mechanism by which ptcy results in donor-recipient tolerance. background: high dose chemotherapy followed by autologous stem cell transplantation (asct) offers a cure in the upfront and relapsed setting in both hodgkin (hl) and non-hodgkin lymphoma (nhl). asct also remains standard of care in previously untreated multiple myeloma (mm) patients after induction therapy, if eligible. the availability of new cellular or other immune therapies that can be used after asct underscores the potential importance of monitoring immune reconstitution after asct. methods: immune reconstitution panels (irp) were evaluated retrospectively in all lymphoma and mm patients over a 5-year span (2012-2017) whom underwent asct at our institution. patients were included if they had a pre-asct measured within 30 days of asct and two other irp at any of the following timepoints (1) day 30-45, (2) day 60-90, and (3) at 1-year post-asct. patients in the lymphoma cohort had their irp excluded if they had additional treatment within the first year post-asct (other than maintenance rituximab). mononuclear cells from peripheral blood were analyzed by flow cytometry for assessment of lymphocyte phenotype and numbers. absolute values were compared using the mann-whitney u test. results: the data on 78 patients were available for analysis (50 mm, 24 nhl, 4 hl) . all lymphoma patients were conditioned with beam. all mm patients were conditioned with a standard high dose melphalan regimen. the median pre-asct absolute cd3 counts in the lymphoma cohort were significantly lower than the mm cohort at 1709 cells/μl vs 3309 cells/μl, respectively (p=0.049). however, the mm cohort exhibited a greater percent reduction in cd3 cells on day 30 at 85.3% vs 63.5%, respectively which continued through day 365 at 79.8% vs 70.3%, respectively. this led to nonsignificant changes in absolute cd3 count by day 365 at 667 cells/μl vs 507 cells/μl, respectively (p=0.39) (figure 1). the median absolute cd4 count pre-asct for mm and lymphoma cohorts were 2101 cells/μl and 863 cells/μl, respectively (p=0.007). similarly, a greater percent reduction in cd4 cells led to comparable absolute counts on day 365 at 310 cells/μl vs 298 cells/μl, respectively (p=0.322). the failure of post-asct cd3 reconstitution to pre-asct levels was driven by lack of cd4+ cell recovery, namely cd4+cd45ra+ cells with a median of 21 cells/μl and 20 cells/μl in the mm and lymphoma cohorts, respectively at day 365 (figures 2 and 3). this led to markedly diminished cd4:cd8 ratios through day 365 (figure 4). [[p531 image] 1. conclusions: impaired t-cell reconstitution in both lymphoma and mm continues through 1-year post-asct. as shown, a larger percent reduction in median cd3 and cd4 counts through day 365 was appreciated in mm compared to lymphoma leading to the nonsignificant differences in the post-asct absolute counts despite significantly higher pre-asct counts in the mm cohort. impaired recovery of cd4 t-cells may increase the risk of opportunistic infections, decrease the response to vaccination and lead to ineffective anti-tumor response. further prospective and larger retrospective studies like this should continue in the modern-era as they may help predict responses to further interventions requiring a robust t-cell repertoire for maximal efficacy such as car-t cell and bite therapies. disclosure: nothing to declare p532 peri-transplant detection of measurable residual disease by multicolor flow cytometry is highly predictive for relapse in acute myeloid leukemia patients background: presence of measurable residual disease (mrd) prior to allo-sct has been shown to be predictive for survival in patients in hematological cr of aml. in this study we analyzed the impact of mrd in such patients measured by 8-color multiparameter flow cytometry (mfc) prior to and on day +100 post-transplant. methods: the bone marrow samples immediately prior to allo-sct and on day +100 post-transplant were retrospectively analyzed. mrd evaluation was carried out with antibodies against: (1) results: a number of 55 aml patients (male, n=35) with median age of 57 years (23-77) in hematological cr prior to allo-sct were enrolled in the study. we observed lower survival in patients with mrd by mfc pre-transplant (2y os: 61% (41-81%) vs. 92% (82-100%), p=0.018) due to increased relapse incidence (35% (17-53%) vs. 8% (0-18%), p=0.009). in multivariate analysis, mrd pos prior to allo-sct has strong significant impact on os (hr 6.5 (1.5 -30) , p=0.015). of 55 patients, a sample both before and on day +100 after transplantation was available in 33 patients. of those 33 patients, 15 (46%) were mrd negative prior to transplant and on day +100 (mrd neg/neg ); 14 (42%) patients were mrd positive prior to transplant and negative at day +100 (mrd pos/neg ); and 4 (12%) patients were mrd positive at both timepoints (mrd pos/pos ). dfs and os for these three groups were as follow: 2y dfs: mrd neg/neg : 93% (80-100%), mrd pos/neg : 78% (56-100%); mrd pos/pos : 25% (0-75%, p=0.006); 2y os: mrd neg/neg : 100%; mrd pos/neg : 93% (79-100%); mrd pos/pos : 25% (0-75%, p< 0.001). upon multivariate analysis, the mrd status prior to transplant and on day +100 showed strong significant impact on dfs (hr 3.6 (1.3 -9.9), p=0.01) and os ), p=0.017). we did not observe any significant impact of other factors included in the multivariate analysis (patient's age, patient's sex, and recipient/ donor sex constellation). conclusions: mrd positivity prior to allotransplant and at day +100 by mfc is highly predictive for survival after allo-sct. disclosure: nothing to declaire background: immune reconstitution is a critical factor for risk assessment of life threatening infections and long-term survival in patients undergoing allogeneic cell transplantation (hsct). methods: immune cell subsets (cd19, cd4, cd8, cd56, cd25+cd127-) were quantified by flow cytometry. trec and krec were quantified simultaneously using droplet digital pcr (dpcr). a total of 31 patients were evaluated. mean age at transplant was 56 years (range: 18-74 years) samples were obtained before hsct and at day 30, 100 and 365 after hsct. results: absolute numbers of cd3 and cd19 cells remained below pre-transplant levels until day 100, increasing further and eventually reaching pre-transplant levels one year after hsct. absolute counts of cd4 and cd25+cd127-cells remained below pre-transplant levels beyond one year after hsct. cd56 cells were characterized by fast reconstitution kinetics, reaching pre-transplant levels already at day 30. b cells correlated with krec levels at all time-points tested, whereas t cells correlated with trec levels only one year after transplantation. when we compared cell subsets, trec, krec levels and the reconstitution kinetics thereof between patients with reduced intensity conditioning (n=26) or full conditioning (n=5) no significant differences were observed. patients with pre-transplant trec levels above the mean (200 trec copies/ml blood) showed higher trec levels and a faster t-cell reconstitution after hsct suggesting that tcell reconstitution can be predicted by analysing thymic functionality before transplantation. indeed, in patients with a pre-transplant trec above 200 trec copies/ml blood, the positive predictive value for an efficient t-cell reconstitution was 0.889 (p=0.012). we analysed the recovery kinetics of the cell subsets, trec and krec levels in patients with and without transplant-related complications. patients with either acute graft-versus-host disease or severe infections showed a slower trec reconstitution when compared with patients without complications. conclusions: our data suggest that the analysis of immune cell subsets together with trec and krec quantification can be used to evaluate the immune reconstitution process after hsct. pre-transplant trec levels allow t-cell reconstitution efficiency prediction after hsct. disclosure: nothing to declare background: falling donor / mixed chimerism after allogeneic haematopoetic stem cell transplant (sct) is associated with an increased risk of relapse and the potential for graft rejection. donor lymphocyte infusions (dli) are often administered in patients with mixed chimerism to achieve full donor chimerism but there is little data on long term outcomes for dli given for persistent mixed chimerism. methods: a retrospective analysis of all patients administered dli for mixed chimerism between 2008 to january 2017 was performed. all patients were transplanted at the university hospital of wales within the south wales blood and marrow transplant (swbmt) programme. patients were identified by the swbmt database and additional outcome data gathered by review of patients' medical records. results: 58 patients were treated with 111 donor lymphocyte infusions between 2008 and january 2017. thirty one patients treated for relapse (with or without mixed chimerism) were excluded as was a further patient with a mismatched donor. the rest were 10/10 match. twenty six patients received a total of 54 donor lymphocyte infusions for mixed chimerism alone. the median age was 63 years (range: 27-71) with 65% women. fourteen (54%) of the patients had sibling donor transplants and twelve (46%) from matched unrelated donors. indications for transplant were: for aml or saml (n=16), myelofibrosis (n=4), mds (n=3), hodgkin lymphoma (n=1), non-hodgkin lymphoma (n=1) and all (n=1). escalating doses of donor cd3+ t cells were administered commencing at 5×10 5 /kg to 5×10 6 /kg then increased at half log increments according to chimerism results until full donor chimerism was achieved. the median number of doses administered was 2 (range 1-5). the median interval was 94 days (range 48-408). the median dose was 1×10 6 / kg (range 5×10 5 -6×10 7 ). seventeen patients (65%) achieved full donor chimerism and remained so until most recent follow up (median 25 months, range 12-64). one patient continued to receive dli after the study period and later reverted to full donor. two patients had ongoing mixed chimerism with no evidence of relapse. two patients relapsed; one of whom later achieved remission. there were six cases of gvhd; acute gvhd (grade ii n=2, grade iii n=1) and 2 cases of chronic extensive gvhd. one patient had gvhd features consistent with overlap syndrome. a total of five patients died, four due to infection (one in a patient with gvhd) and one due to cardiac toxicity from previous treatment (confirmed on post-mortem). conclusions: the results of our single centre study help reinforce the evidence for dli in establishing full donor chimerism when mixed chimerism is detected in the absence of relapse. incremental dli dosing is an effective strategy and associated with a low relapse rate. caution should still be given to the risk of gvhd following dli, however the risk appears to be low in this study. larger prospective studies are ongoing to address the optimal dosing strategy for dli post-transplant. disclosure: nothing to declare hypomethylating agents for the treatment of relapsed acute myeloid leukemia after allogeneic blood stem cell transplantation: a single center experience mariarita sciume 1 , giorgia saporiti 1 , elena tagliaferri 1 , nicola fracchiolla 1 , federica grifoni 1 , giorgia levati 1 , luca baldini 1 , francesco onida 1 the post-transplant period with well-balanced profile of good efficacy and moderate toxicity. we retrospectively evaluated the safety and efficacy of hma +/-dli in a reallife cohort of aml patients relapsing after allo-sct. methods: data from all patients with aml who underwent allo-sct at our institution in the last 6 years and subsequently received hma as a salvage treatment for disease recurrence or preemptively for loss of complete donor chimerism were collected. results: eleven patients with a median age of 64 years (range 41-66) were identified; median time between allo-sct and time to hma therapy was 10 months (range 4-42). according to eln genetic risk stratification, 2 patients were classified in the favorable group, 3 in the intermediate-i, 2 in the intermediate-ii and 4 in the adverse one. six patients were treated with aza, whereas the remaining 5 patients with dac. the cycles were repeated every 28 days. ten patients (91%) started hma for morphological aml relapse, while one patient received aza as a sequential treatment after dli administered for loss of complete donor chimerism. median number of cycles was 3 (range 1 -20). treatment strategy included combination with dli in 5 patients (2 in the dac cohort, 3 in the aza cohort), while in one case of flt3-itd + aml sorafenib was also associated to dac and dli. no grade 3/4 toxicities and no acute gvhd occurred. a clinically significant response was observed in four patients (36%), all receiving at least 4 cycles of hma therapy; in particular, a complete remission (cr) was achieved in 3/10 patients treated for morphological relapse, including the one who received the dac/dli/sorafenib combination and one (favorable eln risk) who received aza alone (not eligible for dli due to a concomitant lateonset cutaneous grade 2 gvhd). of interest, the latter patient also displayed a resolution of the cutaneous gvhd. full donor chimerism recovery with no gvhd was observed in the patient who received aza for the progressive donor chimerism loss not responding to dli alone. with a median follow-up of 7 months (range 4-29), the median os from hma treatment in responding patients was 16 months (range 4-29); at the time of data collection responses were maintained in all four patients. seven patients had died, six from aml progression and one for severe intestinal gvhd occurring after failure of dli+aza and a following salvage induction chemotherapy treatment. conclusions: although arising from a limited number of patients, our real-life experience of treatment with hmas +/-dli in aml patients relapsing after allo-sct showed a general very good safety profile and promising antileukemic activity, altogether suggesting a facilitation of the graftversus-leukemia effect (gvl) associated to a possible suppression of the gvh reaction. disclosure: nothing to declare conclusions: in this study, cd4-positive cell count and igg value had recovered about 15 months after sct. in our institute, we have achieved a low incidence of infection by education and medication for patients until recovery of cd4-positive cell count and igg. however, we found a higher incidence of infection after recovery of cd4-positive cell count and igg. at 12-15 months after sct, administration of prophylactic medications such as sulfamethoxazole-trimethoprim were terminated and social comeback such as return to school or work were achieved in most patients. it is possible that the high incidence of community-acquired infection was associated with their comeback. thus, we should consider additional prevention of infection for patients in this period and further evaluation of immunological markers is needed. disclosure: no potential conflicts of interest were disclosed. effect of minimal residual disease before transplantation on the outcome of haplo-identical hematopoietic stem cell transplantation for high-risk acute lymphoblastic leukemia yehui tan 1 , sujun gao 1 , xiaoliang liu 1 , long su 1 , wei han 1 , yu liu 1 , yangzhi zhao 1 background: to analyze the effect of haploid hematopoietic stem cell transplantation (hid-hsct) on high-risk acute lymphoblastic leukemia (all), and to explore the effect of minimal residual disease (mrd) before transplant on the prognosis. methods: a retrospective analysis was made on 39 high risk all patients accepted hid-hsct in our hospital from january 2013 to january 2018. the clinical features, stem cell implantation, complications, survival and recurrence were compared between pre-transplant mrd + and mrdpatients. results: all the 39 patients got successfully implanted. the overall survival (os) was 54.67%, the disease free survival (dfs) was 40.96%, the incidence of acute graft versus host disease (agvhd) was 53.8%, including 23.1% ii~iv degree agvhd and 2.6% iii~iv degree agvhd. there was no significant difference in stem cell implantation, gvhd, cytomegalovirus and hemorrhagic cystitis between mrd + and mrdpatients. dfs and os in mrd + patients were significantly lower than those in mrd -patients, and the cumulative rr rate increased significantly, there was no significant difference in cumulative trm. conclusions: hid-hsct was an effective method to treat high risk all, but mrd + patients had high recurrence rate and poor prognosis. strategy adjustment should be considered to reduce tumor residual and the transplantation strategy should be optimized for these kind of high risk patients, so as to improve survival rate. disclosure: nothing to declare background: lymphocytes are responsible for the cellular and humoral immunity and, consequently, its recovery after allo-hsct might be linked with the survival after the procedure. the aim of this study was to analyze this hypothesis in our series of patients. methods: all the 209 allo-hsct performed in our center from january 2015 through july 2018 were included in the analysis. median age was 52 years (range: 7-69). 122 pts were male (58,4%) and 87 were female (41,6%). baseline diseases were: 69 aml, 49 lpd, 31 mds, 28 all, 16 mpd, 10 mm, and 6 bmf. donor was unrelated in 113 (54,1%), and was family in 96 cases (45,9%) (including 31 haplo-identical). stem cell source was pb in 195 (93, 3%) and bm in 14 pts (6,7%). conditioning regimen was reduced in 111 procedures (53,1%) and intensive in 98 (46,9%) (including just one non-myeloablative). overall mortalities at days +100 and +365 (the latter in patients with follow-up superior to 1 year) were 9,1% and 24,9%, respectively. median follow-up was 25 months (range: 4-47). evolution of absolute lymphocyte counts (alc) and subpopulations at pre-hsct and during the first year after allo-hsct were analyzed. results: as shown in table 1, alc and cd4+ lymphocytes decreased after conditioning therapy, and recovered progressively during the post-hsct period. at day +365, majority of patients had >1000 alc/mcl, clearly improved compared to admission values. cd4+ lymphocytes at day +100 was still very low, but at day +365 around half of the series had 200-500/mcl. we found a strong link between alc, cd4+ lymphocytes, and cd19 + lymphocytes at days +30 and day +100 with overall survival at day +365 of the series (table 2) . conclusions: in our series, immunity recovery was a late event for majority of patients undergoing allo-hsct. in addition, in our experience, the precocity and quality of the alc, cd4+, and cd19+ cells recovery was clearly linked with long-term survival. background: the reconstitution of t and natural killer (nk) cells after hematopoietic stem cell transplantation (hsct) strongly influences the outcome of hsct including viral infection and graft versus-host disease (gvhd). the purpose of this study was to investigate the clinical efficacy of immune reconstitution including t and nk cells after hsct in children. methods: we reviewed the records of 30 patients who undergoing allogeneic hsct in department of pediatrics, pusan national university children's hospital, from january 2013 to july 2017. the counts of t lymphocyte subsets and nk cells was monitored in peripheral blood by flow cytometric technique during 1, 3, 6, and 12 months post-hsct. blood samples for cytomegalovirus (cmv) and epstein-barr virus (ebv) monitoring were tested by real-time pcr assay. results: for total of 30 patients, the mean age was 9.1 years (range, 9 months-19 years), 16 of the patients were boys and 14 was girl. out of a total 30 patients without pre-hsct cmv viremia or cmv infection, 10 (33.3%) recipients experienced cmv infection. the number of cd8 + t cells in 3 and 6 months post-hsct was significantly higher in patients with cmv reactivation compared to patients without (median 1066.11/μl vs. 979.0/μl, p=0.004, and 1047.45/μl vs. 551.44/μl, p=0.002) . in 6 (20%) recipients presented acute gvhd, the number of cd4 + t cells in 1 and 3 months post-hsct was significantly lower in patients with acute gvhd compared to patients without (median 239.13/μl vs. 365.0/μl, p=0.045, and 165.2/μl vs. 344.27/μl, p=0.035) . the number of nk cells in 1 months post-hsct was significantly lower in patients with cmv reactivation and acute gvhd compared to patients without (258.5/μl vs. 501.67/μl, p=0.004, and 162.5/μl vs. 464.88/μl, p=0.027, respectively) . in multivariable analysis, acute gvhd was shown to be the decisive factor influencing total t cells (p=0.028) and cmv reactivation was independently associated with cd8 + t cells (p=0.026). the cd4 + t cells counts were associated with prior hsct history and acute gvhd (p=0.042 and p=0.038), and the cd8 + t cells counts were also significantly associated with donor type (p=0.016). conclusions: overall, our study documents that immune reconstitution of cd4 + , cd8 + t cells and nk cells is strongly associated with cmv reactivation and acute gvhd. additionally, we show that acute gvhd is influenced by lack of sufficient numbers of nk cells as well as cd4 + t cells early after sct. cd8 + t cells, on the other hand, significantly increase after cmv-reactivation and most likely play an important role in reactivation. disclosure: nothing to declare background: curative effect of allogeneic hematopoietic stem cell transplantation (allo-hsct) depends on the alloreactive t-cell immune response toward residual malignant cells -graft-versus-leukemia reaction. however, alloreactive population has not been phenotypically defined. recent studies suggest that alloreactive t cells express both costimulatory and inhibitory receptors simultaneously. exhaustion caused by the inhibitory signaling dampens tcell functionality, which could lead to the disease relapse. here we aimed to investigate the expression of costimulatory and inhibitory receptors on antigen-experienced t cells after transplantation, to isolate subpopulation specific for allo-hsct patients and analyze their t-cell receptor (tcr) repertoire. methods: expression of coinhibitory and costimulatory molecules on pbmcs patients at various time points after allo-hsct was analyzed for expression of: cd3, cd8, cd4, cd45ra, ccr7, cd95, cd27, cd28, klrg1, tigit, pd1, cd137 and ox40 by flow cytometry and compared to healthy donors. cd3+cd8+cd95-cd27 +cd28+pd1+tigit+ fraction and cd3+ cd8+ control fractions were separated on facs aria ii cell sorter. double barcoded cdna libraries of tcr beta-chains for both fractions were prepared and analyzed by sequencing on illumina platform. sequencing results were processed by migec, mixcr and vdjtools software. enriched clones were identified by fisher's exact test (p>10 -10 ). results: we did not find any significant differences between patients after allo-hsct and healthy donors in single marker's expression, but, when considering coexpression of co-stimulatory and inhibitory molecules on t cells we found that cd3+cd8+cd95-cd27+cd28 +pd1+tigit+ subpopulation was significantly increased in allo-hsct patients. moreover it increased with the time since the transplantation (fig. 1 ). this population was isolated by cell sorting and alongside with total cd8+ fraction subjected to tcr beta-chain repertoire sequencing. the population contained clones significantly enriched compared with cd8+ fraction representing potentially alloreactive cells. this hypothesis is further supported by the notion that the level of expression of cd27 and cd28 co-stimulatory molecules is lower in the group of patients who subsequently relapsed, compared with the patients with complete remission, while the expression of inhibitory receptors was high in both groups. conclusions: according to our data patients after allo-hsct have a phenotypically distinct t-cell population characterized by simultaneous expression of costimulatory and inhibitory markers. this population contains specifically enriched clones, which may be specific for alloantigens. further functional assays are needed to confirm the alloreactive potential of this subpopulation. besides low expression of costimulatory molecules combined with high expression of inhibitory receptors on antigen-experienced t-cells of patients after allo-hsct might be associated with a disease relapse. fondazione mbbm, monza, italy, 3 ospedale san gerardo, laboratorio stefano verri, monza, italy background: poor graft function (pgf) is a severe complication after hsct, with a high risk of morbidity and mortality, mainly due to infections. donor cd34+ scb seems to offer high chances of haematological recovery, not jeopardized by gvhd. however, pediatric reports remain scarce. methods: 19 out of 215 patients undergoing transplantation in our pediatric unit from 2012 to 2018 have been retrospectively evaluated for at least 2 line persistent cytopenia (hb< 9.5g/dl, plt< 30000/mmc, n< 1000/ mmc) and/or transfusion-dependency beyond 3 months after hsct in the presence of full donor chimerism. bone marrow cellularity was evaluated through biopsy as further indicator of pgf. (1/8) to donate or medical decision (7/8). bone marrow cellularity was < 5% in 50% of the patients who underwent scb for which the histology was available (8 cases), and 25% in those who have not been treated (2/8). at 30 days after scb 10/11 (91%) patients had hematological response, which was complete in 46% and partial in 45% of the patients. only 1 patient had no response. the infusion was always well tolerated with no adverse events, and no worsening of gvhd. haematological recovery occurred spontaneously at 30 days after bone marrow biopsy in a significantly lower proportion of patients (2/8, 25%, p< 0.05) within the non-scb group. in two cases platelets engraftment was significantly delayed, up to one year after bone marrow biopsy and in one case thrombocytopenia persists and the patient is still receiving thrombopoietin agonists and red blood cells transfusions at 9 months after bone marrow biopsy. conclusions: a stem cell boost matched the goal to yield count recovery in our cohort. viral infections and gvhd may be possible risk factors for pgf.bilinear or trilinear cytopenia with transfusion dependency and bom cellularity < 5% and full donor chimerism are good indications for scb, that can provide a significantly earlier hematological reconstitution, without risks of gvhd. due to the proved early efficacy and safety of cd34+ stem cell infusion, we suggest that this procedure should be taken in consideration in children with severe bone marrow hypoplasia and persistent cytopenia after hsct. disclosure background: as allogeneic hematopoietic stem cell transplantation (hsct) is sometimes performed despite erythrocyte's antigens incompatibility and mismatch, it is essential to carefully track patients' genotypes after it. methods: for the study we used erythrocytes (n=189) and dna (n=20) from patients undergoing abo-or rhesus-mismatch hsct and their donors. we used posttransplant no transfused patients on the periods according transplant protocol by hemagglutination methods in plate and tube using monoclonal antibodies to abo and rhesus antigens (hematolog, russia). we extracted dna with dna kit (bag, germany) and conducted pcr-ssp with kits abo-type, rh-type (bag, germany). chimerism was assessed by the str-pcr analysis with cordis plus multiplex kit for amplification of 19 polymorphic strmarkers and amelogenin loci. the fragment analysis was performed on a 3130 genetic analyzer. informative loci were chosen by comparison of pretransplant patient's and donor's dna. the percentage of donor chimerism was calculated using standard formula. precise rhce and abo genotypes were determined by direct sanger sequencing. we revealed 3 patients with unexpected erythrocyte abo, rhesus phenotypes and genotypes after hsct on +30 (2 patients) and +160 days (all patients). chimerism analyses on str showed in a.e.kh. and g.l.v. patients 99% of donor's dna and less than 1% of recipient's one. b.n.a. patient was relapsed and chimerism analysis revealed 95% of recipient's dna and 5% of donor's one. using serological methods and pcr-ssp we revealed genotypes abo * a1b1; rhd+; rhce * ccee in patient a. e.kh. before hsct, abo * a2o1; rhd+; rhce * ccee in her donor, and abo * a1a2; rhd+; rhce * ccee on +30d after hsct. genotype a1a2 was no recipient's neither donor's origin. direct sequencing did not prove this genotype, but revealed donor's one.on +160d serological methods and pcr-ssp also revealed donor's genotype in this patient. patient b.n.a. had genotypes abo * o01/o01; rhd+; rhce * c w cee before hsct, her donor -abo * b1o1; rhd +; rhce * ccee. on +160d this patient relapsed, but rhesus genotype has been detected as rhd+; rhce * c wcee (lack e gene). direct sequencing revealed gene rhce*ee. abo genotype was recipient's origin -o1o1. in patient g.l.v. using serological and pcr-ssp methods we determined genotypes abo * o01/o01; rhd +; rhce * ccee genotype before hsct, and abo * a1/ o01; rhd+; rhce * ccee genotype in her hsc donor. on +30d patient had unexpected genotype abo * o01/o01 (a lack of a antigen); rhce * ccee (a lack of e antigen). in order to explain unexpected patient's genotypes after hsct we sequenced her rhce and abo genes and found donor's genotype ccee; a1o1 that was in agreement with results of str analysis. to resolve discrepancies between serological, pcr-ssp and sequencing analysis data we sequenced patient's rhce cdna and observed only ce allele. at present time the molecular basis of selective inactivation one of the two rhce alleles is not clear. on +160d patient had donor's genotype. conclusions: what kind of mechanisms led to discrepancies between results obtained by different laboratory methods are still not clear. an interesting case of expression of only one rhce allele in patient g.l.v. allows us to suggest involvement of some epigenetic mechanisms like dna methylation or histone modification in this process. clinical background: in relapsed patients with acute b -lymphoblastic leukemia (all-b) who achieved complete remission (cr) after re-induction therapy, minimal residual disease (mrd; ≥10 -3 all-b cells/ul) is often detected. according to available data, such condition varies from 30% to even 50% of cases, as assessed by pcr or flow cytometry (fc), while the presence of mrd is the most important risk factor for all recurrence. in this abstract, we describe our experience with bridging therapy using blinatumomab infusion after re-induction regimens and before the planned allogeneic stem cell transplantation (allo-sct). the procedure was performed in three young men suffering from relapsed ph (-) all-b at the age of 19, 22 and 34 years. in the first case (19yo), relapse with previous mrd accounting for 2.7% occurred 15 months after cr1 mrd neg . in the next patient (22yo) the second relapse with central nervous system (cns) involvement occurred 5 months after allo-sct performed in cr2 (26 months after cr1, mrd neg ), while in the third patient (34yo), recurrence with cns and testis involvement occurred 12 years after cr1 (mrd neg ). all patients underwent chemotherapy (flam, hypercvad and dnr/vcr/pegasp/dexa regimens respectively) followed by one cycle of blinatumomab (at a dose of 9mcg/d on days 1-7, followed by 28mcg/d on days 8-28 in a continuous infusion) and allo-sct (using eto/cy/tbi/atg/ conditioning regimen for ist and iiird patient and bucy2 for iind patient; using matched unrelated donor (mud, ist and iiird patient) or matched related donor (iind patient)). mrd status was assessed after each cycle of blinatumomab by fc. results: all patients achieved cr mrd pos after reinduction therapy followed by clearance of mrd after blinatumomab course (tab. 1). the second patient, due to positive mrd 6 months after allo-sct received 4 donor lymphocyte infusions additionally. during the administration of blinatumomab, no adverse events (aes) were observed in grade 3 or 4. one patient developed cytokine release syndrome in grade 1. the progression free survival, time to positive mrd and follow up are presented in tab 1. conclusions: the use of blinatumomab as a bridging therapy between re-induction regimens and allo-sct in patients with all-b and mrd pos appears to be safe and leads to the clearance of mrd which may be crucial in os and pfs prolongation after following allo-sct. future studies on larger groups of patients are necessary to confirm this thesis. background: haploidentical hematopoietic stem cell transplantation (hsct) is considered an alternative treatment for hematologic malignancies in patients who do not have an hla-identical sibling donor [1] . since infections and disease relapse resulting from delayed immune reconstitution (ir) are the most common causes of mortality among patients undergoing haploidentical-hsct [2], timely ir is essential in the recovery and survival of these patients. the aim of this study is to describe the evolution of ir after haploidentical-hsct and to estimate survival rates in patients with delayed vs. adequate reconstitution in a single center in colombia, south america. methods: a retrospective cohort study was conducted on 26 consecutive adult haploidentical-hsct recipients at a tertiary referral center. cd4+cells, cd8+cells, cd3+cells, and immunoglobulins levels were monitored before hsct, at first month, and then every three months for the first two years post-transplantation. descriptive statistics were used to analyze patient's clinical characteristics. the kaplan-meier method was used to assess overall survival (os) and relapse-free survival (rfs) rates. results: twenty-six patients were included (50% were male), with a median age of 25.5 years (range 16-59). the most common indication for haploidentical hsct was acute lymphoblastic leukemia (n=21, 80.8%), followed by non-hodgkin lymphoma (n=3, 11 .5%) and myelodysplastic syndrome (n=2, 7.7%). all patients received gvhd prophylaxis therapy with cyclophosphamide, tacrolimus, and mycophenolate mofetil. fifteen patients (57.7%) presented cytomegalovirus reactivation (25/26 at risk), 5 patients (19.2%) epstein-barr virus reactivation, and 4 patients (15.4%) developed adenovirus infection. median time to neutrophil engraftment (neutrophils>0.5×10 9 /l) was 15 days (range12-29) for the 23 patients recipients of peripheral blood progenitor cells (pbpcs) and 21 days (range20-27) for the three remaining bone marrow recipients. platelet engraftment, defined as >20,000 platelets/ mm 3 background: daratumumab is a human monoclonal antibody directed against the glycoprotein cd38 that is overexpressed on the surface of plasma cells in multiple myeloma patients. it is approved as second line therapy either as single agent therapy or in combination with lenalidomide or bortezomib for the treatment of patients with relapsed/refractory multiple myeloma. despite the curative potential of an allo-sct, the high relapse rate remains a clinical problem. data addressing the choice of an optimal salvage therapy regime for these heavily pre-treated patients is missing. methods: from april 2016 till november 2018 a total of 22 patients (male, n=10) with the median age of 64 years (40-72) received daratumumab as a salvage therapy for relapse of multiple myeloma after allo-sct at the university of hamburg. prior to allo-sct all but one patient had received an autograft, 9 patients even ≥2 autografts and 4 patients also a 1. allograft. the median number of salvage lines post-transplant and prior to first daratumumab infusion was 2 (0-4). these salvage regimens included cyclophosphamide, etoposide, bortezomib, lenalidomide, pomalidomide and carfilzomib. daratumumab was started at a median of 19 months (0-43) after relapse/ progress and initiated as single agent therapy in all patients. concomitantly, 14 patients received either an immunomodulatory drug (lenalidomid, n=10; pomalidomid, n=1) or a proteasome inhibitor (bortezomib, n=3) during a later course of daratumumab infusions. combination therapy was initiated when a slow rise of paraprotein and/or free light chains or no response to monotherapy was observed (median at the 11 th infusion). results: the median number of infusions was 14 . twenty adverse reactions were observed in 13 of 22 (59%) patients: dyspnea (n=5), bronchospasm (n=2) shivering (n=3) , cough (n=2), musculoskeletal pain (n=4), acute coronary syndrome (n=1), skin rush (n=1), facial edema (n=1), pressure on eyes (n=1). all adverse reactions appeared during the first infusion and were mostly mild or moderate (ctc 1-2, n=19). tolerance of the following infusions improved and in none of the cases therapy had to be stopped due to adverse events. three patients developed late onset infections (pneumonia, n=2; urinary tract infection, n=1) followed by temporarily therapy interruption. with a median follow-up of 14 months after the first administration 20 of 22 patients remain alive .9%). one patient died due to progress of myeloma and another died due to severe infection/sepsis. 15 of 22 patients responded (68%; pr, n=5; vgpr, n=8; cr, n=2) to the therapy with daratumumab. the responses (decrease of paraprotein and/or free light chains ≥50%) occurred at a median of 7 days (4-372) after the first administration and lasted for 5.0 months (0.5-26.6). conclusions: daratumumab shows an encouraging efficacy and acceptable toxicity profile in patients with relapsed/refractory myeloma after allo-sct. further studies are needed to investigate the role of the combination therapy with immunomodulatory drugs or proteasome inhibitors in this setting. disclosure: nothing to declare p546 clonal plasma cell detection by high sensitive flow cytometry in aphaeresis product is poor prognostic and not increased by use of plerixafor alone background: in an earlier from our center we have demonstrated that residual clonal plasma cells (cpc) decrease both overall survival (os) and disease free survival (dfs) (ash 2018).plerixafor is a selective antagonist of cxc4 chemokine receptor (cxcr4) and able to mobilize human peripheral blood stem cell (pbscs) by acting synergistically with g-csf.the purpose of this study was to evaluate the safety and efficacy of plerixafor in myeloma patients who were proven poor mobilizers and specifically to assess the flow cytometric measurement of residual clonal plasma cells in the apheresis products. methods: patients with a diagnosis of mm who underwent auto hsct at our center between january 2008-november 2018 were retrospectively analyzed.out of 164 patients, 16 patients received plerixafor as mobilization regimen due to poor mobilization with g-csf.pbsc grafts were tested for the presence of clonal pcs (cpc) and the number of normal pcs (npc) by multi-parameter flow cytometry (fcm).the acquisition of the cells was performed using the navios flow cytometer beckmancoulter) .upon the daily checks of the instrument, 3x10 6 cells for each sample were acquired and the collected data was analyzed using the kaluza software (beckmancoulter,usa). results: patient demographics are shown in table 1 .the majority of patients were male and median age was 62 years in the plerixafor group.the median interval from time of diagnosis to mobilization and follow-up from mobilization were 3.3 months and 14.3 months in plerixafor group, respectively. cpc contamination in the pbsc grafts was detectable in 32 and 7 patients with counts ranging between 0-8.7x10 -5 and 0-0.1x10 -5 in g-csf alone and g-csf+plerixafor groups, respectively (p=0.116).there were no significant differences in the proportion of the patients with graft contamination between subtypes of mm in both groups. one hundred (gcsf/plerixafor;93/7) patients had pre-asct pet-ct imaging done with 71 (gcsf/plerixafor;64/7) have active lesion at the time of mobilization. statistically significant association could not be demonstrated between the disease < cr status at mobilization and the number of apc in the apheresis product in both groups (p>0.05).twelve of 16 patients from plerixafor treatment arm proceeded to transplantation within median 9.5 months.the best overall response to induction treatment is shown in table 1 .thirtyfour patients from the g-csf alone arm and 2 patients from the g-csf+plerixafor arm died during the follow-up (p=0.52).disease progression was seen in 48 patients from g-csf alone group and 6 patients from g-csf+plerixafor group of the study(p=0.56).estimated mean os was better among patients w/o apc contamination in plerixafor group, respectively (38.9±5.3mos vs 16.8mos; p=0.52). conclusions: our results on 164 and few plerixafor used patients show that clonal plasma cells are detectable by multiparametric flow more frequently when patients are poor mobilizers and require plerixafor.the clonal pc contamination can be attributed to the myeloma biology as manifested by higher number of lines induction regimens and pet positivity among the plerixafor-required patients. the overall and disease survival was impaired by residual clonal pcs in the graft but not by plerixafor per se. neither was the content of clonal pcs differed from others.thus the cxcr4 shared by hsc and myeloma cells do not cause a myeloma mobilization. clinical trial registry: -disclosure: nothing to declare prognostic factors for overall survival after allogeneic hematopoietic cell transplantation in multiple myeloma patients all factors with significant influence on pts survival were included multivariate analysis (cox regression model) but only re-admission in the first 365 days demonstrated impact on os (hr 4, 082; p=0, 005) . conclusions: we analyzed risk factors for survival in mm pts who received allo-hct. our study identified disease-related risk factors like iss and transplantationrelated factors such as hct-ci and pam, hospital readmission, days of hospitalization and cmv reactivation that were associated with worse long-term survival. in our series, the most frequent death and re-admission cause was infection, so focusing the efforts in reduction of infection could have a beneficial impact on improvement of survival in mm undergoing allo-hct. [[p547 image] 1. figure 1 ] disclosure: there is no disclosure. novel protocol for autologous hsct in multiple myeloma: ambulatory chemomobilization and transplantation of fresh hematopoietic stem cells with backup storage background: autologous hematopoietic stem cell transplantation (ahsct) after melphalan conditioning is still a part of standard treatment of multiple myeloma patients. traditional transplantation of frozen stem cells poses additional risk for the patients connected with dmso and central venous catheter. the transplantation of fresh cells is an option -however, most mobilization protocols are either low-efficient (g-csf), expensive (g-csf + plerixafor) or toxic (standard dose chemomobilization) to directly proceed to transplantation in this fragile group of patients. we describe here the novel combination of ambulatory mobilization with very low doses of ara-c and g-csf connected with direct ahsct with fresh cells. methods: the prospectively collected database of patients after ahsct was searched for patients who underwent ahsct after chemomobilization with ara-c and transplantation with fresh cells (fc) and compared with control group of consecutive patients transplanted with standard protocol (sp) (transplantation with frozen cells) between july 2016 and october 2018. protocol of ambulatory mobilization was: 400mg/m² of arac on days +1 and +2 and g-csf at the dose 5 μg/kg/day from day +5 and escalated to 10 μg/kg/day split into two doses +10 to +13, apheresis started on day +14 (or later) and finished when at least 7.5 x 10e6 cd34+ positive cells were collected. the collected cells were split in three equal parts:1) for use as fresh transplant 2) frozen for possible 2 nd transplant 3) frozen as backup. results: there were 48 transplantations with fresh cells and 49 transplantations with frozen cells compared. both groups had same mobilization protocol -ambulatory low dose ara-c. the median age and number of transplanted cells was similar in both groups (56 vs 57, p=0.99; 5.2 vs 5.3 cd34+/kg, p=0.97 conclusions: we present novel approach that allows direct ahsct after chemo mobilization in all patients who are treated with melphalan. we show that it is not only feasible to do ahsct directly after chemomobilization but also the results may favour this approach when compared with current standard. disclosure: nothing to declare background: high dose chemotherapy followed by autologous hematopoietic cell transplantation (hsct) is considered, since the nineties, the standard of care for patients aged less than 70-75 years old with newly diagnosed multiple myeloma (mm). however, the optimal induction treatment prior to hsct to reduce the tumor burden has changed during the last few years. improved regimens have shown to be able to increase the quality of the pre-hsct response, which might subsequently impact on the post-hsct response, which has been proved to be associated with a longer pfs. we recently changed the induction therapy for pts candidates to hsct. in this analysis, we aimed to check if newer regimens impacted on pretransplant responses, and how auto-hsct changed the pre-hsct status. methods: all the 117 auto-hsct for 99 mm patients performed in our center from january 2014 through august 2018 were included in the analysis. median age was 60 years (range: 38-76). 54 pts were male and 45 were female. durie-salmon stage was distributed as follows: i (9.4%), ii (37.6%) and iii (53%); 13% had creatinine > 2 mg/dl. iss was: 1 (41%), 2 (28.2%), and 3 (30.8%). type of monoclonal component was: igg (51.3%), light chains (23.9%), iga (22.2%) , and non-secretory (2.6%). 51.3% had bence jones proteinuria. conditioning regimen was melphalan 200 mg/m 2 in 106 (90.6%), melphalan 100-140 mg/m 2 in 9 (7.7%), and other in 2 (1.7%). results: pre-transplant therapy was: vcd in 60 (mostly in 2014-6), vtd/vrd/krd in 34 (mostly in 2017-8), and others in 23 cases. status of the disease at transplant was: cr/vgpr in 75, pr in 35, and sd in 7. distribution of pretransplant response based on the type of induction is shown in table 1. peri-transplant mortality was 0%. day +100 mortality was 1.7% (2 pts), due to progressive disease. as shown in table 2, all patients (14/14) who obtained cr pre-hsct, maintained the response at day +100 post-hsct. among the patients in vgpr at hsct, 20 (32.8%) became into cr, and 37 (60.7%) maintained the response. the cr rate at post-hsct increased 264% compared to pre-hsct (37 versus 14 pts). altogether, after hsct 44 pts (37.6%) improved and 65 (55.6%) maintained the pre-hsct response. during the last period of time, 37 pts started on post-hsct maintenance/consolidation, mainly with lenalidomide. conclusions: 1) with the new chemotherapeutic schemes, 76.5% of patients underwent hsct in cr or vgpr; 2) majority of pts (93.2%) consolidated or improved the pre-hsct response; 3) cr increased substantially (2.64 times) after transplant; 4) optimized induction regimens, along with auto-hsct followed by the recently licensed use of maintenance therapy with lenalidomide, might result in a better pfs of patients with mm. background: autologous stem cell transplantation (asct) is commonly used in treatment of patients over 65 years with multiple myeloma (mm), however the safety and efficacy of this procedure is debatable. methods: we conducted a retrospective review of mm patients who underwent asct from 2013 to 2018 at our institution. the purpose of this retrospective study was to compare the 100-day mortality, time to engraftment, and incidence of grade 1-4 toxicities in elderly mm patients with younger patients taking into account comorbidity information. other secondary end points measured were overall survival (os) and progression-free survival (pfs). results: a total of 95 patients were analysed and categorized by age as young patients (37 to 64 y; n=70) or elderly (65 to 71 y; n=25). the compared groups did not differ in terms of gender, ecog, hct-specific comorbidity index (hct-ci), and disease status at asct. melphalan in a dose of 200 mg/m 2 was used as preparative regimen in 65% of younger patients, and in 43% of the elderly (p= 0.18). the remaining patients received 140 mg/m2 of melphalan or lower dose (range, 100-140 mg/m 2 ) due to hct-ci >2 or age, on the physician discretion. in the whole study group there were no transplant related deaths within the first 100 days of asct. stratifying by age, there was no statistically significant difference concerning febrile neutropenia (fn) incidence, which was observed in 14% of younger patients, and 17% of elderly. in contrast, fn was observed more frequently in patients with hct-ci > 2 (38% vs 12%, p=0.05). grade 1-2 infections were more frequent in older patients (35% vs 5%, p= 0.005), but no difference was found in grade 3-4 infections incidence rate such as pneumonia, uroinfections and neutropenic enterocolitis (25% vs 35%, p=0.52), nor grade 1-2 and 3-4 noninfectious toxicities (50% vs 38%, p=0.31, and 4% vs 21%, p=0.06, respectively) . the median time to granulocyte engraftment was 11 days (range, 9-19 days) in elderly and was comparable with younger patients. the time to platelet recovery was also similar. after the median follow-up of 12 months for survivors, os at 18 months was 94% in both groups. pfs at 18 moths was 65% for younger patients, and 55% for elderly (p=0.86).however, the association between pfs and the dose of melphalan used in conditioning was observed. pfs probability at 18 months for patients conditioned with the dose of 200 mg/m 2 , 140 mg/m 2 and 100 mg/m 2 was 71%, 58% and 21%, respectively (p=0.012). conclusions: our data show that asct in transplant eligible mm patients ≥ 65 years of age is safe and provides similar outcomes as seen in younger patients. disclosure: nothing to declare is mobilization with chemotherapy effect response in the multiple myeloma? background: high dose melphalan therapy with autologous stem cell support is a standart approach in symptomatic multiple myeloma patients. response rates increased with the novel anti myeloma agents and the use of chemotherapy for stem cell mobilization should be questioned. the purpose of this study is to determine the effect of cyclophosphamide used during stem cell collection on disease response and transplantation results. methods: we retrospectively collect data from 270 myeloma patients who underwent autologous stem cell transplantation (asct) in ankara university medicine faculty, blood and bone marrow transplantation unit between january 2012 and november 2018. 51 patients who received cyclophosphamide protocol for stem cell mobilization were included in the study. disease response were determined according to international myeloma study group (imwg) criteria before and after the cyclophosphamide. transplant responses and their effects on survival were also indicated. results: after the diagnosis of mm, 51 patients (male/ female: 32/19; median age: 59 years (between 40-74 years)) with median follow-up of 59.1 months (between 7,6 -185,8 months) underwent asct at a mean of 21,7 ±6,2 months.. forty-one patients were evaluated before and after cyclophosphamide (table 1). in 69% of the patients, the disease response was not altered by cyclophosphamide therapy, and 22% of the patients improved their response status. post-transplant response rates of patients who underwent stem cell mobilization with cyclophosphamide are also shown in table-1. the mean survival of the patients was 52,7 ±4,8 months. when patients were grouped according to changes in response status before and after cyclophosphamide; there was no statistical difference between mean calculated survival (improved response, disease progression and stable disease; 59,3±9,0 months, 22,7±14,7 months and 46,4±5,0 months respectively, p=0.425) (figure-1) . the rates of 1-year and 3-year overall survival (os) of the patients with no response to cyclophosphamide treatment were as follows; 74,2%±7,9% and 70,5%±8,3% respectively. thirteen patients who were followed up median 27 months after transplantation died at an average of 3,4±2,5 months; 6 of these deaths were caused by the infection after transplantation. conclusions: in our study, it was observed that the use of cyclophosphamide for cd34+ stem cell mobilization did not change the disease response rates by 69%. the posttransplant survival rates of mm patients who had progressive disease after cyclophosphamide use were lower. however, these results warranted confirmed by randomized controlled trials. clinical trial registry: -disclosure: nothing to declare results of a single center experience: an attempt to augment conditioning regimen in first autologous stem cell transplantation treatment of multiple myeloma (mm) continues to evolve in the era of novel agents. the addition of bortezomib to highdose melphalan (bor-hdm) has been reported by several groups, and it has been outcome and toxicity profile is comparable to high dose melphalan (hdm) alone. the aim of this retrospective study was to evaluate the outcome of the bor-hdm conditioning regimen on overall response for patients with mm undergoing first single asct at our institution. methods: this retrospective single center study reviewed 136 consecutive myeloma patients who had received the first asct either with bor-hdm (n=15, m/f= 11/4) or single agent hdm (n=121, m/f= 76/45) conditioning regimen. in the single agent hdm conditioning regimen, melphalan was administered intravenously at a total dose of 200 mg/m2 on day -3 and -2 and stem cells were infused on day 0. in the bor-hdm group, melphalan 200mg/m 2 was administered on day -2. bortezomib was administered intravenously at a dose of 1 mg/m 2 on day's -6, -3, +1, and +4 as described in a phase 2 study by intergroupe francophone du myeĺome (ifm). results: all consecutive patients who underwent single asct from january 2010 to march 2018 using bor-hdm as conditioning or hdm were evaluated. conditioning regimen was hdm in 121 patients and bor-hdm in 15 patients. median age was significantly lower in bor-hdm conditioned asct compared to hdm group (62 years vs 52 years, p=000). there was no significant difference for mm subtype, iss stage at diagnosis, prior treatment line among hdm vs bor-hdm cohorts (p>0.5). after a median of 4 cycles of induction chemotherapy, patients in the bor-hd exhibited ≥vgpr of 53.8% (n=7) compared to 56.7% (n=51) in the hdm group (p= p>0.5). pre-asct immune response (if (-)) was reported in 22.3% of patients treated with hdm, higher than that seen in the bor-hdm group (6.7%) (p=0.3). nine (69.2%) patients achieved post-asct immune response (if (-)) ≥vgpr compared to 92 (78.6%) in the hdm group. at the time of this analysis, ten patients in the bor-hdm group and 87 in the hdm group had already died, respectively (p>0.5). a total of 5 (45.5%) patients in the bor-hdm group and 50 (44.2%) patients in hdm group had already progressed (p>0.5). estimated mean os and pfs was shorter for group treated with bor-hdm; 48.2±6.9 mos and 40.2±8.5 mos vs. 76.7±3.9 mos and 67.3±3.8 mos, respectively (p>0.5) (figure-1) . we could not demonstrate the impact of pre or post transplant ≥vgpr immune response on survival and disease free survival. there was no engraftment failure observed on either treatment group and no worsening peripheral neuropathy was developed in the bortezomib arm. conclusions: this single center experience on a small patient pool was able to repeat the prospective randomized study results of ifm. further studies are warranted to explore this regimen, especially when induction treatment with novel agents are employed, with special emphasis on the high-risk myeloma patients where response rates are good but sustainability remains an issue. disclosure: nothing to disclosure the efficacy and safety of bortezomib plus busulfan/ melphalan as conditioning regimen in multiple myeloma undergoing autologous stem cell transplantation: phase 1/2 study background: bortezomib have a powerful antimyeloma activity and was regarded as backbone of therapy in the past decade but its safety and efficacy as a part of busulfan/ melphalan conditioning regimen of autologous stem cell transplantation is yet to be shown. methods: a phase 1/2 trial to explore the safety and activity of a bortezomib on days -6, -3, and +1 added to a conditioning regimen with busulfan and melphalan (bumel, 3.2 mg/kg/day and busulfan during day -5 and -3, 140 mg/ m 2 /day of melphalan on the day -2), in multiple myeloma (mm) patients who received autologous stem cell transplantation following bortezomib-based induction chemotherapy. in phase 1, escalating doses (0.7, 1.0, and 1.3 mg/m 2 ) of bortezomib with bumel were administered in each group with three patients. with determined maximum tolerated dose of bortzomib at a 1.3mg/m 2 /day, cohort with 41 patients were analyzed for phase 2. results: in phase 1, no dose limiting toxicity was observed at a 1.3mg/m 2 /day of bortezomib. in phase 2, overall responses at 3 months was shown as 75% of very good partial response (vgpr) or better and 55% of complete response (cr), whereas post-transplant overall best response included 83% of vgpr or better, and 68% of cr, respectively. with median follow-up duration of 31.4 months, median progression-free survival (pfs) was 26.8 months. the probabilities of 2 years-pfs and overall survival (os) were 56.5% and not estimable, respectively. especially, high-risk cytogenetics were associated adverse survival outcome compared to standard-risk cytogenetics, respectively (pfs, 12.2 vs. 35.7 months, p=0.039; os, 26.7 vs. 73.3 months, p=0.086) . with median 11 days and 10 days for neutrophil and platelet engraftments, any graft failure or delayed engraft was not observed. the common grade 3 or severe non-hematological adverse events included neutropenic fever (73.2%) and stomatitis (14.6%). except three cases with transplant-related mortality due to sepsis, other adverse events were manageable. conclusions: these results demonstrate that bortezomib is safe and can be a part of conditioning regimen in combination with bumel, for patients with transplanteligible multiple myeloma. clinical background: allogeneic stem-cell transplantation (allo-sct) is one of treatment option for patients with multiple myeloma (mm) refractory to novel agents. the reports on allo-sct for mm are limited and it is an important issue to argue appropriate conditioning regimens and stem-cell sources, and patient population who will benefit from allo-sct. methods: we retrospectively analyzed 25 consecutive patients who received allo-sct for relapsed and refractory multiple myeloma (rrmm) between oct 2009 and july 2018 at japanese red cross medical center. characteristics of patients, progression-free survival (pfs), and overall survival (os) were analyzed. results: median age at allo-sct was 47 (range 31-61). twelve patients were male and 13 were female. myeloma type were igg:14, iga: 3, igd: 2, and bence-jones: 6. stem-cell sources were peripheral blood from hlamatched related donor (rpbsct): 6, bone mallow from hla-matched unrelated donor (mud): 4, bone marrow from hla-mismatched donor (mmud): 7, and cord blood (cb): 8. twenty-three of 25 patients received flu/mel-base, one patient received bu/mel-based, and one patient received etoposide/cyclophosphamide-based conditioning regimens. twenty-two patients who transplanted after 2012 received 8gy of total body irradiation (tbi). responses before allo-sct were cr: 8, vgpr: 6, pr: 6, sd: 5. five-year pfs was 22% (95%ci: 6-45) and 5-year os was 42% (95%ci: 16-66). ten patients died during observation period and causes of death were primary disease: 8 and treatment-related mortality: 2. patients with vgpr or better before allo-sct showed significantly better pfs (p=0.04) and os (p=0.011) as compared with others. female recipients showed significantly better pfs (p=0.0034) and os (p=0.035) as compared with male recipients. recipients of mmud showed significantly better pfs (p=0.0034). among 15 patients surviving, 10 patients received treatments including maintenance therapy. conclusions: the reason for better pfs and os in female recipients is unknown. it is interesting that recipients of mmud showed better pfs, suggesting graft-versusmyeloma effects. allo-sct can be an effective treatment option if patients and stem-cell sources are appropriately selected. disclosure: authors declare that there are no conflicts of interest. second autologous hematopoietic stem cell tranpslant versus chemoimmunotherapy in relapsed multiple myeloma after first transplantation: single center data background: combination therapy, mostly triple, followed by autologous hematopoietic stem cell transplantation (auto-hct) is widely accepted as the first-line standard therapy for multiple myeloma (mm). despite the availability of agents such as new immunomodulatory drugs (imids), proteasome inhibitors (pis), histone-deacetylase inhibitors and antibodies, it is still possible to achieve longer and deeper responses, however, multiple myeloma is still not cured and relapse is inevitable. the availability of these novel agents has increased questions for determining optimal treatment of patients with relapse after the first auto-hct. methods: we retrospectively analyzed 60 patients who relapsed according to international myeloma working group (imwg) criteria after 1st auto-hct. first group [salvage chemotherapy(ct)] (n=27) was treated with only chemoimmunotherapy because of early relapse or refractory first auto-sct (within 6 months), ineligible to second transplantation because of co-morbidity, unwillingness to transplant. second group (n=33) (salvage transplantion) was treated with second auto-hct as a salvage therapy. consolidation and long term maintenance treatments were used in both groups. results: there was no difference in sex and age between salvage ct and auto-sct groups [female/male: 11 vs 13/ 16 vs 20; ]. the best response after salvage auto-sct was complete remission (cr) in 62,5%, partial remission (pr) in 15,6% patients, while cr in 44%, pr in 14,8% patients treated with salvage ct. progression free survival (pfs) were significantly better in second transplant group (pfs; 71 % on the first year; 46,9 % on the second year after transplant vs 59 % on the first year; 17% on the second year after the salvage therapy in chemotherapy patients)[p: 0,001]. overall survival (os) in salvage auto-sct group was longer than salvage ct (42,3 % 23.7%), although it did not reach a statistical significance (p>0.05). time to achieving the best response after salvage auto-sct and salvage ct was 1 (0-9) month versus 6,5(2-15) months [p:0,02]. grade 3 or 4 nonhematological toxicities were similar (auto-sct 19%, salvage ct 13%) in both groups. conclusions: salvage auto-hct may provides longer progression free survival with similar toxicity profile according to chemoimmunotherapy especially in patients with sensitive to first auto-sct. it is suggested that earlier and better responses, long-term progression free survival can be achieved with salvage auto-sct. we believe that there will be statistical significance in os such as pfs by increasing the number of patients. the authors believe that large scale randomized clinical trials are needed for optimal treatment of relapsing multiple myeloma after first auto-sct. disclosure: nothing to declare background: one of the conditions for successful transplantation of autologous hematopoietic stem cells (auto-hsct) in patients with multiple myeloma (mm) is the timely recovery of hematopoiesis, which is associated with the quantitative and qualitative characteristics of the graft. one of the key indicators is the content of cd34+ cells in the autograft, which depends on many factors. some of them are due to previous treatment, others are directly related to the patient: age, stage of the disease, features of the hematopoietic stem cells (hsc) microenvironment. the aim of the study was to assess the influence of the immune response genes on the autograft cellularity in patients with mm. methods: а retrospective analysis of the genotyping results was performed. evaluation of 21 loci in 15 genes immune response and harvesting of autologous hsc in 78 patients with mm has been made. hematopoietic stem cell mobilization regimen included cyclophosphamide 4 g/m 2 with granulocyte colony-stimulating factor. genotyping of the immune response genes polymorphic regions was carried out by the polymerase chain reaction with allelespecific primers. the number of cd34+ cells was counted on a 6-color facs canto ii flow cytometer. results: according to the results of the autologous transplant harvesting, two groups of patients were identified. first included 65 patients with an autograft cellularity of more than 2×10 6 /kg body weight. the second group consisted of 13 patients examined with the number of cd34 + cells in the autograft ≤2×10 6 /kg of the patient's body weight. comparing the identified haplotypes of the immune response genes with the cellularity of the transplantation material, it was found that the presence of the mutant allele in the homo-and heterozygous haplotypes of the il1β gene (t-511c) increased the chances of harvesting cellular material with a higher content of cd34+ cells in 4 times (χ2=5.04, p=0.02), and the carriage of the wild type allele in the homo-and heterozygous state of the tlr2 (arg753gln) gene is more than in 15 times (χ2=5.06, p=0.02). currently, it has been shown that single nucleotide or amino acid substitutions in genes can lead to changes in the expression pattern of their final products: increased secretion of interleukin 1β (il-1β) or changes in the spatial configuration and functionality of the receptors (tlr2). thus, in the presence of mutations in the il1β gene, the enhanced synthesis of il-1β influences on fibroblasts, immunocompetent, endothelial, epithelial and other cells, by activating hemopoiesis. in turn, the mutational status of the arg753gln locus located within the tir domain of the tlr2 receptor in the cytosol, determines the spatial configuration of the tlr2 acting as a co-stimulatory receptor of cd4+ cells, which ensure the engraftment of the graft. conclusions: identified haplotypical features of the il1β and tlr2 genes in patients with mm may act as predictors of the response effectiveness to mobilization of hscs in their carriers, which may contribute to the mobilization regimen optimization and will contribute to harvesting the optimal cellularity of an autologous graft. clinical trial registry: none. disclosure: authors declare no conflict of interest. differentiating diffuse from focal pattern on computed tomography: added values of a radiomics approach background: focal pattern in multiple myeloma (mm) seems to be related to poorer survival and differentiation from diffuse to focal pattern on computed tomography (ct) has inter-reader variability. therefore the purpose of this study is to assess if a radiomic approach could help radiologists in differentiating diffuse from focal patterns. methods: we retrospectively reviewed imaging data of 70 patients with mm between january 2013 and september 2018 of whom 61(27 men and 34 women; mean age 54.2 ±3.7) with ct, pet-ct or mri available before bone marrow transplant. two general radiologist evaluated in consensus only ct images to define a focal (at least one lytic lesion >5mm in diameter) or a diffuse (lesions < 5 mm, not osteoporosis) pattern. radiomic analysis on ct thinslice images was then applied with regions of interest (rois) done by one researcher not expert in medical imaging or mm blindly to the condition of the patients. the reference standard to differentiate diffuse from focal pattern was done by radiological evaluation of two expert musculosketal radiologists blinded to the clinical data reviewing ct, mri and pet-ct images. n=104 radiomics features were extracted and evaluated with an open source software. mann-whitney u test for unpaired data with 1000 bootstraps samples was used to compare radiomics features of diffuse and focal patterns and then feature reduction was done to avoid over-fitting. receiver operator characteristic (roc) analysis with area under the curve was done to compare radiologists and radiomics evaluation against reference standard. reading time to perform radiomic analysis was also estimated. results: the pathological group included: 22 diffuse and 39 focal patterns. after feature reduction, 9 features were different (p< 0.05) in the diffuse and focal patterns (n=2/9 features were shape-based: majoraxislength and sphericity; n=7/9 were gray level run length matrix (glrlm)). 150mg/kg). a number of eleven patients did not receive any additional immunosuppression except of post-cy. results: after a median follow up of 11.4 months (range 3.3 -34.5) 28 patients were alive. the 2-year probabilities of pfs and os were 42% (21-63%) and 65% (47-83%).the cumulative incidences (cis) of relapse and nrm at 2 years were 28% (11-45%) and 22% (8-36%), respectively. lower serum albumin level at transplantation (≤36 g/dl) was associated with increased relapses (hr 3.8 (1.2-12.7), p=0.028) and nrm (hr 7.9 (2-30), p=0.0026) and resulted in poorer pfs ), p=0.001) and os ), p=0.048). mmud and haploidentical donors were associated with poorer nrm (hr 6.2 (1.9-20.3), p=0.0027), and resulted in decreased pfs ), p=0.001). the high-risk cytogenetic at diagnosis showed no impact on survival. the cis of acute (grade ii-iv) at day +100 and chronic gvhd at 2 years were 26% (10-42%) and 48% (26-70%), respectively. absence of immunosuppressive medication beside post-cy was associated with poorer os ), p=0.01). conclusions: the conditioning with bu, tt and post-cy leads to a favorable pfs and os due to low incidences of relapse and nrm for patients with multiple myeloma relapsing after autografting. disclosure: nothing to declare methods: between january 2011 and may 2017, we included 51 patients with mm who underwent asct and received bortezomib/lenalidomide/dexamethasone (vrd) consolidation and maintenance therapy, mainly lenalidomide(r) 10mg/day for 21 days every 28 days. results: the median age at transplant was 55 years (46-75). forty-six (90%) of patients received r maintenance, 3 patients received vrd maintenance for higher risk features. median duration of r maintenance was 22 months . r dose was changed for toxicity (grade i-ii) in 17 (33%) patients. twenty-nine (57%) patients relapsed: 13 (25%) patients were shifted to different treatment protocols (treatment change). 6 patients (11%) were kept on the same r maintenance (observation group) and 10 (20%) patients had increased lenalidomide dose with dexamethasone (r/ d group). 6 patients (37%) of the last 2 groups required change of treatment later. the median follow up was 38 months . median tnt was 32 months (7-62). at 2 years, the estimated pfs and os were 39% and 97.5% respectively. the median os and pfs2 (from change of therapy) were 54 and 29 months for patients in the observation group, versus 52 and 27 months in the r/d group, and 45 and 33 months with treatment change, respectively. no statistically significant difference was noted. conclusions: our small monocentric study is limited by its retrospective design and small sample size. however, it suggests that increasing lenalidomide dose as well as adding dexamethasone in selected patients can postpone change to different lines of treatment without affecting survival. disclosure: nothing to declare can the drugs used before autologous hematopoietic stem cell transplantation have impact on cmv reactivation that results in decreased os in myeloma patients after asct? more intensive treatment regimens, such as proteasome inhibitors (pi) and/or immunomodulatory (imid) agents. we performed a retrospective, single center study to evaluate the incidence, risk factors, and outcomes of cmv infection in patients with mm who underwent asct with a high-dose melphalan-based regimen. methods: this study involved a retrospective review of all patients with who underwent asct between january 2015 and november 2018 at our stem cell transplantation center. a total of 144 consecutive adult patients with a diagnosis of mm (median age at diagnosis: 58, range: 35-77) underwent asct following induction treatment with novel agents (pis and/or imids). all patients received antiviral prophylaxis with acyclovir 600 mg/day (n=104) or valaganciclovir 1000 mg/day (n=36). results: baseline patient characteristics, according to induction treatment, are summarized in table1. one hundred-five of the 144 patients (97.2%) were cmv iggpositive before asct. overall, 14.6% (n=21) of cmvseropositive patients developed at least one episode of cmv viremia (cmv dna >100 copies/ml) after a median 24 months (range; 3-48 mos) follow-up. persistent cmv viremia (detectable cmv dna load in more than 2 sequential plasma specimens) occurred in 4.9% (7 of 144) of the seropositive asct recipients and all of them were preventive treated with ganciclovir (n=5) or valganciclovir (n=2). the time from stem cell infusion to the development of cmv viremia ranged from 3 days to 48 days. none of the patients with untreated viremia developed identifiable cmv sequelae. no case of primary infection in seronegative patients at transplant was observed. adding to that none of the patients developed cmv disease post asct. if we analyzed the subgroups of patients according to induction therapy (pi-based, imids, pi+imid), the incidence of post-asct cmv reactivation was higher but not statistically significant, in patients who received only pi vs pi+imid (13 (61.9%) vs 8 (38.1%); p=1.00). in univariate analysis, we could not demonstrate the importance of induction therapy with novel agents the occurrence of a post-asct cmv reactivation requiring antiviral treatment. however, statistically significant association found between the disease < vgpr status at asct and cmv reactivation (61.1% vs. 38.9%; p=0.035). after a median follow-up 14.3 months (range; 1-45.9 months), there was no significant impact on pfs, however there was significant decrease in estimated mean os who had cmv reactivation when compared to those without cmv reactivation (34.1±4.5 vs. 41.9±1.3; p=0.002) (figure-1) . conclusions: cmv establishes lifelong latency within host cells and in the setting of impaired cellular immunity; cmv may reactivate from latency, disseminate, and directly cause life-threatening disease. our data suggests that mm patients treated with pi-based induction regimens and immunological response < vgpr at time of asct seem to have higher risk of developing symptomatic cmv reactivation. however, further studies on a large number of patients are warranted to clarify these findings. clinical background: high-dose therapy followed by autologous stem cell transplantation (asct) has been shown to prolong survival in patients with multiple myeloma (mm) in randomized trials. however, these trials only include patients aged < 65 years. data regarding safety and outcomes in this patient population is lacking. methods: the aim of this study was to compare safety profile and outcomes in mm patients younger and older than 65 years-old who underwent asct in our unit from july 2014 to october 2018. patient's demographics, clinical characteristics, transplant related variables and probability of admission to the intensive care unit (icu) were analyzed. patients aged < 65 and ≥65 years-old would be called m1 and m2, respectively, from now on. sorror index was used to estimate risk of mortality in the two cohorts. results: a hundred and eleven patients with mm underwent asct in the study period. median age was 60.9 years-old (range 35-73) and 53.2% were male. thirtythree (28,8%) patients were ≥ 65 years. the probability of having a high risk comorbidity index was similar in both groups (m1 11,7 vsm2 12,1%). the median cells obtained in the apheresis procedure was 5.35 x10 6 (1,70-15.92) in m1 compared to 3.77 x10 6 (1.96-10-48) in m2. there were no differences in median admission lenght between the 2 cohorts (m1:19 days vs m2: 20 days). median days for neutrophil recovery above 1000 was 12 days in both groups with a wider range in m1 (11-23) compared to m2 (11-15) . no differences were found in platelet recovery above 100.000 (m1 20 days vs m2 21 days). median packed red blood cells and platelets transfusions were 1 (0-6) and 3 (0-16), respectively, in m1. in m2 cohort, they were 2 (0-5) and 4 (0-14), respectively. the incidence of grade 3-4 mucositis in m1 and m2 was 50.6% and 44.1%, respectively. there were no statistically significant differences in terms of using morphine for pain control between the two cohorts (m1, 63,4% vsm2, 58,8%). none patient requiered total parenteral nutrition (tpn) in group m2 and only one in group m1. the incidence of icu admission was 1.5 times higher in patients aged ≥65 than in patients < 65 years-old 6,1% vs3,9%), but differences were not statistically significant (p = 0.52). there were no deaths during the transplant procedure in any of the 2 cohorts conclusions: 1) in our series, high-dose therapy followed by autologous hematopoietic cell transplantation in mm patients aged ≥65 was feasible. 2) transplant procedure in older patients was as safe as in patients < 65 years-old. 3) no differences were found in terms of graft, transfusion support, transplant related complications and length of admission. 4) age should not be a limiting factor in considering the modality of asct in this patient population disclosure: nothing to declare the correlation between the kinetics of peripheral blood counts and the response to treatment after high-dose melphalan with stem cell support in multiple myeloma patients background: the long-term survival of mm patients has dramatically increased in the last 20 years, particularly for younger patients. this is attributable in part to the introduction and development of high dose chemotherapy with melphalan with stem cell support (hdm-asct). currently, frontline asct is still considered the standard of care for all eligible patients. many prognostic factors pre and post transplantation have been identified, e.g.: age, comorbidities, cytogenitcs, response to treatment and disease status prior to and post transplantation. to our knowledge there is no data correlating between kinetics of counts response to melphalan and prognosis. our aim was to assess the prognostic significance of the neutrophil and platelets decaying counts after high dose melphalan. methods -we retrospectively analyzed our cohort of 159 multiple myeloma patients who underwent hdm-asct at the hadassah medical center bone marrow transplant department, between the years 2007-2015. the kinetics of neutrophil and platelet decay during the first two weeks after melphalan administration was fitted using linear and exponential mathematical models. methods: we retrospectively analyzed our cohort of 159 multiple myeloma patients who underwent hdm-asct at the hadassah medical center bone marrow transplant department, between the years 2007-2015. the kinetics of neutrophil and platelet decay during the first two weeks after melphalan administration was fitted using linear and exponential mathematical models. results: factors associated with prolonged os in univariate analysis were: iss stage 1(p=0.024), ≤2 lines of treatment prior to asct(p< 0.001), favorable cytogenetics(p=0.004), response to treatment (pr or better, p=0.046) and rapid linear neutrophil decay (p = 0.046). in multivariate analysis, only ≤2 lines of treatment before hdm-asct and rapid linear neutrophils count decay remained statistically significant for os prolongation. no predictive threshold value of the neutrophil decay incline was found. improved pfs was associated with ≤2 lines of treatment prior to asct, and the response status after hdm-asct (p=0.003, p=0.019). additionally, toxicity evaluation showed prolonged neutropenia to be associated with inferior os (hr = 1.139, p=0.03) and rapid exponential decay of neutrophil counts to correlate with higher incidence of mucositis (p = 0.013). fast platelet decay was associated with delayed platelet engraftment (p< 0.01) conclusions: we have shown that rapid linear decay in neutrophil counts predicts better os without a significant benefit in pfs in mm patients undergoing hdm-asct. this discrepancy might reflect the problematic estimation in a retrospective analysis of pfs. rapid decrease in neutrophils and platelet counts was associated with more toxicity: higher mucositis rate and delayed engraftment, respectively. therefore a rapid decay of blood counts after hdm-asct appears to be an in-vivo phamacodynamic marker of higher efficacy and toxicity of melphalan. disclosure: nothing to declare p564 do we need to freeze hematopoietic cells for autotransplants in patients with myeloma conditioned with melphalan? daniel garcia belmonte 1 , beatriz aguado bueno 1 , miguel herrero coderch 1 , rafael de la camara 1 background: multiple myeloma (mm) is the most frequent indication of auto-hsct, representing 51% of all auto-hsct in 2016 (passweg jr. bmt 2018; 53:1139-48) . nearly all are performed with peripheral blood progenitor cells (pbpc), and melphalan 200 mg/m2 is considered the gold standard conditioning regimen. the standard procedure consists in obtaining progenitors, cryopreserved with dimethyl sulfoxide (dmso) and stored and subsequently thawed and re-infused in the patient on day 0. the procedure of cryopreservation is expensive and has some inherent toxicities (dmso) and loss of cells during the procedure. several groups have used non-cryopreserved progenitors showing some benefits compared with cryopreserved transplants, mainly a faster engraftment and a shorter length of hospitalization. objective: to compare noncryopreserved vs cryopreserved auto-hsct in mm methods: we perform an unicentric, retrospective study on 23 consecutive first auto-hsct mm patients transplanted with pbpc between nov-2011 and oct-2018, and conditioned with high dose melphalan (200 mg/m2). the median follow-up was 1620 days (range: 44-2413). 9 patients received non-cryopreserved and 14 cryopreserved auto-hsct. patients characteristics, without differences between non-cryopreserved vs cryopreserved: women/men (15/8); median age was 65 years (range 49-72); in the majority auto-hsct was done as consolidation after first line therapy (78%); year of transplant ≤2014 (61%), ≥2015 (39%). the number of infused cd34 cells were not different: median 3.5 x10 /kg (range 1.7-11.5) in noncryopreserved patients and 3.75 x10 /kg (range 2.1-17) in cryopreserved patients. results: we didn´t observe significant differences in the day of engraftment between non-cryopreserved vs cryopreserved although always was a little bit faster in the noncryopreserved group with a tendency to faster platelet engraftment (>50000/mm3): >20000 platelets/mm3 (median day: 12 vs 12.5, p 0.2), >50000 platelets/mm3 (median: 13 vs 16 days, p 0.09); >500 neutrophils/mm3 (median: 13 vs 13.5 days, p 0.3). the media of days of hospitalization was shorter in non-cryopreserved patients (18 vs 21 days) although not statistically significant (p 0.14). transplantrelated mortality at day +100 was 0% in both groups. overall survival at 5 years was not different: 83.3% in in non-cryopreserved vs 61.5% in cryopreserved patients (kaplan-meier, log-rank p 0.27). the accumulative incidence of relapse at the median follow up (1600 days) was similar: 33.3% in non-cryopreserved vs 35.9% in cryopreserved patients. conclusions: in our short experience, auto-hsct with non-cryopreserved pbpc in myeloma patients conditioned with high dose melphalan obtain similar results to those performed with classical cryopreserved pbpc and might has a faster platelet engraftment and shorter length of hospitalization. if no advantages are associated with cryopreservation, the simplicity of using fresh product is appealing. disclosure: nothing to declare p565 abstract withdrawn. . results: a early death is observed in one pt (group 1) and 5 pts(group 2). the median delay of aplasia is 14 days (10-22) and 12 days (9-17) respectively. in group 1, among the evaluable pts, 30/37 (81%) are in cr, 5 pts in pr and 2 refractory. in group 2, cr: 35/43 (81%), pr: 6 and 2 refractory. a relapse is observed in 32 pts/37 (86,5%) in group 1 and 35 pts/43 (81%) in group 2 with a frequency of 54% and 41% respectively in the first 24 months. at 36 months: 13 pts/38 (34%) in group 1 and 19 pts/49 (38%) in group 2 are dead. at 60 months: 20 pts (52%) and 29 pts (59%). at 120 months: 31 pts (81,5%) and 37 pts (75,5%). the overall survival (os) of the group 1 and group 2 pts were 66% and 62% at 36 months; 47.5% and 38.5% at 60 months; 18% and 24,5% at 120 months respectively (without significant difference). the event free survival (efs) of group 1 and 2 pts were 37% and 36,5% at 36 months, 25% and 13% at 60 months and 8% and 15% at 120 months respectively (without significant difference). conclusions: these 2 protocols with equivalent toxicity allow obtaining of long-term equivalent results on the response rate early transplant, on the rate of relapse and on the overall survival. these results are identical to those of fermand (1999) . disclosure: nothing to declare background: dimethylsulfoxide (dmso) is a major intracellular cryoprotectant, used for cryopreservation of stem cells. it is toxic to both cells and patients at temperatures above 0 o c. reduction of this effect is achieved by either washing of cells after thawing or by reduction of dmso during freezing and storage. the latter requires addition of extracellular cryoprotectants to the freezing media. we assessed the effect of low dmso concentration and different hematocrits of the frozen cells on cell viability and hematologic recovery in patients, transplanted for multiple myeloma. methods: cells were non-programmed frozen and stored at -80 o c in a cryoprotectant solution achieving final concentrations of 5% dmso, 3.6% of hydroxyethyl starch (hes, weight average molecular weight 450 000 da) and 3% of human serum albumin. the cell concentration in the frozen product for the first 77 patients (84 transplantations) varied between 100x10 6 and 250x10 6 cells/ml. in an attempt to reduce the amount of dmso infused, for the rest of the patients (n=172; 192 transplantations) we further decreased the volume of the freezing suspension by removal of the entire plasma. the average age of the transplanted patients was 55 (35 -71). the cells were bedside thawed at 37 o c water bath. the average cell dose was 2,95x10 6 /kg (1,3 -9,2x10 6 /kg). results: viability of the stem cells following thawing assessed by trypan blue exclusion was 95,34% . the hematocrit of the frozen cells had no effect on cell viability (94,80%(low) vs 95,52%(high)). the major complaints, if any, during stem cell infusion were coughing and an increase in nausea and vomiting induced by the prior conditioning. the average time for hematological recovery was 11,66 days (between 9 and 19) for the neutrophils, and 12,17 (between 9 and 20) days for the platelets. there was no significant difference in viability and hematologic recovery (11,86 and 12,60 vs 11,59 and 12,02) between patients receiving cells frozen with low or high hematocrit. conclusions: dimethylsulfoxide, despite its cryoprotective properties, is toxic for stem cells at temperatures above zero c and induces many side effects (cardiac, neurologic, respiratory, etc.) in the patients. to reduce those side effects we use lower dmso concentration, high hematocrit resulting in lower volume of the frozen cell suspension, thus reducing the final quantity of dmso to be infused to the patients. this does not affect the cell viability or the hematologic recovery of patients after transplantation. our easily performed method for unprogrammed freezing of stem cells with final dmso concentration 5% at -80 o c is safe, well tolerated, and provides cryopreservation, which allows high viability and stable cell engraftment, while reducing the undesired side effects of dmso. disclosure: nothing to disclose the conditioning regimen consisted of melphalan for most of the patients. the average age at the time of transplantation was 55 years (35 -71). patients were transplanted with an average cell dose of 2,95x10 6 /kg (1,30 -9,20x10 6 /kg) for the first transplantation and 2,36x10 6 /kg (1,64 -3,19x10 6 /kg) for the second one (every patient received the same cell dose as for the first) with average cell viability 95,34 % (70 -99%), with little difference between first and second transplantation. results: the average time for hematological recovery was 11,60 (between 9 and 19) days for the neutrophils, and 12.11 (between 9 and 20) days for the platelets. we found no correlation between the cell dose and the hematological recovery. there was no difference in the hematopoietic recovery between the first and the second transplantation in the patients, who underwent tandem or two transplantations. conclusions: recovery time is considered by some to be a function of the effective stem cell number. we did not find such correlation, probably because in the analyzed group all the patients, except four of them, received a dose greater than 2 x10 6 /kg cell, which is accepted as a safe dose for autologous stem cell transplantation. disclosure: nothing to disclose p569 plerixafor-mobilized patients have a high risk of noninfectious fever during engraftment after autologous peripheral blood stem cell transplantation background: plerixafor enables rapid and efficient mobilization of hematopoietic stem cells. however, its impact on adverse clinical events after autologous peripheral blood stem cell transplantation (pbsct) is not fully understood. fever is one of the major complications in the preengraftment phase of pbsct. in this research, we focused on non-infectious fever around the time of bone marrow recovery and investigated whether plerixafor as mobilization therapy plays a role in engraftment fever. methods: we reviewed 80 autologous pbscts for treatment of multiple myeloma at the japanese red cross medical center between 2012-2018. non-infectious fever was defined as temperature ≥37°c with onset between two days prior to and two days after engraftment without clinical or microbiological documentation of infection. results: patients were mobilized by cyclophosphamide and filgrastim in 57.5% (n = 46) and filgrastim and plerixafor in 42.5% (n = 34). the median number of transfused cd34+ cells were 2.61×10 6 /kg and 3.45×10 6 / kg, respectively (p=0.002). patients transfused with plerixafor-mobilized grafts had a higher risk of noninfectious fever (85.3% vs 63.0%, p< 0.05). cd34+ cell number or cyclophosphamide pretreatment had no relationship to non-infectious fever. the recovery of lymphocytes was more rapid in plerixafor-mobilized patients (p=0.001). however, the number of lymphocytes was not associated with non-infectious fever. conclusions: combination of filgrastim and plerixafor as mobilization therapy resulted in an increased risk of noninfectious fever during engraftment comparing to mobilization with cyclophosphamide and filgrastim. while the mechanism remains unclear and requires further studies, plerixafor-mobilized grafts may result in an unintended increase in engraftment fever. clinicians should be aware of this possibility if patients are transplanted with those grafts. disclosure: ks received honorarium outside the submitted work from janssen, novartis, celgene, ono pharmaceuticals, takeda, fujimoto pharmaceuticals and srl. ti received honorarium outside the submitted work from janssen, celgene, ono pharmaceuticals and takeda. we assessed the efficacy of a new conditioning regimen consisted of decitabine (dec), busulfan (bu), cyclophosphamide (cy), fludarabine (flud) and cytarabine (ara-c) for allo-hsct in patients with mds and mds/ mpn. fifty patients were enrolled, including 46 with mds and 4 with cmml. patients received dec 20 mg/m 2 /day on days -9 to -5, combining bu/cy/ flu/ ara-c modified preparative regimen. results: at a median follow-up of 522 (15-1313) days, the overall survival (os) was 86%, disease-free survival (dfs) was 78%, and relapse incidence was 11%. the incidence of severe acute (grade iii/iv) graft-versus-host disease (gvhd) was 22%, and that of (predominantly mild) chronic gvhd was 40%. os at 2 years was 74% for mds patients with high risk, 86% for mds patients with very high risk, respectively. the survival was delightful in patients with poor-risk mutations, such as tp53 and asxl1, (86%) and with three or more gene mutations (77%). among the total 12 patients with poor-risk mutations in our research, only one patient (8%) with tp53 relapsed and one (8%) with asxl1and tet2 died. result of continuous observation after transplantation, the percentage of nk cells in the peripheral blood of all patients who had received dec/flu/bu/cy/ara-c conditioning increased at day 28, which may essentially contribute to disease control post-transplantation. conclusions: in summary, the addition of a 5-day schedule of decitabine to a flu/bu/cy/ara-c conditioning regimen has proven feasible, with a low level of toxicity and promising early disease control especially in patients with high risk mds. disclosure: there are no conflicts of interest. the sfgm-tc mds score at day 180 is associated with post-transplant outcomes in patients with myelodysplastic syndrome who underwent cd34+ selected allogeneic stem cell transplant conclusions: in patients with mds undergoing tcd-hct, the sfgm-tc score at day 180 is significantly associated with survival. the lower incidence of acute gvhd in recipients of cd34-selected transplants and the use of myeloablative condition regimens, with lower relapse, may explain the difference with the original finding that the sfgm-tc was predictive at day 100 in unmodified grafts. disclosure the most frequent grade 3, 4 toxicities were thromobocytopenia and neutropenia. infections developed in 12 patients (33.3%), neutropenic fever in 8 (19.4%). five patients (13.9%) either developed or experienced exacerbation of acute graft versus host disease (gvhd), nonechronic gvhd. conclusions: azacitidine use is associated with only modest activity in patients who relapse after allo-hsct. however, in patients who respond to treatment it may allow for a durable disease control. disclosure: the authors declare no competing conflicts of interest background: somatic mutations in mds patients are closely related with clinical phenotypes and prognosis in mds patients. but whether mutations are prognostic for outcomes after allogeneic hematopoietic stem-cell transplantation (allo-hsct) remains to be elaborated. methods: targeted mutational analysis were performed on samples obtained before transplantation from 134 patients underwent hsct. we analyzed the relationship of mutations and clinical outcomes. results: all 134 patients carried more than one somatic mutations, most frequently in kmt2d(67.16%), arid1b (61.94%), ccdc168(52.24%), pclo(47.01%), asxl1/2 (46.27%), srcap(44.78%), u2af1(42.54%), dnah2 (41.79%), ush2a(41.04%) and tet2(34.33%). tp53 mutations were associated with higher ipss-r risk, complex karyotype and monosomal karyotype. dnah2 were more frequent in pediatric patients. in univariable analyses, tp53 mutations were related with decreased disease-free survival (p=0.047); dnah2 mutations were related with increased disease-free survival (dfs) (p=0.038). in multivariable analysis including ipss-r stratification, gvhd, hct-ci and candidate genes, dnah2 mutations were independently associated with better dfs(p=0.027). conclusions: dnah2 mutations is independently associated with better outcomes in mds patients treated with allo-hsct while tp53 may predict unfavorable outcomes. accounting for these somatic mutations may help better selection of candidates for allo-hsct among mds patients. disclosure background: there is a controversy among experts if and how patients with mds and saml should receive cytoreductive therapy before transplant. while aiming to reduce disease burden in order to lower the risk of relapse after transplant cytoreductive therapy is associated with several drawbacks. besides a considerable risk for toxicity and mortality preventing patients to proceed to transplant cytoreductive therapy may also favour the selection of resistant clones which may be difficult to treat at relapse. methods: to address this hypothesis we retrospectively analysed the response and survival following salvage therapy in 73 patients with mds and saml who had relapsed in median 5.6 months (1 to 110 months) after allo-sct according to their pre-transplant strategy (upfront transplantation n=32 44%; induction chemotherapy [ctx] n=26 36%; hypomethylating agents [hma] n=15 20%). results: the majority of these 73 patients received salvage therapy with hma (n=58, 79%; aza n=57, dac n=1) mostly in combination with dli, while the remaining received other salvage treatments (intensive chemotherapy n=1, dli alone n=1, 2 nd transplant n=3, bsc n=5, miscellaneous n=2, missing information n=3). when focussing on those patients treated with hma and dli it became apparent that a significantly higher proportion of patients in the upfront group (58%) achieved cr after salvage therapy in comparison to pre-treated patients (10% cr, p=0.0005; ctx group 5% cr; hma group 18% cr). accordingly, overall survival (os) calculated from the time of relapse was significantly longer in patients in the upfront group than in the group of pre-treated patients (2-year os 59% vs. 19%, p=0.0001). conclusions: overall, these findings imply that pretransplant therapy may favour the iatrogenic selection of resistant clones, which poorly respond to salvage therapy with hma and dli in case of relapse after allo-sct. furthermore, the results support the concept that an upfront transplant strategy is a promising alternative for patients with mds and saml that can be augmented by salvage therapy with hma and dli. disclosure: ts and gk received travel support, lecture fees and research funding from celgene gmbh conclusions: in our country, this procedure has shown to be feasible and we hope to improve it, with better infection control and by acquiring more experience related to the management of these patients. background: extramedullar relapse of mds is a rare complication after allogeneic stem cell transplantation. we present the case of a 69-year-old woman who was admitted into hospital because of insecure walking. paresis of both legs, hypaesthesia of the inner thighs, increased effort at urinating, reduced sphincter tonus, central paresis of the right arm and discreet paresis of the right facial nerve were documented at neurological exam. mri showed a large tumour of the dorsal thorax that immured the adjacent ribs and spine, affected the processus transversus of t9-10 and invaded the spinal canal. the patient had undergone ric allogeneic stem cell transplantation five years ago for mds-eb 2 with complex aberrant karyotype. following an uneventful course and no signs of gvhd, she had been off immunosuppression since 4,5 years. at the time of the admission the patient had slightly lowered wbc (2,9 gpt/l) and plt (111 gpt/l) and clearly increased ldh (793 u/l). methods: histology of a ct-based biopsy of the paravertebral tumour showed an infiltration of the muscles by blastous cells that were cd45-, cd34-, pax5-positive, tdt and cd79a were questionably positive. provisonal diagnosis therefore was lymphoblastic lymphoma, pox tested negative. the bone marrow was hypocellular with increased numbers of mature lymphocytes, but no definite signs of malignancy. cerebrospinal fluid revealed 1574 cells/μl with 90% blasts. immunotype was cd34, cd117, cd13, cd33, hla-dr positive, pox and lymphatic markers were negative. because of this we finally suspected meningeosis leucaemica. we completed the diagnostic workup with genetical and chimaerism tests and compared the result to the patients' mds before allogeneic stem cell transplantation. [[p580 image] 1. mri scan of the large thoracic tumour] results: cerebrospinal fluid (csf) cells consisted of 92% recipient cells, whereas peripheral blood cells were 100% donor. high risk mds at transplant displayed a complex caryotype including trisomy 8 and tetrasomy 8, now 10% of the cells in csf showed trisomy 8 and 70% tetrasomy 8. chimerism and fish of the solid tumour could not be performed, coexpression of myeloid markers within the tumour is pending. conclusions: in conclusion the patient has meningeosis as a result of exclusively extramedullary relapse of myeloid blasts originating from the initial high risk mds with blast excess and complex aberrant caryotype. the evolution of a trisomy 8 clone to tetrasomy 8 clone in relapse is linked to extramedullar manifestations. whether the solid tumour represents myeloid sarcoma with coexpression of lymphoid markers, extramedullary relapse of mds with lymphoid differentiation or, less likely, a separate lymphobastic lymphoma, is not yet clear. disclosure background: adoptive t cell therapy with genetically engineered t cells is a potent innovative immunotherapeutic approach for cancer treatment. unfortunately, the use of t cells redirected against tumor antigens, is severely limited by 1) the difficulty in identifying appropriate cell surface antigens, that could be targeted by car t cells and 2) the paucity of tumor-specific t cell receptors (tcrs) against shared, oncogenic antigens. methods: focusing on wilms´tumor 1 (wt1), a tumorassociated antigen overexpressed by acute myeloid leukemia and several solid tumors, we designed and implemented an innovative protocol for the rapid isolation of wt1-specific t cells and for the generation and characterization of a library of wt1-specific tcrs displaying different human leukocyte antigen (hla) restrictions, to be exploited by tcr gene transfer and tcr gene editing. to this aim, we repetitively stimulated t cells with autologous antigen-presenting cells, including immortalized b cells, pulsed with overlapping peptides spanning the entire wt1 protein. t cell recognition was assessed by flow cytometry in terms of cd107a expression and ifnγ production. recognized peptides were mapped by a deconvoluting grid and t cell clonotypes were longitudinally tracked by tcrαβ sequencing. results: we successfully expanded tumor-specific t cells from 14 consecutive healthy donors, in an average of 4 rounds of in vitro stimulations. the ability of wt1specific t cells to recognize naturally processed epitopes and their on-target specificity was demonstrated upon coculture with antigen-expressing targets including primary leukemic blasts. tracking of the tcrαβ repertoire during culture led to the identification of 20 clonotypes that recognize several tumor-associated peptides and are restricted by more than 5 hla alleles, including hla-a*02:01. tcrs were then expressed via genome editing. briefly, simultaneous editing of endogenous tcr α and β chain genes was achieved using crispr/cas9 technology (efficiency >90%), followed by transduction of t cells with lentiviral vectors encoding wt1-specific tcrs (efficiency >95% of cd8 + t cells). phenotypic characterization of edited t lymphocytes showed a major enrichment of cells harboring t stem cell memory properties. functional validation of the edited t cells is currently ongoing. preliminary results of a 6 hours coculture experiment show that tcr edited t cells kill fresh wt1 + leukemic blasts, harvested from hla-matched patients, with an efficiency up to 70% at an effector to target ratio of 5 to 1, while no killing of controls is observed. conclusions: we set up a protocol enabling consistent and efficient hunting for tumor-specific tcrs with no need for labor intensive t cell cloning. tcr genes can be easily and rapidly used to redirect t cell specificity against cancer cells by tcr gene editing. disclosure: chiara bonini: research funding from intellia therapeutics p583 car t cell therapy targeting relapsed or refractory cd19+ lymphoid disease with third-generation vector rv-sfg.cd19.cd28.4-1bbzeta maria-luisa schubert 1 , anita schmitt 1 , leopold sellner 1,2 , brigitte neuber 1 , angela hückelhoven-krauss 1 , kunz alexander 1 , lei wang 1 , gern ulrike 1 , birgit michels 1 , susanne hofmann 1 , carsten mueller-tidow 1,2 , dreger peter 1,2 , michael schmitt 1, 2 background: t cells genetically engineered to express chimeric antigen receptors (carts) directed against cd19 have demonstrated significant efficacy in patients with iymphoid malignancies including relapsed or refractory (r/r) b-lineage acute lymphoblastic leukemia (all) or r/r b-cell non-hodgkin's lymphoma (nhl). access to cart treatment for patients in europe has been limited so far given that the vast majority of cart trials have been performed in the united states and the p. r. of china. here we present the preliminary results of the first investigator-initiated trial (iit) cart trial in germany. hd-car-1 (eudract no. 2016-00 4808-60; nct03676504) is a phase i/ii trial with in-house cart manufacturing which was initiated in september 2018 at the university hospital heidelberg. methods: adult as well as pediatric patients with r/r all and patients with chronic lymphocytic leukemia (cll) or nhl including diffuse large b-cell lymphoma (dlbcl), follicular lymphoma (fl) or mantle cell lymphoma (mcl) are treated with autologous t lymphocytes transduced with a third-generation car retroviral vector (rv-sfg.cd19.cd28.4-1bbzeta) targeting cd19. the main purpose of hd-car-1 is to evaluate safety and feasibility of escalating third-generation car t cell doses (1-20×10 6 transduced cells/m 2 ) after lymphodepletion with fludarabine and cyclophosphamide. patients are monitored for cytokine release syndrome (crs), car-t-cell related encephalopathy syndrome (cres) and/or other toxicities. in vivo function, survival and anti-tumor efficacy of carts are assessed. results: to date, three patients (cll, dlbcl and mcl, respectively) have been enrolled and subjected to leukapheresis. high numbers of transduced carts were harvested on day 10 of culture (82-123x10 6 carts). transduction efficiency ranged between 33 and 42%. cart products were sterile and free from mycoplasms and endotoxins. no production failure occurred and all patients received the cart product. no signs of crs or cres > grade 2 have been observed. assessments of clinical responses are pending and will be presented at the conference along with updated technical results. conclusions: for hd-car-1, gmp-conform leukapheresis as well as cart manufacturing was effective. administration, patient monitoring and follow-up were performed in-house providing independency from transport or production sites outside the university hospital heidelberg, altogether suggesting that academic cart iits are feasible in germany. clinical background: the prognosis of adult patients (pts) with relapsed/refractory (r/r) precursor b-acute lymphoblastic leukemia (all) is dismal, including with allogeneic hematopoietic stem cell transplantation (allo-hsct). blinatumomab, a bispecific cd19-directed cd3 t-cell engager and inotuzumab ozogamycin (io), a cd22-directed antibody-drug conjugate revolutionized the field, improving their outcomes. anti-cd19 chimeric antigen receptor t (cart) cell therapy has led to further progress and improved outcome (jacoby e; am j hematol, 2018). nowadays, patients with r/r b-all can be offered both therapies, but there are limited data on the safety and efficacy of cart -cell therapy post antibody treatment. we detailed our single center experience in this regard. methods: this report is a part of a single center, phase 1b/2 study on therapy of b-cell malignancies with locally produced cart-cells (nct02772198). the approach uses autologous t cells with car construct that is composed of an anti-cd19 single-chain fv, cd28 co-stimulatory and cd3-zeta intracellular domains. cd19 expression on the blasts was documented prior treatment in all pts by flow cytometry. all pts received 1 x 10 6 /kg cart-cells after lymphodepletion with fludarabin and cyclophosphamide. results: six pts (2 males and 4 females) with r/r b-all were enrolled, including one with ph-positive b-all. the median age was 42 years (25-59). median number of prior therapy was 4 (3) (4) (5) . five pts had prior allo-hsct. four pts were given antibodies as the last therapy prior to cart cells. two pts received blinatumomab resulting in pr in one of them. two additional pts received io (1 after failing blinatumomab) achieving mrdpositive cr. cytokine release syndrome occurred in all pts and was severe in only one patient who required tocilizumab treatment. this patient was also the only patient who experienced neurotoxicity (grade 3), and was treated with dexamethasone. this patient eventually died 18 days post infusion of cart cells due to severe pseudomembranous colitis, toxic megacolon and sepsis. all pts had prolonged neutropenia for a median of 14 days (12-18) after the infusion of cart cells. at day 28 after infusion of cart-cells the cr for the entire cohort was 67%: three pts with mrd-negative and one with mrd-positive response. among the four pts who received antibodies prior the cart-cells, one patient had mrd-positive and two pts had mrd-negative response. the patient with ph positive b-all had progressive disease during the treatment. two pts were referred to second allo-hsct from other donors. one patient with mrd-negative response relapsed after the second transplant and was treated by salvage therapy. the second patient with mrdnegative response demonstrated prolonged remission (20 months) even without second transplantation. with a median follow-up of 11 months (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) the median progression-free and overall survival for the entire cohort were 7.5 and 9 months, respectively. conclusions: autologous anti-cd19 car t-cell therapy after debulking treatment with antibodies, including blinatumomab and/or io, is feasible and results in high response rates in pts with r/r b-all. patients may respond to anti-cd19 car t-cell therapy even after failure to their last salvage therapy with blinatumomab, which demonstrates similar mechanism of action. clinical background: genetically engineered t cells expressing a chimeric antigen receptor (car-t) targeting specific antigens present on acute lymphoblastic leukemia (all) blasts have generated promising results in children and adults with relapsed and refractory disease. the below report provides an insight of lineage switch occuring as a result of intense immunological selection after car-t cell therapy, even with a tumor clone that has no potential for this switch. methods: an eight year old caucasian male with precursor b (pb) cell lymphocytic leukemia was treated with cd 19 directed car-t cell therapy in third remission, and after relapse after previous bone marrow transplantation (bmt) . he was diagnosed with t(12;21) pb cell all at 2 years of age and treated with bfm protocol. he relapsed 8 months after completion of maintenance therapy, and had a 9/10 mmud bone marrow transplant after etoposide, tbi and alemtuzumab conditioning therapy. he had cutaneous acute and chronic gvhd but 8 months post-transplant, he relapsed again with pb cell all, with the same cytogenetic and immunophenotypic disease characteristics.. he was treated with lymphodepleting chemotherapy with fludarabine, cyclophosphamide and alemtuzumab followed by infusion of cd19 directed car t cells. he developed cytokine release syndrome of grade 1 severity manifested as persistent fever, associated with car t cell expansion in the blood. after car-t infusion,there was no detectable b cell all clone in the marrow by pcr and the cytogenetics were negative for t(12:21) translocation. 8 months after the car t cell therapy, he was found to have a mrd positive disease which was monitored closely. results: we document clonal evolution from cd19 negative, mrd positive disease to aml, with the same ig rearrangement (the same clonal disease) but with complete myeloid phenotype mpo, cd33, cd13 positive disease. there was cytogenetic evolution of the underlying clone but the original t(12;21) was retained within the evolved karyotype. sadly, our patient developed fludarabineneurotoxcity during an attempt to induce aml remission, and further curative-intent chemotherapy was not possible. conclusions: there are two case reports of mll rearranged b-all acquiring a clonally related myeloid phenotype associated with cd19-negative escape after cd19 directed car t cell therapy,so far. but, this is the first post car-t cell therapy transformation of all to aml with etv6-runx1 mutation, which is not recognised to have such lineage-switch potential. unlike mll, etv6-runx1 translocation in the pathogenesis of acute myeloid leukemia is not been reported in the literature. the theory behind such transformation is an intense immunological selection of the tumor, driving it to myeloid differentiation with additional clonal cytogenetic events. disclosure: nothing to declare p586 ivac-all-1: interim analysis of a phase i/ii clinical study on personalized peptide vaccination based on patient-individual tumor-specific variants in relapsed pediatric acute lymphoblastic leukemia armin rabsteyn 1,2 , christopher mohr 3 , olaf witt 2,4 , roland meisel 2,5 , cristiane chen-santel 6 , tobias feuchtinger 2,7 , christopher schroeder 8 , jakob matthes 8 , background: acute lymphoblastic leukemia (all) is the most common pediatric malignancy. standard chemotherapy is a successful treatment in 80% of patients, only about 20% develop a relapse, however these patients have a dismal prognosis. prevention of relapse after firstline chemotherapy or stem cell transplantation (sct) is therefore an urgent clinical need. we established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient hla types. we started clinical translation of this approach by starting a phase i/ii clinical trial in 2016 (nct03559413). besides feasibility and toxicity assessments, we aim to assess the capability of the peptide vaccination to induce neoantigen-specific t cell responses in high-risk all patients to target residual tumor cells and prevent leukemic relapses. methods: key inclusion criteria are: pediatric patients with all who suffered from second relapse after standard therapy or first relapse after sct. hematological remission has to be reached prior to vaccination. nonsynonymous mutations are identified by whole exome and transcriptome sequencing of patient leukemic blasts and healthy reference tissue. hla binding peptides harboring the altered amino acids are subsequently predicted in silico by algorithms syfpeithi, netmhc and netmhcpan for the patients' individual hla type. vaccine cocktails consisting of 3-5 individual peptides are produced and formulated under gmp conditions. the vaccination schedule is 16 vaccinations over 33 weeks using gm-csf and imiquimod as adjuvants. response to the vaccination is monitored by detection of t cells recognizing the vaccinated peptides occurring over time in peripheral blood of patients by prestimulation and intracellular cytokine staining. results: until now, 23 patients were recruited, for 17 of those, whole exome sequencing was performed to identify all-specific snvs using a comparative bioinformatics pipeline. we found an average of 118.8 mutations per patient on dna level. based on these data, an average of 178 hla binders derived from neoantigens could be predicted per patient. an average expression of 34.4% of mutations was assessed by rna sequencing. in all cases validated mutations could be identified and cocktail design was feasible. until now, 4 patients received vaccinations. the vaccine was generally well tolerated and no or only mild side effects were observed. immune monitoring was performed for 2 patients until now. in the first patient, we observed a transient cd4+ response against one vaccinated mhc class ii ligand and a sustained cd4+ response against the included wildtype control peptide derived from the antigen survivin. in the second patient, immune monitoring was performed for the first 8 vaccination timepoints, a t cell response was not measurable at this timepoint of vaccination. conclusions: whole exome sequencing of pediatric all patients is feasible and yields small amounts of expressed, tumor-specific mutations. these few mutations are sufficient to predict hla-binding peptides that can be used to formulate individualized peptide vaccine cocktails. we currently conduct a clinical phase i/ii trial in a small cohort of high-risk all patients to assess safety, toxicity and immunogenicity. clinical background: chimeric antigen receptor t cells (cart) are considered as gene therapy medicinal products (gtmp) and genetically modified organisms (gmo). hence, carts manufacturing for clinical application is strictly regulated. appropriate methods assessing car transgene copy number (cn) in a cart product and definition of the frequency of carts in treated cart patient are mandatory. although quantitative real-time pcr-based (qpcr) analysis has been used for this purpose, no standardized procedure to minimize systematic errors and enable comparability has been established yet. here, we report on a single copy genebased (scg) duplex (dp) pcr (scg-dp-pcr) for the determination of the vector copy number (vcn) in cart products as well as patient samples following cart administration. scg-dp-pcr was validated and compared to the broadly used absolute copy number qpcr (acn) approach within the framework of a clinical trial treating patients with good manufacturing practice (gmp)-grade carts (hd-car-1). methods: for conventional acn, primers and probe targeting the car vector rv-sfg.cd19.cd28.4-1bbzeta were designed. standard curves were established via serial dilutions of the sfg.car plasmid. amplification of the standard curve as well as target genomic dna for vcndetermination was performed as singleplex (sp) pcr (sp-car) (method a). on the same qpcr plate, duplex (dp) qpcr reactions were carried out. additionally to the components comprised within method a, the experimental setup contained haploid human genomes as well as primers and probe targeting ribonuclease (rnase) p as human scg. the amplifications for the sfg.car plasmid (dp-car) and the rnasep gene (dp-rnasep) were performed simultaneously (scg-dp-pcr; method b). scg-dp-pcr was performed for standard curves and target samples. target-sample dna was extracted from carts prepared from leukapheresis products of three healthy donors (hd). results: for method validation, efficiency and linearity (correlation coefficient) of the qpcr reactions of method a (sp-car) and method b (dp-car, dp-rnasep) were assessed by linear regression of the pcr signal to the reference standard curve. overall, standard curves were only considered valid if a correlation coefficient (r 2 ) of above 0.98 and efficiencies of 100% ± 10% were achieved. vcns applying method a and b to the same target sample were compared. sp-car pcr reaction displayed efficiency of 103.5 ± 7.1%; 104.2% ± 2.1% and 99.3 ± 1.6% efficiencies were achieved for dp-car and dp-rnase pcr reaction, respectively (table 1) . applying scg-dp-pcr using formula for relative cn assessment (2 -δct (dp-car -dp-rnasep) ) on hd samples resulted in an average of 0.8 ± 0.2 increased cn when compared to method a (table 2) . conclusions: in terms of efficiency and linearity by linear regression qpcr reactions were comparable. validation of scg-dp-pcr was achieved and represents an exact and less error-prone method to fulfil regulatory safety release criteria of cart products. besides of accurately assessing vcn of transduced cells, scg-dp-pcr is also a highly robust method to follow-up carts in treated patients. applying this approach, no standard curve is needed, hence significantly economizing required material as well as time. disclosure: nothing to declare background: t-cells' antileukemic responses in aml-pts need to be improved. dc leu effectively activate t-cells against leukemic blasts, resulting in blast-lysis ex-vivo. factors influencing these activities are not known. methods: we generated dc/dc leu from aml-blastcontaining mononuclear cells (n=74) using standard methods (mcm-mimic/ca-ionophore/picibanil/ifn-α, "mnc-methods") and from blast-containing heparinized whole blood (n=35) using modulatory kits (various combinations of 2-3 clinically approved response-modifiers, "wb-kits", patent 102014014993) and correlated statistically (t-/u-test, pearsons correlation, # means significant) proportions of dc-/t-cell-subtypes/cytokine-profiles with t-cells' antileukemic cytotoxicity (ctx), achieved after mixed lymphocyte culture (mlc) with/without mncmethod-("t*dc mnc "/"t*bla mnc ") or wb-kit-treated cultures ("t*dc wb "/t*bla wb "). ctx was given as proportions of cases with achieved or "improved" blast-lysis compared to control and as frequencies of viable blasts (bla via ) after effector-cell-influence. pooled data and data obtained with single methods in different cohorts are given. results: 1. generation of dc/dc leu : with a) mncmethods: ø18±15% dc and ø11±10% dc leu and b) wb-kits: ø13±8% dc and ø11±13% dc leu without induction of blasts' proliferation. t-cell-proliferation increased (vs uncultured t-cells) after mlc with a) mnc-methods: ø34±38% vs ø7±9% and b) wb-kits: ø63±25% vs ø23±20%. 2. antileukemic reactivity (t-effector-cell-cytotoxicity after mlc): pooling all data: a) mnc-methods ("t*dc mnc " vs "t*bla mnc "): we found a1) blast-cytotoxicity in ø70%(vs50%) of cases with ø59%(vs117%) bla via , a2) blast-cytotoxicity was improved (vs control) in 86% with ø decrease of bla via of 78%; b) wb-kits ("t*dc wb " vs "t*bla wb "): we found b1) blastcytotoxicity in ø80%(vs71%) of cases with ø68%(vs56%) bla via , b2) blast-cytotoxicity was improved (vs control) in 67% of cases with ø decrease of bla via of 43%. in general, these results could be confirmed with single methods: best mnc-methods were picibanil and mcm-mimic, best wbkits were kits containing gm-csf+picibanil or prostaglandins. 3. correlations: pooling all data: cases with "improved" lysis (vs "not improved" lysis) were characterized by a) mnc-methods: increased proportions of mature dc/cells (ø46±29% vs ø35±20%), dc leu /cells (ø10±10% vs ø5±4%) and proliferating t-cells (ø35±40% vs ø19 ±14%), b) wb-methods: dc/cells (r=0.781 # ), dc leu /cells (r=0.815 # ), dc leu /bla (ø40±23% vs ø29±16%), dc leu /dc (ø59±22% vs ø51±25%), cd8 + t-cells (ø58±20% vs ø50 ±24%), ifn-γ (r=0.868 #) , mcp-1 (593±242 vs 547±223 pg/ml). conclusions: blasts are regularly converted to dc leu in the presence of mnc-methods and wb-kits (simulating the in vivo microenvironment). t-cells' coculture with dc/ dc leu after mlc induces and improves antileukemic t-cell activation compared to controls. blast-cytotoxicity correlates with proportions of dc/dc leu -and t-cell subtypes and released cytokines. these data support a role of antigen presentation by leukemic cells (dc leu ) for the stimulation of an immune response in aml in vitro and possibly in vivo. disclosure: nothing to declare evaluation 1 year after the launch of the motion comic immuno-t, explaining patients and their caregivers how immunotherapy strategies work background: one year ago, the first version of immuno-t, a motion comic explaining to patients and caregivers how immunotherapy strategies work, was released. people were informed on and inspired to use the application during (inter)national meetings and events for the general public. meanwhile, the motion comic was further refined and adapted into a second version, based on the evaluations we've collected on the first version. adaptations included a multi-language tool (currently 6 languages), increased user friendliness, and a new supporting musical score. also, a website was launched from which the second version could be downloaded on tablet or smartphone (both android and apple) and a new online evaluation form could be filled in. in 10 months, 40 people have evaluated the motion comic online, and these results are presented here, as well as our future plans within the immuno-t program. methods: through an online questionnaire, 41 participants from belgium (n=38) and the netherlands (n=3) have evaluated the dutch version, and 4 belgian participants evaluated the french version of the motion comic. results: the total group (n=45) consisted of patients (n=15) and their families (n=2), general public (n=10), students (n=7), health care professionals (n=4), researchers (n=5) and kindergarten teachers (n=2). participants' age ranged from 19 to 80 years, with an even distribution amongst the different generations. the majority of the evaluators (97,8%, n=44) thought the motion comic is a good way to explain immunotherapy to patients. 23 individuals (51,1%) felt mainly interested after watching immuno-t, and a total of 32 participants (71,1%) felt hopeful or motivated. focussing on the patient group (n=15), all of the responders think the immuno-t motion comic is a good tool to use in a patient-doctor consultation. 11 patients (73,3%) felt hopeful and/or motivated after watching the whole motion comic, while 3 of them (20%) felt combative and 5 (33,3%) felt gripped and intrigued. as for the new musical score, 35 participants (77,7%) think the music is suitable for the app, while 6 evaluators (13,3%) think the new music is not or not at all fitted to support the motion comic. conclusions: the detailed evaluations allow us to further improve immuno-t, and aid us in the development of other motion comics we plan to release under the cancer-t in motion umbrella. with the current version of immuno-t, a single-center pilot study is being set up, to test the efficacy and usability of immuno-t, based on qualitative research during the experience of the tool, and using validated questionnaires. with this study we want to evaluate the impact of immuno-t on patient empowerment, and the decision making process. the study protocol will be presented at ebmt. disclosure: the development of immuno-t was partly financially supported by celyad, calgene, novartis, roche, amgen, bms, but these companies did not by any means influence the contents and development of the motion comic. a therapeutic strategy to trespass the blood brain barrier for adoptive nk cell therapy in glioblastoma multiforme induced rat: a preclinical study background: glioblastoma multiforme (gbm) is among the most common and aggressive primary brain tumors with very poor prognosis. according to the central brain tumor registry of the united states, central nervous system (cns) tumors in pediatric patients (ages between 0-14 years old) are the second most common malignancies after blood-born malignancies, and the first amongst solid tumors, and known the most common cause of cancer-related deaths. although hematopoietic stem cell transplantation has been exploited to treat many kinds of malignancies, currently its success rate in gbm is limited. therefore, the gbm treatment paradigm needs shifting towards more effective treatments such as immune cell therapy. natural killer (nk) cells have been recognized as potential anti-cancer effector cells, as they can recognize and target tumor cells. since a small percentage of blood cells are differentiated as nk cells, the number of this group of cells is hardly enough to fight tumors, and so their multiplication and activation would be a potential effective cancer treatment. methods: this preclinical study was focused on setting up an optimal protocol for expansion and activation of naïve nk cells and assessing their efficacy towards induced gbm in rat models. ex-vivo expanded and interlukin-2 (il-2)and heat shock protein-70 (hsp-70)-treated nk cells have been exploited. after in vitro study and confirming the efficacy of treated cells through cytotoxicity assays, we induced gbm in 6 male wistar rats (weighted 275-300 gr) using c6 tumor cells injection in rat brain through stereotactic surgery. the tumor formation was proven by mri imaging. following tumor establishment, we analyzed the effect of single injection of il-2-and hsp-70-treated nk cells compared with single injection of non-treated nk cells in two groups of rats. results: systemic intravenous delivery of il-2-and hsp-70-treated nk cells through tail's vein resulted in tumor shrinkage in different time intervals and complete remission in the first group of gbm-induced rat models, whereas in the other group of gbm rats receiving untreated nk cells, the tumor progressed. therapeutic efficacy of the treated nk cells was ascertained compared with non-treated nk cells considering tumor shrinkage observed in mri and survival rates between the two model groups. conclusions: the amelioration of tumor which has been confirmed by mri, proved the migration of activated nk cells through blood brain barrier and homing to cns, and finally targeting gbm tumor cells. our data suggest that nk-cells treated with il2/hsp70 may be a promising immune cell-based therapeutic approach towards treating the fatal gbm. disclosure: nothing to declare p592 abstract withdrawn. long term sorafenib response for extramedullary flt3 + aml relapse after allogeneic stem cell transplantation since june 2017, sorafenib dose has been tapered to 200 mg/day, due to mild skin and gi toxicity. after 3 years of treatment, she maintains cr at medullary and extramedulary levels, with no evidence of a disease that had escaped the mechanisms of action of chemo, hsct and dli. conclusions: in our patient, treatment with sorafenib has provided long-term control of this refractory extramedullary disease, even at adjusted doses. further studies are needed to confirm the efficacy of flt3 inhibitors in the control of relapses after allo-hsct, extramedullary disease and its potential role as maintenance agent. disclosure: nothing to declare background: although chemotherapeutic(ct) agents that used in the treatment of acute lymphoblastic leukemia (all) increase survival, the results are still weak. longterm survival with ct's in relapse all cases is difficult and the prognosis is very weak. inotuzumab ozogamicin is an anti-cd 22 monoclonal antibody and it has the potential to reduce the overall toxicity of intensive regimens for all, as well as to possibly increase the number of patients who may achieve a state of minimal residual disease. methods: 26-year-old male patient was diagnosed with b-cell all in december 2017.after the hoelzer kt protocol was started, maintenance treatment was continued. in the fifth month of treatment,flag ct protocol was started cause of recurrence was seen on 5% blast detection in peripheral blood smear. in august 2018, inotuzumab ozogamicin treatment was started and six cures were completed because the patient was not in remission. in september 2018, he had gone haploidentical bone marrow transplantation from his sibling donor(8/10)with defibrotid prophylaxis for veno-occlusive disease(vod)s. he engrafted succesfully and chimerizm was 99.85% in 30th days of transplantation. he is 60th day of transplantation and in a remission. results: bone marrow transplantation cannot be performed since the complete response cannot be achieved in patients with relapse and resistant b-all.in these patients, new therapies targeting malignant lymphoblasts are needed. inotuzumab ozogamicinis a monoclonal antibody drug conjugate that targets cd22 antigen on malignant lymphoblasts.in many studies, it has been shown that inotuzumab ozogamycin is effective and reliable anti-tumor activity in adults with recurrent and resistant cd22 positive all. however, monoclonal antibody drug conjugates have been shown to be associated with vod's.for this purpose, we used defibrotid to protect our patient from vod. conclusions: treatment with combination ct regimens in b-all is suboptimal and long-term survival is achieved in only 30-40% of patients.targeted molecular therapy and new regimens are needed in relapse and resistant patients.at this point, inotuzumab ozogamycin is an anti-cd-22 monoclonal antibody, as in our case, it provides remission in recurrent and resistant b-all patients and allows patients to complete their treatment with an allogeneic transplant from a fully compatible donor. disclosure: nothing to declare background: mesenchymal stem cells (mscs) are an attractive consideration for therapeutic cures of many difficult diseases on the cellular-level. due to the trophic effects of the cytokines and chemokines that they produce, mscs have shown multiple beneficial properties in the field of oncology. in this study, we will be investigating the effect of mscs derived from human bone marrow (bm), adipose tissue (at), and umbilical cord derived mscs (uc-mscs) on ovarian cancer. to differentiate the mscs, we performed a comparative analysis between the various sources for proliferative capacity, surface antigen expression, differentiation ability, tumor marker and paracrine activity, and their influence on ovarian cancer cell proliferation. methods: measurements of ovarian tumor marker proteins were computed by elisa. proliferative effects, immunomodulatory effects, and apoptosis of the mscs were measured by the culture and counting of colony formations. flow cytometry (fcm) was used to measure the variation of the immunophenotyping and cytokine secretions in co-culture, as well as gene expression. results: cells noticeably proliferated without any modifications to their immunophenotype during the third subculture. the colony-forming unit fibroblast (cfu-f) test showed a proliferation of the mscs along with healthy cells and cancer cell lines with no changes in their phenotype. the supernatant of mscs showed an increase in cellular death of the ovcar3 in ovarian cancer cell lines. a reduction in the level of ca-125 (75-90%; p=0.769) with ovcar3 in co-culture, and a decline of ldh (10-20%; p=0.03) and beta-hcg (10-20%; p=0.04) were observed in co-culture in caov3, skov3 and igrov3 cell lines. a decrease in cd24 of the cancer cell lines in co-culture with the msc supernatant showed a reduction of the cancer tumorigenicity and aggressiveness, while the rate of the cd24 and cd44 asserted their stem state. msc supernatant decreased cell proliferation and mmp-2, mmp-9, and ca-125 mrna expression, while increasing timp 1, 2, and 3. this suggests that mscs have a role in cell death and inhibition of ovarian cancer cell proliferation. an increase of anti-inflammatory il-4 and il-10 cytokines, and a decrease in growth factor gm-csf along with their proinflammatory inf-a, tnf-a, il-9, and il-17a cytokines were also noted. conclusions: the gene and cytokine activity indicate a potential therapeutic anti-inflammatory and antiproliferative role of mscs on ovarian cancer despite their sources. the reduction of ca-125, ldh, and beta-hcg in co-culture, along with the decrease in cd24 and amplified cellular apoptosis demonstrate the beneficial effects of stem cells in ovarian cancer cell lines. disclosure background: hematopoietic stem cell transplant (hsct) is the only cure in sickle cell disease (scd) so far. because of the risk of toxicity, its indication in france is restricted to severe patients with match sibling donor. this study compares the incidence of severe acute toxicity after hsct, between children aged less than 13 years and teenagers aged 13 to 18 years old, treated for scd. methods: all patients suffering from scd, aged less than 18 years at transplant, who received hsct in chu robert debré and necker, between 01/01/2005 and 12/31/2017, were included. severe acute toxicity, defined by onset of severe acute gvhd, organ toxicity or infection, was compared between the two groups of age. results: 73 patients (59 children and 14 teenagers) were included. all patients received a myeloablative conditioning regimen. bone marrow from a sibling donor was the main stem cell source (n=65; 89%). neither death nor rejection was observed with a median follow-up of 29.6 months (range, . the incidence of grade iii-iv acute gvhd was 12.3% and was similar between the two groups; no risk factor was identified in univariate analysis. teenagers had more frequently acute skin toxicity (21.4% vs 0%, p=0.006). in univariate analysis, patients presenting severe organ toxicity were significantly older than others (9.3 vs 7.5 years old, p=0.027). teenagers were more frequently treated for bacterial (64.3% vs 32.2%, p=0.035) or bk virus (42.9% vs 6.8%, p=0.002) infections. in univariate analysis, patients who developed infection were also significantly older at time of transplant (respectively 9.7 vs 7.5 years old, p=0.016). no severe sinusoidal obstruction syndrome was observed. regarding long-term toxicity, 2 patients presented an extensive chronic gvhd, they were both aged less than 13 years old. no cut-off of age could have been defined. conclusions: this study confirms the excellent results of hsct in scd, with a 5-year event-free survival and overall survival of 100%. an older age at transplant seems to be associated with more frequent severe acute toxicity. these results are consistent with previous studies and suggest that hsct should be performed as soon as possible, without any defined "best age". prospective studies are needed, in order to define the place of each therapeutic in scd, with the aim of reducing treatment-related toxicity and developing alternative strategies for patients without match sibling donor. disclosure: nothing to declare p599 new insights into risk factors for transplant-associated thrombotic microangiopathy (ta-tma) in paediatric hsct (n=170) was associated with ta-tma in 1% vs 5.8% vs 7.7% respectively (p=0.067). the presence of comorbidities at d0 (n=86) was significantly associated with an increased risk of ta-tma 10.4% vs3.7% in absence of co-morbidity (n=320); p=0.032. use of csa/tac-based gvhd prophylaxis was associated with less ta-tma with an incidence of 4% vs 13% if these agents were not included (p=0.01). in univariate analysis ta-tma was significantly higher among patients with agvhd grade ii-iv (12/138; 8.6%) vs grade 0-i (6/227;2.6%) (p=0.01). pres was recorded among 10 cases and 50% of them developed ta-tma. two out of the 21 patients with ta-tma had pathological gene mutations in their complement pathway. on multivariate analysis the presence of active comorbidity was a risk factor for ta-tma (or:6.6; 95% ci:1.1-37.6; p=0.032) while the use of csa/tac-based gvhd prophylaxis did not increase the risk for ta-tma (or:0.04; ci:0.004-0.39; p=0.005). in the presence of comorbidities the use of defibrotide as prophylaxis or therapy for vod (n=86) was associated with a drop in the incidence of ta-tma from 12% (9/79) in absence of defibrotide to 0% (0/7). 2-year overall survival was significantly lower among ta-tma cases (58%) in comparison to 81.5% in absence of ta-tma (p=0.001) (figure 1). conclusions: active co-morbidity is a significant risk factor for ta-tma. use of defibrotide prophylaxis in patients with co-morbidities at the time of hsct might offer protection against ta-tma. surprisingly the use of csa/tac based gvhd prophylaxis is not a risk factor for ta-tma probably through limiting the development of high grades agvhd. the association between pres and ta-tma suggests a common pathway of endothelial damage background: gonadal impairment is a severe late effect of myeloablative conditioning regimes with significant impact on quality of life of cancer survivors. the aim of this study was to analyze gonadal function after busulfan (bu) or treosulfan (treo) containing regimens with regard to pubertal stage. methods: this was a retrospective, multicenter study involving patients treated in pediatric ebmt centers between 1992-2012. patients receiving myeloablative doses of bu or treo as part of hsct conditioning were eligible for inclusion. analysis was conducted in two groups according to pubertal status at time of hsct. results: 137 patients (pts) were treated in 25 pediatric ebmt with bu or treo before allogeneic hsct. the median age at transplant was 11.04 years (range 5-18); 89/ 137 (65%) were males (m), 48/137 (35%) were females (f). 89/137 (65%) pts were pre-pubertal at hsct (f= 27;m=62) and 48/137(35%) were post pubertal (f=21;m=27). 118/ 137 (86%) patients received bu (f=38;m=80),77/118 (65%) were pre-pubertal. 19/137 (14%)(f=10;m=9) received treo,12/19 (64%) were pre-pubertal ( figure 1 ). females who received treo in pre-pubertal stage (n=6/6) reached more often spontaneous puberty (100% vs 38%; p=0.016) compared to pre-pubertal bu group (n=8/21) and occurrence of menarche was higher in treo group (p< .01) hormonal replacement therapy was given in 13/27 (48%) females transplanted in pre-pubertal stage and in 15/21 (71%) of those transplanted in post-pubertal stage. 77/89 (86%) males were pubertal at last follow-up and 8 of them (10%) performed sperm analysis (5 oligo-azoospermic,3 unknown). three pregnancies were reported in the population group, all received bu. regarding the evaluation of hormonal levels in pubertal patients at time of hormonal dosage (median 19.54 yrs) (66 bu and 15 treo), males treated with treo had significant lower lh levels (p = 0.045) compared to bu group. females treated with treo had significant lower levels of lh and fsh (p= 0.02 and p= 0.0035 respectively). conclusions: gonadal damage related to treo was significantly lower compared to bu. we observed that: females transplanted during pre-pubertal period had spontaneous puberty more frequently after treo compared to bu and that hypogonadism hypergonadotropic was more frequent after bu than treo. these results must be further confirmed on a larger population. background: viral infections significantly contribute to both morbidity and mortality in patients undergoing hematopoietic stem cell transplantation. traditional antiviral therapy is associated with lack of efficacy, potential toxicity, prolonged hospitalization and increased patient costs. viral specific t cells can be manufactured from donor blood to treat viral infections post-transplant, and are associated with increased clinical efficacy and low toxicity. we postulated that direct costs of vst therapy are lower compared to traditional anti-viral medications methods: vsts were manufactured according to local protocols and fda requirements. total drug cost (as per institutional charges per drug) was calculated for patients who required treatment for viral infections post-hsct. manufacturing costs of vsts are fixed per fda requirements. patients who were treated with investigational antiviral medications (including brincidofovir) were excluded from analysis. patients treated with vsts +/anti-viral medications over a 2 year period were compared to patients treated only using traditional anti-viral medications, including cidofovir, ganciclovir, valganciclovir, foscarnet and rituximab. results: demographics are shown in table 1 . there were no major differences between the two groups treated. the number of anti-viral medications used in the vst group was lower compared to the anti-viral treatment group. median cost of vst treatment was significantly lower compared to those threated with traditional anti-viral therapy ($12,757 vs $16,392, p-value=0.03) . conclusions: treatment with vsts post-hsct for viral infections was lower in cost compared to anti-viral medical therapy. it is likely that overall costs are further reduced with vsts due to reduced inpatient hospital time, less monitoring of labs associated with anti-viral medication side-effects and reduced ancillary costs including nursing and pharmacy. more studies are needed to examine these indirect costs further. background: dock8 deficiency is an autosomal recessive primary immunodeficiency (pid) disease caused by loss-offunction mutations in the dock8 gene (1) . patients with dock8 deficiency present with multiple abnormalities of the immune system, including defective t cell function and impaired production of antigen-specific antibodies. these lead to persistent viral infections of the skin, mucocutaneous candidiasis, recurrent sinopulmonary infections, atopic dermatitis, and other allergic disease, malignancies, and sometimes autoimmunity (2). hematopoietic stem cell transplantation (hsct) is currently the only curative treatment option available (3) . methods: we retrospectively evaluated our patients who underwent allogeneic hematopoietic stem cell transplantation due to dock8 deficiency in ege university pediatric stem cell transplantation unit between 2013 and 2018. results: we identified 16 patients transplanted at a median age of 7.8 years (range: 2-15.5 years). of 16 patients; 4 (%25) received hsct from matched sibling, 11 (%68) from unrelated donors and 1 patient from haploidentical donor. we used busulfan-based myeloablative conditioning regimen to 12 patients (%75), reduced toxicity myeloablative regimen with treosulfan to 2 patients (%12.5) and nonmyeloablative regimen to 2 patients (%12.5). eight of the recipients received bone marrow, 6 of the patients received peripheral blood stem cells, 2 of the recipients received cord blood as stem cell source. fifteen of 16 patients (%93) had achieved engraftment and median follow-up of patients was 29 months (1-64). grade iii-iv acute graft versus host disease (gvhd) occurred in 13% of patients and chronic graft versus host disease was seen 18 % of patients. one patient received cord blood from unrelated donor did not engraft and died from septic shock. four patients died from transplant related toxicity. our patient's survival was %68 ; 11/16 patients alive. conclusions: hsct is the only curative treatment option for dock8 deficiency. in particular, patients with high comorbidity scores have a high risk of toxicity and toxic death. therefore, reduced toxicity conditioning regimens should be used for these patients. references : background: eltrombopag, a low-molecular-weight synthetic nonpeptide thrombopoietin receptor agonist (tpo-r), is a second-generation tpo. it is an oral thrombopoietin mimetic licensed in chronic immune thrombocytopenic purpura that induces platelet maturation and release by binding to c-mpl receptors on megakaryocytes. in a recent study; for patients with refractory saa, eltrombopag induced a response (at 12 weeks) in at least one hematologic lineage in 44% patients and 36% no longer required platelet transfusions. and also 24% patients became rbc transfusion independent and 36% had a neutrophil response. trilineage responses were seen in 24% of patients; although surprising, this might indicate stimulation of c-mpl receptors on remaining stem cells. delayed recovery from thrombocytopenia is common after stem cell transplantation. in a study including 12 adult patients, eltrombopag was used to enhance platelet recovery for post-hsct thrombocytopenia. it is well tolerated and efficacious offering transfusion independence. methods: in our retrospective study, eltrombopag (50mg/day) was started in 19 pediatric patients (age ranging from 4 to 16 years with a median age 12.3 years) for posthematopoietic stem cell transplantation (hsct) thrombocytopenia. all patients fulfilled the following 3 criteria: (1) undergone hematopoietic stem cell transplantation (hsct), (2) had improved total leucocyte counts after leucocyte engraftment, (3) had prolonged thrombocytopenia (< 30.000) needing platelet transfusion. results: four of the patients have received ric while 15 patients ma conditioning regimens before hsct. two haploidentic, 2 autologous, 7 mud, 8 msd transplantations were performed. et (50mg/day) was started in 19 patients who had thrombocytopenia despite neutrophil engraftment on the +30th day of hsct a reduction in platelet transfusions and a platelet count of more than 30,000 were the primary endpoints. before et treatment, bone marrow biopsy was checked in 15/19 patients, 10/15 patients had decreased number of megacocyocytes. none of the patients had active bleeding at the start of eltrombopag but they were all at high risk of bleeding. according to the platelet monitoring, 12 patients had a dose increase starting from the second week. the number of patients in need of platelet transfusions was 7 at the end of the first month; and only 2 at the end of the 2nd month. all patients had a thrombocyte count of more than 30.000 in the third month. in 11 patients, et was discontinued after 2-11 months. no dose limiting toxicities have been observed. conclusions: as a conclusion, et was found highly effective for posthsct thrombocytopenia, with no drug related adverse effects. there was a gradual increase in platelet count, and none of the patients had any complication due to thrombocytopenia. disclosure background: isavuconazole (isa) is a new triazole approved for ifi treatment in the adult population. advantages are activity against both moulds and yeasts spp, excellent bioavailability after oral administration without relevant food or gastric ph effect, a water-soluble prodrug developed to facilitate intravenous administration without nephrotoxic excipients such as β-cyclodextrin, potentially poor drug-drug interactions. isa does not currently appear to require tdm. isa safety and efficacy have not been yet established in children and, in particular, no data are available in the pediatric hsct setting. methods: italian association pediatric hematology oncology (aieop) multicentric analysis of a cohort of allogeneic hsct pediatric patients who received isa as ifi treatment or prophylaxis. due to the lack of recommended dosing in pediatric patients and a clear target isa plasma trough-level range, the therapeutic monitoring (tdm) of isa concentrations was applied by a validated liquid chromatography-tandem mass spectrometry (hlpc-ms/ ms) assay technique. isa trough plasma concentrations (c0) and 3 hours after drug intake (c3h) were measured. results: a total of 16 allo-hsct recipients were included, (m/f 12/4); median age: 13,1 years, range 5-19, median body-weight 48,7 kg (range 17-80). isa was used as ifi treatment in 14 cases and as prophylaxis in 2 patients. donors were haploidentical in 9 patients, matched-sibling in 4, allogenic-unrelated in 4 cases. according to eortc criteria, ifi was proven in 3 patients (1 penicilum, 1 mucor, 1 aspergillus fumigatus), probable in 5 and possible in 7 patients. lungs were the main localization (12 cases), associated with cns involvement in 4 cases and paranasal sinuses in 3; 1 patient had possible hepatic candidiasis. all patients, but one, received isa as rescue treatment for previous therapeutic failure (6 ambisome, 3 voriconazole, 1 combination therapy, 1 posaconazole). seven patients received only iv isa, 3 received only oral isa whereas 6 patients received both iv and oral isa. patients under 20 kg body weight received half isa dose (100 mg tid loading dose on days 1 and 2, 100 mg/die manteinance). the others received adult schedule; only 5 patients received loading dose. isa was administered for a median of 83 days (range: 24-240). in 3 patients isa was administered in combination with caspofungin. tdm was applied to 5 patients including 1 patient with severe vod and 1 with renal failure secondary to ta-tma. the median isa concentrations were 3.94 (1.57-5.85) mg/l and 4.7 (1.29-8.37 ) mg/l for c0 and c3h, respectively. ifi complete remission was achieved in 5 cases, partial remission in 3; treatment failure was experienced by 2 patients. in 3 cases fungal lesions remained stable. ctae grade ii-iii toxicity was observed during treatment in 3 patients, with increased transaminase and/or creatinine levels which resolved after temporary isa withdrawal. no drug-drug interactions were observed in 3 patients receiving csa as gvhd prophylaxis and no modification of csa daily dose was needed. conclusions: isavuconazole use may be considered in the pediatric population, even in the hsct setting, for its safety, efficacy, tolerability, no drug-drug interaction. of course these data deserve further evaluation. disclosure: nothing to declare p608 new treosulfan-based conditioning regimens including epigenetic agents in patients with very high-risk neuroblastoma background: the pts aged 18 mos. or older with disseminated nb involving bone and bone marrow constitute a group of pts with very poor prognosis. although the majority of them are responsive to intensive conventional chemotherapy, most eventually relapse with efs at 5 years of < 10%. at the beginning of the year 2018 we came up with a protocol for this very unfavorable group including epigenetic therapy (5-azacitidine) in the phases of consolidation. methods: seven pts with a median age of 4 (2,6-8) years completed the protocol and received hdct with autologous sct as a consolidation. hdct included two different epigenetic agent containing regimes according to tumor response to the induction therapy assessed by 123 i-mibg and mri (ct-scan). three pts revealing large active residual tumor assessed by 123 i-mibg scan or multiple active bone metastases received a conditioning regimen (regimen a) including 131 i-mibg therapy at a dose 4.0-4.4 mbe/kg on d-14, treosulfan 10000 mg/m 2 /d, on d-6,-5 and -4 (total 30000 mg/m 2 ), melphalan 70 mg/m 2 /d, on d-3,-2 (total 140 mg/m 2 ), 5-azacitidine 75 mg/m 2 /d on d-9 to d-5 (total 375 mg/m 2 ). four pts with cr or vgpr received a «split» conditioning regimen (regimen b) including treosulfan 10000 mg/m 2 /d, on d 11 and -10, and on d-4 and d-3 (total 40000 mg/m 2 ), melphalan 70 mg/m 2 /d, on d-3 and -2 (total 140 mg/m 2 ), and 5-azacitidine 75 mg/m 2 /d, on d -14 to d -12 and on d-9 to -d-5 (total 600 mg/m 2 ). a median number of 5.3 (3.7-7) cd34+/kg was infused on d0. results: the median recovery times to wbc>1.0x10 9 /l and to an unsupported plt>20x10 11 /l were 10 (7-12) and 13 (9-17) days, respectively. all pts experienced grade 4 hematological as well as infectious toxicity assessed by nci-ctc score. there were 7 episodes of severe organ toxicity of grade 3 occurring in 4 pts. in 4 cases we observed a severe mucositis, in 2 cases gi toxicity and 1 episode of the erythema multiforme occurred. one pt revealed a lifethreatening episode of hypotension of grade 4. no transplant-related death occurred. the median number of transfused rbc and plt doses was 3 (1-4) and 6 (3-8), respectively. all pts are alive and well without signs of disease progression in complete hematological recovery with a limited follow-up of 5.3 (4-7) mos. from day 0 of hdct. conclusions: although it is rather early to evaluate the efficacy of the epigenetic agent's inclusion in the induction and/or consolidation phases of a very high-risk nb treatment, we can assume that, first, the hdct combining mibg i 131 and/or high dose of treosulfan with epigenetic agent such a 5-azacitidine was feasible and had an acceptable toxicity. second, the "split" modality of the treosulfan use in conditioning regimen would permit to increase the total dose of the alkylating agent with no inacceptable toxicity. disclosure: nothing to declare pre-and-post magnetic resonance imaging of hips and knees for detecting osteonecrosis in children undergoing hematopoietic cell transplantation: in whom is it necessary? ali y suliman 1 , sue c kaste 2 , ying li 1 , dinesh keerthi 1 , guolian kang 1 , brandon m triplett 1 , ashok srinivasan 1 between april 2016 and august 2017 at the university medical center hamburg-eppendorf, germany, were included. total and active ratg plasma levels were analyzed by elisa and flow cytometry, respectively. primary endpoint of the study was exposure to ratg. secondary endpoints included transplant-related mortality, incidence of acute and chronic gvhd, immune reconstitution, chimerism, rejection and viral infections. patients were monitored at least 100 days post transplantation. statistical analyses were performed using ibm spss statistics 24 software, or graphpad prism 5 software. results: median total grafalon™ and thymoglobuline™ peak plasma levels were 419.0 μg/ml and 60.4 μg/ml, respectively; median active grafalon™ and thymoglobu-line™ peak plasma levels (appl) were 77.9 μg/ml and 8.11 μg/ml, respectively. active thymoglobuline™ plasma levels showed highly variable pharmacokinetics compared to grafalon™. neither grafalon™ nor thymoglobuline™ exposure correlated with lymphocyte count prior to transplantation, cell count in the graft (wbc, mnc, t cells), age, body weight or body surface area (bsa). this is indicative for a saturation effect in both groups. to correlate high or low ratg exposure with clinical outcome parameters, we built two groups within each patient cohort by median appl. the incidence of gvhd was not dependent on high or low ratg exposure. until day 100 post hsct, 49 viral infections or reactivations (ebv, cmv, adv, hhv6, bkv) occurred in the 32 patients. interestingly, adv infections affected only children with high ratg exposure. the median time to leukocyte engraftment was not significantly longer in the high ratg groups compared to the low ratg groups (17 to 16 days for grafalon™, and 14 to 16 days for thymoglobuline™). there was a decreased and/or delayed recovery of cd3 + , cd4 + and cd8 + t cell reconstitution, but not of b cells and nk cells in the high thymoglobuline™ exposure group compared to the low thymoglobuline™ exposure group. overall survival was not statistically significant with 80% in the grafalon™ and 95.5% in the thymoglobuline™ group without influence of ratg exposure. conclusions: high and low exposure to grafalon™ or thymoglobuline™ did not result in significant differences in outcome parameters as incidence of survival, agvhd, cgvhd, rejection, or mixed chimerism in this limited cohort. delayed and decreased immune reconstitution in the high ratg exposure groups did not translate into different clinical outcome parameters. adv infections only occurred in the high ratg exposure group. grafalon™ and thymoglobuline™ showed distinct pharmacological and immunological differences in children and larger cohorts are needed to detect clinically significant differences and adjust dosing regimens individually. disclosure: nothing to declare background: the optimal conditioning regimen for allogeneic hematopoietic cell transplantation (allohct) in children with myeloid malignancies remains undefined, particularly when reduced-intensity conditioning (ric) regimens are utilized. methods: we performed a retrospective review of children undergoing allohct for acute myeloid leukemia (aml) and myelodysplasia-related aml (mdr-aml) over a 10 year period (2008-2018) at our institution, comparing the outcomes of those who received either a busulfan (bu)-or melphalan/thiotepa (mel/thio)-based conditioning regimen. results: a total of 49 patients were analyzed. twentyone received fludarabine/melphalan/thiotepa and 28 received myeloablative busulfan-based conditioning, either in combination with cyclophosphamide (n=19) or fludarabine (n=9). atg was used in 27 patients depending on donor. recipients of mel/thio were selected for ric regimens due to pre-transplant comorbidities (cardiac dysfunction, n=9, 3 requiring peri-transplant milrinone), transplant during chemotherapy-induced aplasia (n=5), underlying diagnosis of treatment-related aml (t-aml) and significant pre-allohct chemotherapy exposure (n=5). proportions of patients with de novo aml (mel/ thio, 57%; bu, 78%) and mdr-aml (19% and 21%) were similar between groups; however, recipients of mel/thio were more likely to have t-aml (24% vs 0%). cytogenetic and molecular risk factors were similar between groups. the majority of patients were transplanted in cr1 (mel/thio, 71% vs bu, 68%) or cr2 (24% vs 38%). more recipients of bu conditioning (71% vs 43%) were mrd-negative at the time of allohct; both groups had comparable proportions of patients with ≥m2 marrow (~10%). donor types and stem cell sources were similar between groups, except unrelated umbilical cord blood which was more common in bu recipients (21% vs 10%). there were no graft failures in mel/thio recipients, compared to 14% (n=4) in those receiving bu-based regimens. engraftment kinetics and immune reconstitution were similar. overall acute and chronic gvhd incidence was higher in recipients of mel/ thio compared to bu (39% vs 21%, and 29% vs 18%, respectively), but rates of grade iii-iv acute or extensive chronic gvhd were comparable. vod requiring treatment was diagnosed in 6 (21%) recipients of bu conditioning and no mel/thio recipients. median duration of follow-up was 30 months (range 2-70) in the mel/thio group, and 18 months (range 1-121) in the bu group. transplantrelated mortality (trm) was similar in both groups (1 patient), occurring before day 100. relapse incidence was comparable (mel/thio, 29% vs bu, 32%); however, relapse occurred at a later time in mel/thio recipients (median d +396 vs d+137). overall survival at 1 and 3 years was superior in mel/thio recipients (95% vs 74%, and 75% vs 50%, respectively). conclusions: in our single institution experience, use of a melphalan/thiotepa-based ric regimen was associated with similar outcomes compared to full-intensity bu-based conditioning, despite higher risk patient and disease characteristics. the majority of recipients of mel/thio conditioning had significant pre-transplant comorbidities, which did not translate into higher trm. while mel/thio recipients had less optimal leukemia control at the time of transplant and high-risk leukemia features (e.g. t-aml), relapse was similar between groups, occurring later in mel/ thio recipients, which may have contributed to better overall survival. disclosure: nothing to disclose methods: pubertal development and biological gonadal parameters were assessed in a retrospective monocentric cohort of pre-pubertal patients who underwent hsct after myeloablative conditioning with total body irradiation (tbi) or busulfan between 1981 and 2017. results: seventy-four patients (28 girls and 46 boys) were included. no spontaneous pubertal development was found in 50% of girls and 10% of boys (p < 0.001) and delayed puberty or no spontaneous pubertal development was found in 57% of girls and 24% of boys (p=0.009). hormone replacement therapy was used in 82% of girls and 24% of boys (p < 0.001). in univariate analysis, tbi conditioning (p=0.05), female sex (p < 0.001), acute gvhd (p=0.05), extensive chronic gvhd (p=0.021), steroid treatment >6 months (p=0.016), and malignant diseases (p=0.016) were associated with no spontaneous pubertal development, whereas tbi conditioning (p=0.003) and extensive chronic gvhd (p=0.005) were associated with delayed puberty. in multivariate analysis, factors independently associated with no spontaneous puberty onset were female sex (p=0.001) and age >10 years (p=0.033). factors independently associated with delayed puberty were extensive chronic gvhd (p=0.041) and age >10 years (p=0.031). tbi was not an independent risk factor for pubertal complications. conclusions: this study confirms the toxicity of myeloablative conditioning on pubertal development and the role of older age and female sex in increased pubertal issues, and suggests a possible role of gvhd in delayed puberty. disclosure: nothing to declare p613 abstract already published. neutrophil elastase activity may serve as a marker for neutrophil extracellular traps formation following stem cell transplantation ronit elhasid 1 , sivan berger-achituv 1 , hila rosenfeld-keidar 1 , szilvia baron 1 1 tel aviv sourasky medical center -tel aviv university, tel aviv, israel background: post-transplant infections rise dramatically in patients with quantitative or qualitative neutrophil defects and constitute a major source of morbidity and mortality following hematopoietic stem cell transplantation (hsct). neutrophils protect the host from microorganisms via multiple processes including phagocytosis and formation of neutrophil extracellular traps (nets). although reactive oxygen species (ros) production seems to be essential for nets formation, the key enzymes of the process are neutrophil elastase (ne) and myeloperoxidase (mpo). methods: ne and mpo activity as well as nets formation were investigated following hsct in 11 patients at week 1 to 6 and 30 after neutrophil engraftment. neutrophils were isolated using easysep direct human neutrophil isolation kit (stemcell technologies inc.) by immunomagnetic negative selection. enzymatic activity of ne and mpo were measured using colorimetric assays. nets formation of phorbol 12-myristate 13-acetate (pma)activated neutrophils was investigated by confocal fluorescence microscopy. all results were compared to those of healthy volunteers. statistical significance was calculated using one way-anova with bonferroni post hoc test. results: 11 patients (median age of 6.8 years [range 2-22 years]) were investigated, 6 following allogeneic hsct (2 acute lymphoblastic leukemia, 1 acute myeloblastic leukemia, 2 epidermolysis bullosa, 1 rhabdomyosarcoma) and 5 following autologous hsct (4 ewing sarcoma, 1 desmoplastic small round cell tumor). all patients experienced fever and neutropenia. at engraftment, average ne activity was significantly decreased compared to the average value of 50 healthy individuals. ne activity improved week by week in patients, reached the lower reference range at 4 weeks following transplantation (fig. 1a) and continued to increase. the enzymatic activity of mpo was comparable to the average value of 50 healthy individuals (fig. 1b) and showed no significant difference between the distinct time points. at neutrophil engraftment, nets formation was absent and comparable to those of non-activated neutrophils (fig. 1c) . although nets formation increased week by week, it did not reach the average of 7 normal controls during the monitored time period. also linear correlation between ne activity and nets formation (r 2 =0.978) was demonstrated. conclusions: impaired ne activity following hsct corresponds to decreased nets formation and could serve as a marker for netosis. strategies to accelerate the recovery of ne function post transplantation might improve nets formation and thereby induce better infection control. a) the average of ne activity (n=11) during 30 weeks following hsct. reference range was measured and calculated from measurements of 50 healthy volunteers using. b) the average of mpo activity (n=11) during 30 weeks following hsct. reference ranges were measured and calculated from measurements of 50 healthy volunteers using the quartile method. c) the average of netosis activity after 100 nm pma activation for 3h (n=11 background: to have a better understanding of incidence, treatment, outcome and risk factors of immune cytopenia in children after allogeneic hsct. methods: between january 2010 and september 2018, 105 pediatric allogeneic hsct have been performed in 99 patients at the ghent university hospital (ghent, belgium). autoimmune hemolytic anemia was defined by a positive direct agglutinin test (dat). dat was performed at moment of engraftment and in case of hemolysis or unexplained anemia. platelets antibodies were evaluated in case of no otherwise explained thrombocytopenia. results: the cumulative incidence of post allo sct autoimmune cytopenia is 9.5% (10/105). in 9 cases there were positive antibodies against red blood cells, and one patient against had antibodies against platelets. of these 10 cases, only 4 (3.8%) were clinically relevant and needed treatment. the median observation period post sct for the whole cohort was 36 months (3 -105) . the clinically significant immune cytopenia started at a median time of day+158 and day +113 in the group without symptoms. the patient who presented the autoimmune thrombopenia developed antibodies against anti-gpiib/iiia, this was resolved after 130 days, the treatment consisted intravenous immunoglobulins (ivig). two of the 3 patients with autoimmune hemolytic anemia had igg mediated antibodies, and 1 had complementmediated dat. these 3 patients were treated with ivig, steroids, rapamune and rituximab. one patient has still dat positive after 36 months, but clinical stable. the other two are also dat positive and have some hemolysis, but the follow up is much shorter (2 months). treosulfan-contained conditioning regimens were more frequently used in patients with significant immune cytopenia. conclusions: immune cytopenia is an infrequent complication after allogeneic hsct. however, its treatment can be challenging, and the hemolysis can persist for years. the association of rapamune and rituximab was adequate to treat this problem in our patients. background: approaches to the management of refractory and relapsed classical hodgkin´s lymphoma (r-r chl) are changing and become more effective. the role of anti-cd30 targeted immunochemotherapy with brentuximab vedotin (bv) has been extensively investigated in adults with r-r chl and is only to be elucidated in children. the study included 14 children and adolescents with r-r chl that were sucessfully treated with bv-based therapy prior to hematopoetic stem cell transplantation (hsct). median age of patients was 16 years (9-19), main histological variant -nodular sclerosis (93%, n=13), advanced stage at diagnosis -86% (n=12). most were heavily pre-treated (median number of previous therapies -4) and progression after autologous hsct was documented in 4 (29%). refractory disease was diagnosed in 8 (57%) and relapsed in 6 (43%). among relapsed patients 4 (67%) were with multiple episodes, 1 (16.5%) -early and 1 (16.5%) -late relapse. treatment regimens consisted of bv in monotherapy 1.8 mg/kg triweekly (n=7) or combination of bv 1.8 mg/kg on day 1 with bendamustine 90-120 mg/ m2 on days 1 and 2 of 3-week cycles (n=5) or combination of bv 1.8 mg/kg on day 1 with dhap (n=2). median number of bv infusions was 3.5 (2-7). all selected patients achieved complete (n=5, 36%) or partial remission (n=9, 64%) prior to hsct. consolidation with autologous hsct was performed in 9 (64%) and with allogeneic hsct -in 5 (36%). primary end points were overall (os) and progression free survival (pfs). response to bv was not assessed in the study as only responders to the bv-based treatment were included. results: with median follow-up of 497 days (105-1368) os and pfs for all patients are 68% and 66%, respectively. pfs after autologous hsct and allogeneic hsct are 70% and 60%, respectively (p=0.7) at present moment 10 (71%) patients are alive and are in remission. three patients died (21%): disease progression (n=1), postransplant idiopathic pneumonia syndrome (n=1) and posttransplant pneumonia (n=1). bv was generally well tolerated with only mild polyneuropathy in 3 patients (21%) as the main reversable documented adverse event. conclusions: in prognostically unfavourable heavily pretreated children and adolescents with r-r chl achievement of response to bv-based therapy prior to hsct is assosiated with promising rates of os and pfs. disclosure provides a treatment by restoring thymidine phosphorylase function and improving disease manifestations. here we report the outcomes of 4 affected siblings who underwent transplantation using an unaffected sibling donor to highlight important experiences in the transplant of such a rare condition. methods: four siblings of consanguineous pakistani descent aged 16, 15, 13 and 5 years underwent myeloablative hsct using fully hla-matched (10/10) peripheral blood stem cells harvested in a single apheresis from an unaffected 9 year old sibling. the oldest sibling, a 16 year old male, first presented in 2016 having emigrated from pakistan with a history of growth failure and abnormal movements. biochemical, nerve conduction and imaging studies confirmed a diagnosis of mngie. testing on three other siblings identified similar biochemical abnormalities, though the 2 youngest children had minimal clinical manifestations of the disease. based on the progressive nature of the disease and the availability of a fully matched donor, a decision was made to pursue transplant for all affected siblings. results: due to the severity of their disease, the 2 oldest siblings were transplanted first using a myeloablative conditioning regime of fludarabine, thiotepa and treosulfan with alemtuzumab. neutrophil engraftment occurred on day + 11 for both, with 100% donor chimerism achieved. there were no significant transplant related complications. the post-transplant course of the 15 year old sibling was complicated by a major stroke-like event characterised by dramatic imaging changes and requiring ventilation, though no cause was identified and the patient's neurologic deficits have since resolved. gastrointestinal symptoms have persisted and both remain tpn dependent, though symptomatically have shown gradual improvement. following the neurologic complications in their older sibling, the 2 younger siblings were conditioned with auc-targeted busulfan and fludarabine plus alemtuzumab. neutrophil engraftment occurred on day + 11, with full donor chimerism achieved. progression to enteral feeding has been much more rapid, with nutrition now fully enteral for both. there were no significant transplant related complications. conclusions: stem cell transplantation represents the only curative option for mngie. due to its rarity and relative infancy as a condition, little is known of the expected course following transplant or the best approach to transplantation itself. despite previous challenges with graft failure in mngie recipients, we were able to gain rapid and sustained donor engraftment using 2 different myeloablative conditioning regimes with minimal transplant-related morbidity and no mortality. in keeping with previous reports, resolution of established gastrointestinal symptoms has been slow, though the 2 siblings transplanted earlier in their disease course have shown more rapid improvement supporting the role of early recognition and access to transplant. it is essential moving forward that specialised transplantation centres collaborate so as to guide clinicians in the management of such a challenging condition. disclosure: there are no conflicts of interest to disclose. g6pc3 congenital neutropenia -biology of inflammatory colitis associated with gcsf use, and disease response to allogeneic transplant, a report of 4 cases background: an autosomal recessive disease, glucose-6phosphatase catalytic subunit 3 (g6pc3) deficiency is a relatively recently identified cause of chronic severe neutropenia. there can be a spectrum to the disease and patients may also present with non-haematological features including prominent chest veins, cardiac, endocrine or urogenital abnormalities. we describe in our patient cohort a response to gcsf but an inflammatory, incapacitating, biopsyproven colitis associated with that g-csf response. we have transplanted 3 children with such colitis, and describe a similar colitis with intestinal failure in a fourth. methods: we investigate the biology of the neutropenic colitis, and demonstrate necrosis of the stimulated neutrophils. in vitro studies demonstrated that unstimulated neutrophils from patients with g6pc3d exhibited significantly increased production of il8, reactive oxygen species (ros) and neutrophil extracellular traps (nets) alongside significantly higher expression of cd11b, cd66b and cd14. in contrast, neutrophils from patients with g6pc3d produced significantly less ros, mmp-9, neutrophil elastase and nets upon stimulation. neutrophils from patients with g6pc3d also exhibited significantly accelerated apoptosis and secondary necrosis which was exaggerated upon stimulation with live escherichia coli bacteria but could only be partially rescued with supplemental exogenous glucose. results: 3 patients have undergone hsct for g6pc3 neutropenic enterocolitis (1 unrelated donor and 2 msd) after fludarabine treosulfan and thiotepa conditioning therapy. alemtuzumab was given as as serotherapy. all 3 patients are alive and well, immune suppression has been discotniuned and there is no gvhd with normal organ function, and resolution of colitis. we describe a 4 th patient with no good donor who has continuing intestinal failure with g-csf use. conclusions: we describe the aetiology of intestinal inflammation and failure with an extensive study of neutrophil biology in this metabolic neutropenia. we describe a novel indication for hsct in this "g-csfresponsive neutropenia". disclosure: nothing to declare p620 does body mass index (bmi) pose a risk to outcome for pediatric non-infantile patients undergoing hematopoietic cell transplantation (hsct)? mona al-saleh 1 , khawar siddiqui 1 , amal al-seraihy 1 , abdullah al-jefri 1 , ali al-ahmari 1 , hawazen al-saedi 1 , awatif al-anazi 1 , mouhab ayas 1 , ibrahim al-ghemlas 1 with no evidence of toxicity. as benefits of stoss therapy in hsct remain unknown, and safety has yet to have been studied extensively in the pediatric population, we hypothesize that stoss therapy is an effective and safe method to reach and attain sufficient levels of vd in pediatric patients undergoing hsct. methods: this is an ongoing prospective, randomized clinical control trial at phoenix children's hospital that commenced december 1 st , 2017. following consent, subjects are randomized to the intervention (stoss) or control arm prior to hsct. stoss therapy consists of a single oral dose of vd (ranging 100,000iu -600,000iu), given based on baseline 25-hydroxyvitamin d [25(oh)d] level and age, followed by standard weekly supplementation. subjects enrolled on the control arm receive standard of care based on endocrine society guidelines of weekly vd supplementation. data collection includes demographics, 25(oh)d levels at baseline, day +30, and day +100, vd toxicity (hyperphosphatemia, hyperkalemia and renal calculi), as well comorbidities were collected. at each time point and for each trial arm, the mean 25(oh)d level and changes from baseline were computed with corresponding 95% confidence intervals (cis) to indicate variability. results: presently, 12 subjects have completed baseline assessment, with day +30 and day +100 follow-up completed for 11 and 9 of these, respectively. at baseline, the mean (95% ci) 25(oh)d was 19.8 ng/dl (10.9, 28.7) among stoss patients and 18.9 ng/dl (10. 3 results: total hrqol scores of transplanted patients were significantly improved compared to those on supportive care and also compared to healthy siblings (p < 0.0001 and 0.0002 respectively), the same was true for physical (p < 0.0001 and 0.0003 respectively) and emotional functioning (p < 0.0001 and 0.0073 respectively). social and school functioning of transplanted children were not different from healthy siblings (p 0.5893 and 0.7603 respectively) while were very significantly improved compared to children with st on supportive care (p < 0.0001 in both cases). conclusions: bmt in a lower-middle income setting may be even more impactful compared to high-income regions. our analysis clearly indicates normalization of hrqol in all major areas of children transplanted for st. a possible resilience effect was noted for physical and emotional scores which were improved compared to healthy sibling controls. we could not however quantify the effect of longer-term issues like fertility impairment after bmt which may eventually adversely impact hrqol, particularly in the indian culture. disclosure: none allogeneic hematopoietic stem cell transplantation in ataxia telangiectasia patients without malignancy background: ataxia telangiectasia (a-t) is a primary immunodeficiency with mutations in atm-gene. besides a slowly progressive neurodegenerative course, a-t leads to increased susceptibility to malignancies which affects 25% of patient (median:12.5 years) with a high mortality mainly due tocomplications of conventional radio-chemotherapy. the incidence of cancer correlates with the extent of immunodeficiency. patients often develop severe progressive granulomatous skin disease with evidence of vaccine-strain rubella-virus in the lesions. prolonged survival, neurologic improvement and malignancy prevention was observed in atm-deficient mice after treatment by syngeneic hsct. nevertheless, pre-emptive hsct is not routinely performed in a-t patients due to concerns about neurodegeneration and toxicity. methods: we present three a-t patients with severe immunodeficiency phenotype, undergoing successful hsct as an individual treatment strategy intending to restore immunodeficiency for long-term malignancyprevention (patient-1) and to treat progressive skin/joint granulomas (patients-2 and -3). results: patient-1 underwent a reduced intensity conditioning (ric) regimen at 4 years of age including fludarabine (150 mg/m 2 ), cyclophosphamide (80 mg/kg), and atg-fresenius (20 mg/kg/d) which was tolerated well. hematopoietic engraftment occurred by day +15. there was an expansion of naïve and memory cd4 + t-cells and cd19 + cells. while initially a mixed donor chimerism in patient's pbmcs (10-20% donor) was observed, patient's tcells (cd3 + ) reached over 90% of donor origin over time. at last follow-up (6 years) he is well, without signs of gvhd and organ toxicity, off immunosuppression with normal levels of atm-protein; his granulomas resolved. patient-2 is a 6 year-old male who was transplanted from his hla-identical sibling, conditioned with fludarabine (150 mg/m²), cyclophosphamide (120 mg/kg), and atg-fresenius (20 mg/kg). hematopoietic engraftment was observed by day +10. t-cell reconstitution started by day +40 with >200μl cd3+ t-cells. his mixed chimerism rapidly turned to donor origin (95% donor cd3+) over time. there was no acute toxicity, however, he developed lumbosacral pain episodes with evidence of urine bk-virus with spontaneous remission. an intermittent metapneumovirus associated pulmonary hypertension was observed with pericardial effusion. treatment included sildenafil and oxygen. at last follow-up (9 months) patient is well without immunosuppression. patient-3 suffered from recurrent chest infections, failure to thrive and progressive and debilitating rubella positive progressive granulomas of the skin. she received allohsct from an hla-identical family donor at 6 years of age. conditioning included busulfan (2.2 mg/kg), fludarabine (150 mg/m²), cyclophosphamide (40 mg/kg), and alemtuzumab (1 x 5mg/m², 3 x 10mg/m²). hsct was complicated by intermittent acute renal failure, cmv reactivation and tma. hematopoietic recovery was observed by day +22. t-cell chimerism increased rapidly over time (> 90% donor). at last follow-up (7 months) patient is well, off immunosuppression and ivig. her skin granuloma resolved with scarring residues. conclusions: pre-emptive allohsct is feasible in a-t when reduced intensity conditioning is used and can correct the immunodeficiency. it might be a treatment option for some a-t patients at high risk of hematological malignancy and severe granulomatous skin disease. to what extent the restored immune system and the increase of atm-protein in these patients could prevent the development of other malignancies needs to be evaluated further. disclosure: nothing to disclose p625 abstract withdrawn. hematopoietic stem cell transplantation in diamond blackfan anemia: brazilian experience background: diamond-blackfan anemia (dba) is a rare inherited red cell aplasia caused by an intrinsic defect of erythropoietic progenitors. the main therapeutic approach is based on repeated red blood cell transfusions and/or corticosteroid therapy. hematopoietic stem cell transplantation (hsct), a potentially curative treatment for dba, is indicated for patients that do not respond to first-line therapy. methods: the aim of our retrospective study is to report the outcomes of 30 brazilian dba patients transplanted between 1990 and 2017 in 9 bmt centers. the median age of the patients was 5 ys (range 1-15) and 60% were male. seventeen patients (57%) were transplanted with matched related donors (mrd) and thirteen (43%) from matched and mismatched unrelated donors (mud/mmud). in the mrd group all patients received bone marrow as hsc source, while in the mud/mmud, eight patients received bone marrow and five received cord blood. all patients with incompatibilities (mismatched) were ucb (5/5). nineteen recipients were conditioned with busulfan plus cyclophosphamide, while the remaining 11 received fludarabine and busulfan, which has been the preferred regimen in brazil in the recent years. after transplant, most (n=24) of the mrd and mud recipients received cyclosporine and short course methotrexate as graft versus host disease (gvhd) prophylaxis. results: twenty-two out of the 30 patients were alive and disease-free at a median follow-up of 24 months (range 1 to 213 months). the 3-year overall survival (os) was 71% (ci 51-91%) (fig 1) . similar results have been demonstrated in studies from europe and from the united states. when analyzed according to donor type, os was 73% (ci 55-100%) and 66% (ci 43-100%) in mrd and mud/mmud respectively (fig 2) . three out of the 5 patients who were transplanted with ucb died. these results are in agreement with those of previously published data showing worse results in unrelated ucb transplants. twenty-nine out of the 30 patients engrafted successfully. in 25 of the evaluated patients, the median time to neutrophil engraftment was 20 days (range 10-27). one patient experienced an early death from hemorrhagic shock on day 12, before neutrophil recovery, and another two patients experienced primary graft failure. post-transplant chimerism was available for 22 patients. sixteen had complete chimera (>90% chimerism), while 6 patients presented with mixed chimerism. acute gvhd was observed in 9 patients (32%), 6 of which classified as grade iv. five patients developed chronic gvhd, considered severe in three of them. eight patients died at a mean of 155 days (range 12-728 days) after hsct and the main causes of death were infections and hemorrhagic disorders. conclusions: hsct is a potentially curative treatment option for dba. in the present study, we report the outcomes of 30 patients with dba transplanted in brazil with a os of 71%, with better results in mrd compared to mud, as expected. despite the small numbers, we observed lower survival after mud/mmud ucb transplantation. since dba is a rare disease, international collaborative studies are essential to better understand the benefits of the hsct in the treatment of these patients. disclosure: nothing to declare p627 treatment of the obliterant bronchiolitis in pediatric allogeneic recipients: two periods compared results: in group 1, the therapies administered for bo included prolonged treatment with steroids in all patients, anti-tnf in 1, azatioprine in 4; while in the group 2, all patients received ima, montelukast and azitromicin, and 4 received i.v. mpd. the median duration of imatinib therapy was 4 years (0.3-7.1 years). after a median follow-up of 4.4 years (range 0.5-12.1 yrs), 9/11 patients of group 1 (82%) died with bo in progress for transplant-related causes. while in the group 2, 2/14 (14%) died in presence of worsening bo. the estimated os at 1 year after hsct was 75% (95% ci; 50-89) in group 1 and 83% (95% ci, 27-97) in group 2 (p=0.103) (figure 1 ), while the os after 4 year decreased at 42% (95% ci; 16-66) in the group 1 while remained stable in the group 2. conclusions: this experience shows a relevant improving in prognosis of children with bo with the use of this protocol including ima, since the significant improving of survival obtained, confirming as reported in adult populations. disclosure results: we presented 20 patients with pres, age ranging from 2 months to 19 years with a average of 9.5 years. there were ten patients with thalassemia major, two patient with acute lymphoblastic leukemia, three patients with sickle cell disease and one patient with myelodysplastic syndrome, one patient with immune deficiency, two patients with acute miyeloid leukemia, one patient with aplastic anemia. ten patients were males, ten were female. all patients were treated with csa or tacrolimus and metilprednisolone for the prophylasix of gvhd. pres occurred at a median of 90 days (range 5-625). clinical findings at onset of leukoencephalopathy were hypertension, headache, seizures, visual disturbance, and altered mental function. eighteen patients alive with normal neurological status. mri showed abnormalities in all patients including patchy bilateral cortical and subcortical lesions, especially in parieto-occipital lobes. conclusions: bmt is associated with several neurological complications that may be underlying diseases, bmt procedure, and severe immunosupression. pres is an uncommon but serious complication after bmt. we report 20 cases of pres who received allogeneic bmt for thalassemia major to emphasize the importance of early recognition and institution of appropriate management of pres during bmt. disclosure: nothing to declare p631 continuous complete molecular remission using three different monoclonal antibodies followed by allogeneic bone marrow transplantation in an infant with chemotherapy-refractory acute lymphoblastic leukemia bernd gruhn 1 , susan wittig 1 , thomas ernst 1 , jana ernst 1 1 university of jena, jena, germany, background: a 10-week-old infant was diagnosed with very immature acute lymphoblastic leukemia (all) with myeloid markers in a foreign university hospital. at the end of induction therapy according to the current lal/shop protocol 10% leukemic cells were detectable in the bone marrow. treatment was changed to fludarabine, cytarabine and granulocyte colony-stimulating factor (flag) in combination with liposomal doxorubicin. after this re-induction still 5% leukemic cells were detected in bone marrow, so the bispecific t-cell engager antibody blinatumomab was given. due to an increasing portion of leukemic cells during the continuous infusion, antibody therapy was stopped and a cycle of clofarabine, cyclophosphamide and etoposide was administered. unfortunately, still 31% leukemic cells were detectable afterwards. because of chemotherapy-refractory leukemia a palliative oral treatment with mercaptopurine was started. however, the parents did not accept the palliative situation and searched for alternative therapeutic options in other university hospitals in europe. after plenty of refusals the infant was admitted to our hospital five months after diagnosis. methods: for molecular characterization genomic dna was isolated from leukemic cells. a mll-mllt3/af9 rearrangement as a consequence of the translocation t(9;11) (p22;q23) was detected and used as a marker for minimal residual disease. for further molecular characterization targeted deep next-generation sequencing was performed for a panel of 54 leukemia-associated genes. interestingly, no mutation was found. to allow precise immunophenotyping of the leukemic cells treatment with mercaptopurine was stopped. results: as in the first immunophenotyping the cd33 antigen was found, we administered the anti-cd33 monoclonal antibody gemtuzumab ozogamicin twice within two weeks. because of the detection of cd38+ leukemic cells after infusion of gemtuzumab ozogamicin, the anti-cd38 antibody daratumumab was given alternating twice within two weeks. unfortunately afterwards, leukemic cells reappeared being negative for cd33 und cd38, but positive for cd22. therefore, we administered the third antibody, the anti-cd22 monoclonal antibody inotuzumab ozogamicin, whereupon our patient developed a tumor lysis syndrome and a severe bone marrow aplasia. shortly after, allogeneic bone marrow transplantation from an unrelated donor using a special conditioning regimen consisting of thymoglobulin, busulfan, fludarabine and clofarabine was conducted. clofarabine was added because an additional antileukemic effect especially in infant all with mll rearrangement was described. after transplantation the patient suffered from a severe hepatic sinusoidal obstruction syndrome with massive ascites, renal and pulmonary dysfunction, but finally the patient recovered completely. the first bone marrow examination 30 days after transplantation revealed a donor chimerism of 100% and a complete molecular remission using the mll-mllt3/af9 rearrangement as marker for minimal residual disease. in all follow-up bone marrow samples we observed a complete donor chimerism and a complete molecular remission. currently, eight months after transplantation the patient is in a very good physical condition with normal development according to the age. background: paediatric chronic graft versus host disease (cgvhd) is a debilitating condition associated with substantial morbidity and mortality. to date, there are no approved therapies for paediatric patients with cgvhd, and current treatments often lack sufficient efficacy or lead to severe/life-threatening toxicities that limit their effectiveness. ibrutinib, a first-in-class, once-daily inhibitor of bruton's tyrosine kinase (btk), is approved in the us for the treatment of adult patients with cgvhd after failure of ≥1 lines of systemic therapy. this phase 1/2 study will evaluate the use of ibrutinib in paediatric patients with moderate or severe cgvhd. methods: this open-label, multicenter, international phase 1/2 study (pcyc-1146) includes patients with moderate or severe cgvhd as defined by the 2014 nih consensus development project criteria. it is divided into two parts: part a will determine the recommended paediatric equivalent dose (rped) of ibrutinib in patients aged ≥1 to < 12 years, and part b aims to evaluate the safety and efficacy of ibrutinib in patients age ≥1 to < 22 years. for part a, patients with cgvhd aged ≥1 to < 12 years who have failed ≥1 lines of systemic therapy will receive once daily oral ibrutinib at a starting dose of 120 mg/m 2 to be escalated up to 240 mg/m 2 after 14 days, if no grade ≥3 toxicities occur, until the rped is determined. for part b, patients aged ≥12 to < 22 years with cgvhd who have failed ≥1 lines of systemic therapy or have newly diagnosed cgvhd will receive once daily ibrutinib (420 mg) until one of the following criteria is met: treatment is no longer required; new systemic treatment for cgvhd is initiated; progression of cgvhd; recurrence of underlying disease; or unacceptable toxicity. patients with newly diagnosed cgvhd will receive ibrutinib in addition to daily corticosteroids (0.5-1 mg/kg prednisone). patients < 12 years of age may be enrolled in part b and treated at the rped after it is determined in part a. key exclusion criteria include uncontrolled active systemic infection or active infection requiring systemic treatment; progressive underlying malignant disease or any post-transplant lymphoproliferative disease; or active hepatitis c/hepatitis b virus. patients must have adequate renal, hepatic, and hematologic function to be enrolled. the primary endpoint of part a is the rped of ibrutinib, as based on pharmacokinetic (pk) data; secondary endpoints include safety and pharmacodynamics (btk occupancy). the primary endpoints of part b are pk and safety of ibrutinib in paediatric patients with cgvhd. secondary endpoints for part b include response rate at 24 weeks as defined by the 2014 nih consensus development project criteria; duration of response; overall survival; and late effects on growth, development, and immune reconstitution. results: this global study is currently enrolling. conclusions: this phase 1/2 study will explore the use of ibrutinib in paediatric patients with cgvhd to potentially meet the high unmet need for proven effective therapies for this population. disclosure enzyme replacement therapy (ert) is the treatment of choice in non-neuropathic hunter syndrome, but as the recombinant enzyme does not cross the blood brain barrier and neuropathic hunter syndrome is left untreated. hematopoietic stem cell transplantation (hsct) is the standard of care in patients with severe mucopolysaccharidosis (mps) type i (mpsih, hurler syndrome) as early transplantation halts cognitive decline in these patients and significantly improves survival. only few case studies have been published on the potential benefit of hsct in mps ii and mostly used busulfan-based conditioning regimens. in one comparative non-randomised multicenter study, hsct might to be superior compared to ert. here, we present our experience in hsct in three children with hunter syndrome using a treosulfan-based conditioning regimen. methods: a retrospective chart review was carried out in patients, who underwent hsct for hunter syndrome. the conditioning chemotherapy regimen included fludarabine, treosulfan, thiotepa and atg. all patients received bone marrow of either related and or matched unrelated donors. gvhd prophylaxis was performed with csa and methotrexat. results: three patients with hunter syndrome were transplanted in our department in 2010. the age was six months, two years and four years, respectively. bone marrow donors were related in one patient and matched unrelated in two patients. the conditioning therapy was generally well tolerated. major complications were fever of unknown origin with need for antibiotic therapy and a mucositis. one patient developed a cmv reactivation. all patients engrafted successfully and recovered well from the hsct. there was no case of acute or chronic gvhd. in 2018 all three patients are alive. donor chimerism is complete in one patient; two patients have a mixed donor chimerism. after application of donor lymphocyte infusions in one patient, donor chimerism is stable at a low level of 16%. the donor chimerism of the other patient still slowly declines to currently 50%. after stem cell transplantation, two patients did not show further progression of the disease and even achieved psycho-motor improvements. interestingly, one of these patients is the one with the low donor chimerism of 16%. one patient suffers from a further progression of the underlying disease with psycho-motoric agitation, aggressive behavior and loss of speech, that occurred within the first year following hsct, but neurocognition stabilized thereafter. conclusions: we found a beneficial effect of hsct on the neuropsychological outcome or at least stabilization of neurocognitive function in our patients with a follow-up of eight years. despite low toxicity of the conditioning regimen, increased donor chimerism may further improve the neurological outcome. disclosure: nothing to declare. tandem sct in pediatric solid tumors, other than brain tumors, has no advantage in terms of efs over single procedure-single center experience , germ-cell tumour (gct), ewing sarcoma (es), nefroblastoma. patients were divided into 2 groups according to the number of procedures: 1st group-single sct procedure, 2nd group-multiple procedures. regimens used for stem cell mobilisation were: topo-cy for nbl and epi-tax for gct, followed by g-csf±plerixafor. conditioning regimens: bu-mel and thiotepa-cy for pts with nbl, thio-tax and ice for pts with gct. patients received antibiotic, antiviral and antifungal prophylaxis, parenteral nutrition and supportive treatment. patients received consolidation treatment, followed up monthly in the first year, then yearly. patients were evaluated for residual disease by imaging tests. parents signed informed consent forms. results: we performed 67 sct procedures to 52 patients: 65.3% nbl, 17.3% es, 11.6% gct and 5.8% nefroblastoma. for this study only patients with nbl and gct were considered. in 1st group were 79% of pts, 21% in 2nd group. patients were diagnosed, staged and treated according to international protocols. sex ratio was 18f/34m. age distribution was 1-4 y 38% (20 pts), 4-10 y 35% (18 pts), > 10 y 27% (14 pts). peripheral stem cell (pbsc) mobilisation was more difficult in patients with multiple courses of chemotherapy±radiotherapy. we found no difference in the period of engraftment following a 2nd or 3rd procedure. hospitalization and supportive measures increased in 2nd and 3rd procedures (26 to 29 days). patients with multiple courses of chemotherapy and multiple hospitalizations had increased infectious risk and during the 2nd or 3rd procedures developed various infectious complications.incidence of severe oral mucositis after the first hsct was 17%, after tandem hsct was 69%. nbl patients : 1st group-6/23 patients alive and efs, 1/23 receives anti g2 treatment; 2nd group-1/7 patients-alive, 2/7 patients-not reached timepoint for mibg scan; 4/7 patients-mibg negative at first, relapsed after 6 mo; 1/7 patient deceased due to pulmonary toxicity. gct patients: 1st group-1/5 patients alive and ef, 1/5 high values in afp levels and receives metronomic therapy, 3/5 patients deceased due to progressive disease, but only had 2 sct. only 1/5 patient had one procedure and died due to progressive disease. conclusions: in our study, tandem hsct in children with solid tumours lead to an increase in survival rates, at least in the first 6 months after sct. most patients (90%) had progressive or relapsed/refractory disease when referred to our department. multiple procedures require a higher number of cd34 cells, very hard to achieve in patients with multiple courses of chemo± radiotherapy. new approaches have to be considered in these diseases, especially in high risk group. disclosure: nothing to declare background: antimicrobial prophylaxis for prolonged neutropenia occurred during the pre-engraftment period is a common practice in allogeneic hsct recipients. data on its effectiveness are few and generally from cases series and not from randomized clinical trials, especially in children. methods: all clinical records of allo-hsct performed from january 1 st 2007 to november 30 th 2018 at hsct-unit of istituto g.gaslini, genoa-italy, were retrospectively reviewed. collected data were underlying diseases, type of donor, antibiotic prophylaxis administration and type, development of fever and pathogen isolated from blood culture, if any, during pre-engraftment neutropenia. antibiotic prophylaxis, usually starting together with the conditioning regimen, was categorized in "standard" (with amoxicillin/clavulanate or ampicillin/sulbactam) or "tailored" (when based on previous bacterial isolations or colonizations). results: 246 allo-hscts were performed in 217 pediatric patients (59% males) with a median age at hsct of 8 years (iqr: 4-13; range: 0-22). hscts were performed from alternative donor (ad) in 55% patients, from relative donor (rd) in 25%, and from haploidentical donor in 20%. table 1 shows the pre-engraftment febrile neutropenia episodes according to type of antibiotic prophylaxis. 224 (91%) hscts received standard prophylaxis, while 20 (8%) the tailored one; only in 2 (1%) did not receive any prophylaxis. fever occurred in 194 (87%) of episodes in patients receiving standard prophylaxis, in 19 (95%) of those treated with tailored prophylaxis and 1 (50%) in the group without prophylaxis; only 13% of patients who received prophylaxis did not develop fever.in 38% of patients, the febrile episodes were diagnosed as bloodstream infections: staphylococcus aureus in 2%; cons in 19%; enterococcus spp in 15%; enterobacteriaceae in 25%; pseudomonas aeruginosa in 11%; other non-fermenting gram negatives in 2% and fungi in10%. conclusions: the occurrence of fever in patients who received antibiotic prophylaxis suggested that it could not be effective in prevention of fever related to neutropenia after allo-hsct. the personalization of prophylaxis could be a possible path to follow these patients. disclosure methods: a total of 15 patients with leukemia or neuroblastoma were included in the study. patients' mothers signed an informed consent for participation in the study. six of study participants were boys and 9 girls, all aged 3 to 6 years. the control group consisted of 18 healthy preschool children (2 groups of 9 children aged 5 to 6 years), 8 boys and 10 girls. results: in most of games the role of a doctor was played by a child. only one child declined to impersonate both a patient and a doctor. younger children mostly agreed to have for a "patient" a toy (proposed by psychologist or one of child's own), child's mother or a medical psychologist. the game lasted for 15 -20 minutes. most patients preferred using real medical consumables and instruments (syringes, adhesive tape, winged infusion sets or, more rarely, pills). most often a syringe or an adhesive tape was chosen. as known from their mothers, among medical manipulations most unpleasant for children are injections and changing implanted catheter dressing. also, most healthy preschool children preferred using real medical instruments over toy ones. group 1 more often used a syringe, a winged infusion set, adhesive tape, gauze or pills. group 2 most often chose syringe or gauze. among medical instruments both groups more often chose a phonendoscope or thermometer.one patient refused to cause pain to a "toy patient". other children sympathized with a "toy patient", stroke injection or dressing location sites or used soothing terms ("wait a little", "it's going to be all right"), wished prompt recovery and hugged their "patients". one child was angry over his "patient" wishing him to "get hurt too". first preschool group children were mostly scolding a toy "patient" for "being guilty of getting sick". second group children were mostly compassionate, encouraged a "toy patient" telling that "all the procedures are needed to get healthy". from children's schoolmasters we know that all first group children received vaccination about a week before a test. children from second group had no injections. overall attitude towards toy "patients" was more mild in the second group. conclusions: 1. during a play children mostly use the medical devices which cause them most discomfort and/or pain. 2. manipulating the items children illustrate their own impression of medical procedures, which are most unpleasant. 3. children may express their negative emotions directed towards medical manipulations via their play actions, these negative reactions may be suppressed in different ways by parents or medical staff. 4. the intensity of child's own traumatic experience and an attitude of nearby adults may influence the child's attitude towards other patients. 5. the mother's wish for a child to tolerate all medical procedures with ease exceeds real capabilities of a small child. disclosure: nothing to declare. allogeneic stem cell transplantation in patients with mucopolysaccharidosis type iiia (sanfilippo): a case series methods: allogeneic sct was performed at the ages of 2, 5 and 4 years, respectively. all three patients received intrathecal enzyme replacement therapy within a clinical trial setting prior to hsct. the conditioning regimen consisted of treosulfan, fludarabine, thiotepa and thymoglobuline. gvhd-prophylaxis was carried out with csa and mtx in two patients and csa and mmf in one patient. stem cell source was bone marrow in two patients and peripheral blood stem cells in one patient. results: the conditioning regimen was well tolerated and all three patients successfully engrafted. two of three patients had an uncomplicated course without occurrence of acute or chronic graft-versus-host-disease (gvhd). at last follow-up 12 and 15 months after hsct, both patients are in good condition and show constant progress of psychomotor development. the third patient experienced severe steroid refractory acute gvhd of intestines (stage 4) and skin (stage 3), which resolved under intensive immunosuppression with cyclosporine, mycophenolate and ruxolitinib. around day 110 after hsct, this patient showed clinical and biochemical signs of transplant-associated microangiopathy (tma) with cerebral seizures and acute renal failure. the cerebral mri showed progressive cerebral atrophy and leukoencephalopathy, also consistent with a progress of the mps iiia. at last follow-up 15 months after hsct, this patient had recovered from tma and was in a stable clinical condition. conclusions: in consideration of the small case number and the short follow-up period in our cohort, allogeneic hsct might be considered as a salvage therapy for patients with mps iiia if other therapeutic options are unavailable for children with this otherwise unfavourable prognosis. however, the early psychoneurological course after transplant seems promising compared to the literature and hsct could become a treatment option for this rare disease. disclosure: nothing to declare methods: for the identification of underlying molecular mechanisms leading to the increased sensibility of rms cells, the activation status of different nf-kb signaling pathways were analyzed using western blot analysis and quantitative real time pcr (qpcr). further, flow cytometry was used to analyze the surface expression of death receptors on either sm treated or untreated rms cells. the overall effect on cell death induction was measured by pi/hoechst staining using a fluorescent microscope. results: treatment with sm led to the suspected degradation of iaps. followed by the activation of both the canonical nf-κb signaling pathway, indicated by the phosphorylation of iκbα and p65, and the non-canonical nf-κb signaling pathway, as indicated by the accumulation of nik and the degradation of p100 to p52. determination of selected target gene transcription revealed an upregulation of the inhibitor iκbα, nik, p100, il-8 and at later time points the death receptors trail-r1 and trail-r2. analysis of gene transcription also led to the finding of neither up-nor downregulation of ciap1 and p65. to evaluate the involvement of trail-r1 and trail-r2 in the sm induced sensitization towards nk cell-mediated killing, surface expression of both death receptors was analyzed. treatment with sm led not only to an induced transcription of trail-r1 and trail-r2, but also to an increased surface presentation of trail-r2. subsequent ligation of trail-r2 by either wt-trail or a specific agonistic antibody (etr-2) resulted in a significant increase in cell death induction. the aforementioned analysis of gene transcription hints towards a bimodal feedback mechanism regulating both, the canonical and non-canonical nf-κb signaling pathway. on the one side, the canonical pathway is negatively regulated by the induced transcription of the inhibitor iκbα. on the other side, the induced transcription of nik, p100 and relb points towards a positive feedback loop of the non-canonical pathway. one mechanism of the increased rms cell sensitivity might be the induced transcription and surface presentation of the death receptor trail-r2. the involvement of trail receptors is further validated by the cytotoxicity data, illustrating a sm mediated sensitization towards a trail induced cell death induction. this mode of cell death fits to the previous research, were trail transcription could be induced in nk cells by sm treatment. the graphical abstract shows the transcriptional upregulation of target genes leading to a putative bimodal nf-kb regulation and increased surface presentation of trail-r2 by treatment with smac mimetics. aim: to investigate the outcome of ucb transplantation in pediatric patients with malignant and non malignant diseases methods: data from 30 patients underwent first allogeneic bone marrow transplantation with ucb from 2/1999 until 6/2013 were retrospectively analyzed. eighteen had malignant disease (md), of whom 15 in complete hematologic response, and 12 non malignant disease (nmd) (scid 5, chronic granulomatous disease 1, severe aplastic anemia 2, s.kostmann 1, osteopetrosis 1, wiskott-aldrich1, amegakaryocytic thrombocytopenia 1). the majority of the patients were male, for md and nmd, as well (m:10/f:8, m:8/f:4, respectively), of median age 6.5 years (range 0.8-11.8 years) and 0.8 years (range 0.4-6.5 years), respectively results: all patients but one, received 1 ucb unit. hla compatibility in antigen/allele level was at least 5/6 and only in 3 patients with md was 4/6. conditioning regimens were myeloablative and tbi 12 gy was given in 4/30. gvhd prophylaxis consisted of cyclosporine and atg was given in all patients pre-transplantation. median value of nucleated cells for md was 3.75χ10 7 /kg (range 2-6.3χ10 7 / kg) and for nmd was 11.05χ10 7 /kg (range 2-27.2χ10 7 /kg). neutrophil and platelet engraftment was achieved in 13/18 and 12/18 patients with md respectively, in a median time of 31 days (range 17-44) και 43 days (range 20-60). in patients with nmd, neutrophil and platelet engraftment was achieved in 7/12 and 6/12 with median day of engraftment 21 days (range 18-28) και 29 days (range respectively. acute gvhd grade ιι-ιv presented in 9/18 patients with md and 4/12 with nmd, although none had cgvhd. the incidence of viral infections was 22 cases in 11 patients with md and 9 cases in 6 patients with nmd. disease relapse occured in 9/18 patients with md. after a median time of 10 years follow up, overall survival (os) and event free survival (efs) for children with md were 22% and 18.5% respectively, while for nmd, os and efs were 33%.treatment related mortality at d+100 was 8% for md and 50% for nmd. among 18 patients with md, 4 are still alive, while the rest died from relapse (n: 8), viral infections (n: 4), septicemia (n: 1) and agvhd(n: 1). among 12 patients with nmd, 4 are alive, while the rest died from viral infections (n: 2), septicemia (n:4) and multiple organ failure (n=2). the median time of hospitalization for patients with md was 76 days (range 32-168), whilst for nmd was 63 days (range 28-114). conclusions: transplantation of unrelated ucb in our unit was combined with high trm in children with nmd and higher probability of relapse for md. disclosure: nothing to declare p643 serum levels of 5-s cysteinyldopa is associated with stem cell transplantation related complications yukayo terashita 1 , mamoru honda 1 , minako sugiyama 1 , yuko cho 1 , akihiro iguchi 1 1 hokkaido university hospital, pediatrics, sapporo, japan background: diffuse hyperpigmentation is common in patients who received chemotherapy or stem cell transplantation (sct). however, there are few reports of the relationship between skin reaction such as pigmentation and the other complications. pigmentation of the skin is thought to be the result of melanin stagnating in the dermic layer due to increased synthesis of melanin and destruction of the basement membrane due to inflammation induced chemoradiotherapy. melanin pigments are classified into two types: brown to black eumelanin and yellow to reddishbrown pheomelanin. 5-s cysteinyldopa (5scd) is precursors of pheomelanin, and its serum level has been used specific biochemical marker for malignant melanoma. here, we examined serially 5scd during the course of sct to determine association with sct related complications, because visual evaluation of skin color is difficult, and there have been no reports about 5scd as sct related biomarker. methods: we prospectively analyzed 41(27 males, 14 females) patients who received sct between may 2011 and march 2015 in hokkaido university hospital. the median age at transplantation of the patients was 7.9 years (range, 0-22). indication for sct were acute myelogenous leukemia in 10 patients, acute lymphoblastic leukemia in 9 patients, and other disease in 22 patients; juvenile myelomonocytic leukemia(2), malignant solid tumor(11), immunodeficiency(6), anaplastic anemia(2), and diamond blackfan anemia (1) . 34 patients received allogeneic sct and 7 received autologous sct. myeloablative conditioning (mac) was used for 30 patients and reduced intensity conditioning (ric) was used for 11 patients. sera were obtained from patients before conditioning therapy, on day 0, +5, +10, +15, +25 and +40. all blood samples were centrifuged at 3,000 rpm for 15 min, and stored at -80˚c until used. we also examined sct related complications such as graft-versus-host disease (gvhd), viral infection, and pre-engraft syndrome (pes). statistical analyses were completed using the mann-whitney u test for unpaired samples, and kruskal-wallis test for three samples. each test was performed with a 5% level of significance. results: the average value of 5scd reached two peaks, day0 (21.6 nmol/l) and day5 (21.7 nmol/l), regardless of stem cell source and intensity of conditioning. in all patients, we found that the level of 5scd on day0 was associated with viral reactivation (p=0.049), 5scd on day5 was associated with pes (p=0.034), and 5scd on day40 was associated with malignant disease (p=0.04). similarly, in patient who received allogeneic sct (n=34), the level of 5scd on day0 was associated with viral reactivation (p=0.048), 5scd on day5 was was associated with pes (p=0.034), 5scd on day 40 was associated with malignant disease (p=0.04). in addition, the level of 5scd on day5 was associated with gvhd of skin (p=0.027), the peak level of 5scd was associated with acute gvhd (p=0.04). conclusions: we found that 5scd can be a biomarker for sct-related complications such as aute gvhd. it is presumed that the production of pheomelanin could be induced by inflammatory procedure in sct. disclosure: nothing to declare p644 hsct in children with bone marrow failure: outcomes from a single singapore centre prasad iyer 1 , michaela seng 1 , vijayakumari k 1 , ah moy tan 1 , mei yoke chan 1 , rajat bhattacharyya 1 1 kk women's and children's hospital, paediatric haematology-oncology, singapore, singapore background: children presenting with pancytopenia often present a challenge to the paediatric haematologist. the underlying diagnosis can be hard to establish as many of the inherited bone marrow failure syndromes (ibmfs) can present with protean manifestations. the large majority of patients with bone marrow failure are often diagnosed with idiopathic severe aplastic anaemia (saa) despite extensive testing. we report our experience of hsct in patients treated with primary and acquired bone marrow failure. methods: we reviewed case notes of all the children who underwent hsct for bone marrow failure in our centre. results: a total of fifteen patients underwent eighteen stem cell transplants in our centre between 2003 and 2016. three patients were diagnosed with fanconi anaemia, one with hoyeraal-hreidarsson syndrome, one with paroxysmal nocturnal haemoglobinuria and the remaining ten children had idiopathic saa. eight children had matched sibling donor transplant, 1 had a matched related donor, 1 had a matched unrelated donor, 3 had umbilical cord blood transplants and the remainder 5 were haploidentical transplants. four of the haploidentical transplants were t-cell depleted and one was t-cell replete. one child with fanconi anaemia had primary graft rejection with cord blood transplant and was successfully rescued with a haploidentical transplant. one child with saa had primary graft rejection twice (t-cell depleted graft) and then was rescued with an alternate haploidentical donor with a t-cell replete graft. of the two patients who died, one had a fatal fungal infection ten months after transplant, and the other died due to a severe influenza pneumonitis three and a half years after bmt. conclusions: haematopoietic stem cell transplant outcomes from our centre are comparable to leading centres in the world. the understanding of underlying conditions that present with bone marrow failure has improved our approach and the way we treat bone marrow failure syndromes. clinical trial registry: not applicable. disclosure: nothing to declare. methods: a retrospective study was performed in children treated with hsct who received pos or flu during early neutropenic period until engraftment from january 2000 to december 2017 at siriraj hospital in thailand. the efficacy, safety and tolerability of pos were compared to flu. results: there were 66 hsct recipients (allo-hsct 62.1%, auto-hsct 37.9%) with mean age of 7.6+4.3 years. most of the patients were thalassemia (34.4%) followed by hematologic malignancy (32.2%) and solid tumor (16.7%). seventeen and 49 cases received pos and flu, respectively. all of patients in pos group were allo-hsct whereas 48.9% in flu group were allo-hsct. in pos group, 2 cases were diagnosed with suspected ifi and 2 cases were probable ifi with total 4 cases (23.5%). in flu group, 10 cases were diagnosed with suspected ifi and 2 cases were probable ifi with total 12 cases (24.5%) which compared 2 groups were not statistically significant (p=0.937). no possible and proven ifi in both groups. in flu group patients received empirical antifungal treatment more than pos group but no statistical significance (20.4% vs.11.76%, p=0.498). both groups had similar rate of elevated liver function test (p=0.567). no early discontinuation of antifungal prophylaxis for intolerance was found in both groups. only 26.7% of patients achieved pos target trough level of 0.7 mg/l after 7 days of treatment with started dose 4 mg/kg three times a day. conclusions: pos and flu are comparably effective, safety and tolerability in ifi prophylaxis in neutropenic children treated with hsct. defining dose recommendation of pos in this setting requires larger studies. disclosure background: severe congenital neutropenia (scn) is typically characterized by anc of <500/μl, maturation arrest of bone marrow myeloid precursors at the promyelocyte-myelocyte stage, and susceptibility to lethal pyogenic bacterial and fungal infections. scn is a rare group of disorders resulting from intrinsic defects in myeloid cell proliferation and maturation caused by mutations in several genes; elane, hax1, gfi1, was, and g6pc3 are among the most common ones. almost 10% of patients are refractory to g-csf, and the only definitive curative approach for such patients is allogeneic hsct. the current absolute indications for hsct is failure to respond to g-csf treatment, or the development of mds/leukemia in patients with scn. here, we present the result of 10 children with scn who received allogeneic hsct . methods: we retrospectively assessed 10 allogeneic hsct in children with severe congenital neutropenia. all patients received busulphan (bu) based myeloablative conditioning regimen. busulphan was used according to weight adjusted dose. in addition, all patients received fludarabine 150 mg/m2 in five days or cyclophosphamide 200 mg/kg in 4 days and atg 30 mg/kg in 3 days. cyclosporin-a and mtx were used for graft versus host disease (gvhd) prophylaxis. donor chimerism was evaluated in either bone marrow or peripheral blood on days +30, +100 and +180. results: the median transplantation age of the patients was 49 months (range 11-167 months). six of them are male. two of the donors were matched siblings and 8 were unrelated two of which were 1 ag 1 ag mismatched. stem cell source was bone marrow in 6 patients, peripheral blood in 2 and cord blood in 2 patients. all patients engrafted. the median time of neutrophil and platelet engratment to was 15 (13-34) days and 16(9-90) days, respectively. graft rejection was experienced in 2 patients, one of them had received unrelated cord blood. all patients are alive, eight of which are with full donor chimerism (between 95-100 %) without any complication (no infection, no gvhd) with a median 40 months (range 24-83 months) follow up. probability of disease free and overall survival were found 80% and 100%, respectively. conclusions: we concluded that hsct is a useful treatment for scn patients, especially those who are unresponsive to gcsf treatment and at high risk for leukemic transformation. however, a larger number of scn patients and longer follow-up are necessary to identify appropriate conditioning regimens and long-term prognosis. disclosure: nothing to declare background: prolonged thrombocytopenia (pt) or secondary failure of platelet recovery (sfpr) are a lifethreatening complications that occurs in 20-40% and 12-20% respectively of the patients following allogeneic hematopoietic stem cell transplantation (allo-hsct). management strategies, including the use of growth factors, cd34+-selected stem cell boost, mesenchymal stem cell (msc) transfusion, and second allo-hsct, are not effective or possible for all patients. eltrombopag, is an oral non-peptide thrombopoietin receptor agonist, that leads to signal transduction and results in promoting the proliferation and differentiation of megakaryocytes. some recently studies show that also can promote haematopoiesis along all three lineages. methods: we described our experience in 4 paediatric patients with poor graft function or secondary failure of platelet recovery after allogeneic stem cell transplantation treated with eltrombopag. results: patients characteristics are detailed in table 1 . all the patients received and allo-hst. the median dose of cd34+ cells infusion was 6.43x10e6/kg (3.95-8.29 ). neutrophils engraftment occurred in +15 day (10-21d) and platelets in +26 day (16-42d). all the patients had an hypoplastic bone marrow with complete chimerism. the median duration from transplantation to spcf diagnosis was 10 months (1.5-24m) . one of the patients received a stem cell boost prior to eltrombopag, without response. the time onset from spgf/sfpr diagnosis to initiating eltrombopag was 16 days (8-32d). eltrombopag was started at a dose of 1mg/kg/d, requiring an increase dose in all cases. the median dose was 50 mg/d (25-100mg). the overall response rate was 50% (2/4). two patients achieved complete response (cr), as defined by platelet ≥ 50 × 109/l. both patients already got neutrophil ≥ 1.0 × 109/l without g-csf. the time from eltrombopag initiation to achieving cr was 21 (10-49d) days. the treatment was given for a median of 81 days (8-203). it was discontinued after 96 and 203 days respectively in the two responder patients. both patients maintain stable blood counts after discontinuing the treatment. the non-responders patients had to stop the treatment because of other reasons not related to eltrombopag. patient 4 had to be rescued with a cd34+ cells boost with a good response. two patients that were in treatment with voriconazole for a fungal infection developed hyperbilirubinemia. there were no grade 3-4 toxicities related to eltrombopag. conclusions: in our experience, according to recently published studies, eltrombopag is a safe and efficacy drug in the treatment of secondary failure of platelet recovery post-hsct. it may be used successfully in children. sometimes higher doses may be considered if no response is achieved. further prospective trials are needed to increase the level of evidence and to identify predictors of response. disclosure: nothing to declare very slow clearance of busulfan in a child with infant leukemia background: busulfan is a drug with a high interindividual variability between dose and exposition. therefore, it is recommended to perform therapeutic drug monitoring (tdm) in the context of myeloablative conditioning, especially in children. methods: we report on a 7-month old boy (7.2 kg, 66 cm) of caucasian decent born to non-consanguine parents with mll-rearranged prob-lymphoblastic leukemia. diagnosis was established one month after birth from peripheral blood and csf tap showed cns involvement. primary chemotherapy was commenced according to the interfant-06 protocol. however, mrd remained positive two months under treatment, leading to an indication for allogeneic stem cell transplantation. in the interfant-06 protocol, we opted for a conditioning regimen comprised of fludarabine (1.2 mg/kg for 5 days), busulfan and thiotepa (2x5 mg/kg). in our institution, busulfan is applied once daily with a target auc of 85-95 h*mg/l in this very high risk situation. according to body weight, busulfan was given with 5.1 mg/ kg as a three-hour infusion on the first day. busulfan concentrations in plasma were measured with gas chromatography-mass spectrometry (gc-ms) and auc was calculated using bayesian curve fitting. results: exact busulfan quantification was not possible after the first dose due to technical reasons. as the levels were estimated to be very high, we decided to reduce the second dose of busulfan by 25%. this resulted in a very high auc of 44 h*mg/l for the second dose, so that busulfan was discontinued after two days, because it was calculated that the patient already received busulfan with a cumulative auc of 90 h*mg/ml. trough levels after the first and second dose were 547 and 572 μg/l, respectively. the patient showed a very slow clearance of 2.1 l/h/sqm, while the volume of distribution was in the usual range (0.86 l/kg). bilirubin and liver transaminases were in the normal range at the time of conditioning, while albumin and quick were decreased on day +18 after transplantation the patient developed clinical und biochemical signs of venoocclusive disease (vod). vod symptoms completely resolved under therapy with defibrotide. leukocyte engraftment was established on day +14. unfortunately, the patients suffered from an early relapse of the leukemia from day +62. attempts to induce a second remission with blinatumomab failed. the patient is currently under palliative chemotherapy. conclusions: busulfan tdm is very important especially in infants receiving myeloablative doses of busulfan to prevent under-or over-exposure. there is evidence that high busulfan trough levels contribute to the development of vod, but anti-leukemic activity of busulfan and cns permeability make it a valuable drug for very high risk patients in childhood leukemia. larger patient cohorts are needed to assess the exposure dependent risks of toxicity versus relapse in infants and toddlers. disclosure: nothing to declare blood (ucb) obtained at delivery from three children who received a diagnosis of cerebral palsy. methods: immunophenotyping of the ucb leukocyte fraction was performed using multicolor flow cytometry. the procedure was performed according to the protocol by shatorje and colleagues (1) . briefly, the ucb samples were labeled with specific antibodies and incubated in the dark for 30 minutes. afterwards, the samples were treated with 5ml of bd facs lysing solution for 10 minutes to preserve the leukocyte fraction only. the cells were washed using pbs (roche) and then centrifuged twice (1500 rpm, 4°c, 4 minutes). the results were analyzed using the facsdiva software (becton dickinson). results: we found an increased white blood cell (wbc) count, lymphocyte count, and cd4:cd8 ratio in all ucb samples. one patient had a low nk cell count and percentage, and another had a low b-cell count and percentage. one sample displayed high t (cd3+) and th cell (cd4+) counts, but with percentages within the limits of the reference values. conclusions: we detected elevated wbc and lymphocyte counts in all ucb samples, despite a lack of intrauterine infection symptoms. many authors have described the pathogenesis of hypoxic-ischemic encephalopathy. briefly, after an acute hypoxia-ischemia insult, activated resting microglia show macrophage-like activity. this leads to a break-down of the blood-brain barrier, infiltration by peripheral leukocytes, and brain exposure, which further exacerbates inflammation. the role of systemic inflammation is being evaluated in the animal model. it is known that systemic inflammation plays a role in traumatic brain injury (tbi) and is an independent risk factor for poor outcome in isolated tbi patients. on the other hand, m2-phenotype microglia inhibit inflammation and protect neurons from secondary damage and death. however, anti-inflammatory mechanisms in neonates are immature and expose them to extremely intensive inflammation. therefore, anti-inflammatory agents, including stem cells, may be beneficial in these patients. disclosure: three of four authors are employees of the polish stem cell bank, warsaw, poland reference: background: under the hypothesis that early natural killer cell infusion (nki) following haploidentical stem cell transplantation (haplo-sct) will reduce relapse in the early post-transplant period, we conducted a pilot study to evaluate the safety and feasibility of nki following haplo-sct in children with recurrent neuroblastoma who failed previous tandem high-dose chemotherapy and autologous sct. methods: we used the high-dose 131 i-metaiodo benzylguanidine and cyclophosphamide/fludarabine/antithymocyte globulin regimen for conditioning and infused 3×10 7 /kg of ex-vivo expanded nk cells derived from a haploidentical parent donor on days 2, 9, and 16 posttransplant. results: seven children received a total of 19 nkis, and nki-related acute toxicities were fever (n = 4) followed by chills (n = 3) and hypertension (n = 3); all toxicities were tolerable. grade ≥ii acute gvhd and chronic gvhd developed in two and five patients, respectively. higher amount of nk cell population were detected in peripheral blood until 60 days post-transplant compared with reference cohort. cytomegalovirus and bk virus reactivation occurred in all patients and epstein-barr virus in six patients. six patients died of relapse/progression (n = 5) or treatment-related mortality (n = 1), and one patient remained alive. conclusions: nki following haplo-sct was relatively safe and feasible in patients with recurrent neuroblastoma. further studies to enhance the graft-versus-tumor effect without increasing gvhd are needed. disclosure: nothing to declare regenerative medicine p652 repeated administration of g-csf using stem cell mobilization protocol could induce improvement of cognitive functions of children with cerebral palsy: phase ii randomized placebo-controlled study background: we performed phase ii randomized placebocontrolled clinical study to reveal the safety and feasibility of repeated granulocyte colony-stimulating factor (g-csf) administration for improvement of cognitive functions of children with cerebral palsy. methods: forty-four children with non-severe type of cerebral palsy were enrolled, and their age were 2-10 years old. g-csf (5μg/kg) was administered for 5 days subcutaneously every 3months during 18 months. we compared their cognitive functions with the magnetic resonance imaging (mri) findings and the following tools between before and 18 months after treatment; zoo location and picture memory as working memory index (wmi) in wechsler preschool and primary scale of intelligence (wpssi), receptive and expressive vocabulary test (revt), and visual motor integration (vmi)/visual perception (vp) test. mobilized stem cell count and cytokine levels were measured before (d+0) and after (d+5) g-csf administration for 5 days every 3 months. results: no significant findings in demography was noticed between g-csf (g-) and placebo (p-) groups. no serious adverse events were observed during the whole study period. the non-severe adverse events such as urticaria (n=1), itching sense (n=3), bone pain (n=1), headache (n=1), fever (n=2), and stomatitis (n=1) were tolerable. the parents felt the clinical improvements of cognition in 10 cases of g-group and 4 cases of p-group (p=0.0367), of language in 8 cases of g-group and 3 cases of p-group (p=0.0632). in zoo location test, we can not find out the significant score (expressed as age equivalent) differences between g-and p-groups. however, in picture memory test, there were significant improvement of age equivalent of 10 months (45.60±19.66→55.60±23.27) during 18 months of study period in g-group compared to 3 months in p-group (p=0.0242). in revt, there were significant improvement of 18 months of age equivalent in expressive tests of g-group (57.87±33.66→76.00±43.38, p=0.0198) compared to 8 months in p-group. no significant findings were noted in receptive test. vmi test showed the increasing tendency of 14 months of age equivalent in g-group (47.75±13.22→61.13±17.78, p=0.0746) compared to p-group. the increment of cd34 + cell counts in peripheral blood were significant in ggroup compared to p-group. the changed levels of interleukin (il)-6, il-10, vascular endothelial growth factor (vegf) as well as g-csf were noted in g-group. we also observed the correlation of cognitive function tests and white matter connectoms of several networks using functionally-defined white matter atlases. conclusions: the repeated administration of g-csf using stem cell mobilization protocol is safe and feasible to improve the language and cognitive functions in children with cerebral palsy. further studies for cellular and paracrine effect of g-csf and/or mobilized peripheral blood stem cells would be needed. background: while high-dose chemotherapy (hdct) with autologous hematopoietic stem cell transplantation (auto-hsct) is an integral part of multimodal therapy for highrisk neuroblastoma (hr nb), there are still subgroups, in which the results are extremely poor. for these patients allografting (allo-hsct) may offer some hope. methods: we summarize the experience of 83 consecutive hr nb patients receiving therapy in our pediatric transplant department in 2008-2017. the median age was 4 years (8 months to 22 years). a total of 78 auto-hscts and 20 allo-hscts were performed. all auto-hsct recipients were characterized by one or several high-risk features: age of more than 18 months at disease (onset n=56), primary disseminated disease (n=59), unfavorable biologic variant (n=29), poor 1 st -line therapy response (n=16) or systemic relapse (n=9). most patients (n=75) received bu-mel hdct (in younger patients oral busulfan was replaced by busilvex), in 3 primary resistant cases a 5d/5d regimen was used. a total of 20 patients with 1st (n=11) or 2nd (n=2) chemosensitive relapse, resistant relapse (n=4) or poor mobilizers with locally advanced resistant tumor (n=3) received allo-hsct from haploidentical donor with fludarabine-based ric. in 12 cases the transplant was modified via immunomagnetic positive or negative selection, 8 patients received post-transplant cyclophosphamide (post-cy)-based gvhd prophylaxis. gvhd prophylaxis also consisted of calceneurin inhibitors and sirolimus. thirteen of 20 allo-hsct recipients received posttransplant immunoadoptive (n=6) or targeted (n=7) therapy. results: the 5-year os and efs in auto-hsct recipients was 48% and 36%, accordingly. all but one patient engrafted with a median time of 17 (11-51) days. bu-mel regimen was characterized by acceptable toxicity with most common toxicities being oral mucositis and infectious complications. the vod/sos incidence was only 3%. four patients dies due to infection (n=2), cns hemorrhage (n=1), and secondary leukemia (n=1). according to multivariate analysis the most important prognostic factors were response to 1 st line therapy and post auto-hsct mibg scan results. the prognosis in initially resistant patients with good response to 2 nd or 3 rd -line therapy was still very poor (all patients relapsed with the median efs of 12 months). all patients receiving a second auto-hsct after relapse died due to disease progression. with a median follow up of 6 (1-80) months 9 allo-hsct recipients are alive, 6 of them with no signs of disease progression. all long-term responders received post-transplant therapy. one patient died due to transplant complications, other deaths were caused by disease progression. there was no obvious difference between outcomes in post-cy based and transplant modification-based transplantations. agvhd more often developed in modified transplant recipients (8 patients vs 2 in post-cy group, 4 of these cases gr iii-iv), 3 patients in post-cy group had grade iii-iv hemorrhagic cystitis. the median time to engraftment was longer for ptcm group compared to transplant modification group (d +23 vs. d+17, accordingly). conclusions: while single hsct with auto-hsct is a golden standard in hr nb patients, the relapse rate is still high and the prognosis in relapsed/refractory patients is dismal. the allografting has some limited effectiveness in these cases and post-transplant therapy has a potential for further improvement. disclosure: nothing to declare p654 abstract already published. veno-occlusive liver disease (vod) is frequent but well treatable with early defibrotide administration in children with neuroblastoma receiving high-dose busulfan and melphalan background: using high-dose intravenous busulfan and melphalan (bumel) prior to autologous stem cell transplantation (sct) in children with high-risk neuroblastoma, seems to decrease toxicity of the myeloablative regimen, except for vod. in this multicenter retrospective study we aimed to assess the outcome of bumel-associated vod with early defibrotide treatment intervention. methods: we retrospectively analyzed 64 children with high-risk neuroblastoma who underwent autologous sct with i.v. bumel regimen in slovakia and prague, czech republic in the period 1/2008 -10/2018. busulfan was administered in q6 hour schedule, with therapeutic drug level monitoring in 84% of patients. all vod patients except one were treated with defibrotide starting at a standard dose of 25 mg/kg/day, given in 4 doses per day. 1 patient was treated with supportive therapy only. ursodeoxycholic acid was used as prophylaxis in all patients. vod was established using the modified seattle clinical criteria (corbacioglu, lancet 2012). results: the incidence of vod was 23% (15/64) in patients treated with intravenous busulfan and melphalan. there was no significant difference in busulfan total dose/kg between patients with (19.2 mg/kg (sd=1,8)) and without (18.0 mg/kg (sd=2,4)) vod manifestation. vod developed at a median of 17 days after sct (range 11 -22 days). anicteric forms of vod were documented, although 73% patients with vod (11/15) presented with increased bilirubin. 73% patients with vod (11/15) developed ascites but only 4 patients (27%) required ascites drainage. no vod patient received renal replacement therapy and only one needed mechanical ventilation. importantly, we successfully treated vod in all patients. relapse or progression of neuroblastoma was the cause of death in 4 vod patients (27%) who died. conclusions: despite targeting busulfan levels to decrease toxicity of the regimen, vod is common (we observed vod incidence exactly in the range of the siopen hr nbl-1 multicenter study (ladenstein, 2017) ). early recognition and early treatment with defibrotide seems to be effective in vod associated with bumel regimen -none of our 15 patients died due to vod. disclosure: nothing to declare results of high-dose chemotherapy (hdct) with autologous hematopoietic stem cell transplantation (auto-hsct) in the treatment of ewing sarcoma family tumors (esft) background: while current dose-intense treatment protocols allow achieving 70-80% survival in localized esft patients, the long-term survival in high-risk cases is still unsatisfactory. although there is a considerable body of data on high-dose consolidation the real effectiveness and optimal indications for this option are still not completely clarified. therefore, a large prospective cohort analysis may still yield useful data. methods: the whole cohort includes 73 consecutive highrisk esft patients with median age of 12 (range 1-23) years receiving hdct with auto-hsct in 2007 to 2018 after obtaining 1 st (n=25) or 2 nd (n=3) cr, pr (n=35), or stable disease (n=10). the high-risk features included lung (n=52), bone (n=25) or bone marrow (n=11) involvement, inadequate local control in primary axial tumors (n=52), large lesions volume or poor treatment response (n=52), and chemosensitive relapse (n=9). most patients had several risk factors. disseminated disease patients were also evaluated according to prognostic score by r.ladenstein et al. highdose busulfan-melphalan followed by autologous stem-cell transplantation (hdt/sct) was used. results: the median observation time was 60 (range 3-130) months. the 5-year overall (os) and event-free (efs) survival were 40% and 37%, accordingly. most important outcome predictors were inadequate local control in chemoresistant cases, a primary tumor volume more than 200 ml, more than one bone metastatic site, bone marrow involvement and additional lung metastases. according to prognostic risk score in disseminated disease esft patients identified three groups with 5-year os rates of 49% for score ≤3 (19 patients), 36% for score 3 to 5 (27 patients), and 8% for score ≥5 (12 patients), (p< 0.01). conclusions: while bu-mel hdct with auto-hsct may still be a feasible option with acceptable toxicity for chemosensitive patients with inadequate local control and some primarily disseminated cases it is ineffective in primary resistant or very high-risk metastatic patients. disclosure: nothing to declare efficacy of tandem high-dose chemotherapy with autologous hematopoietic stem cell transplantation in the treatment of infant embryonal brain tumors day +15 (range, 8-83), after the second auto-hsct was day +17 (range, 9-86). two-year overall survival (os) was 76% and disease free survival (dfs) was 68%. dfs was significantly better among patients with mb (95%) and pnet (75%) in compared to children with etmr (55%), pb (33%) and atrt (0%), (p=0,01). dfs in patients who received tandem auto-hsct was 75% in compare to infants who received only one auto-hsct (44%), (p=0,000). complications grade 4 (according to common toxicity criteria 2014) were observed in 14% of cases. conclusions: employment of tandem hdct with auto-hsct in primary infant embryonal brain tumors may be a feasible option for patients after induction treatment. both conditioning regimens had acceptable toxicity. all patients who had tandem hdct with auto-hsct had better os (75%) in compare with single hdct (44%). patients with mb and pnet had better prognosis with os 95% and 75%, respectively, in compare with other embryonal tumors. disclosure: nothing to declare background: metastatic extra ocular retinoblastoma is carrying a poor prognosis. therapeutic intensification with high-dose, marrow-ablative chemotherapy and autologous hsct has been explored, but its role is not yet clear. this study aimed to evaluate the survival outcome of patients with extraocular retinoblastoma post autologous stem cell transplant, treated at single center methods: this is a retrospective study included all patients with metastatic extraocular retinoblastoma (stages 4a and 4b) that underwent autologous hsct at the children cancer hospital egypt (cche) 57357 from november 2010 to july 2017, the treatment protocol was adopted from cog protocol (aret0321) as all patients received 4 cycles induction chemotherapy followed by consolidation myloablative conditioning, cem (vp16 416.6 mg/m2 x3, melphalan: 40 mg/m2 x4, carboplatin: 500 mg/m2 x3) and stem cell rescue. patients data including initial disease characteristics, transplant data, and survival outcomes were collected and analyzed results: a total of 11 cases were included with median age of 1.7 years, and male to female ratio 2.66. nine patients (81%) were initially presented by extra ocular disease, while 2 patients were presented by intra ocular disease and progressed to metastatic disease. according to cog staging of extra ocular disease, 4 patients had stage 4a, and 7 were stage 4b (5 of them had trilateral disease). after induction therapy, 7(63%) showed complete response and 4 (36%) had ≥ partial response. with average cd34 count of 4x10 6 / kg, the median time to anc and platelet engraftment were 10 days and 19 days respectively, and there was no transplant related mortality. post-transplant radiotherapy was given only to 2 patients. with median duration of follow up of 32 months, the overall and event free survival rates of whole patients were 88.9% and 87.5% respectively conclusions: high dose chemotherapy and stem cell transplantation is a promising potential curative option for patients with metastatic extra ocular only two primary gf (1.4%) occurred, both without dsa. 20 patients developed a primary pgf (15%). 3-years os, 3years pfs and 1-year nrm were analyzed according to the presence of dsa in comparison with negative population. no statistically difference was found. no impact of the presence of dsa on the risk of developing gf and pgf was revealed. major outcomes of transplant was analyzed separately in patients with pgf and good graft function (ggf). 3-years os, 3-years pfs and 1year-nrm in ggf and primary pgf populations were 62% vs 20% (p< 0.0001); 53% vs 20% (p< 0.001), 12% vs 40% (p=0.009), respectively. conclusions: the presence of low level of dsa in the absence of desensitization doesn't correlate with the risk of developing gf and pgf. patients who experienced a pgf had worse outcomes in comparison with patients with ggf. disclosure: nothing to declare. the impact of hla-dpb1 mismatch in t-cell replete unrelated donor allogeneic stem cell transplantation background: high resolution matching of donor-recipient hla improves outcome in allogeneic stem cell transplants. matching for hla-a, -b, -c, -drb1 and -dq is mandatory in our transplant centre, to identify 10/10 or 9/10 matched unrelated donors. high resolution matching for dpb1 has been added over the last 10-15 years. however, the role of dpb1 matching is not yet clearly defined. methods: in this study, we retrospectively analyzed the impact of hla-dpb1 matching on the outcome of t-cell replete allogeneic hematopoietic stem cell transplants with cya/mtx-and without atg as gvhd prophylaxis in patients with hematological malignancies at oslo university hospital between 2005 and 2017. 301 patients with an unrelated donor fully matched (10/10) at hla-a, -b, -c, -drb1 and -dqb1 loci were included. further, 87 patientrecipient pairs were also fully matched on dpb1 (12/12); 118 had permissive and 96 had non-permissive mimatches of one or two dpb1 alleles. the three groups were comparable with respect to diagnosis, gender, age, cytomegalovirus serostatus and conditioning regimen. results: cumulative incidence of relapse at 5 years were significantly higher in the dpb1 matched pairs compared with the permissive and non-permissive mimatched ones, at 40% vs 22% and 13% (p< 0.001) respectively. relpase free survival and overall survival were superior in the nonpermissive and permissive dbp1 mismatched groups vs the fully matched, at 60% and 49% vs 29% (p=0.01) and 59% and 53% vs 40% (p=0.09) respectively. no difference in frequency of acute gvhd grade ii-iv between the three groups were found; dp match 43%, permissive mismatch 40% and non-permissive mismatched 48% (p=0.49). neither was there a difference seen in gvhd grade iii-iv; 12% vs 17% vs 11%, respectively. finally, there were similar outcomes between the three groups regarding chronic gvhd and trm. in corrected multivariate analysis, only dp matching had significant influence on mortality and survival. conclusions: our results show a favorable relapse free and overall survival following a mud allotransplant with a dpb1 permissive or non-permissive mismatched donor compared to a fully dpb1 matched. this is likely due to an increased gvl-effect in dpb1 mismatched groups without the counterbalance of increased acute gvhd and trm. disclosure: nothing to declare p662 a haploidentical may be a better choice than a female genoidentical donor to transplant a patient with high risk acute myelogenous leukemia in first remission norbert gorin 1 , myriam labopin 1 , didier blaise 2 , goda choi 3 , gerard socie 4 , jean henri bourhis 5 , fabio ciceri 6 , emmanuelle polge 7 , arnon nagler 8 , mohamad mohty 1 china, 3 the first affiliated hospital of soochow university, hematology, suzhou, china background: despite the incidence of leukemia increases with age, currently the geriatric population is poorly represented in the standards of care concerning that older adults undergoing hematopoietic cell transplant (hct) may experience higher transplant-related mortality (trm). previous studies have demonstrated that donor age is vital for older patients by affecting trm and survival. accordingly a relevant question is whether outcomes can be improved with a younger hla-haploidentical offspring donor rather than an older hla-matched sibling (msd). in our previous multi-center report under atg+g-csf based protocol for haplo hct, offspring donor is correlated with lower trm and higher leukemia free survival (lfs) as compared with older msd in subgroup analysis for recipients >50years although it did not reach statistical significance. on the contrary, in a recent report from ebmt and cibmtr under ptcy modality for haplo hct, among patients aged 55 to 76 years, despite lower chronic graft-versus-host-disease (gvhd), graft failure, trm, and overall mortality were higher after transplant from offspring compared with an msd although there were differences in transplant platforms between the 2 groups. methods: we extended our multi-center dataset and a matched pair analysis was performed. outcomes of 142 acute leukemia patients (>=50 years) transplanted in cr1/ cr2 who received hct from offspring (n=57) or msd (n=85) between jan, 2013 and june, 2017 present in the multi-center database were analyzed. because the patient population was small, a 1:1 ratio matched pair analysis was implemented with the following matching factors: underlying disease (acute myeloid leukemia, acute lymphoblastic leukemia), disease status (cr1/cr2), age and sex of patients, year of transplant, blood group incompatibilities, and sex of donor. results: we were able to match 41 offspring with 41 msd patients. the two matched groups were comparable in baseline characteristics except for donor age due to the family relationship. all patients achieved myeloid recovery with a median time of 14d and 12d for msd cohort and offspring group (p=0.002). the 100d platelet recovery rate was 95% in both groups. the cumulative incidence of grade ii-iv acute gvhd in msd cohort was significantly lower than in offspring group (12% vs 37%, p=0.009) while the incidence of chronic gvhd in msd cohort was significantly higher than in offspring group (51% vs 27%, p=0.013). the 3-year trm (10% vs 31%, p=0.028) were significantly lower in offspring-hct compared with in msd-hct and relapse incidence was comparable (8% vs 14%, p=0.56). as a result, the 3-year overall survival (57% vs 82%, p=0.033) and lfs (55% vs 82%, p=0.024) ( figure 1 ) were significantly higher in offspring-hct compared with in msd-hct. in a multivariate analysis, msd-hct remained a significant factor for decreased overall survival (hr 2.791(1.141-6.824), p=0.024) by increased trm ), p= 0.032) in comparison with offspring-hct. conclusions: these data favor a young offspring over an older msd in patients >50 years. the current analyses confirm non-hla donor characteristics, rather than hla disparity, predominantly influence survival in older acute leukemia patients. validation of these findings requires a prospective trial wherein the transplant platforms can be closely matched. [[p664 image] 1. figure1. lfs in offspring-hct compared with in msd-hct (55% vs 82%, p=0.024)] disclosure: nothing to declare. impact of sibling donor-recipient sex combinations on rejection after hla-matched bone marrow transplantation for severe thalassemia 1 cure2children foundation, florence, italy, 2 sankalp india foundation, bangalore, india, 3 people tree hospitals, bangalore, india, 4 south east asia institute for thalassemia, jaipur, india, 5 pakistan institute of medical sciences, islamabad, pakistan, 6 central asiri hospital, colombo, sri lanka, 7 nawaloka hospital, colombo, sri lanka, 8 kokilaben dhirubhani ambani hospital, mumbai, india background: severe thalassemia (st), i.e. a thalassemia syndrome with inability to keep spontaneous hemoglobin > 7 g/dl, is a common indication for bone marrow transplantation (bmt) in children in the middle east and south east asia. sex mismatch has been associated with increased risk of solid organ rejection but is not generally considered an important transplant-associated risk factor in the context of fully matched sibling bmt for st. methods: a total of 154 consecutive sibling bone marrow transplants carried out between january 2009 and april 2017 after conditioning with busulfan (14 mg/kg oral, not adjusted to serum levels) and cyclophosphamide (200 mg/kg) (2 patients) in addition to either thiotepa (10 mg/kg) (49 patients), or anti-thymocyte globulin (genzyme 4 mg/ kg or fresenius 16 mg/kg on days -12 to -10) (92 patients) and fludarabine 150 mg/m 2 (11 patients) were analysed. all cases received cyclosporine and methotrexate or mycophenolate mofetil as gvhd/rejection prophylaxis. in the thiotepa group methylprednisolone at 0.5 mg/kg/day was also used during the first 30 days after bmt (lucarelli protocol 6i). bone marrow was the source of hematopoietic stem cells in all cases, in the atg group it was g-csfprimed (5 μg/kg/dose twice daily for 3 to 5 days prior to harvest). all patients were considered low risk based on liver size < 2 cm from costal margin and age less than 15 years (median 4.2 years, range 0.9 to 14.5), all sibling pairs where hla-compatible. results: [[p665 image] 1. sibling donor-recipient sex combinations.] the lowest rejection rate (5%) was observed in the sister to sister (s2s) group of 24 cases, followed by brother to brother (b2b) group of 40 cases with 11%. in the sister to brother (s2b) group of 59 cases, rejection rate was 21%, and 26% in the brother to sister (b2s) group of 31 cases. on univariate analysis the only significant difference at the p 0.05 level by log rank test was b2s vs. s2s groups (rejection proportions of 26% and 5% respectively). interestingly, all 3 patients with rejection and persistent pancytopenia were female recipients of male grafts. conclusions: even though several preparative regimens were employed over an 8-year period, our data suggests that sex mismatch among compatible siblings should be considered as a relevant variable related to bmt decisionmaking. we also recommend to consider autologous back up hematopoietic stem cell collection and storage in sibling sex mismatched transplants, particularly in brother to sister bmts. same-sex fully matched related bmt for severe thalassemia might be the best scenario in which reducedintensity preparation strategies aiming at maximizing fertility preservation might be explored. disclosure: nothing to declare. outcomes of t-cell replete hematopoietic cell transplantation from mismatched related or unrelated donors using high dose post-transplant cyclophosphamide based gvhd prophylaxis background: high dose post-transplant cyclophosphamide (ptcy) based gvhd prophylaxis overcomes immunological barriers in hla mismatched donor transplantation. ptcy has been adopted in many centers as de facto standard for hct from haploidentical donors (haplo hct). it's use in mismatched unrelated donor transplant (mmud hct) is less well established. methods: we analyzed retrospectively outcomes of contemporary cohorts of patients who underwent haplo hct or mmud hct using ptcy + cyclosporine (csa) and mycophenolate mofetil (mmf) at our center. we compared these outcomes with outcomes of cohorts of patients who underwent hct from matched unrelated donors (mud) using atg based gvhd prophylaxis or matched sibling donor (msd) with csa and mmf. patients and donors were considered matched if they background: hla-alloantibodies are a major risk factor for engraftment failure in allogeneic hematopoietic stem cell transplantation (hsct). particularly, complement fixing, donor specific antibodies were shown to be associated with early engraftment failure. prospective antibody-screening, although not currently required for donor search, could permit early identification of high risk patients for positive crossmatch. aim of this study is to set the basis for future applicability of antibody-screening-based definition of acceptable mismatches in donor selection, by creating a large prospective antibody-screening database of patients due to receive an hla-mismatched allogeneic hsct. methods: patients (n=2106) diagnosed with mds/mps, nhl, mm, cll, cml, anaemia (aplastic anemia, hemoglobinopathies, pnh) and hl were prospectively screened for hla-antibodies whenever initial donor search indicated that no completely matched donor would be available. screening was performed with an elisa class i +ii screening assay. all positive screening cases were tested for antigen-specific antibody identification with luminex sab, and acceptable mismatches were defined. the results were subsequently considered in donor search and selection. we now report the frequencies of alloimmunization observed in these patients. results: the highest rate of alloimmunization was observed in patients from the anaemia disease group (overall 40.5%) followed by those from the mds/mpn group (overall 24.8%). the lowest immunization rates were observed in cll (overall 6.9%) and hl (7.0%) patients. alloimmunization rates for hla-class i antigens (p< 0.001) were significantly higher compared to hla-class ii antigens. overall hla-class i immunization rates ranged from 2.3% to 33.3%. hla-class ii immunization rates ranged from 4.7% to 19.8% (table 1) . conclusions: our findings suggest that patients with high transfusion burden like anaemia and mds/mpn patients have the highest risk of hla-alloimmunization with 40.5% and 24.8% anti-hla prevalence rates, respectively. analysis of follow-up data, will enable us to confirm whether prospective definition and consideration of acceptable mismatches in donor selection may lead to similar engraftment failure rates between immunized and non-immunized patients undergoing hlamismatched hsct. background: mothers displaying a persistent fetal microchimerism (fm) proved to be the most suitable donor in t cell-depleted haploidentical stem cell transplantation (hhsct) in children. we presumed that fetal cells leave an imprint in the mothers' immune system which positively affects recognition and elimination of malignant cells in the child by maternal effector cells. distinct killer cell immunoglobulin-like receptors (kir)/hla constellations are not only associated with reduced relapse rates after hsct in children, but also supposedly influence the establishment of an fm. methods: after approval by the local irb and obtaining informed consent, we initiated a protocol to elucidate the factors that influence the establishment, persistence and effect of fm. we established a digital droplet pcr (ddpcr) protocol to determine the fetal microchimerism. for differentiation between maternal and fetal cells, biallelic short insertion/deletion polymorphisms were used. kir and hla-c genotyping was performed by ssp-pcr. parental nk cell alloreactivity against the respective leukemic blasts and kir phenotyping were analyzed by flow cytometry. results: we analyzed 45 parents, whose children were treated for hematological diseases at the university medical center hamburg-eppendorf. a fetal microchimerism was detected in 25% of the mothers. the amount of fetal cells varies between individuals (8x 10 -6 -9x 10 -4 ). we observed a positive correlation between a persisting fm and hla-c1 homo-and heterozygous mothers along with a maternal cen a/b and cen b/b genotype. additionally, fm positive mothers showed a higher surface expression of the hla-c1 respective receptors kir2dl2/s2. the percentage of alloreactive maternal nk cells against fetal cells was higher compared to paternal nk cells; while alloreactivity of fm positive maternal nk cells was similar to nk cells from fm negative mothers. conclusions: persistence of fm was more frequent in mothers carrying at least one hla-c1 allele and a centromeric b/x motif. phenotypically, fm positive mothers had higher expression of kir2dl2/s2 indicating a role of these receptors on the persistence of an fm. in vitro, maternal nk cells showed a higher alloreactivity compared to paternal nk cells. there was no difference in alloreactivity whether the mothers were fm positive or negative, suggesting other mechanisms are responsible for the superior outcome in transplantation from fm positive mothers. disclosure: nothing to declare background: although there have been significant improvements with conventional therapies in beta thalassemia major, hematopoietic stem cell transplantation is only curative therapy. related donors are preferred to diminish transplant risks. in lack of identical related donor, identical unrelated donors are second best choice. in this study, thalassemia major patients transplanted from unrelated donors (mud) were compared with thalassemic patients transplanted from relative donor (mrd) retrospectively. methods: 45 patients who were transplanted between june 2016 and december 2017 in bahçelievler medical park hospital pediatric bone marrow transplantation unit were evaluated retrospectively. all patients were classified according to pesaro risk classification. thirty four of 45 received busulfan, fludarabine, cyclophosphamide, thioteopa for conditioning, 11patients received myeloablative preparation regimen with treosulfan, fludarabine, thiotepa, cyclophosphamide. all patients were given atg, cyclosporine and methoteraxate for gvhd prophylaxis. the patients were compared in terms of acute complications in first 100 days, engraftment, chimerism, acute and chronic gvhd after transplantation. results were evaluated with ibm spss statistics 22 (ibm spss) program. results: a total of 45 patients, 26 (57.8%) male and 19 (42.2%) female, aged between 1 and 18 years (median 5 years) were evaluated. patients were evaluated in two groups as "mud" (n = 15) and "mrd" (n = 30) groups. there was no difference between groups about given stem cells (mud 6,16±1,07x10 6 /kg and mrd 6,25±1,24x10 6 / kg). neither significant difference between different pesaro risk groups in terms of developing acute and chronic gvhd and nor decreased chimerism were detected. neutrophil engraftment time (16,40 days) in mrd group was significantly longer than mud group (13,71 days) (p = 0.006) but no difference between platelet engraftments were observed. gvhd ratio was 33.3% in mud donor group and 13.3% in mrd group and no statistically significant difference was found(p> 0.005). the incidence of engraftment loss in mud group was 13.3% and 36.7% in the mrd group, and there was no statistically significant difference (p>0.05). the rate of decreased chimerism was found to be significantly higher in the mrd group (50%) than in the mud group (6.7%) (p:0.010; p< 0.05). the survival rate was 92.9% in the mud group and 96.7% in the mrd group. the disease-free survival rate was 90.9% in the mud group and 50% in the mrd group. the disease-free survival of mud group was significantly higher than mrd group (p:0.010). conclusions: in our study, transplant related complications and success of transplantation with both mud and mrds were found to be similar. it is promising for mud transplantations to found lower decreased chimerism and similar os and dfss. based on these results, it was concluded that hsct from non-family donors, especially for patients incompatible with chelation therapy and had organ damage, transplantation from unrelated identical donors can be a good choice. although the results of our study seem promising, larger patient groups and prospective clinical trials are required. disclosure: nothing to declare background: use of g-csf stimulation of bone marrow (bm) donors is beneficial in many aspects; it can enhance tnc yield, but also have an immunomodulatory effect on donor t cell function, particularly invariant natural killer t (inkt) cells expansion as well as apcs. we analyzed outcomes of 34 consecutive patients receiving bone marrow from hla-haploidentical donors that were stimulated with g-csf prior to harvest. methods: in the time period between 05/2012 and 05/ 2018, 34 patients received bone marrow from donors stimulated with 10 ug/kg bw of g-csf on days -2, -1 and day of bm collection. four patients (12%) received myeloablative (bucy) conditioning, one (3%) received tec ric conditioning while 29 (85%) received nma ("baltimore") conditioning. all patients received posttransplantation cyclophosphamide (ptcy) on days +3 and +4, tacrolimus and mmf were started on day +5. for 2 patients donors were fathers, 11 mothers, 9 siblings and 12 children. results: median age was 43 years (20-63), there were 14 female and 20 male patients. twelve patients had aml, 10 hodgkin lymphoma, 5 all, 3 mds, 3 nhl and 1 cml. median number of infused tnc in graft was 4.7x10 8 /kg bw (1.8-8.2) and cd34+ cells 1.9 x10 6 /kg bw (1-4.5) . after median follow up of 397 days (range 26-2139), overall survival was 57%, with median survival of 71 months. engraftment was established in 29 (85%) patients, 2 (6%) had primary rejection and 3 patients (9%) died in sepsis prior to engraftment. of 29 patients that engrafted, further 3 (9%) patients had secondary rejection, two of them were transplanted again from a haploidentical donor, both using pbsc as a source of graft. median time to neutrophil recovery (anc>500) was 23 days (12-36), while median time to platelet recovery (plt>20x10 9 /l) was 30 days (12-72) in evaluable patients. cumulative incidence of agvhd ii-iv was 27.7% (95% ci, 13-44);of note is that of 9 patients that developed agvhd only one had grade iii, while remaining 8 patients had grade ii. cumulative incidence of cgvhd requiring treatment was 6.9% (95% ci, [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . cumulative incidence of relapse was 36.1% (95% ci, 20-52) and trm was 25.2% (95% ci, 11-41). conclusions: the use of g-csf mobilized bm graft in the hla-haploidentical setting with ptcy has proven to be useful to us, not only in terms of tnc yield which was more than satisfactory and contributed to adequate hematological recovery, but also in the excellent control of both acute and chronic gvhd, with most patients developing agvhd of grade ii and only one grade iii (actually developed only after dli given for decreasing chimerism). comparative studies are of course warranted to prove benefit, but this data contributes to the growing body of evidence that indeed donor stem cell stimulation with g-csf has potentially powerful immunomodulatory effect. disclosure: nothing to disclose p674 other-relative donors as a reliable bank for allogeneic hsct in countries with culturally accepted cousin-cousin marriages: a two-year report from a pediatric center in iran background: although the optimal donors for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-hsct) are fully-matched siblings, the cousincousin (consanguineous) marriages in some countries have extended the chance to find a matched donor for the hsctrecipient. in this study, an outcome analysis of transplanted patients receiving stem cells from their relatives other than siblings (other-relatives or non-sibling donors) is provided. methods: in this retrospective cross-sectional study, a two-year report of patients who received allo-hsct from their other-relative donors during september 2016 to september 2018 at the department of stem cell transplantation of children's medical center in tehran, iran is presented. the patients were followed up until 1 st december 2018. results: during this time period, 178 patients underwent hsct (both autologous and allogeneic) at this center, of which 159 cases received allo-hsct. out of allo-hsct recipients, the donors of stem cells for 51 cases (32.1%) were their other-relatives. the median (range) age at hsct was 6 (1-11) years and the majority of patients were boys (31/51, 60.8%). according to disease class, the patients were most commonly involved with non-malignant hematologic diseases (18/51 patients, 35.3%) (figure) . the source of hscs for most patients (48 cases, 94.1%) was peripheral blood and for only 3 patients the source was bone marrow. the donors for 50 patients were fully matched and only one patient received the hscs from a one-locus mismatched donor. hsct was successful in 50 patients with most of them achieving full chimerism (44 patients, 86.2%) followed by those developing mixed chimerism (6 patients, 11.8%) and only one patient (2%) experienced graft failure. post-hsct complications included cmv infection in 34 patients (66.7%), other infections in 7 (13.7%), hemorrhagic cystitis in 3 (5.9%) and pres in 2 (3.9%). acute gvhd occurred in 22 patients (43.1%) and chronic gvhd in 3 (5.9%). death occurred in 7 cases and 5 of them were transplant-related, while 1 was due to disease relapse and 1 due to graft failure. the median of overall survival was 469 (40-792) days. conclusions: the likelihood of receiving hscs from an hla-matched other-relative donor in one-thirds of children undergoing allo-hsct, with comparable outcomes to sibling and unrelated donors (as evidenced in this study compared with other studies), introduces family bank as a reliable source for pediatric allo-hsct in countries with culturally accepted cousin-cousin marriages. hence, for transplant physicians, parental consanguinity would be an indication of an extended search for a potential matched donor among the patient's family. [[p674 image] 1. distribution of patients according to disease and disease class] disclosure: nothing to declare. abstract already published. update on the hla frequency distribution of the portuguese bone marrow donor registry eduardo espada 1 , dário ligeiro 2 , hélder trindade 2 , joão forjaz de lacerda 1 frequency distribution varied throughout the country, allowing for analyses of molecular variance and generation of relatively geographically accurate graphical representations of genetic distances between regions and districts. conclusions: with the most recent hla analysis of the portuguese bone marrow donor registry we were able to extrapolate high-resolution haplotype frequencies from the most common low-resolution hla-a/-b/-drb1 haplotypes (corresponding to 50% of the estimated haplotypes at that level), which will lead to an optimization of its use, hopefully limiting the time between donor search and allogeneic hematopoietic stem cell transplant. disclosure: nothing to declare. abstract already published. abstract already published. unmanipulated haploidentical donor transplantation compared to identical sibling donor had better antileukemia effect for refractory/relapsed acute myeloid leukemia in not remission status background: patients diagnosed with saa with no sibling donors and who are refractory to immunosupression are candidates to hematopoietic stem cell transplant using alternative donors. haploidentical donor transplants has been reported using cyclophosphamide (cy) post stem cell infusion as immunephrophylaxis. the present study has the objective of evaluating overall survival and engraftment rates after haploidentical stem cell transplant for saa in a reference center. methods: 15 saa adult patients (≥15 yo) received hsct from haploidentical donors from de january/2010 to august/2018. median age was 19 y (15-28); donor was the father in six, mother in five and a brother in four cases. stem cell source was marrow in 13 cases (87%). conditioning: 10 patients (67%) received cy 29mg/kg, fludarabine 30mg/m² e tbi 200cgy. the remaining received the same drugs but radiotherapy dose varied from 300-400cgy, all them received immunephrophylaxis with post transplant cy 100mg/kg, cyclosporine and mmf. median of infused cells (tcn) was 4,94x10 8 /kg (2,07-12,66). results: eight patients engrafted (53%). among seven graft failures four received a second haploidentical transplant and one received an unrelated donor transplant as salvage regimen. two patients were successfully rescued after the second haplo and the others died from infectious complications. three years overall survival was 55%. death causes included: five infections and two lung hemorrhage. median survival was 93 days (17-1065). no patient had acute graft-versus-host-disease (gvhd) and one patient had mild c-gvhd. conclusions: haploidentical transplant was feasible as therapy for saa refractory to immunessupression with an overall survival of 55% in this cohort. graft failure however is still a problem to be addressed in this setting. disclosure: no disclosure stem cell mobilization, collection and engineering p681 abstract already published. key performance and quality indicators for a successful bone marrow collection marco sampaio 1,2 , ana salselas 2 , fátima amado 2 , filipa bordalo 2 , sérgio lopes 2 , catarina pinho 2 , susana roncon 2 and one from ecc (staphylococcus spp.)presented positive microbiological results. conclusions: bm collection is a challenging strategy because it is a one-time procedure and manually operatordependent technique; simultaneously it is more difficult to control the final cellular content of the bm, which is a risk for donor volume depletion. bm collection is feasible even with donor and recipient weight difference. poorer performance may be found when higher tnc are requested. we respond efficaciously when the request is between 200 and 400*10 8 tnc but we fail to accomplish higher tnc values. we must emphasise that icc tnc demanded was generally lower than ecc. deciding the appropriate tnc for each patient remains a dare and an art. disclosure: nothing to declare. impact of adding plerixafor to mobilization protocol in the immune reconstitution of vδt cells after autologous hematopoietic stem cell transplantation efrat luttwak 1,2 , yael chava cohen 1,2 , odelia amit 1,2 , irit avivi 1,2 , svetlana trestman 1,2 , esti rom 1,2 , rinat eshel 1,2 , ram ron 1,2 1 tel aviv medical center, tel aviv, israel, 2 sackler faculty of medicine, tel aviv university, tel aviv, israel background: multiple myeloma has remained an incurable disease even in the era of novel therapies. front line treatment typically comprises of induction chemotherapy with 4-6 cycles of a bortezomib-based regimen, stem cell mobilization, and harvesting of peripheral blood stem cells (pbsc) by apharesis, followed by high dose melphalan with hct. while brotezomib-based induction regimens have demonstrated no adverse impact on hematopoietic cell harvest number and quality, no study analyzed the impact of timing of the last brotezomib dose prior to collection. in this study we aimed to determine the effect of the timing of the last dose of brotezomib before hematopoietic cell collection and the collection yield. methods: this was a single center historical prospective study, including all sequential newly diagnosed patients with myeloma between 2012 and 2017 that were given a bortezomib-based induction therapy (≤6 cycles) followed by pbsc collection. we excluded patients who either received 1 st line vtd-pace or lenalidomide-containing regimens. peripheral blood cd34+ cells were measured on the day of collection. patients with cd34+ levels of >10 cells/ microliter started collection on the same day, while those with lower levels were given plerixafor. we performed regression analyses to analyze the impact of a variety of precollection factors, including days from last bortezomib therapy on the collection yield. results: we identified 75 patients who fulfilled the inclusion criteria, table. median time from last dose of brotezomib to first leukapheresis was 11 (range, 2-27) days. a statistically significant correlation was found between the days from last dose of brotezomib and both the first collection day-cd34+ cells/kg (r=0.466, p< 0.001), and the total collected cd34+ cells/kg(r=0.341, p=0.03), figure. the optimal cutoff point as indicated by the roc curve was 8.5 days according to collection success with sensitivity of 79% and specificity of 74%, youden´s index 0.52. in multivariate analysis included other factors affecting collection yield (age, gender, status of disease at collection, and prior radiation) -timing of last dose of brotizomib remained significantly associated with the total collected cd34+ cells/kg (p=0.01). increasing age, female gender, and prior radiation were associated with lower collection yield (p=0.006, 0.012, 0.043, respectively). based on this, we developed a model to predict the total collected cd34pos cells = 11.76+ 0.13 (timing in days of last dose of brotezomib) -0.1 (age) -1.39 (if female) -0.01 (≥pr) -1.35 (if prior radation). conclusions: timing of last dose of brotezomib is an important factor for predicting a successful collection. a washout period of 9 days is associated with a better collection yield. these results should be further validated in a prospective study. age (median, range) 63 (37-78) gender -male (%) 44 (59) prior radiation treatment (%) 18 (24) disease status at collection (%) ≥pr -51 ( disclosure: nothing to declare p685 mobilization with plerixafor in "poor mobilizer" related and unrelated donors of hpc-a in case of failed mobilization with g-csf background: in the allogeneic hpc transplantation, both from related and unrelated donors, the most commonly used source is peripheral blood after mobilization with g-csf. it is however known that about 2% of donors are "poor mobilizers"; the rescue strategies are: a third apheretic collection; bone marrow donation. methods: in 2016-2017 in italy a procedure to be adopted in case of failed mobilization of peripheral blood stem cells has been defined and shared between ibmdr, cnt (transplant national center) and cns (blood national center) and the scientific societies simti, sidem and gitmo, using plerixafor, a selective reversible antagonist of the cxcr4 receptor with its binder (the stromal derived factor sdf-1), in combination with standard g-csf dose. moreover since 2017, in accordance with this protocol, the competent authority (aifa) has extended the registration of plerixafor (law no.648/96), also for the mobilization in "poor mobilizer" healthy donors. finally, in 2018 the protocol was extended to "poor mobilizer" family donors, making management equivalent in related and unrelated donors. at the time of the donor´s informed consent for the donation of hpc, the hypothesis of the lack of mobilization or inadequate collection was illustrated and the possible actions proposed as "back-up donation" were anticipated. failed mobilization of cse has been defined as the presence of one of the two criteria: a number of cd34+ circulating on peripheral blood lower than 20/μl on the 5th day of stimulation (5d), or the collection of cd34+ < 1.0x106/kg weight of the recipient at first apheresis. in these cases, a single dose of plerixafor is administered subcutaneously by health professionals under medical supervision, 0.24mg/kg of body weight, 6-11 hours before the start of apheresis. in case of use plerixafor due to failed mobilization of hpc-a or collection of an inappropriate number of cd34+, the notification is made by the collection center to ibmdr (for both family and non-family donors), and to the recipient transplant center : both donor and recipient express their consent; finally once the collection is completed, the collection center informs ibmdr, which in turn notifies cnt/cns/simti/sidem/gitmo. any adverse reactions/events are notified in real time, based on the sop specifications and current regulations. results: since the introduction of the national protocol, 7 donors (4 unrelated donors and 3 related donors) were treated, presenting at least one of the two inclusion criteria (cd34 < 20/μl at 5d or cd34 < 1x10e6/kg after first collection)after use of plerixafor in all donors, the required dose of cd34 was obtained to ensure successful transplantation, with a sufficient increase in the cd34+ cells. no side effects or adverse reactions related to the administration of plerixafor occurred. conclusions: in cases of failed mobilization in the related and unrelated donor, the use of plerixafor according to the methods described in the shared protocol between ibmdr, cns, cnt, simti, sidem, gitmo, proved to be safe and effective. this protocol emphasizes the great value of the sharing of procedures between the register, institutions and scientific societies, ensuring the supervision of the process and the protection of the donor and recipient. disclosure: nothing to declare background: autologous stem cells transplantation (asct) is an effective treatment option for young patients with multiple myeloma (mm). a minority of patients may still experience untoward toxicity due to delayed engraftment. thus, the current policy in many centers is aimed to increase the target dose of collected cd34 + cells up to an "optimal" level of 4x10 6 /kg per procedure. therefore, an ideal mobilization, aimed to collect 4 to 8 cd34 + cells/kg in one apheresis, should achieve a number of circulating cd34 + cells >40/mcl (very good mobilizers). plerixafor may help to maximize the cd34 + collection but its use is limited by high cost. we carried out a retrospective analysis aimed to predict the quality of mobilization and develop an algorithm to optimize both timing of collection and use of plerixafor. methods: we retrospectively collected data from 91 mobilization procedure performed in our center between 2006 and 2016 for mm. all received the same mobilization protocol with cyclophosphamide (range 3-4 gr/sqm) and g-csf 10mcg/kg from +5. cd34 + cell count was started when white blood cells (wbc) count exceeded 1x10 9 /l. patients were excluded from this analysis if 1) showed a cd34+ count >40/mcl (target achieved at first day count) and/or 2) cd34 + count on second day was missing and/or 3) plerixafor was administered on first day according to previous policies. sixty-eight patients were evaluable for the study. univariate and multivariate logistic regression analysis to study ccd34 + kinetics and assess predictors impact on mobilization was carried out. ratio cd34 +/wbc in first day count, gender, disease category and time from mobilization chemotherapy were also included results: among the 68 patients included in the analysis, the threshold of 40 cd34+/mcl cells on the second day was reached by 35 (51,47%) of patients (groupa) whilst the remaining 33 (24,7%) failed the target (groupb). median (range) wbc x10 9 /l and cd34 + /mcl counts in group a and b were 2,01 (1-5,01) and 21,33 (7,56 -39,91), 1,08 (1) (2) (3) (4) (5) (6) 46 ) and 3,31 (0,06-31,14) respectively, with a statistically significant differences among group (mann-whitney p= 0,01 and p=0,02 respectively). only cd34 + /wbc ratio and cd34 + /mcl on first day count had an impact on kinetics and optimal mobilization. logistic regression model highlight cd34+/mcl (or=1,270; 95% ci: 1,060 -1,522) on first count as an independent predictor of second day optimal mobilizer, with auc of 0.9% (0, 99) in roc analysis. two cd34 + thresholds were then calculated: < 7,56/mcl (ppv 0,81; npv 1,0) that identified poor mobilizer, and ≥ 24,06/mcl (ppv 1,0; npv0,59) that exclude probability to fail on second day. for those with a cd34 + count between 7,56-24,06 the cd34+/wbc ratio (or=1,768, 95% ci: 1,307-2,391) was a predictor of optimal mobilization (auc 0,94; 0,885-0,996); cut-off value was 5,63 (sensibility 94,28; specificity 84,84) conclusions: assessment of circulating wbc, cd34+ and their ratio at wbc recovery in a chemo-based mobilization is a valid tool to manage the collection strategy and the on-demand use of plerixafor. we have developed an algorithm aimed to the use of plerixafor to both rescue poor mobilizers and boost cd34 + count in intermediate mobilizers. background: successful autologous stem cell transplantation (asct) requires the infusion of a sufficient number of hematopoietic stem cells (hscs). peripheral blood (pb) is the most commonly used source of hscs, therefore, it is important to optimize methods used to mobilize the hscs. the most clinically used chemotherapeutic agents for effective mobilization are cyclophosphamide and etoposide. recent published studies suggest that etoposide has a better mobilization effect than cyclophosphamide even at lower doses, but it is not clear why this difference occurs. in this study, we tried to determine whether there is a difference in the mechanism of mobilization between cyclophosphamide and etoposide. methods: first, in order to confirm the clinical data for efficacy and toxicity of mobilization, we retrospectively analyzed the data of patients who were diagnosed with lymphoma and performed mobilization using cyclophosphamide or etoposide from january 2011 to december 2018. second, mesenchymal stem cells (msc) were primarily cultured from the healthy controls, then treated cyclophosphamide or etoposide at a concentration of 10% inhibition of cell growth, and cytokine analysis was performed to identify cytokines known to be associated with mobilization. third, mobilization mouse model using cyclophosphamide or etoposide was generated, total blood was collected at the time of hscs collection, and cytokine and network analysis (using ingenuity pathway analysis) was performed. results: the mobilization yields for cyclophosphamide or etoposide were analyzed. etoposide miblized a significantly higher median number of cd34+cells than cyclophosphamide. the rate of successful or adequate mobilization was also significanctly higher for etoposide in univariate and multivariate analysis (table 1 ). in the analysis of toxicity during mobilization, the incidence of neutropenic fever was higher in the cyclophosphamide group (p = 0.007). during mobilization, cyclophosphamide maintained lower wbc counts than etoposide and showed a large increase in wbc counts at the start of collection ( figure 1 ). the cumulative dose of cyclophosphamide or etoposide in patients who underwent autologous stem cell transplantation did not affect leukocyte (anc>1000/microl or platelet (plt >100k/microl) engraftment. in msc treated with etoposide at a concentration of 10% inhibition of cell growth, il-8, which is a cytokine that promotes hematopoietic stem cell mobilization, were shown a statistically significant increase (figure 2 ). in the mouse model of mobilization (figure 3 ), the levels of kc, one of the il-8 homologues in mice, had significantly increased in the etoposide-treated group compared with the levels in the cyclophosphamide-treated group. the levels of other il-8 homologues, mip-2 and lix, also showed increases in the etoposide-treated group compared with those in the cyclophosphamide-treated group; these differences, however, were not statistically significant (figure 4 ). network analysis based on in vivo cytokine results identified that etoposide could promote mobilization in association with matrix metalloproteinase as compared to cyclophosphamide ( figure 5) . conclusions: etoposide has a higher mobilization efficacy when compared to cyclophosphamide, which could due to the different mechanisms of mobilization through the elevation of il8 and the activation of matrix metalloproteinase associated therewith. background: high-dose chemotherapy followed by autologous blood stem cell transplantation (asct) is a standard therapy for wide range of hematologic and solid malignancies. although various methods have been introduced to improve the peripheral blood stem cell (pbsc) mobilization, autologous stem cell collection (ascc) is not successful in every patient. furthermore, even if the ascc is complete, not all of them lead to asct. we evaluated the result of ascc and actual use of pbsc grafts in current practical setting. methods: we retrospectively reviewed the all consecutive ascc procedures performed at the department of oncology in asan medical center, seoul, korea, between january 2000 and october 2018. the targeted number of background: fanconi anemia (fa) is a rare inherited genetic bone marrow (bm) failure syndrome. while abnormal bm cells production occurs very early in life, the usual age of diagnosis is 5-10 years old. gene therapy (gt) might be an alternative to hematopoietic stem cells (hsc) transplantation, but harvest a large number of autologous hsc remains a challenge. we started a mobilization assay, fancomob, to evaluate the safety and the efficacy of fa patients' mobilization with granulocyte-colony stimulating factor (g-csf) and plerixafor. this study is part of the fa's european gt project "eurofancolen". methods: four patients with fanca mutations following the inclusion criteria were selected before pancytopenia. to note, fa4 was diagnosed before clinical manifestation through family screening. they received subcutaneous injection of g-csf (12 μg/kg twice a day) from d-1 and plerixafor (0.24 mg/kg/day) from d-4. the collection protocol targeted 3x10e6/kg of cd34+ cells, based on a predicted future weight in 5 years. cd34+ cells and white blood cells (wbc) blood count were monitored tightly along the mobilization. patients with more than 10 cd34 +/μl or between 5 and 10 cd34/μl with a clustered aspect detected by flow cytometry after plerixafor injection underwent apheresis. cd34+ cells were immunoselected from the collection with clinimacs purification system (miltenyi) and cryopreserved for further gt manipulation. results: the mobilization target was not achieved for the first two included patients (fa1 12 years old and fa2 8 years old). the minimum value of cd34+/μl required wasn't obtained for fa1 and the flow cytometry cd34+ aspect was not clustered for fa2. cd34+ cells were mobilized quickly but transitionally after plerixafor injection for the last two patients, fa3 and fa4, 5 and 2 years old respectively. both patients underwent apheresis procedures. no cd34+ cell rebound was observed after the apheresis was stopped.collection target was not achieved after four days of collection for fa3. it was obtained the first day for fa4 (figure 1 ). back-up for hsc transplantation could not be cryopreserved because of the limited number of cd34+ cells collected in patients fa3 and fa4. no short-term adverse events were observed. following cd34+ immunoselection, cd34+ cell purity and recovery were poor but in the normal range described in the literature for fanconi patients (8-20%)( table i) . one month after the collection hemograms were unchanged. conclusions: our clinical study offer new data showing that mobilization of fa patients with g-csf and plerixafor is safe and more efficient for younger patients, especially before clinical manifestations of bm failure. further efforts are required to establish an effective technic to purify the cd34+ cells after harvesting. basal cd34+ cell and platelets count are a strong predictor for mobilized peripheral blood stem cells on the 4th day of g-csf treatment in donors cryopreserved pbscs. this was associated with considerable efforts for the patients and caused additional treatment costs. on the one hand, having the therapeutic option of an autologous transplantation in the future may represent a clinically relevant advantage. however, a huge number of stem cell products are kept in storage for many years without ever been used for transplantation. our study provides cause for a careful reevaluation of the current clinical practice, which may help to focus more precisely on patients who actually benefit from a cryostored autologous stem cell graft. [[p693 image] 1. fig. 1 : absolute numbers and relative distribution of stem cell grafts] disclosure: the authors confirm that there are no potential conflicts of interest to disclose, except the following: katharina kriegsmann: research funding from bms, celgene, and sanofi. patrick wuchter: membership in advisory boards for sanofi-aventis. reduction of dimethylsulfoxide (dmso) concentration from 10% to 5% in criopreservation of stem cells. influence on the kinetics of engraftment and tolerance to infusion patricia lopez-pereira 1 , beatriz aguado 1 , elena sola 1 , carmen cámara 1 , isabel vicuña 1 , lorena vega 1 , adrian alegre 1 1 hospital universitario de la princesa, madrid, spain background: dmso is the cryoprotectant most used in the cryopreservation of stem cells. it is associated with adverse effects during the infusion of the product, its toxicity being proportional to the volume infused. the most common concentration used has been 10%, although recent publications report that reducing it to 5% leads to lower rate of side effects without impact on the product or the graft. we retrospectively analyzed 207 patients recipients of autologous peripheral blood stem cell transplantation (hct) in our hospital from january 2009 to september 2017. they are divided into two groups according to the concentration of dmso used in the freezing (10% until september 2014 or 5 % since october 2014). the baseline characteristics of the patients, the infused product and the graft are shown in table 1 . the cd34 count was performed by flow cytometry. all freezings were performed with a biological freezer for programmed controlled rate cryopreservation and stored in ultrafreezers at -80ºc. results: both population groups are homogeneous. the t-test was used for statistical analysis. regarding the cd34 + variable, no statistically significant differences were observed (p = 0.636). neither for the variables leukocyte recovery and platelet recovery (p = 0.178, p = 0.991 respectively). the difference in the variable viability is 5.61 units (ci95%: [0.61-10.62]) and is statistically significant (p = 0.028) in favor of dmso 5%. regarding adverse effects, 100% (n = 4) of the serious adverse reactions occurred in the 10% dmso group (hypotension and seizures). the mild and moderate ones were similar in both groups, most were mild nausea, vomiting and flushing. overall, no statistically significant differences were observed due to the low rate of adverse effects found. patients starting with 2014 until october 2018, total of 414 patients attempted collection of autologous pbscs, and 38 poor mobilizers recieved plerixafor during first mobilization cycle. in total, 9 patients required repeated mobilization cycles (2,2%) of which 6 were from the plerixafor group. in total 42 patients recieved pleriksafor; 23 females and 19 males, median age 58 (8-71) with following diagnoses: 28 nhl, 5 mh, 5 multiple myeloma, 1 neuroblastoma, 1 nephroblastoma, 1 sarcoma ewing and 1 seminoma. of 6 repeated mobilizations with plerixafor, 2 patients (33,3%) still failed to collect adequate transplant. in this period we had altogether 3 unsuccessful mobilizations (33,3% in repeated cycles, 0,72% in total). this group of patients consisted of 1 male and 2 female patients, median age 42 (33-58), diagnosis of nhl and failure to collect after 4 leukapheresis procedures each. median number of leukapheresis needed for adequate collection was 2 with preemtive plerixafor use, and 4 in repeated mobilizations. conclusions: our expirience shows that preemtive use of plerixafor in poor mobilizers is efficient and has enhached success of the pbsc collections. due to drug high cost each institution needs to develop its own algorythm in management of poor mobilizers. the factors contributing to plerixafor mobilization failure still need to be elucidated. disclosure: nothing to declare. platelets recovered from mobilized leukapheresis units obtained from hla-haploidentical donors fulfill the criteria of a conventional hemocomponent and can be used for transfusion background: central venous catheter (cvc) related complications may lead to high morbidity and mortality. unlike cvc, peripheral cannulation offers a quick and inexpensive method for safe and non-traumatic vascular access (va) thus its utilization is strongly recommended whenever possible. the ultrasound (us) guidance for acquiring peripheral va is a useful tool for reduction or elimination of the need of using cvc for stem cells collection. we have made an attempt to introduce us method in our apheresis unit having no previous experience with us devices. the aim of the study was to measure the decrease of cvc insertions after introducing us and evaluate the quality of va by comparing average flow rate and confirming that the desired blood volume could be processed. methods: the theoretical education involved a free elearning course in peripheral ultrasound-guided va (pugva, usabcd, aarhus, denmark). subsequently, the personnel have implemented knowledge in practical training on gelatine and silicone phantoms and healthy volunteers. the practical activities also included a fiveday course in an apheresis centre with us-guided cannulation experience. the details concerning va were recorded, including va site, cannula size, average inlet flow rate, number of inlet pressure alarms reported by the apheresis device. the procedure details where traditional approach was applied i.e. palpable cannulation and cvcs have been collected. similarly, the necessary data for procedures where veins were assessed with ultrasound prior to apheresis were recorded. results: before introducing ultrasonography, 58 stem cell collections have been performed in 46 patients. of all these procedures, 18 were accomplished with cvc (31%) and 40 with peripheral va (69%). median cubital vein was the vessel of choice. out of the 40 peripheral va procedures, 14 (35%) were problematic, with 10 or more inlet pressure alarms during every procedure. after the training stage, 34 collection procedures were performed in 25 patients. after introducing us we have observed a significant reduction of the number of cvc insertion required for successful apheresis from 31% to 9% (p=0.01; chi-square test with fisher's exact). thirty one procedures were completed with peripheral va (91%). ultrasound device enabled cannulation not only the superficial veins but also for the deeper veins. cannulation sites included upper arm cephalic vein (39%), median cubital vein (35%), upper arm basilic vein (13%), median antebrachial vein (13%). out of the 34 collections, 13 were considered problematic (42%). no difference in an average flow rate was observed between procedures performed peripherally with and without ultrasound usage (p=0.34; u mann-whitney test). conclusions: despite no previous experience in us guidance, we have successfully managed to introduce the new method in our apheresis unit. within 5 months, we have reduced cvc usage threefold and as the personnel is gaining more experience, we suppose that the cvc usage may be reduced to episodic cases. despite slightly higher number of pressure alarms, all procedures with ultrasound guidance were completed as planned. ultrasound guidance is the most important tool for significant increase in peripheral va usage and may become the only option for patients with difficult va. disclosure: nothing to declare. abstract already published. donor blood management in healthy bone marrow donors: a retrospective single institution analysis background: over the last two decades mobilized peripheral blood stem cells (pbsc) have been established as the main source of stem cells because of improved engraftment and no necessity for hospitalization for the donors. nevertheless, due to the introduction of promising new transplant regimens, especially in the haploidentical transplantation setting bone marrow (bm) donations are regaining importance. although for both donation methods severe side effects are rarely described, bm collection is associated with considerable blood loss and hence symptoms of acute blood loss are commonly observed. therefore autologous blood are collected routinely in some institutions before donation. since the collected bone marrow amount depends on the target dose, the wbc yield in the product influences the required bone marrow volume. therefore we sought to investigate the relationship between collection volume, rbc volume removal, drop in hb and indications for blood transfusion. furthermore, we assessed wbc and cd34+yields in relationship to various donor parameters and to product volume, in order to find prediction tools for collection volumes. methods: 489 allogeneic bone marrow harvests from adult donors were performed at our institution and retrospectively analyzed. complete blood counts, serum iron and ferritin were assessed at work-up and 4 weeks after donation. the bone marrow product quality including wbc, hematocrit (hct) and cd34+ cells were assessed by automatic hemocytometry and single-platform flow cytometry with ishage gating. results: besides local pain most of the side effects were related to blood loss. none of the donors received blood transfusions. the mean reduction of hemoglobin levels was 2.5 g/dl with a minimum hemoglobin level of 8.9g/dl and a persistent anemia according to who criteria after 4 weeks in 7.9% and pathologically low ferritin levels in 29%. no donor presented symptoms with indication for blood transfusion. the median wbc concentration of the bm product was 26.5/nl (5-95% percentile:16.77-38.75/ nl) the cd34+cell concentration 186.5/μl (5-95% percentile: 73.92-346.9/μl). in the linear regression analysis leukocyte counts of the donor before donation correlated significantly with wbc concentration in the product. thus in order to collect with 90% certainty the 300 mio wbc which are a typical per-kg dose for an allogeneic recipient, 16.24 ml of bone marrow must be collected. collection volume did not systematically affect wbc or cd34+ cell concentration. conclusions: achieving high wbc yields in the bone marrow product allowed for collection of relatively modest bm volumes, thus protecting donors from excessive blood loss. acute adverse events were acceptable. optimization of perioperative management in healthy bone marrow donors may be achieved by good collection technique and reevaluation of wbc yields of each institution to calculate required bone marrow amount. the collection of autologous blood is not indicated. furthermore stringent pre-and postoperative hemoglobin management is predicted to limit adverse effects. disclosure: nothing to declare. donor-recipient weight ratio predicts successful stem cell mobilization on day four of gcsf mobilization results: in group 1 median age of donor was 41 years (range 19 to 51 years). in group 2 median age of donor was 31years (range21 to 48 years). table] 1. table 1 ] elaborates other parameters analyzed between the two groups. one patient in group 1 developed grade ii acute gvhd whereas 3 patients in group 2 developed acute gvhd grade ii-iv. at the last follow up no (0/9) patient in group 1 has any symptoms of chronic gvhd whereas (2/5) patients in group 2 have features of chronic gvhd (one extensive, one limited). conclusions: our observation suggests that upfront use of plerixafor in combination with gcsf modifies the graft favorably decreasing the risk of graft failure and graft versus host disease both acute and chronic. it also helps the donor by decreasing the total volume processed, amount of acd exposure and the duration of harvest. disclosure: none. impact of vitamin d levels on peripheral stem cell mobilization in autologous hematopoietic stem cell transplant recipients ferda can 1 , zeynep arzu yegin 1 , zubeyde nur ozkurt 1 , orhun akdogan 1 , lale aydın kaynar 1 and total product cd34 + cell count [6.7(02.8-15) vs 5.9 (2.9-17.2); p=0.047] were significantly higher in patients receiving chemotherapy+g-csf than g-csf only. the study group was divided into two groups based on peripheral cd34 (cut-off level: 20x10 6 /kg) as well as product cd34 levels (cut-off level: 5x10 6 /kg). vitamin d levels were found to be similar among these groups (p>0.05). total product cd34 + cell count was found to be relatively lower in patients with vitamin d levels below 10 μg/l [5.6(3.3-10) vs 6.7(2.8-17.2); p=0,09]. (figure 1 ) conclusions: based on its effect on stem cells in in vitro studies, it may be considered that vitamin d may have a favourable impact on stem cell mobilization. statistically insignificant but relatively lower total product cd34 + cell count in patients who had lower vitamin d levels, which may indicate a role for vitamin d in stem cell mobilization, needs to be confirmed with larger studies. considering the high prevalence of vitamin d deficiency in the general population, the possible role of vitamin d in hematopoietic stem cell mobilization deserves further consideration. disclosure: nothing to declare background: one of the factors, affecting efficiency of autologous hematopoietic stem cell transplantation (autohsct) in hodgkin lymphoma (hl) patients is early recovery of graft, depending on cd 34+ cell count and conditions of cell product cryopreservation and storage. it is well known, that dimethylsulfoxide (dmso), used for cryopreservation, can be cardiotoxic and cause diverse gastrointestinal, pulmonary, kidney, liver side effects and acute hemolysis. lethal for animals dose 30-40 mg/kg leads to life threatening arrhythmias and respiratory arrest. in order to improve dmso toxicity different ways of alternative cryoconservation modes are studied -lower dmso concentration (5% vs 10%), temperature -80˚c instead of ultra-low and washing of cell product. aim of the study is to evaluate the influence of dmso washing on hematopoietic recovery after autohsct. methods: retrospective analysis of hematopoietic recovery of 37 relapse/refractory hl patients after autohsct was performed. mobilization regimen included second line chemotherapy for hl (dhap, begev, igev, ice) with consecutive g-csf administration. cd 34+ cells were assessed, using 6-colour flow cytometer facs canto ii while cell collection, thawing and washing. cells with 10% dmso were stored at -196˚and washed in 14 cases of transplantation with human albumin-dextran (reopolyglukin) and centrifugation. statistical data processing was performed by the χ2 method -pearson criterion; p -the level of significance of differences. results: patient groups had no difference in age, disease stage, gender, time from treatment start to autohsct and cd34+ cell count (p>0,05). time to wbc recovery >1х10 9 /л was 9-29 (median 13,6) days vs 10-34 (median 13,7) days, time to platelet>30х10 9 /л recovery was 11-34 (median 16,9) days vs 11-58 (median 17,9) days in groups without and with cell washing respectively (p=0,507). no difference in blood component consumption was observed (p=0,546). in 18 out of 23 (78%) patients during cell reinfusion without washing nausea, vomiting, arterial hypertension was observed, no reactions were detected after cell washing (p = 0,03). conclusions: washing autologous mononuclear cells from cryopreservant dmso does not lead to low hematopoietic recovery rate after autohsct and can avoid toxicity, thus making autohsct more safe. disclosure: authors declare no conflict of interests. quality assesment of hematopoietic stem cells autografts after cryostorage, harvested using plerixafor background: the introduction of high-dose chemotherapy followed by transplantation of autologous hemopoietic stem cells (hscs) into the treatment program for multiple myeloma (mm) has significantly increased the frequency of achieving complete remissions and overall survival in patients. to obtain a sufficient amount of hscs, hematopoiesis is stimulated with granulocyte-macrophage factors (gm-csf) both in mono mode and after the administration of cytostatics followed by cytapheresis sessions (alone or after the cytostatics followed by cytapheresis sessions) . cryopreservation protocols are used to preserve cells in a viable state, followed by long-term storage of transplants in liquid nitrogen. however, in some patients it is not possible to obtain the necessary amount of hscs. the inclusion of plerixafor in standard mobilization schemes allows you to prepare the sufficient quantity of hscs in most patients with mm. methods: the study included 18 samples of autografts from 18 patients with mm from 2015 to 2017 (median 2.1 ± 0.3). hscs mobilization was performed on the background of unstable blood formation after high doses of cyclophosphamide 6 g/m 2 with the subsequent administration of g-csf at a dose of 12-24 μg / kg (9 samples from 9 patients) and with the addition of plerixaphor at a dose of 0.24 μg / kg (9 samples from 9 patients). immunophenotype viability of hscs in autotransplants after cryopreservation were determined by flow cytometry using the ishage protocol on a flow cytometer (facs cantoii, becton dickinson) by expressing surface markers of antibodies against cd34, cd45, cd90, cd38 and staining with 7aminoactinomycin (7-aad). the colony-forming activity of hscs (cfu-cfu-mix, cfu-gm, cfu-g, cfu-m) was evaluated in methylcellulose (methocult h4435, stemcell technologies, canada) for 1x10 5 transplanted cells for 14 days. results: the viability of hscs in autografts (cd45 + / cd34 + / 7add-) after cryopreservation in both groups was 98 ± 0.7%. in the group of samples using plerixaphor, a higher content of primitive hemopoiesis precursors (primitive cells) (cd34 + cd90 + cd38-) was detected compared with the control group (29.2 ± 2.7% and 13.2 ± 12%, respectively). the cfu count (cfu-cfu-mix, cfu-gm, cfu-g, cfu-m) in the plerixafor group was 100 ± 2.5 per 1x105 explanted cells, in the control group -70 ± 1.8 ( figure 1a-d) . conclusions: the use of plerixafor against the background of standard protocols for the mobilization of hscs allows to obtain high-quality graft with a higher content of primitive cells and proliferative activity. disclosure: no conflict of interest. nothing to declare. comparison of effectiveness of plerixafor plus g-csf in poor and very poor-movilizers: efficacy of the combination of plerixafor and g-csf in poor-movilizer background: healthy donors ocassionally show a poor response to mobilization agents. plerixafor+g-csf can be a salvage strategy in poor mobilizers. some series describe the use of plerixafor to collect greater doses of cd34+ cells in hematopoietic stem cell transplantation (hsct) with tcell depletion. plerixafor use in the mobilization protocol could help collecting higher cd34+ dose in indirect t-cell depletion (cd34+ selection) for ex-vivo manipulated haploidentical transplantation, with less number of apheresis and a rapid engraftment. methods: data of fourteen healthy peripheral-blood donors was retrospectively collected. they received 4 days 10 mcg/kg/day g-csf and 0,24 mg/kg/day plerixafor on 4º day as mobilization treatment. fourteen pediatric patients (median age 13 years, range 8-15) diagnosed with malignant and no malignant hematological diseases received haploidentical hsct with cd34+ selection and cd45ra+ depletion between february 2015 and july 2017 . results: one leukoapheresis procedure was performed in all cases. median processed volume was 11 liters (range 6-14). median of cd34+ cells obtained was 13,73x10 6 /kg (range 7,8-26,47) . after positive selection, >4x10 6 /kg cd34+ cells were infused in all cases (figure 1 ). neutrophil engraftment was achieved after a median of 15 days (range 10-17). few donors presented only plerixafor mild secondary effects. conclusions: our experience showed that a mobilization protocol using g-csf and standard dose of plerixafor (compasive use) is a safe strategy that allows collecting great cd34+ dose in one apheresis procedure. this could be useful for haploidentical transplantation with ex-vivo t depletion, especially if there´s a weight disproportion between donor and patient. background: mesenchymal stem cells (mscs) are selfrenewing multipotent progenitor cells with wide differentiation potential. their ease of isolation and expansion in vitro as well as their unique regenerative therapeutic properties suggest the use of msc as an approach for treating several disorders. extra-embryonic tissues as placenta have been proposed as potential sources of mscs due to the absence of ethical problems neither risks for the patients. furthermore, only protocols using fresh placental tissue have been described so far. a protocol for isolating mscs from delayed-manipulated tissue was designed and tested in order to optimize the use of placental mscs (mscs-p) in an advanced therapies context. methods: full term placentas (n=10) were obtained from healthy mothers in hospital universitario central de asturias (spain). informed consent was obtained from each mother prior to delivery. after dissection of 12 gr decidual tissue it was washing with saline (b. braun, germany) and cut into small pieces. these biopsies were conserved 24 hours in dmem media with 1% antibiotic solution 100x (gibco, usausa) until processing. the day after, tissue was mechanically minced and then enzimatically digested with a combination of 80ui/ml dnase i (sigma aldrich, usa) and 0.25% tripsin-edta solution (w/v) (biochrom, germany) at 37ºc for 1 hour. then, the mixture was filtered with 40μm cell strainer (bd bioscience, usa) and centrifuge at 300xg for 5 minutes. finally, cells were resuspended in 5ml of dmem media suplemented with 10% fbs and antibiotic, seeded in 24-cm flask and incubated in forma stericult co 2 incubator (thermo fisher scientific, usa) at 37ºc, 5%co 2 . culture-expanded mscs cells were phenotipically characterized by flow cytometry (facs aria iiu, bd) with antibodies against cd29, cd44, cd73, cd90, cd105, cd166, cd34, cd45, hla-dr cd14 and cd19 using mesenchymal cell kit (immunostep, spain). afterwards, these cells were differentiated to adipogenic, osteogenic and chondrogenic lineages using stemmacs adipodiff media, stemmacs osteodiff media and nh chondrodiff medium (miltenyi biotec, germany) respectively. after three weeks of differentiation cells were fixed in 4% paraformaldehide (merck, usa) and analyzed. adipogenic, osteogenic and chondrogenic differentiation was visualized after staining with oil red o, alkaline phosphatase and hematoxilin-eosin (sigma-aldrich, usa). results: mscs-p isolated cells were characterized according to the isct criteria for mesenchymal stem cells. they were positive for cd29, cd44, cd73, cd90 and cd105 and negative for cd14, cd34, cd45 and hla-dr, indicative of a typical msc phenotype ( figure 1 ). all the markers showed a high percentage of expression between 84.6 and 99.9%, meaning that msc population obtained with the designed method was very homogenous. similarly, staining for the three studied lineages was positive ( figure 1 ). conclusions: the described protocol allows us to obtain mscs from decidual placental tissue stored and processed 24 hours after the biopsy extraction using a unique enzymatic digestion. this circumstance permits to take advantage of placentas that are discarded after delivery giving us the option to obtain mesenchymal cells that could be used in clinical trials. disclosure: nothing to declare outcomes of umbilical cord transplant in high risk relapsed or refractory acute myeloid leukaemia background: high-risk relapsed/refractory acute myeloid leukaemia (aml) is a fatal disease. allogeneic haematopoietic stem cell transplantation represents the only chance of cure. as the transplant relies on the graft-versusleukaemia (gvl) effect, and if different donors exert different gvl effects, then choosing the right donor assumes great importance. in manchester, a large bmt centre in the north of england, our practice in such aml has been to choose unrelated cord blood (ucbt), without serotherapy in the conditioning therapy, as our preferred donor cell source. methods: we report the results of 16 unrelated ucbt in 15 patients (five boys and ten girls) with high-risk aml, defined as relapsed or refractory disease. thirteen patients (81%) received this as a 1 st transplant, two patients (13%) received this as a 2 nd transplant for relapsed aml post matched unrelated donor transplant, and one (6%) received ucbt twice, once in cr1 and once in cr2. nine patients (56%) had mismatched ucbt, and the rest were fully matched at class-i (hla-a, -b, and-c) and class-ii (hla-drb1). conditioning was given as treosulfan, fludarabine and thiotepa in half of the patients (n = 8), other treosulfanbased regimens were used in two patients (12%), and busulfan-based regimens were used in six patients (38%). no serotherapy was given. results: the median age at transplant was 5 years (range, 5months -15years). neutrophil and platelet engraftment were achieved in 15 and 12 patients at a median of 16 and 35 days, respectively. 13 patients (81%) had engraftment syndrome. all engrafted patients achieved 100% donor chimerism, except one patient who had mixed lymphoid chimerism initially, that was corrected spontaneously to 100% at three months after transplant. acute gvhd grade i-ii developed in six patients (38%), and grade iii-iv developed in three patients (19%). all cases resolved, except two patients where acute gvhd evolved into chronic gvhd (one with grade i skin gvhd which fully resolved, and one with grade iii gvhd gut colitis who was parenteral nutrition dependent till death). two more patients developed chronic grade i skin gvhd and resolved (chronic gvhd developed in 25% in total). three patients (17%) developed veno-occlusive disease (vod), that completely resolved with defibrotide treatment and necessitated ascitic drainage in one of them. viral reactivations occurred in five patients (30%) and were successfully treated. at a median follow-up of 20 months (range, seven months -four years), eight patients (50%) died at a median of 79 (range, 24 to 230 days), with a transplantrelated mortality of 25% and relapse-related mortality of 25%. five patients (31%) relapsed post-ucbt; four died and one had a successful second ucbt (event-free survival was 44%). immune reconstitution in alive patients was achieved at a median of eight months. conclusions: very high-risk patients treated with ucbt with good overall survival and event-free survival, similar to aml treatment rate with low-risk disease. disclosure: nothing to declare in haploidentical transplants is the incidence of acute and chronic gvhd strictly related to the stem cell source? results: the odds ratio was 1.78 with a 95% confidence interval of 0.94 -3.37 (p=0.07). conclusions: the risk of infection of the uc is not related to the microbiological status of the ucb. a possible explanation for this is the presence of antibiotics in the medium used for uc, but not ucb, transport. this means that cryopreservation of ucs from which contaminated cord blood has been obtained is justified. comparison of turkish stem cell coordination center (turkok) with istanbul university bone marrow bank (tris); a single center experience in match unrelated donors azize mergen 1 , selime aydoğdu 2 , başak aksoy 3 , yunus emre savcı 2 , gürcan dikme 2 , funda çipe 2 , ceyhun bozkurt 3 , tunç fışgın 1 older patients are increasingly being transplanted, thanks to improvement in allogeneic hematopoietic stem cell transplantation (allo-hsct) techniques. increasing donor age is associated with greater risk for mortality and graftversus-host disease (gvhd). since sibling donors are of similar age to recipients, we hypothesized that, in older patients, a young matched unrelated donor (mud) would be comparable to an hla-matched sibling donor (msd). methods: we retrospectively compared outcomes of allo-hsct from msd (n=1797) and 10/10 hla mud (n=2212) in patients aged ≥ 60 years with hematological malignancies transplanted between 2006-2015. all patients received reduced-intensity conditioning and graft source was peripheral blood. the primary outcome was overall survival. msds served as the reference category and were compared to muds split into three age groups (≤25 [n=524], 25-40 [n=1072], >40 [n=616] years) using univariable analyses and multivariable cox regression models adjusted for patient, disease, and transplantation features. results: the median age of hsct recipients was 64 years and was similar across groups. median donor age for msd was 60 years and 22, 33, and 45 for the mud age groups ≤25, 25-40, and >40 years. acute leukemia was the leading transplant indication (46%) followed by myelodysplastic syndrome, myeloproliferative neoplasms and indolent non-hodgkin lymphoma. disease risk distribution was similar across donor groups (low [48%], intermediate [42%] , and high [10%] in the complete population; p=0.771). time from diagnosis to hsct was longer with mud compared to msd and increased with an older age of mud. in a univariate analysis, overall survival was 49% (msd), 50% (mud≤25), 45% (mud 25-40), and 43% (mud≥40, p=0.002). corresponding non-relapse mortality (nrm) cumulative incidence was 22%, 25%.,31%, and 32.2% (p< 0.001) (figure) . gvhd-relapse-free (grfs) was 29%, 30%, 26%, and 24% (p=0.012). in a multivariable cox model, young mud (≤25) had a similar risk for mortality compared to msd (hr 1.00, p=0.97), while a monotonic increase in risk was observed with an older donor age (mud 25-40y: hr 1.17,p=0.014; mud≥40y: hr 1.26, p=0.001) (table) . findings were confirmed in a propensity score analysis, matched for key covariates. nrm and grade 2-4 acute gvhd were consistently higher with mud, with the greatest risk associated with older muds. the hazard for grfs was higher with mud aged 25 or higher compared to msd; risk was not higher with younger mud. conclusions: in older patients receiving reduced intensity conditioning, msd remain the optimal choice. however, when not available, young mud provide comparable results. disclosure: nothing to declare background: there is growing evidence that community acquired respiratory virus (carv) increase the risk of pulmonary invasive fungal disease (ifd) in recipients of allogeneic hematopoietic stem cell transplantation (allo-hsct). to date, there is a lack of knowledge regarding the rate of ifd, risk factors (rfs) as well as the most critical period for the development of a later ifd after carv infections in allo-hsct recipients. methods: in this prospective observational study, we retrospectively analyzed the effect of carv on the development of a later ifd in a consecutive cohort of 287 allo-hsct adult recipients who developed 597 carv infectious episodes from december 2013 to december 2018. respiratory virus in upper and/or lower respiratory tract specimens were tested using multiplex pcr panel assays. results: overall, 29 out of 287 allo-hsct recipients (10%) developed ifd within 2 months after a carv episode at median of 21 days (range 0-158 days) from the day of carv detection. all the ifds involved the lungs and in 28 cases (97%) the diagnostic was ia accomplishing criteria of probable (n= 26) or proven (n=2). of note, 26 out of 29 ifd (91%) occurred within the first year after transplantation. the overall rate of ifd after carv episodes was 5% whereas this rate was higher in recipients developing carv during the first year of transplant (7%). ifd was diagnosed in 25 out of 203 with carv lower respiratory tract disease (lrtd) episodes (12%) compared to 4 out of 394 carv upper respiratory tract disease (urtd) (1%) (p= 0.0001). twenty-three out of 133 carv episodes involving the lrtd during the first year after transplant (17%) developed ifd. we did not found differences in ifd rates according to the type of carv identified. multivariate analysis identified 4 rfs for ifd: the use of atg as a part of conditioning [odds ratio (or) 2.7, 95% confidence interval (c.i.) 1.2-3.4, p= 0.01], carv lrtd (or 11.8, 95% c.i. 3.8-36, p= 0.0001), carv infection during the first year of transplant (or 5.9, p731 natural killer cell alloreactive haploidentical stem cell transplantation for multiple myeloma patients catharina elssen 1 , lotte wieten 1 , peter von dem borne 2 , ellen meijer 3 , gerard bos 1 1 maastricht university medical center, maastricht, netherlands, 2 leiden university medical center, leiden, netherlands, 3 amsterdam university medical center, location vumc, cancer center, amsterdam, netherlands background: in the past years many new drugs for multiple myeloma (mm) have been developed and are responsible for a increase in survival. notwithstanding such progress, mm remains incurable. results from allogeneic stem cell transplantation (sct), including haploidentical transplantation, in mm has shown clinical results. however, these responses are only observed in a minority of patients. we hypothesize that this observation might be due to differences in natural killer (nk) cell alloreacitvity, since we have shown in in vivo and in vitro models that mismatched alloreactive nk cells hold the capacity to kill mm cells. the aim of this prospective phase 2 study is to evaluate if kir-ligand mismatched haploindentical bone marrow transplantation (bmt) with post-transplant cyclophosphamide will improve progression free survival (pfs) in poor risk mm patients. methods: poor risk mm patients, aged < 66 years were enrolled if they were responsive to their last line of therapy. poor risk was defined as, high-risk cytogenetics, or relapse within a year after autologous sct, or treated with three or more previous lines of therapy. a prerequisite of enrolment was the possibility of an nk cell mismatch and availability of a mismatched family donor. patients were excluded if donor-specific hla-antibodies were present. patients received a haploidentical bmt with a non-myeloablative conditioning regimen and post-transplant cyclophosphamide. primary endpoint is pfs at 1,5 years. secondary endpoints are engraftment, bone marrow reconstitution, nk cell reconstitution and repertoire, graft versus host disease (gvhd), infections and non-relapse mortality (nrm) at 1,5 years. results: in total 12 poor risk patients were included in the study of which 10 could be evaluated for the primary end point. graft failure and disease progression before transplant rendered the remaining two patients not evaluable. at this interim analysis 7 patients have already reached the 1,5 years of follow up, 5 relapsed within 1,5 years and 2 died due to treatment related infections, without showing progression of disease (20% nrm). average time of progression is 105 days (60-270 days). two of the remaining patients at follow up, still show responsive disease (days 210 en 120). the average time to neutrophil reconstitution is 19 days (14-28 days). all evaluated patients (6/10) show nk cell reconstitution with a mature phenotype in the bone marrow and peripheral blood by day 60. three patients developed acute gvhd (25%) of which 2/12 grade i-ii agvhd and 1/12 patient showed a grade iv agvhd. treatment related mortality was 3/12 (25%), which was in all cases due to infectious disease. conclusions: our interim analysis of mismatched haploidentical bmt in mm showed that the treatment is feasible and forms a possible platform for immunotherapeutic strategies. the majority of patients showed an early disease progression. we predefined that with a pfs of 25% at 1,5 years we would qualify this treatment option successful. with only two patients still in remission this goal will not be achieved and we hypothesize that the late nk cell reconstitution (day 60) is responsible for the lack of response. clinical background: mscs are known to have immune modulatory capacity and may be effective in the treatment of patients with acute gvhd. however clinical studies yielded inconclusive results which was in part due to the great heterogeneity of the msc used. the off-the-shelf msc preparation "msc-ffm", generated by a proprietary pooling process, selection by plastic-adherence, expansion for an aggregate four weeks followed by cryopreservation until use, is available in germany through a national marketing authorization. "msc-ffm" is indicated in steroidrefractory agvhd, dosed at 1-2 x10 6 /kg bw i.v. in four doses one week apart. methods: we report seven consecutive pediatric patients (median age 7.49 y), who received "msc-ffm" from unrelated hla disparate donors between december 2017 and november 2018 in our institution. we gave msc infusions to 6 patients with steroid-refractory grades iii-iv agvhd and one patient who had therapy-refractory background: regulatory t cells (treg) are known for their immunosuppressive function and have proven successful as graft-versus-host disease (gvhd) prophylaxis after allogeneic bone marrow transplantation in a number of preclinical as well as first clinical studies without compromising graft-versus leukemia (gvl) effects. in murine models of acute gvhd lymph node homing capacity via cd62l (l-selectin) proved to be essential for disease prevention. yet, treg recruitment from lymph nodes to peripheral sites of ongoing gvhd also seems necessary to achieve maximum protective as well as therapeutic effects. the chemokine receptor ccr4 directs activated t cells to sites of inflammation, thus high ccr4 expression should facilitate treg homing to affected gvhd target organs. with this project we lay the foundation for future in vivo studies of treg therapy for gvhd by upregulation of ccr4 expression. methods: we performed systematic ex vivo analysis of ccr4 expression on murine naive and memory conventional (tconv) and regulatory t cells isolated from spleen, blood, bone marrow, lymph nodes, liver and lung. cells were stained for characteristic surface and intracellular markers and characterized by multiparametric flowcytometric analysis. ccr4 expression kinetics following stimulation were analysed in tconv and treg isolated from murine splenocytes by facs and polyclonally activated by anti-cd3/cd28-coated beads in the presence of exogenous il-2. expression was monitored by daily flow cytometric analysis. ccr4 overexpression was induced by transduction of expanded treg with ccr4 mrna via electroporation. expression kinetics were monitored by facs, receptor function was tested in transwell migration assays using ccr4 ligands ccl-17 and ccl-22. results: systematic analyses showed higher ccr4 expression on memory treg than on their naive counterpart in all examined organs with bone marrow samples displaying the greatest disparity. memory treg showed higher ccr4 expression than memory tconv in all analysed organs, except for lymph nodes where both memory populations revealed equal expression levels. stimulation of in vitro expanded treg and tconv lead to a strong increase in ccr4 expression with maximum levels on d3 and d2 respectively, whereas restimulation (d7) resulted in no further relevant ccr4 expression on treg. we performed systematic optimization of stimulation and mrna-electroporation conditions to reliably achieve highlevel short-term ccr4 expression. transduction of treg on d11 of in vitro expansion resulted in a strong ccr4 expression, with maximum levels 2h after electroporation and strong ccr4 expression being detectable for at least 8h. transwell migration assays showed enhanced migrational properties of mrna-electroporated treg towards ccr4 ligands. analyses performed 2h and 16h after electroporation showed persistent migration even though measured ccr4 surface expression had already declined significantly. conclusions: we showed that high ccr4 expression can be detected on memory treg in all analysed organs. since in vitro stimulation of murine treg did not reliably induce ccr4 expression, we established a protocol for ccr4 mrna-electroporation. electroporated cells showed stable short-term ccr4 expression and enhanced migrational properties towards ccr4 ligands in vitro. future studies will show whether the induction of short-term ccr4 expression will facilitate in vivo homing of adoptively transferred treg to sites of ongoing gvhd and thus mediate long-term inflammation suppression. disclosure: the authors have no conflict to disclose. survival and immune reconstitution of syngeneic, haploidentical and allogeneic hematopoietic stem cell transplantation in atm-deficient mice ruth pia duecker 1 , patrick c. baer 2 , stefan zielen 1 , ralf schubert 1 from allo-hsct. it´s not necessary to do chemotherapy before transplantation for patients with bone marrow blast cells more than 10% at the time of diagnosis. [[p738 image] 1. figure 1 the hci-ct of patients before transplantation and occurance of grade iii-iv agvhd on overall surviv] background: 1. allogeneic haematopoietic stem cell transplantation (sct) offers the chance of cure for patients with transfusion-dependent thalassemia (tdt). based on the non-neoplastic nature of this condition sct approaches urgently require to prove both efficacious and safe. methods: 2. we report on 13 children, adolescents and young adults (median age: 6 years; range 2-28 years) with tdt receiving sct from an hla-matched donor (mud n=8, msd n=3, mfd n=2) in our center from 2011-2017. all patients received the same treosulfan-based conditioning regimen (treosulfan 3x14g/m2, fludarabine 4x40mg/m2, thiotepa 1x8mg/kg). gvhd prophylaxis was based on atg-fresenius™ (3x10mg/kg, if mud or mfd as donor), csa (with taper from day +270) as well as mtx (day 1,3,6,11) in 9/13 and mmf in 4/13 patients with mtx toxicity, respectively. stem cell source was bone marrow in 8, peripheral blood stem cells in 4 and cord blood in 1 patient. prior to transplantation 6 children received cytoreductive treatment with azathioprine, hydroxycarbamide and intensified erythrocyte transfusion. iron elimination therapy was carried out in 12/13 children with deferasirox. among the 11 patients with available ferris-can™ analysis 4 patients showed substantial liver iron overload (liver iron > 8mg/g) despite intensive chelation prior to sct. results: 3. all patients achieved leukocyte engraftment at median day +23 (range 15-39), however two patients required a cd34-selected pbsc boost on day + 36 and day +50 based on delayed platelet and/or erythrocyte engraftment. nine patients exhibited full donor chimerism in the bm at day +30, the other 4 showed mixed chimerism with < 5% autologous cells. on day +360 peripheral blood chimerism was complete in 12/13 patients with the remaining patient exhibiting stable split chimerism with 100% donor-derived erythrocytes and 60-80% autologous myeloid cells. acute gvhd was observed in three patients (grade 2: n=1, grade 3: n=2). however, all patients responded to immunosuppressive therapy with steroids ± ecp and re-initiation of cni (n=1). one patient suffered background: patients with relapsed or refractory acute myeloid leukaemia (aml) have a poor prognosis. allogeneic hematopoietic stem cell transplantation is the only curative option. however, allogeneic transplantation with active leukemia failed to improve significantly the longterm outcome. to improve the outcome of allo-hsct in such high-risk and refractory patients, sequential schedule of cytoreduction therapy followed by nonmyeloablative conditioning has been developed. methods: to evaluate the outcome of sequential intensified conditioning regimen followed by allogeneic hematopoietic stem cell transplantation (allo-hsct) for refractory acute myeloid leukemia (aml). results: a total of 20 patients with primary or secondary refractory aml transplanted between june 2011 to july 2018 were included. refractoriness was defined as primary induction failure, relapse within 6 months from induction/ consolidation chemotherapy or second relapse. median age is 39 years (1 to 61). the salvage chemotherapy administered was flag-ida. two patients did not receive intensive chemotherapy because of no recovery after induction chemotherapy. seven days after the end of flag-ida, a reduced intensity conditioning consisting of fludarabine,60 mg/m2, thiotepa, 5mg/kg and busulfan 6,4 mg/kg i.v. (n=12) for haploidentical donors or fludarabine plus busulfan (n=8) for hla identical sibling or unrelated donors was administered. graft-versus-host disease (gvhd) prophylaxis consisted of tacrolimus and mycophenolate mofetil. the mycophenolate was withdrawn at day +35 post-transplantation and tacrolimus at day +100. donor lymphocytes (dli) were infused in patients without agvhd at day +120 post-transplantation. seventeen patients achieved complete donor chimerism, 3 patients progressed early and 1 patient died before engraftment. one of the patients which recovery was with persistent leukemia reached donor chimerism after immunosupression discontinuation. ten patients are alive in complete remission. median follow-up of survivors is 28 months (range: 6-43). five patients died of leukemic progression, 3 as result of gvhd and 1 suffered intracranial hemorrhage. five patients received prophylactic dli. the incidence of acute moderate-severe gvhd and moderate-severe chronic gvhd were 70% (n=14) and 50% (n=7), respectively. the non-relapse mortality was 21% (n=4), mainly due to acute gvhd (n=3) . the 2-year cumulative incidence of relapse posttransplantation was 36.8%. the probability of relapse was 57%±13%. the 2-year os and dfs were 46% ± 12% and 38% ± 12% conclusions: the strategy of sequential chemotherapy followed by allohsct ± prophylactic dli has an acceptable toxicity profile and improves both the relapse rate and the survival for refractory aml patients. disclosure: nothing to declare background: the concept of immunological intervention to prevent relapse after hematopoietic stem cell transplantation is associated with the assessment of the chimerism status. distinguishing patient and donor hematopoiesis is usually performed by str-pcr, a powerful method developed for forensic purposes. however, this method shows restrictions with respect to detection limit, preciseness, and the possibility of automated read out. digital pcr could circumvent some of these limitations. methods: recently, validated for the bio-rad droplet digital platforms, the biotype mentype digitalquant assay was released. the assay uses indel polymorphisms on chromosomal dna to distinguish patient and donor hematopoiesis on a fret hydrolysis assay basis ("taqman assays"). thus the assay in principle is applicable on the chamber based 3 d digital pcr system (thermo fisher). due to different reaction chemistry and physical properties of thermal transfer between the digital pcr platforms protocols are reasonably not fully compatible which would lead to lower fluorescence intensities and poor signal resolution on the solid chip based thermo fisher 3d platform. an adjusted pcr protocol was established and optimized using representative markers, followed by determination of tolerable and optimal amount of input dna. specificity, sensitivity and reproducibility testing with artificial mixed samples preceded the extensive verification by comparative measurement of clinical samples (n=59) and ring-trial samples (n=20). source to allogenic bm or pbsc. ucb units are immediately available for transplantation as they are frozen and banked with defined hla typing and it has an advantage for patients who need urgent transplantation. in addition, a higher degree of hla mismatch appears to be acceptable with a comparatively lower risk of acute and chronic gvhd. meanwhile, a higher incidence of engraftment failure, delayed neutrophil and platelet recovery, and posttransplant immune disorders including pre-engraftment immune reactions (pir) are major problems in unrelated ucbt. methods: in our institute, gvhd prophylaxis in ucbt was changed after march 2013. between january 2007 and march 2013, thirty-two patients received tacrolimus plus methylprednisolone (tac/mpsl) and between april 2013 and january 2018, thirty-one patients received tac plus methotrexate (tac/mtx) for gvhd prophylaxis. to investigate better gvhd prophylaxis after ucbt, we compared transplant outcomes after ucbt using gvhd prophylaxis with tac/mpsl (n=32) and tac/mtx (n=31) in single-pediatric transplantation center. results: the cumulative incidence of neutrophil engraftment at day 30 in tac/mpsl group was 70.1 % and 90.3 % in tac/mtx group (p=0.09). median time of neutrophil engraftment was 2 days earlier in tac/mtx group (17 days) than tac/mpsl group (19 days). according to pir, and acute gvhd, tac/mtx group showed superior outcomes; the incidence of pir (p=0.020) and the cumulative incidences of acute gvhd at day 100 (38.7 vs 68.8 %, p =0.045 for grade ii-iv, 9.7 vs 34.4 %, p=0.021 for grade iii-iv) was significantly lower in tac/mtx group than in tac/mpsl. however, the incidences of relapse (p=0.921) and cytomegalovirus viremia (p=0.908), and estimated overall survival (p=0.87) and event-free survival (p=0.88) were comparable between two groups. conclusions: our results indicated that gvhd prophylaxis with tac/mtx had favorable effects; reduced incidence of rip and acute gvhd after ucbt without any negative influences. disclosure: nothing to declare transfer of donor regulatory t-cells after atg reconditioning cures severe refractory gvhd and leads to long term persistence of regulatory t-cells in the recipient cells on a clinimacs® plus device (miltenyi biotec). the cell product contained 83% foxp3 + t-cells. the patient received 3,4x 10 5 /kg t reg on day +177. subsequently intestinal gvhd decreased and finally resolved. three months after the first t reg transfer the patient got a second t reg transfer (3,4x 10 5 /kg) on day +281 due to decreasing t reg levels. thereafter t reg persisted and there was no recurrence of gvhd. the patient is well with low dose sirolimus and prednisone as the only immunosuppressants and is particularly recovering intestinal function. conclusions: this case illustrates an unusually severe acute gvhd after matched sibling sct. transfer of donorderived t reg was able to cure severe and refractory gvhd after t-cell ablation by atg. transferred t reg persisted in the recipient for a long period and did not lead to any adverse events. disclosure: no disclosures to declaim allogeneic hsct for patients with transfusion dependent anemia from matched and mismatched donors julia fekadu 1 , andrea jarisch 1 , jan sörensen 1 , emilia salzmann 1 , eva rettinger 1 , andré willasch 1 , shahrzad bakhtiar 1 , thomas klingebiel 1 , peter bader 1 after first asct. the mean harvest for patients receiving dhap was 10,02x10 6 cd34(+) cells/kg, 3,04 x10 6 cd34 (+) cells/kg for cy, 8.56 x10 6 cd34(+) cells/kg for igev, 6,76 x10 6 cd34(+) cells/kg for ice, 10,52 x10 6 cd34(+) cells/kg for choep. the patient mobilized with vtd-pace achieved 2,2 x10 6 cd34(+) cells/kg after 2 apheresis. 19 of the patients achieved the target number of > 2x10 6 /kg cd34+ cells after 1 apheresis, 15 after two, and 2 after three apheresis. the median time to apheresis was 13 days (8-18) without significant difference between the regimens. the mean wbc count at the time of apheresis was 42,8x10 9 /l after dhap, 34,5 x10 9 /l after cy, 21,2 x10 9 /l after igev, 23,1 x10 9 /l after ice, 45,8 x10 9 /l after choep. there was correlation between wbc and cd34 harvested cells (p=0.005). grate 3-4 thrombocytopenia was found in 8 patient (5 dhap, 1 ice, 1igev, 1 vtd-pace). grate 3-4 anemia was registered in 3 patients (2 dhap and 1 vtd-pace). no correlation was found between the cd 34+ harvest and the age, number of previous lines chemotherapy, the response before mobilization, the type of the lymphoma and the clinical stage. conclusions: our results demonstrate that the chemo-g-csf protocols have comparable effectiveness with accepbackground: cytomegalovirus (cmv) may cause severe complications in recipients of allogeneic haematologic stem cell transplantation (allohsct). letermovir (ltv, 480/ 240mg daily without/with co-administration of cyclosporine) was recently licenced only for cmv prophylaxis in adult allohsct-recipients. paediatric data as well as data on cmv therapy are missing so far. methods: we administered letermovir 240mg orally once daily (with no co-administration of cyclosporine a) to 2 paediatric patients after allohsct. edta-plasma were occasionally obtained at different time points and frozen for determination of letermovir levels using liquid chromatography/mass spectrometry (lc-ms/ms). results: for details on patients, treatment and cmv load see table 1. in short periods of letermovir administration, cmv blood levels became negative in both patients. considering the lacking safety data in paediatric patients, we stopped letermovir treatment in both patients, when liver parameters increased. in patient 1 hepatopathy turned out to represent histologically proven graft versus host disease (gvhd). in patient 2 liver parameters further increased despite withdrawal for another 4 weeks, however, hepatopathy was only mild and self-limiting. both patients additionally received other possibly hepatotoxic substances (mycophenolate mofetil and trimethoprim/ sulfamethoxazole). letermovir plasma levels were 11.900ng/ml (2h), 11.600ng/ml (12h), 1.640-2.190ng/ml (median 1.990ng/ ml, n=3, 24h) and 1.050 ng/ml (36h after administration). conclusions: during short letermovir treatment, we observed fast resolution of cmv viraemia as well as rising liver parameters in both patients. while elevated liver parameters represented gvhd in 1 patient, a causal relationship with letermovir might be considered in the other patient. letermovir peak levels after administration of 240mg were within ranges reported in adults after administration of 480mg while trough levels were higher indicating differences in pharmacokinetics in terms of delayed clearance. inguinal lymphadenomegaly. after failure to respond to seven conventional treatment lines: methotrexate, cop (cyclophosphamide, vincristin and prednisone); gemcitabine; puva; interferon; acitretin and extracorporeal photopheresis), allogeneic hsct from an identical hla male donor was indicated. the non-myeloablative conditioning consisted of fludarabine (200mg / m2), cyclophosphamide (50mg / kg) and total body irradiation(tbi) (400cgy). prophylaxis of graft versus host disease (gvhd) was performed with cyclosporine (3mg / kg) and mycophenolate mofetil (30mg/kg). after conditioning, there was improvement of pruritus and involution of the skin. bone marrow infusion occurred on 2/9/2018 (d0). on d + 83 he presented recurrence of skin lesions of fmf. donor lymphocyte infusion (dli) was performed (1 x 10 7 cd3 + cells / kg / recipient). he presented oral lichen and diarrhea respectively as manifestations of gvhd on d + 104 and d + 112. as infectious intercurrence, hemorrhagic cystitis occurred by bk virus 3 months after the first dli and he received conservative treatment and remained without systemic immunosuppression. nine months after hsct, a second dli (5 x 10 7 cd3 + cells / kg / receptor) was performed and at this time the patient is without clinical manifestations of fmf or gvhd. conclusions: the clinical response of the presented case confirms what has been reported in the literature. ctcls appear to be particularly susceptible to gvl effect, which makes hsct a potential cure for advanced ctcls in eligible patients. the timing to perform hsct in the clinical course of the disease remains a matter to be settled clinical trial registry: not applicable disclosure: no conflict of interest p755 abstract already published. methods: we applied next generation sequencing (pgm, ion torrent/ fischer lifetechnologies) to an unselected cohort of 414 patients (176 female, 238 male, median age 59 (2-80) years) who had been referred for allogeneic stem cell transplantation due to the presence of a high-risk myeloid disorder dnmt3a r882h (3.1%) .98), cebpa (18.1%; ceb-pap198s (16.9%) vus) kras (6.5%; krasr161r (3.1%) vus), and kit (3.9%; kitm541l (3.9%) .74). patients displayed a median of 3 sequence variants (aml, mpn and cmml patients each 3; mds, saa and patients with other, non-malignant hematologic diseases each 4 sequence variants found most frequently in aml were cebpap198s (13.2% of all sequence variants in patients with aml, p< 10 -3 , chi² test) in patients suffering cmml, dnmt3ar882h was particularly frequent (4.8% of all sequence variants in cmml, p=.064). asxl1e1102d (2.1% of all sequence variants in other, non-malignant hematologic diseases, p=.699), idh2r140q (2.1%, p=.677) and krasr161r (4.2%, p=.010) were frequent in other, non-malignant hematologic diseases. in saa, nrasg12d (4% of all sequence variants in saa patients; p=.005) was frequently found, as were dnmt3ak456fs* (8%, p=.007), tet2l1721w (8%, p=.080) and tet2i1762v (8%, p=.001). conclusions: taken together, these data show that vus occur with high abundancy in this high-risk cohort of patients, and that they differ in frequency between various myeloid disorders methods: retrospective analysis of patientswho experienced either hematological relapse or progressed to aml after allo-hsct and were treated with azacitidine for this indication at 7 hematological centers in poland. the primary end-point was overall survival (os), the secondary -response rate. results: 36 patients, 22 males (61.1%), median age 52 (range, 15-66), were enrolled. the primary indication for allo-hsct was aml median time from allo-(95%ci: 4.9-11); with 2-year os of 17.2% (95%ci: 5.1-35.2). for patients stratified according to ebmt aza relapse prognostic score 2-year os was: 9 prophylaxis of agvhd: with atg -atgam 60mg/b.w.-39pts(36,8%), posttransplant cyclophosphomide (ptcy) 50mg/b.w. on d+3;d+4-67pts (63,2%) conclusions: unmanipulated haplo-hsct in 1 st -2 nd cr in children and adolescents with high risk al allows achieving the long-term survival in 64,7%. the use of g-csf stimulated unmanipulated haplo-bm is associated with a satisfactory rate of engraftment. the main cause of death in our study was relapse after allo-hsct. the frequency of acute and chronic gvhd was acceptable, 10-years grfs rate of 35,3% in 1 st -2 nd cr represents good quality of life following unmanipulated haplo-hsct and therefore may be recommended as option for use in children and adolescents with high-risk al. disclosure: nothing to declare background: b7-h3 (cd276) is thought to act as an immune checkpoint and regulates t and nk cell responses. it is highly overexpressed on many solid tumors while on healthy tissue protein expression is limited. this makes b7-h3 an interesting target for cancer immunotherapy. in highrisk neuroblastoma patients, targeting disialoganglioside gd2 with the recently approved monoclonal antibody (mab) ch14.18 after autologous or allogeneic sct, significantly improves survival. however, gd2 expression is heterogeneous and ch14.18 causes severe adverse effects. thus, we evaluated b7-h3 as an alternative or additional target antigen. we investigated different anti-b7-h3 mab constructs and mab-cytokine fusions (immunocytokines) for their ability to elicit antibody-dependent cellular cytotoxicity (adcc) using expanded γ/δ t cells of healthy donors, chex5lf-il2 was confirmed to be the most effective mab construct. interestingly, chek5-il2 showed comparable target cell lysis -however, lysis was only transient while chex5lf-il2 mediated permanent target cell lysis. using patient pbmcs after receiving allogeneic sct, chex5lf-il2 and ch14.18 mediated comparable lysis. calculated specific lysis of lan-1 (after 96 hrs.; in ascending order): targets + effectors w/o mab (22 %) conclusions: b7-h3 is a suitable target antigen in case gd2 expression is low or absent. immunocytokines and fcoptimized mabs targeting b7-h3 might increase the efficacy of immunotherapy in gd2-negative tumors and in combinatory approaches. until now, the low-fucose immunocytokine chex5lf-il2 seems to be the most promising anti-b7 great ormond street hospital, nhs foundation trust petersburg/ raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, rehabilitation medicine, saint petersburg, russian federation, 2 first i. pavlov state medical university of st. petersburg/raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, bone marrow transplantation for pediatric solid tumor petersburg/raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, pediatric hsct outpatients, saint petersburg, russian federation 1 university hospital carl gustav carus james`s hospital diagnosis was aml in 52%, all in 36% and mds in 12% of patients. 90% of patients received pbsc grafts, 10% received unmanipulated bone marrow grafts. os at 2 years was 70% in msd/mud-atg, 76% in haplo-ptcy, 80% in mmud-ptcy and 43% in mmud-atg groups (p=0.0027). in a multivariate cox model non-relapse mortality was 17%, 17%, 4.5% and 42% in msd/mud-atg, haplo-ptcy, mmud-ptcy and mmud-atg groups, respectively. cumulative incidence of acute gvhd grade 3 or 4 was 6%, 9.5%, 3% and 12% after in msd/mud-atg, haplo-ptcy cumulative incidence of chronic gvhd was 26% in the msd/mud-atg group uwe platzbecker 3 , verena wais 4 republic of china background: there are two most noteworthy strategies of haploidentical stem cell transplantations (haplo-hsct), the baltimore post-transplantation cyclophosphamide (ptcy) with or without anti-thymoglobulin (atg), and the beijing g-csf primed bone marrow (bm) plus peripheral blood stem cells (pbsc) (giac). however, the comparison of these two modalities is scarce. in this study, we aim to compare these two approaches for hematological malignancies based on the taiwan blood and marrow transplantation registry (tbmtr) with the comparable cd34 infusion amounts, the neutrophil engraftment time were statistically distinct among these three groups [d+12 (group 1) vs. d+15 (group 2) vs. d+17 (group 3), respectively as to the graft-versus-host disease (gvhd), the patients in group 1 had more grade ii-iv but similar grade iii-iv acute gvhd compared with others (grade ii-iv: 60.0% vs. 37.5% vs. 21.6%, respectively conclusions: haplo-hsct with different strategies is a feasible treatment modality for hematologic malignancies in taiwan. regarding the retrospective nature and limited patient numbers of this study republic of china background: we previously presented a low-resolution hla analysis of cedace, the voluntary portuguese bone marrow donor registry (ebmt 2011, poster p1127) and, more recently, its epidemiological characterization (ebmt 2018, poster b348). currently, cedace is one of the largest bone marrow donor registries in the world, including nearly 4% of the country's population, twice the number of donors present in 2011. the current work is an update on the most common hla haplotypes found in cedace results: of the 396545 donors in the cedace registry, 99.51% were typed in at least 3 loci (hla-a/-b/-drb1), 43.91% in 4 (including hla-cw), and 1.28% in 5 (including hla-dqb1)the 5, 25 and 150 most common haplotypes accounted for, respectively, 8.9%, 23.5% and 50% of the haplotypes found in the entire registry. the five most common haplotypes at the low-resolution at the 3 loci, low-resolution level, out of 394621 donors, 167505 individual genotypes were identified, leading to an hla matching probability at this level of 57.6% hid-hsct) have a stronger anti-leukemia effect compared to identical sibling donor hsct(isd-hsct) in high-risk features .but in refractory/relapsed(r/r) aml patients who not in remission status, it is unclear whether it also augments the gvl effect antithymocyte globulin was used in haploidentical hsct. unmanipulated bone marrow and peripheral blood stem cells for all patients. cyclosporine, short-term methotrexate were employed for gvhd prophylaxis. mycophenolate mofetil included in hid-hsct. performed multivariate analysis for all patients of pretransplantation variables and developed a predictive scoring system for survival according to adverse factors. results: the total survivor median period of follow up was 46 (20-73) months. hid -cohort had higher 5-year actuarial of os multivariate analysis showed isd-hsct,standard conditioning regimen and less than 50% proportional reduction in blast percentage pre-≥2. conclusions: haploidentical donor compared to identical sibling donor had better anti-leukemia effect in allo-hsct for r/r aml in nr status conditioning protocol was melphalan 200mg/m2 for mm and beam for nhl. the quantification and characterization of γδt cells in peripheral blood samples were performed by flow cytometry based on the expression of cd3/cd45/vδ1/vδ2/vγ9/cd27 at 30, 60 and 100 days after ahsct. percentage (%) of γδt cells represented the proportion of these cells among all t cells. results: median age at ahsct was 61 (40-70) years, 63% male. median time from diagnosis to mobilization was 8 (4-52) months, after a median number of therapeutic lines of 1 (1-3); 7pts (13.0%) received radiotherapy. seventeen pts (31.5%) were re-mobilized with plerixafor±g-csf (25% mm vs 60% nhl there was no difference in febrile neutropenia incidence (p=0.766), timeto-engraftment (p=0.093), time-to-neutrophils>500/μl (p=0.174) or erythrocyte transfusions (p=0.076). however, there was more time-to-platelets>20,000/μl (4 vs 8 days; subpopulations did not affected pfs. conclusions: our results showed that pts mobilized with plerixafor need more collection volume (less cd34+cells, higher dmso). plerixafor negatively affected platelet recovery, with similar hematologic and immune recovery for the remaining variables. its use was associated with higher %vδ1+, suggesting that it induces an antineoplastic phenotype. more studies with larger samples and follow-up period are needed to evaluate plerixafor results: total 1156 ascc procedures were carried out in 1072 patients over 18 years, once in 985 patients, twice in 83 patients, three times in 3 patients, and four times in 1 patient. non-hodgkin lymphoma (nhl) comprised 56.7% of all cases (n = 655) 001), total number of chemotherapy cycles before ascc (or 1.06, 95% ci 1.004-1.13, p = 0.035), failure to achieve at least partial response before ascc (or 2.97, 95% ci 1.01-8.73, p = 0.048), total number of days receiving g-csf for mobilization (or 1.10, 95% ci 1.002-1.23, p = 0.045), and salvage use of plerixafor (or 3.78, 95% ci 1.92-7.47, p < 0.001) were found to be independent factors associated with failure of ascc. at the end of the study period, 90.9% of successful collections (n = 962/1058) were used for asct, 4.5% (n=48/1056) were in storage awaiting transplantation hôpital necker-enfants malades melf 140 methods: a total 103 ahsct candidates [median age: 57(18-75) years; male/female: 54/49] were included in this study. twenty-seven patients (26.2%) were diagnosed as non hodgkin's lymphoma, 8 patients (7.8%) hodgkin's lymphoma, 52 patients (50.5%) multiple myeloma, 12 patients (11.6%) acute myeloid leukemia, 3 patients (2.9%) plasmocytoma and 1 patient (1%) testis cancer. premobilization serum 25-hydroxy vitamin d (25-oh d) levels were measured with immunoassay method peripheral cd34 + cell count background: some published data suggest a positive effect of iron chelators on the risk of post-transplant relapse, with an improvement in overall survival after allogeneic hematopoietic stem cell transplantation (hsct) conditioning regimen consisted to intravenous bu 130 mg/m 2 and, flu 40 mg/m 2 d-6 to d-3. gvhd prophylaxis included atg 2.5 mg/kg on d-2 and d-1, ciclosporin and methotrexate. all patients received peripheral blood stem cell transplant from an identical hla-related donor. iron chelation consisted on deferasirox (exjade ® ) 10-20 mg/kg/day, started at day 100, if serum ferritin level ≥ 800 -1000 ug/l and stopped when the level decreased below 600 ug/l or normalized at day 100 after transplant, 35/ 40 patients were evaluable (g1= 20 pts), (g2 =15 pts). patients were abo compatible (66%), had major incompatibility (29%), and 6% had minor incompatibility. median serum ferritin level at day 100, were 1200 ug/l (833-4449) and 1200 ug/l (123-1853) in g1 and g2 respectively with a median follow-up of 34 months, (12-68), disease relapse incidence was higher in patients who did not received iron chelation treatment (g1: 39.8%) versus those who received oral deferasirox (g2:15.6%) (figure 1), but the difference was not statistically significant conclusions: these results deserve more investigation choep (n=5), and 1patient received vtd-pace 10 μg/kg depending on the protocol. the aim was to collect at least 2x10 6 cd34(+) cells/kg body weight. results: forty patients all patients were stage iii and iv at diagnosis. 29 of the patients were mobilized after one line of treatment, six after two lines of treatment and five after 3 and more lines of treatment alexandra martínez-roca 1 , gerardo rodriguez-lobato 1 , gonzalo gutierrez-garcia 1,2 , maria suárez-lledó 1 background: hematopoietic stem cell transplantation (hsct) is an established procedure in lymphoma background: the role of inflammatory cascade in tumor microenvironment has been demonstrated in several studies. as a part of this issue, elevated neutrophil/lymphocyte ratio (nlr) was shown to be associated with an adverse prognosis, particularly in solid tumors. the aim of this study is to determine the impact of pre-transplant nlr on early transplant complications, as well as post-transplant relapse and survival.methods: a total of 119 lymphoma patients [median age: 49(18-71) years; male/female: 64/55] who underwent autologous hematopoietic stem cell transplantation (hsct) were included in this retrospective study.results: the initial diagnosis was hodgkin lymphoma (hl) in 35 (29.4%), b-cell non-hodgkin lymphoma (nhl) in 67 (56.3%) and t-cell nhl in 17 (14.3%) patients. of 116 patients who were evaluated for pretransplant disease status, 52 patients (44.8%) were in complete remission, 45 patients (38.8%) were in partial remission and 19 (16.4%) patients had refractory disease. median pre-transplant nlr was found to be 3.42(0-97) . when the study population was divided into two subgroups as "low-" and "high-nlr", based on median nlr value, number of febrile days were found to be relatively higher in the low-nlr group (p=0.051). a positive correlation was demonstrated between nlr and lactate dehydrogenase levels (r=0.218; p=0.03); and nlr and ferritin levels (r=0.277; p=0.003). at a median follow-up of 24 (1-264) months, overall survival (os) was found to be better in the low-nlr group without statistical significance [25 vs 13(1-91) months; p=0.069]. in univariate analysis, pre-transplant nlr represented a significant impact on os (p=0.021). other prognostic factors were age (p=0.041), platelet engraftment (p=0.018), post-transplant relapse (p=0.009) and pre-transplant ferritin level (p< 0.001). the permanent impact of ferritin on os was confirmed in multivariate analysis (p=0.008).conclusions: in this study, an adverse impact of elevated pre-transplant nlr on os was demonstrated in autologous hsct recipients with lymphoma. as a predictor of prognosis, nlr may be considered as a safe and cost-effective parameter. further studies are required in order to use this predictor in routine clinical practice.background: high cure rates in childhood diseases have been achieved by stem cell transplantation (sct). however, there is little knowledge concerning recovery of the immune system and community-acquired infection after sct. here we studied the long-term reconstitution of the immune system and incidences of community-acquired infection after sct.methods: we reviewed medical records for 44 patients (m/f: 31/13, median age: 5 years (range: 1-22years) who were treated in the department of pediatrics, hokkaido university hospital. we analyzed cd4-positive cell counts, serum immunoglobulin g (igg) levels, and incidences of community-acquired infection until 2 years after sct. indications for sct were all in 8 patients, aml in 4, aa in 6, nb in 5, rms in 2, jmml in 1, nhl in 1, cgd in 5, was in 3, xscid in 2, apds in 2, cd40ld in 2, and other solid tumor in 3 patients. stem cell sources were autologous pb/bm in 6, allogenic bm in 23 (9 related and 14 unrelated) and allogenic cb in 15 patients. in this study, we excluded patients who relapsed after sct.results: the duration of cd4-positive cell counts < 500/ ml after sct was 14.7±11.4 months in all patients. the durations were 12.6±7.1 months in patients with hematologic malignancies, 11.5±7.3 months in patients with hematologic disorders such as aplastic anemia and pid, 28.8±25.9 months in patients with solid tumor, 16.0 ±10.4 months in patients who received autologous sct, 25.1±22.2 months in patients who received related bmt/ pbsct, 13.8±6.7 months in patients who received unrelated bmt, and 10.5±5.8 months in patients who received cbt. the durations of igg < 500mg/dl after sct were 14.9±11.5 months in all patients, 15.8±9.7 months in patients with hematologic malignancies, 11.6±5.1 months in patients with hematologic disorders, 11.0±8.7 months in patients with solid tumor, 19.0±18.6 months in patients who received autologous sct, 12.0±8.3 months in patients who received related bmt/pbsct, 11.5±5.7 months in patients who received unrelated bmt, and 15.9±8.7 months in patients who received cbt. there was a significantly higher incidence of community-acquired infection from 6 months after sct. there were significant differences in the incidence of community-acquired infections between patients with cd4-positive cell counts of < 500/ml and background: allogeneic hematopoietic cell transplantation (allo-hct) has the potential to cure subgroups of patients with multiple myeloma (mm) but its role is controversial due to high transplant-related mortality. while autologous hct is well established as consolidation after induction therapy using novel agents, allo-hct is still considered experimental due to excessive early toxicity.methods: we retrospectively studied 45 mm patients (pts) ; 25 (55,6%) were males with a median age of 52 years (range: 32-60), who underwent allo-hsct in our center between 2007 and 2018.median time between diagnosis and allo-hct was 40 months (range: 20-143).results: the international staging system (iss) was iss i (33,3%), iss ii (28,9%), and iss iii (37,8%). twentyeight (62,2%) pts received cells from unrelated donor and 17 pts from identical siblings. stem cell source were: peripheral blood (n=25) and bone marrow (n=20). grouprisk distribution using hct-ci and pam index can be seen in figure 1. response was evaluated at day +100: cr (53,3%), vgpr (11,1%), pr (22,2%) and sd (2,2%); response was not evaluated in 5 pts. regarding hospitalization, 11 (24,4%) pts and 22 (48,9%) pts needed readmission in the first 100 days and 365 days post-allohct, respectively, and median days of hospitalization was 27 days (range: 4-92). reasons for first re-admission were: infection (68,2%), gvhd (27,3%) and renal insufficiency (4,5%). twenty-seven (60%) died and death causes were: infection (35,6%), progression (15,6%), secondary tumor (4,4%) and the remainder, gvhd and intracranial hemorrhage. in addition, 26 pts (57,8%) suffered cytomegalovirus (cmv) reactivation.median overall survival (os) was 44 months (range: 11-76). univariate analysis showed that re-admission in first 100 days (hr 4, 19; p=0, 001) , re-admission in first 365 days (hr 3, 6; p=0, 001) , cmv reactivation (hr 2,38; p=0,038), iss (iss-i vs iss-ii and iii; hr 0,347; p=0,033), days of hospitalization in the first 100 days and response at +100 day (cr plus vgpr vs the remainder; hr 0,143; p=0,04) were predictor factors for os. other factors like karyotype, response before allo-hct, hla-mismatch or baseline cmv serostatus, among others, do not impact on os.median os in pts with low hct-ci were 56 months (range: 0-156) vs 6 months in intermediate-high hct-ci background: allo-sct is a potentially curative therapy for patients with multiple myeloma as it provides a graftversus-myeloma effect and a myeloma-free graft. due to increased nrm and unclear os benefit the recent guidelines suggested allo-sct to be used in context of clinical trials focusing on the high-risk patients and those who relapsed early after autograft. reduced-intensity conditioning regimens may improve rate of nrm; however, optimal conditioning regimen is still to be determined. here we studied conditioning regimen with 3 alkylating agents consisting of thiotepa (tt), busulfan (bu) and gvhd prophylaxis with post-transplant cyclophosphamide (post-cy) in high risk myeloma patients relapsing after autograft.methods: a total of 40 patients (m, n=23) with median age of 55 years (range 40-67) underwent an allo-sct (mud, n=19; mrd, n=12; mmud, n=6, haploidentical, n=3) during a period from 2014 to 2018 in university of hamburg. the majority of patients had advanced disease (stage iiiab, 73%) and high-risk cytogenetics (56%). the median response durartion after autograft was 1.8 years (range 0.5-8. 3 ). the conditioning included tt (cum. dosage 10mg/kg), bu (median cum. dosage 9.6mg/kg i.v. or 6.4 mg/kg in elderly patients) and post-cy (cum. dosage 100mg/kg, day +3 and +4). eight patients (all of them received allografts from mmud or haploidentical donors) received additionally fludarabine (flu, cum. dosage of background: the overall incidence of active cmv infection in patients with multiple myeloma (mm) receiving background: several scoring systems have been developed to estimate outcomes in mds patients who undergo allohct. however, none of them have been specifically validated in t cell depleted grafts. the aim of this study is to investigate the prognostic ability of a recently published scoring system (sfgm-tc) in a cohort of patients with mds who underwent tcd transplants.methods: 109 patients underwent a first tcd allohct for mds from 2007 to 2018. the sfgm-tc score (caulier et al. curr res transl med. 2018 ) is performed at day 100 and ranges from 0-8, discriminating low (0), intermediate (1) (2) (3) , and high risk (4-8). additional analyses were performed at day 180 and day 365. a landmark analysis was done at each time point for the day 100, 180, and 365 analyses, respectively. background: hematopoietic stem cell transplantation (hsct) is the only curative procedure for the treatment of myelodysplastic syndrome (mds), but among several limiting factors for its accomplishment, such as the patient´s performance status, is a very relevant issue, i.e., the availability of a compatible hla donor and, when available, very often the donor´s age and comorbidities also constitute factors that hinder this medical conduct. considering this scenario, the possibility of a haploidentical transplantation (ht) has emerged as an option. in latin america, ht has been included as a treatment option since 2014. since then, these patients have been included in the latin american registry of transplantation in myelodysplastic syndrome, making it possible to analyze the viability and results of these transplants.methods: from october 2012 to october 2018, seventeen (17) patients were transplanted with a haploidentical donor and included in the latin american registry. none of these patients had an identical hla (8/8 match) related or unrelated donor. data were obtained from the latin american registry of hsct in mds. the statistical analyses were performed using the software spss, version 23.1 and graphpad prism version 5.0, with significance being set at p < 0.05.results: table 1 shows the patients and their characteristics. all donors were haploidentical. there was a predominance of reduced intensity conditioning, which was performed in 13 patients (76.5%), whereas the others received the myeloablative conditioning. cell source was peripheral blood in 10 (58.8%) and bone marrow in 7 (41.2%) of the patients. graft-versus-host disease (gvhd) prophylaxis in post-hsct was carried out with cyclophosphamide 50mg / kg on d+3 and d+5, cyclosporin from d0 and mycophenolate from d+1. complete hematologic recovery was achieved in 16 (94.1%) patients. the incidence of grade ii-iv acute gvhd was 11.7%, whereas chronic gvhd was 5.8%. one death occurred due to graft failure and none of the patients showed autologous recovery. three other patients died. one on d+210 due to a fungal infection, the second on d+90 due to sinusoidal obstruction syndrome and a third on d+61 due to pneumonia caused by pseudomonas. regarding overall complications, there was a predominance of mucositis (47%), overall infections (35.2%) and reactivated cmv in 23.5% of cases. of the total number of living patients, 8 (47%) achieved complete remission and 5 (29.4%) showed disease relapse. the mean follow-up was 39 months (ranging from 5 to 72 months). the lowest probability of disease-free survival at 3 years was 79% (95% ci: 71.48 -88.51).background: relapse is the most important cause of failure after allogeneic hematopoietic stem cell transplantation (hsct) for flt3-itd-positive acute myeloid leukemia (aml). treatment with flt3 tyrosine kinase inhibitors (tki) constitutes a promising clinical approach to induce remission without conventional chemotherapy.methods: a 50 year-old woman diagnosed with aml secondary to myelodisplastic syndrome (mds) with npm1 mutation and internal tandem duplications of the flt3 gene (flt3-itd) in october 2013. after achieving complete remission (cr) with conventional chemotherapy, she received a hla sibling allogenic-hsct in february 2014, with bucy. four months later, aml relapsed only at medullary level (flt3 ratio: 0,67 %), treated with chemotherapy and donor lymphocytes infusions (dli). she achieved 2 nd cr and developed limited chronic graft-versushost disease (cgvhd). nine months later (april 2015), she suffered the first extramedullary relapse only, breast and skin. disappearance of the lesions at all levels was achieved with chemo and radiotherapy. she always had full hematologic donor chimerism.in december 2015, she referred atypical precordial pain irradiated to the back. cardiac mri was performed and several masses were visualized in the pericardial sac, up to 5 cm in diameter. bm remained in cr with full donor chimerism.pericardial fluid showed massive infiltration by leukemic-flt3 positive cells (ratio: 0,7%). she was not considered background: ta-tma is a severe complication that can reduce survival after hsct. risk factors have been variably reported in adults although data on children remains scarce. we aimed to identify a risk profile for development of ta-tma in children undergoing hsct.methods: we retrospectively reviewed clinical charts of 398 children who underwent 406 hscts between 2013-16: at great ormond street hospital (gosh) and the great north children's hospital (gnch). ta-tma was defined according to revised criteria (jodele et al. 2013) . risk factors were categorized into patient derived [age, gender, active co-morbidity at d0 of hsct (uncontrolled viral/ bacterial or fungal infection, pulmonary, cardiovascular instability, steroid therapy >0.3mg/kg beyond d0, bcgiosis or autoimmune disease), transplant related factors (conditioning intensity, stem cell source, hla-matching, use of ciclosporin a (csa) or tacrolimus (tac), cd34+ dose, ex-vivo t cell depletion, use of defibrotide) and post-hsct factors (agvhd, post-hsct viral reactivation, venoocclusive disease (vod) and occurrence of posterior reversible leukoencephalopathy (pres).results: at a median of 7 months post-hsct, ta-tma occurred among 21/406 transplants (5.1%). there was no reported centre variation (5.7% vs 4.6% in gosh vs gnch; p=0.6). gender, underlying disease -primary immune deficiency (pid) (n=273) vs haematological disease (n=133), use of myeloablative (n=153) vs reducedor minimal intensity conditioning (n=229), use of serotherapy or mega doses of cd34 ≥10 x10*6/kg did not influence the development of ta-tma. donor type: msd/ mfd(n=100) vs mud (n=129) vs mmud/haplo-hsct background: we evaluated the outcome of haploidentical hct (hhct) using ex vivo αβ t cell-depleted (tcd) grafts after reduced-intensity conditioning (ric) containing low-dose tbi (ld-tbi) in pediatric patients with acute leukemia (al) in complete remission (cr).methods: between may 2012 and october 2018, 36 patients with acute leukemia (17 all and 19 aml) in cr received haploidentical hematopoietic cell transplantation (hhct) using tcrαβ-depleted graft at asan medical center children's hospital. eighteen patients received hhct between 2012 and 2015 (earlier time period) and the remaining 18 between 2016 and 2018 (recent study period). the conditioning regimens, the dose of αβ+ t cells and gvhd prophylaxis are summarized in table. results: all 36 patients achieved a sustained neutrophil engraftment at a median of 10 days (range, 9-13) . of 36 patients, 11 patients (8 all & 4 aml) relapsed at a median of 6 months (range, 3-16) after transplant. of the 11 patients, 10 patients died of disease. one patient died of disseminated tuberculosis at 11 months after transplant, leading to the trm of 4% at 1 year. as of december 2018, 25 of the 36 patients survive free of disease at a median follow-up of 21 months (range, . at a median followup of 35 months (range, 1-80), efs and os at 2 years for all patients were 59% and 67%, respectively. outcome of hhct in the recent study cohort was significantly better than that in the earlier study period (efs of 85% vs efs of 44%, p=0.05). among the 17 patients with all, the efs of 8 patients, who received hhct in early time period after conditioning with tbi of 600 cgy, was significantly worse than that of 9 patients, who received in recent study period after a higher dose of tbi at 800cgy (13% vs 83%, p< 0.05). the efss of aml were similar between the two study groups (70% for earlier cohort vs 86% for recent study, p>0.05).conclusions: in pediatric patients with acute leukemia in cr, our current haploidentical hct using ex vivo αβ tcd graft after ric containing ld-tbi without gvhd prophylaxis is feasible approach with a low trm. the background: anti-hla antibodies (ahab) have been recently recognized as an important risk factor for graft failure (gf), especially in hla-haploidentical stem cell transplantation (haplo-hsct). although, recently, ebmt consensus guidelines have been published [ciurea, bone marrow transplant 2018] , experience in pediatric t-cell depleted (tcd, another well-known risk factor for gf) haplo-hsct is lacking. in the present study, we report our experience on the use of a desensitization approach based on ebmt guidelines.methods: between june 2017 and august 2018, all patients affected by non-malignant diseases and scheduled for a transplant from an hla-haploidentical donor after negative depletion of αβ t and b cells as previously described [li pira, biol blood marrow transplant. 2016 ], were tested for ahab with luminex® solid-phase immunoassay (one lambda, thermo fisher scientific) 1 month before the hsct. all patients with a mfi higher than 5000, which is considered a cutoff predicting for gf, were treated with a desensitization protocol based on the use of anti-cd20 rituximab (375 mg/m 2 before and immediately after the end of plasma-exchange cycle) and plasma-exchange (pe) ± infusion of irradiated buffy coat (bc) (if after pe ahab mfi was still > 5000 mfi). this latter was obtained by the non-target fraction of the αβ t-cell/b-cell-depletion procedure and consisted of 5 * 10 7 irradiated nucleated cells/kg of the recipient; this was infused 2-4 hours before the infusion of the tcd graft. pe was performed with miltenyi life 18 tm apheresis unit (miltenyi, bergish-gladback) .results: in the study period, 37 patients received αβ tand b-cell depleted haplo-hsct. eighteen (48%) resulted positive for ahab (mfi> 1000); 14 (77%) of them had an mfi > 5000 for either anti-class i or ii ahab. these patients (see table i background: osteonecrosis (on) is a debilitating complication in survivors of allogeneic hematopoietic cell transplantation (hct). limited data is available on its course post-transplantation in children. the purpose of our study was to identify recipients of hct in whom pre-and post-magnetic resonance imaging (mri) is indicated.methods: the retrospective cohort consisted of 436 patients who underwent first allogeneic hct from 1998-2014, and prospectively underwent a total of 1092 pre-and post-transplant mri studies of the hips and knees done annually for 3 years regardless of symptoms. surviving patients were followed for a median time of 8.33 (range 3.93-14.10) years. cases of on were compared to controls matched for age, sex, transplant type, and follow up in a 1:4 ratio for the following variables: ethnicity, underlying disease, on pre-hct, conditioning regimen, graft source, bone mineral density z-scores, body mass index, presence or absence of graft-versus-host disease, steroid use and dosage, and survival status.results: thirty (6.9%) patients had mri findings confirming on post-hct. all patients with on except one were more than 10 years of age. twenty (67%) patients were male. on pre-hct (p < 0.0001) was the only factor associated with presence of on post-hct. epiphyseal on was seen in 9 (30%) patients pre-hct, and 26 (87%) post-hct. eighteen (60%) patients had involvement of more than 30% of articular surface, and were more likely to undergo surgery (p = 0.009).conclusions: the incidence of on in this large pediatric cohort was 7%. the only risk factor for on post-hct was pre-existing on. mri evaluation for on pre-hct is indicated in all patients. mri evaluation for on post-hct is only indicated for patients with on pre-hct and symptomatic patients. this will entail cost savings of usd 500,000 per 100 surviving allogeneic hct patients per year. patients with more than 30% involvement of the articular surface need close follow up.clinical trial registry: none disclosure: none impact of rabbit anti thymocyte globulin exposure on immune reconstitution and outcome after stem cell transplantation in children background: rabbit anti thymocyte globulin (ratg) has been frequently used for many years as gvhd prophylaxis in pediatric stem cell transplantation. precise dosing regimens are crucial but remain challenging due to several pharmacological characteristics in children.methods: ratg levels were measured in pediatric patients undergoing allogeneic stem cell transplantation after obtaining approval by the local irb and informed consent by legal guardians. 32 pediatric patients who received either thymoglobuline™ (n=22) or grafalon™ (atg-f) (n=10) as part of their conditioning regimen background: obesity among children is a growing health problem. malnutrition or being over-weight can be of prognostic impact among children who need hsct.scientific literature shows a lot of controversy in terms of effect of bmi at the time of infusion on the outcome of hsct.methods: we reviewed data of patients who underwent hsct at king faisal specialist hospital & research centre between 2010-2016 to correlate bmi with the outcome and complications of hsct. transplant naïve recipients with age at infusion between 2-14 years who received hsct from matched related donor or autologous hsct, were included in the dataset for analysis. a total of 423 patients' profiles were reviewed of whom 51.1% were boys. median age at transplant was 7.5 years. primary indication of disease was malignancy in 41.6% followed by hemoglobinopathies 26.7%, bone marrow failures 17.3% and immune disorders 12.5%. solid tumors accounted for 29.5% among malignant disorders. myeloablative conditioning was used in 97.2% transplants with 11.8% regimens containing total body irradiation. majority of the patients 81.3% underwent allogeneic transplantation using bm as the source in 80.4% and pbsc in the remaining 19.6% cases. donor was hla-identical sibling in 89%, parents in 10.2% and others in the remaining 0.9% patients. median tnc dose was 2.55 x 10^9 and cd34 was 5.63 x 10^6 per kg of the body weight at the time of infusion. age and gender specific bmi percentiles were obtained and classified according to the definition of centers for disease control and prevention (cdc); 24.8% (105) recipients were categorized as under-weight, 61.2% (259) normal, 5.9% (25) over-weight and remaining 8% (34) as obese.results: based on chimeric studies at day-100, our engraftment rate was 98.3% (400) out of 407 evaluable cases. median time to neutrophil recovery was 14-days from infusion and 26-days for platelets. no statistically significant difference was found for engraftment rate on d-100 as it was 100% (25) among 98.8% (247) in normal, 97% (97) in under-weight and 96.9% (31) in the obese (p-value: 0.467). median time to neutrophil recovery from the infusion date was 15-days in over-weight patients and 14 in the remaining three groups (p-value: 0.841). acute graft vs. host disease (agvhd) of any grade at day-100 was recorded in 19.9% (84). any-grade agvhd was more common in over-weight 24% (6), followed by obese with 23.5% (8), 21% (22) in under-weight and 18.6% (48) in normal bmi-category (p-value: 0.770). chronic gvhd was more frequent in over-weight (14.3%, 3), compared to 9) background: hematopoietic stem cell transplantation (hsct) is the standard treatment for children with severe aplastic anemia (saa) who have hla-identical related donor. there is no standard conditioning regimen for children with saa secondary to non-fanconi anemia (fa) constitutional bone marrow failure syndromes such as telomeropathies. we report the outcome of a consistent reduced intensity conditioning regimen in patients with idiopathic saa or inherited bone marrow failure syndromes other than fa who underwent hla matched related hsct methods: children with saa underwent hsct using the following conditioning regimen: fludarabine 175mg/m2, cyclophosphamide 80 mg/m2, and atg (thymoglobulin) (10 mg/kg) . gvhd prophylaxis included cyclosporin and mycophenolate mofetil. donors were all matched related and bone marrow was the stem cell source. all patients had normal chromosomal fragility testresults: a total of 18 children with saa underwent hsct, 12 females and 6 males. average age was 8.1 (range 0.8-13.8 years). all nine patients who were tested for telomere length had short telomeres. pathogenic or likely pathogenic mutations were reported in 5 patients (2 ercc6l2, 1 ankrd26, 1 tinf2, 1 lztfl1). all donors had normal physical examination, normal cbc, and negative genetic testing if patient mutation is known. all 18 patients engrafted successfully, median time to neutrophil engraftment was 16 (range, 11-29 days) and platelet engraftment 19 (range, 13-45 days). median infused nucleated cell dose was 3.3 (range,0.9-7.3 x10 8 /kg) and cd34 cell dose was 6.7 (range, 1.1-13.1 x10 6 /kg). none of our patients had acute gvhd and one patient had mild classic chronic gvhd of the skin that was controlled with topical therapy for a short period. three patients had secondary graft failure in the first-year post transplant. first patient had pancytopenia with loss of donor chimerism and underwent successful second transplant using fludarabine, atg, and melphalan. the second patient had a nonfunctioning graft despite full donor chimerism suggesting that the related donor might be affected and had silent phenotype. the third patient had homozygous ercc6l2 mutation and developed progressive cytopenia with myelodysplastic features few months post-transplant and subsequently underwent myeloablative matched unrelated transplant using busulfan, fludarabine, thiotepa and atg. however, the patient progressed to have acute myeloid leukemia six months post hsct. fifteen patients (83%) have normal cbc and stable donor chimerism. median follow-up duration of 1140 days (range 330 -2595 days). one patient with lztfl1 mutation developed chronic renal impairment five years post hsct.conclusions: hsct using lower dose cyclophosphamide (80mg/kg) as part of fludarabine based regimen was safe and effective in saa patients with shorter telomeres and described genetic abnormalities. optimal conditioning regimen in ercc6l2-associated bone marrow failure needs to be defined. larger study is needed to confirm our results.clinical trial registry: not applicable disclosure: nothing to declare late effects in patients with hemophagocytic lymphohistiocytosis treated with hematopoietic stem cell transplantation: a review of the literature background:hemophagocytic lymphohistiocytosis (hlh) is an inherited or acquired disorder of immunedysregulation. early diagnosis and immunosuppressive treatment can prevent significant organ-failure. the inherited forms, and some acquired cases can only be cured by hematopoietic stem cell transplantation (hsct). with modern transplant practices, a significant number of patients survive. the purpose of this literature review was to collect data on the frequency and type of late effects in hlh patients surviving after hsct and to examine the association with pre-existing hlh conditions (eg. involvement of the central nervous system (cns) before transplant) and with the pre-transplant conditioning regimens.methods: the medline, embase, web of science and pubmed databases were searched, by two librarians at the karolinska institutet, between may and september 2016 according to the preferred reporting items for systematic review and meta-analysis (prisma) statement. the search terms included "hlh", "fhl", "mas", multiple terms for "hsct" and late-effect conditions. inclusion criteria were publications in english that included children between january 1995 and may 2016. authors of this review screened all the abstracts of studies against the inclusion criteria.results: only nine papers published between 2006 and 2016, with information on late effects in hlh patients who had undergone hsct, were identified. three reports include only small numbers of hlh patients. the remaining 6 papers contain data on 261 long-term survivors with a median follow-up of 5.4 years. five papers address neurological sequelae with a reported incidence from 7-57%. the highest incidence was found after a thorough neurological assessment of 21 hlh patients compared to matched sibling controls. however, the association with cns disease before transplant and age at transplant was not clear. patients with ebvassociated hlh seem to have fewer long-term neurological problems. non-neurological late effects are described in 4 papers only, with endocrinological problems, namely short stature, being the most frequent. one paper specifically analyzed poor growth, thyroid dysfunction and vitamin d deficiency in a cohort of patients with non-malignant disorders including hlh who had undergone hsct after a reduced intensity conditioning regimen and found significant abnormalities in all groups.conclusions: data on late effects in hlh patients is scarce and is mostly based on the retrospective evaluation of small national cohorts. the available information indicates that a significant number of patients suffers from problems which affect their daily life, but lack of information does not allow to analyze the association between pre-transplant conditions and long-term sequelae. therefore, a retrospective comprehensive analysis of patients registered in the ebmt and cibmtr registries is currently performed. it will be crucial to better define the frequency and type of late effects in a large cohort. this knowledge will aid counselling prior to hsct, provide guidance for long-term monitoring of these patients, and potentially identify specific risk factors for late effects in this rare patient population.disclosure: nothing to declare. allogeneic stem cell transplantation in patients with mucopolysaccharidosis type ii (morbus hunter)bernd hartz 1 , nicole muschol 2 , matthias bleeke 1 , johanna schrum 1 , ingo müller 1background: the transplantation of hematopoietic stem cells (hsct) is one of the leading methods of treatment in patients with blood system diseases, primary immunodeficiency syndromes and genetic diseases. at the same time, the quality of life in patients in the long-term after hsct significantly differs from the quality of life of healthy people of the same age. deformations in psychosexual development including problems in the gender identity formation cause social isolation of adolescents, which makes their sexual selfrealization impossible and significantly reduces the quality of their life. the purpose of our study was an assessment of the level of gender identity formation of adolescents and psychosexual development correlation to the normal adolescents of the same age.methods: in a prospective single-center study in 2018, on the base of the department of rehabilitation medicine raisa gorbacheva memorial institute of children´s oncology, hematology and transplantation, we conducted a study of 17 families. the respondents were: 1) parents / guardians of patients accompanying them in the process of examination; 2) adolescents who underwent hsct treatment and undergo planned examinations at the clinic in the posttransplant period (after d + 100), (n = 17, of which 8 girls and 9 boys, age 12 -17 years, from the date of hsct 1 -5 years).the following methods were used to assess gender identity: specially developed questionnaires for teenagers and parents; questionnaire by sandra l. bem (sandra l. bem, 1974) ; projective techniques "the human picture", "the non-existent animal"; max lüscher´s color choices test.results: the traditional type of gender identity, which characterizes high masculinity among male respondents and high female gender indicators in 100% of cases, was not revealed. both among girls and among boys, the androgynous type prevails with a tendency toward femininity.on average, adolescents see themselves as a bit more courageous than their mothers, with rare exceptions, regardless of gender. this confirms the thesis that we received in a previous study that parents tend to see and encourage complacency of adolescents of both sexes, passivity instead of leadership, dedication and independence. all 100% of adolescents who participated in the test demonstrate a shift in the theme of aggression, 77% have some signs of preventing sexual self-determination, abandoning their body, gender, and age. 88% of patients do not communicate with their peers. in 47% of them, negative emotions prevail over positive ones. one third of the test participants demonstrate strong support for rest and minimizing their efforts.conclusions: the characteristics of family upbringing of adolescents who have undergone hsct often contribute significantly to limiting their social experience and lead to specific deformities of individuality, including in the sphere of gender identity. we consider advisable to introduce thematic group counseling of parents within the framework of the "patient's school" in psychological treatment support in the clinic. early diagnosis of the personal aspects of the psychosexual development of adolescents after hsct allows for timely identification of individual problems in this area and identification of general trends in the long term after hsct.disclosure: all authors -nothing to disclose. abnormalities in the morphology of the umbilical cord blood obtained at delivery from children who received a diagnosis of cerebral palsy maciej boruczkowski 1 , izabela zdolińska-malinowska 2 , maciej rojek 2 , dariusz boruczkowski 2background: embryonal brain tumors are the most common malignancies in infants less than 48 months of age. histologically characterized as undifferentiated small round cell tumors, all are similarly aggressive, have a tendency to disseminate throughout central nervous system and very poor prognosis. we tried to assess the effectiveness of tandem highdose chemotherapy (hdct) with autologous hematopoietic stem-cell transplantation (auto-hsct) in this patient group. methods: from 2010 to 2018, 52 infants under 48 months with different primary embryonal brain tumors such as medulloblastoma (n=28), different pnet nos (n=10), pineoblastoma (n=3), atypical teratoid rhabdoid tumor (n=3), etmr (n=8) after surgical resection and induction chemotherapy were planned to receive tandem hdct with auto-hsct. nine patients were conducted only single transplantation because of the development of lifethreatening complications after the first hdct (n=4) or the emergence of early disease progression (n=5). at the moment of hdct 31 patients were in complete remission (cr), 20 patients were in partial remission (pr) and 1 patient had stable disease (sd). the conditioning regimen for tandem auto-hsct were: the first hdct was carboplatin and etoposide, the second was thiotepa and cyclophosphamide, both with intraventricular methotrexate.results: the median follow-up is 24 months (range, 6-85). the median time to engraftment after the first auto-hsct was background: a series of findings suggest that optimizing natural killer (nk) cell reactivity could further improve outcome after allogeneic hematopoietic cell transplantation (allohct). this could be achieved by killer cell immunoglobulin-like receptor (kir) genotype informed donor selection. an enhanced receptor-ligand model which used kir2ds1 and kir3dl1 donor genotype information to augment nk cell activation and minimize inhibition demonstrated improved survival in one large aml study (boudreau et al, jco 2017) . likewise, a second model built on the classification of centromeric and telomeric kir haplotype motifs, also predicted mortality after allohct for aml (cooley et al, blood 2010) . this joint ebmt and cibmtr study aimed at validating the two approaches in an independent cohort of patients with mds or secondary aml.methods: donor samples were retrieved from the collaborative biobank (dresden, germany) and mapped to patient outcome data extracted from the ebmt and cibmtr. genotyping of all kir genes by sequencing exons 3, 4, 5, 7, 8, and 9 was performed by high resolution amplicon-based next generation sequencing. the impact of the classifiers on time-to-event outcomes was tested in cause-specific cox regression models adjusted for patient age, a modified disease risk index, performance status, donor age, hla-match, sex match, cmv match, conditioning intensity, type of t-cell depletion and graft type.results: clinical data from 1704 patients and corresponding donor genotype information were analyzed. the median age at allohct was 59.4 years (range, 18.1 to 79.6 years). the indication for allohct was mds for 72% and saml for 28% of patients. disease risk was low/intermediate and high/very high in 41% and 59%, respectively. donors were 10/10 matched for 79% of patients. myeloablative, reducedintensity and non-myeloablative conditioning regimens were used in 31%, 57%, and 12% of patients, respectively. peripheral blood stem cells were the predominant graft source (93% of patients). atg was administered in 56% and alemtuzumab in 9% of patients. during follow-up after allohct 776 patients died. in univariable and multivariable analyses of the whole cohort, overall survival and the cumulative incidence of relapse of patients with kiradvantageous versus disadvantageous donors were not statistically significantly different. we could not replicate the pattern of outcomes predicted by the kir3dl1/ conclusions: relapse incidence and overall survival after unrelated donor allohct could not be predicted using the kir3dl1/kir2ds1-receptor-ligand model and centromeric/telomeric kir-motif model in this large cohort of patients with mds or secondary aml. this points at the possibility of interactions between nk-cell mediated alloreactivity and disease type or procedural variations of allohct. available information on kir-genes, which have been sequenced but not yet analysed, will be investigated in exploratory analyses.disclosure: the authors have nothing to disclose in the absence of an alternative donor, it is recommended that patients undergo desensitization therapy, especially with high dsa levels (>5,000 mfi). the aim of this study is to analyze the impact of dsa on risk of gf and poor graft function (pgf), and on major outcomes in a consecutive cohort of patients who were systematically screened for dsa before haplo-sct. methods: 141 consecutive patients were candidates for unmanipulated haplo-sct with post-transplant cyclophosphamide (pt-cy) at our center from january 2012 to january 2018 and 135 were analyzed for the presence of hla antibodies.results: 134 patients underwent haplo-sct. hla antibodies were detected in 40 patients,19 of them were dsa, while 21 were non-dsa (ndsa). 10 patients out of 19 with dsa were transplanted using the same donor; 2 underwent a desensitization program before transplant.background: a recent study from ebmt comparing matched sibling (msd) versus haploidentical donors transplantations, showed better outcome with msd in adult patients with intermediate risk aml in first remission (cr1). however, a female donor to a male recipient transplant combination is a poor prognostic factor and this study did not address the question whether in this situation, a haploidentical donor transplant might do better. the present study compared the outcomes of allografted male patients according to whether they received stem cells from a female msd or a haploidentical donor, in the intermediate and high risk cytogenetics groups (mrc classification).methods: the study included 1066 male patients with cytogenetics transplanted between january 2007 and june 2017 and reported to ebmt. 834 received stem cells from a msd female donor and 232 from a haploidentical donor (133 male and 99 female). the follow up was 25 months (12-62). we studied separately intermediate and high risk patients. multivariate analysis was adjusted on factors differing significantly between the 2 groups.results: 1-intermediate risk group: 638 male patients received a female msd and 160 a haploidentical transplant. the distribution of group characteristics was even except that in the haploidentical transplant group, donors were younger (39 y versus 51; p< 0.0001), marrow was more frequently used (45% versus 17%, p< 0.0001) and the interval from diagnosis to transplant was longer (5.4 versus 4.5 months, p< 0.0001). by univariate analysis at two years post transplant, cumulative incidence (ci) of nrm post haplo was higher (26% versus 15%, p=0.002) and ci of extensive chronic gvh lower (14% versus 27%; p=0.002). lfs post msd and post haplo were 64% and 51% (p=0.03), os 69% and 60% (ns), grfs (43% and 43%). by multivariate analyses the only significant poor risk factors were the haplo-identical transplant for nrm (hr: 1.7 (1.1-2-61)) and the patient age for os (hr: 1.15 (1.02-1.28; p=0.02). haploidentical transplantation resulted in less chronic gvhd (hr: 0.43 (0.29-0.64); p < 10 -4 ), but a lower lfs (hr: 1.7 (1.1-2.61); p=0.04). 2-high risk group: 196 male patients received a female msd and 72 a haploidentical transplant. in the haploidentical group, donors were younger (38 y versus 54; p< 0.0001), marrow was more frequently used (42% versus 11%, p< 0.0001) and the interval from diagnosis to transplant was longer (5.1 versus 4.3 months, p= 0.003). by multivariate analysis, haploidentical transplants were associated with a lower relapse incidence (hr: 0,40 (0.21-0.75; p = 0.004),a better lfs (hr: 0,46 (0.28-0.77; p = 0.003),os (hr: 0,43 (0.25-0.75; p = 0.003), and grfs (hr: 0,53 (0.34-0.84; p = 0.006)(see figure) . the only other significant prognostic factor was patient age.conclusions: this study shows that in a male patient with intermediate risk aml, a genoidentical sister donor remains associated with a better lfs. in contrast, in a male patient with high risk aml in cr1, a haploidentical donor may be a better choice than an hla genoidentical sister.disclosure: nothing to declare p663 abstract already published. hematopoietic transplant for older acute leukemia patients: improved survival with offspring donor in comparison with older-aged matched siblingsyu wang 1 , sheng-ye lu 1 , qi-fa liu 2 , de-pei wu 3 , xiao-jun huang 1background: post-transplant relapse remains the major cause of death of treatment failure. therapeutic options for relapse after first allogeneic stem cell transplant (1st hsct) include chemotherapy followed by donor lymphocyte infusion or second allo-hsct (2nd hsct) from the original donor or change to another donor. however, there is unclear outcome for different treatment approach. in this retrospective cohort study, we aim to compare the clinical outcome after different treatment strategy for relapse after first allo-hsct.methods: between 1992 jan and 2018 oct, 1493 consecutive patients receiving 1 st hsct registered to the bmt database in national taiwan university hospital were analyzed. among them, 580 cases had relapsed after first allo-hsct. one hundred and three patients who received no treatment after relapse or with incomplete data were excluded. their transplant data was collected following the ebmt registry data collection forms and manuals. overall survival rate and progression free survival rate were performed by the kaplan-meier method. univariate and multivariate analysis were performed using cox proportional hazard regression model.results: of the 477 patients who experienced relapse after 1 st hsct, total 244 patients (51%) received chemotherapy followed by dli or 2 nd hsct from the same donor (no change group), 36 patient (8%) received chemotherapy followed by 2 nd hsct from different donors (change group), and 197 (41%) had conventional chemotherapy alone. the patients in "change group" were younger (median age 29 vs 37, p = .02), and had more patients achieving complete remission (cr) prior to 2 nd hsct (44% vs 10%, p = < .01) than patients in "no change group". after the 2 nd hsct, the cr was 68% for "no change group" and 91% for "change group". the progression-free survival at 3-year and 5-year were 10.5% and 5.9% (fig 1a, p = .0438%), respectively, for "no change group" and 9.4% and 9.4%, respectively, for "change group". while the overall survival (os) at 3-year and 5-year were 15.8% and 9% (fig 1b, p = .3459%), respectively, for "no change group" and 13.5% and 9%, respectively, for "change group". those who achieved cr prior to 2 nd hsct had a trend of better os than those without cr (17.5% vs 8.7% at 3-year; 8.7% vs 9.1% at 5year, p =.0365)( fig 1c) . there were 1 cases survived for more than 10 years in "change donor group" and 6 cases survived more than 10 years in "no change group". only one had developed relapse after 2 nd hsct but achieved subsequent remission again.conclusions: our study shows that change donor had similar poor outcome comparing to those using the same donor after the 1 st hsct. patients who achieved cr before 2nd hsct had a trend of better os than those without remission and the long-term survivors were only those who achieved cr prior to 2 nd hsct. novel therapy for cr induction would be warrant for this poor prognostic population.disclosure: nothing to declare functional relevance of fetal microchimerism in nk cell cytotoxicity against leukemic blasts in children: a role for hla-c1 and kir2dl2/s2?background: allogeneic stem cell transplantation (allo-sct) remains the most effective curative intent therapy for patients with unfavorable risk acute leukemia. various donor options are available for the patient who lacks an hla-matched sibling donor, such as unrelated donors (urd) and hla-mismatched family (haploidentical) donors. in order to discover the exact role of transplantation type, there are many retrospective analysis, which compared these donor sources, have been reported. recent studies showed some promising results of haploidentical donor transplantation (hidt) using post-transplant cyclophosphamide in comparison with unrelated donor. the goal of this study was to compare the outcome of allo-sct from haploidentical versus matched unrelated (mud 10/10) or mismatched unrelated donor at a single hla-locus (mmud 9/10) for patients with acute leukemia in remission.methods: ninety-six adult (18-65 years) patients with acute leukemia in first or second remission who underwent allogeneic transplantation with a minimum 100 days follow-up at florence nightingale hospital hematopoietic stem cell transplantation center between 2011 and 2017 were included in this study. patient characteristics and medical records of all patients were reviewed retrospectively. thirty-eight patients who received haploidentical donor transplantation were compared with 23 patients receiving a mud 10/10 and 35 receiving a mmud 9/10. patients who completed minimum 100 days post-transplantation follow-up were identified as eligible for survival analysis.results: the characteristics of the patients and transplant donors in this study are summarized in table 1 . median age of patients was 39.4±14 years. proportion of male patients was 39.1%, 74.2% and 57.8% for mud 10/ 10, mmud 9/10 and hidt groups, respectively, which is significantly different (p=0.02). the other baseline factors were similar, including patient age, donor age, recipient cytomegalovirus (cmv) status, donor cmv status, graft versus host disease incidence, median neutrophil and platelet engraftment times and disease status at post-transplant 100 th day. no significant difference was identified in survival analysis among the mud 10/10, mmud 9/10 and hidt groups, even if they were classified according to primary disease (aml vs all) and pre-transplant disease status (cr1 vs cr2). also, donor cmv status (cmv igg positivity or negativity) was not an important factor on survival analysis when compared between these three groups (p=0.406).conclusions: in our study population, clinical outcomes of hidt patients were inferior to mud 10/10 and mmud 9/10 groups. when choosing an alternative donor for patients without an available hla-matched sibling, urgency of transplantation and host/donor features should be considered. we believe that hidt might be a feasible alternative choice in this subset of patients.disclosure: nothing to declare p673 g-csf primed bone marrow in hla-haploidentical transplantation using post-transplantation cyclophosphamide (ptcy) could promote tolerance and further reduce risk of gvhd nadira durakovic 1,2 , zinaida perić 1,2 , lana desnica 2 , ranka serventi-seiwerth 2 , mirta mikulić 2 , brian melamed 3 , alen ostojić 2 , dražen pulanić 1,2 , pavle rončević 2 , zorana grubić 2 , radovan vrhovac 1,2 implementation, development, and coordination of unique quality management systems with evaluation audits, intrahospital and international accreditation and certification processes. quality of health care is a major focus for providers, patients, and accreditors; so, in this study, we aim to compare the quality of bm harvested at ipo-collection centre (icc) with the quality of bm received from external collection centres (ecc) during these 6 last years.methods: this retrospective evaluation included the number of total nucleated cells (tnc) requested by the transplant centre, the tnc collected, and the results of bm microbiological analysis performed; donor age, weight and infectious disease markers (idm); patient demographics and diagnosis. bm collection technique in use at icc was validated according to jacie standards.we consider successful a collection (sc) with tnc between 75 and 125% of the requested value, unsuccessful (uc) if lower than 75% and outstanding (oc) if over 125%.results: a total of 106 bm was collected, 99 for allogenic (75 unrelated) and seven for autologous transplant; 21 unrelated bm were received from ecc (nine from germany, seven from usa and five from portugal). patient main diagnosis were severe aplastic anaemia (n=28), acute myeloblastic leukaemia (n=20), and acute lymphoblastic leukaemia (n=15). donors idm were all negative and nonreactive.mean age (±standard deviation, sd) was 33 (±13.2) and 31 (±9.3) years for icc and ecc donors, respectively. at icc, we were asked to collect an average (±sd) of 221.7*10 8 (±136.0) tnc while ecc were asked for 184.7*10 8 (±105.7). we collected 169.7*10 8 (±82.3) and received 204.5*10 8 (±109.2) tnc. correlation between requested and collected tnc was 0.69 at icc and 0.56 for ecc.we had 41.2% sc and 26.8% oc meaning an accomplishment of 68.0%. we failed to collect required tnc in 32.0%. although 85% of received bm fulfil tnc requirements, bm processing lowered this value to 55% due to erythrocyte removal (seven patients with major abo incompatibility) and plasma reduction (two patients with abo minor incompatibility). these steps reduce final tnc available for transplant. weight difference between donor and patient had no significant impact on final tnc collection performance.sixteen bm from icc (seven staphylococcus spp., five propionibacterium acnesand fourcorynebacterium spp.) background: success of peripheral blood stem cell (pbsc) collections depends on patient biological parameters and stable apheresis device performance. peripheral blood cd34+ cell enumeration is the most reliable predictive factor of apheresis yield however, there are some unexpectedly poor cd34+ cell harvests despite successful mobilization. the aim of the study was assess total collections cd34+ yields and factors influencing main apheresis procedure outcomes including collection efficiency (ce).methods: of 2233 consecutive donors covering the period 1-1-2016 to 30-9-2018 were analyzed for the following parameters: pre cd34 count, cd34 yield per procedure, total cd34 dose collected per patient, cd34 collection targets requested by clinical teams. the efficiency of pbsc procedures was determined by calculating the ce and the correlation coefficient between pre cd34 count and yield per procedure. ce was correlated to preprocedure wbc, platelet count, pre cd34 count and blood volume processed. all pbsc collections were performed by optia spectra across 7 units in uk.results: of the 2233 donors, 1611 were autologous and 622 allogeneic. the autologous donors underwent in total 2543 procedures. the median cd34 target dose for these donors was 4x10 6 /kg.799 (50%) achieved the target dose with 1 procedure and 566 (35%) with 2 procedures. the median pre cd34 count was 30/μl. the median cd34 yield per procedure was 2.54x10 6 /kg and the median total cd34 dose collected per donor was 5.38x10 6 /kg. 92 (5.7%) of autologous donors collected a total cd34 dose < 2x10 6 /kg, of those 27 (1.7%) had a pre cd34 count < 10/μl and 65 (4%) >10/μl.the allogeneic donors underwent in total 878 procedures. the median cd34 target dose for these donors was 5x10 6 / kg. 381 allogeneic donors (61%) achieved the target dose with 1 procedure and 221 (36%) with 2 procedures. the median pre cd34 count was 51/μl. the median cd34 yield per procedure was 4.07x10 6 /kg and the median of total cd34 dose collected per donor was 6.70x10 6 /kg. 17 (2.7) % of allogeneic donors collected a total cd34 dose < 2x10 6 / kg, of those 3 (0.5%) had a pre cd34 count < 10/μl and 14 (2.2 %) >10/μl. the median ce for autologous donors was 55% (range 20-166) and for allogeneic donors was 47% (range12-116). the ce was negatively correlated to wbc (r= -0.29 and -0.37) and platelet count (r=-0.14 and -0.06) for auto and allogeneic donors respectively, but did not correlate to the pre cd34 and blood volume processed. the correlation coefficient between pre cd34 count and cd34 yield per procedure was r 2 =0.67 for the autologous and r 2 =0.34 for the allogeneic collections.conclusions: the majority of autologous and allogeneic donors achieved the target cd34 dose with one procedure. 94.3 % of autologous and 97.3 % allogeneic donors collected a transplantable cd34 dose of > 2x10 6 /kg. 4% of autologous and 2.7% of allogeneic donors did not collect a transplantable dose despite a precd34 count of >10/μl indicating suboptimal procedure performance. the ce was variable and was negatively correlated to the preprocedure wbc and platelet count. the ce and correlation coefficient are lower in allogeneic donors compared to autologous donors.disclosure: nothing to declare the outcome of autologous blood stem cell collection and its actual use in real world: the 21st century experiencekyoungmin lee 1 , jung yong hong 1 , dok hyun yoon 1 , jae-lyun lee 1 , shin kim 1 , kyoung min lee 1 , jung sun park 1 , cheolwon suh 1background: mobilized peripheral blood stem cells (pbscs) have largely replaced bone marrow as the graft source for allogeneic stem cell transplantation. pbscs mobilization with g-csf is highly effective even on the 4th day in order to collect enough number of stem cells. a longitudinal, prospective, observational, single-center, cohort study on healthy donors (hds) was designed to identify predictors of cd34+ cells on the 4th day. methods: as potential predictors of mobilization, age, sex, body weight, height, blood volume as well as white blood cell count, peripheral blood (pb) mononuclear cells, platelet count, hematocrit, and hemoglobin levels were considered. two different evaluations of cd34+ cell counts were determined for each donor: baseline (before granulocyte colony-stimulating factor [g-csf] administration) and in pb after g-csf administration on day 4. a total of 122 consecutive hds with a median age of 47.5 years were enrolled.results: the median value of cd34+ on day 4 was 43 cells/μl (iq 23-68). basal wbc, plt and basal cd34+, are significantly higher for group with cd34+ on the 4th day over the median than below. a multivariate quartile regression analysis, adjusted by gender, age, basal cd34 + and basal plt, shows a, progressively steeper, relationship between baseline cd34+, basal plt and cd34+ on the 4th day. the basal cd34+ cut-off for prevision of cd34+ on the 4th day was < =2 cells/μl and >=3 cells/μl whereas basal platelets count was < =229 x 109/l and >=230 x 109/l.conclusions: g-csf can be highly effective in hds on the 4th day in order to collect enough number of stem cells and we have developed a model for predicting the probability to perform pbsc collection after a short course of g-csf.disclosure: nothing to declare p692 pre-apheresis peripheral blood cd34+ cell counts highly correlates to actual stem cells collected background: prediction of stem cell yield on the basis of pre-apheresis cd34+ cell count and the processed blood volume is essential for the planning and executing of the apheresis process.methods: data analyzed included donor weight, complete blood count and cd34+ count on day of collection, total processed blood volume, cd34 + cell dose collected in the apheresis product and the number of aphereses performed. using the method described by pierelli et al, predicted cd34 + yields were calculated: predicted cd34 + yield x 106/kg = (benchmark ce x volume of blood to be processed x peripheral cd34+ count per μl) / (patient's weight in kg x metric conversion factor).results: in 2017 we established the method described by pierelli to predict the cd34+ cell yield. 323 allogenic aphereses were performed in 2017 with this approach. mean processed volume was 13.71 liters. the mean cd34+ peripheral count before apheresis was 68/ul, the mean collected cd34+count per kg bodyweight recipient was 6.35. pearson´s correlation coefficient (r) between predicted yield using pre-apheresis cd34+count and actually collected cd34+ cells per kg bodyweight recipient was 0.967. the mean difference between predicted and collected cell dose was +4.45%.with knowledge of the predicted stem cell count, we were able to adjust apheresis procedure. in case of marginal predicted yield compared to the requested cell dose, we increased the blood volume to be processed. this proceeding led to a significant reduction of second day donations in 2017 by 38% compared to 2016. in only 3 cases we saw more than -40% lower cd34+ doses collected than initially predicted. all 3 donors showed mild iron deficiency with rbc microcytosis, a factor known to affect apheresis procedure.conclusions: pierellis method of calculating the stem cell yield shows a good correlation between pre-apheresis cd34 + count and actual collected stem cells, making planning and adjusting of the apheresis procedure more feasible and reliable. this proceeding led to significant reduction of second day donations. attention should be paid to iron deficiency anemia, leading to lower than estimated cd34+ dose.[ background: for more than a decade many transplant centers routinely collect and cryopreserve two or more peripheral blood stem cell (pbsc) grafts for a tandem and/ or salvage autologous blood stem cell transplantation (absct) in patients with hemato-oncological diseases. however, subsequent high-dose chemotherapy (hd-cht) and absct is in many cases not performed for a variety of reasons, specifically in patients with aml, all, mpn and burkitt lymphoma. data about the actual utilization rate of the cryostored stem cell products are lacking.methods: we retrospectively analyzed the collection, storage and disposal practices of pbsc products from a large cohort of patients who were treated at the university hospital heidelberg or at the university medical center mannheim during a 12-year period. disease entities included acute myeloid leukemia (aml, n=74), acute lymphoblastic leukemia (all, n=22), mpn (n=18; primary myelofibrosis [pmf], n=9; chronic myeloid leukemia [cml], n=7; secondary fibrosis/essential thrombocythemia [et], n=1; not specified, n=1) and burkitt lymphoma (n=18). patients between 2001 and 2012 were included and followed until 2016.results: an adequate stem cell graft was defined as ≥2.0 x10exp6 cd34+ cells /kg body weight. 97% of the patients were able to collect at least one stem cell graft and the median number of grafts per patient was 1 (range 0-5). we could demonstrate that only 31% of all patients who had collected sufficient pbscs for transplant subsequently underwent an absct. among the disease entities the actual use of the stored pbsc grafts varied considerably from 0% to 56% (figure 1) .conclusions: we could identify striking discrepancies between the collection/storage and actual utilization of background: biosimilars (bio) of granulocyte colony stimulating factors (gcsf) were approved several years ago on the basis of some studies that indicated similar efficacy to the already patented gcsf (neupogen®, neu) both in terms of shortening the neutropenic period after chemotherapy as well as peripheral blood stem cell mobilization in patients with lymphoma and multiple myeloma (mm) treated with autologous stem cell transplantation (auto-hct). however, all these studies are retrospective and there are still concerns about the real efficacy of bio and even more about the real benefit on final costs.methods: we have retrospectively compared the characteristics of the mobilization procedure in both patients with mm and lymphomas, and healthy donors that received either neu or bio (with no chemotherapy) in 4 university hospitals in catalunya from december / 2013 to november 2017. bio replaced neu in june 2016 in all 4 institutions. primary objectives were the mobilization rate (defined as the percentage of patients that achieved ≥ 10 x 10 3 /ml cd34 + cells in peripheral blood on day 4) and the use of plerixafor (plex) in each group as pre-emptive strategy. a multivariable analysis of risk factors influencing the use of plex and mobilization failure (defined as collection of < 2 x 10 6 /kg cd34+ cells) was also performed.results: we treated 216 patients (102 lymphomas and 114 mm) and 55 healthy donors. both groups of patients (138 neu and 78 bio) and donors (33 neu and 22 bio) were comparable regarding pre-mobilization general characteristics. there was a trend for a lower median cd34+ peak on day 4 for bio patients (17 vs 20, p value = 0.1). a total of 53 patients received plex, although 7 of them (13.2%) out of strict theoretical indication, cd34+ cells > 10 x 10 3 /ml (range 10.5-13.5) and were removed for further analysis (n = 46, 31 in the neu group and 15 in the bio group). median number of cd34+ cells on day 4 was significantly lower in the group bio who needed plex (2.4 vs 4.8 for neu+plex, p=002), as well as cd34+ cells finally harvested (2.5 vs 3.3x 10 6 /kg, p=0.03). mobilization failure rate was higher in bio group (0 vs 20%, p=0.01). regarding no plex patients, median number of cd34 +cells on day 4 was also significantly lower for bio patients (33.4 vs 23.7, p=0.03). risk factors for plex use were age, basal disease (lymphoma) and number of prior mobilization therapies. the use of bio was the only risk factor for mobilization failure patients receiving plex [hr 10.3 (95%ci 1.3-77.8), p=0.02]. with respect to healthy donor mobilization, none of them needed plex but 2 cases from the bio group (9%) needed more than one apheresis procedures (2 and 3, respectively).conclusions: we found a lower efficacy of bio in the setting of stem cell mobilization of patients with only gcsf both in terms of a lower cd34+ cells peak on day 4 and a lower number of cd34+ cells in final apheresis product. bio gcsf also seems to be less effective in healthy donors.disclosure: no conflict of interest to be declaredbackground: auto-sct is a common treatment in patients with mm or nhl. the aim of the prospective multicenter goa (graft and outcome in autologous transplantation) study was to investigate the impact of mobilization method used on the cellular composition of collected blood grafts and eventually hematological and immune recovery as well as long-term outcome post-transplant. altogether 282 patients with mm or nhl transplanted between 5/2012 and 12/2016 at four university hospitals were included. the long-term goal of the study is to evaluate characteristics of optimal blood grafts in regard to post-transplant recovery and outcome. methods: altogether 147 patients with mm undergoing first auto-sct were compared with 135 patients with nhl. all nhl patients were mobilized with chemotherapy + g-csf, whereas 39 % of mm patients were mobilized with g-csf only (p < 0.001). mobilization data, graft cellular composition including cd34 + cell subsets and lymphocyte subsets of the blood grafts, post-transplant hematological recovery and outcome were evaluated. the median followup time was 46 months in mm patients and 35 months in nhl patients.results: mm patients mobilized cd34 + cells better (median peak blood cd34 + 65 vs. 40 x 10 6 /l, p < 0.001). the median number of aphereses was 2 in both groups (p = 0.09). altogether 26 % of the nhl patients received plerixafor compared to 12 % in mm patients (p = 0.002). the median number of cd34 + cells collected was higher in mm patients (6.4 vs. 3.9 x 10 6 /kg, p < 0.001).the median amount of cd34 + cells (with 7-aminoactinomycin) in the infused graft was 2.35 x 10 6 /kg in mm group and 2.5 x 10 6 /kg in nhl group (p = 0.02). the grafts contained more nk cells (median 10.1 vs. 6.1 x 10 6 /kg, p = 0.01) and cd19 + cells (median 1.69 vs. 0.00 x 10 6 /kg, p < 0.001) in mm patients. neutropenic fever tended to be more common in nhl patients (88 % vs. 79 %, p = 0.06) but mm patients had significantly more bloodstream infections (31 % vs. 15 %, p = 0.003). the median duration of hospitalization was longer in the nhl patients (22 d vs. 18 d, p < 0.001) and the nhl patients had significantly more often icu admissions (4 % vs. 0 %, p = 0.02).post-transplant neutrophil engraftment was faster in the nhl group (median 9 d vs. 12 d, p < 0.001). the median time to platelet engraftment was 11 days in both groups. platelet count was higher in the mm group from day 15 until 1 year after auto-sct. there were significantly more early deaths (< 100 d from the graft infusion) (4 % vs. 0 %, p = 0.02) and non-relapse deaths (6% vs. 0%, p = 0.003) in the nhl group.conclusions: mm and nhl patients differ in terms of cd34 + cell mobilization, graft cellular composition and post-transplant recovery as well as risk of non-relapse death. thus, the optimal graft may be different in nhl and mm patients.disclosure: the study was supported by vtr fund from north savo hospital district and study grant from sanofi. abstract already published. single-center experience in use of plerixafor for autologous stem cell mobilization: change in practice over 10 years background: plerixafor has been proven to mobilize human periferal blood stem cells (pbscs) alone or acting synergistically with granulocyte-colony stimulating factor (g-csf). it has mainly been used for rescue mobilization after failed regimen of chemotherapy plus g-csf, but lately preemptive use in poor mobilizers has been established as cost-effective. we aim to show ten years of experience and change in practice with plerixafor use in our center.methods: we retrospectively evaluated the outcome of mobilization procedures and leukapheresis collections in our center in the period from january 2009 to october 2018. practice from the first 5 years, when plerixafor was mainly used as rescue agent after failed attempt, was compared to period from 2014 till present when preemptive use in poor mobilizers (defined as cd34+ cell counts <10 x 10 6 /l blood) was established.results: in the period from 2009 to 2013, total of 434 patients underwent collection of autologous pbscs, and 55 patients required repeated mobilization cycles (12,7%). g-csf alone was used in 39 patients and 16 patients (29%) recieved combination g-csf with plerixafor. this cohort consisted of 10 males and 6 females with non-hodgkin (nhl) and hodgkin lymphoma (mh); 13 and 3 respectively. we noted 37 unsuccessful mobilizations (67,3% in repeated mobilization, 8,5% in total) of which one patient was from plerixafor group (6,3%).background: transplantation of hla-haploidentical hematopoietic stem cells (haplo-hsct) is an established procedure for the treatment of several different hematological diseases. one possible strategy to reduce the risk of graft-versus-host disease is represented by the selective depletion of ab t-lymphocytes (coupled with the depletion of cd19+ b-cells in order to reduce the risk of ptld) using the clinimacs device (miltenyi, bergish-gladback). before depletion, leukapheresis units are washed by lowspeed centrifugation, resulting in a platelet (plt) rich supernatant (prs) as a by-product generally discarded. we studied the possibility of recovering plt from prs of the haplo donor for transfusion to the recipient during the aplasia period occurring after hsct.methods: hsc donors were mobilized with g-csf (plus plerixafor in 6 out of 24 donors) as previously described [locatelli et al, blood 2017] . leukapheresis units, obtained with a spectra optia device, were washed twice (producing 2 prs bags) at low speed to remove plt before starting the ab t-cell/b-cell depletion. the two prs were leukodepleted by filtration, centrifuged, resuspended and pooled in a total volume ranging from 95 to 360 ml intersol (is-plt) for overnight incubation at 22°c with agitation (60 cycles/min). the is-plt samples were analyzed for the criteria established by the italian transfusion law. is-plt bags were examined for the following parameters: volume >40ml, plt after leukodepletion >2x10 11 , residual leukocytes < 1x10 6 , ph >6.4 at 22°c at the end of the 5-day storage period. the sterility was tested using bd bactec culture vials. the evaluation of the residual leukocytes was performed with the bd leucocount kit. the absolute plt counts were determined using hemocytometer sysmex xn2000.results: prs bags from 24 donors were processed to produce is-plt units. median resuspension volume in intersol was 220 ml (range 95-360). the absolute mean value of plt counts measured at the end of the storage period was 2.1 x10 11 (range 1.0-6.1 x10 11 ). this value was found below the threshold fixed by italian regulation in 8 cases (33.3%). mean value of residual leukocytes was 1.3x10 5 (range 0-9x10 5 ); the ph value was always > 6.4. sterility was observed in all cases. according to the work of slichter et al. conclusions: we demonstrated that plts recovered from leukapheresis bags can be accepted as a conventional hemocomponent according to the parameters fixed by italian transfusion law and thus can be administered to the haplo-hsct recipients early after transplantation. this strategy carries several advantages. indeed, apart from the obvious advantages in terms of reduced costs, is-plt can be used to desensitize the recipient by absorption of anti-hla class i antibodies, if present in the recipient. moreover, this strategy can avoid the risk of sensitizing the transplanted patients with hla alleles that differ from the donor's ones. finally, the is-plt unit is readily available. a clinical study aimed at testing the use of is-plt units in transplant recipients will be performed to confirm the clinical efficacy of the approach.disclosure: nothing to declare p700 ultrasound guidance as a powerful tool in increasing background: peripheral blood stem cells are generally the preferred graft source for allogeneic stem cell transplantation for malignant disease. in most centers first apheresis is performed on day 5 after 8 to 9 doses of granulocyte colony stimulating factor (gcsf) up to 5ug/kg twice daily. the dose of gcsf and the number of apheresis procedures required contribute to symptom, travel and time burden donors are put through during the process. we hypothesized that taking donor-recipient weight differences into consideration may help reduce this burden methods: a total of 261 healthy donors who donated peripheral blood stem cells on day 4 of gcsf mobilization in the period between january 2015 and august 2018 at the university medical center hamburg-eppendorf were included in this quality control evaluation. the donors were divided into two cohorts. the impact of donorrecipient weight ratio on stem cell harvest was tested in the training cohort (2015-2016) and validated in the second cohort (2017) (2018) . for the training cohort, donors were grouped according to donor-recipient weight ratio < 1.0 vs. 1.0-1.2 vs. > 1.2. for the purpose of this analysis a stem cells dose of 5 x 10 6 cd34+/kg recipient weight was set for successful apheresis.results: in the training cohort including 142 donors, 52 (37%), 36 (25%) and 54 (38%) had a donor-recipient weight ratio of < 1.0, 1.0 -1.2 and > 1.2 respectively. the target stem cells count of 5 x 10 6 cd34+/kg recipient weight was achieved in 36 of 52 (69%), 26 of 36 (72%) and 45 of 54 (83%) donors with donor:recipient weight ratio < 1.0, 1.0 -1.2 and > 1.2 respectively. the cut-off for the validation cohort was therefore set at a weight ratio of 1.2.in der validation cohort including 119 donors, 75 (48%) had a weight ratio > 1.2 while 62 (52%) had a weight ratio ≤ 1.2. overall in this cohort target cell count of 5 x 10 6 cd34 +/kg recipient weight was reached in 92 (77%) cases. this target was reached in 43 of 62 (69%) of donors with weight ratio ≤ 1.2 and in 49 of 57 (86%) donors with weight ratio > 1.2, p = 0.03.conclusions: a donor-recipient weight ratio of > 1.2 is seen in about 40% of peripheral blood stem cell donations for allogeneic stem cell transplantation. in these cases apheresis on day 4 after 7 doses of gcsf is reasonable. donors with lower weight ratios should preferentially donate on day 5 after 8 to 9 doses of gcsf.disclosure: all authors declare no conflicts of interest background: the effect of a second mobilization and collection of peripheral blood stem cells (pbsc) on the cell yield is low, as we previously demonstrated. however, donor safety has been poorly addressed with no changes in the clinical practices.methods: second donations of unrelated and related donors performed between 2001 and 2017 were evaluated (n=24), including pbsc+pbsc (n=18), bone marrow (bm)+pbsc (n=5) and pbsc+bm (n=1). analytical parameters including leukocyte, lymphocyte, hemoglobin and ldh quantification, obtained on the pre-harvest evaluation of first and second donation, were retrospectively analyzed and compared for all donors. the portuguese bone marrow donors registry (cedace) recommends a time between donations no lesser than 6 months. it also states that in very urgent situations like graft failure, donor should be clinical and analytical cleared and its safety ensured.in order to evaluate the impact of time between donations, donor population was divided in 3 groups: < 2 months, 2-6 months, >6 months; to determine the influence of donor age, donors were divided in 2 groups: < 40 and ≥40 years.results: among the total of 24 donors, 13 were volunteer donors of cedace and 11 were familiar. fifteen second donations were performed because of recipient graft failure and 9 due to disease progression or relapse. at the time of second collection, median donor age was 37 years (range 23-57). the median delay between both collections was 262 days (37-1519). time between donations did not seem to substantially impact the analytical donor evaluation: leukocytes, lymphocytes, hemoglobin and ldh results are kept within the reference values. however, donors with less than 2 months between donations showed a slight decrease on leukocyte counts (35%) and hemoglobin values (12%), from the first to the second pre-harvest evaluation. donor age showed no significant influence on the analytical evaluation. nevertheless, when considering only the pbsc+pbsc donations, donors with ≥40 years showed a small decrease on lymphocyte counts (19%) .conclusions: this study demonstrated that the analytical parameters, chosen based on literature, had no significant changes between first and second donation. however, particular attention should be paid when time between donations is lesser than 2 months or donor age is ≥40 years.as we concluded that no significant changes were observed in the group of 2-6 months, it is our opinion that the minimum of 6-12 months established by the registries can be shortened to 2 months ensuring donor safety. an accurate donor risk assessment with a larger population should be accomplished in order to strengthen this recommendation.disclosure: nothing to declare. gaurav kharya 1 , atish bakane 1 , pratibha dhiman 1 , anil khetrapal 1 , vikrant bhar 1 background: t cell replete haploidentical stem cell transplant (hhsct) is complicated mainly by increased risk of graft failure (gf) and graft versus host disease (gvhd). conventionally gcsf has been used to mobilize hematopoietic stem cells (hsc). in tcr hhsct gcsf mobilized graft with megadose of cd34+ cells expose the patient to higher doses of alloreactive t cells increasing the risk of gvhd. plerixafor based mobilization gives an advantage of giving high cd34 cell dose limiting exposure to high alloreactive t cell dose. we share our experience of gcsf + plerixafor based mobilization for tcr hhsct. methods: 9 consecutive patients suffering from scd who underwent hhsct between jan 2018 till date along with the respective donors were enrolled in the study (group 1). all 9 underwent pre-transplant immune suppression (ptis) 2 cycles at 3 weekly intervals using fludarabine+cyclophosphamide+dexamethasone.the graft was mobilized using gcsf@10mcg/kg/day(d1-d5)+plerixafor@0.24mg/kg(d5) 6-8 hours before the pbsch. gvhd prophylaxis included ptcy 50 mg/kg/ day on d3 and 4, sirolimus and mmf starting from d5. group 2 included 5 historical controls where graft was mobilized using gcsf@10mcg/kg/day(d1-d5). various parameters pertaining to mobilization, harvest, engraftment, gf and gvhd were assessed between the two groups.background: poor mobilizers (pm) defined as those with a peripheral blood cd34 + count ≤10cells/μl on day+4 is a significant risk factor for mobilization failure. within these, patients with < 5cells/μl are considered as very poor mobilizers (vpm). use of plerixafor in vpm patient is controversial. the aim of our study is to compare mobilizing and engraftment between pm and vpm who received plerixafor plus g-csf (p+g-csf).methods: in our center, mobilization with g-csf at dose of 10μg/kg/day was used in all pts. apheresis were scheduled on day+5. plerixafor (0.24 mg/kg) was added if the number of cd34 + cells on day +4 was < 10/ul for 2x10 6 cd34 + /kg requested (or < 20/ul for 4x10 6 cd34 + /kg), or if the number of cd34 + cells collected in the first apheresis was < 50% of cd34 + cells requested.between january 2016 and september 2018, 37 out of 157pts (23,6%) received plerixafor for mobilization. we retrospectively studied 30 pts who mobilized with p+g-csfdue to the number of cd34 + cells on day +4 was < 10/ul.results: twelve out of 30 pts were pm, 7 were females, median age 55,5 years (range:32-70). patients' baseline diseases were: 10 non-hodgkin lymphoma (nhl) (83,3%), 1 multiple myeloma (mm) and 1 hodgkin lymphoma. median cd34 + cell count on day +4 was 8/ ul (range:6-10).there was no mobilization failure. eighteen out of 30 pts (60%) were vpm, 9 were females, median age 62,5 years (range:34-69). patients' baseline diseases were: 10 nhl (55,5%), 7 mm and 1 solid tumor. median cd34 + cell count on day+4 was 2,5/ul (range:2-5). two out of 18 pts (16,6%) were considered mobilization failure, in 2 of them did not realized apheresis due to cd34 + cell count on day +5 was 2/ul. no difference was seen between both groups regarding gender, age, patients baseline disease or median cd34 + cells count on day +4.vpm needed more apheresis sessions, 5/16 pts required 2 sessions against 1/12 pts in pm (p=not significant (ns). we obtained enough cells to carry asct in 90% pts, although mean number of cd34 + cells obtained in vpm was lower than in pm (4,89x10 6 /kg vs 6,38x10 6 /kg, respectively) (p=ns).twenty-six pts underwent asct and mean number of cd34 + cells infused were 4,27x10 6 /kg in vpm vs have been the only route used for chpc administration. the appearance of other catheters types made us to reconsider the exclusive use of the cvc for the infusion of chpc. we analyzed the use of a peripheral iv cannula (pivc) as an alternative to cvc for the infusion of chpc in patients with cardiovascular diseases.methods: medical records of 65 patients who received an asct for hematological malignant diseases at the hospital clínic of barcelona from january 2017 to february 2018 were reviewed. of those, eight were infused through a pivc due to cardiac impairment related to previous treatments, ischemic cardiomyopathy or amyloid deposition.hpc were obtained from peripheral blood by apheresis after mobilization with g-csf using acd-a as an anticoagulant. cryopreservation was performed with autologous plasma and dmso 10% by mechanical means and stored in liquid nitrogen. analytical controls were performed including hematocrit, total nucleated cells, total polymorphonuclear neutrophils and platelets using the advia 120 analyzer. the cd34 + / cd45 + population was analyzed by flow cytometry following the ishage single-platform protocol. viability of total nucleated cellularity was carried out by vital staining with acridine orange and the specific viability of the cd34 + population through the technique of 7-aminoactinomycin d. thawing was performed bag to bag by immersion in a water bath at 37ºc and transferred to the bedside of the patient for gravity infusion using an infusion set without filter through pivc of 22 gauche. vital sings monitoring performed before, during and the end of the every infusion bag including: blood pressure, heart rate, oxygen saturation, body temperature and central venous pressure. other aspects assessed during the infusion were pain, cold sensation and signs of extravasation in the area of pivc insertion. after the infusion, the recovery time of the granulocyte series and platelet were evaluated.results: median volume and bags administered was 480 (360-600) ml and 4 (3) (4) (5) . the median of total nucleated cells, total nucleated cells / ml and total cd34+ cells/kg was 462.6 x 10 8 (329.5-657.8), 107.3 x 10 8 (64.66-156.7) and 3.24 (2.46-4.6) respectively. vital signs were within the normal range and allowed to perform the infusion in an average of 20-30 minutes/bag. no patient required stopping the infusion due to pain in the area of peripheral catheter insertion and no extravasations were detected. all patients referred some cold sensation in the insertion vein and its path. median hematopoietic recovery was 14 (11-19) days for neutrophils and 11 (0-19) days for platelets, similar to the recovery experienced from patients who received chpc through cvc.conclusions: based on our data, we conclude that the administration of chpc, through pivc and by gravity is safe for the product and for the patient, being the preferred choice for patients suffering from some type of cardiovascular disease.disclosure: nothing to declare background: by selective depletion of potentially alloreactive cd45ra + cells, t memory cells might be retained in the graft and could mediate pathogen specific immunity. however, cd45ra expression is not restricted to naïve t cells, but also available on b cells, nk cells and cd34 + stem cells to some extent. methods: within this project we aim to analyze cd45ra expression on stem-and nk cells by flow cytometric analysis to estimate the eventual loss of these cells during cd45ra-depletion. furthermore, clinimacs depletion following a one-step approach of direct cd45radepletion and a two-step approach with primary cd34selection followed by cd45ra-depletion of the negative fraction was investigated.results: cd45ra expression on cd34 + stem cells was in median 16.9%. with a median of 99.4% cd45ra expression was measurable on nearly all b cells, which obviates depletion via cd19. a comparably high cd45ra expression of in median 96.0% was detected on nk cells. unfortunately, the amount of nk cells in the cd45ra-depleted product was 0.24%. clinimacs depletion following one-step approach resulted in a stem cell recovery of 52.0%. memory t cell recovery was 24.2% following one-step and 42.0% applying two-step approach. depletion quality measured by log-depletion was 3.9 and 3.8 for cd45ra + t cells and 3.2 and 3.6 for cd19 + b cells for one-and two-step approaches, respectively.conclusions: with regard to stem cell recovery, a previous cd34-selection before cd45ra-depletion is recommendable.background: current clinical practice of routine use of filters for infusion of autologous hematopoietic cell transplantation (ahct) at bone marrow transplant centers across north america and europe is not known. the use of "y" administration tubing without a filter could possibly increase the risk of infusion of macro-aggregates and cellular debris, which may result in increased side effects.methods: we carried out a retrospective chart review of patients (pts) at spectrum health who underwent ahct. group a (gp a) pts received ahct using a "y" administration tubing with 170-micron filter from 3/2013 -3/2017. these patients were compared to a control group (gp b) that received ahct without filter administration tubing from 3/2014-4/2016.this change in clinical practice occurred due to a change in policy at our transplant center as a result of inorganic particles noticed during cryopreservation. we compared the neutrophil and platelet engraftment duration between these groups. we also studied the length of hospital stay and the effect of filter use on any immediate side effects after infusion. due to the retrospective nature of the study it was not feasible to evaluate the difference in duration of infusion between these groups results: the two groups were similar in their age, gender, primary disease distribution and median number of cd34 stem cells infused (table) . there was no difference in median neutrophil (11 vs 11 days) or platelet engraftment (19 vs 19 days) duration for the filter group and the nonfilter group respectively. the median length of hospital stay was also comparable (17 days). there was no statistically significant difference in the immediate side effects (fever, cough, dyspnea, fluid overload, flushing, nausea, vomiting, hypertension, hypotension and anaphylaxis) or confirmed post-transplant infections (viral, bacterial, fungal) experienced by these two groups.conclusions: our results show that the routine use of filter does not prolong hospital stay, and neutrophil/ platelet engraftment duration, thereby, suggesting that viable stem cells are not affected. on the other hand, filter use failed to demonstrate any appreciable decline in the immediate side effects experienced after ahct. gp background: allo-hsct from related haplo-identical donors (haplo-hsct) with post-transplant high-dose cyclophosphamide is increasingly employed in patients who lack a matched related or unrelated donor. the current standard is to use bone marrow grafts (bm) as peripheral blood stem cell grafts (pbsc) have been associated with an increased risk of acute and chronic gvhd. thus, the aim of our study was to compare the main transplant outcomes and especially the incidence of acute and chronic gvhd in recipients of bm and pbsc grafts. methods: thirty-five unselected patients with hematologic malignancy who underwent an haploidentical transplant at our unit between 2011 and 2018 and received bm (n = 17) or pbsc (n = 18) grafts after the same tbf conditioning regimen were analysed in order to assess differences in transplant outcomes.our gvhd prophylaxis consisted in cyclosporine a (csa) from day -1 to +180, a methotrexate "short course" and mycophenolate mofetil (mmf) from day +1 to +28.results: no statistically-significant differences were observed between patients who received bm grafts and those who received pbsc grafts. at transplant fourteen patients were in first complete remission (cr), twelve in advanced cr and 9 had active disease. according to sorror's risk, nine patients were low-risk, nine intermediate-risk and seventeen high-risk. twenty-eight cmv+ patients received the graft from twenty-three cmv+ and five cmv-donors, seven cmv-patients received the graft from five cmv+ and two cmvdonors. mean age at transplant was 51 years (range 23-72), mean donor's age 38 years (range18-39) and mean follow-up 16.9 months (range 1.9-56.7). median cd 34+ cell dose was 5.2x10 6 /kg (range1.4-10.4), 3.6 x10 6 /kg (range 1.4-7.7) in bm recipients and 14.0 x10 6 /kg (range 4.2-10.4) in pbsc recipients. median time to neutrophil recovery (>500/μl) was 22 days (range 16-39) posttransplant, 23 days (range 18-27) for bm recipients and 20 (range 16-39) for pbsc recipients. platelet recovery (>20.000/μl) occurred in all patients except one at a median of 17 days (range 10-151) post-transplant, at a median of 20 days (11-151) post-transplant for bm recipients and at a median of 14 days (range (10-26) for pbsc recipients. seven patients never reached platelets >50.000/μl. three patients developed a poor graft function. acute and chronic gvhd incidence was 28.5% and 22.8% and the risks of acute (hazard ratio [hr], 1.04; p = .955) and chronic (hr, 0.85; p = .816) graft-versus-host disease were similar in the two patient groups. in addition, there were no differences in relapse risks post-transplant (hr, 0.90; p = .898); relapse-free survival was better with pbsc grafts but this difference did not reach any statistical significance. finally, no significant differences were noted in overall mortality by graft type (hr, 0.62; p = .441).conclusions: despite in haplo-hsct the incidence of acute and chronic gvhd is reported to be higher with pbsc than with bm our small patient series does not confirm this assumption that should be clarified by additional studies. instead, our data suggest that pbsc background: since 1988, cord blood (cbu) has become an alternative source of stem cells for transplantation, with approximately 35,000 procedures currently performed. with a 2-year gs of 50%.objective: analyze the outcomes from all the patients transplanted with cbu in our hospital unit.methods: retrospective, longitudinal study. all patients transplanted with cbu in our hospital between 2007 and 2017 were included. we analyzed 40 patients with ages from 7 months to 14 years.results: two of them received doubled cord transplantation the ratio male: female was 1.3: 1. the transplanted pathologies were: bone marrow failure 35%, immunodeficiency 25%, aml 20%, all 17.5%, osteopetrosis 2.5%. ric regimens were used in patients with bone marrow failure and immunodeficiency and myeloablative conditioning regimens were used in patients with malignant hematology diseases. antithymocyte rabbit globulin (atg) based serotherapy was used. one case received cbu from a related donor (sister), the rest received unrelated cbu obtained from centro nacional de la trasfusión sanguínea. the infusion of cd34 + was in a range of 0.11 to of 2.7 x10 6 /kg with and average of 0.43 x 10 6 / kg. compatibility was 4/6 in 70%, 5/6 in 20% and 6/6 in 10%. post-thawing cellularity was not measured. the hla-c was not analyzed. forty two point five percent of the patients had a successful engraftment; the average time of engraftment was 30 days. primary graft failure was detected in 57.5% and secondary graft failure in 10%, for a total success of 32.5%. gvhd was detected in 20% of patients, of which 90% was grade i-ii and 10% grade iii-iv. the overall mortality was 52.5%. causes of death were: infection 45% relapse 30%, hemorrhage 20% and gvhd 5%. the cbsct continues to be an essential alternative in our patients who required transplantation knowing that this stem cell source allows the procedure to be done with less histocompatibility requirements and it is available immediately, which facilitates the process considering the great diversity that exists within our population. however, in our experience, the cbsct has shown a higher mortality risk, which can be improved by analyzing the hla c, choosing in this way the units with better compatibility, and improving cellular dosage since this is key in success.disclosure: nothing to declare the risk of infection of the umbilical cord is not related to the microbiological status of the umbilical cord blood methods: the statistical analysis was carried out on data obtained from samples taken in poland between 01-jan-2017 and 31-dec-2017. the samples were collected in hospitals by external midwifes and sent to the pbkm in accordance with the requirements of the american association of blood banks. after arrival in the laboratory, the blood samples were cultured and the ucs were assessed immediately for visual signs of infection, such as odor, altered color, or visible bloom. the status of both kinds of samples was introduced into the pbkm general database. for the purpose of this analysis, the ucs were considered as microbiologically pure if stored, destroyed after storage, or handed over to the pbkm. samples marked as infected or disqualified for unknown reasons (other than termination of the contract with the customer, viral infection of the mother, and lack of cell growth) were considered as infected. at the time of the statistical analysis, the samples of unknown ucb microbiological culture status were removed from the generated report. the data was summarized as percentages and the odds ratio was calculated. statistical significance was considered at p˂0.05.background: match family donors are the preferable options in allogenic stem cell transplant. however, in the absence of donor relatives match unrelated donors have been an option. in this study, the donor screening, transplant preparation phases of turkish stem cell coordination center (turkok) and the i̇stanbul university bone marrow bank (tris), were compared.methods: the unrelated donor scanning data between march 2015 and november 2018 in pediatric stem cell transplantation unit of altınbas university bahcelievler medical park hospital were evaluated. 93 unrelated transplants were performed in total. 65 % of these transplants (n=61) were included from the donors of turkok registration system and 34,4 % of these transplants (n=32) were included by means of tris from donors outside of turkey. 8 patients (5 tris, 3 turkok) were excluded from the study in consequence of screening update and postpone of transplantation. the statistics were carried out on a total of 85 patients, 67,1 % of whom were in turkok (n=57) and 32,9 % were transplanted via tris (n=28). the day of application to stem cell transplantation unit, reply dates and the transplantation dates were examined for the transplant patients.results: in the current study, the average response time of turkok was found as 0,91±2,96 day (median: 0), the average transplant time after receiving a reply was found as 98,24±63,89 (median: 85) day, the average number of days from date of application to date of transplantation of patients was found as 101,12±63,17 (median: 89). the average response time of tris was 18,78±18,36 (median: 15) day, the average transplant time after receiving a reply from tris was 136,82±63,84 (median: 119) day, average number of days from date of application of tris to date of transplantation of patients 155,61±72,23 (median: 140) day.the average response time of turkok, the average transplant time after receiving a reply from turkok and the average number of days from date of application of turkok to date of transplantation of patients was shorter than tris. the difference between them was found statistically significant (p< 0.05).conclusions: in this study, it was determined that the transplantation processes with turkok were progressing more rapidly. the rapid progress of the process was attributed to the fact that all donor hla tissue groups in the turkok database were studied in high resolution. in international scans carried out through tris, it was thought that the examination of the donor castings coming from bone marrow banks and the time differences between the countries prolong the process. it was thought that hla tests of the registered donors in the tris database and some international bone marrow banks were studied in low resolution but not studied the all hla loci, the centers wanted high-resolution hla, and therefore the involvement of social security institutions and payment procedures were among the factors extending this process.disclosure: nothing to declare background: allogeneic hematopoietic stem cell transplantation (ahsct) is being performed for a group of hematologic diseases with a curative intent. outcomes after ahsct are influenced by the type of donor used. haploidentical transplantation is an emerging option when a fullmatched donor is unavailable. methods: we retrospectively analyzed our transplants performed between january 2015 and november 2018, investigating outcomes and complications among haploidentical stem cell recipients.results: one hundred and nineteen patients underwent ahsct, 9 of them (7.5%) were recipient of a haploidentical stem cell and included in this study. one patient diagnosed with acute lymphoblastic leukemia (all) were performed a haploidentical ahsct for two times due to relapse. among those 9 transplants, 4 of them were diagnosed with acute myeloid leukemia, 2 with all, 1 with chronic lymphocytic leukemia, 1 with myelodysplastic syndrome and 1 with hodgkin lymphoma. the mean age of group was 35.7±15.2 years. three patients (2 aml, 1 all) were in remission at the time of transplantation. patients were given a conditioning regimen based mostly on busulfan, fludarabin and total body irradiation with a myeloablative intent. patients were also given a various combinations of post-transplant cyclophosphamide, calcineurin inhibitors, mycophenolate mofetil and antithymocyte globulin for graft versus host disease (gvhd) prophylaxis; post-transplant cyclophosphamide administered on 6 (67%) of those transplantations. peripheral blood was the source of stem cells in all patients. patients were infused with mean 6.15±0.67x10 6 /kg of cd34+ cells. hematological recovery was achieved with neutrophil engraftment at a mean of 19.2±2.1 days and platelet engraftment at a mean of 17.3±2.7 days. after a median 8month (0.2-40.6 months) follow up, the cumulative rates of grade 3-4 gvhd, relapse and non-relapse mortality were 11%, 28% (n=7) and 71%, respectively. one patient died due to relapse, at the end of the follow up two were still alive with remission. only one patient has died due to chronic gvhd affecting serosa and resulting with a fatal tracheoesophageal fistula. the mean overall survival was 6.9±2.6 months in our study.conclusions: haploidentical transplant is a feasible option in hematologic malignancies with novel gvhd prophylaxis approaches, especially post-transplantation cyclophosphamide. however, these results need to be supported with further investigations with a larger patient group.disclosure: nothing to declare results: a total of 25 patients between 19 and 58 years (median age: 33 years). transplant was done for following disease: acute leukemia (n=9, 36%), aplastic anemia (n=5, 20%), lymphoma (n=4, 16%), myelofibrosis (n=3, 12%), myelodisplastic syndrome (n=2, 8%), chronic myeloid leukemia (n=2, 8%). ten donors were from turkey and fifteen donors were from different countries of europe and america. two of 25 donors were 9/10 and the other 23 was 10/10 hla matched. the conditioning regimen was mostly non myeloablative (n=17, 68%) while eight patients were treated with myeloablative regimen. other than two patients who took tacrolimus and mycophenolate mofetile all of them got cyclophosphamide and methotrexate for graft versus host disease (gvhd) prophylaxis. the median time of neutrophil and platelets engrafman were 18 days (range 11-31) and 13,5 days (range 9-36) respectively. acute gvhd was seen nearly half of the patients (47,8%).overall survival was 40% for all patients and 15 of 25 patiens (60%) died within first month to 18 months (median 3 months). the mortality rate was more higher for the recipients who had donor source from countries other than turkey (30% vs 80% p=0,018). transplant related mortality was the most common reason of mortality (n=7/ 15, 46,7%) and other reasons were gvhd (33,3%), infections and cirrhosis respectively.conclusions: we found the mortality rate more higher in the patients whose donors were from out of our country. however, we need to further multicentric and prospective investigations to confirm our hypothesis, it would be related with impact of ethnicity.disclosure: nothing to disclose late-breaking abstracts p725 targeted twice daily busulfan-based ric-conditioning for allogeneic hematopoietic stem cell transplantation in pediatric patients with chronic granulomatous disease: a 10-year experience with the zurich protocol matthias felber 1 , mathias hauri-hohl 1 , ulrike zeilhofer 1 , federica achini 1 , jana pachlopnik-schmid 2 , janine reichenbach 2 , seraina prader 2 , tayfun güngör 1background: chronic granulomatous disease (cgd) needs sufficient myeloablation to avoid graft failure. for this purpose the ebmt inborn errors working party currently recommends treosulfan or busulfan-based conditioning regimens for cgd-sct. we analyzed the last 10 years of targeted busulfan-based ric-conditioning including engraftment, gvhd rates, chimerism and late term effects in our pediatric sct center in zurich. methods: between 2008 and 2018, n=34 consecutive pediatric cgd patients (median age 6 years, range 1-16 years, n=4 female, n=9 autosomal recessive inheritance) have been transplanted. all patients received therapeutic drug monitoring of twice daily administered iv busulfan (3 or 4 hour infusions; d-5 to -d2) to achieve a targeted cumulative auc of 45-70 mg/l*h. fludarabine (180 mg/ sqm; d-8 to -d3) and serotherapy (thymoglobuline 7.5 mg/ kg total, d-5 to d-3) or alemtuzumab (0.5-0.6 mg/kg total; d-8-to d-6) were used for immunoablation. donors were matched unrelated (10/10 hla; n= 19), mismatched unrelated (9/10 hla; n= 7), mismatched unrelated (hla 8/10; n=1), matched sibling (hla 6/6; n= 5) and haploidentical parental (hla 5/10; n=2). for 2 patients with haploidentical donor post-transplant cyclophosphamide (d-3 and d-4 with 50 mg/kg iv each) and upfront atg-grafalon (40 mg/kg -12 to-d-9) was used. stem cell sources were bm (n=29) and pbsc (n=5). gvhdprophylaxis included iv csa (d-3; continuous infusion) and iv. mmf (d-0, in 2-3 doses).results: follow-up was 6 to 118 months. good overall engraftment was noted, with n=1 secondary graft failure followed by successful retransplantation. in one patient a stem cell boost/dli was necessary due to decreasing myeloid donor chimerism during ebv reactivation, resulting in rapid myeloid donor reconstitution after intervention. low rates of gvhd were documented with n=2 agvhd grade iv and n=2mild/limited cgvhd (nih criteria). with exception of one patient, myeloid donor chimerism at last follow-up was over 82%, mostly over 95%. overall survival was 31/34 (91%). deaths were due to gi-gvhd (n=1), autoimmune hemolytic anemia/sepsis (n=1) and thrombotic microangiopathy (n=1).conclusions: precision dosing of iv busulfan in combination with fludarabine and serotherapy results in excellent outcome of hsct for pediatric cgd-patients with good engraftment, low overall cgvhd rates and stable, mostly excellent donor chimerism. graft failure rate was as low as 3%. low dose alemtuzumab prevented gvhd in the majority of patients. this analysis demonstrates that targeted busulfan-based conditioning is a valid option for pediatric cgd-patients. serum alemtuzumab or atg monitoring could further improve gf and gvhd rates in the future.disclosure: the authors declare no conflict of interest. abstract already published. young hla-matched unrelated donors are comparable to matched sibling donors in elderly patients receiving reduced-intensity conditioning: an analysis on behalf of the ebmt scientific council 95% c.i. 1.7-20.6, p= 0.001) and voriconazol prophylaxis during carv (or 4.2, 95% c.i. 1.1-115.6, p= 0.03). conclusions: we provide evidence that ifd after carv infection. allo-hsct recipients developing a carv lrtd during the first year after transplant may benefit from an adequate antifungal prophylaxis and a close monitoring for the development of a later ifd.disclosure: jose luis piñana has received both, advisory for preclinical/clinical research and financial support to assist to the spanish society of hematology annual meeting 2018 from msd. favorable outcome and engraftment following reducedintensity conditioned allo-hsct in children with primary haemophagocytic lymphohistiocytosis (hlh) and high-risk langerhans cell histiocytosis (lch) laura m. moser, emilia salzmann-manrique, andrea jarisch, jan sörensen, shahrzad bakhtiar, peter bader background: primary haemophagocytic lymphohistiocytosis (hlh) and high-risk langerhans cell histiocytosis (lch) represent two major entities of childhood histiocytoses, which are -although only of rare occurrence -severe in their clinical manifestations. patients present with multisystemic uncontrolled inflammation and multi-organ involvement requiring diverse courses of immunosuppressive and chemotherapy regimens. allogeneic haematopoietic stem cell transplantation (allohsct) is the only available curative option; however, the cumulative treatment toxicity and the underlying inflammatory disease often result in high organ toxicity and inflammatory complications of transplantation, such as graft versus host disease (gvhd) and/or graft failure. especially patients with unrelated donors often deal with high transplant-related mortality (trm) in the setting of conventional intensity conditioning.herein, we present the clinical course of the disease and transplant outcome of 14 children diagnosed with primary hlh (n=12) and high-risk lch (n=3) who underwent allohsct at our centre from 02/2003 to 08/2018.methods: the hlh cohort consisted of 7 cases of familial hlh (fhlh), 4 cases of griscelli syndrome, one xiap-deficient patient and one hlh-patient with inconclusive genetic testing. all hlh patients had developed clinical symptoms prior to transplantation and had been treated according to hlh-protocols 94, 2004 hlh-protocols 94, or 2015 median age at transplantation was 10 months (4 to 128 months). stem cells were derived from hla-matched siblings (msd, n= 4), matched unrelated donors (mud, n = 9) or haploidentical donors (n=1).the majority of patients (9/14) received a ric regimen containing fludarabine, melphalan and thiotepa (n=8) and fludarabine plus cyclophosphamide (n=1). myeloablative treatment (5/14) included a treosulfan-based regimen (n=3) and busulfan-containing treatment (n=2). the entire cohort received serotherapy using either muromonab (n=3), atg-fresenius® (n=9) or alemtuzumab (n=2).results: the overall survival of the entire cohort was 78.6% (11/14) on a median follow-up of 9.9 years ( figure 1 a+b) .all lch patients, being treated with fludarabine, melphalan and thiotepa, survived transplantation and showed complete remission (3/3) . within the hlh cohort the overall survival was 72.7% (8/11). fatalities (n=3) included two patients from the myeloablative group and one ric-treated patient. the cause of death were progressive disease activity during the conditioning phase, leading to multi-organ failure on day +15 despite immunosuppressive treatment (n=1) and complicated cerebral seizures followed by lung haemorrhage, possibly due to aspiration pneumonia with evidence of enterococcus faecium, resulting in septic multi-organ failure on day +4 (n=1). a third hlh patient developed a sudden cerebral edema and ensuing respiratory insufficiency on day +4. whether this was caused by acute neurotoxic damage by fludarabine or a consequence of relapsed hlh could not be conclusively specified. none of our patients suffered from transplant failure or nonengraftment. there was neither severe acute gvhd (iii-iv) nor chronic gvhd observed in this cohort.conclusions: primary hlh and high-risk lch are lifethreatening medical conditions needing rapid allohsct. ric regimens are well-tolerated and sufficient for proper engraftment and disease clearance. disclosure: the authors have no conflicts of interest to disclose. abstract withdrawn. thrombocytopenia following allo-sct concomitant to stem cell boosts. steroid refractoriness was defined as: progression after three days or no response after 5 days of steroid treatment. the median time from sct to the onset of agvhd was 22.5 d (range 9-122 d), and 11 d (range 3-40 d) from the onset of agvhd to the first msc infusion, respectively.the majority of our patients (n=5) suffered from a malignant disease and received a graft from a matched unrelated donor (n=6), while one patient had a haploidentical donor. gvhd prophylaxis was performed in all patients except the patient with the haplo-identical graft. all patients with agvhd were treated with steroids and the patient with thrombocytopenia required regularly transfusions and romiplostin therapy. the median msc dose was 1.7x10 6 cells/kg bw (minimum 1.1 x10 6 ; maximum 3 x10 6 ). three patients received 2 msc doses, two patients 4, one patient 5 and another 7 doses.results: at the time point of agvhd manifestation and msc application, two patients had cmv reactivation, one patient adenovirus infection and one patient ebvreactivation. by day 28, 5/6 agvhd patients responded to msc administration: 3 with complete response and 2 with partial response. at the last follow-up (median: 4.42 months, range 0,82-12.06 months), 4 of 6 patients were alive without acute or chronic gvhd. one patient died soon after msc treatment with no obvious response in the course of a systemic hyperinflammation syndrome. the other patient although complete responder to msc-ffm developed fatal adenovirus sepsis. this based on the profound tcell depletion induced by concomitant application of steroids. the overall survival probability at six month was 66.7%. no acute side effects occurred after msc infusions. the previously mentioned patient suffering from thrombocytopenia did not need any further transfusions after receiving 2 doses mscs combined with stem cell boosts while continuing romiplostin application.conclusions: our data confirm excellent tolerability and high efficacy of the licensed off-the-shelf msc preparation "msc-ffm" in pediatric steroid-refractory agvhd. in our center, current treatment algorithms have escalated "msc-ffm" to the second line, i.e. immediately after steroid refractoriness has been established. besides immunoregulatory properties, this product might facilitate hematopoietic stem cell engraftment.disclosure: novartis (consultancy: included expert testimony, speaker bureau, honoraria), medac (research funding, patents and royalties), riemser (research funding), neovii (research funding), amgen (honoraria) expression of ccr4 modulates migrational properties of in vitro expanded murine regulatory t cells laura m. moser 1,2 , ulrike tischler 1 , christin riegel 1 , julia minderjahn 1 , rüdiger eder 1 , jaqueline dirmeier 1 , isabel zimmermann 1 , evelyn röseler 1 , petra hoffmann 1,3 , matthias edinger 1, 3 background: hematopoietic stem cell transplantation (hsct) as it is carried out successfully at other genetic instability syndromes seems to be an encouraging opportunity for a curative therapy to restore immunity and prevent the development of hematologic malignancies in ataxiatelangiectasia (a-t). however, experience in the conditioning regimen is limited and no transplantation strategy for a-t patients exists, especially in an allogeneic setting. conditioning regimen and donor selection are critical factors in the clinical setting of hsct and incur substantial risks, especially in a-t. the aim of this study was (1) to evaluate whether different approaches of hsct including allogeneic hematopoietic hsct are feasible in regard to graft versus host response (gvhd) and sufficient concerning immune reconstitution (2) and to de-escalate the toxic effects of the conditioning regimen by reducing the dose of cyclophosphamide (cp).methods: t cells from syngeneic, allogeneic and haploidentical donor mice were used to determine gvhd induced t cell proliferation in a mixed lymphocyte reaction (mlr). atm-deficient mice were treated with cp or reduced cp in combination with fludarabine (flu) and transplanted with 5x10 6 cd90.2 depleted bone marrow donor cells from 129/svev gfp-transfected wildtype mice (syngeneic) or from mice of the f1 generation of 129/svev wildtype mice and c57bl/6 mice (haploidentical), or from c57bl/6 mice (allogeneic). tracking of gfp-positive donor derived cells was performed using flow cytometry and atm pcr. oxidative stress and damage were detected by a rt profiler pcr array and 8-hydroxy-2′deoxyguanosine.results: mlr resulted in an increased proliferation of allogeneic donor t cells compared to syngeneic and haploidentical donor cells. response was lower on dendritic cells isolated from atm-deficient mice compared to wildtype controls. in vivo results showed the restoration of t cells in atm-deficient mice accompanied by a prolonged life span and through reduction of thymic tumors. however, allogeneic stem cell transplantation was accompanied with a higher mortality rate, compared to the haploidentical and syngeneic setting. decreased antioxidative capacity and a higher dna-damage were seen in cp treated atmdeficient mice.conclusions: haploidentical hsct seems to be a feasible strategy for a-t. our data provided further evidence for the high sensitivity against ros-inducing agents in a-t and this fact needs to be taken into consideration in the choice of the host-conditioning strategy.disclosure: nothing to declare this research received funding from action for a-t charity (14gou01) background: prognosis of pediatric patients and young adults suffering from refractory or high-risk soft tissue sarcomas remains poor with limited improvement over the last decades despite multimodal treatment strategies. replacing the immune system by an allogeneic hematopoietic stem cell transplantation (hsct) in refractory solid malignancies has been proposed as a potentially curative therapy due to its presumable graft versus tumor effect. based on this concept we additionally performed consecutive donor-derived lymphocyte infusions in allogeneic hsct-patients with refractory or relapsed solid malignancy to further increase anti-tumor efficacy post-transplant.methods: pediatric patients with relapsed and/or refractory cancers or with delayed responses to the respective induction therapies were offered donorderived cellular therapies after immune system replacement by an allogeneic hsct. cellular immunotherapies comprised of donor lymphocyte infusions (dli), natural killer (nk) cell or cytokine-induced killer (cik) cell infusions generated from the original stem cell donors. allogeneic nk cells were generated from unstimulated leukapheresis by a two-step purification procedure using immunomagnetic cd3 t cell depletion, followed by nk cell enrichment (cd56+) with or without in vitro il-2 stimulation and expansion for 9-14 days. for cik cell generation peripheral blood mononuclear cells were isolated and activated by in vitro cytokine stimulation (inf-γ, anti-cd3, il-2 and il-15) an expanded over 10-12 days. expanded cik cells represented a heterogeneous population of polyclonal t cells with in part shared phenotypic and functional properties of nk cells.results: between october 1 st 2003 and january 1 st 2014 a total of 18 patients (eight patients with rhabdomyosarcoma, one patient with synovial sarcoma, two patients with ewing sarcoma, five patients with neuroblastoma, one patient with hepatoblastoma, and one patient with nasopharynx carcinoma) were enrolled. seven of 18 (39%) patients in this study had achieved complete remission (cr) before hsct while another 7 of 18 (39%) patients had obtained at least very good partial or partial response (vgpr or pr). dli was applied in 5 patients, nk cell treatment was offered to another 6 patients, while cik cell therapy was given to 7 patients.1.5-year probabilities of overall survival (os) and progression-free survival (pfs) were 16.2% and 15.9% for all patients with a median follow up of 8.5 months (range, 1.5-115.1 months). patients in cr at the time of immune cell therapy (it) showed estimated 1.5-year os and pfs of 49.6% and 42.7%, respectively. the majority of patients relapsed and ultimately succumbed to their diseases with two of 18 (11%) patients still being alive 9.6 and 9.3 years after it. cumulative incidence of relapse was 79.8% at 1.5 years. t cell engraftment and immune reconstitution (ir) was improved by it, and correlated with treatment response. however, two of 18 heavily pretreated patients (11%) died due to cumulative treatment-related mortality (trm). furthermore, acute graft-versus-host-disease (agvhd) occurred in 9 of 18 patients (50%) with agvhd grade i-ii observed in 6 (33%) and agvhd grade iii seen in three (17%) patients.conclusions: altogether, the results of this study indicate that allogeneic donor-derived cellular therapy at its current state offers curative benefit in selected refractory childhood cancers but warrants further improvement. background: allogeneic stem cell transplantation (allo-sct) is the only curative treatment option for a variety of nonmalignant diseases. the success of allo-sct is strongly associated with rapid and sustained immune reconstitution (ir). we analyzed the ir in patients who received an allo-sct for nonmalignant diseases.ir was assessed on days +30, +60, +90, +180 and +365 after allo-sct analyzing leukocytes, lymphocytes, monocytes, cd3 + t cells, cd3 + cd4 + t helper cells, cd3 + cd8 + cytotoxic t cells, cd3 -cd56 + natural killer (nk) cells and cd19 + b cells.methods: we analyzed ir-data of 116 consecutive patients receiving allo-scts between september 2001 and november 2018. indications of allo-sct were hereditary anemias (thalassemia, sickle cell disease, diamond blackfan anemia; ha, n=35, 30%), inherited bone marrow failure syndrome (fanconi anemia, severe aplastic anemia, others; bmfs, n=34, 29%), hemophagocytic lymphohistiocytosis (hlh, n=12, 10%), immunodeficiency (id, n=28, 24%) and metabolic disorders (n=7, 6%). the median age at allo-sct was 7 years (range, 0.2 -26) and at diagnosis 0.8 years (range, -0.5 -17.8).patients received 1 st allo-sct from msd/mfd (n=53, 46%), mud (n=42, 36%), haploidentical mismatch family donors (n=15, 13%) and mmud (n=6, 5%). conditioning regimens were busulphan-based (n=14, 12%), treosulphan-based (n=45, 39%), flu-mel-thio (n=35, 30%) or others (n=22, 19%). graft sources were bm (n=84, 72%) and pbsc (n=32, 28%).in order to consider the age-dependency of ir we normalized each absolute cell count with its corresponding age-specific expected mean values. (huenecke et al.; eur j haematol; 2008) results: the ir pattern was similar between the ha and bmfs groups. the cd4 + t cells were recovering slightly faster in ha patients compared to the recovery of bmfs patients.monocytes and nk cells proliferate very fast. at day +60 half of the patients already reached their respective monocytes reference value except for id patients, who reached 80% of the reference value at the end of the first year.cd3 + cd8 + cytotoxic t cells recovered significantly faster in patients with hematologic diseases compared to patients with hlh (p< 0.001) and id (p=0.047). half of the patients reached the reference value of cytotoxic t cells in the hematologic diseases group at day +365. by far inferior was the ir for the hlh patients. in this group only 50% of the patients reached the 45 th percentile of the healthy age-matched reference. in the id group 50% of all patients reached the 87 th percentile of the age-matched reference group at day +365.b cells are profoundly decreased at day +30 in all groups. however, the longitudinal expansion of b cells was significantly lower both in the id group and hlh group compared to the hematologic diseases group. at day +365 fifty percent of patients with id, hlh and hematologic diseases reached the 37 th percentile, 52 th percentile and the 100 th percentile, respectively (p< 0.001; p=0.027).conclusions: allo-sct is the only curative option for patients with nonmalignant diseases. ir is dynamic and revealed a complex diversity pattern with regard to the original disease. to investigate factors influencing ir after allo-sct is crucial to improve outcome of these patients.disclosure: nothing to declare. allogeneic hematopoietic stem cell transplantation in patients with myelodysplastic syndrome of relatively high-risk groups: curative effect analysis and optimal timing selection results: among the 135 patients, 133 patients were transplanted successfully. the 3-year overall survival (os) rate and disease-free survival (dfs) rate was 76.8% ±4.2% and 76.7%±4.3% respectively. the 3-year cumulative relapse rate (rr) and the non-relapse mortality (nrm) rate was 13.9%±0.1% and 18.4%±0.1% respectively. the incidence of grade ii-iv acute graft versus host disease (agvhd) was 25.2%±0.1%. for the patients who survived more than 100 days after allo-hct, the 2 years cumulative incidence of chronic graft versus host disease (cgvhd) was 33.4%±0.2%. univariate analysis showed that the hematopoietic cell transplantation comorbidity index(hct-ci) and grade iii-iv agvhd are the high risk factors for os(81.2±4.5% vs 62.8±10%, p=0.027 and 81.9±4.4% vs 51.1±11.8%,p <0.001). multivariate analysis demonstrated that grade iii-iv agvhd and hct-ci are independent risk factors for os(hr=6.206, p <0.001, 95% ci:2.529~15.288 and hr=2.651,p=0.025, 95%ci:1.127~6.232). chemotherapy before transplantation did not improve os or dfs for patients with bone marrow blast cells more than 10% at the time of diagnosis.conclusions: allo-hsct is an effective treatment for mds patients of relatively high-risk groups. the physical condition of the patients and occurrence of agvhd are independent risk factors. for intermediate and high risk ipss-r mds patients, transplantation before the disease progressed into very high risk can achieve better prognosis, high-risk group can still benefit from rebound gvhd after cni tapering which was promptly responsive to treatment steroids, fk506 and ecp. aa one year after sct 12/13 patients were without gvhd and off all immunosuppression while one single patient was still on taper of immunosuppressant after rebound acute gvhd. no chronic gvhd occurred. sctrelated toxicity was common with mucositis in all patients (who grade 4: n=5), elevated liver enzymes (≥grade 3: n=9) and impaired renal function (gfr 40-60ml/min: n=8). five patients developed neurologic symptoms (seizures n=2, pres n=1, pseudotumor cerebri n=2) which all resolved without sequelae. overall survival and transfusion-free survival was 100% with a median observation time of 5 (2-8) years.conclusions: 4. treosulfan-based conditioning followed by sct from hla-matched related or unrelated donors represents a highly efficacious treatment approach for children, adolescents and young adults with tdt and exhibits an acceptable but not negligible safety profile. an individual risk-benefit assessment incorporating hazards such as secondary graft failure, gvhd and long-term toxicity including infertility and 2 nd malignancy has to be executed in the informed consent process for every patient and his/her guardians.disclosure: "nothing to declare" p740 abstract already published. early iron chelation with deferasirox might prevent relapse after busulfan plus fludarabine and atg as a myeloablative conditioning for hla-identical sibling allogeneic hct in aml results: we show an excellent concordance between chimerism assessment on bio-rad and 3 d platforms over the complete range of mixture ratios (r 2 >0,9) and proof the lower detection limit (0,1 %) compared to str-pcr.conclusions: our results promote the transfer of the established mentype assay to a more diverse instrument portfolio. that will allow to implement the analysis of patient and donor hematopoiesis by digital pcr methods in our lab.disclosure background: with the immense progress in therapeutic regimens in pediatric oncology and stem cell transplantation the survivor rates increased up to 80%. at the same time the field of reproductive medicine has achieved substantial advances to offer potential options for fertility preservation. therefore it is of great importance to implement fertility counseling and fertility preserving (fp) procedures for patients facing gonadotoxic therapy. in the report on the expert meeting of the paediatric diseases working party (pdwp) of the ebmt in 2017 counseling related to fp opportunities should be offered to each patient receiving stem cell transplantation (sct), as part of the pre-sct workup by a dedicated and trained team. yet there many medical, ethical, structural and financial issues to consider and overcome. we describe the setting up process to enable fertility counseling for all children with newly diagnosed cancer or those facing stem cell transplantation for malignant and nonmalignant diseases in our department of pediatric oncology and immunology/stem cell transplantation.methods: at our tertiary care center we assembled a multidisciplinary team involved in fertility preservation (pediatric hematology/oncology, pediatric immunology/ stem cell transplantation, reproductive medicine, andrology, psychology and pediatric surgery). we developed an internal grading system for recommendations regarding fertility preservation based on the current recommendations for fertility preservation of leading societies in this field like ebmt, gpoh and dggg. it is important to find a consensus within the team for the counseling to ensure reliable counseling. the third step is to implement structures needed for fertility counseling and performance of invasive procedures including legal aspects (amg). a detailed description of this process is given.results: after setting up structures for the counseling process we counseled 140 oncology and stem cell transplant patients (0-18 years) between january 2017 and may 2018. we analysed data of the patients including age subgroups and disease entities and the results of the counseling process. for those patients undergoing stem cell transplantation the risk of gonadotoxicity is very high, therefore even the very young children underwent fertility preserving procedures in alignment with our recommendations if they suffered from a nonmalignant disease. currently we discourage tissue preserving in malignant systemic disease due to possible contamination with malignant cells. postpubertal female patients were more likely to undergo invasive procedures such as ovarian tissue cryopreservation in the case of oncological diseases, while sperm cryopreservation was recommended in all postpubertal male patients. overall a high percentage of the patients and their family followed our recommendations.conclusions: fertility preservation should be considered as a very important part of the treatment plan for newly diagnosed children and young adults with cancer and those facing stem cell transplantation. unfortunately there is still a great need for setting up structures in institutions taking care of these patients. in addition fertility preservation sadly lacks funding by health insurance in some countries. with the presentation of our experience and data we want to facilitate incorporation of fertility counseling in other pediatric care centers to provide counseling for pediatric patients in need for fertility preservation.disclosure: no conflict of interest regulatory t-cells (t reg ) have been shown to play a role not only in autoimmune diseases and solid organ transplantation but also in gvhd. several mouse models showed a decrease of gvhd incidence after t reg administration. the few clinical trials regarding the application of t reg for the treatment of gvhd are encouraging, however the data is limited. methods: patient: a 14-year-old boy underwent allogeneic sct for chronic myeloid leukemia refractory to imatinib, dasatinib and nilotinib treatment from his 10/10 hla identical brother. freshly derived unmanipulated bone marrow was transplanted after conditioning with of fludarabine (40 mg/m²/d, day -7 to -4), thiotepa (2x 5mg/ kg, day -3) and melphalan (160 mg/m², day -2). cyclosporin a (csa) and mycophenolatmofetil (mmf) were used for gvhd prophylaxis. leukocyte regeneration (>1000/μl) was seen on day +15, granulocyte regeneration (>500/μl) on day +16 and thrombocyte regeneration (>50.000/μl) on day +21. on day +30 after sct he developed acute intestinal gvhd that exacerbated to grade iv°(bloody diarrhea, ileus) and did neither respond to steroids, nor to different immunosuppressive drugs such as cyclosporin, tacrolimus, sirolimus, mycophenolatemofetil and ruxolitinib. extracorporal photopheresis and the administration of immunmodulatory antibodies (adalimumab and tocilizumab) did not succeed either.results: by administration of low-dose interleukin-2 (il-2) in vivo induction of t reg was expected but did not succeed. finally antithymocyte globuline (atg, 20 mg/kg/ d) was administered on day +150 to +152 to eliminate the gvhd-triggering cells. hence, the gvhd declined to grade iii. finally, a decision was made to manufacture t reg from his stem cell donor. from an unstimulated leukapheresis t reg were selected by magnetic depletion of cd8 + t-cells and cd19 + b-cells followed by positive selection of cd25 + background: treatment of patients with transfusion dependent anemia like thalassemia major (tm), sickle cell disease (scd) and diamond-blackfan anemia (dba) has improved over the last decades. for the vast majority of patients, allogeneic hematopoietic stem cell transplantation (hsct) is the only available curative therapy. for a long time, hsct has only been performed from hla-identical sibling donors (msd) or matched family donors (mfd). however, approximately only 25-30 % of affected patients do have a matched sibling donor, therefore hsct from 10/ 10 (mud) and even 9/10 (mmud) matched unrelated donors has gained importance in recent years.methods: 36 patients (age range: 2-27 years) with scd (n=7), dba (n=6) or tm (n=23), receiving hsct from a msd, mfd, mud or mmud between 2010 and 2019 were included in our analysis. 23 patients received transplants from msd/mfd, 13 patients from mud/ mmud. 7 patients were identical for hla-a, b, cw, drb1 and dqb1, 6 patients shared only 9/10 genes. we analyzed extended haplotypes including drb3, drb4, drb5 and dpb1 for all patients with thalassemia. 7 pairs showed non-permissive dpb1 mismatch and 1 pair mismatch for dpb1 and drb4.results: median time for granulocyte recovery was 20 days in patients transplanted from msd/mfd and 22 days in patients transplanted from mud/mmud. platelet recovery was reached after 22 days after hsct from msd/mfd and 24 days after hsct from mud/ mmud. 28/36 (80%) patients showed complete donor chimerism in all controls. 5/36 (13%) patients showed low level mixed chimerism up to 20% during follow up. 1 patient died shortly after hsct, 1 patient showed slowly increasing mixed chimerism and finally developed autologous recovery and one patient rejected the graft.cumulative incidence of grade ii-iv acute graft-versushost disease (agvhd) of mud/mmud was 15,2%, whereas only 2 cases of agvhd grade i occurred in patients transplanted from msd/mfd. as 1 patient rejected the graft from a hla-identical parent, 1 patient transplanted from a hla-identical grandparent developed autologous recovery after 1 year and 1 patient transplanted from a mud lost the graft due to hemophagocytosis, the probability of event-free survival was 89,5 % after hsct from msd/mfd and 83,9 % from mud/mmud.altogether 34/36 patients (94,4 %) are alive and transfusion-independent with complete donor chimerism two years after hsct; resulting in an overall survival probability of 93,1%. in contrast, overall survival probability was 100% in the group of patients transplanted from msd/mfd and 80,1 % in patients transplanted from mud/ mmud after 2 years.there were 2 patients with thalassemia (6,9%) who died from transplantation-related causes. the first patient died 13 days after hsct from a mmud due to candida sepsis with pulseless electrical activity resulting from cardiac iron overload. the second patient died 5 months after hsct from a mud due to graft failure.conclusions: hsct from mud and mmud is a feasible therapy option for patients with transfusion dependent anemia. nevertheless, it should be noted that iron overload can cause severe complications; therefore, measurement of liver and heart iron concentration through mri prior to hsct as well as phlebotomy after transplantation are advisable.disclosure: novartis (consultancy: included expert testimony, speaker bureau, honoraria), medac (research funding, patents and royalties), riemser (research funding), neovii (research funding), amgen (honoraria) background: the therapeutic options for patients with hodgkin´s disease who relapse after first high-dose chemotherapy with autologous stem cell (1 st asct) support are limited. allogeneic stem cell transplantation in this setting is associated with a high level of transplant-dependent mortality rates in excess of 50-65%. new agent, such as brentuximab vedotin, have been approved for the treatment of these patients, however, their efficacy to provide longterm control or cure is still unknown. a second autologous stem cell (2 nd asct) has historically been considered as an option only in a small group of patients so the published experience is scarce. we report our institution´s experience with second autologous transplants in this patient population.methods: we evaluated the outcome of 16 adult patients (7 (44%) female and 9 (56%) male), who received an 2 nd asct between 10/2013 and 08/2018. planned tandem asct were excluded. the median age at 2 nd asct was 32 years (range 21-54), 15 (94%) patients had a karnofsky performance score ≥80%. 12 (75%) patients were in complete remission (cr) and 4 (25%) patients were in partial remission (pr) at day 100 after 1 st asct. seven (44%) relapses within 12 months after 1 st asct. patients received a median of 1 (0-3) treatment lines between 1 st asct and 2 nd asct. only 4 (25%) patients received brentuximab vedotin and none of the patients in our series received checkpoint inhibitors as salvage after 1 st asct. the median interval from 1 st asct to relapse/progression was12,9 months (range 2,9-133,3). the median interval from relapse/progression to 2 nd asct was 9,2 months (range 1,4-46,6). all patients received beam as the conditioning regimen for 1 st asct, and beeam as the conditioning regimen for 2 nd asct.results: the median time to neutrophil recovery (>0.5x10 9 /l) after 2 nd asct were 10 days (range 8-17). best response at day 100 following 2 nd asct included cr in 12 (75%) patients and pr in 3 (19%); 1 (6%) had stable disease. 3 (19%) patients received brentuximab vedotin and none of the patients received checkpoint inhibitors after 2 nd asct. 14 (87,5%) patients are currently alive, with a median follow-up 49,8 months (range 4,2-154,2).2 patient died after 2 nd asct. causes of death were hl progression. the 5-year overall survival was 86%.conclusions: the second asct in patients with a longterm response after the first asct may be the optimal therapeutic option, the effectiveness of which can be enhanced by using new drugs, such as brentuximab vedotin, at all stages of treatment.disclosure: nothing to declare effectiveness of chemo-g-csf protocols for mobilization of peripheral stem cells in patients with non-hodgkin lymphomas and hodgkin disease-single center experienceilina micheva 1 , stela dimitrova 1 , vladimir gerov 1 , trifon chervenkov 2 , liana gercheva 1 , igor reznik 1background: high-dose chemotherapy and autologous stem cell transplantation (asct) play an important role in achieving long-term remission in patients with non-hodgkin lymphoma (nhl) and hodgkin disease (hd). granulocyte colony stimulating factor (g-csf) combined with high-dose chemotherapy is a frequently used mobilization approach; however, the optimal mobilization strategy has not been determined.the objective of the study was to analyze the mobilizing potential of different regimens used for the collection of peripheral stem cells in patients with relapsed or refractory (r/r) nhl and hd. methods: we retrospectively analyzed 40 patients with r/r nhl and hd undergoing stem cell collection after chemo-mobilization in the transplant unit at the university hospital, varna. patients were mobilized after dhap letermovir is promising, even as a therapeutic agent. more paediatric data are urgently needed.disclosure: nothing to declare p752 development of paroxysmal nocturnal hemoglobinuria in a patient after mudallohsct due to jak2v617fpositive myelofibrosis-a case of successful treatment with the second transplantation from another donor agnieszka tomaszewska 1 , barbara nasiłowska-adamska 2 , iwona solarska 2 , kazimierz hałaburda 2background: paroxysmal nocturnal hemoglobinuria (pnh) is a very rare disease associated with pig-a gene mutations in hematopoietic stem cells. there are only single case reports on evolving myeloproliferative diseases to pnh in the literature. there is no data concerning development of pnh de novo after allogeneic hematopoietic stem cell transplantation. in our report we describe a patient with recurrence of jak2v617-positive myelofibrosis 5 years after matched unrelated donor allogeneic hematopoietic stem cell transplantation (mudallohsct) with simultaneous development of clinically significant pnh. a 51 year-old-man with a history of mudal-lohsct in may 2011 due to jak2v617-positive myelofibrosis secondary to essential thrombocythemia was admitted to our department 5 years later with mild anemia (hb-11.0 g/dl) and elevated lactate dehydrogenase (1400 u/l). during last 5 years he remained in complete remission of myelofibrosis with jak2v617 mutation negativisation and 100% donor chimerism. suspecting disease recurrence we performed trephine biopsy confirming myelofibrosis (mf2/mf3) with heterozygous jak2v617 mutation and in flow cytometry analysis of bone marrow we identified cell membrane defect in myeloid line (loss of cd66c). we decided to perform detailed diagnostic tests on pnh -multiparametric flow cytometry of peripheral blood revealed 89% granulocytes and 15% red blood cells with loss of gpi-anchored proteins -pnh clone. these results corresponded with donor chimerism -it was only 20% of donor dna in bone marrow and 33% in blood tests. molecular analysis didn't revealed any mutations in genes: calr, asxl1 and mpl. finally the diagnosis of myelofibrosis recurrence after mudallohsct with presence of pnh clone was established. the therapy with eculizumab was unreachable. so the second allohsct from another matched unrelated donor after fludarabinemelphalan-thymoglobuline-tbi 200 cgy conditioning was performed on 21.10.2016. we didn't observe any complications of this procedure, engraftment was slightly delayed: anc>0.5g/l on the 29 day and plt>50g/l on the 50 day.results: at present, more than 2 years after the second mudallopbsct, the patient remains in a very good condition with 100% of the second donor chimerism and without any features of pnh (clone is undetectable) and myelofibrosis.conclusions: presented case is the first in the literature well documented myelofibrosis recurrence after mudal-lohsct with concurrently development of clinically significant paroxysmal nocturnal hemoglobinuria. the second mudallohsct from another donor was safe and successful treatment strategy in this situation.disclosure: nothing to declare. abstract already published. cutaneous refractory t-cell lymphoma treated with allogeneic hematopoietic stem cell transplantationmarcia silva 1 , ercole orlando 1 , maria claudia moreira 1 , simone lermontov 1 , simone maradei 1 , yung gonzaga 1 , leonardo arcuri 1 , renato araujo 1 , decio lerner 1 1 instituto nacional de cancer, cemo, rio de janeiro, brazilbackground: folliculotropic mycosis fungoides (fmf) is an aggressive clinical course variant of cutaneous t-cell lymphoma (ctcl) -classic mycosis fungoides (mf) 1 , with distinct clinical and pathological characteristics, and it is less responsive to skin-directed therapies. for diseases in advanced stages, chemotherapy, autologous hematopoietic stem cell transplantation (hsct) or immunomodulator drugs may provide remissions with limited duration and the treatment remains substantially palliative 2,3 . these dismal results have induced to explore the therapeutical approach with allogeneic hematopoietic stem cell transplantation (hsct) in such patients. early studies have shown encouraging results also in patients with advanced disease, suggesting a major therapeutical role played by the graft versus lymphoma (gvl) effect 4,5,6 . methods: this is a case report of the use of allogeneic hsct as a potential cure for cutaneous refractory t-cell lymphoma type folliculotropic mycosis fungoides .results: case presentation : a 31-year-old male patient with refractory subtype b fmf t-cell lymphoma 7 , diagnosed in 2007, clinically characterized by exfoliative erythroderma, widespread plaques on the trunk and limbs, solitary tumor on the right shoulder, pruritus and bilateral key: cord-005460-ezrn8cva authors: nan title: physicians – poster session date: 2017-07-28 journal: bone marrow transplant doi: 10.1038/bmt.2017.134 sha: doc_id: 5460 cord_uid: ezrn8cva nan hematopoietic stem cell transplant unit, hematology department, hospital universitario de donostia, donostia/san sebastián and 2 informatics and automatics department, university of salamanca, spain the immature platelet fraction (% ipf) is a relatively new parameter that measures young (reticulated) platelets in peripheral blood (pb). ips rise as bone-marrow (bm) production of platelets increases. several clinical utilities of the %ipf have been already proved, as the treatment response monitoring in aplastic anemia or immune thrombocytopenic purpura. in this study, we aimed to found if ip measurement might be useful during the grafting phase of hsct. this study includes 141 patients who underwent allo-hsct in our center during the last 2.5 years. 79 were male (56%) and 62 female (44%). median age was 52 years (range: 7-69). baseline diseases were: acute leukemias (78), lymphoproliferative disorders (22), myelodysplastic syndromes (15), chronic myeloproliferative diseases (12), multiple myeloma (8) and bone marrow failures (6) . donor was unrelated in 79 cases, and related in 62 (including 23 haplo-identical). conditioning regimen was: busulphan-based (93), melphalan-based (17), tbi-based (17) and others (14) . progenitors source was pb in 128, and bm in 13. platelet count, %ipf and absolute ip count (aipc) from day +1 to the day of stable graft were analyzed. 52.4% patients reached plat ⩾ 20 000/mcl at day +14, 82.1% at day +21 and 86.9% at day +28. median first day of plat ⩾ 20 000/mcl was day +14 (range: . median %ipf was 2.6% (range: 0-15.4), 2.5% (range: 0-28.4) and 3.65% (range: 0-15.3) at days +9, +10 and +11, respectively. median aipc was 292/mcl (range: 0-2835), 336 (range: 0-2840) and 504 (range: 0-3660) at days +9, +10 and +11, respectively. among the time points analyzed, aipc at day +11 showed the best positive correlation with platelets counts at day +14 (r = 0.72). interestingly, patients with lower aipc at day +11 showed a delayed platelet graft (see table 1 ). contrarily, patients with higher aipc at day +11 had an earlier platelet graft. absolute immature platelet count before the graft seems to predict the precocity of the platelet graft for the majority of patients undergoing allo-hsct. this finding might help physicians for the patient management (anticipation of hospital discharge and so on). disclosure of conflict of interest: none. [p001] p002 analysis of genetic polymorphism for cardiovascular diseases (cvd) in placental and maternal blood in hypertension and hypercholesterolemia c khalil 1 , a azar 1 and a ibrahim 1,2 1 reviva stem cell research and application center, lebanese university, middle east institute of health hospital and 2 faculty of medical sciences, lebanese university, lebanon cardiovascular diseases are the world's leading cause of death representing 30% of the total global mortality. the genetic polymorphism of the 12 cvd genes, especially the ace: angiotensin converting enzyme gene risky alleles (ins/del) which are associated with a high and inappropriate level of ace can be considered as a genetic model in the development of hypertension and its complications in cvd. we evaluated the mutation impact of the 12 cvd genes in the lebanese population, based on 40 samples derived from placental blood (pb) and 40 samples derived from peripheral blood of postpartum mothers. adult females (age ⩽ 35 years) were divided (n = 20 per group) into group1 (normotensive, normocholesterolemia: nn), and group 2 (hypertension, hypercholesterolemia: hh). buffy coat were extracted from the 40 pb. all tests on pb and maternal blood were done by using the test strip assay to identify the most relevant genetic variations to estimate the risk for cvd. the presence of a double mutation (ins+/del+) related to the ace gene in the hh group was 75%. the presence of a single mutation (ins − /del+) was only associated to the hh by 25%. (ins − /del − ) was absent in 100% of the pb and nn. despite the presence of double mutation ins/del for cvd in maternal blood, pb was free of this mutation. therefore, beyond genetic mutations, other factors can play a major role in the occurrence of cvd. disclosure of conflict of interest: none. s124 b e mt automated red blood cell depletion in abo incompatible grafts in the pediatric setting c del fante, l scudeller, s recupero, g viarengo, f compagno, m zecca and c perotti fondazione irccs policlinico san matteo red blood cell (rbc) depletion by apheresis is employed to reduce the rbc content from abo major or bidirectional mismatch bone marrow (bm) grafts mainly to avoid severe haemolysis 1. rbc depletion results in a significant volume reduction (due to both rbc and plasma depletion) and buffy coat concentration 2.3. in pediatric setting, both rbc depletion and volume reduction before transplantation or cryopreservation can avoid fluid overload and renal impairment, especially in low/very low body weight recipients. the aim of this study was to evaluate the quality of the graft and immediate post infusion complications in rbc depleted bm in major and minor abo mismatch recipients using an automated device. patients and methods: bm aspirates for transplantation in pediatric setting were processed at our centre using the spectra optia (terumo bct) automated device. the initial collection preference was set at level 30 and then was adjusted in order to maintain a haematocrit of 5% (colorgram) in the collection bag. flow speed was set at 120ml/min for 10 cycles. mean recipients' body weight was 31 kg (range:11-72). pre and post procedure bm bag volume, hct%, mononuclear cells (mncs) count, (including b and t lymphocytes), cd34+ cell and cell viability were calculated. moreover, post procedure rbc volume and procedure time were registered. on the patient's side, post infusion complications (renal impairment, fluid overload, fever and haemolitic reactions) and time to engraftment were evaluated. results: a total of 20 rbc depletion procedures were consecutively performed on 20 bm grafts (13 major and 7 minor abo incompatibility, 16 mud and 4 related donors). data about pre and post procedure graft composition are reported in table 1. mean time to engraftment for pmn was 22.6 days (range:17-34) and for plt was 33.5 (range: 21-43). pre and post-procedure cell viability were always 497%. mean procedure time was 80.6 minutes (range:59-115). no bacterial or fungal contamination was detected. no infusion complications were recorded. one graft failure was observed. conclusions the spectra optia automated system is efficient in rbc depletion of abo mismatched grafts, permitting an effective volume reduction and an excellent mncs and cd34+ cell recovery in pediatric setting. automated rbc depletion may be proposed in low/very low body weight recipients both in abo major and minor incompatibility setting to minimize graft infusion side effects. building up a stem cell transplantation program in an emergent country, in the public setting, with limited economic resources, is not an easy work to do. international cooperation may be essential for the development of the program, in training, technological support and implementation of international guidelines. after 20 years, we show an experience of international cooperation between a highly developed center in france (institut paoli calmettes, marseille) and the stem cell transplantation department of hospital maciel, a public assistance service in montevideo, uruguay. fourteen persons between doctors and nurses have been trained in france in stem cell collection and processing, patient's clinical handling, nursing, outpatients care and quality management. french missions of experts have been also received in hospital maciel every year since 2002 for in situ human resources training. in last 5 years we developed a program for optimizing transplant results and reducing transplant related mortality (trm), based on several measures: improvement of patients selection, applying the sorror comorbidy index; adjustment of conditioning regimen doses, in order to reduce toxicity; development of a program to improve interaction with the intensive care unit; protocolization of the standard proceedings treatments; and initiating a program of quality and safety at the national institute of quality of uruguay inacal. 456 adult patients have been treated with autologous (asct) (347) or allogeneic (allosct) (109) sct, with hematological malignancies. different modalities of allosct have been included progressively, becoming the only center accredited by the national regulation authorities (fnr) to perform unrelated donor sct and the haploidentical donor sct. this increased the proportion of allogeneic transplants from the historical 20% until 33% in last 2 years. regarding patients health coverage, 45% comes from the private assistance system and 55% from the public health system. the major indications are lymphoid malignancies and acute leukemia, for asct and allosct, respectively, showing the same trend than cibmtr. three-year overall survival (os) for acute myeloid leukemia after allosct is 61%. considering asct for diffuse large b cell lymphoma, 3 years os after autologous sct is 82% and 52% for chemo sensitive and resistant disease, respectively. threeyears os after asct for hodgkin disease is 87 and 67% for sensitive and resistant disease, respectively. asct in multiple myeloma shows an os of 69 and 50% at 3 and 5 years, respectively. in trm, results during the last 5 years (after the described strategy) are shown in figure 1 . the development of the-program of continuous improvement in quality-and the impact of results was locally recognized by two annual prices from inacal in 2013 (bronze) and 2014 (silver) in the category ‛commitment to public service.' a successful mirna-223 and the level of proangiogenic cytokines: angiopoietin-1 (angpt1), matrix metalloproteinase-9 (mmp-9) and vascular endothelial growth factor (vegf) in patients with lymphoproliferative malignancies prior to autologous hematopoietic stem cell transplantation (hsct) and in early posttransplant period. twenty-four patients were enrolled to the study (11 f,13 m). the median (me) age was 57 years. the investigated group consisted of 19 multiple myeloma and 5 lymphoma patients. the plasma samples were collected on 4 time points: before chemotherapy-‛bc', on the day of hsct -‛0', 7 days after hsct-‛+7' and 14 days after hsct-‛+14. ' the cytokines were evaluated using elisa method, while mirna levels were estimated by qpcr method. the wilcoxon matched-pairs test was used to compare groups of dependent continuous variables: mirna's relative quantification (rq) levels or cytokines expression at two different time points. spearman rank correlation coefficient (r) was used to compare independent variables. we observed continuous decline of cytokines and mirnas level after conditioning treatment. the deepest decrease of expression was marked on ‛+7' day ( table 1) . we noticed a positive correlation between mirna-16, mirnacells in pbsc product. among the autologous transplanted patients between march 2011 and october 2016, we have selected 170 according to diagnosis, conditioning regimen and number of infused bags of cryopreserved pbsc. this group included 77 females and 93 males with median age of 56 (range: . most of them, 105 (61.76%), had multiple myeloma (mm), 41 (24.12%) had non-hodgkin's lymphoma (nhl) and 24 (14.12%) had hodgkin's disease (hd). after harvesting, cd34+ cell and leukocyte number in pbsc product were enumerated on flow cytometer and blood cell counter, respectively. pbsc were cryopreserved with 10% dymethil sulfoxide (dmso) and cell viability was measured with trypan blue exclusion test before and after adding dmso, and as well after thawing in water bath on 37°c. as a conditioning regimen for the mm patients, melphalan was used and for the nhl and hd patients we used beam regimen. all received one bag of cryopreserved pbsc and pegfilgrastim 6 mg on the first or the second post-transplant day. time to hematopoietic recovery was measured; for neutrophils 40.5 × 10 9 /l, leukocytes 41 × 10 9 /l and platelets 420 × 10 9 /l with at least 2 days without platelet transfusion. the median number of total leucocytes infused was 91.88 × 10 9 /l (range: 29.27-284.87 × 10 9 /l) of which cd34+ cells were 2-24.09 × 10 6 /kg of patient's body mass (median 4.75 × 10 6 /kg). pre-freezing cell viability before and after adding dmso was with a median of 100% (74.1-100) and 81, 75% (26.7-100), respectively, and post-thaw viability 57.37% (16.7-100) . the average time to engraftment was 9.8 days (6-26) for neutrophils, 10 days (6-27) for leucocytes and 10.8 days (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) for platelets. our results confirmed the known correlation between the number of infused cd34+ cells and engraftment of neutrophils (po 0.0001), leukocytes (p o0.0001) and platelets (p = 0.0005). we found inverse correlation between the infused leukocytes and cell viability with dmso (p = 0.0035) and after thawing (p = 0.0019). no correlation was found between prefreezing and post-thaw viability with hematopoietic recovery, and also between the cd34+ number and these viabilities. no differences were found considering patients' age, gender, diagnosis, conditioning regimen or day of applying pegfilgrastim. we can indirectly infer good survival of cd34+ cells and higher sensitivity of other nucleated cells to preparation of pbsc product. trypan blue exclusion assay, due to its inability to distinguish type of stained cells, is not relevant for cd34+ cells survival determination. disclosure of conflict of interest: none. chronic granulomatous disease (cgd) is a kind of primary immunodeficiency disorder of phagocytic cells which resulting in failure to kill a defined spectrum of bacteria and fungi and in concomitant chronic granulomatous inflammation. allogeneic hematopoietic stem cell transplantation is the only treatment proved to be potentially curative in cgd. unrelated umbilical cord blood (ucb) is increasingly used as an alternative to bone marrow. methods: unrelated ucbt was performed 14 consecutive cgd children at our center between 2015 and 2016. median age was 18.5 months (range: 5-143 months), median body weight was 10.3 kg (range: 8-34 kg). all patients received myeloablative conditioning regimen consisting of busulfan, fludarabine, cytarabine, cyclophosphamide and g-csf. all patients received tacrolimus as prophylaxis for graft-versus-host disease (gvhd). median nucleated cells were 9.2 × 10 7 /kg (range: 4.5-15.9 × 10 7 /kg), and median cd34+ cells were 3.0 × 10 5 /kg (range: 0.9-7.0 × 10 5 /kg). median follow-up time was 9.5 months (range: 6-23 months) results: 10 of 14 patients engrafted. median time to neutrophil engraftment was 30 days, and median time to platelet engraftment was 33.5 days. 13/14 patients were alive, and 10/14 had full donor engraftment. overall survival rate was 92.8%. disease-free survival was 71.4%. 2 of 14 patients had grades iii-iv acute gvhd. no patients developed chronic gvhd. only one patient died from multi-organ failure related to adenovirus infection. conclusion: unrelated ucbt should be considered as potential curative methods in children with cgd. myeloablative conditioning regimen has improved the engraftments of the ucb. disclosure of conflict of interest: none. reduced muscular mass and excess visceral fat in patients undergoing hsct are associated with higher mortality, longer hospitalization, longer use of immunosuppressive drugs, graftversus-host disease (gvhd) and comorbidities leading to shorter survival time. a recent study of patients undergoing allogeneic hsct showed that occurrence of enlarged areas of visceral and peripheral fat is inversely associated with the disease-free interval after the transplant. reduced muscle mass has also been associated with higher prevalence of chronic gvhd and low rates of success following allogeneic hsct. objectives: to investigate whether amount muscle mass and muscle strength (ms) as well as the amount of visceral fat (vf) of patients undergoing hsct would influence the duration of the engraftment time (en). we evaluated 14 hsct patients (⩾18 years) at hospital israelita albert einstein, são paulo, brazil, on their first day of hospitalization, before hsct. the thickness of the right femoral quadriceps muscle (rfq), measured at 6 cm from the top edge of the patella was measured using ultrasound (us) in b-mode. the dominant upper limb strength of the patients was evaluated by the hand grip test. the vf was measured in the abdominal region, by the thickness of the fat layer between the linea alba and the anterior wall of the aorta. most patients were women (57%) with a mean age of 50 years (±16 years) and 50% of our patients were elderly (⩾60 years). the haploidentical (57%) was the predominant hsct, autologous (36%) and allogeneic (7%). most patients were overweight, with body mass index (bmi) of 27 kg/m 2 (±4 kg/m 2 ). the average time en was 16 days (±6 days). rfq was 1.5 cm (±0.3 cm), ms was 31 kgf (±7.0 kgf) and the vf was 5.3 cm (±1.4 cm). patients with lower rfq had a longer engraftment time that was statistically significant as the negative correlation between rfq and en was rs = 0.8, p o0.05), independent of the age and the hsct type as analyzed by linear regression. no significant correlation between vf or ms with en was found. in this cohort of patients we found that longer engraftment times were significantly correlated to reduced muscle mass but no positive or negative correlation was found with superior limb muscular force or with the amount of visceral fat. disclosure of conflict of interest: none. 1 hematopoietic stem cell transplant unit, hematology department and 2 pharmacy department. university hospital of donostia. donostia/san sebastiań introduction: lymphocytes are the cells responsible for the cellular and humoral immunity and, consequently, critical for hematological patients. the aim of this study was to analyze the eventual conexion between lymphocyte recovery and survival (srv) after allogeneic hematopoietic stem cell transplantation (allo-hsct). patients and methods: we retrospectively analyzed data from 223 consecutive patients who underwent allo-transplants in our unit. in total, 126 patients were male (56.5%) and 97 female (43.5%). median age was 53 years old (range: . baseline disease was: acute leukemia (56.9%), lymphoma (11.2%), myelodysplastic syndrome (10.3%), chronic myelogenous leukemia (8.9%), multiple myeloma (4%), aplastic anemia (3.58%), chronic lymphocytic leukemia (3.13%) and others (1.79%). 55.1% of allo-hscts were from an unrelated donor, and 44.9% from a family donor (25% of them haplo-identical). the sc source was pbsc in 89.6%, and bm in 10,4%. a variety of conditioning regimens were employed, including: busulphan-based (69.5%), melphalan based (10.4%), tbi-based (9.86%) and others (9.86%). evolution of absolute lymphocyte counts (alc) and subpopulations during the first year after allo-hsct were analyzed. results: as shown in table 1 , alc decreased abruptly during conditioning therapy and recovered up to baseline at days +30 and +100; at day +365 median alc had clearly improved compared with admission values. median cd4+ cells were lower than 500/mcl in two thirds of pts at day +100 and in only one third at day +365. as shown in table 2 , we found a significant link between alc at day +30 and srv, as well as between cd4+ cells at day +100 and srv. in our series, immunity recovery was a late event for the majority of patients undergoing allo-hsct. in addition, in our experience, the precocity and quality of the alc and cd4+ recovery was clearly linked with long-term survival. disclosure of conflict of interest: none. although there is experimental evidence suggesting the presence of a common mesoderm cell as origin of both hematopoietic (hsc) and mesenchymal progenitor cells (msc) in an animal model, it is still controversial if durable engraftment of native donor-derived mscs without ex vivo treatment can occur in the recipient of allogeneic hsct. to assess the presence of donor-derived msc following hsct. between july 2015 and july 2016, a total of 33 recipients of hsct were analyzed for hsc and msc chimerism. eighteen patients received bm grafts (54%), 11 patients had peripheral blood as stem cell rescue (33%) and finally 3 patients had a cord blood transplantation (9%). patients received myeloablative (91%) or reduced intensity conditioning (9%) for malignant (91%) or nonmalignant disease (9%). bm aspirate cells were plated and expanded in α-mem with 10% human platelet lysate at 10 000 cells/cm 2 . after 5-7 days, nonadherent cells were removed, while the adherent cells were expanded until they reached confluence. after 2 weeks we quantified msc precursors as colony forming unit fibroblast (cfu-f). finally the amplified sequences were resolved by capillary electrophoresis (3500 ruo genetic analyzer, applied biosystems) and analyzed by comparing genotypes of bmt recipell detachment, nuclear dna was extracted (dneasy blood and tissue kit-applied biosystems) and specific polymorphic tandemly repeated regions (strs) were amplified by means of the polymerase chain reaction(pcr) following the specific manufacturers' instructions. (ampfℓstr identifile kit, applied biosystems following hsct (hsc and msc) to those of donors. we cultured 54 whole bm aspirates from patients following hsct with a median time of 244 day (range: 41-1606). cfu-f/1 × 10 6 growth was observed in a majority of bm the prevalence of human pegivirus in recipients of allogeneic hematopoietic stem cell olga koroleva 1 , e parovichnikova 1 , l kuzmina 1 , m drokov 1 , v vasilyeva 1 , z konova 1 , ekaterina mikhalcova 1 , d dubnyak 1 , n popova 1 , tamara romanova 2 , d tikhomirov 2 , t tupoleva 2 and v savchenko 1 1 bone marrow transplant department, national research center for hematology and 2 virology department, national research center for hematology human pegivirus (hpgv; previously named as gb virus c/hepatitis g virus) was discovered more than 20 years ago. it is an rna virus referred within the genus pegivirus of the family flaviviridae. hpgv rna is found in liver, spleen, bone marrow and peripheral blood mononuclear cell, including t-and b-lymphocytes, nk-cells and monocytes. despite of the fact that it is a molecular structure, mechanism of replication and transmission routes are very well understood but the clinical significance of hpgv is still not determined. recipients of allogeneic hematopoietic stem cell have a high risk infection of hpgv. it is known, that hpgv is a nonpathogenic virus, however, it may play a role in immunocompromised individuals. to investigate the frequency of occurrence of hpgv and its clinical significance in recipients of allogeneic hematopoietic stem cell. blood samples were obtained from 101 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-hsct): all n = 21, aml n = 53, mpn n = 7, cll n = 1, mm n = 1, lpd n = 4, aa n = 6, mds n = 8. a median of age was 33 years (19-64 years) . forty five patients were males and 56 patients were females. conditioning regimen was ric in 75 cases, mac in 26. bone marrow as a graft source was used in 68, pbsc-33. all patients received multiple transfusions of blood components at the previous stages of treatment. hpgv rna had been assayed by polymerase chain reaction real time (rt-pcr) on plasma samples before started pre-transplantation conditioning. despite the diagnosis incidence of hpgv was high 53.5% (rna-hpgv was positively in 54 patients). patients with piercings and tattoos had incidence of hpgv in 64% that was not statistically significant (p-0.5). hpgv is known as nonhepatotropic virus. in our study there was also no statistical reliability of specific changes in liver function test such as elevating the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin due to the rna-hpgv. liver enlargement was also not statistically significant according to ultrasound scan results in patients infected with hpgv. we also analyzed the co-infection with hepatitis b and c virus. results are presented in table 1 . coinfection was not statistically significant. however, only one patient with hepatitis c was coinfected hpgv. leukocytes recovery median was 22 days (14-55). thrombocytes recovery median was 23 (11-82). the presence of rna-hpgv did not affect the recovery of peripheral blood cells in patients after allo-hsct. according to our study the frequency of hpgv infection in recipients of allogeneic bone marrow was quite high (53.5%), and it did not depend on the presence of any other hepatotropic viruses. clinical significance of hpgv infection in recipients of allogeneic hematopoietic stem cell has not been revealed, it is possible due to the short follow-up. it needs further clinical research. disclosure of conflict of interest: none. quantification of cd31+ recent thymic emigrants and t cell receptor excision circles (trecs) in umbilical cord blood transplanted patients v devlia 1,2 , j gridlestone 3 , m raymond 3 , s tulpule 1 , d tewari 1,2 , r hough 4 , c navarrete 3 , a madrigal 1,2 , b shaw 1,2 , r danby 1 and a saudemont 1,2 1 anthony nolan research institute, london, uk; 2 ucl cancer institute, london, uk; 3 nhsbt colindale, london, uk and 4 ucl, london, uk reconstitution of t lymphocytes is a limiting factor in the regeneration of an effective immune system in adult patients following hematopoietic stem cell transplantation. cd31 (pecam-1) is a transmembrane glycoprotein expressed on naive t-cells that have recently emigrated from the thymus into the periphery. in peripheral blood, cd31 + t lymphocytes also contain high numbers of t-cell receptor excision circles (trecs); excision loops of dna excised during t-cell receptor gene rearrangement during t cell maturation within the thymus (1) (2) (3) . however, quantification and correlation of cd31 and trec has not been formally investigated in patients following umbilical cord blood (cb) transplantation. quantification of cd31 and trecs post cb transplant will provide an insight into the immune reconstitution of t cells from the thymus. we therefore sought to measure cd31 and trecs in patients after cb transplant and assess whether these markers provided evidence of thymic recovery. we followed 67 adult patients (median age 52.9 years) who underwent cb transplant in the uk. patient samples were collected 28, 60, 100, 180, 365 and 730 days post transplant. using flow cytometry, we determined absolute counts of cd4+cd31 +cd45ra+ and cd8+cd31+cd45ra+, and quantified the copy numbers of trec genes in peripheral blood mononuclear cells (pbmcs) via real time pcr. results: at the six time points, the number of samples collected were the following: 44, 39, 37, 22, 24 and 14. in all of the samples, the overall median number of cd4+cd31+cd45ra+ was 29 cells/μl (range: 0-827 cells/μl). the median level of cd4+cd31+cd45ra+ cells increases from 16 to 50 cells/μl from day 28 to day 365. absolute counts of cd4+cd31+cd45ra+ at all of the six time points is 10-fold lower compared to healthy controls (median: 279 cells/μl, range: 105-523 cells/μl). the overall median number of cd8+cd31+cd45ra+ cells is 30 cells/μl (range: 0-2222 cells/μl). there is an increase in the median number of cd8+cd31+cd45ra+ cells between days 28 and 720 posttransplant from 2 to 92 cells/μl. however, the absolute median counts of cd8+cd31+cd45ra+ cells in patients are twofold lower, 2 years post transplant, compared to healthy controls (median: 252 cells/μl, range: 133-503 cells/μl). in the majority of the patient samples throughout all time points the trec gene copy numbers were undetected (n = 132). in a few patient samples (n = 9) trec gene copy numbers were quantified but with this limited sample size no correlations can be made between the absolute counts and trec gene copy numbers. our data suggests that cord blood transplant patients within the uk have reduced levels of cd4+cd31 introduction: common variable immunodeficiency (cvid) is a highly heterogeneous group of primary immunodeficiency characterized by defective antibody production, recurrent infections, lymphoproliferation and autoimmunity. autosomal recessive mutations in lrba, encoding lps-responsive beigelike anchor protein were first described as a cause of cvid-like disease in 2012. although hsct is accepted as a standard treatment modality for long-term resolution of severe primary immunodeficiencies, its role is less established in patients with lrba deficiency. patients and methods: whole exome sequencing of patient's genomic dna obtained prior to the hsct revealed a homozygous deletion in lrba (c.5527delt:p. c1843fs). immunological analyses including serum immunoglobulin levels, flow cytometry analyses of lymphocyte subsets, cytotoxicity/proliferation assays, vaccine responses were studied at several time points throughout the disease course, prior to and after hsct. a 14-year-old boy, born to consanguineous healthy parents of turkish origin became symptomatic at the age of 6 months. he hospitalized several times due to recurrent pulmonary infections. he developed pancytopenia, lymphadenopathy, hepatosplenomegaly and autoimmunity (autoimmune hemolytic anemia and thyroiditis) with low serum immunoglobulin levels at the age of 4. as a result, he received several courses of steroid and prophylactic immunoglobulin and wide-spectrum antibiotics. over time he manifested growth failure and diagnosed with ibd-like colitis. due to the cumulating severe cvid-related complications, a hsct was performed at the age of 14 years with the bone marrow stem cells from his hla identical brother after a conditioning regimen including fludarabine, busulfan and atg. severe intractable colitis with hypoalbuminemia continued till the engraftment despite vigorous fluid-electrolyte replacement therapy and accompanied with severe episodes of acute gastrointestinal bleeding. after the achievement of full donor chimerism, diarrhea episodes resolved. he received three doses of abatasept because of persistent cytopenia thinking about unresolved immune dysregulation. he is in complete remission at 1-year post-hsct with no signs of graft versus host disease. allogeneic hsct should be considered in patients with lrba deficiency prior to the development of disease-related severe cumulative manifestations. disclosure of conflict of interest: none. inflammatory bowel disease (ibd) is a chronic disorder of the gastrointestinal tract. very early onset ibd (veo-ibd) represents those severe children with disease onset occurring before 6-years-old. interleukin-10 receptors (il-10ra, il-10rb) mutation are considered to be one of the very important genes for veo-ibd. currently variant treatment, such as steroid medication, immunosuppressive agents and biological agents could not get complete remission. allogeneic hematopoietic stem cell transplantation (allo-hsct) was reported to induce remission in those with veo-ibd. we performed unrelated umbilical cord blood transplantation (ucbt) in five consecutive children with veo-ibd due to il-10 receptor mutation between 2015 and 2016. median age of five children was 15 months (range: 6-46 months), and median body weight was 7 kg (range: 3.2-9.5 kg). all patients received reduced intensity conditioning (ric) regimen consisting of busulfan, fludarabine and cytarabine. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. most patients (80%) received a 1 or 2 hla alleles-mismatched cord unit. median nucleated cells of the cord blood were 14.3 × 10 7 /kg (range: 11.2-51.5 × 10 7 /kg), and median cd34+ cells were 4.5 × 10 5 /kg (range: 3.6-14.9 × 10 5 /kg). median follow-up time was 10 months (range: 6-24 months). all patients engrafted, median time of neutrophil engraftment was 22 days, and median time of platelet engraftment was 27 days. four of five patients were alive with continuous donor engraftment, and achieved complete clinical remissions. colonoscopy at 6 months after transplantation in two children revealed the mucosa healing. two children had grade iii acute graft-versushost disease (gvhd). one child developed severe chronic gvhd of both lungs and died of ards at 6 months after transplantation. it is the first clinical trial that unrelated ucbt was performed in veo-ibd children in china. our data should unrelated ucbt with ric should be considered as a potentially curative therapeutic option in children with veo-ibd. disclosure of conflict of interest: none. patients with refractory primary induction failure and resistant relapse are poor candidates for hematopoetic stem cell transplantation (hsct) . additional attempts at remission induction with various combinations of chemotherapy will unlikely improve the outcome and will contribute to excess toxicity. a major goal of sct has been to develop strategies to reduce the risk of gvhd while maintaining or enhancing gvl. tcrαβ+/cd19+lymphocytes depletion is a technology of graft manipulation with a potential to increase gvl effect and improve gvhd control and immune reconstitution in this group of patients. a total of 31 pts with refractory aml (primary induction failure (n = 10), refractory relapse (n = 21)), 17 female/14 male, median age 9.7 years (1. [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] , underwent allogeneic sct between may 2012 and august 2016, median fu 1.5 years (0.3-3.8). 26 pts were transplanted from haploidentical donors and 5 from mud. all pts had active disease (ad) at the moment of sct and received treosulfanbased high-intensity conditioning regimen. three regimens of gvhd prophylaxis were used. regimen 1 (n = 7): atgam 50 mg/kg with (n = 5) or without (n = 2) post-transplant tacro/ mtx; regimen 2 (n = 9): thymoglobulin 5 mg/kg, rituximab 200 mg/m 2 and post-transplant bortezomib on day+2,+5 (n = 9); regimen 3 (n = 15): tocilizumab 8 mg/kg on day-1 and post-transplant bortezomib (n = 15), 4 pts receive additional abatacept 10 mg/kg on day+2, +7, +14, +28. tcrαβ+/cd19 +-depletion of sct with clinimacs technology was implemented in all cases. the median dose of infused cd34+ cells was 8 × 10 6 /kg (range: 4.2-17), tcra/b-14 × 10 3 /kg (range: 2-52). all engrafted pts received additional post-transplant courses of low-dose chemotherapy, including hypomethylating agents and dli. primary engraftment was achieved in 27 of 31 pts(three pts had disease progression, one died at the moment of engraftment), the median time to neutrophil and platelet recovery was 12 days (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) . early mortality within 100 days was 3.2% (one pt with aml had acute lung injury after engraftment on day +14), 1.5-years ptrm-6.7% (95%ci: 1.7-25) . there were no allergic or infusion-related adverse events associated with tocilizumab or abatacept. ci of gvhd grades ii-iv and iii-iv was 25.8% (95% ci:14-47), and 9.7% (95%ci:3.3-28), respectively. ci of cgvhd was 23% (95% ci:12-42). ci of acute gvhd was lower in a group with prophylaxis regimen without serotherapy: 20% (95% ci:2-28) vs 31.3% (95% ci:7-54) in atg group. no correlation between graft composition, donor type with the incidence of agvhd and cgvhd was noted at 1.5 years ppfs (event = death or relapse or progression) was 37% (95% ci:19-54), 1.5-years pos -48% (95% ci:30-67). median time of fu for survivors is 1.5 years (range: 0. [3] [4] . we confirm that the depletion of tcrαβ +/cd19+lymphocytes from the graft ensures high engraftment rate and low transplant-related mortality in pediatric pts with refractory aml. we suggest that tocilizumab and abatacept can be safety administered to children with acute leukemia in the context of treosulfan-based conditioning regimens. long-term follow-up will demonstrate if the gvhd prophylaxis without serotherapy and combined administration of tocilizumab, abatacept and bortezomib post-tcrαβ+/cd19 +depleted grafting will improve gvl effects without extensive gvhd-related morbidity and mortality in pts with refractory aml. disclosure of conflict of interest: none. the jacie experience at the university hospital of amiens l marie-noelle 1 , w brigitte 2 , f isabelle 3 , g bérengère 3 , h muriel 4 , h anne 3 , v elsa 5 , m jean-pierre 3 and c amandine 3 1 lacassagne; 2 oncopôle, chu amiens picardie; 3 hématologie clinique et thérapie cellulaire-chu amiens picardie; 4 oncopôle-chu amiens picardie and 5 the jacie (joint accreditation committee of isct and ebmt) accreditation aims to improve the management of patients benefiting from autologous or allogenic hematopoietic stem cell (hsc) transplants. usually, candidates' centers for jacie accreditation have already existing clinical activity when they have willingness to comply with jacie standards. here, we present our new experience in the implementation of jacie quality process, at the same time as allograft clinical activity. implementing process autograft clinical activity existed at amiens hospital, since 1991, but in lack of center cellular therapy laboratory and hsc collection that were outsourced. the collection activity for autologous transplant was set up in 2009, the cellular therapy laboratory in november 2011 and then the allogenic transplant was started in july 2012. as early as march 2011, we set up a steering committee with hematological clinicians, managers of each sector, a transplant coordinator nurse, the head of the processing laboratory and a part-time quality engineer recruited part-time. each actor had to become familiar with standards to obtain information from accredited centers in order to evaluate objectives and their prioritization. steering committee decided on deadlines and established a roadmap including the following: the list of jacie required standard operating procedures and their writing; assignment of the tasks for each actors in order to evaluate, writing and approval each document; organization of documents diffusion; information to all staff on the approach; creation of feedback committee for adverse events management; establishment of morbidity and mortality review; formalization of initial and continuing training for medical and paramedical staff; and organization of cross audits with external teams. at the same time, we assessed requirements for starting activity: training for medical and paramedical staff; training for the transplant coordinator nurse; circuits for taking care of donors; the organization of the in-patient department; the organization of follow-up of post-transplant patients; and authorizations of the national regulation agencies for processing facility on manipulations and cellular qualifications and for regulatory collection and transplant. allogenic transplant clinical activity started in july 2012. accreditations jacie visit occurred on 8 and 9 june 2015 and our center has been officially accredited since 16 march 2016. neither quality approach, nor clinical activities were easy to implement. medical and paramedical staff had to get acquainted with a new organization and restrictions. despite difficulties, implementing jacie quality process, concomitantly with allograft activity allowed to create a true team dynamics with a common reflection on the means to be implemented. moreover, quality approach has assured us best ensure to care graft patients. the result is true satisfaction, which be credited to all. disclosure of conflict of interest: none. previously published p024 three-dimensional co-culture of peripheral blood monocytes supports and expands functional hematopoietic stem/progenitor cell without immobilization y xu 1 , x li 1 , b wang 1 , w shan 1 , h chen 1 , s liu 1 , r tie 1 , y long 1 , s cai 1 , h xu 1 , x yu 1 and h huang 1 1 bone marrow transplantation center, the first affiliated hospital, school of medicine, zhejiang university very low numbers of circulating hematopoietic stem/progenitor cells (chspcs) are found in normal human peripheral blood (pb) without mobilization. here, we developed a three dimension co-culture system to seize and expansion chspcs from pb monocytes without mobilization. flow cytometry analysis was carried out to identify chspc phenotypes. multipotential properties of chspcs were determined using colonyforming unit assay in methylcellulose and reconstitution ability in the compromised animals. the critical regulation mechanism underlying chspcs was identified with transcriptome analysis based on next-generation sequencing technology at total or single cell levels. loose cobble stone colonies (lcs), round or vessel-like compact colonies (rccs or vccs) were presented in three dimension co-culture system after about 2 weeks. the colonies lasted for at least six passages with no obvious apoptosis sign, and expanded more than~10 000fold during the period. we studied the niche-mediated regulation mechanism of chspc fate at molecular level compared to the conventional method of two dimension culture. furthermore, chspcs were capable of forming all types of hematopoietic colonies, including cfu-gemm, and especially held short term engraftment capacity for compromised nogs by radiotherapy. transcriptome analysis by deepsage identified 167 genes significantly associated with regulating the function of chspcs. figure 1 : the cellular morphology in three dimension culture system for peripheral blood monocytes without mobilization during the culture for 2-3 weeks. figure 4 : short transplantable potential analysis of chspcs. figure 5 : (a) static of differentially expressed genes between three-and two-dimensional culture systems for peripheral blood cells. (b) go functional analysis classifies those genes by biological process, cellular component and molecular function. (c) the significant differences between the molecular phenotypes of three-and two-dimension chspcs indicating that chspcs from three dimension culture hold stem properties. our system may provide a more ideal and balanced approach which not only seizes circulating chspcs, promotes selfrenewal and expansion of chspcs, but also holds phenotypic and functional attributes of chspcs. 6. to wash or not to wash? comparison of neutrophil and platelet engraftment after infusion of cryopreserved autologous stem cells before and after the implementation of bedside thawing am halldorsdottir 1 , s atladottir 2 , m thorsteinsdottir, na arnason 1 , g runarsson 2 , t jonsson 1 , oe sigurjonsson 1,3 and s reykdal 2 1 the blood bank, landspítali, the national university hospital of iceland; 2 department of hematology, landspítali, the national university hospital of iceland and 3 school of science and engineering, reykjavik university cryopreserved autologous peripheral blood stem cell (pbsc) grafts are widely used after high-dose chemotherapy in the treatment of patients with myeloma or lymphoma. prior to infusion, cryopreserved grafts can be thawed at the bedside, or thawed and washed at the cell therapy laboratory. at our institution the practice of routine washing of stem cell grafts in the laboratory was discontinued in april 2012 and bedside thawing implemented instead. this was done to minimize the time thawed cells are exposed to toxic dmso. this study was performed at a single center, at landspítali-the national university hospital of iceland, which is the only transplant center in iceland. autologous pbsc transplants have been performed in iceland since 2004. the study compares outcome for two groups of patients, who received either; (a) thawed and washed autologous pbsc cell grafts from january 2008 to [p024] april 2012, or (b) autologous pbsc grafts thawed at the bedside from april 2012 to november 2016. the following outcomes were compared; days to neutrophil engraftment (absolute neutrophil count (anc) 40.5 per μl), and platelet engraftment (20 and 50 × 10e9/l). data on mean cd34+ cell content/kg of the infused grafts, measured prior to cryopreservation, were also compared. all patients have received premedication with solucortef, clemastine and ondansetron prior to infusion of the graft. from january 2008 to april 2012 a total of 84 patients received thawed and washed autologous pbsc grafts, and between april 2012 and november 2016 83 patients received autologous pbsc grafts thawed at the bedside. majority of the patients were diagnosed with either multiple myeloma or related disorders (n = 86) or lymphoma (n = 67) whereas the remaining patients (n = 14) had miscellaneous diagnoses. days to engraftment and the dose of cd34+ cells infused are compared in table 1 . there was no significant difference in the mean cd34 content of infused autologous stem cells in the two groups (6.9 vs 6.5 × 10e6 cd34+cells/kg, p = 0.41). there was also no difference in the mean number of days to engraftment of neutrophils (12.8 vs 13.3 days, p = 0.14), platelets at 20 days (19.2 vs 18.1 days, p = 0.64) or platelets at 50 days (33.1 vs 28.1 days, p = 0.62) after transplant. one hundred day mortality was comparable in the two groups or 2.4%. additional data on transfusion requirements, infections and use of granulocyte-colony stimulating factor will be presented. [p025] there was no difference in neutrophil or platelet engraftment after changing the autologous stem cell graft thawing procedure from post-thaw washing in the laboratory to bedside thawing. bedside thawing of stem cells is a safe procedure that results in acceptable cellular engraftment. disclosure of conflict of interest: none. the procedure of autologous hematopoietic stem cell (hsc) transplantation requires cryopreservation of hscs. addition of dmso (dimethyl sulfoxide) is necessary to secure the viability of such cells, but this cryoprotectant causes adverse reaction during infusion into patient. the concentrations of dmso in cryopreservation mixture vary strongly between different transplant centers. usually, the hscs are stored in mixtures containing 10% dmso, however, many centers successfully use lower concentrations. the main aim of the study was to evaluate the clinical impact of different dmso concentrations in cryopreservation mixture (5%, 7.5%, 10%) on reconstitution of hematopoiesis after autologous hsc transplantation. the project was approved by the local bioethics committee. written informed consent obtained from all of patients. the study is registered to clinicaltrials.gov (identifier: nct02452099). between january 2014 and july 2016, 150 consecutive patients with hematological malignancies or solid tumors, referred for autologous hsc transplantation, were recruited in the study. the patients were randomly assigned to one of three study arms (50 patients each). hscs obtained by leukapheresis were cryopreserved in three concentrations of dmso: 5%, 7.5%, 10%, respectively. study groups did not differ significantly with regard to the diagnosis (mostly mm, nhl or hl), age or conditioning regimen (chemo-or radiotherapybased). all patients received granulocyte-colony stimulating factor (g-csf, filgrastim) starting from day +4 after transplantation to support neutrophil recovery. in case of 7 patients, the transplantation was cancelled due to progression or other medical reasons. four patients died shortly after transplantation, due to refractory infections. data for 139 patients were subjected to statistical analysis. the viability of nucleated cells on the day of transplantation was similar in all groups (median 96%, range: 85-99% for 10% dmso group; 97%, range: 85-99% for 7.5% dmso; 97%, range: 90-99% for 5% dmso; p = 0.52). the dose of transplanted cd34+ cells was comparable in all group: (median 4.70 × 10 6 /kg of recipient body weight for 10% dmso, 4.35 × 10 6 /kg for 7.5% dmso and 3.97 × 10 6 for 5% dmso, p = 0.44). the median time to leukocyte recovery, defined as the first day with wbc count exceeding 1.0 × 10 9 /l was 10 days in all groups (ranges: 9-12 for 10% dmso; 7-11 for 7.5% dmso; and 9-12 for 5% dmso; p = 0.36). similar results were obtained in case of neutrophil recovery-the median day, when the anc exceeded 0.5 × 10 9 /l, was 10 in all arms (ranges: 9-12; 8-11 and 9-12, respectively; p = 0.20). the day when the platelets level were greater than or equal to 20 × 10 9 /l (sustained without transfusion within 7 days) was similar in all groups: medians were 15 days in 10%, 7.5% and 5% dmso (ranges: 8-20; 0-19; 0-24; p = 0.61). no serious adverse effects were observed during hscs infusion and during 24 h after transplantation. reduction of dmso concentration from in cryoprotective mixture 10% to 7.5% and 5% has no negative impact on cell viability during cryopreservation and engraftment after auto-hsc transplantation. disclosure of conflict of interest: none. a real-world cost-effectiveness analysis demonstrates that introducing plerixafor to improve mobilization in multiple myeloma patients who behave as poor mobilizers is cost-effective considering the whole mobilization and transplant procedure r touzani 1,2 , a-m stoppa 3 plerixafor, a cxcr4-antagonist, is efficient to improve cd34 + cell mobilization and collection in candidates for autologous transplantation who behave as poor-mobilizers. the cost of the drug is however of concern. published medico-economics studies were mostly conducted in the us, and few including detailed and comprehensive micro-costing of the collection and transplantation process; conclusions may thus not apply to european countries where cost structures are different. to compare costs and effectiveness of plerixafor-free and plerixafor-replete management strategies for multiple myeloma patients who behaved as poor-mobilizers after adequate administration of a standard rhg-csf mobilization regimen. sixty patients diagnosed with multiple myeloma were consecutively identified during years 2009-2011, immediately before and after ema granted marketing authorization for plerixafor. poor-mobilizers were defined as having circulating cd34+ cell counts below 20/μl. plerixafor was introduced or not as a result of the attending physician's decision, reflecting progressive changes in medical practices over this transitional period. the historical and study groups were matched over four criteria: disease stage at diagnosis, age, gender and number of chemotherapy treatments received before mobilization. two cost-effectiveness analyses (cea) were conducted; the primary cea looked at the criterion ‛collecting at least 2 × 10 6 cd34+ cells'; a secondary cea looked at the criterion ‛successful autologous transplant administered'. detailed micro-costing evaluations (2015 figures) did not or did include transplantation costs for the first and second cea, respectively. the two groups were similar in terms of age, sex distribution, disease characteristics or previous treatments. 27/30 and 26/30 patients proceeded to high-dose melphalan and autologous transplantation in the study and historical groups, respectively. there was a trend to a higher number of collected cd34 + cells in the control group; however, the proportion of patients who met the minimal target number of 2 × 10 6 collected cd34 + cells/kg was identical (28/30). length of hospitalization, times to neutrophil and platelet recoveries, numbers of prbc and platelet transfusions were identical in the two groups. mobilization and collection costs per patients were more important in the plerixafor group that in the historical group (8.757 vs 5.460 €, p o0.0001), and proportionally higher in patients who received plerixafor as part of a remobilization treatment rather than pre-emptively (10.401 vs 8.162€, respectively). the main cea concluded to a 3.237€ increase in costs for the same number of patients achieving a minimal target number of 2 × 10 6 collected cd34 + cells/kg. the second cea found a decrease in the cost of transplant, with 12.724€ in the study group vs 13.634€ in the historical group (ns). in total, the 2.035€ increase for the complete procedure cost (22.866€ per successfully autografted patient in the study group vs 20.831€ in the historical group) was not statistically different. cost-effectiveness arguments should not been used against the administration of plerixafor in multiple myeloma patients in the european context. future prospective researches looking at patients reported outcome criteria and labour organization in apheresis facilities are needed. disclosure of conflict of interest: this work was supported by a grant from sanofi s.a.; cc: research support, honorarium & hospitality from sanofi s.a. administration of plerixafor for peripheral blood cd34+ stem cell content of o30 × 10 6 /l for autologous stem cell mobilization leads to decreased apheresis days and increased total yield m kamdar 1 , s abebe 1 , gr gonzalez fontal, l gates 1 , a hammes 2 , d abbott 2 , j gutman 1 , b haverkos 1 , d sherbenou 1 and c smith 1 1 division of hematology and transplantation and 2 department of biostatistics and informatics, university of colorado, denver, colorado, usa autologous stem cell transplantation (asct) is an effective treatment for lymphoma and plasma cell neoplasm (pcn) (multiple myeloma and amyloidosis). granulocyte-colony stimulating factor (g-csf) is the most commonly used upfront mobilizing agent with plerixafor-based higher cost approaches reserved for poor/unsuccessful mobilizers. several mobilization algorithms utilizing g-csf and plerixafor have been published however the most efficient and cost effective strategy is yet to be determined. most transplant centers administer plerixafor for peripheral blood (pb) cd34+ stem cell content of o10 × 10 6 /l on day (d) 4 of g-csf mobilization. at the university of colorado (uch) we changed our programmatic approach in 2015 and administered plerixafor for pb cd34+ count of o30 × 10 6 /l on d4 of g-csf mobilization. in this study we evaluate the impact of this novel mobilization algorithm on apheresis days and total stem cell yield. patients (pts) with lymphoma and pcn who underwent asct at uch until 3/2015 received plerixafor if pb cd34+ cells on d4 of g-csf mobilization was o10 × 10 6 /l. based on our institutional review of poor/unsuccessful mobilizers and using logistic regression analysis this algorithm was revised in 4/2015. in the new algorithm all pts received plerixafor if pb cd34+ cells on d4 of gcsf mobilization was o30 × 10 6 /l. demographics were compared between pts with lymphoma and pcn before (group 1: 9/2013-3/2015) and after (group 2: 4/2015-4/2016) the new algorithm was implemented. the primary goal of this analysis was to assess the total days of apheresis and total stem cell yield between the two groups. we also sought to analyze days to wbc engraftment and platelet engraftment. a total of 131 pts were included in this analysis. group 1 consisted of 77 pts (26 pts had lymphoma and 51 pts had pcn). group 2 consisted of 54 pts (20 pts had lymphoma and 34 pts had pcn). we found that there was a significant increase in total yield (p = 0.0017) in group 2 as compared to group 1. on further disease subtype assessment we noted that pts with pcn in group 2 had a significant increase in total yield (p = 0.0014). in lymphoma pts on univariate analysis group 2 showed a significant decrease in apheresis days (0.468 days, p = 0.044, 95% ci: (−0.91, − 0.026)). on multivariate analysis there was still a marginally significant decrease in group 2 (0.47 days, p = 0.052, 95% ci: (−0.916, − 0.024)) compared to group 1. in pcn pts on univariate analysis group 2 showed a significant decrease in apheresis days (0.387 days, p = 0.025, 95% ci: (−0.718, − 0.056)). on multivariate analysis group 2 continued to show a significant decrease in apheresis days (0.426 days, p = 0.02, 95% ci: (−0.772, − 0.081) compared to group 1. we found no significant difference between the two groups in days to neutrophil engraftment and platelet engraftment. our analysis showed that a mobilization algorithm of administering plerixafor for a pb cd34+ stem cell count of o30 × 10 6 /l on d4 of g-csf mobilization led to a decrease of roughly 0.46 days in the lymphoma cohort and a significant decrease of 0.43 days in the plasma cell neoplasm cohort. we also noted a significantly increased total yield of stem cell collection in group 2. overall our programmatic approach led to decreased chair-time for apheresis and better resource utilization. pharmacoeconomic impact of this approach will be updated at the meeting. disclosure of conflict of interest: mk: speakers bureau, seattle genetics; remaining authors declare no conflict of interest. administration of stem cell boosts (scbs) from the original donor offers a therapeutic option. we report on 50 pediatric patients with pgf who received a total of 61 boosts with cd34 + selected peripheral blood stem cells (pbsc) after transplantation from matched unrelated (n = 25) or mismatched related (n = 25) donors. median time between hsct and infusion of the 61 scbs was 94 days (13-519). boosts contained a median number of 3.15 × 10 6 cd34+ progenitor cells/kg body weight (range: 0.71-27.9 × 10 6 ) with a median number of 2417/kg (range: 100-23 630) residual cd3+ t cells. within 8 weeks after application, a significant increase in median neutrophil counts (600 vs 1516/mm 3 , po 0.05) and a decrease in erythrocytes and thrombocytes transfusion requirement (median frequencies 1 and 7 vs 0, p o0.0001 and o 0.001), were observed, and 78.8% of the patients resolved one or two of their initial cytopenias whereas 36.5% had a complete hematological response. additionally median lymphocyte counts for cd3+, cd3+cd4+, cd19+ and cd56+ increased 8.3 fold, 14.2 fold, 22.3 fold and 1.6 fold, respectively. the rate of de novo acute gvhd grade i-iii was only 6% and resolved completely after treatment. no gvhd iv or chronic gvhd occurred. patients who showed a response to scb displayed a trend toward better overall survival (os) (p = 0.07). administration of cd34+ selected scbs from alternative donors is a safe and effective procedure. we hypothesize that the cd34+ progenitor boosts may have an enhancing effect on maturation of committed lymphoid precursors already present in the host or generate another wave of thymic seeding with accelerated t-cell differentiation process in the absence of any immune suppression. further studies are warranted to better define the impact on immune reconstitution and survival. disclosure of conflict of interest: none. plerixafor plus granulocyte-colony stimulating factor (g-csf) has been shown to mobilize more cd34+ cells than g-csf alone for autologous hematopoietic stem cell transplantation (hsct). however, there are few studies that analyze the impact of this strategy in engraftment. the aim of our study is to compare mobilization and engraftment between patients who received a combination of plerixafor plus g-csf and patients (pts) who mobilized with g-csf alone. a retrospective casecontrol analysis was performed in 24 pts with myeloma who mobilized with plerixafor plus g-csf (group p/g-csf) and was compared with 24matched for sex and age controls who mobilized with g-csf alone (group g-csf). all pts underwent hsct between 2009 and 2015. mobilization with g-csf at dose of 10 μg/kg/day was used in all pts. the aphaeresis was scheduled on day +5. plerixafor (0.24 mg/kg) was added if the number of cd34+cells on day +4 was o10/μl for 2 × 10 6 cd34+/kg requested (or o20/μl for 4 × 10 6 cd34+/kg), or if the number of cd34+cells collected in the first apheresis was o50% of cd34+ requested. conditioning and supportive care were similar in both groups. in p/g-csf group, 13 were male and 11 female. median age was 60.92 years (range: 49-71). in group g-csf, 15 were men and 9 female. median age was 60.67 years (range: 50-73 years). there were no differences between both groups. disease status at time of mobilization was different between groups (p = 0.023). in p/g-csf group: 11 (45.83%) pts were in complete remission (cr), 4 (16.66%) very good partial responses (vgpr), 7 (29.16%) partial response (pr) and 2 (0.08%) had no response to treatment. in g-csf group: 7 (29.16%) pts had reached cr, 13 (51.16%) vgpr and the remainder in pr. sixteen (66.67%) pts in p/g-csf group had received ⩾ 2 lines of treatment vs 9 (37.5%) pts in g-csf group (p = 0.046). no difference was seen on mean day-dose of g-csf (14 μg/kg/24 h in p/g-csf group vs 12 μg/kg/24 h) (p = 0.067). there was no difference on cd34+/kg requested (19/24 pts in p/g-csf were requested 2 × 10 6 /kg vs 18/24 in g-csf group) (p = 0.73). p/g-csf group needed more apheresis sessions, 17 (70.83%) pts required ⩾ 2 sessions against 4 (16.67%) pts in group g-csf (p o0.001). we obtained enough cd34+ cells to carry out hsct in all patients, although mean number of cd34 + cells obtained in p/g-csf group was lower than in g-csf group (2.92 × 10 6 /kg vs 4.98 × 10 6 /kg, respectively) (p o0.001). also, mean number of cd34+ infused in p/g-csf group was lower (2.92 × 10 6 /kg vs 3.55 × 10 6 /kg) (po 0.001). however, engraftment results were similar in both groups, as represented in table 1 . patients who required mobilization with plerixafor plus g-csf got an engraftment as good as patients who do not require the combination despite of worse baseline parameters. given that the number of cd34+ infused in the p/g-csf group has been lower than g-csf group, these results might suggest that the different composition of graft cell with plerixafor plus g-csf mobilization, described in some studies, could impact on engraftment outcomes. high-dose chemotherapy following autologous hematopoietic stem-cell transplantation (autohsct) is an effective method of treatment both recurrent and primary refractory lymphoma patients. however, some patients have mobilization failure (‛poor mobilizers') with inadequate collection of peripheral blood stem cell (pbsc). aim: to evaluate the efficacy and factors influencing pbsc mobilization and collection for the autohsct in patients with lymphomas. thirty patients were included in this study: 17-with hodgkin lymphoma, 7-with non-hodgkin lymphoma, 6-with multiple myeloma; 17 women and 13 men of them. the median age of patients was 36 years (24-64 years). the mobilization of pbsc with only colony-stimulating factors (csf) was carried out for 17 patients, chemotherapy (cyclophosphamide, etoposide) in combination with csf-for 13 patients. only one patient had plerixafor mobilization. the concentration of cd34+ in peripheral blood (pb) was studied on the day of the intended cytapheresis. cytapheresis was commenced when cd34+ concentration had been greater than 0.01 × 10 6 cells/ml. twenty-four patients (80%) from 30 had collection of pbsc. the collection was not performed in six patients (20%) because the concentration of cd34+ in pb on the day of the intended cytapheresis was lower than 0.01 × 10 6 cells/ml. there was no possibility to use plerixafor in these cases for economic reasons. the median concentration of cd34+ in pb on the first day of the intended cytapheresis in the group of patients that had cytapheresis was 0.013 × 10 6 cells/ml whereas in the group of failed-0.005 × 10 6 cells/ml (p o0.05). fifty-nine tests of cd34+ in pb were done. distribution and test results by days from the first day of the intended cytapheresis are presented in table 1 . the total number of the cytapheresis was 36. the majority of patients had 1 procedure of pbsc collection (n = 22), 13 patients had 2 procedures and only 1 had 3. the last patient had had two previous failed cytapheresis procedures and the adding of plerixafor helped him to collect necessary number of cells. the median of cd34+ cells on patient's kilo was 2.85 × 10 6 cells/kg. sex, age, mobilizing regimen, previous radiation therapy, the count of lines of chemotherapy before autohsct were not significantly associated with poor pbsc mobilization and collection. only tumor response before autohsct (complete/ partial response or stabilization) was significantly associated with cd34+ cell count in the product of cytapheresis. patients with complete or partial response had significantly better cd34+ count. [p034] disclosure of conflict of interest: none. factors associated with failure in mobilization of peripheral blood hematopoietic progenitor cells in autologous transplantation je dulon-tarqui, bl acosta-maldonado, l rivera-fong, sa sánchez-guerrero, jf zazueta-pozos, ja padilla-ortega, wj ladines-castro and lm valero-saldaña high dose therapy followed by autologous stem cell transplantation (asct) obtained from peripheral blood is currently the standard model for treatment consolidation in various hematologic malignancies. a global incidence of 5-40% of failure to mobilization is reported, and some factors associated with poor mobilizers in hodgkin's lymphoma (hl), non-hodgkin's lymphoma (nhl) and multiple myeloma (mm) when the yield in peripheral blood stem cells (pbsc) collection is unsatisfactory, the effects for the recipient can be serious. the donor's age, gender, body surface area (bsa), processed blood volume and the method of g-csf dose calculation may affect the cd34+ yield. as g-csf has a low distribution volume in the peripheral blood (pb), it might be appropriate to calculate the doses by using the bsa instead of per kg body weight. 175 consecutive allogeneic pbsc donations performed in 170 healthy donors at the karolinska university hospital in stockholm were included. a complete medical history, physical examination, electrocardiogram, chest x-ray and laboratory testing were done before pbsc donation. relevant data for analysis were collected from the institutional quality database for a retrospective review. the total blood volume was calculated using the formula by nadler et al. the bsa was calculated using the formula by du bois and du bois. the concentration of cd34+ cells in the pb and the processed volume of blood were significantly correlated to cd34+ cells yield (po 0.00005 and po0.001, respectively, see table 1 ). the g-csf dose per m 2 was significantly correlated to the concentration of cd34+ cells in the pb (p = 0.0003) and in the product (p = 0.01, see table 1 ). smaller bsa (p o0.001) and less processed volume (p o0.001) were found among female donors, who were given lesser g-csf dose per m 2 (p o0.001) and showed lower yield compared to men (po0.05). however, multivariate analysis of the yield showed that only the concentration of cd34+ cells in the pb and the processed volume remained independent significant (see table 1 ). [p036] in this study, we found the concentration of cd34+ cells in the pb and the processed volume of blood to be independent predictors of yield. we recommend to get a high concentration of cd34+ cells in the pb, and to process adjusted volumes of blood when needed. an evaluation if the calculation of g-csf dose per m 2 is more appropriate than per kg body weight should be done in future studies. autologous stem cell transplantation (asct) has been widely used in the treatment of hematological malignancies over the last two decades. despite its broad use, some characteristics that might influence engraftment have not been exhaustively investigated, particularly graft purity with respect to contamination by platelets (plts) and white blood cells (wbc). here we report collection characteristics and engraftment kinetics of a single center consecutive series of 510 asct. we retrospectively collected clinical records of 481 patients who underwent leucapheresis procedures (la; followed or not by asct) and data on 510 asct at our institution over 16 years (2000-2016) ( table 1 ). the impact on engraftment kinetics of conditioning chemotherapies, amount of infused cd34+ cells and wbc/plts graft contamination were analyzed. absolute neutrophil count (anc) engraftment was defined as the duration of neutropenia (from day 0 to the first of 3 consecutive days of anc4500/μl post asct). regarding cd34+ cell collection, no impact of mobilizing regimens and wbc count during la was observed. on the other hand, we observed a difference in the number of total cd34+ cells collected among different diagnoses: the median overall collection was 7.2 (0.65-64.06) × 10 6 /kg cd34+ cells for nhl patients, 5.66 (0.71-23.31) × 10 6 /kg for mm patients, 6 .15 (0.51-23.24) × 10 6 /kg for hl patients and 3.56 (0.64-20.3) × 10 6 /kg for aml patients) (p = 0.001). considering cd34 + cells/kg harvested on the first day of la, 59.2% of nhl and hl, 57.5% of mm patients and 34% of aml patients harvested ⩾ 5 × 10 6 /kg cd34+ cells. of note, among aml patients, 40.6% collected o 2.5 × 10 6 /kg. the differences were statistically significant (p = 0.003). moreover, an inverse correlation between collected cd34+ cells and age was shown (p = 0.001). anc recovery after asct was not influenced by conditioning regimen whereas diagnosis impacted on the duration of neutropenia (aml patients displayed a longer aplasia, po0.01). we observed that the median days with anco 500/μl were 10, 11 and 12 in patients who received 45.3 × 10 6 /kg, 3.5-5.3 × 10 6 /kg and o3.5 × 10 6 /kg cd34+ cells, respectively (po0.0001). furthermore, the same finding was observed considering the duration of thrombocytopenia (median number of days with plts o50 000/μl: 15, 18 and 20 in patients who received 45.3 × 10 6 /kg, 3.5-5.3 × 10 6 /kg and o 3.5 × 10 6 cd34+ cells, p o0.0001). looking at the apheresis product, we analyzed the impact of harvest contaminating wbc and plts on engraftment kinetics. notably, when the asct collection contained 4100 × 10 3 /μl wbc, anc engraftment (days with anc o 500/μl) lasted longer (median days 11) compared to patients who received a graft with lower wbc count (po 0.0001). a faster anc engraftment was also observed in patients receiving harvests with plts levels 4600 × 10 3 /μl compared to those who infused a collection bag with plts o600 × 10 3 /μl (p = 0.005). herein, we confirmed that the disease and the amount of infused cd34+ cells significantly influence time of anc and plts engraftment; furthermore, we observed for the first time that quality and purity of the graft have a substantial impact on engraftment kinetics. a combination of chemotherapy with growth factor is a commonly used strategy for hematopoietic stem cell (hsc) mobilization. the collection of timely and adequate numbers of hscs is a prerequisite for proceeding to transplantation. a variety of mobilization strategies are currently used. the knowledge of efficacy, safety and predictability of different hsc mobilization strategies might help blood and marrow transplantation (bmt) programs to effectively schedule patients for mobilization. given the many variables associated with the mobilization of hsc, collecting an adequate stem cell dose in a timely and effective manner is an art and science. factors that might affect the process includes type of disease and mobilization protocol, financial clearance, availability of chemotherapy beds, scheduling various diagnostic procedures and transplant urgency. to evaluate the effectiveness and related coordination efforts of ‛just-in-time' strategy of hsc mobilization and collection, we performed a retrospective study comparing all patients in whom peripheral hsc mobilization was attempted at khcc from january 2005 through november 2016. data collected included the disease type, mobilization protocol, days to and number of collections, cd34+ cell dose, calendar of the mobilization and collection. the records of a total of 1042 mobilizations were reviewed. 364 were of healthy allogeneic donors, and the remaining 678 were of patients undergoing autologous transplantation. table 1 depicts the overall summary of number of days and collection procedures per each protocol. detailed mobilization kinetics per disease type and mobilization protocol were also captured and evaluated. [p037] s140 [p038] detailed analysis of mobilization kinetics comparing different mobilization strategies aids in prediction of number of days of mobilization and anticipated number of collections. this helps in proactively scheduling patients based on collection predictability. a seamless communication through a shared calendar between key parties, primarily bmt physicians and nurse coordinators, bmt and flow cytometry laboratories and chemotherapy unit can be achieved. autologous stem cell transplantation is still a standard of care in the treatment of multiple myeloma. lenalidomide-based regimens are commonly used in both transplant-ineligible as well as -eligible patients. prolonged lenalidomide-exposure is known to affect mobilization of cd34 + cells, although the basic mechanisms are poorly understood. limited prospectively collected data is available on the effect of lenalidomide in the capacity to mobilize cd34 + cells for transplantation as well as graft cellular composition and post-transplant hematological recovery compared to the lenalidomide-naive patients. this prospective study included 60 newly diagnosed myeloma patients who received mobilization with low-dose cyclophosphamide + g-csf, were successfully apheresed and transplanted before the end of 2014. twenty-six patients had received a median of three cycles of lenalidomide-based induction (43 %), whereas 34 patients were lenalidomide-naive and served as the control group. both baseline characteristics and collection targets were similar between the groups. cd34 + mobilization and apheresis yields were analyzed and compared between the groups. blood graft cellular composition was analyzed from the thawed cryopreserved samples with a flow cytometry. graft function was evaluated by collecting engraftment data as well as by total blood counts at day +15 and at 1, 3, 6 and 12 months after post-transplant. the patients in the lenalidomide group had both lower median peak b-cd34 + counts and about 40% lower cd34 + yields of the first apheresis but without statistical significance ( table 1 ). the median number apheresis was significantly higher in the lenalidomide arm (2.0 vs 1.5, p = 0.039). the number of cd34 +cd133+cd38-, cd3+cd4+, cd3+cd8+ cells and nk cells in the cryopreserved grafts were comparable between the arms. time to neutrophil engraftment was 12 days in the both groups. the median time to platelet engraftment was 12 d in the lenalidomide group and 13 d in the control group. hematological recovery was comparable between the groups within 12 months post-transplant. lenalidomide-based induction therapy seems to have an impact in the number of apheresis needed, but not in the total yield of cd34 + cells in the graft. neither the graft cellular composition nor posttransplant recovery in myeloma patients was affected by the limited duration of lenalidomide used before mobilization and collection of blood grafts. between september 2015 and november 2016. siemens hematek 3000 system was used for luc count. luc numbers and percentage was measured before leukapheresis. we used pearson test for the correlation and roc curve for cut off value. patients' characteristics were shown in table1. there was not a correlation between luc number and mobilized cd34 positive stem cell number. but luc percentage was positively correlated with mobilized stem cell number (p:0.01). a count of 5 × 10 6 /kg collected stem cells are optimal for autologous stem cell transplantation. we found 2% luc percentage as a cut-off value for prediction of collecting optimal number of stem cells with 61% sensitivity and 60% specificity. as expected luc percentage was negatively correlated with white blood cell count. there was no correlation between mobilized cd34 positive stem cell number and age. both luc percentage and mobilized cd34 positive stem cell number did not differ with underlying disease. we found only one study in the literature that evaluated luc percentage as a tool for the prediction of successful stem cell collection. they found that baseline luc numbers negatively correlated with stem cell mobilization in healthy donors (1) . but we measure luc on apheresis day and found a positive correlation between luc percentage and stem cell mobilization. and we found a cut-off value for optimal stem cell mobilization with acceptable sensitivity and specificity. in our study we demonstrate that luc percentage measurement on apheresis day may be a very simple and cheap tool for the prediction of optimal stem cell mobilization. the spectra optia (so) apheresis system performs a wide range of therapeutic procedures, including peripheral blood stem cell (pbsc) collection in mobilized donors and patients (pts). the device was studied to evaluate the cellular composition of pbscs harvested in pts with multiple myeloma (mm), non hodgkin's lymphoma (nhl) and hodgkin's lymphoma (hl) planed for autologous peripheral stem cell transplantation (apbsct), and to optimize the collection of pbscs using the cd34+ precount and collection efficiency (ce2) of apheresis device which is calculated as follows: ce2 = total cd34 + cells collected × 10 6 /kg; cd34+ precount/ μl × blood processed (liters). the blood volume processed is calculated as follows: desired cd34+ × 10 6 /kg × recipient weight (kg): ce2 × cd34+ precount/μl in our study enrolled pts undergoing pbsc mobilization and planed for apbsct. we evaluated so system's mononuclear cell (mnc) collection performance, with respect to cd34+ cells and mnc collection efficiency, platelet reduction pre to post apheresis, and product purity in view of using prediction algorithms to optimize the procedure and predict the cd34+ yield, blood volume processed and platelets loss. we also evaluated neutrophil and platelet recovery in pts who underwent apbsct. results: between 30/3/2015 and 30/11/2016, 45 pts underwent pbsc harvesting by so device. median age was 46 years (20-71). there were 19 females and 26 males. diagnosis was mm in 21 pts, hl in 17 pts and nhl in 7 pts. the number of ahereses procedures was 59. mobilization consisted in g-csf alone in36 pts, chemotherapy and g-csf in 8 pts, and g-csf + cxcr4 inhibitor in one patient. median count of cd34 + cells pre-collection was 58/μl (16.5-372) . median total blood volume processed was 12.4l (6.3-19.9 ). median count of cd34 + cells collected was 4.1 × 10 6 /kg (1-23.6). median mnc collection efficacy was 48% .median cd34+ cell collection efficacy was 45.5% (15-95%). median platelet reduction pre to post apheresis was 30% (0-50%). median product hematocrit and granulocytes product was 5% (3-9) and 52% (5-93), respectively. twenty-six of the 45 pts underwent myeloablative high dose chemotherapy followed by apbsct which was performed for mm in 18 pts, hl in 6 pts, and nhl in 2 pts. the median count of cd34+ cells infused was 2.5 × 10 6 / kg (1.15-10.6). all the pts received g-csf post-apsct until neutrophil recovery. the median day for neutrophil recovery was 10 (8) (9) (10) (11) (12) (13) (14) . median duration of severe neutropenia (anc o0.5 × 10 9 /l) was 7 days (4-10). the median day for platelet recovery was 10 (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) . median duration of severe thrombocytopenia (platelets o20 × 10 9 /l) was 5.5 days (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) . conclusion: the study results confirm that the so apheresis system's mnc collection protocol is safe and effective. the neutrophils and platelets recovery in pts auto-transplanted was not inferior compared to historical controls. in addition, this system help to use prediction algorithms for whole blood processing to achieve a desirable and optimal yield based on cd34+ precounts and ce2 of the apheresis device. disclosure of conflict of interest: none. peripheral blood stem cell apheresis in small children is difficult! aa hedayati-asl 1 , m emam-jome, p dinarooni, v fallah, a mehrvar and r zangooei 1 in low-weight children with cancer and healthy donor children, peripheral blood progenitor cells (pbpcs) have largely replaced bone marrow as source of autologous and allogeneic stem cells in part because of their relatively easy collection. however, there is a concern regarding medical, psychosocial and technical difficulties in small children. we retrospectively analyzed peripheral blood stem cell apheresis in 38 collections. 30 patients were with cancer (17 patients = neuroblastoma, 4 patients = retinoblastoma, 5 patients = germ cell tumor, 1 patient = hepatoblastoma, 3 patient = wilm's tumor) and 8 healthy children donors. the study was conducted between 2012 and 2016. peripheral stem cell apheresis was performed in the mahak cancer children's hospital in a nice room for children where the patients stayed with their families. patients s142 were not routinely sedated. pbpc were collected by a cobe spectra cell separator (cobe, denver, co, usa). harvesting was performed after 5 days mobilization. mean body weight was 11.6 kg (range: 8-15 kg) for a median age of 3 years (range: 10 months-5 years). mean duration of harvesting was 205 min (range: 164-270 min). mean volume of stem cell collection was 135 ml (range: 110-240 ml). the mean number of total nucleated cells collected was 5.4 × 10 8 /kg (range: 3.2-9.9 × 10 8 /kg recipients). no side effects occurred. children didn't require an additional haematopoietic progenitor mobilization or additional apheresis in other day. pbsc collection was without transfusion in healthy donor children. pbsc collection may be difficult in small children owing to the large volume apheresis compared to the child's weight. various problems, such as metabolic or haemodynamic disorders may be were seen. peripheral stem cell harvest can be performed in low-weight children under safe and effective conditions even when systematic priming by blood is avoided. processing with increase of blood volume may to increase in the yield by recruiting progenitor cells. disclosure of conflict of interest: none. peripheral blood stem cell collection in low body weight children: a single centre experience g del principe*, g leone, s lazzaro, a meschini, k feri, p marchitelli, d carasso, f locatelli and m montanari department of pediatric hematology/oncology and transfusion medicine, irccs bambino gesù children's hospital, rome, italy pbsc became preferred source for autologous transplantation because of easier collection and faster engraftment. however apheresis for low body weight children ( o12.5 kg) is affected by some issues: venous access, extracorporeal volume, metabolic and hemodynamic complications, citrate toxicity, so is crucial to standardize harvesting procedure both maximizing stem cells collection and reducing adverse events. a dual lumen central venous catheter was used to obtain a minimal blood flow of 10-15 ml /min and pbsc collection was performed with spectra optia mnc v6.1 apheresis system, starting with cd34+ cell ⩾ 20 μl in peripheral blood. the priming of extracorporeal circuit was made with compatible, irradiated, leucodepleted packed red cell to avoid hypovolemic state. citrate dextrose solution a(acd-a), with a ratio of 1:24 to whole blood, and a bolus of heparin 50 ui/kg were used as anticoagulants. all patients, treated without sedation, were monitored by ecg, pulse oximetry and non invasive blood pressure; electrolytes panel (na, k, ca) and act (activated coagulation time) were assessed at the beginning, 30 minutes after and then every hour during apheresis. hypocalcemia was managed by 350 mg calcium gluconate slow infusion. we report our experience of pbsc collection in low body weight children ( o12.5 kg) treated in our apheresis department between january 2015 and november 2016. a total of 37 pbsc collections were performed in 17 children (8 m/9 f, median age 21 months, median weight 10.5 kg) affected by medulloblastoma (n = 4), germ cell tumor (n = 2), neuroblastoma (n = 8), retinoblastoma (n = 2), brain cancer (n = 1). total blood volume processed ranged from 2.0 to 4.55 tbv (median 3.05) and median count of cd34+ collected was 5.5 × 10 6 /kg(range: 1.5-54). all procedures were performed with a median duration time of 199 minutes (range: 114-293 min) and no serious adverse events occurred. in our experience pbsc collection is safe and feasible also in low body weight children using a tailored apheresis procedure. disclosure of conflict of interest: none. plerixafor on demand in the first or in the second attempt of cd34 mobilization j romejko-jarosinska, e paszkiewicz-kozik, l targonski, m szymanski, z pojda and j walewski 1 1 high-dose chemotherapy and autologous hematopoietic cell transplantation (auto-hct) is a recommended strategy for patients with relapsed, refractory or high risk lymphoma. mobilization failure of cd34+ cells after granulocyte colonystimulating factor (g-scf) with or without chemotherapy is a factor limiting patient access to this potentially curative procedure. the use of plerixafor with g-csf may improve cd34+ cell harvest in poor mobilizing patients. we evaluated the clinical effectiveness of plerixafor and g-csf ± chemotherapy administered on demand in the first and second attempt of mobilization in lymphoma or myeloma patients who were eligible for auto-hct. we evaluated data on 59 consecutive patients with hodgkin lymphoma (17), dlbcl (17), mantle cell lymphoma (10) , myeloma (8) and other lymphoma subtypes (7) who were mobilized with plerixafor between january 2011 and october 2016. median (range) age of patients was 47 (27-69). patients received a median of 2 (1-4) chemotherapy lines. radiotherapy was applied in 13 patients. all patients received g-csf (10 μg/kg/day) ± chemotherapy and plerixafor (240 μg/kg/day) on demand in the absence of increase in the number of cd34+ cells in peripheral blood above 10/μl on the day of the scheduled apheresis (within 20 days following the chemotherapy and after at least 4 days of g-csf). plerixafor was given to 36 patients in the first attempt of mobilization and to 23 patients during the second mobilization. the mobilization was considered effective if the harvest cell dose was 2 × 10 6 /kg cd 34 or more. after plerixafor administration circulating cd34+ cells increased to 410/μl in 21 patients (58%) and in 11 patients (43%) in the first and in the second mobilization, respectively (p = 0.26). the cd34+ cell collection was performed in 52/59 patients (88%): in 32/36 (89%) patients in the first and in 20/23 patients (87%) during the second mobilization cycle. the median number of apheresis was 3 (range: 1-6), for both mobilizing cycles. the median (range) cd34 cell dose collected in the first and second cycle was 3.03 (range: 0.77-16.87) × 10 6 /kg and 1.89 (range: 18-11.53) × 10 6 /kg, respectively (p = 0.007). the harvest was successful in 27/32 patients (84%) in the first and in 10/20 patients (50%) in the second cycle (p = 0.007). three patients (9%) who failed the collection with plerixafor in the first attempt, succeeded in the second cycle. additional second mobilization with plerixafor was successful in five patients (25%) who failed the first mobilization. in total, 30/32 (93%) and 15/20 (75%) of patients given plerixafor in the first or in the second mobilizing cycle harvested at least a minimum cd34 cell dose for auto-hct (p = 0.05). these results show that plerixafor administered on demand is an effective rescue strategy for poor mobilizing patients. each mobilization cycle with plerixafor resulted in the increase of circulating cd34 cell count. successful harvest is more frequent if plerixafor is administered in the first than in the second mobilization attempt. the evaluation of the prognostic factors for mobilizing failure with plerixafor is necessary to identify the poor mobilizers precisely. disclosure of conflict of interest: jr-j, ep-k, lt and jw: sanofi (travel grants); ms and zp: none; jw: lecture, honoraria cryopreserved stem cell grafts are still widely used both in the autologous or allogeneic settings. cryopreserved grafts can be thawed at the bedside or thawed and washed at the cell therapy laboratory. we recently reported that post-thaw washing did not impair hematopoietic engraftment, in a cohort of 2930 autologous transplanted patients receiving either unwashed or washed grafts (calmels b et al, bone marrow transplant. 2014). post-thaw washing can be implemented using various methods such as manual centrifugation, automated centrifuge-based (sepax 2, biosafe) or spinningmembrane devices such as lovo (fresenius kabi). we here report a step by step implementation of the lovo biomedical device (bmd) for washing thawed stem cell grafts. having defined a washing program, we aim to compare this protocol to our routine process, using the sepax 2 bmd. we took advantage of 11 apheresis products intended for destruction and cryopreserved in 2 identical bags; after dry-thawing (plasmatherm, barkey), bags were connected to the sepax 2 or to the lovo bmd, diluted volume to volume with +4-8°c 6% hydroxyethylstarch 130/0.4 (voluven, fresenius kabi) and processed using the smartwash program (sepax 2) or a 2cycles standard wash protocol on lovo (a cycle referring to one pass through the spinning membrane). the lovo settings were customized for this application: reduction retentate pump rate 75 ml/min, desired inlet pcv 10%, and automated volume to volume dilution. after processing, cd34 and cd45 absolute counts and viability were evaluated by single platform flow cytometry (stem-kit, beckman coulter) and dmso was quantified by capillary zone electrophoresis (p/ace, beckman coulter). post-wash data show comparable cd34+ cell recovery, viability and effective dmso depletion. we conclude that lovo enables high efficiency dmso depletion while preserving optimal cd34 viability and recovery. comparison with sepax 2, a widely used automated centrifugebased device, reveals comparable efficiency. moreover, the length of the procedure when using the lovo does not significantly delay the process as compared to bedside thawing. we are currently evaluating lovo for the processing of multiple bags and higher cell contents, due to its ability to concentrate large volumes of cells suspension. post-thaw washing using automated cell processing systems have thus to be preferred over bedside thawing, since they provide multiple benefits including a short processing time, efficient dmso and cell debris removal, precise determination of infused cd34+ cell dose, and improved cellular stability. [p047] disclosure of conflict of interest: none. using bone marrow (bm) as the graft source results in lower graft-versus-host disease incidence, which is particularity important in haploidentical (haplo) stem cell transplantations (sct). nonetheless achieving adequate cd34+ cell count might be complicated in cases of donor-recipient weight differences. priming with g-csf may partly solve this problem. also there are reports of immunomodulatory effect of bm priming. in the retrospective study we have evaluate the effect of priming on stem cell yield and the outcomes of sct. patients and methods: 50 patients with primed bm graft were matched in the ratio 1:1 to non-primed grafts. the criteria for matching were type of the donor, age of the recipient, underlying disease and disease status at the time of sct. priming was performed with three injections of filgrastim 5-7 mcg/kg daily for 3 days prior to bm harvesting. median recipient age was 18 years (range: 1-58). 60% of patients received the graft from haplo donor, 40% from matched related donor (mrd). 39% had acute lymphoblastic leukemia, 38% had acute myeloid leukemia, 10% had aplastic anemia, 13% had other malignancies. 68% were classified as salvage patients. 27% received myeloablative conditioning, 73% received reduced intensity. post-transplantation cyclophosphamide (ptcy) was used as graft-versus-host disease prophylaxis in 83% of patients. results: the yield of cd34+ × 10 6 cells /kg of recipient weight was only non-significantly higher in the priming group: 6.0 ± 3.4 vs 5.0 ± 2.5, p = 0.12. the yield of cd34+ cells per kg of donor weight was also not different: 4.3 ± 4.0 vs 4.3 ± 5.8, p = 0.55. there was no difference in the incidence of primary graft failure (14% vs 20%, p = 0.42). median time to neutrophil (21 vs 23 days, p = 0.02) and platelet (19 vs 23 days, p = 0.05) engraftment was shorter in nonpriming group. there was no differences between priming and non-priming groups in the incidence of acute grade ii-iv gvhd (14% vs 4%, p = 0.11), moderate and severe chronic gvhd (12% vs 11%, p = 0.88), 1-year non-relapse mortality (12% vs 18%, p = 0.46), relapse incidence (36% vs 38%, p = 0.91), overall survival (64% vs 54%, p = 0.52), event-free survival (52% vs 44%, p = 0.62) and gvhd-relapsefree survival (38% vs 36%, p = 0.62). conclusions: priming of the bone marrow with reported schedule did not result in higher cd34+ cell yield and was not associated with any differences in the outcomes of sct. nonetheless, these results should be interpreted with caution, because our study included large proportion of pediatric patients, patients with active disease and ptcy as gvhd prophylaxis, and they may not translate to the other groups of patients. disclosure of conflict of interest: none. priming with granulocyte-colony stimulating factor preserves the contents and abundant ifn-γ production capacity of γδ t cells z bian 1 , q fu 1 , m huo 1 , xj huang 1 and j liu 1 1 peking university people's hospital the increasing evidences indicate that removal of αβ t-cell and b-cell from grafts was efficient and reproducible in allogeneic hematopoietic stem cell transplantation (allohsct). γδ t cell is one of the functional subpopulations preserved by this graft manipulation and supposed to play a role in improving the transplant outcomes. thus, comprehensive understanding the subsets and functional capacities of γδ t cells in graft becomes important. although there is increased attention paid on this special t-lymphocyte subpopulation, the contents and cytokine production capacities of peripheral γδ t cells before and after granulocyte-colony stimulating factor (g-csf) mobilization for allohsct have not been reported. peripheral blood (pb) before g-csf treatment, g-csf-primed pb and bone marrow (bm) grafts were obtained from 19 healthy donors. the proportions of total γδ t cells and various γδ t-cell subsets were detected by flow cytometry. furthermore, effects of g-csf on the contents and cytokines production by γδ t-cell subsets were also determined. the percentages of most γδ t-cell subsets including cd27+, cd27-, vδ1+, vδ1+cd27+, vδ1+cd27-, vδ2+, vδ2+cd27+, vδ2 +cd27-, and non-vδ1/δ2 were preserved in the g-csf-primed pb grafts compared with those before g-csf mobilization. interestingly, we found that peripheral γδ t cells and various subsets all predominantly expressed ifn-γ in response to stimulation. this abundant ifn-γ production capacity of peripheral γδ t cells were maintained after g-csf treatment. in contrast, production of il-17 by γδ t cell and its subsets were decreased in the same context. priming with g-csf preserved the contents and abundant ifn-γ production capacity of γδ t cells. our data suggests a reasonable role of γδ t cells in preventing from allohsct associated complications and may help establish an effective γδ t cell-based immunotherapeutic approach to improve the overall survival of allohsct. disclosure of conflict of interest: none. processing of hematopoietic stem cells grafts: towards automation of cryopreservation/thawing steps a-l chateau 1 , j gaude 1 , c malenfant 1 , a autret 2 , c lemarie 1 , c chabannon 1 and b calmels 1 1 centre de thérapie cellulaire-institut paoli-calmettes and 2 unité de biostatistiques-institut paoli-calmettes autologous hematopoietic stem cells (hsc) support is still widely used to allow for high-dose chemotherapy in the context of myeloma and lymphoma treatment. in the autologous setting, mobilized aphereses are systematically cryopreserved. currently, cryopreservation and subsequent thawing rely on manual and largely operator-dependent processes such as manual addition of dmso for cryopreservation or thawing in standard water baths. these operations are thus hampered by significant intra-and inter-facility variability and have to be replaced whenever possible with automated and harmonized processes. the aim of our study was to evaluate a recent, versatile device: smartmax (biosafe, eysins, switzerland), based on the peltier-seebeck effect, for its ability to automatically add the dmso-containing solution to the cell product and to thaw hsc bags. we thus compared three different cryopreservation/thawing protocols ( figure 1 ). we first evaluated the use of the smartmax at the thawing step by comparing 110 cryopreserved apheresis products thawed using our routine device: the plasmatherm (barkey), an automated dry-thawing device that contains water (protocol a), with 51 products thawed with the smartmax (protocol b); after thawing, all products were washed using the smartwash program of the sepax2 (biosafe). we then evaluated the smartmax for its ability to automatically add the dmso solution: 9 autologous grafts were processed with the smartmax, both for cryopreservation and thawing (protocol c); we compared these 9 ‛fully automated processes' to 51 apheresis processed with protocol b. absolute cd34+ and cd45+ cell counts and viability were measured before cryopreservation and after washing using single platform flow cytometry. for all three protocols, the quality of the collected product was comparable in terms of median cd45+ cell and neutrophil contents. when comparing protocols a and b, viable cd34+ cell recovery after thawing and washing was slightly lower in the smartmax group (77%) as compared to the plasmatherm group (82%, p = 0.009). when comparing protocols b and c, viable cd34+ recovery was comparable (p = 0.8) when the cryopreservation solution was automatically added by the smartmax (81%), as compared to the manual technique (77%). these preliminary data need to be validated on larger numbers of procedures, however suggest that smartmax use can safely be substituted both to the manual addition of the cryoprotectant and to the traditional thawing step in water baths; potential advantages include complete water removal from sensitive clean rooms and gmp environments. full automation of previously manual and operatordependent technical processes will ultimately allow for improved standardization and reproducibility across cell processing facilities. [p050] disclosure of conflict of interest: none. reduced efficacy of mobilisation using gdp compared to ive a hunter, w merrison, am martin, k hodgson, f miall, r moore and r lewin university hospitals of leicester, nhs trust the use of ive ± rituximab for relapsed/refractory disease in lymphoma is well established. stem cell mobilisation using g-csf post ive administration has been the standard of care in our unit for 20 years. recent interest in cisplatnin-based treatments has seen a change in practice with the use of gdp ± rituximab increasingly common. we have assessed the success of stem cell mobilisation post gdp and compared it to ive using g-csf. patients were eligible for augmentation with plerixafor if their peripheral blood cd34 levels were between 5-10 × 10 6 cells/l at the time of collection. from sept 15 to oct 16 21 patients with progressive or relapsed lymphoma underwent stem cell collection. patients characteristics: 12 dlbcl, 4 follicular and 5 t-cell nhl. had a median age 65 (37-74 years). 13 received gdp, 8 ive. overall 23% patients failed to mobilise a sufficient cd34 cell dose to proceed to hdt. all the patients who received ive mobilised successfully but 5/13 (38%) patients receiving gdp failed to mobilise. of the patients who did mobilise the average cd 34 collection was higher in the patients who received ive 7.1 (4.5-25.4) and the number of apheresis procedures was lower, median 1 (1-2) compared to 3.4 (2.6-9.1) and 2 (13), respectively, in the gdp group. patients in the gdp group who failed to mobilise were not eligible for plerixafor because cd34 levels were below 5 × 10 6 /l. taking age into account the median age in the ive group was higher 65(48-74) than the gdp group 62 (37-74) and the lines of previous therapy were not different. patients who had successful stem cell collections went on to receive hdt with leam and all patients engrafted. in this small collection of patients we have experienced a higher failure of mobilisation post a cisplatnin-based protocol compared both to our historical controls pre plerixafor usage (data not shown) but also to current patients. further investigation is needed to ascertain the impact of cisplatnin on stem cell mobilisation and its impact of treatment strategies. disclosure of conflict of interest: none. single centre experience of zarziotm biosimilar granulocyte-colony stimulating factor (gcsf) for the mobilisation of healthy donors demonstrates good leukapheresis yields and safety profile at 24 month median follow-up jg taylor 1,2 , t seddon 2 , k alizadeh 2 , c agrawal 2 , l kempster 2 , jg gribben 1,2 and sg agrawal 2,3 1 centre for haemato-oncology, barts cancer institute, 2 dept. haemato-oncology, st bartholomew's hospital, london, uk and 3 experimental pathology, blizard institute, queen mary university of london, uk biosimilars have led to significant improvements in the affordability of growth factors such as granulocyte-colony stimulating factor (gcsf). data has shown similar performance and efficiency to parent drugs but concern has been raised about their use in healthy donors due to lack of data examining adverse effects in this setting. we conducted a retrospective analysis investigating mobilisation and adverse effects in 51 healthy sibling donors of adults undergoing an allogeneic haematopoietic stem cell transplant at st bartholomew's hospital from 2011 to 2014. harvest data were gathered from hospital records. adverse effects data were gathered from hospital records and telephone follow up. 58% of donors were male with a median age at harvest of 46 (13-65). all donors were mobilised using zarziotm biosimilar gcsf at a dose of 10 μg/kg/day. median number of apheresis required was 1 (1) (2) (3) . median cd34+ cell count was 5.6 × 10 6 /kg bodyweight (1.3-13.9) with 1664 × 10 6 cd34+/μl (168-3779) in peripheral blood. the target cd34+ count (45 × 10 6 /kg) was achieved in 59% of donors and an adequate yield (2-5 × 10 6 /kg) in 33%. in four donors (8%), the harvest was deemed to have been unsuccessful as the cd34+ count was o2 × 10 6 /kg. the patients with donor harvest yields o2 × 10 6 /kg proceeded to transplant; all four patients engrafted and one patient had mixed chimerism at day 28 but was fully donor by day 75. median cd3+ cell count was 2.7 × 10 6 /kg bodyweight (0.8-6.4) . median days to neutrophil engraftment (40.5 × 10 9 /l) was 16 . median days to platelet engraftment (420 × 10 9 /l) was 5 (0-23) with one patient never engrafting. forty (80%) of 51 donors were contacted at a median of 24 months (1-54) post mobilisation to establish incidence of adverse effects. three donors were uncontactable as they had moved overseas. eight donors were not contacted to avoid distress as their sibling had died since transplant. among contacted donors 42.5% reported side effects including bone and lower back pain controlled with analgesia, constipation and low mood. other side effects included chest pain which was considered to be musculoskeletal in origin on day 3 of gcsf administration associated with taking an increased dose due to patient error (n = 1) and abdominal contractions like labour while receiving gcsf (n = 1). three (7.5%) reported side effects lasting beyond one month post mobilisation: lower back pain lasting 2 months (n = 1), fatigue of 3 months duration (n = 1), and cough of 8 months duration (n = 1). our data demonstrates good mobilisation using 10 μg/kg/day zarziotm biosimilar gcsf without significant adverse effects at 2 years median follow up. this supports its ongoing use for the mobilisation of healthy donors. disclosure of conflict of interest: sga has received honoraria from sandoz and grant support from sandoz and amgen. stem cell mobilization in poor mobilizers with multiple myeloma (mm) or non-hodgkin lymphoma (nhl) before and after introduction of plerixafor: single center comparative analysis using a cost-efficient single fixed-dose schedule r wäsch 1 , c greil 1 , c kiote-schmidt 1 , s hildenbeutel 1 , k kühbach 1 , r bosse 1 , j duyster 1 and m engelhardt 1 1 department of hematology, oncology and stem cell transplantation, university medical center, freiburg, germany collection of hematopoietic stem cells (hsc) from the peripheral blood (pb) is routinely conducted prior to highdose chemotherapy and autologous transplantation. despite safety and efficiency of current apheresis procedures including mobilizing chemotherapy and granulocyte colony-stimulating factor (g-csf), there is a significant rate of mobilization failures due to different patient-dependent factors necessitating additional agents like plerixafor. while plerixafor is approved for patients with mm or nhl based on prospective studies using steady state mobilization with g-csf − /+ plerixafor, prospective studies using chemo-mobilization are lacking. here we compared the outcome of poor mobilizer from the pre-plerixafor era with poor mobilizers who received additional plerixafor in a real world analysis. we analyzed 50 consecutive patients with mm or nhl who were mobilized at our academic center between 2011 and 2016 and received plerixafor, because they were expected to be poor mobilizers, due to 1. low counts of cd34+ cells in pb samples prior to apheresis, 2. after a first apheresis day with insufficient yield or 3. as a rescue strategy after insufficient harvest with previous mobilizing chemotherapy (greil c,…engelhardt m, wäsch r. leukemia & lymphoma 2017, in press). we examined cd34+ cell counts in pb and in apheresis products to identify those patients who were able to collect a sufficient cd34+ cell count for transplantation after application of plerixafor. we compared these data with 100 consecutive poor mobilizers from the pre-plerixafor era, who were mobilized between 2000 and 2011 without plerixafor. the median pb cd34+/μl count at first apheresis was significantly higher after the first dose of plerixafor when compared to the pre-plerixafor group with 19.9 vs 9.8 (p o0.001). accordingly, the median collected cd34+ cells/d (×10 6 /kg bw) and total cd34+ cells (×10 6 /kg bw) were significantly increased with 1.67 vs 0.88 (p o0.001) and 4.13 vs 2.66 (p o0.001), respectively. the rate of 42 × 10 6 cd34+ cells/kg bw in first apheresis (%) increased from 11% in the pre-plerixafor era group to 38% after the first dose of plerixafor in the plerixafor group. consistently, the successful transplantation rate increased from 58% in the preplerixafor group to 90% in the plerixafor group. successful stem cell mobilization could be achieved with only a single fixed-dose of plerixafor in 62% of poor mobilizers as previously reported by our group. the addition of plerixafor to chemomobilization in poor mobilizers with mm or nhl significantly increased pb cd34+/μl counts, apheresis yields and transplantation rates when compared to poor mobilizers from the pre-plerixafor era. these favorable apheresis results can be obtained using our cost-efficient, single fixed-dose plerixafor schedule in the majority of the patients leading to a 90% transplantation rate in poor mobilizer. disclosure of conflict of interest: rw received research funding, advisory and speaker's honoraria from sanofi-aventis. high-dose chemotherapy followed by autologous peripheral blood stem cells transplantation (pbsct) is the standard of treatment for patients with hematological malignancies. recombinant granulocyte colony-stimulating factors (g-csfs) are widely used alone or in combination with chemotherapy, in order to mobilize patient's stem cells (cd34+) for autologous and allogeneic peripheral blood stem cells transplantation. aim: the aim of our study was to compare effectiveness and safety of different biosimilar products of filgrastim used in autologous pbsc mobilization in patients with hematological malignancies. our retrospective analysis included 282 patients (118 women and 164 men) with median age 54 years ( range: ,who underwent the procedure of autologous pbsct in years 2012-2014 in the haematology, blood neoplasms, and bone marrow transplantation clinic of medical university in wrocław. there were three different biosimilar products of filgrastim used: tevagrastim (teva) in 95 patients, nivestim (hospira) in 92 patients and zarzio (sandoz) in 95 patients. 90 (32%) patients were diagnosed with plasma cell neoplasms, 145 (51%) with hodgkin's and non-hodgkin's lymphomas, 20 (7%) patients had acute myeloid leukemia and 27 (10%) had other hematological malignancies. statistical analysis was conducted using statistica 12 (statsoft polska) statistical software. for quantitative variables arithmetic means and standard deviations were calculated for the estimated parameters in the studied groups. distribution of variables was tested using w-shapiro-wilk test. p0.05). there were also small variations in the mean number of leukapheresis necessary to obtain the minimum cd34+ cell count: 1.32 in zarzio group, 1.37 in nivestim group and 1.66 in tevagrastim group. however, there were no difference between biosimilar g-csfs. the highest rate of successful mobilizations (defined as 42 × 10 6 /kg cd34+ cells collected) was observed in 88.2% patients received zarzio, in 86.2% received nivestim and in 84.9% patients received tevagrastim. the safety profile was comparable between the biosimilar g-csf and included bone pain in 30 (10%) patients and headache in 25 (9%) patients. the results are shown in table 1 . all three used biosimilar g-csfs demonstrated similar efficacy and safety in stem cell mobilization in patients with hematological malignancies. therefore, it seems that all the analyzed products can be used interchangeably. presented observations should be verified with wider prospective research. [p055] disclosure of conflict of interest: none. use of g-csf stimulation of bmt donors might prove to be beneficial in many respects, improving tnc yield but also through immunomodulatory effect on donor t cell function and apcs1. we analyzed outcomes of 14 consecutive patients receiving bone marrow transplants from hla-haploidentical related donors that received g-csf stimulation prior to harvest. fourteen patients received hla-haploidentical bmt with pt-cy between 5/2012 and 10/2016. five donors were siblings, 4 children, 4 mothers and 1 father. donors received g-csf at the dose of 10 mcg/kg bw sc. on days − 2, − 1 and 0 before bm collection. twelve patients received nonmyeloablative conditioning according to baltimore protocol2, while two patients received myeloablative conditioning (bucy). along with post-transplantation cyclophosphamide, all patients received tacrolimus and mmf form day +5, as described earlier 2. median age was 41 years (range: 19-63), 7 female and 7 male patients. eight patients had aml, 1 cml, 4 mh and one all. ten of them were in remission, while 2 mh patients were in pr, and 2 aml patients had residual disease as evident by immunophenotyping. median number of infused tnc was 5.17 × 10 8 /kg bw (range: 1.84-8.21); cd34+ cells 1.88 × 10 6 /kg bw (range: 1-4.47 ) and cd3+ cells 1.35 × 10 7 /kg bw (range: 0.37-6.04). median follow up was 362 days (range: 26-1654). eleven patients engrafted (79%), one patient had primary rejection, one had overt disease relapse at day +35 and one patient died in aplasia due to sepsis. median day to neutrophil recovery (anc 40.5 × 10 9 /l) was 21 (range: 15-29), median days to platelet recovery (plt 420 × 10 9 /l) was 31 (range: 12-45). in all patients mmf was discontinued at d +35. two patients developed acute gvhd in our cohort (18%), one after receiving dli for falling chimerism at day +169. one patient (9%) developed chronic gvhd, after having received dli due to disease progression. at the time of analysis 10 patients are evaluable; 4 patients had disease relapse/progression (40%), 6 patients are alive and in remission. one patient died due to sepsis in aplasia (accounting for 7% non-relapse mortality). one patient that rejected the graft was transplanted again from the same donor, using myeloablative conditioning and peripheral stem cells as graft source and engrafted. overall survival median is 2.7 years, with significantly shorter survival if patient was not in complete remission at time of transplant (p o0.01). even though the experience with g-csf mobilized bm graft in the hlahaploidentical setting with pt-cy is relatively small, in our series it has been beneficial in terms of tnc yield. also, the incidence of acute and chronic gvhd in our patients has been low, particularly agvhd with one case developing only after dli. whether the observation is the result of limited number of patients, or it reflects the immunomodulatory effect of g-csf on bm graft as previously suggested1 remains to be seen as further studies are warranted. autologous transplantation of haematopoietic stem cells (ahsct) is usually perceived as a fully standardized and safe procedure; however, a minority of patients experience a delayed engraftment and seldom even an engraftment failure, possibly related to a poor quality of the graft. therefore the current policy in many centers is aimed to increase the target dose of collected cd34+ cells up to an ‛optimal' level of 4 × 10 6 /kg. plerixafor was introduced in the clinical practice to maximize the mobilization of hsc, in order to collect an optimal number of cd34+ cells in a limited number of collections also in poor and slow mobilisers. we carried out a retrospective analysis of our case series aimed to individuate mobilization predictors optimize the ‛on demand' use of plerixafor. we analyzed 162 patients who underwent mobilization with cyclophophamide (4 g/sqm) and filgrastim 10 mcg/kg from +5 in our unit from 2009 and 2016. diagnosis were multiple myeloma (mm) 74 (45.7%), non-hodgkin lymphoma (nhl) 46 (28.4%), hodgkin lymphoma (hdg) 14 (8.6%) and 28 (17.3%) autoimmune disease (ms 14.8%; ssc 2.5%). median age (range) was 53 years ; male/female ratio 82/80. circulating cd34+ cell count was started at white blood cells (wbc) recovery, which was defined as the first day when their count exceeded 1 × 10 9 /l. the primary goal was to identify at wbc recovery one or more factors predicting a suboptimal mobilization, which was defined as the failure to exceed 40 cd34+/mcl circulating cells in the day after the wbc recovery. patients were excluded from this analysis if 1) showed a cd34+ count 440/mcl at wbc recovery (very good mobilizers) and/or 2) had received plerixafor and/or 3) did not proceed to another cd34+ count the day after wbc recovery. binary logistic regression was used to obtain the factors that increased the odds for an optimal mobilization. overall 80 out 162 (49.4%) patients were shown as very good mobilisers as their cd34+ count exceeded 40/mcl at wbc recovery. on the remaining 82, 7 were excluded for the lack of a second assessment and 2 for the lack of data. among the remaining 73 patients, the threshold of 40 cd34+/mcl cells on the second day was reached by 55 (75.3%) of patients (group a) while the remaining 18 (24.7%) failed the goal (group b). median (range) wbc × 10 9 /l and cd34+/mcl counts in group a and b at wbc recovery were 2.01 (1-6.43) and14.65 (0.70-39.91) and 2.84 (1-20) and 7.39 (0-33), respectively, with a statistically significant differences among group (mann-whitney u test with p = 0.01 and p = 0.02, respectively). wbc (or = 2.193; 95% ci: 1.197-4.019) and cd34+/mcl (or = 0.858; 95% ci: 0.77-0.955) in first day count, but not gender, disease category and time from mobilization chemotherapy to first cd34+ count, were predictors of optimal mobilization. combining these two predictors we found that wbc/cd34+ ratio has a sensitivity of 82.4% with an auc 83.7 in roc analysis. assessment of circulating wbc, cd34+ and their ratio at wbc recovery in a chemo-based mobilization strategy can predict sub-optimal mobilization of hsc and support the decision of adding plerixafor. these data will be prospectively validated in a broader set of patients. disclosure of conflict of interest: none. human platelet lysate (hpl) is rich in growth factors (gf) and nutritive elements and represents a powerful xeno-free alternative to fetal bovine serum (fbs) notably for mesenchymal stem cell (hmsc) proliferation. however, there is a large variability in hpl preparations (various sources, use of different and non-standardized production protocols, with variable and limited number of donors), resulting in discrepancies in product quality, low management of product safety and poor batch-to-batch standardization. we describe here the development and the characterization of a standardized hpl prepared from outdated transfusional grade screened normal human donor platelet concentrates (pcs), manufactured on an industrial scale (batch size of 250 donors) and following a highly qualified process (clean room, trained operators, validated aseptic filtration). pcs were frozen at − 80°c and thawed at +4°c to lyse platelets. cell debris were removed by centrifugation and the supernatant (hpl) was recovered. clinical grade 10l batches of aseptic filtered hpl were characterized. first, we showed that hpl prepared from a limited number of donors displayed a variability in terms of gf contents. on the contrary, we observed a robust standardization between industrial batches of hpl (250 donors) in terms of gf contents (bfgf, egf, vegf, pdgf-ab, tgf-beta1 and igf-1), biochemical analyses (total proteins, albumin, fibrinogen, vitamins and iron) and efficacy on bone marrow (bm)-hmsc proliferation. secondly, we compared expansion and functional characteristics of bm-hmscs grown in clinical grade hpl vs msc-screened fbs batches. we showed a reproducible increase in cell growth kinetics using hpl, a maintenance of bm-hmsc clonogenic potential and membrane marker expression (with however a strong overexpression of cd90). we observed a similar adipogenic and osteogenic differentiation potential and finally that immunosuppressive properties of bm-hmscs (inhibition of t-cell proliferation) cultivated in parallel in both conditions also remained identical. finally, we demonstrated the stability over time of hpl stored at − 80°c and − 20°c. in conclusion, we demonstrated the feasibility to use a standardized, characterized, efficient and clinical grade hpl for research and cell therapy applications. disclosure of conflict of interest: sv, se, lc, pb, tb, al, fg and bd are employees of macopharma. previously published p061 alpha/beta t cell depleted donor lymphocyte infusion m karakukcu, e ünal, l kaynar, s özcan, g tezcan karasu and mb acar the main objective of this project is to improve a safe and efficient new donor lymphocyte infusion (dli) with depletion of αβ+ t cells which cause graft versus host disease (gvhd), and enrichment of anti-leukemic γδ+ t cells, nk cells and dendritic cells to build an effective and permanent anti-tumor effects for patients relapsed hematological cancers after allogeneic hematopoietic stem cell (hsc) transplantation who have blasts and mixed chimerism. this study is conducted with collaboration of erciyes university pediatric and adult hsct units, and bahcesehir university, medical park hospital pediatric hsct unit. the tcr αβ+ t cell depleted dli product that is used in the study was collected and separated at erciyes university apheresis unit. the cell contents obtained for tcr αβ+ t cell depleted dli used for patients were cd3 cells were reduced to 1.4-23.5 × 10 6 cells/kg, γδ+ t cells were reduced to 2.58-23.48 × 10 6 cells/kg, αβ+ t cells were reduced by 99.99%, and were obtained at 5-4100 cells/kg. a total of 10 patients (4 female, 6 male) were included in the study, consisting of an adult and 9 children. nine patients had hematological malignancies. five patients were referred for all, three for aml, one for mds and one for griscelli syndrome. efficiency: the clinical response to the αβ+ t cell depleted dli treatment was achieved in 7/10 patients (70%). in these patients, although the increase of chimerism was limited in 2 patients, no recurrence was occurred. one of the two patients who previously responded to the treatment but experience of decreasing chimerism had relapsed after 2 months, and 9 months later. one of these two patients died after relapse. the other was managed by the second transplant. the most important objective of this study was to show that αβ + t cell depleted dli treatment is reliable. none of the patient showed severe gvhd except one patient with mild grade ii gvhd. despite the presence of severe gvhd after hsct in two patients, reactivation for gvhd was not observed after treatment with αβ+ t cell depleted dli. none of the patients had a bone marrow aplasia. as a result, αβ + t cell depleted dli treatment seems to be highly safe, and effective in selected patients. disclosure of conflict of interest: none. hematopoetic stem cell transplantation (hsct) is associated with several potentially lethal complications; for example, relapse of the malignant disease, graft rejection, infectious complications and graft versus host disease (gvhd). higher levels of cd3+ cells in the graft have clearly been associated with increased risk of gvhd, but also superior gvl effect and less infectious complications. to tackle post-transplant complications such as graft failure and relapse, donor lymphocyte infusion (dli) have successfully been used for decades but with an associated risk of gvhd. to decrease the risk of gvhd but still use facilitating cells in the cell product we performed αβ depletion of grafts for use as stem cell booster after allogeneic hsct to treat infections or poor immune reconstitution. in this study, 11 patients were infused post-hsct with αβ t-cell depleted grafts. the indication for infusion of αβ t-cell depleted graft in all patients was poor immune reconstitution with associated infectious complications. for all 11 patients, the original hsct donor was used for the αβ t-cell depleted boost. to characterize the αβ-depleted stem cell grafts, samples were stained for various cellular subsets and analyzed by flow cytometry. we could show a median log depletion of αβ cells of 4.1 and a median yield of γδ t-cells (%) of 61. 4 . the median cd34+ cell dose (×10 6 /kg) was 5.5. all 11 patients were alive 3 months after infusion. after 1 year only one patient succumbed. despite that the majority of patients suffered from agvhd grade 2 or 3 before infusion of αβ t-cell depleted graft none showed increased symptoms afterwards. in more than 70 % of the patients there was a increase in granulocytes, thrombocytes and white blood cells 3 months after infusion. in conclusion, we describe the use of αβ t-cell depleted grafts as stem cell booster in 11 patients suffering infectious complications due to graft failure after hsct with encouraging results. disclosure of conflict of interest: none. delayed engraftment or graft failure still remains a concern in bone marrow transplantation (bmt). graft composition may predict engraftment after infusion. this study aims to determine which quality control parameters used for the characterization of bone marrow grafts are the most predictive in order to minimize the risk of engraftment delay or graft failure. we conducted a multicenter retrospective study in pediatric patients who underwent first allogenic bmt at two centers in barcelona (catalonia, spain) between 2011 and 2015. quantitative variables considered for the study were: total nucleated cells (tnc), mononucleated cells (mnc), cd34 + cells, cd3+ cells and granulocyte-monocyte (gm) colonies enumeration. qualitative variables considered for the study were viability assessed by flow cytometry and clonogenic efficiency of the cd34+ cells (clonegm) which is the ratio between gm colonies and cd34+ cells. 85 patients were included (median age (range) were 7 years old (0) (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) ). the median tnc(range) was 4.26e8/kg (0.51-29.18e8/kg) and 1.1e8/kg (0.48-5.88e8/kg) for mononuclear cells (mnc). on the other hand, the median (range) cd34+ cell dose was 5.03e6/kg (0.85-24.91e6/kg) and t-cell dose (cd3+) was 0.41e8/kg (0.10-44.86e8/kg). the median (range) colonyforming unit granulocyte macrophage (gm/kg) dose was 4.35e5/kg (0.33-48.62e5/kg). the median (range) of cd34+ cell viability, was 95% (63-99%) and the median(range) of the clonogenic potential of cd34+ cells (clonegm) was 10.35% (1.22-76.17%). the median (range) of engraftment was 22 days for neutrophils and 20(11-38) days for platelets. 1 patient was considered as primary graft failure. in the univariate analysis, cd34+ (p = .049) and mnc (p = .045) cell dose predicted a faster neutrophil engraftment and female donor a slower neutrophil and platelet engraftment (p = .012 and p = .040). cell viability also correlated to a better platelet engraftment (p = .030). in the multivariate analysis we observed a trend for a faster neutrophil recovery according cd34+ cells infused. again, female donor was associated with slower engraftment. in order to establish a safety threshold, we did a quartile analysis of cd34 dose and found 3.18e6/kg (quartile 25) discriminates a faster neutrophil engraftment [median 25 days vs 21 days for those with higher cd34+ cells (p = .026)]. in conclusion, we found an association between mnc and cd34+ cell dose and time to engraft, and established a safety threshold of 3.18e6 cd34+/kg. also, bm grafts from female donors were associated with slower engraftment. no other qualitative parameters were predictive of engraftment. disclosure of conflict of interest: none. plasma cell myeloma (pcm) is currently treated with chemotherapy and autologous stem cell transplantation (asct) but relapse rates remain high. adoptive transfer of mature haploidentical natural killer (nk) cells is a promising approach to provide pcm patients with highly immunocompetent effector cells with anti-myeloma function early post transplantation. here we report on the current clinical phase i/ii trial of multiple preemptive infusions of good manufacturing practice (gmp) expanded nk cells to pcm patients (clinicaltrials.gov nct01040026). ten pcm patients were recruited (seven males, three females, median age: 59y). all patients received four cycles of vtd chemotherapy (reaching cr: 4 × , vgpr: 5 × and pr: 1 × ) before high dose therapy with melphalan 200 mg/m 2 and asct. after successful stem cell mobilization and cryopreservation of patients' stem cells after the third vtd cycle, nk cells from haploidentical family donors were purified from unstimulated leukapheresis by t cell depletion and nk cell selection using clinimacs. highly pure nk cells (mean: 4.8 × 10 8 cells) were obtained with a minimal t cell contamination corresponding to a 6.1 log t cell depletion. nk cells were expanded ex vivo for 19 days in gmp-medium containing autologous irradiated feeder and interleukin-2 and -15. nk cell numbers increased 54-fold (range: 38-76-fold). in three nk cell products t cell contents were 11 × 10 5 cells/kg body weight (bw: 10 × above limit of clinical trial) and were successfully reduced by 2°cd3-depletion to 0.3 × 10 5 cells/kg bw. nk cell products were cryopreserved in escalating doses (1.3 × 10 6 , 1.3 × 10 7 and rest as multiple doses of maximal 1.0 × 10 8 cells/ kg bw). the pcm patients received 65-460 × 10 8 expanded nk cells (median: 3.8 × 10 8 cells/kg bw, range: 0.9-5.7 × 10 8 cells/ kg bw) as 3-8 infusions (median. 6 dlis). the nk-dlis were administered between day 2 and 21 after asct and were well tolerated without any acute adverse events. no signs of acute or chronic graft-versus-host disease were observed in any of the patients after a total of 57 nk-dlis. engraftment occurred between days 13-24 (median: 16 days). infused donor nk cells were monitored by short-tandem repeats pcr. donor nk cells were detected in peripheral blood one and 20 h post infusion (% donor nk of enriched blood nk cells: mean: 30%, range: 9-90%, and mean: 17%, range: 0-33%, respectively) indicating significant nk cell survival in recipients in the absence of il-2 support in vivo. clinical responses at last follow-up compared to a retrospective cohort of matched control patients will be presented. these results demonstrate the feasibility of large-scale gmp expansion and safety and immunotherapy with third-party leukemia-specific t cells (leuk-sts) represents an attractive approach for acute leukemia (al) patients lacking a fully matched donor or relapsing after allogeneic hematopoietic cell transplantation (hct). its application however, is limited by the demand for high numbers of antigen presenting cells (apcs), capable to produce clinically relevant numbers of leuk-sts. low volume, non-transplantable cord blood units (cbus) could theoretically serve as an easily accessible source to generate high numbers of dendritic cells (dcs) and subsequently leuk-sts, providing also the advantage of reduced alloreactivity, even in cases of partial matching. our goal was to generate clinically relevant doses of leuk-sts targeting al-related antigens, the wilms tumor protein (wt1) and the preferentially expressed antigen in melanoma (prame), through the exploitation of non-transplantable cbus. to generate dcs, immunomagnetically enriched cd34+ cells from cbus ⩽ 70ml were cultured in g-rex devices in the presence of scf, gm-csf and il-4. dcs matured by toll-like receptor ligand 3 and 7/8 were immunophenotypically characterized by flow cytometry (fcm). secreted cytokines were measured with elisa. matured dcs were activated with a peptide-mix of wt1 and prame and used as apcs to repeatedly stimulate naive t-cells (derived from the cd34fraction of the same cbu). the phenotype and the specificity of generated leuk-sts were determined by fcm and ifn-γ/ elispot, respectively. starting from mean 4.2 × 10 5 ± 1.1 × 10 5 cd34+ cells, from 4 non-usable cbus, we generated 3.3 × 10 9 (range:1.9-5.7 × 10 9 ) myeloid dcs (cd33+/cd11c+:76.8 ± 5.5%) in 35 days (fold change~11.000). the produced cells highly expressed maturation markers (cd11+/cd40+:79 ± 12%; cd11c+/hla-dr+:78 ± 10%) and secreted high levels of th1cytokines (ιl-12:224 ± 185 pg/ml; il-6:1.9 ± 0.1 × 10 5 pg/ml, tnf-α:5268 ± 1316 pg/ml) and low levels of the th2-cytokine, il-10. the average number of cd34-cell-derived leuk-sts after 4 week-culture was 7.5 ± 3.4 × 10 7 (~2 logs above clinical doses). the produced cells were enriched in cd3+ polyclonal cells (80 ± 7%), comprising of cd4+ (28 ± 10%) and predominantly cd8+ cells (52 ± 17%), expressing effector memory (cd45ra − /cd62l − :52.8 ± 5%) and effector memory ra markers (temra: cd45ra+/cd62l − :46 ± 4%), while containing insignificant numbers of cd4+/cd25+cells (1 ± 0.5%). specificity was seen after the second stimulation at the earliest and was increasing after each stimulation [mean spot forming cells (sfc)/2 × 10 5 cells at second, third, fourth stimulation: 106 ± 33; 422 ± 111; 1335 ± 314; respectively]. in particular, produced cells were highly specific for both targeted antigens (prame:1019 ± 275, wt1:316 ± 55), while they expressed low the programmed cell death protein-1 (cd3+/pd-1+:9 ± 4%), implicating absence of cell exhaustion after repeated stimulations. we report a paradigm of ‛circular economy' in science, by the exploitation of non-usable cbus, towards scalable generation of cb-cd34+-cell-derived dcs and cb-cd34-cellderived leuk-sts from the same cbu and establishment of leuk-sts banks. whether similarly produced leuk-sts could significantly advance the treatment of al or leukemic relapse after hct, will be ultimately determined in vivo. disclosure of conflict of interest: none. comparison of two different methods to generate antifungal-specific t-cells under pre-clinical-scale conditions r geyeregger 1 , s tischer 2, 3 invasive infections with aspergillus fumigatus constitute a major cause of morbidity and mortality in immunocompromised patients after haematopoietic stem cell transplantation. although adoptive immunotherapies against viral pathogens are already in phase i/ii trials, clinical-grade methods for the generation of aspergillus-specific t-cells (asp-t-cells) from healthy transplant donors or even related or unrelated thirdparty donors are still under development. in this study, two different strategies interferon-gamma (ifn-g) cytokine capture system (ccs) vs short-term in vitro expansion (ste) were performed from the same healthy volunteers in order to evaluate the most suitable approaches for the in-time generation of clinical applicable asp-t-cells. pbmcs from leukapheresis of healthy donors (n = 6) were first prepared in hannover for the ifn-g-ccs and then sent to vienna to prepare the ste. all donors belong to the allocell registry (www.allocell.com) of hannover medical school and the frequency of asp-t-cells was pretested by high-throughput ifn-g elispot assay. for the ifn-g-ccs, 1 × 10 7 cells were stimulated for 16 h with gmp-conform aspergillus lysate followed by magnetic selection of ifn-g-producing t cells. cells were characterized for phenotype and function by multicolour flow cytometry. for the ste, 20 × 10 7 cells were cultured in g-rex devices and stimulated for 12 days with either the aspergillus lysate alone or with pooled overlapping pepmixes (catb, crf1, f22, gel1, pmp20, shmt and sot) and il-15. to further characterize the final cell products, multicolour flow cytometry, ifn-g elispot and ifn-g/granzyme b flurospot analyses were performed. ifn-g-ccs: frequency of ifn-g positive asp-t-cells pre-magnetic enrichment ranged between 0.07 and 0.16%. recently we defined t-cell donors as eligible if ⩾ 0.03% specific ifn-g+ t cells are detectable. the purity of asp-t-cells among cd3+ cells, obtained from three donors after magnetic selection was in mean 64% ± 3 (range: 58-69%). the absolute number of selected ifn-g+ cd3+ t-cells was 706 ± 194. this could be approximately multiplied by a factor of 100, if 41 × 10 9 pbmcs are used for the generation of clinically applicable t cells using the ccs and the prodigy device. ste: after 12 days, asp-t-cells (n = 3) showed highly specific activity against the lysate (in mean 1339 ± 79 spot forming colonies (sfc)/105 cells) and pooled pepmixes (in mean 892 ± 276 sfc/105 cells). in both methods (lysate vs pooled pepmixes), predominantly cd4+ t-cells were expanded (84% ± 2.3 vs 82% ± 5.3 of cd3+) compared to cd8+ t-cells (12.6% ± 2,9 vs 14.7% ± 5.3). interestingly, whereas after ste, cd4+ t-cells include mainly central memory t-cells (mean 40%; cd62l+cd45ra − ), cd8+ t-cells include mainly effector memory t-cells (27%; cd62l − cd45ra − ). generated t cells were highly functional and cytotoxic as determined by the secretion of effector molecules granzyme b and ifn-g. based on the purity of up to 69% after the ifn-g-ccs and the high number of sfc received after ste with lysate and pepmixes, both methods seem to be suitable for clinicalscale productions. for patients who are in need for high asp-tcell numbers the application of first in-time ccs-purified asp-t-cells followed by the administration of ste cells might be a promising way to boost antigen-specific t-cell response. disclosure of conflict of interest: none. complete computerization of cell therapy product files (‛zero paper') in the qap 10 software o christéle 1 , r catherine 2 , r aline 3 , k mathias 4 , m lavinia 3 , d vincent 5 , m jean-pierre 6 and l marie-noelle 7, 8 1 hematologie clinique et thérapie cellulaire-chu amiens picardie, 2 simedia-ver, 3 hématologie clinique et thérapie cellulaire-chu amiens picardie, 4 simédia-ver, 5 direction système informatique-chu amiens picardie, 6 hématologie clinique et thérapie cellulaire, 7 lacassagne and 8 the computerized management of cell therapy products (ctp) is an obligation for processing laboratories to meet regulatory requirements. the software used is often independent of institutional systems in view of the specificity of cellular therapies and do not always allow the implementation of the ‛zero paper' policies that are being put in place. we report here our experience with the qap10 software (quality assurance partner) developed by the company simédia (www. qap10.com) in open source (mit license) allowing the management of fully computerized ctp files. the qap10 software has been developed to ensure the traceability of ctp for both preparation and quality control by combining the product preparation environment (personnel, premises, reagents, consumables, equipment). initially, with the help of the company simédia, we parameterized the software in accordance with our procedures for the preparation and quality control of ctp. we built a file that we printed out for archiving on paper. it soon seemed necessary to reverse this mode of operation to add to the software the documents papers to obtain a file completely computerized and to avoid paper archiving. the close collaboration between the cell therapy laboratory staff, the software referent within the information system department of the amiens hospital and the company simédia enabled: set up a document backup server sufficiently proportionate in memory. have simedia carry out the necessary developments so that all documents can be integrated into the software, set up a coherent working circuit, organize the registration of documents, put in place a rigorous verification of the mandatory elements of the file. the reflection on the computer file made it possible to evolve the software to widen its use to all documents of management of the laboratory: maintenance of equipment, control of premises, housekeeping, staff training, quality control of automatons, reagents and consumables, process, reception, distribution. rigorous formalization was mandatory to ensure that the record was organized in a uniform manner. an intermediate paper record is still necessary for a period of about 1 month: from the programming of the graft to the final validity of the injected product. this folder consists only of transient elements that cannot be integrated into the qap 10 software immediately. the transition from the paper file to a computer file took place in several stages, calling into question our functioning. the difficulties of this implementation are of several natures: the heterogeneity of the documents components a cell therapy product file, the impossibility of benefiting from an interface between all computer software used on the hospital, the psychological barrier prompting us to keep a paper copy, work habits, the guarantee of computer backup quality as well as its verification. but the complete computerization of the ctp file has the following advantages: easy and secure accessibility of information, resolution problems archiving paper files, a single backup media folder. disclosure of conflict of interest: none. conditioned media from allogenic mesenchymal stem cell culture (msc-cm) enhances wound healing in an allogenic 3d skin model moyasasr al-shaibani, x wang 1 , p lovat, a tulah and a dickinson 1 newcastle university migration of the epidermal layer towards the wound centre is an important step in the healing process. full thickness in vitro skin models can be used to investigate epidermal migration towards an injury site. since wound healing therapies often require allogenic transplantation of primary keratinocytes, an allogenic 3d skin model was developed to investigate epidermal migration. the effect of mesenchymal stem cell conditioned media (msc-cm) was assessed for wound healing using this in vitro human 3d skin model. human mscs were derived from human hip joints, and characterised using standard protocols. at 80% confluence, msc secretions were collected in serum free medium and referred to as msc-cm which were then analysed for protein content using elisa. fully humanised allogenic 3d skin models were developed (n = 3) and a 3 mm punch was induced into each model followed by daily treatment with msc-cm to investigate the migration of the epidermal layer towards the punch centre over the dermal layer at different time points (1 week, 2 weeks, and 4 weeks). intact and wounded models were characterised structurally by haematoxylin/eosin (h&e) staining and immunofluorescence (if) was used to validate the dermal and epidermal biomarkers such as collagen 3 (col3), cytokeratin 14 (k14), keratin 10 (k10), loricrin and involucrin. mscs were characterised as stipulated by the international society for cell therapy, that is, fibroblast like cells with the ability to differentiate into tri-lineages (adipocyte, chondroblast and osteoblast). phenotypically, over 95% of the cells were able to express phenotypic markers for variant stem cells such as cd73, cd90 and cd105. over 95% of the cells were negative for the expression of cd14, cd19, cd34, cd45 and hla-dr (p = 0.025). msc-cm contained different concentrations of a variety of growth factors such as keratinocyte growth factor (kgf), hepatocyte growth factor (hgf), platelet-derived growth factor (pdgf), stromal-derived factor-1 (sdf-1) and macrophage stimulating protein-1 (msp-1). h&e staining showed that the models had distinct dermal and epidermal layers similar to that of real skin. additionally, if showed that the models expressed dermal and epidermal biomarkers, for example, col3, k14, k10, loricrin and involucrin. after treatment with msc-cm, the epidermal multilayers of the punched models started to migrate towards the punch centre and covered the whole punched area after 4 weeks of treatment with recovered expression of the epidermal biomarkers, for example, k14, k10, loricrin and involucrin. a fully humanised allogenic 3d skin model is a useful tool to mimic the in vivo environment and evaluate the wound healing process. it could also be used as a screening method to test candidate wound healing drugs. allogenic keratinocytes could be used as a cellular sheet to cover the wound area with the ability to migrate towards the wound centre and promote wound healing. a possible explanation for promoting epidermal migration at the injury site is that msc-cm contains cytokines which accelerate cell migration such as kfg, sdf-1 and msp-1, in addition to other cytokines which promote both migration and proliferation of epidermal cells, for example, hgf and pdgf. disclosure of conflict of interest: none. before each freezing and after each thawing, a quality control is performed including a minima: (i) cd34+ quantification; (ii) estimation of the percentage of hsc cd34+ viability, via 7aminoactinomycin-d (7-aad) staining and (iii) evaluation of hsc functional ability to form colony ‛cfu-gm' (colony forming unit-granulocyte macrophage). apoptosis, or programmed cell death, involves complex pathways in part the path fas-fas ligand (fasl), mitochondrial components and caspase enzymes. the involvement of apoptosis dependent on caspases activation pathway in hsc cd34+ after thawing remains unknown. here, we assess the extent of apoptosis caspase-dependent before and after cryoconservation of hsc cd34+, using a fluorescent labeled inhibitor of caspases ‛flica. ' we tested the induction of apoptosis caspasedependent, before and after hsc cd34+ cryoconservation from patients with different hematological malignances: multiple myeloma (n = 21), lymphoma (n = 19). caspases pathway activation status was evaluated by flow cytometry, using a fluorescent labelled inhibitor of caspases ‛flica' staining test, in hsc cd34+, lymphocytes cd3+, monocytes cd14+ and natural killer cells cd56+. in order to assess cell viability, cells were stained in parallel with 7-aad. we determined positive cells %, that is, showing caspase activation in viable cells (flica+ cells), before and after cryoconservation. caspase pathway activation level was then correlated with hsc functional ability to form colony ‛cfu-gm,' and day's number of clinical aplasia. in our cohort, we showed a significant caspases pathway activation, with 18.9% cd34+ flica+ cells after thawing, compared with the 2.4% described in fresh cd34+ cells (p o0.0001). moreover, caspases pathway was significantly activated in thawing cd3+, cd56+ and cd14 + cells: flica+ cells % in thawing cells were, respectively, 16.8%, 31.1% and 6.2% vs 3%, 9.7% and o1% in fresh cells. we also report a significant increase of apoptosis caspasedependent in lymphoma patients (22.6% of cd34+ flica+) in comparison to myeloma patients studied (18.6% of cd34+ flica+) (po 0.0001). in contrast, no correlation has been established between observed caspases pathway activation and hsc cd34+ capacity to form cfu-gm, or still day's number of clinical aplasia. our results show substantial cell death, induced by the increase in caspases pathway activation, secondary to the thawing process, and across all study cell types. this advance of apoptosis caspase-dependent may affect the immune response quality during recipient aplasia, without detecting a clinical impact. moreover, caspases pathway activation through cd3+ and cd56+ subpopulations could modify the therapeutic result of donor lymphocytes infusion dli, though yet untested. thawing process in autologous graft induces apoptosis caspase-dependent in all apheresis product cells, particularly in hsc cd34+, without clinical impact in graft fate. disclosure of conflict of interest: none. donor-derived nk cell infusion combined with hla halpoidentcial blood stem cell transplantation to decrease leukemia relapse for high risk acute myeloid leukemia patients b wu, y huang, j xu, y he, jxm zhang*, z wu* hematology department, zhujiang hospital of southern medical university, guangzhou, china 510280 *shenzheng hank biologoical engineering co.ltd. hla halpoidentcial blood stem cell transplantation have solved the donor deficiency for patient who need to treat by transplantation. the high relapse of leukemia especially for high risk patient post transplantation affect the outcome of haploidentical stem cell transplantation. natural killer (nk) cells are part of the innate immune system and play a scavenger role to detect targets marked by ‛missing self' induced by viral infection or malignant transformation. infusion nk cells into receipt prior to stem cell transplantation could decrease the gvhd in mouse bone marrow transplantation model. in an effort to decrease the leukemia relapse and gvhd after halpoidentical stem cell transplantation for high risk acute myeloid leukemia patients, we evaluated the addition of donor-derived nk killer cells before halpoidentical stem cell transplantation in high risk acute myeloid leukemia patient. here we report interim results for five patients enrolled last year. five high risk acute patients received halpoidentcial stem cell transplantation combined with donor-derived nk cells infusion. all patients received an fbca conditioning regimen, which consisted offludarabine (25 mg/m 2 /day, intravenous) on days − 9 to − 5, busulfan (3.2 mg/kg/day, intravenous) on days − 8 to − 5, cyclophosphamide(60 mg/kg/day, intravenous) on days − 3 to − 2 and rabbit antilymphocyte globulin (atg 2.5 mg/kg/day, intravenous) on days − 5 to − 1. donor-derived nk cells were infused into patient prior to stem cell transplantation. gvhd prophylaxis was a combination of cyclosporine a (csa) and short term methotrexate. five high risk patients (2 patients with aml m5 cr2, 1 patient with aml m5 nr, 1 patient with aml m0 cr2 and 1 patient with aml m2 cr2) enrolled from jan 2015 to nov. 2015; the donors are parents and sibling. hla were mismatched between donor and patients. median cd34+ dose infused was 5.06/kg (range: 2.3-106/kg) and the nk cell dose infused was 1 × 10 8 /kg (0.8-1.2 × 10 8 /kg). all five patients got hematology recovery and achieved hematology cr. only one patient occurred grade ii agvhd post transplantation and controlled by methylprednisolone. at a median time of 12 months (range: 9-16 months) post peripheral blood stem cell transplantation, the incidence of acute gvhd grade ii is 20% (1/5) . no chronic gvhd observed. four patients are still cr and survival with event free survival with median 1 year follow up. one patient with aml m5 who had not achieved remission before transplant relapsed after 6 months and got cr with second nk infusion and still survival. nk infusion prior to transplantation was found to be safe and feasible. there was no increase acute gvhd or chronic gvhd risk. there was a trend towards increased 1-year survival for high risk leukemia patient. the potential benefit on overall survival remains to be further evaluated with additional patient enrollment and longer follow up. however, given the favorable safety profile of nk cells, future strategies to enhance efficacy such as repeat dosing or modification of nk cells are worth potential exploration. disclosure of conflict of interest: none. donor lymphocyte infusion (dli) is a therapeutic option in the treatment or prevention of relapse after allogeneic stem cell transplantation (allohsct).of note, the risk of graft-versus-host disease (gvhd) associated with the graft-versus-tumor (gvt) effect may be influenced by the level of hla disparity between donor and recipient. data on use of dli after unmanipulated haploidentical hsct (haplohsct) with post-transplant cyclophosphamide (pt-cy) are still currently limited. we report 7 patients (pts) receiving dli between 2014 and 2016 for prevention or treatment of relapse after haplohsct. seven pts were given 11 haplodli doses, as treatment for relapsed disease (n = 4) or as preventive therapy of relapse for high risk disease (n = 3). four pts had acute myeloid leukemia (aml), 1 had acute lymphoblastic leukemia and 2 lymphomas -1 hodgkin (hl) and 1 non-hodgkin dlbcl. 1 pt had intermediate risk disease features, 1 adverse risk and 5 pts had refractory disease at time of haplohsct. 4 pts had a previous hsct (2 allogeneic and 2 autologous). 6 of the 7 pts received a ric regimen and the source of stem cells was peripheral blood s153 (n = 5) and bone marrow (n = 2). gvhd prophylaxis was cyclosporine and mycophenolate mofetil (mmf), atg and pt-cy. median follow-up after haplohsct was 27 (range: months. median time to neutrophil and platelet (450g/l) recovery were 16 and 24 days, respectively. after haplohsct, 3 pts developed acute gvhd (agvhd) of grade i (n = 1) or ii (n = 2), at a median of 26 days after haplohsct. the median time from haplohsct to first dli was 204 days (range: 71-624). all pts had full donor chimerism at time of dli. before dli 3 pts relapsed at a median time of 149 days (range:86-177), of whom 2 pts had aml and received salvage chemotherapy and 1 pt with hl being treated by dli alone. of the 3 relapsed pts,1 showed progressive disease after first dli dose and 2 achieved a sustained cr (with duration of cr of 6 and 9 months at last follow-up). the remaining 4 pts were given dli in cr, in 1 case (of aml) associated with azacytidine. 5 pts received 1 dli dose and 2 pts were given 3 dli injections with escalating doses. the first dose of dli was 1 × 10 6 cd3/kg in 4 pts, 5 × 10 5 in 1 pt and 1 × 10 5 in 2 pts. the 2 pts who received 3 dli doses (lymphomas) were given: (1) 5 × 10 5 -1 × 10 6 -5 × 10 6 ; (2) 1 × 10 5 for 2 doses followed by 1 dose of 5 × 10 5 . four pts developed chronic gvhd (cgvhd, 57%) in a median time of 23 days (range:11-42) after dli (3 of them had presented previously agvhd grade i-ii). cgvhd was limited in 1 case, moderate in 1 pt and severe in 2 pts. 3 of these pts presented features of an overlap syndrome (acute/chronic gvhd) with signs of agvhd de grade i,ii and iii in 1 pt each. involved organs were skin/mucosal (n = 4), liver (n = 3), gastrointestinal tract (n = 2), lung (n = 1) and joints (n = 1). all patients experiencing gvhd after dli were treated by systemic corticotherapy, extracorporeal photopheresis and cyclosporine or weekly low dose methotrexate. median follow-up after first dli was 10 months (range: . none of the 4 pts receiving prophylactic dli relapsed during the follow-up period. 2 pts died,1 of relapse and 1 of severe cgvhd. 5 pts were in cr at last follow-up,3 with no signs of gvhd and 2 with limited cgvhd. despite the limited cohort, dli after haplohsct appears to be a therapeutic option in high risk pts allowing enhancement of gvt in the setting of haplohsct with post-cy infusion. disclosure of conflict of interest: none. previously published p075 early and sequential ctla4ig primed donor lymphocyte infusions (dli) following post-transplantation cyclophosphamide (ptcy)-based haploidentical pbsc transplantation for advanced hematological malignancies promote proliferation of mature natural killer (nk) cells with cytotoxic potential and markedly reduces relapse-risk without increase in gvhd sr jaiswal, s zaman, p bhakuni, s bansal, s deb, s bhargava and s chakrbarti 1 we have earlier shown that cd56 enriched cell infusion following ptcy resulted in rapid proliferation of mature nk cells with attenuation of gvhd and early use of prophylactic g-csf mobilized dli resulted in improved disease-free survival. ctla4ig has been shown to be effective in attenuating t cell activation and induce transplantation tolerance in preclinical models. it has recently been employed to induce transplantation tolerance and reduce early alloreactivity in patients with nonmalignant disorders undergoing ptcy-based haploidentical hsct. nk cells on the other hand are resistant to ctla4ig and in fact might demonstrate better anti-tumour effect in presence of ctla4ig as cd86 is a putative activation receptor. to explore this phenomenon, we employed sequential ctla4ig primed dli following ptcy-based haploidentical hsct in patients with relapsed/refractory hematological malignancies.15 patients (12-60 years; aml-6, all-5, nhl-4) received abatacept (ctla4ig) as a part of gvhd prophylaxis at 10 mg/kg on day − 1 followed by pbsc and sequentially on days +6, +20 and +35 followed 12 h later by dli of 5 × 10 6 cd3 cells/kg containing 0.03-1 × 10 6 /kg cd56+ cells. ptcy was administered on days +3 and +4 with cyclosporine from day +5 to day +60 and subsequent rapid tapering. the immune reconstitution of the study group (ctla4ig-dli) was compared with the cohort of patients with both malignant and nonmalignant diseases who received abatacept but not dli (n = 12; ctla4ig group) and those receiving cd56 enriched donor cell infusion on day +7 (n = 10; nki group). results: there were no acute infusion related toxicities. all patients engrafted at a median of 15 days (12-20 days). the incidences of acute and chronic gvhd (all mild) were 20% and 25%, respectively. three patients reactivated cmv and there was only one non-relapse mortality (6.9%). only 4 patients relapsed (27.8%) with a disease-free survival of 72.6% at 1 year. these cells had greater expression of cd107a compared to normal healthy donors. the recovery of cd56+, cd56+16+ and cd56 +16 − cells were similar in the ctla4ig-dli and nki groups at days 28, 60 and 90 post-transplant and this was significantly higher than the ctla4ig group ( figure 1 ). in contrast to ctla4ig group, nk cells recovered at day +28 with predominantly cd56dim cd16+ phenotype with significant population of cells expressing kir+nkg2a phenotype in both ctla4ig and nki groups with higher expression of cd107a. interestingly, the 4 patients who relapsed had attenuated recovery of cd56+16+ cells at 28 and 60 days(21/μl and 15 cells/μl) without cd107a expression, in contrast to the rapid and sustained recovery of this population of nk cells in those not experiencing relapse (cd56+16+ cells 181/μl and 103/μl). however, the recovery of tregs was prompt and sustained in the comparator groups, which remained low in the ctla4ig-dli group until day +90. there were no differences in the recovery of other t cell subsets between the three groups. the study demonstrates the unique ability of ctla4ig to augment nk cell proliferation, maturation and cytotoxicity and reduce relapse with attenuation of t cell activation and gvhd in the context of the early use of ctla4ig primed dli following ptcy-based haploidentical hsct without ex vivo selection or expansion. we hope this novel strategy might offer less expensive and yet a viable alternative in the field of nk cell therapy. [p075] disclosure of conflict of interest: none. enhanced cytotoxicity of γδ-cytokine induced killer cells against hematologic malignancies n bloom, s eldror, s caspi, s teihuman,h vernitsky, e jacoby, b bielorai and a toren cik cells are ex vivo expanded by scheduled addition of anti-cd3 mabs and a cytokine cocktail that contains ifn-γ, il-2 or il-15. cells represent an in vitro generated heterogeneous population consisting of different effector cells-cd3poscd56pos, cd3negcd56pos and cd3poscd56neg-t cells that mainly (495%) express α/β t-cell receptor (tcr) s154 and to a lesser extent (o 5%), tcr γδ phenotype. these nklike t cells product show a dual functional activity, retaining their original t cell specificity and nk cytotoxic capacity via marked up regulation of the nk cell receptor, nkg2d. pre and clinical studies showed that the optimal cytotoxic effect of cik cells against different malignancies (target cells) is achieved at 40:1 e:t ratio, which means high numbers of αβ t-cells that might increase the risk of gvhd. here we produced ciks from αβ tcr depleted cellular products (defined as γδcik) and tested their phenotype expression and in vitro cytotoxic activity against hematological malignancies. fresh apheresis products were processed using the clinimacs depletion reagent, according to manufacturer instructions. target product was cultured with rpmi1640 supplemented with 10% fcs and ex vivo expanded by scheduled addition of cytokine cocktail that contains ifn-γ (1000 iu/ml), anti-cd3 mabs (50 ng/ml) and 500 iu/ml il-2. the cells were cultured for 14 days. cytotoxic activity of the γδcik was evaluated against various target hematological malignant cell lines (k562, reh, jurkat, and u937). after 14 days, the αβ depleted cik cultures resulted in 97.5% γδ t-cells (41 folds expansion) compared to 1.0% of γδ t-cells immediately after depletion, and compared to only 1.6% in non-selected cik cells. the percentage of αβ t cells in γδcik cell cultures started from 0.002% (immediately after depletion) to 0.5% compared to 95.1% αβ t cells were found in non-selected cik cells cultures. γδcik cells produced robust cytotoxic activity at a 10:1 e:t ratio against reh cells (22.6 ± 5.3%), jurkat cells (51 ± 7.9%); u937 (62.5 ± 8.5%) and k562 (43.4 ± 2.0%), compared to nonmanipulated cik cell activity against the same targets (5 ± 1.0%; 8.3 ± 1.4%; 12.4 ± 6.1%; 7.3 ± 2.9%, respectively). we found higher degranulation capacity of γδcik cells compared to non-selected cik cells against reh (45.3 ± 16.1% vs 17.6 ± 3.8%), jurkat (42.3 ± 18.4% vs 6.8 ± 3.5%), u937 (37.6 ± 15.5% vs 17.1 ± 3.1%) and k562 (29.2 ± 16. centre de thérapie cellulaire, institut paoli-calmettes; 2 unité de transplantation et de thérapie cellulaire, institut paoli-calmettes; 3 centre d'immunologie de marseille-luminy and 4 laboratoire d'immunomonitoring, institut paoli-calmettes during the past 15 years, the major improvements in the field of allogeneic hematopoietic stem cell transplantation (hsct) (reduced intensity conditioning regimen, high level hla typing, alternative donors, gvhd prophylaxis…) significantly extended the feasibility of this procedure. in contrast, disease recurrence after hsct remains a main issue. thus, many post-hsct prophylactic interventions are under investigation. unmanipulated donor lymphocyte infusion (dli) remains one of the most frequently used post-hsct treatment, but its potential benefit in increasing gvl effect may be counterbalanced by the induction of gvhd. in this setting, the use of adoptive transfer of ex vivo enriched and activated nk cell infusions from the same donor (dli-nk) may induce gvl effect without causing gvhd. we therefore report on a single-center phase 1 clinical trial (nct01853358) evaluating the safety of ex vivo activated allogeneic nk cells infused between days 60 and 90 after hsct. the aim was to determine the maximum tolerated dose (mtd) of ex vivo highly purified and activated dli-nk after matched related donor hsct. the schedule plan a first phase of 3+3 dose escalation method using 3 dose levels (1.10e6/kg, 5.10e6/kg and 45.10e6/kg). grade 3-4 secondary adverse events according to nctci classification and severe gvhd occurring within 30 days after dli-nk were considered as dose-limiting toxicities (dlt). a second step allowed enrolling patients at the mtd. over a period of 3.5 years, 14 patients with various hematological malignancies (aml, all, hl, nhl, mds) were infused with activated nk cells at a median time of 91 days (range: 61-106) post-hsct. apheresis products were collected from the hsc donor, cd3-depleted and cd56-selected by immunomagnetic separation using clinimacs. selected nk cells were cultured for 7 days in medium supplemented with 10% fetal calf serum in the presence of 1000 u/ml of il-2 in air-permeable cell culture bags. after immunomagnetic separation, cd56enriched products had a median cd56+ cell purity of 94% (range: 77-100) and viability of 96% (93-99). after il-2 activation, the median cd56+ cell dose was 4.8 × 10e6/kg (1.2-21.4) , with a viability of 81% (71-94) and a residual cd3 + cell content of 0.4 × 10 4 /kg (0-1.5 × 10 4 /kg). all release criteria to be met were fulfilled for the 14 preparations infused : viability 490%, negative microbiological testing, cd56 + cell count ⩾ 1 × 10 6 /kg, and cd3 + cell content o5 × 10 4 /kg. standardized quality controls were employed at all steps of the manufacturing process, adding a level of consistency to the product testing before release. activated-nk cells were well tolerated in all 14 patients, with no occurrence of dlt. thus, mtd was not reached. two patients presented with a moderate chronic gvhd, both of them during cyclosporine a dose reduction. relapse occurred in 2 patients with aml. one patient died from idiopathic pneumoniae, without evidence of relapse, gvhd or infectious disease. with a median follow up of 30 months (1-41), 2 year os was 83% ( figure 1 ). therefore, infusion of highly purified, activated-nk cells of donor origin as a substitute to standard dli does not induce gvhd nor other side effects after hsct: the demonstration that modulation of nk cell activity can achieve disease control after hsct deserves to be investigated in larger trials. [p077] disclosure of conflict of interest: none. feasibility, safety, rapid production and efficacy of institution-produced cd19 car staff were trained on site for collection, processing and cryopreservation by regional experts. a total of 101 units were collected and processed as part of the initial validation of the project. ucb units were processed on either axp or sepax systems, and all cryopreserved in bioarchive (an automated, robotic cryopreservation system that can archive up to 3623 units). the characteristics of which as well as the post processing data are depicted in table 1 . [p079] we shared a successful story of establishing the first public cord blood bank in jordan. the first 101 units collected showed excellent sterility, viability, collection volume and total nucleated cells. a very good recovery of both nucleated and cd34+ cells were obtained using axp and sepax cell separation systems. the process of validation of equipments and methodology is complete. we anticipate moving to permeant facility of the cord blood bank in the new expansion in early 2017. we look forward for steady progress in ucb recruitments, hla typing, cryopreservation and adherence to netcord-fact standards as well as participation in international registries. functionally active ifn-gamma secreting cmv pp65 specific t cell therapy as an alternative for clinically urgent cmv related diseases n kim, y-s nam 1 , k-i im 1 , j-y lim, y-w jeon 1 , y song and s-g cho 1 the catholic university of korea, seoul cytomegalovirus (cmv) related diseases are a serious cause of morbidity and mortality following hematopoietic stem cell transplantation (hsct). it has been reported over the last two decades that cmv-specific cytotoxic lymphocytes (cmv-ctls) can provide long-term cmv-specific immunity without major side effects as an alternative to antiviral drugs. however, its application has been limited by prolonged manufacturing process of cell therapy. in this study, we apply the ifn-γ cytokine capture system (ccs) using the fully automated clinimacs prodigy device to rapidly produce cmv-ctls that may be applicable in clinically urgent cmv-related diseases. five validation runs were performed using apheresis samples from randomly selected cmv-seropositive healthy blood donors. then, clinimacs prodigy automatically performed successive processes including antigen stimulation, anti-ifn-γ labelling, magnetic enrichment, and elution which took~13 h. the original apheresis samples consisted of 0.3% ifn-γ secreting cd3+ t cells in response to cmv pp65 antigen (cd3+ifn-γ+ cells) which were mainly cd45ra+cd62l+ naive t cells. following ifn-γ enrichment, the target fraction contained 51.3% cd3+ifn-γ+ cells with reduction in naive t cells and the selection of cd45ra − cd62l − and cd45ra +cd62l − memory t cells. furthermore, extended culture of these isolated cells revealed functional activity including efficient proliferation, sustained antigen-specific ifn-γ secretion and cytotoxicity effect against pp65 pulsed target cells. therefore, we suggest ifn-γ ccs by clinimacs prodigy as a simple and robust approach to produce cmv-ctls, which may be highly feasible and applicable in clinically urgent cmvrelated diseases. disclosure of conflict of interest: none. in vitro generation of tumor antigen-specific t cells from patient and healthy donor stem cells s bonte 1 , s snauwaert 1 , g goetgeluk 2 , b vandekerckhove 2 and t kerre 1 1 hematology, ghent university hospital, ghent, belgium and 2 department of clinical chemistry, microbiology and immunology, ghent university, ghent, belgium acute myeloid leukemia remains a therapeutical challenge, as many patients relapse after chemotherapy. allogeneic stem cell transplantation is in most of these patients the only option for cure, but carries a high risk of morbidity and mortality and a suitable donor may be lacking. recently, advances are being made in the field of t cell immunotherapy. the classical protocol, in which peripheral blood lymphocytes (pbl) are transduced with a tumor antigen-specific t cell receptor (tcr), can generate t cells with low and possibly hazardous specificities (due to mispairing of the endogenous and introduced tcr α and β chains). therefore, we have developed a novel protocol in which we generate tumor antigen-specific t cells from cd34+ hematopoietic stem cells. we have already succeeded in generating large numbers of tumor-specific, naive and resting t cells that only carry the introduced tcr, starting from postnatal thymus and cord blood cd34+ cells. now we are optimizing this protocol for clinically more relevant samples, such as mobilized peripheral blood from healthy stem cell donors and from patients in remission after chemotherapy and/ or other treatments, and leukapheresis samples from patients at diagnosis. in our protocol, cd34+ cells were isolated from hla-a2+ fresh patient and healthy donor samples and cultured on op9-dl1 in the presence of scf, flt3l and il-7, until t cell commitment. subsequently, the cells were transduced with a tumor antigen-specific tcr and again co-cultured until cd4+ cd8+ double positive cells were abundantly present. at that point, agonist peptide was added, which induces maturation. finally, cells were polyclonally expanded on feeder cells. for hla-a2 negative samples, cd4+ cd8+ double positive cells were co-cultured with a cell line (t2 pulsed with the agonist peptide or a cell line with endogenous expression of the agonist peptide) which can present the agonist peptide to the maturing t cells. using the above protocol, we were able to generate tumor antigen-specific t cells from 3 out of 3 healthy donor samples, 1/1 sample from a patient in remission and 2/4 samples from patients at diagnosis, who were all hla-a2+. for most samples, multiple rounds of agonist peptide stimulation were necessary to obtain further maturation. in contrast, generation of mature t cells from cd4+ cd8+ double positive cells in postnatal thymus or cord blood co-cultures, requires only 1 round of agonist peptide stimulation. for the hla-a2 negative samples, we were able to generate an adequate cd4+ cd8+ double positive population from 1/1 healthy donor sample, 3/3 samples from patients in remission and 0/1 sample from a patient at diagnosis. agonist selection using a cell line seems inefficient as cd27 is not upregulated and cells did not mature to cd4+ or cd8+ single positive mature t cells. we are currently co-culturing more samples using our protocol. furthermore, we are investigating the effect of freezing and thawing on the in vitro t cell generation process (cell numbers and efficiency). finally, we are also working on optimizing the protocol for generation of tumor antigen-specific t cells from hla-a2 negative patient and healthy donor samples. disclosure of conflict of interest: none. increase of polyspecific immune responses against leukemia-associated-antigens (laa) and reduction of regulatory cytotoxic t-cell (ctl) responses against malignant cells play a major role in maintaining remission and prolonging overall survival in patients with hematologic malignancies after allogeneic stem cell transplantation (allo-sct) and/or donor lymphocyte infusions (dli). graft versus leukemia (gvl) effects after allogeneic stem cell transplantation and/or dli are considered to be t cell-mediated. many groups described specific t-cell responses against several leukemia associated antigens (laa) in different hematological malignancies. however, t cell responses after allo-sct and dli are not well characterized. in this study, we analyzed laa-specific t cell responses after allo-sct and dli. to this end, we assessed the frequency and diversity of laa-specific cd8+ t cells using elispot analysis and tetramer assays in 12 patients (5 patients (pts) with acute myeloid leukemia, 2 pts with chronic myeloid leukemia, 3 pts with multiple myeloma and 2 pts with chronic lymphatic leukemia) before and after dli. epitopes derived from prame, npm1mut, rhamm, wt-1 and other laa were tested. moreover, the frequency of regulatory t (treg) cells was measured and the course of cytokine profiles before and after dli was analyzed. these immunological findings were correlated to the clinical course in the respective patients. in elispot and tetramer assays, an increase in frequency and diversity of laaspecific t cells was observed in all patients. importantly, there was a significant increase from a median of 1 to 4 laa-derived t cell epitopes (p = 0.03) in clinical responders (r) when compared to non-responders (nr). these positive results in r vs nr where s157 confirmed by tetramer-based flow cytometry assays, where an increase in frequency from 0.5 to 2.3% in the r group of laaspecific t cell/all cd8+ t cells was observed. interestingly, the frequency of tregs in clinical responders decreased significantly from a median 72.9 % to 54.6 % (p = 0.008) while the frequency of tregs kept stable over time in non-responding patients. t cell subset analysis did not reveal significant differences before vs after dli administration. in cytokine assays using elisa for the detection of more than 10 cytokines before and after dli we found a shift towards proinflammatory and t cell stimulating cytokines. taken immunologic surveillance of leukemia is employed for the prevention and treatment of relapse post allohsct. to augment this effect donor lymphocytes are infused (dli) in patients at risk. this procedure is associated with a high risk of agvhd and we believe that this route of administration may not make the direct contact between infused cells and blasts the optimal one. to address these issues, we started delivering donor lymphocytes directly to the bone marrow cavity (ib-dli) in patients post allohsct at relapse. three with aml and one with cll, all relapsed post allohsct: 3 allosib: 50-year-old female aml patient (relapsed 2 years post hsct), 22-year-old aml male 7q31 del (relapsed in 3 years, traumatic brain injury), 25-year-old male aml flt3 itd+ received mud hsct (relapsed 9 months) and 64-year-old cll male, tp53 del, ebv reactivation (progressed 7 years). two patients (26% and 12% blasts in the marrow) received ib-dli up-front and two others due to higher proportions of leukemic cells received either flag (aml case) or anti-cd20 moab (cll case) followed by ib-dli. tcr clonotyping revealed in all 4 patients the presence of the prevailing oligoclonal response on the polyclonal background (characteristic for each individual) which was identified in the marrow and in the blood. however, in two out of 4 patients a distinct oligoclonal peak was seen at first in the marrow and then in the blood. microarray analysis of the transcriptome in the marrows of patients who received three ib-dli courses revealed in all patients preferential use of genes associated with lymphocyte or lymphocyte activation pathways. the patients who responded favorably (cr or pr) clustered with the transcriptomes of normal individuals, but those who failed to respond clustered separately. ib-dli was safe and not associated with gvhd. selective accumulation of cd8+cd279+ as well as the presence of a distinct oligoclonal peak in the marrow suggest that tcrbeta clonotypes may be private to leukemia cells recognition. the response may result in cr or pr and the patients were in a good physical shape during the treatment, which makes it possible to deliver the salvage chemotherapy if required. broad spectrum antibiotics were started. after the orthopedic consultation, the fourth finger was amputated and amputation from the left ankle was recommended. a stem cell transplantation option was offered to patients and their relatives as one of the therapeutic approaches. upon acceptance by patient, 10 μgr/kg of colony stimulating agent was started to patient. when the stem cell was 20/μl, the stem cells were collected. the obtained stem cell product was injected intra-lesionally (picture b). granulation tissue began to develop from the second week in the foot floor of the patient. after from 8th week, the necrotic tissue was disappeared and the granulation tissue was appeared. at 24 weeks, 50% of the lesion healed. at 48th week, there was normal tissue instead of necrotic tissue on plantar surface at left leg (picture c). this case report suggests that diabetic foot/ulcer can be healed with intralesional application of stem cells in patients with diabetes mellitus. [p085] disclosure of conflict of interest: none. and third (n = 5) cell infusions were cryopreserved. cells were infused following conventional chemotherapy (ia, mec, hdac) in 15 cases (44%), chemotherapy plus hypomethylating agents in 8 cases (24%) and hypomethylating agents alone in 11 cases (8 azacytidine, 3 decytabine; 32%). the procedure was well tolerated, with mild and transient ‛haploimmunostorm syndrome' (fever 84%, rash 28%, diarrhea 14%). only the two patients with cmml received corticosteroid. one patient suffered early infusional reaction that was resolved with support treatment. none of the patients showed acute or chronic gvhd or persistent donor engraftment in chimerism tests. four patients had bacterial infections, but no other significant invasive fungal or viral infections were observed. all aml/raeb patients treated achieved complete remission with microhct treatment (13; 87%). only one patient, with cmml, died during microhct induction (7%). four patients relapsed at 7, 9, 10 and 15 months after the infusion; two of them achieved a second sustained complete remission with another micro-hct from a different donor (one of them had developed anti-hla antibodies). as described in figure 1 , median overall survival is 16 months and overall survival at 2 years is 40%. microhct is a well tolerated procedure in elderly aml/mds patients who are not candidates to allogeneic hct. infectious complications are insignificant and the remission rates are very encouraging in very high risk cases, with no evidence of gvhd. patients can undergo a second microhct from a different donor. in addition to the experience by ai et al, we have also shown that microhct can be safely administered following a hypomethylant agent course instead of conventional chemotherapy. a large, international, randomized clinical trial will address the safety and efficacy of microhct for elderly aml/ mds patients (nct02171117). [p086] disclosure of conflict of interest: none. wilms tumor protein 1 (wt1) is expressed in a variety of solid tumors and is found in more than 80% of patients with acute myeloid leukemia which makes it an attractive target for immunotherapy. previously it was shown that t cells recognizing wt1 are suitable for adoptive t-cell therapy by increasing the graft versus leukemia effect. however, the efficiency of this therapeutic strategy is still limited due to the low precursor frequency and specificity of wt1-specific t cells in the peripheral blood of healthy donors. the ubiquitous antioxidant inducible enzyme heme oxygenase-1 (ho-1) and its products have immunomodulatory effects, which render it as a potential target for the modification of t-cell responses. recently, we found that inhibition of ho-1 enzyme activity via tin-mesoporphyrin (snmp) results in activation and proliferation of antiviral t cells from healthy donors. in this study we aimed (1) to identify the mechanism of ho-1 modification in the generation of wt1-specific t cells and (2) to develop strategies for the sufficient generation of wt1specific t cells from healthy donors to augment effective t-cell immunity in leukemia patients and to broaden the applicability of adoptive t-cell therapy to the majority of patients. the frequency of wt1-specific t cells in peripheral blood of healthy donors (n = 50) was examined before and after snmp treatment via ifn-γ elispot using the wt1-overlapping peptide pool (ppwt1). enrichment efficiency of wt1-specific t cells after ho-1 inhibition was verified in response to ppwt1 and the hla-a*02:01-restricted wt1 peptides 37 (vldfappga, wt137) and 126 (rmfpnapyl, wt1126) by ifn-g secretion assay and expression analysis of the t-cell activation marker cd137. phenotypic and functional characterization of wt1specific t cells were further assessed by multicolor flow cytometry, luminex assays and elisa with respect to t-cell subsets, cytotoxicity, proliferative capacity and secretion of effector molecules. in 24% of donors we found specific t cells against ppwt1 by ifn-γ elispot (10 spots/250.000 pbmcs). the frequency of wt1-specific t cells in these donors could be increased fivefold after inhibition of the enzymatic activity of ho-1 via snmp. to assess the possibility that ho-1 modulation might be clinically applicable in conformity with good manufacturing practice, enrichment of snmp-treated wt1-s159 specific t cells was evaluated based on ifn-g secretion and cd137 expression. compared to snmp-untreated cells there was a 3.74-fold higher response of ho-1 modified wt1-specific t cells pre-enrichment and an up to 16-fold higher enrichment efficacy, while snmp treatment did not affected the t-cell functionality. in conclusion, modification of the enzymatic activity of ho-1 resulted in a more effective generation of functionally active wt1-specific t cells suitable for adoptive t-cell therapy. this makes ho-1 a promising therapeutic target to boost antigen-specific t-cell responses for treatment we recently developed and characterized a standardized and clinical grade human platelet lysate (hpl) that constitutes an advantageous substitute for fetal bovine serum (fbs) for human mesenchymal stem cell (hmsc) expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological) and ethical issue. because of the progressive use of pathogen reduced (pr) labile blood components, we evaluated the impact of the novel procedure theraflex uv-platelets for pathogen reduction on hpl quality (growth factors content) and efficacy (as a medium supplement for hmsc expansion). this technology is based on short-wave ultraviolet light (uv-c) and has the main advantage not to need the addition of any photosensitizing additive compounds (that might secondary interfere with hmscs). we applied theraflex uv-platelets procedure on fresh platelet concentrates (pcs) suspended in platelet additive solution and prepared hpl from these treated pcs. we compared the quality of pr-hpl with the corresponding non-pr ones, in terms of growth factor contents. then, we evaluated the efficacy of pr-hpl, in comparison with hpl and msc-screened fbs. we performed large scale culture of hmscs during 3 passages and evaluated the proliferation of cells and the maintenance of their properties: profile of membrane marker expression, clonogenic potential, immunosuppressive properties (inhibition of t-cell proliferation) and potential to differentiate in adipocytes and osteoblasts. we showed no impact on the content in 5 growth factors tested (egf, bfgf, pdgf-ab, vegf and igf) and a significant decrease in tgf-b1 (−21%, n = 16, p o0.01). a large scale culture of hmscs during 3 passages showed that hpl or pr-hpl at 8% triggered comparable hmsc proliferation than fbs at 10% plus bfgf (n = 3). moreover, after proliferation of hmscs in hpl or pr-hpl containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties (inhibition of t-cell proliferation) were maintained, in comparison with hmscs cultured in fbs conditions. the potential to differentiate in adipogenic lineage of hmscs cultured in parallel in the 3 conditions, evaluated using oil red o and nile red stainings and the measurement of triglyceride accumulation, remained quantitatively identical. we also showed that the potential to differentiate in osteoblasts (quantified using alizarin red s and von kossa stainings and alp activity measurement) of hmscs grown in hpl or pr-hpl was not impaired, in comparison with fbs. in conclusion, we demonstrated the feasibility to use uv-c treatment to subsequently obtain pathogen reduced hpl, while preserving its optimal quality and efficacy for hmsc expansion for cell therapy applications. although it is still not used widely in clinical practice. in this paper, we demonstrated a case of ada-scid who received hsct as an adolescent from matched unrelated donor (mudd) after termination of her peg-ada treatment due to severe intractable thrombocytopenia induced by peg-ada. patient showed good engraftment and incremental clinical improvement. her post transplantation course was complicated with multiple complications including: grade i gut gvhd as well as hemorrhagic cystitis (btk related) and ebv infection, additionally, she developed several cns complaints like headache, vomiting and dizziness which were found to be due to increased intracranial pressure with multiple enhancing cerebral lesions found on brain imaging. further investigations for the brain lesions confirmed the diagnosis of malignant diffuse large b cell lymphoma (dlbcl) involving the brain. the lymphoma was highly suggested to be originated from donor cells giving the timing relationship between transplant and establishment of the diagnosis. this lymphoma was successfully treated with full recovery and good final immune reconstitution but with lack of b cell engraftment and need for monthly ivig. we conclude that, peg-ada can rarely induce thrombocytopenia in an autoimmune manner by forming antibodies against platelets and good recovery of thrombocytopenia can be achieved after discontinuation of peg-ada. hsct can be considered as modality of treatment even in older patients with scid due to ada deficiency keeping in mind high possibility of complications including, autoimmunity and malignancy. disclosure of conflict of interest: none. (1) (2) (3) (4) . based on the pre and post-apheresis cd34+ cell counts, the collection efficiency of the apheresis amicus device was median 89.5% (54-170) and of the comtec median 82% (32-95). in mm the apheretic collections were started on median day 5 (4-6), while in lymphoma patients, due to chemotherapy, the day of apheresis start was 12 (9-18). after cryopreservation and thawing, viability (7-aad, bd) was median 87.5% (43-100). with these cell products, up to now we engrafted 9 patients following high-dose chemotherapy (5 mm autografted after mel200, 2 hl and 2 nhl autografted after beam). engraftment was prompt and stable in all with anc 0.5 and 1.0 × 10 9 /l on median day 11 (10-12) and 12.5 (11-15), respectively, and with platelet count 20 and 50 × 10 9 /l on median day 14 (11-17) and 17.5 (13-44), respectively. these results are similar to those obtained by most experienced centers in europe and us, and confirm the fact that autologous transplantation may be implemented also in developing countries when appropriate technology and application of standard procedures are employed. with this experience our center is also developing allogeneic transplantation, and the initial results in thalassemia will be reported in a separate abstract. disclosure of conflict of interest: none. extracorporeal photopheresis (ecp) is a safe and effective immunoregulatory therapy for steroid-refractory graft-versushost disease (gvhd) but its mechanism of action is poorly understood. ecp is a non-immunosupressive therapy whose modulating mechanism is thought to result in an increase in t-regs in the patient and in inversion of the cd4/cd8 ratio at the end of treatment. in this study, we evaluated the effect of ecp on t cell response in a cohort of steroid-refractory gvhd patients. from november 2009 to november 2016, 40 patients (28 con acute gvhd and 12 with chronic gvhd) treated with ecp in our unit, were retrospectively evaluated. patient characteristics are shown in table 1 . we performed an ‛off-line' system ecp using a cell separator (spectra optia, teruno bct) for the cmn apheresis; after 8-methoxypsoralen was added, the product was photoinactivated in the ultraviolet a irradiator (uvamatic-g1, macopharma). ecp procedures were performed for two consecutive days, initially weekly (agvhd), or every two weeks (cgvhd) and afterwards monthly according to clinical response. anthracycline-induced cardiotoxicity (aic) is irreversible, which has limited the use of this anthracycline in cancer chemotherapy. to explore the therapeutic effect and its possible mechanism of bone marrow derived mesenchymal stem cells (bmscs) on cardiac damage induced by anthracyclines in a rat model. study selects sd rats aged 2-3 weeks to isolate and culture bmscs, and flow cytometry was used for phenotypic identification of bmscs. 48 female sd rats were first randomly divided into 6 groups: the sham control, bmscs control, 4.0 mg/kg daunorubicin (dnr), dnr with bmscs, dnr with dexrazoxane (dzr), dnr with bmscs and dzr. left ventricular (lv) function before, during and after chemotherapy were assessed by echocardiography. at the end of 4 weeks, animals were euthanized and organs were collected in 10% buffered formalin for histopathology using hematoxylin and eosin staining and immunohistochemical analysis was used to identify the cellular subpopulations that infiltrate the cardiac tissues. after the construction of microrna-21 (mir-21)modified bmscs with lentiviral vector, 50 sd rats were randomly assigned into 5 groups: the normal control, the empty vector control, dnr, dnr with bmscs, dnr with mir-21-modified bmscs. the density of new blood vessels of rats in each group was detected by immunohistochemical method. mir-21, bcl-2, bax and vegf mrna expressions were detected by qrt-pcr. bcl-2, bax and vegf, cx43, troponin t and bnp protein expressions were detected by western blotting. all procedures performed in studies involving animals were in accordance with the ethical standards of the institutional. an animal model of drug-induced cardiomyopathy was built in the dnr treated rats.lv ejection fraction (lvef) and lv fractional shortening (lvfs) were significantly decreased compared to that of the sham control (p o0.001), and the signs of the myocyte injury (myocytolysis, vacuolization and disruption) in paralleled with the inflammatory infiltrates, marked by cd3 and hla-dr, were observed in the dnr group, while bmscs alone or synergistic with dzr facilitate the anthracycline-induced lv dysfunction returning to the baseline values and the recovery of myocarditis (p o0.001). in the mir-21-modified bmscs transplant group, mir-21 expression, cell migration and proliferation ability were higher than that in the bmscs and empty vector groups (p o0.05). the cardiac regenerative capacity of bmscs following significant myocardial injury were further enhanced by mir-21 compared to that of the dnr group and the control groups (all po 0.05), revealed by the significantly higher density of new blood vessels and upregulation of vegf expressions, during which the pro-apoptotic protein bax were down-regulated and the anti-apoptotic protein bcl-2 function were upregulated in the mir-21 overexpression group compared to that with the bmscs, dnr group and the control groups (all po0.05). western blotting demonstrated that the expression of c × 43 were significantly decreased, while expressions of troponin t and bnp were significantly increased in the mir-21 overexpression group in contrast to that with the dnr group (all p o0.05). these results showed that bmscs could reverse cardiac damage induced by anthracycline, and the cardioprotective efficacy was further enhanced by mirna-21-mediated regulation of apoptosis and angiogenesis. disclosure of conflict of interest: none. effective adoptive t cell therapy against cancer is dependent on long-lived tumor-specific stem cell-like t cells with the ability to self-renew and differentiate into potent effector cells. however, current protocols for ex vivo generation of tumorspecific cd8+ t cells result in terminally differentiated effector t cells. it was found that minor histocompatability antigen (miha)-specific cd8+ t cells with an early memory-like phenotype and long-lived memory transcription profile could be expanded from naive precursors using akt-inhibitor viii1. importantly, these akt-inhibited tumor-specific cd8+ t cells showed a superior expansion capacity and anti-tumor effect multiple myeloma bearing mice. for the clinical exploitation of ex vivo generated akt-inhibited tumor-specific cd8+ t cells, we tested the effect of potential clinical grade akt-inhibitors azd5363, gdc0068, gsk2110183, gsk2141795, mk2206 and triciribine in polyclonal stimulations, allogeneic mixed lymphocyte reactions (mlr), and antigen-specific t cell assays. polyclonal stimulation with anti-cd3/cd28 beads on cd8 naive t cells was used for a first screening of the akt-inhibitors. for all inhibitors, a dose dependent effect on the naiveassociated receptors ccr7, cd62l and cxcr4 was observed. this had limited effect on viability, activation and proliferation except for triciribine, which was therefore excluded for further assays. moreover, in the mlr, treatment of naive cd8+ t cells with remaining akt-inhibitors resulted in a dose dependent effect associated with higher ccr7, cd62l, cxcr4 and cd28 expression. furthermore, the akt-inhibited cd8+ t cell products showed a 10-25 fold increased expansion capacity upon restimulation in vitro. when expanding miha-specific cd8+ t cells from the naive repertoire in the presence of one of the akt-inhibitors, the miha-specific cd8+ t cells showed a more early memory phenotype compared to controls. this was displayed in higher levels of the naive-associated receptor cd62l ( figure 1 ). in addition, these miha-specific cd8+ t cells were shown to be functional, as antigen-specific restimultation resulted in degranulation (cd107a) and ifn-γ production. based on this ifn-γ production, the akt-inhibited antigenspecific cd8+ t cells can be selected using the cytokine capture assay (miltenyi, for enriched infusion in patients suffering from hematological malignancies. using aktinhibition in the generation of tumor-reactive t cells results in a more early memory tumor-specific cd8+ t cell product. this adoptive immunotherapy product retains superior proliferation capacity upon infusion, and its potential selfrenewal capacity could result in a long-term anti-tumor effect in patients suffering from a hematological malignancy. chimeric antigen receptors (cars) are composed of an extracellular domain-derived from a tumour-reactive monoclonal antibody, linked to one or more signalling endodomains. in early clinical trials, cd19 car-t cells have demonstrated impressive anti-tumour activity against different b-cell malignancies, including chronic lymphocytic leukaemia, acute lymphoblastic leukaemia (all) and non-hodgkin lymphoma. conventional alpha-beta car-t cells are however hla-restricted and could cause graft-versus-host disease (gvhd) when used across major mismatches, as expected in the highly anticipated setting of off-the-shelf car-t cells from third-party donors. besides being non-hla restricted, gammadelta t cells possess intrinsic anti-tumour reactivity, making them attractive effectors for next-generation car-t cell therapies. so far, however, attempts at exploiting gammadelta t cells in patients have been largely disappointing, possibly because of sub-optimal ex vivo culture conditions. the aim of our study was to optimise the generation of gammadelta car-t cells and to test their anti-tumour potency both in vitro and in vivo. starting from peripheral blood mononuclear cells of healthy donors, we stimulated gamma-delta t cells with zoledronate and il-2/il-15, and transduced them with retroviral vectors encoding for cd19 cars carrying either cd28.z or 4-1bb.z signalling endodomains. we assessed antitumour activity in vitro by measuring killing, secondary expansion and cytokine production after co-culturing gamma-delta car-t cells with different cd19+ all cell lines, and in vivo in nsg previously engrafted with a b-all semi-cell line. although allogeneic hematopoietic stem cell transplantation (allosct) is a curative option to treat hematologic malignancies, disease recurrence remains a concern in the setting of high risk diseases. thus, post allosct therapeutic strategies are needed to treat and/or prevent disease progression. in this setting, donor lymphocytes infusion (dli) is an option as post allosct immunotherapy aiming to enhance graft versus leukemia (gvl) effect. although dli may induce persistent remission, graft versus host disease (gvhd) is a potential complication following dli. because of the suspected higher incidence of gvhd in the presence of hla mismatches, few series focused on dli following haploidentical stem cell transplantation (haplosct) so far. we therefore report our experience of dli following haplosct using post-transplantation cyclophosphamide (pt-cy) platform. we included in this single center study all consecutive adult patients with hematological malignancies who received dli after haplosct with pt-cy as part of gvhd prophylaxis from 2013 to 2016 (n = 21). conditioning regimens were non-myeloablative (low dose tbi-based) or with reduced toxicity (various dose of busulfan according to disease and patient characteristics). ciclosporine a and mycophenolate mofetil were given as additional gvhd prophylaxis in all cases. dli were given at escalating doses, expressed as cd3+cells/kg, without gvhd prophylaxis, and ranged from 1 × 10 5 our study suggests that dli following haplosct with pt-cy is feasible. gvhd is frequent but with a relatively low incidence of severe forms. no response rate was achieved in the context of hematological relapse, underlining that preemptive or prophylactic strategy might be preferred. indeed, the overall good outcome in patients receiving prophylactic dli is promising taking into account the poor prognostic of the diseases indicated for alternative donor transplantation. further prospective studies are needed in specific disease settings to assess the benefit for using such post allohsct immune-intervention. [p103] disclosure of conflict of interest: none. dual specific cytokine-induced killer cell therapy as a treatment option for life-threatening ptld-a case report of the frankfurt experience l-m pfeffermann 1 infection with epstein-barr virus (ebv) is a frequent complication after allogeneic hematopoietic stem cell transplantation (hsct) and besides relapse remains a significant cause of morbidity. prolonged immunosuppression or delayed t-cell recovery may favor ebv reactivation after transplantation, which under these circumstances can lead to life-threatening lymphoproliferative disease (ptld). consensus is lacking on the optimal treatment of ptld. adoptive immunotherapies with both anti-tumor capacity and restored virus-specific cellular immunity may represent optimal treatment options especially when considered in the context of ptld. in this case report we applied in vitro activated t-cells namely cytokineinduced killer (cik) cells with dual specific cytotoxic capacities transferring both anti-cancer potential and donor t-cell memory against ebv infection for the treatment of ebvassociated ptld which progressed to highly proliferative large b cell lymphoma during delayed t-cell recovery after allogeneic hsct. the reported patient had received an allogeneic hsct for secondary myelodysplastic syndrome following acute myeloid leukemia, and due to delayed t-cell recovery had developed ebv-related ptld two months after transplantation. treatment with rituximab, conventional ebvspecific t-cells and wildtype cik cells failed, therefore the patient was offered ebv-specific cik cells on a compassionate use basis. ebv-specific cik cells were generated from peripheral blood mononuclear donor cells. cells were activated and expanded in the presence of ifn-γ, il-2, anti-cd3 antibody and il-15. on day 0 and 2 of culture an ebv peptide pool was added for additional priming. follow-up analysis included in vitro and in vivo monitoring of ebv-specific cik cells. with above mentioned protocol we were able to generate cik cells containing 1 × 10 4 cd8 + ebv-specific t-cells/kg body weight of the patient. infusion of ebv-specific cik cells resulted in rapid clearance of plasma ebv dna level and sustained disappearance of large (vol. 27cm 3 ) ptld-malignant lymphoma. during one-year follow-up analysis we were able to detect ebv-specific cik cells (cd4 + and cd8 + ) in vivo by flow cytometry using specific mhcdextramers. facs-monitoring of the patient´s blood revealed besides cd8 bright t-cells also an increasing cd8 dim t-cell population with a remarkable percentage of t emra cells within this compartment (up to 95%) indicating virus-specific t-cells. no cytokine release syndrome appeared after ebv-specific cik cell treatment, but cytokine secretion patterns, analyzing serum of the patient, reflected cytotoxic and anti-virus capacity provided by this treatment. cytotoxic potential, as well as t h 1 cell differentiation and function offered by ebvspecific cik cell treatment were further confirmed by in vitro analysis. ebv-specific cik cells revealed an 1. s168 hematologic disease. the global survival ratio in the follow-up was 56.8% (with 66.6%, 60.6% and 42.8% survivals in 3, 6 and 12 months, respectively). the variables significantly associated with greater survival were: type of gvhd (cgvhd), number of affected organs (an organ had to be moderately or severely affected to be included in this category) and steroid dependence as the main reason to initiate ecp (see figure 1 ). there was a trend towards significance for the degree of gvhd and cutaneous involvement to be factors associated to enhanced survival ratios. extracorporeal photopheresis is a safe treatment option for patients with gvhd, generating a response and decreasing immunosuppression in an important percentage of them. the presence of cgvhd rather than agvhd, a lower severity degree of the condition, having a lower number of affected organs, skin as main affected organ and steroid dependence as the reason to start the ecp treatment were all factors associated with greater survival in our sample. disclosure of conflict of interest: none. tx) . we report the case of a patient refractory to chemotherapy treated with ibrutinib as debulking therapy before allo-tx. in june 2014, a 62 years old woman was diagnosed with mcl. the staging performed by whole body ct scan, colonoscopy, egds and bone marrow (bm) biopsy was conclusive for stage iva with bulky lymph node over and below the diaphragm, bm, enteric and peripheral blood (pb) localization. the planned treatment included 3 cycles of r-chop, 1 cycle of high dose (hd) cy, 2 cycles of hd arac and autologous sct. after completion of hd cy, the restaging showed progressive disease, with a thyroid involvement and histologic switch in a blastoid variant. disease continued to progress even after 2 cycles hd arac, so we tried to control the disease with r-bendamustine (90 mg/mq on days 1-2 of 21-d cycle), but after the first cycle the neck circumference increased. we shift to lenalidomide (25 mg on days 1-21 of 28-d cycle) without any response after two cycles. we excluded patient from autologous sct programme because of chemo-refractoriness and we searched matched unrelated donor because no hla identical sibling was available. we started a therapy with ibrutinib (560 mg/die on days 1-21 of 21d cycle). after the first cycle we observed a rapid response with decrease of neck size and the disappearance of superficial lymphnode; we performed 6 cycles of ibrutinib, and we reached a good partial remission with lymphnode of max 4 cm, and a bm and pb involvement of 20%. meantime an unrelated donor with 7/8 hla matching was identified, so in december 2015 we performed allo-tx with reduced-intensity conditioning (thiotepa 5 mg/kg-fludarabine 90 mg/m 2 -melphalan 100 mg/m 2 ) and cyclosporine and short term methotrexate as gvhd prophylaxis. engraftment was at day +17. in the first 100 days after allo tx she experienced a clostridium enteritis, transient cmv reactivation and acute gastrointestinal gvhd on day +60 with rapid response to steroid therapy. main complication happened on day 100, when sudden fever and stupor, progressive to coma, occurred; subsequently pneumococcal encephalitis was diagnosed, with positive csf microbiological exam and two signal alteration in the right cerebral hemisphere at mri. the patient was treated with ampicillin and ceftriaxone with a favourable outcome. the mcl revaluations performed at 2, 5 and 9 months showed complete remission with disappearance of all pathological lymph node and pb involvement. currently the patient is at 1 year post asct, she is enrolled in a rehabilitation program and mcl is in complete remission. our experience seems to indicate that ibrutinib is safe and can be used as bridge to allo-tx therapy in refractory mcl. we will investigate side effects of this platform of therapy, and, given the early occurrence of pneumococcal infection, we will consider to perform capsulated bacteria vaccination before allo-tx. disclosure of conflict of interest: none. [p107] pt 2 was diagnosed with c-all in 2014 and received a mud-hsct (tbi 8gy, cyclosphosphamide) in 1/15 due to persistend mrd. following early rel 6/15, 2 cycles of blina led to mrd+ cr, for which a 2nd hsct from a haploidentical family donor (busulfan, thiotepa, fludarabine) was performed in 10/15. molcr lasted 3 months and rel3 was treated with 3 cycles of weekly io followed by one dli (5x10-4 cd3+ cells/kg), resulting in mrd+ cr and complete donor chimerism. five weeks after the last io cycle, the pt was admitted with ascites, hyperbilirubinemia and reduced general condition. vod was suspected, but diagnostic paracentesis revealed malignant ascites demonstrating fatal progressive disease. conclusion. our data suggest that the sequential use of io and dli is feasible even for heavily pretreated patients with r/r all after hsct and can induce molecular remissions. we observed an unusual case of late onset, severe vod responding to defibrotide and one all relapse manifesting itself with ascites in our patients. we therefore suggest close monitoring of liver function tests in the setting of this therapy and extensive diagnostic work-up for any developing liver abnormalities or ascites. disclosure of conflict of interest: ng: advisory board (pfizer, amgen); research support (amgen); gb: honoraria (amgen). [p108] p108 while allogeneic hematopoietic stem cell transplantation from matched related and unrelated donors has become a standard of care treatment for patients with hematological malignancies, transplantations from mismatched or haploidentical family donors remain challenging. currently t-replete and t-deplete transplantation strategies are applied aiming to improve the outcome after haploidentical transplantation. despite high rates of relapse many centers regard post-transplant cyclophosphamide, a t-replete strategy, as a standard of care approach. we have developed a t-depleted transplant approach where donor lymphocytes selectively depleted of alloreactive t-cells (atir101) using th9402, arhodamide-like dye, are infused after cd34-selected haploidentical hsct, to overcome the challenges of infectious complications, gvhd and relapse. in phase i (cr-air-001) we have demonstrated safe infusion of these lymphocytes at doses up to 2 × 10 6 viable t-cells/kg. recently, we reported a promising 1-year grfs was 57% from a phase ii trial (ash 2016),that is awaiting final results soon. here, we introduce a randomized, multicenter phase 3 study (cr-air-009), where 200 patients with acute myeloid leukemia (aml), acute lymphoblastic leukemia (all) or myelodysplastic syndrome (mds) are planned to undergo a haploidentical hsct with either a t-cell depleted graft and adjunctive treatment with atir101, or with a t-cell repleted graft and use of posttransplant cyclophosphamide. inclusion and exclusion criteria are listed in table 1 . all patients will undergo myeloablative conditioning consisting of either tbi (12 gy) or melphalan/ busulfan, in combination with thiotepa and fludarabine. patients in the atir101 study group will receive atg (2.5 mg/ kg once daily for 4 days) during conditioning and atir101 infusion at a dose of 2 × 10 6 viable t-cells/kg is given between 28 and 32 days after the hsct. patients in the ptcy group will receive cyclophosphamide (50/mg/kg) on day 3 and 4 (or 5) with subsequent use of immune suppression up to 6 months post-hsct. the primary endpoint of the study is gvhd-free, relapse-free survival (grfs). grfs is defined as time from randomization until grade iii/iv acute graft-versus-host disease (gvhd), chronic gvhd requiring systemic immunosuppressive treatment, disease relapse, or death, whichever occurs first. this endpoint captures both safety and efficacy. additional secondary endpoints are overall survival (os), progression-free survival (pfs), relapse-related mortality (rrm) and transplantrelated mortality (trm). patients are planned to be randomized in study centers in europe and north america. a number of 40-50 sites are planned to participate in the study. enrolment is expected to continue until mid-2018 with initial results being available first half 2019. results of this study will determine which transplant regimen provides most clinical benefit in haploidentical donor transplantation, with the promise of an effective regimen without the use of post-transplant immune suppression. disclosure of conflict of interest: jr is an employee of kiadis pharma. multipotent mesenchymal stromal cells (mscs) are used for prevention and treatment of graft versus host disease after allogeneic hematopoietic stem cells transplantation due to their immunomodulatory properties. mscs fate in vivo after infusion is unknown. the aim of this study was to analyze the changes in mscs and allogeneic lymphocytes properties when co-cultured in vitro to simulate their interactions in vivo. the bone marrow from 13 donors (7 male and 6 female aged 22-62 years, median 27 years) was used. mscs were cocultured with allogeneic lymphocytes in a ratio of about 1:10 for 4 days and their basic properties were analyzed over time. lymphocytes were activated by adding to the culture medium 5 mg/ml of pha (pha-lymphocytes). some mscs were treated for 4 h with 500 u/ml ifnγ (γmscs). determination of gene expression levels was performed by reverse transcription polymerase chain reaction in real time (modification of the taq-man) and of antigen expression on mscs and lymphocytes by flow cytometry. significant reduction in the proportion of viable cells was observed in mscs co-cultured with pha-lymphocytes. in γmsc co-cultured with pha-lymphocytes no reduction in the proportion of living cells was revealed. this indicates the sensitization of mscs by ifnγ to factors secreted by pha-lymphocytes. in mscs co-cultured with pha-lymphocytes and lymphocytes mean fluorescent signal intensity level (mfi) of cd90 gradually decreased. ifnγ treatment and co-cultivation with lymphocytes led to significant increase of hla-dr mfi on mscs. co-cultivation with lymphocytes increase the hla-dr mfi on mscs much stronger than ifnγ treatment. relative expression level (rel) of ido1 gene increased dramatically in both mscs and γmscs when co-cultured with lymphocytes. at a day 1 in γmscs rel of ido1 increased 500 fold, and then gradually declined. in mscs cocultured with lymphocytes il-6 rel increased almost 20-fold and then decreased 2-fold at the fourth day. the csf1 rel in γmscs showed twofold increases, upon incubation mscs and γmscs with lymphocytes csf1 rel increased fourfold and sevenfold, respectively. co-culture of msc and γmscs with lymphocytes led to decrease in the proportion of cd25, cd38, cd69, hla-dr, and pd-1 positive cells (both cd4 and cd8) after one day, compared with pha-lymphocytes without mscs. proportion of cd25+, hla-dr+ and pd-1+ cells also decreased after 4 days of co-culturing with msc or γmscs (compared with pha-lymphocytes without mscs), but anyway number of activated cells increased 1.2-3 folds compared with first day. number of cd69+ lymphocytes after 4 days of co-culturing with mscs or γmscs did not vary significantly from control and decreased in comparison with first day. main inhibition of activated lymphocytes by co-culturing with mscs occurs during the first day of their interaction, and then the inhibition became less effective, moreover in mscs decreased the rel of the main immunomodulating factors, and most of them were eliminated. mscs treatment with ifnγ resulted in improved survival and resistance of these cells to lymphocytes action. the results indicate that the effect of mscs injected intravenously to patients is limited to several days. disclosure of conflict of interest: none. autologous adipose-derived mesenchymal stem cells (admscs) embedded in platelet-rich fibrin (prf) promote healing in different types of wounds. by avoiding the needlerelated complications, prf-embedded autologous admscs graft provides a new effective stem cell-based therapeutic strategy for wound healing. adult male (age ⩽ 75yo) were equally divided (n = 5 per group) into group 1 (prf only), and group 2 (prf+admscs). regular dressing (without any agent) was used for both groups with a frequency of changing every 3 days. rpf with or without admscs was patched on the wound (maximum surface area 77 cm 2 ). all patients were followed up until complete healing. a complete healing was noted in both groups; however, the healing in group 1was very slow (after 10 weeks), compared to a quicker one in group 2 (after 6 weeks). control of the moisture was very well noted in group 2, less in group 1. group1 showed a lot of exudates on the wound; less exudate in group2 were noted. infections were absent. both groups had a colonized wound. signs of inflammation were very well noted in group 1; no signs of inflammation in group2. admscs embedded in prf offered rapid wound healing responses then prf alone. keywords: mesenchymal stem cells, platelet-rich fibrin, engineered tissue wound healing disclosure of conflict of interest: none. stem cell source p112 additionally cryopreserved g-csf primed pbsc can substitute the second transplantation for the patients with acute leukemia who lately relapsed after hematopoietic stem cell transplantation y lee 1 , j moon 2 , ih lee 3 and s sohn 2 1 department of hematology, kyungpook national university hospital; 2 department of hematology,kyungpook national university hospital and 3 kyungpook national university hospital although allogeneic hematopoietic cell transplantation (allo-hct) is a potentially curative therapy for acute leukemia, survival outcomes of the patients relapsed after transplantation remains poor with high early mortality and a small percentage of second remission. this study evaluated the efficacy of dli using g-csf primed pbsc additionally cryopreserved for the patients who relapsed after allo-hct. we retrospectively reviewed the medical records of the 255 patients who received allo-hct for acute myelogenous leukemia (aml), myelodysplastic syndrome (mds), and acute lymphoblastic leukemia (all) between 1998 and 2013 in kyungpook national university hospital. among the 93 patients who had relapsed after allo-hct, the 39 patients received dli using the additionally harvested cells. at the time of harvest for the first hct, collecting targeted pbscs (greater than 6 × 10 6 /kg cd34+ cells) allowed us to cryopreserve surplus pbscs, including cd3+ cells with dimethylsulfoxide in a nitrogen tank. then, we analyzed the efficacy of dli for the patients who were classified into early relapse or late relapse group by the median time of relapse after transplant. the median age at transplant was 38.5 years (range 15-68 years) and male was 111 patients (44.4%). primary diseases for allo-hct were aml/mds (n = 175, 70%) and all (n = 75, 30%). one hundred forty three patients (57.2%) were in cr1 (complete remission), 25 (10%) in further cr, and 87 (33.2%) in relapsed and refractory status. one hundred seventy one patients (68.4%) received myeloablative conditioning regimen. the median dose of cd34+ cell was 5.21 × 10 6 /kg (range: 1~20.6 × 10 6 /kg). almost 95% of patients achieved the neutrophil engraftment with a median time of 13 days (range: 9-24days). the 1-year overall survival (os), relapse free survival (rfs), non-relapse mortality (nrm) and graft-versus-host disease (gvhd)-free/relapse-free survival (grfs) since hct was 55.3 ± 3.1%, 66.0 ± 3.2%, 28.2 ± 0.3%, and 32.9 ± 3.1%, respectively. there was no significant difference according to s172 the infused cd34+ cell dose (lower o6 × 10 6 /kg vs higher ⩾ 6 × 10 6 /kg). the incidence of chronic gvhd was more frequent in higher cd34+ group (32.9% vs 48.2%, p = 0.042). median time from hct to relapse was 139 days (range: 21~1801 days). after relapse, 46 patients (49.4%) were treated with salvage chemotherapy, 9 patients (9.6%) with second allo-hct, and 38 patients (40.8%) with dli. the median number of cd3+t cell was 2.95 × 10 7 /kg (range: 0.1~5.43 × 10 7 /kg). fourteen patients (23.6%) achieved dli induced cr, 20 patients progressed, and 6 patients were not evaluable for response. dli induced acute gvhd was observed in 24 patients (63.1%) and chronic gvhd developed in 4 patients (10.5%). in late relapse group, the 1-year os since post-transplant relapse was significantly higher in dli group than non-dli group (figure 1 , 53.4 ± 7.4% and 26.7 ± 7.4%, p = 0.039) but, early relapse group had no difference. the patients treated with dli showed significantly survival benefit in late relapse group (median 286 days vs 103 days, p = 0.002). the incidence of dli-induced gvhd does not differ between two groups. dli for the patients who lately relapsed after allo-hct can be a feasible and an effective option in terms of response, donor convenience and it's cost. in the late relapse group, g-csf primed dli may replace second transplantation. disclosure of conflict of interest: none. cord blood transplantation-19 years of experience c alves 1 , f amado 2 , f bordalo 2 , s ferreira 2 , s lopes 2 , c pinho 2 , t rodrigues 2 , l antunes 3 and s roncon 2 1 serviço de imuno-hemoterapia; 2 serviço de terapia celular and 3 registo oncológico do norte, instituto português de oncologia do porto francisco gentil, epe allogeneic haematopoietic stem cell transplantation (hsct) is a well-established treatment for patients with malignant and non-malignant haematological disorders. cord blood transplantation (cbt) has extended the availability of hsct to patients that would not otherwise be eligible for this curative approach because of the lack of human leucocyte antigen (hla) identical donor. the aim of this retrospective study was to analyse and characterize 19 years of cbt activity in our institution (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) . we examined patient electronic files and created a database in excel to register cord blood unit (cbu) parameters and patient characteristics. after thawing, cbu was washed/diluted with validated procedures; the cellular content was evaluated by immunophenotyping and followed ishage recommendations; sterility was assessed by bacterial/fungal cultures, viability by trypan blue exclusion assay and functionality by clonogenic capacity. the transfusion of blood products after transplant was quantified and the hematological recovery (hr) established using cibmtr criteria. correlation between continuous variables was assessed with spearman coefficient. overall survival (os) was determined according to cellular content and hla-disparity by the kaplan-meier estimator. survival between groups was compared using the log-rank test. a total of 59 cbu were administered to 57 patients (table 1) : 30/57 female, the main diagnosis was acute leukaemia (35/57). a sibling cbu was used for 3 patients; the unrelated were imported from europe (66%), usa (32%) and oceania (2%). hla-matching was 6/6, 5/6, 4/6 and 3/6 for 8, 21, 15 and 6 patients, respectively; 47% were abo-identical. after thawing, 95% were washed and presented no microbial growth. the majority of patients were submitted to a bussulfan-based myeloablative conditioning regimen; graft versus host disease prophylaxis was performed with a calmodulin inhibitor+mycophenolate mofetil. complete and mixed chimerism was verified in 42% and 7% of patients; 5% had graft failure; the rest were unknown results (46%). at the moment, we reported 27 patients alive (22 in complete remission, 5 with evidence of disease relapse) and 30 dead at a median of 6 (0.4-198) months after cbt; the most frequent cause (43%) was recurrence of the initial clinical condition. the correlation between nucleated cells (nc) and cd34+ cells per kg/hr (p = 0.277; p = 0.123) and number of cd34+ cells per kg/os (p = 0.123) was not statistically significant. however, the engraftment and os was associated with hla-mismatch (p = 0.025; p = 0.012) and os was related to nc per kg (p = 0.012). our clinical results suggest that despite increased hla disparity, ucb offers promising results. ucbt is feasible in patients when the unit contains a high number of cells. there are several strategies for the future, related to cbu expansion and homing techniques, nurturing procedures, selection of optimal cbu unit and enhancement of immune recovery, in order to improve the application of cbu. s173 received sirolimus and mmf. median time to neutrophil and platelet engraftment was 10 days (9-13) and 18 days (17-124), respectively. one patient died from parainfluenza pneumonia (d111), one patient from ptld (d 203), one patient from late pulmonary vod (d379), and one patients from relapse (d89). with a median follow up for survivors of 17 months, one year survival is 64%. three patients had grade 2-3 gvhd and none of the survivors have chronic gvhd. though unrelated donor chimerism was dominant early after transplant and contributed to early count recovery, definitive engraftment was dominated by ucb chimerism in all but one patient. conclusion: among older adult patients with hematological malignancies,~20% lack haplo-identical relatives. for these patients, double or single ucb transplant is challenging because of delayed engraftment. cd34 selected partially matched grafts from unrelated donors hasten hematopoietic recovery and are over time outcompeted by ucb grafts which provide robust hematopoiesis with low risk of chronic gvhd. the combination of mismatched unrelated hematopoietic progenitors and ucb grafts provides an attractive alternative for older patients lacking hla-idental donors or haploidentical relatives. in planned trials, mismatched donors may be selected based on kir type to further enhance gvl effects. disclosure of conflict of interest: partially supported by miltenyi biotec. haploidentical stem cell transplantation (haplo-sct) is an attractive option for patients who do not have an hlamatched donor. historically it has been associated with high rates of graft rejection, relapse and low incidence of graft versus host disease (gvhd). to decrease these issues we have considered the use of primed bone marrow as stem cell source, early withdrawal of immunosuppressive therapy and the use of donor lymphocytes infusions (dli) in haplo-sct with high-dose post-transplantation cyclophosphamide (ptcy) as main gvhd prophylaxis. to analyse our incidence of acute and chronic gvhd and overall survival (os) in patients with haplo-sct with short course of inmunosupressive therapy. we retrospectively analyzed a cohort of 28 patients who underwent haplo-sct with primed bone marrow as stem cell source, between years 2012 and 2016 in our centre. gvhd prophylaxis consisted in ptcy (50 mgr/kg on days +3 and +4) and tacrolimus plus mycophenolate (mmf) from day +5 as recommended by baltimore group. mmf was stopped on day +28. tacrolimus was tapered off from day +50 with withdrawal on day +120 in patients without gvhd or with active disease. dli were considered if mixed chimerism, relapse or disease progression appeared. the characteristics of the patients are shown in table 1 . results: there was no primary graft failure. eight of 27 patients (29.6%) developed agvhd (grade ii-iv) and it was severe (grade iii-iv) in 2 patients (7.4%). cutaneous agvhd location was the most common presentation (9 patients (33%)) and it was associated with intestinal gvhd in 2 patients. twenty two patients were evaluable for cgvhd. thirteen patients (59%) developed chronic gvhd that was mild, moderate and severe in: 8 (36.3%), 3 (13.6%) and 2(9%) patients, respectively. the median time of onset cgvhd was 6 months (range: 3-33) and it was related with previous withdrawal of the immunosuppression in 5 (22.7%) patients, tapered off immunosuppression in 6 (27.2%) patients and dli in 2 (9%) patients. systemic treatment was required in 8/13 patients but only 2 patients were treated with high doses of steroids (41 mg/kg/day). the median days of is therapy in patients who developed gvhd was 172 days (range: 81-244). dli were used in 7 (25%) patients because of: relapse/disease progression in 5(17.8%) and secondary graft failure in 2(7.1%). two patients achieved complete remission and 2 patients developed cghvd. the median number of dli per patients were 2 (1-3) with a median cd3+cell of 3 × 10 6 /kg (range: 1 × 10 5 -1 × 10 7 /kg). with a median follow-up of 12 months (range: 1-56), the estimated os at 1 and 2 years after haplo-sct were 74% and 52%, respectively. at the moment of this study 17 patients (60.7%) were alive, 14 patients in complete remission, 2 in partial response and 1 in progression. eleven (39.3%) patients died due to: disease (4), infections (4), pleuropericarditis (1), hepatic veno-occlusive disease (1) and refractory gvhd. five patients (17.8%) are without is therapy and without gvhd symptoms. in our experience, early withdrawal of immunosuppression following haplo-sct with primed bone marrow and posttransplantation cyclophosphamide facilitates the development of chronic gvhd and can decrease the relapses in patients with high-risk hematological malignancies. it is necessary more follow up and more studies to confirm this preliminary results. [p115] disclosure of conflict of interest: none. s174 myeloid malignancy (n = 20 except 1 patient with saa) received fludarabine (flu)/busulfan ± tbi 4gy, while lymphoid malignancies (n = 9) received flu/tbi 8gy or cy/ tbi 12gy. all patients received g-csf-mobilized t-cell replete pbsc from a haplo donor. gvhd prophylaxis was ptcy 50 mg/ kg on day +3/+4, tacrolimus (d+5 to +180), and mycophenolate (d+5 to 35). the median duration of follow up of surviving patients is 21 months. median age was 45 (17-66) years, 17 patients (59%) were male, 14 (48%) were african american, and 14 patients (48%) had comorbidity index (hct-ci) ⩾ 3. all patients had hematological malignancy (except 1 patient with saa) including 12 patients (43%) not in cr. disease risk index was high/very high in 12 (42%) and intermediate in 14 (48%). on the day of transplant, 14 patients (48%) did not receive steroid premedication ( = no-steroid group), while 15 patients (52%) received 125 mg of methylprednisolone 30 minutes prior to pbsc product infusion ( = yes-steroid group). all the following outcomes are described in the ‛no-steroid' vs ‛yessteroid' group, respectively. cumulative rate (ci) of anc engraftment (⩾500/μl) on day +28 was 79% (95% ci 60-100%) and 93% (95% ci 81-100) (p = 0.07). ci of platelet engraftment (⩾20 000/μl) on day +56 was 71% (95% ci 51-99%) and 90% (95% ci 74-100%) (p = 0.2). primary engraftment failure was observed in 3 patients; 1 in yes-steroid and 2 in no-steroid. no primary engraftment failure was observed with myeloablative tbi (8-12 gy) (n = 9). ci of agvhd gii-iv (day+100) was 21% (95% ci: 8-58%) and 28% (95% ci: 12-64%) (p = 0.77). ci cgvhd (1 year) was 42% (95% ci 20-90%) and 49% (95% ci 27-87%) (p = 0.48). ci of relapse (1 year) was 22% (95% ci 7-75%) and 7% (95% ci 1-47%) (p = 0.71). ci of non-relapse mortality (nrm) (1 year) was 51% (95% ci 29-90%) and 38% (95% ci 19-77%) (p = 0.48). post-infusion noninfectious fever (d0 to +3) was observed in 12/14 (86%) and 12/15 patients (80%). median tmax was 103f and 102f (p = 0.38). only 1 patient in the no-steroid group developed life-threatening cytokine release syndrome and survived. no difference of viral reactivation was noted between groups. cmv reactivation occurred in 7 (50%) and 13 patients (87%), bk reactivation in 20% (in both groups), hhv6 in 36% and 40%, ebv in 14% and 7%. the 18-month overall survival was 46% (95% ci 17-74%) and 60% (95% ci 35-85%) (p = 0.53). the 18-month disease-free survival was 41% (95% ci 15-68%) and 53% (28-79%) (p = 0.46). t-cell replete haplo pbsc transplant is effective therapy for patients with high-risk hematological malignancies. high-dose steroid premedication with pbsc infusion neither influences transplant outcome nor prevents post-infusion febrile reaction. disclosure of conflict of interest: as discloses grant support (american porphyria foundation), consultation (medpace inc), research support (astellas and fate therapeutics), honoraria (alxion, and spectrum), and royalty for licensing of intellectual property (incysus biomedical). intrabone transplant of unwashed cb in hematological malignancies: engraftment and safety f giglio 1 , s marktel 1 , r greco 1 , m morelli 1 , mt lupo-stanghellini 1 , e xue 1 , l lazzari 1 , m marcatti 1 , m zambelli 2 , c parisi 2 , r milani 2 , s piemontese 1 , a assanelli 1 , c corti 1 , m bernardi 1 , f ciceri 1 and j peccatori 1 1 unit of hematology and bone marrow transplantation, irccs san raffaele scientific institute, milano, italy and 2 immunohematology and transfusion medicine unit, irccs san raffaele scientific institute, milano, italy cord blood transplant (cbt) in adult patients (pts) is limited by the risk of graft failure or delayed engraftment due to low cell counts. to improve the capacity and speed to engraft, intrabone (ib) cbt technique has been investigated, showing high rate of engraftment and low acute gvhd, also when compared with double cb transplant. cb units washing procedure has been suggested to remove dmso toxic potential effect. we report our experience in 15 adult pts with hematological malignancies receiving ib unwashed cb in an attempt to reduce the loss of progenitor cells and the risks associated with cell-washing procedure. between 2009 and 2016 we performed 15 allogeneic hematopoietic stem cell transplant (hsct) using unwashed cbu as a source and infusing them ib. all pts were adult and suffering from hematological malignancies. this population was characterized by very high-risk and advanced phase disease. all pts received a cb hsct because of unavailability of sibling or matched unrelated donors. eleven pts received a treosulfanbased myeloablative conditioning regimen and a sirolimusbased ghvd prophylaxis; four pts received busulfan-based myeloablative conditioning and a cyclosporine-based ghvd prophylaxis. cb units were thawed and diluted with albumindextran solution immediately before the transplant. this ‛nowash' dilution was implemented to reduce product manipulation that may results in cell loss. furthermore, graft manipulation risks potential contamination, requires increased technologist time, and delays time to infusion. the ib infusion was performed under local anesthesia and with short conscious sedation, at bedsite in the bmt ward. the infusion was preformed monolaterally or bilaterally according to the volume to be infused. starting from a 10% dmso concentration in the cb units before the dilution, the graft products contained a median of 3.6% dmso (range: 2.3-6.9) at ib-hsct. the median cd34+ cells infused were 0.09 × 10 6 /kg b.w. (range: 0.03-0.82). the median mono-nucleated cells were 15.66 × 10 6 /kg b.w. (range: 4.7-36.8). the median cd3+ t-cells were 2.6 × 10 6 /kg b.w. (range: 0-5.54). the median infused volume was 75 ml (range: 60-215). no procedure-related adverse events were observed, nor related to the ib technique, neither to the sedation. of the 15 transplanted pts, 9 were evaluable for engraftment (1 patient rejected the graft and 5 patients died before day +30 because of severe infections). all 9 achieved anc 40.5 × 10 9 /l after a median of 24 days (range: 16-45) and 8 achieved plt 420 × 10 9 /l at a median of 45 days (range: 25-141). three patients developed grade iii-iv acute gvhd grade. according to extreme heterogeneity of the population no correlations with relapse incidence and diseasefree survival could be evaluated. ib infusion of unwashed cb is feasible, safe, easy to perform. no adverse events related to the procedure were documented. no dmso toxicity was documented. engraftment was obtained in all evaluable pts. our data confirm that direct ib cbt overcomes the problem of graft failure even when low numbers of cb cells were transplanted, thus leading to the possibility of using of this technique in a large number of adult pts, for whom this approach represents the sole possibility of long-term survival. the ‛no wash' cb dilution can also help the implementation of ib transplant thanks to the easier graft manipulation. [p117] disclosure of conflict of interest: none. lower incidence of cgvhd after cord blood transplantation for hematological malignancies in comparison with hematopoietic stem cell transplantation from other donors: 20 years' experience in a single institute m yoshino, m obiki, m osakie, s ikeno, t sato, m nasashima, y kagaya, n kawashima, t morishita, y ozawa and k miyamura department of hematology, japanese red cross nagoya first hospital the outcome of cord blood transplantation (cbt) for hematologic malignancy was investigated. however the incidence of gvhd is not accurately known. the goal of this study was to compare the outcome of cbt with allogenic hematopoietic stem cell transplantation (allo-hsct) from other sources, mainly unrelated bone marrow (urbm). patients' characteristics: 755 patients who underwent allo-hsct, between 1990 and 2015 in our hospital were retrospectively analyzed. donor sources were cord blood cell (n = 112), urbm (n = 372), hla matched sibling bone marrow (sibling bm) (n = 166), and hla matched sibling peripheral blood stem cell (sibling pbsc) (n = 105). in cbt, the median age was 38.5 (17-68), and the diagnosis included aml (69), all (15), mds (13), cml (4) and other (11). the disease risk was good in 58 and poor in 54. disease risk was slightly higher in comparison with other sources. prophylaxis of acute gvhd was tacrolimus, short-term methotrexate (88), cyclosporine, short-term methotrexate (22) and others (2) . the 5-year overall survival (os) rate after cbt (1990 cbt ( -2004 10 .0%, engraftment failure 6.7%, acute gvhd 3.3%), relapse 10.0% and other 6.7%. in cbt cases, engraftment failure after allo-sct was observed in 24 cases (21.4%) which is higher than that among urbmt (8.6%), 32 out of 372. 10 cbt cases underwent the second allo-hsct and 9 patients achieved engraftment and 7 patients were alive at 100 days after allo-hsct. 6 of them survived at 2 years after allo-hsct. our results suggest that the outcome of cbt has improved in recent years. moreover, cbt has an advantage in the least cumulative incidence of acute/chronic gvhd. cbt may well create the best outcome in the future. disclosure of conflict of interest: none. chronic active epstein-barr virus (ebv) infection is a major type of ebv-associated t/nk-cell lymphoproliferative disorders (lpd) in childhood. however, young adults rarely develop chronic active ebv infection (caebv), and shows more aggressive features than that of childhood. umbilical cord blood transplant (ucbt) is a possible treatment option for caebv patients who have no hla-matched donor, but there is little information available about the efficacy and safety of ucbt for adult patient with caebv. we analyzed six adult patients with caebv who underwent a single-unit ucbt between 2010 and 2016 at our institute (including a case reported in 2014 [1] ). the diagnosis of caebv was made according to the criteria proposed in 2005 [2] ; persistent infectious mononucleosis (im)-like symptom and detection of increased ebv genomes in peripheral blood mononuclear cells (pbmc). ebv-dna load was measured using real-time quantitative pcr. median patient age at diagnosis was 27 (23-39) years. target cells of ebv-infection were cd4+t cells (n = 4) or nk cells (n = 2). median ebv-dna load was 2.6 × 10 4 copies per microgram of dna in pbmc (range: 1.6 × 10 3 -1.3 × 10 5 ) at the diagnosis. all patients were given prednisolone and cyclosporine, and then etoposide (n = 2) or combination chemotherapy (n = 4) before transplant. ebv load has slightly decreased to a median of 1.3 × 10 4 copies (range: 4.0 × 10 2 -7.1 × 10 4 ), but disease status was active at ucbt in all. median time from the diagnosis to ucbt was 101 days (range: 62-440). one patient received total body irradiation (tbi) 12gy + cyclophosphamide (cy) 120 mg/kg + cytosine arabinoside 8 g/m 2 , and the other five patients received fludarabine (flu) + melphalan (lpam) 80-140 mg/m 2 or cy 120 mg/kg with tbi 4gy before ucbt. umbilical cord blood (ucb) was 4/6 hla-matched to the recipients. median number of infused ucb cd34+ cells was 0.87 × 10 5 /kg (range: 0.76-1.93 × 10 5 ). gvhd prophylaxis was consisted of tacrolimus + methotrexate or mycophenolate mofetil. neutrophil engraftment and complete donor chimerism were achieved in four patients, but two of them developed secondary graft failure (gf) early after engraftment. the other two patients developed primary gf. second ucbt was successfully performed in the 4 patients with gf a median of 31.5 days (range: 28-58) after the first ucbt. ebv genomes in pbmc became undetectable immediately after ucbt. at a median follow-up of 873 days (range: 54-2446), ebv-dna was undetectable or very low, and im-like symptoms were resolved in all cases. however, at 7-9 months after ucbt, two patients developed ebv+ b-cell lpd derived from donor cells, that was successfully treated with rituximab therapy. this study suggested that ucbt could eradicate ebv-infected cd4+ t cell-or nk cell-clones. ucbt can be a treatment option for adults with caebv. rituximab monotherapy was effective for post-transplant lpd from donor b cells. however, a high incidence of gf was observed in patients receiving reduced-s176 intensity conditioning of flu/lpam or cy /low-dose tbi. further studies are needed to find more optimal regimens for stable engraftment of ucb in adult patients with caebv. there is an increased incidence of ab0 incompatibility-50-60%, in allogeneic hematopoietic stem cell transplantation (allohsct) in patients who are russian citizens as a result of the variability of genetic polymorphism in the multi-ethnic population and a significant number of unrelated donors from international bone marrow registries. ab0 incompatibility in different types of allohsct may be an additional aggravating factor for the development of immunological complications and decrease effectiveness of treatment, but the data is still controversial [1] . from may 1999 to december 2015 in raisa gorbacheva memorial institute for children oncology, hematology and transplantation 1131 patients with leukemia, malignancies and hereditary diseases were included to the study, who were performed 1428 hsct: allogeneic unrelated -814 (57%); allogeneic related-344 (24.1%); haploidentical -267 (18.7%); umbilical cord blood in 3 patients (0.2%). age was 0-76, median-25 years. patients were predominantly with acute myeloid leukemia-37% (n = 602), acute lymphoblastic leukemia-30% (n = 501) and chronic myeloid leukemia -6% (n = 94). results: in 54.6% of cases (n = 780) ав0 incompatibility was determined: major-37.8% (n = 295); minor-45.4% (n = 354); combined-16.8% (n = 131). ав0 incompatibility in allohsct did not influence overall survival (p = 0.56) and frequency of acute graft versus host disease (gvhd) (p = 0.2). also there was no difference in overall survival depending on combination of condition regimen and ab0 incompatibility: reduced intensity (ric) or myeloablative (mac) (p = 0.7). an increased frequency of acute gvhd was observed in ric and ав0 incompatibility (30.8%) compared to mac (15.3%, p = 0.002). ab0 incompatibility was not a major factor (log worth 0.87) which influenced the fact and speed of donor's transplant engraftment in comparison to level of hlacompatibility (15.1), hematopoietic stem cell source (7.05) and type of hsct. but the presence of major ab0 incompatibility increase the period of erythroid recovery (p = 0.01) as reflected in the higher amount of blood transfusions. complications caused by ab0 incompatibility were identified in 2.4% of all cases (n = 34) including acute and delayed hemolysis, partial red cell aplasia and immune thrombocytopenia. conclusion. the presence of ав0 incompatibility is not a limiting factor to perform allohsct, however, it demands high quality prophylaxis and accurate transfusion therapy depending on ab0 incompatibility type to prevent immune complications. keywords: allogeneic hsct, ab0-incompatibility. poor graft function or graft failure have become common indications for infusion of immune-selected cd34 + cells (‛boost') or second unprocessed allo-hsct, creating the need for remobilization of the same related or unrelated. we retrospectively compared the results of two consecutive cycles of rh-g-csf treatment and peripheral blood progenitor cell collections in 20 related donors cared for at our institution between 2008 and 2016. mobilization consisted of the administration of rh-g-csf at a dose of 10 μg/kg per day injected in the evening, and apheresis was started in the morning of the fifth day after the fourth dose of rh-g-csf. collection was performed with a spectra or spectra optia cell separator (terumo bct). eleven out of 20 were haplo-mismatched donors and 9 were hla matched donors. four donors were re-collected because of recipient graft failure and 16 because of poor graft function; in the latter situation, immunomagnetic selection of cd34+ cells was performed on the collected cell product prior to infusion into the recipient, using the clinimacs medical device, as previously published. median donor age was 43 years (range: 18-66) at time of first donation, median weight 70kg (45-112) and bmi 24 (18-34). median delay between mobilizations 1 and 2 was 192 days (28-1530). interestingly, the median delay between collections was 119 days (43-701) in the haplomismatched setting and 231 (28-1530) in the matched setting. median number of circulating cd34+ cells/μl after the first 4 injections of rhg-csf was 61 vs 37 at the first and second mobilization cycles (po0.001, table 1 ). seven out of 20 donors (35%) requested more than one apheresis session to obtain the target number of collected cd34+ cells during the first cycle, as compared to 12 out of 20 (55%) for the second cycle: this is largely due to the higher target of cd34 + cells for the second collection, expecting that the median cd34 recovery after immunomagnetic selection is 60% in our experience. our study shows that a second cycle of mobilized peripheral blood progenitor cell collection from related donors is associated with a significant reduction in response to hematopoietic growth factors and mobilization capacity. this information allows planning the number of aphereses at the second cycle-and subsequently the number of immunoselection procedures to be carried out-taking into account the higher cd34+ cell dose target needed for subsequent immunomagnetic selection. cmv reactivation remains one of the main complications after allogeneic stem cell transplantation (hsct), requiring antiviral therapy, causing myelosuppression, prolonged hospitalization, higher treatment costs and mortality. cmv seronegative donors are recommended for cmv seronegative recipients. however data about donor selection for cmv positive (cmv-pos) recipients is not conclusive. some studies showed that selecting cmv-pos donors for cmv-pos patients might be beneficial. cmv-seropositivity is very high in lithuania among healthy blood and bone marrow donors (65%) and even higher among hsct recipients (up to 90%), so donor selection for cmv-pos recipients is an object of interest. retrospective analysis of cmv reactivations in cmv-pos allogeneic hsct recipients (transplanted during 2005-2014 year in vilnius university hospital) who survived at least 6 months post hsct was performed. data about cmv reactivation frequency, time post hsct, duration, maximal cmv dna copy number at each reactivation collected. cmv reactivation was considered when cmv dna copies detected 4500/ml in patient's blood. statistical analysis conducted using sas 9.2; student's tests for statistical significance; kaplan-meier methods for overall survival. among 316 allogeneic hsct recipients 286 (90.5%) were cmv-pos. 286 cmv-pos allo-hsct recipients were further analysed. 150 of them received graft from cmv-pos (pos/pos group) and 136-from cmv-seronegative donors (pos/neg group). more patients in pos/neg group experienced cmv reactivation in first 6 months post hsct in comparison to pos/pos group (83.1% vs 65.3%, p o0.05). pos/neg group patients had more cmv reactivations (2.5 vs 2 times in 6 months post transplant period, po 0.05), reactivations were diagnosed earlier post transplant (50.7 vs 123 days post hsct, p o0.0001), had longer duration (60.9 vs 40.3 days, po0.0001) and larger maximal cmv dna copy number (285107,9 vs 24339.4 copy/ml, p o0.0001) in comparison to pos/pos group patients. pos/pos group patients showed tendency for better survival than pos/neg group patients, however did not reach statistical significance. in a univariate analysis only hla mis-match and donor cmv seronegativity were factors statistically significantly associated with cmv reactivation. donor cmv serostatus is significant factor selecting donor for allo-hsct recipients. according to our findings, selecting cmv-pos donor for cmv-pos recipient may reduce cmv reactivation frequency and duration. disclosure of conflict of interest: none. selection of the best hsc donor when a matched donor is not available is still a matter of debate, and reports in pediatric population are scarce. this is a retrospective study conducted by brazilian society of hsct (sbtmo), including 11 centers, aimed to compare matched unrelated (matched-urd), mismatched unrelated (mm-urd) and unrelated cord blood (ucb) hsct. all or aml/mds patients o 18 y/o who have received first unrelated hsct between 2010-2014 were included. hla 4-digit typing was available for urd; for ucb, hla class-i 2-digit typing. overall survival (os), and cumulative incidence (ci) of agvhd, cgvhd, nrm and relapse were analyzed. on an unplanned analysis, we fitted a lognormal bayesian survival model with random effects, imputing the probabilities of ucb matching at 8 loci. a total of 212 patients were included (93 matched-urd, 47 mm-urd and 72 ucb). median age was 8.7 y/o. most patients had all (61%). proportion of early disease in matched-urd was higher (37%, against 19 and 21%). matched-urd were 10/10 4-digit hla matched (except one, for whom dq was not available), while most of mm-urd (89%) were 9/10 hla matched. ucb were 6loci (58%) or 8-loci typed (42%). based on previous report 1 on extending 6-loci hla typing to 8-loci ucb, we estimated that 23 ucb were 0-1 mismatched at 8 loci, 26 had 2 mismatches and 23 had 3 or more mismatches. conditioning regimens were mainly myeloablative, tbi-(59%) or bu-(33%) based. grafts were in vivo t-cell depleted in 67% of the patients, not balanced between groups (p = 0.01). with median follow-up of 3.3 years, 3y os was 56%, 52% and 39% for matched-urd, mm-urd and ucb, respectively (p = 0.02, log rank test). for matched-urd, mm-urd and ucb, ci of grades iii-iv agvhd at 6 months were 18%, 13% and 17% (p = ns); moderate/severe 3y-cgvhd, 8%, 20% and 4% (po 0.05); 3y-relapse, 26%, 21% and 14% (p = ns); and 1y-nrm, 26%, 24% and 49% (p = 0.06). we found out that primary graft failure occurred in 14 (19%) of ucb, compared to 9% in mm-urd and 3% in matched-urd. when ucb matching probabilities at 8 loci were imputed and analyzed in a bayesian model (controlled for age, gender, disease status and diagnosis, and t-cell depletion), survival was inferior in ucb with 3+ mismatches (9.8-times lower median survival, 95 ci 2.6-39.4), but not with up to 2 mismatches (1.3-time higher median survival, 95 ci 0.4-3.8). of note, in vivo t-cell depletion marginally impaired survival (2.4-times decrease, 95 ci 1.0-6.0). discussion: in our population, overall survival achieved with mm-urd was not different to 10/10 matched-urd, despite higher incidence of moderate/ severe cgvhd. on the other hand, survival with ucb was significantly lower. recent report 2 have shown excellent os with ucb compared to 8/8 hla-matched urd. ucb cohort inferior results may have been due to hla disparity degree, since survival in ucb with up to 2 mismatches (out of 8) was not worse. one limitation of our study is that tnc and cd34 from ucb units were not available, impairing primary graft failure analysis. we have also found that in vivo t-cell depletion might have a detrimental effect on survival and should be studied further in prospective trials. in conclusion, mm-urd, especially hla 9/10, is a suitable option when a fully hla 10/10 matched-urd is not available. ucb matched at least 6/8 may also be a good option. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is a proven treatment for patients with high risk or relapsed hematological malignancy. the probability of having a hla matched family donor is about 30%. in populations with high consanguinity rates, the probability of having non-sibling matched family donor(mfd) is much higher. to explore the impact of msd vs non-sibling mfd on outcome of hsct recipients, we undertook a single center retrospective analysis of pediatric patients transplanted with the diagnosis of hematological malignancy at our center in the last five years. a retrospective cohort from 2011 to current included 113 pediatric patients with hematological malignancies transplanted from family donors, of which 98 were from msd and 15 from non-sibling mfd. hla matched family donors were identified by high resolution allelic typing and were matched 10 of 10 hla loci. diseases were all (n = 58), aml (n = 37), mds (n = 7), jmml (n = 5), kml (n = 2), nhl (n = 1) and hodgkin's disease (n = 3). conditioning regimens were tbi or busulphan-based myeloablative in all patients. the median age of the patients was 9.4 years (range: 5 month-18.9 years). although peripheral stem cell seemed to be used more commonly in non-sibling mfd recipients (64% vs 42%), the difference was not statistically significant. the median follow up time for alive patients 24 months (1-67 months) . two year overall survival and leukemia free survival did not differ between patients with transplantations from msd or nonsibling mfd ( 67% ± 5.0 vs 60 ± 5.1, p = 0.31) similarly, leukemia free survival was not different between msd and non-sibling mfd transplants (58.2% ± 13.1 vs 58.1% ± 13.2, respectively). the incidences of grade ii-iv acute gvhd in msd and nonsibling mfd transplants were % 18 and %13, respectively. the incidences of relapses were 28% in msd transplants and 20% in mfd transplants and the difference was not significant (p = 0.49). these data show that the results of hsct from nonsibling mfd is comparable to hsct of msd in children with hematological malignancy. our data emphasize the need for extended high resolution family typing for patients in regions where there is high rate of consanguinity. disclosure of conflict of interest: none. [p125] p127 donor-recipient rh incompatibility is a risk factor for mortality after pediatric matched related allogeneic hematopoietic stem cell transplantation k ghanem 1 , n hariss 1 , z merabi, n kreidieh 2 , n tarek, r saab 1 , s muwakkit 1 , h el-solh 1 and m abboud 1 1 american university of beirut, department of pediatrics and adolescent medicine and 2 american university of beirut, optimal donor selection is critical to achieve the best outcome after allogeneic hematopoietic stem cell transplantation (allo-hsct). there is no consensus regarding the effect of donor-recipient rh incompatibility on survival after matchedrelated donor (mrd) allo-hsct in children and adolescents. this abstract aims to study this effect in a single-institution cohort over a period of 11 years. this is a retrospective chart review for all patients aged o21 years who underwent allo-hsct at the american university of beirut medical center between august 2005 and june 2016. a total of 70 patients with a median age of 11 years (range: 0.6-21 years) underwent allo-hsct from mrd for the following diseases: leukemia (n = 36), bone marrow failure (n = 15), thalassemia (n = 8), scid (n = 5), metabolic diseases (n = 4) and lymphoma (n = 2). the stem cell source was bone marrow for 56 patients (80%) and mobilized peripheral blood stem cells for 14 patients (20%). the grafts contained a median of 5.3 × 10 6 /kg total cd34 cells. tbi was used in 13 patients (18%). all but 4 patients achieved sustained neutrophil and platelet engraftment. after a median follow-up of 47 months (range: 4-135 months), the 2-year overall survival rate was 70% (95% ci: 57-79%). by multivariate analysis using cox proportional hazard regression model looking at the following factors for overall mortality: diagnosis, recipient's age, donor's age, the use of tbi, stem cell source, cd34 count, donor-recipient abo incompatibility, donorrecipient rh incompatibility, and donor-recipient sex-mismatch, the only statistically significant risk factor for mortality was donor-recipient rh incompatibility (hr: 3.26, p: 0.04). this risk was not statistically significant when looking at transplantrelated mortality (hr: 3.9, p: 017) and relapse-related mortality for malignant diseases (hr: 3, p: 0.12). there was no association between the incidence of acute or chronic gvhd and rh incompatibility. donor-recipient rh incompatibility was associated with an increased risk of mortality in children and adolescents undergoing mrd allo-hsct. further studies with larger number of patients are needed to confirm this finding. disclosure of conflict of interest: none. effect of iron or vitamin b12 deficiencies on in vitro colony forming capacity of peripheral blood-derived hematopoietic stem cells in children ny özbek, mm zabun, y köksal and m özgüner iron deficiency (id), id anemia (ida)and vitamin b12 deficiency (vit-b12d) are common disorders in developing countries. in urgent situations, children with these disorders could be donors before treatment. in this study, we investigated capacity of peripheral blood-derived hematopoietic stem cells to develop colony-forming units (cfu) in children with id and vit-b12, in vitro. patients and methods: we included 102 children (age 6 months-18 years) in the study in 5 groups: children with id (n = 15); children with ida (n = 20); children with vit-b12d (n = 12); children with both id and vit-b12d (i/ vit-b12d; n = 18); and control children (n = 37) who has normal peripheral blood findings, and normal ferritin and vit-b12 levels. from each child complete blood counts (cbc), and levels of ferritin, vit-b12, and cero-reactive protein (crp) have been obtained. who criteria, adjusted for age and sex, have been used for definition of anemia, id, ida and vit-b12d. four ml peripheral blood drawn into tubes with edta has been used for cfu analysis. mononuclear cell suspension (1.5 × 10 6 cell/ml), obtained from peripheral blood by ficoll-hypaque density gradient separation method, has been cultured in dishes containing semi-solid agar culture medium (methocult, h4434 classic, stem cell technologies, canada) in appropriate conditions. after 2 weeks, number of cfu colonies [burst forming erythroid (bfu-e); colony forming unitgranulocyte macrophage (cfu-gm); colony forming unitgranulocyte-erythrocyte-monocyte-megakaryocyte (cfu-gemm)] have been investigated by an inverted microscope. results: statistical analysis showed no difference between groups for age, sex, crp levels, and cfu-e, cfu-gm and cfu-gemm numbers. however, expected differences between groups were present concerning mean values of hemoglobin, ferritin and vit-b12 levels, mean corpuscular volume (mcv), and red cell distribution width (rdw) ( table 1) . discussion: this study shows in vitro proliferation capacity of peripheral stem cells has not been influenced by id, ida, vit-b12d, or i/vit-b12d. our results may indicate normal grafting ability of peripheral stem cells obtained from donors with iron, vit-b12 or i/vit-b12 deficiencies for hematopoietic stem cell transplantation. however, in vivo analysis should also be performed in order to reach a definite conclusion. [p128] disclosure of conflict of interest: none. efficiency of day 4 compared to day 6 stem cell mobilization in allogeneic donors h al-gaithi 1 , s al-mamar 2 , m al-huneini 2 , d dennison 2 , s al-kindi 2 , k al-farsi 2 and m al-khabori 2 1 hematology residency training program, oman medical specialty board; 2 hematology department, sultan qaboos university hospital granulocyte colony stimulating factor (g-csf) given for 4-6 days is commonly used for mobilization of allogeneic stem cell donors. the optimal days of g-csf administration is still debatable. the primary objective of this study is to compare the yield of stem cell mobilization, assessed using cd34+ cell count, between day 4 and day 6. secondary objectives include the assessment of the impact of donor's age, weight, mean corpuscular volume and blood group on the difference in the cd34+ cell count. in this retrospective study we included all allogeneic stem cell donors mobilized with g-csf for 6 days from january 2003 till october 2015 in the bone marrow transplantation unit at sultan qaboos university hospital. of 106 donor records reviewed, 84 were with available data and selected for the study. descriptive and analytical statistics were performed using stata 13.1. we included 84 donors with median age and weight of 19 years and 60 kg, respectively. the median day 4 wbc and cd34+ cell count were 37.4 × 10 9 /l and 54 × 10 6 /l respectively; while the median day 6 wbc and cd34+ cell count were 44.4 × 10 9 /l and 86 × 10 6 /l, respectively, (figure) with a statistically significant difference from day 4 (p o0.001). in the multivariable model, there were no significant impact of donor's age (p = 0.215), weight (p = 0.108), height (p = 0.428) and mean corpuscular volume (p = 0.263) on the difference in cd34+ cell yield. however, donor's blood group ab predicated a significantly higher difference (p = 0.036). six days of g-csf mobilization achieves higher cd34+ cell count than 4 days in allogeneic stem cell donors especially in donors with blood group ab. however, cd34+ cell count on day 4 is high enough to allow for successful mobilization. appropriately designed prospective trial is needed to confirm these results. disclosure of conflict of interest: none. there are known differences between individuals on an unrelated hsc donor register who decide to proceed with verification typing (vt) vs those who choose not to. in the anthony nolan registry, white british donors are more than twice as likely as other ethnic groups to continue with testing at vt (or 2.44; po 0.001 (unpublished data)). the purpose of this study was to explore differences in key characteristics between white british donors and british donors from other ethnic groups with a view to developing interventions to reduce vt stage attrition. study recruitment occurred april 2013-may 2016. all donors not proceeding at vt were invited to participate, and a stratified random sample of those proceeding at vt were recruited to meet pre-determined targets for each ethnic group. data were collected via structured interview (telephone or online). 4 broad categories of participant characteristics were assessed: demographic, culturally related, psychosocial, and donation-related. measures were previously validated scales with established psychometric properties either created for, or used in other donation-related settings. for analyses donors were divided into two groups based on ethnicity: white british (wb), and non-white british (nwb). results: 170 wb donors and 187 nwb donors completed interviews donors proceeding at vt were more likely than their counterparts to participate in the study (66% vs 25%, p o0.001). mean donor age was 33.8 with no difference between ethnic groups and 43% of donors in both groups were female. nwb were statistically more likely to have completed higher education, and have a stronger religious affiliation. in contrast they were less likely to be blood or organ donors. nwb also described greater mistrust of the medical system and of hsc allocation. nwb donors were more likely to have joined the register at a recruitment event (p=0.012) or a place of worship (p=0.012), while wb donors were more likely to have joined online (p=0.013). wb donors reported significantly higher scores regarding feeling well informed about donation both at the point of joining, and at the point of vt and were more likely to remember joining the register and the two donation methods. this study highlights important differences in demographics, culturally related variables and donor interaction with the register between white british donors and donors from other ethnic backgrounds. given the higher rate of vt attrition in nwb donors, these findings could be used to tailor interactions/information given to donors on the register to ensure their priorities are addressed. disclosure of conflict of interest: none. data on mismatched family donor transplants for myelofibrosis are scarce due to the risk of poor engraftment, gvhd and exclusion from trials. outcomes from such transplants performed between 2001 and 2015 reported to the ebmt are presented. sixty-nine patients, median age 58 (27-71) years; 44 (64%) male, 50 (74%) had primary, 18 (27%) had secondary myelofibrosis (6 from et, 5 from prv and 7 others) and unknown 1(2%). jak2 v617f was mutated in 15/25. karnofsky performance status was 470% in 98 %; median time from diagnosis to allograft was 41.4 (range: 0.72-213) months. the donors were predominantly male 47 (68%), median age 42 (22-75) years, hla mismatched at 1 locus in 12 (17%) and 2 or more loci in 48 (70%). donor-recipient serology was cmv − / − in 8 (12%) ± in 4 (6%), − /+ in 15 (22%) and +/+ in 34 (49 %) missing 8 (12%) . bone marrow was used in 34 (49%) and peripheral blood in 35 (51%). the median total nucleated cell count (tnc) was 7.5 × 10 8 /kg (range: 2.3-21 × 10 8 /kg) (n = 17). the median cd34+ cell dose was 6.9 × 10 6 /kg [p130] s182 (range: 1.9-18.18 × 10 6 /kg)(n = 19). patients. conditioning was myeloablative in 48 (70%) and ric in 21 (30%). predominant conditioning regimes were fludarabine, busulphan, atg (fbatg) and thiotepa, busulphan, fludarabine (tbf n = 33). tbi was administered in 8 (12%) and t cell depletion in vivo in 22 (32%) and ex vivo in 5 (7%) patients. gvhd prophylaxis varied with post transplant cyclophosphamide administered in 33/67 (48%) and atg in 19/67 patients (28%).neutrophil engraftment occurred in 53 (82%) patients at a median of 20 days (range: 11-83). primary graft failure ensued in 8 (12%) and secondary graft failure in 4 (6%) patients at a median of 12 (range: 4.5-35) months. eleven patients had a second allograft at a median interval of 4 (1-20) months. responses to the first allograft censoring for a second allograft, data available in 45 patients, showed that complete remission was achieved in 35 patients (78 %), 6 (13 %) were never in cr and 4 (9 %) were not evaluable. relapse occurred in 8 (12%) of patients at a median interval of 3 (2.8-21.8) months. the cumulative incidence (ci) of grade ii-iv acute gvhd (agvhd)was 12% (95% ci 4-21%) and for grade iii-iv agvhd at was 5% (95% ci 3-11%). data for chronic gvhd (cgvhd) was valid in 49 patients of whom 47% developed cgvhd. the ci of cgvhd at 2 years was 58% (95% ci 43-72%):ci of limited cgvhd was 45% (95% ci 31-59%) whereas the ci of extensive cgvhd was 10% (95% ci 2-19%). median follow-up was 24 (95% ci 13-35) months. the 2 and 5 year os was 53% (95% ci 40-66%) and was 40% (95% ci 23-57%). the 2 and 5 year rfs was 43% (95% ci 30-56%) and 31% (95% ci 15-47%). the 2-year ci of relapse was 20% (95% ci 10-30%). the 2 year nrm was 37% (95% ci 25-49%), which increased to 49% (95% ci 32-65%) at 5 years. thirty patients died due to infection (16, 53%), gvhd (7, 23%), organ damage or failure (3, 10%), relapse/disease progression (1, 3%) and secondary malignancy or ptld (1, 3%) unknown 2. there was no significant effect (univariate analysis) of recipient or donor gender, degree of hla mismatch, cmv matching, primary or secondary mf, chronic vs advanced disease at transplant, conditioning intensity or regimen, gvhd prophylaxis with atg or post transplant cyclophosphamide or stem cell source on overall survival. the data are encouraging for patients with myelofibrosis, with engraftment, pfs and os being attained with limited severe chronic gvhd from family mismatched donors. disclosure of conflict of interest: none for all other authors, fc consulting with molmed. feasibility of salvage second allogeneic stem cell transplantation for disease relapse or graft failure: a single centre experience g battipaglia 1,2 , d salvatore 3,1 , r dulery 3 , f giannotti 3 , f malard 2 , e brissot 2 , s sestili 2 , f isnard 2 , s lapusan 2 , a-c mamez 2 despite high rates of toxicity and mortality, a second salvage allogeneic stem cell transplantation (second allohsct) might be an option to consider in patients experiencing disease relapse or graft failure after first allohsct. we retrospectively analyzed outcomes after second allohsct in a cohort of 30 patients (18 males and 12 females) transplanted either for disease relapse (group 1, n = 19) or graft failure (group 2, n = 11) between 2007 and 2015 in a single centre in france. median age at second allohsct was 38 (range: 17-64) years. diagnoses were acute myeloid leukemia (group 1: n = 12; group 2: n = 3), acute lymphoblastic leukemia (group 1: n = 2; group 2: n = 4), myelodysplastic syndrome (group 1: n = 2; group 2: n = 1), myeloproliferative neoplasm (group 1: n = 3; group 2: n = 1), bone marrow failure (group 1: n = 0; group 2: n = 2). median time from first allohsct to second allohsct was 38 (range: 2.5-230) months in group 1 and 1.5 (range: 1-34) months in group 2. graft source for the second allohsct were: haploidentical bone marrow (group 1: n = 3; group 2: n = 1), haploidentical pbscs (group 1: n = 5; group 2: n = 1), cord blood (group 1: n = 8; group 2: n = 8), matched unrelated pbsc (group 1: n = 3; group 2: n = 1). at time of second allohsct, 11 patients were in cr and 8 presented active disease in group 1. conditioning regimen was myeloablative in 5 patients (group 1: n = 3; group 2: n = 2), reduced intensity (ric) in 20 cases (group 1: n = 11; group 2: n = 9). a sequential schema consisting of a combination of thiotepa, etoposide and cyclophosphamide followed by a fludarabine and busulfanbased ric was used in 5 out of 8 patients with active disease in group 1. sixteen patients received atg as part of the conditioning regimen for second allohsct (group 1: n = 10; group 2: n = 6). all but one patient engrafted, at a median time of 18 (range: 6-51) days. cumulative incidence of acute and chronic gvhd were 27 ± 17% and 42 ± 19%, respectively, 2-107) months, non-relapse-mortality (nrm) and relapse incidence (ri) were 24 ± 15% and 27 ± 15%, respectively, while disease-free (dfs) and overall survival (os) were 49 ± 19% and 48 ± 15%, respectively, for the entire cohort. in all, 15 patients died of infections (n = 8), hematological disease (n = 4), gvhd (n = 1), hemorrhage (n = 1) and for unknown causes (n = 1). main outcomes of patients in group 1 were: ri 16 ± 15%, nrm 28 ± 18%, agvhd 26 ± 15%, cgvhd 56 ± 20%, dfs 56 ± 20%, os 55 ± 20%, respectively. main outcomes of patients in group 2 were: ri 45 ± 22%, nrm 18 ± 24%, dfs 36 ± 29%, os 36 ± 29%, agvhd 27 ± 23%, cgvhd 18 ± 23%. historically, a second allohsct was hampered by significant morbidity and mortality. however, the advent of reduced-toxicity conditioning regimens and improved supportive care allowed to significantly improve the results of patients receiving a second allohsct as suggested from the above results. therefore, a second allohsct could be considered as an option to rescue a certain number of patients experiencing disease relapse or graft failure, for which prognosis is very poor. decision is to be discussed on a case-by-case basis. disclosure of conflict of interest: none. haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide for patients with high-risk hematologic malignancies am carella department of oncology and hematology, irccs casa sollievo della sofferenza, san giovanni rotondo allogenic hematopoietic stem cell transplantation (sct) has been increasingly used for treatment of adult with high risk hematologic malignancies. for patients lacking an hlamatched related or unrelated donor, unmanipulated haploidentical (haplo)-sct is a potential alternative. haploidentical transplantation performed with post-transplantation cyclophosphamide (ptcy)-based graft-versus-host disease (gvhd) prophylaxis has been associated with favorable outcomes for patients with acute leukemia and lymphomas we analyzed outcomes of 45 patients with hematologic malignancies who received t-cell-replete haematopoietic stem cells and posttransplantation cyclophosphamide after myeloablative or nonmyeloablative hla-haploidentical donor transplantation. the median age was 37 years (14-68); twelve patients were in first remission (cr1), 4 in second remission (cr2) and 29 had an active disease. ). the diagnosis was acute leukemia (n = 32), myelodisplastic syndrome (n = 3), hodgkin disease (n = 7) non hodgkin lymphoma (n = 2) and multiple myeloma (n = 1). median follow-up was 260 days. stem cell source was bone marrow (bm) for 42 patients, and peripheral blood (pb) for 3. myeloablative conditioning (mac) was used in 37 patients and reduced intensity regimen (ric) in 8 patients. thirty one patients were first grafts, the others underwent previous autologous sct (n = 11) or mud (n = 3). gvhd prophylaxis s183 consisted in pt-cy on days +3 and +4, cyclosporine (from day +5), and mycophenolate (from day +5). the median day for neutrophil engraftment was day +20 (14-29). no graft failure was observed. chimerism was evaluable in 39 patient; on day + 30 all patients had 100% donor chimerism on marrow cells median follow-up was 260 days. the cumulative incidence of acute gvhd grade ii-iv was 22%, grade iii-iv 9% and chronic gvhd 15%. one-and 2-years os was 56.53% and 53.39 %, respectively. with a median follow-up for the surviving patients of 752 days (130-2207), the cumulative incidence of transplant-related mortality (trm) is 22%, and the relapserelated death is 26%. thus, we demonstrate excellent rates of engraftment, gvhd, and trm in adult patients treated with haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide. this approach is a widely available, safe, and feasible option for adult patients with high risk hematologic malignancies, including those with a prior history of myeloablative bmt and/or those with co-morbidities or organ dysfunction, also for patients with active disease at the time of transplant. disclosure of conflict of interest: none. it has recently been shown that t-replete allogeneichematopoietic stem-cell transplantation (allo-hsct) from a haploidentical donor (haplo-id) could be a valid option when a matched donor is not available. unfortunately, the worldwide donor registries comprise mainly donors of caucasian origin and patients of non-caucasian origin have a much lower chance of finding a matched unrelated donor (mud). the lengthy period of international search when required and the financial burden of this process are considered as additional significant limitations. at the american university of beirut medical center (aubmc) in lebanon, we started the mud program in 2011 and haplo-id hsct program in 2014. we report here our experience in this two groups of patients. patients and methods: we have transplanted 21 patients from a haplo-id donor since 2014 and compare their outcome with the 6 patients transplanted from a mud since 2011. the patients and transplant characteristics are listed in the table 1 . the 2 groups were comparable except for conditioning. patients in haplo-id group received two days of posttransplant high-dose cyclophosphamide (pt-hdcy) followed by cyclosporine a (csa) and mycophenolate-mofetil while patients in the mud group received pre-transplant antithymocyte-globulins and csa starting on day-3. all patients engrafted in the mud group, while one patient did not engraft in the haplo-id group, the patient had refractory all transplanted with progressive disease, and died on day +47. the median of anc 4500/mm 3 was 14 days (12-20) vs 17 days (12-29) in the haplo-id and mud groups, respectively. fourteen patients from the haplo-id group developed grade 2 acute graft-versus-host disease (agvhd) vs one after mud-hsct. two patients haplo-id group developed limited cgvhd and none after mud grafts. six patients relapsed in the haplo-id group vs three patients in the mud group. two and three patients died from non-relapse mortality in the haplo-id and mud group, respectively. at the last follow-up, 13 patients are still alive in the haplo group vs 2 patients in mud group and all of them are in cr. we conclude that t-replete haplo-id hsct followed by pt-hd cy is associated with promising results or at least comparable to patients transplanted from mud. haplo-id hsct seemed to be safe and feasible in patients with high risk hematological malignancies. finally, because of the obvious advantage in rapidly finding a donor (21 haplo transplants in three years vs 6 mud transplants in 5 years), development of haplo-id hsct is warranted to satisfy the regional needs. [p134] disclosure of conflict of interest: none. haploidentical hematopoietic stem cell transplantation (haplo-hsct) using t-cell-replete (tcr) grafts and posttransplantation cyclophosphamide (ptcy) provides a curative approach for patients with high-risk mds/aml lacking a conventional hla-matched donor. in children and adults haplo-hsct using ptcy as gvhd prophylaxis seems to be safe with low treatment related morbidity and mortality (trm). however, few data are available for elderly patients with advanced disease. we retrospectively analyzed the outcome of 49 patients with mds (n = 5)/aml (n = 44) age 50-74 years (median age 60 years; 24 patients 50-59 years, 25 patients ⩾ 60 years; 21 male), who underwent tcr haplo-hsct with high-dose ptcy at our institution between january 2009 and november 2016. disease was active in 41 patients while 8 had achieved cr. 12 patients failed previous allo-hsct. pretransplantation risk factors were scored using the hematopoietic cell transplantation-specific comorbidity index (hct-ci) which was ⩾ 3 in 19 patients (median hct-ci = 2, range: 0-8). a sequential therapeutic concept using either flamsa (n = 28) or clofarabine (n = 18) as cytoreduction was used prior to reduced intensity conditioning (ric) in all but 3 patients. ric consisted of fludarabine/cyclophosphamide combined with either melphalan (n = 32), busulfan (n = 1) or 4 gy tbi (n = 12). post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mmf in all patients. 57% received a bone marrow graft. one graft rejection occurred. neutrophil and platelet engraftment was achieved in 95% and 77% of evaluable patients, respectively at a median of 19 (13-89) and 33 (11-103) days. acute gvhd grade i-iii occurred in 29% of the patients whereas no grade iv agvhd was observed. chronic gvhd presented in 33%. it was most frequently assessed as mild to moderate (13 pts). only 3 patients developed severe cgvhd; no gvhd related death was observed. cmv reactivated in 22 of 36 patients at risk, one patient developed cmv disease (pneumonia). no ebv reactivation or ptld occurred. one-year trm was 24%. 12/49 (24%) patients relapsed, three within the first 100 days after haplo-hsct. at a median follow up of 27 months (range: 4-74 months) estimated one-and two-year overall survival (os) was 55/46 %, respectively. when stratified by age, estimated one-and two-year os was 65/42% in patients o60 years and 47/47 % in patients ⩾ 60 years (p = 0.771/p = 0.794). one-and two-year progression-free survival (pfs) was 50/45%, respectively. stratified by age estimated one-and two-year pfs was 53/41% in patients o 60 years and 47/47% in the elderly (p = 0.836/p = 0.887). unmanipulated haploidentical allografting using ptcy-based gvhd prophylaxis in high-risk mds and aml patients aged over 50 years is safe and well tolerated resulting in acceptable trm. a remarkable survival outcome can be achieved in elderly high-risk aml/mds patients with significant comorbidities. disclosure of conflict of interest: none. key performance indicators to assess the quality of a collection facility: experience of a single center s roncon*, c pinho 1 , f bordalo 1 , s lopes 1 , s ferreira 1 and f amado 1 1 allogeneic hematopoietic stem cell transplantation (allo-hsct) has evolved into an effective immunotherapy for the treatment of a variety of disorders. when patients do not have a familiar matching donor, transplant centers (tc) search for an unrelated and volunteer donor. this one must be previously evaluated by the collection center (cc) to donate peripheral blood stem cells (pbsc) or bone marrow (bm); lymphocytes can also be asked after allo-hsct. this work aims to evaluate our performance as cc, ensuring donor safety, quality of cell therapy products (ctp) and the accomplishment of tc requirements. we retrospectively analyzed all the requests of ctp collections sent by the portuguese registry from 2012 to 2016. countries of destination, number and type of ctp were determined. we established eight key performance indicators (kpi) classified into four categories: response time; product quality; satisfaction of patients and donors; and on-site donor motivation. the intended target was defined by the mean result obtained in the first half of 2012 (excluding kpi-7). written comments from donor center (dc) and tc were received by email or written in the local notebook. the donor's answers were obtained through a survey given on the collection day. a total of 349 requests were assessed: 231 pbsc, 61 bm, 16 lymphocytes and 41 cancellations; 84% were sent to europe (98/259 to portugal), 14% to america and 2% to oceania; 30/41 were withdrawn by tc (14 patients died, 14 presented progressive disease and 2 had a better hlamatched donor) and 11/41 by dc (7 donors not cleared and 4 refused). the results obtained with kpi-1, -2, -4 and -7 exceeded the intended target (table 1) . after the first kpi-1 results, we verified a positive evolution. we took an average of 3 days of delay in sending donor clearance. however, there is no holdup in the ctp delivery, as demonstrated by kpi-2. regarding kpi-3 it is important to notice that 60% of ctp with a cell number less than requested were bm and lymphocytes; when pbsc was considered separately, the result increased (87% vs 75%). analyzing kpi-4, 85% (n = 11/13) of the contaminated ctp were bm. concerning kpi-5, -6 acknowledgments and -8 commitment, we recognize that our initial targets were too ambitious (100%). the kpi-6 shows a low number of complaints (n = 4): one due to a misreading of the request and three to communication failures; all were properly examined and rectified. a good general status was guaranteed in almost all the donors (kpi-7). the decrease of kpi-8 is due to the fact that one donor refused to proceed after three postponements of the collection date by tc. table 1 -key performance indicators of the quality of our activity as a cc. the overall good level of our results reflects an extremely professional performance as a cc. we consider that these kpi should be continuously monitored with the purpose of earlier detect any deviation of the stated goals and assess the progress against settled strategies. we further suggest the establishment of universal indicators in order to standardize [p136] practices, share expertise and improve the quality of services and products provided to patients and donors. two year later, the patient had a genoidentical allogenic stem cell transplant (from the bone marrow stem cell of his sister, who was 35 years old, hla compatible). he had a reduced intensity conditioning, associating busulfan, fludarbine and antilymphocyte serum. 6 months from the asct, he was in complete remission with 100% donor chimerism. 4 years after the asct, the patient presented a progressive thrombocytopenia without any other peripheral causes. the bone marrow aspiration initially showed a refactory cytopenia with multilineage dysplasia. the patient was followed up during 12 months, and then a second bone marrow aspiration has shown a refractory anemia with excess blasts2 raeb2. a cytogenetic study has every time demonstrated a female karyotype (44,xx) on 20 mitoses out is 20, and chimerism was 100% donor. the diagnosis of the myelodsplastic syndrome of the donor cells was approved. the patient was treated by azacitidine (75 mg/m 2 , from j1 to j7, j1 = j28). after 6 cycles, the patient was in complete hematologic response (normalization of the platelet count) and a partial bone marrow response (normalization of the blasts rate but persistence of the signs of dysplasia). he received 6 more cycles, and presented hematologic relapse (reemerging of thrombopenia). a phenoidentical allogenic stem cell transplantation was suggested. conclusion the occurrence of mds on the donor cells is rare. these anomalies are secondary to intrinsic factors (of donor) or extrinsic factors )of the transplant recipient). the treatment is not definitely determined. disclosure of conflict of interest: none. nk-cell alloreactivity based on kir/ligand mismatch in the donor vs recipient direction provides better graft-versustumor effect in patients with active hematological malignancies undergoing allogeneic t-replete haploidentical transplantation followed by post-transplant cyclophosphamide a wanquet 1,2 , s bramanti 1,3 , s harbi 1 , s fürst 1 , f legrand 1 , c faucher 1 , a granata 1 , p-j weiller 1,2 , c picard 4 , b calmels 5 , c lemarie 5 , c chabannon 2,5,6 , l castagna 1,3 , d blaise 1,2,6 and r devillier 1, haplo-sct have been developed in the past years with very interesting results in high risk patients. gvhd prophylaxis using post-transplant cyclophosphamide (pt-cy) recently allowed extending the use of unmanipulated haplo-sct. it was shown that nk alloreactivity, triggered by donor-recipient inhibitory kir gene-gene mismatches, could lead to better outcomes and survival in the setting of in t-cell-depleted haplo-sct. however, few data is available on the impact of kir-ligand mismatch on the outcome after t-replete haplo-sct with pt-cy. we thus assessed the impact of nk alloreactivity on the outcome of patients who received haplo-sct followed by pt-cy. we retrospectively collected the data from patients from two centers who were treated for various high risk hematological diseases and underwent a haplo-sct with pt-cy from december 2009 to december 2014. we assessed the kir-binding epitope in hla-c and hla-b molecules for all patients, and we predicted nk cell alloreactivity in the donor vs recipient direction via the immune polymorphism database kir ligand calculator, based on the kir-ligand mismatch between donors and patients. because disease status at the time of haplo-sct is one of the most important predictor of outcome, we separately analyzed two cohorts of patients: those transplanted in complete remission (cr group) and those transplanted with active disease (no cr group). using a multivariate cox model (adjusted by disease type, age and conditioning), we therefore evaluated the impact of nk alloreactivity on outcome in both cr and no cr groups. we analyzed 144 patients with a median age of 54y (20-74). they were mostly transplanted for lymphoma (n = 72, 50%) or aml/ mds (n = 47, 33%). patients mostly received a tbi-based nonmyeloablative conditioning regimen (n = 94, 65%) and pbsc as graft source (n = 91, 63%). eighty one and 63 patients were transplanted in cr and in no cr, respectively. nk alloreactivity was found in 30/81 cr patients (37%) and 22/63 no cr patients (35%). with a median follow up of 30 months (12-77), cr patients had a significantly better outcome than those in the no cr group (2-year pfs 65% vs 32%, respectively, p o0.001). in no cr patients, multivariate analysis showed that nk alloreactivity was significantly associated with reduced the risk of relapse (hr = 0.25, p = 0.019, figure 1a ) with no increase of both acute (hr = 1.30, p = 0.648) and chronic gvhd (hr = 2.61, p = 0.232), and nrm (hr = 0.60, p = 0.277). this led to significantly better pfs (hr = 0.41, p = 0.014, figure 1b ) and a trend for better os (hr = 0.52, p = 0.069). in contrast, in cr patients, we found no difference in outcome according to nk alloreactivity for all end points (acute gvhd: hr = 1.78, p = 0.204; chronic gvhd: hr = 2.11, p = 0.321, nrm: hr = 1.69, p = 0.313, relapse: hr = 0.85, p = 0.762, figure 1c ; pfs: hr = 1.19, p = 0.637, figure 1d ; os: hr = 0.83, p = 0.672). our results suggest that nk alloreactivity provides better disease control with no increase of gvhd, especially in patients transplanted with active disease. thus, donor selection should rely on the prediction of nk alloreactivity. this may contribute to improve outcome of these patients with high risk of relapse after transplantation, underlining the need of a specific strategy of donor search, and the promising perspective of early post-transplant nk-cell-based immunotherapy. haploidentical bone marrow transplantation (haplo-bmt) with post-transplant cyclophosphamide (pt-cy) is being increasingly used, in the last five years, for patients lacking a suitable hla-matched donor. genoa study (eudract number: 2012-000703-32) provides for a modified gvhd prophylaxis platform compared to the original baltimora protocol. aim of the study: in this study we assessed outcomes in 282 consecutive patients transplanted from a haploidentical donor for haematological malignancies. all patients received a uniform gvhd prophylaxis: cyclosporine (csa) starting on day 0, mycophenolate (mmf) starting on day +1, and post transplant cyclophosphamide (pt-cy) 50 mg/kg, on days +3 and +5. all patients received a myeloablative conditioning consisting of thiotepa, fludarabine, busulfan (three doses n = 116 or two doses n = 111), or tbi, fludarabine (n = 55). the median age was 48 years (17-74); at transplant 145 (51%) patients were in remission of disease (cr1 and cr2), and 137 had an active disease (49%); all patients were first grafts. the diagnosis were acute myeloid leukemia (n = 111), myelodisplastic syndrome (n = 31), acute lymphoblastic leukemia (n = 56), myelofibrosis and myeloproliferative diseases (n = 43), non hodgkin lymphoma (n = 19), chronic lymphocytic leukemia (n = 9) and multiple myeloma (n = 13). the median follow up was 562 days (range: 6-2241 days). the median infused mononucleated cells was 3.4 × 10e8/kg (range: 1.1-7.7). seven patients died before engraftment, and 21 (7%) had autologous recovery: 15 (5%) after conditioning with 2 doses of busulfan. full-donor chimerism on day +30 was reached in 254 (90%) patients. the median day for neutrophil engraftment was day +18 (range: 13-60 days). the cumulative incidence of grade ii-iv and iii-iv acute gvhd (agvhd) was 17% (n = 49) and 5% (n = 15), respectively. two years cumulative incidence of moderate-severe chronic gvhd (cgvhd) was 13% (n = 39).sixty one (21%) patients experienced haemorragic cystitis. at 3 years the cumulative incidence of non relapse mortality (nrm), relapse and relapse related death was 17% (n = 47), 32% (n = 91) and 25% (n = 69), respectively. causes of death were infections (n = 34), hemorrhage (n = 7), gvhd (n = 5), secondary neoplasia (n = 1) and relapse (n = 69). at 4 years of follow up overall survival and disease free survival was 55.7% and 47%, respectively. at the same time overall survival rate was 73% for patients in remission and 29% for patients with active disease at transplant(p o0.001). in conclusion, a modified pt-cy as gvhd prophylaxis and ma conditioning regimen followed by haploidentical bmt results in a low risk of agvhd and cgvhd and encouraging rates of trm and dfs. disclosure of conflict of interest: none. the italian bone marrow donor registry (ibmdr), in collaboration with admo (associazione donatori di midollo osseo) since 2009 has implemented, as part of the donor enrollment strategy, public enrollment events (pe). our donor center (dc) has taken part to those events since the first years. one or more clinician (or trained biologist) has been present to pe to inform the potential donors, evaluate the candidates and supervise the collection of biological fluids. all the local permission where obtained. aim: aim of this study was to compare the compliance of the donor enrolled in pe with donors enrolled at our dc institutional site. we prospectively evaluated all the donors recalled for further evaluation and/or for requalification in the years 2014 and 2015 at our dc itmi07. we defined 3 possible results for the call: ‛success' (the donor was eligible and accepted to be evaluated, or only temporarily ineligible) ‛not eligible' (the donor was definitively ineligible) and ‛consent denied'. results: a total of 286 donors were called back in the years 2014 and 2015 (16 not found). eightyfour recalled donors had been enrolled after 2009. among them 53 (63.1%) had been enrolled at the dc and 31 during pe (36.9%). the two populations were not different for age at the call, age at enrollment and gender (table 1) . [p140] when evaluating the probability of obtaining a "success", no significant difference was found between the two populations: 86.8% vs. 83.9% (chi square p=0.23). no significant difference was also found for the "not eligible" and the "consent denied" categories. of note, when we turned to the whole 286 donor population we had called back (median age 34, range 21-55), the probability of "success" and "consent denied" were not related to donor age, and time from enrollment to recall, whereas donor ineligibility was (spearman test p=0.02 and 0.002). public events with the presence of an adequate trained medical team represent a valid option for the enrollment of new unrelated donors. disclosure of conflict of interest: none. the search for hematopoietic stem cell unrelated donors in patients with malignant hemopathies with not-sibling matched family donor: the experience of a center a pérez 1 , r goterris 1 , m gómez 1 , s blanco 1 , a segado 1 , c arbona 1 , jch boluda 1 , m poch 1 and c solano 1 1 hematology department, hospital clínico universitario, valencia unfortunately, as few as 30-35% of patients will have an hlaidentical matched sibling donor available for hematopoietic stem cell (hst) donation. the search for an unrelated donor (urd) (adult or cord blood) is often the best option for those patients lacking a suitable matched donor. below we describe the experience with the search for an unrelated donor in our center. between september 1995 and march 2016 the search for urd was activated for 263 patients. the median age of the patients was 46 years (range: 0.4-69), 10% were under 18 years and 60% were males. acute myeloid leukemia (n = 67), acute lymphoblastic leukemia (n = 43), non-hodgkin's lymphoma (n = 60), chronic/prolymphocytic lymphocytic leukemia (n = 23), hodgkin's lymphoma ((n = 13), multiple myeloma (n = 14), chronic myeloid leukemia (n = 15), philadelphianegative myeloproliferative neoplasms (n = 9), myelodysplastic syndrome (n = 8), aplastic anemia/paroxysmal nocturnal hemoglobinuria (n = 6), others (n = 5). the disease status in hematological malignancies was: first cr (n = 77), 4 second cr (n = 49), pr (n = 46) and refractoriness (n = 82). the donor type requested at the activation of the search was an adult (n = 110), umbilical cord blood (n = 7) and two options (n = 146). results: a compatible donor was found in 197 patients (76% of the series) after a median of 44 days (range: 1-847) from the activation of the search. the degree of adult donor compatibility (not available in 7 cases) was: complete hla identity (8/8: n = 49, 10/10: n = 37); an hla difference (7/8: n = 12, 9/10: n = 31); lower degree of compatibility (n = 13). the degree of umbilical cord blood compatibility: identity ⩾ 4/6 (n = 48). a total of 151 patients (57%) were transplanted, 103 from adult donor and 48 from umbilical cord blood. the median time between the activation of the search and the hst transplantation was 4 months (range: 0.7-29), being 3.2 months for acute leukemia and 5.1 months for other pathologies, and between the location of the donor and the hst transplantation 70 days (range: 5-412), being 45 days for umbilical cord blood and 76 days for an adult donor. there were 108 cancellations of the urd search (41% of the total) for the following reasons: clinical status of the patient (n = 63), performing a haploidentical transplant (n = 20), transplant center does not consider (n = 9), norms of the registry (n = 8) and loss of indication of transplantation (n = 8). the median time from the beginning of the search to its cancellation was 4.5 months (range: 0.3-53). at the time of analysis, the median follow-up of the 263 patients is 17 months. the survival of the series in the 5 years is 37% and 43% for patients transplanted from urd. 76% of the searches activated in our center allowed the localization of a urd with an adequate degree of hla compatibility. however, only 57% of the patients for whom the search was activated were finally transplanted. the most frequent cause of cancellation of the procedure was the clinical deterioration of the patient. disclosure of conflict of interest: none. the leukemic transformation of otherwise healthy donor stem cells provides a useful in vivo model to study the mechanisms involved in leukemogenesis. we report two cases of donor cell-derived haematological malignancy in which wholeexome sequencing (wes) was performed in bone marrow (bm) samples from recipient at different times after allogeneic hematopoietic stem cell transplantation (allo-hsct) in order to study the dynamics of emergence of mutations that precede the development of donor cell leukemia (dcl) and donor cell myelodysplastic syndrome (dc-mds). case 1: a 43-year-old female diagnosed with lymphoblastic leukemia-b t(1;19), who developed acute myeloid leukemia (aml) with normal karyotype, npm1+of donor origin 16 months after unrelated cord blood transplantation (ucbt). case 2: a 65-year-old male diagnosed with mantle cell lymphoma, who developed mds 45,xx,-7,del(12)(p12) of donor origin, 57 months after allogeneic bm transplantation from his hla-identical brother. the donor also developed mds several months later. wes (sureselect-xt human-exon 50mb) was performed by next generation sequencing (hiseq) on donor stem cells (scs) infused as well as on bm samples from recipient after allo-hsct. the exome of donor scs and 5 bm samples, from case 1, were aligned to the human reference genome (grch 37/hg19) and donor scs and 9 bm samples were aligned to grch 38/ hg38 in the second case. in both cases non-synonymous variants in the coding regions or synonymous variants in splice regions of genes related to leukemia were selected. in addition, bm samples were matched to their scs and to prior bm samples to identify the acquired variants. variants meeting such criteria were evaluated with 3 functional predictor software's (sift, polyphen2 and mutation taster). wes analysis revealed progressive emergence of multiple somatic mutations probably related to the development of leukemia in bone marrow samples post allo-hsct ( figure 1 ). both scs showed alterations that may be involved in leukemogenesis. (case 1: sh2b3 and case 2: kmt2c, kmt2a, arhgap26 and monosomy 7). somatic mutations, acquired over time, fall into genes that play well-established roles in signalling pathways (ras-mapk, pre-mrna splicing factor, apoptosis, dna doublestrand break repair, dna replication and so on). mutations in leukemic subclones that disappear after chemotherapy were indentified, as well as the acquisition of new mutations in resistant subclones. we propose a possible model of leukemogenesis in these cases ( figure 2 ). the present study reveals a process of sequential clonal expansions, promoted by the acquisition of additional somatic mutations in donor hematopoietic cells. detection of heritable or acquired gene mutations in donor associated with predisposition to haematological malignancies could have clinical implications for the patients undergoing to allo-hsct. although the cause of donor cell-derived haematological malignancy onset seems to be multifactorial, the infusion of a scu with pre-leukemic potential in a context of residual toxicity in recipient as a result of pre-transplant chemotherapy, a post-transplant environment characterized by a decreased immune surveillance may well have played role in these cases. the study of a greater number of dcl cases by next generation sequencing could help to understand this process and to detect new mutations involved in the emergence of aml. disclosure of conflict of interest: none. the impact of donor and recipient sex in allogeneic stem cell transplantation-single center experience (cic 859) y petrov 1 , p ganeva 1 , g arnaudov 1 , s lozenov 1 , y davidkova 1 , v stoeva 1 , i tonev 1 , m guenova 1 and g mihaylov 1 1 national hospital for active treatment of hematological diseases allogeneic hematopoietic stem cell transplantation (hsct) has been one of the most effective therapeutic modalities for patients with hematological malignancies and bone marrow failure syndromes. optimal donor selection is one of the key factors to enhance the success rate of this procedure. we [p142] s189 retrospectively investigated whether and how donor-recipient sex affects transplantation outcomes of 73 patients transplanted between 2010 and 2015 in our center. the median age of the patients was 37 years (range: 23-51). thirty-nine of the patients (53%) received a pbsc from a hla-identical sibling, and 34 patients (46.5%) received pbsc from matched unrelated donor. forty-six percent were male recipients with male donors (m-m), 11.9% were female recipients with male donors (m-f), 23.8% male recipients with female donors (f-m), and 17.8% female recipients with female donors (f-f). we performed a crosstab analysis and χ 2 tests to observe whether the donor sex affects our study population. patients with male donor had superior overall survival and progression-free survival compared to those with female donor (66.7% vs 29.0% p = 0.001 for os, and 52.3% vs 34.2% p = 0.003 for pfs; cramer`s v = 0.372). we further investigated how the disparity of the donor in the four groups (m-m, m-f, f-m and f-f) affects the os, pfs and nrm. the f-m group had a worse overall and progression-free survival comparing the other groups (11% 4-year os and 17% pfs; p o0.0001).this group had 27% relative increase in the non-relapse mortality compared with m-m group (p = 0.009). for m-m group there was a 2% relative increase in the subdistribution hazard of nrm compared with m-f group (p = 0.02). the f-f group and m-f group had similar subdistribution hazard of nrm (39% vs 40% p = 0.009). the incidence of acute gvhd and chronic gvhd for the groups was: 34% and 41% (m-m), 37% and 32% (m-f), 41% and 40% (f-m), 32% and 7% for the (f-f) group. the appearance of either acute or chronic gvhd did not show statistical significance regarding the os and pfs in the groups (p = 0.07). we examined the effect of donor-recipient sex incompatibility on the outcome of hsct in out center. our results showed inferior os and pfs for f-m group and a higher incidence of nrm compared with other groups. these effects might be associated with allogeneic immune responses against h-y antigens. key words: stem cell transplantation, donor sex, recipient sex, overall and progression-free survival [p143] disclosure of conflict of interest: none. from 2011 to 2014, 66% of the 3834 patients affected by hematological malignancy searching for an unrelated donor through the italian registry successfully identified a suitable donor. this proportion increases up to 71% when searching for a cord blood unit was considered, corresponding to total transplant efficiency of 62%. from april 2006, the rome transplant network adopted a unique policy for the identification of a potential alternative donor, following a hierarchical selection that considered as first choice a volunteer unrelated donor, secondly a cord blood unit and last a haploidentical related donor. before starting the unrelated donor search, a preliminary query through the bone marrow donor worldwide database was performed for all the patients referred to the rome transplant network. based on the low resolution hla typing (a, b and drb1) it was possible to arbitrary assign a good or poor score that might predict the identification of a full matched (8/8 a, b, c and drb1) donor. therefore, aims of the present study were to assess the utility of the preliminary query and the impact of the use of high resolution hla typing since the starting of donor search on the timing for the unrelated donor identification. moreover, the final aim was of comparing donor identification and transplant efficiency between the national registry, that considers only the unrelated donor and the rome transplant network, whose policy includes also haploidentical donor as third choice in the donor search process. at rome transplant network 79% out of 417 adult patients met criteria of a good preliminary query corresponding to a matched unrelated donor identification in 50% of cases vs only 12.5% for patients with poor preliminary query. our policy led to 78% and 74%, respectively, of alternative donor identification and transplant efficiency, significantly higher than the corresponding data of 71% (p = 0.007) and 62% (p o0.0001) reported by the national registry. moreover, the median duration of search process for mud identification has been significantly reduced by the use of hr hla typing patient at the start of the formal search activation from 88 (range: 1-1016) to 66 (range: 8-905) days at ibmdr (po 0.001) and from 61 to 41 days (20-321) at rtn (po 0.001). in conclusion, the preliminary query represents a useful tool to address the search towards the best donor choice and to perform transplant in adequate time. moreover, the timing of donor identification has been significantly reduced with the use of high resolution typing at the start of donor search. a search and selection donor policy should be basically established and should include the haploidentical donor to improve the transplant efficiency. disclosure of conflict of interest: none. the long term prognosis of elderly acute myeloid leukemia (aml) patients remains poor. advances in the uses of alternative donors and reduced intensity conditioning regimens have extended the use of allogeneic hematopoietic stem cell transplantation (hsct) to a wider number of patients. however, few studies have reported data on the efficacy of hsct from alternative donors in elderly aml patients. we retrospectively analyzed the transplantation outcome in 93 consecutive elderly aml patients aged 460 years who received hsct (2005 hsct ( -2015 at the catholic blood and marrow transplantation center. donor types were autologous (n = 18) or hla matched related (mrd, n = 28), unrelated (mud, n = 22), or haploidentical (n = 25). for graft-versus-host disease (gvhd) prophylaxis, methotrexate and cyclosporine (mrd) or tacrolimus (mud/haploidentical donor) were used. mud and haploidentical donors were given antithymocyte globulin. the median age was 63 years, with 23 patients (25%) 465 years. intermediate-or adverse cytogenetic risk was observed in 91% of patients. with a median follow-up of 44.7 months, overall survival (os) and disease-free survival (dfs) at 3 years after transplantation were 37% and 38% for autologous, 40% and 35% for mrd, 67% and 62% for mud, and 67% and 67% for haploidentical hsct, respectively. the 3-year relapse was significantly higher for autologous hsct compared to allogeneic hsct (40% vs 14%, p = 0.012), while it was similar among allogeneic donors: mrd, 13%; mud, 14%; haploidentical, 15% (p = 0.925). the 3-year non-relapse mortality (nrm) for mud (24%) or haploidentical donor (18%) hsct was comparable to that of autologous hsct (22%), while it was relatively higher for mrd hsct (52%, p = 0.056). of the 75 patients receiving allogeneic hsct, the 1-year cumulative incidence of moderate to severe chronic gvhd was significantly increased for mrd (64%) compared to alternative donor hsct (35%, p = 0.001). in multivariate analysis, patient age (hr 0.8, 95% ci 0.8-1.0, p = 0.005) and donor type (hr 3.5 95% ci 1.0-13.0, p = 0.056 for mud; hr 6.2, 95% ci 1.7-22.6, p = 0.006 for mrd compared to haploidentical donor) were significantly associated with the cumulative incidence of moderate to severe chronic gvhd, while female-to-male hsct showed a borderline significance (hr 2.1, 95% ci 0.9-4.7, p = 0.075). incidence of acute gvhd was similar according to donor type. in the multivariate analysis for nrm, patient age (hr 1.4, 95% ci 1.1-1.6, p = 0.001), mrd (hr 4.5, 95% ci 1.4-14.4, p = 0.011), and hematopoietic cell transplantation-comorbidity index high risk (hr 6.4, 95% ci 2.3-17.5, p = 0.001) were significantly associated. in conclusion, our results showed significantly higher relapse rate for elderly aml patients receiving autologous hsct compared to allogeneic hsct, responsible for the lower survival rate in autologous hsct. we observed that nrm rate for mud and haploidentical donors for elderly aml patients were lower than expected and similar to autologous hsct. relatively higher incidence of nrm for mrd hsct seemed responsible for the low long term dfs. these results suggest a need for strengthening of gvhd prophylaxis in mrd hsct for elderly aml patients. our results suggest a potential role of alternative donor hsct to improve long term survival rates in elderly patients with aml. disclosure of conflict of interest: none. for patients with saa, transplantation from an unrelated donor (ud) is usually considered after failure of at least one course of immunosuppression. this strategy is based on a relatively high risk of complications for ud transplant recipients, such as graft rejection, graft-versus-host disease (gvhd) and infections. however, the outcome of unrelated donor transplants has significantly improved in recent years, due to better donor selection, conditioning regimen optimization and better supportive care. the authors describe results from 51 patients with saa who receive unrelated allogeneic transplants in a single reference institution from 1997 to 2014. data was retrieved from the center databasis and there were 30 females and 21 males. median age was 15 years old . median total number of cells infused was 3.4 × 10 8 /kg.61% of the patients have received more than 50 transfusions previously. conditioning regimen were: cy 120 + tbi 1320 ± atg in 16 (31%) patients, bu 12 mg/kg+ cy 120+ atg in 18 (35%), and fludarabine + cy+atg in 8 (16%), fludarabine, cy+tbi 200 in 9 (18%) patients. stem cell source was marrow in 84%, cord blood in 13% and peripheral blood in 3% of patients. transplants were full matched in 32 (62%) patients, had one mismatch (out of 12) in 12 (24%) and 2 mismatches in 7 (14%) patients. engraftment was complete as evaluated by donor chimerism at day 30 and 100 post transplant in 36 patients (71%), partial in 4 (8%) and graft failure was observed in 9 (18%) patients. acute gvhd grade ii-iv was seen in 9 patients ( 18%) and nih moderate to severe chronic gvhd was seen in 8 (16%) patients. median overall survival was 328 days (4-4287) and estimated 5 years overall survival was 55%. risk factors for survival identified were: hla mismatch and stem cell sources other than marrow. unrelated transplants are a feasible salvage therapy for patients with saa refractory to immunosuppression, being hla compatibility and marrow stem cell source factors with a positive impact on survival. disclosure of conflict of interest: none. use of haploidentical stem cell transplantation continues to increase, the 2015 european society for blood and marrow transplant activity survey report jr passweg 1 , h baldomero 1 , p bader 2 c bonini 3 , rf duarte 4 , c dufour 5, , a gennery 6 , n kröger 7 , j kuball 8 , f lanza 9 , s montoto 10 , a nagler 11 , ja snowden 12 , j styczynski 13 and m mohty 14 for the european society for blood and marrow transplantation (ebmt) 1 hematopoietic stem cell transplantation (hsct) is an established procedure for many acquired and congenital disorders of the hematopoietic system, including disorders of the immune system, and as enzyme replacement in metabolic disorders. the annual activity survey of the ebmt describes the status of hsct in europe and affiliated countries and has become an instrument used to observe trends and to monitor changes in technology use. teams were invited to report their transplant activity for 2015 by indication, stem cell source and donor type using a single paged survey. a record number of 42 '171 hsct in 37 '626 patients (16 '030 allogeneic (43%), 21 '596 autologous (57%)) were reported by 655 centers in 48 countries in 2015. trends include continued growth in transplant activity during the period 2005 and 2015, with the highest percentage increase seen in middle income countries (allo 209%, auto 215%), and the lowest in very high income countries (allo 64%, auto 28%), for both allogeneic and autologous hsct. in contrast the absolute growth is highest in the very high income countries (growth allo rates 114 transplants per 10 × 10 6 inhabitants, auto rates 85 for very high income countries; allo rates 35, auto rates 38 for middle income). main indications for hsct were myeloid malignancies 9 '413 (25%; 96 % allogeneic); lymphoid malignancies 24 '304 (67%; 20% allogeneic); solid tumors; 1 '516 (4%; 3% allogeneic); and non-malignant disorders; 2 '208 (6%; 90% allogeneic). remarkable is a decreasing use of allogeneic hsct in cll from 504 patients in 2011 to 255 in 2015 and is most likely due to the development of potentially very effective cll drugs. use of haploidentical donors for allogeneic hsct continues to increase 2 '012 in 2015; a 291% increase since 2005. the highest growth is seen in myeloid malignancies 1 '008, with lymphoid malignancies 636, nonmalignant disorders 316 and 52 others. in aml, haploidentical hsct increases similarly for patients with both advanced disease and those in cr1. both marrow and peripheral blood is used as stem cell source for haploidentical hsct with higher numbers reported for the latter. this year's activity survey shows continued increase in the use of haploidentical hsct across europe within the main indication groups and cell source. it reflects in a timely manner current trends in stem cell transplantation and is an essential tool for health care planning and health policy makers. human bone marrow mesenchymal stromal cells derived exosomes (hbmmdes) are small membrane vesicles secreted from mesenchymal stromal cells that may serve as a vehicle for protein, mrna and microrna (mirna) transfer to distant cells; affecting gene expression, proliferation, and differentiation of the recipient cells. therefore, mdes may possess some of the immunoregulatory properties of their parental cells. in the present study we aim to explore the immunomodulatory function of mdes and understand the molecular mechanisms enabling it. for this purpose, we co-cultured hbmmdes with activated human lymphocytes. using ultracentrifugation, hbmmdes were isolated from expanded human bone marrow derived mesenchymal stromal cells (hbmmscs). using em and zeta sizer, particles were shown to be in the range of 80-120 nm. pha activated human peripheral blood lymphocytes (pbls), r-848/il2 activated b cells and anti cd3/cd28 activated t cells were co cultured with purified mdes. cell proliferation was tested using thymidine incorporation assay. we found that exosomes derived from 1 × 10 3 to 1 × 10 6 mscs exhibited a dose-dependent inhibition of lymphocyte proliferation. exosomes derived from 1 × 10 6 mesenchymal stromal cells co cultured with pha activated pbls, activated b cells and activated t cells showed proliferation inhibition of 53%( p ⩽ 0.001), 34.37% (p ⩽ 0.05) and 47.41 % (p ⩽ 0.01), respectively. in order to understand the molecular mechanism behind the immunomodulatory effect of mdes, we have profiled mde's mir content using illumina hiseq 2500 platform and we are currently profiling co cultured activated lymphocytes mrna content using next-generation sequencing system, illumina. preliminary results demonstrate some higher abundance of specific mscs derived mirs in the mdes. hbmmscs have been shown to serve as immune modulators in patients with acute and chronic graft versus host (gvhd). in the future, mdes may provide an alternative therapy for gvhd. compared with bmmscs, mdes are more stable, have no risk of aneuploidity or ectopic proliferation and have less probability of immune rejection. additional studies are needed to explore the applicability of mdes to serve as modulators of the immune response. disclosure of conflict of interest: none. graft-versus-host disease (gvhd) is the major complication after allogenic haematopoietic stem-cell transplantation s192 (hsct). extra virgin olive oil (evoo) is a source of phenolic compounds such as glycoside oleuropein, hydroxytyrosol and tyrosol. olive oil polyphenols have shown antioxidant, immunomodulatory, antiproliferative, anti-apoptotic and antiinflammatory properties that might be useful in the prophylaxis and treatment of gvhd. polyphenolic extract (pe) of evoo was obtained by the method described by vazquez roncero et al. with some modifications. briefly, fifty grams of evoo (oleoestepa, seville, spain) was extracted with methanol/water (80:20, vol/vol, 125 ml ). the mixture of evoo, methanol and water was decanted and the methanolic extract was concentrated and lyophilized. then, the effect of pe in cell viability and activation of t lymphocytes from healthy donor's buffy coats either resting or activated with anticd3 plus anticd28 was analyzed by flow cytometry after staining with 7aad, anexin-v and cd25. proliferation assays were performed with pkh and the quantification of il-2, il-4, il-6, il-10, tnf-α and ifn-γ cytokines in cell culture supernants with bd cytometric bead array (cba). signaling pathways were analyzed by western blot. finally, in a mouse model of acute gvhd (c57bl/6 in balb/c), mice were randomized into two experimental diet groups: standard diet (2014s harlan laboratories) and standard diet (2014s harlan laboratories) supplemented with 600 ppm of pe obtained of evoo. the severity of gvhd was assessed by a scoring system described by cooke et al. that incorporates five clinical parameters: weight loss, posture (hunching), activity, fur texture, and skin integrity. pe did not affect t cell viability. by contrast, pe decreased t-cell activation and proliferation of t-lymphocytes stimulated with anticd3 plus anticd28. in addition, there was a decreased production of th1 (ifnγ, il-2 and tnf) and th2 cytokines (il-4, il-6 and il-10) in the presence of pe. regarding the signaling pathways analyzed, pe inhibited phosphorylation of akt and nuclear translocation of nfkb in activated t cells. in the mouse model of acute gvhd, animals which received the pe supplemented diet had an increased survival as compared to mice receiving a standard diet. also, gvhd incidence was significantly lower among mice receiving the pe supplemented diet as assessed by both the presence of gvhd signs as well as pathological examination. polyphenols obtained from evoo are an important immunomodulatory agent capable to reduce the proliferation and activation of activated t cells and the production of proinflammatory cytokines. in a mouse model of acute gvhd, pe supplemented diet reduced the incidence and severity of the disease and increased the survival of mice. disclosure of conflict of interest: none. graft-versus-host disease (gvhd) is a leading cause of postallogeneic haematopoietic stem cell transplantation (hsct) morbidity and mortality (1) . extracorporeal photopheresis (ecp) is an alternative therapeutic strategy that appears to act in an immunomodulatory fashion, potentially involving regulatory t lymphocytes and dendritic cells in patients who are refractory to steroids. dendritic cells (dcs) are the most important antigen-presenting cells, playing a pivotal role in t-cell function and in the link between innate and adaptive immunity. moreover, dcs are also critical mediators of immune tolerance and energy. they can be divided into two major subsets, plasmacytoid dcs (pdcs) and myeloid dcs (mdcs) which have distinct functions. pdcs play a pivotal role in peripheral tolerance through generation of regulatory t (treg). on the other side mdcs promote, as well as pdcs, th2 and th0/tr1 responses (1-4). our study was performed to understand the mechanism of action involved in immunomodulatory effect of ecp. as the modulation of dcs and tregs number and function (7, 8) may be a central mechanism of ecp in maintaining self-tolerance, down-regulating immune responses, and limiting inflammation (9). eight patients affected by gvhd were included in this pilot study. in ecp apheresed mononuclear cells are exposed to 8methoxypsoralen and uva radiation. after this photoactivation, which induces dna damage and apoptosis, the cells exposed are re-infused into the patient inducing an immunomodulatory effect. all patients or their legal guardians gave their consent for this study. a sample of peripheral blood (pb) (basal condition), a sample of apheresis pre-uva photoactivation (pre-pa) and a sample of photoactivated apheresis (pa) were collected at the first day of ecp and every week for the first month of treatment. circulating dcs, mdcs (cd14/16-cd85+cd33+), pdcs (cd14/16-cd85+cd123+) and tregs (cd4 +cd25+foxp3+) were directly enumerated and phenotypically characterized. the assays were performed at day+1,+8, +15,+21,+30 data are expressed as mean ± s.d. of absolute number of cells/μl. at day +1 there were no differences in the absolute number of both mdcs and pdcs between pre-pa and pa. consequently there were no differences between pb and pa. from day +8 till +30 we observed an increase of these two cellular populations at every date of treatment. comparing the basal pb of day +1 vs day +30 we observed an increment of 40% and 120%, respectively for mdcs and pdcs (mdc from 11247 cell/μl to 15742 cell/μl; pdc from 6983 cell/μl to 15263 cell/μl). comparing the basal pb of day +1 vs day +30 we observed an increment of 115% of tregs (from 4257 cell/μl to 9142 cell/μl) while we observed a median increment of 34% calculated between pre-pa and pa of each day of treatment from day 1 to day +30. no firm conclusions can be drawn from a clinical point of view, however a biological effect has certainly highlighted. in particular no substantial differences in basal pb mdc or pdc emerged during the first month of treatment while a significant increase of mdc and pdc can be observed since day +15 following uva photoactivation. regarding tregs we observed an increment of 115% of tregs between pb from day +1 to day+30 and a median increment of 34% calculated between pre-pa and pa of each day of treatment. disclosure of conflict of interest: none. [p151] p152 impact of th17 cells on xenogeneic graft-versus-host disease l delens, s servais 1 , g ehx 1 , l vrancken 1 , g fransolet 1 , c gregoire 1 , m hannon 1 , s dubois 1 , c daulne 1 , f baron 1 and y beguin 1 1 giga i3 : hematology, university of liege acute graft-versus-host disease (gvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation. its pathophysiology is complex and not yet fully understood. in particular, the impact of th17 cells on murine acute gvhd has yielded conflicting results, while demonstration of increased levels of th17 cells at the site of acute gvhd provided only indirect evidence of their involvement in humans. here, we assessed the potential implication of th17 cells in a humanized mouse model of xenogeneic gvhd (x-gvhd). methods: x-gvhd was induced by infusing human peripheral blood mononuclear cells (pbmcs) into nod-scid il-2rγnull (nsg) mice given 2.5 gy total body irradiation 1 day prior transplantation. th17 cells were generated by culturing naive cd4+ t cells with anti-cd3/anti-cd28 coated beads under th17-skewing cytokines (tgf-β1, il1-β, il-6, il-21, il-23, neutralizing anti-il-2 and anti-ifnγ antibodies) in hypernatremic conditions (nacl 40 mm). results: after 8 days of culture, a median of 21.75% of il-17a+ cells was obtained. we confirmed the expression of il-17a, rorc and il-23r by these cells by rt-qpcr. we next assessed the co-injection of human pbmcs (1.106) with in vitro differentiated cells under th17 skewing conditions (1 × 10 6 ) (co-injection group, n = 20), in comparison with the injection of pbmcs alone (2 × 10 6 cells, pbmcs group, n = 17). we observed higher x-gvhd score (p60%) of cells expressing both il-17a+ and ifnγ+ cells (th17/ th1-like phenotype) among cd4+ il-17a+ cells while coinjected mice had higher blood concentration of il-17a (p = 0.026) than pbmc mice. these results demonstrate that addition of th17 cells worsened x-gvhd confirming their role in acute gvhd pathogenesis. disclosure of conflict of interest: none. although survival from allogeneic stem cell transplantation (hsct) has significantly improved, acute graft-versus-host disease (gvhd) remains a major cause of death. intestinal dysbiosis has been associated with acute gastrointestinal gvhd and poor outcome after hsct. we reported a correlation between microbiota (gm) composition and short chain fatty acid (scfa) production and gvhd in transplanted children. 1 to assess how the metabolic pathways of gm change during transplantation and identify modulators of immune response, we perform first longitudinal metagenomic analysis in children undergoing hsct. 8 patients (pts) (6male; mean age: 10y) with hematologic malignancies (7 all, 1 aml), who received busulphan-based myeloablative conditioning and t-cell replete bone marrow graft were enrolled. pts were prospectively enrolled in a protocol with at least 3 specimens fecal samples collected: one before and two after hsct, in order to build a proper trajectory. gvhd prophylaxis was cyclosporine for 3 pts receiving a matched related donor and cyclosporine, short-term mtx and atg for 5 pts receiving a matched unrelated donor. non-gvhd and gvhd patients had similar exposures to antibiotics during the stool collection. of these pts, 50% developed gvhd within the first 100 days. we applied shotgun metagenome sequencing to total fecal dna from samples collected. functionalities were assigned by reads mapping at different levels of the kegg database. 2 relative abundance was calculated and statistical analysis was performed. according to our findings, core functional profiles were overall conserved through the time-points in all patients ( figure 1a ), in contrast to the phylogenetic profiles behavior, this finding confirming the overall redundancy of gut microbiome core functionalities. analyzing the single metabolic pathways in subjects who developed gvhd, we found in the pre-hsct period a higher relative abundance of nucleobasis (purine and pyrimidine) metabolism (p o0.05) and branched-chain amino acids biosynthesis (p o0.05). functions related to the production of branched-chain amino acids are involved in the biosynthesis of the cell wall of gram-negative bacteria, microorganisms including subgroups with well know opportunistic pro-inflammatory. in addition, post-hsct samples of gvhd patients showed a lower abundance of genes involved in polysaccharides metabolism, as glycan biosynthesis and glycosaminoglycan degradation (p o0.05) ( figure 1b ). glycosaminoglycan degradation activity gets bacteria able to survive during extreme situations, as fasting using mucus polysaccharides as energy source, contributing to maintain a mutualistic composition of gm and scfa production by the saccharolytic functions of the endogenous mucus polysaccharides. this study detects functional peculiarities in the gm of non-gvhd pts. the gut metagenome configuration of non-gvhd patients is structured to derive scfa after hsct. the production of these metabolites promotes peripheral regulatory t-cell generation 3 , potentially explaining the protective role of gm from gvhd. although intestinal epithelial cells (iecs) are crucial regulators of barrier function and immune homeostasis, they also facilitate inflammation in exaggerate responses to proinflammatory mediators by pretransplant conditioning regimen, which plays a critical role in amplifying graft-versus-host disease (gvhd). thus inhibition of the converting to pathogenic iecs by conditioning may represent a novel approach to inhibit gvhd. aryl hydrocarbon receptor (ahr) is the ligandactivated transcription factor which has the ability to mediate the biochemical, metabolic, and toxic effects of environmental chemicals. recently, it has been demonstrated that ahr is an important regulator of cell development, differentiation, and function of both innate and adaptive immune cells. the ability of ahr is induced by respond to endogenous ligands generated from the host cell, diet, and microbiota. here, we investigated the regulatory role of ahr in iecs under inflammatory responses and its therapeutic activity for modulation of gvhd. ahr and cyp1a1 expression in mouse iecs were determined by real-time pcr. mouse iecs were pretreated with endogenous ahr ligands l-kynurenine (l-kyn, 300 mm) or pbs for 6 h and then stimulated with lps or il-1b for 24 h. cytokine levels were measured using the mouse flex-set cytokine bead array or real-time pcr. b6d2f1 (h-2b/d) recipients were administrated l-kyn daily by i.p. injection for 3 days. then the recipients were lethally irradiated and transplanted with 5 × 10 6 tcd-bm plus 2 × 10 6 t cells from b6 (h-2b) donor. mice were monitored every other day for survival and clinical score. colons were collected and stained with hematoxylin and eosin (h&e) for histopathological scoring. we found that ahr was constitutively expressed in the mouse iecs. cyp1a1 (an ahr target gene) was significantly increased by treatment of l-kyn under un-stimulatory condition. we further observed that l-kyn completely abrogated il-1β-mediated il-6 or lps-mediated tnf-a expression in iecs. administration of bdf1 recipient mice with l-kyn before transplantation significantly reduced the lethality and severity of gvhd. histopathology clearly revealed that treatment of l-kyn inhibited intestinal gvhd. our results demonstrate that 1) ahr is constitutively expressed in iecs, 2) treatment of endogenous ligand l-kyn induce ahr activation in the steady status, 3) ahr activation blocks conversation of the epithelial cells into pathogenic cell type, and 4) pre-administration of ahr ligand reduces gvhd. our study suggests that activation of ahr pathway in iecs before allogeneic hematopoietic stem cell transplantation (hsct) is a possible strategy to reduce intestinal gvhd. disclosure of conflict of interest: none. [p156] s196 relating with acute graft-versus-host disease (gvhd) after allogeneic hematopoietic stem cell transplantation (allo-hsct), protecting endothelial cells (ecs) from damage may be a potent prophylaxis and therapeutic strategy of acute gvhd (agvhd). conventional agvhd therapies may cause many adverse side effects because of their multiple targets. therefore, we explored the therapeutic efficacy of simvastatin, a lipid-lowering drug, which has been demonstrated endothelial protection. our previous clinical observation has found patients with agvhd had lower angiopoietin-1 (ang-1) level at day 7 but higher ang-2 level at day 21 than those without agvhd. in this study, we explored changes in ang-1 and ang-2 expression in an agvhd mouse model and determined whether simvastatin prevents gvhd through regulating ang-1 and ang-2 expression. we preincubated ea.hy926 ecs with simvastatin (1mmol/l) 12 h before stimulated with tnf-a, then ang-1 and ang-2 concentration in the cell supernatant was measured by elisa. ang-1 and ang-2 mrna and protein level of treated and untreated cells were examined simultaneously. in vitro simvastatin increased ang-1 production and release but conversely inhibited ang-2 release from ea.hy926 ecs. donor mice spleen cells were injected along with bone marrow cells into recipient mice after lethal irradiation to induce agvhd. simvastatin was administered orally once daily to mice (10 mg/kg) for 7 days after allo-hsct and started −1 day after allo-hsct. then mice survival time was monitored and organ damage was evaluated. the plasma level of ang-1 and ang-2 was measured by elisa, expressions of ang-1 and ang-2 in aortic endothelium were assessed by immunohistochemistry. simvastatin improved the survival and attenuated the histopathological gvhd grades of agvhd mice. plasma levels of ang-1 were significantly decreased, while plasma levels of ang-2 obviously increased in agvhd mice after transplantation. simvastatin reduced plasma levels of ang-2, elevated the plasma levels of ang-1 as well as the aortic endothelial levels of ang-1 and ang-2. in summary, simvastatin represents a novel approach to combat gvhd by increasing ang-1 production while suppressing ang-2 release to stabilize endothelial cells. there is a growing evidence of safety and efficacy of posttransplantation cyclophosphamide (ptcy) in stem cell transplantations (sct) from different donors and graft sources. still the optimal combination of immunosuppressive agents with ptcy should be elucidated for different types of scts. we report the 2-year update of the prospective nct02294552 single-center trial that evaluated risk-adapted graft-versushost disease (gvhd) prophylaxis with ptcy in related, unrelated and haploidentical scts. 200 adult patients (median age 32 y.o., range: 18-62) with hematologic malignancies, including aml (47.5%), all (26.5%), cml (10.5%), mds (4%), and lymphomas (11.5%), were enrolled in the study. 23% of patients were classified as salvage. 26% received the graft from matched related (mrd), 65% from matched/mismatched unrelated (mud/mmud), and 9% from haploidedntical (haplo) donor. 43% received bone marrow graft (bm) and 57%peripheral blood stem cell (pbsc) graft. 18.5% had myeloablative conditioning and 81.5%-reduced-intensity conditioning. gvhd prophylaxis for matched bm grafts consisted of single-agent ptcy 50 mg/kg days+3,+4, for matched pbsc graft-ptcy+ tacrolimus+ mycophenolate mofetil (mmf) 30 mg/kg days 5-35, and for any mismatched graft-ptcy+ tacrolimus+ mmf 45 mg/kg days 5-35. median follow-up was 20 months (range: 4-40). grade ii-iv (10% vs 18% vs 11%, p = 0.37) and grade iii-iv acute gvhd (4% vs 6% vs 0%, [p157] p = 0.59) were not different in mrd, mud/mmud and haplo groups, respectively. moderate and severe chronic gvhd was infrequent in all groups with slightly lower incidence after mud/mmud graft: and 22% vs 9% vs 21%, p = 0.046. nonrelapse mortality (nrm) was not different after mrd, mud/ mmud and haplo sct (8% vs 14% vs 24%, respectively, p = 0.19), while relapse incidence was higher after mrd and haplo grafts: (45% vs 22% vs 52%, p = 0.0017). 2-year overall survival (os), event-free-survival (efs), and gvhd-relapse free survival (gfrs) were 73% vs 71% vs 44% (p = 0.0015); 48% vs 65% vs 33% (p = 0.0008); 29% vs 56% vs 22% (p = 0.0002) for mrd, mud/mmud and haplo groups, respectively. in the multivariate analysis only disease risk index (hr 2.2 95%ci 1.6-3.0, p = 0.0001), severe sepsis (hr 3.8 95%ci 1.8-8.0, p = 0.0004) and chronic gvhd (hr 0.53 95%ci 0.30-0.93, p = 0.02) were predictive for efs, while type of donor was not a significant factor (hr 1.0 95%ci 0.6-1.7, p = 0.99) (figure 1 ). the incidences of complications were: hemorrhagic cystitis-23%, sepsis-24%, severe sepsis-8%, invasive mycosis-8%, cmv reactivation-45%, veno-occlusive disease-2.5%, transplant-associated microangiopathy-3.5%, grade 3-4 liver toxicity-14%, grade 3-4 kidney toxicity-1%. more than one third of patients experienced poor graft function during 100 days after sct, and in 83% of them cmv, hhv and bk virus reactivations were identified as the cause. the reported risk adapted strategy alleviates the risk of gvhd and nrm after mmud and haplo grafts. the observed differences in the relapse incidence, os and efs were predominantly due to unbalanced disease risks in the groups. the relapse of underlying malignancy with this prophylaxis still significantly influences the outcome. substantial number of patients experience poor graft function, which doesn't translate into nrm. disclosure of conflict of interest: none. a high migratory capacity of donor t-cells in response to the lymph node homing receptor ccr7 increases the incidence and severity of graft-versus-host disease vg garcía de soria 1 , ip sainz 2 , e jiménez 1 , a arriero 1 , c fernández-arandojo 1 , c cuesta 2 , b colom 2 , a marcos 2 , a rosendo 2 and cecilia muñoz calleja 2 1 department of hematology hospital universitario de la princesa and 2 department of inmunology. hospital universitario de la princesa graft-versus-host disease (gvhd) pathogenesis involves migration of the donor t-cells into the secondary lymphoid organs (slo) in the recipient, which is steered by two homing molecules: cd62l and ccr7. therefore we investigated whether the migratory capacity of donor t-cells is associated with gvhd. this single center prospective study included 85 donor-recipient pairs. in vitro chemotaxis assays of the lymphocytes of the apheresis product were performed in parallel with the analysis of cd62l and ccr7 by flow cytometry. the potential of activation of ccr7+ t-cells was assessed through ex vivo activation assays with peripheral blood monuclear cells (pbmc) from healthy donors using anti-cd3 and anti-cd28mabs. the migratory index to the ccr7 ligands, ccl19 and ccl21, was higher in t-cells from donors whose recipients will develop gvhd. these data indicated that the migratory capacity of the donor t-cells is clearly related to the development of gvhd. this prompted us to study the relationship between gvhd and the expression of two of the most relevant molecules in the trafficking of lymphocytes towards slo, cd62l and ccr7,as a subrogate index of the migratory potential of t-cells. consequently, we quantified the numbers of cd62l+ and ccr7+ t-cells in the graft. the initial transversal analysis of our data revealed that the percentage of cd62l+ lymphocytes in the apheresis product was very low compared to healthy lymphocytes. the analysis also confirmed that cd62l undergoes plasma membrane shedding after g-csf mobilization thus making it a non-valid biomarker. the analysis of ccr7 molecule revealed that the acute gvhd group received higher percentage of cd4+ccr7+ t-cells, whereas chronic gvhd patients were transplanted with higher percentage of cd8+ccr7+ t-cells compared to the non gvhd group. these results were confirmed when patients were subdivided into degrees of severity. a multivariate analysis was performed to investigate the real value of ccr7 to predict the development and severity of gvhd, and confirmed that ccr7 expression is a risk factor for the development of gvhd. thus, the percentage of ccr7+cd4+ t-cells increases the probability of developing acute gvhd (or = 1.08, c.i (95%) = 1.01-1.16, p = 0.019) and suffering a higher degree (or = 1.08, c.i (95%) = 1.01-1.15, p = 0.014). similarly, the or of the percentage of ccr7+cd8+ t-cells was 1.17 (c.i (95%) = 1.01-1.36, p = 0.0031) and 1.21 (c.i (95%) = 1.05-1.39, p = 0.006) for the development of chronic gvhd and its degrees, respectively. finally, to study the potential of activation of ccr7+ t-cells, we carried out ex vivo activation assays with pbmc from healthy donors using anti-cd3 and anti-cd28mabs and the expression of cd40l on cd4+ t-cells and of cd69 on cd8+ t-cells as markers of activation, demonstrating that ccr7+ t-cells exhibited higher potential of activation than ccr7-t-cells. to our knowledge this is the first analysis of the influence of the migratory capacity of the donor t-cells on clinical outcome following allogeneic hsct. our data show that ccr7 could be considered a subrogate biomarker of the migratory capacity of the donor lymphocytes for predicting the risk of suffering gvhd. based on the previous findings, we propose that the selective depletion of ccr7 expressing cells could be an effective preventive therapy for gvhd. disclosure of conflict of interest: none. previously published p160 a single center research for outcome in patients receiving imatinib for steroid-refractory chronic gvhd after allogeneic stem cell transplantation l ni, y luo, y tan, y hu, y zhao, j shi and h huang despite of major progress in allogeneic stem cell transplantation over the last decades, steroid-refractory chronic graftversus-host disease (sr-cgvhd) remains a leading cause of late morbidity and mortality. pre-clinical evidence confirms cgvhd has antibodies activating the platelet-derived growth factor receptor (pdgf-r) pathway. since this pathway can be inhibited by imatinib, we performed a study including 16 patients with sr-cgvhd given imatinib at a dose of 300 mg per day. all patients with a median age of 25 years (range: 16-53) underwent allogeneic hematopoietic stem cell transplantation in our single center between 2008 and 2015, and chronic gvhd occurred at a median time of 10 months (range: 3-29) after transplantation. patients had active cgvhd with measurable involvement of skin, lung or other districts and had previously failed in first-line immunosuppressive therapy. the major organs involved were lung (n = 11), skin (n = 10) and mouth (n = 1), including 5 cases involving both lung and skin, 8 cases involving 3 or more organs. according to the 2014 national institutes of health (nih) criteria and nih global severity, 13 patients were evaluated as severe cgvhd, and the other three were moderate. meanwhile, the 2014 nih working group had updated its recommendations for overall responses, consisting of complete remission (cr), partial remission (pr), and lack of response (unchanged, mixed response, progression). cr was defined as resolution of all manifestations in each organ or site, and pr was defined as improvement in at least 1 organ or site without progression in any other organ. after 3 months treatment, 14 patients receiving sufficient dose of imatinib revealed overall response rate (orr) at 78.6%, and orr remained unchanged at 6 months assessment, but with cr rate increased to 28.6%. two patients couldn't meet the response of cr or pr were considered as a lack of response, including one evaluated as unchanged and one mixed response because of pr in lung accompanied by progression in eyes. with a median follow-up of 9 months, 14 patients were alive, with a 1 year estimated overall survival was 87.1%. 2 patients eventually died of pneumonia. except 1 patient discontinued imatinib because of grade 2 toxicity as gastrointestinal discomfort at the first month, no one had imatinib-related grade 3 to 4 toxicity. this study suggests that imatinib is a promising and better tolerated treatment for patients with sr-cgvhd. disclosure of conflict of interest: none. acute graft-versus-host-disease (agvhd) is a major complication after allogenic hematopoietic transplantation (allo-sct). in recent years, a number of tissue-specific proteins have been described as biomarkers that could contribute to anticipate and/or diagnose this complication earlier and more accurately. reg3α (regenerating-islet-derived-3-alpha) has been directly related to gastrointestinal (gi) agvhd. our objective was to analyze plasma levels of reg3α at days +15 and +30 in patients who underwent unmanipulated haploidentical transplantation with reduced conditioning regimen (haplo-ric), and to correlate the results with the development of agvhd. we retrospectively analyzed 63 consecutive patients (2009-2016) who underwent haplo-ric with post-transplant cyclophosfamide (days +3, +4), mmf and csa as gvhd prophylaxis. seven cases were excluded due to early death (before day +30) and 4 cases due lack plasma sample. characteristics of the 52 patients included in the analysis are described in table 1 . reg3α detection was performed by elisa (mbl international corp, woburn, ma) according to manufacturer's instructions on 200 μl of plasma obtained at day +15 and +30. the association of the incidence of agvhd with known clinical variables and plasma reg3a levels were performed by cox regression and mann-whitney u-test, respectively. the determination of the best cut-off of reg3α levels to stratify patients with gi agvhd was performed with roc curves. the stadistical program used was r v2.15.0. the cumulative incidence of grade ii-iv and grade iii-iv agvhd was 52% and 17%, respectively. characteristics of agvhd are shown in table 2 . no association was found between agvhd and usual clinical variables (stem cells source, age, sex, conditioning regimen, donor/recipient sex and number of infused cd34 + cells), and with plasma reg3α levels at day +15.plasma reg3α levels at day +30 were higher in patients who devolved gi agvhd compared to patients who did not showed gi agvhd (median [p161] s199 and range: 2483 (2022-5904) vs 1110 (0-7797) pg/ml, p = 0.14, figure 1 ).the best cut-off selected on day +30 was 1989 pg/ml (s85%, e71%). patients with levels higher than 1989 pg/ml at day +30 had a significantly higher incidence of gi agvhd grade ii-iv (hr 6.9, p = 0.008, figure 2 ). plasma levels of reg3α at day +30 after haplo-ric correlated with the occurrence of gi agvhd grade ii-iv. therefore, plasma levels of reg3α could be use for the prediction and/or diagnosis of gi agvhd. disclosure of conflict of interest: none. anti-fibrotic treatment with pirfenidone in patients with gvhd-associated bronchiolitis obliterans syndrome ke hostettler, s gerull, g nair 1 , j passweg 1 , m tamm 2 and j halter 1 1 hematology, university hospital basel, switzerland and 2 pneumology, university hospital basel, switzerland prognosis of lung gvhd remains poor due to progressive decrease of lung function and repeated infections. pirfenidone exhibits anti-fibrotic effects and has been shown to reduce disease progression in patients with idiopathic pulmonary fibrosis. five patients with established bos (nih criteria 2014) and stable or deteriorating lung function under standard immunosuppressive treatment without active infection were treated with pirfenidone (2403 mg/d) in addition to their current therapy. clinical assessments and pulmonary function tests were performed every three months. five patients (4m, 1f), median age 60y (range: 29-65y) that were diagnosed with bos at a median time of 13.5 months post-transplant started pirfenidone at a median time of 51months (8-102) after diagnosis of bos. two patients are currently still under treatment after 611 and 638 days. two patients had to stop treatment due to financial reasons after 189 and 206 days of therapy. one patient never reached more than 20% of the planned dose due to gastro-intestinal symptoms and was excluded from further analysis. at the start of treatment median fev1 was 0.94l (0.72-1.34); 34.5% predicted (range: 21-44%) and median fvc 2.59 l (1.62-3.24); 46-84 % predicted. median fev1 trajectory was − 0.65 % predicted/ month during median 6months before start of pirfenidone (median − 19ml/month) and +0.33% predicted/month (+9.8ml/month) during treatment with pirfenidon. the treatment was well tolerated except in one patient with gastrointestinal complaints, no phototoxic reactions or serious drugrelated adverse events occurred. in our small number of patients pirfenidone was rather well tolerated and generally safe. the observed, albeit small trend in change of fev1 trajectory justifies further studies of anti-fibrotic therapy as a new therapeutic option in bos after allogeneic hsct. disclosure of conflict of interest: none. anti-thymocyte globulin has been widely used for the prevention of severe graft versus host disease in patients undergoing hsct from unrelated donor. however, the optimal dose remains to be defined. in samsung medical center (seoul, korea), institutional strategy for the atg use has been changed since april 2013, and we hypothesized that the incidence of chronic gvhd may differ by atg strategy. before april 2013, atg 4.5 mg/kg was routinely used in allogeneic hsct from unrelated donor, whereas, the dose of atg was escalated to 7.5 mg/kg since april 2013. in this study, a total of 170 patients who underwent allogeneic hsct from matched or unmatched unrelated donor between jan 2010 and dec 2015 were retrospectively analyzed. peripheral blood was used as the source of stem cells in all patients. after a median follow up of 30.9 months, the cumulative incidence of moderate to severe chronic gvhd was 30.2% (95% confidential interval [ci], 18.3 to 43.0) in the low-atg group and 23.7% (95% ci, 14.5 to 34.1) in the non-atg group (p = 0.655). the rate of 2year overall survival (os) was not significantly different between the groups (49.3% in low-atg group vs 48.1% in high-atg group, p = 0.841), as was the rate of disease free survival (dfs) (40.8% in non-atg group vs 42.3% in atg group, p = 0.867) and cumulative incidence of relapse (cir) (23.9% in non-atg group vs 24.5% in atg group, p = 0.776). in allogeneic hsct from unrelated donor, larger atg dose (7.5 mg/kg) did not reduce the incidence of chronic gvhd when compared to lower atg dose (4.5 mg/kg). disclosure of conflict of interest: none. allogeneic hsct provides a curative chance for patients with hematological fatal disease. however, substantial risks remain for morbidity and mortality caused by disease relapse and graft-versus-host disease. in samsung medical center (seoul, korea), institutional strategy for the atg use has been changed since april 2013, and we hypothesized that the incidence of chronic gvhd may differ by atg strategy. before april 2013, atg was not routinely used in matched sibling donor (msd) transplantation, whereas, atg 5 mg/kg has incorporated into hsct process in transplantation from msd thereafter. in this study, a total of 182 patients who underwent allogeneic hsct from msd between jan 2010 and dec 2015 were retrospectively analyzed. peripheral blood was used as the source of stem cells in all patients. after a median follow up of 40.5 months, the cumulative incidence of moderate to severe chronic gvhd was 22.0% (95% confidential interval [ci], 13.5 to 31.8) in the atg group and 55.2% (95% ci, 42.9 to 65.8) in the non-atg group (p = 0.0018). the rate of 2-year overall survival (os) was not significantly different between the groups (62.5% in non-atg group vs 58.7% in atg group, p = 0.624), as was the rate of disease free survival (dfs) (61.1% in non-atg group vs 53.3% in atg group, p = 0.377) and cumulative incidence of relapse (cir) (23.4% in non-atg group vs 28.3% in atg group, p = 0.463). in allogeneic hsct from msd, atg use was significantly associated with less occurrence of chronic gvhd, but not linked to increasing risk of relapse, with showing similar os and dfs between atg and non-atg group. disclosure of conflict of interest: none. long-term follow-up from the prospective randomized phase iii multicenter trial comparing a standard gvhd prophylaxis with cyclosporine a and methotrexate with or without additional pretransplant atlg (grafalon, previously atg-fresenius s) (given 20 mg/kg/day, days − 3 to − 1) in unrelated donor hematopoietic cell transplantation after myeloablative conditioning resulted in a significant reduction of acute and chronic gvhd without compromising relapse rate and survival [1, 2, 3] . here we report on a subsequent prospective non interventional observational study evaluating the outcome of patients receiving atlg in unrelated donor transplantation in day to day clinical practice without the selective measures of a clinical trial (german clinical trials register drks00004581). thirteen transplant centers included 165 patients with haematological malignancies (median age 54 years, iqr 45-61 years, range: 18-77 years) in early (n = 75, 45%), intermediate (n = 29; 18%) or advanced (n = 61; 37%) disease status receiving marrow (n = 6) or pbsc (n = 159) from 10/10 matched (128; 78%) or mismatched (37; 22%) unrelated donors (n = 4 related) after myeloablative (n = 100, 61%) or ric (n = 65, 39%) conditioning. gvhd prophylaxis consisted of calcineurin inhibitors, mainly csa (n = 154, 93%) with mtx or mmf and atlg. different dosing regimens were allowed according to current practise of centers. median total atlg dose was 46 mg/kg (iqr 32-60 mg/kg, range: 15-91 mg/kg). median follow-up was 12 months (range: 8-14 months). as compared to patients in our randomized phase iii multicenter trial [1, 2, 3] , patients in this study were older; advanced disease status, 10/10 match, pbsc transplantation were more frequent, and given median atlg dose was lower. acute and chronic gvhd, nrm, relapse risk, dfs and os at one year were similar to the results obtained in our randomized trial: incidence of°ii-iv agvhd: 27%, iii-iv agvhd: 13%; moderate/severe cgvhd: 24%; nrm: 17%; risk of relapse: 23%; relapse mortality: 10%; os: 73%. the experience in day to day clinical practice confirms the results shown in our randomized trial, namely the gvhd protective effect of atlg without compromising nrm or relapse rates. baseline calprotectin as a predictor for acute gastrointestinal graft versus host disease (gvhd)-a prospective study n schmidlin 1 , a holbro 1,2 , jp halter 1 , d heim 1 , l infanti 1,2 , a plattner 2 , r plattner 2 , c rothen 3 , a buser 1,2 , c bucher 1 and jr passweg 1 1 division of hematology, university hospital basel, switzerland; 2 blood transfusion center, swiss red cross, basel, switzerland and 3 rothen medical laboratories, basel, switzerland graft versus host disease (gvhd) is a major complication after allogeneic stem cell transplantation. so far there is no good validated predictor for the incidence and severity of gvhd. fecal calprotectin (cpt) is a protein in leukocytes with antibacterial properties. it has been shown to be elevated in acute gastrointestinal gvhd. additionally, cpt may be predictive for treatment response. the aim of the current prospective study was to investigate the role of baseline cpt in predicting incidence and severity of intestinal gvhd. in this prospective study conducted at the university hospital basel, switzerland, we included all adult patients undergoing hsct. the institutional review board approved the study. data were collected prospectively. cpt was measured twice before conditioning and at transplantation. fecal samples for cpt were obtained before conditioning and on the day of transplantation and assessed twice by standard elisa. between march 2012 and april 2016 a total of 194 patients (55% males, 154 patients with both baseline and transplant cpt values) were included. patient, disease and transplant characteristics are described in table 1 . median age at transplant was 55 years (range: 21-73 years). most patients had myeloid neoplasia and 46% received myeloablative conditioning. gvhd prophylaxis consisted mainly of cyclosporine containing regimens (98%). cpt levels ranged from 19 to 1500 μg/g both at baseline (median: 100 μg/g) and at transplantation (median: 101 μg/g), with a good consistency between the two measurements performed (internal quality control). on the other hand, cpt did not correlate with c-reactive protein. the two measurements were taken in median 7 days apart, depending on the conditioning regimen. eighty-five patients had an increase of at least 50 μg/g between baseline and transplantation. overall 61 (31.4%) patients developed acute intestinal gvhd (grade 1: 19; grade 2: 18; grade 3: 16, and grade 4: 8 patients, respectively). cpt both at baseline and at transplantation was not predictive for the incidence of gvhd, acute intestinal gvhd, and for acute intestinal gvhd grade 3-4 ( figure) . additionally, we did not find a significant association between cpt levels and the above mentioned endpoints for patients showing an increase of cpt of at least 50 μg/g between baseline and transplantation. in the current prospective study, we didn't find any correlation between baseline cpt values and the incidence and severity of gvhd and intestinal gvhd. further studies identifying early markers and predictors of gvhd are urgently needed. [p166] disclosure of conflict of interest: none. calcinuerin inhibitor (ci) free graft-versus-host disease (gvhd) prophylaxis: its effects on resource utilization, renal function, and the cost of care m muilenburg 1 , k cole, m abidi 1,2 , s williams and as al-homsi 1,2 1 spectrum health blood and marrow transplantation and 2 michigan state university, college of human medicine effective gvhd prevention following allogeneic hematopoietic stem cell transplantation (ahsct) is vital to reducing transplant morbidity and mortality and improving overall outcomes. several strategies are currently utilized for gvhd prophylaxis including mtx, mmf, cis, post-transplant cyclophosphamide (cy), and proteasome inhibitors. recently, we described the results of a phase i-ii trial of cyclophosphamide (cy) and bortezomib (bor) where patients (pts) received cy (50 mg/kg) on days (d) +3 & +4 and bor on d 0 & +3. the incidences of grade ii-iv and grade iii-iv acute gvhd were 35% and 11%. the incidence of chronic gvhd was 28%. in addition to gvhd, there are other factors that affect patients' quality of life and cost of care and that should be considered. it is well documented that cis have an unfavorable toxicity profile. this includes nephrotoxicity and electrolyte disturbances. furthermore, the cis need serial level monitoring. thus, we endeavored to compare the effects of cybor combination against ci-based regimens by focusing on electrolyte requirements, specifically mg, and renal function. we also sought to better understand financial considerations surrounding the need for ci drug level monitoring. sixteen pts were randomly selected from the cybor group and 16 patients from an internal control group of patients who received mmf and cyclosporine or tacrolimus following reduced-intensity ahsct. the groups were well matched in regards to age, sex, disease status, pam score, and baseline renal function. on each pt, mg results from d 0 to +90 were compiled. based on institutional protocol, a mg replacement value was assigned as well as the corresponding drug and infusion charges. next, the number of immunosuppressant (is) trough levels from d 0 to +90 was tallied and the internal lab charges calculated. to compare renal function, gfr was calculated at baseline, d 0, and d +30. χ 2 tests and wilcoxon rank square tests were used to analyze the data. for the cybor group, median mg value was 1.9 mg/dl (iqr 0.3) vs 1.7 (0.3) in the control group (po0.0001). cybor pts required a median of 7 grams (16) vs 49 grams (55) in the control group (p = 0.001). the cost of mg replacement and infusion was significant (p = 0.001) ( table 1) . for is checks, drug levels were checked a mean of 0.625 times per patient in the cybor group compared to 18.75 times in the control group (po0.0001), which also translates to significant savings ( table 2) . considering these costs, the cybor group saved~$6000. for gfr, 3 cybor pts and 1 control pt had reduced gfr at baseline. on d +30, cybor pts had better renal function in comparison to the control group (p = 0.018) ( figure 1 ). in summary, cybor significantly reduced the use of resources post-transplant and thereby the associated cost related to mg replacement and need for drug level monitoring. furthermore, cybor preserved renal function at d +30. these findings could also impact patient's quality of life. although our cost analysis was restricted to certain aspects of care and did not take into account other factors, it highlights specific important benefits of ci-free gvhd prophylaxis and supplicates further study. a formal prospective comparison of cost and qol is warranted. . related donor n = 11 and unrelated donor n = 5 (compatibility 9/10 n = 1), with conditioning regimen: myeloablative n = 5 and non-myeloablative n = 11. median interval between transplantation and diagnosis of cgvhd of 11 months(5.1-40.2); and between cgvhd diagnosis and sativex 23 months (9.1-105.3), with a median of 4 prior treatment lines (2-9). at the time of beginning, the cgvhd was extensive in all patients, severe cgvhd n = 5 and moderate cghvd n = 11. all patients except one had cutaneous involvement (n = 13 with sclerodermal features). in addition, other organs were affected: digestive n = 2, pulmonary n = 7, hepatic n = 4, ocular n = 8, oral n = 9, genital n = 2 and muscular n = 7. drug was started because of pulmonary affectation in 3 patients and due to sclerodermal/muscular involvement in 13 patients. concomitant therapies during treatment were: topical cutaneous treatment n = 11, topical ocular treatment n = 10, pulmonary n = 7, sirolimus n = 8, tacrolimus n = 3, oral corticosteroids n = 9, extracorporeal photopheresis s n = 4, ruxolitinib n = 2, imatinib n = 1, mesenchymal stem cells n = 1. the mean dose were three puff/day (2) (3) (4) (5) , with good tolerance, only two discontinuations of treatment because of adverse effects. median time of treatment 156 days (45 to 561). at the time of the analysis 11 patients were still under treatment. responses mainly occurred within the first 60 days, s203 with a median time of duration of 106 days (20 in 450). responses after two months of treatment were: 6 partial organ response, 4 mixed responses, 4 unchanged and 2 organ progressions; at 120th day (14/16) only two patients maintained their responses (one pr and one mixed response). it must be pointed out that one patient who reached pr with sativex in monotherapy maintain response after 18 months of treatment. in addition, cramps were resolved in 5 patients. sativex appears to be an effective treatment option in patients with chronic gvhd, particularly in those having cramps, sclerodermal features and pulmonary affectation. as seen in multiple sclerosis context, the main issue with its use is the loss of response in the long-term follow up. the median dose is inferior to the one described in ms, leaving the question if higher doses can deepen the response. these results should be confirmed in prospective trials. disclosure of conflict of interest: none. several risk factors associated with acute and chronic graftversus-host-disease (gvhd) have been identified in multiple studies. most commonly associated factors are human leukocyte antigen (hla), mismatch between recipient and donor, as well as several other characteristics such as age, conditioning regimen and prior acute gvhd. objective: the aim of this study was to evaluate the characteristics of acute and chronic gvhd in patients who underwent an allogenic hematopoetic stem cell transplantation (hsct), identify differences in the profile risk factors for acute and chronic gvhd and their impact in post-transplant morbidity and mortality. this retrospective study included 90 mexican adult patients who received an allogenic hsct between january 2010 and march 2016, at instituto nacional de cancerologia. we analyzed 90 patients with a median age of 30 years (15-64), from which, 60% were male patients. among the participants with hematologic malignancies, 39 were previously diagnosed with acute lymphoblastic leukemia, 20 acute myeloid leukemia, 11 chronic myeloid leukemia, 8 lymphoblastic lymphoma and 3 with myelodysplastic syndrome. because bone marrow transplants are not performed at this institution, all transplants were from peripheral blood stem cell harvest. acute gvhd prophylaxis consisted in a triple immunosuppressive drug regimen for all patients. 86.7% of the patients had high risk disease prior to hsct. myeloablative conditioning represented 82% of the applied regimens, which consisted of iv busulfan in 63.3% of the cases. 44.9% of patients, were transplanted within 12 months from diagnosis. the cumulative incidence of acute gvhd at 100 days was 21.1% (19 patients). patients with acute gvhd had 42% grade a, 15% grade b and 42% grade c, according to the ibmtr grading system. 12 patients had skin involvement, with grade 1-2 acute gvhd in 83% of the cases, 4 patients developed liver involvement and 6 patients had gastrointestinal tract disease. 19% of the patients developed chronic gvhd, from which, 57% were classified as severe, 10.5% as moderate and 21.6% as mild. 36% of the patients who developed chronic gvhd had a single organ involvement, while 26.3% had 3 or more organs/sites. prior acute gvhd was associated with de development of chronic gvhd. the multivariate analysis identified hla unrelated donor as the only risk factor associated with the development of acute gvhd (hr, 5.1; 95% ci, 3.3-7.9, p = 0.043). the overall survival at 5 years was of 69% poor patients who developed acute gvhd and of 34% for those who didn´t (p = 0.065). our analysis showed that the incidence of acute and chronic gvhd at our center is lower than the reported at other centers, but we were not able to identify risk factors usually associated with the development of gvhd, perhaps due to the small population that we evaluated. graft versus host disease (gvhd) remains one of the main obstacles to broader application of allogeneic stem cell transplantation (sct). despite the routine use of prophylactic therapies, chronic gvhd (cgvhd) occurs in 10 to 80% of patients undergoing allogeneic sct. ciclosporin a (csa) remains the backbone for gvhd prophylaxis in both myeloablative (mac) and reduced intensity conditioning (ric) sct. however, in a significant proportion of patients, csa causes important side effects and needs to be discontinued. in this study we have evaluated the impact of substituting csa for mycophenolate mofetil (mmf) as immunosuppression (is), on the incidence of cgvhd. we have compared the outcome of 87 consecutive patients that underwent allogeneic sct from march 2011 to november 2015 at the bmt unit of the hammersmith hospital and received csa as part of the gvhd prophylaxis. of them, 54 patients (62%) remained on csa prophylaxis for the duration of the planned post sct immunosuppression period and 33 patients (38%) required a switch to mmf before day +100. the reason for changing the is was nephrotoxicity in the majority of cases (n = 25, 70%), neurological toxicity (n = 2, 6%), disease relapse (n = 1, 3%), intolerance (n = 2, 6%) or not determined (n = 3, 9%) . we excluded from the analysis those patients whose is was changed due to the presence of acute gvhd. both groups had similar patient and transplant characteristics (see table 1 ). [p171] however, distribution according to diagnosis showed a predominance of aml (43%) in patients that remained on csa and mds (2%) for those that switched to mmf. the mean survival rate of the entire cohort was 902.897 days (±87) . the mean survival of each group was: csa 996.86 days (±109.076) and mmf 602.474 (±94.779). this difference in survival reached statistical significance (p:0.04). we graded cgvhd using the nih scoring system as mild, moderate and severe. out the 54 patients that continued with csa, 55.6 % (n = 30) had no cgvhd; 16.7 % (n = 9) had mild cgvhd; 20.4 % (n = 11) had moderate and 7.4 % (n = 4) had severe cgvhd. in patients that switched to mmf 45.5 % (n = 15) did not develop any cgvhd; 9.1 % (n = 3) developed mild; 24.4 % (n = 8) moderate cgvhd and 21.25 % (n = 7) developed severe cgvhd. (p: 0.093). the cumulative incidence of any cgvhd at 2 years post sct was 58% for the csa/mmf group and 42% for the csa only group (p = 0.04). csa is one of the standards of care for gvhd prophylaxis in both ric and mac sct. in our cohort of patients, those who remained on csa had a better overall survival and a reduced incidence of chronic gvhd compared with those patients that stopped csa and replaced it by mmf. csa toxicity should be prevented to avoid gvhd-related complications. disclosure of conflict of interest: none. although the outcome of allogeneic stem cell transplantation (sct) from an unrelated donor (ud) has considerably improved over the recent years, graft versus host disease (gvhd) still represents a severe and potentially lethal complication. in vivo t-cell depletion with anti-thymocyte globulin (atg) has been shown to significantly decrease the risk of both acute and chronic gvhd without compromising survival, however the optimal dose has not been defined yet. aim of present retrospective study was to evaluate the impact of two different doses of rabbit atg (thymoglobulin) on gvhd incidence, infectious complications and outcome of 156 patients undergoing sct from ud. between february 2004 and september 2015, 40 patients received thymoglobulin 5 mg/kg (atg-5 group) and 116 received thymoglobulin 7 mg/kg (atg-7 group) in addition to cyclosporin and short course mtx as gvhd prophylaxis. the two groups were comparable regarding sex, age, diagnosis and disease phase at transplant, comorbidity index, stem cell source and antimicrobial prophylaxis. conditioning treatment was myeloablative in 90% of atg-5 group patients and in 78% of atg-7 group patients. donor and recipient pairs were 10/10 hla matched in 75% of the cases of the atg-5 group and in 39% of the cases of the atg-7 group (p 0.001). netrophil engraftment occurred in 150 (96%) patients at a median of 17 days post transplant (range: 11-41 days); six patients (2 in the atg-5 group and 4 in the atg-7 group) died before engraftment. overall, 48 patients (31%) developed grade ii-iv acute gvhd, without significant differences between the two groups (atg-5 32% and atg-7 30%, p 0.939). similarly, chronic gvhd was not significantly different between the two groups: moderate to severe chronic gvhd occurred in 30% of the patients in the atg-5 group and in 27% of the patients in the atg-7 group (p 0.846). univariate logistic regression analysis didn't show any significant differences between the two groups respect the incidence of bacteremia, invasive fungal infections acute and chronic gvhd. with a median follow-up of 66.6 months, 84 patients (54%) are alive, 79 in complete remission and 5 after disease relapse. transplant related mortality was superimposable in the two groups (atg-5 17% vs atg-7 20%). kaplan-meier estimates of overall survival and event free survival were 54% and 52%, respectively, without statistically significant differences between the two groups and between hla matched and hla mismatched sct. the results of our study suggest that different doses of atg tailored on hla compatibility might be effective for preventing gvhd with any detrimental effect on overall survival and incidence of infectious complications. a prospective randomized study is mandatory to confirm our preliminary results. disclosure of conflict of interest: none. c-reactive protein levels at acute gvhd diagnosis predict steroid-resistance, treatment related mortality and overall survival after allogeneic hematopoietic stem cell transplantation l minculescu 1 , ls friis 1 , bt kornblit 1 , sl petersen 1 , i schjødt 1 , ns andersen 1 and h sengeløv 1 department of hematology, rigshospitalet, copenhagen, denmark acute graft versus host disease (agvhd) remains an excessive cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (hsct). primary treatment consists of high-dose corticosteroids, but a small group of patients are steroid-resistant and their prognosis is especially poor. a predictor of patients at risk of steroid-failure would aid the decision of additional immunosuppressive treatment at an early stage. there is experimental evidence that co-existing inflammation aggravates agvhd. since c-reactive protein (crp) is a systemic inflammatory marker, we aimed to investigate whether crp levels at agvhd diagnosis could predict the risk of failing first-line therapy and developing steroid-resistance. we retrospectively studied 461 patients transplanted between 2010 and 2015, table 1. acute gvhd was diagnosed in 204 patients, 149 of whom had grade ii-iv. crp, total white blood cell-, lymphocyte-and neutrophil counts were available for all patients at the time of agvhd diagnosis. according to local protocol, patients with failed response to high-dose steroid (2 mg/kg) were treated with the tumor necrosis factor (tnf) alpha inhibitor infliximab and categorized as steroid-resistant. of 149 grade ii-iv agvhd patients 28 (19%) developed steroid resistant disease. crp levels at diagnosis among these where between o1 and 263 mg/l. crp levels where significantly higher in patients who developed steroid resistance compared to patients responding to high-dose corticosteroids, p = 0.001, hr 1.011 (95% ci 1.005, 1.018). this translated into significantly increased transplant-related mortality (trm) and decreased overall survival in patients with high crp levels, figure 1 . total white blood cell-, lymphocyte-and neutrophil counts were not associated with steroid resistance in agvhd patients. cxcr3 is chemokine receptor expressed on activated t lymphocytes, in particular on th1 cells, nk cells, dendritic cells, and subsets of epithelial and endothelial cells. cxcr3 ligands attract th1 cells into inflamed tissues and concomitantly block the migration of th2 cells. furthermore, inhibitory functional autoantibodies against cxcr3 occur in humans which play an important role in cxcr3-dependent immune regulation. in addition, cxcr3 regulates endothelial cell homeostasis. there are two variants of cxcr3: cxcr3-a and cxcr3-b. overexpression of cxcr3-a on endothelial cells is associated with an increase in cell survival, whereas overexpression of cxcr3-b dramatically reduced dna synthesis and up-regulated apoptotic endothelial death. here we have studied if a dysfunctional cxcr3 axis might be involved in gvhd pathogenesis and could link endothelial and t cell pathology in acute gvhd. we assessed concentrations of the cxcr ligands cxcl9, cxcl10 and cxcl11 as well as anti-cxcr3 autoantibodies in 98 patients with high grade (3) (4) acute intestinal gvhd for whom serum was available at gvhd onset. furthermore, anti-cxcr3 autoantibodies and cxcl9 levels were measured in sera stored before conditioning therapy. all variables were tested for influence on post-gvhd survival using cause-specific cox regression analysis. at gvhd onset, we observed a strong inter-correlation of cxcr3 ligands, but no correlation with anti-cxcr3 auto-antibodies. compared with pre-conditioning probes, cxcl9 levels strongly increased (median 303 to 721 pg/ml, p o0.001), whereas antithese results suggest crp levels at diagnosis as a valid predictor of developing steroid resistant disease in agvhd grade ii-iv and survival in allogeneic hematopoietic transplant recipients. [p173] s206 cxcr3 decreased (median 4.4 to 2.6 u/ml, po 0.001). anti-cxcr3 levels before conditioning and at gvhd onset correlated (coeff. 0.497, p o0.001), whereas cxcl9 levels did not. in multivariable analyses, low anti-cxcr3 and high cxcl9 measured at disease onset were strongest predictors of survival after acute gvhd. notably, high levels of the proinflammatory chemokine cxcl9 were particularly prognostic of an adverse outcome of gvhd in the presence of a high endothelial risk as assessed by the previously published easix score, while high anti-cxcr3 levels were most protective in patients with low easix score (that is, low endothelial risk). a score based on cxcl9, anti-cxcr3, and easix allowed an effective prediction of acute gvhd outcome ranging from mortality 490% (high cxcl9 + high easix) to mortality o20% (low cxcl9, low easix, high anti-cxcr3. our data suggest a strong role for the cxcr3 axis in the pathology of acute high grade gvhd. the opposing effects of cxcl9 and anti-cxcr3 indicate a functional, attenuating role for these auto-antibodies. the overall prognostic impact of the immunemodulating cxcr3 axis appears to depend on the underlying integrity of the patients' endothelial homeostasis. despite some progress in acute lymphoblastic leukemia (all) treatment including modern chemotherapy modalities, monoclonal antibodies and newer tyrosine kinase inhibitors (tki) for ph positive cases, the final success is still difficult to reach. allogeneic hematopoietic stem cells transplantation (allohsct) has remained an essential approach in attempts to cure all. tki routinely used for all ph(+) pre-and post-transplant treatment are also described as an alternative and adjunctive approach for chronic gvhd especially with fibrotic features due to their antifibrotic activity targeting the platelet-derived growth factor receptor (pdgfr) pathways. in this study we have tried to estimate the potential influence of pretransplant tki treatment on gvhd occurrence comparing all ph(+) and all ph(− ) cases treated with allohsct. a cohort of 119 all patients consisted of 93 all ph( − ) and 26 all ph(+) cases treated with allohsct was retrospectively analyzed. all patients were transplanted from sibling or unrelated donor (no haploidentical procedures were included). all ph(+) patients achieved pretransplant treatment with imatinib and chemotherapy, and ph( − ) patients with chemotherapy alone. the median age in ph( − ) and ph(+) group was 28 vs 35 (p = 0.04), the percentage of hla mismatched transplantations -4,.3 vs 19.2 (p = 0.00004), the percentage of acute gvhd cases-48.4 vs 76.9 (p = 0.01) and extensive chronic gvhd cases-80.5 vs 50.0, respectively. there were no significant difference between groups in patients sex (f/m-41/52 vs 14/12 respectively), ric/mac conditioning, unrelated/sibling donor, donors age, bm/pbpc transplantation, number of cd34 cells and chronic gvhd incidence. all patients received cyclosporine-and methotrexate-based gvhd prophylaxis. gvhd occurrence was analyzed in subgroups as previously described: all ph( − ) and all ph(+). as mentioned above the incidence of acute gvhd was higher in ph(+) group (higher number of hla mismatched transplantations in this group) but the incidence of extensive chronic gvhd was higher in ph (-) group. cox proportional hazard model analysis revealed death risk caused by gvhd higher in ph negative group (hazard ratio = 2.3; ci 95% = 1.02-5.18; p = 0.04). the analysis of competing events was performed to estimate the probability of death caused by gvhd vs other complications (transplant related mortality, infections and relapse). the impact of conditioning was not significant on gvhd related deaths vs other complications (p = 0.234 vs 0.009, respectivelyfigure 1 ). the same results were achieved with donor cmv status (p = 0.09 vs 0.04figure 2 ). we have not found any significant difference either in gvhd or other complications related deaths taking into account patient s sex/age, donor sex/age, patients cmv status, number of cd34 cells transplanted. on the other hand, the influence of agvhd and chgvhd on deaths related to other complications was not significant (p = 0.242 vs 0.147). cumulative probability of overall survival was higher in ph(+) group but the difference was not significant. the impact of pretransplant treatment with imatinib on gvhd occurrence has not been estimated so far. we are aware of our results to be preliminary and variety of data is still to be evaluated. however our results, if confirmed, may suggest the influence of imatinib on decreasing the extensiveness of chronic gvhd. disclosure of conflict of interest: none. seta-tsukinowa, otsu, 520-2192, japan; 3 department of pathology and laboratory medicine, emory university hospital, atlanta, ga, usa; 4 department of biostatistics and bioinformatics, rollins school of public health, emory university, atlanta, ga, usa; 5 pathology and pediatrics, emory university school of medicine, atlanta, ga, usa, aflac cancer center and blood disorders service, children's healthcare of atlanta, atlanta, ga, usa and 6 bloodworks northwest research institute, seattle, wa, usa more than 90% of allogeneic hematopoietic stem cell transplant (allo-hsct) patients receive red blood cell (rbc) and platelet (plt) transfusions in the peritransplant period. preclinical models indicate that rbc and plt transfusions trigger inflammation, raising the question of whether such transfusions are associated with development of severe acute graft-versus-host disease (grade iii/iv agvhd) and mortality in allo-hsct recipients. we conducted a retrospective analysis of rbc and plt transfusions, agvhd incidence, and mortality among 322 consecutive adult patients receiving non-t celldepleted allogeneic bone marrow (11%) or g-csf-mobilized blood stem cell grafts (89%). common underlying diseases were acute myeloblastic leukemia (41%), myelodysplastic syndrome (14%), and acute lymphoblastic leukemia (12%) . underlying disease risk was ranked as low (41%), intermediate (26%) or high (32%). allografts were obtained from 10/10 hlamatched sibling donors (35%), unrelated donors (43%), or from donors mismatched at 1-2 hla alleles (22%). graft sources were bone marrow (11%) or mobilized pbsc (89%). the cumulative incidences of grade iii-iv agvhd and mortality prior to day 150 without developing grade iii/iv agvhd were estimated using the cumulative incidence function and a cox proportional hazards regression model. covariates included in multivariable analysis was limited to baseline covariates associated with grade iii/iv agvhd at the p median number of rbc or plt transfusions ( figure 1 ). univariate analysis showed a lower hematocrit on admission (median of 5 rbc units transfused (p = 0.001) were significantly associated with the risk of developing grade iii/iv agvhd, while a longer time to neutrophil engraftment was inversely associated with grade iii/iv agvhd ( ⩾ median of 15 days, hr 0.58, p = 0.03). multivariate cox regression analysis showed only larger numbers of rbc units transfused and hla mismatch independently associated with severe agvhd (p = 0.02 and p = 0.008, respectively), while underlying disease risk and larger numbers of transfused rbc units were independently associated with overall survival in a multivariate analysis that excluded agvhd grade. overall mortality rate was lowest for the group with fewer rbc and plt transfusions (43%), and greatest for the group with more rbc and plt transfusions (67%). groups that received more rbc units had higher rates of mortality due to gvhd, while patients who received more plt transfusions and fewer rbc transfusions had greater mortality from relapse ( figure 2 ). these data support the hypothesis that peritransplant rbc transfusions are associated with the risk of developing severe agvhd and worse overall survival following allo-hsct. prospective studies are warranted to whether rbc transfusions promote t-cell activation and inflammation in allo-hsct recipients, leading to increased severe agvhd. disclosure of conflict of interest: none. early high umbilical cord blood cd3 chimerism associated with acute gvhd at time of onset in haplo-cord transplantation h choe 1 , j hsu 1 , s mayer 1 , u gergis 1 , a phillips 1 , t shore 1 and k van besien 1 1 department of hematology/oncology, weill cornell medicine, new york, ny 10065, usa introduction: haplo-cord transplantation is a combined haploidentical and cord blood transplant that allows for more rapid engraftment by the haplo with eventual loss of the haplo graft upon engraftment of the cord. haplo-cord transplants are associated with an approximate 25-43% incidence of agvhd. reported, using chimerism assessments at approximately day 56 after transplant for aml and mds, that lower umbilical cord blood (ucb) chimerism in the cd3 or cd33 lineages were associated with increased rates of relapse. we did not find a statistically significant association between day 56 chimerism and risk for acute gvhd.(2) here we report our analysis of chimerisms at the onset of agvhd. patients and methods we retrospectively reviewed all patients who underwent haplocord sct for all hematologic malignancies between july 2012 and march 2016. ucb for haplo-cord transplants were selected based on hla-typing and cell count. grafts were matched for at least 4 of 8 hla loci by the standard criteria and contained a minimum cell count of 1 ×10 7 nucleated cells per kilogram (kg) of the recipient's body weight before freezing. the haploidentical donor was a relative in the large majority of cases. we identified 90 patients evaluable for agvhd (onset before day 100) without preceding relapse or early death. of the total 90 patients, 31 patients were diagnosed with agvhd of any stage and grade. fractionated chimerisms including cd3 and cd33 components were routinely sent to evaluate for engraftment of the recipient vs haplo vs ucb. chimerism data was collected for both agvhd and no agvhd patients. the two-sided student's t-test was used to compare the agvhd cohort to the no agvhd cohort. chimerisms collected on patients with agvhd were within median ± 4 days of onset of agvhd. the median time to onset of agvhd was 47 days (range: 15-99 days). the median post-transplant chimerism recorded for comparison with the no agvhd patients was 57 days. the agvhd cohort had significantly lower cd3 recipient (p = 0.005) and higher cd3 ucb engraftment (p = 0.006). all other fractions, including the cd33 chimerisms, were not significantly different between the two cohorts. the agvhd vs no agvhd cohorts were further compared by degree of hla mismatch (4-6 out of 8 vs 7-8 out of 8). the frequency of agvhd was similar in the 4-6 out of 8 (23/62, 39%) and the 7-8 out of 8 (8/28, 28%) groups. within these subgroups, cd3 ucb chimerism was higher for those with agvhd (p = 0.03 and p = 0.03, respectively). conclusion the onset of agvhd in haplo-cord transplantation is associated with a significantly higher cd3 ucb chimerism and lower cd3 recipient chimerism. higher ucb chimerism may indicate that full ucb chimerism poses a higher risk of agvhd development. vice-versa persistent recipient chimerism may protect from acute gvhd. il-6 is a pleiotropic cytokine with both pro-and antiinflammatory properties (scheller 2014). the proinflammatory properties are mediated through trans-signaling that depends on the soluble il-6 receptor. il-6 trans-signaling is involved in several autoimmune diseases and in regulation of tissue regeneration of the gi-tract. specific snps in the il-6 receptor have been associated with increased baseline crp levels, severity of autoimmune diseases and response to interleukin-6 inhibition in rheumatoid arthritis. so fare little is known about the role of trans-signaling in graft-versus-host-disease (gvhd). in this study we investigated how 4 specific snps in the il-6 receptor influence pretransplant levels of crp, il-6 sil-6r and the risk of grade ii-iv acute gvhd in allogeneic stem cell transplantation (asct) in patients with family donor. dna was available for 103 patients (65 male, 40 female median age 48, range: 15-70) and 101 donors, that underwent asct with a matched related donor (97 sibling) at haukeland university hospital in the period 2006-2016. the majority received conditioning with either bycy (79) or flubu (17) and only 2 patients were transplanted with tbi-based conditioning. four different snps in the il-6r gene (rs2228145, rs4845617, rs4845618, rs4845374) were chosen on the basis of (i) documented or suspected roles in autoimmune disorders; and (ii) allele frequency between 0.10-0.49 and r 2 o0.5 between the different snps. genotyping was done using kaspar assays with viia7 instrument (life technologies). the overall genotype call rate was 98%. no departures (p-values o0.01) from hardy-weinberg equilibrium were observed. pretransplant serum levels of il-6, sil-6r and were analyzed with bio-plex kits (bio-rad, hercules, usa). both serum and dna analyses were performed in duplicates. patients being homozygous for the rs4845618 minor allele had significantly higher pretransplant serum sil6r levels but lower crp levels compared with patients homozygous for the major allele. the overall incidence of agvhd requiring high-dose steroid treatment (grade ii gastrointestinal, grade iii-iv liver and skin) in the cohort was 48%. when analyzing the conventional clinical and laboratory parameters only transplantation with a non-sibling donor was associated with increased risk of agvhd (p-value o0.01 hr 3,20 confidence interval 1.42-7.17). the presence of the rs4845617 in donor or recipient was associate with a significant increase in the rate of aghvd (p-value 0.027 hr 1.93 confidence interval 1.08-3.47). the snp rs4845617 (p-value 0.04 hr 1.86, confidence interval 1.04-3.35) was also significant in an adjusted model including both donor type and rs4845617. none of the evaluated snps were associated with an increase in early or late trm and did not influence os either. this study suggests that snps in the il-6r influence pretransplant biochemical characteristics and clinical outcomes after asct. future studies investigating the effect of il-6 inhibition as gvhd prophylaxis or treatment should include analyses of il-6 receptor snps to investigate their possible influence on treatment outcomes. graft-versus-host disease (gvhd) continues to be the major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant (allo-hsct). the prophylaxis scheme varies according to the center and the country. in ours institution we use triple-prophylaxis based on cyclosporin a (cya), metrotexate (mtx), and mycophenolate mofetil (mmf). this scheme has been used for more than one decade in asian centers where it has proven adequate effective and safe to prevent gvhd. we evaluated 90 patients undergoing allogeneic hematopoietic stem cell transplantation treated at the national cancer institute from january 2010 to december 2015. the triple-prophylaxis scheme consists in cya (adjusted serum levels, mtx (5 mg/m 2 days +1, +6, +11, +18) and we evaluated different doses of mmf, one of them includes 500 mg bid × 35 days and the other has high doses (15 mg/kg bid × 180 days), as gvhd prophylaxis. the response characteristic was analyzed using the pearson test, fisher's exact test on categorical variables and student's t-test, mann-whitney u on continuous tests. kaplan-meier method was used to estimate the probabilities of os, sle with the differences compared by the log-rank test. we analyzed 90 patients with median of age of 30 years (range: 15-64), 60% male gender, all were transplant with peripheral blood progenitor cells as a source. 52.2% were acute lymphoid leukemia and 25.5% acute myeloid leukemia, 12.2% chronic myeloid leukemia, 3.3% myelodysplastic syndromes, 3.3% aplastic anemias, 2.2% non-hodgkin's lymphomas and 1.1% hodgkin's lymphomas. myeloablative conditioning was used in 82% (bucy, cfm-gat) and 18% reduce intense conditioning (flubu, flucy, flucy-gat), 94.4% related hla compatibility. mmf 500 mg twice daily (bid) for 35 days (group 1) and of mmf 15 mg/kg bid for 180 days (group 2), in the group 2 the 85.3% developed febrile neutropenia vs 64.3% in group 1(p = 0.03). the frequency of gvhd was 19.6% group 1 vs 23.5% group 2 (p = 0.6), chronic gvhd was 23.5% vs 19.6% respectively (p = 0.7). at the moment of analysis 58.9% vs 26.5% were free of disease (p = 0.01). there no difference at 5-year overall survival was 37% (group 1) vs 49% (group 2) (p = 0.85), neither free-survival disease (p = 0.85). the mmf regimen shows noninferiority scheme for gvhd. the low doses and for shorter administration did not show differences in the incidence and severity of acute or chronic gvhd, os, dfs compared to the mmf regimen at 180 days with high doses. the high doses shows higher incidence of febrile neutropenia, but there were no differences in documented infections. disclosure of conflict of interest: none. a protein-losing enteropathy can develop due to conditioning regimen related gut toxicity and can cause albumin decline during peritransplant period in allogeneic stem cell transplantation (allohct). damaged intestinal mucosal barrier results in alloactivation of donor t cells and this situation is considered a primary event in the pathogenesis of acute graft-versus-host disease (agvhd). peritransplant albumin decline, as a result of conditioning regimen related protein-losing enteropathy, may predict agvhd (1) . in this retrospective study we tested this hypothesis. we evaluated 249 patients who received allohct between 2011 and 2016. albumin decline from the day of conditioning initiation until its nadir in the first 2 weeks of post-transplant period was calculated as delta albumin. acute gvhd was proven by biopsy in all patients. chi square and mann-whitney test were used for statistical analysis. patients' characteristics were shown in table-1. acute gvhd was developed in 78 patients and severe agvhd was developed in 15 patients. delta albumin was not different between agvhd patients and no agvhd patients. delta albumin was not related with severe agvhd. delta albumin was not different between patients who received myeloablative and reduced intensity conditioning regimens. when we used a cutoff value of 0.9 gr/dl for delta albumin, we could not find a relation between delta albumin and development of both agvhd and severe agvhd. we repeated the analysis for acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) patients who receive myeloablative conditioning regimen and we found the same results, there was no difference between agvhd patients and no agvhd patients in terms of delta albumin. there was a number of studies that used albumin as a predictive and prognostic marker in the setting of agvhd. but albumin may decrease in patients due to many reasons like malnutrition, proteinuria, enteropathy, liver disease or being negative acute phase reactant. because of albumin value can show variability between patients, albumin decline may be a more objective criterion. rashidi et al. showed that 0.9 gr/dl decline in albumin may be a predictor of severe agvhd in 88 patients who was diagnosed with aml and mds and received myeloablative conditioning regimen. we repeat this analysis in our mds and aml (n = 98) patients but we couldn't find this relation. when we evaluated all our 249 patients, again there was no relation between delta albumin and development of both agvhd and severe agvhd. in conclusion, our study did not support rashidi et al.'s findings. because serum albumin level shows variability due to many reasons, it is hard to use albumin as a predictor of agvhd. sclerodermatous chronic graft-versus-host disease (scl-cgvhd) in its severe manifestation affects the patient quality of life and, due to complex pathomechanism, does not respond to standard immunosuppressive therapy-calcineurin inhibitors (cni) with corticosteroid. methotrexate (mtx) and rituximab appeared to be effective in some patients but the novel strategies, including extracorporeal photopheresis (ecp), imatinib, m-tor inhibitors (for example, sirolimus) and ruxolitinib seem to become the real breakthrough. we retrospectively analysed data of 33 patients with scl-cgvhd, who underwent allogeneic hematopoietic cell transplantation (hct) between 2009-2015 in 5 transplant centres. the study group consisted of 32 patients with haematological malignancies and one with aplastic anaemia, 14 female and 19 male, with the median age 36 (18-64). donors' median age was 40, with predominance of matched sibling donors (21 donors) and even distribution of the donors' gender. in 22 patients (67%) acute gvhd (agvhd) was diagnosed with skin involvement observed in 19 ones. acute gvhd directly progressed to cgvhd in 13 cases. in 11 patients (33%) cgvhd developed ‛de novo' and in 2 cases cgvhd was induced by dli. median time from hct to cgvhd diagnosis was 8 months and to scl-cgvhd diagnosis-32 months. seven patients (21%) were scored as moderate cgvhd and 26 patients (79%) as severe cgvhd according to nih-2014 cgvhd activity classification. in 14 patients sclerotic features had superficial form and in 19 ones deep sclerosis was observed. chronic gvhd manifestation in other organs includes: mouth (94%), joints and fascia (77%), liver (74%), eyes (64%), gi tract (33%) and lungs (21%). 26 patients were treated with ecp and/or sirolimus and /or imatinib with 80% response rate (complete-cr, partial-pr or minimal-mr). median duration of ecp therapy, sirolimus and imatinib treatment was 20 months (2-40), 3 months (1-30), and 5 months (1-18), respectively. sirolimus was added more likely (9 patients) as the first in case of suboptimal response to ecp after median 15 weeks and in 4 patients was subsequently replaced by imatinib with no favourable outcome in 3 cases. in 3 patients imatinib was initially used in combination with ecp therapy, leading to pr or mr. mtx without novel therapies was effective in 4 patients with limited skin involvement, 3 patients responded to mtx plus imatinib and 1 patient to mtx plus sirolimus. two patients, after failure of other therapies, have been receiving ruxolitinib with improvement. only 3 patient (15%) were nonresponsive to ecp (progressive or stable disease), 4 patients (36%) to sirolimus and 4 patients (33%) to imatinib. toxicity incidence was equally observed in case of sirolimus and imatinib and lead to the therapy discontinuation in altogether 4 patients. infectious complications were observed in 20 patients (60%). ecp confirms to be the most effective therapeutic strategy in severe forms of scl-cgvhd with favourable safety profile. imatinib and sirolimus, targeting different fibrotic pathways, both play important role in nonresponsive patients, improving the outcome in ecp and non-ecp group. in case of limited access to ecp, mtx remains to be beneficial in combination therapy of moderate scl-cgvhd and an alternative to cni. disclosure of conflict of interest: none. post-transplant morbidity and mortality are majorly determined by gvl effect counter-balanced by gvhd. treatment with systemic steroids represents the first-line therapy for gvhd, but is associated with increased incidence of infection and relapse. ecp can reduce the extent of gvhd while preserving anti-virus/-tumor activity. to elucidate this clinical phenomenon on an immunological level, we correlated clinical data with immunological findings in 20 patients under ecp treatment. nine patients with acute gvhd (agvhd) of the gut ii-iv suffering from severe diarrhea were treated by ecp in addition to triple-drug immunosuppressive therapy. furthermore, 11 patients with chronic gvhd (cgvhd) of the skin or lung despite triple-drug received ecp treatment. patients were evaluated according to their individual response and clinical condition. phenotypical analysis of different cellular subsets of patients and healthy donors was performed by multicolor flow cytometry. functional properties of virus-specific cd8+ t and nk cells were evaluated by inf-γ-elispot and 51cr-release assay. about 20 patients were treated by ecp in this study. however, two agvhd and two cgvhd patients had to be withdrawn from ecp treatment after a few ecp cycles due to pancytopenia or poor clinical condition. for patients with agvhd 8 up to 25 ecp cycles were needed for response. all patients achieving a complete response (cr) were still alive 1 year after initiating ecp therapy. overall response, that is, cr or partial response (pr) according to nih criteria, was obtained in 5 of 7 patients with agvhd (71.4%) including cr in 3 of 7 (42.8%). out of 9 cgvhd patients 7 (77.8%) reached pr, and 2 (22.2%) remained stable under ecp treatment. after 1 year, overall survival (os) was 60% for agvhd patients responding to ecp, while only 25% for non-responders. os for cgvhd patients was 91%. during intensive ecp treatment for patients with agvhd of the gut, the average stool volume and frequency decreased and consistency changed from loose to formed stool. steroids could be tapered down to a mean of 22% of the initial dosage. cgvhd patients were stabilized under ecp treatment and steroid dosage could be reduced to a mean of 38%. clinically responding patients showed increased numbers of regulatory cells including mdscs, foxp3+cd8+ and foxp3+cd25+cd4+ tregs, as well as cd4 − cd8 − cd3+ t, vδ2+ t cells and regulatory b lymphocytes. furthermore, loss of cd62l expression on effector cells like cd4+ te, cd8+ te, nk and nkt cells was observed under ecp treatment. interestingly, ecp treatment did not dramatically influence the frequency of cd4+cd8+cd3+ t, γδ t cells and nkt cells, which possess anti-virus/-tumor function. elispot and 51cr-release assays revealed stable anti-viral activity of cd8+ t cells as well as functional cytotoxicity of nk cells. moreover, cd8+ t, cd8+ tem, cd62l+cd4+ temra, cd56 +cd3 − nk and cd56brightcd16 − nk cells could serve as reliable biomarkers for prediction of response to ecp. conclusion: ecp treatment might stabilize or even improve clinical situation of patients suffering from gvhd. in clinically responding patients an immunomodulation was observed in terms of increasing numbers of regulatory cells with loss of migratory capacity of effector cells while anti-virus/-leukemia t-cell function was preserved. disclosure of conflict of interest: none. extracorporeal photopheresis affects dendritic cells by reducing total numbers and blunting cytokine production in patients with graft versus host disease tj altmann 1,2 , m bickerton 1 , am flinn 1 , u cytlak 1 , p milne 1 , s pagan 1 , m collin 1,2 , v bigley 1,2 and ar gennery 1,2 1 institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom and 2 newcastle upon tyne hospitals nhs foundation trust, newcastle upon tyne, uk graft versus host disease (gvhd) and concomitant immunosuppression is a leading cause of morbidity and mortality post hematopoietic stem cell transplantation (hsct). the pathophysiology of gvhd is complex, involving presentation of histo-incompatible antigen by activated recipient dendritic cells (dcs), activation and proliferation of donor t cells and resultant tissue damage. extracorporeal photopheresis (ecp) is a second-line treatment for steroid refractory or dependent gvhd that facilitates the reduction of immunosuppression. ecp's mechanism of action is unclear and is likely to be multifaceted. apoptosis of lymphocytes, induction of a th2 favoured environment and increased numbers of regulatory lymphocytes have been implicated 1 . although ecp has been shown to modify the function of in vitro monocyte-derived dcs 2 , its effect on primary (non monocyte-derived) dcs has not been studied. our aim was to determine whether ecp had immediate or long-term affects on primary dc numbers or function. we enumerated monocyte and dc subsets (cdc1, cdc2 myeloid dcs and plasmacytoid dcs) in whole blood before, during and after ecp cycles, and developed a novel dc function assay, suitable for use on clinical samples. four adults with immunosuppression withdrawal gvhd and four children with acute gvhd, received ecp during the study. all received ciclosporin gvhd prophylaxis and corticosteroid treatment at onset of gvhd. children received ⩾ 1 dose of infliximab prior to starting ecp. adults received two ecp treatments (one cycle) every 2 weeks and children received two ecp treatments (one cycle) weekly. whole blood was taken before and after each cycle of ecp. trucount flow cytometry analysis of whole blood was used to enumerate mononuclear leukocytes. to assess function, peripheral blood mononuclear cells were isolated by density centrifugation and stimulated with toll-like receptor agonists. cell-specific cytokine production was then analyzed by flow cytometry. samples were compared to healthy controls and pre-ecp samples. median time to first ecp treatment from gvhd diagnosis was 33.5 days. no gvhd flares were experienced during study period. (1) adult had a cycle of treatment delayed due to intercurrent pneumonia. numbers of cdc2, pdcs and classical monocytes were significantly reduced after each ecp treatment in the adult group. dc numbers followed the same trend after ecp in the paediatric group but were not significantly different before and after ecp. this is perhaps due to initial lower dc count compared to adults in the children before the first ecp cycle. functional analysis showed a reduction in cytokine production in dcs and monocytes in both groups over the course of ecp treatment. our data support a cell-intrinsic effect of ecp on monocytes and dcs, with numerical and functional consequences. this may contribute to the beneficial effect of ecp both through reduction of inflammatory effector function and through modulation of interactions with other immune cells. correlation with immunosuppression withdrawal and clinical events during treatment may provide further insight into the role of monocytes and dcs in gvhd and ecp, which may aid in the development of novel targeted therapies for gvhd. extracorporeal photopheresis as early second-line treatment for patients with steroid-dependent or refractory acute graft-versus-host disease: a single-centre experience i sakellari 1 , i batsis 1 , e gavriilaki, a-k panteliadou, a lazaridou, k leontopoulos, d mallouri, a bouinta, v constantinou, e yannaki, c smias and a anagnostopoulos 1 1 department of hematology bmt unit, g. papanicolaou hospital, thessaloniki, greece acute graft-versus-host disease (agvhd) remains a severe complication of allogeneic haematopoietic cell transplantation (allohct). corticosteroids as the backbone of initial therapy for agvhd result in varied complete responses (25-69%). traditional secondary treatments lead to profound immunosuppression without improved survival. on the basis of our experience in chronic gvhd, we aimed to prospectively assess the role of extracorporeal photopheresis (ecp) as early secondline treatment in steroid-dependent and refractory agvhd. we enrolled consecutive patients with steroid-dependent or refractory grade (gr) ii-iv agvhd post allohct from january 2013 to august 2016. all patients with unrelated or haploidentical donors received thymoglobulin (atg) 5 mg/kg as prophylaxis. post-transplant gvhd prophylaxis included cyclosporine-methotrexate in myeloablative and cyclosporine-mycophenolate mofetil in reduced toxicity or intensity regimens. ecp was commenced after assessment of response to 5 days of steroid treatment according to our protocol: two sessions per week for 1 month, one session per 2 weeks for 3 months, evaluation of response and one session per month for 6 months. we studied 20 patients, aged 35 (18-65), post allohct with myeloablative (14), reduced toxicity (4) and intensity (4) conditioning, from sibling (3), matched (8) or one locus mismatched (8) volunteer unrelated and haploidentical (1) donors. disease risk index was high (10), intermediate (9) and low (1). acute gvhd was observed at day +17 (8-50) in 15 patients, late onset at + 130 (110-160) in 4 patients and induced at +38 post donor lymphocyte infusion in a relapsed aml patient. skin, intestine and liver involvement was evident in 6 patients, skin and intestine in 10 and skin only in 4 patients. nine patients (2 with grii, 7 with griii agvhd) were steroid-dependent and 11 (8 with griii, 3 with griv) steroidrefractory. atg was administered simultaneously with ecp initiation in six refractory patients that further developed ebv reactivation (p = 0.032) treated pre-emptively with rituximab. ecp was commenced at day +51 for 15 (4-20) sessions. the majority of patients (16/20) presented partial (6), very good (9) or complete (1) response to ecp. with 8.3 (1.7-51) months of follow-up, immunosuppression was reduced in 10/20 and ceased in 1 patient. clinically significant bacterial infections were found in 17 patients, fungal in 2, cmv and ebv reactivation in 14 and 13, respectively, and other viral in 5 patients. cumulative incidence (ci) of chronic gvhd was 77.4 at 1 year. one-year ci of agvhd-related mortality was 21%. one-year overall survival (os) was 53% and significantly increased in steroid-dependent vs refractory patients (80% vs 36%, p = 0.039). reduction of immunosuppression (p = 0.008) and response to ecp (p = 0.034) were associated with improved os, irrespectively of other factors. our data indicate that ecp should be considered early in the course of steroiddependent or refractory agvhd, before irreversible end organ damage has been established. optimal timing of intervention, frequency, duration and tapering schedule of ecp remain important unanswered questions. [p186] disclosure of conflict of interest: none. extracorporeal photopheresis for treatment of chronic graft versus host disease m lanska, a zavrelova, j radocha and p zak faculty of medicine, 4th department of internal medicine-hematology, university hospital hradec kralove, czech republic allogeneic stem cell transplantation represents a curative approach to many hematologic disorders. graft versus host disease (gvhd) is a1 complication with significant morbidity, mortality and decreased quality of life. extracorporeal photopheresis (ecp) represents possible treatment approach. mononuclear cells (mnc) collected by apheresis are photosensibilized with 8-methoxypsoralenem ex vivo, irradiated with uva and transfused back to the patient. aim of the study: evaluation of patients treated with ecp for gvhd at our center. thirteen patients (8 females and 5 males, median age 44 years) were treated with ecp. about 12 patients (pts) had matched sibling donor and 1 patient had unrelated donor. about 10 pts had sclerodermic form of gvhd, 2 had concomitant pulmonary gvhd, 2 had pulmonary gvhd alone. one pts had mild, seven moderate and five severe gvhd according to nih. about 11 patients were treated with steroids. mnc separation was prepared on cobe spectra and spectra optia (terumo bct, usa). 8-methoxypsoralen was added, irradiation was done on macogenic g2 (macopharma, mouvaux, france). about 696 procedures in 13 pts were performed (median 41 procedures, 12-120 procedures). the schema was as follows: ecp on 2 consecutive days every 2-3 weeks first 3 months with subsequent increase in interval. median follow up was 35 months. in sclerosing form two pts reached cr, six pts pr, one is stable and one patients progressed. in pulmonary gvhd one reached cr, two partial improvement, one is stable. seven pts are still alive, six died (two due to relapse, one secondary malignancy and three infections). it was possible to withdraw steroids in 10 pts. adverse events were clinically negligible. ecp is an effective treatment for chronic gvhd. especially sclerodermic form responds to ecp very well. it is safe and well tolerated procedure with minimal toxicity. supported by prvouk p-37. disclosure of conflict of interest: none. allogeneic haematopoietic stem cell transplantation (hsct) is a potentially curative treatment option for children with a variety of haematological, oncological and immunological diseases. graft versus host disease (gvhd) represents a major cause of post-transplantation mortality and morbidity affecting multiple organs including skin, gut, liver and lungs. gvhd is considered a succession of inflammation and donor t-cell activation initiated by translocation of gastro-intestinal microorganisms through impaired mucosal barriers after chemotherapeutic conditioning and/or infection. diagnosis of gvhd is based on clinical symptoms and histological findings, necessitating invasive and potentially harmful procedures including endoscopy and biopsy. as yet, no non-invasive markers are available for diagnosis or treatment monitoring in children with gvhd. faecal calprotectin (fc) reflects intestinal mucosal inflammation of any origin. in the setting of allogeneic hsct in adults, fc has shown to be a marker for acute (steroid-resistant) gvhd. we aimed to evaluate the feasibility of prospective fc measurement as a non-invasive marker for diagnosis and treatment in children with gvhd. a prospective, observational, single centre study was started in july 2015. by december 2016, 21 paediatric allogeneic hsct patients (age 0-17 years) were included after informed consent. faecal samples were collected from 2 weeks before to 6 months after hsct. fc levels were measured by elia, according to manufacturer's instructions. clinical symptoms were prospectively evaluated and managed according to local guidelines. if gvhd was suspected on clinical grounds, histological confirmation was obtained. first-line therapy for gvhd consisted of corticosteroids. in case of steroid-resistant disease, more advanced immune modulation was applied. a total of five patients developed histologically confirmed gvhd: acute gvhd of skin and gut (n = 2, one patient with steroidresistant disease), acute gvhd of skin only (n = 1); chronic gvhd of lung only (n = 1) and acute gvhd of skin followed by chronic oromucosal gvhd (n = 1). without exception and regardless of gut involvement, gvhd occurrence was accompanied by rises in fc levels to values 4100 μg/g (range: 108-1600 μg/g). fc levels correlated with clinical and histological grading. moreover, adequate response to therapy was consistently reflected by return of fc levels to values o100 μg/g. sensitivity of fc levels to diagnose gvhd was poor due to increased fc levels in patients with posttransplant complications other than gvhd such as viral reactivation and pulmonary or gastro-intestinal infections. fc levels reflect gvhd occurrence and correlate with clinical and histological grading in paediatric allogeneic hsct patients. fc levels increase in case of gvhd regardless of gut involvement, supporting a central role for (subclinical) intestinal inflammation in gvhd initiation. although, in this interim analysis, fc lacks sensitivity to diagnose gvhd, fc may serve as a noninvasive marker for monitoring therapy response and, thereby, reduce the need for repeated invasive procedures including endoscopy and biopsy. acute graft-versus-host disease (agvhd) is a major complication of allogeneic hematopoietic cell transplantation (hct), and glucocorticoids are typically used as first-line treatment. the aim of our study was to evaluate the effect of first-line ecp +/ − steroid therapy in order to reduce the incidence of infections and toxicity. from december 2010 to january 2016, 48 of 180 pts (27%), were diagnosed with agvhd grade ⩾ 2 following allosct. 40 pts were treated with ecp +/ − steroid as first-line therapy. about 8 (20%) pts were treated with ecp only and 32 (80%) with ecp + steroid 1-2 mg/kg/day. we compare this cohort with an historical group of patients, transplanted between 2001 and 2011, who were treated with steroid only for grade 2-4 agvhd (n = 23 out of 130). the two cohorts were well balanced in terms of median age (p = 0.4), disease type (p = 0.9), disease status (p = 0.09), graft source (p = 0.2), conditioning regimen (p = 0.1) and hct-ci (p = 0.3). there were more female patients (p = 0.03) and more haploidentical transplant (haplo-sct) (p = 0.0005) in the cohort treated with ecp+/ − steroid. ecp was performed using the offline technique, and was started as soon as possible with a treatment schedule consisting of four rounds of two procedures per week, three rounds of two procedures every other week and finally two procedures every month. steroid was tapered as soon as possible after starting ecp. the clinical response was evaluated at day +28. median follow-up for alive patients was 28 months for ecp group and 97 months for control group. there was no difference in terms of median time of agvhd onset (38 vs 39 days) and number of pts with grade 2 or 3-4 agvhd ( figure 1 ). ecp was started after a median of 4 (0-30) days from agvhd diagnosis. every patient underwent a median of 19 (2-83) ecp procedures, during a median time of 6 months. on day 28 after starting agvhd treatment with ecp+/ − steroid, 24 pts (70%) achieved cr or pr, 10 pts did not respond and 2 experienced agvhd relapse to front-line therapy. one year cumulative incidence (ci) of agvhd relapsed/refractory was 28.5% for ecp+/ − steroid. these percentages were not different from the cohort receiving steroid alone. ci of moderate-severe cgvhd was lower in the ecp group, probably due to the higher frequency of haplo-sct with pt-cy in the ecp group. about 100 days after agvhd onset, ci of infection (49% vs 74%), especially cmv reactivation (34% vs 67%), was lower in the ecp group, but was not statistical significant. ecp allowed a faster taper of steroid: 17 (3-98) vs 75 days (23-338) (p o0.0001). overall survival, progression-free survival, non-relapse mortality and ci of relapse rates did not differ in the two groups. in multivariate analysis, visceral involvement by agvhd was associated with an increased risk of failure to front-line therapy (hr: 5.5; range: 0.7-41; p = 0.09). this observational study suggests that the overall response rate of ecp +/ − steroids is similar to steroid alone for front-line treatment of grade 2-4 agvhd, but is potentially associated with lower incidence of infection, and in particular of cmv reactivation. a prospective phase 2 clinical trial is warranted to address whether augmentation with ecp may be beneficial for agvhd frontline treatment. [p189] a 4-year-old girl with neuroblastoma received autologous stem cell transplant (asct), followed by antibiotic prophylaxis and filgrastim. her transplant preparative regimen consisted of busulfan and melphalan. engraftment of neutrophil took place on day 11 after asct. twentieth day after asct, she experienced nausea and diarrhea. there was neither skin rash nor elevation in liver enzymes. the diarrhea continued to worsen day by day and reached to a daily volume of 1500 ml/ m 2 . infectious studies for stool and blood including testing for influenza a and b, parainfluenza, adenovirus, epstein-barr virus, amebiasis, cryptosporidium parvum, cytomegalovirus, clostridium difficile, salmonella, campylobacter, yersinia and shigella were all negative. colonoscopy and endoscopy were performed by an experienced pediatric gastroenterologist and findings were suspicious for severe graft versus host disease (gvhd). colonoscopy and rectoscopy revealed severe inflammatory changes, friability and patchy dark exudates on the mucosa of rectum. endoscopy revealed erosions, ulcers in the esophagus and a pale mucosal surface with reticulated submucosal vessels accompanied with erosion and erythema in the antrum of stomach. grade 3 gvhd was confirmed by pathologic analysis that revealed diffuse crypt dropout and mucosal erosion on rectal mucosal biopsy. mucosal erosions, apoptosis of epithelial cells and small lymphocytic infiltration of the lamina propria were found on duodenal biopsy. after these results, we started methylprednisolone intravenously at a dosage of 2 mg/kg/day. on the fourth day of treatment we increased the dosage to 5 mg/kg/day and added cyclosporine to treatment. because of unresponsiveness to treatment we decided to administer thirdparty mesenchymal stem cells (msc) (1 × 10 6 cd73+/cd105+ cells per kg). these were given intravenously at day +49 asct as single infusion. the second dose was given at day +56. within 5 days after first application of mscs, the frequency of diarrhea decreased to one-third. at day +16 after second dose of mscs, the patient's stool became nearly normal. we tapered the steroids first and stopped cyclosporine at +92th days after asct. discussion and conclusion: to our knowledge, this is the second case report of spontaneous severe autologous gvhd in a child with a solid tumor malignancy. regarding the pathogenesis of autologous graft-versus-host disease, there may have multiple causes for the loss of tolerance to self because of disrupted immune system. alteration of t regulatory cells by previous chemotherapy may be key point. endogenous cells that survive conditioning and assist in post-transplant maintenance of self-tolerance may be affected. microchimerism due to maternal cells transmitted during fetal development and persisting throughout adult life has also been postulated as a cause. however it is not very clear for factors that may contribute to the pathogenesis of this rare disease. autologous gvhd has the potential to cause critical illness in the hematopoietic stem cell transplantation patient population. in patients with multiple myeloma some experts report pathologically verified gastrointestinal gvhd as high as 6%. responses to steroids are variable. however, a significant proportion improve dramatically after early therapeutic intervention. so clinicians and pathologists should be aware in suspecting and recognizing gvhd in patients with diarrhea to guide therapy as soon as possible. disclosure of conflict of interest: none. haplosct patients did not receive additional gvhd prophylaxis aside from the ex vivo t-cell depletion (tcd) with clinimacs system. of the bud bmts 36 out of 39 patients engrafted, 92% of which had gvhd. the 8% who did not have any gvhd, relapsed. eighty one percent had agvhd, of which majority (51%) were grade 2 ( table 1) three patients did not have agvhd (10/10 mud and two cord blood grafts), but developed cgvhd. one of the patients who had grade 4 agvhd died and one still has intermittent cgvhd 12 years post bmt. there were 55% who had cgvhd. currently, all of our haplosct receive a cd3/cd45ra tcd grafts (n = 14). the depletion techniques for the 14 others were either cd3tcd, cd3/ cd45ra /tcrab tcd+cd34 +/cd45ra tcd; tcrab tcd. all 28 patients who received haplosct engrafted and 57% had agvhd (56% grade 1) ( table 1 ). we noted that some of the patients presented with nonclassical agvhd signs (upper gut gvhd, oral gvhd, blood). there were no patients who presented with grade 4 agvhd. cgvhd in the cohort was 35%. of note, 35% of patients did not have any gvhd and did not receive any form of immunosuppression post bmt. only eight patients received further immunosuppression for agvhd, median duration 131 (range: 91-344) days. at the time of this report, there are four patients with agvhd still receiving immunosuppression all o100 days and all on tapering doses. haplosct using ex vivo tcd techniques has a lower risk of gvhd with comparable, if not superior outcome to bud. the degree and duration of immunosuppression is also much less. this may translate to earlier immune reconstitution and less viral reactivation. [p191] disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (allo-hsct) offers a potential cure for several hematological diseases, but it is burdened by severe life-threatening complications, being gvhd the major cause of morbility and mortality. recently, more have been understood of the physio-pathologic relationships between endothelium and graft-versus-host disease (gvhd), showing that vascular endothelium is an early phase target of gvhd. in recent years, the direct count of circulating endothelial cells (cec) has emerged as a valuable biomarker of endothelial damage in a variety of disorders. however, due to their rareness and complex phenotype, different published techniques have showed variable degrees of uncertainty, reporting a wide range of cec values in healthy subjects. by means of the commercially available rare cell isolation platform cellsearch system, for cec identification and count, we correlated cec count changes to gvhd onset and response to treatment in allo-hsct patients. cec were analysed in 90 allo-hsct patients (37 aml, 15 all, 11 hd, 4 nhl, 4 cll, 5 mds, 4 cms, 8 mm, 2 saa) at the following time points: t1 (pre-conditioning), t2 (pre-transplant), t3 (engraftment), t4 (day+28 or onset of gvhd), t5 (1 week after steroid treatment). the median cec/ml at t1 was 24 (range: 2-786), in comparison to a value of 2 (range: 1-14) in healthy controls (p 0%: or 4.2, 95% ci 1.6-10.8; p = 0.002). we confirm that cec count represent a valid biomarker to monitor endothelial damage in patients undergoing allo-hsct and can be a valuable tool in supporting the diagnostic definition of gvhd and in monitoring responsiveness to treatment. moreover, the use of the cellsearch system can be crucial in order to move routinely cec monitoring into clinical practice of allo-hsct. reference clinicaltrials.gov nct02064972. disclosure of conflict of interest: this research was conducted with the support of the investigator-initiated study program of janssen diagnostics, llc to ca. kb is employee of janssen diagnostics. results of hla mismatched unrelated donor (mmud) hematopoietic cell transplants (hct) are worse than results of fully matched hct due to higher risk of gvhd, infection and graft failure. atg during conditioning reduces incidence of gvhd but can increase risk of infection and relapse. high doses posttransplant cyclophosphamide (2 × 50 mg/kg) prevent gvhd in haploidentical hct. we initiated this approach instead of atg in hct from one alelle or antigen mismatched unrelated (7/8)mmud-hct in 2014. here we present outcome of 21 patients (cy-group) transplanted between 2014 and 2016, comparing it with outcome of 54 patients transplanted between 2010 and 2016 from 7/8 mmud with atg-f (fresenius) 40 mg/kg given during conditioning. 21 patients in cy-group (12 males, 9 females) were transplanted from mmud mismatched for hla ( a-8, b-3, c-5, dr-5). about 14 patients had aml, 2 mds, 2 cll, 1 cml, 1 all and 1 mps. med. age of patients was 43 years (25-60). about 16 patients received myeloablative (flu-175 mg/m + iv bu 12.8 mg/kg) and 5 nonmyeloablative(flu-175 mg/m+mel 100-140 mg/kg +-tt 5 mg/kg) conditioning. about 18 patients received pbpc and 3 bm as a graft. graft versus host prophylaxis consisted of cyclophosphamide (50 mg/kg aibw) on d+3 and +5, cyclosporine a from d 0 and mmf from d+1. all patients received antibacterial, antifungal, hsv and pcp prophylaxis. historical control (atg) group consisted of 54 patients (32 males, 22 females), med. age 54 y (19-65) who had mmud-hct for aml-21, mds-9, nhl-5, mf-5, all-4, cll-4, cml-2, h.d-1, saa-1, mps-1 and mm-1. there were 13 mismatches for a, 11 for b, 20 for c and 10 for dr. myeloblative conditioning was used in 32 and nonmyeloablative in 22 patients. all patients received atg-f 20 mg/kg × 2 given d-2 and-1. cyclosporin was initiated d-2 and mmf d-1. all patients received anti-infectious prophylaxis as described previously. three of 21 patients from cy group died so far. two of them due to relapse and one due to toxicity and infection during aplasia. five patients relapsed . two achieved cr after dli and one is alive in relapse expecting second hct. about 18 patients are alive, 17 of them in cr. eight patients experienced agvhd (gr.i-3, gr.ii-5,gr.iii-0, gr. iv-0) and eight developed clinically mild cgvhd). about 22 patients from atg group are alive 11-80 m (med. 32 m) posthct. about 32 patients died 1-74 m postransplant (med. 16 m) due to vod, gvhd, infections and relapse. 100-day mortality is 5% (1/21) in cy group and 19% (10/54) in atg group. one year mortality is 14% (2/14) in cy and 30% (13/44) in atg group. patients from cy group have 78% probability of os at 24 months posthct vs 47% from atg group. cyclophosphamide 2 × 50 mg/kg instead of atg fresenius(40 mg/kg) for gvhd prophylaxis reduced 100-day and 1-year mortality, and improved probability of 24 m os significantly in our cohort of patients. this approach seems to be safe and effective in 7/8 mmud-hct. disclosure of conflict of interest: none. high transplanted cd34+ cells are not associated with beneficial effect on graft-versus-host disease-free, relapse-free survival (grfs) after allogeneic hematopoietic cell transplantation y lee 1 and ih lee 2 1 department of hematology, kyungpook national university hospital and 2 inhee lee the success of allogeneic hematopoietic cell transplantation (allo-hct) is comprehensively assessed by individual comorbidity, relapse, graft-versus-host disease (gvhd) and death. besides, inconsistent results have been reported regarding the dose of cd34+ cells. in the current study we have addressed the issue of the potential effect of stem cell dose on the of gvhd-free/relapse-free (grfs) associated with cd34+ cells doses. we retrospectively reviewed the medical records of the 255 patients who received allo-hct for acute myelogenous leukemia (aml), myelodysplastic syndrome (mds) and acute lymphoblastic leukemia (all) between 1998 and 2013 in kyungpook national university hospital. the grfs included grade 3-4 acute gvhd, systemic therapy-requiring chronic gvhd, relapse or death. the patients were reclassified into two groups according to the targeted cd34+ cell doses (6 × 10 6 per kg) by knuh protocol. a lower cd34+ group (n = 165, 64.7%), patients who underwent allo-hct with cd34+ cell dose o6 × 10 6 per kg; and a higher cd34+ group (n = 90, 35.3%) patients who underwent allo-hct with cd34+ cell ⩾ 6 × 10 per kg. the median age at transplant was 38.5 years (range: 15-68 years) and male was 111 patients (44.4%). primary diseases for allo-hct were aml/mds (n = 175, 70%) and all (n = 75, 30%). one hundred forty-three patients (57.2%) were in cr1 (complete remission), 25 (10%) in further cr and 87 (33.2%) in relapsed and refractory status. one hundred seventy-one patients (68.4%) received myeloablative conditioning regimen. gvhd prophylaxis consisted of methotrexate and cyclosporine a or mtx and tacrolimus. the median dose of cd34+ cell was 3.94 × 10 6 per kg (range: 0.46-6 × 10 6 per kg) in lower cd34+ group and 7.54 × 10 6 per kg (range: 6.01-20.6 × 10 6 per kg) in higher cd34+ group. there was no significant difference in neutrophil, platelet engraftment between two groups. the incidence of chronic gvhd was more frequent in higher cd34 + group (32.9% vs 48.2%, p = 0.042). the median follow-up duration was 18.1 months, with a range of 0.2-209.7 months. the 1-year overall survival (os), relapse free survival (rfs), nonrelapse mortality (nrm) and graft-versus-host disease (gvhd)free/relapse-free survival (grfs) since hct was 55.3 ± 3.1%, 66.0 ± 3.2%, 28.2 ± 0.3% and 32.9 ± 3.1%, respectively. there was no significant difference according to the infused cd34+ cell dose (figure1). the relapse rate was not proportionally affected by the cell dose (22.4% vs 26.7%, p = 0.712). and there was no significant correlation between the number of cd3+ and cd34+ cells infused (spearman correlation coefficient: p = 0.307). in a univariate analysis, patients transplanted with the higher cd34+ cell doses and higher cd3+ cell doses had no increased grfs (p = 0.623 and p = 0.158). an independent factor associated with worse grfs was risk status at transplant (hr = 1.782, 95% ci:1.267-2.509, p = 0.001). these results suggest that careful assessing the cd3+ and cd34+ graft content and tailoring the cell dose infused may help in reducing cgvhd risk without negative impact on grfs. a large and prospective study in a homogenous population will be needed to confirm the effect of stem cell dose. disclosure of conflict of interest: none. imatinib associated with extracorporeal photopheresis can fully reverse severe sclerotic-type lesions in patients with chronic graft-versus-host disease: the lille university hospital experience l magro 1 , j gauthier, b catteau 1 , l mannone 2 , a lionet 1 , v coiteux and i yakoub-agha 1 1 lille university hospital and 2 nice university hospital severe sclerotic-type chronic graft-versus-host disease (cgvhd) is difficult to reverse and can dramatically alter the quality of life of patients after allogeneic hematopoietic cell transplantation (allo-hct). imatinib or extracorporeal photopheresis (ecp) used separately yield sustained responses in s218 only about 30% of patients with steroid-refractory cgvhd. given their respective modest efficacy we hypothesized that the combination of imatinib with ecp could lead to higher response rates. we are reporting here on seven patients with severe steroid-refractory sclerotic-type cgvhd treated at our institution using this combination. we retrospectively analysed all patients treated at our institution (n = 7) with the combination of imatinib with ecp for severe steroidrefractory scgvhd. imatinib was started at 200 mg/day and increased to 400 mg/day if well tolerated. the cellex closed system was used for ecp. ecp was initiated twice weekly during 4 weeks. after this « induction » period, ecp sessions were scheduled less frequently according to the response to treatment. additional immunosuppressants were tapered gradually in responding patients. initial grading and response evaluation was determined according to the nih 2014 criteria. steroid-refractoriness was defined as progression of gvhd on high-dose steroids (⩾1 mg/kg) or progression during corticosteroid tapering. patient characteristics are displayed in table 1 . patients received an allo-hct between may 2004 and april 2011. median age at allo-hct was 47 (range: 23-57). a variety of myeloablative (n = 2) and non-myeloablative conditioning regimens were used (n = 5). antithymocyte globulin was used before allo-hct in one patient. gvhd prophylaxis consisted of ciclosporine and methotrexate in six patients. one patient received tacrolimus and methotrexate. five patients had prior history of acute gvhd. nih global severity grade was severe in all patients (n = 7) due to severe sclerotic features. the median number of previous therapies was 3 (range: 2-5). all patients were steroid-refractory. after a median follow-up of 54 months (range: 13-76 months) the overall response rate was 100%. the complete response rate was 57%. median time on ecp associated with imatinib was 36 months (range: 4-60 months). median time to best response was 7 months (range: 3-22 months). corticosteroids could be discontinued in all patients after a median time of 8 months (range: 6-44 months). patients #1, #5 and #6 received maintenance therapy with ecp upon discontinuation of imatinib. in four patients, both ecp and imatinib led to complete response and could be discontinued after 38, 74, 4 and 53 months for patients #3, #4, #5 and #7, respectively. patient #4 and #6 passed away after due to a myocardial infarction and the development of a solid tumour, respectively. patient #4 was off therapy while patient #6 remained on maintenance with ecp. both remained in complete response. patient #2 remained in response during 25 months before progression of cgvhd while on imatinib and ecp. none of our patients experienced adverse events related to either imatinib or ecp. despite the limited number of patients in this report, we observed that the combination of imatinib and ecp can lead to complete and sustained reversal of severe steroid-refractory sclerotic-type cgvhd. these encouraging results should be confirmed in a larger cohort. disclosure of conflict of interest: lm: therakos (honorarium). allogeneic hematopoietic cell transplantation (allo-hsct) is an established treatment modality that is potentially curative for many patients (pts) with acute myeloid leukemia (aml). aml itself is the most common indication for pts undergoing hsct nowadays. for pts with high-risk disease, allo-hsct is, perhaps, the most effective curative treatment and is considered the standard post-remission therapy in first complete remission (first cr). this is a retrospective study to analyze those variables which were associated with patients' overall survival (os) after allo-hsct. the study population consisted of 31 pts who were diagnosed of aml from january 2010 to july 2016 at the hospital universitario central asturias, and submitted to allo-hsct in first cr. risk status based on validated cytogenetics and molecular abnormalities following recommendations of european leukemianet was performed. sixteen (51.6%) were male. median age was 42 years old (range: 1-64). clinical characteristics at transplantation are represented in table 1 . median follow-up was 27 months (5-75). considering the donor type, os at 1 year was higher in pts receiving sd (91.8%) compared to 66% in those who received urd (p = 0.012). regarding graft source, os at 1 year was 88.9% who received pbsc compared to 48% in pts receiving bmsc (p = 0.012). gender also showed significant association with os, which was higher among men, os at 1 year was 100%, compared to 47.4% for women) (p = 0.002). the presence of minimal residual disease (mrd) detected using multiparametric flow cytometry was performed prospectively after induction and consolidation, and before transplantation. thirteen pts had negative mrd before transplantation. median os was greater in pts with negative mrd before transplantation compared to the group with positive mrd (67 vs 27 months, respectively) (p = 0.24). this difference did not reach statistical significance probably because the low number of the sample. thirteen pts developed agvhd. only 4 (28.6%) pts receiving sd developed agvhd compared to 8 (50%) pts among those who had an urd; however this association was not statistically significant (p = 0.23). also, we observed higher incidence of agvhd in bmsc group (6 pts; 60%) whereas only 7 (36.8%) in pbsc group developed agvhd. this tender did not reach significant association (p = 0.2). one year os was 59.8% in pts who developed agvhd and 87.1% who did not (p = 0.05). all factors that had a significant influence on pts survival were included in a multivariate analysis (cox regression model): graft source, donor type, pts gender and agvhd development. developing agvhd kept an independent association with mortality (or 6.12, 95% ci 1.39-27.29, po0.001) and male gender also persisted as an independent protective factor (or 0.12, 95% ci 0.02-0.06, p = 0.003). in our series, agvhd has shown a significant and independent association with os over other parameters such as graft source, type of donor or mrd before transplantation. identifying reliable predictors for agvhd development, controlling well known risk factors for this disease, as well as improving management of immunosupressors should still be the key to potentiate longer os in our patients. larger studies are needed to confirm our results. acute and chronic graft-versus-host diseases (gvhd) are associated with increased morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-hsct). older patients undergoing allo-hsct may experience a high degree of transplant-related complications and this concern has historically limited the use of allo-hsct for some older patients. in many studies, age has been shown to be a negative prognostic factor for survival and associated with higher transplant-related mortality (trm). however, in others, age was not shown to be a significant factor if appropriate adjustments for other comorbidities are incorporated in the analyses. there are very few studies that evaluated the relationship between patient's age, the presence of gvhd and long-term transplantation outcome. the aim of this study is to evaluate the impact of age in patients who develop acute and/or chronic gvhd after allo-hsct for hematological malignancies on the trm incidence. we included in the study 595 patients with hematological malignancies who received allo-hsct and were followed in our center between january 2008 and january 2016. for the purpose of this study, only patients who developed grade ii-iv acute gvhd and/or limited or extensive chronic gvhd where considered for analysis (n = 306). patients were split into three homogeneous groups according to age at transplantation taking into consideration the underlying disease, type of conditioning and disease response at transplantation. group 1 (younger) included patients aged o40 years (n = 103), group 2 (intermediate) included patients aged between 40 and 55 years (n = 102) and group 3 (older) included patients older than 55 years (n = 101). gvhd evolution over time was followed as well as the cumulative incidence of trm was calculated in case of acute or chronic gvhd in each group. thirty seven percent of grade ii gvhd occurred in the younger group (n = 29), 32% (n = 25) in the intermediate group and 31% (n = 24) in the older group; majority (69%) resolved in the younger group as well as in 24 and 46% in the latter two groups, respectively, while trm rates at 1 year were 10%, 30% and 30%, respectively, sdhr = 4.8, p = 0.01. among patients who had acute gvhd grade ii, 51, 36 and 59% in the three respective groups developed chronic gvhd later. grade iii-iv gvhd occurred in 21% (n = 20) in the younger group, 38% (n = 38) in the intermediate group and 38% (n = 35) in the older group; with a respective resolution in 20%, 26% and 17% of patients and were associated with comparable trm rates at 1 year of 39%, 40% and 47%, respectively, p = 0.5. among patients who had acute gvhd grade iii-iv, 40, 37 and 38% in the three respective groups developed chronic gvhd later. de novo chronic gvhd was observed with a higher rate in the intermediate and in the older group (table) while patients with extensive chronic gvhd older than 55 years had significantly higher trm at 2 years (47%) compared to 32% in those younger than 55 years, sdhr = 1.9, p = 0.04. patients who develop acute gvhd grade iii-iv could incur over 40% of trm at 1 year independently of age. resolution of acute gvhd grade ii was significantly better in younger patients while older patients with grade ii acute gvhd or with extensive gvhd had higher mortality compared to younger ones. in addition to an adapted prophylaxis, a better preemptive gvhd strategy should be warranted in older patients. [p197] disclosure of conflict of interest: none. in vivo effects of nilotinib on lymphocyte subpopulation and function following allogeneic stem cell transplantation e marinelli busilacchi 1 , a costantini 2 , j olivieri 1 , n viola 2 , s coluzzi 3 , e pirro 4 , g mancini 5 chronic graft versus host disease (cgvhd) is a major complication of allogeneic stem cell transplantation and is characterized by frequent multiorgan involvement resembling autoimmune diseases; its pathogenesis is still incompletely defined and a standard treatment is lacking. donor-derived cd4+ and cd8+ t lymphocytes have been considered the main effector cells mediating cgvhd pathogenesis; however, recent studies suggest that b cells might also play an important role. in vitro data indicate that tyrosine kinase inhibitors (tkis) such as imatinib and nilotinib affects both innate and adaptive immune response by interacting with different cell populations (t cells, b cells, dendritic cells, mast cells and macrophages). we sought to evaluate the impact of different doses of nilotinib on the distribution and function of lymphocyte subpopulations. we analyzed 44 samples obtained from 15 patient with steroiddependent/refractory cgvhd enrolled in a phase 1-2 study with nilotinib in steroid-refractory cgvhd (nct01810718): triplets of patient were treated with escalating doses starting from 200 mg/die (5), 300 mg/die (5), up to 400 mg/die (5) . blood and plasma were collected at baseline and at day 90 and 180 of therapy. trough plasma nilotinib concentrations had been previously determined by hplc (abstract c039, haematologica: evaluation of nilotinib safety in patients with steroid-refractory chronic graft-versus-host disease: a phase i-ii gitmo study). peripheral blood mononuclear cells were isolated by density gradient centrifugation using ficoll biocoll. six color flow cytometry analysis (facs canto ii) was performed using conjugated antibodies (anti-cd3, cd4, cd25, cd16, cd56, cd19). inflammatory cytokine analysis was performed on plasma samples according to the instruction of bioplex pro human cytokine 17plex assay (bio-rad). statistical analysis was performed by 2-tailed student's t-test; differences were considered statistically significant for po0.05. flow cytometry analysis showed that nilotinib did not exert any significant impact neither on the proportion of t lymphocytes subpopulation (cd3+cd4+ t helper, cd3+cd4+cd25+ t regulatory, cd3 +cd4 − t cytotoxic), nor on b lymphocytes and nk cells. on the contrary, a statistically significant and dose-independent decrease of pro-inflammatory and th-17 cytokine production was observed ( figure 1 ): reduction of il2 (po0.02), il10 (po0.05) and ifnγ (po0.02) were already significant after 90 days; decreases of il17 (po0.05) and tnfα (po0.02) become significant after 180 days. interestingly, after 180 days of therapy, among the 21 patients enrolled (according to the itt criteria) ten patients showed cgvhd improvement and the other five remained stable. this study shows that therapeutic doses of nilotinib can reduce plasma levels of inflammatory cytokines without affecting the proportions of lymphocyte subpopulations. these findings correlate with clinical response and suggest that besides the previously demonstrated anti-fibrotic effects, nilotinib has also potent anti-inflammatory and immune regulatory properties, supporting its role in patients with cgvhd. disclosure of conflict of interest: none. [p198] previously published p200 infectious gastro-enteritis after allogeneic hematopoietic transplantation after reduced intensity conditioning (allo-ric): incidence and possible role in gastro-intestinal acute gvhd i garcía-cadenas 1 , r martino 1 , a esquirol 1 , a bosch 1 , n rabella 2 , s saavedra 1 , c muñoz 2 , j briones 1 , s brunet 1 and j sierra 1 1 hematology and microbiology departments and 2 hospital de la santa creu i sant pau, autonomous university of barcelona, spain enterotoxigenic c. difficile-associated associated disease or infection (cdi) is a common cause of diarrhea after hematopoietic stem cell transplantation (sct). recent studies have suggested the relationship of cdi with gastro-intestinal (gi) graft-versus-host disease (gvhd). the possible role of other types of infectious gastro-enterocolitis (g-ec) in gvhd development has not been studied. as a prior investigation to a national prospective observational study on this issue, we conducted a single-center retrospective analysis including all adult patients who received an allo-ric sct between january 2010 and march 2016. the aim was describing the cause(s) (if known), timing and outcomes of recipients with possible g-ec (defined as new onset acute diarrhea grade ⩾ 2) in the first year after sct. of the 123 patients studied (median age: 54 years, 62% male, 49% aml or mds as underlying disease), 97 (79%) had a total of 148 episodes of acute diarrhea, with 35 (28%) developing more than one event. these acute diarrheas occurred at a median of 39 days (range: 1-363) after sct. overall, a g-ec causing pathogen was identified in 33 of 148 stool specimens (22%) and included: cdi (7), c. jejuni (7), rotavirus (7), adenovirus (2), norovirus (2), b. hominis (3), s. stercoralis, g. lamblia, a. caviae, salmonella enterica and cryptococcus (one in each case). most postransplant diarrheas (68/148; 46%) occurred during the 4 weeks after infusion and were attributable to mucosal damage caused by the ric (negative microbial screening and no evidence of gvhd).the rate of infectious g-ec among the diarrheas occurring after day +30 was 41% (33/80). the overall incidences of enteric infection were 12.7% (95% ci: 6.5-18.9) and 17.6% (95% ci:10.4-24.8) at +6 and +12 months after sct, respectively. all the infected patients had mild to moderate disease, and no deaths were attributable to this complication. there were no differences in 2 year-os and nrm between the infected and uninfected patients (81% vs 73%, p = 0.6 and 16% vs 19%, p = 0.7, respectively). in univariate analysis age o50 years, prior sct, donor type, atg administration and prior grade 2-4 agvhd were associated with development of infectious gastro-enteritis. in multivariate analysis, unrelated donor and grade 2-4 agvhd were the only factors significantly associated with gastrointestinal infection (hr 2.7; 95% ci: 1.1-6.5, p = 0.02 and hr 3.6, 95% ci 1.5-8.5, p = 0.004; respectively). acute gvhd occurred in 46% of patients (n = 56), with a median onset of 54 days (range: 4-231). the cumulative incidences of 2-4 acute gvhd at 100 days and 6 months post-sct were 21% (95% ci: 15.3-32%) and 32.6% (95% ci: 23.4-42%), respectively, and there was a trend toward a higher risk of 2-4 gvhd in the group of patients with an enteric pathogen (48.2% vs 27% at 1 year, p = 0.06). more importantly, an enteric infection occurred just before the onset or aggravation of gvhd in 12/33 infected patients in our study (36%) at a median interval of 8 days after the infection (range: 0-24). in summary, our results confirm that enteric infections are a common complication after allo-ric, representing at least 20% of the episodes of acute diarrhea during the first year post-sct. a possible interplay between infectious g-ec and gvhd was observed in this study. disclosure of conflict of interest: none. long-term efficacy of extracorporeal photopheresis in chronic graft versus host disease m nygaard 1 , t karlsmark 1 , n smedegaard andersen 2 , i schjødt 2 , s lykke petersen 2 , l smidstrup friis 2 , b kornblit 2 and h sengeløv 2 1 department of dermatology, bispebjerg hospital, copenhagen, denmark and 2 department of hematology, rigshospitalet, copenhagen, denmark chronic graft versus host disease (cgvhd) activity is known to fluctuate over time, so we evaluated cgvhd continuously throughout the extracorporeal photopheresis (ecp) treatment course and after stopping ecp. patients with at least 1 year follow-up, who were treated with ecp at department of dermatology, bispebjerg hospital between 2009 and 2015 were evaluated. a single investigator retrospectively evaluated response to ecp monthly for 6 months, every 3 months until 2 years and every 6 months until 3 years. prednisolone doses were recorded every 3 months. responses were defined as complete remission (cr) if no symptoms of cgvhd were present, partial remission (pr) as improvement in cgvhd or stationary cgvhd with more than 50% reduction in prednisolone, no change (nc) as no difference in symptom burden and o50% reduction in prednisolone. progressive disease (pd) was defined as worsening of symptoms with unchanged or intensified immunosuppressive medication. ecp was performed with therakos uvar xts or cellex. there were 45 evaluable patients with moderate (n = 20) or severe (n = 25) steroid-refractory, dependent or -intolerant cgvhd. the median age was 58 years (range: 19-71) and there were 22 females and 23 males. conditioning regimen was myeloablative (n = 10) and non-myeloablative (n = 35). seventeen had related donors and 28 had unrelated. stem cell source was peripheral blood (n = 36), bone marrow (n = 7) or umbilical cord blood (n = 2). number of organs affected by cgvhd was one (n = 8), two (n = 16), three (n = 11), four (n = 9) or five (n = 1) and involved organs were skin (n = 36), eyes (n = 26), mouth (n = 24), lungs (n = 8), genitals (n = 6), liver (n = 6), musculoskeletal system (n = 5) or gastrointestinal tract (n = 2). time from diagnosis of cgvhd to first ecp was median 444 days (range: 11-2760) and time from referral to ecp and the first ecp procedure was median 52 days (range: 11-178). at the time of the first ecp procedure patients were also treated with prednisolone (n = 43), sirolimus (n = 21), calcineurininhibitor (n = 18), mycophenolate mofetil (n = 5), imatinib (n = 4), methotrexate (n = 1) or rituximab (n = 1). one patient received no immunosuppression. total number of ecp cycles was median 20 (range: 1-61). responses over time are shown in figure 1 . overall response to ecp was seen in 25 (56%) of the patients. most responses were seen after more than 3 months ecp treatment. in univariate analysis of possible baseline predictors of response, no significant associations were found. prednisolone dose was significantly reduced at every 3 months after start of ecp (p o0.01). additional cgvhd treatment was administered to 14 (31%) patients during ecp treatment (sirolimus n = 6, calcineurininhibitor n = 6, uva1 n = 4, methotrexate n = 3, rituximab n = 3, mycophenolate mofetil n = 2). about 6 (13%) patients had more than 1 additional treatment. prednisolone dose was increased at least once in 20 (44%) patients during ecp treatment. overall survival at 5 years was 80%. follow up was median 694 days (range: 62-2416). more than half the patients with cgvhd (56%) improve overall after treatment with ecp, but flares in cgvhd activity still occur. prednisolone dose is significantly reduced at all time points after starting ecp, but short term increased doses or additional immunosuppression was necessary in more than one-third of the patients. larger prospective studies with long-term end points are warranted. disclosure of conflict of interest: marietta nygaard has received a travel grant and speaker's fee from therakos/ malinckrodt. chronic graft versus host disease (cgvhd) remains a major cause of morbidity and mortality after hematopoietic stem cells transplantation despite the improvement of the immunosuppressive prophylaxis. skin, buccal, lacrymal and hepatic disorders are the most frequent. sclerotic gvhd remains a severe form and often refractory to standard treatment lines such as corticosteroids and calcineurin inhibitors. the antifibrotic activity of imatinib by the inhibition of pdgf-r and tgfb-β pathways has been used in the treatment of refractory gvhc with sclerotic features and systemic scleroderma. here, we report the results of imatinib treatment in 28 patients (pts) with refractory cgvhd. over a period of 13 years (january 2000-december 2012), 1308 pts received allogeneic stem cells transplantation from related donors, 28 of whom were treated with imatinib for refractory cgvhd: 24 pts for malignant diseases (14 cml, 9 aml, 1 nhl) and 4 pts for aplastic anemia. the median age is 31 years (6-55), the sex ratio m/f: 2.1. conditioning regimen used with chemotherapy alone: myeloablative (14 pts with gvhd prophylaxis combining ciclosporin and methotrexate), reduced intensity (14 pts with prophylaxis combining ciclosporin-mycophenolate mofetil). all pts received peripheral blood stem cell transplant with an average of cd34 cell count: 7.1 × 10 6 /kg (1.23-21.8). the median duration of the cgvhd is 8 months (3-27). the firstline treatment consisted of the combination of steroidsciclosporin with or without mycophelonate mofetil. imatinib was administered to these pts after median treatment duration of 48 months (9-108) for moderate (2 pts) and severe (26 pts) cgvhd according to the nih classification. treatment with imatinib, at doses ranging from 100 to 400 mg/d, was introduced in the second line for all pts. the evaluation is conducted in october 2016 after a median follow-up of 128 months (76-189). tolerance was good except in a one pt with severe thrombocytopenia that led to a transient cessation of treatment. after 6 months, analysis of pts who received imatinib according to couriel criteria and nih criteria: complete remission (cr): 1 pt (18%), partial remission (pr): 20 pts (71%), stable disease (sd): 5 pts, failure: 2 pts (7%). a long-term evaluation performed after a mean duration of treatment 62 months (6-91) finds similar results with a cr: 4 pts (14%), pr: 18 pts (64%), sd: 2 pts (7%) and failure: 4 pts (14%). corticosteroids were tapered or discontinued in 12 pts (cr or pr). at october 2016, 26 pts (93%) were alive and 2 pts (7%) died of severe infections. treatment with imatinib seems to be a good therapeutic option in the treatment of cgvhd in its moderate or severe form refractory to a minimum of two immunosuppressive agents according to the nih criteria as shown by our results in terms of response and survival with good tolerance. disclosure of conflict of interest: none. atg significantly reduces the risk of cgvhd both in unrelated and in hla identical sibling. the finke's study 1 randomised pts undergoing an allogeneic unrelated stem cell transplant (sct) after a myeloablative regimen to receive or not 60 mg/kg atg-grafalon reporting a significant reduction of cgvhd without increase of relapse and no os and dfs effect. however a successive study 2 didn't confirm those results (significant reduction of acute and chronic gvhd but poorer survival mainly due to higher relapse probability in the atg arm). the conflicting data reported on urd sct have several explanations, one is about the dose and the timing of atg. the timing of atg infusion has been demonstrated to be crucial for cb transplant 3 : an earlier administration is still active in preventing gvhd while ensuring engraftment and low hampering of immune reconstitution. here we report a large (193 sct) retrospective monocentric analysis on low atg doses (and 15-25 mg/kg for bm according to the degree of hla matching and 30 mg/kg for all pbsc sct) given early (from day − 6 to − 2). pts in the study were aml (n = 112, 58%), all (n = 57, 30%), hr mds (n = 21, 11%), cr1 (n = 111, 66%) cr2 or 4(n = 31, 18%), active disease (n = 27, 16%) for al; median age was 46 (range: 18-66). myeloablative conditioning were bu-cy120 (n = 72, 37%), bu-flu (n = 61, 32%), edx-tbi (n = 20, 10%), other (n = 40, 21%); pbsc was used in 41% (n = 80); sct were performed between 2005 and 2015 at the bologna transplant center. sct were performed from hla 10/10 identical urd (n = 63, 33%), or from 9/10 (n = 93, 48%), 8/10 (n = 30, 16%) and o8/10 (n = 7, 4%). median follow up was 55 months. overall, grade 2-4 agvhd was 26%, grade 3-4 agvhd 9%; cumulative incidence (ci) of cgvhd of any severity was 25%, for moderatesevere cgvhd 18%. ci of relapse and nrm was 28% and 21%, respectively. the 3-year overall and disease-free survival were 60% (95% ci: 52-67%) and 60% (95% ci: 51-68%). the gvhd (agvhd grade 3-4 and moderate-severe cgvhd) and relapse free survival (grfs) of the entire population ( figure 1 ) was 45% at 3 years (95% ci: 37-52%). restricting the analysis to patients in cr1-2, we found that cgvhd (any severity), gfrs and os at 3 years were 23%, 50% and 64%, respectively. comparing transplants with 10/10 urd to mismatched ones (9/10 or less) we found a trend for increased mod/sev cgvhd in pts undergoing transplant with mismatched urd (shr 2.32, 95% ci: 0.95-5.65, p = 0.06); agvhd grade 3-4 and cgvhd overall were not significantly increased; relapse incidence according to hla mismatches resulted 33% and 26% in 10/10 and ⩽ 9/10, respectively; grfs was 52% in 10/10 and 41% in ⩽ 9/10. the data reported show that low and early administration of atg is able to effectively prevent acute and chronic gvhd without increasing relapse thus ensuring really convincing grfs, even for o10/10 matched urd transplants. graft versus host disease (gvhd) is one of serious complications in patients after allogeneic hematopoietic stem cell transplantation. the application of mesenchymal stem cells (msc) represents a promising method for the treatment of severe steroid refractory gvhd. we present the data from an interim analysis of clinical trial, within which we applied msc in 28 patients with acute or chronic gvhd after allogeneic transplantation. the diagnoses included aml (12 pts), mds (5 pts), all (2 pts), cll/nhl (5 pts), mpn (4 pts). the patients underwent sibling hla-compatible (7), haploidentical (4), unrelated hla-compatible (13) or hla-mismatched (4) transplants. the median interval between the transplantation and msc was 6 months (1-95). the indications for msc infusion were steroid resistant acute gvhd (11 pts), steroid-dependent gvhd (agvhd 3 pts, cgvhd 11 pts) or chronic gvhd with the need for long-term immunosuppression and corticosteroid intolerance (3 pts). msc were applied as a single infusion at a median dose of 3.45 (0.9-5.0) × 10 6 /kg. response to treatment was assessed on day 14, 30, 60 and 100. the severity of gvhd prior to msc was graded as clinical stage 3 (2-3) in acute and stage 2 (1-3) in chronic gvhd, respectively. the median dose of corticosteroids was 0.92 (0.3-1.2) mg/kg/day in agvhd and s224 0.25 (0.1-0.5) mg/kg/day in cgvhd patients. on day +14 the partial response (pr) was achieved in 85% of patients with agvhd, the stabilisation of gvhd (sd) was found in 85% of patients with cgvhd. the dose of corticosteroids was reduced in most patients with agvhd (to 62% of the starting dose; 30-97%), while the early reduction was possible only in 36% of cgvhd patients. on day+100 only 19 patients were evaluable. the agvhd patients (7 pts) achieved a significant clinical response: 4 pr, cr 3 and dose of corticosteroids was reduced in all of them (to 17%; 10-60%). the minor responses were achieved in cgvhd patient (12 pts.) with 3 pr and 9 sd. however the dose of corticosteroid was reduced in 83% of these cases (to 56% of the initial dose; 21-71%). a total of 10 patients died because of infectious complications. most of them (8/10) were agvhd patients who expired early up to day +60. there were observed no side effects of msc application neither during the infusion nor later during the follow-up of 16 (2-45) months. the analysis of lymphocyte reconstitution revealed the changes of kinetics of some subsets as compared to the day +0 benchmark. the b-lymphocyte count tended to decrease in 82% of patients from chronic gvhd subgroup (vs 60% in agvhd). conversely nk cells declined in most agvhd patients (80% vs 36% in cgvhd). also the pro-inflammatory th17 cell was affected especially in agvhd (decrease in 63% pts vs 50% pts in cgvhd). the counts of myeloid/plasmocytoid dendritic cell increased in 80%/80% agvhd and 50%/91% ccvhd patients. the screening testing of cytokines (raybiotech, 42 cytokines, 6 pts, day +0 to +60) revealed changes of some analytes after msc infusion, including a decrease of proinflammatory cytokines such as ifn-γ, tnf-α, il-6. our experience with the treatment of gvhd using msc confirmed the safety of this immunotherapy. the favourable clinical effect with reduction of severity of gvhd and steroids dose was observed, especially in patients with acute form of gvhd. methotrexate day +1 omission is not associated with higher incidence of acute graft-versus-host-disease mm rivera franco, e leon rodriguez 1 and a campos castro 1 1 allogeneic hematopoietic stem cell transplantation (allo-hsct) remains a high-risk procedure due to its related morbimortality, limiting the broader application of this important treatment modality. despite extensive research over the years, acute graft-versus-host-disease (agvhd) affects the majority of patients undergoing allo-hsct, and up to 50% will develop clinically significant grades (ii). over the years, several methods for gvhd prophylaxis have been implemented, including immunosuppresive agents. methotrexate (mtx) is one of the earliest drugs used for gvhd prophylaxis. frequently, a short course of intravenous methotrexate (given on days +1, +3, +6 and +11 after hsct) is combined with a 6-month tapered course of cyclosporine. there is no consensus on which drugs and schedules for prevention of gvhd are best and clinical practice varies by institution. further, it is not clear whether omission of the day +1 dose of mtx has a negative effect on outcome in terms of morbidity. to describe the frequency of acute and chronic gvhd, mucositis and engraftment in patients receiving methotrexate (plus csa) as prophylaxis, omitting day +1. ninety-five consecutive patients who underwent allo-hsct from 1999 to september 2016, and received mtx as immunosuppressive prophylaxis were included. all patients received three doses of mtx, always excluding day +1. mtx was administered iv, either 10 mg/m 2 day +3, +6, +11 or 15 mg/m 2 day +3, and 10 mg/m 2 during days +6 and +11. we included 95 patients (55% male). the most frequent underlying diseases were aplastic anemia (21%) and acute lymphocytic leukemia (21%). ninety-nine percent of patients had a matched related donor. forty patients (42%) had gender disparity with their donor, and 13% presented abo incompatibility (major in 75%). most of the patients received myeloablative conditioning regimens (n = 73, 77%). the median of cd34+ infused cells were 2 × 10 6 (range: 0.8-6.8). the median neutrophil and platelet engraftment was 20 (11-43) and 15 (range: 5-46) days, respectively. from all the cohort, only 15 patients (16%) developed acute gvhd (53% grades i-ii) ( figure 1 ). thirty patients (32%) developed chronic gvhd, which was limited in 73%. most of the patients, 83% (n = 79), presented acute toxicity after the conditioning regimen, from which 76% (n = 60) corresponded to superior mucositis (50% grade i-ii and 50% grade iii-iv. the 10-year overall survival was 59% and the 10-year relapse free survival was 68%. our results showed a low incidence of acute gvhd, mostly grades i-ii, and similar survivals compared to previously reported studies, proposing that the administration of day +1 mtx as gvhd prophylaxis is not mandatory, however, prospective studies might be necessary to test our results. [p205] disclosure of conflict of interest: none. outcome of refractory graft versus host disease (gvhd) treated with extracorporeal photopheresis (ecp) as second line: a single-center experience j cornago navascues, b aguado bueno, av arriero garcía, edc jimenez barral, i vicuña andres and a alegre amor hematology department, university hospital la princesa, madrid, spain gvhd is a common and, sometimes, life-threatening entity related to hematopoietic stem cell transplantation (hsct). steroids remain the first-line therapy but they are not always enough to control it, or their side effects are simply unacceptable. both acute and chronic gvhd are responsible of impairment occurred in different organs that can lead to increase morbidity and mortality in our patients. different options are available as second line, but it is a well known fact that ecp, due to its inmunomodulatory mechanism, yields satisfactory response rates and presents excellent safety profile. from may 2012 to october 2016, 30 patients with steroid-dependent or refractory gvhd have been treated in our centre with ecp. we have performed 305 ecp procedures with the therakos cellex device, an integrated 'on line' system. the transplant was from a sibling donor in 13 cases and 17 from an unrelated donor. the median of cd3+ infused was 247.35 × 10 6 cd3/kg. eight patients (16.7%) presented agvhd, 17 (56.7%) cgvhd and finally, 5 (16.7%) had an overlap gvhd syndrome. most of patients (87.5%) with agvhd had a severe intestinal involvement as the main manifestation of the disease. however, all patients with cgvhd had a multiorgan involvement with a median of four organs affected, being skin, mouth, eyes and lungs the most common implicated. ten patients in our series have died, 7 for gvhd complications or infections and 3 due to relapse of aml. as firstline treatment they all received steroids and cyclosporine or mycophenolate mofetil. median ecp per patient has been 18 (2-31). ecp procedures were performed for 2 consecutive days, in initial phase weekly (in those with agvhd), or every 2 weeks (cgvhd) and then monthly according to clinical response, evaluated by clinical assessment and reduction in immunosuppression. about 75% of patients with agvhd had a significant clinical response to ecp so that steroid doses could be tapered and even in 37.5% of them withdrawal was possible. in the cgvhd group overall response rate (orr) to ecp was 94.1%. in 35% of these patients steroids could be suspended after a median of 8.5 ecp procedures. all patients who responded to ecp in cgvhd are still alive. independently of gvhd type, 81.4% of patients responded to ecp and 37% of them even could stop steroid therapy. those who had no response are dead. in cgvhd, 82.35% of patients remain alive, in contrast with agvhd or overlap syndrome patients whom survival is around 40%. about adverse events, 60% of patients did not present any complication associated with ecp. complications were mostly related to central venous catheter, with 12 cases of bacteremia and 2 thrombosis, easily recovered. in our experience, ecp is effective as second line treatment in gvhd, obtaining the best results in the chronic gvhd group. in fact, cgvhd patients with a good clinical response to ecp, specially when steroid doses can be tapered, have the better outcomes and longer survival. the tolerance to the procedure is excellent without severe adverse events. more experience is required to determine the best scheme of ecp and its role as prophylactic treatment. mesenchymal stromal cells (mscs) possess immunomodulatory properties and may play important roles in graft-versushost disease (gvhd) and engraftment. this study examined co-transplantation of mscs and hscs (hematopoietic stem cells). we investigated co-administration of ex vivo expanded mscs along with hla-identical sibling-matched hscs in β thalassemia major patients. we recruited 70 patients from january 2010 to january 2015 in our study. all participants received cyclophosphamide-based or fludarabine-based conditioning regimens and short-course methotrexate and cyclosporine as gvhd prophylaxis. mscs were administered intravenously (1.0-2.0 × 10 6 /kg) into patients (n = 41) 4 h before infusion of hscs. the outcomes were then compared to those of 29 patients transplanted with hscs alone. the median follow-up in the msc and non-msc group was 2.98 and 2.62 years, respectively. median time to wbc engraftment 40.5 × 10 9 /l was17.7 days (range: 15-20 days) in both groups (p-value = 0.83) and median time to platelet engraftment 420 × 10 9 /l was 27.2 days (range: 22-31 days) in the msc group, while it was 36.6 days (range: 22-50 days) in the non-msc group (p-value = 0.26). fifty-six percent of patients had acute gvhd in the msc group compared to the non-msc group where 65.5% developed acute gvhd (p-value = 0.42). meanwhile, chronic gvhd was 21% in the msc group and 37% in the non-msc group (p-value = 0.14). although the incidence of acute and chronic gvhd was lower in co-transplantation of hscs and mscs, no statistically significant difference was noted between the two groups. three-year overall survival rate was 70% and 61% in the msc and non-msc group, respectively (p-value = 0.78). three-year thalassemia-free survival rate was 54% in the msc group and 61% in the non-mscs group, showing no statistically significant difference (p-value = 0.35). the 3-year rejection incidence in the msc and non-msc group was 19% and 3 %, respectively (p-value = 0.07). there was no statistically significant difference between the two groups in terms of 3-year transplant-related mortality (pvalue = 0.79). this study indicates that co-transplantation of hla-identical sibling hscs with mscs does not inflict harm on bone marrow transplantation procedure and seems to be safe and secure. on the other hand, differences between the two groups in acute and chronic gvhd, engraftment, overall survival, thalassemia-free survival and rejection incidence did not reach statistical significance. therefore, despite the immunomodulatory activity of mscs and their role in gvhd amelioration and engraftment improvement resulted from in vitro studies, their efficacy in the clinical setting has not been conclusively proven which indicates further multicenter randomized clinical trials are required. keywords: β-thalassemia major, co-transplantation of mesenchymal and hematopoietic stem cells, engraftment, graft-versus-host disease. hematology-oncology and stem cell transplantation research center, tehran university of medical sciences, shariati hospital, tehran, iran. disclosure of conflict of interest: none. a number of studies were published with contradictory results comparing tacrolimus (tac) and cyclosporine a (csa) for graftversus-host disease (gvhd) prophylaxis, but there are only few that accounted for pharmacokinetic (pk) parameters. in this retrospective study we have identified pk parameters that affected gvhd incidence and incorporated them in the s226 multivariate comparison of tac-and csa-based prophylaxis. the retrospective study included 95 consecutive patients with csa and 239 consecutive patients with tac prophylaxis. 36% were grafted from matched related donor (mrd) and 64% from unrelated donor (ud). about 30% received busulfanbased myeloablative conditioning (mac) and 70% reducedintensity conditioning (ric). second agent for gvhd prophylaxis was short-course methotrexate (mtx) 10-15 mg/m 2 on days +1, 3, 6, 11 in 66% of patients and mycophenolate mofetil 30 mg/kg days − 1 to +30 in 34%. unrelated graft recipients also received antithymocyte globulin (atgam, pfizer, ny, usa) 60 mg/kg. the pk parameters analyzed were mean and median concentrations, pk variability parameters and number of concentrations below the targeted limit (nlow) within 21, 30 and 50 days after hsct. for tac the highest predictive value for acute gvhd was observed for median concentration during first 21 days (auc = 0.575), and for absolute skewness (auc = 0.567) of concentration data. for csa parameters with highest predictive value were median concentration (auc = 0.547) and variability coefficient (auc = 0.736) during first 30 days. nlow was also a significant parameter for tac current gvhd prevention regimens are partially effective, delay immune reconstitution, impair graft versus tumor effect and are cumbersome to use. therefore, there is a pressing need to develop innovative approaches for the prevention of gvhd. we completed a phase i-ii study employing a calcineurin and mtor inhibitor-free regimen based on a combination of post-transplant cyclophosphamide and bortezomib (cybor) in patients receiving fludarabine and busulfanbased reduced-intensity conditioning followed by peripheral blood, matched related or unrelated transplant. patients receiving grafts from unrelated donors also received ratg. we reported that the regimen was feasible and safe and yielded promising outcomes. (1,2) herein, we compare the results to those of a quasi-contemporaneous matched group of patients receiving a calcineurin-based gvhd prophylaxis. the experimental and control groups were well-matched in terms of age, sex, donor type, disease status, renal function and pam score. the cybor group (n = 28) was treated during a timeframe spanning from 2012 to 2016 and the control group (n = 15) from 2013 to 2016. gvhd prophylaxis for the control group was mmf and csa (n = 2) or tacrolimus (n = 13). both groups received supportive care according to standard institutional protocols. median follow-up for the cybor group was 28.7 months as opposed to 11.2 for the control group. median times to neutrophil engraftment for the cybor and control groups were 16 days (13-23) and 12 (10-21), respectively (p = 0.001). two patients from the cybor arm died before achieving platelet engraftment. for the remaining s227 patients, median time to platelet engraftment was 27 days (15-38). for the control group, five patients never dropped their platelet count below 20 × 109/l. for the remaining patients, median time to platelet engraftment was 17 days (10-29) (p = 0.002). there was no primary or secondary graft failure in either of the two groups. the incidences of acute grade ii-iv and grade iii-iv for the cybor group were 35.7 and 10.7%. for the control group, the incidences were 60 (p = 0.12) and 20% (p = 0.4). the incidence of chronic gvhd for the cybor and control groups were 28% and 14.3%, respectively (p = 0.62). treatment-related mortality was 14.3% and 20% for the cybor and control groups, respectively (p = 0.13). the incidences of cmv, ebv and bk reactivation for the cybor group were 57.1%, 32.1% and 17.9%, respectively. for the control group, the incidences were 46.7% (p = 0.49), 26.7% (p = 0.68), 0% (p = 0.09). the 2-year progression free survival and overall survival were 49.0% and 49.9% for cybor group and 18.8% and 31.3% for the control group ( figure 1 ). the 2year gvhd and disease-free survival (grfs) were 45.6% and 20%, respectively. despite the limitations of our study that include its size and its design and the delayed neutrophil and platelet engraftment associated with the cybor regimen in comparison to calcineurin-based prophylaxis, our data confirm the promising outcomes previously reported with the cybor combination and reaffirm the need for a large randomized study comparing cybor to a standard calcineurin-based regimen. clostridium difficile infection (cdi) causing enterocolitis may represent a serious clinical problem in patients undergoing allogeneic hematopoietic cell transplantation (allo hct). the reported prevalence varies substantially among heterogeneous patient cohorts. although cdi has been proposed as a risk factor for the development of gastrointestinal (gi) acute graft-versus-host-disease (agvhd), limited knowledge on the prevalence of cdi, occurrence of gi agvhd in cdi patients, relapse incidence and mortality of cdi patients in large patient cohorts is available. the aim of this analysis was to study the implications of cdi in a homogenous cohort of patients with either aml or mds undergoing allo hct. at our center all patients undergo stool test once a week for clostridium difficile antigen while in aplasia until discharge, irrespective of clinical symptoms for enterocolitis. patients with positive stool antigen tests (that is, toxin test) in the absence of clinical symptoms were referred to as cd+, in contrast to patients without a positive test and without clinical symptoms which were referred to as cd − . we retrospectively analyzed the data of a total of n = 727 patients with either aml or mds undergoing allo hct in our institution between 2004 and 2015. overall survival (os) was measured from allo hct to the date of death or last follow-up. after hsct, relapse and nonrelapse mortality were considered as competing events. eventprobabilities were calculated according to kaplan-meier for os and using competing event statistics for the cumulative incidence of relapse (cir), non-relapse mortality (nrm) and agvhd. 95% confidence intervals (ci) were provided for major endpoints. statistical analyses were performed using the r environment for statistical computing version 3.1.3 (r core team 2015, vienna, austria, www.r-project.org). from a total of n = 727 patients with either aml or mds who underwent allo hct, we identified n = 528 (73%) who were cd − , n = 103 (14%) who were cd+, and n = 96 (13%) who had cdi. interestingly, n = 33 (34%) of patients with cdi were diagnosed having gi agvhd as compared to n = 13 (13%) of patients who were cd+ and compared to n = 95 (18%) of patients who were cd − , p = 0.001. the three groups harbored no differences when comparing incidences of liver and skin agvhd or chronic gvhd, respectively. when dissecting gi agvhd according to ctcae criteria, only n = 8 (24 %) of cdi patients vs n = 7 (54 %) of cd+ patients, and n = 53 (56 %) of cd − patients had grade 3-4 gi agvhd, p = 0.007. with regard to os and trm, no statistical differences were observed between the three groups. the cir was 13% for patients with cdi, 15% for cd+ patients and 9% for cd − patients, p = 0.02, respectively. this analysis represents the largest published analysis of clostridium difficile in patients with aml or mds who underwent allo hct. the prevalence of cdi in this patient cohort was 13%. patients with cdi developed significantly more often gi agvhd as compared to patients who were either cd+ or cd − , respectively. however, this did not translate into differences in os or trm. disclosure of conflict of interest: friedrich stölzel has received research funding from astellas. the majority of studies on cytokines in allogeneic hsct were performed with classical gvhd prophylaxis, consisting of nonspecific immunosuppressive agents. with this type of prophylaxis almost in all studies published, higher levels of proinflammatory cytokines are associated with development of acute gvhd, while lower levels indicate the success of immunosuppressive agents in abrogation of alloreactive response. currently, there is no data, whether the dynamics of cytokines after ptcy is similar to the situation of classical gvhd prophylaxis. out of 192 adult patients transplanted at first state medical university with ptcy between 2014 and 2015 we have identified 20 cases with acute gvhd and plasma samples available. these patients were matched in the ratio 1:2 to patients who did not develop acute gvhd. the study group was comprised of 60 adult patients with hematological malignancies who underwent hsct. all patients received ptcy-based gvhd prophylaxis. five plasma biomarkers were studied by elisa: il-17a, il-6, il-8, tnf-α and ifn-γ. blood samples were obtained from patients on days − 7, 0, +7, +14. the fifth time point varied between day +21 and +28 to represent the sample after engraftment, but before onset of acute gvhd. about 10 (50%) out of 20 gvhd patients had a grade i, 7 (35%) grade ii, 5 (25%) grade iii agvhd, 6 (30%) patients developed multiorgan agvhd. about 13 patients (21.6%) had chronic gvhd. there was no difference between gvhd + and gvhd − groups in any of the clinical parameters. the median of engraftment for all patients was 21 (9-43: range). the median agvhd was 30 days (23-92: range). neither of the cytokine levels was significantly different in patients with agvhd grades i − iv and without gvhd. however, for patients with agvhd grade ii − iv we found that low levels of il-8 on day +7 (126.83 ± 43.794 vs 276.89 ± 310.51 pg/ml, p = 0.04) and ifn-γ on day +21-28 (34.70 ± 23.71 vs 60.96 ± 41.37 pg/ml, p = 0.03) were associated with increased risk of gvhd. the roc analysis was performed to determine the cut off values for il-8-133.56 pg/ml (auc = 0.714) and ifn-γ -35.94 pg/ml (auc = 0.720). the incidence of agvhd grade ii-iv was significantly higher in patients with levels of cytokines lower than cut off (40% vs 5.7%, p = 0.008 and 43.7%, p = 0.012 for il-8 and ifn-γ, respectively). the same pattern was observed for patients with agvhd grade iii-iv. low levels of il-8 (96.12 ± 39.79 vs 303.52 ± 346.19 pg/ml, p = 0.008) and ifn-γ (21.69 ± 14.78 vs 58.80 ± 39.92 pg/ml, p = 0.012) on day +28 were especially predictive. the cut off values for il-8 was 147.09 pg/ml (auc = 0.869) and for ifn-γ-25.71 pg/ml (auc = 0.858). the incidence of agvhd grade ii-iv was also significantly higher in patients with levels of cytokines lower than cut off (p = 0.004 and p = 0.0006 for il-8 and ifn-γ, respectively). for chronic gvhd only higher level of il-17 at day +28 (209.17 ± 329.59 vs 106.06 ± 210.65 pg/ml, p = 0.037 for patient with and without gvhd, respectively) was significantly predictive. in this pilot trial we have demonstrated that dynamics of cytokines after gvhd prophylaxis with ptcy may be different from conventional one, and well-known predictive biomarkers might not work after ptcy. further large prospective trials are warranted to elucidate reliable biomarkers for gvhd after this type of prophylaxis. disclosure of conflict of interest: none. graft versus host disease (gvhd) remains one of the main obstacles to broader application of allogeneic transplantation. gvhd prevention and treatment techniques are poorly standardized. the 1st-line treatment of newly diagnosed chronic (c) gvhd is corticosteroid. there is no standard 2ndline treatment for cgvhd. approximately 50-60% of patients (pts) with cgvhd require secondary treatment within 2 y after initial systemic treatment. recently the jak1/2 inhibitor ruxolitinib emerged as an efficacious treatment for corticosteroid-refractory (sr) acute and c-gvhd with a 24% of sr-cgvhd patients reporting a long lasting immunosuppression-free complete response. the current study seeks to analyse the efficacy and safety of ruxolitinib in highly pre-treated sr-cgvhd pts in our centre. ruxolitinib treatment was given off label after provision of an informed signed consent and in the absence of alternative therapeutic options including clinical trials. we analysed data prospectively collected at our long-term follow-up clinic between 2015 and 2016. a written consent was given by pts allowing the use of medical records for research in accordance with the declaration of helsinki. overall 5 pts (median age 57 y-range: 39-67 years; mean karnofsky score 70%) with sr-cgvhd were treated s229 with ruxolitinib. median time from transplant was 46 months (range: 9-68). ruxolitinib was initiated at a starting dose of 5 mg twice daily-median time on ruxolitinib 4 months (range: 2-15)-4/5 pts increased the dose up to 10 mg twice daily. four pts had a classic and 1 an overlap sr-cgvhd. all of them had skin sclerodermatous involvement and 4/5 joint and fascia involvement with significant decrease of range of motion and limitation of adl. all pts were previously treated with several lines of immunosuppression (3-11) including high-dose prednisone in 1st line (5/5), rapamycin (5/4), tk-inhibitor imatinib (4/5), extracorporeal photopheresis (4/5). all pts were pre-screened for risk of infection and regularly checked on a fortnightly basis. all pts were under active prophylaxis according to recommendation for gvhd pts and ruxolitinib therapy. after a cumulative follow-up of 867 days we reported only one serious adverse event represented by a cmv pneumonia requiring hospitalization with complete recovery. early time point evaluation (5/5 pts evaluable) at +1 month underlined how all pts were reporting subjective improvement at the patient global ratings according to nih 2014. data were confirmed at the health care provider global ratings. month 3 evaluation (3/5) confirmed meaningful responses (partial responses 3/3) according to nih 2014, with both patient and health care provider global ratings improvement and concomitant enhancement in lee skin symptoms score and sf-36 health-related qol. at last followup no evidence of myelosuppression, infections, pml, nonmelanoma skin cancer was registered. considering the concomitant treatment (with reference to azoles and rapamycin or cyclosporine) no cases of toxicity due to drug-druginteraction was reported. ruxolitinib is well tolerated in highly pre-treated sr-cgvhd. its safety profile seems to be reassuring. the efficacy data observed also at this early time point is preliminary but promising in this subset of pts with a long history (⩾3 lines) of treatment for cgvhd. confirmatory study in a larger number of patients is underway on a multicentre basis. disclosure of conflict of interest: none. severe acute enteral graft-versus-host-disease (gvhd) is a lifethreatening complication of allogeneic bone marrow transplantation. in case of resistance to corticosteroids as the firstline treatment severe enteral gvhd harbors a high morbidity and mortality. retrospective analyses indicate efficacy of the jak1/2-inhibitor ruxolitinib in the treatment of acute or chronic gvhd in adults, but experience in paediatric patients is limited. here, we report a small cohort of paediatric patients with stage 4 steroid-refractory gvhd of the gut who received ruxolitinib as salvage therapy within a multimodal immunosuppressive regimen. we retrospectively analysed four patients aged 8-16 years with severe, steroid-resistant acute gvhd of the gut who were treated with ruxolitinib in our institution. all patients were transplanted for non-malignant haematologic disorders, graft source was 2 × mmud, 1 × mud, 1 × msd. the conditioning regimen consisted of treosulfan, fludarabine and thiotepa. serotherapy with thymoglobuline was administered in all patients transplanted from unrelated donors. all patients received mtx and cyclosporine as gvhdprophylaxis. gvhd was staged according to the glucksberg-scale. ruxolitinib was added to the immunosuppressive regimen when acute stage 4 gvhd was reached and became resistant to treatment with methylprednisolone (2 mg/kg/day) as well as infliximab and mycophenolate (mmf) as second-line immunosuppressants. acute stage 4 enteral gvhd developed at a median of 38 days after transplant (30-58 days) and ruxolitinib was started at a median of 53 days post-transplant (48-85 days). the starting dose varied between 10 mg/day and 40 mg/day, that is, 0.25-0.5 mg/kg/day, taking into account the expectedly low bioavailability of the oral drug during severe diarrhea. upon improvement of gvhd symptoms and/ or increasing side effects the dose was gradually tapered and ruxolitinib was discontinued after a median of 39 treatmentdays (19-83 days) . after addition of ruxolitinib to the immunosuppressive regimen, the symptoms of acute gut gvhd gradually improved in all four patients with decreasing abdominal pain and stool volumes. immunosuppression with steroids and mmf could slowly be tapered. all patients are alive after a median follow-up of 392 days (95-571 days) from diagnosis of acute stage 4 gut gvhd. the most prominent side effect attributable to ruxolitinib was thrombocytopenia with a nadir in platelet counts after 30 days of ruxolitinib treatment in 3/4 patients. platelets recovered within 2 weeks after ruxolitinib was discontinued. neutropenia was observed in one patient with anc dropping o0.5/nl after 30 days of ruxolitinib treatment. mild to moderate elevation of liver transaminases was observed in all four patients during ruxolitinib treatment. one patient developed imminent acute renal failure, another patient showed symptoms of hemolytic uraemic syndrome. however, due to the multimodal treatment of these critically ill patients, these complications could not clearly be attributed to ruxolitinib. ruxolitinib is potentially beneficial in severe acute enteral gvhd in children refractory to corticosteroids as well as second-line immunosuppressants. however, randomized trials are warranted to verify safety and efficacy of ruxolitinib in this patient cohort. disclosure of conflict of interest: none. steroid refractory acute gvhd is a major cause of mortality after allogeneic stem cell transplantation. until date, no agent or treatment strategy has demonstrated superior efficacy in this patient group. the dose and duration of steroid treatment is associated with several short and long-term side effects, therefore concepts facilitating rapid steroid taper may be beneficial. both ruxolitinib and ecp have been reported to be effective in treatment of steroid refractory (sr) agvhd. we analyzed data from consecutive adult patients who received ruxolitinib for sr agvhd between march 2015 and august 2016 in our institution overall, 19 patients (male n = 12; female n = 7) with a median age of 58 years (range: 18-74) were included. donors for allogeneic sct were msd (n = 3), mud (n = 12) and mmud (n = 3). median time to gvhd onset after stem cell transplantation was 29 days (range: 7-154 days). about 14 patients had agvhd grade iii or iv (all with gi involvement), while 5 patients had skin grade 3 involvement. sr agvhd was diagnosed if agvhd manifestations were progressive after 3 days or persistent and without improvement after 7 days or no partial remission after 14 days of treatment with 2 mg/kg bw of systemic steroids. patients received additional ecp (n = 11), if response to ruxolitinib was lacking or slow (n = 9) or instead of ruxolitinib due to cytopenias (n = 2). ruxolitinib was first-line treatment for sragvhd in 11 patients (58%). median initial dose of ruxolitinib was 10 mg (range: 5-15 mg) twice daily. steroids were tapered and stopped, even if agvhd was still active. primary end point was non-relapse mortality at 6 months. secondary end point was response on day 28 after initiation of ruxolitinib. response occurred relatively slowly, resulting in a day 28 overall response rate of 58% (cr = 6, pr = 5). however, a total nine patients (47%) attained a complete response (cr), five with ruxolitinib alone and four others in combination with ecp. about 12 patients (63%) required dose reduction or interruption of ruxolitinib mainly due to cytopenias. after a median follow-up of 210 days, 8 patients are alive. causes of death were relapse of malignant disease (n = 1), gvhd (n = 2), infections (n = 7) and other (n = 1). median survival from diagnosis of sr agvhd was 61 days for non-responders and 252 days for responders ( figure 1 , p = 0.0096). in univariate analysis, non-response was associated with higher risk of nonrelapse mortality (rr; 5.6, 95% ci: 1.51-20.6, p = 0.01). ruxolitinib and ecp are two effective promising treatment options, which may be complementary in patients with sr agvhd. cytopenia is the most frequent side effect of ruxolitinib while infections remain the major cause of death. [p215] disclosure of conflict of interest: ayuk-therakos: honoraria; kröger: novartis: honoraria, research funding. steroid-refractory acute graft-versus-host disease (sr-agvhd) is associated with a dismal outcome. janus kinase (jak) 1/2 signaling has been shown to be instrumental in multiple steps leading to inflammation and tissue damage in gvhd. jak1/2 inhibitor ruxolitinib was studied in the treatment of sr-gvhd by zeiser et al. (leukemia 2015) , and the overall response rate was reported to be 81.5%. we have now studied ruxolitinib in the treatment of six adult patients with steroid-refractory, grade iii-iv, intestinal agvhd. all the patients were male. the median age of the patients was 50 (range: 19-59) years. three of the patients were transplanted for aml, one for all, mds and mm each. all the patients had been given a myeloablative conditioning treatment (cytbi 2, treosulfan+fludarabine 4). two patients had a sibling donor and four a matched unrelated donor. the graft was from peripheral blood in all the patients. gvhd prophylaxis consisted of cyclosporine and a short course of methotrexate, and in addition antithymocyte globulin in the unrelated donor setting and methylprednisolone in one sibling recipient. agvhd of the intestine manifested on days + 17, +25, +39, +60, +63 and +136 with diarrhea. in two patients it was preceded by agvhd of the skin by 7 and 49 days, respectively. gi-biopsy showed acute gvhd of grade iii and of grade iv in three patients each. treatment of intestinal gvhd was started with methylprednisolone 10 mg/ kg/day, tapering the dose to 5 and 2 mg/kg after 12 doses each. gastroduodenoscopy and colonoscopy were performed at the onset of symptoms indicating intestinal gvhd. biopsy confirmed the diagnosis in all cases. because the diarrhea continued in spite of methylprednisolone treatment, ruxolitinib was started 7, 9, 10, 10, 20 and 90 days from the first day of diarrhea. the dose of ruxolitinib was 10 mg × 2 per day orally. four patients showed a clear response to ruxolinitib, normalization of bowel function, after 3, 4, 16 and 27 days from the start of ruxolitinib treatment. the healing of the intestinal lesions was verified by biopsy. two of these patients had received extracorporeal photopheresis simultaneously. two patients did not benefit from ruxolinitib treatment. one of them had continuous infectious complications and therefore ruxolitinib was only started after 90 days from the start of diarrhea. the other patient died of fulminant diarrhea after 3 weeks of ruxolitinib treatment. cmv reactivation was detected in three of the responders, and two of them had also polyoma virus cystitis. one patient developed a pulmonary aspergilloma, which is under control with drugs. corticosteroid-resistant gastrointestinal acute gvhd was treated in six patients, out of whom four showed a good response. disclosure of conflict of interest: none. although methotrexate (mtx) is commonly used in the prophylaxis of graft-versus-host disease (gvhd) after allogeneic hematopoietic stem cell transplantation (allo-hsct), some small studies have also reported its use in the treatment of chronic gvhd. the aim of this study was to evaluate the efficacy and safety profile of low-dose mtx for treatment of sclerodermatous chronic gvhd (sgvhd) after the failure of first and second line treatments. we retrospectively evaluated 23 adult patients who received low-dose mtx as salvage treatment of sgvhd during the period elapsed between june 2006 and june 2016 in a tertiary referral university hospital in spain. there were 17 (73%) males and 6 (27%) females. the median age was 54 years (range: 28-69). all had received an allo-hsct for hematologic malignancies. the median time from allo-hsct to sgvhd was 666 days (range: 334-2679). thirteen patients (56%) had presented previous acute skin gvdh. superficial skin lesions mimicking lichen planus (lichenoid gvhd) were diagnosed in 19 (82%) patients, while lesions resembling lichen sclerosus, morphea or fasciitis (sgvhd) where seen in all 23 (100%) patients. the total body surface area was affected by more than 50% in 15 patients (65%). besides the skin, other organs/tissues involved were the eyes (65%), mouth (52%), nails (34%), lungs (17%), liver (8%) and gastrointestinal tract (4%). treatment lines prior to mtx administration were: prednisone (pdn) in 23 patients (100%), phototherapy (pht) in 4 (17 %), cyclosporine (cya) in 2 (8%), mycophenolate mofetil (mfm) in 2 (8%), pht + pdn + cya in 3 (13%), pdn + mfm + cya in 3 (13%), extracorporeal photopheresis + pdn in 1 (4%). the median time from sgvhd onset to mtx treatment was 308 days (range: 19-937 days). mtx was administered subcutaneously in 21 patients (91%) and orally in 2 patients (9%). median dose of mtx was 13.74 mg/week (range: 7.73-18.48 mg/week) and median length of treatment was 61 weeks (range: 2-148 weeks). in two patients (8%) early withdrawal of mtx occurred (one due to early death secondary to septic shock and other due to rapid disease progression). mtx-related toxicity occurred in three patients (13%): megaloblastic anemia, asymptomatic increase of liver enzymes and mucositis, respectively. response to mtx was evaluated in the 21 patients (91%) s231 who did not suffer early mtx discontinuation. seventeen patients (73%) presented a partial response; of them, two are still under mtx treatment for 26 and 59 weeks, respectively. fourteen patients (60%) received pdn concomitantly to mtx (median dose 20 mg/day, range: 5-60); 1 year after mtx treatment, only four patients were receiving pdn (median dose 5 mg/day, range 5-15). seven patients have finished mtx treatment without reappearance of symptoms, receiving only topical treatment with emollients, tacrolimus or corticoids for short periods. in four patients (26%) sgvhd progressed despite mtx administration. our data suggest that mtx is a safe, inexpensive and effective alternative for refractory sgvhd. its potential used in earlier phases of sgvhd deserves further investigation. disclosure of conflict of interest: none. severe chronic gvhd has a major influence on late morbidity and mortality after hematopoietic stem cells transplantation (hsct). ecp is a good approach to treat refractory-gvhd: leucocytes are obtained from peripheral blood by apheresis, incubated with 8-mop, irradiated and then infused to the patient where they undergo apoptosis and induce tolerance. it is a promising alternative that reduces doses of immunosuppressive therapy and their side effects in the treatment of gvhd. this study shows its efficacy in persistent refractory cgvhd. the procedure was applied to three patients (pts) aged 24, 28 and 32 years (two aml and one cml), sex ratio (1m/2f) who underwent allogeneic-hsct with myeloablative conditioning regimen based on chemotherapy alone from a peripheral blood stem cells with cd34 levels: 1.24, 7.6 and 8.23 × 10 6 /kg respectively. prophylaxis of gvhd combined ciclosporin and methotrexate in short cycle. severe extensive cgvhd (according to nih criteria) was observed in the three cases after an average delay of 5.3 months (3-7) with involvement of 1-6 organs (mouth, eyes, skin, liver, joints and lungs). all pts are refractory to three lines of immunosuppressive agents (ciclosporin-corticosteroids, mmf and imatinib), with an average of 3 thrusts/pt (2-4) over an average period of 65 months (09-103). ecp was performed under the open system or dissociated system (macopharma) for two sessions per week for 4 weeks, one session per week for 8 weeks, one session every 2 weeks for 12 weeks and one session per month for 3 months. after a median period of 6 months (3-10), an average of 22 sessions/pt (15-26) was performed. in terms of tolerance, a red blood cell transfusion was required in one pt, spontaneously resolved lymphopenia was observed in another pt, and a poor venous approach led to the pause of a central catheterization in one pt. the 3month and 6-month evaluation according to the couriel response criteria shows a partial response observed as of the first month with net improvement especially on skin sclerotic features and joints retractions initially refractory to all therapeutic lines. this allowed gradual reduction doses of corticosteroids. pce is recommended in the curative treatment for refractory chronic gvhd from the second line. this encouraging study on a small series shows its efficacy in persistent and late refractory forms. it is nevertheless necessary to evaluate it on a larger number of pts. disclosure of conflict of interest: none. successful treatment of steroid-refractory acute gastrointestinal graft-versus-host-disease by fecal microbiota transplantation p neumeister 3 steroid-refractory acute gastrointestinal (gi) graft-versus-host disease (agvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation (allo-hsct) associated with a high mortality rate. loss of intestinal bacterial diversity is thought to be associated with severity of gi-agvhd and an impaired intestinal microbiota with reduced diversity is an independent predictive factor for mortality. fecal microbiota transplantation (fmt) is the application of a fecal suspension derived from a healthy donor into a patient's gi tract. it has been successfully applied in recurrent clostridium difficile associated diarrhea including patients who underwent allo-hsct. we report the complete resolution of lower gi-agvhd following colonoscopic fmts in three patients that had been refractory to 4-6 lines of immunosuppressive therapies. microbiota analysis by 16s rdna before fmts revealed a severely depleted microbiota in all patients. donors (different persons for each patient) were healthy adult subjects. repetitive (1-6) colonoscopic fmts were necessary to permanently establish the donor's microbiome. all patients responded clinically by reduction/normalisation of stoll volumes, stopping total parenteral nutrition and tapering of steroids. a possible causative relationship of fmt in the reversal of severe intestinal dysbiosis and subsequent resolution of gi-agvhd can therefore be hypothesized. the establishment of donors' microbiota and increase in bacterial richness was associated with disease control. no immediate procedure-related infections or other side effects were observed. besides restoration of an initially severely reduced microbial richness by fmts, response of gi-agvhd was sustained despite reduction and discontinuation of concomitant immunosuppressive treatments. restoration of dysbiosis by fmt might represent a promising novel therapeutic approach for refractory lower gi-agvhd. disclosure of conflict of interest: none. tear film proteomics reveals important differences between patients with chronic ocular gvhd and healthy controls k plattner 1 , n gerber-hollbach 1 , s moes 2 , p jenoe 2 , d goldblum and j halter 1 ophthalmology, university hospital basel, ch-4031 basel and 2 proteomics core facility of the biozentrum, university of basel, ch-4056, basel chronic gvhd frequently involves the eyes, leads to important decrease of quality of life and may threaten visual capacity. sicca syndrome is one of the hallmark of ocular cgvhd. analysis of tear protein composition may help to identify biomarkers for early diagnosis and prognosis of ocular cgvhd. tear fluid of 42 patients with ocular cgvhd were compared with 10 healthy individuals in this single center study. results of the first 10 patients are reported here. tryptic digests from schirmer strips were analyzed on an orbitrap mass analyzer. clinical examinations included slit lamp examination, fluorescein staining, schirmer test, break-up-time (but) and a quality of life questionnaire (osdi). outcome measures were differences and consistency of proteins in human tear fluid s232 between patients with ocular gvhd and healthy controls. statistical analysis was performed by one sample wilcoxon-tests, p-values o 0.01 was considered significant. ten patients (eight males, two females) with a median age of 47 years (range: 24-69) were analyzed. all underwent pbsct, eight from an unrelated donor. cgvhd overall score was moderate in three and severe in seven. eye organ score was 2 in six and 3 in four patients. all patients had more than one organ manifestation of cgvhd. eight were under systemic immunosuppressive therapy at the time of analysis, two had topical treatments only. in total 306 different proteins were detected in tears analyzed. compared to controls, 172 were differentially expressed in ocular cgvhd. expression was highly significantly different in 75 proteins. compared to controls, expression of 41 proteins was at least 10-fold increased, representing 11 different categories. among them, more than 75% of all proteins belong to one of three categories: cytoskeletal proteins, nucleic acid binding or structural proteins. albumin, cluster or keratin (keratin type i-iii) and cluster of pyruvate kinase were most highly overexpressed. expression of 14 proteins was decreased to o1 to 50%, belonging to 12 different protein classes. half of them belong to defense/immunity proteins, enzyme modulators, hydrolases, nucleic acid binding and carrier proteins. expression of lactotransferrin, proline-rich protein and prolactin-inducible protein was most profoundly decreased. compared to healthy controls, a high number of protein is found to be differentially expressed in tears in ocular cgvhd. among them high expressions are observed for proteins that may indicate disturbed integrity of ocular surface and leakage of conjunctival capillaries. most profoundly decreased proteins include proteins with important functions in host defense and immunomodulation. more detailed pathway analysis is necessary to identify biomarkers for ocular cgvhd. disclosure of conflict of interest: none. steroid-dependent chronic gvhd after allogeneic peripheral blood stem cell transplantation is a great problem. nonresponders to corticosteroid therapy are at high risk of mortality. we hypothesized that such patients could benefit from treatment strategy using in patients with primary severe autoimmune diseases like multiple sclerosis and crohn's disease. patient z., 4 y.o. was diagnosed in july 2011 with myelomonocytic leukemia (jmml). initially he was treated with low-dose of cytarabine and epigenetic agents. in september 2013, jmml progression was observed with leukocytosis, thrombocytopenia, splenomegaly. bone marrow aspirate showed 19.2% monocytes and 19.8% blast cells. splenectomy was performed in november 2013 due to refractoriness to blood components transfusions. in december 2014 unmanipulated haploidentical peripheral blood stem cell transplantation from mother with 3.2 × 10 6 /kg cd34+ and 3.5 × 10 8 /kg cd3+ was performed. the conditioning regimen was myeloablative including melphalan 100 mg/m 2 day − 5 and treosulfan 14 000 mg/m 2 days − 4, − 3, − 2. no organ toxicity 4grade 2 was observed. gvhd prophylaxis consisted of hatg 10 mg/kg on days − 5, − 3, − 1, +1, i.v. tacrolimus from d − 1 and mmf 25 mg/kg from d 9. engrafted was fast and prompt (100% donor) with wbc41.0 × 10 9 /l on d +8, plts420 × 10 9 /l on d +10. acute gvhd of stage ii was observed in early posttransplant period and treated with steroids and tnf-α inhibitor (infliximab). patient also received five procedures of ecp. all attempts of immunosupression tapering failed and the patient was staying on high dose of tacrolimus, mmf and courses of steroids till october 2015. in october 2015, gvhd stage ii flare with blood eosinophilia occurred after another attempt of steroids withdrawal. clinical examination showed that the patient was in complete remission with full donor chimerism. mild response of gvhd to steroids was observed. in april and may 2016 patient received two doses of rituximab 375 mg/m 2 with no significant response. in order to restore naive immune system first course of chemotherapy with cyclophosphamide 2000 mg/m 2 was performed in the end of may 2016. no toxicity 4grade 2 was observed. the patient recovered wbc41.0 × 10 9 /l on d +12, plts420 × 10 9 /l on d +11. in the phase of hematological recovery he was mobilized with g-csf and two leukaphereses of pbscs were performed. in june 2016 our patient was transplanted with previously collected 0.5 × 10 6 cd 34+/kg following nonmyeloablative regimen including cyclophosphamide 1500 mg/m 2 on day − 3 and fludarabine 10 mg/kg, on days − 3, − 2, − 1. second dose of cyclophosphamide was not administered because of severe hyponatriemia with seizures due to the cpm administration. no other significant toxicity was observed. the patient did not require either blood product or i.v. antibiotics. doses of tacrolimus and mmf were picked on 2 months late and no more steroids were given. the patient is well in cr with no signs of gvhd for 7 months. we speculate that pbsc collection from patients under massive immunosupression underwent allogeneic transplant is difficult but feasible. the nonmyeloablative regimens in such group of patients could be well tolerated and ensure the restoration of naive recipient immune system. this option could be discussed as an attractive alternative for treating resistant gvhd in steroid resistant patients. disclosure of conflict of interest: none. severe acute gi-gvhd is a serious early complication of allotransplants, and still remains a clinical diagnosis.(1) endoscopic biopsies provide the best supportive evidence, but are invasive and morbid in patients who are already medically compromised. 18 f-fdg pet/ct may be able to stratify patients who require endoscopy and biopsy. to evaluate the performance of 18 fdg pet/ct in differentiating moderate to severe gi-gvhd from no or mild disease in pediatric patients with suspected gi-gvhd. retrospective chart review of all paediatric allo-transplant patients referred for 18 f-fdg pet/ct with suspected gi-gvhd from 2009 to 2015. clinical follow-up, endoscopy and biopsy findings were correlated with 18 f-fdg pet/ct. regional suv parameters were extracted by placing rois around stomach, duodenum, distal ilium, caecum, ascending, transverse, descending colon, recto-sigmoid colon and rectum. regional, and average large and small bowel suv data were statistically compared between patients with no or mild git-gvhd vs moderate to severe disease. the clinical and biopsy-supported diagnosis of acute gi-gvhd was taken as the true positive diagnosis for acute gi-gvhd. roc curves were generated for whole bowel suvmax values. about 50 scans in 34 patients, median age of 9 years (6 mths to 18 y), were performed at a median of 71 days post bmt. there were 15 stage 1, 13 stage 2-4 and 22 with no acute gi-gvhd. transverse colon suvmax was significantly higher in the stage 2-4 gi-gvhd compared to no or stage 1 disease (mann-whitney-u-test p o0.05). there was a non-significant trend for average large bowel suvmax to be higher in the stage 2-4 group than the no or stage 1 disease group (mean suvmax 4.16 compared to 2.94, p = 0.07). a cut off whole bowel suvmax 2.74 had a sensitivity of 79% and specificity of 61% for detecting moderate to severe gi-gvhd. 18 f-fdg pet/ct is a feasible and potentially useful non-invasive tool in the diagnosis and monitoring of therapeutic efficacy in acute gi-gvhd (2) . large bowel suvmax may be higher in patients with stage 2-4 gi-gvhd, and transverse colon suvmax could have the ability to differentiate children with no or stage 1 gi-gvhd from those with stage 2-4 disease. a negative 18 fdg-pet/ct could serve as a criteria to avoid invasive endoscopic procedures and observe for the persistence of gastrointestinal symptoms before subjecting these patients to an imageguided biopsy. in patients too unwell for endoscopy, suvmax 44 (roc curve specificity 75%) and a high suvmax in the transverse colon could serve as supportive evidence for moderate to severe acute gvhd, in the absence of biopsy findings. a major advantage of a pre-endoscopic 18f-fdg pet/ ct is to guide the procedurals to sample areas with the best diagnostic yield. prospective controlled studies are needed. oral mucosal progenitor cells (omlp-pcs) possess immunomodulatory and antibacterial properties, suggestive of their in vivo function in healthy tissue and their potential contribution to scarless wound healing in the buccal mucosa (2, 3). our aim was to establish whether the function of oral stromal progenitors is impaired in chronic graft versus host disease (cgvhd) and restored with response to treatment. a patient with grade 3 oral cgvhd was treated with systemic thalidomide for 9 weeks (200 mg/day). punch biopsies of buccal mucosa were taken before and after treatment. oral progenitor cells were isolated and expanded in vitro. numbers of progenitors was assessed using colony forming unitfibroblast (cfu-f) assays. stem cell markers (cd90, cd105, cd73, cd29, cd34, cd45, hla i and ii) were evaluated by flow cytometry. wound healing and antibacterial potential were assessed using a collagen gel lattice assay and bacterial cocultures as previously described (2, 4) . secreted levels of relevant cyto-and chemokines associated with wound healing were assessed by elisa. significant clinical improvement with reduced inflammation in the oral mucosa and healing of ulcers was seen after 3 weeks of thalidomide treatment, with continued improvement after 9 weeks. cell surface expression of cd90 and cd105 on omlp-pcs was elevated postthalidomide; markers correlated with stemness and angiogenesis in mesenchymal stromal cells. this correlated with a restoration of wound healing potential and antibacterial function after thalidomide treatment ( figure 1 ). figure 1 : antibacterial testing demonstrated a loss of antibacterial function against (a) gram positive and (b) gram negative micro-organisms in the cgvhd omlp-pcs that could be completely or partially restored to levels comparable with healthy controls after thalidomide treatment. *p ⩽ 0.05, **p ⩽ 0.01, ***p ⩽ 0.001. we demonstrate, for the first time a correlation between clinical improvement of oral cgvhd with thalidomide treatment and restoration of endogenous progenitor cell function. this study highlights the importance of a dysfunctional oral mucosal stroma in the pathogenesis of cgvhd. further studies should focus on the role of the stroma in promoting cgvhd and the precise mechanisms by which thalidomide is able to restore its functions. chronic graft-versus-host disease (cgvhd) is a major cause of late morbidity and treatment-related mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (hsct). cgvhd is driven by a th2 biased t-cell mediated alloreactive immune response that leads to chronic inflammation and fibrosis in various organs. thymic stromal lymphopoietin (tslp) is an epithelial cell-derived cytokine that mainly affects myeloid cells. upon stimulation with tslp, dendritic cells are polarized towards a dc2 phenotype driving th2 biased immune response. we hypothesized that tslp is involved in the pathogenesis of cgvhd and that elevated levels of tslp post-transplant may lead to an increased risk of cgvhd. in the present study, we measured plasma tslp levels during hsct to study associations with clinical outcomes including cgvhd. about 38 adult patients undergoing myeloablative hsct at rigshospitalet, denmark, from 2011 to 2013 were included. diagnoses included aml (n = 15), all (n = 11), myelodysplastic syndrome (n = 6), other malignancies (n = 4) and anemias (n = 2). donors were either hla matching siblings (n = 11) or mud (n = 27). grafts were either bmsc (n = 24) or pbsc (n = 14). conditioning included tbi (n = 31) or high-dose chemotherapy alone (n = 7). plasma tslp was measured by elisa (abcam) before transplantation, at the day of transplantation and at day +7, +14, +21 and +90 post-hsct. monocytes were counted daily, and t, b and nk cells were measured at day +30 and +90 using flow cytometry. about 35 (92%) patients engrafted; acute gvhd grade 2-4 was seen in 20 (53%) patients, and 16 (42%) patients developed severe cgvhd. oas was 57.9 %, trm 26.3% and relapse rate 15.8%. median plasma tslp levels increased from before conditioning (101 pg/ml) to reach a peak at day +21 (313 pg/ ml, p = 0.03), followed by a gradual decline. the plasma levels of tslp at day +21 were positively correlated with same-day monocyte counts (ρ = 0.58, p = 0.006). approximately half of the patients (n = 14) experienced an overall rise in tslp from baseline (100 pg/ml) to day +90 (328 pg/ml). this increase in tslp was not significantly associated with any transplantrelated baseline characteristics. however, patients, who had an increase in tslp levels from baseline to day +90, had a significantly higher risk of extensive cgvhd compared to those in whom tslp levels at day +90 were similar or below baseline levels (cumulative incidence of cgvhd: 77% (increased tslp at day +90) vs 38% (normal/low tslp at day +90), p = 0.01). development of cgvhd was also associated with the nucleated cell dose infused (p = 0.04) and transplant using pbsc (p = 0.08). tslp plasma levels were not associated with acute gvhd, oas, trm, relapse rate or numbers of t cell, b cell or nk cells posttransplant. we have found that increased levels of tslp from baseline to day +90 were associated with an increased risk of extensive cgvhd. this association may be due to the ability of tslp to polarize the immune system toward a th2 response. importantly, the increase in plasma tslp levels was not associated with any transplant-related characteristics suggesting that tslp may be an independent predictor of cgvhd. these findings indicate that anti-tslp treatment may be a new approach to fight severe cgvhd. disclosure of conflict of interest: none. thymopoiesis following hct: a retrospective review comparing interventions for agvhd in a paediatric cohort c roberts 1 , am flinn 1 , ma slatter 1,2 , r skinner 2 , h robson 2 , j lawrence 2 , j guest 2 and ar gennery 1,2 1 institute of cellular medicine, newcastle university and 2 great north children's hospital, newcastle-upon-tyne, uk acute graft-versus-host disease (agvhd) is a life threatening complication of allogeneic haematopoietic cell transplantation (hct), treated with topical and/or systemic corticosteroids. in steroid-refractory agvhd extracorporeal photopheresis (ecp) can be effective. ecp exposes apheresed mononuclear cells to 8-methoxypsoralen and ultra-violet radiation. systemic corticosteroids and agvhd are damaging to thymic tissue. delayed immune reconstitution, especially of the t lymphocyte compartment, is associated with increased morbidity and mortality. 1 therefore, management strategies must be effective in treating agvhd but endeavour to minimise resulting thymic damage. we compare the effect of topical steroid therapy, corticosteroids and ecp on thymic reconstitution following hct in paediatric patients. statistical analysis was performed using the kruskal-wallis test. about 155 paediatric allogeneic hcts were performed between june 2010 and april 2016, at the great north children's hospital, newcastle for malignant and non-malignant disease. we reviewed computerised records to categorise patients into four groups: no agvhd, mild agvhd treated with topical steroid, agvhd treated with systemic steroid, agvhd treated with ecp. laboratory data were reviewed to provide values of naive (cd4+ and cd4 − )cd45ra+cd27+ t-lymphocytes at 3, 6, 9 and 12 months post-hct. values for thymic output for the ecp group were additionally recorded at 3, 6, 9 and 12 months during ecp. excluded were patients with no available data, those with o12 months follow-up, those with chronic gvhd, recipient of 41 hct or received dli post-hct. about 104 patients were included, 42 (40.4%) had no agvhd, 49 (47.1%) had agvhd treated topically or systemically, 13 (12.5%) had agvhd and received ecp. for analysis, the group treated with steroids were divided into those treated with topical therapy and those given systemic steroids. the median values of all groups at each time point (3, 6, 9 and 12 months) are shown ( figure 1 ). there was a significant difference between the rate of thymopoiesis (measured by the addition of cd4+ and cd4 − cd25+cd27+ cells) between all groups (no agvhd, agvhd treated with topical or systemic steroids, and agvhd treated with ecp) at 3, 6, 9 and 12 months post-transplant (p = 0.002, p o0.001, p o0.001, p = 0.001 respectively). further analysis excluded those treated with ecp (so including the no gvhd (n = 42), topical treatment (n = 23) and systemic steroid treatment group (n = 26)). at each time point p = 0.001, p = 0.019, p = 0.021and p = 0.019, respectively, demonstrating a statistically significant difference in time to thymopoiesis between those that had developed agvhd and those that had not. acute graft-versus-host disease (agvhd) is a significant cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. which despite first line treatment is well-established (esteroids), second line is not well defined. evaluate the results with different second line treatment used and the risk factors associated with of sr-agvhd. we review the clinical records of 281 consecutive patients undergoing allogenic hsct from 2005 to 2015 in our hospital. about 53% presented agvhd. sr-agvhd was defined as progression after 3 days, no clinical change in 5 days or incomplete response after 14 days of treatment. about 34 patients (25%) met s235 criteria for sr-agvhd. there were no significant differences between both groups (sr-agvhd vs no sr-agvhd) respect to age (recipient/donor), unrelated donor, prophylaxis of gvhd, cd3 lymphocyte and cd34 cell. the median time between transplantation and agvhd diagnosis was 25 days (7-123). patients who did not respond on fifth day of steroid treatment have an 80% rate mortality vs 33% on no sr-agvhd group (p = 0.03). sr-agvhd: 34 patients presented sr-agvhd and this was related to: hla mismatch (35% sr-agvhd vs 15% no sr-agvhd, p = 0.008), male recipient/female donor (38% sr-agvhd vs 17% no sr-agvhd, p = 0.02) and advanced underlying disease (56% sr-agvhd vs 22% no sr-agvhd, p = 0.001). second line: basiliximab (82.4%); extracorporeal photopheresis (ep) (2.9%), timoglobulin (8.8%) and others therapies (5.9%). two patients (6%) obtained complete response (cr) and 10 patients (29%) partial response (pr). global response (cr, pr) after second line (mainly basiliximab) showed better overall survival (p = 0.009). third line: basiliximab (8.3%); ep (41.7%), mesenchymal cells (msc) (8.3%), ruxolitinib (16,7%) and others (24.9%). ruxolitinib improve gvhd cutaneous and hepatic but not intestinal. the best results were achieved with ep (2 cr, 1 pr) and basiliximab/msc (1 pr, respectively). only patients who achieved cr survived. fourth line: msc (50%)/ ruxolitinib (50%) does not improve the prognosis. no serious adverse effects were observed with msc therapy, basiliximab and ep. about 14% of patients showed cmv reactivation with basiliximab. about 27 patients died (80%), 21 patients with early mortality ( o6 months) due to refractory agvhd (40%) or secondary infections (60%). overall survival at 6 months and 2 year was 28 ± 8% and 0%, respectively. in multivariate analysis the main factor for trm was the steroid-refractory vs steroidsensitive (hr 2.00, 95% ci 0.91-4.39; p = 0.083) and was unfavorable the association of hepatic and intestinal agvhd (hr 2.24, 95% ci 0.90-5.57; p = 0.082) no sr-agvhd: 115 patients. trm-100 was 18% (n = 7), mainly due to infection (71%). trm-1 year was 37% (n = 15), mainly by gvhd (40%) and infections (40%). median follow-up of 26 months, os-6 months and 2 year were 84 ± 3%/75 ± 4%, respectively. trm was associated with not obtained cr/pr after second line (p = 0.001), no cr after third line (p = 0.018) and relapse of gvhd despite achieving cr initially (p = 0.004). in our series only the patients that obtained cr/pr after second-line or cr after third-line improved os. the best results in sr-agvhd were obtained with basiliximab and extracorporeal photopheresis. trm was associated with relapse of gvhd and advanced disease to the transplant. randomized clinical trials are needed to assess different treatment modalities for sr-agvhd. [p226] disclosure of conflict of interest: none. extracorporeal photopheresis (ecp) is a therapy for steroidrefractory chronic graft versus host disease (cgvhd). therapeutic response to ecp has been linked with a progressive increase in circulating granulocytic myeloid-derived suppressor cells (g-mdsc) in acute gvhd, but not in cgvhd 1 . low density neutrophils (ldn) phenotypically resembling g-mdsc (putative g-mdsc) show marked flux in cgvhd patients receiving ecp, and a reduction in their frequency is associated with a sustained therapeutic response to ecp 2 . recent data has identified lectin-type oxidized ldl receptor-1 (lox-1) as a specific marker of ldn with t-cell (tc) suppressive activity 3 . using this marker we have conducted a cross-sectional study to assess whether putative g-mdscs in this patient cohort have suppressive activity. about 15 patients with steroid refractory or steroid-dependent cgvhd (mean treatment duration of 9 months) receiving ecp and 8 healthy controls were recruited. patients had gvhd affecting skin (15/15), liver (3/15) and gut (2/15). pbmc were isolated and immunophenotyped by flow cytometry for markers of g-mdscs (cd14 − ve , cd16, cd66b, hla-dr − ve , cd33 int ) and lox-1 expression. suppressive function was determined by measuring the inhibition of proliferation of anti-cd3/cd28-activated purified cd3 tc from healthy donors by 4-day co-culture with g-mdscs from patients. statistical analysis was conducted using graphpad 6. ecp patients had substantially greater frequencies of circulating putative g-mdsc than healthy controls (p o0.0001; median: 13% and iqr 2%-32% vs 0.2% and iqr 0.1%-0.6%, respectively). while there were substantially greater frequencies of circulating lox-1 + cells in pbmc from ecp patients than healthy controls (p o0.0001; median: 1.5% and iqr 0.39%-35% vs 0.053% and iqr 0.029%-0.062%, respectively), these were mainly the minority population within the putative g-mdsc fraction with no significant difference between ecp patients and healthy controls in the proportion of lox-1 + cells (29% ± 16% vs 21% ± 9%, respectively). ecp had no significant effect on circulating putative g-mdsc frequency measured before and the day after treatment (median: 8.4% and iqr 4%-44% vs 16% and iqr 6%-25%; n = 11, respectively) nor on lox-1 frequency (median: 1% and iqr 0.29%-12% vs 2.8% and iqr 0.88%-7.3%; n = 9, respectively). at a tc:g-mdsc ratio of 1:1, isolated g-mdscs from ecp patients suppressed cd3 tc proliferation (mean ± sd: 52% ± 23 %; n = 14). however, the potency of suppression was highly variable (min-max: 18-82%). the pattern of lox-1 expression suggests that only a subset of putative g-mdscs in ecp patients are suppressive and may explain why suppressive function in this cell fraction is so highly variable. however, the relatively high frequency of lox-1 cells in this patient cohort might contribute to immunosuppression resulting in increased susceptibility to opportunistic infections. according to the revised eortc/msg criterion, 29 patients were diagnosed with cns-ifd. among those patients without cns-ifd (3826 patients), cns-ifd were matched in a 1:3 ratio for analyzing the risk factors of cns-ifd. and among 594 (15.4%) patients who occurred pulmonary ifd without cns involvement, 87 patients were selected as control group for analyzing the risk factors associated with involvement of cns in pulmonary ifd. we selected the control group using a 1:3 ratio matched-pair method with the variates of (1) age; (2) sex; (3) underlying disease. we retrospectively reviewed 29 patients complicated with cns-ifd after hsct in our single center during a 10 years period. most patients received haploidentical stem cell transplantation. the median onset time of cns-ifd was 173 (24-972) after hsct, and most (82.7%) of them have prior pulmonary ifd. the most frequent pathogen was aspergillus, while no crypoccosis and candidas were found. the most common clinical presentation was space-occupying symptoms and signs. brain abscess were the most common imaging finding. prior pulmonary ifd (po 0.001, hr 62.746(95% ci, 14.28-275.27)) was the only risk factors associated with occurrence of cns-ifd. while poor response at 6 weeks (p = 0.045, hr 2.574 (95% confidence interval: 1.021-6.487)) was the only risk factor predicting the involvement of cns in pulmonary ifd. the response (complete and partial response) at 12 weeks and last follow-up was 27.6% and 20.6%, respectively. the overall survival was 24.2% at the last follow-up with a median 289 (27-3341) days after transplantation. in conclusion, patients with pulmonary ifd had higher risk of cns-ifd, especially in those with poor response after 6 weeks of treatment. and the prognosis of cns-ifd was very poor after hsct. disclosure of conflict of interest: none. adv may cause severe infections in hsct recipients, especially from unrelated donors or cord blood particularly in pediatrics. disseminated infections usually occur after digestive reactivations. at 3 mo.post-hsct, the incidence of adv digestive infection and viremia in pediatric hsct is about 30% and 15%, respectively. therapeutic strategies to control adv infections are limited to the use of infusion of cidofovir (cdv) or ex vivo anti-adv selected cytotoxic lymphocytes (ctl). however cdv is not labeled for adv treatment, presents a renal toxicity and has shown limited efficacy. specific-ctl remain difficult to produce. brincidofovir (cmx001, bcv, chimerix, usa) is an orally-available lipid conjugate of the nucleotide analog cdv that has demonstrated broad clinical antiviral activity against double-stranded dna viruses (that is, herpes-, adeno-, orthopox-and polyomaviruses. the drug has an increased bioavailability compared to cdv and has shown encouraging results. we report here the results obtained with this compound in 20 patients treated in six centers from january 2015. there were 20 pts (8m/12f), median age at hsct: 65 mo. (15-202). hsct indication was all in nine, pid in six, aml in two, fa in two and ibmf in one. donor was 9/10 or 10/10 mud in four and six pts, respectively; haplo-identical familial donor in 4; 5/6 or 6/6 unrelated cb in two and three pts, respectively; msd in one. stem cell source was bm for 11 pts, cb in 5 and pbsc in 4. two pts underwent a second hsct. cond' regimen were mac in 16 pts. all pts received either ex vivo or in vivo t-cell depletion. three pts presented with adv-disseminated disease, seven pts with blood + other site (throat, urine or stools) adv infection, three with adv-related gut disease, three with blood infection and three with gut infection. the remaining patient received bcv for jc viremia with fever. median time for virus infection diagnosis was d20 post-hsct (range: d-126 to d+300). about 13 pts experienced 24 other viral infection episodes after hsct (cmv: 8; ebv: 5; bk: 5; hsv: 3; hhv6: 2; influenzae: (1). about 12 pts received 1-6 injections of cdv prior to bcv treatment. one pt received specific-adv ctl before bcv without efficacy. the reason to switch from cdv to bcv was uncontrolled adv infection (n = 11) or cdvinduced renal failure (n = 1). two additional pts experienced renal impairment after cdv. about 14 pts received 1-4 lines of immunosuppressive therapies (including ecp) in addition to calcineurin inhibitor at time of bcv therapy due to grade iii and iv acute gvhd in seven and seven pts, respectively. median adv load at time of bcv initiation was 4.5log copies/ ml (range: 3-9) in blood and 5log copies/ml (3.3-7.5) in stools. median duration for bcv therapy was 3 weeks (range: 1-18). about seven pts with blood adv infection or disseminated disease experienced adv disappearance as well as four pts with gut disease or infection. three of them experienced adv infection relapse and received thereafter cdv, bcv or advspecific ctl. five pts presented with grade 2-4 diarrhea during bcv treatment. about 13 were alive at end point where seven died from septis (n = 2), multi-organ failure (n = 2), gvhd (n = 2) and adv disseminated infection (n = 1). adenovirus infections occur often in immunocompromized pts receiving concomitant nephrotoxic drugs that may avoid cdv use.bcv appears as efficient therapy against adenovirus infection in such pediatric pts since here 13 out of 20 pts where alive after adv infection and bcv treatment. in this study we retrospectively analyzed cmv reactivation determined by pcr and response to pre-emptive therapy in patients receiving an haplo (n = 68, 24%) comparing them with a control group of non haplohsct (110 mrd and 106 mud) (n = 216, 76%). median age was 52 years (range: 16-71), 56 for haplohsct and 52 for control group. conditioning regimen was myeloabaltive (mac) in 33.5% and reduced intensity (ric) in 66.5%. haplohsct characteristics: haplo conditioning was fludarabine (30 mg/m 2 or 50 mg/m 2 × 4 days in ric or ma regimen) and busulfan (3.2 mg/kg × 2 in ric or 3 days in ma) (19.1% mac, and 80.9% ric). cyclophosphamide-post was used for gvhd prophylaxis in 94%. median of days to reach more than 500 × 10 9 granulocytes and more than 20 × 10 9 platelets were 17 (13-31) and 22 (0-103), respectively. incidence of acute gvhd was 70% (grade i-ii 64.2%, and iii-iv 5.7%), with two steroid-refractory cases. cmv reactivation: 66.2% of haplohsct patients presented cmv reactivation, vs 29.8% in control group (p = 0.000). median number of cmv reactivation episodes was 1 in both groups. median time to cmv pcr detection was 35 days (4-70) and 40 (0-186) in haplohsct and control group respectively (p = 0.042). average maximum cmv iu by pcr was 17.499 in haplo vs 8.206 in the control group (p = 0.035). first antiviral pre-emptive therapy (valganciclovir in 65.7%) was effective in 82% in haplohsct vs 65% in control group (p = 0.064). main reason for antiviral treatment switch was failure in cmv iu reduction, and foscarnet was the most used therapy in refractory cases. twenty patients developed cmv disease (5 in haplo and 15 in control group) (gi disease 90% and pulmonary disease 10% in both groups). in a multivariate cox-regression model, receiving an haplohsct, serological cmv status (positive patient/negative donor), mac regimen and development of acute gvhd grade i/ii or grade ii/iv were variables associated with a higher risk of cmv reactivation. based on these results, haplohsct is associated with a higher cmv reactivation compared to non-haplohsct, despite a lower incidence of all other risk factors as agvhd or mac in the haplo group. although it is not statistically significant, response to pre-emptive therapy is higher in haplohcst and no differences in cmv disease were observed. disclosure of conflict of interest: none. although a number of patients with hiv infection and hematological disease have successfully undergone allogeneic hsct together with combination anti-retroviral therapy (cart), short and long-term outcomes remain not well known. we report the spanish experience treating hiv-infected adult patients with high-risk hematological malignancies with allogeneic hsct. we retrospectively reviewed 17 hiv-positive patients who received allogeneic hsct in three institutions in spain within geth (grupo español de trasplante hematopoyético y terapia cellular). seventeen patients have been transplanted between 1999 and 2015. median age was 44 (30-57), 82% male. diagnosis and transplant characteristics are summarized in table 1 . cumulative incidence of neutrophil and platelet engraftment were 88% at 30 days (median 15 days), and 76% at 60 days (median 13 days), respectively. with a median follow-up of 42 months (22-87), os and efs were 35%. trm was 17% at 12 months and 32% at 36 months. grade ii-iv agvhd rate was 41%, and moderate/severe cgvhd rate was 41%. all patients received cart. two patients showed severe toxicity related to interaction of immunosuppressive s238 drugs and protease inhibitors. about 76% of patients showed infectious complications. viral infections were the most frequent cause: cmv (9), bk (2), adv (1), hhv-1 (2), hcv (1), hhv-8 (1), parainfluenza (1). two patients had invasive aspergillosis and one patient presented disseminated tuberculosis. causes of death were: relapse (4), infection (3), gvhd (2) and toxicity (1). all surviving patients showed undetectable hiv load after hsct. allogeneic hsct is an effective therapy for high-risk haematological malignancies in patients with hiv infection, and long-term hiv suppression with cart is feasible. however, interactions between immunosuppressive agents and anti-retroviral drugs, high rates of significant gvhd, and frequent infectious complications account for a complex procedure in this population. selected hiv-infected patients with hematologic malignancies should be considered for allo-hsct when indicated, in experienced centers. disclosure of conflict of interest: none. clostridium difficile infection (cdi) is one of the most common causes of nosocomial infectious diarrhea in europe and usa, which results in high morbidity and mortality among hospitalized patients. allogenic hematopoietic stem cell transplant (hsct) recipients remain at high risk for cdi. incidence rate ranges from 2 to 27%. numerous risk factors including acute graft-versus-host disease (agvhd), hla matching status, conditioning-intensity, use of total body irradiation (tbi) may play an important role in the course of cdi in these patients. the aim of this study was to evaluate the prevalence of cdi in children, and to assess the influence of such factors as gender, age, diagnosis, hla matching status, conditioning-intensity, use tbi-containing regimen, source of graft (bone marrow/bm/ vs peripheral blood/pb/)or agvhd on course, duration of treatment and outcome in children undergoing hsct. between 2014 and 2015 a total of 342 hscts were performed in five polish pediatric transplant centers, including 267 allogeneic and 75 autologous. all patients were followed up to 6 months post hsct. we analyzed retrospectively 29 episodes of cdi infection in the group of 342 children. twenty-one of 29 children were diagnosed with hematological malignancies: acute lymphoblastic leukemia (all), acute myeloblastic leukemia (aml), myelodysplastic syndrome (mds), two were diagnosed with severe aplastic anemia (saa), one with chronic granulomatous disease (cgd) and 5 of them-with solid tumors. the median age was 9.3 years (range: 2.5-19.0 years). majority of patients underwent myeloablative conditioning protocol (24/29). in allogeneic setting 21/24 patients underwent mud-hsct, 2/24 pts msd-hsct and one patient was given a haploidentical pbsct. in this series, in 7 out of 24 cases bm was a transplant source, and pb in 17 out of 24. cdi was defined as having diarrhea that tested positive for c. difficile via pcr, cytotoxin assay, or dual enzyme immunoassays. kruskal-wallis test, wilcoxon test and χ 2 -test were used to estimate the influence of risk factors on severity of disease, duration of treatment and outcome. we observed 29 episodes of cdi (8.5 %) in hsct recipients: in 24 allotransplant recipients (8.9% of all transplants) and in 5 autotransplant recipients (6.7% of all auto-hsct). nine patients responded to therapy with metronidazole, seven patients responded to vancomycin alone, and in two patients rifaximine was administered. six children required adding second drug: vancomycin or metronidazole, five patients were not given any medications. there was no significant correlation between such factors as diagnosis, gender, age, conditioning regimen, hla matching, agvhd and severity of disease, and duration of treatment. recurrence rate was difficult to assess due to lack of data. we observed three deaths. one of them was connected with cdi. there was one 17-year-old boy with saa (mud-pbsct, hla 10/10) with no agvhd. the other two deaths were due to progression of s239 disease. cdi occurred in nearly 9% of pediatric patients undergoing hsct, surprisingly often in autologous hsct too (6.7%). almost all patients experienced mild cdi with adequate response to antibiotic therapy. cdi is a rare cause of death among transplant recipients. disclosure of conflict of interest: none. antifungal prophylaxis in high-risk paediatric patients with haematological malignancies: a monocentric experience k perruccio, i capolsini 1 , a carotti 2 , n albi 3 , l pitzurra 4 , a velardi and m caniglia 1 pediatric haematology oncology section; 2 haematology and clinical immunology section; 3 transfusion medicine and 4 the choice of antifungal prophylaxis in high risk paediatric haematological patients (according to the latest ecil6/seifem guidelines) remains an open question. a recent retrospective survey from associazione italiana ematologia oncologia pediatrica (aieop) showed that, in these patient categories, the only variable which significantly impacted on invasive fungal infection (ifi) occurrence was the presence or not of antifungal prophylaxis at the ifi onset (unpublished data). from january 2012, in our pediatric hematology oncology unit, 31 allogeneic hematopoietic stem cell transplantations (hsct) were performed (median age: 10 years; range: 6 months-23 years), mainly for acute leukaemia (median follow-up: 24 months; range: 4-50). patients received liposomal amphotericine b (n = 20), micafungin (n = 10), or fluconazole (n = 1) as primary antifungal prophylaxis until neutrophil recovery (41 × 109/l). seven patients developed acute gvhd (22%) which evolved in gvhd in 5 (16%). as outpatients, they continued with posaconazole (n = 17), voriconazole (n = 4), or micafungin (n = 10) until cd4+t-cell recovery (4200/cmm) or gvhd immune suppressive prophylaxis/treatment withdrawn. during the last year, according to ecil6/seifem guidelines1, we administered primary antifungal prophylaxis also to 10/12 high risk (hr) acute leukaemia patients. two patients with aml were treated with posaconazole, four patients with hr-all received micafungin, four relapsed all patients received micafungin (n = 2), or liposomal amphotericine b (n = 1), or posaconazole (n = 1). one aml patient was then transplanted; all relapsed patients are waiting for transplant. no differences were observed in terms of breakthrough proven/probable (pp)-ifi incidence, according to antifungal prophylaxis in the various patient groups. in particular, in the early phase, we observed a pp-ifi incidence of 10% in both treatment arms (micafungin vs liposomal amphotericine b, p = ns). in the late phase, we observed 1 case of pp-ifi who were receiving posaconazole as prophylaxis. overall survival (os) was 96%, with 4% mortality rate. in hr leukaemia patient group, we observed pp-ifi in the only two patients who were not receiving any antifungal prophylaxis at the ifi onset. antifungal prophylaxis is strongly recommended in paediatric patients with haematological malignancies who are at high risk of ifi. the choice of antifungal drug depends on the treatment phase, drug interactions (particularly for azoles), patient compliance and clinical conditions which interfere with intestinal absorption. in our experience, as no differences were observed in term of efficacy, micafungin resulted the best choice in terms of tolerability, toxicity, compliance and cost saving. antifungal prophylaxis with micafungin and bridging to inhaled liposomal amphotericin b after engraftment in patients undergoing allogeneic hematopoietic stem cell transplantation d rivera* ,1 , c de ramon 1 , a avendaño 1 , j carrillo 1 , d caballero 1 , l lopez 1 , i ormazabal 1 , a navarro 1 , a martin 1 micafungin is an effective antifungal for prophylaxis, active against candida spp. including those resistant to other antifungals (c. glabrata and c. krusei) and also active against aspergillus spp. guidelines focused on antifungal prophylaxis, recommend its use during preengraftment and early postengraftment period in allogeneic hematopoietic stem cell transplant (allo-hsct) recipients. moreover, its profile of low drug interactions and side effects, makes it a suitable alternative for patients who need concomitant treatments, present hepatic insufficiency and in those who do not tolerate oral drug administration. the addition of inhaled liposomal amphotericin b (lamb) after engraftment, provides an alternative way to effectively prevent mold infections, that are acquired mainly by inhalation. inhaled lamb has good tolerance with absence of drug interactions and low toxicity. the aim of this study was to describe the experience in the hsct unit of the university hospital of salamanca with micafungin and lamb as primary prophylaxis in patients undergoing allo-hsct with reduced intensity conditioning (ric) and graft-versus-host disease (gvhd) prophylaxis with tacrolimus and sirolimus. thus evaluating efficacy and tolerability in our population. retrospective observational study from january 2013 to august 2016, including all adult patients undergoing allo-hsct with ric and gvhd prophylaxis with tacrolimus and sirolimus, in whom an azole derivative is not indicated, due to drug interactions. therefore received prophylaxis with micafungin during the preengraftment period and bridging to lamb after engraftment at discharge, and continuing it during the first 100 days post-transplant. data from 106 patients from our hsct unit. ten (9.4%) patients who had invasive fungal infection before undergoing allo-hsct, and 16 (14.8%) patients who received prophylaxis with drugs other than micafungin-lamb were excluded. underlying disease was grouped by leukemia in 44 (41.5%) patients, lymphoma in 23 (21.7%), myelodysplastic syndromes in 18 (17%), multiple myeloma in 11 (10.4%) and other diseases in 10 (9.3%) patients. eighty patients underwent peripheral blood allo-hsct, of whom were related donor in 40 (50%) patients and unrelated donor in 40 (50%). prophylaxis with micafungin in 80 (75.5%) patients, dose of 50 mg per day, with a mean of 19 days (±6 days) with postengraftment bridging with lamb 24 mg weekly, continuing it during the 100 days posttransplant. days of neutropenia during preengraftment, o14 days in 24 (31.2%) patients, 14-28 days in 49 (63.7%), more than 28 days in 4 (5.2%). during follow-up there were three cases (3.8%), two catheter related candida infection, and one esophageal candidiasis. there were no reported aspergillosis cases (possible, probable or proven), according to the european organization for research and treatment of cancer (eortc) criteria. finally, prophylaxis with micafungin and inhaled lamb, was considered an effective and safe strategy in 77 (96.3%) patients, with no side effects reported. according to our experience with micafungin and the addition of inhaled liposomal amphotericin b, the results indicate that, this is an appropriate alternative for antifungal prophylaxis, in patients undergoing allo-hsct, because of their efficacy, few side effects and drug interactions. disclosure of conflict of interest: none. recipients of allogeneic hematopoietic cell transplantation (allohct) are at high risk of developing invasive fungal infections (ifi). in the early phase (o100 days) after allohct, the use of antimold prophylaxis has been generalized, although there is no consensus on the best therapeutic strategy. the use of nebulized liposomal amphotericin b and fluconazole has been shown to be effective, safe and associated with low economic costs in lung transplantation 1 . however, the use of this prophylactic strategy in the early phase of allohct setting has not been evaluated. we included all consecutive patients who received their first allohct in our center from january 2013 to august 2016 and who underwent antifungal prophylaxis according to the prospective ambineb protocol (nebulized liposomal amphotericin b 24 mg administered three times per week as loading dose and once per week and fluconazole 200 mg per day until day +90). patients with a previous ifi were excluded. patients with graft-versus-host disease (gvhd) receiving high dose corticosteroids were allowed to be changed to voriconazole or posaconazole at physician's discretion. the primary objective of the study was the incidence of ifi at day +180. the secondary objectives were to assess adherence and toxicity of the ambineb protocol. only cases with proven or probable ifi according to eortc-msg criteria were considered. a multidisciplinary team of experts in hematology, infectious diseases, microbiology and radiology prospectively evaluated and categorized each case. we included 102 patients with a median age of 50 years (range: 18-70) and a median follow-up for survivor of 14 months (range: 2-47). patients received allohct mainly for acute leukemia (55%), non-hodgkin lymphoma (16%) and myelodysplastic syndrome (12%) . patients received allohct from hla unrelated (59%) or related donors (28%) mostly using a reduced intensity conditioning (57%). graft-versushost disease (gvhd) prophylaxis was performed with calcineurin inhibitors mainly in combination with sirolimus (57%) or methotrexate (17%). after the comprehensive review, only one case of proven or probable ifi at day +180 was diagnosed. prophylaxis with ambineb was completed in 75 patients (74%) while 27 (26%) stopped the treatment. the most frequent causes of discontinuation were possible ifi (6%), gvhd (5%), admission in intensive care unit (5%) and toxicity (6%) (figure) . ninety-four patients (92%) did not have adverse advents associated with ambineb. eight patients presented organ toxicity which was at least partially attributed to ambineb, including gastrointestinal symptoms (n = 4), liver function test abnormalities related to fluconazole (n = 3) and cough (n = 1). of the 27 patients who discontinued ambineb, 22 (81%) were switched to other antifungal drugs including (echinocandins (12, 54%) posaconazole (4, 18%), voriconazole (2, 9%) or others (4, 18%)). overall survival and non-relapse mortality of all patients at median follow-up were 64% (95% ci 58-69) and 25% (95% ci 20-30), respectively. the combination of nebulized ambisome and fluconazole is effective in preventing ifi in the early phase of allo-hct and is associated with high adherence and low toxicity. neutropenia-related infections is a common complication of apsct in patients (pts) with mm and dlbcl. our study aims are to: (1) assess antibacterial susceptibility patterns of isolated organisms, to guide antibiotic prophylaxis (2) identify the epidemiology of bacteremia with susceptibility patterns to direct empiric therapy of febrile neutropenia (3) assess the interval between the occurrence of neutropenia and the isolation of resistant bacteremia to identify the best timing to start prophylaxis (4) identify contributing factors for the development of bacteremia and mortality. our retrospective study included 191 adult pts who underwent apsct for mm and dlbcl between 2005 and 2015. we recorded the following: age, gender, basic illness, comorbidities, number of cd34 + cells infused, a central venous catheter, duration of neutropenia, diarrhea and mucositis, mechanical ventilation, positive bacterial cultures with susceptibility profiles and history of broad-spectrum antibiotic intake for more than 72 h for the past 3 months on hospital setting, and mortality. statistical analysis was carried out using spss (version 19). about 102 isolates were obtained: urine (46.5%), blood (28.5%), sputum (9%), wound (7%), venous catheter (7%) and stool (2%). gram-negative (gn) species were predominant (73.5%) with e. coli (32.5%), klebsiella (k) (11%) and pseudomonas (pseudo) (11%). isolates sensitive to third generation cephalosporins (3gc) represented 83% of the enterobacteriaceae (entero) including 78% in e. coli and 73% in k. all entero isolated were susceptible to carbapenems (carba), pipercillin/ tazobactam (pip/taz), amikacin and ciprofloxacin (cipro). all pseudo (n = 11) and acinetobacter (n = 4) isolates were susceptible to carba, pip/taz, amikacin, cipro, colistin and tigecycline. as for gram-positive (gp) bacteria (26.5%), coagulase negative staphylococci (cns) were predominant (14%) . oxacillin susceptibility reached 29% and two isolates methicillin resistant s. aureus were identified. all gp were susceptible to glycopeptides. a total of 29 bacterial isolates were identified (29 episodes of bacteremia) from 24 pts. gn were predominant (72.5%) with e. coli being most common (24%). all gn were susceptible to 3gc, carba, pip/taz, amikacin and cipro. as for gp (27.5%), cns predominated (24%) including 28% oxacillin-susceptible causing seven episodes of bacteremia with six central line-associated. no glycopeptide resistance was identified. none of the clinical features and pts' characteristics reached statistical significance as risk factor for bacteremia. however, the need for mechanical ventilation and mortality were higher in bacteremic vs non-bacteremic pts (16.7% vs 3%, p = 0.004, and 12.5% vs 3%, p = 0.036, respectively). all bacteremic episodes occurred after developing neutropenia (median = 2.5 days, range: 1-9) except for one case of clabsi caused by e. coli occurring 1 day before neutropenia. pip/taz was prescribed in 21% of bacteremia episodes followed by quinolones (11%) and carbapenems (4%). no previous use of third and fourth generation cephalosporins was observed. we recommend quinolone prophylaxis in apsct pts. for empiric therapy, antibiotics recommended by international guidelines including, cefepime and pip/taz still fit. thus, we could spare the use of carba and other last-resort antibiotics to other conditions. we also recommend continuous surveillance of resistance. disclosure of conflict of interest: none. fever is an almost universal complication in patients undergoing autologous stem cell transplantation (asct), however, microbiological documentation is only achieved in 20-30% of such febrile episodes (fe). 1 this low diagnostic efficiency makes epidemiological assessment in transplant units difficult, and may lead to a suboptimal empirical treatment. we have studied the utility of strict blood culture (bc) extraction as a mechanism to improve microbiological documentation of fe in these patients. we conducted a retrospective study over 66 consecutive asct performed in our centre between june 2015 and may 2016 (1 year). about 33 patients were male and 27 female, with age between 27 and 72 years (mean 57.8). diagnosis was 4 hodgkin lymphoma, 27 non hodgkin lymphoma and 35 multiple myeloma. ascts were performed in reverse isolation conditions, in rooms equipped with hepa and pall filters. prophylaxis against herpes virus and p. jirovecii with acyclovir and pentamidine was used. no prophylaxis against gram negative bacteria or filamentous fungi was performed. bc were extracted at the beginning of every fe and every 48-72 h if fever persisted (or more frequently, following clinical criteria). blood samples from intravascular devices and peripheral blood were collected in two bactec bottles each (for aerobic and anaerobic microorganisms). complementary diagnostic techniques and empiric antibiotic therapy were performed following our institution's guidelines. fe were classified in microbiologically documented infections (mdi), clinically documented infection (cdi) and fever of unknown origin (fuo) following his criteria. 2 85 fe were studied (average 1.28 fe per patient, 6.04 days of fever duration per fe). about 28% of fe were classified as mdi, 26% as cdi and 46% as fuo. mdis were cause by gram positive bacteria (56%), gram negative bacteria (26%) and polymicrobial infections (17%). no viral or fungal infections were observed. an average of 7.2 bc per fe and 7.6 per patient were extracted. the proportion of fe classified as mdi was related to the number of blood cultures extracted during the episode. only 8% of fe with three or less bc extracted were classified as mdi, 21% if 4-6 bc were extracted, and 55% if 7-9 bc were extracted (p o0.05). no significant difference in proportion of mdi classified fe between the extraction of 10 or more bc and 7-9. all patients were discarded in good clinical conditions. according to our experience, a strict 7-9 blood culture extraction is related to a high rate of microbiological documentation of febrile episodes. moreover, we have not observed the rise in gram negative bacteria reported by other studies and gram positive cocci persist as the main infection cause in our centre. candida which has been traditionally related to duration of neutropenia, emerges as a pathogen beyond the aplastic period in allogeneic haematopoietic cell transplantation (allohct). in the setting of alternative transplants and aggressive immunosuppressive therapy, these infections are a challenging problem. there is scarcity of data regarding the significance of breakthrough candidaemia in allohct. to that end, we aimed to determine the incidence, clinical and microbiological characteristics and outcome of candidaemia in allohct recipients. we studied consecutive allohct recipients from january 2014 to june 2016. blood cultures were obtained from peripheral vein or central venous catheters (cvcs) routinely and on febrile patients. well-known risk factors for candidaemia were studied: neutropenia, type of transplant, moderate to severe graft-versus-host disease (gvhd) and coexisting infections. among 108 allohct recipients, we identified seven patients with candidaemia: five post matched unrelated (four myeloablative and one reduced intensity conditioning) and two post haploidentical transplant. in median time of 3.5(1.3-8) months, 20 episodes of candidaemia were noted, despite antifungal prophylaxis with echinocandins or azoles. infections with non-albicans candida spp. occurred more frequently (19/20) and c. parapsilosis was the predominant microorganism (11/19). other species were isolated: c. famata (5), c. krusei (2) and c. haemulonii (1). all candida spp. isolates were phenotypically susceptible to antifungal agents already administered to patients. there was no resistance to echinocandins indicated by minimum inhibitory concentrations (mics). all patients had severe acute or late-onset gvhd with intestinal involvement and cvcs prior to candidaemia. although cvcs were removed in 7/7 and patients were treated with echinocandins, new cvcs were re-contaminated in 4/7 with the same or other species. all patients presented well known risk factors for candidaemia (use of broad spectrum antibiotics due to severe bacterial infections, total parenteral nutrition due malnutrition, long-term high-dose corticosteroids and other immunosuppression), but no neutropenia. one patient survived, whereas five patients succumbed to gvhd and multi-organ failure and one patient to sepsis due to bacteremia. candidaemia was observed in non-neutropenic patients with agvhd and cvcs on antifungal prophylaxis, despite difficulties in diagnosis due to poor sensitivity of blood cultures. the epidemiology of candidaemia has changed in the last decade and its risk is more diverse and complex. the irreversible intestinal gvhd lesions might be the main source of candida in patients receiving antifungal prophylaxis. our data show that candidemia remains an important issue in profoundly immunosuppressed patients contributing to excessive morbidity. our aim was to compare the rate of neutropenic sepsis, defined as fever of 438 o c and a neutrophil count of o0.5 × 10 9 /l, before and after the introduction of ciprofloxacin prophylaxis. one hundred and eight adult patients, of which 60 had acute myeloid leukaemia, 17 had acute lymphoblastic leukaemia, 12 had lymphoma and 19 had other haematological malignancies, were identified through our admission database. of these 108 patients, 48 received oral ciprofloxacin during their neutropenic phase. the median duration of neutropenia was 13 days in both the no-prophylaxis and ciprofloxacin groups. there was a significant reduction in the rate of neutropenic sepsis from 88.3% (53/60) in the no-prophylaxis group to 68.8% (33/48) in the ciprofloxacin group (p = 0.012). prolonged infection, suggested by the use of broad-spectrum antibiotic treatment for more than 10 days, was more common in the group which did not receive prior ciprofloxacin prophylaxis (45.0% vs 20.8%, p = 0.009). rate of intensive care admission (15.0% vs 0.0%, p = 0.005) was also reduced by the use of ciprofloxacin. however, there was no significant difference in the length of stay (mean of 28 vs 25 days, p = 0.187), or in the 30-day infection-related readmission rate (17.9% vs 13.3%, p = 0.536) between the two groups. in terms of the cause of neutropenic sepsis, escherichia coli, klebsiella pneumoniae and pseudomonas aeruginosa were the most common bacteria isolated from cultures in the no-prophylaxis group. eighty percent of these organisms showed sensitivity to ciprofloxacin. in the ciprofloxacin group, staphylococcus epidermidis was the most frequently found bacteria. with regards to treatment related adverse effects, none of the patients who received ciprofloxacin prophylaxis developed clostridium difficile diarrhoea. in conclusion, ciprofloxacin is still an effective antibacterial prophylaxis during neutropenia following allogeneic stem cell transplantation. clinicians should have a high suspicion of a gram-positive infection in patients who develop neutropenic sepsis on ciprofloxacin prophylaxis. disclosure of conflict of interest: none. s243 hematopoietic stem cell transplantation (haplohsct) to cure leukemia, malignancy and some inherited diseases, different additional reasons interfere microbiota metabolism and integrity. among them are radiation and chemotherapy, mucositis, infection and graft versus host disease (gvhd). the curative mechanism of fmt is based on the ability of donor intestinal microbiota to substitute and to provide all necessary functions of altered patient's microbiota. three patients (3, 10 and 28 years old) after haplohsct, who observed pseudomembranous colitis (toxin b-positive) as gvhd of intestine outcome, were enrolled to the study and performed fmt. relatives (mother, father and brother) were used as microbiota donors. donor and patient examination have included routine clinical and biochemistry laboratory data, microbiota cultural methods, pcr of most common intestinal microorganisms. additional for patient-level of fecal calprotectin by elisa was tested, identification of drug resistant bacteria and histology of intestine were made. patient's preparation for fmt included-probiotic (inulin) administration 72 h prior procedure, discontinuation of all antibiotics 24 h prior procedure and antiemetics (5ht3agonist), prokinetics and proton pump inhibitor. delivery of donor's microbiota was performed in two consecutive steps under total intravenous anesthesia: with esophagogastroduodenoscopy-to the duodenum; with colonoscopy-to the caecum. all patients observed complete clinical response in 14-28 days after fmt (table 1 ). in 10 days we have revealed significant quantitative and qualitative changes in microbiota composition, which was matched to donor's microbiota. in 35 days after fmt we identify microbiota changes in oropharynx and urogenital tract similar to donor microbiota. this leads to substitution of multidrug resistant klebsiella pneumoniae strains by drug sensitive microorganisms and helps to treat severe infection complications after haplohsct. platelet aphereses were carried out in 83 donors (50 males and 33 females with median age 38.5 years) using haemonetics instrument with simultaneous leucoreduction. quantitative detection of cmv, ebv and hhv-6 dna was performed by multiplex real-time quantitative pcr kit (interlabservice, russia) in donors' whole pb, plasma and platelet aphereses at the time of platelet collections. viral load in hsct recipients was monitored weekly after hsct and 7 days before hsct by the same pcr kit. lower limit of detection (llod) of the applied kit for all viruses was 500 copies/ml. in specimens of platelet donors we additionally performed ultra-sensitive pcr with llod 100 copies/ml. only one patient (2.4%) was cmv-positive by pcr prior to hsct. cmv reactivation after hsct ⩾ 500 copies/ml was noted in whole pb of 9 patients (22.0%) with median time of 41 days (range: 0-71). donor source in cmv-reactivated patients was as follows (3 mud, 1 msd, 3 haplo). cmv viremia ⩾ 1000 copies per ml was detected in seven patients (17.1%). cmv disease was found in five cases (12.2%). none of patients were positive by pcr for ebv or hhv-6 prior to hsct. ebv reactivation ⩾ 500 copies/ml was found in six cases (14.6%), ⩾ 1000 copies/ml in 5 (12.2%) with median time of 27 days (range: 20-56). no signs of ptld or other ebv-dependent clinical symptoms were observed. hhv-6 level after hsct ⩾ 500 copies/ml was detected in 17 patients (41.5%), ⩾ 1000 copies/ml in 11 ones (26.8%) with median time of reactivation 19 days (range: 13-36). hhv-6 disease was observed in one patient. none of platelet donors were cmv-positive in plasma, whole blood or platelet aphereses products. ebv ⩾ 500 copies/ml was detected in whole pb specimens of five platelet donors (6.0%). application of ultra-sensitive pcr revealed low level of ebv-viremia in additional 11 pb cases with median ebv level 260 copies/ml (range: 115-410). none of platelet donors have any clinical signs of ebv disease. there is no any ebv-positive case among platelet concentrate specimens. in two cases low levels of hhv-6 was found in a whole pb (110 and 180 copies/ ml). none of hhv-6-positive case was observed among plasma and platelet concentrate specimens. despite high incidence of cmv, ebv and hhv-6 reactivation after hsct in pediatric patients we could not show that source of viral reactivation was contamination of platelet apheresis products by donorderived herpes viruses. disclosure of conflict of interest: none. conventional respiratory virus (crv) infections are known to be major causes of morbi-mortality after stem cell transplantation (sct) due to the increased risk of progression to lower respiratory tract infection (lrti) in this setting. risk of developing severe lrti is mostly related to factors specific to the patient and the underlying disease, although the intrinsic virulence of crvs may also determine their outcomes. we conducted a single-center retrospective study including all adult sct recipients who had crv disease (defined as patients with symptomatic respiratory disease and crv identification) during a 7-year period (2009-2016) with the main objective of evaluating epidemiological changes over time and their association with infection outcomes. during the study period 137 episodes of crv disease were diagnosed in 104 patients (median age: 48 years, 58% male, 49% aml or mds as baseline disease). 83 patients (80%) received an allogeneic-sct (allo-sct) (30% had a prior sct) and 21 (20%) an autologous sct. crv disease was diagnosed at a median of 343 days after sct (range: 1-4361), with 33 cases (24%) occurring before day +100. during the infectious episode 31 of 83 allo-sct recipients (37%) had active gvhd and 39 (47%) were under s244 prednisone (pdn)415 mg/kg. most of the patients (84%) had symptoms compatible with an upper respiratory tract infection (urti), with 15 of them (11%) progressing to a lrti, while 7 (5%) had a lrti only. hospital admission was required in 46 episodes (33%) with a median duration of hospitalization of 11 days (range: 1-30), 17% required supplemental oxygen, 6% were transferred to the intensive care unit and 4% required mechanical ventilation. the most commonly identified pathogens over time are shown in figure 1 . twenty-four cases (17%) had concomitant bacterial or fungal infections. influenza a virus was the most frequent crv detected (49 episodes, 36%) followed by human respiratory syncytial virus (37 episodes (27%) and human parainfluenza virus type 3 (18 episodes, 13%). during the 2009 flu pandemic, only 2 of the 13 crv infections diagnosed in sct recipients (15%) were associated to influenza a virus h1n1. antiviral treatment was started in 62 episodes (45%), antibiotics in 56% and combined therapy in 25% during a median of 7 days (range: 4-21). the rv resolved in 129 cases (94%) at a median of 12 days (2-72) from onset, with crv being considered the leading cause of death in only 3 patients (3% of all cases and 3/22 (14%) in those with a lrti). predictors of severe crv infection (including icu admission, need for supplemental oxygen, need for mechanical ventilation requirement or death) in multivariate analysis were lymphocyte count o 200 cells/μl (hr: 4.2, 95% ci: 4-26, p = 0.01) and co-infection with other pathogens (hr: 4.8, ci95%: 1.5-16, p = 0.00). no specific crv nor period post-sct of the infection influenced the risk of severe infection. our results confirm that crv infections are a frequent cause of morbidity after sct with a high need for hospital-based care. temporal changes in the principal circulating crvs has been identified during the 7-year study period, with influenza a virus being the most common. profound lymphocytopenia and presence of co-pathogens are associated with infection severity. [p246] disclosure of conflict of interest: none. the 127 consecutive hsct performed from 2012 to 2016 are being analysed retrospectively. out of them 60 (47%) performed in hepa filter room and 67 (53%) in non-hepa filter room, criterion was purely financial to make this decision. 96/127 (76%) were allogeneic and 31/127 (24%) were autologous hscts. blood cultures both bacterial and fungal were taken at onset of fever and with every change of antibiotics till patient became afebrile. chest x-ray and if required hrct chest was done for all patients who had respiratory complaints. we did not use antibacterial prophylaxis; however, antifungal prophylaxis was administered along with conditioning; and at the onset of fever systemic antibiotics were started. antifungal agents were added if fever persisted for 3 days pre empatively. extremely well trained nurses were looking after both the groups. all treatment protocols, antibiotic/antifungal policies were same in both the groups. median time for neutrophil engraftment was 12 days in hepa filter room and 13 days in non-hepa filter room. total 4/127 (3 %) patients did not engraft till 30 days. out of them 02/60 (3 %) were in hepa filter room and 2 /67 (2.9 %) in non hepa filter room. blood cultures were positive in total 20/127 (16%) patients, 17 were positive for bacterial and 3 for fungal organisms. in hepa filter hsct 11/60 (18%) were positive and in non-hepa filter hsct were 09/76 (13%) positive. total 34/127 (27%) patients developed pneumonia, out of them 14/60 (23%) were in hepa filter and 20/67 (30%) hsct were in non-hepa filter room. statistically not significant. no central venous access cather issues or infections were documented in any groups gr 2-4 agvhd : hepa rooms 11/60 (18.3%),non hepa rooms 14/67 (20.8%) :was not statistically significant the 30-day mortality was 25/127 (20%), 07/60 (12%) patients were from hepa filter rooms and 18/67 (27%) were from non-hepa filter rooms. cost : average cost of allogeneic hsct in hepa room : usd 22 000. average cost of allogeneic hsct in non hepa room: usd 13 700. average cost of auto hsct in hepa room: usd 12 000. average cost of auto hsct in non hepa room : usd 8500. incidence of blood culture positivity & incidence of pneumonia was not different. these are two very important issues in outcome of hsct. agvhd incidence did not depend on the room type. these are significant findings from this study. results were slightly better in hepa filter rooms compare to non-hepa filter rooms, which was statistically insignificant. our study had few confounding factors hence we could not be concluded that hepa-filtered rooms are not necessary. nevertheless, our experience suggests that availability of dedicated hepa units with special air-handling equipment should not be considered a critical and essential precondition for providing allogeneic hsct to patients even in developing world with financial constraints. these would otherwise succumb to potentially curable hematological illnesses with background of financial constraints and wait list of hepa filter rooms. early hsct in a clean patient in non hepa rooms is extremely cost effective with comparable outcomes. nursing care, experience of the team, experience in hsct program & well established protocols are more important in outcome of hscts. disclosure of conflict of interest: none. we present five cases of cytomegalovirus (cmv) pneumonitis occurring in patients after recent allogeneic stem cell transplantation (allohsct). these cases were complicated by an organising pneumonia (during the recovery period) with a predominantly central peribronchial pattern. all patients presented with evidence of active cmv pneumonitis which was treated successfully with anti-viral therapy but was followed by persistent severe dyspnoea, cough and hypoxia. high resolution computed tomography (hrct) imaging showed widespread central peribronchial consolidation with traction bronchiectasis. in most cases there was a marked clinical and physiological improvement after treatment with systemic corticosteroids. however, in all patients the lung function remained abnormal and in some cases imaging revealed a fibrosing lung disease. these cases represent a previously undescribed central peribronchial pattern of organising pneumonia complicating cmv pneumonitis that can result in chronic lung damage. disclosure of conflict of interest: none. cytomegalovirus reactivation in pediatric acute leukemia after stem cell transplantation has an effect on relapse and survival in aml but not in b-precursor all j-s kühl 1 , l sparkuhl 1 and s voigt 1 department of pediatric oncology/hematology/sct, charité universitätsmedizin berlin, berlin, germany several studies have indicated better survival after stem cell transplantation (sct) for acute leukemias, especially acute myeloid leukemia (aml), in case of cytomegalovirus (cmv) reactivation. here, we investigated if cmv reactivation had an impact on survival after sct for aml or acute lymphoid leukemia (all) in children. 177 pediatric allogeneic stem cell transplant recipients from our institution who received myeloablative conditioning were included. transplant indications included aml, t-all and b-precursor all. cmv reactivation was correlated with relapse, mortality as well as acute graft-versus-host disease (gvhd) and was analyzed by fisher's exact test or χ 2 -test (if n4100). from the 177 patients included, 42 were transplanted for aml (24%), 22 for t-all (12%), and 113 for b-precursor all (64%). mortality and relapse rates (27-37% and 18-26%, respectively), cmv reactivation rates (21-36%) as well as numbers of negative cmv serology status (19-32%) of donor and recipient were comparable between different acute leukemias. when patients were analyzed altogether, cmv reactivation had no effect on relapse rates or mortality. however, a tendency towards fewer relapses after cmv reactivation was observed in aml patients (no relapse (0%) with cmv reactivation vs 11 relapse cases (33%) without cmv reactivation; p = 0.083). in those 128 leukemia patients capable of reactivating cmv (that is, donor or recipient cmv seropositive prior to sct), cmv reactivation had a protective effect on relapse rates in aml (no relapse (0%) with cmv reactivation vs 11 relapse cases (44%) without cmv reactivation; p = 0.017). a similar tendency could be seen in t-all whereas no effect in patients with b-precursor all was documented. numbers of acute gvhd cases grade 4i between aml and t-all with or without cmv reactivation were similar. different effects of cmv on relapse rates and mortality in aml vs b-precursor all were noticed in 79 patients who were either not capable of cmv reactivation or who did reactivate cmv post sct. in 17 aml patients, there were no relapses (0%) and 2 deaths (12%) in contrast to 17 relapse cases (27%) and 24 deaths (39%) in 62 children with b-precursor all (p = 0.017 and p = 0.044, resp.). latently cmv infected aml patients without documented cmv reactivation after sct have a significant worse prognosis compared with all other aml patients. this is also likely to be the case in patients with t-all, however, patient numbers in our cohort were too few. the protective effect of cmv reactivation in aml and possibly t-all does not appear to be gvh-related since the rate of relevant acute gvhd cases was comparable. cmv reactivation after sct for b-precursor all lacks significance. disclosure of conflict of interest: none. infections are among the most frequent and relevant complications of hematopoietic stem cell transplantation (hsct). little is known about the role of dental foci for the prevalence of infections in hsct. dental status was prospectively evaluated in all patients at our center before undergoing hsct. a total of 132 different patients before undergoing 163 hsct (83 allogeneic and 80 autologous), with a median age of 52 years (range: 4-70 years) were evaluated. for evaluation a panoramic x-ray evaluation was performed. dental findings included the status of third molars and root fillings as well as caries, periodontitis, destructed teeth and apical bone loss. as non-dental parameters we used age, sex, type and status of central venous line, mucositis, and type of transplantation. these were correlated with neutropenic fever, bacteremia and pneumonia in a bivariate manner before a multivariate analysis was performed. no correlation of initial dental status to neutropenic fever, bacteremia or pneumonia was found. however, bacteremia and suspected infection of central venous lines was a significant predictor of neutropenic fever. in conclusion, dental surgery should only be performed prior to hsct if urgently required and limited to those individuals with overt infection. disclosure of conflict of interest: none. [p250] early experience with clinimacs prodigy ccs method in generation of virus-specific t-cells for pediatric patients with severe viral infections after hematopoietic stem cell transplantation k kallay 1 viral reactivation especially in children is a frequent complication of allogeneic hematopoietic stem cell transplantation. most of these episodes can effectively be controlled by an antiviral or antibody therapy; in refractory cases a novel virusspecific t-cell therapy could be a promising management option. in our pediatric cohort of 43 allogeneic transplantation during 1 year 9 patients fulfilled criteria for virus-specific t-cell therapy (5 boys, 4 girls, median age of 11 (1.5-16) years). six patients were transplanted because of hematological malignancies and 3 for inborn errors. donor distribution was the following: 7 matched unrelated, 1 sibling and 1 haploidentical donor. in 5 cases bone marrow, 3 cases peripheral blood and 1 case cord blood was used as a stem cell source. the underlying viral illness was cmv in 3, ebv in 2 and adenovirus in 1 case, while more than one virus was detected in 3 cases (cmv+adenovirus 2 cases, cmv+ebv 1 cases). viral diseases necessitating a t-cell therapy were cmv pneumonitis and colitis, adenovirus enteritis and cystitis and ptld. patients initially received cidofovir for adenovirus, rituximab for ebv and a combination of gancyclovir and foscarnet for cmv infections. the indication for t-cell therapy was progressive viral disease in 8 of the 9 cases and uncontrollable viral load in 1 case. the procedure was performed on a median of 63 (49-113) day post transplant. donors were 1st degree relatives in 5 cases, 2nd degree relatives in 3 cases and an unrelated person in 1 case, the best hla match was haploidentical. the median age of the donors was 47 (33-60) years. cells were produced by the clinimacs prodigy cytokine capture system (ccs) method after mononuclear leukapheresis. the system produced a median of 9.9 (6.7-25) times 103/kg cd4+ and a median of 32.6 (16-125.1) times 103/kg cd8+ interferon producing cells while the non-interferon producing cells were far below gvhd limit with a median of 3.6 (1.5-12.4) times 103/kg cd4+ and a median of 3.85 (1.4-4.5) times 103/kg cd8 + cells. the t-cell products were administered uneventfully in all but one case. we observed a manageable cytokine storm in one patient. glucocorticoid treatment was ongoing due to acute gvhd in 5 children; however we could manage to keep the steroid dose below 1 mg/kg in all cases. eight patients became completely asymptomatic, while 7 also cleared the virus. we experienced decreasing viral load in all cases, the first negative viral results were achieved on a median day of 13 (13-55). six patients are alive without viral illness or sequale, and complete viral dna clearance in peripheral blood with a median follow up of 329 (144-580) days. one patient with cmv pneumonitis improved during the first week but deteriorated on the second week and died of respiratory insufficiency despite of mechanical ventilation. in 2 cases the viral illness improved or cleared, but the patients died of invasive aspergillosis. no cases of gvhd, rejection, organ toxicity or recurrent infection were noticed. virus-specific t-cell therapy produced by the clinimacs prodigy ccs is a feasible, fast, safe and effective way to control resistant viral diseases after pediatric hematopoietic stem cell transplantation. this treatment can be implemented within a week in most cases. in order to define the appropriate place of this approach for patients with viral reactivations more data should be collected. disclosure of conflict of interest: none. central venous catheter (cvc) is essential for the treatment of recipients of stem-cell transplant. it is usually placed for the administration of conditioning regimen, stem cell infusion, intravenous antibiotics, immunoglobulins, electrolyte and nutritional support and blood concentrates. this patient group is at high risk for catheter-related bloodstream infections that can result in substantial morbidity and mortality. the neutropenia secondary to the conditioning regimen determines the risk of catheter-related infections, which may serve as an entry into the blood circulation, leading to bacteremia, fungemia, and consequently to septic shock and death. the risks of infection and the spectrum of infectious syndromes differ according to the type of transplant, conditioning regimen, type of implant of stem cells and therapies used after the procedure. gram-positive bacteria, particularly coagulase-negative staphylococcus spp, remain the leading cause of catheter-related bloodstream infection, although an increase in gram-negative bacteria as the causative agent has been noted. aim of the study: to evaluate the impact of the early cvc removal on the frequency of febrile episodes and infections in our group of patients. during a 15 years period we have treated 351 patients with hematologic neoplasm with high-dose chemotherapy and stem cells transplantation. patients were treated in sterile room conditioned with hepa filtration. in every patient was introduced double-lumen cvc (321 subclavia, 7 jugular, and 23 femoral). 44% were febrile (10% fuo), catheter-related infection was present in 9%, while positive culture from cvc was present in 28%. the most frequent isolated bacteria from cvc were gram positive-staphylococcus coagulasa negative. the catheter was removed on the day of discharge. trm is 2.4%. from 01 january 2016 to 01 november 2016 we have transplanted additional 40 patients. to aim to decrease infection related mortality we perform strategy to remove cvc on day +2 after stem cell transplantation. the febrile episodes decreased on 25% (10/40), there were no early post-transplant mortality due to infection. early removal of the cvc and adequate handling from the nursing staff is essential for outcome of this patient population in regard of infective complications efficient prevention, early diagnosis, and effective treatment of catheter related infection are essential to providing the best care to these patients and can minimized morbidity and mortality. disclosure of conflict of interest: none. fever in patients with agranulocytosis during autologous hematopoietic stem cell transplantation (autohsct) can be associated with non-infectious causes due to g-csf, vancomycin, engraftment syndrome. in this case biochemical markers, such as presepsin (psp), procalcitonin (pct) and c-reactive protein (c-rp), can help in differential diagnosis of fever of infectious and non-infectious genesis. psp, pct and c-rp were assessed on the day of admission to the hospital (da), on d+1, on d+3, on d+7 and on the day of discharge (dd). if patients developed neutropenic fever (nf), the markers were assessed at the beginning of the fever, 6 h after, then on the second, third, fourth days after. if patients developed nf immediate empirical antibiotic therapy (at) was implemented with meropenem. in cases of ineffective 1st line ab, 2nd line at was added or totally changed. there were 100 patients included in the study: 41 patients with hodgkin lymphoma, 27 with non hodgkin's lymphoma, 32 with multiple myeloma, out of 100 patients there were 51 women and 49 men. the median age was 41 years (18-66 years). the conditioning regimens were cbv, beeac or hd melphalan. 69 patients developed infectious complications (ic): 2 of them had sepsis and others-nf. the median of nf development was 5.5 days. depending on the efficiency of at therapy patients were divided into two groups: group 1: patients that have had effective at (they 've had fast clinical response and they haven't needed to change medicine (n = 45)); group 2: patients that have had ineffective 1st line at, they haven't had response to 1st line at and they've needed to change another at (n = 24)). there were significant differences in psp levels on the third day after ab had been admitted: 365.3 pg/ml in group 1 and 750.6 pg/ml in group 2 (p = 0.0019). similar differences between the analyzed groups were observed on the fourth day: 350.5 and 569.7 pg/ml, respectively (p = 0.024). pct and c-rp didn't show any significant changes between group 1 and 2 on each day of the study (table 1) . disclosure of conflict of interest: none. enterovirus related immune reconstitution inflammatory syndrome (iris) following haploidentical stem cell transplantation in an mhc class ii deficient child r shah 1 , s waugh 2 , k foong ng 1 , z nademi 1 , t flood 1 , m abinun 1 , s hambleton 1 , a gennery 1 , m slatter 1 and a cant 1 1 paediatric immunology and bmt, great north children's hospital, newcastle upon tyne, uk and 2 department of virology, great north children's hospital, newcastle upon tyne, uk immune reconstitution inflammatory syndrome (iris) has been described after hsct in association with fungal, viral and bcg infections. we describe a case of post-hsct iris associated with enterovirus infection. case: a girl with mhc ii deficiency (rfxp2c.362 mutation) underwent treosulfan/fludarabine/ thiotepa/atg conditioned tcrαβcd3+ depleted haploidentical hsct at 1.8 years of age. pre-transplant work up did not reveal any viral or fungal infections except norovirus in stool. cyclosporine (csa) was given as gvhd prophylaxis. neutrophil and platelet engraftment occurred on d+15 and d+9, respectively. on d+6, her stool was tested positive for enterovirus (taqman pcr), however; she was asymptomatic. the child started having fevers and irritability from d+21 which persisted despite the use of antimicrobials. no evidence of fungal or bacterial infection was found. enterovirus pcr in blood was found positive on d+31 (cycle threshold value, ct 32.4) and further typing showed it to be echovirus 13. at this time, symptoms progressed with diarrhoea, developmental regression and signs of radiculopathy. mri (brain and spine) was normal and csf showed pleocytosis (815 wbc/mcl-100% lymphocytes, protein 4.23 g/l) with positive enterovirus pcr (ct 17). subsequently, immunoglobulin prophylaxis was increased to 0.5 g/kg bi-weekly, and with supportive measures, the patient slowly recovered. blood enterovirus pcr remained positive. with no evidence of gvhd, csa was tapered off by day+105 and child was discharged on d+112 on a bi-weekly ivig replacement. she presented 5 days later with signs of raised intracranial pressure. mri showed hydrocephalus, and vp shunt was placed and broad spectrum antibiotics administered. csf showed wbc o1/mcl, protein 0.23 g/l and enterovirus positive. methylprednisolone 2 mg/kg/day was started suspecting iris. in subsequent csf testing 3 days later, enterovirus was negative. enterovirus pcr remained positive in blood during this period. patient's clinical deterioration correlated with a rise in cd4/cd8 counts and c reactive protein with clearance of enterovirus from csf, blood and stool ( figure 1 ). subsequently, the child showed gradual but marked improvement and discharged home. discussion: the clinical features of index case fits into criteria for iris 1 . markedly raised crp suggests high il-6 levels without any bacterial or fungal pathogens being isolated. in addition, iris occurs at the site of prior active infection (brain in index case) and viral clearance and clinical recovery demonstrated with the continuation of steroids. the incidence of enterovirus infection in hsct recipients is around 10% 2 . iris, in this case, had a temporal correlation with discontinuation of csa, and it has been shown that discontinuation of immunosuppression is associated with higher risk of iris. a high index of suspicion for iris is necessary during immune recovery post-hsct especially when immunosuppression is being tapered in a patient with pre-existing infection. aggressive antiviral treatment (when available) and judicious immunosuppression are the keys to managing iris complications. posttransplantation lymphoproliferative disease (ptld) is a significant cause of morbidity and mortality in allogeneic stem cell transplant patients. identifying high risk patients, routine pcr screening, early diagnosis and therapy are crucial for successful management. patients and methods primary objectives of this study were to describe epidemiology of ebv associated ptld and to assess risk factors in our paediatric cohort. additionally, role of immunoglobulin (ig) levels as a possible diagnostic/prognostic marker was analyzed. between 1 january 1 2011 and 30 june 2016, 140 allogeneic transplantations were performed in 118 pediatric patients (82 boys and 36 girls) at our center. median age was 7.68 years (0.03-18). underlying diseases were hematological malignancies (68%), nonmalignant hematological conditions (13%), immunodeficiencies (11%) and others (8%). stem cell source was bone marrow (62%), peripheral blood (19%) and cord blood (19%). donors were unrelated (75%), sibling (18%), haploidentical (4%) or other matched family donors (3%). routine ebv pcr screening and ig level detection were performed weekly. rituximab prophylaxis was given only in nine cases. results ebv dnaemia was found in 16/118 patients (13.6%), while ptld was diagnosed in 11/118 patients (9.3%). all ptld cases were related to ebv infection, median of highest viral load was 11 790 copies/ml (688-6 670 000). diagnosis was confirmed by biopsy in 6/11 cases, further five fulfilled criteria of probable ptld (positive pcr with appropriate clinical symptoms). ptlds occurred at a median of day +48 (19-85) after transplantation. all patients received rituximab treatment along with a reduction of immunosuppressive therapy. four patients died of ptld (mortality 36%), all confirmed by autopsy. a higher incidence of male gender (10/11; 90.9% vs 67.3%), bone marrow graft (10/11; 90.9% vs 59.8%), hematological malignancy (10/11; 90.9% vs 67.3%) and second transplantation (5/11; 45.5% vs 14%) could be detected among ptld patients when compared to the non-ptld group. elevated igg, iga or igm levels were observed in 13/118 patients. nine out of 13 had positive ebv pcr testing, eight of them developed ptld. five of the ptld patients had monoclonal or biclonal immunoglobulin elevation, two of them died. in 3 cases, elevated ig level preceded the positive ebv pcr results by at least 1 week. conclusion: at our centre incidence and mortality of ptld was similar to published data. we observed a tendency that a higher representation of male gender, hematological malignancy, bone marrow graft and second transplantation could be confirmed in the ptld group however due to small number of patients, a correlation and statistical significance could not be calculated. elevation of immunoglobulin levels do not seem to be specific for ptld but in selected cases it could predict ebv disease earlier than pcr testing. disclosure of conflict of interest: none. autologous peripheral hematopoietic stem-cell transplantation is a procedure of a stem cell rescue with patients' own previously collected hematopoietic stem cells, after myelotoxic therapy. the purpose of stem cell reinfusion is to ensure adequate recovery of hematopoiesis, shorten the period of profound neutropenia and to reduce the risk of infections. the transplantation itself carries a moderate risk for infection but some patients have higher risk due to the nature of underlying disease, earlier treatment and in case of severe mucositis. for these reasons, all treated patients are in isolated clean rooms and receive ciprofloxacin, fluconazole and acyclovir prophylaxis. in the 3.5-year period, 177 autologous transplantations were performed. the patients were 20-72 years old, with median of 55.18 years. of all transplanted patients, 106 or 59.88% had multiple myeloma, 66 or 37.3% had lymphoma and 5 or 2.82% had acute myeloid leukemia. all of the patients received pegfilgrastim 6 mg on the first or the second posttransplant day. febrile neutropenia (ne o0.5 × 10 9 /l) was reported if patient's temperature was above 38.3°c in one measurement or above 38°c in two consecutive measurements. these patients were treated empirically with piperacillin/tazobactam 4.5 g four times a day with the addition of vancomycin in the case of severe mucositis or pulmonary infiltrates. in all cases blood and urine cultures were performed, as well as testing for seasonal flu. time to neutrophil recovery (ne40.5 × 10 9 /l) was 7-20 days, with a median of 10 days, and average of 10.25 days. febrile neutropenia was reported in 76 patients (42.94%) and in 39 (51.31%) patient's samples pathogen was isolated. gramm negative bacteria caused sepsis in 54.29% of patients. we had to change empirical therapy according to antibiogram in 37.2% patients. in 1 month follow-up period, there were two (1.12%) infection related deaths. our data on incidence of infections is consistent with literature data but large number of papers show satisfactory results of safety of patients discharged from hospital immediately after the autologous stem cell transplantation and who were treated at home during the phase of profound neutropenia. there is still an ongoing debate whether it is possible to conduct this procedure in such manner in our health system. disclosure of conflict of interest: none. fluconazole was equal to mold-active drugs in preventing early invasive fungal disease after allogeneic stem cell transplantation regardless of transplantation type y sun, j hu, h huang, j chen, j-y li and x-j huang there are still controversies that whether mold-active drugs is better than fluconazole in preventing invasive fungal disease (ifd) after allogeneic stem cell transplantation (hsct). we hypothesis that the optimal prophylaxis might be different in patients with different risk profile, such as in different time period after hsct or received alternative donor transplantation. in the prospective china assessment of antifungal therapy in haematological disease (caesar) study database, 661 out of 1401 patients received primary antifungal prophylaxis were analyzed. the ifd incidence of different time period after transplantation (early, late and very late) and survival were compared among different drug groups. in patients with fluconazole, itraconazole, voriconazole or micafungin prophylaxis, the overall incidence of ifd after transplantation were 7.2%, 12.6%,1.4% and 5.2%, respectively (p = 0.0379). however, there is no difference in early ifd (o40 days post hsct) among 4 groups of patients. the risk factors associated with occurrence of ifd were neutropenia duration 414 days (po0.01, or 3.73 (1.66-8.36)), adult (p = 0.02, or 3.37 (1.23-9.18)) and alternative donor (unrelated donor or haploidentical donor) transplantation (p = 0.01, or 5.88 (1.48-23.32)). in the sub-group analysis with only alternative donor (unrelated donor and haploidentical donor), it also demonstrated that fluconazole is equal to other mold-active drugs in preventing early ifd. patients received fluconazole prophylaxis has even better overall survival. the overall survival in patients with fluconazole, itraconazole, voriconazole or micafungin prophylaxis were 88.3%, 83.5%,78.9% and 72.4%, respectively (p = 0.0047). our current [p259] study suggests that fluconazole is equal to mold-active drugs to prevent early ifd in hsct patients, even in high-risk patients received transplantation from alternative donors. however, further prospective randomized study was warranted to confirm this conclusion. disclosure of conflict of interest: none. (9.5%) received autologous hsct and 38 (90.5%) allogeneic hsct. sixty-five out of 107 patients (60.7%) were affected by different haematological diseases: 36 by lymphoma, 11 by multiple myeloma, 7 by chronic lymphocytic leukaemia and 11 by others diseases including mastocytosis, amyloidosis and essential thrombocythemia. hbv reactivation prophylaxis prescribed was entecavir for 4 hbsag+ inactive carrier patients and prolonged lamivudine (lmv) course for 103 (96%) patients. in 6 patients (5.6%) lmv prophylaxis was withdrawn 12-18 months after the end of immunosuppressive therapy. eight out of 107 patients (7.4%) experienced hbv reactivation: 4 of them during lmv treatment and then they were switched to entecavir or tenofovir therapy, 4 patients reactivate hbv after lmv interruption (3.7%). in these patients reactivation was observed after an average time of 4 months (range: 3-6) after discontinuation of lmv prophylaxis. median duration of prophylaxis was 49 months (range: 40-60) after the end of immuno-suppression. three out of 4 patients (75%) underwent allogeneic hsct and 3 patients (75%) received rituximab. one out of 4 (25%) seroreverted in hbsag positive and hbsab negative status, with hbv-dna42000 ui/ml (table 1) . two patients out of 4 (50%) experienced hbv-dna detection below 20 ui/ml. disclosure of conflict of interest: none. [p260] [p260] the severity is measured on grades (grade 1: microscopic hematuria to grado 4: clots cause urinary tract obstruction). the treatment is based on support measures: hyperhydration, continuous bladder irrigation, instillation of topical agents and in severe cases must be performed a cystoscopy for clot evacuation. in the case of the presence of poliomavirus virus (bk virus) the use of cidofovir had been demonstrated in vitro studies to have activity against bk virus. we performed 42 allogenic transplants of which 10 are haploidentical from 2010 to october 2016. we realized a retrospective case study to analyses the experienced in the management of hc. results: of a total of 42 allogenic transplants realized, developed a hc: 1 patient received an identical hla transplant and the 4 patients remaining haploidentical allotrasplant. all cases were male, with an age range of 18-42 years. the status of the disease was: 3 were in complete remission and 1 had visible disease. 3 of the 4 patients received cyclophosphamide as immunosuppressive therapy and all patients received cyclosporine and mofetil micofenolate also. the onset of the symptomatology was between day 9 and day 82 post transplant and the range of duration was from 14 to 45 days. the four patients precised continuous bladder irrigation but because of the poor response they received instillation of hialuronic acid (4 doses). two patients required the use of cidofovir (3 doses). one of the four patients required urinary tract catheterization because of hydronephrosis and renal impairment. in our review we confirmed that this entity is more frequent in the haploidentical transplant and bkv is the most prevalent cause in the late hc. -the three patients received 3 doses of cidofovir (1mg/kg) without probenecid and had a good response. -three patients present acute renal failure associated to hc. the four patients needed bladder instillations with saline but they had poor response and received at least 4 doses of hyaluronic acid. disclosure of conflict of interest: none. hsv infection in allo-hsct setting is mostly reactivation of latent virus. hsv disease commonly presents as mucocutaneous lesions of the oral cavity. however some patients develop serious fatal visceral dissemination. prophylactic use of acyclovir has markedly reduced the incidence of hsv disease during the period of neutropenia after allo-hsct. in this study, our aim is to demonstrate the incidence, clinical outcome and risk factors for hsv disease in adult allo-hsct. between 2015 and 2016, 89 patients who underwent allo-hsct in our center were included to the study. all hsct candidates and donors were tested for hsv-1/2 immunoglobulin g (igg) antibodies prior to transplantation. all patients received acyclovir prophylaxis (related transplants 400mg tid, unrelated transplants 800mg tid) during conditioning and after allo-hsct up to 3 months. chlorhexidine oral solution as well as bioadherent oral protective gels was used for oral hygiene. all patients were followed for symptoms of reactivation. hsv1/2 igg seropositivity was detected in 66 recipients (74%) and 48 donors (54%). the distribution of hsv status was as follows: recipient and donor seropositive in 34 (38%), recipient and donor seronegative in 9 (10%), recipient seropositive and donor seronegative in 32 (36%), recipient seronegative and donor seropositive in 14 (16%) transplants. the median age of the patients was 41 (range: 18-67), 48 patients were male (54%) and 79 (89%) had malign disease. the stem cell source was peripheral blood in 73 (82%) patients and 48 (54%) received grafts from related donors. sixty four patients (72%) received myeloablative conditioning regimen. the most common graft-vs-host disease (gvhd) prophylaxis administered was cyclosporine (csa) and methotrexate (mtx) in 60 patients (67%). acute graft vs host disease was detected in 29 patients (33%).four patients from seropositive 66 patients (6%) had hsv reactivation, the patient characteristics are given in the table. all patients had hsv reactivation within 1 month of allo-hsct except one patient had symptoms at sixth month posttransplant when he suffered from oral gvhd. all patients s252 and donors were seropositive prior to allo-hsct and responded well to antiviral treatment. the incidence of hsv reactivation in allo-hsct was detected as 6% which is lower to previous studies. successful primary prophylaxis and oral hygiene might reduce the incidence. all patients were responded to antiviral treatment and no visceral dissemination was detected. disclosure of conflict of interest: none. patients who have received hematopoietic stem cell transplantation (hsct) may suffer, to some extent, losses in humoral and cell immunity against antigens to which they had been previously exposed naturally (infection caused by wild microorganisms) or artificially (through vaccination). the conditioning regimen for hsct replaces the patient's immune system and involves the loss of previous immunity. this study analyzed patients included in the vaccination program for hsct recipients in the salamanca health care complex during the period 2010-2016. we assessed the serological status prior to hsct for the following immunopreventable diseases (hepatitis b, hepatitis a, varicella), and the study after hsct also included measles, rubella and parotitis, prior to their inclusion in the hsct vaccination program. the study included 302 patients, 53.8% of which (n = 168) were men. 83.3% of the patients (n = 260) were allogeneic hsct recipients with an average age of 47 ± 16 years, and 16.7% (52) were autologous hsct recipients with an average age of 43 ± 16 years. prior to hsct, 40% of the patients showed immunity against hepatitis b (hbv antibodies 410 ui/l), 82.6% against hepatitis a (positive for hav igg) and 85% against varicella (positive for varicella igg). no statistically significant differences were observed regarding this variable hepatitis b anti-hbs 410 ui/l, hepatitis a, igg positive, varicella igg positive, measles igg positive, rubella igg positive, parotitis igg positive. table 1 compares the serological status before and after transplantation. in the pre-transplant serological study we observed that less than half of the patients are protected against hepatitis b, while over 80% of them are protected against hepatitis a and varicella. regarding the diseases in which we know the serological status before and after transplantation (hepatitis a, hepatitis b and varicella), we observed that most patients maintain immunity. in the case of rubella, measles and parotitis we only have access to the serological status after transplantation, and we observed that parotitis is the disease with the lowest seroprotection. therefore, vaccination would be indicated, just as in the case of hepatitis b. the clinical results support the need to adapt the vaccination schedule to the immunological status of the patients after hsct individually. disclosure of conflict of interest: none. impact of cumulative steroid dose on infectious diseases after allogeneic hematopoietic cell transplantation m watanabe 1 , j kanda, t kitano, t kondou, k yamashita and a takaori-kondo 1 after allogenic hematopoietic cell transplantation (hct), highdose steroids are used to treat transplantation-related complications such as graft-versus-host disease (gvhd). however, the use of high-dose steroids is associated with an elevated risk of infectious diseases. information on the association between cumulative steroid dose and infectious diseases after hct is scarce. a total of 238 patients who underwent their first hct in kyoto university hospital from 2005 to 2015 and survived at least 30 days after transplantation were included in this study. we analyzed the association between cumulative steroid dose used within 30 days after transplantation and the occurrence of infectious diseases, including invasive fungal infection (possible/probable/proven cases), cytomegalovirus (cmv) antigenemia, and bacteremia through 180 days after transplantation. sixty-three patients received transplantation from a related donor, 114 received unrelated bone marrow grafts, and 61 received unrelated cord blood units. their median age was 51 (range: 17-66) years and median day of neutrophil engraftment after transplantation was 21. patients were categorized into 3 groups according to their cumulative steroid dose within 30 days: no steroid administration (n = 183), low-dose cumulative steroid administration under 500 mg of prednisolone in total (n = 27), and high-dose cumulative steroid administration over 500 mg of prednisolone in total (n = 28). reasons for steroid administration were treatment for gvhd in 35 patients, engraftment syndrome in 11, and other reasons including lung complications in 9. the rate of invasive fungal infection was 6% (12 possible cases with pneumonia and 1 proven case of candida blood stream infection) and we found no apparent association between fungal infection and steroid use regardless of dose. cmv antigenemia was diagnosed in 41%, 67% and 60% of patients in the 3 groups respectively, and both low-dose and high-dose steroid groups were significantly associated with a high risk of cmv antigenemia (low-dose group, adjusted hazard ratio (ahr), 2.37, p = 0.004; high-dose group, ahr 1.84, p = 0.01). bacteremia was diagnosed in 9.8%, 11% and 21% of patients in the 3 groups, respectively. high-dose steroid use was a risk factor for bacteremia (ahr 1.70, p = 0.027). seven patients died from infection (fungal, 2; viral, 2; bacterial, 3). two of three bacterial infection-related deaths occurred in the highdose steroid group, although the number of events was too small to analyze. our data confirmed that steroid administration is itself a risk factor for cmv antigenemia and close observation to detect cmv antigenemia is mandatory for patients using steroids regardless of its cumulative dose. high-dose cumulative steroid use is a risk factor for bacteremia. contrary to our expectations, steroid administration showed no apparent association with invasive fungal infection in our study, perhaps because of its generally low incidence in our hospital. disclosure of conflict of interest: none. impact of different t-cell depletion techniques on the incidence of infectious complications after allogenic hematopoietic stem cell transplantation k aikaterini 1 , s federico 1 , d-l vu 2 , e boely 2 , c dantin 1 , a pradier 1 , y tirefort 1 , a-c mamez 1 , o tsopra 1 , c stephan 1 , y beauverd 1 , e roosnek 1 , s masouridi-levrat 1 , c van delden 2 , y chalandon 1 1 department of oncology, hematology unit, university hospital of geneva and 2 department of medicine specializations, infectious diseases, university hospital of geneva t-cell depletion (tcd), obtained by either in vivo antithymocyte globulin (atg) administration or ex vivo depletion, is a well-established strategy for graft-versus-host-disease (gvhd) prevention after allogeneic hematopoietic stem cell transplantation (hsct)1-4. however, the prolonged lymphopenia associated with tcd can result in increased incidence of disease relapse1 and infections. although many studies investigated the impact of tcd on disease relapse 1-4, little is known about the impact of tcd strategies on the incidence of infectious complications after allogeneic hsct. we retrospectively evaluated the incidence of infectious complications in 236 consecutive patients who underwent allogeneic hsct at our center from september 2010 to december 2015. 100 patients received tcd grafts obtained by in vivo atg administration as part of the conditioning regimen (atg group). 17 patients received partially tcd grafts obtained through incubation with alemtuzumab in vitro washed before infusion followed on day +1 by an add-back of donor t cd3+ cells5 (ptcd group). 60 patients received grafts tcd by both methods combined. 59 patients did not receive any form of tcd (no-tcd group). cumulative incidence estimates of infectious complications were calculated and compared using the gray test. given the increased risk of infection associated with gvhd and its treatments, gvhd or death from other causes were defined as competitive events in the analysis. we didn't observe any significant difference in the 1-year cumulative incidence of bacterial infections in patients receiving tcd by atg (45% (95% ci 35-54.5%)) ptcd (58.8% (95% ci 31.2-78.5%)) or both (55% (95% ci 41.4-66.7%)) compared with patients receiving no tcd (52.5% (95% ci 38.9-64.5%)). similarly, the 1-year cumulative incidence of viral infections or reactivations was comparable in patients receiving no-tcd grafts (80.3% (95% ci 66.8-88.8%)) compared with patients receiving tcd grafts (atg: 82.2% (95% ci 72.9-88.6%); ptcd: 76.5% (95% ci 45.7-91.2%); atg+ptcd: 81.7% (95% ci 68.9-89.6%)). finally, no significant impact of tcd was observed on 1-year cumulative incidence of fungal (no-tcd: 6.8% (95% ci 2.2-15.2%); atg: 18.1% (95% ci 11.2-26.3%); ptcd: 11.8% (95% ci 1.8-31.9%); atg+ptcd: 18.3% (95% ci 9.7-29.1%)) and parasitic (no-tcd: 1.7% (95% ci 0.1-8%); atg: 1% (95% ci 0.1-4.9%); ptcd: 5.9% (95% ci 0.3-24.2%); atg +ptcd: 1.7% (0.1-8.3%)) infections. image/graph: 1-year cumulative incidence estimates of infectious complications depending on the tcd strategy employed. the results of our retrospective analysis indicate that the cumulative incidence of bacterial, viral, fungal and parasitic infectious diseases are similar in patients receiving tcd grafts compared to those receiving no-tcd graft, suggesting a favorable toxicity profile of different tcd strategy in respect of infections. these results should be confirmed by similar analysis in large scale, prospective clinical trials assessing the potential benefits of tcd on transplantation outcomes. 57 patients with aml were considered eligible for hsct, 7 died before transplantation. 13 patients (26%) underwent transplantation from hla-identical sibling, 13 (26%) from haploidentical family donor and 19 (38%) from matched unrelated donor, while 5 patients (10%) received unrelated cord blood cells. twenty (35%) out of 57 eligible patients have had an ifi episode before transplant: 6 were proven, 4 probable and 10 possible; (9 (47%) pneumonia, 4 (19%) gastroenteritis, 3 (15%) sinusitis, 2 (10%) candida sepsis, 1(5%) meningitis and 1(5%) cutaneous abscess were registered). five (25%) out of 20 patients with a previous ifi and 2 (5%) out of 37 without previous ifi did not receive hsct (or 5.83 95% ci 1.02-33.96, fisher test p: 0.04). the majority (55%) of patients with a previous ifi waited hsct more than 6 months from the date of eligibility in comparison with those without a previous ifi (55% vs 30%; or 0.37, 95% ci 0.12-1.13, p-value 0.08 fisher test) overall a post transplant ifi episode was diagnosed in 13 (26%) of transplanted patient; 4 (26%) had a relapse of a past ifi vs 9 (25%) of the patients without a previous ifi who had a new episode. (or 1.53, 95% ci 0.39-5.90, p-value 0.4 yates test).a higher number of patients with ifi (10 out of 15, 66%) respect to those without a previous ifi (10 out of 35, 30%) died in a median time of 160 days(range: 22-480 ) after hsct. furthermore, those who had a previous ifi had a lower median survival (317 days (range: 22-1095)) compared to patients without a previous ifi (530 days period (range: 20-1490)) (student's t-test p: 0.014)). a previous ifi episode in the pre transplant period slows and limits the accessibility to hsct, and is significantly associated with an increased mortality. disclosure of conflict of interest: none. s254 delayed immune reconstitution has been described for haploidentical hematopoietic stem cell transplantation (hsct) compared to conventional hsct, nevertheless the incidence of invasive aspergillosis infections (iai) in haploidentical sct and the efficacy of primary prophylaxis are not well defined. our objective is to describe the incidence, risk factors and mortality of iai in our patients, using as prophylaxis micafungin during the conditioning and neutropenia period, switched to posaconazole or voriconazole when oral intake is feasible. we retrospectively analyzed 40 consecutive patients from 2014 to 2016 who received haploidentical grafts: unmanipulated for 20 adults, tcrab depleted in 6 children and cd45ra depleted in 14 children. the stem cell source was peripheral blood in all cases. adults (22-70 yo) were treated for aml/mds (n = 8), all (n = 2) and lymphoma (n = 10). children (6 mo-15 yo) were treated for aml (n = 6), all (n = 9), aplastic anemia (n = 2) and immunodeficiencies (n = 3). conditioning regimen was bu-flu-cy (n = 18, adults), thio-bu-flu (n = 2, adults), flu-mel-thio for all pediatric patients; atg was used in 6 children and tli in 14 children. median follow up was 12 months (1-30) for adults and 7 months (1-28) for children. we used eortc criteria for iai and analyzed probable or definite as cases. there were 6 events of iai, with a bimodal presentation: 3 events (7.5%) during neutropenia period and 3 (7.5%) after 6 months of hsct ( figure 1 ). five of them were probable and one definite (aspergillus niger). site of infection was mainly pulmonar; cns was suspected in two adult patients and skin was proven in one adult patient. all 3 patients at the late period had chronic gvhd at diagnosis. one patient had primary graft failure. severe cmv disease (hepatitis and colitis) was present in one adult. mortality related to iai was high (5/6), patients died at a median of 35 days. figure 1 . iai patients characteristics the global incidence of iai in haploidentical hsct is similar to conventional hcst. primary prophylaxis with micafungin switching to oral triazole is successful (92.5%) during the early period. late cases (7.5%) had clearly known risk factors (chronic gvhd, steroids and cmv), and primary prophylaxis had been modified due to toxicity or interactions. iai mortality in our patients is very high (84%) despite effort in prophylaxis, diagnosis and treatment. visceral intractable abdominal pain prior to skin lesions from herpes zoster can be misdiagnosed as gvhd post stem cell transplantation which may lead to initial increase in immunosuppression and hence high mortality if we don't suspect. case report and literature review through pubmed results: 13year-old male with relapsed all post mud pbsct (10/10) transplant in following cy tbi atg conditioning presented at day +117 with intractable diffuse abdominal pain with constipation. no history of nausea, vomiting or skin rash. on physical examination his abdomen was soft, diffuse tenderness but no rigidity, muscle guarding and rebound tenderness. laboratory tests including liver function test, amylase, lipase were normal. usg abdomen and mri abdomen showed no abnormalities, except for presence of fecolith. during the stay his pain worsened needing morphine infusion, pca and later ketamine. he had previous history of acute gut gvhd controlled on budesonide and cyclosporine which was later being weaned once his symptoms were controlled. in view of previous history of gvhd, gi consultation was sought and he underwent ugi endoscopy and biopsy which was non-significant. on day 7 of his admission he developed a pustular skin lesion on thigh and scrapping from that showed vzv and his blood pcr was also positive, he was started on intravenous acyclovir. his lesions improved and crusted and his abdominal pain subsided after 72 h of acyclovir and was discharged on oral acyclovir after 14 days of intravenous therapy. review of literature illustrated in table 1 . severe abdominal pain in patients who received an allogeneic stem cell transplant has a broad differential. here we describe a case of vzv presenting with intractable abdominal pain needing opioids. because of the poor prognosis and life-threatening nature of disseminated vzv disease, it should be considered and included in the patient's workup. intravesical cidofovir (5 mg/kg, diluted in 90 ml sterile water) was once weekly applied until symptom control for 60 min. via a transurethral catheter, i.v. cidofovir was initiated if no symptom control was achieved after 3 local applications. in patients with hc 3 or 4 a lavage catheter was added. bkv cystitis (dysuria (n = 8) or dysuria combined with hematuria (n = 10)) developed in 18 out of 152 transplants (12%). median age was 54 years, 89% were female and 50% received a mismatch transplantation after 1 mac or 17 ric conditioning regimens. in 67% of bkv cystitis cases also cmv reactivation within the first 180 days could be detected. 67% had acute gvhd ii°-iv°at the onset of bkv cystitis and 83% received steroid medication. the median time to symptom occurrence was day +40 after hsct (iqr 25-75: 31-136). 5 patients (3 with dysuria and one either hc 1°and 2°) didn´t require therapy due to self limiting symptoms. 3 (23%) of 13 treated patients showed only dysuria, 3 (23%) hc 1°, 5 (38%) hc 2°, 1 (8%) hc 3°and 1 (8%) hc 4°. the first patient was treated with i.v. cidofovir twice and symptoms relieved. all the following 12 patients were exposed to intravesical cidofovir as 1st line therapy. 8 patients (67%) achieved a complete remission with a median of 2 intravesical procedures (range: 1-3). 1 patient showed symptom improvement and all 9 patients didn´t require further therapy. 3 patients had to be switched to i.v. application due to bladder spasms during intravesical application (n = 1) or to insufficient symptom control (n = 2). 2 out of these 3 responded to i.v. treatment, whereas 1 patient receiving 2nd transplant didn´t respond at all. in patients with spontaneous symptom relieve the median bkv concentration at the time of symptom onset was 2 log lower compared to those requiring antiviral therapy. local therapy reduced bkv viruria by 2 log. pain during cidofovir instillation in 50% of patients was the only significant side effect of local therapy compared to creatinine increases by 450% in 66.6% of i.v. treated patients. intravesical treatment of symptomatic bkv cystitis with cidofovir (5 mg/kg) is safe and effective with an 75% symptom improvement rate and no systemic side effects. in patients without sufficient symptom or bleeding control i.v. cidofovir is still an option, which however induces significant renal toxicity. we therefore recommend intravesical cidofovir as 1st line therapy in case of dysuria or hematuria induced by bkv after hsct. disclosure of conflict of interest: none. haemorrhagic cystitis is a recognised complication of stem cell transplant (sct), with a reported incidence of 5-25% of cases (1) . the majority of cases are associated with bk polyomavirus (bkv), and less often adenovirus and cytomegalovirus. there are a lack of high quality studies on the optimal prevention and management of haemorrhagic cystitis. treatment options are restricted by conditioning toxicity, immunosuppression and other co-morbidities such as renal impairment. cidofovir has an inhibitory effect on bkv replication and has been used extensively in the treatment of haemorrhagic cystitis. however, severe nephrotoxicity limits routine intravenous use in sct patients. alternative options include using low dose intravenous cidofovir or intravesical administration. we conducted a retrospective case review of post sct patients presenting with bk virus associated haemorrhagic cystitis in our institution between january 2010 and november 2016. we identified 5 patients in total (4 male,1 female). the indications for stem cell transplant were as follows: severe aplastic anaemia 1 high risk aml 1 relapsed aml 1 relapsed all 2 onset of symptoms (haematuria and painful micturition) ranged from day − 4 to day +27, and the time to resolution of symptoms varied from 16 days to 68 days. four of the patients were treated with intravesical cidofovir only, with the number of doses required varying from 3 to 7. one patient received combination treatment with both intravenous (7 doses), and intravesical cidofovir (5 doses). all patients had a good clinical response with complete resolution of symptoms and no major complications. however, the level of bk virus in the urine did not always correlate with clinical response. some of the patients did not tolerate urethral catheterisation and required a general anaesthetic for the placement of the urethral catheter; 1 patient required a supra-pubic catheter. currently 3 out of 5 patients are alive and well; 2 patients died from causes not related to bk virus associated haemorrhagic cystitis. our experience shows that intravesical administration of cidofovir is a safe and effective option for the treatment of bk virus associated haemorrhagic cystitis. an allogeneic stem cell graft from a cytomegalovirus (cmv) seronegative donor puts recipients at high risk of cmv reactivation which can lead to cmv disease and mortality. based on the immunogenicity of cmv phosphoprotein 65 (cmvpp65) we initiated a clinical phase i trial with a novel vaccine designed by our group: a cmvpp65-derived peptide in water-in-oil emulsion (montanide) plus administration of granulocyte-macrophage colony stimulating factor. ten patients received four vaccines s.c. at a biweekly interval after allogeneic stem cell transplantation. we monitored the patients for their clinical outcome and cmvpp65 antigenemia. multi-color flow cytometry test were performed to assess cmvspecific cd8+ and gamma-delta t cells. novel neutralizing anti-cmv antibody assays were established and correlated to clinical parameters. findings: in general, patients tolerated the peptide vaccination well, no drug-related adverse events others than rash or induration at the site of injection were detected. seven of nine patients with cmvpp65 antigenemia cleared the cmv after four vaccinations and were hitherto free from antigenemia. two patients with cmv reactivation showed persisting cmv antigenemia. one of these two refractory patients received additional four injections and remained hitherto free from cmv antigen. another patient obtained a prophylactic vaccination and did not develop antigenemia. an up to six-fold increase in frequency of both cmv-specific cd8+ t cells or vdelta2-gamma-delta t cells was detected in five patients. moreover, titers of neutralizing antibodies increased in four patients up to 10-fold over the time of vaccination. humoral and cellular immune responses correlated with clearance of the cmv load. cmvpp65 peptide vaccination was safe and well tolerated in patients after allogeneic stem cell transplantation at high risk for cmv reactivation. the vaccine showed encouraging immunological and clinical results. a prophylaxis study using the vaccine in solid-organ transplant patients is ongoing. disclosure of conflict of interest: none. sporopachydermia cereana is a rare yeast found in necrotic cactus tissue, predominantly in the americas. infection in humans has only been reported in 4 neutropenic patients with fatal course, either directly from the pathogen or other complications of immunosuppression. treatment is complicated by difficulties in pathogen-identification with conventional diagnostic techniques and by resistance to echinocandins. here we present a patient with acute myeloid leukemia (aml) and s. cereana infection. this is the first patient who was successfully treated with antifungal therapy and who survived s. cereana infection. case presentation we present the case of a 50-year-old female patient who was diagnosed with normal karyotype aml with dnmt3a and idh2 mutations in december 2015. she achieved complete remission after two cycles induction chemotherapy. during the 2 nd induction cycle the patient developed persistent fever in neutropenia despite broad-spectrum antibiotics and the replacement of prophylactic fluconazole to caspofungin. blood cultures showed growth of s. cereana, shown to be sensitive to azoles (mic fluconazole o1 mg/l, mic voriconazole o0.12 mg/l) as well as amphotericin b (mic o0.25 mg/l), but resistant to caspofungin (mic44 mg/l). following the susceptibility profile the treatment was changed first to liposomal amphotericin b, and with the availability of mic results to voriconazole. metastatic fungal infection (that is, endocarditis, endophthalmitis, hepatosplenic candidiasis) was excluded. after regeneration of peripheral blood values the treatment was switched to oral voriconazole. a ct scan of the chest and abdomen prior to allo-hsct after 6 weeks of treatment with voriconazole revealed new multiple necrotic mesenteric lymph nodes. an ultrasound-guided biopsy of a node revealed no growth on fungal cultures, a grocott stain revealed no hyphae or spores. a panfungal pcr of an its (internal transcribed spacer) fragment revealed fungal dna, which could be confirmed as s. cereana. at this time the level of voriconazole in serum was found to be sub-therapeutic (0.4 mg/l), and the dosage was increased accordingly. subsequent ct scans 4 and 6 weeks later revealed a regression of the affected abdominal lymph nodes. in the further course non-myeloablative conditioning with fludarabine and busulfan prior to allo-hsct using pbsc from her hla-matched brother was performed. under prophylaxis with cyclosporine, methotrexate and antithymocyte globulins (atg) graft-versus-host disease (gvhd) remained absent. the allo-hsct was performed under voriconazole treatment with no further complications and the patient engrafted at day 20. the treatment was changed to fluconazole 400 mg daily before discharge. due to the complete radiological regression of the infection in follow-up scans and excellent general condition of the patient 5 months after hsct, fluconazole was discontinued. the patient remains in morphological complete remission 6 months after hsct and has a 100% donor chimerism. the first published case of survival of infection with s. cereana exemplifies the continual progress made in treating infections in the severely immunocompromised patient. diagnosis via its sequence-analysis seems reliable but a high index of suspicion is required for neutropenic patients who do not respond well to standard antimycotic therapy. the increased availability of the technology may lead to more frequent diagnoses in the future. disclosure of conflict of interest: none. neutropenic enterocolitis (ne) is a clinical syndrome characterized by fever and abdominal pain in patients who received chemotherapy for hematological malignancies and who treated with stem cell transplantation (sct). the aim of this study was to determine the incidence, risk factors and outcome of ne after autologous sct (auto-sct). we retrospectively evaluated 226 patients with non-hodgkin lymphoma (nhl), hodgkin lymphoma (hl) and multiple myeloma (mm) who underwent auto-sct between january 2013 and december 2016 in our center. patients with lymphoma were conditioned with carmustine, etoposide, cytarabine, melphalan (beam) or thiotepa, etoposide, cytarabine, cyclophosphamide, melphalan (tecam). patients with multiple myeloma were treated with melphalan as conditioning. diagnosis of ne was established in case of neutropenic fever, abdominal pain or diarrhea, and bowel wall thickening 44 mm on abdominal ultrasonography. febrile neutropenia was seen in 199 (88%) patients of all. the median time from transplantation to neutropenia was 4.5 days (range: 0-9 days). ne occurred in 22 (9.7%) in all neutropenic patients. the median time to ne after auto-sct was 7 days (range: 2-9 days). the median neutrophil engraftment time was 12.5 days (range: 9-18 days). abdominal pain was seen in all patients with ne. twenty one patients (95%) had diarrhea. ileus was seen in 1 (4.5%) patient and septic shock was developed in 3 (13.6%) patients. five (22.7%) of 22 patients had bloodstream infection. klebsiella pneumoniae in 1, pseudomonas aeruginosa in 1, escherichia coli in 1, staphylococcus aureus in 1 and coagulasenegative staphylococcus in 1 patient were documented in patient's blood stream. early diagnosis was made by abdominal ultrasonography in all patients at a day of median 7 days (range: 2-9). twenty (91%) patients were resolved completely with good supportive care and proper antibiotherapy. two (9%) patients died of septic shock and ileus. ne is a rare but serious complication in patients underwent high dose chemotherapy followed by auto-sct. gramnegative bacteria are the main causative pathogens. abdominal ultrasonography is the simple, cheap, fast diagnostic and noninvasive procedure that allows the early diagnosis and effective treatment. disclosure of conflict of interest: none. [p274] neutrophil transfusions in the treatment of neutropenic patients submitted to allogeneic hsct: possible role on graft failure s giammarco, p chiusolo 1 , l laurenti 1 , f sorà 2 , n piccirillo 2 , l teofili 2 and s sica 2 1 hematology department, università cattolica del sacro cuore and 2 hematology departement, università cattolica del sacro cuore granulocyte transfusions (gtx) from g-csf-stimulated donors have been shown to increase the absolute neutrophil count (anc) before expected haematopoietic recovery in neutropenic patients after chemotherapy or haemopoietic sct. thus gt offers a therapeutic option along with antimicrobial agents and growth factors to improve clinical outcome of neutropenic patients with severe infections. the primary limitations of gt include low component cell dose and leukocyte incompatibility. the transfusion of g-csf-mobilized, hla-matched granulocyte components resulted in sustained anc increments, but the efficacy of this procedure has not been established by convincing randomized control trials. aim: we focused our attention on gt in the setting of allogeneic hsct, in particular on the feasibility and safety of this procedure on the rate of engraftment. between 2006 and 2016 our centre performed 211 allogeneic hsct. we analyze data from 59 transplanted patients receiving gt at some point during their disease. indication for gt was severe sepsis mainly due to mdr gram-bacteria. patients received a median of 4 gt (1-34), in different phase: 24 patients during induction therapy, 18 during hsct, 9 at diagnosis and during hsct and 8 after hsct. patients' characteristics are summarized in table 1 . median cd34+ cells dose was 6.4 × 10 6 /kg (range: 1.2-24). donor source was in 49 patients g-csf mobilized peripheral blood, 6 bone marrow and 4 cord blood. median neutrophil recovery (4500/mmc) was 16 days and platelet recovery (420 000/ mmc) was 14 days. sepsis were documented in 36 pts and 45 pts developed fuo. relapse was documented in 21 pts (35%). twenty-two pts are still alive and in complete remission (37%), death occurred in 37 pts: 19 due to trm and the remaining 18 for disease relapse. graft failure occurred in 16 of the 211 pts submitted to hsct. among the 11 patients (18%) who experienced graft failure, six (54%) received gt before hsct, because of sepsis during the induction therapy, and the remaining 5 after hsct, during aplasia period. in the remaining group (152 pts) not receiving gt, only 5 (3%) graft failure were observed. thus a statistically difference (p = 0.0005 fisher's exact test) increase in the rate of graft failure was detected in patients receiving gt. the role of gt in the treatment of infections in neutropenic patients remain still unclear for several reasons including the lack of clinical trials convincingly and consistently demonstrating efficacy, by availability of gt donors and by center's experience. gt has been successfully used in our center in patients with severe sepsis from mdr gram-bacteria during severe neutropenia but an increase number of graft failure has been registered in patients subsequently receiving hsct. alloimmunization to hla antigens in patients receiving gt might lead to an excess of graft failure requiring hla antibodies detection and attempt to reduce titer prior to hcst and maximizing stem cell dose. disclosure of conflict of interest: none. viridans streptococci are microorganisms frequently isolated from blood cultures of patients undergoing myeloablative allogeneic hematopoietic cell transplantation (allohct). poor dentition status has been associated with an increased risk of streptococcal bacteremia in the immediate post-allohct neutropenic period. the objective of this study was to evaluate the impact of oral health status on bacteremia risk in a cohort of patients undergoing therapy for acute myeloid leukemia (aml). a retrospective study was conducted in patients with aml treated at dana-farber/brigham and women's cancer center (df/bwcc) from 2007 to 2011. there was no formal dental assessment prior to aml induction therapy. all patients underwent protocol directed pre-allohct dental evaluation that included a standardized examination, comprehensive dental radiographs, and detailed treatment planning guidelines. poor oral health status was defined as presence of acute or chronic odontogenic infection, and it was assumed that oral health status at the time of induction therapy was the same as the pre-allohct evaluation findings. oral health status at the time of allohct was determined by the completion of required dental treatment. positive blood cultures were recorded from aml induction to day +60 post allohct. organisms that caused bacteremia were classified as 'of possible oral source' by a blinded microbiologist. two-sided fisher's exact test was used to compare the oral health status of the entire cohort to patients with blood cultures of potential oral source. from january 2007 to january 2011, 181 patients with aml underwent myeloablative allohct at df/bwcc and were s260 followed through today +60, and of these, 92 patients met the inclusion criteria and were included in the cohort. the median age was 48 years (range: 24-66) and there was similar distribution of genders. the most common aml induction regimen was daunorubicin and cytarabine (63/92; 68%) and of those that received consolidation therapy (49/92; 53%), almost all patients were treated with cytarabine. nearly all patients (90/92; 98%) received cyclophosphamide and total body irradiation for allohct conditioning and the majority of patients (83/92, 90%) received tacrolimus/methotrexate (n = 51) or tacrolimus/sirolimus (n = 32) for gvhd prophylaxis. over half of patients (51/92, 54%) experienced mucositis during their course of therapy for aml. pre-allohct dental evaluations were completed in 91/92 (99%) of patients. of the 13/91 (14%) patients identified as having poor oral health status, 13/13 (100%) completed all required dental treatment prior to allohct. bacteremias occurred in 63/92 (68%) patients, and 12/63 (19%) had positive blood cultures of potential oral source. of the 12 patients with positive blood cultures of potential oral source, 1/12 (8%) patient developed bacteremia during induction and 11/12 (92%) patients developed bacteremia during allohct. of the 13/91 (14%) patients identified as having poor oral health status, one patient (1/13; 8%) had a positive blood culture with a bacteria of potential oral source during induction/consolidation (p = 0.68). oral health status was not associated with risk of bacteremia of potential oral source at either aml induction/consolidation or allohct. risk of such bacteremia in the setting of myeloablative allohct may be related more to overall gastrointestinal translocation. disclosure of conflict of interest: none. is one of the main alternatives to trimethoprimsulfamethoxazole (tmp-smx) for prophylaxis of pneumocystis pneumonia (pcp)(maertens et al. jac 2016). ato is less effective than tmp-smx to prevent pcp1 but the reasons of this lower efficacy are not well understood. ato acts on pneumocystis, plasmodia and toxoplasma species by inhibiting mitochondrial pyrimidine biosynthesis. ato is highly lipophilic and its absorption in volunteers is improved by a fatty meal. there is a wide inter-individual variability in bioavailability and many drug interferences. the aim of this study was to assess the plasma concentrations of ato in patients under pcp prophylaxis with ato oral suspension and explore the factors which might impact its bioavailability. all adult patients receiving ato for pcp prophylaxis in the hematology and clinical immunology wards between may and september 2016 were included in the study. the prescribed dose was 750 mg of oral suspension twice a day. blood samples were collected around 12 h after the evening dose (cmin) and 1-5 h after the morning dose (cmax). plasma was immediately separated after each sample and frozen at − 20°c until proceeding to the assay. ato plasma levels were measured by uv-high-performance liquid chromatography. clinical and biological data, exact timing and modalities of intake (during a meal or not), and concomitant medications were collected. cmin and cmax results are presented as median (iqr 25-75%) and compared by mann-whitney u-test or signed rank test when appropriate. patients: a total of 85 measurements were performed in 33 patients (allogeneic hsct patients: 19; hematology non-transplanted patients: 6; hiv-infected patients: 7). the mean age (range) was 53 years (33-75), the m/f ratio was 21/12. only two patients were neutropenic. the median cmin was 11.3 μg/ml (6.2-27.8) and the median cmax was 13.4 μg/ml (6.0-28.3). thirteen of the 33 (39%) patients had a cmin. disclosure of conflict of interest: none. presepsin as a marker of infectious complications during high-dose chemotherapy following autologous hematopoietic stem cell transplantation in lymphoma patients y dubinina, v sarzhevskiy and v melnichenko national pirogov medical surgical center lymphoma patients, who undergo high-dose chemotherapy following autologous hematopoietic stem cell transplantation (autohsct), are at high risk of developing infectious complications (ic). mortality from ic during the transplantation, according to various data ranges from 12 to 42%. thus the development of models of early prognosis of ic during autohsct has become more urgent. it's reasonable to include the dynamics of biochemical markers of inflammation in these models. presepsin (psp), procalcitonin (pct) and c-reactive protein (c-rp) were assessed on the day of admission to the hospital (da), on d+1, d+3, d+7 and on the day of discharge (dd). if patients developed neutropenic fever (nf), the markers were assessed at the beginning of the fever, 6 h after, then on the second, third, fourth days after. there were 100 patients included in the study: 41 patients with hodgkin lymphoma, 27with non-hodgkin's lymphoma, 32with multiple myeloma, out of 100 patients there were 51 women and 49. the median age was 41 years (18-66). the conditioning regimens were cbv, beeac or hd melphalan. depending on the presence of ic, the patients were divided into 2 groups: group 1patients without infectious complications (n = 31), group 2patients with the development of infectious complications (n = 69). the median of the nf development was 5.5 days. 53 patients from group 2 had no microorganism growth in blood stream, either in repeated studies. gram+ flora was detected in 12 patients, 1 patient had gram-, 2 patients had mixed flora and 1 patient had pneumocystis jirovecii infection with respiratory insufficiency grade 3. significant differences in psp level between groups 1 and 2 were determined on d+3, on d+7 and the dd after autohsct. considering the median day of the nf appearance (5.5 days), it's supposed both the prognostic value (differences on d+3, that is, 2 days before the clinical manifestation of infection) and the diagnostic value of psp (differences on d+7 and on the dd) ( table 1 , graph 1). [p278] disclosure of conflict of interest: none. hc is often a serious complication and occurs in 70% of allo-hsct recipients. early bleeding is usually the result of chemotherapy toxicity however late occurring hc is multifactorial. bk virus infection has been shown to be related with hc. most studies demonstrate bk virus at the time of bleeding therefore not allowing the risk imposed by asymptomatic infection to be estimated. in this study, our aim is to show the effect of risk factors as well as pre-transplant bk viral load in asymptomatic recipients on development of hc in allo-hsct. between 2014 and 2016, we prospectively evaluated 59 allo-hsct. in order to detect the bk viral load, we performed quantitative bk virus pcr (altona diagnostics, germany) from blood samples at days 0, 30, 60 and 90 after allo-hsct. informed consents were obtained from all participants. bk virus pcr was considered positive if any number of copies were detected above the analytical sensitivity of the tests. the patients were monitored for signs and symptoms of hs. the risk factors for the development of hs were evaluated by univariate and multivariate analysis. p o0.05 was considered statistically significant. the median age of the group was 41 (range: 22-71), 18 of the patients (31%) were aged 450. male to female ratio was 1.36 (34/25). fifty two patients (88%) had diagnosis of malign hematological disease. stem cell source was peripheral blood in 51 (86%), bone marrow in 8 (14%) allo-hsct. patients received stem cells from 26 related donors (44%) vs 33 (56%) unrelated or haplo donors. myeloablative conditioning was administered in 47 patients (80%). forty-four of the conditioning regimens (75%) included cyclophosphamide. hc was diagnosed in 22 patients (37%) at a mean of 100 days (range: 0-367), early hc was detected in 4 of 22 patients (18%). the frequency of bk viremia and number of viral copies are given in detail in table. the frequency of bk viremia increases during transplantation in relation to clinical hc (66%, 66%, 87%, 100%; p = 0.007). acute graft vs host disease (agvhd) was diagnosed in 37 patients (63%) at a median time of posttransplant day 67: grade i-ii gastrointestinal/skin/liver in 31 (84%), grade iii-iv gastrointestinal/ skin/liver in 6 patients (16%). the most common gvhd prophylaxis preferred was cyclosporine and methotrexate in 50 patients (85%). in univariate and multivariate analysis (age450, sex, diagnosis, stem cell source, donor type, conditioning regimen, agvhd, cy administration, bk virus pcr at days 0, 30, 60) bk virus titer positivity at day 0, 30, 60 (p = 0.008, p o0.001, p o0.001), myeloablative conditioning (p = 0.018), the presence of agvhd after day 30 (p = 0.018) and conditioning regimen that includes cyclophosphamide (p = 0.024) are found to be related with increased risk of hs. patients with hc and clots were treated with continuous bladder irrigation as well as 8 of patients with bk viremia received cidofovir and six of them responded to treatment (75%). our study showed that, bk titer positivity, myeloablative conditioning, presence of agvhd, cyclophosphamide containing conditioning are associated with hc. detection of bk viremia in later transplant period is more sensitive for clinically proven hc. prophylactic treatment might be considered in patients with asymptomatic bk viremia in pretransplant period. [p279] disclosure of conflict of interest: none. this project has been granted by ankara university scientific research committee numbered as 15b0230007. high-dose chemotherapy with peripheral blood progenitor cell (pbpc) collection followed by a myeloablative conditioning and autologous stem cell transplantation (asct) is considered the standard of care of relapsed/refractory non hodgkin/hodgkin lymphoma (nhl/hl). a widely adopted conditioning regimen is the combination of carmustine etoposide cytarabine and melphalan (beam), whose feasibility and efficacy has been largely demonstrated. high dose fotemustine plus etoposide, cytarabine and melphalan (feam) has in some cases replaced beam conditioning. neutropenic enterocolitis (nec) is a life threatening complication of patients (pts) treated with chemotherapy (cht) with mortality rate up to 50%. it's a clinical syndrome in neutropenic patients (pts) characterized by abdominal pain (ap), fever (f) and diarrhoea (d). ultrasound (us) was used to evaluate bowel-wall thickening (bwt), and 44 mm is considered diagnostic of nec. early diagnosis is crucial to start conservative medical management (cmm), which appears the optimal strategy for most cases. objective: 1. to evaluate if nec incidence and outcome differs in beam vs feam and 2. to evaluate prospectively if bed-side-us (bus) can detect early signs of nec and guide a prompt treatment (cmm or surgical) in order to reduce mortality. in the last 5 years all pts with nhl/hl admitted in our bmt unit wards at university of pisa (italy), undergoing asct were prospectively enrolled. abdominal us was performed, baseline before treatment, and as only one symptom (or a combination) appeared within 12 h from onset: f and/or d and/or ap in cht-related neutropenic pts. 95 pts were conditioned with beam and 52 pts with feam. nec was diagnosed in n = 19/52 feam and in n = 25/95 beam patients. incidence was 36% and 25% respectively, without a statistically significant difference (p = 0.234). two pts died/19 in feam arm (10.5%) and 2pts/24 in beam arm (8.3%), without a statistically significant difference (p = 0.778). at time of diagnosis (dx) symptoms were: f+ap+d 45%, f+d 4%, f+ap 3%, ap+d 35%,d 3%,ap 10%. f alone was never present at diagnosis of nec. at dx, f was absent in 18/44 nec episodes (40%). all pts were treated promptly as bus allowed diagnosis with cmm except one 1 pts who underwent surgery, guided by us features, during neutropenia. the likelihood of nec dx in a discriminant st model (bayes theorem) for pts with bwt and ap = 98.8%, ap+d = 99.9%, ap+d+f = 100%, ap+f = 99.9%, d+f = 5%. bus allowed to detect early signs of nec and to start prompt treatment in this life threatening complication, of nhl/hl pts undergoing asct. this is a prospective study thus the true incidence of nec in nhl/hl undergoing asct should not be underestimated. there is not a statistically significant difference in incidence and outcome of nec in pts conditioned with beam in respect to feam. with bus pts do not live the isolation room. fever is not a condition sine qua non for nec diagnosis. early diagnosis allows most of pts to be treated with cmm. images of bus and ct were superimposable with lower costs, and less radiation exposure. a low mortality rate in pts with a 25-36% chance of developing this life threatening complication suggests that a prompt bus in neutropenic patients as just one symptom presents allows to make early diagnosis of this life threatening complication and guide prompt treatment (conservative or surgical), reducing mortality. disclosure of conflict of interest: none. quantiferon-cmv in the evaluation of cmv-specific immunity after autologous and allogeneic hsct j moreno 1 , ltesta 1 , l zanetti 1 , l serra 1 , b pereira 2 , m souza 1 , a carolina souza 2 , mp souza 1 , vr colturato 1 and cm machado 1,2 1 hsct program, amaral carvalho foundation and 2 virology laboratory, institute of tropical medicine, university of são paulo cytomegalovirus (cmv) is a major cause of morbidity and mortality after allogeneic hsct. the same is not observed in autologous hsct recipients who do not need to receive immunosuppression after transplantation. in the present study, we compared the reconstitution of cmv-specific immunity in autologous and allogeneic hsct recipients. patients were invited to participate in the study and signed the informed consent. cmv surveillance with the antigenemia (ag) test (cmv brite, biotest, germany) was done weekly in the first 3 months of transplant in allogeneic hsct recipients. preemptive ganciclovir therapy was initiated whenever a positive antigenemia was detected. the presence or absence of cmvimmunity was determined by a commercial interferon (inf) gamma release assay (quantiferon cmv, qiagen) before hsct and monthly thereafter up to d+90. from january to october 2016, 106 hsct recipients (29 auto and 77 allo) were included in the study. ag was positive in 54 (70%) of the allohsct recipients at a median of 39 (range: 14-146) days. ag recurrences occurred at a median of 81.5 (38-249) days, in 15 of the 54 pts (27.8%) who had at least one episode of positive ag. 103 hsct recipients were included in the analysis of qtf-cmv. in the pre-hsct sample, qtf-cmv was reactive in 60 of the 85 allohsct (70.6%) and in 25 of the 28 autohsct (89.3%). significantly less allo hsct recipients recovered cmvimmunity at day +30 (31.8%) and day+60 (59.3%) in comparison with autohsct (95% and 100%, respectively, p o0.01). up to day +90, all autohsct have recovered cmvimmunity, in comparison to 68% of the allohsct recipients (p = 0.096, figure 1 ). the qtf-cmv test performed at d+30, d +60 and d+90 did not predict the risk of cmv reactivation in the following month. similarly, the test did not anticipate the risk of ag recurrences: 80% of the hsct recipients with undetermined or non-reactive qtf-cmv test at d+60 had ag recurrence after this period, in comparison with 70% of the patients with a reactive result (p = 0.56). in the present study, the qtf-cmv test alone could not predict the risk of cmv reactivation or recurrences. [p281] disclosure of conflict of interest: qiagen. recovery of vδ2+ γδ t cells is critical to epstein-barr virus reactivation after haploidentical hematopoietic stem cell transplantation j liu, z bian, q fu, l xu, x zhang, y wang and x-j huang peking university people's hospital, peking university institute of hematology, beijing, china epstein-barr virus (ebv) reactivation and its related disease are life-threatening complications in patients undergone haploidentical hematopoietic stem cell transplantation (haplohsct). our previous studies found that impaired cd4 − cd8 − t-cell recovery correlated to the increased occurrence of ebv infection after haplohsct. γδt cells make up 50-90% of cd4 − cd8 − t cells in the peripheral blood of healthy donors. expansion of vδ1+ γδt t cells after hsct has been reported and this subset could respond against autologous ebv-lcl in vitro. selective activation and expansion of vγ9vδ2-t cell could inhibit ebv-lpd development in humanized mice. however, the association of γδ t-cell recovery with ebv reactivation after allohsct remains unknown. this is a prospective cohort study including 110 consecutive patients who were diagnosed as hematological malignancy and underwent haplohsct. recovery of t lymphocyte and a panel of subsets, including cd3+, cd4+, cd8+, cd4-cd8-, tcrαβ+, tcrγδ+, vδ1+, and vδ2+ t cells, were determined by flowcytometry at 30, 60, 90, 180 days after haplohsct. all recipients and donors were tested negative for ebv dna in the peripheral blood before transplantation. recipients were monitored weekly for ebv dna load until day 100 after transplantation. recipients with peripheral blood plasma ebv dna load 41000 copies/ml at least on two consecutive occasions were diagnosed as ebv reactivation (ebv +). ebv − cohort generally represents patients whose ebv dna loado1000 copies/ml in peripheral blood. within 100 days after haplohsct, 17 of 110 (15.5%) recipients were diagnosed as ebv reactivation. compared to recipients with negative ebv dna load, the counts of cd3+, cd8+, and tcrαβ+ t cells were not statistically different in the ebv+ cohort from 30 to 180 days after haplohsct. in contrast, recoveries of cd4+ and cd4-cd8-t cells in ebv+ patients were significantly hampered at 30 days after transplantation (p = 0.021 and p = 0.046, respectively). although the tcrγδ+ t-cell counts were also decreased at 30 and 60 days in the ebv+ cohort, the comparisons did not reach the statistical significance (p = 0.072 and p = 0.082, respectively). notably, recoveries of vδ2+ γδ t cells at 30, 60 and 90 days were continuously delayed in recipients with ebv reactivation (p = 0.029, p = 0.001 and p = 0.046, respectively). whereas the counts of vδ1+ γδ t cells were similar between the two groups from 30 to 180 days in this context. in this prospective and large cohort study, we showed that the occurrence of epstein-barr virus (ebv) reactivation was associated with the hampered recovery of vδ2+ rather than vδ1+ γδ t cells after haplohsct. our findings will help explore γδt subset-dependent therapeutic strategies to control the serious complications due to ebv infection post transplantation and improve the overall survival of haplohsct recipients. disclosure of conflict of interest: none. in particular, bloodstream infection (bsi)is a frequent complication in the pre-engraftment phase with an impact on the morbidity and mortality of these patients. objectives: to analyze the incidence of bsi in patients undergoing hsct in our center, and to identify predisposing factors for the development of bsi in pre-engraftment phase patients after hsct. fifty-one consecutive patients undergoing hsct were analyzed retrospectively in our center during the period of 1 july 2015 and 30 june 2016. the characteristics of the sample are shown in table 1 . we have reported all the bsi between day 0 and day 30 after stem cell infusion. 70.5% (36 patients) received antibacterial prophylaxis with ciprofloxacin, five patients with broad spectrum antibiotics and five did not received any drug. the average days of fever have been 3.30 days (0-12 days). a total of 184 blood cultures has been collected (3.6 per patient). there have been 15 bsi (21.5% of the patients) with 8 (53.4 %) of cases caused by gram-negative organism (4 escherichia coli, klebsiella pneumoniae, acinetobacter baumanii, proteus vulgaris and delftia acidovorans) and 7 (46.6%) by gram-positives (1 enterococcus faecium, 1 enterococcus faecalis, 3 staphylococcus epidermidis, streptococcus mitis and streptococcus viridians group). one patient presented 3 different episodes of bsi, two patients 2 independent episodes and the rest eight, only one microorganism isolated. we have identified two bsi by extended-spectrum betalactamases (esbl-producing organism) and one isolation of carbapenem-resistant gram-negative bacteria. the rate of quinolone-resistant is 80% in all the sample. in univariate analysis, several factors like presence of comorbidities, presence of severe mucosits, type of catheter and antibacterial prophylaxis modality don't increased the risk to develop bsi (p40.05). the place where the procedure is performed does not influence the development of bsi. although the presence of previous infections is not a risk factor, hospitalization for infection in the 90 days before hsct does influence the development of bsi with statistical significance (po0.05). the crude mortality rate of the sample has been very low (2%), with only one death related to bloodstream infection. bsi are a common relative complication in the patient undergoing hsct but with an extremely low mortality in our sample. hospitalization for infection in the 90 days before hsct does influence the development of bsi. it is important to note that outpatient model and conventional rooms don't increased the incidence of bsi. although the use of quinolones in prophylaxis does not result in an increase in infections caused by multiresistant micro-organisms (esbl and carbapenemias) with acceptable resistance rates (80%), it also does not reduce the incidence of bsi in our sample. according to our analysis, his routine employment still throws light and shadows. [p283] disclosure of conflict of interest: none. septic episodes with multiple bacterial strains during antithymocyte globulin (atg) therapy for conditioning for allogeneic stem cell transplantation under rifaximin gut decontamination d markel 1 , c schultze-florey 1 , t brockmeyer 1 , v panagiota 1 , c lück 1 , a schwarzer 1 , m beck 1 , e dammann 1 , a ganser 1 , g beutel and m eder 1 recent evidence demonstrates the importance of the enteric microbiome for the development of gastrointestinal graftversus-host disease (gvhd) and mortality after allogeneic stem cell transplantation (sct) (1, 2) . accordingly, the usage of the non-absorbed rifamycin derivate rifaximin for gut decontamination has been reported to preserve the intestinal microbiota composition with a positive effect on overall survival in a single centre retrospective analysis (3). we here report severe septicaemia requiring therapy at the intensive care unit (icu) during atg application for conditioning in three patients with rifaximin used as single agent for gut decontamination within 6 months. after changing our gut decontamination from a chinolon-metronidazole regimen to rifaximin, three cases of severe septicaemia by gram-negative and gram-positive bacteria during atg treatment occurred within 6 months. patient #1 was a 52-year-old woman with tmds/aml after breast cancer conditioned according to the flamsa-bu protocol. the second (#2) and third (#3) patient were 55and 37-year-old males with a complex karyotype secondary aml after omf and relapsed inv(16) aml with meningeosis leucaemica, respectively. patients #2 and #3 were treated with flamsa-bu and flamsa-tbi, respectively. all patients received rabbit atg (atg fresenius/grafalon) at a dose of 3 × 10 mg/kg body weight and rifaximin (2 × 200 mg) for gut decontamination. patient #1 developed severe escherichia coli and pseudomonas aeruginosa septicaemia on day − 3 of the conditioning regimen and had to be transferred to the icu with septic cardiomyopathy for therapy with vasopressants and levosimendan. in patient #2 escherichia coli, klebsiella oxytoca, staphylococcus hemolyticus and staphylococcus epidermidis were simultaneously detected in blood cultures at day − 2. the patient was transferred to the icu and treated with vasopressants for septic shock. patient #3 developed septic shock due to klebsiella pneumoniae and enterobacter cloacae on day − 4 under atg therapy. mechanical ventilation and vasopressor therapy were required. fortunately, all three patients survived and completely recovered without any sepsis related disabilities under escalated anti-infective and intensive care therapy. all were discharged from the hospital in the outpatient clinics. interestingly, all isolated gram-negative pathogens were found to be sensible for a chinolon based gut decontamination. the reasons for these septic complications under atg therapy are not exactly understood but raise a note of caution on the use of rifaximin as single agent gut decontaminant during atg application in conditioning for allogeneic sct. infections with mycobacterium genavense were described for the first time in 1990. since then, several cases have been reported, but almost exclusively in patients with aids. most patients who underwent hsct have insufficient cellular immunity. here we report a mycobacterium genavense infection in a patient mimicking a lymphoma-relapse after hsct. a 58year-old female patient was diagnosed in july 2013 with stage ivb alk-negative anaplastic t-cell-lymphoma with cervical, retro-/supraclavicular, mediastinal, axillary and retroperitoneal lymphadenopathy as well as pulmonary manifestation. two chemotherapy treatment lines and autologous stem cell transplantation resulted in a partial remission. to improve remission prior to hsct the patient received 2 courses of brentuximab-vedotin. after conditioning therapy with fludarabine, busulfan, cyclophosphamide and atg, hsct from a hla compatible unrelated donor was performed in april 2014. a pet-ct-scan in november confirmed complete remission. after hsct the patient remained lymphocytopenic with cell count of cd4+ cellso100/μl. after acute stage iii gastrointestinal graft-versus-host disease (gvhd) low dose immunosuppressive therapy was maintained due to mild chronic gvhd of the liver and the upper gastrointestinal tract. beginning in june 2015 the patient experienced increasing fatigue, general weakness, loss of appetite, nausea, night sweating and fever. abdominal ultrasound, urine and blood culture as well as ct scans revealed no focus of infection. different lines of empirical antibiotic therapy resulted only in short term improvement. several blood culture tests remained sterile. a fdg-pet-ct scan showed a paraaortal and parailiacal lymphadenopathy with a high fdg uptake (suv between 19.7 and 20.1), highly suggestive of lymphoma relapse. endoscopic evaluations revealed two polypoid lesions in the bulbus duodeni. histology of duodenal biopsies revealed a massive accumulation of weakly pas-positive bacilli. pcr analysis confirmed an infection with mycobacterium genavense. despite several attempts mycobacteria were not recoverable on solid media even by long term culture. treatment was started with rifampicin, ethambutol, ciprofloxacin and clarithromycin. lymph node manifestation responded to therapy with decreasing fdg-uptake (suv 8.2) in a control fdg-pet-ct scan 3 months later. after 9 months treatment was terminated due to therapy refractory nausea. lymphocytopenia was persisting with cd4+ cellso100/μl. six weeks after stopping the antibiotic therapy, symptoms as fever and weakness reappeared. duodenal biopsy could not confirm persistent mycobacterial infection. fdg-positive intraabdominal lymph nodes (suv 11.2) and spleen (suv 8.2) were detected in a control fdg-pet-ct-scan. five lymphnodes were surgically removed. immunohistology detected histiocytic cell proliferation with no sign of lymphoma relapse. pcr confirmed the presence of mycobacteria-dna. consequently, antibiotic treatment was resumed. mycobacterium genavense can present with all the symptoms of a lymphoma relapse and should be considered in immune compromised patients. reliable diagnosis can only be obtained from lymph node biopsies and/or endoscopic evaluation. treatment has to be accompanied by restoring cellular immunity and should only be stopped after pcr-negative biopsies. disclosure of conflict of interest: none. stratification of patients with multiple myeloma and lymphoma undergoing autologous hematopoietic stem cell transplantation in term of antifungal prophylaxis r moghnieh, s khaldieh, l awad, d abdallah, n droubi, a youssef, a mougharbel, t jisr and a ibrahiim makassed university hospital, beirut, lebanon autologous hematopoietic stem cell transplantation (ahsct) is at intermediate risk for invasive fungal infections (ifi). the recommendations of international scientific societies are not homologous regarding prophylaxis against ifi in patients (pts) undergoing ahsct. the primary end point was to assess risk factors for the need of empiric/preemptive antifungal therapy in ahsct recipients, and to extrapolate to the subgroup of pts that requires antifungal prophylaxis in our population of ahsct pts. the secondary endpoint was to determine the fungal species distribution infecting or colonizing the pts. our study included adult pts (418 yo) who underwent ahsct for lymphoma and multiple myeloma (mm) between 2005 and 2015. all febrile neutropenic pts are being managed according to the 2010 infectious diseases society of america (idsa) guidelines regarding the use of antimicrobial agents in neutropenic pts with cancer. eligible pts were divided into two groups: those who received empirical antifungal therapy and those who did not need it. we recorded demographic and baseline clinical characteristics including: age, gender, comorbidities, stage, disease status at ahsct, high-dose therapy regimen, the presence of mucositis and its grade, the number of cd34 + cells transfused, the presence of central line or portacath, the need for mechanical ventilation, the presence of diarrhea, the duration of neutropenia, and the presence of bloodstream infections. pts who had lung infiltrates suggestive of ifi were analyzed separately. the causative fungal pathogens and colonizers were analyzed. univariate and multivariate analysis of potential risk factors to assess further significance was performed using spss. 190 patients were included.106 pts (56%) had lymphoma and 84 pts (44%) had mm. the need of empiric antifungal therapy was statistically more significant in lymphoma than mm pts (po0.01).the presence of mucositis grade ⩾ 3 showed a statistical significance for the need of antifungal therapy (p = 0.02). in the lymphoma group, remission status (pr vs cr) was not a significant factor for the need of empiric antifungal therapy (p = 0.49).the presence of mucositis grade ⩾ 3 was at the limit of significance ( p = 0.05). in the mm group, remission status (pr vs cr) did not affect the need of empiric antifungal therapy (p = 1). however, mucositis grade ⩾ 3 was found to be a significant risk factor for the need of empiric antifungal therapy (p = 0.02). following factors: the number of cd34 + cells transfused, the presence of central line and portacath, the need for mechanical ventilation, the presence of diarrhea, the duration of neutropenia, and bloodstream infections did not show any significance for the need of antifungal prophylaxis in both groups. all recovered fungal isolates (n = 14) were not from deep seated tissues biopsies or blood, and were identified as candida albicans in 7 with lymphoma, and in 7 with mm. they reflected the candida ecology in this pts series rather than deep seated fungal infections. we suggest to give antifungal prophylaxis to all lymphoma pts because of the higher need of empirical antifungal therapy, and give antifungal prophylaxis to mm pts having a predisposition for severe mucositis. fluconazole is the antifungal of choice for prophylaxis since all the fungal isolates were candida albicans. keywords: autologous hematopoietic stem cell transplantation, antifungal prophylaxis. disclosure of conflict of interest: none. a 48-year-old previously fit woman from a rural area of eastern europe was admitted to the hospital for severe aplastic anemia. steroids, csa, antinfective prophylaxis and supportive therapy were administered without response; therefore rabbit atg was then administered, with minor response; the year later, she underwent allogeneic-hsct (mud 9/10, ric: tbi, cyclophosphamide and fludarabine; gvhd prophylaxis: atg, csa, mtx). several days after transplantation she developed left migraine with ipsilateral back-eye pain. brain mri and ct showed a diffuse opacification of paranasal sinuses, mainly in the sphenoid sinus. the symptoms gradually improved with a specific treatment. the patient achieved a quick and complete haematological recovery and she was discharged. at follow-up visits she complained a flare of the migraine, with a left-sided headache that did not improve with nsaids. the headache gradually intensified until vision in the left eye became blurred with conjunctival injection. after consultation with ophthalmologist, for suspected toxoplasma retinitis, administration of intravitreal steroids and clindamicine was begun with partial benefit. however 15 days after (d +90) she was admitted in hospital because of worsening headache, irradiated in the occipital area, and weakness in the right hemibody. tests on csf were negative for neurotropic pathogens. an mri showed a complete occlusion of the intracranial tract of left internal carotid artery, with likely infectious material localized in the left lateral cerebral fissure. a chest tc showed a nodule with initial excavation in the right superior pulmonary lobe. for suspected tuberculosis she started antitubercular therapy. despite a second lumbar puncture confirmed pleocytosis compatible with acute purulent meningitis, microbiological research for bacteria, fungi and bk were negative. so antitubercular and antitoxoplasma therapy were stopped and the patient underwent surgical biopsy within the sphenoid sinus. pathological examination of the biopsy specimens showed acute and chronic inflammation of the respiratory mucosa, periodic acid. schiff and grocott staining ( figure 1 ) highlighted several septate fungal hyphae. cultural analysis revealed colonies of scedosporium apiospermum so the patient started targeted voriconazole intravenous therapy. nevertheless, 10 days later, she developed aphasia and right hemiparesis. a brain angio-mri confirmed the appearance of new lesions compatible with infectious localizations associated to an increased defect of left internal carotid artery vascularisation and complete left choroid detachment. after 2 weeks of voriconazole a significant clinical improvement have been observed and she was discharged, continuing oral antifungal therapy with voriconazole. at the last follow-up she achieved a complete resolution of neurologic symptoms, with permanent left eye blindness. 8 months later (d +370) she was asymptomatic, with normal haematological and neurological conditions and was able to stop the antifungal therapy. this case-report confirms that the risk of invasive fungal infection (ifi) is relevant in patients receiving hsct for aa, probably due to the prolonged neutropenia and association of other risk factors such as the immunosuppressive therapy and the iron overload. in this very poor prognosis infection, the early diagnosis of cns ifi remains challenging, but the administration of voriconazole was extremely effective. disclosure of conflict of interest: none. in this study, we aim to present the seroprevalence of ebv and incidence of posttranplant lymphoproliferative disease as well as to evaluate the relation with gvhd. between 2006 and 2015, the ebv serology of 364 patients that underwent allogeneic hematopoietic stem cell transplantation and their donors were evaluated in the study. ebv ig g (vca-igg, ebna ig g, ea-igg) and igm (vca-igm) antibodies were detected by chemolluminesance method (abbott, abd). all patients were followed for reactivation. ebv igg seropositivity was detected in 338 patients (93%) and 238 donors (77.7%). there was no statistically difference in related vs unrelated transplants in seropositivity. the median age of the patients was 37 (range: , 217 patients were male (60%) and 295 (81%) had malign disease. the stem cell source was peripheral blood in 299 (82%) patients and 258 (71%) received grafts from related donors. myeloablative conditioning regimen was received by 273 of patients (75%) (table) . all patients received acyclovir prophylaxis (related transplants 400mg tid, unrelated transplants 800 mg tid) during and after allo-hsct up to 3 months. twenty six-yearold pretransplant ebv seropositive aplastic anemia patient had ebv ig m positivity after 3 months of allo-hsct and developed lymphoproliferative disease. he was in complete remission after 4 courses of rituximab and methylprednisolone. three patients were ebv igm seropositive in 4th, 9th and 24th months of allo-hsct and received symptomatic treatment. acute gvhd was detected in 223 patients (61%) whereas 285 patients (78%) had chronic gvhd. acute gvhd and chronic gvhd incidences were similar in comparison of donor ebv seropositive vs seronegative status (78% vs 22%, p = 0.72; 80% vs 20%, p = 0.199). ebv seropositivity was detected in 92.8% of patients. the donor ebv serology was not related with acute or chronic gvhd. [p290] disclosure of conflict of interest: none. the umc utrecht pediatric experience with brincidofovir after allo hsct ca lindemans, m bierings and jj boelens pediatric blood and marrow program, dept. of pediatrics, university medical center utrecht, the netherlands viral reactivation with dna viruses form a considerable complication of allogeneic hematopoietic stem cell transplantation (hsct). there are little effective antiviral therapies and most have considerable toxicity. especially for adenovirus, there is no satisfactory therapeutic option. recently a new oral antiviral agent, the cidofovir prodrug brincidofovir became available to european patients only on the basis of urgent medical need and after a case by case approval by the health authorities. the aim was to describe our single center experience with brincidofovir in the pediatric allogeneic hsct setting. in the umc utrecht, pediatric patients receive t-replete bone marrow or unrelated cord blood (ucb) as the donor source after mostly myeloablative conditioning regimens (+ serotherapy in unrelated-hct). as gvhd prophylaxis patients receive cyclosporine a (csa) and mtx for bone marrow, csa and prednisone for ucb. patients are by standard weekly monitored for the presence of adenovirus, ebv, cmv en hhv6 viremia by rt pcrs in the plasma. extensive immune reconstitution measurements are performed every 2 weeks. since 2015, patients that developed viral reactivation with adenovirus, or a combination of other dna viruses (cmv, bk or hhv6) were offered brincidofovir if the viremia was progressive or in the context of poor immune reconstitution. brincidofovir was given in suspension (10 mg/ml) at the dose of 2 mg/kg biw, or 100 mg biw for larger children. de drug was discontinued when the viral load was below detection level. in total, six pediatric patients (age range: 0-18) received brincidofovir (2 patients tablets, 4 the suspension). four received it for adenovirus reactivation, a 5th patient for cmv and bk and a 6th patient for cmv en hhv6. the median day post-hsct of the first administration was 29 days post hsct (range: − 4 to 101), the median day post detection of viral reactivation 14 days . the median duration of administration was 36 days (10-98) with two patients being discontinued because of death. in no patient the drug was discontinued due to toxicity issues. the patients that died had multi-organ failure due to a combination of severe agvhd and multiple infectious issues. the patients were discontinued when the viral load was low and when they had cd4 counts of at least 50/μl. none of the four alive patients reactivated after the drug was discontinued. urgent medical need administration of brincidofovir is feasible. in our limited series we found the drug was well tolerated. disclosure of conflict of interest: i am a medical consultant for brincidofovir (chimerix). reactivation of herpes simplex virus 1 (hsv-1) or varicellazoster virus (vzv) occurs frequently after allogeneic stem cell transplantation (asct). here, we report three unusual cases, two with reactivation of hsv-1 and one with vzv. patients and methods: patient (pt) 1 (50-year-old, male) was allografted for high risk acute lymphoblastic leukemia in first complete remission after conditioning with total body irradiation (12 gy) and etoposide (60 mg/kg). graft-versus-host disease (gvhd) prophylaxis was performed using cyclosporine a, short course methotrexate and anti t-lymphocyte globulin (atg). pts 2 (44year-old, female) and 3 (67-year-old, male) were allografted for acute myeloid leukemia in second and first complete remission, respectively. conditioning regimens used were flamsa-ric in pt 2 and fludarabine/busulfan in pt 3. in both cases, gvhd prophylaxis consisted of cyclosporine a, mycophenolate mofetil, and atg. pts 1 and 2 had already experienced hsv-1-positive oral mucositis following induction chemotherapy and had successfully been treated with acyclovir. both developed hsv-1-positive oral mucositis again after asct. in both cases, initial therapy with acyclovir i.v. at a dose of up to 10 mg/kg t.i.d. was ineffective. to explore the mechanism leading to clinical acyclovir resistance, the thymidine kinase genes of both viral strains were sequenced. pt 3 presented with severe abdominal pain and nausea 11 months after asct. in this case, acyclovir prophylaxis post asct had been stopped 2 months before due to side effects. moreover, low dose prednisolone therapy was necessary for chronic gvhd. the hsv-1-strain from pt 1 showed a single base pair deletion in the region from nucleotide position 430 to 436 of the thymidine kinase gene (which consists of a guanosine repeat). in pt 2 a single base pair insertion in the same region was found. both genetic alterations lead to a loss of enzyme activity and acyclovir resistance. in both pts treatment was changed to foscarnet which led to rapid improvement. in the case of pt 3, multiple mucosal erosions were found on endoscopy of the esophagus. in these vzv dna was detected by polymerase chain reaction (pcr). only 4 days later, a vesicular skin eruption developed, which did not follow a dermatomal distribution. again, in the vesicular fluid vzv dna was detected by pcr. in this patient, acyclovir (10 mg/kg i.v., t.i.d.) resulted in rapid improvement. reactivation of hsv-1 and vzv after asct is a frequent finding. usually, hsv-1 strains respond well to acyclovir. in some cases, resistance can develop, especially in patients that had been treated with acyclovir before. acyclovir resistance of hsv-1 caused by mutations in the thymidine kinase gene can be overcome by treatment with foscarnet which directly inhibits the viral dna polymerase. disseminated vzv reactivations after asct have been described. clinical presentation can be misleading, for example, beginning with severe abdominal pain that precedes the vesicular eruption by several days. disclosure of conflict of interest: none. toxoplasmosis is a rare but severe complication after hematopoietic stem cell transplantation (hsct) (1) . it can involve the central nervous system alone or can manifest as a disseminated disease. in the paediatric population the mortality rate is high and sequelae are often severe. new diagnostic tools, such as the pcr assay, may allow for rapid diagnosis and preemptive therapy (2, 3) . we retrospectively analysed all children who underwent allogeneic hsct in our centre between january 2011 and december 2015. patients lost to follow up before day +100 were excluded. patients and donors were tested before transplant in order to assess their immunological status against t. gondii. a total of 187 allo-hsct were analysed. before transplant, 28.8% of recipients (r) were toxo-igg positive and 71.2% were toxo-igg negative. among donors (d), serology was available only for 152/187: 23% were toxo-igg positive, 77% were toxo-igg negative. we found a high number of not tested donors (18.7%, 35/187) which included, in most cases, mud from foreign registries. the group at higher risk for toxoplasmosis, d − /r+, included 21.7% pairs, whereas d − /r − were 55.2%, d+/r-were 15.1% and d +/r+ were 7.9%. in our series the cumulative incidence of toxoplasmosis disease was 2.1%, with 4 cases out of 187 transplants. two of them (case 1 and 3) had cerebral toxoplasmosis, one (case 2) had disseminated toxoplasmosis and case 4 had toxoplasmic chorioretinitis. mortality rate was 50%: two patients died because of multiorgan failure and disseminated toxoplasmosis respectively. in no case localized cerebral toxoplasmosis was the main cause of death. no complications were seen in surviving patients. all patients who developed toxoplasmosis were toxo-igg positive before hsct and three of them were transplanted from a toxoplasma igg negative donor (fourth donor not tested). in the two fatal cases the interferon-gamma releasing assay (igra) never became positive, confirming the absence of specific cellular immunity. toxoplasmosis disease can affect hsct outcome in paediatric recipients and pre-hsct seropositivity is the most important risk factor for toxoplasma disease in the post transplant period. in our cohort seroprevalence was higher than expected, probably due to the high number of patients coming from eastern europe. in order to reduce the burden of toxoplasmosis disease in our population we decided to implement a real-time pcr screening protocol for d − /r+ pairs, to provide rapid diagnosis and early therapy. all positive recipients with a seronegative donor will undergo real-time pcr screening starting on the day of stem cells infusion, and regularly until cd4+ t cell recovery. in the future we will analyse the impact of this strategy in this particular subset of immunocompromised patients. treatment with brincidofovir for adenovirus disease in pediatric hematopoietic transplants introduction adenovirus may cause serious morbidity and mortality after allogeneic hematopoietic transplants in children. severe lymphopenia is the main risk factor associated with progression to disseminated and often fatal disease. treatment with unlicensed cidofovir is based on monitoring of plasma viral load by pcr. however, cidofovir is only moderately effective at controlling adenovirus and it is associated with significant renal toxicity. brincidofovir is a lipid conjugate of cidofovir. it has a good oral bioavailability and achieves higher intracellular levels of active drug than cidofovir with a better safety profile. it is a potent inhibitor of viral dna synthesis so it could be indicated in immunocompromised patients with adenovirus disease. patients and methods we present three children of 3, 5 and 9 years old diagnosed of acute lymphoblastic leukemia (all) in 2nd complete remission (the first two patients) and severe aplastic anemia the last one. there were 2 girls and 1 boy. they underwent a peripheral blood hematopoietic stem cell transplantation using αβ/cd19 depletion with a haploidentical donor in the two patients with all and cd45ra depletion with a matched unrelated donor in the other patient. patients that underwent haploidentical transplants developed early acute graft versus host disease grade iii with gut and skin involvement so immunosuppressive treatment with corticoids was started. they developed severe lymphopenia ( o300/ mm 3 ). in the first month after transplant an adenovirus disease was diagnosed in the three patients from the weekly monitoring of plasma viral load by pcr. adenovirus was also tested in stools, urine and respiratory sample. in all patients adenovirus was also detected in urine sample. in one of them adenovirus was detected in nasal exudate too and in the other the virus was isolated in stools and in a skin biopsy. results: all of them were initially treated with cidofovir with poor results. foscarnet and gancyclovir was also used without improvement. finally they started a treatment by compassionate use with oral brincidofovir twice a week. with the first dose of brincidofovir plasma viral load started to go down until its complete disappearance. brincidofovir tolerance was good with only mild and limited diarrhea in two cases in the day they were taking brincidofovir. two of the three patients were alive without signs of adenovirus disease. in the other patient blood adenovirus load by pcr decreased below 1000/ml, but remain high in urine. she died of respiratory failure due to pulmonary graft versus host disease. conclusion brincidofovir may be a promising therapeutic option for the treatment of severe adenovirus disease in immunocompromised patients with a good toxicity profile. disclosure of conflict of interest: none. table 1 . all patients were transplanted with pbsc for haematological malignancy, and s269 received reduced intensity conditioning (ric) regimens with in vivo t-cell depletion. the proportion of patients with baseline and post-vaccination hi titres ⩾ 1:40 were 28.6 and 25% for a(h1n1)pdm09, 14.3% at both time points for a (h3n2), and 32.1 and 25% for b/phuket. pre and postvaccination geometric mean titres gmt) were higher by mn than hi for a(h1n1)pdm09 and a(h3n2), but lower for b/ phuket (p = 0.05). no post-vaccination seroconversions were detected by hi, while a single seroconversion to a(h1n1) pdm09 was detected by mn in a patient vaccinated at 0-3 months. the mn assay did not detect any additional low-titre seroresponses (negative to detectable titre) below hi threshold. none of patient age, lymphocyte count, days from transplant to vaccination, donor type, and gvhd or ist at vaccination correlated with baseline or post-vaccination titres by either assay. response to iiv was virtually absent throughout the first year post-hsct, with a single seroconversion to a(h1n1)pdm09 detected by mn but not hi, although the sample size was small and half of patients were vaccinated at 0-3 months. there is a clear need for a novel, immunogenic seasonal iiv and/or novel vaccination regimens in this population. vaccination of recipients' relatives and close contacts, and hsct healthcare workers should be strongly encouraged. pre-and post-transplant iron overload (io) has been associated with considerable long-term morbidity and mortality in pts undergoing transplantation. classically, management of io in the post-allo-hsct setting has been based in the performance of therapeutic phlebotomies (tp), which are inconvenient for the patient and are often not feasible due to ongoing anemia. we recently published the first prospective study of deferasirox in adult allo-hsct pts with io (vallejo, et al. haematologica 2014). in this retrospective analysis, we analyzed the real-life management of io in the post-allotransplant setting. this study includes the last 113 pts with a minimum follow-up of 6 weeks, who underwent allo-hsct in our center (october 2014-october 2016). 63 pts were male (55.8%) and 44 female (44.2%). median age was 53 years (range: 7-69). baseline diseases were: aml (44.2%), lymphoproliferative disorders (16.8%), mds (12.4%), all (8.8%), chronic myeloproliferative diseases (7.1%), mm (5.3%), and bm failures (5.3%). donor was unrelated in 61 cases (54%; 14 of them hla mismatched), and related in 52 (46%; 21 of them haplo-identical). conditioning regimen was: busulphan-based (68.1%), melphalan-based (13.3%), tbi-based (7.1%), and others (11.5%). progenitors source was pb in 102 (90.3%), and bm in 12 (9.7%). pre-hsct: pts had been transfused with a median of 23 prbc (range: 0-147), and their median serum ferritin (sf) was 1359 ng/ml (range: 22-5116). day +180 post-hsct: 15 pts had died, and 24 pts had not reached that day yet, so 74 pts were evaluable. they had been transfused with a median of 31 prbc (range: 0-157), and their median serum ferritin (sf) was 1127 ng/ml (range: 56-7993). 55% pts had sf superior to 1000 ng/ml. liver mri (by sir method) to assess liver iron concentration (lic) was performed in 44 pts at day +180. seven pts (15.9%) had no io (lic 0-2 mg/g), 12 pts (27.3%) had moderate io (lic 2.1-4.4 mg/g), and 25 pts (56.8%) had severe io (lic superior to 4.5 mg/g). median lic was 4.66 mg/g (range: 0.6-11.34). among the 29 cases with history of more than 20 prbc transfused and sf higher than 1000 ng/ml at day +180, 28 (96.6%) were proved to have liver io by mri; the other pt had io in spleen. 30 pts started some kind of therapy to treat the io: 6 pts with severe io initiated a tp program and 24 pts (6 out of 12 with moderate io, and 18 out of 25 with severe io) initiated chelation therapy with deferasirox. the drug was started at low dose (2.5-5 mg/kg/ day), and was increased if tolerated up to a maximum of 20 mg/kg/day. of note, the majority of pts were also taken a number of medications (immunosuppressants, statins, antimicrobials, etc). 3 of those 24 pts (12.5%) did not tolerate the drug, and were changed to tp. for more details, see the table. (1) the combination of the history of prbc transfusions and serum ferritin levels was, in the majority of cases, enough to assess the io in the post-allo-hsct setting. (2) liver mri (by sir method) helped to assess io in doubtful cases. (3) deferasirox, initiated at low doses and increased if tolerated, was safe and its use helped to avoid the need of therapeutic phlebotomies for the majority of patients. this study reproduces, in a real-life setting, our previous findings in a prospective clinical assay. [p297] disclosure of conflict of interest: none. a case-control study of risk factors of primary graft failure with a focus on associated early-onset severe infections v alcazer 1 , a conrad 2 , f-e nicolini 1 , s ducastelle-lepretre 1 , f barraco 1 , x thomas 1 graft failure (gf) is a rare but devastating event after allogeneic haematopoietic stem cell transplantation (ahsct), exposing the recipient to disease relapse, drawbacks of marrow aplasia, infections and death. the aim of this study was to analyse the risk factors associated with graft failure after ahsct, with a specific focus on early-onset severe infections (esi). we conducted a retrospective, observational, single-centre, matched case-control (1:2) study among adult s270 ahsct recipients transplanted at the haematology department of our institution between 2008 and 2015, with a subsequent follow-up of 12 months. engraftment was assessed at day+42 post-ahsct. gf cases were classified as primary gf (pgf), defined as failure to achieve donor-derived absolute neutrophil count (anc) ⩾ 0.5 × 109/l or lasting more than 3 consecutive days without evidence of disease relapse and early-secondary gf (esgf), referring to the loss by day 42 post-ahsct of a previously functioning graft associated without evidence of disease relapse. each case was matched with two controls according to underlying haematological disease, hla matching, stem cell source, intensity of conditioning and temporal proximity of ahsct. demographics, haematological and graft characteristics as well as esi report were retrieved. esi were classified in invasive fungal infections, viral infections (cmv, ebv, hhv-6, other viruses), toxoplasmosis and severe sepsis of bacterial origin. during the study period, 598 ahsct were performed at our center. seventeen (3.1%) gf cases were identified, of which 15 pgf and 2 esgf, and were matched with 34 controls. in the descriptive analysis, gf and control populations did not significantly differ when considering demographics, haematological characteristics and hematopoietic stem cell source. regarding pretransplantation status and graft characteristics, only disease status (progressive disease) and cell dose (both cd34+ and cd3+ cells number/ kg) were associated with graft failure. the proportion of patients with ⩾ 1 esi before day 42 was significantly higher in cases than in controls (11/17 vs 11/34, p = 0.038), with an overall number of esi events of 19 and 12 among cases and controls, respectively. five cases had ⩾ 2 concurrent esi. the median time from ahsct to the first esi event for gf cases was 17 days (interquartile range (iqr), 11-24) vs 15 (iqr, 8-34) days for controls (p = 0.779). in the gf setting, the most prevalent infections were herpesviridae infections (n = 7 including hhv-6 n = 4, ebv n = 2, cmv n = 1), probable ifi (n = 4), severe sepsis of documented bacterial origin (n = 3), toxoplasmosis (n = 2) among whom one patient developed haemophagocytic syndrome. when further analysing subsets of esi using logistic regression, only toxoplasmosis was a significant risk factor for gf (p = 0.018). death related to an infection was proven for 8 gf patients vs 5 control patients (p = 0.012). the overall survival probability at 12 months was significantly lower in the gf setting than in control patients (hr = 2.59 (95% ci 1.25 − 5.36), p = 0.01). the survival rates at 12 months were 35.3% and 57.7% for gf and control patients, respectively. at our center, graft failure is statistically associated with early-onset severe infections, and already known graft characteristics such as cell dose and disease status. however, our study would need more power to increase its significance. disclosure of conflict of interest: none. allogeneic stem cell transplantation (asct) is a curative option for hematological disorders, especially malignancies. in immunosuppressed women after asct, the progression from cervical dysplasia to invasive carcinoma is accelerated, and cervical cancer is likely a more aggressive disease. therefore, follow-up protocols after asct should include regular gynecologic evaluation with papanicolaou (pap) smears. we retrospectively evaluated 32 pap smears in 20 women who underwent asct and searched the risk factors for abnormal cervical cytology. the median age at transplantation was 44.5 years (range: 22-65 years). the most frequent indication for asct was leukemia (70%), and 85% of the patients received a transplant from a sibling hla-matched donor. stem cell source was peripheral blood in all patients. myeloablative conditioning regimen was used in 50% of patients. cyclophosphamide, busulfan and fludarabin were used in 20 (100%), 18 (90%) and 10 (50%) patients, respectively. acute graft versus host disease (gvhd) occurred in 7 patients (35%) and chronic gvhd in 4 patients (20%). secondary cancer (1 breast cancer) was reported in only one patient at 40 months after asct. the follow-up time was 23 months (range: 3-104 months). after asct, benign and abnormal pap smears were found in 12 (60%) and 8 (40%) women, respectively. the median time between asct and development of abnormal cytology was 2 months (range: 1-11 months). four (20%) women had at least one smear with atypical squamous cells of unknown significance (asc-us), one (5%) had a low-grade squamous intraepithelial lesion (lsil), one (5%) had atypical squamous cells/high-grade lesion (asc-h) and one (5%) had asc-us and asc-h. one (5%) patient had malign smear. two patients with asc-h showed high-grade atypia mimicking cancer but had a negative follow-up. patient who had malign smear died because of aorta dissection. cervical biopsy showed cervical intraepithelial neoplasia (cin) i in 3 (15%) women who had asc-us or asc-h. one patient was hpv-positive. we did not find any relationship between cervical cytological abnormality and clinical factors. after asct, patients are high risk for abnormal cervical cytology and secondary gynecological cancer. regular surveillance of patients is the most important factor for decreasing the risk of developing cervical and other secondary cancers. gynecologic examinations and cervical cytological testing after asct allows early diagnosis and effective management of cervical abnormalities. disclosure of conflict of interest: none. kidney dysfunction is a frequent complication of allogeneic stem cell transplantation (sct) and contributes to the morbidity and mortality of the procedure. incidence of severe acute kidney injury (aki) in patients undergoing nonmyeloablative allogeneic sct for malignant diseases ranges from 14 to 47%. lymphoma patients are often heavily pretreated through both chemotherapy and autologous sct and may be at increased risk of developing kidney injury. we performed a retrospective analysis of 108 consecutive patients with lymphoma undergoing nonmyeloablative allogeneic sct between 2004 and 2016 (table 1) . acute kidney injury (aki) within 100 days of allogeneic sct was diagnosed and staged according to rifle-criteria, and severe aki was defined as rifle stage i-e (4doubling of creatinine or 450% decrease of egfr). chronic kidney disease was defined as an estimated glomerular filtration rate (egfr) o60 ml/min/1.73 m 2 1 year after allogeneic sct. we performed multivariate logistic regression to evaluate potential risk factors for severe aki. severe aki developed in 75 patients (69.4%). reduced overall survival was observed in these patients, although not statistically significant. no significant associations were seen with age at transplantation, baseline kidney function or prior autologous sct. severe aki was associated with acute graft versus host disease (gvhd) (or 2.8, p = 0.026) and the use of an unrelated donor (or 2.8, p = 0.025). chronic kidney disease was observed in 20 (18.5%) of patients alive after 1 year. we report a substantially higher incidence of severe aki after nonmyeloablative allogeneic sct for lymphoma than has been reported for other malignancies. acute gvhd and unrelated donor stem cell s271 source were associated with severe aki, while prior autologous sct, age and baseline kidney function were not. [p301] disclosure of conflict of interest: none. patients with acute myeloid leukemia who are treated with conventional chemotherapy still have a substantial risk of relapse. we, therefore, retrospectively analyzed data to investigate the effects and some risk factors of allogeneic hematopoietic stem cell transplantation in relapsed and refractory acute myeloid leukemia patients, and to provide some suggestion for the clinical treatment. a total of 84 refractory and relapsed acute myeloid leukemia patients receiving allogeneic hematopoietic stem cell transplantation in our center between february 2005 and december 2014 were retrospectively analyzed, including 23 patients in no-remission (nr) and 61 patients in second complete remission (cr2) at the time of transplant. the median age was 35 years (range: 9-55). conditioning was myeloablative using cyclophosphamide, busulfan and total-body irradiation (bu/cy, n = 44; tbi/cy, n = 3), and others were underwent nonmyeloablative stem cell transplantation. 81 patients had successful engraftment. acute-gvhd and chronic-gvhd appeared in 47and 37 patients. the 3year overall survival (os), relapse rate and disease-free survival (dfs) of the cases was 50 ± 6.0%, 44.8 ± 5.8% and 37.1 ± 0.3%, respectively. the 3-year dfs were higher for patients in cr patients (52.2 ± 6.7%) than in nr patients (21.3 ± 10.1%), and the relapse rate in nr group and cr group were 60.4 ± 1.6% and 29.7 ± 0.4% respectively. there was no significant difference in treatment-related mortality compared cr group with nr group. sex, age, related-donor graft were not independent factors affecting os, dfs and relapse rate. it is concluded that allo-hsct is an effective salvage therapy for patients with refractory and relapsed aml. non-remission before transplant and severe agvhd are high risk factors of poor prognosis for allo-hsct. patients in cr group who accept reinduction chemotherapy before transplantation have better prognosis than those in nr. the overall outcome seems related to the disease status. hsct during refractory and relapsed can achieve long-term survival in selected patients with individual therapy. disclosure of conflict of interest: none. the incidence of most hematologic malignancies increases with age. aging is related with a greater prevalence of impaired functional status and comorbidities. although cure of malignant and non-malignant hematological diseases is potentially possible with allo-hsct, it could lead to significant transplant-related mortality. decision making about referral to allo-hsct in older adults is a challenging task. in this study we aim to present our geriatric allo-hscts. from 2007 to 2016, 33 [p303] patients (age 3 60) underwent allo-hsct in our center included to this retrospective study. pre-transplant status as well as posttransplant toxicities, complications and outcomes were determined. the age distribution of the group: 27 patients was aged 3 60 and o65, 5 patients was aged 465 and o70, 1 patient was 71 years old. the median age of donors was 49 (range: 21-73). the pre-transplant patients' characteristics are given in the table. remission was achieved in twenty-three (70%) patients. twenty-six patients (79%) had neutrophil engraftment (40.5 × 10 9 /l) at a median day of 19 (range: 10-41) and platelet engraftment (20 × 10 9 /l) at a median day of 20 (range: 14-54). post-transplant complications are detailed in the table. acute graft vs host disease (gvhd) was occurred in 10 patients (31%) and chronic gvhd in 12 patients (36%). eight patients (24%) were diagnosed with a relapse and 1 year relapse-free survival was 15%. the 1-year and 2-year os were detected as 30% and 12%. the most common reason for mortality was sepsis. the 1-year os was higher in patients who had reduced intensity conditioning regimen and remission status pre-transplant however they were not statistically significant (30% vs 21%, p = 0.6; 31% vs 25%, p = 0.9) (figure) . since increasing number of older patients being diagnosed with hematologic malignancies, this trend of increasing number of allo-hsct will continue. tolerability and effectiveness are lesser, toxicity is higher in older adults. although study population is relatively small, reduced-intensity conditioning and pre-transplant remission status may be related to better survival. comprehensive geriatric assessment may be considered prior to allo-hsct for global evaluation. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (asct) is a procedure with high morbidity and mortality (10-20%) requiring a complex hospital infrastructure. improved support measures and development of homecare units has allowed that asct at-home programs may be possible. our center has launched a pioneering program in our country in patients with asct to perform at home the following of aplasia, control of immunosuppressive therapy (ist) and intravenous support from the d+1 of asct until the engraftment and independent ambulatory patient. to evaluate the patient safety, we compared the group of patients at-home (asct-op) with a cohort of asct 'in patient' with similar characteristics (asct-ip). 26 asct patients between january 2014 and october 2016 at the hospital clinic of barcelona. 13 patients performed asct-op and 13 had an asct-ip. all patients received conditioning (myeloablative-mac-or reduce intensity-ric-) in the hospital with fludarabine 40 mg/m 2 (d1-4) and busulphan 3.2 mg/kg (2-4 doses), prophylaxis of gvhd was performed with tacrolimus/mycophenolate (mmf) in asct-op group and cyclosporine(csa) and methotrexate (mtx) or mmf in asct-ip group. in all patients, the infectious prophylaxis was conventional (levofloxacin, fluconazole and acyclovir). moreover, the asct-op group received prophylaxis with ceftriaxone 1 g intravenous (iv) once daily and liposomal amphotericin b inhaled 25 mg twice a week during neutropenia. the asct-op group from d+1 received a nurse visit once daily and physician visits twice a week in the hospital. baseline characteristics were analyzed those related to toxicity and patient outcomes. the median age (range) was 56 years (23-69), male/female 16/10; (62% male). the source of the progenitors was peripheral blood in all cases and analysis of the results detailed in the table: disclosure of conflict of interest: none. an increase in rdw-sd after allogeneic hematopoietic transplantation is associated with a poor prognosis s leotta, a cupri, a di marco, a spadaro, l scalise, g sapienza, mg camuglia, g avola, g moschetti 1 and g milone 1 istituto oncologico del mediterraneo red cell distribution width (rdw), is an erythrocyte index influenced by stress erythropoiesis, inflammation and antioxidants. rdw predict mortality in sepsis, chronic kidney diseases and in cardiovascular disease. no data are available on rdw after hematopoietic transplantation. in a retrospective study we collected data on changes of rdw-sd in a group of 81 patients who received allogeneic hematopoietic transplantation. fortyeight patients were affected by acute leukemia, 13 by lymphoma, 9 by mm, and 11 by other diagnosis. rdw was studied at baseline and monthly for the first 3 months. a subset of 34 patients were studied prospectively for clinical and laboratory signs of microangiopathy. at baseline before the transplant a rdw-sd higher than normal upper limit was observed in 43% of allogeneic candidates. a high co-morbidity score (htc-ci score 2-5) at the pre-transplant screening was a factor associated to high rdw-sd (χ 2 p = 0.01). a value of rdw-sd higher than normal range, at baseline, was not associated to any other factors, such as age, diagnosis, phase of the disease, previous transplantation, c-reactive protein, bilirubin, creatinine and arterial hypertension. early after allogeneic transplant we noticed at day +30 a significant reduction of rdw-sd but subsequently (at day +60) the proportion of patients showing an abnormal rdw-sd increased to 63%. an abnormal rdw-sd at s274 day +60 was registered in 70% of allogeneic transplant patients who presented an acute gvhd while in only 30% of patients who did not presented during the first 3 months an acute gvhd (χ 2 p = 0.02). in allogeneic transplantation group, patient who, at day +60, had a rdw-sd higher than normal value had a inferior outcome in respect to patients having a rdw-sd within normal ranges (os was 70% vs 30%; logrank: p = 0.04;), (ci of trm: 45% vs 18%).these two groups were not significantly different for pretransplant features in the subset of patients studied prospectively, abnormal rdw-sd was associated to presence of schystocytes in pb (chi test: 0.004) and patients having ⩾ 2% schystocytes had a median rdw-sd of 71 (iqr 31) vs a median rdw-sd of 46 (iqr 12.2) in patients who did not show schystocytes in pb (mann-whitney u-test p = 0.004). rdw-sd was significantly correlated also to serum triglycerides (r = +0.4, p = 0.0004) and to red blood cell mean corpuscular volume (r = +0.32, p = 0.02). abnormal rdw-sd is frequent after allogeneic transplantation. abnormal rdw-sd is associated to acute gvhd and its value obtained at day +60 marks a group of patients with poor prognosis because of high trm. this simple parameter warrant further studies to determine its clinical usefulness in monitoring of patients suffering acute-gvhd and in diagnosis and monitoring transplant associated microangiopathy. [p305] disclosure of conflict of interest: none. sickle cell disease (scd) poses a lot of psychological burden for the patient and the caregiver. it also poses a significant financial burden over the family. ohaeri et al. developed a 16 point questionnaire to asses sickle cell disease burden called as sickle cell disease burden index (scdbi) and its impact on caregiver's quality of life (qol). we used this questionnaire to assess the impact of hematopoietic stem cell transplant (hsct) on caregiver's qol. 16 point questionnaire was sent to 15 set of parents whose child underwent hsct between january 2016 and june 2016. scdbi contained 16 questions in various domains (3:family finances, 3:family interactions, 5:routine family activity and 5:parental coping ability). answers were graded on a score of 0-3 (0:never occurred and 3:occurred regularly or had a severe impact on the family). the results were interpreted in two headings a. family finances and interactions (0: no impact; 1-3: insignificant impact; 4-6: moderate impact; 7-9: severe impact) and b. routine family activity and parental coping ability (0: no impact; 1-5: insignificant impact; 6-10: moderate impact; 11-15: severe impact). all these domains were assessed before and after hsct. ten parents replied with duly filled questionnaire. mean age at hsct was 8.1 years (range: 1-14), m/f:7/3. all were symptomatic for 46 months before hsct with 90% having more than 2 hospital admissions. majority of parents were from middle class with median family income of 30 000 usd per annum (range 16 000-200 000 usd). median score for family finances and interactions (a) before hsct was 6 (range: [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] which decreased to 0 (range: 0-3) after hsct. median score for routine family activities and parental coping ability (b) before hsct was 13 (range: which decreased to 0 (range: 0-6) after hsct. our results suggest that before hsct there was a moderate impact on family finances and interactions which reduced to no impact after hsct. similarly there was severe impact on family activities and parental coping ability before hsct which changed to no impact after hsct. our study suggests that hsct not only improves the qol of the child but also of the caregivers. chronic graft versus host disease (cgvhd) is a late complication of allogenic hematopoietic stem cell transplantation (hsct) that affects many tissues and organs and manifests with polymorphic clinical features similar to autoimmune diseases. poorly understood pathophysiological mechanisms are implicated in inflammation and tissue fibrosis which is a hallmark of cgvhd. the affection of lachrymal glands is frequent and contributes to ocular manifestations presenting as dry eye syndrome. autologous serum eye drops (aesds) are used topically to facilitate tissue healing and ease the symptoms in a variety of ocular diagnosis. it is unclear if the serum of a patient with cgvhd is suitable for remedy preparation and if the transplanted patient himself can meet the criteria for autologous donation. aim is to show the safety, feasibility and efficacy of autologous serum preparations in ocular lesions after allogenic hsct. donors should meet criteria for autologous blood donation (infectious disease status, complete blood count hgb4110 g/l, hct433%, adequate venous access). aesds are prepared from 150 ml of autologous blood left to clot, irradiated and centrifuged to separate serum which is diluted with saline in 1:5 ratio or 1:1 if requested. product is dispensed into 1.5 ml ampules, stored at − 20°c and a 3-month supply is released to the patient after receiving negative results of sterility testing. in period from 2005 to 2016. in the aesds program 12 patients (4 female, 8 male) with ocular symptoms were included. all met required predonation criteria. of 29 collections performed, one failed due to venous access problem and one product had to be discarded due to hemolysis. cgvdh global nih score of the patients at start of the program was: 4 severe, 6 moderate, 1 mild and 1 not scored. all patients presented with moderate to severe dry eye symptoms. in 6 (50%) patients aesds alleviated dry eye symptoms. in 3 (25%) out of 12 patients referred to aesd program, more than 3 autologous blood collections were performed (range: 4-10) and aesds were used regularly through period of 10-36 months, which points to the beneficial effect of the long-term use of the serum. three patients dropped out because aesds showed no advantage compared to commercial lubricant eye drops preparations. one patient dropped out because of a venous access problem, 4 patients had disease s275 progression and needed other therapies: 3 cases of amniotic membrane application of which 2 continued with aesds to facilitate the healing effect. one patient was recently included and the effect of aesd is still evaluated. autologous donations in cgvhd patients are feasible, safe and autologous serum preparations can help relieve symptoms of dry eyes. it needs to be further elucidated specifically in which patients and at what point of the disease course the effect of the aesds is the most beneficial to make optimal use of these preparations. disclosure of conflict of interest: none. idiopathic pneumonia syndrome (ips) is a non-infectious pulmonary complication with diffuse lung injury that develops in 5-10% of patients who undergo hematopoietic cell transplantation (hct) and the mortality rate remains high at 80%1. the major aim of this study was to identify prognostic biomarkers for ips and establish positive and negative predictive values (ppv and npv) of ips. in a case-control study, we compared 41 patients with ips with available samples (transplanted between 1988 and 2014 at fhcrc) with 162 hct control recipients who did not require bronchoscopic examination and who did not grow any bacterial or fungal blood cultures. for each subject, plasma samples at day 7 post hct and onset of ips or matched time points for controls were analyzed. the 'onset sample' for controls was the sample closest to day 24 (median day of onset for patients with ips). we measured six proteins by elisa: suppressor of tumorigenicity 2 (st2), tumor necrosis factor receptor 1 (tnfr1), interleukin-6 (il-6), lymphocyte vessel endothelial receptor (lyve)-1, endothelial protein c receptor (epcr), and herpes virus entry mediator (hvem). multivariable logistic regression models were used to evaluate the association of each protein with ips vs controls. cytokine cutoff values that maximized discrimination between ips and controls were identified using receiver operating characteristic (roc) analysis. ppv and npv of ips were calculated using the identified cytokine cutoffs across a range of hypothetical ips prevalence values (0-15%) day 200 weighted kaplan-meier survival curves were estimated for high/low cytokine subgroups. similarly, a weighted log-rank test was used to evaluate p-values. a multivariable logistic regression model including six cytokines showed that st2 and il-6 were significantly important markers to identify ips at the onset (table 1) . st2 value at day 7 post hct was significantly associated with occurrence of ips and il-6 had a marginal association. predictive values for ips by a plausible percentage of the actual hct population (up to 15%) are shown in figure 1 . of the six proteins, st2 showed the highest ppv both at onset and day 7 post hct followed by tnfr1, and il-6. npv were high in all the markers. to analyze whether st2 and il-6 at day 7 after hct can predict survival following ips, we dichotomized the patients into cytokine high and low groups (cutoff level: st2, 19 ng/ml; il-6, 35 pg/ml) and compared survival after downweighting the observations to represent a plausible percentage of the actual population (ips prevalence, 5%). day 200 survival rate were significantly lower in st2 high value group than in st2 low value group (80% vs 88%, p = 0.015). similarly, il-6 high value was associated with high mortality (day 200 survival rate, 78% vs 88%, p = 0.003). st2, il-6, and tnfr1 were good prognostic markers for occurrence ips. especially, st2 and il-6 at day 7 after hct can be a predictor for both ips occurrence and survival following ips. these results require validation in an independent prospective hct population. body composition parameters are sensitive nutritional indicators that influence response to treatment and mortality in cancer patients. research is not conclusive on the changes in muscle attenuation and adipose tissue areas in the stem cell transplantation (sct) phases. objective is to assess the changes in adipose tissues, skeletal muscle index (smi) and waist circumference (wc) among stem cell recipients in the peri-transplantation phase. study design: institutional review board approved this retrospective study with 61 adult patients (age416 years) having b and t lymphoma who underwent sct. each patient was imaged by pet/ct scan pre-sct and 3 months post transplantation. a cross sectional image was analyzed at the level of the l3 to calculate total adipose tissue (tat), visceral adipose tissue (vat), intra-muscular fat (imf), smi and wc. data was analyzed by gender since body composition parameters differed significantly between the two categories in the literature. the study sample consisted of 61 patients (mean age: 38.2 ± 13.7 years, 35 (57%) males, 51(83.6%) autologous sct, median overall survival in months: 39.8 in males and 40.5 in females). death was observed in 6 (17.1%) males and 1(3.8%) female. patient characteristics were similar for males and females except for weights (kg) and body mass index (kg/m 2 ): 86.7 and 28.6 vs 63.5 and 24.1 in males and females respectively. changes from pre-sct to 3 months post sct revealed that tat, vat, smi and wc decreased with mean differences of 42 ± 61.2 cm 2 , 18.28 ± 37.6, 3.3 ± 7.5 cm 2 /m 2 and 4.58 ± 5.4 cm, respectively in males (po0.01). in females, tat and wc significantly decreased with mean differences of 18.4 ± 37 cm 2 and 3.1 ± 4.3 cm, respectively (po0.01). in females, vat and smi decreased clinically but did not reach clinical significance. in multivariate analysis, no significant associations were shown with mortality and progression rates. this study fills a research gap by providing data on the evolution of body composition parameters in the peri-transplantation phase. tat, vat, smi and wc decrease 3 months post transplantation. future studies should evaluate the associations of these parameters with major outcomes on larger sample sizes. [p310] disclosure of conflict of interest: none. . 5 patients received tbicontaining preparative regimen. all these patients were exposed to calcineurin inhibitors for prevention and treatment of gvhd. 16 patients suffered from cgvhd-grade moderate or severe. all patients required systemic corticosteroids, because of gvhd (16pts) or during basic treatment of lymphoma (2 pts). all patients had deficient states of vitamin d initially and required replacement. all of them, except for 2 patients, had balanced adrenal insufficiencies and 2 patients had balanced hypothyroidism. all women had premature ovarian failure (2 received hrt). according to measurements of bone mineral density (bmd), low bone mass was detected in 15 patients; osteopenia (11pts), osteoporosis (4pts). bone loss of femoral neck (8-osteopenia, 3-osteoporosis) occurred more often than lumbar vertebral ( 6-osteopenia, 2-osteoporosis) or radius (3osteopenia, 1-osteoporosis). presence of avascular necrosis of bone (avn), confirmed by mri, was detected in 12 patients and the most common site of involvement was the femoral head(all patients), knee(3 pts) and shoulder(1 pts). one of the first symptoms of avn was pain and functional limitation. all patients required intensive analgesic treatment, usually nsaids and 4 patientsfentanyl. fractures occurred in 12 patients. the femoral neck (7 pts) and thoracic or lumbar vertebral (3 pts) were two most common fracture sites. all patients were qualified for surgery; 6 patients required hip replacement, 6patients still awaited to perform surgery or were disqualified because of severe, skin cgvhd. bone complications may occur in about 17% of allo-hct survivors (including 30% patients with gvhd, and up to 60% patients with severe or moderate cgvhd) within first 4 years after allotransplantation. bone loss, particularly at the femoral head, is the most common complication. avascular necrosis usually requires surgical intervention because of fractures. exposure to higher doses of corticosteroids (during treatment of gvhd) increases risk of bone complications. early diagnosis by mri and dxa may help to detect bone complications. (41%) and (e) 3 (21%). a total of 41 (30%) pts failed to meet these criteria but remained alive on day 5 and 39 (28%) died before day 5. the overall survival (figure 1 ) for the 58 pts was 28% at 3 years with an overall mortality in icu of 33% (19/58) compared to 71% (29/41) for those who did not meet our criteria. the overall survival for pts that met our criteria at fifth day and were discharged to the haematology ward (n = 39), was 49% at 3 years. in this study, 50% of patients survived their icu admission. patients could be stratified according to the reason for admission and given an individualized 5-day trial: those who met our criteria for successful icu trial (42%) had a low icu mortality (33%) and those who were subsequently discharged home had a overall survival of 49% at 3 years. this study raises the possibility of offering a short-term icu stay to oncohematologic patients and perhaps allows for the ceiling of intensive care for those who fail these criteria. [p312] disclosure of conflict of interest: none. in contrast, only 2.98% of patients without cgvhd showed a thromboembolic complication in the later time course, with one patient showing an additional thrombotic risk factor. in multivariate analysis cgvhd was an independent risk factor for thromboembolic complications after hsct. 1.8% of patients with thrombosis before hsct showed one afterwards. thrombosis before hsct was not found as risk factor for thromboembolic complication after hsct. our retrospective analysis showed an increased risk for thromboembolic complications after allogeneic hsct, with substantial higher risk in patients with chronic gvhd (8.9%). in ongoing studies we currently investigate a vascular screening procedure with additional biomarkers according to inflammation and endothelial damage in patients with cgvhd prospectively. we hope to identify patients at risk for thromboembolism and prevent future complications on an individualized basis. disclosure of conflict of interest: none. (1). pts with cns involvement received intrathecal therapy with cytarabine and in one case additional cns irradiation was applied. 10/11 pts died after a median time of 9 mts (range: 1-25 mts) due to resistant systemic relapse, infectious complications or extensive graft-versus-host disease following allohsct. 1 patient remains alive and disease-free at 88+ mts following secondary allohsct. conclusions: our data indicate that em disease following allohsct affects a significant proportion of pts with aml. sites of em relapses vary widely among the pts with skin and cns being frequently involved. an aggressive approach combined of local and systemic therapy including secondary allohsct may produce favorable response in a small proportion of pts, however, overall prognosis for pts with isolated em relapses still remains poor. due to the lack of effective treatment strategies, there is a need for novel approaches to manage isolated em relapses after allohsct. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct)-associated thrombotic microangiopathy (tma) is a multifactorial complication, and has variable incidence in study populations due to different diagnostic criteria. aim: our aim was to identify pediatric patients with hsct associated tma using 4 different diagnostic tma criteria published in literature and to compare the various groups for tma parameters and outcomes. we enrolled 33 pediatric patients who underwent allogeneic hsct using treosulfan based or reduced intensity conditioning therapy. 4 different tma diagnostic criteria, the bmt ctn toxicity committee consensus definition (1), the overall thrombotic microangiopathy grouping (2), the diagnostic criteria created by city of hope (3) and the criteria proposed by jodele et al. (4) were used to startify the patients. we determined and registered the following tma activity markers: presence or development of increased ldh and decreased haptoglobin levels, new onset anemia, thrombocytopenia, fragmentocytes, coombs test, kidney function, proteinuria, hypertension and terminal complement complex (sc5b-9). complement pathway activities, components and sc5b-9 were measured during early hsct period. two/33 (1), 7/33 (2), 3/33 (3) and 10/33 (4) subjects met the different tma diagnostic criteria according to the four different systems on day 12 and 34 (1) and on median 44 (2), 43 (3), 61 (4) post-hsct days. all of the 6/10 patients who were defined with the first three criteria, met the forth definition. due to normal haptoglobin levels and kidney function, 4/10 patients fulfilled only the forth criteria. tma coexisted with acute graft-versus-host disease in 7/10 cases (7/10 vs 4/23; p o0.01). patients who met any of the different tma diagnostic criteria had higher sc5b-9 level on day 28 (411 vs 201 ng/ml; p = 0.003) compared to those without. all of the 10/33 subjects defined with tma had elevated sc5b-9 (4250 ng/ml) level during the early hsct period. two patient died before day 100 after hsct, out of which one patient met all of the four tma diagnostic criteria. after a median 2.29 (1.2-3.1) year follow-up time, overall survival was 24/33. 8/10 patients with tma survived, compared to 16/23 patients without tma. relapse related mortality was the most common cause of death (n = 7/9, po0.05), while tma was not a significant cause of mortality after reduced toxicity conditioning therapy. hsct-associated tma has a variable and complex pathophysiology. using the different diagnostic criteria may influence the incidence and the time of diagnosis of this transplant-related complication. monitoring all of the published tma activity parameters, including complement terminal pathway activation marker, may help to guide physicians to recognise tma after hsct. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) is associated with a risk of non-relapse mortality (nrm). it's important to assess the risk of complications and mortality before the hsct. some indexes quantify the impact of patients' comorbidities on hsct outcome. the most frequency used is the hct comorbidity index (hct-ci) and the european group for blood and marrow transplantation score (ebmts). this study tried to determine which of the two indexes best predicts the outcome in a series of patients submitted to hsct in a single center. between 2011 and 2015, 259 hsct were performed in our center. a total of 215 hsct have been analyzed (we excluded patients o18 years (yr), 2 nd hsct, haploidentical donors and hsct for specific diseases with very low number ( o3%) of hsct performed: aplastic anemia, cll, prolymphocytic leukemia, mycosis fungoides, sezary syndrome, dendritic cell neoplasia, plasma cell leukemia and poems syndrome). the hct-ci and ebmts were calculated retrospectively (yr 2011-2013) and prospectively (yr 2014-2015). overall survival (os), relapse incidence (ri) and nrm were analyzed in the overall series and separately according to the type of hsct: autologous hsct (auto-hsct) or allogeneic hsct (allo-hsct). male: 89 (59%) patients. median age: 54 yr (range: 18-71). diseases: aml 54 (25%), all 19 (9%), mm 67 (31%), nhl 41 (19%), hl 12 (5%), mds 17 (8%), cmpd 5 (3%). disease status: 1 st complete remission (cr) or 1 st chronic phase 102 (48%), ⩾ second cr 30 (14%), first partial remission (pr) 55 (26%), ⩾ second pr 12 (5%), no response 11 (5%) and without previous treatment 5 (2%). auto-hsct in 120 patients (56%) and allo-hsct in 95 (44%) patients. related and unrelated donor were 52 (55%) and 43 (45%), respectively. the conditioning regimen was standard in 57 (60%) cases and reduced intensity in 38 (40%). hct-ci and ebmts grouped 0-2, 3 and ⩾ 4 were 57 (48%), 29 (24%), 34 (28%) and 38 (32%), 51 (42%), 31 (26%) in auto-hcct and 61 (64%), 18 (19%), 16 (17%) and 34 (36%), 32 (34%), 29 (30%) in allo-hsct, respectively. median follow-up was 3.15 yr (0.66; 5.73) for the overall series, 3.18 yr (0.66; 5.73) for auto-hsct and 2.60 yr (0.97; 5.70) for allo-hsct. significant differences in os and nrm were found according to the ebmts in patients submitted to auto-hsct. one-yr-os and 3-yr-os were 91% (95% ci: 85%; 97%) and 68% (95% ci: 57%; 79%), respectively, in patients with ebmts 0-3, vs 73% (95% ci: 57%; 87%) and 56% (95% ci: 37%; 75%), respectively, in patients with ebmts ⩾ 4 (p = 0.033). one-yr-nrm and 3-yr-nrm were 2% (95% ci: 0%; 7%) in patients with ebmts 0-3, vs 13% (95% ci: 4%; 28%) in patients with ebmts ⩾ 4 (p = 0.015). no significant differences were observed for ri according to ebmts in patients submitted to auto-hsct. no significant differences in os, ri and nrm were observed according to ebmts in patients submitted to allo-hsct. no significant differences regarding os, ri or nrm were found when the hct-ci was assessed. in our series, only the ebmts was predictive of os and nrm in patients submitted to auto-hsct. failure to find statistically significant differences for the hct-ci and for ebmts in allo-hsct recipients could be due to an insufficient number of patients or to a partial retrospective collection of data. infertility is common after hct predominantly as a result of the chemoradiotherapy used in conditioning. nonetheless, some patients do retain or recover fertility. newer reduced intensity regimens may be less gonadatoxic. in addition, patients are increasingly encouraged to store gametes, or embryos before transplant. we sent questionnaires to 602 ebmt centers requesting retrospective details of number of pregnancies and pregnancy outcome for all patients treated between 1995-2015. 27 centers responded from 13 countries detailing 234 patients who became pregnant/partners conceived. the most frequent underlying diagnoses were acquired bone marrow failure (n = 44, 25f) aml (n = 42, 15f), hd (n = 26, 15f), cml (n = 25, 7f), all (n = 19, 4f) and b nhl (n = 18, 10f). other diagnoses included mds, mps, solid tumours, autoimmune disease, cll, t-nhl, haemoglobinopathy. of 110 females (f), 35 (32%) involved assisted reproductive techniques (art). 30f had tbi ( seven o4 gy) of which 16 (50%) had art. 25f had reduced intensity conditioning of whom 6 (24%) had art. 70f were specified as having standard conditioning of whom 24 (34%) had art. 73f had allogeneic (26 art, 36%) and 37f had autologous transplants (9 art, 24%). of 124 men (m) whose partners conceived, 61 (49%) had art. 54m received tbi of which 36 (67%) had art. where specified, 19 had reduced intensity hct (3 art, 16%) and 94 had standard conditioning (48 art, 51%) .93 had had allogeneic hct (43 art) and 31 autologous (12 art). 19 men had reduced intensity transplants. 53 men received tbi (two o4 gy) of whom 36 (68%) had art compared to 69 men without tbi, 16 (23%)of whom had art. data on return of menstruation was available for 84. 64 indicated yes and 12 (19%) had art. 20 indicated amenorrhoea of whom 14 (70%) had art. 224 specifying number of children had 324 live births (lb) and 87 (39%) patients had more than one child after hct. 146 lb occurred in female patients (41 art, 28%) and 178 lb were in partners of male patients (88 art, 49%). the median gestational age for 61 female patients was 39 weeks (range: 22-42) and the median birth weight was 3 kg (range: 0.3-4.19). there were 3/80 congenital anomalies. the median follow up of the offspring was 5 y (range: 0-15). developmental problems were indicated for 1/71 (fine motor skills) and learning difficulties in 1/70 (adhd). in partners of male patients the median gestational age for 62 offspring was 39 weeks (range: 26-43). the median birth weight for 56 offspring was 3 kg (range: 0.87-4.16). congenital malformations occurred in 4/89. one infant died of pulmonary infection. in women, several methods of assisted conception were used including hormone stimulation, ivf, cryopreserved embryos, donor embryos and cryopreserved ovarian tissue. the most frequent method was use of donor embryos (22/35) in which a minimum of 30 attempts led to 21 lb. the median number of attempts was 1 (range: [1] [2] [3] [4] [5] . art were frequently used in this group of posttransplant patients particularly in male patients vs female, tbi vs non-tbi, amenorrhoeic vs menstruating women, standard conditioning vs ric. in patients who conceive after hct, successful pregnancy leading to healthy offspring is the likely outcome. disclosure of conflict of interest: none. reduction of trm after sct was observed over the transplant periods and supportive care with danaparoid was found to be significantly effective to reduce trm. therefore, prophylactic administration of danaparoid is considered to be a reasonable option to improve the transplant outcomes for children. [p320] disclosure of conflict of interest: none. the attainment of transfusion independence after transplant is sometimes hampered by a combination of factors, ranging from infections to the need of combined therapy for clinical complications, as well as control of gvhd. iron overload is frequently observed in hematological patients before and after hematopoietic stem cell transplantation (hsct). whereas several reports have focused on iron overload before transplant, up to now, this is the only report that show full recovery of hematopoiesis and correlate this to deferasirox chelation performed on this particular subset of patients. we report on 19 patients, transplanted for hematological diseases (17 acute leukemia, 1 aplastic anemia, 1 multiple myeloma) heavily transfused before transplant that, considering the iron overload, were treated with deferasirox after hsct. before starting deferasirox, the patients were fully engrafted and in complete remission, although transfusion dependent, and with incomplete hematological reconstitution after allogeneic hsct. patients were selected according to the following inclusion criteria: (1) transfused pre-transplant with more than 20 rbc units; (2) incomplete hematological recovery; (3) transfusion-dependence; (4) serum ferritin 41000 ng/ml; (5) normal creatinine value. the workup for other aetiologies resulted negative. all patients received an initial dose of deferasirox 10 mg/kg/day, later adjusted according to side effects. all patients experienced an increase in hemoglobin levels, with a reduction in the frequency of rbc transfusions, followed by transfusion independence (median time: 24 days from the first dose of deferasirox). in addition, it was promptly (median time: 27 days) associated with hematological improvement, with sustained values and no further platelet support or growth factors administration. no relevant modifications with immunosuppressive or myelosuppressive drugs were made during deferasirox treatment. deferasirox was well tolerated. basing on our results, we think that deferasirox determined stimulatory, and/or depressive effects on hematopoiesis after allo-hsct. this clinical experience raises the possibility of a potential additive benefit on hematopoiesis after transplant following iron chelation therapy with oral deferasirox. further long term studies, in larger cohorts of patients are needed to confirm these data and to design an efficient strategy to reduce iron loading after transplant. disclosure of conflict of interest: none. supported in part by ail pesaro onlus. (4) hypertension (n = 2). all five patients had normal adamts13 levels and negative testing for shiga toxin. complement mutation genetic studies were obtained for four patients including 10 genes (n = 2) and 12 genes (n = 2) and were all negative. testing for complement pathway including c5b-9 were obtained for 2 patients and were normal. all five patients were treated with eculizumab with induction treatment at 900 mg weekly × 4 doses, followed by one dose of 1200 mg on the fifth week, and 1200 mg every 2 weeks thereafter. patients had a recovery of hemoglobin and platelets and a rise in haptoglobin and a normalization of ldh within 4-6 weeks from the start of eculizumab. eculizumab was discontinued for 3 of the 5 patients without recurrence of their tma; they are now 18-24 months since the discontinuation of eculizumab. in summary, there is a subacute syndrome of thrombotic s282 microangiopathy that can occur late post transplant. this syndrome appears to be complement mediated as shown by its response to a terminal complement inhibitor. it also appears to be transient without recurrence following treatment discontinuation. disclosure of conflict of interest: none. transplant associated microangiopathy (tam) is a very severe complication occurring after allogeneic bone marrow transplantation (bmt), burdened by a high case-fatality rate. it is characterized by abnormal complement activation, triggered by various agents (calcineurin inhibitors, acute gvhd, infections) with subsequent endothelial damage. in the literature, 6 cases of mutations in recipient complement genes are described, but none in donor dna. here we describe for the first time 3 patients affected by tam, carrying mutations in donor complement genes. in our lab, we studied 6 patients affected by tam; they were screened for cfh autoantibodies, adamts13 function and variants and macro-rearrangements in cfh (and related), cfi, cfb, cd46, c3, dgke, thbd genes and at-risk haplotype (cfh-h3 and mcpggaac) by means of next-generation sequencing (ngs) and multiplex ligationdependent probe amplification analysis (salsa mlpa p236 armd mix-1; mrc holland). ngs was used to sequence dna by haloplex kit (agilent) on a miseq (illumina) platform with 450-fold coverage of every target base. the bioinformatic analysis was performed using sophia genetics and the pathogenicity was assessed by means of in silico predictions (polyphen2, sift, mutationtaster, aligngvgd). all of the predicted pathogenetic variants were confirmed using sanger sequencing. the same genetic screening was extended also to donor dna in all 6 cases. the screening for known causes of tam revealed mutations in recipient complement genes in one case; no mutations were found neither in recipient nor in donor dna in two cases; instead, donor genetic alterations were found in 3 patients whose characteristics are summarized in table s283 donor hematopoietic cells. in the three cases presented, tam was relatively delayed with respect to hsct, in particular in two cases (6 months) and this timing is compatible with the concept of reticulo-endothelial 're-population' by donor cells of monocytic lineage, responsible for the production of regulatory proteins of the alternative pathway of the complement. we also underline the response to anti-c5 inhibition in the 2 patients who were treated with eculizumab; this fact further supports the hypothesis that the disease was related to complement dysregulation. we therefore suggest that both the recipient and the donor should be screened for complement gene mutations, so that more cases could be identified and the pathogenesis of tam could be further clarified. among these we observed one autologous engraftment, one death due to septic shock before engraftment and two primary gf. we used a desensitization treatment based on 4 plasma exchange procedures, intravenous immunoglobulin (1 g/kg) and rituximab (375 mg/sm) in 2 patients. one of these patients (aml, haploidentical donor) had dsa against hla-b50 (mfi 900). she experienced primary gf with increasing titles of dsa (maximum mfi 10 500); so, on day 38, a second transplant from the same donor was performed after a desensitization treatment. a progressive decrease in dsa was documented (up to mfi ⩽ 200). on day 12 patient achieved neutrophil count over 500/μl and on day 23 platelet count over 20 000/μl. the second patient (mds, haploidentical donor), instead, received a desensitization procedure before the first transplant. she had dsa against hla-a24 (mfi 3700), and after desensitization dsa levels decreased and reached 0. on day 20 patient achieved neutrophil count over 500/μl and on day 38 platelet count over 20 000/μl. dsa were detected in 1/9 of usct candidates (11%) and 4/17 of haplosct candidates (24%) and they were associated with failure to obtain allogeneic engrafment in 3 cases. desensitization treatment achieved dsa clearance and engraftment in the 2 patients in which it was performed, underlining the potential benefit of this procedure in the setting of hsct with dsa that has to be validated by prospective and controlled studies. disclosure of conflict of interest: none. early complications and late effects and quality of life at myeloma multiplex patients z trajkovska-anchevska, a pivkova, s genadieva-stavrich, l chadievski, z stojanoski, l chevreska and b georgievski university hematology clinic, skopje, macedonia the subject of this research is the quality of life at patients with myeloma multiplex at diagnosis and during therapy within 6-12 months. the research aims to analyze patients to be able to continue activities which will contribute for improving their quality of life as a priority task placed before the patient, his family, health institutions and social environment. this research was conducted at the university clinic for hematology skopje in the period from june 2009 to march 2012. it covers patients infected with multiple myeloma, diagnosed and treated during this period. a total of 80 patients analyzed, using the eortc qlq c30 ver. 3.0 standardized questionnaires for hr quality of life that analyzed the physical, cognitive, emotional, personal and social functions related to the patients. it also analyzed and general health and quality of life. analysis of physical functioning at diagnosis is 27.5 during treatment 59.5, significantly improved. personal functioning at patients at the diagnosis is 17.9, during therapy − 36.4. analyzing emotional functioning in patients at diagnosis is 39.9, during the therapy over 73.3 significantly improved. in examining the cognitive functioning is also a significant difference at diagnosis 55.2, during treatment 72.5. social functioning of the patients was 26.2 at the diagnosis; during the treatment grow to 50.8. significant improvement was notices in these patients' symptoms like fatigue, nausea and vomiting, pain, dyspnea, insomnia, loss of appetite, constipation and diarrhea. the analysis of the financial difficulties of patients at diagnosis is 76.2 and 72.5 during treatment, meaning no significant difference in the time given. the analysis of the overall health and quality of life at patient has a value of 23.9, and during therapy 58.8. quality of life at patients with myeloma multiplex that makes the research group was significantly improved as a result of on time diagnosis and treatment with modern medicaments and the role of social worker with the application of certain social skills, continuous counseling, guidance and education for their reintegration in the community. installing the quality of life as a separate category and investigating the factors that affect its expression in the daily functioning of the patients within the changed framework of action, as like this example for malignant disease. the needs of clearly defined interactions patient illness and treatment, quality of life and specifying the segments where it can effectively act and improve in order to achieve positive progression towards improving the qualitative features of this category is a clear and primary objective that must be inserted into the current approaches to monitoring patients with malignant hematological diseases. acute graft-versus-host disease (agvhd) is a common and severe complication after allogeneic stem cell transplantation. since the current first-line treatment is based on treatment with systemic glucocorticoids (gc), steroid-induced hyperglycaemia develops frequently in patients with (agvhd) potentially impacting on their outcome. we performed a retrospective analysis on 104 patients who received systemic gc for agvhd and thoroughly investigated the consequences of aberrant glucose metabolism. in particular, we focused on glucose parameters early after initiation of gc. with a median of 50 (range: 4-513) blood glucose measurements during gc treatment, increasing mean, median and maximum glucose levels as well as the need for insulin treatment were associated with decreased overall survival (os) in simple and multiple survival analysis. early hyperglycaemia, as defined by mean blood glucose levels 4125 mg/dl during the first 3 days of gc therapy, was also found to be highly associated with adverse outcome: in multivariate analysis, the hazard ratio (hr) for death was 2.5 (95% ci 1.32-4.87, p = 0.005) in patients with early hyperglycaemia. while the risk of death due to relapse was not increased, the hr for death due to non-relapse mortality was 3.26 (95% ci 1.53-6.92, p = 0.0021) in a competing risk analysis. a score based on early hyperglycaemia and non-response to gc within 7 days allowed the identification of three risk groups: patients with both risk factors had an inferior os at 5 years of 4.1% as compared to 75.4% in patients with none. patients with one risk factor had a 5-year os rate of 32.0% (p = 0.0002 for trend). in this retrospective study, we identified early hyperglycaemia after gc initiation as a prominent factor predicting increased nonrelapse mortality in agvhd patients. in addition, a score based on early hyperglycaemia and lack of response to gc was highly predictive for overall survival in these patients. disclosure of conflict of interest: none. early toxicity because of infectious complications not relapse is the main cause of death after allogeneic transplantation in aplasia for patients with refractory or relapsed acute myeloid leukemia high-dose cytarabin was given in 13/25 pts with induction failure. the search for a stem cell donor was started immediately after results of high-risk cytogenetic, no achievement of bone marrow aplasia on day 14 of induction therapy, or immediately after diagnosis of relapse. four patients had a related 10/10 donor, for 17 patients a 10/10 matched unrelated donor was identified and 10 patients received a transplant from a 9/10 unrelated donor. the interval between diagnosis of primary disease or relapse and tx was 3 (1-7) months (mo) for both groups . in 24 patients melphalan (100-140 mg/m 2 ) was used to induce an aplasia before starting conditioning therapy. the interval between melphalan and conditioning therapy was 13 (9-21) days. three pts started the conditioning therapy while in aplasia after previous chemotherapy. the conditioning therapy was of reduced intensity in all pts. and consisted of treosulfan (30 g/m 2 )/fludarabin(flu) in 19 pts, tbi(8gy)/flu in 7 pts and busulfan(8 m/(kg)/flu in 5pts, respectively.atg was given to all pts with an unrelated donor. most pts (21/31) had a severe neutropenia with a median of 0.3/nl ( 0.1-5.2) before starting melphalan because of refractory leukemia. after a median follow-up of 21 (4-68) mo 11 pts (35%) were alive without relapse. 6 (19%) pts died because of a relapse after a median of 6 (3-25) mo. the nonrelapse mortality was 45% (14/31 pts). most of these pts (10/14, 71%) died because of infectious complications early after transplantation (med 1; 0-19mo). in 4 pts graft versus host disease was the main cause of mortality. in this retrospective 'real-life' analysis, we showed that an early allogeneic transplantation is feasible for patients with primary refractory or relapsed aml. a reduced intensity conditioning after induction of aplasia with melphalan offers a chance of long-term relapse-free survival for about 30% of patients with an otherwise dismal prognosis. nrm is high, especially because of infectious complications early after transplantation, probably related to the long period of severe neutropenia. therefore, the focus has to be set on early recognition and intervention of infectious complications. disclosure of conflict of interest: none. recent evidence supports the effector role of complement activation in several types of thrombotic microangiopathythe atypical hemolytic uremic syndrome (ahus) as well as the transplantation-associated thrombotic microangiopathy (ta-tma). the blockade of the terminal complement complex formation by anti-c5 monoclonal antibody eculizumab provides an effective treatment option in severe and devastating cases of ta-tma. the experience with the use of eculizumab in this indication is slowly accumulating in the hsct community, however the published data originate from small case series or uncontrolled trials and sharing of emerging real-life observations may be valued. on case reports of two pediatric patients treated with eculizumab for ta-tma with very detailed followup of multiple complement parameters, including terminal complex sc5b-9 and eculizumab drug levels we would like to demonstrate: (1) achieving therapeutic levels of eculizumab (499 μg/ml) may be unsuccessful even with initially intensified dosing interval. furthermore, we documented rapid eculizumab clearance from circulation which allowed only for short periods ( o48 h) of efficient drug levels during the weekly dosing. (2) we did not observe tightly correlated sc5b-9 and eculizumab levels within the dosing intervals; however the long-term sc5b-9 formation suppression was achieved concomitantly with improved eculizumab levels and slowed drug clearance. (3) classical complement pathway activity assay (ch50) may not reliably substitute for therapeutic efficiency monitoring in case of hypocomplementaemia due to protein losses (profound diarrhea, proteinuria, gi bleeding, catabolism). this holds true also for the alternative pathway activity which remained low during treatment in both patients. (4) mycotic infections may represent serious therapy related risks in eculizumab treatment after hsct (both patients achieved control of complement activation after multiple doses of eculizumab, however suffered fatal infections subsequently). besides, we observed a significant increase in c3a concentrations correlated with clinical onset of infection which invites for further investigation of this complement cascade product as early indicator of mycotic infection. in conclusion, we would like to highlight the great added value of timely available complement assay results, including sc5b-9 and especially eculizumab drug level values-to be used together with detailed clinical parameters for directing effectively these highly personalized (and also costly) treatments. [p329] disclosure of conflict of interest: none. table 1 . no difference in terms of drug-related adverse events was observed in the three patient cohorts with no reported serious adverse events. similar results were obtained performing two separate sub-analysis only for lymphoma or myeloma patients. despite the limitations due to the non-randomized nature of the study, from our data on a large cohort of patients s286 with a long-term follow-up biosimilar filgrastim (zarzio®) could be considered substantially equivalent in terms of efficacy and safety to lenograstim (myelostim®) and peg-filgrastim (neulasta®), when used for hematological recovery and febrile neutropenia prophylaxis after asct in adult patients with hematologic malignancies. disclosure of conflict of interest: none. we studied all 107 adults who underwent allo-hsct during a 23-month period (01 january 2015 to 22 november 2016) in our center. a total of 6 pts (5.6%) received epag for pfg with thrombocytopenia. three pts were male, and three female. median age was 59 years (24-67). the baseline diagnoses were: alm (2), mds-raeb (1), idiopathic myelofibrosis (1), aa (1), and cll (1). three transplants were from family donor (all of them haplo-identical), and 3 from unrelated donor (the three of them hla 9/10). sc source was pb in 5 cases, and bm in 1. epag was started at 50 mg/day and escalated each 2 weeks to 75, 125 and 150 mg if platelet count was o50 × 10 9 /l. we analysed the platelets, anc, and hgb at epag initiation and 90 days after being with the maximum dose. median time between allo-hsct and eltrombopag initiation was 120 days (17-155). median maximum dose used of epag was 150 mg/day (125-150). median platelets, anc and hgb before starting treatment were 13 × 10 9 /l (5-28), 1 × 10 9 /l (0.07-11.2) and 8.6 g/dl (7.6-12.1), respectively. five patients (83%) were severely thrombocytopenic (platelet count ⩽ 20 × 10 9 /l), 4 (67%) were anemic (hbg o 10 g/dl), and 3 (50%) were neutropenic (anc o1.0 × 10 9 /l). median platelets, anc and hgb at day +90 of maximum dose were: 37 × 10 9 /l (8-108), 2.4 × 10 9 /l (0.93-9.62) and 11.8 g/dl (7.9-14.5), respectively. the 5 thrombocytopenic pts (100%) responded to epag, with increases of 120 00, 25 000, 28 000, 39 000 and 96 000 × 10 9 /l in the platelet count. three anemic pts (75%) responded and achieved increases of hgb of 1.1, 4.7 and 6.8 g/dl. finally, the 2 neutropenic pts (66.6%) responded and achieved increases of anc of 4080 and 9550 × 10 9 /l. at the moment of the analysis close, pts are at a median of +11.5 months post-hsct (8) (9) (10) (11) (12) (13) (14) (15) (16) , and all but one (who died from a septic shock) are alive and outpatient. this survival is striking for subjects who develop a complication with such a high expected mortality as pfg. pgf is a life-threatening complication, relatively frequent after alternative donor hsct, whose treatment has been very disappointed. we report our experience in pts who developed pgf during the last 2 years. epag induced responses in platelets in all pts of the studied group. bilineal and trilineal responses were also seen. in our opinion, prospective studies are warranted in order to confirm epag as a new efficient treatment of post-hsct poor graft function. disclosure of conflict of interest: none. s287 ing 19 patients who developed es with an equal number of patients who did not between january 2013 and november 2016. we analyzed variables such as cd34+ cells per kg infused, use of granulocyte colony-stimulating factor (csf-g) and engraftment day. analytical data, including baseline and maximum determination of serum glutamic oxaloacetic transaminase (got) and glutamic pyruvic transaminase (gpt), c-reactive protein (crp) and procalcitonin (pct), as well as clinical data fever, weight gain, digestive and respiratory symptoms, pulmonary infiltrates were analyzed. sixty-eight patients were women. median age was 59 years old (range: 39-73). patients were conditioned with beam (52%), melphalan 200 mg/m 2 (42%) and bcnu-tt (5%). nineteen patients developed es in our series, which correspond to eight percent of all asct. case and control groups were matched according to age, sex, diagnosis and conditioning regimen. the most prevalent baseline disease in the group with es was myeloma (42.1%), followed by mantle cell lymphoma (26.3%). all patients who developed es had fever, 79% skin rash, 37% respiratory symptoms, 16% pulmonary infiltrate an 9% digestive symptoms. a summary of the comparison of data analyzed in subgroups is shown in table 1 . we found significant difference in the percentage of weight gain (p = 0.007), increase of tgo (p = 0.0001), increase of tgp (p = 0.001) and increase the number of cd34+ cells per kg infused (p = 0.043), we found an inverse correlation between the number of cd34+ cells per kg infused and incidence of es. however, in terms of post-transplant csf therapy (p = 0.075) and crp and pct valor (p = 0.85 and p = 0.25, respectively) we did not find significantly difference to develop es. in our series, weight gain and tgo and tgp rise were risk factors for es development. therefore, we should be aware of es in patients who develop fever, elevated liver enzymes and weight gain during graft phase. we did not find a significant difference in crp and pct suggested in other studies. further studies are required to better characterize risk factors of es development. busulphan-based (155), melphalan-based (122), beam (58), tbi-based (22), and others (43). weight at hospital discharge was significantly lower than at admission (5.6% in allo-hsct, and 5.4% in auto-hsct). weight at day +100 was also significantly decreased compared with the admission (8.6% in allo-hsct, and 6.7% in auto-hsct). weight at day +100 was lower than the ideal for their sex and height in the allo-hsct setting. contrarily, among the patients undergoing auto-hsct, the weight at day +100 remained higher than the ideal for their sex and height in a high proportion of cases. regarding serum albumin, it was significantly decreased at discharge (9% in allo-hsct, and 17.5% in auto-hsct), but recovered values similar to admission at day +100. in the auto-hsct setting, prealbumin levels were significantly reduced at discharge (39%), and in lower proportion at day +100 (8%), compared with admission values. in the allo-hsct patients, prealbumin levels were significantly reduced at discharge (5%), but had been recovered at day +100, compared with admission values. disclosure of conflict of interest: none. recently, blood and marrow transplant clinical trials network has proposed a composite endpoint: gvhd-free, relapse-free survival (grfs) for hsct outcomes. this endpoint includes as event: iii-iv acute gvhd (agvhd), relapse, death or chronic gvhd (cgvhd) requiring systemic treatment. in the last embt annual meeting a redefinition of this endpoint was proposed changing cgvhd event from those patients with cgvhd requiring systemic treatment (the original one) to those with just severe cgvhd (the redefined one). we retrospectively analysed 603 patients consecutively transplanted (1995-2014) excluding non-malignant diseases, second allo-sct and those o16 years old age. we had generated two composite endpoints: in both iii-iv agvhd, relapse or death were considerated events but we defined grfs1 as the one with cgvhd event including those who required systemic treatment (as the original one) and in grfs2 just those with severe cgvhd (the ebmt redefined one). the median age was 49 years (16-69) and 59% (362) were males. other characteristics of patients are resumed in table 1 . with a median follow up for patients alive of 39 months , the median estimated survival in months and the % at +1 year and +2 years was: 114 months, 71% and 62% overall survival (os); 24 months, 58% and 50% event free survival (efs); 6 months, 35% and 26% grfs1; 11 months, 46% and 38% grfs2. 138 (23%) and 210 (35%) hadn't any event in grfs1 and in grfs2, respectively. in grfs1, event's incidence was: 90 (15%) for iii-iv agvhd, 170 (28%) for cgvhd, 151 (25%) for relapse and 54 (9%) for death; in grfs2 was 90 (15%), 65 (11%), 173 (28%) and 65 (11%), respectively. considering those patients with cgvhd as event in grfs1, 105 of them hadn't the event as cgvhd at the same time in grfs2 (since they had cgvhd requiring systemic treatment but not severe cgvhd). for these patients, the alternative event in grfs2 was: 72 without any event, 22 relapsed and 11 died. in the multivariate, the factors associated with better outcomes were: in grfs1 early ebmt stage (p o0.001 with early as reference; intermediate p factor with more impact in both, but it is interesting to point it out that haploidentical donor had an advantage in grfs1. these results are being validated in a large series and the definitive results will be available at the moment of the meeting congress. [p334] disclosure of conflict of interest: none. steroid refractory acute graft-versus-host disease (gvhd) remains a major complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). affected patients have a very poor prognosis. gvhd has been associated with transplant-associated thrombotic microangiopathy (ta-tam). endothelial damage mediated by radiation, viral reactivation, drug exposure or alloreactivity results in exposure of subendothelial collagen, activation of coagulation and small vessel occlusion to a degree that results in organ failure. complement is thought to be a major mediator of endothelial damage. although a consensus exists about the exceedingly high morbidity and mortality of ta-tam and diagnostic criteria have been converging to a consensus, no biomarkers to diagnose tam and predict outcome have been established. we hypothesize that a ta-tma, related to dysregulation of the alternative complement pathway correlates with organ damage. a retrospective analysis of 660 consecutive patients with hematological malignancies receiving an allo-hsct at the university hospital basel in the period from 2003 to 2013 was performed. data on the occurrence, risk factors and outcome of patients with ta-tma and the correlation with acute gvhd was collected. available biopsies of organs suspected to be affected by tam and/or gvhd will be performed. routine bone marrow biopsies for histological, immunohistochemical signs of ta-tam and complement activation will be analyzed. serum samples will be used to characterize markers of complement activation using plasma levels of c5b-9 and c5b-9 deposition in tissues biopsies. 660 patients (aml n = 260; all n = 152; mds/mpn n = 93; lymphoid neoplasm n = 85; plasma cell disorder n = 53; bone marrow failure n = 17) underwent myeloablative (n = 432) and non-myeloablative (n = 228) allo-hsct at a median age of 47 years (range: 19-71 years). forty-eight (7.3%) patients matched the established diagnostic criteria for tam (increased ldh, platelet count o50 g/l or o 50% of normal baseline, schistocytes 42 per high power field, creatinine increase). the median time to onset of tam was 36 days post-transplant (range: 22-67 days). subjects with ta-tam had significantly higher 3-year nonrelapse mortality compared to those without (47.8% vs 18.2%, p o0.001). grades 2-4 agvhd and cytomegalovirus viremia were independent risk factors for ta-tam, and serum ldh level 4500 u/l as well as arterial hypertension were early signs of ta-tma occurrence. patients with clinically relevant agvhd (⩾ grade 2) had more ta-tam than patients without agvhd (45% vs 24%; po 0.001). tam correlated with agvhd severity; the higher the agvhd grade, the more the patients who suffered from tam. allo-hsct recipients with grades 2-4 agvhd or cytomegalovirus viremia should be closely monitored for the presence of ta-tma. at the meeting first results of histological, immunohistochemical and complement activation analyses will be presented. disclosure of conflict of interest: none. hemorrhagic cystitis (hc) after stem cell transplantation (sct) can cause significant morbidity and prolonged hospitalization. early bleeding occurs almost exclusively when using cyclophosphamide (cy) (5-25% of cases), while late onset hc are classically attributed to bkv infection, and occurs up to 58% of patients (pts) receiving myeloablative haplo-sct who had positive bk viruria (1, 2). we retrospectively studied hc cases among pts submitted to haplo-hsct in our department. thirty-eight pts receiving an haplo-sct with post-transplant cy (pt-cy) were included (table 1) . prophylaxis for cy included hyperhydratation (3 l/m 2 of 0.9% saline) and mesna administration (200 mg for each 1000 mg of cy/daily divided into three doses). hematuria was graded as follows: grade i, microscopic; grade ii, macroscopic; grade iii, with clots; and grade iv, leading to urinary retention or requiring surgical intervention (1). pts with hc and clots were treated with continuous bladder irrigation. twenty-three pts (60.5%) developed hc at a median of 9.5 days post-sct (range: 1-57). clinical severity was grade i in 6 cases (26.1%), grade ii in 13 cases (56.5%), grade iii in 2 cases (8.7%) and grade iv in 2 cases (8.7%). at the onset of hc diagnosis, bk viruria was investigated in 13/23 pts. five pts (38.5%) had bkv negative (bkv − ) hc and 8 pts (61.5%) bkv positive (bkv+) hc. bkv-hc occurred after a median of 5 days (range: 5-52) while bkv+ hc after 14.5 days (range: 5-57), respectively (p = 0.06). among bkv+ pts, 4 received iv cidofovir 5 mg/kg once a week for 2 weeks and then once every 2 weeks. median number of administrations was 3 (range: 2-4). oral probenecid was given at the dose of 2 g 3 h before and 1 g 2 and 8 h after cidofovir administration. two pts obtained a complete response (cr) after 70 and 110 days, respectively, one patient reached a partial response after 31 days and one pt failed to obtain a response. no pts developed renal toxicity during treatment. one pt received ganciclovir for concurrent cmv viremia and bkv+ hc resolved in 55 days. three patients did not receive any treatment for mild or asymptomatic cystitis. all of them achieved remission after a median of 10 days from the onset (range: 5-57.) among bkv-hc, 3 pts obtained spontaneous resolution after a median of 4 days (range: 1-52), while two pts died early after sct. finally, among pts for whom bk viruria was not available, a remission was reached in 6 of them after a median of 28.5 days (range: 12-43), while 4 pts died early after sct. in our cohort of pts, hc occurrence was of 60.5% and bkv was responsible for the 61.5% of cases. contrary to its high incidence, hc showed a relative benign course, with an overall remission rate of 87.5%, regardless of treatment. finally, we found a trend for a longer interval between sct and hc onset in pts with bkv+ hc, as compared to cy-related hc (p = 0.06). sos is a rare and serious complication of hematopoietic stem cell transplantation (hsct). it is diagnosed using the modified baltimore criteria of hyperbilirubinemia, weight gain or ascites 45% over baseline, hepatomegaly or right upper quadrant pain of liver origin. only defibrotide has been approved for the treatment of veno-oclusive disease. hdmp has been described as effective sos therapy in a few case series (1, 2). we describe our experience of treating adult sos using hdmp. objective is to retrospectively analyze the treatment efficacy and overall survival of patients diagnosed with sos after hsct and treated with hdmp. we used vilnius university hospital data base to identify patients diagnosed with sos under baltimore criteria and treated with hdmp over 2007-2016 period. patient demographics, transplant and clinical data, response, survival (kaplan-meier survival analysis) and hdmp infusion related complications were analyzed. we identified 11 patients (9 males) of whom 10 had had allogeneic hsct (6 reduced intensity conditioning) and one had received a double autologous hsct. sos was diagnosed on the median day +22 (+7 to +81 days). the median bilirubin value was 61.7 μmol/l (11.1-137 μmol/l). all patients had liver enlargement of median 210 mm (160-235 mm) on ultrasound. two patients had normal bilirubin values but displayed the remaining signs and symptoms of sos at diagnosis. patients received intravenous methylprednisolone 500 mg/m 2 every 12 h for 3 days. none received defibrotide. seven (64%) patients responded on median day +12 (+3 to +20 days) after the start of hdmp. four responded by decrease in serum bilirubin by 50% and resolution of symptoms without the need of further treatment. the remaining three responders received maintenance treatment after one course of hdmp with reduced doses of methylprednisolone until resolution of symptoms. four patients failed to respond and died of multiorgan failure on median day +12 (+5 to +41). the median observation time was 6 months (0-44 months). the median overall survival for the sos group was 8 months (range: 0-18) and it was 27 months among the responders. no adverse reactions related to hdmp infusion were observed. hdmp therapy in adult sos results in clinically relevant response rate. further prospective trials are required to assess hdmp efficacy in comparison to defibrotide or as add on therapy. prevalence of hypertension (ht) in general pediatric population is~4%, while in children treated with hematopoietic stem cell transplantation (hsct) it is up to 30%. we assessed factors contributing to the development of ht in children treated with hsct and usefulness of ambulatory blood pressure monitoring (abpm) in this population of patients. the study included 30 children (21 boys, 9 girls; mean age 10.9 years) treated with hsct for neoplasms (n = 22; 73%) or non-neoplastic disorders (n = 8; 27%). control group included 19 children (8 boys, 11 girls; mean age 12 years). abpm measurements (spacelab device) were performed before hsct and after a mean of 7 months after hsct (in 16 of the 30 children). blood samples were collected from 10 children treated with hsct and all controls. total rna extraction was performed and microarray analysis was conducted using genechip human gene 1.0 st arrays (affymetrix). in patients after hsct no antihypertensive treatment was used. mean systolic blood pressures (sbp) before and after hsct did not differ significantly from the control group. mean diastolic blood pressures (dbp) before and after hsct were 68.7 ± 12.2 mm hg and 65.4 ± 9.42 mm hg, respectively, and mean dbp percentiles were 77.1 ± 7.9 and 78.3 ± 6.7, respectively; the differences between the study group and the control group were significantly higher before hsct. mean 24-hour arterial pressure (map) percentiles were 83.3 ± 11.1 and 79.2 ± 8.7, respectively; the differences between the study group and the control group were significantly higher before hsct. before hsct and after the procedure, the european society of hypertension criteria for high normal blood pressure (bp) and ht were fulfilled in 16%/12% patients and 20%/0% patients, respectively. nocturnal bp decrease o 10% was found in 46%/ 53% patients and 420% nocturnal bp decrease in 3%/7% patients, respectively. in the control group o 10% nocturnal bp decrease was found in 10% of children and 420% nocturnal bp decrease in 5% of children. when the groups of patients before and after hsct were compared, highly significant differences were found in gene expression levels for mthfr ( in children referred for hsct a trend towards higher bp values was seen. in children assessed 6 months after hsct more abnormalities in nocturnal bp measurements were seen, which may be a predictor of ht. in children treated with hsct significant differences in the expression of ht-related genes were found. abpm was useful in bp monitoring in children treated with hsct. hypothyroidism may complicate of allogeneic hematopoietic stem cell transplantation (allo-hsct); risk factors are analysed. we studied 229 patients with aml who underwent an allo-hsct between 2003 and 2013 with different conditioning regimens (myeloablative, reduced-intensity, chemotherapybased, total body irradiation-based). thyroid stimulating hormone (tsh) and free thyroxin levels (ft4) were available in 104 patients before and after allo-hsct. median age at transplantation (n = 104) was 47 years (iqr 40-59), 37 (35.6%) were female and overall mortality was 34.6% (n = 36) ( table 1) ursodeoxycholic acid (udca) has been shown to have a protective effect in the liver complications after allogeneic stem cell transplantation (allo-sct), but it also has other immunomodulatory effects; it has been described also a potential benefice as graft-versus-host disease (gvhd) protection. we retrospectively analysed 618 patients consecutively transplanted between 1995-2014 excluding second allo-sct and those o16 years old. we analysed the differences between those with and without prophylactic udca using spps v20. results: the median age was 49 years (16-69) and 59% (362) were males. other patient characteristics are resumed in table 1 objective of study was to evaluate the impact of disease status on the outcomes of allogeneic hematopoietic stem cell transplantation (allo-hsct) in the treatment of patients with refractory and relapsed acute lymphoblastic leukemia(all). 52 patients with refractory and relapsed all, including 19 cases in advanced stage (nonremission, nr) and 33 cases in more than or equal to second complete remission(⩾ cr2), received allo-hsct after myeloablative conditioning regimen in our department. results: 51 patients engrafted successfully. the transplantation-related mortality (trm) rate of nr and ⩾ cr2 was 10.5% vs 12.1% (p = 0.815). the incidence of agvhd was 52.6% vs 57.6% (p = 0.730), including 42.1% vs 33.3% (p = 0.527) with mild (grade i-ii) and 10.5% vs 24.3% (p = 0.399) with severe (grade iii-iv) agvhd. the incidence of cgvhd was similar also(41.6% vs 57.9%, p = 0.660). with a median follow-up of 12(1.8-44.5) months, the cumulative relapse rate of nr and ⩾ cr2 was 47% vs 34.3%(p = 0.425), respectively. the estimated 2 year overall survival (os) and 2 year leukemia-free survival (lfs) rate were 42.6% vs 45.7% (p = 0.487) and 46.3% vs 46.2% (p = 0.571), respectively. multivariate analysis results showed that cgvhd was independent favorable risk factor for os and lfs of r/r all. for relapsed patients, os was significantly better with first cr duration 46 months and time to transplant ⩽2 months. alio-hsct is an effective salvage treatment option for patients with refractory and relapsed all. our retrospective analysis showed that r/r all with different status prior transplant had similar outcome post transplantation. disclosure of conflict of interest: none. the deleterious effect of intensive care unit (icu) admission during hematopoietic stem cell transplantation (sct) on patient survival is well established. however, it is unknown whether admission into the icu during the chemotherapy for the underlying disease has any impact on survival after sct. we reviewed patients who had received a first sct between the years 2000 and 2016 in our institution, and we compared the overall survival (os), relapse incidence (ri) and non-relapse mortality (nrm) between patients who required icu admission during the chemotherapy prior to the sct (icu group) with matched patients (1:2) who did not need it (no-icu group). sixty-six patients were included, 22 of them in the icu group and 44 in the no-icu group. as shown in table 1 , the main clinic-biologic variables and the sct procedure were comparable between the patient groups. the causes of icu admission for the icu group patients were: 11 (50%) respiratory failure, 4 (18%) septic shock, 4 (18%) neurological disturbance, 2 (9%) post-surgery and 1 (5%) tumor lysis syndrome. seventeen patients (77%) needed mechanical ventilation. the median time between icu admission and the sct procedure was 144 days (range: 106-1097), and the median days of icu stay were 12.5 . with a median follow-up after sct of 5.47 years (0.50-16.22) for the icu group and 4.52 (0.73-15.85) for the no-icu group, the 5 year os (ic 95%) probabilities were 49% (28-70%) and 45% (29-61%) in the icu and no-icu patients (p = 0.353), the 5-yr probabilities of relapse were 34% (14-56%) and 42% (25-58%)(p = 0.755) and the 5-yr probabilities of nrm were 32% (14-52%) and 24% (12-38%)(p = 0.333), respectively. there were no differences in either os, ri or nrm between icu and no-icu in the allogeneic or autologous subgroups considered separately. at the moment of the study, s295 12 (54%) of icu and 22 (50%) of no-icu group had died. the causes of death in the icu group were: relapse in 5 (42%), infection in 4 (33%), gvhd in 1 (8%) and gvhd plus infection in 2 (17%). the causes of death in the no-icu group were: relapse in 8 (36.4%), infection in 4 (18.2%), gvhd in 3 (13.6%), gvhd plus infection in 5 (22.7%) and veno-occlusive disease and secondary malignancy, one each (4.55%). in this series, admission to the icu before sct did not have an impact on outcomes after sct. these results suggest that these patients benefit from this treatment as much as the other patients, without expecting worse outcomes as a result of a previous icu admission. supported in part by grants rd12/0036/0029 (rticc, fejer), pi14/01971, instituto carlos iii, and sgr225 (gre), spain. disclosure of conflict of interest: none. autologous stem cell transplant (asct) is a well established treatment for several haematologic and non haematologic malignancies, either as front-line or rescue therapy. however it is associated with toxicity and complications which might lead to treatment-related mortality (trm). although decrease in trm has been reported, data about the precise reduction and detailed analysis of causes of mortality throughout years are scanty. the aim of this study was to evaluate early trm and its causes in patients who underwent an asct in a single center along the last three decades. data of all consecutive adults (415 years old) asct recipients were prospectively collected at a single center from december 1988 to august 2016 and then retrospectively analysed. trm was defined as mortality happened into the 100 days post asct or during conditioning treatment due to any cause except relapse or progression of main diagnosis. demographic characteristics, diagnosis, conditioning regimen and cause of death were analysed. data were compared for two periods: from december 1988 to december 2000 and from january 2001 to august 2016. a total of 849 patients were included, median age was 45 years (16-71) and 50.3% were male. diagnoses were: lymphoma (n = 391), multiple myeloma (mm) (n = 216), acute myeloid leukaemia (aml) (n = 93), amyloidosis (al) (n = 15), acute lymphoblastic leukaemia (all) (n = 39), solid tumours (including breast cancer and germ-cell tumours) (n = 89), chronic myeloid leukemia (cml) (n = 3), thrombotic thrombocytopenic purpura (ttp) (n = 2) and autoimmune disease (n = 1). the most frequent indication for asct was lymphoma (46.1%) and mm (25.5%). twenty patients died within 100 d from asct (trm). demographic characteristics and causes of death for this patients are shown in table1. the cumulative incidence of trm at day +100 was 2.8% (95% ci 1.9-4.1). comparing both periods, trm cumulative incidence was 7.9% (95% ci 4.9-11.8) in first period (1988-2000) vs 0.8% (95% ci 0.3-1.8) in last period (2001-2016) po 0.001. (figure 1 ). according to main diagnosis trm cumulative incidence was higher in patients diagnosed with solid tumour 6.7% (95% ci 2.7-13.2) and al 6.7% (95% ci 0.4-26.9) followed by acute leukaemia (aml/all) 4.5% (95% ci 1.9-9.1), mm 2.8% (95% ci 1.1-5.6) and lymphoma 1.3% (95% ci 0.5-2.8) p o0.03 (figure 2 ). sepsis (65%) was the main cause of death in both periods of time, and the only one cause of death in the last period. the second cause was sinusoidal obstruction syndrome (sos/vod) (20%), which only appeared in the first period. this study shows a low incidence of trm in asct recipients, with a significant decrease in the last period (2001-2016), despite the higher risk in some groups of patients such as those with amyloidosis and solid tumours. in our experience, infection is the main cause of early death in asct recipients and sos/vod has disappeared in last years as a cause of early transplant related mortality. disclosure of conflict of interest: none. incidence and risk factors for hepatic sinusoidal obstruction syndrome after allogeneic hematopoietic stem cell transplantation: a retrospective multicenter study of turkish hematology research and education group (threg) hepatic sinusoidal obstruction syndrome (hsos) is a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). the mean incidence of hsos was found to be 13.7% (0-40%) in the literature. we examined the incidence and risk factors for hsos after allo-sct. eight centers from turkey were enrolled in the study. we retrospectively evaluated the medical records of patients who were treated with allo-sct between january 2012 and december 2015. a baltimore criterion was used for assessment of hsos. two hundred eighty three (96%) of 295 patients who were treated with prophylaxis with defibrotide alone or one or more of the n-acetylcysteine, diuretics and heparin used defibrotide (10-25 mg/kg/day). the study included 889 patients (514 males/348 females) with median age of 37 (15-71) years. the demographic and clinical characteristics of patients were summarized in table 1 . the incidence of hsos was 9.3% (83). prophylaxis for hsos was used in 40 (48.1%) of patients, who developed hsos. defibrotide as prophylaxis was received by 32 of 40 (80%) of patients. hsos developed in a median of 13 (0-34) days after stem cell infusion. seventy-five (90.3%) of patients who developed hsos had infection at the time of diagnosis. forty-five of them had ascites, 63 had hepatomegaly and, 74 had weight gain. seventy-two (86.7%) of patients with hsos were treated with defibrotide after diagnosis. the median time of starting defibrotide in these patients was 14 (2-29) days. thirty-six (43.3%) of patients with hsos recovered completely and forty-seven (56.7%) of them died as a result of multi organ failure. the incidence of hsosrelated mortality in allo-hsct cohort was found to be 5.3%. in univariate analysis, statistically significant associations were not found between hsos incidence and age/sex of recipient, type of conditioning regimen, stem cell source and type of gvhd prophylaxis. on the other hand primary diagnosis of myelofibrosis, donor type, engraftment status and prophylaxis for hsos were significantly associated with hsos development. hsos prophylaxis was significantly decreased hsosassociated mortality (p = 0.006). hsos still remains a serious life-threatening complication of allo-sct. although the incidence is low, hsos is associated with increased 100-day nonrelapse mortality. hsos prophylaxis especially with defibrotide, seems to reduce hsos associated mortality in high risk patients. [p346] disclosure of conflict of interest: none. hemorrhagic cystitis (hc) is a serious complication occurring after allogenic hematopoietic stem cell transplantation (hsct) more frequent on haploidentical (haplo) hsct, with an incidence of 10-70% 1 associated mainly with the effect of cytotoxic agents such as cyclophosphamyde (cy). the conditioning regimen, bkpyv infection and graft versus host disease have an implication in the incidence. other authors related the reactivation of cmv and a previous transplantation as risk factors to hc development2. with this study we aim to describe the hc incidence and risk factors in all haplo-hsct performed in the canary islands. we analyzed all consecutive haplo-hsct from family donors performed at our hospital between 2013 and 2016. the conditioning regimen used for the transplant was the hopkins haplo protocol with high dose cy (50 mg/kg on days 3 and 4) posttransplantation (ptcy). we used as hc prophylaxis intense hydratation on the cy administration day and the following 24 h (using bladder wash only in 1 patient with cardiac dysfunction) and perfused mesna at 100% of cy dose beginning 15 min before the cy administration on 16 pts and at 20% of the last dose at 0, 4 and 8 h on all pts. we used spss v.23 to determine the cumulative incidence (ci) of hc. we performed 20 haplo-hsct, of which 10 were males (1 was transplanted 3 times) and 8 were women. the mean age was 40 (range: 16-64). the pts presented the following diagnosis: aml (10), all (1), eh (5), nhl (3), am (1). 45% of pts received the haplo-hsct in remission, 50% with refractory disease and 5% of pts did not receive previous treatment. 6 pts developed hc (36.5% ci at day +80) (figure 1a ) with a median time from haplo-hsct to onset of 23 days (range: 3-42), 1 (17%) was grade i, 4 (66%) grade ii and 1 (17%) grade iv. the grade i case did not received the mesna infusion like most of the other pts. no pts died due to hc and all cases resolved without sequelae. 12 pts received cy pre-and post-transplant and only 8 pts received ptcy. the ci at day +80 for the pts with ptcy was 33.3% and for cy preand post-transplant 38.3% (figure 1b) . we found no statistically significant difference on the ci of hc between these two groups. the development of hc was related to cy in 1 patient, who suffered from this complication on the second and third haplo-hsct. for the rest of the pts (after day +30) the hc was related to bkpyv infection, as a consequence of the immunosuppression state of the patient, we also observed all these pts had positive serum viral load for cmv. the incidence of hc associated to post-hsct high cy dose in our series is 15% lower than other ones. most of them on grade 1 or 2 and without mortality associated. the risk of hc is high, particularly in the setting of highly pre-treated patients (especially those undergoing a 2nd transplant). the development of hc after day +30 is evidently associated to bkpyv as a contributing factor for continuous inflammation and cmv reactivation (as an immunosuppression marker). in our study, hc did not have an impact on mortality of high-risk patients after haplo-hsct. the hc remains frequent with a high morbidity in particular when it is severe, often causing prolonged hospitalization and resource use. we need further studies to recognize the at-risk population early. [p347] ta-tam is not a rare post-transplant complication and it is potentially fatal. in survivors, it was often associated with longterm morbidity and chronic organ damage, mostly to the kidney with poor renal prognosis. our retrospective study showed ta-tam associated risk factors included t reg haplo hsct as the incidence was highest in this group, tbi-based conditioning or tbi based conditioning plus cyclophosphamide. although acute gvhd and infection were associated with ta-tam in retrospective studies, no association emerged between acute gvhd or infection preceding diagnosis in our series of patients. in order to prevent ta-tam we need to understand its underlying biological mechanism so we are investigating its pathogenesis by means of cytokine assays, histology and murine models. disclosure of conflict of interest: none. mortality in children requiring invasive mechanical ventilation (imv) after allogeneic hematopoietic stem cell transplantation (hsct) is known to be high. little is known about the longterm outcome of those who survive imv. we therefore reviewed the medical records of 55 children who survived s298 imv after they had received a hsct between 2000 and 2012 in the two pediatric hsct centers in the netherlands. retrospective multi-center cohort study in two university hospitals that perform all pediatric hscts in the netherlands. long-term survival of hsct recipients who had received imv was assessed. health status was reviewed more in detail for those who were still alive 2 years after discharge from the pediatric intensive care unit (picu). in the absence of standardized use of quality of life questionnaires, health status was expressed as the number of affected domains (cardio-respiratory, motor and miscellaneous, regardless of the degree of dysfunction) and level of education. health status was categorized as follows: no health problems when all four domains were normal; mild health problem when there was an abnormal score in one of the four domains; moderate health problems when there was an abnormal score in two domains; severe health problem when there were abnormal scores in three or all four domains. between january 2000 and december 2012, 641 patients underwent a hsct in the two study centers together. a total of 89 hsct recipients received imv within 1 year after hsct (14% of all hsct recipients). median time between hsct and picu admission was 59 days (iqr 17-102 days). the most common indication to start imv was respiratory failure (73%). median duration of imv was 10 days (iqr 5-18 days). 34 patients (38%) died during their picu admission. of the 55 patients who were discharged alive from picu, 27 patients were still alive 2 years after picu discharge (49% of those who survived picu admission). health status of these long-term survivors was assessed in december 2014 by hospital database review, using the most recent hospital contact. follow-up time varied from 2 to 11 years (median 6.5 years) after picu discharge. two patients (8%) had no health impairment, eight patients (33%) had mild health problems, five patients (21%) had moderate health problems, and nine patients (38%) had severe health related problems. very little is known about long-term mortality and morbidity of hsct recipients who survived imv. survival of picu treatment in pediatric hsct recipients is limited. however, long-term outcome of those who survive picu treatment seems promising: a considerable proportion of them still is alive 2 years later without obvious sequelae. this is the first study which assessed long-term outcome of imv after hsct. further studies in this population are urgently required to counsel parents and to optimize quality of life outcomes in these children. disclosure of conflict of interest: none. long-term surveillance data support lack of increase in mortality or cancer risk in brincidofovir clinical trial participants m morrison, k fitzgerald 1 , t brundage, a harrison and wg nichols 1 chimerix brincidofovir (bcv) is an orally bioavailable lipid conjugate of cidofovir (cdv), with broad-spectrum activity against doublestranded dna viruses, including cytomegalovirus (cmv), adenoviruses (adv), polyomaviruses (bk and jc viruses), and orthopoxviruses. bcv is being evaluated for prevention of cmv and other dna viruses in high-risk hematopoietic cell transplant (hct) recipients, and for the treatment of serious adv infection. bcv is also being developed for the treatment of smallpox under the us fda's animal efficacy rule. because bcv, cdv, and other marketed nucleoside analogs are reported to be carcinogenic in rodents, a registry was established to evaluate the long-term safety of bcv in subjects who have participated in bcv clinical studies. to date, the registry includes prior participants from study 301 (a placebo (pbo)-controlled study of bcv for cmv prevention) and study 304 (a single-arm study of bcv for adv treatment). subjects are encouraged to consent for long-term follow-up in the registry following participation in bcv clinical studies. registry participants are followed at 6-month intervals for a minimum of 3 years from the time of completion of the parent study. development of malignancies (new or relapsed), lifethreatening or fatal adverse events (aes) assessed as potentially related to bcv, and subjects' vital status are collected. a total of 649 subjects were enrolled in the parent studies (302 bcv and 148 pbo from study 301, 199 bcv from study 304). of these, 291 are enrolled in the registry as of 24 october 2016 (155 bcv and 80 pbo from study 301, 56 bcv from study 304). bcv recipients in the registry are 60% male, 84% white, with a median age of 47 ( o1-76) years, similar to the bcv recipients in the parent studies. the median duration of follow-up is 12 (0-30) months, with 80% of subjects continuing in follow-up at the time of analysis. all-cause mortality from the time of first dose in the parent study through current registry follow-up is 25% for bcv vs 22% for pbo (p = 0.559) in study 301, and 51% for bcv in study 304. all-cause mortality in the registry since completion of the parent study is 21% bcv vs 24% pbo for subjects from study 301 (p = 0.618) and 14% bcv for subjects from study 304 (figure 1) . the incidence of a new malignancy in registry subjects from study 301 is 17% bcv vs 23% pbo (p = 0.295), and the incidence of relapsed primary malignancy is 12% bcv vs 21% pbo (p = 0.055). in registry subjects from study 304, 7% developed a new malignancy and 4% had a relapse of the primary malignancy. no bcv-related life-threatening or fatal aes have been reported to date in the registry. registry data collected to date support no increase in late mortality or increased risk for carcinogenicity in patients treated with bcv. long-term surveillance for cancer risk and other drivers of mortality is important for novel compounds undergoing development in hct and other immunocompromised patient populations, with high background risk for these outcomes. [p351] disclosure of conflict of interest: all authors of this abstract are employees and stockholders of chimerix. hematopoietic stem cell transplantation (hsct) is a medical procedure that allows the cure of many paediatric diseases. it has been described an increased risk of new malignancies in this population and it represents an important cause of late mortality. we analyzed the late evolution of 371 patients submitted at pediatric age to hematopoietic transplantation (hsct) (allogeneic or autologous) in santa creu i sant pau hospital between 1984 and 2013. a total of 434 hsct was analyzed. it has been calculated the cumulative incidence of secondary malignancies at 30 years of follow-up. it has been done univariate and multivariate analysis of risk factors through χ 2 -test and binary logistic regression method (odds s299 ratio, or). it has been studied the relative risk (rr) for new malignancies through comparison of observed cases in our cohort with the expected cases in the general population. we observed 19 cases of secondary malignancies with a cumulative incidence of 6% at 15 years, 12% at 20 years and 36% at 30 years of follow-up. the risk was higher of expected in general population for each tumor type and in the different range of age, being the rr for malignancies in our cohort of 51.4 at 30 years of follow-up. solid tumors were the most prevalent malignancies (16 out of 19 cases). the median time of latency from hsct to diagnosis of malignancy was 16 years (1-31 years) . the thyroid tumors were the later ones and hematologic malignancies the earliest to be developed. chronic graft versus host disease was a statistically significant risk factor in univariate (or = 16; p = 0.006) and multivariate analysis (or = 15.4; p = 0.000). total body irradiation of conditioning was a significant risk factor only in multivariate analysis (or = 4.3; p = 0.03). previous radiotherapy was a significant risk factor only in univariate analysis (or = 3.1; p = 0.04). mortality was 42% (8 out of 19) between patients with a new malignancy and it was the cause of death for all the cases. we observed an incidence of secondary malignancies after hsct of 5.1% that is significantly higher compared to the expected in the general population (p = 0.000). the factors that have been related to an increased risk were chronic gvhd, tbi and previous radiotherapy. microalbuminuria defined, as urinary albumin: creatinine ratio (acr) of 30-300 mg albumin/g creatinine is a marker of endothelial dysfunction and inflammation. in general populations albuminuria predicts development of chronic kidney disease (ckd) and cardiovascular disease (1). in the general population microalbuminuria is associated with the metabolic syndrome (2) . in patients with hypertension, diabetes and the critically ill, it is a marker of adverse events and poor outcomes. following hematopoietic cell transplant (hct), microalbuminuria at day 100 was associated with a four-fold increased risk of chronic kidney disease (ckd) at 1 year in a single centre study; macroalbuminuria at day 100 was associated with a 6.8-fold increased risk of non-relapse mortality (3) . international guidelines for adult and children survivors of hct recommend that proteinuria is assessed at least annually (4, 5) . there is a paucity of data on the prevalence and implications of micro and macroalbuminuria in long-term survivors (410 years) of adult hct, however. this was a single-centre retrospective study conducted in patients attending a dedicated clinic for long-term (minimum 10 years) survivors of hct. we investigated prevalence of albuminuria and its association with renal disease, cardiac disease and the metabolic syndrome. of 55 patients, 8 were treated for acute leukemia, 2 for aplastic anaemia and 46 for cml. the median follow up time was 24 years (range: 13-37 years) and the median age at follow up was 55 years (range: 24-81 years). for 36/55 urinalysis was normal (group a) and 19 (34%) had microalbuminuria (group b). none had macroalbuminuria. group b were significantly more likely to have ckd grade 2-4 (egfr o 60) compared to those in group a (p = 0.001). group b patients were significantly more likely to have diabetes or impaired glucose tolerance 7/19 (37%) vs 2/35 (6%) in group a (p = 0.005). group b patients were also significantly more likely to have dyslipidaemia (p = 0.019 ) with 14/19 (70%) affected vs 23/35 (37%) in group a. cardiac disease and hypertension were more frequent in group b, 4/19 (21%) and 7/19 (37%), respectively vs group a 3/35 (9%) and 8/35 (22%) but these data were not statistically significant. the more features of the metabolic syndrome present, however, (elevated hba1c, /glucose, dyslipidaemia, hypertension) the more likely a patient was to have microalbuminuria (p = 0.007). our data demonstrates that microalbuminuria is a frequent finding in long term survivors of hct. patients with microalbuminuria are more likely to have ckd grade 2 or below. they are also more likely to have diabetes and dyslipidaemia. as this was a retrospective study we are not in a position to comment on whether microalbuminuria is predictive of the development of renal disease, metabolic syndrome or cardiovascular disease in this group of patients. this warrants further study as intervention, for example with ace inhibitors, may have the potential to reduce morbidity. the purpose of the study is the improvement of transplantation techniques and supportive care lead to an increasing number of long-term survivors after allogeneic hematopoietic stem cell transplantation (ahsct). recipients of ahsct have a higher prevalence of cardiovascular risk factors. ambulatory blood pressure measurement (abpm) is the 'gold standard' to diagnose arterial hypertension (ht). the prevalence and treatment control of ht by abpm is unknown in ahsct patients (pts). this prospective single center study at university hospital basel included all pts ⩾ 1 year after ahsct in complete hematological remission during annual follow-up consultation. office blood pressure (obp) was measured on both arms after 5 minutes rest. abpm by noninvasive continuous bp monitoring (pulse transit time method) was performed on the same day. ht was defined as obp ⩾ 140/90 mm hg, mean systolic bp ⩾ 130mm hg on abpm s300 (bp 24 ) and/or current use of antihypertensive drugs. 175 pts (39% female) were included with median age of 53 years (range: 19-75) and 9 years (range: 1-33) after transplantation. 108 (62%) pts received total body irradiation-based conditioning, 70 (40%) pts had chronic graft-versus-host disease, and 39 (22%) required immunosuppression. mean bmi (kg/m 2 ) (± sd) was 25 ±5, with 22 (13%) pts 430. twenty-seven (15.4%) pts were current smokers. fourty-three (25%) pts had chronic kidney disease (egfr o60 ml/min/1.73 m 2 ) and 17 (10%) diabetes. 82 (47%) pts were on antihypertensive drugs consisting of ace/at-ii-inhibitors in 55 (31%), calciumchannel blockers in 18 (10%), beta-blockers in 32 (18%) and diuretics in 24 (14%) pts. thirty-nine (22%) pts were on ⩾ 2 drugs. among our cohort 47 (27%) pts were normotensive without antihypertensive treatment (mean age 46 ± 13 years, 62% female and mean bp 24 (systolic/ diastolic bp) 113 ± 8/76 ± 8 mm hg). 128 (73%) pts were hypertensive and/or on antihypertensive treatment. untreated ht was diagnosed in 46 (26%) pts (mean age 52 ± 13 years, 41% female and mean bp 24 of 147 ± 22/91 ± 12 mm hg), including 14 (8%) with white-coat hypertension and 9 (5%) masked hypertension (normal obp, high abpm). in the group of pts with current antihypertensive medication 32/82 (39%) were controlled (mean age 55 ± 13 years, 25% female, and mean bp 24 119 ± 9/77 ± 7 mm hg) whereas 50/82 (61%) were hypertensive on abpm (mean age 55 ± 11 years, 24% female, mean bp 24 146 ± 13/90 ± 10 mm hg). thirty-four (68%) pts with uncontrolled ht were already hypertensive at obp. although long-term survivors after ahsct are known to be at elevated cardiovascular risk, diagnosis of arterial hypertension was missed in every fifth patient. the proportion of controlled hypertension is poor with only 39%. disclosure of conflict of interest: none. myasthenia gravis (mg) is a rare complication of allogeneic stem cell transplantation (sct) and is often associated with graft-versus-host disease (gvhd). we report a 49-year-old man who presented with oculobulbar and neck weakness 30 months after an unrelated donor, allogeneic sct for chronic myeloid leukaemia (cml). he was diagnosed in 2009 with chronic phase cml. this responded poorly to tyrosine kinase inhibitors (tkis) and he was found to carry the t315i mutation with additional monosomy 7. he underwent a fully hla matched unrelated donor sct with y 90 -anti cd66 targeted radiotherapy, fludarabine, melphalan and alemtuzumab conditioning. he had grade 1 cutaneous gvhd on ciclosporin withdrawal but no other significant gvhd. he has an immune mediated neutropenia since 4 months post sct and has reduced immune reconstitution as demonstrated by a sub-normal absolute cd4 level. he remains on pneumocystis prophylaxis and has not experienced increased infection. chemotherapeutic agents have a cytotoxic effect on the oral mucosa and is a major problem following cancer treatment. cooling the oral mucosa in conjunction with chemotherapy infusion, using ice chips, is known to reduce the severity of oral mucositis (1, 2) . although effective, ice chips are perceived as uncomfortable. the aim of the present study was to determine the optimal cooling temperature to prevent adverse effect of chemotherapeutic agents using tissue engineered oral mucosal models (teom). teom were incubated at 35°c, 30°c, 25°c or 20°c for 30 min followed by exposure to 162 μg/ml of 5-fu for 2 h (control models were incubated at 35°c). teom were then washed and further incubated for 48 h at 37°c co 2 . cell viability and inflammatory cytokine production (il-6 and tnf-α) were measured using (prestoblue) and (elisa), respectively this study demonstrates an increased capacity to restore cell viability with decreasing temperature (figure 1a ). teom treated with 5-fu further showed an increased secretion of the pro-inflammatory cytokines tnf-α and il-6 at all temperatures compared to un-treated controls. for il-6, secretion increased markedly when cells were incubated with 162 μg/ml 5-fu at 35°c and 30°c compared to cells incubated with medium alone at 35°c (figure 1b) . for tnf-α, secretion was significantly higher (p o0.05) in cells treated with 162 μg/ml 5fu at 35°c compared to untreated mucosal models and mucosal models treated with162 μg/ml 5fu but incubated at 20°c (figure 1c ). teom models incubated at 20°c has an increased capacity to restore cell viability following exposure to 5-fu. incubation at 20°c further reduces the release of pro-inflammatory cytokine compared to those incubated at 35°c. (2) and one received fludarabine and cyclophosphamide. all patients received campath-1h as part of the conditioning regimen. stem cell source: peripheral blood stem cells 73 patients and 3 bm. comorbidity was assessed using the haematopoietic cell transplantation co-morbidity index (hct-ci), with 16 patients (21%) having no co-morbidities, 35 (46%) a co-morbidity index of 1-3 and 25 (33%) had a score ⩾ 4. follow up of survivors ranged from 1 to 148 months (median: 32 months). at the specified end point 29 patients had relapsed (38%) with an actuarial 3-year relapse rate of 55%. there were 34 deaths (44%). relapse (23) was the main cause of death with transplant related mortality of 5% (4) at day 100, 8% (6) at 6 months and 13% (10) at 1 year. the actuarial os at 3 years was 48%, with a 3-year dfs of 39%. of the surviving relapsed patients all received chemotherapy and donor lymphocyte infusions resulting in effective recovery of remission, showing the utility of this approach. in terms of co-morbidity, actuarial survival rates were 60% in those with an hct-ci index of 0, 39% with an index of 1-3 and 50% with an index ⩾ 4. the results of this retrospective study indicate that allosct using reduced intensity conditioning regimens can be an effective treatment strategy for older patients with high risk myeloid malignancies including those with significant co-morbidities. relapse remains the main cause of treatment failure and strategies to reduce relapse risk are required. patients that relapse post allosct may respond to further treatment such as azacytidine or intensive chemotherapy and donor lymphocyte infusions. (3) whether patient-related variables were associated with disagreement. this is a secondary analysis of a cross-sectional multicenter study where patients and clinicians completed an identical qol questionnaire (fact-bmt) at day 90. clinical and demographic variables as well as anxiety and depression (hads) were collected. agreement was analyzed with the intraclass coefficient correlation (icc). rates of under-and over-estimation were calculated. logistic regression models identified predictors of disagreement. we analyzed 96 pairs of questionnaires, filled in by 96 patients and 11 clinicians. patients' median age was 54 years, 50 (52%) were men, and 50 (52%) received an allogeneic hct. clinicians' median age was 42 years, 7 were men and had worked on the transplant field for a median of 12 years (range: 3-23). agreement on qol was moderate (icc = 436). exploratory analyses revealed that agreement for emotional (icc = 092) and social (icc = 270) wellbeing was poor, whereas it was moderate for physical (icc = 457), functional (icc = 451) and bmt concerns (icc = 445). patients' wellbeing was overestimated in 41-59% of the categories of wellbeing parameters, and underestimated in 10-24%. patient-related variables explained 12-17% of the variance on disagreement across scales. specifically, anxiety contributed to disagreement in all subscales, except in social wellbeing, where non-significant univariate associations were observed (p40.05). type of transplant (allogeneic vs autologous), performance status, and graft-versus-host disease were not associated with disagreement (p40.05). patients and clinicians agreement on qol is suboptimal, particularly on emotional and social wellbeing. patients' wellbeing cannot be estimated from other sources than themselves. these results highlight the unmet needs of hct recipients with respect to qol-related issues; an outcome that must be addressed by hct programs since their wellbeing is as important as survival endpoints. disclosure of conflict of interest: none. . we wanted to test the function and the safety of picc device as alternative to standard cvc in patients submitted to autologous stem cell transplantation (abmt). the primary end point of the study was to individuate the cause leading to the failure of picc (its removal or the needing of another cvc during the abmt procedure). secondary end points were the correct function of the device and its praticity. twenty patients submitted to abmt for multiple myeloma (18) or lymphoma (2) experienced a double lumen picc device (17) or a single lumen (3) if the patient already carried a permanent single lumen cvc such as hickman or port-a-cath. we excluded from this experience patients with high risk of life-threatening situation or high risk of intensive care already before abmt. picc devices were placed from a specialistic nurse team by ultrasound identification of a deep venous vessel in upper arms. melphalan 200 or ceam were the standard conditioning regimens employed in myeloma and lymphoma abmt respectively. we considered a failure all the causes leading to the removal of picc or requiring another cvc before the end of the transplant procedure. at last we collected nurses and clinicians opinions about the picc functionality. no complication has been recorded in positioning phase. 19/20 patients maintained the picc device for all the time of transplant procedure. only one patient needed to remove the device for infection. the opinion of nurses and clinicians about the picc device was a significatively slower speed of infusion and resistance to the flow; in fact, 11/20 patients needed an infusional pump. the idraulic resistance of the catheter was particular evident against cellular fluids (stem cells suspension, transfusions of blood and platelets). for this reason picc seems to be less indicated in patients requiring many endovenous infusions (nurses' opinion). the rate of infection of picc devices seems to be lower compared to cvc, but the number of cases tested in this experience is too limited for definitive conclusions about it. for other aspects picc is similar to other cvcs. picc seems to be a valid alternative to standard cvc in patients who do not require intensive care, and in particular in patients with low intensity abmt who do not present a high number of endovenous infusions. maybe picc is less burdened of infections respect to normal cvc. this fact, summed to the lower risk during the positioning of the device, leads to consider the use of this device in abmt setting for standard risk patients. disclosure of conflict of interest: none. there are only few algorithms for the selection of hlamismatched unrelated donors, when no fully matched donor is available. indirect recognition of hla-mismatches can be predicted using the model of 'predicted indirectly recognizable hla epitopes' (pirche). the pirche model is a recently developed computer-based strategy, which classifies hladerived epitopes that are potentially presented by patientdonor shared hla-molecules. we performed a multicenter retrospective study evaluating the impact of pirche on outcome after allogeneic stem cell transplantation from hla 9/10 matched unrelated donors. the study cohort included 1997 adult patients who had undergone allogeneic stem cell transplantation for aml or mds. pirche scores were computed for 424 recipients of hla 9/10 matched unrelated donor transplants (9/10mud) using a web-based tool. primary endpoint was overall survival at 2 years. patients with a 9/10 mud were divided into 2 groups according to the sum of pirche i+ii values (pirche score). eighty-five (85) patients had a pirche score of 0 (no pirche detected), 339 a pirche score 40. km estimate of 2 year os was higher for 9/10 mud with pirche score = 0 compared to pirche score 40: 57% (95% ci: 51-63%) vs 47% (95% ci 41-53%), p = 0.04. os was similar for 9/10 mud with pirche score = 0 and 10/10 mud (57% vs 55%). cox regression analysis revealed poorer os for pirche scores 40 (rr 1.5, 95% ci: 1.0-2.1, p = 0.03). cumulative incidence of nrm at 2 years was lower for 9/10 mud with pirche score = 0 compared to pirche score40 (20% vs 32%, p = 0.05). multivariate cox regression analysis revealed poorer nrm for pirche score40 (rr 1.7, 95% ci: 1.0-2.9, p = 0.03). cumulative incidence of agvhd grade 2-4 at 6 months was not significantly different for 9/10 mud with pirche score 0 compared to pirche score40 (23% vs 30%, p = 0.2). cumulative incidence of cgvhd at 2 years was lower for 9/10 mud with pirche score 0 compared to pirche score40 (31% vs 49%, p = 0.04. our findings require confirmation, ideally in a large prospective cohort study. if validated, the pirche model would allow selection of permissible hla-mismatches that may be associated with an improved transplant outcome in terms of reduced nrm and better os. [p364] disclosure of conflict of interest: none. this study was supported by a research grant from pirche-ag to the university medical center, hamburg-eppendorf. pretransplant liver dysfunction has been recognized as a risk factor for complications and mortality after allogeneic hematopoietic cell transplantation (allo-hct). however, there is no consensus on the optimal way to evaluate liver function in hct candidates. transient elastography (te) is a noninvasive method for diagnosing liver damage and cirrhosis. while elastography is widely used in the setting of viral hepatitis, its possible role in allo-hct recipients has not been deeply evaluated. patients receiving allo-hct in our center from may 2014 are scheduled to receive pretransplant evaluation by a hepatologist under a prospective protocol. the evaluation includes a hepatologist consultation, liver function and infectious serology tests and te. all patients receive ursodiol from hct admission to day +30. this study constitutes the first evaluation of the ongoing protocol for patients receiving their first allo-hct from may 2014 to august 2016. sixty patients received a first allo-hct during the study period. sixteen patients did not undergo hepatologist evaluation due to timing issues (n = 6), unstable medical condition (n = 4) or other reasons (n = 6). finally, 44 patients received pretransplant evaluation by a hepatologist under the current protocol and constitute the study population. median age at transplantation was 51 years (range: 21-69). most patients received a transplant for acute leukemia (n = 23, 52%) or non-hodgkin's lymphoma (n = 10, 23%) mainly from hla matched unrelated donors (n = 21, 48%). thirty-two patients received reduced-toxicity regimens (73%). graft-versus-host disease (gvhd) prophylaxis consisted of tacrolimus in combination with another agent. median follow-up for survivors of 14 months (range: 3-29). median elastography was 5.6 kpa (range: 2.9-13.7). considering the hct-ci categories on hepatic dysfunction, 38, 6 and 0 patients scored 0, 1 and 3 points, respectively. there were two cases of veno-oclusive disease (vod). overall survival and non-relapse mortality of all patients at median follow-up were 76% (95% ci 69-83) and 22% (95% ci 14-30), respectively. in the univariate analysis, median elastography was not associated with a higher risk of nrm (p-value = 0.13), os (p-value = 0.11) or hepatic chronic gvhd (p-value = 0.32). the two patients with vod had normal pre-hct transaminase levels and te. this first analysis of an ongoing protocol with universal pre-hct evaluation of hepatic function indicates that increased values of transient elastography are not associated with higher nrm or lower os after the procedure. further studies including a larger number of patients are needed in order to clarify the possible role of elastography in the hct setting. disclosure of conflict of interest: none. allogeneic hematopoetic stem cell transplantation (hsct) remains associated with a high morbidity and mortality in spite of advances in hsct management. specifically, pulmonary complications account for a substantial proportion of deaths within the first 100 days after hsct. therefore, identification of lung dysfunction and additional comorbidities are crucial for preventive strategies in hsct. given the inconsistent association of pretransplant lung function s305 parameters on mortality after hsct and the significant changes in hsct care over the last decades, the aim of our study was to assess the effect of pulmonary function and comorbid conditions on mortality in patients undergoing hsct for hematological disorders. we retrieved relevant clinical data of all consecutive patients at the hematology division of the basel university hospital with a transplant for hematological disorders between 2008 and 2015. we examined the lung function at baseline and 3, 6 and 12 months after hsct-including the 1 s forced expiratory volume (fev1% of predicted), fev1/vcmax and diffusing capacity for carbon monoxide (dlco, adjusted for hemoglobin concentration). in addition, we assessed pretransplant conditions such as age, sex, karnofsky performance status (kps), donor type and various risk scores in hsct (hematopoietic cell transplantation comorbidity index (hct-ci), european society for blood and marrow transplantation (ebmt), revised pretransplant assessment of mortality score (pam)). using uni-and multivariate cox proportional-hazards regression analysis, we evaluated patient-and transplant-related risk factors for all-cause mortality by including the following categorical candidate variables: fev1 (⩾80% vs 50-79% vs o50% of predicted), kps ( o90% vs ⩾ 90%), age ( o54 vs ⩾ 54 years), conditioning intensity and donor type (matched-related vs mismatchedrelated vs matched-unrelated vs mismatched-unrelated). within the study period, 429 patients with predominantly acute leukemia (64%) or lymphoproliferative disorders (28%) underwent myeloablative (n = 330) and non-myeloablative hepatic veno-occlusive disease/sinusoidal obstruction syndrome (vod/sos) is a potentially life-threatening complication of conditioning for hematopoietic stem cell transplantations (hsct). recombinant thrombomodulin (rtm) is a new drug for treating disseminated intravascular coagulation (dic) and is an endothelial anticoagulant cofactor that promotes the thrombin-mediated formation of activated protein c (apc). rtm has been used to treat vod/sos, but its ability to prevent vod/sos has not been established. we evaluated the cases of 19 pediatric hematology and oncology patients (8 (43%) acute myeloid leukemia, 3 (16%) acute lymphoblastic leukemia, and 4 (21%) neuroblastoma patients, and 1 (5%) patient each with myelodysplastic syndrome, rhabdomyosarcoma, hemophagocytic syndrome (hlh), and wiskott-aldrich syndrome) who underwent hsct at our institution between 2007 and 2014 and had ≧ 1 risk factors for vod/sos. these risk factors included previous treatment with gemtuzumab ozogamicin (go), receiving 42 hsct, undergoing conditioning with busulfan (bu), and being diagnosed with hlh. the patients who received hsct after 2012 (n = 8; rtm group) were treated with rtm as a prophylaxis against vod/sos (380 u/kg per day for 7 days; from days 7 to 13) together with ursodeoxycholic acid (urso) and low-molecular-weight heparin (lmwh), and the others (n = 11; control group) were only treated with urso and lmwh. the incidence of vod/sos was evaluated, and various coagulation parameters and markers of endothelial injury (plasminogen activator inhibitor type (pai-1) and apc) were measured in both groups. the patients' median age was 2 (range: 0-18) years, and 11 (58%) were male. clinical characteristics, including vod/sos risk factors, were wellmatched in both groups. the risk factors possessed by the patients included receiving 42 hsct (9/19, 47%), previous go treatment (6/19, 32%), conditioning with bu (3/19, 16%), and a diagnosis of hlh (1/19, 5%). although vod/sos occurred by post-hsct day +35 in 3 (27%) patients in the control group, vod/sos was not seen in the rtm group. two of the former 3 patients (2: previous treatment with go, 1: a diagnosis of hlh) suffered severe vod/sos, and 1 (a diagnosis of hlh) died of the condition. no grade 3/4 adverse events involving bleeding or severe organ damage were reported in the rtm group. interestingly, the mean peak value of pai-1 and apc (markers of endothelial injury) were significantly lower in the rtm group (table 1) . [p367] disclosure of conflict of interest: none. protective effect of early human cytomegalovirus reactivation on relapse of myeloproliferative disorders after allogeneic hematopoietic stem cell transplantation z peric 1,2 , j wilson 2 , n durakovic 1,2 , l desnica 2 , a ostojic 2 , vv rezo 2 , v marekovic 1,2 , r serventi-seiwerth 2 and r vrhovac 1,2 1 school of medicine, university of zagreb, zagreb, croatia and 2 university hospital centre, zagreb, zagreb, croatia there have been conflicting results regarding the association between early cytomegalovirus (cmv) reactivation and decreased incidence of relapse after allogeneic hematopoietic stem cell transplantation (allo-hsct). this prompted us to retrospectively evaluate the potential impact of cmv reactivation on transplantation outcomes in a study population of 161 consecutive adult patients who underwent allo-hsct in our institution and were treated and followed in a homogenous manner. patients were monitored for cmv reactivation once weekly for the first 100 days after allo-hsct. monitoring was done with a real time qpcr with lower limit of detection of 150 genome copies per ml of blood. when cmv viremia was detected, all patients were treated with intravenous ganciclovir or oral valganciclovir untill two consecutive negative qpcr assays. univariate and multivariable proportional hazards models using the fine and gray approach were considered to evaluate the variables for relapse, treating death as competing event. between 2011 and 2014, 97 male and 64 female patients underwent allo-hsct at a median age of 46 years (range: 18-64). among them, most patients were treated for myeloid malignancies (74 aml, 11 mds and 24 mpn with 11 cml, 11 mf and 2 cmml), while the rest had lymphoproliferative disorders (24 all, 10 nhl, 6 mm, 5 mh and 6 cll) and one patient had aplastic anemia. the donors were unrelated in 79 cases, related in 77 patients and haploidentical in 5 patients. most of the patients (70%) received peripheral blood stem cells after a reduced-intensity conditioning regimen (56%). with a median follow-up of 23 months, early cmv reactivation occured in 62% patients at a median of 27 days after transplantation and did not affect relapse incidence in patients with lymphoproliferative disorders. on the contrary, the cumulative incidence (ci) of hematologic relapse in patients with myeloproliferative disorders (aml and mpn) at 20 months after allo-hsct was 36% (95% ci, 21-52%) in patients without, opposed to 18% (95% ci, 10-29%) in patients with cmv reactivation (p = 0.04). however, cmv reactivation did not significantly affect (p = 0.21) overall survival between patients with (64%; 95% ci 53-77%) and without cmv reactivation (48%, 95% ci 34-68%). a striking and previously unreported correlation between cmv reactivation and relapse was found in patients with mpn; the ci of relapse was 50% (95% ci, 12-80%) in patients without, opposed to only 6% (95% ci, 25-100%) in patients with cmv reactivation (p = 0.01). a substantial and independent reduction of the relapse risk in myeloproliferative disorders (aml+mpn) associated with early cmv reactivation was confirmed by multivariate analysis using time-dependent covariate functions for high-risk disease, use of atg, chronic graft-versus-host disease (hazard ratio 3.33; 95% ci, 1.09-10.09, p = 0.03), and cmv reactivation (hazard ratio 2.37; 95% ci, 1.05-5.37, p = 0.04). in summary, this report supports an independent role of cmv reactivation on relapse in patients with myeloproliferative disorders. to our knowledge, we are the first to show a significant reduction of relapse incidence in patients with mpn, even though our findings are based on a relatively small number of patients. however, this putative virus-versus-myeloproliferation effect definitely warrants further research. [p368] disclosure of conflict of interest: none. final result of fact-bmt is score ranged 0-148 point (the higher the score, the better qol). for qualitative assessment of donor-recipient relationship, the adult sibling relationship questionnaire (asqr) in polish version was used. the asrq-s consists of 47 items which are spread over eight scales designed to investigate three factors: warmth, conflict and rivalry. the questionnaires were given to both subgroups, donors and recipients of msd-hsct and the results were compared to each other. the overall result of the fact-bmt questionnaire was 109.0 ± 7.5 points, which means that the examined group generally described their qol as 'quite good'. the best results were found in functional well-being (25.6 ± 0.9), while the worst in emotional well-being (20.7 ± 0.5) dimension. statistically, the qol score was not influenced by age at hsct (p = 0.256), current age (p = 0.378) or gender (p = 0.117) of the respondents. the recipients scored highest on warm factor (62.6 ± 7.8), while donor respondents scored slightly higher rivarly (60.3 ± 6.0) than warm (45.7 ± 5.4). the second dimension scored by recipients was rivarly (40.7 ± 6.8). conflict scores were lowest, although donor respondents scored higher on these than recipient respondents (38.6 ± 5.5 in donors vs 32.9 ± 3.6 in recipients). statistical analysis revealed that the being a donor or recipient of msd-hsct determines the level of rivarly in the sibling relationship (p = 0.007) with no impact on warm and conflict dimension. health-related qol in transplanted patients is quite good. sibling donor-recipient relationship is unbalanced with recipient respondents being more likely to assess a warm relationship, while rivalry was more likely to be present among donor. further multicenter studies based on larger cohort of patients are necessary to assess sibling relationship after transplantation life experience. disclosure of conflict of interest: the authors have nothing to disclose. this work was supported by grant from poznan university of medical sciences (502-14-01104119-10398). rate of re-admission in patients undergoing allogeneic transplants from identical siblings, unrelated donors or haploidentical donors f sora 1 , s sica, l laurenti, p chiusolo, s giammarco, i innocenti, e metafuni, a corbingi and a bacigalupo 1 department of hematology, catholic university of rome hla identical siblings (sib), unrelated (ud) and family hla haploidentical donors (haplo) are currently being used for patients undergoing an allogeneic transplant (hsct) for hematologic disorders. gvhd prophylaxis is usually different, and is commonly based on a calcineurin inhibitor (cni) and methotrexate (mtx) with or without atg for sibs and uds, wheres post-transplant cyclophosphamide (pt-cy)+a cni and mycophenolate (mmf) is used for haplos. we will refer as sib, ud, haplo platform, the combination of a given donor and a given gvhd prophylaxis. the outcome of these three different platforms is usually measured in terms of gvhd, non relapse mortality (nrm) and survival. days of admission and readmissions are important in terms of morbidity, but also of costs, and are usually not reported. aim of the study: assess the duration of the first admission and the incidence of a new re-admissions, in the first 100 days after the transplant. we retrospectively analyzed 151 patients from 2012 to2016. sixtyone received peripheral blood stem cell graft from an ud, and gvhd prophylaxis with cya+mtx+atg; 54 received a peripheral stem cell graft from a sib and gvhd prophylaxis with cya +mtx; 36 patients received bone marrow hsct from haplorelated donor and pt-cy+cya+mtmf. patients characteristics are shown in table 1 . relapses were excluded from the readmission analysis. the median time from the transplantation to discharge was 25 days for ud, 27 for haplo and 21 days for sib: there was no significant difference between haplo vs ud (p = 0.6), whereas the admission of both haplo and ud was longer than sibs (po 0.01). first readmission. fiftyone patient out of 151 required of a new admission for complications after tranplant (28 out of 61 after mud (46%), 13 out of 54 (24%) using a sibling donor and 10 out of 36 using an haploidentical donor (28%)). there were significantly more re-admissions in the ud vs sib group (0.01) and a trend for more ud readmissions vs haplo (p = 0.08); siblings had the lowest number of readmissions. time to neutrophil engraftment was comparable in haplo vs ud patients (p = 0.1) and in sib vs ud (p = 0.1); the time was longer in haplo vs sibs (p o0.01). the reason to re-admitted the patients in the hospital after tranplantation was fever in 14 out of 28 (50%) new admissions in ud setting,11 out of 13 (85%) in sib and 7 out of 10 (70%) in haplo; acute gvhd was the cause for re-admission in 5 out of 28 (18%) ud, 1 out of 13 (8%) sib and none in haplo. the other causes for re-admission in the hospital were hemorragic cistitis, thoracic or abdominal pain. second re-admission. of hospitalization is registered in 10 out of 61 patients in ud (7 for aghvd and 3 fever), 2 out of 54 (4%) in sib (2 episodes of fever) and 1 out of 36 (3%) patients in haplo (1 for fever and 1 progressive disease). also for second episodes, ud grafts had significantly more admissions compared to haplo and sibs. third re-admission was recorded only in ud patients (5 out of 61-8%). this study shows a comparable duration of admission for transplant for haplo and ud patients, both significantly longer than sib grafts. the number of re-admissions is comparable in haplo vs sibs and there is a trend for lower number of re-admission as compared to uds. we interpret this outcomes with caution given the relatively small sample size and heterogeneous disease population included. future studies need to confirme our results. disclosure of conflict of interest: none. prolonged thrombocytopenia (pt) is frequent event after allogeneic haematopoietic stem cell transplantation (hsct), especically in haploidentical transplantation, which could be up to 15% according to our previous report. pt has significant negative impact on long-term outcomes, mainly due to increased non-relapse mortality. however, there are no efficious treatment. in this study, we report the preliminary results of recombinant human thrombopoietin (rhtpo) in treating this kind of patients. from 2016.7 to 2016.10, 16 patients were enrolled under the following inclusion criterion: (1) diagnosed with dpe or sfpr after allogeneic stem cell transplantion; (2) no sign of minimal residual disease or recurrence of hematological malignancy; (3) not using other tpo receptor agonist or il-11 within 1 month of enrollment. pt include delayled platelet engraftment (dpe) and secondary failure of platelet recovery (sfpr). the former was defined as failure to achieve platelet counts ⩾ 20 000/μl for 7 consecutive without transfusion until 35 days after transplantation, while the latter was defined as a decline in platelet counts below 20 000/μl for 7 consecutive days, or requiring transfusion support after achieving sustained counts without transfusions for 7 consecutive days after hsct. the prescription of rhtpo was 15 000 iu once daily for 28 days, or if patients achieve platelet ⩾ 50 000/μl for 3 consecutive days with a duration o28 days. response was defined as success of achieve platelet counts ⩾ 20 000/μl for 7 consecutive days. the response time was defined as the first day achieve response from the start of prescription. the primary end point was response rate, and the secondary end point was reponse time. a total of 16 patients were enrolled, including 7 males and 9 females. the median age was 30 (18-50) years. all patients received haploidentical transplantation. among these patients, 10 patients were dpe and 6 were sfpr. all patients received a 28-day prescription. the overall response rate was 50% (8 out of 16) in the overall population, while 60% (6 out of 10) in dpe and 33.3% (2 out of 6) in sfpr, respectively. among the 10 patients with response, the median response time was 21 (10-28) days from the first dose of rhtpo. after 4 weeks of the last dose of rhtpo, none of the responsed patient lose response. since the followup time is too short, the impact of relapse, gvhd were not reported. this single-arm preliminary result suggest that rhtpo could be a efficious method to manage pt after stem cell transplantation. however, these result need further confirmation. disclosure of conflict of interest: none. reproductive health in long-term female survivors after allogeneic hematopoietic stem cell transplantation z peric 1,2 , a samardzic 2 , n durakovic 1,2 , d tina 1,2 , l desnica 2 , r serventi-seiwerth 2 and r vrhovac 1,2 1 school of medicine, university of zagreb, zagreb, croatia and 2 university hospital centre zagreb, zagreb, croatia most female recipients of allogeneic hematopoietic stem cell transplantation (allo-hsct) suffer from premature menopause, infertility and endocrine imbalance owing to gonadal damage from myeloablative conditioning. in order to evaluate ovarian recovery and long-term endocrine complications in our institution, we performed a retrospective study of female patients who received a myeloablative allo-hsct during their reproductive age. we identified 50 female patients who underwent myeloablative allo-hsct in our institution between 1983 and 2009 and were still alive with available follow-up at the time of this study. among them, 37 patients accepted to participate and responded to a query designed for this s308 purpose. the median age of our patients at transplantation was 32 years (range: 12-47 years). they were interviewed at a median of 20 years (range: 7-33 years) post allo-hsct. the majority of patients were transplanted for a myeloid malignancy (14 acute myeloid leukemia, 7 chronic myeloid leukemia, 3 myelodysplastic syndromes and 1 chronic myelofibrosis), while 7 patients had aplastic anemia and 5 had acute lymphoblastic leukemia. all patients received bone marrow transplant from a hla-matched related donor after a myeloablative conditioning. conditioning regimen consisted of cyclophosphamide with or without total body irradiation (tbi) or in combination with busulfan. only 6 patients (16%) resumed a normal menstrual cycle after allo-hsct, without the need for hormonal replacement therapy (hrt). all these patients were transplanted for aplastic anemia and none of them received tbi in the conditioning regimen. eight patients (22%) remained amenorrheic indefinitely and never started hrt, even though most of these women were transplanted under the age of 40 years. 25% of these patients were diagnosed with osteoporosis later in life. the remaining 23 patients (62%) started hrt at a median of 11 months after allo-hsct (range: 3-27 months). however, only seven patients on hrt (30%) resumed regular menstrual cycle. a median duration of hrt therapy was 6 years (range: 3-20 years). none of the women receiving long-term hrt had severe cardiovascular complications or breast cancer. finally, five women gave birth to eight healthy children in our study population. three unassisted pregnancies were observed in two female patients after spontaneous recovery of ovarian function (both patients with aplastic anemia). the remaining two patients restored ovarian function with the use of hrt and gave birth after an assisted pregnancy (one woman gave birth to triplets after an in vitro fertilization (ivf), while other became pregnant with a donated oocyte). in spite of the fact that almost all women who undergo allo-hsct develop an ovarian failure, spontaneous recovery is sometimes possible, particularly following conditioning regimen without tbi. in patients without spontaneous recovery, hrt should be initiated promptly to prevent the early and late unwanted effects related to estrogen deficiency. moreover, recovery of normal ovarian function and even a viable pregnancy is a realistic possibility in patients placed on hrt, particularly with the use of potential therapeutic interventions as ivf or oocyte cryopreservation. it is therefore crucial to provide adapted pre-transplant counselling and recommendations for regular post-transplant follow-up in female patients who undergo allo-hsct. disclosure of conflict of interest: none. transplant-associated thrombotic microangiopathy (ta-tma) is a multifactorial disorder caused by systemic vascular endothelial injury leading to end-organ damage often involving the kidney. ta-tma occurs in up to 30% of patients undergoing hsct, and may be associated with poor outcome. although pathogenesis has not been fully clarified, activation of the complement system has been suggested to play a central role, and eculizumab, a monoclonal antibody (mab) that mediates terminal complement blockade, has shown therapeutic benefit in cases unresponsive to immunosuppression modulation. we report the case of a pediatric allogeneic hsct recipient with severe ta-tma, who did not tolerate treatment with eculizumab, now successfully treated with oms721, a novel human mab targeted to the mannan-binding lectin-associated serine protease-2 (masp-2), a molecule central to the activation of the lectin pathway of complement. a 14-year-old girl received an allogeneic hsct from a hla-compatible unrelated donor for the treatment of diamond-blackfan anemia. at month +5 of the posttransplant course, she developed progressive deterioration of renal function, microhematuria and serositis, that prompted the cyclosporine discontinuation. from month +7, the patient experienced progressive trilinear cytopenia, elevated ldh, schistocytes, undetectable haptoglobin, hypertension, increased serum creatinine, nephrotic range proteinuria, and serositis, and a diagnosis of ta-tma was established. laboratory investigations documented no abnormalities in the patient but identified a stop-codon heterozygous 43 variant in cfhr5 c.485_489dupaa (p.glu163lysfs*10) in the donor's dna. the patient was initially treated with eculizumab, but she developed acute pulmonary edema soon after eculizumab administration as the consequence of a possible reaction to the drug which had to be discontinued. the patient was subsequently treated with plasma exchange, with only limited benefit. upon ta-tma relapse at month +11, eculizumab was re-administered at lower doses, but she developed a new episode of acute pulmonary edema, preventing further eculizumab continuation. renal function progressively deteriorated and she was started on hemodialysis, reaching a 3 times weekly regimen. the patient received oms721, kindly provided on a compassionate use basis by omeros corporation, seattle, usa, starting with an iv dosing schedule. she did not experience any adverse events, and was able to tolerate the treatment well. at 2 months from oms721 initiation, she has shown improvement in ldh and haptoglobin levels, and, more importantly, her creatinine levels have normalized, allowing for complete discontinuation of hemodialysis and partial outpatient management. anti-masp-2 mab oms721 is a promising new option for the treatment of ta-tma occurring after hsct, and seems to have a safe profile also in the pediatric/adolescent setting. disclosure of conflict of interest: none. severe cytokine release syndrome after t-cell replete haploidentical transplantation with post-transplant cyclophosphamide is associated with increased death rate d taurino 1 , j mariotti 1 , b sarina 1 , l morabito 1 , s bramanti 1 , c carlo-stella 2 , a santoro 2 and l castagna 1 1 bone marrow unit, humanitas cancer center, istituto clinico humanitas, rozzano, italy and 2 hematology department, humanitas cancer center, istituto clinico humanitas, rozzano, italy haploidentical stem cell transplant (haplo-sct) represents a potential curative strategy for several hematological malignancies. haplo-sct may represent an alternative option when a hla matched-identical sibling (hlaid) or a matched unrelated donor (mud) is not available. the syndrome of systemic inflammation, characterized by fevers, vascular leak, hypotension, and respiratory and renal insufficiency, in the context of elevated inflammatory markers and cytokine levels was previously described as cytokine-release syndrome (crs)1. recent publications have elicited the occurrence of crs after haploidentical transplant, especially after peripheral blood stem cell graft, and its high-related mortality 2-4. here we report the experience of our institution with crs after haplo-sct. between march 2014 and october 2016, we treated 29 patients with haplo-sct with a graft source represented by peripheral blood stem cells. we monitored the occurrence of crs symptoms and utilize a previously described grading system 1, 4 starting from day 0, up to day 14 after transplant. severe crs is defined as grade 3 or higher because it requires aggressive interventions and is characterized by oxygen requirement ⩾ 40%, 43 l nasal cannula, hypotension requiring high dose or multiple vasopressors, grade 3 renal toxicity or grade 4 transaminitis. other characteristics comprise newonset altered mental status without other explanation and new cardiomyopathy without wall motion abnormality. results: 27 out of 29 patients experienced fever between day 0 and day 14 post transplant with most episodes (24 patients) occurring between day 0 and day 4. on day 7 after transplant, 3 patients had grade 3, 6 grade 2 and 19 grade 0 crs, respectively. by day 14 post haplo-sct, 5 patients had crs grade 43, 5 grade 2 and 1 grade 1. overall, the incidence of crs any grade was 43% (95% ci 21-55%). 1 year after transplant 8 patients died because of non-relapse related side effects. with a median follow-up for alive subjects of 10 months, 1-year overall survival (os) was 64% (95% ci: 42-80%). 1-year os was 73% for patients with a crs 3 on day 7 (p = 0.007). conclusions: crs represent an important complication after haplo-sct. crs score 43 on day 7 after hst apparently correlates with long-term survival. better strategies need to be implemented for an early detection of severe crs in order to develop effective treatments, such as tocilzumab, for this important side effect. further studies are ongoing at our institution in order to correlate post-haplo crs with graft composition, laboratory parameters and immunereconstitution. hematopoietic cell transplantation (hct) is associated with significant morbidity that impairs survivor's sexual functioning. however, few studies have specifically addressed it. thus, we examined (1) sexual functioning during the first year post hct, (2) differences between allogeneic and autologous hct, and (3) whether demographic, clinical and psychological variables were associated with sexual functioning. this is a prospective multicenter study assessing patients before hct, at day 90, 180 and 360. sexual functioning was assessed with the changes in sexual functioning questionnaire, which yields a total score, along with scores for the dimensions of frequency, pleasure, orgasm, desire and arousal. anxiety and depression (hads) were also collected. we included 159 consecutive hct recipients: 91 (53%) were men, with a median age of 51 years (range: 18-71), 93 (58%) received an allogeneic hct and 66 (42%) an autologous hct. sexual functioning was significantly affected: 86% of the sample reported impairment at pre-hct, 91% at day 90, 87% at day 180 and 86% at day 360. mixed model analysis indicated that sexual functioning was not associated with time from hct (p = 0.802) or hct type (p = 0.538). however, there was an interaction between these two variables (p = 0.022), particularly at day 90, since sexual functioning had improved among autologous survivors and worsened among allogeneic survivors leading to nonsignificant differences between hct type (p = 0.082). frequency of sexual functioning improved during the study period (po 0.001), and no differences were observed between hct type (p = 0.111). again, there was a borderline interaction between post-hct time and hct type (p = 0.059), since autologous survivors reached higher frequencies than allogeneic survivors, with significant differences at day 90 (p = 0.003). pleasure significantly improved during the study period (p = 0.035), without observing differences between hct groups (p = 0.121). again, however, autologous survivors reported significant improvements in pleasure at day 90 (p o0.001) and a trend at day 180 (p = .093) when compared with allogeneic survivors. orgasm did not improve during the study period (p = 0.837), and no differences were obtained between hct groups (p = 0.413). allogeneic survivors had higher orgasm scores at pre-hct (p = 0.020), which worsened during the study period, particularly at day 90 (p = 0.028). in contrast, autologous survivors reported improvements in orgasm by day 90. non significant results were obtained in the sphere of sexual desire and arousal (p40.1). bivariate analyses indicated that women, older age and depression were associated with impaired sexual functioning at all assessed time-points (p o0.05). chronic graft-versus-host disease (gvhd) was associated with worse sexual functioning at day 180 (p = 0.045) and 360 (p = 0.020). no differences were obtained when considering diagnosis, having received previous hct, intensity of the conditioning regimen and whether patients lived with a partner (p40.05). stepwise multivariate regression analyses indicated that gender (p = 0.001) and extensive chronic gvhd (p = 0.012) predicted for worse sexual functioning at day 360. sexual functioning should be routinely assessed and considered for eventual targeted intervention in both hct populations, particularly during the first year post transplant. additional clinical efforts should focus on patients more vulnerable to impaired sexual functioning. disclosure of conflict of interest: none. significant improvement of qol by using atg as part of the conditioning regimen followed by hla-identical peripheral stem cell transplantation in acute leukemia patients. results from a prospective, randomized phase iii study (atg family study) b francesca 1 , s carlos 2 , w christine 3 , s mariarosaria 1 , p massimo 4 , s carmine 5 , m giuseppe 6 , b wolfgang 7 , cm angelo 8 , p francesca 9 , m nicola 10 cgvhd is a major complication after allogeneic sct. we previously demonstrated that the addition of anti-tlymphocyte globulin (atlg neovii, formerly atg-fresenius) to a myeloablative preparing regimen followed by peripheralblood sct from an hla-identical sibling for pts with acute leukemia resulted in a significant reduction of cgvhd, without increasing the risk of relapse or infection. 1 the study protocol included quality of life (qol) questionnaires (eortc qlq-30 and hdc29) before and after sct (day+ 100, 6, 12 and 24 mos). the qlq-c30 includes a global qol scale, five functional scales (physical, role, emotional, cognitive and social function) and nine symptom scales (fatigue, nausea-vomiting, pain, dyspnea, insomnia, appetite loss, constipation, diarrhea and financial problems). the qlq-hdc29 includes six multi-item scales and eight single items that describe impairment through highdose treatment. mixed models for repeated measures (mmrm) and linear mixed models (lmm) were used to analyze the time courses and the slopes of the outcomes depending on treatment arm (atg vs non atg), age, country, sex, and cgvhd. (clinicaltrials.gov: nct00678275). pts with a qol form returned decreased by visit (70% pre-sct, 45% at 100 days and 29% at 24 mos after sct). forty-nine percent in the atg and 60% in non atg arm provided any qol forms after sct. return of any post-sct qol forms by country was 68% for germany, 62% for italy and 25% for spain. pts with cgvhd were more likely to return qol questionnaires (66% vs 45% w/out cgvhd) while neither age nor sex were closely associated with qol form return. the majority of subscales of the qlq-30 indicated an average improvement of qol and reduction of symptoms over time, notably in the atg group. in an mmrm model controlling for country, age, sex and cgvhd, pts treated with atg showed significantly more pronounced improvement of global health status/qol over time compared to non-atlg (p = 0.02), with a treatment group difference of 2.8 ± 3.9 points (marginal mean ± sem) at day 100 and increasing to 10.5 ± 5.3 points at month 24 favoring atg. significant superiority of atg (po0.05) was also observed for four of the five functional scales as well as for several symptom scales scores including appetite loss, insomnia, nausea-vomiting and dyspnea. for the qlq-hdc29, significant treatment effects favoring atg were observed for gi side effects and impact on family. lmm analyses of qol by country indicate that patients from italy generally gave more favorable ratings for all functional scales and lower scores for most symptom scales than those from germany while the time courses and slopes were similar for most scales. these results underline the importance of the habits and cultural environment which are distinctive of each country. males and females showed similar qol ratings at pre-and post-sct. patients up to 34 years tended to provide more favorable functional ratings and less severe symptom scores than older patients and also showed more pronounced improvements of qol. pts receiving atg in a randomized study have significantly less cgvhd and improved grfs, resulting in an improved qol regarding global health status and most functional scales. notably, we also observed a significant difference in qol assessment between pts from germany and italy. oral mucositis (om) is a well-known side effect of high-dose chemotherapy and radiotherapy in hematological patients, which influences the health-related quality of life (hrqol) of the affected patients. the purpose of this study is to demonstrate the impact of om on hrqol in stem cell transplanted patients in routine care. prospective, noninterventional single-center observational study was performed at a german university hospital. inpatient allogenic and autologous stem cell transplant patients ⩾ 18 years with high-dose chemotherapy. om was assessed with the who oral toxicity scale, pain using the numeric rating scale (nrs) and the performance status with the ecog score. hrqol was captured with the eortc qlq-c30 and the qlq-oh15 questionnaires (3 days before hematopoietic stem cell transplantation (hsct); 7 days after hsct; 14 days after hsct). statistical significance was assumed p o0.05. a total of 20 patients (11 autologous and 9 allogenic) was included from august to december 2016. a total of 11 (55%) patients developed om. of these 11 patients, 3 suffered from grade 1, 3 from grade 2, 4 from grade 3 and 1 from grade 4 om. three days before hsct, the mean qol of all 20 patients was 45%, the mean qlq-c30 summary score 60.3% and the mean oral health related quality of life 82.3%. most of the patients suffered from om around day 7 after hsct. after 7 days, quality of life (qol) was higher in patients with no om (32.3%) than in patients with om (30.0%). the qlq-c30 summary score was significantly (p = 0.004) lower in patients affected by om (43.1%) than in patients who did not develop an om (65.8%). om affected patients had significantly more limitations in emotional (no om 84.4%; om 56.7%; p = 0.038) and cognitive functioning (no om 93.8%; om 51.7%; p = 0.002) and in fatigue (no om 58.3%; om 80%; p = 0.045), pain (no om 14.6%; om 55%; p = 0.005) and insomnia (no om 12.5%; om 60%; p = 0.001), they had a significantly higher rate of problems. oral health-related quality of life was significantly (p = 0.003) lower in patients who were affected by om (57.9%) compared to patients who did not develop an om (83.9%) and patients with an om had significantly more problems with a sore mouth (no om 8.3%; om 46.7%; p = 0.028), sticky saliva (no om 29.2%; om 63.3%; p = 0.045) and sensitive mouth (no om 8.3%; om 56.7%; p = 0.003). after 14 days, qol was higher in patients with no om (47.9%) compared to patients with om (46.7%). patients with no development of om had a higher but not significant physical functioning, cognitive functioning and social functioning. patients affected by om had higher levels of fatigue and pain and more often suffered from a sore mouth. oral health-related quality of life was higher in patients without om (75%) compared to patients with om (68.3%). comparing all assessed days patients with om had higher scores on the nrs increasing with a higher grade of om (mean nrs score grade 1; 2-2.3, grade 3; 4-4.5), the ecog index was higher in om affected patients during episodes with om (mean ecog score-2.3) compared to episodes without om (mean ecog score-1.9). om has a major impact on the hrqol, health-related symptoms and functionality. in the future, there has to be a higher awareness from clinicians and patients of the prevention, assessment und causes of om. more research has to be initiated to ease the symptomatology and to improve patients' quality of life. disclosure of conflict of interest: none. according to ebmt data, chronic gvhd (cgvhd) occurs in 40-70% of all patients after allogenic hematopoietic stem cell transplantation (allo-hsct). pulmonary cgvhd is the most severe form. but it is very unpredictable to use due to the fact that many factors can affect it (breath-dependent; need experience not only from physician but from patients also and so on). here we show that routine software-based image analysis algorithm can provide data that highly correlated with pft results and have excellent sensitivity and specificity in pulmonary cgvhd diagnosis. we blindly analyzed 120 ct scans (made without additional expiration) in 24 allo-hsct patients at different time points. all scans were performed on ct scanner aquilion 64, toshiba, japan. according to hounsfield units (hu) definition, − 1000 hu ('air') have approximate density at 0 g/ml; 0 hu ('water') have approximate density at 1 g/ml. the analysis of ct scans (heart, vessels and bronchi were excluded from analysis) was based on automated software conversion (image-analysis algorithm providing by multivox software, msu, moscow, russia) of each ct-image pixel from hu to density units (g/ml). pft were performed using standard procedures at same as ct scans time points (spirolab iii, italy). all patients with hematological malignancies (acute leukemia-17, aplastic anemia-1, chronic myeloid leukemia-1, t-cell lymphoma-1, chronic myeloproliferative disorder-1, myelodysplastic syndrome-3) were transplanted in national research center for hematology between 2012 and 2015. median of age was 41.5 years (range: 19-60 years). eight patients were males, 16-females. seventeen received reduced-intensity and 7-myeloablative conditioning regimen. graft from match unrelated donor (mud) were used in 17 cases, 'mismatch' mud-2, match related donor (mrd)-9, 'mismatch' mrd-1. median follow-up is 44.8 months. we analyzed lung tissue experimental density in patients before and after allo-hsct at different time points. median of lung tissue experimental density were 0.178 (interquartile range (iqr), 0.148-0.186), 0.17 (iqr, 0.153-0.185) and 0.147 (iqr, 0.131-0.172) for patients before allo-hsct, after allo-hsct with cgvhd (except pulmonary cgvhd) and with pulmonary cgvhd, respectively. mann-whitney u test was used to reveal significant differences between these groups (see figure 1 ). also, we found strong correlation between pft and experimental density (spearman's correlation coefficient r = 0.537) (see figure 2 ). forty-five ct scans of patients with pulmonary cgvhd and 59 ct scans of patients without pulmonary cgvhd at the time of ct scan as control subjects were included in roc analysis to assess the clinical values of our model. we generated an roc curve and found that the area under the curve (auc) was 0.77 (95% ci, 0.67-0. 86) (p o0.0001) (figure 3 ). standard ct scan is presented as easy to perform, breath-independent, standardized and wide spread method for every patient after allo-hsct. it can be performed many times during all their post-hsct life. ct scan with a simple software analysis allows to select a group with high probability of pulmonary cgvhd and who can be suspected of cgvhd development by this method with sensitivity-60% and specificity-81.36%. disclosure of conflict of interest: none. the choice of effectiveness criteria affects conclusions of economic evaluation of newer allogeneic bone marrow transplantation modalities :example based on a randomised multicenter trial comparing two reduced intensity conditioning regimen (flu-bu-atg) vs (flu-tbi) for matched related allo-sct s le corroller*, anne-gaelle 1 , c siani 2,1 , r tabrizi a re-evaluation of the per-diem hospitalization cost was performed in 2016 and included the utilization of hospital technical facilities and a more precise estimation of overheads costs. we performed three separated cost-effectiveness analysis, using, respectively, pfs, os and qaly as end point. when using pfs as effectiveness, relapse costs were not included. weighting coefficients for the cost per qaly analysis came from the literature. at 5 years, os and pfs were 41% and 29%, respectively, and did not statistically differ between groups. the mean total cost per patient was not statistically different between groups (111725€ for fba vs 98316€ for ftbi, ns). using pfs as end point, the icer of fba compared to ftbi is 35 034€ per year of pfs gained. using os, the icer became non-statistically significant, signifying that when handling uncertainty, no difference in term of cost-effectiveness was observed between fba and ftbi with os as end point. using s312 qaly, the icer was statistically ns again, showing no advantage in terms of cost per qaly of one conditioning regimen over the other. this result was obtained both considering three weighted health states (dfs, progression and death) and four weighted health states (dfs without gvhd, dfs with gvhd, progression and death) for the qaly calculation. using os and qaly, the two conditioning regimens were not different in terms of cost-effectiveness, while fba may be considered as more cost-effective using pfs as effectiveness criterion. using intermediary end points allows economic evaluation to be available earlier in the life cycle of an innovation. however, it implies strong hypotheses about the predictive value of the pfs over the os. longer period evaluation and qaly may reverse preliminary results. this situation is likely to exist in the hematology setting where alternatives between chances of cure and toxicities of treatment are often observed. research about allogeneic sct modalities is archetypical of such situations and decisions makers should be aware of the necessity of further economic re-evaluation along the development and diffusion process of innovative treatments. disclosure of conflict of interest: none. the impact of corticosteroids prophylaxis for the engraftment syndrome incidence during autologous stem cell transplantation in multiple myeloma and amyloidosis the es is a complication of asct characterized by an inflammatory response during peripheral blood recovery. the standard treatment is based on corticosteroid therapy. the incidence of es after asct increases in chemotherapy lowtreated patients such as those with multiple myeloma (mm) and amyloidosis (al).moreover, the es is associated with the use of g-csf after infusion of stem cells. therefore, our bmt team does not use g-csf since 2009 in this population reducing the incidence and severity of es. therefore, it makes sense to use low-dose prednisone to prevent this complication. in this study, we compared two consecutive cohorts of patients with mm/al that performed an asct while evaluating the corticosteroids prophylaxis (cp) in the es incidence and its effect on other clinical variables. we included 120 patients with mm (n = 96; 80%) and al (n = 24; 20%) that performed an asct between january 2011 and november 2016 in a single institution. the median age (range) was 56.7 (33.8-71.7) years. during the procedure, all patients received melphalan as conditioning chemotherapy and none received g-csf. fortyseven patients (39%) received intravenous methylprednisolone or oral prednisone 0.5 mg/kg/day from day +7 until reaching a neutrophil count ⩾ 500 per mm 3 for 3 consecutive days (cs group), and 73 (61%) patients did not receive corticosteroids (noncs group). the characteristics of patients in both groups (age, gender, status performance and previous treatment were similar (p40.05)). the cs group, received higher doses of cd34 + than the noncs group (3.68 × 10 6 /kg vs 2.92 × 10 6 /kg, respectively, p = 0.006). the median (range) days of neutropenia ( o 500 per mm 3 ) was 9 (4-25) days. es was diagnosed in 43 (36%) patients. fifty-seven (48%) patients had fever, showing infectious focus or microbiological isolation in 24 (20%) cases, whereas the incidence of grade iii-iv oral mucositis and relevant gastrointestinal toxicity was 8% and 2.2%, respectively. the complete analysis between groups (cs versus noncs) for the whole series and in the mm/al subgroups is detailed in table 1 . the administration of corticosteroids as prophylaxis seems to reduce the incidence of es in the overall series or in the analysis for the subgroups (mm and al) without increasing infection. [p381] disclosure of conflict of interest: none. chronic gvhd is a condition that might occur after allo-hsct and has been proved to impair long-term survival and quality of life of patients. graft failure is also a major potential complication for patients undergoing transplant for an aplastic anemia/bone marrow failure (bmf). partial in vivo t-cell depletion, employing anti-thymocyte globulin (atg) during conditioning, has been proved to successfully prevent the mentioned potentially life-threatening complications in highrisk patients. however, the possibility of developing epstein-barr virus (ebv)-induced post-transplant lymphoproliferative disorders (ptlp) has been a limiting factor to use atg. this study includes the last 100 pts with a minimum follow-up of 100 days, who underwent allo-hsct in our center (november 2014-august 2016). a total of 56 pts were male and 44 female. median age was 53 years (range: 7-69). baseline diseases were: acute leukemias (54), lymphoproliferative disorders (17), myelodysplastic syndromes (12), chronic myeloproliferative diseases (7), multiple myeloma (5) and bone marrow failures (5) . donor was unrelated in 57 cases, and related in 43 (including 18 haplo-identical). conditioning regimen was: busulphan-based (70), melphalan-based (13), tbi-based (8) and others (9). progenitors source was pb in 89 and bm in 11. patient/donor ebv pre-transplant serology was: +/+ in 94 cases, +/ − in 5 and − /+ in 1. rabbit atg (thymoglobuline) was employed in 58 cases: 54 at 4.5-6 mg/kg (urd transplants) (low dose), and 4 cases at 7.5 mg/kg (all of them pts with bmf) (intermediate dose). family donor (including haplo-identical) transplants of those pts with diagnosis different from bmf (42 cases) did not receive atg. systematic monitoring of ebv using quantitative pcr was employed. ebv reactivation was considered when dnaemia was superior to 1000 copies per ml. a total of 5 pts presented ebv reactivation: 0/42 (0%) in cases without atg, 4/54 (7.4%) in cases with low-dose atg and 1/4 (25%) in cases with intermediate-dose atg. median time of reactivation was the day +34 (range: +32 to +151). there was one single case of ebv-induced ptld which belonged to the intermediate-dose atg group. all cases (including the one with ptlp) were successfully treated with rituximab at 375 mg/m 2 /week. median number of doses employed were 3 (range: [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . mortality due to ebv was 0% in our series. limited donor availability in the form of either matchedrelated or unrelated donors drew attention to haplo-hct. donors of haplo-hct shares an exact haplotype with the recipient but is mismatched for hla genes on the unshared haplotype. most studies have shown promising results in terms of graft success and survival. in this study our aim is to present the early and late outcome of our haplo-hct patients. between 2012 and 2016, we retrospectively evaluated 16 haplo-hct in terms of post-transplant outcome, survival and complications who diagnosed and followed in our center. the median age of patients was 37 (range: 19-61), 10 (63%) of them were male recipients. the patient characteristics were given in table 1 . thirteen patients (81%) had pre-transplant active disease. neutrophil and platelet engraftment was achieved in 7 patients (44%) at a median day of 21 (range: 16-40) and 34 (range: 13-116). eight of 16 patients (50%) died within 1 month after transplant because of sepsis without achieving engraftment. haplo-hct is the second transplant in four of 16 patients (25%): 1 patient relapsed after full-matched related transplant, 1 patient relapsed after 9/10 matched unrelated transplant, 1 patient had engraftment failure after full-matched unrelated transplant, 1 patient underwent haplo-hct in another center, followed in remission for 2 years and relapsed. acute graft vs host disase (agvhd) was diagnosed in 6 patients (37%), whereas chronic gvhd in 4 patients (25%). four patients were relapsed (25%) during follow-up with median rfs of 6 months. three patient had bk virus-positive hemorrhagic cystitis (18%). the distribution of infections is shown in figure, viral infections were detected later than fungal and bacterial infections. previous history of invasive pulmoner aspergillosis was detected in 5 of the patients (31%) (2 of them were re-transplanted) and received secondary prophylaxis. overall survival (os) of 6 months and 1 year were 25% and 18%, respectively. the choice between alternative graft sources depends on the urgency of the transplant on each institutional preference. higher complication and infection rates in addition to decreased survival compared with previous studies since our patient population consisted of refractory patients with comorbidities. preferable patient profiles undergoing haplo-hct may have better outcomes. disclosure of conflict of interest: none. the third month risk factor score: detection of disease at day +100 of allogeneic stem cell transplantation is the most important risk factor of worse prognosis m celis 1 , c fernández 1 , l yáñez 1,2 , a bermúdez 1,2 , a insunza 1 , m colorado 1 , m lópez-duarte 1 , i romón 1 , s garcía-ávila 1 , a cabero 1 , a casado 1 , m sánchez-escamilla 1 , c richard 1 and e conde 1,2 1 hematology department, hospital universitario marqués de valdecilla and 2 university of cantabria before allogeneic stem cell transplant (sct), several index can provide prognostic information (ebmt risk score and hcti score). however, there is scarce data for the impact of the procedure during the first 100 days of transplant, in which opportunistic infections and the acute graft versus host disease (gvhd) can induce harmful effects. our purpose is to create a risk factor score, measured at day +100 post sct, to give information about the prognosis of the patient. we retrospectively analyzed seven clinical (disease, fungal and cmv infection, acute gvhd, treatment with corticosteroids, karnosfsky status and length of hospitalization) and eight analytical (related to immune status, liver and lung function, nutritional status, iron overload and platelet count) risk factors in 131 patients who underwent sct in our center between 2011 and 2015 and were alive at day +100. data were collected as categorical variables and compared by χ 2 -test. significant variables (p o0.05) were evaluated in a multivariate logistic regression model. those who maintained statistical significance were then assigned a point value calculated with their β-coefficient. summation of the points resulted in a weighted risk score. median age was 51 years (range: 4-73) and 80 were males (61.1%). the most frequent disease was aml, 49 patients (37.4%). the conditioning regimen was myeloablative in 97 patients (74%) and bone marrow was the principal stem cell source (71%). donor was mrd in 39 (29.8%), mud in 51 (38.9%) and mmd in 41 (31.3%). the median followup was 26 months (range: 3-66). the univariant model identified five prognostic variables: detection of disease by molecular, cytogenetic or flow cytometry asses in leukemias, myelodisplastic syndrome and multiple myeloma or image (ct scan ± pet) in lymphoma, dose of corticosteroids ⩾ 0.5 mg/kg/ day, ferritin 42500 ng/ml, albumin o3.0 g/dl and platelet o100 000 per mm 3 . table 1 shows variables evaluated. in the multivariate model, the detection of disease (hr 3.80, 95% ci 1.90-7.61, p 2500 ng/ml (hr 2.39, 95% ci 1.30-4.42, p = 0.005), and platelet o100 000 per mm 3 (hr 2.06, 95% ci 1.11-3.89, p = 0.022) were associated with higher risk of death and according with their-coefficient 4, 2 and 2 points were, respectively, assigned. the third month risk score (tmrs) was calculated in all patients and they were stratified into three groups: low risk of death (a, 0-2 points), intermediate risk (b, 4 points) and high risk (c, ⩾ 6 points). at 2 years post sct, the estimated overall survival according with the tmrs was 78.9% ± 4.3 in group a, 31.0% ± 8.6 in group b and 36.4% ± 14.5 in group c, po 0.001. although the harmful effect of the first 3 months of transplant can impact in the survival, the detection of disease at day +100 is the most determinant risk factor of death. this fact gives us the need of transplant in the best response and, in those who cannot, to plan promptly rescue strategies. the next objective is to confirm our risk score in a validation group. disclosure of conflict of interest: none. recombinant human soluble thrombomoduline alpha (rhtm) is a novel anticoagulant agent and approved for disseminated intravascular coagulation in japan. the aim of the study is to evaluate the therapeutic potential of rhtm for sinusoidal obstructive syndrome/hepatic veno-occlusive disease (sos/ vod). we retrospectively studied 878 times of allogeneic hematopoietic cell transplantation in toranomon hospital from june 2008 to june 2015. we extracted the patients who used rhtm for dic and satisfied the diagnostic criteria of sos/ vod around the same time, because the use of rhtm for sos/ vod alone is off-label. data on the patients who used rhtm for 43 days within 100 days after transplantation were analyzed. the patients who were already treated with rhtm before the emergence of the first symptom or sign of sos/vod, and who started rhtm over 30 days after the emergence of the first symptom or sign of sos/vod, were excluded from the [p383] analysis. to diagnose classical sos/vod (⩽21 days after transplantation), we used two classical criteria of the modified seattle and the baltimore. for late-onset sos/vod (4day 22 of transplantation), we used the criteria of ebmt. we defined as severe sos/vod, if the patients had renal (cr ⩾ 2 times of baseline), respiratory (spo2 ⩽ 90% or the need for positive pressure) or central nervous system failure until 2 weeks after the diagnosis of sos/vod. complete response (cr) was defined as the resolution of all the symptoms and the signs in sos/vod diagnostic criteria. a total of 39 patients were extracted. the median age was 60 years (range: 27-72) and 27 patients (69%) was male. donor cell sources were ucb (n = 34) and ubm (n = 5). most of the prophylaxis regimen was the combination of ursodeoxycholic acid and dalteparin in 36 patients (92%). classical sos/vod was diagnosed in 3 (8%) and 8 patients (21%) by the criteria of the modified seattle and the baltimore at the median day of 14 (range: 11-14) and 16 (range: 11-20), respectively. twenty-eight patients (72%) were diagnosed as late-onset sos/vod at the median day of 44 (range: 22-89). severe sos/vod developed in 33 patients (85%) (renal, n = 32; respiratory, n = 7; central nervous system, n = 15). the elevation of transaminase was observed in 18 patients (46%). the median interval from the emergence of the first symptom or signs of sos/vod to rhtm administration was 7 days (range: 0-23). the median duration of rhtm use was 11 days (range: 3-63). rhtm was used alone in 20 patients (51%), in combination with dalteparin in 7 (18%), with atiii in 5 (13%), with dalteparin and atiii in 3 (8%), with atiii and pge1 in 2 (5%), and with pge1 in 2 (5%). corticosteroid was used concomitantly in 32 patients (82%). finally, 13 patients achieved cr of sos/vod. the cumulative incidence of cr of sos/vod was 33.3 % at 1 year after the administration of rhtm (95% confidence interval, 18.5-48.9%). the median interval from the administration of rhtm to cr of sos/vod was 51 days (range: 6-141). at 1 year after transplantation, overall survival was 25.6% (95% confidence interval, 13.3-69.9%). from the administration of rhtm to 2 weeks after the cessation of rhtm, 23 hemorrhagic adverse events were observed. seven out of 23 events were at grade 3-5, and 5 out of 7 events were fatal (intra-abdominal in 2, gastrointestinal in 1, lung in 1 and brain in 1). we concluded that rhtm had a therapeutic potential for sos/vod. disclosure of conflict of interest: none. thrombopoietin receptor agonists for delayed and prolonged clinically-relevant severe thrombocytopenia after allogeneic hematopoietic stem cell transplantation v bosch vilaseca 1 , i garcía cadenas 1 , e roldán 2 , s novelli 1 , r martino 1 , p barba 2 , a esquirol, l díaz polo 1 , g orti 2 , d valcárcel 2 and j sierra 1 1 hematology department, hospital de sant pau, barcelona, spain and 2 hematology department, hospital de la vall d'hebron, barcelona, spain persistent thrombocytopenia is a common complication after allogeneic stem cell transplantation (allosct), which dramatically increases the patients' dependence on hospital-based healthcare. thrombopoietin receptor agonists (tpoa) increase platelet counts in other clinical settings; however, the experience regarding their use after allosct is limited. we retrospectively evaluated tpoa efficacy in 15 consecutive adult allosct recipients who received tpoa as a compassionate use for severe thrombocytopenia post-engraftment. five patients (33%) had primary and prolonged failure of platelet recovery, while 10 had secondary thrombocytopenia: in seven of these cases, gvhd and/or a viral infection were the 'attributed' cause, while three were classified as post-allosct itp. all 15 patients were dependent on platelet transfusions (median: 2 times per week, range 1-5), with severe bleeding episodes in nine cases (60%) before tpoa onset. tpoa was started at a median of 160 days after allosct (range: 65-1041). romiplostim was used in 13 (87%) cases. the median starting dose was 2 μg/kg once a week (range: 1-5 μg/kg), while the final dose identified as most beneficial was 4 μg/kg (range 1-10 μg/kg). eltrombopag was used in 2 cases (13%), with an initial dose of 50 mg daily; while the final doses were 100 and 150 mg daily. overall, 9/15 patients responded to tpoa therapy (defined as a stable platelet recovery to ⩾ 30 000/μl without transfusion support). the 180-day cumulative incidence of successful platelet recovery to ⩾ 30 000/μl and ⩾ 50 000/μl was 70% (95% ci, 67-73%) and 56% (95% ci, 42-69%), respectively, which were reached at a median of 21 and 48 days from start of therapy. five of the 9 patients (56%) with severe bleeding at onset responded to tpoa (4 of them without further hemorrhages) at a median of 93 days (range: 1-299). at a median follow-up of 511 days from start of therapy, three patients who responded continue tpoa treatment, while four other responders were able to discontinue it without recurrence of thrombocytopenia. among these 4 patients, s317 the median total duration of treatment was 201 days (range: 113-300). one patient lost his response within 5 months after tpoa onset when he developed thrombotic microangiopathy associated with progressive gvhd. the remaining responder experienced disease relapse on day +88 after allosct. among the 6 non-responders, 1 had leukemia relapse during tpoa treatment, 1 switched from romiplostim to eltrombopag without success and the remaining cases had active severe infections at tpoa onset (2 hemorrhagic cystitis and 1 cmv colitis) or non-controllable intestinal bleeding due to progressive gvhd. tpoa were well tolerated, with only 2 patients showing adverse events (grade 3 liver toxicity and grade 3 fatigue), which did not lead to any change in therapy. six patients (40%) underwent follow-up bone marrow biopsies that did not display any increase in marrow fibrosis, including the 1 patient who had myelofibrosis prior to allosct. although six patients in the study had active gvhd when tpoa was started, no patients showed worsening of gvhd. our results support the safety and efficacy of tpoa for the treatment of persistent thrombocytopenia in allosct recipients. further studies should compare the efficacy of romiplostim and eltrombopag and identify surrogate clinical and laboratory variables that are predictive of response to one (or both) of these tpoa. disclosure of conflict of interest: none. . clinical response in both groups was defined as improvement of organ function (no neurological residues; normalization of kidney function) and independence of red blood cell and platelet transfusions. results: the median time of ta-tma onset was 8.2 months (0.5-32.8) after hsct. thirty-five of 37 patients (95%) were under treatment with calcineurin-inhibitors or sirolimus at the time the ta-tma occurred. in all cases, the immunosuppressive drug was stopped promptly. in 28 patients, classical treatment was the primary therapy with a response rate of 52% (including four patients who switched to ec), whereas the response rate to ec treatment was significantly higher with 92% (p = 0.017). all patients receiving ec showed sufficient blockade of the terminal complement pathway after the second ec application (ch50 o 10%). despite the increased response rate for ec therapy, there was no difference seen between these two groups according to overall survival in weeks: classical treatment 9 (95% ci 0-19.3) vs ec treatment 14.9 (95% ci 6.9-22.7) p = 0.84. the main cause of death differed significantly between this two treatment approaches with a therapy-related mortality due to infection with 70% in the ec group during tma therapy and none seen in the classical treatment group (p = 0.001). progressive gvhd was identified as an adverse prognostic factor in both groups (p = 0.048 and p = 0.005). conclusion: in our analysis, we show that ec shows a significant higher response rate in severe ta-tma patients compared to the classical treatment approach. however, in both groups the outcome remained very poor. since most patients presented with advanced, severe ta-tma, especially in the ec group, we hypothesize that earlier diagnosis and treatment of ta-tma and more effective prevention and treatment of infections will improve the outcome of patients with this complication. however, randomized studies are essential for comparison of these two treatment strategies to identify patient groups that benefit from a treatment with ec. disclosure of conflict of interest: none. tocilizumab as an effective treatment in cytokine release syndrome as an early peri-transplant complications in patients subjected to allogeneic stem cell transplantationproinfammatory/autoimmune patient/donor hla haplotype life-threatening early allogeneic hsct complication risk factor hypothesis m-g patrycja 1,2 , p-j beata 1,2 , s marcin 1 , k ksenia 1 , s-k agnieszka 1 and sb aleksander 1,2 1 bone marrow transplantation unit, department of haematology, krakow university hospital and 2 jagiellonian university collegium medicum cytokine release syndrome (crs) is classical complication of car t cells therapy, but also can be connected with early peritransplant complications in patients subjected to allogeneic stem cell transplantation. it can be connected with atg infusion, but also with inflammatory response during periengraftmetnt period (pre-engraftment syndrome and engraftment syndrome) and septic infections. severity of these complication can differ depending on patient's performance status and therapeutic options from just observation and vigilance to mechanical ventilation need. we would like to present small patient series (n = 5) subjected to msd (n = 1) and mud (n = 4) with early transplant-related complications treated with combination of steroids (dexamethason) and tocilizumab. in two of them, tocilizumab was used after second dose of atg. both patients present hypotonia with decreased urine output, prompt increase of creatinine level and presence of acute inflammatory parameters crp, beta2microglonulin and procalcitonin level, fluid retention and decreased oxygen saturation. in another one patient, these symptoms were connected with pbsc infusion from unrelated donor. in later two patients, we observe almost the same clinical presentation in preengraftment phase. in every of patients infection was ruled out-blood cultures were negative. all these patients were treated with tocilizumab in a single dose of 8 mg/kg. in all patients, we observed prompt response-normalization of clinical state, renal function, oxygen saturation and decrease of inflammatory factors-crp, procalcitonin and beta2microglobuline. discussion: crs is a rare complication connected with early phase of allogeneic stem cell transplantation. there were no results of treatment with steroids, reduction of a dose of cyclosporine a according to decreased renal function, but all patient completely/fully recovered after single dose tocilizumab treatment. all our patients were subjected to reduced intensity protocols, what might be a risk factor to develop crs because non complete depletion of the patient origin monocytes/macrophages active population. we also analyzed other factor connected with crs in early peritransplant period finding possible connection with s318 proinflammatory hla phenotype. it was obvious in the patient one our patients with peri-engraftment phase crs-he was diagnosed previously with rheumatoid arthritis b27 pos, dr4. in three of five, we have found sle predisposition in hla phenotype (drb1*1501/dqb1*0602 or drb1*0301/ dqb1*0201), in later one-ra associated hla antigen drb1 0101.these patients were analyzed correlating with historical cohort of additional five patients with mortal and another three with very severe early peri-transplant complications and in all we have found the same 'sle or ra hla phenotype'. because small number of analyzed patients and documented high frequency of these haplotype in population, this is still an opened question is proinflammatory/autoimmune hla phenotype connected pathogenically with predisposition to develop severe transplant complications and are we able to treat all these patients with combination of steroids with tocilizumab. further analysis is needed. disclosure of conflict of interest: none. transplant-associated thrombotic microangiopathy (ta-tma) is a severe complication post haematopoietic cell transplantation (hct) leading to high mortality rates. however, outstanding questions regarding its diagnosis, pathophysiology and treatment remain in the literature. recent studies suggest evidence of complement activation, implicating that complement inhibition may be an effective alternative treatment strategy in refractory patients. therefore, we hypothesized that increased complement activation can be detected in ta-tma patients using a functional assay, the modified ham test. we enrolled consecutive patients with ta-tma according to the international working group criteria from january 2015 to june 2016. as controls, we studied patients with graft-versushost-disease (gvhd). complement activation was detected using the modified ham test, a cell proliferation assay based on the susceptibility of a pnh-like cell line to complement activated serum. normal human serum was used as a negative control and lipopolysaccharides(lps)-incubated normal serum as a positive control. all samples were tested in triplicates and twice. we studied 10 ta-tma patients transplanted from unrelated 8/8 matched (4) or 7/8 mis-matched (3) donors, identical (2) and haploidentical (1) siblings. all patients presented severe acute and/or chronic gvhd. ta-tma presented at median +109 (9-930) day post-transplant. in the control group, we studied two patients with steroidsensitive grii and two with steroid-refractory griv acute gvhd. we were able to detect significantly increased complement activation in the serum of ta-tma compared to gvhd patients (p = 0.039). based on previous studies and present controls, percentage of non-viable cells higher than 20% was considered a positive modified ham test, indicating increased complement activation in four ta-tma patients. regarding treatment outcomes, two patients with a negative modified ham test responded to cyclosporine cessation and steroid administration. plasma infusion with/without plasma exchange was initiated in seven patients. however, only three of them responded to second-line treatment. the modified ham test result was significantly increased in refractory patients (p = 0.048). the terminal complement inhibitor eculizumab was administered in one refractory patient with a positive modified ham test and renal failure at presentation. despite delayed initiation (28 days post ta-tma diagnosis), response was observed after three doses of eculizumab including evidence of reduced hemolysis, schistocytosis and transfusion needs. however, the patient succumbed to complications of end-stage renal disease (54 days post ta-tma diagnosis). among 10 ta-tma patients, 8 succumbed at a median +215 (73-430) day to transplant-associated complications, related to gvhd and infections from multi-resistant pathogens. ta-tma is associated with increased morbidity, mortality and severe complications, including gvhd. unlike gvhd, increased complement activation was observed in a significant portion of ta-tma patients. complement inhibition seems an encouraging therapeutic option in these patients. given the lack of robust functional assays for complement activation, the modified ham test may be useful for early recognition of patients that would benefit from complement inhibition. . this proposal includes, along with the 'classical sos' (cases diagnosed before day +21), the new type 'late onset sos' (cases diagnosed afterwards). new ebmt criteria for severy grading classify cases of sos into four grades (mild, moderate, severe, and very severe). the aim of this retrospective study is to analyze the cases of severe/very severe, both classical and late onset sos, occurred in our unit during the most recent period of time. we studied the last 100 pts, with a minimum follow-up of 100 days, who underwent allo-hsct in our center (november 2014-august 2016). 56 pts were male and 44 female. median age was 53 years (range: 7-69). baseline diseases were: acute leukemias (54), lymphoproliferative disorders (17), myelodysplastic syndromes (12), chronic myeloproliferative diseases (7), multiple myeloma (5), and bone marrow failures (5) . donor was unrelated in 57 cases, and related in 43 (including 18 haplo-identical). conditioning regimen was: busulphan-based (70), melphalan-based (13), tbi-based (8) , and others (9). all patient received prophylactic [p390] s320 ursodeoxycholic acid. progenitors source was pb in 89, and bm in 11. five patients developed severe/very severe sos (5% incidence); 3 were classical (at days +8, +11 and +20), and 2 were late onset (at days +34 and +44) (see table 1 ). four cases had received conditioning with a busulphan (iv)-based regimen (doses from 6.4 to 12.8 mg/kg), and one case with tbi plus cyclofosfamide at high doses. all cases presented with right upper quadrant pain, jaundice, ascites, weight gain, hiperbilirrubinemia, and renal function impairment. all but one had increased transaminases. the five cases were treated with defibrotide, in spite of which all of them died. considering that overall day +100 mortality was 9%, severe/ very severe sos was the most important cause of death of the series. [p391] although milder forms of sos might resolves within weeks, the most severe forms are still associated with a very high mortality rate. prophylaxis with defibrotide (the drug currently licensed for treatment) for high-risk patients has not been sufficiently studied yet. therefore, a high index of suspicion, early detection and early therapy are the only ways to try to reduce mortality due to sos in the hsct setting. disclosure of conflict of interest: this research has been performed entirely with public financial support. the royal marsden hospital, sutton, uk; 2 anthony nolan research institute, london, uk and 3 university college london, london, uk secondary poor graft function (spgf) complicates up to 15% allogeneic hcts, and is associated with increased mortality and poor quality of life due to recurrent infections and the need for ongoing blood product support. potential interventions include a second allograft using further conditioning, however many patients with spgf have a reduced performance status and are at an increased risk of complications from this procedure. unconditioned haematopoeitic progenitor cell (hpc) top-ups are associated with a high risk of gvhd if unmanipulated cellular products are used. cd34+ selection offers an attractive alternative, but incurs a loss of up to 20% hpcs and is an expensive procedure, unavailable to many centers internationally. alemtuzumab, a monoclonal anti-cd52 antibody, is routinely used in allogeneic transplant conditioning in the uk to prevent gvhd. we report the results of a retrospective study examining the efficacy of alemtuzumab conditioned hpc top-ups for spgf. data pertaining to patients who had undergone a second infusion of hpcs from their original donor were identified from our hospital-specific promise database. those who met the criteria of spgf defined as ⩾ 2 of hemoglobin 20 × 10 9 /l without support. 10 patients (4 pediatric, 6 adult) who underwent initial allogeneic transplants for malignancy (8) or bone marrow failure (2) received an alemtuzumab conditioned hpc top-up for spgf at our center 2005-2016. the diagnosis of spgf was made at a median 2.6 months post allograft (range 2-60) with trilineage cytopenias in 7 patients and bilineage cytopenias in 3 patients. all patients had received transplants from 10/10 (7 patients) or 9/10 (3 patients) matched unrelated donors. the median interval between initial transplant and top-up was 187 days (range 87-1853), and a median cd34 dose of 2.7 × 10 6 /kg recipient weight (range 0.3-7.63) was infused. 90% patients achieved haematological improvement (hi) at a median 20 days post-top-up (range 13-179), with the only failure to achieve hi seen in the patient who had received the lowest cd34 dose (0.3 × 10 6 /kg). one patient developed grade i agvhd post top-up but no grade ii-iv agvhd was observed. 1 year os was 80% and 2 year os 60% following hpc top-up. 3 deaths occurred due to infection at 1, 5 and 23 months post top-up, and one due to relapse of a prior non-haematological malignancy. 9 patients had an aplastic or hypocellular bm trephine pre-top up, which was repeated at 100 days post topup in 6 patients, of whom 5 had a normocellular bm trephine, while 1 remained hypocellular. alemtuzumab conditioned hpc top-up appears an effective intervention for spgf with results comparable to those of cd34 selected top-ups, and therefore represents a feasible alternative. larger studies are needed to exclude complications including viral reactivation and to investigate immune reconstitution following this procedure. disclosure of conflict of interest: none. high dose chemotherapy (hdt) followed by autologous stem cell transplantation (asct) has shown to improve outcome in patients with relapsed/refractory diffuse large b cell lymphoma (dlbcl). in the rituximab era, the benefit of asct has been debatable as prior study (coral study) has shown that patients who received r-chop as induction chemotherapy & responded to salvage chemotherapy had a poorer outcome following asct compared with those who received chop alone. in addition, it remains unclear whether addition of rituximab to standard high dose beam regimen provides any additional benefit. we retrospectively analyzed 63 dlbcl patients receiving high dose beam (n = 27) or rituximab +beam (r-beam) (n = 36) followed by asct for relapsed/ refractory dlbcl since 2002. all patients who received chop (n = 15) ± rituximab (n = 48) as first line therapy and who received ⩽ 2 lines of salvage chemotherapy before asct were analyzed. rituximab was given at the dose of 375 mg/m 2 on day +1 and +8 of asct. twenty-two (81%) patients in beam group and all the patients (100%) in r-beam group received rituximab-based salvage chemotherapy prior to asct. the 10year overall survival (os) was 71% and event-free survival (os) was 67% for the whole cohort. r-chop induced patients did not fare any worst after asct than chop induced patients (10 year os 73 vs 68 %; p = 0.91). there was a trend towards better survival in patients with pre-transplant disease free interval (dfi) 412 months compared to those with dfi 500/μl) time was 10 days and 11 days, respectively. median platelet recovery (420 000/μl) time was 17 days and 11 days, respectively (p = 0.11). ten year os (67% r-beam vs 77% s321 beam, p = 0.38) and efs (65% r-beam vs 73% beam, p = 0.28) were also comparable between both groups. hdt with beam and asct remains beneficial for patients with relapsed/ refractory dlbcl. it should be offered to all patients who respond to salvage chemotherapy with the expectation that they fare no worse than patients who do not receive rituximab in the induction chemotherapy. addition of rituximab following the standard beam for hdt and asct does not compromise haematopoietic recovery, but does not result in improved outcome in our study. prior use of rituximab during first-line or salvage therapy in most of the patients of r-beam group might have negated the beneficial effect of r-beam over beam. (1) . in this study, we aimed to develop a cns targeted chemotherapy regimen, which has lower toxicity and higher complete remission rates, in combination therapy. eight patients with secondary cns lymphoma (scnsl) and two with primary cns lymphoma (pcnsl), followed between the years 2010 and 2015, were included in the study, retrospectively. the patients were histologically diagnosed with biopsy and underwent autologous stem cell transplantation (apkht). all patients were treated with r-idaram/ rt (radiotherapy)/subsequently autologous stem cell transplantation (apsct) with r-beam protocol. the r-idaram regime consists of the following substances: rituximab 375 mg/m 2 , 50 cc/h infusion, day 1; cytosine arabinoside 1.0 gr/m 2 i.v., 1 h infusion, days 2 and 3; dexamethasone 100 mg, 12 h infusion, days 2, 3 and 4; idarubicin 10 mg/m 2 i.v.,15 min infusion, days 2 and 3; methotrexate 3 gr/m 2 (2 gr/m 2 at 445 years old-patients), 6 h infusion, day 4; and cytosine arabinoside 70 mg plus methotrexate 12 mg, intrathecally, days 2 and 8. the patients included seven males and three females. the median age was 44 years (range: 23-67). six scnsl patients were diagnosed in the application and two of them were diagnosed during r-chop chemotherapy (ct) protocol. five patients (57%) were stage ivb, and the others (43%) were stage iiib at diagnosis. after two or three chemotherapy cycles, patients were mobilized with growth factor support and median 5.510 6 cells per kg (range: 4-8) stem cells were collected. then, at a dose of 3600-4140 cgy cranial rt was administered for 14 days. after the third cycle of r/idaram, the state of remission was evaluated by cranial mri and lumbar puncture (lp). all patients achieved complete remission. neutrophil engraftment occurred at a median of 12 days (range: 10-18) and platelet engraftment occurred at a median 16 days (range: 11-21). after apkht, three patients relapsed and died at the fourth, ninth, and thirteenth months. grade i-ii manageable neurological toxicity occurred in two patients. the median follow-up time was 24 (range: 2-74) months. the five-year overallsurvival (os) was 63%. serious signs of infection were not observed in patients during transplantation. in pcnsl and scnsl, a standard treatment regimen has not yet been found. apsct with r-beam following modified r/idaram/rt is a curative and applicable therapeutic regimen with low toxicity, which can provide high rates of long-term survival and disease-free survival. despite the advent of novel therapies, autologous hematopoietic stem cell transplantation (ahsct) following melphalan (m)-based conditioning remains the standard of care for patients with multiple myeloma who are eligible. still, the majority of patients experience disease progression and ultimately succumb to their disease. we hypothesize that integrating novel agents in the conditioning is feasible and safe and may increase complete remission rates and overall survival. we completed a phase i, dose escalation study of carfilzomib (c) added to a backbone of bendamustine (b) and melphalan. all patients received a fixed dose (20 mg/m 2 ) of c on days (d) − 29, − 28, − 22, − 21, − 15 and − 14. in addition, patients were conditioned as described in table1. due to dose-limiting toxicity in cohort 2, the study was amended after the first 6 patients. subsequently, the dose of m was reduced to 140 mg/m 2 and the d +6 dose of c was omitted, per oversight of a data safety monitoring board. fifteen patients were enrolled, 9 males and 6 females. median age was 56 years (39-68). performance status was ⩾ 80% (kps) in all patients. per the international staging system (iss), 3 patients had stage i disease, 5 had stage ii, 6 had stage iii, and 1 had unknown staging. three patients had high-risk cytogenetics: 2 with t(4;14) and 1 with deletion 17p. four patients had undergone a prior ahsct. disease status at enrollment was stable disease (sd) (n = 3), partial response (pr) (n = 8), or very good partial response (vgpr) (n = 4). median cd34+ cell dose infused was 3.11 × 10 6 /kg (2.23 − 6.92 × 106). median follow-up was 18.2 months (1.4-28.7). all fifteen patients are evaluable s322 for engraftment. median time to neutrophil engraftment was 12 d (11-15). one patient died before achieving platelet engraftment. for the remaining patients, median time to platelet engraftment was 16 d (12-20) . non-hematologic toxicities included grade 3 acute mucositis (n = 1), lower gi complications (n = 7), electrolyte disturbances (n = 7), transaminase elevation (n = 1) renal insufficiency (n = 1), atrial fibrillation (n = 1), hypoxia (n = 1), prolongation of the qtc interval (n = 1), and grade 4 acute sepsis (n = 2), including 1 death (cohort 2) on d +44. eight patients went on to receive maintenance therapy: 3 with bortezomib, 3 with lenalidomide, and 2 with lenalidomide, dexamethasone, and c. posttransplant disease status was assessed per protocol by spep, spif, serum free light chains, and light chain ratio. twelve patients were evaluable on d +100. two patients had sd, 7 had vgpr, and 3 had complete response (cr). eight patients were evaluable on d +365. two patients had progressive disease, 1 had pr, 3 had vgpr, and 2 had cr. the combination of cbm prior to ahsct appears feasible, with manageable toxicities, at the doses described in cohort 3b. a prolonged follow-up and a phase ii study are warranted to determine response rates and long-term outcomes. disclosure of conflict of interest: none. beam (carmustine, etoposide, cytarabine, melphalan) is the most frequently used high-dose chemotherapy regimen for patients with lymphoma referred for autologous hematopoietic cell transplantation (autohct). in recent years a novel conditioning protocol containing bendamustine instead of carmustine (beeam) has been proposed in order to potentially increase the efficacy. so far, however data on its safety are limited. the aim of this study was to retrospectively compare the safety profile of beam and beeam based on single center experience. 174 consecutive patients with lymphoma treated with beam and 63 patients treated with beeam between year 2011 and 2016 were included in the analysis. the median age was 47 (19-69) years and 46 (22-73) years, respectively (p = ns). clinical characteristics of both groups were comparable. patients with hodgkin's lymphoma constituted 49% in the beam group and 40% in the beeam. among those with non-hodgkin lymphoma the diagnosis of dlbcl predominated. beam treatment consisted of carmustine 300 mg/m 2 on day − 6, etoposide 400 mg/m 2 /d on days − 5 to − 2, cytarabine 400 mg/m 2 /d on days − 5 to − 2, and melphalan 140 mg/m 2 on day − 1. in the beeam regimen carmustine was substituted by bendamustine administered on days − 7, − 6 at the total dose of 400 mg/m 2 i.v. peripheral blood was used as a source of stem cells. cd34+ cell dose was 5.1 (1.5-42.6) × 10 6 /kg in the beam group and 4.1 (2 − 16.8) × 10 6 /kg in the beeam group (p = ns). time to engraftment and the rates of adverse events up to day +100 after autohct were the study endpoints. all patients engrafted in both study groups. median time to neutrophil 40.5 × 10 9 recovery was 11 (7-37) days after beam and 10 (7-12) days after beeam (p = 0.13). median time to achieve platelet count 450 × 10 9 was 13 (7-44) days and 14 (7-33) days, respectively (p = 0.29). two patients died without progression before day +100 in the beam group, both due to bacterial infections. no early deaths were reported in the beeam group. the rates of grade 3 or 4 adverse events were comparable (see: table 1 ). administration of bendamustine instead of carmustin as part of conditioning does not affect engraftment as well as toxicity profile of the regimen. therefore beeam may be safely used in patients with lymphoma undergoing autohct. its efficacy requires evaluation in prospective studies focused on homogenous patient populations. [p398] disclosure of conflict of interest: none. the baltimore group reported a low dose tbi-based nonmyeloablative conditioning regimen followed by t cell replete bone marrow, with post-transplantation cyclophosphamide (pt-cy) to control gvhd and graft rejection. based on the fact that in our facility conventional low dose tbi was not available, we wanted to explore whether tmi/tli could be a potential substitute the aims of our study was to explore if tmi/tli can be considered an effective substitute of tbi in terms of os, pfs and nrm. retrospective analysis was applied in 159 cases of haploidentical hsct from april 2009 to october 2016. all patients underwent baltimore conditioning associating fludarabine (30 mg/m 2 /day) day − 6 to − 2, cy (14.5 mg/kg/day) on days − 6 and − 5, and tbi 2 gy in 135 patients and tmi/tli 2 gy in 24 patient at day − 1. unmanipulated bone marrow graft was infused at day 0. postgrafting immunosuppression consisted of cy (50 mg/kg/day) on day +3 and +4, and mycophenolate mofetil for 30 days, and tacrolimus or cyclosporine. no differences between the two groups was observed in term of age, gender diagnosis, disease status and donor type. 93% of patients engrafted in both arm (23/24 and 125/135). in tbi cohort vs tmi/tli cohort, the median time to anc 4500/μl and platelet recovery 420 000/μl was not different (22 and 27 days vs 20 and 27.5 days, p = 0.14 and 0.32, respectively). in all tmi/tli evaluable patients, full chimerism was observed at days +30. after a median followup of 17 months in tmi/tli cohort and 34 months in tbi arm, 1-year nrm was 25.9% and 13.9% (p = 0.16), respectively. the 1 years os and pfs were not statistically different in the two groups 70% vs 57.1%, p = 0.12 and 62.5% vs 48.1%, p 0.09, respectively). the 1-year relapse incidence was 26% in tmi/tli group and 23.6% in tbi group, p = 0.61. no difference in incidence of both agvhd and cgvhd was observed between the two groups. this retrospective analysis suggests that tmi/ tli could be considered an effective substitute of low dose tbi, with a sufficient degree of immunesuppression of recipient, allowing engraftment and full chimerism. the gvhd both acute and chronic as well as the 1-y nrm were not different. disclosure of conflict of interest: none. comparison of the beeam conditioning regimen and the beam conditioning regimen in the autologous transplantation for hl and nhl s lozenov 1 , p ganeva 1 , y petrov 1 , g arnaudov 1 and g mihaylov 1 1 the beam has established itself as a standard of care conditioning regimen in the autologous lymphoma hsct setting for most transplant centres in europe. yet however various other regimens are being compared with it in order to achieved better safety profile, better os and dfs, in order to improve results with chemoresistant and unfavourable patients. one such regimen is beeam (bendamustine, etoposide, cytarabine, melphalan).we aimed to compare the efficacy of the beam and beeam conditioning regimens and to compare their myelotoxicity profile. we evaluated retrospectively 114 adult patients (mean age 41.1197 with sd 11.12404), receiving auto-hsct at the national specialized hospital for active treatment of hematological diseases in sofia, bulgaria for relapsed/refractory hl or nhl (of them mh -57, dlbcl -26, pmbcl -15, fl -3, lbl -3, ptcl-nos -3, aitl -2, alcl -2, mcl -2, mzl -1) for the period from 1.01.2013 to 1.07.2016 with a follow-up of patients up to 1.11.2016. ninety-two of the patients received the beam (as previously described -bcnu 300 mg/m 2 i.v. day − 6, etoposide 200 mg/m 2 i.v. days − 5 to − 2, cytarabine 400 mg/m 2 i.v. days − 5 to − 2, and melphalan 140 mg/m 2 i.v. day − 1) regimen and 22 received beeam regimen (bendamustine on days − 7 and − 6 (160 mg/m 2 ); cytarabine, 400 mg/m 2 intravenously daily, from day − 5 to day − 2; etoposide, 200 mg/m 2 intravenously daily, from day − 5 to day − 2; and melphalan, 140 mg/m 2 intravenously on day − 1). the overall survival at the second and third years of follow-up (os-2, os-3) and dfs at the third year, the cr rates and the average time periods to hematological recovery, were compared. the os at 2 and 3 years, respectively, was 86.1% and 86.1%, for beeam and 78.8 % and 71% for beam, the dfs at 3 years was 76.4% for beeam and 73.2% for beam, provided that the differences did not have statistical significance (p 0.851 for os and p 0.890 for the dfs). the cr rate was 63.63% in the beeam group versus 50% in the beam group. from the patients who received autologous hsct in stable disease or progression pre-transplant status (chemoresistnat patients), 22.72% of the patients receiving beeam achieved cr at the first post-transplant evaluation versus 10.86% respectively for the beam group. the mean time to hematological recovery for neutrophils was 11.1765 ± 4.91471 days (beeam) versus 10.2469 ± 3.56216 days (beam) and 12.6471 ± 6.04091 days (beeam) versus 11.1235 ± 2.52677 days (beam) for platelets. beeam appears to be a non-inferior alternative conditioning regimen to the standard beam, it shows a trend towards higher myelotoxicity, but also a trend towards better response rates in chemoresistant patients. [p400] disclosure of conflict of interest: none. autologous hematopoietic stem cell transplantation (asct) is widely used as a consolidation therapy in aggressive non-hodgkin's lymphoma (nhl) and recurrent or refractory classic hodgkin's lymphoma (hl). in mexico, the use of carmustine (bcnu) in the conditioning regimen of these patients is limited due to the lack of access to the drug and its high costs. this study aims to compare results in terms of toxicity, disease-free and overall survival between a group of patients treated with the standard regimen beam and another group treated with a scheme in which carmustine was replaced by cisplatin (peam regimen). a comparison of two groups with lymphoma was performed and the clinical aspects of cisplatin 100 mg/m 2 d . the characteristics were well balanced between the two groups. the mean time for neutrophil grafting (4500 per mm 3 ) was significantly slower with beam than with peam (12 vs 11 days, p = 0.001), hospitalization time was longer with beam compared to peam (25 vs 22, p = 0.015). on the other hand, proportion of patients who require red blood cell s324 transfusion was significantly higher in beam group (58%) versus peam group (20%) (po 0.001), but total amount of platelet transfusion did not differ between groups. about the toxicity, beam patients had significantly more frequent incidence and severity of nausea/vomiting (95% vs 53.8%) and diarrhea (61.5% vs 90%) compared to peam (p o 0.01). no significantly differences were observed in incidence of mucositis (p = 0.65). at the moment of the analyses, 75% of patient of the peam group were in complete response versus 59% of the patients treated with beam, but it did not represent a significant difference. disease-free survival and 5-year overall survival in the peam vs beam scheme were similar with 64% vs 59% (p = 0.69) and 84% vs 76% (p = 0.35) respectively but with less toxicity using the peam scheme. peam regiment is not inferior scheme compared with beam, because it shows similar outcomes in disease-free survival and overall survival. additionally, peam is a well-tolerated regime and beam scheme was associated with greater gastrointestinal toxicity such as nausea, vomiting and diarrhea, also greater hematology toxicity such as more requirement of red blood cell transfusion. [p401] disclosure of conflict of interest: none. cumulative busulfan exposure is associated with relapse following busulfan and cyclophosphamide myeloablative allogeneic stem cell transplantation for acute myeloid leukaemia e wong, d kliman 1 , m chau 2 , j szer 2 , c nath 1 , p shaw 1 , d ritchie 2 , d gottlieb 1 and a bajel 2 1 westmead hospital, new south wales, australia and 2 royal melbourne hospital, victoria, australia the optimal busulfan exposure to reduce disease relapse in adult patients with acute myeloid leukaemia (aml) undergoing busulfan/cyclophosphamide myeloablative allogeneic stem cell transplant (allosct) is poorly defined. we retrospectively analysed busulphan pharmacokinetics (pk) and outcomes of patients who underwent busulfan/cyclophosphamide conditioned allosct for aml from 2010 to 2016. busulfan was administered intravenously over 5 days (1.6 mg/ kg/d for 2 days followed by 3.2 mg/kg for 3 days). peripheral blood was obtained for busulfan pk after the first dose. subsequent doses of busulfan were decreased if daily busulfan exposure (area under the curve; auc) was anticipated to exceed 5000 μm per min/day. cyclophosphamide was dosed at 120 mg/kg. the primary outcome was the cumulative incidence of relapse (cir) accounting for non-relapse mortality (nrm) as a competing risk. independent variables analysed included age, sex, cytogenetic risk group, disease risk index (dri), donor type, stem cell source, t-cell depletion, and cumulative busulfan auc (cumauc) calculated as previously described. 1 (figure 1) . t-cell depletion was also associated with increased cir (hr 3.3; p = 0.0049). patient age, sex, cytogenetic risk, dri and graft type were not significantly associated with cir. on multivariate analysis, cumauc 415 000 μm per min remained significantly and independently associated with lower cir (hr 0.38; p = 0.036). cumauc was not associated with nrm, rfs, os, or the incidence of acute or chronic gvhd. figure 1 . cumulative incidence of relapse in patients stratified by total busulfan exposure. [p402] cumulative busulfan exposure 415 000 μm per min is independently associated with reduced relapse following busulfan/cyclophosphamide allosct for adults with aml. these findings support further evaluation of the optimal busulfan exposure to reduce aml relapse in a prospective clinical trial, whereby patients could be randomised to target cumauc 415 000 μmol per min versus standard practice. hematopoietic stem cell transplant with busulfan and cyclophosphamide (bucy) based conditioning has a relatively high incidence of liver toxicity and sinusoidal obstruction syndrome (sos). busulfan and cyclophosphamide metabolites share the same glutathione conjugation in the liver metabolism. a small number of studies addressed different sequence of both drugs bucy vs cybu during conditioning. differences in liver toxicity, sos, transplant related mortality (trm), relapse incidence (ri) and overall survival (os) were reported favoring cybu conditioning. we decided to address the above issues at the umc ljubljana, slovenia. this was a retrospective study following patients with myeloid malignancies (aml, mds, mpn) with bucy (n = 30) and cybu (n = 14) conditioning through a three year period in a single institution. primary endpoint was detecting difference in liver toxicity by measuring levels of liver enzymes. secondary endpoints were incidence of sos, difference in trm, ri and os. patients characteristics between groups at the time of the transplant did not differ significantly. we observed significantly higher liver toxicity through elevated bilirubin and alt in the bucy 73.3% than cybu 64.3% patient group (picture 1). the highest probability of liver toxicity was around d0 in the bucy group and in the second week after the transplant in the cybu group. the incidence of sos, trm and ri were comparable between the groups. there was no difference in os between the patient groups during the 40-month follow-up. bucy conditioning for hematopoietic stem cell transplant causes higher incidence of liver toxicity compared to cybu conditioning. there is no difference in sos frequency, trm, ri and os between bucy and cybu conditioning. prospective controlled comparison would be needed for further study of the subject. disclosure of conflict of interest: none. early monocyte recovery is associated with better overall survival after busulfan containing myeloablative conditioning allogeneic hematopoietic cell transplantation in patients with acute myeloid leukemia a lojko-dankowska 1 the outcomes of allogeneic hematopoietic cell transplantation (allohct) in acute myeloid leukemia (aml) depend on different patient-, disease-and transplant related factors, including the dose and combination of agents used for conditioning. the aim of the study was to analyze the outcomes of allohct in patients with intermediate or high risk aml according to disease risk index (dri) who received myeloablative conditioning consisted of intravenous busulfan (9.6-12.8 mg/kg) combined with cyclophosphamide (120 mg/ kg) or fludarabine (150 mg/m 2 ) between 2010 and 2016 in our institution. the published data indicate that the combination of busulfan (bu) and fludarabine (flu) seems to have more favorable toxicity profile than combination of bu and cyclophosphamide (cy), so bucy regimen has been substituted with buflu as the myeloablative conditioning for aml patients in our institution practice since 2014. we evaluated the influence of type of regimen on transplant outcomes along with the impact of other potential prognostic factors, including age of patient, dri, donor type, hla and gender mismatches, stem cells source, and lymphocyte and monocyte recovery. the study group consisted of 71 aml patients, median age 37 years (range: 19-59), classified as intermediate (n = 57) or high (n = 14) risk according to the dri, who were conditioned with bucy (n = 38) or buflu (n = 33) followed by allohct from hla identical sibling (n = 23) or 9-10/10 matched unrelated donor (n = 48). the stem cell were collected from peripheral blood (n = 44) or bone marrow (n = 27). gvhd prophylaxis consisted of calcineurin inhibitor combined with mtx plus atg in allohct from unrelated donors. engraftment was observed in all patients. the median time to neutrophil count (0.5 g/l) and platelet count (20 g/l) recovery was shorter after buflu in comparison with bucy (18 days vs 22 days; p = 0.045 and 13 days vs 20 days; p o0.001), however peripheral blood stem cells were used more often after buflu regimen than after bucy (88% vs 40%, p340/mm 3 on+21 day after transplant (2-year os 77% vs 55%, p = 0.034) and intermediate vs high dri (2-year os 78% vs 53%, p = 0.068). in multivariate analysis higher amc after allohct remained the only independent favorable prognostic factor for os (rr 0.35 (95% ci 0.13-0.97), p = 0.04). our results suggest that early monocyte recovery after myeloablative bu containing conditioning allohct is significant favorable predictor of outcome. in our experience both bucy and buflu myeloablative regimens result in similar long-term survival after allohct in aml patients. [p403] disclosure of conflict of interest: none. the use of t-cell depletion as part of the conditioning protocol has the potential to improve the tolerability of allogeneic stem cell transplantation (hsct) through the reduction in graft versus host disease (gvhd). despite the wide spread adoption of this practice in many parts of the uk and europe, definitive recommendations regarding the most appropriate dose remain elusive. previous experience by our group with 100 mg of alemtuzumab combined with fludarabine and busulfan based conditioning demonstrated good long-term outcomes with low rates of gvhd. however, due to concerns of high relapse risk especially in patients with high-risk myelodsypastic syndrome and acute myeloid leukaemia, we instituted a policy change in 2013 to reduce the dose of alemtuzumab in the conditioning protocol from a total of 100-60 mg. we conducted a retrospective analysis of all consecutive patients undergoing reduced intensity unrelated allogenic stem cell transplantation with fludarabine (150 mg/m 2 ), busulfan (6.4 mg/kg iv or 12.8 mg/kg iv) and alemtuzumab (fb2c or fb4c, respectively) conditioning for neoplastic myeloid disorders between 2010 and 2016. patients were subsequently analysed in two cohorts; those receiving 60 mg of alemtuzumab (n = 84) and those receiving 100 mg of alemtuzumab (n = 95). apart from a decreased proportion of females in the 60 mg alemtuzumab group, the cohort was balanced across the different dose levels ( table 1 ). the longterm overall survival (os) of the entire cohort was good with a 5 year os of 51%. no significant differences in overall outcomes across the two groups were observed with a 5 year os of 51% in the 60 mg group vs 49% in the 100 mg group (p = 0.39). cumulative incidence of relapse (cir) and nonrelapse mortality (nrm) was 42% and 21% and 39% and 24% in the 60 mg and 100 mg groups, respectively. interestingly, age had a significant effect on nrm in the 100 mg (7% age o50, 28% age 50-65 and 41% age 465 p = 0.04), but not in the 60 mg group (11% age o50, 19% age 50-65 and 24% age 465 p = 0.8). the effect on relapse rate was not significant in either group (p = 0.6 and p = 0.11, respectively). this retrospective analysis did not demonstrate an overall improvement in transplant outcomes with dose de-escalation of alemtuzumab from 100 to 60 mg. in particular, we did not see the anticipated improvement in relapse rate in this cohort. notably older patients seem to tolerate the 60 mg dose better due to the lower nrm. prospective trials with accompanying translational work are required to determine the optimal dosing and schedule for this group of patients. disclosure of conflict of interest: none. bendamustine was given at 200 mg/m 2 /d for the first 4 pts then 100 mg/m 2 /d for the 4 subsequent pts and finally at 120 mg/m 2 /d for the remaining pts (22 pts). among the beam group, 68% had non-hodgkin's lymphoma (nhl) and 32% hodgkin's lymphoma (hl) compared to 87% and 13%, respectively, in the beeam group (p = 0.014). hhv-6 detection was performed by pcr for symptomatic pts (fever, rash or prolonged cytopenia). patients were housed in single bedrooms with air filtration and received the same supportive care. median age was 50 (18-66) and 56 (20-67) in the beam and beeam groups respectively and median of previous chemotherapy regimens was 2 (range: 1-5). fifty two out of 90 patients were male (37/60 in the beam group and 15/30 in the beeam group). pts were in cr (46.7% vs 56.7%) or pr (53.3% vs 43.3%) at time of transplant. there was no difference in terms of hematologic recovery (median = 11 days (range: 7-22)), blood and platelets transfusion, mucositis toxicity. there was no statistical difference in the incidence of acute renal failure when comparing the two groups. however, there was a very striking difference when considering the highest dose of bendamustine when compared as well to the two others doses of bendamustine (po 0.00001) as to the beam group (p = 0.005). additionally, we also observed a high incidence of symptomatic hhv-6 infections (53.3% vs 8.3%, p o0.00001), digestive toxicity (36.6% vs 15%, p = 0.03) and a longer hospitalization duration (25 days (range: 18-59) vs 21 days (range: 18-32), p = 0.001) for patients in the beeam group overall. with a median follow up of 18.3 and 9.7 months for beam and beeam respectively, overall survival (93% vs 86%), transplant related mortality (0% vs 3%) and event free survival (83% vs 78%) were comparable. overall, beeam regimen was associated with longer duration of hospitalization, higher rate of digestive toxicity and increased risk of symptomatic hhv-6 infection as compared to the beam regimen. in addition, higher doses of bendamustine (200 mg/m 2 /d for two consecutive days) were associated with unacceptable high rate of acute renal toxicity. with a still short follow-up, the absence of benefit on disease control together with higher short term toxicity does not allow to recommend the use of beam instead of classical beam. should it be used, we suggest that pts should be carefully monitored for renal toxicity and for hhv-6 infection in case of symptoms. disclosure of conflict of interest: none. high-dose treosulfan and melphalan for consolidation therapy in high-risk ewing sarcoma me abate, a paioli, a longhi, m cesari, e palmerini and s ferrari musculoskeletal department, rizzoli orthopaedic institutes, bologna, italy common toxicities observed after high dose chemotherapy with busulfan and melphalan for high risk ewing sarcoma (es) are generally well managed by current supportive care but some patients can develop severe complications. treosulfan is an alkylating agent that has recently been used as a substitute of busulfan to prevent potential serious complications related to busulfan. medical records of 7 es patients undergoing autologous peripheral blood stem cell (apbsc) transplantation after intravenous treosulfan (treo) and melphalan (mel) from 2011/6/1 to 2016/2/29 were analyzed with regard to toxicity and outcome. patients were included into the study if they were eligible for the protocols activated in our institution for es and presented reasons that did predict potential complications related to busulfan, such as previous radiotherapy on axial skeleton/pelvis or coexistence of high risk of epilepsy. as consolidation treatment patients received intravenous treo 12 g/m 2 over 3 days and mel 140 mg/sqm with support of apbsc transplant and use of granulocyte colony stimulating factor. in those patients with lung metastases total lung irradiation was performed at least 2 months after treomel. frequency of toxicity for treomel was recorded with at least 6 months of follow-up and was evaluated according to nci ctg common toxicity criteria. the median age at diagnosis of patients receiving treomel was 16 years (range 13-25 years), 3 males and 4 females. 5 patients had localized disease at diagnosis with poor radiological or histological response to standard chemotherapy; one patient had lung metastases at diagnosis and one patient had relapsed disease with lung metastases. before receiving treomel the primitive tumour underwent radiation therapy in 5 cases (3 pelvis, 1 cervical vertebra, 1 sacrum), surgical resection in one case(tibia) and surgical resection plus radiation therapy in one case (fibula). 2 patients showed eeg abnormalities at high risk of developing epilepsy. the median number of infused cd34+ cells was 5.9 × 10 6 /kg (range 3.4-15.9). febrile neutropenia occurred in 5/7 patients and lasted one day in 3 patients and 2 days in 2 patients. median time to granulocyte engraftment was 10 days (range 10-12 days); median time to platelet engraftment 420 000 was 10 days (range 9-16 days). only one patient needed 2 red blood cells transfusions; 5 patients needed 1 platelet transfusion and 2 patients needed 2 platelet transfusions. none developed grade 3-4 stomatitis or grade 3-4 [p406] hematuria or grade 3-4 liver toxicity. surprisingly, a patient became pregnant after 1 year and 10 months from transplantation. with a median follow-up of 24 months (range 8-65 months) 5 patients are alive in complete remission, one patient is alive with relapsed disease and one patient died for disease progression. these results, related to a limited cohort of patients, confirm the lower toxicity observed for treosulfan with respect to busulfan. although more data are needed to clarify the role of treosulfan in es, the impact of potential severe complications observed with busulfan, including infertility, should suggest its replacement with treosulfan in selected cases. disclosure of conflict of interest: none. immunoadsorption procedures prior to haploidentical allogeneic pbsct could prevent graft failure in patients with hematological malignancies displaying anti-donorspecific hla antibodies donor-specific anti-hla antibodies (dsa) have been shown to be associated with a high risk of rejection in solid organ transplantation and with graft failure (gf) in allogeneic hematopoietic stem cell transplantation. a combination of anti-cd20 (rituximab), plasma exchange (pe), and ivig in 12 patients with additional buffy coat infusion in 5 among them prevented graft loss in all patients that became c1q negative before sct. we addressed the question whether immunoadsorption in combination with rituximab can also be applied in patients with dsa to prevent graft failure in haploidentical pbsct. four patients with acute myelocytic leukemia or myeloma in second complete remission were enrolled. the presence of dsa was determined by luminex at pre bmt checking. immunoadsorption was performed with polyclonal sheep anti-human igg adsorbers (miltenyi biotec gmbh, germany) on life 18 apheresis system. in addition all patients received rituximab 375 mg/kg bw in a single dose. patients were conditioned with a reduced intensity regimen comprising tbi 2 gy, cyclophosphamide 29 mg/kg, and fludarabin 150 mg/m 2 . all patients received cyclophosphamide post bmt (ptcy) 100 mg/kg. non-t-cell depleted pbsct were transfused in a sequential manner in 2 doses each. the data of the patients and treatments is summarized in table 1 . two patients had a normal hematopoietic reconstitution and are alive at +21 and +15 months post-transplantation, one with hepatic gvhd; chimerism was 100% in peripheral blood on last follow up. one patient died following a graft failure. by a combination of rituximab and repeated immunoadsorption prior to allogeneic pbsct the titer of dsa could be lowered sufficiently to enable engraftment. ia turned out to be a safe procedure without relevant clinical side effects. hematopoietic reconstitution was in the normal range in 2 of 3 evaluable patients. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative option for patients with beta thalassemia major. however, the availability of hla-matched related donor remains the main obstacle for allogenic hsct. although, a few studies have been reported, experience with hla matched unrelated donors is limited. we present the result of 35 children with beta thalassemia major who received allogeneic hsct from hlamatched unrelated donors with using a novel conditioning regimen. we retrospectively assessed 35 unrelated hsct in children with beta thalassemia major. all patients received busulphan (bu) based myeloablative conditioning regimen. busulphan was used according to weight adjusted doses. in addition, all patients received fludarabine 150 mg/m 2 in 5 days, cylophosphamide 120 mg/kg in 3 days, thiotepa 10 mg/kg in one day and atg 30 mg/kg in 3 days. cyclosporin-a and mtx were used for graft versus host disease (gvhd) prophylaxis. donor chimerism was evaluated in the peripheral blood on days +30, +100 and +180. the median age of the patients was 6.9 years (range 14 month-15 year). two of the patients were grouped in class i and rest of them were class ii. the median serum ferritin level was 1.255 ng/ml (range, 585-5832). all of s329 the donors were matched 10/10 with high-resolution hla typing in gvhd direction but three of them 9/10 with graft failure direction. twenty-three of them received bm (median tnc: 6.2x108/kg) and 12 pbsc (median mnc:7.3x108/kg) with median cd34+ cell number 7.20x106/kg. the median neutrophil and platelet engraftment days were 13 and 14 days in pbsc and 17 and 20 days in bm group, respectively. grade i-iv acute gvhd was observed in 7 patients (26%) and only one experienced limited chronic gvhd with only skin involvement. mild to moderate vod was seen in 13 patients (37%) and treated with defibrotide successfully. all patients except one are alive with full donor chimerism (between 95-100 %) with a median 12 months (range 3-49 months) follow-up. one patient died because of cmv pneumonia. these data show that the results of hsct from unrelated donors in selected low risk thalassemia patients may be comparable to hsct of matched sibling donors. however, it needs further studies with long term follow up and larger study population. disclosure of conflict of interest: none. table 1 . data about cytogenetic risk of group 1 patients were available only in 9 individuals. differences between groups were analyzed by t-student and chi square tests. survival was analyzed by kaplan-meier method and differences in survival between groups were evaluated by log rank test. no differences were found between groups regarding gender, sc source, disease status at sct, type of donor and number of cd34+ cells infused. patients in group 2 were significantly older (median age for groups 1 and 2: 32 vs 38, p = 0.021). gvhd prophylaxis protocols included atg in a higher frequency in group 2. no differences between groups 1 and 2 were observed in neutrophils recovery (median days to anc4500/μl: 9 vs 8 respectively, p = 0.78) and platelets recovery (median days to platelets 420 000/μl: 8 vs 5 respectively, p = 0.51). patients in group 1 required more red cell transfusions (median packed rbc: 3 vs 1, p = 0.048). no differences were observed regarding platelets transfusion requirements or length of hospitalization. post-sct os was significantly better in group 2 (3 years-os group 1: 23%; group 2: 61%; p = 0.029) (figure 1). there were no significantly differences between groups regarding frequency of mucositis, diffuse alveolar haemorrage, sepsis, acute and chronic gvhd. vod was more frequent in group 1 (4/21 vs 0/20, p = 0.03). trm mortality was higher in group 1 (6/21 vs 3/20), being this difference no statistically significant (p = 0.29). as it was reported by others, the use of fludarabine-based conditioning regimen was associated with a significantly better post-sct os and a reduced frequency of vod in aml patients. reduction in trm and differences in the frequency of described complications are not statistically significant probably due to the small size of this sample. since march 2016, given the limited availability of melphalan, we administer the beac regimen (carmustine, etoposide, cytarabine, and cyclophosphamide), instead of the gold standard conditioning regimen beam, followed by autologous haematopoietic cell transplantation (ahct) in relapsed or refractory hodgkin (hl) and non-hodgkin (nhl) lymphoma patients. the primary goal of this analysis was to assess the immediate related toxicity of this alternative regimen. we used beac (carmustine 300 mg/m 2 , etoposide 800 mg/m 2 , cytarabine 800 mg/m 2 , and cyclophosphamide 140 mg/kg) in 22 consecutive lymphoma patients (13 hl, 9 nhl) who underwent ahct for relapsed or refractory disease. the median age of the patients was 42.5 years (17-64). they all received peripheral stem cell grafts with a median cd34+ cell dose of 5.13 × 10 6 /kg cells (2.07-15.78). disease status post salvage treatment (at ahct) was complete remission (cr) in 6, partial remission (pr) in 11 and progressive disease in 5. the disease was chemosensitive to salvage therapy in 17/21 patients. median follow up was 112 days (31-224). toxicity was assessed according to the who toxicity scale grading. all patients engrafted successfully. median time for engraftment was day +10 (d+10) for neutrophils (4500/mm 3 ) and d+11 for platelets (420 000/mm 3 , without transfusion within the previous 3 days). patients were hospitalized for a median of 19 days (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) . no treatment-related mortality occurred. two patients died due to disease progression (both nhl patients, on d+143 and d+63). toxicity assessment until d+30 is presented in table 1 : moreover, no hemorrhagic cystitis or macroscopic hematuria, and no cardiac events were encountered. febrile neutropenia was recorded in 4 and bacteremia in 9 patients (7 gram+, 2 gram-, 2/9 related to central venous catheter), with fever ≤ grade 2 in all cases. during d+30-100 two patients presented fever of unknown origin, and 3 patients had upper or lower respiratory infections, with no other adverse events being recorded. in terms of disease best response within 3 months post ahct (18/22 patients evaluated), 11 patients achieved or sustained cr, 5 pr (1 of these patients eventually died due to disease progression), 1 relapsed and 1 succumbed due to disease progression (no response). according to our preliminary results, the early toxicity profile of beac is very low, the regimen is easily tolerable for the patients, and without any treatment-related mortality. its use as an alternative conditioning regimen in ahct for lymphoma patients seems feasible. further investigation including more patients and comparative analysis to other conditioning regimens are warranted for reliable conclusions on the toxicity and efficacy of beac. disclosure of conflict of interest: none. veino-occlusive disease (vod) is a potentially fatal adverse event caused by intravenous (iv) busulfan used in bone marrow transplantation (bmt) conditioning. the objective of this study was to identify determinants of vod in children treated by iv busulfan. this was a retrospective analysis of data collected in 293 children from two bmt centers over 10 years. vod was diagnosed according to modified seattle criteria. individual pharmacokinetic data, including busulfan area under the concentration-time curve (auc) and maximal concentration (cmax) were estimated in all children by using a validated bayesian approach. 1 we examined the relationships between the occurrence of vod and available data in a learning (n = 243 patients) and validation set (n = 50 patients) obtained by random splitting. logistic regression was used as a continuous statistical model. in addition, we used classification and regression tree (cart) analysis, a machine learning and binary partitioning technique, to identify determinants of vod and their optimal cut-off values. the predictive performance of variables within both models was assessed by these results are compared with historical data from our service using beam as conditioning followed by auto-sct in lymphoma patients. nine patients were enrolled to receive neam: mitoxantrone 6 mg/m 2 day -6 to -4, etoposide 100 mg/m 2 every 12 hours and cytarabine 100 mg/m 2 every 12 hours day -6 to -3, and melphalan 140 mg/m 2 day -2, followed by auto-sct. the median age was 51 years (19-63); five non-hodgkin lymphomas (nhl) and four hodgkin lymphomas (lh). six patients were in partial remission (pr), two in complete remission (cr), and one with progressive disease at time of auto-sct. neam patients were compared with a historical control group of patients receiving beam regimen (n = 167). differences between groups were analyzed by t-student and χ 2 -tests. median cd 34+ cells infused in neam and beam groups was 3.48 × 10⁶/kg (3.09-8.72) and 4.14 × 10⁶/ kg (1.02 -25.6), respectively (p = 0.42). the median time to neutrophil recovery (4500/μl) was 16 days (11-22) and 10 days (3-30) (p = 0.000017) and median time for platelets recovery (420 000/μl) was and 15 days (9-30) and 8 days (1-49) (p = 0.018) respectively, for neam and beam patients. median duration of hospitalization was 34 days (24-55) with neam and 27 days (15-59) with beam (p = 0.002). among neam patients, 88% had one or more febrile episodes during neutropenia. no case of grade iii or iv mucositis was described. there was no transplant-related mortality (trm: 0%) associated with the use of neam regimen. at the present, all neam patients are alive, two of them in relapse (22%). due the difficulties in obtaining carmustine in our region, neam can be considered as a feasible alternative to beam. however, despite the sample was small enough to draw conclusions, we find that neam presents prolonged aplasia of significant value, we are currently exploring conditioning regimens followed by auto-sct in hodgkin's and non-hodgkin's lymphomas based on bendamustine, etoposide, cytarabine and melphalan. disclosure of conflict of interest: none. at present, decision-making about conditioning regimens for allogeneic hsct is based on patient's and donor's features, and disease characteristics. during the last years, terms as 'myeloablative/non-myeloablative/reduced-intensity' have been frequently employed in a confusing and unequal way among the different centers. knowing the expected intensity and myeloablative effect from each regimen is very useful and constitutes the aim of this analysis. we have analysed the severe neutropenia (anc o500 per mcl), and thrombocytopenia durations (platelets o20 000 per mcl), the need for platelet concentrates transfusion and the duration of the inpatient period of the 223 allo-hsct carried out during the last four years in our centre. these data are reported according to the conditioning regimen used and to the type of transplant performed. then, they are compared among them in order to stablish intensity ranks. results: population characteristics are described in table 1 : conventional intensive regimens (bu-cy2, cy-bu2, tbi-cy) reported more days of severe neutropenia and greater need of platelet concentrates transfusion. the regimens with less days of severe neutropenia, less need of platelet concentrates transfusion and fewer days of admission were flu-bu3 and flu-bu2. allotransplants carried out with stem cells from bm presented more days of severe neutropenia and longer hospital stay. similar platelet transfusion need was reported. haplo-identical allotransplants reported more days of severe thrombocytopenia, but were not asociated to longer neutropenia or longer hospital stay than the others. these data are described in detail in table 2 . flu-bu: the number (4, 3 or 2) expresses the doses of busulphan administered at 3.2 mg/kg/day. pc: platelet concretates data is expressed in medians. within the analysed conditioning regimens, an intensity rank is stablished regarding the myelosupression induced (in descending order): conventional intensive regimens (cy-bu2, bu-cy2, tbi-cy), flu-mel, flu-bu4, flu-bu3 and flu-bu2. all of them induced severe neutropenia and thrombocytopenia, and for that reason they must be considered myeloablative regimens. disclosure of conflict of interest: none. myeloablative allogeneic stem cell transplant for aml and mds: the impact of advanced age in the outcome m sánchez-escamilla* 1,2 , s garcía-ávila 1 , l yáñez san segundo 1,2 , ma bermudez rodriguez 3,2 , mm colorado araujo 1,2 , a casado diez 1 , m celis alvarez 1 , a cabero martinez 1 , c fernandez martinez 1 , a insunza garminde 1,2 , c richard espiga 1,2 and e conde garcía 1,2 1 allogeneic hematopoietic stem cell transplantation (allo-hsct) is the only curative option in high risk myeloid hematological malignancies. myeloablative conditioning (mac) regimen has been proven to be effective in the control of high risk diseases in advanced age patients. objective: the aim of this study was to analyze the efficacy of myeloablative allo-hsct in two cohorts of patients considering their age at transplant. we also analyzed the incidence of acute and chronic graft versus host disease (gvhd) and procedure related outcomes who underwent to myeloablative allo-hsct were retrospectively analyzed. the median age was 49 years (iqr 36-57). both groups were divided regarding their age at allo-hsct [group 1, age ⩾ 55 years (n = 41) and group 2, age o55 years (n = 93)]. patient´s characteristics are shown in picture 1. data were collected as either continuous data and compared by two-tailed unpaired t-test or mann-whitney test, or as categorical variables and compared by chi-square. the procedure related outcomes were analyzed with the kaplan-meier test. the incidence of acute gvhd grade ii-iv was similar in both groups (43.9% in group 1 and 42.2% in group 2, p = 0.761 ). the mean day to acute gvhd (grade ii-iv) development was 38 days in group 1 and 40 days in group 2. the most involved organs in both groups were skin (group 1: 94.4% and group 2: 72.1% [p = 0.258]) and gut (group 1: 55.6% and group 2: 62.8% [p = 0.598]). at day +100 post-transplant 123 patients were alive and evaluable for chronic gvhd. the incidence of cgvhd development was similar between group 1 and 2 (61.1% versus 68.4%, respectively, p = 0.140). however, severe grade s333 of cgvhd was high in group 1 patients (25.0% versus 18.4%). with a median follow up of 43 months (iqr, 9-70) the probability of os was significantly low (p = 0.004) in group 1 (46.3% +-7.8) compared with group 2 (67.0% ± 4.9). pfs was also significantly low (p = 0.003) in group 1 (41.5% +-7.7) compared with group 2 (66.3% ± 4.9). trm at 43 months was higher in group 1 compared with group 2 (34.1% versus 17.2%). mortality due to relapses was also higher in group 1 (17.0% versus 12.9%). most of the patients died during the first 24 month. comparing both groups at this time (24 months post-transplant), trm was higher in group 1 compared with group 2 (26.8% versus 14.0%). deaths due to relapse were also higher in this group (17.1% versus 10.7%). in our series, myeloablative conditioning regimen provides good survival rates and disease control in high risk hematopoietic diseases, however in patients aged ⩾ 55 years confers high toxicity. it may be necessary to evaluate other strategies in this group of patients. disclosure of conflict of interest: none. allogenic hematopoietic cell transplantation (hct) is reserved for a group of high risk multiple myeloma (mm) patients having relapsed after high-dose melphalan and autologous transplantation. in general, reduced-intensity conditioning (ric) regimen are applied in this patient group with the aim of reducing transplant related mortality (trm). however, relapse of disease remains a major challenge after allogeneic hct. to address this issue, we added radioimmunotherapy (rit) to a conventional ric regimen. we have used a 188 rhenium anti cd66 antibody in combination with a ric conditioning regimen. this ß-emitter leads to a so called 'cross-fire' effect allowing for bone marrow doses of 20 gy and in parallel may target cd66 on myeloma cells. we hypothesized that this strategy may decrease the incidence of relapse. so far, we have treated nine patients with high risk relapsed multiple myeloma. all patients had received one (n = 7), two (n = 1) or three (n = 1) high-dose regimens and autologous hct. conditioning therapy was flu/mel (n = 5), flu/ bu (n = 2) or flu/treo (n = 1). flu/cy and 2 gy tbi were applied before haploidentical transplantation. patients received g-csf mobilized pbsc from unrelated (n = 8) or haploidentical (n = 1) s334 donors. either tacrolimus/ methotrexate/ bortezomib or cyclosporine a/ methotrexate were used for gvhd prophylaxis. early extramedullary toxicity was limited. neutrophil and platelet engraftment was timely and complete in time in seven of nine cases. all patients achieved full donor chimerism around day fifteen after hct. severe acute graft-versus-host-disease (gvhd grade iii-iv) occurred in two patients and was lethal in both cases. two patients have experienced extramedullary relapse, one of them in the central nervous system and the other in the soft tissue. in two patients, a transplantation-associated thrombotic microangiopathy (ta-tma) was diagnosed. four patients are alive and in complete remission. we conclude that the combination of a ric regimen with a 188 rhenium anti-cd66 radioimmunotherapy is save and feasible. the incidence of gvhd, ta-tma and extramedullary relapse will be monitored closely and will be presented in a larger patient cohort. disclosure of conflict of interest: none. the sirolimus/tacrolimus (sir/tac) combination has been associated with a better outcome after allogeneic hematopoietic stem cell transplantation (allo-hsct) when compared with conventional prophylaxis for graft vs host disease (gvhd) as cyclosporine/methotrexate in the true reduced-intensity conditioning (ric) setting but not in the myeloablative setting. in moderate-intensity regimens as thiotepa/busulphan/fludarabine (tbf), the sir/tac combination has not been evaluated. from january 2009 to december 2015, all consecutive ric-allo-hsct recipients who received sir/tac combination to prevent gvhd in three spanish institutions were included in the study. the reduced-toxicity regimens used in this study where: (a) intravenous busulphan (6.4 mg/kg) and fludarabine 150 mg/ m 2 (bf), or (b) thiotepa days -4 and -3 and 10 mg/kg if 455 yrs old or 5 mg/kg if o55 yrs old) on days -6 and -5 added to the bf regimen (tbf). the gvhd prophylaxis with sir/tac was given as detailed elsewhere (cutler c blood 2014) and was consistent within the 3 center. the outcomes of the procedure according to the conditioning regimen were analyzed. overall, 125 patients were included: 66 tbf and 59 patients in the bf group, with a median follow-up of 22 months (range 3-83) and no difference in the median age (56 vs 58 years old). there were more males (71% vs 47%, p = 0.007) and more female donors to male recipients (35% vs 13%, p = 0.006) and more patients with lymphoid diseases and previous asct in the tbf group (27% vs 12%, p = 0.03), whereas there were more unrelated donors in the bf group (56% vs 76%, p = 0.02). other baseline characteristics were balanced between the 2 groups (table 1) . sir/tac prophylaxis had to be discontinued in 48% and 65% patients in the tbf and bf groups, respectively. toxicity was the main reason for discontinuation in the tbf group. the most frequent toxicities were renal injury (tbf 30% and bf 10%) and neurologic impairments (tbf 6%, bf 5%). in the bf group, the main reason of discontinuation was relapse or a mixed chimera. patients who received tbf presented higher incidence of extensive chronic gvhd (65% vs 39%, p = 0.02), higher nrm at 100 days (21% vs 4%) and at 2 years (42% vs 13%, p = 0.001). there were no differences in os (2 years) between both groups (49 ± 6.9% vs 80 ± 5.5%, p = 0.001) (figure) . there were no differences regarding to acute gvhd 2-4 (39% vs 36%, p = 0.31), acute gvhd 3-4 (23% vs 13%, p = 0.08), or relapse (up to 2 years, 22% vs 14%, p = 0.3) between the 2 groups, either. the combination of sir/tac as gvhd prophylaxis was associated with higher incidence of chronic gvhd and nrm in patients receiving conditioning regimen with tbf compared to those receiving bf. there were no differences in os between both groups. [p418] cr complete remission, pr partial remission, nr non remission, hct-ci hematopoyetic cell transplant comorbility index. disclosure of conflict of interest: none. reduced-intensity conditioning regimen with fractionated total body irradiation of 6 gy and cyclophosphamide 60 mg/kg for allogeneic hematopoietic stem cell transplant is well tolerated and offers a potential disease control as treatment of acute leukemia and lymphoproliferative disorders m adler, t girinsky 1 , s koscielny, g ferini, s wittnebel, s mayeur, c chahine, m vanghele, s pilorge, c castilla-llorente and j-h bourhis 1 the use of reduced-intensity conditioning regimens (ric) before allo-hsct is widely extended since it preserves the graft-versus-leukemia effect but reduces treatment related mortality. however, there exist different ric regimens with diverse outcomes and the choice of the ric regimen relies on the type of disease treated, experience of the center and previous therapies. this is a retrospective study of patients treated in our institution within 01/2000 and 12/2015. the ric regimen consisted of fractionated total body irradiation (ftbi) of 6 gy administered in 3 consecutive days (2 gy/day) and cyclophosphamide 60 mg/kg given in 2 days (30 mg/kg/day). post-transplant immunosuppression consisted of csa started the day before allo-hsct and short mtx on days 1, 3 and 6 after transplantation. for patients receiving transplant from unrelated donors, anti-thymocyte globulin at a dose of 5 mg/ kg (2.5 mg/kg/day for 2 days at day -2 and -1) was used as part of the immunosuppressant therapy. 78 patients (median age: 54 years: range: 36-64 years) were included. the median hct-ci was 0.5 (range: 0-4). primary disease was multiple myeloma (mm) in 45 (58%), al/mds in 14 (18%), cll in 10 (13%), nhl in 9 cases (12%) . 51 patients (65%) received transplant from matched related donors, 22 (28%) from matched unrelated donors and 5 (6%) from mismatched unrelated donors. female to male mismatch incidence was 23% (n = 18). most of the patients (n = 77) received a peripheral blood graft. 1 patient received a second allogeneic transplant. mm patients were transplanted in a "tandem" autologous-allogeneic hsct program in 42 cases. the median number of chemotherapy lines prior to transplant was 3.5 in cll, 2.8 in mm and 2.5 in nhl. 62 patients (91%) engrafted by day 28 post transplant. neutrophil engraftment occured at a median of 19 days (range: 14-35 days) and platelet engraftment at a median of 18.5 days (range: 9-103 days). full donor chimerism was observed in 62 out of 67 patients (92%) having survived by day 180. primary graft rejection was observed in 4 patients. treatment related toxicities consisted of grade 3/4 mucositis in 53 patients (68%), grade 3 (range: 2-4) cardiac toxicity in 6 patients (8%), grade 3 (range: 3-4) hemorrhagic complications in 4 patients (6%) including 3 cases of hemorrhagic cystitis and secondary malignancies in 4 patients, this within a median follow-up of 6.6 years. infectious complications during aplasia included fever of unknown origin (n = 52), bacteremia (n = 17) with 3 cases of bacteremia with severe sepsis and 8 cases of infections defined by bacterial foci. incidence of agvhd was 33% with 3 cases of grade 3/4 refractory agvhd. cgvhd occurred in 30 pts (39%). the non-relapse mortality (nrm) at 100 days was 5% including 2 cases of septic shock, 1 case of acute cardiac toxicity and 1 case of agvhd. the nrm at 1 year was 10%. 1-year survival rates were 60% in al, 80% in cll and 88% in nhl with extended survival benefit. in al patients, the relapse incidence was 36% comprising 2 patients who progressed during conditioning. the 1-year survival rate in mm patients was 77%. in mm patients who were in complete response prior to transplant, median overall survival was 4.6 years. the used ric regimen resulted in durable donor engraftment with an acceptable toxicity profile permitting efficient disease control in the described cohort. disclosure of conflict of interest: none. graft manipulation using selective depletion of αβ-t cells provides a source for haploidentical hematopoietic stem cell transplantation (haplo-hsct) enriched in effector cells. initial reports demonstrated safety and rapid immune reconstitution using this method, in malignant and non-malignant disorders using several proposed conditioning regimens. no specific considerations were given to hematologic malignancies. we reviewed a total of twenty seven pediatric patients who underwent haplo-hsct using αβ-t cell depletion between 2013-2016 in a single tertiary referral center. we report the results of 22 procedures performed in eighteen patients transplanted for malignancies. twenty two haplo-hsct were performed in eighteen patients. the indication for hsct was acute leukemia in sixteen (all = 11, aml = 5) and neuroblastoma in two. median age at hsct was 5.6 years. six patients had failed a prior hsct, and the remainder had no matched donor. the initial reduced-toxicity conditioning regimen consisted of melphalan, fludarabine, thiotepa and atg, and resulted in a high rate of graft rejections (7 of 9). thus, a totalbody irradiation (tbi)-based regimen was implemented, with prompt engraftment in all the patients. we observed rapid neutrophil and platelet engraftment kinetics (median time to engraft, 10 days and 11.5 days, respectively). significant treatment related complications were all due to graft failure in patients receiving reduced-toxicity conditioning, with two infection-related mortalities in the presence of prolonged neutropenia. none of the patients developed hepatic sinusoidal-obstruction syndrome, and no grade 3-4 acute graft-versus-host disease (gvhd) or chronic gvhd were observed with either regimen. importantly, the majority of patients with acute leukemia were free of immunosuppression in the first 100 days post hsct. the 2-year actuarial event-free and overall survival of the entire cohort were 34% and 55% respectively, with results for tbi-based conditioned patients being 58% and 88%. overall, we demonstrated that a tbibased conditioning for haplo-hsct using αβ-t cell depletion for malignant diseases resulted in acceptable outcomes in these high-risk patients without increased toxicity. disclosure of conflict of interest: none. high-dose chemotherapy conditioning regimens followed by autologous stem cell transplantation (auto-sct) generally provide good results in relapsed and refractory lymphomas. we evaluated the efficacy and safety of tecam regimen as conditioning with autologous stem cell support in patients with relapsed/refractory lymphomas. thirty-two (16 patients were refractory, 15 patients were relapse and one frontline treated) patients (21 m, 11 f) with lymphoma at various stages (stage ii, 19%; stage iii, 22%; stage iv, 59%) who underwent asct were included in this retrospective study. the median age at transplantation was 52.5 years (range, 28-69 years). the diagnosis were as follows: 9 diffuse large b-cell non-hodgkin lyphoma (nhl), 9 hodgkin lymphoma (hl), 5 mantle cell lymphoma, 3 follicular lymphoma, 3 marginal zone lymphoma and 3 t-cell nhl. all patients received tecam as conditioning regimen that consist of thiotepa (40 mg/m 2 × 4 days), etoposide (200 mg/m 2 × 4 days), cyclophosphamide (60 mg/ kg × 1 day), ara-c (200 mg/m 2 × 4 days) and melphalan (60 mg/m 2 × 2 days). median cd34(+) cells were 6.7 × 10 6 /kg (range; 1.9-19.3 × 106/kg) which were infused at day 0, followed by recombinant human granulocyte colonystimulating factor (rhug-csf) at a dose of 5 μg/kg/day. the median time between mobilization and auto-sct was 2 months (range; 1-13 months). the median time to recovery of absolute neutrophil and platelet counts independent of transfusion were 11 (range; 9-19) and 14 (range; 10-41) days, respectively. the median stay in hospital was 28 days (range, 11-108 days). bacterial, sitomegalovirus and invasive fungal infection were detected in 11 (34%), 4 (13%) and 2 (6%) patients, respectively in first 100 days of auto-sct. three s336 patients (9.3%) died from transplant-related complications. the overall response rate was 75% (22 cr, 68.8%; 2 pr, 6.2%) after auto-sct. relapse developed in 7 patients during median follow-up period of 6.5 months (range; 1-21 months) after auto-sct. the 1-year estimated dfs ( figure 1 ) and os were 70% and 45%, respectively. no statistical significance was observed for os and pfs in terms of gender, patient age ( o60 and ⩾ 60 years) and nhl and hl lymphoma group (p ⩾ 0.05). the tecam regimen for auto-sct in lymphoma seems to provide encouraging results in terms of response and its good tolerance with acceptable toxicity. [p421] disclosure of conflict of interest: none. allogeneic hematopoietic cell transplantation (allosct) is the only curative treatment for myelofibrosis. however, its widespread use is limited by early non-relapse mortality (nrm). the optimal modalities of the conditioning regimen are a major unmet clinical need. in an attempt to reduce early nrm, we used a tbf conditioning regimen (thiotepa, busulfan (bu), fludarabine (flu) and antithymocyte globulin (atg)). our aim was to reduce nrm and improve engraftment by using such tbf conditioning. thirty consecutive patients with a median age of 56 years (range, 32-69) who underwent allosct for primary (n = 18) or secondary (n = 12) myelofibrosis were included. according to the refined dynamic international prognostic scoring system (dipss-plus), patients were stratified as intermediate-1 (n = 3), intermediate-2 (n = 6), and high (n = 16) risk. five patients had blast transformation. ruxolitinib was given to 14 patients (47%) prior to allosct. graft source was pbscs in 26 patients (87%) and bm in 4 patients (13%). donors were matched related (mrd, n = 6), unrelated (n = 19) and haploidentical (n = 5). conditioning regimen was tbf in 18 patients (60%). in our historical cohort 8 patients (27%) received fb (flu, bu, atg). in addition, 4 patients received a 'tec-ric' sequential conditioning (thiotepa, etoposide, cyclophosphamide, and after 3 days rest, flu, bu and atg) for blast transformation (n = 2) or refractory proliferative myelofibrosis (n = 2). gvhd prophylaxis consisted of cyclosporine (csa) and mycophenolate mofetil in 26 patients (87%), csa and short course methotrexate in 3 patients (10%) with abo mismatch and csa alone in 1 patient (3%) with mrd. high dose posttransplant cy (pt-cy) was added in haplo cases. no significant difference was observed between tbf, fb and tec ric patients in terms of age, gender, karnofsky score, comorbidity index, number of previous treatment line, history of ruxolitinib administration and source of stem cells. median follow-up was 20 months (range, 3-75). two tbf patients died of septic shock before engraftment at day +12 and +19 after allosct, respectively. one fb patient died of graft failure at day +108 post allosct. median time to neutrophils and platelets (420 g/ l) recovery was 15 days (range, 9-28) and 20 days (range, 1-55) with tbf, 17 days (range, 14-53) and 18 days (range, 7-50) with fb, and 19 days (range, 15-24) and 14 days (range, 14-58) with tec ric. grade ii-iv acute gvhd occurred in 27.8% of tbf patients, 37.5% of fb patients, and 25% of tec ric patients (p = 0.90). moderate chronic gvhd developed in 1/13 evaluable tbf, 2/7 fb and 0/4 tec ric patients. no severe forms of chronic gvhd were observed. at last follow-up, 1 patient relapsed, 12 died and 18 are still alive. main causes of death were disease progression (n = 1), infection (n = 9) and gvhd (n = 2). nrm at 2 years was 33.3% in tbf patients, 50% in fb patients, and 25% in tec ric patients. the 2-year os were 66.7% in tbf patients, 37.5% in fb patients, 75% in tec ric patients, respectively. cd34+-selected stem cell boost without further conditioning allowed to 4 patients for poor graft function, with significant hematological improvement in 3 patients. tbf conditioning regimen seems to be efficient in allosct for patients with myelofibrosis and compares favorably with previously published fb regimens. these preliminary results give a rationale to support a prospective evaluation of this platform. disclosure of conflict of interest: none. we proposed here to compare the outcome of patients receiving either thymoglobuline (atg), a rabbit anti-human thymocyte immunoglobulin or campath, a recombinant dna-derived humanized monoclonal antibody directed against cd52. campath and atg are both commonly used as in vivo tcd before hsct, respectively in united kingdom and france but very few comparing data are available. all consecutive patients with acute myeloblastic leukemia (aml), myelodysplastic syndrome (mds) or myeloproliferative neoplasia (mpn) who received a reduced intensity hsct from an unrelated donor transplanted between 2006 and 2015 were included in this study. a propensity score was used to identify and control potential confounding to relate the treatment group to the outcomes. in the matched sample, cox regression model was used to describe the association between treatment and outcomes. 322 patients have been included. all patients received fludarabine and busulfan with either atg (n = 153) or campath (n = 169). patients treated by atg received cyclosporine plus mycophenolate mofetil or methotrexate and patients treated by campath received cyclosporine alone as gvhd prophylaxis. comparing patient and transplant characteristics, atg patients were older (62 vs 60 years), had less often aml (52 vs 69%), had higher disease risk (adverse dri: 14 vs 9%; poor cytogenetics: 25 vs 11%; high cibmtr score: 41 vs 28%), were less often in complete remission at time of transplant (62 vs 76%) and were transplanted less often from a mismatched hla donor (16 vs 26%). cumulative incidence of sustained engraftment was in 98% and 99% campath and atg patients. time to neutrophil engraftment was longer in atg patients (19 vs 13 days). acute gvhd ii to iv rate were higher after atg (44% vs 19%) as well as chronic extensive gvhd (26% vs 13%). relapse rate was higher after campath (44% vs 27%). disease-free survival (dfs) was higher after atg (53 vs 37%) and the gvhd-free relapse free survival (grfs) was similar (35% vs 32%). according to the prognostic factors for outcome, a propensity score was developed selecting 234 patients from the original cohort. the estimation of tcd effect was than studied. relapse risk was higher in patients treated by campath while there is a non-significant advantage for atg in dfs (table 1) . [p423] tcd with atg or campath gives similar os, dfs and grfs. severe acute or chronic gvhd is lowered by campath but the higher relapse risk counterbalances the potential benefit of campath finally given similar os. nevertheless, lower risk disease patient might benefit from campath while higher risk patients might benefit from atg. disclosure of conflict of interest: none. high-dose chemotherapy (hdt) with autologous stem cell transplantation is the standard of care for relapsed/refractory (rr) or high grade non-hodgkin-lymphoma (nhl) and hodgkin-lymphoma (hl)2. the standard hdt in autologous stem cell transplantation (asct) for lymphoma is carmustinebased hdt using a combination of carmustine, etoposide, cytarabine and melphalan (beam); this standard conditioning programme is used by most groups worldwide1. we have designed novels hdt regimens in which carmustine was substituited by an equal dose of fotemustine (feam)3 or thiotepa (team) and we compared these two hdt regimens in terms of engraftment times, toxicity, tolerability and frequency of relapse after asct. from february 2011 to september 2016 we consider a total of 67 relapsed/refractory patients affected by hl and nhl respectively 18 hl and 49 nhl with different grade of initial disease (grade i-iv) and different response to prior treatments. the all other drugs were administered according to a standard beam regimen1. after a day of rest, autologous peripheral blood progenitor cells were infused on day 0, followed by s.c. g-csf (5 mg/kg) from day 1 of asct until 2 consecutive days when the ancs were 4500 × 109/l3. the primary objectives of the study were to assess the feasibility and safety of the feam and team regimens in terms of acute toxicity, grade of mucositis, hemopoietic engraftment and relapse after asct. acute toxicity include chemotherapy-induced nausea and vomiting, diarrhea, hepatotoxicity, nephrotoxicity and infection complication. in all 67 patients cd34+ cells were collected from peripheral blood and the median number of infused cells per patient was 5.79 × 10e6/kg. the median time of engraftment was 9 days for neutrophil recovery (n4500 × 10 9 /l) and 11 days for plt recovery (420 000 × 10 9 /l). acute toxicity occurred in 14 total patients (20.8%), mucositis grade 3-4 occurred in 34 patients (50% of cases). frequency of relapse in all 67 cases was 43.2%. feam conditioning regimen was used in 41 cases showing a median time of neytrophil recovery of 10 days and a median time of plt recovery of 11 days. acute toxicity occurred in 4 of these cases (9.7%), mucositis grade 3-4 occurred in 18 patients (43.9% of cases). frequency of relapse in feam group of patients was 41.4%. team conditioning regimen was used in 26 cases showing a median time of neytrophil recovery of 9 days and a median time of plt recovery of 11 days. acute toxicity occurred in 10 of these cases (38.4%), mucositis grade 3-4 occurred in 16 patients (61.5% of cases). frequency of relapse in team group of patients was 50%. relapse/progression of lymphoma and conditioning regimen toxicities remain limitations to treatment success. the two novels hdt regimens feam and team are safe and feasible and show similar engraftment times, tolerability and frequency of relapse. maybe the team regimen shows toxicity slightly higher than feam regimen but longer follow-up is needed to evaluate fully its efficacy and long-term safety. disclosure of conflict of interest: none. treosulfan is a prodrug of a bifunctional alkylating cytotoxic agent. there are few reports regarding toxicological side effects of treosulfan-based conditioning prior to hsct. here we report on incidence of early potential treosulfan-related toxicity in 118 patients treated with treosulfan-based conditioning before hsct. treosulfan was given at a dose of 14 g/m 2 /d for 3 days in combination with fludarabin 30 mg/m 2 /d for 5 days prior to hsct. most patients (n = 93) had a haematological malignancy, while 25 patients had a non-malignant disorder as hsct indication. an hla-a, -b and -dr matched unrelated donor (mud) was used in 80 cases, 33 patients had a hla-identical sibling donor and 5 received an hla-a, -b or -dr allele mismatched unrelated donor. as graft versus host-disease (gvhd) prophylaxis, most patients (n = 93) received cyclosporine and methotrexate. patients medical records were scrutinized retrospectively to collect laboratory tests (aspartate aminotransferase (ast), alanine aminotransferase (alt), creatinine) before hsct and then weekly until 5 weeks after hsct. levels of ast and alt were significantly increased 1 week after hsct compared to before hsct. however, only a few patients had transaminase levels over 2 or 3 times the upper normal level (unl) levels decreased sharply after the first week. most of the cases with high levels of ast/alt at one week had normal or close to normal levels before hsct. creatinine levels increased after week 2 but no patient had levels ⩾ 2 × unl. clinical features of all oral mucositis (om) were recorded using the world health organization (who) scoring system. most patients (68%) had no or very limited (grade i) om, 22% had grade ii and 10% had grade iii or iv of om. according to our toxicological results this is low-toxic protocol. however, all patients became neutropenic, 61% already at the time of graft infusion, indicating that the protocol has a myelo-toxic effect comparable to conventional mac protocols. all patients engrafted, except three patients who died very early. median time to neutrophil and platelet engraftment was 18 (range 10-31) and 15 days (9-55), retrospectively, which is significantly later when compared to engraftment data for other ric protocols used at our centre (data not shown). median duration of neutropenia (o0.5 × 10 9 /l) was 17 days , comparable to what is expected after conventional mac conditioning. secondary graft failure (gf) occurred in 8 (6.8%) patients, all having a nonmalignant disorder and 6/8 having a urd. non-relapse mortality (nrm) was 7.5% (95% ci 3.7-13.3%) at 100 days and 11.9% (6.8-18.5%) at one year after hsct. causes of death within one year after hsct were: relapse 7, epstein-barr virus associated posttransplant lymphoproliferative disease (ptld) 4, other infections 4, organ failure 2, gvhd 2, hemophagocytic lymphohistiocytosis (hlh) 1. other infections occurring within 100 days after hsct were cytomegalovirus (cmv) reactivation 46 (39%), invasive fungal infection 6 (5.1%) and blood stream infection 47 (40%). veno-occlusive disease of the liver or sinusoidal obstruction syndrome (vod/sos) occurred in one patient and haemorrhagic cystitis in two patients. this study shows that early regimen-related toxicity after hsct was low despite similar marrow toxicities compared to mac regimens. disclosure of conflict of interest: none. allogeneic stem cell transplantation from haploidentical donors (haplosct) is an increasingly adopted option for patients (pts) with high-risk hematological malignancies. in our institution, we previously described a platform for unmanipulated peripheral blood stem cell (pbsc) haplosct using a calcineurin-free gvhd prophylaxis with sirolimus, micophenolate and anti-human t-lymphocyte immunoglobulin (atg) after conditioning with treosulfan and fludarabine (trramm; peccatori et al., leukemia 2015) . as an attempt to decrease relapse rate, especially in advanced-phase diseases, we designed a new phase ii prospective clinical trial intensifying conditioning regimen with the addition of 4gy total-body irradiation (tbi) (trramm4gy; eudract#2011-001534-42). we report results on 75 pts. 75 pts affected by aml (n = 49), other myeloid (n = 8) and lymphoid (n = 18) malignancies were prospectively enrolled from may 2010 to june 2015. median pts age was 45 y (range 17-67). revised disease risk index (r-dri) was low or intermediate in 31 pts, high in 34 pts and very-high in 10 pts. twenty-five pts had previously received an allogeneic stem-cell transplantation with a median time from 1st to 2nd sct of 17 months . median hct-comorbidity index by sorror et al. was 1 (0) (1) (2) (3) (4) (5) . pts received a myeloablative conditioning regimen consisting of treosulfan (14 g/m 2 /d from -6 to -4), fludarabine (30 mg/m 2 /d from − 6 to − 2) and tbi 4gy (fractionated in 2 doses, from − 1 to 0). source of stem cells were unmanipulated g-csf-mobilized pbsc from haploidentical donors. gvhd prophylaxis consisted of atg-fresenius (grafalon, neovii) 10 mg/kg/d from − 4 to − 2, rituximab 200 mg/m 2 on − 1, mycophenolate mofetil 10 mg/ kg from − 1 to +30 and sirolimus (target concentration 8-15 ng/ ml) from − 7. median infused cd34+ and cd3+ cell doses were 6.9 × 10⁶/kg and 2.15 × 10⁸/kg, respectively. median follow-up for survivors was 48 months (5-74). neutrophil engraftment occurred in 95% of pts with a median of 17 d (9-47), platelet engraftment was reached in 76% of pts with a median of 17 d (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) . the 100-d cumulative incidence (ci) of grade ⩾ 2 acute gvhd (agvhd) was 31 ± 10% and of grade ⩾ 3 agvhd 27 ± 10%; the 2 years ci of chronic gvhd was 34 ± 11%. the ci of transplant-related mortality (trm) at 1 y and 4 y were 31 ± 10% and 34 ± 11%, respectively. the ci of relapse at 1 y and 4 y were 27 ± 10% and 34 ± 11%, respectively, with a median time to relapse of 90 d . interestingly, we did not observe any extramedullary relapse; loss of mismatched hla-haplotype occurred in 33% of relapses. among 36 pts who were in active disease at time of haplosct and who were evaluable, 94% achieved complete remission (cr) and full donor chimerism at day+30. the 1y and 4y probabilities of disease-free survival (dfs) were 42 ± 11% and 33 ± 11%, respectively. at 4 y, 27% of pts are alive, disease-free and immunosuppression-free minimal residual disease, tolerance, chimerism and immune reconstitution the number of human leukocyte antigen (hla)-mismatched hematopoietic cell transplantation (hct), including cord blood transplantation, has been increasing. hla-flow method can discriminate mismatched hla antigens between donor and recipient by using flow cytometry, and can evaluate minimal residual disease (mrd) or chimerism after hla-mismatched hct. by developing more simple methodology, hla-flow might be more widely applicable. we have developed modified 6-colorbased hla-flow method. the aim of this study is to evaluate the utility of the 6-color-based hla-flow for monitoring of mrd and chimerism after hla-mismatched hct in children. from june 2013 to november 2016, serial monitoring of mrd or chimerism by the 6-color-based hla-flow was performed in twelve patients undergoing hla-mismatched hct (46 tests). nucleated cells obtained from bone marrow were stained by immunofluorescent antibodies against hla antigens mismatched between donors and recipients. these cells were also stained by immunofluorescent antibodies against surface antigens such as cd3, cd19, cd33, cd34 and cd16/cd56 for determining lineage of the cells. these surface antigens were also used as a marker of leukemic blasts in the mrd study. we used 6-color-based flow cytometry (facs-navious) and the data were analyzed with flow jo. erythroblasts and dead cells were excluded from the analysis. in each study, at least 105 cells were analyzed. for mrd analysis, we concurrently tested real-time quantitative polymerase chain reaction (pcr) of peripheral wt1 mrna or leukemia-specific fusion genes. pcr of polymorphic short tandem repeats or fluorescent in situ hybridization of x/y chromosomes was concurrently tested for chimerism study. age of patients ranged from 0 to 16 years. donor sources included bone marrow (n = 9) and cord blood (n = 3). for mrd monitoring of acute leukemia (n = 9), the 6-color-based hla-flow could detect mrd in three patients. five patients have not experienced relapse. no discordance with other mrd markers was observed in these patients. hla-flow could not separate donor-derived cells from recipient-derived ones in one patient receiving bone marrow transplantation. as for chimerism testing (n = 3), the 6-colorbased hla-flow could successfully evaluate quantitative lineagespecific chimerism in all patients. there is no discrepancy between hla-flow and other methods. we could complete evaluation of the 6-color-based hla-flow within two days in all tests. the 6-color-based hla-flow is a simple, quick and useful method for the quantitative evaluation of mrd and lineagespecific chimerism after hla-mismatched hct in children, irrespective of donor sources. it is thought that our method is applicable in all institutions owing 6-color-based flow cytometry. acknowledgement: we thank drs. nobukazu watanabe, eri watanabe and natsuko sato (university of tokyo) for their technical advices. we also thank drs. tomoko okunushi (chiba university), hidefumi hiramatsu and katsutsugu umeda (kyoto university) for their care of patients involved in this study. disclosure of conflict of interest: none. survival (pfs), cumulative incidence of relapse (cir) and acute/ chronic gvhd incidence in aml patients (pts) submitted to allo-hsct at our institution between january 2003 and december 2014. this retrospective study evaluated 122 aml pts submitted to allo-hsct from 56 matched sibling donors (msd) and 66 matched unrelated donors (mud) who provided bone marrow (bm) or peripheral blood as stem cell grafts.ir was evaluated at 100, 180 and 365 days post-transplant in all 122 pts. cmv-dna copies were determined in peripheral blood by quantitative pcr twice weekly in the first 100 days post-transplant and subsequently once weekly. cmvi/r was analyzed as a timedependent covariate.effect of cmvi/r on os and pfs was estimated by cox proportional hazard model. cir and gvhd incidence were analized with a competing risk approach, considering death from any cause as a competing event. effect of cmvi/r on cir and gvhd were evaluated by fine & gray model. median age at allo-hsct was 50.7 years (19.1-68.9 ). in our population 68% of donors were seropositive for a previous cmv infection and pts transplanted from these donors showed a significantly lower cumulative incidence of cmvi/r than pts transplanted with seronegative ones (shr = 0.56, 95%,ci:0. our study demonstrates that cmvi/r influences the success of allo-hsct by determining a better ir characterized by a higher cd8+ cell number that might exert an immune protective control on disease outcome by improving os,pfs and cir with no effect on gvhd. another factor of utmost importance to achieve this same goal might be constituted by the significantly increased nk cell number six months after allo-hsct. to assess the dynamics of molecular response to treatment in aml adult patients with concomitant flt3 and npm1 mutations. this retrospective single center studystudy was approved by the institutional review board of american university of beirut medical center. twelve consecutive newly diagnosed (n = 11) or relapsed (n = 1) aml patients received idarubicin/cytarabine induction and one or two consolidation (s) ( table 1) . seven patients received allogeneic stem cell transplant (allo-sct) and 3 had haploidentical-sct (hap-lo_sct); all followed by post-transplant sorafenib maintenance. median follow-up was 11.5 (6-27) months. all transplanted patients remain alive and disease free.flt3 mutation was tested on dna using a qualitative method with a sensitivity of 0.01%. npm-1 mutation was tested on cdna using a qualitative or a quantitative rt-pcr with a sensitivity of 0.01% and 0.008 ncn respectively. patients were tested at diagnosis, after induction, after each consolidation, before and s341 at days 30, 60 and 100 after allo-sct for kinetics of npm1 and flt3 molecular response. after induction, flt3 became negative in all tested patients (n = 10). after first consolidation, flt-3 was not tested in 3 patients who had a negative result after induction, was negative in 8 patients including the 2 patients who were not tested after induction, whereas a molecular relapse was noted in one patient who developed a hematological relapse and rapidly died. another patient died after the third consolidation, in complete remission, due to septic shock. no molecular positivity for flt-3 was noted later on, whether after second consolidation or post-transplant. conversely, npm-1 mutation became negative in 2 out of 12 tested patients after induction, in 1 additional patient after first consolidation and in 7 additional patients after sct, mostly after starting sorafenib. npm-1 mrd value remained elevated in 3 out of 4 patients with quantitative assessment at diagnosis and post induction (figure1). flt3 become negative early after induction while npm1 negativity lags behind. persistent npm-1 mrd does not seem to predict post-transplant outcome and may indeed become negative after sorafenib. these results need confirmation in larger studies. disclosure of conflict of interest: none. in allogeneic stem cell transplantation (allo-sct), an early detection of the transplant outcomes such as overall survival (os), event-free survival (efs), cumulative incidence of relapse (cir) and non-relapse mortality (nrm) is fundamental regarding the use in time of additional therapy after sct. therefore, we investigated the association between early immune reconstitution (ir) on day +30 after allo-sct and outcomes in children suffering from acute leukemia or myelodysplastic syndrome (mds). this study collected data from 188 allo-sct from january 2005 until december 2014 in our institution. the median survival follow-up was 38 months. indications of allo-sct were all (n = 113, 60%), aml (n = 44, 23%) and mds (n = 31, 17%). the median age was 10 years (range, 0.6 -18). patients were transplanted in cr (n = 131, 70%) and pr/nr (n = 57, 30%). patients included in the study received 1st sct (n = 170, 90%), 2nd sct (n = 15, 8%) or 42 sct (n = 3, 2%). grafts were from sibling (msd; n = 42, 22%), matched unrelated (mud; n = 95, 51%), haploidentical (n = 45, 24%) or mismatched unrelated (mmud; n = 6, 3%). conditioning regimens were tbi-based (n = 87, 46%) or chemo-based (n = 101, 54%). stem cells were from bone marrow (n = 118, 63%) or peripheral blood (n = 70, 37%). we analyzed the absolute count of lymphocytes (alc), monocytes, cd3+ t cells, cd3 +cd4+ t-helper cells, cd3+cd8+ cytotoxic t-cells, cd3-cd56+ natural killer (nk) cells and cd19+ b cells assessed on day 30 ± 5 after sct. we used the percentiles of the lymphocyte subsets of the same cohort to categorize the samples throughout the study. patients with alc over the 50th percentile of alc (alc o 327 cells/μl) had a 1.97-fold increased hazard ratio (hr) to develop relapse (p = 0.017). nk cell counts on day 30 after sct were strong associated with os, efs, cir and nrm. patients with nk cell count over the 75th percentile (nk 4 268.8 cells/μl) had increased hr for os (hr = 1.8, p = 0.01) and efs (hr = 2.01, p = 0.017) compared to patients with nk count under the 75th percentile. patients with nk cells over the 25th percentile (nk o52.5 cells/μl) had a hr = 3.3 (p = 0.009) for relapse and hr = 0.364 (p = 0.016) for nrm compared to patients with nk cell count under the 25th percentile. monocyte cell count on day 30 correlated with os, efs and cir. patients with cd14+ cells count under the 15th percentile of cd14+ (cd14+ o242 cells/μl) has an increased hazard ratio for os, efs and relapse compared to patients with cd14+ cell counts over the 15th percentile. no association between absolute cell count of cd3+, cd4+, cd8+ and cd19+ on day +30 after allo-sct and any outcomes either os, efs, cir or nrm was found. the study confirms the strong association between early ir and outcomes after allo-sct in children. our study suggests that especially nk cell and monocyte cell count on day +30 may have significant prognostic implications. our findings suggest that the cells count of alc, nk cells and monocytes on day +30 after allo-sct could be useful to predict outcomes after allo-sct and should be taken into account in considering alternative treatment. disclosure of conflict of interest: none. early immune reconstitution (eir) has proven to be a significant determinant for the outcome of allogeneic hematopoietic stem cell transplantation. in the setting of unmanipulated haploidentical transplantation (haplo-hsct), some groups have identified the absolute leukocyte count on day +30 (alc30) as an independent prognostic factor in terms of overall survival (os), disease free survival (dfs) and infectious mortality (im). the aim of this study was to evaluate the impact of eir on os, dfs and im among patients who underwent haplo-hsct with postransplant cyclophosphamide (ptcy) at our institution. from july 2011 to april 2016, 83 haplo-sct were performed at our institution. threedied before day 30 after haplo-sct, and 13 patients had missing data. conditioning regimen consisted of fludarabine, cyclophosphamide and busulfan. twenty-nine patients received a reduced intensity conditioning regimen (1-2 days of busulfan) while 37 a myeloablative regimen (3-4 days of busulfan). gvhd prophylaxis comprised ptcy, cyclosporine and mycophenolate mofetil. patients were assessed for eir by means of alc30, cd3+ t lymphocyte count on +30 (cd3), nk lymphocyte count on +30 (nk) and nk cd56 bright percentage on +30(cd56 br ). we analyzed 66 pts, with a median follow-up of 21 months (9-36). the median age of the pts was 43 (range 30-57), 76%men. diagnosis were: aml(32%), hl (23%)non-hl (17%), all (8%),mds(8%), cml(6%), others(6%). 55% were in complete remission at the time of transplant, 21% in partial remission and 24% had overt disease. in terms of infectious complications, cmv reactivation was documented in 76% of the pts, 1% developed a proven invasive fungal infection and 26% suffered from bk+hemorrhagic cystitis. median os and dfs were 21 (9-36) and 17 months (7-31), respectively. im rate was 21% at the end of follow up. median follow-up was 21 months (9-36). roc curves were used to determine the optimal cut-off values for each of the studied parameters: 300 cells/μl for alc30, 120 cells/μl for cd3, 41 cells/μl for nk and 83% for cd56 br were chosen. pts with alc30 ⩾ 300/μl had better os (p = 0.005) and dfs (p = 0.05), than those with alc30 o 300/μl. median os and dfs were 25 months vs not reached (nr) and 22 months vs nr, respectively. pts with cd56 br ⩾ 83% had better os (p = 0.04) than those with lower values. median os was 25 months vs nr; however no difference was seen in terms of dfs. we didn´t observe statistically significant differences in os or dfs, among pts with different levels of cd3 and nk on +30. cumulative incidence of im was significantly lower in pts with an alc30 ⩾ 300 (p = 0.04), pts with cd3 ⩾ 120/μl (p = 0.006) and pts with nk ⩾ 41 (p = 0.04); patients with cd56 br ⩾ 83% showed tendency to have lower cumulative incidence of infectious mortality (p = 0.24, non-significant). cumulative incidence of relapse was not affected by alc30, cd3, nk or cd56 br . our study supports the independent prognostic value of early immune reconstitution after unmanipulated haploidentical transplantation with ptcy, especially in terms of lower infectious mortality. os and dfs were better among patients with alc30 ⩾ 300 cells/μl. pts with cd56 br ⩾ 83% also showed better os. no correlation was found between cd3 or nk on +30 with os or dfs. cumulative incidence of infectious mortality was affected by alc30, cd3 and nk on +30; while cd56 br seems to have less impact. [p431] disclosure of conflict of interest: none. an early absolute lymphocyte count (alc) recovery after autologous stem cell transplantation (asct) for hematologic malignancies has been related with an improved transplant outcome due to a faster autologous immune restoration. in this retrospective study we analyze post-transplant survival of non hodgkin lymphoma (nhl) patients and its relation with alc at day +15 post-asct. we analyzed 53 consecutive adult nhl patients who underwent asct at the hematology and sct department of hospital maciel (montevideo, uruguay). only individuals with at least 6 months post-transplant follow up were included. all patients received beam (bcnu, etoposide, cytarabine and melphalan) conditioning regimen followed by peripheral blood stem cells previously collected by apheresis. median cd34+ cell dose was 4.13 × 10e6/kg (1.62-12.58). median alc at day +15 was 500/μl. patients were divided into two groups: alc at day +15 inferior than 500/ul (group 1) and alc at day +15 superior or equal than 500/ul (group 2). differences between groups were analyzed using t-student and chi-square tests, with statistical significance determined at p o0.05. disease free survival (dfs) and overall survival (os) were analyzed by kaplan meier method. differences in survival between groups were determined by log-rank test. no differences were observed between groups regarding gender, histology, disease status at transplant and cell dose. patients in group 1 were older and more heavily pretreated. neutrophils and platelets engraftment were significantly faster in group 2 (table 1) . after a median follow up of 36 months, disease-free survival (dfs) and overall survival (os) were superior in group 2. median dfs was 47 months and not reached (p = 0.019) and os was 51 months and not reached (p = 0.016) in groups 1 and 2 respectively (figure 1 ). an early alc recovery after asct was associated with a superior dfs and os in nhl patients. individuals with alc major or equal than 500/ul had also a shorter time to neutrophils and platelets recovery and a shorter hospital stay. in this study, cd34+ cell dose does not seems to be a determinant factor for lymphocyte recovery. the load of previous treatment may influence lymphocyte recovery after asct. these results support the association between early post-asct lymphocyte recovery and improved survival in nlh patients. [p432] disclosure of conflict of interest: none. t cell depletion (tcd) reduces the risk of graft versus host disease (gvhd) but also the graft versus leukaemia (gvl) effect, thus increasing the risk of relapse. donor lymphocyte infusions (dli) can be given to boost donor chimerism, with the intention of enhancing the gvl effect. 1 it is not currently known whether giving dli based on bone marrow chimerism (bmc) influences survival, or whether certain groups of patients benefit more from dli than other groups. in addition, it is not known whether the overall aim of achieving 100% bmc associates with improved survival. we investigated whether day 100 (d100) bmc was predictive of survival, and whether giving dli based on this result was associated with improved overall survival. data were retrospectively collected from case notes and laboratory reports for patients who underwent allogeneic stem cell transplant (allosct) for aml or mds at the northern centre for bone marrow transplantation between 2010 and 2015. patients who died prior to d100 were excluded from the analysis. of the 147 patients analysed (aml 117, mds 30), 68% were male and 38% female. the median age was 59 years (range 22-74). conditioning was with flu/bu/ alemtuzumab (63), flu/mel/alemtuzumab (45), cy/tbi alemtuzumab (7), flamsa tbi/bu atg/alemtuzumab (27), other (5) . 103 (70%) received a graft from an unrelated donor, 42 (29%) a matched sibling donor and 2 (1%) another source. 143 (97%) received mobilised pbscs, 3 (2%) bone marrow and 1 (1%) cord blood. statistics were performed using graphpad prism. p values were calculated using the chi square test and taking po0.05 to determine significance. bmc was divided into 3 groups 100%, 90-99% and o 90%. 100% bmc at d100 was associated with a significant increase in 2 year overall survival (os) (74.6% vs 57.3% and 33.4% for 90-99% and o90%, respectively, p o0.0012). patients with a d100 bmc o80% had a 2 year os of o10% (with relapse the cause of death in 90%). in patients whose d100 bmc was o 90%, there was a significant improvement in 2 year os seen in those who received dli (61.7% survival at 2 years vs 0% with no dli, po0.0026) (figure 1 : os by d100 bm chimerism (with and without dli). attainment of 100% bmc at a subsequent time point also significantly improved survival in those with a d100 bmc of 90-99% (79.4% 2 year survival vs 33.6% who never attained 100%, p o0.001) and o90% (100% 2 year survival vs 14.7%, p o0.006). we found d100 bmc to be predictive of os in this population. in addition, dli was associated with an improvement in os, especially in patients whose bmc at d100 was o90%. there was also a statistically significant improvement in os seen in patients who subsequently attained a 100% bmc, where it was o100% at d100. the objectives of this analysis were to examine the optimal alc recovery cutoff utilizing receiver operator characteristics (roc) analysis and to examine infused allograft characteristics associated with early alc recovery. after due irb approval, patients (pts) with aml and all who underwent hct at our institution between 2010-2015 were identified. pts with t-cell depletion or maintenance post hct were excluded. data were collected retrospectively from the patient's records. cellular contents of infused products (cd34, cd3, tnc, mnc, alc and amc) in addition to alc post hct were analyzed and optimal cutoff, if present, was established using roc analysis for the end point of relapse. time to end point analysis was computed using the kaplan-meier with log ranks. for competing events, cumulative incidence was computed using grey's model. univariable and multivariable analyses were performed using cox proportional hazard regression. a total of 72 pts met the inclusion criteria and were analyzed. optimal alc cutoff by roc analysis was established to be on day +14 (d14) with alc 40.3 × 10 9 /l and was subsequently defined as early lymphocyte recovery (erl). pts with alc ⩽ 0.3 × 10 9 /l were deemed to have delayed lymphocyte recovery (dlr). patients were subsequently stratified accordingly and patient, disease and transplant related factors were well balanced between the groups. median follow up of the entire cohort was 17 (2-64.8) months. graft characteristics: roc analysis established optimal cellular cutoff, if present to predict elr. pts in the elr group were more likely to receive cd 34 × 10 6 /kg o6 (0.018), cd3 4 24 × 10 7 /kg (0.017) and alc 41.3 × 10 8 /kg (p = 0.015). we did not find a significant threshold for other allograft variables i.e. (tnc, mnc or amc). post hct outcomes: at 2 years, corresponding cumulative incidence of relapse (cir) and non-relapse mortality (nrm) was16.9% vs 46.9% (p = 0.025) and 23.2% vs 14.2% (p = 0.51), for elr and dlr cohorts, respectively. there was a trend towards improved progression free survival (pfs) and overall survival (os) in favor of elr vs dlr at 61.9% vs 40.1% (p = 0.09) and 70.1% vs 53.9% (p = 0.12), respectively. median time to neutrophil and platelet engraftment was 17 and 24 days, respectively for both groups. incidence of acute graft vs host disease (agvhd) was similar (p = 0.4); however, chronic gvhd (cgvhd) was more prevalent in the elr group at 70% vs 27%, respectively (p = 0.0006). on s344 multivariable analysis for relapse, elr retained its prognostic significance with hr 0.27 (0.05-0.94; p = 0.038). cgvhd and first complete remission (cr1) at the time of hct were also protective factors from relapse in multivariable analysis. we observed that elr is an independent predictor for relapse in patients receiving allogeneic hct for acute leukemia with a trend towards improved os. this is possibly related to higher incidence of cgvhd. elr was influenced by infused allograft characteristics particularly cd34 count. given the sample size and retrospective nature of the analysis, these important observations should be examined prospectively. disclosure of conflict of interest: none. allosct is the only curative option for the treatment of hematological disorders with depression of hematopoiesis and primary immunodeficiencies.non-myeloablative conditioning (mac) regimens lead to long persistence of mixed chimaerism (mc) in the majority of patients. purpose: to estimate the relationship between type of hematopoietic chimaerism and appearance of gvhd in patients with non-malignant diseases after allosct eleven patients (8 boys and 3 girls) with median age of 9 years (range 3-17) were included in the current study. among them there were 7 patients with severe aplastic anemia (saa), 2 with fancony anemia (fa), 1 with thalassemia, 1 with nijmengen syndrome, treated in our center from 2008 to 2016. donors' sources were as follows: siblings in 7 cases, mud (10/10) in 4 ones. in 5 cases bone marrow aspirate were used, in 6 mobilized peripheral blood hematopoietic stem cells. conditioning regimens included fludarabin, cyclophosphamide and horse atg for saa patients, in fa and nijmengen syndrome patients this scheme was augmented by low-dose busulfan. in thalassemia patient we used mac with busulfan, fludarabin and horse atg. in majority of case gvhd prophylaxis consisted of tacrolimus and methotrexate combination. when allosct was performed form mud patients were additionally administered with mycophenolate mofetil. median of follow-up period was 32 mo (range 7-93). quantitative evaluation of chimerism was done by multiplex amplification of str loci with subsequent fragment analysis using «cordis plus» kit («gordiz llc», russia). we analyzed whole bone marrow and peripheral blood together with cd3+ and cd19+ lymphocyte subpopulations. presence of ⩾ 99% donors' hematopoietic cells was considered as complete donor chimerism (cc), less than 99% was considered as mc. all patients engrafted in time and all of them are alive at the time of current analysis. there were no severe life-threatening complications, infections or graft rejections. only 2 patients achieved cc at day +28. at day +100 only these 2 patients stayed in cc. at this time point mc was mainly revealed in cd3 + lymphocytes. in 1 year after hsct proportion of cc patients enlarged to 82% (2 patients did not achieved this time point). there is no any correlation between time of engraftment and chimerism value at day +28, either between the dose of transplanted cd34+ cells and chimerism level ((p40.05 in both cases). severe gvhd was noted only in 2 female patients with cc at day +28. in the first case it was acute gvhd grade iii after hsct from mud, in the second case extensive form of chronic gvhd in 1 year after hsct from sibling was observed. there are no other cases of grade iii-iv acute gvhd in the observed cohort of patients. localized form of chronic gvhd [p434] was revealed in 4 (36%) patients. in other patients there were no signs of chronic gvhd. despite limited number of observations we assumed that fast achievement of cc corresponds to severe gvhd. and vice versa, long persistence of mc prevented emergence of gvhd. however our findings need to be confirmed in a larger group of patients and preferably in a multicenter setting. disclosure of conflict of interest: none. interest of quantitative assessment of hematopoietic chimerism by real-time quantitative polymerase chain reaction after hematopoietic cell transplantation for hematological malignancies: a retrospective analysis on 347 adult patients from rennes university hospital g laure 1 , c mathilde 2 , b marc 1 , n stanilsas 1 , d charles 1 , l christine 2 , a mehdi 2 , lb laetitia, s gilbert 3 , l thierry 1 and r virginie 2 1 department of hematology, chu pontchaillou, rennes; 2 immunogenetics and histocompatibility laboratory, etablissement français du sang, rennes and 3 etablissement français du sang-bretagne chimerism (percentage of recipient versus donor-derived blood cells) is used to document engraftment after hematopoietic stem cells transplantation (hsct). detection of persistant host cells, 1-2 as well as an increase in recipient cells chimerism has been associated with impaired dfs and os. 3 quantitative real time pcr (qrt-pcr) 4 is a highly sensitive, reproducible method, which can detect very low levels of recipient cells. the aim of this study was to evaluate the prognostic impact of early chimerism 30 days (d30) and 100 days (d100)) after hsct and the meaning of detection of an increase of chimerism, even at low levels, during follow-up. 347 adult patients who underwent hsct in rennes between 2002 and 2013 were included in this retrospective study. all chimerism analyses were done with qrt-pcr using whole blood sample. complete chimerism (cc) was defined by less than 0.1% recipient cells detected. with a median follow-up was 653 days, 101 patients relapsed with a median time of 116.5 days after hsct. both d30 and d100 mixed chimerism (mc) (40.1 % recipient cells detected) were associated with an increased relapse risk (p = 0.0003 and p o0.00001 respectively) compared to patients with cc in univariate analysis. however, when looking at subgroups analysis, d30 and d100 mc vs cc was significantly associated with increased relapse risk in this cohort for myeloid diseases (p = 0.0049 and p o0.0001) but not for lymphoid diseases (p = 0.506 and p = 0.059). no difference in os was observed (p = 0.32 and p = 0.34). more important, detection of an increased of mc (imc) was associated with an increased relapse risk in univariate and multivariate analysis (or = 9.69 [5.42; 17 .34], or = 10.05 [5.35; 18 .90]), (po0.0001), as well as impaired os (p = 0.0043) and dfs (p o0.0001). among the 103 patients with aml and at least 2 chimerism analysis available, only 3 relapsed without imc detected but the patients' last available chimerism analysis was 75, 84 and 138 days before relapse respectively. median levels of recipient cells detected was 1.2 %. altogether, these results indicate that serial analyses of chimerism with qrt-pcr are a useful tool for post-transplant monitoring and might help identify patients at highest risk to relapse after transplant, especially in myeloid disease. 3 monitoring frequency is critical in order to obtain the highest clinical impact, and the timing of monitoring as well as the safety and type of pre-emptive interventions still need to be explored. considering the kinetics of the disease, frequent analysis in myeloid pathology might improve the detection of impending relapse. although an approximately 1-log higher sensitivity of quantitative pcr (qpcr) compared to short tandem repeat (str) has been documented in different studies, the latter remains the standard procedure for hc assessment to date. we hypothesized that qpcr could be superior to str for monitoring the molecular kinetics of donor cell engraftment, response to donor lymphocyte infusions (dli) and development of relapse post-hct. we analyzed 30 patients (pts) who underwent mainly 10/10 hla-matched unrelated hct mostly for acute myeloid or lymphatic leukemia at the university hospital essen between 2006 and 2013. transplant conditioning was mostly myeloablative and gvhd prophylaxis was by cyclosporin a and methotrexate without anti-thymocyte globulin (atg). median follow-up of pts was 1504 days (d) (317-2891). cytomegalovirus (cmv) reactivation in the first 100d posttransplant was measured by pp65 (n = 22) or pcr (n = 8). a total of 459 retrospective genomic dna samples from peripheral blood (pb; n = 364) or bone marrow (n = 95) collected between d21 and d2302 post-transplant were available for hc analysis in parallel by str (mentype chimera, biotype) and qpcr (alleleseq, abbott). threshold for hc positivity in qpcr was set at 0.1% following published protocols. concordance in hc analysis between qpcr and str was found in 365/459 (79.5%) samples, with all 94 discordant cases positive in qpcr but negative in str. engraftment could be assessed in 110 samples drawn at d21-d213 from 18 pts without relapse in the first 6 months post-hct. these samples showed concordant negative or positive qpcr and str results reflective of full donor engraftment or persistent mixed chimerism (pmc) in 5 and 3 pts, respectively. in 2 pts, qpcr but not str documented delayed conversion to full donor chimerism until d200. in the remaining 8 pts, positive results in qpcr but not str during early engraftment were observed exclusively in bm, in particular those drawn before d35 post-hct. qpcr but not str was also able to document the kinetics of conversion to full donor chimerism which took 126d and 196d in 2 pts receiving dli for treatment of pmc and relapse, respectively. 8 informative relapses could be assessed in 33 samples drawn at least 192d before onset. 6/6 bm and 23/26 pb were positive in qpcr, compared to 2/6 bm and 2/23 pb in str, with a sensitivity of 8/8 (100%) and 3/8 (37.5%) relapses, respectively. consistent with previous reports on a protective effect of early cmv reactivation on relapse in gvhd prophylaxis regimens without atg, relapse occurred in 1/10 (10%) pts who experienced cmv reactivation in the first 100d post-transplant, compared to 10/20 (50%) pts who did not. no apparent associations were observed between early cmv reactivation and engraftment kinetics post-hct. hc assessment by qpcr is highly concordant with str, but markedly superior for molecular monitoring of engraftment kinetics and relapse. positive qpcr results in bm should be interpreted with caution during early engraftment, while both bm and pb were highly informative for relapse in our series. these results advocate the feasibility and clinical utility of qpcr for post-hct hc monitoring in routine use. disclosure of conflict of interest: the commercial assays for qpcr chimerism analysis were provided by abbott molecular free of charge. tyrosine kinase inhibitor (tki) has become the standard of care in patients (pts) with chronic myeloid leukemia (cml) and an unavoidable tool in the combined therapy for pts with philadelphia chromosome positive (ph+) acute lymphoblastic leukemia (all). nevertheless, allogeneic stem cell transplantation (hsct) remains the standard therapy of all ph+ and of cml pts failing 1st line therapy with tki, with failure or insufficient response or intolerance or mutations resistant to 2nd generation tki, or in the advanced phase at diagnosis. in the past decade the feasibility and safety of post-hsct imatinib administration as prophylactic or therapeutic strategy was confirmed. second and 3rd generation tki administration after hsct is under investigation. here we are reporting our experience in post-hsct treatment with the 3rd generation tki ponatinib in 5 pts treated between 2011 and 2016 at our institution. pts data and information were collected from institutional database and chapters revision. a written consent was given by pts allowing the use of medical records for research in accordance with the declaration of helsinki. pts and diseases features are reported in table 1 . pre-transplant treatment for the all ph+ patient consisted of chemotherapy combined with dasatinib, followed by a 1st mud hsct and dasatinib in maintenance. the patient relapsed 1 year after hsct with documentation of mutation v299l. ponatinib was introduced as salvage treatment to bridge 2nd haplo hsct. pre-transplant treatment for the cml patients consisted of tki therapy with combination of chemotherapy in case of uncontrolled progression of disease. two pts received a 1st mud hsct but relapsed respectively 5 months and 4 years later. ponatinib was introduced as salvage treatment to bridge 2nd haplo hsct. four pts received ponatinib 45 mg daily before the last hsct: one patient achieved sustained major molecular response, 4 pts obtained transient response. all pts were presenting 2nd generation tki resistant mutations (table 1) . ponatinib was started at a median of 157 days after hsct (range, 117-583) as salvage treatment in overt relapse (3 cases), prophylaxis (1 case) or preemptive therapy (1 case). acute gvhd was diagnosed in 4 pts before ponatinib administration, 2 of them also experienced chronic gvhd. no new cases of gvhd were observed after initiation of ponatinib. immunosuppressive treatment and azoles treatment were discontinued before ponatinib in all but one patient who was under combined treatment for chronic gvhd: therapeutic drug monitoring was closely performed without evidence of drug-drug interaction. pts were regularly evaluated for toxicities and monitored for cardiovascular events. no serious adverse events were reported in our experience: we administered ponatinib at a median maximum dosage of 30 mg daily (range, 15-45 mg), for a median of 24 weeks (range, 4-132 weeks). at last evaluation one patient maintained the status of molecularly undetectable leukemia (follow-up post hsct: 34 months) and one major molecular response (follow-up post hsct 29 months). three patients who received therapeutic ponatinib in overt relapse did not respond and died for progressive disease. ponatinib is safe and well tolerated as bridge to hsct and to maintain disease control after transplant. prophylaxis targeted therapy and preemptive therapy with ponatinib may lead to the reduction of disease relapse for high-risk ph+ leukemia. disclosure of conflict of interest: none. at nuh singapore we have adopted the cd45 ra depletion to ameliorate graft versus host disease. materials and methods: we have transplanted 14 leukemia patients with cd3 depleted hsct followed by cd45ra depleted donor lymphocyte infusion. no additional gvhd prophylaxis or gcsf was used. results: 100% patients achieved primary engraftment. median time for neutrophil engraftment (4500/μl without gcsf) was 14 days (range 7-17 days), platelet engraftment (450 000) was 13 days (range 10 to 29 days). immune reconstitution was rapid with median cd4 and cd8 cell counts 4200/μl at day 30. by day 200 median cd4 count was 390/μl (range 312-817/μl). no patient developed grade iv acute gvhd. there was a significantly reduced incidence of invasive viral infections as compared to conventional transplants. importantly, all patients achieved complete remission (cr) on day +21 and remained in cr for longer time as compared to conventional transplants. conclusion: our preliminary data suggests that rapid immune-reconstitution of nk cells and t cells with this strategy correlates with reduced infection related mortality without loss of graft versus leukemia effects. disclosure of conflict of interest: none. the use of post-transplantation high-dose cyclophosphamide (pt-cy) has overcome the need for extensive depletion of t lymphocytes from haploidentical donor grafts, which traditionally resulted in severe and prolonged immunosuppression. it is therefore relevant to investigate the degree and the tempo of immune reconstitution after t cell replete haploidentical stem cell transplantation (haplo-sct) by use of pt-cy. we prospectively monitored cellular immunity in 15 consecutive adult patients (male/female: 9/6), who underwent haplo-sct with pt-cy for myeloid (n = 12) or lymphoid (n = 3) malignancies. the median age at transplant was 56 (range, 27-68) years. the conditioning regimen was myeloablative in 10, reduced-intensity in 4, and non-myeloablative in 1 case. the source of the graft was peripheral blood (n = 7) or bone marrow (n = 8). in addition to pt-cy, graft-versus-host disease (gvhd) prophylaxis included tacrolimus and mycophenolate mofetil. absolute counts of cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ cells were measured by flow cytometry at 1, 3, 6, 9, 12, 18 and 24 months post transplant. the median doses of infused cd34+ and cd3+ cells were 4.13 (range, 1.16-9.61) × 10 6 /kg and 4.45 (range, 2.2-38) × 10 7 /kg, respectively. neutrophil engraftment (4500/ul) was achieved at a median of 18 (range, 16-35) days, whereas platelet engraftment (420 000/ul) was observed at a median of 23 (range, 13-54) days. seven patients developed acute gvhd (grade i/ii: 6, grade iii: 1). chronic gvhd occurred in 3 patients, and was extensive in the 2 of them. cytomegalovirus infection was detected in 9/15 cases at a median interval of 42 (range, 30-89) days post transplant. two patients were administered rituximab for epstein-barr virus reactivation at 10 months, whereas one patient developed bk virus-associated hemorrhagic cystitis at 2.5 months following haplo-sct. there was 1 death due to gvhd and infection at 7 months post transplant. at a median follow-up of 12 (range, 3-33) months, 14/15 patients remain alive and disease-free. the absolute counts of t and b cells were extremely low early post transplant, while nk cells recovered from the first month (mean count, 254/μl). the number of cd8+ t cells started to increase beyond the first month, and exceeded lower normal limit at 3 months (mean count, 296/μl). cd4+ t cells remained in general low ( o100/μl) for the first 3 months, increased moderately by 6 months (mean count, 186/μl), and approached lower normal values at 9 months (mean count, 325/μl) [ figure 1 ]. of note, cd4+cd45ra+ naïve t cells remained significantly impaired (absolute count range, 4-52/μl) in all 7 patients in which they were assessed beyond the 1st year from transplant. cd19+ b cells were suppressed for the entire first trimester (mean count at 3 months, 68/μl), but increased rapidly between 6 and 9 months (mean counts, 165/μl and 366/μl, respectively). in haplo-sct with pt-cy, reconstitution of cellular immunity can be achieved at adequate levels by 6-9 months following transplant. the observed deficit in the recovery of naïve t-helper cells may be related to a possible effect of pt-cy on thymopoiesis and warrants further investigation. [p440] disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) is a curative therapy for patients with sickle cell disease (scd). hemoglobin a in scd ameliorates the manifestations of the disease and this could be achieved with stable mixed chimerism after a reduced intensity hsct. this study aims to estimate the proportion of patients who develop mixed chimerism after hsct for scd and to characterize its progression in patients who develop it. this is a retrospective cohort study conducted at sultan qaboos university hospital (squh) bone marrow transplant unit in oman. we included all patients with scd who received hsct over the course of 10 years between may 2007 to may 2016. patients who received second hsct were excluded. short tandem repeat polymerase chain reaction was used for chimerism assessment. mixed chimerism was defined as 5 − 95% chimerism at 6 months from hsct. the data was analyzed by r program 3.1.2. χ 2 or student t-test were used to assess the impact of acute graft versus host disease (agvhd) prophylaxis, age at transplantation, gender, red blood cell antigen alloimmunization, preparative regimen, and ferritin on the development of mixed chimerism. we included 56 eligible patients. the median follow-up time after hsct was 26 months (interquartile range: 17.3 − 50.3 months). the mean age at transplant was 19.9 years (standard deviation: 8.44). fifty-nine percent of patients were male. most patients had s/s genotype (77%), followed by s/beta-thalassemia mutation (20%). the indications for bmt were: stroke in 7%, acute chest syndrome (acs) in 9%, recurrent vaso-occlusive crisis (voc) in 38%, stroke and acs in 7%, acs and voc in 31%, orbital compression syndrome in 2%, stroke and moyamoya disease in 4%, and moyamoya disease in 2%. the two most frequently used preparative regimens were busulfan/fludarabine/atg in 49% and thiotepa/treosulfan/fludarabine in 42%. twenty-five percent of patients developed mixed chimerism at six months after hsct. on follow up of patients with mixed chimerism, 10% rejected the graft, 20% developed complete chimerism, and 70% continued to be in mixed chimerism. preparative regimen and the development of agvhd were statistically significant predictors of mixed chimerism at 6 months (p values: 0.00079 and 0.01817 respectively). age at transplant, gender, red blood cell antigen alloimmunization, and ferritin were not statistically significant predictors of the mixed chimerism (p40.05). the study confirmed that mixed chimerism can commonly be achieved in patients with scd after hsct and in majority, it remains stable on long-term follow-up. reduced intensity preparative regimen and lack of agvhd predicts the development of mixed chimerism. larger prospective studies are needed to confirm these results. disclosure of conflict of interest: none. there was a majority of male patients (69%). the median hbf level was 15% (0-77), median monocyte count was 5.109/l (range: 0-74.8), median platelet count was 64.109/l (4-377), median marrow blasts was 5% (0-37) above 96 patients who were explored by marrow karyotype, 27% of them had a monosomy 7. mutations in ptpn11 were detected in 33 patients. fifty patients (43%) were treated with the bu/cy/mel regimen, whereas 22 patients (19%) received the bu/flu/mel regimen. at 6 years, the overall survival (os) was 62% (95% ci: 53-72). nineteen and 9 patients developed vod after bu/cy/ mel and bu/flu/mel conditioning regimen, respectively. the cumulative incidence of agvhd 3-4 was 26% (95% ci: 19-35). the 6-year cumulative incidence of relapse and non-relapse mortality was 30% (95% ci: 22-39) and 18% (95% ci: 11-25), respectively. the median delay of relapse was 90 days (range 15-1330). among relapsing patients, 16 were transplanted twice and one underwent 3 hsct. in multivariate analysis, female donor to male recipient sex-mismatch, cmv status, total body irradiation and ras-double mutation/other additional mutation predicted poorer outcomes. the bu/flu/mel conditioning regimen was associated with a decreased risk of relapse. however, there was no statistical difference for os between the two main preparative regimens, bu/cy/mel vs bu/ flu/mel. our results show that allogeneic hsct may cure approximately 60% of patients with jmml and are similar to the best results published by other groups. relapse represents the main cause of treatment failure and a second hsct should be proposed. despite a decreased risk of relapse with the bu/ flu/mel regimen, there was no statistical difference in terms of os between the two main conditioning regimens, bu/flu/mel vs bu/cy/mel. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (ahsct) is the only curative treatment modality for the majority of pediatric patients with myelodysplastic syndrome (mds) and myeloproliferative neoplasms (mpn). the purpose of this study is to evaluate overall (os) and failure (relapse or death from any cause) free survivals (ffs), non-relapse mortality (nrm) and relapse incidence in children who underwent ahsct for mds or mpn in a single center from turkey. we retrospectively analyzed 45 ahsct carried out in 43 patients (median age: 9.2 years; range: 0.4-18; 24 males). thirty four had primary mds and 9 had secondary mds. according to the modified who mds and mpn classification, 18 had refractory cytopenia (rc), 12, refractory anemia with excess blast (raeb), 1, refractory anemia with excess blast in transformation (raeb-t) and 12, juvenile myelomonocytic leukemia (jmml). amongst patients with secondary mds, 4 had been treated for acute myeloid leukemia, 2 had been treated for non-hodgkin's lymphoma and 1 had been treated for acute lymphoblastic leukemia, retinoblastoma and osteosarcoma, each, previously. donors were related in 18 transplantation (5 haploidentical transplantation) and the stem cell resources were bone marrow (n = 27), peripheral blood (n = 14), cord blood (n = 2) and bone marrow +peripheral blood (n = 2). three-year ffs and os for patients with mds were 55% and 57.0%, respectively; and for patients with jmml, 50% and 64%, respectively. crude incidence of nrm and relapse for entire group were 33% and 22%, respectively. ahsct offers durable ffs and os for a significant group of pediatric patients with mds and mpn. less toxic conditioning regimens could result in better results in some patients. disclosure of conflict of interest: none. allogeneic stem cell transplantation in children with autism z antonella, g alessandra, ma beatriz and r vanderson bone marrow transplantation unit, hospital sirio-libanes, são paulo, brazil autism spectrum disorders (asd) are severe heterogeneous neurodevelopmental abnormalities characterized by dysfunctions in social interactions and communication skills, restricted interests, repetitive, and stereotypic verbal and non-verbal behaviors. the etiology of asd remains unknown, but recent studies suggest a possible association with altered immune responses and asd. inflammation in the brain and central nervous system has been reported with microglia activation and increased cytokine production in postmortem brain specimens of individuals with asd. other studies have established a correlation between asd and family history of autoimmune diseases, associations with mhc complex haplotypes, and abnormal levels of various inflammatory cytokines and immunological markers in the blood. the paracrine, secretome, and immunomodulatory effects of stem cells would appear to be the likely mechanisms of application for asd therapeutics. we describe two cases of patients with asd who underwent hsct for acute lymphoblastic leukemia (all) and whose symptoms were markedly decreased like an improvement of social interaction, communication, and behaviors. the first patient is an 11-year-old girl with asd who was diagnosis with ph-positive all in october 2011 (at the end of treatment, bcr-abl remained positive). she underwent a matched sibling hsct in march 2015. the conditioning regimen was total body irradiation (tbi) and cyclophosphamide. during the 20-month follow-up period, we observed improvement in social interaction, communication, and behaviors (according to the childhood autism rating scale-cars). the second case is a 7-year-old boy with asd, asperger syndrome, who was diagnosis with all in september 2012. he presented with bone marrow and testicular relapse in may 2015 and underwent a matched unrelated hsct in november 2015. the conditioning regimen used was etoposide, atg and tbi. during the 12-month follow-up period, we observed improvement in social interaction, communication, and behaviors (according to cars). there is no treatment for asd thus every effort to minimize the symptoms are valuable. in both cases, social interaction was significantly increased, and the aggressive behaviors decreased. clinical cases have reported responses in autistic children receiving cord blood cd34+ cells. several incurable neurological disorders have shown benefits with cellular therapy. thus, autism should be explored as an indication. clinical studies are an immediate need to fully explore its potential in autism. disclosure of conflict of interest: none. conditioning is the initial phase of hematopoietic stem cell transplantation, based on high dose chemotherapy, combined or not with total body irradiation, aiming to eradicate the disease and prepare the environment of the bone marrow for the new cells. conditioning regimens can be characterized as myeloablative or non-myeloablative. during the period of conditioning and immunological reconstitution, signs and symptoms of the gastrointestinal tract are frequent, negatively influencing oral food intake, and may require the use of complementary nutritional therapies, aiming at an adequate caloric intake with the objective of avoiding decreasing in the nutritional status. the study aims to describe the association between the regiment intensity and the nutritional aspects during hospitalization of children and adolescents undergoing allogeneic hematopoietic stem cell transplantation (hsct) at a tertiary hospital. a retrospective study with medical records of patients undergoing allogeneic hsct, aged between 0 and 19 years of age (incomplete) between january 2009 and december 2014. data were collected (regimen intensity, clinical signs of mucositis and nutritional therapies used) during the hospitalization and analyzed by the relative risk (rr). sixty-three patients were evaluated, being 56% male, with a median age of 10 years. nineteen types of conditioning protocols were used. of these, 64% were high intensive regimen and 36% were low intensive regimen. the four most applied (59% of cases) were bucy (busulfan + cyclophosphamide) with and without atg (thymoglobulin), as well as cytb (cyclophosphamide+total body irradiation), also with and without atg. mucositis were observed in 83% of patients, being 50% grade 3 and grade 4. the association was positive when analyzed the regimen intensity (myeloablative) with mucositis (rr = 1.51 (1.10-2.08)) as well with the use of parenteral nutrition (rr = 2.49 (1.17-5.13)). patients showed high prevalence of mucositis during hospitalization decreasing food intake, being necessary to use the parenteral nutrition. myeloablative regimen needed more nutritional therapy intervention when compared to non-mieloablative regiment. results demonstrate that an appropriate nutritional screening tool considering these aspects could help to intervene earlier maintaining an adequate nutritional status. autoimmune cytopenia (aic) is a potentially serious complication of hematopoietic stem cell transplantation (sct). autoimmune hemolytic anemia (aiha) is the most common aic, followed by immune thrombocytopenic purpura and autoimmune neutropenia. aic after sct is considered difficult to treat and associated with high morbidity and mortality. the aim of this cohort study is to evaluate incidence, outcome, potential risk factors and current treatment strategies and to explore the immune dysregulation predisposing to aic. the ebmt-promise database was accessed to identify all pediatric scts between 2000 and 2016 complicated by aic at our center. potential risk factors (i.e., age, gender, diagnosis, donor type, stem cell source, conditioning regimen) for aic after sct were assessed using univariate and multivariate cox regression analysis. in addition, we summarized treatment decisions of all aic patients. a nested matched case-control study was performed to search for possible biomarkers for aic. of 531 consecutive scts, 27 were complicated by the development of aic (cumulative incidence 5.2%) at a median of 5 months post-sct (figure) . aiha was the most common aic (48%), followed by combinations of two or more aics (evans syndrome, 33%). non-malignant disease, young age, alemtuzumab serotherapy pre-sct, non-tbi based conditioning regimen and cmv reactivation were associated with aic in univariate analyses. using multivariate cox regression analysis, non-malignant disease (hr 3.6, p = 0.028), alemtuzumab use (hr 2.4, p = 0.035) and cmv reactivation (hr 3.7, p = 0.013) were independently associated with aic (figure) . for patients with cmv reactivation, diagnosis of aic was made at a median of 4 months (iqr [1] [2] [3] [4] [5] [6] [7] [8] ) after detection of maximum viral load. in our nested case-control analysis, serum levels of individual anti-and proinflammatory, and regulatory cytokines did not differ significantly between patients and controls. however, the cytokine profile of aic patients appeared to skew towards a more pronounced th2 response, compared to controls. firstline treatment, usually with prednisone and/or ivig, or a waitand-see approach led to resolution of aic in 9 (33%) cases. second and subsequent-line therapies, often in combination with continuation of other treatments, consisted of rituximab (n = 16), bortezomib (n = 7) or sirolimus (n = 3) and eventually led to resolution of aic in 44%, 57% and 100% of cases, respectively. overall survival of aic patients was 78%. in this retrospective cohort study, we identified cmv reactivation post-sct, alemtuzumab use and non-malignant disease as independently associated clinical risk factors for the development of aic. treatment with first-line therapy was mostly insufficient. for patients with severe aic, rituximab, bortezomib or sirolimus can be regarded as promising step-up therapies. figure (bkv) may cause polyomavirus-associated nephropathy or polyoma virus-associated hemorrhagic cystitis in bone marrow-transplant patients.we present 19 patients with bk polyoma virus (bkv) ascociated hemorrhagic cystitis and 2 patients with bk polyoma virus associated hemorrhagic cystitis and nephritis. between 2013 and 2016, 124 patients received an allogeneic bmt at acıbadem adana hospital pediatric bone marrow transplantation unit. 21 patients occurred bkv associated hemorrhagic cystitis and nephritis. bkv was detected in the urine analysis and blood by pcr (polymerase chain reaction) in all patients. we presented 21 patients with bkv infection, age ranging from 3 to 20 with a average of 13.1 years. they underwent allogeneic bmt due to thalassemia major (13 patients), aplastic anemia (4 patients) and acute lymphoblastic leukemia (4 patients). the patients were treated with hydration, continuous bladder irrigation, ciprofloxacin, and weekly intravesical hyaluronic acid instillation for four weeks, and cidofovir. fourteen patients showed complete resolution of hematuria. one patient with refractory above these therapy also received hyperbaric oxygen and recombinant factor viia (rfviia, novoseven; novo nordisk,bagsvaerd, denmark). hemodialysis was performed in two patients who developed renal failure due to nephritis. bkv is ubiquitously present in the general population. 1 reactivation of infection occurs under conditions of immunosuppression, particularly hsct or renal transplantation, and causes late-onset hc. bkv the management of bkv cystitis and nephritis sometimes may be very difficult and refractory all treatments, we presented our experience of bkv infection and management in transplanted patients in our center. patients with high-risk hematologic malignancies (hrhm) are among those in the highest risk group for developing invasive fungal disease (ifd), especially mold infections. allogenic hematopoietic stem cell transplantation (alsct), acute myeloid leukemia (aml), refractory and relapsed acute lymphoblastic leukemia (all), myelodysplastic syndromes and chronic extensive graft-versus-host disease are considered hrhm. ifd are a major cause of morbidity and mortality in these patients, however, the optimal strategy for antifungal p448 s352 prophylaxis in this population is not well defined yet. we performed a retrospective, observational study to investigate documented ifd during antifungal prophylaxis in children with hrhm who were admitted in our unit between 2010 and 2016. demographic and clinical data were collected from patient's electronic medical records. all patients were treated with prophylactic voriconazole (vcz) according to our local practice. oral administration was preferred when available. vcz therapeutic drug monitoring (tdm) was not available in our center until june 2016. breakthrough ifd was defined as occurrence of a proven or probable ifd according to eortc/ msg criteria while on vcz prophylaxis (⩾7 days of treatment) or within 15 days after discontinuation of prophylaxis. during the study period, 75 hrhm patients were treated with prophylactic vcz in our unit. 4 patients out of 75 developed a breakthrough ifd. patient's demographic characteristics, main diagnosis and treatment are collected in table 1 . initial and maintenance vcz doses are adjusted by weight in all patients except in patient-4 (adjusted according to vcz plasma level). adherence and tolerance to treatment was excellent in all patients. disclosure of conflict of interest: none. (3) stable mixed chimerism (smc) when fluctuations of ac were o5%; and (4) mixed progressive chimerism (pmc) when ac were ⩾ 15%. 2-3 101 hscts performed in 85 patients (pts) were included: 72 children with a median age of 2.01 yrs (iqr 0.62-7.35 yrs) at diagnosis and 6.2 yrs (iqr 2-11 yrs) at hsct received one hsct (84.7%),10 pts two hsct (11.8%), and 3 pts three hscts. primary diagnosis were bone marrow failures in 37 pts (43.5%), primary immunodeficiencies in 25 (29.4%), inborn errors of metabolism in 15 (17.5%) and haemoglobinopathies in 8 (9.41%). the donor was match related in 23 (23%) procedures, match unrelated in 63 (62%), and haploidentical in 15 (15%); stem cell source was bone marrow in 55 (54%), peripheral blood in (26%) and cord blood in 20 (20%). conditioning regimen (cr) included busulfan in 19 hscts (18.8%), treosulfan in 33 (32.7%), while 48 hscts (47.5%) were conditioned with reduced intensity crs (including low dose of tbi in 9); 1 pt did not received cr. gvhd prophylaxis was based on csa/mtx (or mmf) in association with atg (69) or alentuzumab (16) ; recipients of tcrαβ/cd19 depleted haploidentical graft did not received post transplant immunosuppression. engraftment was observed in 87 hscts (79 after 1 st , 7 after 2 nd and 1 after 3 rd hsct) after a median of 18 day (iqr 14-23 days). acute gvhd occurred in 45 hscts at risk (52%), and it was severe (gr. iii-iv) in 20 (23%), chronic gvhd in 31 (31%). at last follow-up (median 4.35 yrs), 75 (88%) pts were alive, while 10 pts are dead for infections (n = 5), vod (n = 1), c-gvhd (n = 3) and vascular event (n = 1). figure 1 reported the evolution of chimerism over time. in our experience in children with non malignant disease, cc increased from 36% to 67% at subsequent analyses. 60% of pts with mc at 1 st evaluation became cc, 16% remained smc, 5% evolved in pmc, and 19% rejected. only 2 pts with cc at first time point rejected the graft. this study highlight the extreme variability of chimerism in the early post transplant course of children with non malignant disease and confirmed the relevance of performing serial analysis to monitor and, if necessary, improve graft function. naive t-cells identified by cd45ra expression are believed to cause graft-versus-host-disease (gvhd), while cd45ra-t-cells are memory cells that provide anti-infection and anti-tumoral effects. depleting cd45ra+ naïve cells and retaining memory t-cells in the graft is a novel approach to haploidentical hsct for children. 18 children with high risk leukemia (6 aml, 12 all) received cd45ra-depleted haploidentical hsct following non-myeloablative conditioning. cell-selection performed on g-csf-mobilized peripheral blood. two cellular products obtained using clinimacs device, infused to each patient: a cd34 selection and a cd45ra depletion from the cd34negative fraction. product infused contained a median of 6.04 (range 4.04-9.93) x106/kg cd34+ cells and a median of 6.5 (range 1.3-490) × 10 3 /kg of cd3+ cells in the cd34-selected s353 graft. the second product was the cd45ra depletion, cd45ra +/kg was a median of 6.15 × 10 3 /kg (range 0-498 × 10 3 /kg) and a median 4.70 (range 2.21-6.37) depletion log of cd45ra + cells. median dose of cd45ro+ cells (memory t-cells) infused was 8 (range 3.8-102) × 10 7 /kg. seventeen patients achieved neutrophil engraftment at median of 10 days (range 8-12) post-transplant. one patient could not achieve engraftment, died at day +8 due to sepsis. two patients presented secondary graft failure (day +18 and +20), both received a second hsct. three patients developed agvhd 4grade ii with gastrointestinal tract involvement, all steroids responsive. three patients presented clinical features of cgvhd. patients have an extensive skin involvement, with hepatic findings in one and pulmonary affection in other, at day +315, +130 and +330 post. ten of 18 patients (55.5%) remain alive in remission with median follow-up 156 (range 8-597) days post-transplant. eight patients died, 3 due progression at day +128, +117, +162 (2 presented positive minimal residual disease at hsct), 4 due to infectious complications (days +8, +44, +50, +55) and 1 due to cardiogenic shock at day +253. four patients relapsed, 3 of them died afterward with progressive disease. t-cells led immune recovery, achieved values higher than 500, 600, 1500 and 2400 cells/mcl at day 30, 60, 90 and 210 respectively. most of t cells were cd8+cd45ra-(median of 288, 370 and 2334 × 10 6 /mm 3 respectively on day +30, +60 and +90) and cd4+cd45ra-t cells (median of 129, 161 and 767 × 10 6 /mm 3 respectively on day +30, +60 and +90), while cd8+45ra+ and cd4+45ra+ cells remained low recapitulating the cd45ra depleted graft composition. six patients presented cytomegalovirus reactivation, one progressed to cmv disease. five patients with hhv-6 encephalitis. probable aspergillosis in 1 patient (aml-m7 with secondary graft failure) at day +16 after second hcst. two cases of toxoplasmosis (1 cns, 1 pulmonary). cd45ra-depleted haplo-hct showed acceptable tolerability with rapid and sustained engraftment as well as a full donor chimerism, minimal risk of acute gvhd and accelerated inmunologic reconstitution. to note the high incidence of hhv-6 encephalitis seen. disclosure of conflict of interest: none. collection of peripheral blood stem cells in teenager sibling donors: a single center experience c carvalho 1 , f amado, f bordalo, s ferreira, s lopes, c pinho and s roncon 1 serviço de terapia celular, instituto português de oncologia do porto francisco gentil, epe human leukocyte antigen (hla) compatibility is important in allogeneic haematopoietic stem cell transplantation in order to reduce post-transplant complications; however, siblings only present a 25% chance of hla-match with the patient. the well-known advantages and the low risk of complications associated to peripheral blood stem cells (pbsc) collected by apheresis made this procedure the first option in teenagers. the aim of this retrospective study was to analyse and characterize the paediatric sibling pbsc donor population assuring safety during the collection procedure, providing a high-quality product and accomplishing patient needs. we consulted the clinical files of donors under 18 years old since 1995-2015; a database in excel ® was created to register population characteristics, collection parameters and graft requirements. the informed consent was obtained from parents before procedure. the leukapheresis were performed with a cobe spectra system; since 2009, we use a spectra optia apheresis system. the donor/patient weight ratio (proposed by styczynski et al.) was determined for each pair. the collection was programed based on clinical and analytical donor's features as well as transplant requirements. the analytical assays were done by a certified laboratory. we performed 29 pbsc apheresis in 23 healthy donors, 10 females and 13 males ( table 1) . all of them started on the 5 th day after mobilization with granulocyte colony-stimulating factor (g-csf) administered subcutaneously, bidaily. the weight ratio was o1 in eight situations. most of donations were performed by peripheral vein; a central venous catheter (cvc) was placed into a femoral vein in six adolescents. a median of 4 (3) (4) (5) blood volemias were processed during 174(115-318) minutes; the anticoagulation used was citrate+heparin (ratio 25:1). in general, one-collection day was enough to obtain the number of cd34+ cells required; six donors had to perform a 2 nd collection. in 19 cases, we cryopreserved the exceeding cells after graft infusion. the procedure was well tolerated, with only 2 adverse reactions registered (one hematoma in the puncture local; one paraesthesia due to hypocalcaemia induced by citrate). no blood products were used after the procedure or needed for the priming of the extracorporeal circuit. so far, no serious long-term adverse events were observed. table 1 . median (range) of donors and leukapheresis products data. our long lasting experience shows that pbsc collection in the teenage population is safe and feasible, allowing us to obtain a high-quality product for the patients. there were no adverse events associated with the g-csf mobilization or cvc placement which is different from the experience of other groups. we recognize that leukapheresis by peripheral vein is a lengthy procedure but no complaint was reported to the collection team. [p451] disclosure of conflict of interest: none. correspondence between clinical and hystological grading of gastro-intestinal grading acute graft versus host disease in children m faraci 1 , a rizzo 2 , p gandullia 3 , s arrigo 4,3 , a barabino 3 , e lanino 1 , s giardino 1 and c coccia 5 1 hematopoetic stem cell transplant unit, institute g gaslini, genoa, italy; 2 pediatric department, institute g gaslini, genoa, italy; 3 gastroenterology and digestive endoscopy unit, institute g gaslini, genoa, italy; 4 gastroenterology and digestive endoscopy unit, institute g gaslini, genova and 5 department of pathology, institute g gaslini, genoa, italy diagnosis of gastro-intestinal acute graft versus host disease (gi-agvhd) is frequently confirmed by apoptosis findings on mucosal biopsies. 1 aims of this single center retrospective study is to evaluate the correlation between clinical and histological grading of gi-agvhd in children undergoing allogeneic haematopoietic stem cell transplantation (allo-hsct), and to describe histological findings obtained by gi endoscopies in order to evaluate usefulness in the diagnosis of gi-agvhd. 348 allo-hscts were performed in our department between january 2000 and december 2015. gi biopsies were performed in 26 pts (7.4%) because of suspected gi-agvhd. 14 pts were transplanted for malignant (53.8%) and 12 for not malignant diseases. the median age at hsct was 9.5 years (0.5-16.9).14 pts (54%) received myeloablative and 12 (46%) reduced intensity conditioning regimen. 21 pts (80.7%) received an unrelated donor (ud), 4 pts a related donor (rd) (15.3%), and 1 an haploidentical donor (3.8%). at onset of diarrhea, microbiological examinations of stool were performed and pcr research for cmv, adenovirus, hhv6, ebv were evaluated in blood and in mucosal biopsies. mucosal biopsies were obtained with esophago-gastro-duodenoscopy in 4 pts (15.3%),esophago-gastro-duodeno-colonscopy in 3 (11.5%), pancolonscopy in 11 (42.3%), flexible sigmoidoscopy in 3 (11.5%), and rectal suction biopsy in 5 pts (19.2%). all mucosal biopsies, except in case of rectal suction, were obtained under sedation. the interval between mucosal biopsies and onset of gi acute symptoms was 23 days (from − 66 to 103 days). biopsies were taken from different sites in the gi tract, were stained using hematoxylin-eosin and evaluated using histological grading of agvhd. 1 in these 26 pts the maximum grade of agvhd was: grade 2 in one (4%), grade 3 in 14 (54%), and grade 4 in 11 pts (42%). at time of histological evaluation, diarrhea was the most common gi symptom (84.6%); 2 children had also cutaneous agvhd and 5 hepatic agvhd. pcr-cmv was positive in 2 mucosal biopsies obtained with pancolonscopy, pcr-adenovirus in other 2 obtained with upper and pancolonscopy, pcr-hhv6 in 2 rectal biopsies, and pcr-ebv in one with upper and pancoloscopy. the comparison between clinical and histology grading of gi-agvhd is shown in table 1 . mucosal biopsies were positive in 1/4 pts evaluated with esophago-gastroduodenoscopy (25%) (grade 1 agvhd), in 3/3 pts undergone esophago-gastro-duodeno-colonscopy (grade 1 in 2 and grade 3 in 1), in 8/11 (73%) who received a pancolonscopy (grade 1 in 5, grade 2 in 1, grade 4 in 2), and 7/8 (87%) of rectal biopsy obtained by sigmoidoscopy or rectal suction biopsy (grade 1 in 3, grade 2 in 1, grade 3 in 1, and grade 4 in 2). one patient developed duodenal intraparietal hematoma after upper endoscopy. in our experience, we did not demonstrated a overall correlation between clinical and histological grading of agvhd showing that hystological examinations underestimated the grade mild or moderate of agvhd. we confirmed 2,3 that rectal biopsies represent to be more effective and safe diagnostic method for the confirm of diagnosis of gi-agi. during the past few decades, hematopoietic cell transplantation (hct) as a treatment modality for primary immunodeficiencies (pid) has undergone remarkable advancement mainly due to better availability of alternate donors resulting in increase in not just matched unrelated donor (mud) but also increased haploidentical (haplo) and cord blood transplants (cbt). additionally, refinement of the conditioning regimens and better graft versus host prophylaxis have presumably led to better survival outcomes. however, a literature gap is identified in evaluation of these outcomes in general with respect to donor and conditioning regimens. we conducted a systematic review by performing a comprehensive search of the pubmed and ovid library from its inception to august 2016. mesh terms included 'primary immunodeficiency (immunodeficiencies)', 'stem cell transplant', 'bone marrow transplant' and 'hematopoietic cell transplant'. all pid studies which used hct as a treatment modality were included. experimental cellular therapies were excluded. both cellular immunodeficiencies (e.g. scid, was, a-t), and innate immunity disorders (e.g. ifngr, cgd) were included in the search. reviews, case reports, meta-analysis and non-english language articles were excluded from our electronic search. publication bias was excluded by performing a methodological search of unpublished conference abstracts from the annual meetings of cis, aspho, asbmt, ebmt, and siop from 2000 to 2016. the data were analyzed considering the outcomes -overall survival and gvhd. 21 studies fulfilled the strict selection criteria for the electronic search comprising of 1010 pid patients. in majority of the hcts, a myeloablative conditioning regimen (mac) was utilized (47% of the evaluable) but a shift towards more reduced intensity conditioning (ric) was observed in the later years. 120 cbts were identified. 27% of patients developed some degree of acute gvhd, whereas chronic gvhd was identified in 15% of the patients. total number of haplos was 317. overall survival was found to be 71% post-hct. a meta-analysis could not be performed due to the heterogeneity of both the predictor variable data (combined stem cell sources were also used for hct) and due to the extremely small number of the patients when categorized in subgroups (e.g. for omenn syndrome, rag deficiencies). this is the largest study of hcts in pid, and we observe that alternate donor hcts have increased significantly over the past decade for the treatment of pid. while the incidence of chronic gvhd was low, acute gvhd still remains a problem in about a third of the pid patients transplanted. disclosure of conflict of interest: none. hepatic veno-occlusive disease (vod) is a common and serious complication of hemotopoietic stem cell transplantation (hsct) in children. we aimed to assess prospectively the use of prophylactic defibrotide in pediatric patients undergoing hsct. in this study, 113 patients who underwent hsct were given defibrotide prophylaxis as 25 mg/kg per day in four divided intravenous infusions over 2 h, starting on the same day as the pretransplantation conditioning regimen. the mean duration of use of defibrotide is 20 days as a prophylaxis. in this study, 113 patients were recruited, 66 male patients and 47 female patients, with the average of 9.1 years, range 1-20; 8% infants, 55% children and 37% adolescent. there were 50 patients with thalassemia major, 41 patients with leukemia, 11 patients with aplastic anemia, one patient with diamond blackfan anemia, two patients with congenitale dyserythropoetic anemia, one patient with osteopetrosis, four patients with famial hemophagocytic lymphohistiocytosis, two patienrs with severe immune deficiency and one patient with kostman syndrome. all transplants were allogeneic. no serious side effects were seen. in eight patients developed clinical vod (seattle criteria). in these patients, defibrotide dose was increased to a treatment dose of 40-60 mg/kg per day. one infant patient with kostman syndrome died due to hepatic and pulmonary veno-occlusive disease. after 36 months of follow up, 7 patients who developed vod are being well and no patient have transplant related complications. hepatic veno-occlusive disease, which is caused by hepatocyte and sinusoidal vessel endothelium damage, can ocur early after hsct, and in its severe form, may lead to liver faillure, hepatorenal syndrome, portal hypertension, and eventually death from multiorgan faillure. in this prospective study, we demonstrated that the use of defibrotide is safe and effective in preventing and treating vod in pediatric patients at high risk. immune reconstitution (ir) is critical for clinical outcome after allogeneic hematopoietic stem cell transplantation (hsct). host and proceeding-related factors affect the ir dynamics and survival. isolated ir parameters are commonly correlated and proposed to predict clinical outcomes after hsct, but these approaches only confer prognostic value at single time points or for single markers. we aim to demonstrate an appropriate methodology to assess the capability of combined serial measurements of lymphocyte subsets to reflect the impact of infections on ir after paediatric hsct. retrospective data of patients receiving a first hsct for any indication with any cell source in the paediatric hsct program from 2006 to 2015 were included. to characterize the kinetics of immune reconstitution, cd3+, cd4+, cd8 + t-cells, b-cells, nk-cells and their naive and memory subsets were measured and analysed at various time points at 2 years post-hsct to stablish a joint model for the evolution of cell subpopulations. slope per month (cellular increase or decrease) of each lymphocyte subsets were calculated and compared with clinical outcomes and cumulative risk of infections. a total of 88 children (range from 0-15 y.o. median 5 y.o.) were included, with cb (n = 19) pb (n = 22) and bm (n = 47) as cell sources. the cumulative incidences after early period were 45% for viral infections (ebv 27%, cmv 22%, bk 11%, adv 4%) and 30% for bacterial infections. data on ir were available for 77%, of the diseasefree survivors. in a exploratory multivariate analysis we detected mainly differences in cd8+, cd8+cd45ro+ memory and nk cells at 1 year after hsct, with dependent tendency according on the cell source and hla compatibility. analysis of the slope tendency patterns were stablished for the analysis of the impact of infections in the ir. delay in cd8+ and cd8+ra+ appearance (mean slope/m = − 7.1% and − 1.8% respectively) remarks the ir profile for bacterial infections, and delayed in nk, cd8 and cd8ro+ (−8.4%, − 5.8%, − 26% respectively) for overall viral infections. additional correlations allow differences in ebv (cd8+ra+ high mean slope/m = 15.9%), cmv (delayed in cd8ro+ slope/m = 10%), and bk infection (cd8+ra + plus cd4ro+ and nk high mean slope/m = 24.9%, 36% 22%). understanding the dynamics of reconstitution by integrating information from the monitoring of lymphocyte subpopulations allows the establishment of kinetic profiles that may help to evaluate the risk of infections and adjust infection prophylaxis in the follow-up of transplanted patients. mortality rate in hsct patients admitted to intensive care unit (icu) is still as high as 20% to 70%. this rate increases when respiratory complications progress to acute respiratory failure (arf) requiring mechanical ventilation (mv). 1 the aim of this study was to determine the feasibility and effectiveness of early continuous positive airway pressure (cpap) delivered in a pediatric hematology-oncology ward to prevent occurrence of arf requiring mv. we retrospectively analysed children treated with cpap in our pediatric hematology-oncology ward between october 2011 and october 2016. thirty-two patients received cpap delivered with helmet during the study period. data were available for 26 patients, 15 males and 11 females, median age 12 years [range 2-20]. eighteen patients underwent allogenic hsct: 1 from sibling donor, 11 from matched unrelated donor, 4 from haploidentical family donor, 2 from cord blood unit. seven patients had a malignant disease: 5 all, 1 aml,1 ewing sarcoma. infectious pneumonia was the main cause of arf in 16/26 patients (61.5%): 9 viral pneumonitis (4 rhinovirus, 3 parainfluenzae virus, 1 respiratory syncitial virus and 1 cmv). five patients had proven/ probable invasive fungal infection according to eortc criteria (3 aspergillosis and 2 mucormycoses). other causative agents were pneumocystis jiroveci (1), bacillus of calmette and guerein (1), toxoplasma gondii (1) and st. mitis (1) . non infective causes of arf were acute transfusion related lung injury (2), hemorragic alveolitis (2), cryptogenic organizing pneumonia (3), tumor lysis syndrome (1), and alveolar oedema due to renal failure (1). according to chest imaging, 13/26 patients (50%) presented with pulmonary consolidations, while 31% had both interstitial infiltrates and pulmonary consolidations. at baseline median neutrophil count was 2.05 × 10 3 /μl (range 0-21.0 × 10 3 /μl), mean heart rate 128 bpm, pulsiossimetry saturation in room air 86%. h-cpap was applied in 19/26 patient with a curative aim, in 7/26 patients as palliative support to reduce respiratory distress. median positive pressure delivered was 10 cmh 2 o (7-12 cmh 2 o), median fio 2 was 40% (30-100%). h-cpap was applied for a median of 11 days . no patient failed h-cpap because of agitation or adverse events (skin breakdown, conjunctivitis, gastric distension or epistaxis). ten patients were transferred to icu (34.6%), 8/10 because of hsct complications. median icu stay was 8.7 days (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) . only 3 patients required mechanical ventilation, in 2 cases associated to ecmo. nether psao 2 in room air (p 0.98 ci 95%) nor cpap level (p 0.76 ci 95%) correlated with the need of icu admission. patients requiring higher fio 2 during cpap demonstrated a not statistically significant trend to higher icu admission rate (p = 0.149).there was a higher rate of mv in patients with higher cpap fio 2 level (p = 0.008). mv prolonged icu stay (p 0.0049). cumulative mortality was 34.6% (9/26); only 1 patient died in icu (10%), because of post hsct parainfluenza virus pneumonitis requiring ecmo. helmet cpap delivery in pediatric hsct ward is feasible and safe, both for curative and palliative aim. if applied early, cpap could reduce icu admission rate for mv and icu mortality. veno occlusive disease (vod) and graft versus host disease (gvhd) are both dreadful complications of hematopoietic stem cell transplantation (hsct). although they have different clinical signs, it is suggested that they share similar pathophysiological pathway. defibrotide is used in the treatment of vod for a long time but it is very less known about its effect on gvhd. in this study, we analyzed a 'high risk' patient population in pediatric hsct to show the effect of defibrotide on acute gvhd. between june 2014-august 2016 totally 75 'high risk' pediatric allogenic hsct procedures were enrolled in this study. 'high risk' definition involves busulphan/ melphalan usage in conditioning regimen, second myeloablative hsct, pre-existing liver disease, allogenic hsct for leukemia with second relapse and diagnosis of hemophagocytic lymphohistiocytosis (hlh) or osteopetrosis. defibrotide prophylaxis group (n = 22) received 25 mg/kg/day per day and continued for at least 14 days after transplantation. the control group (n = 53) received only continuous infusion of low-dose heparin until 30 days after transplantation. for the comparison between groups, the fisher's exact test was used. all analyses were performed using spss 20 and p-value of 0.05 was considered statistically significant. we analyzed totally 75 hsct procedures with different diagnosis; 17 beta thalassemia major, 14 leukemia, 9 hlh, 18 primary immunodeficiencies, 3 osteopetrosis, 4 fanconi aplastic anemia (faa), 2 myelodysplastic syndrome, 2 neuroblastoma, 1 congenital amegakaryocytic thrombocytopenia, 1 krabbe disease, 1 aplastic anemia, 1 hypereosinophilic syndrome and 1 sickle cell disease. all the procedures meet the 'high risk' definition; most of them (n = 50) have busulphan for conditioning, also there are 9 hlh and 3 osteopetrosis patients, 2 neuroblastoma patients had the second myeloablative regimen, faa and aplastic anemia patients had pre-existing liver disease, and 2 of the leukemias had beyond second relapse. the mean age was 6.7 years old (0.25-19.6), 27 hsct performed from match sibling donor (msd) (36%), 3 hsct from match family donor (mfd) (4%), 43 hsct from match unrelated donor (mud) (57%) and 2 hsct from haploidentical mother (3%). we especially focused on gvhd and vod. totally 13 vod cases (17%) in these 75 hsct procedures were detected. only two of them detected in the prophlaxis group (9%) and 11 cases in the control group (20%). there were 32 cmv reactivation cases detected in 75 hsct procedures (42%). in the prophlaxis group there were 11 cases (50%) and in the control group 21 cases (39%). we detected 36 acute grade i-iv gvhd cases in 75 hsct procedures (48%). only 4 of them were in the prophlaxis group (18%) and 32 cases were in control group (60%). the prophlaxis group's agvhd ratio was significantly lower than the control group (p = 0.001). defibrotide for vod prophylaxis is confirmed by several studies, but its benefits for agvhd is not clear. in this study, we show the significant effect of defibrotide on agvhd. we speculate that the protective effect of defibrotide on both vod and agvhd could be explained by the similar pathophysiology of these complications. we need larger studies on the pathophysiological pathways, then we could invent more effective interventions. disclosure of conflict of interest: none. conventional extracorporeal photopheresis (ecp) has proven efficient for the treatment of graft-versus-host-disease (gvhd) but is limited to patients with sufficient body weight. a mini buffy coat ecp (mini-ecp) 'off line' technique that allows treatment of small children has been developed, using various methods for mononuclear cell (mnc) separation from whole blood. we present treatment of low body weight child with mini-ecp 'off line' technique using sepax system for mononuclear cell (mnc) selection and macogenic irradiator. a toddler with juvenile myelomonocytic leukemia (jmml) developed acute gvhd, after a matched unrelated stem cell transplantation (sct) performed at the age of 12 months. acute gvhd of the skin occurred three months after sct and responded to high dose steroids, but recurred six months after sct (biopsy of the skin confirmed acute gvhd) together with gvhd of the liver. because of the resistance to steroids and cyclosporine, mini-ecp was introduced as therapy. the patient weighed 8 kilograms. blood was collected from tunneled central venous catheter, and collected volume was replaced with saline infusion. the cord blood collection bag (macopharma, france), which contains 21 ml citrate phosphate dextrose (cpd) anticoagulant solution was used for whole blood collection. whole blood was processed using sepax system separator (biosafe, switzerland), and final volume of buffy coat was set on 25 ml. extracted buffy coat was transferred into the uv-a illumination eva bag (macopharma, france) and diluted with saline solution up to 200 ml. 8methoxypsoralen (gerot, austria) was injected directly into the uv-a illumination bag, and cells were irradiated by the uv-a illumination device macogenic 2 (macopharma, france). irradiated cells and autologous residual blood were reinfused back to the patient. during the whole procedure patient's vital signs were monitored. ecp procedures were performed 3 times per week for 4 weeks, followed by 2 times per week at 2 weeks intervals for 2 months. in 3 month period 28 mini-ecp procedures were performed. median of collected whole blood was 92 ml (range 52-100). median of total nucleated cell (tnc), and mononuclear cell recovery after sepax separation were 88.3% (range 66.7-104), and 90.8% (range 63-102), respectively. median of hematocrit in final irradiated product was 4% (range 3.8-6%). patient was reinfused with median of 1.0 (range 0.66-1.8) tnc × 10 8 /kg bw, and median of 4.95 (range 3.62-13.72) lymphocyte × 10 8 /kg bw. after one month of ecp together with steroids and cyclosporine, gvhd of the skin improved, and the steroids were gradually weaned, with no recurrence. gvhd of the liver showed no improvement, and other therapies had to be introduced, but without steroids. for the first time in croatia, mini-ecp was performed in a child with gvhd, in whom conventional ecp could not be used because of insufficient body weight. mnc separation using automated closed system sepax separator has proven efficient and safe. mini-ecp treatment was continued for three months, without technical difficulties. positive effect was experienced concerning the skin gvhd, but not the liver gvhd. after the first experience in our country, in future we plan to use this technique for low-weight patients or patients with contraindications for apheresis, which are in need of second-or third-line therapy for gvhd. disclosure of conflict of interest: none. gonadal failure represents one of the late effects of haematopoietic cell transplantation (hsct) with a negative impact on quality of life in young patients (pts) undergoing hsct1,2. the aim of this retrospective multicentre ebmt study was to assess gonadal function in untreated pts undergoing allogeneic hsct between 5 to 20 years (yrs) of age, after a preparative regimen with busulphan (bu) or treosulfan (treo). eighty-seven pts (32 females, 55 males) were reported from 17 out of 123 contacted ebmt centers: 26/87 (30%) received allogeneic hsct during pre-pubertal and 61/87 (70%) in pubertal phase. of the 87 pts, 76 (87.4%) received bu in myeloablative dose [25 pre-pubertal, (median age of 6.7 yrs) and 51 pubertal, (median age of 13.4 yrs)] and 11 pts (12.6%) received treo (1 in pre-pubertal and 10 in pubertal period). underlying diseases were primary immunodeficiency (34.5%), chronic myeloid leukemia (33.3%), myelodisplastic syndrome (24.1%), familial haemophagocytic lymphohistiocytosis (6.9%) and shwachman-diamond syndrome (1.1%). 17/26 of prepubertal pts (71%) developed spontaneous puberty (69.5% in the bu group and 100% in treo group). 21/28 (75%) females undergoing hsct during puberty completed their pubertal development (71.4% in bu group and 100% in treo group). none of females (4/4) with bu during pre-pubertal phase developed spontaneous menarche (sm), while 33.3 %(7/21) of females who received bu in pubertal period had sm. all females (n = 5) treated with treo during pubertal phase had sm (100%). for both conditioning regimens, the 42.8% (12/28) s358 of females treated during the puberty experienced sm. among the remaining 14 females (for 2 pts the information is missing) who did not developed sm, 13 received hrt 2.5 yrs after hsct and 5 of them had ovarian recovery after a median of 2.3 yrs from hsct (1.43-6.72). the median age at last follow up was 15.8 and 13.2 yrs in bu and treo pre-pubertal group, and 22.2 and 19.9 yrs in bu and treo pubertal group respectively. in the pubertal group, 18 females (69.5%) are still receiving hormonal replacement therapy (hrt) (16 in the bu group and 2 in treo group). 2 pts (7.4%) had spontaneous pregnancy. no problems in newborns are reported. sperm analysis was performed in 18.2% of pubertal pts (6/33) of males, and 66% (n = 4/6 treated with bu) were azoospermic (data regarding 2 pts were missing). the sperm analysis was repeated in half of the males. until now no paternity was reported. in this experience, the pubertal development in pts who received treo (n = 6) was normal, and in the bu group the majority of females (70%) had normal puberty. the rate of sm is higher (100%) in females after treo than bu (28%). the hrt is ongoing at last follow-up in 76% of females treated with bu and in 40% of those who received treo. our data suggests that treo may have a better outcome than bu in young girls receiving allogeneic hsct and larger studies are warranted. male patients require longer follow-up. prevention in patients transplanted from partially matched donors. we report the single centre experience in haploidentical sct. in years 1999-2016 in the department of pediatric bmt, oncology and hematology at wroclaw medical university, 72 children underwent sct from partially matched, haploidentical parental donors. graft manipulation in 38 patients consisted of cd34sel, in 22 patients the cd3 immunomagnetic depletion (tcd-cd3) was performed, and in 12 -tcr alpha-beta depletion (tcd-ab). we analysed the impact of type of manipulation procedure, conditioning regimen, demographic factors, and kir genotype on survival and probability of neutrophil recovery. the probability of engraftment and neutrophil recovery was 86% vs 77% in cd34sel group (p = ns). probability of 5 year overall survival in the tcd group was similar to the cd34sel group (45% vs 34%, p = ns). in the tcd patients, neither use of busulfan vs treosulfan, nor kir genotype, nor donor sex had noticeable impact on sct result and survival. patients transplanted after tcd due to non-malignant disease had higher survival probability, than those with malignancies (69% vs 28%, p = 0.04). the trm in tcd patients was reduced in comparison to cd34sel (24 vs 58%, p = 0.003). the trm after tcd resulted mostly from severe viral infections in tcd-cd3 patients. in 4/34 tcd patients spontaneous acute, skin (stage 2) gvhd was diagnosed and successfully treated. two patients received unmanipulated donor lymphocyte infusions (dli) and developed severe acute steroid-resistant (grade 4) gvhd, in one of them with fatal outcome. tcd methods are superior to cd34sel due to significant reduction in treatment related mortality. haploidentical sct after tcd can result in durable engraftment, but warrants intensive post-transplant monitoring for infectious complications and cautious approach to dli therapy. disclosure of conflict of interest: none. median of 15 days for neutrophils in both groups, 17 for platelets (23 in ptcy,12 in αβ+cd3+/cd19+depleted, p 0.005). donor chimerism was complete in 22 patients (84.6%). in αβ +cd3+/cd19+depleted group, 4 patient rejected (33.3%:1 primary and 3 secondary reject, 22, 28, 55 and 195 days after haplo, respectively) and were rescued with a second transplant. seven patients (50%) developed acute (a-) gvhd in ptcy group (grade 1-2 in 4; grade 3-4 in 3), compared to one (8.3%: grade 4) in αβ+cd19+depleted group (p 0.02). among patients at risk, 3 out of 9 in ptcy group developed chronic (c-) gvhd (33.3%:1 score-3, 1 overlap, 1 score-1), compared to 0/9 patients in αβ+cd3+/cd19+depleted group (p 0.08). the cumulative risk of cmv-reactivation was 72% and 58% in ptcy and αβ+cd3+/cd19+depleted groups, respectively (p 0.63). t-cell reconstitution was significantly different in the two groups,with a median absolute number of cd16+56 +cd3-and γδ+cd3+ higher in αβ+cd3+/cd19+depleted group on day +120 (p 0.03) and a median number of cd3 +cd8+ higher in ptcy group on day+180 (p 0.04). length of hospitalization was shorter in the αβ+cd3+/cd19+depleted group, with a median time from haplo to discharge of 23 days compared to 31 days in the ptcy group (p 0.003). some children have not donor and an urgent need to proceed to transplantation because of disease status. we reviewed the role of haploidentical transplantation in children and report our single center experience. ten children were transplanted from haploidentical family members donors (median age:12.5 years). we performed alfa beta t cell depleted transplantation in three patients and unmanipulated bone marrow transplantation with posttransplant cyclophosphamide in seven patients. the diagnosis were eight high risk leukemias (three all and five aml) and two severe aplastic anemia. patients were myeloablative conditioned with cyclophosphamide, fludarabine and total body irradiation in aplastic anemia received alfa beta t cell depleted grafts with a median cd34 cell dose of 2.9 × 10 6 /kg (range:2.6-3.2) and busulphan, cyclophosphamide in high risk leukemias received unmanipulated bone marrow grafts with posttransplant cyclophosphamide in 3rd and 5th day of posttransplant with a median cd34 cell dose of 7.4 × 10 6 /kg (range:2.38-16.1). median follow up of our patients 10 months. six of 10 patients are alive and in disease free follow up. four patients were relapsed and dead median 5.5 months of transplantation. the rate of relapse was 50 % for leukemia patients in remission and 50% for patients with active disease. myeloablative conditioning regimen followed by haploidentical bone marrow transplantation with posttransplant cyclophosphamide may be an option in high risk leukemia patients need urgent transplantation because of desease status who have not donor. table 1 . all patients received hd-cy 50 mg/kg on d+3 and d +4. cyclosporine a 3 mg/kg/d i.v., then 6 mg/kg/d p.o. adjusting for blood levels 200-400 ng/ml and mycophenolate mofetil 15 mg/kg 2 times daily po were started on d+5. mmf was discontinuated on d+35, csa-after d+100. all pts received anti-microbial prophylaxis for bacteria, fungal, herpes infection and pneumocystis according to institutional practices. analysis for donor chimerism and mrd were performed at d+30, +60, +100, +180. pts, donors and stem-cell harvest characteristics are described in table 1 . 4 pts had high risk hematological malignancies, and 1 relapsed after auto-sct neuroblastoma (hr-nb). 1 pt was transplanted in 1st cr (aml m7) and others in 2nd cr. 3 pts had full engraftment (neutrophil engraftment at 17,18 and 22 days). 1 pt (hr-nb) was concerned as a primary failure for achieving neutrophil and platelet engraftment by d+30, despite of complete donor chimerism in bone marrow. he was transplanted additionally with the same donor at d+49 after 1st transplant. 1 pt died before engraftment at d+20 (fulminant ps. aeruginosa-sepsis). 2 pts remain alive in cr (2ndcr-aml and 1st cr-m7 aml) with follow-up of 375 and 164 days (05/12/2016) without cgvhd with complete donor chimerism. 2 pts relapsed after d+100 ( were transplanted in 2nd cr-flt3 aml and 2nd cr-nb) and died. 1 pt died because of infectious complication at d+20 (transplanted in 3d cr-all). 4/5 pts had grade 2 acute gvhd. hla-haploidentical hsct with post-transplant t-cell in vivo repletion grafts by using hd-cy is feasible and effective in children with hr-haematological malignancies. [p463] who were match unrelated donor. thalassemic reconstitution occurred in three patients. acute graft-versus-host disease (gvhd) of grade ii-iv occurred in 17 % and chronic gvhd in 12%. acute and chronic gvhd were seen more frequently in patients with class 2-3 compared to class 1. mortality rate was also higher in these groups. seven patients died. one patient died on post-transplant day 26 due to intracranial bleeding. the other 6 patients with chronic gvhd died between 182 and 257 days, on average 219 days post-transplant. these data suggest that allogeneic bmt remains an important treatment option for children with beta-thalassaemia major, particularly when compliance with iron chelation is poor. the society to support children suffering from cancer, also known as mahak, was set up in 1991 as a non-governmental and non-profit organization. in the past two decades, the organization has attracted a vast public support and fulfilled a great part of its mission which is to support children with cancer, reduce the child mortality rate and create an appropriate environment that empowers families who have children with cancer. pediatric stem cell transplant also is used to treat many types of conditions affecting children and adolescent, including cancer and certain hematologic, immune reconstitution inflammatory syndrome (iris) is a clinical condition emerging after immune recovery of an immunocompromised status, mostly after the initiation antiretroviral therapy (art) in human immunodeficiency virus (hiv) infected patients, but also in several other settings, such as the recovery from the severe combined immunodeficiency (scid) status after hematopoietic stem cell transplantation (hsct). herein, we report a patient transplanted for scid who developed iris for two times, namely shortly after transplantation and after donor lymphocyte infusion (dli) ( table 1 ) in our patient, t cells passing from the donor probably contributed to the immediate post-transplant increase in the size of granulomas. this inflammatory response waned after the institution of immunosuppressive and methylprednisolone therapy. however, immunosuppressives were stopped due to lowered chimerism at follow-up, and the inflammatory response re-appeared after administering stem cell support containing a large amount of t cell from the donor for dli purpose. although the mechanism by which dli results in clinical responses is unclear, it is presumed to be a t cellmediated process. several studies have been performed to strengthen our understanding of the immunopathogenesis of iris. while some of those studies put forth t cell-associated causes, others implicated cytokines and non-t cells. the reaction that developed in our patient is suggestive of t cellassociated causes. immune reconstitution inflammatory syndrome remains a poorly understood entity. the dli procedure in our case provides a unique clue supporting a t cell mediated process. pediatric transplant teams need to be s364 aware of the previous iris phenomenon of bcg-adenitis while making the decision of dlis. [p469] disclosure of conflict of interest: none. pediatric patients treated with a hematopoietic stem cell transplantation (hsct) often suffer from late side effects caused by the treatment. the aim of this study is to investigate the late effects of a hsct on dental development. in addition, patients and parents awareness on this topic was investigated. 42 young adults treated and followed at the ghent university hospital who were under the age of 12 y at the time of hsct were examined clinically and radiographically (planmeca promax 2d). transplants (11 autologous/31 allogeneic) were done for malignant disease in 34 pts. eight patients received a hsct for a non-malignant disease. twelve patients underwent a conditioning regimen with total body irradiation (tbi), 21 patients with busulfan and 9 patients with other chemotherapeutic agents. 16 patients were o3 y, 9 patients were 3-6 years and 17 patients were 4 6 years at hsct. every patient was evaluated on dental agenesis, microdontia and rootcrown ratio. patients and their parents were asked about their knowledge and interest for dental screening at the follow-up clinic using a questionnaire. overall, the prevalence of agenesis and microdontia of one or more dental elements is respectively 51.3% and 46.2% in our study population. 76.3% of patients have a strongly aberrant root-crown ratio of at least one element. patients treated o3 years of age show significantly more microdontia (76.9%; po0.001) as well as agenesis (92.3%; p o0.001) compared to patients treated at an older age. the first premolar of the mandible is the most vulnerable element for agenesis as well as for microdontia. more microdontia is found in patients treated with a busulfan conditioning regimen compared to the other conditioning regimens (68.4% versus 25%). patients older than 6 years, treated with busulfan have statististically more microdontia compared to patients 46 y treated with tbi conditioning regimen (p = 0.044). there was no difference of the conditioning regimens on agenesis nor on root-crown ratio. almost all patients/parents find it important to receive information about the dental late effects of a hsct and are interested in dental screening at the follow-up clinic. treatment with hsct has an explicit negative impact on dental development. the degree of this effect depends on age at hsct and used conditioningregimen. dental follow-up of these patients is essential and should be incorporated in the follow-up program. disclosure of conflict of interest: none. importance of body composition in the outcome of hematopoietic stem cell transplantation in elderly patients l koch 1 , n hamerschlak 1 , r garcia 1 , c prado 1 , c silva 1 and a pereira 1 hospital israelita albert einstein the loss of muscular mass is a well recognized cause of the decline in muscle strength and functionality that accompany the aging process. in 1989, irwin rosenberg proposed the term 'sarcopenia' to describe the decline in muscle mass associated with aging. changes in body composition after hematopoietic stem cell transplantation (hsct) have been the subject of previous studies. immunosuppressive therapy and corticosteroids are known to alter skeletal muscle metabolism. infections and graft-versus-host disease (gvhd) that can occur after hsct may also affect body weight and composition. therefore, both the treatment and complications after hsct exert large negative effects on lean muscle mass, especially in elderly patients. patients with hematologic malignancies are usually well nourished before undergoing hsct. objective: the aim of this study is to determine in an elderly population whether parameters of body composition could be correlated to outcomes after hsct. we performed a retrospective longitudinal study through review of medical records of 48 patients ⩾ 60 years old undergoing hsct at hospital israelita albert s365 einstein, from 2013 to 2015, that were subject to tomography scans (cts) in a period ranging from 60 days before and 15 days after hsct. table 1 . there were no differences between groups with respect to age, gender, diagnosis, stage of disease, and source of stem cells. in ly patients, the quantity of peripheral cd34+ cell dose (×10 6 /kg) infused was different between groups (group ly-ct the incidence of nf was significantly higher in group mm-g (19 (59.4%) vs 19 (37.3%); p = 0.049). no differences were observed in the incidence and severity of mucositis, first day and duration of fever, documented bacterial infections or readmission rate between mm patients groups. this study suggests that in at home asct, the use of piperacillintazobactam prophylaxis significantly reduces the incidence of neutropenic fever and hospital readmission in patients with ly, and also that no administration of g-csf in mm patients reduces significantly the incidence of neutropenic fever. disclosure of conflict of interest: none. [p472] allogeneic stem cell transplantation (sct) has been recognized as a curative treatment for patients with wiskott-aldrich syndrome (was). in sct for was, myeloaberative conditioning (mac) has been indicated to avoid a mixed chimera. however, risk factors for a mixed chimera in patients with was who have received sct have not been evaluated. here, we analyzed the outcomes of sct and risk factors for a mixed chimera in 108 patients with was who underwent sct in japan since 1985. we reviewed medical records of 108 consecutive was patients who received sct since january 1985 who were registered with the japan society for hematopoietic cell transplantation. the age of the patients at transplantation ranged from 3 months to 23 years, and the mean age was 3.81 years. the origin of the stem cells was related bone marrow (bm) or peripheral blood stem cells (pbsc), unrelated bm or pbsc, and unrelated cord blood (cb) for 36, 41 and 27 patients, respectively. a preparative conditioning regimen consisting of mac was provided to 76 patients, and reduced-intensity conditioning was provided to 30 patients. fifty-one patients received prophylaxis against graft-versus-host disease (gvhd) with cyclosporine in combination with methotrexate (mtx) or a steroid, and 51 patients received tacrolimus (tac) with mtx or a steroid. chimerism analysis had been performed in 91 patients. neutrophil engraftment was achieved in 91 patients (82.7%). the engraftment rate was significantly higher in patients who received tac for gvhd prophylaxis, (p = 0.0001) overall survival rate was significantly higher in patients with complete chimerism than in patients with mixed chimerism (88.2 ± 6.1% and 66.7 ± 9.9%, respectively, p = 0.003), though there was no significant difference in stem cell sources. using multivariate analysis, the rate of complete chimerism in patients who received mac including cyclophosphamide (cy) at more than 200 mg/kg was significantly higher (p = 0.02) than the other conditioning. not only patients with mixed chimerism but also patients with complete chimerism were complicated with auto-immune diseases. in this study, achievement of complete chimerism after sct was important for survival in patients with was. we found that patients who underwent mac including cy at more than 200 mg/kg had a higher rate of complete chimerism. we also found a higher neutrophil engraftment rate in patients who received tac for gvhd prophylaxis. thus, mac including cy at more than 200 mg/kg and tac for gvhd prophylaxis are optimal conditions of sct for patients with was. disclosure of conflict of interest: none. adenosine deaminase (ada) deficiency is an inherited autosomal recessive immunodeficiency which represents about 10-15% of scid. since 1992 we diagnosed 29 patients affected by ada-scid: 10 underwent hematopoietic stem cell transplantation (hsct), 10 were treated with replacement therapy with peg-ada and 4 received gene therapy; 5 patients died before or after treatment. maternal t lymphocyte engraftment is frequently detected in scid patients, but this is never been found in ada deficient patients. a 3-months-old italian girl, from non-consanguineous parents, presented to our hospital with a history of frequent bronchiolitis associated with dermatitis, mycosis, hypogammaglobulinaemia, marked lymphopenia (t cells cd3, 171/mmc; cd3/cd4, 158/mmc; cd3/ cd8, 8/mmc, b cells 15.2/mmc, and nk cells, 110/mmc) and in vitro absence of proliferative response to pha. level of immunoglobulins was almost normal (igg 439 mg/dl, iga 87 mg/dl, igm 57 mg/dl). high levels of toxic metabolites were found: axp, 1.573 micromol/ml rbc; daxp, 0.629 micromol/ml rbc; %daxp, 28.5. ada activity in rbc lysates was abnormally high for scid-ada (0.54 u/g hb). molecular analysis confirmed diagnosis: the sequencing of exon 10 revealed two mutations: a missense mutation previously reported called p.ser291leu (c. 872c4t) and a new missense mutation defined p. leu298pro (c.893t4c). t-cells str analysis of patient showed 54.1% maternal t lymphocytes engraftment never reported before in ada-scid patients. the girl was transferred to the isolated bmt unit and the respiratory symptoms progressively improvement. replacement therapy with peg-ada was started immediately at a dose of 30 u/kg/twice per week. ig therapy was started at a dose of 200 mg/kg every two weeks. after three months of treatment patient showed an increase in t cells count (cd3, 411/mmc), and a decrease of toxic metabolites: axp, 1.652 micromol/ml rbc; daxp, 0.011 micromol/ml rbc; %daxp, 0.7 maternal t-cell engraftment persists, despite a good response to the peg-ada therapy. the last examination before hsct reveals maternal t-cell engraftment of 9.2%. patient underwent hsct from mud hlaidentical donor after a myelo-ablative reduced intensity conditioning regimen protocol d ebmt/esid guidelines. the number of infused cd34+ cells was 14.29 × 10 6 /kg and 69.47 × 10 6 cd3/kg. she is actually at day+108 post hsct, is doing well and shows 100% engraftment of donor cells. disclosure of conflict of interest: none. graft versus host disease (gvhd) is a frequent complication in patients undergoing haematopoietic stem cell transplantation. while the exact pathophysiology of gvhd is not known, the gut microbiome has been implicated in its development since it was shown that total gut decontamination (tgd) decreases the incidence of gvhd. with this study we aim to get insight into the diversity of the gut microbiota before, during and after total gut decontamination in comparison with selective gut decontamination (sgd). secondly, we want to identify changes in microbiota composition that relate to the occurrence of graft-versus-host disease. for this prospective cohort study we recruited 22 children (o 18y) that were eligible for a stem cell transplantation at the leiden university medical center between january and december 2015. of these, 64% (n = 14) received tgd (consisting of piperacillin/ tazobactam and oral amphotericin b), whereas the other 36% (n = 8) received selective gut decontamination with polymyxin /neomycin and oral amphotericin b. in total, 129 fecal samples were collected, weekly during admission for the stem cell transplantation and monthly thereafter up to 6 months after transplantation. also samples were collected from family stem cell donors as healthy controls. samples were processed within 24 hours and stored in the -80 freezer, after which 16s v4 amplicon sequencing (illumina hiseq, rapid mode, 250 bp read length) was applied. data analysis (taxonomy and shannon diversity) was primarily done using qiime (ref). compared to microbiota diversity in stem cell donors (mean shannon index (si) 3.43), we observed an overall lower mean si during tgd (1.90) and slightly higher mean si during sgd (2.43). microbiota diversity months after sgd (2.45) was similar to diversity during sgd (2.43), while diversity months after tgd (2.63) was higher than during tgd (1.90). further analysis of repopulation dynamics demonstrated no differences in repopulation duration after both decontamination strategies. however, we did observe differences in the type of bacteria that repopulated, with bacteroidales being more prominent in sgd and lactobacillales more prominent in tgd patients. actinomycetales (genus rothia) was exclusively present in tgd patients during decontamination. also, the clostridiales (blautia, lachnospiraceae and peptostreptococcaceae) were bacteria that appeared after the decontamination period. four patients (18%) in this cohort developed gvhd grade 1 or more. in these patients we did observe individual compositional changes of the gut microbiota at the time of ghvd diagnosis, e.g very low diversity or dominance of enterobacteriales. considerable microbiota diversity is observed in patients that received tgd. different repopulation dynamics were observed between tgd and sgd. no common pattern was found in the gvhd cases. disclosure of conflict of interest: none. minimal residual disease (mrd) pre-and post-hct for children with aml is highly predictive of event-free survival: a pediatric blood and marrow transplant consortium study d jacobsohn 1 johns hopkins all children's hospital, 16 children's hospital los angeles, usc keck school of medicine multicenter data regarding the significance of mrd in children with aml pre-and early post-hct are lacking. we hypothesized that pre-and post-hct mrd assessments using wt1 pcr combined with multi-dimensional flow cytometry (mdf) would be predictive of disease relapse and event-free survival (efs) at 2-yrs post-hct. subjects were o21 yrs with aml in morphologic cr undergoing ma allogeneic hct. stem cell sources included bm, pbsc, or cb. bm and pb samples were collected at 3 time points: baseline ( o3 weeks prior to preparative regimen); day+42 (±14 days); and day+100 (±20 days). bm samples were analyzed for both wt1 expression and mdf mrd (single reference lab using a 'difference from normal' approach without access to diagnostic phenotype); pb samples were analyzed for wt1 only. mdf detection limit was 0.02%; however, we required that 2 independent analysts certify that the abnormal population was aml. in addition, sorted mrd+ cells were tested for chimerism. wt1 positivity was defined as ⩾ 1300 copies for bm and ⩾ 200 copies for pb. results were not available to the treating clinician. 150 subjects were enrolled at 34 centers in us and canada. 20 enrolled subjects did not undergo hct and 6 were excluded for progression prior to hct or other ineligibility. in 124 eligible subjects, 2-yr efs and os were 52% and 61%, respectively. the 2-yr ci of relapse and trm were 36% and 13%, respectively. mdf identified 7 subjects pre-hct having 0.2-14% residual disease. the 2-yr relapse rate in subjects with +mrd by mdf pre-hct was 100% vs 32% (23-40%) in those who were negative. 2-yr dfs and os were 0% and 29% (4-65%) for positive mdf pre-hct, and 54% (45-63%) and 62% (53-71%) for negative mdf. pre-hct mdf sensitivity for 2-yr dfs was 10%; specificity was 100%. mdf mrd at days 42 and 100 were similarly predictive of outcome. sorted mrd+ cells from 19 post-hct samples were all noted to be of recipient origin. pb wt1 had no correlation with dfs or relapse; bm wt1 at day+100 correlated with 2-yr os (79% (68-88%) low/neg vs 57% (39-75%) high). other wt1 cutoffs studied demonstrated no correlation with outcomes. figure 1 : relapse probability by flow cytometry mrd at 3 time points. mdf mrd pre-hct and at days +42 and +100 was significantly associated with lower efs and os in children with aml undergoing hct. mdf is specific but not sensitive, as many negative mdf patients relapsed. our goal was to define a reproducible assay that did not depend on having the initial aml profile. this would facilitate multi-institutional studies aimed at decreasing relapse. given that constraint, we were able to detect clear mdf mrd in a small percentage of patients that was highly predictive and can be used in trials. wt1 level was not predictive in this multi-institutional trial. the sensitivity of flow was significantly affected by not having the initial flow available. future attempts to improve sensitivity should include initial flow and/or test higher channel flow or molecular pcr techniques. in addition, we confirmed that mrd + cells obtained by cell sorting post-hct were of recipient origin. future testing of 'suspicious' sorted cells by fish, molecular, or comparative genomic hybridization could possibly increase mfd sensitivity. novel cellular or targeted therapies should be tested in clinical trials to improve outcomes in patients with mfd mrd noted either pre-or post-hct. [p480] disclosure of conflict of interest: none. novel mutations were identified with ngs and low intensity of conditional regimen succeeded in children with fanconi anemia who received allo-hsct s hu 1 , h hou, j lu, p xiao, x bian, h liu, y hu, j ling, l li, l kong, z zhai and y yao 1 children's hospital of soochow university to explore the possiblity of applying next-generation sequencing (ngs) to diagnose the disease of fanconi anemia (fa) and evaluate the efficiency and safety of low intensity conditional regimen on children with fa receiving allogenic hematopoietic stem cells transplantation (allo-hsct). five patients initially suspected as severe aplastic anemia were diagnosed as fa by the method of next-generation sequencing (ngs)-based genetic diagnosis panel. one patient received hla-identical sibling donor hematopoietic stem cell transplantation (mrd), three patients underwent unrelated donor matched (ud) hsct, and one patient received unrelated cord blood transplantation (ucb). the conditional regimen consisted of either 300 cgy tbi or 3.2-3.6 mg/kg of busulfan with 20-40 mg/kg of cyclophosphamide. meanwhile, atg at 10 mg/kg and fludarabin at 140-180 mg/m 2 were included as well. cyclosporin or tacrolimus as well as mycophenolate mofetil (mmf) were used for the prophylaxis of graft versus host disease (gvhd). engraftment of neutrophil and platelet and complications followed transplantation such as infection, gvhd, and hemorrhagic cystitis (hc) were observed. of 5 cases diagnosed as fa by ngs, only 1 case showed the abnormality of chromosome fragility test which has been regarded as golden criteria in the diagnosis of fa. meanwhile, we found 5 novel mutations in 3 cases of fa which enriched chinese national database with data of rare diseases by ngs. the counts of mononuclear cells (mnc) were (3.87-18.57) × 10 8 /kg for non-ucb and 9.83 × 10 7 /kg for ucb. the counts of cd34+ were (4.01-9.57) × 10 6 /kg for non-ucb and 2.56 × 10 5 /kg for ucb. all 5 cases succeeded in allo-hsct with the low intensity of conditional regimen. the median time for neutrophils engraftment was 11 days (range 9~15 days), median time to platelets (plt) engraftment was 14 days (range 8~28days). one case occurred with grade i of agvhd, 2 cases with hemorrhagic cystitis. after transplantation, all patients were monitored the copies of ebv-dna and cmv-dna of whole blood, and five case with ebv positive and 3 cases with cmv positive. no patient suffered of ebv or cmv disease. the hepatic veno-occlusive disease (vod) and hc were observed in 5 fa patients after transplantation. ngs showed much more specific and facilitated for the diagnosis of fa. low intensity of conditional regimen is efficient and safe which should be recommended for the treatment of fa patients. disclosure of conflict of interest: none. outcome of alternate donor stem cell transplantation in children: an indian experience sp yadav 1,2 , n rastogi 1,2 , d thakkar 1,2 , s kohli 1,2 , s nivargi 1,2 , r misra 1 and s katewa 2 1 in india due to lack of donor registries and cord blood banks very few alternate donor stem cell transplants (sct) are performed. haploidentical sct has become feasible with availability of post-transplant cyclophosphamide (ptcy) technique. here we present our experience of setting up alternate donor program for sct for children in india and report the outcomes of the same. we collected data retrospectively of all children who underwent alternate donor sct during jan 2013 to dec 2016 in two centres. a total of 47 sct were performed for 43 children; median age 6 years (1-18 years) and 37 were boys and 6 girls. of these, 41 underwent haploidentical (35 ptcy and 6 tcr alpha-beta/cd19 depleted), 4 matched unrelated donors (mud) and 2 unrelated cord blood (ucb) sct. the diagnosis was: primary immunodeficiency-10, thalassemia major-14, sickle cell disease-3, inherited bone marrow failure-4, acquired aplastic anemia-3, acute lymphoblastic leukemia-3, acute myeloid leukemia-4, neuroblastoma-4, ewings sarcoma-1 & leukodystrophy-1. the conditioning was with fludarabine, cyclophosphamide and total body irradiation backbone in 36 children (thiotepa added in 24), fludarabine and treosulfan in 5, fludarabine and busulfan in 2, busulfan and cyclophosphamide in 4. serotherapy was part of conditioning, rabbit anti-thymoglobulin 4.5 mg/kg in 38 and campath 1 mg/kg in 9. graft vs host disease (gvhd) prophylaxis was ptcy along with tacrolimus and mycophenolate mofetil in 37 patients (35 haploidentical, 1 mud & 1 ucb) and ex-vivo tcr alpha-beta depletion in 6 and cyclosporine and methotrexate in 4. all were transplanted after a signed informed consent. a median of 8 million of cd34cells/kg was infused (range 5-24 million/kg).graft source was peripheral blood in 39 and bone marrow in 6 and ucb in 2. five children died before engraftment. the remaining 42 had neutrophil engraftment by median of 14 days (range 8-35) and platelet engraftment by median of 14 days (range 9-48). chimerism at day+100 was available in 34 cases; 31 of them had full donor hematopoiesis. one had mixed chimerism and 3 fully recipient. four children underwent a second haploidentical sct after rejection of which 2 are alive and disease free. the median follow-up of remaining patients is 9 months (range 1-40); the cumulative incidence of graft versus host disease (gvhd) acute and chronic extensive was 26% and 20% respectively. grade-iii-iv acute gvhd was seen in 3 patients. a total of 15 patients have died (sepsis-4, stroke-1, gvhd-3, vod-3, encephalitis-3 and progressive disease-1). among encephalitis deaths, one child had undergone ucb with ptcy and another tcr alpha-beta depleted second sct.; both had bk virus in the csf.there were 11/41 deaths in haploidentical (ptcy-10/35 & tcr alpha-beta-1/6), 3/4 in mud and 1/2 in ucb sct. overall survival is 68% and disease free survival is 66% at last follow up. alternate donor sct is an acceptable curative option for children lacking a matched sibling donor. haploidentical donor sct is more feasible in the setting of lack of donor registries having indian ethnicity donor. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) from an unrelated donor (ud) is largely used for pediatric patients with all in second complete remission (cr) lacking an hlaidentical sibling. in this study, we retrospectively analyzed outcome of patients (pts) given ud-hsct in centers affiliated to the associazione italiana di ematologia ed oncologia pediatrica (aieop) network between 2000 and 2013. three hundred fifty-six pts with all in second cr experiencing either bone marrow (bm), isolated extramedullary or combined relapse were included in the study; 139 were males and 217 females, median age at hsct being 4.8 years (range 0.5-19). bm, peripheral blood (pb) and cord blood (cb) were the stem cell source in 64%, 16% and 20% pts, respectively. all children received a myeloablative conditioning regimen, either tbi-(293 pts) or chemotherapy-based (63 pts). as gvhd prophylaxis, the combination of cyclosporine a, short-term mtx and atg was employed in most pts. according to the berlin-frankfurt-munster (bfm) classification of first leukemia recurrence, 59% and 42% of pts were assigned to the s1+s2 and s3 +s4 groups, respectively. level of pre-hsct minimal residual disease (mrd), measured within 30 days before hsct through flow cytometry (fcm) in the laboratory of padova, is available in 37 children; more data will be presented during the s372 meeting. with a medium follow-up of 6.5 years (range 0.5-15), the overall survival (os) was 52%, while the event-free survival (efs) was 50%. the cumulative incidence of transplant-related mortality (trm) and leukemia recurrence were 24% and 25%, respectively. the efs probability for children transplanted in the time period [2000] [2001] [2002] [2003] [2004] [2005] [2006] [2007] [2008] [2009] and 2010-2013 was 45%, 56% and 52%, respectively (p = ns). patients who received a tbi-based conditioning regimen had a significantly better outcome in comparison to children who received chemotherapy-based treatment, efs being 53% and 36%, respectively (p = 0.01). efs of pts belonging to s1+s2 and s3 +s4 groups was 66% and 35% respectively (p = 0.0001). the difference in efs is largely explained by an increased incidence of leukemia recurrence in s3+s4 (34%) compared to s1+s2 pts (23%) (p = 0.0002). efs of pts who experienced grade ii acute gvhd was 68%, while that of pts with either absent/grade i acute gvhd or grade iii-iv acute gvhd was 52% and 11%, respectively (p = 0.0001). pts with limited chronic gvhd had a better efs as compared to those with either extensive or absent chronic gvhd (77%, 35% and 61%, respectively; p = 0.039). the choice of stem cell source (bm, pbsc, cb) did not influence the probability of efs, which was 56%, 46%, 40% respectively (p = ns). importantly, among pts with evaluable mrd before hsct (n 37), the group with detectable levels 0.001% (n 9), respectively 54% and 17% (p = 0.038). conclusions. outcome of children with 2nd cr all who underwent transplant from an ud is significantly influenced by the presence of tbi in the conditioning regimen, limited severity of acute and chronic gvhd and bfm classification at time of 1st relapse. notably, mrd level before transplant, namely with a cut-off of 0.001%, influences efs. disclosure of conflict of interest: none. the median mononuclear cell dose was 5.5 × 10 8 /kg. the median time to reach absolute neutrophil count 40.5 × 10 9 /l was 13 days, and the median time to platelet count 420 × 10 9 was 16 days. grade 2 and grade 3 mucositis was seen in 61% of our patients. transplant-related mortality at 100 days not occurred. only three patients relapsed 15, 18 and 30 months after transplant (mean 21.5 m.). with a median follow-up of 39 months (4-48 months) after transplant the event free survival were 84%. only one patient had death, two years after transplantation. no significant different between cbv group vs ceam group in engraftment day. high-dose therapy with stem cell rescue can lead to durable remissions in children and adolescents with advanced hd. future investigations should focus on strategies designed to decrease relapse after auto-transplantation, particularly in patients at high risk for relapse. our analysis suggests that these regimens (ceam, cbv) are feasible in pediatric patients with acceptable engraftment and toxicity. pbsc collection may be difficult in small children owing to the large volume apheresis compared to the child's weight. various problems, such as metabolic or haemodynamic disorders may be were seen. peripheral stem cell harvest can be performed in lowweight children under safe and effective conditions even when systematic priming by blood is avoided. processing with increase of blood volume may to increase in the yield by recruiting progenitor cells. disclosure of conflict of interest: none. outcomes of children with hemophagocytic lymphohistiocytosis given allogeneic hematopoietic stem cell transplantation in italy allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment for patients with familial hemophagocytic lymphohistiocytiosis (hlh) or relapsed/ refractory hlh. we analyzed outcomes of a cohort of 109 patients (65 m, 44 f) with hlh given hsct between 2000 and 2014. median age at hsct was 2 years (range 0.4-20). genetic testing was performed for 94/109 patients (86%). mutation of prf1 was found in 31 patients (32%), of unc13d in 32 (33%), of stxbp2 in 2 (2%), of rab27a in 6 (6%), of sh2d1a in 5 (5%), of birc4 in 2 (2%) and of lyst in 1 patient (1%). no known gene abnormality was found in 15 patients who had recurrent/ refractory hlh. central nervous system (cns) involvement at diagnosis was recorded for 79 patients (72%) and was present in 30 of them (38%). the primary endpoint was event-free survival (efs), defined as the probability of being alive and in continuous complete remission (cr) at last follow-up. in order to determine efs, death from any cause, relapse or graft failure were considered events. ninety-five patients received one transplant, while 14 received more than one hsct, because of rejection in 8 patients or disease relapse in 6 (preceded by rejection in 1 case): 2 hsct were performed in 12 cases, while 3 and 4 hsct were performed in 1 case each. donor for first transplant was an hla-matched sibling for 25 patients (23%), an unrelated donor for 73 patients (67%) and a partially matched family donor for 11 patients (10%). conditioning regimen was busulfan-based for 61 patients (56%), treosulfan-based for 21 patients (20%) and fludarabine-based for 26 patients (24%). the 5-year probability of overall and efs were 71% and 60% respectively (fig. 1) . twenty-six (24%) patients died due to transplant-related causes, while 14 (13%) and 10 (9%) patients experienced graft rejection and/or relapse, respectively (see also fig. 1 ). twelve out of 14 children (86%) given a 2nd hsct after graft failure/relapse are alive and disease-free. active disease at hsct was not statistically associated with adverse outcomes, while patients had a trend for a worse outcome if the interval between diagnosis and hsct was 46 months. patients transplanted from partiallymatched family donors (pmfd) had a significantly worse efs (9%) than recipients of a matched family donor transplant (73%) or a matched unrelated donor allograft (63%, po 0.001). the main reason for the dismal efs of pmfd recipients was graft rejection, which, however, was largely rescued by a 2nd hsct. patients given peripheral blood stem cell transplantation had a lower efs probability (39%) as compared to bone marrow (60%) or cord blood recipients (76%, p = 0.0185). children given hsct o o/u46 months from diagnosis had a better efs as compared to those transplanted 46 months from diagnosis (69% vs 50%, p = 0.069). in multivariate analysis, only the use of a pmfd predicted a worse efs probability (relative risk:12.26, p = 0.0008). these data suggest that in patients with hlh allogeneic hsct is able to cure 2/3 of patients. haploidentical hsct in patients with hlh is currently associated with unsatisfactory rate of engraftment, new approaches being needed to ameliorate this outcome. active disease does not preclude the chance of benefiting from transplantation, which should be ideally performed within 6 months from diagnosis. [p485] defibrotide shows efficacy in the prevention of sinusoidal obstruction syndrome (sos) after allogeneic hematopoietic stem cell transplantation: a retrospective study on 237 patients. disclosure of conflict of interest: none. standard gvhd prophylaxis regimens impair the graft-versustumor (gvt) effect, delay immune reconstitution and are associated with high rate of infections. high-dose posttransplantation cyclophosphamide (ptcy) targets alloreactive donor t cells proliferating early after bmt, promotes regulatory t cell and permits rapid immune reconstitution. in this pilot trial we evaluate the safety and effects of ptcy in unmanipulated haploidentical and matched unrelated transplantation (mud) in pediatric patients with all. fifteen pediatric patients with high risk all underwent unmanipulated allogeneic bone marrow (bm) (n = 11) or peripheral blood stem cell (pbsc) (n = 4) transplantation followed by ptcy between april 2014 and march 2016 with a median follow-up of 24 months (7-27). eight patients were transplanted from haploidentical donors and 7 from mud. the median age was 9.5 years (range 1.9-17) and were in complete remission (cr) at the moment of bmt. in 2 patients this was a second bmt. all pts. received myeloablative conditioning regimen (treosulfan-based n = 14, tbi based n = 1) and ptcy on day +3, +4, posttransplant prophylaxis consisted of tacrolimus from day +5 (n = 5), tacrolimus/mmf (n = 8), atg (rabbit, thymoglobuline) at 5 mg/kg without other posttransplant prophylaxis(n = 2, both from mud). primary engraftment was achieved in 100% of pts., the median time to neutrophil recovery was 20 days and to platelet recovery was 22 (7-69) days. all pts. had full donor chimerism on day +30. causes of death included viral infections (n = 1); gvhd and viral infection (n = 1). cumulative incidence (ci) of acute gvhd grade ⩾ ii was 33% (95% ci: 16-68), grade iii-iv-13.3% (95% ci: 3.7-48) and chronic gvhd-21.2% (95% ci: 7.7-58.4). two-year event-free survival (efs) and overall survival (os) were 86.7% (95% ci: 69.5-100) and were equal. median time of follow-up for survivors is 2 years (range 0.7-2.3). we demonstrate that unmanipulated hsct and posttransplantation cyclophosphamide allows for high rate of engraftment with acceptable transplant-related mortality in pediatric patients with all. all major outcomes were equivalent between transplantation from unrelated and haploidentical donor. gvhd prophylaxis including ptcy was effective. event-free survival was high despite chemotherapybased conditioning in most patients. disclosure of conflict of interest: none. serotherapy with atg is frequently used in allogeneic hsct to prevent gvhd and rejection. however, the choice of the two most frequently used rabbit atg brands depends on country, disease protocol, national recommendations and/or physician's preference. atg-genzyme (atg-g, thymoglobulin) is prepared by immunizing rabbits with human thymocytes, whereas rabbits are immunized with a jurkat cell line for production of atg-fresenius (atg-f, recently named as antihuman t-lymphocyte immunoglobulin atlg, grafalon, noveii biotech). the recommended dose of both brands differs a factor 4-5. we have previously reported the pharmacokinetics/ pharmacodynamics (pkpd) of atg-g in a large cohort of pediatric hsct recipients and concluded that the clearance of the active component of atg, which is the portion of atg binding to lymphocytes, had a major impact on immune recovery post-hsct, while total atg did not. 1 both atg brands have frequently been compared according to disease outcome, without detailed analysis of composition and clearance of the active components. in the present study, we compared clearance of the active component and immune recovery after atg-g and atg-f, respectively. the serum concentrations of total and active atg were measured longitudinally after hsct in 56 children (40 atg-g, 16 atg-f), transplanted with bm or pbsc of unrelated donors for all or aml between january 2010 and june 2016 in leiden (n = 36) or copenhagen (n = 20). atg-g treated patients received a total dose of 6-10 mg/kg and atg-f was given at a total dose of 30-60 mg/kg in both cohorts administration was divided over 3-4 days. serum samples (pre-conditioning, day of hsct, +1; +2; +3; +4 and +6 weeks and +2 and +3 months after hsct) were analyzed by elisa for total atg and by quantitative flow cytometry on hut78 cells for active (lymphocyte binding) atg. lymphocyte (sub-)populations were analyzed at +1, +2 and +3 months post-hsct by flow cytometry. as reference group for immune recovery, 22 children transplanted for all or aml with an hla-identical donor and not receiving serotherapy were included. the median serum concentration of total atg at the day of hsct was 6 times higher for atg-f (atg-g 67 μg/ml, atg-f 403 μg/ ml; figure a) as the result of the higher dose of atg-f given. the active atg concentration was twice as high for atg-f (atg-g 11.1 au/ml, atg-f 23.4 au/ml figure b ). three weeks later at the expected time of engraftment, the total atg concentration was decreased with the same factor for both atg brands (atg-g from 67 to 25 μg/ml, factor 2.7; atg-f from 403 to 139 μg/ml, factor 2.9). however, the active atg concentration showed a much faster decline for atg-f (atg-g from 11.1 to 1.03 iu/ml, factor 10.8; atg-f from 23.4 to 0.23 iu/ml, factor 100). correspondingly, the number of cd3 t-cells at 1 month post-hsct was higher after atg-f than after atg-g (atg-g, atg-f and no-serotherapy 41, 169 and 386 cells/μl, respectively. figure c) . this is the first study to compare the pkpd of total and active atg-genzyme and atg-fresenius. active atg-f showed a much faster clearance than atg-g, which was associated with a significantly faster cd3 t-cell recovery at 1 month post hsct. thus, atg-f is not only quantitatively but also qualitatively very different from atg-g, which will clearly impact hsct outcomes. reduced toxicity myeloablative conditioning regimen in pediatric hematologic malignancies not associated with improved outcomes s chaudhury 1,2 , i helenowski 2 , r duerst 1,2 , wt tse 1,2 , m kletzel 1,2 , j schneiderman 1,2 and d jacobsohn 3 1 ann and robert h. lurie children's hospital of chicago; 2 northwestern university feinberg school of medicine, chicago and 3 children's national health system, washington dc allogeneic (allo) hematopoietic cell transplantation (hct) is the only curative potential therapy in refractory and relapsed pediatric leukemias. poor outcomes in allo hct are associated with treatment-related mortality (trm), mostly due to regimen-related toxicities (rrt) and graft-versus-host disease (gvhd) after myeloablative conditionings (mac), but high relapse rate with reduced-intensity or nonmyeloablative regimens. 1 to improve trm, without compromising conditioning intensity, we prospectively explored the feasibility and efficacy of a mac but reduced-toxicity conditioning (rtc) regimen, consisting of fludarabine 30 mg/m 2 /d (given first) × 5d, daily busulfan dosed to target an auc of 4000 microm*min/d × 4, ratg 1.5 mg/kg/d × 3 and 400cgy of total body irradiation in 30 patients (table 1) with hematologic malignancies. gvhd prophylaxis was cyclosporine and mmf. all patients tolerated the rtc well, with no graft failures. rrt included moderate mucositis (67%), infections (bacterial 33%, viral reactivation 63%, fungal 20%) and 3 cases of venoocclusive disease (vod). cumulative incidence d100 ⩾ gr 3 acute gvhd was 32% (95% confidence interval [ci], 16-50), extensive chronic gvhd was 3.5% (95% ci, 0.2-16). mortality at 100 days was 10.7% (95% ci 3-25), 2 due to infections with agvhd and 1 vod. with a median follow-up of 1.5 y (range, 0.6-5), the cumulative incidences of relapse at 1 years was 29% (95% ci, 14-47). mortality due to severe agvhd was 90%. overall survival (os) and progression-free survivals (pfs) for 1year was 63% (95% ci, 42-78), and 56 % (95% ci, 36-72) respectively. on univariate analysis there was no association of outcomes with donor type, graft source, disease or busulfan exposure except significantly higher cgvhd in unrelated donors, agvhd severity with peripheral blood. in summary, the use of the myeloablative rtc resulted in comparable trm, with high relapse rate was high, including in those developing chronic gvhd. this suggested a less robust graft-versusleukemia effect resulting in poor pfs and os. nonetheless, this regimen may be used as a lower-trm platform to combine with other strategies, intensive disease monitoring pre and post hct, addition of post hct maintenance therapy in combination with marrow as the stem cell source to decrease relapse or gvhd. specific immune response to vaccinations decline after hematopoetic stem cell transplantion (hsct). re-vaccination of all hsct recipients is recommended in all guidelines but bcg vaccination is not recommended due to safety concerns after hsct. mycobacterium tuberculosis can cause severe disease in children including meningitis and milliary tuberculosis (tb). the bacille-carmette-guerin (bcg) is a liveattenuated vaccine with documented efficacy against milliary disease and meningitis. routine vaccination of all infants residing in countries with high tb incidence is recommended by world health organization. however, there is no data in literature regarding its safety in post hsct setting. here, we report 34 children who underwent matched related allogeneic hsct at ankara university pediatric bone marrow transplant (bmt) unit and received bcg 24-months post-transplant. all patients were free of graft versus host disease (gvhd) and immunosuppressive therapy (ist) and had negative ppd skin test prior to vaccination. none of the recipients developed local or disseminated tuberculosis as a complication of bcg with a median follow up of 10 years. we conclude that the bcg vaccine is safe in the post hsct period when administered at least 24 months out of transplant to a selected group of patients who are free of gvhd and ist. disclosure of conflict of interest: none. single centre experience of harvesting bone marrow from donors o3 years of age r raj, r uppuluri 1 , d subburaj 1 , d jayaraman 1 , k mullanfiroze 1 , v swaminathan and l vaidhyanathan 1 1 department of paediatric blood and marrow transplantation, apollo speciality hospital harvesting bone marrow for allogeneic marrow transplantation from donors o15 kg presents special challenges. we present data on 67 sibling donors from our institution between 2006 and 2016. the mean age was 23 months with a range between 8 months to 48 months. children less than one year accounted for 25% of our donors with the youngest being 8 months of age and the smallest donor weighed 5.5 kg. all aspirations were performed from iliac crests and all donors were given general anaesthesia by a paediatric anaesthetist. irradiated blood was transfused in 97 % of the donors during the procedure. the volume of marrow obtained ranged from 5 to a maximum of 20 ml/kg donor weight. the product contained an average cd34 count of 5.5 × 10 6 /kg recipient weight with a range from 1.6 to 12 × 10 6 /kg. only on one occasion was a second harvest needed, where the donor weighed 12 kg and recipient 42 kg with major blood group incompatibility requiring red cell reduction. the yield of cd34 cells per ml of bone marrow was on average 22% higher than children above 3 years of age. all recipients showed brisk engraftment in 2 weeks. none of these donors experienced major difficulties following the aspiration procedure. thus, very young children may safely donate marrow for allogeneic transplantation and the yield of stem cells obtained is substantial. this data is particularly relevant in transplantation for haemoglobinopathies like thalassaemia major and sickle cell anaemia, where families are being counselled about a target of 15 kg for the donor in order to plan transplantation. disclosure of conflict of interest: none. sinusoidal obstruction syndrome-veno-occlusive disease in pediatric patients given either autologous or allogeneic hematopoietic stem cell transplantation (hsct). a retrospective study of the aieop-sct (italian haematology-oncology association-stem cell transplantation) group m faraci 1 , r luksch, e calore 2 , f saglio 3 , a prete 4 , mc menconi 5 , v trevisan 6 , g de simone 7 , v tintori 8 , s cesaro 9 , s santarone 10 , mg orofino 11 , e lanino 1 , m zecca 12 and a bertaina 13 sinusoidal obstruction syndrome (sos), known as venoocclusive disease (vod), is a potentially life threatening complication that can develop after hsct. although sos progressively resolves within few weeks in most patients, the severe forms result associated with multi-organ dysfunction and high mortality rate (480%). aim of this survey is to evaluate incidence and management of sos in a large cohort of children receiving either allogeneic or autologous hsct. we retrospectively reviewed pediatric hscts performed in 12 (46%) out of 26 aieop affiliated centers, between january 2000 and april 2016. new ebmt criteria have been used for the diagnosis of sos (serum total bilirubin ⩾ 2 mg/dl and 2 of the following criteria: painful hepatomegaly, weight gain 45%, and ascites) and for the classification of severity grading. 1,2 among a total number of 6208 hsct procedures (2980 autologous and 3228 allogeneic), we identified 94 (1.5%) patients with sos. this complication occurred in 37 and 55 cases after autologous and allogeneic hsct, respectively. fiftytwo pts (55%) received iv busulphan (bu) at myeloablative dose, 28 (30%) oral bu, while 14 (15%) were treated with different conditioning regimen. the median time of sos occurrence was 16 days after hsct. details about prophylaxis and therapy are reported in figure 1 . out of the 92 children, 54 (59%) fulfilled all sos-ebmt criteria. bilirubin ⩾ 2 mg/dl, gain of weight 45%, ascites, and painful hepatomegaly did not occurred in 16, 3, 4 and 2 patients, respectively. thrombocytopenia was present in 85 pts (92%), thickening of gallbladder in 63 (68%) and abnormalities of coagulation parameters in 78 (84%). according to sos ebmt severity grading, levels of transaminases were mild in 18 pts (19%), moderate in 21 (22%), severe in 13 (14.1%), and very severe in 42 (45.6%). notably, creatinine was mild in 67 pts (71%), while 5 (5.4%), 10 (10.8%), and 12 (13%) children showed moderate, severe and very severe grade of renal failure. thirty-three pts (36%) had respiratory failure, and 28 (85%) of them experienced right pleural effusion. six out of the 25 patients who developed acute kidney injury, required dialysis. severe encephalopathy occurred in 8 pts (8.6%) and 22 (24%) out of the 92 pts evaluated, were admitted in intensive care unit. as therapy of sos, 69 pts received defibrotideâ (df); the dosage was 25 mg/ kg/day in 63% of them. the median duration of df treatment was 15.5 days (range 4-104). thirty-three (35%) pts received methylprednisolone (median dose of 2 mg/kg). fifteen pts (16.3%) died due to mof (2 in moderate, 6 in severe, and 7 in very severe group) at a median time of 6 days from sos diagnosis (range 3-75 gg). our multicenter survey showed that, at least in our experience, there is a significant variability in the management approaches to sos/vod in children, while, diagnostic evaluations are more homogeneous. interestingly, in our cohort, the increase of bilirubin may be an absent criteria, while thrombocytopenia and abnormalities of coagulation parameters are more frequent. as expected, mof occurred mostly in patients experiencing severe sos. df represents first strategy to treat sos in the majority of patients, even if steroids and ursodeoxycholic acid are still used. the hyper-ige syndromes are characterized by marked elevations in plasma ige levels and eosinophilia with impairment in t cells which clinically results in combined immune deficiency. dock8 deficiency, the autosomal recessive form, brings about allergic/atopic manifestations and unusual susceptibility to infections with herpesvirus family members (herpes simplex virus, human papilloma virus) and molluscum contagiosum. symptoms in patients with dock8 deficiency typically emerge during childhood, and the majority results in death because of infections and malignancy by the third decade. hematopoietic stem cell transplantation (hsct) is now considered a standard of care for dock8 deficiency when an appropriate donor is available. in this study, we present the 5 unrelated hsct results of 4 children with dock8 mutation. the demographic and clinical data of the 4 patients with 5 transplantations studied are shown in table 1 . hsct was administered between august 2013 and august 2015 at bahçeşehir university medical park antalya hospital and the clinical data of the hscts are presented in table 2 . all patients were transplanted from unrelated donors with bone marrow, except one with cord blood. the cord blood transplantation´s regimen was non-myeloablative which resulted with rejection. despite existence of serious morbid problems before transplantation, all the patients engrafted successfully. majority of the complications mentioned in the table 2 were improved and they are in the follow-up in an outpatient basis. discussion dock8 deficiency has high mortality, and hsct should be considered as early as possible before development of significant organ damage. despite myeloablative conditioning and high morbidity before the transplantation, survival was very good in our patients. myeloablative and nonmyeloablative transplants have been performed from related and unrelated donors and have reported successful results even without the preparative regimen. in our center, all transplants performed from unrelated donors by myeloablative regimen have been successful but have resulted in transplant rejection with cord blood transplantation after nonmyeloablative regimen. in all of our patients, stable full chimerism has been detected, however mixed chimerism have also been shown to be useful in several reports. whether hsct also cures the autoimmune complications and reduces the risk of cancers is as yet undetermined. however, a myeloablative conditioning regimen followed by allogeneic hematopoietic stem cell transplantation from unrelated donors in dock8 deficiency results in improvement of the clinical phenotype with a low incidence of regimen-related toxicity. disclosure of conflict of interest: none. successful bone marrow transplantation after myeloablative conditioning in a child with ipex syndrome b kuşkonmaz 1 , d ayvaz 2 , mh abur 3 , fv okur 1 , g karagüzel 4 , f orhan 5 , i̇ tezcan 2 and du çetinkaya 1 immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome is a rare disorder. although most patients present in infancy with a clinical triad of intractable diarrhea, insulin-dependent diabetes, and eczematous dermatitis, some patients present with severe food allergies and other autoimmune manifestations. the disease is caused by mutations in the forkhead box p3(foxp3) gene, a transcription factor that is essential for the development and function of regulatory t (treg) cells. this cells plays an essential role in controlling immune responses and preventing autoimmunity. patients usually die in the first years of life without treatment. the only effective cure is hematopoietic stem cell transplantation (hsct). here we report a patient with ipex syndrome who underwent hsct after myeloablative conditioning. 9 months of age boy with the history of diarrhea, insulin-dependent diabetes, eczematous dermatitis, pneumonia, coombs positive hemolytic anemia, referred to our hospital for investigation of immunodeficiency. on admission physical examination showed eczematous skin rash, submandibular lymphadenopathy, hepatosplenomegaly. before hsct the patients treated with immunosuppressive agents including methylprednisolone, mycophenolate mofetil and monthly intravenous immunoglobulin. complete blood count revealed anemia (hb: 7.7 g/dl), and eosinophilia (1900/mm 3 ). serum immunoglobulins were: ig g: 1550 mg/dl (463-1006), igm: 172 mg/dl (46-159), iga: 60.9 mg/dl (17-69), ige :1538 iu/ml. lymphocyte subset analysis showed cd3 64%, cd4 22%, cd8 40%, cd16+56 13%, cd19 19%. foxp3 gene analysis showed c.748_750delaag mutation. at the age of 1 year, patient underwent hsct from his hla matched sibling. myeloablative conditioning regimen including busulfan (20.4 mg/kg) and fludarabine (160 mg/m 2 ) was given to the patient. cyclosporine a and methotrexate (day +1, day +3, day +6) were used as graft versus host disease prophylaxis. bone marrow was used as the stem cell source and the number of cd34+ cells was 4.5 × 10 6 /kg. neutrophil and platelet engraftment were achieved on day +13 and +35 [p493] s378 respectively. acute and chronic gvhd were not observed, but patient developed veno-occlusive disease treated with defibrotide, sepsis treated with broad spectrum antibiotics. chimerism analysis showed %98 donor profile at the third month of hsct. after hsct, autoimmune hemolytic anemia, eczematous dermatitis, food allergies, diarrhea and type 1 diabetes resolved completely within two months after hsct. now the patient is in good clinical condition without any symptoms 5 months after hsct. early hsct provides better outcome in patients with ipex, before the organ damage due to autoimmunity and/or adverse effects of immunosuppressive therapy. myeloablative conditioning is associated with substantial transplantation-related mortality whereas nonmyeloablative conditioning carries an increased risk of rejection because of dysregulated effector t-cell function. in this patients, myeloablative conditioning was preferred because of the risk of rejection. although the required levels of donor chimerism and conditioning intensity are unknown, engraftment of donor treg cells seems to be sufficient to control the disease. the patient is well without any symptoms of ipex after hsct with full donor chimerism. disclosure of conflict of interest: none. interferon gamma receptor 1 deficiency (ifnr1) is a rare autosomal recessive immune deficiency disorder associated with very poor outcome secondary to severe and disseminated mycobacterial infections. hematopoietic stem cell transplantation (hsct) has been proposed as a curative option. however, hsct for these patients is particularly difficult owing to a high rate of graft rejection. the use of a non t-cell depleted transplant from an hla-identical sibling and fully myeloablative conditioning regimen has been shown to have improved outcomes. we report the first successful hsct with a t-depleted haplo-identical donor, performed in a girl with severe ifnr1 deficiency. we reviewed the medical chart of a 2-year-old hispanic girl with ifnr1 deficiency who was diagnosed at birth, since her brother had previously been diagnosed with the same complete ifnr1 deficiency. they were found to have a novel mutation variant detected at c.201-1g4t. as expected with this disorder, she developed disseminated infection with mycobacterium abscessus infection at 2 months of age and was subsequently found to have mycobacterium abscessus osteomyelitis. she was treated with multiple antibiotics including: amikacin, linezolid, meropenem and clarithromycin while tigecycline was added a few weeks prior to admission for hsct. she was continued on this therapy until day + 30 following which antimicrobials were gradually weaned off. she was enrolled on the bp-004 trial, a multicenter, prospective phase i-ii trial (enrolling both malignant and non-malignant diseases) evaluating αβtcr +/cd19+ depleted haplo-transplantation followed by administration of bpx-501 t cells containing the ic9 suicide gene, (clinicaltrials.gov nct02065869). her conditioning regimen included busulfan (4 mg/kg/day for 4 days) and cyclophosphamide (50 mg/kg/day for 4 days). fludaragbne, tli (900 cgy) . gvh prophylaxis with atg/rituximab. the patient received a graft with: tnc-9.98 × 10 8 cells/kg, cd34+ cells-16 × 10 6 cells/kg, and αβtcr+ t cell content of 4.78 × 10 4 cells/kg. as per protocol, since the αβ tcr+ t cells in the product was below threshold of 1 × 10 5 cells/kg, she did not receive any post-transplant immune suppression. bone marrow recovery occurred at day +10 with anc 4500/mm 3 and platelet recovery at day +18. full engraftment with 100% donor chimerism based on cytogenetic analysis was observed at day +28 after transplantation and has remained stable. she is currently 14 months post-transplant, and has done well without major complications and or signs of mycobacterial infection. there is limited data in patients receiving hsct for ifnr1 deficiency with very poor outcomes either relating to graft failure, transplant complications and progressive mycobacterial infection. to our knowledge, this is the first patient with ifnr1 deficiency transplanted successfully with a haploidentical donor and alive without any active mycobacterial infection. this report suggests that using a highly immunopotent graft depleted of only αβtcr+ t cells while retaining other immune effectors might offer a potential strategy to engraft these high risk patients using haplo-identical donors thereby allowing access to virtually all patients in need. disclosure of conflict of interest: none. tandem autologous stem cell transplantations for high risk pediatric embryonal central nervous system tumors: a single center experience k rosenfeld 1 , r dvir 1 , s constantini, j roth 2 , s edelman 1 , a tal 1 , d levin 1 , m manisterski 1 , s achituv 1 and r elhasid 1,3 1 department of pediatric hematology-oncology, tel aviv medical center; 2 department of pediatric neurosurgery, tel aviv medical center and 3 sackler faculty of medicine, tel aviv university pediatric embryonal central nervous system tumors are highly malignant tumors, which tend to disseminate through the cerebrospinal fluid to the brain and spinal cord and include: medulloblastoma, pinealoblastoma and primitive neuroectodermal tumors (pnets). the recommended treatment for these tumors is a complete surgical excision, craniospinal radiation and chemotherapy. the use of high dose chemotherapy with tandem autologous hematopoietic stem cell transplantation (hsct) has been advocated for high risk patients, and infants who could not be irradiated. between july 2010 and november 2016, 16 pediatric patients (11 males, 5 females) suffering from high risk medulloblastoma, pnet or pinealoblastoma underwent tandem autologous hsct. they were treated according to two protocols: group a consisted of ten patients with median age of 8.2 years (range 3.9-15.5 years) received the st jude sjmb03 protocol, while group b consisted of six patients with median age of 2.1 years (range 1.4-3.5 years) who received the children's oncology group -acns0334 protocol. all patients engrafted with median time for neutrophil engraftment of 11 days (range 7-14 days) and for platelets engraftment (420 000) of 13 days (range 13-24 days). median follow-up was 3.5 years (range 1 week-6 years). neurological toxicity: two group a patients had convulsions episodes, one occurred during infusion of cryopreserved stem cells, and the other was a result of progressive disease during the last course of hsct. gastrointestinal toxicity: seven patients required total parenteral nutrition due to mucositis. diarrhea occurred in seven patients, two of them were diagnosed with rota virus and two with clostridium difficile. infectious complications: all patients suffered from at least one episode of neutropenic fever which was treated with broad spectrum antibiotics. there were 7 documented bacteremia in 6 patients. (1 klebsiella pneumonia, 1 proteus mirabilis, 3 staphylococcus aureus, 1 streptococcus viridans and 1 staphylococcus epidermidis). metabolic complications: four patients in group a developed reversible syndrome of inappropriate anti-diuretic hormone secretion (siadh) during chemotherapy, and all group a patients developed hypomagnezemia. four patients died, one due to progressive disease, one due to early relapse 3 months post treatment, one due to late relapse 5 years post treatment and one due to sepsis 4 months post treatment. another patient relapsed 1.5 years s379 post treatment, underwent surgery and radiotherapy and is now 3 years post therapy. late effects: four group a patients developed endocrinological sequelae at a median of 20 months (range 17-21 months) and require hormone replacement therapy. tandem autologous hsct is a feasible treatment for pediatric high risk embryonal tumors, with good engraftment and acceptable toxicities using sjmb03 and acns0334 protocols, with overall survival of 75%. long follow-up is needed in order to diagnose and treat late effects. disclosure of conflict of interest: none. the diagnostic role of liver stiffness measurement in predicting hepatic veno-occlusive disease (vod) in pediatric hematopoietic stem cell transplantation (hsct) k kleinschmidt 1 , f ravaioli 2 , r rondelli 1 , g marasco 2 , r masetti 1 , a prete 1 , a colecchia 2 , d festi 2 and a pession 1 1 pediatric oncology and hematology unit, department of pediatrics, university of bologna, sant 'orsola-malpighi hospital and 2 department of medical and surgical sciences, university of bologna vod is a potentially life-threatening complication associated with hsct in which immediate therapeutic action is crucial for patients' outcome. liver stiffness measurement (lsm) using fibroscan represents a non-invasive method to detect the grade of liver fibrosis and portal hypertension as in case of vod. to evaluate the predictive potential of lsm in pediatric patients (pts) at risk for developing vod, a prospective, ongoing, single-center study has been performed at the university hospital of bologna. lsm was performed by using the fibroscan device, which consists of a 3.5 mhz ultrasound transducer probe that transmits low-frequency vibrations (50 hz) to the liver tissue. the propagation velocity is proportional to the stiffness (elasticity) of tissue. lsm will obtain pathological high values (47.5 kpa) when the tissue is altered like in liver fibrosis, or post-sinusoidal portal hypertension. from november 2014 -september 2016, 25 pediatric pts (18 male, 7 female), aged 3-20 years (mean 11.7), affected by hemato-oncologic disease, eligible to allogeneic (22) or autologous (3) sct conditioned with busulfan-based chemotherapy, were enrolled. pts were scheduled for 4 study examinations with lsm: at t0 (baseline) before chemotherapy, t1 (day 7-10 after sct), t2 (day 17-20) and t3 (day 27-30). the diagnosis of vod was defined according to modified seattle/baltimore criteria. twenty-five pts were enrolled in the protocol, 22 of which were evaluable for the study (pts characteristics table 1 ). 4 out of 22 pts (18%) developed vod. the cumulative incidence (se) of vod in our setting was 19 % (8.6). baseline lsm values on t0 of all pts were normal (7.5 kpa at t2 (p = 0.002) and t3 (p = 0.004). from our observations, an anticipating pattern of pathological lsm in presence of clinical and laboratory parameters within normal ranges in patients who develop vod can be derived. preliminary data indicate a high predictive potential of lsm in the diagnosis of vod, however the number of cases is not sufficiently representative to draw definitive conclusions. to optimize the predictive potential of the method, more frequent (daily) measurement in the critical time frame are currently investigated. [p497] all= acute lymphoblastic leukemia, aml= acute myeloid leukemia, bu= busulfan, treo=treosulfan, fluda= fludarabine. disclosure of conflict of interest: none. [p497] the exact role of extra-corporeal photopheresis in children with gvhd: an unanswered question ss anak, h bilgen 1,2,3,4,5,6,7 , y yaman, et saribeyoglu, k ozdilli, v hazar, m elli, am kokrek, h hizli and k payalan ecp continues to be a controversial treatment, probably due to the mechanism of action not being identified, the varying photopheresis procedures and treatment schedules, and the difficulty of conducting trials on relatively rare diseases with involvement of clinically heterogeneous organs. ecp was performed in our pediatric transplant center to 8 patients mean age of 12 years ( 4-18) diagnosed to have all ( 3 pts), thalassemia ( 2 pts), aplastic anemia (1), blacfan diamond (1), refractory hodgkin disease (1) following our internal protocol for 152 ecp sessions. five of the patients had mud, 3 had hla id sibling transplants. chronic gvhd was diagnosed in 2 of the patients 6 had acute gvhd. skin was involved in all the patients, liver in 6 of the patients, lung in 3, gut in 6 and mucous membranes in 7 patients. the ecp treatment consisted essentially of three steps: (1) collection of mncs from the patient, (2) processing of mnc buffy coat, and (3) return of mncs to the patient. collection was performed using a cell separator (haemonetics mcs plus), processing two blood volumes. our protocol provides for a maximum final mnc volume to be collected at 150 ml, with a hematocrit (hct) value below 5%. the maximum procedure time was set at 180 min. the mncs collected were adjusted to a constant volume of 300 ml by the addition of saline and 3 ml of 8-mop in aqueous solution, to always obtain a final concentration of the drug of 200 ng/ml. the diluted buffy coat was transferred into a special uv-a-permeable bag (pit-kit medtech solutions), and uv-a radiation at 2 j/cm 2 was performed (uva-pit irradiator). the photoactivated mncs were returned to the patient within 30 minutes using a blood transfusion set. during ecp procedure, patients' vital signs were monitored. anticoagulation consisted in acidcitrate-dextrose formula a set at a variable ratio (1:14-1:20) according to the patient's characteristics (clinical conditions, body weight, coagulation values) and platelet (plt) count. prophylaxis of hypocalcemia consisted of the administration of calcium gluconate (5 ml diluted in 5-10 ml saline) every 30 to 45 minutes. all procedure related side effects were recorded. during the reinfusion and postreinfusion phases, the patients were monitored for fever, chills, headache, rash, erythema, urticaria, itching and edema. no serious complication was detected. all the patients had also steroids, 4 had concurrent mesenchimal stem cells. ecp was applied on 2 consecutive days every 2-4 weeks which is continued for approximately 6 months followed by a maintenance schedule tapered to an every 2-to 4-weeks. the mean session cycle was 19 ( 6-51) between february 2015 to november 2016 . the most commonly involved organ was the skin which demonstrated a response rate of 75%, followed by liver (66%), lung (25%), gut (18%) and mucous membranes (68%) the concurrent immunosuppression could be reduced during ecp therapy, and no increase in opportunistic infections was detected. 1/8 patient died after a relapse, 7/8 are alive with chronic mild gvhd. however, despite our good response rates, our understanding of ecp remains limited. patients who suffer from acute and chronic gvhd have limited treatment options. ecp remains an important therapeutic option. future basic, translational, and clinical research studies will provide a better understanding of its mechanism of action and optimize its therapeutic potential. disclosure of conflict of interest: none. tolerability and responses to ex vivo il2 activated nk cells from haploidentical parental donors in paediatric patients with refractory leukaemia/lymphoma pl tan 1 prognosis for patients with refractory leukaemia/lymphoma ineligible for transplants and those who relapse posttransplant is poor. in adult settings, adoptive transfers of ex vivo il2 activated natural killer ('ank') cells from nk alloreactive donors, especially for nk sensitive cancers, has been successful in bridging patients to curative transplants.(1) this approach has not been reported in paediatric patients. we report our experience in 8 consecutive patients, of median age 9 (range, 1-15) years, with refractory leukaemia/ lymphoma (aml, 2; all, 2; mixed phenotype acute leukaemia, 2; lymphoma, 2) who received 9 treatments with 'ank' from haploidentical parental donors on institutional protocol, between aug 2012 and 2016. parents/legal guardians/patients provided informed consents as per institutional guidelines for donors and patients procedures. donor lymphocytes harvested at steady state were cd3 depleted followed by overnight culture in il2 before being infused into patients lymphodepleted with fludarabine and cyclophosphamide. additional rituximab were given to 3 patients and another received tbi 2 gy. subcutaneous il2 injections at doses 1-3 mu/m 2 /dose started on d-1 and were planned for 6 doses, as tolerated. nk alloreactive donors (kir-ligand mismatch) and kir b/x genotype were available to all except 2 patients. two patients were treated for post-transplant relapse; 1 of whom also received 'ank' pre-transplant; other 6 patients had failed best conventional therapy including cd19/cd3 bispecific t cell engager (blinatumomab) in 1. lymphodepletion was well tolerated. a median tnc and cd56+ dose of 9.8 (range, 2.9 to 38) × 10 7 /kg and 1.8 (range, 1.2-18) × 107/kg, respectively were administered. cytokine release syndrome (crs) was observed in 8 of 9 treatments (6 grade 1, 1 grade 3, 1 grade 4). the patient with dock8 deficiency, disseminated ebv+ cerebral lymphoma had grade 4 crs and robust tumour lysis syndrome but succumbed to neurotoxicity. of the 9 treatments, there were 7 responses, including the 2 given posttransplant. excluding the 2 treatments given post-transplant and 2 non-responders, median peak donor chimerism was 93% (range, 7-100%) occurring at a median of 12 (range, 7-22) days. five patients (4 responders, 1 non-responder) proceeded to transplants at a median of 43 (range, 35-96) days after 'ank.' responders had longer survival time compared to nonresponders (median 314 vs 88 days). two responders (25%) achieved sustained minimal residual disease (mrd) remission after transplants and are alive 138 and 380 days from 'ank.' five eventually died of their primary leukaemia/lymphoma; 1 from crs. our preliminary experience in a small cohort of 8 paediatric patients with refractory leukaemia/ lymphoma showed that adoptive transfers of ex vivo il2 activated nk cells from haploidentical parental donors were tolerable; with responses seen in 75% of patients; and 25% achieving prolonged mrd remissions after transplants. patients with cerebral diseases might be at increased risks of neurotoxicity with this approach; and care must be taken in patient selection and the design of the lymphodepletion therapy. alternative donor choices are limited in multi-racial, multiethnic societies with small families such as singapore. unrelated cord blood transplant provides a feasible alternative to patients lacking adult stem cell donors in children with primary immunodeficiency diseases. method: we describe our experience using unrelated cord blood transplant (ucbt) for 9 children with pid from august 2005 to november 2014. during this period we performed hsct for 12 children with pid: 9 with unrelated cord blood (75%); 2 with msd and i mud. out of 9 cases of ucbt there were 5 severe combined immunodeficency (scid), 2 chronic granulomatous disease (gcd), 1 hyperigm syndrome and 1 wiskott aldrich syndrome (was). the median age of transplant was 13.7 months (range 1.3 to 83.3 months). all presented with multiple infections ranging from disseminated bcg infection to parainfluenza /rsv /rotavirus infection to pseudomonas sepsis, staphylococcal endocarditis to pulmonary aspergillosis for scid. hyper igm presented with pnemocystitis carini pneumonia while cgd conditions presented with perianal abscess and fungal pneumonia. the child with was had life threatening git bleeding and a hemorrrhage trachaebronchial cast removed after a failed initial extubation for gastroscopy. conditioning regimes consisted of reduced intensive (fludarabine based) conditioning regime for scid and myeloablative regime for the rest. the median tnc dose was 12.7 × 10(7)/kg (range 4.2 to22.5) and median cd34+ cells dose was 3.68 × 10(5)/kg (range 1 to 8.9). results: all engrafted well except for one graft failure in cgd. he refused 2nd transplant and died 1.5 years post transplant from fungal pneumonia. median engraftment time for neutrophil was 21 days (range 13 to 33) and platelet was 30 days (range 18 to 65 days). grade 1 skin aghvd occurred in one patient while another patient died of agvhd of liver and lungs. chronic gvhd was found skin and liver in one patient. trm was 11 % (due to agvhd). median follow up was 1255 days (ranged 327 to 4109). overall 5 years survival was 78%. post-transplant complication with life threatening puemonitis was not uncommon. one patient developed biopsy -proven idiopathic interstitial pneumonitis and required ecmo for one month. he received immunosupressive drugs including methylprednisolone, infliximab, oral imatinib (tk inhibitor), azithromycin and nebulised becotide. he was weaned off oxygen after 2-3 months. conclusion: our limited experience showed unrelated cord blood is good source of stem cell for transplant in pid in a multiracial population. one case of graft failure was likely due too low cell dose cd34 +cells dose 1 × 10(5)/kg. the expertise in icu has enabled us to support several patients who presented with infective pneumonia pre-transplant and post -transplant. with better technology like alpha/beta depletion haploidentical transplant may be a better option to achieve engraftment earlier so as to avoid stormy post-transplant infections seen in unrelated cord blood setting. disclosure of conflict of interest: none. in spite of these recommendations, literature from developing countries suggest that pbscs are used more and more frequently without compromising the transplant results, as they seem to be preferred graft source for donors in many countries incl. poland. therefore we analyzed the efficacy of mud-hsct in children with saa transplanted in our centre. clinical data of 44 saa and pnh children and adolescents (27 boys and 17 girls), who underwent mud-hsct between october 2000 and july 2016 were retrospectively analyzed. the median age was 11.2 years (range 0.7-20 years) according to the graft source, the patients were divided into pbsct group (37 patients) and bm group (7 patients). four patients required second mud transplant due to graft rejection. overall survival for all patients was 66 %. estimated 5-year overall survival (os) was not statistically different between pbsct group and bm group [(68% vs 54% ) p = 0.42]. there was no significant difference in os between group who had ist before transplant and the group, who had an upfront transplant as a first line of therapy [65% vs 62%, p = 0.79].the time to neutrophil and platelet engraftment was statistically longer in bm group than in pbsc group [(anc 16 vs 15 days, plt 29 vs 16 days, respectively) p = 0.017]. the incidence of grade iii-iv acute graft-versus-host disease (gvhd) in pbsct group was similar to that in bm group [37% (14/37) vs 29% (2/7)]. the incidence of chronic gvhd in pbsct group was similar to that in bmt group [8% (3/37 ) vs 14% (1/7)]. other transplantrelated complications like heart failure, central nervous bleeding, incidence of infections were comparable within the two regimens. there were 15 deaths in the whole group. the main reason of death were infectious complications or multiorgan failure (mof) in severely pretransfused patients in this historical cohort of patients. unrelated donor pbsct in children and adolescents with saa seems to be not inferior to unrelated donor bmt. the incidence of chronic gvhd was surprisingly low in saa recipients of mud pbsc. increased morbidity and mortality due to infections was due to individual poor clinical situation of patients before transplant (i.e. fungal infections, contamination with resistant bacteria, prolonged neutropenia). disclosure of conflict of interest: none. dyskeratosis congenita (dc) is characterized by the clinical triad of reticular skin pigmentation, nail dystrophy, and oral leukoplakia. the majority of patients with dc develop bone marrow failure (bmf), which is the main cause of death in dc patients. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment for bmf associated with dc. transplant-related morbidity/mortality is common, especially after myeloablative conditioning regimens. hsct has been introduced into the management of dc, which has had remarkable clinical results. we report our experience in 4 children with dc who underwent allogeneic transplantation at a single medical center. patients received a fludarabine-based reduced intensity conditioning (ric), and the graft source was unrelated peripheral blood stem cells. median age at the time of hsct was 5.5 years (range, 4-13 years). the numbers of infused mononuclear cells and cd34+ cells were 15.62 ± 3.04 × 10 8 /kg and 5.80 ± 3.37 × 10 6 /kg, respectively. the median time of neutrophil and platelet recovery were 13.5 days (range, 12-17 days) and 21.5 days (range, 19-26 days). two patients experienced grade ii-iii acute graftversus-host disease (gvhd), and chronic gvhd was only observed in one patient. all four patients remained alive and transfusion independent at the median follow-up of 18.5 months (range, 9-31 months). correction of previously existing physical defects was observed in two patients. unrelated peripheral blood hsct can be a curative option for dc. ric based on the type of disease is important to s382 achieve successful hsct. a larger sample size and extended follow-up of this rare patient population are needed to determine whether the changes in therapy will improve longterm survival. disclosure of conflict of interest: none. autosomal recessive hyper-ige syndrome due to dock8 mutation is a combined primary immunodeficiency, characterized by severe eczema, recurrent infections, and susceptibility to autoimmunity, malignancy, and multiple allergies, in addition to unusual high serum ige level. dock8 patients tend to have a progressive severe clinical course with mostly fatal outcome during second to third decade of life without hematopoietic stem cell transplantation (hsct). in our center we have a large number of dock8 patients. during a period of 11 years (2006-2016), we transplanted 14 patients with documented dock-8 mutation confirmed by molecular genetics. one patient did not receive any conditioning because of poor clinical condition and he died from severe cutaneous and gut gvhd and another patient received cbt with bu/flu with zero engraftment. the rest of the patients received hsct from hla full matched donor with chemoablation with bu/cy for all with 100% lymphoid and myeloid engraftment (str). among those patients who received chemoablation, gvhd developed in 6 patients mostly grade i and ii. in addition 3 patients died: one died of severe gvhd and the other two died of sepsis. for dock8 patients we highly recommend early hsct if fully matched donor is available to prevent the high mortality associated with the disease. alloreactivity triggered by interactions between killer cell immunoglobulin-like receptors (kir) and natural killer (nk) cells plays a role in graft-versus-tumor (gvt) effects after hematopoietic stem cell transplantations (sct). in particular, kir-ligand mismatching between the donor and recipient might promote nk cell alloreactivity after unrelated cord blood transplantations (ucbt) in adult patients with acute myeloid leukemia (aml). recently, it has been suggested that allogeneic nk cells could be the effector cells that mediate gvt effects after mismatched allogeneic transplants for refractory childhood solid tumors. however, there are few reports about the efficacy of kir-ligand mismatched sct in pediatric cases. here, we report the excellent outcomes of kirligand mismatched cbt (kir-cbt) in pediatric patients with refractory malignant disease. we evaluated the cases of 9 pediatric hematology and oncology patients [4 (44%) aml, 1 (12%) myelodysplastic syndrome [mds] , and 4 (44%) neuroblastoma [nbl] patients] who underwent kir-cbt between 2010 and 2016 at our institution. among the 4 aml cases, one involved refractory disease (induction failure), and the other three involved relapsed aml (one patient relapsed after the 1st sct because of 5q-). all 4 nbl patients underwent kir-cbt followed by auto-peripheral blood stem cell transplantation (pbsct) because of stage 4 disease. the mds patient underwent kir-cbt because of refractory anemia with excess blasts. kir mismatching was defined as incompatibility between the donor kir and recipient kir ligand, and only inhibitory kir that interacted with human leukocyte antigen (hla)-bw4, -c1, or -c2 group ligands were considered. the median age of the patients was 4 (range 2-18) years. all 4 of the aml patients were in complete remission (cr) at the time of the hsct (cr1 = one case, cr2 = 3 cases). the mds patient was in a non-cr state, and all of the nbl patients were in their 1st cr at the time of the hsct. the aml patients received total body irradiation (tbi)-based conditioning (12 gy tbi and 120 mg/kg cyclophosphamide [cy]), and the mds patient received busulfan (bu)-based conditioning (19.2 mg/kg bu and 120 mg/kg cy). the nbl patients received reducedintensity conditioning regimens (125 mg/m 2 fludarabine, 140 mg/m 2 l-pam, and 2 gy tbi). the cb exhibited hla 2-4 locus mismatches (dna typing), including at least one inhibitory kir gene mismatch. the prophylaxis for graftversus-host disease (gvhd) consisted of tacrolimus and shortterm methotrexate. anti-thymocyte globulin (atg) was not used as a gvhd prophylaxis in any case. after the median follow-up period of 25 months (range: 4 -54 months), all 9 patients were alive, and none of them had relapsed after the kir-cbt. although grade ii-iv gvhd was observed in 6 patients (67%), it was controlled with prednisolone. chronic gvhd was not seen in any case. the present findings suggested that nk cell alloreactivity plays a role in preventing childhood myeloid leukemia and nbl relapse after kir-cbt. although our results are limited, this report provides novel data to support further investigations into the use of kir-cbt for the treatment of pediatric refractory malignant disease. disclosure of conflict of interest: none. impact of fcm-based minimal residual disease on transplant outcomes in patients with aml in hematological complete remission t oka, j kanda 1 , k ohmori 2 , m hishizawa 3 , t kitano 4 , t kondo 5 , k yamashita 6 it is reported that the presence of minimal residual disease (mrd) before hematopoietic stem cell transplantation (hsct) is associated with poor overall survival in patients with acute myelogenous leukemia (aml) in hematological complete remission (cr). we retrospectively analyzed the association between flowcytometry (fcm)-based detection of mrd and transplant outcomes. we included 56 adult patients with aml in hematological cr, who underwent their first allogeneic hsct between april 2005 and may 2015 at kyoto university hospital. mrd of bone marrow before hsct was measured using fcm. to search for target antigens to detect mrd, threecolor fcm analyses were performed using a differential panel for every disease and patient, which allowed us to detect ⩾ 0.1% of mrd. of the 56 patients (median age: 48.5, range: 18-66), 41 patients were included in the mrd-negative group (mrd o0.1%), whereas 15 were included in the mrd-positive group (mrd ⩾ 0.1%). in the latter group, 6 patients were included in the mrd-low group (mrd o0.6%), and 9 were included in the mrd-high group (mrd ⩾ 0.6%). there was no significant difference in the patient background between the mrd-negative and mrd-positive groups. the 3-year overall survival rates for the mrd-negative, mrd-low, and mrd-high groups were 82%, 63%, and 30%, respectively (p = 0.007, figure 1 ). in a multiple regression analysis, the mrd-high group was significantly associated with higher overall mortality than the mrd-negative group (mrd-low vs mrd-negative, hazard ration [hr] 1.62, p = 0.554; mrd-high vs mrd-negative, hr 8.47, po 0.001). the 3-year relapse rates for the mrdnegative, mrd-low, and mrd-high groups were 15%, 0, and 67%, respectively (p o0.001). there were no significant differences in non-relapse mortality among the three groups. the analysis of fcm-based detection of mrd revealed that an mrd positivity of ⩾ 0.6% was significantly associated with high risk of relapse and death even in patients with aml with hematological cr. the stronger consolidation or conditioning therapy before hsct based on mrd could improve transplant outcomes in these patients. [p506] disclosure of conflict of interest: none. post-transplant cyclophosphamide (ptcy) and megadose t cell depleted (tcd) haplohsct for tolerance induction f aversa 1 , e bachar-lustig 2 , n or-geva 3 , y zlotnikov klionsky 2 , l prezioso 1 , s bonomini 1 , a monti 1 , i manfra 1 , c schifano 1 , s pratissoli 4 , f lohr 5 , r lamanna 6 , v sgobba 6 , n giuliani 7 and y reisner 2 1 hematology and bmt unit, university hospital of parma, italy; 2 department of immunology, weizmann institute of science, rehovot, israel; 3 neurology department, stanford school of medicine, stanford, california; 4 radiotherapy unit, university hospital of modena, italy; 5 radiotherapy unit, university hospital of modena, italy; 6 genetic unit, university hospital of parma, italy and 7 hematology and bmt unit, university hospital of parma, italy the use of ptcy is associated with reduced risk for gvhd in t cell replete nma haplo-hsct; however, this intervention is still not sufficiently safe to justify treatment of non-malignant diseases or as a platform for organ transplantation. experimental data: in a total of 66 mice, we showed that combining the power of megadose tcd hsct with high dose ptcy (fig.1a) , enables marked and durable chimerism following nma conditioning, while each modality alone was ineffective (figure 1a) . chimerism included all myeloid and lymphoid lineages, and lda analysis of alloreactive t cells revealed specific immune tolerance towards donor stimulators (fig.1b) , also associated with acceptance of donor but not 3 rd party skin. clinical trial: a similar protocol was developed for clinical use. the first patient, a 54 yr old male with high-risk multiple myeloma in cr after autohsct, received megadose (15.4 x10 6 cd34+ cells/kg) cd3/cd19 depleted (1.17 x10 5 cd3+t cells/kg) haploidentical pbpcs after atg, fludarabine and 2 gy single frcation tbi. ptcy was given to control both hvg and gvh reactions (fig. 1c) . hematopoietic engraftment was achieved at day +15 with over 97% donor type chimerism during the first 6 months in the myeloid and b cell lineages. t cells during this period were predominantly of host type (10-23% donor type), gradually increasing to 63-72% at 9-12 months post transplant (fig. 1d) . the patient overcame cmv and subsequently ebv reactivation without any treatment (fig. 1e-1g) . dextramer facs analysis revealed that cmv and ebv specific cd8 t cells were exclusively of host origin (fig. 1f-1h) . at +18 months, cr and normal free light chain ratio were confirmed. the second patient, a 50 year-old male with high risk heavily pretreated multiple myeloma (tandem auto-hsct, 3 yr maintenance with lenalidomide, salvage therapy with vd) received a similar hsct (10.8 x10 6 cd34+ cells/kg, 1.2x10 5 cd3 +t cells/kg). despite transient engraftment (50% donor cell on day +17), graft failure with autologous recovery (0.04% donortype chimerism) was documented on day +30. this may be due to the extended treatment (3 yrs) with lenalidomide, but rejection cannot be excluded. after 5 months, this patient tolerated a second haplo-hsct (different donor) after myeloablative conditioning (atg, treosulfan, thiotepa and fludarabine) and alfa/beta tcr/cd19-depleted pbpcs. at 8 month follow up, he shows no sign of gvhd, good immunological reconstitution, excellent quality of life, and remains in complete remission. collectively, our murine proof of concept data supported by clinical experience in the first high risk mm patient. the marked level of host t cells persisting over the first year after hsct can provide anti-viral immune protection until thymus-derived donor t cells are generated. avoiding additional post transplant immune suppression ensures a robust anti-viral immunity and a graft vs tumor effect. the rejection experienced by the 2 nd patient, although corrected by a 2 nd myeloablative tcd hsct, indicates that the conditioning must be fine-tuned to optimize engraftment in every patient. we are therefore testing, increasing tbi from 2 gy to 3 gy. further studies will determine the efficacy of this approach in elderly mm patients, in non-malignant hematopoietic diseases, or as a prelude for organ transplantation and cell therapy. over the last decade the addition of alemtuzumab to fludarabine-based reduced intensity conditioning regimen is common practice in the unrelated donor allograft setting. in recent years, however, its use has extended to reduced intensity hla-identical sibling donor allografts with the aim of providing an additional prophylaxis against gvhd. it is difficult to assess though whether this practice has any negative influence in the relapse rate or whether it has any net benefit or disadvantage in terms of overall survival. in this retrospective study we have analysed a historical cohort of 65 patients [41 males, 24 females, mean age 59.05 (40-73)] who s385 received a ric fully matched unrelated donor (49 patients) or sibling donor (16) hsct as consolidation treatment for hr aml in 2 transplant centres in uk and greece. the conditioning regimen included fludarabine in all cases, together with melphalan and alemtuzumab(29 patients), busulphan and campath (21 patients), busulphan and thiotepa (1 patient), melphalan (3 patients), busulphan with and without atg (9 patients) total body irradiation (200 cgy, 2 patients). in total, 50 patients received alemtuzumab (35 mud 50 mg alemtuzumab and 15 sibling donor hsct recipients 30 mg alemtuzumab) and 9 patients (7 mud and 2 sibling donor hsct recipient) received atg with 5 patients receiving t replete allografts. gvhd prophylaxis was ciclosporin for patients receiving alemtuzumab based or atg based regimen and ciclosporin with low dose methotrexate for t-replete allografts. the median follow up was 32.3 months (range 3-154 months).all but four patients were transplanted in cr1 overall, patients receiving conditioning without alemtuzumab suffered more frequent (po0.0001) and more severe (po0.0001) acute gvhd. this group, however, had a significantly (po 0.05) lower relapse rate. the overall survival remained unaffected. the subgroup of patients receiving allografts from mud had a clear benefit in terms of a lower incidence (p o0.0001) and severity (p o0.0001) of acute gvhd: none of the patientsreceiving alemtuzumab experienced grade iv agvhd, but up to 5/14 patients not receiving alemtuzumab suffered severe grade iv gvhd. however, the use of campath was associated with a significantly higher rate of relapse or progression of the aml (po 0.02), so that none of the 6 mud recipients not having campath relapsed, while 9/26 patients having alemtuzumab relapsed. although none of these factors had a net impact on survival, there was a nonsignificant (p = 0.07) trend towards a higher survival in patients who received alemtuzumab. in the sibling donor allograft setting, alemtuzumab had no significant impact on the incidence of acute gvhd, relapse or survival. finally, in diseases where cytogenetic or molecular markers of high risk were available, our results showed a better overall survival (po0.06) in ric alemtuzumab conditioning undergoing fully matched unrelated donor hsct, probably as a result of the protection against graft versus host disease while maintaining graft versus leukaemia effect. overall, alemtuzumab is a highly protective agent against agvhd in mud hsct recipients while it maintains the graft versus leukaemia effect.however it did not show any clear benefit of its use in the identical sibling donor setting. larger prospective studies are required in order to determine the need for this agent in this particular setting. disclosure of conflict of interest: none. blastic plasmacytoid dendritic cell neoplasm (bpdcn) is a rare disease which constitutes o1% of all hematologic neoplasms annually. majority of bpdcn present with diverse skin involvement prior to leukemic dissemination, whereas a minority (~10%) have systemic involvement at diagnosis. there are no established therapies for bpdcn and most pts receive acute leukemia, myeloid or lymphoblastic, induction regimens; but responses are short-lived and prognosis is poor upon relapse. allogeneic hematopoietic cell transplantation (allo-hct) is offered to bpdcn cases based on small retrospective or registry case series. we retrospectively analyzed outcomes of bpdcn pts who received an allo-hct at 5 transplant centers in the usa. a total of 20 pts were eligible for analysis ( table 1 ). the primary endpoint was overall survival (os). twenty patients (m = 18, 90%), median age of 51 (14-71) yrs, received an allo-hct from a matched related (n = 9, 45%), matched unrelated (n = 7, 35%), mismatched-unrelated (n = 2, 10%), umbilical cord (n = 1, 5%) or haploidentical (n = 1, 5%) donor using myeloablative (mac) (n = 13, 65%) or reducedintensity (ric) (n = 7, 35%) conditioning. fifteen pts received hyper-cvad as pre-allograft therapy (front-line = 14, salvage = 1). the majority (n = 17, 85%) were allografted in cr1. median f/u for survivors was 26.3 (3.9-128.8) months. median time-to-neutrophil and platelet engraftments were 16 (12-26) days and 15 (7-45) days, respectively. five pts never dropped s386 platelet counts below 20 000/μl. three pts (mac = 2, ric = 1) relapsed at 6, 7, and 99 months, respectively. all 3 relapsed with marrow involvement (1 had also skin involved). mean os was 81.9 (53.1-110.8) months. one-year and 3-year os were 85% (95% ci = 64-95%) and 70% (95% ci = 48-85%), respectively. there was no difference in 3-year os when comparing mac versus. ric (hr = 1.68 (95% ci = 0.34, 8.4), p = 0.53). median time to onset of acute gvhd was 35 (10-117) days; grade ii-iv acute gvhd occured in 6 cases. chronic gvhd was seen in 5 cases (mild = 2, mod/severe = 3). allo-hct is an effective therapy for bpdcn resulting in durable remissions. encouraging outcomes observed in this analysis may be explained by offering allo-hct early in the disease course and in the setting of complete remission. larger studies are needed to better understand risk factors for relapse to develop post-transplant strategies to improve outcomes. disclosure of conflict of interest: none. a risk-factor analysis for overall survival in patients with acute leukemia that relapse following t-replete haploidentical transplantation: on behalf of the acute leukemia working party of the european society for blood and marrow transplantation s piemontese 1,2 , m labopin 2,3 , f ciceri 1,2 , c schmid 2,4 , a ruggeri 2,3 , w arcese 5 , z gulbas 6 , y koc 7 , j tischer 8 , b bruno 9 , w depei 10 , d blaise 11 , d beelen 12 , g ehninger 13 , a boumendil 2,3 , m houhou 2,3 , m mohty 2,3 and a nagler 2, 14 relapse of acute leukemia is the leading cause of transplantation failure with devastating results. relapse post t-replete haploidentical transplantations (haplo-sct) is not well characterized. the objective of this study was to identify riskfactors for overall survival in patients with al that relapsed after a haplo-sct. from 2007 to 2014, 1660 haplo-sct were performed in 186 ebmt centers as first allogeneic transplantations for adults with acute leukemia. out of 657 patients for whom we were able to receive updated data, 208 relapsed and were included in this analysis. median follow-up among survivors was 25 months after haplo-sct (2-97) and 7.2 months (1-71) after relapse. median time from haplo-sct to relapse was 5 months (11 d-36m). diagnosis was acute myeloid leukemia (aml) in 72% and acute lymphoblastic leukemia (all) in 28% of the patients, respectively .fifty-two (25%) patients were transplanted in first complete remission (cr1), 42 (20%) in cr2 or cr3, while 114 (55%) were transplanted in active disease. ric regimen was used in 116 (57%) patients and 85 (41%) received bone marrow as stem cell source. post-transplant cyclophosphamide (pt-cy) was used for graft-versus-host disease (gvhd) prophylaxis in 121 patients (58%). fifty-two (25%) of the patients who relapsed post haplo-sct experienced previously acute gvhd and 42 (21%) chronic gvhd post transplantation. treatment of relapse varied and included: none in 42 (21%), ist withdrawal only in 15 (8%), chemotherapy (ct) only in 59 (30%), tyrosine-kinase inhibitor (tki) only in'(2%), tki and ct in 6 (3%), dli only in 9 (4%), subsequent transplant in 12 (6%), ct and dli in 37 (19%), ct and subsequent transplant in 8 (4%), tki ct and subsequent transplant in 1 (0.5%), dli and subsequent transplant in 5 (2.5%) patients. donors for second allogeneic transplant were unrelated (n = 1), haploidentical (n = 23) and cord blood (n = 2). second transplant was performed in cr for 8 patients and in relapse for 18 patients. only 2 patients who received a second haplo were alive at 47 and 69 months post second transplant. the majority of patients who received dli were in relapse at time of dli (81%), and 26% achieved cr after dli. os 1y after dli was 27%, 7 patients being alive at a median time of 18 mo (4-55) post dli. overall, the one-year overall survival (os) following relapse was 17% (95% ci: 11.6-22.3). in univariate analysis disease status at haplo-sct (cr vs active disease), cytogenetics (good/intermediate vs poor) and median time from haplo-sct to relapse (4 or o 5.03 months) were associated to a higher os at one year after relapse: 27% (p = 0.003), 27% (p = 0.03) and 25% (p o10 -4 ), respectively. in multivariate analysis complete remission at haplo-sct (p = 0.004; hr 0.64; ci:0.47-0.87) and time from haplo-sct to relapse higher than 5.03 months (p = 0.001; hr 0.58; ci: 0.41-0.81) were risk factors for a higher os after relapse. in the 58 patients transplanted in cr and relapsing more than 5 month after haplo, 1 and 2 y os were respectively 33% and 20%. these findings suggest that similar to other transplantation setting os for acute leukemia that relapse post haplo-sct is dismal. disease status at transplant and time from transplant to relapse are the two important prognostic factors that can predict somewhat better survival. indication for second transplant should be carefully evaluated. integrations with novel therapies are in unmet need to prevent and treat relapse post haplo-sct. disclosure of conflict of interest: none. patients (pts) with aml who relapse after autologous stem cell transplantation (asct) have a dismal outcome but some can be rescued with an allogeneic transplantation (allohsct). yet, available evidence presently stems from analyses of limited patient numbers. we decided to analyze the ebmt registry to evaluate the outcome and determine the prognostic factors in a large series of such pts. the ebmt registry was screened for adult pts with de novo aml (non-apl) who received an allograft in cr2 or first relapse (2000-2015) after being autografted in cr1. pts receiving ex vivo t cell depletion (tcd) were included only if they received a haploidentical allohsct. inclusion criteria were met by 537 pts (48% female, median age 45 [range 18-78] years). median time from asct to relapse was 10 (range 0.6-176, iqr 5.8-19.1) months. at allohsct, pts were in 1 st relapse (25%) or cr2 (75%). donors were matched sibling (18%), unrelated (57%), haploidentical (13%), or cord blood (12%), respectively. conditioning was myeloablative in 46% and reduced intensity in 54% of the pts, respectively. the median follow up was 52 months (range o1-167 months). at 3 years post allograft (figure), leukemia free survival ( 4 -18] of the pts. all factors significantly associated with ⩾ 1 endpoint in univariate analysis were entered in a multivariate cox regression model (table 1) . ri was lower in pts transplanted in cr2 rather than in relapse (31.1% vs 44.7%; hr 1.76, p = 0.004) and in pts who relapsed later (410 months, median value) as opposed to those who relapsed early post asct (25.5% vs 43.2%; hr (per month) 0.97, p o10 -3 ). ri was lower in pts transplanted with an unrelated donor (ud) in comparison to those transplanted from a matched sibling donor (29.1% vs 50.5%; msd, hr: 0.49, p o10 -3 ). patient age, poor cytogenetics, transplantation in relapse, previous tbi for asct, myeloablative conditioning (mac) vs reduced intensity (ric) and ud, haplo or cbt vs msd all significantly increased nrm. lfs was significantly better in pts with good risk (47.3%) than in pts with intermediate risk or poor risk cytogenetics (29%; hr 0.62, p = 0.007) or in pts who relapsed late (per month: hr 0.99, p = 0.003) post asct. lfs was worse in pts who previously had received tbi (20% vs 45%; hr = 1.82; po10 -3 ). the same prognostic factors were significant for os. haploidentical (hr 2.25, p = 10 -3 ) and cord blood (hr 2.09, p = 0.003) transplants resulted in lower os than those from msd. finally, date of transplant significantly influenced os which was higher in pts transplanted after january 2008 vs those allografted before; 48.2% vs 31.7%, hr (per year) 0.96, p = 0.02). about one third of adult patients with aml who relapse post asct can be rescued with an allogeneic transplantation, especially if the duration of persisting cr post asct is long and no tbi was received in the past. transplantation from an msd while in cr2 rather than at relapse offers the best outcome. disclosure of conflict of interest: none. high incidences of graft-versus-host disease (gvhd) and relapse have seriously impeded the widespread application of haploidentical hematopoietic stem cell transplantation (haplo-hsct) for high-risk acute leukemia lacking conventional hla-matched donors. one hundred and ten high-risk acute leukemia patients underwent haplo-hsct with idarubicin (ida) intensified conditioning regimen (ida intensified bucy2 for acute myelocytic leukemia (aml) and ida intensified tbi-cy for acute lymphoblastic leukemia (all)). for donor-recipient hla 3/6 or 4/6 transplant, we separately administered a total of 9 mg/kg or 6 mg/kg antithymocyte globulin (atg) and basiliximab for gvhd prophylaxis. all enrolled patients were observed longitudinally until death or lost to follow-up. the 100-day cumulative incidences of ⅱ-ⅳ and ⅲ-ⅳ agvhd for all patients were 30.3%, 14.7%, respectively. the 2-year cumulative incidence of extensive cgvhd was 12.2%. the relapse rate was 18.2%. the 3-year probability of overall survival (os) reached 63.1%. the patients in non-complete remission (nr) showed significantly higher relapse and worse survival than complete remission (cr) minimal residual disease (mrd) (-) and cr mrd (+) patients. however, the relapse, 3y-os and disease-free survival (dfs) of cr mrd (-) did not differ from cr mrd (+) patients, indicating our intensified transplant technique could overcome the poor prognosis of mrd. for whatever aml or all patients, the relapse rates, agvhd, cgvhd and the estimated 3-year os and dfs between two atg group were equivalent, except that all patients in atg 9 mg/kg experienced higher relapse (33.0% vs 19.2%, p = 0.226). although the incidence of cytomegalovirus (cmv) reactivation in atg 9 mg/kg and 6 mg/kg was 77.4%, 72.9%, the average episodes of cmv reactivation were remarkably [p512] higher in 9 mg/kg. our ida intensified haplo-hsct technique could improve the outcome of high-risk acute leukemia and could be recommended as a good alternate for patients lacking hla-matched sibling donors. diagnosed secondary aml were randomized 1:1 to cpx-351 or standard 7+3 therapy. cpx-351 induction was 100 units/m 2 on days 1, 3, 5 (first induction) and days 1, 3 (reinduction); 7+3 first induction was cytarabine 100 mg/m 2 /day × 7 days and daunorubicin 60 mg/m 2 on days 1, 2, 3, and reinduction was cytarabine 100 mg/m 2 /day × 5 days and daunorubicin 60 mg/ m 2 on days 1, 2. a dynamic allocation procedure stratified patients by age group (60-69 or 70-75 years) for each study arm. patients with complete response (cr) or cr with incomplete platelet or neutrophil recovery were considered for allogeneic hct, based on institutional criteria. overall survival (os) landmarked at the time of hct was assessed. a total of 309 patients were enrolled on the induction trial. . patient and aml characteristics in the hct age subgroups were generally similar between arms. in both age subgroups of patients receiving hct, median os was longer in the cpx-351 arm than in the 7+3 arm (table 1 ). in the 60-69 group, serious adverse events (saes) prior to hct in the cpx-351 and 7+3 arms occurred in 28% and 22% of patients, respectively; in the 70-75 group, in 13% and 67%, respectively. the most common sae was febrile neutropenia (cpx-351, 11.5%; 7+3, 5.3%), occurring in all age groups. relapse after allogeneic haematopoietic stem cell transplant (allo-hsct) for acute myeloid leukaemia (aml) and myelodysplastic syndrome (mds) remains the main cause of treatment failure. it is associated with dismal prognosis and short survival. proposed salvage strategies are tapering of immunosuppressive therapy, re-induction with chemotherapy and consolidation with donor lymphocyte infusion (dli) or second allo-hsct, although, results remain disappointing. azacitidine (aza) and dli has proved to be an effective and well-tolerated outpatient approach in this setting, and results in at least temporary disease control in the majority of patients, thus, representing a valuable alternative to current treatments. between january 2010 and november 2016, 16 patients with relapsed aml or mds after allo-hsct were treated with subcutaneous aza 100 mg/m 2 days 1-5 every 28 days and escalating doses of dli if feasible at manchester royal infirmary, uk. aza was continued until cr or disease progression. patients characteristics: median age 60 (range 45-69) years, 56% males, diagnoses were aml (n = 12) and mds (n = 4). five (31%) patients had either monosomal or complex karyotype. fifty percent of patients were in cr1 before transplant, 12.5% in cr2, 12.5% had a partial response and 25% did not receive any chemotherapy before the transplant. fifteen out of 16 received fludarabine-base reduced intensity conditioning regimen and all but one had a t-cell depleted graft. at relapse 88% had mixed donor chimerism. median time to relapse was 9.5 (range 2-21) months after allo-hsct. with a median follow up of 6.5 (range 1-25) months a median of 5 (range 1-16) courses of aza were administered and median of 2 (range 1-6) dli were infused. doses of dli were administered starting at 0.1 x107/kg and escalating by log5. aza and dli infusions were well tolerated; only two patients withdrew due to intolerance. seven patients were admitted at least once due to infections (86%) or progressive disease. only two patients developed mild gvhd grade 1. complete remission was achieved in 12.5% patients and stable disease in 56%. patients in cr had full donor chimerism. median overall survival for patients in cr was 25 months compared to 9 months for those who did not respond (p = 0.031). patients with more than 20% blasts on bone marrow at time of relapse after allo-hsct had a worse outcome than those with less than 20% blasts (11 months and 25 months respectively, p = 0.09). no differences were seen when compared time to relapse ( o 6 months vs ⩾ 6 months) s390 and outcome, or disease and overall response, although numbers in this series are small. image/graph: overall survival following azacitidine and dli, patients in complete remission, stable disease and disease progression. azacitidine and dli can provide long term remissions in patients with relapsed aml/mds post allo-hsct with low toxicity. lower disease burden at relapse carries better outcomes. low rates of gvhd are seen following azacitidine and dli most likely showing the immunomodulatory effect of azacitidine described by other groups. acute myeloid leukemia (aml) is a frequent complication in patients affected by telomere maintenance disorders ('telomeropathies') such as dyskeratosis congenita (dkc). treatment of aml in dkc patients by chemotherapy and hematopoietic stem cell transplantation is characterized by frequent remission failure, high organ toxicity and poor outcome. a 27-yearold patient with aml was admitted to our hospital in december 2014. he had been treated with 6 cycles beacopp for hodgkin´s lymphoma (hl) in 2009. on admission, the patient presented clinical signs of premature aging with hair greying and lack of fully recovered hair growth after chemotherapy (cx) for hl. flow-fish analysis revealed tl below the 1% percentile within leucocytes in line with the suspected diagnosis of telomeropathy. retrospective tl analysis by confocal q-fish from bm at hl diagnosis confirmed short tl before the start of any chemotherapy. he received standard aml induction cx (3+7), but follow-up revealed persistence of aml. salvage cx with flag-ida was applied resulting in partial remission with only weak regeneration of normal hematopoiesis. the patient received an allogeneic stem cell transplantation (asct) after conditioning with 140 mg/m 2 melphalan and fludarabin from his hlamatched brother whose tl was found to be normal. after asct, he developed sinusoidal obstructive syndrome and progressive liver failure treated with defibrotide and he was admitted to icu for sepsis. leucocyte count showed sufficient engraftment on day 14; however, liver function recovered only partially. during critical care treatment, the patient showed cardiomyopathy, renal failure and extensive wound healing problems without epithelial proliferation indicative of severe replicative exhaustion. finally, he died due to sepsis with acute liver failure on day 91 after asct. aml arising from dkc is a rare event with substantial impact on patients´prognosis. therapy remains challenging due to poor bm function and high risk of organ toxicity, especially liver failure and lung fibrosis. dose reduction of alkylating agents and avoidance of total body irradiation are necessary in conditioning prior to asct in patients with dkc and aml, however no clear data or recommendations exist for the management of these patients. tl screening can help to identify patients with suspected dkc related bm failure or aml and to identify family donors without telomeropathy. physicians should be aware of possible dkc related aml, especially in familial cases of aml or bone marrow failure, impaired or prolonged recovery following cytoreductive treatment or coincidence of solid (e.g. oral cavity carcinomas) and hematological malignancies. disclosure of conflict of interest: none. chronic graft-versus-host disease and donor lymphocyte infusions in patients with non-de novo acute myeloid leukemia or advanced myelodysplastic syndromes after allogeneic stem cell transplantation pg hemmati 1 , k pfeifer 1 , lg vuong 1 , cf jehn 1 , p le coutre 1 , b dörken 1 and r arnold 1 1 medizinische klinik mit schwerpunkt hämatologie, onkologie und tumorimmunologie, charité-universitätsmedizin berlin, campus virchow-klinikum, berlin, deutschland aml with myelodysplasia-related changes and therapy-related aml (taml), collectively termed secondary aml (saml) in daily clinical routine, represent distinct subgroups in the 2016 revised who classification of myeloid neoplasm and leukemias. as compared to de novo-aml, saml is associated with a poor survival when using conventional chemotherapy approaches. this is mainly due to unfavorable cytogenetics, older age and/or the presence of comorbidities as well as poor response to induction therapy. furthermore, cumulative organ toxicity resulting from treatment of the antecedent solid malignancy in patients with therapy-related disease has to be taken into account. allogeneic stem cell transplantation (allosct) represents the only option to achieve long-term disease control and definitive cure. we retrospectively analyzed 204 patients with saml or advanced mds (eb-2 according to who) transplanted at our center between 1995 and 2015. at the time of allosct, 98 patients (48%) were in complete hematologic remission (chr), whereas 106 patients (52%) had active disease. cytogenetic risk was categorized according to the swog/ecog classification and was favorable (n = 3; 2%), intermediate (n = 94; 46%), unfavorable (n = 84; 41%), or unknown/undetermined (n = 23; 11%). standard myeloablative conditioning (mac) using 12 gy total body irradiation (tbi) and cyclophosphamide was used in 41 patients (20%), whereas fludarabin/busulfan/atg-based reduced intensity conditioning (ric) was applied in 163 patients (80%). grafts were from related (n = 51; 25%) or unrelated (matched: n = 112; 55% or mismatched: n = 41; 20%) donors. the median follow-up of the surviving patients was 46 (5-24) months. a graft failure occurred in 5/204 patients (3%). at last day of follow-up 72/204 patients (35%) were alive and in chr. relapse occurred in 77/204 patients (38%) after a median interval of 4.6 (range: 0.1-135) months. cause of death were either relapse or nrm (gvhd and/or infections) in 69/204 patients (34%) or 56/204 patients (28%). at 1, 3, 5, and 10 years after allosct overall survival (os) or disease-free survival (dfs) of the entire cohort was 56%, 46%, 38%, and 29% or 50%, 38%, 36%, and 27 %, respectively. at the same time points, the cumulative incidence of relapse (ci-r) or non-relapse mortality (ci-nrm) was 30%, 37%, 37%, and 40% or 20%, 25%, 27%, and 33%, respectively. extensive uni-und multivariate analyses revealed a number of factors associated with inferior outcome, e.g. poor-risk cytogenetics, the presence of taml, advanced age, reduced physical performance, and comorbidities, whereas donor type (unrelated versus unrelated), and remission status had no significant impact on overall outcome. furthermore, the development of gvhd, especially the presence of cgvhd, and the use of donor-lymphocyte infusions (dli), either in a prophylactic or pre-emptive setting, were identified as independent predictors for a reduced relapse incidence, which in turn, led to an improved os and dfs. our results indicate that allosct represents an important treatment option for patients with saml. however, a relapse rate of 30% at 12 months prompts the development of novel approaches to prevent early disease recurrence. strategies to augment the graft-versus-leukemia (gvl) effect of allosct may help to improve the results. disclosure of conflict of interest: none. myeloid sarcoma (ms) is a rare hematologic myeloid neoplasm that can involve any site of the body. it can occur as an exclusively extramedullary form or it can be associated with an acute myeloid leukemia (aml), a chronic myeloproliferative neoplasm (mpn) or a myelodysplastic syndrome (mds) at onset or at relapse. the rarity of ms does not enable prospective clinical trials and therefore a specific multicenter register can be useful for the clinical and biological studies of this rare disease. we report the clinical characteristics and outcome of 48 histologically confirmed ms, diagnosed and treated in 9 italian hematological centers in the last 10 years. the patient's median age was 46 years. there were 9/48 de novo extramedullary ms, 24/48 de novo aml-related ms and 15/48 were secondary aml-related ms. the most common extramedullary anatomic sites of disease were: skin, lymph nodes and soft tissues. forty-three patients (90%) underwent a program of intensive chemotherapy including flai, hdac-ida, hypercvad and mec schemes, with a cr rate of 44% (19/43). twenty-two (46%) patients underwent allogeneic sct, 13 from a mud, 8 from an hla-identical sibling donor and 1 from an haploidentical donor. the median os of the whole population (48 pts) was 16.7 months. the os probability at 1, 2 and 5 years was 64%, 39% and 33%, respectively. the os was better in patients that underwent an intensive therapeutic program (median os: 18 months vs 5 months). among the intensively treated patients, in univariate analysis, the os was better in young patients (p = 0.008), in patients that underwent allo-sct (p = 0.009) and in patients that achieved a cr during treatment (p = 0.001), and was worse in pts with secondary aml-related ms (p = 0.007). age, response to intensive chemotherapy and allo-sct were the only three variables that significantly influenced dfs (p = 0.02, p = 0.01 and p = 0.04, respectively). in multivariable analysis, allo-sct and response to intensive chemotherapy remained significant in predicting a better os (p = 0.04 and p = 0.001, respectively), and response to intensive chemotherapy was the only significant variable in predicting dfs (p = 0.01). after allo-sct we observe a survival advantage in patients who achieved a pre-transplant cr (p = 0.008) and in those who developed a chronic gvhd (p = 0.05). patients with ms, both with de novo and secondary forms, still have a very unfavorable outcome and require an intensive therapeutic program, that includes allo-sct, whenever possible. the outcome after allo-sct is positively influenced by the development of chronic gvhd suggesting a graft versus ms effect. disclosure of conflict of interest: none. relapse of acute lymphoblastic leukemia (all) after allogeneic stem cell transplantation (sct) is associated with poor prognosis. blinatumomab may enhance the efficacy of donor lymphocyte infusions (dli) in this specific situation but data on the concurrent use of dli and blinatumomab are sparse. the patient presented here was diagnosed with standard risk pre-b-all (presence of t(3;9); bcr-abl and cd20 negative) at the age of 23. during treatment according to the german multicenter all-study group (gmall) protocol he presented with molecular relapse and 8 months after initial diagnosis he received a tbi-based myeloablative sct from an unrelated hla-identical (10/10) donor. post sct he was negative for minimal residual disease (mrd) with 100% donor engraftment. given the high relapse risk he received 3 prophylactic dli without occurrence of graft-versus-host disease (gvhd). one year after 1st sct he presented with an extramedullary (testes) and molecular relapse. after remission induction resulting in negative mrd he received a 2nd sct from an alternative, hlaidentical (10/10) donor after reduced intensity conditioning. this again resulted in negative mrd with 100% donor chimerism without any gvhd. six months after 2nd sct he presented with bone marrow relapse. we decided on the concurrent use of blinatumomab and dli. the first cycle of blinatumomab was initiated at standard dose including dose escalation without relevant toxicities. on day 40 of the 2nd cycle, i. e. in the infusion-free interval before the 3rd cycle the patient received the first dli at 1x107 cd3/kg. no toxicities or gvhd occurred. the 3rd cycle of blinatumomab was initiated and a second dli at 2.5x107 cd3/kg was applied on day 3 of the 3rd cycle. on day 32 of the 3rd cycle, i. e. day 29 after 2nd dli the patient presented with signs of overlap gvhd (mouth, skin) and topical steroids were started. upon progression of clinical gvhd systemic steroids were initiated with immediate response. steroids were rapidly tapered and a 4th cycle of blinatumomab was started. gvhd did not recur. current staging after the 4th cycle blinatumomab, i.e. on day +413 after 2nd sct and 7 months after initiation of blinatumomab treatment revealed complete remission with negative mrd, 100% donor chimerism and no signs of extramedullary relapse. counts of cd4-cells at that time point were 147/μl. no relevant infections or relevant blinatumomab-associated toxicities were present during the entire course after the 2nd sct. in this case concurrent treatment of blinatumomab and dli resulted in the longest disease-free interval for our patient compared to preceding chemotherapy or dli alone. together with the small number of reported cases (ueda et al.) this supports the concept of concurrent blinatumomab and dli as an effective post sct treatment. the objective of the study is to evaluate the clinical efficacy and safety of decitabine (dac) in combination with haag regimen [homoharringtonine (hht), cytarabine (ara-c), doxorubicin (acla) and recombinant human granulocyte colony stimulating factor (g-csf)] for advanced patients with acute myeloid leukemia (aml). thirty-six patients with advanced aml receiving dac combined with haag chemotherapy in our center from december 2012 to august 2015 were enrolled in this study. eighteen of them were refractory or relapsed aml, and another 18 patients were those who didn't achieve complete remission (cr) after a course of induction chemotherapy. the therapeutic responses, side effects and longtime survival were retrospectively analyzed. after a course of treatment, the rate of cr and partial response (pr) was 58.3% (21/36) and 22.2% (8/36) respectively, while the overall response rate (orr) was 80.6% (29/36) in the cohort. for the patients with refractory or relapse aml, cr was 61.0% (11/18), pr was 22.2% (4/18), and orr was 83.3% (15/18). while for the other not getting cr after a course of induction chemotherapy, cr was 55.6% (10/18), pr was 22.2% (4/18), and orr was 77.8% (14/18). grade 4 hematological toxicities were observed in all patients, and 72.2% cases experienced infection. and all non hematological side effects were mild and well-tolerated. with a median follow-up of 7.5 (0.5~33.3) months, the 1-year overall survival (os) rate was 43.3%, 24.2% for the refractory or relapsed aml patients, and 61.6% for those not achieving cr after a course of induction chemotherapy. the difference was significantly (p = 0.01). conclusion dac combined with haag regimen is safe and effective salvage treatment for advanced stage aml patients. disclosure of conflict of interest: none. aml patients harboring flt3-itd mutation are associated with decreased survival compared to patients without flt3-itd mutation. nevertheless, whether flt-itd mutation also has negative impact on the post-transplant survival is less clear. for flt3-itd mutated aml, a decreased leukemia-free survival (lfs) after allogeneic hsct was observed in ebmt analysis but not cibmtr. in this study, unlike studies of ebmt or cibmtr which only pre-specified populations of patients were analyzed (cr1 in ebmt, cr1+cr2 in cibmtr), we examined the prognostic impact of flt3-itd mutation on post-transplant outcome of "all" the adult aml patients reported to taiwan bone marrow transplant registry (tbmtr). tbmtr is a research collaboration affiliated to the taiwan society of blood and bone marrow transplantation. it comprises all the 14 transplantation centers in taiwan that contribute detailed data on hsct. adults aged ⩾ 18 years with a diagnosis of aml and with known flt-itd mutation status in the registry were included. patient characteristics and transplant outcome following allogeneic hsct for flt3-itd mutated and nonmutated aml were compared. kaplan-meier estimates were used to calculate the probability of lfs and overall survival (os). multivariable analyses for lfs and os were performed using cox proportional hazards model. 365 patients who met the eligibility criteria were enrolled for analysis. the median follow-up of survivors was 21 months. of the 365 patients, 94 (25.8%) were positive and 271 (74.2%) were negative for flt3-itd mutation. flt3-mutated patients had significantly more transplantation at cr1 (57.4%), shorter time interval between diagnosis and hsct (5.6 months), and higher wbc count at diagnosis (51.7 × 10 9 /l) comparing to patients without flt3 mutation (43.5% at cr1, 6.5 months from diagnosis to hsct, and 11.8 × 10 9 /l wbc count at diagnosis). significant more flt3 mutated patients had intermediate-risk (80.9%) and normal (64.9%) karyotype at diagnosis. the age, donor type, stem cell source, conditioning regimen, and atg use were not significant different between flt3-mutated and non-mutated patients. of the whole population, flt3 mutation status did not negatively impact the transplant outcome (2 years os for flt3 mutated and non-mutated patients: 45.2% vs 50%, log rank p = 0.624; 2 years lfs for flt3 mutated and non-mutated patients: 40.2% vs 32.4%, log rank p = 0.192). when different pre-transplant conditions (cr1, subsequent cr, and no cr) were analyzed separately, flt3-itd mutation status is still not a significant prognostic factor of os and lfs for patients in cr1 (equally good) and no cr (equally bad). however, for patients in subsequent cr, flt3-itd mutation is the only significant factor predicting poor os and lfs in multi-variable analysis (median os and lfs for flt3 mutated and nonmutated patients: 378 vs 1252 days, log rank p = 0.005; 204 vs 1049 days, log rank po 0.001 respectively). the incidence of non-relapse mortality, grade 3/4 acute gvhd and extensive chronic gvhd is comparable between flt3-mutated and nonmutated patients. flt3-itd mutation is a significant and strong predictor of poor survival for aml patients in subsequent cr at hsct. for flt3-itd non-mutated aml, a sizable portion of patients can have disease free survival after allogeneic hsct at subsequent cr. however, allogeneic hsct at cr1 should be strongly recommended for flt3-itd mutated aml. [p523] disclosure of conflict of interest: none. allogeneic stem cell transplantation (asct) is a curative strategy in acute myeloblastic leukemia (aml) and myelodysplastic syndrome (mds). however, relapse keeps being the main cause of treatment failure. extramedullary relapse (er) is a rare event and its management is not well standardized. we retrospectively analyzed patients who received asct from 2006 to 2016 and developed er in our centre. we performed a descriptive study to analyze characteristic of these patients, post-relapse treatment and survival. statistic analysis was performed using spss v.22. we found a total of 18 patients with er, one of them with 2 er after 2 consecutive asct, so we analyzed 19 cases of er. patient and transplant characteristics are summarized in table 1 . at day +100, 95% of patients were in complete response (cr). er occurred after a median of 13 (2-98) months post-asct. eleven patients (58%) presented with a bone marrow relapse concomitant with the er. er affected central nervous system (cns) in 7 patients (36.8%), bone in 4 patients (21%), skin or soft tissue in 3 patients (15.8%), mama in 2 patients (10.5%), ocular globe in 2 patients (10.5%) and teste in 2 patients (10.5%). two of them presented with multiple sites affected. between the 7 patients who developed cns relapse, 2 of them had received intrathecal prophylaxis. regarding post-er management, immune modulation was conducted in 16 patients (immunosupression tapering in 9, donor lymphocyte infusions in 4 and both strategies in 3). all patients except one received systemic treatment (salvage chemotherapy in 11, azacitidine in 5, low dose arac in 1 and atra in 1 patient with a promyelocytic leukemia). together with systemic treatment, 12 received radiotherapy and intrathecal therapy was used in all 7 patients with cns involvement. response: 12 out 18 patients treated, 12 (63.2%) achieved cr and 6 (31.6%) progressed. two responding patients received a 2 nd asct. after a median follow-up of 67 months (15-123), 8 patients are alive and disease free, with an estimated overall survival of 45% at 5 years. patients receiving salvage chemotherapy followed or not by a 2 nd asct experienced a significantly better os than those receiving other therapies (median os 92 vs 10 months; p = 0.005). patients with bone marrow involvement at relapse show a worse prognosis (median os 39 vs 54 months; p = 0.23) although not statistically significant due to small number of patients (image 1). ten patients died due to disease progression. er must be considered in patients receiving an asct in case of organ symptoms. patients can be rescued with salvage chemotherapy followed or not by a 2 nd asct achieving good results in terms of long term os. it seems that involvement of bone marrow at relapse confers a worse prognosis, what should be confirmed in a larger series of patients. [p524] disclosure of conflict of interest: none. flag-ida regimen as bridge therapy to allotransplant in refractory/relapsed aml patients: a single-center experience c pasciolla, m delia, d pastore, p carluccio, a ricco, a russo rossi, a mestice, f albano and g specchia university of bari, italy although treatment outcome in acute myeloid leukemia (aml) adult patient has improved over the past decade, relapse still occurs in up to 50-70% of cases. furthermore, 15-30% of patients fail to achieve complete remission (cr) because of treatment-resistance. the management of primary refractory and/or relapsed disease remainschallenging for clinicians. in our study, we reviewed the outcome of 116 refractory and/or relapsed aml patients who underwent salvage therapy with the flag-ida regimen between 2005 and 2015 at our institution. the study aim was to determine the efficacy of the flag-ida regimen in order to clarify which variables (who ps, ldh, bone marrow, peripheral blood blasts and platelets counts, white blood cells (wbc), pmn, molecular-cytogentic risk, duration of response and relapsed or refractory disease), present before starting flag-ida treatment, might have an impact both on cr and on os. we analyzed 116 consecutive adult patients (56 males, 60 females; median age 48 years, range 17-72) with newly diagnosed acute myeloid leukemia refractory to standard induction regimens or relapsed after cr, who received the flag-ida protocol as salvage therapy between january 2005 and december 2015. sixty-eight of the 116 patients (58%) were in first relapse, forty-seven patients (42%) were refractory to conventional chemotherapy. median wbc count before salvage therapy was 10.1 x109/l (range 0.56-88). median bone marrow and peripheral blasts counts were 52 and 20%, respectively; median platelets count was 91x10e3/μl. according to the fab classification, 14 patients had m0, 5 m1, 53 m2, 16 m4, 22 m5, 4 m6, 2 had biphenotype acute leukemia. according to molecular-cytogenetic risk stratification 51 (44%), 44 (38%) and 21 (18%) patients belonged to poor, intermediate and good risk group, respectively. sixty-nine of 116 patients (59%) achieved complete remission (cr); forty-seven 41%) patients were refractory to the salvage therapy. in multivariable analysis, variables with positive impact on response rate were lower wbc counts (o10e3/μl, p = 0.0047), higher platlets counts (450x10e3/μl, p = 0.046), molecular-cytogenetic risk (p = 0.032), duration of response in relapsed aml (p = 0.006) and relapsed rather than primary refractory disease (p = 0.042), respectively. median os was 17 months (m). cox regression analysis confirmed that both higher platlets counts, p = 0.002 (17 (450x10e3/μl) vs 11 m (o50x10e37ul), log rank, p = 0.05) and relapsed disease, p = 0.041 (23 (relapsed) vs 17 m (refractory), gehan-breslow, p = 0.021) correlated with better survival. of note, molecular-cytogenetic risk evaluated before starting treatment was associated with cr, while no correlation was found with os. our data seem to confirm the value of flag-ida in relapsed amland may suggest its best usage as bridge-therapy in patients awaiting allotransplantation. disclosure of conflict of interest: none. s395 leukemia relapse is the major cause of death in patients received allogeneic hematopoietic stem cell transplantation (allo-hsct). the precise etiological mechanisms of leukemia relapse remain unclear. both leukemia cells themselves and hematogenesis micro-environment are involved in the relapse event. in our previous study, we reported a case of donor derived relapse of acute myeloid leukemia (aml) after allo-hsct. the patient and his donor-sister both harbored a germline mutation(c.584-589dup) in cebpa gene. donor hematopoietic cells transformed to aml by developing two somatic cebpa mutations (247dupc and 914-916dup) in the patient's microenvironment. hence we suspect that 584-589dup mutation of cebpa gene may altered hematopoiesis microenvironment and increased the survival of aml cells. to conform our hypothesis, we transfected mesenchyme stem cells (mscs) with cebpa 584-589dup or wide type and took vector as control. aml cell line hl60 cells were co-cultured with transfected mscs and then treated with 40ng/ml doxorubicin. apoptosis and cell cycle were detected at day 3. mscs protected hl60 cells from toxicity of doxorubicin. this protection was enhanced by overexpression of cebpa 584-589dup . apoptosis rates of hl60 cells in group of msc-vector and msc-cebpa 584-589dup were 61.7 ± 5.8% vs 30.9 ± 5.6% (p＜0.05). a larger part of hl60 cells remains quiescent with s396 higher rate of g0/g1 phase in msc-cebpa 584-589dup group, which may reduce the sensibility of hl60 cells to doxorubicin. to explore mechanisms involved in the alteration of microenvironment, we performed rna sequence with each group of mscs. we found that col1a1, col1a2 and col3a1 were upregulated in msc-cebpa 584-589dup group compared with msc-cebpa wt group (col1a1:cebpa wt vs cebpa 584-589dup was 1713.65 vs 2317.88, p = 4.70e-19; col1a2:cebpa wt vs cebpa 584-589dup was 2260.02 vs 2755.81, p = 2.76e-10; col3a1: cebpa wt vs cebpa 584-589dup was 746.20 vs 964.82, p = 1.06e-06). furthermore, we found that ddit3 and herpud1 genes, which were important factors in cellular unfolded protein response(upr) and to topologically incorrect protein, failed to augment in cebpa 584-589dup group (ddit3 : vector vs cebpa wt the cure rate of childhood acute lymphoblastic leukemia (all) has improved considerably and approaches 80% today. however, the outcomes of patients who suffer from leukemic relapse remain unsatisfactory. despite the high cure rate of children and adolescents with all a subgroup of patients benefit from allogeneic hsct. allo hsct remains the standard treatment for intermediate/high risk aml patients. 59 patients, all = 42 and aml = 17 age 1 to 20 years with median age 11 years, m/f = 33/26(m/f all = 15/18, aml = 8/9) underwent sct in our hospital (from 2012 to 2016). fifty-eight patients transplanted allo hsct and 1pt aml auto hsct. conditioning regimens consisted of busulfan (iv) +cyclophosphamide for allo and cyclophosphamide + vp16 +cytarabine for auto hsct. peripheral blood (pb) was the source of progenitor cells in 47 patients, bone marrow (bm) in 11 patients and cord blood in one patient. in allo hsct, 50 patient transplanted 6/6 matched and 8 patients 5/6 matched. gvhd prophylaxis regimen was cyclosporine + mtx. all patients engrafted. in allogeneic pbsct all patients' median time to absolute neutrophil count (anc) 40.5 × 109/l was 12 days, and the median time to platelet count 420 × 109 was 15 days vs 17 and 21 days in allo bm all patients. in allogeneic pbsct aml patients median time to anc 40.5 × 10 9 /l was 12 days, and the median time to platelet count 420 × 109 was 14 days. (all patients with aml transplanted with pb). at present 47 pts are alive (36 all, 11 aml) and 12 pts died due to ards, vod, hemorrhagic stroke, sepsis and relapse. trm was 9% at 100 days. median time of death after transplantation was 195 days in all and 153 in aml. in allo pbsct all patients hospitalization period were 36 days vs 45 in allo bm all patients. acute gvhd appeared in 78% pts. chronic gvhd appeared in 55% pts. with a median follow-up of 35 months (3-51 months) after transplant the event-free survival were 73% and four years overall survival 75% in all patients. a median follow-up of 32.5months (4-48months) after transplant the event-free survival were 68% and three years overall survival 63% in aml patients. hematopoietic stem cell transplantation can lead to durable remissions in children and adolescents with leukemia and increase in survival of children. pbsct in childhood all was consistent with significant faster anc and platelet recovery in allogeneic pbsct, hospitalization was shorter. longer follow-up is required to evaluate fully efficacy and long-term results. disclosure of conflict of interest: none. group hla-c1/c2 subtypes were defined as previously practiced. median age of patients was 41, 47% of them were male. allo-hscts were performed from 60% unrelated donors vs 40% related donors. remission status was detected in 67% of patients whereas 12% had active disease pre-transplant. stem cell sources were as follows: 88% peripheral blood, 7% bone marrow, 3% cord blood, 2% bone marrow plus peripheral blood. the most frequent fab subtype was aml-m4. patients were grouped by hla-c status: 23% c1/c1 homozygote, 57% c1/c2 heterozygote and 20% c2/c2 homozygote. the frequency of hla c donor/recipient mismatch allo-hscts was 19%. relapse was detected in 26% of patients. the relapse risk was significantly lower in c1/c1 homozygote patients compared to c2/c2 homozygotes (13% vs 36%, p = 0.02). lfs was similar between c1/c1 homozygote group and c2/c2 homozygote group (p = 0.324) (figure 1 ). in multivariate analysis (age, sex, remission status, related/ unrelated transplant, aml subtype), lfs was increased by pre-transplant remission status (p1 year). there was no difference detected between 2-years os in c1/c1 homozygote group and other groups (57% vs 50%, p = 0.51) (figure 2) . when similar analysis were repeated with donor hla type results were not significant. our results confirm two earlier published reports on aml and all. even in the absence of kir genotyping, hla group c1 has a protective effect. if hla matched donor is not possible a donor-recipient hla-c mismatch favoring c1 to c2 may be preferable. disclosure of conflict of interest: none. immunomodulatory kits do not induce aml-blasts' proliferation ex vivo: ipo-38 is an appropriate and reliable marker to detect and quantify proliferating blasts c plett, dc amberger, a rabe, d deen, z stankova, a hirn, y vokac, j-o werner, j schmohl, d krämer, a rank, c schmid and h schmetzer aml-blasts can be converted to dcleu by immunomodulatory 'kits' (ex vivo). t-cells' energy can be overcome after stimulation with dc/dcleu and results in antileukemic reactivity. a potential induction of blast-proliferation (e.g. by immunomodulatory kit-application in vivo) in aml-pts has to be excluded. 8 kits containing combinations of gm-csf with 1-2 additional factors (pge-1/2, picibanil, ifnα, tnfα, calciumionophore) were studied with respect to the generation of dc/dcleu from blasts, mediation of antileukemic reactivity (after dc/dcleu-stimulation) and with respect to their potential to induce blast-proliferation in a whole blood (wb) culture-system. we studied 3 different markers (ipo-38, ki-67, cd71) and quantified blast proliferation before/after culture. we correlated findings with (ex vivo) antileukemic functionality, with disease-entities and the course of the disease. dc-generation: we could generate dc/dcleu regularly from wb culture from 36 aml-pts. detection of blast proliferation: ø65.6 % (range46-82%) of uncultured blasts expressed ipo-38, 33.1% (16-50%) cd71, 25.4% (8-43%) ki-67. induction of blast proliferation: pooling all results we found lowest amounts of proliferating blasts after culture with kit i (gm-csf+picibanil, 10% ± 13.32), kit k (gm-csf+pge2, 9.14% ± 12.01), kit m (gm-csf+pge1, 7.67% ± 11.79). amounts of proliferating blasts were lower compared to uncultured cells. highest expression of proliferating blasts was found with ipo-38 followed by cd71 and ki-67.we found few individual aml-samples with increased blast-proliferation after ex vivo kit-culture. antileukemic activity: t-cells stimulated with dcleu (generated with kits) improved antileukemic activity. correlations between blast-proliferation and antileukemic activity will be presented. clinical correlations: pts with bad (vs good) cytogenetic risk were characterized by higher proportions of proliferating blasts in uncultured blasts; in some pts with iron-deficiency anemia (ida) proportions of cd71+unculured blasts were lower than of ipo-38/ki-67+ blasts. ipo-38 is a stable marker to be used to quantify proliferating blasts in aml-pts. cd71 is also a good marker, although not suitable for some pts with ida, ki-67 is no reliable marker for every given pt. subtypes of pts correlated with proportions of proliferating blasts. in general kit treatment of blasts did only exceptionally induce blast proliferation ex vivo. in general lowest risk for blast proliferation was seen after culture with kit i, k and m. t-cellstimulation with dc/dcleu generated after kit-treatment resulted regularly in antileukemic reactivity. we conclude that an in vivo treatment of aml-pts with kits i, k or m might be safe (no induction of blast proliferation). disclosure of conflict of interest: none. the occurrence of additional cytogenetic abnormalities (acas) is common in philadelphia chromosome-positive acute lymphoblastic leukemia (ph+ all), but is of unknown significance in the tyrosine kinase inhibitor era. recent study [aldoss et al., 2015] has revealed the acas appear to have a significant deleterious effect on outcomes post-hsct in adult ph+ all patients only. we retrospectively analyzed data from adult and pediatric patients with ph+ all who had undergone allogeneic hematopoietic stem cell transplantation (allo-hsct) at our university between 2008 and 2015. among 65 patients with ph+ all, 53 patients had available data on conventional cytogenetics before allo-hsct. all patients and transplant characteristics are listed in table i . thirty-three of 53 patients (51%) had isolated t(9;22). acas were revealed in 20/53 (31%) pts, including 13/53 (20%) pts with ⩾ 3 cytogenetic abnormalities (with complex karyotype, ck). the median follow-up was 645 (26-2461) days. overall survival (os) and event free survival (efs) were 48% (95% ci 7-33) and 30% (95% ci 17-44) at 4 years, respectively. in univariate analysis, prognostic factors associated with increased os and efs were donor type (match related/match unrelated vs haploidentical; p = 0.02 for both), the disease status at transplant (cr1 vs beyond cr1; p = 0.01, only for efs), acas (aca-vs aca+; p = 0.04, only for os) and, especially, the complex karyotype (ck-vs ck+; p = 0.01, only for os) (figure 1 ). multivariate analysis showed that the independent prognostic factors for os and efs remained the complex karyotype (hr-2.79, 95% ci, 1.23-6.34; p = 0.01) and disease status at transplant (hr-2.15, 95% ci, 1.13-4.09; p = 0.01), respectively. the study demonstrates the acas and disease status at allo-hsct to be independent prognostic factors not only for adult, but for pediatric ph+ all patients too. up to 20% of newly diagnosed acute myeloid leukemia (aml) patients (pts) present initially with hyperleukocytosis consequently placing them at increased risk for morbidity and mortality during induction. 1,2 whereas early publications 3 have indicated that hyperleukocytosis is an adverse prognostic factor associated with poor long term outcome, it is currently unknown whether hyperleukocytosis still retains prognostic value for aml patients undergoing allogeneic stem cell transplantation. furthermore, it is unknown whether hyperleukocytosis retains prognostic value when modern molecular markers such as flt3 and npm1 are accounted for. we hypothesized that hyperleukocytosis at initial diagnosis is still an independent adverse prognostic factor influencing long term outcome in aml pts undergoing allogeneic stem cell transplantation. we performed a retrospective analysis using the multicenter registry of the acute leukemia working party (alwp) of the european society for blood and marrow transplantation (ebmt). pts included in the analysis were over 18 years of age, with de-novo non-m3 aml, a presenting white blood cell count of over 100k, with an hla matched related or unrelated donor, transplanted between 2005 and 2014. clinical outcome indices of hyperleukocyotosis pts namely, non-relapse mortality (nrm), graft versus host disease (gvhd), relapse incidence (ri), leukemia free survival (lfs), overall survival (os) and gvhd-free/relapse-free survival (grfs) were compared to a cohort of pts without presenting leukocytosis. multivariate analyses were used to assess whether hyperleukocytosis was independently associated with ri, nrm, os, lfs, and grfs. age, gender, number of chemotherapy inductions, cytogenetics, donor type, fms-like tyrosine kinase-3 (flt3) status, nucleophosmin (npm1) status, and conditioning intensity were covariates for regression modeling. a cohort of 357 pts with hyperleukocytosis (159 patients with wbc over 50k and less than 100k, and 198 patients with wbc over 100k) was compared to 918 pts without hyperleukocytosis. pts with hyperleukocytosis were younger, had an increased rate of favorable risk cytogenetics, were more likely to be flt3 and npm1 mutated, and had an increased rate of myeloablative conditioning. on univariate analysis pts with hyperleukocytosis had an increased rate of ri (30% vs 22.7%, p = 0.013), and decreased incidence of grfs (36.6% vs 45.3%, p = 0.022). in multivariate regression analysis, hyperleukocytosis was significantly associated with increased ri (hazard ratio [hr] of 1.55, 95% confidence interval [ci], 1.145-2.124; p = 0.004), s399 poorer lfs (hr of 1.38, 95% ci, 1.071-1.785; p = 0.013), decreased grfs (hr of 1.38, 95% ci, 1.117-1.71; p = 0.002), and poorer os (figure 1) (hr of 1.4, 95% ci, 1.073-1.846; p = 0.013). image/graph hyperleukocytosis at initial presentation retains a significant prognostic role for aml patients undergoing allogeneic stem cell transplantation even in the current era of advanced molecular prognostication. outcome after hematopoietic stem cell transplantation for philadelphia-positive aml: relatively favorable outcome in patients allografted in first complete response; a survey from the acute leukemia working party of the european society for blood and marrow transplantation (ebmt) v lazarevic 1 , m labopin 2 , w deipei 3 , i yakoub-agha 4 , a huynh 5 , p ljungman 6 , n schaap 7 , d blaise 8 , jj cornelissen 9 , n maillard 10 , p pioltelli 11 , t gedde-dahl 12 , s lenhoff 1 , m houhou 2 , j esteve 13 , m mohty 2 and a nagler 2,14 aml with t(9;22) and bcr-abl rearrangement (ph-pos aml) is a very rare aml subtype, recognized as a new provisional entity in the recent who 2016 classification. the role of stem cell transplantation (sct) in the era of abl tyrosine-kinase inhibitors (tkis) is mostly unknown. we analyzed long-term outcome in patients ⩾ 18 years after allogeneic or autologous sct performed between 2000-2013 in ebmt centers responding to a designated survey. patients with blast crisis cml and philadelphia-positive all were excluded. primary end-point was os. secondary end-points were nrm, acute gvhd, chronic gvhd, lfs, ri, and the effect of tki on outcome. 65 patients (median age, 48 years, range: 19-67; 31 males and 34 females) with ph-pos aml undergoing sct (allogeneic, 57; autologous, 8) were identified. median wbc count at diagnosis was 57x10 9 /l (1.2-366) and 25% had splenomegaly (data missing on 10 patients). translocation t(9;22) was the sole abnormality in 36 patients (55.5%). the majority of the patients received one or two courses of chemotherapy before transplant and 79% attained cr after one course. the majority (70%) received a tki (mostly imatinib, 34/43) before transplant, with a median period of exposition of 86 days (iqr 60-173), while 21 (34%) received tki after the transplant either as maintenance (n = 11) or treatment for relapse (n = 9). at time of transplant, 56 patients were in complete response (cr1-53, including all autosct; cr2-3) and the remaining 9 patients were allografted in advanced phase. among 40 patients with available information, 14 achieved a mrd negative status at transplant (ratio bcr-abl/abl o10 4 ).regarding allosct, conditioning regimen was myeloablative (mac) in 29/30 (96%) patients o50 years, while in patients 450 years 15 received a reduced intensity regimen (ric) and 9 mac. cell source was peripheral blood stem cells in 39 and bone marrow in 18 allogeneic transplants. the donor was a hla matched sibling (msd) in 32 cases and unrelated (ud) in 25, amongst whom 18 were 10/10 and 7 were 9/10 hla matched, respectively. in the 8 patients undergoing autologous sct the majority received busulfanbased conditioning (n = 6) and peripheral stem cells (n = 7). median follow-up was 6. 36.4%, 46.5%, 59.4%, and 34.9%, respectively. by the univariate analysis, age ( o50 vs ⩾ 50) was associated with ri (5-yr: 22.7 vs 50%), lfs (5-yr: 61.9 vs 31.8%, and grfs (5-yr: 52.4 vs 18.2%), whereas mrd-negative status before allosct was associated with an improved grfs (38.9 vs 16.7%). in the 8 patients autografted, ri, nrm, lfs and os at 5-year was 50% (12.5-79.4), 0%, 50% (15.4-84.6), and 62.5% (29-96), respectively. outcome of patients with ph-pos aml who received allosct in cr in recent years was relatively favorable, especially among younger patients, probably reflecting the beneficial effect of tki. disclosure of conflict of interest: none. outcome of allografting for aml-cr2 is equivalent across the bsbmt and ebmt and is associated with encouraging os and dfs across all age groups j byrne, j perry 1 , k kirkland 1 , r pearce 1 and g jackson 2 1 bsbmt and 2 newcastle university and freeman hospital, newcastle relapsed aml has a very poor prognosis with a high mortality, even if a second cr2 is achieved. the only curative treatment is with an allogeneic hsct but allografts for aml in cr2 are considered to have a worse outcome compared to those performed in cr1, especially in older patients for whom this therapy may not be considered. the bsbmt undertook a bench-marking study analysing the outcomes for all patients allografted for aml-cr2 from 2006 to 2011. the uk outcomes from 534 paediatric and adult patients were compared to 3070 non-uk patients transplanted for the same indication reported to the ebmt during the same period. allogeneic transplants for aml-cr2 represent an important part of any allograft program and numbers referred for allograft were stable between 2006-2011 in both programs. the median age of patients was 45.8 yrs and 43.3 years in the bsbmt and ebmt cohorts respectively, with 14% and 15% of patients aged o 18 years and 19% and 16% aged 4 60 years in the 2 groups. the length of first remission was missing in many of the ebmt registrations so time from diagnosis to transplant was used as a surrogate for this and was similar in both cohorts (18 m and 19 m respectively). similarly the presence of comorbidities was poorly reported in both databases but was similar. the bsbmt cohort included fewer patients undergoing ric conditioning protocols (55% vs 66%), fewer sibling transplants (25% vs 38%) and more pbsc allografts (79% vs 20%). transplant related morbidity and mortality were similar across the two cohorts (bsbmt v ebmt) with rates of severe acute gvhd (grade iii and iv) 6% v 10%, limited chronic gvhd 35% v 28%, relapse at 1 year 22% v 20% and death in cr at 1 year 20% v 18%. chronic gvhd (both limited and extensive) appeared more common in the bsbmt cohort (50% v 36%) although reporting of cgvhd was more comprehensive and complete within the bsbmt registry and may be under-reported in the ebmt registry. outcomes were excellent in both cohorts with outstanding rates of leukaemia free survival in this high risk cohort at day 100 (bsbmt v ebmt) 86% v 83%, at 1 year 63% v 67%, at 3 years 49% v 53% and at 5 years 41% v 43%. although os and lfs was significantly shorter in patients aged 4 60 years, at 37% and 30% the results in this high risk age group are acceptable and warrant its continued use. multivariate analysis of the combined cohorts showed that age at transplant ( o 18 yrs/18-60/460 yrs), time from diagnosis to transplant and the presence of agvhd were important factors affecting dfs. risk factors for relapse included the type of conditioning used, presence of agvhd and time from diagnosis to transplant, whereas those for trm included age, agvhd, source of stem cells and time to transplant. there was no significant difference in outcomes between the bsbmt and ebmt for this indication. outcomes for patients allografted for aml-cr2 are excellent and appear superior to those reported for patients not undergoing an allograft in both the bsbmt and ebmt cohorts. the os and dfs observed are comparable to those reported for allografts in aml-cr1 and, although this study has not considered outcomes for patients who did not achieve a 2 nd cr, it nevertheless supports the practice of risk stratification of aml patients such that only high risk patients are offered an allograft in cr1 with the remainder being offered an allograft as salvage after relapse. disclosure of conflict of interest: none. to evaluate the efficacy and safety of intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation (allo-hsct) for atl. a total of 21 evaluable atl patients treated at our center from 1995 to 2015 are retrospectively analyzed. the htlv-i proviral dna load in peripheral blood mononuclear cells using pcr assay was developed, in comparison with htlv-1 tax protein expression measured by western-blot, to confirm the diagnoses and monitor the disease control. 13 patients were male and 8 patients were female. median age was 50.8 (range 28-66) years. all obtained patient samples were subjected to flow cytometric examination and karyotype analysis. 19 patients received chemotherapy as the induction therapy while 2 quit at the time of diagnosis, 5 with da-edoch regimen while 14 with other regimens such as chop, vcap and amp. da-edoch regimen is a variation of dose-adjusted epoch regimen with the replacement of prednisone (60 mg/m 2 per day) by dexamethasone (15 mg/m 2 per day). before the conventional regimens bucy followed by prophylaxis donor lymphocyte infusion, both received a course of salvage chemotherapy including fludarabine and cytarabine for 5 days registered on http://clinicaltrials.gov (nct02328950). the gvhd prophylaxis consisted of atg, csa and mtx. the patient characteristics, therapeutic effect and survival data were collected. all patients came from the coastal area in the south-east of china. subtype classification of these 21 atll were 18 acute, 2 lymphoma and 1 chronic type. the main manifestations were characterized with cutaneous and respiratory involvement, hepatosplenomegaly, lymphadenopathy and the laboratory abnormalities as leukocytosis with atl cells, hypercalcemia and elevated serum ldh. typical morphological characterisitic of "flower cells" were observed in 85.7% cases and most of the atll cells are cd4+cd8-. chromosomal abnormalities were detected in 4 cases. all 14 patients who didn't receive da-edoch regimen died of disease progress, while among 5 patients with da-edoch regimen, 2 achieved cr, 2 pr and 1 died. with a median follow-up of 18.6 (10-24.1) months, 3 patients respond to da-edoch are still alive. 2 patients in cr achieved successful engraftment with complete donor chimerism in one month post haplo-identical transplant. both were received prophylaxis donor lymphocyte infusion and the immunosuppressive agents were abruptly discontinued for induction of a graft-verus-atl (gvl) effect. they keep remain alive and disease free longer than 2 years so far without severe graftversus-host (gvhd), and the htlv-1 proviral dna became undetectable after allo-hsct. conclusion: it shows great promise of da-edoch regimen followed by allo-hsct to the long-term cure of atl with apparent clearance of the virus. haplo-identical transplantation can be an alternative option for the atl patients without increasing non-relapse mortality. [p537] disclosure of conflict of interest: none. in the absence of an hla-matched related donor or a good matched unrelated donor in time, haploidentical stem cell transplantation (haplo-sct) is an option for patients requiring an allogeneic hematopoietic stem cell transplant. substantial progress has been made in the last two decades with a dramatic improvement in patient outcomes, with some groups reporting preliminary beneficial effects similar to the ones in hla matched unrelated donor and cord blood transplant. several strategies have been adopted through the years for the procedure. the two strategies used in haplo-sct are ex vivo t-cell depletion and t-cell replete transplantation. the latter can be done with a combination of immunosupressive drugs ( beijing approach) or with post-transplantation highdose cyclophosphamide (post-cy). due to of its lower cost, feasibility and practicality, post-cy has become the most often used platform for haplo-sct in the majority of allogeneic transplantation units worldwide. we analyzed our experience in haplo-sct, since the first one in march 2104 to the last one that has just been done in october 2016. we collected all complications reported, also mortality related to treatment and to the disease. we analyzed the overall survival (os). 9 transplants were treated, with different sct indications, the most common being acute myeloblastic leukemia (n = 5, 55%), the rest of indications are exposed in figure 1. all our patients, independent of the conditioning receive post-cy as t cell depletion measure and stem cells were collected from peripheral blood. age at the time of transplant was 40.6 years, 77% were males, 23% females, the rest of patient characteristics are listed in table 1 . in our series the treatment related mortality (trm) was low with only 1 patient (11%) that died before the day +100. as complications, we reported 33% of hemorrhagic cystitis, 22% of sinusoidal obstruction syndrome, 22% reports systemic inflammatory response syndrome, 55% of citomegalovirus (cmv) reactivation. neutrophils graft is 18.2 days (r = 14-23) and the platelets graft is 24.2 days (r = 13-34). in our series we haven't reported any case of graft failure, one of the transplantes the patient had antihla antibodies, this was treated with a plasmapheresis previous the stem cell infusion, and was infused with a high number of cd34+ cells (8 × 10 6 /kg), no graft problems, and has had no complications since then with the graft. the os for the whole group is 22 months, with a median not reach at 36 months, with 2 patient's dead at time of analysis. two patients had a relapse after the haplo-sct (22%), both of them received lymphocyte donor infusion, sadly, neither of them responded. the haplo-sct procedure is been adopted by many centers for high risk hematology malignancies, mainly because the fast availability of donors, and because of the preliminary results that have been reported place it as good as the unrelated donor or cord unit transplant. our center is getting experience in these types of transplants, and our results reflect similar outcomes as larger studies. with longer follow ups we will be able to keep the trend of good results both in procedure safety and disease efficacy. os, toxicity and trm are expected for these high risk malignancies. in overall, the haplo-sct seems a reasonable technique that is reflecting in our short series, the results being reported in studies worldwide at bigger scale. disclosure of conflict of interest: none. complex karyotype, the presence of flt-3 itd and losses of genetic material in chromosome 7, are all considered high risk markers in aml. patients bearing these abnormalities could undergo a myeloablative allogeneic transplantation in cr1 whenever this is possible, although this could significatly reduce the chances for cure in older patients due to increased transplant related mortality. ric allografts could be performed in older patients in order to overcome the deleterious effects of these individual abnormalities but its effect still remains controversial in the high risk group. between 2005 and 2015, a total 65 patients [41 males, 24 females, mean age 59.05 (40-73)] received a ric allograft (49 from fully matched unrelated donors, and 16 from an identical sibling) for high risk aml in 2 transplant centres. high risk disease was classified according to their response to treatment, the presence of complex karyotype, the presence of individual cytogenetic/molecular abnormalities or a combination of these. in particular, 24 patients presented one single karyotype abnormality and 17 presented three or more. ten patients presented alterations of chromosome 7 and 7 patients presented flt-3 itr. the conditioning regimes included fludarabine in all cases, together with melphalan and campath (29 patients), busulphan and campath (21 patients), busulphan and thiotepa (1 patient), melphalan (3 patients), busulphan with and without atg (9 patients) total body irradiation (200 cgy, 2 patients). graft versus host disease prophylaxis was ciclosporin for patients receiving alemtuzumab or atg but for patients who had a t-replete allograft ciclosporin and low dose methotrexate was the preferred prophylaxis. all but four patients were transplanted in cr1 patients were followed-up for a mean 32.3months (range 3-150). thirty-one (47%) patients remain alive. the causes of death (34 cases) include relapse or progression of the original disease (13), transplant-related causes (17) and unknown in 4 cases. the influence of the genetic abnormalities on survival was analysed, showing there were no significant differences between patients with normal karyotype, single chromosomal abnormalities and two or more abnormalities. likewise, the kaplan-meier survival analysis of patients bearing flt-3 itd was not significantly different to the rest of the cohort (p = 0.4; 3/7 died, with only one case being related to relapse). patients bearing chromosome 7 abnormalities (with or without other chromosomal aberrations) had a comparable, not significantly (p = 0.6) different survival to the rest of the cohort: 6/10 patients bearing this abnormality died although, most interestingly, none of these deaths were related to relapse. our results indicate ric allografts can provide an adequate consolidating effect in hr aml with complex karyotype, alteration of chromosome 7 or flt-3 itr, yielding clinical results that are comparable to those obtained in patients with aml allografted for other indications. this is particularly important as these alterations are more frequent in patients whose age prevents them from having myeloablative grafts. disclosure of conflict of interest: none. early detection of inapparent replicative human cytomegalovirus (hcmv) infection together with its preemptive antiviral treatment has led to a marked reduction of life-threatening hcmv disease after allogeneic hematopoietic stem cell transplantation (allosct). we first reported in a retrospectively performed study that hcmv reactivation is associated with a reduced risk for relapse in patients with aml after transplant. now, we evaluate the impact of early hcmv replication on the risk for leukemic relapse in patients with aml after t cell depleted transplantation in a prospectively performed observational study (registration trial drks00004300). between january 2012 and march 2015 we enrolled 251 patients with aml in this trial who were consecutively transplanted at the university hospital of essen. 239 patients received a myeloablative regimen (tbi based conditioning n = 83, chemotherapy based conditioning n = 156) and 12 patients a ric (n = 12 chemotherapy based regimen). patients were transplanted in 1.cr (n = 123), 2.cr (n = 51) or more progressive disease stages (n = 77) from hla-identical sibling donors (n = 60) or hlaidentical unrelated donors (urd) (n = 126) or mismatched unrelated donors (n = 65). patients who received a second transplant were excluded from the study. the median age of patients was 54 years (range 18-72) and that of the donors 35 years (range 14-69). gvhd prophylaxis was performed with mtx and csa, or csa and mmf with (n = 182) or without atg (n = 69) (30-60 mg total dose). the incidence of acute gvhd grade 2-4 was statistically not different in both groups (46% versus 40%). hcmv-reactivation (hcmv-r) detected by pcr occurred between 23 and 87 days (median 45 days) after allo sct. only patients surviving day 60 after transplant were included in the study for estimation of relapse incidence (cir) or overall survival (os). hcmv status of recipients (r) or donors (d) were in 29% r-/d-, 12% r-/ d+; 28% d+/r-and 31% r+/d+. patients with a documented hcmv-r had a cir at 4-year after transplant of only 30% as compared to 47% in patients without a hcmv-r (p = 0.016). estimates for 4-year os were in favor for patients with hcmv-r (61% for patients with hcmv-r versus only 48% for patients without hcmv-r), but this did not achieve statistical significance. non-relapse mortality was greater in patients with hcmv reactivation (23% versus 12%, ns) a substantial and independent reduction of relapse risk associated with early hcmv replication was confirmed by multivariate analysis including competitive factors as unfavorable cytogenetics according to eln, advanced disease stages of aml, hla-identical donor versus mismatched donor, sibling versus unrelated donor, presence of acute gvhd grades ii-iv, chronic gvhd, and hcmv-r [(hazard ratio: 0.61, 95% ci: 0.38-0.98, p o0.042) for occurrence of hcmv-r]. the final result of this first prospective performed study confirms an independent advantageous effect of early hcmv replication on the leukemic relapse risk in patients with aml after transplant. disclosure of conflict of interest: none. . primary engraftment of wbc and platelets was achieved in 64 pts, one patient (pt) died at day +3 after hsct, and one had a secondary graft failure. a median time to wbc engraftment was 13 days (9-27), to platelets -14 days (9-32). cumulative incidence (ci) of acute gvhd (agvhd) grade ⩾ ii was 25% (95% ci:16-38): from haplo-23%(12-45), from mud 26% (15-45),p = 0.7. ci of agvhd4iii 8%(95% ci:3-18): haplo vs mud -7% (95% ci:2-25) vs 9%(95% ci:3-25), respectively,p = 0.7. ci of chronic gvhd (cgvhd)-16% (95% ci:9-28). ci of agvhd was significantly lower in a group with regimen 2 (2014-2016) of gvhd prophylaxis: 10% (95% ci:4-25) vs 32%(95% ci:18-57), po 0.0001. regimen 2 was also effective in prevention of cgvhd: ci at 2 year after hsct was 10% vs 24%, p = 0.14. сi of trm was 12%(95% ci:6-24): haplo-7%(2-25), mud -14% (6-32). early mortality (before +100-day) was relatively low (n = 3): 1 pt died from bacterial sepsis; two pts died due to adv infection. thirteen pts died after 100 days: 9 pts relapsed and died due to complications of second hsct; bacterial sepsis in 1 pt and viral infection (adv and cmv) in 3 pts (2 with extensive chronic gvhd). ci of relapse was 24% (95% ci:15-38) at 2 year: from haplo-12% (95% ci:4-36), from mud-32% (95% ci:20-52),p = 0.086. event-free survival (efs) at 2 years was 64% (95% ci: 52-77): haplo -81%(95% ci: 66-97), mud -54% (95% ci:37-70), p = 0.06. os was 70% (95% ci: 58-82) at 2 years: haplo-77% (95% ci: 57-77), mud -65% (95% ci: 48-81), p = 0.3. relapse-gvhd-free survival at 2 years was different among recipients of haplo and mud hsct: 51%(95% ci: 28-75) vs 32% (95% ci: , p = 0.3. we confirm that the depletion of tcrαβ+/cd19+ depletion from the graft ensures high engraftment rate and acceptable transplant-related mortality in pediatric aml pts. there is a trend towards better efs for haploidentical transplantation. gvhd prophylaxis including ratg/rituximab/bortezomib improves gvhd control s404 in recipients of tcrαβ+/cd19+depleted grafts in comparison to hatg/tacro/mtx apparently without loss of anti-leukemic activity. disclosure of conflict of interest: none. results of t-cell depleted haploidentical stem cell transplantation in adults with acute leukemia improve with time: a study from the acute leukemia working party of the european society of blood and marrow transplantation (ebmt) s simona 1,2 , m labopin 3 , a ruggeri 1,3,4 , a velardi 5 , f ciceri 6 , j maertens 7 , l kanz 8 , f aversa 9 , d bron 10 , d bunjes 11 , m mohty 1,3 and a nagler 3, 12 t cell-depleted (tcd) transplants from haploidentical donors are increasingly used in the absence of a hla full-matched donor for patients (pts) with high risk acute leukemia (al). progress has been made in optimization of conditioning regimens and post-transplant cellular therapy to potentiate the graft-versus-leukemia effect with no graft-versus-host disease (gvhd). however, relapse incidence (ri) and non relapse mortality (nrm) remain the main obstacles for pts outcomes. we report 308 adults with de novo al, given a tcd haplo from 2005 to 2015. to analyze the effect of transplant period on tcd haplo outcomes, pts were analyzed in two separate groups: 2005-2011 (n = 191) and 2012-2015 (n = 117). tcd haplo were performed in 66 ebmt centers. median follow-up was 11 months with no difference according to transplant periods. median age was different between groups, being 41 and 46 years respectively (p = 0.044). the majority of pts had acute myeloid leukemia (aml) (75% vs 69%, p = 0.261) and disease status at haplo was first complete remission (cr1) in 55% and 64% of pts respectively (p = 0.115). pts transplanted before 2012 had more frequently a karnofsky performance status o90% (19% vs 10%, p = 0.041). conditioning was myeloablative in 76% and 77% of tcd haplo before and after 2012 (p = 0.935), mainly based on fludarabine(flu)-tbi, flu-melphalan-thiotepa or cyclophosphamide-tbi. as for ric it was flu-melphalan-thiotepa, flu-tbi or cyclophosphamide-tbi. the cumulative incidence (ci) of neutrophil engraftment, grade ii-iv acute gvhd and chronic gvhd were not different according to transplant period, being 93% and 90%, p = 0.302; 20% and 22%, p = 0.667; 19% and 11%, p = 0.119, respectively. in the whole population 2-year ri and nrm were 20% and 48%, with no difference before and after 2012 (21% vs 19%, p = 0.722; 54% vs 38%, p = 0.109, respectively). ri was 20% before 2012 versus 15% after 2012. the main cause of nrm was infection, with no difference over time (46% vs 50%, p = 0.316). in multivariate analysis, disease status was the only factor associated with ri (hr 2.05, 95% ci 1.09-3.84, p = 0.025). tcd haplo after 2012 (hr 0.57, 95% ci 0.38-0.86, p = 0.008), younger age (hr 0.82, 95% ci 0.63-0.98, p = 0.023) and ric (hr 0.53, 95% ci 0.32-0.88, p = 0.014) were independently associated with lower nrm. 2-year os was 36% with a marked improvement for tcd haplo performed after 2012 (47% vs 29%, p = 0.024), while lfs and grfs were 31% and 24%, respectively. according to disease status, 2-year lfs, os and grfs were higher for pts transplanted in cr1 (38% vs 22%, p o0.001; 41% vs 29%, p = 0.006; 30% vs 17%, respectively p = 0.004). in multivariate analysis, tcd to further establish the role of haplosct in high-risk aml, we performed a retrospective matched-pair comparison of hlamatched sct vs haplosct/ptcy in two german centers. highrisk aml was defined by any of the following criteria: refractory or relapsed aml, secondary aml, or genetic aberrations classified as intermediate-high or adverse accordingly to the eln classification. all consecutive adults, who fulfilled ⩾ 1 of these criteria before either hla-matched or haplosct/ptcy were included (n = 200). recipients of haplosct were pair-matched with patients receiving a matched donor sct. matching variables were (1) stage at sct, (2) genetic subgroups accordingly to eln, and (3) age (±5y). 39 patients (pts) undergoing haplosct/ptcy could be successfully pairmatched (p = 1.0 for stage and genetic subgroup, 0.9 for age) with 39 recipients of matched sct (12 family, 27 unrelated sct). within the entire cohort, median patient age was 57y (24-70). at start of conditioning, 20% of patients were in cr, 18% had refractory, 52% had relapsed, and 10% had untreated disease. genetics were favorable (16%), intermediate i (51%), intermediate ii (12%) and unfavorable (21%). hla-matched sct was uniformly performed following flamsa-ric. 34 recipients of haplosct/ptcy (87%) received cytoreductive chemotherapy with flamsa (n = 16) or clofarabine (n = 18) before ric was started. median follow-up among survivors was 33 months. overall cr rate at d+30 was 95%, 4 patients suffered from refractory disease or early death, (n = 2 each). overall-survival (os) for the entire cohort was 74%/53.9% at 1y/3y from sct. the corresponding 1y/3y leukemia-free survival (lfs) was 63.5%/46.6%. median time to engraftment was 18.0 and 17.5 days after matched and haplosct, respectively (p = 0.8). with respect to outcome, no difference was observed between the two groups: os at 1y/3y was 78.5%/54.5% after matched sct, and 61.5%/55.2% after haplosct/ptcy (p = 0,71, figure 1 ). lfs at 1y/3y was 76.2%/ 42.6% within the hla-matched group and 56.4%/50.8% within the haploidentical group (p = 0,99). recipients of haplosct showed a higher incidence of agvhd ⩾ ii°(46% vs 18%, p = 0.014), as well as a trend towards increased 1y-nrm (18% vs 5%, p = 0.08), whereas 1y-relapse rates were comparable (23% after haplosct/ptcy vs 26% after matched sct, p = 0.5). relapse was the most frequent cause of death in both cohorts, main causes of nrm were gvhd and infections (no difference between the two groups). allogeneic sct following sequential conditioning can achieve excellent results in high-risk aml. in our study, results after haploidentical transplantation were comparable to those obtained after hla-matched sct. hence, haplosct/ptcy following sequential conditioning can be considered as a reasonable option for patients with high-riaml. [p544] disclosure of conflict of interest: none. a significant proportion of patients with acute myeloid leukemia ( aml) will either be refractory to initial chemotherapy or will suffer refractory relapse. the role of allogeneic transplantation (hct) in active disease is contentious. there is a growing body of literature that sequential chemotherapy, pioneered by the german flamsa regimen, followed by ric hct is a safe and efficacious modality in these patients, and there have been numerous modifications of this regimen, especially as amsacrine is not widely available. fludarabine, cytarabine and etoposide (vp16) (flav) have been reported as an an effective salvage regimen. here we report on single center outcomes of a variation of the flamsa regimen, substituting amsacrine for etoposide with mainly myeloablative conditioning. patients were consented for a clinical protocol if they had aml that was refractory to 2 cycles of chemotherapy, or 1 cycle and considered to be at risk of complications of a second cycle, and if they had a matched related donor. patients with myelodysplasia received flav if they had high or very high risk cytogenetics. cytoreductive chemotherapy consisted of fludarabine 30 mg/m 2 / day × 4 days, cytarabine 2g/m 2 /day × 4 days, etoposide 100 mg/m 2 /day × 3days, commenced simultaneously. after 3 days of rest, conditioning chemotherapy consisted of fludarabine 30 mg/m 2 × 2 days and and iv busulfan 0.8 mg/ m 2 q 6 hours; the number of busulfan doses varied between 8-12, depending on patient comorbidity. ten patients (76%) received myeloablative doses of busulfan. patient received 2 doses of atg at 2.5 mg/m 2 /day on day -3 and -2. patients received gcsf mobilized peripheral blood hematopoietic cells. post-transplant gvhd prophylaxis was csa and mmf. csa was tapered from day+60 and stopped at day +90 in the absence of gvhd. mmf was discontinued between day +30 and day +40. donor lymphocyte infusions were collected for planned prophylactic dli. thirteen patients received a flav-sct between march 2014 and october 2016. the median age was 39(15-57); male:female ratio was(7: 6). 10 patients (77%) had aml and 3 (23%) pts had mds. all patients had detectable disease prior to flav. the median time for plt engraftment was 19 days (9-50). the median time for anc engraftment was15 days (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) . cytogenetic cr rate on a day 28-30 bone marrow was 46%, and morphological cr was 60%.3 patients (23 %) developed veno-occlusive disease. acute gvhd grade ii-iv occured in 4 pts ( 30%). 3 (23%) patients developed chronic gvhd. death was due to disease relapse in 5 (38%) and nrm in 1(7.7%) patients, resulting from h1n1 pneumonia. 5 patients (38%) received dli for post transplant relapse, and one of these is in molecular remission. at a median follow up of 4.8 months post transplant (1.6-29m), 1 year dfs was 23%. the 1 year and 2 year os was 51% (±14%) (figure 1 ). our experience, consistent with published data, demonstrates that for patients with active aml refractory to chemotherapy, transplantation is an effective modality of disease control and may be the only curative therapy in a significant proportion. etoposide appears to be a suitable alternative to amsacrine. our patients tolerated busulfan at myeloablative doses, and this may be required for adequate disease control. our report is limited by small numbers and relatively short follow-up, but is encouraging enough for us to continue offering flav hct for these high-risk patients. [p545] disclosure of conflict of interest: none. sequential high-dose chemotherapy reinduction followed by haploidentical transplantation in acute leukemias l brunello 1,2 , cm dellacasa 1 , l giaccone 1,2 , e audisio 3 , d ferrero 3,2 , s d'ardia 3 , b allione 3 , s aydin 3 outcomes after t-cell replete haploidentical stem cell transplantation (haplo hsct) have been encouraging and haplo hsct has become an alternative option for patients without a hla-identical related or unrelated donor. 1,2 as previously published, the sequential use of intensive chemotherapy and allogeneic transplantation represents a possible approach to the treatment of high-risk acute myeloid leukemia (aml). 3, 4 between 2010 and 2015, 19 acute leukemia (al) patients received sequential therapy (s.t.) consisting of high-dose chemotherapy reinduction and haplo hsct during the chemotherapy-induced neutropenia. median age at transplant was 50 years (range: 21-62); median number of previous therapy lines was 2 (range: 1-3) and 5/19 (26%) patients had received a previous allogeneic hsct. twelve and 5 out of 19 patients had de-novo aml and secondary aml, respectively; furthermore two patients presented blastic crises of chronic myeloid leukemia. all patients had active disease at the time of s.t. and median marrow blast count before reinduction was 25% (range: 6-88%). hematopoietic cell transplantation comorbidity index was ≥ 3 in 12/19 patients (63%). all patients received high-dose cytarabine (≳1 g/sqm) containing regimens as reinduction therapy. the conditioning regimen was started after a median of 9 days from the end of reinduction (range: 4-15). sixty-eight percent of patients (13/19) were conditioned with a myeloablative regimen (thiotepa tot. 10 mg/kg, busulfan tot. 9.6 mg/kg, fludarabine tot. 150 mg/ sqm), while 6/19 (32%) patients received a non-myeloablative conditioning. bone marrow was used as stem cell source in 18/19 (94%) patients. graft-versus-host disease (gvhd) prophylaxis consisted of post-transplant cyclophosphamide with calcineurine inhibitors and mycophenolic acid. all patients engrafted but one, who was rescued with a second haplo-hsct with peripheral blood stem cells from the same donor. median day of neutrophil recovery was day +18 (range: 14-24). median follow-up of survivors was 4.2 years (range: 1.6-5.4); 1-year overall and event-free survivals were 37% and 32%, while 1-year relapse incidence and non relapse mortality were 42% and 26%, respectively. overall cumulative incidences of acute and chronic gvhd were 39% and 38% at day +100 and +400. among patients who developed gvhd, 2 grade iii-iv acute gvhd and 2 moderate-severe chronic gvhd were observed. at 4.2 years post haplo-hsct, 20% of patients are alive and disease free. in our cohort of heavily pre-treated and high-risk patients, s.t. with a myeloablative conditioning was safely used to reduce leukemic burden pre-transplant and enhance graft-versus-leukemia effects. only the prompt availability of a haploidentical donor allowed to implement this treatment modality. though small the patient cohort, our findings suggest that transplant-related toxicity was acceptable and early relapse was the major treatment-failure. however, long-term survival and disease-free rates of 20% in these very poor prognosis patients are highly encouraging. in elderly patients with acute lymphoblastic leukemia (all) and kinase activating lesions allogeneic stem cell transplantation (allo-sct) is considered to be the only curative option. 1 however, high risk of relapse and non-relapse mortality (nrm) often withholds elderly patients with existing comorbidities from definitive therapy. 2 even though, age alone as the most important eligibility criterion for allo-sct has become less important. 3 a 61-year old patient with a medical history of adipositas, hypothyreosis, arterial hypertension, hypercholesterolemia and coronary artery disease with stent implantation was diagnosed with common-b-cell-all based on immunophenotype. cytogenetic analysis showed two coexisting clones with an abnormal karyotype of 47,xx; t(1;9)(q22;q34), +17 [3] and 46,xx; t(1;9)(q22;q34),der(6)(6;17)(p23;q21) [2] and no evidence of bcr-abl1 positive disease using fluorescence in situ hybridization (fish) technique. rt-pcr and sequencing of the fusion transcript was performed to validate the rcsd1-abl1 t(1;9)(q22;q34) fusion between exon 3 of rcsd1 and exon 4 of abl1. western analysis of phosphorylated abl1 and its downstream target crkl was performed to investigate the in vivo activity of dasatinib. clinical monitoring of minimal residual disease (mrd) levels has been performed via rt-pcr of the rcsd1-abl1 fusion transcript followed by nested pcr of the amplicon to detect early relapse or mrd. as positive control the plasmid pcr2.1-topo_rcsd1-abl1(626bp) was synthesized encoding rcsd1-abl1 amplicon with the fusion site. our patient was enrolled on gmall elderly (01/2003) and treated accordingly. thereby, no sustained remission could be achieved. flag-ida re-induction and study treatment with an oral pi3k/mtor inhibitor remained futile. cytogenetics and verification of the rcsd1-abl1 fusion gene prompted salvage treatment with the tyrosine kinase inhibitor (tki) dasatinib as single agent. the in vivo activity of dasatinib was highlighted by a decrease of rcsd1-abl1 amplicon and inhibition of phosphorylated abl1 and its downstream target crkl was shown. clofarabine and cyclophosphamide complemented treatment and mrd negative remission was achieved due to administration of the bi-specific t-cell engager blinatumomab. consolidation with allo-sct was performed. ongoing remission has been achieved for more than 30 months now. we demonstrate that monotherapy with tki like dasatinib is effective in refractory rcsd1-abl1 positive all. to the best of our knowledge, this is the first elderly patient with rcsd1-abl1 positive all with a sustained and ongoing complete remission. thereby, we suggest allo-sct after successful tki even for elderly patients with existing comorbidities and uncommon cytogenetics. relapse is the most important cause of failure of allogeneic hematopoietic stem cell transplantation (hsct) for flt3-itdpositive acute myeloid leukemia (aml). in those cases, treatment with flt3 tyrosine kinase inhibitors (tki) constitutes a promising clinical approach to induce remission without conventional chemotherapy. forty-eight-year-old woman was diagnosed of aml secondary to myelodisplastic syndrome with npm1 mutation and internal tandem duplications of the flt3 gene (flt3-itd). after achieving complete remission (cr), she received a sibling allogenic-hsct. four months later, aml relapsed at the medullary level, without central nervous system (cns) involvement, treated with conventional chemotherapy and donor lymphocytes infusions (dli). she achieved second cr and developed chronic graft-versus-host disease (cgvhd). nine months later, she suffered the first extramedullary relapse, at the mammary, cutaneous and probably pericardial levels. there was not medullary involvement. disappearance of the lesions at all levels was achieved with conventional chemotherapy and radiotherapy, and full donor chimerism. eight months later, she referred atypical precordial pain irradiated to the back. cardiac mri was performed in which several masses were visualized in a pericardial sac up to 5 cm in diameter (dec-15). bm was maintained in cr. in study of pericardial fluid, infiltration by leukemic flt3 positive cells was observed. the patient was not considered candidate for further systemic chemotherapy nor radiotherapy treatment. then, treatment with the flt3 sorafenib inhibitor was started, by compassionate use, at dosage of 200 mg/12 h, which maintains after one year. after first month with sorafenib, pericardial lesions decreased considerably, ranging from 5 cm in diameter to 1.7 cm (jan-16). in the subsequent ct controls, progressive decrease of the lesions has been observed and no new lesions have appeared in other locations. in the last ct (oct-16) pericardial thickening is almost non-existent, without new lesions. after one year of treatment, she maintains cr at medullary and extramedullary levels images. in our patient, treatment with sorafenib has achieved sustained control of extramedullary disease, which had escaped the mechanisms of action of conventional chemotherapy, allotransplant and dli. further studies are needed to corroborate the efficacy of flt3 inhibitors in the control of aml extramedullary disease and in the treatment of relapses after allo-hsct. all pts with tbi-based regimen received rabbit atg. tcrαβ+/cd19+ depletion of hsct with clinimacs technology was implemented in all cases according to manufacturer's recommendations. the median dose of cd34+ cells in transplant was 10 × 10 6 /kg (range: 3.9-18.8), tcrα/β-23 × 10 3 /kg (range: 0.2-300). primary engraftment was achieved in 68 of 71 pts. (2 pts died before engraftment, one received second hsct), the median time to neutrophil and platelet recovery was 13 and 14 days, respectively. early (100 day) mortality was 7%(95% ci: 3-17), 2-year ptrm-17% (95% ci: 10-30). the three early deaths were due to bacterial sepsis (n = 2) and viral infections(n = 1), seven late: viral infection in 4 pts (adv = 2, adv+cmv = 1, cmv = 1), bacterial sepsis in 2 pts and rhinocerebral mucormycosis in 1 pt, all late deaths were associated with cgvhd and prolonged corticosteroid therapy. ci of acute gvhd grades ii-iv and iii-iv was 22.5% (95% ci: 9.6-53), and 7% (95% ci: 1.3-37), respectively. ci of cgvhd was 17.6% (95% ci: 6.4-48). regimen 2 was more effective in prevention of agvhd ii-iv: ci at 2 year after hsct was 14% vs 35,7% in regimen 1, p = 0.033 and in cgvhd 7% vs 35.7%, p = 0.006. ci of relapse at 2 years was 32% (95%ci:15.7-64.5). two-year pefs(event = death or relapse) was 49.9% (95% ci: 37-62), 2-year pos-54% (95% ci:41-67). in patients, who received tbi-based conditioning pefs was 59% (95% ci: 38-81), as compared with treosulfan-based 47% (95% ci: 32-61), p = 0.65. median time of follow-up for survivors was 2.2 years (range: 0.3-4.4). we confirm that the depletion of tcr-alpha/ beta and cd19 lymphocytes from the graft ensures high engraftment rate and acceptable transplant-related mortality in pediatric all patients. viral infections and leukemia relapse await further improvement of control. all major outcomes were equivalent between transplantation from unrelated and haplo donor. disclosure of conflict of interest: none. the prognosis of patients (pts) with relapsed/refractory acute lymphoblastic leukemia (all), especially after allogeneic hematopoietic stem cell transplantation (allo-hsct), is very poor. therapeutic options for these pts are limited. blinatumomab is a bispecific t-cell engager (bite) antibody construct with dual specificity for cd19 and cd3. bite therapy may help to overcome the resistance to chemotherapy (ct) with minimal toxicity, and may be a bridge to allo-hsct. we analyzed data of 34 pts from 4 hematologic centers in russian federation with relapsed cd19 positive all, who received bite. the median age was 22 (range: 1-62) years, 9 (26%) pts o18 yrs, 25 (74%) pts ≥ 18 yrs. the diagnosis was all b-i (egil) subtype in 11 pts, b-ii-in 16, b-iii-in 5 pts, and 1 patient had mixed phenotype leukemia (m5 (fab) and b-all). three (9%) pts had philadelphia positive (ph+) all. in 11 pts it was the first relapse of all, in 13-second, in 10-third. thirty pts had isolated bone marrow relapse, 4 pts-combined relapse (bone marrow and extramedullary sites). the bone marrow blast infiltration was o 50% in 15 pts, 450% in 19 pts. disease relapse was revealed after ct in 19 pts (7 (37%) pts received allo-hsct after the therapy of relapse), after allo-hsct (3-from related, 9-from unrelated, 3-from haploidentical donors)-in 15 pts (4 pts received second allo-hsct after the therapy). in 8 pts with posttransplant relapse donor lymphocyte infusion (dli) was used in combination with bite. every patient received from 1 to 7 cycles (median 2) of bite. complete remission (cr) was achieved in 18 (53%) pts (in 14 (41%) pts it was minimal residual negative remission): in 8 (42%) pts with all relapse after ct, in 10 (67%) pts-after allo-hsct. pts with less than 50% blasts in bone-marrow at baseline experienced substantially higher response rates compared with patients with 50% blasts or higher (67% (10/15) vs 52% (10/19)). response rates were similar although the number of relapse-45% (5/11) in first relapse, 61% (8/13) in second relapse and 70% (7/10) in third relapse. pts with posttransplant all relapse who received bite in combination with dli had higher response rate than pts, who received bite as monotherapy: 87.5% (7/8) vs 43% (3/7), respectively. one-year os was 50% (95% ci 23-77%). one-year dfs was 48% (95% ci 21-75%). grade ≥ 3 hematological toxicity (neutropenia, thrombocytopenia) was observed in 13 (38%) pts, grade ≥ 3 liver toxicityin 4 (12%) pts. five patients (15%) developed toxic neuropathy during the therapy. cytokine release syndrome occurred in 3 (9%) pts. one patient after allo-hsct (but not after dli) developed grade i agvhd. there were no fatal treatment related toxicity. tree (17%) pts who responded to bite had relapse. eighteen (53%) pts died: 14 pts-of disease relapse/ progression. the treatment of relapsed/refractory all with bite is effective and has acceptable toxicity. we demonstrated high efficacy in therapy of posttransplant all relapses, especially when bite was combined with dli, perhaps, due to additional immunological effect of the transplant. disclosure of conflict of interest: none. the mechanism of sorafenib anti-leukaemic effect seen in aml patients relapsing post allohsct involves the augmentation of alloreactivity which includes infiltration of the affected marrow by cd8+ cells having pd-1 receptor which presence characterize lymphocytes with antitumour potential multikinase inhibitor (mki) sorafenib is clinically active in acute myeloid leukaemia (aml) patients, especially in those with flt3 itd who receive allohsct as a part of their primary treatment. to throw some light on the mechanism of this antileukemic post-transplant sorafenib effect we studied the fate of two patients (flt3 itd-positive, npm1-positive) who relapsed 56 (53-year-old male) and 256 (50-year-old female) days post-transplant and their salvage treatment included sorafenib. the multikinase inhibitor (400 mg twice daily) was given either together with flag or da 2+5. the response was prompt. the patients became, after completion of the chemotherapy, leukaemia free. both patients continued the sorafenib (200 mg twice daily) treatment together with the aml standard maintenance chemotherapy (female case) or without any chemotherapy (male patient, substantial comorbidity and liver toxicity). (1) . the patients responded well to the therapy and were free of leukaemia for 16 and 32+ months after initiation of the mki treatment (flt3 itd negative, 100% chimerism documented in the blood and in the marrow). (2) . in both patients, 3 and 25 months on sorafenib, skin lesions appeared either in the context of cgvhd, which progressed to a life-threatening level in a male patient or as a photodermatitis-like cheek eruption. histopathology revealed the presence of severe cd3+ cells infiltration in affected tissues in both patients. (3) . cd8 positive lymphocytes colonized the marrow of both patients. these marrows infiltrating cells co-expressed cd279 (pd-1 receptor) in proportions which were higher than those seen in the blood (14.72% ± 1.45% vs. 3.63% ± 1.21%, p = 0.002). a similar observation was made for cd8+cd69+ cells (37.26% ± 3.50% vs. 1.58 ± 0.43%, po 0.002). 5. transcriptome analysis of the marrow cells, which addressed the genes involved in lymphocyte activation, revealed the presence of proinflammatory profile which included a higher expression of tlr9 and il-12. (1) sorafenib given with or without moderate chemotherapy was effective in two patients in maintaining the anti-leukaemic effect of salvage chemotherapy. (2) this was associated with the presence of alloreactivity (affected tissues infiltration with cd3+ cells) clinically seen as a severe fatal cgvhd aggravated by sorafenib treatment associated unwanted effects in one cases and with rather mild skin lesions appearing later during the treatment. the outcome of elder patients with acute myeloid leukemia or high risk myelodysplastic syndrome treated with allogeneic hematopoietic stem cell transplantation ch tsai 1,2,3 1 division of hematology, department of internal medicine, national taiwan university hospital; 2 tai-cheng stem cell therapy center, national taiwan university and 3 genome and systems biology degree program, national taiwan university allogeneic hematopoietic stem cell transplantation (hsct) is a curative-intent treatment for patients with high-risk hematologic diseases, including acute myeloid leukemia (aml) and myelodysplastic syndrome (mds). both the incidences of aml and mds increase with age and patients elder than 60 years of age were traditionally excluded from hsct because of high possibilities of therapy related morbidity/mortality. recently, with the introduction of reduced intensity conditioning regimens and the improvement of hsct care, more and more elder patients could undergo hsct for consolidation or salvage purposes. however, literature regarding the treatment outcome of elder patients receiving hsct is scarce. patients diagnosed as aml or high risk mds aged equal or more than 60 years were recruited consecutively at national taiwan university hospital. the high risk mds was defined to include myelodysplastic syndrome with excess of blasts-1 and 2 according to the 2016 world health organization (who) criteria. the cytogenetic risk stratification was based on original medical research council classification. from 2008 to 2016, a total of 51 patients were enrolled consecutively. the median age was 63.1 (range: 60-73) years and the gender distribution was even. among them, 11 (21.6%) patients had high risk mds, 27 (53%) had de novo aml, 10 (19.6%) had secondary aml, and three (5.9%) had therapy related aml. at diagnosis, four (7.8%) patients had extramedullary disease. nine (17.6%) had unfavorable-risk cytogenetics, 12 (23.5%) had either unfavorable-risk cytogenetics or intermediate-risk cytogenetics but with flt3-itd mutations. regarding the pre-hsct disease status, nine patients had the first complete remission (cr), 11 had the second cr, and 31 patients were treatment naive or had refractory disease. the graft-versushost-disease(gvhd)-free/relapse-free survival (grfs) in which events include grade 3-4 acute gvhd, chronic gvhd with moderate severity according to cibmtr criteria, relapse, or death of any cause. with median follow-up of 38 months (range: 0.8-85.2), the median overall survival (os) for all patients was 13.9 months, the relapse-free survival (rfs) was 11.3 months, and the grfs was 9.0 months. in univariate analysis for os and rfs, high-risk mds was a favorable prognostic factor but secondary or therapy related disease (p = 0.013 for os and 0.007 for rfs, respectively), unfavorablerisk cytogenetics or intermediate-risk cytogenetics but with s410 flt3-itd mutations (p = 0.005 and 0.002, respectively), pre-hsct refractory disease (p = 0.018 and 0.037, respectively), and grade 3-4 acute gvhd (p = 0.001 and 0.002, respectively) were unfavorable prognostic factors. however, for grfs, only unfavorable-risk cytogenetics or intermediate-risk cytogenetics but with flt3-itd (p = 0.020) and pre-hsct refractory disease (p = 0.018) were unfavorable prognostic factors. in multivariate cox proportional hazards regression analysis for os and rfs, grade 3-4 acute gvhd was a significant unfavorable risk factor; for gfrs, pre-hsct refractory disease status was a significant unfavorable risk factor. our results showed that the choice of hsct should not solely based on the age factor and pre-hsct disease status. incorporating cytogenetics and genetic mutation status could risk-stratify elder patients with hsct. further prospective trials are warranted to validate these findings. disclosure of conflict of interest: none. children affected with acute lymphoblastic t cell leukaemia (t-all) and relapse after allogeneic stem cell transplantation (sct) have limited treatment options and a poor prognosis. immune checkpoint inhibitors targeting the programmed death (pd-1) receptor pathway may enhance the graftversus-leukaemia (gvl) effect by blockade of inhibitory signals to t cells mediated by its ligand pd-l1. we report a 9-year old girl with refractory t-all after allogeneic sct, who was treated off-label with the pd-1 inhibitor pembrolizumab. the girl was diagnosed with t-all (8.2 g/l wbc, 82% bone marrow infiltration, cns negative, t (6;11)) and underwent hlahaploidentical bone marrow transplantation from her mother with post-transplant cyclophosphamide since she failed to achieve molecular remission despite an intensified chemotherapeutic regimen. on day 100 post sct, she had a 100% donor chimerism and decreasing minimal residual disease (mrd) marker (minimal 1 × 10 − 6 ). 140 days post sct she had a molecular relapse with an mrd of 5 × 10 − 3 and a subsequent morphological relapse as well as mixed donor chimerism. further treatment regimens included chemotherapy, intrathecal therapy and four donor lymphocyte infusions (dlis). initially, she displayed a good morphological response to dlis but the leukaemic burden eventually remained stable with an mrd of 2 × 10 − 2 . considering 54.2% pd-1 expression on cd3+ t cells in the patient's bone marrow and the encouraging data in other hematologic malignancies an off-label therapy with the pd-1 inhibitor pembrolizumab1-4 was initiated. the patient and her parents gave informed consent and she received a single dose of pembrolizumab at 3.3 mg/kg 343 days after sct. one week after administration of pembrolizumab, the patient developed acute gvhd grade iv of the skin, mucosa, liver, lung, central nervous system and eyes. she had a severe generalized inflammatory reaction with high inflammatory markers, increased hepatic transaminases and lymphocytic infiltration of the liver, cerebrospinal fluid and bronchoalveolar lavage fluid. magnetic resonance imaging (mri) of the brain revealed periventricular white matter lesions and hyperintensities of basal ganglia and bilateral temporal lobe consistent with autoimmune encephalitis. treatment with high-dose corticosteroids, cyclosporine and the anti-interleukin 6 receptor antibody tocilizumab slightly improved her clinical condition. her mrd value significantly decreased to 4 × 10 − 4 two weeks after administration and she achieved a 100% donor chimerism in bone marrow. despite this promising response her medical condition deteriorated and the severe inflammatory reaction caused fatal multi-organ failure. this is to our knowledge the first report on a remarkable and fast response to pd-1 inhibition in a patient with pediatric t-all refractory to multiple lines of therapy including allogeneic sct. this case illustrates the potential risk of checkpoint inhibitors to trigger severe gvhd that is not responsive to steroids. induction of inflammatory gvl responses without causing severe gvhd by therapeutic checkpoint inhibition needs to be addressed in future clinical trials. in recent years, there is a remarkable trend in the use of haploidentical-related hematopoietic cell transplantations (haplo-hct) in patients who do not have a hla matched related or unrelated donor. here, we report our single-center experience, in patients who underwent haplo-hct for acute leukemia. between 2011 and 2016 seventeen consecutive adult patients, seven males and ten females, median age 42 years (range: 18-61 years) with high-risk acute leukemia underwent unmanipulated, bm or pbsc transplantation from an haploidentical family donor. eleven patients transplanted for acute myeloid leukemia (5 in cr1, 1 in cr2, 1 in minimal active disease after cr1, 1 second trasplant in cr2, 1 transformed mds in cr1, 2 aml secondary to myelofibrosis in cr1), 5 for acute lymphoblastic leukemia (2 in cr1, 3 in active disease) and 1 mastcell leukemia (secondary to aml) in active disease. sixteen patients received myeloablative conditioning, and 1 reduced intensity, respectively. in five patients stem cells source was bm, in 12 were g-csf mobilized pbsc. the median infused cd34+ cell dose was 4.47 × 10 6 (range: 1.0 × 10 6 -8.2 × 10 6 ). conditioning regimens were: bu-flu-mac (n = 9), tbf-mac (n = 7), tbf-ric (n = 1) the regimens for gvhd prophylaxis were: ptcy as sole gvhd prophylaxis (n = 1), mtx-csa-atg (n = 9), methylpred-atg-tacrolimus (n = 6), atg-csa-mtx-mmf (n = 1). sustained trilineage engraftment occurred in 15 patients (88%), two patients died of transplantation-related complications before day 21 after transplantation without myeloid recovery. for patients receiving bm or pbsc grafts, the median time to 4500 neutrophils recovery was 16 days (range: 10-36), and 420,000 platelets recovery was 16 days (range: 13-37). 7/15 patients (46.6%) and 2/15 (13.3%) had ii-iv and iii-iv grade of acute gvhd, respectively.the incidence of grade ii-iv cgvhd was 27%. after a median follow-up of 11 months, 4/17 patients (23.5%), 4 out 5 patients transplanted in cr1, are alive and disease free at 50, 28, 19, 16 months (inclusing the patient transplanted for aml after imf). the 2-year probability of overall and progression-free survival was 40% (95% ci, 4.0-58.0%) and 28.6% (95% ci, 2.0-20.0%), respectively. causes of death were: sepsis (n = 1 ), fatal agvhd (n = 1), pneumonia (n = 1), toxicity (n = 2), progression (n = 4) and relapse (n = 4). in our experience unmanipulated bm or pbsc transplantation from haploidentical family donor is feasible approach with high engraftment rates and acceptable trm (23%) and rate of grade iii-iv agvhd, associated with durable remission in a proportion of patients with high-risk acute leukemia, specially in patients with aml transplanted in first remission. it is generally recognized that allogeneic hematopoietic stem cell transplantation (allo-hsct) should not be administrated to patients with severe aplastic anemia (saa) or very severe aplastic anemia (vsaa), when they got active infection. however, without neutrophil, severe infection is usually difficult to control and even fatal. under these circumstances, rapid recovery of neutrophil by allo-hsct might be an alternative to control infection. from january 2002 to december 2015, there were 175 young patients received allo-hsct for saa or vsaa at shanghai children's medical center in china. among them, 22 patients (11 males and 11 females) with a median age of 7.0 years (range: 3.0-14.0 years) received allo-hsct with refractory active infections. refractory active infection was defined as persistent neutropenic fever with nonresponse to standard doses of broad-spectrum antibacterial agents and antifungal agents for more than three weeks, with or without definite focus of infection. prior to allo-hsct, four patients had persistent fever of unknown origin, 11 patients with singlesite infection, and 7 patients with multiple-site infections. sites of infection included lung, sinus, cellular tissue, peritoneum, liver, spleen and skin. the conditioning regimen consisted of fludarabine, cyclophosphamide and rabbit-antithymocyte globulin with or without total body irradiation (tbi) (2-3 gy). twelve patients were transplanted from mismatched unrelated donors, 3 from matched sibling donors, and 7 from haploidentical donors. sixteen patients received g-csf mobilized peripheral blood stem cells, three patients g-csf mobilized peripheral blood stem cells plus g-csf primed bone marrow stem cells, two patients bone marrow stem cells, and 1 patient umbilical cord blood stem cells. a median of 11.4 × 10 8 /kg mononuclear cells with 4.6 × 10 6 /kg cd34+ cells were transfused, except the patient who underwent ucbt with a total of 1.3 × 10 8 /kg mononuclear cells and 1.5 × 10 6 /kg cd34+ cells transfused. eighteen patients achieved recovery of neutrophil and finally control of infections, including one patient who suffered primary graft failure and had autologous marrow recovery. three patients died of infection and one patients died of acute renal failure before recovery of neutrophil. one patient died of pneumonia 8 months after allo-hsct. one patient become thrombocytopenia after allo-hsct. the other 16 patients are all disease-free. there were five patients developing grade i-ii acute gvhd, and 4 patient grade iii-iv acute gvhd. all were cured at last. three patients had localized chronic gvhd and one patient had extensive chronic gvhd. with a median of 2 years follow-up, the overall survival rate and disease-free survival rate are 77.3% ± 8.9% and 71.3% ± 10%, respectively. allo-hsct could be a feasible way to control infection for children with saa or vsaa in the present of refractory active infections. disclosure of conflict of interest: none. paroxysmal nocturnal hemoglobinuria (pnh) may present hemolysis isolated (classical pnh) or associated with aplastic anemia (aa; aa/pnh syndrome). while classical pnh patients require anti-complement treatment (eculizumab), the treatment of aa/pnh patients should target their underlying aa by immunosuppression (ist), or even bone marrow transplantation (bmt). however, in a few patients clinically meaningful aa and hemolysis may be concomitant, eventually justifying both ist and eculizumab. to date there is no standard treatment for s413 this rare condition. amongst a large cohort of 145 pnh patients (between 2007 and 2016) at our reference centers, st. louis hospital (paris) and federico ii university (naples), we retrospectively assessed characteristics and outcomes of patients diagnosed with aa/pnh who received intensive ist during or immediately before (3-6 months) eculizumab treatment. nine patients were identified. eight patients fulfilled the criteria of severe aa, and one had an immunemediated isolated agranulocytosis. since no patient had a hla-matched related donor for bmt, all patients received intensive ist according to institutional guidelines. six out of 9 patients were already on eculizumab treatment at the moment of starting intensive ist (concomitant treatment) whereas 3 patients received ist in the 3-6 months (median time of 3 months) before the introduction of anti-complement therapy (sequential treatment). for all patients already on treatment, eculizumab was not discontinued to minimize the risk of rebound intravascular hemolysis and thrombotic complications. eculizumab was administered at the standard dose of 900 mg fortnightly in all but one patient, who needed an increased dose (1200 mg) because of pharmacokinetic breakthrough. six patients (5 aa and 1 agranulocytosis), including the three undergoing a sequential treatment, received standard ist with horse-antithymocyte globulin (h-atg, 40 mg/kg for four consecutive days) combined with cyclosporine a (csa). the remaining three aa patients received alemtuzumab (3-10-30-30 mg subcutaneously in four consecutive days) and csa within the prospective trial nct00895739; one of these patients a few months later also received a second ist course with rabbit-atg (3.5 mg/kg for five consecutive days) and csa. all the patients completed the scheduled treatment without any side effect, including infusion-related reactions. lymphocyte depletion (o 100/μl) was observed in all patients irrespective of sustained therapeutic complement blockade. all the patients were available for response assessment at 6 months. among the six patients receiving a concomitant treatment we observed one partial response (pr) and two complete responses (cr), whereas the three remain patients were non-responders (nr). of them one was rescued with an unrelated bmt, while two remained on eculizumab treatment. one of the cr relapsed at 3 years showing clonal evolution and finally died. all the other patients are alive, keeping their hematological response. patients receiving a sequential therapy were one in pr and two in cr 6 months after introduction of eculizumab. in conclusion, for patients diagnosed with severe aa/pnh syndrome intensive ist and eculizumab treatment, can be safely delivered either concurrently or sequentially, with an overall response rate of nearly 70%. this is the first systematic description of the management of severe aa in hemolytic pnh patients receiving eculizumab treatment. disclosure of conflict of interest: none. (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) in aa, 12 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) in rcc, and 8 (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) years in rcmd. sixty-five patients underwent bmt from hla-matched (related 41, unrelated 24) and 23 from hla-mismatched (related 6, unrelated 17) donors. conditioning regimens were used as follows; cyclophosphamide (cy)+antithymocyte globulin (atg) ± total body irradiation (tbi) (n = 29), fludarabine (flu)+cy ± atg ± tbi (n = 42), and flu +melphalan (mel) ± atg ± tbi (n = 17). all patients got engraftment after bmt. however, late graft failure was found in 6 patients with rcc, and 7 with rcmd, but none with aa. out of 13 patients who developed late graft failure, 8 patients used flu+cy ± atg ± tbi, 3 used cy ± atg ± tbi, and 2 used flu +mel ± atg ± tbi for conditioning regimens. five-year cumulative incidence (ci) of graft failure was higher in rcmd (36 ± 6.0%) than in aa (0%) and rcc (20 ± 1.8%), significantly (po0.01). five-year ci of graft failure tended to be higher in flu regimen (23 ± 2.2%) than in cy+atg ± tbi regimen (10 ± 0.65%), but not significant (p = 0.20). five-year ci of graft failure did not differ between with (21 ± 2.1%) or without tbi (29 ± 6.3%) (p = 0.57). multivariate analysis revealed that the morphological classification was a significant risk factor for graft failure (po0.01). five-year failure free survival rate (63 ± 11%) in rcmd was significantly lower than in aa (96 ± 3.8%) and rcc (74 ± 7.9%) (p = 0.02). graft failure, second malignancy, and death were considered as failure events. one patient with aa died of infection, four with rcc died of infection (n = 2), bleeding (n = 1) and myocarditis (n = 1), and one with rcmd died of infection. five-year overall survival rates were not different among 3 groups (aa, 96 ± 3.9%; rcc, 89 ± 5.2%; rcmd, 95 ± 4.7%) (p = 0.53). high incidence of graft failure in rcmd may be due to higher bm cellularity than in aa and rcc. the optimal conditioning regimen of bmt should be established for children with abmf based on the bm cellularity and morphological classification. disclosure of conflict of interest: none. recent studies have suggested inferior outcome of patients treated with rabbit atg (thymoglobulin, sanofi) as compared to horse atg (atgam,pfizer or lymphoglobulin, genzyme), and a higher early mortality with rabbit atg has been suggested to explain this difference. aim: to assess early mortality, response rates at 3,6 and 12 months and long term outcome, in a large cohort of aa pts, treated in europe or asia with rabbit atg and cyclosporin, as first line treatment. eligible for this study were pts with aa, treated with thymoglobulin between 2001 and 2012 in europe (n = 519) or asia (n = 457). median year of treatment, was 2008: characteristics were comparable : median age 20 and 21 years, interval diagnosis treatment (23 and 25 days) and severity of the disease (46% and 48% with vsaa). early mortality was analyzed for all 976 patients.long term outcome was also analyzed for 800 pts for who response data (no, pr, cr) were available. mortality o90 days was 5.5% and 2.1%, respectively, in the time period 2001-2008 and 2009-2012 (p = 0.007). in these 2 time periods, early mortality for patients aged 0-60, was reduced from 3.5% to 1.4% and for patients over 60, from 22% to 9%. overall response was recorded in 800 patients. at 6 months the cumulative incidence of response was comparable in the 2 time periods: 62% vs 66%, and at 1 year, 73% vs 75% (p = 0.8). response rates at 6 months were age dependent: 68%, 66%, 62%, 40% respectively in patients aged 0-20, 21-40, 41-60, 460 (p = 0.0006). when non responders at 3 months were reevaluated at 1 year, 59% had responded, 26% were non responders, 5% had died, and 10% had received other treatment. responses at 6 months, were 60%, 66%, 74%, in pts with very severe, severe and non severe aa (p = 0.0001). the actuarial 10 year survival for the entire population was 71%, and 70%, when pts were censored as surviving at transplant. actuarial 10 year survival in univariate analysis was as follows: 89% vs 61% for day 90 responders vs non responders (p o0.01), 68% vs 80% for males versus females (p = 0.07); 82%, 72%, 66%, 27% in pts aged 0-20, 21-40, 41-60, 460 years (p o0.001); 67%, 78%, 76% in pts with neutrophils o0.2 × 10 9 /l, 02-05 × 10 9 /l and 40.5 × 10 9 /l (p o0.001); 77%, 75%, 68% for pts with an interval diagnosis-treatment of o30 days, 31-60 days or 4 60 days (p = 0.002). finally pts treated 42008 had a 5 year survival superior to pts treated before 2008 (84% vs 77%, p = 0.01). survival at 5 years, in the recent period (2009-2012), was 83% for pts aged 1-60 and 60% for pts over 60 years. in multivariate cox analysis the following variables remained independent predictors of survival: patient age, year of treatment, severity of the disease, interval diagnosis treatment, and gender. thymoglobulin +csa is effective and safe in patients with aa. the outcome is mainly age dependent. the inrerval between diagnosis and treatment remains a strong predictor: the earlier the better. for pts o60 years old current early mortality. 4. for pts 460 years of age, current early mortality is higher (9%), response rate (40%) and 5 year survival (70%) are lower. 5. the actuarial 10 year survival for the enire population was 71%. survival at 5 years has improved from 77% (o o/u42008) to 84% (2009-2012), especially for pts over 60 years (37% vs 70%, p = 0.006). [p560] s415 disclosure of conflict of interest: we thank centers for providing up date follow up of their patients . this study was supported by a grant of sanofi-genzyme. both of the patients have extremely low anc o0.05 × 10 9 /l, reto0.5%, plt o20 × 10 9 /l. both was given antibiotic treatment with carbapenem, vancomycin/linezolid, voriconazole or amphotericin b liposome and got no response. no pathogenic bacteria or fungus was found from either of the patients. both of them had no full sibling or matched unrelated donor and had their father as their haploidentical donor. bone marrow combined with peripheral blood stem cell (pbsc) was adopted. conditioning: fludarabine days − 5 through − 2, (40 mg/m 2 × 4), intravenous busulfan (1.1 mg/kg q6h) on days − 4 to − 2. gvhd prophylaxis: high-dose cyclophosphamide 40 mg/kg on days +3 and +4, mmf and tacrolimus since days +5. rabbit anti-thymocyte globulin (thymoglobulin) 2.5 mg/kg on days − 4 to − 2. stable neutrophil engraftment (anc 40.5 × 10 9 /l) occurred on day +13 and day+19 respectively. platelet achieved 20 × 10 9 /l on day +11 and day +55, respectively. both transplant course was complicated by febrile neutropenia without detected etiology, while both children have no fever since the first day anc 40.5 × 10 9 /l.the facial swelling was resolved in both patients except for palatal fistula and fistula of maxillary sinus as the sequela of severe nasosinusitis. no acute or chronic gvhd. case1 had hemorrhagic cystitis on day +30 which last for about 30 days, and suspected thrombotic microangiopathy (tma) with hypertension, thrombocytopenia, elevated ldh and creatine on day +51 which was resolved soon after discontinue of tacrolimus. case2 had delayed engraftment of platelets and herpes simplex virus 6 encephalitis on days +40 which was cured by ganciclovir and high dose intravenous immunoglobulin. now they are 9 and 7months post-hsct respectively and are doing well with 100% chimerism and no gvhd. alternative donor hsct may be considered as the first line salvage therapy for patients of vsaa with extremely low anc and active infection. fast reconstruction of neutrophil helped to control the infection. hallo-identical hsct make sure nearly every patients can find a donor. ptcy is proved to be efficient and safety in gvhd prophylaxis and facilitating engraftment in these two challenging cases. disclosure of conflict of interest: none. long-term outcome of patients with severe aplastic anemia receiving allogeneic hematopoietic cell transplantation using nonmyeloablative conditioning with fludarabine and low dose total-body irradiation l cheryl xiu qi 1 , l yeh ching 2 , p michelle li mei 1 , t lip kun 1 , h william ying khee 2 , g yeow tee 2 , t patrick huat chye 2 and k liang piu 1 1 department of haematology-oncology, national university cancer institute, singapore and 2 department of haematology, singapore general hospital, singapore allogenic haematopoeitic stem cell transplant (ahct) offers the best prospect of cure in patients with severe aplastic anaemia (saa). the use of myeloablative hct is however limited by the toxicity of preparative regimens, the lack of matched sibling donors, transplant related mortality and graft rejection. the introduction of non-myeloablative (nm) conditioning offers the possibility of extending this potentially curative treatment to patients in whom ahct was previously contradindicated. in 2006, we reported the outcome of 8 patients with saa who have received ahct using nonmyeloablative conditioning comprising of 3 days of fludarabine at 25mg/m 2 and total body irradiation at 2 gy (flu + tbi 2gy). here, we report a longer follow-up, with 6 additional patients who had recevied ahct with this regimen. fourteen patients with a median age of 37 years old (range: 17-48 years old) received filgastrim-mobilised peripheral blood stem cell transplant from either hla identical sibilings (n = 12) or matched unrelated donor (n = 2) after receiving nm conditioning consisting of flu + tbi 2gy. the first two patients received cyclosporine (cya) and mycophenolate mofetil (mmf) for the post-transplant immunosuppessive therapy. the remainining 12 patients received cya, mmf and a short course of methotrexate (mtx) for additional graft-versus-host dsease (gvhd) prophylaxis. results all patients achieved prompt engraftment. the median time for engraftment of neutrophils (40.5 × 10 9 /l) and platelets (420 × 10 9 ) were 16 days (range: 13-20 days) and 13 days (range: 8-25 days), respectively. chimerism analysis on day 28 and subsequently showed 495% donor cells in all patients except 1, who developed secondary graft failure at 3 months and required salvage hct. none of the patients experienced grade 3 and above regimenrelated toxicity. five patients developed grade ii-iv acute gvhd and 2 patients developed limited chronic gvhd. with a median follow-up of 8.8 years (range: 0.54-14.52 years), the estimated overall survival and event free survival were 86% and 79% respectively. the two patients who did not receive mtx developed acute gvhd of the liver and succumbed to infective complications. the remaining 12 patients who had received triple immunosuppressive therapy were well, with limited chronic gvhd seen only in 2. our results suggest that the nm conditioning regimen comprising of flu + tbi 2gy provides sufficient immunosuppression to allow prompt and stable engraftment with minimal regimen-related toxicity. it is an attractive option for patients with saa who require ahct but are at increased risk of regimen-related complications from more intensive cyclophosphamide-based regimens. disclosure of conflict of interest: none. paroxysmal nocturnal hemoglobinuria (pnh) is a rare acquired clonal disorder of hematopoietic cells characterized by the triad of hemolytic anemia, cytopenias and high risk of venous thrombosis. due to the rarity of the disease, most reported data derive from multicenter studies. we describe the natural history of the disease in a 30-year (yrs) long single center series of pnh patients (pts). we performed a retrospective analysis of 42 consecutive pts followed at our center from 1985 to 2016. since 1985, the diagnosis was made by ham test; starting from 2000, flow cytometry (fc) analysis was used to diagnose new pts and to confirm pnh in pts previously diagnosed by the ham test. at diagnosis, 26 pts had classic pnh, 9 aplastic pnh and 7 intermediate form. the cumulative incidences of thrombosis, cytopenia and clonal neoplasm were 39%, 18% and 10%, respectively. except for 1 pt with aplasia, no severe infections were diagnosed, nor renal failures or pulmonary hypertention. the 30 yrs overall survival (os) was 84%. a nonsignificant better os was associated to the absence of thrombotic events (96% vs 80%) and to a diagnosis made during the last decade (100% vs 90% vs 75%).up to 2005 the treatment options were supportive care or allogenic bone marrow transplantation. since 2005, eculizumab was used in transfusion-dependent patients and/or in case of a thrombotic history. overall, 14 pts were transfusion-independent for the entire period of the illness, 28 were transfusion-dependent and/or had thrombotic events(8pts). six of the latter pts never received eculizumab but only transfusion support (3 pts) or allogeneic bone marrow transplant (3 pts), while 22 pts received eculizumab (the first 4 pts were included in the phase iii triumph and shepherd trials).considering the increased risk of meningococcus infection for pts on eculizumab, vaccination with conjugated anti-meningococcus serotypes acwy was employed and, since 2016, conjugated antimeningococcus serotype b was added. overall, 18 pts treated with eculizumab became transfusion-independent and four remained transfusion-dependent. no thrombotic event was observed after eculizumab, even if 8 pts had recurrent thromboembolisms prior to receiving the drug. no severe infection was documented. one patient developed extravascular hemolysis and receive a successfully selective splenic artery embolization. the 10 yrs os in the eculizumab group was 92%.no pnh-associated death occurred. our study confirms that thrombosis is a major complication in pnh pts not receiving eculizumab, influencing os. the better os in the last decade is probably due to the use of eculizumab and to lack of thrombotic events. in particular, for 22 pts on eculizumab the 10 year os was 92%, even though half of the pts had thromboembolism and diagnosis made prior to the last decade. although kidney failure and lung hypertension have been reported, we did not observe these complications in our long follow-up case series. we can assume that the availability of a dedicated emergency room at our center allows to perform, promptly, hyper-hydration or transfusion support in case of hemoglobinuria crisis, reducing the risk or organ damage. no infections have been observed after eculizumab, probably due to the vaccination program schedule recommended in the literature, plus the addition of conjugated anti-meningococcus serotype b. however, shared guidelines are needed. disclosure of conflict of interest: none. mortality following hsct in saa pts over the age of 40 is reported to be in the order of 50%, without taking in to account long term sequelae such as chronic gvhd, known to be more frequent in older patients. this has prompted international guidelines to recommend first line immunosuppressive therapy above 40 years of age. the question is whether this is still true in 2017. the aim of the study is to assess whether trm in saa patients grafted 2010-2015 is reduced,as compared to the era 2001-2009. we used the wpsaa ebmt registry, and identified 748 pts aged 40 years or more, with acquired saa, grafted between 2001 and 2015. we divided pts in 2 transplant eras:2001-2009 (n = 327) and 2010-2015 (n = 407). in the more recent period (2010-2015) pts were older (53 vs 49 year, p o0.01), were more often grafted from alternative donors (alt) (64% vs 43%, po0.01), with a greater use of bm (54% vs 41%, p o0.01), and with a longer interval dx-tx (317 vs 258 days, p0.01), and more often received a fludarabine containing regimen (55% vs 42%, p o0.01). the os 5 year of pts grafted in 2001-2009 was 57%, compared with 55% for pts grafted 2010-2015 (p = 0.7). in multivariate analysis, including the interval diagnosis transplant, patient's age, donor type, stem cell source and conditioning regimen, the lack of improved survival in 2010-2015 was confirmed (p = 0.3). a very strong age effect was shown both in univariate and multivariate analysis: survival of pts aged 40-50 years, 51-60 years and 461 years, was respectively 64%, 54%, 41% (p o0.0001) and this was confirmed in multivariate analysis. the conditioning regimen, also proved to be a significant predictor, with improved survival for alt transplants receiving flu containing regimens (56% vs 46%, po0.001). in general pts receiving either cy200 or a flu containing regimen, did significantly better than pts receiving other preparative regimens (58% vs 50%, p = 0.02). the use of a sibling donor (sib) did not prove to predict survival in multivariate analysis. pts receiving campath in the conditioning,did significantly better than pts not receiving campath (65% vs 54% po0.01); similarly survival of patients with atg was superior 59% vs 41% compared to patients not receiving atg (p o0.01). when pts receiving either campath or atg (n = 564) were compared to patients not receiving either (n = 161), the difference in survival was 61% vs 41% (p o0.0001), and this was significant also in multivariate analysis. combined primary and secondary graft failure was reduced from 16% to 12% in the two time periods (p = 0.02), acute gvhd grade ii-iv was reduced from 15% to 11% (p = 0.1) and chronic gvhd was also reduced from 32% to 26% (p = 0.04) infections remain the leading cause of death in both transplant eras (18% and 22% respectively), followed by gvhd (5% and 4%) and graft failure (5% and 2%), whereas ptld have been reduced from 3% to 0.5%. hsct in pts with acquired saa aged 40 and over, continues to carry a significant risk of trm also in 2010-2015, ranging from 36% in younger pts (40-50) to 59% in older pts (460 years). survival is predicted in multivariate analysis, by two crucial predictors: patients' age and the use of either campath or atg,the latter giving a 20% survival advantage over no campath/atg. alt and sib donors produce similar survival. this study gives further support to current guidelines, suggesting first line therapy with atg+csa, in pts over the age of 40. [p565] disclosure of conflict of interest: none. autoimmune diseases p566 allogeneic haematopoetic stem cell transplantation as curative therapy for early-onset, refractory crohn's disease e groene 1 , p bufler 1 , k krohn 1 , s immler 1 , g marckmann 2 , t feuchtinger 1 , s koletzko 1 and m albert 1 1 dr. von hauner university children's hospital and 2 institute of ethics, history and theory of medicine, lmu results of a recent randomized trial suggest that autologous hsct is an option in adult patients with severe, therapyrefractory crohn's disease (cd) with an associated mortality risk of 4%. however, relapse of the disease is frequent (1). in contrast allogeneic hsct has resulted in long-term cure of cd in affected patients transplanted because of haematological malignancy (2). we report a 17 year old girl who was diagnosed with severe cd at age seven (paris classification l3, l4a, b1). neither next generation sequencing nor immunological work up identified a monogenetic cause of cd. progressive chronic inflammation manifesting ubiquitously in the gastrointestinal tract resulted in severe complications, such as perianal fistulas with rectal stenosis, intestinal abscesses, dysphagia, severe weight loss, failure to thrive, delayed puberty and the need for ileostomy and long-term exclusive enteral nutrition via tube feeding. despite multiple lines of therapy, including repeated nutritional therapy, steroids, immunosuppressants (methotrexate, azathioprine) and biologicals (infliximab, adalimumab, certolizumab) a lasting remission could not be achieved resulting in poor quality of life. after careful risk/benefit assessment including ethical counselling allogeneic hsct was offered. she underwent allogeneic hsct from a matched (10/10) unrelated bone marrow donor (4.3 × 10 8 /kg total nuclear cells). conditioning was performed according to a protocol successfully applied in adolescents with chronic granulomatous disease (3) with alemtuzumab (3 × 0.2 mg/kg/d), targeted busulfan (tauc 53770 ng × h/ml) and fludarabine (6 × 30 mg/m 2 ). cyclosporine a and mycophenolate mofetil were used as gvhd prophylaxis. neutrophil and platelet engraftment were observed on days +20 and +24, respectively. the post hsct course was complicated by grade i acute skin gvhd treated with topical steroids and toxic megacolon secondary to scarring stenosis on both ends of the unused colon on day +130 requiring surgery and a colostomy. at 12 months post hsct the patient is well, off immunosuppressive medication, without gvhd and exhibiting 495% donor chimerism. the cd is in complete clinical and histological remission as proven by endoscopy and biopsies. stoma reversal with restitution of intestinal continuity is planned for the next 12 months. refractory cd can lead to life-threatening complications and severely reduced quality of life. although long-term outcome in our patient will need to be carefully assessed, allogeneic hsct may offer a curative therapy in children and young adults with severe cd, even in the absence of an identified monogenetic cause. current ebmt recommendations include consideration of ahsct in exceptional circumstances for patients with severe refractory cd. the only randomised trial of ahsct in cd (astic) confirmed substantial short-term benefits but failed to meet its primary 1 year endpoint. to further clarify the longterm safety and efficacy of ahsct in cd we performed a retrospective analysis of patients undergoing ahsct for cd outside the astic trial using the ebmt registry. patients were identified from the ebmt registry. all adult patients undergoing ahsct for a primary diagnosis of cd from 1997 to 2015 were eligible for inclusion. patients who were enrolled in the astic trial were excluded. from a total of 99 patients (across 27 centres) on the ebmt registry, data were obtained from 76 patients transplanted in 14 centres in 7 countries. median patient age was 30 yrs (range: 20-51) and 63% were female. median age at first diagnosis of cd was 18yrs (range: 2-48). patients were heavily pre-treated, having failed or been intolerant to a median of 6 previous lines of therapy (range: 3-10). 55% had received experimental therapy prior to auto-hsct. 80% of patients had undergone at least 1 operation. the median time from first diagnosis of cd to auto-hsct was 12.3 years (range: 1.3-25.8). all patients received peripheral blood stem cells following conditioning with cyclophosphamide 200 mg/kg and 84% received anti-thymocyte globulin (atg). the median cd34+ dose infused was 5.5 (range: 2.4-40.6) × 10 6 /kg. twelve percent of patients underwent cd34+ selection. neutrophil and platelet engraftment occurred at a median of day 10 (range: 6-18) and day 9 (range: 1-44), respectively. sixety-one percent received post transplant g-csf. median length of follow-up following auto-hsct was 42 months (range: 6-174). at 100 days post auto-hsct, 64% of patients were in clinical remission (cr), defined as no abdominal pain and normal stool frequency. a further 27% experienced significant improvement, defined as improvement in abdominal pain and stool frequency. for 5% there was no appreciable change in disease and in 4% the disease worsened compared to baseline. at 1 year post auto-hsct, 39% were in cr, 19% were improved, 20% were unchanged and 22% had worsened. at last follow-up, 37% were in cr, 23% were improved, 25% were unchanged and 15% had worsened. overall 74% restarted medical therapy post auto-hsct and 38% required further surgery. overall 26% developed an infection requiring treatment post auto-hsct (11% bacterial, 12% viral). ebv and cmv reactivation occurred in 7% and 4% respectively and herpes zoster occurred in 4%. a secondary autoimmune disease developed in 13%, most commonly thyroid disease (63%). malignancy developed in 5%, of which skin cancer accounted for 75% of cases. one patient died at 56 days post auto-hsct due to cmv infection, sepsis and multiorgan failure. this large retrospective series further supports the safety and efficacy of ahsct in a population with severe and treatment-refractory crohn's disease, 60% of patients experienced complete remission or significant improvement in cd symptoms with long-term follow-up. trm observed was similar to ahsct for other indications. in summary, ahsct appears to be an extremely promising therapy for severe refractory cd. further follow up of astic patients and future randomised trials are warranted. disclosure of conflict of interest: none. memory stem t cells (tscm) are long living self-renewing memory t cells with long-term persistence capacity, which play a relevant role in immunological memory and protection against infectious diseases and cancer1,2,3,4,5,6. the aim of this work is to investigate the potential role of tscm as a reservoir of arthritogenic t cells in rheumatoid arthritis (ra). we analysed the dynamics of circulating tscm (here identified as cd45ra+ cd62l+ cd95+ t cells) and other memory t-cell subpopulations by multiparametric 11-color flow cytometry in 27 patients with active ra and in 14 of them also during treatment with anti-tnfα biological agents (etanercept). to analyse cytokine productions, functional assays were performed stimulating peripheral blood mononuclear cells (pbmcs) with pma/ionomycin and brefeldin a. after the stimulation, cells were stained for surface markers, fixed and stained for intracellular cytokines. we traced circulating antigen specific cd4+ t cells for the vimentine-derived citrullinated peptide (vimcit) 65savracitssvpgvr77 7,8 in hla-drb1 × 04:01 ra patients before and during the anti-tnfα treatment using custom mhc class ii tetramers. viral antigen specific cd8+ t cells were traced using mhc class i dextramers. age-matched healthy donors (hds) were used as control for all the experiments. we found a significant expansion of cd4+ tscm in patients with active ra both in terms of frequency and absolute counts. notably, cd4+ tscm significantly contracted upon anti-tnfα treatment, suggesting a role of tnfα in tscm accumulation. in contrast to cd4 +t cells, cd8 compartment did not show significative alterations compared to (hds). furthermore, cd4+tscm in ra patients displayed an enrichment in the th17 phenotype, largely implied in autoimmune disorders, while the other t cell subpopulation were not enriched in the th17 phenotype. at the antigen specific level, we were able to trace in hla-drb1 × 04:01 patients antigen specific cd4+ t cells, comprising tscm, specific for the vimentin-derived citrullinated peptide. of notice, citrullinated vimentin specific cd4+ t cells, including tscm, contracted during anti-tnfα administration, while viral-specific cd4+ t cells (ebvbhrf-1) and antiviral cd8 specificities (cmvpp65, flump, ebvbmlf-1) were not affected by etanercept administration, thus suggesting a possible role of cd4+ tscm as reservoir of arthritogenic autoreactive t cells. overall, our results suggest that tscm, by representing a long-term reservoir of undesired specificities, might play a non redundant role in sustaining ra and possibly other t cell mediated disorders, thus representing novel biomarkers as well as therapeutic targets. ongoing experiments will characterize the tcr repertoire on sorted tscm and cd4+ memory subsets in order to identify a possible oligoclonality in tscm repertoire. in conclusion, the analysis of tscm dynamics in autoimmune disorders could have relevant clinical implications as new biomarkers and for devising innovative therapeutic strategies. ebv and cmv reactivation following autologous haematopoietic stem cell transplantation (hsct) for autoimmune neurological diseases resolves spontaneously and rarely requires treatment c mapplebeck 1 , b sharrack 1 , h kaur 1 , y ezaydi 1 , h jessop 1 , l pickersgill 1 , l scott 1 , m raza 1 and ja snowden 1 1 departments of haematology, neurology and virology, sheffield teaching hospitals nhs foundation trust, sheffield, uk autologous haematopoietic stem cell transplantation (hsct) for severe autoimmune diseases involves immunosuppressive conditioning regimens and current guidelines recommend monitoring for viral reactivation of cytomegalovirus (cmv) and epstein barr virus (ebv) (snowden et al 2012) . however, the incidence, degree and management of viral reactivation are not established. we performed a retrospective observational service evaluation study of all patients receiving cyclophosphamide 200 mg/kg + rabbit anti-thymocyte globulin 6 mg/kg (atg, thymoglobulin) followed by autologous hsct for various autoimmune neurological diseases between 2011 and 2016 at our centre. data collected included the baseline serological status of the patient prior to transplant and serial blood pcr quantitation (copies/ml). if ebv and cmv reactivation occurred details of further management was collected and descriptive statistics were used to summarise outcomes. twenty-three patients received autologous hsct between january 2011 and october 2016; 21 patients with multiple sclerosis (ms), 1 with chronic inflammatory demyelinating polyneuropathy (cidp) and 1 with stiff person syndrome. twenty-two patients had positive ebv igg serology prior to transplant and 1 patient had an equivocal result. seventeen patients had evidence of ebv reactivation and a further patient had ebv dna detected post-transplant but with less than 250 copies/ml. the average time to peak ebv pcr was 26.5 (range: 12-44) days post-transplant and a range: in ebv pcr peak level from 623 to 577 000 copies/ml. the 4 patients who had ebv pcr results of over 100 000 copies/ml had ct scans of chest, abdomen and pelvis performed which did not demonstrate significant lymphadenopathy or hepatosplenomegaly. in all patients monitored for a detectable ebv reactivation, the ebv pcr spontaneously began to fall within 2 months (average 36 days, range: 18-60 days) post-transplant and no specific treatment was required. one patient had late ebv reactivation of 3480 copies/ml at 6 months post-hsct associated with chronic tonsillitis and tonsillectomy specimens showed follicular hyperplasia without evidence of post-transplant lymphoproliferative disorder (ptld) and ebv pcr levels normalised without other treatment. 8 (35%) patients had positive cmv igg serology prior to transplant and one patient had an equivocal result. only 1 of 23 patients had a significant reactivation of cmv with 51 300 copies/ml at 21 days post-transplant, successfully treated with intravenous immunoglobulins and valganciclovir. two other patients had low level cmv reactivation with 94 and 476 copies/ml, respectively which resolved spontaneously without treatment. ebv reactivation in patients with neurological autoimmune disease undergoing autologous hsct is common and usually resolves spontaneously without treatment. asymptomatic cmv reactivation occurs in approximately 13% of patients in this setting and may require treatment. autologous hematopoietic stem cell transplantation (hsct) has been utilised for the treatment of severe multiple sclerosis (ms). it results in significant improvement of neurological function, although patients can experience exacerbations of ms-related symptoms during the procedure. we reviewed 17 patients with ms who underwent stem cell mobilisation and collection from march to november 2016. the median age was 40 years (24-55). nine patients (53%) were male. the interval from diagnosis to hsct was 114.3 months (range: 11.6-128.3). 9 patients (53%) had relapsing-remitting (rrms), six patients (35%) secondaryprogressive (spms) and two patients (12%) primary-progressive (ppms) multiple sclerosis. only 2 patients (12%) had not received any prior treatment, whereas 10 patients (59%) received two prior treatments, three patients (17%) received three treatments and two patients (12%) received four treatments. the median expanded disability status scale (edss) score was 6 (range: 2-6). peripheral blood stem cells were mobilised with cyclophosphamide (cy) 2 g/m 2 on day +1 and daily gcsf (5 μg/kg subcutaneously) from day +3 until the completion of the harvest. hsct was performed at a median of 33 days after mobilisation (range: 25-59). the conditioning regimen consisted of cy (50 mg/kg/day from day − 5 to − 2) and atg (2 mg/kg/day from day − 4 to − 2). exacerbation of ms symptoms was defined as the appearance of new or worsening of old symptoms for at least 24 h duration in a previously stable (4 weeks) patient. of the total cohort, 13 patients (76%) underwent mobilisation with cy+gcsf uneventfully. only two patients (12%) had an exacerbation of ms requiring hospital admission after collection (one with fatigue and increase of spasticity, other with worsening weakness). no patient required hospital admission during the mobilisation procedure. the median cd34+ cell dose was 8.39 × 10 6 /kg (range: 2.2-24). the median number of apheresis was 1(1-2). a total of 14 patients have undergone hsct at the time of this analysis. during transplant a total of 11 patients (78%) experienced an exacerbation of ms. of these, 54% (n = 6) before day 0 and 46% (n = 5) between day +4 and +7. symptoms of exacerbation were: muscle spasms in 4 patients (36%), weakness and reduced power of limbs in 4 patients (36%), increase instability and tremor in two patients (18%) and one patient (10%) with worsening of neuropathic pain. only three patients (28%) received treatment with methylprednisolone for ms exacerbation and symptoms had fully resolved by discharge in all patients. other transplant complications included neutropenic fever in all, invasive fungal infection in 1, fluid overload in 9 (64%) and atg related complications in 11 (78%) such as fever (n = 10) and pericarditis/serositis (n = 1). the median time to neutrophil engraftment was 10 days (10-14) and the median duration of hospital admission was 20 days (15-25). exacerbation of ms symptoms is common during hsct and can also occur during mobilisation. in our hands, after cy and gcsf mobilisation only two patients (11%) developed an exacerbation of ms symptoms compared with 11 patients (78%) after ct and atg conditioned hsct. it is possible that the addition of atg to cy triggers an immunological response involved in this transient deterioration of the ms symptoms. further studies are required to confirm this hypothesis. disclosure of conflict of interest: none. inflammatory immune response syndrome (iris) is a noninfectious worsening of neurological condition during immune recovery and has been documented to occur in hiv and in multiple sclerosis following alemtuzumab. the manifestation of iris includes headache, nausea, weakness, neurologic deficits, and mri enhancing lesion. we report three cases of iris after autologous non-myeloablative hematopoietic stem cell transplantation (hsct) in patients for which the transplant indication was an inflammatory neurologic disease: neuromyelitis optica (nmo), chronic relapsing inflammatory optic neuritis (crion), and multiple sclerosis (ms). mobilization was with cyclophosphamide 2 gr/m 2 and gcsf. conditioned regimen was 200 mg/kg cyclophosphamide (50 mg/kg/d) and 6.0 mg/kg ratg (thymoglobulin). the conditioning regimen for nmo and crion also included 1000 mg rituximab. case 1. a 22 years old african-american female with systemic lupus erythematosus (sle) and nmo was discharged day 10 and readmitted on day 14 for fever, headache, progressive altered mental status with dysarthria and legs. brain mri had numerous t2/flair hyperintense and enhancing lesions in the subcortical and periventricular white matter. a lumbar puncture was negative for infection including jcv. complete recovery occurred after treatment included high dose of steroids and plasmapheresis. case2. a 48 years old female with crion experienced blindness, weakness and slurring of speech three months post hsct. mri showed a large enhancing brain stem lesion. lumbar puncture was jcv negative. complete recovery occurred after solumedrol and rituximab. mri 6 months later demonstrated complete resolution of the enhancement with return of vision to baseline. case3. a ten year-old boy diagnosed with paediatric ms developed hemichorea seven days after hsc reinfusion. brain mri revealed a gadolinium-enhancing lesion in the contralateral basal ganglia. lumbar puncture was negative for infection including jc virus. symptoms resolved spontaneously after seventeen days. the appearance of new neurologic symptoms and mri enhancing lesions early after autologous hsct is unexpected and may be related to lymphocytes in the graft, immune recovery post engraftment, and or persistent auto-antibodies. it is mandatory to perform a lumbar puncture to exclude the possibility of infections including progressive multifocal leukoencelopathy (pml) due to jcv. the timing of presentation, the negativity of jc viral load, and the complete recovery with or without immune suppression suggest the hypothesis of iris, as an epiphenomenon of the immune reconstitution following autologous hsct for neurologic diseases disclosure of conflict of interest: none. hematopoietic stem cell transplantation is the effective method of therapy for cns autoimmune disorders in children. long-term outcomes and late effects estimation required. the aim of the study is to estimate long-term outcomes and late effects at children underwent auto-hsct for multiple sclerosis (ms) and allo-hsct for neuromyelitis optica (nmo). twelve pts. with ms and 3 pts. with nmo were included to the analysis. ms pts. gender: female -75% (n = 9), male -25% (n = 3 allogeneic haematopoietic stem cell transplantation (hsct) remains the sole curative option for patients with myelofibrosis (mf). although a spectrum of conditioning regimens has been used, the optimal preparative treatment before hsct remains to be defined. we did a phase ii randomized study at 21 transplant centers in italy with the aim of comparing the reduced-intensity conditioning (ric) fludarabine-busulfan (fb) (conventional arm), that had been already tested in the prospective ebmt study (1) with the ric fludarabine-thiotepa (ft) (experimental arm), that has been widely used in italy in the last two decades (2) . eligible to this study were patients with primary mf or a mf subsequent to a previous essential thrombocytemia or polycyhemia vera, an age ≥ 18 ≤ 70 years, a karnofsky performance status 4 60, a comorbidity index o o/u4 5 and with at least one of the following unfavorable prognostic factors: anemia (hb o 10 g/dl), leukocitosis (25 × 10 9 /l), circulating blasts 41% or constitutional symptoms. patients were randomized to receive intravenous busulfan 0.8 mg/kg for 10 doses or thiotepa 6 mg/kg for two doses associated to fludarabine 30 mg/m 2 for impact of pre-transplant ruxolitinib in myelofibrosis patients on outcome after allogeneic stem cell transplantation syed abd kadir, sharifah shahnaz, author 1,2 , zabelina, tatjana, co-author 1 , christopeit, maximilian, co-author 1 , wulf, gerald, co-author 3 , wagner, eva, co-author 4 , bornhaeuser, martin, co-author 5 , schroeder, thomas, co-author 6 , crysandt, martina, co-author 7 , mayer, karin tina, co-author 8 , stelljes, matthias, co-author 9 , badbaran, anita, co-author 1 , wolschke, christine, co-author 1 , ayuk ayuketang, francis, co-author 1 , triviai, ioanna, co-author 1 , wolf, dominik, co-author 8 ruxolitinib (rux) is the first approved drug for treatment of myelofibrosis. because spleen size and constitutional symptoms may influence outcome after allogeneic stem cell transplantation (asct), rux is recommended before stem cell transplantation in order to reduce therapy-related morbidity and mortality and improve outcome (ebmt/eln recommendation, leukemia 2015) the aim of this retrospective study was to evaluate the impact of pretreatment with rux in comparison to transplantation of rux-naïve mf patients with regard to outcome after asct. we included 149 myelofibrosis patients (pts) with a median age of 59 years (range: 28-74) who received asct between 2000 and 2015 from related (n = 23), matched (n = 86) or mismatched (n = 50) unrelated donor. all patients received busulfan-based reduced intensity conditioning. while 113 pts (66%) did not receive rux, 46 pts (34%) received rux at any time point prior to asct. the median daily dose of rux was 30 mg (range: 10-40 mg) and the median duration of treatment was 28 days (range: 12-159 days). in 11 pts rux was stopped before stem cell transplantation because of no response or loss of response, while in 35 pts rux was given until start of conditioning. gvhd prophylaxis consisted of cni plus short course mtx or mmf and anti-lymphocyte globulin. according to dynamic ipss (dipss) (n = 170) the patients were either low (n = 2), intermediate-1 (n = 36), intermediat-2 (n = 72), or high risk (n = 36). as the median follow up was shorter for patients treated with rux (15 vs 73 months, po0.001). primary graft failure was seen in 2 pts in the rux and three in the non-rux group. the median leukocyte engraftment was 13 days (range: 9-32) in the ruxolitinib and 14 days (range: 7-34) in the non rux group (p = 0.7). the incidence of acute gvhd grade i to iv was significantly lower in the rux group (49% vs 64%, p = 0.05), while agvhd grade ii-iv (33% vs 44%, p = 0.14) and grade iii/iv (23% vs 25%, p = 0.48), did not differ significantly. the ci of nrm at 1 year was 18% (95% ci: 6-30%) for the rux group and 22% (95% ci: 14-30%) for the non-rux group (p = 0.58), and the ci of relapse at 2 years was 8% (95% ci: 0-16%) vs 20% (95%ci: 12-28%, p = 0.25). the 2 years rfs and os was 66% (95%ci: 50-82%) and 69% (95%ci: 51-87) for the rux group and 59% (95% ci: 49-69%) and 70% (95% ci:62-78%) for the non-rux group (p = 0.29 and p = 0.45, respectively). within the rux group (n = 53), 24 pts responded to rux (more than 25% spleen size reduction), while 29 pts did not respond or lost response prior to stem cell transplantation. here, no significant difference could be seen between the responding and non-responding group for nrm (19% vs 17%, p = 0.69), relapse (4% vs 13%, p = 0.62), rfs (61% vs 72%, p = 0.81) and os (63% vs 75%, p = 0.89). in a multivariate analysis including rux treatment as variable there was a non-significant trend in favor for in the tyrosine kinase inhibitor (tki) era, allogeneic haemopoietic stem cell transplantation (allo-hsct) has become the later-line therapy but still remains the only known curative treatment for chronic myeloid leukemia (cml). since the introduction of tki in our centre in 2004, the trend of allo-hsct among our cml cohort has changed over time. the purpose of this study is to examine hsct outcomes of our cml cohort who was either tki naïve or has received tki therapy prior hsct. between may 1999 and december 2015, 98 cml patients in our center received allo-hsct with 39% were tki naïve. the time of diagnosis to transplant was significant shorter among the tki naive group as compared to those received tki prior hsct (17.29 ± 7.29 months versus 42.33 ± 31.92 months, respectively). there were no gender different (60% males) but the median age at hsct was younger among tki naïve group, 29.50 years (range: 14-44 years) versus 33.50 years (range: 16-59 years) respectively. malays remained majority ethnic group but the percentage was reduced among patients received tki prior hsct (60.5% versus 46.7% respectively). the disease phase at hsct was significant different whereby majority of tki naïve group was in first chronic phase (cp1) (60.5%) as compare to patients with prior tki exposure (35.0%). all the patients in the tki naïve group received hla-matched related siblings donor (mrd) with 81.6% marrow stem cell source whereas only 88.8% of patients who have prior tki exposure received mrd with 93.3% were from peripheral blood stem cell (pbsc). all patients in the tki naïve group but only 73.3% among patients who have prior tki exposure received full myeloablative conditioning regimen. there was slower neutrophil and platelet engraftment (19.97 ± 4.50 days versus 15.02 ± 3.55 days and 20.03 ± 6.72 days versus 13.93 ± 4.70 days respectively) among tki naive group. at 30 june 2016, the 1-year overall survival (os) of cml at all disease status was 50% in tki naive group versus 32% for patients who have prior tki exposure and transplanted in more advance disease stage. in general, patients in cp1 have the best os. there was higher incident of grade 2 to 4 acute graft-versus-host-disease (gvhd) among the tki naïve group (48.6% versus 16.7%, respectively) likely due to intensity of conditioning regimen with no significant different in chronic gvhd incident. similarly, there was higher relapse rate among tki naive patients (44.7% versus 16.7%, respectively) as upfront post transplant tki was not routinely given to this group of patients in the past. further multivariate analysis to ascertain predictors of transplant outcome among this cohort of patients included disease status, donor-recipient gender combination, ethnic difference will be presented. in conclusion, despite emergent of effective and potent next generation tki, hsct still has it role as curative modality for patients who failed tki. as showed in our data, the transplant outcome is excellent for patients who remain in cp1 at the time of hsct and it is important to identify patients earlier, before disease progression, especially young patients, in order to optimize transplantation outcomes. disclosure of conflict of interest: none. the purpose of this analysis was to provide 10-year follow-up of the gcllsg cll3x trial which aimed at evaluating reducedintensity conditioning (ric) hsct in patients with poor-risk cll. the cll3x trial included 100 patients (median age 53 (27-65) years), of whom 90 patients were allografted with blood stem cells from related (40%) or unrelated donors (60%) using fludarabine-alkylator-based ric regimens. 24% had refractory cll at hsct, and 35% had a tp53 deletion and/or mutation. the 6-year follow-up of the trial including the observation that genetic risk factors such as tp53 lesions and sf3b1 and notch1 mutations had no prognostic impact has been previously reported. survival and relapse information was requested for all patients who underwent hsct within the cll3x trial in 9 german centres (the canadian centre was unavailable for follow-up) and were alive at the 6-year followup. results: follow-up information was received for 37/44 patients (84%) alive at the 6-year follow-up. of these, 5 patients had died (3 cll, 1 chronic gvhd, 1 secondary cancer), and 3 had experienced disease recurrence. with a median follow-up of survivors of 9.7 (0.6-15.2) years, 10-year nrm, relapse incidence (rel), event-free survival (efs), and overall survival (os) of all 90 patients allografted was 25%, 55%, 31% and 51%, respectively, without significant effects of tp53 lesions on outcome. absence of minimal residual disease (mrd) at the 12-month landmark post hsct was highly predictive for a reduced relapse risk, in particular if mrd eradication occurred only after immunosuppression withdrawal, suggesting of effective graft-versus-leukemia activity (gvl; 10-year rel 12%). in the 32 patients who were alive and event-free 6 years post allohct, nrm, rel, efs, and os 4 years after this landmark (or 10 years after transplant) was 3.4%, 18%, 79%, and 94% with a median follow-up of 4.3 years (1.2-9.2) after the 6-year landmark. notably, no relapse event occurred beyond 10 years post hsct. of those who remained event-free beyond 10 years, all 8 patients who were available for mrd assessment at their most recent follow-up were mrdnegative. altogether 39 of the 90 allografted patients had cll recurrence after transplant; 34 between 2003 and 2010, and 5 from 2011 onwards. whilst the median survival of those patients who relapsed during the earlier period was 19 months, all 5 patients with late relapse are currently alive 4-62 (median 28) months after the event. conclusions: long-term observation of patients allografted in the cll3x trial confirms that ric hsct can provide gvl-mediated sustained disease control in a sizable proportion of patients with poor-risk cll independent of the tp53 status. patients who are in mrdnegative remission one year after hsct have an 87% probability of remaining disease-free at least for 10 years. however, late relapses do occur but may benefit from strategies involving innovative pathway inhibitors. hallek: consultancy and speakers bureau for pharmacyclics, llc and an abbvie company; speakers bureau for janssen; m. kneba: consultancy, honoraria, travel grants and research funding from gilead and roche; consultancy, honoraria and travel grants from abbvie and janssen-cilag; research funding from amgen; travel grants from glaxo-smithkline; p.dreger: consultancy for roche and janssen; consultancy and speakers bureau for novartis and gilead. no evidence for an increased gvhd risk associated with post-transplant idelalisib given for relapse of chronic lymphocytic leukemia or lymphoma: first results of a survey by the ebmt chronic malignancy and lymphoma working parties p dreger 1,2 , a boumendil, l koster 2 , c scheid 3 idelalisib is a kinase inhibitor (ki) approved for the treatment of cll and follicular lymphoma (fl). idelalisib has a specific adverse effect profile including immune-mediated inflammatory conditions such as colitis and pneumonitis, raising concern about the safety of this ki if administered for treatment of malignancy recurrence after allogeneic hematopoietic cell transplantation (allohct). the purpose of this ongoing study is to provide information on the safety and efficacy of idelalisib in this setting. we included in this study adult patients who had been registered with the ebmt for an allohct for cll or lymphoma and who received idelalisib for treating disease relapse or persistence at any time after transplant as indicated by participating investigators upon request by the ebmt study office in leiden. baseline patient, disease, and transplant data were collected from med-a forms. centers were requested to provide additional treatment and follow-up information. as of november 29, 2016, a total of 19 patients have been registered, of whom a full dataset as required for this analysis was available for 14 patients (cll 9, fl 2, diffuse large b-cell lymphoma (dlbcl) 1, peripheral t-cell lymphoma 1, unspecified 1) who had undergone allohct between july 2009 and april 2015. all patients except one were male. median age at transplantation was 52 (36-63) years and the median interval from diagnosis to allohct was 3.5 (0.8-12.2) years. prior to allohct, 3 patients (1 cll and 2 lymphoma) had received an autohct and two other patients had been exposed to ki (idelalsib 1, ibrutinib 1). disease status at allohct was sensitive in 71% of the patients. conditioning was reduced-intensity in 71% of the transplants and included in vivo t cell depletion in the majority of cases (71%). donors were identical siblings in 43% with pbsc being the stem cell source in all cases. the interval between hct and idelalisib commencement was 18 (2-68) months in the cll group but only 3 (1-57) months in the lymphoma group. prior to idelalisib, grade ii-iv acute gvhd and chronic gvhd had been observed in 7% and 36% of the patients, but was still active at the time of idelalisib commencement in only two cases (14%) . four patients with cll had already failed ibrutinib given for post-hct relapse prior to idelalisib. the median time on idelalisib until documented withdrawal or event (progression, retreatment, death) was 237 (9-569) days. after start of idelalisib, one patient developed grade 2 acute gvhd and subsequently chronic gvhd, however, in this patient idelalisib was started as early as 30 days after transplant. efficacy of idelalisib in this high-risk patient sample was limited with only one pr in the cll group (stable disease 4, progressive disease 1, not available 3; lymphoma not available), translating into a median event-free survival after start of idelalisib of 240 days. five patients with cll underwent a subsequent treatment with an alternate ki (ibrutinib 3, venetoclax 2). altogether, there were five deaths, all due to diease progression (cll 2, lymphoma 3). median overall survival was 360 days for the whole sample and not reached for cll. this preliminary data does not support concerns about the safety of idelalisib in the post allohct setting. updated results of this ongoing study will be presented at the meeting. tested patients (67%) achieved a ccyr and at least a mmolr, respectively. the response to transplant by day 30 assessment correlated significantly with the disease status before transplant. a higher percentage of patients who experienced cytogenetic response before transplant experienced molecular response post-transplant (77%) compared with those who did not (61%; p = 0.027). for the entire group, the 1-year cumulative incidence (ci) of acute gvhd grade ii-iv and grade iii-iv were 41% and 15%, respectively; 5-year ci of extensive chronic gvhd was 31%. there was no significant difference in the ci of severe acute or chronic gvhd between donor types. the ci of nrm at 100 days and 1 year was 14% and 30%, respectively. the ci of cytogenetic and molecular relapse at 5 years was 22% and 31%, respectively. overall the 5-year os, pfs and gvhd-free, relapse-free survival (grfs) were 49%, 34%, and 22%, respectively. in multivariable analysis for grfs, transplant in cp2 and the use of haploidentical donor significantly associated with better grfs. the 5-year grfs of patients in cp2, ap and bp before transplant was 24%, 16% and 14%, respectively (p = 0.013). ( figure 1a ) patients receiving a haploidentical donor had a better 5-year grfs when compared with hla matched transplants (53% vs 21%, p = 0.019). ( figure 1b ) for pfs, transplantation in cp2, using a haploidentical donor and mac regimen associated with better pfs while age, cytogenetic and molecular response before transplant did not predict survival. ahsct is curative for a proportion of patients with advanced cml. pfs and grfs are favorably influenced by percentage of bm blasts and donor type, with haploidentical donor having at least as good outcomes as hla matched donors, while molecular and cytogenetic response before transplant do not appear to correlate with survival posttransplant disclosure of conflict of interest: none. allogeneic stem cell transplantation (sct) has been considered as the treatment of choice for younger patients (pts) with high-risk chronic lymphocytic leukemia (cll). role of allogeneic sct in era of novel drugs is widely discussed. here we present our results after sequential use of chemotherapy and reduced-intensity conditioning (ric) in cohort of 25 high-risk cll pts. high-risk cll was defined by one of the following: disease refractory to purine analogs, short response or early relapse (within 24 months) after purine analog combination treatment, and/or progressive disease with unfavorable genetic abnormalities (del [17p]/tp53 mutation). we analyzed 25 pts with high-risk cll undergoing chemotherapy and ric sct in our centre from august 2007 to june 2016. the median of pretransplant lines were 3 (range: 2-4), novel drugs (idelalisib, ibrutinib) were used in 20% of pts (5/25). fludarabine (30 mg/m 2 ) and cytarabine (2 g/m 2 ) for 4 days (fc) were used for cytoreduction in all pts. after 3 days of rest, ric consisting of 4 gy tbi, anti-thymocyte globulin 10-20 mg/ kg/day for 3 days, and cyclophosphamide 40-60 mg/kg/day for 2 days followed. median age of pts was 53 years (range: five-year overall (os) and relapse-free survival (rfs) was 59% and 57%. ci for cgvhd in pts surviving more than 3 months post-hct was 35% after 5 years and 49% after 10 years. in a multivariate cox-regression model the occurrence of cgvhd independently improved os (p = 0.001, hr 0.27; 95% ci 0.12-0.59%) as well as rfs (po 0.001, hr 0.19; 95% ci 0.08-0.46). high risk dipss plus score demonstrated significant inferior survival compared to intermediate-2 (os p = 0.006; rfs p = 0.009), int-1 (os p = 0.037; rfs p = 0.042) and low risk (os p = 0.021; rfs p = 0.014) which could be confirmed in multivariate analysis for os (p = 0.001, hr 3.13; 95% ci 1.56-6.3) and rfs (p o0.001, hr 4.84; 95% ci 2.05-11.43). rfs additionally was improved by splenomegaly (n = 60) vs. normal spleen size (n = 11) at time of hct (p = 0.01, hr 0.29; 95% ci 0.1-0.75). ruxolitinib (n = 20) or none (n = 45) pre-treatment compared to other drug therapy (n = 19) resulted in improved os (p = 0.013) and rfs (p = 0.031) and was an independent factor for rfs in multivariate analysis (p = 0.046, hr 0.39; 95% ci 0.16-0.99). non-relapse mortality (nmr) was significantly determined by high-risk dipss plus score (p = 0.001) or dipss high and int-2 (p = 0.009). relapse incidence was significantly lower in pts with splenomegaly compared to asplenic or normalspleensized pts prior to hct (p = 0.027). our data point out that pre-therapy and dipss or dipss plus score are relevant pre-transplant outcome factors while chronic gvhd is the most important independent hct-related factor. furthermore, splenomegaly at hct reduces risk of relapse and therefore improves rfs. [p582] disclosure of conflict of interest: none. thalassaemia major affects 10 000 new babies in india each year and haematopoietic stem cell transplantation offers the only chance of cure. we present data on 179 children with thalassaemia major aged between 9 months and 17 years using a uniform conditioning regimen consisting of thiotepa 8 mg/kg, treosulphan 42 gm/m 2 and fludarabine 160 mg/m 2 . equine antithymocyte globulin at a dose of 45 mg/kg was added to children who were undergoing transplantation from an unrelated donor source. there were eight deaths before engraftment due to sepsis or bleeding and two related to graft versus host disease. all patients showed complete chimerism on day 28. however, in 21 children there was an acute drop in donor chimerism between day 60 and 120 post transplantation. immunosuppression was abruptly stopped when donor chimerism dropped below 95% in all children. seven children responded well and re-established complete chimerism with this measure. seven children progressed to develop complete graft loss. donor lymphocyte infusion (dli) in the form of small aliquots of peripheral whole blood from the donor was administered in seven children. dli was used in a graded fashion every 2 weeks starting from 1 × 10 4 /kg of cd3, followed by 1 × 10 5 /kg and 5 × 10 5 /kg. all of them continued to maintain their graft with these interventions. drop in chimerism was seen particularly in children less than 3 years at the time of transplantation comprising 14 out of 21 children. older children with lucarelli class iii were also prone to rejection in our earlier series and this complication has now been eliminated with pre-transplant immunosuppression and hypertransfusion. children above the age of 7 years were more prone to graft versus host disease and required on average 18 months of immunosuppression. treosulphan based protocol has been equally well tolerated by all age groups, all lucarelli classes of children with thalassaemia major and different donor sources. the transplant related mortality and graft rejection rates have been low at 5.5% and 3.9%, respectively. however, children less than 3 years need to be monitored carefully during the first 4 months of transplantation as early withdrawal of immunosuppression can prevent graft rejection resulting in excellent outcomes. disclosure of conflict of interest: none. 16 institute of cellular medicine, newcastle university, newcastle-upon-tyne, uk hemophagocytic lymphohistiocytosis (hlh; hemophagocytic syndrome) is a rare syndrome of potentially fatal, uncontrolled hyperinflammation. allogeneic stem cell transplantation (allosct) is indicated in familial, recurrent or progressive hlh. additional recommendations include central nervous system involvement and unknown triggering factor. while data for allosct outcome are available for the pediatric setting, information for adults is very limited. the aim of this study was to retrospectively analyze the information from the ebmt databases about adult hlh patients who underwent allogeneic stem cell transplantation. we obtained data of 67 adult (≥18 years of age) patients transplanted due to hlh. additionally, an hlh-oriented questionnaire was sent to the clinical centers, with 23 responses received so far. median age at transplantation was 28 (range: 18-65). there was a slight male predominance 43/67 (64%). the majority of patients were reported with secondary hlh 33/67 (49%), the familial disease was reported in 29/67 (43%) patients. in two patients triggering factor was attributed to malignancy. the majority of patients received stem cells obtained from the peripheral blood (52/67; 78%) while for the remaining ones it was bone marrow. reduced intensity conditioning was used since 2004 in 23/63 (37%) of patients. thirteen (19%) patients received tbi. donor choice was: 33 matched unrelated (50%), 7 mismatched unrelated (11%), 26 identical sibling (39%). engraftment was observed in 55/61 (77%). the cumulative incidence of acute graft versus host disease (gvhd) at 100 days was 26% (95% ci 15-37%). the cumulative incidence of chronic gvhd at 1 year after allosct was 13% (95% ci 2-23%) and increased to 31% at 3 years (95% ci 16-47%). the 3-year probability of overall survival is shown in fig.1 . the median survival time was 8 months. the 3-year os was 41% (95% ci 28-55%). for patients who survived until 3 months, this proportion was more favorable with an os of 62% (95% ci 45-78%) at 3 years after transplantation. among 19 patients with observation times longer than 15 months, only one patient died (in the 48th month after allosct due to relapse which occurred in the 12th month. after 12 months no more relapses of hlh were recorded-the cumulative incidence reached 19%. the non-relapse mortality reached 42% after 15 months. the familial disease was associated with a better prognosis than secondary hlh (p = 0.04). unlike the pediatric population, where reduced intensity conditioning (ric) was associated with higher survival, in adult patients there was no difference between the conditioning types. data form the 23 questionnaires confirm clinical picture typical for hlh at the diagnosis: fever in 21/22 (95%), splenomegaly in 19/20 (95%), hemophagocytosis in 18/20 (90%) and hyperferritinemia with median concentration of 4215 ng/ml (range: 63-260 160). image fig.1 overall survival after allogeneic stem cell transplantation for adult hlh patients until 36 months (95% confidence intervals are shown in grey). the number of patients at risk is indicated below the time axis at the corresponding time points. to our knowledge, this is the largest group of adult patients with hlh who underwent allogeneic stem cell transplantation. relatively low relapse incidence shows that allosct can effectively cure hlh. patients who survive the first period after this procedure can expect a long disease-free survival. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative option for children suffering from various life-threatening inherited non-malignant diseases with best results using hla-identical family donor. hsct from unrelated or mismatched family donors is associated with increased risk of agvhd and graft rejection.use of post-transplantation cyclophosphamide (ptcy) with or without additional immunosuppression has been shown to be effective prophylaxis against gvhd in patients with hematological malignancies. there are limited reports of hsct using pt cy for patients with non-malignant disorders. we retrospectively analyzed results of 18 hsct in patients with life-threatening non-malignant diseases using ptcy-based gvhd prophylaxis. patients characteristics are presented in table 1 . thirteen patients (72.2%) were transplanted upfront, 5 patients (27,8%) were rescued after primary or secondary graft failure after first hsct. donors were hla-matched (n = 8) or mismatched (8-9/10) (n = 4) unrelated, haploidentical (n = 5) or hla-identical family (n = 1). bone marrow was used as graft source in 13 (72.2%) patients and peripheral blood stem cell in 5 (27.8%). median cd34+/kg recipient weight-7.25 × 10 6 (3.0-13.5), cd3+/kg-9.785 × 10 7 (0.78-90.6). the conditioning regimen was myeloablative in 12 patients (conventional-3, reduced toxicity-9), reduced intensity-6. the gvhd prophylaxis consisted of a combination of ptcy at dose of 50 mg/kg on days +3 and +4 with calcineurin inhibitors (tacrolimus-6 pts, cyclosporine a-10 pts) or sirolimus (1 pt) and mmf (15 pts) starting on day +5. all but one patients received also serotherapy with rabbit (12 pts) or horse atg (5 pst) and rituximab (4 pts). with a median follow-up of 8 months (range: 1-33), the kaplan-meier estimates of os − 81.5%. one patient with thalassemia died before engraftment on day+11 from severe vod. 15/17 pts (88%) achieved engraftment. the median time for neutrophil and platelet engraftment was 23 (16-28) and 19 days (12-32), respectively. primary graft failure was observed in 2 patients (1 was successfully retransplanted from another haploidenticle donor, 1 was not eligible for a second transplantation, but alive). at last follow up, 10 (67%) patients had full donor chimerism, 4 (27%) had stable mixed chimerism without signs of disease progression. one patien with wiscott-aldrich syndrome had secondary graft failure with progressive loss of donor chimerism and were successfully rescued with second haploidentical transplant from the same donor. of 15 engrafted patients, agvhd ii-iv was seen in 4 (26.7%) patients. one patient developed grade ii (gut stage ii) agvhd, which resolved with systemic steroids. severe (griii-iv) agvhd was observed in 2 pts after second hsct, both had calcineurin and mtor-inhibitors induced toxicity leading to discontinuation of this drugs, but responded on combined (steroids and ruxolitinib) therapy. one patient with was developed grade iii gvhd (gut stage 4) after severe cmv-colitis and died on day from multiple organ failure (suspected tma). one patient developed extensive chronic gvhd of kidney (minimal change [p587] disease) after tapering of immunosupression. one patient with hurler syndrome had seizures, died on day+29 from multiple organ dysfunction syndrome. conclusion: ptcy is a promising option for agvhd prophylaxis in patient with non-malignant disease, lacking an hla-matched family donor. disclosure of conflict of interest: none. an exploratory, open-label study to evaluate the safety and feasibility of atir201, a t-lymphocyte enriched leukocyte preparation depleted ex vivo of host alloreactive t-cells (using photodynamic treatment), as adjuvant treatment to a t-cell depleted haploidentical hematopoietic stem cell transplantation in patients with beta-thalassemia major c selim 2 , w rob 3 , l sarah 4 , f josu de la 5 previous studies demonstrated that donor lymphocytes, selectively depleted of alloreactive t-cells (atir), could be given safely in adult patients receiving a haploidentical hsct. in 42 patients a single dose of atir, at doses up to 2 × 10 6 viable t-cells/kg, was given and no grade iii/iv acute gvhd has been reported. this confirms the efficacy of the elimination method of allo-reactive t-cells and attributes to its beneficial safety profile. in an ongoing phase 2 study, cr-air-007 (nct01794299), infusion of atir101 at 28 days post-hsct results in a reduction of transplant-related mortality (trm) and improvement of overall survival and event-free survival. adjunctive treatment with donor lymphocytes in patients receiving a t-cell depleted, haploidentical hsct for nonmalignant diseases such as beta thalassemia major, could provide early immunological support and better immune reconstitution in the absence of gvhd. in an open-label, multicenter phase 2 study (cr-bd-001; eudract 2016-002959-17), 10 patients age ≥ 2 years and ≤ 25 years with beta thalassemia major will undergo a haploidentical hsct with adjunctive administration of atir201. patients will receive a t-cell depleted graft (cd34-selected, or cd3/cd19 depleted, or tcr-αβ depleted, depending on the experience of the study center) from a related, haploidentical donor, patient conditioning will be myeloablative following standard practices at the study center. atir201 infusion at a dose of 2 × 10 6 viable t-cells/kg is given between 28 and 32 days after the hsct. to assess safety, patients will be evaluated for the occurrence of dose limiting toxicity (dlt), defined as acute gvhd grade iii/iv within 180 days post hsct. efficacy will be primarily evaluated by transfusion-free survival (tfs), occurrence of severe infections, and time to t-cell reconstitution, taking into account hematologic and sustained engraftment. all patients will be closely monitored for cmv, ebv and adenovirus titers, with initiation of pre-emptive treatment upon rising blood titers. regulatory authorities in the united kingdom and germany have approved this clinical study protocol. enrolment of the study is expected to continue during 2017, with first report of safety of atir201 to be expected first half 2018. disclosure of conflict of interest: j. rovers is employee of kiadis pharma, sponsor of the study. sickle cell disease (scd) can be cured with haematopoietic cell transplantation (hct), yet progress in the practice and research of hct for scd has not come without risks and uncertainty. the information and decisions that families and physicians encounter in this field are complex and hanging. in this hermeneutic study, we analyze the case of one family who advocated for hcts for two of their four children knowing the potential risks. these experiences have had a profound impact on both the family and the medical team. this study was conducted through the research method of hermeneutic phenomenology. hermeneutic inquiry is described as the practice and theory of interpretation and understanding in human contexts and aims to make sense of the particulars of these contexts and arrive at deeper understandings. data collection: in-depth interviews were conducted with the mother of the family, the hct nurse coordinator, and the hct physician. the interviews were audiotaped and transcribed verbatim. the transcribed interviews were later reviewed by the physician, who then wrote an additional reflection. this work culminated in approximately 70 pages of single spaced data in textual form. in hermeneutics, interpretation takes place through a careful reading and re-reading of the data, looking for statements and instances that resonate with the researcher. initial individual interpretations of researchers are then raised to another level of interpretive analysis in the research team's communal attention to the data. particular criteria guide the analysis: agreement, coherence, comprehensiveness, potential, and penetration. the following excerpts and interpretations are provided as examples of the analysis, with names changed for confidentiality. "being heard" arose repeatedly in this family's experience, including at the time of their request for a transplant without meeting the traditional criteria for hct. they persisted in their belief that their children would benefit from hct. "they gave us options to see if there was a chance for a transplant...how life would look…. and then we figured…a transplant for him was better at the time…worth the risks…. and you wouldn't even know. he plays basketball now, he plays sports, he's active and he can exercise and run. i never had any regrets because i felt it was better and the most important thing is his organs were really intact; none of the organs were destroyed…so i think it's the right decision we made" (mother). "this family has changed my career, and my life as a result. they challenged my practice and way of thinking. they did so in a considerate way, out of a duty to advocate for their children. we worked through the tension of different viewpoints with respect and all of us grew in the process. at least i can say our team did. i certainly did... i am humbled by their trust and respect…i am grateful to them" (physician). patients and providers are deeply impacted by their interactions. dr. robert buckman stated that it was the individual case that changed his practice always. he claimed he could not walk into a new patient's room without his practice being forever changed. in presenting this hermaneutic analysis, we aim to remind ourselves of the opportunity for growth that can result from reflection on this sacred patientprovider relationship. disclosure of conflict of interest: none. defibrotide (df) prophylaxis and adjustment of busulfan schedule to prevent veno-occlusive disease and thrombotic microangiopathy in an infant with a membrane cofactor protein (mcp) gene mutation and metachromatic leukodystrophy undergoing hematopoietic stem cell gene therapy (hsc-gt) v calbi 1,2 , f fumagalli 1,3,4 , r penati 3 , g consiglieri 3 , m migliavacca 3,1 , d redaelli 1 , s acquati 1 , v attanasio 1 , r chiesa 5 , f ferrua 1,3 , f barzaghi 1,3 , m cicalese 3,1 , a assanelli 3,6 , p silvani 7 , s tedeschi, r arora 8 , a soman 8 , f ciotti 3 , m sarzana 3 , g antonioli 3,1 , c baldoli 9 , s martino 10 , gl ardissino 11 , mg natali sora 4 , l naldini 1,12 , f ciceri 12,13 , a aiuti 1,12,3 and me bernardo hepatic veno-occlusive disease (vod) and thrombotic microangiopathy (tma) are life-threatening complications that can occur after hsc transplantation. expert consensus guidelines support use of df for treatment and prophylaxis of vod due to its ability to restore thrombo-fibrinolytic balance and protect endothelial cells. presymptomatic monozygous twins affected by late infantile metachromatic leukodystrophy (mld) underwent investigational hsc-gt after conditioning with busulfan. no comorbidities were evident at baseline. the dose of transduced cd34+ cells was similar in both patients (18.2 × 10 6 cd34+/kg for patient 1 and 14.1 × 10 6 cd34+/kg for patient 2). patient 1 (p1): at 8 months of age, received conditioning with iv busulfan 80 mg/m 2 /dose for 4 doses (target auc 85 mg × h/l). on day (d) +18 after gt, he developed severe vod and was treated with diuretics, fresh frozen plasma, paracentesis and df. on d+39 schistocytes in peripheral blood, marked proteinuria, complement factor consumption, and increases ldh and bilirubin were observed. the patient's condition worsened, with reduced urine output and generalized oedema with pleural effusion. stool, urine and blood cultures were negative and adamts13 activity was 35%; anti-complement factor h (cfh) antibodies (ab) were positive (1371 ui/ml). these findings led to the diagnosis of atypical hemolytic uremic syndrome (ahus; a form of tma) and eculizumab (300 mg/weekly dose) was started on d +40. patient subsequently developed pulmonary oedema and needed non-invasive ventilation. molecular analysis revealed a heterozygous deletion of cfhr3-r1 and ala353val mutation in the mcp gene, a defect previously shown to be associated with ahus. due to the presence of ab anti-cfh and antiplatelet, 4 weekly doses of rituximab (375 mg/m 2 ) were administered. after treatment, p1 progressively improved although he showed prolonged severe anaemia and thrombocytopenia and bone marrow (bm) hypoplasia, secondary bleeding which required reinfusion of unmanipulated autologous bm cells on d +66. nine months after hsct-gt p1 has shown good hematopoietic recovery, stable engraftment of the transduced hscs, no signs of renal damage or complement activation, albeit with neurodevelopmental delay. patient 2 (p2): given his twin history and genetics, this 9 month old infant was considered at increased risk of vod, so prophylaxis with df (25 mg/kg/d) was administered from d-4 to d+30 and the busulfan conditioning was modified by adjusting the auc to a lower target (1 mg/kg/dose for 14 doses; target auc 67.2 mg × h/l). the child had a good clinical recovery and didn't develop signs of vod or tma after hsc-gt. on d+12 and +14, respectively, anti-cfh and anti-platelet ab were positive. considering the history of the twin, 4 weekly doses of rituximab were administered. p2 is currently 8 months after gt with persistent engraftment of transduced hscs and no signs of tma. data from this case-control report of monozygous twins diagnosed with mld, and subsequently shown to also harbor mutations in complement regulator gene, suggest that df prophylaxis and busulfan adjustment may have helped prevent systemic microangiopathic damage in the second twin. patients with rare disease may have mutations in genes in addition to those that cause their disease. patients at risk of post-transplant tma following hsc-gt for genetic diseases may require tailored df prophylaxis and treatment. disclosure of conflict of interest: a. aiuti is the principal investigator of the tiget-mld clinical trial of gene therapy. the mld gene therapy was licensed to glaxosmithkline (gsk) in 2014 and gsk became the financial sponsor of the trial. all authors declare no other competing interests. hematopoietic stem cell transplantation (hsct) using an optimized conditioning regimen is essential for the longterm survival of patients with inherited bone marrow failure syndromes (ibmfs). we report hsct in 25 children with fanconi anemia (fa, n = 12), diamond-blackfan anemia (dba, n = 7), dyskeratosis congenita (dc, n = 5) and shwachman-diamond syndrome (sds, n = 1) from a single hsct center. the graft source was peripheral blood stem cells (n = 20) or cord blood stem cells (n = 5). fa, dc and sds patients received reduced-intensity conditioning, while dba patients had myeloablative conditioning. the median numbers of infused mononuclear cells and cd34+ cells were 14.4 × 10 8 /kg and 4.5 × 10 6 /kg, respectively. the median time for neutrophil and platelet recovery was 12 and 17 days, respectively. there was one primary graft failure. after median follow up 3 years the overall survival was 96%. the incidence of grade ii-iii acute and chronic graft versus host disease (gvhd) was 32% and 16% respectively. in a multivariate analysis, the type of conditioning regimen was the only factor identified as significantly associated with grade ii-iii acute gvhd (p = 0.01). we conclude that hsct can be a curative option for patients with ibmfs. disease specific conditioning regimen was important to disease the transplant-related mortality. [p591] disclosure of conflict of interest: none. homozygous sickle cell disease (scd) patients suffering from end-stage renal disease (esrd) show a variable outcome after kidney transplantation as underlying disease can cause poor allograft survival and disease-specific problems. we present a case of a 27-year old patient with severe scd and esrd who underwent haploidentical bone marrow transplantation (bmt) with consecutive living kidney transplantion (lkt). the patient suffered from multiple complications of scd including stroke with secondary hemorrhage, symptomatic epilepsy, esrd and uncontrolled hypertension. the patient had been on hydroxyurea without success and required regular blood transfusion due to severe renal anemia. the rationale for bmt was uncontrollable iron overload. a reduced intensity conditioning regimen was used with (fludarabine, cyclophosphamide and 2gy of tbi, dose-adjusted to esrd). graft-versus-host disease (gvhd) prophylaxis consisted of post-transplant high-dose cyclophosphamide, cyclosporine a (cya) and mycophenolate mofetil (mmf). the donor was her 56-year old mother with hbs trait, the stem cell source was bone marrow, the cell dose 4.74 × 10 8 nucleated cells/kg. during conditioning daily hemodialysis was performed to keep drug levels stable. neutrophil engraftment occurred on day +26, chimerism at day +19 was 98%. hbs increased from 1.3% pre-hsct to 40.0% 6 months after hsct. hemoglobin values increased from 70 g/l pre-hsct to 110 g/l post-hsct and reticulocytes from 16 g/l to 124 g/l. erythropoietin levels increased from 2.3 iu/l pre-hsct to 178 iu/l 6 months after hsct. during the follow-up, the patient did not show any sign of acute gvhd or vaso-occlusive crisis, hemolysis or sickling. relevant complications were disease-related (therapy resistant hypertension and epileptic seizure due to former brain damage). on day +151 a lkt from the same donor was performed. the initial immunosuppressive treatment with mmf was continued, cya was switched to tacrolimus and steroids were added for 3 months. the post-transplant period was uneventful. currently, 12 months after haploidentical bmt and 6 months after lkt there are no signs of gvhd, the blood chimerism is 100%, the kidney allograft function is very good (gfr 73 ml/min/1.73 m 2 ) and immunosuppression is withdrawn. iron overload is being corrected by regular phlebotomies. the patient no longer requires antihypertensive medication and there is evidence of vascular remodeling. this is the first report of a successful haploidentical bmt followed by kidney transplantation from the same donor in a patient with scd. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) can cure non-malignant diseases, such as primary immune deficiency (pid), severe aplastic anemia (saa) and osteopetrosis (op). in the absence of a well-matched donor, transplantation from a haplo-identical donor maybe considered. post-transplant cyclophosphamide (ptcy) is a new strategy derived from the treatment of malignant diseases in adults that has been little studied in high-risk pediatric nonmalignant diseases. fifteen children (2.2 years, range: 0.19-10.88) underwent hsct in the pediatric immunology and hematology unit of necker hospital, paris, between december 2014 and september 2016. these children were suffering from op (n = 3), saa (n = 2), hemophagocytic lymphohistiocytosis (hlh) (n = 3), immunodysregulation polyendocrinopathy enteropathy x-linked (ipex) syndrome (n = 2), combined immune deficiency (n = 4) and leukocyte adhesion molecule deficiency (n = 1). three patients received a low-intensity conditioning regimen (cr) (based on fludarabine, cyclophosphamide, and total body irradiation) whereas the other 12 received myeloablative cr (based on busulfan auc targeted and fludarabine). fourteen patients received serotherapy before hsct. post-transplant cyclophosphamide (50 mg/kg/ day) was given on d3 and d4 and graft versus host disease (gvhd) prophylaxis with cyclosporine and mycophenolate mofetil was initiated on d5. the transplanted stem cells were obtained from bone marrow in all cases. engraftment with full donor chimerism was observed in 13 patients. the median cd34+ cell dose was 14.8 × 10 6 cells/kg body weight (range: 7.4-35.9 × 10 6 ). neutrophils recovered after a median of 18 days (range: 14-32), and overall survival (os) was 80% after a median follow-up of 1 year (range: 0.18-1.98). three patients died due to graft failure (n = 2) or infectious complications related to gvhd (n = 1). grade ii acute gvhd occurred in 8 of the 13 patients displaying engraftment (62%), and chronic gvhd and/or autoimmune complications were observed in four patients (31%). viral complications were frequent, occurring in 10 patients (77%) with cmv infection (n = 8) /disease (n = 1), adenovirus disease (n = 1) and bk virus cystitis (n = 4). haploidentical transplant with ptcy is feasible in high-risk patients with non-malignant diseases. chronic gvhd and autoimmunity were more frequent than for more conventional approaches in such patients. infection rates were high. disclosure of conflict of interest: none. sickle cell disease (scd) remains associated with high risks of morbidity and early death. even best of supportive care fails to improve quality of life. hematopoietic stem cell transplant (hsct) can be considered for selected group of patients. in long run it is not just economical but also substantially improves quality of life (qol). we report our experience with hsct for scd from india. seventy three consecutive patients suffering from scd who underwent hsct between january 2006 and november 2016 were included in the study. fifty two underwent matched sibling donor (msd), 2 matched family donor (mfd), 3 matched unrelated (91/0 or 10/10), 2 cord blood transplant cbt (1 matched sibling cord blood and 1 matched unrelated) and 15 patient underwent haploidentical transplant. different conditioning regimens were used and so was the graft versus host disease prophylaxis depending on institutional protocols as depicted in table 1. a total of 73 patients underwent sct. the median age was 9 years (10 months-29 years). m/f ratio was 45/28. majority of patients were either from african union or oman. all patients suffered from one or other severe symptoms making them eligible for sct. graft source was bone marrow (bm) in 30 with median cd34 count of 5.3x106/kg (0.92-10.7), peripheral blood (pb) in 36 with median cd34 count of 8.5x106/kg (3.9-20.18), cord blood in 2 with median cd34 count of 1.27x105/kg (0.44-2.1) and combined bm & pb in 5 with median cd34 count of 6.37x106/kg (1.5-23.3). of the 73 patients, 61 are alive and disease free with lansky/karnofsky scores of 100. there were 8 deaths (4 msd/mfd/mud; 3 haploidentical and 1 matched unrelated cbt). four patients rejected the graft (2 haploidentical and 2 msd/mfd/mud). at the last follow up, the probabilities of survival, scd-free survival, and transplantrelated mortality were 89%, 83.5%, and 11%, respectively. outcome of hsct in scd has improved significantly. with better conditioning regimens, improved supportive care, the outcome of alternative donor transplant and adult scd has improved and matches sibling donor transplant. hsct should be strongly considered as a curative modality for selected patients suffering from scd. disclosure of conflict of interest: none. s434 staff jointly defined more than 50 local standard operating procedures. patients with low-risk characteristics (age ≤ 7 years, liver size ≤ 2 cm below costal margin) and a hla matched sibling donor were considered eligible in this initial phase of activity. a downstaging protocol with hydroxyurea and deferoxamine or deferasirox was adopted. conditioning regimen included iv busulfan and cyclophosphamide. gvhd and rejection prophylaxis included atg from day − 12 to − 10 and csa, mtx and methylprednisolone. gcs-f primed bone marrow was chosen as stem cell source. the first allogeneic hsct of the whole iraq was performed in a child with thalassemia at hiwa hospital in october 2016. up to now, 3 patients (2 females, 1 male) underwent hsct; median age at transplantation was 2 years; median infused tnc 18.5 × 10 8 /kg, cd34+ 10.1 × 10 6 /kg. all of them engrafted. no major complication were observed. one of them developed grade ii agvhd (skin only) which resolved after increasing the dose of steroids. a huge number of patients with low-risk thalassemia are now in the waiting list and some of them have already started downstaging having planned hsct in a short time. a matched sibling transplant program in children with thalassemia is feasible and safe in kurdistan. such a program can provide many advantages: far less psychosocial and financial burden for the families and significant saving for the government. the estimated costs of performing locally hsct are much less than in the countries where patients were previously referred. the continuation of cooperation is of paramount relevance for further implementing the activity and extending the transplant accessibility to patients with other hemato-oncological disorders of childhood. disclosure of conflict of interest: none. long term follow-up after reduced-intensity conditioning and stem cell transplantation for thalassemia major r rihani, a natsheh, sm abu, e khattab, r najjar, f sheab, s sharma, n hussein, a tbakhi and m sarhan bone marrow and stem transplantation program-king hussein cancer center, amman, jordan hematopoietic stem cell transplantation (hsct) is the only curative treatment for thalassemia major (tm). reducedintensity conditioning (ric) before hsct for high risk tm patients results in fewer complications, when compared with myeloablative regimens. one hundred and three tm patients received hscts from an hla-identical related donor at king hussein cancer center, between january 2002 and november 2016. of those, 62 were high risk tm (60%) who received ric hscts. in this report, we describe follow-up beyond 2 years (median, 42; 24.6-126 months) post ric hscts. forty-four class ii-iii patients (58%) were identified (25% with hepatitis c); with a median age of 14 (13.4-28) years. females accounted for 59% (n = 26). conditioning regimen consisted of oral busulfan 8 mg/kg, fludarabine 175mg/m 2 ,tli 500cgy and atg followed by pbsct. gvhd prophylaxis consisted of mmf and csa. median infused stem cell dose was 5.4 × 10 6 /kg. all patients attained neutrophil and platelet engraftment (median, 15.3 and 21.3 days, respectively). persistent mixed donor or full donor chimerism were observed in 95.5% (n = 42) and 4.5% (n = 2), respectively. immune-suppressive therapy for gvhd treatment was required in 16 (36.4%) patients (agvhd, n = 8; cgvhd, n = 8). moreover, veno-occlusive disease occurred in 4 patients (9%) that resolved completely. secondary graft failure was noted in 11 (25%) patients. the 5-year overall survival was 100%, while the 5-year probability of thalassemia-free survival was 72.2%. other factors evaluated include: growth parameters, endocrine and other organ functions, in addition to functional status. this report confirms the safety and efficacy of ric regimens in hscts for high risk tm patients. those regimens are associated with excellent engraftment and sustained mixed donor chimerism; and lead to excellent thalassemia-free and overall survival rates. [p596] disclosure of conflict of interest: none. in-time hsct for pts. with hurler syndrome (hs) can significantly improve the results. long-term follow-up and late effects estimation required to prepare a special observation and rehabilitation programs. aim. to analyze our experience with hsct for hs in the field of special observation and rehabilitation programs. forty hsct during the 2004-2016 were performed for 38 pts. with hs. median age at the diagnosis was 15 months (3-38 months), at hsct-24 months (9-48 months). bm used in 62.5% (n = 25), pbsc-32.5% (n = 13), cb-5.0% (n = 2). mac conditioning was used for 29 hsct, ric-for 11. ric regimen: flu+mel+atg, mac: bu/treo +flu+thio/mel (bu was used in early 2000) and atg +rituximab (in case of mud hsct). all pts. with ric received mud hsct, pts. with mac mud-18 pts., mrd-5 pts. pts. received csa/tacro-based gvhd prophylaxis. mmf/mtx was additionally added in all cases. in 3 ric hsct immunomagnetic сd3/сd19+ depletion of pbsc (by clinimacs) was used. a special observation protocol including somatic and neurocognitive estimation was developed. all pts. engrafted with full donor chimerism on d+30. median of engraftment day-20 (11-29 days). thirty three pts. survived. reasons of death-mac: infections-3 pts., ric: trali-1 pt., agvhd-1 pt. trm improved, over the years, with improving of supportive care and donor selection as well as pre-transplant screening. no early severe toxicity revealed. pulmonary infection episodes was registered in 30% of pts. in our study. gvhd: grade iideveloped 14 pts., grade iii-iv-2 pts. (after ric), local cgvhd-3 pts. (ric). no extensive cgvhd. 5 pts. rejected (mac and ric rejection rate was same). at median follow up of 60 months (8-160 months), the estimated 5 years pos was 81%. best response correlated with early hsct (and better status before hsct) and higher level of aidu after. late effects estimation showed that 47.4% (n = 18) of patients experienced late effects: cardio-vascular-16 pts., skeletal-15 pts., endocrine-3 pts. all pts. with cardio-vascular effects received mac. skeletal effects affected patients of older age, pts. transplanted in younger age do not have such effects. median period of late effects arising after hsct was 15 month (3-27 months). only 3 pts. experienced serious pulmonary late effects (infections), all episodes was before 2010. no pts. in our study have progressive retinal degeneration. 80% of pts. improved in the neurosensory component and all pts. improved in neurocognitive status and development after hsct. best response correlated with neurocognitive rehabilitation based on unique computer model used by our group in russian national rehabilitation center "russkoe pole." in-time hsct is an effective and safe way to stop neurodegenerative process for pts. with hs. both mac and ric regimens can be used with the same effectiveness. mac regimens associated with bigger number of cardiovascular late effects. long-time follow-up showed that these patients require the special observational protocol including estimation of cardio-vascular, skeletal, endocrine and neurocognitive risks. better neurocognitive response correlated with intensive rehabilitation using computer model. russian joint study showed effective cooperation for treatment pts. with hs in the national setting. disclosure of conflict of interest: none. little is known about pathogenesis of solid tumors after hsct but, intensive cytotoxic conditioning therapy with defective dna repair of persisting stem cells/stromal cells, viral infection, and immunosuppression may play a role. 3/6 patients with solid tumors had a melphalan-based conditioning. melphalan was linked to sarcoma and lung cancer in animal model. there are few data linking parotid mec to infection by cmv and hhv6 which can remain dormant in the salivary glands. both affected patients had hhv6 during the transplant period. p8 and p11 had a family history of solid tumor pointing to a possible genetic factor. whilst secondary malignancy post-hsct for patients with malignant disorders is well recognised, non-ptld malignancy post-hsct for pid has not previously been reported. a larger study is needed to evaluate incidence and risks. allogeneic hsct is a treatment of choice for the bone marrow failure in patients with sds. hsct from unrelated or mismatched family donors is associated with higher morbidity and mortality compared with matched sibling. combined pgd and hla antigen testing is a possible option to preselect a compatible donor for an affected sibling requiring hsct. we describe a case report demonstrating first successful hsct for 6 years girl with sds by using preimplantation genetic diagnosis and hla matching. diagnosis of sds was suspected at 5 m.o., based on clinical features, family history, laboratory studies. at 6 m.o., bone marrow (bm) aspiration revealed hypocellular marrow with signs of dysplasia and expansion of blasts (15.6% blasts). the sanger sequencing of sbds gene showed c.183-184ta4ct and c.258+2t4c mutations. the patient had recurrent infections, including bilateral pneumonia caused by phaeohyphomyces, bloodstream infection, cmvdisease. due to the lack of matched related or unrelated donors, hsct with ric (flu, mel, atg) from haploidentical father was performed at 16 months of age. after the 1st allo-hsct, engraftment was achieved on d+13, initial str study showed full donor chimerism. post-transplant period was complicated with severe cmv-infection and signs of secondary hlh. at d+135, graft rejection was registered. the girl became dependent on regular rbc and platelet transfusions, bm examination revealed hypocellularity with moderate signs of myelodysplasia without elevated blast count. due to lack of available hla-compatible donors, an option of in vitro fertilization (ivf) with preimplantation selection of a normal hla-matched embryo was considered. after controlled ovarian hyperstimulation 2 embryos were hla-compatible and healthy (first, wild-type; second, heterozygous for sbds gene mutation c.183-184ta4ct). hence, the only unaffected hla-identical embryo was transferred resulting into full-term pregnancy. at the age of 5.5 years after 1st hsct, the 2nd s437 transplant was performed with a combination of cb and bm as a source of hematopoietic stem cells. the donors' age was 2 years a reduced toxicity conditioning regimen (rtc) based on flu150 mg/m 2 , treo42 g/m 2 , thiotepa 10 mg/kg with serotherapy (thymoglobuline 7.5 mg/kg) was used. because of neurotoxicity, arterial hypertension, since d+1 csa was changed to sirolimus +mmf for gvhd prophylaxis. the total number of infused nc was 2.5 × 10 8 /kg; cd34+, 3.85 × 10 6 /kg; cd3+, 2.4 × 10 7 /kg. engraftment was achieved on d+28. any signs of gvhd, severe infectious or toxic complications were not observed. eight months later, the patient is alive, has full donor chimerism in bm and is not transfusion-dependent. in the absence of hla-identical donor, ivf with preliminary pgd and hla-typing could be a chance for matched donor to cure patients with non-malignant genetic diseases. in case of low cord blood cellularity, a combination of cb and bm from the same sibling could be used. our experience showed a successful engraftment of sds patient and stable donor chimerism after second hsct of cb and bm from pgd-selected sibling with rtc. disclosure of conflict of interest: none. the safety and efficacy of familial haploidentical (fhi) stem cell transplantation utilizing cd34 enrichment and cd3 addback in patients with high risk sickle cell disease (scd) ( figure 1a ). probability of 1yr efs is 87.4% (ci95: 58-97%) ( figure 1b ). immune cell reconstitution has been robust and similar to rtc and msd allosct in scd (table 1 ). there have been 3 deaths, vod, steroid refractory agvhd and cgvhd. mac followed by fhi utilizing cd34 enrichment and t-cell addback in patients with high-risk scd is safe, tolerable and results in long-term donor chimerism and absence of scd symptoms or complications. a larger cohort and follow-up will be required to confirm these preliminary findings. disclosure of conflict of interest: none. supported by r01fd004090-01a1. [p601] s438 lymphoma p602 a clinical prognostic index for assessing patients aged 460 being considered for high-dose therapy and autologous stem-cell transplant in relapsed or refractory high-grade non-hodgkin lymphoma d edwards 1 , k kirkland 1 , r pearce, s robinson 1 and g cook 1 bsbmt patients with relapsed high-grade nhl or disease refractory to first-line therapy can still be cured with high-dose therapy and autologous stem cell transplant if they respond to salvage chemotherapy. this aggressive algorithm is accepted in younger patients but is less well established in the elderly. age 460 has a negative predictive score in the international prognostic index (ipi) and there are concerns that the outcomes of hdt in these patients are significantly worse. deciding which older patients will benefit from hdt is challenging and there are no established predictive tools to guide physicians. we present a clinical prognostic index derived from information readily available at the time a patient is being assessed for asct the bsbmt audited the outcomes for uk patients aged 460 transplanted between 2004-2009 (n = 371) and benchmarked against the european bone marrow transplant (ebmt) database for the same period (n = 2695). the primary outcome was progression-free survival (pfs) but data was also analysed for overall survival (os), relapse rate (rr) and non-relapse mortality (nrm). we included all patients with a diagnosis of high grade nhl and the following demographic features were also analysed: age at diagnosis; age at transplant; m/f; year of transplant; cr/not cr at transplant; no. of prior therapies; no. of cells infused; clinical staging; karnofsky status at transplant; histology; ipi at diagnosis; mobilising regime and conditioning regime. candidate prognostic indices were factors achieving significance in univariate and multivariate analyses of the main outcomes by regression analysis. the best prognostic index was selected based on the bsbmt dataset and then applied to the rest of the ebmt dataset (the validation dataset). there were no significant differences in patient characteristics between the uk and non-uk groups nor in outcomes of pfs, os, rr or nrm. (figure 1 ). in both univariate and multivariate analysis the following features were associated with a significantly worse outcome for pfs, os, rr and nrm : age466, karnofsky score. disclosure of conflict of interest: none. underwent an allo-sct at our center after a treosulfan-based conditioning regimen. eleven pts received a mrd, 19 pts a mud, and 11 pts a haplo unmanipulated pbsc allo-sct. at allo-sct 17 pts were in cr, 10 pts were in pr, and 14 pts had sd/pd. hct-ci was evaluable for 34 pts, 18 had a score ≥ 3. the backbone conditioning regimen consisted of treosulfan 14g/m 2 from day − 6 to − 4, and fludarabine 30 mg/m 2 from day − 6 to − 2; twenty-five pts were treated with this reduced toxicity conditioning (rtc) regimen. intensification with other alkylating agent (melphalan, thiotepa, or cyclophosphamide) or radiotherapy (4gy total dose) was applied on the remaining 16 pts (myeloablative conditioning, mac). gvhd prophylaxis was based on cyclosporine a and methotrexate (17 pts) or rapamycin and mycophenolate mofetil (24 pts), plus anti-thymocyte globulin or post-transplant cyclophosphamide accordingly to donor type. median numbers of infused cd34 +/kg and cd3+/kg were 6.66 × 10 6 (range: 2.72-12.24) and 2.34 × 10 8 (range: 0.03-6.89), respectively. median follow-up was 61 months (range: 18-139). thirty-nine pts were evaluable for engraftment; median time to neutrophil ≥ 0.5 × 10 9 /l was 16 days (range: 10-30), and 16 days (range: 10-59) to platelet ≥ 20 × 10 9 /l. treosulfan conditioning provided a cr in 3 and 6 pts respectively in pr and sd/pd at transplant. no graft failure was observed. one and 5 years overall survival (os) was 58.5% and 52.6%, respectively. progression free survival (pfs) and gvhd-free/relapse-free survival (grfs) were respectively 51.2% and 41.5% at 1 year, 44.3% and 23% at 5 years. one and 5 years relapse/progression incidence (ri) was 29.3% and 36.1%, respectively. transplant related mortality (trm) was 14.6% at 100 days, 19.5% at 1 year and for the entire follow-up. the 100-day cumulative incidence (ci) of agvhd grade ≥ 2 was 4.9%; ci of moderate to severe cgvhd was 24.8% at 2 years. the outcome of pts in cr at 5 years was significantly better compared to that of pts with active disease in terms of both os (82.4% vs 33.3%, p o0.005), pfs (68.6% vs 25%, p o0.005), grfs (36.3% vs 12.5%, p o0.005), and ri (19.6% vs 50%, p o0.05). no statistical differences in os, pfs, and ri were found when pts were stratified according to donor type and [p602] the use of rtc or mac regimen. at last follow-up, 22 patients are alive and disease free; 3 of them obtained a durable cr using chemotherapy and/or dli for disease progression after allo-sct. treosulfan-based conditioning regimen is effective and well-tolerated in patients with advanced b-nhl undetgoing allo-sct. disclosure of conflict of interest: none. systemic anaplastic large cell lymphoma (salcl) is a very infrequent well-defined histological entity that comprises around 11% of all t-cell non-hodgkin lymphoma. in the absence of prospective clinical trials, autologous stem cell transplantation (autosct) is considered the standard of care as consolidation therapy after first line therapy for those patients not expressing the alk protein (alk neg salcl) and for patients with relapsed disease. the objective of this retrospective analysis was to analyse the long-term outcome of patients diagnosed with salcl and being treated with autosct during the course of the disease, making special emphasis on the potential impact of the administration of brentuximab vedotin (bv). eligible for this study were patients 18 years or above with salcl who underwent autosct between 2010 to 2014 and were reported to the ebmt. baseline patient, disease, and transplant data were collected from ebmt med-a standard forms. centers with potentially eligible patients were contacted to provide additional treatment and follow-up information including a written histopathology report for central review. seventy-nine patients (48 males) with a median age at diagnosis of 43 years (range: 14-70) and at transplantation of 45 years were included in the final analysis. thirty-nine patients were alk negative, 38 alk positive and in 2 patients expression of alk protein was unknown. at diagnosis, 60 patients (76%) presented with advanced stage and 48 (61%), with b symptoms. sixty-three patients (80%) received 1-2 lines of therapy before autosct. ten patients were treated with bv at some point before autosct; two patients as second line therapy, three as third line, one as fourth line and four as fifth line therapy. the median number of bv doses was 5 (range: [3] [4] [5] [6] [7] [8] . the median time between diagnosis and transplantation was 12 months (range: . most patients had chemosensitive disease at autosct [65 patients (82%)] and in all but 2 patients peripheral blood was used as the source of stem cells. conditioning regimen consisted on beam / beam-like protocols in 72 patients (91%). all patients engrafted. with a median follow up for surviving patients of 34 months (range: 2-71), 57 patients are alive (72%), 20 patients died (25%) and 2 patients (3%) are lost for follow up. disease relapse after transplantation was the most frequent cause of death after the procedure. cumulative incidence of non-relapse mortality for the whole series was 3% (95% ci, 0.5-9) at 100 days, 1 year and 3 years. cumulative incidence of relapse was 27% (95% ci 17-27) and 32% (95%ci 21-44) at 1 and 3 years, respectively. 1 and 3years progression free survival (pfs) was 70% (95% ci 60-82) and 65% (95% ci 54-78), respectively and 1 and 3-years overall survival (os) was 82% (95% ci 73-91) and 71% (95% ci 61-83), respectively. there were no significant differences in any of the outcomes between bv treated and non-treated patients. autosct results in a promising pfs and os in patients with salcl. the potential impact of the administration of bv as salvage strategy before the procedure needs to be further elucidated. disclosure of conflict of interest: none. coeliac disease (cd) is a t-cell immune-mediated enteropathy to dietary gluten, characterized by small bowel villous atrophy resulting in malabsortion. the enteropathy is reversible with a gluten-free diet (gfd), however symptoms and signs which persist 41 year are defined as refractory coeliac disease (rcd). rcd is divided into type i and ii, depending on absence/ presence respectively of clonal intra-epithelial t-lymphocytes (iels) with an aberrant phenotype (cytcd3 pos, membranous cd3, cd4 and cd8 neg). rcdii patients have a 5 year survival of 1.0, plts 420) was successful at a median of 11.5 (range: 10-15) days and no transplant-related mortality occurred. all patients achieved a clinical complete remission, with normalization of nutritional indices at 100 days, but persistently abnormal iels and clonal t-cells on duodenal biopsy. with a median follow-up of 42.5 (range: 22-56) months, 5 patients remain in clinical remission, 1 patient relapsed with rcd and no patient progressed to eatl. chemotherapy and asct is a safe and effective strategy for the treatment of rcd offering the possibility of sustained clinical responses. clonal tcr in duodenal biopsy/blood and iel flow cytometry form part of the patient evaluation prior to the chemotherapy/asct program. most patients with hodgkin lymphoma (hl) are cured with conventional chemotherapy. however, approximately 20% of patients relapse after primary treatment. for those, high-dose chemotherapy (hdc) followed by autologous stem cell transplantation (asct) is the standard of care. fifty seven adult patients with relapsed or refractory hl submitted to asct between 2000 and 2015 were reviewed. variables examined were sex, age, ann arbor stage (i-ii vs iii-iv), b symptoms, bulky disease, extranodal involvement, nodal areas involved (≥3vs12vs ≤ 12 months) and response to the treatment prior to asct. log-rank test was used to compare differences in survival for each factor. patients median age was 31 (17-64) years at diagnosis. ann arbor stage iii-iv in 35 (61%) patients, b symptoms in 25 (44%), extranodal involvement in 20 (35%) and bulky disease in 13 (23%). all patients were treated according to the abvd protocol in first line. indications for asct were relapsed disease (n = 30, 52.6%) and lack of complete response (cr) or progressive disease with 1st line treatment (n = 26, 45.6%). there were a median of 2 (2-5) treatment-lines before asct (protocols eshap, ice, beacoop, gvd and others). the disease was chemosensitive in 86% cases: cr in 24 and partial response (pr) in 25 patients prior to asct. refractory disease (rd) in 14% (n = 9). in 84.2% patients, the hematopoietic cells mobilization was performed under stimulation with granulocyte-colony stimulating factor in hematologic recovery after the cycle of 2nd line chemotherapy, and most of which required 1 (1-4) apheresis. conditioning regimens were beam (93%) and gmb (7%). the median time to hematologic recovery was 11 days (8-14) for neutrophils4500/ul and 13 days (9-25) for platelets420,000/ ul. three months after asct, thirty-nine (68.4%) patients had cr, one (1.8%) patient maintained pr and 6 (10.5%) patients had disease progression. status unknown in 7 patients and four (7%) patients died. relapse rate 32% (n = 15/47). with a median follow-up time after asct of 52 (0-169) months, median disease-free survival (dfs) was 26 (0-169) months and overall survival (os) was 52 (0-169) months. there were 19 deaths (33.3%), four (7%) related to early infectious complications of asct, two (2.6%) due to late infectious complications, eleven (21.1%) due to disease progression and 1 (1.8%) in context of secondary acute myeloid leukemia. response to the treatment prior to asct was the only factor with survival influence. the dfs and os differed significantly in chemosensitive disease compared with rd (dfs mean: 51 vs 21 months,p = 0.025, os mean: 64 vs 22 months, p = 0.007). the response to salvage treatment prior to asct is the main prognostic factor for survival after asct. prognosis remains poor in patients with rd or early and disseminated relapses. for these patients, the therapeutic approach should include intensive treatment with tandem hdc and stem cell transplantation, allogeneic transplant or early consolidation with brentuximab-vedotin after asct. hodgkin's lymphoma (hl), although considered a curable neoplasm in adults, could be associated with a very poor prognosis when refractory to primary induction therapy or when it relapses within 12 months from an autologous stem cells transplant (auto-sct). the optimal treatment of patients with heavily pretreated/refractory hl is controversial. brentuximab vedotin (bv) is an active single agent in this context; unfortunately, there are no well established therapies when patients fail to respond or progress after bv. encouraging results were recently described with checkpoint inhibitors. similarly, data pertaining to efficacy of bendamustine (benda) shows encouraging activity in various refractory lymphomas. we included in this study adult patients with hl who relapsed post auto-sct and were refractory to or progressed after salvage bv and were treated with benda as salvage therapy with an intention to proceed with an allo-sct. this study was [p606] conducted in two major centers in lebanon, the american university of beirut medical center (aubmc) and makassed university hospital. we identified 12 eligible cases. the primary study endpoint was objective response rate (orr). the secondary endpoint evaluated successful rate of bridging into an allo-sct. the median follow-up times from auto-sct and from benda salvage were 35 (14-59) and 10 (4-35) months, respectively. the median age of patients was 27 years (17-54). all patients had bv as salvage therapy post auto-sct, and all of them progressed after a median of 4 (3-6) cycles. clinical characteristics are outlined in table 1 . patients received a median of 6 cycles (2-8) of benda. the treatment was well tolerated, with rather infrequent adverse events and transient and manageable toxicities. the orr was 75%, in 9 of 12 patients, with 43% obtaining a complete response. eventually, 6 of 9 proceeded to allo-sct using a matched related donor, and the remaining 3 patients are planned for allo-sct. only one patient died from disease progression after 24 months post allo-sct. two of 3 patients who progressed following benda received salvage therapy with nivolumab and are being planned for haplo-identical transplant while the third one is being planned for therapy with nivolumab. from the initiation of benda, the median duration of response for the 9 patients was 10 months (4-29); all these patients had maintained a continuous response at the last follow-up examination. conclusion: notwithstanding the limitations associated with our analysis, namely a small sample size and its retrospective nature, these results suggest a role for bendamustine in post bv failures. these findings also provide the basis to evaluate the concept of benda as a bridge to allo-sct in a large prospective study. [p607] disclosure of conflict of interest: none. brentuximab vedotin for relapsed or refractory hodgkin lymphoma, single center experience king faisal specialist hospital and research center, riyadh, kingdom of saudi arabia ms rauf 1 , i maghfoor 1 , a badran 1 , mn zahir 1 and s akhtar 1 1 hodgkin lymphoma (hl) patients with relapsed or progressive disease after high dose chemotherapy (hdc) and auto-sct have limited curative options. fda granted approval of brentuximab vedotin (bv) for the treatment of hl and anaplastic large cell lymphoma (alcl) patients who fail auto-sct or have had at least 2 prior multiagent chemotherapy regimens and are not candidates for auto-sct. we are reporting single center experience of bv usage in this "approved" setting. medical records were reviewed to collect required data. kaplan-meier (km) method was used to calculate overall survival (os) and progression free survival (pfs) from date of first dose of bv. from 2013-2015, 25 patients received bv. 24/25 had hl (22 classic hl-nodular sclerosis, 2 hl-mixed cellularity) and 1 alcl. 19/25 (76%) pts were primary refractory or had early relapse after initial treatment. 15/25 (60%) pts received bv were refractory to the last treatment. all the baseline characteristics of patients are mentioned in table 1 . median bv cycles administered were 6 (2-16). overall response rate (orr) was 40% (10 patients): cr in 6 (24%), pr in 4 (16%) (5/10 were primary refractory or early relapsed). median pfs for whole group was 5 months (95% ci, 3.6-6.3). km estimated 1-year os was 74% and 2 year was 68%, median os has not been reached yet. for 10 patients who responded, pfs at 12 months was 68% (95% ci, 38%-98%), median pfs not reached. for 15/25 patients with progressive disease (pd) or non responders after bv, median pfs was only 3 months (95% ci, 1.1-4.8). there was no difference in os between patients with responders and non responders. median os has not yet been reached in either group as mentioned in survival curves. at the median follow up of 19 months (range: 4-55 months) 18 patients are alive, 10 patients are alive without disease, 4 patients received consolidation bone morrow transplant (2 auto-sct and 2 allo-sct). 2 patients completed 16 courses and achieved cr. rest of 4 patients who are alive without disease; they had pd on bv but achieved cr with other treatments. 8 patients are alive with disease; 1 patient is on bv and 7 are on another treatment. 7 patients have died, 2 because of pneumonia while being on bv and 5 due to pd. 3/15 patients who received bv, achieved cr after failing all previous treatments and are in cr. peripheral sensory neuropathy developed in 3 patients; one required dose reduction. 1 patient stopped treatment due to pulmonary toxicity. we are reporting largest single center data from middle east which confirms that bv as a single agent is effective and safe. overall response rate is lower as compare to pivotal trial but cr rate is comparable to other reported case series. this analysis also concludes that bv can be used as bridge to transplant in patients who don't respond salvage chemotherapy. disclosure of conflict of interest: none. was used to diagnose hiv infections. cox proportional hazards models were used to evaluate risk factors of overall mortality. fifty-six patients with nhl (1.4%) and 23 patients with mm (0.8%) were positive for hiv antibody. in patients with nhl, overall survival was significantly lower in the hiv-infected patients than in the hiv-negative patients [5year overall survival: hiv-infected patients, 44% (95% confidence interval, 29%-58%) vs. hiv-negative patients, 65% (95% confidence interval, 63%-67%), p o0.001)]. in a multivariate analysis, hiv infection was significantly associated with an increased risk of mortality (hazard ratio 2.39, p o0.001), and this effect was consistent regardless of transplant year. on the other hand, overall survival in patients with mm was similar between the 2 groups [61% (95% confidence interval, 31%-82%) vs. 63% (95% confidence interval, 63%-67%), p = 0.988]. previous studies in europe and the united states showed comparable survival rates between hiv-infected and hiv-negative patients with nhl. however, our study showed that hiv infection was associated with a higher risk of mortality in patients with nhl in japan. suppression of t cell-mediated immunity or hiv related diseases might affect transplant outcomes in japanese patients. [p609] disclosure of conflict of interest: none. while beam and beac regimens (bcnu, etoposide, cytosar in both regimens and melphalan or cytoxan, respectively) are commonly used as conditioning high-dose therapy (hdt) in patients with non-hodgkin lymphoma (nhl), there have been few reports comparing these regimens. a retrospective analysis found the superiority of beam over beac in terms of overall survival (os) and event-free survival (efs). toxicities were similar, except that beam was associated with more frequent lower gastrointestinal (gi) mucositis. other studies reported that these regimens had similar efficacy and outcome. recently, a concern regarding cardiotoxicity of beac has risen. the current study aimed to compare efficacy and toxicity of beac and beam as consolidation hdt in young patients with mantle cell lymphoma (mcl) undergoing autologous stem cell transplantation (asct). this is a retrospective analysis of outcomes in mcl patients who received hdt with beam or beac followed by asct at 3 bone marrow transplant centers in israel. os, disease-(dfs) and progressionfree survival (pfs) and regimen toxicity were compared. seventy seven mcl patients who were diagnosed between 1995-1/2016 and received consolidation with beac or beam were included in the analysis. forty nine patients were treated with beam and 28 patients-with beac. no significant differences between the groups were revealed in terms of age, sex, the mantle cell lymphoma international prognostic index (mipi) risk score, induction protocol and% of patients transplanted in first complete response (cr1) (mean age 57 yrs in beam vs 59 yrs in beac group; 69% of patients in beam group had mipi risk score 2-3 vs 62% in beac group; 68% of patients in beam group were transplanted in cr1 vs 71% in beac group). the amount of infused cd34 cells was significantly higher in the beam group (median cd34 cells/ kg: 8.2 in beam vs 4.6 in beac groups; p = .001); the number of days to platelet engraftment was significantly greater in the beac group (median 12 days in beam vs 14 days in beac group; p = .02). there were no differences in the number of blood transfusions or hospitalization days between the groups. the rate of grade 3-4 upper mucositis was significantly higher in the beam group (41% in beam vs 18% in beac group; p = .046); no other differences in toxicity (grade 3-4 lower mucositis, pulmonary congestion, infections) were observed between the regimens. non-relapse mortality by day 30 posttransplant was 0% in both groups. a median follow-up was 29 (range: 1-119) months. the 3-yr dfs in beam and beac groups was 58% and 64%, respectively (p = .65). there was no difference in the 3-yr os between the groups (70% in beam and 84% in beac group; p = .51). there was a trend to improved dfs and os in patients transplanted in cr1 receiving beam (p = .09, figure) . in multivariate analysis, low-to-intermediate mipi and transplant in cr1 were found to significantly increase pfs (p = .04 and.01, respectively), while the hdt regimen did not affect pfs. beac and beam hdt regimens followed by asct had similar efficacy in mcl patients. there was a trend to improved dfs and os in patients transplanted in cr1 and treated with beam vs beac. the toxicity profile was similar in both groups, except a significantly higher rate of grade 3-4 upper gi mucositis. [p610] disclosure of conflict of interest: none. early or refractory relapsed ( o1 year) diffuse large b-cell lymphoma has a very poor prognosis especially for those not responding to salvage chemotherapy. allogeneic stem cell transplantation is potentially curative. even though this is less likely in those not responding or having frank progression pretransplantation. methods: at our institution we identified all patients with aggressive b-cell lymphoma (diffuse-large b-cell lymphoma and blastoid mantle cell lymphoma) who were refractory or progressive to salvage chemotherapy with r-dhap and who had peripheral blood stem cells (42 × 10 6 cd34+/kg body weight) collected after the 1st or 2nd cycle. after high-dose melphalane and autologous stem cell transplantation 13 patients had a partial and 6 a complete remission. 1 patient died due to neutropenic infection, 2 patients died due to progressive disease leading to a transplant related mortality of 3.5%. median progression-free survival after autologous transplantation was 4.6 months. 24 proceeded to allogeneic stem cell transplantation. 8 patients had a matched related sibling, 9 had a matched unrelated donor and 7 had a mismatched unrelated donor. transplant related mortality was 42% in this heavily pretreated population. 2-year overall survival of all patients intended for treatment is 21%. one of these patients with relapsed mediastinal lymphoma after allogeneic transplantation was cured by salvage radiotherapy and is in long-term remission (42 years). conclusions: salvage high-dose melphalane and autologous peripheral blood stem cell transplantation for diffuse large b-cell lymphoma as a bridge to allogeneic transplantation is potentially curative for a minor fraction of these patients. however, the remission rate of 79% (46% pr, 21% cr) and progression-free survival of 4.6 months after high-dose melphalane and autologous stem cell transplantation provides a window of opportunity to use new drugs and cellular therapies in these poor prognosis patients. high dose chemotherapy and autologous stem cell transplantation is the treatment of choice for patients with relapsed refractory hodgkin lymphoma. several factors including number of chemotherapy lines received before conditioning, time of relapse and remission status before transplantation can predict survival and pfs in patients undergoing autologous stem cell transplantation. in 2012, we reported on a 63 patients who underwent high dose chemotherapy followed by autologous stem cell transplantation from 2003 to 2008. all patients with relapsed or refractory hodgkin lymphoma in the period of 2009-2013, who underwent high dose chemotherapy followed by autologous transplantation were retrospectively analyzed. the main outcomes of the study were complete remission (cr) at day 100, overall survival (os) and relapse-free survival (rfs). the impact of the following variables on os and rfs: (a) disease status at the time of transplant, (b) number of chemotherapy lines prior to conditioning and (c) time of relapse 12 months and (d) age. a total of 78 patients were identified. the median age was 31 year. there were 50.6% females and 49.4% males. complete remission (cr) was achieved in 48.7% of patients and 49.9% with chemotherapy sensitive disease at the time of transplantation. prior to conditioning regimen, 43.2% received two chemotherapy lines, and 56.8% received more than two lines. 41% relapsed in less than 12 months and 57% relapsed more than 12 months after completion of therapy cr at day 100 was 69.2%. the median os for the whole group was 62.0 months; the median rfs was 10,6 months. the number of chemotherapy lines significantly impacted os and efs. cr status before conditioning, favorably influenced os and efs with a trend toward better os in favor of those who underwent abmt while in complete remission. the time of relapse and the age did not affect survival outcomes. [p614] the outcome of patients with relapsed or refractory hodgkin lymphoma is favorable and the number of chemotherapy lines received before conditioning is the only factor that had a statistically significant impact on os and efs. since the identification of human immunodeficiency virus (hiv), a clear association between hiv and specific malignancies has been recognized. high-grade b cell lymphomas are the most common malignancy complicating hiv infection and one of three aids defining malignancies. diffuse large b cell lymphoma (dlbcl) accounts for 80% of cases. before 1996, lymphomas were the cause of 16% of all deaths attributable to aids. after the introduction of highly active antiretroviral therapy (haart) overall incidence of adm declined, however longer survival and exposure to environmental risk factors have increased the incidence of non adm (adm) such as hodgkin's lymphoma (hl). since haart has improved overall survival substantially, the aim of chemotherapy should be complete remission rather than palliation with careful consideration of drug interactions and side of haart. between 2011 and 2016 a total of 510 patients were detected hiv positive. twenty-one of these patients were diagnosed with a malignancy and 8 patients referred to our department with a hematologic malignancy were evaluated retrospectively. diagnosis, stage, treatment, survival data were recorded. haart during chemotherapy, nadir cd4 count and cd4 count at diagnosis of malignancy was evaluated. four patients were diagnosed with high grade b cell lymphoma, 2 patients with primary central nervous system lymphoma (pcnsl), 1 patient with hl and 1 patient with multiple myeloma (mm). all patients were male and median age at diagnosis was 40.5 (24-63). hiv seropositivity was identified during evaluation of malignancy in both pcnsl patients. median duration of hiv seropositivity before diagnosis of malignancy was 18 months for the remaining patients. patient characteristics, treatment modification and cd4 counts are summarized in tables 1 and 2 . lymphoma was fatal in 5 and the cause of death was identified as lymphoma progression in all patients including one patient diagnosed with hodgkin's lymphoma. a patient presented with multiple plazmositomas was diagnosed with multiple myeloma is currently receiving induction treatment together with haart. hiv related lymphoma patients frequently present with extra nodal disease, incidence of central nervous system involvement is also higher and prognostic score tends to be in the intermediate or high-risk groups. prognosis is also worse than hiv negative population. degree of immunosuppression is implicated and the duration of immunosuppression is directly correlated with the risk of developing lymphoma rather than hiv itself. haart allowed the use of aggressive chemotherapy since it improved immune system and decreased infectious complications. multiple myeloma is a rare neoplasm observed in hiv infection and the treatment is based on data obtained from hiv negative patients. treatment of such patients as well as lymphomas should take into consideration the toxic effects of haart combined with chemotherapy. since hiv positive [p615] patients are excluded from most studies, there are no guidelines to direct treatment and avoid toxicities. drug interactions should be monitored closely and modifications should be made accordingly. interruption of haart may not be mandatory since studies have shown safety of continuation of haart during chemotherapy. for newly diagnosed hiv and malignancy, careful clinical and laboratory evaluation should be made before postponing haart until after chemotherapy. disclosure of conflict of interest: none. the outcome of hdct and asct in refractory hodgkin lymphoma (r-hl) is not as encouraging as in relapsed hl. ten years ago we analyzed and reported outcomes of asct in our r-hl patients, however the follow-up was short. now we a reporting long term outcomes in r-hl after asct in one of the largest numbers reported to date. between 1996 and 2014, patients with hl who underwent hdc and asct for r-hl in adult medical oncology (age 4 14 years) were identified. r-hl is defined as partial response (pr), no response (nr), stable disease (sd), progressive disease (pd), relapsing within 3 months (relapse o3 m) of finishing the planned (chemotherapy + radiation therapy (xrt)) treatment or refractory to salvage chemotherapy. kaplan-meier (km) method was used to estimate progression free survival (pfs) and overall survival (os) from the day 0 of asct while progression is defined as progression of disease, relapse and death from any cause. all percentages are rounded to nearest. 307 patients underwent hdc and asct during 1996-2014 and 177 of them met the criteria of r-hl. male 97 (55%), female 80 (45%), median age at diagnosis was 22.2 years (8-61 years) and at asct was 24 years (14-62 years). initial therapy was abvd in 153 (86.4%), mopp/copp alternating with abv or abvd in 11 (6%) and others in 13 (7%). 49 (28%) had xrt after initial chemotherapy. response to initial chemo + xrt was pr in 80 (45%), pd in 51 (29%), cr in 38 (21%) (28/38 relapsed within 3 months and others have refractory relapse) and no response in 4 (2%) and others in 4 (2%). prior to salvage chemotherapy, 119 (67%) had stage iii-iv, 90 (51%) extra-nodal involvement, 43(24%) bulky disease and 31 (18%) had b symptoms, spleen involvement in 43(24%), performance status 0, 1 in 155 (88%). eshap was used as first line salvage in 153 (86%) or 3 rd line 13 (7%). post salvage / prior to hdc and asct disease status was pr in 112 (63%), cr in 53 (30%) and nr/sd in 12 (7%). 111 (63%) patients had a fdg-pet scan prior to asct, 45 (25%) were in cr. beam was used as conditioning regimen. median follow-up for all alive patients is 81 months (15-224) from asct. response rate post asct: cr in 105 (59.3%), pr in 16 (9%), nr/sd in 1 (0.6%) and pd in 45 (25.4%) patients, others /unknown in 10 (5.6%). 61 (35%) patients had xrt post auto-sct. type of first post hdc auto-sct event was no event in 81 (46%), persistent disease in 17 (10%), pd in 45 (25%), relapsed disease in 23 (13%), treatment related mortality in 6 (3%) and died of other cause 5 (3%). at last follow-up in november 2016, 94 patients (53%) are alive with no disease, 6 (3%) alive with disease, 64 (36%) died of disease and 13 (7%) died of treatment related mortality or other causes. for entire group, km estimated median os is 129 months, 1,2,3,5 and 7 year survival is 83%:73%:64%:59%:55% respectively. median pfs is 48.5 month, 1,2,3,5 and 7 year pfs is 57.6%:53.6%:51%:50%:47% respectively. we are reporting a very high risk group of patients with a very long follow-up. in patients with r-hl, eshap + beam combination resulted in high response rate (68.3%). these remissions are durable. a 5 year os survival of greater than 50% in our population is higher than most reports with similar numbers. although our cohort has a 7 year os survival of 55%, 45% patients have either relapsed or died underscoring need for improvements in the management refractory hl. [p616] disclosure of conflict of interest: none. here we update the previously reported results of our reduced-intensity conditioning (ric) allo-hsct experimental program, initiated in 2002. as of november 2016, in our centre 34 patients underwent ric allo-hsct. donors were hla-identical sibling in 16, fullymatched unrelated in 7, 1 or 2-mismatch-unrelated in 9 and haploidentical relative in 2. median age was 53 years (range: 19-66). all patients (22 m and 12 f) had stage iib/iv refractory mf (n = 22) or refractory ss (n = 12). median number of previous treatment lines was 6 (range: 2-12). source of stem cells was peripheral blood in 31 patients and bone marrow in 3. median time from diagnosis to hsct was 46 months (range: 13-264). conditioning included flu/ctx/tbi200, pentostatin +tbi200 and flu/mel in case of hla-identical or unrelated donor, whereas the tt/flu/ctx/tbi200 regimen was used in the haplo setting. gvhd prophylaxis included csa/mmf in all patients, with the addition of atg in cases with unrelated donor and post-transplant ctx (50 mg/kg giorni +3 e +4) in cases with haploidentical donor. full donor chimerism was obtained in 28/33 of the evaluable patients, in a median time of 2 months (range: 1-12). grade ii-iv acute gvhd occurred in 16 patients (57%), while grade iii-iv was observed in 8 patients (28%). chronic gvhd occurred in 10 patients (36%), being extensive in 4 (14%), all transplanted from hla-identical sibling (no atg). following transplantation, a complete remission (cr) was achieved in 22 out of the 33 evaluable patients (67%), of whom 2 experienced relapse at +25 and +35 months, respectively. transplant-related death occurred in 6 patients (17%), of whom 4 were in cr. out of the 11 patients who did not achieve cr, 9 died from progressive disease (median follow-up of 12 months, range: 3-31), 1 from a secondary malignancy and 1 is still alive with disease 41 months after transplant. of note, all pts who died in progression had chemoresistant disease at time of transplant. at the last follow-up, 18 patients were alive and 16 (89%) maintained cr after a median time of 66 months (range: 4-189). in the whole population, the 5-year overall survival was 52% (95% ci 34-70) and the 5-year disease-free survival (dfs) was 44% (95% ci 27-62). however, when mf and ss were analysed separately, 5-yrs dfs were 32% (95% ci 12-51) and 69% (95% ci 38-99), respectively (figure) . apart from diagnosis, outcome appeared to be primarily associated with the disease status at transplantation, with a 5-yr dfs of 100% in the group of patients (n = 8) who were in cr before starting the conditioning. after a median follow-up longer than 5 years, we confirm the efficacy of ric allo-hsct as a powerful therapeutic strategy in inducing and maintaining remission in selected patients with chemosensitive advanced-stage ctcl, with results particularly encouraging in ss. [p617] disclosure of conflict of interest: none. outcomes of allogeneic hematopoietic stem cell transplantation for hodgkin lymphomas: a retrospective multicenter experience of the rete ematologica pugliese (rep) f gaudio 1 , p mazza 2 , am carella 3 , d pastore 1 , g pisapia 2 , a mele 4 , p galieni 5 , n cascavilla 3 , g specchia 1 and v pavone 4 1 hematology, university of bari, bari, italy; 2 hematology, ospedale "san giuseppe moscati", taranto, italy; 3 hematology, ospedale "casa sollievo della sofferenza", san giovanni rotondo, fg, italy and 4 hematology, ospedale "cardinale panico", tricase, le, italy; 5 hematology, ospedale "c. g. mazzoni", ascoli piceno, italy hodgkin's lymphoma (hl) is a potentially curable disease, and modern therapy is expected to successfully cure more than 80% of the patients. second-line salvage high-dose chemotherapy and autologous stem cell transplantation (sct) have an established role in the management of refractory and relapsed hl, leading to long-lasting responses in approximately 50% of relapsed patients and a minority of refractory patients. patients progressing after intensive treatments, such as autologous sct, have a very poor outcome. allogeneic sct represents the only strategy with a curative potential for these patients; this study reports a retrospective multicenter experience of the rete ematologica pugliese (rep) over the past 16 years aiming to define the impact of patient, disease, and transplant-related characteristics on outcomes. 67 patients with histologically confirmed diagnosis of hl who received allogeneic sct from 2000 to 2016 were retrospectively studied. the median age was 34 years (range: 16-57 years) and 36 (54%) were male. the majority of patients (84%) had had a prior autologous sct. at the time of allogeneic sct, 28 (42%) patients had a chemosensitive disease and 39 (58%) were chemorefractory. most (93%) patients received reduced-intensity conditioning, 54% received matched sibling donor and 46% matched-unrelated donor grafts. the disease status at day 100 post-transplant was reported in 62 out of 67 evaluable patients. of the 26 patients with chemosensitive disease, 18 (70%) achieved a cr, 7 (27%) had a pr or stable disease and 1 (3%) had progressive disease. of the 36 patients with chemorefractory disease 7 achieved a cr (20%), 26 had a pr or stable disease (72%) and 3 (8%) had progressive disease. although the overall survival has improved significantly in mantle cell lymphoma (mcl) according to advanced treatment options, relapsed or refractory disease remains a challenge. recently, lots of targeted agents actively have been tried clinical studies and adapted to clinical practice in indolent lymphoma. however, the role of frontline autologous hematopoietic stem cell transplantation (auto-hsct) has not been fully understood in patients with mcl, compared with a few impressive published data about auto-hsct as salvage treatment option for patients with relapsed mcl. so, we retrospectively evaluated consecutive patients diagnosed mcl, and compared the clinical outcomes of high-dose chemotherapy followed by auto-hsct and conventional chemotherapy alone. between january 2003 and december 2014, consecutive patients with newly diagnosed with mcl at catholic blood and marrow transplantation center in south korea were included in this study. all of the patients received high-dose cytarabine-containing regimen or chop with/without rituximab regimen for induction therapy regardless of transplant eligibility. the treatment approach in our institution for patients was based on the physician discretion for transplant eligibility or ineligibility that depend on patient age, comorbidities, and disease status. seventy patients were included in the analysis. initial chemotherapy regimens were consisted of chop (n = 12, 17%), r-chop (n = 44, 63%), r-hypercvad (n = 10,14%), and hypercavd (n = 4, 6%). demographics and disease characteristics of both groups are shown in table1. patients received auto-hsct were superior s449 overall survival (os; p = 0.015) and progression-free survival (pfs; po0.001). the subgroup analysis according to high-risk of mcl international prognostic index (mipi) or bone marrow involvement was performed. between the two treatment arms among the high-risk mcl group, the clinical parameters were not different. the high-risk mcl patients with frontline auto-hsct showed superior os (p = 0.0216) and pfs (po0.001) compared with conventional chemotherapy alone. although mcl is classified within indolent lymphoma, frontline auto-hsct can be considered for patients diagnosed with mcl in the group of high-risk mipi or bm involvement with the favorable survival outcomes. disclosure of conflict of interest: none. nasal type extranodal nk/t-cell lymphoma (enktl) is a very rare and agressive malignancy characterized by a poor outcome. current standard therapy is not yet established. the role of high dose therapy followed by haematopoietic stem cell transplantation (hsct) is still controversial. we evaluated the outcomes of all the enktl patients undergoing hsct in a multicenter analysis on patients registered by the société francophone de greffe de moelle et de thérapie cellulaire (sfgm-tc) and compared them with a population of french patients who received chemotherapy alone. sixty four enktl (48 males and 16 females) received hsct, including 19 allogeneic (allosct) and 45 autologous transplantations (autosct). median age at the time of hsct was 43 years (range: 17 to 70 years). overall, 57% of the patients presented with disseminated disease (64% and 55% in the allosct and autosct, respectively), 61% were in complete response (cr) at the time of hsct (74% and 61% in allosct and autosct groups, respectively) and 82% had received l-asparaginase regimen prior to hsct (73% and 84% in allosct and autosct groups, respectively). five (26%) and 20 (44%) patients of the allosct and autosct groups underwent upfront hsct therapy, respectively. four patients received tandem autologous/ allogeneic transplants. in allosct, stem cell source was a matched related donor in 13 patients, an unrelated donor in 3 patients and an umbilical cord blood in 3 patients. reduced intensity conditioning regimens (based on fludarabine-busulfan combination) and beam regimen were used in 42% and 84% of patients from the allosct and autosct groups, respectively. median overall survival for the whole cohort was 17.1 months (range: 1 to 131 months). the 3-year non-relapse mortality was 16.1% and 22.7% in the allosct and autosct groups, respectively (p = 0.58). the 3-year overall survival (os) and progression free survival (pfs) were 47.5% and 40.8% in the autosct and 43.4% and 47.4% in the allosct group, s450 respectively ( figure 1a) . the absence of cr prior to hsct was associated with a poor prognosis (p = 0.008). as compared to allosct, autosct resulted in a better outcome in patients who didn't achieve cr before transplant (p = 0.002) and tended to have better outcome in high pink risk score (figure 1 b-c) . finally, at 3 years pfs and os of patients who have been treated by chemotherapy alone (ct) (n = 55) or followed by allosct (n = 12) or autosct (n = 17) in cr1 were 55%, 81% and 50%, 54% and 44%,50%, respectively ( figure 1d ). in this french cohort, more patients received autologous hsct in upfront therapy than allogeneic hsct. in cr1, there is no evidence suggesting that transplantation is associated to a better outcome than chemotherapy alone. however, a precise matching based on the pink score will be evaluated to ensure that patients who were intensified were not of worst prognosis. in refractory patients there is also no clear advantage to perform allosct when compared to autosct. however, in relapsing disease after ct or autosct allosct, allowed to obtained durable control of the disease. disclosure of conflict of interest: none. high relapse rate is one of concerns for allo-sct in pts with relapsed/refractory aggressive lymphoma. an optimal conditioning regimen designed for aggressive lymphoma may reduce relapse, especially during early post-transplantation period. however, it is not established yet. results of a german phase 2 study of allo-sct with conditioning regimen of fludarabine, busulfan (12 mg/kg po or 9.6 mg/kg iv), and cyclophosphamide with or without post-transplantation rituximab for relapsed/refractory aggressive lymphoma suggested the role of myeloablative busulfan-containing regimen in reducing relapse rate in pts with aggressive lymphoma. based on these results, we conducted a single institution prospective study to explore feasibility of the bmf regimen consisted of full-dose busulfan, melphalan, and fludarabine in pts with relapsed/refractory aggressive lymphoma (umin000013940). patients with aggressive lymphoma who achieved at least sd with salvage chemotherapy after experiencing either pd during first-line therapy, early relapse ( o12 mo) after firstline therapy, late relapse (≥12 mo) but refractory to salvage therapy, relapse after auto-sct,; age 20-65; ecog ps of 0-2; and without severe organ dysfunction were eligible. donor source could be 6/6 matched related or unrelated donor pb/bm or cb with ≤ 2 antigen mismatch; the bfm regimen was consisted of busulfan 12.8 mg/kg iv, fludarabine 180 mg/m 2 , and melphalan 80 mg/m 2 (yamamoto h. bbmt 2016). gvhd prophylaxis was csa + mtx (related pb), tac + mtx (unrelated bm), and tac + mmf (cb). primary end point of the study was survival with engraftment at day 60, and secondary end points were engraftment rate at day 100; nrm and relapse rate at day 100 and 1 y; progression free survival (pfs), overall survival (os), and gvhd at 1 y. protocol was approved by irb and written ic was obtained from all pts. twelve pts (male 10, female 2) with a median age of 55 y (33-63) were enrolled. ps was 0-1 in 11 pts. diagnosis were dlbcl (n = 4), transformed fl (n = 3), enktcl (n = 3), ptcl (n = 1), and aitl (n = 1). median number of previous line of therapy was 3.5 (2-5) and 5 pts had failed previous auto-sct. diseases status at transplantation was cr (n = 6), pr (n = 4), and sd (n = 2). donor source was cb (n = 6), unrelated bm (n = 5), and related pb (n = 1). survival with engraftment at day 60, primary endpoint of the study, was achieved in 100%. neutrophil engraftment was achieved at a median of day 18 (13-32). full donor chimerism at day 30 was achieved in all of the 11 pts evaluated. two pts developed vod which was manageable. with a median follow-up of 20 mo, 3 pts had progression of lymphoma at 2, 5, 6 mo. five pts died and cause of death were progression of lymphoma in 3, interstitial pneumonitis in 1 (at 5 mo), systemic adenovirus infection in 1 (at 5 mo), and agvhd in 1 (at 4 mo). os and pfs at 1y were 54% and 46%, respectively. relapse and nrm rates were 8% and 0% (day 100), and 27% and 27% (1y), respectively. agvhd of grade ii-iv was observed in 7/12 pts and 2 pts developed limited cgvhd. this prospective study shows that allo-sct using myeloablative conditioning regimen with full-dose busulfan, melphalan, and fludarabine for relapsed/refractory aggressive lymphoma is feasible and deserves further evaluation. disclosure of conflict of interest: none. for patients with advanced ctcl, the allogeneic hsct seems to be curative with graft versus lymphoma effect playing a major therapeutical role. in this retrospective study, 16 patients with a median age of 52 years (range: 24-67) affected by ctcl underwent allogeneic hsct after a median of 4 (range: 1-8) lines of chemotherapy, including autologous transplant for 2 of them. the median time from diagnosis to hsct was 46 months (range: 9-309). the diagnoses were: sezary syndrome (ss, n = 8). mycosis fungoides (mf, n = 2), primary cutaneous cd30+ lymphoma (n = 4), panniculitis-like t-cell lymphoma (n = 1), nk t cell lymphoma (n = 1). at time of hsct, 2 patients (12.5%) were in complete remission (cr), 9 (56.3%) in partial remission (pr) and 5 (31.2%) had active disease. the patients were transplanted from an hla-identical (n = 7), mismatched (n = 1) or haploidentical (n = 1) sibling, from matched unrelated donor (n = 5) or from a single cord blood unit (n = 2). different pre-transplant regimens were used as myeloablative (mac) in 6 (th-bu-flu, n = 4; bu-cy, n = 2) or as reduced intensity (ric) in 10 (th-flu-cy, n = 7; th-bu-flu, n = 3). al patients engrafted for neutrophils at a median of 17 days (range: 12-46) and 14 patients engrafted for platelets at a median of 14 days (range: 12-77). acute gvhd was of grade 0-i in 9 patients and ii-iv in 7 (40.7%). skin was the most common organ involved. five of 11 evaluable patients experienced chronic gvhd which was mild in 3 and severe in 2. at a median of 3 months (range: 1-11), 7 patients died (4 mac and 3 ric) because of gvhd (n = 4), vod (n = 1), pneumonia (n = 1) or multiorgan failure (n = 1). all 12 patients surviving at 3 months from transplant were in cr. only patients prepared with a ric (n = 4) relapsed respectively at 3, 9, 10 and 11 months from hsct. these patients received dli associated or not to chemotherapy. three achieved cr, which remained stable in 2, while one patient died in cr from post dli acute gvhd. one patient (nk-t cell) not achieving cr is still alive with active disease. for all 16 patients the median survival was 10 months (range1-130). with a median follow up of 76 months (range: 4-130), 9 patients (2 mac, 7 ric) are alive, 8 in cr and 1 with active disease. at 10 years, the os was 54 ± 13%; at 5 years dfs was 34 ± 12%. according with the median time (46 months) from diagnosis to transplant, the 10-year os was 88 ± 12% for patients transplanted early and 25 ± 15% for the others (p o0.04), while dfs was respectively s451 58 ± 19% and 13 ± 12% (p o0.05). despite the small number of patients, our results confirm the high susceptibility of ctcl to the graft versus lymphoma effect and point out the time to transplant as a crucial prognostic factors for the outcome. finally, the long-term follow up of our series strongly supports hsct for the cure of ctcl. disclosure of conflict of interest: none. recently, a new prognostic score, the nccn-international prognostic index (ipi) has been developed 1 to stratify patients affected by diffuse large b cell lymphoma (dlbcl), and in high-intermediate and high risk groups the survival was equal or less than 50%. the aim of this analysis was to evaluate the outcome of a cohort of dlbcl patients undergoing high dose chemotherapy (hdc) as consolidation following first line chemo-immunotherapy, after their re-classification according to the nccn-ipi. we performed a retrospective study on 221 patients diagnosed with dlbcl, with a high/intermediate or high-risk disease according to the ipi (2-5), who received upfront hdc with asct, in 2 institutions. the patients were then re-stratified according to the nccn-ipi and arbitrarily classified in 2 groups: low risk (nccn-ipi ≤ 3) and high risk (nccn-ipi ≥ 4). the pre-transplantation disease status was assessed by positron emission tomography (pet) or computed tomography (ct). the primary endpoints were non-relapse mortality, progression-free survival (pfs), overall survival (os) and relapse risk. the estimated 3-year pfs for all patients was 80.1% (95% confidence interval [ci] 74.2-86.0) and the 3-year os was 91.0% (95% ci 86.7-95.3). of these patients, 93 had a low risk ipi score (ipi = 2) and 128 were considered high risk (ipi ≥ 3). subsequently, the whole cohort was re-stratified according to the nccn-ipi: 133 patients were allocated to the high-risk (nccn-ipi ≥ 4) group, and 88 to the low-risk group (nccn-ipi ≤ 3). the analyses were then carried out for both groups. the 3-year pfs was 92.0% (95% ci 85.7-98.3) in the low-risk group and 71.2% (95% ci, 62.2-80.2) in the highrisk group (po 0.01), whereas the 3-year os was 98.8% (95% ci 96.4-100) in the low-risk group and 85.0% (95% ci 77.9-92.1) in the high-risk group (p = 0.02). the significant difference in os and pfs between the two groups was mainly due to the cumulative incidence of relapse at 3 years (graph 1): 8.0% (95% ci 3.2-15.7) in the low-risk group and 27.1% (95% ci 18.7-36.2) in the high-risk group (po 0.01). non-relapse mortality was comparable in both cohorts: 1% (95% ci 0.2-3.3) for all patients. figure 1 : cumulative incidence of relapse following hdc and according to nccn-ipi. patients affected by high-risk dlbcl still have an unsatisfactory prognosis after treatment with conventional therapy regimens, even in the rituximab era. the 5-year os and pfs in patients with nccn-ipi score ≥ 4 range: from 33% to 64% and from 30% to 51% respectively 1 . although this is a retrospective analysis subject to all related biases, our results suggest that upfront intensive therapy with autologous stem cell transplantation may significantly improve the outcome of these patients compared to conventional chemotherapy. the role of hdc in the treatment of dlbcl is controversial. however, new entities or new risk stratifications, as the one reported here, could allow to identify high risk subpopulations that could benefit from this approach. enteropathy-associated t-cell lymphoma (eatl) is an exceedingly rare and often rapidly fatal subtype of peripheral t-cell lymphoma, arising from intraepithelial lymphocytes. eatl type i is associated with celiac disease; type ii occurs in patients without inflammatory pre-conditions (according to who 2016 classification now called monomorphic epitheliotropic intestinal t-cell lymphoma (meitl)). surgical debulking and anthracyclinebased chemotherapy (ctx) followed by high-dose chemotherapy (hdctx) and autologous cell rescue (asct) are pursued when possible in this often malnourished and frail patient cohort. yet, even with intensive consolidation relapse occurs in 40-70% of patients. the value of allogeneic hematopoietic cell transplantation (hct) is not clarified as of today due to limited reports. here, we report on a patient with meitl who was rescued with an allo-hct for his 2nd relapse following prior asct. moreover, we summarize the available literature on the use and outcomes of allo-hct for eatl and meitl. a 48y old man with spontaneous intestinal perforation was diagnosed with meitl following emergency partial resection of the small intestine. histology revealed infiltration by monotonous medium-sized lymphocytes with abnormal immunophenotype (cd3+, cd56+, cd8+, cd5-, cd30-, tia-1+) consistent with type ii eatl. post-surgical 18f-fdg pet-ct scan showed abnormal uptake in gastric antrum and pyloric region but no other manifestations. ctx with cho(e)p (6 × ) followed by beam hdctx and asct was performed and achieved a complete remission (cr1). however, 9m post asct disease relapsed and was treated with 2 × dhap, and 4 × dhaox. cr2 was achieved after the 3rd cycle of salvage therapy. due to anthracyclineinduced cardiopathy allo-hct could not be performed at that time. 5m after completion of salvage therapy, disease relapsed again, and was progressive under pralatrexat treatment (1 cycle, 6 infusions). by then cardiac function had recovered and therapy was switched to dose-reduced mini-beam (2 × ). in cr3 reduced intensity conditioning (ric; fludarabine, busulfan, atg) and allogeneic hct from a matched sibling donor was performed. ciclosporin a (csa) and mycophenolate mofetil (mmf) were given as gvhd prophylaxis. prompt engraftment in blood (day+14) and full donor chimerism in the marrow (d+100) were achieved. immunosuppression was tapered and discontinued on d+55 (mmf) and d+193 (csa), respectively. 18f-fdg pet-ct scan at 3m post-hct showed cr, but at 6m relapse was suspected (under work-up). only few cases of patients with eatl/meitl treated with allo-hct are reported in the literature (n = 9, table 1 ), and the value of this highly aggressive therapy is not clear at this point. of note, the patients listed in table 1 were given allo-hct instead of asct. long-term complete remission (cr) could be achieved in 4/9 patients, while 5 patients suffered from early relapse and died of the disease (n = 4 before d+100 post allo-hct). asct following surgery and ctx appears to cure 33-60% of patients in available series. no treatment concept is available for relapse following asct, and no published data are available for allo-hct for relapse post asct. the disease is exceedingly rare and is afflicted with very poor outcomes. therefore, patients given this aggressive treatment should be reported, even when treatment outcomes are not positive. disclosure of conflict of interest: none. strong graft versus lymphoma effects with low toxicicty of haploidentical hematopoietic stem cell transplantation comparing with hla-identical in t cell lymphomas: a retrospective multicenter study s bramanti, r devillier, s fuerst, b reda, a granata, s harbi, c faucher, i legrand, a santoro, d blaise and l castagna istituto clinico humanitas rozzano consolidation treatment of relapsed/ refractory t-nhl with allogeneic stem cell transplant (sct) is considered a curative options but few patients manage to undergo this procedure, due to the highly refractory nature of the disease. the primary aim of this work is to evaluate the gvl effects among t-nhl with both hla identical and haploidentical donors. we have retrospectively analized the long term outcome of 43 consecutive patients affected by t-nhl, received hlaidentical or t-cell replete haplo-sct with pt-cy, in 2 european centers, between february 2010 and october 2015. the patients received nonmyeloablative (nmac) or reduced intensity (ric) conditioning regimen. gvhd prophylaxis consisted of 50 mg/kg of pt-cy (day +3 and +4) in haplo setting and atg plus cyclosporine a in the hla identical setting. patients characteristics were reported in the table 1 . no differences were founded in the two groups . most of the patients were transplanted in complete remissions but only 8 as consolidation of first line. no graft failure occurred. the cumulative incidence of acute gvhd grade 3-4 was 5% in the haplo setting vs 11% in the hla id .extensive chronic gvhd was seen in 7% of haplo, and in 28% in the hla id . 9 patients had cmv reactivation, 3 hemorrhagic cystitis, and 1 ebv reactivation. after a median follow-up of 3 years os was 83% and 71% and pfs was 77% and 64% in the haplo vs hla id group see figure 1 . nrm was 5% in haplo setting and 11% in hla identical one. the 3 years cir is 16% and 23% in haplo and hla id setting respectively. this study confirm a strong anti-lymphoma effect of allo hsct without prohibitive toxicities. haplo-hsct with pt-cy shows low rate of cgvhd in a contest of poor prognosis t-nhl patients. [p625] disclosure of conflict of interest: none. ibrutinib is the first-in class bruton tyrosine kinase inhibitor that has been approved for the treatment of relapsed mantle cell lymphoma. however, despite the high response rate of 68% including 21% of complete response, the median duration of response is relatively short with an overall survival of 58% at 18 months (wang ml, et al, n engl j. med 2013). we report a single experience of 3 patients with relapsed mcl who underwent allogeneic stem cell transplant (allosct) after ibrutinib monotherapy salvage. all 3 patients had previous autologous stem-cell transplantation (asct) before and were given ibrutinib at a dose of 560 mg daily after the second or subsequent relapse. all patients had to be at least in pr according to cheson 2014 criteria before allosct. patients had an unrelated 10/10 (2) or 9/10 (1) allo-sct from peripheral hematopoietic stem cells after a reduced conditioning regimen with busilvex, fludarabine and antithymocyte globulin in association with zevalin according to our recent published phase 2 study protocol (bouabdallah k, et al. ann oncol 2015). graft versus host disease (gvhd) prophylaxis consisted on ciclosporine and methotrexate. patients (2m/1f) were aged from 62 to 65 years and received between 2 and 3 previous chemotherapy regimens including asct in their last treatment strategy before introduction of ibrutinib. all patients had extensive disease with gastric involvement in 2 patients and pulmonary localization in 1 patient. median time between diagnosis and ibrutinib introduction was 5 years (3-7) and the median time between asct and allosct was 4 years (4) (5) . median duration of ibrutinib treatment was 5 months (5-6) and it was stopped one week before proceeding to allo-sct. it was not planned to restart btk inhibitor after transplant. patients were assessed for response after at least 3 months of treatment with ibrutinib. at time of evaluation, all patients were in complete (2) or very good partial response (1) before allosct. the patient in partial response had 92% tumor reduction with persistent gastric ulcer where histology examination shows cd5+ but negative cycline d1 lymphoid cells. all patients engrafted (median duration of pnn o500g/l = 11 days (10-15) and median duration of platelets o20g/l = 2 days (0-14)) with fulldonor chimerism at 1 month. one patient had a grade ii cutaneous chronic gvhd (cgvhd) with favorable outcome and developed 8 months later a bronchopulmonary obstruction syndrome related to cgvhd. with a median follow-up of 9 months (9-23) after allo-sct, all 3 patients are alive in cr. one patient, in complete metabolic response before transplant had a gastric relapse 3 months later but achieved again a cr 3 months after reintroduction of ibrutinib. after the first case reported by furtado m et al (leuk lymphoma 2016), we report here 3 additional cases with longer follow-up after allogeneic transplantation. the excellent tumor control after treatment with ibrutinib together with a very good outcome after allosct should drive to consider this approach in young patients with mcl relapse after asct. disclosure of conflict of interest: none. autologous hematopoietic stem cell transplantation (autosct) is considered the standard approach for high risk or relapsed/ refractory non-hodgkin and hodgkin lymphoma. although a large variety of conditioning regimens are available, including the widely used beam (carmustine, etoposide, cytarabine, melphalan), there is no consensus regarding a standard approach. in the context of carmustine shortage, we have chosen to replace it by thiotepa. however, clinical data about thiotepa-based autosct conditioning are still sparse, except some retrospective data for primary central nervous system lymphoma. thus, we designed a multicenter prospective study (nct02504190) to assess the efficacy and toxicity of a team (thiotepa, etoposide, cytarabine, melphalan) conditioning regimen. team regimen consisted in total dose thiotepa of 8 mg/kg on day-6; etoposide 100 mg/m 2 /12 h and cytarabine 200 mg/m 2 /12 h (day-5 to -2); melphalan 200 mg/m 2 on day-1. patients underwent autosct with team conditioning, and were included in this analysis if they have fullfilled the following criteria: age older than 18 years, biopsy-proven hodgkin or non-hodgkin lymphoma, hiv seronegative, and first autosct. thirty-three male and nine female with a median age of 59 years (range: 19-72) were analyzed thus far. karnofsky score was 20g/l was 13 days (range: 8-48). of note, 5 patients received thrombopoietic agents after engraftment because of persisting thrombocytopenia. the most significant regimen-related toxicities were mucositis in 100% of patients (median grade = 3, range: 2-4) and diarrhea in 98% of patients (median grade = 1, range: 0-3). other non-hematologic grade 3 adverse events occurred in 13 patients (31%) and no grade 4 adverse events were observed. central line-associated bloodstream infection occurred in 11 patients (26%). surprisingly, 16/36 evaluable patients (44%) developed human herpesvirus 6 reactivation. only 2 patients required intensive care unit transfer. the median duration of hospital stay was 29 days (range: 20-62). after a median follow-up of 8 months (range: 2-20), the non-relapse mortality (nrm) was 0%. only one patient relapsed of refractory aitl 2 months after autosct and died 1 month after. the estimated 1-year overall survival and progression-free survival were 98% and 98%, respectively. a team conditioning regimen seems to be a safe and valid platform in autosct for patients with high-risk or relapsed/ refractory lymphoma. although mucositis and diarrhea were frequent, there were no grade 4 adverse events and no deaths related to the treatment. updated results with updated followup will be presented. disclosure of conflict of interest: none. the cell of origin has no prognostic impact on high-dose chemotherapy with r-beam and autologous stem cell transplant for diffuse large b cell lymphoma s lozano cerrada, r saliba, s srour, s ahmed, c hosing, r champlin and y nieto university of texas, md anderson cancer center, department of stem cell transplantation and cellular therapy diffuse large b-cell lymphoma (dlbcl) is a biologically heterogeneous disease that can be classified according to its cell-of-origin (coo). the germinal center b-cell (gcb) subtype has better outcome with frontline r-chop than the activated b cell (abc) subtype. however, the prognosis of these two types of dlbcl after high-dose chemotherapy and autologous stem cell transplant (asct) is less clear. the purpose of our study was to evaluate progression-free survival (pfs), event-free survival (efs) and overall survival (os) in a cohort of dlbcl patients treated with r-beam (rituximab, carmustine, etoposide, cytarabine, melphalan) and asct according to coo. we have the dicep regimen effectively reverses the poor outcome for lymphoma patients with suboptimal response or failure post 1st salvage treatment p kaloyannidis 1 the outcome of patients (pts) with refractory hodgkin's (hl) and non-hodgkin lymphomas (nhl) post 1st salvage treatment (salv1) is considered poor. the published data, have shown extremely low survival rates (15-20%) even after 2nd salvage treatment (salv2) followed by autologous stem cell transplantation (asct), due to the low response rates post salv2 and the high relapse rates post asct, confirming that the management of these pts remains a major challenge. we herein evaluated the dicep regimen [dose intesified cyclophoshamide (1750 gr/m 2 ), etoposide (350 mg/m 2 ) and cisplatin (35 mg/m 2 ), days 1-3] as a salv2 treatment, in terms of safety and efficacy regarding disease response and stem cell mobilization/collection. moreover, we evaluated pts' long term outcome post asct. we retrospectively analyzed the data of 27 (11 hl, 16 nhl) pts, with a median age of 32 (16-61) yrs. twenty-one had suboptimal response (75% reduction): 4 and minor response (≤50% reduction): 2). three pts had stable disease while 3 experienced progression. overall 23/27 pts underwent asct after a median of 44 days (range: 22-70) post dicep. no pt was considered ineligible for the asct due to unacceptable toxicity post dicep; 4 did not undergo asct because of progressive (n = 3) or stable (n = 1) disease. the 5-yr overall survival (os) was 70% for the whole cohort of pts (71% for hl and 66% for nhl, p = ns) while the 4-yr progression free survival (pfs) from dicep administration (± asct) was 62% (60% for hl and 64% for nhl p = ns). in particular, for the 23 autografted pts, the 5-yr os was 70% (80% for hl, 70% nhl p = ns) and the 4-yr pfs was similar, 70% (65% for hl, 72% for nhl, p = ns) our data demonstrate that dicep is an effective salvage regimen with acceptable toxicity and no negative impact on the cd34+ collection. the promising response rates post dicep in combination with the very encouraging pfs rates achieved post asct, in this unfavorable and heavily pretreated group of patients, strongly support the rationale for using dicep as 1st line salvage regimen in selected pts in order to proceed to a successful asct. for the treatment of aggressive lymphoma, high dose chemotherapy followed by autologous stem cell transplant (asct) is an important component. however, the role of upfront asct in patients with diffuse large b cell lymphoma (dlbcl) is still controversial. furthermore, there is currently no consensus on a single best conditioning regimen for asct in patients with dlbcl. we retrospectively analyzed the records of 184 patients with dlbcl who underwent upfront asct in state of complete remission (cr) or partial remission (pr) from 17 institutions in korea. we evaluated the outcomes and prognostic factors of upfront asct in patients with dlbcl. we compared the outcomes of most widely used two conditioning regimens for asct; carmustine based regimens and busulfan containing regimens. total 141 patients (81.0%) achieved cr after asct and overall response rate (orr) was 86.7%. with median follow up of 34 months, 65 patients (35.3%) had progression or relapse. the 3-year overall survival (os) rates and progression free survival (pfs) rates were 75% and 65%, respectively. infection events were found in 99 patients (54.5%) and treatment related mortality was 3.8%. these outcomes were comparable with the results of previous other studies. cox multivariate analysis for os showed that eastern cooperative oncology group performance status (ecog ps) ≥ 2 (p = 0.003) and rituximab based induction therapy (p = 0.062) were significant prognostic factors. in addition, the following factors were significantly associated with pfs in multivariate analysis; female (p = 0.031), ps ≥ 2 (p = 0.018) elevated β2-microglobulin (p = 0.008), failure to achieve cr with induction chemotherapy (p = 0.004), carmustine based conditioning regimen (p = 0.012) and melphalan based conditioning regimen (p = 0.031). there were no significant differences in os and pfs according to stage, b symptom, bulky disease, high lactate dehydrogenase, bone marrow involvement, high or high-intermediate international prognostic index (ipi), absolute lymphocyte count and absolute monocyte count. therefore, it is considered that upfront asct can overcome the poor prognosis in patients with advanced stage or high risk ipi. in the analysis with conditioning regimen, neutrophil and platelet engraftment were slower in the carmustine group compared to the busulfan group. there were no significant differences in os between busulfan group and carmustine groups with 3-year os rates of 74.4% and 77.9%, respectively (p = 0.797). pfs at 3years was 66.0% in busulfan group versus 59.9% in carmustine group (p = 0.241). however, carmustine based conditioning regimen was poor prognostic factors for pfs in multivariate [p631] s457 analysis (p = 0.004). in subgroup analysis, busulfan group had significantly higher pfs compared to the carmustine group especially in female patients (67.9 months vs. 7 months, p = 0.002), with b symptom (67.9 months vs. 7.4 months, p = 0.033) and abnormal serum ldh level (70.2 months vs. 25 .9 months, p = 0.045). the outcomes of upfront asct in patients with dlbcl after induction therapy were acceptable. it is considerable in selected high risk patients who achieve cr with induction treatment, and have good performance status at diagnosis. in cases of conditioning regimen, busulfan based regimen resulted in improved outcomes compared with carmustine based regimen especially in patients with disseminated disease or female patients. disclosure of conflict of interest: none. no heavy chain was present in 25%). the predominant light chain was kappa (60%). 80 patients had bence-jones positive myeloma. 109 received bortezomib as induction therapy before transplant. we analyzed overall survival (os) and progression-free survival (pfs) in 5 groups of patients. we separated the groups according to improvement in grade of response from preasct to postasct. the post-asct grade of response was measured 3 months after asct. the os and pfs were estimated by the kaplan-meier method. pfs was measured from diagnosis to disease relapse and os was measured from diagnosis to death by any cause. results by subgroups of patients are detailed in table 1 . median os and pfs of the whole group was 8 years and 54 months, respectively. if we analyze groups only by their grade of response before asct we find the following results: rc (5years os rate 78.9%, median pfs 95 months); pr/vgpr (median os 7.31 years and pfs 40 months); sd/progression (median os 6.46 years and pfs 21 months). according grade of response after asct, instead: rc (5 years os rate 78%, median pfs: 66 months); pr/vgpr (median os 7,31 years, and pfs 40 months); sd/progression (median os 1.05 years and pfs 11 months). in our experience, the grade of response before asct is a capital predicting factor for patients os and pfs. patient in cr before asct that preserve it after transplant, have a median pfs of 95 months, the 5 years os rate being 80.4%. patients in situation of progression after asct have a very dismal prognosis (median os 1.05 years, pfs: 11 months), however, patients who change from sd/progression to pr after asct have a median pfs of 29 months and a os of 6.4 years. comparing these results we observe that this second group is particularly benefited by transplant. autologous peripheral blood hematopoietic stem cell transplantation in elderly patients with multiple myeloma as a standard therapeutic procedure. is it feasible? a single-center experience l cadievski, s genadieva stavric, z stojanoski, a pivkova veljanovska, d miloska, b kocoski, l cevreska and b georgievski university clinic of hematology, department for hematopoietic stem cell transplantation, university ss. cyril and methodius, skoje, republic of macedonia autologous peripheral blood stem cell transplantation (pbsct) represents a standard therapeutic approach in the treatment protocol of myeloma patients. it is known that multiple myeloma is a hematological disease that is a characteristic for the older population. autologous pbsct ideally should be performed in every myeloma patient, but with the elderly myeloma patients the procedure might be risky if know the possible comorbidities, or the possibility of the body to fully compensate the side effects of the conditioning regimen, the procedure or its possible complications. we present our experience in using high dose conditioning with melphalan 200 mg/m 2 followed by autologous pbsct for elderly myeloma patients, using the age limit od 65 years. our retrospective analysis of our data during 16 years of experience, shows that we have performed autologous pbsct on 12 patients with myeloma at the age of 65 or older. 11 males (91.6%), and 1 female (8.4%). 6 patients (50%), were diagnosed with igg type myeloma, 5 patients (41%) with iga myeloma, and 1 patient (9%) with light chain myeloma. median age of the patients was 66.9 years (65-73). all patients were initially treated with cy-thal-dex regimen. in 5 (41%) patients complete response (cr) was achieved, in 5 (41%) very good partial response (vgpr), and in 2 (18%), partial response (pr). in all patients the mobilisation of hematopoietic stem cells was performed with g-csf, and a median of 2 apheresis procedures were performed, and the average number of collected cells was 3.11 × 10 8 /kg tt mononuclear cells (range: 6.0-2.1). days to confirmed engraftment in our group of patients was 11.5 (range: 9-15). the number of blood transfusions was on average 2.8 (range: 0-6), and the number of transfusion of thrombocytes 38.1 units (range: 10-74). in the majority of patients, mainly after the year 2012 (that represents 10 patients of the whole group), we used noncryopreserved hematopoietic stem cells, kept under the temperature of 40c, for median of 2 days, thus avoiding the toxicity of dmso. additionally, we used central venous catheter inserted in the femoral vein for apheresis and application of the stem cells afterwards. the day after, the catheter was removed, thus avoiding catheter associated infections. all patients received standard infectious prophylaxis with fluconasole 200 mg/daily, ciprofloxacin 500 mg/ two times daily, acyclovir 200mg/ three times daily, cefixime 400 mg/once daily, and ursodeoxycholic acid for vod prevention. no serious infectious complications were reported. our transplant related mortality was 0%. in the group with noncryopreserved stem cells no graft failure was reported. in two patients we even performed tandem autologous pbsct with no major complications. of the group of 12 patients, the majority, 8 patients (66%), had hta as comorbidity, 1 (8%) with cardiomyopathy, and 1 (8%) with inserted prosthetic aortic valves. three patients (24%) have died because of relapse of the disease. our oldest patents were 72 and 73 years old, and are still alive 1 year posttransplant, in cr. we can conclude the performing autologous pbsct in elderly myeloma patients can be safe and effective therapeutic option, but with careful selection of the patients, balancing the risk profile of the patient and the benefit, or the risk of the procedure. affective supportive care, monitoring and reducing the risk of complications is an imperative to a good result. disclosure of conflict of interest: none. autologous stem cell transplantation program for patients with multiple myeloma in an outpatient setting k lisenko 1 , s sauer 1 , g egerer 1 , j schmier 1 , m witzens-harig 1 , a schmitt 1 , ad ho 1 , h goldschmidt 1,2 , j hillengass 1 and p wuchter 1,3 1 department of medicine v, heidelberg university hospital, heidelberg, germany; 2 national center for tumor diseases heidelberg (nct), heidelberg university, heidelberg, germany and 3 institute of transfusion medicine and immunology, mannheim, german red cross blood service baden-württemberg-hessen, medical faculty mannheim, heidelberg university, germany the first and second authors contributed equally. high-dose chemotherapy with melphalan and autologous blood stem cell transplantation (absct) for treatment of symptomatic multiple myeloma (mm) is performed in the usa and canada mostly on an outpatient basis, whereas in germany and western europe an inpatient setting is the standard. we report on a german single-centre program to offer the procedure on an outpatient basis to selected patients. major inclusion and exclusion criteria for eligibility were defined as follows: patients had to be able to reach the hospital within 45 minutes, had reliable support from their family at home, had an ecog performance score of 0-1 and were willing and able to comply with the demands of the program. patients with severe co-morbidities were not included. all patients were treated on our outpatients' clinic and examined on daily visits by a team of physicians. feedback from patients was obtained by means of a questionnaire. from september 2012 to september 2016, 26 patients with mm stage iiia were enrolled. all engrafted within the expected time range: (median time to leukocyte 41,000 /μl and neutrophil recovery 4500 μ/l: 14 days; median time to platelet recovery 420/nl: 10 days, 450 /nl: 14 days). twenty patients (77%) had an episode of neutropenic fever but only in 5 patients (19%) blood cultures were found to be positive. there occurred no cases of infection with multiresistent bacteria. although rather liberal criteria for hospital admission were applied, 18 of 26 patients (69%) could be treated entirely on an outpatient basis. eight patients (31%) were temporarily admitted for inpatient treatment with a median duration of 4.5 days (range: 2-18 days), mainly because of neutropenic fever. no severe adverse events occurred. feedback from patients revealed a high level of satisfaction with the outpatient setting. high-dose chemotherapy and absct on an outpatient basis is safe and feasible if conducted in a comprehensive surveillance program. the feedback from patients was very positive, thus encouraging further continuation and expansion of the program. disclosure of conflict of interest: none. high dose of melphalan (bor-mel). we retrospectively analyzed 69 patients with mm who underwent asct between january 2014 and march 2016. in these patients, conditioning regimen consisted of a high dose of melphalan (140-200 mg/ m 2 ) intravenously on day -2 and two doses of intravenous bortezomib at 1.3 mg/m 2 administered on days − 1 and +2. this cohort was compared with patients underwent asct between 2003 and 2013, conditioned with high dose of melphalan alone. response rate was evaluated according to imwg criteria. all patients were evaluated after induction therapy and 3 months after asct. all patients were followed until death or reference date (november, 2016). results: patients' demographics and baseline disease-related characteristics are shown in table 1 . [p636] no difference was found in terms of neutrophil and platelet engrafment, hospitalization days (p=0.723) and use of mechanical invasive ventilation (p=0.415). bor-mel regimen did not enhance severity of preexisting peripheral neuropathy (pn) in any patients, and only one presented de novo grade 2 pn. non relapsed mortality was 1.4% and 1% in the bor-mel and mel cohorts, respectively (p=0.673). complete response rate after transplant was significantly better in the bor-mel cohort than in the mel cohort (65.2% vs. 40.7%; p=0.001) ( figure 1b) . when the analysis was restricted to patients who received bortezomib-based therapy, this difference was also statistically significant (64.1% vs. 44.4%; p=0.024) ( figure 1d ). median of follow-up was 12 months in the bor-mel vs. 42 months in the mel cohort. no difference was found in terms of overall survival (os) and progression free survival (pfs) between both groups. for all patients, a post-transplant deeper response was associated with better os and pfs (p=0.008 and p o0.001, respectively). our results are in line with previous studies demonstrating that bortezomib combined with melphalan is a well tolerated conditioning regimen and may enhance the response rate after transplant, even in patients receiving bortezomib in the induction therapy. these results should be confirmed in a randomized trial. for newly diagnosed patients (pts) with multiple myeloma (mm), the triple-agent induction treatment based on bortezomib plus dexamethasone in combination with cyclophosphamide (vcd) or lenalidomide (vrd) represent extremely reliable regimens, which in combination with early autologous stem cell transplantation (asct) result in high response rates and prolonged long-term outcomes. however, though both regimens are widely used, there are extremely limited studies that compare the vrd vs. vcd in terms of safety and efficacy. in the present study we compared the outcomes of 19 newly diagnosed mm pts who received induction treatment vrd (n = 10) or vcd (n = 9) and proceeded early to asct. the vrd and vcd pts groups were similar regarding age at diagnosis (52 vs.54 ys, p = ns), interval between diagnosis-asct (5,8 vs. 5,7 months, p = ns) and maintenance treatment post asct (8 vs. 6 pts, p = ns). per revised international scoring system (riss), the vrd-group had slightly more advanced disease (stage i: 1, stage ii: 7 and stage iii: 2), compared to vcd-group (stage i: 3, stage ii: 5 and stage iii: 1), however this difference was not statistical significant. the conditioning regimen consisted of single agent melphalan: 200mg/m 2 . the t-test, kaplan-meir and cox regression were utilized for the statistical analysis. following a median of 4 cycles of treatment (range: 4-6 for vrd vs. 2-6 for vcd, p = ns), in the vrd-group 4 pts achieved complete remission (cr), 5 pts very good partial remission (vgpr ≥ 75% reduction of m-band) and 1 pt partial remission (pr: 50-75% reduction of m-band) while in the vcd-group cr:3, vgpr:2 and pr:3 pts (p = ns). the toxicities in terms of peripheral neuropathy, myelosuppression, liver and renal function were well tolerated and no patient discontinued treatment due to severe side effects. the 5-yr overall survival (os) was 100% for the vrdgroup vs. 75% for the vcd-group; nevertheless, the difference was not significant due to the size sample of the pt groups. the stage at diagnosis, the disease status pre-asct and the maintenance post-asct did not influence the os. interestingly, the 4-yr progression free survival (pfs) was significantly superior for patients who had been induced with the vrd regimen (75% vs. 36% p = 0.05) and for patients who achieved cr or vgpr before asct (pfs: 50%) while no pts with pr pre-asct was progression-free 2 yrs post asct (p = 0.001). in multivariate analysis, only the cr or vgpr status before asct favorably affected the long term pfs. our results are in line with the limited published data from other studies with larger series of patients. in our study, very low disease burden before asct proven to be an independent factor for prolonged pfs. taking into consideration that vrd resulted in more cr or vgpr status, it is reasonable to conclude that vrd is a highly effective regimen and could be first treatment choice for newly diagnosed mm patients who are fit for early asct post induction. lenalidomide cohort and 64 in the maintenance bortezomib cohort. baseline characteristics and outcome data were obtained via chart review. the primary outcome was pfs. the secondary outcomes were overall survival (os) and treatment-related toxicities. the median follow-up time was 33 months. median time to death (4.28 years vs 5.77, p = 0.47) and median time to progression (1.71 years vs 1.74, p = 0.77) were not significantly different in the maintenance lenalidomide cohort compared to the maintenance bortezomib cohort. in the multivariate analysis, pfs was worse in patients at international staging system (iss) stage 3 at diagnosis compared to those at iss stages 1 and 2 (hr, 1.86; 95% ci, 1.11 to 3.12; p = 0.02) and worse in patients with less than very good partial response (vgpr) to last prior therapy compared to those with a response to prior therapy of at least vgpr (hr, 2.05; 95% ci, 1.14 to 3.69; p = 0.02) [see figure 1 ]. pfs was improved in patients with more than two years of maintenance therapy compared to those with less than two s464 years of maintenance therapy (hr, 0.40; 95% ci, 0.22-0.70; po0.01), but this result does not account for patients who ended maintenance therapy due to disease progression. os was worse in patients at iss stage 3 at diagnosis compared to those at iss stages 1 and 2 (hr, 3.87; 95% ci, 1.44 to 10.39; p = 0.01). peripheral neuropathy was more common in the bortezomib cohort (39% vs 9%, p o0.01), while cytopenias were more common in the lenalidomide cohort (30% vs 3%, po0.01). figure 1 kaplan-meier curve for pfs for the maintenance lenalidomide group versus the maintenance bortezomib group by log-rank test (p = 0.16). lenalidomide and bortezomib maintenance after transplantation have equal efficacy in prolonging progression-free and overall survival in patients with multiple myeloma. iss stage significantly affects time to progression and overall survival, and response to last prior therapy affects time to progression. length of maintenance therapy may be a significant predictor and warrants further analysis. these findings suggest that both lenalidomide and bortezomib are acceptable maintenance therapy options for post-transplantation multiple myeloma patients. autologous stem cells transplantation (auto-hct) is an accepted method in multiple myeloma (mm) patients, but usually it is not curative. the issue of allogeneic hematopoietic stem cells transplantation (allo-hct) is challenging yet for myeloma. we investigated allo-hct in mm and compared with auto-hct. in this retrospective study, we recruited 272 patients from january 2011 to january 2015 (218 (80.15%) patients in autologous group and 54 (19.85%) in allogeneic group). we performed allogeneic hct with peripheral blood stem cells source in our center for patients who are relatively young (less than 55 years old) with good performance, have match sibling donor and accepted allogeneic hct. the conditioning regimens in autologous group was melphalan 200 mg/m 2 only and in allogeneic groups was fludarabine 30 mg/m 2 plus melphalan 140 mg/m 2 in 5 consequent days. gvhd prophylaxis consisted of methotrexate and cyclosporine. the outcomes then compared between two groups using log-rank and gray tests and cox proportional hazard regression. the median follow-up in the autologous and allogeneic group was 17.02 months. three years disease-free survival of auto-hct was 38.61% (ci: 27.37%, 49.72%) and 68.88% (ci: 50.74%, 81.47%) for allo-hct patients (p value = 0.0363). three years overall survival of auto-hct was 77.26% (ci: 66.08%, 85.16%) and 82.15% (ci: 64.92%, 91.44%) for allo-hct patients (p value = 0.6363) showing no significant statistical difference between two groups. mortality rate was 11.01% for auto-hct and for allo-hct was 12.96%. the most common cause of death between two groups was relapse of primary disease. three year relapse incidence was 20.83% (ci: 9.04%, 35.30%) for allo-hct and 54.33% (ci: 42.02%, 65.09%) for auto-hct (gray's test p value = 0.018). the three year trm incidence was 10.36% (ci: 2.92%, 23.33%) and 7.01% (ci: 3.14%, 12.98%) in allogeneic and autologous patients respectively (gray's test p value = 0.42). despite there was no statistically significant difference between two groups in terms of os but dfs and relapse incidence was meaningfully better in allogeneic group. so, perhaps the reason of non-significant os improvement in allogeneic group is higher early death due to higher trm. we suggest that this study needs longer follow up to see whether allo-hct resulted in os improvement. disclosure of conflict of interest: none. myeloablative allogeneic hematopoietic stem cell transplantation from unrelated donors for patients with relapsed or refractory multiple myeloma n tsukada 1 , s shingaki, m ikeda, t ishida and k suzuki 1 division of hematology, japanese red cross medical center allogeneic hematopoietic stem cell transplantation (allo-sct) for patients with multiple myeloma (mm) is increasing in number despite in the era of novel agents, especially as a second line treatment and beyond. it has been reported that allo-sct for patients with mm resulted in high incidence of treatment related mortality (trm). high incidence of disease relapse is also a major problem especially after reducedintensity stem cell transplantation (rist). it is an important issue to reduce the incidence of trm while preventing disease relapse. the use of stem cells from unrelated donors is required for those without hla-matched sibling donors. the purpose of this study is to evaluate the feasibility of an intensified conditioning regimen incorporating both 140 mg/ m 2 of melphalan and 8 gy of total body irradiation (tbi), followed by allo-sct from unrelated donors for patients with relapsed or refractory mm. we retrospectively analyzed eight consecutive patients who received allo-sct from unrelated donors with the conditioning regimen including 8gy of tbi, fludarabine 25 mg/m 2 for five days, and melphalan 140 mg/m 2 between april 2013 and july 2015 at the japanese red cross medical center. six patients received unrelated bone marrow transplantation (bmt) and two patients received cord blood transplantation (cbt). graft-versus-host disease (gvhd) prophylaxis was consisted of tacrolimus and short term methotrexate. the median age at allo-sct, the time from diagnosis of myeloma to allo-sct, and the numbers of prior treatment lines were 48.5 years (range: 31-60 years), 38.5 months (range: 8-64 months), and 3.5 lines (range: 1-7 lines), respectively. five patients are female. no episode of either grade ≥ iii toxicity or non-relapsed mortality was documented during the median follow-up period of over two years. cumulative incidence of grade ≥ ii acute and severe chronic graftversus-host disease were 37.5% (95% confidence interval [ci] 7.2%-69.4%) at 100 days and 25.0% (95% ci 2.9%-58.1%) at 180 days, respectively. probabilities of progression-free survival and overall survival were 22.5% (95% ci 0.0%-58.7%) and 58.3% (95% ci 22.0%-94.7%), at 3 years, respectively. the results suggest that allo-sct conditioned with this intensified regimen may be tolerable for patients with relapsed or refractory mm. disclosure of conflict of interest: none. the role of allogeneic stem cell transplantation (allosct) in the era of novel myeloma drugs remains controversial. it is the only curative treatment option but non-relapse mortality makes the decision making difficult as opposed to achievements with autologous sct and new mm drugs by which the median survival is nowadays nearing 10 years. aim of this study was retrospectively evaluate the outcome of allosct for mm performed at our institute, including evaluation of factors affecting survival. all 66 consecutive patients allotransplanted for mm between 1986 and 2014 were included. the data were collected from our transplant registry. frequencies and medians were produced as appropriate. kaplan-meier method was used to calculate os and pfs and log rank test for comparisons. univariate analysis for factors affecting survival was performed with cox proportional hazard model. median age of all 66 patients was 55 (36-66) years. half of the patients had igg myeloma, 23% had iss score 3 (score available for 30 patients), and 33% had high-risk cytogenetics (data available for 39 patients). response to treatment at sct was at least vgpr in 80% of patients, and transplant timing was early (within 15 months from dg) in 58% of patients. sibling donors were used in 58% and muds in 42% of transplants, and conditioning was ma for 50% and ric for another 50% of patients. acgvhd grade 0-2 occurred in 78% and grade 3-4 in 22% of patients; 32% of patients had extensive chrgvhd. posttransplant cr rate was 83%. 45% of pattients have relapsed after allosct, and 42% are alive with the median follow-up of 5,6 years. non-relapse mortality has been 30% (35% until 2005, 27% since then). the median survival of patients up to age of 60 years is 4.9 years vs 3.0 years for patients 460 years (n = 8) with survival plateau after 6 years at 40% level. transplant period, cytogenetics, donor type, conditioning intensity or occurrence of chrgvhd had no statistical impact on survival. significant differences in os were observed between disease status at scr 4 vgpr vs o15 months from dg vs later), grade of acgvhd 0-2 vs 3-4, and best resonse post-transplant cr vs not less than cr. the respective differences for pfs were in sct timing, grade of chrgvhd, and best post-transplant response. in univariate cox regression analysis the only significant factors for os were severity of acgvhd and cr vs other responses after sct, and for pfs allosct timing, severity of chrgvhd, and best response to sct. with allosct ca. 40% of mm patients can be cured but at the cost of high non-relapse mortality. the occurrence of grade 3-4 acgvhd and less than cr response to sct predict poor survival. considering the increasing survival expectations with modern standard therapy for mm, allosct may be recommended for younger patients with high-risk features, and allosct should be done early in the disease course. disclosure of conflict of interest: none. angiogenesis plays an important role in the pathophysiology of hematological malignancies including plasma cell myeloma (pcm). microrna-21 (mir-21) is overexpressed and displays oncogenic activity in cancers. however, little is known about the role of mir-21 in pcm. the aim of the present study is to examine the expression level of peripheral mir-21 in pcm patients and to determine its role in angiogenesis. vegf serum levels and mir-21 in pbmcs was measured in 93 patients with pcm directly before melphalan 200 mg/m 2 followed by autologous hematopoietic stem cell transplantation (auto-hsct) and 2 months after hsct; and 35 healthy controls. the study population was divided into two groups after therapy: responders (stringent complete response, complete response, very good partial response, partial response) and nonresponders (stable disease, progressive disease). gene expression of mir-21 was quantified by sybr green real-time fluorescent quantitative pcr. further tube formation of huvecs and vegf secretion was measured in mir-21 mimic or inhibitor transfected human plasma cell myeloma cell lines h929 and rpmi-8226. the expression level of mir-21 was significantly increased (2.7 ± 0.55 versus 0.78 ± 0.22; p o0.01) in pbmcs of pcm patients compared with healthy controls. further, serum vegf levels were increased in pcm patients (477 ± 145 pg/ml versus 178 ± 78 pg/ml in normal controls; p o0.01). after auto-hsct, the expression level of mir-21 was significantly different in responders compared to nonresponders. responders had a lower expression of mir-21 compared to non-responders. further, serum vegf levels decreased in responders to auto-hsct compared to nonresponders. vegf expression was increased in the supernatant from mir-21 mimic transfected human pcm cell lines h929 and rpmi-8226 compared with the negative control, while s467 vegf was decreased in the mir-21 inhibitor transfected cell lines. the angiogenic ability of huvecs was increased under pretreatment with the supernatant from h929 and rpmi-8226 cells transfected with mir-21 mimic compared with negative controls and decreased when pretreated with mir-21 inhibitor transfected cells (fig. 1) . this study demonstrated that mir-21 was upregulated in pcm patients. responders to auto-hsct had a decrease of mir-21 expression and vegf levels. further, mir-21 regulated angiogenesis. therefore inactivation of mir-21 or activation of its target gene may be a potential therapeutic approach in pcm. fig. 1 : in vitro matrigel tube formation assay. (i), normal control (ii, iii), mir-21 mimic transfected h929 cells (iv), mir-21 mimic transfected rpmi-8226 cells (v), mir-21 inhibitor transfected h929 cells. original magnification × 100. disclosure of conflict of interest: none. [p647] pilot study of busulfan/thiotepa as conditioning regimen followed by allografting and post transplantation cyclophosphamide in advanced relapsed myeloma patients c wolschke, e klyuchnikov, d janson, m heinzelmann, m christopeit, f ayuk and n kröger university medical center hamburg-eppendorf despite the significant improvement in outcomes has been observed for myeloma patients, the disease still remains incurable. due to limitations, such as trm and gvhd, the role of allogeneic stem cell transplantation as salvage therapy in this setting remains unclear. in present pilot study we provide data on the use of post cyclophosphamide (ptcy) as gvhd prophylaxis after a busulfan/thiotepa based conditioning regimen in patients who relapsed after autologous stem cell transplantation. between 11/2014 and 08/16 17 myeloma patients (male n = 10, female n = 7) with a median age of 55 years (range: 45-66) (pts), who relapsed after autologous stem cell transplantation received allogeneic stem transplantation with with ptcy as gvhd prophylaxis after busulfan (9.6 mg/kg for age 460y and 6.4 mg/kg for age 460years) and thiotepa (10 mg/ m 2 )and for haploidentical and mmud additional fludarabin (90 mg/m 2 ). all pts. were relapsed after one or two autologous stem cell transplantations. donors were haploidentical (n = 1), mmud (n = 4), mud (n = 6) and hla-identical sibling (n = 6). stem cell source was pbsc (n = 15) or bm (n = 2). all patients received cyclophosphamide 50 mg/kg of body weight on day +3 and +4, which was in 10 pts (n = 5 mrd, n = 5 mud) the only gvhd prophylaxis, while patients with mmud and haploidentical donor received also cyclosporine a from day +5 and mmf (until day 35) and 2 patients (mrd and mud) received additional cyclosporine. we observed no primary or secondary graft failure. the median time for neutrophil and platelet engraftment was 19 (range: 15-24) and 51 days (range: 22-279), respectively. major toxicities grade 3 and 4 were: renal (n = 1) and mucositis (n = 1). major infectious complications were: cmv: n = 10 cmv-reactivations (n = 10), sepsis (n = 5), pneumonia (n = 3) rsv-(n = 2) and hsv (n = 1). acute gvhd grade ii to iv and ii/iv was noted in 29% and 24%, respectively and mainly seen in patients with cyclophosphamide as single gvhd prophylaxis. remission rate were n = 8 complete remission, n = 7 vgpr, n = 1 partial remission, n = 1 n.a. after a median follow up of 12 months 3 pts progressed and 7 patients (n = 1 relapse, n = 6 trm) died. the 1 year pfs was 18% (n = 3). busulfan/thiotepa is an active conditioning regimen for advanced relapsed myeloma patients. post cyclophosphamide might increase anti-myeloma activity, but as single gvhd prophylaxis it causes significant agvhd in mrd and mud and additional immunosuppressive agents such as cyclosporine should be added. disclosure of conflict of interest: none. magnetic resonance imaging (mri) for multiple myeloma (mm) is a sensitive, non-invasive and non-toxic method for detecting myeloma lesions. the goal of the study was to assess whether quantitative mri metrics can detect treatment response and replacement of neoplastic cells by fat marrow. the study was hipaa-compliant and irb-approved. we retrospectively identified all patients who achieved a complete response (cr) after induction therapy between 2000 and 2014. inclusion criteria for the study was total spine mri imaging at diagnosis and after achieving cr. cr was determined using the imwg criteria. spinal vertebrae t12 through l5 were outlined with imagej software. fractures and lesions were excluded. images were analyzed using histogram-based (entropy, skewness, kurtosis) and texture-based statistics. a two-sided t-test was used to compare quantitative mri metrics from before therapy and after achieving cr. cox regression was used to explore the association between progression free survival (pfs) and change in each quantitative mri metric based on a median split. pfs was defined as the time from the second mri to death or progression of disease. nineteen patients met the above criteria. median age was 61.5 years (range: 37.5-72.2). majority of patients (68%) were male. majority of patients had iss stage 1 disease (57.9%) and standard-risk cytogenetics (89.5%). an induction regimen containing an imid and/or a proteasome inhibitor was commonly used (73.7%). all patients received an autologous stem cell transplant (asct) consisting of high dose melphalan followed by autologous stem cell rescue. three patients received a planned second asct. seven patients (36.8%) were in cr before asct. nine patients (47.4%) were treated with imid maintenance after planned initial therapy. median time to repeat mri imaging after cr was 10 months (range: 4.4-19.8). mean change in measurements of kurtosis, skewness, entropy and 6 texture analyses are shown in table 1 . no significant change was detected between preand post-cr mri. furthermore, no significant association was seen between the change in any quantitative metric and pfs. [p649] despite promising results by other groups, we could not find a significant association between quantitative t1 image analysis and cr or pfs. there was heterogeneity in the time of repeat mri imaging which may have limited our ability to study interval change. although no definitive conclusions can be made from this small sample, correlation between pfs and kurtosis or texture d may be promising and should be investigated in a larger group prospectively. multiple myeloma (mm) treatment (tx) has evolved in recent years. solid data on the impact of new tx on patient (pt) outcomes outside clinical trials, however, is lacking. this study aimed at investigating tx practices, pt journeys, and outcomes in the real-world in countries with different access to new tx. the study was conducted between 04/2015 and 06/2016 in bulgaria, croatia, czech republic, poland, romania, and slovakia. it consisted of a cross-sectional (x) and a retrospective (r) phase. for the x-phase, investigators included all symptomatic mm pts seen during a 3-week counting phase to provide a snapshot of where in the pathway pts were at a given moment. for r-phase, investigators collected data on current and past tx, including symptoms, dosages, administration schedule, tx durations, tx interruption, reasons for change/discontinuation, and tx response. pts were selected in reverse chronological order with a quota of a maximum of 3 pts who completed first-line (1l) tx within the past 3 months (mo), 4 pts in second-line (2l) and 7 pts in third or higher lines (3+l). pts included in the x-phase could also be included in the r-phase, if they met the respective inclusion criteria. in total, 39 physicians included 522 pts in the x-and 35 physicians included 277 pts in the r-phase. in the x-phase, 52% of pts were o 65, 36% were 65-75, and 12% were 475 years; the median time since diagnosis was 27 mo. 57% of pts were currently undergoing tx, 41% were previously treated and 2% had never been treated. of currently-treated pts, 37% received 1l, 30% 2l, 19% 3l and 14% 4+l. in the r-phase, 47% of pts were o65 years. of pts receiving 1l, 59% continued to 2l, 33% to 3l, 15% to 4l and 8% to 5l. of the 38% of pts eligible for stem cell transplantation (sct), 55% ( = 21% of all pts) received sct at 1l; these proportions were similar across countries. the most frequently-used regimens in 1l and 2l were bortezomib-based (57% and 53%, respectively), in 3l and 4+l lenalidomide-based (47% and 35%, respectively). median duration of 1l was 6 mo, followed by a median disease-free interval (dfi) of 4 mo. median dfi was longer in pts with sct than in those without (6.5 mo vs 1.5 mo). time to progression (ttp) decreased with later tx lines, from median 9 mo at 1l to 4 mo at 3l. depth of response, as assessed by the treating physician, decreased with each additional line of tx: 50% of pts achieved at least very good partial response (≥ vgpr) in 1l, while only 25% achieved ≥ vgpr at 3+l. ttp was longer in pts with better response levels: in 1l, median ttp for pts with ≥ vgpr was 22 mo versus 6 mo for pts with 6 mo for pts with ovgpr. the most common ( ≥20%, all grades) adverse events (aes) and co-morbidities in 1l were anemia (42%), thrombocytopenia (29%), neutropenia (25%), neuropathy (25%), and fatigue (22%). these aes disrupted treatment in 57% in 1l, 41% in 2l and 55% in 3+l. the study found that of sct eligible pts, only slightly more than half were transplanted. poorer outcomes and increasing ae incidence with each tx line highlight the challenges of mm tx. information on real-world pt management may be valuable for physicians to plan their tx strategies and can provide input for health economic evaluations of existing and new tx. disclosure of conflict of interest: daniel coriu declares to have received consulting fees or other remuneration (payment) from novartis, amgen, pfizer, takeda, janssen. ivan spicka declares to have received research grants from celgene, consulting fees or other remuneration (payment) from bms, takeda, celgene, janssen-cilag, and amgen, and to be a member of the speakers bureau of celgene, janssen-cilag, amgen, and bms. zdenka stefanikova declares to have received consulting fees or other remuneration (payment) from amgen, celgene, and takeda and be a member of the speakers bureau of amgen, celgene, and takeda. daniela niepel, krisztian szabolcs toka, and paul schoen are amgen employees and hold amgen stock. dominik dytfeld, and georgi mihaylov have nothing to declare. safety and efficacy of autologous stem cell transplantation in elderly patients with multiple myeloma t maia 1 , c marini 1 , p medeiros 1 , e aguiar 1 , j cancela pires 1 , r bergantim 1 , f trigo 1 and je guimarães 1,2 1 hematology department, centro hospitalar de são joão and 2 faculty of medicine, university of porto autologous stem cell transplantation (asct) is considered standard treatment for multiple myeloma (mm) patients under the age of 65 years, but its safety and efficacy still uncertain for patients over this age. retrospective analysis from one single centre concerning mm patients under, equal or over 65 years who underwent asct between january/2010 and july/2016. it was also compared to 65-70 years old mm patients diagnosed in this period of time who were not transplanted. we analysed a total of 160 patients, 135 of which underwent asct. onehundred-and-six of the transplanted patients were aged 65 years or less (median 56, iqr 10 years), 29 patients were aged more than 65 years (median 67, iqr 2 years) and 25 patients were non transplanted (median 68, iqr 4). the conditioning regimen for younger patients who underwent asct consisted mainly of melphalan 200mg/m 2 (mel200) while half of the elder patients received melphalan 140mg/m 2 (mel140). regarding transplant-related myelotoxicity there were no statistical differences between patients aged 65 years or less and over 65 years old, however the first group needed less days of g-csf (p = 0.04). non-hematopoietic toxicity measured by infections and mucositis was not influenced by age. patients 465 years conditioned with mel200 had more days of aplasia (p = 0.05), greater need of g-csf (p = 0.01) and transfusional support (p = 0.04) than patients ≤ 65 years. there were no differences on non-hematopoietic toxicity. in the elderly group, patients conditioned with mel200 presented more aplasia days (po0.01), higher grade of mucositis (p = 0.03) and more days of iv antibiotics (p = 0.02) than those transplanted with reduced dose of melphalan. comorbidities had no effect on transplant-related toxicity, either by age or by dose of melphalan. days of hospitalization and post-transplant complications did not differ according to age group. transplant related mortality was 2% at day 100 posttransplant. survival after transplant in patients 65 years old or under vs older patients (median follow-up time, 30 months), was not influenced by age (os, 83mo vs 59mo, p = 0.17; pfs, 38mo vs 37mo, p = 0.59). regarding the non-transplanted elderly group, these are patients with more renal disease (p = 0.02) and poorer performance status (p = 0.04) than the transplanted cohort. there is also higher cytogenetic risk (p = 0.01). induction regimens were similar in transplanted group and non-transplanted group 465 years old, and response to first line therapy (before asct of transplanted group) revealed no differences. infections were the most common complication in both groups. transplanted patients needed less days of hospitalization (p = 0.04). comparing the long term outcome of these two groups, survival curves of the elderly patients transplanted were clearly superior to the nontransplanted (os, 62mo vs 21mo, p o0.01; pfs, 45mo vs 20mo, p o0.01) although one has to consider that the non-transplant group has worse features than the elderly transplant group. transplantation in the elderly still debatable but this study shows that it might bring benefit. globally, transplant related toxicity is not influenced by age. regarding dose of melphalan, higher dose in elderly patients has higher toxicity, without apparent benefit in survival. therefore, age should not restrict the access to asct, but instead selection must be based on individual clinical and functional status. disclosure of conflict of interest: none. second autologous stem cell transplantation for relapse after allografting in multiple myeloma using cd 34+ selected donor cells without immunosuppression p novak 1 number of patients receiving a second allogeneic stem cell transplantation (sct) in europe is increasing despite high treatment related mortality (trm). in multiple myeloma only very few reports of second allogeneic sct exist with limited number of patients and substantial mortality. while in most hematological malignancies, the donor cell chimerism is dropping down if patients are relapsing, in myeloma donor cell chimerism remains complete despite relapse. to reduce trm we thought that full donor cell chimerism may allow us to perform a second high dose busulfan based chemotherapy followed by "autologous transplantation" after stem cell mobilization and collection. however, because two consecutive patients failed to collect sufficient cd34+ cells for an autologous transplantation even with plerixafor, we used donor t cell depleted cd34+ selected cells and transplanted those patients in an "autologous" fashion without any immunosuppression. to enhance graft-versus-myeloma effect, we added donor lymphocyte infusion (dli) at day 100. we report here on 11 myeloma patients with a median age of 58 years (range: 48-68) who relapsed after allogeneic sct and underwent a second "autologous" sct with cd34+ selected donor cells. all patients had received one (n = 8) or two (n = 3) autologous sct before 1. allografting. 6 patients received an upfront auto-allo protocol and 5 patients received 1. allogeneic sct as a salvage therapy. 73% of patients received a reduced intensity melphalan based conditioning regimen for 1. allogeneic sct and the median pfs was 39 months (range: 22-56). before 2. allograft patients had received overall a median of 5 (range: 3-7) treatment lines. at the time of 2. allogeneic sct all patients had a full or nearly full donor cell chimerism and remission status was very good partial remission (n = 1), partial remission (n = 5), stable disease (n = 4), progressive disease (n = 1). 82% of patients received a myeloablative busulfan based conditioning regimen and all received cd 34+ selected stem cells with a median number of 5.3 × 10 6 /kg cd34+ cells (range: 1.4-7.5) and 5 × 10 3 /kg cd3+ cells (range: 1.6-6). engraftment was noted in 100% at a median of 10 days (range: 9-14). no further graft-versus-host disease (gvhd) prophylaxis was performed and no acute gvhd (agvhd) was observed. according to treatment plan, 9 patients received escalating dli around day +100, starting with a median dose of 2 × 10 6 /kg (range: 0.5-5) in combination with lenalidomide maintenance in 6 patients. 4 patients experienced agvhd ii-iv after dli. two patients had a severe gvhd (grade iii) which resolved completely after steroid therapy. no nonrelapse mortality after sct and dli was observed. after a median follow up of 43 months (range: 6-49) the median pfs was 16 months (range: 8-24) which translates into a pfs for all patients of 61% at 1 year and 13% at 2 years. median os was 31 months (range: 13-48) and an os of 69% at 2 years and 27% at 3 years was observed. for patients with advanced multiple myeloma relapsing after allografting, a second "autologous" sct with cd34+ selected donor cells without immunosuppression followed by dli is an encouraging treatment option with low toxicity. disclosure of conflict of interest: none. second autologous stem cell transplantation as treatment option for relapsed patients with multiple myeloma: a single center experience (cic 859) p ganeva, y petrov 1 , m mincheff 2 , i tonev 3 , m guenova, l gartcheva 4 , a michova 5 , g arnaudov 6 and g mihaylov 7 1 ya. petrov; 2 m. mincheff; 3 i. tonev; 4 l. garcheva; 5 a. michova; 6 g. arnaudov and 7 g. mihaylov the use of modern therapies such as bortezomib, lenalidomide, thalidomide coupled with upfront high-dose therapy and autologous stem cell transplantation (asct) has resulted in improved survival in patients with newly diagnosed multiple myeloma (mm). the role of second asct as salvage therapy for relapse is unclear because of the availability of new agents to treat progression in multiple myeloma (mm). as the treatment options for management of patients with relapsed mm has become increasingly complex, physicians must consider both disease-and patient-related factors when choosing the appropriate therapeutic approach, with the goal of improving efficacy while minimizing toxicity. we retrospectively reviewed all mm patients who received a second asct as salvage therapy at our center from 2009 to december 2015. for this period we performed 211 transplants for mm patients. twenty five (11.8%) patients received second asct (18 patients were relapsed) and for 7 patients asct was performed as tandem transplant. we analyzed only second asct for relapse. the median time to relapse after first transplant was 18.8 months (range: 8-50 months). all patients received reinduction therapy before the second asct. conditioning was performed with melphalan with two different doses (200 mg/m 2 and 140 mg/m 2 ). the median age at second transplant was 51.8 years (range: 40-67 years), and female/man ratio was 4/14. median interval between first and second asct was 28.3 months (range: 12-60 months). we have no observed early deaths. until now 8 (45%) patients are dead because of progression disease. response rate was assessed three months after asct, nine (50%) patients achieved vgpr, three (16.6%) patients achieved at least a partial response, three (16.6%) had sd and three (16.6%) progressed despite salvage asct.median overall survival (os) was 35.6 months ( relapse ≥ 24months = 47.7; ≤ 24 months = 20). second asct is a feasible and safe option for salvage therapy in mm, especially in bulgaria where the possibility of using novel agents such as carfilzomib, lenalidomide, daratumomab for relapsed patients is limited to clinical trials, because of no reimbursement. the best results were observed in patients whose time to progression was more than 24 months after first asct. advances in treatment of multiple myeloma (mm) has improved overall survival in these patients (pts). a steady increase in the risk of secondary malignancies has been reported over the last decades in mm survivors. estimated incidence of secondary acute myelogenous leukemia or myelodysplastic syndrome (t-mds/aml) after treatment with alkylating agents is 1%-1.5% per year 2-10 years after primary chemotherapy. no specific risk factor has been recognized, but genetic instability, natural history of the disease as well as induction therapy and autologous stem cell transplantation (hct) have been implicated. recently, novel anti-myeloma treatments have been linked with an increase in secondary malignancies, but no solid relationship has been established yet. in a retrospective study, we analyzed the incidence of secondary malignancies (t-aml/mds and solid tumors) in patients suffering from mm who had undergone autologous hct using high-dose melphalan conditioning regimen in our bmt unit. study population consisted of 192 consecutive pts with median age of 55 years (29-70), 56.5% of them being male, who were transplanted during a period of 28 years . type of myeloma was igg/a/d in 56%, 18.8% and 0.5% respectively, while 17.2% was light chain and 7% nonsecretory. the majority of pts presented with k light chain myeloma (62.8%). there was almost equal distribution between iss stages i and ii (45%/38.5%) and only 16.6% were diagnosed with advanced stage myeloma. most pts received two lines of chemotherapy (60%) and all of them more than one. treatment regimens before autologous hct included vad (63pts), bortezomib-based (133pts), dcep (8pts) and rd (29pts) and 34 pts received radiotherapy. chemotherapy administration for mobilization was used in 18 pts (9.3%). conditioning regimen before autologous hct consisted of high-dose melphalan (200 mg/m 2 ) and in case of renal insufficiency 140 mg/m 2 . incidence of a secondary malignancy was 5.7% after a median follow up period of 46 months. t-aml /mds was diagnosed in 9 (4.68%) pts and 2 (1.02%) were diagnosed with breast and lung cancer respectively. pts diagnosed with secondary malignancy were previously exposed in induction therapy to melphalan (6), vad (3), bortezomib (3), high-dose cyclophosphamide as mobilization treatment (4) and radiotherapy (4) . cytogenetic analysis was available in 6 patients diagnosed with t-mds/aml and the majority (4/6) presented complex karyotype. abnormalities mainly observed were deletions and insertions in chromosomes 5,7,17. pts with secondary malignancies had an overall survival of 68 months (26-178), however, after malignancy diagnosis, survival was very poor, four months only . secondary malignancies in pts with multiple myeloma after autologous hct occur with a substantial frequency and have a dismal prognosis. the role of novel treatment agents has to be elucidated. further studies are needed to identify new risk factors and develop better surveillance strategies. [p654] disclosure of conflict of interest: none. survival analysis after allogeneic hematopoietic stem cell transplantation in patients diagnosed with multiple myeloma: a single center experience p patricia hernandez* 1 , r maria calbacho 2 , a laura posada 3 , g fabio augusto ruiz 2 , r anabelle chinea 2 1 hospital universitario nuestra señora de candelaria, santa cruz de tenerife (spain); 2 hospital universitario ramón y cajal, madrid (spain) and 3 hospital universitario cruces, barakaldo (spain) allogeneic hematopoietic stem cell transplantation (allohsct) may provide long term remission cures for patients diagnosed with high-risk multiple myeloma. however, its use is limited since it has a high rate of treatment-related mortality (trm), and because its efficacy compared to autologous hsct is not fully established. we studied 16 patients that underwent allohsct between 2002-2015. population characteristics are in table 1 . all patients were treated at least with one prior therapy lines (1) (2) (3) (4) (5) , all including autohsct (43.75% underwent 2 prior autohsct). 83% had 2 or 3 prior therapy lines. 11 of them received bortezomib as part of treatment regimens. donor characteristics: 2 non-related; 14 hla-identical. gvhd prophylaxis: methotrexate plus a calcineurine-inhibitor: 12 cyclosporine and 4 tacrolimus. median follow-up 15.5 months (1.3-174.5), average was 35.9 months. seven patients died (43.75%); 2 because of progression (12.5%), and 5 (31.25%) due to trm, including infections and immediate complications of transplantation, such as toxicities, icu admission and agvhd: infections: 7 cmv reactivations, 3 invasive fungal and 7 bacterial infections. disease status: 6 patients were in cr prior to allohsct. 3 of them maintained it after. remaining patients died before disease was evaluated. seven patients were in pr prior to transplant, and 4 reached cr after allohsct. one had progressive disease and reached cr after the procedure. two had stable disease and progressed after allo; one of them is in cr after additional therapy lines, and the other one died 4 months after due to it. donor characteristics: hla-identical sibling donors: 87.5% (1 hla-mismatch, passed away 2.7 months after allo due to trm). one of the nonrelated donors, had an hla-mismatch, and died 4 months after allohsct due to trm, the other one is alive after 21 months. gvhd: 10 (62.5%) developed agvhd and 3 of them maintained it chronically. two suffer from cgvhd, plus 3 that initiated it as agvhd. 9 were refractory to steroids. longterm survivors: 3 patients had overcome three years after allohsct. they were among 39 and 50 years old at the time transplant was performed. none of them received bortezomib as part of therapy protocols for the disease. all had 2 therapy lines prior allograft. 2 were submitted to 2 prior autologous hsct. relapse: 4 patients relapsed after allohsct (25%, median time to relapse 6.2 months), being alive 50% at the end of the study. allogeneic hsct is associated with a high incidence of nrm and a low incidence of relapse. rates of acute and chronic gvhd are high. in our cohort, besides that more than 50% are alive until now, they suffer from extensive chronic gvhd and are in need of treatment. long-term survival may be related with patient factors such as young age, but also low tumor burden, or less prior therapy lines; in our group there are no differences in this aspect. studies including high-risk abnormal cytogenetics should help to define which patients are best candidates to allohsct. high-dose melphalan followed by autologous haematopoietic cell transplantation (ahct) remains the standard of care for eligible multiple myeloma (mm) patients. the majority of patients in clinical practice and trials receive a melphalan dose of 200 mg/m 2 (mel200), but a reduced dose of 140 mg/m 2 (mel140) is often used in patients perceived to be unable to tolerate mel200. it remains to be determined whether this considerable dose difference results in different clinical outcomes. we therefore analysed 1978 patients with mm who underwent a first single mel140 or mel200-conditioned ahct between january 2008 and december 2012. all patients were included in the calm study, an analysis of a prospectively defined cohort of patients with data reported retrospectively to the ebmt, covering ahcts for mm and lymphoma. patients in the mel140 group were older than patients in the mel200 group at the time of ahct (median 64 years [range: 28-73] vs 59 years ; p o.001). compared to the mel200 group (n = 1733, 87.6%), fewer patients in the mel140 group (n = 245, 12.4%) were overweight or obese (49.5% vs 63.9%; p = .003). compared to the mel200 group, more patients in the mel140 group had received proteasome inhibitor-containing induction therapy (71.7% vs 57.5%; p = .001), had a karnofsky score of ≤ 80 (38.2% vs 28.1%; p = .002), and were transplanted in less than pr (13.0% vs 7.8%; p = .025). overall survival (os) from the time of ahct was not significantly different between the mel140 and mel200 group (6significantly different between mel140 and mel200 (12 days in both groups for neutrophil recovery; 16 vs 15 days for platelet recovery). however, late neutrophil recovery was noted in a small proportion of patients in the mel200 group. neutrophil recovery 421 days post ahct was not observed in any engrafting patient in the mel140 group, but occurred in 37 (2.2%) engrafting patients in the mel200 group (p = .011). a cox proportional hazards model that included melphalan dose, age, and remission status at ahct showed that melphalan dose had no effect on os, pfs and relapse risk. the findings suggest that mel140 is not inferior to mel200 in younger and older mm patients and may reduce the risk of delayed haematological recovery in some patients. further analyses in relevant subgroups such as patients with high-risk features or renal impairment are required. disclosure of conflict of interest: none. high-dose therapy (hdt) followed by autologous stem cell transplantation (asct) remains the standard of care for patients younger than 65 years of age with multiple myeloma (mm). different agents are being used to control the disease before asct, including the older thalidomide based combination or the newer bortezomib and lenalidomide based combination. the relation between the initial induction regimen and outcomes after asct is not completely clear. to evaluate the effect of different induction regimens on asct outcome, we retropsectively evaluated the outcomes of a low cost older regimen of thalidomide based combination in doublets or triplets with newer novel agents like bortizomib or lenalidomide based combination in a low resources country in transplant-elegible patients with multiple myeloma who underwent autologous stem cell transplantation at king hussein cancer center bmt program we retrospectively reviewed the files of patients diagnosed with mm from january 2008 till december 2015, who received induction treatment followed by hdt and asct and followed up in a single institution. we compared the effects of different induction regimens, disease stage, and remission status before transplantation on over-all survival (os), event free survival (efs) and progression free survival (pfs) using kaplan meier curves. a total of 94 patients were included, 54 (57.4%) of them received thalidomide based induction (group 1) and 40 (42.6%) received bortezomib and lenalidomide based induction (group 2). patients also offered no consolidation nor maintenance therapy. 35 (37.2%) patients were stage i, 34 (36.2%) stage ii and 15 (16%) were stage iii. stage was not documented for 10 (10,6%) of cases. 58 (61.7%) were in complete remission (cr) and 36 (38.3%) were in partial remission (pr). the estimated 5-year os for the whole group was 57.7%. there was no statistically significant difference between both groups in regards to initial iss stage of disease (p = 0.332) or cr status before asct. 32 patients (59.3%) in group 1 achieved complete remission ( cr ) or very good partial response (vgpr), while 25 (62.5%) patient in group 2 achieved cr or vgpr. there was no statistically significant difference between group 1 and group 2 in 5-years os ( 5-year os was 60% vs 57%, p = 0.5007), efs (39.6% vs 52.6%, p = 0.8029) or pfs (45.2% vs 57.8, p = 0.8033). the use of an old, low-cost, thalidomide-based regimen in a low-resources country achieved a favorable transplantation outcomes in patients with multiple myeloma who received hdt and asct. double autologous stem cell transplantation (asct) is a useful treatment for multiple myeloma (mm) patients. we can make the second asct (2asct) without reinduction treatment (tandem regimen) or after a reinduction treatment after first asct (1asct) relapse (salvage regimen). we have conducted a retrospective study over 61 mm patients undergoing a double asct performed in our centre from 1996 to 2016. we have compared the different conditioning regimens used, and if there are any difference between tandem or salvage asct. we do not use maintenance treatment systematically. characteristics of patients and conditioning regimens in table 1 . the overall survivals (os) of our patients are 139 months (m) from treatment start till last control. the most important prognostic factors are the duration of the progression free survival (pfs) after 1asct (hr: 0.96 (0.94-0.99); p = 0.006), and the use of bumel like conditioning regimen at the 1asct or at the 2asct vs another conditioning regimens (hr: 3.43 (1.4-8.39); p = 0.007). today there are 27 patients alive (43%), but only 10 (37%) are free of mm now. the 25 patients who were treated with tandem have a little better os than salvage patients (166m vs 103 m; p = 0.55. not significative). patients at tandem group who received different conditioning regimen at the 1asct and at the 2asct live more time than patients treated only with melphalan 200 (mel200) at both asct. at salvage group the duration of pfs after 1asct is better than the pfs after 2asct (28 m vs 13 m). the use of the same conditioning regimen at the both asct has worse results than if we use different treatment. patients who were treated with s474 bumel at the 1asct or 2asct have better os than patients treated with cbv or mel200. patients who not responded to reinduction treatment before 2asct have worst pfs after 2asct (rc:29 m, response; 19m and not response; only 10 m). attention is drawn to the fact that patients who received bumel at 1asct have large os, but they are very few (8) patients. only one patient has died during the 2asct, and was a patient of salvage group treated with bumel. double autologous transplantation continues to be a useful treatment despite the new mm treatments, and allows to prolonged the os. tandem asct probably is a useful treatment in high risk mm patients. salvage treatment is most useful in patients with a large pfs after 1asct, and good response to reinduction treatment. although mel200 continue to be the standard conditioning regimen for asct in mm patients, we have observed that patients treated with different conditioning regimen at 1asct and 2asct have better prognostic, and bumel has the best results in our serie. disclosure of conflict of interest: none. allogeneic haematopoietic stem cell transplantation (allo-hsct) is an effective treatment for myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). the prognosis of elderly patients with mds and aml after chemotherapy is poor. allo-hsct is feasible in these patients; however the management of elderly patients with mds and aml for allo-hsct is difficult. we performed a retrospective survey of allo-hsct for elderly patients with mds and aml in our institution. we retrospectively analyzed the records of elderly patients ≥ 60 years with mds and aml who underwent allo-hsct in our hospital between january 2011 and december 2015. in this study, we assessed the ipss-r (revised international prognosis scoring system) cytogenetic score and the ipss-r score against the outcome of elderly mds and aml patients who treated with allo-hsct. fifty-one elderly patients with mds and aml were treated with allo-hsct in our institution, 47 patients with mds (29 with mds overt aml) and 4 with de novo aml. ages ranged from 60 to 71 years (median 64), 18 patients were female and 33 were male. there was a history of malignant disease in 14 patients. according to the ipss-r cytogenetic scores of mds patients, 10 patients fell in the good risk group, 7 were in the intermediate risk group, 7 were in the poor risk group, and 23 were in the very poor risk group. regarding the ipss-r score, 1 patient fell in the low risk group, 5 in the intermediate risk group, 6 in the high risk group, and 35 in the very high risk group. sixteen patients were in 1st complete remission (cr), 1 patient was in 2nd cr, 9 patients were in partial remission, and 25 patients were not in remission (nr) upon administration of allo-hsct. all patients received a reduced intensity conditioning regimen. 45 patients [p658] were treated with fludarabine (flu), melphalan and low dose tbi-containing regimens; 5 patients were treated with flu, intravenous busulfan and low dose tbi; and one patient was treated with flu, cyclophosphamide and low dose tli. graftversus-host disease (gvhd) prophylaxis consisted of tacrolimus plus methotrexate in 46 patients, and tacrolimus, methotrexate and mycophenolate mofetil in 5 patients. thirty-four patients received anti-thymocyte globulin (atg). the donor source was sibling bone marrow (bm) in 1 patient, sibling peripheral blood stem cell in 7, unrelated bm in 36 and unrelated cord blood in 7. relapse-free survival (rfs) and overall survival (os) were 40.7% (95% confidence interval (ci): 27.2-53.8%) and 49.7% (95% ci: 35.1-62.7%) at 1 year, 31.4% (95% ci: 18.2-45.5%) and 33.6% (95% ci: 19.2-48.5%) at 3 years, respectively (figure 1.) . in this study, 4 patients died before engraftment. non-relapse mortality (nrm) was 19.6% at day 100. twenty-five patients developed chronic gvhd (3 patients limited and 22 extensive). the causes of death were disease progression (10 patients), treatment-related mortality (13 patients), infection (4 patients) and other causes (3 patients). we suggest that many elderly allo-hsct patients with mds and aml were in the very poor risk group when the ipss-r cytogenetics score was assigned, in the very high risk group when the ipss-r score was assigned and nr upon administration of allo-hsct. rfs and os were 31.4% and 33.6% at 3 years, respectively. there is a need for novel treatment strategies to manage elderly mds and aml patients for allo-hsct. [p659] disclosure of conflict of interest: none. counting bone marrow blasts as a percentage of nonerythroid cells provides superior risk stratification for mds patients with erythroid predominance a sun 1 , y yu 1 , t zhang 1 , q wang 1 , d liu 1 and s chen 1 1 the first affiliated hospital of soochow university, jiangsu institute of hematology, suzhou, china patients with erythroid predominance (≥50% erythroblasts, mds-erythroid) compose a significant proportion of patients with mds. the erythroid/myeloid subtype was divided from the aml category into mds-erythroid by the 2016 who classification of myeloid neoplasms. at that time, there was no consensus on a more appropriate way of enumerating bone marrow (bm) blasts from tncs or necs in mds-erythroid patients. to clarify these questions, 1283 mds patients were retrospectively analyzed in our center. mds-erythoid was observed in 27.0% of patients (346/1283), and these patients had similar clinico-pathological features and overall survival, with 937 cases of mds with o50% encs. by calculating the percentage of bm blasts from necs, 73 of 200 patients (36.5%) with mds-erythroid who were diagnosed within who subtypes without excess blasts (eb) were moved into higherrisk categories and showed shorter os than those who remained in the initial categories (p = 0.041). recalculating the international prognostic scoring system-revised (ipss-r) by enumerating blasts from necs, 40 of 168 (23.8%) mdserythroid patients with relatively lower risk were re-classified as higher-risk and had significantly poorer survival than those who remained in the lower-risk category (p = 0.030). this was especially true for the intermediate risk group that was stratified by ipss-r (unchanged patients vs. shifted patients, p = 0.007). however, the impact of enumerating bm blasts from necs on classification and prognostication was not evident in all mds patients. in conclusion, our results suggested that enumerating the percentage of bm blasts from necs significantly improved the prognostic assessment of mds-erythroid, especially for patients within the intermediate risk group stratified by ipss-r. disclosure of conflict of interest: none. myelodisplastic syndrome (mds) is a group of clonal and heterogeneous diseases, characterized by ineffective hematopoesis. the incidence of mds is about 5% of all blood disorders in children, approximately 40% of them develops acute leukemia. allogeneic hematopoietic stem cell transplantation (allo-hsct) is effective curative treatment of mds in children, but depends on disease status, type of clonal chromosomal abnormalities presented at the time of allo-hsct and graft quality. the aim of this study: to analyze the influence of graft quality on the outcome of childhood mds after allo-hsct. allo-hsct were performed in 58 patients (pts) p662 hypomethylating agents vs. allogeneic sct in elderly patients with advanced myelodysplastic syndromes: a single center study j cermak, a vitek, m markova-šťastná, j soukupova-maaloufova and p cetkovsky institute of hematology and blood transfusion, prague, czech republic a group of 26 patients older than 50 years of age with myelodysplastic syndrome (mds) raeb ii or with acute myeloid leukemia with multilineage dysplasia with less than 30% of bone marrow blasts (mds raeb-t according to the fab classification) was treated with hypomethylating agents (hma) and the results were compared to those obtained in an age and diagnosis matched group of 16 patients who underwent allogeneic stem cell transplantation (sct). in the hma group, 22 patients received azacytidine (vidaza) in the dose of 75 mg/ m 2 × 7 every 28 days and 4 patients were treated with decitabine (dacogen) in the dose of 20 mg/m 2 × 5 every 28 days. median number of cycles administered was 10.8 (range: 3-31). in the transplanted group, 8 patients were transplanted upfront and 10 patients were pretretated with combination chemotherapy, 8 patients received myeloablative conditioning and 8 patients were transplanted after reduced conditioning regimen. a hematologic response to hma (cr,pr, hematologic improvement) was observed in 15 patients (61.5%), cr was achieved in 8 patients (31.8%). in sct group, engraftment was achieved in 14 out of 16 patients, 8 patients died after sct ( 5 on complications related to sct, 3 patients relapsed). no difference in 1 year survival was observed between both the groups (65.6% for hma vs. 62.5% for sct), however, median overall survival (os) was 19.0 months in hma treated group compared to 47.6 months in sct group (p = 0.03). in a recent analysis performed at 48 months after starting the treatment, 2 patients treated with hma (7.7%) and 6 transplanted patients (37.5%) were alive, 16 patients in hma group and 3 patients in sct group relapsed. estimated 5 years survival was 31.3% in sct group and only 3.8% in hma group (p = 0.001). no significant differences in results and adverse effects of treatment were observed between patients aged 51-60 years and those older than 60 years in both hma and sct groups. our results confirm previous observations showing that despite a promising effect of hma resulting in hematologic response in more than 50% of elderly patients with advanced mds, allogeneic sct still represents the only potentially curative treatment connected with long-term survival in a significant number of patients even in this mds patients subgroup. disclosure of conflict of interest: none. immunophenotypic assessment of erythroid dysplasia by a simplified cocktail in myelodysplastic syndromes in taiwan c-c li 1 , p-f weng 1 , c-t lin 1 , j-l tang hypomethylating agent (hma) is commonly used as a bridge therapy to prevent leukemic transformation prior to selection of a donor for allogeneic stem cell transplantation (sct) in patients with myelodysplastic syndrome (mds), and showed low toxicity. although its roles are known, the underlying genetics and clonal dynamics upon hma treatment has not been systematically examined using serial samples, especially in allogeneic stem cell transplantation (sct) setting. in this study, we performed targeted serial sequencing on 66 bone marrow samples from 22 mds patients treated with hma for bridging of allogeneic sct. to perform targeted deep sequencing, bm mononuclear cells before hma treatment and, and fractionated t-cell samples (cd3+ fraction) as controls were taken before hma treatment. analysis of genetic mutations were performed using targeted resequencing by illumina hiseq 2000 (sureselect custom probe set targeting [p664] entire exon regions of a myeloid panel consisting of 84 genes). all 22 patients received hma (decitabine: 15, azacitidine: 7), and the median number of cycles was four (range: 2-12). the overall response rate for hma pre-treatment was 55%: there were four cases of complete remission (cr) (18%), six cases of marrow cr (27%), and two cases of hematologic improvement (9%). targeted sequencing revealed 37 mutations in 16 patients (16/22, 73%) with median of 2 mutations per patient (range: 2-5). mutated genes were then grouped into 8 biological pathways, defined in the cancer genome atlas (tcga) aml study. the most frequent biological pathway at diagnosis was dna methylation (32%), followed by activated signaling (27%), chromatin modifiers (18%), tumor suppressors (18%), spliceosome (14%), cohesin complex (9%), npm1 (4%), and myeloid transcription factors (tfs) (4%). when assessing the difference in pattern of variant allele frequency (vaf), we found the significant reduction of vafs in four (25%) patients after hma. with a median follow-up of 63.4 months, 5-year overall survival (os) were 69.6% (95% ci, 49.0-90.2). there was no significant difference in os according to the presence of mutations in each biological mutational pathway (all, p40.05). to identify prognostic value of mutational dynamics, we subclassified the change of variant allele frequencies (vafs) after median fourth cycles of hma [no mutated or reduction of vafs (11 patients) vs. stable or increased (11 patients)]. however, there was no significant association between the dynamic of mutation and os (p = 0.374). these data show that hma using as bridge therapy for allogeneic sct in mds patients is insufficient to achieve the sufficient molecular responses and, mutational pathway and dynamics may not prognostic in this clinical setting. to clearly demonstrate the role of hma pre-treatment in mds, systematic assessment on a larger cohort is necessary. disclosure of conflict of interest: none. any role of high-dose chemotherapy in mediastinal nonseminoma germ cell tumors? p pedrazzoli, s secondino, a necchi 1 , f lanza 2 and g rosti 1 istituto nazionale tumori, milano and 2 ospedale santa maria delle croci, ravenna among germ cell tumors, primary mediastinal nonseminoma germ-cell tumors (pmnsgcts) have the poorest outcome with 5-year overall survival ranging from 40 to 45%. indeed, the presence of mediastinal location defines per se a "poor prognosis" category according to the igcccg classification. this clinically and biologically distinct disease entity is associated with lower complete response rates to chemotherapy (ct), high rates of relapse and disappointing results from salvage ct. current standard first line treatment for patients with mediastinal primary location is still four cycles of peb, as for all igcccg poor-prognosis patients. we have reviewed available data present in the literature, including recommendations and expert opinions, on the use of high-dose chemotherapy (hdc) with autologous stem cell support in pmnsgcts. the use of hdc as both early intensification (that is, first-line setting) and at disease recurrence (salvage setting) have been reported in small cohorts of patients. according to the largest retrospective comparison, it has been suggested that hdc, given up-front, may produce a 15% to 20% absolute improvement in survival compared with standard dose ct. studies of the ebmt suggest that responsive disease after induction therapy may have a better outcome. mediastinal primary had salvage rates by hdct of less than 15% based on an international multicenter analysis and an ebmt study. the use of hdct in pmnsgcts warrants further investigation, preferably with the use of modern hdct strategies (that is, multiple carboplatin and etoposide courses). while hdc cannot be routinely recommended in pmnsgcts, selected patients with chemosensitive disease may benefit from early intensification. a retrospective analysis evaluating the large ebmt database is ongoing; results will be presented at the meeting. disclosure of conflict of interest: none. high dose therapy and autologous stem cell transplantation in gynaecological malignancies: a monocentric retrospective study m nderlita 1 , i vergote 1 and d dierickx 2 1 university hospitals leuven, department of gynaecology; university hospitals leuven and 2 department of hematology high-dose chemotherapy (hdt) followed by autologous stem cell transplantation (asct) has been established as a treatment option in many relapsed hematologic malignancies. however, in spite of many small trials, there still is no proven role for this treatment in solid tumors including most gynaecological epithelial carcinomas. however, in some recurrent non-epithelial ovarian cancers, such as sex cord stromal tumors, germ cell tumors, neuroendocrine gynaecological tumors and gestational trophoblastic disease, some studies suggest a possible role for hdt followed by asct. we performed a monocentric retrospective descriptive analysis of all patients diagnosed with gynaecological malignancies and treated with hdct followed by asct in our center. clinical, laboratory, pathological and imaging data were collected and analysed, together with information on treatment and outcome. eleven patients were included in this analysis, with a median age of 29 years (range: 14-56) at time of diagnosis. eight patients suffered from ovarian neoplasia. at time of diagnoses 6 patients showed metastatic disease. first line therapy consisted of surgery (n = 4), chemotherapy (n = 2) or a combination of both (n = 5). median time to progression after first line therapy was 39.8 months (range: 0-192) with a median time between primary diagnosis and start hdt of 54.7 months (range: 4-306). three patients underwent single ast, whereas the other 8 patients had a tandem ast, with a median time of 2 months between first and second hdt (range: 1-4). treatment related toxicity was manageable, although there was 1 treatment-related death. at last follow up 5 patients (45%) were still alive with a median follow up of 3.9 years (range: 0.25-15.1) after last asct for all patients. of the 6 deceased patients 5 died with progressive disease. although the number of patients is very small, this retrospective study shows that hdt and asct is feasible in heavily pretreated patients with relapsed/refractory gynecological malignancies, although further studies are mandatory for optimal selection of patients, histological subtype and timing of hdt during the disease course. disclosure of conflict of interest: none. the human endogenous retroviruses (hervs) are remnants of ancient exogenous retroviral infections of the humans: they represent about 8% of the human genome 1 . the basic genes of hervs are group-specific antigen (gag), polymerase (pol) and envelope (env); there are also two regulatory regions, long terminal repeat (ltr), located at 5' and 3' ends. several reports have shown that hervs may play a role in the development of autoimmune diseases, such as multiple sclerosis 2 . additionally the existence of a strong relationship between hervs expression and cancer, based on the mrna expression profile of hervs in normal and cancer tissues has been suggested 2 . the increased level of expression level of herv-h in colorectal cancer (crc), a major cause of cancer death worldwide has been already shown. the aim of the study was to analyse the expressions of env genes of herv-r, herv-h, herv-k and herv-p in the peripheral blood mononuclear cells (pbmcs), in the tumor and in the adjacent normal tissues of 20 colorectal cancer patients. a group of control composed by pbmcs from 46 healthy subjects was also included. rna was isolated from the biological samples and a reverse transcription assay was conducted. quantitative real time pcr was performed to evaluate the expression of the hervs env gene. all the env genes were related to the expression of an housekeeping gene, gapdh. the quantification was carried out using comparative ct method and the difference between the levels of env gene expression in pbmcs, cancer and adjacent normal tissue was given by fold difference. fold difference values were relative to a calibrator: first the pbmcs of patients and then pbmcs of control healthy group. δct values were analysed using the paired sample t-test, followed by a bonferroni correction. higher levels of expression of herv-h, herv-k and in particular herv-p were found in tumor tissues, as compared to pbmcs and to adjacent normal tissues of patients, with an increase of 24-, 10-and 78folds, respectively. the δct distribution of herv-h, herv-k and herv-p in cancer tissues were statistically significant (po0.05) ( table 1 ). the expression of herv-h, herv-k and herv-p env gene resulted increased in the colorectal tumor tissues also when compared with the pbmcs of the healthy controls (5-, 15-and 26-folds, respectively). the δct distribution of herv-h, herv-k and herv-p in tumor tissues were statistically significant ( ρ < 0.05). no difference of expression was observed between pbmcs of healthy controls, pbmcs and normal adjacent tissues of patients (figure 1 ). hervs env gene expression cannot be used as a diagnostic biomarker, but it is conceivable that hervs are directly involved in the pathogenic process of cell transformation and, if the protein expression will be demonstrated, the protein of hervs env gene could be the target for new immunotherapy strategies against colorectal cancer. [p668] disclosure of conflict of interest: none. a biosimiliar g-csf filgrastim is as effective as a reference drug however itis not as cost effectiveas it supposed to be and by the way no impact on the health care system m kurt yüksel, g pekcan, u sahin, s bozdag, s toprak, p topcuoglu, o arslan, g gurman and m beksac ankara university school of medicine biosimiliars are up to 1000 times the size of small molecule generic drugs and far more structurally complex. additionally biosimiliars are manufactured in living cell lines using processes that cannot be exactly replicated from one manufacturer to the next. a biosimiliar cannot be identical to its reference biologic drug. with 67billion dollars in global sales of biologic medicines anticipated to go off patent by 2020.this lead to fast production of biosimiliar drugs. besides, it is expected that biosimiliar drugs will be more cost effective than the reference drugs and will have a meaningful impact on health care systems around the world. aim: to compare biosimiliarfilgrastim (leucostim) with two reference g-csf filgrastim (neupogen) and lenograstim(granocyte) in the context of safety, efficacy and cost effectivity. records of patients with multiplmyeloma(mm) whom underwent autologous stem cell transplantation(asct) and received g-csf sc5mikrogram/kg/day from +day 5 until engraftment were [p668] retrospectively evaluated 60 mm patients were treated with high dose melphalan and asct at the ankara university school of medicine bone marrow transplantation unit between 2013 and 2016. the median age was 59 (38-75 years) with 55% male. patients were divided into three groups (n = 20) whom received reference filgrastim (neupogen), lenograstim (granocyte) and biosimiliarfilgrastim (leucostim): groups a, b and c respectively. the total cost of each g-csf in dollars was calculated by one package of g-csf multiplied by total used days . chi-square, mann-whitney u and kruskal-wallis tests were used for analyses of variance. the percentage of patients who received melphalan 200 mg/m 2 were%80, 85, 80in groups a, b, c respectively (p = 0.9).there was no statistically significant difference between the engraftment day of neutrophil 500 and 1000; platelet 20 000 and 50 000 in the groups. (p = 0.07, p = 0.55, p = 0.33, p = 0.81 respectively) themedian numbers of g-csf administered days were 7(5-18), 8 (5-12), 7 (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) in groups a, b, c, respectively .eventhough there was no statistical difference between the numbers of days( p = 0.23), the total cost in dollars was statistically difference between a vs b and c vs b (both p o0.0001) and there was no statistical difference between a vs c (p = 0.89), total cost in dollars as follows: 155$ (112-288$), 416$(260-624$) and 166$(81-250$) for the group a, b and c respectively. our results demonstrate that biosimiliar gcsf leucostim is highly similar to existing licenced biologic products in turkey with no clinically meaningful difference interms of safety and efficacy. on the other hand it as a biosimiliardoes not have a meaningful impact on the cost savings to the health care system as expected when compared with reference filgrastim. disclosure of conflict of interest: none. in this study, we investigated the roles of prx ii, one of 3 critical peroxidases besides catalase and gpxs, in cml primary cells at diagnosis and remission while patients were treated with sti (signal transduction inhibitor) and tested the same roles in imatinib(im) sensitive ph+ cell lines and resistant cell lines as well. newly diagnosed cml cells, im resistant k562 cells and parental k562 cells were treated stis and analyzed western blot assay to detect bcr-abl, phosphorylated bcr-abl and prx2 protein expression level. we added n-acetylcysteine (0-5mm, 6hr) to k562 cells to show antioxidant effect of imatinib and analyzed dcf-da detection for intracellular ros level and western blot for prx2 protein level. mtt assay was performed to detect cell death by nac time-dependent treatment of 5mm nac(0, 24, 40, 48hr). imatinib resistant k562 cells were established by treatment of gradual increment of imatinib. we also repeatedly investigated the effects of im therapy using prxii overexpressed k562 cells by transfection. at diagnosis of cml, ros level was elevated and prx ii was either absent or significantly suppressed. as ph chromosomes were decreased with stis, suppressed or absent prxs levels were restored to the level of normal individuals. these findings were also inversely correlated with the level of ph chromosomes in the cases of disease progression and re-remission with further treatment. when sti were treated in ph positive cell line, we found deceased cell survival and ros level by mtt assay and dcf-da methods respectively, but elevated prx ii by western blot. by the treatment of nac into ph+ cell lines, the level of dcf-da was decreased and mtt level was down, but prx ii level was elevated. interestingly, the level of bcr-abl oncogene were decreased in prx ii tranfected cells. meanwhile, we observed that prx ii restoration was mild or weak in imatinib resistant k-562, which we established in our lab. the importance of the roles of ros and its prx ii, antioxydant enzymes in cml is further established by our work. our finding may contribute to find a new pathway on which tkis are working besides the mechanisms of atp binding competitively, blocking the binding of abl-bcr kinase to the substrate resulting apoptosis of ph+ cells. furthermore, our finding may be useful to overcome the stis resistant cml in the clinics in the future. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation for the treatment of mucopolysaccharidosis f shunqiao, s xiaodong and l junhui department of hematology, capital institute of pediatrics, china mucopolysaccharidosis (mps) is a lysosomal storage disorder caused by deficient activity of the iduronate-sulfatase.this leads to accumulation of glycosaminoglycans(gags) in the lysosomes of various cells,which causes progressive multisystem involvement with ensuing death.the aim of this study was to exploit the effect of treatment with allogenic hematopoietic stem cell transplantation and administration of high doses of cyclophosphamide early after haplobmt in these cases. we retrospectively reviewed data from 3 mps patients (2 cases mps ii, and 1 case mps i). the two mps ii patients were 44-month-old and 35-month-old boy and the mps i patient is a 84-month-old girl at the time of transplantation. the reduced-intensity of bu+flu conditioning regimen in allo-hsct for these patients was as follows: busulfan 4 mg/kg at 5-2 days before transplantion,fludarabine 40 mg/m 2 at 6-3 days before transplantion.graft-versus-host disease(gvhd) prophylaxis:rabbit antithymocyte globulin 2.5 mg/kg daily at 5-3 days before transplantation,shortcourse methotrexate,posttransplantation high-dose cyclophosphamide on days +3 and +4 was followed by mycophenolate mofetil and cyclosporine.the donors all were their hlahaploidentical father. these three patients' neutrophil engraftment occurred on +14d, +12d and +15d after transplantation respectively, platelet engraftment occurred on day +14d, +10d and +15d after transplantation respectively.complete donor type engraftment was confirmed by short tandem repeat-polymerase chain reaction(str-pcr) on day 14 after transplantation. no regimen-related toxicity occurred,gvhd and graft failure were not observed. 1 month after transplantation, the activity of the iduronate-sulfatase was increased to normal. the motion of metacarpophalangeal joints ameliorated, regression of hepatosplenomegaly, the neurocognitive function improved. allogeneic hematopoietic stem cell transplantation is an effective measure to treat patient with mps at least mps ii and mps i. the reduced-intensity conditioning regimen was helpful to decrease the regimen-related toxicity. posttransplant cyclophosphamide approach successfully used and reduced the incidence of gvhd. this study aimed to evaluate the feasibility of alternative donor hematopoietic stem cell transplantation (hsct) using busulfan, fludarabine, and thymoglobulin conditioning for patients with chronic granulomatous disease (cgd) who lack an hla-matched familial donor. medical records of 11 consecutive patients who received alternative donor hsct between may 2010 and may 2016 were reviewed, and the transplant-related outcome measures were analyzed retrospectively. the donor source was unrelated peripheral blood (pb) in 4, unrelated cord blood (cb) in 4, and haploidentical father in 3 patients. only 2 transplants (8/8 allele-matched unrelated pb) were hla-matched according to current standards relevant to the donor type. the conditioning regimen was uniform; fludarabine 40 mg/m 2 on days -8 to -4, busulfan 3.2 mg/kg/d (or 120 mg/m 2 /d) on days -6 to -3, and thymoglobulin 2.5 mg/kg/d on days -3 to -1 (or on days -8 to -6 in cb recipients). all but one patient were male and their median age at transplantation was 6.5 y (range: 1.1-26.3). one patient who received a cord blood graft suffered from primary engraftment failure, while the other 10 patients were successfully engrafted with their chimerism levels ranging from 66% to 100% (median 100%) at 1 month post-transplant. the median days to neutrophil and platelet engraftment were 12.5 (range: 11-22) and 27 (range: 11-47), respectively. among the 10 patients engrafted, one patient experienced secondary graft failure which was rescued by a second transplantation. the remaining one patient who failed to engraft was also rescued with a haploidentical graft from his mother. eight patients (73%) developed cmv antigenemia, and one of those patients developed cmv hepatitis. three patients developed grade 3 acute gvhd which were manageable. one patient who developed grade 4 hepatic gvhd eventually died. two patients developed extensive chronic gvhd, but became free of immunosuppressants after a complete resolution in one and with remaining stable mild joint contractures in the other. including 2 patients who were rescued by additional transplantation, 10 patients are alive with their latest chimerism levels ranging from 86.8% to 100% (median 100%). the estimated 5-y overall survival rate was 85.7% with a median follow-up of 49 months (range: 6-72). even though the majority of our cohort underwent a mismatched transplantation, the survival rate was excellent. while conditioning with busulfan, fludarabine, and thymoglobulin seems feasible for alternative donor hsct in patients with cgd, special attention needs to be payed on cmv infection and severe gvhd which might offset the high survival rate. disclosure of conflict of interest: none. diarrhea is a common infectious complication in patients who had hematopoetic stem cell transplantation 2 so, we aimed to detect entamoeba histolytica ratio before engraftment, amoung 375 patients who had diarrhea after periferic hematopoetic stem cell transplantation (phsct) in our clinic. allogenic phsct patients had a median age of 29 (range: 15-63) and autolog phsct patients had a median age of 54 (range: 18-74). diarrhea is described as an abnormal increase in the frequency (3 or more times per day), volume or liquidity of stools. we based upon this description in this study. we made stool examination in the first day of diarrhea. as stool examination, we used direct microscopic evaluation and adhesin antigen test specific for e.hystolytica with enzyme linked immunosorbent assay (elisa), e. histolytica ii, techlab, blacksburg, usa). we accepted e.hystolytica positivity as detecting cyst or/and trophosoit in stool and antigen test positivity at the same time. in our study, 185 of 375 patients had diarrhea in the first 28 days of phsct. diarrhea was found in 139 of 242 in autologous phsct patients (%57), 21 of 63 patients in allogenic phsct with non-myeloablative conditioning regiment (%33) and 25 of 70 patients in allo phsct with myeloablative conditioning regiment (%36). diarrhea occured at +8th day of transplantation and the median duration of diarrhea was 3 days. e. histolytica positivity was found 46 of 185 patients (25%) who underwent phsct within first 28 days of transplantation. infection is an important mortality and morbidity factor for patients who had hematopoetic stem cell transplantation, when especially before engrafment (between 0-30 days). 1 autologous phsct patients were elderly, with poor self-care and low socioeconomic status individuals. e. hystolytica is a frequent pathogen in posttransplant diarrhea at endemic regions. prophylactic metronidasole treatment should be used routinely for autologous phsct as in allogenic phsct. 3 patients and companions sholud be tested for e.hystolytica before autologus/allogenic phsct in endemic regions. prophylactic treatment for amebiasis and scanning patient/companions could be a part of solution for post phsct diarrhea. despite the emergence of disease modifying therapies (dmts) for multiple sclerosis (ms) a cohort of patients with aggressive disease have ongoing progression/relapse, associated with progressive disability. autologous haematopoietic stem cell transplantation (ahsct) has been used worldwide for aggressive ms with inflammatory changes on mri. we update on a uk single centre experience of ahsct in ms. a retrospective audit of ahsct performed for ms from 2012 to 2016 at 1 uk centre (king's college hospital) was undertaken. patients were selected for transplantation based on persistent clinical relapses (relapsing-remitting ms) or secondary progressive neurological disability with mri lesion activity despite use of at least 1 dmt. primary progressive patients were also eligible if new/active mri lesions were demonstrable. followup included clinical evaluation, edss assessment and mri scanning. we report our preliminary findings. as of november 2016, 30 patients (16 female, 14 male, 18 rrms, 10 spms, 2 ppms) had received ahsct. mean age at transplant was 40.6 years (range: 22-57). the mean baseline edss was 5.3 (range: 2.5-8.0). 29 patients underwent cyclophosphamide/atg conditioning, while 1 received beam/atg. whilst conditioning and stem cell infusion were well tolerated there was a high rate of infections, with 23/30 patients developing a culture confirmed infection. reactivation of ebv and cmv were observed in a number of patients (21 and 8, respectively) while a number of delayed herpes zoster infections were also seen (4 cases of shingles and 2 of disseminated varicella infection in patients who had previously experienced it in childhood). median follow-up was for 361 days (63-1479). of patients with a formal 6 month assessment (n = 16), 4 had a stable edss, 6 had an improved score (median improvement 0.5, range: 0.5-2.5) and 6 had a deterioration in their score (median 0.5, range: 0.5-1.0). at 12 months (n = 11), 1 had a stable edss, 4 had an improved score (median 0.75, range: 0.5-1) and 6 had a deterioration in score (median 0.5, range: 0.5-1.5). at 24 months, two patients assessed both had improvements in edss scores (median 1, range: 0.5-1.5). for patients who underwent mri at 6 month follow-up (n = 14), 10 had a stable lesion load, 2 demonstrated improvement in lesions and 1 had a new lesion (the remaining mri was difficult to read due to a high baseline lesion load in this patient). 4 patients had mri's at 12 months; 3 were stable and 1 demonstrated a reduction in lesion load. to date, no patients have developed secondary malignancies or autoimmune diseases. of patients with followup data, 4/18 rrms patients experienced suspected clinical relapses following hsct-only one had a new lesion on mri (with no gadolinium enhancement). 3 of the 4 received steroids to treat these relapses (it is unclear if the remaining patient received treatment). 1 patient tried a new disease modifying therapy (1 dose of rituximab) following hsct. ahsct in this cohort was feasible with universal mobilisation and harvest. whilst conditioning and stem cell infusion were well tolerated, there was a high rate of infectious complications in the neutropenic phase. however, the transplant related mortality was 0% despite significant levels of disability amongst this patient cohort. ahsct remains a treatment option to be further investigated in this difficult cohort of patients. disclosure of conflict of interest: none. peripheral blood (pb) stem cells (scs) mobilized with g-csf are the first-choice source for allogeneic transplantation. we carried out a prospective study on healthy donors (hds), to identify donor characteristics that could influence the effectiveness of mobilization with special focus on the value of the basal cd34+ cell count. sibling hds were analyzed in a prospective study. we tested somatic variables (sex, age, weight, height, volemia) and, basal blood counts (white blood cell, peripheral blood mononuclear cell, platelets, hematocrit, hemoglobin, cd34+ cell). hds received g-csf subcutaneously at a dose of 10 μg/kg day. two different determinations of cd34+ cells were done in each donor: baseline (before g-csf administration) and in pb on the morning of the fifth day (after g-csf administration). 128 consecutive hds (65 males) with a median age of 43 years were enrolled. the mean value of cd34+on day 5 was 90.8 cells/μl, while the median value was 75.5 cells/μl. we performed two multivariate analyses either by using median regression (to predict the median value of cd34+on day 5) according to the values of cd34+ at baseline, the first adjusted by gender, age and blood volume and the second by gender, age and bmi. results of both models indicate that from basal cd34+ values o = 1 to values ranging between 3 and 4 cells/μl, predicted median values of cd34+ on day 5 significantly increase, from 54.6 to 92.8 cells/μl for model adjusted by blood volume, and from 49.9 to 92 cells/μl for model adjusted by bmi. baseline, pb cd34+ cell count correlated with the effectiveness of allogeneic pbscs mobilization and could be useful to plan the collection. disclosure of conflict of interest: none. comparison of efficacy between chemotherapy plus granulocyte colony stimulating factor (g-csf) and chemotherapy plus g-csf and granulocyte-macrophage colony stimulating factor (gm-csf) for mobilization of peripheral blood stem cells (pbsc) and hematological recovery post-transplantation in patients with multiple myeloma (mm). a retrospective study of autologous peripheral blood stem cell (apbsc) mobilization data of 56 mm patients who treated with chemotherapy plus g-csf or chemotherapy plus g-csf and gm-csf from may 2008 to july 2016. the mobilization efficacy and hematopoietic recovery were analyzed. a total of 65 stem cell mobilizations were performed in 56 mm patients. in the univariate analysis, successful collection rate of single harvest in female and in patients at iss stage iii, r-iss ii/iii and chemotherapy plus g-csf was lower(po0.05). however, age(≦60 yrs vs 460 yrs), subtype, d-s staging (i+ii vs iii), cycles of chemotherapy before mobilization (≦6 cycles vs 46 cycles), disease phase before mobilization (pr vs cr) and interval diagnosismobilization (≦18 months vs 418 months) were not correlated with the cd34+ cell collection and successful mobilization rate(p＞0.05). in the multivariate model, rate of successful mobilization in patients who received chemotherapy plus g-csf+gm-csf mobilization regimen was high (or = 12.009, 95%ci 1.961-73.537). the effect of mobilization regimen remained significantly (p = 0.007). all patients successfully underwent hematopoietic reconstruction without transplantation-related mortality. chemotherapy plus g-csf +gm-csf mobilization regimen can significantly increase the effect of apbsc mobilization and ensure the reconstruction of hematopoietic function after transplantation. this mobilization regimen is a safe and effective method of mobilizing apbsc. disclosure of conflict of interest: none. clinical efficacy of bk virus specific t-cells in treatment of severe refractory hemorrhagic cystitis after hla haploidentical transplantation om pello 1 , a bradshaw 2 , a innes 2 , s-a finn 2 , s uddin, e bray 2 , e olavarria, jf apperley and j pavlu 1 centre for haematology, imperial college at hammersmith hospital, london, uk and 2 department of clinical haematology, hammersmith hospital, imperial college healthcare nhs trust, london, united kingdom hemorrhagic cystitis caused by bk virus (bkv) is a significant complication of allogeneic hematopoietic cell transplantation (hct). it is particularly common in the setting of hla haploidentical transplantation and can be challenging to manage. here we present a post haploidentical hct patient who developed severe bkv haemorrhagic cystitis resistant to standard therapy and who responded to adoptive transfer of donor t cells enriched with anti-bkv specific cells. a 40 year old man underwent hct for acute myeloid leukaemia with inversion of chromosome 3 and monosomy of chromosome 7 while in first complete remission. as he had no related or unrelated hla identical donor, cells from his hla haploidentical sister were used. on day +32 of this procedure he developed haemorrhagic cystitis. supportive treatment was initiated and cystoscopy showed diffuse bleeding from his urinary bladder with blood clots. urine pcr for bkv showed 5.2 billion copies/ml. despite bladder irrigation, local therapy to s483 bladder mucosa and intravenous hydration, he failed to improve, so treatment with weekly intravenous cidofovir was initiated on day +38. his symptoms improved, but on day +72 he again deteriorated on weekly infusions of cidofovir. his immunosuppression was slowly tapered off without any graft versus host disease (gvhd) but without any significant effect on his hemorrhagic cystitis. he underwent bladder diathermy, was treated with intravesicular hyaluronate and with intravenous cidofovir, but continued to have frank haematuria with blood clots and significant lower abdominal pain. although there was no evidence of obstruction his renal function deteriorated on cidofovir therapy. hence we elected to trial adoptive anti bkv therapy. a leukoapheretic lymphocyte collection was used to prepare an anti-bkv t cell enriched product using the clinimacs prodigy and the cytokine capture system from miltenyi biotec. the eluted product contained 50% and 5% of cd4+ and cd8+ lymphocytes expressing ifng+ respectively and the cd4+/cd8+ dose adoptively transferred on day +86 of transplantation was 0.34 × 10 4 /kg. in vivo expansion of anti-bkv t cells in the patient was analysed weekly for the first month using the research grade peptivators bkv lt and bkv vp1 and the rapid cytokine inspector (cd4/cd8 t cell) kit. bk viral load was monitored by pcr in urine samples twice weekly. ifng+ anti-bkv reactive t cells were undetectable in the patient for the first two weeks after adoptively transfer of donor t cells. twenty days after the adoptive transfer an increase in the cd4+ ifng+ population was observed, in response to the bkv vp1 peptivator. this observation correlated in time with a substantial decrease of the urine bkv viremia from 3.3 million copies/ml to 1360 copies/ml and a complete resolution of patient's symptoms. no gvhd, recurrence of urinary symptoms or any other problems have been observed to date (day +260 of transplantation, +174 days after the adoptive transfer). we are not aware of any other reports of successful adoptive anti bkv cellular therapy. our experience suggests safety and efficiency of the use of anti-bkv t cell enriched products using the clinimacs prodigy and the ifng capture system in hla haploidentical hct where bkv cystitis constitutes a significant complication. this opens the possibility of further clinical trials. disclosure of conflict of interest: none. haploidentical donor (hd) has been used as an alternative stem cell source when patients do not have a hla-matched related or unrelated donor. to overcome the hla barrier, haploidentical stem cell transplantation (haplosct) using post-transplantation cyclophosphamide (ptcy) has been conducted. here, we compared the clinical outcomes of haplosct using ptcy with those of unrelated donor transplantation. eighty-two patients (28 from hd and 54 from unrelated donor [ud]) who underwent allogeneic hematopoietic stem cell transplantation (hsct) in seoul national university children's hospital from january 2013 to june 2016, were analyzed. there were no significant differences between hd and ud patients with respect to median age of patients, sex distribution, and diagnosis [42. 6%], p = 0.081). the conditioning regimen of haplosct included targeted busulfan, fludarabine and cyclophosphamide using ptcy, tacrolimus and mycophenolate mofetil for graftversus-host disease (gvhd) prophylaxis. all patients showed engraftment except for a patient who underwent unrelated hsct. neutrophil engraftment of ud was faster than hd (median 11 days versus 15.5 days, respectively, po0.001). however, there was no significant difference of platelet engraftment. incidences of complications, such as hepatic venoocclusive disease, cmv infection, and hepatic dysfunction, between both groups, were comparable, except hemorrhagic cystitis (hd: 32.1%, ud: 7.4%, p = 0.004). moreover, cumulative incidence of acute gvhd (hd: 32.3%, ud: 44.7%, p = 0.260), severe chronic gvhd (hd: 8.4%, ud: 26.7%, p = 0.059), relapse (hd: 28.6%, ud: 25.1%, 7p = 0.323) and non-relapse mortality (hd: 0%, ud: 9.7%, p = 0.151) were not significantly different. the overall and event-free survival of hd and ud were 85.4% vs 86.2% (p = 0.703) and 75.0% vs 75.9% (p = 0.509), respectively. the clinical outcomes of haplosct using ptcy were comparable with those of ud, and a trend of lower cumulative incidence of severe chronic gvhd and non-relapse mortality was encouraging. it could be a promising alternative therapeutic option in pediatric hsct. disclosure of conflict of interest: none. , median number of apheresis procedures was 2,15 (1-6), median amount od dmso infused 20 ml (7-60). time to engraftment was median 11 days (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) . statistical comparison between cryopreserved pbsc grafts and bm showed benefit for pbsc in the terms (po0.01) of faster engraftment, less infective complications, less transfusion support and less hospital stay. in 253 patients (86%) dmso related events were not registered during graft administration. in 41 patients (14%) mild to moderate dmso related events were registered, as nausea in 34 patients (83.3%), vomitus in 20 patients (50%), tachycardia in 8 (20.8%), hematuria in 6 patients (16%) and 2 patients (4.16%) with bradycardia, hypotension, fever and high temperature during graft infusion. cryopreservation of stem cells is a feasible procedure at our institution. there are some issues that have to be improved. the process is standardized with achieved engraftment in all transplanted patients. disclosure of conflict of interest: none. effectivity of a fludarabine based conditioning regimen in allogenic hematopoietic stem cell tranplantation for patients with severe aplastic anemia and over twenty years old p mustafa 2,3 , k melya pelin 1 , s handan haydaroglu 2 and g ilknur 1 1 gaziantep university, faculty of medicine, department of internal medicine; 2 hematology and 3 bone marrow tranplantation unit, gaziantep, turkey severe aplastic anemia (saa) is an anemia with bone marrow hypocellularity and caused by hematopoietic stem cell failure (1) . allogenic periferic hematopoietic stem cell transplantation (aphsct) is a curative treatment choice (2) . although cyclophosphamide (cyc) and anti thymocyte globulin(atg) is accepted as standart conditioning regimen, especially for patients with high rejection risk, using fludarabin (fu) based regimens show increased successful engraftment ratio with minimal toxic side effects (3) . to the study, 20 saa patiens who were transplanted from hla matched sibling donors between the years 2010-2015 were included. the patients comprised of 13 male (%65) and 7 female (%35). median age was 22 (range: 20-42). the median time from diagnosis to transplantation was 3 (range: 2-108) months. conditioning regimen consisted of cyc (1200 mg/m 2 ), fu (120 mg/m 2 ), atg (fresenius rabbit, 15 mg/kg). the median dose of stem cells was 7 × 10 6 stem cell/kg (range: 5-12). methotrexate (10 mg/ m 2 given four days) and cyclosporine (cyca) (3-5 mg/kg given 18 months) were applied for graft versus host disease (gvhd) prophylaxis. all 20 patients ecog performance status were good (0-1). prior to transplantation only one of the patients received atg-csa, the others received only supportive treatment. after aphsct, neutrophil engraftment was occured at a median of 16 days (range: 11-20) and thrombocyte engraftment was occured at a median of 14 days (range: 11-20). posttransplant graft failure was observed in only one patient at tenth month and this patient had aphsct again from the same donor with the same conditioning regimen. acute gvhd didnot occur in any patient. the 5 (%25) of patients had common chronic skin/oral mucosa gvhd. these 5 patients received methylprednisolone (mp) and/or mycophenolate mofetil (mmf) in addition to the cyclosporine treatment. extracorporal photopheresis was applied to the two patients with chronic gvhd. all chronic gvhd patients had complete response to the immunsupresive treatment with a median follow up time 46 months (range: 1-64). one patient died from sepsis. at 5 year overall-survival rate was 90%. fu based conditioning regimen in aphsct with young saa patients has favorable results. fu based regimen might be a gold-standard treatment in the future. cgvhd; tgfb-induced factor homeobox 1, interleukin 2 receptor gamma, tetra trico peptide repeat domain 37, carbonic anhydrase i, serpin peptidase inhibitor clade a and myod family inhibitor. we established a 3-gene model (myod family inhibitor, tgfb-induced factor homeobox 1, tetra trico peptide repeat domain 37) to diagnose cgvhd. our 3-gene model showed 81.00% sensitivity, 90.40% specificity, 80.8% precision, 81.03% accuracy and 86.90% roc area in diagnosing cgvhd. tgfb-induced factor homeobox 1 increased in expression after rituximab treatment in responders. myod family inhibitor was found to be able to predict rituximab responses in steroid-refractory cgvhd patients. we could demonstrate that gene expression studies were useful in the diagnosis of cgvhd after allo-hsct. we developed a 3-gene model to diagnose cgvhd. hematopoietic stem cell transplantation (sct) is physically and psychosocially demanding. however, exercise interventions may have positive impact on sentiment and psychological well-being in patients undergoing sct. we report on a prospective, randomized study comparing the influence of a multimedia sensor-based practice with classical physiotherapeutic treatment (pt) on psychological aspects and quality of life (qol). patients undergoing sct were randomized into the control group (n = 23) receiving pt or the experimental group exercising on the nintendo-wii (n = 19). patients of both groups performed the exercises under the supervision of a physiotherapist and completed the functional assessment of cancer therapy -bone marrow transplantation (fact-bmt), hospital anxiety and depression scale (hads-d) and distress thermometer at the date of hospital admission (t1) and on day 14 (t2), 28 (t3) and 100 (t4) after sct. questionnaires were completed by the participants independently and without supervision. groups were compared using the mann-whitney u-test. a p value o0.05 was considered statistically significant. the median age of patients was 59 years in the control group and 57 years in the experimental group. results of fact-bmt generally showed a decline of the qol domains measured on t2 and t3 and a raise at t4 in both groups. physical well-being (pwb) showed the strongest fluctuation of all domains. it declined significantly between t1-t2 in both groups (pt p = 0.015, wii p = 0.019), followed by a significant increase between t2-t4 (both groups p = 0.001). however, only in wii-group results of pwb at t4 ranked significantly above t1 (p = 0.028). highest scores were proved for social and emotional well-being (swb/ewb) in both groups. in wii-group ewb increased significantly between t1-t4 (p = 0.015) and ranked above pt-group at all times. functional well-being (fwb) scored lowest in both groups at all times. the score of bone marrow transplant scale (bmts), the second lowest score in both groups, was always higher in wii-group. the level of distress was comparable between both groups. however, at t2 distress increased above the cut-off level of 5 in both groups (wii-group p = 0.006, pt-group p = 0.276). this was accompanied by an increase of anxiety (p = 0.705) and depression (p = 0.006) in the pt-group, while both parameters decreased in the wii-group (p = 0.087 and p = 0.220), respectively. anxiety in intervention group 5,8/4,4/5,0/,4 at t1/t2/t3/t4 stayed below standard group 5,9/6,4/5,9/6,4 at all times. depression averaged out at 4,9/6,5/5,4/5,7 in physiotherapy group and 5,5/4,3/5,9/3,8 in wii-group. to the best of our knowledge, this is the first study to show that exergaming using the nintendo-wii is feasible in the immediate phase after hsct. exergaming may be regarded as beneficial since our data indicate less psychological distress and higher qol in sct recipients exercising with nintendo-wii. therefore, it may be used in addition to pt. disclosure of conflict of interest: none. acute graft versus host disease (agvhd) is the most frequent and serious complication following haematopoietic stem cell transplantation (hsct), with a high mortality rate. a clearer understanding of the molecular pathogenesis may allow for improved therapeutic options or guide personalised prophylactic protocols. circulating micrornas are expressed in body fluids and have recently been associated with the etiology of agvhd, but global expression profiling in a hsct setting is lacking. this study profiled expression of n = 799 mature micrornas in patient serum, using the nanostring platform, to identify micrornas that were dysregulated at agvhd diagnosis. selected micrornas (n = 10) were replicated in independent cohorts of serum samples taken at agvhd diagnosis (n = 42) and prior to disease onset (day 14 post-hsct, n = 47) to assess their prognostic potential. sera from patients without agvhd were used as controls. dysregulated micrornas were investigated in silico for predicted networks and mrna targets. profiling identified 61 micrornas that were differentially expressed at agvhd diagnosis. mir-146a (p = 0.03), mir-30b-5p (p = 0.007), mir-374-5p (p = 0.02), mir-181a (p = 0.03), mir-20a (p = 0.03) and mir-15a (p = 0.03) were significantly verified in an independent cohort (n = 42). mir-146a (p = 0.01), mir-20a (p = 0.03), mir-18 (p = 0.03), mir-19a (p = 003), mir-19b (0.02) and mir-451 (p = 0.01) were differentially expressed 14 days post-hsct in patients who later developed agvhd (n = 47). high mir-19b expression was associated with improved overall survival (os) (p = 0.008), while high mir-20a and mir-30b-5p were associated with lower rates of non-relapse mortality (p = 0.05 and p = 0.008) and improved os (p = 0.016 and p = 0.021). pathway analysis associated the candidate micrornas with haematological and inflammatory disease. circulating biofluid micrornas are dysregulated at agvhd onset and have the capacity to act as prognostic and diagnostic biomarkers. their differential expression in serum suggests a role for circulatory micrornas in agvhd pathology, which warrants further investigation. disclosure of conflict of interest: none. factors associated with medication adherence amongst allogeneic hematopoietic stem cell transplantation recipients j lehrer 1 , e brissot 2,3 , a ruggeri 2,4 , r dulery 2 , a vekhoff 2 , g battipaglia 2 , f giannotti 2 , c fernandez 1,5,6 , m mohty 2,3 and m antignac 1 1 ap-hp, hôpital saint-antoine, service de pharmacie, paris, f-75012; 2 service d'hématologie et de thérapie cellulaire, hôpital saint antoine, assistance publique-hôpitaux de paris; 3 sorbonne patients with median ages of 23 years (range: 11-54 years) between december 2006 and march 2016, which including 7 case of primary hlh (homozygous missense mutation in unc13d: n = 3; homozygous missense mutation in prf1: n = 1; heterozygous missense mutation in prf1 in the combination with hemizygous missense mutation in sh2d1a: n = 1; mutation in rab27a: n =1; mutation in itk: n = 1). 5 cases of unknown causes hlh, 10 cases of lympgoma -hlh (nk/t-cell lymphoma: n = 6, primary γδt cell lymphoma in skin: n = 1; subcutaneous panniculitis-like t cell lymphoma: n = 2; primary t cell lymphoma in skin: n = 1) and 39 cases of ebv associated hlh. 41 patients achieved cr+pr before hsct, and 20 patients nr. 47 patients were transplanted from hla-haploidentical family donors, 13 from hla-identical sibling donors, and 1 from a matched unrelated donor. conditioning regimen include tbi and non-tbi. the median overall survival rate was 65.6% with a median survival time of 38 months (range: 5-119 months). os of primary hlh is 85.7%, os of unknown causes hlh is 60%, os of lymphoma-associated hlh is 60%, os of ebv-hlh is 64.1%. os of cr+pr is 80.5%, os of nr is 35.0% 6 patients without engraftment died because of 2 graft failure and 4 toxicity of conditioning regimen. 15 patients with engraftment died. of those, 1 patient died of hsct-associated tma, 3 patient died of grade iv agvdh, 5 patients died of relapsed hlh or organ failure as results from unsuccessful treatment of the progressively elevated ebv-dna load. 2 patient died of tumor relapse, and 4 patient died of infection. acute gvdh occurred in 42 patients with grade i-ii agvdh in25 patients and grade iii-iv agvdh in 17 patients; chronic gvdh occurred in 19 patients. 46 patients achieved completed chimerism, 9 patients appeared with mixed chimerism,and2 patient presented with graft failure. of 34 ebv-hlh with engraftment, reactivated ebv infection was found in 33 (97%) with the whole blood ebv-dna load at 103-107 copy numbers per ml. ptld occurred in 3 patients confirmed by pathology. after reduced immunosuppressors, negative result of ebv infection was obtained while patients developed gvdh. for 39 ebv-hlh, patients who carry with ebv loading ebvdna ≤ 105 copies/ml before transplantation, overall survival rate was significantly higher than that of ebvdna4105 copies/ml (po0.05); who achieved cr+pr os was significantly higher than that of nr (po0.05); who range: from diagnosis to transplantion ≤ 6 months os was significantly higher than that of 46 months (po0.05). allogeneic hematopoietic stem cell is an effective method for primary hlh and lymphoma-hlh, ebv-hlh,even haploid transplantation. the remission status before transplantation is decisive for the prognosis. disclosure of conflict of interest: none. hepatic veno-occlusive disease after allogeneic hematopoietic stem cell transplantation in a single centre: revised diagnosis and incidence according to new ebmt classification s santarone, a natale, p olioso, g papalinetti, t bonfini, p accorsi, s angelini, g iannetti, m di ianni and p di bartolomeo dipartimento di ematologia, medicina trasfusionale e biotecnologie, bmt center, ospedale civile, pescara, italy sinusoidal obstruction syndrome, also known as venoocclusive disease (sos/vod), is a potentially life threatening complication that can develop early after hematopoietic cell transplantation (hct). in this study we retrospectively investigated the incidence, risk factors and outcomes of sos/vod in 978 transplants, performed in 896 patients between march 1982 and may 2016, on the basis of the new diagnostic criteria and classification of the ebmt. the patient's median age was 31 years (1 to 71). of them, 536 were males and 442 females. 896 patients received one transplant and 82 two transplants. a diagnosis of hematological malignant and nonmalignant disease was present in 784 and 194 cases, respectively. the disease risk at hct was standard in 397 cases, intermediate in 237 and high in 344. an hla identical sibling donor was used in in 691 cases, an unrelated donor in 166 and a haploidentical family donor in 121. conditioning was myeloablative in 813 transplants and at reduced intensity in 165. source of hematopoietic stem cells was bone marrow in 680 transplants and peripheral blood in 298. we did not limit the diagnosis of sos/vod to the classical 21 days after hct, but all suspicious cases appearing in the first 100 days were evaluated. sos/vod was diagnosed in 56 cases, of which 47 in the first 21 days after transplant and 9 between day 22 and 50 (median day 9). their main clinical characteristics are shown in table 1 . the severity of sos/vod was mild in 5 patients (9%), moderate in 6 (11%), severe in 8 (14%) and very severe in 37 (66%). the cumulative incidence (ci) of sos/vod was 5.7+0.005%. among the most relevant variables studied in univariate analysis (recipient age and gender, ferritin level at hct, type of hematological disease, disease risk at hct, type of donor, number of transplants, time of transplant, drugs used in the conditioning regimen, intensity of the conditioning regimen, source of stem cells), there was no factor with an adverse impact on sos/vod incidence. of 56 patients with diagnosis of sos/vod, 41 (73%) died. sos/vod was the main cause of death in 9 patients and a relevant contributing cause of death in 10. of relevance, 6 of 8 patients (75%) with severe sos/vod and 34 of 37 patients (92%) with very severe sos/vod died, whereas only one patient with moderate sos/vod died and no patient with mild sos/vod died. among 56 patients with sos/vod, 19 received defibrotide therapy and 37 the best supportive available therapy. defibrotide was given for a median of 20 consecutive days (range: 5 to 87), starting at day 18 post-hct (range: 3 to 49) with a median total bilirubin level of 3,16 mg/dl (range: 1.4-20.7). the 1-year overall survival (os) of patients treated with defibrotide was better as compared to that of patients who received the supportive therapy (47% versus 27%) although the difference doesn't reach the significance (p = 0.25). the occurrence of sos/vod does influence significantly the 1-yr os considering that it was 72 +1.5% for patients without sos/vod and 33+6% for patients with sos/vod (p = 0.0001). in conclusion, the new ebmt diagnostic and severity criteria for sos/vod has been very useful in identifying patients with severe and very severe forms of this complication. if validated in prospective studies, these criteria will allow an earlier selection of patients requiring immediate therapeutic intervention. [p692] disclosure of conflict of interest: none. the prognosis of patients with newly diagnosed ewing's sarcoma family of tumors (esft) has improved significantly over the last few decades. nonetheless, the long-term survival is still below 35% patients with high risk features.the role of s490 high dose chemotherapy and autologous stem cell transplantation (hdct and asct) for high risk and relapsed esft was analyzed. a retrospective medical chart review was done on patients with efst who underwent hdct and asct between september 1998 and january 2015 at seoul national university children's hospital. indications for hdct and asct included metastasis at diagnosis, bulky primary tumor (4100 ml), axial/ central primary site, and relapsed disease. single hdct and asct was performed in the earlier period, and the regimen was changed from mec (melphalan, etoposide, carboplatin), to topothiocarbo (topotecan, thiotepa, carboplatin), and to bumel (busulfan, melphalan). tandem hdct and asct was performed in the recent period, 1st hdct with bumel and 2nd hdct with modified mec (melphalan, etoposide, carboplatin). twenty-one patients who were diagnosed with esft at a median age of 8.7 years old underwent conventional chemotherapy, radiation therapy and/or surgery and received hdct and asct in complete response (n = 14) or partial response (n = 7). the overall survival of the patients was 70.0% at median 3.6 years and the event free survival (efs) of the patients was 55.0% at median 2.9 years from the last asct. the efs of the patients who underwent single hdct and asct with mec (n = 11), topothiocarbo (n = 1), and bumel (n = 4) was 54.5%, 0.0% and 75% respectively. the efs of the patients who underwent tandem hdct and asct (n = 5) was 50.0%. seven patients relapsed at median 6.6 months from the last asct. despite further treatment, 5 patients died of disease and 2 patients are currently alive without disease. one patient developed acute myeloid leukemia at 17.8 months from the last asct and is currently alive without disease after additional chemotherapy, hla-haploidentical stem cell transplantation and donor lymphocyte infusions. one patient died of transplantation-related mortality due to septic shock and lung infection. hdct and asct may be a promising treatment option for patients with high risk or relapsed esft. further refinements may be needed to identify the optimal regimen and number of hdct and asct. disclosure of conflict of interest: none. post transplant cyclophosphamide (pt-cy) has expanded the use of unmanipulated haploidentical grafts which have a high hla disparity between host and donor. one of the consequences of hla disparity is the development of engraftment syndrome (es). this is an immunological reaction characterized by non-infectious fever and skin erythema that develops after neutrophil engraftment. es resembles an infectious process but treatment involves the use of high dose steroids. our hypothesis is that pts undergoing haploidentical transplants (haplo) with pt-cy should have a high rate of es given the high hla disparity between donor and recipient. objectives: to determine the incidence, symptoms, morbidity and mortality of es in patients undergoing haplo with pt-cy at our institution. retrospective analysis of 22 patients with highrisk hematological diseases undergoing haplo with pt-cy at clinica santa maria between november 2012 and august 2016. es was diagnosed using the spitzer criteria (1). es was diagnosed if pts met 3 major criteria or 2 major plus 1 minor criterion. symptoms could occur prior to or after neutrophil engraftment (neutrophils over 500 cells / ul). all patients signed informed consent and the study was reviewed by our institutional review board. 22 patients received haploidentical grafts ( table 1) . all patients had neutrophil engraftment at a median of 18 days. 9/22 patients (41%) had symptoms that met criteria for es ( table 2 ). 2/9 were transferred to icu due to hypoxemia and 1 patient died after diagnosis of es. 5/9 pts were treated steroids. all patients received broad spectrum antibiotics during the febrile period and neutropenia. blood cultures, ebv and cmv pcr were negative in all es pts. there were no significant differences in hospital stay or one-year overall survival (os) between patients who developed and pts who did not develop es (median 37 vs. 35 days respectively, p = 0.68; one-year os 56% vs. 57%, p = 0.86, respectively). es is a frequent complication in patients undergoing hsct haplo with pt-cy. the incidence of es in our study was higher when compared to historical full match related donors series and lower when compared to cord blood transplant studies (2) there was no increased morbidity and mortality associated with es diagnosis. prompt institution of steroids is recommended in es patients after ruling out an underlying infectious process to avoid further complications. haploidentical allogenic hematopoietic stem cell transplants (haplo-hsct) is an alternative transplant procedure for patients with hematologic malignancies that are in need of transplant and do not have a compatible donor. due to the broad hla disparity, the haplo-hsct can be performed with t-cell depletion and megadose of cd34+. alternatively haplo-hsct can be performed with non t-cell depleted transplants (t-replete) either in combination with anti-thymoglobuline serum (atg) or post-transplant cyclophosphamide (pt-cy) as gvhd prophylaxis strategy. center effect is a known risk factor for outcomes of haplo-hsct in both t-cell depleted (tcd) and t-replete settings. however, many centers tend to specialize in one gvhd prophylaxis strategy making it difficult to differentiate the treatment effect from the center effect. the objective was to investigate the role of center effects in gvhd prevention strategy, on leukemia-free survival (lfs) and overall survival (os) in a population of adult patients with acute leukemia receiving haplo-hsct. a retrospective multicenter study was conducted on patients reported to the ebmt registry. inclusion criteria were: age 418 years, lymphoblastic or myeloid acute leukemia (all or aml) in first or second complete remission (cr1 or cr2), receiving a haplo-hsct between 2005 and 2014. in this population (n = 606), in order to assess the interaction between center and gvhd prevention treatment, we then included in the study selected patients from the centers that had performed more than 20% of both tcd and t-replete haplo-hsct during the study period. center effects on the outcomes consisted of 1) center effect on the baseline risk of event and 2) interaction between center and strategy of gvhd prevention. these center effects were estimated using cox mixed-effects models and tested using permutation tests. all models were adjusted on age, cmv statuses, disease (all or aml), secondary leukemia, previous autologous transplant, disease status (cr1 or cr2), peripheral blood vs. bone marrow transplant, conditioning regimen. after selection, 226 patients were available across 29 centers in europe. one hundred and one (45%) patients received tcd, 125 t-replete haplo-hsct (62 (27%) using atg and 63 (28%) using pt-cy). overall, 175 (77%) patients had aml. there were 86 (69%) peripheral blood transplants in the tcd group and 92 (91%) in t-replete. median follow-up was 2.7 years. in adjusted analyses, without accounting for center effect, t-replete tended to be associated with better lfs (hazard ratio (hr): 0.70 (95%ci 0.45-1.07), p = 0.10) and os (hr = 0.67 (95%ci 0.43-1.04), p = 0.076). when center effects were included, there was significant heterogeneity across centers on the baseline risk of both outcomes (lfs: p = 0.036 and os: p = 0.048). when accounting for interaction between center by strategy for gvhd prevention, the effect of t-replete vs. tcd on the outcomes did vary across centers (p = 0.065 and p = 0.031 for interactions in lfs and os, respectively) ( figure 1 ). we found an interaction between center and strategy for gvhd prevention on outcomes of patients who received a haplo-hsct. the difference between the 2 strategies (tcd or t-replete) varied across centers, in size and direction. this could be in part related to the increase in expertise with each technique in some centers and with the different management of complications, such as infections-related and relapse. disclosure of conflict of interest: none. adherence included recognition of spuriously high levels (typically from contaminated lines) and delayed dose adjustment due to late reporting of levels by the laboratory. the most common cause of unjustifiable non-adherence was failure to increase the dose in response to a low level. inadequate or excessive dose adjustments may be due to lack of experience or unfamiliarity with the sop. two interventions were launched with the aim of improving adherence to the sop for therapeutic tacrolimus dosing. firstly, to provide a rapid and user-friendly calculation method, we developed a mobile phone application (tacrocalc, a dose calculator based upon the sop algorithm) for android and ios devices using python and swift, respectively. secondly, to reduce the number of spuriously high levels, all nurses responsible for specimen collection participated in an educational module delivered by medical and senior nursing staff. key messages included the need to: use only the dedicated colour-coded tacrolimus lumen to infuse iv tacrolimus; avoid sampling from this lumen; sample peripherally when other lumens are known to be contaminated (reasons for this are being explored); suspend infusion of iv tacrolimus 15 minutes before taking a level; send only immediately pre-dose levels for oral tacrolimus. initial re-audit of 16 episodes post intervention (data collection is ongoing) demonstrated a 40% increase in sop adherence (p = 0.03; fisher's exact test), with no cases of unjustifiable non-adherence and a significant reduction in spuriously high levels. in conclusion, the use of tacrocalc by doctors and the implementation of targeted teaching for nurses dramatically improved adherence to the tacrolimus sop. this should ultimately improve therapeutic dosing whilst avoiding toxicity, which may result in better transplant outcomes. tacrocalc is now being adapted to include an option for paediatric dosing, with the potential to dose related medications such as cyclosporine. disclosure of conflict of interest: none. king's college hospital, 2 imperial healthcare, charing cross hospital and 3 imperial healthcare, hammersmith hospital managed with calcium and vitamin d alone in 28/43 cases (65%) and together with bisphosphonates in 9/43 (21%). osteoporosis was managed with bisphosphonates ± calcium/ vit d in 19/36 and with calcium/vit d alone in 10/36. 8 /34 indicated that they would give bisphosphonates in the absence of osteoporosis, if a patient with osteopenia was receiving long term steroids. dissemination and implementation of existing guidance on investigation and managing low bmd post hct appeared to be poor amongst respondents to our survey. routine dxa scanning was underused; the trigger for dxa in the context of steroids is inappropriately high at many centres at 1 mg/kg daily for 3 months; in established osteoporosis, bisphosphonates were used less frequently than would be anticipated. these findings may reflect the limited data on which current recommendations have been made, or the large number of non-transplant guidelines for investigating and managing low bmd which confound management of this post-hct patient group. hematopoietic stem cell transplantation (hsct) still remains as the most efficient therapy for adult patients with acute myeloid leukemia. for older patients and those lacking a hlacompatible donor, autologous hematopoietic stem cell transplantation (auto-hsct) is a valid therapeutic option. 1 authors aimed for determining the effect of auto-hsct for acute myeloid leukemia patients and analyze group of patients who underwent auto-hsct. the study has been set as a retrospective single center study. clinical information included age, gender, aml type and cytogenetic risk. pretransplantation treatment, mobilization and conditioning were analyzed and thus subsequently authors used kaplan and meier method to calculate the actuarial overall survival rate. table 1 describes patients' characteristics. majority of patients received similar induction therapy based on combination of cytarabine and anthracycline. timespan from the diagnosis to auto-hsct varied from 74 days to 1791 days, median was 175 days. seventy (88,6%) patients received a preparative regimen consisting of busulfan at 1mg/kg orally, four times daily for 4 days for a total dose of 16 mg/kg administered on day -6 through day -3 and melphalan 100-150 mg/m 2 intravenously for over 4 hours on day -2. patients achieved an absolute neutrophile count (anc) of ≥ 0.5 × 109/l in between 10 to 40 days; median was 14 days. patients achieved not transfused platelet count ≥ 20 × 109/l in between 10 to 209 days; median was 19 days. median of patients' discharge from hospital was 19 days (range: from 13 to 44 days) since auto-hsct. hundred day mortality after autologous transplant was at 6.32% (5/79). on the date of our evaluation (april 30, 2016), 48 patients were alive and in continued cr. the relapse rate was 39.5% (32 patients) and 7 patients (8.6%) were lost from follow-up. the 5-year overall survival (os) was 60.8%, so the target median of overall survival has not been reached. [p701] the development of dyslipidaemia is commonly observed after haematopoietic stem cell transplantation (hsct). few data are available concerning lipid profiles over a long followup period or with regard to the different transplantation types (autologous vs. allogeneic) or the effect of multiple transplantations on the development of dyslipidaemia. a retrospective, single center cohort study including 1239 adult patients (416 years) who underwent hsct at the university hospital basel s496 1973-2013 and who survived ≥ 100 days was performed. patients with at least a baseline lipid measurement were included (n = 1096) and grouped according to the type of their first hsct (autologous or allogeneic). for the examination of the effect of subsequent hscts, patients with consecutive transplantations of the same type were included and other patients were censored when a different transplantation type was performed. serial lipid profiles (total-, ldl-and hdl-cholesterol and triglycerides) before and after transplantation were examined. of the 1096 patients, 407 underwent a first, and 89 of these at least one subsequent autologous hsct. 689 underwent a first, and 85 of these at least one subsequent allogeneic hsct. median age of patients at autologous hsct was 52y (iqr 39-61) and 43y (32-53) at allogeneic hsct. 62% and 58% were males, median bmi pre-transplant was 25 (22-28) and 24 (22-27). the majority of patients underwent s497 intensive conditioning before hsct. median follow-up time was 3.0 years in the autologous and 4.8 years in the allogeneic group, with a maximum follow up time of 26.1 and 34.3 years, respectively. table 1 shows the number and percentage of patients with dyslipidaemia (1st autologous and allogeneic transplants). the distribution of exact total cholesterol values along with comparisons with baseline measurements according to group are presented in the figure 1 . *% based on number of measurements available total, ldl-and hdl-cholesterol and tg increased within 3 months of transplantation, regardless whether autologous or allogenic transplantation or a first or a subsequent transplantation was performed. the percentage of patients with dyslipidaemia accordingly rose significantly within 3 months of transplantation and persisted throughout follow-up. although patients undergoing an autologous hsct presented with higher baseline values of total cholesterol, a significantly greater increase post-transplant was observed after allogeneic hsct. first and subsequent transplantations seem to behave similarly with respect to changes in lipid profiles. disclosure of conflict of interest: none. nuremberg, erlangen, germany; 4 department of cancer immunology, institute for cancer research, oslo university hospital, radiumhospital, oslo, norway; 5 kg jebsen center for cancer immunotherapy, institute of clinical medicine, university of oslo, oslo, norway; 6 department of haematology and oncology, university hospital of the goethe university, frankfurt, germany and 7 childrens hospital, goethe university, frankfurt, germany natural killer (nk) cells are lymphocytes of the innate immunity with a potent anti-tumor capacity. in tumor patients, such as multiple myeloma (mm) patients, an elevated number of nk cells correlates with a higher overall survival (os) rate. our study adressed nk cells characteristics and anti-tumor ability in mm patients. especially cytotoxicity of patientderived, cytokine-stimulated nk cells against mm cells has been analyzed at various time points (at diagnosis, before/ after chemotherapy and/or auto-sct). nk cells from patients were analyzed by facs after pbmcs isolation via ficoll separation at different time points: tp1, before the start of high dose chemotherapy (hdc)/auto-sct; tp2, after early leukocyte recovery (leukocytes 41000/μl) and tp3: at least 2 weeks after tp2. for testing nk cell cytotoxicity against mm cells, nk cells were purified via negative selection and expanded in vitro for 1-2 weeks in low doses il-2 and il-15. nk cells were divided into the cd56 ++ cd16 − or cd16 + and cd56 + cd16 ++ subsets. while the major nk cell subset at tp1 was the cd56 + cd16 ++ nk cell subpopulation (71.86%), after leukocyte recovery at tp2 cd56 ++ cd16 − /+ nk cells were the main subsets (cd16 − : 22.85%; cd16 + : 36.51%). we further evaluated the nk cell function upon tumor interaction at the defined time points. cd56 ++ cd16 − nk cells were the main subset to produce ifn-γ upon interaction with k562 cells at all different time points. the percentage of ifn-γ-positive cd56 + + cd16 − nk cells was slightly decreased at tp2 compared to tp1 but significantly increased from tp2 to tp3 (p-value: 0.0008). similarly, mip-1β-and cd107a-positive cd56 ++ cd16 − cells remained constant between tp1 and tp2, whereas their percentages increased from tp2 to tp3 [p-values: 0.0056 (mip 1β) and 0.0232 (cd107a)]. moreover, in a small group of mm patients, we isolated nk cells and expanded them for 1-2 weeks prior to the functional assays. as expected, the expansion rate was reduced after chemotherapy compared to nk cells from healthy controls, but the patients nk cells increased their ability to kill mm cells due to the ex vivo cytokine expansion. conclusion: our data demonstrate that nk cells have an altered phenotype and function after hdc/auto-sct. remarkably, these nk cells were able to secrete cytokines and still displayed cytotoxic capacity against different types of tumor cells. however, as the proliferative capacity of nk cells seemed to be reduced following chemotherapy, innovative nk cell therapeutic approaches further improve the patients nk cell activity by an ex vivo cytokine stimulation procedure. finally, we suggest that an additive cell therapy with cytokinestimulated autologeous nk cells might improve the outcome of mm patients. lymphoid and myeloid acute leukemia are the most frequent type of cancer and the most frequent cause of cancer related death in children. relapse and refractory disease are the main clinical problems that current therapies are still unable to solve. one of the main nk cell activating receptors is nk cell group 2d (nkg2d). nkg2d receptor recognizes human mica/ulbp1-6 ligands. these nkg2d ligands are expressed in leukemia cells and constitute suitable targets for immunotherapy. the expression of nkg2d ligands was analyzed in peripheral blood mononuclear cells from 61 pediatric patients suffering from acute leukemia (21 acute myeloid leukemia, 25 b cell acute lymphoid leukemia and 15 t cell acute lymphoid leukemia), as well as in 7 leukemia cell lines (k562, rs4-11, jurkat, nalm-6, molt-3, reh and cem), by flow cytometry using specific monoclonal antibodies directed against mica, micab, ulbp-1, ulbp-2, ulbp-3 and ulbp-4, and by quantitative pcr using taqman probes. peripheral blood mononuclear cells from healthy donors were labeled with cd45ra microbeads and depleted using automacs device. the hl20i4r-mndanticd19bbz lentiviral vector was derived from the clinical vector cl20i4r-ef1a-hgcopt27 but contained the extracellular domain of nkg2d, the hinge region of cd8a and the signaling domains of 4-1bb and cd3-z. the cassette was driven by mnd promoter. viral supernatant was produced by transient transfection of hek293t cells with the vector genome plasmid and lentiviral packaging helper plasmids pcagg-hivgpco, pcagg-vsvg and pcag4-rtr2. cytogenetic studies and array comparative genomic hybridization were performed to analyze the genetic stability of lentiviral-transduced memory t cells. the in vitro cytotoxicity of cd45ra − t cells against leukemia cells, healthy pbmc and mesenchymal stem cells (msc) was evaluated by performing conventional 4-hour europium-tda release assays or by flow cytometry using cfse and 7aad labeling of target cells. nkg2dl were heterogeneously expressed in leukemia primary cells and cell lines. for b cell all primary samples, we found expression of mica/b, mica and ulbp1 decreased in refractory disease compared to remission (p = 0.01, p = 0.03 and p = 0.02, respectively). lentiviral transduction of nkg2d-4-1bb-cd3z markedly increased nkg2d surface expression in cd45ra − memory t cells, which became consistently more cytotoxic than untransduced cells against leukemia cells. additionally, no chromosomal aberrations nor cytotoxic activity against healthy pbmc or mesenchymal stem cells was observed in nkg2d car expressing t cells. our results demonstrate nkg2d-car redirected cd45ramemory t cells target nkg2dl expressing leukemia cells in vitro and could be a promising and safe immunotherapeutic approach for acute leukemia patients. peripheral blood stem cell mobilization and collection from elderly patients (≥65 years) with multiple myeloma: a single center experience g cengiz seval 1 , sk toprak 2 , s civriz bozdag 2 , m kurt yuksel 2 , p topcuoglu 2 , o arslan 2 , m ozcan 2 , t demirer 2 , g gurman 2 , h akan 2 , m beksac 2 and o ilhan 2 1 clinic of hematology, yildirim beyazit university yenimahalle education and research hospital and 2 department of hematology, ankara university school of medicine high-dose melphalan followed by autologous hematopoietic cell transplantation (auto hsct) has become the standard procedure for patients with symptomatic multiple myeloma (mm). the ability to mobilize stem cells from healthy donors shows little deterioration with age, the influence of patients' age on auto hsct is uncertain and studies in patients' ≥ 65 years are scarce. severe studies specific to mm have failed to show an independent effect of patient age on cd34+ mobilization. we retrospectively compared myeloma patients below the age of 65 with patients above 65 years of age, analyzing cd34 mobilization into peripheral blood and the number of leukapheresis needed to collect at least one single stem cell graft. material and methods: from february 1999 through april 2016, data from 501 myeloma patients below the age of 65 were compared to 52 myeloma patients above 65 years of age. all these data were obtained from the ankara university faculty of medicine center for therapeutic apheresis and written informed consent was signed according to our institution regulations. most of the patients received only gcsf at a dose of 5 μg/kg bw twice-daily s.c. until stem cell procurement. patients underwent further pbsc collections until we obtained the target dose 420 cd34+ cells/μl blood. a maximum of 3 collections were performed in the first mobilization; if the cell dose was not achieved, we submitted patients to a second mobilization. fifty two of 553 patients were above 65 years of age (median age 66, range: 65-73) and 501 patients were below the age of 65 (median age 54, range: 29-64). baseline characteristics of the older and younger patient cohorts are summarized in table 1 . mobilization regimens for the younger patient population were cyclophosphamide based (n: 122), g-csf only (n: 372) and +plerixafor (n: 7). mobilization in the older population was with cyclophosphamide based (n: 10), g-csf only (n: 41) and +plerixafor (n = 1). the chemotherapy regimens were not statistically different between both age groups. there were no significant statistical differences in time from diagnosis to mobilization, number of prior therapies or disease status between both patient groups. the number of cd34+ circulating cells before scheduled leukapheresis was mean 69.28 cells/μl (median 49 cells/ μl, range: 2-397; sem ± 46.875) in all patients (including patients who failed mobilization). our data support the observation that after a standard mobilization regimen with anti-myeloma chemotherapy and once-daily growth factor support, patients above 65 years of age show an impaired cd34 mobilization into peripheral blood compared to a younger population. this can be overcome by an increased number of leukaphereses. still the number of progenitor cells in the actual graft is inferior compared to the younger population. [p712] disclosure of conflict of interest: none. donor and/or recipient citomegalovirus (cmv) seropositivity has been associated with a poor overall survival (os) in patients who have received an allogeneic hematopoietic stem cell transplantation (allohsct). in comparison with seronegative donors, hsct from seropositive donors has been associated with decreased disease-free survival (dfs) and increased non-relapse-related mortality (nrm). we analyzed the prognostic impact of cmv serology status (donor/ recipient) in patients diagnosed with acute leukemia (al) [p713] s503 who had received an allohsct in our institution. retrospective unicentric study of patients diagnosed with al between 2001 and 2015 who received allohsct.the following outcomes were studies: os, dfs, and cumulated incidences of relapse (ri), nrm, acute graft-versus host disease (agvhd) and chronic gvhd (cgvhd). the series included 163 patients (86 males, 77 females), median age of 44 years . al type: 42 (26%) all, 121 (74%) aml. type of transplant: 88 (54%) related donor, 42 (26%) unrelated donor and 33 (20%) unrelated umbilical cord blood. the majority, 111 (68%), received myeloablative conditioning. stem cells source: peripheral blood 124 (76%), cord blood 33 (20%) and bone marrow 6 (4%). cmv serology status: positive receptor 124 (76%), negative receptor 39 cases (24%); positive donor 100 (61%), negative donor 63 (39%). serology status combinations (d/r): +/+ 84 (52%), +/ − 40 (24%), − / − 23 (14%), − /+ 16 (10%). 56 patients developed agvhd and 14 (9%) cgvhd. the impact of donor/recipient cmv serology status on os, dfs, ri, nrm and incidence of agvhd and cgvhd for the overall series is reported in table 1 . no statistically significant differences were detected in any of the analyzed variables. in this study, donor/recipient cmv serology showed no influence on the analyzed variables os, dfs, al relapse, nrm, acute and chronic gvhd. however, the sample size limits the validity of the results. disclosure of conflict of interest: none. supported in part with the grants pi10/01417 from fondo de investigaciones sanitarias and rd12/0036/0029 from rticc, instituto carlos iii and 2014sgr225(gre), generalitat de catalunya, spain. petersburg, russia during the last two decades ahsct has been used as a treatment option for ms with promising outcomes. qol is an important outcome of ms treatment. its assessment gives the patient's perspective on the overall effect of treatment. we aimed to study qol in ms patients before and after ahsct and search the value of the data obtained for decision-making. a total of 135 patients with different types of ms were enrolled in the study: mean age-34 (range-17-54) years old; male/ female-53/82; mean edss-3.5 (range: 1.5-8.5). all patients were treated by ahsct. reduced-intensity beam-like conditioning was used (bcnu 300 mg/m 2 , etoposide 100 mg/m 2 , ara-c 100 mg/m 2 and melphalan 100 mg/m 2 ). mean follow-up was 24 months (range: 12-53 months). qol was assessed using generic questionnaire sf-36. for comparisons t-test for independent samples or mann-whitney test was used. qol parameters in ms patients at 12 months after ahsct improved in comparison to base-line: physical functioning-66.3 vs 52.6, role-physical functioning-62. 8 6 . further qol improvement was registered at long-term follow-up: integral qol index exhibited 0.50 at long-term follow-up as compared to 0.32 at base-line. qol improvement was more dramatic in relapsing-remitting ms than in progressive ms. we found a significant increase of all eight sf-36 scales in a year posttransplant as compared with base-line in relapsing-remitting ms patients (po 0.05). in progressive ms patients statistically significant improvement was registered for six out of eight sf-36 scales (except bodily pain and role-emotional functioning) (p o0.05). improved qol parameters were preserved over the entire study period in all the patients who did not have disease progression or relapse. in conclusion, qol monitoring in ms patients after ahsct provides clinicians with the unique information regarding the changes in physical, psychological and social well-being of patients who have been treated with this new treatment modality. it allows to evaluate risks/ benefits of ms patients undergoing ahsct and might influence decision-making. further studies are needed to examine the trajectory of qol changes in this patient population to better define treatment outcomes after ahsct. disclosure of conflict of interest: none. pediatric patients with leukocyte adhesion deficiency type-i (lad-i), a rare autosomal recessive primary immunodeficiency disorder, experience severe and recurrent lifethreatening bacterial infections. allogeneic haematopoietic stem cell transplantation (hsct) offers the possibility of curative therapy although the conditioning regimen used for hsct in lad-i is still a controversial issue. this study provides evaluation of outcome of the lad-i pediatric patients who underwent reduced-intensity conditioning (ric) hsct. twenty four patients (14 female) with severe lad-i who received 26 hscts between februay 2007 and september 2016 at our center were enrolled. the median age at hsct was 30 months (range:4 months-14 years). patients received bone marrow (n = 9), peripheral blood progenitor cells (n = 14) or umbilical cord blood grafts (n = 3) from hla-matched related donors (n = 18), mismatched related or unrelated donors (n = 4), unrelated fully matched donors (n = 1) and haploidentical relative donors (n = 1). ric regimen was provided with fludarabine, melphalan and anti-thymocyte immunoglobulin. cyclosporine a and prednisolon were used as graft-versus-host disease (gvhd) prophylaxis. engraftment occurred in 23/26, of which one patient experienced graft rejection.the median times to neutrophil and platelet engraftments were 12 days (range: 10-23days) and 15 days (range: 10-32days), respectively. with a median follow-up of 43 months (range: 2-95months), overall survival (os) was 70.8%.the main causes of death were gvhd and infection. acute gvhd occurred in ten patients (4 grade i-ii, 6 grade iii-iv) and 3 patients also developed chronic gvhd. there were no significant differences in acute gvhd occurence and also os regarding to the stem cell sources. at this time,10 patients with full chimerism and 6 patients with mixed chimerism are alive and disease free. conclusion: hsct offers long term benefit in lad-1 and should be considered as an early therapeutic option if a suitable hla-matched stem cell donation is available. as pretransplant infections in primary immunodeficient patients especially those affected by lad-1 lead to rise in mortality rate, ric regimen is found to be safe and mixed donor chimersim appears sufficient to prevent significant symptoms. disclosure of conflict of interest: none. tregs based immunotherapy may be beneficial in several immune mediated diseases including graft versus host disease (gvhd). the possibility of cryopreserving tregs might lead to the administration of multiple doses, thus potentially increasing their efficacy in chronic diseases. however, there are few and controversial data on the functionality of tregs after cryopreservation. here, we evaluated the phenotype and the inhibitory capacity of thawed tregs. tregs were purified from leukapheresis of normal donors (n = 3) by double immunomagnetic depletion (cd8 and cd19) followed by cd25 enrichment using the clinimacs system (miltenyi biotec) under gmp condition. the cells were cryopreserved in saline solution containing 10% human serum albumin (hsa) and 10% dmso with a controlled-rate freezing. cell viability was assessed by 7-aad staining. number/phenotype and function were evaluated on fresh and thawed tregs. cryopreserved autologous t effector (teff) cells were used in mlr assays. before cryopreservation the tregs enriched product mean viability was 95 ± 4% and the mean percentage of cd45+cd4 +cd25+cd127low and cd45+cd4+cd25+cd127lowfoxp3+ cells was 74 ± 13% and 66 ± 10%, respectively. we then analysed the tregs enriched product after thawing. mean viability of thawed tregs, by 7-aad staining, was 85 ± 7%. the viable tregs were almost totally cd4+cd25+ (97 ± 2%). the mean percentage of cd4+cd25+cd127low and cd4+cd25 +cd127lowfoxp3+ thawed cells was 73 ± 14% and 71 ± 20% respectively. the contaminant cells present in the treg enriched product were mostly cd4+cd25+cd127+ (around 18%). we further characterized the phenotype of the cd4 +cd25+cd127low population. this population was almost totally foxp3+ (93 ± 6%) and expressed selected markers at various degree (cd62l (50 ± 2%), cd15s (6 ± 2%), cd45ra+ (19 ± 3%), hla-dr+ (15 ± 10%), ccr7+ (74 ± 5%), cd49d (52 ± 14%), cd26+ (1 ± 0.4%), cd196+cd161+ (4 ± 1%). notably, viable thawed tregs were able to induce inhibition of autologous teff cells in a 1:2 tregs:teff ratio as freshly isolated tregs: 44 ± 16% (thawed) vs 55 ± 24% (fresh) of inhibiton (p4 0.1). in conclusion, here we demonstrated that thawed tregs from healthy donors mantain a stable phenotype. in addition, in our hands tregs show good suppressive ability after thawing despite lower expression of cd62l and cd15s relapsed and refractory malignant b cell diseases: evidence for therapeutic efficacy via subcutaneous administration of anti-cd20 × anti-cd3 antibody lymphomun r buhmann, p ruf, j hess, h lindhofer, u jacob 1 and m dreyling 1 the trifunctional antibody anti-cd20 × anti-cd3 lymphomun represents a chimeric immunoglobulin scaffold (mouse igg2a/ rat igg2b) with promising treatment outcome in patients suffering from malignant b cell diseases. by changing the lymphomun administration route from intravenous (i.v.) to subcutaneous (s.c.) the proinflammatory cytokine-mediated side effects were considerably slighter and generally welltolerated. most importantly, s.c. lymphomun showed outstanding responses in b cell depletion even in the absence of elevated cytokine levels (e.g. il-6) that are required for cytotoxic t cell activation. in summary, the clinical tolerability of s.c. lymphomun may result in a considerable improvement of the subjective well-being and in enhanced mobility due to decreased pain symptomatology. intestinal microbiota disruption is associated with acute gastrointestinal (gi) gvhd and inferior outcome in patients after allogeneic stem cell transplantation (asct). the wide use of systemic broad spectrum antibiotics adds a further risk factor contributing to major microbiota shifts. here, in a retrospective analysis of 200 patients undergoing asct at the regensburg university medical center we assessed the relative expression of paneth cell antimicrobial peptides (amps) in 292 human intestinal biopsies in relation to acute gi gvhd and systemic antibiotic treatment. the relative expression of paneth cell amps was significantly higher in biopsies of the upper gi tract than in the lower gi tract for reg3α (p ≤ 0.001), human defensin (hd) 5 (p ≤ 0.001) and hd6 (p ≤ 0.001). regarding the distribution of paneth cell amps in the gi tract we observed significantly higher expressions of all three paneth cell amps in the duodenum, jejunum and ileum compared to the stomach, colon and rectum (po0.001, figure 1 ). in the presence of acute gi gvhd, paneth cell amps reacted contrarily in the upper and lower gi tract: we observed a decrease of hd5, hd6 and reg3α in the upper gi tract (p ≤ 0.01), similarly paneth cell count dropped in case of severe gi gvhd stage 2-4 (po0.001). however in the lower gi tract severe acute gi gvhd was associated with an increase of paneth cell amps (p ≤ 0.03). initiation of additional systemic antibiotic treatment prior to day 10 after asct correlated with a significantly higher expression of hd5 (p = 0.002) and reg3α (p = 0.01) in intestinal biopsies compared to patients without or with initiation of systemic antibiosis after day 10. however, no significant differences were found in terms of hd6 expression in intestinal biopsies and start of systemic antibiotic therapy. the expressions of hd5, hd6 and reg3α in intestinal biopsies seem to respond to major microbiota disruptions caused by acute gi gvhd or systemic antibiotic treatment. while observations in the upper gi tract seem to reflect paneth cell damage, the relative increase in the lower gi tract may indicate inflammatory induction of amps in colonic epithelial cells in the course of gvhd. [p718] disclosure of conflict of interest: none. patients (55%) were in complete remission at the time of pcy haplo-sct. hematopoietic cell transplantation-comorbidity index was ≥ 3 in 20 patients (74%). thirteen patients (48%) received non-myeloablative conditioning regimen (as baltimore schema, luznik et al. bbmt 2008) prior to haplosct while remaining patients received busulfan-based regimen. all patients were given pcy and both csa and mmf as gvhd prophylaxis. day+100 cumulative incidence of grade 2 to 4 and 3 to 4 acute gvhd was 15% and 7%. 2-year cumulative incidence of chronic gvhd was 12%. the cumulative incidence of non-relapse mortality and relapse at 2 years were 38% and 27%, respectively. with a median follow up of 25 months (range: 4-63), 2-year progression-free and overall survivals were 36% and 39%, respectively. disease status at the time of haplosct was a major determinant for outcome. indeed, 2year nrm and os were 58% and 25% in patients transplanted with active disease, respectively, while corresponding values in patients transplanted in cr were 21% (p = 0.036) and 49% (p = 0.041), respectively ( figure 1a and 1b) . we can conclude that in selected patients who could be candidate for second transplantation, haplosct is feasible and may represent a curative option. the overall incidence of relapse of 27% is promising in this situation for which no alternative for cure is available, with relatively good survival in patients transplanted in cr. however, the very high nrm (58%) in refractory patients should make us consider second transplant with caution in this setting. for these patients, specific developments are needed to avoid procedure-related toxicity. [p722] disclosure of conflict of interest: none. secondary solid tumors following hematopoietic cell transplantation for thalassemia major a natale, s santarone, a meloni, a pepe, m di ianni, s angelini, p di bartolomeo 1 1 dipartimento di ematologia, medicina trasfusionale e biotecnologie-ospedale civile, pescara, italy secondary solid tumors (sst) have been described after hct, in particular for patients affected by hematologic malignancies. there is limited information about the incidence of sst following hct for thalassemia major (tm). the aim of this study was to determine the incidence of sst in 134 patients with tm who received hct in our center between 1983 and 2013. 117 patients survived more than 3 years after hct and were enrolled in the study. of them, 57 were males and 60 females. their median age at time of hct was 10 years (1-29). as conditioning regimen, they received busulfan (14 mg/kg) and cyclophosphamide (200 mg/kg). the gvhd prophylaxis included cyclosporine and methotrexate. all patients received bone marrow cells from an hla identical donor. at time of this report, 112 patients were cured, whereas 5 patients rejected their graft and are now under regular transfusion treatment. overall, the median follow-up after hct was 24 years (3-34). seven patients developed a malignancy 3.2 to 28 years (median 16.4 years) after hct including 2 carcinomas of the tongue, 1 oral squamous cell carcinoma, 1 colorectal cancer, 1 thyroid carcinoma, 1 carcinoma of the uterine cervix, and 1 parotid carcinoma. the 30-yr cumulative incidence (ci) of developing sst was 10+0.17%. all patients underwent surgical resection of the tumor and in addition 4 of them received chemotherapy and/or radiotherapy. of relevance, the 3 patients with cancer of the oral cavity were affected by severe chronic gvhd with buccal cavity involvement. 2 patients (1 with parotid and 1 with tongue carcinoma) died of tumor progression and 5 are living. we compared these results with 2 case control populations. first of all, we investigated the occurrence of solid tumors in the 117 individuals (64 males, median age 10 years at time of marrow donation), who served as stem cell donors for hct. one donor developed breast cancer 29 years after marrow donation at age of 38. the 30-yr ci of developing solid tumor for donors was 4.5+0.21% with a statistically significant difference (p = 0.03) as compared to that of transplanted patients. the second case control population consisted of 117 patients affected by tm treated with transfusions and iron chelation. the matching technique applied was based on the variables age and sex. one control per case (transplanted patient) was randomly selected from the miot (myocardial iron overload in thalassemia) registry and matched by sex and age with the transplanted patient population. 2 patients developed an hepatocellular carcinoma (hcc) at age of 39 and 44 years, respectively. one patient died and one is living. using the event rate measure, we observed an event rate of 0.102 at 30 years for the transplant group and 0.041 for the nontransplant group (p = 0.106). this study shows that the magnitude of increased risk of sst is twofold to threefold for patients treated with hct as compared with an age-and sex matched nontransplant tm patients or with stem cell donors. notably, among the transplanted patients we didn't observe any case of hcc, which is one of the most frequent solid tumor in nontransplant tm patients, whereas we observed 4 cases of head/neck cancers. in our series, cgvhd seems to be a strong risk factor in the development of new solid tumors. patients with cgvhd, especially those with involvement of the oral cavity, must receive a very long careful monitoring and surveillance in order to prevent the development of secondary cancers. disclosure of conflict of interest: none. sequential treatment with bortezomib plus thalidomide plus dexamethasone followed by autologous hematopoietic stem cell transplantation (hsct); consolidation and maintenance therapy in patients with multiple myeloma a bachiri 1 , ma bekadja 2 , s talhi 2 , s abderrahmani 1 , h ouldjeriouat 3 and r bouhass 2 1 department of hematology, hmru oran, oran, algeria; 2 department of hematology and cell therapy, ehu1st november, oran, algeria and 3 department of hematology and cell therapy, oran, algeria the management of multiple myeloma (mm) has been significantly improved in recent years in young patients, where ahsct and advent of new molecules was introduced as first line treatment. the sequential treatment (induction followed by autologous hematopoietic stem cell transplantation; consolidation and maintenance therapy) has increased rates response (cr and vgpr) as well as the overall survival. our purpose was to assess the efficacy and adverse effects of sequential treatment with vtd chemotherapy and autologous hsct followed by consolidation and maintenance therapy. in a prospective multicenter study, we evaluated this mm management strategy at oran, in two hematology centers. patients aged under 65 years with de novo mm, were treated with induction included: bortezomib (1.3 mg/m 2 , d1-d4-d8-d11), thalidomide (100 mg/ m 2 d1-d21) and dexamethasone (40 mg, d1-d4; d8-d11). a total of 3 to 4 cycles where delivered every 21 days. autologous stem cell was mobilized using g-csf alone (15 μg/kg/day for 5 days). leukapheresis to harvest stem cells were performed on day -2 and -1. the conditioning regimen consisted of melphalan 200 mg/m 2 . a consolidation phase was initiated two months later with the same protocol (vtd), followed by a maintenance treatment with thalidomide 50 mg/day given orally for 12 months. this study was done over a 6-years period (january 2010-december 2015). fifty patients were included. they include 19 women and 31 men (sex ratio = 1.63). the median age at diagnosis was 53 years (32-64). according to durie salmon staging, 80% of patients were in stage iii, while 38% were in stages iii according to iss staging. the monoclonal component was igg in 56% of patients. postinduction overall response rate in the 50 eligible patients was 100%, including 52% vgpr and 38% cr/ and 10% pr rates. the median of cd34 + rate was 3.88x10 6 /kg (1.41 to 11). all patients had engraftment on the median of day 10 (range; 7 to 14) and platelet transfusion independence on the median of day 13 (range; 9 to 19). there was no graft failure. one patient died following the procedure (trm). posttransplantation on day 100, cr and at least vgpr remained significantly higher (98%). in the 49 evaluable patients, the estimated os at 79 months was 82%, the estimated dfs at 43 months was 66% and the pfs at 78 months was 66.5%. at the 30/09/2016, 43 (86%) patients are alive and 39 (80%) without disease activity after a median follow-up of 33 months (range; 3-79). the main hematological toxicities post transpland (grade 3/4) were thrombocytopenia (49%), neutropenia (50%), and anemia (8%). the most frequently observed nonhematological toxicities (all grades) included peripheral neuropathy (66%). our experience suggests that the sequential protocol used in first line produce a better outcome with fewer adverse events and is an interesting therapeutic option in term of efficacy and tolerance. disclosure of conflict of interest: none. micrornas are small, non-coding single-stranded rnas and regulate approximately 50% of all genes by repressing translation. they are present in bodily fluids, where they are protected from rnase-mediated degradation by encapsulation into extracellular vesicles (evs) and demonstrate a novel capacity to regulate the cellular differentiation of blood cells and immune function. candidate micrornas mir-377, mir--199, mir-93* and mir-423 have previously been associated with acute graft versus host disease (agvhd) in posthematopoietic stem cell transplant (hsct) patient plasma. however, validation in independent cohorts is necessary, and their presence within extracellular vesicles (evs) has not been explored. microrna expression was evaluated in a prognostic cohort (n = 81) of day 14 (d14) post-hsct patient serum samples by taqman qrt-pcr. further assessment in an independent cohort of serum samples taken at the time of agvhd diagnosis was also performed. expression was also assessed in serum evs at sequential time points (pre-hsct, d0, d7 and d14) and an independent verification cohort of d14 serum samples by ev isolation, rna extraction and taqman qrt-pcr analysis. this study replicated elevated serum expression of mir-423 (po 0.001), mir-199 (p = 0.04), mir-93* (p o0.001) and mir-377 (p = 0.03) in agvhd, in a prognostic cohort of d14 post-hsct patient samples (n = 81). expression was also associated with disease severity. further analysis at agvhd diagnosis in an independent cohort (n = 65) confirmed high expression of mir-423 (p = 0.02), mir-199 (p = 0.007) and mir-93* (p = 0.004) at disease onset. investigation of microrna expression patterns during early hsct at sequential time points (pre-hsct to d28) identified elevated micrornas at d7 post-hsct in all transplant patients. in a novel investigation of microrna expression in serum evs (n = 15), mir-423 (p = 0.09), mir-199 (p = 0.008) and mir-93* (p = 0.001) levels were lower at d14 in patients who later developed agvhd, and this was replicated for mir-423 (p = 0.02) and mir-199 (p = 0.04) (n = 47). comparing serum to circulating evs, at d14 patients remaining agvhd-free had significantly higher expression of mir-423 (p = 0.03), mir-199 (p o0.001) and mir-93* (p = 0.001) in the ev fraction. results validate the capacity for circulating serum mir-423, mir-199 and mir-93* to act as diagnostic and prognostic biomarkers for agvhd. novel findings of differential expression between whole serum and the ev compartment prior to disease onset suggest a role for ev micrornas in the biology of agvhd, which warrants further investigation. disclosure of conflict of interest: none. prior data indicate similar outcomes after transplants from hla-haplotype-matched relatives, hla-idntical siblings and hla-matched unrelated donors. we used our dataset to answer a clinically important question: who is the best donor for a person with acute leukemia. we analyzed data from persons with acute leukaemia in 1 st complete remission treated in a prospective, multi-centre study. patients were randomly divided into training (n = 611) and validation (n = 588) sets. 1199 consecutive subjects received a transplant from an hla-haplotype-matched relative (n = 685) or an hlaidentical sibling (n = 514). 3-year leukaemia-free survivals (lfss) were 75% (95% confidence interval [ci], 72, 78%) and 74% (70, 78%; p = 0.95). the multivariate model identified 3 major risk factors for transplant-related-mortality (trm): older donor/recipient age (donor440years/recipient430 years; hazard ratio [hr] = 1.88; [1.05, 3.35]; p = 0.03), female-to-male transplants (hr = 2.11; [1.50, 2.98; p = 0.01) and donor-recipient abo major-mismatch transplants (hr = 1.55 [1.08, 2.23; p = 0.02). a risk score was developed based on these three features. trms were 8% (5, 10%), 15% (12, 18%) and 31% (19, 43%) for subjects with scores of 0-1, 2 and 3 (p o0.001). 3 year lfs were 78% (75, 81%), 74% (70, 78%), and 58% (45, 71%; p = 0.003). the risk score was validated in an independent cohort. in recipients 450years, lfss were 69% and 86% (p = 0.08) after transplants from identical-sibling or children. our data confirm donor source or degree of hla-disparity is not significantly correlated with transplant outcomes. selection of the best donor needs to consider donor-recipient age, sex-matching and abo-incompatibility amongst persons with acute leukemia receiving transplants from family members. [p726] disclosure of conflict of interest: none. synergistic effect of kir ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation for treatment of myeloid malignancies d cardozo, a marangon, r da silva, fj aranha, j visentainer, s bonon, s costa, e miranda, c souza and f guimarães. the lack of one or more hla class i alleles, whose protein products are the ligands for kir receptors, has been exploited as a prognostic factor for the outcome of patients with haematological malignancies treated by haematopoietic stem cell transplantation (hsct). although it has been accepted that kir-hla interactions may influence the outcome of the hlamismatched hsct, there is no consensus regarding the settings of hla-matched transplantation. there are studies that have reported either benefits, or no effects, under the influence of inhibitory kir-hla interactions. additionally, certain activating kirs and/or reactivation of cytomegalovirus (cmv) infection have been reported to affect the outcome of hla-matched transplantation. the goal of this study was to evaluate the influence of kir-hla genotypes on the outcome of patients undergoing treatment for haematological malignancies by non-t-depleted lymphocyte haematopoietic stem cell transplantation (hsct) from hla-matched sibling donors. the prospective study was conducted at the center of hematology, university of campinas, and 50 patients and their donors were followed up from 2008 to 2014. kir and hla class i genes were genotyped and patients grouped based on the presence of kir ligands combined with kir genotype of their respective donors. patients with all kir ligands present (n = 13) had a significantly higher (p = 0.04) incidence of acute graft-versus-host-disease (gvhd) than patients with one or more kir ligands missing (n = 37). the overall survival following transplantation of patients with myeloid malignancies (n = 27) was significantly higher (p = 0.035) in the group with one or more kir ligands missing (n = 18) than in the group with all ligands present (n = 9). presence of kir2ds2 was associated with a worsening of hsct outcome while reactivation of cytomegalovirus (cmv) infection improved the outcome of patients with one or more kir ligands missing. our results indicate that kir-hla interactions affect the outcome of the hla-matched transplantation, particularly in patients with myeloid malignancies. disclosure of conflict of interest: none. p = 0.015), lower disease-free survival (p = 0.015 and p = 0.015) and lower overall survival (p = 0.01 and p = 0.014). one-year cir of the above two groups were 5.6 ± 5.4% vs. 57.1 ± 18.7% in mrd negative and positive patients, respectively (p = 0.001). in addition, those who had consistent positive mrd prior to hlamatched sibling hsct showed even worse outcomes compared to patients without pre-mrd. unmanipulated haploidentical hsct might have the stronger graft-versus-leukemia effect compared to hla-matched sibling hsct. it suggested that those who received unmanipulated haploidentical hsct with pre-mrd might not need more intensive relapse intervention after transplantation. disclosure of conflict of interest: none. the retrospective study of allogeneic hematopoietic cell transplantation for 36 patients with mixed-phenotype acute leukemia in toranomon hospital, japan in the real clinical setting, however, there are substantial number of patients who can not achieve cr after chemotherapy. we conducted a retrospective study including such patients to elucidate the outcome of allogeneic hct in toranomon hospital, japan. we studied the patients with mpal diagnosed from july 2008 to september 2015. mpal was diagnosed according to who classification in 2008. from june 2013, we examined cytoplasmic myelo-peroxydase (cmpo) routinely for flowcytometric analysis in all the patients, to distinguish mpal from acute lymphoblastic leukemia (all). we included the patients who were diagnosed as mpal in toranomon hospital, regardless of their diagnosis or clinical course in the previous hospitals. a total of 36 mpal patients underwent their first allogeneic hct with related bone marrow or peripheral blood stem cells (r-bm/pb) (n = 9), unrelated bone marrow (u-bm) (n = 6), and unrelated umbilical cord blood (u-cb)(n = 21). the median patient age was 41 years (range:17-69). the immunophenotype of leukemia cells included 23 cases of b and myeloid (b/my) (64%) and 13 cases of t and myeloid (t/my) cell lineage(36%).eleven patients(31%) harbored philadelphia chromosome. the remission induction chemotherapy was performed with all-type regimens in 31 patients, and acute myeloid leukemia (aml)type regimens in 5 of 36 patients, 18 patients(50%) were not in cr at the time of transplantation. myeloablative conditioning (mac) regimens were used in 30 pantients(83%). the 2-year overall survival (os) rate was 43.1% (95% confidence interval (ci), 25.7-59.4%). to identify the factors that influenced os, we performed univariate analysis and compared the following pre-transplantation factors: age at the time of transplantation ( o41 vs.4 = 41 years), committed immunophenotype (b/my vs.t/my), karyotype (philadelphia chromosome (ph vs.non-ph), disease status at the time of transplantation (cr vs.non-cr), donor cell source (r-bm/pb vs.u-bm vs.u-cb, cb vs.non-cb), and conditioning regimen (mac vs.reduced intensity conditioning). cr at the time of transplantation was extracted as a significant predictive factor for the better os(2-year os; cr vs. non-cr, 63.0% (95% ci, 32.1-82.8%) vs.22.2% (95% ci, 6.9-42.9%), p = 0.009). the cumulative incidence of relapse rate (rr) at 2 years after transplantation was 53.3% (95% ci, 31.8-70.8%). to identify the factors that influenced relapse rate, we performed univariate analysis and compared pretransplantation factors same as above. harboring philadelphia chromosome was extracted as a significant predictive factor for lower relapse rate (2-year rr; ph vs.non-ph, 27.3%(95% ci, 0.1-77.3%) vs. 66.5%(95% ci, 41.4-82.8%), p = 0.003). the older patients(p = 0.07) and the patients in cr (p = 0.05) also showed a trend towards lower relapse rate. allogeneic hct provided 63.0% of 2-year os for mpal patients in cr at the time of transplantation. on the other hand, for patients not in cr, 2year os was approximately 20%. the use of tyrosine kinase inhibitors along with chemotherapy before transplantation might prevent relapse after transplantation in mpal patients with ph chromosome. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (allo-hsct) is a standard of treatment for many patients with hematological malignancies. however, the disease relapse and graft failure after first allo-hsct (1 st allo-hsct) lead to poor outcomes almost in all cases. second allo-hsct (2 nd allo-hsct) is one of primary options that can decrease the mortality in this group of patients. here we report our experience of 15 patients who underwent 2 nd allo-hsct. the aim of the study was to estimate a clinical efficiency and practicability of 2 nd allo-hsct. we included 15 patients (9 males/6 females) with acute myeloid leukemia (aml, n = 10), acute lymphoblastic leukemia (all, n = 3) and myeloproliferative disease (mpd, n = 2) who underwent 2 nd allo-hsct for relapse (66,7%) or graft failure (33,3%) from the same (n = 8) or another donor (n = 7) between november 2012 and october 2016. median age was 31 years (range: 18-42 years). three (20%) patients had a matched related donor (mrd), nine (60%) patients had a matched unrelated donor (mud) and three (20%) patients had a mismatched unrelated (mmud) at the second transplant. to evaluate time gap affecting outcomes all patients were divided into two groups: who underwent 2 nd allo-hsct in more/less than 6 months after 1 st allo-hsct. in "less than 6 months" group three patients were re-transplanted for relapse and one-for graft failure, in other group there were seven patients who received 2 nd allo-hsct for disease relapse and four-for graft failure. fisher's exact test were performed to exclude probability of imbalance between groups (p40.05). median of overall survival (os) and disease-free survival (dfs) after 2 nd allo-hsct was 13.5 months and 10.59 months respectively. (see figure 1a ,1c) two patients (13.3%) developed graft failure and three relapsed (20%). acute graft-versus host disease (agvhd) incidence was extremely low as 13.3% (n = 2) even despite use of mud/mmud in 80% of cases. mortality rate were 53.3% in a group of 2 nd allo-hsct. it should be noted that only 3 (20%) patients died because of disease progression. five patients (33.3%) died in complete remission due to severe infections or previous toxicity (e.g. heart failure). the effect of donor change on dfs was not significant (p = 0,88). our statistical analysis reveal significantly differences in os in patient with long-term interval (46 months) between 1 st and 2 nd allo-hsct. median of os in patients who underwent 2 nd allo-hsct in more/less than 6 months after 1 st allo-hsct was 20,59 vs 7,02 months respectively. (see figure 1b ,1d) for hazard ratio (hr) estimation mantel-haneszel approach were used hr for group who were transplanted in less than 6 months from 1 st allo-hsct was 8.36, (95% ci, 1.054 s511 to 66.38, p = 0.04). as for dfs difference was not significant (p = 0.07). according to our analysis, performing 2 nd allo-hsct in a period less than 6 months after 1 st allo-hsct seemed not very reasonable due to extremely high mortality even in young patients (hr-8.36, p = 0.04). as for "more than 6 months" group it can be considered even despite hla-disparity between donor-recipient pair due to extremely low agvhd rate (13.3%). donor change was not associated with better outcome (p = 0.88) disclosure of conflict of interest: none. hemopoietic stem cell transplantation (hsct) is an effective treatment for many hematologic disorders, and globally over 70 000 procedures/year are performed in more than 70 countries. however, not all the countries have enough resources and expertise to establish an hsct program, and patients are often forced to emigrate for transplantation, with heavy social and economic consequences. in the year 2015 the iuc (an italian ngo) identified the hiwa cancer hospital (hch) in sulaymaniya (iraqi kurdistan) as a possible site for the establishment of a new hsct transplant center. a hsct expert from italy (mi) following a visit to the hch, reported a positive conclusion on the feasibility of an hsct project. this was mainly due to the fact that many of the required technologies were already available at hch, including a 6-bed positivepressure, hepa-filtered-air clinical unit, 2 last-generation cell separators and a well equipped hla laboratory. following this preliminary survey, a capacity building project was rapidly made and submitted to the italian agency for development cooperation, that approved and funded it in march 2016 with the specific aim to cure thalassemia patients either of kurdistan and of the refugees population from syria and other parts of iraq. in april 2016, the joint italian and kurdish team started the project. a first autologous transplant was done in june 2016 followed by 8 more autografts (overall, 5 myeloma and 4 lymphoma patients). in october, following appropriate downstaging, a first low-risk thalassemia patient was allografted from her hla-id sibling, followed by 2 more patients. all the patients engrafted promptly, with one death occurring on day +23 with acute cardiac failure and a major toxicity recorded in a single patient (nhl, severe enterocolitis with perforation) that was successfully treated. the full process for the start-up included the following activities developed during 8-month time: (1) s512 of transplants, the hch group also submitted to ebmt an application for full membership, that was promptly approved. in all this project, the italian counterpart provided over 30 highly-experienced volunteer specialists (physicians, nurses, technicians and one physicist), each with a specific mission plan. despite the many difficulties and obstacles encountered, the clinical results obtained so far appear encouraging, though there is still need to furtherly support the hch in order to make it totally independent. following this intervention, the hch is the only one center performing both auto and allo hsct not only in the iraqi kurdistan region, but also in all the iraqi nation. we conclude that international cooperation may be fruitful also in the field of high-technology medicine, and may contribute to improve the capabilities of centers even in critical geographic areas, representing a valuable instrument also to implement nation-to-nation scientific exchanges. disclosure of conflict of interest: none. the use of plerixafor with g-csf in conditioning regimen for hematopoietic stem cell transplantations with tcr alpha/beta and cd19 depletion of graft in wiscott-aldrich syndrome patients: a single-center experience b dmitry 1 , l alexandra 2 , s larisa 1 , g elena 1 , s irina 1 , t pavel 1 , k rimma 1 , n galina 3 , m michael 1 and m alexei 1 grade 2 acute gvhd (agvhd) was 13% (2 pts). no pt experienced a grade 42 agvhd. three patients presented a limited form of chronic gvhd (21%). incidence of oral mucositis and gastrointestinal/liver toxicity has been extremely low in this population of patients, even in those with active disease and heavily treated at the time of transplant. eight out of fifteen pts (53%) are alive with a median follow-up of 31 months (range: 12 -48 m). seven (47%) are in cytogenetic/molecular remission. six out the eight patients who were transplanted in cr1 or cr2 are alive (75%), while two out the seven patients who were transplanted in advanced phase are alive (29%). in this preliminary clinical experience, we find that unmanipulated haploidentical transplants with post-transplant cyclophosphamide are a valid alternative and have outcome comparable to unrelated and match sibling transplants, in pts with hematologic malignancies. advanced disease is the only adverse factor for diseasefree survival. we therefore consider this therapeutic option when a match sibling or a 10/10 ag mud donor is not immediately available. disclosure of conflict of interest: none. autism spectrum disorder (asd) is a group of neurodevelopmental disorders characterized by impaired social communication and interactions with restricted and repetitive behaviors. although asd is suspected to have either heritable or sporadic genetic basis, its fundamental etiology and pathogenesis are poorly understood. recently researchers have suggested that stem cells have therapeutic potential for asd. wharton's jelly-derived msc (wj-msc) from third-party donors (tpd) have high proliferation and differentiation potential. this cell population has also non-immunogenic and immunomodulatory properties, thus seem to be a promising treatment stem cell source. the polish stem cell bank (pbkm) has provided wj-msc for clinical application in a medical therapeutic experiment for children with asd. twenty-three patients (pts) with asd aged from 3 to 18. 10/12 (median age: 7 years and 7 months), after bioethical committee approval, received intravenous injections of wj-msc, obtained from tpd. the cells were previously collected from healthy newborns, then processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapour. wj-msc immunophenotype was confirmed using flow cytometry assay. the pts received from 1 to 5 injections in intervals from 4 to 12 weeks. the average cell dose per infusion was 1.01 × 10^6/kg of body weight (bw). each pt was examined by the same neurologist at the day of infusion. comorbidities present in some patients: unspecified speech disturbances, flaccid paralysis, flaccid tetraplegia, unspecified encephalopathy, epilepsy, sensorineural hearing loss. one patient was diagnosed with 2 comorbidities: conductive hearing loss and intellectual disability. almost 80% of pts, after their treatment with wj-msc, revealed positive changes in neurological examination. an improvement in speech was observed in 10 pts and improvement of cognitive functions ensued in 7 pts. what is more, 26% of children showed progress in self-reliance, social interactions and improved their ability to concentrate. there was a reduction of aggressive behavior in 4 pts and 2 pts have experienced better quality of sleep. there was only one adverse event after wj-msc infusions -psychomotor agitation occurred in 24 hours after the administration. five follow-ups have not yet been completed. the administration of thirdparty donor wj-msc seems to be safe and efficient procedure with promising preliminary results in patients with asd. hematopoietic stem cell transplantation between red cell incompatible donor-recipient pairs red blood cell depletion from bone marrow and peripheral blood buffy coat: a comparison of two new and three established technologies human bone marrow processing using a new continuous-flow cell separation device disclosure of conflict of interest: none. references 1. zama d, et al. gut microbiota and hematopoietic stem cell transplantation: where do we stand? bmt the kyoto encyclopedia of genes and genomes-kegg metabolites produced by commensal bacteria promote peripheral regulatory t-cell generation disclosure of conflict of interest: none antifungal prophylaxis in hematopoietic stem cell transplant recipients: the unfinished tale of imperfect success guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective differences in aspergillus-specific immune recovery between t-cell-replete and t-cell-depleted hematopoietic transplants toxoplasmosis following allogeneic hematopoietic stem cell transplantation diagnosis of toxoplasmosis after allogeneic stem cell transplantation: results of dna detection and serological techniques implementation of molecular surveillance after a cluster of fatal toxoplasmosis at 2 neighboring transplant centers management of high blood pressure genes for blood pressure a prospective studyon the predictive value of plasma bk virus-dna load for hemorrhagic cystitis in pediatric patients after stem cell translantation cidofovir for bk virusassociated hemorrhagic cystitis:a retrospective study hemorrhagic cystitis after bone marrow transplantation bcsh/bsbmt guideline: diagnosis and management of veno-occlusive disease (sinusoidal obstruction syndrome) following haematopoietic stem cell transplantation drug safety evaluation of defibrotide defibrotide for prophylaxis of hepatic veno-occlusive disease in paediatric haemopoietic stem-cell transplantation: an open-label, phase 3, randomised controlled trial safety and effects of prophylactic defibrotide for sinusoidal obstruction syndrome in hematopoietic stem cell transplantation disclosure of conflict of interest: none university children's hospital basel, division of paediatric oncology/haematology late complications subcommittee of translated related complications and quality of life wp; 5 clinic of paediatric haemato-oncology, department of women's and children's health, university of padova, italy; 6 department of surgery, division of transplantation division of blood and marrow transplantation, the children's hospital at westmead ovarian function after bone marrow transplantation during childhood pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning haploidentical hematopoietic transplantation:current status and future perspectives t-cell replete haploidentical donor transplantation using post-transplant cy: an emerging standard-of-care option for patients who lack an hla-identical sibling donor hematopoietic stem cell transplantation in thalassemia major and sickle cell disease: indications and management recommendations from an international expert panel allogeneic stem cell transplantation for thalassemia major killer-cell immunoglobulin-like receptors reactivity and outcome of stem cell transplant kir b haplotype donors confer a reduced risk for relapse after haploidentical transplantation in children with all kir/hla interactions negatively affect rituximab-but not ga101 (obinutuzumab)-induced antibody-dependent cellular cytotoxicity reduction of minimal residual disease in pediatric b-lineage acute lymphoblastic leukemia by an fcoptimized cd19 antibody diagnoses: hodgkin's lymphoma(hl)-38 pts (refractory 22; relapsed 16); non-hodgkin's lymphoma(nhl disease status before asct: 1st (after refractority prior radiotherapy to the mediastinum -19/48 (39.6%); heavily pretreated patients with advanced disease (x 3 lines previous treatment) 18/48 (37.5%). grafts: pbsc -43/48 pts with median of cd34+cells-3 ccnu dose: 47/48 pts 300mg/m2; 1pt 400 mg/m2. engraftment: anc>500 cells/mkl: median=d+11(8÷24), 47/48pts. plt>50 000 cells/ mkl: median=d+ 28(13÷122), 43/48pts. full engrafted 43/ 48pts /8 pt required a short-term mechanical ventilation (2 of them died because of lung infection ad d+68 and d+82) aeruginosa associated sepsis on a background of graft failure); 2pts (4.2%)-d+68 and d+82 (pulmonary toxicities +infection; both had prior mediastinal radiotherapy). relapse/ progression after asct-8/48 pts (16.6%), 6 of them died. 1 pt achieved secondary mds (diagnosed 4.5 mo after asct). for this group of pts with relapsed/refractory lymphomas (n=48) 11-year os=0 for nhl(n=10) efs=0.88 (se ± 0.2) lomustine-containing conditioning regimen cem (lomustine, etoposide, melphalan) is effective and feasible in autologous stem cell transplant efficacy and toxicity of a ccnu-containing high-dose chemotherapy regimen followed by autologous hematopoietic cell transplantation in relapsed or refractory hodgkin's disease champlin re reduced-toxicity conditioning therapy with allogeneic stem cell transplantation for acute leukemia idarubicin-intensified bucy2 conditioning regimen improved survival in high-risk acute myeloid, but not lymphocytic leukemia patients undergoing allogeneic hematopoietic stem cell transplantation: a retrospective comparative study comparison of outcomes of idarubicin intensified tbi-cy and traditional tbi-cy conditioning regimen for high-risk acute lymphoblastic leukemia undergoing allogeneic hematopoietic stem cell transplantation: a single center experience inhibition of cd25 (il-2r alpha) expression and t-cell proliferation by polyclonal anti-thymocyte globulins csf-primed bone marrow transplantation for patients with high-risk hematologic malignancies in an exploratory analysis, os after hct appeared to be longer in the cpx-351 arm in both age groups. these results suggest that cpx-351 may provide an effective bridge to successful transplant for a high-risk subgroup of aml patients. support: celator pharmaceuticals, inc., a subsidiary of jazz pharmaceuticals plc consulting ambit biosciences, amphivena therapeutics, ariad, astellas pharma sunesis, tolero; institutional research funding abbvie chiarella and louie: employment celator/jazz; stock jazz pharmaceuticals plc. hoering disclosure of conflict of interest: none. inkt-/nk-/cik-cell (subsets) are important for immunesurveillance. antibody 6b11 targets the vα24-jα18-invariant-t-cell-receptor (tcr) in the cdr3-region, which is semiinvariantly rearranged in inkt-cells. we characterized: i.) inkt-/nk-/cik-subsets in pb-samples from healthy donors (n = 9 subsets under stimulation with dendritic-cells of leukemic origin (dc leu ), generated from aml-blasts in mononuclear cells(mnc) and whole-blood (wb, containing soluble/cellular components of pts' pb) with 'cocktails' (dc-generating-methods/kits). 1.1) compared to healthy mnc (significantly) lower proportions of inkt-cells comparable correlations were seen in adultall-and cll-pts. 2.1) we quantified inkt-/nk-/cik-subsets before/after mixed-lymphocytecultures (mlc) of t-cell-enriched immune-reactive cells stimulated with mnc/wb (with or without pretreatment 'cocktails' inducing blasts' conversion to dc leu ) from aml-pts. our findings show, that 1)inkt-/nk-/cik-cells increase after mlc independent of the stimulator-cells-suspension (under the influence of il-2); 2) pretreatment of mnc/wb-blasts with 'cocktails' increases inkt-counts and induces a shift in the composition of inkt-/nk-/cik-subsets after mlc, that might correlate with an improved antileukemic potential; 3) individual samples showed varying, however higher inkt-, cik-cell-counts after pretreatment with different (especially prostaglandin-containing) 'cocktails'; 4) dc-/inkt-/nk-/cikcells-values after mlc were comparable in physiological hypoxia vs normoxia; 5) in cases with antileukemic blast-lytic activity after mlc t-/inkt-/nk-/cik-cells were significantly increased-pointing to an involvement of these cells in antileukemic reactions. in summary: (1) healthy mnc present with significantly higher inkt-/nk-/cik-cells compared to aml/all/cll-leukemic mnc. (2) subtypes of inkt-cells differ in healthy vs leukemic samples, resembling a shift in the composition of inkt-cells. (3) amounts of inkt-/ nk-/cik-cells in aml/all/cll-mnc-samples correlate with prognosis. (4) 'cocktail'-treated aml-blasts (resulting in dc leu ) lead to a shift in t-,inkt-/nk-/cik-cell-counts/compositions, what correlates with improved antileukemic activity against aml-blastspointing to a cross-talk of these cells. proportions of inkt-/ nk-/cik-cells management of philadelphia chromosome-positive acute lymphoblastic leukemia (ph+ all) outcome of allogeneic stem cell transplantation for aml and myelodysplastic syndrome in elderly patients (⩾60 years) comorbidity-age index: a clinical measure of biologic age before allogeneic hematopoietic cell transplantation high rate of hematological responses to sorafenib in flt3-itd acute myeloid leukemia relapsed after allogeneic hematopoietic stem cell transplantation phase i trial of maintenance sorafenib after allogeneic hematopoietic stem cell transplantation for fms-like tyrosine kinase 3 internal tandem duplication acute myeloid leukemia haematopoietic cell transplantation with and without sorafenib maintenance for patients with flt3-itd acute myeloid leukaemia in first complete remission quantitative monitoring of minimal residual disease (mrd) after sct was performed by four-colour flow cytometry and/or real time pcr. the median time of neutrophil engraftment (above 0.5 × 10e9/l) was 16 days, 96% of pts (24/25) engrafted, one patient died in aplazia. non-relapse mortality (nrm) after 1 year and 2 years was 8% (2/25) and 13% (3/25). causes of death were refractory gvhd (n = 1), infection (n = 1) and multiorgan failure (n = 1). incidence of acute gvhd was evaluated in 23 pts: 52% (12/23) of pts had gvhd (grade i+ii in 7 pts, grade iii in 5 pts). incidence of chronic gvhd was evaluated in 22 pts, 41% (9/22) of pts had gvhd with median follow-up from sct 37 months (range: 4-90), 64% of all pts (16/25) were alive (14 in remission of cll with mrd negativity, 2 with relapse), 9 pts died (3 from nrm, 6 from cll relapse/progression), 8 relapses (32%; 8/25) occurred. sequential use of chemotherapy and ric regimen with allogeneic sct is safety and effective treatment of high-risk cll with reponse rate 86% and low nrm. progression-free survival and overall survival at 3 years from sct were 56% and 64% 1 department of hematology, hemostasis, oncology and stem cell transplantation hannover deutsche klinik für diagnostik helios klinik wiesbaden, germany; 19 imperial college london at hammersmith hospital du cane road centre for haematology london disclosure of conflict of interest: none. references 1. sibon d, brice p. optimal treatment for relapsing patients with at our institution, pr-hl is defined as partial response (pr), no response (nr), stable disease (sd), progressive disease (pd), relapsing within 3 months of finishing the planned treatment. progression free (pfs) and overall survival (os) from the day 0 of auto-sct was estimated by kaplan-meier (km) method. from 1996 to 2014, 234 patients with aethera trial criteria were identified. male 121 (52%), female 113 (48%), median age at diagnosis:22 yrs (12-61), at auto-sct: 24.3 yrs (13.8-63)(90% o40 yrs). initial chemo: abvd in 194 (83%). 70 (30%) had radiation therapy (xrt) after initial chemo. response to initial chemo + xrt was refractory disease:152 (65%), relapse between 3-12 months:49 (21%) and relapse after 12 months:33 (14%) aethera had 28% stable disease before sct vs we have only 6%. aethera 24 months pfs (45% control arm, 65% brentuximab arm, investigator assessment) and our 56.4% is not much different. despite having similar selection criteria, our median pfs is higher than both aethera trial placebo and experimental arm. clinically, rate of progression in both studies are very high and comparable at 24 months. given the very high cost of this drug and while waiting for survival fifty-nine (82%) and 13 (18%) patients had relapsed and primary refractory chemosensitive dlbcl, respectively. secondary ipi was 0-1 in 23 (44%) patients, 2 in 13 (25%) patients and 3-4 in 16 (31%) patients. fifty-one (71%) and 21 (29% patients had gcb and abc tumors, respectively. abc patients received more prior lines of chemotherapy than gcb patients (76% vs 48% received 4 2 lines of chemotherapy, p = 0.03). the rest of characteristics were equally distributed between both groups (table 1) disclosure of conflict of interest: none disclosure of conflict of interest: none. p631 upfront autologous stem cell transplantation in patients with diffuse large b cell lymphoma: focused on risk factors for survival and conditioning regimens ds kim 1 association between complete response and outcomes in transplant-eligible myeloma patients in the era of novel agents e jantunen 1 and v varmavuo 12 1 department of medicine disclosure of conflict of interest: none. and 6 hematology department lenalidomide after stem-cell transplantation for multiple myeloma bortezomib induction and maintenance treatment in patients with newly diagnosed multiple myeloma: results of the randomized phase iii hovon-65/gmmg-hd4 trial disclosure of conflict of interest: none we performed a retrospective study to investigate survival outcomes and toxicities of l maintenance therapy compared with b maintenance in mm patients post-ahct. this study included 156 patients who received ahct for mm between 2008 and 2015 after induction with l-or b-based therapy. all patients received ahct within 12 months of mm diagnosis and received melphalan 200 mg/m 2 conditioning. patients who received tandem transplantations (autologous or allogeneic) were excluded. only patients initiating maintenance therapy within 6 months post-ahct were included. maintenance therapy was defined as monotherapy with either l or b. the primary outcome was pfs. secondary outcomes were overall survival (os) and treatment-related toxicities. 92 patients received l maintenance and 64 b maintenance post-ahct. at baseline there were no differences in iss stage, ds stage or cytogenetic risk between maintenance cohorts. at time of analysis, 49% (n = 45) receiving l maintenance and 52% (n = 33) on b maintenance experienced disease progression. median time to progression (1.71 vs 1.74 yrs, p = 0.77) was not significantly different between cohorts. by multivariable analysis, choice of maintenance (l vs b) was not significant for pfs or os. variables significant for improved pfs were iss stage i disease response improved while on maintenance in 38% (n = 24) with l and 34% (n = 31) with b. median os was not statistically different between maintenance cohorts (4.28 vs 5.77 yrs, p = 0.47). iss stage i/ii vs iii while cytopenias were more common in the l cohort (30% vs 3%, p o 0.01). the median follow-up time for survivors was 33 months. these findings suggest that both lenalidomide and bortezomib are equivocal maintenance therapy options for post-transplantation mm patients. choice of maintenance therapy post-ahct for mm did not demonstrate a difference in survival outcomes. based on these data, maintenance choice should be guided by patient specific anticipated tolerance rather than drug type alone. iss stage and post-ahct disease response continue to be significant predictors for outcomes. toxicities recorded on maintenance were as anticipated. length of maintenance therapy may be a significant predictor and warrants further analysis. the analysis was underpowered to disclosure of conflict of interest: none. p650 real-world multiple myeloma management practice patterns and outcomes in six central and eastern european countries d coriu 1 , d dytfeld 2 , d niepel 3 , i spicka 4 second autologous stem cell transplantation as salvage therapy for multiple myeloma: impact on progression free survival and overall survival second autologous stem cell transplantation: an effective therapy for relapsed multiple myeloma second auto asct for treatment of relapsed multiple myeloma the role of second autografts in the management of myeloma at first relapse moving beyond autologous transplantation in multiple myeloma ebmt data office bortezomib-based versus nonbortezomib-based induction treatment before autologous stem-cell transplantation in patients with previously untreated multiple myeloma: a meta-analysis of phase iii randomized, controlled trials first-line therapy with thalidomide and dexamethasone in preparation for autologous stem cell transplantation for multiple myeloma mechanism of action of bortezomib and the new proteasome inhibitors on myeloma cells and the bone microenvironment: impact on myeloma-induced alterations of bone remodeling boys; 19 girls) with following mds types: refractory cytopenia of childhood-11 (19%), refractory anemia with excess blasts -14 pts (24%), refractory anemia with excess blasts in transformation-22 pts (38%), juvenile myelomonocytic leukemia in 11 pts (19%). the median of age was 7 years (1-19 years) mac consisted busulfan (bu) 16 mg/kg + cyclophosphamide 120 mg/kg. ric included fludarabine (flu) 150 mg/m 2 + melphalan (mel) 140 mg/m 2 , flu 150-180 mg/m 2 + bu 8mg/ kg. the bone marrow (bm) was used in 38 pts (66%), peripheral blood stem cells (pbsc) in 13 pts (22%), combination of bm and pbsc in 7 pts (12%). 5-years overall survival (os) was 45% os was in pbsc group -28%; bm group-50%, combination of bm and pbsc-25% there were two cases of mds, eb-2, although erythroid aberrancy can not be found, fc did disclose significant aberrancy on myelomonocytic lineages. on the other hand, all the normal control bm samples revealed no any erythoid phenotypic abnormality. our study suggests this simplified cocktail of 2-tube, 4-color, fc is very sensitive and useful in the assessment of erythroid phenotypic abnormalities in mds we analyzed 62 consecutive patients (44% were female, median age of 48 (range: 18-70) allografted for mds (median ebmt risk score of 3, median disease risk index of intermediate risk) over a 19-year period (1998-2016) with mac conditioning for 66% and ric for 34% pfs 40 ± 14%, grfs 26 ± 12%, ri 37 ± 13% and trm similarly, there was not difference between tdep and non tdep patients for 3-years pfs (48 ± 18% and 28 ± 20%, p value 0.321), 3-years gfrs (32 ± 17 vs 19 ± 17, p value 0.111) (graph), 3-years ri (36 ± 18% and 37 ± 20%, p value 0.622) and 3-years trm (26 ± 16% and 23 ± 18%, p value 0.933). finally, tdep had no significant impact on 3-years grade 2-4 agvhd when compared to the non tdep (26 ± 18% and 31 ± 16%, p value 0.656). it had not either on 3-years cgvhd (26 ± 18% and 28 ± 34%, p value 0.637). our study shows that tdep is feasible on patients undergoing hsct for mds disclosure of conflict of interest: none. p665 mutational pathway and dynamics may not be prognostic in patients with myelodysplastic syndrome receiving hypomethylating agent pre-treatment for allogeneic stem cell transplantation republic of korea; 3 department of computer science; 4 the donnelly center for cellular and biomolecular research amebiazis after bone marrow transplantation use of a five-agent gvhd prevention regimen in recipients of unrelated donor marrow impact of age on outcomes after bone marrow transplantation for acquired aplastic anemia using hla-matched sibling donors treatment of acquired severe aplastic anemia: bone marrow transplantation compared with immunosuppressive therapy-the european group for blood and marrow transplantation experience disclosure of conflict of interest: none. leukemia, myelodisplastic syndrome, juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia s bondarenko hla-mismatched unrelated (n = 5, 10%), and haploidentical (n = 8, 15%) donors. response was achieved in 30% (n = 16) of pts after 1-5 (median 3) courses of hma therapy: complete remission (cr) in 2 (4%), partial remission (pr) in 14(26%) of pts. stabilization (s) was documented in 30 (56%) pts, in 8 (14%) pts there was disease progression (p) after beginning of hma therapy mismanaging the gift of life: noncompliance in the context of adult stem cell transplantation l'adhésion thérapeutique et at. des lieux en allogreffe de cellules souches hématopoïétiques (csh) dans des services de pédiatrie et d'adulte. rapport de la sfgm-tc predictive validity of a medication adherence measure in an outpatient setting data is limited to small case series, transplant registries and a single prospective multicenter observational study. here we report our institutional experience with auto-hct in patients with hrl. twenty patients with hrl [non-hodgkin = 14 (70%), hodgkin = 6 (30%)] and treatable hiv infection underwent hdt consisting of carmustine, etoposide, cytarabine and melphalan (beam) followed by peripheral blood auto-hct from 04/2006 to 07/2015. in 2 cases rituximab was administered as part of the preparative regimen. patient-, disease-, and transplant-related characteristics are summarized in table 1. median age was 48 years (range: 35-61). the median follow-up for surviving patients was 42 months (range: 6-110) abbreviation: n: number of patients; m: male gender; auto-hct: autologous hematopoietic cell transplant; nos: not otherwise specified; dlbc: diffuse large b-cell lymphoma ara-c), melphalan; cr1: first complete remission; cr2 :second complete remission disclosure of conflict of interest: none. p696 incidence of secondary primary malignancies (spm) in patients with multiple myeloma m curly 22 , g laurent 23 and k nicolaus 24 1 city of hope igm 21 (0.6%), lines of induction regimens prior to hsct one in 2003 pts (53%), two in 724 pts ( 19.3%), 4 2 in 348 pts (9.3%), and missing in 682 pts (18%). induction regimens included imids and proteasome inhibitor (pi)s with alkylating agents in 1266 pts (33.7%), imids and pis with no alkylating agents in 1328 (35.5%), and alkylating agents with no imids or pis in 478 (12.7%) and missing data in 685 (18%). radiotherapy was used pre hsct in 614 pts (16.3%), no radiation in 2461 pts (66%) and missing data in 682 (18.2%). plerixafor (p) was administered mostly for poor hsc mobilization as defined by the centers number of hsc collected o3 × 10 6 in 239 pts (6.4%), 3-5 in 397 pts ( 10.6%), 45 × 10 6 in 1394 pts (37%), and data missing in 1727 (46%). the number of cd 34+ hsc infused o3 × 10 6 in 760 pts (20%), 3-5 × 10 6 in 1055 pts (28%0, 45 × 10 6 in 799 pts (21%), and missing in 1143 (30%). a total of 141 pts developed spm with cumulative incidence of 5.4% (95%ci 4.4,6.3) at 72 mo. data are missing in 414 pts (11%) use of radiotherapy, type of induction, hsc cell dose did not influence the cumulative conflict of interest: f. sahebi, none declared, s. iacobelli, none declared, l. koster none declared l. gardaret none declared, n. kroger received research fund from sanofi, curly morris, none declared p697 interaction between center effect and strategy for gvhd prophylaxis on outcome of t-cell depleted and t-cell replete haploidentical transplant inserm u1153 ecstra team expanding transplant options to patients over 50 years-improved outcome after reduced intensity conditioning mismatched-unrelated donor transplantation for patients with acute myeloid leukemia: a report from the acute leukemia working party of the ebmt nkg2d ligands in tumor immunity comprehensive analysis of nkg2d ligand expression and release in leukemia: implications for nkg2d-mediated nk cell responses nkg2d cars as therapy for cancer russian federation high incidence of mixed chimerism with impaired graft function remains a significant issue in patients with wiskott-aldrich syndrome (was) after hsct. simultaneous use of plerixafor with g-scf is efficient in inducing stem cell release and opening of bone marrow niches. the use of plerixafor/g-csf in conditioning demonstrates better levels of donor chimerism in patients with acute myeloid leukemia. we report our experience of plerixafor/g-csf usage in patients with was as an addition to myeloablateive conditioning to improve stem cell engraftment p = 0,85. events were considered: death in 2 patients, graft rejection in 5 patients, mixed myeloid chimerism (less than 20% donor) in 2 patients. median time of event was 3,2 months after hsct (1.23-8.6) all patients are alive, median fu is 3 months, range: 0.43-6.3. 2 patients had acute gvhd: 1-grade 2 (gut), 1-grade 1 (skin), in both cases resolved after a short course of steroids. all patients had more than 95% donor chimerism monthly till the time of last fu. the comparison of peripheral blood chimerism (% of donor cells) in was patients transplanted with and without plerixafor/g-csf in conditioning is shown (figure 1). the additional use of plerixafor with g-csf references 1. moratto et al disclosure of conflict of interest: none. is undesirable. fifteen pts (3 males, 12 females, median age 48, range: 17 -65 years) with high risk hematologic malignancies ( acute myeloid leukemia n.10, 66%; acute lymphoblastic leukemia n 2, 13% pretransplant conditioning regimen consisted of thiotepa 10 mg/kg in two days, busulfan 9.6 mg/kg in three days, and fludarabine. source of stem cells was g-csf stimulated bone marrow in all. dose of marrow nucleated cells and cd34+ were 5.4 (range: 3.4-6.7) × 10 8 /kg and 3.5 (range: 2.1-5.8) × 10 6 /kg respectively. post-transplant cyclophosphamide at 50 mg/kg/ day was given on days 3 and 5 after transplantation, together with cyclosporine (starting at day − 1 until day 180 posttransplant) and mycofenolate (from day +1 to day +20) modeling autism spectrum disorders with human neurons autism spectrum disorders neurobiology and genetics of autism: a developmental perspective. the development of autism: perspectives from theory and research wharton's jelly-derived mesenchymal stem cells treatment in children with cerebral palsy: our second preliminary results of the clinical application in poland a mucha 1 , k kosterna 1 , m chroscinska-krawczyk 2 , m kotarska 2 , k mitosek-szewczyk 2 , m murzyn 3 the polish stem cell bank cases application potential of bone marrow mesenchymal stem cell (bmscs) based tissueengineering for spinal cord defect repair in rat fetuses with spina bifida aperta sensory neuron differentiation potential of in utero mesenchymal stem cell transplantation in rat fetuses with spina bifida aperta: sensory neuron differentiation of in utero mscs analysis of post allo-hct relapse in acute leukaemia patients, a comparative on second allo-hct and donor lymphocyte infusions g orti 1 , j sanz 2 , i garcia-cadenas 3 , i sanchez-ortega 4 , mj jimenez 5 , p barba 1 , c ferra 6 , r parody 4 , j sierra 3 , ma sanz 2 , s querol 7 and d valcarcel 1 1 hospital universitari vall d´hebron; 2 hospital universitario la fe; 3 hospital de sant pau i la santa creu; 4 hospital duran i reynals ico, 5 hospital germans trias i pujol ico; 6 hospital germans trias i pujol and 7 banc de sang i teixits acute leukaemia relapse after allogeneic hematopoietic cell transplantation (allo-hct) associates poor prognosis. in this scenario, lowering the tumour burden prior to a second allo-hct (2 nd allo-hct) or donor lymphocyte infusions (dli) is essential to improve survival. thus, patients that respond to chemotherapy and subsequently receive a dli or 2 nd allo-hct appear to associate better outcomes compared to patients receiving only chemotherapy, but data regarding this particular group of patients is lacking. we retrospectively analysed a cohort of post allo-hct relapsed acute leukaemia patients, who, after tumour reduction, were treated with either a 2 nd allo-hct or dli. data was collected from 5 centers, 42 patients were consecutively included from 1995 to 2016. patients were treated to reduce the tumour burden and received the 2 nd allo-hct or dli on morphological remission or postchemotherapy aplasia. 26 patients (62%) were diagnosed with aml and 16 (38%) with all. 23 patients (55%) underwent 2 nd allo-hct and 19 (45%) received dli. median patient age was 38 (4-66) years. the median follow-up was 674 (9-5823) days. since data regarding time from first allo-hct to relapse was unavailable, we calculated the time from allo-hct to 2 nd allo-hct or dli (time to 2 nd allo-hct or dli). median time to 2 nd allo-hct/dli was 336 (9-8823) days, and was 674 days and 336 days for 2nd allo-hct and dli respectively (p = 0.004). regarding the dli group, the median dli dose was 1.1x10 7 / cd3+ (0.01-10x10 7 ) cells and the mean number of infused dli was 1.4/patient. one-year os was 51% (se ± 8%). in os univariate analysis, longer time to 2 nd allo-hct/dli associated better survival rates (p = 0.003). the 1-year dfs was 39% (se ± 8%). a longer time to 2 nd allo-hct/dli (p = 0.006) and 2 nd allo-hct compared to dli (p = 0.047) (figure 3 ) associated better dfs. the 1-year nrm was 24% (se ± 8%). univariate analysis identified pb as stem cell source as linked to better nrm (p = 0.086). the 1-year relapse incidence (ri) was 35% (se ± 9%). ri univariate analysis related longer time to 2 nd allo-hct/dli (p = 0.013) to lower ri. on os multivariate analysis, longer time to 2 nd allo-hct/dli was associated to better survival (p = 0.042). this association was also observed on dfs multivariate analysis (p = 0.017). table 1 summarizes 2 nd allo-hct and dli univariate analysis. grade ii-iv acute gvhd was diagnosed in 8 (35%) and 5 (26%) patients post 2 nd allo-hct and dli, respectively. chronic gvhd was diagnosed in 8 (4 extensive) and 3 patients after a 2 nd allo-hct and dli, respectively. in this study, longer time to 2 nd allo-hct/dli associated better dfs. 2 nd allo-hct (compared to dli) associated better dfs on univariate analysis, but this association was not observed on multivariate analysis. of note, the 2 nd allo-hct group included more patients with longer time to 2 nd allo-hct/dli. this might be explained by 2 nd allo-hct patients relapsing later or by the fact that the preparation of a 2 nd allo-hct might require longer time than dli. results of this analysis warrant further study with larger number of patients.advancing age is associated with worse prognosis in acute myeloid leukemia (aml). intensive induction chemotherapy in patients aged ⩾ 60 years results in lower aml remission rates with increased induction mortality vs younger patients. cpx-351 is a liposomal formulation of cytarabine and daunorubicin encapsulated at a 5:1 molar ratio. a phase iii, randomized, open-label study of cpx-351 vs 7+3 (cytarabine and daunorubicin) in newly diagnosed older patients with high-risk secondary aml showed superior survival in the cpx-351 arm (hazard ratio 0.69; p = 0.005). in that trial, eligible patients went on to allogeneic hematopoietic cell transplantation (hct). an exploratory analysis of those patients by age strata is reported here. patients aged 60 to 75 years with newly p534 number, composition and/or antileukemic activity of (dc-stimulated) invariant nkt-, nk-and cik-cells is predictive for outcome of patients with aml, all and cll cl boeck 1# , dc amberger 1# , f doraneh-gard 1 , w sutanto 1 , t guenther 1 , j schmohl 2 , f schuster 3 , h salih 2 , f babor 3 , a borkhardt 3 myelofibrosis (mf) is a hematolgic malignancy which is characterised by extramedullary hematopoiesis due to bone marrow fibrosis resulting in spleno-and/or hepatomegaly. allogeneic stem cell transplantation (allo-hsct) is the only curative treatment for mf but is associated with therapy related morbidity and mortality. retrospective studies suggested an increase of liver toxcicity in mf patients in comparison to other diseases following allo-hsct. the aim of this prospective study was to evaluate the impact of liver stiffness measured by transient elastography (fibroscan) on liver toxicity after allo-hsct. between 2013 and 2015 we included 39 patients (male 64%, female 36%) who underwent allo-hsct due to primary mf(72%), postpv/et-mf (23%) or mf in transformation (5%). the median age of the patients was 62 y@@@ears (range: 35-74). conditioning regimen was mainly busulfan based reduced intensity. all patients received atg. gvhd prophylaxis was csa/mmf in all patients. stem cell source was peripheral blood in 95% and bone marrow in 5% of the patients. donor sources were as follows: mrd (18%), mud (77%) and haploidentical relative (5%). fibroscan was performed prior to conditioning. elevated liver enzymes, bilirubin above the normal value or the onset of veno-occlusive disesae (vod) from the time of conditioning start and within the first 100 post-transplant days were considered as indicators for liver toxicity. the median stiffness of the liver measured by fibroscan on the day before conditioning treatment start was 7.6 kpa (range: 4.4-39.7). six patients (15%) had prior liver diseases such as cirrhosis (n = 1), viral hepatitis (n = 3), steatosis (n = 1), or vod (n = 1). the median onset of liver toxicity was day 0 (range: − 2 until +92). the median bilirubin level of all 39 patients was 4 mg/dl (range: 0-17). the median ap level was 153 u/l (range: 80-833), the median ggt level was 343 u/l (range: 88-1647), the median alt level was 108 u/l (range: and the median ast level was 67 u/l (range: 20-12292). the pearson-test revealed a positive correlation between liver stiffness and the elevation of the ap (r = 0.55, p = 0.001) and ggt levels (r = 0.54, p = 0.008). the comparison of the median maximum enzyme and bilirubin levels is shown in table 1 . in two patients who developed severe vod requiering defibrotide, the liver stiffness level was 6.9 kpa and 13.8 kpa, respectively. the patient with the highest stiffness level (39.7 kpa) developed acute gvhd of the liver, which completely resolved after steroid treatment. only one of those five patients who had stiffness levels 413 kpa died due to liver toxcity and concurrent septic shock, he suffered from viral hepatitis prior to transplantation. liver stiffness measured by transient elastography (fibroscan) positively correlates with the elevation of the cholestatic enzymes ap and ggt in myelofibrosis patients after allo-hsct and may predict liver toxicity. disclosure of conflict of interest: none.[p574]in the era of tyrosine kinase inhibitors (tki) as superior first line treatment in the therapy of cml, the concept of allogeneic hsct has been pushed to the role of salvage therapy. to date, data on allogeneic hsct after tki-therapy are scarcely available. in this study, we report single center data on the outcome of 52 cml patients, for the most part pretreated with tki, who underwent allogeneic hsct between 1999 and 2015 with a follow-up of 12 months to 17 years. upon obtaining written informed consent 48 patients diagnosed with bcr-abl-positive cml and 4 patients with bcr-abl-negative atypical cml were included in this analysis. the majority of patients underwent myeloablative conditioning regimen. the median age at time of hsct was 47 years with a range: from 19 to 67 years. twenty-one patients were transplanted from a matched related donor, and 31 received stem cell grafts from an unrelated hla-compatible donor. 36/48 patients received tki-therapy before transplantation, 23 patients received more than 1 tki prior to hsct. 10/48 patients were treated with interferon prior to hsct. twenty-two patients were transplanted due to acceleration or blast crisis. twenty-six patients received an allogeneic hsct in chronic phase (cp, n = 16) or complete hematologic (chr, n = 7) or cytogenetic remission (ccyr, n = 3). kinase domain mutations could be identified in seven patients including t315i-mutation in four patients. seven patients showed "major route" cytogenetic aberrations. next to advanced disease status, tki intolerance (n = 4) and tki resistance (n = 11) were the main indications for hsct after 2001. after a median follow up of 5 years and 3 months, those 26 patients transplanted in cp, chr or ccyr showed an overall survival (os) of 79%. 3/27 patients died in remission and two patients died after cml relapse. after 2004 none of the 15 patients transplanted in cp, chr or ccyr died or relapsed so far, with a median follow-up of 1672 days. all of these patients received tki therapy prior to transplant. twenty-two patients transplanted in advanced stage cml (bc and ap) had after a median follow up of 5 years an os of 44%. the difference between survival curves is significant (log rank test p = 0.041; hr 0.3407, 95% ci of ratio 0.1211-0.9585). prior to transplantation 19 of these patients received a tki-therapy. in this group, four patients died due to cml relapse, one died after development of donor cell leukemia and five patients died in remission. one patient with atypical cml was transplanted in bc and died of progressive disease shortly after transplantation. the other three patients with atypical cml were transplanted in cp-phase. with a median follow-up of 648 days these patients are in ongoing remission. even in times of tkitherapy allogeneic hsct remains a successful and safe therapy option for cml patients with tki intolerance or resistance. patients transplanted in cp or complete remission had an excellent long-term outcome. allogeneic hsct should be considered in tki resistance or intolerance before the development of blast crisis. despite tki therapy, overall survival deteriorates in patients with advanced disease. however, this treatment modality can improve survival rates substantially compared to other available therapies. tkimaintenance therapy could be a possible strategy to prevent cml relapse, although randomized data on tki-maintenance therapy after allogeneic hsct are still lacking.[p583]disclosure of conflict of interest: none. use of first or second generation tki for cml after allogeneic hematopoietic stem cell transplantation: a study by the cmwp of the ebmt y chalandon, s iacobelli 1 , j hoek 2 , l koster 2 , l volin 3 , j finke 4 , jj cornelissen 5 , i yakoub-agha 6 patients (pts) relapsing with cml after allogeneic hematopoietic stem cell transplantation (allohsct) may be treated with tki and/or dli. as nowadays the majority of cml pts would have received at least imatinib prior to transplantation, we were interested in analizing (a) the type of tki used after allohsct, (b) the indication for tki treatment, (c) the outcome of this treatment and d) the temporal relationship with dli if given. 435 pts received tki after first allogeneic hsct for cml. transplants had been performed in cp1, n = 194, ap, n = 60 or for more advanced disease (bc/4 cp1, n = 177) from hla identical siblings (n = 231) or ud (n = 204) between 2000 and 2013. tki given prior to transplant was imatinib (n = 268), dasatinib (n = 162), nilotinib (n = 88), bosutinib (n = 4) and ponatinib (n = 7). median age at transplant was 44 (18.5-68) years, 274 pts (63%) were male. tki post allohsct were given between 2000 and 2015. first tki given was either imatinib (n = 223), dasatinib (n = 131), nilotinib (n = 67), bosutinib (n = 2) or ponatinib (12). the indications for tki therapy were the same as for transplantation (n = 25), for relapse/progression/ persistent disease (n = 246), for prophylaxis/pre-emptive (n = 147), planned (n = 5), others (n = 8) and missing (n = 4). median follow-up from start of tki was 55 (1-171) months. the median time interval from transplant to tki was 6 (0.2-165) months. it was longer for tki given for relapse/progression with 15 (1-89) months and shorter for tki given for prophylaxis/pre-emptive with 1.6 (0. haematopoietic cell transplant (hct) is the only curative approach for scd. due to concerns regarding the toxicities associated with myeloablative conditioning regimens in adults, a non-myeloablative protocol was developed by hsieh et al. (national institutes of health, nih protocol). the use of this novel regimen was able to achieve a curative degree of mixed donor chimerisms with minimal transplant-related complications. the alberta children's hospital (ach) has adopted this conditioning regimen in children due to the efficacy and low rates of toxicities published by the nih group. with generally lower rates of gvhd in younger recipients, our group had no reason to believe rates of toxicities would be greater in a younger population with fewer comorbidities secondary to scd than those described in the nih cohort. to our knowledge, there is no published literature describing the utilization of the nih protocol in a paediatric population. we describe our experience in children with scd who underwent matched sibling donor (msd) peripheral blood hct using nih protocol. this retrospective cohort describes outcomes of msd hct in children with scd who underwent hct with the nih conditioning regimen between 2013-2017. a total of 13 potential subjects were identified. eight subjects have consented to the analysis to date. msds with either normal haemoglobin or sickle cell trait were considered appropriate for donation. the transplant procedure: the conditioning regimen consisted of alemtuzumab 0.2 mg/kg/dose administered subcutaneously daily for five days (days − 7 to day − 3). patients received a tbi dose of 300 cgy on day -2, with testicular shielding for male recipients. gvhd prophylaxis consisted of a sirolimus load of 3 mg/ m 2 /dose (po) on day − 1, followed by 1 mg/m 2 /dose once a day starting on day 0. unmanipulated peripheral blood stem cells were infused on day 0. sirolimus was used for gvhd prophylaxis post-hct and continued until at least one year. weaning of sirolimus was initiated no earlier than 1 year post-hct and if donor t-cell chimerisms were greater than 50%. institutional supportive care protocols for scd hct were followed. patients were eligible for early discharge post-hct even prior to neutrophil engraftment. eight patients (5 f, 3 m) have been registered on this retrospective study. follow-up ranges from 1 to 52 months post-hct. there were no failed stem cell mobilizations. all patients had donor neutrophil engraftment at a median of 21 days. all patients are currently alive. there have been no cases of graft failure to date and no sickling crises post-hct. one patient has dropping myeloid chimerisms but still 4 20% donor. no cases of veno-occlusive disease, idiopathic pneumonia syndrome, cerebral hemorrhage, pres, or posttransplant lymphoproliferative disease were observed. three cases of cytomegalovirus (cmv) reactivation required pre-emptive therapy. only one patient did not initiate sirolimus weaning at 1 year post-hct due to donor t-cell chimerisms of 42%; this patient is 52 months post-hct and is likely to start weaning sirolimus soon. there have been no cases of acute or chronic graft-versus-host disease. nonmyeloablative conditioning regimen is safe and effective as curative therapy for scd. long-term follow-up of these children to assess organ function post-hct is underway. disclosure of conflict of interest: none.the number of new hiv/aids cases has been declining in developed countries, whereas it is still increasing in japan, with the cumulative number reaching 26,607 as of june 28, 2016. hiv infection is associated with an increased risk of hematological malignancies such as non-hodgkin lymphoma (nhl). autologous hematopoietic cell transplantation (auto-hct) is a treatment option for hiv-infected patients with nhl and multiple myeloma (mm). however, the prognosis after auto-hct in hiv-infected japanese patients remains unclear. the aim of this study is to evaluate the effect of hiv infection on transplant outcomes after auto-hct in japan. using the national database of the japan society for hematopoietic cell transplantation, we retrospectively evaluated patients with nhl (n = 3862) and mm (n = 2670) who underwent their first auto-hct between 2001 and 2014. presence of hiv antibodyperipheral t-cell lymphomas (ptcl) comprise a heterogeneous group of diseases among which ptcl-not otherwise specified (ptcl-nos) represents the most common histology. patients with ptcl are typically offered high-dose chemotherapy followed by autologous hematopoietic cell transplantation (auto-hct) as front-line consolidation. allogeneic hct (allo-hct) is generally offered in the relapsed setting; however, in selected cases it is also offered as front-line consolidation. no randomized controlled trial (rct) have been performed to date comparing offering an allo-hct versus other treatment modalities either in the front-line or in the relapsed setting. thus, we performed this systematic review/meta-analysis to assess the totality of evidence pertaining to the role of allo-hct in ptcl. search of the literature was undertaken via pubmed and web of science from inception until september 6, 2016. no search limits were applied but studies presented only in abstract form were excluded. data were collected on treatment benefits (complete remission (cr), progression-free survival (pfs), overall survival (os)) and harms (non-relapse mortality (nrm), grade ii-iv acute graft-versus-host disease (gvhd), and chronic gvhd). the search identified 1271 references; however, only 17 studies (6 in front-line (n = 132 pts), 11 in relapsed/refractory setting (n = 330 pts)) were eligible based on our inclusion criteria and had extractable data. three studies included both frontline and relapsed/ refractory cases but data for certain outcomes were reported separately. the median follow-up time for studies evaluating allo-hct in the front-line or relapsed/refractory setting ranged from 30-45 months and 12-85 months, respectively. in the front-line setting, allo-hct resulted in cr rates of 64% ((95%ci = 50-77%), 2 studies, n = 49 pts), pfs rate of 64% ((95% ci = 49-78%), 5 studies, n = 100 pts), and os rate of 72% ((95% ci = 62-81%), 5 studies, n = 95 pts). nrm rate was 6% ((95% ci = 0-15%), 3 studies, n = 68 pts). acute (grade ii-iv) and chronic gvhd rates were 39% (95% ci = 24-56%), 2 studies, n = 38 pts) and 33% (95% ci = 16-53%), 3 studies, n = 64 pts), respectively. in the relapsed/refractory setting, allo-hct resulted in cr rates of 68% ((95% ci = 70-97%), 5 studies, n = 75 pts), pfs rate of 39% ((95% ci = 31-47%), 6 studies, n = 203 pts), and os rate of 52% ((95% ci = 43-60%), 6 studies, n = 307 pts). nrm rate was 21% ((95% ci = 13-31%), 7 studies, n = 194 pts). acute (grade ii-iv) and chronic gvhd rates were 34% (95% ci = 23-46%), 7 studies, n = 197 pts) and 37% (95% ci = 30-44%), 8 studies, n = 199 pts), respectively. notwithstanding the need to perform a rct to compare the efficacy of allo-hct versus auto-hct as front-line consolidation in ptcl, the results of this systematic review/meta-analysis show very encouraging os rates of 72% following allo-hct. moreover, allo-hct also offers an encouraging os rate of 52% in patients with ptcl in the relapsed/refractory setting. the higher nrm rate in the relapsed/refractory setting probably reflects the adverse effect of a higher number of prior prescribed therapies. one of the limitations of our analysis is the inability to analyze outcomes for individual histologic subtypes due to the aggregate nature of the published data. disclosure of conflict of interest: none. high-risk patients with relapse or refractory hodgkin lymphoma do significantly better after hdc auto-sct compared to control arm of aethera trial. mature results from a cohort of 234 patients s akhtar, s rauf, tam elhassan and i maghfoor king faisal specialist hospital and research center, riyadh, kingdom of saudi arabia brentuximab vedotin use in hodgkin lymphoma (hl) patients who had hdc auto-sct has been reported to improve progression free survival (pfs) but not the overall survival (os) in a phase 3 trial (lancet 2015;385:1853-62). in this trial, after hdc auto-sct, 329 high risk hl patients were randomized to receive placebo (control gp) vs brentuximab (experimental gp) as consolidation therapy. we are reporting our experience of patients with similar selection criteria as control gp. hl patients z14 yrs who received hdc auto-sct with similar selection criteria as defined in aethera trial were identified that is, patients had at least one of the following risk advanced lymphomas still represent a therapeutic challenge and allo-hsct is among treatment options. between march 2007 and august 2016, seventy-three patients (pts) affected by r/r lymphomas (34 nhl and 39 hl) underwent an allo-hsct after a treosulfan-based conditioning regimen and sirolimus as calcineurin-inhibitor-free prophylaxis of gvhd. six pts received a mrd, 18 pts a mud, and 49 pts a haplo unmanipulated pbsc. at allo-sct 30 pts were in cr, 13 pts were in pr, and 30 pts had sd/pd; sixty patients underwent autologous sct before allo-hsct. hct-ci was evaluable for 64 pts, 33 had a score ≥3. thirty-three pts received treosulfan and fludarabine reduced toxicity conditioning regimen (rtc) and intensification with other alkylating agent or with 4 gy total body irradiation was added on the remaining 40 pts (myeloablative conditioning, mac). all pts received a backbone gvhd prophylaxis with sirolimus and mycophenolate mofetil; atg or pt-cy or both were added in 41, 25, and 3 pts respectively. median numbers of infused cd34+/kg and cd3 +/kg were 6.01 × 10 6 (range: 2.72-9.06) and 2.39 × 10 8 (range: 0.3-6.89), respectively. median follow-up was 44 months (range: 3-111); median time to neutrophil ≥ 0.5 × 10 9 /l was 17 days, and 20 days to platelet ≥ 20 × 10 9 /l. sixteen out of 43 patients with pre-transplant active disease obtained a cr after treosulfan conditioning; nine of them (6 hl and 3 b-nhl) achieved durable cr without post transplant treatment. oneand 3-years os was 62% and 48%, pfs was 47% and 37% at 1 and 3 years respectively; cumulative incidence of relapse/ progression was 32% and 42% at 1 and 3 years. grfs was 31% and 19% at 1 and 3 years, respectively. transplant related mortality (trm) was 15% at 100 days, 21% at 1 year and for the entire follow-up. the 100-day cumulative incidence (ci) of agvhd grade ≥ 2 was 19% and ci of agvhd grade ≥ 3 was 10%; ci of moderate to severe cgvhd was 23% at 2 years and for the entire follow-up. no differences in ci of agvhd or cgvhd were found if pts were stratified according to donor type, but ci of moderate-severe cgvhd was significantly higher in pts after mac regimens (p o0.0005). as expected, the outcome of pts in cr was significantly better compared with active disease, in terms of os (p = 0.0061), pfs (p = 0.00022), ri (p = 0.0028). in multivariate analysis, intensity of conditioning regimen (rtc vs mac), gvhd prophylaxis (use of atg, pt-cy or none), donor sex and age at allo-sct did not impact the transplant outcomes; both os and pfs were reduced by active disease at allo-hsct (hr = 4.37, ci 95% 1.76-10.86, p = 0.01 and hr = 4.37, ci 95% 1.97-9.7, p = 0.00, respectively) and by nhl histology (hr = 3.88, ci 95% 1.65-9.19, p = 0.02 and hr = 2.43, ci 95% 1.09-5.42, p = 0.03, respectively); grfs and ri were impacted only by active disease (hr = 2.25, ci95% 1.19-4.25 and hr = 4.89, ci 95% 1.87-12.62, p = 0.01, respectively). allo-hsct after treosulfan conditioning and sirolimus gvhd prophylaxis is feasible even in heavily pretreated pts affected by lymphomas. complete remission status before transplant remains crucial for better outcomes and in the era of new targeted treatments should be pursued. disclosure of conflict of interest: none.university of eastern finland, kuopio, finland and 12 department of medicine, kymenlaakso central hospital, kotka, finland autologous stem cell transplantation continues to have an important role in the treatment of patients with multiple myeloma (mm). in mm patients the most commonly used mobilization method is granulocyte-colony stimulating factor (g-csf) ± cyclophosphamide (cy). generally, up to 10-20% patients mobilize poorly with these methods and plerixafor may be used to enhance mobilization. the most important parameter of graft quality has usually been the number of cd34+ cells, but there are also significant numbers of other cell subsets in the grafts and they may also be of special interest in regard to post-transplant recovery and outcome. for example, a higher number of lymphocytes and nk cells in the grafts has been associated with improved lymphocyte as well as nk cell recovery, respectively. the mobilization methods used seem to affect the graft composition. however, there is currently no prospective data on the effects of plerixafor on the graft composition, post-transplant hematological and immune recovery or outcome in patients with mm. altogether eighty-seven patients with mm were included into this prospective study. seventy-seven patients were mobilized with g-csf ± cy (control group) and ten patients received also plerixafor due to poor mobilization (plerixafor group). in the control group 57/77 (74%) and in the plerixafor group 3/10 (30%) of patients were mobilized with g-csf+cy (p = 0.009). there were no statistically significant differences between the groups according to age, gender, paraprotein type, initial iss, induction therapy used or disease status at the time of mobilization. by imwg risk stratification, there were more high risk patients in the plerixafor group (5/10 vs. 13/77, p = 0.066). cryopreserved graft samples were analyzed with flow cytometry for t and b cells (cd3/cd8/cd45/cd19) as well as for nk cells (cd3/cd16+cd56). also, cd34+ cell subclasses were analyzed (cd34/cd38/cd133). complete blood counts were evaluated at +15 days, 1, 3, 6 and 12 months posttransplant. to evaluate immune reconstitution, flow cytometry of lymphocyte subsets (t, b, nk) was performed in a subset of patients at 1, 3 and 6 months after the graft infusion using the same antibody panel as for graft analysis. there were no significant differences between the groups in the number of cd34+ cells in the grafts. also, the median number of aphereses was two in the both groups (p = 0.086). the proportion of the more primitive cd34+ cells (cd34 + cd133 + cd38 -) was significantly higher in the plerixafor group (p = 0.001). in addition, the number of various lymphocyte subsets analysed was significantly higher in plerixafor group table 1 ). there were no statistically significant differences in the course of hematological recovery. the recovery of blood cd3+cd4+ t cells was significantly faster in the plerixafor group at one at three moths post-transplant. there was no significant difference in the progression-free survival (pfs) (log rank, p = 0.408) with the median follow-up time of 703 days in the plerixafor group and 882 days in the control group (0.099). in the present study plerixafor added to g-csf ± cy seemed to significantly alter the cellular composition of autologous blood grafts in poorly mobilizing mm patients. hematological recovery was comparable but the cd3+cd4+ t lymphocyte recovery was faster in the plerixafor group. the pfs was comparable between the groups. disclosure of conflict of interest: dr. valtola has received honoraria from sanofi and jansen-cilag. dr. silvennoinen has received a research grant from celgene and janssen, honoraria from genzyme and sanofi and participated in advisory board organized by amgen, janssen and takeda. dr. siitonen has received honoraria from amgen and celgene. dr. jantunen has received honoraria from genzyme, amgen and sanofi and has participated in eu leadership meeting organized by genzyme as well as medical advisory board meeting organized by genzyme and amgen. dr. varmavuo has received consultancy fees from abbvie, roche, celgene, amgen and sanofi. the other authors declare no conflicts of interest. bortezomib after high-dose melphalan as conditioning regimen before autologous stem cell transplantation in patients with multiple myeloma: a comparison with the historical conditioning regimen with melphalan alone ga ferini; ja arbelbide; al basquiera; e nucifora; n schutz; v otero; d fantl hospital italiano d buenos aires, buenos aires, argentinahigh dose of melphalan followed by autologous stem cell transplantation (asct) is the standard of care for younger patients with multiple myeloma (mm). to enhance the efficacy of the conditioning regimen, the intergroupe francophone du myelome added bortezomib to melphalan showing improved response rates, without significant toxicity. bortezomib has shown synergistic effects with melphalan, mainly if the bortezomib is administered 24 hours after the melphalan. since 2014, we have changed our conditioning regimen for patients with mm undergoing asct by adding bortezomib toallogeneic stem cell transplantation (allosct) is a potencially curative option for patients with multiple myeloma (mm). despite the improvement of reduced-intensity-conditioning (ric), transplant-related mortality (trm) remains high. there is no consensus on which graft versus host disease (gvhd) prophylaxis regimen is superior. some studies have suggested that tacrolimus-based prophylaxis is more effective than cyclosporine (csa) in terms of lower incidence of severe acute gvhd (agvhd), with no impact on overall survival (os). herein, efficacy and toxicity between two gvhd prophylaxis regimens is analyzed. we retrospectively analyzed 14 patients (pts) with relapsed mm who received allosct ric in the period from 2003 to 2015 in a single centre (table 1) . population: age, 51 years (40-73); median follow-up: 19 months (1-175). conditioning regimen: allo-ric (fludarabine + busulfan or melphalan regimens) and 100% was bortezomib-based in the tacrolimus group. donor: matched related (11 pts), unrelated (1), mismatch unrelated (1) and haploidentical (1) donor. gvhd prophylaxis: all patients received a short course of methotrexate + csa (9 pts, 64%) or tacrolimus (5 pts, 36%). complete response at transplant was 33% at csa group and 60% at tacrolimus group. all pts underwent toxicity related to chemotherapy (mainly mucositis and neutropenic fever) with organ impairment (renal or liver) in 100% tacrolimus arm as well as 4 pts in csa group. the incidence of agvhd was 80% and 77.8% in tacrolimus and cyclosporine groups, respectively (p = 0.99). grade iii-iv agvhd were reported in 2 pts (40%,tacrolimus) and 4 pts (44%, cyclosporine), with severe gastrointestinal and liver involvement. glucocorticoid resistance was observed in 75% in both groups. patients with refractory agvhd received other immunosuppressive therapies: more than 3 second-lines agents (3) (4) (5) (6) were necessary in fifty percent of pts in both groups to control gvhd. two patients had to interrupt tacrolimus due to neurological toxicity and suspected thrombotic thrombocytopenic purpura. no patients had to discontinue treatment in the csa arm because of toxicity. the 12-months os was 78.6% (80% in tacrolimus vs 66.7% in csa (p = 0.78)) and the 24-months was 68.8%. a total of 4 pts died because of gvhd. during follow-up, only 2 patients relapsed (10 and 126 months after allosct, respectively) in csa group. no relapse were seen in tacro group. in our experience, no significant differences were observed between both calcineurin inhibitor in terms of os, toxicity and gvhd incidence. an explanation could be our small number of patients. allosct is an effective therapy for selected patients but it is associated with high rates of gvhd and trm. a long-termsafety and effective prophylactic regimen is necessary as main objective.[p638]disclosure of conflict of interest: none. several parameters, including early lymphocyte, neutrophil, platelet recovery, and infused dose of cd34+ cells, have been associated with clinical outcome of patients with haematological malignancies. however, their prognostic significance remains uncertain. the aim of current study was to evaluate prognostic significance of clinical and laboratory parameters that might influence survival after autologous stem cell transplantation (asct) in hodgkin lymphoma (hl) and multiple myeloma (mm). this retrospective study included a total of 90 with hl and 114 mm patients (median age 32 years, 55 years, respectively) who underwent asct between november 2005 and june 2016. hl patients were conditioned with beam (84.4%) and cbv (15.6%) regimen, while mm patients received conditioning with high dose of melphalan. high ips (international prognostic score) at diagnosis had 68.9% hl patients and high iss (international scoring system) had 27.2% of mm patients, of which 8.8% had renal impairment. the average of transplanted cd34+ cells in hl patients was 7.15 × 10 6 /kg (range: 2-25.0 × 10 6 /kg), and 6.6 × 10 6 /kg (range: 2-15.51 × 10 6 / kg) in mm patients. after asct, favourable treatment response (partial/complete remission) achieved 83.3% hl patients, of whom 22.7% had infused o5 × 10 6 /kg cd34+ cells. median time to recovery of absolute lymphocyte count 500 × 10 6 /l or greater (alc500) was 16 days (range: 9-31 days), recovery of absolute neutrophil count ≥ 500 × 10 6 /l (anc500) was 12 (range: 6-26 days), and platelet recovery ≥ 20 × 10 6 /l (plt20) was 12 days (range: 5-44 days). after asct, 93.6% mm patients achieved favourable treatment response, of whom 26.5% had infused cd34+ cell dose48.7 × 10 6 /kg. median time to alc500 was 15 days (range: 9-23 days), anc500 was 13 (range: 9-24 days), and plt20 was 11 days (range: 5-26 days). median follow up of patients with hl was 67 months, while after asct, median event free survival (efs) was 20 months, and overall survival (os) was 38 months. treatment response after asct strongly influenced both efs and os after asct (po0.0001). in patients who achieved favourable treatment response, os and efs after asct were influenced by infused cd34+ cell dose (o5 × 10 6 /kg vs. ≥ 5 × 10 6 /kg), prolonged recovery of alc500 by day+20, plt by day +13, and achieving of anc500 by day +11(po0.05). multivariate analysis among significant variables showed that infused cd34+ cell dose was the most important parameter that influenced os and efs (po0.05). median follow up of mm patients was 50 months, while after asct, median efs was 26 months and os was 34 months. regarding patients who achieved favourable treatment response, os and efs after asct were influenced by the presence of renal impairment, infused cd34+ cell dose (≤8.7 × 10 6 /kg vs. 48.7 × 10 6 /kg) and plt20 recovery by day +13 (po0.05). among these significant parameters, multivariate analysis pointed out infused cd34+ cell dose as the most important parameter that influenced both os and efs (po0.05). these data suggest that number of infused cd34+ cells is an independent factor that may contribute to outcome of patients with hl and mm. disclosure of conflict of interest: none. high-dose therapy with autologous stem cell transplantation (asct) has become the treatment of choice for symptomatic eligible patients with multiple myeloma (mm). we studied an induction regimen of cyclophosphamide, bortezomib and dexamethasone (cybord) and showed rapid and deep responses after 4 cycles in patients with newly diagnosed mm and we subsequently done asct with melphalan (mel) conditioning. cost is the major limiting factor in developing world.all the drugs used are generic brands manufactured in india. a total of 25 mm patients (median age: 54.5 years, 76% male and 24% female) were transplanted between 2012 and 2016. in all, patients had igg kappa-12 (48%), igg lambda-03 (12%), iga lambda-05 (20%), iga kappa-02 (08%), kappa light chain 02 (08%), lambda light chain 01 (04%) patients. prior to autograft, all cases had received cybord with generic medicines. median time diagnosis to asct was 7.5 months (5 to 21 months). stem cell mobilization was done with g-csf alone in 17 (68%), g-csf plus plerixafor in 07 (28%) and chemo mobilization in 01 (04%) patients. all patients received asct support after conditioning with 200 mg/m 2 generic melphalan alone (dose adjustment was done according to renal status). all patients received thalidomide maintenance from march 2012. bortezomib used was manufactured by dr. reddy's lab, hyderabad and melphalan used was manufactured by emcure pharmaceuticals, pune, india. 68 patients from 1999 to 2011 received cyclophosphamide, vincristine, adriamycin and dexamethasone (cvad) protocol of originator medicines followed by originator melphalan conditioning and asct (cvad-mel-asct). at the time of autograft, 21 (84%) of patients were in complete remission, 02 (08%) in partial remission, 02 (08%) very good pr. median day of engraftment was 10 for neutrophils and 14 for platelet. transplant related mortality was 16% (4/25) out of which 2 died of infection and 2 deaths of cardiac events. the pfs and os rates were 80% and 84% at median follow up of 18.6 months. patients who were treated with cvad-mel-asct had efs of 74% at 2yrs and 52% at 5yrs. cost of bortezomib showed significant difference, generic was 4000usd where as for originator drug was 20000usd for 4cycles of chemotherapy. cost of melphalan also showed difference with 450usd for generic and 2000usd for originator drug. generic cybord showed excellent response rate and allows excellent stem cell collection and transplantation which can further consolidate response and improve outcome. cybord induction and melphalan conditioning with generic medicines can be considered a standard regimen for transplant-eligible patients with newly diagnosed mm in resource constraint situation. generic cybord-mel-asct is more cost effective than originator cvad-mel-asct. generic medicines produced in india are of good quality and cost effective. this study needs long term follow up to assess survival parameters at a median. disclosure of conflict of interest: none. and an extra copy of one or more odd-numbered chromosomes and as intermediate risk(ir) if they had t(4;14) or del(13) (q).overall survival (os) and relapse-free survival (rfs) were calculated from the time of allo hsct and auto hsct on day 0, from diagnosis to death or disease progression. the median age at presentation was 53.86 (range: 20-80) years, and 72 (63.7%) were men. at a median follow-up time of 18 months, 73% were alive.45 of the 113 patients with available fish samples underwent auto hsct. 24 patients (53.3%) achieved cr and 21 patients (46.7%) relapsed. of the 13 patients who had received allo hsct, five patients (38.5%) achieved cr and five patients (38.5%) remained alive. in patients who received auto hsct, the risk of relapse was 56% less than those never transplanted (p = 0.02), but the difference was not significant in patients who received allo hsct. the relapse-free survival in hr patients was 6 months (po 0.001), in ir was 11 months (p o0.001) and in sr was 37.67 months (p o0.001). in transplant patients, rfs in hr patients was 5.73 times more than sr group (po0.001) and in ir group was 3.35 times more than sr (p o0.001). the survival time in transplant patients was significantly better than non-transplanted patients (p o0.001). the median overall survival (os) in hr patients was 25.45 months, in standard risk group 30 months and in sr patients was 31 months. cytogenetic abnormalities detected by fish are of significant value in classification, risk stratification and management of patients with mm. we can use cytogenetic data to provide prognostic information and also to guide management and clinical practice. these data indicate that autologous stem cell transplantation could potentially be of benefit to myeloma patients. disclosure of conflict of interest: none. chronic graft-vs-host disease (cgvhd) is the most troublesome complication developing after allogeneic hematopoietic stem cell transplantation (allo-hsct). diagnosis of cgvhd has largely been based on clinical features only. we previously reported gene expression profiles in patients with cgvhd after allo-hsct. we extended our study to develop a molecular diagnostic method of cgvhd. we selected six most commonly expressed genes from the former dna expression study. and, a home-made 6-gene pcr array were used to evaluate gene expression profiles in the peripheral blood mononuclear cells of 39 patients given allo-hsct (20 cgvhd patients, 19 non-cgvhd patients) and 19 normal controls. the gene expressions of the allo-transplanted patients were compared to those of the stem cell donors. sybr green qpcr and multiplexqpcr were performed to confirm the usefulness of the selected genes in the diagnosis of cgvhd. infogainattributeeal and ranker were used to develop a gene model to diagnose cgvhd. k-nearest neighbor model and weka classifiers lazy ibk module were applied to evaluate the performance of the gene model. in another 21 steroid-refractory cgvhd patients (14 responders, 7 non-responders), the gene expression changes were analysed using our 6-gene pcr array before and 57 days after rituximab treatment. we identified six genes most accurately delineating cgvhd patients from those without treatments after allogeneic hematopoietic stem cell transplantation (hsct) are long and constraining for patients. medical adherence in hsct patients is of major concern in daily practice but it has been not yet described. 1, 2 the aims of our study were to evaluate treatment adherence and to identify factors associated with adherence behaviors. an observational single-center study was based on self-reported questionnaires completed by patients in a hematology day hospital between november 2015 and july 2016. the patientreported adherence was evaluated using the eight-item morisky medication adherence scale (mmas-8). 3, 4 individual item scores were summed: patients with a score of 8/8 were considered as good adherents to medication whereas a poor adherence referred to a score under 8. among the latter, medium adherence ranged to a score of 6-8, while a score of o6 was considered low adherence. socio-demographic and medical characteristics were collected by health records. a univariate model was used to evaluate if some of patients' characteristics were associated with adherence. statistical analysis was performed using r software (version 0.98.1103 -2009-2014 rstudio, inc). fifty-six patients were included in the current study. median age at transplantation was 55 years (range: 16-72 years). diagnosis were aml (n = 28), all (n = 10), myelofibrosis (n = 8) and other hematological diseases (n = 10). 24 patients received a hsct from a related donor (13 haploidentical). myeloablative conditioning was used in 18 patients and reduced intensity regimen in 38 patients. a total of 64.3% (36/56) of the patients were poor adherent according to mmas-8. among these patients, 6/36 were low adherent and 30/36 were medium adherent. the results of univariate analysis showed that a poor adherence was associated with a longer time since hsct and discharge at home. however elderly patients, patients treated with cyclosporine and patients with daily hydration at home were associated with a better adherence (po 0.05). our study presents the first data on adherence among patients undergoing hsct. risk factors associated with a poor adherence have been identified in order to determine patients' profiles that will benefit more from interventions to improve adherence. particular attention has to be paid to younger patients. efforts to establish a regular follow-up of these patients are needed in order to sustain patients in the treatment adherence to prevent the occurrence of severe complications. we studied the effect of basic fibroblast growth factor (fgfb) and dexamethason on expansion and immune modulation of mscs in patients with lymphomas. mscs were generated from bone marrow aspirates obtained from the patients with hodgkin's lymphoma (hl; n = 8) and non-hodgkin's lymphoma (nhl; n = 4). the adherent fraction of marrow aspirate was cultivated with/without the basic fibroblast growth factor (fgf-b, 10 ng/ml) or dexamethason (10 − 5 м or 10 − 8 м) to reach 80-90% confluence. then mscs were passaged with accutase and used for experiments after 1-2 passages. the number of msc precursors (cfu-f) in bone marrow of lymphoma patients was found to be significantly decreased both in patients with nhl (17 ± 5, p o0.01) or hl (26 ± 5, p o0.05). the time until 80-90% confluence was significantly increased and took on average 26 ± 2 days (vs 15.4 ± 0.6 in donors). finally, the immunosuppressive ability of patient msc was significantly lowered and was only registered at the high concentrations of mscs (1:1 and 1:2). the expansion of patient mscs was significantly promoted with fgfb resulting in a significant decrease of primary cell cultivation (from 25.4 ± 1.52 to 18.6 ± 1.21 days; p = 0.041) and a statistically significant twofold increase in the number of cells received at the first passaging. in addition, in cultures with fgfb there was a decrease in the relative amount of resting mscs and a threefold increase of cycled cells in cd73+ mscs. dexamethasone has also provided a moderate stimulating effect on the msc growth. in fact, the use of 10 − 5 м of dexamethasone resulted in the increase of the cell yield by 1.6 times and of 10 −8 м-by 1.9 times. however, fgfb and dexamethasone differed in their effect on the msc ability to inhibit the proliferative response of t lymphocytes upon stimulation with mitogens or alloantigens. indeed, fgfb failed to correct the impaired immunosuppressive activity of patient mscs, and median percentage of suppression still remained lowered-17% vs 16% without fgfb. in contrast to fgfb, dexamethason could increase the immunosuppressive activity of patient mscs by 1.5 times (in dose of 10 − 5 м) and by 2.1 times (for 10 − 8 м). our data indicate that fgfb and dexamethasone used during the generation of mscs exert a stimulating effect on the msc expansion. in contrast to fgfb, dexamethasone, in the broad range: of doses, was able to enhance the suppressive properties of mscs that are initially reduced in patients with lymphoma. these findings suggest the existence of at least two mechanisms of impairments in immunoregulatory function of mscs in lymphomas-dependent and independent of the msc proliferation. disclosure of conflict of interest: none. free nonabsorbable antibacterial digestive decontamination is associated with a low incidence of gastrointestinal acute gvhd and better gvhd-free/ relapse-free survival (grfs) in the atg-based conditioning regimens nabil yafour 1 commonly antibacterial prophylaxis based of oral no absorbable antibiotic such as (neomycin colistin, gentamicin, vancomycin) used before and after engraftment, other fluoroquinolone such as levofloxacine were recently used to prevent invasive infection. however the exact interaction with gastrointestinal acute graft versus host disease (gi-agvhd) remains unclear. the objective of this study was to evaluate a novel composite endpoint of gvhd-free/relapse-free survival (grfs), in which events include grades 3-4 gi agvhd, chronic gvhd requiring systemic therapy, relapse, or death in atg based-conditioning regimens, with free no absorbable antibacterial digestive decontamination prophylaxis. a total of 39 evaluable consecutive patients with hematological disease were included in period of february 2013 to mai 2016. patients with malignancies disease (n = 33) received myeloablative conditioning regimens plus atg (5 mg/kg); including once daily busulfan (130 mg/m 2 ,-6d to -3d, iv ) + fludarabine (40 mg/m 2 /d, -6d to -3d, iv) (aml = 26, all = 3, cml = 1) or melphalan (140 mg/m 2 , d-1, iv) (all = 3). six patients received cy/atg for saa. gvh prophylaxis consisted to; ciclosporine a (csa) + mtx. csa was maintain levels between 150-400 ng/ml and tapered at the discretion of the treating physician. all patients were received peripheral blood stem cells (pbsc) graft from a matched related donor. since december 2015 levofloxacine and voriconazole was administered as antibacterial and antifungal prophylaxis. diagnostic, clinical grading and treatment of gi-agvhd and gi-cgvhd were performed according to established criteria and nih recommendations. probability of grfs was estimate by kaplan-meier method. median age was 35 years (range: 6-60). median dose of cd34+ and cd3+ cell doses were 4.9 × 10 6 (range: 2.5-7) and 1.84 × 10 8 (range: 0,036-5,28). the median time to neutrophil and platelet recovery were13 days (range: 6-33) and 14 days (range: 10-43) respectively. at time of transplant 12/39 (31%) had an intestinal colonization with extended-spectrum betalactamase (esbl) producing bacteria. only 5/39 (13%) developed infectious diarrhea during the period of transplant. incidence of grade iii/iv gi-agvhd and gi-cgvhd requiring systemic therapy were 5% and 5% respectively. for patients with malignancies diseases (n = 33), 23 (70%) were alive at a median follow up of 12 months (range: 5-43). incidence of relapse, disease free survival rates were 30%, 67% respectively. the grfs rate as defined previously was 48% at 30 months. these results confirm that free no absorbable antibacterial digestive decontamination and atg-based conditioning regimens were associated with very low incidence of gi-gvhd and better grfs in patients with malignancies diseases. diverse bacterial populations of the gastrointestinal tract remain important factors to promote immune tolerance after allogeneic sct. disclosure of conflict of interest: none. g-csf primed hla haploidentical transplantation from maternal or collateral donor using atg plus reduced dose of posttransplantation cyclophosphamide: results of a phase ii prospective trial y wang 1 , x-j huang 1 1 peking university people's hospital, peking university institute of hematologythe transplantation milieu using granulocyte colony-stimulating factor (g-csf), and anti-thymocyte globulin (atg) for hlahaplotype-mismatched transplants from related donors has resulted in favourable outcomes with low transplant-related mortality (trm), without increased relapse rate. however, in this transplant modality, the poorer outcome owing to high incidence of graft-versus host disease (gvhd) related to maternal donor or collateral donor remains a concern. meanwhile the use of post-transplant cyclophosphamide (pt/cy) in recent years appears to be protective against severe acute and chronic gvhd. we performed a prospective pilot study of hla haploidentical stem cell transplantation (sct) from maternal or collateral donors with intensified conditioning including g-csf and atg, followed by two lower doses of pt/cy (14.5 mg/kg × 2 doses). outcomes were compared with those of 160 controls from matched-pair analysis who undergone haploidentical sct from other donors than mother or collateral relatives at the same time period. a total of 40 patients with myelodysplastic syndrome (mds) or leukaemia undergoing haploidenticla sct from maternal or collateral donors were enrolled in the study. incidence of grade ii-iv and grade iii-iv acute gvhd at day 100 were comparable between the study group and the control group (17.5% vs. 33.3%, p = 0.07; 5.0% vs. 12.5%, p = 0.24). incidence of cmv and ebv reactivation at day 100 were also comparable between the study group and the control group (75.0% vs. 85.0%, p = 0.16; 15.0% vs. 29.2%, p = 0.09). after a median follow-up of 303 days and 341 days, the incidence of trm and relapse at 1 year were comparable between the study group and the control group (5.0% vs. 13.3%, p = 0.16; 10.0% vs. 6.7%, p = 0.54); the probability of overall survival and lfs at 1 year were comparable between the study group and the control group (84.2% vs. 79.8%; p = 0.24; 83.0% vs. 77.7%, p = 0.48). in conclusion, conditioning with atg and low-dose pt/ cy might be a feasible option for patients undergoing hla haploidentical, t-cell replete sct from maternal or collateral donors. trial registration: the study is registered at www. clinicaltrial.gov as nct02412423. disclosure of conflict of interest: none. hematopoetic stem cell transplantation (hct) is a lifesaving treatment option for eligible patients with hematological malignancies. hct is inherently associated with a risk of nonrelapse mortality that varies greatly depending on transplant and patient characteristics. the assessment of the risk of complications and mortality before the procedure is extremely important. the hct comorbidity index (hct-ci) introduced by sorror m. is one of the tools proved to predict hct outcomes and was shown to be significant in various disease and hct settings. the objective is to evaluate hct-ci index of hct recipients, determine impact of different variables on ci score, particularly those, showing pulmonary and cardiac function. data of hct-ci of autologous (auto) and allogeneic (allo) hct recipients, transplanted during period january 2015-october 2016 were analyzed. impact of pulmonary and cardiac function values on ci score was evaluated: dlco (diffusing capacity of the lung for carbon monoxide), fev1 (forced expiratory volume) and ef (cardiac ejection fraction) are parameters, reflecting pulmonary and cardiac function, which values are included into hct-ci score. the statistical data analysis was conducted using spss program. the differences were considered statistically significant at p ≤ 0.05. records of 100 allo and 220 auto hct recipients, transplanted during 01.2015 -10.2016 in vilnius university hospital were revised. median age of allo hct and auto hsc recipients was 50 (19-75) and 58 (19-74) years respectively. main indication for allo hct was acute myeloid leukemia 48 (48%) patients and for auto hct -multiple myeloma 133 (60.5%) patients. hct-ci was completely calculated (no values missing) in 64 allo and 102 auto hct recipients. only patients with available complete hct-ci data were further analyzed. hct-ci in hct recipients was as shown in table 1 . hct-ci score o3 was calculated in 37 (57.8%) and ≥ 3 in 27 (42.2%) allo hct recipients. hct-ci score o3 was calculated in 45 (44.1%) and ≥ 3 in 57 (55.9%) auto hct recipients. hct-ci score did not differ statistically significant between male and female recipients in both hct categories as well as in different age groups of patients (below and above 40 years in allo and below and above 60 years in auto hct). dlco was found to be below normal values (o80%) in 43 (67.19%) allo hct and in 67 (65.7%) auto hct recipients. fev1 was less affected and found to be lower 80% in 6 (9.3%) allo hct and in 17 (16.7%) auto hct recipients. ef below 50% detected in 1 (1,6%) allo hct and in 6 (5.9%) auto hct recipients. low dlco was found to cause the greatest impact on hct-ci score and was statistically significantly associated with higher hct-ci (po0.001). the most common hct-ci in both hct groups was score 3. dlco was found to be below normal ranges in relatively large patient group and had the greatest impact on hct-ci score. further studies on reasons of pulmonary function impairment and it's impact on hct outcomes are warranted.[p690]disclosure of conflict of interest: none. haemophagocytic lymphohistiocytosis (hlh), a life-threatening hyper-inflammation syndrome, is classified into primary and secondary forms. primary hlh is caused by gene mutations resulting in impaired cytotoxicity of natural killer (nk) cells and cytotoxic t lymphocytes (ctls). secondary hlh arises in the setting of autoimmunity, infection, malignancy, or less commonly, may be idiopathic. treatment of hlh has two major goals: halting the triggering event and controlling the overactive immune system. however, patients with primary or recurrent secondary hlh should subsequently undergo allogeneic hct for long lasting disease remission. we retrospectively evaluated 61 hematopoietic stem cell transplantation (hsct) might be a valid treatment option for adults suffering from aggressive t-cell malignancies providing long term disease control. since a suitable hla-matched donor cannot be identified for all patients (pts) in need for transplantation, alternative donors graft sources such as related hla-haploidentical donors are considered. through introduction of t-cell-replete (tcr) hlahaploidentical transplantation (haplo-hsct) using post transplantation cyclophosphamide (ptcy) successful treatment with low non-relapse mortality rate (nrm) has been observed in lymphoma patients (luznik et al., bmt, 2008) . however, less data are available on the outcome of this haplo-approach in the treatment of t-cell malignancies, in particular when disease is refractory. we retrospectively evaluated the outcome of haplo-hsct using tcr grafts and ptcy in 8 pts with peripheral t-cell lymphoma treated between 2010 and 2015 at our institution (t-nhl = 6, t-all = 2; male n = 5; median age: 37 years). disease was refractory/active at time of transplantation in 7 pts, while one had achieved second cr. all patients received at least 2 prior treatment lines and one patient failed previous allogeneic transplantation. while fludarabine and cyclophosphamide served as backbone for conditioning, 3 pts received a tbi-based and 5 a drug-based conditioning regimen which was myeloablative in 50%. if disease was active at time of haplo-hsct, a sequential therapeutic concept was performed involving intensive chemotherapy (clofarabine n = 6) shortly preceding conditioning (zoellner ak et al., bmt, 2015) . post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mycophenolate mofetil in all patients. graft source was bone marrow in 3 pts. no primary graft rejection occurred; 7/8 pts engrafted, one died early in aplasia. neutrophil/platelet engraftment was achieved at a median of 20 (range: 14-36) and 42 (range: 17-117) days, respectively. acute gvhd grade ii-iii was observed in 4 pts, whereas no patient developed grade iv agvhd. mild chronic gvhd occurred in one patient. 50% of the pts developed grade ii-iii treatment-related toxicities most commonly diarrhea (33%) and mucositis (25%); grade iv toxicity (mucositis) was observed in one patient only. no vod occurred. cmv reactivated in 4/5 pts at risk, whereas no ptld was seen. proven invasive aspergillosis was diagnosed in one patient. at day +30 seven pts achieved cr. 3 pts relapsed and 3 died (relapse n = 1, infection n = 2). 1-year nrm was 25%. at a median follow up of 46 months (range: 15-76) the estimated 1-year and 3-year overall survival (os) and progression-free survival (pfs) were 63%/63% and 50%/33%, respectively. three pts received haploidentical dlt pre-emptively (n = 2) and therapeutic (n = 1), leading to sustained cr in two, while no severe gvhd occurred. sequential therapy in the setting of tcr haplo-hsct using ptcy as gvhd prophylaxis is feasible, well tolerated and shows low rates of gvhd and acceptable nrm in patients with relapsed/refractory t-cell lymphoma/ leukemia providing an effective anti-lymphoma/leukemic activity. thus, we suggest that intensified tcr hapo-hsct using ptcy should be considered as an alternative for patients suffering from aggressive t-cell malignancies, lacking hlamatched donors. disclosure of conflict of interest: none. tacrolimus is a calcineurin inhibitor increasingly used as immunosuppression following allogeneic stem cell transplantation; maintenance of therapeutic serum levels is essential to reduce the risk of graft rejection and graft versus host disease. however, tacrolimus can be associated with serious side effects and potential drug interactions. regular monitoring of serum levels and appropriate dose adjustment is essential to ensure therapeutic levels and to avoid toxicity. in our adult bmt unit, an established standard operating procedure (sop) provides a prescriptive dosing algorithm for: (i) initiation of tacrolimus therapy; (ii) conversion between iv and oral routes; (iii) dose adjustment based upon tacrolimus serum level and interacting medications. we performed an audit assessing adherence to the sop dosing algorithm. 97 inpatient tacrolimus dosing episodes from five consecutive haploidentical transplants were retrospectively analysed. 17 episodes were excluded due to insufficient records. for the remaining 80 episodes, tacrolimus serum levels and corresponding doses were identified. the response of the medical team to each serum level was compared with the sop dosing recommendation. to account for sensible rounding of doses, a margin of error of ± 10% was permitted. adherence to sop dosing was 54%. non-adherence to the sop (46%) was subcategorised as justifiable (21%) or unjustifiable (25%). justifiable non-autologous hsct is currently being explored for its efficacy and safety in the treatment of multiple sclerosis (ms). as more experience is gained in treating this cohort, treatment related mortality has steadily improved although the procedure still carries a degree of risk. ebv reactivation is well described in allogenic stem cell transplants although less so in autologous transplantation. we investigated the frequency of ebv reactivation in patients with ms undergoing autologous hsct at a single uk site. 30 patients underwent autologous hsct for treatment of ms at king's college hospital between feb 2012 and aug 2016. all were mobilised with cyclophosphamide 4g/ m 2 and g-csf. 29 were conditioned with cyclophosphamide and atg, and one with beam/atg. previous exposure to ebv (ebv igg) was assessed prior to transplant and local posttransplant ebv monitoring was performed on whole blood samples by means of quantitative pcr in 26 patients. data was collected retrospectively. all 26 (100%) patients were positive for ebv igg pre-transplant. overall, 194 samples were tested for monitoring post-transplant. 20 (76.9%) patients demonstrated positive pcr post-transplant on local testing with one further patient being negative on local tests but later becoming positive on testing in their parent hospital (full results unavailable). of these 20, the median time to positive testing post-transplant was 24 days (7-91). maximal ebv dna titre was reached at a median time of 40 days post-transplant (7-101) with a mean maximum titre of 5.17 log (3.3-8.2). 2 patients experienced symptomatic reactivation with an associated large paraproteinemia. one of these developed hyper-viscosity requiring plasma exchange and developed neurological symptoms mimicking an ms relapse (max ebv titre of 8.2 log). this patient received rituximab and ebv level is declining, the other was observed carefully but developed right leg weakness which is slowly improving. the patient with raised ebv at their parent hospital also received rituximab (unclear if this reactivation was symptomatic). we have developed a protocol to pre-emptively treat ebv reactivation with rituximab once a 6 log titre is reached and one patient has so far been treated according to this. of the 18 patients with locally confirmed reactivation who did not receive rituximab, 9 (50%) self-resolved at a median time of 98 days (44-182), 5 (33.3%) have ongoing re-activation (4 with improving, 1 with stable titres) and 4 (22.2%) have not had any local bloods performed ≥ 6 months. 1 patient with selflimiting reactivation later had a further positive titre (370 days post-transplant and 182 days post initial resolution). ebv reactivation appears to be common in patients with ms in the first 3 months post autologous hsct. unlike in other patient groups such as aplastic anaemia patients receiving allogeneic transplants it can cause significant neurological symptoms which may be confused with ms relapse. the mechanism of this reactivation is probably related to atg administration but may be exacerbated by prior immune suppression in this heavily pre-treated group, the majority of whom have received highly active disease modifying therapies in the past. these results demonstrate the importance of monitoring for ebv reactivation following autologous hsct and the consideration of pre-emptive therapy. disclosure of conflict of interest: none. reduced bone mineral density (bmd) is a well recognised complication of hct. guidelines recommend scanning by dual energy x-ray absorptiometry (dxa) one year after transplant in all hct patients 1 or else specific groups of high risk patients. 2, 3 it is recognised that both dose and duration of steroids are risk factors for low bmd and it is recommended that prednisolone doses greater than or equal to 5mg/day for more than 3 months should prompt a dxa scan. for patients with osteopenia it is recommended that calcium/vitamin d supplements are given together with lifestyle advice including diet, smoking cessation and weight bearing exercise. 1 in this survey we have investigated the current practise in investigating and managing bone health in the context of hct. a survey was sent to all 453 centres including 45 countries registered with ebmt as of november 2016. 63 centres replied from 14 countries. response numbers to each question were variable and are indicated by the denominators. 5/36 used a national guideline to guide their practise, and 3/34 used an international guideline. no single guideline was quoted more than once. 25 low testosterone has been demonstrated to be an independent determinant of endothelial (dys)function in men. graftversus-host disease (gvhd) is a major contributor to nonrelapse mortality (nrm) after allogeneic stem cell transplantation (allosct). vulnerability of the recipients' endothelial cell system is a novel concept to explain why a proportion of patients with acute gvhd fail to respond to escalating immunosuppressive therapy and ultimately succumb to gvhd and related complications. this retrospective study investigated the prognostic impact of pre-transplant testosterone levels on nrm after allosct in male patients. between 2002 and 2014, a total of 277 male patients undergoing allosct at heidelberg university (median age 55 years) provided informed consent to participate in this observational study (training cohort). a total of 71 patients (26%) received transplants from related donors (rd). diagnoses were aml (48%), mds (29%), lymphoid malignancies (33%) and multiple myeloma (12%) . a total of 176 patients (78%) received statin treatment post allosct as per institutional standard policy. for validation, an independent patient cohort of 205 men allografted for aml and mds (median age 57 years, 18% rd, no statin treatment) at essen university was analysed. pretransplant serum samples were collected between 0 and 2 months before allosct and cryopreserved at − 80°c. testosterone and suppressor of tumorigenicity-2 (st2) levels were measured by radioimmunoassay and elisa, respectively. median pre-transplant testosterone level in the training and validation cohort was 13.6 nmol/l (range: 0.3-41.7 nmol/l) and 16.0 nmol/l (0.8-38.1 nmol/l), respectively. in the training cohort, lower pre-transplant testosterone as continuous variable was associated with shorter os (p = 0.009). lower testosterone levels showed a trend towards higher nrm (p = 0.09) and a significant association with nrm after onset of acute gvhd (p = 0.02). multivariate analysis confirmed lower pre-transplant testosterone levels as a significant predictor of an increased nrm risk after gvhd onset (p = 0.03). in the subgroup of patients not receiving statins post-transplant, lower testosterone levels were associated with increased incidence of transplant-associated microangiopathy (p = 0.01), and, in addition, with higher pre-transplant st2 levels indicating endothelial vulnerability. in the validation cohort, similar results with regard to overall survival (os, p = 0.02), nrm (p = 0.04), nrm after acute gvhd onset (p = 0.03) in univariate analysis, and to nrm after gvhd onset (p = 0.02) in multivariable analysis could be observed. the association of pre-transplant testosterone levels (in quartiles) and incidence of nrm after gvhd onset in the training and validation cohort is depicted in figure 1a and 1b, respectively. our study suggests that low pre-transplant testosterone is associated with serological and clinical evidence for endothelial damage and is an independent risk factor for a fatal outcome of gvhd. prospective studies in the allosct setting investigating testosterone and testosterone supplementation in deficient patients are highly warranted. disclosure of conflict of interest: none. nk cells anti-tumor ability in multiple myeloma patients s tognarelli 1,2 , b jacobs 3,4,5 , i von metzler 6 , h serve 6 , p bader, t klingebiel 7 , a mackensen 3 and e ullrich 1,2 1 department of pediatric stem cell transplantation and immunology, childrens hospital, goethe university, frankfurt, germany; 2 cellular immunology, loewe centre for cell and gene therapy, goethe university, frankfurt, germany; 3 department of hematology and oncology, university hospital erlangen-busulfan is one of essential drugs for hematopoietic stem cell transplantation (hsct). because of its narrow therapeutic range: targeted busulfan using therapeutic drug monitoring (tdm) has been used. generally, the initial dose of busulfan is determined by patients' body surface area as 120 mg/m 2 except for infants (80 mg/m 2 ). however, pharmacokinetic evidence of these initial doses is scarce. therefore, we investigated the full pharmacokinetics of busulfan in infant and child, and attempted to validate that these initial doses are acceptable. one hundred ninety-five pediatric patients undergoing hsct using four-day targeted busulfan were enrolled. of them, 6 patients received hsct when their age was ≤ 1 year old (infant group [ig]), and 19 patients received when 1-2 years old (toddler group [tg]). the remaining 170 patients were defined as a child group (cg). busulfan was administered intravenously once daily for 4 consecutive days. tg and cg received 120 mg/m 2 as the first dose, and ig received 80 mg/m 2 . using daily tdm, we adjusted the next dose of busulfan. target daily and total area under the curve (auc) were 18 750 μg*h/l/day and 74 000-76 000 μg × h/l, respectively. median first-day busulfan auc of ig, tg, and cg were 18 416, 22 529 and 20 410 μg × h/l, respectively, which was significantly different (p = 0.031). however, there was no significant difference in median total busulfan auc (ig; 74 180, tg; 73 406, and cg; 74 482 μg × h/l, respectively, p = 0.089). the coefficient of variance (cv) of four-day busulfan aucs in ig and cg was similar (median cv: 22.1% and 24.7%, respectively), whereas cv of tg was 40.4%. in sub-analysis of tg and cg who received equally 120 mg/m 2 as the first dose, there was an inverse correlation between age and first-day busulfan auc (r = − 0.148, p = 0.042), as well as between age and cv of four-day busulfan aucs (r = − 0.210, p = 0.004). initial busulfan dose as 80 mg/m 2 for infant could be acceptable in aspect of first-day auc and cv of four-day busulfan aucs. however, higher first-day auc and cv were shown in tg. although target total busulfan auc could be achieved safely by tdm, we suggest that reduction of initial dose less than 120 mg/m 2 is also necessary to patients with 1-2 years old to lower the relatively higher first-day auc. taken together, tdm is highly recommended to reduce busulfan toxicity, especially in younger children. disclosure of conflict of interest: none. post-induction treatment strategy of acute myeloid leukemia (aml) is currently driven by european leukemia net (eln) risk assessment at diagnosis. if it is well established that patients belonging to favourable-risk group can be treated with chemotherapy and/or autologous stem cell transplantation (sct) and that those belonging to the unfavourable-risk group should be addressed to allogeneic (allo) sct, for patients included in the intermediate-risk groups the best post-induction treatment has not been established yet. we report here a 6years (2010-2015) allo-sct single center experience in 78 aml patients. median age was 53 years (range: 20 -68), 17%, 23%, 9% and 51% were grouped in the eln favourable, intermediate-i, intermediate-ii and unfavourable risk category, respectively and 47% of the patients were allotransplanted in advanced disease-phase (2nd complete remission). half of the patients received a sibling hla compatible donor, 76% of the cases received peripheral blood stem cells and half of the patients received a myeloablative conditioning regimen. graft versus host disease prophylaxis was conventionally based on cyclosporine and shor-course methotrexate, with the addition of antilymphocyte immunoglobulin in case of matched unrelated donor. the clinical and transplant characteristics of the patients according to the eln-risk group were well balanced. with a median follow up of 20 months (range: 8-58 months), the projected 2 years overall survival (os) and disease free survival (dfs) is 45% (95% ci: 32-57%) and 43% (95% ci: 30-54%). the median os and dfs in favourable/intermediate-i vs intermediate-ii/unfavourable is 21.8 and 14.8 months ( figure 1a ; p = 0,67) vs 18 and 14,8 months ( figure 1b ; p = 0.66). the relapse rate (rr) and the non relapse mortality (nrm) at two years are 38% (95% ci: 26-50%), and 15% (95% ci: 8-26%), respectively. non differences were observed comparing the 2 years rr and the 2 years nrm of patients in the favourable/intermediate-i vs intermediate-ii/unfavourable eln risk group (36% vs 40%; p = 0.66 and 16% vs 18%; p = 0.95). interestingly, the percentage of patients allotransplanted in advanced phase of the disease was higher in those included in low/intermediate-i with respect to intermediate-ii/unfavourable eln-risk group (73% vs 43%; p = 0.001). our data suggest that allo-sct can cure approximately 40-50% of aml patients, with no difference within the eln risk groups. disease recurrence remains the major problem and this is highly correlated to the percentage of patients in advanced phase of the disease at transplant, particularly in eln favourable/intermediate-i patients. we are currently collecting the data on minimal residual disease (mrd) status of these patients during chemotherapy and before transplant using moelcular biology on target genes and/or multiparametric flow cytometry on leukemia associated immunophenotype, in order to assess if the prognosis of these patients may be refine by the prospective application of mrd data.[p707]disclosure of conflict of interest: none. outcome of allogeneic stem cell transplantation for patients with high-risk acute leukemia according to donor type and graft-versus-host disease prophylaxis s lindner, t berg 1 , j riemann 1 , s ajib 1 , z jedlickova 1 , s gueller 1 , f lang 1 , a sackmann 1,2 , n goekbuget 1,2 , h martin 1 , a bacigalupo 3 , h serve 1,2 and g bug 1 1 department of medicine ii, hematology and oncology, university hospital frankfurt, goethe university, germany; s500 2 german cancer consortium (dktk), german cancer research center, heidelberg, germany and 3 università cattolica del sacro cuore, fondazione policlinico universitario gemelli, roma, italyin high-risk acute leukemia (hr-al), allogeneic hematopoietic stem cell transplantation (hsct) is the only potentially curative treatment. increasingly, hsct is being performed utilizing alternative donors. we retrospectively analyzed the outcome of 148 consecutive patients (pts) with hr-al (aml/all, n = 118/30) undergoing first allogeneic hsct in our transplant unit between 1/2011 and 6/2015 according to donor type and graft-versus-host disease (gvhd) prophylaxis: in the matched related donor group (mrd, n = 26), hsct was performed with standard immunosuppression (is), that is, calcineurin inhibitor (cni) plus methotrexate or mycophenolate mofetil (mmf). for 10/10 hla-allele matched unrelated donors (10/10 mud, n = 84) or 9/10 hla-allele mud (9/10 mud, n = 24) we used is and anti-thymocyte globulin (atg fresenius/neovii). hsct with a haploidentical family donor or an 8/10 hla-allele mismatched unrelated donor was performed using is with cni plus mmf and post-transplant cyclophosphamide (pt-cy, n = 14). a myeloablative (n = 75) or reduced-intensity (n = 73) conditioning regimen was applied in complete remission (cr, n = 106) or active disease (n = 42). pts had a median age of 52 years (range: 19-72) and hematopoietic cell transplantation comorbidity index of 3 (range: 0-11). patient and treatment characteristics were well balanced between the groups except for a higher percentage of pts transplanted in cr in the pt-cy group (93% vs. 54-76%, p = 0.03). peripheral blood stem cells were preferred for mrd, 10/10 mud and 9/10 mud (81%, 95% and 96%, respectively) and bone marrow for 93% of pt-cy based hsct. all pts engrafted. with a median follow-up of 28 months (range: 1-60), probability of overall survival (os) at 3 years was 54 ± 10% for the mrd, 65 ± 6% for the 10/10 mud, 41 ± 11% for the 9/10 mud and 93 ± 7% for the pt-cy group, without significant differences (p = 0.05). however, the probability of achieving the combined endpoint gvhd-and relapse-free survival (grfs) at 3 years varied significantly between the groups (mrd 8 ± 5%, 10/10 mud 43 ± 6%, 9/10 mud 29 ± 10% and pt-cy 57 ± 13%, po0.01), reflecting the high cumulative incidence (ci) of chronic moderate and severe gvhd at 1 year in the mrd (58 ± 11%) as opposed to the other groups (10/10 mud 12 ± 4%, 9/10 mud 12 ± 8% and pt-cy 33 ± 14%, p o0.01). of note, donor type had no impact on ci of transplant-related mortality (trm) at 3 years (12 ± 3%), acute gvhd g3-4 at day +100 (10 ± 3%) or leukemic relapse at 3 years (34 ± 4%). overall, aml pts 460 years of age had a significantly inferior relapse-free survival compared to younger pts (50 ± 9% vs. 71 ± 6%, respectively, p o0.01) without a higher ci of trm (p = 0.37). median time to aml relapse was 6 months. our results suggest that pt-cy-based alternative donor hsct is safe in hr-al pts and provides a solid basis for a randomized clinical trial comparing hsct from haploidentical family donors and 9/10 mud, currently in preparation. while os did not vary between groups, grfs was dismal after mrd transplants without atg, due to high rates of severe chronic gvhd, consistent with published data. as leukemic relapse remains the major cause for treatment failure especially in elderly pts, maintenance strategies using novel drugs or cellular therapies are warranted. disclosure of conflict of interest: none. relapse following hematopoietic stem cell transplant (hsct) is the leading indication for a second transplant in patients with malignant disease. hsct has been shown to be superior to chemotherapy alone or palliative measures in these patients. for non-malignant disease a second transplant may be considered for graft failure after first transplant. data regarding the outcome of a second hsct for non-malignant disease is scarce. we retrospectively analyzed 29 patients who underwent a second hsct, for survival and toxicity data. twentynine patients (age 0-19 years) who received a second hsct at our institution during 1998-2015 were included in the analysis. thirteen patients had an underlying malignancy and 16 patients were transplanted for non-malignant indications, including inborn errors of metabolism, non-malignant hematologic diseases and immune deficiency. median follow up was 14 months (range: 1-180). there were 10 deaths (77%) in the malignant group, 7 (53%) were due to disease relapse and 3 (23%) were transplant related. fifty percent of deaths occurred within the first year following the second hsct. in the non-malignant group there were 5 deaths (31%), of which 2 (12%) were attributed to the underlying disease and 3 (18%) were transplant related. all deaths but one occurred within the first year post hsct. treatment related mortality following second hsct is higher compared to first transplant. the higher survival rate in the non-malignant group suggests that transplant following graft failure should be considered ins501 patients with otherwise incurable underlying disease. though the outcome for patients with relapse of malignant disease following hsct is poor, a second transplant may benefit a subset of these patients. attempts to achieve complete remission prior to transplant should be made to improve outcome. due to the small number of patients in our cohort, further multi-center trials are needed. disclosure of conflict of interest: none. disseminated bcg infection (bcg-osis) is a rare but most serious complication in vaccinized especially immunocompromised children. severe combined immunodeficiency disorder (scid) is probably the commonest primary immunodeficiency associated with bcg-osis, though there is no such definitive data as most of the cases described in literature are in the form of reports. hematopoietic stem cell transplantation (hsct) is a life-saving treatment for patients with scid, especially if therapy is instituted early, prior to onset of infections.as bcg vaccine is routinely given to all iranian children at birth, the likelihood of having an active infection at the time of transplant would be significantly high. the main objective of this study was to evaluate the outcomes of hsct in scid patients with disseminated bcg infection . sixteen scid patients underwent hsct in our center since 2007 to 2016, of which nine patients (7 male, 2 female) were enrolled in this analysis. all the 9 patients had received bcg vaccination according to the national vaccination protocol, and had undergone anti-tuberculosis (tb) treatment prior to transplant due to disseminated bcg infection. the mean age at hsct was 9.3 months (range: 6-13 months). patients received bone marrow (n = 1), peripheral blood progenitor cells (n = 6) or umbilical cord blood grafts (n = 2) from hla-matched related donors (n = 7) and mismatched unrelated donors (n = 2). three patients received unconditioned matched sibling donor transplants and ric regimen was provided with fludarabine, melphalan and rabbit anti-thymocyte immunoglobulin (thymoglobulin) in others . cyclosporine a and prednisolon were used as graft-versus-host disease (gvhd) prophylaxis. they also continued to receive anti-tb treatment. all patients but one engrafted. the median times to neutrophil and platelet engraftments were 12 days (range: 11-39), and 18 days (range: 17-90), respectively. engraftment with full chimerism (495%) occurred in 5 patients and the other 3 patients had mixed chimerism. with a median follow-up of 24 months (range: 3 -48 months), overall survival was 66.7%. the main cause of death was disseminated bcg infection.three out of 8 patients who achieved engraftment, developed acute gvhd (grade i-ii), while one patient developed extensive chronic gvhd. although anti-tb treatment continued, tuberculous dactylitis occurred in 3 patients post-hsct that were successfully treated. on last post-hsct follow-up, 4 patients with full chimerism and 2 with mixed chimerism are alive and disease free. scid is called as a pediatric emergency as it invariably leads to fatality in infancy without early aggressive therapy and hsct. in hsct recipients, the impaired cellular immunity renders these patients more susceptible to infection. as previous reports suggest, our study demonstrates that with appropriate anti-tb cover, immunological reconstitution with complete recovery from bcg infection can be achieved by early hsct. disclosure of conflict of interest: none. paraproteinemia occurrence after allogeneic hematopoietic stem cell transplant as a possible marker for chronic gvhd onset f monaco 1 , s tamiazzo 1 , f dallavalle 1 , l calcagno 2 , m pini 1 and m ladetto 1 1 hematology and 2 transfusion medicine, azienda ospedaliera ss. antonio e biagio e cesare arrigo, alessandria, italy transient monoclonal gammopathy is commonly reported after solid organ or stem cells transplant (sct) for hematologic malignancies. however the clinical significance of a paraproteinemia appearance is not fully understood, because the attempts to correlate its effect on survival rates, graft versus host disease (gvhd) occurrence and viral reactivations have led to controversial results. starting from these reports we decided to evaluate among our allogeneic transplanted patients the incidence of m-component and its possible relationship with chronic gvhd. one-hundred and one patients undergoing allosct at the hematology unit of alessandria (italy) between 2006 and 2015 were evaluated. 55% of patients were male and 45% were females. pretransplantation diagnosis included: 62 acute myeloid leukaemia/ high-risk myelodisplastic sindromes (62%), 14 acute lymphoblastic leukaemia (14%), 13 lymphoproliferative disorders (13%) and 12 other less common malignancies (12%) . patients with multiple myeloma were excluded from the study. all patients had, at least, two pre-transplantation serum electrophoresis with no evidence of pre-existing monoclonal component. serum electrophoresis was scheduled to be performed at 90, 180 and 360 days and 2 years after transplantation. forty-nine patients were submitted to allo-sct from a sibling donor and 52 from a matched related donor (mud); in vivo t-cell depletion with anti-thymocyte globulin was used in 63 patients. thirty-four patients relapsed after allosct, 52 (52%) developed chronic gvhd and 56 patients (56%) are currently alive at the last follow-up. posttransplantation follow up ranged from 81 to 2695 days with a median of 496 days. paraproteins were detected in 52 out of 101 patients (52%), being monoclonal in 28 patients, and bi or tri-clonal in the remaining cases; the immunoglobulin subclass most commonly observed was igg. ten-year overall survival of the whole population was 50%; splitting the population in two cohorts (with or without paraproteinemia) we did not detect any statistical differences in overall survival, gvhd development and relapse incidence at +90 and +180 days posttransplant; viceversa, after 360 days, a statistically significant difference was observed in chronic gvhd occurrence in patients with or without paraproteinemia (85% vs 42%, respectively, po 0.001). ten-year overall survival curves were significantly better in patients with paraproteinemia as compared with the paraprotein-free group (59% vs 45%, p = 0.04), and an even more evident significance was seen in ten-year relapse free survival curves (66% for patients with paraprotein vs 48% for patients without paraprotein, p = 0.009). monoclonal gammopathy, also in our experience, is frequent following allo-sct. we observed a strong correlation between the occurrence of paraproteinemia, chronic gvhd and a significantly better overall and relapse-free survival. recently many evidences showed that b cells are involved in the pathogenesis of chronic gvhd (cgvhd) and anti-b-cell therapy has been suggested for the treatment of cgvhd. we speculate that the presence of a monoclonal gammopathy after allogeneic transplant is expression of the activation of the b-cell compartment. a prospective study with a larger population should be considered, in order to confirm our results and assay post-transplantation monoclonal gammopathy as an early marker for gvhd development. disclosure of conflict of interest: none.inherited bone marrow failure (ibmf) syndromes are rare pediatric disorders that characteristically associate physical abnormalities, progressive bone marrow failure and predisposition to cancer. the most common of these disorders is fanconi anemia (fa). stem cell transplantation (sct) using related or unrelated donors are the only curative therapeutically approach when severe marrow failure is established. the aim of the study was to analyze the results of sct for patients with ibmfs in a single center. we performed a retrospective study in pediatric patients with ibmf admitted in pediatric hematology and bone marrow transplant department, fundeni clinical institute between january 2000 and september 2016. diagnosis and severityof ibmfs were established based on hematological results, bone marrow biopsy and clinical findings. genetic testing for ibmfs is not currently available in our country. indication for sct was established when patients developed moderate/severe aplastic anemia and became transfusion dependent.in case of dba, sct indication was established for steroid resistant disease. the donors were selected from family members or unrelated donors, 10/10 matched.the conditioning regimens used were reduced intensity (fludarabine 120-150 mg/m 2 , cy 20 mg/kg, f-atg 40 mg/kg) for af, dc and myeloablative (busulfan i.v., fludarabine 150 mg/m 2 , thiotepa 20 mg/kg, f-atg 30 mg/kg) for dba. gvhd prophylaxis consisted of standard methotrexate and csa/tacrolimus. all parents signed informed consent forms. in our center, between 2000 and 2016, 20 patients with ibmf were diagnosed: 10 (50%) patients with fa, 6 (30%) patients with diamond blackfan anemia (dba), 2 (10%) patients with diskeratosis congenita (dc), and 2(10%) patients with not classifiable ibmfs. the patient data is available in table 1 . seven out of 20 patients (35%) performed sct procedures: sibling 3 patients (2 patients with af, 1 patient with dc), mud 4patients (3 patients with af, 1 patient with dba). all patients (100%) engrafted for pmn (median = 17, range: 12-29 days) and platelet (median 21, range: 13-46 days).2/7 (42%) presented reactivation of cmv and received valganciclovir, 1/7 developed cmv disease (encephalitis and pneumonia), 2/7 (28%) developed bkv cystitis and required extensive hydration and levofloxacin. 4/7 (57%) developed grade i-ii skin acute gvhd day +100, which responded to topical treatment and low dose of corticosteroids. 1/7 (14%) developed grade iii intestinal acute gvhd, which responded to high-dose corticosteroids.1 /7 (14%) developed grade iv intestinal chronic gvhd (day +160), without response to high-dose corticosteroids, mmf and later died on day +221, due to infectious complications (severe pulmonary and cerebral aspergillosis). 6/7 patients (85%) are alive, with 100% donor chimerism 4/6(66%) or stable mixed chimerism 2/6 (33%). median follow-up for sct patients was 515 days (26 days-5y 6mo). conclusions in our study we observed a low incidence of severe complications associated with low mortality rate (14%) . sct is a procedure that associates multiple risk situations, but it remains the only curative cytomegalovirus (cmv) infections remain a significant cause of morbidity and mortality in patients whose immune systems are compromised, including hematopoietic stem cell transplant (hsct) recipients. although the adoptive transfer of third party cmv-specific t cells has proven both safe and clinically beneficial in treating even drug-refractory infections/disease, broader implementation and commercialization of this strategy has been hampered by (i) the postulated need for extensive cell banks generated from donors representing diverse hla profiles, and (ii) lack of large scale t cell manufacturing processes. to address these limitations we have developed a proprietary decision tool (cytomatch™) to identify a small panel of healthy donors who should provide almost universal hla coverage; and optimized a simple, scalable manufacturing process to generate large numbers of cmv-specific t cells. to assess the robustness of our strategy we generated a bank of cmv-specific t cells (viralym-c™) from 8 carefully selected healthy donors. the lines were polyclonal, comprising both cd4 + (21.3 ± 6.7%) and cd8 + (74.8 ± 6.9%) t cells, expressed central cd45ro+/cd62l+ (58.5 ± 4.2%) and effector memory markers cd45ro+/cd62l-(35.3 ± 12.2%), and were specific for the immunodominant cmv antigens ie1 and pp65 (ie1: 419 ± 100; pp65 1070 ± 31 sfc/ 2 × 10 5 , n = 8). a fixed-dose (2 × 10 7 cells/m 2 ) phase i clinical trial was subsequently initiated to test the safety and efficacy of these "ready to administer" t cells in pediatric and adult hsct recipients with drug-refractory cmv infections. using our bank of just 8 lines, we have identified a suitable line for 21 of 22 patients screened. of these, 7 patients have been treated with viralym-c cells; 6 received a single infusion and 1 patient required 2 infusions for sustained benefit. there were no immediate infusion-related toxicities; and despite the hla disparity between the viralym-c™ lines and the patients infused, there were no cases of de novo or recurrent graft versus host disease (gvhd). based on viral load (measured by quantitative pcr) and/or symptom resolution, viralym-c cells controlled infections in all patients with 5 complete (cr) and 2 partial responses (pr) achieved within 4 weeks of infusion. one patient with cmv retinitis had complete resolution of symptoms following viralym-c™ infusion. our results demonstrate the feasibility, preliminary safety and efficacy of "ready to administer" viralym-c™ cells that have been generated from a small panel of healthy, eligible cmv seropositive donors identified by our decision support tool. these data suggest that cost-effective, broadly applicable t cell anti-viral therapy may be feasible for patients following hsct and potentially other conditions. disclosure of conflict of interest: drs. juan vera, ann leen and brett giroir hold equity and drs. ifigeneia tzannou, sunitha kakarla are employed by viracyte. haploidentical stem cell transplantation (hsct) protocols utilizing ex vivo t-cell depleted grafts have been proven efficient in preventing graft versus host disease (gvhd), but cause a delay in early t-cell recovery that increases the risk of graft rejection, leukemia relapse and viral infections. conventional donor lymphocyte infusion (dli) after hsct transplantation is conditioned because of the high prevalence of gvhd even with low dose of t cells. here we present preliminary data of escalating cd45ro+ memory t cells as dli in three patients that received a selective graft depleted of naïve (cd45ra+) t-cells. three children that were transplanted following nonmyeloablative conditioning regimen with a graft consisting of cd34+ and cd45ra-cells, with mixed chimerism, lymphopenic and viral/opportunistic infections and minimal residual disease positive before hsct received dose scalating cryopreserved haploidentical cd45ra-memory t cell starting with a initial dose of 1 × 10 5 /kg, until a maximal dose of 1 × 10 8 /kg with a 21 days interval. we infused 10 products with a naïve (45ra+) t-cell dose less than 1 × 10 4 /kg with 499.9% purity of cd3 + cd45ro+ memory t-cells in all cases. all infusions were well tolerated without any side effect during infusions neither gvhd. following the dli, a progressive increase in t cell counts was observed. our preliminary data suggest that dose escalating of haploidentical memory t cells (45ro+) as dli provides a safety platform, even with high dose of t cells (1 × 10 8 /kg), for adoptive immunotherapy in haploidentical 45ra+ depleted grafts with no gvhd complications, and allows an increase in t cell reconstitution. however, efficacy of this strategy requires longer studies. relapse after allogeneic hematopoietic stem cell transplantation (allohsct) remains a major therapeutic challenge: outcome is very poor, without curative option in most cases. second allohsct may be considered in few selected patients because of anticipated limitations: (1) donor availability; (2) high toxicity due to previous treatments; (3) low efficacy considering the very advanced disease situation. we hypothesized that the use of post transplantation cyclophosphamide (pcy) haplo-sct after relapse following allohsct may deal in part with these limitations. in particular, the presence of full haplotype hla mismatch could provide a decisive antileukemic effect relative to alloreactivity. in absence of large series in this setting, we report here the outcome after haplosct for patients who relapse after a first allohsct. we retrospectively studied adult patients, who received a second pcy haplo-sct for hematological malignancies. patients were treated between 2009 and 2016. the objective was to assess both the feasibility and the efficacy of haplosct in this setting. twenty seven patients were included: median time between first allohsct and relapse was 11 months (range: 1-82). median age at second transplantation was 49 years old (range: 21-61). most of patients had acute myeloid leukemia (n = 12, 44%) or hodgkin lymphoma (n = 6 patients, 22%). fifteen the impact of minimal residual disease and its kinetics prior to different types of allogeneic hematopoietic stem cell transplantation on clinical outcomes in patients with acute myeloid leukemia z xiao-su 1,2 , l yan-rong 1 , yan-hong 1 , p xu-ying 1 , qian-jiang 1 , hao-jiang 1 , lan-ping, xu 1 , xiao-hui zhang 1,2 , yu-wang 1,2 , h xiao-jun 1,2 and c ying-jun 1 this study investigated the impact of minimal residual disease (mrd) and its kinetics prior to different types of allogeneic hematopoietic stem cell transplantation (hsct) on clinical outcomes in patients with acute myeloid leukemia (aml) in complete remission (n = 132). 107 patients who received unmanipulated haploidentical hsct and 25 patients who received hla-matched sibling hsct were enrolled. mrd measured using 8-color flow cytometry (fcm) at fixed time points before transplantation was retrospectively analyzed. the patients were divided into four groups based on mrd kinetics before transplantation: consistent negative, positive to negative, negative to positive and consistent positive. during the follow-up, total twenty (15.2%) patients underwent relapse. through unique variate analysis, none of mrd status at various time points before unmaipulated haploidentical transplantation was associated with clinical outcomes, as well as the dynamic change of mrd before hsct (p40.05), although the patients with consistent positive mrd before hsct seemed to have a relatively higher incidence of relapse (p = 0.151). one-year cumulative incidence of relapse (cir) were 11.2 ± 4.7% vs. 31.1 ± 13.8% in mrd consistent negative and consistent positive groups, respectively (p = 0.202). however, patients with positive mrd after the second chemotherapy or pre-mrd before hla-matched sibling hsct showed a significant poor outcomes including higher cir (p = 0.015 and both neuroblastoma (nrb) and rhabdomyosarcoma (rms) in childhood are the aggressive malignant disease with higher mortality. this paper aims to study the efficacy of autologous peripheral blood stem cell transplantation (apbsct) in the treatment of high risk advanced nrb and rms. 34 patients with high-risk stage iv nrb and 9 patients with advanced childhood rms were treated by apbsct in our hospital from october of 2010 to may of 2016. in the subgroup of nrb patients, 16 patients got complete remission (cr) and 1 patient got cru while 17 patients had tumor residual disease after intensive induction therapy before asct. the median age was 5.55 (1-9) years old. primary sites of the tumors included submaxilla (n = 2), cervical (n = 5), adrenal gland (n = 16)and retroperitoneal (n = 11). the conditioning regimen consisted of busulfan and melphalan (busulfan 1 mg/kg × 4d, melphalan 140mg/m 2 x1d) or cem regimen (carboplatin 600 mg/m 2 × 3d, etoposide 500 mg/m 2 × 3d, cyclophosphamide 1800 mg/ m 2 × 2d); the pathology of 9 stage iii childhood rms patients was embryonal rhabdomyosarcoma. there were 8 cases in cr and 1 case in partial remission (pr). the median age was 6.56 (3-13) years old. primary sites of the tumors included bladder (n = 1), left forearm (n = 3), retroperitoneal (n = 1), pelvic (n = 3) s513 and talus (n = 1). the conditioning regimen consisted of melphalan, cyclophosphamide and dactinomycin (melphalan 60 mg/m 2 × 3d, cyclophosphamide 1800 mg/m 2 × 2d, dactinomycin 0.0 13 mg/kg × 3d).there were 13 double apbsct cases (nrb n = 12, rms n = 1). all the relapse patients were treated with chemotherapy and radiation therapy. all the patients successfully underwent mobilization, collection and reinfusion. the time of hematopoietic reconstitution was (11. 0 ± 3) days, no severe toxicity was observed, no transplant-related death was found. with a median follow-up of 25.35(2-60) months, one of the patients was lost to follow-up. in the subgroup of nrb patients (n = 33): the 2-year event-free survival and total survival rate of all patients were 66.2% and 79.4%, respectively. the survival time of no recurrence was significantly different between the double transplantation group and single transplantation group (p o0.05). in the subgroup of rms patients (n = 9), 1 patient died, 6 patients live without pd(1 patients had double apbsct), 2 patients suffered recurrence but still alive. apbsct achieved good outcome in patients with high risk advanced nrb and rms. transplantation-related toxicities were tolerable. double apbsct significantly improved the depth of remission. disclosure of conflict of interest: none. transplantation outcomes of a once-daily intravenous busulfan and fludarabine conditioning for allogeneic hematopoietic stem cell transplantation in pediatric aml and high risk mds: single center experience in korea y-t lim, e-j yang and k-m park department of pediatrics, pusan national university children's hospital, yangsan, koreathere have recently been some reports suggesting that oncedaily intravenous busulfan as a conditioning regimen for hematopoietic stem cell transplantation (hsct) possibly reduces the toxicities without influencing the clinical outcome as compared with the traditional 4 times daily dosage schedule. but until recently there has been little research and limited data available on the safety and efficacy of oncedaily intravenous busulfan and fludarabine in pediatric allogeneic hsct. we report the outcomes for allogeneic hsc recipients, evaluating engraftment status, regimen related toxicities (rrt), and event free survivals (efs) after use of oncedaily intravenous busulfan and fludarabine conditioning for allogeneic hsct in children with aml and high risk mds in a single pediatric center of korea. from january 2005 to december 2015, 22 aml and 2 high risk mds children who received once daily iv busulfan/fludarabine based conditioning regimen for allogeneic hsct were reviewed, bu/flu ± atg consist of intravenous fludarabine (40 mg/m 2 ) and busulfan (110~130 mg/m 2 , once daily iv) on days -6 to -3, and antithymocyte globulin (atg) (3 mg/kg) on days -3 to -1. all patients received tacrolimus and mini-dose methotrexate (5 mg/m(2)) for graft versus host disease (gvhd) prophylaxis. 17 boys and 9 girls were enrolled with median age of 10.1 years (range: 0.6-17.9 years). the median period from diagnosis to transplantation was 7 months (range: 5-49 months). more than half of the patients had a matched sibling donor (n = 16, 62%), 27% patients (n = 7) had a matched unrelated donor, 8% patients (n = 2) had a mismatched unrelated donor, and the remaining 1 patient had a mismatched family donor. as a stem cell source, peripheral blood stem cells (pbsc) were 22 cases (85%), bone marrow and cord blood were 2 cases in each. the median follow-up for patients was 40 months. the median number of infused total nucleated cells and cd 34+ cells except cord blood transplantation were 9.2 × 10(8)/kg and 7.8 × 10(6)/kg. all patients including who received cord blood were successfully engrafted. the median time to absolute neutrophil count (anc) recovery (anc 4 500 × 10(6)/l) and platelet recovery (platelet4 20,000 × 10(6)/l) were 13 days, 18 days in each. the incidence of acute gvhd was 19.2%, while severe grade iii/iv gvhd was observed in only 2 patient (7.7%). there were only two cases (8.7%) of extensive chronic gvhd in this study. transplant-related toxicities were acceptable, there was no case with cns toxicity, eleven patients (42.3%) developed grade ii,iii mucositis and grade i-iii hepatic toxicity in twenty four (92.3%), but transient. there was 3 clinically diagnosed veno-occlusive disease (vod), but most recovered by fluid restriction and diuretics. nine patients (36%) showed positive cytomegalovirus (cmv) antigen/pcr but only one patient developed cmv colitis. eight patients died: 7 due to relapse/disease progression, 1 due to extensive chronic gvhd. the 5-year efs and overall survival were 62.2% and 66.1% respectively. at 3 year, the cumulative incidence of relapse was 19.2%. overall, once-daily intravenous busulfan and fludarabine was less toxic and effective as conditioning regimen in aml and high risk mds patients undergoing allogeneic transplantation in children. disclosure of conflict of interest: none. haploidentical stem cell transplantation from unmanipulated graft has becoming a practiced option for high risk hematological malignancies who lack a matched related or unrelated donor. lack of a matched sibling or unrelated donor (mud) can be a significant barrier to allogeneic transplantation in patients who stand to benefit from this procedure. hlahaploidentical donors are readily available for nearly all such patients. haplo transplantation has inherent advantages over mud transplantation including the lower cost of graft acquisition, greater availability of donors for ethnic minorities, and immediate access to the donor in patients in whom delay cerebral palsy (cp) is a heterogeneous group of conditions that result in permanent motor disability. it may occur due to perinatal hypoxic insults, developmental brain abnormalities, genetic diseases, traumatic or infectious causes. in general the condition is non-progressive, but improvement over time is rarely seen. various treatment methods have been used for the management of this disorder. however, there has been no absolute cure for cp. the ultimate goal of stem cells therapy is to use the regenerative capacity of the stem cells causing a formation of new tissues to replace the damaged tissue. the polish stem cell bank (pbkm) has provided wharton's jellyderived msc (wj-msc) for medical therapeutic experiment application in children with cp. wj-msc from third party donors were administered to 27 patients (pts) with cp aged from 1.6/12 to 16.9/12 (median age: 6 years and 1 month). twenty two pts have received infusions intravenously (i.v.), 1 pt intrathecally (i.t.), and 4 pts via both routes (first i.v., next i.t.). the cells were previously collected from healthy newborns, processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapour. msc immunophenotype was confirmed using flow cytometry assay. the pts have received from 1 to 6 infusions in intervals from 4 weeks to 6 months. median i.t. dose was 15 × 10 6 cells per infusion, while median i.v. dose was 1 × 10 6 cells/kg of body weight per infusion. each patient has been examined by the same neurologist at the day of each infusion and the result of examination has been described in a follow-up. twelve patients were diagnosed with epilepsy as comorbidity. eighteen pts (67%) showed positive changes in neurological examination after their treatment with wj-msc. almost half of the children experienced improvement of cognitive functions (12 out of 27 pts). muscle tension was reduced in 6 pts. improvement in the ability to concentrate, better contact with others and improved social interactions were observed in 19% of pts. correction of motility was noticed in 5 pts, 2 pts have experienced better quality of sleep. in 3 cases there has been a reduction in the number of epileptic seizures (1 pt even discontinued some of his medicines). there were no s515 noticeable changes in neurological examination of 2 patients. seven follow-up forms have been not received yet. the experiment data provide evidence that third-party donor wj-msc are suitable and efficient stem cells for treatment in patients with cp. however further and more extensive examination, with a greater number of patients is needed, which will be beneficial for far-reaching results. spina bifida (sb) is a congenital malformation resulting from failure of fusion in the spinal neural tube during embryogenesis. despite surgical repair of the defect, most patients who survive with spina bifida have multiple system damage due to neuron deficiency in the spinal cord. it has been confirmed that the mesenchymal stem cells (mscs) have the ability to survive, migrate and differentiate into cells of a neural lineage. wharton's jelly-derived mscs (wj-mscs) from third-party donors have high proliferation and differentiation potential along with non-immunogenic features, thus seem to be a promising stem cell source. the polish stem cell bank (pbkm) has provided wj-msc for clinical application in a medical therapeutic experiment for children with sb. eleven patients (pts) were qualified for administration of wj-mscs. three pts have been waiting so far for their therapy after bioethical committee approval. seven pts were in the middle of stem cell therapy (after 1 or 2 injections), 1 pt had finished one cycle of stem cell therapy (5 injections -ijs) and resumed therapy by administering a first dose of wj-mscs. the cells were previously collected from healthy newborns, processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapors. six pts have received infusions intravenously (median dose: 1.01 × 10 6 /kg body weight per infusion), and 1pt was given 1 injection of 40 × 10 6 cells intrathecally. each patient has been examined by the same neurologist at the day of each infusion and the result of examination has been written in a follow-up. there were 6 pts, who received at least 2 doses of wj-msc, and all of them showed positive changes in neurological examination. the important improvement, declared by pts, was in areas: pronunciation and/or self-reliance (3pts), movement of arms and/or legs (4pts), quality of life (3pts), core stabilization (1pt). only one adverse event occurred after third injection of wj-msc: 1 pt had nausea and a fever. in case of other pts it was too early to provide reliable feedback. the transplantation of wj-mscs could stimulate the mscs to differentiate towards sensory neurons. this could be one of the reasons of observed improvement of many vital functions in patients, after mscs treatment. this approach might have value in the experimental treatment of sensory neuron deficiency in spina bifida.